results about the author(s) asmaa m. zahran department of clinical pathology, south egypt cancer institute, assiut university, assiut, egypt hanaa nafady-hego department of microbiology and immunology, faculty of medicine, assiut university, assiut, egypt sawsan m. moeen department of internal medicine, clinical haematology unit, faculty of medicine, assiut university, assiut, egypt hanan a. eltyb department of medical oncology, south egypt cancer institute, assiut university, assiut, egypt mohammed m. wahman department of clinical oncology, south valley university, qena, egypt asmaa nafady department of clinical and chemical pathology, qena faculty of medicine, south valley university, qena, egypt citation zahran am, nafady-hego h, moeen sm, eltyb ha, wahman mm, nafady a. corrigendum: higher proportion of non-classical and intermediate monocytes in newly diagnosed multiple myeloma patients in egypt: a possible prognostic marker. afr j lab med. 2022;11(1), a1713. https://doi.org/10.4102/ajlm.v11i1.1713 note: doi of original article published: https://doi.org/10.4102/ajlm.v10i1.1296. correction corrigendum: higher proportion of non-classical and intermediate monocytes in newly diagnosed multiple myeloma patients in egypt: a possible prognostic marker asmaa m. zahran, hanaa nafady-hego, sawsan m. moeen, hanan a. eltyb, mohammed m. wahman, asmaa nafady published: 25 mar. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. in the published article, zahran am, nafady-hego h, moeen sm, eltyb ha, wahman mm, nafady a. higher proportion of non-classical and intermediate monocytes in newly diagnosed multiple myeloma patients in egypt: a possible prognostic marker. afr j lab med. 2021;10(1), a1296. https://doi.org/10.4102/ajlm.v10i1.1296, there was an error on page 3 under the ‘results’ section. the mean age of the mm patients should be 63.5 as per table 1 instead of 6.35. the first paragraph under the ‘results’ section is updated to: results baseline characteristics of newly diagnosed multiple myeloma patients and healthy controls the mean age of the mm patients was 63.5 ± 4.07 years, and the number of men (13) was double that of women (7) (table 1). the proportion of plasma cells in bone marrow was 39.7% ± 3.7% and that of the monoclonal band (m-protein) was 4.44 g/dl ± 2.8 g/dl. the authors apologise for this error. the correction does not change the study’s findings of significance or overall interpretation of the study’s results or the scientific conclusions of the article in any way. reviewer acknowledgement open accesshttp://www.ajlmonline.org page 1 of 1 we would like to take this opportunity to thank all of those who provided scientific and logistical support for this special issue of the african journal of laboratory medicine: should names have inadvertently been excluded from this list, the publisher apologises and undertakes to amend the exclusion in the next issue. ajlm african journal of laboratory medicine in an effort to facilitate the selection of appropriate peer reviewers for the african journal of laboratory medicine, we ask that you take a moment to update your electronic portfolio on www. ajlmonline.org for our files, allowing us better access to your areas of interest and expertise, in order to match reviewers with submitted manuscripts. if you would like to become a reviewer, please visit the journal website to register. to access your details on the website, you will need to follow these steps: 1. log into the online journal at http://www. ajlmonline.org 2. in your ‘user home’ [http://www.ajlmonline. org/index.php/ajlm/ user] select ‘edit my profile’ under the heading ‘my account’ and insert all relevant details, bio statement and reviewing interest. 3. it is good practice as reviewer to update your personal details regularly to ensure contact with you throughout your professional term as reviewer to the african journal of laboratory medicine. please do not hesitate to contact me if you require assistance in performing this task. chantal parkins submissions@ajlmonline. org tel: +27 (0)21 975 2602 fax: +27 (0)21 975 4635 182 heidi albert george alemnji timothy amukele linda andiric rosemary audu ronald ballard debi boeras rachel crane karidia diallo dennis ellenberger paula fernandes glen fine peter fonjungo thomas gachuki emily griswold eduardo gudo emmanuel idigbe zachary katz luciana kohatsu paul lee kitty linde talkmore maruta martin matu barbara mckinney fausta mosha sikhulile moyo christina mwangi jane mwangi nqobile ndlovu clement ndongmo john nkengasong michael noble alaine nyaruhirira bryan nyary phoebe nzombe chin yi ou elde mel paladar chantal parkins linda parsons bethanie rammer philip rotz lee schroeder connie sexton judith shang ritu shrivastava edwin shumba beth skaggs penny smorenburg suzanne taylor hamilton tilley ralph timperi corey white shanita williams isatta wurie tun ye this research has been supported by the us president’s emergency plan for aids relief (pepfar) through the us centers for disease control and prevention. the findings and conclusions in this article are those of the authors and do not necessarily represent the official position of the us centers for disease control and prevention. use of trade names is for identification purposes only and does not constitute endorsement by the us centers for disease control and prevention. slmta steering committee: eduardo gudo emmanuel idigbe ismae whyms jean-bosco ndihokubwayo john n. nkengasong (chair) michael wanga nguyen thi thuong trevor peter tsehaynesh messele slmta technical advisory group: ana malla de abreu anthony emeribe fausta mosha george alemnji judith shang katy yao kelebeletse mokobela luciana kohatsu nqobile ndlovu sibongile zimuto talkmore maruta (chair) vireak voeurng http://www.ajlmonline.org open access page 1 of 1 reviewer acknowledgement acknowledgement to reviewers in an effort to facilitate the selection of appropriate peer reviewers for the african journal of laboratory medicine, we ask that you take a moment to update your electronic portfolio on https://ajlmonline.org for our files, allowing us better access to your areas of interest and expertise, in order to match reviewers with submitted manuscripts. if you would like to become a reviewer, please visit the journal website and register as a user. in order to be considered, please email submissions@ajlmonline. org indicating your intention to register as a reviewer for the journal. to access your details on the website, you will need to follow these steps: 1. log into the online journal at https:// ajlmonline.org 2. in your ‘user home’ [https://ajlmonline.org/ index.php/ajlm/user] select ‘edit my profile’ under the heading ‘my account’ and insert all relevant details, bio statement and reviewing interest(s). 3. it is good practice as a reviewer to update your personal details regularly to ensure contact with you throughout your professional term as reviewer to the african journal of laboratory medicine. please do not hesitate to contact us if you require assistance in performing this task. publisher: publishing@aosis.co.za tel: +27 21 975 2602 the editorial team of the african journal of laboratory medicine recognises the value and importance of the peer reviewer in the overall publication process – not only in shaping the individual manuscript, but also in shaping the credibility and reputation of our journal. we are committed to the timely publication of all original, innovative contributions submitted for publication. as such, the identification and selection of reviewers who have expertise and interest in the topics appropriate to each manuscript are essential elements in ensuring a timely, productive peer review process. we would like to take this opportunity to thank and recognise the following reviewers for their precious time and dedication, regardless of whether the papers they reviewed were finally published. we apologise for any names that have been inadvertently left out. these individuals provided their services to the journal as a reviewer from 01 october 2022 to 30 september 2023. adama a. ahmad francis ajeneye moses o. akiibinu abdul-azeez a. anjorin george aryee seth a. attoh oluwatoyin a. babalola ebi c. bile macdonald m. chaava bettina chale-matsau adon chawe yazied chothia modibo coulibaly rotimi a. dada mohammed b. danlami tjeerd datema ibrahim i. daud alpha a diallo yakhya dieye adetoun ejilemele uchenna s. ezenkwa eyiuche d. ezigbo raymond d. fatchu tusabe fred renuka gadde kerusha govender lisa graham horace gumba samson m. haumba leriska haupt sandra havyarimana lucius c. imoh emmanuel o. irek prenika jaglal lavania joseph sudhakar kalvatala abera a. kitaba jean f. kloppers winny koster modupe a. kuti sylvester y. lokpo ako-egbe louis talkmore maruta gerald mboowa margaret mokomane gene morse shaheen mowla sikhulile moyo takudzwa j. mtisi timothy m. mtonga wilson mtotela graham mutandi leonard mutema jane mwangi koumpingnin nebie lucy o. nyangau simon o. nyangena collins o. odhiambo joseph m.o. oduor abiodun r. ojewuyi onyekachukwu f.i. okeke olajumoke oladipo ahmed olowo-okere adesuyi a. omoare nyamongo onkoba augustine a. onyeaghala japheth a. opintan sophia osawe joachim potgieter pasipanodya i. ruredzo kwabena a.n. sarpong lee schroeder francois smit wendy l. solomon ajaykumar k. thirumala fabio timeus ralph timperi lara vojnov reubina wadee musau wakabongo james w. wambugu camila m. wilhelm sheila m. woodcock azza a. zulfu http://www.ajlmonline.org� https://ajlmonline.org mailto:submissions@ajlmonline.org mailto:submissions@ajlmonline.org https://ajlmonline.org https://ajlmonline.org https://ajlmonline.org/index.php/ajlm/user https://ajlmonline.org/index.php/ajlm/user mailto:publishing@aosis.co.za acknowledgement to reviewers article information authors: mary n. mataranyika1 landine k. beukes1 affiliations: 1ministry of health and social services, namibia correspondence to: mary mataranyika postal address: private bag 70046, khomasdal, windhoek, namibia dates: received: 14 may 2014 accepted: 15 sept. 2014 published: 03 nov. 2014 how to cite this article: mataranyika mn, beukes lk. view from the top: involvement of namibia’s health ministry in laboratory quality improvement. afr j lab med. 2014;3(2), art. #195, 2 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.195 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. view from the top: involvement of namibia’s health ministry in laboratory quality improvement in this commentary... open access • introduction • implementing laboratory quality management in namibia    • lessons learned    • sustainability • acknowledgements    • competing interests    • authors’ contributions • references introduction top ↑ laboratories form the backbone of health systems, providing critical test results that aid diagnoses and identify the causes of disease outbreaks. accurate laboratory results improve medical interventions, minimise surgical and treatment errors, decrease the length of hospital stays and reduce the cost of hospitalisation.1 weak reporting systems and delayed or inaccurate processing of test results contribute to increased morbidity and mortality from major diseases of public health concern, such as tuberculosis, malaria, cholera and hiv.2in 2012, namibia’s ministry of health and social services (mohss), working with stakeholders from both the private and public sectors, developed namibia’s first national public health laboratory policy3 and strategic plan4 (approved by the cabinet and launched in 2013) to guide the delivery of quality laboratory improvements and services. according to the laboratory policy, the mohss is mandated to offer high-quality laboratory services; support clinical care providers in disease treatment and prevention; provide disease surveillance and response; and develop policies that adhere to the national health principles of service equity, accessibility, affordability and sustainability.3,4 in order to address issues of poor reporting systems and inaccurate diagnostic results, the mohss provides oversight over the namibia institute of pathology, the blood transfusion service of namibia and all private laboratories. the government of namibia endorses the world health organization (who) resolutions and programs aimed at laboratory improvements, including the 2008 maputo declaration to strengthen laboratory systems,5 the who regional office for africa’s (afro) stepwise laboratory quality improvement process towards accreditation (slipta) framework6 and the strengthening laboratory management toward accreditation (slmta) programme.7 implementing laboratory quality management in namibia top ↑ in 2010, the mohss, with support from partners, began to implement the slmta programme and slipta framework. the national public health laboratory policy encourages all laboratories to embrace slmta and slipta, and a concerted effort is now being planned to assist the country's 64 laboratories to implement quality improvement. in june 2012, the mohss supported eight people from six namibian laboratories to participate in a series of slmta workshops held at the zimbabwe national quality assurance programme in harare, zimbabwe. slmta in namibia follows the standard three-workshop model, with improvement project implementation periods of approximately two to four months following each workshop.7 progress of the enrolled laboratories is monitored through audits using the slipta checklist. in addition to overall score and star ratings on a zeroto five-star scale, the checklist evaluates 12 quality areas. the african society for laboratory medicine (aslm) and the clinical and laboratory standards institute (clsi) conducted official slipta audits for four of the laboratories post-slmta; the remaining two laboratories did not receive audits because of lack of permission. one laboratory achieved three stars, two laboratories achieved two stars and one laboratory received zero stars. the highest scoring quality areas were client management, facilities and safety and purchasing and inventory. the lowest scores were observed in internal audit, management reviews and corrective action. internal audits were either not being carried out or were conducted inconsistently. management reviews did not address all appropriate topics and did not include action plans, responsibilities, timelines or status information. most corrective actions and action plans were conducted by untrained staff. lessons learned to impact health outcomes and improve laboratory services, all stakeholders – including government agencies, funding bodies and staff – must be involved in and committed to achieving shared laboratory improvement goals. it is suggested that ministries of health adopt quality practices and standards as an integral part of their programmes and understand that external reviews and the accreditation of medical laboratories involve more than the mere assessment of conformance to standards for organisational processes. in namibia, there is a need for the mohss and the audited laboratories, with the assistance of aslm, to jointly develop and follow up on a plan of action to address the gaps identified in the audits. amongst the gaps in staff knowledge identified during these audits were how to manage non-conforming events; how to conduct internal audits and root cause analysis; and how to carry out preventive and corrective actions and monitor their effectiveness. in addition, staff lacked necessary skills in management of quality control and monitoring for trends, biases and shifts, as well as mulitirule (also called ‘westgard rules’) violations and client/customer survey feedback. nonconformities should be shared routinely with staff so that they can implement corrective actions. furthermore, on-site quality assurance responsibilities for the chief medical laboratory technologists/technologists in charge were overloaded. additional leaders should be trained in international organization for standardization (iso) 15189:2012 standard requirements and management responsibilities. it would be advisable for the mohss to develop a system of assessing all laboratories and addressing gaps identified, and then to use those assessments as a means of ensuring that quality becomes a condition for re-registration or licensing to operate a laboratory. sustainability an essential component in implementing a new programme is to plan for sustainability. the mohss is taking ownership of the quality improvement programmes by budgeting for future implementation of slmta and slipta activities throughout the country. the mohss plans to build sustainability into its laboratory quality initiatives by increasing the number of in-country auditors, mentors and trainers, as well as by hosting training-of-trainers workshops so as to remove dependency on costly, external implementers. it is anticipated that this strategy will build capacity and promote self-sufficiency in namibian medical laboratories. the mohss plans to build capacity for writing grant applications to support slmta and slipta, trainings, operational research, dissemination of information and integration of all laboratory services with the aim of attaining accreditation to iso standards in select laboratories. it would be advisable for the mohss to set up a national structure to audit and recognise laboratories; encourage development of innovative procedures and methods; allocate funding for equipment maintenance; and collaborate with accreditation bodies and other institutions in order to implement laboratory quality improvements. as quality improvements become institutionalised in hospital laboratories, it is becoming evident that entire hospital systems are in dire need of similar quality improvement programmes. the namibia mohss calls on international agencies to develop and adapt programmes such as slipta and slmta for use throughout hospital systems so as to ensure continuous quality patient care. acknowledgements top ↑ we would like to acknowledge the mohss permanent secretary, mr. andrew ndishishi; the mohss director, ms. p.k. nghipandulwa; and the us centers for disease control and prevention (cdc) office in namibia, for their continuous encouragement toward this cause. we would also like to thank mr. harold kaura of the namibia institute of pathology, mr. theron slinger of maxi medical laboratory, dr. van finckenstein of the national blood transfusion service of namibia, caprivi pathology centre laboratory management; and the permanent secretary of the ministry of defence for allowing their staff to participate in this programme. much appreciation is also given to talkmore maruta (aslm) and patrick mateta (clsi) for constructive criticism of the mohss during the audits. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions m.n.m. (mohss) was the lead author and researcher for this article. l.k.b. (mohss) was co-author and reviewed and revised the manuscript. references top ↑ 1. nkengasong jn, nsubuga p, nwanyanwu o, et al. laboratory systems and service are critical in global health: time to end the neglect? am j clin pathol. 2010;134(3):368–373. http://dx.doi.org/10.1309/ajcpmpsinq9brmu62. peter tf, rotz pd, blair dh, et al. impact of laboratory accreditation on patient care in the health system. am j clin pathol. 2010;134(4):550–555. http://dx.doi.org/10.1309/ajcph1skq1hnwghf 3. namibia ministry of health and social services, national public health laboratory policy (establishing a strong public health laboratory system) [document on the internet]. c2012 [cited 2014 oct 14]. available from: http://www.mhss.gov.na/files/downloads/dcc_3182_nphl_policy_final_new%20copy.pdf 4. namibia ministry of health and social services, national public health laboratory strategic plan (establishing a strong public health laboratory system) [document on the internet]. c2012 [cited 2014 oct 14]. available from: http://www.mhss.gov.na/files/downloads/687_3184_nphl_strategic%20plan_final_new%20copy.pdf 5. world health organization regional office for africa. the maputo declaration on strengthening of laboratory systems: [document on the internet]. c2008 [cited 2013 jan 02]. available from: http://www.who.int/diagnostics_laboratory/maputo-declaration_2008.pdf 6. world health organization’s regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation in the african region (with checklist) [document on the internet]. c2012 [cited 2014 oct 20]. available from: http://www.afro.who.int/en/clusters-a-programmes/hss/blood-safety-laboratories-a-health-technology/blt-highlights/3859-who-guide-for-the-stepwise-laboratory-improvement-process-towards-accreditation-in-the-african-region-with-checklist.html 7. yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin path. 2010;134(3):401–409. http://dx.doi.org/10.1309/ajcpnbbl53fwuiqj introduction meeting structure and details opportunities and threats highlighted in interviews with key stakeholders summary and next steps references about the author(s) timothy amukele department of pathology, johns hopkins university school of medicine, united states citation amukele t. africa cdc: establishing integrated surveillance and laboratory networks for rapid disease detection and response, control, prevention, and clinical care in africa. afr j lab med. 2017;6(1), a638. https://doi.org/10.4102/ajlm.v6i1.638 editorial africa cdc: establishing integrated surveillance and laboratory networks for rapid disease detection and response, control, prevention, and clinical care in africa timothy amukele copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction there is little similarity between asbestos, hiv, and the severe acute respiratory syndrome coronavirus. however, their histories illustrate the three keys for effective detection and control of disease: clinical identification, laboratory detection, and public response. in diseases where all elements of this three-step framework were in place, the time from initial observation to appropriate public response tended to occur quickly. for example, in late november 2002, an atypical pneumonia that ultimately came to be known as severe acute respiratory syndrome made its appearance in china and a few other countries in east asia. once the world health organization was notified in february 20031, a massive coordinated response was deployed. the causative agent was identified within two weeks2 and the disease was halted by effective quarantine in 42 days3. if we want to support similar rapid responses to current and future epidemics, we need to create systems that foster the aforementioned three keys: clinical identification of disease, laboratory detection of disease, and public response to disease. the central piece of these three pillars is laboratory detection, which requires a good laboratory workforce and networks. in keeping with this central role, workforce and laboratory network development were the twin goals of the three-day africa centres for disease control (cdc) workshop held in addis ababa from 27 to 29 march 2017. meeting structure and details the workshop, titled ‘innovative approaches to establishing and strengthening regional integrated surveillance and laboratory networks for disease control, prevention, and clinical care’, was jointly supported by the africa cdc, the world health organization (who), and the african society for laboratory medicine (figure 1). the workshop had participants from both the private and public sectors who had a role in the laboratory sector in africa. each day was divided into brief morning talks focused on sharing best practices and case studies, and afternoon breakout sessions focused on addressing a list of key questions that were relevant to each day’s topic. figure 1: attendees of the ‘innovative approaches to establishing and strengthening regional integrated surveillance and laboratory networks for disease control, prevention, and clinical care’ workshop. the workshop was jointly supported by the africa cdc, the world health organization, and the african society for laboratory medicine, and included participants from both the private and public sectors with a role in the laboratory sector in africa. the first day of the meeting focused on laboratory networks currently in africa. two themes that were highlighted in the morning talks were the need for mapping of laboratories, as well as success stories in laboratory mapping from different african regions. briefly, dr pascale ondoa presented the labnet scorecard4, which measures laboratory network preparedness, maturity, and adherence to regulations. dr brian wood presented a report on a video-conferencing technology that trains primary care providers on how to treat complex diseases — the ‘extension for community healthcare outcomes (echo)’ model5. dr rebecca martin shared the experience of establishing the east african public health laboratory network. mr adjane kossivi koura shared the impressive work of resaolab, which has over 700 laboratories in its laboratory-strengthening network spread over seven west african countries6, and dr naima el mdaghri talked about laboratory networks in north africa. the second day of the meeting focused on antimicrobial resistance (amr). four of six talks shared country examples of amr best practices and the two others were by international partners. dr christopher larson gave the first of the international-partner talks with an introduction of the who’s global antimicrobial resistance surveillance system (glass). glass, started in may 2015, is a harmonised system to collect worldwide amr data. the second talk, given by dr john wilson, was an introduction to the fleming fund, a uk-funded initiative focused on helping lowand middle-income countries perform amr surveillance as well as build capacity. the rest of the morning talks were examples of best practices from various african regions. dr tenaw andualem discussed the amr control strategy in ethiopia, with emphasis on the key role that engaging the media plays in public education, dr kone louis penali shared the amr update and control strategy from côte d’ivoire, dr mohamed genedy presented the amr update and control strategy in egypt, and dr ondoa shared a talk from the cdc’s senegal regional collaborating centre titled ‘what can a framework for surveillance laboratory networks for antimicrobial resistance in five regions of africa look like? role of africa cdc regional collaborating centres’. the afternoon breakout sessions were also on the topic of amr and were guided conversations focused on various aspects of establishing amr surveillance, intervention and control, including the role of private and international partners. all groups returned with clear recommendations on the role africa cdc could play in amr going forward. day three of the symposium focused on two topics: laboratory workforce training and the role of the private sector (non-governmental organisations and private laboratories) in public health. as with the prior days of the workshops, the talks focused on best practices from various african regions. dr jane carter discussed the critical role that non-governmental organisations play in advancing laboratory medicine and public health in africa, through the framework of experiences from the african medical and research foundation’s 50-year history. this was followed by dr tamrat bekele, founder and ceo of international clinical laboratory (icl), who discussed icl’s support of the public healthcare sector in ethiopia. icl is a joint commission-accredited laboratory that has been in operation for 13 years in addis ababa. one example of many was the provision of full laboratory testing to debrebirhan hospital, located about 100 km east of addis ababa, as well as expanding microbiological culture to those public hospitals without the capacity to provide this service. dr trevor peter next talked about the laboratory workforce development programmes. he introduced the audience to a new programme developed by the african society for laboratory medicine for a community laboratory quality corps, which is focused on ensuring quality at the lower cadres of the health care system. dr samantha dittrich shared the american public health laboratory’s ‘lab manager training’ programme, and dr patrick nguku closed the presentations by presenting his work developing an epidemiology workforce through the field epidemiology and laboratory training program in nigeria7. opportunities and threats highlighted in interviews with key stakeholders there was a palpable air of excitement and cautious optimism at this inaugural laboratory workshop of the africa cdc. i interviewed two key stakeholders in an effort to capture some of their voices about the workshop and the way forward. the following were their responses to the question, ‘what do you see as the way forward for strengthening laboratory networks and workforce development in africa’? dr john nkengasong (inaugural director, africa cdc) the first outcome of this meeting is the launch of the africa cdc regional integrated surveillance laboratory network (rislnet). this will be a vehicle to drive networking in the five geographical regions. the five rislnets will be in zambia, kenya, gabon, nigeria, and senegal. the goal is to have national public health institutes in each region anchored within the rislnet. the close networking of the rislnets with national players will help to drive regionally appropriate plans for amr, pandemic preparedness, and rapid response, among others. the second outcome of this meeting is the establishment of the africa cdc anti-microbial resistance and surveillance network (africa cdc amrsnet). the goal would be to standardise the approach to amr. the africa cdc amrsnet will work closely with who’s glass. africa cdc amrsnet will strive to achieve quality data across the region, establish mentorship programmes, and centres of excellence to drive amr work. the third outcome of this meeting is to highlight and improve the role of the private sector laboratories in supporting public health work in africa. the private sector is well equipped to extend the reach and capacity of the public health role that africa cdc is serving in the community. the most important thing for me is: ‘is the african patient being served?’ professor alash’le abimiku (chair of the board, african society for laboratory medicine) i am hoping that africa cdc will use the wide array of best practices and resources that have been highlighted during this meeting to improve what it does as far as disease surveillance, prevention, control of outbreaks, laboratory workforce development, etc. knowledge of the tools and possibilities out there will help africa cdc to advocate more appropriately, based on a true understanding of the opportunities and challenges on the ground. secondly, i am hoping for better harmonisation of policies and programmes across borders. even though this might require modification of what individuals and institutions are doing, this work is critical because we as africans will have more political traction globally as a collective voice rather than singly. a good example of such collaboration is how we utilise the three to four biosafety level 4 laboratories in the continent. if we all work together within our respective regions we can take advantage of existing infrastructure rather than building from scratch. we all need the mindset that harmonisation, networking, and collaboration are value-added. the africa cdc has an incredible leader that is taking the advantage of this meeting to bring the best minds and ideas to the table. the success of the regional collaborating centres will depend largely on their ability to continue to think in this open-minded, proactive, and inclusive way. summary and next steps the goal of the africa cdc is to, in their own words, ‘safeguard africa’s health’. achieving this goal on a population level requires three things: clinical acumen to diagnose disease, coordinated laboratory networks to diagnose disease, and an appropriate response to control disease. the workshop in march in addis ababa focused primarily on building and sustaining laboratory networks to aid disease detection and control. while there were many excellent ideas that came out of the breakout sessions suggesting various ways forward for the africa cdc, what struck me was the abundance of positive examples from various regions of africa. these examples from the private and public sector are already effective in the region, and have in some cases been sustained for decades. success is inevitable if the africa cdc serves as the custodian of these success models. the african journal of laboratory medicine encourages submissions that highlight these examples of best practices. references world health organization multicentre collaborative network for severe acute respiratory syndrome diagnosis. a multicentre collaboration to investigate the cause of severe acute respiratory syndrome. lancet lond engl. 2003 may 17;361(9370):1730–3. peiris jsm, lai st, poon llm, guan y, yam lyc, lim w, et al. coronavirus as a possible cause of severe acute respiratory syndrome. lancet lond engl. 2003 apr 19;361(9366):1319–25. parashar ud, anderson lj. severe acute respiratory syndrome: review and lessons of the 2003 outbreak. int j epidemiol. 2004 aug;33(4):628–34. ondoa p, datema t, keita-sow m-s, oskam l, ndihokubwayo j-b, isadore j, et al. a new matrix for scoring the functionality of national laboratory networks in africa: introducing the labnet scorecard. afr j lab med. 2016 oct 31;5(3): 9 pages. arora s, thornton k, murata g, deming p, kalishman s, dion d, et al. outcomes of treatment for hepatitis c virus infection by primary care providers. n engl j med. 2011 jun 9;364(23):2199–207. l d, jl m, i s, r d, j s, a n, et al. [resaolab: west african network of laboratories to enhance the quality of clinical biology]. bull soc pathol exot 1990. 2015 feb;108(1):36–40. nsubuga p, johnson k, tetteh c, oundo j, weathers a, vaughan j, et al. field epidemiology and laboratory training programs in sub-saharan africa from 2004 to 2010: need, the process, and prospects. pan afr med j. 2011;10:24. article information authors: philip h. fortgens1,2 fierdoz omar1,2 affiliations: 1department of clinical laboratory sciences, division of chemical pathology, university of cape town, south africa2national health laboratory service, groote schuur hospital, south africa correspondence to: philip fortgens postal address: division of chemical pathology, university of cape town/national health laboratory service, groote schuur hospital, anzio road, observatory, cape town, 7925 dates: received: 09 dec. 2011 accepted: 20 sep. 2013 published: 03 dec. 2013 how to cite this article: fortgens ph, omar f. cardiac troponin t quantitative assay failure as a result of antibody interference. afr j lab med. 2013;2(1), art. #23, 3 pages. http://dx.doi.org/10.4102/ ajlm.v2i1.23 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. cardiac troponin t quantitative assay failure as a result of antibody interference in this oorspronklike navorsing: wiskundeonderrig... open access • abstract • introduction • research method and design    • case    • interference experiments • results • discussion • conclusion • acknowledgements    • competing interest    • authors’ contributions • references abstract top ↑ background: immunoassays are prone to interference by various substances which may cause inaccurate results. this type of interference is difficult to detect analytically.objective: a case of cardiac troponin t quantitative reader (roche diagnostics) assay failure was detected and investigated in order to ascertain the likely cause. method: patient whole blood was mixed with cardiac troponin t-positive blood, patient and control sera were denuded of immunoglobulin g by protein a-affinity chromatography and patient sera were mixed with mouse serum. samples were analysed on a cardiac troponin t quantitative reader. results: a mixture of patient whole blood and cardiac troponin t-positive blood resulted in assay failure; removal of immunoglobulin g from patient sera reversed the cardiac troponin t assay failure; the addition of mouse serum as a heterophile antibody blocking agent had no effect. conclusion: it is proposed that the interference resulting in assay failure may not be because of a heterophile antibody, but rather a result of a circulating autoantibody to cardiac troponin t, which may compete with antibody assay reagents for binding sites. introduction top ↑ a consensus document released by the european society of cardiology, the american college of cardiology, the american heart association and the world heart federation task force in 2012 has proposed cardiac troponin (ctn) as the preferred biomarker for myocardial necrosis because of its superlative myocardial tissue specificity and high clinical sensitivity.1 furthermore, ctn has also been shown to have value for the prediction of adverse cardiovascular events in patients presenting with acute coronary syndrome.2 cardiac troponin t (ctnt) appears to be an important marker of coronary heart disease, mortality and risk of heart failure in a healthy population without manifest cardiovascular disease.3measurement of cardiac troponins is achieved by immunoassay. despite extensive experience with this methodology, however, immunoassays are occasionally subject to interfering substances that compromise their accuracy – indeed, it is estimated that antibody interference affects approximately one in 2000 immunoassay results.4 we report a novel case of assay failure using the cardiac troponin t quantitative reader (roche diagnostics). research method and design top ↑ case a 61-year-old female, with a history of ischaemic heart disease and hypertension, presented to the emergency unit on two occasions 12 days apart with chest discomfort. repeated attempts by the diagnostic laboratory to obtain ctnt measurements failed, as reflected by the absence of a positive control line on test strips (cardiac troponin t quantitative reader, roche diagnostics; figure 1). as the creatinine kinase level was within normal limits (26–140 u/l) at both visits and the myoglobin was normal (7–64 ng/l) when measured at the second visit, the patient was discharged with follow-up. figure 1: absence of a control line on the roche cardiac troponin t quantitative test strip. interference experiments antibody interference was suspected and the following investigation was thus performed. prior ethics approval was not obtained as the investigation would lead to improvement in this patient’s management. firstly, a 1:1 mixture of the patient’s sample and a recently-assayed anonymous sample positive for ctnt (both heparinised whole bloods), was assayed for ctnt.5 secondly, patient and control plasma samples were depleted of immunoglobulin g (igg) using protein a-affinity chromatography.6 these samples were analysed for ctnt prior to and after igg depletion. the cardiac troponin t quantitative reader is a lateral flow immunoassay, utilising the sandwich principle on a test strip with two murine monoclonal anti-ctnt antibodies.7 thirdly, in order to exclude the presence of interfering human anti-mouse antibodies (hama), mouse serum was added to the patient plasma (1:4) and the mixture was incubated for one hour at room temperature, following which the ctnt was measured. lastly, to determine whether the automated ctnt assay on the roche elecsys e170 analyser was subject to the same interference, dilutions of a known ctnt-positive plasma sample mixed with the patient plasma were assayed for ctnt. results top ↑ the mixture of whole blood patient sample and a ctnt-positive specimen inhibited the formation of the control line on the ctnt reagent strip, supporting our suspicion of an interfering substance. whilst only the control sample elicited a control line prior to igg depletion (ctnt < 0.03 ng/ml), both the patient and control samples elicited control lines after igg depletion (ctnt < 0.03 ng/ml), suggesting that igg was the interfering substance. test-strips contain hama-blocking antibodies,7 but despite the presence of additional blocking agent (mouse serum), the control line did not develop, which suggested strongly that the interfering igg was not an hama (table 1). dilutions of a known ctnt-positive plasma sample with the patient plasma showed a linear response when assayed for ctnt on the roche elecsys e170 analyser, suggesting that this platform is not subject to the same autoantibody interference. table 1: cardiac troponin t quantitative test strip performance. discussion top ↑ there is increasing use of ctn measurement in the diagnosis of myocardial injury and any inaccuracy with regard to measured values is more likely to have serious clinical impact than would be the case with immunoassays for many other analytes. several immunochemical interferences have been well described, including heterophile antibodies,8 rheumatoid factor9 and circulating troponin autoantibodies.10,11 recent studies have shown that 15.9% of samples from cohorts of normal blood donors were positive for autoantibodies to troponin t or troponin i and 10.9% were positive for both.12 autoantibody–antigen macrocomplexes have been well described for a number of biomarkers such as salivary amylase,13 creatine kinase,14 aspartate aminotransferase15 and lactate dehyrogenase,16 but there is no evidence to suggest that these complexes contribute to a pathological process. in contrast, ctn autoantibodies have been shown to contribute to the progression toward heart failure in mice and a similar process may occur in humans.17 furthermore, it has been proposed that the reduced clearance of immunoglobulin–cardiac troponin complexes from the circulation may result in increased levels of measured troponins.18 this phenomenon does not, therefore, represent assay interference, but rather an analytically-true result, albeit misleading. this study shows that the removal of igg from patient serum reverses the ctnt assay failure and that the addition of mouse serum as a heterophile antibody-blocking agent has no effect. this, together with the quantifiable result of the myoglobin assay (roche diagnostics), which uses the same test principle, species of monoclonal antibody and reader as the ctnt assay, suggests that the interference may not be because of a heterophile antibody, but rather because of an autoantibody to ctnt. such an antibody may compete with the mouse anti-ctnt–gold complex for binding to immobilised ctnt on the test-strip, which normally serves as a test control. in the case of the myoglobin assay, the immobilised control antigen (myoglobin) is different, which may explain why it is unaffected. an autoantibody could also compete with reagent anti-ctnt antibodies for binding sites, thereby preventing the development of a test line (figure 2). there have been several reports of positive interference in the ctnt assay by heterophilic antibodies with the cardiac troponin t quantitative test7 and the elecsys e1708 automated platform but, to the best of our knowledge, this is the first report of ctnt assay failure on the former platform. we conclude that the failure of this assay is a result of putative autoantibodies to ctnt. assay failure also allowed for the immediate detection of potential interference, but the majority of cases of interference remain undetected at the analytical level. it is crucial, therefore, that any discrepancies between results and the clinical picture be addressed by clinicians with the laboratory, to avoid further potentially-invasive and expensive investigations. figure 2: possible effect of blocking human anti-cardiac troponin t antibodies on the roche cardiac troponin t quantitative lateral flow immunoassay. conclusion top ↑ a case of assay failure was detected in the cardiac troponin t quantitative reader. immunoassays are subject to interference and it is important to investigate anomalous results to exclude such assay interference. after removing igg from patient serum and re-assaying the ctnt, assay failure was found to reverse. this case highlights the fact that immunoassay interference remains a persistent problem and vigilance is encouraged in order to minimise its impact. furthermore, in the case of troponins, interference may be caused by circulating autoantibodies to ctn. acknowledgements top ↑ we thank noel markgraaf for mouse serum, roche diagnostics for the elecsys e170 troponin t assay kit and dr judy king for her critical appraisal of the manuscript.this work was submitted as a presentation at the federation of south african pathology societies 50th annual congress, september 2010 and as a poster at the international federation of clinical chemistry and laboratory medicine (ifcc) worldlab and euromedlab congress, berlin, 2011. competing interest the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions f.o. (university of cape town/nhls) initiated the study, contributed to project design and critically revised the manuscript. p.h.f. (university of cape town/nhls) contributed to study design, performed the experiments and wrote the manuscript. references top ↑ 1. thygesen k, alpert js, jaffe as, et al. on behalf of the joint esc/accf/aha/whf task force for the universal definition of myocardial infarction. third universal definition of myocardial infarction. eur heart j. 2012;33:2551–2567. http://dx.doi.org/10.1093/eurheartj/ehs184, pmid:22922414 2. aldous sj, florkowski cm, crozier ig, et al. high sensitivity troponin outperforms contemporary assays in predicting major adverse cardiac events up to two years in patients with chest pain. ann clin biochem. 2011;48(pt 3):249–255. http://dx.doi.org/10.1258/acb.2010.010220, pmid:21441393 3. saunders jt, nambi v, de lemos ja, et al. cardiac troponin t measured by a highly sensitive assay predicts coronary heart disease, heart failure, and mortality in the atherosclerosis risk in communities study. circulation. 2011;123(13):1367–1376. http://dx.doi.org/10.1161/circulationaha.110.005264, pmid:21422391, pmcid:pmc3072024 4. levinson ss, miller jj. towards a better understanding of heterophile (and the like) antibody interference with modern immunoassays. clin chim acta. 2002;325(1–2):1–15. http://dx.doi.org/10.1016/s0009-8981(02)00275-9 5. chan ao, chan jp, choi kl, et al. a patient with an increased troponin level without evidence of ischaemic cardiac injury. hong kong med j. 2004;10(4):277–279. pmid:15299174 6. selby c. interference in immunoassay. ann clin biochem. 1999;36(pt 6):704–721. pmid:10586307 7. white gh, tideman pa. heterophilic antibody interference with cardiac t quantitative rapid assay [letter]. clin chem. 2002;48(1):201–202. pmid:11751561 8. shayanfar n, bestmann l, schulthess g, et al. false-positive cardiac troponin t due to assay interference with heterophilic antibodies [letter]. swiss med wkly. 2008;138(31–32):470. pmid:18690561 9. onuska kd, hill sa. effect of rheumatoid factor on cardiac troponin i measurement using two commercial measurement systems [letter]. clin chem. 2000;46(2):307–308. pmid:10657400 10. bohner j, von pape kw, hannes w, et al. false-negative immunoassay results for cardiac troponin i probably due to circulating troponin i autoantibodies [letter]. clin chem. 1996;42(12):2046. pmid:8969651 11. legendre-bazydlo la, haverstick dm, kennedy jl, et al. persistent increase of cardiac troponin i in plasma without evidence of cardiac injury. clin chem. 2010;56(5):702–705. http://dx.doi.org/10.1373/clinchem.2009.138164, pmid:20427735 12. adamczyk m, brashear rj, mattingly pg. coprevalence of autoantibodies to cardiac troponin i and t in normal blood donors [letter]. clin chem. 2010;56(4):676–677. http://dx.doi.org/10.1373/clinchem.2009.138099, pmid:20093555 13. berk je, kizu h, wilding p, et al. macroamylasemia: a newly recognized cause for elevated serum amylase activity. n engl j med. 1967;277(18):941–946. http://dx.doi.org/10.1056/nejm196711022771801, pmid:4167531 14. lee kn, csako g, bernhardt p, et al. relevance of macro creatine kinase type 1 and type 2 isoenzymes to laboratory and clinical data. clin chem. 1994;40(7 pt 1):1278–1283. pmid:8013099 15. krishnamurthy s, korenblat km, scott mg. persistent increase in aspartate aminotransferase in an asymptomatic patient. clin chem. 2009;55(8):1573–1575. http://dx.doi.org/10.1373/clinchem.2008.120782, pmid:19638492 16. fujita k, takeya c, saito t, et al. macro lactate dehydrogenase: an ldh–immunoglobulin m complex that inhibits lactate dehydrogenase activity in a patient’s serum. clin chim acta. 1984;140(2):183–195. http://dx.doi.org/10.1016/0009-8981(84)90343-7 17. wu ah. cardiac troponin: friend of the cardiac physician, foe to the cardiac patient? circulation. 2006;114(16):1673–1675. http://dx.doi.org/10.1161/circulationaha.106.652123, pmid:17043176 18. pettersson k, eriksson s, wittfooth s, et al. autoantibodies to cardiac troponin associate with higher initial concentrations and longer release of troponin i in acute coronary syndrome patients. clin chem. 2009;55(5):938–945. http://dx.doi.org/10.1373/clinchem.2008.115469, pmid:19264856 hiv situation in zimbabwe laboratory infrastructure for hiv-related testing in zimbabwe quality assurance in zimbabwe existing quality assurance programmes and lessons learnt national quality assurance programme for hiv acknowledgements references about the author(s) sibongile zimuto zimbabwe national quality assurance programme (zinqap), harare, zimbabwe agrippa mtambara ministry of health and child care, harare, zimbabwe ben cheng international diagnostics centre, london school of hygiene & tropical medicine, london, united kingdom brad cunningham systemone, johannesburg, south africa rodney taruvinga zimbabwe national quality assurance programme (zinqap), harare, zimbabwe debrah i. boeras international diagnostics centre, london school of hygiene & tropical medicine, london, united kingdom raiva simbi ministry of health and child care, harare, zimbabwe citation zimuto s, mtabara a, cheng b, et al. quality assurance for point-of-care testing in zimbabwe. afr j lab med. 2016;5(2), a448. http://dx.doi.org/10.4102/ajlm.v5i2.448 country profile quality assurance for point-of-care testing in zimbabwe sibongile zimuto, agrippa mtambara, ben cheng, brad cunningham, rodney taruvinga, debrah i. boeras, raiva simbi received: 25 mar. 2016; accepted: 11 aug. 2016; published: 17 oct. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. hiv situation in zimbabwe zimbabwe was one of the sub-saharan countries most severely affected by hiv and aids. the first aids case was reported in 1985. hiv prevalence increased sharply from 1985 to the mid-90s, peaking at 27.7% in 1997; thereafter, it started to decline (figure 1).1 according to the national aids council, hiv prevalence was estimated to be 15.0% as of the end of 2014.2 zimbabwe has a population of 15.25 million and has a generalised, feminised and homogenous hiv epidemic which continues to decline in terms of new infection rates, prevalence and aids-related mortality1. however, there are localised areas (11 districts) of high hiv transmission, described as hot spots, which include border districts, growth points, small-scale mining areas, fishing camps and commercial farming settlements. figure 1: hiv prevalence in zimbabwe 1970–2016. the number of people living with hiv in zimbabwe is estimated to be 1 390 211, with an incidence of 0.98, for the 15–49-year age group (table 1).1 new infections are estimated to be 69 105 and annual hiv deaths 63 853; 905 368 people are in of need antiretroviral therapy. nearly 80.0% of adults have access to antiretroviral therapy, while only 40.5% of children have access to treatment. table 1: zimbabwe hiv country statistics laboratory infrastructure for hiv-related testing in zimbabwe zimbabwe has a tiered laboratory network comprising three reference laboratories, five central hospital laboratories, eight provincial hospital laboratories and 66 district laboratories (figure 2). according to the ministry of health and child care aids and tuberculosis unit, there are 4291 hiv rapid testing sites, which may be voluntary counselling and testing or prevention of mother-to-child-transmission of hiv sites. these sites are located at various levels of the zimbabwe health delivery system. cd4 tests are conducted using conventional equipment as well as point-of-care (poc) devices. there are 138 conventional cd4 analysers and 360 operational cd4 poc testing sites. figure 2: zimbabwe’s tiered laboratory network. early infant diagnosis (eid) is conducted at three sites, strategically located in different geographical locations to allow easier access to testing. the eid testing sites are in harare at the national microbiology reference laboratory, in the north of the country, mpilo central hospital laboratory in the south and mutare provincial hospital laboratory in the east. health facilities send their eid samples to any one of the three laboratories, depending on their geographical location. based on the number of hiv-positive mothers presenting at prevention of mother-to-child transmission screening that have accessed eid, zimbabwe’s unmet eid needs are quite low. zimbabwe has developed a national viral load scale-up plan in support of the 90-90-90 goals.3 zimbabwe is in the process of procuring eight viral load instruments – two biomérieux nuclisens® six abbott m2000 instruments – through global fund. the abbott instruments are due for delivery in october 2016. partners such as the united states agency for international development have pledged to support human resources and other accessories. zimbabwe has recently completed an evaluation of the cepheid genexpert® viral load platform and the data are being analysed. they also evaluated the drw samba i platform, but there are reservations with this method and the country would like to evaluate samba ii. zimbabwe plans to roll out poc viral load testing at the district level in view of the complexity of the method and the increased workload on clinical health personnel at lower levels of the health system brought on by task shifting. it is envisaged that poc tests will provide access to viral load testing at low-volume sites in remote settings. zimbabwe revised its laboratory’s strategic plan in october 2015 (unpublished), detailing the focus areas for laboratory services in zimbabwe. the strategic plan will run until 2018. quality assurance in zimbabwe the zimbabwe national quality assurance programme (zinqap) is the national eqa provider. zinqap provides an accredited proficiency testing programme as well as training and mentorship in quality systems. zinqap provides its services to both publicand private-sector laboratories on a cost recovery basis. the zinqap eqa programme covers all the major disciplines of laboratory medicine, including microbiology, clinical chemistry, immunology, haematology and blood transfusion. on-site support and supervisory visits are conducted to assist poor performers to determine the cause of the poor performance and implement corrective and preventive actions. according to the zimbabwe medical laboratory guideline, medical laboratories are required to participate in a proficiency testing programme as a pre-requisite for registration.4 however, due to funding limitations, this is not yet been fully implemented. the ministry of health and child care has developed a policy on poc testing, which guides the selection, validation deployment and quality assurance for poc testing. this policy has yet to be approved and distributed. the eqa implementation models for hiv and related testing are as follows: eqa for hiv rapid testing is composed of proficiency testing panels prepared in-house by zinqap and distributed to participating sites on a monthly basis. however, less than 5% of sites are currently on the eqa programme, due to funding limitations. cd4 eqa for conventional cd4 analysers is also run by zinqap and is also composed of in-house prepared samples, which are distributed six times a year to participating sites. eqa for cd4 poc testing is prepared by qasi, an initiative of the public health agency of canada, which was previously distributed by the national microbiology reference laboratory, before being transitioned to zinqap. there are 266 poc testing sites on the qasi poc cd4 eqa programme. the london school of hygiene and tropical medicine is conducting a project in zimbabwe to determine the cost of a national eqa system in collaboration with the ministry of health and zinqap. this project includes building capacity for zinqap to provide eqa to all poc cd4 sites (figure 3). this model will be used to expand eqa to other poc testing platforms as they are introduced to the country. there are funding constraints for the full roll out of poc eqa. zimbabwe is engaging all partners and stakeholders to ensure that the future introduction of poc devices includes eqa. figure 3: zimbabwe external quality assessment model. poc testing poses new challenges to eqa, especially taking into account that hundreds of devices are being deployed for use by non-skilled, non-laboratory personnel with limited knowledge of quality practices.5,6 eqa for poc testing is an important tool which can provide information on the quality of the whole poc testing process. the connectivity project being piloted in zimbabwe, which includes a proof of concept for automated reporting of eqa results, presents a possible solution to the eqa challenge for poc testing. automated connectivity solutions can be used to transmit eqa performance data to the eqa provider for data analysis, so that corrective action can be taken, if necessary, in a timely manner. this overcomes the current problem of a low percentage of sites reporting eqa results. connectivity can also be perceived as being complementary to eqa, as it not only conveys information on test results but also operational data from the device, which can provide key equipment performance characteristics and quality indicators as part of a continuous quality assurance process. the system can also provide alerts when stocks are low, hence improving supply chain management. the three eid sites participate in the eqa run by the national institute for communicable diseases, south africa. the viral load eqa programme is organised by the us centers of disease control and prevention, atlanta; currently there is one laboratory site on this programme. existing quality assurance programmes and lessons learnt zimbabwe has been participating in eqa programmes since the mid-1980s. proficiency testing services were obtained from the ukneqas, before the establishment of zinqap in 1998. over the years, several lessons have been learnt regarding proficiency testing and eqa. an analysis of proficiency testing participation and performance records provides useful insights that can give guidance for policy makers and implementers. local ownership of the programme is very important, as a home-grown programme is well versed in local challenges and best placed to come up with interventions to address challenges faced. a locally-coordinated programme also has the advantage of quicker turnaround times and can provide on-site support to poor performers. local production of panels results in panels with characterisation that is similar to patient samples, which can best assess the testing systems. financing of eqa programmes is of critical importance, as they provide important information on the quality of testing and therefore the reliability of the results obtained.7 the national hiv response is based on testing; therefore, it is of critical importance to have a system of monitoring the quality of testing. it is important to ensure that there is adequate funding for the initiation and maintenance of the eqa programme. initial establishment of the eqa programme can be through partner support, however, it is important to ensure the future sustainability and maintenance of the programme with financial support from its beneficiaries. depending only on partner funding is not sustainable and poses the risk of collapse of the programme when funding is no longer available. it is also very important to have a mechanism that enforces eqa participation, with consequences for sites that do not participate. clear understanding and appreciation of the value of eqa is critical to sustainability – it is important to establish a culture of quality. national quality assurance programme for hiv a national quality assurance programme for hiv poc testing should provide clear guidance on selection and deployment of test equipment and methods.8 there should be a system of validation and/or verification of test methods, equipment and devices before they are put into use. policies, standard operating procedures and job aides need to be in place to provide the necessary guidance. training and competency assessments of the personnel conducting the testing are necessary to ensure that tests are carried out correctly and the results obtained are accurate and reliable. an internal quality control system must be in place and documented. a supply chain management system to ensure uninterrupted service delivery is also important. there is also a need to have a proficiency testing programme to independently assess the quality of testing. in addition, it is necessary to have a system to provide on-site support and supervision to poor performers, including root cause analysis, corrective and preventive actions. all testing sites must participate in the programme. a locally-coordinated or run proficiency testing programme is of greater value, as it provides samples that are similar to patient samples. funding and sustainability of the programme are of critical importance. billions of dollars are spent globally on test equipment, reagents and consumables for hiv. the hiv response is based on results obtained from various testing systems and it is thus of critical importance to ensure that there is a system of assessing and confirming that the tests conducted are accurate and reliable, so as to ensure quality patient care and efficient utilisation of resources. acknowledgements competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. sources of support none. references joint united nations programme on hiv/aids (unaids). global aids response progress report 2015 [document on the internet]. c2015 [cited 2016 sep 27]. available from: http://www.unaids.org/sites/default/files/country/documents/zwe_narrative_report_2015.pdf national aids council (nac), zimbabwe. hiv & aids situation [page on the internet]. c2011 [cited 2016 sep 27]. available from: http://www.nac.org.zw/about/hiv-aids-situation unaids. 90–90–90 – an ambitious treatment target to help end the aids epidemic [document on the internet]. c2014 [cited 2016 sep 27]. available from: http://www.unaids.org/en/resources/documents/2014/90-90-90 ministry of health & child welfare, zimbabwe. zimbabwe national guidelines on hiv testing and counselling [document on the internet]. c2005 [cited 2016 sep 27]. available from: https://www.k4health.org/sites/default/files/zimbabwe%20national%20guidelines%20on%20hiv%20testing%20and%20counselling.pdf nkengasong jn, nsubuga p, nwanyanwu o, et al. laboratory systems and services are critical in global health: time to end the neglect? am j clin pathol. 2010;134(3):368–373. http://dx.doi.org/10.1309/ajcpmpsinq9brmu6 birx d, de souza m, nkengasong jn. laboratory challenges in scaling up of hiv, tb and malaria programs: the interaction of health and laboratory systems, clinical research, and service delivery. am j clin pathol. 2009;131(6):849–851. http://dx.doi.org/10.1309/ajcpgh89qdswfons the world bank. zimbabwe economic update: results-based financing of health clinics helps zimbabwe to improve service delivery and weather economic headwinds [page on the internet]. c2016 [cited 2016 sep 27]. available from: http://www.worldbank.org/en/country/zimbabwe/publication/zimbabwe-economic-update-changing-growth-patterns-improving-health-outcomes zimbabwe ministry of health & child welfare. zimbabwe national hiv and aids strategic plan 2015–2018. harare [document on the internet]. c2015 [cited 2016 sep 27]. http://www.nac.org.zw/sites/default/files/znasp%20iii%20final%20(1).pdf abstract introduction methods results discussion acknowledgements references about the author(s) barbara i. nabaigwa international health sciences university, kampala, uganda bashir mwambi international health sciences university, kampala, uganda john okiria international health sciences university, kampala, uganda caesar oyet international health sciences university, kampala, uganda citation nabaigwa bi, mwambi b, okiria j, oyet c. common uropathogens among diabetic patients with urinary tract infection at jinja regional referral hospital, uganda. afr j lab med. 2018;7(1), a621. https://doi.org/10.4102/ajlm.v7i1.621 brief report common uropathogens among diabetic patients with urinary tract infection at jinja regional referral hospital, uganda barbara i. nabaigwa, bashir mwambi, john okiria, caesar oyet received: 21 feb. 2017; accepted: 08 sept. 2017; published: 09 feb. 2017 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract between june 2015 and october 2015, 159 mid-stream urine samples from diabetic patients were cultured. the prevalence of urinary tract infection was high at 22% and women were more affected compared with men (p = 0.017). factors associated with urinary tract infection in these patients were age, sex and high blood glucose levels. diabetic patients should be screened periodically for urinary tract infection. introduction urinary tract infection (uti) is common among both adults and children. according to tandogdu and wagenlehner,1 prevalence of uti varies greatly worldwide from 0.7% to 20%. patients with diabetes mellitus are highly susceptible to uti,2,3 and up to 35% of diabetic patients experience a uti. a number of factors predispose patients with diabetes to uti. these factors include weak host immune systems with impaired neutrophil function, depressed t-cell-mediated immune response, decreased production of prostaglandin e, thromboxane b2 and leukotriene b44 and depressed antioxidant systems,5 all of which expose such patients to infection. urinary incontinence due to disorders of the autonomic nervous system leads to incomplete bladder emptying, which in turn allows uropathogens to colonise and invade the urogenital niches.4 presence of glucose in urine of the diabetic patients, coupled with poor metabolic control, provides a conducive environment for pathogenic bacteria to flourish and cause utis.6 several uropathogens have been implicated in diabetic patient infections. the most common uropathogens isolated from diabetic patients are escherichia coli, klebsiella spp., staphylococcus aureus and candida albicans.7 this study was conducted to establish which uti aetiological agents are most common among diabetic patients attending the diabetes clinic at jinja regional referral hospital. risk factors for utis by these pathogens were also evaluated. methods ethical considerations this study was approved by the institutional review board of the international health sciences university in kampala, uganda (approval number: ugs 420-2016). research staff explained the study to eligible participants. patients who agreed to participate in the study signed consent forms. study design this was a cross-sectional study conducted between june 2015 and october 2015. a total of 210 diabetic men and women aged between 18 to 70 years who presented with a medical history of painful urination, urinary incontinence and lower abdominal pain at the time of the study and who provided informed consent to participate were recruited by systematic sampling. upon arrival, patients were requested to pick a card from a box containing three numbers: 1, 2 or 3. every fourth patient was enrolled after a patient picked a card with the number 2. participants were instructed to collect about 20 ml of mid-stream urine into a pre-labelled sterile screw-capped, graduated, wide-mouth plastic container. urine specimens were transported to the laboratory at 4°c within two hours of collection. all the specimens were subjected to a leukocyte esterase and nitrite test using a dipstick rapid test from cypress diagnostics (langdorp, belgium). urine specimens positive for nitrites or leukocyte esterase were subjected to bacterial culture. using a 0.002 ml, calibrated wire loop, urine specimens positive for nitrites or leukocytes were aseptically inoculated on cystine-lactose-electrolyte deficient medium (oxoid ltd, basingstoke, hampshire, united kingdom) and blood agar (oxoid ltd, basingstoke, hampshire, united kingdom), then incubated aerobically overnight at 37°c. pure growth with > 105 colony-forming units (cfu) per millilitre of urine was considered positive. mixed cultures and growth with < 105 cfu were considered negative. standard reference strains, staphylococcus aureus (atcc25923), escherichia coli (atcc25922) and pseudomonas aeruginosa (atcc 27853) were used as controls for testing the quality of culture media. a non-fasting 5-ml blood specimen was collected from each participant in oxalate/fluoride vacutainers (becton, dickinson and company, franklin lakes, new jersey, united states). each specimen was analysed on a cobas integra 400 analyzer (roche diagnostics, indianapolis, indiana, united states) for blood glucose level. a blood glucose level of > 7.8 mmol/l was considered to be hyperglycaemic.8 data management and analysis data were analysed using stata, version 11 (statacorp, llc, college station, texas, united states). a 95% confidence level was used for the analyses. multivariate logistic regression was used to evaluate associations of urinary tract infection with age group and blood glucose. the chi-square test was used to compare occurrence of urinary tract infection between men and women. p-values of less than 0.05 were considered statistically significant. results a total of 210 diabetic patients consented and were recruited, of whom 122 (76.7%) were women. the rapid dipstick test showed that a total of 159 (75.7%) of the urine specimens were positive for either nitrites, leukocytes or both; these were cultured and included in the analysis. thirty-five of the 159 cultures were positive for bacterial growth (> 105 cfu/ml of urine) giving the prevalence of uti at 22.0%. of the 122 female participants, a total of 29 (23.8%) had a uti compared with 14 (15.9%) of 88 men (p = 0.017). also, 22 out of the 35 positive culture were from patients aged over 50 years (p = 0.003) (table 1), and 28 of 35 (80%) uropathogens were isolated from participants with hyperglycaemia (p = 0.0026). only 7 of the 35 (20%) isolates were obtained from participants with a normal glucose level, whereas no isolate was obtained from participants with low fasting blood glucose (p < 0.0001). eighteen of the 35 (51.4%) isolates were staphylococcus saprophyticus, 12 (34.3%) were escherichia coli, 3 (8.6 %) were klebsiella spp., 1 (2.85%) was citrobacter spp. and 1 (2.85%) were enterococci. table 1: distribution of isolates by participant age (n = 159). discussion urinary tract infection, defined as the presence of > 105 cfu of bacteria per ml of fresh urine,9 is a common type of infection. the risk of uti is two to three times higher among patients with diabetes than among their non-diabetic counterparts.9 the overall prevalence of uti in our study was 22.0%, which is high compared to a study conducted among romanian patients where the prevalence of uti was 12.0%.10 this difference in prevalence could be due to, among other causes, variations in socioeconomic status.2 diabetes management is expensive; thus, low-income countries such as uganda are more prone to advanced effects, including uti, compared with middle-income countries such as romania. however, the prevalence of uti in our study was similar to that of a study in sudan where prevalence was 19.5%12 and the socio-demographic environment is similar to uganda. these findings call for earlier interventions to control diabetes in low-income countries. more women had uti compared to men (29/35, p = 0.017). this finding agrees with observations from other studies, in which female patients had a higher risk of uti than male patients.1 the cause of higher prevalence of uti among women is thought to be due to anatomical predisposition compared with men. women have a short and wide urethra with close proximity to the anus, allowing intestinal organisms easier access to the urethra. another possible cause may be due to changes in the physiological environment of the vagina among diabetic women, such as decreases in normal vaginal flora and a less acidic ph of the vaginal surface.14 this may be exacerbated among women with poor hygiene.18 our study also found that the number of uti cases increased with increased age. participants older than 50 years were more affected (22/35) compared with other age groups. another study, by wilke et al., found similar associations between age and uti.17 higher numbers of cases of uti among older diabetic patients could be the result of decreased urinary flow, coupled with incomplete bladder emptying resulting from neuropathy, reductions in oestrogen with loss of vaginal flora among women and prostate disease in men.17 our study revealed that hyperglycaemia was positively associated with uti among diabetic patients. up to 80% of the isolates were from participants with high blood glucose levels (> 7.8 mmol/l) compared with participants with normal glucose levels (< 7.8 mmol/l). the high uti prevalence among hyperglycaemic patients is most likely due to poor contraction of a dysfunctional bladder leading to static urine pools; this, together with glycosuria, creates a suitable environment for bacterial growth. however, some studies have not detected associations between uti and blood glucose level.13 this may be because a single blood glucose measurement may not represent glycaemic control over time, which would predispose diabetic patients to uti. the most common uropathogen in our study was s. saphrophyticus, followed by e. coli. this finding is not in agreement with several studies conducted on diabetic patients. a study in bangladesh showed that bacteria of the enterobacteriacae family, especially e. coli and klebsiella spp., are the most common uropathogens in diabetic patients.19 another study also demonstrated e. coli as the most common uropathogen responsible for uti in diabetic patients.7 the explanation for this discrepancy may be differences in study design and sample size or the fact that s. saprophyticus infections often yield < 105 cfu/ml of urine even in suprapubic aspirated urine samples. this would mean that in many instances growth of this organism is considered non-significant in routine urine culture performed on mid-stream urine. our study had a smaller sample size with a higher proportion of women, who harbour s. saprophyticus. the small sample size could account for our low rates of e. coli. another reason could be differences in the population distribution of the organisms, since most studies have been performed outside of uganda and there is little literature on the distribution of these organisms among diabetic patients in uganda. recommendations we recommend periodic screening of diabetic patients for uti. a study with larger sample size and power should be conducted to evaluate the distribution of uropathogens among diabetic patients in uganda. limitations a limitation to our study that should be considered when interpreting its results is that the sample size was small. this limited the power of the study for some analyses. conclusions there is high prevalence of uti among diabetic patients in uganda. age, sex and high blood glucose were associated with uti in this group of diabetic patients. acknowledgements the authors wish to extend their heartfelt appreciation to the patients who agreed to participate in the study. we would like to categorically acknowledge the immense contribution of the staff of jinja regional referral hospital and central public health laboratory. the manuscript was edited by ms bethanie rammer for english-language usage. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support none. authors’ contributions c.o. conceived the topic and wrote the protocol, b.i.n. collected the data, b.m. analysed the data, c.o. drafted the manuscript and j.o. edited the manuscript. references tandogdu z, wagenlehner fm. global epidemiology of urinary tract infections. curr opin infect dis. 2016;29(1):73–9. funfstuck r, nicolle le, hanefeld m, et al. urinary tract infection in patients with diabetes mellitus. clin nephrol. 2012;77(1):40–8. bateganya mh, luie jr, nambuya ap, et al. morbidity and mortality among diabetic patients admitted to mulago hospital, uganda. malawi med j. 2003;15(3):91–4. boyko ej, fihn sd, scholes d, et al. risk of urinary tract infection and asymptomatic bacteriuria among diabetic and nondiabetic postmenopausal women. am j epidemiol. 2005;161(6):557–64. joshi n, caputo gm, weitekamp mr, et al. infections in patients with diabetes mellitus. n engl j med. 1999;341(25):1906–12. benfield t, jensen js, nordestgaard bg. influence of diabetes and hyperglycaemia on infectious disease hospitalisation and outcome. diabetologia. 2007;50(3):549–54. sewify m, nair s, warsame s, et al. prevalence of urinary tract infection and antimicrobial susceptibility among diabetic patients with controlled and uncontrolled glycemia in kuwait. j diabetes res. 2016;2016:6573215. american diabetes association. diagnosis and classification of diabetes mellitus. diabetes care. 2010;33(suppl 1):s62–9. abrahamian fm, moran gj, talan da. urinary tract infections in the emergency department. infect dis clin north am. 2008;22(1):73–87, vi. nitzan o, elias m, chazan b, et al. urinary tract infections in patients with type 2 diabetes mellitus: review of prevalence, diagnosis, and management. diabetes metab syndr obes. 2015;8:129–36. chita t, timar b, muntean d, et al. urinary tract infections in romanian patients with diabetes: prevalence, etiology, and risk factors. ther clin risk manag. 2017;13:1–7. hamdan hz, kubbara e, adam am, et al. urinary tract infections and antimicrobial sensitivity among diabetic patients at khartoum, sudan. ann clin microbiol antimicrob. 2015;14:26. raoofi a, ghavami m, shahhamzeh m, et al. the impact of demographic factors and blood sugar control on the incidence of urinary tract infections in khorramabad in 2013. iran red crescent med j. 2016;18(5):e21942. yu s, fu az, qiu y, et al. disease burden of urinary tract infections among type 2 diabetes mellitus patients in the u.s. j diabetes complications. 2014;28(5):621–6. mohammed a, abdelfattah m, ibraheem a, et al. a study of asymptomatic bacteriuria in egyptian school-going children. afr. health sci 2016;16(1):69–74. yeshitela b, gebre-selassie s, feleke y. asymptomatic bacteriuria and symptomatic urinary tract infections (uti) in patients with diabetes mellitus in tikur anbessa specialized university hospital, addis ababa, ethiopia. ethiop med j 2012;50(3):239–49. wilke t, boettger b, berg b, et al. epidemiology of urinary tract infections in type 2 diabetes mellitus patients: an analysis based on a large sample of 456,586 german t2dm patients. j diabetes complications 2015;29(8):1015–23. leydon gm, turner s, smith h, little p. women’s views about management and cause of urinary tract infection: qualitative interview study. bmj (clinical research ed). 2010;340:c279. shill mc, huda nh, moain fb, karmakar uk. prevalence of uropathogens in diabetic patients and their corresponding resistance pattern: results of a survey conducted at diagnostic centers in dhaka, bangladesh. oman med j. 2010;25(4):282–5. abstract introduction reagents for testing reagent procurement training workshops quality assessment preliminary summary data of expansion capacity acknowledgements references about the author(s) brett whitaker division of viral diseases, national center for immunization and respiratory diseases, centers for disease control and prevention, atlanta, georgia, united states karen a. alroy division of viral diseases, national center for immunization and respiratory diseases, centers for disease control and prevention, atlanta, georgia, united states erica guthrie influenza division, national center for immunization and respiratory diseases, centers for disease control and prevention, atlanta, georgia, united states sarah schildecker influenza division, national center for immunization and respiratory diseases, centers for disease control and prevention, atlanta, georgia, united states susan hiers office of the director, national center for immunization and respiratory diseases, centers for disease control and prevention, atlanta, georgia, united states jill woodard office of the director, national center for immunization and respiratory diseases, centers for disease control and prevention, atlanta, georgia, united states s. arunmozhi balajee division of viral diseases, national center for immunization and respiratory diseases, centers for disease control and prevention, atlanta, georgia, united states citation whitaker b, alroy ka, guthrie e, et al. strengthening laboratory capacity for detection of respiratory viral pathogens through the global health security agenda (ghsa) framework. afr j lab med. 2019;8(1), a861. https://doi.org/10.4102/ajlm.v8i1.861 lessons from the field strengthening laboratory capacity for detection of respiratory viral pathogens through the global health security agenda (ghsa) framework brett whitaker, karen a. alroy, erica guthrie, sarah schildecker, susan hiers, jill woodard, s. arunmozhi balajee received: 10 july 2018; accepted: 17 jan. 2019; published: 18 july 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: endemic and emerging respiratory viruses are a threat to public health, and a robust public health laboratory system is essential to ensure global health security. objective: this program sought to expand molecular laboratory testing capacity to detect a broad range of respiratory pathogens in clinical respiratory specimens collected during disease surveillance and outbreak investigations. methods: as a part of the global health security agenda (ghsa), the united states centers for disease control and prevention utilised the equipment and training infrastructure already in place at the world health organization national influenza centers to expand testing capacity for respiratory viruses in laboratories in ghsa partner countries. this was done through the provision of quality assured reagents, including multiplex platforms and technical guidance for laboratory staff, as well as the assessment of laboratory testing accuracy. conclusion: early findings illustrated that ghsa laboratories have been able to expand testing capacity using specimens from routine surveillance, as well as from outbreak situations. keywords: public health laboratory; multiplex; respiratory viruses; global health security; polymerase chain reaction. introduction acute respiratory infections have threatened public health for decades.1,2,3,4 in 2003, when severe acute respiratory syndrome emerged out of hong kong, it illustrated the speed with which respiratory pathogens could spread globally as well as their potential for worldwide economic impact.5 two respiratory viruses have recently emerged as public health threats: influenza a/h7n9 and middle east respiratory syndrome coronavirus.6,7 according to the international health regulations established by the world health organization (who), countries should develop capacities for timely detection and rapid public health response to emerging and re-emerging threats, and these capacities rely in part on a robust public health laboratory system.8 the global health security agenda (ghsa) (www.ghsagenda.org) is a partnership of countries and international organisations launched in 2014 to strengthen global capacity to detect, prevent and respond to disease threats that occur naturally, accidentally or deliberately. one of ghsa’s primary areas of focus, known as an ‘action package’, is an emphasis on strengthening national laboratory systems (action package: detect 1).9 through this action package, ghsa enables countries to enhance real-time bio-surveillance and ensure that laboratories are capable of safe, accurate detection and characterisation of epidemic-prone pathogens, including both known and novel threats.9 in order for public health laboratories to prepare for outbreaks and emerging pandemic threats, they should demonstrate ongoing capacities for routine diagnostics. for almost a decade, the united states centers for disease control and prevention (cdc) influenza division has developed laboratory capacity for seasonal influenza virus and pandemic influenza preparedness. as a who collaborating center, cdc provides technical assistance for influenza testing in a network of global laboratories, known as national influenza centers (nics), which have functional links to their assigned who reference laboratory and access to quality assured reagents from the cdc influenza division through the international reagent resource (irr) (https://www.internationalreagentresource.org). the nics often have standardised, state-of-the-art equipment, in addition to well-trained molecular biologists. specimens for routine testing at nics are received from influenza surveillance sites that include regional hospitals and clinics using the severe acute respiratory infection (sari) or influenza-like illness case definitions. frequently, sari and influenza-like illness sentinel systems are models of successful surveillance in resource-limited countries where epidemiologic data are linked to specimen collection and laboratory testing.10 an opportunity exists to expand the existing surveillance systems at nics in ghsa countries by expanding testing capacity for other priority agents. accordingly, as a part of the ghsa, the united states cdc strengthened and expanded its existing capacity to test for respiratory viral pathogens in laboratories of select countries (table 1). these activities, detailed below, can serve as a model for other countries with nics and laboratories with moderate technical capabilities that are looking to expand their capacities beyond influenza. table 1: countries with expanded non-influenza virus reagent access that engaged in strengthening activities (february 2016–september 2017). reagents for testing in order to expand its laboratory capacity to test for respiratory viruses beyond influenza, cdc’s division of viral diseases deployed a panel of in-house singleplex real-time reverse transcriptase polymerase chain reaction (rrt-pcr) assays (table 2).11,12,13,14,15 specifically, a subset of seven cdc in-house singleplex rrt-pcr assays and one specimen quality control assay rnase p (rnp) was distributed as a kit for sari surveillance and outbreak investigations. all cdc in-house assays utilise taqman® probes (applied biosystems, foster city, california, united states) with a 5’ fam fluorophore and either an internally linked bhq-1® (biosearch technologies, novato, california, united states) or 3’ terminal bhq-1® (biosearch technologies, novato, california, united states) quencher. the rnp control allows laboratory scientists to know if the quality of nucleic acid has been compromised, potentially confounding any rrt-pcr results. these assays have been previously validated to work with several commercial one-step rrt-pcr enzyme kits such as the agpath-idtm one-step rt-pcr kit (thermo fisher, waltham, massachusetts, united states) and the qscript® one-step qrt-pcr kit, low rox (quantabio, beverly, massachusetts, united states). real-time pcr instruments evaluated by cdc to be compatible with this reagent panel include the 7500 fast dxtm (applied biosystems, foster city, california, united states), mx3000p® and mx3005p® (agilent, santa clara, california, united states), cfx96tm and cfx96tm touch (bio-rad, hercules, california, united states), and the viiatm 7 in both taqmantm array card and 96-well pcr plate formats (thermo fisher, waltham, massachusetts, united states).11,12,13,14,15,16 table 2: human respiratory pathogens targeted by centers for disease control and prevention assays and fast-track diagnostics respiratory pathogens panel. when encountering an outbreak of unknown etiology, a multiplex platform may enable laboratories to rapidly identify the potential pathogen causing illness; therefore, cdc provided countries access to a multiplex assay kit known as ftd33 respiratory pathogens® (fast-track diagnostics, esch-sur-alzette, luxembourg), a commercially available multiplex assay that can detect up to 33 common respiratory viruses and bacteria. the ftd33 comes with its own proprietary primers, probes, pcr buffer, enzyme mix and controls needed to perform the tests. a previous version of the ftd33 kit, the ftd21 respiratory pathogens kit, was validated against the cdc respiratory virus singleplex assay panel.17 table 2 compares the pathogens targeted with the cdc seven-pathogen non-influenza respiratory virus panel and the ftd33. ethical considerations this article followed all ethical standards for a research without direct contact with human or animal subjects. reagent procurement quality assured reagents are essential for accuracy in diagnostic testing. in addition, a reliable method for procurement and distribution of available reagents is necessary to order and receive laboratory materials, particularly when many of the reagents are temperature sensitive. utilising ghsa funds, the online reagent portal, the irr, was expanded to include 14 new custom products including the ftd33 kit, the cdc-developed respiratory virus rrt-pcr assay kits and controls, as well as ancillary commercial products such as rrt-pcr enzyme kits and specimen extraction kits required to run the cdc respiratory virus rrt-pcr assays. the irr is a programme established by cdc to provide registered users with various reagents and information for the study and detection of influenza virus. it was chosen as the delivery mechanism for these reagents, because the selected laboratories for ghsa were already using or in the process of being approved to use the irr for the purpose of who influenza surveillance, eliminating the need to establish a new or duplicative reagent distribution plan. training workshops in addition to enhancing laboratory capacity through the availability of quality reagents, cdc has enhanced its technical capacity through partnerships to conduct workforce training. working with the association of public health laboratories, laboratory training took place in atlanta in july of 2016. nine countries participated and were trained in the expanded respiratory viral pathogen detection, including bangladesh, burkina faso, cameroon, côte d’ivoire, pakistan, senegal, uganda, tanzania and vietnam. the pasteur institute of paris, france, conducted similar laboratory training in yaoundé, cameroon, in april 2017 with representatives from benin, guinea-bissau, mali, mauritania, the democratic republic of congo and togo. table 1 lists countries that have received training up to december 2017. the training curriculum included lectures from subject matter experts and hands-on laboratory exercises in order to familiarise attendees with the cdc rrt-pcr assays and the use of the ftd33. didactic lectures covered a range of topics including an overview of respiratory viruses, quality assurance and biosafety guidelines, setting up a molecular diagnostics laboratory, and performing rrt-pcr assays on human clinical respiratory specimens. the hands-on exercises covered total nucleic acid isolation from clinical respiratory specimens, setting up rrt-pcr reactions for cdc singleplex and ftd33 multiplex assays, as well as analysis of the rrt-pcr data. laboratory subject matter experts from cdc and the association of public health laboratories guided participants through the exercises ensuring everyone followed proper techniques. the specimens used for training were mock respiratory specimens spiked with inactivated respiratory viruses. by pre-characterising these specimens with the cdc respiratory virus panel, the participants’ results could be used as a metric for successful learning. quality assessment in order to test each country’s laboratory capabilities and availability of reagents following the training, cdc is preparing to provide laboratories with external quality assessment panels. these panels, produced by quality control for molecular diagnostics (http://www.qcmd.org), enable laboratories to process and test proficiency samples using the same protocol they use for clinical specimens. the results will be reported back to quality control for molecular diagnostics for scoring and the compilation of performance reports. confidential individual reports will be returned to the respective laboratories. based on their individual performances, cdc may recommend additional training and technical support, if necessary. for laboratories expanding their viral testing capacity, the availability of external quality assessment panels ensures quality performance. preliminary summary data of expansion capacity from february 2016 to september 2017, the irr shipped 502 individual products, supporting respiratory virus rrt-pcr testing, to 25 different laboratories in 19 countries, and scientists from 15 countries received training (table 1). preliminary data from two of these laboratories indicated that a total of 362 respiratory specimens were tested, 158 by cdc singleplex respiratory virus assays and 204 by the ftd33 multiplex assay. it is hoped that in the future, the public health laboratory workforce in ghsa countries will collaborate with their epidemiology counterparts and utilise these reagents for the purposes of understanding the diversity of circulating respiratory viral pathogens, calculating the burden of disease for priority pathogens, and using multiplex platforms for outbreak detection. next steps and conclusion the ghsa measures its success according to metrics defined by the who’s joint external evaluation tool.18 in the arena of national laboratory systems, two of these indicators include the capacity for the detection of priority diseases, as well as for effective and modern diagnostics. the training and materials provided through cdc’s engagement in ghsa laboratory strengthening has provided tools and building blocks to more than one-third of all ghsa countries to more readily achieve their goals in laboratory capacity. data generated from expanded testing will allow for a more complete understanding of the burden of disease in their country. a routine system of collection, transportation, testing and results sharing in a quality assured environment will enable the public health system to respond to outbreaks when they occur. lessons learned through the experiences described above, we identified key challenges and lessons learned where greater attention and investment may be warranted for future laboratory capacity building. the irr is an important tool in the rapid expansion of testing capacities for the ghsa countries. introducing new laboratory testing reagents through the irr is a way of supporting countries that do not normally perform routine surveillance testing beyond influenza virus detection, and enables them to generate baseline data for additional pathogens. centralising the distribution of custom cdc testing reagents along with commercial products through the irr ensured that laboratories had a streamlined, single-stop source for vital, quality-assured laboratory reagents. this eliminates the need to get separate quotes for the same products specific to all the different laboratories. additionally, by using the irr as a reagent distribution mechanism, it is easy and fast to deploy new reagents for emergency situations, such as outbreaks of newly emergent disease. developing and deploying training programmes with standardised curricula enables multiple laboratories and countries to practise uniform testing procedures, thereby allowing for better direct comparisons between countries and regions, and ultimately offering a better resolution in understanding the behaviour of respiratory viruses around the globe. in combination with strong epidemiological data, multiplex platforms such as ftd33 or other comparable products may provide valuable diagnostic information to better understand possible etiologies of respiratory disease, and ultimately improve detection capacities. additionally, some multipathogen detection assays and platforms may still be cost prohibitive for many countries and public health systems, especially when used as a surveillance tool where the quantity of specimens needing testing can be quite high. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions b.w. and s.a.b. provided the article framework. k.a.a., e.g., s.s., s.h. and j.w. added clarifications and additional text where appropriate based on their involvement and expertise. source of support none. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the findings and 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https://www.who.int/ihr/publications/who_hse_gcr_2018_2/en/. abstract introduction methods results discussion acknowledgements references about the author(s) anthere murangwa rwanda military hospital, kigali, rwanda kanan t. desai division of cancer epidemiology & genetics, national cancer institute, national institutes of health, rockville, maryland, united states julia c. gage division of cancer epidemiology & genetics, national cancer institute, national institutes of health, rockville, maryland, united states gad murenzi rwanda military hospital, kigali, rwanda patrick tuyisenge rwanda military hospital, kigali, rwanda faustin kanyabwisha rwanda military hospital, kigali, rwanda aimable musafili department of paediatrics and child health, school of medicine and pharmacy, college of medicine and health sciences, university of rwanda, kigali, rwanda gallican kubwimana rwanda military hospital, kigali, rwanda research for development (rd rwanda), kigali, rwanda leon mutesa research for development (rd rwanda), kigali, rwanda centre for human genetics, college of medicine and health sciences, university of rwanda, kigali, rwanda kathryn anastos departments of medicine and epidemiology & population health, albert einstein college of medicine and montefiore health systems, bronx, new york, united states hae-young kim department of public health, new york medical college, valhalla, new york, united states philip e. castle division of cancer epidemiology & genetics, national cancer institute, national institutes of health, rockville, maryland, united states division of cancer prevention, national cancer institute, national institutes of health, rockville, maryland, united states citation murangwa a, desai kt, gage jc, et al. agreement between xpert and ampfire tests for high-risk human papillomavirus among hiv-positive women in rwanda. afr j lab med. 2022;11(1), a1827. https://doi.org/10.4102/ajlm.v11i1.1827 original research agreement between xpert and ampfire tests for high-risk human papillomavirus among hiv-positive women in rwanda anthere murangwa, kanan t. desai, julia c. gage, gad murenzi, patrick tuyisenge, faustin kanyabwisha, aimable musafili, gallican kubwimana, leon mutesa, kathryn anastos, hae-young kim, philip e. castle received: 06 jan. 2022; accepted: 26 may 2022; published: 19 oct. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: high-risk human papillomavirus (hrhpv) may cause more than 99% of cervical cancers worldwide. little is known about performance differences in tests for hrhpv. objective: this study analysed agreement for detection of hrhpv between the established, clinically validated xpert hpv assay and the novel isothermal amplification-based ampfire hpv genotyping assay. methods: this study was nested in a larger project on cervical cancer screening among approximately 5000 women living with hiv in kigali, rwanda. this sub-study included 298 participants who underwent initial screening for cervical cancer using the xpert hpv assay and visual inspection with acetic acid in 2017 and tested positive by either or both. participants were rescreened using colposcopy, and cervical samples were collected between june 2018 and june 2019. samples were then tested for hpv using the xpert hpv assay and ampfire hpv genotyping assay. agreement between results from both tests was analysed using an exact version of mcnemar test and chi-square test. results: overall agreement and kappa value for detection of hrhpv by xpert and ampfire were 89% and 0.77 (95% confidence interval: 0.70–0.85). ampfire was marginally more likely to diagnose hrhpv-positive than xpert (p = 0.05), due primarily to the extra positivity for hpv16 (p < 0.001). conclusion: overall, there was good to excellent agreement between the xpert and ampfire when testing hrhpv types among women living with hiv. ampfire was more likely to test extra cases of hpv16, the most carcinogenic hpv type, but the clinical meaning of detecting additional hpv16 infections remains unknown. keywords: xpert hpv assay; ampfire hpv genotyping assay; high-risk human papillomavirus types; women living with hiv; cervical cancer screening. introduction high-risk human papillomavirus (hrhpv) causes virtually all cervical cancer-related cases worldwide.1 the elimination of cervical cancer relies on primary prevention, involving the immunisation against human papillomavirus (hpv), and on secondary prevention based on the screening and treatment of cervical precancer lesions.2 evidence to date indicates that hpv genotyping can help with risk stratification for further triage and management of hrhpv-positive women.3,4 unfortunately, hpv assays commonly used in the screening programmes are of higher cost, with longer turnaround time, and offer genotyping limited to hpv16 and 18 only. many rapid, low-cost assays designed for providing extended hpv genotyping have been developed in recent decades. however, they have variable analytical sensitivity and specificity for different hpv types with limited clinical validation data.5,6,7 xpert is a world health organization pre-qualified test,8,9 which uses a single integrated cartridge, prefilled with primers and fluorescent probes, as well as two reagents meant for the extraction, amplification and detection of some hpv dna regions, on the xpert platform. xpert uses 1 ml of cervical specimen, which is collected in preservcyt, to test for 14 types of hrhpv using real-time polymerase chain reaction in six color channels contain primers and probes for the detection of specific genotypes or pooled results as follows: ‘sac: primary’ for the samples adequacy control, ‘hpv 16; primary’ for hpv 16, ‘hpv 18_45; primary’ for the hpv 18/45 pooled result, ‘p3: primary’ for the pooled result of any of hpv types 31, 33, 35, 52 or 58, ‘p4: primary’ for the pooled result of either of hpv types 51 or 59, and ‘p5; primary’ for the pooled result of any of hpv types 39, 56, 66 or 68. the turnaround time for the assay is approximately 1 h.3,5,6 the ampfire is a promising new lower-cost assay, using isothermal amplification along with a real-time fluorescence system in four channels to individually detect 15 types of hrhpv, including hpv16, 18, 31, 33 35, 39, 45, 51, 52, 56, 58, 59 and 68, plus intermediate-risk hpv53 and 66. the ampfire assay uses a simple processing protocol to detect hpv directly without prior dna extraction from clinical samples, including dry samples. the assay has a small footprint and a sample-to-result time of approximately 1 h.7,10,11 we recently completed a cervical cancer screening study of approximately 5000 rwandan women living with hiv. in a subset of women who underwent colposcopy, we compared the analytic performance for hpv detection by the xpert and ampfire systems. methods ethical considerations this sub-study derived from a larger study on cervical cancer screening among rwandan women with hiv,12 which was approved by the rwanda national ethics committee and albert einstein college of medicine institutional review board (no. 878/rnec/2016). participation in the parent study was voluntary. women who participated in this research were informed about the study purpose and procedures before consenting to participate. confidentiality was consistently observed when handling data. study design and sampling the participants included 298 women living with hiv who were recruited from a larger study of cervical cancer screening, which was carried out among approximately 5000 rwandan women living with hiv. all women were initially screened by either the xpert hpv assay (cepheid, sunnyvale, california, united states) or visual inspection with acetic acid and those positive by either or both methods were referred for colposcopy. a detailed protocol related to the study population and recruitment was further developed in the parent study.12 the participants included women who were lost to follow-up in the parent study for a period of 6–38 months. after retrieving them, they were enrolled in the present study, for which specimens were collected between june 2018 and june 2019. these women with a delay in colposcopy attendance underwent colposcopy, during which cervical specimens were collected using a luckmedical cervical brush (luck medical consumables co., jiangsu, china). these samples were put into 20 ml of the preservcyt medium (hologic, bedford, massachusetts, united states). setting all women included in the study lived in kigali, which is the capital and biggest city of rwanda. the latest population and housing census, dated 2012, indicated that kigali was populated with 1 132 686 inhabitants.13 the study was implemented at rwanda military hospital, which is a tertiary referral hospital located in kigali. the recruitment and screening of participants for hpv as well as the data collection were carried out in the department of hiv, where women living with hiv were usually followed up. laboratory testing cervical specimens were mixed with preservcyt before being tested for hrhpv. a volume of 1 ml of the mixed sample was added to the xpert cartridge, which was then placed in the xpert assay for the analysis. results from this analysis were available within an hour. the sample of cervical specimens mixed with preservcyt was also tested for hrhpv using the ampfire (atila biosystems, mountain view, california, united states). a volume of 1 ml of this sample was added to a 1.5 ml-micro-centrifuge tube and centrifuged for 10 min at 10 000 revolutions per minute.14 the supernatant was removed from the micro-centrifuge tube while the pellet underwent further treatment by mixing it with the lysis buffer before it was incubated for 10 min at 95 °c. after the incubation, 2 µl of the treated pellet were mixed with a prepared solution of 12 µl of reaction mix and 11 µl of primer mixes (atila biosystems, mountain view, california, united states), which were the ampfire genotyping reagents. the mixed sample was placed in the ampfire for the analysis, which took an hour before the results were available. the cut-off point values of positivity applied when using xpert and ampfire assays were defined according to the manufacturers’ instructions.15,16 statistical analysis agreement statistics (positive, negative and overall agreement, and kappa values) for 14 hpv types, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68, detected by both tests, were calculated. results for hpv53, detected by ampfire but not by xpert, were excluded. results from ampfire were grouped according to the hpv groups as detected by the xpert hpv assay. firstly, hpv groups were considered non-hierarchically, recognising that a given specimen could test positive for more than one group. secondly, the two tests were also compared using risk-based hierarchical hpv group types, considering that different hpv groups are associated with different risks of cervical cancer: hpv16 positive, else positive for hpv18/45, else positive for hpv 31/33/35/52/58, else positive for other high-risk hpv types (hpv51/59/39/56/66/68), or else negative; hpv51/59 and hpv39/56/66/68 have similarly lower risk for invasive cervical cancer and were grouped together. mcnemar test and chi-square test was applied to assess significant differences (p < 0.05) in test positivity. data entry was performed using epi-info 7 (version march 2015, centers for disease control, atlanta, georgia, united states). afterwards, data were transferred to the statistical package for the social sciences statistics 20 (ibm corp., armonk, new york, united states) for the analysis. results overall agreement between the two test results for the 14 hpv types was 89% and the kappa value was 0.77 (95% confidence interval: 0.70–0.85). ampfire was marginally more likely to test positive for the 13 types of hpv (p = 0.05) than xpert (table 1). agreement ranged from 92% (hpv31/33/35/52/58) to 98% (hpv51/59), and kappa values ranged from 0.68 (hpv39/56/66/68) to 0.84 (hpv18/45) for hpv groups. ampfire was more likely to test positive for hpv16 than xpert (p < 0.001). table 1: agreement for detection of human papillomavirus overall and hpv types grouped according to the xpert hpv assay between xpert hpv assay and ampfire hpv genotyping assay, rwanda, june 2018 june 2019. on the hierarchical analysis, the two assays had good agreement with an overall unweighted kappa value of 0.77 (95% confidence interval: 0.70–0.85) (table 2). the hierarchical analysis also showed a similar pattern to that of non-hierarchical analysis for hpv 16 and hpv 18/45, with extra cases detected by ampfire. however, for hpv31/33/35/52/58, more extra cases were detected by xpert than by ampfire while for other hrhpv types, both tests detected similar numbers of extra cases. overall, ampfire was more likely to rank hpv results in categories with higher risk than xpert (p < 0.001). table 2: pairwise ampfire and xpert results, categorised hierarchically according to hpv risk groups, rwanda, june 2018 june 2019. more than 80% of the hpv18/33/35/45/52/58/59 detected by ampfire were positive in the xpert channel that tested for that type (as a group of types) (table 3). by contrast, only 54.4% of hpv51 and 42.9% of hpv39 detected by ampfire were positive in the xpert channel that tested for that type (as a group of types). table 3: concordance of individual hpv types detected by ampfire hpv genotyping assay with hpv groups detected by xpert hpv assay (non-hierarchical), rwanda, june 2018 june 2019. discussion this study sought to compare the xpert and ampfire among women living with hiv in rwanda. we found an overall good to excellent agreement between the two methods for the detection of hrhpv. notably, ampfire was more likely to test positive cases for hpv16 than xpert but the clinical meaning of the detection of these additional hpv16 infections was unknown. the comparable findings between xpert and ampfire hpv regarding the detection of hrhpv may be explained by the well-known performance of these tests. previous studies have shown that ampfire could detect all hpv genotypes with sensitivity and specificity of more than 95%.5 in addition, when compared to other tests, including the roche cobas® and linear array, an excellent concordance between these tests and ampfire has been demonstrated for the detection of 15 hrhpv.16 further, xpert has been widely used for the detection of various types of hpv, including hrhpv types. in zimbabwe, an overall good concordance (77.2%, kappa = 0.698) between xpert and seegene anyplex ii hpv hr detection kit was found when testing hrhpv among women aged from 30 to 60 years.17 in south africa, xpert was combined with linear array to optimise the screening of cervical cancer. this combination has resulted in increased sensitivity and specificity for the detection of hrhpv, highlighting the consistent high performance of xpert even when combined with other valid tests.15 the xpert was also considered a world health organization pre-qualified test, stressing its validity.9 despite the overall agreement between the xpert and ampfire, differences in the detection of some types of hrhpv were noticed. these differences could be reflected by the extra cases of hpv16 and other hrhpv, including hpv18 and 45, which were detected by the ampfire compared to xpert. however, we could not find any scientific reason that could be attributed to the extra positivity by ampfire, especially for hpv16. in contrast, xpert has shown greater potential of detecting more cases of hpv31/33/35/52/58 than ampfire. this extra detection of some hrhpv by xpert compared to ampfire could probably be explained by its greater performance of detecting concurrent infections by hrhpv. recommendations as explained earlier, both assays perform as expected and use simple procedures with less equipment, and a rapid turnaround time, which make them highly recommendable as point-of-care screening for cervical precancer and cancer in lowto middle-income countries. strengths this was the first study to have analysed the agreement between xpert and ampfire for the detection of hrhpv among women living with hiv in rwanda. thus, our findings may provide new insights regarding the performance of the two assays when applied in low-resource settings. limitations the authors could not determine why there was extra positivity by ampfire, especially for the detection of hpv16. further studies, involving larger numbers of specimens and multiple sites, or histopathologic analyses could perhaps be relevant to address this knowledge gap. conclusion this study revealed good to excellent agreement between xpert and ampfire when testing hrhpv types among women living with hiv in a rwandan setting. it has also shown an extra positivity by ampfire compared to xpert, especially for hpv16, which requires further studies or adjudication by histopathologic tests. acknowledgements authors thank the study participants for having consented to take part in the study. they also acknowledge the data collectors who have been involved in the fieldwork. authors are also indebted to the facilitation by the management of the health centres where the initial cervical cancer screening took place. competing interests this study received xpert hpv tests at a reduced cost from cepheid (sunnyvale, california, united states). authors’ contributions a. murangwa and p.e.c. conceived the study and received feedback from k.t.d. and j.c.g. a. musafili and k.t.d. analysed and interpreted data with substantial inputs from p.e.c., j.c.g., k.a., l.m., g.m., g.k., p.t., f.k. and h.-y.k. a. murangwa and k.t.d. drafted the manuscript, which was critically reviewed by other authors. all authors approved the final version of the manuscript before submission. sources of support this study was funded by grants from the national cancer institute of the united states national institutes of health (5u54ca19016304, 5u54ca254568). data availability the data sets used or analysed during the current study are available from the corresponding author, a. murangwa, on reasonable request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references walboomers j, jacobs m, manos m, et al. human papillomavirus is a necessary cause. j pathol. 1999;189:12–19. world health organization. preparing for the introduction of vaccines: policy and programme guidance for countries. geneva: who; 2006. de sanjose s, quint wgv, alemany l, et al. human papillomavirus genotype attribution in invasive cervical cancer: a retrospective 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2020;37(183):1–10. fitzpatrick mb, dube rs, katzenstein da, et al. hrhpv prevalence and type distribution in rural zimbabwe: a community-based self-collection study using near-point-of-care genexpert hpv testing. int j infect dis. 2019;82:21–29. https://doi.org/10.1016/j.ijid.2019.02.022 article information authors: laura t. eno1 terence asong1 elive ngale2 beatrice mangwa2 juliana ndasi2 maurice mouladje3 remmie lekunze4 victor mbome5 patrick njukeng2 judith shang1 affiliations: 1us centers for disease control and prevention, cameroon2global health systems solution, cameroon 3buea regional hospital, cameroon 4cameroon development corporation, central clinic, tiko 5regional delegation for health, buea, cameroon correspondence to: laura eno postal address: po box 817, yaoundé, cameroon dates: received: 07 aug. 2014 accepted: 22 sept. 2014 published: 03 nov. 2014 how to cite this article: eno lt, asong t, ngale e, et al. driving hospital transformation with slmta in a regional hospital in cameroon. afr j lab med. 2014;3(2), art. #221, 5 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.221 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. driving hospital transformation with slmta in a regional hospital in cameroon in this original research... open access • abstract • introduction • research methods and design    • study area    • establishment of the quality improvement task force    • tailoring slmta for the entire hospital • results • discussion    • limitations of the study    • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: inspired by the transformation of the regional hospital buea laboratory through implementation of the strengthening laboratory management toward accreditation (slmta) programme, hospital management adapted the slmta toolkit to drive hospital-wide quality improvement.objective: this paper describes changes in the hospital following the quality improvement activities in hygiene and sanitation, the outpatient waiting area and the surgical and maternity wards. methods: in march 2011, hospital management established a quality improvement task force and created a hospital-wide quality improvement roadmap, following the slmta model. the roadmap comprised improvement projects, accountability plans, patient feedback forms and log books to track quality indicators including patient wait time, satisfaction level, infection rates, birth outcomes and hospital revenue. results: there was steady improvement in service delivery during the 11 months after the introduction of the quality improvement initiatives: patient wait time at the reception was reduced from three hours to less than 30 minutes and patient satisfaction increased from 15% to 60%. treatment protocols were developed and documented for various units, infrastructure and workflow processes were improved and there was increased staff awareness of the importance of providing quality services. maternal infection rates dropped from 3% to 0.5% and stillbirths from 5% to < 1%. the number of patients increased as a result of improved services, leading to a 25% increase in hospital revenue. conclusion: the slmta programme was adapted successfully to meet the needs of the entire hospital. such a programme has the potential to impact positively on hospital quality systems; consideration should be made for development of a formal slmta-like programme for hospital quality improvement. introduction top ↑ high-quality healthcare service delivery is essential for improving patient outcomes and reducing mortality. despite advances in expanding and strengthening public health services in developing countries, many hospitals continue to struggle to maintain quality systems.11,2 in order to meet accreditation standards that have been set forth to ensure uniform healthcare, comprehensive quality management systems (qms) must be established throughout the hospital system.13,4 that being said, no hospital qms has previously been piloted or implemented in cameroon.in the clinical laboratory setting, however, a proven quality improvement tool already exists, namely, the strengthening laboratory management toward accreditation (slmta) programme. slmta is a competency-based management training programme designed to achieve immediate and measurable improvements in laboratory quality systems.15 launched in kigali in 2009, slmta has spread rapidly and, by the end of 2013, had been implemented in 617 laboratories in 47 countries.16 practical approaches to addressing daily challenges, task-based methods and hands-on learning on management practices are amongst the innovative features of the slmta curriculum. progress is measured using the world health organization regional office for africa’s (who afro) stepwise laboratory quality improvement process towards accreditation (slipta) checklist,17 which is divided into 12 sections corresponding to the quality system essentials.18 resulting audit percentage scores determine the quality ratings on a zero(< 55%) to five(≥ 95%) star scale. the laboratory at regional hospital buea (rhb) was one of five pilot laboratories selected by the ministry of health (moh) to implement the slmta programme in cameroon. a baseline audit of the rhb laboratory was performed in november 2009 by a who afro-trained local auditor. three slmta training workshops were conducted, starting in october 2010 and interspersed with improvement projects and on-site mentoring. the exit audit was conducted in september 2012 and showed that rhb’s laboratory had made a marked improvement, rising from 18% (zero stars) at baseline to 67% (two stars) at the exit audit. improvement was observed in 11 out of 12 quality system essential sections. the successful implementation of slmta at rhb’s laboratory attracted the attention of hospital management, who were interested in building qms throughout the facility. in march 2011, it was proposed that the slmta toolkit be tailored into an improvement programme for the entire hospital. this study describes how rhb adapted the slmta programme in order to strengthen hospital quality systems and to achieve measurable outcomes. research methods and design top ↑ study area rhb is located in the south west region of cameroon, with a capacity of 120 beds and a workforce of 200 staff serving a population of 90 088. the hospital consults an average of approximately 30 000 patients annually, 40% of whom require laboratory services. in addition to laboratory analyses, the hospital's services include: medicine, paediatrics, obstetrics, gynaecology, surgery, dentistry, eye care, radiology and haemodialysis. rhb also serves as a teaching hospital for the faculty of health sciences at the university of buea. establishment of the quality improvement task force hospital leadership participated in one of the slmta workshops conducted for laboratory staff and proposed that other hospital units be engaged in similar quality improvement activities. to coordinate this effort, hospital management formed the quality improvement task force (quitaf).quitaf was chaired by the hospital director, in collaboration with the general superintendent of the hospital as well as the laboratory director, who served as the quality assurance officer. unit heads were appointed as quality officers, whilst clinicians were also engaged as members of quitaf. quitaf was mandated to develop a measurable hospital quality improvement plan so as to strengthen the delivery of hospital services, with the goal of improving patient care. the responsibilities and accountability of quitaf members were defined. quality officers were primarily in charge of daily operations, implementation and maintenance of quality systems in their respective units. the quality assurance officer and the general superintendent provided high-level direction and oversaw the overall improvement activities, whilst technical guidance was provided by two slmta trainers and laboratory mentors from the us centers for disease control and prevention (cdc) cameroon office. the hospital director reviewed and approved all technical and quality management plans developed by the staff and made financial decisions when necessary. tailoring slmta for the entire hospital laboratory staff members who had been trained in slmta were assigned to work with quitaf members to develop a hospital-wide quality improvement programme. three training modules from the slmta toolkit (cross-cutting, productivity management and work area management) were modified in order to address hospital-wide issues concerning turnaround time, floor plans and standardised procedures. ten hospital staff members were trained for three days in april 2011 followed by mentorship provided by laboratory staff and the two cdc-cameroon slmta trainers. subsequent workshops were conducted in the various units based on specific gaps. participants were encouraged to develop improvement projects in order to address major challenges in their units using minimal financial resources. selection, implementation and results of improvement projects were discussed in the quitaf meetings.a quality improvement roadmap was created outlining action items for each key improvement area, with expected outcomes and deliverables (table 1). this plan was reviewed during weekly team meetings in order to assess progress made, address key challenges and capture lessons learnt. the almoner team, which is responsible for all information concerning the various hospital units, services available and amounts charged per service delivery, disseminated pertinent information on the quality improvement plan to all. table 1: quality improvement roadmap for regional hospital buea. for evaluation purposes, baseline data for the number of patients served per month and patient wait times were established using past records from the patient log books and direct observation. indicators were measured non-systematically using logs and reports from key informants. maximum patient wait times were estimated by scanning log books in order to determine the total time from arrival of the patient in the outpatient reception area to when they left the room (patient wait time at reception) or when they left the laboratory with their results (overall patient wait time). the proportion of public toilets that were functional in the facility was assessed as a measure of hospital hygiene and sanitation. the infection rate as a result of poor hygiene was estimated by the theatre nurse, who reviewed admission records in the surgical ward before and after the improvement project. the overall infection rate was counted from the patient records; the number of infections related to poor sterilisation was estimated before and after program improvements. stillborn and maternal infection rates were estimated by the midwife of the maternity ward using birth records. the hospital director provided an estimate of the number of patients served annually and the hospital revenue; he also initiated a staff survey to assess their awareness of quality improvement activities. patients were asked to provide feedback on service quality. one thousand (500 each in march and september 2011) patient feedback forms were made available with suggestion boxes; patient satisfaction was estimated based on the proportion of returned forms with positive comments. results top ↑ patient wait time at reception decreased from three hours to less than half an hour (table 2) and total patient wait time decreased from greater than three days to less than one day. staff awareness of quality improvement programmes in the hospital increased from 10% to 75%. overall patient satisfaction increased from 15% to 60%. the percentage of patients who said they would return to the rhb for consultation rose from 60% at baseline to 90% six months later; and 85% said they would recommend the hospital to their friends or relatives. the proportion of functioning public toilets increased from 10% to 75%. table 2: improvement projects at the regional hospital buea selected by each unit head in collaboration with the entire quality improvement task force (quitaf maternal) team from april 2011 to february 2012. other key indicators also showed marked progress: the maternal infection rate dropped from 3% to 0.5%; the number of stillbirths decreased from 5% to < 1%; and the number of patients served grew and hospital revenue increased by 25%. to improve sterilisation practices, a window was introduced between the sterilisation room and the preparation room in the theatre, minimising the exposure of surgical supplies to potential contamination by transporting them through the crowded hospital corridors. new systems were developed such as: a calendar for sterilisation of equipment; a log book for recording and counting sterilised forceps and disposable gowns; and placement of additional dust bins around the hospital. signage was posted to indicate the location of units and to encourage proper hygiene habits within the hospital. sessions were conducted to educate patients on proper use of the public toilets. these changes led to less foot traffic and increased cleanliness in the overall hospital environment. discussion top ↑ hospital management at rhb successfully adapted the slmta toolkit for a multi-disciplinary hospital-wide improvement programme. the quitaf facilitated team work and held the units accountable for quality. increased collaboration between clinicians and laboratory staff ensured that quality improvement efforts were patient-centred. effective communication ensured that the entire hospital was aware of the changes and helped to foster a culture of quality. during quitaf meetings, experiences and challenges were shared, leading to positive changes such as timeliness and consistency in the reporting of results. the implementation of a hospital-wide quality improvement plan allowed hospital management to evaluate its staff’s strengths, establish procedures and communicate more effectively.the quality change implemented in the entire hospital and advances in hospital processes led to critical improvements in patient care, as reported in the reduction of wait times, infections and stillbirths. the public took notice; as patient satisfaction increased, so did the number of patients and hospital revenue. implementation of hospital-wide improvements in conjunction with slmta further enhanced the laboratory’s quality strengthening efforts. for example, the presence of laboratory staff during clinical rounds allowed critical issues to be discussed in real time. clinicians began to better understand laboratory delays and hospital staff expressed increased confidence in relying on the hospital laboratory for testing, rather than on privately-owned facilities. in august 2014, an official who afro slipta audit was conducted in the rhb laboratory by the african society for laboratory medicine. the laboratory earned two slipta stars (with an audit score of 69%), indicating that it is well on its way to developing an effective qms. the audit report highlighted the complementary role played by quitaf in supporting both laboratory and hospital quality improvement.19 through quitaf, the entire hospital management and staff were mobilised to support and contribute to sustaining the quality culture. following the implementation of the quality improvement programme, hospital management is now working to develop targets for the annual number of patients served, minimum revenue expected, maximum number of deaths due to poor practices and maximum number of customer complaints. limitations of the study this assessment is subject to several limitations. modifying the slmta toolkit for the entire hospital was not always easy, as tools to measure improvement in non-laboratory settings were lacking. patient feedback forms were created and, whilst written comments on the forms were useful, they were not easily quantifiable; more thorough qualitative analysis of this feedback is warranted. most critically, many of the indicators reported were not observed directly or collected systematically, but were based on expert report and retrospective review of log books. furthermore, because of logistical barriers, some variables were not measured before implementation, limiting our conclusions. the data presented here represent convenient, readily-available programmatic data, using pre-existing sources or those developed during the quality improvement initiative itself. whilst results are encouraging, validation in other facilities using thorough scientific techniques is needed in order to draw solid conclusions. conclusion modification of the slmta programme for the wider hospital setting allowed hospital management and staff to identify gaps and develop a plan to address them. the director of the rhb testified that, ‘[q]uality can be improved by making changes within the healthcare system without necessarily increasing resources’ and also that:‘slmta is an invaluable tool for every lab director, every hospital manager and health policy maker because of its value in ensuring quality improvement within the laboratory and its potential in contributing to strengthening the entire health system at little or no cost.’ expressing a similar opinion, the ministry of health and social services (mohss) laboratory director of namibia said: ‘as quality improvements become institutionalised in hospital laboratories, it is becoming evident that entire hospital systems are in dire need of similar quality improvement programmes. the namibia mohss calls on international agencies to develop and adapt programmes such as slipta and slmta for use throughout hospital systems so as to ensure continuous quality patient care.’110 given the positive experience at rhb, we second this call and recommend that a hands-on competency-based curriculum be developed fully in order to support hospital quality management, extending the strengthening laboratory management toward accreditation programme into one for strengthening hospital management toward accreditation. acknowledgements top ↑ this research was supported by the us president’s emergency plan for aids relief (pepfar) through the cdc, global health systems solution (ghss) serving as mentors and the entire management, staff and health committee of the rhb. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions l.t.e. (cdc, cameroon) was the main author, quality assurance advisor and slmta master trainer, and provided technical assistance to the laboratory and the quitaf team. t.a.1 (cdc, cameroon) was a mentor, evaluated improvement projects within rhb, and reviewed the manuscript. e.n.1 (ghss) was a mentor and slmta trainer and provided guidance in implementing the quitaf project. b.m.1 (ghss) was a mentor and assisted in training the hospital quitaf team. j.n.1 (ghss) was a slmta master trainer and mentor, facilitated the deployment of mentors, and assisted in the design and review of the manuscript. m.m. (buea regional hospital) lead the quitaf project and administered the questionnaire for the study. r.l. (cameroon development corporation) provided training to hospital staff and was responsible for laboratory quality improvement projects and data collection. v.m. (regional delegation for health, buea) championed the extension of slmta to the entire hospital, and reviewed and provided guidance on the manuscript. p.n.1 (ghss) provided technical assistance. j.s.1 (cdc, cameroon) conducted upper management advocacy, provided technical assistance, and reviewed and revised the manuscript. references top ↑ 1. birx d, de souza m, nkengasong jn. laboratory challenges in the scaling up of hiv, tb, and malaria programs: the interaction of health and laboratory systems, clinical research, and service delivery. am j clin pathol. 2009;131(6):849–851. http://dx.doi.org/10.1309/ajcpgh89qdswfons2. vitoria m, granich r, gilks cf, et al. the global fight against hiv/aids, tuberculosis, and malaria: current status and future perspectives. am j clin pathol. 2009;131(6):844–848. http://dx.doi.org/10.1309/ajcp5xhdb1pnaeyt 3. tabrizi js, gharibi f, wilson aj. advantages and disadvantages of health care accreditation models. health promot perspect. 2011;1(1):1–31. 4. shroeder lf, amukele t medical laboratories in sub-saharan africa that meet international quality standards. am j clin pathol. 2014;141(6):791–795. http://dx.doi.org/10.1309/ajcpq5ktkagsscfn 5. yao k, maruta t, luman et, nkengasong jn. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. 2014;3(2), art. #194, 8 pages. http://dx.doi.org/10.4102/ajlm.v3i2.194 6. yao k, luman et, slmta collaborating authors. evidence from 617 laboratories in 47 countries for slmta-driven improvement in quality management systems. afr j lab med. 2014;3(2), art. #262, 11 pages. http://dx.doi.org/10.4102/ajlm.v3i2.262 7.world health organization’s regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation in the african region (with checklist) [document on the internet]. c2011 [cited 2013 aug 09]. available from: http://www.afro.who.int/en/clusters-a-programmes/hss/blood-safety-laboratories-a-health-technology/blt-highlights/3859-who-guide-for-the-stepwise-laboratory-improvement-process-towards-accreditation-in-the-african-region-with-checklist.html 8.clinical and laboratory standards institute. application of a quality management system model for laboratory services; approved guidelines – third edition. clsi document gp26-a3. wayne, pa: clinical and laboratory standards institute; 2004. 9.who afro slipta cameroon report 26–30 august 2013. african society for laboratory medicine debrief (unpublished) (aslm); 2013. 10.mataranyika mn, beukes lk. view from the top: involvement of namibia’s health ministry in laboratory quality improvement. afr j lab med. 2014;3(2), art. #195, 2 pages. http://dx.doi.org/10.4102/ajlm.v3i2.195 article information authors: robyn marshall1,2 jenifer vaughan1,2 ria david1,3 elise schapkaitz1,2 sergio carmona1,2 tracey wiggill1,2 affiliations: 1department of molecular medicine and haematology, university of the witwatersrand, johannesburg, south africa 2national health laboratory service, johannesburg, south africa 3division of medical oncology, department of internal medicine, university of the witwatersrand, johannesburg, south africa correspondence to: robyn marshall email: dr.rmarshall@gmail.com postal address: university of the witwatersrand medical school, room 3b22, 7 york road, johannesburg, south africa dates: received: 11 dec. 2014 accepted: 18 aug. 2015 published: 23 nov. 2015 how to cite this article: marshall r, vaughan j, david r, schapkaitz e, carmona s, wiggill t. primary plasma cell leukaemia in a 22-year-old woman: a case report. afr j lab med. 2015;4(1), art. #289, 5 pages. http://dx.doi.org/10.4102/ajlm.v4i1.289 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. primary plasma cell leukaemia in a 22-year-old woman: a case report in this case studies... open access • abstract • introduction • ethical considerations    • potential benefits and hazards    • recruitment procedures    • informed consent    • data protection • case presentation • management and outcome • discussion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ introduction: primary plasma cell leukaemia is a rare and highly aggressive disease that is commonly diagnosed a decade earlier than multiple myeloma, at a median age of 55 years. however, it has also been described in younger patients, as documented in this case report. it often presents with hepatosplenomegaly and lymphadenopathy, whilst the presence of bony lesions are less-commonly seen when compared to multiple myeloma. case presentation: this report describes the case of a young woman who presented with symptoms of anaemia and a history of menorrhagia. on further careful examination, she was found to have additional signs and symptoms and was later diagnosed with primary plasma cell leukaemia. management and outcome: on admission, the patient received supportive care measures, including blood products. at diagnosis, a specific chemotherapy regimen was commenced; however, this failed to induce remission. the decision to continue with supportive care only was made and the patient died seven months later. discussion: this case study is presented because of its rarity, the young age of the patient at presentation and the unusual clinical and laboratory findings. persistent anaemia unresponsive to standard treatment should raise the index of suspicion and further investigations directed to exclude malignancies should be considered. introduction top ↑ plasma cell leukaemia (pcl) is a rare and aggressive plasma cell dyscrasia,1 which is divided into primary and secondary subtypes. the distinction from multiple myeloma (mm) and other plasma cell dyscrasia is based on the presence of a circulating peripheral blood plasma cell count of > 20% or > 2 × 109/l.2,3 primary pcl presents de novo in the leukaemic phase and comprises 60% of all pcl.3,4 it has a median age of diagnosis of 55 years, a decade earlier than mm and secondary pcl, with the symptoms often mimicking those of acute leukaemia.5 prognostic parameters include a low serum albumin; hypercalcaemia and elevated β2 microglobulin, serum lactate dehydrogenase and serum c-reactive protein; an absolute peripheral blood plasma cell count of > 4 × 109/l; thrombocytopenia and an increased percentage of s-phase plasma cells; advanced age (age at diagnosis of 60 years or older) and poor performance status (eastern cooperative oncology group grade of ≥ 2),6,7 based on a patient's ability to perform the normal activities of daily living. specific cytogenetic results associated with lower overall survival rates include a complex karyotype, hypodiploidy, as well as several deletions and translocations.6 ethical considerations top ↑ consent was obtained from the patient with ethical clearance from the university of the witwatersrand human research ethics committee. the ethics clearance number is m130269. potential benefits and hazards there were no risks to the subject involved in this case report; and no potential physical or psychological dangers were anticipated. there was no perceived benefit to this patient. no information that could identify the patient has been published and the authors have endeavoured to maintain the patient's anonymity. it is hoped that other patients with a similar presentation of severe anaemia and a serious underlying condition may be clinically managed more quickly. recruitment procedures as this was a case study that highlighted a rare and interesting case, the patient was requested to provide consent to make use of her clinical information, including examination findings, special investigation results and treatment strategies applied. entitlement to withdraw consent would lapse once the case report was submitted for publication. informed consent the patient was requested to provide written consent to allow for all or any part of this material collected (other than unique patient identifiers) to appear as an abstract, a case study, or an article in a journal, and any other works or products, in any form or medium. data protection the patient's name has not been published with the material and the authors have endeavoured to assure anonymity. however, it is understood that despite the best efforts of the authors, the possibility that someone, for example, members of the patient's family or the healthcare staff, may recognise the patient from the images and/or the accompanying text. all data collected for this case report was available only to the authors of the case report and no other party had access to any of the patient's individual identifiers. case presentation top ↑ a 22-year-old female patient presented at a peripheral clinic in september 2012 with a history of heavy menses after receiving medroxyprogesterone acetate for contraceptive purposes in july 2012. at this time she complained of fatigue, dizziness, lower back pain and poor appetite; iron supplements were prescribed. in november 2012 she presented at a local hospital complaining of severe back pain and persistent vaginal bleeding. her past medical history revealed that this had been ongoing for more than one month. on admission she was found to have no significant lymphadenopathy or hepatosplenomegaly. she was also tachycardic and an echocardiogram revealed a functional ejection systolic murmur. a radiographic skeletal survey showed multiple lytic lesions (figure 1). all pertinent laboratory investigations are summarised in table 1. figure 1: radiographic skeletal survey showing multiple lytic lesions although no pathological fractures were present. (a) the lesions involved the ribs, the shoulders bilaterally and the spine. (b) the pelvis and the upper femora also reveal lytic lesions. a number of baseline and definitive special investigations were performed (table 1). a bone marrow investigation revealed hypercellular marrow with extensive infiltration by a population of abnormal plasma cells similar to that described in the peripheral blood (figure 2a, 2b and 2c). the infiltrate was positive for immunohistochemical stains, including cd38 (figure 2d), cd56 and mum-1, but was negative for cyclin-d1, cd20 and cd45. fewer than 20% of cells were positive for ki-67. immunophenotypic analysis revealed a population of ~60% – 70% large, more complex cells that expressed bright cd38, moderate cd138 and bright aberrant cd56 with dim kappa light-chain restriction. figure 2: bone marrow aspirate and trephine samples of primary plasma cell leukaemia. (a) tumour cells were variable in size, ranging from small to intermediate. the nuclei were rounded and eccentric and a peri-nuclear hoff was also noted (giemsa staining. magnification: 50×). (b) occasional, more primitive binucleate forms were also observed (giemsa staining. magnification: 50×). (c) the chromatin appeared clumped but had a more primitive appearance in the larger cells with occasional nucleoli (giemsa staining. magnification: 50×). (d) normal haemopoiesis was mostly displaced by the extensive diffuse infiltrate of cd38 positive cells (anti-cd38 staining. magnification: 40×). fluorescence in situ hybridisation analysis revealed that the cells were positive for a deletion at 13q14.3, showed trisomy of chromosome 18 and loss of the fibroblast growth factor receptor (fgfr3) gene. chromosomal analysis of the bone marrow cultures revealed a complex karyotype (table 1). features of note were numerical aberrations, including monosomy of chromosomes 12, 13 and 14 and trisomy of chromosome 18. structural aberrations included partial deletion of chromosome 1p and an extra chromosome 1 with a partial deletion on the p-arm, leading to an additional copy of the 1q region. there was a translocation involving chromosomes 3 and 14, leading to rearrangement of the immunoglobulin heavy chain locus (igh@) gene. terminal deletion of chromosome 4p confirmed the deletion of the fgfr3 gene. table 1: summary of relevant laboratory investigations performed on this patient. management and outcome top ↑ on admission, the patient was stabilised and given fresh frozen plasma, platelets and packed cells. with confirmation of the diagnosis, the patient was started on a chemotherapy regimen which included: vincristine 3.6 mg/m2 intravenously on days 1–4 (d1–d4), doxorubicin 9 mg/m2 intravenously on d1–d4, dexamethasone 40 mg orally on d1–d4, dexamethasone 40 mg orally on d9–d12, dexamethasone 40 mg orally on d17–d20. she received one cycle of therapy; however, this induction failed to induce remission. discussion top ↑ de novo pcl is a rare disease and presentation in patients aged younger than 40 years is exceptionally rare. to the best of our knowledge, only two case reports of such patients, in whom primary pcl presented at aged 30 and 21 years, have been reported in the literature.8,9 as is often reported, this patient presented with symptoms suggestive of an acute leukaemia. however, some of her presenting symptoms would more commonly be seen in secondary pcl and mm, including the presence of bone pain and lytic lesions. there was no evidence of extra-medullary deposits and there was an absence of hepatomegaly, splenomegaly and lymphadenopathy. there was no evidence of a pleural effusion and no renal dysfunction, which are commonly described in primary pcl.5,7,10,11 in addition to some of the unusual clinical features noted, atypical laboratory features were also found, including the presence of bright cd56 expression on flow cytometric assessment and the absence of cd20 on immunohistochemical staining.10 cytogenetic abnormalities are a common feature of pcl, with 70% of patients with primary pcl and 100% of patients with secondary pcl presenting with abnormal karyotypes.5 a recent study that provided a genomic characterisation of patients with primary pcl revealed a significant overlap with characteristics seen in mm, although tp53 deletions, complex karyotypes, hypodiploidy and igh@ translocations were more frequently present in pcl.4,12,13 these igh@ translocations were identified in 87% of primary pcl cases, del(13q) in 74% of cases and del(17p) in 35% of cases.4 in addition, abnormalities in chromosome 1 are frequent in pcl, particularly 1q21 amplification and del(1p), a deletion that has been associated with shorter overall survival.14,15 both monosomy 13 and trisomy 18 are common in pcl, with monosomy 13 occurring in up to 85% of cases and trisomy 18 in 43%.7,16 all of the aberrations described above were part of this patient's karyotype, except for the tp53 deletions. monosomy 12 and 14 have also been reported in primary pcl in the form of case reports.17 poor prognostic factors in primary pcl include both clinical and laboratory parameters.6,7 of these, the case study patient presented with a performance status score of ≥ 2, an absolute peripheral blood plasma cell count of > 4 × 109/l, thrombocytopenia, diffuse marrow infiltration, specific cytogenetic abnormalities, including a complex karyotype, elevated lactate dehydrogenase, elevated β2 microglobulin and hypercalcaemia. induction therapy failed to induce remission. survival is known to be poor in this category of plasma cell dyscrasia, with 28% of patients dying in the first month following diagnosis.10 the average survival for primary pcl is 11.2 months.5 in general, treatment is aimed at improving quality of life and prolonging survival. therapy initiation should begin promptly and aim for rapid disease control in an attempt to prevent early death.11 chemotherapy regimens have previously been based on those used for mm, with no specific standard protocol available. of these, intensive multidrug regimens with an alkylating agent as a base have been used with limited success and more recently bortezomib-based regimens are recommended, followed by autologous stem cell transplantation, if feasible.6,10 allogeneic transplantation can be considered in younger patients.6 our patient received one cycle of induction therapy with a modified vincristine, doxorubicin, dexamethasone regimen, as bortezomib therapy was not available. with the regimen provided, she never obtained complete remission and autologous transplant was not possible. she subsequently received palliative care and died in hospital seven months after the initial diagnosis. this case was presented because of its rarity and the young age of the patient at presentation, as well as its unusual laboratory and clinical features. this was an unexpected diagnosis and, not surprisingly, the diagnosis was not made at first presentation. a history of recent contraceptive use, menorrhagia and a resultant iron deficiency was not an uncommon finding. however, additional symptoms were also noted and the patient had no improvement, despite iron supplementation. a high index of suspicion, together with early referral and the use of basic first line investigations, such as differential count, should be advocated. the inaccessibility of the recommended drug therapy was also likely to have affected survival and diminished chances for possible stem-cell transplant. it is vital to make an early diagnosis of haematological and other malignancies in our setting where treatment options are already limited and delayed diagnosis may negatively impact prognosis. acknowledgements top ↑ karyotyping and fluorescence in situ hybridisation analyses were performed in the somatic cell genetics unit, department of molecular medicine and haematology, university of the witwatersrand, johannesburg, south africa. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions r.m. (university of the witwatersrand and national health laboratory service) was involved with the concept and design, data collection and writing of the article and takes on the overall responsibility for the article. j.v. and e.s. (university of the witwatersrand and national health laboratory service) were responsible for data collection and critical revision of the article. s.c. and t.w. (university of the witwatersrand and national health laboratory service) were responsible for critical revision and final approval of the article. r.d. (university of the witwatersrand) was responsible for data collection and critical revision of the article. references top ↑ dimopoulos ma, palumbo a, delasalle kb, alexanian r. primary plasma cell leukaemia. br j haematol. 1994;88(4):754–759. pmid: 7819100. kyle ra, maldonado je, bayrd ed. plasma cell leukemia. report on 17 cases. arch intern med. 1974;133(5):813–818. pmid: 4821776. international myeloma working group.criteria for the classification of monoclonal gammopathies, multiple myeloma and related disorders: a report of the international myeloma working group. br j haematol. 2003;121(5):749–757. pmid: 12780789. mosca l, musto p, todoerti k, et al. genome-wide analysis of primary plasma cell leukemia identifies recurrent imbalances associated with changes in transcriptional profiles. am j hematol. 2013;88(1):16–23. pmid: 23044976, http://dx.doi.org/10.1002/ajh.23339 tiedemann re, gonzalez-paz n, kyle ra, et al. genetic aberrations and survival in plasma cell leukemia. leukemia. 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leukaemia as demonstrated by interphase fluorescence in situ hybridisation. br j haematol. 2006;134(6):613–615. pmid: 16889615, http://dx.doi.org/10.1111/j.1365-2141.2006.06237.x avet-loiseau h, daviet a, brigaudeau c, et al. cytogenetic, interphase, and multicolor fluorescence in situ hybridization analyses in primary plasma cell leukemia: a study of 40 patients at diagnosis, on behalf of the intergroupe francophone du myélome and the groupe français de cytogénétique hématologique. blood. 2001;97(3):822–825. pmid: 11157506, http://dx.doi.org/10.1182/blood.v97.3.822 taniwaki m, nishida k, takashima t, et al. nonrandom chromosomal rearrangements of 14q32.3 and 19p13.3 and preferential deletion of 1p in 21 patients with multiple myeloma and plasma cell leukemia. blood. 1994;84(7):2283–2290. pmid: 7919347. background on hiv in south africa current laboratory infrastructure and hiv-related testing in south africa current quality assurance framework and policy for hiv testing in south africa national quality assurance programme for point-of-care testing in south africa acknowledgments references about the author(s) natasha m. gous national health laboratory service, johannesburg, south africa national priority program of the nhls, johannesburg, south africa leigh berrie national health laboratory service, johannesburg, south africa national priority program of the nhls, johannesburg, south africa patience dabula national health laboratory service, johannesburg, south africa wendy stevens national health laboratory service, johannesburg, south africa national priority program of the nhls, johannesburg, south africa department of molecular medicine and haematology, school of pathology, university of the witwatersrand, johannesburg, south africa citation gous nm, berrie l, dabula p, stevens w. south africa’s experience with provision of quality hiv diagnostic services. afr j lab med. 2016;5(2), a436. http://dx.doi.org/10.4102/ajlm.v5i2.436 country profile south africa’s experience with provision of quality hiv diagnostic services natasha m. gous, leigh berrie, patience dabula, wendy stevens received: 11 mar. 2016; accepted: 11 aug. 2016; published: 17 oct. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. background on hiv in south africa south africa is an upper–middle-income country with the second-largest economy in africa.1 in mid 2015, the population was estimated at 54.96 million; 51.0% were women and 30.2% were younger than 15 years of age.2 the population density, age and gender structures, however, vary significantly. south africans have a high burden of communicable diseases such as hiv and tuberculosis, as well as non-communicable chronic diseases such as diabetes, hypertension and cancer.3 life expectancy has seen progressive increases over the years, in part due to the rapid and effective scale-up of hiv and tuberculosis care in the country.2,4 in 2015, life expectancy was 60.6 years for men and 64.3 years for women.2 however, maternal and child mortality rates are relatively high compared to other middle-income countries.4,5 the latest unaids estimates for 2015 indicated that almost 7 million [6.7–7.4 million] south africans were living with hiv.6 in the 15–49-year age group, prevalence was as high as 19.2%, with women the worst affected.6 in the 0–14-year age group, 240 000 children are estimated to be living with hiv.6 antiretroviral therapy coverage is expanding, with approximately 3.4 million south africans currently receiving antiretroviral therapy and close to 4 million predicted to be on antiretroviral therapy in the 2016 and 2017 fiscal year.3 current laboratory infrastructure and hiv-related testing in south africa healthcare in south africa is two-tiered, consisting of a public sector serving over 80% of the population and a smaller private sector catering to the middleand upper-income population, largely through medical insurance. the south african national department of health (ndoh) has overall responsibility for healthcare, but is particularly responsible for the public sector. south africa is divided into nine provincial departments of health; each is responsible for managing and providing comprehensive healthcare services through a district-based model.1 an approximate 4420 primary public healthcare facilities are available, of which 3991 provide hiv treatment services.1,7 diagnostic testing in the public sector is the mandate of the national health laboratory service (nhls), the largest pathology service provider in the country. the nhls is a national public entity established through the amalgamation of a number of public-sector laboratory service providers and various provincial department of health laboratories.8 the nhls serves more than 80% of the population through a network of over 260 laboratories throughout the nine provinces. the national priority program was established in 2010 to provide support to the nhls and ndoh through the management, coordination, standardisation and implementation of a number of national programmes, including hiv viral load, early infant diagnosis (eid), tuberculosis, cd4 and, more recently, hiv drug resistance. the national priority program now supports 16 regional laboratories for hiv viral load testing, nine laboratories for eid, 52 regional laboratories for cd4 testing, 211 genexpert® mtb/rif testing sites and five hiv drug resistance testing laboratories (figure 1). figure 1: gis map of testing laboratories implemented by the national priority program throughout south africa. there are currently (a) 52 cd4 (yellow), (b) 16 viral load (red) and (c) 211 genexpert mtb/rif (green) testing laboratories. further infrastructure supporting the nhls programme includes significant investment into the nhls laboratory information system using trakcare or disalab, to which all analysers within the nhls are interfaced. all national test results are collected centrally and archived within a single central data warehouse, which is a large server able to store, manage and analyse all laboratory information system data from all tests generated. for cd4 testing, the routine method employed is panleucogating, a cost-effective technology developed within south africa and licensed to beckman coulter.9,10 approximately 3.6 million panleucogating cd4 tests were performed within the nhls in 2015. south africa has also evaluated the alere pima™ cd4 technology and other point-of-care (poc) devices.11,12,13 a number of pimas have been implemented by non-government organisations14 and department of health facilities in certain provinces for pilot studies, but the south african nhls has not yet adopted pima or any other poc technology for wide-spread implementation. this has mainly been due to current reliable cd4 services with good laboratory turnaround times7 and the prohibitive cost of implementing widespread cd4 testing at the poc.15 however, with increasing testing demands, expansion of cd4 testing services is required. a newly-proposed cd4 testing model, the integrated tiered service delivery model7 (figure 2), provides a service delivery system that enables high-volume testing on one end, whilst integrating and extending laboratory services into small laboratories with existing infrastructure, to rapidly scale up services, to lower volume sites on the other end. placement of testing equipment is therefore, in accordance with service needs; in a community laboratory, for example, small automated equipment is placed to test 100–150 samples per day, covering the service needs of all the local clinics in the area,7 whereas in very remote sites, without any reasonable access to a laboratory and where less than 30 tests per day are needed, multiple poc technologies can be used to supplement and extend laboratory services, facilitating total service coverage7 (figure 2). figure 2: integrated tiered service delivery model. the proposed nhls cd4 tiered laboratory network structure comprises six service tiers, which support decreasing service test volumes in increasingly remote sites. tier 1 (servicing a single site) and tier 2/‘poc hub’ (servicing up to 10 remote clinics) utilise multiple poc technologies to extend laboratory services (hiv, tuberculosis) in remote, hard-to-reach areas, beyond a reasonable distance to a tier-3 laboratory; tier 3 represents a community laboratory that serves > 10 < 50 clinic sites; tier 4 and tier 5 are regional laboratories or ‘metro’ centralised laboratories performing high-volume testing; tier 6 represents coordinated, harmonising national support from an expert team or reference laboratory.7 the integrated tiered service delivery model is currently being implemented in the nhls7 and has demonstrated success16 in substantially reducing turnaround times of reporting in remote areas of south africa, thus ensuring total service coverage and rapid turnaround times, irrespective of where services are provided7 (figure 2). costs of the integrated tiered service delivery model are contained and remain fixed across a network, whilst still providing reasonable access to service. although the decentralised approach using poc (at the tier 1 or tier 2 level) has been shown to cost five to seven times more than a conventional laboratory-based service,15 these higher costs can be cross-subsidised by the majority of national service requirements (in south africa > 90%) being met by significantly more cost-efficient conventional laboratories7,15 at tiers 3, 4 and 5.7 the integrated tiered service delivery model approach further enables a network for technical and quality support for lower tier sites, with lower tier sites supported within a defined locality by the nearest higher tier sites. hiv viral load testing within the nhls relies on centralised, high throughput, laboratory-based testing. the tender is awarded every three years through a highly competitive selection process and for the last two rounds has used two suppliers, namely abbott molecular and roche molecular diagnostics. within the 16 hiv viral load laboratories nationwide, 13 laboratories utilise either the roche cobas® ampliprep/cobas® taqman® version 2 or the high throughput cobas® 8800/6800 systems and three use the abbott m2000 realtime™ hiv-1 system. all instruments are fully automated, interfaced, real-time platforms which facilitate a faster result turnaround time. in 2015 alone, over 3.5 million viral load tests were performed and this number is anticipated to increase to almost 5 million if testing targets are met for the next fiscal year (april 2016–march 2017). in addition to scaling up centralised testing, the nhls is also considering adopting a tiered viral load testing model using a combined approach of high throughput, mid-throughput and poc platforms and is embarking on a pilot project to evaluate the feasibility of such a model in collaboration with the ndoh. eid is performed in nine laboratories on the roche cobas ampliprep/cobas taqman platform using the qualitative cobas ampliprep/cobas taqman hiv-1 test, v2. during 2015, approximately 450 000 hiv pcr tests for eid were performed. due to changes in the clinical algorithm, which includes birth testing and follow-up testing to avert early mortality prior to three months of age, these numbers should theoretically increase significantly and poc technologies may have a place for niche testing, but will require further investigation. the south african ndoh estimates that 60% – 70% of all hiv-positive persons are also co-infected with tuberculosis.3 thus, together with the nhls, the department of health have been global leaders in rolling out genexpert® mtb/rif testing in south africa. a total of 314 genexpert instruments of varying sizes have been placed in 211 sites, in both urban and rural settings, with expansion of the programme to special risk populations such as correctional facilities and peri-mining communities. in future, genexpert® mtb/rif testing laboratories may be used for decentralisation and expansion of viral load testing services. current quality assurance framework and policy for hiv testing in south africa the nhls has implemented a quality management system in compliance with various standards (iso 15189, iso 17025, iso 9001 and iso 17043) and the competence of all medical testing laboratories is, therefore, in accordance with the relevant iso standard and guidelines for good laboratory practice. clinical pathology laboratories operate according to the requirements of iso15189:2012. the nhls national quality assurance division is the national quality-related policy-setting body and is responsible for establishing, documenting, implementing, maintaining and continually improving the quality management system. the national quality assurance division manages the in-house production and distribution of external quality assessment (eqa) material. these proficiency testing schemes are offered both internally and externally, to private laboratories in the country and also to 23 countries outside of south africa, in several pathology disciplines, and are operated in accordance with iso/iec 17043:2010. the national quality assurance division is also responsible for ensuring that nhls laboratories are accredited by the south african national accreditation system through implementation of standard requirements, including participation in eqa programmes/proficiency testing and auditing against iso 15189 for medical testing laboratories. cd4 laboratories participate in the nhls eqa programme and beckman coulter 3-iqap and also monitor internal quality measures (flow count rates) to ensure ongoing excellence of service. all viral load testing laboratories participate in the quality control for molecular diagnostics eqa programme and centers for disease control dry test tube programme; both programmes are coordinated by the nhls quality assurance division. eid testing laboratories participate in the nhls dried blood spot eqa program. specifically for the cd4, hiv and tuberculosis programmes, the national priority program provides monitoring through: monthly meetings with suppliers to identify problem areas, monitor monthly turnaround times, stock control, instrument breakdowns and throughput. site monitoring, including monthly indicator reports detailing test volumes, errors and training needs and which are reviewed and actioned accordingly. for hiv viral load, a process of continuous quality monitoring is used through remote connectivity: the use of the abbott mview software for all hiv viral load laboratories utilising the abbott platform. the use of remote connectivity software (axeda) for all hiv viral load laboratories utilising the roche platform. the use of an antiretroviral therapy dashboard for continuous monitoring of laboratory performance. this dashboard was developed together with the nhls central data warehouse and generates monthly reports for both internal and external stakeholders in terms of test volumes and result ranges from national and provincial down, to district level for hiv viral load, cd4 and eid. laboratory site visits and assistance for accreditation. national quality assurance programme for point-of-care testing in south africa a final policy draft is being vetted for quality assurance of poc testing and the nhls will play a pivotal role in the management and support of poc testing services to ensure it performs to the same quality standards as current diagnostic testing. the nhls should take full responsibility for implementation of technology, training, monitoring and evaluation, procurement and stock control.7 poc testing sites will implicitly follow iso 22780 and nhls will manage relevant accreditation procedures for sites performing poc testing.17 a key component of the quality assurance programme will be the inclusion of internal and external quality control procedures and management. the provision of quality assurance schemes for poc testing will be the responsibility of the nhls, which already has the capacity, expertise and proven experience to establish and implement new programmes. as an example, the nhls assisted the ndoh in ensuring the quality of the approximate 8 million rapid hiv tests performed per year, through development of materials, test kit monitoring and training. this included development of a quality assurance plan and quality improvement programme in collaboration with the centers for disease control, which has been adopted by the ndoh. as part of these activities, post-market surveillance of rapid-test kit lots is conducted prior to national release. the quality assurance programme for rapid hiv testing is still an area in need of improvement. some of the key challenges experienced during implementation of the rapid hiv testing quality assurance programme included: staff not implementing what was taught during training; lack of understanding of quality management principles; staff not adhering to quality assurance testing procedures due to high workload; and lack of knowledge transfer following training. many of these challenges have been overcome using the following strategies: initiation of ‘train-the-trainer’ workshops. development of a draft quality plan which is made available to sites. training of provincial coordinators who have overall site management and can follow up on problem sites. introducing more follow-up site visits to check compliance. the genexpert® mtb/rif dried culture spot eqa programme, developed in collaboration with university of the witwatersrand in 2011,18,19 is another example of nhls expertise. the dried culture spot eqa has become an integral component to the genexpert® mtb/rif programme and now supports ~391 sites in 24 countries. the material is manufactured and distributed in-house and is easy, safe, stable and cost effective. the programme is supported by an automated, real-time reporting and quality monitoring web-based tool, tbgx monitor™ (www.tbgxmonitor.com), which remotely collects and analyses eqa data by uploading the data from individual genexpert modules. this programme has also shown success in non-laboratory users18 and is being expanded to other molecular tuberculosis diagnostic platforms20 as well as adaptation to other diseases. a similar quality assurance model will likely be developed for poc technologies. conclusion if poc testing is expected to improve and support diagnostic and clinical services in south africa, the laboratory needs to play a major role in ensuring success of the programme through a poc quality assurance framework, much the same as for laboratory testing. the nhls has demonstrated proven success in conceptualisation and implementation of quality management systems for national programmes and will adopt a similar strategy for poc testing through the integrated tiered service delivery model. acknowledgments the authors wish to acknowledge the ndoh, nhls, national priority program (npp) and quality assurance division for the work which contributed to this publication. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support none. references southafrica.info. healthcare in south africa [page on the internet]. c2012 [cited 2016 feb 18]. available from: http://www.southafrica.info/about/health/health.htm#.vswwh0axhyg statistics south africa. statistical release p0302: mid-year population estimates 2015 [document on the internet]. c2015 [cited 2016 sept 14]. available from: http://beta2.statssa.gov.za/publications/p0302/p03022015.pdf south african national department of health. national department of health annual performance plan 2014/15–2016/17 [document on the internet]. c2014 [cited 2015 mar 15]. available from: http://www.hst.org.za/publications/national-department-health-annual-performance-plan-201415-201617 world health organization. south africa: country health profile [page on the internet]. c2015 [cited 2016 feb 18]. available from: http://www.afro.who.int/en/south-africa/country-health-profile.html countdown to 2015 [now countdown to 2030]. a decade of tracking progress for maternal, newborn and child survival. country profiles: south africa [document on the internet]. c2015 [cited 2016 feb 18]. available from: http://www.countdown2015mnch.org/country-profiles unaids. south africa: hiv and aids estimates 2015 [page on the internet]. c2015 [cited 2016 sept 14]. available from: http://www.unaids.org/en/regionscountries/countries/southafrica glencross dk, coetzee lm, cassim n. an integrated tiered service delivery model (itsdm) based on local cd4 testing demands can improve turn-around times and save costs whilst ensuring accessible and scalable cd4 services across a national programme. plos one 2014;9(12):e114727. http://dx.doi.org/10.1371/journal.pone.0114727.ecollection 2014. national health laboratory service. genexpert remote connectivity [page on the internet]. c2014 [cited 2014 mar]. available from: http://www.nhls.ac.za/?page=cepheid_genexpert_interfacing&id=71 glencross dk, scott le, jani iv, et al. cd45-assisted panleucogating for accurate, cost-effective dual-platform cd4+ t-cell enumeration. cytometry. 2002;50(2):69–77. glencross dk, janossy g, coetzee lm, et al. large-scale affordable panleucogated cd4+ testing with proactive internal and external quality assessment: in support of the south african national comprehensive care, treatment and management programme for hiv and aids. cytometry b clin cytom. 2008;74(suppl 1):s40–51. http://dx.doi.org/10.1002/cyto.b.20384 bergeron m, daneau g, ding t, et al. performance of the pointcare now system for cd4 counting in hiv patients based on five independent evaluations. plos one. 2012;7(8):e41166. http://dx.doi.org/10.1371/journal.pone.0041166 epub 2012 aug 9. glencross dk, coetzee lm, faal m, et al. performance evaluation of the pima™ point-of-care cd4 analyser using capillary blood sampling in field tests in south africa. j int aids soc. 2012;15(1):3. http://dx.doi.org/10.1186/1758-2652-15-3 gous nm, scott le, potgieter j, et al. implementation and operational research: implementation of multiple point-of-care testing in 2 hiv antiretroviral treatment clinics in south africa. j acquir immune defic syndr. 2016;71(2):e34–43. http://dx.doi.org/10.1097/qai.0000000000000872 fajardo e, metcalf c, piriou e, et al. errors generated by a point-of-care cd4+ t-lymphocyte analyser: a retrospective observational study in nine countries. bull world health organ. 2015;93(9):623–630. http://dx.doi.org/10.2471/blt.14.146480 cassim n, coetzee lm, schnippel k, et al. estimating implementation and operational costs of an integrated tiered cd4 service including laboratory and point of care testing in a remote health district in south africa. plos one. 2014;9(12):e115420. http://dx.doi.org/10.1371/journal.pone.0115420 ecollection 2014. coetzee l, cassim n, glencross d. implementation of a new ‘community’ laboratory cd4 service in a rural health district in south africa extends laboratory services and substantially improves local reporting turnaround time. s afr med j. 2016;106(1):82–87. http://dx.doi.org/10.7196/samj.2016.v106i1.10081 world health organization. improving the quality of hiv-related point-of-care testing: ensuring reliability and accuracy of test results [document on the internet]. c2015 [cited 2016 feb 19]. available from: http://www.who.int/hiv/pub/toolkits/handbook-point-of-care-testing/en/ gous n, cunningham b, kana b, et al. performance monitoring of mycobacterium tuberculosis dried culture spots for use with the genexpert system within a national program in south africa. j clin microbiol. 2013;51(12):4018–4021. http://dx.doi.org/10.1128/jcm.01715-13 epub 2013 sep 25. scott le, gous n, cunningham be, et al. dried culture spots for xpert mtb/rif external quality assessment: results of a phase 1 pilot study in south africa. j clin microbiol. 2011;49(12):4356–4360. http://dx.doi.org/10.1128/jcm.05167-11 gous n, isherwood le, david a, et al. a pilot evaluation of external quality assessment of genotype mtbdrplus versions 1 and 2 using dried culture spot material. j clin microbiol. 2015;53(4):1365–1367. http://dx.doi.org/10.1128/jcm.03340-14 epub 2015 jan 21. abstract introduction methods results discussion acknowledgements references about the author(s) nora m. said department of clinical pathology, faculty of medicine, zagazig university, zagazig, egypt enas m. mekkawy department of rheumatology and rehabilitation, faculty of medicine, zagazig university, zagazig, egypt dina said department of rheumatology and rehabilitation, faculty of medicine, zagazig university, zagazig, egypt dina g. abdelhamed department of clinical pathology, faculty of medicine, zagazig university, zagazig, egypt alia a. el shahawy department of medical microbiology and immunology, faculty of medicine, zagazig university, zagazig, egypt eman a. abdelaziz department of medical biochemistry, faculty of medicine, zagazig university, zagazig, egypt fatma z. kamel department of medical microbiology and immunology, faculty of medicine, zagazig university, zagazig, egypt alshymaa a. ahmed department of clinical pathology, faculty of medicine, zagazig university, zagazig, egypt citation said nm, mekkawy em, said d, et al. genetic copy number variation of tlr7, t-bet, il12b, and fcγriiib in egyptian ankylosing spondylitis patients. afr j lab med. 2025;14(1), a2916. https://doi.org/10.4102/ajlm.v14i1.2916 original research genetic copy number variation of tlr7, t-bet, il12b, and fcγriiib in egyptian ankylosing spondylitis patients nora m. said, enas m. mekkawy, dina said, dina g. abdelhamed, alia a. el shahawy, eman a. abdelaziz, fatma z. kamel, alshymaa a. ahmed received: 20 june 2025; accepted: 11 oct. 2025; published: 29 nov. 2025 copyright: © 2025. the authors. licensee: aosis. this work is licensed under the creative commons attribution 4.0 international (cc by 4.0) license (https://creativecommons.org/licenses/by/4.0/). abstract background: ankylosing spondylitis (as) is a polygenic disorder. copy number variations (cnvs) alter the expression of tlr7, t-bet, il-12b, and fcγriiib genes, contributing to as development via their immune system roles. objective: the current study aimed to assess the correlation between as susceptibility and cnvs of tlr7, t-bet, il12b, and fcγriiib, as well as their influence on disease activity. methods: the study involved 42 healthy controls and 72 patients with as, recruited from the rheumatology and rehabilitation clinic of zagazig university hospitals, zagazig, egypt, from 01 november 2023 to 30 october 2024. sociodemographic, clinical data and blood samples were collected from all participants. copy number estimations for tlr7, t-bet, il12b, and fcγriiib genes were performed using sybr green real-time polymerase chain reaction. results: of 72 cases aged 19 to 52 years, 47 (65.2%) were men and 25 (34.8%) were women. controls included 42 participants aged 27 to 55 years, 22 (52.4%) men and 20 (47.6%) women. higher il12b and fcγriiib gene copy numbers were significantly associated with a higher risk of as (p = 0.001, odds ratio [or]: 3.8, 95% confidence interval [ci]: 1.7–8.07, and p < 0.001, or: 5.5, 95% ci: 2.31–13.08, respectively). while t-bet and tlr7 gene cnvs showed no significant association with as risk. no significant association was observed between the studied cnvs and as activity. conclusion: high copy numbers of il12b and fcγriiib genes may be associated with increased as risk. however, no significant correlation was found between as risk and tlr7 or t-bet cnvs. what this study adds: the findings of this study revealed that genetic copy number variations may contribute to the risk of as. keywords: ankylosing spondylitis; copy number variations; tlr7; t-bet; il12b; fcγriiib. introduction ankylosing spondylitis (as) is a common seronegative autoimmune arthropathy, primarily characterised by axial skeleton and sacroiliac joint inflammation. in severe cases, inflammation may progress to fibrosis and calcification, reducing spinal flexibility and resulting in fusion and restricted mobility.1 the primary clinical symptoms include back pain, progressive stiffness of the spine, and joint inflammation. furthermore, extra-articular manifestations, particularly acute anterior uveitis and inflammatory bowel disease, may further complicate the clinical course.2 the aetiology of as is multifactorial, involving numerous factors, such as genetic factors, environmental influences, prior infections, and socio-economic status.3 the most substantial genetic correlation is found with hla-b27, a key genetic factor accounting for 16% – 50% of disease genetic susceptibility.4,5 previous genetic studies on as have primarily focused on disease susceptibility. despite its significance, investigating genetic factors associated with radiographic damage and disease activity has been largely overlooked.4,6 recent findings have highlighted the substantial role of copy number variations (cnvs) in the progression of as.3 copy number variations are structural genomic alterations involving genome-wide deletions or duplications that affect one kilobase to several megabases.7 copy number variations can significantly alter gene expression by altering the copy number (cn) of dosage-sensitive loci, thereby perturbing gene expression.8 moreover, cnvs may reposition genes and regulatory regions, affecting gene expression.9 research increasingly emphasises the significance of cnvs in various complex immune and nonimmune disorders.10,11 gene cnvs for fc gamma receptor iiib (fcγriiib), t-box expressed in t-cells (t-bet), interleukin-12b (il12b), and toll-like receptor 7 (tlr7) have been identified as risk factors for the onset of several autoimmune disorders, such as rheumatoid arthritis, as, and systemic lupus erythematosus.12,13,14,15 lower cns of fcγriiib genes have been linked to an increased risk of developing as.16 a high cn of the t-bet gene has been associated with increased susceptibility to as, along with acute anterior uveitis, indicating the potential impact of the t-bet gene cnv on the clinical course of the disease.15 a previous study demonstrated that extra gene copies are associated with higher messenger rna levels of t-bet and il-12b,17 increasing il-12b expression, which has been demonstrated to exacerbate the severity of as.18 a chinese study indicated that cnvs in the tlr7 gene are linked to as, with a reduced cn posing a risk for men and a protective effect for women.19 furthermore, a number of studies have connected several gene polymorphisms to the progression of as and the activity of the disease. the ankylosing spondylitis disease activity score based on c-reactive protein is the standard method for evaluating as disease activity. this validated composite index combines c-reactive protein levels with patient-reported measures (back pain, morning stiffness length, and global disease activity), and categorises disease activity as inactive (< 1.3), low (1.3–2.1), high (2.1–3.5), or extremely high (≥ 3.5), based on the 2018 criteria.20 the link between cnvs and as risk has been the subject of several studies in developed countries, but far less is known in north african countries with limited resources, especially egypt. moreover, to our knowledge, the influence of these cnvs on disease activity has not been previously examined. thus, the purpose of this study was to examine the relationship between the risk of developing as in egyptian patients and cnvs in the fcγriiib, t-bet, il12b, and tlr7 genes. we also aimed to assess the influence of these cnvs on as activity. methods ethical considerations this study was approved by the zagazig university institutional review board (approval number: 11207) on 25 october 2023. all participants provided written informed consent prior to participating. procedures were followed in accordance with the helsinki declaration. the patient’s information had been concealed from the lab staff as the data records and sample tubes were coded. subjects this study was a case-control study conducted at zagazig university hospital, zagazig, egypt, from 01 november 2023 to 30 october 2024. it comprised 72 consecutive patients with as, recruited from the rheumatology and rehabilitation clinic of the zagazig university hospitals, zagazig, egypt. the control group comprised 42 healthy volunteers of the same ethnicity, matched for age and gender, devoid of any autoimmune or malignant disorders. sample size of this study was calculated using epi info version 3.01 (centers for disease control and prevention [cdc], atlanta, georgia, united states), at 95% confidence interval (ci), based on the previously published data.12,15,19 eligibility criteria of participating patients included fulfilling the modified new york criteria for as diagnosis.21 patients with other coexisting immunological or malignant conditions were excluded from the study. the diagnosis of as and the evaluation of disease activity have been conducted by two expert rheumatologists based on clinical examination, radiographic evidence, and laboratory investigations. a verbal questionnaire was given to the participants in order to gather sociodemographic data, including age, ethnicity, disease duration and family history. the following were obtained from patients’ records: hla-b27 status, c-reactive protein, and erythrocyte sedimentation rate levels. disease activity was assessed using asdas.20 extraction of genomic dna venous blood samples were collected in tubes containing ethylenediaminetetraacetic acid. total dna was extracted according to the manufacturer’s instructions using dna isolation kits (qiaamp dna minikit; qiagen gmbh, hilden, germany) and stored at −20 °c. agarose gel electrophoresis was used to roughly assess the extracted dna concentration before storage.22 copy number estimation real-time quantitative polymerase chain reaction (pcr) (rtqpcr, sybr green; enzynomics, life technologies, delhi, india) was utilised to assess the cnvs of fcγriiib, t-bet, il12b, and tlr7, following the previously described procedure.23 the concentration of the extracted dna was accurately measured using a qubit fluorometer immediately prior to the real-time pcr run for mixture preparation.24 the pcr reactions were conducted using an applied biosystems 7500 real-time pcr machine, following the manufacturer’s instructions for the pcr mixture and cycling conditions. forkhead box protein p2 (foxp2) gene (a non-cnv gene) served as the reference for normalisation. table 1 lists the primer sequences used to estimate cnvs for the genes under study and the reference gene foxp2. table 1: primer sequences for estimating copy number variations for the reference gene foxp2 and the genes under study, zagazig university hospitals, zagazig, egypt, november 2023 – october 2024. copy numbers (cns) were calculated using the comparative cycle threshold (2-^^ct) method.23 copy numbers for fcγriiib, t-bet, il12b, and tlr7 were categorised as low cn (< 2), normal cn (= 2), and high cn (> 2). data analysis data were tabulated and analysed using spss-22 (ibm corporation, armonk, new york, united states). student’s t-test and one-way analysis of variation with least significant difference post-hoc test were used to compare means, and the kruskal-wallis test was used to compare medians. chi-square and fisher’s exact tests were used to compare frequencies. the level of statistical significance was set at a p-value of < 0.05. results demographic and clinical characteristics the study included 114 participants: 72 as patients and 42 healthy controls. among the 72 as patients, aged 19 to 52 years, 47 (65.2%) were men and 25 (34.8%) were women. the control group consisted of 42 individuals aged 27 to 55 years, with 22 (52.4%) men and 20 (47.6%) women. among patients with as, 11.1% exhibited low disease activity, 58.3% demonstrated high activity, and 30.6% presented with very high activity. table 2 and table 3 summarise the demographics and clinical characteristics of the study participants. table 2: demographic characteristics of the study participants, zagazig university hospitals, zagazig, egypt, november 2023 – october 2024. table 3: clinical characteristics of the study participants, zagazig university hospitals, zagazig, egypt, november 2023 – october 2024. frequency distribution of fcγriiib, t-bet, il12b, and tlr7 genes copy number variations patients and healthy controls were compared regarding the frequency of cnv in the genes under investigation (table 4). table 4: fcγriiib, t-bet, il12b, and tlr7 gene copy number variation distribution in ankylosing spondylitis patients and healthy controls, zagazig university hospitals, zagazig, egypt, november 2023 – october 2024. elevated cns of the il12b and fcγriiib genes (> 2) were associated with an increased risk of as (> 2) (il12b: p = 0.001, odds ratio [or]: 3.8, 95% ci: 1.7–8.07; fcγriiib: p < 0.001, or: 5.5, 95% ci: 2.31–13.08). while low cns of il12b and fcγriiib genes (< 2) were found to be associated with reduced risk of as (il12b: p = 0.02, or: 0.38, 95% ci: 0.16–0.87; fcγriiib: p = 0.001, or: 0.22, 95% ci: 0.09–0.54). however, neither t-bet nor tlr7 gene cnvs were significantly associated with the risk of as. association between ankylosing spondylitis activity and copy number variations in fcγriiib, t-bet, il12b, and tlr7 genes to determine the association between cnvs of the examined genes and as activity, we analysed the differences in cases concerning cnvs and the erythrocyte sedimentation rate, c-reactive protein, hla-b27, and ankylosing spondylitis disease activity score – c-reactive protein grade (table 5). however, we could not identify any correlation between the clinical manifestations of as and fcγriiib, t-bet, il12b, and tlr7 cnvs. table 5: ankylosing spondylitis disease activity score – c-reactive protein grade and copy number variation frequencies of fcγriiib, t-bet, il12b, and tlr7, zagazig university hospitals, zagazig, egypt, november 2023 – october 2024. discussion the study examined the association between the risk of as and cnvs in the fcγriiib, t-bet, il12b, and tlr7 genes among egyptian patients, as well as their impact on disease activity. the results of the current investigation showed a strong correlation between elevated fcγriiib and il12b copy numbers (> 2 copies) and a higher risk of as, with 3.8-fold and 5.5-fold increases in risk associated with each. as for the t-bet gene cnv, there was no significant distinction between as patients and healthy controls, even though as patients had a higher frequency (51.4%) of increased cns (> 2 copies). furthermore, as patients had a higher frequency of low tlr7 cn (59.7%). nevertheless, no significant link between as risk and tlr7 cnvs was found. meanwhile, we found no association between as activity and cnvs of tlr7, il12b, t-bet, or fcγriiib. in contrast to our findings, the analysis of fcγriiib cnv frequency distribution in a study of algerian as patients in 2019 showed no statistically significant difference between patients and controls.28 a chinese study in 2016 indicated that a low cn (≤ 3) of the fcγriiib gene may be a risk factor for as.12 the observed discrepancy in results may be attributed to variations in the distribution of cn groups, as our study defined three copies as a high cn. to the best of our knowledge, this is the first study to investigate the association of il12b cnv and the risk of as. previous research, however, including a chinese study in 2024 and a bulgarian study in 2019, found a strong correlation between il12b snps and an increased genetic risk for as.29,30 additionally, il12b has been identified as a possible therapeutic target for as in two recent chinese studies conducted in 2024.30,31 in contrast to our findings, a study conducted in china in 2016 identified a higher prevalence of a high t-bet cn in as patients, which was significantly correlated with disease risk.15 a chinese study in 2018 identified a significant correlation between low tlr7 cn and the risk of as. nevertheless, the cns found in this investigation were restricted to one or two copies, in contrast to our analysis, which found that more than two copies of the gene were found in 51.4% of as patients and 42.9% of healthy controls.19 however, our findings aligned with those of the chinese 2018 study by wang et al., which indicated no association between clinical manifestations and tlr7 cnvs.19 no prior research has examined the relationship between as activity and fcγriiib, t-bet, and il12b cnvs. limitations it is important to take into account the limitations of this study when evaluating the results. first, the study was conducted at a single centre and had a relatively small sample size. therefore, subsequent multicentre studies with larger cohorts may be necessary to corroborate the results, as they might be lacking sufficient power. second, despite the established impact of genetic cnvs on the level of gene expression, which may influence the outcomes of therapy, the study did not address the effect of the genes under investigation on the response to therapy. conclusion the study’s findings indicate that possessing more than two copies of both il12b and fcγriiib may increase the risk of developing as. however, as risk was not significantly correlated with tlr7 or t-bet cnvs. acknowledgements we express our gratitude to all of the willing participants in this study, as well as to the staff at zagazig university hospitals, particularly the medical laboratory department and the rheumatology and rehabilitation clinic in zagazig, egypt, for their technical assistance throughout this study. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions n.m.s. and a.a.a. helped to conceptualise the study. n.m.s., e.m.m., d.s., and d.g.a. all made contributions to the study’s investigation. contributions to data curation came from e.m.m., d.s., d.g.a. and a.a.a. d.g.a., a.a.a., a.a.e.s., e.a.a., and f.z.k. all made contributions to the methodology. the project administration and original draft preparation were handled by n.m.s. the article was reviewed and edited with assistance from e.m.m., d.s., f.z.k., e.a.a., a.a.e.s. and a.a.a. f.z.k., e.a.a., and a.a.e.s. assisted with the study supervision. all of the authors read and approved the article. 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department, st. francis mission hospital, katete, zambia ruth l. mfune department of clinical sciences, faculty of medicine, michael chilufya school of medicine, copperbelt university, ndola, zambia paul m. syapiila department of clinical sciences, faculty of medicine, michael chilufya school of medicine, copperbelt university, ndola, zambia sharon d. zimba department of clinical sciences, faculty of medicine, chikankata college of biomedical sciences, chikankata, zambia pipina a. vlahakis department of basic science, faculty of medicine, michael chilufya school of medicine, copperbelt university, ndola, zambia samson mwale department of biomedical sciences, tropical diseases research centre, ndola, zambia kapambwe mwape department of clinical sciences, faculty of medicine, michael chilufya school of medicine, copperbelt university, ndola, zambia memory chirambo-kalolekesha department of clinical sciences, faculty of medicine, michael chilufya school of medicine, copperbelt university, ndola, zambia misheck chileshe laboratory department, mary begg health services, ndola, zambia joseph mutale laboratory department, kabompo district hospital, kabompo, zambia tobela mudenda department of pathology, ndola teaching hospital, ndola, zambia grace manda laboratory department, kalomo district hospital, kalomo, zambia victor daka department of clinical sciences, faculty of medicine, michael chilufya school of medicine, copperbelt university, ndola, zambia citation chawe a, mfune rl, syapiila pm, et al. knowledge, attitude and practices of covid-19 among medical laboratory professionals in zambia. afr j lab med. 2021;10(1), a1403. https://doi.org/10.4102/ajlm.v10i1.1403 original research knowledge, attitude and practices of covid-19 among medical laboratory professionals in zambia adon chawe, ruth l. mfune, paul m. syapiila, sharon d. zimba, pipina a. vlahakis, samson mwale, kapambwe mwape, memory chirambo-kalolekesha, misheck chileshe, joseph mutale, tobela mudenda, grace manda, victor daka received: 21 sept. 2020; accepted: 06 jan. 2021; published: 04 mar. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: coronavirus disease 2019 (covid-19) is a novel disease that has spread to nearly every country worldwide. medical laboratory professionals are key in the fight against covid-19 as they provide confirmatory diagnosis for subsequent management and mitigation of the disease. objective: this study investigated the knowledge, attitude and practices of covid-19 and their predictors among medical laboratory personnel in zambia. methods: we conducted a cross-sectional study among medical laboratory professionals in zambia from 10 to 29 june 2020. data were collected using google forms and exported to statistical package for social sciences version 23 for statistical analysis. independent predictors of covid-19 knowledge and practices were determined. adjusted odds ratios (aor) and their 95% confidence intervals (ci) are reported. results: a total of 208 medical laboratory professionals, 58.2% male, participated in the study. the majority of respondents had good knowledge (84.1%) and practice (75.0%) regarding covid-19. predictors of good knowledge included having a bachelor’s degree (aor: 5.0, ci: 1.13–22.19) and having prior covid-19 related training (aor: 8.83, ci: 2.03–38.44). predictors of good practice included having a master’s or doctor of philosophy (phd) qualification (aor: 5.23, ci: 1.15–23.87) and having prior covid-19 related training (aor: 14.01, ci: 6.47–30.36). conclusion: our findings revealed that medical laboratory professionals in zambia have good knowledge regarding covid-19. there is need for continuous professional development to ensure that medical laboratory professionals are well informed and aware of best practices to aid in curbing the pandemic. keywords: covid-19; medical laboratory professional; knowledge; attitude; practices. introduction coronavirus disease 2019 (covid-19) is a respiratory disease caused by severe acute respiratory syndrome coronavirus 2.1 coronaviruses have been known to affect humans, infecting the respiratory tract and causing infections ranging from mild to severe.2 in the past, different strains of coronaviruses have caused severe acute respiratory syndrome and middle east respiratory syndrome. research has shown that covid-19 is more contagious compared to the previous outbreaks, but less lethal.3 transmission occurs through the inhalation of droplets or contact with surfaces that have been contaminated with the severe acute respiratory syndrome coronavirus 2. as of this writing, there is currently no known vaccine or documented specific treatment for covid-19 disease.4 drugs that show potential to treat critically ill patients are still being investigated for safety and efficacy.4,5,6 prevention and control of the spread of covid-19 is done by social distancing, wearing face masks to prevent both the inhalation and transmission of infectious droplets, as well as effective hand hygiene by regularly washing hands or using alcohol-based hand sanitisers.7,8 the first cases of covid-19 were reported in december 2019 in wuhan, hubei province, central china. since then, the disease has spread to nearly every country worldwide leading to the world health organization declaring covid-19 a pandemic on 11 march 2020.9 as of 27 october 2020 there were 44 055 642 confirmed cases and 1 168 306 deaths reported globally, with 1 740 720 confirmed cases and 38 830 deaths reported in africa.10 in zambia, 16 243 confirmed cases and 348 deaths were reported as of 27 october 2020, including a high proportion of community deaths.11,12 few publications and national situation reports exist that detail the number of healthcare workers (hcws) including medical laboratory personnel around the world infected with covid-19.13 in china where the disease first started, 2055 hcws were infected as of 24 march 2020.14 in the united states, 9282 hcws were infected as of 14 april 2020.15 the world health organization estimates that over 1000 hcws in africa had been infected with covid-19 by 23 july 2020.16 information on the source of infection among hcws in countries that have recorded the disease remains limited. medical laboratory professionals are key personnel in the diagnosis of covid-19 in zambia. although they are not in the front line, which precludes them from prominence, their role in providing confirmatory diagnosis is the main basis upon which cases are identified and clinical management is instituted. the work areas of biomedical laboratory professionals are very hazardous due to both suspected and unsuspected infectious agents. lack of knowledge and good attitude, as well as poor laboratory practices, can have a twofold effect. firstly, a wrong diagnosis leading to wrong patient management could portend severe consequences for the patient as well as undermine transmission prevention efforts; this is particularly true with covid-19. secondly, poor attitude and practices could result in safety incidents (such as infection transmission) which could be deleterious to both the concerned staff and their immediate environment, including co-workers, families and patients or laboratory clients.17 therefore, this study aimed at evaluating the knowledge, attitudes and practices of medical laboratory professionals in zambia who are integral in the diagnosis of covid-19. methods ethical considerations ethical clearance for this study was obtained from the tropical diseases research centre institution review board (registration number: 00002911). the questionnaire contained an information sheet regarding the study and an informed consent statement for participants to agree to participate or not. all participants who declined to take part in the study were immediately withdrawn and could not proceed to respond to the questionnaire. access to the online portal for the questionnaire was limited to investigators who were assigned user rights by the principal investigator to ensure confidentiality of collected data. sample size determination we used the methods for calculating the sample size for prevalence studies as described by pourhoseingholi and others.18 assuming an 82% prevalence of good knowledge and practice regarding covid-19 obtained from a similar study among hcws,19 a confidence level of 95% and a precision level of 5%, we obtained a minimum required sample size of 227 participants. by extrapolating to a finite population of 1900 medical laboratory personnel officially recognised under the medical laboratory register of the biomedical society of zambia, we obtained a final sample size of 204 participants. study design this cross-sectional survey was conducted among 208 medical laboratory professionals in zambia. due to covid-19 related restrictions imposed during this period, it was not feasible to conduct face-to-face interviews and therefore we administered an online questionnaire using google forms (alphabet, inc., mountain view, california, united states). data collection content validation of the questionnaire was done by administering the questionnaire to faculty in the biomedical sciences unit at the copperbelt university, ndola, zambia. the results from the pre-test were not included in the final analysis but were used to modify the questionnaire based on feedback. the questionnaire had two main components: demographics and knowledge, attitudes and practices. the demographic section had 8 questions while the knowledge, attitudes and practices section was divided into the following subsections: knowledge (8 questions), attitude (5 questions) and practices (13 questions). to ensure that only target respondents participated in the survey we distributed the link to the survey through the email database and whatsapp (facebook inc, menlo park, california, united states) group facilitated by the biomedical society of zambia during data collection from 10 to 29 june 2020. a total of 750 participants were availed the link for the questionnaire. there were 210 medical laboratory personnel who participated in the study, giving a response rate of 28%. participants who consented to participate in the study by checking the consent box in the preliminary page of the questionnaire were allowed to proceed with the rest of the questionnaire. only participants who completed the questionnaire by clicking on the ‘submit’ button had their responses recorded. data management and statistical analysis collected data were downloaded and cleaned in microsoft excel (microsoft corp., redmond, washington, united states) and exported to statistical package for social sciences version 23 (ibm corp., armonk, new york, united states) for statistical analysis. to ensure the internal consistency and reliability of the data, we used cronbach’s alpha coefficient according to methods described in a previous study.20 we obtained an alpha value of 0.645, indicating adequate reliability.21 the outcome variables in this study were covid-19 knowledge levels and practice towards covid-19. attitude was investigated but limited to descriptive analysis due to the limited number of questions. eight questions were used to assess covid-19 knowledge, each scoring 1 point for a correct answer. thirteen questions were asked to determine whether a participant had good practice towards covid-19 or not, each scoring 1 point for positive practice and 0 for negative practice. bloom’s cut-off of 80%22 was used to determine good knowledge and good practice. questions were adapted from a previous study.23 to identify factors that predict good knowledge and practice towards covid-19, bivariate analysis was performed. all factors that were statistically significant as reported by 95% confidence interval (ci) in bivariate logistic regression were included in a forward stepwise multivariate logistic regression model to identify factors that were independently associated with good knowledge and practice. results demographic characteristics a total of 208 medical laboratory professionals from seven provinces of zambia took part in this study. there were more men (n = 121, 58.2%) than women. half of the participants were aged 20–29 years while more than half had a diploma. most of the respondents were from a hospital laboratory (n = 134, 64.4%) (table 1). table 1: demographic characteristics of medical laboratory professionals, zambia, june 2020. participants showed good knowledge regarding awareness of practical measures to stop the spread of covid-19 (n = 208, 100.0%), the techniques used to test for covid-19 (n = 208, 100.0%) and symptomatic management of covid-19 (n = 202, 97.1%) (table 2 and table 3). one-quarter (n = 52, 25.0%) of the participants reported being involved in sampling for covid-19 while slightly over a third (n = 73, 35.1%) reported having been trained in sample handing and transportation. table 2: characteristics of responses for knowledge and practice towards covid-19, zambia, june 2020.† table 3: characteristics of responses for knowledge and practice towards covid-19, zambia, june 2020.† factors associated with covid-19 knowledge a high proportion of respondents were knowledgeable about covid-19 (n = 175, 84.1%). bivariate logistic regression analysis showed that having a bachelor’s degree compared to having a certificate or diploma (crude odds ratio (or): 4.68, ci: 1.07–20.44) and covid-19 training (crude or: 8.72, ci: 2.02–37.65) among participants were associated with covid-19 knowledge. therefore, participants with higher academic qualifications and covid-19 training were 4.68 and 8.72 times more likely to have good covid-19 knowledge (table 4). table 4: covid-19 knowledge of participants and associated factors, zambia, june 2020. practice towards covid-19 by medical laboratory personnel poor practices towards covid-19 were recorded among three-quarters of the participants. having a master’s degree or a doctor of philosophy compared to having a certificate or diploma (crude or: 4.51, 95% ci: 1.30–15.70), private or research laboratories compared to clinic or health centre laboratories (crude or: 3.09, 95% ci: 1.01–9.45) and covid-19 training (crude or: 12.97, 95% ci: 6.19–27.18) were associated with good covid-19 practices (table 5). table 5: covid-19 practices of participants and associated factors, zambia, june 2020. attitude towards covid-19 among medical laboratory personnel about 93.8% of participants reported that they would accept isolation from the community if diagnosed with covid-19. a few (46.6%) would accept to be vaccinated against covid-19 if a vaccine was available. on the other hand, many participants (97.6%) were ready to take part in anti-epidemic community activities. our study revealed that 77.9% were confident that zambia could win the battle against the covid-19 virus (table 6). table 6: attitude of biomedical professionals towards coronavirus disease 2019, zambia, june 2020. factors independently associated with good knowledge and practice about covid-19 after controlling for possible confounding factors, having a higher current qualification (bachelor’s degree) and covid-19 training were independently associated with good covid-19 knowledge among biomedical professionals in zambia. similarly, having a higher current qualification (master’s degree or a doctor of philosophy) and covid-19 training were independently associated with good covid-19 practice (table 7). table 7: factors independently associated with good covid-19 knowledge and practice, zambia, june 2020. discussion very few studies worldwide have documented knowledge, attitudes and practices among hcws towards covid-19 due to the novel nature of the disease.22 our study findings showed that the majority of medical laboratory professionals in zambia had good knowledge of covid-19. the level of knowledge on covid-19 among participants was similar irrespective of their gender, age and laboratory facility. these findings are encouraging as they indicate that there are no inherent differences in knowledge of covid-19 among groups based on unique demographic characteristics in the population. the majority of participants exhibited poor practices towards covid-19 contrary to findings from uganda and nepal.19,22 differences in knowledge and practice regarding covid-19 have been reported before in a study by asemahagn.24 most participants with poor practices were those who had certificate qualifications, those without prior covid-19 training and those from clinic and health centre laboratories. this could be attributed to limited resources, health information and laboratory materials in most clinic and health centre laboratories found in rural areas. poor practices can lead to delayed or wrong laboratory diagnosis, leading to poor patient management or safety incidents that could harm the personnel and their immediate co-workers, families and patients or laboratory clients.25 current qualification and covid-19 training among participants were significantly associated with good covid-19 knowledge, a finding similar to that obtained by a study in vietnam,26 but contrary to the findings of bhagavathula and others.27 our findings show that participants with higher academic qualifications and covid-19 training were 4.68 and 8.72 times more likely to have good covid-19 knowledge. the current qualification was also significantly associated with good covid-19 practices which agrees with study findings from uganda.22 on the other hand, the type of laboratory facility and covid-19 training were significantly associated with good covid-19 practices. this shows that participants who received covid-19 training were 12.97 times more likely to have good practices towards covid-19 and general infection prevention; this is in agreement with similar studies in nepal and ethiopia.19,28 limitations the study may be susceptible to self-presentation bias as it was based on an online questionnaire. the study was limited by the level of responses available and could have been strengthened by increasing the range of answers using, for example, a five-point likert scale. the questions on attitude were limited and therefore not powerful enough to generate meaningful conclusions on the attitude of respondents. the study could not capture dropouts and respondents who refused consent as only individuals who consented and submitted the questionnaire had their responses recorded. conclusion our study found that medical laboratory professionals in zambia have good knowledge regarding covid-19. the current qualification and covid-19 training were independently associated with covid-19 knowledge and practice. as cases of covid-19 continue to be recorded in the country, there is need for continuous professional development among medical laboratory personnel as a key intervention in improving their contribution to covid-19 control efforts. acknowledgements the authors are grateful to the biomedical society of zambia and its members for facilitating the distribution of questionnaires to the medical laboratory personnel. competing interests the authors declare that they have no financial or personal relationships that may have influenced the writing of this article. authors’ contributions a.c., r.l.m., g.m. and v.d. conceptualised the study. a.c., s.d.z., p.a.v., s.m., k.m., m.c.-k., j.m. and t.m. developed the data collection tools. a.c., p.m.s., m.c. and v.d. performed the formal analysis and interpretation. a.c., r.l.m., p.a.v., k.m. and v.d. wrote the first draft manuscript. all authors read and approved the final manuscript. sources of support this research was not supported by any external 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– structure and aims college constitution strategic plan curriculum sources of funding achievements to date future plans acknowledgements references about the author(s) shahin sayed department of pathology and laboratory medicine, aga khan university hospital, nairobi, kenya rudo mutasa department of pathology, university of zimbabwe, harare, zimbabwe ephata kaaya department of pathology, muhimbili university of health and allied sciences, dar es salaam, united republic of tanzania victor mudenda department of pathology, university teaching hospital, lusaka, zambia erasmus rajiv department of chemical pathology, stellenbosch university, stellenbosch, south africa edda vuhahula department of pathology, muhimbili university of health and allied sciences, dar es salaam, united republic of tanzania jamilla rajab department of human pathology, university of nairobi, kenyatta national hospital, nairobi, kenya robert lukande department of pathology, makerere university, kampala, uganda edwin walong department of human pathology, university of nairobi, kenyatta national hospital, nairobi, kenya angela mutuku college of pathologists of east central and southern africa, ecsa health community, arusha, united republic of tanzania kenneth fleming green templeton college, university of oxford, oxford, united kingdom citation sayed s, mutasa r, kaaya e, et al. establishing the college of pathologists of east, central and southern africa – the regional east central and southern africa college of pathology. afr j lab med. 2020;9(1), a979. https://doi.org/10.4102/ajlm.v9i1.979 lessons from the field establishing the college of pathologists of east, central and southern africa – the regional east central and southern africa college of pathology shahin sayed, rudo mutasa, ephata kaaya, victor mudenda, erasmus rajiv, edda vuhahula, jamilla rajab, robert lukande, edwin walong, angela mutuku, kenneth fleming received: 22 jan. 2019; accepted: 04 mar. 2020; published: 03 june 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract issues: the scarcity of pathologists in sub-saharan africa is a well established fact that is attributable to few training programmes in the region; this is further compounded by the lack of harmonised curricula, training and exams within and without member countries. description of the intervention: through the association of pathologists of east, central and southern africa, the college of pathologists of east, central and southern africa (copecsa) was formed with the clear-cut goal of establishing a regional and internationally recognised college to support and inform good quality medical and laboratory practice by promoting leadership, mentorship and excellence in the safe practice of pathology through training, exams, accreditation, advocacy and professional development for health. lessons learnt: since its inception in 2010, copecsa has conferred fellowships to 120 practising pathologists in the east, central and southern africa in partnership with international organisations; the college has been awarded five competitive grants and conducted several quality improvement workshops. recommendations: this paper describes the journey that copecsa has made towards standardising the practice and training of pathology in the east central and southern africa region. keywords: copecsa; ecsa; pathology; sub-saharan africa; college. issues pathologists are medical doctors specialising in the study of the cause of disease and how it affects the body. they study these by examining changes in the cells, tissues, blood and other body fluids. pathology is, therefore, a wide area of medical practice that encompasses several specialties including, but not restricted to: anatomical and surgical pathology, cytopathology, microbiology, haematology, chemical pathology, forensic pathology, immunopathology, molecular pathology, and genetic pathology. the list keeps expanding with advances in medicine. pathology is crucial to the practice of medicine and therefore touches every facet thereof. pathologists guide medical doctors on the right path for treating disease(s) and significantly contribute to research that advances medicine and devises new treatment. in summary, pathologists investigate the potential, presence, cause, severity, and progress of the disease. additionally, they also contribute to monitoring the effects of treatment on diseases.1 it has been well established for some time now that there is a scarcity of pathologists in sub-saharan africa.2 this scarcity is compounded by a shortage of training programmes, with only about 80 cellular pathologists being trained each year.3 in addition, the variable standards of training in pathology have further increased the gap in the delivery of quality pathology and laboratory services. as an example, some training programmes in anatomical pathology suffer from a lack of student exposure to sufficient numbers of cases and case mix4 due to a paucity of standardised guidelines for competency training. this situation is further exacerbated by the unrealistic expectations placed on newly-qualified pathologists who are posted to large public hospitals without appropriate mentorship and supervision and are thus ill-prepared to manage complex laboratory functions. the founding of the regional college of pathology for east central and southern africa (ecsa) was hence conceptualised in an effort to address both issues. description of the intervention the idea of a regional college of pathology in the ecsa region is probably as old as the association of pathologists of east, central and southern africa (apecsa), which was established in 1990.5 subsequently, in the united nations development programme’s review of member countries’ progress towards the attainment of the millennium development goals at the half-way stage (2007), it was acknowledged that, although the ecsa nations had made varied progress on millennium development goals pertaining to the health of citizens, member countries were behind on the realisation of targets and that much more in terms of action, allocation of resources and sustained commitment was needed.6 in response to this, with respect to the training of pathologists, the members of apecsa decided to move towards the establishment of the college of pathologists of east, central and southern africa (copecsa). a steering committee was constituted in 2008 to spearhead the establishment of the college and work on the constitution. the steering committee comprised members from seven of the nine ecsa countries. the proposed name of the college was adopted as the ‘college of pathologists of east central and southern africa’ with the acronym ‘copecsa’. the steering committee organised a consultative meeting on the establishment of copecsa on 4 august, 2009, at fairview hotel, nairobi, through sponsorship from the british division of the international academy of pathology (bdiap) and the united kingdom’s royal college of pathologists (rcpath). members of the steering committee, apecsa executive officers and representatives from apecsa countries attended the meeting. in addition to expertise from overseas; expertise was sought from two other existing african colleges: the college of pathologists of south africa and the west africa college of pathologists. the host organisation, east, central and southern african – health community, was also represented. the meeting laid the foundation for the formation of copecsa by deliberating on the constitution, the structures of the future college, membership, registration fees, timelines for operationalisation and linkages with other colleges and institutions. it was agreed that copecsa would be an autonomous body and the steering committee was proposed to act as an interim copecsa council. the steering committee presented a final draft constitution and organisational structure for the college, which was adopted by the apecsa 2010 annual general meeting in kampala, uganda, where a resolution was passed that paved the way for the establishment of the college. the constitution provided for pathologists in apecsa to be founder fellows. the college was formally inaugurated at the same apecsa meeting in kampala in september 2010; an executive council and members of the copecsa council were elected. a six-member executive committee, who form part of the 21-member council drawn from the ecsa region, governs the college. the executive committee oversees the functions of the education, examination and credentials committee and the general purpose and finance committee (figure 1). a part-time development officer, a position that has been funded by the rcpath and bdiap, runs the day-to-day administrative functions for the college. figure 1: the college of pathologists of east, central and southern africa organogram. college of pathologists of east, central and southern africa – structure and aims the college is a professional body that draws its membership from pathologists registered and practising in the 16 member countries, namely: kenya, tanzania, uganda, rwanda, burundi, zambia, zimbabwe, ethiopia, eritrea, south africa, malawi, seychelles, madagascar, botswana, zanzibar and mauritius (table 1). to foster collaboration among the pathology profession on the african continent, an executive decision was made to invite eligible pathologists from the west african college of pathologists to become fellows of the college. furthermore, in recognition of their contribution to the establishment of the college, honorary fellowships were bestowed upon individual fellows of the rcpath and the bdiap. the college was established ‘to develop leadership and promote regional excellence in the practice of pathology and to be responsible for maintaining standards through training, examinations and professional development’.5 with an overarching aim for supporting the practice of pathology in the region, copecsa strives to be recognised as a college that provides structure and leadership in the fields of both anatomical and clinical pathology in sub-saharan africa. table 1: number of college of pathologists of east, central and southern africa fellows distributed across countries. through ‘excellence in pathology for better health’ copecsa seeks to contribute to the quality of health care services for citizens in each of the member countries and beyond, especially by means of the health-related millenium development goals and, now, sustainable development goals. the goal of copecsa is to establish a regional and internationally recognised college that supports and informs medical and laboratory practice and ensures quality pathology in africa. to make this vital contribution to healthcare and the provision of quality health services, copecsa is committed through its vision: ‘to develop and promote leadership and excellence in the safe practice of pathology through training, accreditation, advocacy and professional development for quality health’.8 further, copecsa as a college endeavours in practice to realise its critical mandate as stated in its mission: to develop a highly competent and ethical specialist workforce in pathology able to provide quality laboratory services for diagnosis, prevention, and management of disease and also for research to international standards. [https://www.copecsa.org/about-us/].8 from the outset, the college recognised the importance of setting structures in place to operationalise functions. this process involved the development of the college constitution, a strategic plan, a robust competency-based curriculum, financial considerations and providing opportunities for research and training through leveraging its partnerships with various international organisations. the following sections highlight the various activities the college has undertaken, lessons learned along the way and achievements to date. college constitution the constitution highlights copecsa’s articles of association where, ‘pathology’ has been construed in the fullest and most inclusive sense, outlining in detail the college governance.6 strategic plan anchored on the college’s vision and mission statements, the 5-year strategic plan (2012/3–2017/8) charted the path for the future growth of copecsa. this plan was focused on priorities that align goals towards upholding the core value of maintaining international standards in the safe practice of pathology within the region. the college identified its main strengths and weaknesses, while recognising opportunities for growth (box 1). during the preparation of the strategic plan, arious approaches were deliberated upon in order to address the core issues identified and to mitigate the threats it faces in the current environment (table 2). box 1: college of pathologists of east, central and southern africa strengths, opportunities, weaknesses and threats analysis, 2012. table 2: college of pathologists of east, central and southern africa strategic plan (2012–2018). curriculum the harmonised curriculum sets out the minimum standards and competencies for training and residency programmes in anatomical, clinical and general pathology. the curriculum seeks to harmonise the knowledge, skills and behaviours that respective trainees should acquire during their training and residency in the region.8 the required qualification to become a fellow is an undergraduate medical degree (bachelor of medicine and chiropody [mbchb], bachelor of medicine and surgery [mbbs], or equivalent) qualification. after training in pathology at accredited training institutions, the trainee is certified and conferred a fellowship upon successfully passing the copecsa exams. in order to kickstart the college, founding fellows had to be inaugurated by nomination in order to have a cohort of well-known and recognised academics in the region who could lay the foundation. hence, the inaugural fellows were capped in arusha, tanzania, by senior officials of international sister bodies that had helped set up the college. however, it was soon realised that the original cohort was not broad enough to represent: (1) all the regions of sub-saharan africa and (2) all the specialties in pathology that would allow the college to mount examinations in all. for this reason, a second cohort of fellows was capped in kigali, rwanda, in 2016. going forward, however, the thrust of the college and award of fellowship will largely be by examination. it has taken the college three years to initiate examinations in order to ensure that the standard of the exam is set at the right level from the very beginning. all the necessary stakeholders have been consulted and due diligence performed. the college is now ready to mount its first examination before the end of 2020. in an accompanying article9, we articulate the need for a harmonised curriculum that can serve the region, as has been demonstrated by the lessons learned from the zambia mmed programme in cellular pathology. it is notable that copecsa’s anatomical pathology curriculum has recently been adopted in full by the ministry of health and ministry of education in zambia. in tanzania, copecsa’s curricula have been presented to the tanganyika medical council for consideration and in kenya, the ministry of health and the kenya medical practitioners and dentists board are working with stakeholders to draft a policy paper for the adoption of a collegiate system for all medical specialities. however, much remains to be done to ensure adoption and recognition throughout the ecsa region. sources of funding the college of pathologists of east, central and southern africa is currently dependent on its membership subscriptions. however, the college has been supported in its various activities by both the rcpath and bdiap. both bdiap and rcpath have also supported the position of a part-time development officer for the college. this was with the understanding that the college must become self-sustaining/sufficient in the near future. achievements to date conferment the college held its first conferment ceremony in 2014 in arusha, tanzania, where 64 pathologists across the ecsa region had their degrees conferred (figure 2). subsequently in 2016, the college conferred fellowships on 56 pathologists in kigali, rwanda. figure 2: college of pathologists of east, central and southern africa achievements. development of a curriculum for the region the education examination and credentials committee of the college has worked towards developing curricula in general pathology, anatomical pathology and clinical pathology. the curriculum development process included convening a curriculum development committee to identify critical issues, trends in the content area and assess regional needs. once the programme and course goals and objectives were set, deliberations were held to identify resource materials for programme implementation. assessment tools and instruments were developed to measure student progress. the college is currently in the process of implementing its curriculum. partnerships and projects in partnership with various international organisations (box 2), copecsa has successfully implemented programmes and projects in which more than 100 pathologists have been trained in various aspects of pathology and laboratory medicine service. box 2: current active college of pathologists of east, central and southern africa partnerships. programmes and projects the labskills africa programme ($1 100 000.00): this programme was designed to improve the standards and quality of both pathology and laboratory medicine services. the college partnered with rcpath, bdiap, aga khan university hospital nairobi, stellenbosch university, ecsa health community and five in-country associations (https://www.rcpath.org/international/projects/labskills-africa.html). this programme was a 30-month health systems strengthening initiative funded by the united kingdom’s department for international development, whereby 20 laboratories in kenya, uganda, tanzania, zambia and zimbabwe participated in the use of integrated skills training, knowledge transfer, leadership development and mentoring for the following key tests: rapid hiv, rapid malaria, peripheral blood film, haemoglobin/haematocrit estimation, urinalysis and tuberculosis. at the culmination of the labskills africa programme, a combined population of 110 million people was served, and 1.7 million tests performed in the laboratories (https://www.rcpath.org/international/projects/labskills-africa.html). also, the programme was able to train and mentor 100 pathologists, biomedical scientists, laboratory technologists and technicians across the 20 laboratories. the copecsa/rcpath partnership was feted as a recipient of the times higher education award in the category international collaboration of the year (https://www.timeshighereducation.com/news/times-higher-education-awards-2016).10 national cancer institute program announcement reviewed by an institution 15–155 ($56 000.00): in 2016–2017, copecsa, in partnership with the university of colorado cancer centre and african strategies for advancing pathology, was the recipient of a national cancer institute program announcement reviewed by an institution (15–155) initiative, a regional programme for improving anatomical pathology to support cancer care. this was a 17-month research strategy that focused on evaluating the best approaches to training pathologists and senior residents in ecsa with high quality standardised cancer diagnosis in order to determine which approach is most effective at improving the expertise of the pathology workforce in lowand middle-income countries, and share the lessons learned to contribute to future training efforts.6 this programme engaged 17 pathology departments and trained 52 pathologists and senior residents in institutions from: zimbabwe, kenya, uganda, tanzania, zambia, rwanda, burundi, malawi, madagascar, mozambique, and botswana. a follow-up training workshop was held on 17–19 november, 2018 in nairobi, kenya, funding for which was awarded under the national institutes of health r13 grant mechanism.6 an additional 20 practising pathologists and senior pathology residents from eight countries were trained in staging of breast, cervical, ovarian, colorectal, stomach, endometrial and head and neck cancers. the programme was unique in that international faculty members were paired with local faculty members in the planning and designing of the course curriculum in order to build the capacity of the local faculty to deliver the course content in future workshops.11,12 cytopathology short course: the college, in partnership with the international academy of cytology, trained a total of 27 practising pathologists from the region in image-guided fine-needle aspiration biopsy in nairobi, kenya. faculty members from the university of california, san francisco (united states) and notre dame university, sydney (australia) led the course. national institutes of health – sickle pan-african research consortium collaboration ($634 807.00): copecsa supported the application for a national institutes of health grant by one of its fellows. the sickle pan-african research consortium brings together clinicians, academicians and scientists with existing sickle cell disease programmes, from the hub in east africa (tanzania) and collaborative consortium sites in west africa (ghana, nigeria), which will expand to form the sickle pan-african network by involving 22 sites in 17 countries. the main goal is to reduce the public health burden (mortality and morbidity) of sickle cell disease in africa while at the same time establishing the capacity for further research. the aim is for this research is to contribute to scientific knowledge in order to find a cure for sickle cell disease by striving to reach an understanding on how a monogenic disease can have such heterogeneity in the region. it has taken copecsa more than 5 years to get off the ground. many lessons have been learnt along the way as the college begins to establish its footprint. currently, copecsa covers 13 countries in sub-saharan africa, which may have been an overly ambitious plan. political instability in some member states and communication challenges across borders have been problematic. the engagement of stakeholders at multiple levels in member countries has been a slow and onerous process due to limited funding sources and the lack of a dedicated full-time secretariat. the dependence of the college on international partner support for running the affairs of copecsa has been fraught with challenges as the college, over the years, has become overly reliant on external funding to support its day-to-day functions. furthermore, there have been varying levels of success in persuading university authorities in some member countries of the benefits of adopting a harmonised curriculum and examination process. uncertainties regarding the recognition and registration of fellows by individual country medical boards has further impeded progress. currently, the unpredictable financial sustainability of copecsa and a lack of adequate visibility and assurance of employment opportunities have been barriers to attracting fellows to the college. in order to mitigate some of these challenges, in some member countries, like kenya and tanzania, the college is working with other ecsa colleges to ratify their curricula and examinations with the countries’ medical boards. for a long time, the executive committee were under the misperception that college curricula were regulated under the commission of university education rules and therefore needed to be approved by the commission of university education before roll out. the commission of university education requirements include appropriate physical infrastructure, which was almost impossible to fulfil as the collegiate system was envisioned to be based on apprenticeship at existing accredited training facilities followed by a final common exit exam upon completion of college curricular requirements. a lot of time was thus wasted in the process of understanding the regulatory framework for each country. the recognition that pathology is a highly-specialised discipline meant that only select training facilities across the ecsa region would have the faculty, volumes, case mix and equipment for adequate exposure to specialised techniques such as immunohistochemistry, cytology, and medical and forensic autopsies. prospective candidates would thus be expected to take responsibility for their own training costs at these accredited centres, while the college provides logistical support for visa and accommodation options. to avoid overburdening the current few training facilities with new students, a decision was made by the executive committee to restrict fellowship exams to recent mmed pathology graduates and practising pathologists. the executive committee also noted that the current lack of clarity on cross-border recognition of college credentials is one of the issues that needs to be addressed, in order to attract more members to copecsa. future plans examinations the first sitting of copecsa’s fellowship examinations will take place in july 2020. before july 2020, copecsa proposed to undertake the following activities in preparation for the first sitting: publish and promote the copecsa curricula. recruit and train copecsa examiners. launch a series of webinars to provide key stakeholders, institutions, trainees and prospective candidates with an introduction and overview to copecsa’s curricula, training standards and examinations. establish a network of recognised training institutions and examination centres throughout the region. develop an accreditation process for training institutions, wishing to be accredited by copecsa. launch copecsa’s trainee membership category. applications to sit the 2020 copecsa fellowship examinations would open at the end of february 2020. recommendations the establishment of a regional college of pathology in ecsa is a first step in harmonising pathology training and standardising the practice of pathology within the region. through sharing of resources among member states, the college aims to maximise scarce human resources and infrastructure. furthermore, the proposed accreditation of training sites lends itself to improvement in the quality of pathology training and subsequent practice. the college strongly recommends partnerships with like-minded regional and international pathology associations as a strategy that is vital for the successful implementation of the college agenda. a key recommendation of the college is measurement of improvement in health services that can be ascribed to the establishment of copecsa. the value of a pathologist in clinical practice has already been highlighted. the college plans to institute monitoring and evaluation activities to measure its impact on improved health service provision and overall health outcomes within the ecsa region. acknowledgements we would like to thank the east central and southern africa health community (ecsa-hc) and the department of pathology at the aga khan university hospital, nairobi for supporting copecsa’s secretariat. we thank the royal college of pathology (rcpath) and the british division of the international academy of pathology (bdiap) for financial support to the college towards standardising the practice of pathology in ecsa region. we thank dr ann nelson, dr alec howatt, dr archie prentice, dr dan milner, rosemary emodi, umo young for their support towards the establishment of copecsa. we also thank gaylord mwangi for technical assistance in formatting the manuscript. permission to publish this manuscript was obtained from the executive council of copecsa. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions authors s.s., r.m., e.k. and k.f. contributed to the design of the manuscript. all authors contributed to the writing and approved the final version of this manuscript. ethical considerations ethical approval was not required for this article. sources of support we are grateful to the royal college of pathology (rcpath), british division of the international academy of pathology (bdiap) for financial support and the east central and southern africa health community (ecsa-hc) and the department of pathology, aga khan university for supporting our secretariat. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views expressed in the submitted article are those of the authors and the college and not an official position of the institutions or funders. references the royal college of pathologists of australasia. what is pathology? [homepage on the internet]. 2019 [cited 2019 apr 25]. available from: https://www.rcpa.edu.au/pathology-careers/what-is-pathology nelson am, milner da, rebbeck tr, iliyasu y. oncologic care and pathology resources in africa: survey and recommendations. j clin oncol. 2016;34(1):20–26. https://doi.org/10.1200/jco.2015.61.9767 wilson ml, fleming ka, kuti ma, looi lm, lago n, ru k. access to pathology and laboratory medicine services: a crucial gap. lancet. 2018;391(10133):1927–1938. https://doi.org/10.1016/s0140-6736(18)30458-6 sayed s, cherniak w, lawler m, et al. improving pathology and laboratory medicine in low-income and middle-income countries: roadmap to solutions. lancet. 2018;391(10133):1939–1952. https://doi.org/10.1016/s0140-6736(18)30459-8 nelson am, hale m, diomande mij-m, et al. training the next generation of african pathologists. clin lab med. 2018;38(1):37–51. https://doi.org/10.1016/j.cll.2017.10.004 lomazzi m, borisch b, laaser u. the millennium development goals: experiences, achievements and what’s next. glob health action. 2014;7:23695. https://doi.org/10.3402/gha.v7.23695 african strategies for advancing pathology | [homepage on the internet]. [cited 2018 dec 28]. available from: https://www.pathologyinafrica.org/ copecsa – pathologists of east, central & south africa copecsa [homepage on the internet]. [cited 2018 dec 28]. available from: https://www.copecsa.org/about-us/ mudenda v, malyangu e, sayed s, fleming k. addressing the shortage of pathologists in africa: creation of a mmed programme in pathology in zambia. afr j lab med. 2020;9(1), a974 https://doi.org/10.4102/ajlm.v9i1.974 winners of times higher education awards 2016 announced | the news [homepage on the internet]. [cited 2018 dec 28]. available from: https://www.timeshighereducation.com/news/winners-times-higher-education-awards-2016-announced sayed s, lester sc, wilson m, et al. creation and pilot testing of cases for case-based learning: a pedagogical approach for pathology cancer diagnosis. afr j lab med. 2017;6(1):7. https://doi.org/10.4102/ajlm.v6i1.637 wilson ml, ayers s, berney d, et al. improving anatomic pathology in sub-saharan africa to support cancer care. am j clin pathol [serial online]. 2018 mar 7 [cited 2018 dec 28];149(4):310–315. available from: https://academic.oup.com/ajcp/article/149/4/310/4872377 country situation, including hiv status laboratory infrastructure and hiv related testing in malawi malawi’s quality assurance framework and policy for hiv laboratory and point-of-care testing existing quality assurance programmes and lessons learnt acknowledgements references about the author(s) lutho i. zungu ministry of health directorate of health technical services – diagnostics, lilongwe, malawi termson magombo clinton health access initiative, lilongwe, malawi tarsizio chikaonda university of north carolina (unc) hiv/aids research project laboratory, lilongwe, malawi rachel thomas clinton health access initiative, lilongwe, malawi reuben mwenda ministry of health directorate of health technical services – diagnostics, lilongwe, malawi james kandulu ministry of health directorate of health technical services – diagnostics, lilongwe, malawi benson chilima national reference laboratories, community health sciences unit (chsu), lilongwe, malawi mavuto chiwaula national reference laboratories, community health sciences unit (chsu), lilongwe, malawi alwin mbene northern health zone, ministry of health, lilongwe, malawi emmanuel saka unicef, lilongwe, malawi citation zungu li, magombo t, chikaonda t, et al. a national quality assurance programme for point-of-care testing in malawi. afr j lab med. 2016;5(2), a540. http://dx.doi.org/10.4102/ajlm.v5i2.540 country profile a national quality assurance programme for point-of-care testing in malawi lutho i. zungu, termson magombo, tarsizio chikaonda, rachel thomas, reuben mwenda, james kandulu, benson chilima, mavuto chiwaula, alwin mbene, emmanuel saka received: 05 aug. 2016; accepted: 11 aug. 2016; published: 17 oct. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. country situation, including hiv status malawi is a landlocked country, stretching over 94 084 square kilometres of land and 24 404 square kilometres of fresh water, whose economic backbone is agriculture. malawi’s hiv positivity rate is estimated at 10.0% (table 1).1 hiv prevalence varies widely by geographic regions, where it is 14.5% in southern malawi, 7.6% in central malawi and 6.6% in northern malawi. prevalence also differs by level of urbanisation in line with population density. rural malawi has a positivity rate of 8.9%, which is half the rate of the 17.4% found in urban areas.1 table 1: malawi key statistics: brief demographic summary. laboratory infrastructure and hiv related testing in malawi malawi has nine molecular laboratories that serve as hubs for pcr testing and process all hiv viral load and early infant diagnosis (eid) samples collected on dried blood spots (figure 1). malawi is piloting point-of-care (poc) testing for eid. seven health facilities are participating in this pilot. table 2 shows the number of cd4 testing devices at facilities at different levels of the public health care system, as well as at partner and private laboratories. figure 1: distribution of pcr laboratories and early infant diagnosis point-of-care testing in malawi. (a) red balloons show the nine conventional pcr laboratories. (b) red balloons show the seven pilot sites for early infant diagnosis point-of-care testing. table 2: distribution of laboratory and point-of-care cd4 testing in malawi table 3 shows the number of cd4, hiv eid and viral load tests performed on both laboratory and poc platforms. the low percentages for the targets in all categories demonstrate that there is a large gap between demand and needs met. table 3: summary of cd4, early infant diagnosis, and viral load testing in 2015. besides the ongoing implementation pilot of poc testing for eid in tertiary, secondary and primary care settings, malawi is also considering poc testing for hiv viral load in 2016 and 2017 on the most relevant platforms available on the market. malawi’s quality assurance framework and policy for hiv laboratory and point-of-care testing poc testing has a significant part to play in the delivery of efficient healthcare services, as rapid availability of test results can lead to increased clinical effectiveness, less loss to follow-up and improved outcomes for patients. poc testing has also helped expand laboratory services to hard-to-reach areas that lack trained technicians or constant electricity supply.2 however, along with the scale-up of poc/near-poc diagnostics, there is a critical need to ensure the quality and accuracy of testing results through proper training and innovative quality assurance activities. a formal policy defining the principal role of the diagnostic division of the health technical services directorate in malawi has become indispensable. this ensures that the whole process of testing is conducted in accordance with the fundamental principles of clinical governance and national, as well as international, accreditation standards to improve and sustain quality. currently, the professional partnership that exists between the diagnostics division, clinical, and implementing partners in malawi ensures that poc testing equipment is suitable for its intended use, is adequately supported (in terms of consumables and maintenance), safety and quality standards are adequately met, results of investigations performed are recorded, and that it is operated only by well-trained staff. the diagnostic technical working group has established a poc testing subcommittee to provide technical advice about equipment and consumables, user training, internal quality assurance and external quality assurance (eqa) implementation, support and accreditation. to systematically guide this process, the following documents have been developed: poc guidelines. sample transportation guidelines. hiv testing and counseling guidelines. genexpert guidelines. quality assurance manual. hiv clinical management guidelines for adults and children. procurement guidelines. training certification guidelines. to monitor and improve the quality and accuracy of test results, various tools have been developed and are now being implemented as part of the quality framework. these tools include the following: the dried tube specimen-based national proficiency testing programme. standardised hiv logbooks for national quality assurance programme. hiv rapid testing quality improvement initiative. registration for enrollment on international eqa schemes. this quality framework is summarised in figure 2. figure 2: malawi’s centralised quality assurance framework. the central level includes community health sciences unit and health technical services and support -diagnostics. together with partners, the central level is responsible for: preparation of quality control materials; identifying and correcting problems; evaluating kits and reagents; standardisation of procedures and developing standard operating procedures; preparing reagents; training; collating reports; analysing data; offering feedback; conducting disease surveillance; and evaluating and standardising equipment. the national poc testing task force (subcommittee) reports to national diagnostics technical working group, which in turn reports to the ministry of health. zones hold a support/coordination role. at the zonal level (5 zones), laboratory supervisors follow quality assurance issues, including supervision, mentorship, training, eqa, corrective actions, etcetera. district laboratories supervise the health centres and follow up on quality assurance issues, in the same manner as the laboratory supervisors. existing quality assurance programmes and lessons learnt currently eqa schemes (both international and national) to which testing sites are registered and subscribed include the following: national health laboratory services: this is a south african scheme that provides proficiency testing samples in chemistry, haematology, and cell morphology (monthly). in parasitology, samples for blood and stool parasites are provided three times a year. for tuberculosis microscopy, quality control materials are provided three times a year, whereas for bacteriology cultures, the scheme sends eqa panels three times a year. ukneqas: the scheme sends eqa panels three times per year in the areas of cd4, tuberculosis microbiology, haematology and chemistry. afriqualab: under this scheme, the samples are provided three times a year, for cd4, tuberculosis microbiology, haematology, chemistry and hiv. qasi, canada: this scheme provides free proficiency testing panels three times a year for approximately 60% of cd4 poc testing sites. however, current efforts to scale the scheme up to 100% have slowed down because malawi has changed direction in order to adopt a universal testing and treat policy during the second quarter of 2016. at the local level, there are also national quality assurance schemes organised and provided by the public health and reference laboratory: hiv proficiency testing (biannual). malawi blood transfusion services. central reference laboratory for tuberculosis microscopy and genexpert®; and microbiology quality control materials. lessons learnt and challenges training of poc testers to understand the need for quality assurance is key. where implementation is coordinated multilaterally (donors, partners, and government functionaries), great success is achieved, for instance in cd4 and viral load testing implementation. in addition, sample transportation systems are of great importance, which are being conducted by riders for health in malawi. participation in eqa schemes is improving, even though some participating laboratories/sites delay submission of eqa results. inadequate knowledge of some testers with respect to interpretation of eqa results is an issue in some schemes. where schemes are not well understood, some laboratories are enrolled in two or three schemes for the same assay, yet have limited interest in performing eqa testing. inconsistent supply of laboratory reagents due to stock outs or poor management of the supply chain has negatively impacted eqa implementation. in addition, long periods of equipment breakdown also negatively impact eqa. some further challenges are that not all tests have been enrolled in eqa schemes, which is often due to the cost of enrollment. the lack of availability of internet connectivity in many sites also hinders success of eqa schemes. finally, the existence of vertical disease control programmes compromises collaborative efforts aimed at leveraging resources to maximise productivity and achievement. national quality assurance programme for point-of-care testing in malawi while the principles of quality assurance are the same for poc testing and conventional, laboratory-based testing, the methods by which they are applied differ, depending on many factors. these factors may include clinical setting, testing frequency, complexity of the device and internal controls, cost and practicality of providing the quality assurance system, and whether laboratory or non-laboratory end users are the ones operating the devices.3 currently, the malawi poc testing qa framework follows the structure of the national qa framework (figure 3). figure 3: schematic diagram of malawi’s point-of-care testing testing quality assurance operational framework showing components that support the quality assurance programme. manufacturers have recognised that the main consumers of poc tests are healthcare workers with non-laboratory backgrounds. hence, poc testing units have been developed with built-in quality control checks and connectivity to allow for real-time oversight of testing. eqa programmes are a component of a continuous quality assurance and improvement cycle, and under international organization for standardization (iso) standards, are a mandatory requirement for medical laboratories.4,5 the requirement is reflected in the international standard, iso 22870 poct – requirements for quality and competence.5 conclusion malawi has thus far provided reasonable coverage for eid and viral load testing through its corridor of nine molecular laboratories that serve as hubs. the hub system and dried blood spots have allowed for increased coverage. malawi is currently piloting poc testing for eid to reach even more remote settings. the professional partnerships that malawi ministry of health has fostered between the diagnostics division, clinical, and implementing partners has supported the necessary guidance that will allow for continuous monitoring and further improvements. this partnership has been realised with the poc task force. in addition, a formal poc policy defining the principal roles of partners has become indispensable. similarly, malawi has various partnerships with numerous eqa providers to support quality testing. while participation in eqa schemes is improving, we are looking to use the results from eqa to identify and improve on the inconsistent supply of laboratory reagents due to stock-outs or poor management of the supply chain. acknowledgements competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. sources of support none. references national aids commission, malawi. malawi aids response progress report, april 2015 [document on the internet]. c2015 [cited 2016 sep 26]. available from: http://www.unaids.org/sites/default/files/country/documents/mwi_narrative_report_2015.pdf ministry of health, malawi. hiv viral load scale up strategic and implementation plan, 2015–2018. lilongwe, malawi; 2016. shephard m. point-of-care testing comes of age in australia. aust prescr. 2010;33:6–9. http://dx.doi.org/10.18773/austprescr.2010.003 national pathology accreditation advisory council. requirements for participation in external quality assessment. 4th ed. canberra: australian government department of health and ageing; 2009. international organization for standardization (iso). point-of-care testing (poct) – requirements for quality and competence. document iso 22870:2006. geneva, switzerland: iso, 2006; p. 11. abstract introduction methods results discussion acknowledgements references about the author(s) mark w. kieh partnership for research on ebola virus in liberia (prevail), new kru town, monrovia, liberia sarah m. browne partnership for research on ebola virus in liberia (prevail), new kru town, monrovia, liberia greg a. grandits division of biostatistics, university of minnesota, minneapolis, minnesota, united states julie blie partnership for research on ebola virus in liberia (prevail), new kru town, monrovia, liberia jestina w. doe-anderson leidos biochemical research, fredrick, maryland, united states marie l. hoover advanced biomedical laboratories, cinnaminson, new jersey, united states bionca davis division of biostatistics, university of minnesota, minneapolis, minnesota, united states cavan s. reilly division of biostatistics, university of minnesota, minneapolis, minnesota, united states james d. neaton division of biostatistics, university of minnesota, minneapolis, minnesota, united states h. clifford lane division of clinical research, national institute of allergy and infectious diseases, national institute of health, bethesda, maryland, united states stephen b. kennedy partnership for research on ebola virus in liberia (prevail), new kru town, monrovia, liberialiberian college of physicians and surgeons, monrovia, liberia citation kieh mw, browne sm, grandits ga, et al. adult and paediatric haematology and clinical chemistry laboratory reference limits for liberia. afr j lab med. 2020;9(1), a1080. https://doi.org/10.4102/ajlm.v9i1.1080 original research adult and paediatric haematology and clinical chemistry laboratory reference limits for liberia mark w. kieh, sarah m. browne, greg a. grandits, julie blie, jestina w. doe-anderson, marie l. hoover, bionca davis, cavan s. reilly, james d. neaton, h. clifford lane, stephen b. kennedy received: 20 aug. 2019; accepted: 12 aug. 2020; published: 25 nov. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: as more research is conducted in liberia, there is a need for laboratory reference limits for common chemistry and haematology values based on a healthy population. reference limits from the united states may not be applicable. objective: the aim of this study was to present laboratory reference ranges from a liberian population and compare them to united states ranges. methods: serum chemistry and haematology values from 2529 adults and 694 children and adolescents obtained from two studies conducted in liberia between 2015 to 2017 were used to determine reference limits. after removing outliers, the reference limits defined by the 2.5th and 97.5th percentiles were determined by sex in three age groups (6–11, 12–17, and 18+ years). results: the median (interquartile range) of adults was 29 (23, 37) years; 44% were female. the median (interquartile range) for children and adolescents was 12 (9, 15) years; 53% were female. several reference ranges determined using liberian participants differed from those in the us. for chemistries, a high percentage of both adults and children/adolescents had high serum chloride levels based on united states ranges. for haematology, a high percentage of liberian participants had haemoglobin and related assays below the lower limit of united states ranges. conclusion: chemistry and haematology reference intervals determined for a liberian population of healthy individuals should be considered for establishing eligibility criteria and monitoring of laboratory adverse events for clinical trials as well as for use in clinical settings in liberia and perhaps for other countries in western africa. keywords: reference ranges; liberia; chemistries; haematology; paediatric. introduction reference limits for clinical laboratory tests for healthy adults and children in liberia are not available based on a liberian population. in a clinical setting, reference limits determined from a healthy population can provide useful information for making decisions based on laboratory reports. in addition, in a research setting, reference limits are often used for inclusion and exclusion criteria, for assessing possible adverse effects of treatment, and for the diagnosis of outcomes. differences in reference limits for haematology and chemistry measurements have been reported between united states (us) reference intervals and reference intervals determined for healthy individuals in other african countries.1,2,3,4,5,6,7,8,9,10,11,12,13,14 these differences have been attributed to differences in socioeconomic status, diet, physical exercise, environmental pathogens and altitude. the partnership for research on ebola virus in liberia (prevail) initiated several studies beginning in 2015. our experience carrying out this research motivated the determination of reference limits based on healthy people living in liberia who enrolled in two of the studies. for example, in one of these studies, a vaccine trial for the prevention of ebola virus disease (evd), a large percentage of apparently healthy participants had initial laboratory test result values outside the limits considered ‘normal’. the purpose of this paper is three-fold: (1) to describe chemistry and haematology test results for a large number of apparently healthy adults and children in liberia; (2) to use that data to define reference limits for use in future research projects in liberia; and (3) to compare the reference limits determined based on liberian participants with those based on us participants. methods ethical considerations we used data from two studies. prevail i and prevail iii, to determine the reference limits. all participants aged 18 years and older provided written informed consent. a parent or guardian signed a written informed consent for all participants under age 18 years, and children aged 9 years and older also signed a written assent. both study protocols were approved by the national research ethics board of liberia and the institutional review board of the united states national institutes of health; protocol identification number 15-i-n071 (prevail i) and 15-i-0122 (prevail iii). study design and sample selection the prevail i study was a phase 2 placebo-controlled randomised trial that evaluated the efficacy and safety of two vaccines to prevent evd. the prevail iii study was a cohort study of evd survivors and their close contacts. close contacts were identified by survivors and either lived with the survivor at the time of diagnosis or after discharge from the ebola treatment unit, or were sexual partners after discharge. the study design, methods and results of prevail i and prevail iii have been described elsewhere.15,16,17 study participants used to determine reference limits in prevail i, volunteers aged 18 years or older were enrolled over a three-month period beginning in february 2015 at redemption hospital in monrovia, liberia. the trial excluded participants with a history of evd, those with a temperature of more than 38 ºc, and women who were pregnant or breast-feeding. the following additional exclusions were made for these analyses: (1) participants with a history of high blood pressure, diabetes or cancer; (2) participants with hiv or syphilis infection based on blood testing; and (3) participants with antibody levels against the ebola virus surface glycoprotein greater than or equal to 548 enzyme-linked immunosorbent assay units (eu)/ml which was considered indicative of past ebola infection. these additional exclusions were made to remove potentially unhealthy participants that could have abnormal laboratory values as a result of medical conditions. in prevail iii, close contacts of evd survivors of any age, identified by evd survivors, were enrolled between 2015 and 2017. close contacts were enrolled at three sites: john f. kennedy (jfk) medical centre and duport road clinic (both in monrovia), and c.h. rennie hospital (a more rural site about 70 km north of monrovia). for participants in prevail iii, the following additional exclusions were made for these analyses: (1) participants with a history of high blood pressure, diabetes, cancer, stroke or ischemic heart disease; (2) participants with hiv or syphilis based on blood testing; and (3) participants with antibody levels against the ebola virus surface glycoprotein greater than or equal to 548 eu/ml. a map showing the locations of the sites where participants were enrolled in prevail i and iii is provided in figure 1. monrovia is a coastal city located on the atlantic coast, with elevation just above sea level. figure 1: map of liberia and location of study sites. laboratory measurements laboratories were established at redemption hospital, jfk medical centre, and c.h. rennie hospital. for participants enrolled in prevail i, all laboratory testing was done at redemption hospital. for participants enrolled in prevail iii, the testing was done at jfk hospital (jfk participants) or c.h. rennie hospital (c.h. rennie and duport road participants), with occasional backup testing at redemption hospital. the same laboratory equipment and methods for blood drawing, specimen labelling and testing were used by all three laboratories, with written documentation of all procedures provided in standard operating procedures. all samples were typically analysed within 4 h. calibrators and controls (vender and third-party) were run daily at each site to maintain quality assurance. the vendors provided precision data for each analyte. calibrators and control samples were run before the study samples, and results needed to be within the accepted range in order for the study samples to be analysed. in both studies, the participants were seen in the morning for their baseline clinic visits, during which non-fasting venous blood specimens were obtained. blood was collected in serum separator tubes for chemistry analyses and ethylenediaminetetraacetic acid tubes for haematology. specimens were then transferred to the respective laboratory. all samples were accessioned, centrifuged (if required) and then analysed on benchtop instrumentation: haematology using cell dyn ruby (abbott diagnostics, abbott park, illinois, united states), and chemistries using trademark name for alfa wassermann’s first chemistry analyzer (ace) alera or ace axcel (alfa wassermann, west caldwell, new jersey, united states), with alera or axcel used interchangeably. in addition to assessing hiv and syphilis serostatus, a complete blood count was obtained with differential and platelet count, aspirate aminotransferase, alanine aminotransferase, creatinine, potassium, chloride and sodium. alcohol intake was not ascertained in either study. each day reports were generated giving chemistry and haematology results for each participant and indicating values that were outside ‘normal’ limits based on us values. data analysis laboratory data for prevail i and iii were combined for these analyses. as a first step, outliers for each test were removed after box-cox transformation (restricting transformation to the identity, square root or log) using a method proposed by tukey:18 values 1.5 × interquartile range above the 75th percentile or 1.5 × interquartile range below the 25th percentile were removed. this was done because the information collected to exclude participants with chronic conditions was minimal and the goal was to identify a ‘healthy’ population. following this step, for each laboratory test, the reference limits defined by the 2.5th and 97.5th percentiles were determined based on a commonly used non-parametric approach.19,20 percentile values were back-transformed to the original scale. because it is important that reference ranges consider age and sex, this process was performed separately for each sex and age group (6–11, 12–17, and 18 years and over). the median and reference ranges are cited for male and female individuals in each of the three age groups. the percentage of participants both below and above published us reference ranges21 are cited for each laboratory value. the results found in this study and for other countries in west africa are given for reader reference. no formal statistical tests were made for these comparisons. non-parametric tests were conducted for differences in medians between male and female participants and among the three age groups by sex. all analyses were performed using sas version 9.4 (sas institute, cary, north carolina, united states). p-values < 0.001 were regarded as statistically significant. results of the 3986 participants enrolled in prevail i and iii, 3223 met the eligibility criteria for these analyses (figure 2). the adults enrolled in prevail i that are included in these analyses had a median age of 29 years, 33% were female, and the median body mass index was 21.6 kg/m2 (table 1). the adults in prevail iii that are included in these analyses had similar age distributions as those of the prevail i adults, but included a higher percentage of women and had a slightly higher median body mass index. the median age for the children/adolescents was 12 years, 53% were female, and the median body mass index was 17.4 kg/m2. figure 2: flow diagram for inclusion of participants for determination of reference levels: monrovia, liberia 2015–2017. table 1: baseline characteristics of study participants in laboratory analysis: monrovia, liberia 2015–2017. laboratory median levels and reference limits by age and gender many laboratory median levels and corresponding reference ranges differed significantly by sex and age (table 2). for example, the median and reference ranges for white blood cell (wbc) count in male participants aged 6–11 years was 7.18 (4.61–11.92) x 103/μl compared to 5.77 (3.63–9.72) x 103/μl in adult female participants. table 2: median and reference ranges for chemistries and haematology by gender and age group: monrovia, liberia 2015–2017. for chemistry, adult men had higher liver enzymes, creatinine, and potassium levels, with lower serum chloride and sodium levels than adult women (p < 0.001). in general, the differences between sexes were less pronounced in children/adolescents. for example, the median serum creatinine levels were nearly identical for male and female individuals aged 6–11 years and only slightly higher for male than female individuals aged 12–17 years. for haematology values, adult men had lower wbc counts but higher red blood cell counts and haemoglobin levels than adult women (p < 0.001). platelet counts were higher in adult women than men. the differences in haematology values between sexes were much smaller in the youngest age group (ages 6–11 years) and were not statistically significant. for serum chemistry, the median aminotransferase was inversely related to age for both male and female participants, whereas serum creatinine was strongly positively related to age for both sexes (p < 0.001). the median serum sodium increased with age for both sexes. for haematology, the wbc counts were highest for ages 6–11 years for both sexes and the red blood cell counts increased with age for male but not female participants. the median haemoglobin levels increased with age in both sexes but the relationship was stronger in male participants. platelets decreased with age for both male and female participants. comparison with united states reference ranges for chemistries, a high percentage of both adults and children/adolescents would be classified as having high serum chloride levels based on the us reference standard (table 3). similarly, several haematology factors would be classified as out of range for both adults and children/adolescents using the us standard. a high percentage of both children/adolescent and adult participants had haemoglobin, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, and haematocrit levels below the lower limit. in addition, 20% – 25% of liberian adults had wbc counts and neutrophils below the us reference standard. table 3: percentage of study participants outside of us1 reference intervals: monrovia, liberia 2015-2017 reference ranges for other west african countries medians and reference limits for our liberian study showed some differences compared to reference limits reported for ghana, mali, and gambia (table 4);4,10,13,14 chemistry values were only available from ghana. liver enzymes were lower in liberia than in ghana. potassium, sodium, and chloride levels were also lower in the liberian population than in the ghanaian population. the median and reference limits for wbc counts were similar for men in liberia and mali. the median wbc counts for women in liberia were higher than in the other west african countries. the red blood cell counts for men in liberia were similar to both ghana and mali, while the red blood cell counts for women in liberia were higher than those in ghana. the haemoglobin levels were similar in the liberian population as compared to the other west african countries. platelet counts differed among countries but were always higher in women than men in each country. comparison of medians among countries were not always consistent with the reference ranges, suggesting differences in variability between populations. table 4: comparisons of adult liberian reference ranges with other adult african countries reference ranges, monrovia, liberia 2015–2017. discussion to determine reference limits for common clinical laboratory test results for people in liberia by age and sex, we used baseline data collected at the time of enrolment in two research studies conducted during the west african ebola epidemic. one study enrolled healthy adults in a vaccine trial16 and the other enrolled adults and children who were close contacts of individuals who survived ebola.17 ‘unhealthy’ participants were excluded based on medical history and for positive hiv or syphilis tests. our goal was to select individuals for determining reference intervals and to use recommended statistical methods for determining these intervals as outlined by the clinical and laboratory standards institute22 that could be used to interpret an individual’s laboratory test results and could be used in the design of future clinical research studies. we found that for several laboratory tests, the reference limits based on the data from this liberian population differed greatly from the us-based reference limits. for example, the haemoglobin reference ranges were much lower in this study than the us-based reference values. based on the us reference limits, one could imply that a high proportion of liberians have ‘abnormal’ low haemoglobin levels. other reference limits that differed considerably were chloride, for which the liberian population had higher reference limits, and mean corpuscular volume, mean corpuscular haemoglobin, haematocrit and mean platelet volume, for which the liberian population had lower reference limits. differences between values found in this study compared to us levels could be due to several reasons, including genetics, environmental factors, subclinical disease, and laboratory equipment and/or methods. the observed differences are unlikely to be because of laboratory differences, as the laboratories set up in liberia used standard equipment that is also used in the us for chemistries and haematology. the reference limits from our study also confirm that separate values for many laboratory parameters should be considered for men and women and for adults and children/adolescents. the adult women in our study had significantly higher wbc counts than men. the children/adolescents also had higher wbc counts, lower creatinine levels, and lower haemoglobin levels than adults. many clinical trials use reference limits and tables for grading laboratory toxicities, such as the division of aids table for grading the severity of adult and paediatric adverse events,23 to define eligibility criteria and to grade adverse events during follow-up. the difference in reference limits between those estimated for liberia and us limits could impact the number of participants found to be eligible for a trial, as well as the percentage developing adverse events based on laboratory test results. for trials conducted in other parts of africa this has been the case. eller et al.9 studied the impact of using us reference limits in uganda to screen participants for an hiv vaccine trial. they found that us reference limits led to more exclusions during screening for a phase 1 vaccine trial than the use of their reference limits derived from people living in uganda. they also noted that the division of aids toxicity table did not reflect locally established reference limits, and the lower limit for neutrophils they had estimated would qualify as a grade 2 adverse event. segolodi et al.7 reported that many healthy volunteers for an hiv pre-exposure prophylaxis trial conducted in botswana had abnormal amylase results according to us-derived reference values. zeh et al.24 reported that over 58% of participants would have been excluded from a trial in kenya using us reference limits as compared to reference limits determined for the local population. in addition, 40% of otherwise healthy study participants would have been considered to have a grade 1–4 laboratory-based adverse event based on the division of aids toxicity table. there may be pros and cons to using liberian versus us reference limits for reporting adverse events. the burden associated with reporting lower-severity grade events,25 particularly those based solely on laboratory results and not associated with symptoms, would suggest using reference limits from local populations. on the other hand, in early phase research of novel treatments such as the ebola studies on which this research is based, it may be more prudent to use more conservative limits such as the us reference limits until safety is established. a strength of this study is the large number of participants (> 2500 adults and nearly 700 children/adolescents) studied with common laboratory methods in two research studies, allowing for more precise estimates of laboratory percentiles on which the reference limits are based. to our knowledge, this is the first report of laboratory reference limits from a liberian population. we recommend that additional laboratory-based studies be conducted to establish suitable laboratory reference limits for liberia. limitations a limitation to this study is that we based our definition of ‘healthy’ participants largely on a self-reported medical history. while we used statistical methods that attempted to remove outliers to establish a ‘healthy’ population, and that typically removed about 1% – 3% of participants from the group where normal ranges were calculated using 2.5% and 97.5% percentiles, our cohort is likely to include some participants with undiagnosed illnesses. conclusion in conclusion, we present the chemistry and haematology reference intervals from a liberian population of healthy individuals for men and women and for children/adolescents and adults. these levels should be considered for screening and monitoring participants in clinical trials in liberia and perhaps other countries in west africa. acknowledgements the authors would like to thank the many participants in the prevail i and prevail iii studies whose data were used for this manuscript. competing interests the authors have declared that no competing interests exist. authors’ contributions h.c.l. and s.b.k. conceived and designed the studies; m.w.k., j.d.n. and g.a.g. wrote the article; g.a.g. and c.s.r. analysed the data; m.l.h. set up and conducted the laboratory testing in liberia. clinical support and data collection were provided by m.w.k., s.m.b. and j.b; j.w.d.-a. and b.d. contributed to the editing of the manuscript. j.w.d.-a. and b.d. were also involved in the collection of data and monitoring of the study. source of support united states national institute of allergy and infectious diseases. p1 principal investigators: stephen kennedy, fotorma bolay, and h. clifford lane, p3 principal investigators: mosoka fallah and michael sneller. data availability statement the data used for this manuscript is available upon request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references mekonnen z, amuamuta a, mulu w, et al. clinical chemistry reference intervals of healthy adult populations in gojjam 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https://doi.org/10.1093/clinchem/17.4.275 kratz a, ferraro m, sluss pm, lewandrowski kb. laboratory reference values. n engl j med. 2004;351;1548–1563. https://doi.org/10.1056/nejmcpc049016 clinical and laboratory institute (clsi). defining, establishing, and verifying reference intervals in the clinical laboratory; approved guideline – third edition. clsi document ep28-a3c (isbn 1-56238-683-4). wayne, pa: clinical and laboratory standards institute; 2008. u.s. department of health and human services, national institutes of health, national institute of allergy and infectious diseases, division of aids (daids). table for grading the severity of adult and pediatric adverse events, version 2.0 [homepage on the internet]. c2014 [cited 2019 aug 15]. available from: https://rsc.niaid.nih.gov/sites/default/files/daids-aegrading-table-v2-nov2014.pdf zeh ce, odhiambo co, mills la. laboratory reference intervals in africa. london, uk: intech open; 2012. https://doi.org/10.5772/48250 chou vb, omer sb, hussian h, et al. the costs associated with adverse event procedures for an international hiv clinical trial determined by activity-based costing. j acquir immune defic syndr. 2007;46(4):426–432. https://doi.org/10.1097/qai.0b013e318156ee37 abstract introduction methods results discussion acknowledgements references about the author(s) edwards kasonkanji university of north carolina project-malawi, kamuzu central hospital, lilongwe, malawi tarsizious chikaonda university of north carolina project-malawi, kamuzu central hospital, lilongwe, malawi luke eastburg division of hematology, department of medicine, school of medicine, university of north carolina at chapel hill, chapel hill, north carolina, united states maurice mulenga university of north carolina project-malawi, kamuzu central hospital, lilongwe, malawi tamiwe tomoka university of north carolina project-malawi, kamuzu central hospital, lilongwe, malawi yuri fedoriw department of pathology and laboratory medicine, school of medicine, university of north carolina at chapel hill, chapel hill, north carolina, united states matthew painschab division of hematology, department of medicine, school of medicine, university of north carolina at chapel hill, chapel hill, north carolina, united states amy brownlee department of pathology and laboratory medicine, school of medicine, university of north carolina at chapel hill, chapel hill, north carolina, united states citation kasonkanji e, chikaonda t, eastburg l, et al. diagnostic adequacy of adult bone marrow biopsies in malawi: a retrospective cohort study. afr j lab med. 2025;14(1), a2966. https://doi.org/10.4102/ajlm.v14i1.2966 original research diagnostic adequacy of adult bone marrow biopsies in malawi: a retrospective cohort study edwards kasonkanji, tarsizious chikaonda, luke eastburg, maurice mulenga, tamiwe tomoka, yuri fedoriw, matthew painschab, amy brownlee received: 11 aug. 2025; accepted: 23 sept. 2025; published: 30 nov. 2025 copyright: © 2025. the authors. licensee: aosis. this work is licensed under the creative commons attribution 4.0 international (cc by 4.0) license (https://creativecommons.org/licenses/by/4.0/). abstract background: bone marrow (bm) sampling is a critical component of clinical care, informing the diagnosis of benign and malignant haematologic disorders. objective: we aimed to assess adequacy and characterise the spectrum of diagnoses rendered for bm samples reviewed at kamuzu central hospital in lilongwe, malawi. methods: we reviewed clinical and pathologic data from all adult bm specimens seen at kamuzu central hospital from 01 june 2012, through 30 june 2024, and utilised r to summarise clinical and diagnostic data. results: we identified 602 bm samples, most of which were biopsy-only specimens (n = 335, 56%). most patients were male (n = 345, 57%), with a median age of 42 years. of the 539 (90%) patients with known hiv status, 372 (69%) were hiv-uninfected. the most frequent indications for bm sampling were anaemia (n = 188, 31%) and cancer staging (n = 118, 20%). there was no documented indication for 72 cases (12%). excluding bm samples obtained for cancer staging (n = 118, 20%), there were 403 (83%) diagnostic samples. of these, 156 (39%) were malignant, with 50 (32%) acute leukaemias and 38 (24%) lymphomas. conclusion: we found a high diagnostic yield (85%) of bm samples in this cohort, along with a considerable proportion of new cancer diagnoses (39%) in diagnostic samples taken for reasons other than cancer staging. non-malignant diagnoses were predominant, yet resources for subcategorising these are limited in our setting. what this study adds: this study highlights the high diagnostic yield of adult bm samples at kamuzu central hospital and characterises these diagnoses, revealing not only a substantial burden of malignancies but also important gaps in diagnostic capacity. keywords: bone marrow biopsy; diagnostic adequacy; haematologic disorders; lymphoproliferative disorders; pathology; malawi. introduction bone marrow (bm) sampling is an essential component of diagnosis for numerous haematologic and non-haematologic disorders, allowing for the examination of cellular composition and morphology, structure, and function. repeated bm evaluation is also necessary for ongoing management of patients with certain haematologic malignancies. quality of bm sampling can significantly affect the accuracy of diagnostic interpretation, and recommendations for sampling have been published.1 for example, the world health organization recommends that bm core biopsies measure at least 1.5 cm in length, and provides guidance on how best to interpret bm aspirates – differential counts should be performed closely adjacent to particles of undiluted samples.2 anaemia and pancytopaenia, known indications for bm examination, are common in malawi.3,4 however, resources for identifying the causes of cytopaenia beyond malaria are limited. for example, nutritional supplementation for iron, folate, and vitamin b12 can be attempted. however, specific laboratory testing and examination of tissue biopsies for other causes is typically not available, especially at more outlying health centres.5 bone marrow samples can be interpreted at kamuzu central hospital in lilongwe, malawi, because of ongoing collaborative research and laboratory development efforts between the malawi ministry of health, kamuzu central hospital, and the university of north carolina at chapel hill.6 of note, kamuzu central hospital is the referral hospital for the northern and central regions of malawi, with a catchment area of approximately 10 million people. it serves as the national cancer center, and the kamuzu central hospital pathology laboratory is the only public pathology lab in malawi. the diagnostic yield and underlying pathology seen in bm biopsies being done in malawi has not been investigated. given this gap, we aimed to assess diagnostic adequacy and characterise the diagnoses from bm samples reviewed at kamuzu central hospital over a 12-year period. given our pathology laboratory capacity and previous observations, we hypothesised that bm sampling in this setting would yield a diagnosis in most (> 50%) cases, and that diagnostic yield would be related to the type and quality of bm sampling. methods ethical considerations our study was approved by the malawian national health sciences research committee and the university of north carolina institutional review board. waiver of consent was approved for this study since it involved a retrospective analysis of de-identified pathology data from the kamuzu central hospital pathology laboratory. study design and setting we conducted a retrospective study of adult (patients ≥ 18 years) bm aspirate and biopsy samples received and interpreted in the pathology laboratory at kamuzu central hospital from 01 june 2012, through 30 june 2024. sample and data collection relevant clinical and pathological data was extracted from the electronic laboratory information system. clinical data in the laboratory information system are transcribed from handwritten requisition forms that are filled out by the submitting provider. these forms include age, sex, hiv status, indication for sampling, provider name, clinic where the biopsy was performed, and a free text space which typically includes presenting symptoms and/or peripheral blood counts. as these were done during routine clinical care, the completeness of the forms was inconsistent. bone marrow core biopsy specimens were fixed in 10% buffered formalin solution, decalcified, and processed with a leica tp1020 automatic benchtop tissue processor (leica biosystems, nussloch, germany). sections (3 µm – 5 µm) of the resulting formalin-fixed paraffin-embedded tissue blocks were stained with haematoxylin and eosin, cover-slipped, and examined, per routine clinical protocol. bone marrow aspirates were smeared onto glass slides and stained with diff-quik before cover-slipping. laboratory personnel conducted routine monitoring and maintenance of all laboratory equipment. the quality of slides and immunohistochemical stains was evaluated by the local pathologists during routine workflow and during weekly teleconference meetings between cancer clinicians and pathologists at kamuzu central hospital and university of north carolina, as we have described previously.7 primary diagnoses were made by local pathologists, and the vast majority of cases were discussed during weekly multidisciplinary telepathology conferences.7,8,9 diagnoses were based on morphologic assessment as well as utilisation of a limited number of immunohistochemical stains (cd45, ck ae1/ae3, mpo, tdt, cd20, cd3, cd138, blc2, and cd30) as described previously, and acid-fast stain for mycobacteria.10,11 no flow cytometry, molecular sequencing, or cytogenetic testing was available. data analysis exported data from the laboratory information system were analysed using r version 4.4.2 (november 2024).12 descriptive statistics were completed to summarise data, including proportions for categorical variables, means and standard deviations for normally distributed data, and medians and interquartile ranges for non-normally distributed data. results we identified 602 adult bm samples that were interpreted at the kamuzu central hospital pathology laboratory from 01 june 2012, through 30 june 2024 (table 1). most specimens were biopsy-only (n = 335, 56%), and most patients were male (n = 345, 57%), with a median age of 42 years (interquartile range 30–54 years). hiv-status was known in 539 cases. of these, 372 (69%) were hiv-uninfected. the most common indications for bm sampling were anaemia (n = 188, 31%), cancer staging (n = 118, 20%), and pancytopaenia (n = 73, 12%). indication was not documented in 72 (12%) cases. table 1: clinical and specimen characteristics associated with bone marrow samples (n = 602) evaluated at kamuzu central hospital pathology laboratory, june 2012 – june 2024. most specimens (n = 512, 85%) were of sufficient quality to receive a discrete diagnosis, and the most common diagnosis was benign or reactive, not otherwise specified (n = 313, 52%) (table 2). of the 181 (30%) malignant cases, the most common diagnoses were acute leukaemia (n = 65, 11%), with 33 (5%) cases of acute lymphoblastic leukaemia and 32 (5%) cases of acute myeloid leukaemia, and lymphoma (n = 45, 7%), most commonly diffuse large b cell lymphoma (n = 16, 3%). when we excluded bm samples obtained for cancer staging (n = 118, 20%), there were 403 (83%) diagnostic samples, 156 of which (39%) were malignant, with 50 (32%) acute leukaemias and 38 (24%) lymphomas. table 2: summary of diagnoses from bone marrow samples (n = 602) evaluated at kamuzu central hospital pathology laboratory, june 2012 – june 2024. based on indication, the highest diagnostic yield was seen in biopsies done for cancer staging (92% yield), and the lowest diagnostic yield was seen in those done for b-symptoms (67% yield) (figure 1). biopsies done for suspected leukaemia (n = 24, 4%) and hepatosplenomegaly (n = 48, 8%) had the highest proportion of malignant diagnoses (63% for suspected leukaemia, and 51% for hepatosplenomegaly), while biopsies done for cancer staging (n = 118, 20%) and lymphadenopathy (n = 43, 7%) groups had the lowest proportion of malignant diagnoses (23% for cancer staging, and 26% for lymphadenopathy). figure 2 illustrates the distribution of malignant, non-malignant, and non-diagnostic cases based on indication for bm sampling. figure 1: proportion of (a) diagnostic and non-diagnostic cases and (b) malignant and non-malignant diagnoses within each indication for bone marrow sampling for samples evaluated at kamuzu central hospital pathology laboratory, june 2012 – june 2024 (n = 602). figure 2: distribution of diagnoses made on bone marrow samples (n = 602) based on indication for sampling, kamuzu central hospital pathology laboratory, june 2012 – june 2024. we stratified the cohort by hiv status (table 2, figure 3, and figure 4). the two most common indications for bm sampling in hiv-positive (n = 167) and hiv-negative (n = 372) patients were anaemia (32% for hiv-positive, and 30% for hiv-negative) and cancer staging (26% for hiv-positive, and 19% for hiv-negative). the third most common indication for hiv-positive patients was lymphadenopathy (13%), and for hiv-negative patients, pancytopaenia (13%). the hiv-negative patients had a higher proportion of malignant diagnoses both when samples obtained for cancer staging were included (37% vs 17%) and excluded (39% vs 20%). of the malignant diagnoses, acute leukaemia was more prominently represented in the hiv-negative group (13% vs 4%). however, when samples obtained for cancer staging were excluded, this difference decreased (32% vs 28%). lymphoma was the most common malignant diagnosis within the hiv-positive group (n = 10, 34%). however, when cancer staging cases were excluded, acute leukaemia (n = 7, 28%) and lymphoma (n = 7, 28%) accounted for the same proportion of cases. figure 3: distribution of indications for bone marrow sampling (n = 602 cases) stratified by hiv infection status for bone marrow samples evaluated at kamuzu central hospital pathology laboratory, june 2012 – june 2024. figure 4: distribution of malignant diagnoses (n = 181 cases) stratified by hiv infection status for bone marrow samples evaluated at kamuzu central hospital pathology laboratory, june 2012–june 2024. bone marrow samples that included both a biopsy and an aspirate (n = 197) had the highest diagnostic yield (91%, p < 0.001) compared to samples that were either aspirate (n = 70, 71%) or biopsy-only (n = 335, 84%) (table 3). for cases with a bm biopsy, the median biopsy length was 1.0 cm (interquartile range 0.5–1.5), and 38% (n = 186 of 491) were ≥ 1.5 cm. when comparing diagnostic and non-diagnostic specimens, there was no difference in median biopsy length (p = 0.50) or number of aspirate slides reviewed (p = 0.44). figure 5 shows images of representative diagnostic and non-diagnostic bm biopsies. figure 5: lowand high-power images of representative (a) non-diagnostic and (b) diagnostic bone marrow samples evaluated at kamuzu central hospital pathology laboratory, between june 2012 and june 2024. the non-diagnostic biopsy shows a short, fragmented bone marrow biopsy with features suggestive of subcortical sampling. the diagnostic biopsy shows a hypercellular, reactive bone marrow. table 3: proportion of diagnostic bone marrow samples (n = 602) depending on specimen type: aspirate only, biopsy-only, or biopsy with aspirate, kamuzu central hospital pathology laboratory, june 2012 – june 2024. discussion bone marrow characterisation is essential for diagnosing, understanding, and treating haematological disorders.13 our objective was to characterise adult bm samples evaluated under routine clinical care at kamuzu central hospital pathology laboratory and to assess their adequacy for diagnostic yield. in our setting, 85.0% (n = 512) of all bm samples received a discrete diagnosis. of these cases, 54% (n = 331) were non-malignant, including most of the samples taken for cancer staging (n = 84 of 118, 77%), while 30% (n = 181) were malignant. the diagnostic yield of bm samples submitted to kamuzu central hospital was excellent compared to similar studies, some conducted in high-resource settings, reporting inadequacy rates of up to 85%.14,15,16,17,18,19,20 this is particularly notable when one considers the inherent resource limitations in malawi, and sparse knowledge of clinical context because of absence of electronic medical records, incomplete requisition forms (12% in our study), and diverse care providers obtaining samples across a large referral area. in addition, the median core biopsy length of bm samples reviewed at kamuzu central hospital was 1.0 cm (interquartile range 0.5 cm – 1.5 cm), which falls shy of the at least 1.5 cm length recommended by the world health organization2,15 and the american society for clinical pathology to ensure reliable interpretation and diagnosis of bm specimens.1,14,19,21 despite these issues, 88.0% (n = 530) of our cases had a known biopsy indication, and, as has been reported previously,22,23 we have a robust multidisciplinary approach to diagnosing patients with suspected haematologic disorders in this setting. both factors likely contribute to our high diagnostic yield. nevertheless, ongoing quality assurance measures are needed to optimise specimen quality, ensure accurate clinical information is provided to the laboratory, and standardise diagnostic examination of reporting.24,25 the most common diagnosis in our study was benign or reactive, not otherwise specified (n = 313, 52%) which accounted for 95% of the non-malignant diagnoses (n = 331). increased diagnostic granularity of these cases is an important area of opportunity for the laboratory. while our current system can largely rule out malignancy, we may not be providing a clear or clinically actionable explanation for patients with non-malignant disorders affecting the bm. proper evaluation requires integrating clinical data with morphologic evaluation of peripheral blood and bm samples, as well as additional ancillary studies. important ancillary testing techniques not available at the kamuzu central hospital pathology laboratory include a larger immunohistochemical staining panel for both infectious and malignant diseases, flow cytometry, and molecular and cytogenetic testing. in addition, reliable clinical testing for nutritional deficiencies, autoimmune disease, and infectious aetiologies are lacking. strengthening this capacity is essential for providing more granular diagnoses, as well as increasing understanding and improving management of benign haematologic disorders, including neglected diseases in this region such as tropical splenomegaly. the most common malignant diagnosis in our study was acute leukaemia (n = 65 of 181, 36%), with an equal number of acute lymphoblastic and acute myeloid leukaemias. this is similar to data from other retrospective studies conducted in india (2017) and pakistan (2016), where leukaemia was diagnosed in at least 21% of bm specimens.24,25 in addition, a variety of other malignant disorders were identified in our study, including non-hodgkin lymphoma, hodgkin lymphoma, chronic myeloid leukaemia, and myelodysplastic syndrome. overall, there is a significant burden of aggressive malignant diseases in our patient population. effective, potentially curative treatments are available for many of these malignancies, as we have shown previously.26,27,28 however, there is an ongoing need to support and expand these treatment modalities, a significant challenge in a resource-limited setting. in addition, more research is needed to further understand local disease pathogenesis, natural history, and optimal management. the most common indication for bm sampling in this study was anaemia (n = 188, 31%), and the majority (n = 331, 55%) of bm samples were interpreted as non-malignant. this aligns with our anecdotal impression that a large proportion of the kamuzu central hospital haematology patients are referred for chronic, unexplained anaemia, with or without splenomegaly, and with no definite features of malignancy on clinical exam. in addition, our findings are comparable to a 6-year retrospective study conducted at the oncology center – mansoura university, egypt, which found that 44.9% (n = 92) of bm samples were interpreted as reactive.9,29 limitations while we were able to determine a diagnostic yield of bm samples reviewed at kamuzu central hospital and to highlight the distribution of malignant and non-malignant diagnoses within these samples, these findings must be interpreted alongside the limitations of this study. diagnostic yield was used as a surrogate for bm sample quality, and only total biopsy length was provided instead of total evaluable biopsy length. moving forward, quality assurance measures can be implemented on the clinical and laboratory sides to maintain optimal sample collection and processing techniques as well as record key variables for ongoing assessment of bm sample adequacy and quality.22,30,31 clinical data provided with bm samples were not only limited, but may also be somewhat unreliable, given the nature of sample submission. lastly, given that this is a hospital-based cohort study, there is inherent selection and referral bias compared to population-based studies. opportunities to more fully characterise disorders requiring bm sampling across the country of malawi need to be explored. conclusion pathologic evaluation of bm samples is essential in the care of patients with both benign and malignant haematologic disorders. we demonstrate a high diagnostic yield of bm biopsies reviewed at kamuzu central hospital, despite significant challenges inherent in our setting, including limited resources and minimal access to clinical data. one of the major strengths of our system is a multidisciplinary approach to diagnosis and patient care. however, identifying ways to increase accessibility to accurate clinical information is needed. in addition, most bm samples in this study were interpreted as benign or reactive, yet we have limited ability to robustly evaluate non-malignant samples. there is an urgent need to develop additional innovative diagnostic resources to better characterise these diseases in malawi. acknowledgements we thank dr george liomba for his invaluable contributions to the launch of the pathology laboratory at kamuzu central hospital in lilongwe, malawi, and his role in patient care. this article is based on research previously presented in abstract form at the 12th annual symposium of global cancer research, held virtually on 26 july 2024. the abstract has since been developed into a full article, which has been expanded and revised for journal publication. this republication is done with permission from the conference organisers. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions edwards kasonkanji, matthew painschab, yuri fedoriw, and amy brownlee conceptualised and designed the study. edwards kasonkanji and amy brownlee prepared the article. tarsizious chikaonda reviewed and analysed the data. yuri fedoriw, amy brownlee, edwards kasonkanji, matthew painschab, luke eastburg, maurice mulenga, and tamiwe tomoka contributed to collecting and interpreting the clinical data as well as diagnosing the bone marrow samples. amy brownlee, yuri fedoriw, and matthew painschab revised the article and supervised the study. all authors contributed to the article, discussed the results, and approved the final version for submission and publication. sources of support this work was supported by grant funding from the national institutes of health (grant numbers: u54 ca254564-01 and d43 ca260641). data availability the data that support the findings of this study are available on request from the corresponding author, amy brownlee the data are not publicly available due to privacy reasons. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. the article does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the authors are responsible for this article’s results, findings, and content. references marinelli lm, fang h, howard mt, et al. bone marrow biopsy operator experience and impact on aspirate, biopsy, and ancillary testing quality. mayo clin proc innov qual outcomes. 2018;2(3):241–247. https://doi.org/10.1016/j.mayocpiqo.2018.06.007 vardiman jw, thiele j, arber da, et al. 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adults in malawi. jco glob oncol. 2022;8:e2100388. https://doi.org/10.1200/go.21.00388 painschab ms, westmoreland kd, kasonkanji e, et al. prospective study of burkitt lymphoma treatment in adolescents and adults in malawi. blood adv. 2019;3(4):612–620. https://doi.org/10.1182/bloodadvances.2018029199 stanley cc, westmoreland kd, heimlich bj, et al. outcomes for paediatric burkitt lymphoma treated with anthracycline-based therapy in malawi. br j haematol. 2016;173(5):705–712. https://doi.org/10.1111/bjh.13986 hasserjian rp. reactive versus neoplastic bone marrow: problems and pitfalls. arch pathol lab med. 2008;132(4):587–594. https://doi.org/10.5858/2008-132-587-rvnbmp sever c, abbott cl, de baca me, et al. bone marrow synoptic reporting for hematologic neoplasms: guideline from the college of american pathologists pathology and laboratory quality center. arch pathol lab med. 2016;140(9):932–949. https://doi.org/10.5858/arpa.2015-0450-sa lee sh, erber wn, porwit a, tomonaga m, peterson lc. icsh guidelines for the standardization of bone marrow specimens and reports. int j lab hematol. 2008;30(5):349–364. https://doi.org/10.1111/j.1751-553x.2008.01100.x abstract introduction methods results discussion acknowledgements references about the author(s) jasmine ramiah department of haematology, national health laboratory services, inkosi albert luthuli central hospital, durban, south africa department of haematology, school of laboratory medicine, university of kwazulu-natal, durban, south africa dashini pillay department of haematology, national health laboratory services, inkosi albert luthuli central hospital, durban, south africa department of haematology, school of laboratory medicine, university of kwazulu-natal, durban, south africa nadine rapiti department of haematology, national health laboratory services, inkosi albert luthuli central hospital, durban, south africa department of haematology, school of laboratory medicine, university of kwazulu-natal, durban, south africa citation ramiah j, pillay d, rapiti n. performance of the automated sysmex xn-3000 analyser for detecting white blood cell abnormalities in south africa. afr j lab med. 2023;12(1), a2140. https://doi.org/10.4102/ajlm.v12i1.2140 original research performance of the automated sysmex xn-3000 analyser for detecting white blood cell abnormalities in south africa jasmine ramiah, dashini pillay, nadine rapiti received: 07 dec. 2022; accepted: 11 may 2023; published: 19 july 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: automated haematology analysers such as the sysmex xn-3000 (sysmex corporation, kobe, japan) utilise white blood cell (wbc) flags to identify quantitative and qualitative abnormalities. owing to clinical and biological factors, the sensitivity and specificity of the flags vary when compared to microscopy, the gold-standard method for assessing peripheral blood smear (pbs) morphology. objective: this study assessed the performance of the sysmex xn-3000 haematology analyser in comparison to pbs microscopy for the detection of wbc abnormalities. methods: we collected 250 random full blood count samples from the haematology laboratory at inkosi albert luthuli central hospital, durban, kwazulu-natal, south africa, from march 2022 to april 2022. the performance of the automated wbc flags of the sysmex xn-3000 was assessed in comparison to pbs microscopy, and the impact of established clinical variables on the performance of the flags was determined. results: the sensitivity of the ‘blast’ flag was 96.3%, and the specificity was 84.9%. the efficiency of the flag was adversely impacted by low white cell counts (< 1.5 × 109/l; p < 0.001), chemotherapy (p = 0.002), malignancy (p = 0.02), and infection (p = 0.02). the ‘abnormal lymphocyte’ flag demonstrated a sensitivity of 90% and a specificity of 96.2%, and its performance was adversely impacted by chemotherapy exposure (p = 0.03). three cases (1.2%) erroneously flagged as ‘monocytosis’ demonstrated blasts on microscopy. conclusion: in our setting, pbs microscopy remains necessary to confirm blasts, abnormal lymphocytes, and monocytosis in patients with malignancy, current chemotherapy exposure, low white cell counts, and infection. what this study adds: this study adds evidence that pbs morphology remains the gold standard for confirming wbc abnormalities in patients with a history of malignancy, chemotherapy, and leucopenia. keywords: sysmex analyser; xn-series; automated analysers; fbc; flagging; white blood cell flags; clinical and laboratory standards institute guidelines. introduction the microscopic assessment of a well-prepared and well-stained peripheral blood smear (pbs) is the gold standard for identifying white blood cell (wbc) abnormalities.1 this manual pbs assessment requires skilled and trained morphologists.2 automated full blood count (fbc) haematology analysers can flag suspected quantitative and qualitative abnormalities,1 and the wbc flags can potentially alert the laboratory technical staff to abnormal blood samples prior to the review of the pbs, thus improving workflow, particularly in resource-constrained settings. globally, restricted laboratory budgets result in a reduction in the number of experienced morphologists.3 a laboratory system with automated technology that can prioritise a blood sample with abnormal wbc features is thus advantageous. however, there are limitations in the specificity and sensitivity of these flags.4 the sysmex xn-3000 automated fbc analyser (sysmex corporation, kobe, japan) has white cell differential, white cell nucleated, and white cell precursor (wpc) channels, with the wpc channel demonstrated to possess an improved sensitivity for detecting blasts.5 the national health laboratory services haematology laboratory at inkosi albert luthuli central hospital, located in durban, kwazulu-natal, south africa, processes approximately 9000 fbc samples and 300–600 pbs samples monthly. the inkosi albert luthuli central hospital is a referral hospital that provides medical care to patients with benign and malignant haematological conditions.6 the purpose of this study was to assess the performance of the wbc flags of the sysmex xn-3000 analyser (sysmex corporation, kobe, japan) for the detection of wbc abnormalities in comparison to pbs microscopy, which is used as the gold-standard method in our setting. we also aimed to identify the clinical variables in our hospital setting that may be associated with false-positive (fp) and false-negative (fn) wbc flags.2 methods ethical considerations the study was approved by the university of kwazulu-natal biomedical research ethics committee (ethical clearance number: brec/00003769/2022). informed consent was not required by the ethics approval committee as these samples were routinely performed by the clinician. the clinician had obtained consent for all laboratory procedures prior to admission of the patients and the laboratory had waived need for consent as no additional tests were performed. full brec ethics approval was obtained for this study. informed consent was not obtained as these samples were submitted to the laboratory for routine fbc analysis and pbs microscopy by the treating clinician. patient confidentiality was maintained during the study by the allocation of unique and anonymised study codes. the research data were stored electronically on password-protected devices accessible only by the researchers. study design this was a laboratory-based prospective study conducted in the national health laboratory services, durban, kwazulu-natal, south africa. the study comprised 250 blood samples, which were representative of the normal and abnormal adult patient population (> 12 years of age) sent to the laboratory for routine fbc analysis in dipotassium ethylene diamine tetra-acetate tubes (becton dickinson, berlin, germany). the sample size was calculated using stata® v15.1 statistical software (statacorp llc, college station, texas, united states) in consultation with a statistician who audited the number of monthly fbc requests. the samples were collected from march 2022 to april 2022. the results were quality assured by assessing the instrument’s daily internal quality controls and external quality assurance, according to the department’s standard operating procedure.6 automated white blood cell analysis the anticoagulated samples were drawn and analysed within 24 h of sampling on the sysmex xn-3000 analyser (sysmex corporation, kobe, japan) for fbc and wbc differential count analysis. if the sample demonstrated ‘blast’, ‘atypical lymphocytes’, or ‘abnormal lymphocytes’, the wpc channel was reflexed and run according to the routine procedure.5 samples that were clotted or haemolysed, had insufficient volume, were older than 24 h, or were collected in an incorrect tube or erroneously labelled were excluded from the study. the sysmex xn-3000 analyser uses the principles of fluorescent flow cytometry to identify wbc sub-populations based on the intensity of the side-fluorescent light and side-scattered light.5,7 the initial reflex or warning is identified by the white cell differential, which generates an internal protocol message or flag. this flag is further confirmed or removed by the more sensitive wpc channel.5,8 peripheral blood smear analysis a pbs was prepared from each blood sample and stained according to the may-grunwald giemsa technique using the automated slide stainer (sysmex p10 slide maker and stainer, sysmex corporation, kobe, japan) according to the clinical and laboratory standards institute recommendations.9 each pbs was microscopically assessed (olympus cx23, evident corporation, tokyo, japan) at 10× and 50× magnification with oil by the principal investigator and a trained haematology laboratory technologist. a 100 wbc differential count was performed for each sample. the machine differential and manual differential counts were entered into microsoft® excel 2016 spreadsheets (microsoft®, redmond, washington, united states). clinical information we collected the following clinical information from the hospital and laboratory information systems: age, sex, white cell count (wcc), presence of an infection, coronavirus disease 2019 active infection or previous coronavirus disease 2019 infection, clinical diagnosis, transplant status, current chemotherapy exposure, and hiv status. infection, as recorded in the clinical information, was determined by a temperature of ≥ 38 °c or culture of an organism (bacterial, viral, etc.) from any clinical site, or a clinical site of infection.10 the reference range for wcc in our setting ranges from 3.92 to 10.40 × 109/l for male patients and from 3.90 to 12.60 × 109/l for female patients. in our study, a low wcc was defined as a count less than 1.5 × 109/l.6 data analysis statistical data were captured using microsoft® excel 2016 (microsoft®, redmond, washington, united states) and analysed using r statistical computing software of the r core team, 2020, version 3.63 (microsoft, windows [32/64 bit], redmond, washington, united states). all the numeric variables were skewed, and the descriptive statistics were consequently presented as medians with lower and upper quartiles. the categorical variables were presented as counts and percentage frequencies. the association between the numeric variables was determined using pearson’s correlation test. all the statistical tests were conducted at a 5% level of significance. a p-value of < 0.05 was considered significant for this study. sensitivity and specificity analyses were calculated using the ‘epi.tests’ r function from the epir package (https://cran.r-project.org/package=epir) while the mcnemar test was performed using the epi.kappa function also from the epir package. a ‘true-positive’ (tp) was defined as a wbc abnormality detected by the analyser and confirmed by pbs microscopy. a ‘false positive’ (fp) was defined as a wbc abnormality detected by the analyser but not observed on pbs microscopy. a ‘true-negative’ (tn) was defined as a wbc abnormality not detected by the analyser and not observed on pbs microscopy. a ‘false negative’ (fn) was defined as a wbc abnormality not detected by the analyser but observed on pbs microscopy. the equations used for the sensitivity, specificity, positive predictive value (ppv), and negative predictive value (npv) calculations using the epir package are shown below: the impact of the clinical variables (presence of infection, malignancy, current chemotherapy usage and low wcc [< 1.5 × 109/l]) on the wbc flags was assessed using the mcnemar test when the wbc flag did not correlate (fp or fn) with the microscopy results. results study population this study included 250 adult patient samples (table 1). one hundred and thirteen (45.2%) of the patients were men and 137 (54.8%) were women. one hundred and forty-five samples (56%) were from the haematology or oncology clinic or ward. of the total 250 study samples, there were 125 normal (no quantitative or qualitative wbc abnormalities present) and 125 abnormal (flagged by the analyser as having wbc abnormalities) samples. table 1: demographic characteristics and clinical information of the study population, inkosi albert luthuli central hospital, durban, kwazulu-natal, south africa, march 2022 – april 2022. clinical characteristics of the 112 (44.8%) patients with a haematological malignancy, 51 (20.4%) had acute myeloid leukaemia, 36 (14.4%) had acute lymphoblastic leukaemia or lymphoma, 10 (4%) had a lymphoproliferative disorder, and 8 (3.2%) had a plasma cell dyscrasia. one hundred and three (41.2%) patients were on chemotherapy, 74 (29.6%) had an infection, and 8 (3.2%) patients had received a haematopoietic stem cell transplant. twenty-two (8.8%) samples were from patients with benign haematological disorders such as thalassaemia major, aplastic anaemia and sickle cell disease. among the 28 (11.2%) hiv-positive patients, the cd4 count ranged from 35 × 109/l to 1233 × 109/l, and viral load ranged from 0 copies/ml to 656 000 copies/ml. white cell count the wcc among the study subjects ranged from 0.1 × 109/l to 145.5 × 109/l (median: 5.24 × 109/l; interquartile range: 2.43–8.67 × 109/l). thirty-seven samples (14.8%) demonstrated a wcc ≤ 1.5 × 109/l, 169 (67.6%) demonstrated a wcc between 1.6–11.9 × 109/l, and 44 samples (17.6%) demonstrated a wcc of ≥ 12 × 109/l. the blast percentage ranged from 0% to 78%. there were agreements (p < 0.001) between the pbs microscopy and the numerical wbc values noted on the sysmex xn-3000 analyser for the median differential counts of neutrophils (54 × 109/l vs 55.4 × 109/l), lymphocytes (26 × 109/l vs 29.1 × 109/l), immature granulocytes (0 × 109/l vs 0.50 × 109/l), and monocytes (7 × 109/l vs 7.55 × 109/l) (table 2). the basophils, however, did not demonstrate an agreement as the median count was 0 × 109/l by pbs and 0.40 × 109/l by the automated analyser (p = 0.494). table 2: correlation between white blood cell differential count by the sysmex xn-3000 analyser and white blood cell differential count by microscopy, inkosi albert luthuli central hospital, durban, kwazulu-natal, south africa, march 2022 – april 2022. sixty-two (24.8%) samples were flagged for ‘blast’ by the analyser, 47 (18.8%) were flagged for ‘immature granulocytes’, and 43 (17.2%) were flagged for ‘abnormal lymphocytes’. the efficiencies of the ‘conspicuous eosinophil’, ‘lymphocytosis’, ‘atypical lymphocyte’, and ‘immature granulocyte’ flags were not impacted by the clinical parameters (p > 0.05) (table 3). table 3: performance of the sysmex xn 3000 analyser for the detection of white blood cell abnormalities and the impact of clinical variables on the performance of wbc flags, inkosi albert luthuli central hospital, durban, kwazulu-natal, south africa, march 2022 – april 2022. the ‘abnormal lymphocyte’ wbc flag generated by the instrument demonstrated a sensitivity of 90% (95% confidence interval [ci]: 76.3% – 97.2%), a specificity of 96.2% (95% ci: 92.6% – 98.3%), a ppv of 81.8%, and an npv of 98.1%. on comparing the analyser results to microscopy, four samples (1.6%) that were not flagged for ‘abnormal lymphocytes’ by the analyser were determined to be fn, and eight (3.2%) samples were fp for ‘abnormal lymphocytes’. the ‘blast’ flag generated by the instrument demonstrated a sensitivity of 96.3% (95% ci: 81% – 99.9%), a specificity of 84.9% (95% ci: 79.5% – 89.4%), a ppv of 44.1%, and an npv of 99.5%. one (0.4%) sample was fn for ‘blast’, and 33 (13.2%) were fp. the blast count ranged from 0% to 78%. the accuracy of the ‘blast’ flag was influenced by a low wcc (p < 0.001), the presence of malignancy (p = 0.002), current chemotherapy exposure (p = 0.02), and infection (p = 0.02). the monocytosis ‘flag’ generated by the instrument demonstrated a sensitivity of 84.3% (95% ci: 60.4% – 96.6%), a specificity of 97.0% (95% ci: 93.9% – 98.8%), a ppv of 69.6%, and an npv of 98.7%. seven (2.8%) samples flagged for monocytosis by the automated analyser were determined to be fp for monocytosis and three (1.2%) samples not flagged by the analyser were determined to be fn. these three (1.2%) samples had > 20% blasts on manual microscopic assessment of the pbs. the ‘left shift’ flag generated by the instrument demonstrated a sensitivity of 59.7% (95% ci: 47.5% – 71.1%), specificity of 95.1% (95% ci: 96% – 99.9%), a ppv of 95.6%, and an npv of 85.9%. twenty-nine (11.6%) samples were determined to be fn and two (0.8%) samples were fp. the accuracy of this flag was influenced by current chemotherapy exposure (p = 0.005). discussion we evaluated the performance of the sysmex xn-3000 automated analyser for the detection of wbc abnormalities in 250 routine adult blood samples at the national health laboratory services laboratory at inkosi albert luthuli central hospital, durban, kwazulu-natal, south africa. the majority of the samples were from patients diagnosed with a malignancy and patients receiving chemotherapy, which was expected as this is a tertiary referral centre for haematological and oncological disorders. congruent with reports in the literature, the accuracy of the ‘blast’, ‘abnormal lymphocytes’, ‘left shift’ and ‘monocytosis’ automated wbc flags were impacted by clinical parameters such as current infection, low wcc (≤ 1.5 × 109/l), malignancy, and current treatment with chemotherapy.11 failure to identify malignant conditions portends significant clinical implications and is one of the greatest concerns for laboratories. therefore, in these instances, laboratories should rely on the confirmatory result of a detailed microscopic assessment, which is the gold standard.12 there was an agreement between the sysmex xn-3000 analyser and pbs microscopy for the differential counts of neutrophils, lymphocytes, immature granulocytes, and monocytes (p < 0.001). however, there was no agreement between the sysmex xn-3000 analyser and pbs microscopy for the differential count of basophils. the automated analyser does not quantify the presence of blasts and abnormal or atypical lymphocytes; it only provides a qualitative assessment through the presence of flags. therefore, a quantitative agreement between microscopy and the analyser could not be determined. the ‘blast’ flag indicates the suspected presence of malignancy (myeloid or lymphoid).13 although the wpc, employed by the xn-series, has improved the detection of blasts, the literature demonstrates a low specificity and variable sensitivity (60% – 100%).4 a study conducted in denmark in 2020 revealed that the wpc produced a high number of fp (14.5%) and fn (12.8%) results when detecting blasts, possibly due to leucopenia and a low number of circulating blast cells.4 our study demonstrated a sensitivity of 96.3% and a specificity of 84.9% for the blast flag, and the blast count ranged from 0% to 78%. the sensitivity and specificity of the blast wbc flag were determined to be influenced by leucopenia (wcc of ≤ 1.5 × 109/l). the lower the wcc, the higher the likelihood of erroneous blast detection by the analyser. this is similar to a previous report in korea in 2021 where the sensitivity increased as the wbc rose to > 6 × 109/l.2 although in our study, the automated analyser did not detect the presence of blasts in one sample (0.4%) from a patient with leucopenia (wcc: < 1.5 × 109/l), malignancy and current chemotherapy exposure, 21% blasts were detected by microscopy in that sample. in the korean study, the sensitivity of the wpc in detecting blasts in leucopenic samples (< 1.5 × 109/l) was 56%.2 this may be explained by patients receiving chemotherapy or transplantation, which may alter the side-fluorescent light.2 in the case of leucopenia, blasts may be further missed due to infrequency.4 our study demonstrated that microscopy remains the gold standard in the detection of blasts in patients with a low wcc, patients with a malignancy, and patients receiving chemotherapy. the ‘atypical lymphocyte’ flag indicates reactive disorders (infections or inflammatory states).14 the morphology may be heterogenous, including larger size, round nucleus, larger nucleolus, abundant deep basophilic cytoplasm with tenting, or the presence of intracytoplasmic granules.15 according to a local study conducted at charlotte maxeke johannesburg academic hospital, south africa, in 2018, the ‘atypical lymphocyte’ flag was associated with a high fp rate of 41.74%.7 inflammatory states such as hiv may affect the sensitivity of the atypical lymphocyte and abnormal lymphocyte channel of the automated analyser.7 this, however, was not proven in our study possibly due to the low number of hiv-positive patients in the study (11.2%), which is lower than the 27% prevalence of hiv in kwazulu-natal.16 schapkaitz and khoza previously demonstrated the sensitivity and specificity of the ‘atypical lymphocyte’ flag in patients with hiv in the south african setting to be 100% and 74.67%.7 our study revealed the reverse: a sensitivity of 26.1% and a specificity of 99.5%. our study also demonstrated that the accuracy of the atypical lymphocyte flag was not significantly influenced by the clinical parameters. ‘abnormal lymphocytes’ indicates a malignant lymphoid process which could be acute, chronic, indolent, or high grade.17 distinguishing reactive lymphocytes secondary to a viral aetiology from those arising from a lymphoproliferative disorder is challenging due to morphological heterogeneity.7 according to a local study conducted in charlotte maxeke johannesburg academic hospital, south africa, in 2018, the fp rate for abnormal lymphocyte flags using the sysmex xn-9000 was 55.56% due to the presence of hiv, opportunistic infections, and lymphopenia.7 the study also demonstrated a sensitivity of 88.46% and a specificity of 74.6% for the ‘abnormal lymphocyte’ flag. in our study, the fp rate was 3.2%, and we observed a higher sensitivity (90%) and specificity (96.2%). in our study, four (1.6%) samples confirmed to be positive for abnormal lymphocytes using pbs microscopy were fn for ‘abnormal lymphocytes’ on the analyser. the clinical parameters in those samples included recent chemotherapy exposure, malignancy, and infection; two samples were unexplained. our study also demonstrated reduced reliability of the ‘abnormal lymphocyte’ flag in the presence of chemotherapy. in centres treating and monitoring haematological disorders, a high sensitivity for ‘abnormal lymphocytes’ is required. thus, in these instances, laboratories should rely on the microscopic review as the gold standard. the common reactive causes of monocytosis are chronic infections such as tuberculosis, inflammatory states, autoimmune disorders, and regenerating bone marrow post-insult.18 south africa has the highest incidence of tuberculosis, with a prevalence of 852 cases (95% ci: 679–1026) per 100 000 population.19 three (1.2%) samples flagged for ‘monocytosis’ by the analyser were determined to be fn. a microscopic examination of the pbs in these samples, however, revealed > 20% blasts in these samples. this is clinically relevant as these samples were from patients with malignancies, low wccs, and recent chemotherapy exposure. adding the ‘monocyte workflow optimisation’ rule set in the extended information processing unit of the analyser adds the function to distinguish reactive conditions from malignant monocytic conditions based on the presence of dysplasia and persistent (> 30 days) monocytosis (> 1.5 × 109/l), and to recommend a blood smear examination in only malignant monocytic conditions.18 furthermore, the combination of a monocyte count of > 1.5 × 109/l with low haemoglobin (< 10 g/dl) or an abnormal or blast flag may warrant urgent pbs review to exclude a malignant aetiology. a microscopic review may be necessary in the clinical context in monocytosis cases with a low wcc, malignancy, and chemotherapy usage to exclude the presence of relapsed or residual disease.18 overall, this study did not show a significant influence of the presence of hiv, previous or current coronavirus disease 2019 infection, or transplantation (solid organ or haematopoietic stem cell) on the sensitivity or specificity of the analyser, probably due to the low study numbers. limitations the study may be limited by the subjectivity of the primary investigator. this limitation was improved by adopting the assessment of a second observer. to the best of our knowledge, this is the first prospective study of this nature in the local population. conclusion the sysmex xn-3000 automated fbc analyser generates valuable numerical (through the white cell differential and wpc channels) and qualitative (through internal protocol messages or flags) information that may assist laboratory staff in accurately assessing and interpreting pbs results and help pathologists and clinicians reach timeous and correct diagnoses.15 our study confirmed that in laboratories focused on patients with haematological conditions, low wcc (< 1.5 × 109/l) and undergoing chemotherapy or haematopoietic stem cell transplants, microscopy review remains the gold standard for the detection of blasts, abnormal lymphocytes, and monocytosis. acknowledgements the authors thank the management and technologists of the national health laboratory services kwazulu-natal as well as the management of inkosi albert luthuli central hospital and catherine connolly and partson tinarwo, at the university of kwazulu-natal biostatistics department, for their assistance with the study. competing interests the authors wish to declare that they have no personal or financial interests that may have influenced the writing of this article. authors’ contributions j.r. and d.p. conceived the research concepts and developed the theory, methodology, and protocol for this study. j.r. collected and analysed the data. n.r. reviewed the work and advised on the study design and implementation of the research. d.p. supervised and n.r. co-supervised the project. all authors contributed to the analysis of the results and the writing of the final manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability data supporting this study is available from the corresponding author, j.r., on request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references joubert j, weyers r, raubenheimer je. reducing unnecessary blood smear examinations: can sysmex blood cell analysers help? med technol sa [serial online]. 2014;28(1):6–12. available from: https://hdl.handle.net/10520/ejc157717 lee n, jun j hyun, lee ds. decreased sensitivity of the automatic white precursor cell channel (wpc) for blast flagging in patients with leukopenia. clin biochem. 2016;49(9):675–681. https://doi.org/10.1016/j.clinbiochem.2016.01.016 briggs cj, linssen j, longair i, machin sj. improved flagging rates on the sysmex xe-5000 compared with the xe-2100 reduce the number of manual film reviews and increase laboratory productivity. am j clin pathol. 2011;136(2):309–316. https://doi.org/10.1309/ajcpdlr4kgkafw4w sejrup j, pedersen dm, phillipsen jp, et al. performance of the sysmex white precursor channel to discover circulating leukemic blast cells. int j lab hematol. 2020;2020(1):1–10. https://doi.org/10.1111/ijlh.13274 blomme s, boeckx n, brusselmans c, claerhout h, van laer c. the integration of sysmex xn-9100’ wpc channel reflex testing in the detection of reactive versus malignant blood samples. int j lab hematol 2020;43(2):191–198. https://doi.org/10.1111/ijlh.13352 mbongwa, nompumelelo dys. haematology sysmex xn3000 analyzer and workflow (hae1798). durban: nompumelelo, mbongwa; 2019. schapkaitz e, khoza p. performance evaluation of the white cell precursor channel on the sysmex xn hematology analyzer in hiv specimens. j clin lab anal. 2018;32(4):1–5. https://doi.org/10.1002/jcla.22335 kim h, hur m, choi sg, oh km, moon hw, yun ym. comparison of white blood cell counts by wnr, wdf, and wpc channels in sysmex xn hematology analyzer. int j lab hematol. 2015;37(6):869–875. https://doi.org/10.1111/ijlh.12421 clinical and laboratory standards institute (clsi). reference leukocyte (wbc) differential count (proportional) and evaluation of instrmental methods; approved standard-second edition [homepage on the internet]. clsi document h20-a2. 2007. available from: https://clsi.org/media/1400/h20a2_sample.pdf bennett je, dolin r, blaser mj. infectious disease essentials. amsterdam: elsevier; 2015:138–149. petrone j, frater j. blast flagging of the sysmex xn-10 hematology analyzer with supervised cell image analysis: impact on quantity parameters. int j lab hematol. 2019;41(5):601–606. https://doi.org/10.1111/ijlh.13069 white blood cell functionality [homepage on the internet]. whitepaper. 2017. pp. 1–7. available from: www.sysmex-europe.com/whitepapers sukhacheva e. comments re article on comparison of performance and abnormal cell flagging of two automated hematology analyzers: sysmex xn 3000 and beckman coulter dxh 800. int j lab hematol. 2020;42(3):e96–e97. https://doi.org/10.1111/ijlh.13132 weinberg se, behdad a, ji p. atypical lymphocytes in peripheral blood of patients with covid-19. br j haematol. 2020;190(1):36–39. https://doi.org/10.1111/bjh.16848 enhancement se. seed haematology. syxmex. 2012;(march):3–6. vandormael a, cuadros d, kim hy, bärnighausen t, tanser f. the state of the hiv epidemic in rural kwazulu-natal, south africa: a novel application of disease metrics to assess trajectories and highlight areas for intervention. int j epidemiol. 2021;49(2):666–675. https://doi.org/10.1093/ije/dyz269 refaat l, abdellateif ms, bayoumi a, khafagy m, kandeel ez, nooh ha. detection of abnormal lymphocytes in the peripheral blood of covid-19 cancer patients: diagnostic and prognostic possibility. hematol (united kingdom). 2022;27(1):745–756. https://doi.org/10.1080/16078454.2022.2089830 kocaturk e, kusku kiraz z, uskudar teke h, demir ss, alatas io. a new approach for diagnosing hematological malignancies using monocytosis workflow optimization and abnormal lymphocyte/blast flag of sysmex xn series of blood count analyzers. int j lab hematol. 2020;(june):1–6. https://doi.org/10.1111/ijlh.13281 ayles h, mureithi l, simwinga m. the state of tuberculosis in south africa: what does the first national tuberculosis prevalence survey teach us? lancet infect dis. 2022;22(8):1094–1096. https://doi.org/10.1016/s1473-3099(22)00286-9 abstract introduction ethical considerations case presentation management and outcomes discussion acknowledgements references about the author(s) nicolene steyn department of chemical pathology, faculty of health sciences, university of pretoria, pretoria, south africa national health laboratory service, tshwane academic division, pretoria, south africa bettina chale-matsau department of chemical pathology, faculty of health sciences, university of pretoria, pretoria, south africa national health laboratory service, tshwane academic division, pretoria, south africa aron b. abera inqaba biotechnical industries (pty) ltd, pretoria, south africa gertruida van biljon national health laboratory service, tshwane academic division, pretoria, south africa division of paediatric nephrology, department of paediatrics, faculty of health sciences, university of pretoria, pretoria, south africa tahir s. pillay department of chemical pathology, faculty of health sciences, university of pretoria, pretoria, south africa national health laboratory service, tshwane academic division, pretoria, south africa citation steyn n, chale-matsau b, abera ab, van biljon g, pillay ts. neonatal presentation of a patient with liddle syndrome, south africa. afr j lab med. 2023;12(1), a1998. https://doi.org/10.4102/ajlm.v12i1.1998 case study neonatal presentation of a patient with liddle syndrome, south africa nicolene steyn, bettina chale-matsau, aron b. abera, gertruida van biljon, tahir s. pillay received: 29 june 2022; accepted: 03 feb. 2023; published: 14 apr. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract introduction: liddle syndrome is an autosomal dominantly inherited disorder usually arising from single mutations of the genes that encode for the alpha, beta and gamma epithelial sodium channel (enac) subunits. this leads to refractory hypertension, hypokalaemia, metabolic alkalosis, hyporeninaemia and hypoaldosteronism, through over-activation of the enac. case presentation: we describe a 5-day old neonate who presented with severe hypernatraemic dehydration requiring admission to steve biko academic hospital in south africa in 2012. further evaluation revealed features in keeping with liddle syndrome. two compound heterozygous mutations located at different subunits encoding the enac were detected following genetic sequencing done in 2020. the severe clinical phenotype observed here could be attributed to the synergistic effect of these known pathological mutations, but may also indicate that one of the other variants detected has hitherto undocumented pathological effects. management and outcome: this child’s treatment course was complicated by poor adherence to therapy, requiring numerous admissions over the years. adequate blood pressure control was achieved only after the addition of amiloride at the end of 2018, which raised the suspicion of an enac abnormality. conclusion: to our knowledge, this is the first liddle syndrome case where a combined effect from mutations resulted in severe disease. this highlights the importance of early recognition and management of this highly treatable genetic disease to prevent the grave sequelae associated with long-standing hypertension. whole exome sequencing may assist in the detection of known mutations, but may also unveil new potentially pathological variants. what this study adds: this study highlights the importance of developing a high index of suspicion of tubulopathy such as liddle syndrome for any child presenting with persistent hypertension associated with hypokalaemic metabolic alkalosis. keywords: liddle syndrome; epithelial sodium channels; genetic sequencing; hypertension; hyporeninaemia; hypoaldosteronism. introduction liddle syndrome, the most common monogenic cause of hypertension,1 is an autosomal dominantly inherited disorder typified by salt-sensitive hypertension, hyporeninaemia, hypoaldosteronism, metabolic alkalosis and variable hypokalaemia.2 even though symptoms and signs may present in infancy, the diagnosis is often significantly delayed.3 sodium reabsorption in the epithelial cells of the distal renal tubule is regulated by the epithelial sodium channel (enac).4 liddle syndrome arises from activating mutations of the scnn1a, scnn1b and scnn1g genes,5 which encode for the intracellular carboxy-terminal domains of the alpha, beta and gamma enac subunits. this results in an elevated number of channels and markedly increased independent activity with consequent sodium and water retention, hypertension and negative feedback suppression of renin and aldosterone secretion.2,3 ethical considerations written consent was obtained from the child’s parents and ethics approval obtained from the research and ethics committee at the university of pretoria (no. 536/2020). confidentiality was ensured in the preparation of this case study. case presentation a male patient of ethiopian descent, born in november 2012, presented on day five of life with severe hypernatraemic dehydration and acute renal failure requiring admission. on examination, he appeared severely wasted and dehydrated with absent femoral pulses. abdominal ultrasound revealed a thrombus in the aorta, attributed to the hyperviscosity associated with the severe dehydration. the thrombus resolved following heparin therapy and the child was discharged upon resolution of his renal failure with rehydration in december 2012. he was reviewed a week after discharge and found to have hypertension. during subsequent admissions over the years of follow-up, no clotting abnormalities were found and renal ultrasonography revealed no new thrombus nor renal abnormalities. no cardiac abnormalities were detected on sonography. there was no history of consanguinity. laboratory results (analysed on an abbott architect ci8200 (abbott laboratories, chicago, illinois, united states) up to 8 years of age revealed potassium values ranging from 2.0 mmol/l to 3.0 mmol/l (reference interval [ri]: 3.7 mmol/l – 5.9 mmol/l) and sodium levels 159 mmol/l – 171 mmol/l (ri: 136 mmol/l – 145 mmol/l) (table 1). metabolic alkalosis was also present (hco3− = 29 mmol/l to 34 mmol/l [ri: 23 mmol/l – 29 mmol/l]). random urine potassium was elevated (12.0 mmol/l – 22.0 mmol/l [ri: < 10 mmol/l]) despite the low serum potassium on presentation. table 1: laboratory results trends during follow-up at steve biko academic hospital in south africa, 2020. initial investigations in 2019, for the specific r563q mutation and mutations on exon 13 of the beta subunit of the enac (most common causes of liddle syndrome in south africa), were negative. further investigation was undertaken, with the aid of external funding, of all exons and exon-intron boundaries of the alpha (scnn1a [genbank nm_001038.5]), beta (scnn1b [genbank nm_000336.2]) and gamma (scnn1g [genbank nm_001039.3]) subunits encoding for the enac; these were amplified by polymerase chain reaction and sequenced using the nimagen, brilliantdye™ terminator cycle sequencing kit v3.1, brd3-100/1000 (nimagen, nijmegen, the netherlands), according to manufacturer’s instructions, in 2020. two sets of compound heterozygous transition mutations were found in the coding regions of the scnn1a and scnn1b genes (figure 1). in scnn1a, c.1000g>a in exon 5 resulted in ala334thr substitution and c.1987a>g in exon 13 led to thr663ala amino acid change. in the scnn1b gene, there was a c.7g>a mutation in exon 2 leading to a val3met substitution, and a c.1325g>t mutation in exon 9 leading to a gly442val substitution. no mutations were detected in the coding region of the scnn1g gene. figure 1: sanger sequencing electropherogram results of subunits scnn1a and scnn1b of the enac indicating mutations (arrow). (a) sequence electropherogram showing a heterozygous c.1000g>a mutation (chr12:6355415 [grch38.p14]) in exon 5 of scnn1a. (b) sequence electropherogram showing a heterozygous c.1987a>g mutation (chr12:6347896 [grch38.p14]) in exon 13 of scnn1a. (c) sequence electropherogram showing a heterozygous of scnn1a c.7g>a (chr16:23348606 [grch38.p14] in exon 2 of scnn1b. (d) sequence electropherogram showing a heterozygous c1325g>t mutation (chr16:23377219 [grch38.p14]) in exon 9 of scnn1b. management and outcomes the patient was initially managed on multiple antihypertensive drugs and potassium supplementation, but the treatment course was complicated by poor adherence to therapy and follow-up, resulting in numerous re-admissions over the years to achieve blood pressure control. effective blood pressure control was only achieved on commencement of amiloride in 2018, years after the initial presentation. this prompted the investigations for an enac abnormality. discussion in a neonate presenting with hypernatraemia and hypokalaemic metabolic alkalosis, the use of diuretics, persistent vomiting, nasogastric free drainage losses or a tubular disorder such as gitelman or bartter syndrome should be considered. if hypertension is also present, secondary causes such as congenital adrenal hyperplasia, primary hyperaldosteronism, syndrome of apparent mineralocorticoid excess, glucocorticoid-remediable aldosteronism, renal artery stenosis and a deoxycorticosterone-producing tumour should be included in the differential diagnosis.6 the presence of persistent hypertension with hypokalaemic metabolic alkalosis should raise the suspicion of unregulated enac activation.4 sodium reabsorption in the epithelial cells of the distal renal tubule is regulated by the enac, which is activated through the renin-angiotensin-aldosterone system6 (figure 2). it is important to determine if the hypertension is associated with low renin levels. in this instance, both serum aldosterone (< 27.0 pmol/l [ri: 49–643 pmol/l, supine], measured by diagnostic products corporation aldosterone coat-a-count kit, diagnostic products corporation, los angeles, california, united states) and plasma renin concentration (0.5 miu/l [ri: 6.5–36.2 miu/l, supine], measured by cis bio active renin assay, cisbio bioassays, codolet, france) levels were suppressed. figure 2: renin-angiotensin aldosterone system physiology. when the enac is activated independently of aldosterone stimulation, treatment with aldosterone antagonists has no effect. however, the use of amiloride or triamterene can lead to complete resolution of symptoms, as they are direct antagonists of the renal tubular enac and cause natriuresis while being both potassium and magnesium sparing.3 if there is a suspicion of an enac mutation based on the response to these drugs,3 whole exome sequencing should be undertaken. the very rare missense single nucleotide variant (for which our patient is heterozygous), that causes the substitution of glycine to valine (p.gly442val), has been linked to hypertension with increased enac activity.7,8 the effect of this polymorphism has been assessed by measuring the urine-aldosterone to -potassium ratio.7 increased enac activity would decrease this ratio as excess sodium absorption results in reduced aldosterone production and elevated urinary potassium excretion.7 this phenomenon has been confirmed in liddle syndrome patients where the urine-aldosterone to -potassium ratio was lower in subjects with the polymorphism than in normal subjects.9 the association of the alpha enac polymorphism (for which our patient is heterozygous), that causes the substitution of alanine to threonine (pala334thr), has been related to hypertension in certain studies10 and is associated with increased enac activity of 1.6-fold10 in functional studies. these findings in the current patient indicate that the severe clinical phenotype observed could be attributed to the compound heterozygous mutations located at different subunits of the enac and may indicate the presence of a yet unrecognised pathological variant. analogous to disorders caused by mineralocorticoid excess, liddle syndrome classically presents with hypertension, hypokalaemia and metabolic alkalosis. these findings are not always present, which may lead to under-diagnosis of the syndrome.11 identification of this condition is challenging, as the differential diagnosis for secondary hypertension is broad and the syndrome may present atypically. patients may have marked variations in phenotype, even with the same genotype.3 liddle syndrome principally arises from a transport impairment causing increased sodium reabsorption and excretion of potassium and hydrogen ions in the distal renal tubule. invariably, this leads to hypertension due to sodium and fluid retention with consequent hypokalaemic metabolic alkalosis and the suppression of renin and aldosterone through negative feedback. rare variants may, autonomously or cumulatively, cause hereditary disorders. liu and colleagues observed substantial differences in serum potassium levels and symptom onset in rare and non-rare scnn1b and scnn1g variant carriers, suggesting potential pathogenicity of some variants.12 to date, approximately 31 different liddle syndrome-causing alleles have been described in 72 families from four continents.3,10,13 interpretation of results from next-generation sequencing technologies are challenging as they have increased not only the diagnostic sensitivity, but also the number of variants with uncertain clinical significance. it is feasible that additional mutations that increase enac activity and result in phenotypical liddle syndrome will be discovered. in a study including patients with the liddle syndrome phenotype from kenya, nigeria and south africa, most patients had variants of a number of diverse genes that affect the enac channel.13 the authors speculated that certain patients may have combinations of variants that predispose to both increased aldosterone secretion and increased activity of the enac.13 in the assessment of patients with hyporeninaemic hypertension, investigation for a mutation in the enac subunits is recommended since early diagnosis and correct management of these patients may improve outcomes. delayed treatment is associated with failure to thrive and hypertension-related morbidity and mortality from cardiovascular disease, cerebrovascular disease, nephrosclerosis and progressive renal failure. furthermore, mutation studies permit clinicians to advocate for family screening based on the proband to identify carriers.14 this is the first case where a combined effect from mutations resulted in severe disease. genetic testing (including whole exome sequencing) should be performed, when possible, to identify mutations in patients with suspected secondary hypertension and unusual presentation.14 conclusion liddle syndrome is a poorly understood, but treatable genetic disease. misor late diagnosis may lead to unfavourable clinical sequelae. thus, any infant presenting with hypertension and metabolic alkalosis, with or without hypokalaemia, should raise suspicion for liddle syndrome, even in patients without a family history of hypertension. more functional studies are needed to characterise the numerous variants associated with the syndrome and their potential pathogenicity. acknowledgements christiaan labuschagne and erika viljoen of inqaba biotechnical industries for technical assistance with sequencing studies. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions n.s. drafted the original manuscript; b.c.-m. conceptualised and edited the manuscript; a.b.a. performed genetic studies and interpretation; g.v.b. undertook patient examination and investigation; and t.s.p. supervised the study. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability data supporting the findings of this study are available within the article. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any 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abstract introduction methodology results discussion acknowledgements references about the author(s) victor n. fondoh bamenda regional hospital laboratory, bamenda, north-west region, cameroon njong a. mom faculty of economics and management sciences, university of bamenda, bamenda, north-west cameroon citation fondoh vn, mom na. mother-to-child transmission of hiv and its predictors among hiv-exposed infants at the bamenda regional hospital, cameroon. africa. afr j lab med. 2017;6(1), a589. https://doi.org/10.4102/ajlm.v6i1.589 original research mother-to-child transmission of hiv and its predictors among hiv-exposed infants at bamenda regional hospital, cameroon victor n. fondoh, njong a. mom received: 03 nov. 2016; accepted: 27 may 2017; published: 14 dec. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: mother-to-child transmission (mtct) of hiv, has been a major global public health burden. despite the use of antiretroviral prophylaxis by hiv-positive pregnant women and their infants, safe obstetric practice and safe infant feeding habits in the prevention of mtct of hiv, the prevalence of hiv among hiv-exposed infants is still high in cameroon. objective: our objectives were to determine the prevalence, assess the predictors and determine the effect of combination antiretroviral therapy (cart) on mtct of hiv at the regional hospital in bamenda, cameroon. methods: this was a retrospective study. secondary data from 877 hiv-exposed infants aged ≤ 72 weeks were extracted from the records between january 2008 and december 2014. the predictors and effect of cart on mtct of hiv were analysed using a multivariable logistic regression model and risk analysis, respectively. results: out of 877 hiv-exposed infants, 62 were positive for hiv, giving a prevalence of 7.1%. maternal antiretroviral intervention and infant age group were statistically significant predictors of mtct of hiv. hiv-positive mothers who were on cart were 2.49 times less likely to transmit hiv than those who were not on cart. conclusion: in order to reduce the prevalence of hiv among hiv-exposed infants, maternal antiretroviral intervention should be targeted and the use of cart by hiv-positive pregnant women should be encouraged. introduction mother-to-child transmission (mtct) of hiv refers to vertical transmission of hiv from an hiv-positive mother to her baby at one or more of the following stages: pregnancy, labour, delivery or breastfeeding.1,2 globally, this accounts for 90% of hiv infections in children under the age of 15 years.3 in 2016, 90% of the estimated four million children living with hiv resided in sub-saharan africa.4 it is also estimated that 5% – 10% of mtct occurs during pregnancy, 10% – 20% during labour and delivery and 10% – 20% through breastfeeding. the risk of transmission is 15% – 45% without intervention. with intervention, the risk is reduced to 5% in breastfeeding populations5 and less than 2% in non-breastfeeding populations.3 one-third of hiv-positive children die within one year of birth and half before their second birthday without intervention.6,7 in 2012, cameroon registered an 8.4% hiv prevalence in hiv-exposed infants with the north-west region registering 3.7%.8 interventions for the prevention of mother to child transmission (pmtct) of hiv include: antiretroviral prophylaxis given to women during pregnancy and labour and to their infants within the first weeks of life, safe obstetric practices and safe infant feeding habits.9,11 these interventions were being implemented globally by the pmtct programme initiated by the world health organization in order to meet goal four of the millennium development goals which centers on the reduction of child mortality. the implementation of the pmtct programme was started at the regional hospital bamenda (rhb), cameroon, in 2008 by the elizabeth glaser pediatric aids foundation of the cameroon baptist convention health board. this programme is being funded by the united states president’s emergency plan for aids relief.12 there are many reports on the outcomes of the pmtct interventions.13,14 our objectives were to determine the prevalence of mtct of hiv among hiv-exposed infants in cameroon, assess its predictors and determine the efficacy of combination antiretroviral therapy (cart) as an intervention. methodology ethical considerations authorisation to conduct this research was obtained from the catholic university of cameroon’s institutional review board (no. 020/hepm/catuc-irb/15). approval to carry out the research at rhb was obtained from the rhb institutional review board (no. 001/app/rdph/rhb/irb). authorisation to collect data was obtained from the rhb paediatric treatment and care centre (rhb-ptcc) head of department. consent for the hiv-exposed infants enrolled for follow-up at the rhb-ptcc was given by their mother or caregiver orally. study area the study site was the rhb, situated in bamenda, in the north-west region of cameroon. the rhb was chosen because it is a level-two hospital for five district hospitals serving a population of 302 749 inhabitants. it also has one of the largest ante-natal clinics that implements the pmtct programme, an extended programme on immunisation centre for referral of hiv-exposed infants for follow-up and the rhb-ptcc, where hiv-exposed infants are enrolled and followed up. research design this was a retrospective study. secondary data from routine follow-up records of hiv-exposed infants were reviewed and collected from the rhb-ptcc from the period january 2008 to december 2014. follow-up of hiv-positive mothers and their infants was based on the world health organization guidelines that evolved over the duration of the study period in response to changes in international guidelines and newly available information.15 during care at the ante-natal clinic, pregnant women enrolled in the pmtct programme were tested for hiv by serology using determine hiv-1/2 ag/ab combo (alere medical co., ltd, matsuhidai, matsudo-shi, chiba; first line) and immunocomb ii hiv 1&2 bispot (orgenics ltd, yavne, israel; second line) kits. pregnant women with no test records and those who had never known their hiv status were tested at the maternity ward upon presentation for delivery. those who were hiv-positive were placed on antiretroviral therapy during pregnancy, labour and delivery depending on when their status was identified. the antiretroviral drugs were usually used singly or as cart, depending on availability and/or clinical conditions. the medications used were: nevirapine, zidovudine, lamivudine, tenofovir, efavirenz, abacavir, lopinavir/ritonavir, atazanavir/ritonavir. the cart could be one of the following regimens: zidovudine + lamivudine + nevirapine, zidovudine + lamivudine + efavirenz, abacavir + lamivudine + efavirenz, tenofovir + lamivudine + efavirenz, tenofovir + lamivudine + lopinavir/ritonavir, tenofovir + lamivudine + atazanavir/ritonavir. the women were also counseled on feeding interventions for their infants. hiv-exposed infants were placed on nevirapine or zidovudine, (depending on availability, immediately) after birth at the maternity ward and as scheduled by the rhb-ptcc.15 the hiv status of the infants were determined at six weeks of age or as early as possible after enrolment in the centre. a heel-prick dried blood spot was collected from the infants at the rhb laboratory and sent to the early infant diagnosis laboratory in mutengene (from 2008 to 2011) or the tuberculosis reference laboratory, bamenda (from 2012 to 2014), all in cameroon, for qualitative analysis of hiv dna by polymerase chain reaction (pcr) testing. infants were declared positive for hiv if the hiv dna pcr test was positive. inclusion criteria included in the studies were hiv-exposed infants up to 72 weeks old, who had hiv results (including that of their respective mothers) registered in the hiv dna pcr register and whose mothers gave consent (orally). exclusion criteria excluded from the studies were infants who came in for a second hiv dna pcr test, were older than 72 weeks of age, had no test results (including that of their respective mothers) registered in the hiv dna pcr register, and those whose mothers did not consent (orally). data collection data collection was conducted by three trained reviewers. the following records were reviewed: rhb-ptcc results register, pmtct register and follow-up forms of each registered infant. the data from each participant were cross-checked for consistency in the three registers. detailed information on the records and management of the infants was obtained from key informants (health care providers) working at the rhb-ptcc. data were collected using a structured data collection format. data were coded and entered into a microsoft excel 2010 spreadsheet (microsoft corporation, redmond, washington, united states). the variables were selected based on the pmtct of hiv interventions (methods) that directly affect the physiology of the mother and child. the dependent variable was the outcome of interest: hiv transmission (the hiv status of exposed infant). the independent variables were infant age at hiv diagnosis (0–6 weeks or > 6–72 weeks), sex (male or female), maternal antiretroviral intervention (on cart or not on cart), obstetric intervention (vaginal or caesarean section), infant antiretroviral intervention at time of diagnosis (nevirapine or zidovudine) and the infant feeding intervention (breastfed or formula) at time of diagnosis. data were double-checked by a second reviewer in order to guarantee quality. mothers of twins were counted twice. data analysis analyses were conducted using r statistical software (r, version 3.3.3; r foundation for statistical computing, vienna, austria). descriptive statistics were done using frequency distribution. univariable analysis was used to determine the association between the pmtct interventions and hiv transmission. the predictors of mtct of hiv were assessed by building a multivariable logistic regression model, adding the variables in a forward selection process in order, starting with those with the smallest p-value from the univariable analysis. variables with a p-value greater than or equal to 0.2 on the univariable analysis were not included in the multivariable analysis. the final model was based on the selection of the smallest akaike information criterion,14,16,17 and risk analysis was used to determine the effect of cart. analyses were conducted using 95% confidence intervals, and a p-value of less than 0.05 was considered statistically significant. results data from 1221 hiv-exposed infants (including 1221 of their respective hiv-positive mothers) were collected between january 2008 and december 2014 (figure 1). a total of 877 hiv-exposed infants (including 877 of their respective hiv-positive mothers) with hiv results were included in the analysis, and 344 were excluded (three infants came in for a second hiv testing; two infants were older than 72 weeks and 339 infants did not have complete data for hiv status). of the 877 hiv-exposed infants, 62 were hiv-positive giving an overall hiv prevalence of 7.1% among hiv-exposed infants (figure 2). figure 1: flow chart of representation of the population used to estimate the prevalence of hiv in hiv exposed infants in the regional hospital bamenda. figure 2: prevalence of hiv in hiv-exposed infants over the years 2008–2014. of the 877 hiv-exposed infants included in the analysis, 418 (47.7%) were male, of whom 28 (6.7%) were hiv-positive (table 1). the median age at hiv diagnosis was 6 weeks (range = 2–48 weeks), and 601 infants (69.0%) were in the age group 0–6 weeks, of which 30 (5.0%) were positive for hiv. of the 877 hiv-positive mothers, data for 824 were analysed for maternal antiretroviral intervention; 53 had incomplete data and were excluded (figure 1). twenty (3.5%) of the 564 mothers on cart were hiv-positive. of the 877 hiv-exposed infants, data for 641 were analysed for infant obstetric intervention, and 236 with incomplete data were excluded. thirty-seven (6.7%) of the 556 infants delivered vaginally were hiv-positive. of the 877 hiv-exposed infants, data for 842 were analysed for infant antiretroviral intervention, and 35 with incomplete data were excluded. forty (8.0%) of the 501 infants on nevirapine were hiv-positive. of the 877 hiv-exposed infants, data for 868 were analysed for infant feeding intervention, and nine with incomplete data were excluded. fifty-one (7.6%) of the 668 infants who were breastfed were hiv-positive. table 1: hiv transmission among hiv-exposed infants – univariable analysis our univariable analysis indicated that there were statistically significant associations between hiv transmission and both age group (chi-square: 12.550, p = 0.001) and maternal antiretroviral intervention (chi square: 15.473, p < 0.001) (table 2). there were no statistically significant associations between hiv transmission and infant sex (chi-square: 0.167, p = 0.683), infant feeding intervention (chi-square: 1.635, p = 0.201), infant antiretroviral intervention (chi-square: 1.891, p = 0.089) or obstetric intervention (chi-square: 0.019, p = 0.890). table 2: multivariable logistic regression of predictors of hiv transmission among hiv-exposed infants following our multi-variable logistic regression model, obstetric intervention, although not statistically significant, was retained as it improved the overall fit of the model based on the akaike information criterion. infant sex, infant feeding intervention and infant antiretroviral intervention did not improve the overall fit of the model and thus were excluded. the final model included three variables: maternal antiretroviral intervention, infant age group and obstetric intervention. based on this model, maternal antiretroviral intervention (p = 0.001) and infant age group (p = 0.017) were statistically significant predictors of mtct of hiv (table 2). our risk analysis revealed that mothers on cart were less likely to transmit hiv to their infants (adjusted odds ratio (or) = 2.49, 95% confidence interval (ci): 1.23–5.02) compared to those who were not on cart. similarly, infants aged 0–6 weeks at the time of hiv diagnosis were less likely to be infected with hiv compared to those aged > 6–72 weeks (adjusted or = 2.34, 95% ci: 1.61–4.72). discussion our study revealed that the prevalence of hiv among hiv-exposed infants in bamenda, cameroon, was 7.1%, which is still a burden to society. this may be due to non-adherence to antiretroviral protocols by mothers and infants, vaginal delivery or breastfeeding.10,18,19 a similar result (7.0%) was reported from nigeria between 2011 and 2012.20 lower prevalence was reported in south india (6.5% in 2010)21, south africa (5.8% between 2004 and 200823, 5.4% between 2008 and 201022) and in high-income countries (2.9% between 2001 and 2003 in europe16, less than 2% in united states, europe, brazil and bahamas between 1997 and 200011). this may be due to the universal use of cart, elective caesarean sections and avoidance of breastfeeding in developed countries24. such preventive approaches are limited in poor countries (including cameroon) due to poor funding and social and cultural norms.24 higher prevalence was reported in zambia (12.2% between 2007 and 2010)26 and nigeria (9.1% in 2010).27 this might suggest better management of the pmtct programme at our study site. the north-west region registered a prevalence of 3.7% in 20128, far lower than the prevalence of 7.1% in our study. the high prevalence might be due to the fact that, rhb is a level-two referral hospital with a high influx of patients resulting from the availability of specialised and satisfactory services including (but not limited to) the laboratory, paediatric, obstetric and gynecological services). our multiple logistic regression model revealed that infant age at hiv diagnosis and maternal antiretroviral intervention were statistically significant predictors of mtct of hiv. our observations are similar to those observed in other models in ethiopia and europe, where infant age group at diagnosis and maternal antiretroviral intervention, respectively, were statistically significant predictors of mtct of hiv.13,16 in our study, infants aged > 6– 72 weeks were at higher risk of hiv transmission compared to those aged 0–6 weeks. this might be because most of the infants who were diagnosed at older than six weeks of age were those who were sick, coupled with the fact that some were not on antiretroviral drugs. late diagnosis at the study site might have resulted from poor communication, stigmatisation and discrimination, poor training of health care providers and lack of stock of antiretroviral drugs. poor communication between the ante-natal clinic, delivery and postnatal facilities and lack of good information systems may lead to late diagnosis of hiv-exposed infants.28,29 at the rhb, there is no traceable standard system or network (software or internet) to link the ante-natal clinic, maternity, postnatal care, immunisation centre, the rhb-ptcc and the community. there is a communication link between the caregiver and the rhb-ptcc, but this link is established during enrolment at the centre. there may be some gaps in communication if an infant’s mother is critically ill or dies. due to inadequate counselling and education during antenatal visits, some mothers are unsure of the type of tests or activities to be done on their infants at the early infant diagnosis services. thus, they may not be motivated to take their child for follow-up treatment and care services.30 fear of stigmatisation and discrimination, especially at the immunisation center (where some of these infants are identified) increases maternal reluctance to identify themselves for proper follow-up referral.30,31,32 during the vaccination process at the rhb vaccination center, the vaccinators identify the cards of hiv-exposed infants and invite them for a discussion after the process, which creates a stigma on the mothers. secondly, the nature of the infrastructure may also contribute to stigmatisation. all infants identified for dried blood spot collection are referred to the blood bank at the rhb laboratory. babies brought to the blood bank are assumed to be an hiv-exposed child, which is a stigma. we found that hiv-positive women who were not on cart were 2.49 times more likely to transmit hiv to their babies compared to those who were on cart. all the mothers in our study who were not on cart were either on nevirapine or zidovudine. nevirapine and zidovudine are single-drug regimens, which are not as effective as cart. however, this justifies the assertion that cart decreases the risk of mtct of hiv33 and is more effective than nevirapine or zidovudine.34 several studies have reported viral resistance to nevirapine in pregnant women, including women in cameroon.35 poor training of health care provides, stock-outs of cart or delays by some hiv-positive mothers to start cart may be other reasons for the high risk of mtct of hiv. this may be caused by administrative bottlenecks or poor management at some or all levels. a report from cameroon in 2012 indicated that there was inadequate training of health care providers, stock-out of antiretroviral drugs, poor antenatal coverage, fewer pregnant women going for hiv testing, low coverage of hiv-positive women on antiretroviral drugs, weak follow-up of hiv-exposed infants and fewer hiv-exposed infants tested at six weeks of age, which led to ineffective implementation of the pmtct programme.8,36 the risk of hiv transmission by caesarean section delivery in this study was 2.34 times higher than vaginal delivery. some of the caesarean sections were elective and some were a result of complication during delivery, but all were counted as caesarean section. this might have accounted for the high risk of mtct of hiv through caesarean section deliveries that resulted due to complications during labour, including membrane rupture before the caesarean section was done and subsequent blood contact with hiv-contaminated blood.37 recommendations in order to reduce the spread of hiv, particular attention should be directed to the infant age group at diagnosis and maternal antiretroviral intervention. all pregnant hiv-positive women in cameroon should be placed on cart. antiretroviral drugs reduce maternal viral load and risk of hiv transmission to the baby and partner.38 these recommendations can only be achieved through a joint endeavour by the government, the institutions, the communities and individuals. the government should provide resources including continuous supply of cart to all pregnant women, improve the diagnostic services for maternal and infant diagnosis, subsidise delivery kits, train and employ more health caregivers and improve on infrastructure. a ‘one-stop shop’ infrastructure with network and a web-based medical record system to link the ante-natal clinic to the maternity ward, postnatal, immunisation centre, the rhb-ptcc, the laboratory and the community should be created at the rhb to better manage the hiv-exposed infants, improve confidentiality and reduce stigmatisation. the institutions should improve on the protocols of treatment and encourage hiv-positive pregnant women to take cart during pregnancy in order to keep their baby safe, ensure hiv dna pcr testing for infants at six weeks, encourage vaginal delivery (discourage the use of caesarean section except when it is unavoidable) and encourage formula feeding of hiv-exposed infants. the community should avoid stigmatisation of hiv-positive individuals. hiv-positive mothers and hiv-exposed babies should adhere to their routines: take their drugs regularly and report any unusual development in their systems timeously, accept their identity and come out of stigmatisation. limitations since we used secondary data from the records of the rhb-ptcc, it was difficult to control the inconsistencies of missing data from one or more of the parameters. although missing data did not affect our results since it was considered during analysis, it could have increased our sample size for better interpretation and reduction of the risk of bias. since this was not a clinical trial, it was difficult to have a clear-cut distinction between infants who were exclusively breastfed or formula-fed because other food supplements or drugs including traditional medicine might have been given to the infants. we could not distinguish between those who had elective caesarean sections from those who had caesarean sections due to complications in pregnancy. thus all were grouped as caesarean section. since all the mothers and infants were said to be on antiretroviral drugs, it was not possible to distinctively assess those who had never been on antiretroviral drugs. we could not distinguish the specific regimen taken by the pregnant women on cart. thus we considered all as cart. we also could not also distinguish those who were on a specific regimen because of availability or their clinical condition. the data for all variables were not available. this study was to assess the predictors of the pmtct interventions and these predictors were available. conclusion our study concludes that the prevalence of hiv among hiv-exposed infants was 7.1%. infant age group at diagnosis and maternal antiretroviral intervention were statistically significant predictors of mtct of hiv. the risk of mtct of hiv when hiv-positive mothers were on cart was 2.49 times lower compared to those who were not on cart. acknowledgements we acknowledge the administration of the catholic university of cameroon, bamenda for introducing this programme for scholars in bamenda, cameroon. we sincerely thank prof. wilfred mbacham (head of programme – health, economics policy and management), dr sunjoh frida, dr kinge thompson njie, dr awasom charles nde, ms muluh claris, mrs mary-teresia ghumbemsitia, dr kum clatus, mrs mildred njimnsi, mrs brenda allo, mrs amina saidu, ms tchiazah ngwe, mrs akoko elisabeth, mr aseh promise, dr ndong ignatius, mr bong roland, ma kila kilo, mrs tih helen, mr mbeleck manleck, miss andin ntungen, mrs pafoule edith, dr njimented godfrey, dr yongabi kenneth, mrs fondoh rachel, mama grace fondoh, dr richard fondoh, dr frankline nkongho egbe and dr hermann ngouakam for their contributions towards the success of this work. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support none. authors’ contributions v.n.f. was the lead author responsible for the conceptualisation of the study, study design, data collection and final writing up of the manuscript. n.a.m. was the co-author responsible for the specification of the analytical model, data analysis and interpretation of the results. references rgopoulos, et al., aids in pregnancy part 1: epidemiology, testing, effect on disease progression, opportunistic infections, and the risk of vertical transmission. skinmed, 2007. 6: p. 18–23. https://doi.org/10.1111/j.1540-9740.2007.05762.x mark, crepaz, and jansen, estimating sexual transmission of hiv from persons aware and unaware that they are infected with the hiv virus in usa. aids, 2006. 20: p. 1447–52. https://doi.org/10.1097/01.aids.0000233579.79714.8d de cock, k.m., et al., prevention of mother-to-child hiv transmission in resource-poor countries: translating research into policy and practice. jama, 2000. 283(9): p. 1175–1182. 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the malawi bridge project. journal of the association of nurses in aids care, 2011. 22(3): p. 215–228. https://doi.org/10.1016/j.jana.2010.08.005 watts, d., r. balasubramanian, and r. maupin, maternal toxicity and pregnancy complications in hiv-infected women receiving antiretroviral therapy: an analysis of the pactg 316 study. american journal obstetric gynecolology, 2004. 190: p. 506–516. https://doi.org/10.1016/j.ajog.2003.07.018 orlando, s., et al., cost-effectiveness of using haart in prevention of mother-to-child transmission in the dream-project malawi. journal of acquired immune deficiency syndromes, 2010. 55(5): p. 631–634. https://doi.org/10.1097/qai.0b013e3181f9f9f5 ayouba, a., et al., low rate of mother-to-child transmission of hiv-1 after nevirapine intervention in a pilot public health program in yaounde, cameroon. journal of acquired immune deficiciency syndrome, 2003. 34(3): p. 274–280. https://doi.org/10.1097/00126334-200311010-00003 cdc, national operational plan for scaling up of early infant diagnosis(eid) continuum of care 2013 2014 (draft one). 2012, center for disease control and prevention: cameroon. p. 35. gibb, d.m., et al., uptake of interventions to reduce mother-to-child transmission of hiv in the united kingdom and ireland. aids, 1997. 11(7): p. f53–f58. https://doi.org/10.1097/00002030-199707000-00001 al, d.c.k.e., prevention of mother-to-child hiv transmission in resource-poor countries: translating research into policy and practice. journal of the american medical association, 2000. 283: p. 1175–1182. https://doi.org/10.1001/jama.283.9.1175 article information authors: bernard nkrumah1 beatrice van der puije2 veronica bekoe3 rowland adukpo4 nii a. kotey2 katy yao5 peter n. fonjungo5 elizabeth t. luman5 samuel duh2 patrick a. njukeng6 nii a. addo3 fazle n. khan7 celia j.i. woodfill1 affiliations: 1us centers for disease control and prevention, us embassy, ghana2global health systems solutions, c75/20 amanfro street, abelenkpe, ghana 3national aids control program, ghana health service, ghana 4national public health reference laboratory, ghana health service, ghana 5us centers for disease control and prevention, atlanta, united states 6global health systems solutions, cameroon 7us centers for disease control and prevention, cote d’ivoire correspondence to: bernard nkrumah postal address: us centers for disease control and prevention, us embassy, number 4 forth circular road, cantonments, ghana dates: received: 15 july 2014 accepted: 06 aug. 2014 published: 03 nov. 2014 how to cite this article: nkrumah b, van der puije b, bekoe v, et al. building local human resources to implement slmta with limited donor funding: the ghana experience. afr j lab med. 2014;3(2), art. #214, 7 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.214 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. building local human resources to implement slmta with limited donor funding: the ghana experience in this original research... open access • abstract • introduction • research method and design    • programme implementation approach    • baseline audits and site selection    • development of a local mentorship programme    • slmta programme implementation    • programme monitoring and evaluation    • preparation for slmta expansion    • data entry and analysis • results    • measuring the impact of slmta    • local capacity building and cost of slmta implementation • discussion    • limitations of the study    • recommendations    • conclusion • acknowledgements    • competing interests    • disclaimer    • authors’ contributions • references abstract top ↑ background: in 2009, ghana adopted the strengthening laboratory management toward accreditation (slmta) programme in order to improve laboratory quality. the programme was implemented successfully with limited donor funding and local human resources.objectives: to demonstrate how ghana, which received very limited pepfar funding, was able to achieve marked quality improvement using local human resources. method: local partners led the slmta implementation and local mentors were embedded in each laboratory. an in-country training-of-trainers workshop was conducted in order to increase the pool of local slmta implementers. three laboratory cohorts were enrolled in slmta in 2011, 2012 and 2013. participants from each cohort attended in a series of three workshops interspersed with improvement projects and mentorship. supplemental training on internal audit was provided. baseline, exit and follow-up audits were conducted using the stepwise laboratory quality improvement process towards accreditation (slipta) checklist. in november 2013, four laboratories underwent official slipta audits by the african society for laboratory medicine (aslm). results: the local slmta team successfully implemented three cohorts of slmta in 15 laboratories. seven out of the nine laboratories that underwent follow-up audits have reached at least one star. three out of the four laboratories that underwent official aslm audits were awarded four stars. patient satisfaction increased from 25% to 70% and sample rejection rates decreased from 32% to 10%. on average, $40 000 was spent per laboratory to cover mentors’ salaries, slmta training and improvement project support. conclusion: building in-country capacity through local partners is a sustainable model for improving service quality in resource-constrained countries such as ghana. such models promote country ownership, capacity building and the use of local human resources for the expansion of slmta. introduction top ↑ the recent drive by the world health organization’s regional office for africa (who afro) and the us president’s emergency plan for aids relief (pepfar) toward strengthening laboratory systems in africa is a historic step in the improvement of health systems. however, this effort is hampered by the lack of locally-qualified laboratory personnel.1,2 the need to strengthen weak laboratory networks, systems and services in developing countries was highlighted in 2008 in a series of advocacy meetings: the maputo declaration (january 2008) for strengthening laboratory health systems,1 the lyon statement (april 2008) on the need for developing countries to establish practical quality management systems,3 and the yaoundé resolution (september 2008) issued by who afro in recognition of the dilapidated state of the laboratory health systems and the need to strengthen all laboratory tiers in order to fight multiple diseases.4 in the following year in kigali (july 2009), who afro launched a stepwise laboratory accreditation preparation scheme, which recognises and encourages incremental progress toward fulfilment of the requirements of the international organization for standardisation (iso) 15189 standard; and, at the 59th session of the who afro regional committee (september 2009), member states adopted resolutions afr/rc59/r2 and afr/rc59/wp/3 aimed at strengthening public health laboratories and other centres of excellence in order to improve disease prevention and control.4 also launched in kigali was the strengthening laboratory management toward accreditation (slmta) programme, developed by the us centers for disease control and prevention (cdc) and partners so as to guide countries toward achieving the called-for improvements. as pepfar’s flagship programme for strengthening laboratory systems, slmta has been implemented in 47 countries, demonstrating measurable and positive impact.5ghana has more than 420 public sector laboratories organised into a four-tier laboratory system: national/central, regional/zonal, district and subdistrict levels. nationaland regional-level laboratories provide technical assistance and supportive supervision for the district and subdistrict level laboratories. in 2012, the ministry of health (moh) and the ghana health service (ghs) drafted a five-year national laboratory strategic plan. this strategic plan clearly addressed local capacity building and highlighted the government’s commitment to fulfil their mandate of providing quality healthcare through the adoption of slmta as the systematic approach for implementation of laboratory quality management systems (qms) in ghana. in conjunction with the strategic plan, ghana drafted a national laboratory accreditation policy. accreditation ensures the quality, precision, dependability and timeliness of laboratory testing results.6,7,8 two private laboratories in ghana are accredited, but none of the public sector laboratories, which perform the bulk of patient testing, are accredited to any national or international standards. called the largest health initiative ever implemented by a single country to address a disease, since 2003 pepfar has provided financial and technical support to developing countries to fight hiv, saving millions of lives.9 laboratory strengthening is a critical component of the pepfar strategy. in 2013, the institute of medicine report on pepfar noted that ‘its substantial support for laboratory strengthening has had fundamentally positive effects for the response to hiv and has been leveraged to improve the functioning of entire health systems’.9 ghana is one of pepfar’s targeted assistance countries, which receive limited financial support for key populations or priority technical areas, capacity building and/or technical assistance.10 ghana’s annual laboratory budget from pepfar is about $1.1m (8% of the total ghana pepfar funding). this notwithstanding, pepfar is a major source of support for ghana’s laboratory system strengthening programmes. given the limited funding, ghana sought a strategy to implement the slmta programme for laboratory system strengthening in an economical and sustainable manner. this paper describes how a country like ghana, which receives very limited funding, was able to achieve marked improvement in laboratory quality management by empowering local partners to implement the slmta programme. research method and design top ↑ programme implementation approach a top-down programme implementation approach was adopted. under this model, the country sought to first build capacity and strengthen the quality of laboratory services within the nationaland regionallevel laboratories. in turn, these higher-level laboratories would be equipped to support capacity building and strengthen quality laboratory services at the lower tier levels. two local implementing partners, the governmental agency ghs and a non-government not-for-profit organisation, global health systems solutions (ghss), were engaged by the cdc’s ghana office to implement slmta through cooperative agreements. baseline audits and site selection eighteen public sector laboratories (three national, 12 regional and three district level) were considered initially for enrolment into the slmta programme. ghs conducted a baseline audit of all 18 laboratories using the stepwise laboratory quality improvement process towards accreditation (slipta) checklist, which provides a quantitative measure of adherence to iso 15189 requirements. the scored checklist quantifies a laboratory’s quality status using a zeroto five-star rating: 0–141 points (< 55%) = zero stars, 142–166 points (55% – 64%) = one star, 167–192 points (65% – 74%) = two stars, 193–218 points (75% – 84%) = three stars, 219–243 points (85% – 94%) = four stars and 244–258 points (95% – 100%) = five stars.11fifteen of the 18 audited laboratories were selected to enrol in the slmta programme based on several factors: their baseline scores; infrastructural availability and suitability; geographical distribution; staffing; and management and staff willingness to participate. the 15 laboratories comprised two national-level laboratories, 12 regional-level laboratories and one district-level laboratory (table 1). geographically, these laboratories cover all 10 regions in ghana (figure 1). these laboratories were grouped further into three cohorts: cohort 1 comprised four laboratories; cohort 2, five laboratories; and cohort 3, six laboratories. table 1: level and function of laboratories in the slmta programme. figure 1: geographic location of laboratories involved in slmta implementation in ghana, 2011–2013. development of a local mentorship programme in october 2011, ghss, working with ghs and other stakeholders, initiated a local mentorship programme, training and placing full-time local mentors at each of the selected laboratories. potential mentors were recruited through newspaper advertisements in an open competitive process; minimum qualifications included a degree in medical laboratory technology or equivalent and previous experience in working with ghs. their training included classroom-based lectures on topics such as qms and the 12 quality system essentials (qses); iso 15189 requirements; conducting audits using the slipta checklist; and project management. trainee mentors then spent one to three weeks with ghss senior mentors during a field practicum before they were sent to their post. mentors were closely supervised by ghss, ghs and cdc by means of a weekly and monthly reporting systems. slmta programme implementation the first cohort of laboratories implemented slmta from april 2011 to april 2012, the second from may 2012 to may 2013 and the third from february 2013 to november 2013. four staff members from each enrolled laboratory, namely, the laboratory manager, quality manager and two other staff members, participated in the three slmta workshops which were coordinated by ghss and ghs. laboratories selected their own participants, with assistance from their upper management, by following the above-mentioned selection criteria. slmta implementation in the three cohorts followed the prescribed slmta process,12 with the three workshops taught by qualified slmta facilitators from ghs and ghss, implementation of improvement projects after each workshop and quarterly site visits to monitor progress, track quality indicators and provide technical assistance. participants also attended complementary training on iso 15189 and internal audit conducted by the ghana standards authority (gsa). site visits were conducted using the slmta site visit monitoring tool by either ghss alone or by a combined technical team from ghs, cdc’s ghana office and ghss. written reports detailing observations, technical assistance provided and recommendations were delivered to the laboratory, the regional directors of health and hospital directors in order to ensure that management within each region were well informed regarding the programme and the progress made by their laboratories. programme monitoring and evaluation intermediate audits were conducted semi-annually in order to measure the progress made by the laboratories whilst helping partners to review their work plan, implementation approach and the mentorship programme. the audits were conducted by trained in-country auditors using the slipta checklist and results were communicated to the participating laboratories so as to guide corrective actions. mentors who were cross-trained as auditors did not conduct audits in laboratories that they mentored. an exit audit was conducted for each laboratory in the three cohorts at the end of the slmta training. follow-up audits were conducted six months after the exit audits in order to monitor the performance of the laboratories and to ensure that recommendations from the exit audits were addressed. one laboratory from cohort 3 (l12) did not receive an intermediate audit because of delayed communication to the site. another laboratory in cohort 3 (l15) did not receive an exit audit because of its high scores at baseline and intermediate audits; in november 2013, this laboratory and the 3 highest-performing laboratories from cohort 1 underwent official slipta audits by the african society for laboratory medicine (aslm).additional indicators, such as specimen rejection rates and patient satisfaction, were also tracked in cohort 1 laboratories so as to assess the progress and impact of implementation. specimen rejection rates were calculated as a percentage of samples rejected as a result of non-conformity to specimen acceptance criteria. because of a lack of data on the specimen rejection rate in ghanaian laboratories prior to the implementation of slmta, specimen rejection rates were only monitored after baseline, during slmta implementation and thereafter for three years. patient satisfaction was measured using questionnaires given to patients and comments received from patients through the suggestion box. the cost in us dollars for the implementation of slmta was reported by the implementing partners. estimated costs included mentor salaries, slmta training and improvement project support. salaries of mentors were determined by the implementing partners, in accordance with ghana’s labour laws. slmta training costs were calculated based on four personnel from each laboratory and two trainers participating in the three five-day workshops. costs included per diem, local transportation to the training venue, training materials for all participants and the workshop venue package. workshop trainers were staff members from the implementing partner; their salaries were considered to be an in-kind contribution and were not included in the estimates. similarly, salary and time missed from work for participants were not included. expenditures sustained by the partner in order to support laboratory improvement projects, such as colour-coded bin liners, emergency eye-wash kits and iso training for internal auditors, were also estimated. preparation for slmta expansion in april 2013, a laboratory auditors training course was organised by the clinical and laboratory standards institute (clsi) and aslm for selected professionals with experience in qms, iso 15189 and slmta. the training programme equipped the participants to plan, prepare and conduct independent laboratory quality audits based on the slipta checklist and iso 15189 requirements. the training format consisted of classroom didactic presentations and a field practicum through mock audits.in september 2013, a slmta training-of-trainers (tot) workshop was conducted in order to increase the pool of local trainers and implementers for nationwide slmta scale-up. this workshop was led by two slmta master trainers from ghana and one from nigeria, all previously trained to lead tot workshops.13 local laboratory professionals who had implemented slmta were selected for the training. data entry and analysis the results of the audits were entered into a microsoft® excel spreadsheet (microsoft® 2010). statistical analysis was done using stata se 12.1 (stanford university it services 2012) after the data had been cleaned and exported. continuous variables such as slipta scores were summarised and presented as medians. percentage scores were determined by dividing the respective scores by the maximum possible points and expressing the results as a percentage. results top ↑ measuring the impact of slmta laboratories in all three cohorts demonstrated a steady improvement in the implementation of qms, as was illustrated by the median slipta scores at each audit (figure 2). median improvements from baseline to exit were 23 percentage points for cohort 1, 29 percentage points for cohort 2 and 20 percentage points for cohort 3. the most improved laboratory (l5) demonstrated an increase from 2% at baseline to 50% at exit, which increased further to 59% at the follow-up audit six months later. laboratory 2, on the other hand, scored 38% at baseline and 46% at exit, but decreased to 36% at the follow-up audit (figure 3). at baseline, only one of the 15 laboratories was at the one-star level. by the exit audit, three laboratories had reached at least one star; of the eight laboratories that conducted follow-up audits, seven had reached at least one star. figure 2: median slipta scores for the three ghana slmta cohorts. each of the 12 qses improved from baseline to exit. the most improved areas were process control and internal and external quality assessment (54%); customer service (50%); organisation and personnel (48%); and documents and records (44%) (figure 4). information management showed the least improvement (9%), followed by purchasing and inventory (18%) and internal audit (20%). the areas with the lowest median exit audit scores were internal audit (20%), occurrence management (25%), corrective action (33%) and management reviews (35%). figure 4: median performance of all 15 ghana slmta laboratories across the 12 quality systems essentials, as measured by the slipta checklist at the baseline and exit audits. the three highest-performing laboratories at exit audit (l1, l3 and l4, all from cohort 1), plus l15 from cohort 3, which did not receive an exit audit but earned three stars at both the baseline and intermediate audits, underwent official slipta audits by aslm. official slipta audit results overall were slightly higher than exit audit results (figure 3). three of these laboratories (l1, l3 and l15) earned four official slipta stars and one (l4) earned one star. figure 3: performance of 15 ghana slmta laboratories at baseline, intermediate, exit, follow-up and official slipta audit. average specimen rejection rates across the four slmta laboratories in cohort 1 decreased from 32% (range 23% – 44%) in 2011 to 25% (range 12% – 35%) in 2012 and 10% (range 3%–12%) in 2013. patient satisfaction increased from 25% to 70% over the same time period (figure 5). figure 5: specimen rejection and customer satisfaction trends for slmta cohort 1 laboratories. local capacity building and cost of slmta implementation the ghana slmta team presently comprises 18 slmta trainers, 15 mentors, 11 clsi/aslm trained auditors and two master trainers. this team has been responsible for the successful implementation of three cohorts of slmta with a total of 15 laboratories, training 60 laboratory professionals in qms. to date, two senior mentors and six trainers have completed phase one of the slipta auditor training conducted by aslm and clsi.the cost for slmta implementation for each laboratory was incurred in the areas of project management, slmta trainings and embedded mentorship. taken together, the implementing partners reported a total expenditure of $600 000 to implement slmta in the 15 laboratories. on average, $40 000 total was spent per laboratory to cover mentors’ salaries ($24 000), slmta training ($6000) and improvement project support ($10 000). discussion top ↑ the ghana national laboratory strategic plan prioritises the development of local capacity as a sustainable way to support the delivery of quality results for improved patient care and treatment. in 2009, ghana adopted slmta using limited funding and only local human resources to drive the programme forward. results have been remarkable in the 15 laboratories enrolled in slmta – audit scores doubled from baseline to exit and more than half of those laboratories reached one or more stars, including three laboratories that have achieved four stars. the success of the ghana slmta programme can be attributed to three strong factors: management engagement and commitment, the use of local partners and implementation of a local mentorship programme.management engagement and commitment have been shown to be critical elements in promoting quality laboratory services.14 in ghana, management was engaged at three levels: central, regional and facility. in june 2012, shortly after completion of the first slmta cohort, ghs organised a one-day meeting, chaired by the director general of ghs, to sensitise regional health directors, hospital medical directors and laboratory managers from all 10 regions of the country on the slmta programme and how it could benefit their facilities. a medical director whose facility had recently completed slmta gave a presentation on how the programme had transformed his facility. this medical director became the ‘slmta ambassador’ amongst his peers. the meeting served as a catalyst in the acceleration of slmta implementation in ghana; afterward, regional directors of health engaged regularly with participating laboratories by means of site visits, courtesy calls and review of audit reports. some medical directors at the facility level even joined in-house slmta trainings, site visits and debriefing activities. others personally took on responsibilities to oversee some improvement projects. this high-level engagement created tremendous enthusiasm within the facilities and contributed to staff morale. with a limited budget, it was important for ghana to incorporate cost-effective and results-oriented approaches into its programme implementation. to achieve the country’s priority of developing sustainable human capacity, local partners were engaged from the beginning, which proved to be advantageous in several respects: (1) they were familiar with the local administration, culture and language; (2) they were well accepted into the facilities as peers; and (3) their service was less expensive than international partners. local partners contributed directly to workforce development through the training and hiring of local human resources. care was taken to ensure that hiring was done through a merit-based process and was well specified in the organisations’ policies and procedures. key components included training, capacity building, salary structure and working conditions for technical, administrative and financial staff. to ensure that programme objectives and targets were met, partners were assisted in adhering to reporting requirements in a timely and consistent manner. mentorship is an important vehicle to establish and solidify qms and to help laboratories achieve their quality improvement goals.15 guidance regarding the implementation of a structured laboratory mentorship programme has been documented.15 similar to the lesotho mentorship approaches,16,17 ghana adopted a full-time, resident mentorship approach. in this approach, each mentor was assigned to a laboratory and resided within the locality where the laboratory was situated. effective mentoring requires full understanding of a laboratory’s culture, processes, procedures and people; we found that this embedded mentorship approach was further enhanced with the placement of indigenous professionals who already understood the in-country laboratory culture. it has been suggested that mentorship visits of four to eight weeks may be more effective than shorter periods.17 in our embedded mentorship approach, mentors stayed at the facility full time for the duration of the programme (18–24 months) and worked with laboratory staff to help raise the laboratory’s level of performance. one positive result of slmta implementation in ghana was improved patient satisfaction in the quality of service delivery and a reduction in the rejection of specimens. these results are consistent with previously-published findings.7 patient satisfaction was improved by the introduction of: customer service managers; suggestion boxes; client satisfaction surveys; and posters which displayed in bold text the cost of tests, expected turnaround time for tests and other vital information to patients. specimen rejection was reduced through three interventions: development of quality manuals that defined clearly the policies, processes and procedures for all the laboratories; development of a clinicians’ handbook that defined in detail to all clinicians, nurses and midwives the specimen acceptance and rejection criteria for the laboratory; and consistent training for both laboratory and non-laboratory staff. despite the improvements, challenges remain in the slmta laboratories. some qses, such as internal audit, occurrence management, corrective actions and management reviews, scored a median of ≤ 35% at the exit audit, indicating that these critical areas are not functioning adequately. these qses are common low-scoring areas, as reported in lesotho16 and other countries.18 in ghana, these deficiencies were largely a result of a lack of internal audit skills, inadequate staffing, limited experience of mentors, inefficient communication channels and institutional bottlenecks, such as administrative and procurement7 processes. as a result of these findings, training on internal audit has been conducted for all laboratory and quality managers, in collaboration with gsa. inadequate staffing levels and lack of motivation amongst some staff members, coupled with increased workloads, may have contributed to delays in the implementation of improvement projects. these issues are not easy to address, although advocacy continues for the prioritisation of laboratory human resource needs and identification of incentive options for overworked staff. many of the mentors used in these three cohorts had been trained recently and this was their first mentoring experience; whilst some settled in quickly, others needed more time to adjust to their new environment and tasks assigned. with time, these mentors have gained tremendous experience and are more effective in assisting laboratories to implement quality systems. once slmta roll-out is complete, these mentors will have skills that will make them valuable assets as quality managers. finally, inefficient communication and institutional bottlenecks remain a challenge; however at some facilities, especially in those where management was engaged in the slmta process, efforts are being made to simplify administrative processes and streamline communication channels. most countries implementing slmta have relied heavily on pepfar funding for implementation support. limited pepfar funding in ghana meant building local capacity that could be sustained and replicated as the slmta program grows. because slmta activities such as training, mentoring, monitoring and auditing are inter-related, we adopted a cross-training approach such that slmta trainers were also trained as mentors and certified as auditors, in an effort to maximise their potential. limitations of the study one limitation of this study was the absence of control laboratories that were followed over the same period of time. as a result we could not compare the improvements with laboratories that were not enrolled in slmta. whilst it is possible that some improvements observed in this programme were a result of secular influences or random factors, the magnitude of the observed impact strongly suggests a positive impact of the slmta programme. recommendations local partners may be considered for in-country programme implementation. however, capacity building of partner staff in administrative, technical and financial areas must be an integral part of the programme to ensure utmost compliance with reporting requirements. conclusion the slmta programme in ghana has shown substantial laboratory improvements as evidenced by progress in 15 laboratories, including four that have been audited officially by aslm. this experience demonstrates that local partners, when supported and managed adequately, can achieve great results at a reasonable cost. our programme also demonstrates the feasibility of indigenous capacity building and sustainability in an era of reduced pepfar funding, as countries are encouraged to do more with less. acknowledgements top ↑ we are grateful to the leadership of the moh, ghs and the national aids/sti control program. we appreciate the assistance of our implementing partner ghss and all participating laboratories. this activity has been supported by pepfar through cdc under the terms of cooperative agreement numbers ps002987 and ps002762. the findings and conclusions in this paper are those of the authors and do not necessarily represent the views of the cdc. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. disclaimer the findings and conclusions in this article are those of the authors and do not necessarily represent the official position of the cdc. authors’ contributions b.n. (cdc, ghana) was the activity manager and headed the writing of the manuscript; b.v.d.p. (ghss, ghana), v.b. (national aids control program), r.a. (national public health reference laboratory) and n.a.k. (ghss, ghana) conducted slmta trainings, mentoring and contributed to the writing of the manuscript; k.y., p.n.f. and e.t.l. (all cdc, atlanta) provided technical assistance and contributed to the writing of the manuscript; s.d. (ghss, ghana), p.a.n. (ghss, cameroon), n.a.a. (national aids control program), f.n.k. (cdc, cote d’ivoire) and c.j.i.w. (cdc, ghana) managed the programme and contributed to the writing of the manuscript. all authors have read and agreed to this manuscript. references top ↑ 1. world health organization’s regional office for africa. the maputo declaration on strengthening of laboratory systems [document on the internet]. c2008 [cited 2013 jan 02]. available from: http://www.who.int/diagnostics_laboratory/maputo-declaration_2008.pdf2. fonjungo pn, kebede y, arneson w, et al. preservice laboratory education strengthening enhances sustainable laboratory workforce in ethiopia. hum resour health. 2013;11:56. http://dx.doi.org/10.1186/1478-4491-11-56 3. world health organization. joint who–cdc conference on health laboratory quality systems. who/hse/ihr/lyo/2008.3 [document on the internet]. c2008 [cited 2013 jan 03]. available from: http://www.who.int/ihr/lyon/report20080409.pdf 4. gershy-damet gm, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. http://dx.doi.org/10.1309/ajcptuuc2v1wjqbm 5. yao k, luman et, slmta collaborating authors. evidence from 617 laboratories in 47 countries for slmta-driven improvement in quality management systems. afr j lab med. 2014;3(2), art. #262, 11 pages. http://dx.doi.org/10.4102/ajlm.v3i2.262 6. cobbina e, agbezudor jy, amuzu ps, et al. the current status and future of medical laboratory quality regulation and accreditation in ghana. accred qual assur. 2012;17:613–619. http://dx.doi.org/10.1007/s00769-012-0927-x 7. zeh ce, inzaule sc, magero vo, et al. field experience in implementing iso 15189 in kisumu, kenya. am j clin pathol. 2010;134(3):410–418. http://dx.doi.org/10.1309/ajcpzirkdus5lk2d 8. peter tf, rotz pd, blair dh, et al. impact of laboratory accreditation on patient care and the health system. am j clin pathol. 2010;134(4):550–555. http://dx.doi.org/10.1309/ajcph1skq1hnwghf 9. institute of medicine. evaluation of pepfar. washington, dc: the national academies press; 2013. 10. the president’s emergency plan for aids relief. fy 2014 country operational plan guidance. version 2 [document on the internet]. c2013 [cited 2013 dec 15]. available from: http://www.pepfar.gov/documents/organization/217765.pdf 11. world health organization’s regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation in the african region (with checklist) [document on the internet]. c2011 [cited 2013 jan]. available from: http://www.afro.who.int/index.php?option=com_docman&task=doc_download&gid=8642&itemid=2593 12. yao k, maruta t, luman et, nkengasong jn. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. 2014;3(2), art. #194, 8 pages. http://dx.doi.org/10.4102/ajlm.v3i2.194 13. maruta t, yao k, ndlovu n, moyo s. training-of-trainers: a strategy to build country capacity for slmta expansion and sustainability. afr j lab med. 2014;3(2), art. #196, 7 pages. http://dx.doi.org/10.4102/ajlm.v3i2.196 14. opio a, wafula w, amone j, et al. country leadership and policy are critical factors for implementing laboratory accreditation in developing countries: a study on uganda. am j clin pathol. 2010;134(3):381–387. http://dx.doi.org/10.1309/ajcp6kmotclisgj3 15. maruta t, rotz p, trevor p. setting up a structured laboratory mentoring programme. afr j lab med. 2013;2(1), art. #77, 7 pages. 16. mothabeng d, maruta t, lebina m, et al. strengthening laboratory management towards accreditation: the lesotho experience. afr j lab med. 2012;1(1), art. #9, 7 pages. 17. maruta t, motebang d, wanyoike j, et al. impact of mentorship on who-afro strengthening laboratory quality improvement process towards accreditation (slipta). afr j lab med. 2012;1(1), art. #6, 8 pages. 18. luman et, yao k, nkengasong jn. a comprehensive review of the slmta literature part 2: measuring success. afr j lab med. 2014;3(2), art. #276, 8 pages. http://dx.doi.org/10.4102/ajlm.v3i2.276 introduction clinical manifestations and disease spectrum patient outcomes laboratory diagnosis of corynebacterium diphtheriae barriers and practical implications paradigm shift in the laboratory approach to corynebacterium diphtheriae conclusion acknowledgements references about the author(s) kgaogelo r. masemola department of medical microbiology, national health laboratory service, durban, south africa school of laboratory medicine, college of health sciences, university of kwazulu-natal, durban, south africa department of medical microbiology, prince mshiyeni memorial hospital, durban, south africa citation masemola kr. from neglect to necessity: the case for routine work-up of nontoxigenic corynebacterium diphtheriae in clinical microbiology. afr j lab med. 2025;14(1), a2802. https://doi.org/10.4102/ajlm.v14i1.2802 opinion paper from neglect to necessity: the case for routine work-up of nontoxigenic corynebacterium diphtheriae in clinical microbiology kgaogelo r. masemola received: 03 mar. 2025; accepted: 04 aug. 2025; published: 17 sept. 2025 copyright: © 2025. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction toxigenic corynebacterium diphtheriae is well known for causing classical respiratory diphtheria, a life-threatening disease controlled by widespread toxoid vaccination.1,2 nontoxigenic c. diphtheriae strains (those lacking diphtheria toxin production), on the other hand, have received less attention.1,3 however, nontoxigenic c. diphtheriae infections are being increasingly reported globally in recent times.4 for years, clinical laboratories often dismissed corynebacteria from non-respiratory sites as ‘diphtheroids’ (skin or mucosal commensals/contaminants), and did not identify them to species level.5 this practice has led to under-recognition of infections by nontoxigenic c. diphtheriae.1 with improvements in identification methods and accumulated case reports, nontoxigenic c. diphtheriae is now understood as an emerging pathogen capable of causing invasive disease, even in immunised populations.5,6 the aim of this article is to underscore why ‘diphtheroids’ should no longer be routinely overlooked as insignificant commensals dismissed as contaminants, and to encourage laboratories and clinicians to recognise and appropriately manage nontoxigenic c. diphtheriae infections.5 global perspective nontoxigenic c. diphtheriae infections have been increasingly reported since the late 20th century.1,4 these infections often occur in socioeconomically marginalised groups and settings (table 1) where the organism can colonise damaged skin.7 in the 1980s and 1990s, outbreaks and case clusters were documented in various regions.4,8 by the mid-1990s, experts were already warning of the resurgence of c. diphtheriae and the rise of nontoxigenic c. diphtheriae in hospital settings.4 studies and surveillance reports have indicated that nontoxigenic c. diphtheriae infections are rising, often linked to intravenous drug use, homelessness, and skin infections.7,9 table 1: selected reported outbreaks and case series of nontoxigenic corynebacterium diphtheriae. african perspective african cases of nontoxigenic c. diphtheriae infections are less well documented, likely because of underreporting.1,2 despite this, there is growing recognition that these infections also occur in africa and can be severe.1 south africa has reported sporadic invasive cases over the years such as an outbreak of five fatal nontoxigenic endocarditis cases in 2021.1 unlike europe and north america, where nontoxigenic c. diphtheriae surveillance is more documented, african epidemiological data remain sparse. the true burden of disease is therefore unknown. clinical manifestations and disease spectrum nontoxigenic c. diphtheriae infections can present in a variety of ways. cutaneous diphtheria manifests as chronic, non-healing ulcers, or sores, which often serve as reservoirs for transmission.10,14 invasive bloodstream infection and endocarditis are the most severe form of nontoxigenic c. diphtheriae disease, with a high mortality rate.1,8 septic arthritis and osteomyelitis are less common, but are serious infections which often occur in the context of trauma or injection drug use.16 nontoxigenic strains do not cause classic respiratory diphtheria, but they can colonise the throat and cause sore throat or tonsillitis.6 other rare manifestations of nontoxigenic c. diphtheriae are pneumonia, urinary tract infections (especially in catheterised patients), and other localised infections.3,15 patient outcomes the mortality associated with nontoxigenic c. diphtheriae infections varies depending on the type and severity of the infection, as well as patient-specific factors.3 in a study in seattle, 9 (20%) of 44 patients with c. diphtheriae detections had death documented in the medical records.3 nontoxigenic c. diphtheriae infective endocarditis has a mortality rate ranging from 35% to 66%.3 in one cluster of endocarditis cases, the 30-day mortality rate was 80%.1 three of the four endocarditis patients in the seattle study died from endocarditis-related complications within 1 week of presentation.3 laboratory diagnosis of corynebacterium diphtheriae the laboratory work-up for c. diphtheriae is typically undertaken when there is a strong clinical suspicion of diphtheria or in the context of an outbreak. for example, during the 2015 kwazulu-natal diphtheria outbreak, laboratories in south africa were advised to incorporate selective media for the isolation of c. diphtheriae when processing throat swabs.2 however, in the absence of these defined scenarios, coryneform bacteria are seldom investigated beyond being classified as ‘diphtheroids’ or ‘coryneform bacteria’, with many isolates dismissed as contaminants.5 incidental identification of c. diphtheriae has increased with the widespread adoption of advanced technologies such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof ms), which provides rapid species-level identification.3 in cases where further work-up is pursued, laboratory protocols typically involve: gram staining to identify pleomorphic, gram-positive bacilli with a club-shaped morphology, often arranged in angular ‘chinese character’ formations.8,9,13 standard culture media, including 5% sheep’s blood agar, chocolate agar, and macconkey agar, are inoculated, with additional biochemical testing performed on suspect colonies.8,13 some laboratories opt to subculture onto selective media such as cystine tellurite blood agar or tinsdale medium, where c. diphtheriae produces characteristic black colonies with brown halos.17 genus and species identification is then confirmed using commercial systems such as analytical profile index assays or maldi-tof ms, with isolates in some countries being referred to national reference laboratories for confirmation and toxin determination.17 the modified elek test is used for phenotypic expression, while polymerase chain reaction detects the tox gene.17 traditionally, antimicrobial susceptibility testing is not routinely performed. the european committee on antimicrobial susceptibility testing body has published guidelines for susceptibility testing of c. diphtheria and c. ulcerans.18 barriers and practical implications while this article is framed as an advocacy argument for the routine species-level identification of corynebacteria, the author fully acknowledges that there are practical and cost considerations to be kept in mind. these include: lack of access to diagnostic advances such as maldi-tof ms, analytical profile index coryne kits and molecular diagnostics lack of microbiology reference laboratory support cost of selective culture media and confirmation testing many so-called diphtheroids may actually be unrelated skin or environmental bacteria, not true corynebacteria increased workload in clinical laboratories because of routine work-up of coryneform bacteria difficulty in ascertaining the clinical significance of corynebacteria in polymicrobial cultures lack of clinical information accompanying culture requests. these practical considerations are not insignificant, and any potential change must account for this. it is within this context that a shift in approach is being suggested. paradigm shift in the laboratory approach to corynebacterium diphtheriae a transition is required in the approach to coryneform bacteria within clinical microbiology laboratories. this shift must also include improved communication between clinicians and laboratory staff. this will ensure that relevant clinical context accompanies specimen submissions and therefore guide appropriate interpretation of findings. the identification of c. diphtheriae can no longer be relegated to incidental findings.5 if this remains the case, its epidemiology will remain poorly understood, and the true burden of disease will continue to be underestimated. to address this emerging threat, the following steps are critical: microbiology laboratories should have a heightened index of suspicion for nontoxigenic c. diphtheriae. public health agencies should integrate nontoxigenic c. diphtheriae into antimicrobial resistance surveillance programmes to track trends in antimicrobial resistance and virulence. investment in cost-effective diagnostic tools for resource-limited settings is essential for improving detection. the longstanding practice of routinely dismissing coryneform species-level identification must be reconsidered. to ensure accurate identification and reporting, laboratories should consider diagnostic workflows that incorporate selective media, species confirmation using maldi-tof ms or analytical profile index, and toxin determination through reference laboratory testing (table 2). table 2: recommendations for the identification, confirmation, and surveillance of corynebacterium diphtheriae. conclusion routine species-level identification of c. diphtheriae should be prioritised in laboratories, moving beyond classification of diphtheroids as mere contaminants. diagnostic workflows should incorporate selective media and advanced identification techniques to enhance detection accuracy. national reference laboratories should expand surveillance efforts to track nontoxigenic c. diphtheriae cases, enabling a better understanding of disease burden and informing public health policies. clinicians must remain aware of c. diphtheriae as a potential pathogen in bloodstream infections, endocarditis, and soft tissue infections to ensure timely diagnosis and appropriate management. further research should focus on exploring the molecular epidemiology of nontoxigenic c. diphtheriae and standardising detection and treatment protocols. acknowledgements the author expresses sincere gratitude to colleagues at the national health laboratory service, department of medical microbiology, kwazulu-natal, south africa, whose insights, and discussions provided the inspiration for this article. competing interests the author declares that he has no financial or personal relationships that may have inappropriately influenced him in writing this article. author’s contribution k.r.m. is the sole author of this article. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support the author received no financial support for the research, authorship, and/or publication of this article. data availability the authors confirm that the data supporting the findings of this study are available within the article and its references. disclaimer the views and opinions expressed in this article are those of the author and are the product of professional research. the article does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the author is responsible for this article’s results, findings, and content. references lovelock t, du plessis m, van der westhuizen c, et al. non-toxigenic corynebacterium diphtheriae endocarditis: a cluster of five cases. s afr j infect dis. 2024;39(1):539. https://doi.org/10.4102/sajid.v39i1.539 du plessis m, wolter n, allam m, et al. molecular characterization of corynebacterium diphtheriae outbreak isolates, south africa, march–june 2015. emerg infect dis. 2017;23(8):1308–1315. https://doi.org/10.3201/eid2308.162039 karmarkar en, fitzpatrick t, himmelfarb st, et al. cluster of nontoxigenic corynebacterium diphtheriae infective endocarditis and rising background c. diphtheriae cases–seattle, washington, 2020–2023. clin infect dis. 2024;78(5):1214–1221. https://doi.org/10.1093/cid/ciae094 reacher m, ramsay m, white j, et al. nontoxigenic corynebacterium diphtheriae: an emerging pathogen in england and wales? emerg infect dis. 2000;6(6):640–645. https://doi.org/10.3201/eid0606.000614 el-hazmi mm. late-onset prosthetic valve endocarditis caused by nontoxigenic corynebacterium diphtheriae. j infect dev ctries. 2015;9(8):905–909. https://doi.org/10.3855/jidc.5976 zou j, chorlton sd, romney mg, et al. phenotypic and genotypic correlates of penicillin susceptibility in nontoxigenic corynebacterium diphtheriae, british columbia, canada, 2015–2018. emerg infect dis. 2020;26:97–103. https://doi.org/10.3201/eid2601.191241 dangel a, berger a, konrad r, bischoff h, sing a. geographically diverse clusters of nontoxigenic corynebacterium diphtheriae infection, germany, 2016–2017. emerg infect dis. 2018;24(7):1239–1245. https://doi.org/10.3201/eid2407.172026 romney mg, roscoe dl, bernard k, lai s, efstratiou a, clarke am. emergence of an invasive clone of nontoxigenic corynebacterium diphtheriae in the urban poor population of vancouver, canada. j clin microbiol. 2006;44(5):1625–1629. https://doi.org/10.1128/jcm.44.5.1625-1629.2006 wojewoda cm, koval ce, wilson da, chakos mh, harrington sm. bloodstream infection caused by nontoxigenic corynebacterium diphtheriae in an immunocompromised host in the united states. j clin microbiol. 2012;50(6):2170–2172. https://doi.org/10.1128/jcm.00237-12 harnisch jp, tronca e, nolan cm, turck m, holmes kk. diphtheria among alcoholic urban adults. a decade of experience in seattle. ann intern med. 1989;111(1):71–82. https://doi.org/10.7326/0003-4819-111-1-71 gubler j, huber-schneider c, gruner e, altwegg m. an outbreak of nontoxigenic corynebacterium diphtheriae infection: single bacterial clone causing invasive infection among swiss drug users. clin infect dis. 1998;27(5):1295–1298. https://doi.org/10.1086/514997 patey o, bimet f, riegel p, et al. clinical and molecular study of corynebacterium diphtheriae systemic infections in france. coryne study group. j clin microbiol. 1997;35(2):441–445. https://doi.org/10.1128/jcm.35.2.441-445.1997 tiley sm, kociuba kr, heron lg, munro r. infective endocarditis due to nontoxigenic corynebacterium diphtheriae: report of seven cases and review. clin infect dis. 1993;16(2):271–275. https://doi.org/10.1093/clind/16.2.271 zasada aa. nontoxigenic highly pathogenic clone of corynebacterium diphtheriae, poland, 2004–2012. emerg infect dis. 2013;19(11):1870–1872. https://doi.org/10.3201/eid1911.130297 xie ag, yomogida k, berry i, et al. notes from the field: increase in nontoxigenic corynebacterium diphtheriae – washington, 2018–2023. mmwr morb mortal wkly rep. 2024;73(17):405–407. https://doi.org/10.15585/mmwr.mm7317a4 funke g, altwegg m, frommelt l, von graevenitz a. emergence of related nontoxigenic corynebacterium diphtheriae biotype mitis strains in western europe. emerg infect dis. 1999;5(3):477–480. https://doi.org/10.3201/eid0503.990326 leber a. respiratory tract cultures. in: church dl, editor. clinical microbiology procedures handbook. 4th ed. washington dc: asm press, 2016; p. 3.11.1.1–3.11.9.4. the european committee on antimicrobial susceptibility testing. breakpoint tables for interpretation of mics and zone diameters [homepage on the internet]. 2025; version 15.0 [cited 2025 feb 27]. available from: https://www.eucast.org/clinical_breakpoints abstract introduction methods results discussion acknowledgements references about the author(s) zoliswa mayekiso department of laboratory medicine & pathology, faculty of health sciences, walter sisulu university, mthatha, south africa department of public health, faculty of health sciences, walter sisulu university, mthatha, south africa kelechi e. oladimeji ezintsha, faculty of health sciences, university of the witwatersrand, johannesburg, south africa guillermo a. pulido estrada department of public health, faculty of health sciences, walter sisulu university, mthatha, south africa charles hongoro developmental, capable and ethical state division, human sciences research council (hsrc), pretoria, south africa school of health systems and public health, university of pretoria, pretoria, south africa teke r. apalata department of laboratory medicine & pathology, faculty of health sciences, walter sisulu university, mthatha, south africa citation mayekiso z, oladimeji ke, estrada gap, hongoro c, apalata tr. impact of novel software on laboratory expenditure at an academic hospital in south africa. afr j lab med. 2023;12(1), a2159. https://doi.org/10.4102/ajlm.v12i1.2159 note: additional supporting information may be found in the online version of this article as online supplementary document 1. original research impact of novel software on laboratory expenditure at an academic hospital in south africa zoliswa mayekiso, kelechi e. oladimeji, guillermo a. pulido estrada, charles hongoro, teke r. apalata received: 12 jan. 2023; accepted: 28 aug. 2023; published: 30 nov. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: countries across the globe report an increase in expenditure associated with medical laboratory testing. in 2020, the united states department of health and human services reported that laboratory test expenditures increased by $459 million us dollars (usd) from $7.1 billion usd in 2018. in south africa, laboratory testing expenditure in the public sector increased from $415 million usd in 2014 to $723 million usd in 2021. objective: this study aimed to evaluate the impact of an innovative software, electronic gatekeeping (egk), on medical laboratory test expenditures at nelson mandela academic hospital, in the eastern cape, south africa. methods: in this cross-sectional study, an interrupted time series analysis technique was used to evaluate trends in expenditure during a 48-month study period. to measure the impact of egk on laboratory expenditure, we analysed laboratory expenditure over two study periods: a period of 24 months occurring before egk implementation (01 june 2013 to 31 may 2015) and a period of 24 months occurring during egk implementation (01 june 2015 to 30 may 2017). results: there was a significant reduction (211 928 fewer tests) in the number of tests performed during the intervention (434 790) compared to before the intervention (646 718). laboratory test expenditure was $1 663 756.72 usd before the intervention period and $1 105 036.88 usd during the intervention period, demonstrating a cost savings of $558 719.84 usd. conclusion: electronic gatekeeping is a cost-effective intervention for managing medical laboratory expenditures. we recommend that the health sector scale up this intervention nationally. what this study adds: using an interrupted time series interval, the authors determined that egk is a cost-effective intervention for managing medical laboratory expenditures at a tertiary hospital. this study’s findings can promote and contribute to improved laboratory systems and test investigations. keywords: electronic gatekeeping; medical laboratory expenditure; interrupted time series analysis; cost-effectiveness; intervention; rural; academic hospital; south africa. introduction on a global scale, countries report an increase in the cost of healthcare and medical laboratory testing.1 in 2020, the united states department of health and human services reported an increase of $459 million united states dollars (usd) in laboratory test expenditure in 2018 – an upswing from the previous year’s expenditure of $7.1 billion usd. the primary sources of the increased spending were identified as an increase in the number of genetic tests requested, the cessation of discounts for specific chemistry tests, and a change in payment rates.2 similarly, in the united kingdom, the national health service reported an increase in expenditure from £4 billion british pounds to £5 billion british pounds within a five-year period.3 canada has also reported a similar trend in medical laboratory expenditure, with an annual increase of 5.3% attributable to increased laboratory testing based on unnecessary and repeated medical laboratory investigations as well as a hesitancy to develop advanced technological and digital interventions.4 in tandem with the increases in medical laboratory expenditures reported by these developed countries, the south africa national health laboratory service (nhls) reported a 45% increase between 2010/2011 and 2013/2014, totalling r4.5 billion south african rand.5 laboratory expenditure rose to r7.1 billion in 2016/2017,6,7,8,9 r8.2 billion in 2017/2018, and r10.7 billion in 2020/2021.10,11,12,13 to address these increasing rates of expenditure by the medical laboratory sector, healthcare settings, specifically in the developed parts of the world, have considered various approaches. strategies that have been implemented include but are not confined to computerised or electronic order entry systems,14 laboratory test profiles,15 education16 and minimum re-testing intervals.17 there is variable evidence of the effectiveness of these strategies as they are effective in some contexts and ineffective in others.18 in addition, some hospital settings have implemented a single strategy while others have implemented a multi-pronged approach. except for south africa, most african countries lack published data on medical laboratory expenditures that could facilitate the implementation of cost-effective strategies to manage the demand for laboratory test investigations on the african continent. furthermore, the published reports in south africa are insufficient to establish clear patterns to guide the implementation of effective interventions. in the south african public health sector, the nhls and department of health developed a financing mechanism that levies a fee for its services: the fee for service payment model. this model has been identified as a contributor to the escalating costs in the healthcare system and a barrier to integrated care by promoting fragmentation and higher spending.19 to overcome this challenge, in 2010, a demand management system known as electronic gatekeeping (egk) was introduced and piloted at a tertiary academic hospital in the western cape. in 2015, the intervention was rolled out in other provinces in the country, including four tertiary academic hospitals in the eastern cape. thus far, only two studies conducted in urban tertiary academic hospitals have attempted to provide evidence of the effectiveness of egk as a demand management system. one of these studies20 concluded that the egk intervention was an effective method of demand management, while the other study21 found the intervention not to have a substantial effect on the way clinicians request laboratory investigations. given the varying findings from the only two published studies on the effectiveness of egk as an intervention in south africa, this study was conducted to evaluate the impact of egk on laboratory expenditure at a tertiary hospital in the eastern cape, south africa. methods ethical considerations the walter sisulu university human research ethics and biosafety committee in south africa reviewed and approved this study (062/2019). the study only involved the use of secondary data on expenditure, primarily collected electronically by the nhls and supplied to nmah. patient consent was not required and thus waived by the ethics committee because data were secondary and lacked any identifiers linking to any patient. data protection in place included first obtaining necessary approvals from the ethics committee and hospital management, and ensuring that the data remained anonymous and were only accessed by the researcher. study design this cross-sectional study used an interrupted time series analysis (itsa) to determine the impact of the egk intervention on laboratory expenditure, comparing the period before (24 months from 01 june 2013 to 31 may 2015) and during (24 months from 01 june 2015 to 31 may 2017) the egk intervention. the egk is a demand management system embedded in a laboratory interface system. the system uses a decision tree as an analytical tool to approve or reject laboratory tests requested by clinicians for patients based on the protocols for patient management devised by hospital medical specialists and consultants22 (figure 1). this decision tree is simple and transparent, representing a series of actions and possible outcomes that can help determine whether a decision should be made and reveal the optimum choice for each scenario.23 before the egk intervention, there was no other intervention in place to improve laboratory expenditure in the country. figure 1: decision tree applied using the electronic gatekeeping demand management system for the approval of electronic gatekeeping-subjected laboratory tests that were rejected at nelson mandela academic hospital, south africa, between 01 june 2015 and 31 may 2017. study setting this study was conducted at the nelson mandela academic hospital (nmah), which is situated in mthatha, a small rural town in the or tambo district municipality in the eastern cape province of south africa. in 2019, the district was estimated to have a population of 1 514 306 million.24 the nmah has three academic arms: patient management, medical training and research, with the nhls pathology laboratory attached to it. the nmah is the region’s referral hospital, which provides specialist medical services and has a 576-bed capacity with inpatient wards, outpatient clinics and theatres.25 study data and variables the study included 28 conveniently sampled laboratory tests that were subjected to egk. secondary data on the number, type and costs of tests performed before and during egk intervention were obtained from nhls monthly reports, which were generated electronically and provided on spreadsheets using microsoft excel 2016 (microsoft corporation, redmond, washington, united states). these reports are routinely supplied by the nhls to nmah to monitor and evaluate laboratory accounts. the variables in the reports include the type of tests, number of tests, expenditure on the tests, and dates the tests were performed (month and year). economic analysis for this study’s economic evaluation, only the costs of the tests analysed over the four-year period were included in the main analysis. all monetary values were converted from south african rand to usd using a conversion rate of r14.92 to $1.00 usd as per the south african reserve bank exchange rate on 17 march 2021.26 statistical analysis statistical analysis involved the use of an itsa to analyse changes in expenditure trends. the itsa, also termed a regression model, is most suited to comparing the impact of interventions before, during and after the event27,28,29 (figure 2). this allows for the evaluation of interventions that have been rolled out at well-defined time points or at well-spaced intervals before, during and after interventions.30,31 statistical analyses were conducted using stata version 14.2 (statacorp, college station, texas, united states). newey-west standard errors were reported to account for autocorrelation. we assumed that the outcome variable would not be altered without the egk intervention, that is, the pre-intervention trend would continue unchanged. figure 2: interrupted time series analysis model equation used to estimate the effects of electronic gatekeeping intervention on expenditure at nelson mandela academic hospital, south africa, between 01 june 2015 and 31 may 2017. results regression analysis on expenditure before and during electronic gatekeeping intervention of the 28 tests analysed, 20 tests demonstrated an increasing expenditure trend before the implementation of the intervention, while the remaining eight tests showed a decreasing expenditure trend. these eight tests were phosphorus, cholesterol, triglycerides, high-density lipoprotein, hepatitis a immunoglobulin m, hepatitis b surface antigen, hepatitis b surface antibody, and hepatitis c antibody tests. in the first month of the intervention, 27 of the 28 tests demonstrated a decreasing expenditure trend, with only hepatitis b surface antibody demonstrating an increasing trend. in the months following the first month of the intervention – june 2015 to may 2017 – the trajectory of the expenditure for 64% (18/28) of the tests continued a decreasing trend, whereas the remaining 36% (10/28) continued an increasing trend. these tests were glycated haemoglobin, phosphorus, cholesterol, triglycerides, c-reactive protein, thyroid stimulating hormone, hepatitis a immunoglobulin m, hepatitis b surface antigen, hepatitis b surface antibody, and hepatitis c antibody. for ease of interpretation of the results, all tests presented hereafter are categorised according to their clinical significance. evaluation of trends in expenditure before and during electronic gatekeeping intervention kidney function tests – urea and creatinine the expenditure on kidney function-related tests (creatinine and urea) demonstrated a significant monthly increase prior to the intervention. in the first month of the intervention, the expenditure decreased significantly for creatinine ($856.95 usd reduction, p = 0.002) and urea ($817.60 usd reduction, p = 0.002). this was followed by an insignificant decreasing trend in monthly expenditure for both tests. after the first month of the intervention, the expenditures for both creatinine ($36.29 usd increase, p = 0.007) and urea ($32.11 usd increase, p = 0.011) were estimated to increase significantly every month (online supplementary figure 1 and online supplementary table 1). bone and mineral metabolism-related tests – calcium, magnesium and phosphorus before the intervention, the expenditures on calcium and magnesium tests demonstrated statistically insignificant increases every month, while expenditure on phosphate tests decreased insignificantly. in the first month of the intervention, there was a notable decrease in expenditure on all these three tests, with a statistically significant decrease in expenditure on calcium tests ($667.38 usd decrease, p < 0.001) and magnesium tests ($754.78 usd decrease, p < 0.001). expenditure on phosphate tests decreased ($408.63 usd decrease, p = 0.178) as well but not significantly. after the first month of the intervention, expenditures on all three tests were estimated to decrease insignificantly every month for calcium ($7.25 usd decrease, p = 0.139), magnesium ($4.13 usd decrease, p = 0.38) and phosphate ($4.46 usd decrease, p = 0.290) (online supplementary figure 2 and online supplementary table 2). indicator test for long-term glycaemic control – glycated haemoglobin there was a statistically insignificant increase in monthly expenditure for glycated haemoglobin tests before the intervention. in the first month of the intervention, a statistically significant increase in expenditure was observed in the first month of the intervention, a statistically significant increase in expenditure was observed ($13.50 usd increase, p < 0.001). after the first month of the intervention, the monthly test expenditure was estimated to increase significantly ($13.62 usd increase, p = 0.00). (online supplementary table 3 and online supplementary figure 3). figure 3: expenditure trends on tests conducted at the nelson mandela academic hospital, south africa, before (01 june 2013 to 31 may 2015) and during (01 june 2015 to 31 may 2017) electronic gatekeeping intervention. the graph shows a clear reduction in expenditure during the electronic gatekeeping intervention period compared to the period before the intervention. liver function-related tests before the intervention, expenditures on total protein and albumin tests increased insignificantly every month. in the first month of the intervention, both test expenditures demonstrated a significant decrease (total protein: $914.47 usd decrease, p < 0.001; albumin: $1813.12 usd decrease, p < 0.001). after the first month of the intervention, expenditure for total protein tests was estimated to decrease insignificantly ($3.98 usd decrease, p = 0.522) and expenditure for albumin was estimated to decrease significantly ($30.71 usd decrease, p = 0.026) (supplementary figure 4 and supplementary table 4). total expenditure for total bilirubin and conjugated bilirubin tests increased insignificantly every month before the intervention. in the first month of the intervention, expenditure on both tests decreased significantly (total bilirubin: $1259.99 usd decrease, p < 0.001; conjugated bilirubin: $810.57 usd decrease, p < 0.001). after the first month of the intervention, both test expenditures were estimated to decrease monthly (total bilirubin: $5.07 usd decrease, p = 0.596; conjugated bilirubin: $26.62 usd decrease, p = 0.002) (online supplementary figure 5 and online supplementary table 5). expenditure on both alanine transaminase and aspartate aminotransferase tests increased insignificantly every month before the intervention. in the first month of the intervention, expenditure for both tests demonstrated a statistically significant decrease (alanine transaminase: $1283.30 usd decrease, p < 0.001; aspartate aminotransferase: $1288.06 usd decrease, p < 0.001). after the first month of the intervention, the alanine transaminase expenditure increased monthly but not significantly ($2.74 usd increase, p = 0.683), whereas the aspartate aminotransferase expenditure decreased significantly ($32.08 usd decrease, p = 0.004) (online supplementary figure 6 and online supplementary table 6). in the first month of the intervention, there were significant decreases in expenditure for both the alkaline phosphatase ($1183.30 usd decrease, p < 0.001) and gamma-glutamyl transferase ($1272.27 usd decrease; p < 0.001) tests. after the first month of the intervention, the alkaline phosphatase expenditure was estimated to decrease significantly monthly ($37.27 usd decrease, p = 0.001) and gamma-glutamyl transferase expenditure was estimated to decrease insignificantly ($3.74 usd decrease, p = 0.704) (online supplementary figure 7 and online supplementary table 7). inflammatory marker-related tests – c-reactive protein in the first month of the intervention, the c-reactive protein expenditure decreased significantly ($1784.05 usd decrease, p < 0.001). however, after the first month of the intervention, the c-reactive protein expenditure was estimated to increase significantly every month ($89.26 usd increase, p = 0.000) (online supplementary figure 8 and online supplementary table 8). lipidaemia-related tests – cholesterol, triglycerides and high-density lipoprotein expenditure on all three lipidaemia-related tests decreased insignificantly every month before the intervention. in the first month of the intervention, all three tests demonstrated significant decreases in expenditure (cholesterol: $116.26 usd decrease, p < 0.001; triglycerides: $116.33 usd decrease, p = 0.026; high-density lipoprotein: $276.38 usd decrease, p < 0.001). after the first month of the intervention, expenditure on cholesterol ($0.92 usd increase, p = 0.658) and triglycerides ($0.34 usd increase, p = 0.937) were estimated to increase monthly but not significantly. the expenditure for high-density lipoprotein was estimated to decrease insignificantly ($4.55 usd decrease, p = 0.093) (online supplementary figure 9 and online supplementary table 9). prostate cancer-related tests – prostate-specific antigen expenditure for the prostate-specific antigen test increased significantly every month before the intervention. in the first month of the intervention, there was a significant decrease ($278.20 usd decrease, p < 0.001) in expenditure for this test. it was estimated that after the first month of the intervention, the monthly expenditure for prostate-specific antigen would increase significantly ($8.05 usd increase, p = 0.005) (online supplementary figure 10 and online supplementary table 10). thyroid function-related tests – thyroid stimulating hormone, thyroxine and triiodothyronine we noted insignificant increasing trends in monthly expenditures for the thyroid stimulating hormone and thyroxine tests before the implementation of the intervention. during the same period, the monthly expenditure for triiodothyronine increased significantly. in the first month of the intervention, expenditure decreased significantly for thyroid stimulating hormone ($500.21 usd decrease, p = 0.041) and triiodothyronine ($506.62 decrease, p < 0.001), whereas for thyroxine, expenditure decreased insignificantly ($352.00 usd decrease, p = 0.053). after the first month of the intervention, the thyroid stimulating hormone expenditure was estimated to increase significantly ($26.26 usd increase, p = 0.000), whereas the thyroxine expenditure was estimated to decrease insignificantly ($7.06 usd decrease, p = 0.114). during the same period, the triiodothyronine expenditure was estimated to decrease significantly ($20.32 usd decrease, p = 0.001) (online supplementary figure 11 and online supplementary table 11). anaemia, leukaemia, and abnormal bleeding-related tests – full blood count and differential count expenditures on anaemia, leukaemia, and abnormal bleeding-related tests increased every month before the intervention. the observed increase was statistically significant for full blood count tests and insignificant for differential count tests. in the first month of the intervention, there were significant reductions in expenditures for both tests (full blood count: $3518.21 usd decrease, p < 0.001; and differential count: $5103.73 usd decrease, p < 0.001). after the first month of the intervention, both test expenditures were estimated to increase insignificantly (full blood count: $50.89 usd increase, p = 0.264; differential count: $3.23 usd increase, p = 0.848) (online supplementary figure 12 and online supplementary table 12). liver inflammation-related tests – hepatitis a glycated haemoglobin, hepatitis b surface antigen, hepatitis b surface antibody and hepatitis c antibody before the intervention, monthly expenditure for the ham and hepatitis c antibody tests demonstrated significant decreasing trends, whereas the expenditure for hepatitis b surface antigen and hepatitis b surface antibody showed insignificant decreasing trends. in the first month of the intervention, the monthly expenditure decreased insignificantly for ham ($238.47 usd decrease, p = 0.673), hepatitis b surface antigen ($177.74 usd decrease, p = 0.281) and hepatitis c antibody ($10.58 usd decrease, p = 0.776). in the same period, hepatitis b surface antibody expenditure showed a statistically insignificant increase ($52.07 usd increase, p = 0.776). after the first month of the intervention, expenditure for ham, hepatitis b surface antibody, and hepatitis c antibody were estimated to decrease significantly every month (ham: $14.16 usd decrease, p = 0.000; hepatitis b surface antibody: $19.74 usd decrease, p = 0.023; hepatitis c antibody: $15.31 usd decrease, p = 0.007) (online supplementary figure 13 and online supplementary table 13). impact of electronic gatekeeping intervention on patient volumes more tests were conducted before the egk intervention (646 718) than during the intervention (434 790). before the intervention, the expenditure trend showed irregular fluctuations, whereas during the intervention, the expenditure trend was somewhat more stable (figure 3). total expenditure was also greater before the intervention ($1 663 756.72) than during the intervention ($1 105 036.88). in the period before the intervention, the lowest number of tests conducted per month was 12 258, for which the expenditure was $30 285.50. during the same period, the highest number of tests conducted per month was 36 962, for which the expenditure was $96 054.60. in the intervention period, the lowest number of tests conducted per month was 15 215, for which the expenditure was $41 306.86. during the same period, the highest number of tests conducted was 22 696, for which the expenditure was $55 701.49 (online supplementary table 14). we also conducted analyses to assess the possible impact of patient volumes on laboratory expenditure. we noted that 348 804 patients were admitted to the facility in the 24 months during the intervention, while slightly more than the 336 283 were admitted in the 24 months before the intervention. similarly, the outpatient headcount during the 24-month intervention was 339 711, which was slightly more than the 258 267 outpatients received at the facility during the 24 months before the intervention. discussion analysis of the findings revealed that the introduction and implementation of egk at nmah worked well to manage demand for laboratory tests when compared to the periods when there was no egk implementation. this is better understood when observing the logistic regression analysis of the test expenditures before, during and after the egk intervention periods. in the period before the intervention, majority of the tests showed an increase in expenditure patterns. in the period during the intervention, the majority of tests showed a decrease in expenditure patterns, and post intervention, the linear trend assumed that although the overall expenditure will continue to decrease during this period, expenditure may increase for some of the tests that showed a decrease in expenditure during the intervention period. in the regression analysis of laboratory expenditure in the period before the intervention, 71% (20/28) of the tests analysed demonstrated an increasing expenditure trend, six of which showed a statistically significant increase in expenditure, including creatinine, urea, c-reactive protein, prostate-specific antigen, triiodothyronine and full blood count. the remaining 29% (8/28) of the tests showed a decreasing expenditure trend, with seven of the eight showing a statistically insignificant decrease, including phosphate, cholesterol, triglycerides, high-density protein, hepatitis b surface antigen, hepatitis b surface antigen, hepatitis b surface antibody, and hepatitis c antibody. during the egk intervention, 96.4% (27/28) of the tests demonstrated decreasing expenditure trends, which were attributed to or associated with the implementation of the intervention. only one of the 28 tests (3.6%) – hepatitis b surface antigen – demonstrated an increasing expenditure trend during this period. these findings demonstrate the importance of interventions to curtail unnecessary testing and reduce expenditure. in the months following the intervention, 64% (18/28) of the tests continued a decreasing expenditure trend, while 36% (10/28) showed an increased expenditure trend. it was noted that one of the factors that contributed to the observed change (increase) in expenditure was that over time, clinicians found ways to circumvent the rules of egk intervention to access the tests they desired. to ensure the robustness of the intervention, this identified vulnerability needs to be addressed by the stakeholders. further analyses of the expenditure trends after the intervention revealed that expenditure was likely to decrease for 57% (16/28) of the tests (25% significantly and 32% insignificantly) and to increase for the remaining 43% (12/28) (21.5% significantly and 21.5% insignificantly). these results and projections highlight the magnitude of unnecessary laboratory testing commonly performed in the absence of an intervention such as egk. in a retrospective study conducted at a cape town hospital in south africa to determine the number of chemistry tests rejected by egk and subsequently restored over a six-month period, it was shown that out of 68 480 tests that were subjected to egk, only 6.7% were rejected, while 14.7% of these were restored when the clinician made that request.20 the authors in that study concluded that the majority of the rejected tests were unnecessary and real cost savings were made, applauding egk as a simple, effective and sustainable method of demand management. they also showed that the egk rejections did not have a negative impact on patient care. our results are in concordance with these findings and demonstrated that egk is an effective method of reducing unnecessary laboratory test requests to save costs. however, another 22-month retrospective study carried out at a pretoria hospital in south africa to determine the number of chemistry tests rejected by egk found that of the 1 445 782 tests that were subjected to egk, only 3.33% were rejected, while 1.3% of these were restored when a request was made by a clinician. the authors reported the cost savings as ‘modest’, concluding that egk does not have a substantial effect on the clinician test requesting pattern.21 when we evaluated trends in expenditure and test volumes before and during the intervention, we noted that more money was spent on laboratory investigations before the intervention compared to the intervention period. there were more tests analysed before the intervention compared to during the intervention. it is important to note that in some instances, increases in expenditure for certain tests during the egk intervention period could also be attributed to inflation. on evaluation of the impact of patient volumes on laboratory expenditure, we noted that there were more patients at the hospital during the intervention period compared to before the intervention. since test volumes and expenditures remained lower during the intervention compared to the period before the intervention, we concluded that the increase in inpatient and outpatient days during the 24-month period of the egk intervention did not affect the test volumes or expenditure during this period. the lower test volumes and expenditure may thus be attributed to the implementation of egk at the facility. we further state that the robust results provided by our estimates through itsa may provide foundations for public policy decision-making within the south african national health system, especially regarding interventions with significant potential for the reduction of laboratory expenditure related to unnecessary testing. limitations the study was limited to nmah and the results may not be representative of other health facilities that might have implemented egk in south africa. to the best of our knowledge, this is the first egk study in south africa to use an itsa design to assess the impact of the intervention. as opposed to the other two published reports on egk effectiveness in south africa,20,21 which only evaluated the costs and effectiveness of the egk intervention based on rejected tests, our study design had the advantage of assessing trends in longitudinal data and thus revealing pre-intervention trends through regression modelling.28,29,32 given this scarcity of evidence in the study setting and geographical region, as well as the uniqueness of our methodological approach, our findings could not be compared to other studies for similarities or differences. our study did not also investigate the clinical impact of egk rejections on patient care. conclusion by assessing expenditure trends before and during the egk intervention, we have established that egk is a cost-saving and effective intervention for managing the demand for laboratory testing. the results obtained in this study highlight the importance of interventions to restrict unnecessary medical laboratory testing, which would reduce wastage and associated costs. this study is unique and has provided strong evidence in support of introducing and implementing egk in laboratories as a means for the department of health to maintain affordable and sustainable costs of laboratory testing. acknowledgements the authors would like to thank indiphile qubule for designing figure 1. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions z.m. and t.r.a conceptualised and implemented the study. k.e.o. and g.a.p.e. reviewed and provided analysis. c.h. provided the health economic expertise to the data curation and analysis. t.r.a. supervised the research and k.e.o co-supervised the research. z.m wrote the first draft, reviewed by k.e.o and all authors reviewed and approved final version of the article. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability data supporting the findings of this study are available from the corresponding author, t.r.a. disclaimer the views expressed in this manuscript are those of the authors and not those of the institutions they are affiliated with. references konger lr, ndekwe p, jones g, et al. reduction in unnecessary clinical laboratory testing through utilization management at a us government veteran’s affairs hospital. am j clin pathol. 2016;145(3):355–364. https://doi.org/10.1093/ajcp/aqv092 u.s.department of health and human services. medicare laboratory test 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2015;35:1–4. https://doi.org/10.1136/bmj.h2750 article information authors: moustapha mbow1,3 ndèye s.s. santos1 makhtar camara1 awa ba1 aliou niang2 géraldine daneau3 djibril wade3 abdou a. diallo1 maxim toupane1 maïmouna diakhaté1 nafissatou lèye1 papa a. diaw1 souleymane mboup1 luc kestens3 tandakha n. dieye1 affiliations: 1laboratory of bacteriology and virology, aristide le dantec university hospital, dakar, sénégal2department of pneumo-phthisiology, fann university hospital, dakar, sénégal 3institute of tropical medicine, unit of immunology, department of biomedical sciences, antwerp, belgium correspondence to: moustapha mbow postal address: 30, avenue pasteur bp 7325, dakar, sénégal dates: received: 30 june 2012 accepted: 09 apr. 2013 published: 02 sept. 2013 how to cite this article: mbow m, santos nss, camara m, et al. hiv and tuberculosis co-infection impacts t-cell activation markers but not the numbers subset of regulatory t-cells in hiv-1 infected patients. afr j lab med. 2013;2(1), art. #41, 8 pages. http://dx.doi.org/10.4102/ ajlm.v2i1.76 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. hiv and tuberculosis co-infection impacts t-cell activation markers but not the numbers subset of regulatory t-cells in hiv-1 infected patients in this original research... open access • abstract • introduction • research method and design    • study population and diagnostic tools    • cd4+ and cd8+ t-cell counts    • analysis of regulatory and activation markers by flow cytometry    • statistical analysis • results    • study subjects    • cd4+ and cd8+ t-cell counts    • cd38 and hla-dr expression on t-cells    • impact of tuberculosis on expression of tregs in hiv-infected patients    • correlation between cd4+ t-cell counts and treg cells and activation markers • ethical considerations    • potential benefits and hazards    • recruitment procedures    • informed consent    • data protection • trustworthiness    • reliability    • validity • discussion • limitations of the study • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: tuberculosis (tb) has been shown to accelerate the clinical course of hiv infection, but the mechanisms by which this occurs are not well understood. regulatory t-cells (tregs) are known to dampen hyperactivation of the immune cells, but it remains unclear whether hyperactivation of t-cells in hiv infection is associated with a decrease of tregs and what the effect mycobacterium tuberculosis (mtb) co-infection has on t-cell activation and tregs. objectives: in this study, we aim to evaluate whether active tb is associated with the increased expression of t-cell activation markers and reduced number of treg cells in hiv-1-infected patients. methods: this study was conducted on 69 subjects consisting of 20 hiv-infected patients, 20 hiv and mtb co-infected patients, 19 mtb-infected patients and 10 uninfected control subjects negative for both mtb and hiv. the frequencies of t-cell activation markers (cd38 and hla-dr) and treg cells (cd4+cd25+cd127-) were measured by flow cytometry. results: significantly higher expression of cd38 and hla-dr on cd4+ and cd8+ t-cells was found in mtb and hiv co-infected patients compared with hiv-infected patients. however, no significant difference in the percentage of treg cells was reported between hiv patients with tb and those without. the study also showed a negative correlation between regulatory t-cells frequency and cd4+ t-cell counts. conclusion: these results suggest that tb enhances the expression of peripheral t-cell activation markers during hiv infection, whilst having no impact on the percentages of treg cells. introduction top ↑ hiv infection and tuberculosis (tb) are serious public health problems, especially in africa. co-infection with mycobacterium tuberculosis (mtb) and hiv leads to alteration in the clinical course of both diseases.1 since the 1990s, the global burden of tb has been markedly exacerbated by hiv, which is one of the leading causes of the resurgence of tb in developed countries.2 chronic activation, dysfunction of the immune system and loss of cd4+ t-cells3 favour the emergence of active tb in hiv patients.4 reciprocally, the immune response of the host to tb has been shown to increase hiv-1 replication4,5 and to accelerate the natural progression to aids.6,7 the proportional expression of the hla-dr activation marker has been shown to increase in tb and/or hiv dual-infected patients as compared with tb single-infected patients.8 today, tb remains one of the leading causes of death amongst hiv-positive patients.1the mechanism by which tb accelerates the clinical course of hiv infection remains unclear. however, accumulating data suggest that t-cell activation in hiv-infected patients is a predictor for clinical disease progression9,10,11,12. cd38 and hla-dr expression levels on cd4+ and cd8+ t-cells, both markers of t-cell activation, are increased in hiv-infected patients and their levels of expression are associated with the hiv disease stage in untreated patients.13 furthermore, elevated cd38 expression on cd8+ t-cells is a strong marker for the risk of chronic hiv disease progression to aids and, eventually, death.14 there is some evidence that aberrant immune activation, at least in part, leads to t-cell depletion through activation-induced cell death or enhanced hiv replication.15,16 regulatory t-cells (tregs) influence the outcome of various infections17 and have been shown to be involved in the regulation of the immune response during hiv infection.18,19 whilst tregs suppressing hyperactivation may slow disease progression, their expansion has been associated with disease progression20,21 and a worsening of the immune deficiency that is characteristic of hiv infection.22,23,24 several studies have reported conflicting results as levels of treg cells were found to be unaffected,25 increased26 or decreased27 with disease progression in hiv-infected patients. there is still controversy about cd4+cd25+cd127low/– and cd4+cd25+foxp3+ identifying the same subset of treg cells. lui et al. suggested that they could be used interchangeably28 whilst klein et al. stated that those subsets were not identical.29 however, other studies confirmed that both subsets can be used to identify tregs.30,31,32 to our knowledge, there have been no previously-published studies regarding the issue of whether tb is associated with the expression of treg cells in hiv-infected patients. this study aimed to investigate the association between levels of treg cells and t-cell activation in hiv-infected patients with and without tb. research method and design top ↑ study population and diagnostic tools recruitment of study participants and collection of clinical information took place at the department of pneumophthisiology at the national hospital center of fann. in total, 69 adult patients (≥18 years old) seen by one physician between january 2010 and june 2011 were enrolled in this study. only mtb-infected patients who had not received tb treatment two weeks prior to blood collection were included. hiv-infected patients did not receive any antiretroviral treatment. pregnant women were excluded from the study.hiv was diagnosed by means of an enzyme-linked immunosorbent assay (elisa) serological test (genscreen hiv ag/ab, bio-rad, marnes la coquette, france) and all positive hiv serology tests were confirmed by western blot (hiv blot 2.2, diagnostic biotechnology ltd., usa). tb was diagnosed by microscopic examination and/or sputum culture on solid medium lowenstein-jensen slopes (active pulmonary tb) or by biopsy, microscopic examination and/or culture fluid effusion (active extrapulmonary tb). study participants were distributed into four groups based on hiv and tb status. cd4+ and cd8+ t-cell counts laboratory testing was conducted at the immunology unit in the laboratory of virology and bacteriology at aristide le dantec university hospital. for investigation of immunological status, cd4+ and cd8+ t-lymphocytes were counted by flow cytometry using a facscount instrument (becton dickinson, san jose, ca, usa). in short, 50 µl of blood collected in a k3edta-vacutainer tube was added to each of the tubes, containing the cd4 or cd8 reagents. after incubation for one hour at room temperature in the dark, 50 μl of paraformaldehyde was added to each of the tubes, which were then analysed as laid out below. analysis of regulatory and activation markers by flow cytometry to assess regulatory and activation status of the tlymphocyte population, cell-surface staining was performed using three different panels of fluorochrome-conjugated monoclonal antibodies – panel 1: fitc-conjugated anti-cd3, percp-conjugated anti-cd8 and pe-conjugated anti-cd38; panel 2: fitc-conjugated anti-cd3, percp-conjugated anti-cd8 and pe-conjugated anti-hla-dr; and panel 3: percp-conjugated anti-cd4, apc-conjugated anti-cd25 and pe-conjugated anti-cd127. all antibodies and reagents were obtained from bd biosciences (san jose, ca, usa). fifty µl of whole blood was added to a 5 ml facs tube for panels 1 and 2, whereas 100 µl of whole blood was used for panel 3. the 3 tubes were then vortexed and incubated for 15 minutes at room temperature in the dark. subsequently, 900 μl of lysing solution was added into tubes 1 and 2, and 1800 μl into tube 3. preparations were vortexed again and incubated for an additional 15 minutes at room temperature in the dark. finally, supernatants were removed by centrifugation at 1800 rpm, washed once with 1 ml of pbs/1%bsa/0.05%nan3 and once again with 1 ml of pbs/1%bsa. the pellets were re-suspended in 400 μl of fixation solution (pbs/1% bsa/pfa) and the percentages of cd38 and hla-dr expressing t-cells and tregs were counted and analysed on a facscaliburtm flow cytometer (becton dickinson, san jose, ca, usa). dot plots were analysed using cellquesttm pro 5.1 software. statistical analysis data were collected in excel and analysed with spss 17 (spss inc, chicago, il, usa). graphs were created in prism 5 (graphpad, la jolla, ca, usa). differences between groups were assessed for statistical difference using nonparametric kruskal-wallis h and mann-whitney u tests and student’s t-tests. we used the pairwise test to assess the similarity levels of matched cd4+ counts. correlations were calculated using the nonparametric spearman test. the level of significance for all statistical tests was set at p < 0.05. results top ↑ study subjects the study analysed blood samples from 69 participants distributed throughout 4 groups: tb–hiv– (controls) (n = 10), tb+hiv+ (n = 20), tb+hiv– (n = 19) and tb–hiv+ (n = 20). the median age in years [minimum–maximum] for each group was: tb–hiv– (35 [18–45]), tb+hiv+ (43 [30–65]), tb+hiv– (32 [19–65]) and tb–hiv+ (28 [18–65]). cd4+ and cd8+ t-cell counts immunological status was assessed using cd4+ t-cell counts. we found a classic decrease of cd4+ t-cells in hiv-infected subjects compared with hiv-free individuals. we also found a stronger degree of immunodeficiency in the tb+hiv+ group (median 89 cells/mm3 [interquartile range (iqr): 27–217]) compared with the tb–hiv+ group (median 311 cells/mm3 [iqr: 96–504]) (p = 0.04) (figure 1). moreover, 75% of the tb+hiv+ patients had cd4+ cell counts under 200 cells/mm3, whilst only 40% of the hiv single-infected group had reached this level. however, cd8+ t-cell counts were not statistically different between tb+ and tb– hiv patients (p = 0.08) (figure 1). figure 1: t-cell lymphocyte counts. representative box-and-whisker plots of counts of (a) cd4+ and (b) cd8+ t-cells in tb–hiv– (n = 10), tb+hiv– (n = 19), tb–hiv+ (n = 20), and tb+hiv+ (n = 20) groups are shown. in order to avoid a bias by comparing t-cell activation markers and treg cells between tb+ and tb– hiv infected patients with different cd4+ counts, patients were matched two by two for absolute cd4+ counts, and 12 cd4-matched patients were found within the groups tb–hiv+ (189.0 cells/mm3) and tb+hiv+ (190.3 cells/mm3). cd38 and hla-dr expression on t-cells to assess t-cell activation in the different groups, we calculated the percentages of cd8+ t-cells and cd4+ t-cells expressing cd38 and hla-dr. the gating strategy is shown in figure 2. the percentage of cd4+ t-cells expressing cd38 was significantly higher in the tb+hiv+ group (median 68.5% [iqr: 59.6–88.5]) compared with the tb–hiv– (median 46.8% [iqr: 43.5–55.0]) (p < 0.001) and tb+hiv– (median 62.2% [iqr: 45.7–69.3]) groups (p = 0.03) (figure 3a). however, the difference between the tb+hiv+ and tb–hiv+ groups did not reach statistical significance (p = 0.14). figure 2: gating strategy for assessing t-cell activation markers and regulatory t-cells. figure 3: t-cell activation markers. representative column bars show percentages of (a) cd4+cd38+, (b) cd8+cd38+, (c) cd4+hla-dr+ and (d) cd8+hla-dr+ t-cells in the tb–hiv– (n = 10), tb+hiv– (n = 19), tb–hiv+ (n = 20) and tb+hiv+ (n = 20) groups. the percentage of cd8+ t-cells expressing cd38 was significantly higher in the tb+hiv+ group (median 88.6% [iqr: 79.9–94.4]) compared with the tb–hiv+ group (median 65.5% [iqr: 47.4–88.3]) (p = 0.01) and also compared with both hiv-seronegative groups (tb+hiv– [p < 0.001] and tb–hiv– [p < 0.001]) (figure 3b).with regard to hla-dr expression in t-cells, we found that the tb+hiv+ group displayed a significantly higher percentage of cd4+ cells expressing hla-dr (median 41.2% [iqr: 19.0–59.2]) as compared with the tb–hiv– (p < 0.001) and tb+hiv– groups (p < 0.001) (figure 3c). however, the difference between the tb+hiv+ and tb–hiv+ groups did not reach statistical significance (p = 0.12). finally, the percentage of cd8+ t-cells expressing hla-dr was significantly higher in the tb+hiv+ group (median 66.2% [iqr: 61.2–79.9]) than in the tb–hiv+ group (median 45.8% [iqr: 32.6–64.9]) (p = 0.006) as well as both hiv-seronegative groups (figure 3d). to correct for the bias due to differences in absolute cd4 counts between the two hiv+ groups (tb+ and tb–), patients were matched for absolute cd4+ counts in the tb–hiv+ and tb+hiv+ groups. cd4 values from the tb–hiv+ group have been linked to the approximately similar cd4 values from the tb+hiv+ group, with the following maximum differences in cd4 counts between the two matched subjects for each of four pre-established ranges: maximum difference of 20 cells for cd4 counts < 100 cells/mm3, maximum difference of 40 cells between 100 and 200 cells/mm3, maximum difference of 60 cells between 200 and 500 cells/mm3 and maximum difference of 120 cells for cd4 counts > 500 cells/mm3. following these criteria, we found 12 cd4-matched patients in the tb–hiv+ (mean: 189.0 cells/mm3) and tb+hiv+ (mean: 190.3 cells/mm3) groups (paired difference of the means = 1.08; p = 0.989). cd38 and hla-dr expression on cd4+ and cd8+ t-cells were significantly higher in the tb+hiv+ as compared with tb–hiv+ groups (figure 4). figure 4: activation markers and treg cells matched for absolute cd4 counts. representative column bars of percentage of t-cell activation markers and treg cells in the tb–hiv+ (n = 12) and tb+hiv+ (n = 12) group that were matched for absolute cd4+ t-cell counts (mean cd4+ was 189.0 cells/mm3 and 190.3 cells/ mm3 for tb–hiv+ and tb+hiv+ respectively). impact of tuberculosis on expression of tregs in hiv-infected patients the tb–hiv+ group showed a significantly higher percentage of treg cells compared with both hiv-seronegative groups (p < 0.001) (figure 5a), as did the tb+hiv+ group (p < 0.001). however, there was no statistically-significant difference between the tb+hiv+ and tb–hiv+ groups (p = 0.587). figure 5: regulatory t-cells. representative box-and-whisker plot of percentage of cd4+cd25+cd127– in the tb–hiv– (n = 10), tb+hiv– (n = 19), tb–hiv+ (n = 20) and tb+hiv+ (n = 20) groups. when matching for absolute cd4 counts between the tb–hiv+ and tb+hiv+ group, no significant difference in the percentage of tregs was found between the two groups (figure 4). correlation between cd4+ t-cell counts and treg cells and activation markers cd4+ t-cell counts were inversely correlated with fractions of tregs in both the tb–hiv+ (r = –0.663; p = 0.001) and tb+hiv+ (r = –0.447; p = 0.048) groups (figure 6a). figure 6: correlation between cd4+ t-cell counts and frequencies of treg cells. negative correlation between cd4+cd25+cd127– t-cells and cd4+ t-cell count (a) in tb–hiv+ group (r = –0.663, p = 0.001) and (b) in tb+hiv+ group (r = –0.447, p = 0.048). in the hiv-infected group (tb–hiv+), tregs were also negatively correlated with both cd8+cd38+ (r = –0.50; p = 0.022) and cd4+hla-dr+ (r = 0.74; p < 0.0001) (figure not shown). ethical considerations top ↑ the study protocol has been approved by le comité national d’ethique de la recherche en santé of senegal (permit number: 000015msas/dprs/cners) and the review board of the institute of tropical medicine of antwerp (permit number: irb/ab/ac/066). potential benefits and hazards there was no direct risk related to this study since the samples used were taken for routine biological monitoring (for hivand tb-infected patients) and for voluntary hiv testing (for controls) that are collected independent of any research activity. the participants were followed up regularly at the department of pneumo-phthisiology of fann university hospital where the ministry of health and the national aids programme support all medical and social aspects including care and protection. healthy controls consisted of volunteers enrolled from the same department where the national aids programme also supports the voluntary hiv testing. recruitment procedures participation was totally voluntary and written informed consent was obtained from each subject. informed consent through an informed consent presentation by a social worker, participants were informed about the study and how it would be conducted, including the benefits and risks, voluntary participation and confidentiality. data protection results as well as clinical and social information are strictly confidential. participants’ names or any other information that may allow identification of the subjects were not – and will not be – used in written or oral reports or in scientific publications. it was also specified in the consent form that samples will be kept for at least 10 years after the study for eventual future investigations and that any participant had the right to refuse storage of the sample and ask the biological material be destroyed. trustworthiness top ↑ this study was conducted at the laboratory of bacteriology and virology of aristide le dantec university hospital which is a un/aids reference centre for the biological monitoring of hiv patients. all the units comprising the laboratory (virology, bacteriology, immunology, and molecular biology have subscribed to external quality control programmes to ensure their quality approach and the trustworthiness of their results. reliability a default facscalibur template was drawn up using optimal compensation for accurate detection of the parameters of interest. for any new sample, t-cell markers were assessed using this template without changing any of the parameters. calibration and laser alignment of the facscaliburtm instrument used were performed periodically in order to ensure reliability of the system. validity staining controls were used when assessing t-cell markers by flow cytometry. for parameters that do not show clear separation between positive and negative populations, the positive population was set based on fmo (fluorescence minus one) control panels that lacked one of the researchers’ desired fluorochrome-associated markers in order to assess the negative limit. discussion top ↑ hiv single-infected and hiv and/or tb co-infected patients displayed increased t-cell activation compared with hiv-uninfected subjects. these results are consistent with findings that hiv infection and hiv and/or tb co-infection are associated with peripheral activation of the immune system.8,33,34,35,36,37 immune activation caused by hiv mediates increased proliferation of cd4+ cells and makes these cells more vulnerable to infection and destruction by hiv, accelerating the progressive exhaustion of cd4+ cells.38,39 our results showed that the expression of both cd38 and hla-dr on cd8+ t-cells, but not on cd4+ t-cells, was significantly higher in tb+hiv+ patients than in the tb–hiv+ patients. after matching for absolute cd4 counts, cd38 and hla-dr expression were significantly higher on both cd4+ and cd8+ t-cells in tb+hiv+ patients as compared with those who were tb–hiv+. this indicates that hiv and/or tb co-infection is associated with additional t-cell activation as compared with hiv infection alone, which may favour hiv progression. patients with tb alone also displayed higher expression of peripheral cd38 on cd4+ and cd8+ t-cells than the uninfected controls, although the differences were not statistically significant. these results are, however, in line with previous findings, suggesting that infection with tb is associated with immune activation.40 for the purposes of this study, tregs were defined as being cd4+cd25+cd127– t-cells. there is still controversy about cd4+cd25+cd127low/– and cd4+cd25+foxp3+ identifying the same subset of treg cells; however, a recent study published in cytometry has confirmed that both subsets can be used to identify tregs.32 our results show that hiv-infected patients had a significantly higher percentage of tregs than the controls. this is consistent with previous studies that have shown an increase of treg cells in hiv-infected patients.41,42 moreover, the inverse correlation between the percentage of tregs and the absolute cd4+ t-cell counts amongst hiv-infected patients suggests that relative increase of cd4+cd25+cd127– t-cells is a marker of disease severity, as reported recently in a study using the cd4+cd25+foxp3+ treg subset.20 an important question to address is whether active tb has an impact on the expression of tregs; this may help to understand, at least partly, the mechanisms by which tb alters the clinical course of hiv. it has been proposed that treg cells may be upregulated during hiv infection to avoid overactive immunity and therefore protect against viral replication17 and tissue damage,43 and that tb, which is implicated in faster progression of hiv infection, may be associated with lower levels of treg cells. on the other hand, another group has reported that higher levels of treg cells may lead to reduced immune responses and favour the pathogen over the host, and that immunodeficiency is expected to be aggravated if treg cells are present in a greater amount.23,24 this study did not find any significant difference in the expression of treg cells between hiv single-infected patients and tb and/or hiv co-infected subjects – neither in the whole study group nor in groups matched for absolute cd4 counts – suggesting that tb has no additional effect on the expression of tregs in hiv-infected patients. thus, our results seem to indicate that the mechanisms by which tb acts on hiv progression do not affect the number of tregs. however, a limitation of this study is that treg functionality was not assessed. because treg cells are a subset of cd4+ t-cells, a fraction of studied tregs may have been lost or became nonfunctional following immunosuppression or immune activation associated with hiv infection. investigation of cd4+ and cd8+ t-cell counts revealed significantly fewer cd4+ t-cells in tb and hiv co-infected patients compared with single-infected subjects with either tb or hiv alone, suggesting a more advanced immunodeficiency in co-infected patients. similar results have been reported by ddo et al. who found that tb contributes to the decrease in cd4+ t-cell count during tb and/or hiv co-infection.37 several studies have reported an association of tb with the acceleration of immunodeficiency and increased virus replication in hiv infection.4,6,40,44,45,46 with this in mind, our finding that 75% of co-infected patients had reached the stage of clinical aids (cd4 < 200/mm3), compared with 40% of the hiv single-infected patients, is not surprising. in summary, we found that expression of cd38 and hla-dr on cd4+ and cd8+ t-cells was higher in tb and/or hiv co-infected patients as compared with hiv single-infected individuals. however, tb and/or hiv co-infected patients did not express treg cells differently from hiv single-infected patients. these results suggest that tb accelerates hiv disease progression mainly through its impact on cd4+ and cd8+ t-cell activation, but not by changing the percentages of tregs. in order to better clarify the role of t-cell activation markers and treg cells in hiv disease progression amongst tb and/or hiv co-infected patients, additional investigations considering the compartments studied, the stage of the disease, the hiv viral loads and the functionality of treg cells are warranted. limitations of the study top ↑ this study was subject to some limitations, one being that the study groups used were relatively small; a larger sample size may be required to discern statistically significant differences in certain categories between certain groups. hiv viral loads were not measured at all and cd8+ t-cell counts were not looked at amongst the controls. as mentioned previously, treg functionality was not assessed. acknowledgements top ↑ we are grateful to and would like to thank all the participants of this study for their collaboration. this study would not have been possible without the medical staff of the department of pneumo-phthisiology of fann hospital and the staff of the immunology unit of the laboratory of bacteriology and virology, who are also deserving of our thanks. this study was co-funded by the direction générale pour le développement (dgd) de belgique. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. authors’ contributions t.n.d. (university of dakar), s.m. (university of dakar), l.k. (institute of tropical medicine of antwerp, and university of antwerp), g.d. (institute of tropical medicine of antwerp) and a.n. (clinic of infectious disease, fann hospital) conceived and designed the study. m.c. (university of dakar), a.a.d. (laboratory of bacteriology and virology, aristide le dantec university hospital) and p.a.d. (laboratory of bacteriology and virology, aristide le dantec university hospital) designed the experiments. n.s.s.s. and d.w. (laboratory of bacteriology and virology, aristide le dantec university hospital) performed the experiments. m.m. (laboratory of bacteriology and virology, aristide le dantec university hospital), n.s.s.s. and m.c. analysed and interpreted the data. s.m., l.k. and t.n.d. contributed to reagents/materials/analysis tools. m.m. and n.s.s.s. wrote/drafted the manuscript. a.b., m.t., m.d. and n.l. 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http://dx.doi.org/10.1097/01.aids.0000171401.23243.56, pmid:15905668 27. apoil pa, puissant b, roubinet f, et al. foxp3 mrna levels are decreased in peripheral blood cd4+ lymphocytes from hiv-positive patients. j acquir immune defic syndr. 2005;39(4):381–385. http://dx.doi.org/10.1097/01.qai.0000169662.30783.2d, pmid:16010156 28. liu w, putnam al, xu-yu z, et al. cd127 expression inversely correlates with foxp3 and suppressive function of human cd4+ t reg cells. j exp med. 2006;203(7):1701–1711. http://dx.doi.org/10.1084/jem.20060772, pmid:16818678, pmcid:pmc2118339 29. klein s, kretz cc, krammer ph, et al. cd127(low/-) and foxp3(+) expression levels characterize different regulatory t-cell populations in human peripheral blood. j invest dermatol. 2010;130(2):492–499. http://dx.doi.org/10.1038/jid.2009.313, pmid:19940860 30. hartigan-o’connor dj, poon c, sinclair e, et al. human cd4+ regulatory t cells express lower levels of the il-7 receptor alpha chain (cd127), allowing consistent identification and sorting of live cells. j immunol methods. 2007;319(1–2):41–52. http://dx.doi.org/10.1016/j.jim.2006.10.008, pmid:17173927 31. seddiki n, santner-nanan b, martinson j, et al. expression of interleukin (il)-2 and il-7 receptors discriminates between human regulatory and activated t cells. j exp med. 2006;203(7):1693–1700. http://dx.doi.org/10.1084/jem.20060468, pmid:16818676, pmcid:pmc2118333 32. saison j, demaret j, venet f, et al. cd4+cd25+cd127assessment as a surrogate phenotype for foxp31 regulatory t cells in hiv-1 infected viremic and aviremic subjects. cytometry b clin cytom. 2013;84(1):50–54. http://dx.doi.org/10.1002/cyto.b.21047, pmid:23019018 33. catalfamo m, di mascio m, hu z, et al. hiv infection-associated immune activation occurs by two distinct pathways that differentially affect cd4 and cd8 t cells. proc natl acad sci usa. 2008;105(50):19851–19856. http://dx.doi.org/10.1073/pnas.0810032105, pmid:19060209, pmcid:pmc2596741 34. cohen stuart jw, hazebergh md, hamann d, et al. the dominant source of cd4+ and cd8+ t-cell activation in hiv infection is antigenic stimulation. j acquir immune defic syndr. 2000;25(3):203–211. http://dx.doi.org/10.1097/00126334-200011010-00001, pmid:11115950 35. hunt pw, brenchley j, sinclair e, et al. relationship between t cell activation and cd4+ t cell count in hiv-seropositive individuals with undetectable plasma hiv rna levels in the absence of therapy. j infect dis. 2008;197(1):126–133. http://dx.doi.org/10.1086/524143, pmid:18171295, pmcid:pmc3466592 36. hertoghe t, wajja a, ntambi l, et al. t cell activation, apoptosis and cytokine dysregulation in the (co)pathogenesis of hiv and pulmonary tuberculosis (tb). clin exp immunol. 2000;122(3):350–357. http://dx.doi.org/10.1046/j.1365-2249.2000.01385.x, pmid:11122240, pmcid:pmc1905783 37. ddo sr, de cunha rm, kallas eg, et al. distribution of naive and memory/effector cd4 + t lymphocytes and expression of cd38 on cd8 + t lymphocytes in aids patients with tuberculosis. braz j infect dis. 2003;7(2):161–165. http://dx.doi.org/10.1590/s1413-86702003000200010 38. orendi jm, bloem ac, borleffs jc, et al. activation and cell cycle antigens in cd4+ and cd8+ t cells correlate with plasma human immunodeficiency virus (hiv-1) rna level in hiv-1 infection. j infect dis. 1998;178(5):1279–1287. http://dx.doi.org/10.1086/314451, pmid:9780247 39. fleury s, de boer rj, rizzardi gp, et al. limited cd4+ t-cell renewal in early hiv-1 infection: effect of highly active antiretroviral therapy. nat med. 1998;4(7):794–801. http://dx.doi.org/10.1038/nm0798-794, pmid:9662370 40. rodrigues ds, medeiros ea, weckx ly, et al. immunophenotypic characterization of peripheral t lymphocytes in mycobacterium tuberculosis infection and disease. clin exp immunol. 2002;128(1):149–154. http://dx.doi.org/10.1046/j.1365-2249.2002.01809.x, pmid:11982602, pmcid:pmc1906372 41. kolte l, gaardbo jc, skogstrand k, et al. increased levels of regulatory t cells (tregs) in human immunodeficiency virus-infected patients after 5 years of highly active anti-retroviral therapy may be due to increased thymic production of naive tregs. clin exp immunol. 2009;155(1):44–52. http://dx.doi.org/10.1111/j.1365-2249.2008.03803.x, pmid:19016807, pmcid:pmc2665678 42. tenorio ar, spritzler j, martinson j, et al. the effect of aging on t-regulatory cell frequency in hiv infection. clin immunol. 2009;130(3):298–303. http://dx.doi.org/10.1016/j.clim.2008.10.001, pmid:19008157, pmcid:pmc2662473 43. weiss l, piketty c, assoumou l, et al. relationship between regulatory t cells and immune activation in human immunodeficiency virus-infected patients interrupting antiretroviral therapy. plos one. 2010;5(7):e11659. http://dx.doi.org/10.1371/journal.pone.0011659, pmid:20657770, pmcid:pmc2908121 44. hane aa, thiam d, cissokho s, et al. [hematologic abnormalities and immunodepression in hiv]. bull soc pathol exot. 199l;92(3):161–163. 45. kony sj, hane aa, larouzé b, et al. tuberculosis-associated severe cd4+ t-lymphocytopenia in hiv-seronegative patients from dakar. sidak research group. j infect. 2000;41(2):167–171. http://dx.doi.org/10.1053/jinf.2000.0721, pmid:11023763 46. morris l, martin dj, bredell h, et al. human immunodeficiency virus-1 rna levels and cd4 lymphocyte counts, during treatment for active tuberculosis, in south african patients. j infect dis. 2003;187(12):1967–1971. http://dx.doi.org/10.1086/375346, pmid:12792875 abstract introduction laboratory structures, role of public health institutions and need for integration integration of public health institutions and laboratories through the implementation of international health regulations and the integrated disease surveillance and response framework global health security agenda integration of laboratory networks, surveillance and public health institutions conclusion acknowledgements reference about the author(s) philip c. onyebujoh who afro tb laboratories focal point, world health organization, harare, zimbabwe ajay k. thirumala independent consultant, horamavu, bangalore, india jean-bosco ndihokubwayo lab systems, who africa regional office, brazzaville, congo citation onyebujoh pc, thirumala ak, ndihokubwayo j-b. integrating laboratory networks, surveillance systems and public health institutes in africa. afr j lab med. 2016;5(3), a431. http://dx.doi.org/10.4102/ajlm.v5i3.431 opinion paper integrating laboratory networks, surveillance systems and public health institutes in africa philip c. onyebujoh, ajay k. thirumala, jean-bosco ndihokubwayo received: 03 mar. 2016; accepted: 15 aug. 2016; published: 31 oct. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract the ebola outbreak in west africa underlined the urgent need for integration of public health systems, including the establishment of national laboratory networks, surveillance systems, and health research institutions at all levels of service delivery. the integration schema presented here would assist in driving the immediate steps needed for integration of public health systems, particularly laboratory networks, in support of the implementation of international health regulations and the global health security agenda in the african region. increased funding, political willingness from countries, and coordination through enhanced technical assistance from international partners, are critical in achieving this objective. introduction the recent ebola virus disease crisis in three west african countries – guinea, liberia and sierra leone – highlighted several concerns with respect to the functioning of public health systems in the resource-constrained settings of africa.1,2,3 key concerns included: (1) lack of capacity for timely clinical screening, referral, diagnosis, notification, treatment and containment of people with suspected infection; (2) lack of effective surveillance and response systems and trained workforce; (3) rapid breakdown of existing public health services due to the ebola virus disease outbreak; (4) inadequate investment in building integrated, cross-cutting systems with the capacity to respond to public health emergencies; (5) challenges in implementing the world health organization’s (who) international health regulations (ihr); and (6) insufficient research and development capacity at the country and regional levels for new drugs, diagnostics and treatment strategies. these concerns and challenges are not limited to the three countries that experienced the recent crisis. in general, challenges faced by public health systems in africa, especially at the primary care level, include: (1) lack of laboratory facilities integrated across diseases and services; (2) poor funding and uptake of new rapid diagnostic technologies; (3) overemphasis on resourcing vertical disease-control programmes; (4) patient and health system delays; and (5) lack of fully-integrated electronic disease surveillance systems. laboratory structures, role of public health institutions and need for integration laboratories play a critical role in the prompt diagnosis of diseases and treatment monitoring. the specific administrative structures of public health laboratories within ministries of health differ among countries in africa. in the majority of countries in the african region, laboratories fall under a directorate, department or unit for national laboratory services, which are housed within the ministry of health. difficulties in coordination of laboratory services with disease control programmes have been observed in such conventional administrative structures. district and below district-level hospital laboratories act as the default ‘integrated’ laboratories, due to inadequate staff levels, skill sets and infrastructure. intermediate-level laboratories are practically district-level laboratories and act as regional laboratories, but without the necessary functional dichotomy. primary health centre laboratories are poorly equipped for rapid diagnosis or rapid response; and national-level laboratories are not fully networked with district-level laboratories for the functional management of laboratory networks.4 donors and partners in several countries promote ‘parallel’, disease-specific laboratories, especially for the major diseases of poverty (tuberculosis, hiv and malaria), as governments fail to address deficiencies. in a few countries, government-run centres-of-excellence were developed as public health research institutes with support from international partners.5 in some countries, environmental health protection and food safety services, as well as national drug testing laboratories, are also administered under public health services. the lack of adequate resources, both financial and technical, has contributed to undue prioritisation of key diseases, which has in turn resulted in vertical disease control programmes and laboratories (e.g., tuberculosis, malaria and hiv). the absence of the integration of policy formulation, strategy and budgeting have been compounded by competing disease funding priorities and has created disjointed laboratory services in several developing countries.6 this has resulted in disparate and loosely-organised skill deployment, resource wastage and redundancies within the system. laboratory workers with the cross-cutting skills to effect integrated services have been systematically under-utilised, as they are increasingly engaged in services for single-disease programmes.7 some of the key issues plaguing public health laboratories8,9 include: (1) under-resourced infrastructure, including equipment; (2) poor laboratory linkages to clinical services, resulting in low test demand and suboptimal utilisation of modern diagnostics for clinical decisions; (3) poor laboratory quality control and assurance systems; (4) a paucity of leadership to provide adequate policy intervention, technical guidance and supportive supervision; and (5) inadequate or absent national laboratory networks. these issues impact staff motivation and the overall credibility of laboratory services. in spite of the functional challenges in the delivery of services, there is an urgent need to integrate already-existing public health technical capacity within countries in the african region. effective linkages between public health institutes and laboratories, in addition to optimisation of services, would facilitate early detection of emerging antibiotic resistance, cancers and other non-communicable diseases, as well as periodic epidemiological disease surveys, which would inform policy changes. the network of african national public health institutes10 works to facilitate the collaboration and strengthening of public health research institutes affiliated to the network of african national public health institutes in africa.11 present membership of the network comprises 16 institutions, including the kenya medical research institute, the uganda viral research institute, and the south african national institute for communicable diseases. in addition, in some countries in africa, private-sector clinical pathology laboratories, either corporate chains, or individual stand-alone laboratories, offer services to people who can afford quality care for a premium (such as medical aid/insurance, or out-of-pocket expenses). systematically engaging african national public health research institutions and centres of excellence, as well as private-sector laboratories, with national laboratory systems and integrated surveillances systems would enhance utilisation of available laboratory and research capacities, and management of resources. integration of public health institutions and laboratories through the implementation of international health regulations and the integrated disease surveillance and response framework in 2005, the ihr mandated countries to detect, assess and respond to all events that may constitute public health emergencies of international concern, and report the events to the who.12 the integrated disease surveillance and response (idsr) programme, a comprehensive regional framework for strengthening national public health surveillance and response systems in africa, was initiated in 1998. in 2006, who regional office for africa (who afro) member states recommended that ihr 2005 be implemented using the idsr framework. the idsr strategy is aimed at integrating the collection, analysis, and reporting of data on 40 priority diseases and conditions at different levels of the health system, including relevant laboratory data. over the past decade, the idsr strategy has successfully reduced parallel disease-specific surveillance programmes, increased the capacity of central laboratories, and unified disease reporting matrices among implementing countries in the african region. it has enhanced adherence to ihr by encouraging and establishing early disease warning and real-time event management systems at the national level, including prompt reporting to the who through national idsr focal point. ihr core capacity target dates have been revised twice, first to 2014 and then to 2019 after the ebola virus disease outbreak in west africa. as of 2014, two-thirds of countries had not met their core capacity requirements and 48 countries had not responded to who queries with respect to their state of readiness.13,14 in hindsight, such shortcomings may have contributed to the state of unpreparedness within the west african sub-region that increased vulnerability to the recent ebola virus disease outbreaks. recent successes in efforts to strengthen african laboratory networks, laboratory infrastructure, rapid diagnostic technologies, and quality management systems need to be comprehensively integrated into the core functions of the idsr through new regional structures or coordination mechanisms. furthermore, enhanced research and development support through already-existing public health research institutes and centres of excellence would not only provide inter-disciplinary technical skills and capacity to staff for quality services and accurate diagnosis, but also assist in the systematic introduction of new and rapid diagnostic technologies and the provision of periodic epidemiological research surveys that will inform efficient planning for health services. surveillance mechanisms need high-level coordination at the sub-regional and national levels, with frequent modification.15 provision of this level of coordination for active surveillance, timely analyses and response, especially for events of public health significance, are anticipated through the establishment of the centres for disease control and prevention in africa.16 global health security agenda the global health security agenda (ghsa), formed in 2014 in response to the ebola virus disease crisis and supported by high-level international collaboration, is a multi-country concerted effort toward creating a world safe and secure from infectious disease threats.17 to effect coordination, 11 action packages were designed and entrusted to contributing countries for enlisting political leadership and action. action packages are bound by a prevent–detect–respond framework. there is a need to develop in-country mechanisms and responsibilities to address the ghsa. integration of health systems across diseases and structures would provide a path for this. it is envisaged that the capacity to respond to the objectives of the ghsa would depend on maximising detection and surveillance capabilities through integrated laboratory frameworks within target countries. integration of laboratory networks, surveillance and public health institutions integration envisages a process whereby regional and country-level governmental services, including multi-dimensional functions such as health financing, human resources, strategic planning, and others, along with international developmental partners and donors, work closely to promptly reach and sustain the goal of health security for all. in the resource-limited settings of africa, integrating and coordinating laboratory services and different service components of national health structures would lead to efficient use of resources and building of laboratory networks, as well as ensuring that laboratories contribute to national disease surveillance and control at all levels of health services (figure 1).18 figure 1: key crosscutting elements of a national health laboratory system. in principle, integration of laboratory services should be achieved first at the country level, followed by the sub-regional level, as integration aims to best utilise the available resources in an unbiased fashion. figure 2 depicts a conceptual, regional integration schema with functions. to achieve the desired goal, complete structural, operational and functional cohesion are required. in practice, this could be achieved by forging multi-level coordination, including appointment of officers with sufficient authority to oversee and coordinate functions in line with a defined strategy at the country and sub-regional levels. at the sub-regional level, the coordination mechanism will encompass intergovernmental bodies, relevant functions within sub-regional economic blocs, and partners. countryand regional-level functionaries would jointly develop a response strategy to ensure that planned activities are implemented. a concerted effort to integrate modern diagnostic platforms, specifically those that use a single piece of equipment for diagnosis of multiple diseases, are needed within this regional schema. information gathering, analyses, monitoring and evaluation of activities will be part of the action package and response at this level. the who and partners, including the us centers for disease control and prevention, the u.s. president’s emergency plan for aids relief, the african society for laboratory medicine, the african union and others, would provide oversight for country–country cooperation and ensure that networks of laboratories, surveillance systems and public health institutes are functional. the schema proposes integration of the ghsa packages at the country and sub-regional levels. while it is proposed to entrust relevant disease and disorder detection capabilities to the integrated laboratory networks, response would be entrusted to the idsrs, and prevention to public health institutions. figure 2: integration as conceptual schema with three key components – laboratory networks, surveillance systems, and public health institutions. several elements of each component are listed for integration to achieve the goal of public health laboratories. this helps with prompt adjustment to policy changes and timely detection through accurate technologies, and ensures prevent/respond measures against diseases. achieving national and regional collaboration enhances countries’ capacities to meet the integrated disease surveillance and response and international health regulations targets. conclusion the ebola virus disease crisis has reinforced the need to act decisively on a new regional and global structure, as well as the necessity for integrating in-country and regional health systems under one unified, rapid service delivery mechanism, with early warning, detection and response capabilities. political will and fiscal and administrative commitments are urgently required from governments to ensure that cross-cutting integration of health systems happens and that the ihr core requirements and the ghsa are met. acknowledgements the authors wish to acknowledge the support received from the who afro inter-country support team in the form of a number of field visits made for laboratory assessments in the years 2013 to 2015. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support none. authors contribution p.c.o. was the lead focal point for the project, j-b.n. and a.k.t. provided technical assistance to laboratories through world health organization assessment missions and recommendations. p.c.o., j-b.n. and a.k.t. contributed equally to the gap analysis of the laboratory systems, and the design and development this opinion article. reference alexander ka, sanderson ce, marathe m, et al. what factors might have led to the emergence of ebola in west africa? plos negl trop dis. 2015;9(6):e0003652. http://dx.doi.org/10.1371/journal.pntd.0003652 moon s, sridhar d, pate ma, 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internet]. c2015 [cited 2015 nov 25]. available from: http://apps.who.int/iris/handle/10665/204630 introduction planning for low and medium-income country laboratories to embrace sustainability in laboratory medicine implementation of sustainable practices and economic benefits acknowledgements references about the author(s) tony badrick royal college of pathologists of australasia quality assurance program, new south wales, australia john anetor department of chemical pathology, college of medicine, university of ibadan, ibadan, nigeria citation badrick t, anetor j. preparing medical laboratories in lowand middle-income countries for a sustainable future. afr j lab med. 2025;14(1), a2734. https://doi.org/10.4102/ajlm.v14i1.2734 opinion paper preparing medical laboratories in lowand middle-income countries for a sustainable future tony badrick, john anetor received: 21 dec. 2024; accepted: 24 june 2025; published: 16 sept. 2025 copyright: © 2025. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction hospitals and healthcare facilities generate significant waste and environmental impact.1 these effects include generating large quantities of solid and liquid waste, using hazardous materials, consuming large amounts of energy and water, and producing large quantities of wastewater and greenhouse gases.2 because of the specialised nature of the equipment used and the need to tightly control temperature and humidity in medical laboratories, they typically use three to six times more energy per unit surface area. they also consume 60% – 80% more energy than a typical office building.3 medical laboratories, in addition, generate large quantities of chemical (toxic) wastes, clinical glassware, culture plates, stock cultures, and highly infectious wastes in massive amounts. of particular concern is radioactive wastes, hazardous and infectious wastes that require packaging, handling, and special treatment methods.4 inappropriate management of these dangerous wastes can result in ill health in staff in healthcare facilities, patients, waste collectors, and the public at large, because of exposure to these health risks.5 within the laboratory, there is a high level of understanding of the risks, especially in higher-income countries. in contrast, in other laboratories, particularly in the lowand medium-income countries,6 understanding of the broader risks to the community of laboratory waste is less well appreciated. while guidelines and educational campaigns are available, there is still only a rudimentary understanding of how sustainability guidelines can apply to diagnostics laboratories and equipment.7,8,9,10 in the low-and middle-income countries,6 regulations and guidelines are being used to reduce the industry’s environmental impact, but there is still a poor understanding of how sustainability guidelines can apply to medical laboratories in most developing countries especially in africa. modifying existing products to meet sustainability requirements is rarely an option, leaving new product development as the only alternative long-term approach to an immediate problem.2 the problems are multiplied manyfold in lowand middle-income countries where there are numerous issues, including scarcity of financial resources, priority of the country, natural calamities, and conflict between governments and trade unions. organisational profit-making schemes with sustainable technologies, corruption at various levels, lack of interest, and efforts to incorporate sustainable development goals are other major obstacles to enabling sustainability in laboratory medicine in the lowand middle-income countries.11,12 recognising these issues, the international federation of clinical chemistry and laboratory medicine (ifcc) responded by creating a task force on the environmental impact of laboratory medicine. the ifcc has been active in this area, with the formation in 2012 of an earlier ad hoc ifcc panel on the environmental responsibility of clinical laboratories.8 the ifcc panel produced a set of proposals for mitigating the environmental impact of clinical laboratories. the new ifcc task force on the environmental impact of laboratory medicine was created with the following goals, and an intended focus on developing countries: ‘identify existing peer-reviewed, high-quality publications that describe the impact of laboratory medicine operations on the environment and actions that laboratories and manufacturers can take to reduce the negative impact. develop recommendations and practice guidelines that laboratories in both developed and developing countries can implement to reduce the environmental impact of laboratory operations without compromising the quality of services provided to patients. develop a plan to share information and educate ifcc national societies and corporate members. identify existing peer-reviewed, high-quality publications that describe the laboratory testing that can be performed to measure both levels and biological effects of toxic environmental chemicals in human biological material, including studies to demonstrate the concentrations of chemicals found in human biological tissues (human biomonitoring).’ as required, the task force will establish a formal collaborative link with the european federation of laboratory medicine task force on green labs and other ifcc groups and organisations involved in related activities.13 planning for low and medium-income country laboratories to embrace sustainability in laboratory medicine understanding the cultural, organisational and behavioural changes are necessary to implement long-term and sustainable changes. it requires strong and unwavering leadership with a well-defined and documented plan. it would also require external support from trusted mentors and internal champions who understand the importance of the task ahead. it may also be important to stress that there is a long-term economic benefit in implementing sustainable laboratory medicine practice. the plan would include the following components:8 establish and maintain procedures that specify environmental objectives and targets and direct efforts towards continual improvement. these need to be realistic, given the resource constraints that many laboratories face in these countries. minimise appliance energy and water consumption. there are often relatively easy ways to reduce energy usage, such as simply switching equipment or lights off when they are not in use (https://greenlabs.eflm.eu/documents/presentation_energy.pdf). reduce waste by reducing the use of resources and embrace the reuse or recycling of materials (https://greenlabs.eflm.eu/documents/presentation_waste.pdf). review environmental practices at least annually. this ensures that the plan is current and updated with new projects added as appropriate. engage in advocacy by encouraging customers, suppliers and other stakeholders to mirror the organisation’s commitment to environmental responsibility. green purchasing policy purchasing should be incorporated into purchase contracts ahead of final purchase of major equipment and consumables. environmental managers may be appointed by each laboratory to take charge of toxic chemical use and appropriate disposal as well as possible recycling (https://ifcc.org/executive-board-and-council/eb-task-forces/task-force-environmental-impact-of-laboratory-medicine-tf-eilm/). implementation of sustainable practices and economic benefits many universities and institutes across the globe have implemented sustainability projects. one of the most prominent ones is the university of groningen, faculty of science and engineering, university of groningen, the netherlands.14,15 the integration of sustainable practices in clinical laboratories as part of a global scientific community embracing sustainability in science is important for mitigating environmental impact promoting resource conservation, and in ensuring the long-term viability of healthcare systems. collaboration and innovation in laboratories can lead the way toward a healthier and more sustainable environment. the economic benefit of sustainable laboratory practice is evident from the examination of standaedised data from selected hospitals and other institutions that have implemented sustainable programmes. the data revealed that economic benefits of intervention exceeded $5.4 billion dollars over a 5-year period and $15 billion dollars over a 10-year period with an estimated gross net savings of over $15.2 billion dollars in the long run. returns were almost tripled over 10 years.16,17 briefly, the barriers to embracing sustainable practices are erroneous phobias about initial investment.16 these can be addressed by innovative and committed leadership, as well as education and persuasion, as elegantly demonstrated by freese et al.15 appropriate leadership will lead to policy formulation and regulation that will guide organisations, also elegantly discussed by freese and his colleagues.15 sustainability has enormous scientific, social and economic benefits that can only be achieved or embraced by persuasion at this time. regulations that are enforceable may follow. in an opinion paper such as this, it is not possible to include too many details, but again, the paper by freese et al.,15 and several others, have comprehensively addressed aspects of the technology.18 it is, therefore, a public health and laboratory medicine priority in many lowand medium-income countries, especially those in africa, to put measures in place to ensure sustainable laboratory medicine practices. in the science of toxicology, there is a well-recognised axiom in pollution science and exposure control: ‘poison one part of the globe and the whole world is poisoned’.19 pollution has no respect for international boundaries; this is why international partners must join hands with lowand medium-income countries in enthroning sustainable laboratory medicine practices. the medical laboratory community must form a partnership with lowand medium-income counties to eradicate laboratory medicine-associated adverse environmental impacts and ensure green laboratory medicine practice for all for the future. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. j.a. serves as an editorial board member of this journal. the peer review process for this submission was handled independently, and the author had no involvement in the editorial decision-making process for this manuscript. the authors have no other competing interests to declare. authors’ contributions both authors, j.a. and t.b., contributed equally to this work. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. the article does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the authors are responsible for this article’s results, findings, and content. references janik-karpinska e, brancaleoni r, niemcewicz m, et al. healthcare waste – a serious problem for global health. healthcare (switzerland). 2023;11(2):242. https://doi.org/10.3390/healthcare11020242 schneider e, prosser b. the pathologist. lab sustainability: a chance for change. knutsford, cheshire: texere publishing; 2023. lopez jb, jackson d, gammie a, badrick t. reducing the environmental impact of clinical laboratories. clin biochem rev. 2017;38(1):3–11. pruss a, giroult e, rushbrook p. safe management of wastes from health-care activities [homepage on the internet]. geneva: world health organization; 1999 [cited 2024 sept 12]. available from: https://iris.who.int/bitstream/handle/10665/42175/9241545259.pdf who. health-care waste. who factsheet [homepage on the internet]. 2018 [cited 2024 sept 12]. available from: https://www.who.int/news-room/fact-sheets/detail/health-care-waste street a, vernooij e, rogers mh. diagnostics waste: whose responsibility? global health. 2022;18:30. https://doi.org/10.1186/s12992-022-00823-7 building a global culture of sustainability in science [homepage on the internet]. 2024 [cited 2024 sept 12]. available from: https://www.mygreenlab.org/ lopez jb, badrick t. proposals for the mitigation of the environmental impact of clinical laboratories. clin chem lab med. 2012;50(9):1559–1564. https://doi.org/10.1515/cclm-2011-0932 eflm. green labs. eflm guidelines for green and sustainable medical laboratories [homepage on the internet]. 2022 [cited 2024 sept 12]. available from: https://www.eflm.eu/upload/docs/eflm-green-lab-booklet.pdf england nhs. greener nhs [homepage on the internet]. 2023 [cited 2024 sept 12]. available from: https://www.england.nhs.uk/greenernhs/ dhandapani k, venkataraman h. the state of sustainability in developing countries. in: brinkmann r, editor. the palgrave handbook of global sustainability. cham: palgrave macmillan, 2023; p. 911–937. united nations industrial development organization. industrial development report: industrialization, environment and the millennium development goals in sub-saharan africa – the new frontier in the fight against poverty [homepage on the internet]. united nations 2005 [cited 2024 sept 14]. available from: https://www.un-ilibrary.org/content/books/9789210451253 international federation of clinical chemistry and laboratory medicine (ifcc). new taskforce on environmental impact of laboratory medicine and invitro diagnostics. milan: federation of clinical chemistry and laboratory medicine; 2023. freese t, kat r, lanooij sd, bollersen tc, de rooi cm, elinga m, et al. a guide for sustainability in laboratories. active molecular systems. faculty of science and engineering, groningen: university of groningen; 2013. freese t, elzinga n, heinemann m, lerch mm, feringa bl. the relevance of sustainable laboratory practices. rsc sustain. 2024;2:1300. https://doi.org/10.1039/d4su00056k kaplan s, sadler b, little k, franz c, orris sp. can sustainable hospitals help bend the healthcare cost curve? commonw fund. 2012;29:1–14. buffoli m, capolongo m, bottero m, cavagliato e, spernza s, volpatti a. sustainable healthcare: how to assess and improve and improve healthcare structure sustainability’s sustainability. ann ig. 2013;25:411–118. badrick t, majid h, mannisto t, anetor j. environmental issues in clinical laboratories: pragmatic pathways to sustainability. ejifcc. 2025;36:187–192. anetor j, anetor g. green laboratory medicine in africa: what is the contribution to environmental toxicity and how prepared is the continent for sustainable laboratory medicine? clinica chimica acta. 2024;558(suppl 1):118320. https://doi.org/10.1016/j.cca.2024.118320 abstract introduction methods results discussion acknowledgements references about the author(s) sumayya abdullahi department of medical laboratory science, ahmadu bello university, zaria, nigeria idris n. abdullahi department of medical laboratory science, ahmadu bello university, zaria, nigeria hafeez a. adekola department of molecular biology and biotechnology, nigeria institute of medical research, lagos, nigeria nicholas baamlong department of family medicine, university of abuja teaching hospital, abuja, nigeria amos dangana national reference laboratory, nigeria center for disease control and prevention, abuja, nigeria yahaya usman department of medical laboratory science, ahmadu bello university, zaria, nigeria abdurrahman e. ahmad department of medical laboratory science, ahmadu bello university, zaria, nigeria sumaiya salisu department of medical laboratory science, ahmadu bello university, zaria, nigeria mukhtar m. abdulaziz department of medical microbiology, ahmadu bello university, zaria, nigeria citation abdullahi s, abdullahi in, adekola ha, et al. nasal carriage rate and multiple antimicrobial resistance indices of staphylococcus aureus among healthcare students at the ahmadu bello university, nigeria. afr j lab med. 2025;14(1), a2667. https://doi.org/10.4102/ajlm.v14i1.2667 original research nasal carriage rate and multiple antimicrobial resistance indices of staphylococcus aureus among healthcare students at the ahmadu bello university, nigeria sumayya abdullahi, idris n. abdullahi, hafeez a. adekola, nicholas baamlong, amos dangana, yahaya usman, abdurrahman e. ahmad, sumaiya salisu, mukhtar m. abdulaziz received: 26 oct. 2024; accepted: 07 apr. 2025; published: 27 june 2025 copyright: © 2025. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: healthcare students could harbour multidrug-resistant (mdr) and methicillin-resistant staphylococcus aureus (mrsa). there is a need to understand the extent and factors associated with nasal carriage of these strains. objective: this study determined the frequency and risk of nasal s. aureus, and multiple antimicrobial resistance indices among students at ahmadu bello university, zaria, nigeria. methods: this comparative cross-sectional study collected nasal samples from 02 january 2024 to 31 july 2024 from healthcare students at ahmadu bello university, nigeria, which were processed for s. aureus identification. antimicrobial resistance phenotype was determined by the disk diffusion method. structured questionnaires were used to collect participants’ sociodemographic and risk factor data. results: a total of 251 students participated, including 126 (50.2%) men and 125 (49.8%) women (aged 17–44 years). the nasal carriage of s. aureus was 31.5% (79/251) and mrsa was 23.5% (59/251). clinical-phase students had a higher frequency of nasal mrsa (25%) than preclinical-phase students (22.1%). staphylococcus aureus resistance against non-beta-lactams was highest for tetracycline (49.4%) and ciprofloxacin (29.1%), with 39.2% (31/79) showing mdr. medical and pharmacy students had statistically significant higher nasal carriage of mdr-s. aureus (p < 0.05). students residing in households of 5–8 individuals had the highest nasal mdr-s. aureus carriage (p = 0.0044). staphylococcus aureus isolates with multiple antimicrobial resistance indices of 0.2 (29.1%) and 0.3 (24%) were the most predominant. conclusion: high levels of nasal mrsa and mdr-s. aureus were obtained from this study. the predominance of strains with high antimicrobial resistance indicates sources with high antibiotic use. what this study adds: to our knowledge, this is the first epidemiological study on the multiple antimicrobial resistance indices of nasal s. aureus in healthcare students in africa. moreover, this is the first report to categorises subgroup variation of nasal mdr-s. aureus carriage by the six major groups of healthcare students. keywords: staphylococcus aureus; multidrug resistance; multiple antibiotic resistance index; medical students; nigeria. introduction staphylococcus aureus is a member of the nasal microbiota, and a leading source of healthcareand community-associated infections in people, especially the methicillin-resistant s. aureus (mrsa) strains, resulting in morbidity and death.1 staphylococcus aureus and mrsa may colonise a variety of places, including the skin and mucosal surfaces of humans and animals, and can contaminate environmental surfaces, food, and water.1,2,3,4,5 it is often present in healthy individuals without producing illness, but it is a risk factor for developing staphylococcal infections.6 in the healthcare setting, mrsa is of substantial concern, as it can be transmitted across patients, healthcare workers, students, and visitors.7,8 when mrsa is transmitted to immunocompromised patients, it can cause a variety of illnesses, ranging from minor skin infections to serious disorders such as endocarditis, osteomyelitis, and sepsis.9 often, the overuse and misuse of antimicrobial agents selects for the emergence of mrsa and multidrug-resistant (mdr)-s. aureus (strains that are resistant to three or more classes of antimicrobial agents).10 these can complicate the use of antimicrobial chemotherapy against infectious diseases caused by s. aureus when the strains present with the mdr phenotype.10 healthcare centres generally have sick individuals (humans or animals).11 hence, students who receive clinical training in these settings could be at high risk of contracting clinically resistant s. aureus strains.12 conversely, students colonised with mrsa in their nasal passages have the potential to transmit these strains to sick people and animals (for veterinary students) in healthcare centres during clinical posting and ward rounds,8,13 consequently increasing the burden or morbidity to the patients. nevertheless, mrsa can also be acquired from both the community and animals (zoonosis).1,2 healthcare students can harbour mdr and mrsa in their nostrils and can be the source of transmission to hospitalised patients and other individuals in healthcare facilities. it has been shown that nasal colonisation by s. aureus among these categories of students could be very high.8 collectively, this indicates the need for stringent infection prevention and control measures among medical and health sciences students. it is important to note that these colonisation rates could vary, depending on the duration of contact with hospital settings, and on the type or form of contact with patients. hence, the need to undertake the present study to determine the level of the indicated epidemiological parameters among various groups of healthcare students (either preclinical or clinical phases). there are numerous studies, including many from most african countries and cities, that have identified the ecology of mrsa in medical students, as indicated in a systematic review and meta-analysis.13 in the global meta-analysis, a pooled prevalence of 28% nasal mrsa carriage in medical students was estimated.13 furthermore, the study found out that a higher pooled prevalence was obtained in students at the clinical phase of training (33%) than in those at the preclinical phase of training (25%).13 however, there are very scarce data on subgroup analyses of mrsa carriage rates among students from the other major healthcare disciplines, and the multiple antimicrobial resistance (mar) indices of the isolates. this comparative cross-sectional study aims to determine the nasal carriage of s. aureus, its antimicrobial resistance phenotypes and mar indices, and to assess the factors that could be associated with nasal s. aureus and mdr-s. aureus carriage among medical, veterinary, and health sciences students at ahmadu bello university, zaria, nigeria. methods ethical considerations all study protocols were submitted for ethical review and were approved by the human research ethical committee of ahmadu bello university teaching hospital (approval no.: nhrec/tr/abuth-nhrec/01/02/23). all data generated were treated with the utmost confidence and analysed anonymously. all participants gave written informed consent before enrolment into the study. samples and data collection from participants were performed according to helsinki’s declaration as revised in 2024. data collected were stored in a password-protected computer, only accessible by the principal investigator. study area this study was conducted from 02 january 2024 to 31 july 2024 at the ahmadu bello university and ahmadu bello university teaching hospital, kaduna state, nigeria. the university and teaching hospital serve as the main tertiary institution and reference hospital in the northwest geopolitical zone of nigeria. moreover, they are the oldest and largest institutions for the training of medical and other healthcare professionals in northern nigeria. study design, sample size, data collection this was a cross-sectional study, and the sample size was calculated from the only available previous cross-sectional study on a healthy population of medical students in nigeria.14 using the 14% prevalence of nasal s. aureus from the study of adesida et al.,14 a minimum sample size of 190 was calculated. however, for statistical credence, 251 healthy students who consented to participate were enrolled. sociodemographic variables and other data such as prior use of antibiotics, household or hostel room density, and prolonged contact with animals were collated from the participants through structured, paper-based questionnaires administered by the researchers (interviewers). sample collection nasal samples from both nostrils of eligible participants were collected by some of the investigators in batches and preserved in amies transport media (himedia laboratories, maharashtra, india). within 4 h of collection, the nasal samples were transported to the laboratory on cold packs (2 °c – 8 °c) by some of the investigators, where they were processed for s. aureus recovery. isolation and identification of isolates were carried out based on the colonies’ colour, size, shape, tube coagulase test, strong mannitol fermentation, and gram staining at the microbiology laboratory of ahmadu bello university teaching hospital, zaria, nigeria. laboratory experiments the nasal swabs were inoculated onto mannitol salt agar (himedia laboratories, maharashtra, india). after incubation (24 h at 37 ºc), a colony was collected from each sample. the colony was reisolated to obtain a pure s. aureus culture for identification. a rapid in-house tube coagulation reaction and production of bright (strong) yellow colours from mannitol fermentation were used to identify the s. aureus strains. ecologically, s. aureus is the only coagulase-positive staphylococcus that readily colonises the nostrils of humans.15 other coagulase-positive staphylococci are specifically adapted to dogs, cats, and horses.16 furthermore, other coagulase-positive staphylococci (such as staphylococcus delphini, staphylococcus ursi, staphylococcus coagulans, and staphylococcus cornubiensis) do not ferment mannitol, while staphylococcus pseudintermedius and, staphylococcus intermedius weakly ferment mannitol after a prolonged incubation of > 24 h.17 hence, the use of tube coagulation tests in addition to strong mannitol fermentation was considered sufficient to identify s. aureus in healthcare students. the susceptibility of all identified s. aureus strains to different antimicrobial agents was performed using kirby-bauer’s disk diffusion method on mueller-hinton agar (oxoid, manchester, united kingdom). this was done using the following oxoid® antibiotic discs (µg/disc): penicillin (1), cefoxitin (30), mupirocin (200), gentamicin (10), clindamycin (2), erythromycin (15), ciprofloxacin (5), chloramphenicol (30), tetracycline (30), and linezolid (10). the 2024 european committee on antimicrobial susceptibility testing criteria were used for breakpoint interpretation.18 methicillin resistance was determined by the identification of the cefoxitin resistance phenotype on the mueller-hinton agar plate. for epidemiological purposes, the mar index was defined as the number of antibiotics to which an isolate was resistant, divided by the total number of antibiotics tested. staphylococcus aureus strains with mar indices of greater than two (> 2) were used to indicate areas with high antibiotic pressure, as previously described.19 furthermore, an isolate was considered mdr if it was resistant to ≥ 3 classes of the antimicrobial agents tested.20 data analysis epiinfo® (centers for disease control and prevention, atlanta, united states) was used to digitise data collected on paper questionnaires. categorical sociodemographic variables were expressed as frequencies and compared with the detection rates of nasal s. aureus using the chi-square (χ2) test. furthermore, bivariate analyses were performed to determine the association between risk factors and sociodemographic variables with s. aureus and mdr-s. aureus carriage rates by medcalc version 23.0.2 (medcalc, ostend, belgium). all analysis outputs with p < 0.05 at 95% confidence interval were considered statistically significant. results the 251 medical and health science students were enrolled in different undergraduate programmes. the age range of the participants was from 17 years to 44 years with a mean ± standard deviation of 23.1 ± 3.5. about 50.2% were men and 124 of the students were in the clinical phase of training (exposed to healthcare settings). most of the students in their clinical phase were in their first year (n = 50, 40.3%), followed by those in their third year (n = 39, 31.5%) (table 1). table 1: sociodemographic variables of study participants in zaria, nigeria, january 2024 to july 2024 (n = 251). one hundred and eighty-five (73.7%) of the students were residing in urban settlements, 31 (12.4%) resided in rural areas, and 35 (13.9%) in suburban settlements. one hundred and twenty-six of the students resided in houses with a density of 5–8 individuals (50.2%). one hundred and fifteen (45.8%) consumed antibiotics within the last 3 months before their enrolment into the study (table 1). prevalence of staphylococcus aureus and methicillin-resistant staphylococcus aureus among healthcare students the nasal carriage rate of s. aureus and mrsa among the 251 participants was 31.5% (n = 79) for s. aureus and 23.5% (n = 59) for mrsa (figure 1a). specifically, the prevalence of s. aureus was highest among the bachelor of nursing science students (13/32, 40.6%), followed by doctor of veterinary medicine (18/53, 34%), bachelor of pharmacy (3/40, 32.5%), bachelor of radiography students (7/23, 30.4%), bachelor of medicine, bachelor of surgery (mbbs) (15/53, 28.3%), and bachelor of medical laboratory science students (12/50, 24%). furthermore, the prevalence of mrsa was highest among the bachelor of nursing science (10/32, 31.3%), followed by bachelor of pharmacy (10/40, 25%), doctor of veterinary medicine (13/53, 24.5%), bachelor of medical laboratory science (10/50, 20%), mbbs students (10/53, 18.9%), and bachelor of radiography students (4/23, 17.4%). students in the clinical phase had a relatively higher frequency of nasal mrsa (31/124, 25%) than those in the preclinical phase (28/127, 22.1%) (odds ratio = 1.1786, p = 0.5814) (figure 1b). there was no statistically significant association between the nasal carriage rate of s. aureus (χ2 = 2.97, p = 0.705) and mrsa (χ2 = 6.695, p = 0.2443) with the programme of study of the participants (p > 0.05) (figure 1b). figure 1: (a) prevalence of nasal staphylococcus aureus and methicillin-resistant s. aureus carriage and (b) association between the phase of study and antibiotic use with the prevalence of nasal methicillin-resistant s. aureus among among health sciences students of the ahmadu bello university; zaria, nigeria, january 2024 to july 2024. prevalence and factors associated with nasal staphylococcus aureus carriage the frequency of nasal s. aureus carriage in female students (33/126, 32.8%) was relatively higher than in male students (41/125, 30.2%). students within the age range of 28–33 years had the highest prevalence of nasal s. aureus (4/6, 66.7%), followed by those 16–21 years old (28/75, 37.3%), and those 22–27 years old (47/167, 28.1%). none of the students within the 34–45 range had s. aureus carriage (table 2). students aged 28–33 years had the highest odds of nasal s. aureus carriage (odds ratio = 5.106, 95% confidence interval: 0.9047–28.82) (table 2). students who resided in rural areas had the highest prevalence of nasal s. aureus (13/31, 41.9%), followed by those in the suburbs (12/35, 34.3%) and those in urban settlements (54/185, 29.2%). students in the preclinical phase of their training had a relatively higher frequency of nasal s. aureus carriage (41/127, 32.3%) than those in the clinical phase (38/124, 30.6%). of those in the clinical phase of their training, those who had been on clinical and/or laboratory posting for 3 years (15/39, 38.5%) had the highest prevalence of s. aureus, while the lowest was recorded among students who had been on posting for 1 year (12/50, 24%) (table 2). students who resided in houses with densities of 5–8 individuals (51/75, 40.7%) had the highest prevalence of s. aureus, followed by those who resided in houses with ≥ 9 individuals (16/51, 23.9%) (table 2). students who resided in houses with a density of 5–8 individuals had statistically significant odds of nasal s. aureus carriage (odds ratio = 0.3836, 95% confidence interval: 0.19–0.81, p = 0.0086) (table 2). table 2: prevalence of staphylococcus aureus culture-positive and factors associated with nasal carriage in the healthcare students in zaria, nigeria, january 2024 to july 2024. students who used antibiotics within the last 3 months before their enrolment into the study had a closely similar frequency of nasal s. aureus carriage (31.6%) compared with those with no antibiotic use 3 months before enrolment in the study (31.3%). students who had prolonged contact with pets or livestock had a relatively lower frequency of nasal s. aureus carriage (41/142, 28.9%), than those who did not (38/109, 34.9%) (table 2). factors associated with nasal carriage of multidrug resistant-staphylococcus aureus the frequency of multidrug resistant-s. aureus was highest among bachelor of pharmacy students (9/14, 64.3%), followed by mbbs (7/15, 46.7%), doctor of veterinary medicine (7/18, 38.9%), and bachelor of nursing science students (5/13, 38.5%). the frequency of nasal mdr-s. aureus in female students (19/41, 46.3%) was relatively higher than in male students (12/38, 31.6%). students within the age range of 22–27 years had the highest prevalence of nasal mdr-s. aureus (19/47, 40.4%), followed by those 16–21 years (11/28, 39.2%), and those 28–33 years, (1/4, 25%) (table 3). students who resided in suburban areas had the highest prevalence of nasal mdr-s. aureus (6/12, 50%), followed by those living in rural areas (5/13, 38.5%), and those in urban settlements (20/54, 37%). students in the clinical phase of their training had a closely similar frequency of nasal mdr-s. aureus carriage (15/38, 39.4%) to those in the preclinical phase (16/41, 39%). of those in the clinical phase of their training, those that had been on clinical/laboratory posting for 4 years (1/2, 50%) had the highest frequency of mdr-s. aureus, while the lowest was recorded in students who had been on posting for 2 years (3/10, 30%) (table 3). students who resided in houses with densities of 5–8 individuals (24/51, 47.1%) had the highest frequency of mdr-s. aureus, followed by those that resided in houses with 1–4 individuals (4/12, 33.9%). students who used antibiotics within the last 3 months before their enrolment into the study had a relatively higher frequency of nasal mdr-s. aureus carriage (44.4%), compared with those with no antibiotic use 3 months before enrolment in the study (34.9%). students who had prolonged contact with pets or livestock had a relatively higher frequency of nasal mdr-s. aureus carriage (17/41, 41.5%) than those who did not (14/38, 36.8%). both mbbs and bachelor of pharmacy students had statistically significant high frequencies of nasal mdr-s. aureus (both p < 0.05). furthermore, students who resided in houses with a density of 5–8 individuals had the highest odds of nasal mdr-s. aureus carriage (odds ratio = 3.85, 95% confidence interval: 0.99–13.66, p = 0.0044) (table 3). table 3: frequency of culture-positive mdr-staphylococcus aureus and factors associated with its nasal carriage in the healthcare students in zaria, nigeria, from january 2024 to july 2024. antimicrobial resistance rates in staphylococcus aureus isolates out of the 79 s. aureus strains identified, the pattern of resistance to tested antimicrobial agents, in descending frequency, was as follows: penicillin (93.7%), cefoxitin (73.4%), tetracycline (49.4%), ciprofloxacin (29.1%), gentamicin (10.1%), clindamycin (8.7%), erythromycin (7.6%), chloramphenicol (3.8%), erythromycin-constitutive clindamycin (2.5%), erythromycin-inducible clindamycin resistance (1.3%), high-level mupirocin (1.3%). all strains were susceptible to linezolid, and one strain (1.3%) was completely susceptible to all the antibiotics tested (figure 2). about 39.2% of strains presented the mdr phenotype, of which one strain presented an erythromycin-inducible clindamycin resistance (figure 2). over 30% of the s. aureus strains had similar mdr phenotypes (table 2). figure 2: antimicrobial resistance rates in 79 staphylococcus aureus strains obtained from the participants in zaria, nigeria, january 2024 to july 2024. eight (10.1%) of the s. aureus strains were solely resistant to penicillin, and one (1.3%) to cefoxitin, with a mar index of 0.1 (table 4). two strains had the highest antimicrobial resistance profile, with a mar index of 0.6 for resistance to penicillin-cefoxitin-tetracycline-clindamycin-gentamicin-ciprofloxacin and penicillin-cefoxitin-tetracycline-erythromycin-clindamycin-ciprofloxacin (table 4). strains with a mar index of 0.2 were the most predominant (29.1%), followed by strains with a mar index of 0.3 (24%), 0.4 (18.9%), 0.5 (12.6%), and 0.6 (2.5%) (figure 3). table 4: antimicrobial resistance profile and multiple antimicrobial resistance indices of the staphylococcus aureus isolates from the study participants in zaria, nigeria, january 2024 to july 2024. figure 3: levels and frequency of multiple antimicrobial resistance indices of staphylococcus aureus isolates in zaria, nigeria, january 2024 to july 2024. discussion an overall prevalence of 31.5% for carriage of s. aureus and 23.5% for carriage of mrsa were obtained in this study. subgroup analysis showed that nursing science students had the highest prevalence of both s. aureus and mrsa carriage. to our knowledge, this is the first epidemiological study on the mar indices of nasal s. aureus colonisation rate in nigeria. the 31.5% prevalence of s. aureus recorded in this study is similar to the results of studies done among healthcare students identified in medical and health science students in plateau and delta states of nigeria in 2018 (37.3%) and 2024 (38.5%).21,22 however, the finding from the present study is similar to the nasal s. aureus carriage reported in other countries such as nepal in 2011 (35%), and poland in 2018 (30%).23,24 a relatively lower prevalence of 16.8% was reported in medical students at kaduna state university (nigeria) in 2024, and 14% at babcock university (nigeria) in 2020.25,26 furthermore, lower prevalence values were reported in china (23.1%), thailand (29.7%), ethiopia (27.1%), turkey (21.4%), germany (22.3%) and saudi arabia (25.3%) from 2011 to 2022.27,28,29,30,31,32 much higher nasal s. aureus carriage was reported by ogefere and ogunleye (54.7%)33 and solomon et al. (66.6%),34 both in 2019 in nigeria, and 50% by rosales-gonzález et al.12 in 2024 in spain. the variations observed in the nasal s. aureus carriage from these studies could be because of differences in the study protocols, sample size, laboratory methods for s. aureus identification, and levels of antibiotic use by the students. in the present study, there were higher odds of nasal s. aureus carriage with age and household density of the students. a similar finding was reported in china in 2017, where medical students of the age group 20–30 years were found to have a higher nasal s. aureus carriage.35 it was previously reported that the nasal s. aureus carriage tends to increase with age and population density.36 households and communities in congested conditions are more likely to be exposed to s. aureus colonisation, with an increased transmission rate.37,38 although not statistically significant, there was a trend for higher s. aureus colonisation in the nasal cavity in female students, and those who resided in rural settlements. a similar study in madagascar previously reported higher s. aureus nasal carriage in female medical students and healthcare workers in 2016.39 the higher rate in female students could be because of biological reasons or differences in susceptibility to s. aureus colonisation as a result of circulating steroid hormones.40 the higher s. aureus rate among students who were rural residents could be because of the relatively higher contact rate of the students with animals in the rural areas, and poor personal and environmental hygiene that could facilitate the increased transmission of s. aureus.41 this is similar to the findings of ansari et al.42 in 2016, who reported a higher nasal s. aureus carriage rate in medical students who were rural residents than in their urban counterparts. the overall prevalence of mrsa recorded in this study is 22.7%, and comparable to 26.2% (delta state of nigeria), and 27.1% (ethiopia).22,31 however, a higher prevalence of nasal mrsa (48.8%) was reported by solomon et al.,34 while a lower value (12.2%) was reported by rosales-gonzález et al.12 several factors have been linked to increased s. aureus carrier status, including sinusitis, antimicrobial treatments, prolonged contact with animals, smoking, and contact with healthcare professionals.29,43,44 although not statistically significant, nasal mrsa carriage was higher in students with antibiotic usage in the previous 3 months. this was similar to studies in ethiopia (2023) and malaysia (2012) that found that recent antibiotic usage was associated with higher nasal carriage of mrsa.45,46 furthermore, students who reported prolonged contact with animals (pets or livestock) had a relatively higher frequency of nasal mdr-s. aureus carriage than those who did not. this finding is not unexpected, as occupational or companionship contact with animals is a major risk factor for mdr-s. aureus.47 it is important to remark that the prevalence of mdr-s. aureus was statistically significantly higher in pharmacy and mbbs students. similar findings were reported by obajuluwa, parom and kubau (2024) in nigeria.25 moreover, mdr-s. aureus was significantly associated with households with 5–8 individuals. these reflect the degree of contact with patients in the clinical phase of training, as students who had 4 years in clinical posting had the highest mdr-s. aureus carriage. this finding indicates that once healthcare students start interacting directly with patients in a clinical setting, their likelihood of carrying mdr-s. aureus is much higher compared to before their clinical exposure as a result of the increased contact with other potential mdr-s. aureus carriers in healthcare settings, such as healthcare professionals (especially physicians and nurses), patients, and contaminated environment surfaces and equipment. furthermore, high household density can facilitate the transmission of mdr strain because of increased contact with many individual carriers within the same rooms and houses. the strong antibiotic selective pressure among congested populations produces a suitable condition that could facilitate the emergence and efficient dissemination of many mdr bacteria, such as mdr-s. aureus.48 generally, up to 30% of the human population is asymptomatic and permanently colonised with s. aureus in their nostrils.49 however, mdr-s. aureus strains, especially mrsa, can be translocated from the nose to other parts of the body, causing infection or facilitating transmission to other humans, animals, and the environment through nasal secretion droplets during sneezing, coughing, kissing, or handshaking.50,51,52 most of the s. aureus isolates were resistant to penicillin and ciprofloxacin. this could be explained by the widespread and indiscriminate use of these classes of antibiotics in the treatment of s. aureus.49 similar findings were found in many other studies in nigeria (2011 to 2024).24,25,34,53 the presence of mupirocin-resistant s. aureus (1.4%) may render mupirocin less effective as a nasal decoloniser or as topical treatment for superficial skin infections.54 fortunately, linezolid resistance was not detected in any of the s. aureus strains and very low resistance (only one student) to chloramphenicol was recorded. however, tetracycline resistance was high. this suggests that our research participants had prior contact with livestock, as tetracycline resistance is more frequent in livestock breeders than in healthy humans with no occupational contact with livestock.55 this is because tetracycline is a very common antimicrobial agent used in livestock husbandry, prophylaxis, and as a first-line treatment of most bacterial infections. consequently, bacteria (including s. aureus) found in farm settings (including farmers) exhibit high selective pressure against tetracycline.55 remarkably, one strain presented a characteristic erythromycin-inducible clindamycin resistance. this phenotypic feature could be useful as a marker for the surveillance of s. aureus cc398 lineage.55 however, this finding is subject to further molecular confirmation, such as the screening for the ermt and ermc genes.55,56 multidrug resistance was high in the s. aureus carriers, as some isolates presented resistance against five or six classes of antibiotics (mar index ≥ 5). therefore, mdr-s. aureus may limit the available chemotherapeutic options against staphylococcal and many other gram-positive bacterial infections.53 this shows that most of the s. aureus strains originated from areas with high antibiotic pressure.16 recommendations first, it is recommended to consider the improvement of infection prevention and control measures in the wards, clinics, laboratories, pharmacies, and veterinary hospitals where healthcare students undertake training. furthermore, the implementation of active hospital and antimicrobial resistance surveillance systems should include all medical, and other groups of healthcare students as potential reservoirs of nasal mdr-s. aureus and mrsa. second, it will be interesting to determine the clonality of mrsa strains with similar antimicrobial resistance profiles from different students. hence, pulse-field gel electrophoresis and/or whole phylogenomic analysis could provide insight into the potential transmission route of these strains. limitations this study is not without limitations. first, the use of phenotypic identification of antimicrobial resistance using disk diffusion may not account for some antimicrobial agents that could be phenotypically susceptible when tested by the disk diffusion method but could still carry the antimicrobial resistance genes. for instance, linezolid resistance is best identified by the determination of minimum inhibition concentrations through broth dilution tests or e-tests. second, the use of automated bacterial identification systems such as bactec and vitek ii compact, and advanced equipment such as the matrix-assisted laser desorption ionisation-time of flight (mass spectrometry could have allowed the possibility to determine the performance characteristics of combined tube coagulase test and strong mannitol fermentation in the identification of s. aureus. conclusion high levels of nasal mrsa and mdr-s. aureus were obtained from this study. the predominance of strains with high mar indices indicates that strains were from locations with much antibiotic pressure. furthermore, many strains with similar antimicrobial resistance profiles could indicate potential interhost transmission within human clusters either at the community level or clinics and laboratories where these students were performing their postings. collectively, these students could serve as vectors of transmission of mdr and virulent strains to the patients or the community. based on this, it could be inferred that the medical and health science students are a potential source of mrsa strains at ahmadu bello university, zaria, nigeria.7 acknowledgements we appreciate the technical support provided by medical laboratory scientists of the microbiology laboratory of ahmadu bello university teaching hospital, zaria, nigeria. this article is partially based on the author’s thesis entitled, ‘nasal carriage and antimicrobial resistance pattern of staphylococcus aureus among health sciences students in ahmadu bello university, zaria’, towards the degree of bachelor of medical laboratory science in the department of medical laboratory science, ahmadu bello university, zaria, nigeria, received on 19 december 2024, with supervisor, dr idris nasir abdullahi. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions i.n.a. and s.a. were involved in conceptualisation, methodology, validation, software, formal analysis, data curation, funding acquisition and data visualisation, as well as writing the original draft, and reviewing and editing of the final submission. h.a.a., n.b., a.d., s.s., y.u., a.e.a., m.m.a. were involved with validation, and formal analysis, as well as writing the original draft, and reviewing and editing of the final article. i.n.a. was responsible for supervision and project administration. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the authors confirm that the data supporting the findings of this study are available within the article. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. the article does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the authors are responsible for this article’s results, findings, and content. references 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emma kalk1 sonjiha khan1 gayle sherman1,2,3 affiliations: 1paediatric hiv diagnostic syndicate, wits health consortium, johannesburg south africa 2department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa 3national health and laboratory service, johannesburg, south africa correspondence to: kapila bhowan postal address: po box 1474, glenvista 2058, south africa dates: received: 31 may 2011 accepted: 17 nov. 2011 published: 15 dec. 2011 how to cite this article: bhowan k, kalk e, khan s, sherman g. identifying hiv infection in south african women: how does a fourth generation hiv rapid test perform? afr j lab med. 2011;1(1), art. #4, 5 pages. http://dx.doi.org/10.4102/ ajlm.v1i1.4 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. identifying hiv infection in south african women: how does a fourth generation hiv rapid test perform? in this original research... open access • abstract • introduction • methods    • study participants    • sample size    • hiv testing    • interpretation of rapid test    • analysis • results • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: hiv rapid tests (rt) play an important role in tackling the hiv pandemic in south africa. third generation rt that detect hiv antibodies are currently used to diagnose hiv infection at the point of care. determine combo (dc) is the first fourth generation rt that detects both p24 antigen (p24ag) and hiv antibodies (ab), theoretically reducing the window period and increasing detection rates. early detection of maternal hiv infection is important to mitigate the highrisk of vertical transmission associated with acute maternal infection. objectives: we assessed the performance of the dc rt against third generation rt in antenatal and post-partum women. methods: third generation rt advance quality and acon were used in a serial algorithm to diagnose hiv infection in antenatal and post-partum women over six months at a tertiary hospital in johannesburg, south africa. this data provided the reference against which the dc rt was compared on plasma and whole bloodsamples. results: the 1019 participants comprised 345 (34%) antenatal and 674 (66%) post-partum women. ninety women (8.8%) tested hiv-positive of whom 59 (66%)were tested antenatally, and 31 (34%) post-partum yielding prevalence rates of 17.1% and 4.6% respectively. the sensitivity and specificity of the ab component of dc on plasma antenatally was 100% (93.8% – 100%) and 100% (98.6% – 100%) respectively and post-partum was 100% (88.9% – 100%) and 99.6% (98.8% – 99.9%) respectively. one false positive and not a single true positive p24ag was detected. of 505 post-partum women who tested hiv-negative 6–12 months prior toenrolment, 12 (2.4%) seroconverted. conclusion: the fourth generation dc offered no advantage over current third generation rt in the diagnosis of hiv infection. introduction top ↑ hiv rapid tests (rt) play an important role in addressing the hiv and aids pandemic in south africa. they can be conducted at the point of care because theyare easy to perform and require no special instrumentation. the advantage of point of care rt is that the patient can receive their hiv test result at the same clinic visit, which reduces loss to follow-up and fast tracking patients into care.1 rt are less costly than laboratory-based assays for antibody (ab) detection namely hiv enzyme-linked immunosorbent assays (elisa) and viral detection namely hiv dna or rna or p24 antigen (p24ag). in south africa, pregnant women are offered counselling and testing for hiv at their first antenatal clinic visit and at 34 weeks of pregnancy if their initial hiv test was negative.2 women at the rahima moosa mother and child hospital (rmmch) in johannesburg, south africa are also offered an hiv test immediately post-partum if their hiv status is unknown or more than six weeks have elapsed since their last negative hiv test. the importance of hiv retesting has been demonstrated by a south african study in which 3.4% of women who tested hiv-negative at their first antenatal visit, subsequently seroconverted during pregnancy or within a year after delivery.3 the advance quality hiv rapid test (in tec products, inc. xianen, china) and acon hiv 1/2/0 tri-line rapid test (acon laboratories, inc., san diego, usa) are currently used to diagnose hiv infection in women in prevention of mother-to-child transmission (pmtct) programmes in gauteng province, south africa. these third generation rt detect hiv ab that are produced in response to the virus by a serial testing algorithm as recommended by the south african pmtct guidelines2 (figure 1). advance quality is used to screen for hiv ab and if positive, the acon test is performed to confirm hiv status. laboratory-based hiv elisa and, less commonly, viral detection assays can be used as a tiebreaker to confirm an hiv status if serial rt results are discordant.2 the determine combo hiv-1/2 ag/ab combo test (dc) (inverness medical, japan co., ltd) is the first fourth generation rt and can be performed on either plasma or whole blood samples.4 it is an enhancement of the third generation tests as it can detect both hiv ab and p24ag in a single test.5 the p24ag is a marker of early hiv infection and is detectable in blood during the window period before hiv ab become detectable.6 the dc rt is reported to have the potential to identify hiv infection five days (range 2–20 days) earlier than third generation rt. the reported sensitivity and specificity of the dc ab component is 100% (95% confidence interval 98% – 100%) and 100% (95% confidence interval 98.2% – 100%) respectively and the sensitivity of the p24ag component is 86.6% (95% confidence interval 76% – 93.7%).7 detection of early hiv infection during the window period of third generation rt would allow more maternal hiv infections to be detected. furthermore, during early maternal hiv infection the levels of the virus in the blood are at their peak and the risk of transmission to the infant during birth and breast-feeding is very high.8 detection of early infection would allow more women and their infants at a high risk of vertical transmission to access pmtct. the performance of the fourth generation hiv dc rt in diagnosing hiv infection status antenatally and in the early post-partum period in comparison to the third generation hiv rt in routine use at public healthcare facilities in gauteng, south africa was assessed. the advantage offered by the dc rt over third generation rt of detecting hiv infection earlier to increase identification of women at a high risk of vertical transmission for pmtct was investigated. figure 1: study methodology: the determine combo rapid test (dc rt) was performed at the same time as the routine algorithm. methods top ↑ study participants women attending the antenatal clinic and delivery unit at rmmch in johannesburg were invited to test for hiv infection. counsellors interviewed the participants to establish their hiv status. women with an unknown hiv status, those who had tested hiv-negative more than 6 weeks previously and those who reported an hiv-positive status but had no documented evidence thereof on their maternal card were invited to participate in the study. women with a documented hiv-positive status were excluded. written informed consent was obtained from all participants who agreed to test for hiv infection. ethics approval (m091119) for the study was granted by the human research ethics committee at the university of the witwatersrand, johannesburg. sample size the prevalence of hiv infection amongst antenatal women in gauteng province, south africa in 2008 was 29.9% (95% confidence interval 28.4% – 31.2%)9; however, the prevalence of hiv infection amongst women testing antenatally and shortly after delivery at rmmch in 2008 was lower at 15% and 4.2% respectively since women with a known, documented hiv-positive status are excluded and proportionately more women fall into this category after delivery than antenatally.10 from rmmch hiv testing records we expected approximately 200 women to present for testing per month with a prevalence that depended on the proportion of women testing before or after delivery. a convenience sample of all women eligible for hiv testing at rmmch who agreed to participate in the study was chosen to assess the number of additional women the dc rt could identify as being infected over a 6-month period on plasma samples. for assessment of whole blood samples, the centers for disease control and prevention recommendations to include samples from at least 20 hiv-infected and 80 hiv-uninfected women were followed.11 hiv testing five millilitres of whole blood was drawn into an ethylenediaminetetra acetic (edta) tube for testing at the study site. samples were centrifuged to obtain plasma on which the rt were performed. dc rt were performed on whole blood prior to centrifugation. all rt were performed within one hour of blood sampling by a single laboratory technician according to the manufacturer’s instructions. third generation rt advance quality and acon were used serially on plasma according to the national testing algorithm2 to diagnose hiv infection (figure 1) and were the reference standards against which the dc results were compared. interpretation of rapid test the dc is a qualitative immunochromatographic test which is read visually. the test strip is divided into an hiv ab window and an hiv p24ag window. the presence of a pink line in either or both of the windows is indicative of hiv infection. each test strip incorporates a procedural positive control and the test is considered valid only if the positive control is detected. the women received their rt results and post-test counselling within four hours of blood sampling. patients that were hiv-positive on both third generation rt were referred to antiretroviral treatment clinics. the results of the dc rt were not disclosed to the patient. however, where the third generation and dc hiv ab rt results were discordant, samples were referred for confirmatory fourth generation elisa (architecht® hiv ag/ab combo assay; abbott diagnostics; wiesbaden, germany). patients that had detectable p24 ag on dc were followed up with three confirmatory tests that is viral detection assay (vironostika hiv-1 antigen; biomerieux; bosiend, the netherlands) , viral load testing (nuclisens easyq-easymag hiv-1, version 1.2 assay; biomerieux; boxtel, the netherlands) as well as fourth generation elisa. disclosure of the patient’s hiv status was delayed for 48 hours. analysis likelihood ratios were calculated instead of predictive values because predictive values depend on prevalence and a difference in hiv prevalence was anticipated between women tested in the antenatal and post-partum period. a positive likelihood ratio (sensitivity/(1 – specificity)) > 10 strongly predicts hiv-infection, whereas a negative likelihood ratio ((1 – sensitivity)/specificity) < 0.1 virtually excludes the condition. results top ↑ between march and august 2010, 1019 (92.7%) of the 1099 women eligible for hiv testing at rmmch were enrolled in the study. of the 1019 participants, 345 (33.9%) were tested antenatally and 674 (66.1%) post-partum. according to the routine third generation rt diagnostic algorithm, 90 (8.8%) of the 1019 patients tested positive for hiv infection without the need to use a tiebreaker. of the 90 hiv-infected women, 59 (65.6%) were antenatal and 31 (34.4%) were post-partum. the hiv prevalence amongst the women tested antenatally was 17.1% and those tested post-partum was 4.6%. knowledge of the women’s hiv status prior to undergoing hiv testing on the study was documented for those tested in the early post-partum period only (table 1). of the 505 women that had reported or tested hiv-negative between 6 and 12 weeks prior to study enrolment, 12 (2.4%) tested positive demonstrating that new hiv infections were occurring in this population. not all of these women had their negative hiv status documented on their maternal record and the possibility that some reported their status incorrectly cannot be excluded. the dc rt was performed on plasma samples of the 1019 women and on whole blood samples on a subset of 380 women. all 1399 tests performed demonstrated positive control strips therefore no dc rt was invalid. sensitivity, specificity and likelihood ratios for the dc rt were calculated separately for women who were tested antenatally and post-partum (table 2). the sensitivity of the dc rt ab component was 100% in all groups of women tested irrespective of the sample type. the specificity of the dc rt ab component in women tested post-partum was less than those tested antenatally. the dc rt results concurred with the third generation rt results in all patients except for three post-partum women on whom both plasma and whole blood samples were tested. in two patients the ab component of the dc rt was positive on both their plasma and whole blood samples but negative on both third generation rt. laboratory based fourth generation elisa tests on both cases were negative confirming two false positive dc hiv ab results. the dc p24ag was reactive on plasma and the whole blood of only one patient of all 1019 women tested. this patient was suspected of having an early infection since both third generation rt and the dc ab component tested negative. however the laboratory based fourth generation hiv elisa was negative and the viral load was undetectable indicating a false positive dc p24ag result. no p24ag bands on dc were obtained on any of the 90 hiv-infected women nor was a single case of early hiv infection detected. table 1: hiv status of post-partum women prior to enrolment and after testing with the national testing algorithm. table 2: performance of determine combo rapid test hiv antibody (dc rt ab) component in plasma and whole blood samples in women tested antenatally and post-partum. discussion top ↑ considering the high enrolment rate, the sample of women tested in this study is likely to be representative of women who are tested for hiv-infection at rmmch over half a year. furthermore, the hiv prevalence of 17.1% and 4.6% in women testing antenatally and immediately post-partum respectively is similar to a previous description at rmmch.10 an hiv prevalence of 24.1% in women of unknown hiv status is also comparable to the 28% prevalence previously described in 2008. however, the seroconversion rate in post-partum women of 2.4% is less than the previously described rate of 4.5%.10 the implication that new maternal infections are occurring in this population remains. in practice, a positive p24ag dc test would require confirmation of early seroconversion by fourth generation elisa or nucleic acid testing which may delay initiation of pmtct. in contrast to a study that reported 16% invalid dc rt tests due to failure to detect the control,12 all dc rt in this study were valid possibly because we used fresh, not stored samples. sensitivity and specificity of the dc rt was comparable to plasma and whole blood in contrast to a previous report that demonstrated lower sensitivity of the dc rt in whole blood compared to serum samples.12 the sensitivity of the fourth generation dc rt in detecting hiv ab was comparable to that of the reference third generation rt in antenatal and post-partum women in plasma and whole blood samples. the specificity of the dc rt in detecting hiv ab was slightly reduced in whole blood and plasma in post-partum women owing to the false positive results in two women, but was still within the world health organization recommended range of more than 98%.13 the fourth generation dc rt did not detect a single true positive p24ag, even in the 90 hiv-infected women. the sensitivity in detecting p24ag in hiv-infected women was 0% as compared with the claim of 86.6% obtained on hiv-infected samples.7 the reason for this may be that p24ag forms immune complexes with hiv ab and thus no free p24ag is present for detection by the dc.6 the dc rt therefore did not identify any new cases of maternal hiv infection over the 6 month study period. possible reasons for this include that the sensitivity of the dc p24 ag component is poor or that no women with acute hiv infection were enrolled. the former concurs with previous reports that the dc rt p24 ag component lacks sensitivity particularly where levels of p24 ag are below 400 pg/ml.12,14 additionally, the p24ag component of the dc rt has reduced sensitivity in comparison to other fourth generation viral detection assays,7 some of which are able to detect p24ag at levels of 4 pg/ml – 5 pg/ml.15 the initial laboratory based p24ag assays also demonstrated poor sensitivity that was subsequently improved by denaturation of the immune complex and signal amplification to enhance p24ag detection.16 a limitation of this study is that the incidence of hiv infection in women undergoing hiv testing at rmmch is unknown therefore, although new maternal infections were demonstrated, it is possible that no women with acute hiv infection were enrolled during the short window period before hiv ab and subsequent immune complex formation. nevertheless, the dc rt did not identify any new infections over those identified by the 3rd generation rt assays over six months in the rmmch pmtct programme. conclusion top ↑ the dc rt failed to demonstrate any advantage over third generation rt currently in use in our setting in either determining hiv infection status or in identifying recently infected women. improved sensitivity of p24 ag detection is required before fourth generation rt will offer an advantage over their third generation counterparts in the field. acknowledgements top ↑ funding for this study was provided by the national health laboratory services. we thank all the women who participated and the clinical staff who were involved. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. authors’ contributions all of the authors have made substantial contribution to the manuscript. g.s. (wits health consortium) conceptualised and planned the study as well as performed critical revision of the manuscript. k.b. (wits health consortium) analysed and interpreted the study data and prepared the manuscript. s.k. (wits health consortium) performed the testing of samples, maintained quality assurance in the laboratory and performed critical revision of the manuscript. e.k. (wits health consortium) assisted with patient recruitment and management in the field as well as contributed to the writing and editing of the manuscript. references top ↑ 1.branson b. point-of-care rapid tests for hiv antibody [homepage on the internet]. c2003 [cited 2011 jan 02]. available from: http://www.cdc.gov/hiv/topics/testing/resources/journal_article/j_lab_med_20031.htm 2.national department of health. clinical guidelines: pmtct (prevention of mother-to-child transmission) [document on the internet]. c2010 [cited 2011 apr 26]. available from: http://www.doh.gov.za/docs/factsheets/guidelines/pmtct.pdf 3.moodley d, esterhuizen t, reddy l, et al. incident hiv infection in pregnant and lactating women and its effect on mother-to-child transmission in south africa. j infect dis. 2011;203(9):1231–1234. http://dx.doi.org/10.1093/infdis/jir017, pmid:21398393 4.inverness medical. determine hiv-1/2 ag/ab combo test package insert. in: package insert i. japan: inverness medical group, 2009; p. 1–7. 5.guidasci t. fourth generation point of care hiv screening. cli clinical laboratory international [homepage on the internet]. c2009 [cited 2011 apr 28]. available from: http://www.cli-online.com/index.php?id=2715 6.mcrae b, lange j, ascher m, et al. immune response to hiv p24 core protein during the early phases of human immunodeficiency virus infection. aids res hum retroviruses. 1991;7(8):637–643. http://dx.doi.org/10.1089/aid.1991.7.637, pmid:1931233 7.beelaert g, fransen k. evaluation of a rapid and simple fourth-generation hiv screening assay for qualitative detection of hiv p24 antigen and/or antibodies to hiv-1 and hiv-2. j virol methods. 2010;168(1–2):218–222. http://dx.doi.org/10.1016/j.jviromet.2010.06.002, pmid:20561542 8.volmink j, sigfried nl, merwe l, brocklehurst p. antiretrovirals for reducing the risk of mother-to-child transmission of hiv infection. [cochrane review] in: the cochrane library, issue 1, 2007. oxford: update software. pmid:17253490 9.department of health. national antenatal sentinel hiv and syphilis prevalence survey in south africa, 2009. pretoria: national department of health; 2010. 10.technau kg. can a routine peri-partum hiv counselling and testing service for women improve access to hiv prevention, early testing and treatment of children? msc dissertation, johannesburg, university of witwatersrand, 2009. 11.centers for disease control and prevention. guidelines for appropriate evaluations of hiv testing technologies in africa. department of health and human services; 2001. 12.pavie j, rachline a, loze b, et al., sensitivity of five rapid hiv tests on oral fluid or finger-stick whole blood: a real-time comparison in a healthcare setting. plos one. 2010;5(7):e11581. http://dx.doi.org/10.1371/journal.pone.0011581, pmid:20657834, pmcid:2906506 13.world health organization. guidelines for hiv diagnosis and monitoring of antiretroviral therapy: revised version 2009. geneva, switzerland: who; 2009. 14.tardy j. [hiv rapid testing of fourth generation]. journee nationale d’infectionology lyon [document on the internet]. c2009 [cited 2011 apr 27]. available from: http://www.infectiologie.com/site/medias/jni/jni09/vih/tardy-depist-jni09.pdf. french. 15.schupbach j, boni j. quantitative and sensitive detection of immune-complexed and free hiv antigen after boiling of serum. j virol methods. 1993;43(2):247–256. http://dx.doi.org/10.1016/0166-0934(93)90080-b 16.schupbach j. measurement of hiv-1 p24 antigen by signal-amplification-boosted elisa of heat-denatured plasma is a simple and inexpensive alternative to tests for viral rna. aids rev. 2002;4(2):83–92. pmid:12152521 abstract introduction a pathway to outbreak use in vitro diagnostics outbreak recognition development of in vitro diagnostics evaluation of in vitro diagnostics emergency use authorisation and emergency use assessment and listing country assessment previously unassessed in vitro diagnostics implementation monitoring conclusion acknowledgements references about the author(s) elliot p. cowan partners in diagnostics, llc, rockville, maryland, united states citation cowan ep. a framework for the assessment and implementation of diagnostics in outbreak situations. afr j lab med. 2016;5(3), a494. http://dx.doi.org/10.4102/ajlm.v5i3.494 lessons from the field a framework for the assessment and implementation of diagnostics in outbreak situations elliot p. cowan received: 16 may 2016; accepted: 12 aug. 2016; published: 31 oct. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract observation: outbreak situations require in vitro diagnostics (ivds) to identify those who are infected and to track the infectious agent in the population. however, such ivds are typically not available and must be developed. in addition, the process of ivd development, assessment, and implementation are very time and resource intensive. recognising the extraordinary public health need for ivds in an outbreak situation, streamlined processes are needed to provide tests that meet the standard of a reasonable assurance of safety and effectiveness in the shortest amount of time. these ivds are designated for outbreak use. addressing issues: this paper presents a pathway to the outbreak use of ivds that can be considered by countries experiencing an outbreak situation. it takes into account recognition of the outbreak, product development, regulatory evaluation, implementation, and monitoring of the outbreak-use test. streamlined assessment programmes for emergency-use tests have been established by the us food and drug administration and the world health organization. these programmes take into account test requirements for the country in which the outbreak exists. therefore, countries can consider adopting these tests without the need to conduct expensive and time consuming assessments, such as performance studies. key responsible parties are identified for each step of the pathway, recognising that transparency and communication among all parties are critical. introduction medical devices used for in vitro diagnostics (ivds) are at the frontline of medical decision-making, which ranges from individual patient management to addressing public health crises. this central role for ivds means that they must be safe, effective and reliable. regulatory oversight of ivds provides assurance by assessing both the performance claims made by manufacturer and the systems they have in place in their facilities to maintain product quality. however, all ivds are not regulated in the same way. typically, the higher the risk associated with an ivd, the more detailed the assessment. this approach should be flexible in an outbreak situation. outbreaks are associated with infectious agents that pose a grave danger to the population, and therefore the risk associated with an ivd that will be used to assist in the diagnosis of affected individuals is high. however, there is also an urgent need to make the ivd available as quickly as possible. time is of the essence to limit the spread of the infectious agent and its associated morbidity and mortality. in this case, it is not possible for the assessment stringency to be consistent with the risk. there is an urgent need, then, to dramatically shorten the review process while ensuring the benefits of the ivd outweigh its risks. this paper describes a pathway for the evaluation and regulation of ivds essential to respond to an infectious disease outbreak when no adequate, approved diagnostic tests for that infectious agent are currently available. a pathway to outbreak use in vitro diagnostics an outbreak-use ivd is an ivd that is restricted to use during an outbreak situation to meet the critical public health need posed by the outbreak. it is a test that has been determined to have a reasonable assurance of safety and effectiveness in the shortest possible time. ‘reasonable assurance’ means the information provided for assessment will be: (1) limited only to that necessary to demonstrate that the ivd is capable of fulfilling the goal of correctly identifying as many infected individuals as the product technology is capable of; and (2) sufficient to show that the benefits of using the product outweigh its risks. ‘shortest possible time’ means identifying and overcoming test development and performance assessment barriers and using a process that has a clear, direct path, recognising the extraordinary threat to public health posed by the outbreak. outbreak-use ivds should be restricted to use during the outbreak. when the outbreak has officially ended, outbreak-use ivds should be neither distributed, nor procured, nor used for clinical purposes. rather, the manufacturer is encouraged to seek a general clinical use claim for its test. however, valuable information on test performance can be obtained during outbreaks to support a conventional regulatory assessment. figure 1 shows the overall approach for implementing ivds for outbreak use. figure 1: pathway toward implementation of outbreak-use in vitro diagnostics. each of these steps will be examined in the context of the development of tests that have a reasonable assurance of safety and effectiveness. this pathway is not specific to a particular infectious agent, but rather is a general framework intended to be applicable to any outbreak situation for which there is not a previously-accepted ivd. outbreak recognition an outbreak situation must be recognised and the infectious agent that is responsible for the outbreak identified to enable a cascade of actions that will facilitate both the development of tests and the regulatory assessment of those tests. this is a starting point for the process of ivd development, involving several parties. the united states department of health and human services, through the food and drug administration (fda), and the world health organization (who), each has a mechanism to evaluate/authorise products for use in emergency situations and these authorised products can play a critical role in an outbreak (see below). however, the fda, with input from either the us centers for disease control and prevention (cdc) or other us government agencies, and who must each officially recognise that a public health outbreak situation exists in order to invoke their respective review and performance evaluation mechanisms. therefore, public health authorities in the country experiencing the outbreak must make the cdc (or other us agencies) and the who aware of the outbreak to enable them to recognise the outbreak situation as soon as possible to enable their respective streamlined emergency test assessment procedures to be officially declared. manufacturers must receive from global public health authorities, such as the cdc and who, accurate information on the nature of the outbreak infectious agent, its biology and clinical manifestations. this is necessary to identify target(s) that are most appropriate for detection in infected individuals. regulatory authorities require this information to assess safety and effectiveness. therefore, consistent, accurate and detailed information about the infectious agent must be widely disseminated. in addition, manufacturers require clinical specimens to aid in test development and validate test performance. regulatory authorities require these specimens to aid them in their assessment and post-market activities. therefore, well-characterised clinical specimens must be made available and accessible from endemic areas through coordination by public health agencies working together with ministries of health. at the end of this phase: there should be a conclusive identification of the specific infectious agent, with as many accompanying details as possible. this should include the virulence of the organism and the potential danger to individuals obtaining and testing specimens. all appropriate stakeholders, including ivd manufacturers, will be informed of the outbreak and all available information on the infectious agent. the fda and who will recognise that an outbreak situation exists, to enable emergency authorisations for tests. the most appropriate diagnostic targets, intended use population(s), testing setting(s) and specimen type(s) will be identified. clinical specimens will be available to test manufacturers for test development, and there will be no administrative barriers to obtaining those specimens. development of in vitro diagnostics next, candidate ivds for the outbreak infectious agent are developed. however, this process takes time and cannot be expected to meet the immediate need. therefore, initial tests developed and deployed by public health agencies, such as the cdc, for research and surveillance purposes, once rapidly demonstrated to have diagnostic performance capabilities, will play a critical role in identifying and diagnosing infected individuals at the beginning of the outbreak. this will allow commercial test manufacturers the time needed to rapidly develop new tests to meet the outbreak need or modify existing test platforms to detect the outbreak infection and meet the needs of a potential surge in newly-infected individuals. existing test platforms may or may not have undergone a regulatory assessment. a target product profile must be developed that identifies the specific characteristics required for the outbreak use ivd and communicate it to potential manufacturers. potential test developers/manufacturers should be identified that are most likely to develop and produce an outbreak use ivd in the shortest possible time, preferably to have the capability to consistently produce a quality test at a high volume and have adequate distribution channels in the endemic areas. they should be provided with incentives to support an acceptable business case for taking on test development. these may include funding to offset the cost of well-characterised specimen procurement and in-field studies, if required, which are often the major costs in bringing diagnostic products to market. there are a number of additional challenges that must be addressed at this step, including technical barriers (for example, it is much easier to develop a nucleic acid test than a serological test); the time required to develop a test that is adequate to meet the needs of the outbreak situation; access to specimens from endemic areas for test development; the ability to manufacture the test at a scale sufficient to meet the need; and identifying manufacturers willing to develop and produce tests that will likely only be used during an outbreak situation. when successful, manufacturers will be prepared to submit their outbreak-use ivds for emergency regulatory assessment. evaluation of in vitro diagnostics all ivds for outbreak use should be assessed for safety and effectiveness to determine that the benefits of using the ivd outweigh the risks, and to provide some assurance that the ivd is manufactured under an appropriate quality system to assure that the products are produced, stored, and distributed in compliance with current good manufacturing practices. this is necessary to reasonably assure two equally important and essential elements: the test is capable of detecting the outbreak infectious agent; and it will consistently perform as expected. this is done through the review of scientific and surveillance information submitted by the test developer both before and after the regulatory evaluation. we can consider three routes to acceptance of an ivd for outbreak use: assessment of an ivd that has received an fda emergency use authorisation [eua], a mechanism that assesses medical products for use in designated public health emergency situations, including ivds for use in outbreaks.1 assessment of an ivd that has been found acceptable through the who emergency use assessment and listing (eual) mechanism, which also assesses medical products for use in designated public health emergency situations, including ivds for use in outbreaks.2 country assessment of an ivd that has not been previously assessed. figure 2 is an overview of these routes and the overall recommended steps a country may follow for acceptance of an outbreak-use ivd for each of the pathways. figure 2: assessment scheme for adoption of outbreak-use in vitro diagnostics. emergency use authorisation and emergency use assessment and listing as discussed above, the fda and/or who must recognise an outbreak situation in order to use its respective emergency assessment declaration mechanism for ivds. when that happens, the fda eua and who eual pathways are each designed to provide reasonable assurance of safety and performance for an ivd for outbreak diagnostic use (and limited to such use) in the regions where the outbreak has occurred. both the eua and the eual take into account such factors as outbreak agent diversity, the ability of the product to function (ease of use) and its stability in the outbreak area environment, and potential interfering conditions, among others, consistent with the target product profile. therefore, tests that have been successfully evaluated by the eua or eual processes should be considered appropriate for outbreak use without further assessment. a country should, however, obtain from the test manufacturer: official documentation from the fda or who to verify eua or eual status. a signed statement to attest that the outbreak use product provided to the country is identical to that evaluated for eua or eual (the same manufacturing site, the same product version, and the same manufacturing procedures). tests could then be accepted for outbreak use immediately upon verification of eua or eual status. however, it is important to note that the eua process does not assess stability of the ivd at appropriate conditions of temperature, humidity, dust, altitude and ruggedness for a particular country other than the united states. countries that have the ability and resources should conduct these studies for an eua product. countries that do not would be best served by implementing tests that have eual status, since the who process includes this necessary stability evaluation. country assessment previously unassessed in vitro diagnostics ivd manufacturers often seek acceptance for outbreak use directly from the country experiencing the outbreak without having eua or eual status for its test. in this case, countries should assess the candidate tests according to the level of stringency used by the eua and eual processes, which rely heavily on information submitted by the manufacturer. the country may choose to conduct its own performance assessment; however, these studies should be very limited and designed only to confirm that the test performs as expected. studies done by the country should not be intended to establish test performance. these studies consume valuable time and are unnecessary at this stage. rather, the focus should be on a limited verification of key manufacturer claims, including appropriate stability studies, as discussed above. it should be noted that countries affected by outbreaks often have limited capacity to assess ivds on their own and require the assistance of public health agencies to do so. when faced with an outbreak situation, countries should consider that both the fda and the who have established fast-track ‘emergency use’ review pathways to evaluate the known and potential benefits of tests, both commercial and non-commercial (such as those tests developed by the cdc), which can be used to diagnose emerging, life-threatening, infectious diseases when no available fda-approved/cleared test or who-prequalified test is available to healthcare providers. the authorisation for these tests is based on the totality of available scientific evidence presented to the fda or the who by the test developer and is available on-line to public health authorities assessing which test is the most suitable for use in their country.1,2 tests that have not been reviewed by a recognised regulatory body may certainly be assessed in-country, but it should be recognised that appropriate, well-designed studies may be costly and can delay the implementation of the ivd when it is critically needed. implementation while much of the responsibility for implementation lies with procurement bodies and agencies responsible for maintaining the supply chain, there are a number of actions affecting implementation that are related to the regulation of the outbreak-use ivd. communication of what tests are acceptable for outbreak use is necessary to ensure that only tests that have met acceptable criteria are used in the outbreak. for example, the fda establishes ‘conditions of authorisation’ for every eua test it evaluates. this may refer to who may distribute the test, who may use the test and which individuals should be tested. users must be trained in the operation of the test, including specimen preparation, running the test, and interpreting the test result. training must also include clearly communicating the limitations and capabilities of the test to ensure that test operators use the test properly and only with allowable specimens. successful implementation will see a distribution system that supplies adequate numbers of tests to areas affected by the outbreak, and users will be qualified to conduct and interpret the test. monitoring given the critical role of the ivd in the outbreak and the limited studies that supported its acceptance, the ivd should be monitored for performance after it is introduced into clinical use. monitoring can be addressed in a number of ways. each country must decide which of these options is appropriate to monitor tests in clinical use during the outbreak situation. adverse event reporting there is an expectation that the manufacturer will track adverse events (including any product quality issues, especially those that lead to incorrect test results) associated with its test. this is required for both eua and eual products, with reporting of events to the fda and who, respectively, and should be a requirement by countries assessing tests for outbreak use. to support this, while each country should generate a reporting system for adverse events and a monitoring plan for the outbreak use ivds it implements, there should also be a regional adverse event reporting system that coordinates responses to issues that arise quickly and effectively. however, adverse event reporting systems cannot work unless test users are trained to understand what constitutes an adverse event and how to identify and report product-related issues. there must also be a culture that encourages test users to report adverse events and any product-related issues, and the test labeling must have clear instructions on what to do in the case of an adverse event. periodic panel testing users periodically test panels of well-characterised specimens. this will assess the proficiency of test users and monitor ongoing test performance. lot testing newly-received test kit lots are tested using a panel of specimens as a condition for acceptance into the country for outbreak use. surveillance testing clinical specimens are periodically sent to a reference laboratory for testing to determine agreement with field test results (both specimens positive for the outbreak agent and specimens negative for the outbreak agent) to monitor for false positive and false negative test results. this is typically done from predetermined sites designated as surveillance sites. accumulation of performance data to support world health organization prequalification as a condition of eual, a manufacturer commits to filing for prequalification for its test. this means that the manufacturer will gather test performance data while the test is being used in the outbreak situation. fda similarly encourages developers to work towards gathering eua test performance data to obtain permanent status of clearance or approval for their test as, after the epidemic is declared over, the eua version of a test is no longer allowed to be on the market. monitoring should be implemented as soon as possible after introduction of the ivd into clinical use and continue throughout its use during the outbreak. conclusion each portion of the pathway that ultimately leads to the introduction of an outbreak-use ivd requires the involvement and cooperation of multiple bodies, each with critical roles and responsibilities. this includes public health agencies, ministries of health, regional regulatory bodies (such as the pan-african harmonisation working party3 ), the test user, and the test manufacturer. table 1 shows proposed roles and responsibilities. note that nearly every party is involved in some way in each of the steps of the outbreak-use pathway. this means that, whereas each participant in the pathway, by virtue of its expertise, has a particular and essential role in bringing forward and maintaining the quality of outbreak use ivds, success depends upon all of the participants working together. the foundation of this cooperative approach is transparency and communication. table 1: summary of roles and responsibilities for outbreak-use in vitro diagnostics a transparent process is one in which information is freely available to as many people as possible. in the context of an outbreak situation, this includes the identification of the outbreak agent, the clinical course of the disease, and availability of potential targets around which a test could be developed (i.e., nucleic acid sequences, antigens, etc.), variants of the agent, epidemiology (location, movement, etc.), well-characterised specimens for test development and monitoring of test performance once the outbreak-use ivd is in clinical use, and simple and clear policies and procedures to accept a test for outbreak use. communication is the tool for transparency, and is necessary at all levels. this includes communication among those identifying the outbreak source, between those identifying the outbreak source and the product developers, the product developers and the regulators, public health authorities and clinicians, and all involved parties and the public. of course, there are limits to transparency and communication. for example, a test manufacturer should not be expected to disclose proprietary information for its test. in general, though, a culture of information and specimen sharing by the countries most affected, and eventually the availability panels of well-characterised specimens for assay-to-assay comparison studies, are critical to the development, performance validation, performance review, and implementation of outbreak-use ivds. box 1: lessons learned. acknowledgements the author thanks dr sally hojvat for valuable discussions and a critical review of the manuscript. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. sources of support none. references us food and drug administration. emergency use authorization [page on the internet]. c2016 [updated 2016 may 13; cited 2016 may 15]. available from: http://www.fda.gov/emergencypreparedness/counterterrorism/ucm182568.htm world health organization. emergency use assessment and listing (eual) procedure for ivds [page on the internet]. n.d. [cited 2016 may 15]. available from: http://www.who.int/diagnostics_laboratory/eual/emergency/en/ pan african harmonisation working party on medical devices and diagnostics [homepage on the internet]. c2015 [cited 2016 may 15]. available from: http://www.pahwp.org abstract introduction methods results discussion acknowledgements references about the author(s) aderonke o. oluwo dental division, massey street children’s hospital, lagos, nigeria mary a. lawal medical division, massey street children’s hospital, lagos, nigeria cecilia a. mabogunje medical division, massey street children’s hospital, lagos, nigeria olubunmi t. okurame laboratory unit, massey street children’s hospital, lagos, nigeria citation oluwo ao, lawal ma, mabogunje ca, okurame ot. antibiotic susceptibility pattern among children admitted to a hospital in nigeria: a retrospective study. afr j lab med. 2024;13(1), a2362. https://doi.org/10.4102/ajlm.v13i1.2362 original research antibiotic susceptibility pattern among children admitted to a hospital in nigeria: a retrospective study aderonke o. oluwo, mary a. lawal, cecilia a. mabogunje, olubunmi t. okurame received: 06 nov. 2023; accepted: 24 may 2024; published: 20 aug. 2024 copyright: © 2024. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the impact of antimicrobial resistance on children living in resource-limited countries has been underreported, despite its established global threat. objective: this retrospective study aimed to describe the trend of antibiotic susceptibility in the paediatric age group. methods: sensitivity test report data consisting of 300 paediatric patients aged 18 hours to 192 months were retrieved from the microbiology laboratory records at a state-owned children’s hospital in nigeria over a period of 4 months starting from december 2021 to march 2022. five genera (escherichia coli, klebsiella spp., pseudomonas spp., staphylococcus aureus and streptococcus spp.) were cultured as recommended by the clinical laboratory standard institute, using the kirby bauer disc diffusion method. antimicrobial susceptibility testing was carried out on isolates using 15 different antibiotics. results: staphylococcus aureus was the most frequent pathogen isolated 32.1% (50/156) and pseudomonas spp. was the least frequent pathogen isolated 7.1% (11/156) in all samples. the isolates with the highest rate of resistance to the tested antibiotics were s. aureus 32.1% (50/156), e. coli 28.2% (44/156) and klebsiella spp. 20.5% (32/156). isolates in all age groups were more resistant to ampicillin, amoxicillin + clavulanic acid, cefuroxime and cefepime. conclusion: antibiotic resistance is high, especially the younger nigerian children. strict antibiotic protocols should be adhered to especially in the use of empirical antibiotic therapy in hospitals. what this study adds: our study reveals a higher trend of antibiotic resistance, especially in younger children. it further shows that the pathogens are most resistant to the most available empirical antibiotics in nigeria. keywords: antibiotic resistance; children; bacteria pathogens; susceptibility pattern; antibiotic therapy. introduction antimicrobial resistance (amr) is a global public health issue for both epidemiologic and economic reasons.1 the world health organization (who) estimates about 700 000 deaths yearly due to multi-drug resistance, with 200 000 deaths occurring in neonates.2 antimicrobial resistance, described as a global crisis, is causing difficulty in the treatment of common bacterial infections in children and is, in some instances, making treatment impossible.1,2 this has led to fatal outcomes as evident in an increase in mortality and morbidity among the paediatric age group.1,3 antibiotics are among the most prescribed drugs, both in the hospital and within the community, in the paediatric age group.3 the factors that guide such prescriptions are often invalid, especially in sub-saharan africa, where self-medication is high and the use of unmeasured herbal medication is common practice.4 the inadequate microbiology laboratories and equipment often justifies the empirical use of antibiotics in children.5,6,7 the development of bacterial resistance mechanisms is a normal process, regardless of resistance gene acquisition or gene mutation; however, inappropriate or unregulated use is believed to hasten the process.1 abuse of antibacterial agents in humans provides a selective pressure that promotes and spreads resistant strains.1 unnecessary prescriptions used in the management of viral infections and other non-communicable diseases, coupled with inappropriate dosage, have contributed to an increase in the development of multi-drug resistant bacteria species.5,6,7 a lack of equipped laboratories, coupled with non-adherence to infection prevention and control practices, increases the risk of amr, reduces the quality of care, and increases paediatric morbidity and mortality.5,8,9,10 in the west african region, absence of monitoring of licensed pharmaceutical vendors has given way to availability of many firstand second-line antibiotics as over-the-counter medications. this unguided practice, alongside the widespread patronage of drug hawkers, reduces the strength of antimicrobial surveillance within the region. likewise, the pattern of antibiotic resistance is not well studied in children, despite the high usage of antibiotics in this age group. the african continent has a major setback in accrued data on amr.9 the who considers africa to have a wide gap on recorded prevalence of amr as compared to most other continents.11 this can be attributed to weak national surveillance systems in many african nations. for instance, in a 2023 who survey,12 the one health approach created by the organisation was found to be efficient in only eight african countries.12 the one health approach aims to prevent and control amr by employing stakeholders in different disciplines and sectors to work together through surveillance and shared databases.12 an earlier report on central and east african nations clearly defined the inadequacy in antimicrobial susceptibility testing.13 this report also indicated that the amr pattern in these regions may be more ubiquitous than documented.13 recent efforts to define the map of amr (especially in the paediatric group) in sub-saharan africa have proven difficult because of limited data across west african states, thus affecting the formation of comprehensive national guidelines and protocol.6,10 the paucity of knowledge on the trends of amr in nigerian children has necessitated this research. this study therefore aims to determine the trend of antibiotic susceptibility of identified pathogens cultured from different clinical specimens in a sample of hospitalised nigerian paediatric patients. methods ethical considerations ethical approval was obtained from the lagos state university teaching hospital health research and ethics committee (lrec/06/10/2193). the ethical approval was obtained in a written format after submission of a detailed study proposal and before data collection. all data-collecting procedures followed the guidelines of the national health research ethics committee, nigeria (www.nhrec.net). as this retrospective study involved data of collected specimens with laboratory codes from routine clinical practices, informed consent was waived by the lagos state university teaching hospital health research and ethics committee according to the declaration of helsinki. data access authorisation and restriction of sensitive records were duly observed according to the national data protection and privacy regulation. authorised data records were kept in a locked file cabinet accessible only to the principal investigator. study design and population this was a retrospective study to describe the pattern of susceptibility to antibacterial agents in children over the period from december 2021 to march 2022. the study population comprised inpatients admitted at the massey street children’s hospital in lagos, south-western nigeria. the hospital is currently the only public children’s hospital in the state. it is located in a densely populated suburb of the state and cares for patients from all over the state and its environs. clinical care and laboratory procedures clinical specimens, including blood, cerebrospinal fluid, ear swabs, urine, and stool, were collected from hospitalised patients, according to their disease condition, as part of clinical care by the managing physician, and submitted to the microbiological testing unit. at the study site, cultures including urine, ear, and cerebrospinal fluid followed standard microbiological methods such as morphology on culture media, gram staining, and conventional biochemical testing. blood culture was performed according to the bd bactectm automated blood culture systems (becton, dickinson and company, sparks, maryland, united states). all cultures were cultured aerobically, at 37 °c for 18 h – 24 h, and negative cultures were incubated for up to 5 days for bacteria growth before being reported as negative. antimicrobial susceptibility testing the in-vitro antibiotic susceptibility of the identified pathogens was performed as recommended by the clinical laboratory standard institute,14 using the kirby bauer disc diffusion method on mueller-hinton agar (himedia laboratories pvt, mumbai, india). five genera (escherichia coli, klebsiella spp., pseudomonas spp., staphylococcus aureus, and streptococcus spp.) were isolated and tested for antibiotic susceptibility using 15 antibiotics by disc diffusion method. the following antibiotic agents were tested: ampicillin (10 µg), amoxicillin/clavulanic acid (20/10 µg), cefotaxime (30 µg), cefepime (30 µg), oxacillin (1 µg), azithromycin (2 µg), clindamycin (2 µg), cefuroxime (30 µg), ceftriaxone (30 µg), erythromycin (15 µg), cefoxitin (10 µg), meropenem (10 µg), ciprofloxacin (5 µg), ofloxacin (5 µg), and ampicillin + sulbactam (10 µg). gram-negative bacteria e. coli and klebsiella spp. were screened for extended-spectrum beta-lactamase production using ceftazidime (30 µg). the double-disk synergy test for ceftazidime (30 µg), and co-amoxicillin/clavulanic acid (20/10 µg) was used to confirm extended-spectrum beta-lactamase production. methicillin-resistant s. aureus was detected using the cefoxitin (30 µg) disc diffusion test. the susceptibility interpretation (susceptible, intermediate, resistant) of zone clearance was according to the clinical and laboratory standards institute guidelines.14 the american type culture collection e. coli 25922 and s. aureus 25923 were used as quality control strains for all laboratory methods. isolates with intermediate or resistant results on antibiotic susceptibility were classified as resistant strains during data analysis. data collection we extracted data from case record files and microbiology laboratory medical records for 300 paediatric patients aged 18 hours to 14 years, who were admitted and tested in the laboratory unit of the hospital during the study dates. information on patients’ socio-demographic characteristics, including age and gender, was obtained from the patient’s medical records. patients with missing registration numbers, incomplete medical records, and those attending the highly active antiretroviral therapy clinic were excluded from the study. antimicrobial sensitivity test report data were collected from the microbiology records of the laboratory unit of the hospital. bacterial profiles were obtained by reviewing the laboratory culture database. data analysis we entered the participants’ demographic characteristics, pathogen isolated, and susceptibility pattern of all isolates into a microsoft excel spreadsheet (v.16.0, redmond, washington, united states). participants were divided into 5 subgroups according to their paediatric age: neonate (0–1 month), infant (˃ 1 month – 12 months), preschooler (˃ 12 months – 60 months), school age (˃ 60 months – 120 months), adolescent (˃ 120 months – 192 months). data were analysed using ibm® spss® version 29 (armonk, new york, united states). the variables (socio-demographic and clinical characteristics, bacterial isolates, and antibiotic susceptibility) were expressed as frequencies, percentages, mean ± s.d., and ratios, as considered applicable. results a total of 300 participants were enrolled in this study (table 1). there were 169 (56.4%) boys and 131 (43.6%) girls, with a ratio of 1.3:1. the mean age of the participants was 41.13 ± 52.69 months (range 0–192 months) with the largest number in the neonatal group, and the smallest number in the adolescent group. blood (51.7%) and urine (30.3%) cultures accounted for the most requested microbiological test. of the 300 culture results, 156 (52.0%) were culture positive while 144 (48.0%) were culture negative. the highest percentage of positive samples was found in the neonatal group (34.6%). overall, children in the younger age groups, including neonates (18.0%), infants (10.7%), and preschool children (11.3%) accounted for most of the positive samples (table 1). table 1: socio-demographic and clinical data of subjects in lagos, south-western nigeria, from december 2021 to march 2022. age distribution among culture-positive samples of the 156 (52.0%) positive samples, boys had a higher percentage of positive samples in the neonate (37.8%), preschool (23.2%), and adolescent (11.5%) groups; the infant (23.0%) and school-age (18.9%) groups recorded a slightly higher percentage of positive samples among girls (table 2). staphylococcus aureus was the most frequent organism isolated (32.1%), and pseudomonas spp. was the least frequent organism isolated (7.1%) among all samples (table 2). staphylococcus aureus was the most frequently isolated organism in the neonatal (13.5%) and adolescent (3.2%) groups, whereas e. coli was the most isolated organism in all the age groups except for the neonatal (7.1%) adolescent (0.6%) age groups. pseudomonas spp. was not isolated among infants and was infrequently detected in the neonatal and adolescent groups (both 0.6%). streptococcus spp. was not detected in the adolescent group as compared to the younger age groups. table 2: age distribution among culture positive samples and in relation to gender, lagos, nigeria, from december 2021 to march 2022. pattern of drug resistance among detected isolates the most frequently recovered isolates, including s. aureus (n = 50), e. coli (n = 44) and klebsiella spp. (n = 32), were also the most often resistant to the tested antibiotics (table 3). isolates were most often resistant to ampicillin (n = 58), amoxicillin + clavulanic acid (n = 54), and cefepime (n = 53), compared to the other antibiotics tested. table 3: pattern of drug resistance in culture positive samples of children in lagos, south-western nigeria, from december 2021 to march 2022. antibiotic susceptibility pattern among different age groups antibiotic susceptibility patterns varied by age group and isolate (table 4). younger age groups were more resistant to the antibiotics tested as compared to isolates from the older school-age and adolescent groups. isolates were resistant to 11 of 15 drugs tested in the neonatal group in contrast to isolates from the school-age group, which were more sensitive to 10 of 15 drugs tested. among all the drugs tested, isolates demonstrated more resistance to ampicillin (neonate 73.7%, infant 88.7%, preschool 82.4%, school age 85.7%) and cefuroxime (neonate 56.7%, infant 73.3%, preschool 52.4%). in contrast, isolates showed more sensitivity to meropenem (neonate 84.6%, infant 88.2%, preschool 84.2%, school age 85.7%, adolescent 100.0%) and erythromycin (neonate 81.2%, infant 100.0%, preschool 66.7%, school age 100.0%, adolescent 75.0%) in the majority of the age groups. isolates from the preschool group were more resistant to amoxicillin + clavulanic acid (58.3%), cefotaxime (54.5%), and cefuroxime (52.4%). this differed from the infant group which exhibited more resistance pattern to cefepime (52.0%), azithromycin (66.7%), and cefuroxime (73.3%). likewise, isolates detected in the neonatal group were more resistant to ceftriaxone (57.1%) and ofloxacin (66.7%), as compared to the rest of the subgroups. table 4: susceptibility pattern from positive samples among different age groups from lagos, south-western nigeria, from december 2021 to march 2022. discussion this retrospective study demonstrated the varying pattern of antibiotic susceptibility in children. the bacterial species studied have shown strong associations with drug resistance globally and are part of the most important consideration of the who’s research for new antimicrobial discovery and development.15 our results show that the highest number of positive samples were from the neonatal group, and blood specimens contributed to more than half of specimens processed. this observation probably points to the alarming rate of neonatal sepsis and the rising cases of septicaemia among children, especially in sub-saharan africa. our findings are in agreement with previous reports from nigeria and gambia.16,17 the pathogenic organisms studied are part of the list of emerging bacteria threats as highlighted by fair et al.18 we agree with this report, as our findings revealed similar trends, with s. aureus, e. coli and klebsiella spp. having the highest resistance profile among the drugs tested.18 staphylococcus aureus, which was the most isolated organism among all the subjects studied, also had the highest resistance profile. cassat et al.19 in their review emphasised the rapidly evolving nature of s. aureus, producing aggressive resistant strains.19 this organism is also believed to be the most prevalent infective paediatric pathogen in several parts of the world, posing a significant threat without newer therapeutic options.18,19 the isolates included in this study also exhibited more resistance to ampicillin, amoxicillin + clavulanic acid, cefuroxime and cefepime. previous african studies have reported high resistance to all the drugs except for cefepime.20,21,22,23 the high prevalence observed in our study may be explained by the fact that information obtained were from records of patients on admission who were more likely to require antimicrobials compared with outpatients. likewise, the observed high rate of resistance could be associated with pre-exposure to a wide variety of antibiotics with optimal or sub-optimal dosages before their referral to our hospital. we also note that ampicillin, amoxicillin + clavulanic acid, cefuroxime and cefepime are all under the who access, watch, reserve classification of antibiotics to support antimicrobial stewardship programmes in the hospitals.24 these drugs, recommended by the who, are the most prescribed to children currently by physicians, especially in suburban and rural nigeria. resistance to these drugs suggests a disturbing shift to antibiotics considered reserved under the who classification intended for severe infections.24,25 we recorded a higher resistance pattern in younger children compared with school aged children and adolescents. this is similar to previous documented reports from different parts of africa.3,10,17,26,27,28,29 children, especially those under 5 years of age, receive more antibiotics than adults due to frequent bouts of infections. furthermore, blurring insights to mechanism of paediatric pathogenic organism, coupled with scarce data on resistance patterns in this age group, may increase antibiotic resistance in these age groups.30 this may be especially true in sub-saharan africa, which is considered a resource-poor region. this study also noted the high resistance profile of isolates within the neonatal group. this observation is in resonance with previous reports on the mounting resistance pattern in neonatal sepsis.31,32 these authors have also noted that e. coli and klebsiella spp. are dominant resistant pathogens isolated in neonatal sepsis. from our results, meropenem remains an available option as it shows a high sensitivity rate; however, this drug has been designated by who as a reserved drug and should be used under strict antibiotic policy.24 limitations the data used in this retrospective study were from records of hospitalised children in our facility who may have been infected with multi-drug resistant bacterial pathogens not reported in our study. the presence of additional multi-drug resistant pathogens not included in our analyses could have altered the susceptibility pattern reported here. however, our findings can be used as part of ongoing arguments on national awareness programmes on antimicrobial resistance in nigeria. conclusion our study demonstrated a higher antibiotic resistance in younger children compared with school aged children and adolescents. the pathogens studied were resistant in all age groups to the most available and empirically used antibacterial agents in nigeria: ampicillin, amoxicillin + clavulanic acid, cefuroxime, and cefepime. in awareness of this emerging threat, we believe this is a stimulation towards strengthening antimicrobial stewardship programmes and effective surveillance systems following current who guidelines at local, state and national levels in nigeria. acknowledgements the authors would like to thank the microbiology unit of the laboratory department, and the health information and medical records department at massey street children’s hospital for their contributions. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions this work was carried out in collaboration between all authors. all of the authors have accepted responsibility for the entire content of this submitted article and approved the submission. a.o.o. and m.a.l. conceived, designed and planned the study. a.o.o. collated the data and performed the analysis and acted for all correspondence. a.o.o. and o.t.o. designed the methods. a.o.o., m.a.l., o.t.o., and c.a.m. participated in drafting the article and have thoroughly checked and revised the article for necessary changes in format, grammar and english standard. all authors read and approved the final version of the article. all authors read and approved the article. sources of support the authors received no financial support for the research, authorship, and/or publication of this article. data availability the authors confirm that the data supporting the findings of this study are available within the article. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. it does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the authors are responsible for this article’s results, findings, and content. references antoñanzas f, goossens h. the economics of antibiotic resistance: a claim for personalised treatments. eur j health econ. 2019;20:483–485. https://doi.org/10.1007/s10198-018-1021-z fight antimicrobial resistance: protect mothers and newborns. in 4th global conference of women deliver. copenhagen: who regional office for europe; 2016. fadare j, olatunya o, oluwayemi o, ogundare o. drug prescribing pattern for under-fives in a paediatric clinic in south-western nigeria. ethiopian j health sci. 2015;25(1):73–78. https://doi.org/10.4314/ejhs.v25i1.10 james pb, wardle j, steel a, adams j. traditional, complementary and alternative 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https://doi.org/10.3389/fcimb.2023.1287051 rallis d, giapros v, serbis a, kosmeri c, baltogianni m. fighting antimicrobial resistance in neonatal intensive care units: rational use of antibiotics in neonatal sepsis. antibiotics. 2023;12(3):508. https://doi.org/10.3390/antibiotics12030508 tam py, bekker a, bolaji ob, et al. neonatal sepsis and antimicrobial resistance in africa. lancet child adolesc health. 2023;7(10):677–679. https://doi.org/10.1016/s2352-4642(23)00167-0 abstract introduction methods results discussion acknowledgements references about the author(s) modisa s. motswaledi department of biomedical sciences, faculty of health and wellness sciences, cape peninsula university of technology, cape town, south africa department of medical laboratory sciences, faculty of health sciences, university of botswana, gaborone, botswana ishmael kasvosve department of medical laboratory sciences, faculty of health sciences, university of botswana, gaborone, botswana oluwafemi o. oguntibeju department of biomedical sciences, faculty of health and wellness sciences, cape peninsula university of technology, cape town, south africa citation motswaledi ms, kasvosve i, oguntibeju oo. potential role of lu/bcam in hiv-related atherosclerosis. afr j lab med. 2019;8(1), a792. https://doi.org/10.4102/ajlm.v8i1.792 brief report potential role of lu/bcam in hiv-related atherosclerosis modisa s. motswaledi, ishmael kasvosve, oluwafemi o. oguntibeju received: 16 feb. 2018; accepted: 22 feb. 2019; published: 30 sept. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract atheromatous lesions are formed by macrophages and low-density lipoprotein cholesterol invading the vascular intima. here we show that increasing cholesterol levels are associated with peripheral monocyte depletion and this imbalance is aggravated by carriage of lu/bcam leukocyte adhesion molecules. this is true only in hiv infection and probably explains the risk of atherosclerosis observed in hiv-positive patients. keywords: cholesterol; non-high-density lipoprotein cholesterol; lu/bcam; atherosclerosis. introduction the pathology of atherosclerosis is tightly linked to three main factors; leukocytes, plasma lipids and vascular injury or chronic inflammation.1,2 leukocytes such as neutrophils, activated monocytes or monocyte-derived macrophages and platelets accumulate in the vascular sub-endothelial matrix by use of adhesion molecules. the lutheran blood group, also known as the basal cell adhesion molecule (lu/bcam), is one such molecule and is expressed on both monocytes and endothelial cells.3 it is the natural ligand for laminin, an extracellular basement membrane protein that facilitates both adhesion and extravasation of monocytes.3 in addition, monocytes and neutrophils also constitutively express α4β1 integrin,4 another ligand for lu/bcam. these molecules enable these cells to adhere to the endothelium and have been implicated in the pathology of crescentic sickle cell disease,4 as well as in several cancers, suggesting more efficient tumour tethering and greater tumour size.5,6 in hiv-positive individuals, the viral tat protein accumulates in the extracellular matrix of blood vessels and facilitates integrin-mediated adhesion7 with the consequence that hiv-infected monocytes have enhanced extravasation.8 hiv infection is also characterised by chronic inflammation resulting from the activation of t-cells, macrophages, neutrophils and natural killer cells – all of which release pro-inflammatory cytokines that create a state of persistent inflammation.9 this chronic inflammation involves neutrophils which produce reactive oxygen species thought to contribute to the oxidation of low-density lipoprotein cholesterol.10 cholesterol enrichment of neutrophil membranes results in more stabilised rolling and adhesion on endothelial cells,2 where they may recruit monocytes10 to promote atherogenesis. in particular, non-high-density lipoprotein (hdl) cholesterol has been documented as one of the more important promoters of atherosclerosis in hiv-positive patients.11 the atheromatous lesion forms by gradual accumulation of oxidized low-density lipoprotein cholesterol and infiltration by macrophages, smooth muscle cells and platelets.12 this may ultimately narrow the vascular lumen, causing tissue hypoxia distal to the atheroma. moreover, the release of rantes (ccl5) by activated platelets in the atheroma leads to further recruitment of monocytes and neutrophils.13 a high monocyte count has thus been reported to be a long-term predictor of plaque formation,14 while neutrophil enzymes such as elastase and myeloperoxidase in the atheroma may destabilise it,15 leading to its fragmentation and subsequent generation of emboli that may cause vascular occlusion at distant sites. in our previous study, the lutheran blood group, lub, was associated with a three-fold risk for hiv infection,16 and it was suspected that this could be due to its adhesiveness, which promotes trans-endothelial migration and the spread of infected monocytes to distant sites. the current study therefore sought to determine the effect, if any, of carriage of the lub antigen as an adhesion molecule on circulating monocytes and neutrophils, as well as how these parameters related to cholesterol measurements. the results indicate that hiv does affect the relationship between monocytes and cholesterol, and that this relationship is accentuated by expression of the lub antigen. methods ethical considerations ethical clearance for the study was obtained from the university of botswana’s office of research and development (permit no. urb/irb/1365), botswana’s ministry of health and wellness health research development committee (permit no. hpdme 13/18/1) and the cape peninsula university of technology, faculty of health and wellness research ethics committee (permit no. cput/hw-rec 2015/h11). informed consent was not necessary, since de-identified, residual samples were used,17 and the study was approved to use residual samples. study design the study was conducted at the julia molefe clinic, gaborone, botswana, from december 2016 to february 2017. one hundred blood samples comprising 58 female patients and 42 male patients were enrolled in the study. the patients had all tested positive for hiv and were being prepared for enrolment into the antiretroviral therapy programme. patients were enrolled sequentially upon receipt of an adequate residual sample to perform requisite tests. a full blood count was done using the sysmex® xt1800i haematology analyser (sysmex corporation, kobe, japan). the samples were phenotyped for the lutheran antigens (lua and lub) using specific antisera (fortress diagnostics, antrim, united kingdom) according to the manufacturer’s instructions. the reactivity of the antisera was confirmed by use of antigen-positive and negative cells selected from an antibody identification panel (diapanel®, lot 45241.88.1, bio-rad®, cressier fr, switzerland). total and hdl cholesterol measurements were also made using the au480 chemistry analyser (beckman-coulter, brea, california, united states). non-hdl cholesterol was calculated as the difference between the two measured values. we also revisited a previous dataset of 261 normal individuals as a control group to examine the same relationships in healthy, hiv-negative individuals18 statistical analysis the results were analysed using ibm spss version 24 (ibm corporation, armonk, new york, united states) statistical software. statistical analyses included comparison of means according to carriage of the lutheran antigens, as well as the correlation of data to explore relationships between variables. results were considered significant only if p < 0.05. results the mean total cholesterol did not differ between controls and hiv-positive patients (mean ± s.d. = 4.02 ± 0.85 vs 3.96 ± 1 mmol/l, p = 0.571) (table 1). likewise, the mean non-hdl values did not differ between the groups (2.68 ± 0.85 vs 2.88, ± 0.588 mmol/l, p = 0.089). table 1: comparison of total and non-high-density lipoprotein cholesterol in hiv-positive patients and hiv-negative controls, julia molefe clinic, gaborone, botswana, december 2016 – february 2017. among hiv-positive patients, total cholesterol correlated weakly and negatively with monocytes but not with neutrophils (r = −0.208, p = 0.038 vs r = −0.090, p = 0.372). however, with respect to the atherogenic non-hdl cholesterol, a stronger depletive effect was observed with monocytes (r = −0.401, p = 0.006), but not neutrophils (r = −0.226, p = 0.136). among hiv-negative controls, there was no correlation between monocytes and total cholesterol or non-hdl cholesterol. however, a very weak positive relationship was observed between total cholesterol and neutrophils (r = 0.142, p = 0.023). when patients were segregated according to their lub phenotypes, the correlation between non-hdl cholesterol with phagocytes improved for both monocytes (r = −0.442, p = 0.018; figure 1) and neutrophils (r = −0.369, p = 0.053; figure 2). in contrast, this relationship was absent for both phagocytes in lub-negative patients (r = −0.259, p = 0.315 for monocytes vs. r = 0.143, p = 0.584 for neutrophils). overall, lub expression was associated with lower absolute monocyte counts (0.45 ± 0.18 vs 0.54 ± 0.22 × 109/l, p = 0.023). figure 1: monocyte depletion in hiv infection was inversely associated with increased non-hdl cholesterol, julia molefe clinic. this relationship was weaker in lub antigen-negative patients (a) but enhanced in lub-positives (b). (a: r = −0.259, p = 0.315 versus b: r = −0.442, p = 0.018). julia molefe clinic, gaborone, botswana, december 2016 — february 2017. figure 2: in lub-positive patients (a), nonhigh-density lipoprotein cholesterol was associated with neutrophil depletion. this effect was totally absent in lub-negative patients (b). (a: r = −0.369, p = 0.053, versus b: r = 0.143, p = 0.584). julia molefe clinic, gaborone, botswana, december 2016 february 2017. discussion we studied the relationship between cholesterol and phagocytes in hiv-positive and hiv-negative individuals in an attempt to determine the influence, if any, of the lu/bcam on phagocyte counts. we hypothesised that carriage of the lub antigen, an adhesion molecule, would enhance margination of leukocytes leading to a peripheral deficiency of the affected leukocytes. indeed, we observed a significantly lower absolute monocyte count in lub-positive individuals compared to lub-negative individuals. we also observed a negative correlation between cholesterol and phagocytes (neutrophils and monocytes), which corroborates the findings by other investigators that membrane cholesterol, which is in equilibrium with plasma cholesterol,19 increases leukocyte adhesion and extravasation.1,2 moreover, phagocyte counts were negatively correlated with non-hdl cholesterol, which is known to promote adhesion and atherogenesis.1,12 the monocyte depletive effect of cholesterol was accentuated in lub-positive individuals but totally absent in the lub-negative individuals. while the difference in absolute monocyte counts may be considered to be small, the difference translates to 90 million cells per litre. the resulting statistical significance underlines a consistent finding of monocyte depletion in lub-positive individuals, and may explain the insidious development characteristic of atherosclerosis. we conclude that higher levels of cholesterol are associated with lower peripheral monocyte and neutrophil counts and that this depletion is accentuated in individuals expressing the lub antigen. this depletion is an effect of hiv infection, since it was not observed in the hiv-negative population and probably involves the particular role of the hiv-tat protein’s enhancement of vascular adhesion and trans-endothelial migration of monocytes reported by other investigators.20 hiv-infected monocytes are known to migrate more efficiently across vessel walls, while maintaining their infectivity.8 we conclude that the lub blood group contributes to the depletion of circulating phagocytes, possibly by enhancing their margination and extravasation.2 working from the established fact that cholesterol, especially non-hdl cholesterol, promotes the adhesion of phagocytes,1,2 with the possible reduction of peripheral counts, we note that in hiv infection, even normal cholesterol levels are associated with diminishing circulating phagocytic cells. this could be due to the chronic inflammatory status occasioned by hiv infection. it appears, therefore, that hiv-positive individuals are at higher risk of developing cholesterol-related monocytopenia and neutropenia, which, in the presence of increased non-hdl cholesterol, points to the peculiar vulnerability of hiv-positive patients to cardiovascular diseases. although these observations were generally expected considering that cholesterol promotes tethering of phagocytes,2,8 it was interesting that only in hiv infection was the phagocyte-depleting effect of cholesterol significant. when cholesterol components were examined, non-hdl cholesterol negatively affected phagocyte counts consistently, especially the monocyte counts, which corroborates previous reports by others11 linking non-hdl cholesterol to atherosclerosis in hiv infection. limitations a limitation of our study was that we did not directly measure phagocyte migration but rather inferred it from diminished peripheral cell counts. future studies need to focus on direct migration measurements of phenotyped phagocytes. moreover, the ages of the patients included in the study were not available, making it impossible to include the effect of age in the cholesterol analysis. this notwithstanding, the results provide critical information for hiv-associated atherosclerosis. conclusion our findings suggest that the risk of cardiovascular disease in hiv-1-positive patients may in part be due to an interplay between cholesterol, phagocytes and lub-mediated adhesion, all of which tend to recruit phagocytes out of circulation. while a genetic predisposition for atherogenesis has always been alluded to, our results suggest that the lub adhesion molecule may be a potentially important genetic link. the results also suggest a need to manage cholesterol in hiv-positive patients to achieve lower levels than the general public, especially for those who are lub-positive. moreover, the vulnerability occasioned by lub-positive status may provide an opportunity for personalised care in lub-positive, hiv-positive individuals. acknowledgements the authors wish to express gratitude to the staff and management of the gaborone district health management team, especially those at the julia molefe clinic, where samples were obtained and haematology and chemistry tests were performed. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions m.s.m. conceived the idea, carried out the experiments, analysed results and drafted the article. o.o.o. and i.k. provided experimental guidance, participated in data analysis, critiqued the content and gave final approval of the version to be published. sources of support this research project was supported by a research grant from the office of research and development of the university of botswana, (granted to m.s.m.), and from the cape peninsula university of technology and national research foundation south africa (cput/rj23), granted to o.o.o. however, the funders did not contribute in any way to the design of the study, data collection, analysis, or interpretation of data nor in the writing of the manuscript. data availability statement data sharing regarding this article is available upon written request to the corresponding 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mungmungpuntipantip, viroj wiwanitkit african journal of laboratory medicine | vol 10, no 1 | a1471 | 24 march 2021 97 104 112 120 126 134 141 148 158 scientific letter biobanking of covid-19 specimens during the pandemic: the need for enhanced biosafety olayinka s. ilesanmi, aanuoluwapo a. afolabi african journal of laboratory medicine | vol 10, no 1 | a1379 | 21 october 2021 lessons from the field from benchmarking to best practices: lessons from the laboratory quality improvement programme at the military teaching hospital in cotonou, benin alban zohoun, tatiana b. agbodandé, angélique kpadé, raliatou o. goga, rené gainsi, paul balè, bibata m. sambo, remi charlebois, rachel crane, michele merkel, ludovic anani, ekaterina milgotina african journal of laboratory medicine | vol 10, no 1 | a1057 | 11 february 2021 lessons from the field the deployment of mobile diagnostic laboratories for ebola virus disease diagnostics in sierra leone and guinea lance d. presser, jeanette coffin, lamine koivogui, allan campbell, julian campbell, fatmata barrie, jone ngobeh, zein souma, samuel sorie, doris harding, alimou camara, pepe tohonamou, basala traore, frank a. hamill, joe bogan, sharon altmann, casey ross, jay mansheim, robert hegerty, scott poynter, scott shearrer, carmen asbun, brendan karlstrand, phil davis, jane alam, david roberts, paul d. stamper, jean ndjomou, nadia wauquier, mohamed koroma, alhaji munu, jason mcclintock, mar mar, true burns, stephen krcha african journal of laboratory medicine | vol 10, no 1 | a1414 | 22 october 2021 lessons from the field setting up a molecular diagnostic laboratory for sars-cov-2 testing: experience of a single centre in a resource-constrained setting iriagbonse i. osaigbovo, isaac o. igbarumah, ekene b. muoebonam, darlington e. obaseki african journal of laboratory medicine | vol 10, no 1 | a1326 | 30 march 2021 lessons from the field adaptation of an electronic dashboard to monitor hiv viral load testing in côte d’ivoire mary kirk, paul h. assoa, casey iiams-hauser, yves-rolland kouabenan, jennifer antilla, caleb steele-lane, greg rossum, pascal komena, patricia sadate ngatchou, nadine abiola, alain kouakou, adama pongathie, jean b. koffi, christiane adje, lucy a. perrone african journal of laboratory medicine | vol 10, no 1 | a1284 | 17 may 2021 lessons from the field strategic site selection for placement of hiv early infant diagnosis pointof-care technology within a national diagnostic network in lesotho anafi mataka, esther a.j. tumbare, tsietso motsoane, david holtzman, monkoe leqheka, kolisang phatsoane, emma sacks, anthony isavwa, appolinaire tiam african journal of laboratory medicine | vol 10, no 1 | a1156 | 24 august 2021 lessons from the field african countries established covid-19 testing in one month: here’s how they did it timothy amukele, ryland n. spence african journal of laboratory medicine | vol 10, no 1 | a1457 | 15 december 2021 case study emphysematous pyelonephritis in an infant from sokoto, north-western nigeria fatima b. jiya, paul k. ibitoye, nma m. jiya, maryam amodu-sanni, yahaya mohammed, dada m. aquib, lukman k. coker african journal of laboratory medicine | vol 10, no 1 | a1181 | 26 april 2021 160 162 169 175 182 188 194 200 page ii of iii http://www.ajlmonline.org open access table of contents brief report epstein-barr virus, human papillomavirus and herpes simplex virus 2 co-presence severely dysregulates mirna expression jude o. okoye, anthony a. ngokere, charles c. onyenekwe, olaposi omotuyi, deborah i. dada african journal of laboratory medicine | vol 10, no 1 | a975 | 16 march 2021 brief report performance of taqman probes for the detection of sexually transmitted infections in south african women nireshni mitchev, ravesh singh, nigel garrett, veron ramsuran, abraham j. niehaus, koleka p. mlisana african journal of laboratory medicine | vol 10, no 1 | a1124 | 31 march 2021 brief report cross-validation of a high-performance liquid chromatography nevirapine plasma assay in a resource-limited setting in zimbabwe faithful makita-chingombe, anthony t. podany, timothy mykris, farai muzambi, richard w. browne, andrew j. ocque, robin difrancesco, lee c. winchester, courtney v. fletcher, tinashe mudzviti, charles c. maponga, gene d. morse african journal of laboratory medicine | vol 10, no 1 | a1264 | 08 july 2021 204 214 218 brief report re-decontamination of liquid mycobacterial cultures: additional mycobacterium tuberculosis yield in the era of xpert mtb/rif ultra in cape town, south africa dawood da costa, pieter nel african journal of laboratory medicine | vol 10, no 1 | a1529 | 10 december 2021 correction corrigendum: brucellosis – laboratory workers’ nightmare come true: a case study lebogang skosana, farzana ismail, nontombi mbelle, mohamed said african journal of laboratory medicine | vol 10, no 1 | a1690 | 22 november 2021 reviewer acknowledgement african journal of laboratory medicine | vol 10, no 1 | a1812 | 22 december 2021 223 226 227 page iii of iii introduction going forward: from now to the future acknowledgements references about the author(s) isatta wurie african society for laboratory medicine, addis ababa, ethiopia association of public health laboratories, silver spring, maryland, united states citation wurie i. sierra leone laboratory systems – now and future. afr j lab med. 2016;5(3), a549. http://dx.doi.org/10.4102/ajlm.v5i3.549 commentary sierra leone laboratory systems – now and future isatta wurie received: 23 aug. 2016; accepted: 30 aug. 2016; published: 31 oct. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction the impact of the 2013–2016 ebola virus disease outbreak in the mano river union region, which includes liberia, sierra leone and guinea,1 is a direct indicator of the acute limitations in national healthcare systems and the critical role played by diagnostic and public health laboratories. despite having a central public health reference laboratory, sierra leone lacked the capacity to provide the biosafety level 3 laboratory support that is required to effectively direct responses and that could have offset such a prolonged and costly health systems war against the outbreak. before the outbreak, sierra leone had in place both a national medical laboratory policy and a five-year strategic plan for 2010–2015,2,3 and had achieved some notable successes. these included the establishment of the first laboratory medicine/science undergraduate degree course to increase the human resource pool of laboratorians, the development of national norms and standards to inform laboratory support for the national health package, and the activation of the central public health reference laboratory. the central public health reference laboratory formed the apex to institute a public health laboratory network to diagnose key national priority diseases, such as influenza, yellow fever and measles, support the restructuring of laboratory tier systems, and support the integration of surveillance activities, especially for hiv molecular testing and the national health demographics health survey. however, during the implementation of the strategic plan, the country experienced two major disease outbreaks – cholera in 20124 and ebola virus disease in 20131 – that crippled the implementation of other key interventions. sierra leone is now focused on building a resilient healthcare delivery system. this will require major changes to improve routine services at clinical and public health laboratories throughout the country. the strategy is to develop comprehensive multi-programme integration, including tuberculosis, malaria and hiv, as well as disease surveillance and preparedness combined with best practices for successful healthcare delivery adapted to suit the country’s infrastructure, capability and environment. going forward: from now to the future for laboratory services to reach the goal of supporting an improved healthcare system, the following restructuring and strengthening framework platform is being developed, where it is lacking, or in an accelerated and strengthened implementation mode, where it existed. leadership in the reconstruction of a national healthcare laboratory system, two key components are: (1) political will at the ministry level of the national government; and (2) health-sector technical leadership to motivate and drive the concepts and importance of laboratories in healthcare delivery. using the ebola virus disease outbreak as an example, there were 16 international laboratories supporting the outbreak response. coordinating the activities of these partners was a major challenge, because the government did not have a defined leadership plan for dealing with such competing issues. therefore, the operations of the international laboratories were independent, with all but one having an exit plan via training of local experts. moving forward, the government has put in place a service-level agreement that ensures health-sector commitments and coordinated support for laboratory services. first, political will was secured by including laboratory service indicators for improved health into the health ministry’s development goals. next, a directorate or unit charged with policy direction and monitoring under the office of the director of hospital and laboratory services is being strengthened, with more staff linking with directorate of health systems strengthening. to manage integration of clinical and public health laboratory and disease surveillance services, operational leads will define the strategic scope of work for key professional positions through the health commission. finally, a team of specialists in laboratory science, process management, laboratory-related programmes, epidemiology, surveillance and health development partnership will be established to provide technical oversight in an advisory capacity, advocate for laboratory resources and act as monitors to ensure an integrated approach is taken toward achieving the national health development goals. national direction health sector guidance tools laboratory services form an integral part of quality healthcare planning and delivery. although national documents existed in sierra leone,2,3 their integration and coordination was limited. with the restructuring of departments within the ministry of health and sanitation, the health systems strengthening unit instituted and with emphasis by the director of hospital and laboratory services recognises that reliable, integrated and well-structured laboratory services are essential if the national basic package of essential health services delivery is to be achieved. this health systems strengthening–director of hospital and laboratory services combination is now charged with developing and consolidating tools and processes to guide health sector reconstruction and development for a strengthened and resilient health service. the health sector guidance tools comprise six essential elements: (1) the basic essential health package defines the level of laboratory support required at all tiers of the healthcare system and was developed with community-based input taken into consideration; (2) the national laboratory policy defines the scope of operations of laboratories within the mandate of the health sector; (3) the national laboratory strategic plan translates the policy into actionable services through strategic interventions; (4) the national integrated disease surveillance plan specifically defines the responsibility of laboratory support in the areas of public health, outbreak response and epidemic monitoring; (5) the national implementation framework and log plan outlines the annual plan that will provide the basis for smart monitoring and evaluations; and finally, (6) the laboratory operations standards define the minimum standards required for laboratory functions to support clinical and public healthcare facilities. functional health sector laboratories defining functional laboratory systems the function of a laboratory system is defined and tailored according the health needs of a particular country, taking into account regional and other international health regulations. the overall aim is to reduce mortality and morbidity through prompt diagnosis for effective treatment, protection of the community from outbreaks by giving special focus to select, epidemic-prone diseases relevant to the country, ensure efficiency of services with appropriate laboratory testing and, above all, ensure the confidence of patients, thereby facilitating positive health-seeking behaviour. a structured and organised laboratory service will strive to ensure staff are motivated, thereby improving quality staff recruitment and their retention. core laboratory services for sierra leone, core laboratory services were recommended based on their role in quality laboratory service delivery in the context of the entire healthcare system. the first and most obvious is diagnosis. clinicians require accurate and timely laboratory results to inform appropriate patient management and monitor treatment outcomes. thus, the clinical services to be offered through sierra leone’s network of clinical facilities and across all tiers of the national healthcare system will be selected for their ability to improve diagnostic accuracy, reduce misdiagnosis and provide prompt intervention. services associated with surveillance will be recommended based on their capacity to provide effective disease carrier identification, contact tracing and linkages with inter-sectorial integrated interventions. treatment services will be more targeted, with the goal of using drugs selectively and thus minimising potential antimicrobial resistance, in addition to providing effective monitoring of treatment progress and side effects. other factors that are considered include the role of services in public health, namely, monitoring of epidemic trends and early diagnosis of pathogens to support containment and infection prevention. health assessments and screening capabilities are also key factors, as constant monitoring of key populations, such as pregnant women and infants for wellness status or potential referral and screening of donor blood, are critically important. finally, support of a laboratory information system is crucial to providing empirical data for assessment of risk factors and monitoring the general health of the country’s population through prevalence and incidence indicators. sustaining laboratory operations minimum laboratory standards governance and leadership coordination are also required to push the daily operations of laboratories through a coordinated allocation of appropriate resources to meet the minimum standards necessary for the functioning of clinical and public health laboratories. the standards and the priority levels set for their implementation in sierra leone, through its national health sector strategic plan 2015–2020,5 are depicted in table 1. table 1: minimum laboratory standards and implementation priorities in sierra leone, 2016. to institute these basic minimum standards for laboratory operations, reconstruction of sierra leone’s laboratory system is being coordinated using a multi-faceted approach to ensure that all possible alternatives are explored (figure 1).6 ultimately, more than one approach may be required to achieve effective services. for example, to ensure adequate water supplies, both national and local efforts will be needed. solutions may vary by location, where purification of a surface water source may be practical for a low-volume remote laboratory, but drilling a new well or a bore hole with filter action treatment may be required for a high-volume, referral laboratory. figure 1: sustainable reconstruction framework. finally, in order for a laboratory system to be truly sustainable, it is important to define and allocate resources for ‘hidden’ costs. hidden operational costs are easily overlooked because of more obvious and tangible costs related to diagnostic capacity. however, it is important to realise that the entire function of a laboratory is dependent on these costs. for example, housekeeping, security, caretaking, cleaning and building maintenance are all necessary and important general functions that must be maintained. similarly, infrastructure costs, such as communications, utilities and waste disposal, are important for general functioning of the laboratory, and staffing and training-related costs, such as career development, staff workload ratios, training and continuing education, are critical for staff proficiency and retention. more obvious are equipment and supply-related costs, such as service contracts, inventory systems, safety compliance and certifications. public health laboratory emergency preparedness during public health emergencies, laboratory services are a critical component of the response for directing timely actions and interventions. through the provision of empirical data, laboratories set the pace to confirm diagnosis and support the testing of epidemiologically-significant specimens with potential public health implications. to achieve the objective of prompt response to contain an outbreak, laboratories in sierra leone are part of an integrated national rapid response core team (figure 2). the rapid response core team works with surveillance experts, health communicators, clinicians and zoonotic experts in the investigation and identification of the causative agents of outbreaks. the integrated action flow involves: (1) notification and logistics preparedness; (2) specimen management, which includes collection, storage and transportation of biosamples; (3) testing – and ensuring a quality assured result – at the facility; (4) laboratory-based surveillance and confirmation within the network of public health reference laboratories; and (5) reporting of results for action by clinicians and for public health interventions, and monitoring of trends by surveillance and epidemiology experts. figure 2: framework for the reconstruction of laboratory system preparedness. integrated laboratory data management in sierra leone, the network of public health reference laboratories systems network, as part of the national health institute or agency, will serve as the national focal point for capturing the laboratory data that is necessary for monitoring disease trends, ensuring that prevention and control strategies are undertaken properly – activities that also are essential for policy and decision making. in addition, this agency will maintain and communicate laboratory data and information to assist in identification, understanding and controlling of disease outbreaks. conclusion although much remains to be done, since the official end of the ebola virus disease outbreak, progress has been made toward the reconstruction of a sustainable healthcare laboratory system in sierra leone. next steps will involve developing and strengthening partnerships among the countrywide network of laboratories and developing a national map of laboratory resources to assist these partnerships and improve communication and training. acknowledgements i would like to acknowledge the following: director of hospital and laboratory services, dr victor matt-lebby, who leads the resilience laboratory service drive to future improved services; the chief medical officer, dr brima kargbo, for reactivation of the national laboratory technical working group that operated the national laboratory coordination centre for the ebola outbreak; the ebola response laboratory technical working core group, namely, professor sahr gevao, dr yvonne harding, dr abdul kamara (national laboratory manager), mr abu george, dr kadi yulla; supporting partners in options consultancy, namely, dr mohamed yillah, dr mohamed abass, mohamed fofanah and austin adeyemo; uk ebola response, namely, lt col rob brown, captain marc white; the laboratory epidemiology and surveillance technical working group leads, dr amara jambai and dr foday dafae; and supporting laboratory partners, namely, the us and china centres for disease control and prevention, the world health organization–sierra leone and ehealth africa. competing interests the author declares that she has no financial or personal relationship(s) that may have inappropriately influenced her in writing this article. sources of support this article was developed as part of experience working independently and voluntarily as part of the national laboratory technical working group. part of it was supported through the african society for laboratory medicine with the us centres for disease control and prevention foundation, and the association of public health laboratories. references world health organization ebola response team. ebola virus disease in west africa – the first 9 months of the epidemic and forward projections. n engl j med. 2014;371(16):1481–1495. http://dx.doi.org/10.1056/nejmoa1411100 government of sierra leone, ministry of health and sanitation. national laboratory policy, sierra leone. internal document. freetown, sierra leone: directorate of hospital and laboratory services; n.d. government of sierra leone, ministry of health and sanitation. national health sector strategic plan 2010–2015 [document on the internet]. c2009 [cited 2016 oct 19]. available from: http://www.ministerial-leadership.org/sites/default/files/resources_and_tools/nhssp%202010-2015.pdf world health organization global task force on cholera control. cholera country profile: sierra leone [document on the internet]. c2013 [cited 2016 aug 24]. available from: http://www.who.int/cholera/countries/sierraleonecountryprofile2013.pdf?ua=1 government of sierra leone, ministry of health and sanitation. national health sector strategic plan 2015–2020. internal document. freetown, sierra leone: directorate of hospital and laboratory services; 2014. government of sierra leone, ministry of health and sanitation. national ebola recovery strategy for sierra leone 2015–2017 [document on the internet]. c2015 [cited 2016 oct 19]. available from: https://ebolaresponse.un.org/sites/default/files/sierra_leone_recovery_strategy_en.pdf article information authors: racheal w. kimani1 anne w.t. muigai2 willie sang3 john n. kiiru3 samuel kariuki3 affiliations: 1institute of tropical medicine and infectious disease, jomo kenyatta university of agriculture and technology, kenya 2faculty of science, jomo kenyatta university of agriculture and technology, kenya 3centre for microbiology research, kenya medical research institute, kenya correspondence to: racheal kimani postal address: po box 6056-00200, thika, kenya dates: received: 26 apr. 2012 accepted: 09 apr. 2013 published: 17 oct. 2014 how to cite this article: kimani rw, muigai awt, sang w, kiiru jn, kariuki s. virulence factors in environmental and clinical vibrio cholerae from endemic areas in kenya. afr j lab med. 2014;3(1), art. #41, 7 pages. http://dx.doi.org/10.4102/ ajlm.v3i1.41 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. virulence factors in environmental and clinical vibrio cholerae from endemic areas in kenya in this original research... open access • abstract • introduction • research method and design    • detection of virulence factors from environmental and clinical v. cholerae    • amplification • results    • isolation of v. cholerae from environmental sources    • detection of virulence genes from environmental and clinical v. cholerae isolates • ethical considerations    • potential benefits and hazards • trustworthiness    • reliability    • validity • discussion    • isolation of environmental v. cholerae from western and coastal regions of kenya    • prevalence of virulence factors in environmental and clinical isolates of v. cholerae • conclusion • acknowledgements    • conflicting interests    • authors’ contributions • references abstract top ↑ background: since 1971, kenya has had repeated cholera outbreaks. however, the cause of seasonal epidemics of cholera is not fully understood and neither are the factors that drive epidemics, both in kenya and globally.objectives: the objectives of the study were to determine the environmental reservoirs of v. cholerae during an interepidemic period in kenya and to characterise their virulence factors. methods: one hundred (50 clinical, 50 environmental) samples were tested for v. cholerae isolates using both simplex and multiplex polymerase chain reaction. results: both sediments and algae from fishing and landing bays yielded isolates of v. cholerae. clinical strains were characterised along with the environmental strains for comparison. all clinical strains harboured ctxa, tcpa (el tor), ompu, zot, ace, toxr, hyla (el tor) and tcpi genes. prevalence for virulence genes in environmental strains was hyla (el tor) (10%), toxr (24%), zot (22%), ctxa (12%), tcpi (8%), hyla (26%) and tcpa (12%). conclusion: the study sites, including landing bays and beaches, contained environmental v. cholerae, suggesting that these may be reservoirs for frequent epidemics. improved hygiene and fish-handling techniques will be important in reducing the persistence of reservoirs. introduction top ↑ since 1971, kenya has suffered repeated cholera outbreaks. from 1974 to 1989, outbreaks were reported every year with an average case fatality rate of 3.6%.1 more cases have been reported in kenya since 2005 and an outbreak in 2007 had a case fatality of up to 5.6%.2 in 2011, 60 cholera cases, including 10 laboratory-confirmed cases and one refugee death, were reported in the world’s largest refugee camp in dadaab, kenya.3significant advances have been made in understanding the molecular basis of vibrio cholerae pathogenicity, including the identification of environmental reservoirs for the microorganism.4 it has also been shown that a 7th cholera pandemic spread from the bay of bengal in at least three independent but overlapping waves with a common ancestor in the 1950s and several transcontinental transmission events have been identified.5 the main reservoirs of v. cholerae are humans and aquatic sources such as brackish water and estuaries.6 ahmed et al.7 showed that multiple genetic lineages of v. cholerae were simultaneously infecting persons in kenya. this finding is consistent with the simultaneous emergence of multiple distinct genetic lineages of v. cholerae from endemic environmental reservoirs rather than recent introduction and spread by travelers. however, the cause of seasonal epidemics of cholera is not fully understood and neither are the factors that drive epidemics, both in kenya and globally. the objectives of this study were to determine the environmental reservoirs of v. cholera during an interepidemic period in kenya and to characterise their virulence factors. research method and design top ↑ the study was conducted in the coastal and western regions of kenya from february 2010 to november 2010. the coastal region sampling points were distributed in the cholera-endemic districts of kwale, malindi, mombasa and kilifi (figure 1). at each sampling point, preference was given to watering points and where there was human activity, as recommended by local health officials. the western region sampling points included the districts of kisumu, siaya, rachuonyo, homa bay, nandi hills, nyando, bondo, vihiga and busia. one sample of water, zooplankton, phytoplankton, sediments and/or floating vegetation was collected at each site as available, using the method described by huq et al.8 figure 1: coastal and western regions of kenya where environmental samples were collected. isolation began on the first day after collection of the environmental samples. one ml each of the zooplankton and phytoplankton homogenates was enriched in 10 ml (1x) alkaline peptone water. ten ml of each plant homogenate and sediment sample were also enriched in 5 ml triple-strength alkaline peptone water. on the second day, subculturing was done from the previous day’s alkaline peptone water inoculates on plates of thiosulphate citrate bile salts (tcbs). on day three, the presence of yellow mucoid colonies was considered presumptive for v. cholerae. presumptive colonies were tested by measuring their reaction to an oxidase reagent (1% dimethyl-p-phenylene-diamine dihydrochloride), along with other biochemical tests (voges proskauer, indole and carbohydrate fermentation). the environmental strains agglutinated with polyvalent o antisera and were sucrose fermenters. they formed large yellow mucoid colonies on the tcbs. colonies were also gram stained to confirm the morphological characteristics of the isolates. detection of virulence factors from environmental and clinical v. cholerae archived clinical isolates were sampled from the kenya medical research institute (kemri) laboratory. these isolates were mostly from outbreaks which occurred in the years 2009–2010. systematic random sampling was carried out in order to choose clinical strains. from a list of whole population strain numbers, a random strain number was chosen as the start strain. thereafter, every third strain from the whole population was sampled until a total of 50 samples were chosen, which is equal to the number of environmental strains. amplification deoxyribonucleic acid (dna) was isolated from freshly-cultured environmental and clinical v. cholerae isolates. the protocol was as follows: on the first day, bacterial isolates were grown at 37 °c overnight in mueller-hinton plates. cells were harvested and suspended in distilled water on the second day. the suspension was heated in a water bath at 95 °c for five minutes to lyse the cells. this was then centrifuged at 14 000 x g for six minutes in order to remove the protein debris. a sample supernatant was then stored at -20 °c for further use. polymerase chain reaction (pcr) was performed in a total volume of 32 µl containing 5 µl (5 ng/µl) of dna template, 1.0 µl (0.1 mm) each of forward and reverse primers, healthcare mastermix (ge healthcare, uk) and 25 µl nuclease-free water.all strains were then screened for the presence of genes encoding virulence determinants in v. cholerae including cholera toxin (ctxa), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), haemolysin (hlya), and nag-specific heat-stable toxin (st). detection of the tcpa gene specific to the el tor and classical biotypes was performed using a common forward primer and biotype-specific reverse primers. similarly, two forward primers were used for the detection of the biotype-specific haemolysin gene (hyla). pcr conditions and primers used for the detection of tcpa, ompu, tcpi, toxr and hyla genes were similar to those described previously by rivera et al.9 (table 1 and table 2), whilst detection of the ctxa gene was done using primers and conditions described before by fields et al.10 all pcr assays were performed using an automated dynax thermal cycler. pcr products were analysed by electrophoresis on 1.5% agarose gels, stained with ethidium bromide, visualised under uv light and recorded with the aid of a gel-documentation system. table 1: primers used in the study. table 2: primers pooled for multiplex pcr analysis. results top ↑ isolation of v. cholerae from environmental sources the coastal region had a total of 23 positive isolates out of 207 collected environmental samples (11%) (table 3). kwale had the most positive isolates, accounting for 43% of positive isolates collected in this region, followed by kilifi (35%) and mombasa (22%). no samples collected in malindi had positive isolates. the western region had 27 positive isolates out of 199 collected samples (12%). homa bay had the most positive isolates, accounting for 30% of positive isolates collected in this region, followed by kisumu, siaya and rachuonyo (15% each); nandi hills and nyando (7% each) and bondo, vihiga and busia (~4% each). table 3: sources of v. cholerae samples collected in coastal and western kenya by region. detection of virulence genes from environmental and clinical v. cholerae isolates all clinical strains (50 of 50) were pcr positive for the gene representing the el tor fragment for haemolysin (hyla) (figure 2), whilst only 10% (5 of 50) of the environmental strains were positive (table 4). in addition, all clinical strains were pcr positive for the gene representing the classical fragment for haemolysin as compared with 13 out of 50 (26%) of the environmental strains. for environmental v. cholerae strains, the results of the multiplex pcr yielded positive results for tcpa (el tor) at 12% (6 of 50), ctxa at 8% (4 of 50), zot at 24% (12 of 50), tcpi at 8% (4 of 50), toxr at 22% (11 of 50) and ompu at 0% (0 of 50) in the environmental strains. all clinical and environmental v. cholerae isolates (100%) were negative for the st gene. figure 2: example amplification of ompu, toxr and hyla genes in clinical vibrio cholera. table 4: results of polymerase chain reaction. table 4 part 2: results of polymerase chain reaction. ethical considerations top ↑ ethical approval was provided by the kenya national ethics review committee (approval number kemri/res/7/3/1). potential benefits and hazards there were no potential benefits or hazards to human subjects as they were not involved in the study. trustworthiness top ↑ reliability reliability is associated with accuracy, stability, consistency and reproducibility of the research. this was ensured by peer examination and open discussion with all participants. validity validity entails ensuring that the datasets gathered or items used are pertinent or relevant to the research conducted. this was ensured by use of appropriate research design and methods. discussion top ↑ isolation of environmental v. cholerae from western and coastal regions of kenya knowledge regarding environmental reservoirs of v. cholerae is of critical epidemiological and public health importance. we identified environmental reservoirs of v. cholera in kenya in a disease climate consisting of few reported cases. it is therefore likely that the v. cholerae existing in the environment amplify during the off-season to cause epidemics. prevalence of virulence factors in environmental and clinical isolates of v. cholerae in this study, the occurrence and distribution of selected virulence-associated genes in environmental and clinical strains of v. cholerae collected in kenya were demonstrated.environmental studies of v. cholerae have been done with the expectation that the v. cholerae strains possessing the entire complement of virulence genes would be isolated. in this study, as in previous similar studies, this did not happen. this can be explained by the fact that virulence genes are dispersed amongst environmental strains of v. cholerae and may be ferried about in mobile genetic elements. these genotypes of environmental v. cholerae are said to act as reservoirs for the virulence factors and potential mixing and matching leads to the formation of new pathogenic strains.11 two multiplex pcr assays revealed that all of the clinical strains were positive for ctx, zot, tcpi, and ace, as well as hlya (both el tor and classical), ompu and the toxr gene, suggesting the presence of an intact core toxin region in all isolates. these genes are found together and represent the genome of filamentous bacteriophage ctxφ. this implies that they possessed a ctxφ and tcp pathogenicity island. the pilus colonisation factor tcp acts as a receptor for ctxφ, which can infect non-toxigenic v. cholerae, leading to the emergence of new toxigenic strains of v. cholerae. the genes that encode the cholera toxin subunits ctxa and ctxb are localised to a ctx genetic element which is made up of a 4.6 kbp central core region and a 2.4 kbp repetitive sequence known as rs2.12 classical strains of v. cholerae o1 contain two copies of the ctx element, one on each chromosome. waldor and mekalanos13 were able to show that ctx is a filamentous bacteriophage related to the m13 coliphage.12 faruque, alberts and mekalanos14 reported that an environmental v. cholerae and two v. mimicus strains, all of which lacked tcp, were capable of being infected by ctxф. the v. cholerae pathogenicity island (vpi) is 39.5 kb in size and contains genes associated with virulence (tcp-acf cluster), the regulation of virulence (toxt and tcpp/h), the regulation of chemotaxis (tcpi or acfb) and mobility (int and orfi).9 the genes encoding tcp have been suggested to be part of a larger genetic element consisting of a cluster of genes. the tcpa el tor gene was positive in all clinical v. cholerae isolates and in 12% of environmental isolates in the current study, and the tcpa classical gene was negative in all isolates. harkey et al.15 suggested that regulators such as tcpi that act downstream of toxr and toxt may function to fine tune the expression of the tcp virulence determinant throughout the pathogenic cycle of v. cholerae. the tcpi encoding gene present in 8% of our environmental strains may thus have some other physiological functions as well.16 the outer membrane protein, ompu, was reported to be a potential adherence factor for v. cholerae. in this study, the gene was present in all of the v. cholerae strains of clinical origin that were tested, and negative in v. cholerae strains isolated from the environment. the haemolysin gene has been employed to differentiate between the two biotypes of v. cholera o1. two primer sets for the classical and el tor biotypes were used in this study. the v. cholerae strains of clinical origin from both biotypes were positive for the hyla gene. environmental isolates were 26% positive for haemolysin of the classical biotype and 10% for the el tor biotype. this phenomenon of detecting both fragments encoding for hyla specific to both the classical and the el tor biotypes in one isolate was observed in a similar study.17 nag-st in v. cholera o1 strains that is closely related to the heat-stable toxins produced by enterotoxigenic e. coli and other enteric pathogens was defined.18 the heat-stable enterotoxin production by the st genes was tested and none of the v. cholerae strains (of either environmental or clinical origin) were positive. the regulation and expression of genes for growth and survival depend on the regulon toxr, which is regulated by a cascade mechanism involving three known components: toxr, toxs and toxt. toxr, a 32-kda transmembrane protein, is the master regulator and its expression is dependent upon environmental growth conditions (incubation temperature, ph, osmolarity, bile salts, oxygen tension, hydrostatic pressure and amino acid composition of the medium.19 the toxr gene encodes a transcriptional activator controlling ct gene expression (ctxa), tcp biogenesis (tcpa), outer membrane protein expression (ompu), and at least 17 distinct genes in the o139 and o1 strains.20 in this study, the presence of the toxr gene was verified in all v. cholerae isolates of clinical origin and in 24% of the environmental isolates. conclusion top ↑ the implications of finding v. cholerae in environmental sources with virulence genes also found in clinical isolates are significant. the possibility is that this may lead to an epidemic strain arising through gene transfer that links genes that facilitate transmission in human populations, whilst those that foster persistence are proliferating in nearby environmental niches. gene interactions are therefore important to understanding the epidemiology and persistence of virulence factors and subsequent epidemic outbreaks. proper hygiene and fish-handling techniques should be ensured to cut the transmission from environmental sources to humans. acknowledgements top ↑ the authors would wish to thank the disease surveillance unit of the ministry of public health and sanitation, kenya for providing information on past cholera outbreaks. we also thank kemri–cmr members of staff for their support in this work. conflicting interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions r.k. (institute of tropical medicine and infectious disease, jomo kenyatta university of agriculture and technology [jkuat]) analysed the data and drafted the manuscript. s.k. (cmr–kemri), w.s. (cmr-kemri) and a.w.t.m. (faculty of science, jkuat) supervised the carrying out of this work. j.n.k. (cmr–kemri) supervised the laboratory protocols and execution of experiments. references top ↑ 1.world health organization. weekly epidemiol rec. 2004;79:236–244.2.world health organization. global task force on cholera control. cholera country profile [document on the internet]. [cited april]. available from: http://www.who.int/cholera/countries/kenyacountryprofile2010.pdf 3.nehebay s. cholera breaks out in kenya's dadaab refugee camp: un [page on the internet]. c2011 [cited 2012 sep 12]. available from http://www.reuters.com/article/2011/11/15/ozatp-kenya-un-refugees-idafjoe7ae0dr20111115 4.colwell rr, tamlin ml, brayton pr, et al. environmental aspects of vibrio cholerae in transmission of cholera. in: sack rb, zinnaka y, editors. advances in research on cholera and related diarrhoea. 7th ed. tokyo: ktk scientific publishers, 1990; pp. 327–343. 5.mutreja a, kim dw, thomson nr, et al. evidence for several waves of global transmission in the seventh cholera pandemic. nature 2011;477(7365):462–465. http://dx.doi.org/10.1038/nature10392 6.world health organization. cholera annual report 2011. weekly epidemiol rec. 2012;87(31–32), 289–304. 7.mohamed aa, oundo j, kariuki sm, et al. molecular epidemiology of geographically dispersed vibrio cholerae, kenya, january 2009–may 2010. emerging infectious diseases. 2012;18(6):n.p.. 8.huq a, sack rb, nizam a, et al. critical factors influencing the occurrence of vibrio cholerae in the environment of bangladesh. appl environ microbiol. 2005;71(8):4645–4654. http://dx.doi.org/10.1128/aem.71.8.4645-4654.2005 9.rivera in, chun j, huq a, et al. genotypes associated with virulence in environmental isolates of vibrio cholerae. appl environ microbiol. 2001;67(6):2421–2429. http://dx.doi.org/10.1128/aem.67.6.2421-2429.2001 10.fields pi, popovic t, wachsmuth k, et al. use of polymerase chain reaction for detection of toxigenic vibrio cholerae o1 strains from the latin american cholera epidemic. j clin microbiol. 1992;30(8):2118–2121. 11.karaolis dkr. a vibrio cholerae pathogenicity island associated with epidemic and pandemic strains. proc natl acad sci u s a. 1998;95(6):3134–3139. http://dx.doi.org/10.1073/pnas.95.6.3134 12.garg p, nandy rk, chaudhury p, et al. emergence of vibrio cholerae o1 biotype el tor serotype inaba from the prevailing o1 ogawa serotype strains in india. j clin microbiol. 2000;38(11):4249–4253. 13.waldor mk, mekalanos jj. lysogenic conversion by a filamentous phage encoding cholera toxin. science. 1996;272:1910–1914. http://dx.doi.org/10.1126/science.272.5270.1910 14.faruque sm, albert mj and mekalanos jj. epidemiology, genetics and ecology of toxigenic vibrio cholerae. microbiol mol biol rev. 1998;62:1301–1314. 15.harkey cw, everiss kd, peterson km. the vibrio cholerae toxin-coregulated-pilus gene tcpi encodes a homolog of methyl-accepting chemotaxis proteins. infect immun. 1994;62(7):2669–2678. 16.chakraborty s, mukhopadhyay ak, bhadra rk, et al. virulence genes in environmental strains of vibrio cholerae. appl environ microbiol. 2000:66(9):4022–4028. http://dx.doi.org/10.1128/aem.66.9.4022-4028.2000 17.singh dv, matte mh, matte gr, et al. molecular analysis of vibrio cholerae o1, o139, non-o1, and non-o139 strains: clonal relationships between clinical and environmental isolates. appl environ microbiol. 2001;67(2):910–921. http://dx.doi.org/10.1128/aem.67.2.910-921.2001 18.arita mt, honda t, miwatani t, et al. purification and characterization of a heat-stable enterotoxin of vibrio mimicus. fems microbiol lett. 1991;63(1):105–110. http://dx.doi.org/10.1111/j.1574-6968.1991.tb04513.x 19.dirita vj. co-ordinate expression of virulence genes by toxr in vibrio cholerae. mol microbiol. 1992;6(4):451–458. http://dx.doi.org/10.1111/j.1365-2958.1992.tb01489.x 20.herrington da. toxin, toxin-coregulated pili, and the toxr regulon are essential for vibrio cholerae pathogenesis in humans. j exp med. 1998;168(4):1487–1492. http://dx.doi.org/10.1084/jem.168.4.1487 21.shi l, miyoshi s, hiura m, et al. 1998. detection of genes encoding cholera toxin (ct), zonula occludens toxin (zot), accessory cholera enterotoxin (ace) and heat-stable enterotoxin (st) in vibrio mimicus clinical strains. microbiol immunol. 1998;42(12):823–828. http://dx.doi.org/10.1111/j.1348-0421.1998.tb02357.x 22.vicente ac, coelho am, salles ca. detection of vibrio cholerae and v. mimicus heat-stable toxin gene sequence by pcr. j med microbiol. 1997;46(5):398–402. http://dx.doi.org/10.1099/00222615-46-5-398 23.bhanumathi r, sabeena f, isac sr, et al. molecular characterization of vibrio cholerae o139 bengal isolated from water and the aquatic plant eichhornia crassipes in the river ganga, varanasi, india. appl environ microbiol. 2003;69:2389–2394. http://dx.doi.org/10.1128/aem.69.4.2389-2394.2003 abstract introduction methods results discussion acknowledgements references about the author(s) frantz jean louis centers for disease control and prevention, port-au-prince, haiti jennifer y. huang centers for diseases control and prevention, atlanta, georgia, united states yacouba k. nebie world health organization, conakry, guinea lamine koivogui institut national de sante publique, conakry, guinea gayatri jayaraman world health organization, conakry, guinea nadine abiola centers for diseases control and prevention, kinshasa, congo amanda vansteelandt centers for diseases control and prevention, atlanta, georgia, united states mary c. worrel centers for diseases control and prevention, atlanta, georgia, united states judith shang centers for diseases control and prevention, yaoundé, cameroon louise b. murphy centers for diseases control and prevention, atlanta, georgia, united states david l. fitter centers for diseases control and prevention, atlanta, georgia, united states barbara j. marston centers for diseases control and prevention, atlanta, georgia, united states lise martel centers for diseases control and prevention, conakry, guinea citation jean louis f, huang jk, nebie yk, et al. implementation of broad screening with ebola rapid diagnostic tests in forécariah, guinea. afr j lab med. 2017;6(1), a484. https://doi.org/10.4102/ajlm.v6i1.484 lessons from the field implementation of broad screening with ebola rapid diagnostic tests in forécariah, guinea frantz jean louis, jennifer y. huang, yacouba k. nebie, lamine koivogui, gayatri jayaraman, nadine abiola, amanda vansteelandt, mary c. worrel, judith shang, louise b. murphy, david l. fitter, barbara j. marston, lise martel received: 04 may 2016; accepted: 15 nov. 2016; published: 31 mar. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: laboratory-enhanced surveillance is critical for rapidly detecting the potential re-emergence of ebola virus disease. rapid diagnostic tests (rdt) for ebola antigens could expand diagnostic capacity for ebola virus disease. objectives: the guinean national coordination for ebola response conducted a pilot implementation to determine the feasibility of broad screening of patients and corpses with the oraquick® ebola rdt. methods: the implementation team developed protocols and trained healthcare workers to screen patients and corpses in forécariah prefecture, guinea, from 15 october to 30 november 2015. data collected included number of consultations, number of fevers reported or measured, number of tests performed for patients or corpses and results of confirmatory rt-pcr testing. data on malaria rdt results were collected for comparison. feedback from ebola rdt users was collected informally during supervision visits and forums. results: there were 3738 consultations at the 15 selected healthcare facilities; 74.6% of consultations were for febrile illness. among 2787 eligible febrile patients, 2633 were tested for malaria and 1628 oraquick® ebola rdts were performed. a total of 322 oraquick® ebola rdts were conducted on corpses. all ebola tests on eligible patients were negative. conclusions: access to ebola testing was expanded by the implementation of rdts in an emergency situation. feedback from ebola rdt users and lessons learned will contribute to improving quality for rdt expansion. introduction the ebola virus disease (evd) outbreak in guinea started in december 2013 and affected 25 of the 34 administrative prefectures of the country.1 laboratory-enhanced surveillance is critical for rapid detection of the potential re-emergence of evd. the capacity of the laboratory system in guinea is characterised by poor infrastructure, insufficient numbers of qualified personnel, lack of instrumentation and a limited quality assurance system. to support the diagnostic capacity to adequately respond to the evd outbreak, multiple countries and organisations deployed mobile laboratories and/or diagnostic equipment for rapid detection of the ebola virus. while these laboratories were critical to ensuring prompt and effective case management during the response,2 many are downsizing or ending operations. yet the need for consistent and ongoing capacity for diagnosis of evd still exists in guinea. testing capacity is limited throughout the country, and guinea currently lacks a timely and reliable specimen referral system for the safe transfer of specimens to centralised testing facilities. current evd diagnosis relies heavily on reverse transcription polymerase chain reaction (rt-pcr) technology. while rt-pcr is highly sensitive and specific and is considered the gold standard for evd diagnosis, it requires skilled technicians and an appropriate laboratory infrastructure, including stable power supply, controlled temperature, and appropriate biosafety procedures. rapid diagnostic tests (rdts), such as lateral flow assays that detect ebola antigens, could address many of the challenges of relying on laboratory-based rt-pcr. compared to pcr, antigen-based rdts are better adapted to use in the field, can be designed to require limited or no cold chain, require less training and equipment and can provide results in minutes.3 in november 2014, the world health organization (who) issued a call for ‘rapid, sensitive, safe and simple ebola diagnostic tests’ adapted for severely resource-constrained settings.4 in response, several companies developed antigen-based rdts. as of late 2015, two of these had received regulatory approval by the who or the us food and drug administration for emergency use authorization. the reebov™ antigen rapid test (corgenix, broomfield, colorado, united states) was approved by who and was issued an emergency use authorization for use with whole blood by the us food and drug administration in february 2015. the oraquick® ebola rapid antigen test (oraquick® ebola rdt; orasure technologies, inc., bethlehem, pennsylvania, united states) received an emergency use authorization from the us food and drug administration in july 2015 for whole blood testing.5,6,7 the oraquick® ebola rdt has a manufacturer-reported sensitivity of 84% (95% confidence interval [ci]: 63.92–95.46) and a specificity of 98.0% (95% ci: 89.35–99.95) for whole blood. by late 2015, there were no data on the performance of the oraquick® ebola rdt for oral fluid and overall very limited data and experience with field implementation of the rapid tests. although the yield of the ebola rdt was expected to be low (given the low prevalence of ebola at this stage of the epidemic), the identification of an unknown ebola transmission chain was considered a high priority, given the duration and mortality rate of the epidemic. the implementation of ebola rdts in this emergency situation would allow broader screening of a population that had limited access to ebola rt-pcr testing and a high prevalence of diseases with ebola-like symptoms, such as malaria. the ebola response coordination in guinea determined that while rapid tests could play an important role in surveillance for evd, additional information about their performance was needed. laboratories in guinea conducted additional testing of several rapid tests, including the oraquick® ebola rdt, to improve local familiarity with the tests and confirm their performance characteristics in terms of sensitivity and specificity (unpublished data). in collaboration with the who, the guinean red cross (rc), the us centers for disease control and prevention, and the guinean national institute of public health developed plans to evaluate the potential to screen more broadly with the oraquick® ebola rdt, based on its performance results and the emergency use authorization from the us food and drug administration. forécariah prefecture was chosen for the oraquick® ebola rdt field evaluation pilot, given the high impact of evd in this prefecture in 2015 and the prefecture’s contiguity with sierra leone. forécariah is located in western guinea, with an estimated population of 244 649 people.8 the prefecture was one of the most highly affected in guinea during the 2013–2015 west african evd epidemic; the cumulative incidence of evd in forécariah was nearly six times greater than that of guinea overall (forécariah = 198 cases per 100 000 population; guinea = 36 cases per 100 000 population).9 as one of the most affected areas, forécariah was included in a march 2015 presidential health emergency declaration that required secure burial practices for all deaths.10 during initial planning for the evaluation, evd transmission had been controlled in forécariah, and the prefecture was considered an example of a recently-affected area. however, evd was reintroduced in forécariah in september 2015, resulting in limited additional transmission.9 during the epidemic, the response infrastructure in forécariah grew to meet the prefecture’s increasing case load, and by april 2015 included an ebola treatment unit (etu) and a laboratory with rt-pcr capacity (k-plan laboratory).11 here, we report the initial results of the pilot implementation of the oraquick® ebola rdt in forécariah prefecture from 15 october to 30 november 2015. our objectives were to document the implementation process and to describe the results of ebola rdts, initial feedback from rdt users and major barriers to implementation. methods ethical considerations the protocol was approved as a non-research programme evaluation activity at the us centres for disease control and prevention. the activity was authorised by the guinean national coordination for ebola response. planning two strategies were developed for the ebola rdt implementation pilot plan: testing on live patients and testing corpses (figure 1). all partners involved in laboratory-based surveillance activities participated in the elaboration of the oraquick® ebola rdt implementation plan. the laboratory cluster working group for the ebola response, led by the director of the guinean national institute of public health, coordinated the activities and helped define roles and responsibilities for all stakeholders. the who, the guinean national institute of public health and the us centers for disease control and prevention developed the training materials; rc volunteers were responsible for screening corpses from both the community and hospitals and for ensuring safe burials, while the k-plan laboratory in forécariah played a key role in pcr testing for confirmation of oraquick® ebola rdt results. prefectural epidemiologists from the us centers for disease control and prevention, the who and the guinean ministry of health were responsible for data collection and for supervising the selected health facilities to monitor the oraquick® ebola rdt implementation in the field. figure 1: oraquick® ebola rdt pilot study implementation strategies in forécariah, guinea, 15 october to 30 november 2015. health facility selection initially, all health posts (n = 36) and health centres (n = 10) in forécariah were designated to implement the rdt pilot. however, to improve the feasibility of the pilot, 15 sites were ultimately selected based on location and patient volume. at least one health facility was selected in each of the nine sub-prefectures of forécariah. additional facilities were chosen along the border with sierra leone (figure 2). figure 2: map of health facilities selected for the oraquick® ebola rdt pilot in forécariah†. eligibility criteria and patient and corpse management the eligibility criteria for testing were designed to be sensitive and to capture all patients with possible unknown evd contact (figure 1). given the low sensitivity of the oraquick® ebola rdt at low viral load (initial phase of illness), known and monitored contacts of persons with evd were not eligible for rdts. instead, any monitored contact who developed symptoms compatible with ebola was to be transferred to an etu for evaluation. patients with a temperature > 38 °c or with history of fever in the 48 hours preceding consultation were eligible for the oraquick® ebola rdt. given the similarities between ebola and malaria symptoms (e.g., fever, chills, body aches, nausea, and vomiting), evaluation procedures specified concurrent testing with a malaria rdt.12 given their identical testing criteria, the malaria rdt is a valuable comparator for the utilisation rate of ebola rdts and for identifying any ebola rdt-specific implementation issues. patients with a positive ebola rdt were referred to an etu for rt-pcr confirmation using the real star filovirus screen rt-pcr kit 1.0 (altona diagnostics, hamburg, germany), and the light-cycler 480 (roche molecular diagnostics, pleasanton, california, united states). all community and hospital deaths were also eligible for screening using oraquick® ebola rdt. because the oraquick® ebola rdt had not been approved for swabs, a second swab was also collected for rt-pcr confirmation and programme decisions. for example, the decision whether to trace the contacts of the deceased was driven by the rt-pcr results. these rdt data on swabs were sent to who and the us centers for disease control and prevention for validation. by presidential edict, all burials were secured by the rc without waiting for test results. training materials a set of presentations was prepared to cover the following topics: protocol for the implementation of the ebola rdt and eligibility criteria to screen patients and corpses; principles and use of the oraquick® ebola rdt; quality assurance for using the oraquick® ebola rdt; communication practices around testing results; use of personal protective equipment; waste management; data collection; and supervision tools. the training was designed for healthcare workers (i.e., physicians, nurses, health agents) and for rc volunteers. communication around ebola rapid diagnostic tests healthcare workers were given talking points to help them explain and inform patients about the differences between the rdts for malaria and ebola and the ebola rt-pcr test, as well as the steps that would be taken in response to a test result. communication around swabs of corpses focused on targeted messaging for the rc volunteers to give to the families of the deceased and for healthcare workers to give to patients. because secure burials were required regardless of test results, and concurrent pcr testing was done for all deaths, it was agreed among partners not to provide the results of negative rdts to families. personal protective equipment and waste disposal use of personal protective equipment, including safety goggles and/or face shields, masks or respiratory equipment, disposable gowning, boots and gloves, was recommended for performing ebola rdts. used rdts were decontaminated in 3% chlorine solution before disposal. at the 15 selected health facilities, used personal protective equipment was either incinerated or buried deeply, depending on available resources for waste management. used personal protective equipment from the burial teams was transported safely to an rc facility for incineration and burial. test kit distribution and quality control ebola rdt kits and consumables were distributed to the selected health facilities during the training. data on stock management and replenishment of kits and consumables were collected at each supervision visit. the protocol called for all positive ebola rdts and 10% of negative samples from live patients to be selected by convenience sampling for confirmatory testing by rt-pcr. all samples collected from corpses were tested by rt-pcr. health facilities supervision and data collection a standardised supervision checklist was developed for use at initial and follow-up visits to evaluate the following elements at each selected facility: laboratory infrastructure; personnel competencies; documentation; and storage and stock management capacity. the checklist was also designed to evaluate specimen and waste management in accordance with recommended biosafety standards, and the basic elements of quality assurance (proper documentation, positive and negative controls, availability of standard operating procedures, and respect of rdt reading time). following initial training, prefectural epidemiologists conducted weekly supervision and collected data on the oraquick® ebola rdt implementation. data were collected by phone when logistical constraints prevented the epidemiology team from visiting the health facilities. during the epidemiologists’ visits, clinic registries were reviewed for several variables of interest (i.e., number of consultations; number of fevers [reported by patient as ‘i have a fever’ or ‘i feel feverish’ (recorded), number of fevers (measured as > 38 °c)]; number of patients tested with the oraquick® ebola rdt; number of corpses screened with the oraquick® ebola rdt; number of specimens confirmed by rt-pcr; number of malaria rdts used; and number of positive malaria rdts). teams also collected informal qualitative information from practitioners about their experiences with the ebola rdt and lessons learned, through forums with healthcare workers and rc volunteers and discussions in the field during this pilot. results pre-implementation during the initial training in september 2015, 166 participants were trained (101 healthcare workers and 65 rc volunteers) over three days. live patient rapid diagnostic tests a total of 28 patients were tested for evd in forécariah during the six weeks prior to the pilot. between 15 october 2015 and 30 november 2015, there were 3738 consultations at the 15 selected healthcare facilities. of these, 74.6% (n = 2787) of consultations were for febrile illness (reported or measured), of which 58.4% (n = 1628 of 2787) were screened for evd (table 1). only 14% (n = 393 of 2787) of the reported fever cases had an actual measured fever of over 38°c. during the same period of time, 94.5% (n = 2633 of 2787) of reported fevers were tested for malaria. table 1: key results of the ebola rapid diagnostic test pilot study at 15 sites in forécariah, guinea, 15 october to 30 november 2015. during the evaluation, there was one false positive ebola rdt, which was from a person who was a high-risk contact of a patient confirmed to have evd who had initially refused transfer to an etu, but presented to one of the health facilities participating in the pilot. she was tested by rdt independent of the evaluation protocol and then immediately transferred to an etu, where the result was confirmed negative by rt-pcr. a total of 163 (10%) negative rdt test results were confirmed negative by rt-pcr as part of the quality assurance process put in place. the ratio of ebola rdts to malaria rdts averaged 0.62 (range 0.19 to 1.08). the proportion of positive malaria rdts averaged 67.3% (n = 1771) of all patients seeking care for febrile illness. feedback from healthcare workers the gap between malaria rdt and ebola rdt use may be explained by healthcare workers’ misunderstanding of the testing algorithm (some healthcare personnel believed that they were only to conduct the oraquick® ebola rdt when the patient temperature was at least 38 °c) and also by temporary stock-outs of oraquick® ebola rdt kits. other reasons why the ebola rdt was not conducted included the following: patient declined; healthcare personnel forgot to conduct the test or decided that only a test for malaria was indicated; or the person responsible for conducting the test was not at work at that time. based on reports from workers at multiple sites, healthcare personnel did not regularly inform the patient when administering the ebola rdt. the most commonly-cited reason for not informing the patient was concern that patients would refuse evd testing. some healthcare workers were concerned that if the community became aware that the local clinic was testing for evd, the patients would avoid the clinic because of negative associations with the disease. several healthcare workers also reported that patients coming from villages that had been affected by evd were more receptive to the test than those coming from unaffected villages. logistical constraints and the high turnover of implementing partners in the field hindered regular supervision of the healthcare facilities, which in turn affected the completeness of data collection, distribution of ebola rdt kits and controls, onsite technical assistance and overall quality assurance. community deaths a total of 332 deaths were reported during the study period and 97% (n = 322) were tested for evd. all 322 corpses screened with oraquick ebola rdt were negative. these samples were all confirmed negative by rt-pcr. no healthcare workers or rc volunteers reported problems related to handling or testing using oraquick® ebola rdt. during a refresher training session in late november, rc volunteers reported no difficulties with families of the deceased resulting from use of the rdt during secure burials. discussion neither healthcare providers nor rc volunteers reported serious problems performing the oraquick® ebola rdt. during the pilot period, the broader testing criteria for the ebola rdt increased the number of people being tested for evd, even though only a little more than half of the eligible patients were tested. the number of live patients tested for evd in forécariah increased more than 20-fold and 97% of reported deaths were screened. thus, rdts appear to offer an important tool for expansion of surveillance for evd, in line with who recommendations.13 importantly, there were no serious problems with false positive results; about 10% of negative samples were collected randomly and retested using rt-pcr, with all results confirmed negative. in the one case, when the initial screening test was positive, either falsely or because of incorrect techniques, it was straightforward to conduct follow-up testing with rt-pcr and resolve the situation. while all febrile patients should have been eligible for both malaria and ebola rdt tests, most febrile patients had a malaria rdt done, but only about half of febrile patients were tested for evd. this gap is likely explained by a combination of rdt user error (misunderstanding or forgetting the rdt protocol), logistics (stock out, lack of personnel), or patient refusal. the utilisation rate could be improved with regular supervision, improved job aids, onsite technical assistance and better support for rdt stock management. the usage and positivity rate of the malaria rdt were higher in the 15 health facilities than reported previously in guinea.14 the high proportion of positive malaria tests could reflect season or altered patterns of care-seeking behavior.14 the biggest challenges for the implementation of oraquick® ebola rdt in forécariah were data collection, poor rdt stock management causing frequent stock-outs, and logistic and environmental constraints. the travel time from the centre of forécariah to the health facilities was as much as several hours, depending on the weather and road conditions. the inconsistent connectivity, either through cell phone reception or electricity, was a barrier to receiving data by phone or using electronic transmission. some of the health facilities were staffed by only one person, and both data collection and management require time and training. finally, implementing partners in the field had high staff turnover, which hampered regular monitoring and evaluation of the project, as well as site supervision, implementation of the standardised supervision checklist, kits and control distribution, and technical assistance that would improve tracking of implementation progress, as well as measurable quality assurance in the programme. broader implementation of the rdt will have to address multiple health communications issues, including the stigma attached to evd, public mistrust of facilities performing ebola testing, and preand post-test counseling for patients undergoing an ebola rdt. lessons learned from hiv rdt implementation will be a useful model. costs for the oraquick® ebola rdt are currently high, which may limit more widespread roll out of the test and further validation of test performance is necessary. limitations the main limitation of this study is the lack of follow-up on tools put in place to ensure quality; positive and negative controls were merely implemented, and supervision to ensure that standard operating procedures are followed was lacking. continuous training will also be needed to integrate ebola rdt as part as a routine testing for eligible patients in guinea. conclusions the use of rdts facilitated a marked increase in the numbers of suspected patients and corpses tested for ebola in forécariah, contributing to the critical objective of maintaining vigilant surveillance for ebola in the context of the recently controlled epidemic. the implementation programme was made possible by the high political commitment of the national coordination for the control of evd, as well as the support from the guinean national institute of public health and all stakeholders. the comparison of ebola rdt and malaria rdt utilisation allowed field teams to identify ebola rdt specific issues. feedback from ebola rdt users contributed to the development of new protocols for improved quality assurance and project tracking during the expansion to other testing sites. lessons learned from this pilot will guide the expansion of oraquick® ebola rdt throughout the country to support surveillance, to identify potential undetected cases and prevent future outbreaks. box 1: lessons learned. acknowledgements the authors thank the field epidemiology training program graduates who were involved, including marcel sefu, alain magazani, cullen seaton, henriette bulambo, lon kightlinger, eric mensah, michele balihe and eva de vallescar. disclaimer the findings and conclusion of this report are those of the author(s) and do not necessarily represent the official position of the centers for disease control and prevention. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support none. authors’ contributions f.j.l. led the conception and design of the work, as well as the writing of the paper. y.k.n. and l.k. contributed substantially on the conception and design of the work and training. j.y.h., g.j., m.c.w. and l.b.m. contributed to the acquisition, analysis, and interpretation of data for the work. a.v., j.s. and n.a. contributed to the implementation of the work and the training of all healthcare workers on ebola rapid disease testing. l.m., b.j.m. and d.l.f. contributed to drafting the manuscript and revised it critically for important intellectual content. references world health organization. emergencies preparedness, response: ebola maps 2015 [page on the internet]. c2015 [cited 2015 dec]. available from: https://www.who.int/csr/disease/ebola/maps-2015/en/ diers j, kouriba b, ladan fofana l, et al. [mobile laboratories for rapid deployment and their contribution to the containment of emerging diseases in sub-saharan africa, illustrated by the example of ebola virus disease] [article in french]. med sante trop. 2015;25(3):229–233. https://doi.org/10.1684/mst.2015.0485 nouvellet p, garske t, mills hl, et al. the role of rapid diagnostics in managing ebola epidemics. nature. 2015;528(7580):s109–116. https://doi.org/10.1038/nature16041 world health organization. urgently needed: rapid, sensitive, safe and simple ebola diagnostic tests [page on the internet]. c2014 [cited 2015 dec]. available from: https://www.who.int/mediacentre/news/ebola/18-november-2014-diagnostics/en/ broadhurst mj, kelly jd, miller a, et al. reebov antigen rapid test kit for point-of-care and laboratory-based testing for ebola virus disease: a field validation study. lancet. 2015;386(9996):867–874. https://doi.org/10.1016/s0140-6736(15)61042-x world health organization. who emergency use assessment and listing for ebola virus disease ivds: public report [document on the internet]. c2015 [cited 205 dec]. available from: https://www.who.int/diagnostics_laboratory/procurement/150219_reebov_antigen_rapid_test_public_report.pdf us food and drug administration. emergency use authorizations [page on the internet]. c2015 [cited 2015 dec]. available from: https://www.fda.gov/medicaldevices/safety/emergencysituations/ucm161496.htm institut national de la statistique. répartition population et densité par région et prefecture [page on the internet]. c2015 [cited 2015 dec 21]. available from: https://www.stat-guinee.org/index.php/statistiques/donnees-structurelles/demographie/31-population-densite-region-prefecture world health organization. ebola data and statistics [page on the internet]. c2015 [cited 2015 dec]. available from: https://apps.who.int/gho/data/view.ebola-sitrep.ebola-summary-20151211?lang=en bureau de presse de la présidence de guinee. ebola: état d’urgence renforcé pour les préfectures de forécariah, coyah, dubréka, boffa et kindia pour une période de 45 jours [page on the internet]. c2015 [cited 2015 dec]. available from: https://guineelive.com/2015/03/29/ebola-etat-durgence-renforce-pour-les-prefectures-de-forecariah-coyah-dubreka-boffa-et-kindia-pour-une-periode-de-45-jours/ croix rouge francaise. ebola: la riposte se déplace à forécariah [page on the internet]. c2015 [cited 2015 dec 21]. available from: https://www.croix-rouge.fr/actualite/lutte-contre-ebola/ebola-la-riposte-se-deplace-a-forecariah-1861 dixon mg, schafer ij. ebola viral disease outbreak – west africa, 2014. mmwr. 2014;63(25):548–551. world health organization. ebola response phase 3: framework for achieving and sustaining a resilient zero [document on the internet]. c2015 [cited 2015 dec]. available from: https://apps.who.int/iris/bitstream/10665/184693/1/ebola_resilientzero_eng.pdf?ua=1 plucinski mm, guilavogui t, sidikiba s, et al. effect of the ebola-virus-disease epidemic on malaria case management in guinea, 2014: a cross-sectional survey of health facilities. lancet infect dis. 2015;15(9):1017–1023. abstract introduction review method findings discussion conclusion acknowledgements references about the author(s) asa’ah nkohkwo n-s technomed, london, united kingdom gabriel agbor department of biochemistry, institute of medical research and medicinal plants studies, yaoundé, cameroon emmanuel asongalem department of biomedical sciences, faculty of health sciences, university of buea and toxicology society, buea, cameroon claude tagny haematology and blood transfusion service, university teaching hospital, yaoundé, cameroon tazoacha asonganyi department of biochemistry and immunology, faculty of medicine and biomedical sciences, university of yaoundé 1, yaoundé, cameroon citation nkohkwo a, agbor g, asongalem e, tagny c, asonganyi t. whole blood pathogen reduction technology and blood safety in sub-saharan africa: a systematic review with regional discussion. afr j lab med. 2016;5(1), a363. http://dx.doi.org/10.4102/ajlm.v5i1.363 review article whole blood pathogen reduction technology and blood safety in sub-saharan africa: a systematic review with regional discussion asa’ah nkohkwo, gabriel agbor, emmanuel asongalem, claude tagny, tazoacha asonganyi received: 12 sept. 2015; accepted: 01 mar. 2016; published: 09 june 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: despite vast improvements in transfusion services in sub-saharan africa over the last decade, there remain serious concerns on the safety and adequacy of the blood supply across the region. objective: this review paper ascertains the role of pathogen reduction technology (prt) in improving blood safety and supply adequacy in the region. method: the state of blood safety in sub-saharan africa was reviewed. meetings, seminars and correspondence were undertaken with key clinicians, scientists and professional bodies in the region, including the world health organization’s regional office for africa, to examine the suitability of prt for improving the safety of whole blood transfusion, a prevalent transfusion format in the region. results: existing literature suggests that combining prt with current blood safety measures (such as serology) would improve the safety and adequacy of the blood supply for transfusions in sub-saharan africa. this was echoed by the findings of the stakeholder meetings. conclusion: following a detailed appraisal of two leading prt systems, the mirasol® prt system and the cerus s-303 system, we suggest that companies conduct comprehensive toxicological evaluation of the agents used for prt and publish this in the scientific literature. we also recommend that the safety and efficacy of these technologies should be established in a randomised clinical trial conducted in sub-saharan africa. introduction the united nations millennium declaration, issued in 2000, led to the establishment of eight international development goals, which became known as the millennium development goals.1 the intention of the 193 member states of the un was to meet these goals by 2015 by: (1) reducing child mortality rates; (2) improving maternal health; and (3) combatting hiv, malaria and other diseases.1 establishing an adequate supply of safe blood for transfusion would improve the likelihood of achieving these three goals, especially in lowand middle-income countries, many of which are situated in sub-saharan africa.2 the world health organization (who) estimates that there are 3.9 whole blood donations per 1000 inhabitants in low-income countries; far below the minimum who requirement of 10 whole blood donations per 1000 inhabitants.2 the who has estimated that in low-income countries the prevalence of infectious diseases such as hiv is 425 times greater than in high-income countries.3,4,5 testing methodologies also vary significantly between these regions. in high-income countries, almost all blood samples are tested with the highly-sensitive nucleic acid testing (nat),3,4,5 whereas lowand middle-income countries still rely on various serological methods and rapid tests, which are less sensitive and thus have a greater window period than nat screening. moreover, most laboratories in low-income countries do not perform tests in a quality-assured manner.3,4,5 indeed, this has necessitated accreditation and mentoring initiatives, such as the establishment of the african society for laboratory medicine in 2011. thus, patients in lowand middle-income countries are at higher risk for transmission of infection through blood transfusion than patients in high-income countries.3,4,5 whilst improvements have been made in the past, a high residual risk of transfusion-transmitted infections (ttis) remains because of viruses, bacteria, protozoa and residual contaminating leukocytes, despite decades of blood safety programmes.3,4,5,6,7,8 finally, whereas in high-income countries almost all blood is fractionated into plasma, platelets and red blood cells (rbcs),9,10 in sub-saharan africa, approximately two million of the three million units per year are still transfused as whole blood.9,10 where they are componentised, the primary requirement is for rbcs, followed by plasma.9,10 in 2010, only 31.5% of blood centres in the who’s african region prepared blood components.9,10 blood safety in sub-saharan africa in recent years, there has been a vast improvement in the organisation, management, clinical and technical aspects of transfusion services in sub-saharan africa,9,10,11 although serious concerns remain with regard to the safety and adequacy of the blood supply. according to the who recommended minimum of 10 units of donated blood per 1000 catchment population, an estimated 8–9 million units of blood are currently needed per year for transfusion in africa.11 in sub-saharan africa, 44% of maternal deaths are attributed to severe bleeding during pregnancy and childbirth.11 thus, the majority of blood products are used for treating pregnancy-related bleeding.11 another major group that receives blood transfusions are children; 50% of paediatric transfusions are for malaria-induced anaemia in children and other severe morbidities, notably anaemia from sickle-cell disease.12 improving access to safe blood leads to improved healthcare. in malawi, safer blood led to a 60% decrease in mortality amongst seriously ill children and a 50% decrease in mortality amongst pregnant women with severe blood loss.13 many factors in sub-saharan africa contribute to the low availability of safe blood. two major factors are low donation rates and a high prevalence of ttis.2,3,4,5,8,9 the blood donation rate in sub-saharan africa is generally low, with 4–5 per 1000 population8,9 compared with 30 donations per 1000 in developed countries.2 thus, only 40% of targeted blood supply needs were realised by collection services in 2010.2,9,14 according to who data,8,9 blood transfusion from regular voluntary non-remunerated blood donors (vnrbds) have the lowest risk of ttis. however, vnrbds represent less than 50% of whole blood donations in low-income countries compared with 76% – 100% in high-income countries.8,9 the low vnrbd levels in some african countries may be a result not only of lack of organisation and financial resources, but also, to some extent, of socio-cultural barriers such as limited levels of education, religious and mystic beliefs and misconceptions about blood use.15 the high prevalence of ttis, such as infection with hiv (5% amongst adults), hepatitis b virus (8%) and hepatitis c virus (10%),9 contributes to the poor supply, in terms of adequacy and safety.2,3,4,9,10 also important to note is that, of the estimated one million annual deaths resulting from malaria globally, 90% occur in sub-saharan africa.7,9 this suggests a high carrier rate of malarial parasite in this region and hence in the pool of potential blood donors. according to the respondents of a 2010 survey of the status of blood safety in the who african region, an estimated 7.5% of blood units were discarded to avoid the risks of ttis.9 in high-income countries, donor screening and deferral procedures, as well as serologic testing and nat, have helped to make blood a safer product, drastically reducing the risk of classical tti agents such as hiv and hepatitis viruses.3,4 however, whilst highly-sensitive techniques such nat are available in high-income countries, screening and testing in lowand middle-income countries where the risk is highest, are limited to a combination of donor screening and/or deferral and serological procedures. high residual risks have been reported in african blood banks where nat is not performed.4 nat would allow identification of contaminated blood from donors who are in pre-serological phases of disease. however, this additional screening test is usually too expensive for the limited budgets of african blood services. another major difference between blood transfusion in lowand middle-income countries and high-income countries is that only 41.2% of all collections in africa (1.4 million of 3.4 million) were transfused as components in 2010.9 although there is increased use of components in sub-saharan africa, with the greatest need being for rbcs, it is expected that whole blood transfusions and red cell concentrates will continue to be the most requested units in the near future.10,12 aim the aim of this review was to determine the suitability of pathogen reduction technology (prt) for improving the safety of whole blood transfusions in sub-saharan africa, as well as for improving the adequacy of the blood supply available for transfusions. this technology could offer a way forward toward addressing the millennium development goals in sub-saharan africa through helping to ensure an adequate and safe blood supply. review method during the period september 2012 to january 2015, we searched for articles in the pubmed index using keywords and combinations of keywords, including ‘pathogen reduction/inactivation’, ‘blood safety’, ‘transfusion-transmitted infections’, ‘sub-saharan africa’, ‘mirasol’, ‘s303’, ‘riboflavin’ and ‘amustaline’. we also sought out articles from emerging key opinion leaders. we then applied the grading of recommendations, assessment, development and evaluation (grade)16 guidelines to undertake a qualified review of the collected publications17 on the challenges of blood safety in sub-saharan africa. we considered the potential of nascent prt as a possible solution for the challenges.18,19,20 the two most promising novel interventions, the mirasol® prt and the cerus s-303 prt, were examined, as these two systems could offer additional solutions to ensure safer transfusion of whole blood, potentially leading to a paradigm shift for healthcare in lowand middle-income countries.18,19,20 the use of grade enabled us to avoid bias and be as objective as possible in considering the information and literature we assessed. the dearth of randomised controlled trials, as recommended by grade, meant that the literature findings had to be verified. hence, to triangulate our observations, and given the target consumer region of the two leading prts, we engaged key stakeholders in the region. we sought expert opinions by discussing the findings of our literature search with clinicians, scientists and various professional bodies, including the who’s regional office for africa, at seminars in brazzaville, republic of congo and yaoundé, cameroon.21,22,23 findings the challenges of an inadequate and unsafe supply of blood for transfusions the situation described in the who 2010 survey9 was reinforced by the statements and presentations at the meetings in brazzaville and yaoundé21,22,23 and is typified by the current state of affairs in cameroon,24 ghana,5 nigeria7 and the rest of sub-saharan africa.5,15 two key challenges in the region were highlighted: the overall inadequacy of the blood supply and the lack of safe blood for transfusions. these challenges are faced not only by women with pregnancy-related problems and children with paediatric malaria-induced anaemia,8,9 but also by individuals with other severe, debilitating conditions such as sickle-cell disease. sickle-cell disease is a genetic haemoglobinopathy, manifesting typically as life-threatening episodes or ‘crises’ (including a combination of moderate to severe pain, anaemia and other acute complications) related to venous occlusion.25,26 with an estimated 200 000 infants born every year in africa with sickle-cell disease and an estimated prevalence of 2%,27 there are approximately 20 million people living with sickle-cell disease in sub-saharan africa. because of the region’s growing population, this number will continue to increase. the effective management of sickle-cell disease in other regions suggests that 10% of a given population of sickle-cell disease patients require a life-saving transfusion each year to prevent strokes in children or to address acute complications in other groups.27,28 prognosis is poor across sub-saharan africa, with an 80% five-year mortality rate amongst children with sickle-cell disease, resulting from the lack of resources to ensure appropriate and safe management of life-threatening anaemia, amongst other issues.27,29 thus, added to the wider iatrogenic risks such as ttis associated with transfusions,5,6,7,8,9 the shortage of blood denies sickle-cell disease and many other patients in sub-saharan africa the adequate healthcare that is taken virtually for granted in europe and north america. the potential of pathogen reduction technology there are a number of current technologies for the reduction of blood-borne pathogens.19,20,21 these include systems for platelet concentrates and plasma, such as the cerus intercept system, which is based on psoralen; the theraflex system, which is based on uvc irradiation; the mirasol system, which is based on riboflavin and ultraviolet light; and the cerus s-303 system, which is based on a dna crosslinker. presently, only two of the most prominent prt platforms have the potential for whole blood treatment: the mirasol prt system for whole blood30 and the cerus s-303 system.31 a phase-iii study has recently been completed for the mirasol system for whole blood.32,33 thus, given the prominence of the whole blood transfusion format in sub-saharan africa, only the mirasol prt and cerus s-303 systems were considered potentially suitable at the brazzaville and yaoundé stakeholder meetings.18,19,20 discussion ensuring blood safety in sub-saharan africa three key principles18,19,20,34 would protect recipients from donor blood pathogens: improved donor selection/exclusion measures; improved and standardised methodology of testing; and reduction/removal of pathogens from the blood. with regard to donor selection and/or exclusion, it should be pointed out that the who promotes vnrbd.35 the who also sees the practice of family replacement donations, as exemplified in cameroon,24 as a risk to the blood supply because of the perceived potential of paid blood donation. however, there is some disagreement on the risk of this practice within the context of low-income countries, as some view the primary problem more as lack of available blood.36 family donation may not increase the risk to the blood supply in comparison to vnrbds.36 furthermore, the establishment of vnrbds as the only source of blood, as a policy, may not only increase the cost of a unit of blood by twoto five-fold and exacerbate the pre-existing blood shortage, but would hardly meet local blood supply needs.36 it is also questionable whether hospital-based family replacement blood banks in africa would ever be able to maintain adequate blood supplies to meet local needs. some have reported that first-time vnrds are no safer than family/replacement donors.35 as for the testing of donated blood, the current practice in high-income countries is to employ the following layers of protection: donor screening, serology, nat, leukoreduction, gamma irradiation and bacterial detection. donors in high-income countries have a much lower prevalence of ttis.37,38 the outcome is that risk for ttis in high-income countries is low for tested pathogens.37,38 typically, residual risk ranged from 1 in 300 000 units for hepatitis b virus in the united states, to 1 in 8 million units for hiv in canada, to 1 in 11 million units for hepatitis c virus in germany.37,38 in contrast, in sub-saharan africa, residual risks were 4.3 in 1000 units for hepatitis b virus, 1 in 1000 units for hiv and 2.5 in 1000 units for hepatitis c virus from blood transfusion.39 the who further reported inadequate quality assurance of donated blood, including unreliable vetting of the donor and serological testing of the donated blood, coupled with the practice of whole blood transfusion.13 considering all of these factors, the current status of highly risky transfusion in sub-saharan africa requires a new paradigm approach. to start a combination of new technologies is needed that would be effective for whole blood transfusion – a significant format of transfusion in sub-saharan africa – and that could enhance the current testing solution for improved safety. given the difficulty of recruiting sufficient donations, the proposed way forward should also have the potential to reduce supply wastage by rendering safe blood that would otherwise be rejected for testing positive. amongst other attributes, the appropriate technology would need to exhibit: safety, effectiveness, clinical efficacy, physiological integrity of biological components, operational efficiency and competitive health economics. the various platforms encompassing prt represent such a novel approach.18,19,20 prt platforms are already in use for platelets and plasma in many countries throughout europe,30,31 asia and the americas. with the emergence of platforms suitable for whole blood use,18,19,20,30,40 prt now has the potential to offer an additional step in the standard blood safety value chain in sub-saharan africa. moreover, the ability to inactivate pathogens in whole blood offers the flexibility of either using the blood as whole or, after inactivation, to subsequently separate the blood into components for storage.40 moving forward: validation and safety of pathogen reduction technology we examined two prt platforms in detail, the mirasol prt system for whole blood and the cerus s-303 system. both systems are based on disrupting the nucleic acid in the pathogen. the mirasol system achieves this through electron transfer reactions, whereas the cerus s-303 system does so through irreversible crosslinking of the dna. we focused specifically on the toxicology of the systems to laboratory staff and patients and the potential usefulness of these two systems for whole blood transfusion. the mirasol prt system for whole blood30,38 the system: the mirasol prt system is manufactured by terumo bct (lakewood, colorado, united states) and was granted the ce mark in 2008.30 the system employs riboflavin (vitamin b2) and ultraviolet illumination to inactivate a range of known and unknown pathogens.41 riboflavin first binds with the nucleic acids in the pathogen, then the ultraviolet illumination of the vitamin b2–pathogen complex causes an irreversible chemical reduction (specifically, guanine oxidation), disrupting the dna and consequently inactivating the pathogen (figure 1). figure 1: (a) mode of action of the mirasol prt system; (b) structure of riboflavin. safety: riboflavin is a safe, non-toxic, non-mutagenic, water-soluble vitamin necessary for normal cell function, growth and energy production in humans.42 used in vital metabolic processes, it is found in most animal and plant tissues and is considered an essential part of the human diet.42 the institute of medicine of the united states national academy of sciences recommends a dietary intake for riboflavin of 1.1 to 1.3 milligrams per day for adults.42 studies have shown that excess riboflavin is rapidly excreted in the urine; consequently, a minimal amount is stored in the body.42 riboflavin deficiency may contribute to increased concentrations of plasma homocysteine, which is associated with an increased risk of cardiovascular disease.42 amongst other morbidities, it may also be associated with impaired handling of iron and night blindness. riboflavin and its photoproducts produced using the mirasol system are present in untreated human blood.43 this has been demonstrated using high performance liquid chromatography where mirasol-treated platelet concentrates and untreated platelet concentrates were compared.43,44 the analysis indicated that no new photoproducts were formed during the mirasol system process.43,44 extensive toxicology studies have been performed to confirm the safety profile of riboflavin.42 in addition, riboflavin photoproducts that are generated from the mirasol process, such as lumichrome, can be transfused at several degrees of magnitude lower than the toxic concentration. further, a large, well-controlled study of mirasol-treated platelets, as well as a haemovigilance study on mirasol-treated products, have shown no adverse events attributed to the use of mirasol. of importance is a study at the institute of health and tropical medicine in warsaw, poland,43 that followed six patients with thrombotic thrombocytopenic purpura who received a total of 711 mirasol-treated fresh frozen plasma units. the massive transfusions of mirasol-treated fresh frozen plasma were found to be safe and effective when used for therapeutic plasma exchange in the treatment of thrombotic thrombocytopenic purpura.43 the chemistry of the base technology for the mirasol prt platform remains the same for whole blood, plasma and platelets.30,38 concerns have been expressed that photo-treatment is limited in its application to materials containing rbcs. the absorption of light by haemoglobin in several regions of the ultraviolet and visible spectra results in the photo-treatment of rbcs and alters the cells in a similar manner. however, most of the applied energy occurs in the uvb range (280–315 nm) with peak wavelength at 313 nm, which is different from the absorbance energy of mitochondrial enzymes (370–450 nm). thus, the oxidative phosphorylation pathway is not affected.45,46 indeed, photo-treatment does not have any effect on the quality of rbcs for transfusion.47,48 strengths and challenges: the mirasol platform has been licensed and is being used across europe for plasma and platelet transfusions.30 it has also recently been tested in a phase iii trial in ghana for preventing malaria associated with whole blood transfusion.32,33 in ghana, up to 28% of all blood is contaminated with plasmodium spp. parasites.5 the phase iii trial reported that mirasol-treated whole blood showed a statisticallyand clinically-significant reduction in transfusion-transmitted malaria compared with the control group that received untreated whole blood.32,33 in addition, the number of transfusion-related adverse events from the transfused blood products was similar between the treated and control groups.32,33 this clinical trial demonstrated the potential of prt for sub-saharan africa, showing that even for untested pathogens, prt can significantly reduce the incidence of ttis. because the mirasol prt is a nascent technology, the blood-banking sector, long-accustomed to routine traditional testing, would need to take into consideration the substantial paradigm shift and the training of personnel toward ensuring its safe and effective implementation. in addition, the extra cost of combining the new technology with current routine testing remains a key limitation. the cerus s-303 blood system49,50,51 the system: the cerus s-303 system is manufactured by cerus corporation (concord, california, united states) and a ce mark was granted in 2002.31 the cerus system (figure 2) employs a chemical compound, s-303 (amustaline), which intercalates with the nucleic acids in the pathogen with no activation of the material.49,50,51 the system uses the s-303 frangible anchor linker effector (frale) method, which includes a nucleic acid binding component that serves as anchor, a nucleic acid reactive group (the effector) and a hydrolysable linker.49,50,51 the frale compound targets nucleic acids, whereas the effector moiety, a nitrogen mustard, results in crosslinking (through the effector) of the nucleic acid, thus preventing replication and inactivating the pathogen. after the reaction, the material decomposes through a ph-dependent process into an unreactive product, s-300.51 glutathione is used to quench the side reactions of the effector with other biological materials.49 figure 2: mode of action of the cerus s-303 system. safety: the cerus platform uses a xenobiotic/foreign chemical, s-303, as the pathogen reduction agent, which in turn results in xenobiotic breakdown products, including s-300 and products of the ‘heteroalkyl’ group.52 quinacrine and quinacrine mustard are structural analogues of s-303 (figures 3 and 4). the pharmacokinetic properties of quinacrine are well known.53 following administration, it is rapidly absorbed and distributed in the body. plasma levels remain low compared with tissue concentrations,54 with high concentrations in the liver, spleen, lungs and adrenal glands. liver concentrations can reach 20 000 times concentrations in plasma.55,56,57 the brain, heart and skeletal muscles have low concentrations of quinacrine;55,56 and significant deposits are found in the skin, fingernails and hair.57 over 80% – 90% of quinacrine is bound to plasma proteins, whereas cerebrospinal fluid levels are 1% – 5% of plasma levels with a half-life of 5–14 hours, depending on dosage.58 synthetic structural analogues of quinacrine, such as quinacrine mustard and s-303 and the latter’s metabolic product (s-300), would probably share some of these attributes.59 the s-300 study report51 did not measure the amount of s-300 deposited in various organs and tissue. such short-term toxicological studies are not likely to reveal rare or slow effects, such as carcinogenicity, or other possible consequences of genotoxicity. figure 3: s-303 reaction product: s-300. figure 4: members of the acridine family of compounds: (a) structure of acridine; (b) structure of acridine family member quinacrine; (c) structure of acridine family member quinacrine mustard; (d) structure of acridine family member s-303. furthermore, quinacrine, quinacrine mustard, s-303 and s-300 are heterocyclic amines, which are known for their mutagenicity.60,61 quinacrine and quinacrine mustard interact with bovine heart mitochondrial f1-atpase,62 with quinacrine mustard potently inactivating the enzyme.63 further, quinacrine and quinacrine mustard bind to mouse muscle nicotinic receptors of acetylcholine, irreversibly inhibiting response,63 as well as to axonal membranes of nerve cells,64 interfering with the conduction of nerve impulses. quinacrine mustard also has cytostatic and cytotoxic properties.65 it is well known that compounds with structures similar to a pharmacologically-active drug are often themselves biologically active.59 therefore, we are concerned about the striking structural similarity of s-303 to substances like quinacrine mustard, quinacrine and acridine orange that are known to be either in the mustard group of compounds or in the heterocyclic amine group, which is also known for having cytotoxic and genotoxic properties. some patients transfused with s-303-treated rbcs developed positive cross-match reactions to the rbcs with the first generation of the cerus platform. as a result, the second generation system includes glutathione as a quencher.65 as stated by the manufacturers, glutathione binds to s-30065, the reaction product, to prevent it from reacting with proteins. glutathione does not enter rbcs, but remains in the external environment where it prevents s-300 from reacting with plasma proteins.52 however, in vivo, glutathione is found predominantly in rbcs, not in plasma.51 if whole blood treated with s-303 were to be transfused into patients, the glutathione in the treated sample would be diluted in vivo and no longer available to prevent s-300 from reacting with the vital biological components of the extracellular environment. strengths and challenges: the cerus intercept platform version of the cerus s-303 is currently available for platelets and plasma. a phase iii clinical trial for rbcs has been completed in europe.20,50 thus, similar to the mirasol prt system, the cerus s-303 platform is potentially useful for whole blood applications, making it another strong contender for reducing ttis in situations where whole blood transfusion is traditional, as is the case in sub-saharan africa. that said, there is a need for more appropriate experimental approaches to determine adverse effects such as immunotoxicity, carcinogenicity and genotoxicity. importantly, the current absence of a removal step in the whole blood prt process proposed for packed rbc treatment for the s-303 platform only emphasises the need for such detailed toxicological evaluations. indeed, further extensive toxicological evaluation of s-303 and its breakdown products, in phase iii of the evaluation process, would ensure not only safety but also a reproducible and user-friendly technology for resource-limited settings. such technology should be adapted, not only to current recommended blood safety regulations, but should include ethical considerations, affordability and economical sustainability for blood services.66 thus, as in with the mirasol system, addressing toxicity considerations in addition to the extra cost of combining the cerus s-303 technology with current routine testing remain key limitations. pathogen reduction technology and the pathogen detection window the above limitations of toxicity (cerus s-303 platform) and extra cost (both the mirasol and cerus s-303 platforms) notwithstanding, based on the underpinning pathogen nucleic binding chemistry, both prt formats are potentially effective against a range of pathogens known and unknown, on white blood cells, as long as they contain nucleic acids. moreover, by virtue of the underlying analytical sensitivity of the strategy, prt has the potential of further closing the pathogen detection window period – when blood levels of specific pathogen markers are still below the detection limits of other methods (figure 5). figure 5: the principle of combining pathogen reduction technology with existing testing methods. this highlights the potential of prt to markedly improve the safety of blood through a synergistic combination with the diagnostic window of testing. this conclusion came out of the seminars held with stakeholders, including clinicians, scientists and the who’s regional office for africa, during the period of this study.21,22,23 conclusion whole blood transfusion and adequacy of the blood supply given the high cost of establishing and running a blood componentisation programme, it might be beneficial for lowand middle-income countries to keep their focus on whole blood transfusions, increase blood donations and decrease the risk of exposure to pathogens in the blood supply, rather than on extensive componentisation. however, facilities permitting, the need for blood components might be met through apheresis, allowing blood banks to produce specific blood components based on the demand of the prescribing physician. that said, reduction of blood transfusion to their indispensable indications and appropriate use of blood components are critical to meet safe blood needs. this includes early diagnosis of diseases requiring blood transfusion, the use of surgical techniques and of medication aimed at limiting blood loss, as well as respect for national directives. in 2009, less than one out of 10 african countries had a proper policy in place for clinical use of blood.9 inappropriate use of blood leads to blood shortages and increased risk of ttis. a new safety paradigm toward whole blood pathogen reduction technology the provision of an adequate blood supply and safe transfusion of blood as recommended in the united nations’ millennium development goals remains a major challenge for sub-saharan africa. current blood-borne pathogen testing arrangements, hampered by lack of adequate testing resources, result in ttis at an estimated risk of 10% – 50% per transfusion. moreover, because of poor blood donation rates, blood supplies at blood banks are low. this review supports the position that prt may offer a solution for ensuring that the regionally dominant format of whole blood transfusion is rendered safer by removing ttis. we suggest that a new paradigm toward combining prt with standard quality testing would improve the safety of transfusion. the existing literature suggests that combining prt with current routine blood screening methods (such as serology) would improve the safety of transfusions in sub-saharan africa. following a detailed review of two of the leading prt methods, the terumo bct mirasol prt whole blood system and the cerus s-303 system, we suggest that manufacturers conduct a comprehensive toxicological evaluation of the agents used for prt and publish their findings in the scientific literature. we also recommend that the safety and efficacy of prt technology should be established in a randomised clinical trial conducted in sub-saharan africa, if the technology is adopted in the region. acknowledgements we would like to acknowledge the contributions of the delegates of the cameroon prt round-table panel, yaoundé, 28–29 january 2015, including: dr noah owona (ministry of public health and permanent secretary of the national blood safety programme, yaoundé, cameroon); professor dieudonné adiogo (vice dean, faculty of medicine, university of douala); dr biboum balogog (head of the blood bank, che/cnps essos, yaoundé); colonel dr celestin ayangma (head, laboratories and blood bank, garnison military hospital, yaoundé); professor dora mbanya (faculty of medicine and biomedical sciences & head, haematology laboratory & blood bank, chu, yaoundé); dr maurice mouladje (the blood bank, regional hospital, buea). competing interests the authors declare an unconditional financial support from terumo bct (manufacturers of the mirasol prt system) during this study. the statements made are the independent collective position of the authors on the respective issues discussed. authors’ contributions a.n. 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department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa manfred e. tepper national health laboratory service (nhls), johannesburg, south africa lindi m. coetzee national health laboratory service (nhls), johannesburg, south africa department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa deborah k. glencross national health laboratory service (nhls), johannesburg, south africa department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa citation cassim n, tepper me, coetzee lm, glencross dk. timely delivery of laboratory efficiency information, part i: developing an interactive turnaround time dashboard at a high-volume laboratory. afr j lab med. 2020;9(2), a947. https://doi.org/10.4102/ajlm.v9i2.947 lessons from the field timely delivery of laboratory efficiency information, part i: developing an interactive turnaround time dashboard at a high-volume laboratory naseem cassim, manfred e. tepper, lindi m. coetzee, deborah k. glencross received: 18 dec. 2018; accepted: 05 feb. 2020; published: 29 apr. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: mean turn-around time (tat) reporting for testing laboratories in a national network is typically static and not immediately available for meaningful corrective action and does not allow for test-by-test or site-by-site interrogation of individual laboratory performance. objective: the aim of this study was to develop an easy-to-use, visual dashboard to report interactive graphical tat data to provide a weekly snapshot of tat efficiency. methods: an interactive dashboard was developed by staff from the national priority programme and central data warehouse of the national health laboratory service, johannesburg, south africa, during 2018. steps required to develop the dashboard were summarised in a flowchart. to illustrate the dashboard, one week of data from a busy laboratory for a specific set of tests was analysed using annual performance plan tat cut-offs. data were extracted and prepared to deliver an aggregate extract, with statistical measures provided, including test volumes, global percentage of tests that were within tat cut-offs and percentile statistics. results: nine steps were used to develop the dashboard iteratively with continuous feedback for each step. the data warehouse environment conformed and stored laboratory information system data in two formats: (1) fact and (2) dimension. queries were developed to generate an aggregate tat data extract to create the dashboard. the dashboard successfully delivered weekly tat reports. conclusion: implementation of a tat dashboard can successfully enable the delivery of near real-time information and provide a weekly snapshot of efficiency in the form of tat performance to identify and quantitate bottlenecks in service delivery. keywords: turn-around time; laboratory efficiency; interactive dashboard; indicators; performance assessment. introduction turn-around time (tat) is an important performance indicator for a laboratory service. it refers to the time from first registration in a laboratory to a result released on the laboratory information system (lis).1 historically, within the national health laboratory service (nhls) of south africa, tat reporting was provided in annual and quarterly static management reports generated by the lis, trakcare,2 which also provided ad hoc reporting for use at the laboratory level. these reports are printed to provide a snapshot of tat reporting and are suited for staff working at the laboratory level. at a national level, the corporate data warehouse (cdw) of the nhls collated global tat data from over 266 testing laboratories based on predetermined, annual performance plan cut-offs. national tat cut-offs are set by expert committees of different pathology disciplines with final confirmation from senior management before implementation. these cut-offs are set with provisions for all levels of service laboratory: from low-volume laboratories with limited test repertoires to high-volume testing laboratories with extensive test offerings, including specialised testing, such as viral load testing. however, a large percentage of nhls laboratories have 24-hour service and have emergency units in the hospitals in which they are housed; such laboratories have locally stricter tat cut-offs for emergency and other local tests than are reflected in the national cut-offs for all samples. historically, the nhls cdw tat reports generated were static and reported only the mean tat. turn-around time data have a positive skewness, that is, a long tail to the right, meaning that the mean will be greater than the median. this implies that tat data reported previously,1,3 reporting the mean tat, masks good performance, while concealing poor efficiency. further, neither the current lis nor cdw reports enable detailed analysis of the information or drilling down to laboratory or test level data for additional information about tat efficiencies. data presented at the first conference of the african society for laboratory medicine in cape town, south africa, in 2012, reported daily laboratory test volumes and mean tat for authorised results, stratified by individual laboratories,4 providing a snapshot of performance. this enabled review of cd4 laboratory efficiency for a national programme and provided important insights into laboratory operations. a recent evaluation of tat for hiv programmes reported a methodology to further categorise laboratory tat performance using three additional measures:3 (1) median tat, (2) 75th percentile tat (tail size) and (3) percentage within cut-off tat. these data were graphically presented using a scatter plot of percentage of samples within the tat cut-off (x-axis) against the tat 75th percentile (y-axis), categorised into four quadrants of performance to help identify the level of laboratory performance in a national programme. this approach made it easier to identify both good performers and outliers in the same analysis.3 the report was generated in excel and included the raw monthly data, a scatter and bar graph and a summary table of all laboratories per business region, the 75th percentile, the percentage within cut-off values and tat component 75th percentile values. this report was primarily distributed to managers of all hiv-related operational programmes, that is, cd4, viral load and tuberculosis testing and early infant diagnosis for review and intervention, and not shared across the network of testing laboratories. senior management in gauteng, south africa, expressed a need for a tat monitoring tool that would enable them to better manage their laboratories and identify sites with poor tat performance. given the static nature of the historic tat reporting in the organisation, an interactive system that offered information to enable review of performance, including outliers (tail size assessment), while confirming that sites were meeting cut-offs, would be a useful tool to enable business and laboratory managers alike to monitor their efficiency via tat performance in real time. the concepts already developed and in use as excel reports for the hiv programmes were the starting point for developing a reporting dashboard for use across multiple disciplines and tests done throughout the network of 241 testing laboratories of the nhls in south africa. the aim of this study was to develop an easy-to-use information system, in a dashboard format. this would enable weekly reporting of tat data as a snapshot of performance. to achieve this, a number of changes to current tat reporting had to be addressed. these changes included (1) moving from programme-specific, single-test tat reporting previously used3 to a specific set of high-volume tests, (2) adopting tat measures reported by coetzee et al.,3 (3) identifying dashboard software to use and (4) identifying the target users.3 the specific set of tests (or ‘basket’ of commonly requested tests) should be representative across the primary pathology disciplines. this article sets out to describe the process followed to develop the tat dashboard, using available software, that could provide a weekly summary of national, business unit and laboratory level tat performance for a basket of tests. for the purposes of this article, data from a single participating tertiary laboratory were used to illustrate the data distribution, as it represents an example of a testing facility that performed all tests reviewed in the prescribed national basket. this was done to show the respective levels of drilling functionality of the dashboard and to iterate the interactive properties, while demonstrating how the dashboard can be used to assess performance and identify outliers for intervention. methods ethical considerations ethics clearance for this study was obtained from the university of the witwatersrand (m1706108). only anonymised laboratory data were used for the study and did not contain any patient identifiers. study design a retrospective descriptive study design was used to analyse and report laboratory tat data for a specific set of tests (table 1). table 1: sample of a turn-around time dashboard table that lists the outcomes for the basket of tests, south africa, 2018. steps to developing a turn-around time dashboard the various steps required to develop the dashboard are summarised in a flowchart (figure 1). figure 1: flowchart depicting all steps required to develop a turn-around time dashboard, south africa, 2018. sample population and turn-around time definition using convenience sampling, data were selected from among the tests performed at a single busy academic laboratory in gauteng for one week during 2018. aside from global tat reporting, the dashboard should adopt the three tat measures reported by coetzee et al.3 these include pre-analytical, analytical and post-analytical components of tat components, namely: (1) the time from first registration at the source laboratory to registration of the referral at the testing laboratory (lab-to-lab tat), (2) time from registration at the testing laboratory to results being populated by the lis interface (testing tat) and (3) time from result population by the lis interface to manual review and authorisation by senior laboratory staff (review tat). test basket development and inclusion and exclusion criteria focus group meetings were arranged with local area and business managers to define a test basket for the dashboard. the principles adopted were as follows: (1) measure a limited number of tests with a focus on the tests with the highest volumes of tests performed, (2) measure data for the indicator analyte (as a proxy) for specific panel tests (for example, the creatinine test was used as an indicator for assessment of urea and electrolyte test performance), (3) use the annual performance plan tat cut-offs and (4) deliver dashboard files via email (due to bandwidth constraints). all samples within this organisation test basket were included in the example analysis and included the most commonly requested tests selected from haematology, coagulation, hiv-tuberculosis and chemistry (table 1). a mapping table was developed to identify the lis test sets and items to be reported. for each test, the tat cut-off was also stipulated. the mapping table was used to guide the data extract. data extraction for the purposes of demonstrating how the data were manipulated to create the dashboard, data were extracted for the week of 2–8 september 2018 from the cdw from four data sources: (1) the operational data store that contained the original lis data (figure 2), (2) the ‘cdw fact’ that reported test volumes, (3) the test method dimension5 (provides details on the test such as a unique identifier, discipline, test method code and name and national number from the cdw) and (4) tat cut-off dimension (captures annual performance plan cut-offs) (figure 1).6 using an outer join, data from these four data sources were prepared as a temporary detailed table. the first temporary table limited data to the test basket, adding the tat cut-offs and provided information using the laboratory hierarchy (region, business unit and laboratory). because this table would be too large to use for the dashboard and assuming email delivery of the final report, two additional steps were used to create a smaller aggregate data set. the mean, standard deviation, 75th percentile and percentage within tat cut-off were added. all tat data were reported in hours. the final temporary table was exported as a microsoft excel (redmond, washington, united states) worksheet and imported into the microstrategy desktop analytics tool (providence, virginia, united states).7,8 after the data were imported, the respective dashboard sheets were developed to include relevant tat information for all levels of management. figure 2: visual representation of data preparation steps to transform and move raw turn-around time data, south africa, 2018. data were moved from the operational data store (the laboratory information system) to a production server which consists of facts and dimensions. the production server structures the data by setting sets of data to specific target areas to facilitate final reporting in dashboard format. criteria for an effective dashboard for any dashboard to be effective, it needs to adhere to a number of key outputs: it should (1) be visually engaging and easy to view and understand the tat data displayed, (2) enable dynamic drilling down from a bird’s-eye view to a local perspective, that is, from national or provincial level down to the laboratory level per test, (3) provide a report on a weekly basis for a tat snapshot view and (4) highlight tat outliers for laboratory managers to follow-up and direct corrective action. from a more technical perspective, the dashboard also had to include additional features that included (1) conditional formatting to highlight good, average and poor performance, (2) provision of various reporting formats such as bubble charts, tables and bar charts, (3) ability to import data for a variety of formats and (4) ability to send the weekly dashboard data file via email, that is, small file format (≤ 6 mb). data analysis and visual dashboard display the dashboard displays (sheets) developed were as follows: (1) a bubble chart reporting the percentage within tat cut-offs and 75th percentiles, (2) a table (table 1) displaying the bubble chart data and (3) the 75th percentile for each phase of the component tat reported by the test method. a bubble chart dashboard sheet was created to include: (1) the 75th percentile tat (y-axis), (2) the percentage within tat cut-off (x-axis), (3) the test volumes (size by and colour by) and (4) the test method (colour by and break by). the region codes and laboratory names were added to the dashboard as filters (radio buttons and search box display styles). an 85% within tat cut-off reference line was added to aid identification of specific tests and associated laboratories with tat that were outside of the tat cut-offs. the data used to generate the bubble chart were also reported as a table in a separate sheet. the table listed the test name, total number of tests, tat cut-off, the percentage within tat cut-off and 75th percentile tat for the basket of tests reported on. the table uses ‘stop highlighting’ to denote the different percentage within tat cut-off as follows: (1) 85% or higher in green, (2) 75% – 84% in orange and (3) under 75% in red. lower percentage within tat cut-off and higher 75th percentiles indicate an increased risk that any given laboratory is not adequately delivering patient reports that will enable timely clinical intervention. a component tat sheet was created as a clustered horizontal bar chart to display the component tat as follows: (1) test method name (y-axis) and (2) component 75th percentile tat (lab-lab tat, testing tat and review tat), differentiated by colour. the testing laboratory name was added to enable refining and filtering data down to the laboratory level. results the successfully developed dashboard enabled delivery of weekly tat data. data from 45 599 reported samples for the week 2–8 september 2018 were utilised to demonstrate the dashboard development described here. the 75th percentile and the percentage of tests within stipulated cut-off for each test in the basket were visualised on the dashboard landing screen. this allowed the user to view data by test at the national, provincial and laboratory levels to visually identify outlying tests. the dashboard contained three individual sheets: the bubble chart, the tat table and the component tat sheets. figure 3 shows typical weekly tat data presentation outcomes as a bubble chart dashboard. in this example data set, only one test method, rapid plasma reagin (syphilis), failed to meet the 85% tat cut-off and is reported as a small grey dot on the bubble chart dashboard. for this test, the reported percentage within cut-off was 83.8%, within the 75% and 84% category highlighted as orange in table 1. a cluster of tests in the bottom right reported 90% or higher within tat cut-off with a 75th percentile tat of 8 hours or less. only one test reported a percentage within tat cut-off between 85% and 89%: total cholesterol (red dot). higher test volumes were reported for the hiv viral load (n = 19 055) and creatinine (n = 8857) tests. figure 3: an example of the microstrategy desktop bubble dashboard chart used to report total turn-around time data for an example site’s week’s data, south africa, 2018. the percentage within cut-off turn-around time is reported on the x-axis with the 75th percentile turn-around time on the y-axis. the bubble size indicates test volumes. reference lines were added at 85% within stipulated turn-around time cut-off on the x-axis. each test within the test basket is colour coded with the key provided on the right. outlying tests are immediately visible. for the tat table, results for the bubble chart are summarised (table 1) per test. at the 75th percentile tat, no test exceeded the cut-off tat. a 100% within cut-off tat was reported for three tests: activated partial thromboplastin time, full blood count and platelet count. similarly, six tests reported a percentage within cut-off tat between 95% and 99%. the dashboard also reports component tat in hours (figure 4), including (1) lab-to-lab, (2) testing and (3) review times, with the tail size in hours for the distribution of each component tat. in any given laboratory, some samples tested are local (from the immediately adjacent hospital), while other samples are referred for testing from nearby hospitals where these tests are not available. as such, a zero lab-to-lab component indicates that the samples were not referred but are samples collected and tested locally. for referred samples included in the example data set (see figure 4, cd4 antiretrovirals, d-dimer, hiv viral load among others), the lab-to-lab component tat 75th percentile represents the inter-laboratory referral time, ranging in this instance from 12 to 23 hours (figure 4). in the testing phase, tat ranged from 0.25 to 63 hours (where 63 hours represented a single test, the rapid plasma reagin, syphilis, that was regarded locally as an outlier; see table 1 for detail). the 75th percentile review tat was 2 hours or less across all tests. figure 4: microstrategy desktop dashboard bar chart used to report the component turn-around time data for an example site’s for the week 2–8 september 2018, south africa. the components reported are lab-lab (inter-laboratory referral time), testing (time from registration to testing) and review (time from testing to review) turn-around time components. discussion access to information in an interactive dashboard format has previously enabled retrieval of health data for immediate clinical use in the nhls in south africa.8 a similar approach has been applied and demonstrated in this work for tat data. the dashboard described here provides an interactive, weekly snapshot of tat performance together with information about tat distribution, tail size (outlier) assessment,1,3 to varying levels of laboratory managers across the nhls, to enable timely intervention where poor service delivery is identified. the dashboard is comprised of a few basic parameters that act together to provide information about tat. date-stamping of samples in the lis is a prerequisite to provide the basic information necessary to detail tat linked to any given sample. together with relevant sample identification datalogged, data is transferred to a central database for careful curation. later tat data extraction is performed using standard data query tools. in the instance of a wider network of laboratories operating within the same organisation, such as the south african nhls, lis data is stored using a decentralised architecture. aggregate data, in the format described above, can then be collated and used to develop national tat dashboards. the dashboard described in this study simplifies presentation of complex data by enabling visualisation of any given laboratory’s efficiency. for the purpose of this study and to demonstrate the effectiveness and simple format of the dashboard developed, data from a single busy laboratory were used to illustrate the different outputs of the dashboard (graphs and table). the example data used here reveal how the dashboard can be used to identify tests that are not meeting national (or local) cut-off criteria. in the example presented, rapid plasma reagin (syphilis) testing was noted as an outlier as it did not meet the organisation-stipulated 85% within cut-off tat. the summary table (example shown in table 1) also provides a spreadsheet format table of the relevant tests either meeting, or failing to meet, the national cut-off criteria. the additional information on tat component analysis further assists management to identify those areas of laboratory testing, within the respective pre-analytical, analytical and post-analytical components, that may need investigation for improvement. the dashboard was successfully rolled out to all nhls testing laboratories; weekly data are currently received by these laboratories for review. the dashboard development included a drilling down function into the performance of a particular test to see results by testing laboratory, or business unit. the addition of tail size measures1,3 has also enabled managers to identify less efficient areas of their laboratory services with outlying performance, seen in the example case described in this article (rapid plasma reagin – syphilis), which would have been otherwise missed using conventional reporting alone (using mean tat reporting). in addition, to enable practical sample-by-sample audit, individual samples that did not meet cut-off criteria were identified for follow-up in an additional summary table sheet (added at the request of laboratory managers to enable better intervention, not shown). use of the dashboard has also led to laboratory process changes with improved individual component tat. for example, post-analytical tat improvements included implementation of ‘auto review’.9,10,11 with respect to analytical delays identified, testing delays could be correlated with instrument breakdown logs from the laboratories or instrument suppliers to identify reasons for prolonged testing tat.9 the impact of dashboard usage on improving tat is described in detail in the companion article in this issue.9 risk management teaches that not all errors can be predicted.12 however, it is only through active review of quality processes that delays, errors and problems can be detected earlier to enable corrective action. thus, critical to managing risk is the continuous and ongoing evaluation and assessment of procedures and processes to ensure that the same errors are not repeated. here, human capital is key to the sustainability and success of any dashboard implementation. noble et al.12 reported that only the persistence and interest of laboratory personnel to maintain quality can ensure smooth and rapid progress of error detection (and correction back to quality) that is fast and sustainable. one of the fundamental lessons learnt from the development of the dashboard described here is that providing tools to assess tat performance does not in itself imply corrective action or improvement. the dashboard is merely a tool that enables managers to effectively and efficiently ensure procedural excellence. nkengasong et al.13 also suggest that in order for innovation to be adopted and sustainable, innovation and performance enablers should both energise and incentivise laboratories across four pillars: implementation, measurement, reward and improvement. a culture of diligence and willingness on the part of managers to meaningfully use information provided in the dashboard is thus important to enable making consequential changes at the laboratory level. political will and strong senior leadership are also needed to make systems, such as those introduced with the dashboard described here, both functional and sustainable.13 this can be done by appropriately recognising and rewarding laboratories and personnel who use the tools provided. pre-analytical errors should not be underestimated, as they can increase both testing errors and tat.13 in the example laboratory performance reported here, all four referred tests tat outcomes were compromised due to pre-analytical delays. documenting these delays and acting to reduce pre-analytical time, including travel time, and time spent in receiving centres prior to sample registration, can be used to streamline services. another outcome reported by managers using the dashboard was that the information could be documented week-by-week to provide objective evidence to document and motivate for additional resources required to achieve tat cut-offs, for example additional sample collection schedules, increasing testing capacity, and motivation for auto review and authorisation modules.9 it is important in the context of a resource-poor setting to highlight that the dashboard described here was developed without specific funding, relying only on the collaborative effort of nhls staff (the authors) with data management or microstrategy skills. data is routinely transferred from the lis to the cdw, where is it collated and carefully curated for downstream research and operational needs. initial formats were undertaken using cdw extracted data analysed in ms excel to create simple charts plotting 75th percentile and median tat, by laboratory, for annualised or quarterly aggregated tat data. thereafter, analyses were extended to create week-by-week practical and usable worksheets so that individual laboratories could view current data. using microstrategy, a freely available software program, a dashboard was developed to enable automatic presentation of the data in a visible interactive format (with the snapshot aggregate data file emailed to users weekly) to facilitate automated more immediate access to current tat data. future planning includes providing live data in the dashboard, facilitated by extending local bandwidth capacity and immediate real-time analysis of data within the cdw itself. conclusion this article outlines the database management and methods used for the development of a dashboard that enables presentation of weekly tat data to relevant business and laboratory managers, as part of the overall quality management portfolio of the organisation. this novel approach ensures the delivery of quality, timely pathology reporting by the south african nhls and, ultimately, better patient care. training on the use of the dashboard is essential to ensure that users are competent. users need to both understand the principles applied in the dashboard as well as the functionality embedded in the dashboard. political will and leadership are vital to ensure that deficiencies identified by the dashboard lead to better quality and more efficient and timely laboratory services. as african laboratories move toward increasing the number of centres that prepare for or achieve accreditation,13 it is vital that laboratories are aware of the commitment needed to continually monitor, evaluate and re-assess their status quo. such commitment will ensure that the quality of the laboratory services they offer shows improvement over time. it is therefore important to consider what is required to achieve and maintain the quality of testing to avoid costly pitfalls14 and inaccurate or delayed result reporting. in this regard, although much of the focus of quality management is placed on quality of tests themselves, time management in a laboratory is as crucial as assuring the quality of the tests performed. without timely delivery of patient results, appropriate and meaningful clinical management of patients cannot be accomplished. limitations the data presented in this study focus on the within-laboratory network tat and did not record or assess delays outside the laboratory capture net. pre-analytical tat referred to in this work denotes the time taken to transport a sample from a receiving laboratory to a testing laboratory. ideally, sample tracking systems that relay tracking data to the central data warehouse, linked to discrete samples,will enable total end-to-end service assessment of tat. lessons from the field the dashboard subsequently developed has been extended to the top 22 highest volume tests performed across the organisation but does not report data for pathology sections like microbiology or anatomical pathology disciplines or the more specialised units like cytogenetics or immunology. plans are underway to broaden the test basket and to additionally include critical tests such as cardiac troponin levels, shown in other work (not reported here) to have tat that currently falls beyond meaningful clinical impact. the data presented provide only a weekly snapshot. as technology permits, it is important to extend and broaden development of this dashboard at the database warehouse level using business intelligence analytics tools that enable reporting real-time data. it is envisaged that laboratories could use large screens within laboratories themselves to track real-time progress for immediate response and corrective action, where required. alternatively, remote management could be facilitated using specially developed mobile devices to display live tat performance. the currently reported dashboard data does not distinguish between different levels of service (i.e. tertiary versus primary and secondary hospitals) with different levels of patient care (intensive care unit, stat-lab, trauma departments). data is aggregated and compared to the national cut-off for each test in the dashboard presented here. however, individual laboratories have established locally-relevant tat cut-offs for emergency and routine contexts depending on the level of care (primary versus tertiary). acknowledgements the authors thank area, business and laboratory managers in gauteng for their participation in the pilot project. the test basket used in the development of this dashboard was determined in consultation with this group. the authors also thank mr bahule motlonye of the national health laboratory service for his input during the pilot phase of the dashboard development undertaken during 2017. competing interests the authors declare no conflict of interest. authors’ contributions d.k.g. supervised the study by providing leadership and oversight as the project leader. n.c., m.e.t. and l.m.c. designed the study, developed the methodology and conducted the research. m.e.t. developed the data systems to deliver a dashboard. m.e.t., n.c. and l.m.c. conducted the data analysis. d.k.g. reviewed the data, provided editorial comments and technical input. all authors contributed to the manuscript development. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references hawkins rc. laboratory turnaround time. clin biochem rev. 2007;28(4):179–194. intersystems. trakcare lab enterprise versus standard lims [homepage on the internet]. cambridge, ma: intersystems; 2018 [cited 2018 dec 03]. available from: https://www.intersystems.com/products/trakcare/trakcare-lab-enterprise/#features coetzee lm, cassim n, glencross dk. using laboratory data to categorise cd4 laboratory turn-around-time performance across a national programme. afr j lab med. 2018;7(1):a665. https://doi.org/10.4102/ajlm.v7i1.665 drury s, coetzee lm, cassim n, glencross d. using central data warehouse (cdw) reports for monitoring cd4 laboratory workload and related turn-around-time (tat). 1st international conference of the african society for laboratory medicine (aslm 2012); cape town, south africa; 1–7 december 2012. lifewire. facts versus dimensions tables in a database [homepage on the internet]. new york: lifewire; 2018 [cited 2018 dec 03]. available from: https://www.lifewire.com/facts-vs-dimensions-1019646 carmona s, macleod w. development of paediatric, vl and cd4 dashboards and results for action reports [homepage on the internet]. funded by usaid. 2017 [cited 2018 dec 03]. available from: http://www.righttocare.org/wp-content/uploads/2016/11/programme.pdf microsoft corporation. microsoft office [homepage on the internet]. redmond, wa: microsoft corporation; 2018 [cited 2018 dec 03]. available from: https://products.office.com/en-za/products microstrategy. microstrategy desktop [homepage on the internet]. va: microstrategy; 2018 [cited 2018 dec 03]. available from: https://www.microstrategy.com/us/get-started/desktop cassim n, tepper meet, coetzee lm, perelson l, glencross dk. evaluating the impact of an interactive turn-around-time (tat) dashboard to improve performance at a busy high test volume laboratory. afr j lab med. in press 2018. krasowski md, davis sr, drees d, et al. autoverification in a core clinical chemistry laboratory at an academic medical center. j pathol inform. 2014;5(13):15. https://doi.org/10.4103/2153-3539.129450 cassim n, tepper meet, coetzee lm, perelson l, glencross dk. impact of use of a tat dashboard in improving laboratory test turn around times. afr j lab med. in press 2018. noble ma, martin r, ndihokubwayo jb. making great strides in medical laboratory quality. afr j lab med. 2014;3(2), art. 256, 2 pages. https://doi.org/10.4102/ajlm.v3i2.256 nkengasong jn, birx d. quality matters in strengthening global laboratory medicine. afr j lab med. 2014;3(2), art. 239, 2 pages. https://doi.org/10.4102/ajlm.v3i2.239 okeke in. building reasources to meet evolving laboratory medicine challenges in africa. afr j lab med. 2018;7(1):a915. https://doi.org/10.4102/ajlm.v7i1.915 article information authors: ankie van den broek1 coosje j. tuijn2 lisette van ’t klooster2 elizabeth msoka3 marion sumari-de boer3 jaffu chilongola4 linda oskam2 affiliations: 1royal tropical institute (kit) health, the netherlands2royal tropical institute (kit), biomedical research, the netherlands 3kilimanjaro clinical research institute (kcri), tanzania 4kilimanjaro christian medical university college, tumaini university makumira correspondence to: ankie van den broek postal address: royal tropical institute, kit health, po 95001, 1090 ha, amsterdam, the netherlands dates: received: 10 july 2013 accepted: 25 feb. 2014 published: 24 july 2014 how to cite this article: van den broek a, tuijn cj, van ’t klooster l, msoka e, sumari-de boer m, chilongola j, oskam l. understanding the interface between clinical and laboratory staff. afr j lab med. 2014;3(1), art. #127, 6 pages. http://dx.doi.org/10.4102/ ajlm.v3i1.127 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. understanding the interface between clinical and laboratory staff in this original research... open access • abstract • introduction • research method and design    • literature search    • analysis of guidelines    • testing • results    • the conceptual model       • inner circle: three phases where communication takes place       • outer circle: factors influencing relationships and communication       • organisational factors       • personal factors       • relationship between organisational factors and personal factors       • context       • analytical framework • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: the interface between clinicians and laboratory staff is where the two meet and work together to provide quality care to their clients (patients). effectiveness of the interface depends on the way the two groups of professionals relate to and communicate with each other. the number and type of tests requested and the use of the test results for clinical decision making can be influenced by the interface between clinicians and laboratory staff. a model to understand the factors and dynamics around the interface is lacking.objectives: to propose a new conceptual model to gain insight and analyse factors that influence the laboratory–clinical staff interface. methods: to develop the conceptual model, a literature study was performed, regulatory guidelines and standards for laboratories were analysed and discussions were held with experts on the topic. result: a conceptual model and analytical framework provided good guidance in understanding and assessing the organisational and personal factors shaping the interface. the model was based on three elements: (1) the three phases of communication (pre-analytical, analytical and post-analytical); (2) the organisational and personal factors of interaction; and (3) the socio-political, economic and cultural context in which clinicians and laboratory staff operate. conclusion: assessment of the interface between clinicians and laboratory workers can be performed in a systematic way. applying this model will provide information to managers of health institutions and heads of laboratories and clinical departments about what happens when clinicians and laboratory staff interact, thus aiding them in designing strategies to improve this interface. introduction top ↑ diagnostic tests requested by clinicians are performed by laboratory staff and provide clinicians and patients with the test results that are required for clinical decision making. the contribution of laboratory services to clinical decision making not only depends on the performance of the laboratory itself, but also on the behaviour of clinicians with regard to requesting tests and using the results.1 request behaviour is influenced by the interactions between these two health cadres.2 a study by bridges et al.3 on interprofessional collaboration highlighted factors that shape the interface, including responsibility, accountability, coordination, communication, cooperation, assertiveness, autonomy, mutual trust and respect. in low-income countries, little research has been performed to understand elements that shape the quality of the interface between clinicians and laboratory workers and their influence on the quality of care. carter et al.4 mention the lack of communication between clinicians and laboratory services, giving various reasons for this from the perspective of the clinicians and the laboratory staff. according to this article, clinicians are not accustomed to teamwork and the laboratory staff may not recognise the clinical importance of their findings, either for clinical decision making or for patient management.4assessment of the interface between clinicians and laboratory workers will help health workers and their managers understand how factors related to the organisational culture and the personalities of the staff members have an impact on the interface and, therefore, the effectiveness and quality of service delivery. research method and design top ↑ the conceptual model depicted in figure 1 was developed after completion of a literature search and an analysis of regulatory guidelines and standards for laboratories, followed by a thorough discussion between clinical and laboratory experts within the team. following this discussion, the study team returned to the literature to search for missing information. figure 1: conceptual model of the various factors that shape the interface between clinical and laboratory staff. literature search a literature search was performed using a non-systematic approach, as it was known beforehand that minimal peer-reviewed literature would be available and important data could be found in grey literature. the first literature search (scopus) yielded 59 relevant peer-reviewed articles, from which 14 were selected as appropriate. articles were searched for that could provide information about the interactions and interface between laboratory and clinical services by using different search terms referring to the services or to the staff working at these services. the search strategy included the following terms: ‘laboratory services’ and ‘health systems’; ‘laboratory services’ and ‘human resources’ or ‘laboratory personnel’ or ‘laboratory staff’ or ‘laboratory workforce’; ‘laboratory services’ and (‘role’ or ‘impact’) and ‘health care’ or ‘health services’; ‘laboratory services’ and (‘physicians’ or ‘clinician’ or ‘nurses’ or ‘health manpower’ or ‘health personnel’ or ‘medical staff’ or ‘nursing staff’ or ‘patients’) and (‘consumer satisfaction’ or ‘satisfaction’ or ‘dissatisfaction’ or ‘interaction’ or ‘opinion’ or ‘attitude of health personnel’); ‘laboratory services’ and (‘essential’ or ‘rational’) and ‘health care’ or ‘hospital’ or ‘hospitals’ or ‘clinic’ or ‘clinics’ or ‘medical centre’ or ‘medical centres’. the second search performed focused on peer-reviewed articles and grey literature from 1995 onwards using google scholar, isi web of knowledge, pubmed, the royal tropical institute website, science direct, scopus and the world health organization website. the following search terms were used: ‘attitude of health personnel’; ‘laboratories/utilization’; ‘physicians/psychology’; ‘trust’ and ‘clinician’ or ‘health workers’; ‘laboratory quality’; ‘national laboratory guideline’; ‘laboratory strengthening declaration’. the term ‘interface’ was not used in the search strategy as it was not expected to increase the number of articles related to the interface between laboratory workforce and clinicians; when both types of health workers are mentioned, all articles concerning this interface appear. adding the term ‘interface’ would yield the retrieval of articles that discuss the interface between the functioning of the laboratory and computerised systems used in the laboratory. this search was followed by the snowball literature search method. analysis of guidelines the analysis of regulatory guidelines and standards for laboratories (international organization for standardization [iso] 15189,5 iso 228696 and iso 9001;7 clinical and laboratory standards institute [clsi] gp 26;8 joint commission international [jci]9) provided information on the proposed daily practices for human resource management and relationship building between the laboratory and clinical departments. the interface between the clinicians and laboratory staff is addressed in these documents. an analysis showed that maintaining the relations with customers and monitoring of the customer satisfaction (clinicians are considered to be customers of the laboratory) are both included in several of these regulatory guidelines (table 1). table 1: issues in iso 15189, iso 22869, iso 9001, clsi gp 26 and jci guidelines that refer to the interface between the laboratory and clinical departments. the importance of including the interface in regulatory guidelines and standards for laboratories is also recognised by yao et al.,10 who mention the consultation of the client and client satisfaction surveys as being key areas on the road to laboratory accreditation. based on the findings from the literature search, intensive discussions were held amongst the clinical and laboratory experts in the study team. it was determined that the quality of the interface is related to the moment when interaction happens, the organisational culture of the health facility and the personalities of the clinicians and the laboratory staff. testing the conceptual model was tested during a mixed-method field study in four health facilities in moshi district of kilimanjaro region, in tanzania. this field study is described in ‘the interface between clinicians and laboratory staff: a field study in northern tanzania’ by tuijn et al.11 results top ↑ the conceptual model the conceptual model is based on three elements: (1) the phases during which communication takes place; (2) the organisational and personal factors that influence the interface; and (3) the social, political, cultural and economic context in which the health facility operates. inner circle: three phases where communication takes place plebani12 argues that clinicians and laboratory staff interact during the preand post-analytical phases; this concept was taken as being the starting point of the framework. a third phase was added to plebani’s concept, namely, the analytical phase. application of plebani’s concept to the analytical phase refers to communication that takes place during the performance of a test or a range of tests; for example, a laboratory worker may ask for clarification on the sample type, sample volume, or information regarding the patient’s situation in order to understand the test results or to suggest additional tests. incidentally, the clinician can be present when a test is performed and the outcome can be discussed based on observations by both health cadres (although this does not happen often). in this way, the phases are linked to the time frame in which the clinician (on behalf of the patient) asks for the diagnostic test, waits for the test results and develops a plan after receiving the results. in all three phases, information sharing between clinicians and laboratory staff can take place. during these phases, a number of activities and interactions take place, as are listed in table 2. this list of activities is based on the knowledge and experience of clinicians and laboratory scientists in the research team. table 2: activities and interactions taking place between clinicians and laboratory staff during the pre-analytical, analytical and the post-analytical phases. outer circle: factors influencing relationships and communication communication between clinicians and laboratory staff is influenced by: (1) organisational factors, such as the management (rules, guidelines, meetings) and the identity of the organisation; and (2) personal factors (knowledge, attitude, competencies) visualised in the outer circle of the conceptual model. in some cases, no literature could be found to support the opinion that certain factors influence the dynamics in the interface. as explained in the methodology, the factors adopted in the model were based on the experiences of the study coordinators. organisational factors identity of the organisation: identity can influence many aspects in an organisation such as conditions for thinking and learning; possibilities for open communication between management and staff; and communication between health cadres working in various departments of the health facility. for this article, the identity of a health organisation in a low-income country was defined based on its position in the health system (primary, secondary or tertiary level); the ownership of the organisation (public, private – faith-based or secular); and the organisation’s profit status (not-for-profit or corporate). it was assumed that the identity of the organisation has a bearing on the level of knowledge of clinicians and laboratory staff. for example, the level of education and opportunities for continuous professional development are different for staff working in referral hospitals compared with staff working in primary healthcare facilities. monitoring and supervision of health staff can be organised differently in public and private health facilities.the identity of an organisation can also relate to the availability of resources, such as equipment, consumables and human resources, as well as to leadership and management styles, thus influencing the motivation of the health staff. it can also influence the personalities and the relationships between people working in an organisation: in faith-based organisations, it is likely that staff have activities in common outside working hours (e.g. church activities) that can influence the communication channels in the health facility. although the literature study did not provide information related to the identity of the organisation, this factor was added to the framework as it was assumed that this is an important variable for the interface between different departments in a health institution. style of management: the management can influence the interface between the clinical and laboratory departments in a health institution in three ways: 1. provision of rules and guidelines for the requesting of diagnostic tests (selecting the correct test, following correct test requisition methods, being aware of the availability of tests) and reporting of test results. here. the role of an ‘intermediate health worker’, often a nurse, is important to take into consideration, but was not noted in the literature search. however from the field study it was learned that a nurse is often responsible for taking samples to the laboratory and collecting the test results, as well as transferring them to the clinician. 2. facilitation of mandatory and voluntary meetings in which clinical and laboratory staff participate and interact. 3. the style of management that influences the performance and motivation of health workers by mechanisms such as availability of job descriptions, supportive supervision of staff, implementation of staff appraisals, provision of opportunities for continuous education and career development and demonstration of appreciation for the staff’s work. these mechanisms also influence the interaction between different cadres of health workers that need each other’s competencies in order to perform their work. personal factors the personal factors that influence the interface are the individual competencies of the health workers, including knowledge, attitude and skills, as well as issues relating to the professionalism and professional education of the clinical and laboratory staff. in the literature, several examples are provided regarding personal factors that influence the laboratory–clinician interface. table 3 provides an overview of the evidence found in the literature regarding these personal factors. table 3: evidence found in peer-reviewed and grey literature on personal factors that have an influence on the interface between clinicians and laboratory staff. phases. the individual competencies of clinical and laboratory staff can be insufficient because of lack of updates on national policies and guidelines, lack of supervision and coaching and lack of motivation to understand the impact of inaccuracy on the quality of care. in the field study described by tuijn et al.,11evidence was found suggesting that lack of competencies can also be a result of a low level of education, especially amongst laboratory workers. many tasks in the laboratory are performed by laboratory attendants without formal training. during the field study, it was determined that the needs and wishes of patients could influence clinicians’ test-requesting behaviour or their use of test results. when waiting times at the laboratory were long, clinicians did not ask for a repeat test, as it could be inconvenient for the patient. when a clinician was influenced in his request behaviour, it was classified as a personal factor. however, management can have an impact on personal factors when, through guidelines and supervision, these issues are discussed. issues related to professional education and professionalism are: (1) the different professional viewpoints of clinicians and laboratory workers: the laboratory worker is focused on the outcome of the test as the gold standard for treatment whilst the clinician values it as additional information to confirm or complete the clinical diagnosis; (2) the institutionalised professional positions of both cadres in the health system and health facility in which clinicians occupy a higher position in the hierarchy; and (3) trust in the quality of the work of the other health professional: for the clinician to make decisions regarding diagnosis and treatment and for the laboratory worker to make decisions regarding the test procedures and interpretation of results, each needs to rely on the competence of the other. relationship between organisational factors and personal factors organisational factors and personal factors are partly interdependent. management of the health facility can influence the individual factors and professionalism through attrition and deployment policies, supervisory mechanisms and opportunities for continuous professional education. the management style can also impact staff motivation and the acceptance and ability of the various health professionals to communicate with persons in different positions and different fields of expertise. the identity of the health facility can also have an influence on the type of job applicants and on personal contacts between staff members (e.g. workers at faith-based hospitals often meet during church activities). context in the outer square of the conceptual model is the context in which the health institution operates. social, cultural, political and economic factors have an impact on the hospital and its health workers. many low-income countries experience a severe shortage of human resources for health, leading to understaffing or employment of insufficiently-qualified staff in several departments of the hospital.13,14,15,16,17,18,19,20 the field study confirmed the shortage of qualified laboratory staff, leading to a situation where staff with less education take on responsibilities that include communicating with highly-educated clinicians. analytical framework we developed an analytical framework that serves as a guide when assessing the interface between laboratory and clinical staff. in this framework, all the factors and activities that influence the dynamics around the interface in the three phases are brought together. this analytical framework was used and is explained in more detail in the field study, ‘the interface between clinicians and laboratory staff: a field study in northern tanzania’, by tuijn et al.11 discussion top ↑ this model is new and was developed for health services in low-income countries, which face challenges with regard to service delivery in resource-constrained settings. the conceptual model and the framework provide an overview of factors that determine the interface between clinicians and laboratory workers. all of the factors that provided the basis for this conceptual model informed the study team about the complexity of this interface, but also showed that a well-functioning interface can contribute to quality of care. in low-income countries, little attention is given to this interface, even though the high workload for many of the health workers requires the efficient use of all services and thus efficient cooperation between services to provide quality care. by creating awareness in both groups and improving the interface between the clinicians and laboratory staff, use of laboratory services can be optimised, enabling clinicians to make better diagnoses and treatment plans for their patients. in developing this model, it was determined that the identified peer-reviewed articles do not provide information regarding all factors that influence an effective interface; for example, the interdepartmental management was hardly discussed. however, the importance of interdepartmental management is partly addressed in the reviewed regulatory standards for laboratories, which mention development and/or monitoring of relationships with clients, including physicians, as well as policies regarding interdepartmental meetings. several factors which were included in the model were not mentioned in either peer-reviewed articles or regulatory standards, for example, general personal factors such as age and gender, and cultural factors such as hierarchy in relationships or family ties, but it was assumed that these factors influence staff attitudes and they were thus included in the framework. the pilot field study provided indications that these factors influence the communication between clinicians and laboratory staff, but the study was too small to make firm conclusions about this issue. the complexity of the interface, which is influenced by organisational and personal factors as well as the health facility’s context, calls for a holistic analysis involving all stakeholders (clinicians, laboratory staff, intermediate health workers such as nurses, the management and patients or clients). this first field study has demonstrated the robustness of this conceptual model. it enables analysis of the factors that shape an effective interface. the plan is to perform an assessment of the interface with a sample of 20 health facilities to increase the evidence on these factors. outcomes of such a study may motivate managers of health institutions and heads of laboratories and clinical departments to invest in analysis of interdepartmental interaction so that, based on their findings, they can design strategies to improve the interface in their settings. conclusion top ↑ a new conceptual model has been developed to assess the interface between laboratory and clinical staff in low income countries. acknowledgements top ↑ we kindly acknowledge marjolein dieleman, royal tropical institute (kit) health, development policy and practice, for her valuable input and critical assessment of this article. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. authors’ contributions a.vdb. (royal tropical institute [kit] health), c.j.t. (royal tropical institute [kit], biomedical research), l.vtk. (royal tropical institute [kit], biomedical research) and l.o. (royal tropical institute [kit], biomedical research) developed the conceptual model. a.vdb., c.j.t, e.m. (kilimanjaro clinical research institute), m.s-db (kilimanjaro clinical research institute) and j.c. (kilimanjaro christian medical university college) participated in the field study to test the model. a.vdb. drafted the article. all authors contributed to and approved the final manuscript. references top ↑ 1.cohen gm. access to diagnostics in support of hiv/aids and tuberculosis treatment in developing countries. aids. 2007;21(suppl 4):s81–s87. http://dx.doi.org/10.1097/01.aids.0000279710.47298.5c 2.polage cr, bedu-addo g, owusu-ofori a, et al. laboratory use in ghana: physician perception and practice. am j trop med hyg. 2006;75(3):526–531. 3.bridges dr, davidson ra, odegard ps, et al. interprofessional collaboration: three best practice models of interprofessional education. med educ online. 2011;16:10. http://dx.doi.org/10.3402/meo.v16i0.6035 4.carter j, müller-stöver i, östensen h, et al. good clinical diagnostic practice: a guide for clinicians in developing countries to the clinical diagnosis of disease and to making proper use of clinical diagnostic services. world health organization regional office for the eastern mediterranean: cairo; 2005. isbn: 978-92-9021-393-2. available from: http://www.scribd.com/doc/160701374/dsa236-goof-clinical-diagnostic-practice-who-pdf 5.international organization for standardization. iso15189:2007. medical laboratories – particular requirements for quality and competence [page on the internet]. 2007 [cited 2014 mar 30]. available from: http://www.iso.org/iso/catalogue_detail?csnumber=42641 6.international organization for standardization. iso/tr 22869:2005. medical laboratories – guidance on laboratory implementation of iso 15189:2003 [page on the internet]. 2005 [cited 2014 mar 30]. available from: http://www.iso.org/iso/home/store/catalogue_tc/catalogue_detail.htm?csnumber=35172 7.international organization for standardization. iso 9001:2008. quality management systems – requirements [page on internet]. 2008 [cited 2014 mar 30]. available from: http://www.iso.org/iso/catalogue_detail?csnumber=4648 8.linical laboratory standards institute. gp26-a3. application of a quality management system model for laboratory services, approved guideline – third edition. nccls: wayne, pa; 2004. available from: http://isoforlab.com/phocadownload/csli/gp26-a3.pdf 9.joint commission international. accreditation standards for clinical laboratories. 2nd ed. joint commission international: oakbrook terrace, il; 2010. 10.yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin pathol. 2010;134(3):401–409. http://dx.doi.org/10.1309/ajcpnbbl53fwuiqj 11.tuijn cj, msoka e, mushi dl, et al. the interface between clinicians and laboratory staff: a field study in northern tanzania. afr j lab med. 2014;3(1), art. in press. 12.plebani m. exploring the iceberg of errors in laboratory medicine. clin chim acta. 2009;404(1):16–23. http://dx.doi.org/10.1016/j.cca.2009.03.022 13.nuwaha f. the challenge of chloroquine-resistant malaria in sub-saharan africa. health policy plan. 2001:16(1):1–12. http://dx.doi.org/10.1093/heapol/16.1.1 14.mepham so, squire sb, chisuwo l, et al. utilisation of laboratory services by health workers in a district hospital in malawi. j clin pathol. 2009;62(10):935–938. doi: 10.1136/jcp.2009.069062938 15.world health organization. strategic approach for the strengthening of laboratory services for tuberculosis control, 2006–2009 [document on the internet]. 2006 [cited 2014 mar 30]. available from: http://apps.who.int/iris/bitstream/10665/69303/1/who_htm_tb_2006.364_eng.pdf?ua=1 http://dx.doi.org/10.1093/heapol/czm046 17.butao d, chafulumira f, felling b, et al. malawi: laboratory services and supply chain assessment. usaid/deliver project: arlington, va; 2009. 18.barat l, chipipa j, kolczak m, et al. does the availability of blood slide microscopy for malaria at health centers improve the management of persons with fever in zambia? am j trop med hyg. 1999;60(6):1024–1030. 19.may ta, clancy m, critchfield j, et al. reducing unnecessary inpatient laboratory testing in a teaching hospital. am j clin pathol. 2006;126(2):200–206. http://dx.doi.org/10.1309/wp59ym73l6cegx2f 20.world health organization. the world health report 2006 – working together for health. who: geneva; 2006. available from: http://www.who.int/whr/2006/en/ acknowledgements references about the author(s) francesco marinucci abbott gmbh, wiesbaden, germany jens dhein abbott gmbh, wiesbaden, germany citation marinucci f, dhein j. the time to address diagnostic needs in universal health coverage is now: leveraging the scale up of national testing capacity for hiv viral load and sars-cov-2. afr j lab med. 2022;11(1), a1685. https://doi.org/10.4102/ajlm.v11i1.1685 opinion paper the time to address diagnostic needs in universal health coverage is now: leveraging the scale up of national testing capacity for hiv viral load and sars-cov-2 francesco marinucci, jens dhein received: 28 july 2021; accepted: 01 mar. 2022; published: 22 june 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the goals of universal health coverage (uhc) are to ensure that all people can access the quality health services that they need, to protect people from public health threats and to avoid catastrophic health expenditure.1 the range of essential services under uhc is country-specific and depends on factors such as the existing health system, the burden of different diseases and the resources available. as countries embark on defining their guaranteed package of services, the existing laboratory network represents an extraordinary opportunity to use the already existing equipment at its install base and full capacity across programmes, and to expand the laboratory testing menu in the uhc package. this article focuses on diagnostics and describes the reasons why flexible and high-throughput molecular platforms are critical to ensure access to quality testing and to improve diagnostic service coverage in the context of uhc initiatives. integrated models of service delivery are instrumental for ensuring impactful and sustainable uhc interventions. horizontal integration links healthcare professionals operating at the same level of care and ensures multiple diagnostic tests are available in the same laboratory tier. the focus of vertical integration is to increase coordination among testing sites and to improve communication within the different tiers of the laboratory network (figure 1). as a result of the laboratory network optimisation, the laboratory system can fulfil the multiple needs of patients in more efficient and sustainable ways.2 figure 1: vertical and horizontal integration of diagnostic services across the laboratory network allows all patients to access the same testing menu regardless of their entry point to the health system. most testing has been carried out in central laboratories serving a large network of health facilities. unfortunately, the role of laboratory-based platforms that allow the expansion of laboratories’ menus of assays (polyvalence) on the same analyser across different public health programmes has been undervalued, resulting in inadequate investments in vertical integration. this lack of investment has exacerbated the persistent issues related to communications between health facilities with the consequence of long turn-around times to get actionable results back to clinicians. instead of strengthening the infrastructural gaps, the focus of different vertical programmes switched to bringing diagnostic services closer to patients, which increased the divides within laboratory networks. the development of diagnostics that can generate results in primary healthcare settings has favoured the decentralisation of diagnostic services in several areas, including tuberculosis, hiv, viral hepatitis and malaria, with the main result being that diagnostic capacity is now closer to the patients.3 global and national guidelines accompanied this transition to lower-level health facilities; however, many programmes for major diseases in lowand middle-income countries are still organised in vertical silos and lack the vision of combining platforms allowing menu expansion in different tiers of the laboratory network.4 combined vertical and horizonal integration of diagnostic services across diseases has the potential to bring numerous benefits. for patients this results in more comprehensive care and services, added convenience due to time savings and reduced out-of-pocket expenses. for programmes, this results in cost savings from a more efficient use of resources, earlier treatment initiation and hence reallocation of resources to additional services. lastly, this would increase the resilience of the health system to adapt to the onset of new diseases and public health threats that happen unexpectedly, such as outbreaks and epidemics. the recent coronavirus disease 2019 (covid-19) pandemic exacerbated the need for expanded diagnostic capacity in such situations and the importance of a quick response time to address the increased demand for testing.5 a significant number of countries, including lowand middle-income countries, have built up their infrastructure in a short time. diagnostics manufacturers responded to the pressing need for covid-19 testing with rapid development and the launch of new covid-19 tests. the increased awareness of the importance of diagnostic testing provides the opportunity to look at other diseases that can be eliminated and where diagnostics plays a key role. tuberculosis, hiv, cervical cancer and viral hepatitis, although treatable, are still underdiagnosed in many high burden countries. considering these concerns, over the years the world health organization has released several global strategies to end these diseases6 (figure 2). covid-19 has demonstrated the advantages and limitations of current technologies to diagnose infections. the polymerase chain reaction is regarded as the gold standard for the detection of severe acute respiratory syndrome coronavirus 2 as a result of its high sensitivity for specific genetic sequences. polymerase chain reaction-based techniques amplify this genetic material through multiple amplification cycles allowing the detection of small quantities of pathogens. over the last 17 years many countries have scaled up their hiv viral load testing capacity with financial support from the united states president’s emergency plan for aids relief and the global fund. the covid-19 pandemic has pushed countries to further scale up their polymerase chain reaction testing capacities for severe acute respiratory syndrome coronavirus 2 by installing automated and high-throughput molecular diagnostic systems. many of these systems have random access capability and allow the use of multiple polymerase chain reaction assays on the same platform. while demand for hiv viral load testing will remain stable in the near future, the demand for severe acute respiratory syndrome coronavirus 2 testing will likely decline, leaving molecular diagnostic testing platforms free and available for testing of other disease markers. with the equipment, reagents, skilled personnel, connectivity to the laboratory information system, training, and the technical support already in place, such automated platforms may help to achieve meeting the targets defined in the global strategies and uhc initiatives (figure 2). previous limitations would be mitigated since the increased level of automation reduces complexity and makes it easier for laboratories to perform testing. random access systems eliminate the need for the sorting and batching of samples, provide continuous loading capability, and ready-to-use reagents. larger on-board assay storage capacities and random access allow testing of low, medium and high-volume assays side-by-side. testing capabilities could be rapidly adapted in response to increasing demand during a pandemic or in response to seasonal changes caused by respiratory viruses.7 short sample-to-answer turn-around times allow same day reporting of results, with some of the molecular diagnostic platforms providing capability for sample prioritisation (true random access function) without disrupting routine sample processing.8 menus comprising multiplex polymerase chain reaction assays allow differentiation of several pathogens in a single test, such as for respiratory or sexually transmitted infections. multiplex assays enable laboratories to process more tests on demand in a given period of time. this conserves important testing materials that may be in short supply during an outbreak situation, and it saves sample volume that can be used for additional analyses. the addition of middleware solutions to the automated molecular diagnostic platforms could provide auto-verification capabilities that automate data transfer, improve turn-around time, reduce manual labour and the potential for human error. centralised statistics and consolidated reports provided from the laboratories can give public health officials information for their ongoing surveillance programmes to control the spread of diseases and to monitor programme successes. taken together, the hiv viral load and the covid-19-driven uptake of molecular diagnostic platforms with expanded menus of assays provides a unique opportunity for laboratories to scale up molecular testing for hiv, viral hepatitis, tuberculosis, respiratory viruses, and high-risk human papillomavirus. the high automation level would enable laboratories to become more efficient in delivering high-quality results to support global health strategies in high burden countries. furthermore, the flexibility of such platforms, both in terms of volumes and testing menus, place them at the forefront of regional initiatives for coordinated outbreak response and other initiatives of concern to public health.9 simply increasing the number of shifts in the laboratory allows increases in the number of samples run on the platform. this flexibility is key for countries’ ability to rapidly cope with an increased demand for testing due to outbreaks and epidemics. the capability to use laboratory-developed tests on automated molecular diagnostic platforms can further strengthen the laboratories’ capabilities to respond in a timely manner to newly emerging diseases or variants. figure 2: link between global health strategies and automated molecular platforms. while enabling scalability to react in an outbreak situation, an expanded menu of assays is a strong basis to continue expanding uhc packages. countries can leverage existing sample referral networks, such as for hiv viral load testing, to serve multiple disease programmes by using the current infrastructure to move different specimen types from peripheral sites to the testing laboratory.10 the incremental cost-effectiveness of adding assays to the same high-throughput molecular diagnostic platform and the transportation of multiple specimens to the same laboratory would provide better use of scarce resources.11 this is particularly effective for infections like viral hepatitis c and human papillomavirus, where related diseases progress slowly, and access to results in a follow up visit does not impact clinical management. if required, results can be reported rapidly in situations where immediate clinical decisions must be made.8 good coordination across multiple programmes is the foundation for more integrated testing to combine the advantages of both point-of-care tests and high-throughput molecular diagnostic platforms. ideally, vertical and horizontal integration should occur simultaneously as part of the laboratory network optimisation supported by united states president’s emergency plan for aids relief, the african society for laboratory medicine and the africa centres for disease control and prevention, with the ultimate goal of deploying laboratory-based platforms and point-of-care technologies where they are needed the most. high-throughput molecular diagnostic platforms, when used at full capacity, allow better use of resources across the laboratory network, which is central for achieving more people-centred care. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions f.m. and j.d. contributed equally to the conceptualisation and writing of the manuscript. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references kieny mp, bekedam h, dovlo d, et al. strengthening health systems for universal health coverage and sustainable development. bull world health organ. 2017;95(7):537–539. https://doi.org/10.2471/blt.16.187476 ondoa p, ndlovu n, keita ms, et al. preparing national tiered laboratory systems and networks to advance diagnostics in africa and meet the continent’s health agenda: insights into priority areas for improvement. afr j lab med. 2020;9(2):1103. https://doi.org/10.4102/ajlm.v9i2.1103 parsons lm, somoskovi a, lee e, et al. global health: integrating national laboratory health systems and services in resource-limited settings. afr j lab med. 2012;1(1):11. https://doi.org/10.4102/ajlm.v1i1.11 considerations for adoption and use of multidisease testing devices in integrated laboratory networks [homepage on the internet]. world health organization information note 2017. who/htm/tb/2017.06. [cited 2021 may 07]. available from: https://www.who.int/tb/publications/2017/considerations_multidisease_testing_devices_2017/en/ ondoa p, kebede y, loembe mm, et al. covid-19 testing in africa: lessons learnt. lancet microbe. 2020;1(3):e103–e104. https://doi.org/10.1016/s2666-5247(20)30068-9 pai m, walia k, boehme cc. essential medicines and essential diagnostics: a package deal. lancet public health. 2019 oct;4(10):e492. https://doi.org/10.1016/s2468-2667(19)30165-3 venkatesan p. covid-19 diagnostics-not at the expense of other diseases. lancet microbe. 2020;1(2):e64. https://doi.org/10.1016/s2666-5247(20)30041-0 obermeier m, pacenti m, ehret r, et al. improved molecular laboratory productivity by consolidation of testing on the new random-access analyzer alinity m. j lab med. 2020;44(6):319–328. https://doi.org/10.1515/labmed-2020-0102 amukele t. africa cdc: establishing integrated surveillance and laboratory networks for rapid disease detection and response, control, prevention, and clinical care in africa. afr j lab med. 2017;6(1):a638. https://doi.org/10.4102/ajlm.v6i1.638 fonjungo pn, alemnji ga, kebede y, et al. combatting global infectious diseases: a network effect of specimen referral systems. clin infect dis. 2017;64(6):796–803. https://doi.org/10.1093/cid/ciw817 williams j, edgil d, wattleworth m, et al. the network approach to laboratory procurement and supply chain management: addressing the system issues to enhance hiv viral load scale-up. afr j lab med. 2020;9(1):a1022. https://doi.org/10.4102/ajlm.v9i1.1022 ajlm 4_1 reviewer acknolegdement.indd reviewer acknowledgement open accesshttp://www.ajlmonline.org page 1 of 1 we would like to take this opportunity to thank all of those who provided scientific and logistical support for this issue of the african journal of laboratory medicine: should names have inadvertently been excluded from this list, the publisher apologises and undertakes to amend the exclusion in the next issue. ajlm african journal of laboratory medicine in an effort to facilitate the selection of appropriate peer reviewers for the african journal of laboratory medicine, we ask that you take a moment to update your electronic portfolio on www. ajlmonline.org for our files, allowing us better access to your areas of interest and expertise, in order to match reviewers with submitted manuscripts. if you would like to become a reviewer, please visit the journal website to register. to access your details on the website, you will need to follow these steps: 1. log into the online journal at http://www. ajlmonline.org 2. in your ‘user home’ [http://www.ajlmonline. org/index.php/ajlm/ user] select ‘edit my profile’ under the heading ‘my account’ and insert all relevant details, bio statement and reviewing interest. 3. it is good practice as reviewer to update your personal details regularly to ensure contact with you throughout your professional term as reviewer for the african journal of laboratory medicine. please do not hesitate to contact us if you require assistance in performing this task. publisher: publishing@aosis.co.za tel: +27 21 975 2602 fax: +27 21 975 4635 adeola tomi-olugbodi alpha diallo amy piatek belete tegbaru bharat parekh brenda okech chris murrill christophe longuet dennis mok elisabet manasanch elisabeth gerwing-adima gajendran sivakumar heather alexander jack nyamongo john n. nkengasong joseph d. kitukulu kekoura kourouma kyle degruy lara vojnov lila rahalison linda parsons maja kodani michael z. wang musau wakabongo nicaise ndembi nwadiuto esiobu paolo ferrinho pascale ondoa pat riley patrick adam raquel villegas robert n. maina segundo r. leon sharon martin shirley lecher tjeerd datema yeshitila friew zirra m. mangoro abstract introduction methods results discussion acknowledgements references about the author(s) asmaa m. zahran department of clinical pathology, south egypt cancer institute, assiut university, assiut, egypt hanaa nafady-hego department of microbiology and immunology, faculty of medicine, assiut university, assiut, egypt sawsan m. moeen department of internal medicine, clinical haematology unit, faculty of medicine, assiut university, assiut, egypt hanan a. eltyb department of medical oncology, south egypt cancer institute, assiut university assiut, egypt mohammed m. wahman department of clinical oncology, south valley university, qena, egypt asmaa nafady department of clinical and chemical pathology, qena faculty of medicine, south valley university, qena, egypt citation zahran am, nafady-hego h, moeen sm, eltyb ha, wahman mm, nafady a. higher proportion of non-classical and intermediate monocytes in newly diagnosed multiple myeloma patients in egypt: a possible prognostic marker. afr j lab med. 2021;10(1), a1296. https://doi.org/10.4102/ajlm.v10i1.1296 original research higher proportion of non-classical and intermediate monocytes in newly diagnosed multiple myeloma patients in egypt: a possible prognostic marker asmaa m. zahran, hanaa nafady-hego, sawsan m. moeen, hanan a. eltyb, mohammed m. wahman, asmaa nafady received: 09 june 2020; accepted: 11 may 2021; published: 25 aug. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: interaction between multiple myeloma (mm) cells and proximal monocytes is expected during plasma cell proliferation. however, the role of monocyte subsets in the disease progression is unknown. objective: this study evaluated circulating monocyte populations in mm patients and their correlation with disease severity. methods: peripheral monocytes from 20 patients with mm attending assiut university hospital in assiut, egypt, between october 2018 and august 2019 were processed using a flow cytometry procedure and stratified using the intensity of expression of cd14 and cd16 into classical (cd16−cd14++), intermediate (cd16+cd14++), and non-classical (cd16++cd14+) subsets. the data were compared with data from 20 healthy control participants with comparable age and sex. results: in patients with mm, the percentage of classical monocytes was significantly lower (mean ± standard error: 77.24 ± 0.66 vs 83.75 ± 0.5), while those of non-classical (12.44 ± 0.5 vs 8.9 ± 0.34) and intermediate (10.3 ± 0.24 vs 7.4 ± 0.29) monocytes were significantly higher when compared with those of controls (all p < 0.0001). proportions of non-classical and intermediate monocytes correlated positively with serum levels of plasma cells, m-protein, calcium, creatinine and lactate dehydrogenase, and correlated negatively with the serum albumin level. proportions of classical monocytes correlated positively with albumin level and negatively correlated with serum levels of m-protein, plasma cells, calcium, creatinine, and lactate dehydrogenase. conclusion: circulating monocyte subpopulations are skewed towards non-classical and intermediate monocytes in mm patients, and the intensity of this skewness increases with disease severity. keywords: multiple myeloma; classical monocytes; intermediate monocytes; non-classical monocytes; flow cytometry. introduction multiple myeloma (mm) is a cancer that affects b-cells, particularly plasma cells, leading to their expansion and the excessive production of immunoglobulins. malignant plasma cells may control the quality of the immune cells in the bone marrow and produce immunoglobulins that can act as tumour-associated antigens which are further processed by antigen-presenting cells to stimulate specific anti-tumour t-cell responses.1 monocytes and macrophages play a fundamental role in this inflammatory response, possibly through bone marrow expansion and cytokine production in myeloma patients.2,3,4,5 this is corroborated by findings from in-vitro studies.6,7 the increased peripheral monocytes in mm patients may inhibit antitumor immune responses and increase tumour aggression.2,3 human peripheral blood monocytes are a heterogeneous population, as they differ in their phenotype, surface marker expression, and function.8 they can be classified based on the fluorescent intensities of cd16 and cd14 into three distinct populations, including classical monocytes (cd16−cd14++), intermediate monocytes (cd16+cd14++), and non-classical monocytes (cd16++cd14+).9 circulating non-classical monocytes have been observed to increase in some autoimmune diseases such as lupus and rheumatoid arthritis,10,11 and the frequency of intermediate monocytes has been associated with bone erosion in patients with psoriatic arthritis.12 in oncology settings, the intermediate monocytes have been reported to play a crucial role in patients with mm,9,13 and the proportion of intermediate monocytes increased proportionately with the tumour load.14,15 myeloma cells produce toll-like receptor ligands that cause the expansion of non-classical monocytes in mm patients.16 in addition, circulating monocytes correlate with the well-known prognostic factors for mm, and evaluating monocyte subpopulations can enhance our understanding of mm mechanisms and aid patient follow-up. although bone marrow aspirates and trephine biopsies provide high diagnostic yields for mm detection,17 finding a less invasive diagnostic procedure such as flow cytometry will be a clinical breakthrough. this study aimed to describe the immunophenotypic profile of the peripheral blood monocytes in newly diagnosed mm patients at the assiut university hospital, assiut, egypt. methods ethical considerations the human research ethics committee of the south egypt cancer institute, assiut university, approved this study (seci-irb-iorg0006563, 37:10/1/2018). written informed consent was obtained from patients and healthy volunteers in accordance with the declaration of helsinki. patients and controls provided written informed consent and participated in the study after study clarification. patients got the maximum care, and the study procedures were in accordance with relevant guidelines and the declaration of helsinki. investigators and researchers collected data using paper data collection forms. patients were identified by serial study codes, which were linked with patient names and hospital file numbers on a separate sheet and stored in a locked safe cabinet. data analysis was performed on the de-identified data. study population a prospective case-control study was conducted at the clinical haematology unit, internal medicine department, assiut university hospital, and medical oncology department, south egypt cancer institute, assiut university, in assiut, egypt, from october 2018 to august 2019. twenty newly diagnosed untreated mm patients diagnosed according to the world health organization criteria for mm, and 20 matched (age and sex) healthy volunteers were included in this study. patients who were under treatment or refused to participate were not included. multiple myeloma patients were classified using the durie-salmon staging system18 that assesses tumour burden based on three laboratory parameters and evaluates bone involvement based on a radiographic skeletal survey. patients who had haemoglobin levels over 10 g/dl, normal serum calcium or less than 10.5 mg/dl, normal bone x-ray or solitary lytic lesion, monoclonal immunoglobulin g under 5 g/dl, immunoglobulin a under 3 g/dl, and bence jones protein under 4 g/24 h were classified as stage i. stage ii patients were patients with a moderate number of myeloma cells that did not meet either stage i or stage iii criteria. stage iii patients were those who met one or more of the following criteria: haemoglobin under 8.5 g/dl, serum calcium value over 12 mg/dl, advanced lytic bone lesions, monoclonal immunoglobulin g over 7 g/dl, immunoglobulin a over 5 g/dl, and bence jones protein over 12 g/24 h. as the durie-salmon scoring may not provide good prognostic discrimination, the international staging system was used in this study to group mm patients according to the serum β2-microglobulin (β2m) and serum albumin into 3 stages: stage i characterised by serum β2m less than 3.5 mg/l and serum albumin 3.5 g/dl or higher, stage ii in which serum β2m was less than 3.5 mg/l and serum albumin was less than 3.5 g/dl or β2m was between 3.5 mg/l and 5.5 mg/l, and stage iii in which serum β2m was 5.5 mg/l or higher.19 patient data were also collected for the study; these included age, sex, and clinical history of bone pain, pathological fracture, weakness and fatigue, recurrent infection, manifestation of hypercalcemia such as thirst, polyuria, manifestation of hyperviscosity (blurring of vision, mental confusion), bleeding tendency, and neuropathy. clinical signs of anaemia, oedema, and renal impairment were carefully assessed in all patients. a radiographic skeletal survey of the skull and long bones was also done to detect lytic bone lesions, pathological fractures, and abdominal ultrasound. complete blood count with differential was done using the cell-dyn ruby fully automated blood counter (cell-dyn ruby system, abbott diagnostics, chicago, illinois, united states; serial number: 36026bg). examination of peripheral blood smear for rouleaux formation was done as part of routine care. blood urea, serum creatinine, total protein, albumin, and lactate dehydrogenase (ldh) tests were done using the cobas integra 400 automated chemical analyser (roche diagnostics gmbh, mannheim, germany; serial number: 500558). serum protein electrophoresis with immunofixation was performed to identify monoclonal protein (m-protein) types among patients at diagnosis using the pretty interlab device (interlab, rome, italy; serial number: 38405301). bone marrow aspiration and biopsy to evaluate and quantify bone marrow plasma cell infiltration were done on all recruited patients. all these investigations were done as routine services for newly diagnosed mm cases. flow cytometric detection of monocytes subtypes fifty microlitres of remnant routine complete blood count samples were added to 5 µl of cd14 fluoroisothiocyanate-conjugated and cd16 phycoerythrinconjugated (becton dickinson [bd] biosciences, san jose, california, united states). after 15 min of incubation at room temperature in the dark, lysis and washing of erythrocytes was done. the cells were then re-suspended in phosphate-buffered saline, and cells were acquired using the bd facscalibur flow cytometry system (bd biosciences, san jose, california, united states), with data analysis done using the cellquest software (bd biosciences, san jose, california, united states). an anti-human immunoglobulin g isotype-matched negative control was used for each flow cytometry experiment. forward and side scatter histograms were used to define monocytes. afterwards, the expression of cd14 and cd16 were assessed in the monocyte population. the classical subset was cd14++cd16-, intermediate subset was cd14++cd16+, and non-classical subset was cd14+cd16++ (figure 1). monocyte subset data are expressed as percentages of the total monocytes. figure 1: monocyte subsets in multiple myeloma patients and healthy controls at assiut university, assiut, egypt from october 2018 to august 2019. (a) flow cytometric detection of monocytes subsets. forward scatter and side scatter dot was used to detect monocyte region (r1) (left). cd14-fluoroisothiocyanateand cd16-phycoerythrin-positive cells were identified among monocytes and classified as cl-mo (classical monocytes, cd14++cd16-), in-mo (intermediate monocytes (cd14++cd16+) and non-cl-mo (non-classical monocytes cd14+cd16++) (right). (b) the frequency of the monocytes subsets varies significantly between multiple myeloma patients and healthy controls (classical monocytes: mean ± standard error 77.24 ± 0.66 in multiple myeloma patients vs 83.75 ± 0.5 among controls, p < 0.0001; non-classical monocytes: 12.44 ± 0.5 in multiple myeloma patients vs 8.9 ± 0.34 among controls, p < 0.0001; and intermediate monocytes: 10.3 ± 0.24 in multiple myeloma patients vs 7.4 ± 0.29 among controls, p < 0.0001). statistical analysis we used statistical package for the social sciences version 21 (ibm corporation, armonk, new york, united states) for data analysis. quantitative data are presented as mean ± standard error while qualitative data are presented as frequencies and percentages. mann-whitney test analysis was performed to determine the statistical significance and differences between the study groups. spearman’s correlation was performed to determine the association between monocytes subtypes and laboratory parameters. results were considered significant if p was 0.05 or less. results baseline characteristics of newly diagnosed multiple myeloma patients and healthy controls the mean age of the mm patients was 6.35 ± 4.07 years, and the number of men (13) was double that of women (7) (table 1). the proportion of plasma cells in bone marrow was 39.7 ± 3.7% and that of the monoclonal band (m-protein) was 4.44 ± 2.8 g/dl. table 1: baseline characteristics of studied newly diagnosed multiple myeloma patients and healthy controls, assiut, egypt, october 2018 – august 2019. laboratory investigations showed that platelet counts, total leukocytic counts, serum total protein, and urea levels were comparable between patients and healthy controls (p > 0.05). multiple myeloma patients had significantly reduced haemoglobin (9.3 ± 2.4 g/dl in mm vs 12.8 ± 1.97 g/dl in healthy controls; p = 0.045) and serum albumin (2.46 ± 0.9 g/dl in mm patients vs 4.63 ± 1.1 g/dl in healthy controls; p < 0.0001) when compared to healthy controls. on the other hand, after comparing the mean levels of serum calcium, ldh, and serum creatinine between mm patients and healthy controls, we found statistically significant increased levels in mm patients (calcium: 11.75 ± 0.4 mg/dl in mm patients vs 8.8 ± 0.1 mg/dl in healthy controls, p < 0.0001; ldh: 886.86 ± 92.6 units per litre in mm vs 155.8 ± 8.23 units per litre in controls, p < 0.0001; serum creatinine: 1.9 ± 0.2 mg/dl in mm patients vs 0.97 ± 0.1 mg/dl in healthy controls, p < 0.0001). based on the durie-salmon staging system, 12 (60%) of the 20 newly diagnosed untreated mm patients had stage i disease, 6/20 patients (30%) had stage ii disease, and 2/20 patients (10%) had stage iii disease. whereas according to the international staging system, 70% (n = 14) of mm patients had stage i disease, 20% (n = 4) had stage ii, and 10% (n = 2) had stage iii. phenotypic characterisation of monocytes in multiple myeloma patients monocytes present in the peripheral blood displayed a range of intensities of cd16 and cd14 expression. they were divided into classical (cd16−cd14++), intermediate (cd16+cd14++), and non-classical (cd16++cd14+) based on these ranges. in healthy controls, classical monocytes represented the major component among total monocytes (83.8%), followed by non-classical monocytes (8.9%) and intermediate monocytes (7.4%). multiple myeloma patients had statistically significant lower proportions of classical monocytes (77.24% ± 0.66 in mm patients vs 83.75% ± 0.5 in controls), and higher proportions of non-classical (12.44% ± 0.5 in mm patients vs 8.9% ± 0.34 in controls) and intermediate (10.3% ± 0.24 in mm patients vs 7.4% ± 0.29 in controls) monocytes compared to healthy controls (all with p < 0.0001) (figure 1). correlation between monocyte subsets and laboratory parameters in newly diagnosed untreated multiple myeloma patients in newly diagnosed untreated mm patients, the proportion (%) of classical monocytes correlated positively with the serum albumin level (r2 = 0.731; p < 0.0001), and correlated negatively with serum levels of serum creatinine (r2 = 0.389, p < 0.0001), ldh (r2 = 0.545, p < 0.0010), calcium (r2 = 0.582, p < 0.0001), m-protein (r2 = 0.222, p = 0.005), and with the percentage of bone marrow plasma cells (r2 = 0.347, p < 0.0001), while urea and total protein did not show significant correlation (figure 2). figure 2: correlation of classical monocytes with laboratory parameters in multiple myeloma patients and healthy controls at assiut university, assiut, egypt, from october 2018 to august 2019. the percentage of classical monocytes (cd16−cd14++) and (a) creatinine, (b) urea, (c) lactate dehydrogenase, (d) calcium, (e) total protein, (f) albumin, (g) m-protein, and (h) bone marrow plasma cells % were correlated using spearman’s correlation test and correlated positively with level of serum albumin (r2 = 0.731, p < 0.0001) (f), and correlated negatively with levels of serum creatinine (r2 = 0.389, p < 0.0001) (a), lactate dehydrogenase (r2 = 0.545, p < 0.0001) (e), calcium (r2 = 0.582, p < 0.0001) (d), m-protein (r2 = 0.222, p = 0.05) (g), and the percentage of bone marrow plasma cells (r2 = 0.347, p = 0.005) (h). in contrast, the proportion (%) of non-classical monocytes in newly diagnosed untreated mm patients correlated negatively with the serum albumin level (r2 = 0.624, p < 0.0001), and correlated positively with serum levels of creatinine (r2 = 0.413, p < 0.0001), ldh (r2 = 0.533, p < 0.0001), calcium (r2 = 0.575, p < 0.0001), m-protein (r2 = 0.28, p = 0.024), and with the percentage of bone marrow plasma cells (r2 = 0.27, p = 0.018), while urea and total protein did not show significant correlation (figure 3). figure 3: correlation of non-classical monocytes with laboratory parameters in multiple myeloma patients and healthy controls at assiut university, assiut, egypt, from october 2018 to august 2019. the percentage of non-classical monocytes (cd16++cd14+) and (a) creatinine, (b) urea, (c) lactate dehydrogenase, (d) calcium, (e) total protein, (f) albumin, (g) m-protein, and (h) bone marrow plasma cells % were correlated using spearman’s correlation test and correlated negatively with level of serum albumin (r2 = 0.624, p < 0.0001) (f), and correlated positively with higher levels of creatinine (r2 = 0.413, p < 0.0001) (a), lactate dehydrogenase (r2 = 0.533, p < 0.0001) (c), serum calcium (r2 = 0.575, p < 0.0001) (d), m-protein (r2 = 0.28, p = 0.024) (g), and the percentage of bone marrow plasma cells (r2 = 0.27, p = 0.018) (h). similarly, the proportion (%) of intermediate monocytes in newly diagnosed untreated mm patients correlated negatively with serum albumin level (r2 = 0.649, p < 0.0001) and correlated positively with the serum levels of creatinine (r2 = 0.222, p = 0.002), ldh (r2 = 0.338, p < 0.0001), calcium (r2 = 0.398, p < 0.0001), and with the percentage of bone marrow plasma cells (r2 = 0.301, p = 0.016), while urea, total protein, and m-protein did not show significant correlation (figure 4). figure 4: correlation of intermediate monocytes with laboratory parameters in multiple myeloma patients and healthy controls at assiut university, assiut, egypt, from october 2018 to august 2019. the percentage of intermediate monocytes (cd16+cd14++) and (a) creatinine, (b) urea, (c) lactate dehydrogenase, (d) calcium, (e) total protein, (f) albumin, (g) m-protein, and (h) bone marrow plasma cells % were correlated using spearman’s correlation test and correlated negatively with level of serum albumin (r2 = 0.649, p < 0.0001) (f), and correlated positively with levels of serum creatinine (r2 = 0.222, p = 0.002) (a), lactate dehydrogenase (r2 = 0.338, p < 0.0001) (c), calcium (r2 = 0.398, p < 0.0001) (d), and the percentage of bone marrow plasma cells (r2 = 0.301, p = 0.016) (h). discussion this study found that the percentage of classical monocytes was significantly lower in patients with mm compared to controls, while the percentages of non-classical and intermediate monocytes were significantly higher. moreover, the proportions of non-classical and intermediate monocytes were positively correlated with serum levels of plasma cells, m-protein, calcium, creatinine, and lactate dehydrogenase, and correlated negatively with the serum albumin level. proportions of classical monocytes correlated positively with albumin level and correlated negatively with serum levels of m-protein, plasma cells, calcium, creatinine, and lactate dehydrogenase. multiple myeloma is a plasma cell cancer that exploits monocytes and macrophages for growth and expansion16 through the production of inflammatory interleukins such as il-6 that promote persistence of malignant plasma cells4,16 and enhance tumour progression.5 monocytes,20 especially non-classical monocytes,16 increase in mm patients. in this study, we analysed circulating monocytes in 20 newly identified untreated mm patients to discover their role in disease progression and postulate their possible use as a prognostic factor during patient follow-up. we found significantly increased proportions of non-classical (cd16++ cd14+) and intermediate (cd16+ cd14++) circulating monocytes in newly diagnosed untreated mm patients compared to healthy controls. our results are similar to those of a study in 2015 in france that found an increased frequency of cd14+/++ cd16+ monocytes in 20 mm patients compared to healthy volunteers; however, they did not find a correlation between the monocyte subsets and circulating or bone marrow plasma cell counts.15 similarly, a study in norway in 2015 found that the proportion of non-classical monocytes increased with tumour load in mm patients (n = 12) and suggested that non-classical monocytes could maintain the development of myeloma cells.16 however, the study noted that the increased proportion of non-classical monocytes might be caused by numerous mechanisms. by the end of the study, the investigators concluded that the non-classical subset of monocytes in patients with mm are functional monocytes that could be stimulated to produce cytokines, and may enhance myeloma expansion, contributing to the disease process. furthermore, malignant plasma cells yield increased levels of transforming growth factor beta 1,21 which hypothetically increases cd16 expression. therefore, it is probable that in patients with high plasma cell levels, cd14-high cells under the effect of transforming growth factor beta 1 settle into the cd16+cd14dim cell regions. the different monocyte subset proportions between mm patients and healthy controls could be explained by several mechanisms. nucleic acids and immune complexes produced during disease mainly encourage the production of intermediate and non-classical monocyte subsets, and these yield interleukins to maintain myeloma growth in mm patients.14 also, higher proportions of the non-classical subset of monocytes could be produced by numerous mechanisms such as stimulation by m-protein, which is produced by the malignant plasma cells,16 as myeloma cells secrete higher quantities of immunoglobulins.9 further studies are necessary to understand the mechanisms behind these skewed monocyte subpopulation frequencies and whether it is a cause of disease severity. multiple myeloma patients had lower levels of haemoglobin, albumin, and calcium when compared to healthy control volunteers (p = 0.045, p < 0.0001, and p < 0.0001). parts of these findings are in agreement with a study conducted in austria in 2010 which reported that most mm cases had anaemia of variable severity.22 similarly, in a study conducted in the united states in 1982,23 we found no differences between the platelet count and total leukocytic count in mm cases and healthy controls. the urea levels were comparable among mm patients and controls, while the serum creatinine levels were significantly higher in mm patients compared to controls (p < 0.0001); this is in contrast to previous reports that found variable degrees of renal impairment in patients with mm.24,25 lactate dehydrogenase was significantly increased in mm patients versus controls (p < 0.0001), as previously reported.26 the proportion of plasma cells in bone marrow was 39.7 ± 3.7% and that of the monoclonal band (m-protein) was 44.4 ± 2.8 g/l, comparable with that of previous reports.16 limitations one limitation of this study was the small sample size, but these initial results provide new perspectives on the diagnosis and monitoring of mm. conclusion the higher frequencies of non-classical and intermediate monocytes in newly diagnosed untreated mm patients suggest a possible role of these cells rather than classical monocytes in the disease process. the participation of these monocyte subsets in mm pathogenesis should thus be part of future investigations as they may provide important information for the diagnosis and follow-up of mm patients. more studies are needed to address the possibility of using flow cytometry results as a primary staging criterion. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions s.m.m., h.n.-h., h.a.e., m.m.w. and a.m.z. performed the research, designed the study, analysed the data, and wrote the manuscript. a.m.z. and a.n. performed the flowcytometric study design and analysis. s.m.m., a.n, h.n.-h. m.m.w. and a.m.z. revised the article and approved the final manuscript. sources of support this research received no specific grants from any funding agency in the public, commercial, or not-for-profit sectors. data availability data are available from the corresponding author upon request after approval of the authors’ governmental university leaders. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or 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med. 1990;150(8):1693–1695. https://doi.org/10.1001/archinte.1990.00040031693017 dimopoulos ma, kastritis e, rosinol l, et al. pathogenesis and treatment of renal failure in multiple myeloma. leukemia. 2008;22(8):1485–1493. https://doi.org/10.1038/leu.2008.131 simonsson b, brenning g, kallander c, et al. prognostic value of serum lactic dehydrogenase (s-ldh) in multiple myeloma. eur j clin invest. 1987;17(4):336–339. https://doi.org/10.1111/j.1365-2362.1987.tb02197.x abstract introduction methods results discussion acknowledgements references about the author(s) leanne swart department of molecular medicine and haematology, charlotte maxeke johannesburg academic hospital, national health laboratory service, johannesburg, south africa department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa melanie pretorius department of molecular medicine and haematology, charlotte maxeke johannesburg academic hospital, national health laboratory service, johannesburg, south africa department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa denise lawrie department of molecular medicine and haematology, charlotte maxeke johannesburg academic hospital, national health laboratory service, johannesburg, south africa department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa deborah k. glencross department of molecular medicine and haematology, charlotte maxeke johannesburg academic hospital, national health laboratory service, johannesburg, south africa department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa citation swart l, pretorius m, lawrie d, glencross dk. commercial duraclone panels for extending the repertoire of multicolour immunophenotypic panels in an academic flow cytometry laboratory in south africa. afr j lab med. 2022;11(1), a1720. https://doi.org/10.4102/ajlm.v11i1.1720 original research commercial duraclone panels for extending the repertoire of multicolour immunophenotypic panels in an academic flow cytometry laboratory in south africa leanne swart, melanie pretorius, denise lawrie, deborah k. glencross received: 31 aug. 2021; accepted: 28 july 2022; published: 29 nov. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: commercial multicolour fixed immunophenotyping panels can improve flow cytometric diagnostic immunophenotyping repertoire. objective: this study validated the commercially available, standardised beckman coulter lyophilised duraclone re panels to discriminate specific haematolymphoid subtypes. methods: we compared the diagnostic capability of the duraclone acute leukaemia b (alb), chronic leukaemia b (clb), and plasma cells (pc) panels to the predicate second-line panels in charlotte maxeke johannesburg academic hospital, johannesburg, south africa, from april to august 2020. clinical diagnostic concordance between the in-house second-line immunophenotyping (the predicate method) and duraclone was established. the alb panels tested for precursor b-cell acute lymphoblastic leukaemia (n = 11) or normal bone marrow haematogones (n = 9); clb panels established haematolymphoid subtypes of mature b-cell lymphoproliferative disorders (b-lpd) (n = 20), while pc panels detected plasma cell dyscrasias (pcd) (n = 17). flow cytometer setup and data interpretation to discriminate normal and aberrant immunophenotypes were per manufacturer’s instructions. results: there was 100% clinical diagnostic concordance between the predicate and the test panels for second-line diagnostic investigation of b-all (with additional cd56), mature b-lpd (with additional discernment of cd81, ror-1, cd79b and cd43) and pcd. conclusion: the duraclone clb exceeded the predicate second-line performance, offering extended second-line diagnostic discernment of mature b-lpd subtypes and discernment of cd5+ b-lpd from other non-cd5+ (or cd5–) b-lpd; likewise, the pc panels enabled discovery of pcd. while alb testing offered no additional diagnostic advantage over existing predicate investigation, cd58 did offer additional information to discern haematogones from b-all. keywords: flow cytometry; multicolour; immunophenotyping; lymphoma; leukaemia; plasma cell dyscrasia; acute lymphoblastic leukaemia; b-cell lymphoproliferative disorder; lyophilised reagent; fixed panels. introduction flow cytometry immunophenotypic analysis is a powerful diagnostic and research tool for investigating haemato-lymphoid malignancies, such as leukaemia, lymphoma, and plasma cell dyscrasias (pcd). recently, multicolour flow cytometric immunophenotyping has become invaluable for improving detection and detailed immunophenotyping during diagnosis and detection of low frequencies of abnormal or aberrant cell populations during minimal residual disease assessment.1,2 aberrant cell phenotypes are distinguished from normal developmental counterparts2 and normal background leucocyte populations3 by analysing and comparing several simultaneous cell surface and cytoplasmic antigen expression profiles. multicolour fluorochrome-conjugated antibody panels generally require antibody concentrations and fluorescence intensity optimisation through titration techniques.4,5 these assays can be technically challenging and error-prone due to the addition of multiple individual liquid reagents requiring manual pipetting,4,6 thus requiring laboratory technicians skilled in titration technique and flow cytometry,4,7 including fluorescence-minus-one, colour compensation, and other technical flow cytometry issues. commercially available, standardised, lyophilised, fixed and pre-titrated antibody panels,8,9,10,11,12,13 like the beckman coulter (bc) duraclone inventory, including the identification of normal and abnormal b progenitor cells (re alb),14 various chronic b-cell lymphoproliferative disorders including cd5+ chronic lymphocytic leukaemia (re clb)15 and normal or abnormal plasma cells (re pc) panels, offer an attractive alternative to the locally developed, in-house multicolour panels described. commercial multicolour panels also usually offer the advantage of a standardised instrument and analysis protocols; preset software-guided controls are available to establish optimal standardised instrument settings, verified with abnormal and normal stabilised blood material controls.9 in addition, these commercial panels are typically fixed panels supplied in a stable, dry (lyophilised) format with predetermined antibody combinations in a single tube9; these qualities enhance laboratory efficiency, reduce technical laboratory error and simplify reagent inventory. some commercial panels also offer some flexibility in marker combinations by providing ‘open’ fluorescence channels14,15 so that liquid drop-in markers can be added to the ‘fixed’ panels to accommodate unique and specific diagnostic requirements of the investigating laboratory. our laboratory previously established the positive impact on quality, laboratory workflow and diagnostic usefulness of the commercially available, fixed and standardised bc clearllab 10c16 multicolour panel system (beckman coulter, miami, florida, united states). although this method provides excellent coverage to discover most acute leukaemias,9,16 the clearllab 10c system lacks additional markers to further identify and discern specific haematolymphoid subtypes, especially within the group of mature b-cell lymphoproliferative disorders and pcd. specifically, although the bc clearllab 10c can easily discern normal b-cell precursors or haematogones from similar immunophenotypes present in minimal residual b-cell acute lymphoblastic leukaemia (b-all) disease, the cd58 marker is not included. however, it may be helpful to discriminate disease from normal b-cell precursors during minimal b-all disease assessment.1,2 the clearllab 10c system also lacks markers to discern cd5-positive and cd5-negative mature b-cell lymphoproliferative disorders (lpd), using markers such as cd23, cd43, cd79b, cd81, and ror-1 recommended for classification by rawstron et al.17,18 investigation of pcd is also limited if investigated only with the clearllab 10c system which lacks cd138, an absolute requirement for identifying plasma cells together with cd3819. in our laboratory, we have developed several 2–4-colour in-house, second-line immunophenotyping panels to identify the specific haematolymphoid subtypes mentioned, including markers such as cd23 and fmc-7 for mature b-cell lpd and cd138 with cd56 and cd200, to diagnose pcd. these, however, require a separate repertoire of liquid reagents and necessary flow cytometry technical expertise to set up and run the tests. for a busy site, such as our unit, which processes over 400 samples per month, standardised commercially available panels that can supplement second-line immunophenotypic investigation after clearllab10c investigation could dramatically increase the repertoire of markers tested and also improve workflow efficiency in our site. existing predicate second-line investigation utilises locally established, in-house, non-standardised and manually assembled liquid fluorochrome-conjugated antibody panels. the primary aim of this study was to evaluate the fixed and standardised, pre-titrated commercially available duraclone re alb, re clb and re pc panels (beckman coulter, mumbai, india) as a comprehensive and compact alternative to our existing second-line immunophenotypic investigation of haematological neoplasms. there were two parts to this study. first, we compared the duraclone clb and pc tubes to the laboratory’s in-house predicate 2–4-colour method for the following markers: combinations of cd10, cd23, fmc7 and cd22 to discern mature b-cell lymphoproliferative disorders, or cd19, cd38, cd56, cd200 and cd138 to characterise plasma cells. secondly, we verified manufacturer-described expression for those additional markers that were not included in the older predicate 2–4-colour method, such as verifying the expression of cd43, cd79b, cd81 and ror-1 in the clb tube and cd38 and cd138 with cd27, cd81 and cd56 included in the duraclone pc tube against expected expression in normal leucocyte population counterparts. we also validated the overall diagnostic outcome of the alb tube; here, we asked whether the cd58 included in the alb tubes offered any additional diagnostic advantage in identifying a b-all that had been identified in the first-line clearllab 10c investigation. methods ethical considerations the university of the witwatersrand health research ethics committee approved the study (ethics clearance number m1704129). the study’s objective was to validate the commercially available beckman coulter duraclone pc, clb and pc fixed-tube, pre-titrated panels as an alternative second-line diagnostic workup to our existing in-house second-line immunophenotypic investigation. the interpretation of flow cytometric data and consequent clinical diagnostic outcomes of the existing in-house, second-line, non-standardised panels were compared to the overall clinical diagnostic outcomes of the duraclone analyses. study design and site this study was a prospective observational cohort study conducted according to the standards for reporting diagnostics accuracy.20 the work was undertaken from april 2020 to august 2020 and conducted in the national health laboratory service flow cytometry laboratory at the charlotte maxeke johannesburg academic hospital in johannesburg, south africa. this laboratory performs routine immunophenotypic investigation of leukaemias and lymphomas for both children and adults, referred from charlotte maxeke johannesburg academic hospital and other hospitals within the university of the witwatersrand service precinct, as well as from other centres around south africa. morphological assessment of peripheral blood, bone marrow and trephine samples (from the same patients whose samples are tested in the flow cytometry unit) is performed in the sister haematology laboratory; auxiliary molecular and cytogenetic investigations are also performed on site. the laboratory participates in the united kingdom national external quality assurance scheme (uk neqas, sheffield, united kingdom) leukaemia immunophenotyping part 1 and part 2 (interpretation) proficiency testing scheme.21 testing approach immunophenotypic testing of all samples at the charlotte maxeke johannesburg academic hospital flow cytometry unit is two-tiered. firstly, an initial first-line workup is performed with bc clearllab 10-c (beckman coulter, miami, florida, united states). the first workup identifies most acute myeloid and lymphoid leukaemia subtypes, distinguishes early and mature t-cell or b-cell lymphoproliferative disorders, and hints at the presence of a pcd (if a population of brightly expressing cd38 cells is noted). a limitation of this clearllab 10c system is that further immunophenotypic characterisation of mature b-cell lymphoproliferative disorders17 or pcd19,22 requires second-line testing using markers that are not included in the first-line testing. for this study, in-house second-line testing specifically assessed cd23 and fmc-7 expression on mature b-cells or cd38 and cd138 expression on plasma cells. specifically, in the context of b-lpd and in comparison to our in-house second-line investigation that included cd23 and fmc-7, could duraclone clb testing with ror-1, cd20, cd43, cd79b, and 81 replace cd23 and fmc-7 in discerning cd5 b-cell chronic lymphocytic leukaemia (cll) from other cd5 and non-cd5 expressing b-lpd.17,18 samples after all routine testing with clearllab 10c and in-house second-line testing (table 1), samples having sufficient remnant prepared-cell-concentrate or at least 1 ml remaining of the whole sample were re-tested using one of the test methods (either duraclone re alb, re clb or re pc). the aim was to collect at least 20 clinical specimens for each validation (estimated to be 60 samples). all peripheral blood and bone marrow samples were collected by attending physicians into ethylenediaminetetraacetic acid vacutainer tubes; the single body fluid sample was not collected into an anticoagulant but submitted in a vacutainer tube without anticoagulant. at the end of the study period, a total of 57 anonymised samples were identified for second-line duraclone comparison, including peripheral blood (n = 13), bone marrow (n = 43), and a pleural fluid sample (n = 1) collected from paediatric and adult patients. for duraclone alb testing, 20 bone marrow aspirate samples were tested. eleven of these patients had a diagnosis of b-all (n = 11), while nine had immunophenotypically normal haematogones. one of the nine was a follow-up of a b-all with normal haematogones and no evidence of residual disease. twenty samples diagnosed with a b-cell lymphoproliferative disorder by predicate clearllab 10c and in-house second-line were referred for duraclone clb testing. the clb testing set comprised 12 bone marrow aspirate samples, seven peripheral blood samples, and a single pleural fluid sample. due to the decommissioning of the older facscalibur flow cytometer during the period of study (the instrument used to undertake the predicate in-house second-line testing), only 17 samples with a diagnosis of a pcd by clearllab 10-c and in-house second-line investigation were tested by duraclone pc panels. table 1: possible reagent selection for method comparison according to suspected target population undertaken from april 2020 to august 2020 at an academic pathology service in johannesburg, south africa. instruments the naviostm (bc, miami, florida, united states) and the facscalibur flow cytometer (becton dickinson biosciences, san jose, california, united states) were used during this study. the naviostm flow cytometer was used to analyse the predicate clearllab 10c as well as the test duraclone panels. internal quality control on the naviostm included daily assessment of background contamination, cellular events carryover between tubes, and fluorospheres acquisition to verify the flow cytometer optical alignment and fluidics (flow-check pro, bc, lismeehan, ireland). in addition, clearllab™ normal and abnormal process control cells (bc, lismeehan, ireland) were used to verify sample processing, acquisition, and analysis. clearllab 10c panel acquisition setup was achieved by applying target values set in the manufacturer manual and using bc flowset pro beads to adjust the voltages that enabled optimal detection and separation of dim and bright antigens. after pilot testing (data not shown), the naviostm instrument clearllab 10c settings were deemed appropriate for the duraclone multicolour data acquisition and analysis. the facscalibur flow cytometer was used to acquire the predicate in-house second-line 2–4-colour fluorescence panels using cellquest software (bd, san jose, california, united states). quality control performed on the bd facscalibur included daily assessment of background contamination, carryover, acquisition, and analysis of manufacturer-recommended 3-colour and apc calibrite beads (becton dickinson biosciences, san jose, california, united states). further acquisition and analysis of immunotroltm process control (bc, brea, california, united states) using four monoclonal antibodies, cd14 fitc, cd13 pe, cd45 percp and cd3 apc (all becton dickinson biosciences, san jose, california, united states), was done. sample preparation sample preparation included lysing two or more 0.5 ml sample aliquots (dependent on the initial white cell count) with 14.5 ml isotonic ammonium chloride ph 7.1–7.4 (8.99% nh4cl, 0.84% nahco3 and 0.0372% ethylenediaminetetraacetic acid; merck, darmstadt, germany) in a 15 ml conical centrifuge tube for 15 min at room temperature. after incubation, samples were spun at 3000 g for 3 min, the supernatant was decanted, while the pellet was washed four times with 14 ml phosphate-buffered saline at ph 7.3 ± 2 (oxoid ltd, basingstoke, united kingdom) containing 0.09% sodium azide (nan3) (merck, darmstadt, germany) and 0.2% bovine serum albumin (biowest, nuaille, france). following washing, samples were reconstituted to 0.5 ml with phosphate-buffered saline, and the white cell count was determined. cells were then diluted (or concentrated) to achieve the recommended cellular concentration of ≤ 10 000 cells/µl for monoclonal antibody incubation. subsequently, 100 µl of this cell concentrate, which contained approximately 106 cells, was added to the clearllab 10c and in-house panels (see table 1). if the sample was deemed to be suitable for inclusion in the study (i.e. had an established diagnosis by predicate clearllab 10c), and if there was sufficient remaining material, 100 µl of the remaining cell concentrate was tested with either duraclone alb, clb or pc panels (table 1). details of the markers included in these duraclone panels are outlined in table 2. each duraclone panel has a ‘spare’ capacity for additional markers per the investigator’s need. in our study, in two cases of precursor b-all, the marker cd22 (apc) (bc, miami, florida, united states) was added in liquid format to the alb panel to occupy the free apc channel (see table 2, duraclone re alb panel) to verify cd22 expression in the context of a b-all. in a single case of a suspected pcd, liquid cd117 ecd (bc, miami, florida, united states) was added to the duraclone re pc tube in the ‘free’ ecd channel to demonstrate expression of cd117 in a case of multiple myeloma. after adding the cell aliquot into panels, and, where applicable, the addition of liquid monoclonal reagent, all samples were incubated at room temperature (average 20 °c – 22 °c) in the dark for 15 min. after incubation, all samples were washed once with phosphate-buffered saline and reconstituted to 0.5 ml. table 2: possible reagent selection for method comparison according to suspected target population validated at the charlotte maxeke johannesburg academic hospital flow cytometry laboratory in johannesburg, south africa, april 2020 to august 2020. data acquisition and interpretation of flow cytometric case data all samples were acquired on a flow cytometer to acquire raw flow cytometric listmode data. for the predicate in-house panels acquired on the facscalibur, 5000 (.fcs data) events were collected. paint-a-gate software (becton dickinson biosciences, san jose, california, united states) was used for analysis of raw facscalibur flow cytometric data with a primary gating focus using cd45 and side scatter. for both the clearllab 10c and duraclone panels, at least 50 000 (listmode data) events were acquired on the navios. kaluza c™ version 1.1 (bc, miami, florida, united states) software was used to analyse all raw listmode data to facilitate clinical interpretation. kaluza c™ clearllab b, t, m1 and m2, and duraclone panel analysis protocols were developed according to manufacturer specifications.23 first, doublets, debris and unlysed red blood cells were excluded, and, where possible, a neoplastic target population was identified in a primary gate using cd45 and side scatter. thereafter, secondary gating focused on identifying the same target immunophenotype across each of the t, m1 and m2 panels.9,16 finally, simultaneous identification of normal populations in the background was based on local in-house developed gating strategies to identify granulocytes, monocytes and mature lymphocytes using cd45, side scatter characteristics and specific regular expression of the markers studied (figure 1 and figure 2). figure 1: relative expression of cd81 relative to normal t-cells and granulocytes in the clb tube validated at the charlotte maxeke johannesburg academic hospital flow cytometry laboratory in johannesburg, south africa, april 2020 to august 2020. each plot (a, b and c) shows cd81 expression (x-axis) versus frequency (y-axis). in a, there is relative (weak) under-expression of cd81 in a patient with cll gated on cd19+|cd5+; in b, there is relative over-expression of cd81 in a patient with follicular lymphoma gated on cd19+|cd10+ c is a control experiment of a paediatric sample with 20% haematogones showing bright expression of cd81 on normal precursor b-cells gated on weak cd45+| cd19+. orange represents the b-cells identified with cd19; the blue population represents background granulocytes identified with cd45 and side scatter, and red represents background t-cells gated on cd5 (all markers are included in the clb panel). figure 2: identification of plasma cells using the duraclone pc tube validated at the charlotte maxeke johannesburg academic hospital flow cytometry laboratory in johannesburg, south africa, april 2020 to august 2020. in histogram a, negative cd45 expression of plasma cells is shown (purple), with cd45 positive (background) expression noted amongst granulocytes (blue), monocytes (green) and mature lymphocytes (red). the target plasma cell population were primary-gated on bright cd138 and cd38 to reveal aberrant cd117 expression (b) and under-expression of cd81 (c) and cd27 (d). a similar approach was used for each of the comparative second-line duraclone re alb, re clb, and re pc listmode data analysis but additionally incorporated manufacturer-recommended gating strategies for clinical interpretation of data.23 the relative intensity of expression of cell surface antigens was used to discriminate between normal and aberrant immunophenotypes according to manufacturer specifications. positivity (or no expression) of individual markers was established, and overall haematolymphoid immunophenotypes sub-classified according to the established positive markers outlined in the world health organization (who) 2016 classification of tumours of haemopoietic and lymphoid tissues.24 specifically, expression of cd27, cd43, cd56, cd58, and cd81, which were not included in the predicate clearllab 10c and in-house second-line panel analysis, were verified on background (normal) populations as evidence of a satisfactory internal positive control and proof that the reagents met the manufacturer’s performance specifications. in the alb analyses, typical secondary backbone markers, including co-expression of cd19/cd10/cd34 enabled further characterisation of markers cd20, cd38, and cd58 and established the presence of normal precursor b-cells (haematogones) or abnormal precursor b-cells. likewise, for the duraclone re clb, dual expression of either cd19/cd5 or cd19/cd20 was used to identify the ‘target’ or neoplastic b-cell population before specific characterisation of cd43, cd79b, cd81 and ror1 expression. plasma cells were defined in the duraclone re pc panel by the dual expression of both cd38 and cd138; a normal or malignant plasma cell immunophenotype was subsequently noted following interrogation of markers including cd81, cd27, cd19, cd200, cd56, and cd45. statistical analysis clinical diagnostic outcomes were collated into microsoft excel (redmond, washington, united states) spreadsheets. marker expressions and specific clinical diagnoses noted for the duraclone re alb, re clb, and re pc panels were compared to the clinical diagnostic outcomes from the existing in-house 2–4-colour antibody panels described (table 2). the agreement between methods (comparing the final diagnostic outcome) was assessed using contingency tables to determine sensitivity and specificity. true positives were regarded as clinical diagnostic outcomes matching the predicate clearllab 10-c with in-house panels, and test system outcomes using alb, clb or pc. for example, when a b-cell cll was diagnosed on the initial clearllab investigation with cd23 expression by predicate second-line testing, a diagnosis of a b-cell cll was confirmed in the clb tube with positive ror-1 and cd43 expression. true negatives were those cases where there was no disease noted, either by predicate clearllab and second-line testing or by test method with duraclone panel testing. false positives were defined as those cases where there was no disease noted on predicate clearllab with in-house second-line testing, but the disease was noted by respective duraclone analysis. likewise, false negatives were defined as disease on predicate first-line clearllab and second-line testing, but no disease by respective duraclone panel testing. results normal and abnormal b progenitor cells panel twenty samples were tested with the duraclone alb panel. the duraclone re alb evaluation on both routine diagnostic precursor b-all samples, and patient samples who were being followed up after therapy for precursor b-all, revealed 100% positive and negative agreement to clearllab 10c reported outcomes (table 3). the estimated sensitivity and specificity rates were both 100%. cd58 expression (an additional marker included in the duraclone alb tube, but not clearllab tubes) enabled further confirmation of the diagnostic outcome reported; here, there was consistent under-expression of cd58 noted on haematogones and consistent over-expression seen on abnormal precursor b-cells (blasts), verifying manufacturer-described cd58 expression. cd22 apc expression (that was added as an additional marker panel in two precursor b-all cases to demonstrate that the addition was possible) was verified against the expected disease outcome (positive in two cases of b-all) and met the manufacturer’s specifications in the duraclone re alb (table 3). table 3: comparison of predicate reagents and duraclone re alb reagent during the assessment of normal and abnormal precursor b-cells undertaken at the charlotte maxeke johannesburg academic hospital flow cytometry laboratory in johannesburg, south africa, april 2020 to august 2020. chronic b-cell lymphoproliferative disorder panel the duraclone re clb panel identified the b-cell target population using a combination of cd19 and cd5 or cd20, as well as surface cd81, ror1, cd79b, and cd43 expression. twenty samples were tested with the duraclone clb panel. the calculated sensitivity and specificity were 100%, with full diagnostic concordance noted across all cases evaluated (n = 20). all cll cases (n = 16/20) showed under-expression (weak) of cd81, cd79b and cd20 (table 4). further, surface ror1 expression was detected in 15 of the 16 (93.75%) cd5-expressing cll cases; all cll showed expression of cd43. a single case of follicular lymphoma (n = 1) showed a bright expression of cd79b. three cases (15%) evaluated could not be definitively sub-classified with the in-house second-line panel analysis or the duraclone re clb reagents. an additional control sample (not included in table 2), with confirmed 20% of b-cell precursors, revealed and confirmed bright cd81 on the normal precursor b-cells (figure 1). table 4: comparison of predicate reagents and duraclone re clb reagent during the assessment of mature b-cell lymphoid proliferations undertaken at the charlotte maxeke johannesburg academic hospital flow cytometry laboratory in johannesburg, south africa, april 2020 to august 2020. normal and abnormal plasma cells panel seventeen samples were tested with the duraclone pc panel. a plasma cell population was identified by backbone markers cd138 and cd38 in the duraclone re pc panel, with subsequent determination of expression of cd19, cd27, cd45, cd56 and cd200. there was cross-panel marker equivalency to the existing in-house panels (table 5) and clinical outcomes, with 100% agreement achieved, and estimated sensitivity and specificity rates of 100%. in addition, the assessment of cd27 and cd81 on target plasma cells revealed consistent under-expression of cd81 or cd27 (n = 14/14) (figure 2). a single (n = 1) pcd had positive surface cd117 expression (figure 2) on the aberrant identified plasma cell population, in agreement with cd117 expression noted in the matching clearllab 10c m2 analysis. table 5: comparison of predicate reagents and duraclone re pc reagent during assessment of normal and abnormal plasma cell populations undertaken at the charlotte maxeke johannesburg academic hospital flow cytometry laboratory in johannesburg, south africa, april 2020 to august 2020. discussion the recently evaluated,9,16 fixed-tube, standardised, pre-titrated reagent system, clearllab 10c (including the b-cell, t-cell and m1 and m2 tubes), and clearllab ls11 were recently implemented to replace our outdated facscalibur/paint-a-gate method for routine immunophenotypic investigation of haematological neoplasms at our laboratory. this new system provides flow cytometric workup for haematological neoplasms and enables processing up to 400 samples a month. previously, leukaemia flow cytometry testing was limited to simple 2–4-colour analysis; sample setup was manually intensive and error-prone. systems previously implemented into our laboratory streamlined standardised and automated testing and improved workflow and quality of reporting.25,26,27 similarly, implementing the clearllab 10c and ls system in our laboratory16 has markedly improved workflow and minimised errors while providing standardised and substantially better quality-controlled sample and data acquisition and comprehensive data. the latter allows for improved target population identification, especially for small populations. complementary and supplementary multicolour panel options are published3,4,10,19,28 and could theoretically be assembled as required in the lab to provide additional marker panels needed to complete full patient immunophenotypic investigation. however, setting up quality-controlled multicolour panels in lowand middle-income countries is both challenging and less reliable16. the clearllab system facilitates detailed diagnosis across a broad range of acute leukaemias, and is sufficiently sensitive to detect the presence of a mature b-cell lpd, but it lacks specificity to discern and diagnose different mature b-cell lpd9 or confirm pcds. for example, although the b-cell panel is useful to differentiate precursor b-cell from more mature b-cell lpd, the panel does not accommodate sufficient markers to discern different types of mature lpds, including cd23, cd43, cd79b and fmc-7 markers necessary to distinguish b-cell lpd per the who classification.24 likewise, markers, such as cd58, for diagnosis for subsequent follow-up of precursor b-all, or cd79a and cd81, would be of value in the b-cell panel to discern neoplasm from haematogones. plasma cell dyscrasias are also not easily identified in the clearllab system, which lacks cd138 and other markers useful for diagnoses and follow-up of pcd.22,29,30 although cd38, cd56, and cd45, used to identify plasma cells, are included in the clearllab panels, plasma cell identification is not definitive. specifically, cd56, included in the t-cell panel, is assessed separately from cd38 (included in the b-cell and m2 panels). a pre-titrated-monoclonal panel including all three markers would suitably enhance the clearllab system; the duraclone panels used in this study provided this supplement format. normal and abnormal b progenitor cells panel the expression of cd10, cd19, cd34, cd38, cd20 and cd45 in the duraclone re alb panel showed excellent cross-panel marker expression equivalency and concordant clinical diagnostic outcome when compared to clearllab 10c b-cell panel expression. cd58 together with cd38, cd10, cd19, cd34, cd20 and cd45 has potential for sensitive minimal residual disease assessment31,32 along with cd81 expression.31 cd58 in the index test alb tube proved to be an excellent adjunct for assessing precursor b-cells and was helpful in discerning abnormal precursor b-cells from normal precursor b-cells (haematogones)31. distinct discordant cd123 and cd34 expression patterns have also been described on haematogones and can be used in a strategy to determine b-all minimal residual disease.32 cd123 was not however included in the alb tube. in our predicate method, the clearllab 10c system, co-expression of cd123 with cd34 and cd19 assessment within the clearllab m2 panel, used to identify precursor b-cells, together with cd19, cd10 and cd34 co-expression in the clearllab b-cell tube, therefore offers more diagnostic information than the duraclone alb tube alone for the follow-up of precursor b-all and residual disease assessment. the clearllab 10c system was developed to diagnose early and later b-cell lymphoproliferative disorders; these 10-colour panels are fixed. thus, additional markers cannot be tested for in the panels. however, additional markers can be added to the re alb, fine-tuned toward detecting early b-cell precursors only. cd22 and cd123 could be valuable additions to the alb panel. for example, a liquid reagent drop-in of cd22-apc in the free apc fluorescence channel of the duraclone re alb panel could provide a baseline assessment of cd22 expression on b-all blasts, which would be useful for deciding on anti-cd22 targeted therapy in b-all patients. cd123 could also be added in a second available channel to enable further discrimination of disease (b-all) from normal haematogones (see table 2). chronic b-cell lymphoproliferative disorder panel the expression of cd5, cd19, cd20 and cd45 in the duraclone re clb reagent showed excellent cross-panel marker expression, equivalent to clearllab 10c b panel expression, with 100% (n = 20/20) and clinical diagnostic outcomes agreement. the additional markers in the duraclone re clb panel, including cd81, ror1, cd79b and cd43 which are not included in the laboratory predicate clearllab system, specifically the b-cell tube, met the requirements for intended use and the manufacturer performance specifications. the duraclone re clb panel proved to be an excellent fixed supplementary panel to replace our outdated second-line investigation for the workup of mature b-cell lymphoid proliferations and proved to be especially useful for discerning cd19/cd5 positive haematolymphoid disease subtypes. the surface cd81, cd79b and cd20 showed consistent under-expression in cll, while the presence of ror1 was also helpful in discerning cll over mantle cell lymphoma as described in previous studies,17,18,33 especially when interpreted in conjunction with cd200 expression in the clearllab b-cell panel.34,35 in the event of a cd19/cd10 co-expressing mature b-cell lymphoid proliferation, cd43 in the duraclone re clb panel was shown to be a useful cell surface marker to discriminate follicular lymphoma from a high-grade b-cell lymphoma. similarly, cd79b within the duraclone re clb panel, read together with cd38 expression in the clearllab b-cell tube, identifies a mature b-cell lymphoid proliferation with plasmacytoid differentiation. as for the alb panel, free channels in the panel allow for additional flexibility to investigate related markers that are regarded as useful to discern mature b-cell haematolymphoid subtypes. a liquid reagent drop-in of cd23-ecd in the duraclone re clb panel could further confirm b-cell cll and discern b-cell cll from other cd19/cd5 co-expressing target populations.17,18,33 normal and abnormal plasma cells panel the cd38, cd138, cd45, cd19, cd56, and cd200 expressions in the duraclone re pc panel showed excellent cross-panel marker expression and equivalent diagnostic outcome and facscalibur/pag analysis outcomes, with 100% (n = 17/17) clinical diagnostic concordance (agreement). in addition, as previously described,29 cd27 and cd8130 proved to be valuable markers to discern aberrant plasma cells over normal plasma cells. finally, the usefulness of the re pc panel can be further extended by adding cd117-ecd30 reagents. for one sample, we added cd117-ecd reagents to the pc tube, confirming the presence of malignant plasma cells and minimal residual disease assessment.14,30. limitations firstly, the sample size per panel evaluated is small, and further studies are needed to confirm the outcomes reported here. secondly, the fluorochromes used in the panels are specifically designed for use on a bc navios instrument. therefore, the products could be used on alternative instruments only if the respective filter setups23 (of the particular laboratory’s flow cytometer) can accommodate data collection from the fluorochromes used in the clearllab 10c or duraclone panels. conclusion this study confirms that the bc duraclone alb and clb panels are suitable to provide additional second-line immunophenotypic workup of precursor b-all and mature b-cell lymphoproliferative disorders resepectively, at disease presentation and are suitable to supplement first-line clearllab 10c laboratory predicate method testing. the under-expression of cd81, cd79b, and positive cd43 and ror1 expression, noted in the duraclone clb panel specifically assists in distinguishing b-cell cll from mantle cell lymphoma and other cd5 negative b-cell lpds. the duraclone re pc panel is suitable for the second-line investigation of pcd. cd27, cd56, cd81 and cd200, together with cd19, cd38 and cd138 in a single analysis, were efficient in identifying aberrant plasma cells. although there was diagnostic and individual marker concordance, we did not find the alb more useful for diagnosing b-all over our existing clearllab system (utilising the b-cell and m2 tubes). the inclusion of cd5831 in the re alb tube may however be a valuable adjunct for discerning normal reactive b-cell precursors from the minimal residual disease at follow-up. lastly, the potential benefit of using commercialised multicolour lyophilised fixed panel preparations having stable, standardised antibody reagents reduces the risks of technical error, improves laboratory efficiency, and simplifies reagent inventory. in addition, the duraclone ‘free fluorescence channels’ provide some additional flexibility for a laboratory to use their preferred markers to establish diseases (or not) of their choice. acknowledgements d.k.g. and d.l. thank mrs merriam machaba at charlotte maxeke johannesburg academic hospital flow cytometry. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions d.k.g. conceptualised, led and funded the study, provided project administration and project supervision as well as supervision of all laboratory testing and data analysis. d.l. and d.k.g. oversaw technical aspects and the setup of protocols for flow cytometric data acquisition and data analysis. l.s. and m.p. analysed flow cytometric data and recorded the outcomes in spreadsheets for analysis comparison. d.k.g. undertook final checking of data and compiled the final laboratory validation report. l.s. and d.k.g. translated the validation report into a first draft for publication format. all subsequent drafts were written by d.k.g. sources of support funding for this study was made possible with research incentive funds accrued to d.k.g. through the university of the witwatersrand. data availability all outcomes are anonymised and published in the maunscript. individal raw flow cytometric listmode data files are available on request from the corresponding author, d.k.g. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any of the affiliated agencies of the authors. references borowitz mj, wood bl, 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lymphocytic leukemia. cytometry b clin cytom. 2019;96(2):143–148. https://doi.org/10.1002/cyto.b.21722 abstract background description of the intervention lessons learnt recommendations acknowledgements references about the author(s) valerie f. donkeng-donfack mycobacteriology unit, national tuberculosis reference laboratory, centre pasteur du cameroun, yaoundé, cameroon suzanne m. ongoulal mycobacteriology unit, national tuberculosis reference laboratory, centre pasteur du cameroun, yaoundé, cameroon yvonne j. djieugoue mycobacteriology unit, national tuberculosis reference laboratory, centre pasteur du cameroun, yaoundé, cameroon yannick kamdem simo mycobacteriology unit, national tuberculosis reference laboratory, centre pasteur du cameroun, yaoundé, cameroon henri manga national tuberculosis control program, yaoundé, cameroon danielle a.d. tollo national tuberculosis control program, yaoundé, cameroon edwige m.a. belinga national tuberculosis control program, yaoundé, cameroon vincent mbassa national tuberculosis control program, yaoundé, cameroon jean l. abena national tuberculosis control program, yaoundé, cameroon sara eyangoh centre pasteur du cameroun, yaoundé, cameroon citation donkeng-donfack vf, ongoulal sm, djieugoue yj, et al. tuberculosis-loop-mediated isothermal amplification implementation in cameroon: challenges, lessons learned and recommendations. afr j lab med. 2022;11(1), a1792. https://doi.org/10.4102/ajlm.v11i1.1792 lessons from the field tuberculosis-loop-mediated isothermal amplification implementation in cameroon: challenges, lessons learned and recommendations valerie f. donkeng-donfack, suzanne m. ongoulal, yvonne j. djieugoue, yannick kamdem simo, henri manga, danielle a.d. tollo, edwige m.a. belinga, vincent mbassa, jean l. abena, sara eyangoh received: 23 nov. 2021; accepted: 20 apr. 2022; published: 26 aug. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: until 2016, microscopy was the main tool for the early detection of pulmonary tuberculosis in cameroon, especially in remote settings. due to the poor sensitivity of microscopy, there was a need to implement a molecular assay in order to improve tuberculosis case detection. intervention: in 2017, tuberculosis loop-mediated isothermal amplification (tb-lamp), a molecular rapid diagnostic test recommended by the world health organization, was implemented in cameroon as a replacement test of microscopy for initial diagnosis of pulmonary tuberculosis and also as a follow-on test to microscopy for smear-negative sputum specimens. a roll out plan for tb-lamp implementation in cameroon had been developed from january 2017 to april 2017, followed by initial implementation at four sites in may 2017. additional sites were added progressively. lessons learnt: the use of tb-lamp as a follow-on test to microscopy for smear-negative sputum specimens helped in the detection of tuberculosis in 14.77% of those who were sputum-smear negative in 2019. tuberculosis-loop-mediated isothermal amplification usage as an initial test, followed by testing with xpert mtb/rif for rapid tuberculosis and rifampicin resistance detection during tuberculosis mass screening campaigns, reduced the turn-around time by 73.23% as compared to when the gene xpert instrument was used alone. recommendations: the implementation and scaling up of tb-lamp in cameroon contributed to increase access to tuberculosis molecular diagnosis in remote settings and as such improved tuberculosis case notification. however, to better enhance this notification and optimise the use of a tb-lamp instrument, a suitable sample transport system is recommended. keywords: tb-lamp; molecular test; implementation; roll out; notification. background tuberculosis remains a major public health problem worldwide and tuberculosis detection cases remain a challenge and a priority for tuberculosis care and control.1,2 however, microscopy which is the main tool for the early detection of tuberculosis, especially in resource-limited countries with a higher burden of tuberculosis, has a sensitivity of about 50% – 60%.3 this sensitivity is even lower in children, people living with hiv and people with extrapulmonary tuberculosis.4 in addition, this sensitivity is lower compared to molecular tests such as tuberculosis loop-mediated isothermal amplification (tb-lamp) and genexpert.5 culture, which is the gold standard method for mycobacterium tuberculosis complex diagnosis, is time consuming due to the slow growth rate of m. tuberculosis complex and requires the use of sophisticated instruments such as the biosafety cabinet which is expensive, requires a lot of training and has a high cost of maintenance.6 accordingly, to rapidly detect tuberculosis cases as well as improve case detection including negative smear cases, the implementation of molecular world health organization (who)-recommended rapid diagnostic assays as tuberculosis initial diagnostic test is recommended to improve tuberculosis testing efficiency and increase tuberculosis bacteriologically confirmed cases.7 in 2012, the national tuberculosis control program (ntcp) of cameroon introduced xpert® mtb/rif (cepheid, sunnyvale, california, united states), an automated semi-quantitative nested real-time polymerase chain reaction for the rapid and simultaneous detection of m. tuberculosis complex and rifampicin resistance.8 however, xpert® mtb/rif equipment was placed at the regional laboratories and recommended for tuberculosis diagnosis in specific categories of presumptive tuberculosis cases including: children (0–5 years), people living with hiv, contact patients with multidrugor rifampicin-resistant tuberculosis, prisoners, refugees, patients previously treated for tuberculosis or those with treatment failures. in 2016, tb-lamp was endorsed by the who to be used as a replacement test for sputum-smear microscopy. the who recommended to use tb-lamp as the initial diagnosis test for the detection of pulmonary tuberculosis (ptb) in adults who present signs and symptoms consistent with ptb, and also as an add-on test following negative smear microscopy, particularly when additional testing of smear-negative sputum specimens is necessary.9 cameroon is a tuberculosis burden country10 with an estimated annual incidence of 103 new cases per 100 000 inhabitants in 2017. the expected number of new cases each year in cameroon is 47 000. however, only around 50% of tuberculosis cases are reported annually, meaning that around 50% of tuberculosis cases are still missing.11 accordingly, the ministry of public health of cameroon, through the ntcp, decided to implement tb-lamp (eiken chemical, tokyo, japan) in 2017, with the goal to increase access to molecular diagnosis and ultimately improve tuberculosis case findings.9 tuberculosis-lamp is the only molecular test recommended by the who to be used in primary health care for the diagnosis of active tuberculosis.12 this test is a manual assay that does not need an air-conditioned room or sophisticated equipment and is not affected by short power intervals or variations. up to 14 samples can be tested at once using tb-lamp equipment, up to 70 tests can be performed in 8 h,9 and the results can be read with the naked eye under ultraviolet light.9 implementation of the tb-lamp assay requires minimal laboratory infrastructure and few biosafety requirements. the tb-lamp test is a less expensive test,13 with high sensitivity compared to microscopy and a similar sensitivity and specificity to xpert mtb/rif.5,14,15,16 before cameroon, only countries from asia, including the philippines, vietnam, cambodia and myanmar, performed pilot implementation of tb-lamp with no big issues and they received project funding. since the endorsement of tb-lamp by the who, some african countries other than cameroon, such as nigeria, zambia, kenya, uganda, angola, ivory coast, uganda, gambia and senegal, performed pilot implementation of tb-lamp and they would be preparing for the extension phase. however, amongst african countries, cameroon is the country where the implementation process started, in 2017. this paper describes the challenges, lessons learned and recommendations for tb-lamp implementation in cameroon. description of the intervention ethical considerations the ministry of health through the national tuberculosis control program conducted the implementation of tb-lamp in cameroon. as such, no ethical approval was required. policy development in 2014, with the support of the foundation for innovative new diagnostics, cameroon participated in the evaluation of tb-lamp. the centre pasteur du cameroun, through the national reference laboratory, conducted this evaluation at jamot hospital in yaoundé. the results showed that tb-lamp displayed higher sensitivity (82.6%; 95% confidence interval [ci]: 76.9–87.2) compared to smear microscopy with 53.6% (95% ci: 46.8–60.3). tuberculosis-lamp showed a specificity of 96.0% (95% ci: 93.2–97.7) and microscopy, 99.0% (95% ci: 97.1–99.7). meanwhile, the sensitivity and specificity of tb‑lamp were similar to genexpert® (89.9%; 95% ci: 85.0–93.3 and 97.0%; 95% ci: 94.4–98.4, respectively).16 the overall results of this evaluation was used by the who to approve the tb-lamp assay in 2016 as a follow-on and replacement test for sputum-smear microscopy in the diagnosis of ptb in adults with signs and symptoms consistent with tuberculosis.9 therefore, the ministry of health, through the ntcp, reviewed the policy about to be implemented and decided to introduce this new molecular diagnostic technique in cameroon. evidence base tuberculosis-lamp implementation in cameroon started in may 2017 with a 2-month pilot phase. four laboratories in two regions (centre and south) were selected for this phase based on their workload. the centre and the south regions represent, respectively, about 18.0% and 3.5% of the total population of cameroon, with a tuberculosis prevalence of about 229/100 000 and 129/100 000 inhabitants in 2017. the centre region is amongst the three regions with the highest number of tuberculosis case notifications in cameroon. the four laboratories involved during this pilot phase include jamot hospital, mbalmayo and bafia district hospital in the centre region and ebolowa regional hospital in the south region. these laboratories were selected because of their higher workload during the first semester of 2017, compared to other laboratories in their respective regions. in addition, theses laboratories were not far from the national reference laboratory that oversaw training and monthly monitoring during this pilot phase. nine laboratory technicians were trained, and each laboratory was equipped with a tb-lamp machine and an uninterruptible power supply. in these four laboratories, only the tb-lamp assay was used for routine diagnosis of ptb in adults. data obtained after two months of implementation (june and july 2017) at the four pilot sites were compared to the results obtained at the same sites over the same period of the previous year (june 2016 and july 2016), where only microscopy was used for initial ptb diagnosis. the compared data showed a 30.0% increase in positivity rate between 2016 and 2017. table 1 shows the number of patients who accessed the tests from the four pilot sites during the months of june and july 2017 and the proportion of positive to negative cases. these results served as an evidence base to guide and reinforce cameroon’s decision to go ahead with the scale up of tb-lamp in the country. table 1: number of patients tested with tuberculosis loop-mediated isothermal amplification as initial tuberculosis diagnosis test in june 2017 and july 2017 in four diagnostic and treatment centres in cameroon. search for funds in order to scale up the implementation of tb-lamp in cameroon, the results obtained during the pilot phase were used by the ntcp to address cameroon’s 2018–2020 request for global fund (gf) support. in 2018, this request was validated by gf to support tb-lamp implementation in cameroon. in addition to the equipment and reagents, the gf support also covered training of laboratory technicians and supervisors of the tb-lamp sites. however, the allocated funds had neither fees for humaloop t instrument installations nor fees to conduct related activities such as meetings with clinicians. furthermore, these funds did not consider additional materials not provided by the kits, including scissors, gloves, marker pens and tissue paper. fortunately, these additional fees were covered by the government through the tuberculosis national reference laboratory (tb-nrl) hosted at centre pasteur du cameroun for an optimal implementation of the new technique. in addition, the government also supported sample transportation. selection of settings twenty-one tuberculosis diagnostic and treatment centers (dtcs) out of the 260 dtcs present in the country were selected in the 10 regions for the scaling up of tb-lamp. many reasons guided the selection of these dtcs. nineteen of the dtcs are at remote settings and had only microscopy as a tuberculosis diagnostic tool. two of the dtcs are tuberculosis regional reference laboratories. the workload of the selected dtcs was higher compared to the others. all 21 dtcs have electricity and easy access for sample transportation. this selection of sites for tb-lamp extension was performed prior to the training of staff. procurement, revision of tuberculosis national guidelines and tuberculosis diagnosis algorithms the procurement of reagents and equipment (humaloop t and uninterruptible power supply) was done through the global drugs facility (gdf). during the procurement period, the ntcp revised the tuberculosis national guidelines and tuberculosis diagnosis algorithms in may 2018 to include tb-lamp assay. on the revised tuberculosis national guidelines, the ntcp recommended the use of tb-lamp at the dtcs where it was implemented as a replacement test for sputum-smear microscopy for initial diagnosis of ptb in adults with signs and symptoms consistent with tuberculosis. however, at the microscopy testing centres, tb-lamp was recommended to be used as a follow-on test to smear microscopy for smear-negative sputum specimens in adults with signs and symptoms consistent with ptb. steps for tuberculosis-loop-mediated isothermal amplification scaling up in cameroon the scaling up of tb-lamp in cameroon was performed in four steps including trainings, equipment installation, stakeholder engagement and sample transportation. training trainings started with the training of trainers organised by the tb-nrl at the centre pasteur du cameroun. five trainers, including three from the tb-nrl and two from the pilot sites (jamot hospital and ebolowa regional hospital), were trained over five days. they were then involved in the training of about 50 laboratory technicians. trainings included theoretical and practical sessions (figure 1). the objective of these trainings was to improve the technical capacities of laboratory technicians for better diagnosis of tuberculosis in the laboratory using humaloop t equipment. due to budget constraints, only four sessions of three days each were organised for the training in the country. a minimum of 12 personnel were trained per session by two trainers with one humaloop t instrument. the training was laborious for the trainers because of the high number of trainees per session. figure 1: tuberculosis loop-mediated isothermal amplification trainings during scaling up in cameroon, june 2019 and august 2019. (a) transfer of sputum sample to heating tube. (b) removal of heating tube from the heating block of the humaloop t instrument. (c) dna extraction. (d) labelling of reaction tubes. douala tb regional reference laboratory 08-08-2019 (a, b) and garoua tb regional reference laboratory 20-06-2019 (c, d). installation of humaloop t instrument at selected sites after training sessions, humaloop t instruments with uninterruptible power supplies were installed at the 21 selected sites. the role of the uninterruptible power supply is to allow the equipment to keep running for at least a period when the primary power source is lost. the installations were performed in the week following the training to enable technicians to quickly start tb-lamp testing in their respective settings, with a fresh memory of the training. the activity of humaloop t instruments installation was not funded by the gf. the government covered this activity through the tb-nrl. stakeholder engagement in order to ensure a successful implementation of tb-lamp in cameroon, the ministry of health, through the ntcp, had organised a sensitisation meeting in march 2018, on world tb day, on the role of tb-lamp in improving tuberculosis diagnosis in cameroon. the meeting was chaired by the minister of health, together with the head of the ntcp and the general manager of the centre pasteur du cameroun. this meeting involved community leaders from high burden communities, tuberculosis active case finders, physicians, laboratory technicians, directors of tb-dtcs, the ministry of health, other stakeholders, and both national and international media. in addition to this meeting, regional sensitisation meetings were also organised. attendees included physicians, nurses, bikers involved in sample transportation and other health staff involved in tuberculosis patient care. during the meetings, participants were informed about the introduction of the new tuberculosis diagnosis assay at the dtc and regional level, as well the role and the advantages of the new technology. also, tb-lamp algorithms were presented and discussed. the government funded these national and regional sensitisation meetings through the tb-nrl. sample transportation the sample transportation system of smear-negative sputum specimens from microscopy centres to tb-lamp centres for further testing was organised in all regions in 2019 in order to give all ptb presumptive cases the benefit of the tb-lamp assay. according to the number of tb-lamp laboratories in a region, microscopy laboratories were organised into pools, and with a tb-lamp laboratory. this activity of smear-negative sputum specimen transportation was funded by the government. bikers and public transport were used for transportation. city transportation used bikers whereas intercity transportation used intercity public transportation. furthermore, laboratories were equipped with phones and communication credits to ensure communication between them and the bikers for sample collections and results deliveries, to reduce the turn-around time. the government funded this activity. the challenges were insufficient funds as well as the small number of bikers. challenges the implementation of tb-lamp in cameroon took two years (from may 2017 to may 2019). during these two years, 27 dtcs were gradually equipped with a humaloop t instrument. the implementation started with four machines in 2017, followed by two machines in 2018 and 21 machines in 2019. the government funded the first six machines and the gf the other 21. from the four pilot sites in 2017, the ntcp established a 3-year extension plan (2018–2020), supported by the gf. many challenges accompanied this implementation. make pipette-60 set material available on the global drugs facility catalogue the procurement of reagents and equipment (humaloop t and uninterruptible power supply) through the gdf took about 10 months between placement of order and delivery. the issue was due to the absence of the pipette set on the gdf catalogue. therefore, human and eiken companies negotiated with the gdf to include pipette-60 set material on the gdf catalogue, to facilitate procurement. getting additional funds for tuberculosis-loop-mediated isothermal amplification implementation-related activities and for additional materials for an efficient implementation of tb-lamp, we requested additional funds from the government to conduct related activities, such as meetings with clinicians, and to purchase supplementary materials such as scissors, gloves, marker pens and tissue paper needed for tb-lamp assays, but not provided with the kits. trainings of laboratory technicians organising a 3-day training session for a minimum of 12 technicians with only one humaloop t instrument was very challenging because of the reduced number of days and the high number of trainees. accordingly, the tb-nrl proposed to the ntcp to reduce the number of trainees per session for the next year and to train technicians directly in their laboratory. increasing tuberculosis case notification to increase tuberculosis case notification using a humaloop t instrument, the ntcp implemented the use of tb-lamp in 2019 as a follow-on test to smear microscopy for smear-negative sputum specimens in adults with signs and symptoms consistent with ptb. this activity was done through an organised sample transportation system for transportation of smear-negative sputum specimens to tb-lamp sites. this activity helped to detect up to 388 (14.77%) tuberculosis patients out of 3061 smear-negative sputum specimens transferred to tb-lamp sites. these patients could have been released into the community and, as such, could have continued to spread the disease. in addition, the ntcp organised tuberculosis mass screening campaigns at 34 prisons in 2019 and the tb-lamp assay was used as initial tuberculosis diagnosis followed by testing with xpert mtb/rif for tuberculosis and rifampicin resistance detection. tuberculosis mass screening campaigns helped in the detection of 123 tuberculosis cases out of 3672 presumptive tuberculosis cases at the 34 prisons, and three rifampicin resistance cases. table 2 presents the summary stats, by year, for the four initial sites and the number of positive cases picked up by tb-lamp. table 2: summary statistics for the four initial sites by year and number of positive cases picked up by tuberculosis loop-mediated isothermal amplification, cameroon, 2017–2019. lessons learnt the implementation of tb-lamp in cameroon offered an opportunity for us to draw several lessons for an effective implementation. these lessons learned will be useful for countries that will start with tb-lamp implementation after us. pulmonary tuberculosis-loop-mediated isothermal amplification materials and its use the tb-lamp assay needs additional materials such as scissors, gloves, marker pens and tissue paper, that are not provided with the kits. moreover, tb-lamp assays required a positive and a negative control for each run. these controls provided by the kits include one tube with 0.4 ml of positive control and three tubes of 0.5 ml of negative control. the available volume of positive control is necessary for 12 runs of six samples/run. however, when a laboratory performs less than six samples/run, they will perform more than 12 runs and the 0.4 ml of positive control will not be sufficient. also, using less than six samples/run leads to the consumption of more extraction and detection kits for controls. this situation could therefore lead to a stock shortage if the estimations did not consider it. education of tuberculosis-loop-mediated isothermal amplification users the use of tb-lamp does not require a background in molecular biology. during tb-lamp implementation in cameroon, most of the laboratory technicians trained at the remote setting on the use of tb-lamp for tuberculosis diagnosis were the ones performing microscopy with no background in molecular biology. infrastructure tuberculosis-lamp has been implemented in the laboratories performing microscopy as a routine test for tuberculosis diagnosis. therefore, the same infrastructure used for microscopy was applied. there was no need to adjust or to modify infrastructure. reduction of turn-around time during tuberculosis mass screening campaigns in 2019, a diagnostic algorithm based on an initial testing with tb-lamp, followed by testing with xpert mtb/rif to diagnose tuberculosis, reduced the turn-around time by 73.23% during mass campaigns in 34 prisons compared to xpert mtb/rif when used alone. recommendations search for funding implementation of tb-lamp requires consideration, during forecasting, fees of related activities such as meetings and sample transportation, as well as additional materials not provided by the kits. training trainings should include a maximum of four trainees for a 3-day session with one humaloop t instrument, and sufficient financial resources should be allocated for these training activities, so as to train technicians in their various laboratories with the use of their instrument. pulmonary tuberculosis-loop-mediated isothermal amplification settings the robustness of the humaloop t instrument makes it good for peripheral laboratories receiving large numbers of samples. this will avoid the use of less than six samples/run. with the help of solar panels, a humaloop t instrument can be used for tuberculosis active case finding in communities where there is no electricity. implementation of a diagnostic algorithm based on an initial testing with tuberculosis-loop-mediated isothermal amplification followed by testing with xpert mtb/rif to diagnose tuberculosis this approach improved early and rapid tuberculosis detection with an added advantage of providing rifampicin resistance status. data management datatocare, a simple, customisable, and patient-centric application developed by savics srl (brussels, belgium), could help to record patient, physician and laboratory operator information, send results directly to the physician via sms or email, and easily generate weekly, quarterly and annual reports. conclusion despite some difficulties, the implementation of tb-lamp in cameroon was well conducted, and the tb-lamp test is used as a who-recommended initial rapid diagnostic test for all people with signs and symptoms of tuberculosis. the challenges, lessons learned and recommendations resulting from this implementation will help other countries to have a more efficient implementation. acknowledgements the authors would like to thank: the ministry of public health and the national tuberculosis control program for fundraising and the revision of national tuberculosis guideline as well as tuberculosis diagnosis algorithm; the global fund for providing necessary funds for the implementation; centre pasteur du cameroun who conducted this implementation and provided additional funds to support tb-lamp equipment installation as well as organisation of meetings with clinicians; all the national tuberculosis reference laboratory staff for their active participation on the training of laboratory technicians. all the national tuberculosis control program staff for their participation at all the implementation steps; all the laboratory technicians for using tb-lamp test for tuberculosis diagnosis; and all the physicians for requesting tb-lamp as an initial test for ptb diagnosis. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions v.f.d.-d., v.m., e.m.a.b., h.m., j.l.a. and s.e. contributed to the design and implementation of the study. v.f.d.-d. and s.e. contributed to the analysis of the results, and to the writing of the manuscript with input from all authors. v.f.d.-d., s.m.o., y.j.d. and y.k.s. contributed to the training of laboratory technicians and installation of equipment. v.f.d.-d. and d.a.d.t. contributed to quantifying of the reagents, consumables and equipment needed for the implementation and scaling up of the technique. sources of support the global fund through new funding model 2018–2020 (nfm3 2018–2020) supported this work. data availability the authors confirm that the data supporting the findings of this study are available within the article. disclaimer the views expressed in the submitted article are the authors’ own, not an official position of the institution or funder. references world health organization. global tuberculosis report [homepage on the internet]. 2018 [cited 2019 may 15]. available from: http://apps.who.int/iris/bitstream/handle/10665/274453/9789241565646-eng.pdf?sequence=1&isallowed=y world health organization. xpert mtb/rif implementation manual – technical and operational ‘how-to’: practical considerations [homepage on the internet]. 2014 [cited 2015 jan 12]. available from: https://apps.who.int/iris/bitstream/handle/10665/112469/9789241506700_eng.pdf?sequence=1&isallowed=y zijenah ls. the world health organization recommended tb diagnostic tool [homepage on the internet]. 2018 [cited 2019 mar 18]. available from: https://www.researchgate.net/publication/327984940 nahid p, kim sp, evans ca, et al. clinical research and development of tuberculosis diagnostics: moving from silos to synergy. j infect dis. 2012;205(2):159–168. https://doi.org/10.1093/infdis/jis194 donfack vf, ngando l, pefura ew, et al. comparative study of loopamp™ mycobacterium tuberculosis complex kit for rapid detection of mycobacterium tuberculosis complex in cameroon. biomed biotechnol res j. 2018;2(1):46–52. https://doi.org/10.4103/bbrj.bbrj_86_17 khairunisa s, erica l. an activist’s guide to tuberculosis diagnostic tools [homepage on the internet]. 2017 [cited 2020 mar 20]. available from: https://www.treatmentactiongroup.org/wp-content/uploads/2017/04/tb-diagnostics-guide.pdf world health organization. framework of indicators and targets for laboratory strengthening under the end tb strategy [homepage on the internet]. 2016 [cited 2017 jun 25]. available from: https://www.who.int/publications/i/item/9789241511438 boehme cc, nabeta p, hillemann d, et al. rapid molecular detection of tuberculosis and rifampin resistance. n engl j med. 2010;363(11):1005–1015. https://doi.org/10.1056/nejmoa0907847 world health organization. the use of loop-mediated isothermal amplification (tb-lamp) for the diagnosis of pulmonary tuberculosis: policy guidance. world health organization [homepage on the internet]. 2016 [cited 2017 jan 15]. available from: https://apps.who.int/iris/bitstream/handle/10665/249154/9789241511186-eng.pdf?sequence=1&isallowed=y world health organization. global lists of high burden countries for tb, multidrug/rifampicin-resistant tb (mdr/rr-tb) and tb/hiv, 2021–2025 [homepage on the internet]. geneva: world health organization; 2021 [cited 2021 sept 18]. available from: https://cdn.who.int/media/docs/default-source/hq-tuberculosis/who_globalhbcliststb_2021-2025_backgrounddocument.pdf world health organization. global tb report 2017. [homepage on the internet]. 2017 [cited 2018 nov 18]. available from: https://www.who.int/publications/i/item/9789241565516 world health organization. model list of essential in vitro diagnostics [homepage on the internet]. 2018 [cited 2020 apr 18]. available from: https://aslm.org/wp-content/uploads/2018/05/who_edl_2018.pdf tb-lamp test now available for $6 through global drug facility [homepage on the internet]. 2020 [cited 2020 apr 18]. available from: https://www.stoptb.org/news/tb-lamp-test-now-available-6-through-global-drug-facility shete pb, farr k, strnad l, gray cm, cattamanchi a. diagnostic accuracy of tb-lamp for pulmonary tuberculosis: a systematic review and meta-analysis. bmc infect dis. 2019;19:268. https://doi.org/10.1186/s12879-019-3881-y pham th, peter j, mello fcq, et al. performance of the tb-lamp diagnostic assay in reference laboratories: results from a multicentre study. int j infect dis. 2018;68:44–49. https://doi.org/10.1016/j.ijid.2018.01.005 yadav r, sharma n, khaneja r, et al. evaluation of the tb-lamp assay for the rapid diagnosis of pulmonary tuberculosis in northern india. int j tuberc lung dis. 2017;21(10):1150–1153. https://doi.org/10.5588/ijtld.17.0035 abstract introduction methods results discussion acknowledgements references about the author(s) frank ssedyabane department of medical laboratory science, faculty of medicine, mbarara university of science and technology, mbarara, uganda thomas c. randall department of global health and social medicine, massachusetts general hospital, harvard medical school, boston, massachusetts, united states joseph ngonzi department of obstetrics and gynaecology, faculty of medicine, mbarara university of science and technology, mbarara, uganda rogers kajabwangu department of obstetrics and gynaecology, faculty of medicine, mbarara university of science and technology, mbarara, uganda alexcer namuli department of obstetrics and gynaecology, mbarara regional referral hospital, mbarara, uganda joy muhumuza department of obstetrics and gynaecology, faculty of medicine, mbarara university of science and technology, mbarara, uganda josephine n. najjuma department of nursing, faculty of medicine, mbarara university of science and technology, mbarara, uganda deusdedit tusubira department of biochemistry, faculty of medicine, mbarara university of science and technology, mbarara, uganda citation ssedyabane f, randall tc, ngonzi j, et al. association between dyslipidemia and cervical intraepithelial neoplasia: a case-control study in south-western uganda. afr j lab med. 2024;13(1), a2374. https://doi.org/10.4102/ajlm.v13i1.2374 original research association between dyslipidemia and cervical intraepithelial neoplasia: a case-control study in south-western uganda frank ssedyabane, thomas c. randall, joseph ngonzi, rogers kajabwangu, alexcer namuli, joy muhumuza, josephine n. najjuma, deusdedit tusubira received: 20 nov. 2023; accepted: 20 may 2024; published: 16 july 2024 copyright: © 2024. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: altered lipid levels may be associated with the development of a number of malignancies, including cancer of the cervix. however, there is limited understanding of this relationship in the rural ugandan context. objective: we investigated the connection between dyslipidaemias and cervical intraepithelial neoplasia (cin) among women attending the cervical cancer clinic at mbarara regional referral hospital in south-western uganda. methods: this unmatched case-control study was conducted between december 2022 and february 2023 and included women with cin (cases) and women without intraepithelial lesions (controls) in a 1:1 ratio. participants were selected based on cytology and/or histology results, and after obtaining written informed consent. demographic data were collected, and venous blood was drawn for lipid profile analysis. dyslipidaemia was defined as: total cholesterol > 200 mg/dl, low-density lipoprotein > 160 mg/dl, triglycerides > 150 mg/dl, or high-density lipoprotein < 40 mg/dl. at diagnosis, cases were categorised as either cin1 (low grade) or cin2+ (high grade). results: among the 93 cases, 81 had cin1, while 12 had cin2+. controls had a 13.9% (13/93) prevalence of high triglycerides and cases had a prevalence of 3.2% (3/93; p = 0.016). reduced high-density lipoprotein was the most prevalent dyslipidaemia among cases (40.9%; 38/93). statistically significant associations were found between high serum triglycerides and cin (odds ratio: 1.395, 95% confidence interval: 0.084–1.851, p = 0.007). conclusion: a notable association was observed between triglyceride dyslipidemia and cin. further studies into biochemical processes and interactions between lipids and cervical carcinogenesis are recommended through prospective cohort studies. what this study adds: this research provides additional information on the potential role of lipids in cervical carcinogenesis among women in rural uganda. it also presents the possible prevalence of multimorbidity involving cervical cancer and cardiovascular diseases, particularly in low-resource settings lacking preventive measures against the increasing prevalence of dyslipidaemia. keywords: cervical intraepithelial neoplasia; dyslipidaemia; total cholesterol; low-density lipoprotein; high-density lipoprotein; triglycerides; uganda; cervical cancer. introduction globally, there are 604 127 new cases of cervical cancer, ranking it as the fourth most common cancer, with 341 831 cervical cancer-related deaths worldwide in the year 2020.1 there has been an increasing prevalence of cervical cancer in africa, especially the sub-saharan region.2 it is the second most common cancer (with an incidence of 40.1/100 000 and mortality of 28.6/100 000), and the primary cause of cancer-related deaths among women in the east african region.1 developed countries, implementing screening programmes for women aged 25 years and above, have witnessed a significant reduction in cases by more than a third, a trend not mirrored in less-developed nations,3 probably as a result of inadequate interventions, including vaccination against human papillomavirus (hpv). it is a scientific fact that hpv is a necessary, albeit not standalone, factor in cervical cancer development.4,5 as a result, there is increasing research in the area of immunotherapy for advanced cervical cancer, especially the assessment of vaccine-related therapies, t-cell therapy and immune-checkpoint inhibitors in patients diagnosed with advanced cervical cancer.6 cervical cancer development occurs through a number of stages, starting with a persistent infection with any of the hpv genotypes that are high risk, then cervical intraepithelial neoplasia (cin), finally leading to invasive cervical cancer.7,8 noteworthy cofactors, including hiv infection, chlamydia trachomatis infection, smoking, gravidity, oral contraceptive use, and other forms of immunosuppression, also contribute to development of cervical cancer.8,9,10,11 our prior study revealed a potential link between cin and obesity, a component of metabolic syndrome.12 other components of metabolic syndrome include dyslipidaemia and insulin resistance, which have been shown to be associated with treatment outcomes of cervical lesions.13 lipids play a very critical role in metabolism, potentially serving as cofactors in the development of cin and invasive cervical cancer.14,15,16 alterations in lipids has been linked to development of a wide range of disease conditions.17,18,19,20,21 moreover, dysregulated lipids have been shown to occur in development of malignancies,16,17,22 with recent evidence connecting disordered lipid levels to cervical cancer and its progression.21,23 our previous study in an urban setting in uganda showed a significant association between dyslipidaemia and cancerous cervical lesions.24 however, information on any potential association between lipid levels and cin remains limited, especially in rural regions of uganda. this study was therefore conducted to investigate whether there is an association between serum lipid levels and cin among women attending the cervical cancer prevention clinic at mbarara regional referral hospital in south-western uganda. methods ethical considerations ethical approval for this study was granted by the mbarara university of science and technology research ethics committee (must-2022-396) and the uganda national council for science and technology (hs2395es). additionally, administrative clearance from the hospital director at hospital director at mbarara regional referral hospital was sought before commencing the study. we obtained written informed consent from each participant and/or their legal guardian prior to their involvement in the study. in the case of illiterate participants, written informed consent was acquired from their authorised representatives. to maintain confidentiality, study numbers were used instead of names on all data collection tools, and participant identifiable information was separated during data analysis. interactions between participants and the research team occurred in a private and comfortable setting, free from disturbances and accessible to only one participant at a time. women diagnosed with cervical intraepithelial lesions (cases) received the standard care package at the cervical cancer clinic. the nurse at the clinic provided participants with their visual inspection with acetic acid, pap smear, histology, and lipid profile results. all methods employed in this study adhered to relevant guidelines and regulations. study setting this study was conducted at the cervical cancer clinic of mbarara regional referral hospital, between 01 december 2022 and 27 february 2023. mbarara regional referral hospital is a regional referral hospital for the entire south-western uganda and it is located in mbarara city, approximately 260 km from the capital city, kampala. this clinic offers services to an average of 75 women per week. it receives women from over 13 districts of the entire south-western region of uganda plus the neighbouring countries including tanzania, rwanda and democratic republic of congo. study design we conducted this unmatched case-control study including women who visited the mbarara regional referral hospital cervical cancer clinic. cases were defined as women with a positive visual inspection with acetic acid result who underwent pap smears and histology, subsequently confirmed to have cin in accordance with the clinic’s standard care. controls consisted of women with a negative visual inspection with acetic acid result and those confirmed to be devoid of cervical lesions based on cytology and histology. sample size determination we calculated our sample size using the online openepi, version 3, open-source calculator-sscc (openepi – sample size for unmatched case-control studies). for this calculation, we considered a two-sided confidence level of 95, a study power of 80% and a case-to-control ratio of 1. we took into account a proportion of cases with dyslipidaemia of 40.99%,25 along with a proportion of controls with dyslipidaemia of 18.8%,26 together with a least extreme odds ratio of 3.0. this is based on previous studies which have demonstrated a high variability in prevalence of dyslipidaemia in africa, ranging from 5.2% to 89%.27 these studies offer the most accurate depiction of the cervical cancer study population that we could find.25 this gave a total of 75 cases and 75 controls using the fleiss module with continuity correction.28 after factoring in the possible 15% attrition rate, the resultant sample size came to at least 86 cases and 86 controls. sampling procedure we prospectively selected cases and controls by using purposive sampling to recruit cases after cytology or histology specimen collection and following a positive screening test. then we recruited a control each time a case was identified, that is, the incidence density sampling method as described previously.29 eligibility criteria we included women aged 21 years and above, who visited the study site during the time of the study and who consented to participate. we excluded any woman who was undergoing any form of treatment with any lipid-altering drugs such as antihypertensive and cholesterol-lowering drugs. data collection demographic data demographic information was gathered by use of a pretested enrollment form administered by research assistants, specifically the midwives from the mbarara regional referral hospital cervical cancer clinic. upon obtaining written informed consent, participants received assistance in completing the enrollment form to record their demographic details. the collected data encompassed information such as age, district and village of residence, history of blood pressure and diabetes, marital status, highest attained level of education, hiv status, smoking habits, oral contraceptive use, family history of cervical cancer, age at sexual debut, number of lifetime sexual partners, and the number of full-term births. blood collection and lipid measurements four millilitres (4 ml) of venous blood was drawn aseptically by venipuncture from the mid-cubital vein, into plain vacutainers following consent and completion of the enrollment form. specimens were given identification numbers (codes) and allowed to clot at room temperature, after which they were transferred to the laboratory for centrifugation (3000 revolutions per minute for 5 min) with the aim of separating serum from blood cells. the serum was then transferred into cryovial tubes using a micropipette. lipid concentrations were measured using a fully automated analyser, cobas 6000 clinical chemistry analyzer (roche diagnostics, indianapolis, indiana, united states) following standard operating procedures. interpretation of lipid profile serum lipid measurements were interpreted as follows using adult reference ranges according to the national cholesterol education program.30 we defined dyslipidaemia as total cholesterol concentration above 200 mg/dl, low-density lipoprotein (ldl) concentration above 160 mg/dl, triglyceride concentration of more than 150 mg/dl, or high-density lipoprotein (hdl) concentration below 40 mg/dl. histology and cytology using a cytobrush, cytological specimens were taken from the ectocervical and endocervical regions, smears made on spotless, marked glass slides and quickly fixed with 95% alcohol. for staining and analysis, fixed pap smears were brought to the mbarara university of science and technology histopathology lab. using the papanicolaou stains, all of the pap smears were stained following standard operating procedures. the principal investigator and a pathologist performed the examination, and the bethesda system for reporting cervical cytology31 was followed throughout the reporting process. cone/punch biopsy samples were taken for histological analysis. after being fixed in 10% formaldehyde for 48 h, the samples were processed in an open system tissue processor for 14 h, following a standard operating procedure. after sectioning, haematoxylin and eosin staining was applied to the tissues. stained slides were examined by the principal investigator together with a pathologist and reported as either cin1, cin2 or cin3. cin1 is considered ‘low grade’ and represents abnormal changes observed in less than one-third of the thickness of the cervical epithelium. cin2 represents abnormal changes observed in between one-third and two-thirds of the thickness of the cervical epithelium, while cin3 represents abnormal changes affecting more than two-thirds of the cervical epithelium. cin2 and cin3 are considered ‘high grade’. data management and analysis after collection by research assistants and the principal investigator, data were entered into an excel spreadsheet (microsoft office professional plus 2013, version 15.0.4675.1003, microsoft inc., redmond, washington, united states) and then imported into stata 13 (statacorp llc, college station, lakeway, texas, united states) software. we used descriptive statistics including frequencies, means ± standard deviations, median values and interquartile ranges to characterise the population. differences between respective groups were tested using the standard t-tests, chi-square tests and the fisher’s exact tests. associations between cin grades and specific dyslipidaemias were derived using logistic regression analysis. cases with cin2 or cin3 were combined into a single group called ‘cin2+’, because of the small number of cases in each group. all statistical outcomes were reported with 95% confidence intervals and p-values of < 0.05 were considered significant. we performed both bivariate and multivariate logistic regression analysis. factors which were biologically plausible in cervical cancer and those whose p-values were ≤ 0.2 at bivariate analysis were considered for multivariate logistic regression analysis. these factors included age, smoking, contraceptive use, level of education, history of high blood pressure and history of diabetes. results study participants’ characteristics in this case-control study, we enrolled 93 cases and 93 controls (table 1). there was a statistically significant variation in age between the two groups (p < 0.001); the mean age of participants control group was 38.87 years and in the case group was 34.57 years. additionally, more than half of the participants fell within the age group of 30 years – 49 years and this observation was statistically significant (p = 0.044). there were notable statistically significant differences in the highest level of education attained among study groups, with 13 out of 93 (13.9%) controls and 3 out of 93 cases (3.2%) reporting never having studied. contraceptive use was reported by 38 out of 93 (40.9%) controls and 56 out of 93 (60.2%) cases. among the cases, 7 out of 93 reported being smokers, resulting in a smoking prevalence of 7.6% among cases. table 1: demographic characteristics of study participants at mbarara regional referral hospital between december 2022 and february 2023. distribution of dyslipidaemias across cervical intraepithelial neoplasia grades out of the 93 cases, 81 had cin1 while 12 had cin2+. the mean triglyceride levels among cases was 95.9 mg/dl and among controls was 123.7 mg/dl, demonstrating a statistically significant difference (p = 0.001). high triglycerides showed a prevalence of 13.9% (12/93) among the controls and a lower prevalence of 3.7% (3/81) among cases, though this difference in distribution was not statistically significant (p = 0.08). high ldl showed a prevalence of 12.9% (12/93) among controls and a prevalence of 3.7% (3/81) among cases, and this distribution was also not statistically significant (p = 0.08). the prevalence of low hdl was higher in cin2+ cases (66.7%; 8/12) although not statistically significant when compared to controls (p = 0.086) (table 2). table 2: distribution of dyslipidaemias across cytology/histology results of study participants at mbarara regional referral hospital between december 2022 and february 2023. association between dyslipidaemias and cervical intraepithelial neoplasia from univariate logistic regression analysis, both high serum triglycerides and high serum ldl were found to be associated with cin (table 3). however, in multivariate logistic regression analysis, with adjustments made for age, smoking, contraceptive use, level of education, history of high blood pressure and diabetes, only high serum triglycerides was significantly associated with cin (odds ratio: 1.395, 95% confidence interval: 0.084–1.851, p = 0.007). on the other hand, high serum ldl showed borderline association with cin (odds ratio: 0.251, 95% confidence interval: 0.061–1.047, p = 0.058). table 3: logistic regression analysis showing association between dyslipidaemias and cervical intraepithelial neoplasia among study participants at mbarara regional referral hospital between december 2022 and february 2023. discussion we present a high prevalence of reduced hdl in cin (40.9%), especially those with cin2+ (66.7%). we also present the overall prevalence of increased serum triglycerides as 3.2% in cin and 3.7% specifically for cin1. high serum triglycerides was also significantly associated with cin in our study population. dyslipidaemia entails a variety of lipid abnormalities and may involve a combination of: extremely high cholesterol level of above 200 mg/dl, ldl cholesterol greater than 160 mg/dl, hypertriglyceridaemia of more than 150 mg/dl or significantly reduced hdl cholesterol of ≤ 40 mg/dl.32 it is one of the five metabolic syndrome components33 and it increases one’s risk of developing cardiovascular and other non-communicable diseases, including cancer of the cervix.34 it is therefore important to detect metabolic syndrome components in cin in order to prevent heart and other cardiovascular diseases. from our study, we observed that reduced hdl cholesterol was the most prevalent dyslipidaemia, an observation that has been reported in many previous studies.35,36 a study among south africans in the vaal region, between 2008 and 2011, reported similar findings on reduced hdl and increased triglycerides.37 a systematic review in 2018 revealed a reduced hdl prevalence of 41.4% and a prevalence of increased triglycerides of 16.5% in the general african adult population which is consistent with our findings.38 this shows an increased risk of cardiovascular diseases among the study population. we did not observe any significant association between reduced hdl cholesterol and cin. this is contrary to previous findings. for instance, frontela-noda et al., through their study at cuba’s national institute of oncology and radiobiology, among adult women from 2018 to 2020, reported a significant association between high grade squamous intraepithelial lesion and low hdl cholesterol.39 however, from a survey conducted from 1999 to 2010, a reduced serum hdl concentration was reported to be positively correlated with cervical cancer among united states citizens.40 this also indicated an increased likelihood of development of cardiovascular diseases among the study population. this study found a statistically significant association between increased serum triglycerides and cin. this observation is consistent with previous reports on the link between triglycerides and cin. though we studied cin, a precursor stage for cervical cancer, previous cohort studies among icelanders and austrians showed that increased serum triglyceride concentration is associated with an increased cervical cancer prevalence.41,42 it is important, however, to note that observations in cin cannot just be extrapolated to cervical cancer, even if one leads to the other. another case-control study among united states adult women revealed that serum triglyceride concentrations are correlated with increased risk of cancer of the cervix.40 dyslipidaemia is said to go hand in hand with other components of metabolic syndrome, including obesity, whose contribution to cervical cancer risk is not only reflected in elevated oestrogen levels43 but also in increased risk of hpv infection.44,45 metabolic syndrome components are said to act as cofactors in cervical carcinogenesis, which is based on the positive correlation of oestrogen and adipokine levels with cervical cancer.46,47 increased serum triglycerides and low hdl are said to be risk factors for cervical cancer.48 they are known to stimulate proliferation of cancer cells and induce anti apoptotic capacity by activation of reactive oxygen species.49 it is worth noting that a good fraction of our participants in both case and control groups reported a history of diabetes and hypertension. previous studies have demonstrated that hypertension results in hypoxia, which results in synthesis of hypoxia-induced factor-1, which in turn promotes angiogenesis.50 it is hypoxia-induced factor-1 that is said to support development of hpv-induced lesions.51 the fact that controls also had a history of hypertension and diabetes, therefore, emphasises the multifactorial requirement for development of cervical cancer other than hpv infection. this case-control study was designed with adequate power to derive associations. the other strength of this study was that we used internationally acceptable cut-off values for categorising dyslipidaemias. also, the presence of cin was confirmed with either pap smear or histology, the gold standard, following national guidelines. for controls, absence of lesions was ascertained by a negative visual inspection with acetic acid test result, the routine screening test as recommended by national guidelines. implications for clinical practice this study demonstrates the association between triglyceride dyslipidaemia and cin. with increasing prevalence of non-communicable diseases, patients would benefit from combined screening for cervical cancer as well as dyslipidaemia, preferably on a single visit. just as for cervical cancer screening, a test and treat approach would be more impactful if coupled with screening for dyslipidaemia. limitations a major limitation of this study was the fact that controls were not matched with cases. some confounders may still have had an effect on statistics. this further presents a shortcoming for case-control studies, in addition to their inability to ascertain causation. however, to the best of our knowledge, we gathered information on all factors known to be associated with cervical lesions and performed logistic regression analysis which generated a logistic regression model with all the factors included. we also expect our study to have suffered from selection bias as well as recall bias during participant enrollment. during collection of data, participants were required to remember information on a number of variables including history of diabetes and hypertension. these could have led to false conclusions about the study population since a lot of data may have been left out or improperly reported during data collection. however, for most of the variables that required participants to recall, we did our best to compare the provided information with participants’ charts. we also observe that the sample size used for this study was smaller than what is observed in other studies, despite the adequate power. this therefore might have a negative impact on the generalisability of results to the general population. conclusion we observed a significant association between dyslipidaemia, especially triglyceride dyslipidaemia, and cin. this underscores the importance of expanding screening efforts for dyslipidaemia to effectively manage potential complications and other non-communicable diseases associated with dyslipidaemia. furthermore, we advocate for prospective cohort studies, with larger sample sizes, to deepen our understanding of the relationship between dyslipidaemia and cin, as well as its broader implications for cervical cancer. adequate management of dyslipidaemia could stop progression of cin to cervical cancer. molecular studies could be carried out to understand the biochemical processes and interactions behind lipids and cervical carcinogenesis. acknowledgements we acknowledge the nurses, midwives and clinicians of mbarara regional referral hospital, especially the cervical cancer clinic. we also acknowledge the research assistants who participated in data collection. special acknowledgement goes to expert laboratories and the mbarara university of science and technology histopathology laboratory where lipid estimation and histopathology analysis were done, respectively. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions f.s. conceived the idea and developed the protocol and the first draft of the manuscript. co-authors t.c.r., d.t. and j.n. participated in protocol writing, and supervised from data collection to analysis. they reviewed and provided overall guidance in the entire write-up and approved the final version prior to submission. j.n.n., r.k., a.n. and j.m. participated in data collection and analysis, and reviewed and approved the final version of the manuscript. all authors are accountable for all aspects of this manuscript. sources of support research reported in this publication was supported by the fogarty international centre of the national institutes of health under award number d43tw011632. the content is solely the responsibility 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laboratory in western kenya john n. waitumbi, esther omuseni, josphat nyataya, clement masakhwe, faith sigei, allan lemtudo, george awinda, eric muthanje, brian andika, rachel githii, rehema liyai, gathii kimita, beth mutai african journal of laboratory medicine | vol 11, no 1 | a1737 | 22 july 2022 lessons from the field tuberculosis-loop-mediated isothermal amplification implementation in cameroon: challenges, lessons learned and recommendations valerie f. donkeng-donfack, suzanne m. ongoulal, yvonne j. djieugoue, yannick kamdem simo, henri manga, danielle a.d. tollo, edwige m.a. belinga, vincent mbassa, jean l. abena, sara eyangoh african journal of laboratory medicine | vol 11, no 1 | a1792 | 26 august 2022 review article beta-lactamase resistance genes in enterobacteriaceae from nigeria babafela b. awosile, michael agbaje, oluwawemimo adebowale, olugbenga kehinde, ezekiel omoshaba african journal of laboratory medicine | vol 11, no 1 | a1371 | 22 february 2022 review article microbiology laboratories involved in disease and antimicrobial resistance surveillance: strengths and challenges of the central african states passoret vounba, severin loul, ludovic f. tamadea, joël f.d. siawaya african journal of laboratory medicine | vol 11, no 1 | a1570 | 31 march 2022 review article low-level viraemia: an emerging concern among people living with hiv in uganda and across sub-saharan africa nicholus nanyeenya, noah kiwanuka, damalie nakanjako, gertrude nakigozi, simon p.s. kibira, susan nabadda, charles kiyaga, isaac sewanyana, esther nasuuna, fredrick makumbi african journal of laboratory medicine | vol 11, no 1 | a1899 | 20 october 2022 original research impact of pre-covid-19 epidemic preparedness on the trajectory of the pandemic in african countries talkmore maruta, sikhulile moyo african journal of laboratory medicine | vol 11, no 1 | a1571 | 31 march 2022 original research prevalence of alpha and beta haemolysin among blood group o donors in bamenda, cameroon victor n. fondoh, nobert ndzenjempuh, tamunjoh stella, richard m. fondoh, charles n. awasom, rebecca enow-tanjong, egbe p. egbengu, robert leke, njini f.n. rose, denis nsame african journal of laboratory medicine | vol 11, no 1 | a1432 | 19 april 2022 original research red cell distribution width as a surrogate marker of haemoglobinopathies in western kenya benard m. mutua, george sowayi, patrick okoth african journal of laboratory medicine | vol 11, no 1 | a1644 | 29 april 2022 36 42 49 59 69 74 83 89 page i of iv table of contents editorial cop27 climate change conference: urgent action needed for africa and the world lukoye atwoli, gregory e. erhabor, aiah a. gbakima, abraham haileamlak, jean-marie kayembe ntumba, james kigera, laurie laybourn-langton, robert mash, joy muhia, fhumulani m. mulaudzi, david ofori-adjei, friday okonofua, arash rashidian, maha el-adawy, siaka sidibé, abdelmadjid snouber, james tumwine, sahar yassien mohammad, paul yonga, lilia zakhama, chris zielinski african journal of laboratory medicine | vol 11, no 1 | a2080 | 04 november 2022 editorial point-of-care testing: connecting communities in africa and ensuring equity in access to health and diagnostics rajiv t. erasmus african journal of laboratory medicine | vol 11, no 1 | a2072 | 13 december 2022 opinion paper challenges and complexities in evaluating severe acute respiratory syndrome coronavirus 2 molecular diagnostics during the covid-19 pandemic lesley e. scott, lara d. noble, ashika singh-moodley, trish kahamba, diana r. hardie, wolfgang preiser, wendy s. stevens african journal of laboratory medicine | vol 11, no 1 | a1429 | 26 april 2022 opinion paper to break the cycle of covid-19 lockdowns and safely open up economies, we must ensure equitable access to diagnosis and treatment nqobile ndlovu, yenew k. tebeje, renuka gadde, trevor peter african journal of laboratory medicine | vol 11, no 1 | a1650 | 27 may 2022 opinion paper the time to address diagnostic needs in universal health coverage is now: leveraging the scale up of national testing capacity for hiv viral load and sars-cov-2 francesco marinucci, jens dhein african journal of laboratory medicine | vol 11, no 1 | a1685 | 22 june 2022 lessons from the field strengthening laboratory networks in the central africa region: a milestone for epidemic preparedness and response patrick a. njukeng, charles njumkeng, callistus ntongowa, mohammed abdulaziz african journal of laboratory medicine | vol 11, no 1 | a1492 | 19 may 2022 lessons from the field practical tips to using formalin-fixed paraffin-embedded tissue archives for molecular diagnostics in a south african setting barbara s. van deventer, lorraine du toit-prinsloo, chantal van niekerk african journal of laboratory medicine | vol 11, no 1 | a1587 | 23 june 2022 lessons from the field challenges with the pursuit of iso 15189 accreditation in a public health laboratory in ghana seth attoh, francis k.m. tetteh, mary mcaddy, kingsley ackah, richmond kyei, marcus moroti, cynthia boateng, laurinda adusu-donkor, joseph boafo, alhassan yakubu, sarah kwao, emmanuel sarkodie, nana-banyin koranteng, monica a. addo, frederick hobenu, kwasi agyeman-bediako, raymond d. fatchu african journal of laboratory medicine | vol 11, no 1 | a1448 | 19 july 2022 1 4 6 11 14 18 23 29 vol 11, no 1 (2022) issn: 2225-2002 (print) | issn: 2225-2010 (online)african journal of laboratory medicine http://www.ajlmonline.org open access table of contents original research appropriate use of plasma glucose tests for diagnosis of diabetes mellitus in ibadan, nigeria modupe a. kuti, olabisi t. bamidele, chioma t. udeh, bola j. eseile, olajumoke a. ogundeji african journal of laboratory medicine | vol 11, no 1 | a1433 | 29 april 2022 original research high-risk human papillomavirus-associated vulvar neoplasia among women living with human immunodeficiency virus in zambia fred maate, peter julius, stepfanie siyumbwa, leeya pinder, trevor kaile, mulindi mwanahamuntu, groesbeck parham african journal of laboratory medicine | vol 11, no 1 | a1563 | 12 may 2022 original research diagnostic challenges with accurate identification of listeria monocytogenes isolates from food and environmental samples in south africa teena s.m. thomas, juno thomas, karren le roux, sanelisiwe t. duze, faith mkhwanazi, adriano duse african journal of laboratory medicine | vol 11, no 1 | a1482 | 23 may 2022 original research south african study of blast phase chronic myeloid leukaemia: a poor prognostic outlook katherine e. hodkinson, nikki bouwer, jenifer vaughan african journal of laboratory medicine | vol 11, no 1 | a1578 | 31 may 2022 original research enhancing accreditation outcomes for medical laboratories on the strengthening laboratory management toward accreditation programme in kenya via a rapid results initiative ernest p. makokha, raphael o. ondondo, daniel k. kimani, thomas gachuki, frank basiye, mercy njeru, muthoni junghae, marie downer, mamo umuro, margaret mburu, jane mwangi african journal of laboratory medicine | vol 11, no 1 | a1614 | 31 may 2022 original research aetiology of pancytopenia: experience of a south african tertiary academic centre erica-mari nell, zivanai c. chapanduka african journal of laboratory medicine | vol 11, no 1 | a1645 | 31 may 2022 original research newly implemented community cd4 service in tshwaragano, northern cape province, south africa, positively impacts result turn-around time lindi-marie coetzee, naseem cassim, deborah k. glencross african journal of laboratory medicine | vol 11, no 1 | a1376 | 03 june 2022 original research childhood aplastic anaemia with paroxysmal nocturnal haemoglobinuria clones: a retrospective single-centre study in south africa candice l. hendricks, ashen naidoo, rajendra thejpal, nadine rapiti, beverley neethling, yasmin goga, suvarna buldeo african journal of laboratory medicine | vol 11, no 1 | a1537 | 06 june 2022 original research implementation of a customised antimicrobial resistance laboratory scorecard in cameroon, ethiopia and kenya andré trollip, renuka gadde, tjeerd datema, kamau gatwechi, linda oskam, zachary katz, andrew whitelaw, peter kinyanjui, patrick njukeng, dawit a. wendifraw, ibrahimm mugerwa, grace najjuka, nicholas dayie, japheth a. opintan, heidi albert african journal of laboratory medicine | vol 11, no 1 | a1476 | 20 june 2022 97 102 112 118 125 133 141 150 157 original research the application of sigma metrics in the laboratory to assess quality control processes in south africa marli van heerden, jaya a. george, siyabonga khoza african journal of laboratory medicine | vol 11, no 1 | a1344 | 22 june 2022 original research delays in hiv-1 infant polymerase chain reaction testing may leave children without confirmed diagnoses in the western cape province, south africa kamela l. mahlakwane, wolfgang preiser, nokwazi nkosi, nasheen naidoo, gert van zyl african journal of laboratory medicine | vol 11, no 1 | a1485 | 23 june 2022 original research the togo national proficiency test pilot programme for basic clinical chemistry tests kafui c. kouassi, améyo m. dorkenoo, komivi gbada, yaovi-gameli afanyibo, minogblon têko, adjane koura african journal of laboratory medicine | vol 11, no 1 | a1565 | 24 june 2022 original research implementing an intensified mentorship approach towards accelerated medical laboratory accreditation in 10 counties in kenya susan k. musau, christina mwachari, elvis kirui, junghae muthoni, taylor lascko, natalia blanco, alash’le abimiku, emily koech african journal of laboratory medicine | vol 11, no 1 | a1814 | 01 july 2022 original research cost of running a full-service receiving office at a centralised testing laboratory in south africa naseem cassim, neeshan ramdin, sadhaseevan moodly, deborah k. glencross african journal of laboratory medicine | vol 11, no 1 | a1504 | 13 july 2022 original research evaluation of fixed-panel, multicolour clearllab 10c at an academic flow cytometry laboratory in johannesburg, south africa deborah k. glencross, leanne swart, melanie pretorius, denise lawrie african journal of laboratory medicine | vol 11, no 1 | a1458 | 15 july 2022 original research formulation of phage cocktails and evaluation of their interaction with antibiotics in inhibiting carbapenemase-producing klebsiella pneumoniae in vitro in kenya noutin f. michodigni, atunga nyachieo, juliah k. akhwale, gabriel magoma, abdoul-salam ouédraogo, andrew n. kimang’a african journal of laboratory medicine | vol 11, no 1 | a1803 | 18 july 2022 original research occurrence and antimicrobial susceptibility patterns of salmonella species from poultry farms in ibadan, nigeria terese g. orum, olayinka o. ishola, oluwawemimo o. adebowale african journal of laboratory medicine | vol 11, no 1 | a1606 | 20 july 2022 original research therapeutic drug monitoring of phenytoin and valproic acid in critically ill patients at windhoek central hospital, namibia bonifasius s. singu, helen morrison, lydia irengeya, roger k. verbeeck african journal of laboratory medicine | vol 11, no 1 | a1628 | 21 july 2022 166 173 180 186 192 200 210 218 224 page ii of iv http://www.ajlmonline.org open access table of contents original research prevalence of hepatitis b virus core antibodies among blood donors in nigeria: implications for blood safety foluke a. fasola, adeola a. fowotade, adedayo o. faneye, adeyeni adeleke african journal of laboratory medicine | vol 11, no 1 | a1434 | 26 july 2022 original research efficacy of diversely isolated lytic phages against multi-drug resistant enterobacter cloacae isolates in kenya ivy j. mutai, angela a. juma, martin i. inyimili, atunga nyachieo, anthony k. nyamache african journal of laboratory medicine | vol 11, no 1 | a1673 | 11 august 2022 original research establishment of haemoglobin a2 reference intervals in pretoria, south africa: a retrospective secondary data analysis cailin nieuwenhuizen, tshiphiri netshidzivhani, johan potgieter african journal of laboratory medicine | vol 11, no 1 | a1841 | 12 august 2022 original research hepatitis b virus infection and hbeag positivity among pregnant women in south west uganda naome mugabiirwe, rogers kalyetsi, richard ayella, james obote, frank ssedyabane african journal of laboratory medicine | vol 11, no 1 | a1784 | 18 august 2022 original research bacteriological profile and antibiogram of blood culture isolates from bloodstream infections in a rural tertiary hospital in nigeria oluwalana t. oyekale, bola o. ojo, adewale t. olajide, oluwatoyin i. oyekale african journal of laboratory medicine | vol 11, no 1 | a1807 | 24 august 2022 original research genexpert rollout in three high-burden tuberculosis countries in africa: a review of pulmonary tuberculosis diagnosis and outcomes from 2001 to 2019 victor williams, marianne calnan, bassey edem, chukwuemeka onwuchekwa, chika okoro, christine candari, rhodora cruz, kennedy otwombe african journal of laboratory medicine | vol 11, no 1 | a1811 | 30 august 2022 original research global antimicrobial resistance and use surveillance system on the african continent: early implementation 2017–2019 barbara tornimbene, sergey eremin, reuben abednego, elamin o. abualas, ilhem boutiba, abiodun egwuenu, walter fuller, laetitia gahimbare, susan githii, watipaso kasambara, chileshe lukwesa-musyani, fidy a. miamina, sekesai mtapuri-zinyowera, grace najjuka, olga perovic, bassem zayed, yahaya a. ahmed, maha t. ismail, carmem l. pessoa da silva african journal of laboratory medicine | vol 11, no 1 | a1594 | 31 august 2022 original research effect of sputum quality and role of xpert® mtb/rif assay for detection of smear-negative pulmonary tuberculosis in same-day diagnosis strategy in addis ababa, ethiopia waganeh sinshaw, abebaw kebede, adane bitew, mengistu tadesse, zemedu mehamed, ayinalem alemu, bazezew yenew, misikir amare, biniyam dagne, getu diriba, ephrem tesfaye, dinka f. gamtesa, yeshiwork abebaw, helina mollalign, getachew seid, muluwork getahun african journal of laboratory medicine | vol 11, no 1 | a1671 | 31 august 2022 229 237 246 251 257 264 272 283 original research human herpes virus type-6 is associated with central nervous system infections in children in sudan nada a. abdelrahim, nahla mohamed, magnus evander, clas ahlm, imad m. fadl-elmula african journal of laboratory medicine | vol 11, no 1 | a1718 | 22 september 2022 original research audit of amylase and lipase requests in suspected acute pancreatitis and cost implications, south africa annie e. cook, thumeka p. jalavu, annalise e. zemlin african journal of laboratory medicine | vol 11, no 1 | a1834 | 26 september 2022 original research red blood cell alloimmunisation in multi-transfused patients from an haemodialysis service in burkina faso koumpingnin nebie, salam sawadogo, salifo sawadogo, jérôme koulidiati, habi y.a. lengani, abdoul g. sawadogo, jérôme babinet, mohammed khalloufi, saliou diop, eléonore kafando african journal of laboratory medicine | vol 11, no 1 | a1625 | 26 september 2022 original research comparison of commercial assays and two-step approach to detect clostridioides difficile in south africa sarishna singh, mae newton-foot, pieter nel, colette pienaar african journal of laboratory medicine | vol 11, no 1 | a1809 | 29 september 2022 original research molecular detection of hepatitis b virus genotype e with immune escape mutations in chronic hepatitis b patients on long-term antiviral therapy in jos, nigeria joseph anejo-okopi, edith okeke, pantong m. davwar, chika onwuamah, harris onywera, patience omaiye, mary duguru, ocheme j. okojokwu, otobo i. ujah, bulus jonathan, chima a. george, ramyil s. crown, fiyaktu b. yakubu, judith o. sokei, leona c. okoli, onyemocho audu, seth c. inzaule, isaac o. abah, patricia agaba, oche o. agbaji, atiene s. sagay, claudia hawkins african journal of laboratory medicine | vol 11, no 1 | a1677 | 18 october 2022 original research agreement between xpert and ampfire tests for high-risk human papillomavirus among hiv-positive women in rwanda anthere murangwa, kanan t. desai, julia c. gage, gad murenzi, patrick tuyisenge, faustin kanyabwisha, aimable musafili, gallican kubwimana, leon mutesa, kathryn anastos, hae-young kim, philip e. castle african journal of laboratory medicine | vol 11, no 1 | a1827 | 19 october 2022 original research optimising courier specimen collection time improves patient access to hiv viral load testing in south africa sarah j. girdwood, thomas crompton, naseem cassim, floyd olsen, portia sejake, karidia diallo, leigh berrie, dorman chimhamhiwa, wendy stevens, brooke nichols african journal of laboratory medicine | vol 11, no 1 | a1725 | 25 october 2022 original research practices and barriers to screening for hyperglycaemia in pregnancy among providers of antenatal care in jos, nigeria lucius c. imoh, abdulazis s. longwap, favour e. haruna, oghale j. asieba, joy p. istifanus, joy a. imoh, mathilda e. banwat african journal of laboratory medicine | vol 11, no 1 | a1845 | 31 october 2022 290 296 301 307 313 320 325 331 page iii of iv http://www.ajlmonline.org open access table of contents original research causes of death and post-mortem testing for sars-cov-2 in a tertiary hospital during the covid-19 pandemic in ghana edward asumanu, seth attoh, raymond x. servor, clement laryea, mary mcaddy, fred hobenu, raymond factchu, kwesi agyemang-bediako, edward o. nyarko, godwin k. nyarko, marcus k. moroti, lawrence edusei african journal of laboratory medicine | vol 11, no 1 | a1766 | 23 november 2022 original research malaria an opportunistic infection in hiv/aids patients? – a nigerian experience joseph n. enuma, felix o. sanni, malau b. matur, njab e. jean, tosan erhabor, iheukwumere i. egbulefu african journal of laboratory medicine | vol 11, no 1 | a1842 | 24 november 2022 original research oral human papilloma virus infection among dental clinic attendees in ibadan, nigeria adedayo o. faneye, oyeteju s. babalola, georgina n. odaibo, juwon arotiba, olufemi d. olaleye african journal of laboratory medicine | vol 11, no 1 | a1555 | 25 november 2022 original research impact of rapid centrifugation on routine coagulation assays in south africa reola haripersadh, dashini pillay, nadine rapiti african journal of laboratory medicine | vol 11, no 1 | a1901 | 28 november 2022 original research commercial duraclone panels for extending the repertoire of multicolour immunophenotypic panels in an academic flow cytometry laboratory in south africa leanne swart, melanie pretorius, denise lawrie, deborah k. glencross african journal of laboratory medicine | vol 11, no 1 | a1720 | 29 november 2022 340 348 354 359 366 scientific letter appropriate disposal of waste in the laboratory: neglected but not forgotten christoffel j. opperman, sarishna singh, francois barton african journal of laboratory medicine | vol 11, no 1 | a1786 | 14 july 2022 case study extranodal presentation of a lymphoma with precursor b-cell phenotype and translocation t(8;14) in south africa katherine e. hodkinson, yvonne perner, deborah k. glencross, tracey wiggill, adam botha, janet poole african journal of laboratory medicine | vol 11, no 1 | a1355 | 31 january 2022 brief report retrospective analysis of vitek®2 performance compared to manual broth micro-dilution for colistin susceptibility testing of acinetobacter baumannii complex isolates in south africa vuyolwethu fadana, teena thomas, nina von knorring african journal of laboratory medicine | vol 11, no 1 | a1597 | 28 february 2022 correction corrigendum: higher proportion of non-classical and intermediate monocytes in newly diagnosed multiple myeloma patients in egypt: a possible prognostic marker asmaa m. zahran, hanaa nafady-hego, sawsan m. moeen, hanan a. eltyb, mohammed m. wahman, asmaa nafady african journal of laboratory medicine | vol 11, no 1 | a1713 | 25 march 2022 reviewer acknowledgement african journal of laboratory medicine | vol 11, no 1 | a2096 | 19 december 2022 375 377 382 386 387 page iv of iv article information authors: rosemary a. audu1 rosemary n. okoye2 chika k. onwuamah1 fehintola a. ige1 adesola z. musa3 nkiruka n. odunukwe4 daniel i. onwujekwe4 oliver c. ezechi4 emmanuel o. idigbe1 phyllis j. kanki5 affiliations: 1human virology laboratory, nigerian institute of medical research, lagos, nigeria 2clinical diagnostic laboratory, nigerian institute of medical research, lagos, nigeria 3monitoring and evaluation unit, nigerian institute of medical research, lagos, nigeria 4clinical sciences division, nigerian institute of medical research, lagos, nigeria 5harvard school of public health, boston, massachusetts, united states correspondence to: rosemary audu email: rosemaryaudu@yahoo.com postal address: human virology laboratory, nigerian institute of medical research, 06 edmond crescent, pmb 2013 yaba, lagos, nigeria dates: received: 17 mar. 2014 accepted: 11 aug. 2015 published: 30 sept. 2015 how to cite this article: audu ra, okoye rn, onwuamah ck, et al. potential for false-positive hiv test results using rapid hiv testing algorithms. afr j lab med. 2015;4(1), art. #178, 5 pages. http://dx.doi.org/10.4102/ajlm.v4i1.178 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. potential for false-positive hiv test results using rapid hiv testing algorithms in this original research... open access • abstract • introduction • methods    • study setting    • national testing algorithm    • study design    • data abstraction and inclusion criteria    • laboratory methods    • statistical analyses    • ethical considerations • results • discussion    • limitations    • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: in order to scale up access to hiv counselling and testing in nigeria, an hiv diagnostic algorithm based on rapid testing was adopted. however, there was the need to further evaluate the testing strategy in order to better assess its performance, because of the potential for false positivity. objectives: the objective of this study was to compare positive hiv test results obtained from the approved rapid testing algorithm with results from western blot tests performed on samples from the same patient. methodology: a retrospective review was conducted of hiv screening and confirmatory results for patients seen between 2007 and 2008. rapid test and western blot results were extracted and compared for concordance. discordant results were further reviewed using a combination of hiv-1 rna viral load and cd4+ cell count test results and clinical presentation from medical records. results: analysis of 2228 western blot results showed that 98.3% (n = 2191) were positive for hiv-1, 0.4% (n = 8) were positive for hiv-2 and 0.3% (n = 7) were dual infections (positive for both hiv-1 and hiv-2); 0.6% (n = 13) were indeterminate and 0.4% (n = 9) were negative. further investigation of the 13 indeterminate results showed nine to be hiv-1 positive and four to be hiv-negative, for a total of 13 negative results. the positive predictive value of the hiv counselling and testing algorithm was 99.4%. conclusion: using the rapid testing algorithm alone, false positives were detected. therefore, effective measures such as training and retraining of staff should be prioritised in order to minimise false-positive diagnoses and the associated potential for long-term psychological and financial impact on the patients. introduction top ↑ the diagnosis of hiv infection is most often based on the detection of serum antibodies to hiv. these serological tests could be classified either as screening tests, such as rapid tests or enzyme linked immuno-sorbent assay (elisa), or confirmatory tests, such as western blot (wb). prior to the availability of rapid testing, same-day results were not obtainable and an estimated one third of individuals tested did not return to learn their hiv status.1 compared with elisa and wb, rapid test assays are cheaper, easier to perform and the results are readily available on the same day. in 1999, the world health organization and the joint united nations programme on hiv/aids recommended the use of a combination of rapid test assays or elisas to confirm positive results, employing a highly sensitive test as the first screening test and a second, highly specific confirmatory test to verify the detection of antibodies specific to hiv. this was considered to be as reliable for confirmation as wb but at a much lower cost.2 in 2007, the government of nigeria adopted a serial testing algorithm with three combinations of the following rapid test assays: determine® (abbott, tokyo, japan), stat-pak® (chembio diagnostic systems, medford, new york, united states) or uni-gold™ (trinity biotech, jamestown, new york, united states). samples with discordant results were further tested with bundi rt (bundi international diagnostics ltd, aba, nigeria) as the tiebreaker.3 the adopted algorithm could be used in any of the combinations shown in table 1. because of quality issues with the bundi test kits, the use of the kit was suspended; the third combination was retained and is currently in use in the country. table 1: algorithm adopted in 2007 for hiv rapid testing in nigeria. as with all screening assays, hiv rapid test screening has a certain degree of false-positive results, irrespective of the algorithm in use.4 for this reason, a confirmatory test is necessary for a definitive diagnosis. evaluations of hiv rapid testing strategies have been reported in some african countries where the results have been used to formulate alternative hiv testing strategies.5,6 regardless of which rapid test algorithm is used, false-positive results may still occur;7,8 1% – 2% of reactive rapid test results have been found to be negative with an hiv nucleic acid test.7 since the introduction of the 2007 hiv rapid test strategy in nigeria, its performance has not been evaluated. therefore, the assessment of the algorithm’s performance remains an important priority to better inform national policy and ensure accurate hiv diagnosis and surveillance. the aim of this study was to compare positive hiv test results obtained from the approved rapid testing algorithm with results from wb tests performed on samples from the same patient. methods top ↑ study setting the study was conducted at the nigerian institute of medical research (nimr), lagos, a parastatal of the federal ministry of health. nimr has an hiv treatment centre that currently provides comprehensive hiv care, antiretroviral treatment (art) and support for over 18 000 patients. the centre is supported by the government of nigeria and the us president’s emergency plan for aids relief-funded programme for art; as a result, all services are provided free to patients. patients who screen positive for hiv at the hiv counselling and testing (hct) centre or any government of nigeria-approved hiv counselling and testing centre are referred to the treatment programme for further management. these patients are clinically assessed and sent to the nimr virology laboratory for baseline laboratory assessment. the nimr virology laboratory is an iso 9001:2008 certified laboratory and employs well trained and competent personnel to perform the laboratory assays. quality assurance measures undertaken by the laboratory include: participation in external quality assessment for all assays; equipment maintenance; and several measures to verify pre-analytical, analytical and post-analytical processes. national testing algorithm the approved national serial testing algorithm used for this study was: determine, unigold and stat-pak as tie breaker. this algorithm is currently in use in nigeria; thus, the results of this study are relevant to the current situation in the country. study design this was a retrospective study, which analysed data from the programme’s electronic database. cases with discordant results were further reviewed using a combination of plasma viral load, cd4+ cell counts and clinical data to determine a presumptive hiv diagnosis or progression of infection. hiv test results generated between january 2007 and august 2008 were analysed. discordant results were reviewed until december 2011. data abstraction and inclusion criteria data were abstracted from records of patients aged 18 years or older who had tested positive for hiv using the 2007 nigerian rapid test algorithm at the nimr hct, satellite sites or private clinics. data for patients sent to the nimr virology laboratory for confirmation of hiv diagnosis by wb were abstracted for this study. data from all patients who had given informed consent to participate in research studies were included. wb test result data included all positive, negative and indeterminate results. indeterminate results were defined as reactivity profiles that were not compatible with either a positive or a negative interpretation. laboratory methods the nimr hct site conducted hiv testing using the national testing algorithm; this required the use of non-cold chain dependent rapid test kits supplied by the government of nigeria through the central medical stores. the satellite sites and private clinics who referred hiv-positive patients for care and treatment also indicated the rapid test kits used in obtaining the positive hiv results. at the nimr virology laboratory, the baseline assessment included: hiv confirmation using the wb technique (immunetics inc., boston, massachusetts, united states) to detect specific viral proteins for both hiv-1 and hiv-2; cd4+ cell count (cells/µl) using the flow cytometry technique employed by the partec cyflow counter ii (gorlitz, germany) to determine eligibility for the initiation of art; and quantitation of hiv-1 rna viral load in plasma (roche amplicor v1.5, mannheim, germany) with a lower limit of detection of 400 copies/ml. the viral load assay was further used to monitor the effectiveness of therapy and for timely identification of poor adherence and possible virologic failure. statistical analyses the positive predictive value was calculated based on the results of the rapid tests and wb using standard formulae. exact 95% confidence intervals for these proportions were calculated. analyses were conducted using statistical package for social sciences version 17 for windows (ibm corporation, chicago, illinois, united states). ethical considerations ethical approval for this study was obtained from the nimr and harvard school of public health institutional review boards. written informed consent (dated 14 november 2006) was obtained prior to enrolment of patients for both services and research. results top ↑ results from 2228 different patients meeting the inclusion criteria were extracted from the programme’s electronic database. there was a male: female ratio of 1.9:1 and the mean age was 38.8 ± 9.0 years. the nimr hct facility accounted for 967 (43.4%) of the results abstracted, whereas 1261 (56.6%) were from satellite sites and private clinics. the analyses of the wb results showed that 98.3% were positive for hiv-1, 0.4% were positive for hiv-2 and 0.3% had hiv-1 and hiv-2 dual infections. there were also 13 (0.6%) indeterminate and nine (0.4%) negative results (table 2). the 13 indeterminate results were further investigated using plasma viral load levels and nine (69.2%) were found to be infected with hiv-1 based on very high hiv-1 viral load levels (median = 310 377 rna copies/ml; range = 2150–991 706 rna copies/ml), whereas four (30.8%) were considered to be hiv-negative based on undetectable viral load results (<400 rna copies/ml). thus, a total of 13 results that had been positive according to the rapid test algorithm were actually negative. this yielded a false-positive rate of 0.6%. table 2: hiv-positive rapid test results obtained by western blot technique, nimr, january 2007 − august 2008. clinical records of cases with negative wb results were reviewed. after a minimum of 12 months with no clinical evidence of infection or laboratory evidence of hiv disease, these cases were discontinued from the clinic. the four cases with indeterminate wb results and undetectable viral load results were monitored for an additional two years. in the absence of any clinical manifestation of hiv infection and repeat undetectable viral load results, they were discharged from the clinic. taking both the true positives and false positives into consideration, the positive predictive value was calculated to be 99.4% (99.0% – 99.6% ci). the satellite sites and private clinics were responsible for 46.2% (6/13) of the false-positive results. upon further investigation, we found that all patients at these facilities (100%) had been screened using determine and stat-pak test kits, including those with false-positive results. since both kits produced concordant results, a third kit was not required as a tie breaker for any of these cases. the immunological and virological data of these false positives were analysed and it was found that their median cd4+ cell count was 668 cells/µl (range = 50–1500 cells/µl) and all had an undetectable viral load (< 400 rna copies/ml). discussion top ↑ rapid hiv antibody tests help to improve access to testing in both clinical and nonclinical settings, as well as increasing the proportion of people who receive their results once tested. as good as the rapid hiv screening assays may be, a confirmatory test is still required for a definitive diagnosis because of the possibility of false-positive results. false positives may result from numerous causes, including the inherent characteristics of the assay, or may be because of the genetic diversity of the virus.4 the percentage of false positivity reported in this study was 0.6%, which is similar to that reported in other studies,3,9 but is lower than the 2% reported in cameroon.10 as part of nigeria’s global aids response progress report in 2012, 11.7% of the population of 162 265 000 had received an hiv test within the last 12 months and knew their hiv status.11 this corresponds to 18 985 005 tests performed in nigeria within the year. given the false-positive rate of 0.6% found in this study, approximately 113 910 people per year who are not hiv-positive receive a false hiv-positive diagnosis. the us president’s emergency plan for aids relief reported the yearly cost of management of an hiv patient to be us $338.12 these 113 910 false-positive diagnoses could result in us $38.5m per year in inappropriate but significant costs for hiv management. these resources could be better used for the estimated 1 512 720 hiv-infected individuals still requiring art. possible ways to identify patients with false-positive results include: lack of risk factors for hiv, normal cd4+ cell count and undetectable viral load.13 in nigeria, the cd4+ cell count reference value has been reported to be 365–1571 cells/µl.14 causes of false-positive results include: fictitious hiv exposure/infection15 and hiv vaccination,16 the latter being unlikely because hiv vaccine trials have not been conducted in nigeria. other likely reasons for false-positive results could be either clerical or technical. the possibility of these errors in the nimr virology reference laboratory is low because of the laboratory’s stringent quality management system. none of the laboratories that carried out the hct for the results included in this study had implemented a quality management system. this is not unusual, considering the dearth of knowledge about quality management systems in most laboratories in africa, including nigeria.17 the inconsistent or incorrect use of the rapid test algorithm has also been reported as a possible cause of false-positive results.18 in addition, false-positive results could also be a result of poor algorithm planning or implementation.19 the incorrect interpretation of weak-positive test lines and non-usage of tie-breaker algorithms, which occurs in our setting, could explain the latter. the use of confirmatory wb allowed for the diagnosis of hiv-1 and hiv-2, between which these particular rapid test assays cannot discriminate. as seen in previous studies,20 hiv-1 was the major hiv type amongst patients accessing hiv care in our clinic. the very low rate of hiv-2 and hiv-1/2 dual infections observed in this study could be attributed to the decline in its prevalence, as reported in other west african countries,21 resulting in part from the inefficiency of transmission of hiv-2.22 in this study, men had a higher uptake of testing than women. this is similar to observations made between 2003 and 2007 (about the same timeframe as this study), but by 2011, this finding had been reversed.11 this is most likely attributable to the increased availability of prevention of mother-to-child transmission treatment, as well as treatment including paediatric therapy. the delivery of a false-positive hiv result to a patient can be devastating, as it often results in psychological stress and trauma, particularly with subsequent stigmatisation. in a study from three african countries, namely, the democratic republic of congo, burundi and ethiopia, some patients who were falsely diagnosed as hiv-positive had been abandoned by their partners after they started on art or prophylaxis.23 there are also substantial financial costs related to misdiagnosis, where patients might lose time and compensation from work in order to attend clinic visits. in addition, the possible side effects and toxicities of unnecessary art need to be considered. in our study, patients with false-positive results were found to have high median cd4+ cell counts and undetectable viral loads. neither of these conditions can rule out the possibility of hiv infection, particularly if patients are natural elite controllers24 or receiving art. it has also been reported that there are apparently healthy nigerians who have cd4+ cell counts below 350 cells/µl.17,25 it is important to note that the cost of side effects, psychological trauma and lifelong treatment is unquantifiable. there have been reports of suicides resulting from a diagnosis of hiv,26 hence the gravity of false-positive results should be well-recognised. it is therefore essential that great care and caution is exercised in hiv diagnosis. in spite of the challenge of false-positive results, the use of rapid test kits doubled the proportion of individuals who had been tested and received their test results between 2003 and 2007.11 the calculated positive predictive value of 99.4% in this study is similar to the established national positive predictive value of 99.5% (96.8% – 99.9% ci) based on the national algorithm’s sensitivity of 100% and specificity of 99.7%.3 this implies that the use of this algorithm is still very essential in the expansion of hiv testing in a highly-populated country such as nigeria, if the spread of this virus is to be controlled. limitations this study is, however, not without limitations. the sensitivity, specificity and negative predictive values could not be estimated, because only hiv-positive patients were referred to the treatment centre for further follow up, including the wb re-testing. in like manner, not all cases referred from the hct centres eventually reported to the treatment centre. it is also important to note that although wb is a confirmatory test, it does have a window period of six weeks; thus, its usefulness for the detection of acute infections is limited,27,28 as there is a possibility of missing early infections. although rapid test kits also have a window period, the higher sensitivity assay is used initially, then followed by a very specific test. the long follow up of patients with discordant test results and repeat testing should have reduced the effect of the six-week window period. the wb assay also generates some indeterminate results. however, an indeterminate result is still acceptable in comparison to a false-positive result, as it allows for further evaluation.29 conclusion in this study, we found a 0.6% rate of hiv false positives with the implementation of the 2007 nigerian hiv rapid testing algorithm. it is therefore recommended that clinicians should refer suspicious false-positive patients for wb confirmation. all possible measures should be taken to prevent false-positive diagnoses associated with rapid testing alone, as the long-term costs of treatment, side effects and psychological trauma to patients may be considerable. acknowledgements top ↑ we are grateful to the harvard/aids prevention initiative in nigeria (apin) us president’s emergency plan for aids relief (pepfar) programme, which provided the financial support for this project. this work was supported, in part, by the us department of health and human services, health resources and services administration (u51ha02522). the contents are solely the responsibility of the authors and do not represent the official views of the funding institutions. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions r.a.a (nigerian institute of medical research) was the project leader. r.n.o., c.k.o. and f.a.i. (nigerian institute of medical research) performed most of the experiments and retrieval of records. a.z.m. (nigerian institute of medical research) was the data manager and responsible for data analysis. n.n.o., d.i.o. and o.c.e. (nigerian institute of medical research) were responsible for clinical management and manuscript preparation. e.o.i. (nigerian institute of medical research) made conceptual contributions, whilst p.j.k. (harvard school of public health) was responsible for project design and manuscript preparation. references top ↑ us centers for disease control and prevention, advancing hiv prevention: new strategies for a changing epidemic – united states, 2003. mmwr. 2003;52(15):329–332. unaids/world health organization. operational characteristics of commercially available assays to determine antibodies to hiv-1 and/or hiv-2 in human sera. report 11. geneva: world health organization; 1999. federal ministry of health, nigeria. laboratory based hiv rapid test validation in nigeria, phase 1, april 2007. nigeria: the nigeria hiv rapid test evaluation working group; 2007. cordes rj, ryan me. pitfalls in hiv testing. application and limitations of current tests. postgrad med. 1995;98(5):177–180, 185–186, 189. lyamuya ef, aboud s, urassa wk, et al. evaluation of simple rapid hiv assays and development of national rapid hiv test algorithms in dar es salaam, tanzania. bmc infect dis. 2009;9:19. http://dx.doi.org/10.1186/1471-2334-9-19 andersson s, da silva z, norrgren h, et al. field evaluation of alternative testing strategies for diagnosis and differentiation of hiv-1 and hiv-2 infections in an hiv-1 and hiv-2-prevalent area. aids. 1997;11(15):1815–1822. http://dx.doi.org/10.1097/00002030-199715000-00005 wesolowski lg, delaney kp, meyer wa, et al. use of rapid hiv assays as supplemental tests in specimens with repeatedly reactive screening immunoassay results not confirmed by hiv-1 western blot. j clin virol. 2013;58(1):240-244. http://dx.doi.org/10.1016/j.jcv.2013.06.019 baveewo s, kamya mr, mayanja-kizza h, et al. potential for false positive hiv test results with the serial rapid hiv testing algorithm. bmc res notes. 2012;5:154. http://dx.doi.org/10.1186/1756-0500-5-154 anzala o, sanders ej, kamali a, et al. sensitivity and specificity of hiv rapid tests used for research and voluntary counselling and testing. east afr med j. 2008; 85(10):500–504. aghokeng af, mpoudi-ngole e, dimodi h, et al. inaccurate diagnosis of hiv-1 group m and o is a key challenge for ongoing universal access to antiretroviral treatment and hiv prevention in cameroon. plos one. 2009;4(11):e7702. http://dx.doi.org/10.1371/journal.pone.0007702 national agency for the control of aids, federal republic of nigeria global aids response, country progress report [document on the internet]. c2012 [cited 2014 sep 12]. available from: http://www.unaids.org/en/dataanalysis/knowyourresponse/countryprogressreports/2012countries/nigeria2012garprreportrevised.pdf [updated link viewed 2015 sep 07, from: http://www.unaids.org/sites/default/files/en/dataanalysis/knowyourresponse/countryprogressreports/2012countries/nigeria%202012%20garpr%20report%20revised.pdf] holmes cb, atun r, avila c, et al. expanding the generation and use of economic and financial data to improve hiv program planning and efficiency: a global perspective. j acquir immune defic syndr. 2011;57(suppl 2):s104–s108. http://dx.doi.org/10.1097/qai.0b013e31821fa12d mylonakis e, paliou m, greenbough tc, et al. report of a false-positive hiv test result and the potential use of additional tests in establishing hiv serostatus. arch intern med. 2000;160 (15):2386–2388. http://dx.doi.org/10.1001/archinte.160.15.2386 oladepo dk, idigbe eo, audu ra, et al. establishment of reference values of cd4 and cd8 lymphocyte subsets in healthy nigerian adults. clin vaccine immunol. 2009;16(9):1374–1377. http://dx.doi.org/10.1128/cvi.00378-08 craven de, steger ka, la chapelle r, et al. factitious hiv infection: the importance of documenting infection. ann intern med. 1994;121(10):763–766. http://dx.doi.org/10.7326/0003-4819-121-10-199411150-00006 belshe rb, clements ml, keefer mc, et al. interpreting hiv serodiagnostic test results in the 1990s: social risks of hiv vaccine studies in uninfected volunteers. niaid aids vaccine clinical trials group. ann intern med. 1994;121(8):584–589. http://dx.doi.org/10.7326/0003-4819-121-8-199410150-00005 gershy-damet g, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. http://dx.doi.org/10.1309/ajcptuuc2v1wjqbm aniedobe mn, onwuamah ck, onubogu cc, et al. proficiency of private laboratories in diagnosis of hiv in nigeria. oral presentation at the 18th union conference, africa region, 3rd – 5th march 2011. available from: http://www.afro.who.int/en/nigeria/press-materials/item/2884-the-18th-conference-of-the-international-union-against-tuberculosis-and-lung-disease-iuatld-african-region-held-from-3-5th-march-2011-abuja-nigeria.html klarkowski d, o’brien dp, shanks l, et al. causes of false-positive hiv rapid diagnostic test results. expert rev anti infect ther. 2014;12(1):49–62. http://dx.doi.org/10.1586/14787210.2014.866516 kashyap b, gautam h, chadha s, et al. delayed progression and inefficient transmission of hiv-2. southeast asian j trop med publ health. 2010;41(3): 570–573. madec y, boufassa f, porter k, et al. spontaneous control of viral load and cd4 cell count progression among hiv-1 seroconverters. aids. 2005;19(17):2001–2007. http://dx.doi.org/10.1097/01.aids.0000194134.28135.cd us centers for disease control and prevention. interpretation and use of the western blot assay for serodiagnosis of human immunodeficiency virus type 1 infections. mmwr. 1989;38(suppl 7):1–7. owen sm, yang c, spira t, et al. alternative algorithms for human immunodeficiency virus infection diagnosis using tests that are licensed in the united states. j clin microbiol. 2008;46(5):1588–1595. http://dx.doi.org/10.1128/jcm.02196-07 shanks l, klarkowski d, o’brien dp. false positive hiv diagnoses in resource limited settings: operational lessons learned for hiv programmes. plos one. 2013;8(3):e59906. http://dx.doi.org/10.1371/journal.pone.0059906 audu ra, idigbe eo, akanmu as, et al. values of cd4+ t lymphocyte in apparently healthy individuals in lagos, nigeria. ejsr. 2007;16(2):168–173. keiser o, spoerri a, brinkhof mw, et al.; swiss hiv cohort study; swiss national cohort. suicide in hiv-infected individuals and the general population in switzerland, 1988–2008. am j psychiatry. 2010;167(2):143–150. http://dx.doi.org/10.1176/appi.ajp.2009.09050651 olaleye od, bernstein l, ekweozor cc, et al. prevalence of human immunodeficiency virus types 1 and 2 infections in nigeria. j infect dis. 1993;167(3):710–714. http://dx.doi.org/10.1093/infdis/167.3.710 bock p, markovitz d. infection with hiv-2. aids. 2001;15(suppl 5):s35–s45. http://dx.doi.org/10.1097/00002030-200100005-00006 ngan ccl, thoe sys, chan kp, et al. alternative strategies for confirmation of human immunodeficiency virus infection require judicious use. j. clin. microbiol. 2002;40(1):314–315. http://dx.doi.org/10.1128/jcm.40.1.314-315.2002 abstract introduction methodology results discussion acknowledgements references about the author(s) yahaya mohammed department of medical microbiology and parasitology, faculty of basic clinical sciences, college of health sciences, usmanu danfodiyo university, sokoto, nigeria aaron o. aboderin department of medical microbiology and parasitology, college of health sciences, obafemi awolowo university, ile-ife, nigeria iruka n. okeke department of pharmaceutical microbiology, faculty of pharmacy, university of ibadan, ibadan, nigeria adebola t. olayinka department of medical microbiology and parasitology, faculty of medicine, ahmadu bello university, zaria, nigeria citation mohammed y, aboderin ao, okeke in, olayinka at. antimicrobial resistance of vibrio cholerae from sub-saharan africa: a systematic review. afr j lab med. 2018;7(2), a778. https://doi.org/10.4102/ajlm.v7i2.778 review article antimicrobial resistance of vibrio cholerae from sub-saharan africa: a systematic review yahaya mohammed, aaron o. aboderin, iruka n. okeke, adebola t. olayinka received: 30 jan. 2018; accepted: 27 sept. 2018; published: 06 dec. 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the world health assembly adopted the global action plan on antimicrobial resistance, which includes improving the knowledge base through surveillance and research. noteworthily, the world health organization has advocated a global antimicrobial resistance surveillance system to address the plan’s surveillance objective, with most african countries enrolling in or after 2017. aim: the aim of this article was to review prior data on antimicrobial resistance of vibrio cholerae from sub-saharan africa with a view for future control and intervention strategies. methods: we used the preferred reporting items for systematic review and meta-analysis (or ‘prisma’) guidelines to search the pubmed and african journals online databases, as well as additional articles provided by the nigeria centre for disease control, for articles reporting on the antibiotic susceptibility of v. cholerae between january 2000 and december 2017. results: we identified 340 publications, of which only 25 (reporting from 16 countries within the sub-saharan african region) were eligible. the majority (20; 80.0%) of the cholera toxigenic v. cholerae isolates were of the serogroup o1 of the el tor biotype with ogawa and inaba serotypes predominating. resistance was predominantly documented to trimethoprim-sulphamethoxazole (50% of the studies), ampicillin (43.3% of the studies), chloramphenicol (43.3% of the studies) and streptomycin (30% of the studies). resistance mechanisms were reported in 40% of the studies. conclusion: our results demonstrate a documented antimicrobial resistance of v. cholerae to multiple antibiotic classes, including cell wall active agents and antimetabolites with evidence of phenotypic/genotypic resistance to fluoroquinolones. introduction vibrio cholerae are gram-negative and curved bacilli. certain members of this species are associated with a severe acute watery diarrhoea that is the most distinctive sign of a clinical condition called cholera.1 there exist numerous v. cholerae serogroups, but the o1 and o139 strains stand out as the major agents for the major outbreaks of cholera globally. the serogroup o139 is majorly restricted to some parts of asia; however, serogroup o1 v. cholerae, further subdivided into the el tor and classical biotypes, are distributed worldwide.2 the v. cholerae o1 el tor biotype was responsible for the seventh cholera pandemic, which started in indonesia and spread rapidly to bangladesh, india, iran and iraq.3 cholera was imported to africa in the 1970s from these countries during this seventh pandemic. it entered from west africa from where it spread to east, central and south africa.4 v. cholerae virulence and drug resistance evolved during the course of the seventh pandemic and a new variant cholera biotype emerged.2 this variant is called the ‘hybrid’ or ‘atypical’ biotype and it has mixed markers of the classical and el tor biotypes. hybrid v. cholerae have the el tor biotype, but with the non-el tor ctxb toxigenic allele. this atypical el tor biotype is associated with higher virulence and more widespread antibiotic resistance.2 atypical v. cholerae carry mobile genetic elements like the integrative/conjugative elements (ices), which are capable of self-transfer and integration into host chromosomes, facilitating rapid spread and stable acquisition.5 recently, the occurrence of new variant pathogenic strains of v. cholerae has been attributed to new ctx prophage rearrangements.6 resistant v. cholerae have disseminated globally and now threaten the effective treatment and control of cholera, especially in the low and middle-income countries.1,5 recent evidence suggests that cholera is exacting a very high burden on the african continent in this era.6 however, few data are available about the nature and extent of outbreaks or the properties of strains.7 multi-country or global studies generally have less input from africa.8 upon commissioning from the nigeria centre for disease control (ncdc), we set out to review data on the antimicrobial resistance of v. cholerae from publications done in sub-saharan africa, in order to provide evidence that may serve as a yardstick for future control programmes and interventions. methodology overview of study protocol this systematic review was done using the preferred reporting items for systematic reviews and meta-analysis (or ‘prisma’) guidelines.9 the protocol for the study was developed in conjunction with the ncdc panel of experts and a more detailed version of the protocol is available on request. search strategy we searched medline using pubmed for articles published in english between 01 january 2000 and 31 december 2017 with the search terms ‘antimicrobial resistance’, ‘antibiotic resistance’, ‘vibrio cholerae’ and the names of the individual countries of sub-saharan africa. additional searches were done in the african journals online database, using an additional search term of ‘antimicrobial susceptibility’. we also scanned a list of articles obtained from the ncdc to select eligible articles that conformed with our search terms. study selection criteria articles were included for this review provided they reported on the antimicrobial susceptibility profile of v. cholerae isolates from clinical specimens in sub-saharan africa and were published between january 2000 and december 2017. we included articles irrespective of whether the isolates were obtained as part of an outbreak investigation or from a hospital-based site using cross-sectional survey, provided they were from human specimens. selection procedure the titles and abstracts of all search results were listed and were thereafter reviewed to identify papers for full text review. the selection procedure is outlined in figure 1. forty-five papers were excluded, because all attempts to secure full text versions were unsuccessful. names of authors from articles were not blinded before or after the full text review. we used predetermined inclusion and exclusion criteria to select papers for full review. papers selected for full review after abstract review were retrieved as full manuscript papers through pubmed, hinari, from the ncdc or by personal communication with the corresponding authors. seventy-six review articles were excluded, and 45 papers did not report on the susceptibility pattern of v. cholerae and were also excluded. twenty-eight papers were on environmental samples; hence, they were excluded. nineteen articles were not from sub-saharan africa and 15 articles evaluated susceptibility to plant extracts and, consequently, they were all excluded. eight articles were in french and six articles used isolates from animal sources and they were all excluded. figure 1: summary of study selection procedure. data extraction a database was created in which the study name, study period, susceptibility pattern, biochemical properties, genes and virulence factors of the v. cholerae isolates from countries of sub-saharan africa were recorded where applicable (table 1). we could not adequately carry out a quantitative study, because most of the susceptibility patterns of the isolates were not recorded in actual numerical values. most of the studies also did not report on the quality control procedure they used and some of the studies relied only on molecular detection of resistance genes. table 1: characterisation of eligible articles that sought resistance of vibrio cholerae from countries in sub-saharan africa. attempt to reduce bias an attempt to reduce bias within studies and between individual studies was done. the review was also conducted in a group with materials, articles and the papers double-checked by at least two members of the group. analysis approach the extracted data were reported as outlined by the authors using the susceptibility categorisation of the pathogens into sensitive, intermediate and resistant. we reported on the biochemical characteristics of the v. cholerae in terms of serogroup, biotype and serotype. we also reported on the clinical diagnosis for isolates in studies where diagnosis was stated. we also highlighted the use of molecular methods, genotyping or virulence features for the characterisation of isolates wherever such data were available. results general characteristics of the studies included in the analysis our search generated 267 articles after removal of duplicates. during abstract review, we excluded 242 articles, because they did not meet our inclusion criteria. twenty-five articles were included in the final analysis. one article reported resistance of v. cholerae from five sub-saharan african countries and was therefore divided into five for convenience. in all, the articles obtained reported on 16 of the 47 countries within the sub-saharan african region. one study (3.4% for each) was obtained from each of the following countries: angola, chad, madagascar, namibia, senegal, south africa and togo. two studies (6.9% for each) were from each of cote d’ivoire, democratic republic of the congo, ghana, guinea bissau, mozambique, tanzania and zambia. three (10.3%) studies were from kenya and five (17.2%) were from nigeria. twenty-four (82.8%) of the studies reported serogroup o1 as the only serogroup, while two (6.9%) studies reported the o1 serogroup coexisting with the non-o1/non-o139 serogroup, but even in those studies the o1 serogroup dominated. three (10.3%) studies did not report on the serogroup status. none of the studies reported the o139 serogroup from any country in the region of sub-saharan africa. twenty two (75.9%) studies reported the el tor biotype, while one (3.4%) study reported the existence of the el tor and the atypical el tor biotype. six (20.7%) of the studies did not biotype their isolates. there was no report of the classical biotype from any of the studies. eight (27.6%) studies reported the ogawa serotype as the predominant serotype, three (10.3%) studies reported on inaba existing alone and six (20.7%) reported the ogawa/inaba coexisting together. the coexistence of inaba/ogawa/hikojima was reported from one (3.4%) study. eleven (37.9%) of the studies did not report on any of the serotypes. seventeen studies detected or confirmed cholera toxin and toxin-co-regulated pilus genes (ctxb, ctxa and tcpa). table 1 shows the characteristics of eligible articles that investigated resistance of v. cholerae from sub-saharan africa. resistance was documented to trimethoprim-sulphamethoxazole (50% of the studies), ampicillin (43.3%), chloramphenicol (43.3%), streptomycin (30%), nalidixic acid (30%), nitrofurantoin (26.7%), ceftriaxone (20%), spectinomycin (10%), sulfonamide (6.7%), penicillin g (6.7%) and cloxacillin (3.3%). the antibiotics to which susceptible strains were reported were: tetracycline (46.7% of the studies), amoxicilin/clavulanic acid (6.7%), florfenicol (3.3%), azithromycin (3.3%), imipenem (3.3%), ciprofloxacin (3.3%), ofloxacin (3.3%) and erythromycin (3.3%). mutations in antibiotic resistance determinants (gyra, parc, flor, stra, and strb) were detected in nine (31.1%) of the studies, while twenty (68.9%) studies did not conduct genotypic studies on the isolates. the icevchang2 and icevchind5 were reported from seven (24.1%) of the studies while the other twenty-two (75.9%) studies did not perform this genotypic analysis. summary of the resistance studies on the vibrio cholerae isolates from the various studies the average prevalence of resistance to cell wall active agents by the v. cholerae organisms from 20 studies was 68.8% (100–0%). however, 25 studies reported on the resistance to fluoroquinolones with their total average of 44.0% (100–0%), while the average prevalence of resistance to inhibitors of nucleic acid, predominantly the sulphonamide and co-trimozaxole, was 92.0% from 25 studies (table 1). twenty-seven studies reported 43.5% (100–0%) prevalence of resistance to protein synthesis inhibitors of 30s subunit, while the average prevalence of resistance to protein synthesis inhibitors of 50s subunit (table 1) was 62.5% (100–0%) from 26 eligible studies. specific characteristics of the studies included in the analysis in angola, ceccarelli et al. performed a retrospective study on v. cholerae o1 el tor strains responsible for the 2006 outbreak. the isolates were resistant to all the major groups of antimicrobials tested and they demonstrated the appearance of a novel v. cholerae epidemic variant in africa with a new ctxφ arrangement previously described only in the indian subcontinent.6 kaas et al. investigated the 2010/2011 v. cholerae outbreak in lake chad basin around cameroon. the outbreak strains were resistant to all the antimicrobials tested and, in addition, possessed the integrative conjugative element icevchind5. this is said to be clonal and clustered, distant from the other african strains.10 kacou-n’douba et al. documented resistance to chlorampenicol and cotrimoxazole during a cholera epidemic in 2011 from cote d’ivoire.11 smith and colleagues characterised v. cholerae o1 from cote d’ivoire, democratic republic of the congo, guinea bissau, mozambique and namibia. all the isolates were of ogawa serotype and positive for the ctxa gene. there was generalised resistance to nalidixic acid, chloramphenicol and cotrimoxazole in all isolates from the five countries.12 in democratic republic of the congo, miwanda et al. documented resistance to cotrimoxazole, erythromycin and chloramphenicol. however, no resistance to fluoroquinolones was reported from this study.13 in two separate studies from ghana, eibach et al.14 and opintan et al.15 documented resistance to trimethoprim-sulphamethoxazole, ampicillin and nalidixic acid. dalsgaard and colleagues4 from guinea bissau demonstrated resistance to ampicillin, aminoglycosides, cotrimoxazole and tetracycline. only colistin remained effective from their study. they also demonstrated that resistant isolates possessed a multi-resistance transmissible plasmid that encoded trimethoprim (dhfrxii) and aminoglycoside resistance (ant(3”)-1a).4 in kenya, urassa et al.16 documented resistance to ciprofloxacin, tetracycline, ampicillin, erythromycin and chloramphenicol by v. cholerae during two separate outbreaks.16 another study from kenya by scrascia et al.17 showed resistance of v. cholerae to chloramphenicol, streptomycin and cotrimoxazole.17 sang and colleagues18 in kenya were able to demonstrate resistance to ciprofloxacin, tetracycline, ampicillin, erythromycin and chloramphenicol among v. cholerae isolates.18 dromigny and colleagues19 conducted a study in madagascar in 2002 among v. cholerae isolates and documented resistance to tetracycline, ampicillin, nalidixic acid and nitrofurantoin.19 dengo-baloi et al.20 performed a study in mozambique to determine the antibiotic resistance patterns of v. cholerae o1 ogawa. the isolates were resistant to ampicillin, azithromycin, sulphamethoxazole, nalidixic acid and nitrofurantoin. genes for cholera toxin (ctxa, rstr2, tcpa) and the virulence factors of icevchban9 and icevchind5 were elaborated.20 smith from namibia21 characterised isolates of v. cholerae from a 2006/2007 outbreak and the isolates were all resistant to trimethoprim, sulphamethoxazole and streptomycin. the isolates possessing either the sfii or noti digestion sites were further analysed using advanced molecular techniques and it was demonstrated that they all have the same origin.21 okeke and colleagues22 investigated an outbreak of acute gastroenteritis from niger state, north-central nigeria, where eight v. cholerae organisms were isolated. they all had the o1-serogroup and el tor biotype. all of them were sensitive to tetracycline but resistant to trimethoprim, sulphonamide, spectinomycin and chloramphenicol.22 opajobi et al.23 detected 34 strains of v. cholerae in jos university teaching hospital over a one-year period. they were all of the o1 serogroup, el tor biotype and inaba serotype. they were all resistant to chloramphenicol, ampicillin, cloxacillin and penicillin g, but sensitive to tetracycline, ofloxacin and erythromycin.23 a study done by quilici et al.24 using the v. cholerae isolates from the september/october 2009 outbreak of acute watery diarrhoea in north-eastern nigeria and northern cameroon implicated the serogroup o1 of the el tor biotype and ogawa serotype as the causative serotypes. the toxigenic genes of ctxa and ctxb were elaborated, in addition to detected mutations in the genes responsible for quinolone resistance. the ctxb gene was similar to the one detected in india. all of them were resistant to trimethoprim-sulphamethoxazole, ciprofloxacin, sulphonamide and nalidixic acid. all the isolates were resistant to tetracycline, but moderately susceptible to chloramphenicol and ampicillin.24 in 2013, marin and colleagues25 described v. cholerae that were isolated from cases of acute watery diarrhoea outbreaks in nigeria from 2009 to 2010. they reported that these toxigenic v. cholerae isolates were mostly of o1 serotype, and that atypical el tor strains with the integrative conjugative element (ice) of the sulfamethoxazole and trimethoprim (sxt) element, gyra, cholera toxin (ctx) phage and cytidine triphosphate (ctp) synthetase clusters showed reduced susceptibility to ciprofloxacin and chloramphenicol. another study by marin et al.26 in 2014 characterised the whole genome of 13 strains of v. cholerae that were obtained from the 2010 outbreak. they all harboured an ice (icevchnig1) that was characterised and shown to possess genes for trimethoprim, sulfamethoxazole, streptomycin and chloramphenicol resistance. they were found to have the same gene content and gene order with similar elements detected over 20 years ago in haiti,27 angola6 and bangladesh.28 dutilh and colleagues29 used an innovative model to characterise the genomic variation of microbial genome with specific reference to v. cholerae isolates obtained worldwide with nigeria inclusive. they were able to outline mobile functions of phages, prophages, transposable elements, and plasmids. they constructed a phylogenetic tree that revealed that the v. cholerae strain isolated in 2010 from nigeria was closely related to strains already circulating in nepal and haiti.29 in senegal, the study by sambe-ba et al.30 identified atypical el tor v. cholerae o1 ogawa that were resistant to streptomycin and cotrimoxazole. ismail and colleagues31 from south africa characterised a multi-resistant v. cholerae with resistance to ampicillin, cotrimoxazole, nalidixic acid, tetracycline, kanamycin and streptomycin. the isolates were positive for the sxt element, had quinolone resistance-determining mutations in the genes encoding gyra (ser83-ile) and parc (ser85-leu) and produced tem-63-β-lactamase.31 a study in tanzania by moyo et al.32 documented resistance to ampicillin, amoxicillin-clavulanate, erythromycin, chloramphenicol, tetracycline, gentamicin and cephalothin. another study in tanzania by mercy et al.33 identified resistance of v. cholerae to furazoline, trimethoprim-sulphamethoxazole, polymyxin-b and streptomycin. the cholera virulence determinant genes ctxa, tcpa, ctxb and rtxc were also elaborated. in zambia, mwansa and colleagues34 documented resistance of v. cholerae to trimethoprim, sulphamethoxazole, tetracycline and furazolidine. the cholera toxin and the virulence genes ctxa, rstr2, rfbo1 and tcpa were also elaborated.34 chiyangi et al.,35 also from zambia, detected resistance to cotrimoxazole, nalidixic acid and nitrofurantoin.35 discussion cholera outbreaks have been ongoing within sub-saharan african countries for the past four decades. unfortunately, the specific strains responsible and their antibiotic resistance patterns are not well studied and elucidated.36 this consequently impacts negatively on the control programmes for cholera across the continent. despite our extensive database searches, we could only find a few articles that exclusively met our inclusion criteria of reporting antibiotic resistance profiles of v. cholerae from sub-saharan africa. this reflects a worrisome neglect of research on v. cholerae resistance trends from sub-saharan africa, despite the high prevalence of v. cholerae and its almost seasonal occurrence.29,37 we were able to retrieve some of the identified full text reviews from major databases by personally contacting the corresponding authors, which we recommend for researchers from developing countries like ours. there is a changing pattern of vibrio cholerae serogroups and biotypes responsible for cholera outbreaks worldwide.2 during the seventh pandemic, the typical el tor strain was responsible for outbreaks in asia, caribbean countries and africa. however, studies have now highlighted the fact that the multi-drug resistant atypical el tor and non-o1/non-o139 v. cholerae strains are the major drivers worldwide with sub-saharan africa included.2 the study done by marin et al.22 signifies that the 2009/2010 outbreaks were caused by a highly multi-drug resistant atypical el tor strain carrying major virulence determinants.22 our review revealed only v. cholerae isolates of the o1 and the non-o1/non-o139 serogroup with absence of the o139 serogroup from sub-saharan africa. this is consistent with literature evidence that reflects the occurrence of the v. cholerae o139 to be mostly restricted to bangladesh and parts of india.37 none of the studies in our review detected the ‘classical’ biotype of v. cholerae. the classical biotype has ceased to be implicated in cholera outbreaks globally, has been replaced by the el tor since after the sixth pandemic and is now mostly restricted to bangladesh.38 the stand-alone existence of the hikojima serotype was not detected by any of the studies from our review. this is not surprising as the hikojima serotype is rare and contains all the major antigens (a, b and c). one current hypothesis is that the hikojima serotype is an unstable serotype that represents a transitional state between ogawa to inaba serotype.39 v. cholerae displayed an increasingly complex resistance phenotype to various antimicrobial drugs from our review. the majority of the studies we reviewed did not state the guidelines they followed in conducting the antimicrobial susceptibility testing; nevertheless, they reported variable levels of resistance to the fluoroquinolones. the existence of quinolone resistance-determining mutations in gyra and parc in most of the isolates studied provides a genetic basis for fluoroquinolone resistance. the isolated vibrio cholerae strains that harboured the fluoroquinolone resistant genes were resistant to multiple antimicrobial agents, which has important implications for the antimicrobial-based epidemic control strategies that are still the mainstay within countries in sub-saharan africa.40 an increasing trend of resistance to cotrimoxazole was observed from many studies. kacou-n’douba11 and smith et al.12 documented resistance to chloramphenicol and cotrimoxazole from their studies in cote d’ivoire, democratic republic of the congo, guinea bissau, mozambique and namibia.11,12 this is worrisome, because, until now, cotrimoxazole was considered the drug of choice against v. cholerae. this study provides support for the more recently advocated vaccine-based strategies, which have a better chance of reducing outbreak size and case fatality rates. as access to cholera vaccine stockpiles is dependent on laboratory confirmation of cholera, public health laboratory strengthening is essential.41 in angola, ceccarelli et al. demonstrated the appearance of a novel v. cholerae epidemic variant in africa with a new ctxφ arrangement previously described only on the indian subcontinent.6 similarly, quilici et al.24 identified a strain of v. cholerae from nigeria with resistant ctxb clones that are similar to a strain identified earlier in india. this possibly indicates a trans-continental transmission of resistant organisms and this has implication for global health for appropriate international control.42 the finding of transferable resistance to almost all of the antibiotics commonly used to treat cholera was documented from many studies. some of this was documented by ceccarelli,1 kaas,2 dalsgaard4 and ismail.31 this is of great public health concern and a cause of alarm for the continent. this finding also highlights the need to develop africa’s capacity in terms of national reference laboratories, because the use of serotyping and bio-typing is inadequate for tracking the origin and clonality of v. cholerae isolates. genotypic analysis, multi-locus sequence analysis, pulse field gel electrophoresis or whole genome sequence analyses are needed to track clonality. unfortunately, these methods are more advanced and only available in a few reference laboratories within the continent.4 limitations the most appropriate pictorial representation for meta-analytic data is the forest plot. however, we could not construct one because the studies we included did not provide the component data that is essential for a forest plot. conclusion antimicrobial resistance exists among v. cholerae isolates from sub-saharan africa and includes the most feared fluoroquinolone resistance variety as well as resistance to the cell wall active agents and antimetabolites. the volume of research from countries in sub-saharan africa on antimicrobial resistance trends in v. cholerae needs to be expanded and better explored. guidelines on antimicrobial chemotherapy and standardisation of antimicrobial susceptibility testing need to be strictly adhered to. acknowledgements we thank the nigeria antimicrobial resistance technical group for review of an earlier version of this manuscript and we are grateful to nigeria centre for disease control for commissioning and supporting the research. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. sources of support the study was supported by the nigeria centre for disease control, abuja. the authors carried out the assignment as part of their routine work. i.n.o is a uk medical research council and department for international development-supported african research leader. authors’ contributions y.m. was the project leader, a.o.a., i.n.o. and a.t.o. were responsible for experimental and project design. y.m. performed most of the experiments. a.o.a., i.n.o. and a.t.o. made conceptual contributions and performed some of the experiments. a.o.a., i.n.o and a.t.o. prepared the samples and calculations were performed by y.m. references salim a, lan r, reeves pr. pathogenic clones. emerg infect dis. 2005;11:1758–1760. https://doi.org/10.3201/eid1111.041170 safa a, nair gb, kong ryc. evolution of new variants of vibrio cholerae o1. trends microbiol [serial online]. 2010 [cited 2017 jan 25];18:46–54. available from: http://www.ncbi.nlm.nih.gov/pubmed/19942436 who. cholera outbreak–assessing the outbreak response and improving preparedness [homepage on the internet]. global task force on cholera control, who/cds/cpe/zfk. 2004 [cited 2017 jan 25]. available from: www.who.int/cholera dalsgaard a, forslund a, petersen a, et al. class 1 integron-borne, multiple-antibiotic resistance encoded by a 150-kilobase conjugative plasmid in epidemic vibrio cholerae o1 strains isolated in guinea-bissau. j clin microbiol. 2000;38:3774–3777. kitaoka m, 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opintan ja, okeke in. fluoroquinolone-resistant enteric bacteria in sub-saharan africa: clones, implications and research needs. front microbiol. 2016;7:5. https://doi.org/10.3389/fmicb.2016.00558 abstract introduction methods results discussion acknowledgements references about the author(s) mostafa vaghari-tabari department of clinical biochemistry and laboratory medicine, tabriz university of medical sciences, tabriz, iran liver and gastrointestinal diseases research center, tabriz university of medical sciences, tabriz, iran soheila moein molecular medicine research center, hormozgan university of medical sciences, bandar abbas, iran department of biochemistry, faculty of medicine, hormozgan university of medical sciences, bandar abbas, iran durdi qujeq cellular and molecular biology research center (cmbrc), health research insititute, babol university of medical sciences, babol, iran department of clinical biochemistry, babol university of medical sciences, babol, iran mehrdad kashifard department of internal medicine, gastroenterology division, ayatollah rouhani hospital, babol university of medical sciences, babol, iran haydeh alaoddolehei department of hematology and medical laboratory sciences, para medical faculty, babol university of medical sciences, babol, iran karimollah hajian-tilaki department of biostatistics and epidemiology, babol university of medical sciences, babol, iran citation vaghari-tabari m, moein s, qujeq d, kashifard m, alaoddolehei h, hajian-tilaki k. sensitivity and specificity of mean platelet volume as a laboratory marker for irritable bowel syndrome: can it be added to rome criteria? afr j lab med. 2020;9(1), a1001. https://doi.org/10.4102/ajlm.v9i1.1001 original research sensitivity and specificity of mean platelet volume as a laboratory marker for irritable bowel syndrome: can it be added to rome criteria? mostafa vaghari-tabari, soheila moein, durdi qujeq, mehrdad kashifard, haydeh alaoddolehei, karimollah hajian-tilaki received: 24 feb. 2019; accepted: 14 sept. 2020; published: 21 dec. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: irritable bowel syndrome (ibs) is a functional gastrointestinal disorder. objective: this study aimed to evaluate red blood cell distribution width (rdw) and mean platelet volume (mpv) as laboratory markers to discriminate ibs patients from both healthy controls and patients with inflammatory bowel disease (ibd). methods: this case-control study enrolled patients referred to ayatollah rouhani hospital, endoscopy department, babol, iran, for colonoscopy examination from 2015 to 2017. fifty ibs patients were selected from among patients who had undergone a normal colonoscopy and showed symptoms matching the rome iii criteria. fifty healthy participants and 50 ibd patients, matched for sex and age, were also enrolled in this study. both rdw and mpv were measured and analysed by independent sample t-test and receiver operating characteristic curve analysis. a p-value of less than 0.05 was considered statistically significant. results: while rdw was higher and mpv was lower among ibs patients compared to healthy controls (p = 0.047 and p = 0.001), there were no significant differences in rdw or mpv levels between ibs and ibd patients. the area under the curve of rdw in the discrimination between ibs and ibd was 0.620 (p = 0.039), and the area under the curve of mpv in the discrimination between healthy controls and ibs patients was 0.801 (p = 0.001). conclusion: mean platelet volume is potentially a useful laboratory marker for distinguishing between ibs patients and healthy individuals. red blood cell distribution width should be considered as a potential marker to distinguish among ibs and ibd patients. keywords: ibs; irritable bowel syndrome; inflammatory bowel disease; red blood cell distribution width; mean platelet volume. introduction bowel disorders have been categorised into organic and functional diseases. organic diseases have observable and measurable disease processes. for example, these diseases may be associated with tissue damage. inflammatory bowel disease (ibd) and colorectal cancer are examples of important organic intestinal diseases. there are no organic pathologies such as masses and ulcers in intestinal functional disorders.1 irritable bowel syndrome (ibs) is one of the most common functional gastrointestinal disorders associated with abdominal pain and a range of other symptoms like stomach cramps, bloating, diarrhoea, constipation, or alternate periods of diarrhoea and constipation.2 the exact pathogenesis of ibs is not clear but it is believed that impairment in the brain-gut axis causes ibs. it seems that multiple factors, including environmental factors such as diet, stress, and intrinsic factors, such as epigenetics and genetics, are involved in ibs pathogenesis and can affect the brain-gut axis.2 irritable bowel syndrome symptoms and severity vary from one individual to another. irritable bowel syndrome is commonly diagnosed by clinical signs and symptoms according to the rome iii criteria.3 although colonoscopy is not required in the diagnosis of ibs, a colonoscopy can be requested to rule out organic diseases such as ibd in patients with rectal bleeding. based on the rome iii criteria proposed in 2006, an ibs patient is one who has had recurrent discomfort or abdominal pain 3 days per month in the last 3 months and who has met two or more of the following criteria: decrease in pain or discomfort after defecation, change in stool frequency or change in stool form.4 according to signs and symptoms, ibs is divided into four groups, namely: ibs-d—diarrhoea is the predominant symptom; ibs-c—constipation is the predominant symptom; ibs-m—alternating periods of diarrhoea and constipation is a predominant symptom; ibs-u—no predominant symptom is experinced.5,6 in addition to the rome iii criteria, a simple laboratory test with acceptable sensitivity and specificity will aid the diagnosis of ibs. the effectiveness of laboratory tests for ibs diagnosis is poorly investigated. however, some studies have shown that red blood cell distribution width (rdw) and mean platelet volume (mpv) may be useful for ibs diagnosis.7,8 both rdw and mpv are hematologic markers with clinical significance and are routinely included in the complete blood count test. not only is rdw an indicator of erythrocyte size variation and conventionally used for categorisation of anaemia,9 it is also high in some clinical conditions such as autoimmune disease, liver disorder and sickle cell disease.9,10 some studies have demonstrated high rdw in ibd.11,12 meanwhile, mpv has been traditionally used for examination of platelet production status in the bone marrow and has clinical importance in some circumstances13; mpv levels may also be altered in hypertension, diabetes and ibd.14 irritable bowel syndrome is an organic intestinal disease with two major subtypes: ulcerative colitis and crohn’s disease.15 its clinical signs and symptoms have been shown to have a significant overlap with ibs.5 some studies of ibd have demonstrated that mpv can be used for assessment of disease activity.14,16,17 although ibs is conventionally diagnosed based on clinical symptoms, a further laboratory test can be done to increase the validity of the diagnosis. additionally, patients may need a colonoscopy, for example colonoscopy can be requested for patients who have a family history of ibd. however, this could be avoided if an available laboratory test that accurately discriminates ibs from an organic disorder like ibd. this would also reduce the incidences of colonoscopy. thus this study evaluated the utility of rdw and mpv as potential laboratory biomarkers to discriminate between ibs and healthy controls as well as for distinguishing among ibs and ibd patients. methods ethical considerations this retrospective study is a part of master of science (msc) thesis project (no. 6793) and has conformed to the standards of the world medical association, as embodied in the declaration of helsinki and the protocol was approved by the hormozgan university of medical sciences ethical committee (ir.hums.rec.94.182). all patients signed the written informed consent forms provided by hormozgan university of medical sciences and agreed that their medical information could be used in this study. these forms contained information about the project and how patient information was used. to protect patients’ personal information, a special code was assigned to each patient’s information. study design and sample selection all participants were over 18 years of age and were referred to a gastroenterologist at the ayatollah rouhani hospital, babol in northern iran. the consultation period was from september 2015 to january 2017. irritable bowel syndrome case group these were patients whose clinical symptoms matched the rome iii criteria, who had normal c-reactive protein (crp) and erythrocyte sedimentation rate without organic disorders, confirmed by colonoscopy examination. patients’ symptoms included abdominal pain, diarrhoea, constipation and other ibs symptoms. primary reasons for colonoscopy included rectal bleeding, family history of colorectal malignancy and positive stool occult blood test. colonoscopy examination was done by expert gastroenterologists using an olympus colonoscopy instrument (olympus, tokyo, japan). irritable bowel syndrome patients with any of the following criteria were excluded: abnormal haemoglobin level, iron deficiency, abnormal blood smear microscopic analysis, any type of cancer, diverticular disease, history of colorectal surgery, any type of blood disease, diabetes, cardiovascular dysfunction, liver and kidney disease, any type of infectious disease, any type of congenital disease or use of non-steroidal anti-inflammatory drugs (e.g. using aspirin before blood sampling and colonoscopy examination). a case group of 50 patients, 23 women and 27 men, met the inclusion criteria and were enrolled as ibs patients. among these ibs patients, 18 patients had ibs-d, 17 patients had ibs-c and 15 patients had ibs-m. inflammatory bowel disease case group these were patients diagnosed with ibd by colonoscopy and approved by histopathologic methods. inflammatory bowel disease patients who met the following criteria were excluded: previous colorectal surgery, diverticular disease, cardiovascular disease, cancers, infectious disease, liver diseases, kidney diseases, congenital blood disorders, diabetes and taking non-steroidal anti-inflammatory drugs before blood sampling. fifty ibd patients (14 of them had crohn’s disease and others had ulcerative colitis) were matched for sex and age. among these ibd patients, 25 patients had active disease and 25 patients were in clinical remission. control group the control group consisted of 50 healthy volunteers, matched for sex and age, who were referred to the laboratory of ayatollah rouhani hospital, in babol in northern iran. the healthy participants were selected after consultation with a gastroenterologist. all did not have a colonoscopy examination, clinical signs and symptoms of ibs or a history of ibd. healthy individuals were excluded if they had: any systemic condition, any type of anaemia or blood disorder, abnormal haemoglobin, abnormal crp or erythrocyte sedimentation rate levels, abnormal blood smear, abnormal iron levels or ibs sign or symptoms. laboratory measurements a venous blood sample was taken from each participant’s arm into ethylenediaminetetraacetic acid-tubes for complete blood count testing. for a precise selection of patients and controls routine laboratory analysis including complete blood count, erythrocyte sedimentation rate, crp, iron profile and blood smear microscopic analysis was performed for all participants. red blood cell distribution width and mpv were determined simultaneously with complete blood count in a sysmex cell counter instrument (sysmex, kobe, japan). the crp, serum iron and total iron-binding (tibc) levels were quantitatively measured in an auto-analyser instrument (hitachi, tokyo, japan). the following assay kits were used: bionic crp kit (bionic, tehran, iran), serum iron kit (darman faraz kave, tehran, iran) and tibc kit (darman faraz kave, tehran, iran). serum ferritin level was measured by the enzyme-linked immunosorbent assay method according to the ferritin enzyme-linked immunosorbent assay kit instructions (pishtaz teb, tehran, iran) and using an rt2100c enzyme-linked immunosorbent assay reader instrument (raytolife, hamburg, germany). erythrocyte sedimentation rate was measured by the conventional westergren method. in this method, 2 ml of blood sample collected in a tube containing 0.4 ml of sodium citrate was transferred to a standard westergren-katz tube. the tube was set to stand for 1 h, for erythrocytes to settle. the column of separated plasma was measured, along with the rate of settling in millimetres per hour. statistical analysis the data obtained were analysed using an ibm statistical package for the social sciences (spss software version 17, armonk, new york, united states). the independent sample t-test was used for comparing variable means between groups. the receiver operating characteristic analysis was also used for comparing the utility of mpv and rdw for discrimination between ibs patients and healthy controls as well as for discrimination between ibs and ibd patients. p-values less than 0.05 were considered as statistically significant. results our data demonstrated that the mpv level was significantly reduced while the rdw level was significantly elevated in ibs patients when compared with those of healthy subjects, but the levels of the same parameters were not significantly different between ibs and ibd patients (table 1). table 1: demographic and laboratory characteristics of patient and control groups, babol, iran, 2015–2017. receiver operating characteristic analysis showed that mpv has a larger area under the curve (0. 801, p = 0.001) than rdw (figure 1, figure 2, table 2) in differentiating between the healthy control and ibs patients. mean platelet volume had a 68% sensitivity and 88% specificity at 9.55 femtoliter. figure 1: receiver operating characteristic curve analysis to evaluate mean platelet volume utility in discriminating between healthy controls and irritable bowel syndrome patients, babol, iran, 2015–2017. figure 2: receiver operating characteristic curve analysis to evaluate red blood cell distribution width utility in discriminating between irritable bowel syndrome patients and healthy controls, babol, iran, 2015–2017. table 2: receiver operating characteristic curves for comparison of mean platelet volume and red blood cell distribution width utility in discriminating between healthy controls and ibs patients, babol, iran, 2015–2017. receiver operating characteristic analysis showed that rdw compared to mpv had a larger but not significant area under the curve of 0.620 (p = 0.039) for differentiating between ibs and ibd patients (figure 3, figure 4, table 3). the best cut-off point for rdw was a value of 13.05%, with 72% sensitivity and 56% specificity, to help discriminate ibd patients from ibs patients. figure 3: receiver operating characteristic curve analysis to evaluate mean platelet volume utility in discriminating between irritable bowel syndrome and inflammatory bowel disease patients, babol, iran, 2015–2017. figure 4: receiver operating characteristic curve analysis to evaluate red blood cell distribution width utility in discriminating between inflammatory bowel disease and irritable bowel syndrome patients, babol, iran 2015–2017. table 3: receiver operating characteristic curves for comparison of mean platelet volume and red blood cell distribution width utility in discriminating between irritable bowel syndrome and inflammatory bowel disease patients, babol, iran, 2015–2017. discussion our results showed that there was not a significant difference in rdw and mpv between ibs and ibd patients. although average rdw among ibs patients was lower compared with rdw among ibd patients, the difference was not statistically significant (p = 0.068). however, this difference could become meaningful if the sample size were increased. our data also showed that, in comparison with healthy controls, the mean mpv was lower while rdw was higher among ibs patients, which could be due to subclinical inflammation in ibs patients. subclinical inflammation in ibs has been previously reported in some studies,18,19 and it is well known that mpv and rdw can be altered in inflammatory conditions.11,12,14 our findings augment the hypothesis that subclinical inflammation has a role to play in ibs. however, further studies in this regard are needed. our findings, in contrast to a study conducted in turkey, showed that the mpv levels were higher among ibs patients than among healthy controls.7 this inconsistency may be due to the difference in study populations or inclusion criteria. in our study, the results of receiver operating characteristic curve analysis showed that the area under the curve, sensitivity and specificity of mpv for ibs diagnosis are acceptable. the use of receiver operating characteristic curve analysis to evaluate the utility of mpv in ibs diagnosis has not been previously presented by other studies; thus, a direct comparison of our results with other studies, results was not possible. inflammatory bowel disease is a complex gastrointestinal disease and multiple factors are involved in its pathogenesis.20,21,22 according to our results, rdw has a relatively low specificity for differentiating between ibd and ibs patients. our results are similar to a study conducted in hungary that reported 92% specificity and 78% sensitivity for rdw (cut-off: 13.4%) in active crohn’s disease diagnosis. these researchers also reported that in the majority of ibs patients rdw was normal. they however did not evaluate the specificity and sensitivity of rdw in discriminating between ibd and ibs.23 further studies are needed to evaluate the utility of rdw as a diagnostic marker. there are very few studies investigating laboratory diagnosis of ibs and it is proposed that the mpv may be used as an ibs biomarker. a mean platelet volume test in addition to the clinical parameters of the rome criteria can improve the diagnosis of ibs. limitations our study has some limitations. firstly, a major study limitation is that our sample size was relatively low because the aim of the study was primarily to evaluate the utility of rdw and mpv for laboratory diagnosis of ibs. budget and time limitations further limited the possible sample size. the second limitation of our study was that we could not assess the other laboratory factors to discriminate between healthy controls and ibs patients. however, we used rigorous inclusion and exclusion criteria for patient selection in an attempt to mitigate the influence of confounding conditions. conclusion red blood cell distribution width was higher while the mpv level was lower among ibs patients compared to healthy controls, although the same parameters did not differ significantly when compared with ibd patients. mean platelet volume is a potential marker with adequate sensitivity and specificity to discriminate healthy controls from ibs patients. however, it is not useful for discriminating between ibd and ibs patients. red blood cell distribution width could be a potential marker for differentiation between ibd and ibs. further studies with a sufficiently large sample size are needed for a comprehensive evaluation of the utility of these potential biomarkers to discriminate between healthy controls, ibs patients and ibd patients. acknowledgements the authors express their thanks to hormozgan university of medical sciences. the authors also thank the personnel of the endoscopy department of ayatollah rouhani hospital and the laboratory personnel. moreover, the authors wish to express their gratitude to dr javad shokri-shirvani, dr seyed saeid mohammadi, tooba yousefi, koroush rasoulpour, hamed ghasem tabar and maryam ghasemnejad for their helpful and professional assistance. competing interests the authors have declared that no competing interests exist. authors’ contributions d.q. designed the experiments. m.v.-t. performed the experiments. k.h.-t. analysed the data and s.m. contributed to the writing and revising of the manuscript. m.k. selected the patients based on colonoscopy examination and h.a. helped in the performance of haematology tests and analysed these tests. sources of support this project was funded by prof. soheila moein. also, this investigation was partly supported by the grant no. 9437 from the research council of hormozgan university of medical sciences. data availability statement our data are available from the corresponding author upon reasonable request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references drossman da. functional gi disorders: what’s in a name? gastroenterology. 2005;128(7):1771–1772. https://doi.org/10.1053/j.gastro.2005.04.020 gazouli m, wouters mm, kapur-pojskic l, et al. lessons learned-resolving the enigma of genetic factors in ibs. nat rev gastroenterol hepatol. 2016;13(2):77–87. https://doi.org/10.1038/nrgastro.2015.206 saha l. irritable bowel syndrome: pathogenesis, diagnosis, treatment, and evidence-based medicine. world j gastroenterol. 2014;20(22):6759–6773. https://doi.org/10.3748/wjg.v20.i22.6759 shih dq, kwan ly. all roads lead to rome: update on rome iii criteria and new treatment options. gastroenterol rep. 2007;1(2):56–65. lacy be, mearin f, chang l, et al. bowel disorders. gastroenterology. 2016;150(6):1393–1407. https://doi.org/10.1053/j.gastro.2016.02.031 enck p, aziz q, barbara g, et al. irritable bowel syndrome. nat rev dis prim. 2016;2:16014. https://doi.org/10.1038/nrdp.2016.14 aktas g, alcelik a, tekce bk, tekelioglu v, sit m, savli h. red cell distribution width and mean platelet volume in patients with irritable bowel syndrome. prz gastroenterol. 2014;9(3):160–163. https://doi.org/10.5114/pg.2014.43578 coskun a, yavasoglu i, sargin g, et al. the role of mean platelet volume in patients with non-specific abdominal pain in an emergency department. prz gastroenterol. 2015;10(3):156–159. https://doi.org/10.5114/pg.2015.49042 lippi g, plebani m. red blood cell distribution width (rdw) and human pathology. one size fits all. clin chem lab med. 2014;52(9):1247–1249. https://doi.org/10.1515/cclm-2014-0585 sahli ca, bibi a, ouali f, et al. red cell indices: differentiation between β-thalassemia trait and iron deficiency anemia and application to sickle-cell disease and sickle-cell thalassemia. clin chem lab med. 2013;51(11):2115–2124. https://doi.org/10.1515/cclm-2013-0354 arhan m, önal i̇k, taş a, et al. the role of red cell distribution width as a marker in inflammatory bowel disease. turk j med sci. 2011;41(2):227–234. yeşil a, senateş e, bayoğlu i, erdem ed, demirtunç r, kurdaş övünç a. red cell distribution width: a novel marker of activity in inflammatory bowel disease. gut liver. 2011;5(4):460–467. https://doi.org/10.5009/gnl.2011.5.4.460 bancroft aj, abel ew, mclaren m, belch jj. mean platelet volume is a useful parameter: a reproducible routine method using a modified coulter thrombocytometer. platelets. 2000;11(7):379–387. https://doi.org/10.1080/09537100020008311 gasparyan ay, ayvazyan l, mikhailidis dp, kitas gd. mean platelet volume: a link between thrombosis and inflammation? curr pharm des. 2011;17(1):47–58. https://doi.org/10.2174/138161211795049804 moein s, vaghari-tabari m, qujeq d, majidinia m, nabavi sm, yousefi b. mirnas and inflammatory bowel disease: an interesting new story. j cell physiol. 2019;234(4):3277–3293. https://doi.org/10.1002/jcp.27173 kapsoritakis an, koukourakis mi, sfiridaki a, et al. mean platelet volume: a useful marker of inflammatory bowel disease activity. am j gastroenterol. 2001;96(3):776–781. https://doi.org/10.1111/j.1572-0241.2001.03621.x yüksel o, helvacı k, başar ö, et al. an overlooked indicator of disease activity in ulcerative colitis: mean platelet volume. platelets. 2009;20(4):277–281. https://doi.org/10.1080/09537100902856781 de silva ap, nandasiri sd, hewavisenthi j, et al. subclinical mucosal inflammation in diarrhea-predominant irritable bowel syndrome (ibs) in a tropical setting. scand j gastroenterol. 2012;47(6):619–624. https://doi.org/10.3109/00365521.2012.666672 tornblom h, lindberg g, nyberg b, veress b. full-thickness biopsy of the jejunum reveals inflammation and enteric neuropathy in irritable bowel syndrome. gastroenterology. 2002;123(6):1972–1979. https://doi.org/10.1053/gast.2002.37059 vaghari-tabari m, moein s, qujeq d, kashifard m, hajian-tilaki k. positive correlation of fecal calprotectin with serum antioxidant enzymes in patients with inflammatory bowel disease: accidental numerical correlation or a new finding? am j med sci. 2018;355(5):449–455. https://doi.org/10.1016/j.amjms.2017.12.009 mohammadi e, qujeq d, taheri h, hajian-tilaki k. evaluation of serum trace element levels and superoxide dismutase activity in patients with inflammatory bowel disease: translating basic research into clinical application. biol trace elem res. 2017;177(2):235–240. https://doi.org/10.1007/s12011-016-0891-0 de souza hs, fiocchi c. immunopathogenesis of ibd: current state of the art. nat rev gastroenterol hepatol. 2016;13(1):13–27. https://doi.org/10.1038/nrgastro.2015.186 farkas k, papp m, nyári t, et al. red blood cell distribution width in combination with serological markers can help in the differentiation between crohn’s disease and ulcerative colitis. open gastroenterol j. 2010;4:1–4. https://doi.org/10.2174/1874259901004010001 acknowledgements references about the author(s) iruka n. okeke department of pharmaceutical microbiology, university of ibadan, ibadan, nigeria citation okeke in. extending the breadth of african laboratory medicine. afr j lab med. 2019;8(1), a1128. https://doi.org/10.4102/ajlm.v8i1.1128 editorial extending the breadth of african laboratory medicine iruka n. okeke copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the african journal of laboratory medicine’s (ajlm) eighth volume contains almost 30 articles, our largest issue yet. a few areas of laboratory medicine are not represented, but the impressively expansive scope of the volume extends from bacteriology (including mycobacteriology), clinical chemistry, clinical pharmacology, haematology, histopathology and virology to informatics, laboratory development and quality management. while appreciating that growth has come with good subject area representation, we have to be just as intentionally concerned with the diversity of matters other than content. if nothing is done, some estimates suggest that it will be over 200 years before gender parity is reached throughout society1 and other forms of imbalance may be even more entrenched. ajlm is therefore not alone in its current introspection and quest for diversity. in a bid to accelerate the quality improvements that will come with inclusiveness, a number of editors, journals and independent researchers have published their recent observations and reflections on the gender and geographic balance of editors, reviewers and authors.2,3,4 almost universally, they find that representation in scientific journals is slanted towards the male northern bias that permeates most of science, even if the tilt angles differ. as a journal owned by an african scientific society, with both editorial and publishing offices based on the continent, ajlm has an uncommon position in this discourse. firstly, women are severely underrepresented in african science5 and globally women play a prominent role in healthcare delivery but are much less visible in its leadership.6 both of these deficits could well influence representation in a laboratory medicine journal with a lens on africa, the continent that is home to the fewest scientists per capita. secondly, as a regional journal with global reach, we receive and publish a large number of scientific articles from african authors. we simultaneously provide an owned perspective and a global view of african science, a dual role that is rare in scientific publishing.2,4,7 this vantage-point notwithstanding, ajlm also represents a powerful mouthpiece for speaking to african scientists and therefore we cannot take for granted that our editorial processes and authorship will reflect our readership. the vision and content of a journal is shaped by its editors. as of october 2019, ajlm’s editorial team comprised a female editor-in-chief, five section editors (two female) and a 22-person editorial board that included only three women. this team had affiliations on four continents with a third of us working in africa. with three of our six editors being women (not counting our managing editor, who is also female), we merit only 60% by bhaumik and jagnoor’s8 composite editorial board diversity score. but, given our focus on africa, we have a greater proportion of africa-based editors than any of the 27 global health journals that were assessed in that study. we lag most noticeably in geographic diversity. scientists from all over the world work with laboratory medicine experts on our continent to better health, and findings from studies in or on africa will inform the practice of laboratory medicine anywhere. so, our editorial board is deliberately global even though we do seek editors who are familiar with science in africa. we, therefore, would benefit from representation from australasia, latin america and the caribbean, which is currently absent from our editorial board. even though this is not the case with respect to african biomedical research overall,5 the vast majority of our articles are wholly or predominantly authored by africans. in spite of the well-documented male skew in african science,5 ajlm vol. 7(1) 2018 comprised articles with nine female and five male first authors (with one indeterminate). first authors on articles in vol. 8 include 11 men and nine women (4 indeterminate based on first name and online searches). therefore, ajlm is an important venue for publishing female-led work, particularly from africa. we are also pleased to find that ajlm continues to feature articles from a range of african countries and from beyond them and that it addresses health issues of different populations, including ethnic minorities or those living in rural or remote areas. unfortunately, while we have good representation from africa, we find that there is less diversity in the geographic origin of ajlm articles within the continent. five 2018 corresponding authors listed south african addresses and one each from rwanda, ethiopia, the netherlands, kenya, cameroon, barbados, haiti, namibia, nigeria and canada. in 2019, we published six articles with corresponding authors from south africa, four each from nigeria and the united states (us), one with a double nigeria-us affiliation, two from kenya and saudi arabia and one each from belgium, botswana, cameroon, ethiopia, ghana and zimbabwe. while preponderance of anglophone corresponding authors is expected for an english-language journal, the underrepresentation of submissions from western, central, north and east africa in our recent volumes is obvious and concerning. if ajlm has uncommon success in publishing high-quality african research, it should do so for those parts of the continent least represented in science. what could ajlm do to enhance diverse representation in our journal going forward? firstly, we need to maintain the diversity that we do have and to ensure that with the growth in our size and reputation, minority contributors do not become overshadowed or, worse, replaced. ajlm will continue to encourage female scientists to submit to, review for and edit for the journal as well as to support those that work with us. we will also continue to offer a mentored review process and copy-editing support for accepted articles, which allows us to feature work by new authors and non-anglophone scientists. however, we will have to do much more to bring in manuscripts from across the continent and explicitly wish to call for papers from countries not featured in our last two volumes. from time to time, ajlm offers heavily subscribed manuscript writing training workshops and in future, we will prioritise early-career participants from african countries who are underrepresented in the journal or who are female. we will also endeavour to invite more female scientists to review for the journal and take places on our editorial board. finally, with this editorial, ajlm is in the process of keeping track of the demographics of our authors, reviewers and board because, unless we do, we will have no idea how far we are from our goal of being the primary outlet for truly balanced scientific work in laboratory medicine across the continent. acknowledgements i am grateful to el-shama monu-nwoko for collating data. competing interests i declare that i have no financial or personal relationships that may have inappropriately influenced me in writing this article. references gates m. gender equality is within our reach. harv bus rev. 2019. briggs rc, weathers s. gender and location in african politics scholarship: the other white man’s burden? afr aff. 2016;115(460):466–489. https://doi.org/10.1093/afraf/adw009 espin j, palmas s, carrasco-rueda f, et al. a persistent lack of international representation on editorial boards in environmental biology. plos biol. 2017;15(12):e2002760. https://doi.org/10.1371/journal.pbio.2002760 chersich mf, blaauw d, dumbaugh m, et al. local and foreign authorship of maternal health interventional research in low-and middle-income countries: systematic mapping of publications 2000–2012. glob health. 2016;12(1):35. https://doi.org/10.1186/s12992-016-0172-x okeke in, babalola cp, byarugaba dk, djimde a, osoniyi or. broadening participation in the sciences within and from africa: purpose, challenges, and prospects. cbe life sci educ. 2017;16(2):es2. https://doi.org/10.1187/cbe.15-12-0265 world health organization. delivered by women, led by men: a gender and equity analysis of the global health and social workforce. who: geneva;2019. abimbola s. the foreign gaze: authorship in academic global health. bmj glob health. 2019;4(5):e002068. https://doi.org/10.1136/bmjgh-2019-002068 bhaumik s, jagnoor j. diversity in the editorial boards of global health journals. bmj glob health. 2019;4(5):e001909. https://doi.org/10.1136/bmjgh-2019-002068 introduction challenges in expanding and enhancing yellow fever laboratory diagnostics building the lassa fever laboratory network during an epidemic access to diagnostics and laboratory systems integration acknowledgements references about the author(s) dhamari naidoo who health emergency program, infectious hazard management, world health organisation, abuja, nigeria chikwe ihekweazu nigeria centre for disease control, abuja, nigeria citation naidoo d, ihekweazu c. nigeria’s efforts to strengthen laboratory diagnostics – why access to reliable and affordable diagnostics is key to building resilient laboratory systems. afr j lab med. 2020;9(2), a1019 https://doi.org/10.4102/ajlm.v9i2.1019 opinion paper nigeria’s efforts to strengthen laboratory diagnostics – why access to reliable and affordable diagnostics is key to building resilient laboratory systems dhamari naidoo, chikwe ihekweazu received: 30 mar. 2019; accepted: 27 may 2020; published: 26 aug. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction since 2016, the nigeria centre for disease control (ncdc) has been strengthening its technical capacities as the country’s national public health institute.1 one key area receiving considerable attention has been the strengthening of laboratory diagnostic and networking capabilities for diseases of public health importance. previous investments in strengthening and financing laboratory capacities have focused almost entirely on single disease programmes. in just over two years, the ncdc has made progress in changing this situation with the operationalisation of a new national reference laboratory. the national reference laboratory has the diagnostic capacity for national priority diseases, including yellow fever (yf), lassa fever, monkeypox, cerebrospinal meningitis, cholera, influenza and other enteric pathogens. in addition, a specimen referral system has been established to cover the large geographic expanse of the country and a national laboratory network supporting 41 facilities. the national laboratory network integrates vertical disease networks for the standardisation of testing algorithms and improvement of coordination and functionality.2 in 2017 alone, the ncdc responded to widespread outbreaks of cerebrospinal meningitis, monkeypox, yf, lassa fever and cholera.1 with several public health challenges, including increasing disease outbreaks and emerging antimicrobial resistance,3 efficient coordination and integration of surveillance and laboratory systems is of critical importance. improving access to diagnostics at the country level is a fundamental component of building laboratory systems critical for disease surveillance and timely outbreak detection and containment, as well as improved patient management.4 advocacy on the importance of diagnostic development for outbreak preparedness is occurring through global strategies such as the world health organization’s (who) research and development blueprint and eliminating yellow fever epidemics strategy.5,6 however, challenges still remain in accessing diagnostic tests that are reliable, available through local production or procurement, and easily implementable and scalable during emergencies.7 this is becoming more apparent as countries struggle to leverage laboratory capacity from established vertical disease programmes, such as polio, hiv and tuberculosis, for emerging public health threats. the limited global investments to increase access to diagnostics across a wider range of diseases of public health concern, rather than individual diseases, will continue to limit the capacity of african countries to develop resilient and disease-wide public health systems.4 this report summarises the challenges and early successes of laboratory integration across two disease networks in nigeria. challenges in expanding and enhancing yellow fever laboratory diagnostics since 2016, the re-emergence of yf has caused outbreaks in angola, brazil, chad, the democratic republic of congo, ghana, guinea and uganda.8 this prompted the who to launch the eliminating yellow fever epidemics strategy to reduce the risk of yf in high-risk countries through strengthening outbreak detection, response and prevention.9 in 2017, yf re-emerged in nigeria, 17 years since the last reported case.10 localised outbreaks continue to be reported and, as of march 2019, nigeria had seen approximately 4100 suspect cases, of which 139 were confirmed in 17 states throughout the country11 (figure 1). figure 1: weekly trends of yellow fever outbreak in nigeria from 2017 to 2019. the nigerian yf laboratory network, established in 2006, consists of four subnational laboratories located in lagos, kaduna, abuja and gombe (figure 2), with capacity to implement imunoglobulin m (igm) detection, the standardised african yf laboratory diagnostic algorithm. the national laboratories receive reagents for laboratory-developed, enzyme-linked immunosorbent assays through two sources: the institut pasteur of dakar and the united states centres for disease control and prevention.12 due to operational differences between the laboratory-developed and commercial elisa protocols, implementation is challenging and requires continuous training for the national staff. following recommended guidelines, presumptive positive samples from any of the four national laboratories have to be shipped to the who’s regional reference laboratory at the institut pasteur of dakar in senegal, where additional testing is conducted. this is done to exclude vaccine-induced imunoglobulin m antibody positivity and cross-reactivity because of infections with other flaviviruses – a limitation of serological testing.8 figure 2: geographic location and specimen referral catchment areas for the lassa fever and yellow fever laboratories. (a) lassa fever laboratories, (b) yellow fever laboratories. the response to the ongoing outbreak has been challenged by limited access to reagents. between 2016 and august 2017, no yellow fever testing occurred in nigeria because of a lack of reagents.7 when the outbreak was first detected in kwara state, nigeria, in august 2017, the same month that reagents became available, it took one month to confirm the laboratory result at the regional reference laboratory. the index case reported the onset of symptoms on 16 august 2017 and the final laboratory confirmation was only received on 12 september 2017.9 delayed outbreak detection and reporting of cases has an impact on the approval of vaccine requests from the international coordination group on vaccine provision13 and planning for reactive vaccination campaigns for outbreak control. the importance of rapid confirmation was recently highlighted during the widespread yf outbreak in edo state, nigeria, in 2018. clusters of cases were reported from four local government areas on 14 november 2018. on 21 november 2018, the ncdc was informed of nine patient samples that had tested positive for yf by quantitative reverse transcription polymerase chain reaction (qrt-pcr) at the african center of excellence for genomics of infectious disease, a non-yf-network laboratory. these yf cases were found in patients initially referred to the irrua specialist teaching hospital with suspected lassa fever. after lassa fever tests were negative, the patients were tested for yf following continued clinical presentation with typical viral haemorrhagic signs and symptoms.14 immediately after receiving the report of positive results, the samples were sent to the institut pasteur of dakar on 29 november 2018 for re-confirmation. the first set of results yf confirming both imunoglobulin m and qrt-pcr results were received from the institut pasteur of dakar on 7 december 2018. the plaque reduction neutralisation test results were shared on 31 december 2018. however, the outbreak was declared on 24 november 2018, based on the qrt-pcr results. declaring the outbreak using the molecular results allowed for the immediate deployment of rapid response teams and the initiation of a request for vaccines from the international coordination group on vaccine provision. however, the challenge faced by national authorities was two fold – accepting test results carried out by non-who-recognised yf-network laboratory; and by qrt-pcr, a method not routinely used for confirmation at the national level. molecular testing has not been effectively operationalised in the african region.4,15 in response, the national yf testing algorithm was updated to include molecular testing, and the national lassa fever laboratories were registered in a yf qrt-pcr quality-assurance programme, a critical step required to demonstrate technical capacity to detect yf virus infection. overall, two critical bottlenecks hamper the strengthening of the national yf laboratory network. these are limited access to clinically validated or regulatory approved molecular and serologic tests either through the who network or through commercial manufacturers; and the limited capacity to perform in-country confirmatory diagnosis by plaque reduction neutralisation test and differential diagnosis for flaviviruses.8 the eliminating yellow fever epidemics strategy is currently addressing the shortfalls in diagnostics through updating diagnostic algorithms, working with industries and partners to fast-track diagnostic development and evaluations, and creating an international procurement and supply chain. to operationalise these changes at the country level, increased support and funding is required for training, procurement of diagnostic tests for yf and other flaviviruses, and access to quality control materials for the development of national external quality assessment activities. building the lassa fever laboratory network during an epidemic in 2018, nigeria recorded an unusually active epidemic season of lassa fever. a total of 3498 suspect cases were reported, of which 633 were laboratory confirmed. several factors, such as increased disease awareness, improved surveillance and laboratory capacity, were thought to have contributed to the increased number of confirmed cases.3 before establishing national diagnostic capacity, testing was performed outside nigeria, in kenema, sierra leone. between 2005 and 2012, diagnostic capacity was established at the lagos university teaching hospital and the irrua specialist teaching hospital. the irrua specialist teaching hospital accounted for over 90% of the testing workload, as it provided diagnostic support for an endemic region in nigeria.16 the 2018 epidemic season marked the beginning of a collaboration with the international community under the framework of the who’s research and development blueprint to improve lassa fever detection, treatment and prevention.17 one of the biggest gains made in 2018 was the rapid expansion of national diagnostic capacity, with the establishment of two additional testing facilities at the ncdc national reference laboratory in abuja and the virology laboratory at the federal teaching hospital in abakaliki, ebonyi state (figure 2). the expansion was attributable to the availability of funding during the outbreak from donors and the who through the federal government to the ncdc. this enabled the procurement of equipment and reagents, the building of infrastructure and increased political commitment from the federal and state governments of nigeria. a key factor in the early success of the network was the adoption of a standardised testing algorithm based on a 2-gene target testing strategy using qrt-pcr, with a combination of both commercial(realstar® lassa fever rt pcr kit version 1.0, altona diagnostics, hamburg, germany) and laboratory-developed tests being used to detect all known lineages of lassa fever virus.16 published diagnostic literatures showed limited availability of commercial assays for lassa fever diagnosis; none was who approved.17 however, through the expedited who research and development pathway, a new kit-based format of the laboratory-developed test was evaluated in nigeria and submitted for review under the who’s expert review panel for diagnostics at the end of 2018. because of comparable analytical data between the laboratory-developed and commercial kits, improved usability and an easier procurement process, the new kit format (realstar® lassa fever rt pcr kit version 2.0, altona diagnostics, hamburg, germany) was adopted within the laboratory network despite the need to perform the assay as two singleplex tests. despite the remaining challenges, such as difficulties in importing large quantities of diagnostic kits considered as dual-use by german and european union legislation, and sustainable funding to maintain national testing capacity at the current levels, the collaborations with technical partners such as the bernhard nocht institute of tropical medicine, the foundation for innovative new diagnostics and the who under the who research and development blueprint initiative, proved to be instrumental in capacity building and development of national priorities for operational research. this multi-partner collaboration has delivered tangible benefits to the country by improving diagnostic preparedness for lassa fever and demonstrating that investments made during outbreaks improve health systems. access to diagnostics and laboratory systems integration as the ncdc continues its journey to deliver on its mandate, stronger integration of surveillance and laboratory systems becomes critical for outbreak detection and response. laboratory network integration will facilitate integrated electronic reporting systems, improve the use of diagnostic testing to guide patient care across all levels of the health system, and allow for better financial planning to allocate resources required to maintain operational activities in the face of dwindling external funding. to improve nigeria’s yf and lassa fever outbreak detection and response, there is the need to integrate both systems networks into an efficient and effective laboratory surveillance system. to achieve this, yf would need to be included as part of a panel of recommended tests for a differential diagnosis on patients who test negative for lassa fever. to operationalise this differential testing, access to commercial yf molecular assays with clinical validation data is required.8 further product development is needed for safe and reliable lassa fever point-of-care testing to improve access to diagnostic testing for patients in all hospital facilities across nigeria. the experience of nigeria has shown that restrictive access to diagnostic tests as a result of unequal global investment in diagnostic development hinders the building of resilient and integrated laboratory systems. stronger leadership is required from the who to address these challenges. greater progress will be made in building laboratory systems at the country level when the framework is built on (1) multi-partner engagement with joint leadership and collaboration with the ministries of health and national public health institutes of affected countries, (2) international and domestic funding that ensures the sustainability of diagnostic testing to promote continuous product development and (3) a revised african regional laboratory strategy that promotes diagnostic stewardship and the uptake of new diagnostics for multi-pathogen testing to reduce vertical disease structures. this is not only important for nigeria but for the entire continent. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions d.n. and c.i. contributed equally to the writing of this manuscript. ethical considerations ethical clearance is not required for this opinion article. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references njidda am, oyebanji o, obasanya j, et al. the nigeria centre for disease control. bmj glob health. 2018;3(2):e000712. https://doi.org/10.1136/bmjgh-2018-000712 nigeria centre for disease control [homepage on the internet]. c2018 [cited 2020 jan 19]. available from: https://ncdc.gov.ng/reports/annualreports ilori ea, frank c, dan-nwafor cc, et al. increase in lassa fever cases in nigeria, january–march 2018. emerg infect dis. 2019;24(5):1026–1027. https://doi.org/10.3201/eid2505.181247 onyebujoh pc, thirumala ak, ndihokubwayo j-b. integrating laboratory networks, surveillance systems and public health institutes in africa. afr j lab med. 2016;5(3):a431. https://doi.org/10.4102/ajlm.v5i3.431 kieny mp, salama p. who r&d blueprint: a global coordination mechanism for r&d preparedness. lancet. 2017;389(10088):2469–2470. https://doi.org/10.1016/s0140-6736(17)31635-5 world health organization (who). the eye strategy addresses three important global health agendas [homepage on the internet]. c2017 [cited 2020 jan 19]. available from: https://www.who.int/csr/disease/yellowfev/eye-strategy-and-global-agendas/en/ kelly-cirino cd, nkengasong j, kettler h, et al. importance of diagnostics in epidemic and pandemic preparedness. bmj glob heal. 2019;4(suppl 2):e001179. https://doi.org/10.1136/bmjgh-2018-001179 domingo c, charrel rn, schmidt-chanasit j, zeller h, reusken c. yellow fever in the diagnostics laboratory. emerg microbes infect. 2018;7(1):1–15. https://doi.org/10.1038/s41426-018-0128-8 world health organization (who). weekly epidemiological record, 10 august 2018, vol. 93, 32 (pp. 409–416) [homepage on the internet]. c2018 [cited 2019 mar 23]. available from: https://www.who.int/wer/2018/wer9332/en/ world health organization (who). 2000 – yellow fever in nigeria [homepage on the internet]. c2015 [cited 2019 mar 25]. available from: https://www.who.int/csr/don/2000_05_19/en/ nigeria centre for disease control. an update on yellow fever outbreak in nigeria [homepage on the internet]. c2018 [cited 2019 mar 23]. available from: https://ncdc.gov.ng/diseases/sitreps/?cat=10&name=anupdateofyellowfeveroutbreakinnigeria martin da, muth da, brown t, johnson aj, karabatsos n, roehrig jt. standardization of immunoglobulin m capture enzyme-linked immunosorbent assays for routine diagnosis of arboviral infections. j clin microbiol. 2000;38(5):1823–1826. https://doi.org/10.1128/jcm.38.5.1823-1826.2000 world health organization (who). international coordinating group (icg) on vaccine provision [homepage on the internet]. c2016 [cited 2020 jan 19]. available from: https://www.who.int/csr/disease/icg/qa/en/ nigeria centre for disease control. response to yellow fever cases in edo state [homepage on the internet]. c2018 [cited 2019 mar 17]. available from: https://ncdc.gov.ng/news/157/response-to-yellow-fever-cases-in-edo-state best m, sakande j. practical recommendations for strengthening national and regional laboratory networks in africa in the global health security era. afr j lab med. 2016;5(3):a471. https://doi.org/10.4102/ajlm.v5i3.471 asogun da, adomeh di, ehimuan j, et al. molecular diagnostics for lassa fever at irrua specialist teaching hospital, nigeria: lessons learnt from two years of laboratory operation. plos negl trop dis. 2012;6(9):e1839. https://doi.org/10.1371/journal.pntd.0001839 mazzola lt, kelly-cirino c. diagnostics for lassa fever virus: a genetically diverse pathogen found in low-resource settings. bmj glob health. 2019;4(suppl 2):e001116. http://doi.org/10.1136/bmjgh-2018-001116 acknowledgements references about the author(s) iruka n. okeke department of pharmaceutical microbiology, faculty of pharmacy, university of ibadan, ibadan, nigeria citation okeke in. african laboratory medicine in the time of covid-19. afr j lab med. 2020;9(1), a1447. https://doi.org/10.4102/ajlm.v9i1.1447 editorial african laboratory medicine in the time of covid-19 iruka n. okeke copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 2020 has been a turbulent year but one that fixed the importance of laboratory medicine in the eye of the global public. public health experts’ worst fears surrounding a hypothetical ‘agent x’ that spurred thousands of calls for ‘pandemic preparedness’ in the last half-decade have become eerily true and the importance of ‘testing’ is now broadly acknowledged. by the end of the first quarter of 2020, following its emergence in wuhan, china, agent x was named severe acute respiratory syndrome coronavirus 2 (sars-cov-2); it had caused infections in almost every country, and one of the worst pandemics of all time was underway (figure 1). instituting sars-cov-2 testing rapidly and reliably was one of the pivotal distinguishers between countries that got a handle on their coronavirus disease 2019 (covid-19) epidemics and those that did not. figure 1: in the time of covid-19. the importance of laboratory medicine has been made more visible by covid-19 than at any time this century. no country in the world was ready by the time covid-19 overwhelmed each national surveillance and health system. across africa, pandemic response planning began early and, as sars-cov-2 entered our continent relatively late, most african countries were able to deploy testing before the virus arrived.1,2 to their tremendous credit, african countries leveraged this testing advantage to implement pandemic infection control policies that likely combined with as yet unknown biological features to yield what are still now relatively temperate versions of covid-19 devastation. african countries have also continued to grow testing capacity so that, while we are not the necessary step ahead of the virus, most countries in africa are, to some degree, keeping pace with it. national epidemic responses depend on very local activities. published this year in the african journal of laboratory medicine (ajlm) is an exemplary local response to a covid-19 crisis in an african sars-cov-2 testing laboratory. the crisis was a laboratory outbreak that not only endangered the medical laboratory scientists, but also disabled the testing service when the need was greatest.3 among other things, the affected laboratory implemented heightened infection prevention and control measures and increased physical distancing. it also increased automation of sample processing, which reduced contact between staff and infected specimens, and will improve operations in the years to come. as another article from skosana et al.4 demonstrates, workplace infections are an ever-present risk in laboratories and so lessons learned from this pandemic will have broad application. at the patient level, a significant proportion of covid-19 survivors live for months with disabling sequelae in ‘long-covid’; the ultimate prognosis for this new disease remains unclear.5 covid-19 also has unclear long-haul implications on communities, on countries and on healthcare systems and their laboratories. the pandemic is a frontal challenge to weak systems, but it also offers opportunities to develop them and build resilience, if the response is implemented with this in mind.6 for example, future procurement could be templated on more generally applicable network approaches to procurement and supply chain management outlined by williams et al. for hiv, forestalling the procurement crises experienced in africa with covid-19 testing and personal protective equipment supplies.7,8 sars-cov-2 is not the only epidemic pathogen that circulated in africa in 2020. the democratic republic of congo declared a 21-month ebola outbreak that ended on 14 may 2020, but on 01 june 2020, a new outbreak began. nigeria has worked to contain lassa fever virus epidemics for most of the duration of the covid-19 pandemic. while these three feared viruses ravage, measles, cholera and other epidemics are also in play.8 one of the things that has become increasingly visible in the course of these epidemics is the central role laboratory medicine must play to contain them. volume 9, issue 1 of ajlm goes beyond chronicling a broad range of infectious disease catastrophes to outlining lessons learned that will strengthen health systems.3,9,10,11,12,13,14,15,16,17,18 while most pandemic activity has focused on reverse transcription polymerase chain reaction and other forms of testing to support the identification of cases and transmission chains,10 other domains of laboratory medicine have made important contributions that shed more light on the pathogenesis of the disease, and therefore how best to improve treatment.19,20 as clinical and laboratory health workers and the scientific community engaged in discoveries to combat the pandemic, another equally frenetic behind-the-scenes response took place in editorial offices as we, gratefully helped by volunteer reviewers, struggled under the mound of covid-19 submissions to bring to the fore discoveries that are most worthy of priority and long-term attention. in mid-may, relatively early in the pandemic, a science commentary reported that covid-19 researchers and policymakers were becoming overwhelmed by the literature in this brand new field with a 20-day doubling time on the number of articles indexed by major databases.21 by early november, that curve had flattened somewhat but, still, over 70 000 covid-19 articles were indexed in pubmed or posted on pre-print servers, awaiting assessment. the flood has spurred the creation of artificial intelligence approaches for curating the literature.21 but the bulk of the work required to review, improve and present these works to the drowning community is done manually by editors and volunteer reviewers. unsurprisingly, the pandemic has seen highly publicised discourse on research article quality, as well as some very high-profile retractions. the strain on peer review has also led to some misleading information driving covid-19 policy, prevention and therapeutics, some of which have been amplified by influential non-scientists. among the many lessons learned on the fly in this pandemic is that health crises bring an influx of emergency-care patients, specimens requiring immediate testing and a flood of literature. resilience is needed as much in scientific publishing as in health systems. pandemic preparedness must include plans for rapidly but effectively sifting through the literature flood to retrieve the knowledge most likely to reign in the emergency, which is the only way to stop the overwhelm on both fronts. as a regional journal addressing laboratory medicine on a continent that has seen multiple epidemics this year, we at ajlm quickly found that our standard workflows would not manage submissions fast and rigorously enough for a helpful pandemic response. at the same time, it was important for us to continue to process manuscripts across the journal’s scope. we did manage to ensure that the journal could contribute in valuable ways towards addressing the pandemic while at the same time ensuring that important articles not focused on the pressing problem of covid-19 were published. indeed, by responding to covid-19 but not succumbing to ’coviddisation’,22 we observed that articles submitted before the pandemic was declared were key to the covid-19 response. among these are the future of diagnostics special issue article by preiser and van zyl23 on pooled testing, with a focus on hiv, which included critical knowledge for aligning test-and-trace covid-19 needs with the extreme resource limitations that almost every country has seen associated with polymerase chain reaction testing. additional articles in that special issue also have direct relevance to strengthening testing capabilities in the pandemic.24,25 to meet the double demands of maintaining peer review and production of regular articles, the submission rate of which continues to rise, and processing pandemic submissions, the pandemic pushed us to alter our operations. ajlm’s new normal allows for a regular submission track, and a new fast track for articles containing knowledge that could be applied to the ongoing covid-19, lassa fever and ebola haemorrhagic fever outbreaks. this year, these ran in addition to the call to our special issue (volume 9, issue 2) on the future of diagnostics, which was guest edited by timothy amukele, noah fongwen and rosanna peeling.26 our fast track remains open and will include the many excellent articles we are processing now for volume 10. altogether, ajlm received 47 submissions through its viral epidemic fast track between april 2020 when we opened the track and october of the same year. only two of these epidemic articles addressed lassa fever, and none addressed ebola haemorrhagic fever, pointing to the disproportionately low scientific activity on local african epidemics. so far, just 10 of the covid-19 fast-tracked articles have been accepted. this acceptance rate compares with that for regular submissions but is unexpectedly low, because we expected predominantly pressing issues worthy of publication to come via the fast track. similar high submission, low acceptance rates have been reported by editors of other journals.27 do the low acceptance rates make all the extra hard work done on pandemic submissions by the editorial offices worth it? this question is important to us, because all three epidemics are still in play, our journal’s overall submission rate has continued to rise and our fast track is still open. the answer is undoubtedly yes. firstly, by overseeing the peer review of the manuscripts we received, we did help to shield frontline health workers and policymakers from the even larger deluge of covid-19 preprints that they would otherwise have had to navigate. this is an important function of peer review in general. secondly and more pointedly, we are proud to be publishing key articles that influence the course of the pandemic, particularly on the african continent, where the dynamics are different, the response, while variable, is largely commendable and resources are severely limited. given the sacrifices made in so many areas towards containing this pandemic, this is the least we could do. acknowledgements i thank the authors, reviewers, editorial office and publishers’ staff that made african journal of laboratory medicine’s necessary capacity surge in this pandemic possible. i am also grateful to frontline medical laboratory science and other health professionals for their selfless service through this pandemic. competing interests the author has declared that no competing interests exist. author’s contributions i conceived and wrote the editorial. disclaimer the views and opinions expressed in this article are those of the author and do not necessarily reflect the official policy or position of any affiliated agency of the author. references kapata n, ihekweazu c, ntoumi f, et al. is africa prepared for tackling the covid-19 (sars-cov-2) epidemic. lessons from past outbreaks, ongoing pan-african public health efforts, and implications for the future. int j infect dis. 2020;93:233–236. https://doi.org/10.1016/j.ijid.2020.02.049 senghore m, savi mk, gnangnon b, hanage wp, okeke in. leveraging africa’s preparedness towards the next phase of the covid-19 pandemic. lancet glob health. 2020;8(7):e884–e885. https://doi.org/10.1016/s2214-109x(20)30234-5 opperman cj, marais gjk, naidoo m, hsiao m, samodien n. response to a cluster of severe acute respiratory syndrome coronavirus 2 cases at a diagnostic laboratory. afr j lab med. 2020;9(1):a1307. https://doi.org/10.4102/ajlm.v9i1.1307 skosana l, ismail f, mbelle n, said m. brucellosis – laboratory workers’ nightmare come true: a case study. afr j lab med. 2020;9(1):a1114. https://doi.org/10.4102/ajlm.v9i1.1114 honigsbaum m, krishnan l. taking pandemic sequelae seriously: from the russian influenza to covid-19 long-haulers. lancet. 2020;396(10260):1389–1391. https://doi.org/10.1016/s0140-6736(20)32134-6 umaru fa. scaling up testing for covid-19 in africa: responding to the pandemic in ways that strengthen health systems. afr j lab med. 2020;9(1):a1244. https://doi.org/10.4102/ajlm.v9i1.1244 nkengasong jn, mankoula w. looming threat of covid-19 infection in africa: act collectively, and fast. lancet. 2020;395(10227):841–842. https://doi.org/10.1016/s0140-6736(20)30464-5 nkengasong jn, tessema sk. africa needs a new public health order to tackle infectious disease threats. cell. 2020;183(2):296–300. https://doi.org/10.1016/j.cell.2020.09.041 egyir b, obeng-nkrumah n, kyei gb. covid-19 pandemic and antimicrobial resistance: another call to strengthen laboratory diagnostic capacity in africa. afr j lab med. 2020;9(1):a1302. https://doi.org/10.4102/ajlm.v9i1.1302 olalekan a, iwalokun b, akinloye om, popoola o, samuel ta, akinloye o. covid-19 rapid diagnostic test could contain transmission in lowand middle-income countries. afr j lab med. 2020;9(1):a1255. https://doi.org/10.4102/ajlm.v9i1.1255 mitton b, rule r, mbelle n, van hougenhouck-tulleken w, said m. post-procedural bacillus cereus septic arthritis in a patient with systemic lupus erythematosus. afr j lab med. 2020;9(1):a1119. https://doi.org/10.4102/ajlm.v9i1.1119 govender s, mbambo l, nyirenda m, sebitloane m, abbai n. herpes simplex virus-2 infections in pregnant women from south africa: evaluation of the immunoflow rapid test. afr j lab med. 2020;9(1):a854. https://doi.org/10.4102/ajlm.v9i1.854 haumba sm, toda m, jeffries r, et al. prevalence of cryptococcal antigen (crag) among hiv-positive patients in eswatini, 2014–2015. afr j lab med. 2020;9(1):a933. https://doi.org/10.4102/ajlm.v9i1.933 madeira cm, azam ki, sato dn, khosa c, bhatt n, viegas so. evaluation of the ogawa-kudoh method for tuberculosis isolation in two health units in mozambique. afr j lab med. 2020;9(1):a929. https://doi.org/10.4102/ajlm.v9i1.929 pasipamire m, broughton e, mkhontfo m, maphalala g, simelane-vilane b, haumba s. detecting tuberculosis in pregnant and postpartum women in eswatini. afr j lab med. 2020;9(1):a837. https://doi.org/10.4102/ajlm.v9i1.837 sayed s, mutasa r, kaaya e, et al. establishing the college of pathologists of east, central and southern africa – the regional east central and southern africa college of pathology. afr j lab med. 2020;9(1):a979. https://doi.org/10.4102/ajlm.v9i1.979 mudenda v, malyangu e, sayed s, fleming k. addressing the shortage of pathologists in africa: creation of a mmed programme in pathology in zambia. afr j lab med. 2020;9(1):a974. https://doi.org/10.4102/ajlm.v9i1.974 demba rn, aradi sm, mwau m, mwanda wo. kaposi’s sarcoma-associated herpesvirus protein orf75 among hiv-1 patients in kenya. afr j lab med. 2020;9(1), a939. https://doi.org/10.4102/ajlm.v9i1.939 attoh sa, hobenu f, edusei l, et al. postmortem diagnosis of covid-19: antemortem challenges of three cases at the 37 military hospital, accra, ghana. afr j lab med. 2020;9(1):a1290. https://doi.org/10.4102/ajlm.v9i1.1290 admou b, hachimi a, samkaoui ma. how can clinical immunology laboratories contribute to the management of severe covid-19 cases in limited resource contexts? afr j lab med. 2020;9(1), a1282. https://doi.org/10.4102/ajlm.v9i1.1282 brainard j. scientists are drowning in covid-19 papers. can new tools keep them afloat. science. 2020 (13 may). https://doi.org/10.1126/science.abc7839 pai m. covidization of research: what are the risks? nat med. 2020;26(8):1159. https://doi.org/10.1038/s41591-020-1015-0 preiser w, van zyl gu. pooled testing: a tool to increase efficiency of infant hiv diagnosis and virological monitoring. afr j lab med. 2020;9(2):a1035. https://doi.org/10.4102/ajlm.v9i2.1035 emperador dm, mazzola lt, kelly-cirino c. an open-source molecular diagnostic platform approach for outbreak and epidemic preparedness. afr j lab med. 2020;9(2):a1017. https://doi.org/10.4102/ajlm.v9i2.1017 naidoo d, ihekweazu c. nigeria’s efforts to strengthen laboratory diagnostics – why access to reliable and affordable diagnostics is key to building resilient laboratory systems. afr j lab med. 2020;9(2):a1019. https://doi.org/10.4102/ajlm.v9i2.1019 fongwen n, boeras d, peeling rw, amukele t. connected diagnostics systems: the future of disease control in africa. afr j lab med. 2020;9(2):a1365. in press. the lancet global health. publishing in the time of covid-19. lancet glob health. 2020;8(7):e860. https://doi.org/10.1016/s2214-109x(20)30260-6 abstract introduction research method and design results discussion acknowledgements references about the author(s) joseph m. ochieng’ oduor institute of primate research (ipr), nairobi, kenya school of medicine, kenyatta university, nairobi, kenya nyamongo onkoba institute of primate research (ipr), nairobi, kenya fredrick maloba school of pure and applied sciences, kenyatta university, nairobi, kenya atunga nyachieo institute of primate research (ipr), nairobi, kenya citation ochieng’ oduor jm, onkoba n, maloba f, nyachieo a. experimental phage therapy against haematogenous multi-drug resistant staphylococcus aureus pneumonia in mice. afr j lab med. 2016;5(1), a435. http://dx.doi.org/10.4102/ajlm.v5i1.435 note: this article was republished with the cfu and pfu dosages missing superscripts ‘108 cfu/ml and 108 pfu/ml’ corrected to ‘108 cfu/ml and 108 pfu/ml’ in the abstract. the publisher apologises for any inconvenience caused. original research experimental phage therapy against haematogenous multi-drug resistant staphylococcus aureus pneumonia in mice joseph m. ochieng’ oduor, nyamongo onkoba, fredrick maloba, atunga nyachieo received: 10 mar. 2016; accepted: 02 aug. 2016; published: 30 sept. 2016; republished: 26 aug. 2022 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: community-acquired haematogenous staphylococcus aureus pneumonia is a rare infection, though it can be acquired nosocomially. currently, antibiotics used against s. aureus pneumonia have shown reduced efficacy. thus, there is need for an alternative therapy against multidrug-resistant s. aureus (mdrsa) strains in the community. objective: we sought to determine the efficacy of environmentally-obtained s. aureus lytic phage against haematogenous mdrsa pneumonia in mice. methods: phages and mdrsa were isolated from sewage samples collected within nairobi county, kenya. isolated s. aureus bacteria were screened for resistance against ceftazidime, oxacillin, vancomycin, netilmicin, gentamicin, erythromycin, trimethroprim-sulfamethoxazole and cefuroxime. thirty balb/c mice aged six to eight weeks were randomly assigned into three groups: the mdrsa-infection group (n = 20), the phage-infection group (n = 5) and the non-infection group (n = 5). mice were infected with either mdrsa or phage (108 cfu/ml) and treated after 72 hours with a single dose of clindamycin (8 mg/kg/bwt) or 108 pfu/ml of phage or a combination therapy (clindamycin and phage). the efficacy of phage, clindamycin or clindamycin with phage combination was determined using resolution of lung pathology and bacterial load in lung homogenates. results: the viable mdrsa count was 0.5 ± 0.2 log10 cfu/gm in the phage-treated group, 4.4 ± 0.2 log10 cfu/gm in the clindamycin-treated group and 4.0 ± 0.2 log10 cfu/gm in the combination-treated group. the efficacy of phage therapy was significantly different from other therapeutic modes (p = 0 < 0.0001). histology showed that the mice treated with phage did not develop pneumonia. conclusion: phage therapy is effective against haematogenous mdrsa infection. thus, it can be explored as an alternative treatment method. introduction during lung infection with bacteria and viruses, the lung tissue becomes inflamed, deflated and occupied by pus and mucus, and the bronchial walls thicken due to accumulation of lymphocytes.1 streptococcus pneumoniae, haemophilus influenzae, mycoplasma pneumoniae and staphylococcus aureus all cause pneumonia.2 s. aureus pneumonia is usually associated with nosocomial infections common with patients in intensive care units.3 community-acquired pneumonia, especially haematogenous (blood-borne) s. aureus pneumonia is rare; however, it has been documented to have high morbidity and mortality rates world-wide.4 haematogenous pneumonia is common in children, immunocompromised persons and the elderly who have chronic skin and soft tissue infections.5 globally, there has been an emergence of multidrug-resistant bacteria, including methicillin resistant s. aureus and multidrug-resistant s. aureus (mdrsa), which are difficult to treat with currently available antibiotics.6,7 these ‘super bugs’ have made the treatment of haematogenous pneumonia and other associated infections difficult.8,9,10,11 thus, there is need for an alternative therapeutic measure, such as phage therapy, to combat super bugs. phage therapy refers to the use of bacteriophages (or phages) as antibacterial agents against bacterial pathogens.12 currently, the use of phage therapy is common primarily in georgia and russia, despite once being practised globally before the advent of antibiotics.13,14,15 its use ceased in western europe, the united states and canada in the 1940s due to poor understanding of the nature, safety, therapeutics and pharmacology of phages.16 phage therapy is cheap in terms of development and use as a therapeutic agent. phages are abundant in nature and are capable of in vivo auto-dosing, that is to say, they are capable of multiplying into several copies at the infection site; hence, only a small dose is required for treatment.17 in kenya, community-acquired mdrsa infections are commonly reported among hiv patients and at public hospitals that are located in informal settlements or slums.18 thus, there is a need to characterise the profile of bacterial strains that are currently circulating in environmental sources. this will provide necessary information that can be used in management of infections caused by mdrsa bacteria. this study sought to determine the therapeutic potential of environmentally-obtained lytic phages in nairobi, kenya, against haematogenous mdrsa pneumonia in balb/c mice. research method and design ethical considerations all experimental protocols and procedures of the study were reviewed and approved by the institutional review committee on animals ethics of the institute of primate research (ref no: irc/02/14) ethical committee on the use of laboratory animals for biomedical science in accordance with the international guidelines on animal care, handling and use for biomedical research. the experiments are reported in accordance with arrive guidelines.19 study animals the study included 30 balb/c mice of mixed sexes aged 6 to 8 weeks which were sourced from the institute of primate research rodent house, karen-nairobi, kenya, and maintained at their rodent facility. the mice were fed on antibiotic-free food rations (obtained from unga feeds, nairobi, kenya) and water was provided ad libitum. isolation of mdrsa s. aureus bacterial strains were isolated from 20 sewage samples collected from informal settlements, sewage treatment plants and abattoirs within nairobi county, kenya. specimens from all the samples were streaked on selective mannitol salt agar (liofilchem®, roseto degli abruzzi, italy) supplemented with 4 µg of ciprofloxacin [liofilchem®, roseto degli abruzzi, italy]. single colonies were amplified in nutrient agar (himedia, mumbai, india). staphylococcus strains were identified using microscopy, physiological tests and an analytical profile index of staphylococcus system [biomérieux, marcy l’etoile, france]. we screened isolated s. aureus bacteria for antimicrobial resistance to the following drugs: ceftazidime 30 µg, oxacillin 1 µg, vancomycin 30 µg, netilmicin 30 µg, gentamicin 10 µg, erythromycin 15 µg, trimethroprim-sulfamethoxazole 25 µg and cefuroxime 30 µg (liofilchem®, roseto degli abruzzi, italy), according to the clinical laboratory standard institute protocol.20 isolates were considered to be mdrsa, if they were non-susceptible to more than one class of antibiotics. colonies identified as mdrsa were re-suspended in 50% glycerol-nutrient broth and stored at -20 °c until use. phage isolation we analysed half a litre of sewage samples collected from 10 sites (nairobi city sewage treatment plant, informal settlements and an abattoir) within nairobi county, kenya. twenty specific s. aureus lytic phages were isolated from these samples using the modified method of anany et al.21 briefly, equal volumes of ultra-filtered sewage samples and nutrient broth, plus 1 ml of 18-hour-old mdrsa culture were mixed and incubated overnight at 37 °c while shaking at 120 rpm (lab-line® incubator-shaker, waltham, massachusetts, united states). the following day, the culture was centrifuged at 10 000 g for 10 minutes (fisher centrific® centrifuge, waltham, massachusetts, united states), and the supernatant was filtered through a 0.22 µm filtration unit (cambridge, massachusetts, united states), then screened for the presence of phages using a double-layer plaque assay. individual plaques were selected and sub-cultured in 2 ml of nutrient broth containing a sensitive bacterial host (106 cfu/ml). the isolated phages were then tested against the previously-isolated mdrsa by spot assay. the virulence of the phages was determined by using an efficiency of plating analysis,22 whereby the strain with numerous plaques on mdrsa lawn was considered the most virulent. the virulent phage was selected and used as a therapeutic agent against mdrsa in mice. experimental design thirty mice were randomly assigned to one of three groups: the mdrsa infection group (n = 20), the non-infection group (n = 5) and the phage infection group (n = 5). twenty mice were infected with 108 cfu/ml of mdrsa isolate intravenously by means of the tail vein and then sub-divided into four sub-groups: mdrsa with clindamycin (clindar, indi pharma, mumbai, india) (8 mg/kg body weight) treatment (n = 5); mdrsa with phage (108 pfu/ml) treatment (n = 5); mdrsa with combination treatment (clindamycin [8 mg/kg body weight] and phage [108 pfu/ml]) (n = 5); and mdrsa with no treatment as the control (n = 5) (figure 1). presence of infection was determined by observing the physical appearance of the mice and assessing the bacteraemia level. the bacteraemia count was assessed by culturing blood samples from each mouse by using the pour plate method. fifty microlitres (50 µl) of blood was obtained from each mouse from the tail vein and mixed with 950 µl sterile normal saline. this mixture (blood-normal saline) was added to molten nutrient broth, allowed to cool, then incubated overnight at 37 °c. the bacterial colony count was recorded per millilitre for each mouse. a similar procedure was used to determine the bacteraemia for each mouse after treatment was initiated. treatment with clindamycin, phage or combination was administered at 72 hours post-mdrsa infection when the bacterial levels in the blood began to rise. each mouse in the treatment groups received a single dose of the mentioned therapeutic agents. the experiments were repeated three times; thus, the total number of mice used in the study was 90. figure 1: design of the in vivo experimental study. the study was repeated three times and a total number of 90 mice were used. viable bacteria levels in mouse lung homogenates upon necropsy, lungs were obtained aseptically and homogenised for bacterial culture as follows: 500 µl of homogenised tissue was diluted with normal saline at a ratio of 1:20 (homogenised lung tissue to saline). the diluted, homogenised tissue was then plated on 7.5% sodium chloride nutrient agar to select for mdrsa and incubated at 37 °c for 18–20 hours. histopathology phage safety and mdrsa pathogenicity were determined by euthanising the mice when they exhibited poor physical appearance and breathing difficulties in order to alleviate pain and suffering. surviving mice were euthanised at 10 days post-infection. lung samples were collected in 10% formaldehyde for histological analysis. levels of inflammation were scored using categories as described by schünemann et al.23 briefly, the categories worst, worse, bad, good, better and best were assigned based on a scale of 5 to 0. the ‘worst’ category was assigned for severely inflammed septa, numerous collapsed alveoli, large pockets of pneumonia, mucus-congested alveoli, presence of perivascular fibrosis blood vessels and lack of ventilation (score = 5). the ‘worse’ category included all conditions in the worst category but with poor ventilation (score = 4). the ‘bad’ category was assigned for minor inflammation of septa, few pockets of pneumonia and moderate ventilation (score = 3). the ‘good’ category was assigned for minor inflammation of septa, few pockets of mucus-congested alveoli and good ventilation (score = 2). the ‘better’ category was assigned for no inflammed septa and a few pockets of mucus-congested alveoli (score = 1). the ‘best’/normal category was assigned for no inflammation and good ventilation (score = 0). statistical analysis bacterial and phage counts were represented as mean ± standard error of the mean. the statistical significance of differences between groups was determined by one-way analysis of variance, followed by tukey’s multiple comparison test, using graph pad prism 5.0.1 (graph pad software, san diego, california, united states). a p-value of less than 0.05 was considered statistically significant. results multidrug-resistant s. aureus isolation the environmentally-isolated s. aureus bacterium was resistant to β-lactam antibiotics (ceftazidime, cefuroxime and oxacillin). in addition, it was non-susceptible to glycopeptide (vancomycin), aminoglycoside (netilmicin and gentamicin) and macrolide (erythromycin) antibiotics. however, it was susceptible to nucleic acid synthesis inhibitors (co-trimoxazole [trimethroprim-sulfamethoxazole]) (figure 2). thus, the bacterial isolate was considered to be multidrug-resistant, as it was non-susceptible to more than one class of antibiotics. figure 2: antibiogram test of s. aureus isolate. the isolate was resistant to: ceftazidime, 30 µg (1); oxacillin, 1 µg (2); vancomycin, 30 µg (4); netilmicin, 30 µg (6); gentamicin, 10 µg (7); erythromycin, 15 µg (8); and cefuroxime, 30 µg (5). however, it was sensitive to trimethroprim-sulfamethoxazole, 25 µg (3). phage isolation and in vitro activities spot assays were used to identify 10 s. aureus lytic phages which produced large clear plaques on the mdrsa lawn (figure 3) while other isolates failed. of the 10 phage isolates, one created a larger patch on the mdrsa lawn. its efficiency of plating analysis showed that it had more plaques than other phages. figure 3: spot assay showing most virulent phage isolate. therapeutic effects in mice two groups of mice had 100% survivorship (n = 5), namely, the non-infected/non-treated group and the phage-infected mice. only three mice survived in the mdrsa-infected group before treatment (72 hours post-infection) commenced. all the mice that were treated with either clindamycin, phage or the combination therapy (clindamycin plus phage) survived at day 7 post-infection and each group had three mice. mice that had phage infection (phage control group) were more active than mice in other treatment groups (clindamycin, phage and combination). only one mouse in the mdrsa-infected control group survived at day 7 post-infection and it was weak (table 1). table 1: number of surviving mice at 72 hours post-infection and 7 days post-treatment, nairobi, kenya. viable bacteria levels in mouse lung homogenates the number of viable mdrsa in mouse lung homogenates was significantly lower in mice treated with phage only (log10 cfu/gm = 0.5 ± 0.2) compared with those in the mdrsa-infected, non-treated control group (log10 cfu/gm = 8.0 ± 0.2) (p < 0.0001) (figure 4). compared with the mdrsa-infected, non-treated group, statistically-significantly differences were observed in the viable bacterial counts in the mdrsa-infected, clindamycin-treated (log10 cfu/gm = 4.4 ± 0.2; p < 0.05) and mdrsa-infected, combination-treated groups (log10 cfu/gm = 4.0 ± 0.2; p < 0.001). the differences in counts between the phage-treated group and groups treated with clindamycin and the combination therapy were also statistically significant (p < 0.0001). figure 4: mean bacterial count (mean log10 cfu/gm ± se) in lung tissue from mdrsa-infected mice. controls received no treatment, remaining infected mice were treated with clindamycin, phage, or a combination therapy (clindamycin plus phage). each group had five mice. levels of significance: *p < 0.05; **p < 0.001; and ***p < 0.0001; each was compared with mdrsa control. histopathological examination the lung tissues of mice in the phage-infected group were relatively normal, with minor focal congestion compared with tissue from mice in the mdrsa-infected non-treated group, which had deflated alveoli congested with mucus, lymphocyte-infiltrated septa and pockets of serous fluid. the lung tissue of mdrsa-infected, non-treated mice were similar to lung tissue from clindamycin-treated mice. however, lung tissue from the clindamycin-treated group (n = 3) overall was graded as bad, since they had less congestion, fewer pockets of serous fluid and deflated alveoli compared with the non-treated mdrsa infected mice. mice that received the combination therapy (n = 3) showed inflammation of the septa and blood vessel walls, moderately deflated alveoli and several serous fluid pockets compared with the non-infected (n = 5) and phage-infected mice (n = 5) (p < 0.0001) (figure 5). figure 5: mouse lung tissue slides (40x magnification). tissue from non-infected, non-treated mice (a) and phage-infected, non-treated mice (b) were well ventilated. tissue from mdrsa-infected, non-treated control mice (c) had pockets of serous fluid (pneumonia) (1), lymphocyte-infiltrated (inflamed) septa (2), deflated alveoli (3) and alveoli full of mucus (4). tissue from mdrsa-infected, clindamycin-treated mice (d) had pockets of pneumonia (1), inflamed septa and perivascular fibrosis of blood vessels (5). tissue from mdrsa-infected, phage-treated mice (e) was well ventilated with resolved inflammation. tissue from mdrsa-infected mice treated with a combination of clindamycin and phage (f) had pockets of pneumonia (1), inflamed septa (2) and deflated alveoli (3). discussion this study found that s. aureus isolates collected from sewage water and found to be resistant to multiple drug therapies caused haematogenous pneumonia in mice, which was characterised by infiltration of immune cells and accumulation of serous fluid in the lungs. this confirmed that the isolated bacterium was pathogenic and able to cause sepsis, then was further confirmed by isolation of viable mdrsa bacteria from the lung homogenates. the accumulation of serous fluid and pus in the lungs indicated that the mice induced an innate immune response toward multiplying mdrsa bacteria.24 this inflammatory immunological phenomenon may have led to the inflammation observed in the bronchi, bronchioles, alveoli and septa, as well as blood vessels. our findings agree with other studies in which mice were infected intravenously with methicillin-resistant s. aureus, then administered a dose of antibiotic on a daily basis for a week.25,26,27 however, in our study we used a single dose of s. aureus-specific lytic phage. the use of phage as a treatment method attained 100% efficacy, an indication that the single dose administered was enough to clear the bacteria from the infected mice. this is probably due to the auto-dosing properties of phages, whereby they increase in numbers at the site of an infection.28 the mode of phage administration, concentration, dosage and timing of treatments and resistance to phages by the pathogenic bacteria are some of the factors that determine the efficacy and safety of phage therapy.29 these factors may be responsible for the differences between our findings and those of other studies,30,31 in which methicillin-resistant s. aureus-infected mice were treated immediately or six hours after infection with phage to achieve a 100% efficacy. the timing of the initiation of treatment may be the crucial factor in achieving 100% efficacy. however, we cannot rule out the role of immunity in clearing the bacteria, considering that yao et al.32 established that within 72 hours of infection the mouse immune system has the capacity to clear bacteria from the system. in our study, the combination therapy (clindamycin plus phage) reduced the efficacy of the phage to about 60%, indicating that clindamycin may have an antagonist effect on phage lytic activity. clindamycin is a bacteriostatic antibiotic; thus, it may interfere with phage protein synthesis, which would interrupt the phage auto-dosing mechanism. limitations of the study our study results were generated in mice. while our findings provide vital information on the antimicrobial resistance profile in kenya, they cannot be translated directly to humans. however, comparative animal models can be utilised to ascertain the safety and therapeutic potential of environmentally-available phages against infections caused by multidrug-resistant bacteria. conclusion in conclusion, we report for the first time that a single dose of mdrsa-specific lytic phage is efficacious against haematogenous pneumonia caused by multidrug-resistant s. aureus in mice. acknowledgements we acknowledge the institute of primate research staff for providing logistical support during experimentation; dr geoffrey omuse and dr allan njoroge (aga khan university hospital, nairobi, kenya) for donating atcc 43300; prof w. jaoko and mr j. oloo (university of nairobi, medical school, kenya) for donating atcc 29213; and prof. b. weber-dabrowska and mr m. lusiak-szelachowska (institute of immunology and experimental therapy, wroclaw, poland) for providing phage isolation protocols. this study was approved by the ethics committees of the institute of primate research (ref no: irc/02/14). all institutional and national guidelines for the care and use of animals were followed. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. sources of support none. authors’ contributions j.m.o.o. made conceptual contributions. j.m.o.o. and a.n. designed the study and the experimental protocols. j.m.o.o. collected the samples, performed the experimentation, and collected and analysed the data. f.m. assisted in reading the histology slides. j.m.o.o. and n.o. wrote the article. all authors read and approved the final article for publication. references alcón a, fàbregas n, torres a. 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of mice against lethal staphylococcus aureus infection by novel bacteriophage phi mr11. j infect dis. 2003;187(4):613–624. epub 2003 feb 7. http://dx.doi.org/10.1086/374001 thiel k. old dogma, new tricks—21st century phage therapy. nat biotechnol. 2004;22(1):31–36. http://dx.doi.org/10.1038/nbt0104-31 yao l, berman jw, factor sm, et al. correlation of histopathologic and bacteriologic changes with cytokine expression in an experimental murine model of bacteremic staphylococcus aureus infection. infect immun. 1997;65(9):3889–3895. abstract introduction methods results discussion acknowledgements references about the author(s) dawood da costa division of medical microbiology and immunology, department of pathology, faculty of medical and health sciences, stellenbosch university, cape town, south africa national health laboratory service, tygerberg hospital, cape town, south africa pieter nel division of medical microbiology and immunology, department of pathology, faculty of medical and health sciences, stellenbosch university, cape town, south africa national health laboratory service, tygerberg hospital, cape town, south africa citation da costa d, nel p. re-decontamination of liquid mycobacterial cultures: additional mycobacterium tuberculosis yield in the era of xpert mtb/rif ultra in cape town, south africa. afr j lab med. 2021;10(1), a1529 https://doi.org/10.4102/ajlm.v10i1.1529 brief report re-decontamination of liquid mycobacterial cultures: additional mycobacterium tuberculosis yield in the era of xpert mtb/rif ultra in cape town, south africa dawood da costa, pieter nel received: 15 jan. 2021; accepted: 26 july 2021; published: 10 dec. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract a retrospective review of liquid mycobacterial cultures was performed at a laboratory in south africa from 01 january 2018 to 31 december 2018 to assess the increased yield in detecting mycobacterium tuberculosis complex following sample re-decontamination. only 9 of 99 (9%) re-decontaminated samples were culture positive for m. tuberculosis complex. xpert mtb/rif ultra, concurrently performed on 7 of the 9 samples, detected m. tuberculosis complex in all but 1 sample. re-decontamination of non-sterile samples did not increase the m. tuberculosis complex yield enough to offset the financial costs and additional labour in a laboratory that utilises the xpert mtb/rif ultra system as a first-line diagnostic modality. keywords: tuberculosis culture, liquid mycobacterial growth indicator tube culture, decontamination, re-decontamination, xpert mtb/rif ultra. introduction tuberculosis is caused by mycobacterium tuberculosis, which belongs to a group of closely related, slow-growing mycobacteria collectively referred to as the m. tuberculosis complex. in 2018, there were an estimated 10 million cases of tuberculosis globally, with seven million cases notified and 1.45 million tuberculosis-related deaths1. south africa, in the same year, had an estimated 301 000 new tuberculosis cases1. detection of m. tuberculosis complex by culture method remains the gold standard for confirming tuberculosis disease.2 it can detect as few as 10 m. tuberculosis complex colony-forming units (cfu) per ml in clinical specimens.3 liquid culture increases the number of bacteriologically confirmed cases of tuberculosis by 20% – 30%, even when rapid, sensitive nucleic acid amplification tests such as the xpert ultra (cepheid, sunnyvale, california, united states)3,4 are used. culture is advantageous as it allows for phenotypic drug susceptibility testing of the isolates. processing of specimens for culture is, however, costly, labour intensive, expertise requiring, and time-consuming (requiring up to six weeks to process to completion) owing to the slow proliferation time of m. tuberculosis complex.5,6,7 the xpert ultra assay is a closed, cartridge-based, nucleic acid real-time polymerase chain reaction system that allows for the simultaneous detection of m. tuberculosis complex and rifampicin susceptibility in under 2 h. it can be performed directly on clinical samples and has a 5 cfu/ml – 25 cfu/ml limit of detection; but, it has a lower sensitivity than tuberculosis culture in samples from people living with hiv and on samples where no acid-fast bacilli (afb) are visualised on microscopy (smear negative).3,4 clinical specimens from non-sterile sites that are submitted to the mycobacteriology laboratory may be contaminated by other more rapid-growing bacteria.5,8 these specimens undergo a digestion-decontamination procedure as recommended by the world health organization. the n-acetyl-l-cysteine sodium hydroxide digestion-decontamination allows m. tuberculosis to be cultured in a liquid culture medium despite reducing the organism viability by between 20% and 30%.3,6,8,9 a proportion of cultures will however remain contaminated despite standard decontamination procedures. an acceptable rate for contaminated cultures in liquid media is 5% – 8%.5,6,7,8,10 global recommendations for re-decontamination exist,7 but no published data on an acceptable compliance level for re-decontamination could be found. the world health organization recommends that re-decontamination of contaminated liquid culture be performed when the first of two submitted cultures is ‘positive for m. tuberculosis and contaminated’ and the specimen requires drug susceptibility testing, while the second culture is negative for m. tuberculosis, and if the mycobacterial protein antigen 64 result is indeterminate, owing to the presence of contaminants. the aims and objectives of the study were to determine the contamination rate at the tygerberg hospital (tbh) mycobacteriology laboratory; assess the increase in m. tuberculosis complex yield following re-decontamination in samples that had undergone xpert ultra testing; to assess laboratory non-compliance rates with regard to the recommended sample re-decontamination protocol; and to perform a cost analysis of the re-decontamination of specimens. methods ethical considerations ethical approval for this laboratory-based study was obtained from the health research ethics committee, stellenbosch university, south africa, (project identification: 14939, hrec x20/04/016). informed consent was waived by the stellenbosch university ethics committee for this laboratory-based study. data remained confidential throughout the study. study setting this study was conducted at south africa’s national health laboratory service (nhls) division of medical microbiology and immunology mycobacteriology laboratory located in tbh. annually, the tbh mycobacteriology laboratory processes approximately 10 000 specimens for m. tuberculosis complex culture from tbh. all samples from non-sterile sites for m. tuberculosis complex culture undergo decontamination with 1.25% n-acetyl-l-cysteine sodium hydroxide and the decontaminated samples are processed according to the becton-dickinson mycobacterial growth indicator tube (mgit) testing protocol.5 specimen re-decontamination is performed on specimens from anatomical sites that are not easily obtainable or contaminated specimens that are microscopy positive for afb. easily obtainable anatomical specimens from non-sterile sites, such as urine and sputum specimens, are not re-decontaminated.11,12 study design and data analysis a retrospective study was conducted to determine the number of m. tuberculosis complex cultures performed from 01 january 2018 to 31 december 2018. data were electronically extracted from the nhls central data warehouse into a microsoft excel (microsoft office 2016, microsoft corporation, redmond, washington, united states) datasheet. results were anonymised and stratified according to culture status: negative, positive or contaminated. contaminated samples underwent further analysis to determine eligibility for re-decontamination; compliance rates were calculated, and results were verified on the nhls database, trakcare webview (trakcare lab version l6.10, 2012, intersystems corporation, cambridge, massachusetts, united states). results a total of 9585 m. tuberculosis complex cultures were performed; 8049 (82.1%) were culture-negative, 912 (9.3%) were positive for m. tuberculosis complex, 31 (0.3%) were positive for non-tuberculous mycobacteria, and 593 (6.0%) were contaminated. a total of 139 samples were assessed for re-decontamination, of which 99 (71%) were appropriately re-decontaminated, 37 (29%) were appropriately denied re-decontamination (due to having multiple samples incubating), and 3 (2.2%) were inappropriately denied re-decontamination. three samples were eligible for re-decontamination but did not undergo re-decontamination: two were sputum samples, in which afb was observed on the contaminated culture and had xpert ultra testing which detected rifampicin-susceptible m. tuberculosis complex, and one was a bronchoalveolar lavage sample in which a second sample was contaminated. non-compliance to re-decontamination was low, with 97.8% of samples correctly assessed for re-decontamination. of the 99 re-decontaminated samples, 75 (76%) were culture-negative, 5 (5%) contaminated, 10 (10%) positive for non-tuberculous mycobacteria and nine (9%) were positive for m. tuberculosis complex. of 99 re-decontaminated samples, 89 samples were from anatomical sites not easily obtainable and 10 were samples that were microscopy positive for afb. of the 10 contaminated samples that were microscopy positive for afb and that underwent re-decontamination, all were sputum samples; non-tuberculosis mycobacteria were isolated from three samples and m. tuberculosis was isolated from seven. only six of the seven samples that had m. tuberculosis complex isolated on culture underwent xpert ultra testing and were all positive for m. tuberculosis complex. in summary, only 9 of 99 (9%) re-decontaminated samples were culture positive for m. tuberculosis complex. on xpert ultra testing, 6 of the 9 (67%) tested positive for m. tuberculosis complex, 1 (11%) tested negative for m. tuberculosis complex, and 2 (22%) did not undergo xpert ultra testing (but were smear positive for afb on an auramine stain). discussion we found that 6% of all specimens undergoing mycobacterial culture at the tbh mycobacteriology laboratory were contaminated, which is in keeping with internationally accepted contamination standards of 5% – 8% in liquid media.5,6,7,8,10 non-compliance to the recommended re-decontamination standard operating procedure was low at 2.2%. these three samples were not re-decontaminated as the xpert ultra had detected m. tuberculosis complex on two samples, and the third sample had additional specimens still incubating. we found that of the seven re-decontaminated samples that were microscopy positive for afb and culture positive for m. tuberculosis complex, six (86%) were also xpert ultra positive for m. tuberculosis complex. a diagnostic accuracy study of the xpert ultra by dorman et al., with study participants from south africa, found the sensitivity for smear-positive m. tuberculosis complex to be 99%4 suggesting that xpert ultra would likely have detected m. tuberculosis complex in the sample that was not tested. in our setting, re-decontamination of samples that have undergone xpert ultra testing only yielded one additional m. tuberculosis complex isolate and although the sample size to identify the additional positive m. tuberculosis complex yield is small, to our knowledge this is the first study assessing the additional m. tuberculosis complex yield in re-decontaminated samples. the correlation between xpert ultra and m. tuberculosis complex positivity in re-decontaminated samples also reflects the excellent utility of xpert ultra testing as the initial diagnostic test in the south african national tuberculosis-testing algorithm. this finding is likely due to the xpert ultra’s lower m. tuberculosis complex detection limit of 5 cfu/ml – 25 cfu/ml compared to its predecessor xpert mtb/rif.3 currently, the cost of re-decontamination and additional liquid mgit culture at tbh nhls amounts to r79.22 (south african rand; approximately, $5.00 united states dollars) per sample. when considering the low additional m. tuberculosis complex yield, added labour, and long turnaround time to final culture result, re-decontamination is not a cost-effective option in the setting where xpert ultra is used as the initial diagnostic test. limitations this was a laboratory-based study using routinely available data. the treatment status of patients who submitted samples could not be obtained, which may have impacted on m. tuberculosis complex yield following re-decontamination. owing to the small sample size eligible for re-decontamination, and varying laboratory decontamination protocols, the findings in this study may not allow generalisation of our findings to other centres. conclusion the poor increase in yield of m. tuberculosis complex after re-decontamination of samples reflects the efficiency of the south africa tuberculosis-testing algorithm, which employs xpert ultra testing, that has low limit of detection. in our high-burden tuberculosis setting, routine re-decontamination is not cost-effective and not recommended in specimens that have undergone xpert ultra testing. acknowledgements we thank the nhls for access to their electronic central data warehouse and mr j. goodway for electronic data extraction. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions d.d.c. was responsible for the concept and study design, analysis and interpretation of data, and writing of the manuscript. d.d.c. and p.n. were involved in the critical revision and approval of the final manuscript. sources of support the authors received no financial support for the research, authorship or publication of this article. data availability the data that support the findings of this study are available from the corresponding author, d.d.c., upon reasonable request. disclaimer the views expressed in the submitted article are the authors’ own and not an official position of the institution. references world health organization. global tuberculosis report. geneva, switzerland: who; 2019. lewinsohn dm, leonard mk, lobue pa, et al. official american thoracic society/infectious diseases society of america/centers for disease control and prevention clinical practice guidelines: diagnosis of tuberculosis in adults and children. clin infect dis. 2017;64(2):e1–e33. https://doi/org/10.1093/cid/ciw694 chakravorty s, simmons am, rowneki m, et al. the new xpert mtb/rif ultra: improving detection of mycobacterium tuberculosis and resistance to rifampin in an assay suitable for point-of-care testing. mbio. 2017;8(4):1–12. https://doi/org/10.1128/mbio.00812-17 dorman se, schumacher sg, alland d, et al. xpert mtb/rif ultra for detection of mycobacterium tuberculosis and rifampicin resistance: a prospective multicentre diagnostic accuracy study. lancet infect dis. 2018;18(1):76–84. https://doi/org/10.1016/s1473-3099(17)30691-6 siddiqi sh, rüsch-gerdes s. mgit procedure manual for bactec mgit960 tb system. geneva, switzerland: foundation for innovative new diagnostics; 2006. kent pt, kubica gp. public health mycobacteriology: a guide for the level iii laboratory. atlanta, georgia: centre for disease control; 1985. fitz-gerald m, lumb r, who global tb programme. laboratory safety – the handbook global edition [homepage on the internet]. geneva: gli working group secretariat; 2019. [cited 2020 oct 23]. available from: http://www.stoptb.org/wg/gli garcia ls. clinical microbiology procedures handbook. third edit. (garcia ls, ed.). santa monica, california: asm press; 2007. mtafya b, sabiiti w, sabi i, et al. molecular bacterial load assays concurs with culture on naoh-induced loss of mycobacterium tuberculosis viability. j clin microbiol. 57(7). https://doi/org/10.1093/jac/dku415 stop tb partnership (world health organization). childhood tb subgroup. global laboratory initiative advancing tb diagnosis. mycobacteriology laboratory manual. first edit. geneva, switzerland: global laboratory initiative; 2014. goodway j, rautenbach c. processing of positive mgit vials: national health laboratory service tygerberg laboratory standard operating procedure mic1223. cape town, south africa: nhls; 2019. rautenbach c, goodway j. tb cultures: national health laboratory service tygerberg laboratory standard operating procedure mic1222. cape town, south africa: nhls; 2019. abstract introduction methods results discussion acknowledgements references about the author(s) anthony p. oyom department of medical laboratory sciences, faculty of allied health, clarke international university (formerly international health sciences university), kampala, uganda emmanuel okello department of medical laboratory sciences, faculty of allied health, clarke international university (formerly international health sciences university), kampala, uganda victoria acam department of medical laboratory sciences, faculty of allied health, clarke international university (formerly international health sciences university), kampala, uganda christine aramo department of medical laboratory sciences, faculty of allied health, clarke international university (formerly international health sciences university), kampala, uganda bashir mwambi department of medical laboratory sciences, faculty of allied health, clarke international university (formerly international health sciences university), kampala, uganda john c. okiria department of clinical medicine and community health, faculty of allied health, clarke international university (formerly international health sciences university), kampala, uganda caesar oyet department of medical laboratory sciences, faculty of allied health, clarke international university (formerly international health sciences university), kampala, uganda citation oyom ap, okello e, acam v, et al. prevalence and antifungal susceptibility of gastrointestinal candidiasis among diabetic patients: a cross-sectional study. afr j lab med. 2020;9(1), a997. https://doi.org/10.4102/ajlm.v9i1.997 original research prevalence and antifungal susceptibility of gastrointestinal candidiasis among diabetic patients: a cross-sectional study anthony p. oyom, emmanuel okello, victoria acam, christine aramo, bashir mwambi, john c. okiria, caesar oyet received: 13 feb. 2019; accepted: 12 aug. 2020; published: 10 dec. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: gastrointestinal candidiasis is the most predominant opportunistic human mycosis, especially in diabetic patients. there is a global increase in antifungal resistance coupled with a rarity of information on antifungal susceptibility profiles in uganda. objective: this study aimed to determine the occurrence and antifungal susceptibility of gastrointestinal candidiasis in diabetic patients. methods: stool and fasting blood specimens were obtained from randomly sampled consenting patients with diabetes mellitus at st. francis hospital nsambya in kampala, uganda to determine candida infection, fasting blood glucose and glycated haemoglobin levels. susceptibility testing was performed on muller hilton agar supplemented with 2% glucose and 0.2 µg of methylene blue, using the e-test diffusion method. results: among the 241 patients included in the analyses, the overall prevalence of gastrointestinal candidiasis was 15.4% (n = 37). candida albicans (62.16%, n = 23) was the predominant species, followed by candida glabrata (18.92%, n = 7), candida tropicalis (8.11%, n = 3), candida krusei (5.41%, n = 2) and candida dublinensis (5.41%, n = 2). resistance was observed with miconazole (48.65%), clotrimazole (18.92%) and fluconazole (8.11%). no resistance to itraconazole and nystatin was observed. gastrointestinal candidiasis was associated with poor glucose control (p ≤ 0.001), prior use of antibiotics (p ≤ 0.001), antifungals (p ≤ 0.001) and corticosteroids (p ≤ 0.001) and was more common among female patients (p = 0.01). conclusion: occurrence of gastrointestinal candidiasis was relatively low among our participants, and infection was associated with poor glucose control, female sex and use of antifungals, antibiotics and corticosteroids. keywords: candidiasis; diabetes; gastrointestinal; antifungal; susceptibility. introduction candida species reside in the human gastrointestinal tract as part of the body’s microbiota. due to changes in host environment (such as immunosuppression, metabolic imbalances and dysbiosis), they can proliferate as opportunistic pathogens.1,2 they are the predominant cause of opportunistic human mycoses, and are capable of causing superficial as well as invasive mycoses.3 candida proliferation, especially with heavy growth, within the gut may result in diarrhoea and abdominal discomfort.4,5 patients with diabetes mellitus (dm) are also susceptible to intestinal candidiasis,6,7 due to the effects of the hyperglycaemic state on the immune system such as dysfunction in the microbicidal activity, chemotaxis and the phagocytosis ability of the neutrophils.8 additionally, there is increased death of leukocytes and decreased response to moderators of inflammation such as histamine and bradykinin among dm patients,9 resulting in a reduction in the capability of their immune systems to combat gastrointestinal infections. any of the candida species can cause gastrointestinal candidiasis among diabetic patients; however, candida albicans is the most common.10 studies reporting prevalence of gastrointestinal candidiasis at the global and continental levels are scarce, and studies in various countries have yielded varying results. in poland, kowalewska et al.2 reported a prevalence of gastrointestinal candidiasis to be 75.47% among type 1 dm patients; however, in india, the prevalence varied from 2.45% in north india to 9.7% in goa.10 a study in turkey reported prevalence of gastrointestinal candidiasis to be between 25% and 40%.11 a review of studies conducted across the african continent reported a non-uniform prevalence of gastrointestinal candidiasis with a continental prevalence of 12.42% but with a slightly higher prevalence in sub-saharan africa at 12.8%.9 the range of antifungal drugs available for treatment of gastrointestinal candidiasis is limited, with azoles, polyenes, allylamines, echinocandins and flucytosine as the available options.3 both c. albicans and non-albicans candida species such as candida tropicalis, candida glabrata and cand ida krusei have shown high rates of intrinsic and acquired forms of antifungal resistance.5,12,13,14 there are suggested factors that predispose diabetic patients to gastrointestinal candidiasis and these factors include superimposed immunosuppression, use of steroids, use of antibiotics and poor glycaemic control.15 despite the increase in the number of patients with dm, a non-communicable disease with the potential to induce conditions that increase the risk of mycosis,3,7 gastrointestinal candidiasis among diabetic patients remains an understudied condition,2,5 with few guidelines on the identification and treatment of such infections.16,17 in uganda the problem is compounded by a lack of adequate microbiology facilities in most health laboratories, which inhibits the timely diagnosis and treatment of such infections. we aimed to determine antifungal susceptibility of gastrointestinal candida isolates from dm patients with persistent diarrhoea at st. francis hospital nsambya. methods ethical considerations this study was approved by the institutional ethics committee and institutional review board of international health sciences university: approval number ihsu-rec/0046. all participants provided written informed consent before the enrolment, and for participants younger than age 18 years, written informed consent was provided by a parent or legal guardian. study design this was a cross-sectional study carried out at the diabetes clinic of st. francis hospital nsambya, kampala, uganda. two hundred and eighty dm patients attending the hospital’s diabetes treatment clinic were assessed by a medical officer for clinical presentation consistent with diarrhoea. inclusion criteria patients with diabetes who had signs and symptoms of gastrointestinal infection such as diarrhoea, abdominal pain, bloating and heartburn were consented and enrolled in the study. exclusion criteria diabetic patients who had other immunosuppressive diseases, chronic diseases and those admitted in wards were not included in the study. participants were randomly selected and informed consent was obtained as previously described.18,19 briefly, a list of all the patients attending treatment at the clinic was obtained. microsoft excel (microsoft corporation, redmond, washington, united states) was used to generate numbers ranging between 1 and 500 to three digits. the numbers were typed on cards and the cards were placed in a box. eligible patients who picked cards with numbers that were multiples of three were enrolled in the study. the cards were reshuffled each time a card was picked and picked cards were not replaced. where repeated numbers were generated, only one card with such numbers was left in the box. data and sample collection clinical data were obtained from the consenting participants’ medical records for demographic characteristics such as age, sex, type of diabetes and date of diabetes diagnosis plus information on use of antibiotics, corticosteroids, anti-diabetes and antifungal medication. stool specimens and blood samples were collected from the recruited participants according to the united states centers for disease control and prevention 2014 guidelines.16 in brief, a sterile, wide-mouthed spoon fitted with a graduated stool container was labelled for each participant and participants were instructed to produce about 10 ml of stool. the specimens were immediately delivered to the laboratory for subsequent analysis. one 4-ml fasting blood specimen was collected from each participant into fluoride/oxalate tubes and the plasma separated from the cells within 30 min after collection. sample analyses potassium hydroxide wet mounts and smears for gram staining were prepared from each stool specimen to examine them for candida blastoconidia and pseudohyphae. specimens were cultured on saboraud dextrose agar (laboratorios conda, madrid, spain) for colony counts and candida differential agar (himedia laboratories, mumbai, india) for species identification; cultures were incubated at 37 °c for 24–72 h and checked for growth.16 the germ tube test and growth test at 45 °c were performed to distinguish between c. albicans and c. dublinensis. colonies on sabouraud dextrose agar were enumerated and counts above 105 cfu/ml were interpreted as overgrowth indicative of infection, based on reviewed literature.2,16. following species identification of isolates, susceptibility testing was performed using the kirby-bauer disk diffusion test on a mueller hinton agar supplemented with 2% glucose and methylene blue (himedia laboratories, mumbai, india). inoculum was prepared by picking five distinct colonies of approximately 1 mm from 24-h-old culture grown on sabouraud dextrose agar. colonies were suspended in 5 ml of sterile 0.85% saline and turbidity adjusted to 0.5 mcfarland standard which corresponds to an approximate yeast density of 1 × 106 to 5 × 106 cells/ml. the surface of the muller hilton agar was dried and seeded with the yeast suspension using a sterile cotton swab by streaking the entire agar surface of the plate with the swab three times, turning the plate 60 degrees between each streaking. the inoculum was allowed to dry for 5 min – 15 min with the lid in place, the antifungal discs were placed on the agar surface aseptically and then the plates were incubated at 35 °c ± 2 °c within 15 min after the discs were applied. the plates were examined for susceptibility after 20–24 h of incubation or at 48 h when insufficient growth was observed after 24 h incubation. inhibitory zone diameters were measured in millimetres at the transitional point where growth abruptly decreased, as determined by a marked reduction in colony sizes.20 antifungal susceptibility disks were used to test for susceptibility to two triazoles (fluconazole and itraconazole), two imidazoles (clotrimazole and miconazole) and one polyene (nystatin) (laboratorios conda, madrid, spain). fasting blood glucose levels were tested using a point-of-care accu-chek glucose meter (roche diabetes care, inc., ängelholm, sweden) and glycated hemoglobin levels were determined using an automated glycated hemoglobin 501 analyser (hemocue ab, ängelholm, sweden). quality control growth testing for all culture media was performed using reference candida strains (candida albicans atcc 10231, candida glabrata atcc 15126, candida krusei atcc 24408, candida tropicalis atcc 750) and negative control strains (escherichia coli atcc 25922 and staphylococcus aureus atcc 25923) as recommended by the manufacturers. susceptibility testing was performed in adherence to national committee for clinical laboratory standards guidelines.20 procedures for gram staining, serial dilutions and culture techniques were performed according to protocols developed by the american society for microbiology.21 blood glucose and glycated hemoglobin determination were performed on calibrated devices following the manufacturer’s manual. statistical analysis data were entered in an excel spreadsheet and analysed using stata special edition, version 10.0 (statacorp, college station, texas, united states). data regarding study population characteristics, proportion of patients with gastrointestinal candidiasis, species distribution and susceptibility profiles were analysed using frequency distributions and 95% confidence intervals. bivariate analysis through the use of chi-square tables, and multivariate logistic regression were used to analyse associations between risk factors and gastrointestinal candidiasis. statistical significance was assumed to exist if p-values were less than 0.05. the risk factors studied were age of the participants, sex, glycaemic control (‘good’ control = glycated haemoglobin level < 7.0%; ‘poor’ control = glycated haemoglobin > 7.0%), history of antibiotic therapy, history of corticosteroid therapy and type of dm. participants were analysed in four age groups: children (< 18 years), youth (18–40 years), adult (41–65 years) and elderly (> 65 years). diabetes type was classified based on diagnostic information on the patient’s form into type 1 and type 2 dm. the history of therapies was classified as yes if the patient had treatment for more than 2 weeks but less than 1 month earlier or no if the patient had therapies either for less than 2 weeks or more than 1 month earlier. results two hundred and forty-one study participants were recruited into the study (table 1). one hundred and seventeen (48.5) of the participants were female patients; 82 (34.02%) patients had type 1 dm and the rest had type 2 dm. the mean age of the participants was 43 years (95% confidence interval: 41–46); among type 1 dm patients the mean age was 21 years (95% confidence interval: 19–22 years) and in type 2 dm patients this was 55 years (95% ci: 53–57 years). the average fasting blood glucose levels in the study participants was 9.9 mmol/l (standard deviation = 2.3 mmol/l), and average glycated haemoglobin level was 9.3% (standard deviation = 1.6%). table 1: characteristics of the study participants at enrolment (n = 241), kampala, uganda, march 2017 to december 2017. proportion of diabetes mellitus patients with gastrointestinal candidiasis cultures from 37 (15.4%) patients had colony counts consistent with gastrointestinal candidiasis (≥ 1 × 105 colony-forming units/ml of stool). among infected patients, 11 (29.7%) had type 1 dm, and 26 (70.3%) had type 2 dm (p = 0.706). about one-third of patients with gastrointestinal candidiasis (25, 67.6%) were female, and 12 (32.4%) were male (p = 0.009). the majority of the isolates were c. albicans (n = 23, 62.2%), followed by c. glabrata (n = 7, 18.9%), c. tropicalis (n = 3, 8.1%), candida dublinensis (n = 2, 5.4%) and c. krusei (n = 2, 5.4%). among female patients, only c. albicans and c. dubliniensis were isolated (figure 1). c. glabrata, c. krusei and c. tropicalis were isolated exclusively from male patients. figure 1: distribution of candida species by sex (n = 37), kampala, uganda, march 2017 to december 2017. antifungal susceptibility of gastrointestinal candida isolates a total of 25 of the 37 isolates (67.6%) were susceptible to fluconazole and 3/37 (8.1%) isolates were resistant to fluconazole. itraconazole susceptibility was observed in 33 (89.2%) isolates; 15 (40.5%) isolates were susceptible to clotrimazole, while 7 (18.9%) were resistant and 18 (48.6%) isolates were resistant to miconazole, while 8 (21.6%) were sensitive. all isolates were susceptible to nystatin (table 2). table 2: antifungal susceptibility profile of candida isolates from diabetes mellitus patients (n = 37 isolates), kampala, uganda, march 2017 to december 2017. patient factors associated with gastrointestinal candidiasis there were 12 (34.4%) male, culture-positive patients and 25 (67.6%) female, culture-positive patients (p = 0.01). twenty-five (67.6%) of the culture-positive patients reported prior use of antifungal drugs, whereas the rest had no history of antifungal drug use in the past weeks (p < 0.001); 26 (70.3%) culture-positive patients had used antibiotics and 11 (29.7%) had not used antibiotics in the past weeks; 14 (37.8%) culture-positive patients had used corticosteroids and the rest had not used corticosteroids in the past weeks (p < 0.001) (table 3). thirty-five had poor glucose control, and 20 were hyperglycaemic. based on glycated haemoglobin results, 2 (5.4%) culture-positive patients had good glycaemic control and the remaining 35 (94.6%) culture-positive patients had poor glycaemic control (p < 0.001). table 3: relationship between patient factors and gastrointestinal candidiasis (n = 37), kampala, uganda, march 2017 to december 2017. discussion the overall prevalence of intestinal candidiasis in this study was 15.4% (37 isolates found among 241 patients). this is considerably lower than other findings such as 21.5% in cameroon,22 75.47% in poland in 20152 and 41.1% in mexico.23 the prevalence in this study was also lower than the overall prevalence in sub-saharan africa, which was estimated to be 23.4%.8 gurleen and savio24 in india, however, reported a lower prevalence (9.7%). previous authors have noted a wide variation in the prevalence of candidiasis depending on region, population surveyed and even the research methods used.3 whereas most studies used similar culture-based methods, the quantification threshold for colony-forming units of the culture colonies have varied. in this study, a threshold range of 105 cfu was considered significant for growth, whereas in the study in poland,2 a wider range (103 cfu – 106 cfu) was considered. this would have accounted for the significantly higher prevalence in their study. in the current study, female patients had a higher prevalence rate compared to male patients (21.4% vs 9.7%; p = 0.009). a study conducted in vienna, austria, on burn patients found a female predisposition in systemic and related candidiasis.25 the predilection of gastrointestinal candidiasis in female patients is poorly understood and there is no proper explanation for the predisposition.26 species distribution of candida isolates c. albicans accounted for the majority of isolates in the current study. this is consistent with other studies that identified it as the most common isolate from clinical materials,27,28 and could be attributed to the fact that c. albicans is highly adapted to the human mucosal surfaces and possesses virulence factors such as protease production and biofilm formation.3,29 these enhance its chances of survival in the gastrointestinal tract. omrani et al.9 in their review of african studies also found a predominance of c. albicans. non-albicans species in general accounted for a larger number of isolates in studies by banerjee et al.30 in india, as well as in brazil and chile.31 no mixed species infections were encountered in this study, unlike the studies in poland2 and india.30 among non-albicans species in this study c. glabrata was predominant, accounting for half (7/14) of all non-albicans isolates. this was followed by c. tropicalis (3/14), c. krusei (2/14) and c. dubliniensis (2/14). species diversity was greater in male patients and type 2 dm patients; however, the latter could simply be a reflection of the fact that the type 2 dm sub-group accounted for the majority of patients. the relationship could perhaps be better established with a cohort study design. antifungal susceptibility patterns of candida isolates there are relatively fewer options for the treatment of mycoses compared with antibiotics. fluconazole is a narrow spectrum fungistatic azole with good activity against yeasts; however, from the mid-1990s concerns about resistance have persisted.3 this study found that 87% of c. albicans isolates were fluconazole-susceptible. similarly, the study in poland2 and a study conducted in 2013 in india32 also reported fluconazole susceptibility in over 80% of c. albicans isolates. fluconazole resistance among non-albicans species was found to be 21%. this was considerably lower than observations from the polish study2 where 56% of non-albicans species were susceptible to fluconazole. this has implications for clinical therapy since the guidelines for the treatment of non-albicans infection in uganda require fluconazole. intrinsic resistance in non-albicans species has been documented in species such as c. krusei, and reduced susceptibility in c. glabrata and c. guilliermondii has been reported.33,34 candida tropicalis has been known to exhibit fluconazole resistance up to 31.3%28; however, this could be due to the use of a panel that mostly comprised high-potency drugs normally reserved for systemic infections, such as amphotericin b and voriconazole. in this study c. krusei, was also the most fluconazole-resistant strain. this strain has been known to exhibit intrinsic fluconazole resistance; sanguinetti et al.28 reported an estimated global fluconazole resistance of 78% in c. krusei. itraconazole has a wider spectrum of activity than fluconazole, and is normally effective against both fluconazole-susceptible and fluconazole-resistant candida strains.17 lesser susceptibility, however, was observed in 2 c. glabrata and both c. krusei isolates. the polish study,2 however, found that only 28% of c. albicans and 11% of non-albicans species were itraconazole susceptible. higher rates of resistance were observed in the imidazoles; 18.9% of isolates were resistant to clotrimazole and nearly half were resistant to miconazole. it is worth noting that a number of patients in this study had prior exposure to antifungal medications, especially azoles, which were mostly used to treat dermatomycoses and candida. clotrimazole and miconazole are commonly used over-the-counter antifungals, and these could therefore be a driver for the reduced susceptibility and resistance patterns observed in some patient isolates. all isolates in this study were susceptible to nystatin, which is consistent with reports that document low rates of polyene-class antifungal resistance.3,17,33 nystatin carries the additional advantage of being a low-cost medicine and is widely available in many formulations including tablets, suspension and topical preparations. in uganda, the treatment plan for oropharyngeal candidiasis and gastrointestinal candidiasis may require the use of nystatin preparations, which replaces the expensive and less available medicines such as caspofungins. patient factors associated with gastrointestinal candidiasis there was no significant association between the diabetes type and gastrointestinal candidiasis. gosiewski et al.34 also found no association, despite a higher prevalence among those with type 2 dm patients. this was contrary to kumar et al.,35 who found a higher prevalence among type 1 dm. gastrointestinal candidiasis was more common among female patients than among male patients (p = 0.01) and among patients with poor glycaemic control (p ≤ 0.001) than among those with good glycaemic control. the use of antifungals (p ≤ 0.001), antibiotics (p ≤ 0.001) and corticosteroids (p ≤ 0.001) was a predisposing factor to gastrointestinal candidiasis. similar findings were reported by banerjee et al.,30 in india, where a majority of patients were on antibiotic and corticosteroid therapy. antibiotics, especially the broad-spectrum variety, are known to interfere with the balance of gut microbiota in the human body.27,36,37 in doing so, they create a favourable environment for the proliferation of yeasts, and some studies have reported a possible influence on drug resistance profiles. ben-ami et al.,37 found a significant association between the use of antibiotics such as carbapenems, clindamycin and colistin and fluconazole resistance, but in this study the class of antibiotics used by diabetic patients was not reported. use of antifungal therapy could also select for more resistant strains. this was demonstrated by lortholary et al.,38 using data from a prospective surveillance programme; the authors observed high rates of isolation of fluconazole-resistant candida species following the recent use of the drug. in this study, poor antifungal susceptibility at baseline level and inappropriate doses of antifungal drugs could be responsible for the association that was observed. increased age is usually associated with decreased effectiveness of the immune response.1,17 the elderly patients and adults accounted for the majority of infections, but there was no significant association between age and gastrointestinal candidiasis. therefore, this observation could simply be a reflection of the study population, most of which comprised adults and elderly patients. in contrast, banerjee et al.30 observed a higher isolation rate in children (0–12 years), and attributed it to the weaker immune systems in such populations. given the adult: paediatric ratio in that study (1:1.9), however, the sampling method may have also had an influence on this result. corticosteroid use was also associated with gastrointestinal candidiasis. this has also been observed by glavey et al.,39 in their cross-sectional study, and madhumati and rajendran.40 kakeya et al.41 and fardet et al.42 reported an increased risk of susceptibility to opportunistic infections in patients exposed to corticosteroids. most patients with gastrointestinal candidiasis in this study had poor glucose control. studies investigating the association between the two have yielded varying results. findings from this study are similar to the report by olczak-kowalczyk et al.,43 but contrary to studies conducted by suarez et al.,44 kowalewska et al.2 and arslan et al.29 which did not find association between poor glycaemic control and candidiasis. recommendations as a package for the diagnosis and management of gastrointestinal candidiasis, speciation of candida isolates should be performed, and antifungal susceptibility profiles established to guide patient therapy. the speciation is important, since species within the candida genus differ widely, both in their ability to cause infection and also in their susceptibility to antifungal agents. nystatin was shown to have excellent antifungal activity in this study and could be considered for empirical therapy in settings where inadequate resources may inhibit antifungal susceptibility testing. limitations this study enrolled only participants who complained of gastrointestinal symptoms and several asymptomatic patients could have been left out. this might have reduced the prevalence of gastrointestinal candidiasis. routine culture and sensitivity testing was conducted and very sensitive techniques such as molecular techniques were not performed. this could have reduced the detection of candida organisms. conclusion prevalence of gastrointestinal candidiasis was relatively low among the participants of this study. the infection is associated with female sex, poor glycaemic control and previous use of antifungals, antibiotics and corticosteroids. nystatin can be a drug of choice in the treatment of gastrointestinal candidiasis, if the suspension or tablet formulations can be made available. acknowledgements the authors are highly grateful to the diabetic patients who accepted to participate in the study. the author wishes to sincerely acknowledge the contributions of the administration and staff of st. francis hospital nsambya diabetes centre, the staff of the international health sciences university laboratory and teaching staff, especially mr taremwa ivan mugisha. competing interests the authors have declared that no competing interest exists. authors’ contributions c.o. and a.p.o. conceived the topic and collected the data, c.o. analysed the data, b.m. and e.o. drafted the manuscript, and j.c.o., v.a. and c.a. edited the manuscript. all authors read and approved the contents of the manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy of any affiliated agency of the authors. references mahon rc, lehman cd, manuselis g. textbook of diagnostic microbiology. 5th ed. st louis. mo: saunders elsevier; 2015. kowalewska b, kawko m, zorena k, myśliwiec m. yeast-like fungi in the gastrointestinal tract in children and adolescents with diabetes type 1. pediatr endocrinol diabetes metab. 2014;20(4):170–177. https://doi.org/10.18544/pedm-20.04.0017 anaissie ej, 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dermatol. 2013;88(2):222–225. https://doi.org/10.1590/s0365-05962013000200006 gurleen k, savio r. prevalence of candida in diarrhoeal stools. j dent medi sci. 2016;15(4):47–49. rubaihayo j, tumwessigye nm, konde-lule j, wamani h, nakku-joloba e, makumbi f. frequency and distribution patterns of opportunistic infections associated with hiv/aids in uganda. bmc res notes. 2016;9(1):501. https://doi.org/10.1186/s13104-016-2317-7 loster je, wieczorek a, loster bw. correlation between age and candida species infections of complete denture wearers: a retrospective analysis. clin interv aging. 2016;2016(11):1707–1714. https://doi.org/10.2147/cia.s116658 flevari a, theodorakopoulou m, velegraki a, armaganidis a, dimopoulos g. treatment of invasive candidiasis in the elderly: a review. clin interv aging. 2013;2013:8:1199. https://doi.org/10.2147/cia.s39120 sanguinetti m, posteraro b, lass-flörl c. antifungal drug resistance among candida species: mechanisms and clinical impact. mycoses. 2015;58(suppl 2):2–13. https://doi.org/10.1111/myc.12330 arslan s, koç an, sekerci ae, et al. genotypes and virulence fac+tors of candida species isolated from oral cavities of patients with type 2 diabetes mellitus. turk j med sci. 2016;46(1):18–27. https://doi.org/10.3906/sag-1405-73 banerjee p, kaur r, uppal b. study of fungal isolates in patients with chronic diarrhea at a tertiary care hospital in north india. j mycol med. 2013;23(1):21–26. https://doi.org/10.1016/j.mycmed.2012.12.002 sardi ocj, scorzoni l, bernardi t, fusco-almeida ma, mendes gsjm. candida species: current epidemiology, pathogenicity, biofilm formation, natural antifungal products and new therapeutic options. j med microbiol. 2013;62(1):10–24. https://doi.org/10.1099/jmm.0.045054-0 premkumar j, ramani p, chandrasekar t, natesan a, premkumar p. detection of species diversity in oral candida colonization and anti-fungal susceptibility among non-oral habit adult diabetic patients. j nat sci biol med. 2014;5(1):148. https://doi.org/10.4103/0976-9668.127315 spampinato c, leonardi d. candida infections, causes, targets, and resistance mechanisms: traditional and alternative antifungal agents. biomed res int. 2013;2013:204237. https://doi.org/10.1155/2013/204237 gosiewski t, salamon d, szopa m, sroka a, malecki mt, bulanda m. quantitative evaluation of fungi of the genus candida in the feces of adult patients with type 1 and 2 diabetes-a pilot study. gut pathog. 2014;6(1):1. https://doi.org/10.1186/s13099-014-0043-z kumar bv, padshetty ns, bai ky, rao ms. prevalence of candida in the oral cavity of diabetic subjects. j assoc physicians india. 2005;53:599–602. vaishnavi c, kaur s, prakash s. speciation of fecal candida isolates in antibiotic-associated diarrhea in non-hiv patients. jpn j infect dis. 2008;61(1):1–4. ben-ami r, olshtain-pops k, krieger m, et al. antibiotic exposure as a risk factor for fluconazole-resistant candida bloodstream infection. antimicrob agents chemother. 2012;56(5):2518–2523. https://doi.org/10.1128/aac.05947-11 lortholary o, desnos-ollivier m, sitbon k, fontanet a, bretagne s, dromer f. recent exposure to caspofungin or fluconazole influences the epidemiology of candidemia: a prospective multicenter study involving 2,441 patients. antimicrob agents chemother. 2011;55(2):532–538. https://doi.org/10.1128/aac.01128-10 glavey sv, keane n, power m, o’regan aw. posterior pharyngeal candidiasis in the absence of clinically overt oral involvement: a cross-sectional study. lung. 2013;191(6):663–668. https://doi.org/10.1007/s00408-013-9503-3 madhumati b, rajendran r. evaluation of chrom agar in speciation of candida species from various clinical samples in a tertiary care hospital. int j curr microbiol app sci. 2015;4(9):463–472. kakeya h, izumikawa k, yamada k, et al. concurrent subcutaneous candida. 2014. fardet l, petersen i, nazareth i. common infections in patients prescribed systemic glucocorticoids in primary care: a population-based cohort study. plos med. 2016;13(5):e1002024. https://doi.org/10.1371/journal.pmed.1002024 olczak-kowalczyk d, pyrżak b, dąbkowska m, et al. candida spp. and gingivitis in children with nephrotic syndrome or type 1 diabetes. bmc oral health. 2015;15(1):57. https://doi.org/10.1186/s12903-015-0042-6 suárez lb, alvarez mi, de bernal m, collazos a. candida species and other yeasts in the oral cavities of type 2 diabetic patients. colombia méd. 2013;44(1):26–30. https://doi.org/10.25100/cm.v44i1.1040 abstract introduction immunity to covid-19 disease dynamics of covid-19 in sub-saharan african countries infectious diseases in sub-saharan africa immune response to microbial infections possible reasons for a presumed immune sufficiency in sub-saharan africa projections into the future of covid-19 and disease processes conclusion acknowledgements references about the author(s) abel o. idowu department of pharmaceutical microbiology and biotechnology, faculty of pharmacy, college of medicine, university of lagos, lagos, nigeria yusuf o. omosun department of microbiology, biochemistry and immunology, morehouse school of medicine, atlanta, georgia, united states joseph u. igietseme department of microbiology, biochemistry and immunology, morehouse school of medicine, atlanta, georgia, united states centers for disease control and prevention (cdc), atlanta, georgia, united states anthony a. azenabor department of pharmaceutical microbiology and biotechnology, faculty of pharmacy, college of medicine, university of lagos, lagos, nigeria citation idowu ao, omosun yo, igietseme ju, azenabor aa. the covid-19 pandemic in sub-saharan africa: the significance of presumed immune sufficiency. afr j lab med. 2023;12(1), a1964. https://doi.org/10.4102/ajlm.v12i1.1964 review article the covid-19 pandemic in sub-saharan africa: the significance of presumed immune sufficiency abel o. idowu, yusuf o. omosun, joseph u. igietseme, anthony a. azenabor received: 01 june 2022; accepted: 24 oct. 2022; published: 30 jan. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract a novel coronavirus known as severe acute respiratory syndrome coronavirus 2 (sars-cov-2) was first reported in china in 2019 and later ignited a global pandemic. contrary to expectations, the effect of the pandemic was not as devastating to africa and its young population compared to the rest of the world. to provide insight into the possible reasons for the presumed immune sufficiency to coronavirus disease 2019 (covid-19) in africa, this review critically examines literature published from 2020 onwards on the dynamics of covid-19 infection and immunity and how other prevalent infectious diseases in africa might have influenced the outcome of covid-19. studies characterising the immune response in patients with covid-19 show that the correlates of protection in infected individuals are t-cell responses against the sars-cov-2 spike protein and neutralising titres of immunoglobin g and immunoglobin a antibodies. in some other studies, substantial pre-existing t-cell reactivity to sars-cov-2 was detected in many people from diverse geographical locations without a history of exposure. certain studies also suggest that innate immune memory, which offers protection against reinfection with the same or another pathogen, might influence the severity of covid-19. in addition, an initial analysis of epidemiological data showed that covid‑19 cases were not severe in some countries that implemented universal bacillus calmette–guerin (bcg) vaccination policies, thus supporting the potential of bcg vaccination to boost innate immunity. the high burden of infectious diseases and the extensive vaccination campaigns previously conducted in africa could have induced specific and non-specific protective immunity to infectious pathogens in africans. keywords: covid-19; coronavirus; immune response; sub-saharan africa; infectious diseases. introduction in december 2019, a group of pneumonia cases of undetermined origin where patients presented with clinical signs that resemble viral pneumonia was first described in the city of wuhan, hubei, china.1 analysis of samples from the lower respiratory tract by deep sequencing implicated a novel coronavirus that was named 2019-ncov.1,2 on 11 march 2020, the world health organization (who) declared the coronavirus disease 2019 (covid-19), caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), a worldwide pandemic. as of 07 september 2021, sars-cov-2 had cumulatively infected over 224 686 497 people and caused 4 566 167 deaths in 225 countries, with a case fatality rate of 2.1%.3 the spectrum of covid-19 manifestation may vary from a self-limiting mild respiratory tract infection to a severe pneumonia that could lead to the failure of multiple organs and death.4 the common symptoms, including fever, cough, and difficulty in breathing, typically emerge within 2–14 days after exposure.4,5 certain individuals may, however, exhibit minimal or no symptoms even with a positive reverse transcription polymerase chain reaction test.6,7,8,9 the risk of death is greater among older or immunocompromised patients,10 and the ability of asymptomatic individuals to efficiently transmit the virus11 has made it difficult to control the epidemic.6 coronaviruses are characteristically single-stranded, positive-sense rna viruses that disseminate widely in birds, humans, and other mammals, and can cause enteric, respiratory, neurological, and hepatic diseases.12,13 six species of coronavirus species cause human diseases, and four of them, nl63, hku1, hcov-229e, and oc43, are predominantly responsible for mild respiratory infections.14 the first sars-cov, which emerged in 2002,15,16,17 and middle east respiratory syndrome coronavirus, which became known in 2012,18 are two deadly novel coronaviruses that cause severe pneumonia and have appeared occasionally in different areas. however, in contrast to sars-cov-2, the number of confirmed sars-cov and middle east respiratory syndrome coronavirus cases are smaller (8100 and 2500) because of limited transmission from person to person. the probability of the periodic emergence of novel coronaviruses is high as a result of its extensive dissemination among humans and animal species, high genetic variability and recurrent genomic recombination, in addition to other factors such as growing human and animal interactions, regular cross-species infections, and rare spillover events.19,20 the sequencing of the 2019-ncov genome showed that it is phylogenetically similar to particular beta-coronaviruses found in bats that belong to the sarbecovirus subgenus in the coronaviridae family.2 contrary to expectations that the african region would quickly succumb to the devastation of the rampaging covid-19 pandemic, fewer cases, as shown by the number of cases per million people, are being reported in africa compared to other parts of the world (figure 13). besides, a comparison of the incidence of cases and the case fatality ratio among some selected african and western countries (table 13) showed that the case fatality ratio and number of cases in africa were relatively low. this suggests that africa was mildly impacted by the pandemic despite the weak health infrastructure. this review summarises current literature on the dynamics of infection and immunity to sars-cov-2 and other infective diseases in africa and the unique characteristics of the sub-saharan african population that might have impacted the course and severity of the pandemic. we also describe the host immune response mechanisms to other microbial infections and the possible reasons for a presumed immune sufficiency to covid-19 in the african population. figure 1: global distribution of covid-19 cases per million people as of 07 september 2021. table 1: cumulative total number of covid-19 cases, deaths and case fatality ratio reported in some western and african countries between 05 february 2020 and 07 september 2021. the terms ‘epidemiology and immunity to covid-19’, ‘covid-19 and associated coronaviruses’, ‘prevalent infectious diseases in africa’, ‘host immune response mechanisms to viral and other microbial infections’, and ‘immune sufficiency to covid-19 in the african population’ were used to carry out a systematic search on relevant search engines and websites such as google, google scholar, pubmed, african journals online, world health organization, center for disease control and prevention, nigeria centre for disease control, and africa centre for disease control and prevention. we included all relevant studies published in the english language between 2000 and 2021. immunity to covid-19 the specific aspects of immunity that define both the protective and pathogenic reactions to sars-cov-2 infection are not clear. though some components of the immune response to sars-cov-2 infection appear to be unique, some aspects are similar to the mechanisms of immunity to other human and animal infections. an understanding of the protective components of the immune reaction to sars-cov-2 infections is important for effective vaccine development. as is the case with many infectious diseases, an immune reaction to sars-cov-2 involves both cell-mediated and antibody-facilitated responses.21 t-cell responses against the viral spike protein have been shown to correlate well with the neutralising titres of immunoglobin g and immunoglobin a antibodies.22,23,24 this observation could have important implications for vaccine design and the development of durable immunity. as the primary inflammatory cells, the cd8+ t-cells play a crucial function in clearing the virus. the level of inflammation during sars-cov-2 infection is significantly reliant on the association between the cd4+/cd8+ ratio and the total lymphocytes, namely cd4+ t-cells, cd8+ t-cells, and b-cells and natural killer cells.10 in both mild and severe cases, there is a decrease in the absolute numbers of t lymphocytes, cd4+ t-cells, and cd8+ t-cells, but the decrease is more in severe than in mild cases.10 the implication of the lymphopenia observed is that it reduces the ability of the immune system to fight the infection and increases the chances of severe illness. multivariate data analysis showed that a reduction in b-cells and cd8+ t-cells, and a rise in the cd4+/cd8+ ratio can independently predict a poor treatment outcome.10 in the context of the african population where immune sufficiency is presumed, it is expected that a reduction in the numbers of t lymphocytes would be limited but this needs to be investigated. interferon-γ expression by cd4+ t-cell induction also tends to reduce in severe cases compared to moderate cases.4 it takes between 10 and 21 days after infection for antibody response to develop in most sars-cov-2-infected persons, and it takes between 6 and 15 days after the onset of disease for the immunoglobin m and immunoglobin g antibodies to sars-cov-2 to develop.25,26,27,28,29 in mild cases, antibody development can take up to 4 weeks or longer and may even be undetectable in a small number of cases. the basis of protective immunity from a successive infection is the immune memory derived from either primary infection or immunisation.30,31,32 recently, a predominantly cross-sectional study, which also included a longitudinal component of 188 recovered covid-19 cases, assessed the involvement of the cd4+ t-cell, cd8+ t-cell, and humoral components of adaptive immunity in the immune memory. the result showed that covid-19 infection is followed by the generation of considerable immune memory involving all types of adaptive immune components,21 thus suggesting that activation of antibodies or t-cell reactivity are correlates of protection against covid-19 infection. the question is: how long does the immune protection last? in other coronaviruses, antibody levels decline between 12 and 52 weeks from the inception of symptoms, resulting in homologous reinfections.33 in contrast to sars-cov-1 infections where immunoglobin g antibody levels could be sustained for two years in 90% of patients and for three years in 50% of patients,34 immunoglobin m and immunoglobulin g antibody levels in sars-cov-2 could only be sustained for over seven weeks35 or until day 49 in at least 80% of the cases.36 a recent study showed that 95% of study subjects retained immune memory lasting approximately six months after infection.21 disease dynamics of covid-19 in sub-saharan african countries the incidence of sars-cov-2 infections in sub-saharan africa was initially low but increased rapidly over time until it became widespread in virtually all countries. as of 07 september 2021, the total number of covid-19 cases reported in 55 african countries was 7 926 999, with 200 045 deaths and a case fatality ratio of 2.5%.37 this corresponds to 3.6% of the total number of cases and 4.4% of the total number of deaths reported globally. within africa, the total number of covid-19 cases and deaths have varied between countries and with time37 (figure 23). as of 07 september 2021, south africa, which is the epicentre of the pandemic in africa, had recorded the highest incidence of validated covid-19 infections (2 819 945) in sub-saharan africa, followed by ethiopia (314 984), kenya (240 172), zambia (207 114), nigeria (195 511), and botswana (162 186).37 although nigeria is the most populous african nation, it has the fifth largest case count in sub-saharan africa and the largest outbreak in west africa.37 the number of cases being reported in africa compared to the other regions of the globe may have been partly influenced by the low level of testing,37 suggesting a high likelihood of many undetected cases. the level of testing in africa has been hindered by factors that include limited laboratory capacity, availability and accessibility of testing sites, cost of testing, low health literacy, and the stigma associated with testing positive.38 also, certain characteristics peculiar to africa in terms of age, health, lifestyle, and previous experiences with other infectious disease outbreaks might have impacted how the pandemic is playing out in africa. figure 2: cumulative total number of covid-19 cases and deaths reported in africa between 05 february 2020 and 07 september 2021. with a median age of under 20 years, africa has a relatively young population compared to european union countries (43 years) and china and the united states (38 years).39 besides, only 3% of the african population is above 65 years, which is the threshold for significant chances of complications and deaths due to covid-19.40 in nigeria, the most populous country in africa, over 44% of the population is below the age of 15 years, the age group with the fewest number of reported cases and mortality.41 however, there are fears that other demographic features peculiar to africa such as frequent migration across borders, a high number of displaced persons, rapid urbanisation, and large households41 could complicate prevention and mitigation efforts, thereby driving up the case count and the number of deaths. in addition, factors like the worsening burden of existing health conditions such as tuberculosis and hiv/aids42,43 with their attendant effects on the population’s immune status, as well as the high incidence of non-communicable diseases such as hypertension and heart diseases, increase the chances of complications from covid-19 infections. infectious diseases in sub-saharan africa infectious diseases, responsible for a minimum of 69% of deaths in africa,44 are a serious challenge in africa due to various underlying factors such as poverty, poor environmental hygiene, overcrowded housing, limited access to affordable healthcare, and lack of basic health education.45,46,47 malaria, hiv/aids, tuberculosis, ebola disease, lassa fever, guinea worm, elephantiasis, river blindness, lower respiratory tract infection, and diarrheal disease are prevalent or have had outbreaks in africa.48 there are six infectious diseases in the 2019 who report of the top 10 causes of disability-adjusted life years in sub-saharan africa.49 these include malaria, aids, lower respiratory tract infections, diarrheal infections, meningitis and tuberculosis.49 the spread of these diseases could occur more easily in countries with inadequate infrastructure and fragile health systems. malaria, caused by plasmodium species, is a vector-borne parasitic disease that infects humans via the bites of infected female anopheles mosquitoes.50 in 2020, the who estimated that 3.2 billion people across 96 countries run the risk of having malaria, with the highest burden being in sub-saharan africa. this region accounts for 94% of global cases and 94% of deaths, 67% of which are in children aged under five years.50 the hiv is a retrovirus that progressively destroys and weakens the immune system of an infected person until it becomes vulnerable to opportunistic infections. if untreated, the hiv infection can progress to the most complex stage of the disease referred to as aids, resulting in death.51,52 over 37.7 million people are living with hiv globally, with 680 000 annual deaths.53 sub-saharan africa is the worst affected, being home to 67% of people living with hiv and 39% of new hiv cases.53 although most people living with hiv have higher co-morbidities and experience more severe outcomes from covid-19 than people without hiv, most of them did not have access to covid-19 vaccines as of mid-2021.53 tuberculosis is caused by the bacterium mycobacterium tuberculosis and is an infectious disease that commonly affects the lungs and is transmissible from person to person through coughing, sneezing, or spitting.54,55 in 2019, there were 10 million cases of tuberculosis (208 000 were in hiv-positive people) and over 1.2 million deaths due to tuberculosis (15% of all deaths were related to hiv).56 africa ranked second in the global burden of new tuberculosis cases (25%) in 2019.56 ebola virus causes an acute, contagious, and deadly disease that is transmitted from person to person through close contact with fluids from the body of symptomatic and asymptomatic living patients or dead victims.57,58 periodic, remote outbreaks of ebola have occurred in many central african countries.59,60 the most widespread outbreak of ebola occurred in guinea, west africa, in 2014 from where it spread to neighbouring sierra leone and liberia, killing an estimated 11 000 people by april 2016.61,62,63 immune response to microbial infections the human body protects itself from microbial infections through the immune system made up of innate and adaptive arms that are not only linked but consist of an extraordinarily diverse compilation of cells (figure 3; compiled in biorender, https://biorender.com/). the innate immune system is the host’s first-line defence against invading pathogens, while adaptive immune responses are triggered by the host’s immune recognition controls.64 with the help of molecular patterns that are pathogen-related, the innate immune system recognises repetitive evolutionarily conserved molecules on pathogens by using germline-encoded pattern recognition receptors such as c-type lectin receptors, toll-like receptors, nucleotide-binding oligomerisation domain-like receptors, and retinoic acid-inducible gene i-like receptors.65 the immune cells that mediate innate immune defences such as natural killer cells, complement proteins, and phagocytic cells like monocytes, macrophages, and neutrophils do not have effector mechanisms that induce immunological memory.66 figure 3: cells involved in innate and adaptive immune responses against pathogens. the immune cells from innate immunity which provide the first line of defence are generated by myeloid lineage cells and they include monocytes, macrophages, erythrocytes, platelets, and granulocytes. cells from the adaptive immune system are responsible for the second line of defence and consist of natural killer cells, b-cells, and t-cells that come from lymphoid progenitor cells. recognition of external antigens by the macrophages and dendritic cells of the innate immune system triggers the naive cd8+ and cd4+ t-cells of the adaptive immune system. adaptive immunity comprises cell-mediated and humoral immunity, which have a wider and fine-tuned range of recognition because of exposure to variable antigens. the unique traits of the adaptive immune system are pathogen-specific immunological memory, immune effector functions, tolerance, regulation of host immune homeostasis, and increased production of inflammatory mediators.67 when the innate immune system is exposed to invading organisms in the form of vaccines (bacillus calmette–guerin [bcg] vaccine, measles vaccine, etc.) or bacterial and fungal complex biomolecules (lipopolysaccharides, β-glucan, etc.), the immune cells undergo metabolic alteration or epigenetic reprogramming.68,69 these changes, described as ‘trained immunity’, elicit an elevated response when challenged afterwards by the first trigger or different unrelated organisms or molecules through tor b-cell-independent responses, leading to increased production of inflammatory mediators.70,71,72 unlike the conventional, more precise, epitope-dependent adaptive immune memory that is mediated by lymphocytes, the control of trained immunity is facilitated by a unique mechanism that is not very specific and lasts only for a short while.73 nevertheless, both fulfil the same major task of eliciting a faster and more robust response that destroys the pathogens and improves the survival of the host. trained immunity could be beneficially applied to develop improved vaccines74,75 and limit the adverse effects of inflammatory diseases.76 possible reasons for a presumed immune sufficiency in sub-saharan africa adaptive immunity-mediated pre-existing immunity to sars-cov-2 covid-19 infections are often asymptomatic or mildly symptomatic but may be severe, mostly among vulnerable patients, presenting as acute respiratory distress.77 the mild pattern of infection in many patients may be due to an exposure-related cross-immunity to other endemic common cold coronaviruses.78 these viruses cause mild self-limiting respiratory infections and circulate widely in the human population where globally, more than 90% are seropositive for at least three of the common cold coronaviruses.79 there has been evidence that supports the likelihood that circulating memory b-cells and neutralising antibodies developed to one type of coronavirus may cross-react with other members of the coronavirus family.78 emerging data from studies conducted in unexposed donors from different geographical locations revealed that 20% – 50% had lymphocytes with significant reactivity to pools of sars-cov-2 antigen peptides.22,23,24,80,81 there is also speculation that the sars-cov-2-specific t-cells in unexposed individuals originated from the memory t-cells obtained from contact with common cold coronaviruses.82,83 in a study conducted in germany, the highest t-cell reactivity occurred against a pool of sars-cov-2 spike peptides that are homologous to common cold coronavirus spike proteins.24 the occurrence of sars-cov-2 pre-existing primed t-cells and neutralising antibodies acquired through previous exposure to endemic coronaviruses could slow down disease transmission and limit the number of cases and mortality.78,84 individuals with a high level of pre-existing cd4+ t-cells with sars-cov-2 recognition memory may elicit a faster and more robust immune response upon exposure, thus limiting the severity of the disease. in such individuals, t follicular helper cd4+ t-cells have a memory that could potentially enhance the antibody-neutralising response against sars-cov-2. the memory of cd4+ and cd8+ t-cells might also enhance direct antiviral immunity in the lungs and nasopharynx early after exposure, consistent with previous knowledge that cd4+ t-cells in the lungs have antiviral activity against the associated sars-cov85 and that memory cd8+ t-cells protect against viral infections. a recent study that used samples collected before the pandemic in africa, europe, and south and north america showed the occurrence of pre-existing humoral immunity against the spike (s) and nucleocapsid (n) proteins of sars‑cov‑2. although antibodies specific to the sars-cov-2 s and n proteins were not common in all populations, the prevalence of n-specific antibodies was higher in the two african study locations, gabon and senegal, than in europe and south and north america.86 in separate studies conducted with samples from gabon (central africa), nigeria, ghana, benin (west africa), tanzania (east africa), and zambia (southern africa), the pre-existing immunity was high against the sars-cov-2 n and, to a lower extent, s proteins,87,88,89,90 which suggests that high sars-cov-2 pre-existing immunity is widespread in africa. in addition, previous studies conducted in france, england, and the united states have also reported low levels of pre-existing sars-cov-2 s protein cross-reactive antibodies in uninfected individuals.91,92,93 although the reason for the underlying sars-cov-2 high seropositivity in african populations has not been established, some have postulated that the immune system could be stimulated to develop cross-reactive antibodies against sars-cov-2 as a result of infections with the common human coronaviruses87,88 due to the wide distribution of the common human coronaviruses throughout the globe.14 in this regard, samples collected from africa, europe, and south africa were used in a recent serological study to demonstrate the existence of antibodies that cross-react against the n protein of common human coronaviruses, but their presence did not show any correlation with the sars-cov-2 n protein cross-reactive antibodies.88 this suggests the possibility that the high cross-reactive immunity reported by the african population is being driven by other main contributors besides the common human coronaviruses. high seropositivity against sars-cov-2 n in samples collected before the pandemic could also be an indication of the circulation of other coronaviruses that have not yet been recognised. the limited number of cases and mortality due to sars-cov-2 infection in africa may be attributed to a high cross-reactive immunity background as observed in sera from african populations. large studies are needed to measure and demonstrate the correlation between pre-existing immunity, prospective infection and disease severity as a way to define the possible function of pre-existing t-cell memory in sars-cov-2 infection. innate immunity-mediated trained immunity and immune tolerance in addition to adaptive immunity-mediated cross-immunity, trained immunity and immune tolerance mediated by the innate immune system could also considerably modify the clinical spectrum and mortality of covid-19 infection. trained immunity is the memory characteristic in the innate immune system of vertebrates that elicits an increased reaction to secondary infections or sterile activation of inflammation.65,94 besides trained immunity, the cells of the innate immune system could also reach a status of immune tolerance or hypo-responsiveness, wherein there is a decrease in the production of pro-inflammatory mediators when these cells encounter secondary heterologous stimuli.70,71,72 for example, epidemiological data, as well as in vivo and in vitro studies with bcg vaccines conducted among humans and in murine-derived peripheral blood mononuclear cells subjected to immune-stimulatory agents such as lipopolysaccharide, β-glucan, and flagellin, provided evidence of either increased or decreased inflammatory reaction to secondary stimuli that corroborates the concept of trained immunity and immune tolerance.70,71,72 in addition, animal studies have shown that immunisation with the bcg vaccine can protect against subsequent infections by candida albicans and m. tuberculosis.95,96 immunisation with the bcg vaccine can also protect against a controlled version of human yellow fever infection97 or malaria98 by augmenting the pro-inflammatory activity of monocytes. it is reasonable to argue that the non-specific nature of this type of cross-protection is not consistent with mediation by adaptive immunity, but it demonstrates that the host defence can develop innate-like immune adaptation mechanisms. several countries have used the bcg vaccine to protect against tuberculosis caused by m. tuberculosis by exposing individuals to a live, attenuated strain of mycobacterium bovis. apart from the partial and possibly variable immunity the bcg vaccine offers against tuberculosis, it also protects in an off-target manner against other non‑tuberculosis infectious diseases.99 for example, bcg vaccination-induced trained immunity offers protection against infections with rna and dna viruses such as herpes and influenza, reduces yellow fever viraemia, and protects against respiratory tract infections.97,100,101,102,103 data from a randomised, double-blind, clinical trial conducted recently showed that there was a reduction in the incidence of respiratory tract infections in bcg-vaccinated patients 65 years and older.104 this protection against a variety of pathogens is achieved through trained immunity.97 most countries with a high incidence of tuberculosis have implemented a comprehensive bcg vaccination policy that encourages children to get the bcg vaccine in early childhood. the protection offered by the bcg vaccine may persist for up to 30–40 years after inoculation as described in a study in norway,105 or up to 50–60 years as shown in a bcg vaccine trial among american indians and alaska natives.106 however, data from these and some other studies have also shown that bcg vaccination-induced protection could also wane over time.107 apart from its reported prospect to enhance innate immunity, preliminary epidemiological analyses have also shown a reduction in covid-19 severity in countries that are implementing the policy of widespread bcg vaccination.108 these observations suggest that the bcg vaccine may protect against sars-cov-2 infection. consequently, in 2020, bcg vaccination was proposed as a strategy to prevent infection and combat the covid‑19 outbreak.108 although bcg vaccination is linked to a reduction in the number of covid-19 cases and mortality,109,110,111 many of the assessments do not consider likely confounding factors such as testing rate, socio-economic factors, and co-morbidities. one epidemiological study that corrected for confounding variables, particularly the testing rate, did not find evidence that the overall bcg vaccination policy correlated with the spread of sars-cov-2 and its associated mortality.112 available information as of 2020 on the bcg atlas (www.bcgatlas.org) showed that all african countries except south sudan have an existing universal bcg vaccination policy. if it is proven that bcg vaccination does indeed offer protection against sars-cov-2, the relatively low number of cases and deaths could be attributed to the long-standing universal bcg vaccination policy in african countries rather than under-reporting or diagnostic limitations. the determinant of whether the innate immune system exhibits either trained immunity or immune tolerance is not known but is thought to be related to the dose and duration of the primary trigger.72,113 trained immunity and immune tolerance have been demonstrated in studies that focused on circulating mononuclear cells with a short lifespan, as well as progenitor stem cells of the bone marrow, epithelial cells, and resident tissue macrophages with long lifespans.70,71 this may imply that the host’s innate immune response to different pathogens could last longer. trained immunity and immune tolerance may have significant implications for covid-19 infection. in sub-saharan africa, vast portions of the population live under unfavourable conditions such as crowded and unhygienic environments, leading to recurrent infections with bacteria, viruses, and eukaryotic parasites, and thus making it possible to attain immune tolerance against a novel pathogen like sars-cov-2. immune tolerance could prevent the innate immune system from reaching a hyper-inflammatory state or a ‘cytokine storm’, which is generally associated with the ultimate fatality of covid-19 disease. people living in developed countries with better sanitary conditions are less exposed to multiple pathogens during their lifetime and thus less likely to attain immune tolerance. this may be partially responsible for the clinical spectrum and the heightened vulnerability to novel pathogens like sars-cov-2 observed in such populations. projections into the future of covid-19 and disease processes against the background of increasing population immunity due to vaccination and high rates of natural infection, many new variants of sars-cov-2 have emerged and have been designated by the who as either variants of concern or variants of interest.114 these variants pose a greater risk to public health because they may be more transmissible or produce more severe disease, escape immune response, cause diagnostic or treatment failure, or reduce the efficacy of vaccines.114,115 the five main variants of concern are: b.1.1.7 (alpha), first described in the united kingdom; b.1.351 (beta), first reported in south africa; p.1 (gamma), originated from brazil; b.1.617.2 (delta), first described in india; and b.1.1.529 (omicron), first detected in south africa. the alpha, beta, and gamma variants share the n501y mutation, which likely makes them more transmissible. the e484k and k417n mutations displayed by both the beta and gamma variants decrease the binding of neutralising antibodies, thus causing partial immune escape, favouring reinfections, and reducing the in vitro efficacy of some antibody therapies or vaccines.116,117 the frequent emergence of these variants globally challenges the use of herd immunity as an approach to managing the pandemic and has been responsible for new waves of the pandemic and thousands of deaths globally.114 the alpha variant was quickly replaced by the highly contagious delta variant, which was soon outcompeted by the omicron variant to become the dominant circulating variant. the omicron variant is more highly transmissible than the other variants, being able to infect 3–6 times more people than the delta variant, including individuals immune to the other variants.117,118 the omicron variant has been detected in 77 countries as of december 2021.119,120 although several aspects of the behaviour of the omicron variant and how it will impact the course of the pandemic have not been well defined, current data indicate that immunisation with existing vaccines could reduce the risk of serious illness, hospitalisation, and death.119,121 however, in vitro studies indicate that the neutralisation efficacy of vaccine sera against omicron reduces greatly compared to the previously circulating delta variant.122 if more data confirm that the omicron variant indeed produces milder forms of the disease, it may suggest a trend that the pandemic is on its way out but the world may have to come to terms with living with the virus. it may require occasional vaccination like in the case of the flu virus to prevent infection. the background of previous exposure to multiple pathogens in the african population may provide a further advantage in conferring protection against the severe form of the disease. in africa, the prospect that new diseases may emerge is high due to the existing burden of infectious diseases and conducive environmental conditions such as high population growth, rapid economic development, increased exploitation of previously untapped natural resources, and increasing interactions between humans and wildlife. a prompt and coordinated global health response would be required to quickly control outbreaks either at the local or regional level as exemplified by the global response to the 2014 ebola epidemic.61,122 while the emergence of new diseases may not have a major repercussion on african population dynamics, it may have serious economic and social consequences. when these diseases emerge, we can draw on scientific advances and previous experience, especially with hiv/aids and ebola, to quickly contain them. africa might be able to withstand some of these emerging diseases owing to enhanced immunity due to prior exposure to numerous pathogens and extensive vaccination programmes within the continent. conclusion this review has shown that pre-existing immunity against sars-cov-2 is widespread in africa and higher than in western countries. this immunity may be related to previous exposure to endemic coronaviruses or other multiple pathogens and could have been responsible for the reduced disease transmission and the limited number of cases and mortality recorded in african countries. the relatively low number of cases and deaths in africa could be attributed to the activation of innate immune-mediated trained immunity or immune tolerance due to frequent exposure to multiple pathogens and the impact of long-standing universal bcg vaccination policies in many african countries rather than under-reporting or diagnostic limitations. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions a.o.i. did extensive literature work and research to concretise the review idea and drafted the manuscript. y.o.o. contributed substantially to revising the draft. j.u.i. contributed to revising the article. a.a.a. conceived the idea, designed and revised the draft manuscript. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy position of any affiliated agency of the authors. references world health organization (who). novel coronavirus – china [homepage on the internet]. 2020 [cited 2021 sept 21]. 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davison nomonde dlamini adetoun ejilemele okechukwu ekenna devy m. emperador uchenna s. ezenkwa renuka gadde lisa gerwing-adima kreshalen govender kerusha govender diana r. hardie omayma m. hassanin samson m. haumba leriska haupt hiroshi ichimura lucius c. imoh emmanuel o. irek prenika jaglal thumeka jalavu feyisayo e. jegede zacceus jeremiah stephen b. kennedy jean f. kloppers elsie c. kruger lynnette t. kyokunda http://www.ajlmonline.org� https://ajlmonline.org mailto:submissions@ajlmonline.org mailto:submissions@ajlmonline.org https://ajlmonline.org https://ajlmonline.org https://ajlmonline.org/index.php/ajlm/user https://ajlmonline.org/index.php/ajlm/user page 2 of 2 reviewer acknowledgement http://www.ajlmonline.org open access 3. it is good practice as a reviewer to update your personal details regularly to ensure contact with you throughout your professional term as reviewer to the african journal of laboratory medicine. please do not hesitate to contact us if you require assistance in performing this task. publisher: publishing@aosis.co.za tel: +27 21 975 2602 raphael lihana fred maate faithful makita-chingombe talkmore maruta gerald mboowa barend mitton modisa s. motswaledi takudzwa j. mtisi timothy m. mtonga stephanie murugan pieter nel apeksha niraula chinwe a. nnabugwu simon o. nyangena abiodun r. ojewuyi olusegun s. ojo olajumoke oladipo sylvia olomu denis omali dayo r. omotoso uvie onakpoya okello e. onen royronald o. ongong’a jessica opie kome otokunefor john oyelakin-ogunbileje sam peters dashini pillay joachim potgieter jennifer a. punt kwabena a.n. sarpong lee schroeder elopi sibanda farouk a. umaru rochelle van wijk lara vojnov reubina wadee musau wakabongo sheila m. woodcock raj n. yadav annalise e. zemlin http://www.ajlmonline.org� mailto:publishing@aosis.co.za acknowledgement to reviewers abstract introduction materials and methods results discussion acknowledgements references about the author(s) jenifer l. vaughan department of molecular medicine and haematology, university of the witwatersrand, johannesburg, gauteng, south africa department of haematology, chris hani baragwanath academic hospital, johannesburg, gauteng, south africa nazeer alli department of molecular medicine and haematology, university of the witwatersrand, johannesburg, gauteng, south africa department of haematology, chris hani baragwanath academic hospital, johannesburg, gauteng, south africa sandra havyarimana excellent medical laboratory services, oshakati, oshana, namibia estee benade department of molecular medicine and haematology, university of the witwatersrand, johannesburg, gauteng, south africa department of haematology, chris hani baragwanath academic hospital, johannesburg, gauteng, south africa citation vaughan jl, alli n, havyarimana s, benade e. assessment of international consensus group for haematology smear review rules among patients with plasmodium falciparum malaria in johannesburg, south africa. afr j lab med. 2018;7(1), a715. https://doi.org/10.4102/ajlm.v7i1.715 brief report assessment of international consensus group for haematology smear review rules among patients with plasmodium falciparum malaria in johannesburg, south africa jenifer l. vaughan, nazeer alli, sandra havyarimana, estee benade received: 10 nov. 2017; accepted: 17 may 2018; published: 29 nov. 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract a peripheral blood smear review is a useful but labour intensive adjunct to the full blood count and differential count. in this study, we retrospectively evaluated the performance of the international consensus group for haematology smear review rules for detection of malaria in 153 samples with plasmodium falciparum parasitaemia. review rules were triggered in 132 (86.3%) samples, including all patients with severe malaria. of the 21 false negative samples, 14 (66.7%) had haemoglobin values ≥ 10g/dl and platelets > 120x109/l. introduction malaria affects in excess of 200 million people annually, with 90% of cases occurring in africa.1 it has a significant mortality rate, with 445 000 malaria-related deaths documented by the world health organization in 2016.1 its diagnosis rests on rapid diagnostic tests, which detect malaria antigen, as well as microscopic identification of parasites on thick and thin smear reviews (sr).1,2 however, a proportion of patients are diagnosed incidentally where malaria is not suspected clinically following a peripheral blood sr from a sample submitted for a routine full blood count (fbc) or differential white cell count (dwcc). as such, ensuring the maximum possible pick-up rate of malaria in samples submitted for a fbc or dwcc is a priority. however, perpheral blood sr is labour intensive and requires skilled morphologists, which are in short supply. careful selection of samples for sr is therefore vital. this was recognised by the international consensus group for haematology (icgh), who compiled a set of evidence-based guidelines directing sr in order to facilitate detection of clinically important morphologic features while reducing the number of sr done.3 these rules have reduced sr rates in many laboratories, but adjustment is often required to accommodate for local pathology patterns, clinician preferences and resource availability. for instance, in laboratories servicing haematology-oncology centres, increased vigilance for the detection of neoplastic cells in the blood is required. many clinical haematologists prefer manual review of all samples collected from patients known to have haematological diseases, but this is not always feasible in laboratories with staffing constraints. laboratory-specific adjustment of the ichg sr guidelines should therefore be undertaken where necessary, with ongoing monitoring in order to facilitate further fine-tuning of the rules and to reduce false negative rates where possible. in particular, every effort should be made to ensure that life-threatening pathologies (including malaria, haemolysis, megaloblastosis and haematologic malignancies) are not missed as a result of implementation of such rules, and where pathology has been found to have been overlooked, adjustments to the sr rules should be considered. at the chris hani baragwanath academic hospital national health laboratory service laboratory in johannesburg, south africa, we validated these rules, and made necessary adjustments to accommodate local peculiarities (table 1). for example, leukopenia is very common in our setting due to the combined effects of benign ethnic neutropenia (which affects up to 60% of black south africans)4 and frequent hiv infection (> 1/3 of the fbcs processed in our laboratory are collected from hiv-positive patients).5 consequently, the low white cell count threshold for smear review was reduced from 4 × 109/l to 2 × 109/l. other changes were made on the basis of manufacturer recommendations (such as review with an immature granulocyte count > 5% as compared to the presence of an immature granulocyte flag +), acceptable false positive rates in our experience (such as nucleated red blood cells > 5/100 white cells as opposed to the presence of a nucleated red blood cell flag +) or to enhance the clinical significance of findings (such as sr when the analyser flags possible platelet clumping only when the platelet count is low as opposed to the presence of a platelet clumping flag with any platelet count). these rules were validated in a representative selection of samples, and shown to have an acceptable false negative rate (< 5%). however, the number of samples collected from patients with malaria for the validation process was limited. this study therefore aimed to retrospectively validate the performance of the chris hani baragwanath academic hospital national health laboratory service sr rules in the detection of this critical pathology. table 1: modified icgh rules implemented at the chris hani baragwanath academic hospital national health laboratory service laboratory. materials and methods ethical considerations the study was approved by the human research ethics committee of the university of the witwatersrand, johannesburg (ref. no. m150160). study design samples requested for malaria testing (plasmodium falciparum antigen along with thick and thin sr) between january and april of 2015 were extracted from the laboratory information system (trakcare, intersystems, cambridge, massachusetts, united states) and those with positive test results identified. samples positive for the p. falciparum antigen but negative on microscopy were excluded. the laboratory information system was then searched for fbcs performed on the same day as the malaria test in order to evaluate whether sr rules were triggered. fbc samples were processed using a sysmex xe-5000 analyser and sysmex information system middleware (sysmex, kobe, japan). all icgh rules were applied to samples with both a fbc and dwcc requested, whereas only parameter-related rules were applied to samples with requests for a fbc alone. sr was also performed in samples when the analyser’s inbuilt malaria suspect flag was triggered. the rules violated for each fbc identified were documented, and the performance of the rules assessed. as a retrospective, laboratory-based study, no clinical information was available. determination of which patients had severe malaria was therefore defined on the basis of laboratory criteria, viz. severe anaemia (hb < 7 g/dl in adults and < 5 g/dl in children aged < 12 years), parasitaemia > 10%, renal failure (urea > 20 mmol/l or creatinine > 265 umol/l) hypoglycaemia (< 2.2 mmol/l) or hyperbilirubinaemia (> 50 umol/l with > 100 000 parasites/ul).6 patients admitted to the intensive care unit were also classified as having severe malaria. statistical analysis continuous data are presented as the median (interquartile range) and categorical data as frequencies and percentages. the mann-whitney u-test was used to compare variables of interest (both ordinal and continuous). the false negative rate was calculated as the proportion of all patients with malaria where sr rules were not triggered. statistical analysis was performed using statistica software, version 12.0 (stat soft (pty) ltd, tulsa, oklahoma, united states). statistical significance was accepted at a two-sided p-value of 0.05. results a total of 153 samples collected from 130 patients met the inclusion criteria. pertinent demographic information is summarised in table 2. most patients had only a fbc performed, with a dwcc being requested in 63 (41.2%) patients. sr rules were triggered in 132 (86.3%) cases, either in the current specimen or in previously submitted samples. of the reviewed samples 63 were submitted for a dwcc (9/63 in previous and 54/63 in current samples) and 69 for a fbc only (16/69 in previous and 53/69 in current samples). the rule trigger rate did not differ between samples requested for a dwcc as compared to those requested only for a fbc (85.7% vs 86.7%; p = 0.87), but was significantly higher among patients classified as having severe malaria as compared to those without severe malaria (100.0% vs 81.9%, p = 0.017). table 2: pertinent demographic data. smear review rules triggered parameter rules were triggered in all samples, the commonest being the thrombocytopenia rule, which was flagged in 105 patients (table 3). common analyser morphology flags included those querying the presence of atypical lymphocytes, immature granulocytes and blasts, but these were not triggered in the absence of parameter flags. table 3: parameter rules triggered among patients with malaria. analysis of patients without smear review rules triggered among the 21 patients in whom no sr rules were triggered, 19 (90.5%) had a haemoglobin level ≥ 10 g/dl, 14 (66.7%) had a platelet count >120 × 109/l and 8 (38.1%) had a platelet count >150 × 109/l. the malaria parasitaemia did not differ statistically between those with and without sr rules triggered (median 1.9% vs 2.1%; p = 0.44), and was < 0.5% in six patients. nine (42.9%) of the samples with no sr rules triggered a request for a dwcc, and all were the first or only sample submitted. assessment of morphology findings when sr was performed, malaria parasites were missed in 13.0% of cases, predominantly when the parasitaemia was low (median parasitaemia was 0.35% in those where parasites were missed vs 3.1% in those with parasites identified). the parasite detection rate was significantly greater in patients with a parasitaemia > 0.5% as compared to those with lower parasite loads (93.1% vs 37.5%; p < 0.0001). discussion in this retrospective study assessing the performance of modified icgh sr rules in patients with malaria, sr was prompted in 86.3% of cases. parameter flags were triggered in all patients, the commonest being the thrombocytopenia flag which was triggered in 68.6% of cases. the false negative rate (12.7%) was higher than that recommended by the icgh (< 5%),3 largely due to the presence of near normal blood counts (haemoglobin > 10 g/dl and platelets >120 × 109/l) in 70% of patients with no sr rules violated. analyser morphology flags were evaluated only among samples with a request for a dwcc, but these did not make a substantial contribution to increasing the sr rate, with no statistical difference between those with and without a dwcc requested (85.7% vs 86.7%; p = 0.87). clearly, a proportion of cases with malaria will inevitably be missed using current sr rules. reassuringly, sr rules were triggered in all patients with severe malaria, thus reducing the likelihood of this condition being missed when not suspected clinically. however, a substantial proportion (13.0%) of cases were missed upon sr, most commonly in cases with very low paraistaemia (< 0.5%). this highlights the poor sensitivity of microscopy for malaria diagnosis, which is highly dependent on operator skill and further compromised by the pressures of large caseloads.7 alternate means of screening blood samples for malaria (where specific malaria testing has not been requested) would be of value. malaria detection technologies offered by several haematology analysers are of interest in this respect, as a fbc is invariably requested in hospitalised patients with fever of uncertain origin. in sysmex analysers, malaria suspect flags are generated on the basis of ‘pseudo-eosinophilia’ (which detects pigment-laden granulocytes or monocytes) or abnormalities of the dwcc and reticulocyte scatterplots. the reported sensitivity of these parameters is variable (46.2% – 69.4% for the abnormal diff scattergram or pseudo-esoinophilia,8,9 60% – 97% for abnormalities of the wbc/baso scatterplot10 and 77% for abnormalities of the reticulocyte scattergram10), with superior performance among patients with p. vivax infection.10,11 in this study, a malaria flag was not triggered by the middleware in any of the cases, most likely due to the rarity of p. vivax infection in johannesburg. this mirrors the poor performance of these parameters in patients with p. falciparum malaria reported elsewhere10,11 and suggests that the currently available malaria-specific sysmex flags are not of significant utility in our setting. two algorithm-based approaches for sysmex analysers were developed by campuzano-zuluaga et al. using novel and research-based parameters (such as an increase in the number of events detected in the lymph-y channel and the number of events within a currently unevaluated area of the wbc/baso scatterplot [designated the wbc/baso {iii} counting area]), and showed good diagnostic accuracy (> 85%) for p. falciparum.10 the utility of one of these algorithms (the n-od1pf model) was confirmed by dubreuil et al., who showed a sensitivity of 77% for p. falciparum detection using this algorithm in a non-endemic area (france).11 further assessment of such models in our setting would be of interest. lastly, the new sysmex xn-30 haematology analyser has novel malaria detection technology12 currently in the testing phase, which reportedly has a sensitivity of 98.4% for p. falciparum detection (personal communication prof. theresa coetzer, dept of molecular medicine and haematology, university of the witwatersrand, johannesburg). the study is ongoing but preliminary results are encouraging. however, it remains to be seen whether this technology could be incorporated on existing routine haematology platforms. other malaria-specific testing modalities carry superior sensitivity rates to conventional microscopy, such as malaria antigen rapid detection kits and various point-of-care kits employing polymerase chain reaction technology (such as the illumigene ®, which targets a 214-base-pair segment of a non-coding region of plasmodium species mitochondrial dna13,14 and has a limit of detection for p. falciparum and p. vivax of 2 and 0.125 parasites/µml, respectively15). while such detection rates are impressive, these modalities are not suitable for incidental detection of malaria, but are of primary value when malaria is suspected clinically. limitations of this study include the paucity of clinical information, as well as the absence of non-falciparum species among the cases included. conclusion the international consensus group for haematology smear review rules have a significant false negative rate for the detection of p. falciparum malaria, but perform well in patients with severe malaria. until more sensitive technologies for incidental malaria detection are widely available, clinical vigilance for this condition is required. acknowledgements competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support none. authors’ contributions j.l.v. was responsible for data collection, analysis and manuscript preparation. n.a. did editorial work and provided analytical input. s.h. and e.b. were involved in data collection and provided editorial input. references world health organization. world malaria report 2017. geneva: who; 2017. licence: cc-by-nc-sa 3.0 igo. blumberg lh. recommendations for the treatment and prevention of malaria: update for the 2015 season in south africa. s afr med j. 2015;105(3):175–178. barnes pw, mcfadden sl, machin sj, simson e. the international consensus group for hematology review: suggested criteria for action following automated cbc and wbc differential analysis. lab hematol. 2005;11(2):83–90. https://doi.org/10.1532/lh96.05019 ramsuran v, kulkarni h, he w, et al. duffy-null-associated low neutrophil counts influence hiv-1 susceptibility in high-risk south african black women. clin infect dis. 2011;52(10):1248–1256. https://doi.org/10.1093/cid/cir119 vaughan jl, wiggil tml, alli n, hodkinson k. the prevalence of hiv seropositivity and associated cytopenias in full blood counts processed at an academic laboratory in soweto, south africa. s afr med j. 2017;107(3):264–269. https://doi.org/10.7196/samj.2017.v107i3.11206 severe malaria. trop med int health. 2014;19 suppl 1:7–131. https://doi.org/10.1111/tmi.12313_2 hanscheid t. current strategies to avoid misdiagnosis of malaria. clin microbiol infect. 2003;9(6):497–504. https://doi.org/10.1046/j.1469-0691.2003.00640.x huh hj, oh gy, huh jw, chae sl. malaria detection with the sysmex xe-2100 hematology analyzer using pseudoeosinophilia and abnormal wbc scattergram. ann hematol. 2008;87(9):755–759. https://doi.org/10.1007/s00277-008-0486-8 yoo jh, song j, lee ka, et al. automated detection of malaria-associated pseudoeosinophilia and abnormal wbc scattergram by the sysmex xe-2100 hematology analyzer: a clinical study with 1,801 patients and real-time quantitative pcr analysis in vivax malaria-endemic area. am j trop med hyg. 2010;82(3):412–414. https://doi.org/10.4269/ajtmh.2010.09-0560 campuzano-zuluaga g, alvarez-sanchez g, escobar-gallo ge, et al. design of malaria diagnostic criteria for the sysmex xe-2100 hematology analyzer. am j trop med hyg. 2010;82(3):402–411. https://doi.org/10.4269/ajtmh.2010.09-0464 dubreuil p, pihet m, cau s, et al. use of sysmex xe-2100 and xe-5000 hematology analyzers for the diagnosis of malaria in a nonendemic country (france). int j lab hematol. 2014;36(2):124–134. https://doi.org/10.1111/ijlh.12145 hamaguchi y, yoshida t, suzuki y, et al. overview of malaria detection technology achieved with automated hematology analyzer xn-30. sysmex j int. 2017;27(1):1–6. notomi t, okayama h, masubuchi h, et al. loop-mediated isothermal amplification of dna. nucleic acids res. 2000;28:e63. https://doi.org/10.1093/nar/28.12.e63 hopkins h, gonzález ij, polley sd, et al. highly sensitive detection of malaria parasitemia in a malaria-endemic setting: performance of a new loop mediated isothermal amplification kit in a remote clinic in uganda. j infect dis. 2013;208:645–652. https://doi.org/10.1093/infdis/jit184 lucchi nw, gaye m, diallo ma, et al. evaluation of the illumigene malaria lamp: a robust molecular diagnostic tool for malaria parasites. sci rep. 2016;6:36808. https://doi.org/10.1038/srep36808 abstract introduction methods results discussion acknowledgements references about the author(s) alshymaa a. ahmed department of clinical pathology, faculty of medicine, zagazig university, zagazig city, egypt alia a. el shahawy department of medical microbiology and immunology, faculty of medicine, zagazig university, zagazig city, egypt heba m. kadry department of medical microbiology and immunology, faculty of medicine, zagazig university, zagazig city, egypt nora m. said department of clinical pathology, faculty of medicine, zagazig university, zagazig city, egypt citation ahmed aa, el shahawy aa, kadry hm, said, nm. performance of two multiplex flow cytometric assays for antibody detection in egyptian patients. afr j lab med. 2023;12(1), a2099. https://doi.org/10.4102/ajlm.v12i1.2099 note: additional supporting information may be found in the online version of this article as online supplementary document 1 original research performance of two multiplex flow cytometric assays for antibody detection in egyptian patients alshymaa a. ahmed, alia a. el shahawy, heba m. kadry, nora m. said received: 14 oct. 2022; accepted: 22 mar. 2023; published: 18 may 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: autoantibodies are vital biomarkers for the diagnosis, assessment and prognostic determination of various autoimmune disorders. objective: this study aimed to evaluate the performance of the two athena multi-lyte® systems for the detection of various autoantibodies. methods: a total of 105 systemic lupus erythematosus patients, 35 patients with other autoimmune diseases (diseased controls), and 30 healthy volunteers (healthy controls) at zagazig university hospitals, zagazig city, al sharqia governorate were tested for anti-double-stranded dna (anti-dsdna) antibodies using indirect immunofluorescence (iif) and the athena multi-lyte® anti-nuclear antibodies-ii system between may 2020 and april 2022. seventy-five patients with clinically suspected autoimmune vasculitis (aiv) and 25 healthy volunteers were also tested for anti-myeloperoxidase and anti-proteinase 3 antibodies using iif, the athena multi-lyte® aiv system, and enzyme-linked immunosorbent assay (elisa). results: the athena anti-dsdna test (98.5%) was more specific than iif (96.9%) for diagnosing systemic lupus erythematosus, but both tests had the same sensitivity (38.1%). combining both methods increased sensitivity to 47.6%, while increasing the cut-off of the athena anti-dsdna test to 134 international units/ml increased specificity to 100%. the athena multi-lyte aiv system exhibited substantial agreement with iif regarding anti-myeloperoxidase testing (κ = 0.65) and almost perfect agreement with elisa (κ = 0.85). the athena multi-lyte® aiv system exhibited perfect agreement with iif (κ = 1) and substantial agreement with elisa for anti-proteinase 3 testing (κ = 0.63). conclusion: athena multi-lyte® systems appear to be reliable for anti-dsdna, anti-myeloperoxidase, and anti-proteinase 3 screening and may be an optimal choice for monitoring anti-dsdna levels. what this study adds: it is necessary to evaluate various autoantibodies detection assays to increase both sensitivity and specificity of autoimmune diseases diagnostic approaches. athena multi-lyte® systems appear to be reliable for anti-dsdna, anti-myeloperoxidase, and anti-proteinase 3 screening and may be an optimal choice for monitoring anti-dsdna levels. keywords: autoantibodies; anti-double-stranded dna; anti-neutrophil cytoplasmic antibodies; multiplex; performance. introduction systemic lupus erythematosus (sle) and systemic vasculitis are multisystem autoimmune diseases characterised by the generation of circulating autoantibodies specific to different target antigens. the incidence and prevalence of both sle and anti-neutrophil cytoplasmic antibody (anca)-associated vasculitis (aav) show wide geographical variations.1 the overall incidence of sle ranges from 0.3 cases per 100 000 population per year in ukraine to 31.5 per 100 000 per year among afro-caribbean people living in the united kingdom.2 in egypt, the estimated prevalence of adult sle is 6.1 cases per 100 000 persons, with a higher prevalence (11.3 per 100 000) reported among women.3 anti-neutrophil cytoplasmic antibody-associated vasculitis inflicts considerable global effects, including elevated mortality rates, poor quality of life, and socio-economic burdens,4 and studies have demonstrated an aav prevalence of between 48 and 184 cases per million individuals.1 in egypt, aav constitutes about 3.6% of vasculitides.5,6 however, there is inadequate data to identify the prevalence of aav in africa and south asia.7 autoantibodies are crucial biomarkers that guide the diagnosis, treatment, and follow-up of autoimmune disorders.8 new automated high-throughput assays are continuously being developed to replace conventional assays for detecting these autoantibodies. however, an accurate evaluation of these new techniques is mandatory to ensure that they are clinically valuable and can improve various medical decisions.9 according to the guidelines provided by the european league against rheumatism and the american college of rheumatology, patients are classified as positive for sle if they have serum anti-nuclear antibody titres ≥ 1:80.10 the presence of antibodies against double-stranded dna (anti-dsdna) is the serological hallmark for a diagnosis of sle. an increased anti-dsdna titre in association with low levels of the complement components c1q, c3, and c4 is indicative of an acute sle exacerbation.11,12 diverse methods have been developed for the detection of anti-dsdna. the crithidia luciliae immunofluorescence test is remarkably specific and yields a high positive predictive value for sle. however, the sensitivity of c. luciliae immunofluorescence test for sle diagnosis is low (ranging from 20% to 55%, depending on the kit supplier), and being a semi-quantitative test, it is not an optimal choice for clinical follow-ups and disease flare predictions.13,14,15 in contrast, the enzyme-linked immunosorbent assay (elisa) is more sensitive (> 60%) than c. luciliae immunofluorescence test but has a lower specificity for sle.15,16 multiplex bead-based assays provide the advantage of assessing multiple antibody specificities simultaneously in small volumes of serum. one such technique is the addressable laser bead immunoassay, as exemplified by the commercially available luminextm platforms. the sensitivity of addressable laser bead immunoassay is similar to that of elisa for the detection of anti-dsdna, while the specificity is comparable to that of c. luciliae immunofluorescence test.17 existing guidelines for the diagnosis and follow-up of sle such as ‘the 2019 european league against rheumatism and the american college of rheumatology classification criteria for systemic lupus erythematosus’18 do not specify a certain assay for anti-dsdna detection. however, the guidelines advise that positive results should be confirmed by indirect immunofluorescence (iif) or a farr assay.15,19 the detection of ancas is vital for the diagnosis of the unique group of small-vessel vasculitic disorders named aav. this group includes three main diseases: granulomatosis with polyangiitis, microscopic polyangiitis, and eosinophilic granulomatosis with polyangiitis. patients with granulomatosis with polyangiitis are mainly proteinase 3-anca positive, while those with microscopic polyangiitis and eosinophilic granulomatosis with polyangiitis are mainly myeloperoxidase-anca positive.20,21 indirect immunofluorescence is used as a screening test for anca in accordance with the 1999 international recommendations for anca detection.22 this guideline advises the use of an antigen-specific assay to confirm a positive iif test. however, the revised 2017 international consensus of anca testing23 stated that the combined use of both iif and antigen-specific immunoassays is not necessary. specifically, a high-quality antigen-specific immunoassay is sufficient and does not need iif confirmation.24,25 international guidelines and recommendations have emphasised the roles of anti-dsdna and anca in the diagnosis and follow-up of autoimmune rheumatic diseases and vasculitic disorders.15,23 with this study, therefore, we aimed to assess the performance of the athena multi-lyte® test systems for the detection of these antibodies. methods ethical considerations ethical approval for this study was obtained from the zagazig university-institutional research board (zu-irb) (approval number: 5926). written informed consent was obtained from participants and parents or guardians of patients younger than 18 years. all procedures were performed according to the principles of the declaration of helsinki.26 patients’ files and samples were de-identified and coded, and all laboratory members were blinded to the participants’ data. study population this study was conducted in the clinical pathology department of the faculty of medicine at zagazig university, zagazig, al sharqia, egypt, from may 2020 to april 2022. all participants were of the same ethnicity. the required sample sizes were calculated using the openepi program version 3 (https://www.openepi.com/samplesize/sscc.htm) at 95% confidence, 90% power, and a 10% dropout rate.27 we assumed the anti-dsdna sensitivity in sle patients to be 33% and that up to 3% of healthy controls and 6% of diseased controls can be anti-dsdna positive.15 we also assumed an anca sensitivity of 46% in vasculitis patients and that up to 6% of healthy controls may be anca positive.28 sera for anti-dsdna testing were collected from 170 subjects, including 105 patients who met the revised criteria (european league against rheumatism and the american college of rheumatology) for sle classification18 and 65 control subjects matched for age and gender. the control subjects included 35 patients diagnosed with autoimmune diseases other than sle (diseased controls) and 30 healthy participants (healthy controls). sera for anca testing were collected from 75 patients with clinically suspected autoimmune vasculitis (aiv) and 25 healthy volunteers matched for age and gender (control group). individuals who were pregnant, had a severe infection or systemic disease,29 or refused to sign the informed consent were excluded from the study. personal data (age, gender, ethnicity) and medical data were collected from patients’ files retrieved from the rheumatology department after obtaining the required permissions. for the diseased control group, the disease diagnosis was confirmed clinically and by the presence of specific autoantibodies. serum rheumatoid factor and anti-ccp measured using the cobas 6000 autoanalyzer (roche diagnostics, forrenstrasse, rotkreuz, switzerland) were used to confirm rheumatoid arthritis. using the anti-nuclear antibodies profile 3 immunoglobulin g (igg) euroline immunoblot assay (euroimmun, seekamp, lübeck, germany), we measured anti-sjögren’s-syndrome-related antigen a and anti-sjögren’s-syndrome-related antigen b for primary sjögrens’ syndrome diagnosis, anti-scl70 for systemic sclerosis diagnosis, and anti-jo1 and anti-pm/scl100 for polymyositis diagnosis. sample collection two millilitres of whole blood were collected from each patient at the time of diagnosis. the blood samples were left to clot at room temperature for 15 min – 30 min and then centrifuged at 1000−2000 × g for 10 min. sera were separated and stored at −80 °c prior to use. multiplex microbead-based immunoassay the athena multi-lyte ana-ii plus test system (zeus scientific, evans way, branchburg, new jersey, united states) was used for the quantitative detection of igg-class anti-dsdna, the qualitative detection of igg-class anti-nuclear antibodies, and the semi-quantitative detection of igg-class antibodies specific for eight different nuclear antigens (sjögren’s-syndrome-related antigen a, sjögren’s-syndrome-related antigen b, sm, rnp, scl-70, jo-1, centromere b, and histone) in the sera of enrolled subjects. the athena multi-lyte aiv plus test system (zeus scientific, evans way, branchburg, new jersey, united states) was used for the semi-quantitative and qualitative detection of igg-class antibodies specific for three analytes (myeloperoxidase, proteinase 3, and glomerular basement membrane) in the sera of enrolled subjects. results > 120 international units/ml were considered positive. the microsphere suspension was prepared according to the manufacturer’s instructions and analysed using the luminex200 platform (a diasorin company, technology boulevard, austin, texas, united states). all samples were run in duplicate. indirect immunofluorescence assay anti-dsdna was detected via iif using nova lite® dsdna c. luciliae kits (inova diagnostics, inc., san diego, california, united states). the screening was performed at a serum dilution of 1/10, after which positive samples were diluted in the range of 1/20 to 1/640 for anti-dsdna titre determination. anti-neutrophil cytoplasmic antibody was detected via iif using nova lite® anca anti-neutrophil cytoplasmic autoantibody kits (inova diagnostics, inc., san diego, california, united states). the screening was performed at a serum dilution of 1/20. both tests were performed according to the manufacturer’s instructions. positive anti-myeloperoxidase appears as perinuclear staining (p-anca) while positive anti-proteinase 3 appears as cytoplasmic staining (c-anca).30 enzyme-linked immunosorbent assay for anti-myeloperoxidase and anti-proteinase 3 the orgentec anti-myeloperoxidase (p-anca) and anti-proteinase 3 (c-anca) elisa kits (orgentec diagnostika gmbh, mainz, germany) were used for the quantitative measurement of igg-class autoantibodies specific for myeloperoxidase and proteinase 3. results ≥ 5 iu/ml were considered positive. all samples were run in duplicate. we performed the assays and calculated the results according to the manufacturer’s instructions. data analysis the spss® 20.0 software package (ibm corporation, armonk, new york, united states) was used for data processing and analysis. the overall detection results are expressed as percentages of the total number of samples. agreement between different assays was assessed using cohen’s kappa (κ) coefficient, with the following levels of agreement: 0.01–0.2 = slight agreement, 0.21–0.4 = fair agreement, 0.41–0.61 = moderate agreement, 0.61–0.8 = substantial agreement, and 0.81–1 = almost perfect or perfect agreement. receiver operating characteristic curves were constructed, and the areas under the curves were calculated at 95% confidence intervals. the t-test and one-way analysis of variance with post hoc tests were used to compare ages, and the chi-square test was used to compare frequencies. the spearman correlation coefficient test (r) was performed to examine the correlation between anti-dsdna concentrations measured by athena multi-lyte® ana-ii (continuous variable) and anti-dsdna titres estimated by iif (ordinal variable).31 results were considered significant at a p-value < 0.05. sensitivity, specificity, accuracy, and positive and negative predictive values were calculated using medcalc (https://www.medcalc.org/) (medcalc software ltd, acacialaan, ostend, belgium).32 in addition to the clinical and analytical performance evaluations conducted, we compared the general characteristics of the assays used, including time taken for the run, estimated cost per test, and the technical experience required. results general characteristics of patients anti-dsdna antibodies were studied in 105 sle patients and participants in control groups (30 healthy participants and 35 patients with autoimmune diseases other than sle). among patients with other autoimmune diseases, 18 had systemic sclerosis, 15 had rheumatoid arthritis, one had polymyositis, and one had primary sjögren syndrome. within the anca group, we examined 75 patients with aiv and 25 healthy participants matched for age and gender. systemic lupus erythematosus patients’ ages ranged from 5 to 60 years (mean ± standard deviation: 32.4 ± 12.8 years), and 80 (76.1%) of them were women (table 1). the ages of the aiv patients ranged from 14 to 77 years (mean ± standard deviation: 38.4 ± 15.7 years), and 50 (66.7%) of them were women. table 1: demographic characteristics of sle patients and control subjects at the zagazig university hospitals, zagazig, al sharqia, egypt, may 2020 – april 2022. the most common symptom observed in the sle group was skin manifestations (86.7%; 91/105) in the form of photosensitivity, malar, or discoid rash (table 2). articular manifestations in the form of arthritis and arthralgia were observed in 82.8% (87/105) of patients, while 75.2% (79/105) of patients had nephritis. autoimmune vasculitis patients presented mainly with constitutional symptoms (80.0%; 60/75) and skin manifestations (57.3%; 43/75) in the form of skin ulcerations and palpable purpura. the lungs (66.7%; 50/75) and cardiovascular system (56.0%; 42/75) were the most frequently affected organs in these patients. anti-nuclear antibodies were detected in all sle patients (100%; 105/105) and 77.1% (27/35) of the diseased control group. other autoantibodies were detected in both groups with variable frequencies. table 2: clinical manifestations and serological findings of sle patients, aiv patients, and diseased controls, zagazig university hospitals, zagazig, al sharqia, egypt, may 2020 – april 2022. anti-double-stranded dna testing by iif and athena multi-lyte ana-ii test overall, 38.1% (40/105) of sle patients were positive for anti-dsdna by iif or athena multi-lyte® ana-ii. additionally, athena multi-lyte® ana-ii yielded equivocal results for 9.5% (10/105) of samples (anti-dsdna 100 iu/ml – 120 iu/ml). all healthy controls were negative for anti-dsdna using both iif and athena multi-lyte® ana-ii. one patient with rheumatoid arthritis had positive anti-dsdna results with iif, and another one had positive results with both methods. there was a moderate correlation between the anti-dsdna concentrations measured using athena multi-lyte ana-ii and the anti-dsdna titres estimated using iif (r = 0.55, p < 0.001). the athena multi-lyte® ana-ii and iif results exhibited substantial agreement with respect to anti-dsdna testing (κ = 0.66, 95% confidence intervals: 0.53–0.79, percent agreement: 87.6%, p < 0.001). at the manufacturer’s cut-off (120 iu/ml), the sensitivity of athena multi-lyte ana-ii for sle diagnosis was 38.1%, and the specificity was 98.5% (online supplementary table 1, figure 1). the best youden’s index was obtained when the cut-off was lowered to 109 iu/ml, at which the sensitivity increased to 46.7% and the specificity decreased to 96.9%. full (100%) specificity was reached by increasing the cut-off to 134 iu/ml, but this caused the sensitivity to decrease to 37.1%. for iif, the sensitivity was 38.1% and the specificity was 96.9%. however, the combination of both methods at the manufacturer’s cut-off (120 iu/ml) increased the sensitivity to 47.6% and the specificity to 96.9%. figure 1: receiver operating characteristic curves showing the clinical performance of iif and athena multi-lyte ana-ii when conducted separately and in combination for the diagnosis of sle among patients attending zagazig university hospitals, zagazig, al sharqia, egypt, between may 2020 and april 2022. the european league against rheumatism and the american college of rheumatology criteria was used as the gold standard. to determine its analytical performance, we compared the results of athena multi-lyte® ana-ii to those of iif, the gold-standard method. athena multi-lyte® ana-ii had a sensitivity of 73.8% (95% confidence intervals: 57.96% – 86.14%), a specificity of 92.19% (86.10% – 96.19%), a positive predictive value of 75.61% (62.47% – 85.24%), a negative predictive value of 91.47% (86.56% – 94.70%), an accuracy of 87.65% (81.74% – 92.19%), and an area under the curves of 0.830 (0.746–0.914) (figure 2). figure 2: receiver operating characteristic curve showing the analytical performance of athena multi-lyte ana-ii in the detection of anti-double-stranded dna among patients with sle attending zagazig university hospitals, zagazig, al sharqia, egypt, between may 2020 and april 2022. indirect immunofluorescence was used as the gold standard. anti-myeloperoxidase and anti-proteinase 3 analyses using iif, athena multi-lyte® iif, and elisa overall, 62.7% (47/75) and 14.7% (11/75) of patients tested positive for p-anca and c-anca by iif, while only 40.0% (30/75) and 14.7% (11/75) tested positive for anti-myeloperoxidase and anti-proteinase 3 by athena multi-lyte aiv. using the elisa method, 32.0% (24/75) of patients were positive for ani-myeloperoxidase, and 28.0% (21/75) were positive for anti-proteinase 3. all healthy volunteers were negative for anti-myeloperoxidase and anti-proteinase 3 using all three methods. all subjects were negative for anti-glomerular basement membrane. regarding the results of anti-myeloperoxidase testing, we observed a substantial agreement between iif and athena multi-lyte aiv (κ = 0.653), a moderate agreement between iif and elisa (κ = 0.525), and an almost perfect agreement between athena multi-lyte aiv and elisa (κ = 0.853) (table 3). for anti-proteinase 3 testing, there was a perfect agreement between athena multi-lyte aiv and iif (κ = 1), and a substantial agreement between elisa and both iif and athena multi-lyte aiv (κ = 0.635). moreover, the elisa results were highly correlated with the athena multi-lyte aiv results for the analyses of both anti-myeloperoxidase (r = 0.606, p < 0.001) and anti-proteinase 3 (r = 0.363, p = 0.006). table 3: agreement between the studied methods (athena multi-lyte aiv, iif, and elisa) for anca detection among aiv patients attending zagazig university hospitals, zagazig, al sharqia, egypt, between may 2020 and april 2022. the clinical performances of the anti-myeloperoxidase and anti-proteinase 3 detection methods could not be assessed because a definitive diagnosis of aiv relies on histopathology, which was not performed routinely for all patients. however, we assessed the analytical performances of athena multi-lyte aiv and elisa versus iif, the gold-standard method. for anti-myeloperoxidase, athena multi-lyte aiv had a higher sensitivity than elisa (63.8% vs 51.6%), while both tests had a specificity of 100% (online supplementary table 2). for anti-proteinase 3, both methods had a sensitivity of 100%, while athena multi-lyte aiv had a higher specificity than elisa (100% vs 88.8%). a comparison of the general characteristics of the methods used showed that iif had the highest estimated cost (240.00 egyptian pounds [egp]) for anti-dsdna testing as further dilutions were required to obtain the antibody titre using iif (table 4). however, for anca testing, elisa had the highest cost (200.00 egp) and time consumption (180 min) due to the need for two separate kits to obtain antigen-specific identification of anti-myeloperoxidase and anti-proteinase 3. in addition to being a quantitative rather than semi-quantitative assay, the athena multi-lyte also required the least amount of technical expertise (a bachelor’s degree in clinical laboratory science), was the least time-consuming (90 min), and had intermediate costs (120.00 egp). table 4: comparison of the general characteristics of athena multi-lyte, iif, and elisa used for anti-dsdna and anca detection among sle and aiv patients attending zagazig university hospitals, zagazig, al sharqia, egypt, between may 2020 and april 2022. discussion this work was designed to evaluate the performance of two commercially available multiplex bead-based flow cytometric assays, the athena multi-lyte ana-ii and athena multi-lyte aiv systems, for the detection of anti-dsdna and ancas. the multiplex assays had reasonable performance compared with traditional methods used for the detection of autoantibodies. for the detection of anti-dsdna, our data demonstrated a substantial agreement between the athena multi-lyte test system and iif. however, we observed a highly significant correlation between anti-dsdna concentration by athena multi-lyte test system and iif antibody titre. our findings are in concordance with the results of previous studies conducted in canada in 201033 and 2013,34 and in italy in 2018.15 these studies used a device from a different manufacturer (bioplex 2200, bio-rad laboratories, hercules, california, united states) and reported the same level of agreement and correlation with disease activity as achieved with iif. according to our data, the athena multi-lyte test system had the same sensitivity as iif for anti-dsdna detection, as well as improved specificity. moreover, the combination of both methods increased the sensitivity to 47.6%. two studies performed by infantino and colleagues in italy in 201514 and 201815 reported that the multiplex bead-based assay was more sensitive than iif but was slightly less specific.15 based on our observations, the athena multi-lyte test system for anti-dsdna detection provides multiple advantages, as it is a simple and rapid method with a reasonable cost and has acceptable clinical and analytical performance. although the combination of athena multi-lyte ana-ii and iif provided better sensitivity, economic factors might hinder the routine use of this combination for screening. our findings favour the use of the athena multi-lyte test for patient follow-ups because this one-step quantitative analysis is simpler than the multiple dilutions required for the semi-quantitative iif determination of antibody titres. the athena multi-lyte test systems exhibited almost perfect agreement with elisa and moderate agreement with iif with respect to anti-myeloperoxidase detection. additionally, the athena multi-lyte assay exhibited an almost perfect agreement with elisa and a perfect agreement with iif for anti-proteinase 3 detection. these results are consistent with the results of previous studies, including one performed in the netherlands in 2007, and another multicentre study performed in 2017 that recruited patients from germany, denmark, the netherlands, and belgium.35,36 only one study, conducted in the united states in 2018,37 reported a moderate agreement between a multiplex bead-based assay and elisa for both anti-myeloperoxidase (κ = 0.35) and anti-proteinase 3 (κ = 0.53) detection. this difference might be attributable to the use of kits produced by a different manufacturer (bio-plex® 2200 testing platform, bio-rad laboratories, hercules, california, united states). the analytical performance of athena multi-lyte aiv was superior to that of elisa for both anti-myeloperoxidase and anti-proteinase 3 detection. for anti-myeloperoxidase testing, athena multi-lyte aiv was more sensitive than elisa, while both methods were equally specific. for anti-proteinase 3 testing, both methods were equally sensitive, whereas athena multi-lyte aiv was more specific. regarding anca detection, histopathology is the definitive diagnosis for aiv.10 however, these data were not available for the patients included in this study. therefore, we were unable to assess the clinical performance of athena multi-lyte aiv and anca iif assays. still, the athena multi-lyte test systems provided good analytical performances and significant agreement with the results of conventional assays, warranting their use for anti-myeloperoxidase and anti-proteinase 3 screening. limitations this was a single-centre study with a relatively small sample size. the study was also limited by a lack of histopathology data for patients with suspected aiv. conclusion the athena multi-lyte test systems are reliable and robust for the simultaneous detection of autoantibodies. these tests aid in the diagnosis and follow-up of systemic rheumatic disorders and the diagnosis of vasculitic disorders. multiplex autoantibody testing is less time-consuming than traditional single-antibody assessment and requires only small sample volumes. future studies that include the definitive diagnosis of aiv according to international standards are required to yield a better understanding of the clinical performances of anti-myeloperoxidase and anti-proteinase 3 tests. we recommend the conduct of a multicentre study with a larger sample size to confirm our findings. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions a.a.a., a.a.e.s., h.m.k. and n.m.s contributed to the study conceptualisation and design. n.m.s. and a.a.a. contributed to sample processing, data analysis and manuscript writing. a.a.e.s. and h.m.k. contributed to patient’s selection, data collection and manuscript revision. finally, the manuscript has been read and approved by all the authors. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability data supporting the 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performance and correlation with systemic lupus erythematosus disease activity. clin biochem. 2013;46(4–5):317–320. https://doi.org/10.1016/j.clinbiochem.2012.12.004 damoiseaux j, vaessen m, knapen y, et al. evaluation of the fidis vasculitis multiplex immunoassay for diagnosis and follow-up of anca-associated vasculitis and goodpasture’s disease. ann n y acad sci. 2007;1109(1):454–463. https://doi.org/10.1196/annals.1398.051 bossuyt x, rasmussen n, van paassen p, et al. a multicentre study to improve clinical interpretation of proteinase-3 and myeloperoxidase anti-neutrophil cytoplasmic antibodies. rheumatology. 2017;56(9):1533–1541. https://doi.org/10.1093/rheumatology/kex170 sun q, calderon b, zhao z. discrepancies between two immunoassays for the determination of mpo and pr3 autoantibodies. clin chim acta. 2017;470:93–96. https://doi.org/10.1016/j.cca.2017.05.008 abstract introduction methods results discussion acknowledgements references about the author(s) annie e. cook department of clinical biochemistry, derriford combined laboratory, university hospitals plymouth national health service trust, derriford hospital, plymouth, united kingdom division of chemical pathology, department of pathology, stellenbosch university and national health laboratory service, tygerberg hospital, cape town, south africa thumeka p. jalavu division of chemical pathology, department of pathology, stellenbosch university and national health laboratory service, tygerberg hospital, cape town, south africa annalise e. zemlin division of chemical pathology, department of pathology, stellenbosch university and national health laboratory service, tygerberg hospital, cape town, south africa citation cook ae, jalavu tp, zemlin ae. audit of amylase and lipase requests in suspected acute pancreatitis and cost implications, south africa. afr j lab med. 2022;11(1), a1834. https://doi.org/10.4102/ajlm.v11i1.1834 original research audit of amylase and lipase requests in suspected acute pancreatitis and cost implications, south africa annie e. cook, thumeka p. jalavu, annalise e. zemlin received: 25 jan. 2022; accepted: 25 may 2022; published: 26 sept. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the internationally accepted criteria for the diagnosis of acute pancreatitis (ap) requires two of the three following features to be present: characteristic abdominal pain, elevated serum amylase and/or lipase enzymes, or consistent imaging results. however, sensitivity and specificity can vary depending on the population and cut-off values used. objective: this study evaluated the suitability of amylase and lipase as first-line diagnostic biomarkers of suspected ap for the local population served by tygerberg hospital, south africa. methods: this retrospective analysis was conducted in june 2019 using all amylase and/or lipase request data from december 2018. patient clinical data were included in sensitivity and specificity analyses of amylase, lipase or dual requests for diagnosis of ap. cost per test data were obtained from the national health laboratory service and used to calculate the total cost of the tests and potential savings. results: sensitivity for lipase was 90.0% compared to 50.0% for amylase. specificity was similar for singular measurements of lipase and amylase. dual measurement of amylase and lipase showed no improvement in sensitivity (83.3%) and only a minor increase in specificity (97.4%) compared with measurement of lipase alone. the estimated savings was r2522.85 ($174.98 usd), with a potential annual cost saving of r84 423.74 ($5855.69 usd). conclusion: lipase was shown to be a more sensitive biomarker compared to amylase for the screening of ap, providing evidence for laboratories to educate local staff and promote improved requesting practices by clinicians. additionally, preventing unnecessary dual requests may reduce costs. keywords: audit; pancreatitis; amylase; lipase; cost. introduction the atlanta symposium in 1992 sought to provide a unified international consensus for the classification of acute pancreatitis (ap). the revised version of the atlanta criteria expands on the previous definition to include a classification of severity, defines imaging morphology and discriminates ap as either intestinal oedematous pancreatitis or necrotising pancreatitis.1 according to the revised atlanta criteria and several other international guidelines, ap diagnosis requires two of the following three criteria to be present: the rapid onset of severe and persistent epigastric pain consistent with ap; serum lipase and/or amylase levels greater than three times the upper limit of normal (uln); or imaging consistent with ap, using contrast-enhanced computed tomography, magnetic resonance imaging or transabdominal ultrasonography.1,2,3,4 the gold standard for diagnosis of ap is often considered to be supporting radiological evidence,5 however, this option is rarely considered suitable as a first-line test, and therefore pancreatic enzyme biomarkers provide an essential criterion for quick and accurate assessment in cases where ap is suspected. currently serum amylase and lipase remain the most commonly requested tests for investigation of ap, which is supported by several international guidelines.1,3,4 the limitations of these biomarkers is that both lipase and amylase can also be raised to levels of greater than three times the uln in non-pancreatic conditions, such as renal disease, appendicitis and cholecystitis.6 liraglutide, a glucagon-like peptide-1 receptor agonist used for the treatment of diabetes, has also been shown to falsely elevate serum amylase and lipase levels.7 additionally, neither enzyme can determine aetiology or severity of ap in adults.8 despite this, lipase is considered to be the preferential diagnostic biomarker.9 this was first suggested in united kingdom guidelines 2005,4 due to the longer half-life of lipase compared with amylase; such that lipase still remains detectable 7–14 days after symptom onset, compared with 3–4 days with amylase.6 lipase is now globally accepted as the superior analyte for the investigation of ap, with many international studies and reviews evidencing its improved sensitivity and specificity compared with that of amylase.5,10,11 sensitivity and specificity can vary depending on the population included and the cut-off values used. the two most common aetiologies of ap internationally are gallstones or alcohol abuse. the most common cause of ap in the tygerberg community where this study was performed is alcohol abuse.11,12 we aimed to determine the relative sensitivity and specificity of lipase compared with amylase for the diagnosis of ap within the local population served by tygerberg hospital for the month of december 2018, and to determine what proportion of these requests were clinically indicated. the recommended test for ap is serum lipase, where available; amylase is used in settings where serum lipase is not available. the practice of requesting both tests (dual testing) in the initial evaluation is not recommended but is seen on a regular basis in our laboratory. this practice is not cost effective; it is therefore important to assess how prevalent it is so that it can be addressed. the final aim, therefore, was to review the absolute numbers of dual versus single amylase and lipase requests for the whole of 2018 and to calculate the potential cost-savings associated through changes in requesting practices since lipase became available in our laboratory. methods ethical considerations ethical approval was obtained to access the clinical data of patients where these analytes had been requested. this was sought in advance of the project start and was granted by the health research ethics committee of the faculty of medicine and health sciences at stellenbosch university (reference number: n19/03/036). patient consent was not obtained as a waiver of consent was awarded by the approving ethics committee. the clinical records of patients were only accessed if they were included in the study and were only accessed by authors of this paper. full anonymity of patients was maintained throughout the study and all data was stored in password protected files. study site this was a retrospective clinical audit of amylase and lipase requesting at tygerberg hospital, cape town, south africa. tygerberg hospital is a tertiary hospital in parow, cape town and provides inpatient and outpatient care to public health-sector users. it is a 1400 bed multidisciplinary teaching hospital affiliated with stellenbosch university. the national health laboratory service (nhls) chemical pathology laboratory at tygerberg hospital provides 24-h diagnostic service. the nhls is the preferred provider of pathology services to the public health sector. data collection pathology information technology provided an anonymised data set for all amylase and lipase requests for 2018 that included the following parameters: patient hospital numbers, ward location, date of birth and the amylase and/or lipase results. data from 1 december 2018 to 31 december 2018 were chosen as an initial subset for analysis and the clinical notes surrounding the dates of the amylase and lipase requests within this month were then individually scrutinised. enterprise content management software (open text ecm, opentext corporation, waterloo, ontario, canada), a web-based electronic patient management system, was used to assess each patient record and to determine whether any consistent imaging was performed, the patient clinical symptoms upon presentation and the final patient diagnosis. a diagnosis of ap was considered confirmed if two of the three atlanta criteria were fulfilled or in most cases, by an explicit statement provided in the clinical notes. requests were only included and analysed where the patient was being managed at tygerberg hospital and the laboratory test(s) had been carried out at the tygerberg nhls laboratory. records were excluded if patients were aged < 13 years old, had a recent history of penetrating or blunt abdominal trauma and where notes were incomplete or unclear. the cost per test data were obtained from the nhls and used to calculate the total cost of the tests over the month of december and potential savings if dual testing were to be eliminated. laboratory analyses samples were analysed for amylase and lipase using an enzymatic colorimetric methodology and performed on the roche® cobas® 6000 analyser (roche diagnostics, mannheim, germany) at the tygerberg hospital nhls laboratory. tygerberg hospital is accredited by the south african national accreditation system that regularly participates in external quality assessment to retain this status. statistical analysis all data used within the study were recorded, sorted and analysed using microsoft excel (microsoft corporation, 2019 [16.0], redmond, washington, united states). the normal ranges used by tygerberg hospital at the time this audit was conducted were 20 u/l – 104 u/l for serum amylase and 13 u/l – 60 u/l for lipase. based on these ranges, patient results for all amylase and/or lipase results for december 2018 were either designated ‘≤ 3uln’ less than or equal to three times the uln; or ‘> 3uln’, if the result was greater than three times the uln. presenting clinical details (if available), final diagnosis and relevant imaging were also recorded and then allocated either ‘yes’, ‘no’ or ‘unknown’ depending on whether they were considered consistent with ap. based on these parameters, amylase and/or lipase were designated either a false positive (fp), false negative (fn), true positive (tp) or true negative (tn) status. the relative sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) were calculated using the following equations: microsoft excel was also used to provide estimated cost savings associated with changes in amylaseand lipase-requesting practices. the sample numbers obtained from the 1 to 31 december dataset were used to extrapolate predicted annual savings. results a total of 268 patients had either a lipase or amylase test (or both) carried out during december 2018 at tygerberg hospital (table 1). forty-six of the 268 requests were excluded from the final data analysis, including duplicate requests (n = 16), requests on patients where notes were incomplete or unclear (n = 10) , patients with a recent history of penetrating or blunt abdominal trauma (n = 9), patients with chronic pancreatitis (n = 9), and patients < 13 years old (n = 2). a total of 222 patients were therefore included in the final data analysis; 12 (5.4%) patients had a final diagnosis of ap. table 1: amylase and lipase results for acute pancreatitis testing obtained from tygerberg hospital, south africa, 1–31 december 2018. specificities were relatively equivocal for single amylase (98.6%) and lipase tests (96.9%) (table 2). sensitivity of amylase showed a significant difference, however, at only 50.0% compared to lipase at 90.0% for the subset of data analysed. dual requesting showed an equivocal specificity of 97.4%, however the sensitivity was calculated to be 83.3%, which was 6.7% less than lipase testing alone. table 2: sensitivity, specificity, positive predictive value, negative predictive value and respective 95% confidence interval for amylase, lipase and dual testing for acute pancreatitis at tygerberg hospital, 1–31 december 2018. in december 2018, lipase was requested singly 151 times compared with amylase, which was requested 16 times (table 3). fifty-five of the 222 patients (25%) had dual testing for amylase and lipase. the price of amylase and lipase tests was r45.87 (south african rand [zar], equivalent to $3.10 united states dollars [usd]) per individual test for the period of december 2018. the cost of dual requests for the month of december was r2522.85 ($174.98 usd); that of unnecessary tests was r733.92 ($50.90 usd) and for those not clinically indicated, r5320.92 ($369.06 usd). the projected potential annual cost saving from all these additional or unnecessary tests is r84 423.74 ($5855.69 usd) (table 3). table 3: amylase and lipase test requests and predictive cost savings, tygerberg hospital, 1–31 december 2018. discussion the aim of this study was to compare the relative sensitivities and specificities of lipase and amylase for the diagnosis of ap across the tygerberg hospital population, and to evaluate this in the context of current requesting practices. the initial hypothesis by the laboratory was that amylase was often requested preferentially to that of lipase; the results of this audit, however, have shown that the opposite is true and that most requests in 2018 were, instead, for lipase. additionally, we examined the cost implications associated with current requesting practices and have estimated significant cost-saving potential with a change to single lipase requesting in place of unnecessary dual requests. lipase and amylase have been shown to have high specificity with respect to the diagnosis of ap,5,10,11 when used at the levels of greater than three times the uln, as recommended in the atlanta criteria.2 in this context, specificity is the ability of these biomarkers to correctly identify patients without ap when they are at levels of less than three times uln as stipulated in the atlanta criteria.2 whilst both biomarkers showed similar specificities from the results of this study, at 98.6% for amylase and 96.9% for lipase, sensitivity between the two analytes differed significantly, which was likely to have been significantly impacted by the small sample size. despite this, the results of this study agreed with the widely accepted international consensus, that lipase is a superior biomarker in cases of suspected ap, with a sensitivity of 90% compared with only 50% for amylase. this study was useful because it provided data based on the local tygerberg hospital population. it is widely acknowledged that the aetiology of ap can have a significant impact on the serum levels of these pancreatic enzymes,11 thus it can provide more relevant data for the local medical community to guide recommendations on appropriate requesting practices. amongst the tygerberg population, alcohol abuse is the primary cause of ap,11,12 which typically exhibits lower levels of amylase and lipase in comparison to the next most common cause of ap, gallstones. this could impact the relative sensitivity and specificity of these analytes according to the levels stipulated in the atlanta criteria.1 the sensitivity and specificity from dual requests showed minimal improvement in specificity when compared to lipase alone, and a reduced sensitivity. there were 513 dual requests for 2018. this represents around 257 unnecessary test requests annually which can be costly and potentially detrimental with respect to reduced sensitivity. a recent 2017 review of nine studies by ismail and bhayana,8 which included studies carried out by chang and chung5 and hofmeyr et al.,11 concluded a negligible difference in sensitivity and specificity from dual testing of lipase and amylase compared with singular testing of lipase.5,8,11 whilst studies by basnayake and ratnam,9 and ismail and bhayana8 have commented that the ratio between amylase and lipase levels could help direct clinicians to the aetiology of ap,8,9 it was generally concluded across several reviews and studies, that dual testing was not a cost-effective option for diagnosis of ap.5,8,11,13 a prospective study by hofmeyr et al.11 concluded that on admission to tygerberg hospital lipase sensitivity was significantly improved at 91% compared with 62% for amylase. the specificity of amylase and lipase testing for this population group were comparable, at 93% and 92%, respectively. the recommendations from this study were to promote lipase as the biomarker of choice locally for the diagnosis of ap.11 a potential strategy for overcoming inappropriate dual requesting and the use of amylase instead of lipase in these patient groups could be to limit or introduce a local procedure for demand management of amylase requests. the largest potential cost-saving identified from this study, however, would be through the promotion of greater clinically led requesting in suspected ap. over half of the requests included within this study were considered inappropriate based on the clinical details and final diagnosis provided in the patient notes. limitations there were several important limitations that should be considered with respect to this study. firstly, and most significantly, only 12 of the 222 patients used for the final data analysis had confirmed ap. despite this small subset of data, sensitivity and specificity of lipase and amylase was consistent with a previous study of these biomarkers within the tygerberg population.11 however, it should be noted that the large sensitivity differences observed between amylase and lipase are likely because of the small sample size used. the second limitation of this study is that requests were only considered clinically relevant if the patient notes mentioned abdominal, epigastric or associated pain or overtly queried ap. it is important to note that there are other disease states where these biomarkers may be requested for use in diagnosis and monitoring, where the patient may not typically present with abdominal pain for example, pancreatic cancer, mumps and cystic fibrosis.14,15 whilst these test requests would be considered clinically relevant, for the purposes of this audit, these cases were not accounted for in the final data and cost analysis. in addition, it is important to note that the main ap aetiology will vary between different population groups and this study only includes the local tygerberg population. another consideration that has not been accounted for as part of this study but may affect the associated sensitivities and specificities of these analytes is assay performance across different analytical platforms and methods. notwithstanding the limitations of this study, even this small dataset supports the existing literature, that lipase shows improved sensitivity when compared with amylase for the diagnosis of ap. ideally, a repeat clinical audit should be conducted to include at least a year of data which would enhance the robustness of these results. indeed, a comparative clinical audit from a united kingdom population, where the most common aetiology of ap differs, would help to demonstrate that lipase is a better marker, regardless of aetiology. the results of this study have helped to better equip the laboratory to inform and promote more clinically led and evidence-based requesting practices amongst local clinicians. whilst these pancreatic enzyme biomarkers can provide an important tool for clinicians trying to detect or eliminate an ap diagnosis, it is also important for requestors to have a full understanding of the potential limitations associated with them. a prompt diagnosis of ap is important for limiting associated complications if left undetected and ultimately improving patient outcomes. conclusion the results of this clinical audit support a growing body of evidence that lipase is superior as a first-line test for suspected ap and that there is little additional clinical value derived from dual requesting of lipase and amylase. despite the small subset of data used within this audit, we have shown that the sensitivity and specificity of lipase and amylase was consistent with a previous study of these biomarkers within the tygerberg population, and that lipase is the superior biomarker in terms of sensitivity. it is therefore recommended that dual requests for amylase and lipase are replaced by singular lipase requests where ap is suspected. acknowledgements this study was conducted at tygerberg hospital by a.e.c. during an elective placement, the travel for which was partially funded through a biochemical society travel grant. we would like to thank mr. w. kleinhans for his help with data acquisition. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions a.e.z. conceived the study and a.e.z. and t.p.j. supervised a.e.c. a.e.c. and t.p.j. performed the audit and analysed the data. a.e.c. wrote the first draft and all authors contributed to the final version of this article. sources of support a.e.c. received a biochemical society (general travel grant), london, united kingdom. data availability data supporting the findings of this study are available from the corresponding author, a.e.z., on request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references banks pa, bollen tl, dervenis c, et al. classification of acute pancreatitis–2012: revision of the atlanta classification and definitions by international consensus. gut. 2013;62(1):102–111. https://doi.org/10.1136/gutjnl-2012-302779 international association of pancreatology; american pancreatic association. iap/apa evidence-based guidelines for the management of acute pancreatitis. pancreatology. 2013;13(4 suppl. 2):e1–e15. https://doi.org/10.1016/j.pan.2013.07.063 tenner s, baillie j, dewitt j, vege ss. american college of gastroenterology guideline: management of acute pancreatitis. am j gastroenterol. 2013;108(9):1400–1415. https://doi.org/10.1038/ajg.2013.218 johnson cd. uk guidelines for the management of acute pancreatitis. gut. 2005;54(suppl. 3):1–9. https://doi.org/10.1136/gut.2004.057026 chang jwy, chung ch. diagnosing acute pancreatitis: amylase or lipase? hong kong j emerg med. 2011;18(1):20–25. https://doi.org/10.1177/102490791101800104 panteghini m, bais r. serum enzymes. in: rifai n, horvath ar, wittwer ct, editors. tietz textbook of clinical chemistry and molecular diagnostics. 6th ed. maryland heights, mo: elsevier, 2018; p. 404–434. steinberg wm, buse jb, ghorbani mlm, ørsted dd, nauck ma. amylase, lipase, and acute pancreatitis in people with type 2 diabetes treated with liraglutide: results from the leader randomized trial. diabetes care. 2017;40:966–972. https://doi.org/10.2337/dc16-2747 ismail oz, bhayana v. lipase or amylase for the diagnosis of acute pancreatitis? clin biochem. 2017;50(18):1275–1280. https://doi.org/10.1016/j.clinbiochem.2017.07.003 basnayake c, ratnam d. blood tests for acute pancreatitis. aust prescr. 2015;aug;38(4):128–130. butler j. towards evidence based emergency medicine: best bets from the manchester royal infirmary edited by k mackway-jones electrical stimulation and bell’s palsy. emerg med j. 2002;19(5):428–434. https://doi.org/10.1136/emj.19.5.428 hofmeyr s, meyer c, warren bl. serum lipase should be the laboratory test of choice for suspected acute pancreatitis. s afr j surg. 2014;52(3):72–75. https://doi.org/10.7196/sajs.2003 anderson f, thomson sr, clarke dl, loots e. acute pancreatitis: demographics, aetiological factors and outcomes in a regional hospital in south africa. s afr j surg. 2008;46(3):83–86. akhtar a, sarode r, agrawal d. measuring both serum amylase and lipase for acute pancreatitis lowers quality and raises cost. cleve clin j med. 2017;84(9):670–672. https://doi.org/10.3949/ccjm.84a.16103 acb. amylase test [homepage on the internet]. lab tests online uk; 2018 [cited 2019 nov 01]. available from: https://labtestsonline.org.uk/tests/amylase-test acb. lipase test [homepage on the internet]. lab tests online uk; 2018 [cited 2019 nov 01]. available from: https://labtestsonline.org.uk/tests/lipase-test abstract introduction methods results discussion acknowledgements references about the author(s) tiyani c. mthombeni department of medicine usage in south africa (musa), faculty of health sciences, north-west university, potchefstroom, south africa johanita r. burger department of medicine usage in south africa (musa), faculty of health sciences, north-west university, potchefstroom, south africa martha s. lubbe department of medicine usage in south africa (musa), faculty of health sciences, north-west university, potchefstroom, south africa marlene julyan department of medicine usage in south africa (musa), faculty of health sciences, north-west university, potchefstroom, south africa molebogeng r. lekalakala-mokaba division of medical microbiology, department of pathology, faculty of health sciences, university of limpopo, polokwane, south africa polokwane laboratory, national health laboratory service, polokwane, south africa citation mthombeni tc, burger jr, lubbe ms, julyan m, lekalakala-mokaba mr. eskape pathogen incidence and antibiotic resistance in patients with bloodstream infections at a referral hospital in limpopo, south africa, 2014–2019: a cross-sectional study. afr j lab med. 2024;13(1), a2519. https://doi.org/10.4102/ajlm.v13i1.2519 note: additional supporting information is available for download alongside the online version of this article as online supplementary materials. original research eskape pathogen incidence and antibiotic resistance in patients with bloodstream infections at a referral hospital in limpopo, south africa, 2014–2019: a cross-sectional study tiyani c. mthombeni, johanita r. burger, martha s. lubbe, marlene julyan, molebogeng r. lekalakala-mokaba received: 03 june 2024; accepted: 01 oct. 2024; published: 29 nov. 2024 copyright: © 2024. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: there is a paucity of research on the incidence and antimicrobial resistance (amr) of enterococcus faecium, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa, and enterobacter spp. (eskape) pathogens in africa because of the inadequate establishment of amr surveillance systems. objective: this study reports on the incidence and amr of bloodstream eskape pathogens at a referral hospital in northern south africa. methods: this retrospective descriptive study used routinely collected bloodstream isolates (pathogen identification and antimicrobial susceptibility testing performed using automated systems) from the south african national health laboratory service, from january 2014 to december 2019. resistant phenotypes analysed included methicillin-resistant s. aureus and carbapenem-resistant a. baumannii. results: the eskape pathogen incidence rate was stable from 2014 to 2019 (p = 0.133). the most isolated pathogens were s. aureus (268/746; 35.9%) and a. baumannii (200/746; 26.8%). staphylococcus aureus increased from 39 isolates in 2014 to 75 in 2019 (p = 0.132). the incidence rate of a. baumannii increased from 11.9% (16/134) in 2015 to 37.8% (68/180) in 2019 (p = 0.009). most isolates (417/746; 55.9%) were from the neonatal ward. carbapenem-resistant a. baumannii increased from 68.8% (11/16) in 2014 to 75.0% (51/68) in 2019 (p = 0.009). methicillin-resistant s. aureus decreased from 56.0% (14/25) in 2016 to 17.3% (13/75) in 2019 (p = 0.260). conclusion: routine data provide essential information on the incidence of eskape pathogens and amr phenotypes, serving as a basis for an antibiogram, a surveillance tool in antibiotic stewardship programmes. what this study adds: the study provided local information on the incidence and amr pattern of eskape pathogens, which is essential when developing empiric treatment protocols for appropriate antibiotic prescribing and infection prevention and control practices. keywords: acinetobacter baumannii; bloodstream infections; antibiotic resistance; klebsiella pneumoniae; south africa; staphylococcus aureus. introduction based on estimates from the global burden of disease study, bacterial infections and antimicrobial resistance (amr) were respectively responsible for 7.7 million and 4.9 million global deaths in 2019, making them the second leading cause of death after ischaemic heart disease.1,2 therefore, amr has become a global public health concern.2,3 the world health organization’s (who) african region is no exception. in 2019, this region had an estimated 1.05 million deaths that were associated with amr.4 however, amr surveillance data in the region are scarce.2,5 the inappropriate use of antibiotics is the leading cause of multidrug-resistant pathogens,3 particularly in lowand middle-income countries, where multidrug-resistant bacteria accounted for 15.5% of healthcare-acquired infections in 2011.6 most multidrug-resistant healthcare-associated infections are caused by six pathogens, namely enterococcus faecium, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa, and enterobacter spp., known as eskape, which are capable of ‘escaping’ the biocidal properties of antibiotics.6,7 the eskape pathogens are characterised as opportunistic infections and are associated with the highest risk of mortality and increased healthcare costs.6,7,8 in 2017, the who released its first edition of the 12 bacterial drug-pathogen priority list – a list of drug-pathogen combination bacteria that pose the greatest threat to human health because of the emergence of multidrug resistance.9 all eskape drug-pathogen resistant combinations – for example critical priority carbapenem-resistant a. baumannii (crab), carbapenem-resistant p. aeruginosa, carbapenem-resistant k. pneumoniae (k. pneumoniae), third-generation cephalosporin-resistant k. pneumoniae (3gcr k. pneumoniae), high priority vancomycin-resistant e. faecium, methicillin-resistant s. aureus (mrsa), third-generation cephalosporinand carbapenem-resistant enterobacter spp., and vancomycin-resistant s. aureus – are on the who’s priority list and serve as important indicators for amr surveillance and research.9 bacterial bloodstream, lower-respiratory tract, and intra-abdominal infections dominated the global and regional burdens of amr in 2019.1,2,4 the six eskape pathogens were among the 10 commonly isolated bloodstream infections responsible for amr-related deaths worldwide in 2019.1,2 lowand middle-income countries, especially in africa, have a high burden of bloodstream infections by eskape pathogens.2,4,8,10 the amr status of specific healthcare settings varies based on the epidemiology of bacterial infections, the antibiotics used, and the geographic area.1,2,11 in south africa, there were 39 000 amr-related deaths in 2019.12 according to national laboratory surveillance data, 40.0% of positive blood cultures in south africa contain one or more eskape pathogens.11 between 2014 and 2019, the bloodstream pathogens isolated the most in neonates in south africa were k. pneumoniae (28.0%), a. baumannii (14.0%), and s. aureus (12.0%).13 similarly, in 2020, from a mixed population of patients, k. pneumoniae was the eskape pathogen isolated the most in the bloodstream in south africa, followed by s. aureus, a. baumannii, e. faecium and p. aeruginosa.11 the susceptibility of k. pneumoniae to meropenem in south africa decreased significantly from 98.0% in 2014 to 89.0% in 2019 (p = 0.033). during the same period, the a. baumannii isolates decreased susceptibility to meropenem (23.0% to 12.0%; p = 0.051).13 the latest (2020) amr data for eskape pathogens in south africa showed that the incidence of extended-spectrum beta-lactamase-producing k. pneumoniae increased from 65.0% in 2016 to 70.0% in 2020, and carbapenem-resistant k. pneumoniae was at 40.0%.11 methicillin-resistant s. aureus decreased from 23.0% in 2016 to 18.0% in 2020. also in 2020, crab was 80.0%, carbapenem-resistant p. aeruginosa was 33.0%, and vancomycin-resistant e. faecium was 1.3%, with provincial variations attributed to differences in the empirical use of different antibiotics.11 previously, south africa conducted sentinel amr surveillance mainly in urban academic hospitals. therefore, there was inadequate amr surveillance data from rural hospitals, particularly those in limpopo province.14,15,16,17 a nationwide public sector study13 and several single-centre bloodstream culture studies from gauteng,18 kwazulu-natal19 and the western cape20,21 show incidence and amr variability of eskape pathogens according to the health sector (public versus private), location, hospital ward, patient age (e.g., neonates versus adults), level of healthcare (i.e., district, regional, and national hospitals), the source of infection (e.g., community-acquired versus hospital-acquired infections), and increased antibiotic resistance of gram-negative bacteria. these observed variations emphasise the importance of local data on the incidence and antibiotic resistance, particularly for local policy and practice interventions such as developing empirical treatment formularies by hospital infection prevention and control and antibiotic stewardship programmes.11,13 the surveillance of eskape pathogen incidence and priority drug-pathogen-resistant combinations must therefore be prioritised and strengthened to understand their magnitude.2,8,10 against this background, this study reports on the incidence of eskape pathogens, selected combinations of who priority drug-pathogen resistance and resistance to reserve (last resort) antibiotics using routinely collected data of patients with bloodstream infections limpopo province, south africa. methods ethical considerations the study obtained ethical clearance from the north-west university health research ethics committee (approval number nwu-00312-20-a1). the national health laboratory services manager for academic affairs and research granted permission to access the data source. the head of the limpopo department of health granted data access permission and use of the study centre name in publications. the north-west university health research ethics committee also granted a patient informed consent waiver since the data set was provided without patient names and other personal details (unique identification codes only). the data set was received in a password-protected microsoft excel® (microsoft corp., redmond, washington, united states) spreadsheet file and was stored on a password-protected computer. this manuscript does not report on the use of any animal or human data or tissue. data collection a retrospective, quantitative descriptive study design was adopted. the data were retrieved from the national health laboratory service (nhls) database, the national repository for all laboratory tests performed by south african public hospitals.22 no patient demographic and clinical data were extracted for the study. extracted study variables include the ward name, specimen type (blood), patient unique identification codes, organism name, year of isolation, and antimicrobial susceptibility test outcome (intermediate, resistant, or sensitive). the data represented all blood culture specimens performed at the 509-bed mankweng tertiary hospital. there were 454 beds designated as either level 1 (district) or level 2 (regional), and 55 as level 3 (tertiary services beds). the hospital is approximately 30 km east of polokwane in the capricorn district of limpopo province, south africa. mankweng tertiary hospital is the primary hospital for the surrounding community (population of ~260 000 to 280 000). in addition, the hospital offers 24-h tertiary services and receives referrals from district and regional hospitals in limpopo province.23 all eskape pathogens isolated from bloodstream infection and their respective antibiotic susceptibility results from january 2014 to december 2019 were included in the study. the study period was chosen to correspond to available antimicrobial consumption data extracted from the limpopo province pharmaceutical depot, which had previously been analysed and published. the data comprised all patients with positive blood cultures. blood cultures are considered the ‘gold standard’ investigation for detecting micro-organisms in blood (bloodstream infections). the nhls used different blood culture bottles to collect blood samples. two blood culture bottles (aerobic and anaerobic) were used for adults, and one for paediatric patients.24 the samples are inverted 8–10 times.24 blood culture samples were stored at 20 °c – 25 °c until the nhls courier collected them from mankweng tertiary hospital and transported to pietersburg tertiary hospital nhls laboratory for further processing (pathogen identification and amr testing). laboratory analyses in the nhls, blood culture bottles were sent to the microbiology laboratory for incubation in the automated blood culture system after inoculation with appropriate blood volumes.24 once blood cultures became positive for growth, they were removed from the machine for manual subculture techniques and gram staining following standard operating procedures.24 the laboratory used the vitek® 2 (biomérieux, marcy l’étoile, france) automated systems for bacterial identification and antimicrobial susceptibility testing, except for colistin, where broth microdilution was used where indicated. the definitions of the who priority list for drug-pathogen combination eskape bacteria were based on the definitions provided by the centers for disease prevention and control (table 1).25 the vitek® 2 machine tests antimicrobial susceptibility using the broth microdilution method and 64-well plastic cards with 17–20 antimicrobial agents. the vitek® 2 ast-n255 cards were used for gram-negative antimicrobial susceptibility testing and the ast-p603 cards for gram-positive. the vitek® 2 antimicrobial test panel comprises amikacin, amoxicillin with clavulanic acid, ampicillin-amoxicillin, cefazolin-cephalexin, cefazolin, cefepime, cefotaxime-ceftriaxone, cefoxitin, ceftazidime, cefuroxime, cephalexin, chloramphenicol, ciprofloxacin, clindamycin, cloxacillin, ertapenem, erythromycin-azithromycin, fusidic acid, gentamicin, imipenem, linezolid, meropenem, moxifloxacin, mupirocin, nalidixic acid, nitrofurantoin, ofloxacin, penicillin, penicillin-ampicillin, piperacillin with tazobactam, sulphamethoxazole with trimethoprim, teicoplanin, tetracycline, tigecycline, tobramycin, and vancomycin. the nhls employed the clinical and laboratory standards institute guidelines m100 for the corresponding year to interpret all antibiotic susceptibility results.26 the clinical and laboratory standards institute m39 guideline (analysis and presentation of cumulative antimicrobial susceptibility test data) recommends empirical therapy with an antibiotic or antibiotics that demonstrated 80.0% to 90.0% susceptibility to the most relevant organisms associated with a specific infection.27 table 1: antimicrobial resistance phenotypes definition provided by the united states centers for disease prevention and control. data analysis duplicates per patient were identified using unique patient identifiers and specimen collection dates (< 28 days). the data set was categorised into relevant variables and descriptive analysis was performed and presented as frequencies and proportions. because of varying antibiotic susceptibility testing done by the nhls over the years for some drug-pathogen combinations, n-values fluctuated. during data analysis, isolates that tested intermediate or resistant to antibiotics were classified as resistant strains according to the phenotypes (table 1). the mann-kendall test with a α-value of 0.05 was used to determine the statistical significance of changes in pathogen incidence and drug-pathogen resistance based on frequencies. the analyses were performed using the r-statistics package version 1.1.5 (r foundation for statistical computing, vienna, austria).28 results the study extracted data on eskape organism name, isolation ward and year, and amr test outcomes, without patient sociodemographic or clinical data. the incidence of eskape pathogens isolated from all positive blood cultures showed no significant variation between 2014 and 2019 (p = 0.133) (table 2). table 2: incidence of eskape pathogens at a referral hospital in limpopo, south africa, 2014–2019. between 2014 and 2019, s. aureus (n = 268/746; 35.9%) was isolated most frequently, followed by a. baumannii (n = 200/746, 26.8%), and k. pneumoniae (n = 118/746; 15.8%) (table 2). staphylococcus aureus increased from 39 isolates in 2014 to 75 in 2019 (p = 0.132). acinetobacter baumannii increased significantly from 11.9% (n = 16/134) in 2015 to 37.8% (n = 68/180) in 2019 (p = 0.009), while k. pneumoniae isolation declined from 46 in 2014 and 68 in 2015 to four isolates in 2016 (p = 0.007). no k. pneumoniae isolates were identified from 2017 to 2019. most isolates (n = 417/746; 55.9%) were from the neonatal ward and the intensive care unit (n = 60/746; 8.0%) (table 3). table 3: number of eskape pathogens in the isolation ward at a referral hospital in limpopo, south africa: 2014–2019. carbapenem-resistant a. baumannii increased significantly from 69.8% (n = 11/16) in 2015 to 75.0% (n = 51/68) in 2019 (p = 0.009) (figure 1). the incidence of mrsa isolates decreased from 14 (56.0%, n = 25) in 2016 to 13 (17.3%, n = 75) in 2019 (p = 0.260). vancomycin-resistant s. aureus was 4.7% (n = 12/256). in general, 3gcr k. pneumoniae incidence was 84.7% (n = 100/118), with no change in incidence rates from 82.6% (n = 38/46) in 2014 to 86.8% (n = 59/68) in 2015 and 75.0% (n = 3/4) in 2016 (p = 0.070), and carbapenem-resistant k. pneumoniae was 5.9% (n = 7/118) (p = 0.176). carbapenem-resistant p. aeruginosa was 8.0% (n = 4/50). figure 1: drug-pathogen resistance of global priority tracer phenotypes at a referral hospital in limpopo province, south africa: 2014–2019. klebsiella pneumoniae (n = 0/36) was not resistant to colistin (see online supplementary table 1). colistin resistance was 1.1% (n = 2/190) in a. baumannii and 2.4% (n = 1/41) in p. aeruginosa. all samples for e. faecium (n = 0/100) and s. aureus (n = 0/193) were susceptible to tigecycline. resistance to tigecycline was 10.7% (n = 20/187) for a. baumannii and 3.8% (n = 4/100) for k. pneumoniae. linezolid resistance was found in 2.0% (n = 2/100) of e. faecium and 0.8% (n = 2/251) of s. aureus isolates. incidence rates for multidrug resistance between a. baumannii and cefepime, ciprofloxacin, gentamycin, meropenem, and piperacillin with tazobactam increased significantly from 2015 to 2019 (p = 0.008) (online supplementary table 2). there was no change in the resistance rate for p. aeruginosa to amikacin (p = 0.242), cefepime (p = 0.060), ciprofloxacin (p = 0.176), meropenem (p = 0.817), and piperacillin with tazobactam (p = 0.159). discussion key findings of our study are that eskape pathogens blood culture isolates showed no significant variation between 2014 and 2019, and that s. aureus, a. baumannii, and k. pneumoniae were the eskape pathogens commonly isolated in mankweng tertiary hospital. most of the eskape isolates were from the neonatal ward, followed by the paediatric ward and the intensive care unit. there was an increase in crab, whereas mrsa decreased. colistin resistance was less than 3.0% for gram-negative pathogens a. baumannii and p. aeruginosa. in our study, the blood culture isolates of the eskape pathogens corresponded to the national trend in south africa.11 between 2018 and 2020, there was no significant variation in the proportion (ranging from 40% to 41%) of eskape pathogens isolated from all public sector positive blood cultures in south africa, despite an increase in the total number of blood cultures tested.11 a national neonatal public sector south african study from 2014 to 2019 also found that despite increased blood culture collection, the annual proportion of positive blood cultures remained unchanged, with an incidence risk of 6.0 per 1000 livebirths (95% confidence interval: 6.0–6.1).13 antimicrobial use is a significant promoter of resistance, giving resistant bacteria a selection advantage over sensitive ones.9 the observed insignificant variation in the incidence of eskape pathogens in our study and across the south african public sector11 could be attributed to the centralised medicines procurement contracts based on the national standard treatment guidelines and essential medicines lists.11 however, there is no definitive evidence to explain the stable incidence of eskape pathogens in our study or across south africa, therefore ecological studies are warranted. despite the overall consistent eskape pathogen incidence, our study showed variation in the incidence among individual eskape pathogens, which could be attributed to antimicrobial selection pressure. staphylococcus aureus, a. baumannii and k. pneumoniae were the most frequently isolated eskape pathogens in our study, similar to a countrywide public sector neonatal population study in south africa between 2014 and 2019,13 as well as a south african national mixed-population report from 2020.11 furthermore, our findings corroborate 2019 global1,2 and other single-centre trends from the limpopo (2016) and kwazulu-natal (2011–2015) provinces in south africa.29,30 the increased number of s. aureus isolates observed in our study is concerning, as global data show that s. aureus was the leading cause of fatal bloodstream infections, resulting in approximately 299 000 deaths and 30 million years of life lost in 2019.1 acinetobacter baumannii was the sixth most isolated bloodstream pathogen globally in 2019.1,2 the high incidence of a. baumannii in this study is furthermore concerning since it is the leading cause of prolonged hospital stay,2,10 increasing the risk of hospital-acquired infections and escalating healthcare costs, and delaying patient access to care owing to bed shortages.7,8,31 despite being one of the bloodstream infections isolated the most in our study, k. pneumoniae isolation decreased significantly during the study. this development was encouraging, since k. pneumoniae was the bloodstream pathogen isolated the most in south africa between 2014 and 2019,11,13 and was responsible for higher mortality from bloodstream infections in sub-saharan africa than s. aureus.1,4 in south africa in 2019, s. aureus infections were associated with 6000 deaths, a. baumannii with 6700, and k. pneumoniae with 3200.12 the increased incidence of multidrug-resistant gram-negative a. baumannii in our study (hospital setting) could be attributed to inefficient infection prevention and control methods and limited antimicrobial stewardship.13 the higher eskape pathogen incidence in the neonatal ward versus other wards could be attributed to the increased risk of neonatal sepsis resulting from risk factors such as prematurity, low birth weight, ruptured membranes, previous surgery, and intrapartum fever.32 these neonatal sepsis risk factors could lead to longer hospital stays, during which horizontal pathogen transmission could occur, particularly in overcrowded neonatal units with inadequate adherence to infection prevention and control measures.13 in addition, the variation in ward incidence could be attributed to differences in the type of healthcare services offered in neonatal, paediatric, and intensive care units, such as premature and low-weight neonates vulnerable to sepsis, vulnerable paediatric populations, and severely ill patients.13,32 the ward variation could also be attributed to differences in sampling practices across various hospital wards, where the causative pathogen(s) in bloodstream infections may go undiagnosed because minimal blood samples are collected.13 this assertion is substantiated by the findings of a 2018 study conducted at a tertiary hospital in gauteng, south africa, which showed that there was inconsistent adherence to blood culture collection guidelines, with over two-thirds (67.0%) of patients who were eligible for a blood culture not receiving one.33 other single-centre neonatal and children’s studies from gauteng, kwazulu-natal and the western cape in south africa have previously identified eskape pathogens as the dominant isolates of bloodstream infections between 2008 and 2018.15,16,17,18,19 the overall high and increasing incidence of crab in this study resembles the 80.0% for south africa observed in 2020,11 and the neonatal population between 2014 and 2019 (a. baumannii isolates declined in susceptibility to meropenem [23.0% to 12.0%; p = 0.051]),13 which could drive the use of colistin since a. baumannii exhibited multidrug resistance in our study.11,15 in 2019, crab caused 50 000 to 100 000 amr-related deaths globally,1 and was associated with the largest mortality impact of healthcare-acquired infections in lowand middle-income countries.8 there is a scarcity of data for a. baumannii-colistin susceptibility and/or resistance in lowand middle-income countries.2 acinetobacter baumannii was susceptible to colistin in our study. in addition, this study demonstrated that gram-negative a. baumannii and p. aeruginosa colistin resistance was below 3.0%, consistent with south african laboratory-based sentinel surveillance and routine data in 2016 to 2017.11,15 this could be attributed to the restricted use of colistin in the south african public sector.11,34,35 a 2020 south african national report showed a. baumannii tigecycline resistance at 15.0%,11 compared to 10.7% (n = 20/187) in our study. in south africa, tigecycline is registered to treat complicated skinand soft-tissue infections, intra-abdominal infections, and community-acquired bacterial pneumonia.36 as multidrug-resistant infections such as crab increase,37 clinicians may use high doses of tigecycline for unregistered indications such as sepsis,37,38 which can lead to secondary bacteraemia because of its bacteriostatic properties.38 according to a south african administrative report, clinicians were concerned with poor clinical outcomes when tigecycline was used alone.11 the emergence of tigecycline-resistant a. baumannii is complex as a result of multiple and multifaceted resistance mechanisms,38 necessitating comprehensive clinical evidence and research literature review before recommending alternate therapeutic choices. a medicine use evaluation study for tigecycline in our study setting is, therefore, recommended. methicillin-resistant s. aureus decreased in this study, similar to south african national surveillance data for 2016, 2017 and 2020,11,14 and previous single-centre studies conducted between 2011 and 2012 in the gauteng and kwazulu-natal provinces of south africa.18,19 this observation contradicts global mrsa data, which showed that mrsa increased by 14.0% from 21.0% in 2016 to 35.0% in 2020.39 the publication of the national guidelines for the prevention and containment of antimicrobial resistance in south african hospitals in 2017 may have contributed to the decrease in mrsa in our study, and in south africa.37 however, more research on the progression of infections is needed to support this assertion.40 the incidence of mrsa remains a concern given the increased incidence of s. aureus in this study, as mrsa has the most significant effect on hospital stay (increasing the stay by 14 days compared to sensitive s. aureus) in lowand middle-income countries.8 in addition, the proportions of mrsa, together with 3gcr escherichia coli, serve as global indicators for the 2030 sustainable development goals to reduce amr and require constant monitoring.41 staphylococcus aureus was susceptible to vancomycin (susceptibility above 90.0%), making it an empirical choice, with tigecycline as a last resort.42 klebsiella pneumoniae was the most isolated eskape pathogen in south africa in 2015 to 2017.11 consequently, the absence of k. pneumoniae isolates during the last 3 years (2017 to 2019) of our study was important. the potential contributing factors were beyond the scope of our study and will require future research to provide practical implications. the resistance of 3gcr k. pneumoniae in this study is comparable to the south african report between 2015 and 2017,11 and other single-centre studies in south africa from the gauteng and western cape provinces of south africa between 2008 and 2013.18,43 however, carbapenem-resistant k. pneumoniae in our study was low (< 10.0%), rendering carbapenems an effective alternative watch antibiotic for the management of 3gcr k. pneumoniae, and reserve antibiotics (colistin and tigecycline) as a last resort.42 the low carbapenem-resistant k. pneumoniae in our study could have accelerated the selection process, leading to extensive carbapenem use in 3gcr k. pneumoniae management, which could have increased crab growth. our study and single-centre studies conducted in the western cape province of south africa between 2008 and 201821,43 have yielded similar findings, not detecting vancomycin-resistant e. faecium. nevertheless, the latest 2020 south african data indicated a vancomycin-resistant e. faecium incidence rate of 1.3%, which remains low.11 based on this finding, vancomycin is considered an empirical choice for the treatment of bloodstream e. faecium infections. at the same time, reserve antibiotics (linezolid and tigecycline) should only be used as a last resort.44 compared to previous south african data from 2020, the incidence rate of carbapenem-resistant p. aeruginosa was low, at 33.0%.11 furthermore, p. aeruginosa had low resistance to aminoglycosides and fluoroquinolones, making access antibiotics (amikacin and gentamycin) and watch antibiotics (ciprofloxacin and meropenem) empirical choices for bloodstream infections, with colistin as a last resort.42 limitations our study had the following limitations. firstly, the study relied on a laboratory-based data set not linked to patient diagnoses and outcomes, limiting inferential analysis. consequently, future patient-level pathogen-specific clinical studies are encouraged to describe morbidity and mortality risk factors associated with these infections, particularly in neonatal, paediatric, and intensive care units. secondly, our study used routine laboratory data to report on phenotypic amr of eskape pathogens. we acknowledge that in an ideal setting, phenotypic and molecular surveillance amr data are used together to classify bacterial isolates and describe the magnitude and mechanism of amr. however, molecular amr surveillance in south africa is limited to the national referral laboratory; decentralisation to rural healthcare facilities is limited by lack of funding. thirdly, the use of retrospective routine data does not allow for the determination of reliability and validity for certain observations. fourthly, the requested annual blood sample data from the study data source were not provided. consequently, due to data limitations, we could not determine the overall or individual incidence rate of bloodstream eskape pathogens. even though routine surveillance data are available in some low-resource settings, limited access to essential variables such as total blood cultures could present a challenge to the estimation of prevalence and surveillance of infectious diseases, as was the case in our study. fifthly, our retrospective study design has the inherent limitation of low external study validity, and our findings could not be generalised to other settings. lastly, other limitations included missing variables and selection bias as not all isolates were subjected to antibiotic susceptibility testing, limiting essential data analysis. future studies should consider confounding variables such as infectious disease diagnostic protocols, antibiotic stewardship programmes, and infection prevention and control measures to improve their contextualisation and interpretation.41 conclusion this study has presented essential information on the incidence of eskape pathogens and antibiotic resistance phenotypes included in the list of global priority drug-pathogen combinations in patients with bloodstream infections in mankweng tertiary hospital in limpopo province, south africa. therefore, it is essential to establish a reliable and timely surveillance system, implement and maintain infection prevention and control measures, and empirical alternative antibiotic treatment regimens. the present information could serve as a basis for the development of an institutional antibiogram that could be used together with institutional formulary and prescribing guidelines for optimal antibiotic use. the surveillance data could provide valuable drug selection information on appropriate empirical therapy before the availability of antibiotic sensitivity testing results for a specific patient. acknowledgements the authors acknowledge the national health laboratory services staff (thembekile shelembe), who assisted with data extraction and preparation. this article is partially based on the author’s thesis entitled ‘antibiotic utilisation in public sector healthcare systems of limpopo province, south africa’ towards the degree of doctor of philosophy in pharmacy in the department of medicine usage in south africa, north-west university, south africa on 2 august 2024, with supervisors, johanita r. burger, martha s. lubbe and marlene julyan. competing interests t.c.m. works at the central provincial office of the limpopo department of health but is not involved in the daily operations of the mankweng tertiary hospital. the other authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions t.c.m. designed the study. j.r.b., m.s.l. and m.j. were involved in conceptualising and supervising the work. t.c.m. processed the data, performed the analysis, and designed tables and graphs. j.r.b., m.s.l., m.j., and m.r.l.-m. assisted in interpreting the results. t.c.m. took the lead in drafting the article. t.c.m., j.r.b., m.s.l., m.j. and m.r.l.-m. provided critical feedback and helped shape the research, data analysis, and article. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the data that support the findings of this study are available from the national health laboratory service. the data were used under license for the current study and are therefore not publicly available. data are, however, available from the corresponding author, t.c.m., upon reasonable request and with permission of the national health laboratory service. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. the article does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the authors are responsible for this article’s results, findings, and content. references ikuta ks, swetschinski lr, robles aguilar g, et al. global mortality associated with 33 bacterial pathogens in 2019: a systematic analysis for the global burden of disease study 2019. lancet. 2022;400(10369):2221–2248. https://doi.org/10.1016/s0140-6736(22)02185-7 murray cjl, ikuta ks, sharara f, et al. global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. lancet. 2022;399(10325):629–655. 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https://www.who.int/data/gho/indicator-metadata-registry/imr-details/5751 simner pj, hindler ja, bhowmick t, et al. what’s new in antibiograms? updating clsi m39 guidance with current trends. j clin microbiol. 2022;60(10):e0221021. https://doi.org/10.1128/jcm.02210-21 dramowski a, cotton mf, rabie h, whitelaw a. trends in paediatric bloodstream infections at a south african referral hospital. bmc pediatr. 2015;15:33. https://doi.org/10.1186/s12887-015-0354-3 thomas r, ondongo-ezhet c, motsoaledi n, et al. incidence, pathogens and antimicrobial resistance of blood and cerebrospinal fluid isolates from a tertiary neonatal unit in south africa: a 10 year retrospective review. plos one. 2024;19(1):e0297371. https://doi.org/10.1371/journal.pone.0297371 abstract introduction methods results discussion acknowledgements references about the author(s) munyaradzi pasipamire research and evaluation, eswatini national aids programme, ministry of health, mbabane, eswatini edward broughton research and evaluation, university research co. llc, chevy chase, maryland, united states international health, johns hopkins bloomberg school of public health, baltimore, maryland, united states mandzisi mkhontfo university research co. llc, mbabane, eswatini gugu maphalala eswatini health laboratory services, mbabane, eswatini batsabile simelane-vilane university research co. llc, mbabane, eswatini samson haumba university research co. llc, mbabane, eswatini citation pasipamire m, broughton e, mkhontfo m, maphalala g, simelane-vilane b, haumba s. detecting tuberculosis in pregnant and postpartum women in eswatini. afr j lab med. 2020;9(1), a837 https://doi.org/10.4102/ajlm.v9i1.837 original research detecting tuberculosis in pregnant and postpartum women in eswatini munyaradzi pasipamire, edward broughton, mandzisi mkhontfo, gugu maphalala, batsabile simelane-vilane, samson haumba received: 19 may 2018; accepted: 17 apr. 2020; published: 30 july 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: tuberculosis diagnosis in pregnancy is complex because tuberculosis symptoms are often masked by physiological symptoms of pregnancy. untreated tuberculosis in pregnant and postpartum women may lead to maternal morbidity and low birth weight. tuberculosis in hiv-positive pregnant women increases the risk of maternal and infant mortality. objective: this study aimed to determine tuberculosis prevalence stratified by hiv status and identify screening algorithms that maximise detection of active tuberculosis among pregnant and postpartum women in eswatini. methods: women were enrolled at antenatal and postnatal clinics in eswatini for tuberculosis screening and diagnostic investigations from 01 april to 30 november 2015 in a cross-sectional study. sputum samples were collected from all participants for tuberculosis diagnostic tests (smear microscopy, genexpert, mgit culture). blood and urine samples were collected from hiv-positive women for cluster-of-differentiation-4 cell count, interferon gamma release assay and tuberculosis lateral flow urine lipoarabinomannan tests. results: we enrolled 990 women; 52% were pregnant and 47% were hiv-positive. the prevalence of tuberculosis among hiv-positive pregnant women was 5% (95% confidence interval [ci]: 2–7) and among postpartum women it was 1% (95%ci: -1–3). tuberculosis prevalence was 2% (95%ci: 0–3) in hiv-negative pregnant women and 1% (95%ci: -1–2) in hiv-negative postpartum women. the national tuberculosis symptom screening tool failed to identify women who tested tuberculosis-culture positive. conclusion: routine tuberculosis symptom screening alone is insufficient to rule out tuberculosis in pregnant and postpartum women. only sputum culture maximised the detection of tuberculosis, indicating a need to balance access and cost in developing countries. keywords: tuberculosis; pregnant women; postpartum women; tuberculosis screening; tuberculosis diagnosis; hiv; eswatini. introduction there has been a major decline in maternal mortality over the past two decades.1 however, pregnancy poses several challenges to tuberculosis management because of its adverse effects on diagnosis and treatment outcomes.2,3,4 the exact burden of tuberculosis among pregnant women is undefined.5 the incidence of tuberculosis among postpartum women is unknown and difficulties with diagnosis suggest underestimation.6 in 2015, the world health organization (who) advocated for research on new diagnostic methods targeting pregnant and postpartum women including hiv-positive women7 and inexpensive tuberculosis screening algorithms for this population.8 the clinical presentation of tuberculosis may be similar to some manifestations of pregnancy, making tuberculosis diagnosis in this population difficult.6 the presence of tuberculosis during pregnancy may result in a threefold increase in adverse birth outcomes such as preterm birth, low weight at birth and foetal growth restriction.8,9 screening for tuberculosis in women of reproductive age is imperative, because concomitant tuberculosis disease causes higher case fatality rates than in men of the same age.10 active tuberculosis disease, especially when treated late or left untreated, is likely to result in severe adverse outcomes affecting both mother and baby,11,12,13 with an estimated 3.4-fold increase in infant mortality.14,15 the who has recommended three options for tuberculosis symptom screening based on availability of xpert mtb/rif assay, chest x-ray and also hiv status of the individual.16 the option one algorithm uses a cough lasting more than 2 weeks to screen positive, option two uses any tuberculosis symptom and option three relies on positive chest x-ray findings.16 option two of the who symptom-screening algorithm gives a positive screen if a cough of any duration or any other tuberculosis symptom is evaluated.16 the national tuberculosis symptom screening tool (ntbss) adapted option one of the who screening algorithm, through which individuals screen positive if there is a cough of at least 2 weeks duration,16 or a cough plus any other symptom of fever or unexplained loss of weight or night sweats, or if any two symptoms are present.16,17 despite developments in screening and diagnosis, emerging data show that the who-recommended four-symptom screen may miss persons with tuberculosis disease.4 studies on hiv-positive pregnant women found that the sensitivity of any one of the four tuberculosis symptoms was 28% in south africa18 and 42.9% in kenya.19 few studies have considered the potential use of tuberculosis lateral flow urine lipoarabinomannan (lf-lam) as an add-on to the tuberculosis screening algorithm for hiv-positive pregnant and postpartum women.20 xpert® mtb/rif testing has been rolled out in eswatini and is the preferred tuberculosis diagnostic method for women. however, a study among hiv-positive pregnant women in kenya reported xpert® mtb/rif sensitivity of 43% and a specificity of 100%, when compared to mycobacterium tuberculosis culture results19 as the gold standard. bactec mgit 960 liquid culture has been shown to have a sensitivity and specificity of 100% and 93.3%.21 tests for latent tuberculosis infection have shown mixed value for determining the presence of the infection.22 existing tests for latent tuberculosis infection include the tuberculin skin test (tst) and the newer interferon-gamma release assays (igras) but both have drawbacks.23,24 the limitations of the tst include low sensitivity and specificity among hiv-positive patients and possibly among pregnant women.11 similarly, a study in kenya using the t-spot tb igra showed a more than threefold increased risk of active tuberculosis or mortality among pregnant women who tested positive using igra.25 in eswatini, tuberculosis prevalence among pregnant women is not documented. use of the recommended who four-symptom screen has identified very few pregnant women with positive results.26 the objectives of this study were to determine the prevalence of bacteriologically confirmed tuberculosis among the study population of hiv-positive and hiv-negative pregnant and postpartum women and to identify effective tuberculosis screening algorithms. methods ethical considerations ethical approval was obtained from the eswatini national health research board (formerly scientific and ethics committee [approval reference: mh/599c]), cdc institutional review board (irb) (reference: cgh-hsr tracking #: 2015-196), and university research co. llc irb (02 march 2015). participants signed informed consent written in their preferred language (siswati or english). participants received no incentives but were reimbursed transport costs for additional visits to read tst. diagnostics tests and treatment, if required, were free of out-of-pocket charges. study design we conducted a cross-sectional study enrolling pregnant and postpartum women, aged 18 years and older, attending antenatal and postnatal care clinics, from 01 april to 30 november 2015 at three public health facilities in three of the four regions of eswatini. sociodemographic and clinical data, including past tuberculosis screening results where applicable, were collected. eswatini is categorised by who as a high tuberculosis/hiv burdened country with a co-infection rate of 70% and a tuberculosis incidence rate of 398 per 100 000 population.27 inclusion criteria participants were pregnant and postpartum women who were not on anti-tuberculosis treatment at enrolment or who had not taken anti-tuberculosis medicines, including isoniazid for tuberculosis preventive therapy, within the 2 months preceding enrolment, based on documented evidence from patient clinical records, and who provided informed consent. four groups of women were enrolled: hiv-positive pregnant, hiv-negative pregnant, hiv-positive postpartum, and hiv-negative postpartum. study population and sample size a sample size of 183 in each group was determined and a full narrative of sample size calculation is fully described in our protocol paper titled ‘screening in maternity to ascertain tuberculosis status (smats) study’.4 participants were consecutively enrolled until the sample size was reached. clinical and laboratory procedures symptom screening all participants were screened using the who-recommended national tuberculosis four-symptom screening (standard ntbss) tool. participants held clinic cards which were checked for evidence of tuberculosis symptom screening at their last clinical encounter to ascertain routine tuberculosis screening coverage at their previous visit. participants were screened as positive using the standard ntbss tool, if they had a cough lasting at least 2 weeks,16 or a cough lasting less than 2 weeks plus any other symptom of fever or unexplained loss of weight or night sweats, or if any two symptoms were present.16,17 enhancements of the tuberculosis screening tool were done by adding to the four symptom screening tool any history of contact with a person on tuberculosis treatment or who had been diagnosed with tuberculosis and the presence of tuberculosis symptoms within the household inhabitants. we measured sensitivity, specificity and predictive values (positive and negative) of the who-recommended four symptom tuberculosis screening tool among hiv-positive and hiv-negative pregnant and postpartum women compared with sputum culture, the gold standard for m. tuberculosis detection. radiological procedures even though chest radiographs are not contraindicated in pregnancy, chest radiographs were only carried out among postpartum women (both hiv-positive and hiv-negative) to eliminate risk of radiation exposure to the foetus. specimen collection and laboratory procedures two samples of sputum (for xpert® mtb/rif, smear microscopy and culture using bactec tm mgit 960) were collected from all participants.4 sputum samples were collected through the production of spontaneous self-expectorated phlegm (preferred method), sputum induction through nebulising with hypertonic saline or, if both the above failed, the participant received a sputum container to take home and attempt to produce an early morning sputum sample and bring it back to the health facility. testing of sputum samples was done at the national tuberculosis reference laboratory in mbabane. tuberculosis culture was the gold standard test and in situations where sputum samples were insufficient for the three tests, culture was prioritised ahead of xpert® mtb/rif and smear microscopy. a urine sample for the lf-lam test and two 4 ml blood samples for igra testing and cluster-of-differentiation-4 (cd4) cell count testing were collected. interferon-gamma release assays and lf-lam were only done for hiv-positive women due to limited evidence on igra use28 and existing who recommendations on lf-lam use in hiv-positive individuals.29 tst was also done and the induration was read after 48 hours – 72 hours. igra and tst procedures were explained to all participants and only hiv-positive participants were then asked which test they preferred between igra and tst. sample collection and storage followed national standard operating procedures for urine and blood collection and manufacturer’s instructions for the determinetm tuberculosis lf-lam test (abbott laboratories, lake bluff, illinois, united states) and igra testing.4 all sputum specimens were analysed by means of: (1) xpert® mtb/rif assay (cepheid, sunnyvale, california, united states), (2) concentrated ziehl-neelsen microscopy, (3) liquid–medium culture method (bactectm mgit 960tm tb diagnostic system; becton, dickinson & company [bd], crystal lake, new jersey, united states), and (4) mgit 960tm dst (bd, crystal lake, new jersey, united states). positive cultures were identified as m. tuberculosis using the tuberculosis ag mpt64 rapid® assay (standard diagnostics, inc., yongin, south korea).30 interferon-gamma release assays and cd4 cell count testing were done at lancet laboratory in johannesburg, south africa. igra blood specimens were collected directly into igra tubes used for quantiferon-tb gold in-tube assay (cellestis ltd, carnegie, victoria, australia).31,32 cd4 cell count tests were conducted by bd facscalibur™ flow cytometry (bd biosciences, san jose, california, united states) using venous blood collected in sterile four millilitre bd vacutainer edta tubes by trained study nurses. data management and data analysis data collection the data collection tools were matched to the tools used for routine data collection at health facilities. demographic fields in the data collection forms were adapted from client cards. data fields for tuberculosis symptom screening were adapted from the national tuberculosis screening tool. patient information was anonymised. data analysis data were entered in epi info™ (centers for disease control and prevention, atlanta, georgia, united states) and research electronic data capture (redcap; vanderbilt university, nashville, tennessee, united states), and data extraction tools were cross-checked to validate conflicting fields. laboratory results were compared with the electronic study results file generated from the laboratory, and participant identity numbers were used to relate the data. we evaluated both option one and option two of the who four symptom screening algorithms. tst numeric readings were recoded as positive, if the length of the induration was ≥ 5 mm for hiv-positive participants or ≥ 10 mm, if the participant was hiv-negative.32,33 all other lengths, including 0 mm, were recoded as negative according to existing literature and cdc guidance on interpretation of tst results.32,33 igra was done at a private laboratory according to manufacturer’s recommendations and the differences in readings between quantiferon-tuberculosis gold in-tube tuberculosis antigen, tuberculosis nil and tuberculosis mitogen were used to interpret positive, negative and indeterminate results, respectively (figure 1).34 figure 1: interpretation of interferon-gamma release assays results. frequencies and proportions were used to describe participant characteristics and related clinical data. diagnostic parameters of sensitivity, specificity and positive and negative predictive values analyses for tuberculosis symptoms, and tuberculosis diagnostic tests were calculated in stata version 13 (© 1985–2013 statacorp llc, college station, texas, united states). using logistic regression, associations between culture-positive tuberculosis and hiv status and pregnancy or postpartum status variables were determined. other sociodemographic and clinical factors were considered for inclusion in the multivariate model if the p-value was ≤ 0.1 on bivariate analysis. factors that perfectly predicted the outcome were excluded. estimates were reported with 95% confidence intervals (cis) and corresponding p-values. results description of study participants of the 990 women who were enrolled, 516 (52%) were pregnant: 101 (10%) in first trimester, 219 (22%) in second trimester and 196 (20%) in third trimester (figure 2). the remaining 474 (48%) were all postpartum women. among the participants, 470 (47%) were hiv-positive and, among them, 434 (92%) were on antiretroviral therapy. figure 2: enrolment categories and culture results of pregnant and postpartum women attending antenatal and postnatal care clinics in eswatini between april and november 2015. most women (790, 80%) had secondary or tertiary education, and 300 (30%) were in formal employment (table 1). the median household density (number of people per room within household) was 1.5 (interquartile range: 1–2 people per room). the median age was 26 (interquartile range: 22–31) years. table 1: demographic characteristics of pregnant and postpartum women attending antenatal and postnatal care clinics in eswatini between april and november 2015. tuberculosis screening and sputum collection of the 990 participants screened, 48 (5%) screened positive for tuberculosis using the ntbss tool. there were 181 (18%) with at least one tuberculosis symptom (table 1). participants who screened positive for specific tuberculosis symptoms included 103 (10%) with a cough of any duration, 38 (4%) with night sweats, 29 (3%) with fever and 48 (5%) with weight loss. among 516 pregnant women, 433 (84%) were screened for tuberculosis at their last clinical encounter compared with 283 (60%) of the 474 postpartum women. in 531 (54%) patients, sputum collection was spontaneous, 158 (16%) by induction and 87 (9%) as early morning samples. however, 214 (22%) participants failed to produce sputum using any of the three methods. in total, 776 (78%) participants produced sputum samples and 758 (98%) had samples available for culture testing and 704 (93%) had valid culture results. however, only 361 (47%) had sputum samples available for xpert® mtb/rif testing and xpert® mtb/rif results were available for 361 (100%) participants, although 18 (5%) of those with xpert® mtb/rif results had no culture results for comparison. prevalence of tuberculosis tuberculosis status was either bacteriologically confirmed or ruled out by culture testing in 704 (93%) participants who had sputum samples available for culture. the overall prevalence of tuberculosis in this cohort of pregnant and postpartum women was 2% (95% ci: 1–3) (table 2). m. tuberculosis was found in 15 (2%) participants and 12 (80%) were pregnant. the prevalence of tuberculosis among pregnant women was 3% (95% ci: 1–5) compared to 1% (95% ci: 0–2) in postpartum women. the prevalence of tuberculosis among those who were hiv-negative was 1% (95% ci: 0–2) compared to a prevalence of 3% (95% ci: 1–5) among those who were hiv-positive (table 2). table 2: prevalence of tuberculosis, by hiv status, among pregnant and postpartum women attending antenatal and postnatal care clinics in eswatini between april and november 2015. acceptability of tuberculosis skin test and interferon-gamma release assay a total of 450 participants chose between tst and igra as their preferred test method. of this number, 277 (62%) chose tst as the preferred method compared to 173 (38%) for igra (data not shown in tables). among the 540 with no decision on preferred method, 512 (95%) were not sure, and 28 (5%) chose not to respond to the question. tuberculin skin tests were done on 961 (97%) participants, of which 659 (69%) came back for reading within 48 h – 78 h, whereas 302 (31%) were lost to follow-up. of the 659 participants who had tst results, 237 (36%) were tst-positive. igras were done on 465 hiv-positive participants. of the 465 igra results, 153 (33%) were positive, 273 (59%) negative, 23 (5%) indeterminate and 16 (3%) were missing. performance of tuberculosis screening and diagnostic tests the standard ntbss tool failed to identify women with tuberculosis disease with a sensitivity of 0% (95% ci: 0–29) among hiv-positive and 0% (90% ci: 0–60) among hiv-negative participants (table 3). the enhanced screening tool, by including history of contact with a person with tuberculosis or presence of tuberculosis symptoms within the household of the participant, did not improve the sensitivity of the standard ntbss tool. only the inclusion of a tuberculosis contact history significantly improved the sensitivity of the algorithm to 18% among hiv-positive women, although specificity decreased from 94% to 62%. table 3: sensitivity, specificity, positive and negative predictive values of tuberculosis screening symptoms and diagnostic tests in pregnant and postpartum women attending antenatal and postnatal care clinics in eswatini between april and november 2015. m. tuberculosis was detected in two (1%) of the 361 xpert mtb/rif results, and no rifampicin resistance was detected in either one. no m. tuberculosis was detected in the remaining 359 (99%) samples. sputum smear microscopy was done on 724 (73%) participants, and 4 (1%) had acid-alcohol-fast bacilli. lf-lam was done on 411 (87%) participants and 327 (80%) had culture results. when compared to culture, the sensitivity of both the tuberculosis screening and the tuberculosis diagnostic tests were less than 50% in both hiv-positive and hiv-negative women (table 3). association between socio-demographic and clinical covariates and tuberculosis culture diagnostic algorithm specific algorithms could not be analysed due to the missing data of tests used to construct the different algorithms, as well as low prevalence of tuberculosis symptoms among our study participants. the study showed that those who were hiv-positive had a threefold risk of culture-positive tuberculosis compared to hiv-negative individuals (odds ratio = 3.23; 95% ci: 1.00–10.40, p = 0.05) (table 4). those residing in the central region (odds ratio = 0.08; 95% ci: 0.01–0.61, p = 0.015) and southern region (odds radio = 0.19; 95% ci: 0.04–0.89, p = 0.035) were independently less likely to have culture-positive tuberculosis compared to the northern region (reference region) and (table 4). table 4: factors associated with culture positive tuberculosis in pregnant and postpartum women attending antenatal and postnatal care clinics in eswatini between april and november 2015. discussion summary of key findings according to our review, this is a unique study in this setting to determine the burden of tuberculosis among pregnant and postpartum women regardless of the presence of tuberculosis symptoms. we observed that higher proportions of pregnant women (84%) were previously screened for tuberculosis during their last clinic visit prior to enrolment compared to postpartum women (60%). however, these were lower than the universal screening (99%) reported among people living with hiv attending antiretroviral therapy clinics in eswatini.35 even though 80% of participants who were confirmed to have tuberculosis disease were pregnant, there were no statistical differences between the prevalence of tuberculosis in pregnant and postpartum women. the highest prevalence of tuberculosis was among hiv-positive pregnant women (5%), which is comparable to the 3.3% observed in neighbouring south africa.11 although 93% of participants who were found to have active tuberculosis reported no tuberculosis symptoms, there were no differences in tuberculosis prevalence between those reporting symptoms and those with no symptoms. a study conducted in south africa found a higher tuberculosis prevalence among patients who did not report symptoms of tuberculosis.18 a study from ethiopia did not find any person with active tuberculosis disease among pregnant women but did not test for tuberculosis among those who did not have tuberculosis symptoms.36 this has significant public health implications for tuberculosis control, considering previous reports that asymptomatic patients with culture-positive tuberculosis can transmit tuberculosis.18 the who four-symptom ntbss screening tool failed to identify women with active tuberculosis disease, as the majority of women with confirmed tuberculosis disease did not have symptoms of tuberculosis. almost a third of participants who had a tst done did not have results, because participants did not come back within the stipulated time for reading, despite the provision of transport imbursements for additional visits for tst reading. pregnancy is known to suppress the t1-helper pro-inflammatory response, resulting in masking of tuberculosis symptoms and increased susceptibility to m. tuberculosis reactivation8 and primary infection with m. tuberculosis.37 women are unlikely to show typical symptoms like sweating at night and fever, and these are further masked by pregnancy.8,17 weight loss can be masked by physiological changes during pregnancy.18 the low sensitivity of the tuberculosis screening tool has been reported in many other studies.18,19,38,39 adapting the screening tool to include a history of tuberculosis contact as an independent indicator of positive tuberculosis screen improved sensitivity by 18% but only in hiv-positive women. this sensitivity (18%) is still too low for effective tuberculosis case finding and ruling out tuberculosis among pregnant and postpartum women. lacourse et al.19 also showed poor performance of the four-symptom screening tool in hiv-positive pregnant women in kenya. tuberculosis diagnosis in pregnancy is often delayed due to atypical symptoms.11,39,40 culture prevailed as the reliable gold standard to diagnose tuberculosis; some authors recommend that it should be mandatory for establishing tuberculosis diagnosis in this group.11,39 a false-negative symptom screening, which is the initial screening method for triaging for tuberculosis diagnostic testing, will often lead to a delay in diagnosis and treatment of active tuberculosis and consequently poor foetal and maternal outcomes.17 a false-positive screening result leads to inconvenient and costly laboratory procedures.17 however, the routine symptom screening tool had high negative predictive values (97% – 99%), which supports its utility in identifying people who are unlikely to have tuberculosis, especially people living with hiv. therefore, those who screen negative with the standard ntbss tool and are at high risk of progressing from latent tuberculosis to active tuberculosis can be given tuberculosis preventive therapy in this setting of high hiv-tuberculosis burden.39,41 we demonstrated that tst had poor performance when used as a screening method for exposure to tuberculosis and its feasibility is further challenged by a third of participants who were lost to follow-up for a reading of skin reaction. however, alternative follow-up methods, including home visits, should be included to complete the tst readings. igra was a less preferred screening method compared to tst, possibly due to the need for blood draw. in addition, igra demands laboratory infrastructure42 compared to tst and is currently available only in private laboratories in this setting. strengths and limitations we considered it a strength that our methodology included participants who did not have the usual symptoms of tuberculosis, minimising the risk of under-reporting of true tuberculosis prevalence in this study population.39,43 we also induced sputum in women unable to produce sputum spontaneously, thus maximising the tuberculosis diagnostic yield.19 in addition to symptom screening, we attempted five tuberculosis tests for all participants. we had planned to test 10 different algorithms using different testing combinations and ordering of individual diagnostic tests. however, given the low agreement, the low number of positives, and the poor performance of the who symptom screening tool (several of the algorithms began with the who symptom screening), the analysis was of no utility, and we did not include these results in this report. although unintended by the study design, more than half of the participants did not have an xpert® mtb/rif assay done, which is a near point-of-care test that allows results to be quickly available (as early as 2 h) to clients and service providers, leading to a quick clinical management decision, unlike culture which may take several weeks. conclusion the four-symptom screening tool appears likely to miss women with active tuberculosis. without sensitive, symptom-based tuberculosis screening in this subpopulation, a high index of suspicion of tuberculosis is necessary and factors such as a history of tuberculosis contact should prompt clinicians to consider tuberculosis in their differential diagnosis, especially in a setting of high hivtuberculosis burden. bold, deliberate decisions to invest in laboratory-based, quality-assured culture testing are required to maximise detection of people who have active tuberculosis disease in countries with a high hivtuberculosis burden in order to end tuberculosis by 2035 as envisaged by the who’s end tb strategy.44 however, the feasibility of increasing access to culture in this setting is confronted by costs related to culture testing, transportation, specimen storage and lengthy waiting periods for culture results by clinicians and patients. therefore, low-income and middle-income countries should strike the right balance to ensure access to culture for those who could benefit from culture and availability of the newer, more sensitive xpert® mtb/rif ultra platform for rapid tuberculosis diagnosis. prevalence of tuberculosis was particularly high (4.5%) among hiv-positive pregnant women and low (0.6%) among hiv-negative postpartum women. although we were unable to test different tuberculosis screening algorithms, the poor performance of the standard ntbss tool that serves as the entry to tuberculosis services highlights the challenge of diagnosing tuberculosis in pregnant and postpartum women. acknowledgements the authors thank ministry of health staff from participating health facilities and colleagues from university research co. llc eswatini: marianne calnan, sandile ginindza and rosanna jeffries, and lani marquez from university research co. llc center for human services. we also thank munamato mirira from usaid eswatini, sikhathele mazibuko and peter preko from cdc eswatini, and surbhi modi from cdc atlanta for their support during the development of the protocol. competing interests the authors have declared that no competing interests exist. authors’ contributions m.p. contributed to the conception of the study, implementation, analysis, interpretation of results, drafting and critical review of intellectual content, and final approval of the manuscript. e.b. contributed to the conception of the study, implementation and analysis, interpretation of results, drafting and critical review of intellectual content and final approval of the manuscript. m.m. contributed to the analysis, interpretation of results, drafting and critical review of intellectual content and final approval of the manuscript. g.m. contributed to the conception of the study, implementation, critical review of intellectual content and final approval of the manuscript. b.s.-v. contributed to the conception of the study, implementation, critical review of intellectual content and final approval of the manuscript. s.h. contributed to the conception of the study, implementation, analysis, interpretation of results, drafting and critical review of intellectual content, and final approval of the manuscript. all of the authors agreed to be accountable for all aspects of the work, including accuracy and integrity. sources of support support for this study was provided by the american people through the united states agency for international development (usaid) applying science to strengthen and improve systems project, managed by university research co., llc under the terms of cooperative agreement number aid-oaa-a-12-00101. funding for this study of tuberculosis diagnosis in eswatini was provided by the us president’s emergency plan for aids relief (pepfar) through usaid. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views expressed in this article are views of the authors and not an official position of the authors’ institutions or funder. references carvajal–aguirre l, amouzou a, mehra v, ziqi m, zaka n, newby h. gap between contact and content in maternal and newborn care: an analysis of data from 20 countries in sub–saharan africa. j global health. 2017;7(2):020501. https://doi.org/10.7189/jogh.07.020501 sugarman j, colvin c, moran ac, oxlade o. tuberculosis in 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2010;362(8):707–716. https://doi.org/10.1056/nejmoa0907488 linas bp, wong ay, freedberg ka, horsburgh jr cr. priorities for screening and treatment of latent tuberculosis infection in the united states. am j respir crit care med. 2011;184(5):590–601. https://doi.org/10.1164/rccm.201101-0181oc gounder cr, wada ni, kensler c, et al. active tuberculosis case-finding among pregnant women presenting to antenatal clinics in soweto, south africa. j acquir immune defic syndr. 2011;57(4):e77–e84. https://doi.org/10.1097/qai.0b013e31821ac9c1 world health organization. the end tb strategy [homepage on the internet]. geneva: who; 2015 [cited 2019 apr 12]. available from: http://www.who.int/tb/end-tb_brochure.pdf?ua=1 abstract introduction ethical considerations case presentation management and outcomes discussion acknowledgements references about the author(s) stephanie j. kennedy department of haematology and cell biology, faculty of health sciences, university of the free state, bloemfontein, south africa department of haematology, national health laboratory service, bloemfontein, south africa anne-cecilia van marle department of haematology and cell biology, faculty of health sciences, university of the free state, bloemfontein, south africa department of haematology, national health laboratory service, bloemfontein, south africa citation kennedy sj, van marle a-c. pseudomonocytosis on a sysmex xn haematology analyser masking the monocytopenia of hairy cell leukaemia in a south african woman. afr j lab med. 2025;14(1), a2617. https://doi.org/10.4102/ajlm.v14i1.2617 case study pseudomonocytosis on a sysmex xn haematology analyser masking the monocytopenia of hairy cell leukaemia in a south african woman stephanie j. kennedy, anne-cecilia van marle received: 30 aug. 2024; accepted: 09 dec. 2024; published: 25 mar. 2025 copyright: © 2025. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract introduction: hairy cell leukaemia (hcl) is a rare b-cell lymphoproliferative disorder characterised by medium-sized villous lymphocytes (‘hairy cells’) and monocytopenia in the peripheral blood. automated full blood count (fbc) haematology analysers may spuriously count ‘hairy cells’ as monocytes, resulting in pseudomonocytosis. case presentation: a 72-year-old woman presented with symptomatic anaemia and massive splenomegaly to a regional hospital in north west province, south africa, in june 2023. an fbc and differential count, performed on a sysmex xn-series haematology analyser, revealed a monocytosis of 42.82 × 109/l. however, a manual differential count, peripheral blood microscopy, and multiparameter flow cytometry confirmed a monocytopenia with numerous ‘hairy cells’. management and outcome: the patient was referred to a tertiary hospital where bone marrow morphology and a brafv600e mutation confirmed a diagnosis of hcl. unfortunately, she demised shortly after admission. conclusion: here, we report a case of hcl where a sysmex xn-series artifactually counted ‘hairy cells’ as monocytes, masking the characteristic monocytopenia. with the recent introduction of sysmex xn-series fbc haematology analysers (sysmex corporation, kobe, japan) in national health laboratory service laboratories across south africa, we urge operators to be cognisant of the inherent limitations of fbc analysers in generating blood counts. what this study adds: even modern automated laboratory analysers with advanced technologies have inherent limitations. this case highlights the importance of a manual differential count and peripheral blood smear review in the era of automation. keywords: hairy cell leukaemia; pseudomonocytosis; monocytopenia; haematology full blood count analyser; sysmex xn-series. introduction hairy cell leukaemia (hcl) is a rare b-cell lymphoproliferative disorder accounting for approximately 2% of lymphoid leukaemias, and usually involves the bone marrow (bm), splenic red pulp, and peripheral blood (pb).1 although lymphocytosis is uncommon in hcl (5% – 10%), circulating ‘hairy cells’, albeit at low numbers, are characteristic (85%).1 variable cytopenias are a frequent laboratory feature, whilst monocytopenia is almost always present (98%).1,2 an accurate automated white blood cell (wbc) differential count demonstrating monocytopenia should trigger a suspicion of hcl and warrants careful morphologic assessment for ‘hairy cells’.3,4 however, certain automated full blood count (fbc) haematology analysers may misidentify ‘hairy cells’ as monocytes, resulting in pseudomonocytosis.5,6 in 2022, the national health laboratory service (nhls) and roche diagnostics (pty) ltd entered into a service level agreement for the placement of sysmex xn-series automated fbc haematology analysers (sysmex corporation, kobe, japan) in nhls laboratories (n = 146) across south africa. this national roll-out involved the placement of the sysmex xn-1000 series in medium (n = 51) and large laboratories (n = 20) and xn-l 530 instruments in smaller laboratories (n = 75).7 automated fbc haematology analysers generate valuable quantitative and qualitative information that can assist laboratory staff with the accurate interpretation of fbc results, as well as alert them to prioritise abnormal samples for pb smear review.8,9 not all nhls laboratories are staffed with pathologists. therefore, technologists and technicians frequently rely on automated analyser data to guide referral for microscopy. ethical considerations ethical clearance was obtained from the health science research ethics committee of the university of the free state (reference no.: ufs-hsd2024/1295). the patient’s results were pseudonymised to ensure patient confidentiality and stored securely on a password-protected computer only accessible by the authors. attempts to locate the patient or their next of kin to obtain permission to publish this report were unsuccessful. case presentation a 72-year-old woman presented with symptomatic anaemia to a regional hospital in north west province, south africa, in june 2023. on clinical examination, she had massive splenomegaly but no lymphadenopathy. an fbc was performed on a sysmex xn-1000 series and showed pancytopenia (haemoglobin 2.9 g/dl, platelet count 97 l × 109/l, neutrophils 0.93 × 109/l), with a leucocytosis of 58 × 109/l. the automated wbc differential count showed a lymphocytosis of 14.16 × 109/l and a marked monocytosis of 42.82 × 109/l. the pb smear was referred to the nhls universitas academic laboratory, south africa, for pathologist review. a manual differential count and pb smear microscopy revealed almost no monocytes. however, abnormal lymphocytes were increased (78%) (figure 1). these cells were intermediate in size, with abundant pale blue cytoplasm and circumferential villi. their nuclei were kidney-shaped with a ground-glass chromatin pattern and inconspicuous nucleoli. figure 1: (a–d) ‘hairy cells’ on the peripheral blood smear of a 72-year-old woman diagnosed with hairy cell leukaemia at national health laboratory service universitas academic laboratory, south africa, in june 2023. wright’s stain (a) 50× objective, (b) 10× objective and (c, d) 100× objective. (e, f) for comparison, images of normal monocytes on a peripheral smear are shown; wright’s stain, 50× objective. these cells lack the circumferential villi and ground-glass chromatin pattern of ‘hairy cells’. multiparameter flow cytometry was performed on the facscanto™ ii (becton, dickinson, and company, franklin lakes, new jersey, united states), and the data analysed on infinicyt™ version 2.0 (cytognos, santa marta de tormes, salamanca, spain) software. analysis revealed an abnormal population of large cells with moderate complexity and bright cd45 expression extending into the ‘monocyte window’ (figure 2). these cells expressed pan-b-cell markers, cd19, cd20, cd22, cd79b, and sigm, with surface kappa light chain restriction. villous markers (cd11c, cd25, cd103, cd123) and cd200 were positive. this immunophenotype is characteristic of hcl.2,6 there was no significant monocyte population. figure 2: flow cytometry dot plots of the pertinent cluster of differentiation markers used in the immunophenotypic diagnosis of hairy cell leukaemia that were performed for our patient at national health laboratory service universitas academic laboratory, south africa, in june 2023. the population of interest is demonstrated in red. the teal green population represents normal t cells, whilst the pink population constitutes neutrophils, and the blue population constitutes natural killer cells. (a, b) the hairy cells are large in size (increased fsc-a) with bright cd45 expression. (c–f) the hairy cells brightly express pan-b-cell markers, cd19, cd20, and cd79b, with surface kappa light chain restriction, confirming clonality. (g–i) the hairy cells express cd200 with co-expression of villous markers, cd11c, cd25, cd103, and cd123. because monocytopenia, rather than monocytosis, is typically seen in hcl, an fbc and automated wbc differential was repeated on the advia® 2120i (siemens healthineers, erlangen, germany) fbc analyser in our laboratory. the same blood sample subsequently revealed a monocytopenia of 0.93 × 109/l, with an increase in the lymphocyte count to 34.51 × 109/l. these counts correlated with the pathologist’s manual differential count and flow cytometry analysis. it was concluded that the sysmex xn-1000 series spuriously counted the ‘hairy cells’ as monocytes, resulting in pseudomonocytosis. management and outcomes the patient was referred to a tertiary hospital. further workup of the case confirmed a diagnosis of hcl, with the detection of the brafv600e mutation and characteristic bm morphology. unfortunately, the patient demised shortly after admission, and further details regarding treatment could not be obtained. discussion monocytopenia in the setting of pancytopenia should trigger a suspicion of hcl.3,4 demonstrating clonal b cells with ‘villous’ markers (cd11c, cd25, cd103, and cd123) on multiparameter flow cytometry is critical for the timely diagnosis of hcl.2,6,10 however, characteristic lymphocytes with circumferential villi (‘hairy cells’) may be sparse on pb morphology.1,10 unless the pathologist has a high index of suspicion on the initial pb smear review, the diagnosis of hcl may be unnecessarily delayed until a bm aspirate and trephine biopsy and molecular studies for the brafv600e mutation are performed. furthermore, bm aspiration is frequently unsuccessful because of bm fibrosis in hcl.2,10 it is important to note that the leucocytosis and abundance of ‘hairy cells’ (78%) that were seen in our case is unusual. lymphocytosis is only reported in 5% – 10% of cases of hcl.1 by comparison, the hcl cases evaluated by tofan et al.3 and furundarena et al.11 revealed an average of 19.6% and 9.4% circulating ‘hairy cells’. villous lymphocytes are not unique to hcl. their differential diagnosis includes artefact and other splenic b-cell lymphomas/leukaemias, namely splenic diffuse red pulp lymphoma, splenic marginal zone lymphoma and hcl variant (an entity retained in the international consensus classification, but reclassified as splenic b-cell lymphoma/leukaemia with prominent nucleoli in the world health organization classification of haematolymphoid tumours 5th edition).12 considering its relative sensitivity and specificity for hcl, the presence or absence of monocytopenia is often relied on during the initial assessment of these lymphomas to assist with the differential diagnosis.3,10 however, the sensitivity for detecting monocytopenia in hcl varies amongst automated fbc haematology analysers, and may be more reliable on instruments using peroxidase staining techniques such as the advia® 2120i.4 instruments utilising fluorescent staining and light scatter, or physical properties of cells, may yield inaccurate monocyte counts in hcl.3,4 sysmex xn-series automated fbc haematology analysers incorporate cytochemical reactions followed by fluorescence flow cytometry using a semiconductor laser to perform a wbc differential count. this method involves the addition of a fluorescent dye to wbcs, which are subsequently separated by their different light scatter properties according to size (forward scatter), complexity (side scatter), and fluorescence signal.3,13,14 scattergrams provide a visual representation of separated cell population in the white cell nucleated-, white cell differential-, and white cell precursor channels.3,13 additionally, internal protocol messages or ‘flags’ assist with the rapid identification of quantitative and qualitative wbc abnormalities.8,9 in our case, the sysmex xn-1000 series correctly flagged ‘neutropenia’, ‘leucocytosis’, ‘lymphocytosis’ and the presence of ‘blasts/abn lymphocytes?’ (sic). the white cell differential scattergram showed a large population of cells in the ‘monocyte window’ (figure 3). based on their similar forward and side scatter properties, the xn-series classified ‘hairy cells’ as monocytes, which resulted in a spurious ‘monocytosis’ flag. no white cell precursor scatterplot was generated by the instrument, suggesting that the white cell precursor channel (which detects abnormal lymphocytes or blasts) was not reflexed. similarly, in five of six hcl cases evaluated by tofan et al.3, a pseudomonocytosis was generated when automated differential counts were performed on the sysmex xn-series. this impaired separation of mononuclear cells was ascribed to the larger size and more abundant cytoplasm of the leukaemic cells.3 figure 3: (a) the white cell differential channel on the xn-series (sysmex corporation, kobe, japan) primarily classifies white blood cells. this normal scattergram displays lymphocytes, monocytes, eosinophils, basophils, neutrophils, and debris.13,14 (b) the white cell differential scattergram for our case, performed on a sysmex xn-1000 at a regional laboratory in north west province, south africa in june 2023, demonstrated a large population of cells in the ‘monocyte window’. the wbc flagging performance of the xn-series has been evaluated in several studies, including two local studies performed in nhls laboratories.8,15,16 ramiah et al.8 demonstrated sensitivities and specificities of 90% and 96.2% for the ‘abnormal lymphocyte’ flag, and 84.3% and 97% for the ‘monocytosis’ flag. three samples (1.2%) were erroneously flagged as ‘monocytosis’ where blasts were demonstrated on microscopy.8 schapkaitz et al.16 evaluated the performance of the sysmex xn-9000 and found the white cell differential and white cell precursor channels to be 100% sensitive for detecting abnormal lymphocytes. however, neither of these studies included hcl cases. the limitations of sysmex automated fbc haematology analysers in flagging abnormal results in hcl have not been widely reported. furundarena et al.11 compared the performance of sysmex xn and xe-5000 analysers in oncohaematologic patients and concluded that both instruments must improve flagging in hcl. both analysers only generated one flag (‘abn lympho/blasts?’) in one out of five hcl samples included in their study.11 considering the rarity of hcl, this neoplasm may be underrepresented in the verification studies of xn-series analysers that have been performed to date. further studies are needed to evaluate the performance of the xn-series, specifically in hcl. conclusion monocytopenia may prompt early investigation for hcl in the right clinical scenario. however, haematology analysers, such as the sysmex xn-series, that rely on cytochemistry with fluorescence flow cytometry may artifactually report ‘monocytosis’ in hcl. with the nationwide introduction of the sysmex xn-series in nhls laboratories, this case serves as a reminder that a manual differential count and morphologic review of suspicious results remain important in the era of automation. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions s.j.k. and a.-c.v.m. contributed to the conceptualisation and design of the case report, and the collection and interpretation of data. s.j.k. and a.-c.v.m. contributed equally to writing and editing the case report. the final article was approved by both s.j.k. and a.-c.v.m. sources of support this article received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability the data that support the findings of this case study are available from the corresponding author, a.-c.v.m., upon reasonable request. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional 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https://doi.org/10.1038/s41375-022-01620-2 sysmex corporation. chapter 15: p. 466–469. in: automated hematology analyzer xn series transportation units (xn-9000/xn-9100). instructions for use. (ver.22). kobe: sysmex corporation; 2021. sysmex corporation. chapter 5: p. 66–71. in: automated hematology analyzer xn-l series (xn-530/xn-430/xn-330). general information (ver.5). kobe: sysmex corporation; 2020. sysmex south africa scientific customer services. customer literature list – white blood cells. 2024(03). 2024 [cited 2024 may 31]. available from: https://www.sysmex.co.za/academy/library/publications/ schapkaitz e, raburabu s. performance evaluation of the new measurement channels on the automated sysmex xn-9000 hematology analyzer. clin biochem. 2018;53:132–138. https://doi.org/10.1016/j.clinbiochem.2018.01.014 references about the author(s) rajiv t. erasmus department of chemical pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa citation erasmus rt. point-of-care testing: connecting communities in africa and ensuring equity in access to health and diagnostics. afr j lab med. 2022;11(1), a2072. https://doi.org/10.4102/ajlm.v11i1.2072 editorial point-of-care testing: connecting communities in africa and ensuring equity in access to health and diagnostics rajiv t. erasmus copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. according to the african society for laboratory medicine, ‘[m]edical laboratories play a pivotal role in global disease diagnosis, surveillance, outbreak investigation, initiation and monitoring of therapy, as well as research and development’. however medical laboratories in africa cannot meet the testing demands of a rapidly growing health delivery service,1 due to a lack of resources and inadequate diagnostic services, which compromise patient care. this leads to misdiagnosis, resulting in underor over-treatment of disease, giving rise to numerous societal health challenges.1 a recent lancet commission review2 revealed a lack of access to diagnostics at the lowest tier of the healthcare system – the community – where the highest population in need of services exists, but only 47% globally have access. in its mapping exercise undertaken between november 2019 and april 2020, the european centre for disease prevention and control3 found that point-of-care (poc) testing could present a possible solution as it plays: a critical role in rapidly diagnosing both infectious and noninfectious diseases and is not only for appropriate and timely treatment but also for the detection of outbreaks and controlling the rapid spread of infectious diseases. (p. 1) in 2013, almost a decade ago, jani and peter4 had stressed ‘how point-of-care testing could drive innovation in global health’. indeed, if one fast-forwards to 2022, poc testing has made great strides in diagnosing and managing the disease burden in africa. according to world health organization,5 the diseases afflicting the african population are responsible for a substantial loss in health, estimated at 704 765 879 disability adjusted life years in 2015 alone. in the world health organization african region, total losses amounted to 629 603 271 disability adjusted life years; 59.1% were from communicable, maternal, perinatal and nutritional conditions with diabetes, anaemia, malaria and syphilis contributing to the greatest burden of disease.5 the international organization for standardization defines poc testing and near-patient testing for any disease (not just infectious diseases) as ‘testing that is performed near or at the site of a patient with the result leading to possible change in the care of the patient’.6 the small footprint of the instrument means that they can be easily transported. according to the united states’ national institutes of health, ‘empowering clinicians to make decisions at the “point-of-care” has the potential to significantly impact healthcare delivery and to address the challenges of health disparities’ and this could result in ‘the success of a potential shift from curative medicine, to predictive, personalised, and pre-emptive medicine’.7 although poc testing is progressively being used for the identification and management of various disease states in africa, controlling various poc instruments at numerous sites can be difficult, particularly when such instruments are run by non-technical staff. however, these challenges can be overcome with recent advances in connectivity and digitisation, as well as standardisation of software and middleware, which allow the quality of testing to be monitored in real time. connectivity allows for poc instruments to hook up with a laboratory or hospital information system, as well as with electronic medical records.8 for this purpose an open-access data management system is critical, permitting connections between instruments from any manufacturer. this process automatically validates and transfers patient results, including those from electronic medical records, as well as monitoring and managing data, multiple testing devices, and operators.8 as these are intimately linked to operator training and competency, harm to the patient is minimised by reducing analytical errors. faster decision-making can also occur due to significantly lower turnaround times.8 with the advent of 5g and artificial intelligence, greater focus on personalised care is likely to occur resulting from big data analysis and development of algorithms; communities in africa stand to benefit from this. wireless technology, the internet of things and big data will not only allow patients in africa to take charge of their health needs, but hugely improve the way health-related data is transmitted and interpreted, making it possible for healthcare delivery to be more efficient, precise and, ultimately, more affordable. in addition, accessibility would mean increased equity in healthcare and diagnostics to communities in africa. smartphone-based operation, paper-based sensing assays, and lab-on-a-chip are being turned into mobile laboratories, which are furthering this trend in remote areas.9 already there are well-established programmes that have changed the health landscape in africa. the most recent cochrane review10 reported that poc viral load testing showed both high sensitivity and specificity for the diagnosis of hiv viral load, at or above the clinical threshold of 1000 copies/ml, among people living with hiv who attended healthcare facilities for their hiv tests. furthermore, poc viral load testing has been reported to enhance viral suppression and retention in hiv care.10,11 rapid antigen lateral flow tests were particularly useful during the coronavirus disease 2019 (covid-19) pandemic and were used for rapid screening and tracing of cases in communities.12 the introduction of poc lateral flow urine lipoarabinomannan assays has improved tuberculosis case detection and lowered diagnostic holdups in people living with hiv and is particularly useful in african communities where traditional methods have a long turnaround time. although education, training, data governance and quality management will remain key to ensure the reliability and accuracy of test results,13,14 these results demonstrate that poc testing can simplify treatment and improve outcomes for hiv-positive adults receiving antiretroviral therapy in resource-limited settings and that poc testing presents a great advantage for disease diagnosis, monitoring and its management for patients in africa. the african union11 has put forward a digital transformation strategy for africa 2020–2030. if it is successfully implemented, it will no doubt further propel poc testing, ensuring optimal governance and quality. thus, africa is in a unique position to use poc and digital technologies, including machine learning and artificial intelligence, into a highly effective strategy for delivering speedy, high-quality, data-driven care to communities in africa. africa must take advantage of the convergence of poc testing and digital technologies, as this will surely enhance diagnostics and healthcare on the continent. references messele t. about aslm [homepage on the internet]. aslm; 2014 [cited n.d.]. available from: https://na.eventscloud.com/ehome/65245/aboutaslm fleming ka, horton s, wilson ml, et al. the lancet commission on diagnostics: transforming access to diagnostics. lancet. 2021;398(10315):1997–2050. https://doi.org/10.1016/s0140-6736(21)00673-5 european centre for disease prevention and control. assessment of point-of-care testing devices for infectious disease surveillance, prevention and control – a mapping exercise. stockholm: european centre for disease prevention and control; 2022. jani iv, peter tf. how point-of-care testing could drive innovation in global health. n engl j med. 2013;368(24):2319–2324. https://doi.org/10.1056/nejmsb1214197 world health organization. a heavy burden: the productivity cost of illness in africa. brazzaville: who regional office for africa; 2019. international organization for standardization. iso 22870: 2016 point-of-care testing (poct) – requirements for quality and competence. [cited 2016 nov]. available from: https://www.iso.org/standard/71119.html omnia health. point-of-care diagnostic testing: empowering the clinicians. omnia health, informa markets; 2020 [cited 2020 jan 16]. available from: https://insights.omnia-health.com/laboratory/point-care-diagnostic-testing-empowering-clinicians erasmus rt, sumedha s, el-sharkawy r. connectivity strategies in managing a poct service. ejifcc. 2021;2:190–194. kumar s, nehra m, khurana s, et al. aspects of point-of-care diagnostics for personalized health wellness. int j nanomed. 2021;16:383–402. https://doi.org/10.2147/ijn.s267212 ochodo ea, olwanda ee, deeks jj, mallett s. point-of-care viral load tests to detect high hiv viral load in people living with hiv/aids attending health facilities. cochrane database syst rev. 2022;3(3):cd013208. https://doi.org/10.1002/14651858.cd013208.pub2 drain pk, dorward j, bender a, et al. point-of-care hiv viral load testing: an essential tool for a sustainable global hiv/aids response. clin microbiol rev. 2019;32(3):e00097-18. https://doi.org/10.1128/cmr.00097-18 crozier a, rajan s, buchan i, mckee m. put to the test: use of rapid testing technologies for covid-19. bmj. 2021;372:n208. https://doi.org/10.1136/bmj.n208 jalavu tp, rensburg m, erasmus r. clinical staff knowledge and awareness of point-of-care-testing best practices at tygerberg hospital, south africa. afr j lab med. 2020;9(1):a853. https://doi.org/10.4102/ajlm.v9i1.853 gous n, boeras di, cheng b, takle j, cunningham b, peeling rw. the impact of digital technologies on point-of-care diagnostics in resource-limited settings. expert rev mol diagn. 2018;18(4):385–397. https://doi.org/10.1080/14737159.2018.1460205 abstract introduction methods results discussion trustworthiness acknowledgements references about the author(s) irith de baetselier institute of tropical medicine, department of clinical sciences, sti reference laboratory, nationalestraat, antwerp, belgium douglas taylor fhi 360, durham, north carolina, united states justin mandala fhi 360, durham, north carolina, united states kavita nanda fhi 360, durham, north carolina, united states christel van campenhout scientific institute of public health, brussels, belgium walter agingu impact research and development organization, kisumu city, kenya lorna madurai global clinical and viral laboratory, kwa-zulu natal, south africa eva-maria barsch pathcare bloemfontein, quantum building, bloemfontein, south africa jennifer deese fhi 360, durham, north carolina, united states lut van damme fhi 360, durham, north carolina, united states bill & melinda gates foundation, seattle, washington, united states tania crucitti institute of tropical medicine, department of clinical sciences, sti reference laboratory, nationalestraat, antwerp, belgium citation de baetselier i, taylor d, mandala j, et al. verification of chemistry reference ranges using a simple method in sub-saharan africa. afr j lab med. 2016;5(1), a404. http://dx.doi.org/10.4102/ajlm.v5i1.404 original research verification of chemistry reference ranges using a simple method in sub-saharan africa irith de baetselier, douglas taylor, justin mandala, kavita nanda, christel van campenhout, walter agingu, lorna madurai, eva-maria barsch, jennifer deese, lut van damme, tania crucitti received: 09 dec. 2015; accepted: 27 july 2016; published: 17 oct. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: chemistry safety assessments are interpreted by using chemistry reference ranges (crrs). verification of crrs is time consuming and often requires a statistical background. objectives: we report on an easy and cost-saving method to verify crrs. methods: using a former method introduced by sigma diagnostics, three study sites in sub-saharan africa, bondo, kenya, and pretoria and bloemfontein, south africa, verified the crrs for hepatic and renal biochemistry assays performed during a clinical trial of hiv antiretroviral pre-exposure prophylaxis. the aspartate aminotransferase/alanine aminotransferase, creatinine and phosphorus results from 10 clinically-healthy participants at the screening visit were used. in the event the crrs did not pass the verification, new crrs had to be calculated based on 40 clinically-healthy participants. results: within a few weeks, the study sites accomplished verification of the crrs without additional costs. the aspartate aminotransferase reference ranges for the bondo, kenya site and the alanine aminotransferase reference ranges for the pretoria, south africa site required adjustment. the phosphorus crr passed verification and the creatinine crr required adjustment at every site. the newly-established crr intervals were narrower than the crrs used previously at these study sites due to decreases in the upper limits of the reference ranges. as a result, more toxicities were detected. conclusion: to ensure the safety of clinical trial participants, verification of crrs should be standard practice in clinical trials conducted in settings where the crr has not been validated for the local population. this verification method is simple, inexpensive, and can be performed by any medical laboratory. introduction high prevalence rates for hiv, tuberculosis, malaria and several other infectious diseases are found in africa.1 these high prevalence rates have led to an increasing number of clinical trials being conducted on the african continent. the majority of these trials aim to evaluate methods and interventions to reduce the burden of these diseases.2,3 to appropriately screen eligible participants and to detect and monitor possible toxicities related to the investigated products, accurate clinical laboratory reference ranges for the study population are required. these ranges were first introduced by gräsbeck and saris to describe fluctuations of blood parameter concentrations in well-characterised groups of individuals.4,5 local clinical laboratories often rely on reference values established by the manufacturer or presented in a textbook, which are rarely specific for african populations.6,7 previous studies have demonstrated that using laboratory reference ranges for haematology and biochemistry obtained from populations other than those under investigation have led to the possible exclusion of healthy participants and an over-reporting of adverse events.6,8 thus, a good practice is to use reference ranges specific to the study population for appropriate and safe management of participants.9 in june 2009, we initiated a multi-centre phase iii clinical trial to assess the safety of the antiretroviral combination tenofovir disproxil fumarate/emtricitabine (tdf/ftc) as pre-exposure prophylaxis for hiv among women in sub-saharan africa (fem-prep trial).10 one of the primary objectives of the trial was to assess the safety of tdf/ftc in healthy women at high risk of acquiring hiv. the primary safety endpoints included confirmed grade 3 or grade 4 toxicity of aspartate aminotransferase/alanine aminotransferase (ast/alt) and phosphorus and grade 2 or higher toxicity of creatinine during and up to four weeks after study product administration. calculation of toxicity grades for the aforementioned parameters of interest required reference to the chemistry reference range (crr).10 abnormal renal and liver function values were exclusion criteria for study participation and were also protocol-required safety criteria for study drug interruption or permanent withdrawal. accurate crrs were therefore essential to guarantee the safety of the participants during the study and to provide adequate classification of adverse events.11 the analysers, the analytical methods and the study populations differed among the study sites in the fem-prep trial, making the transferability of crrs between sites inappropriate. in european and united states-based clinical trials, it is common practice to test all study samples in a central laboratory to limit test variation across study sites.12 however, in multi-centre trials in sub-saharan african settings, the use of a central laboratory is not always recommended due to difficulties related to sample transport, instability of biochemical compounds and undesired delays in reporting of results.13 according to the clinical and laboratory standards institute (clsi), each laboratory should determine its own laboratory reference limits, including the crr.14 the crr should be derived from a healthy population representative of the study population.14 however, determination of a crr can be complex, time-consuming and expensive.15 in short, the clsi guidelines recommend the establishment of crrs with at least 120 reference individuals using a non-parametric ranking method or, as an alternative, a robust method with a minimum of 20 samples from qualified reference individuals when there are sample size constraints. the majority of clinical laboratories collaborating in the fem-prep trial had nationallyor regionally-established crrs or used the manufacturer’s ranges prior to the study. use of manufacturer-defined crrs may not be appropriate for clinical trial target populations due to potentially important differences in socio-demographic characteristics, environmental context, malnutrition, dietary patterns, genetics, or infection with helminths or other parasites (such as malaria or schistosomiasis).6,7,16,17,18,19 notably, recent reference studies conducted in africa have identified differences within populations and sometimes within sub-groups,20 making the applicability of such reference ranges less likely. ultimately, use of non-population-specific crrs could compromise the scientific validity of clinical trial conclusions by underor overestimating the severity of adverse events. our objective was to use a feasible and inexpensive method to verify previously-established crrs and assess the impact of the final revised crrs on the observed safety results in our phase iii clinical trial. methods ethical considerations the fhi 360 protection of human subjects committee (phsc), institutional review boards at all study sites and applicable regulatory committees approved the study. study design women were recruited at four different sites in bondo, kenya; pretoria and bloemfontein, south africa; and arusha, tanzania from june 2009 to april 2011. because of a decision on april 18, 2011 to close the study early due to futility, verification of the crr was not finalised for the arusha site and is therefore not addressed in this paper. women had to be aged between 18 and 35 years, not pregnant, and in general good health to be included in the study. the crrs were verified at each site using a subset of 10 screened participants who were negative for: hiv, hepatitis b virus, chlamydia trachomatis, neisseria gonorrhoeae, trichomonas vaginalis, candida spp. and bacterial vaginosis. sample collection all laboratory activities, including specimen transport, processing, testing, result reporting and storage, were conducted in accordance with good clinical laboratory practices. at each study site, serum was collected in a plastic uncoated serum separation tube at screening, at weeks 4, 12, 24, 36, 52 and 56, and when clinically indicated. samples were immediately taken to the on-site laboratory and were processed within two hours of collection. quantification of ast/alt, phosphorus and serum creatinine was performed according to the procedures described by the manufacturer and documented in site-specific standard operating procedures. the study site in bondo performed the chemistry testing on-site using vitros dt ii (ortho-clinical diagnostics, inc., johnson & johnson, buckinghamshire, united kingdom) until may 2010 and thereafter used the vitros 250 instrument (ortho-clinical diagnostics, inc., johnson & johnson, buckinghamshire, united kingdom). two private laboratories performed the chemistry analysis on the samples collected at the south african study sites. both laboratories were iso 15189 accredited, had excellent infrastructure and had collaborated previously in multi-centre clinical trials. the study site in pretoria shipped serum samples daily for chemistry analysis in temperature-monitored cool boxes with ice packs to the global clinical and viral laboratory (gcvl; durban, south africa). upon arrival, the samples were immediately analysed using the synchron cx5 beckman chemistry analyzer (beckman coulter, inc., fullerton, california, united states). the bloemfontein site transported the serum samples every two hours to pathcare laboratories, located five minutes’ drive from the study site. the samples were transported at ambient temperature and were analysed immediately upon arrival using the synchron cx5 beckman chemistry analyzer. all sample transportation was validated before implementation. laboratory methods after the study began, we verified the crrs for ast, alt, creatinine and phosphorus according to guidelines established previously by sigma diagnostics based on the biological variation of the analytes (no reference available). in brief, the following was performed at each study site. the alt, ast, creatinine and phosphorus values of serum samples collected from 10 clinically-healthy participants obtained at screening were used to calculate a ‘patient mean’, after which the mean of the current reference range was determined. example: manufacturer’s reference range for ast = 9 to 52 u/l: the patient mean was compared with the established reference range mean and the percent difference between the selected samples, and the established reference mean was calculated. example: ast reference range mean = 30.5 u/l selected patient sample mean = 23.4 u/l the percent (%) deviation was compared with the tolerance limit listed in the reference range deviation tolerance limits table (table 1). table 1: reference range verification tolerance limits according to sigma diagnostics. if the percent deviation was within the listed tolerance limits, the current crr could be used and no further action was required. cases in which the percent deviation exceeded the tolerance limit required collection of additional values to adjust the ranges. we determined a priori to collect an additional 30 values in case adjustment was required. the mean and standard deviation (sd) were calculated from the 40 representative values. a range of (mean – 3 sd) to (mean + 3 sd) was set from these data, and any values that fell outside these limits were eliminated. afterwards, the mean and sd were calculated from the remaining values. the reference range was determined to be the mean ± 2 sd. in the event of changes in analyser or reagent/methodology, re-verification of the established crr was done according to the above-mentioned procedures. from all sites, 20 values per analyte were available permitting us to compare the applied verification method with the clsi guidelines.14 according to clsi, the crr is accepted when at least 18 values fall within the original reported limits. if three to four results fall outside these limits, another 20 reference values should be obtained. if no more than two of these new values fall outside the crr, the crr is accepted, otherwise the crr should be corrected using the clsi guidelines. statistical analysis the freeware ‘reference value advisor’ (rva) was used to perform all calculations according to the clsi.15 to assess the impact of the crr verification and adjustment on the toxicity grading, we calculated chemistry grades using both the pre-existing and newly-established crrs. the statistical analysis to assess the impact of crr verification on the toxicity grading used the database of the fem-prep trial.10 we included all women who were randomised, made at least one follow-up visit where chemistries were assessed, and did not return their entire product unused. we graded laboratory chemistry measurements at each site as grade 1, 2, 3 or 4 (for alt, ast and creatinine) or grades 2, 3 or 4 (for phosphorus) according to the division of aids (daids) table.10 in addition to daids guidance, the protocol specified that any creatinine value during follow-up which exceeded 1.5 times baseline be coded as grade 1, even if the absolute measurement was less than 1.1 times the upper limit of normal (uln). the onset date of each abnormality was the date when the abnormality was first detected, regardless of when the highest grade level occurred. likewise, individual abnormalities were considered ongoing until the values returned to normal, even if there was a partial decrease in grade. we included all measurements obtained prior to primary censoring dates (e.g. on or before a participant’s week 52 visit, her seroconversion visit, or an earlier discontinuation visit, whichever came first), irrespective of adherence to treatment regimen. we computed the total number of toxicity events that would have been missed using only the initial crrs. results verification of crr table 2 summarises the initial analytes’ reference ranges used by the study sites and the crr after verification. prior to the study, both south african laboratories were using laboratory-specific reference ranges or those established by a competent national authority, whereas the kenyan study site was using manufacturer crrs. based on the verification results, we had to recalculate the crr for ast at the bondo site and that for alt at the pretoria site. the pre-existing phosphorus reference range did not require revision at any study site. the reference range for creatinine had to be adjusted at all three sites, mainly due to changes in methodology, reagent or standard. the need to adjust the crr was checked using the sigma method and clsi guidelines. results between both methods were in complete concordance except for alt in pretoria, which should not have been corrected according to clsi. table 3 compares the pretorian crr obtained with the sigma method and the non-parametric method as described by clsi, based on the availability of 128 values from the pretorian site. table 2: initial and revised chemistry reference range values for each study site. table 3: comparison of chemistry reference range from the pretoria site obtained with the sigma method and the clinical and laboratory standards institute method. impact of revised crr on number of laboratory toxicities the grading of adverse events based on laboratory abnormalities was performed in accordance with the daids grading table and therefore relied on the uln. the final revised crrs were applied from the time of establishment; previous adverse events were not re-graded during the study. table 4 provides the total number of toxicities found during the trial using the initial crr versus the final reference ranges for ast, alt and creatinine. in our settings, the crrs became narrower after adjustment, with a corresponding lowering of the uln. as a consequence, the overall number of laboratory toxicities that occurred during the trial was higher using the adjusted ranges as compared to the pre-existing ranges. for hepatic toxicity management, 25 grade 2 alt, 13 grade 3 alt, 19 grade 2 ast, and two grade 3 ast results would have been graded differently using the initial crrs. according to the initial crrs, there were no grade 2 creatinine toxicities; however, 14 grade 2 creatinine toxicities were identified using the newly-calculated ranges. table 4: toxicity grades for ast/alt and creatinine using initial versus final reference ranges. the laboratory abnormality frequency for alt, ast, creatinine, and phosphorus between the two study groups (placebo vs. tdf/ftc) using the initial and final crrs were compared and are presented in table 5. although the majority of the missed toxicities were grade 1, half of the participants with a grade 3 or grade 4 hepatic toxicity would have been misclassified, if management had been based on the initial crrs. the impact of the revised creatinine reference ranges was less pronounced. however, all five cases of grade 2 creatinine toxicity would have been misclassified as a grade 1 using the initial crrs. table 5: laboratory abnormality frequency tables based on the initial versus final chemistry reference ranges. table 4 presents the total number of toxicities and table 5 tabulates the number of adverse events based on laboratory abnormalities. table 5 only shows the highest grade of abnormality occurrence even if this occurred after seroconversion, whereas table 4 only includes results obtained on or before the primary censoring date. when comparing tables 4 and 5, an idiosyncrasy in alt data is noted. one participant had a grade 1 alt abnormality before her primary censoring date (i.e., seroconversion visit) but experienced grade 3 ast toxicity 24 weeks after seroconversion according to the old crr. when the new crrs were used, the latter toxicity was reclassified as grade 4. there were no significant differences in the proportion of women experiencing toxicities in the tdf/ftc and placebo groups based on the initial crr (results not shown). using the revised crr, however, a significantly higher percentage of women in the tdf/ftc group experienced alt grade 1 or higher toxicities (p = 0.033),10 and there was also a trend toward a higher percentage of women in the tdf/ftc group experiencing grade 2 or higher ast toxicities (p = 0.069). discussion the clsi guidelines recommend the establishment of crrs with at least 120 reference individuals using a non-parametric ranking method or, as an alternative, a robust method with a minimum of 20 samples from qualified reference individuals when there are sample size constraints. it was not feasible in the fem-prep trial to recruit reference individuals prior to the initiation of the trial, as the study sites were research centres that did not see routine patients. therefore, we verified the existing crrs using specimens collected at screening using the sigma verification procedure. this method is simple, does not require statistical expertise, is less time-consuming, inexpensive and can easily be implemented by any laboratory. we also examined the impact of the new reference ranges on the toxicity grading. when looking at our data, a total of 9 alt/ast grade 3 or higher, 5 serum creatinine grade 2 toxicities and many grade 1 toxicities would have been missed if the original crrs were used. laboratories are essential for both the detection and prevention of diseases. in clinical trials, laboratories play a crucial role in endpoint measurement. in the fem-prep trial, the laboratory safety endpoints were based on chemistry parameters to detect liver and kidney toxicities. there were no additional clinical or laboratory costs involved in the verification process since the chemistry tests were a required screening procedure. the major disadvantage of using specimens collected at screening was that study participants could be excluded or included erroneously through misclassification of toxicity grades during the time of crr verification and adjustment. however, in fem-prep there were no instances of discordant eligibility classification when applying the pre-existing versus final verified crrs due to predefined inclusion criteria, which required that creatinine be < 1.5 mg/dl and hepatic function tests be < 2x uln. immunohaematological reference ranges are now well defined in asia and africa, and different studies have reported the need for population-specific clinical chemistry reference ranges.17 recently, reference value studies among women and/or men in different countries in sub-saharan africa have been conducted.6,16,17,18,19 table 6 summarises crrs established in african countries compared with our final revised crr. previous studies in kenya confirmed our creatinine values,13,16,17 but overall our crrs for ast/alt were narrower than those previously reported. for the south african sites, our ranges were also more compressed than in previous studies but, to date, no comparable studies have been performed in south africa. the narrower ranges are explained by the fact that we calculated the ranges in a specific age group and population, namely, clinically healthy women aged between 18 and 35 years who were at high risk of acquiring hiv infection. table 6: comparison of chemistry reference ranges from the three study sites to those reported in previous publications. limitations our study has several limitations. ideally, verification of reference ranges should be conducted before trial initiation. it is also possible that the number of specimens (10) required for initial verification by this method were too few. for example, clsi recommends a set of 20 reference specimens and replacement of outliers if necessary. we compared the applied method with the clsi guidelines and obtained similar results, except for one parameter (alt) in one study site (pretoria) which should not have been corrected according to clsi. as we had more than 120 reference values available (n = 128), we recalculated the alt crr using rva13 with the clsi method and noted good concordance with the sigma method. conclusion we detected a large number of toxicities that would not have been identified using the pre-existing crrs due to the decrease in the uln for hepatic and renal parameters. overall, we developed more population-appropriate crrs that may have improved the clinical safety management of study participants. in conclusion, establishing local reference ranges is necessary to comply with the high-quality standards of good clinical and laboratory practices. unfortunately, not all laboratories have the resources necessary to establish local reference ranges; therefore, verification of existing reference ranges offers a good alternative. methods such as the former sigma method or freeware including robust, transformation and non-parametrical methods can be applied on reference samples sets without additional costs and in the absence of sophisticated statistics by any laboratory performing chemical analysis. trustworthiness reliability during several laboratory supervision visits, the correctness of the values that were used to verify the crr was checked with the raw data. all laboratories worked according to good clinical laboratory practice guidelines and two laboratories were also iso 15189 accredited. validity we report here on an easy method that uses 10 values to verify the reference ranges and that can be implemented in any laboratory without need for statistical expertise. we also show that determining the crr before the start of a clinical trial is imperative to ensure that all toxicities found in the study are graded correctly. with this study, we also compared the sigma method with the clsi guidelines and obtained similar results. our reported crr are also within range with what has been previously found. acknowledgements this study was conducted in the context of the fem-prep clinical trial. we thank all the women who participated in the clinical trial and all study staff who worked on the study. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. sources of support the main study was made possible through grants funded by the united states agency for international development (usaid), the contraceptive and reproductive health technologies and research utilization program, and the preventive technologies agreement no ghoa000900016-00. early support was also provided by the bill and melinda gates foundation. gilead sciences, inc. donated the tdf-ftf and placebo. views expressed in this publication do not necessarily reflect those of fhi 360 or the agencies funding the study. author’s contributions i.d.b. and t.c. wrote the first draft of the manuscript. d.t., j.m., k.n., j.d. and l.v.d. revised and edited the text. t.c. and c.v.c. created the experimental design, and d.t. performed the statistical analysis. w.a., l.m., and e.-m.b. generated the data. all authors revised and approved the present version of the manuscript. references world health organization. world health statistics 2015 [document on the internet]. c2015 [cited 2016 may 23]. available from: http://apps.who.int/iris/bitstream/10665/170250/1/9789240694439_eng.pdf?ua=1&ua=1 esparza j, osmanov s. hiv vaccines: a global perspective. curr mol med. 2003;3(3):183–193. http://dx.doi.org/10.2174/1566524033479825 jaoko w, nakwagala fn, anzala o, et al. safety and immunogenicity of recombinant low-dosage hiv-1 a vaccine candidates vectored by plasmid pthr dna or 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clinical trials in developing countries. qual assur. 2003;10(2):83–89. alemnji g, nkengasong jn, parekh bs. hiv testing in developing countries: what is required? indian j med res. 2011;134(6):779–786. http://dx.doi.org/10.4103/0971-5916.92625 clinical and laboratory standards institute. ep28-a3c: defining, establishing, and verifying reference intervals in the clinical laboratory; approved guideline – third edition. wayne, pa: clsi; 2010. geffré a, concordet d, braun jp, et al. reference value advisor: a new freeware set of macroinstructions to calculate reference intervals with microsoft excel. vet clin pathol. 2011;40(1):107–112 http://dx.doi.org/10.1111/j.1939-165x.2011.00287.x. epub 2011 feb 7. karita e, ketter n, price ma, et al. clsi-derived hematology and biochemistry reference intervals for healthy adults in eastern and southern africa. plos one. 2009;4(2):e4401. http://dx.doi.org/10.1371/journal.pone.0004401. epub 2009 feb 6. zeh c, amornkul pn, inzaule s, et al. population-based biochemistry, immunologic and hematological reference values for adolescents and young adults in a rural population in western kenya. plos one. 2011;6(6):e21040. http://dx.doi.org/10.1371/journal.pone.0021040. epub 2011 jun 21. kibaya rs, bautista ct, sawe fk, et al. reference ranges for the clinical laboratory derived from a rural population in kericho, kenya. plos one. 2008;3(10):e3327. http://dx.doi.org/10.1371/journal.pone.0003327 dosoo dk, kayan k, adu-gyasi d, et al. haematological and biochemical reference values for healthy adults in the middle belt of ghana. plos one. 2012;7(4):e36308. http://dx.doi.org/10.1371/journal.pone.0036308. epub 2012 apr 27. mine m, moyo s, stevens p, et al. immunohaematological reference values for hiv-negative healthy adults in botswana. afr j lab med. 2012;1(1):5. abstract introduction research method and design results discussion acknowledgements references about the author(s) joseph h.k. bonney noguchi memorial institute for medical research, university of ghana, legon, ghana edward o. nyarko 37 military hospital, public health division, accra, ghana sally-ann ohene world health organization ghana country office, accra, ghana joseph amankwa disease surveillance department, ghana health service, accra, ghana ralph k. ametepi 37 military hospital, public health division, accra, ghana shirley c. nimo-paintsil noguchi memorial institute for medical research, university of ghana, legon, ghana badu sarkodie disease surveillance department, ghana health service, accra, ghana prince agbenohevi 37 military hospital, public health division, accra, ghana michael adjabeng disease surveillance department, ghana health service, accra, ghana nicholas n.a. kyei 37 military hospital, public health division, accra, ghana samuel bel-nono 37 military hospital, public health division, accra, ghana william k. ampofo noguchi memorial institute for medical research, university of ghana, legon, ghana citation bonney jhk, nyarko eo, ohene s-a, et al. molecular confirmation of lassa fever imported into ghana. afr j lab med. 2016;5(1), a288. http://dx.doi.org/10.4102/ajlm.v5i1.288 original research molecular confirmation of lassa fever imported into ghana joseph h.k. bonney, edward o. nyarko, sally-ann ohene, joseph amankwa, ralph k. ametepi, shirley c. nimo-paintsil, badu sarkodie, prince agbenohevi, michael adjabeng, nicholas n.a. kyei, samuel bel-nono, william k. ampofo received: 10 dec. 2014; accepted: 02 feb. 2016; published: 25 apr. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: recent reports have shown an expansion of lassa virus from the area where it was first isolated in nigeria to other areas of west africa. two ghanaian soldiers on a united nations peacekeeping mission in liberia were taken ill with viral haemorrhagic fever syndrome following the death of a sick colleague and were referred to a military hospital in accra, ghana, in may 2013. blood samples from the soldiers and five asymptomatic close contacts were subjected to laboratory investigations. objective: we report the results of these investigations to highlight the importance of molecular diagnostic applications and the need for heightened awareness about lassa fever in west africa. methods: we used molecular assays on sera from the two patients to identify the causative organism. upon detection of positive signals for lassa virus ribonucleic material by two different polymerase chain reaction assays, sequencing and phylogenetic analyses were performed. results: the presence of lassa virus in the soldiers’ blood samples was shown by l-gene segment homology to be the macenta and las803792 strains previously isolated in liberia, with close relationships then confirmed by phylogenetic tree construction. the five asymptomatic close contacts were negative for lassa virus. conclusions: the lassa virus strains identified in the two ghanaian soldiers had molecular epidemiological links to strains from liberia. lassa virus was probably responsible for the outbreak of viral haemorrhagic fever in the military camp. these data confirm lassa fever endemicity in west africa. introduction lassa virus is a single-stranded rna virus and a member of the arenaviridae family and the genus arenavirus. the virus is the aetiologic agent of lassa fever, which is an acute and often fatal illness, endemic in portions of west africa.1 there are an estimated 300 000 to 500 000 cases of lassa fever each year,2 with a reported mortality rate of 15% – 20% for hospitalised patients. rodents of the genus mastomys are the reservoir for the virus, which is transmitted through direct contact with materials contaminated with urine and/or droppings of infected rodents. human-to-human transmission of the virus may give rise to nosocomial or community-based outbreaks. subsequent to its first isolation in lassa, nigeria and its restricted endemicity to two geographical regions in west africa, recent reports have shown expanded areas of spread.3,4 the upturn in global travel activities and international commitments in conflict and disaster situations have made the import of lassa fever and other viral haemorrhagic fevers (vhfs) into non-endemic countries a more likely event than in the past, which has been well documented.5,6,7,8 presently, an epidemic of another vhf, ebola virus disease, has spread throughout guinea and beyond its borders. as of december 6, 2014, a total of 17 895 ebola virus cases, including 6394 deaths, had been reported in six west african countries (guinea, liberia, sierra leone, nigeria, senegal and mali) and the democratic republic of the congo.9 the use of molecular diagnostic tools has resulted in highly sensitive and specific tests for infectious organisms and genetic diseases. these tools, which include polymerase chain reaction (pcr) and molecular sequencing, have come in handy for the rapid identification of lassa virus and other vhf agents from suspected cases. in may 2013, an outbreak of acute febrile illness affecting three military personnel occurred in the ghanaian united nations mission in liberia military contingent in zorzor, liberia. of the three cases, two were evacuated to a military hospital in ghana, where molecular diagnostic methods were used to determine the causative agent. two of the three cases were fatal. research method and design ethical considerations verbal consent was sought from the close relatives of the patients. the officers were also spoken to personally by the medical team at the 37 military hospital. setting and patients the index case was a male soldier with severe symptoms of malaria who died on may 11, after admission to a level ii hospital for about a week. two male colleagues, aged 27 years (patient 1) and 33 years (patient 2), who were living in the same camp, subsequently developed severe fever and myalgia on may 15 (patient 1) and 21 (patient 2). their medical conditions deteriorated, necessitating their medical evacuation to the 37 military hospital, a level iv facility, in accra, ghana, on may 26, 2013 after five days of illness for patient 1 and 11 days for patient 2. patient 1 died two days after admission. at the military hospital in accra, the medical staff ruled out malaria through microscopic examination of thick and thin blood smears and focused on the clinical manifestations, which were classified as vhf. a presumptive clinical diagnosis of lassa fever was made on the strength of previous outbreaks of lassa fever in liberia.10 to investigate these clinical suspicions, blood samples from the patients and five asymptomatic close contacts were analysed. laboratory investigations five millilitres of blood were collected from each of the two patients (patient 1 sample number: 60fsd_28052013; patient 2 sample number: 59_pn_28052013) and five asymptomatic contacts (medical staff who had initially attended the two patients without barrier nursing). the samples were transported to the virology department of the noguchi memorial institute for medical research at the university of ghana in legon, ghana. the blood samples were processed into serum by low-speed centrifugation at 4 °c and the resultant 3 ml aliquots of sera were cryopreserved at -80 °c. real-time reverse transcription-pcr pan filoviridae assay for marburg and ebola viral rna was extracted from 140 µl of the blood samples using the qiaamp viral rna mini kit (qiagen, hilden, germany) according to the manufacturer’s instructions. a diagnostic assay developed by panning et al.,11 for filovirus species and carried out with a onestep rt-pcr reaction kit (qiagen, hilden, germany) was used in a 25 µl total reaction volume, including 3 µl of the extracted rna. the real-time diagnostic assay used five optimised large (l)-gene primers and three probes, as well as an internal control with a separate detection probe. reactions were supplemented with 40 ng/ml bovine serum albumin and 400 mmol/l each dntp. the primers and probes used were designed and published by panning et al.11 (table 1). all probes were synthesized by tib-molbiol (berlin, germany). amplification in a 96-well abi 7300 real time pcr system (life technology holdings, singapore) involved the following steps: 50 °c for 30 minutes; 95 °c for 15 minutes; and 45 cycles of denaturation at 95 °c for 15 seconds, annealing at 52 °c for 25 seconds and extension at 72 °c for 20 seconds. fluorescence was measured at the end of each 52 °c annealing step. table 1: reverse transcription-pcr test assays used in the laboratory investigations, ghana, 2013. gel-based pan flaviviridae assay for yellow fever and dengue fever an endpoint reverse transcription (rt)-pcr protocol for the detection and identification of flaviviruses, developed by pierre, drouet and deubel in 199412 with a set of universal oligonucleotide primers, was used for laboratory investigation of flaviviruses present in the serum samples. these primers correspond to the 3’ non-coding region of the ns5 gene, which is highly conserved amongst the mosquito-borne flaviviruses. the onestep rt-pcr kit (qiagen, hilden, germany) was used with the following cycling conditions: reverse transcription at 50 °c for 30 minutes; initial activation step at 95 °c for 15 minutes; 45 cycles in a 3-step cycling of 95 °c for 30 seconds, 52 °c for 30 seconds and 72 °c for 30 seconds, with a cooling step of 30 seconds at 30 °c. gel-based lassa fever s-gene segment assay a gel-based conventional rt-pcr assay was performed using abi 2720 thermal cycler (life technology holdings, singapore) for the detection of lassa virus with primers specific for regions of the s rna segment. the rt-pcr (45 cycles) contained onestep rt-pcr kit reagents (qiagen, hilden, germany) with the sense primer 36e2 and the antisense primer lvs-339-rev, as described by ölschläger et al.13 (table 1). cycling conditions for the rt-pcr involved the following steps: 50 °c for 30 minutes; 95 °c for 15 minutes; and 45 cycles of 95 °c for 15 seconds, 52 °c for 30 seconds and 72 °c for 30 seconds. the amplification products (expected size: 320 bp) were electrophoresed on a 2% agarose gel (peqlab biotechnologie, erlangen, germany), stained with ethidium bromide and viewed under a kodak gel logic 100 imaging system (cole-parmer int., chicago, illinois, united states). lassa virus l-gene segment amplification by rt-pcr an rt-pcr assay specific to the l-gene segment of the lassa arenavirus on the rna extracted from the processed blood samples. the 45 cycle rt-pcr used a agpath-id one-step rt-pcr kit (ambion, life technologies, thermo fisher scientific, new york, new york, united states) with the primers as described by vieth et al.14 (table 1). the reaction solution consisted of: 2.5 μl nuclease-free h2o, 12 μl 2x rt-pcr buffer, 2 μl of the upstream and downstream primers (10 μm each), 1.5 μl 2x rt-pcr buffer enzyme mix and 5 µl dna. nucleic acid amplification started with a 30 minute rt step at 50 °c, then an initial denaturation step of 2 minutes at 95 °c and a 45-cycle amplification of 20 seconds at 98 °c, 30 seconds at 55 °c and 60 seconds at 72 °c. a gel was used to separate the products of the nucleic acid amplification. images of the dna bands were captured using a gel logic 100 imaging system (eastman kodak company, rochester, new york, united states). the dna bands of nearly 400 bp in size were analysed and purified for sequencing. real-time rt-pcr assay for lassa fever a real-time rt-pcr assay was performed using the abi 7300/7500 real time pcr system (life technology holdings, singapore) for the detection of lassa virus with primers specific for regions of the small (s) rna gene segment. the reagent used in the preparation of the master mix for the 45-cycle rt-pcr was the power sybr® green rna-to-ct™ 1-step kit (life technologies, carlsbad, california, united states) with the sense primer 36e2 and the antisense primer lvs-339-rev, as described in detail by ölschläger et al.13 (table 1). the amplification protocol was as follows: 48 °c for 30 minutes; 95 °c for 10 minutes; and 45 cycles of 95 °c for 15 seconds, 60 °c for 1 minute. at the end of each cycle, fluorescence was measured at 60 °c. sequencing and phylogenetic analysis the pcr products generated in the conventional rt-pcrs from the l-gene segment fragments were purified in accordance with the manufacturer’s instructions using a commercial kit (big dye xterminator purification kit, applied biosystems, carlsbad, california, united states). the purified products were sequenced on both strands using the l-gene pcr primers and an abi prism 3130 genetic analyser (hitachi high technology, singapore). the sequences were assembled with lasergene software (dnastar, thermo fisher scientific, new york, new york, united states) and automated base calling was proofread by visual inspection of the electropherograms. other published lassa virus sequences were obtained from the national center for biotechnology information’s15,16 genbank (table 2) to align with the generated sequences for phylogenetic analysis. with the use of the national library of medicine’s basic local alignment search tool17 (blast) program, we compared the patients’ lassa virus sequences with other reference lassa virus sequences to locate regions that were similar. all sequences obtained were copied into bioedit software (north carolina state university, raleigh, north carolina, united states) and put together into an ideal sequence alignment file with the use of a multiple sequence alignment tool, clustalw18 (version 2; embl-ebi, cambridge, united kingdom). we then used molecular evolutionary genetics analysis (mega) software (version 5.05) in a kimura two-parameter model19 to infer a phylogenetic tree from the aligned nucleotide sequences following a neighbour-joining method. to establish reliability and to infer the strength of similarity between the patient’s sequence and the referenced sequences from the phylogenetic tree, a bootstrap analysis of 1000 replicates was used. table 2: lassa fever viral strains used for homology and phylogenetic analysis of ghana case strains, 2013. results detection and characterisation of nucleic acid in clinical specimens the gel-based rt-pcr flavivirus tests for yellow fever and dengue fever (types 1–4), as well as the real-time rt-pcr filovirus test for marburg and ebola, were negative (table 3). however, the rt-pcr amplification of the s-gene segment of arenaviruses detected a 320 bp dna band in the well with the patients’ clinical specimens. the length and position of these bands were on a par with the positive lassa virus control (an inactivated culture supernatant of cells infected with lassa virus strain csf); no band was observed in the negative control (pcr-grade water) well. the real-time rt-pcr amplification of the s-gene segment of the arenavirus test produced a clear peak with a sigmoid-shaped curve for the patient’s samples, whereas no peak was observed for the negative control, which in the figure is covered by the threshold line, or baseline (figure 1). this two-result signal indicated the presence of lassa virus in the patients’ sera. the samples from the five asymptomatic close contacts were tested in the same assay run and no indication of the presence of lassa virus was observed (data not shown). figure 1: real-time pcr amplification of patient sera and controls. table 3: summary of suspected patient information and their test results, ghana, 2013. lassa virus l-gene segment sequencing and homology analysis the gel-based rt-pcr amplification assay for the large l-gene segment of the arenavirus showed ~400 bp products that matched the expected size (figure 2). the comparison of the nucleotide sequences from the amplified products with the genbank database showed that the patients’ sequences had high similarities to known lassa virus strains. genetically, the sequences were close to strains that had originated in guinea and been reported in liberia. the highest similarity (85% maximum identity in nucleotides) was to lassa virus strain macenta20 and lassa virus strain las803792.14 figure 2: ethidium bromide-stained 2% agarose gel image of the pcr products generated from the two patients samples run in duplicate. phylogenetic tree analysis the phylogenetic analysis with genbank data revealed that the nucleotide sequences from the patients had close phylogenetic relationships with the reported lassa virus strains macenta20 (genbank accession number: ay628200) and las80379214 (genbank accession number: ay693638) (figure 3). the sequence data from the patient who died (patient 1, sample no. 60fsd_28052013) was sent to genbank and assigned accession number kf425246. figure 3: phylogenetic relationship between the nucleotide sequences of reported lassa virus strains and that of the two patients. discussion we identified the aetiological agent responsible for suspected cases of vhf imported into ghana. the patients were soldiers with the ghanaian united nations mission in liberia military contingent in zorzor, liberia, who had had close contact earlier with another soldier suspected of dying from vhf. these patients were medically evacuated, with deteriorating health conditions, to a level iv military hospital in ghana, where viral nucleic acid was detected and characterised from the patients’ blood specimens. the characteristic arenavirus signal indicated on both the gel-based and real time rt-pcrs was confirmed as lassa virus by capillary dna sequencing. these results confirmed lassa virus as the aetiological agent that caused the outbreak in liberia, where lassa fever is known to be endemic.21 it has been documented that lassa fever seems to have two geographically-separate endemic areas: the mano river region in the west (guinea, sierra leone and liberia) and nigeria in the east.22 moreover, literature indicates that, since the initial discovery of lassa fever in nigeria in 1969, nosocomial outbreaks have occurred repeatedly in three localities, specifically zorzor, phebe and ganta in liberia.23 molecular analyses of patients’ samples from the outbreak we report are indicative of clues regarding the source of the aetiological agent. the nucleotide sequences from the samples aligned closely with the lassa fever strain isolated in guinea in 200424 and also reported subsequently in liberia.21 the 85% proportion of alignment between genbank-reported strains and sequences from the patients’ sera suggests a link between the source of the patients’ lassa virus infection and the district of zorzor in liberia from which they were evacuated. in addition, this finding supports the assertion that persons participating in humanitarian missions or peacekeeping activities in the regions comprising sierra leone and liberia are at risk for lassa fever.23,24,25 phylogenetic analysis of our patients’ sequences with genbank-reported lassa virus strains yielded a single parsimonious tree rooted with the prototype lp strain of lassa virus. our results indicate that our patients’ samples had close genetic relationships to the macenta20 and las80379214 strains of lassa virus, both of which were reported in liberia but isolated in guinea in 2004. the sequence data from the patient who died (patient 1; sample no. 60fsd_28052013) had close homology with reported lassa strain las803792, which was isolated from a fatal case in 2004.14 this observation is consistent with a study that suggested that lassa virus strains differ in virulence potential.1 limitations our report was limited by our inability to conduct a battery of tests, including serological assays, for either an ideal suspected case(s) of vhf or for the five asymptomatic close contacts of the evacuated patients. that notwithstanding, this report underscores the importance of preventive measures for all visitors and workers including peacekeeping forces to endemic regions in west africa. this is because there is currently no effective lassa fever vaccine is available. recommendations it is recommended that medical support plans for peacekeeping operations should be built purposefully and in consideration of existing endemicity and history of endemicity in the host nations. such support plans should be duly informed by frequently-updated research on endemic agents and other health concerns in the visiting country. sensitisation of officers and other travellers to infectious agents in the host country and the importance of speedy reporting to health facilities when unwell should be given sufficient emphasis. this report of laboratory investigations of imported cases of lassa fever and other documented medical fatalities on past peacekeeping operations supports the need for essential medical organisational changes in future operations. this would involve a good balance of proximity to medical care and transportation time for medical emergencies. conclusion in conclusion, it is envisaged that the importation of vhfs into non-endemic countries will increase in likelihood as a result of increased travel and international commitments in conflict and disaster situations to vhf-endemic countries in west africa. thus, healthcare providers should: (1) have a high index (low threshold) of suspicion for vhf amongst travellers returning from endemic areas; (2) promptly implement appropriate infection prevention and control measures; and (3) rapidly report suspected cases to avert undue nosocomial transmission. acknowledgements we are most grateful to the medical and para-medical staff of the public health department of the 37 military hospital and the staff of virology department of the noguchi memorial institute for medical research, college of health sciences, university of ghana, legon, particularly acknowledging the technical support by juliana naa dedei aryeequaye. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. sources of support this work was supported by the noguchi memorial institute for medical research, university of ghana, legon. authors’ contributions j.h.k.b. performed the laboratory testing, analysed and interpreted the results and wrote the manuscript. e.o.n. coordinated sample collection and surveillance and edited the manuscript. s.-a.o. coordinated surveillance and edited the manuscript. j.a. supervised surveillance and edited the manuscript. r.k.a. was involved in surveillance and sample collection. s.c.n.-p. participated in sample testing and analysis and edited the manuscript. b.s. took part in the surveillance and edited the manuscript. p.a. participated in sample collection and surveillance. m.a. edited the manuscript and was involved in surveillance. n.n.a.k. was involved in surveillance and sample collection. s.b.-n. was involved in surveillance and edited the manuscript. w.k.a. supervised the design and implementation of the work and edited the manuscript. references günther s, lenz o. lassa virus. crit rev clin lab sci. 2004; 41(4):339–390. http://dx.doi.org/10.1080/10408360490497456 mccormick jb. epidemiology and control of lassa fever. curr top microbiol immunol. 1987;134:69–78. http://dx.doi.org/10.1007/978-3-642-71726-0_3 sogoba n, feldmann h, safronetz d. lassa fever in west africa: evidence for an expanded region of endemicity. zoonoses public health. 2012;59(suppl 2):43–47. http://dx.doi.org/10.1111/j.1863-2378.2012.01469.x dzotsi ek, ohene sa, asiedu-bekoe f, et al. the first cases of lassa fever infection in ghana. ghana med j. 2012;46(3):166–170. unit for surveillance and communication, unit for 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ii. serological and virological findings in hospital patients. trans r soc trop med hyg. 1984;78(5):656–660. http://dx.doi.org/10.1016/0035-9203(84)90232-3 panning m, laue t, ölschlager s, et al. diagnostic reverse-transcription polymerase chain reaction kit for filoviruses based on the strain collections of all european biosafety level 4 laboratories. j infect dis. 2007;196(suppl 2):s199–204. http://dx.doi.org/10.1086/520600 pierre v, drouet mt, deubel v. identification of mosquito-borne flavivirus sequences using universal primers and reverse transcription/polymerase chain reaction. res virol. 1994;145(2):93–104. http://dx.doi.org/10.1016/s0923-2516(07)80011-2 ölschläger s, lelke m, emmerich p, et al. improved detection of lassa virus by reverse transcription-pcr targeting the 5’ region of s rna. j clin microbiol. 2010;48(6):2009–2013. http://dx.doi.org/10.1128/jcm.02351-09 vieth s, drosten c, lenz o, et al. rt-pcr assay for detection of lassa virus and related old world arenaviruses 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a, chain psg, do lh, et al. lassa virus strain macenta segment l, complete sequence. accession number: ay628200; 2004. jahrling pb, frame jd, smith sb, et al. endemic lassa fever in liberia. iii. characterization of lassa virus isolates. trans r soc trop med hyg. 1985;79(3):374–379. http://dx.doi.org/10.1016/0035-9203(85)90386-4 fichet-calvet e, rogers dj. risk maps of lassa fever in west africa. plos negl trop dis. 2009;3(3):e388. http://dx.doi.org/10.1371/journal.pntd.0000388 ter meulen j, koulemou k, wittekindt t, et al. detection of lassa virus antinucleoprotein immunoglobulin g (igg) and igm antibodies by a simple recombinant immunoblot assay for field use. j clin microbiol. 1998;36(11):3143–3148. fair j, jentes e, inapogui a, et al. lassa virus-infected rodents in refugee camps in guinea: a looming threat to public health in a politically unstable region. vector borne zoonotic dis. 2007;7(2):167–171. http://dx.doi.org/10.1089/vbz.2006.0581 khan sh, goba a, chu m, et al. new opportunities for field research on the pathogenesis and treatment of lassa fever. antiviral res. 2008;78(1):103–115. http://dx.doi.org/10.1016/j.antiviral.2007.11.003 conclusion references about the author(s) adetoun ejilemele department of pathology and immunology, baylor college of medicine, texas children’s hospital, houston, texas, united states citation ejilemele a. point-of-care testing and environmental sustainability: balancing patient care and planetary health. afr j lab med. 2025;14(1), a3015. https://doi.org/10.4102/ajlm.v14i1.3015 editorial point-of-care testing and environmental sustainability: balancing patient care and planetary health adetoun ejilemele copyright: © 2025. the author. licensee: aosis. this work is licensed under the creative commons attribution 4.0 international (cc by 4.0) license (https://creativecommons.org/licenses/by/4.0/). point-of-care testing (poct) is testing performed at the site of patient care in a variety of settings, by clinical staff who are generally not laboratory personnel, to allow healthcare providers to make prompt clinical decisions. in lowand middle-income countries (lmics), access to laboratory service can be challenging for various reasons.1,2,3 to improve access to tests, the world health organization recommends implementation of the model list of essential in vitro diagnostics, where the majority of the tests recommended for community hospitals and health facilities without laboratories (designated as tier i) are rapid diagnostic tests or utilise dipstick technology,4 typically performed at the point of care. the goal of the list of essential in vitro diagnostics is to guide countries in prioritising in vitro diagnostics that can be made available at various levels of their healthcare systems, strengthening laboratory capacity, while promoting rational use of tests.4 it is also expected that the list of essential in vitro diagnostics will guide donor nations and manufacturers to focus on globally important health issues.4 implementation of the list of essential in vitro diagnostics is expected to improve access to laboratory testing, reduce reliance on empirical treatment and enhance earlier diagnosis and treatment. most rapid diagnostic tests (rdt) designated for tier 1 settings will be single-use devices and those for noncommunicable disorders will most likely be performed using small analysers with single-use cartridges and other disposable consumables. consequently, it is expected that the use of poct and consumables will increase. for example, 3.9 billion rapid diagnostic tests were delivered between 2010 and 2022 to support malaria diagnosis, and more than 82% of these tests were to sub-saharan african countries.5 despite the benefits and hope that poct offers, it also has negative environmental consequences, owing to the need to maintain a good supply chain reliant on the use of fossil fuels. there is also the predicted need to dispose of single-use plastic devices with limited recyclability that will be processed as biohazardous waste.6 for various reasons, waste produced in healthcare facilities continues to increase yearly by 2% – 3%.7 while 75% – 90% of healthcare waste is non-hazardous, as much as 10% – 25% is considered hazardous.6 in lmics, waste segregation is often limited, and healthcare waste is frequently disposed of entirely as hazardous waste into open dumpsites, usually at suboptimal temperatures, prior to incineration.6 point-of-care test devices typically have high plastic and metal components and contribute to landfills in lmics. about 16 000 tonnes of plastic are produced globally for rapid testing every year,8 and deterioration of plastic in landfills into microand nano plastics is known to contribute over 16 000 chemicals to the environment, of which more than 4200 are known to have adverse long-term effects on human health.9 burning rapid diagnostic tests at suboptimal temperatures contributes to environmental contamination owing to the production of hydrocarbons, dioxins and furans, known carcinogens, which can contaminate the soil.6 noxious gases, vapours, and particulate matter that are released into the atmosphere from burning cause outdoor air pollution – the second-highest risk factor for non-communicable diseases, estimated to have caused more than 4.2 million premature deaths worldwide in 2019, with 89% of those deaths occurring in lmics.10 batteries used in powering poct devices, which contain heavy metals such as mercury and cadmium, are disposed of in open burn sites, which will lead to contamination of soil and groundwater by the residual ash. heavy metal toxicity is associated with damage to the immune system, neurological system, lungs and kidneys.6,11 chaotropic agents made from cyanide derivatives, such as guanidinium thiocyanate, are common components for nucleic extraction in poct devices used for infectious disease testing. they can contaminate water sources and poison aquatic life.12 it is therefore clear that the use of poct in lmics, without adequate planning about how the generated waste will be managed, will most likely worsen the health status of the communities where these devices are used. steps to mitigate the environmental impact should start with the selection of the poct devices to be used. when possible, devices with sustainability certification should be selected because these will probably have less environmental impact. at a global level, regulations and guidelines should be formulated and implemented to ensure that poct devices meet minimum sustainability standards. local manufacturing of devices will become important to reduce the environmental impact of supply chain and, in lmics, will require support through the implementation of favourable government policies. lowand middle-income countries must develop and implement waste management strategies that will reduce environmental contamination. the world health organization document on waste management6 proposes minimum guidelines on segregation, collection, storage, transportation and disposal of healthcare waste, which could be followed where national policies do not exist. development of policies suitable for the local context should be encouraged. at a global level, the target product profiles provided by the world health organization and other bodies should include language on waste management requirements for poct devices. training healthcare staff in the proper handling of healthcare waste will also be an important component of the process to enhance the level of understanding about how healthcare waste should be handled, as well as its effect on health when not managed properly.6,11 avoiding unnecessary testing will reduce the environmental impact of poct by reducing waste production. many poct devices are made from plastic materials derived from non-renewable sources. reducing the environmental impact of such devices will require vendors to consider and implement the use of more sustainable materials during the design phase, such as the pregnancy test designed by lia diagnostics, based on material derived from 100% cellulose structure, is flushable.13 regulations may become necessary to limit the plastic content of poct devices.8 the use of non-chaotropic agents, such as sodium dodecyl sulfate, for nucleic acid extraction, has been investigated and its use in the manufacture of poct devices should be encouraged. take-back schemes for broken equipment should be expanded to lmics to reduce the production of greenhouse gases from the improper disposal of such items. conclusion while expanding poct will be important in providing much-needed laboratory testing coverage, health systems in lmics must consider the environmental effects of these devices and implement policies that will minimise its impact. this will include selecting tests that meet sustainability standards, using local manufacturers where feasible, and localised waste management plans. these steps will be critical in ensuring that enhancing diagnostic capability does not cause adverse environmental effects that will further worsen the health of the communities that are served. references nkengasong jn, nsubuga p, nwanyanwu o, et al. laboratory systems and services are critical in global health: time to end the neglect? am j clin pathol. 2010;134(3):368–373. https://doi.org/10.1309/ajcpmpsinq9brmu6 katoba j, kuupiel d, mashamba-thompson tp. toward improving accessibility of point-of-care diagnostic services for maternal and child health in lowand middle-income countries. point care. 2019;18(1):17–25. https://doi.org/10.1097/poc.0000000000000180 wilson ml, fleming ka, kuti ma, looi lm, lago n, ru k. access to pathology and laboratory medicine services: a crucial gap. lancet. 2018;391(10133):1927–1938. https://doi.org/10.1016/s0140-6736(18)30458-6 world health organization. the selection and use of essential in vitro diagnostics: report of the fourth meeting of the who strategic advisory group of experts on in vitro diagnostics, 2022 (including the fourth who model list of essential in vitro diagnostics). geneva: world health organization; 2023. contract no. 1053. world health organization. who malaria policy advisory group (mpag) report, 18–20 april 2023. geneva: world health organization. world health organization. safe management of wastes from health-care activities [homepage on the internet]. 2nd ed. geneva: world health organization; 2014 [cited 2025 sep 24], p. 329. available from: https://www.who.int/publications/i/item/9789241548564 kaposi a, nagy a, gomori g, kocsis d. analysis of healthcare waste and factors affecting the amount of hazardous healthcare waste in a university hospital. j mater cycles waste manag. 2024;26(2):1169–1180. https://doi.org/10.1007/s10163-024-01890-1 university of edinburgh. experts urge plastic limit for lateral flow tests [homepage on the internet]. 2025 [cited 2025 sep 10]. available from: https://www.ed.ac.uk/news/experts-urge-plastic-limit-for-lateral-flow-tests-0#:~:text=the%20researchers%20urge%20setting%20plastic,range%20of%20illnesses%20and%20conditions wagner ml, monclús l, arp hph, et al. state of the science on plastic chemicals – identifying and addressing chemicals and polymers of concern [homepage on the internet]. 2024 [cited 2025 sep 10]. available from: https://zenodo.org/records/10701706 world health organization. ambient (outdoor) air pollution. who fact sheet [homepage on the internet]. 2024 [updated 2024 oct 24; cited 2025 jun 30]. available from: https://www.who.int/news-room/fact-sheets/detail/ambient-(outdoor)-air-quality-and-health janik-karpinska e, brancaleoni r, niemcewicz m, et al. healthcare waste—a serious problem for global health. healthcare (basel). 2023;11(2):242. https://doi.org/10.3390/healthcare11020242 ongaro ae, ndlovu z, sollier e, et al. engineering a sustainable future for point-of-care diagnostics and single-use microfluidic devices. lab chip. 2022;22(17):3122–3137. https://doi.org/10.1039/d2lc00380e gross p. lia diagnostics has launched its eco-friendly, flushable pregnancy test to consumers [homepage on the internet]. tech.ly; 2021 [cited 2021 mar 15]. available from: https://technical.ly/startups/lia-diagnostics-launched-flushable-pregnancy-test/ article information authors: katy yao1 elizabeth t. luman1 slmta collaborating authors2 affiliations: 1international laboratory branch, division of global hiv/aids, us centers for disease control and prevention (cdc), atlanta, georgia 2see appendix: author information correspondence to: katy yao postal address: 1600 clifton rd, ms g45, atlanta, ga 30329-4018, united states dates: received: 15 sept. 2014 accepted: 15 sept. 2014 published: 03 nov. 2014 how to cite this article: yao k, luman et, slmta collaborating authors. evidence from 617 laboratories in 47 countries for slmta-driven improvement in quality management systems. afr j lab med. 2014;3(2), art. #262, 11 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.262 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. evidence from 617 laboratories in 47 countries for slmta-driven improvement in quality management systems in this original research... open access • abstract • introduction • research methods and design    • slmta implementation and evaluation    • data analysis • results    • programme spread    • audit results    • sustainability    • slmta implications for laboratory testing • discussion    • limitations of the study • conclusion • acknowledgements    • competing interests    • authors’ contributions    • cdc disclaimer • references • appendix abstract top ↑ background: the strengthening laboratory management toward accreditation (slmta) programme is a large-scale effort to improve the quality of laboratories in resource-limited countries. objectives: this study sought to evaluate the first four years (2010–2013) of slmta implementation. methods: country-level data were submitted by slmta programme leads and compiled globally. performance was measured before (baseline) and after (exit) slmta implementation using an audit checklist which results in a percentage score and a rating of zero to five stars. some laboratories continued to monitor performance in post-exit surveillance audits. we evaluated score improvements using two-tailed t-tests for equal variances and estimated the number of tests performed by slmta laboratories based on star level. results: slmta was implemented in 617 laboratories in 47 countries in africa, the caribbean, latin america and southeast asia. at the baseline audit, the laboratories scored an average of 39% on the checklist and 84% of them were rated below one star. as of december 2013, 302 laboratories had completed the slmta programme; mean checklist scores increased from 39% at baseline to 64% at exit (p < 0.001) over an average 16-month programme duration. ninety-two laboratories conducted a surveillance audit at a median of 11 months after their exit audit; 62% further increased their performance. six slmta laboratories have achieved accreditation status. in total, the 617 slmta laboratories conduct an estimated 111 million tests annually. only 16% of these tests were conducted by laboratories with at least one star at baseline, which increased to 68% of tests after slmta training. thus, approximately 23 million tests are conducted annually by laboratories previously at zero stars that now have one to five stars; this number is projected to increase to 58 million when currently-enrolled laboratories complete the programme. conclusion: slmta has transformed the laboratory landscape in resource-limited countries worldwide and has the potential to make a substantial and sustainable impact on the quality of laboratory testing and patient care. introduction top ↑ substantial resources have been invested in management training and development to help scale up and improve the quality of health services in order to reach the united nation’s millennium development goals.1 however, evidence is scarce in terms of the impact that has actually been achieved. an international meeting convened by the world health organization (who) in 2005 on strengthening leadership and management in low-income countries concluded that programme evaluation often focuses on the number of trainees and pre-/post-knowledge-based tests; evaluation of programme impact on managers’ daily work and job outcomes is more the exception than the norm.2 strengthening laboratory management toward accreditation (slmta) is a large-scale effort aimed at improving the quality of laboratory services and patient care in resource-limited settings by developing competent laboratory managers. slmta provides an innovative training curriculum on implementing practical quality management systems (qms) using existing resources with built-in accountability and evaluation that focuses on result-oriented outcome measures.3 launched in 2009 in kigali, rwanda, this programme seeks to engage laboratories in continuous quality improvement and to accelerate their preparations toward accreditation to international standards.4,5 in this article, we present evidence from the first four years of slmta programme implementation (2010–2013). we report data on changes in levels of laboratory compliance with international organization for standardization (iso) 15189 requirements using the who regional office for africa’s (who afro) accreditation preparedness checklist. the findings from this unprecedented study shed light on the widespread success of an innovative programme that has empowered laboratory teams throughout the developing world to strive for continuous quality improvement and work toward accreditation, despite limited resources. research methods and design top ↑ slmta implementation and evaluation the methodology of the slmta programme has been described previously.3,5 briefly, slmta is a competency-based programme that uses a series of short courses and work-based learning projects to effect rapid and measurable laboratory improvement for better patient care through enhanced management skills and implementation of practical quality management systems. the slmta training programme is based on a series of three workshops. after each workshop, participants implement improvement projects supported by regular supervisory visits or on-site mentoring. laboratories are evaluated using who afro’s stepwise laboratory quality improvement process towards accreditation (slipta) checklist,6 which includes 111 items divided into 12 sections that represent the 12 quality system essentials (qses).7 in order to assess progress made by the laboratories, audits are conducted using the slipta checklist at the beginning (baseline) and at the end (exit) of the slmta programme. many countries also conduct intermediate audits to help guide programme implementation, as well as surveillance audits after exiting the programme so as to monitor continued improvement and assess sustainability. after an audit, laboratories receive a score which determines their star rating – from zero to five, with < 55% corresponding to zero stars, 55% – 64% one star, 65% – 74% two stars, 75% – 84% three stars, 85% – 94% four stars and ≥ 95% five stars. when a laboratory achieves a five-star rating, it may be encouraged to seek accreditation. data analysis programme data up to december 2013 were collected from all countries implementing slmta. variables included year of implementation, number and types of laboratories in each cohort (i.e., enrolment in the same training round), number of people trained, audit scores and approximate number of tests conducted by each laboratory. data were collated and analysed in microsoft® excel 2013. descriptive statistics (percentages, medians, ranges) were calculated. statistical significance of improvements were assessed using 2-tailed t-tests for equal variances (f-tests showed equal variances at p > 0.1); comparison of improvements across laboratory types was assessed using one-way analysis of variance (anova). given the wide variations in the time between the baseline audit and the first slmta workshop, programme length was defined as the time from the first slmta workshop to the exit audit. in some countries, results for large national reference laboratories were reported by department rather than for the laboratory as a whole. for consistency, we aggregated the department scores into a single score per laboratory, using median values across laboratory departments. we estimated the number of laboratory tests conducted in slmta laboratories based on country reports for 2012; missing data were imputed using averages by laboratory type. results top ↑ programme spread since its introduction in 2009, slmta has been implemented in 47 countries worldwide, including 23 countries in africa, 12 in the caribbean region, 10 in central and south america, and two in southeast asia (figure 1, table 1). figure 1: global reach of slmta by year of implementation, 20102013 (n = 47 countries). table 1: strengthening laboratory management toward accreditation: training and results, 2010-2013. table 1 (contnues...): strengthening laboratory management toward accreditation: training and results, 2010-2013. table 1 (contnues...): strengthening laboratory management toward accreditation: training and results, 2010-2013. as of december 2013, 65 slmta cohorts had been initiated, with one to 55 laboratories per cohort. thirty were first cohorts within countries, including three regional cohorts which encompassed multiple countries, whilst the remaining 35 were subsequent cohorts in 23 countries as they expanded the programme (table 1). a total of 1923 people from 617 laboratories were trained. thirty-seven of these laboratories (6%) were re-enrolled in a subsequent slmta cohort. eighteen per cent of the 617 laboratories were at the national level, 27% at regional or provincial levels, 38% at district or primary levels, 10% belonged to non-governmental, faith-based or private organisations, 5% were military laboratories and 2% were blood banks. nearly all (98%) of these laboratories provide hiv-related services, such as hiv diagnosis, treatment monitoring, opportunistic infection or tuberculosis testing, and blood bank testing. of these laboratories, 302 (49%) completed the slmta programme and conducted an exit audit, whilst the remaining 315 (51%) were still going through the programme at the time of this analysis. audit results at the baseline audit, the mean score for all 617 slmta-enrolled laboratories was 39% (median 37%) and 84% received zero stars (i.e., score < 55%) on the slipta five-star scale (table 1). for the 302 laboratories that had completed the programme and conducted an exit audit, mean scores increased from 39% at baseline to 64% at exit (p < 0.001). whilst 85% had received zero stars at baseline, only 30% remained at zero stars at exit (figure 2). figure 2: star levels at baseline and exit amongst the 302 laboratories completing the strengthening laboratory management toward accreditation programme, 2010–2013. results based on the stepwise laboratory quality improvement process towards accreditation checklist baseline and exit audit scores. the average improvement from baseline to exit audit was 25 percentage points over a mean programme duration of 16 months. sixty-eight per cent of laboratories improved by at least one star, including 22% that increased by three or more stars. although 32% of the laboratories did not increase their stars, 23% of these more than doubled their scores from baseline to exit. improvements tended to be higher for laboratories with lower baseline scores. those with a starting score below 25% had an average increase of 37 percentage points, whilst those with a starting score of 65% or higher had an average increase of nine percentage points (r = -0.49, p < 0.001) (table 2). table 2: improvement by baseline level based on the stepwise laboratory quality improvement process towards accreditation checklist. baseline and exit scores and improvements were similar between the different types of laboratories (p > 0.05) (figure 3). amongst laboratories that had completed the slmta programme, the 132 laboratories implementing slmta in the first year (2010) had the same mean improvement as the 170 laboratories implementing slmta in 2011 to 2013 (24 percentage points). figure 3: baseline and exit scores by laboratory type for the 302 laboratories completing the strengthening laboratory management toward accreditation programme 2010–2013 based on the stepwise laboratory quality improvement process towards accreditation checklist. sustainability ninety-two laboratories had conducted post-slmta surveillance audits, at a median of 11 months post-exit audit (range five to 28 months). of these laboratories, 62% further increased their score, including 34% whose score increased by > 10 percentage points post-exit audit (figure 4). of the national-level laboratories that conducted post-slmta audits (n = 19), 79% improved their scores, whilst only 56% of district-level laboratories (n = 27) further increased their scores (p = 0.03). overall, there was regression toward the mean, as laboratories that received lower scores at exit were more likely to improve their scores at the post-slmta audit (r = -0.48, p < 0.001). figure 4: post-slmta surveillance audits (n = 92 laboratories), based on the stepwise laboratory quality improvement process towards accreditation checklist. as of september 2014, six slmta laboratories have been accredited to international standards (table 3). these laboratories had baseline scores approximately equal to all slmta laboratories, with a median score of 38% (range 29% to 75%) as compared to a median of 37% for all slmta laboratories; one of the laboratories started at three slipta stars, whilst the other five laboratories began at zero stars. at the exit audit, the six laboratories had a median score of 90%, with an increase of 52 percentage points, as compared to the international average of 64% and 25 percentage points. the median time from slmta initiation to accreditation was 31.5 months (range 17–54). in march 2013, the kenya hiv national reference laboratory became the first slmta laboratory (and the first public medical laboratory in kenya) to earn accreditation.8 the hai duong preventive medicine center in vietnam had the lowest baseline score of the six laboratories at 29%, but achieved the greatest improvement from baseline to exit (58 percentage points); it took the laboratory only 19 months from initiation of slmta to accreditation. table 3: strengthening laboratory management toward accreditation laboratories that have achieved formal accreditation as of september 2014. slmta implications for laboratory testing in total, the 617 slmta laboratories conducted an estimated 111 million tests in 2012. approximately 43.5 million of these tests were conducted by the 302 laboratories that had completed the slmta programme by the time of this analysis. only 16% of these tests were conducted by laboratories with at least one quality star prior to slmta implementation (3% had ≥ 3 stars); after slmta training 68% of tests were performed by laboratories with at least one quality star (28% had ≥ 3 stars). this translates to approximately 23 million tests conducted by laboratories that previously had zero stars and now have one or more stars. furthermore, within the group with zero stars prior to slmta implementation, more than one in three tests were performed by laboratories scoring below 35% on the slipta checklist; after slmta completion this proportion decreased to one in 50 (figure 5). assuming similar quality improvements for the 315 currently-enrolled laboratories, we project the total number of tests conducted by laboratories with one or more stars that had previously been at zero stars to rise to 58 million by the end of 2015. figure 5: estimated proportion of 43.5 million tests performed by star level of laboratories before (a) and after (b) slmta implementation, based on the stepwise laboratory quality improvement process towards accreditation checklist scores for the 302 laboratories that completed the slmta programme. before slmta: 16% of tests were done in laboratories with at least one star. after slmta 68% of tests were done in laboratories with at least one star. discussion top ↑ since its launch in 2009, slmta has achieved wide global coverage and demonstrated measurable improvement using a standardised checklist, transforming the laboratory landscape in many resource-limited countries worldwide. the results have been remarkable, with audit scores increasing 25 percentage points, and two-thirds of the laboratories that completed the slmta programme improving by at least one quality star level. twenty-three countries have implemented a second cohort of training and 23 million tests conducted by laboratories previously lacking any quality management system are now being conducted by laboratories with at least a basic level of quality in place. the slmta programme has the potential to make a substantial and sustainable impact on the quality of laboratory testing and, therefore, patient care. as of august 2014, 102 laboratories from 15 countries in sub-saharan africa had received an official who afro slipta audit by the african society for laboratory medicine (aslm) (maruta t, 2014, personal communication, august 31); ninety-seven (95%) of these laboratories had gone through the slmta programme. it is evident that slmta has emerged as a vital strategy in accelerating laboratory preparedness for accreditation in the developing world and in facilitating the fulfillment of aslm’s 2020 vision, goal 2: ‘by 2020, enroll 2500 laboratories in the who slipta quality improvement programme and enable 250 laboratories to achieve accreditation by international standards’.9 to date, the slmta programme has been primarily funded by the us president’s emergency plan for aids relief (pepfar) through cdc and implementation has been limited primarily to pepfar-supported countries. although hiv-focused, these resources have been leveraged to build wide-spread capacity and strengthen the overall laboratory system.10 slowly, partners are joining ranks to help spread the programme. since 2011, the world bank has funded five countries (burundi, kenya, rwanda, tanzania and uganda) to implement slmta in 32 laboratories through its east africa public health laboratory network project. the southern african development community (sadc) will soon partner with the zimbabwe national quality assurance program in spreading the programme to all 15 member states, some of which are not supported by pepfar. this is the first study to examine the existing level of laboratory quality on a broad international scale. the mean baseline audit score of the 617 slmta-enrolled laboratories was 39%, well below the 55% required to attain even the lowest quality level on the slipta scale, and only 16% of the laboratories met or surpassed the one-star level. these results paint a bleak picture of the current quality of laboratories in much of the developing world. whilst the issue of the lack of quality amongst laboratories in developing countries has been acknowledged in numerous publications4,11,12,13,14,15,16,17 and a series of policy statements,18,19,20,21 the current study begins to quantify the problem and sheds some light on a potential solution. the slmta curriculum was designed to be closely aligned to iso 15189; however, it has proven to be suitable to support other standards as well. for example, a slmta-enrolled laboratory in vietnam that tests environmental and food samples was accredited to iso 17025. two per cent of slmta sites are blood banks or blood-transfusion centres, which use different standards and have their own accreditation programmes. in cameroon, inspired by the transformation achieved by its slmta-enrolled laboratory, a hospital adopted slmta in order to embark on its own quality improvement journey.22 the need for improved quality is so widespread that others have called for the expansion of slmta into a programme that helps guide quality improvement in entire hospital and public health systems.23 according to a may 2013 survey, only two government-owned medical laboratories in the 48 sub-saharan african countries outside of south africa were accredited.11 in 2013 to 2014, three more government-owned laboratories in the region (all located in kenya) were accredited after slmta implementation (table 3). in the same timeframe, two slmta laboratories outside of the sub-saharan region have achieved accreditation, whilst several others are awaiting accreditation assessments. these are only the first of what we anticipate will be many slmta laboratories reaching accreditation as the programme matures, since preparing for accreditation is a multi-year process. however, it is not feasible, either economically or programmatically, for all laboratories to pursue accreditation to international standards. as countries develop laboratory strategic plans, they will need to assess the options and develop realistic goals. the ministry of health in uganda, for example, has set a national target of three stars for general hospital laboratories and five stars for national and regional reference laboratories (lali w, 2014, personal communication, august 31). regardless of the ultimate goal, the slmta programme will provide the tools necessary to guide laboratories in the continuous quality improvement process designed to achieve better patient care. it is outside the scope of this study to examine how factors such as cohort size, programme length, laboratory type, mentorship model (amount, quality and type) and additional training impact the performance of the programme. however, detailed data are being collected as part of the slmta programme, including audit scores for each of the 12 qses and laboratory indicators such as turnaround time, specimen rejection rates, equipment downtime, proficiency testing, customer satisfaction and cost. these data are a potential gold mine of information that could be harnessed to identify causative factors of success and to fine-tune the programme with evidence-based strategies for continued improvement. an electronic-tool that will facilitate collection, management, aggregation, analysis and reporting of slmta programmatic data on a global level is currently under development by cdc and its partners. limitations of the study these study results should be interpreted in light of several limitations. firstly, half of the enrolled laboratories have not yet completed the programme and therefore have no exit audit data. as the programme matures, a higher proportion of the participating laboratories will have full data, reducing the risk of bias. similarly, whilst we found that most laboratories that conducted surveillance audits continued to improve over a time-span of anywhere from five to 28 months after the slmta programme ended, the programme is too young to assess long-term sustainability. another concern is the quality of the audits, especially in the initial years of the slmta programme before formal training and certification of auditors. whilst the slipta checklist was designed to help standardise audit scoring, some variability may remain; assessment of intraand inter-auditor variability is needed. this was an observational programmatic study. many factors were not controlled, such as programme duration, size of cohorts, mentorship model and additional training provided to enrolled laboratories. future studies that compare these factors, as well as results of slmta laboratories with non-slmta laboratories are needed in order to separate the impact of slmta implementation from natural improvement in laboratory quality over the duration of the programme. in addition, estimates of the number of laboratory tests were based on 353 (57%) laboratories that submitted information; missing data were imputed using average volume by laboratory type, but some uncertainty remains. ultimately, a thorough evaluation of the slmta programme will require assessment of programme impact on patient care and health outcomes. systematic evaluation of key indicators is needed, as well as targeted evaluations of programme cost-benefit, the impact of quality improvement on testing error rates and the association of quality services with patient outcomes, so as to determine public and personal health implications. conclusion top ↑ few management and leadership development programmes have been implemented on such a large scale with results-oriented outcome measures. with data collected from 617 laboratories in 47 countries throughout africa, the caribbean, latin america and southeast asia, slmta is truly a global effort. it has demonstrated its ability to transform the laboratory landscape in resource-limited countries worldwide. evidence from this study suggests that the slmta programme has the potential to make a substantial and sustainable impact on the quality of laboratory testing and patient care. acknowledgements top ↑ competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions k.y. (cdc, atlanta) managed the global slmta programme, collected the data and wrote the manuscript. e.t.l. (cdc, atlanta) analysed the data and provided substantial input to the manuscript. the collaborating authors provided the data from their respective countries and reviewed the manuscript. cdc disclaimer the findings and conclusions in this report are those of the authors and do not necessarily reflect the official position of the organisations represented by any of the authors, including the cdc, the department of the navy, the department of defense, or the us government. references top ↑ 1.united nations general assembly. united nations millennium declaration [document on the internet]. c2000 [cited 2014 oct 04]. available from: http://www.un.org/millennium/declaration/ares552e.htm 2.egger d, travis p, dovlo d, et al. strengthening management in low-income countries. making health systems work: working paper no. 1 who/hss/healthsystems/2005.1 [document on the internet]. c2005 [cited 2014 oct 04]. available from: http://www.who.int/management/working_paper_1_en_opt.pdf 3.yao k, maruta 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2012;138(5):639–641. http://dx.doi.org/10.1309/ajcp77jzijaguidr 15.nkengasong jn. a shifting paradigm in strengthening laboratory health systems for global health: acting now, acting collectively, but acting differently. am j clin pathol. 2010;134(3):359–360. http://dx.doi.org/10.1309/ajcpy5asuejyq5rk 16.nkengasong jn, nsubuga p, nwanyanwu o, et al. laboratory systems and services are critical in global health: time to end the neglect? am j clin pathol. 2010;134(3):368–373. http://dx.doi.org/10.1309/ajcpmpsinq9brmu6 17.olmsted s, moore m, meili r, et al. strengthening laboratory systems in resource-limited settings. am j clin pathol. 2010;134(3):374–380. http://dx.doi.org/10.1309/ajcpdqosb7qr5glr 18.world health organization regional office for africa. the maputo declaration on strengthening of laboratory systems: [document on the internet]. c2008 [cited 2014 jul 21]. available from: http://www.who.int/diagnostics_laboratory/maputo-declaration_2008.pdf 19.world health organization. joint who–cdc conference on health laboratory quality systems, lyon, france, 9–11 april 2008 [document on the internet]. c2008 [cited 2014 jul 21]. available from: http://www.who.int/csr/ihr/lyon/report20080409.pdf?ua=1 20.world health organization regional office for africa. who report calls for strengthening public health laboratories [page on the internet]. c2008 [cited 2014 jul 21]. available from: http://www.afro.who.int/en/media-centre/pressreleases/item/679-who-report-calls 21.african society for laboratory medicine. ministerial call for action – strengthening laboratory services in africa. presented at 2012 international conference, cape town, south africa [document on the internet]. c2012 [cited 2014 jul 21]. available from: http://www.aslm2012.org/docs/aslm2012%20ministerial%20call%20for%20action.pdf 22.eno lt, asong t, ngale e, et al. driving hospital transformation with slmta in a regional hospital in cameroon. afr j lab med. 2014;3(2), art. #221, 5 pages. http://dx.doi.org/10.4102/ajlm.v3i2.221 23.mataranyika mn, beukes lk. view from the top: involvement of namibia’s health ministry in laboratory quality improvement. afr j lab med. 2014;3(2), art. #195, 2 pages. http://dx.doi.org/10.4102/ajlm.v3i2.195 appendix top ↑ table: group authorship. abstract introduction methods results discussion acknowledgements references about the author(s) gemeda abebe school of medical laboratory sciences, faculty of health sciences, jimma university, jimma, ethiopia mycobacteriology research center, jimma university, jimma, ethiopia dossegnaw aragaw school of medical laboratory sciences, faculty of health sciences, jimma university, jimma, ethiopia mulualem tadesse school of medical laboratory sciences, faculty of health sciences, jimma university, jimma, ethiopia mycobacteriology research center, jimma university, jimma, ethiopia citation abebe g, aragaw d, tadesse m. fluorescence microscopy for the diagnosis of smear-negative pulmonary tuberculosis in ethiopia. afr j lab med. 2020;9(1), a810. https://doi.org/10.4102/ajlm.v9i1.810 original research fluorescence microscopy for the diagnosis of smear-negative pulmonary tuberculosis in ethiopia gemeda abebe, dossegnaw aragaw, mulualem tadesse received: 04 apr. 2018; accepted: 24 june 2020; published: 28 sept. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: despite its low sensitivity, microscopy remains the main method for the diagnosis of pulmonary tuberculosis in most laboratories in ethiopia. few studies have evaluated the performance of light-emitting diode fluorescent microscopy (led-fm) in bleach-concentrated smear-negative sputum specimens. objective: this study aimed to evaluate the diagnostic performance of led-fm for smear-negative pulmonary tuberculosis in ethiopia. methods: a total of 194 adult patients with a cough lasting for more than two weeks, and who had three direct smear-negative sputum tests for mycobacterium tuberculosis by ziehl-neelsen light microscopy, were included. all direct ziehl-neelsen-stained smear-negative sputum samples were cultured and were also visualised by led-fm. smears for led-fm were performed from bleach-concentrated sputum sediment. the diagnostic performance of the led-fm was compared to the culture method (the reference standard). results: of the 194 smear-negative sputum specimens analysed, 28 (14.4%) were culture-positive and 21 (10.8%) were led-fm-positive for m. tuberculosis. however, only 11 of the 21 (52.4%) led-fm-positive patients had a confirmed tuberculosis diagnosis by culture. light-emitting diode fluorescence microscopy (fm) had a sensitivity of 39.3% (95% confidence interval: 21.2–57.4) and specificity of 93.9% (95% confidence interval: 90.4–97.6). ten led-fm-positive specimens were culture-negative, and all of these specimens had scanty grading (1–19 bacilli per 40 fields on led-fm). conclusion: this study showed that implementation of led-fm on bleach pre-treated and concentrated sputum can significantly improve the diagnosis of smear-negative pulmonary tuberculosis. however, all scanty grade, positive smears by led-fm need to be confirmed by reference culture method. keywords: sputum smear-negative; light-emitting diode fluorescent microscopy; bleach pretreatment; ethiopia; health. introduction ethiopia is among the 30 highest-burden countries for tuberculosis, tuberculosis-hiv coinfection and multidrug-resistant tuberculosis in the world.1 according to the first national population-based tuberculosis survey report, the number of registered smear-negative tuberculosis cases exceeded that of smear-positive cases in 2010/11.2 this prevalence survey showed that smear-positive cases accounted for only 43% of culture-positive tuberculosis cases in ethiopia. although sputum smear-positive cases are more infectious than smear-negatives, studies have reported that 17% of tuberculosis transmissions were due to sputum smear-negative but culture-positive cases.3 moreover, smear-negative pulmonary tuberculosis cases are associated with longer health service delays due to delayed diagnosis. such delays in diagnosis may delay initiation of treatment, and further tuberculosis transmission may occur.4,5 a major challenge in tuberculosis control is the lack of an accurate, cost-effective, widely available point-of-care test. the cepheid xpert® mtb/rif assay (cepheid, sunnyvale, california, united states) is a molecular test with single-use cartridges on the genexpert instrument system. the world health organization (who) has recommended xpert mtb/rif as the initial diagnostic test in individuals with presumptive multidrug-resistant or hiv-associated tuberculosis.6 the who has also recommended that xpert mtb/rif may be used as a follow on test to smear microscopy, especially in further testing of smear-negative specimens. following the who recommendation, the ethiopian ministry of health has started a rollout of the xpert mtb/rif assay in selected laboratories.7 currently, few laboratories have access to xpert mtb/rif machines and smear microscopy remains the most widely used tuberculosis diagnostic tool in ethiopia.7,8 however, ziehl-neelsen (zn) smear microscopy was found to have low sensitivity, ranging from 20% to 50%.8,9,10,11 on the other hand, fluorescence microscopy (fm), both conventional and light-emitting diode (led) using auramine o, has showed increased detection of tuberculosis bacilli by 10%.12,13 however, conventional fm use has been limited by the short life span of mercury vapour light, the need for regular maintenance and the requirement for a dark room.14 as a result, in 2011, the who recommended the replacement of conventional fm by light-emitting diode fluorescent microscopy (led-fm) as an alternative for zn light microscopy.15 led-fm increases the sensitivity of smear microscopy because of the fact that slides can be examined at a lower magnification, allowing the examination of a much larger area per unit of time.12,13,16 this is particularly important in high tuberculosis burden and workload countries such as ethiopia, where technicians spend much of their time on examining smears. there is limited data on the diagnostic performance of led-fm in bleach-treated and concentrated sputum specimens, particularly paucibacillary samples. in this study, we aimed to evaluate the diagnostic performance of led-fm in bleach pre-treated and concentrated sputum specimens for the diagnosis of smear-negative pulmonary tuberculosis in ethiopia. methods ethical considerations this study was reviewed and approved by the institutional review board of jimma university (ref. no: rpgc/510/2014). consent was obtained from all participants for use of routine clinical data for research purposes. laboratory results were reported back to the clinicians for treatment initiation or decision as early as available. study setting and participants this cross-sectional study was conducted at the jimma university specialized hospital, a teaching and referral hospital, in southwest ethiopia from february 2014 to august 2014. adult patients (age > 18 years) presenting with a cough (≥ 2 weeks) and who had had three negative tests for mycobacterium tuberculosis by zn sputum direct smear microscopy, were included in the study. patients taking anti-tuberculosis treatment or prophylaxis in the four weeks before the screening were excluded. study procedures adult presumptive pulmonary tuberculosis cases were requested to submit sputum specimens. inclusion criteria was based on the who case definition for a pulmonary tuberculosis suspect17 and included patients having a persistent cough (≥ 2 weeks), with or without one of the following symptoms: night sweats, unintentional weight loss, fever, chest pain, shortness of breath, loss of appetite, and contact with a tuberculosis patient. as part of routine practice, all patients gave three sputum samples (spot-morning-spot). sputum samples were examined by zn sputum smear microscopy. smears were considered positive if acid-fast bacilli (afb) were seen on the smear from any of the three sputum samples. sputum smear-positive patients were treated for tuberculosis according to the national tuberculosis and leprosy programme treatment guidelines. for the current analysis, patients who had three smear-afb-negative sputum tests by zn microscopy were included in the study. demographic and clinical characteristics were collected through interview by use of a questionnaire. smear microscopy by ziehl-neelsen sputum smear examination by the zn staining technique is the standard method for the diagnosis of pulmonary tuberculosis in ethiopia.8 a portion of patient sputum specimen was used for direct smear microscopy using the zn method and the remaining portion was stored at 2 °c – 8 °c in a refrigerator for the culture and led-fm tests. for the zn method, a direct smear was prepared on the spot during specimen collection on a clean slide. then standard zn staining procedure was applied. the stained smears were examined for afb under the oil immersion objective lens in a light microscope (olympus cx31 light microscope; olympus, tokyo, japan). smear results were reported for the presence or absence of afb using the world health organization/international union against tuberculosis and lung disease scale, with a positive result corresponding to ≥ 1 afb per 100 high-power fields.18 bleach processing and concentration of sputum the morning sputum sample was divided into two equal parts. the first half was transferred to a 15 ml conical centrifuge tube (sarstedt ag & co. kg, nümbrecht, germany) and treated with an equal volume of 5% bleach (chora gas and chemical products factory, addis ababa, ethiopia). after mixing, the tubes were left for 15 minutes at room temperature with shaking for 30 seconds every 5 min. then phosphate buffered saline was added up to 15 ml and centrifuged at 3000 revolutions per minute for 15 min using a simple centrifuge. this is a small, compact, bench-top, non-refrigerated centrifuge that is easy and safe for operation and is available in the peripheral laboratories of ethiopia. after centrifugation, the supernatant was decanted, the sediment was re-suspended with 1 ml of phosphate buffered saline (ph = 6.8), then the smear was prepared for led-fm. light-emitting diode fluorescent microscopy smears were prepared on frosted slides and completely covered with auramine o solution (sigma-aldrich, machelen, belgium). after 20 min, the slides were washed and decolourised with 0.5% acid alcohol solution for 3 min and counter-stained with 0.5% potassium permanganate for 1 min. stained smears were examined under led-fm (primo star iled, carl zeiss, gottingen, germany) with 400x magnification and 40 fields were examined. led-fm results were reported for the presence or absence of afb using the world health organization/international union against tuberculosis and lung disease scale, with a positive result corresponding to ≥ 1 afb per 20x for screening and 40x for confirmation.19 sputum culture and identification culture was done both on lowenstein–jensen (lj) slants and mycobacteria growth indicator tubes (mgit). briefly, the second half of the morning sputum sample was decontaminated using the n-acetyl-l-cysteine and sodium hydroxide (nalc/naoh) method with a final naoh concentration of 1%.20 an equal volume of standard nalc/naoh solution was added to the specimen and incubated for 15 min. after centrifugation for 15 min at 3000 x g, the sediment was re-suspended in 1 ml of sterile phosphate buffered saline. a total of 500 µl of the resulting pellets was inoculated into the mgit and 100 µl onto the lj tubes. in each run, m. tuberculosis strain h37rv was used as a positive control. for the negative control, random lj slants and mgit tubes were inoculated with sterile phosphate buffered saline. differentiation of the m. tuberculosis complex from non-tuberculous mycobacteria was performed using the sd bio line mpt64 tb ag test (standard diagnostics, yongin, south korea). statistical analysis data were analysed using ibm spss statistics for windows, version 20.0. (2011; ibm corp., armonk, new york, united states). descriptive statistics were used for analysis of demographic and clinical characteristics. sensitivity, specificity, and predictive values of the led-fm, and respective 95% confidence interval (95% ci), were calculated with culture method as reference standard. mycobacterium tuberculosis culture positivity was defined as identification of m. tuberculosis on the lj medium and/or mgit culture. we excluded data of patients with contaminated culture results from the diagnostic accuracy analysis. results characteristics of study participants a total of 265 consecutive adult patients with suggestive symptoms of pulmonary tuberculosis were screened. fifty-three patients were excluded from the study (26 were sputum afb smear-positive by zn microscopy, 13 did not provide three sputum samples, 9 had samples with inadequate volume and 5 patients had missing sputum samples). of the remaining 212 patients, 18 were excluded because of contaminated culture or missing led-fm results, leaving 194 patients for the final analysis (figure 1). figure 1: flow diagram of patient recruitment, sample processing and diagnostic test results, ethiopia, 2014. of the 194 presumptive cases, 60.8% (118/194) were male. the median age of patients was 38 years (interquartile range 23–55). the majority (57.2%, 111/194) of patients had received antibiotics from local clinicians before the patients were referred to our hospital. thirty-nine (20%) patients recalled that they had previous contact with known tuberculosis patients. all patients were currently not taking anti-tuberculosis treatment but 26.3% (51/194) of the cases had a history of previous anti-tuberculosis treatment (table 1). table 1: demographic and clinical characteristics of the study participants (n = 194), ethiopia, 2014. all patients included in this study had a cough for at least two weeks. about half (50.5%, 98/194) of the patients were coughing for more than four weeks before admission to our hospital. the most common clinical symptoms observed in these patients were weakness in 81.4% (158/194), night sweats in 70.6% (137/194), loss of appetite in 69.6% (135/194), fever in 66.5% (129/194), and weight loss in 59.3% (115/194). haemoptysis was observed less frequently (13.4%, 26/194) (table 1). we used an unadjusted logistic regression model to determine the predictors of smear-negative, culture-positive cases. none of the predictors analysed were associated with smear-negative pulmonary tuberculosis (p-value > 0.05). accuracy of light-emitting diode fluorescent microscopy sputum was culture positive for m. tuberculosis in 14.4% (28/194) of zn smear-negative patients. twenty-one of the 194 patients with negative sputum (zn method) had positive sputum smear examinations by led-fm from bleach-concentrated sputum, but only 11 of these 21 patients (52.4%) had a confirmed tuberculosis diagnosis by culture (lj and/or mgit). among patients with negative sputum culture, led-fm gave positive results in 10 cases; however, these had scanty grading. patients’ cards for all led-fm-positive, culture-negative patients were retrospectively reviewed to evaluate their treatment outcome. only five of these patients started anti-tuberculosis treatment based on clinical criteria and three of them responded positively to anti-tuberculosis treatments. two led-fm positive patients did not respond to anti-tuberculosis treatment. for the remaining five, treatment outcome information was not available. led-fm gave positive results in 11 of 28 culture-positive samples and in 10 of 166 culture negatives. using culture as the reference standard, the sensitivity of led-fm for diagnosing smear-negative pulmonary tuberculosis was 39.3% (95% ci: 21.2–57.4) and the specificity was 93.9% (95% ci: 90.4–97.6). the positive predictive value for led-fm was 52.4% (95% ci: 31–73.7), and the negative predictive value was 90.2% (95% ci: 85.7–94.6) (table 2). table 2: diagnostic performance of light-emitting diode fluorescence microscopy compared to reference standard (culture), ethiopia, 2014. discussion in this study, we reported a significant increase in detection of smear-negative pulmonary tuberculosis by using led-fm on bleach-concentrated sputum specimens. rapid identification and treatment of tuberculosis cases is the keystone of tuberculosis control. although xpert mtb/rif is the diagnostic test of choice for tuberculosis, only a few laboratories in ethiopia have access to the xpert mtb/rif assay. moreover, in laboratories where xpert mtb/rif is available, there is a continuous stock-out of cartridges. thus, in most laboratories, case detection relies primarily on identification of afb in non-concentrated sputum zn smears using a conventional light microscope.8 conventional microscopy is inexpensive, rapid and highly specific, but has poor sensitivity, resulting in a high rate of missed cases.12,21,22,23,24 light-emitting diode-based fm has been proposed as a technique to increase the sensitivity of smear examination. previous studies showed that led-fm was approximately 10% more sensitive than conventional microscopy using zn and had comparable specificity.13,25 few studies have evaluated the performance of led-fm using bleach-concentrated sputum samples for the diagnosis of smear-negative pulmonary tuberculosis, and none of them were from ethiopia.14,25 bleach, or sodium hypochlorite, is cheap and available almost anywhere as household bleach. as a potent disinfectant, bleach also has the advantage of limiting the risk of laboratory infections. in addition, the relative centrifugal force needed for the concentration of mycobacteria was lower after digestion with bleach and can easily be achieved by a low-cost, table-top centrifuge that can easily be used under existing conditions in tuberculosis laboratories in developing countries such as ethiopia. in the current study, led-fm yielded a positive result in a significant proportion of afb sputum smear-negative patients. light-emitting diode fm detected tuberculosis bacilli in 39% of culture-positive but zn smear-negative patients. light-emitting diode fm was found to be more sensitive than zn smear microscopy. similar results have been reported by different investigators.16,24 possible explanations for increased sensitivity of led-fm may be because of a stronger affinity of auramine than carbol-fuchsin to mycolic acid. with led-fm, slides can be examined at a lower magnification, thus allowing quick examination, which would favour increased sensitivity. another reason for increased sensitivity of led-fm could be related to the fact that led-fm was performed after bleach processing and centrifugation. bleach processing has been reported to improve the identification of bacilli by providing a clearer microscopy field through digestion of mucus and concentrating bacilli by means of centrifugation. had results of led-fm been made available to the clinicians, 11 (39.3%) additional patients could correctly have been started on anti-tuberculosis treatment. even though we observed increased sensitivity of led-fm in bleach-processed and concentrated sputum, there were 17 culture-positive cases that were negative by led-fm in this study. the low number of tuberculosis bacilli present in smear-negative sputum could be the reason for false-negative led-fm test results. a negative result with led-fm does not rule out a diagnosis of smear-negative tuberculosis, given the fact that led-fm was unable to identify 60.7% of patients with culture-confirmed pulmonary tuberculosis in this study. therefore, patients with a strong clinical suspicion of pulmonary tuberculosis despite a negative led-fm should be initiated on anti-tuberculosis treatment. despite this, led-fm can provide a faster turn-around time, minimising loss to follow up during diagnostic evaluation of smear-negative pulmonary tuberculosis patients. in our study, 10 led-fm positive specimens were culture-negative. all of these smears had scanty grading. slightly lower or comparable specificity of led-fm compared with conventional zn microscopy has been reported in previous studies.16,25 on certain occasions, auramine o can stain cellular debris or other artefacts and may lead to false positive led-fm results. on the other hand, negative culture results of these 10 cases may be due to over-decontamination of specimens. specimens with low numbers of tuberculosis bacilli are particularly prone to being over-decontaminated and can result in false-negative cultures.26 it is of paramount importance to ascertain that culture-negative led-fm-positive cases are unambiguously true tuberculosis cases. therefore, we recommend further clinical evaluation study which specifically addresses such cases. limitations this study is not without limitations. light-emitting diode fm was performed only on bleach pre-treated and concentrated sputum samples. ideally, led-fm should be done on both bleach pre-treated and concentrated samples as well as non-bleach pre-treated and concentrated samples. then the effect of bleach pretreatment and concentration in the diagnosis of smear-negative tuberculosis will be evaluated. however, this was not done in the current study. conclusion we concluded that, of those patients found to have a negative result in three consecutive sputum examinations with zn, a significant proportion (10.8%) had a positive result using led-fm after bleach processing and centrifugation. light-emitting diode fm improves the speed and increases the detection rate of smear-negative pulmonary tuberculosis, although all scanty grades need to be confirmed with the standard method. in resource-limited settings where there is no xpert mtb/rif and culture, led-fm in bleach-processed and concentrated sputum can be considered complementary to conventional zn smear microscopy. future studies on the overall yield of led-fm in bleach-processed sputum with a larger sample size are warranted for routine diagnosis of patients suspected of having smear-negative pulmonary tuberculosis in resource-limited countries. acknowledgements the authors are grateful to the patients who consented to take part in this study. we would also like to thank the staff of the mycobacteriology research center of jimma university for the assistance and guidance during data collection. competing interests the authors declare they have no conflicts of interest. authors’ contributions g.a. and m.t. conceived and designed the study. d.a. and m.t. performed the experiments. m.t. and d.a. analysed and interpreted the data. g.a. coordinated the microbiological testing. d.a. wrote the initial draft of the manuscript. all authors reviewed and gave input to the subsequent manuscript drafts. sources of support this study was supported by the college of health sciences, jimma university, ethiopia. the funders had no role in study design, data analysis and interpretation, or the decision to prepare the manuscript and submit for publication. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references world health organization. global tuberculosis report. geneva: who; 2016. kebede a, alebachew z, tsegaye f, et al. the first population-based national tuberculosis prevalence survey in ethiopia, 2010–2011. int j tuberc lung dis. 2014;18(6):635–639. https://doi.org/10.5588/ijtld.13.0417 tostmann a, kik sv, kalisvaart na, et al. tuberculosis transmission by patients with smear-negative pulmonary tuberculosis in a large cohort in the netherlands. clin inf dis. 2008;47(9):1135–1142. https://doi.org/10.1086/591974 behr m, warren s, salamon h, et al. transmission of mycobacterium 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cunningham j. facing the crisis: improving the diagnosis of tuberculosis in the hiv era. j inf dis. 2007;196(suppl 1):15–27. https://doi.org/10.1086/518656 vignesh r, balakrishnan p, shankar em, et al. value of single acid-fast bacilli sputum smears in the diagnosis of tuberculosis in hiv-positive subjects. j med microbiol. 2007;56(12):1709–1710. https://doi.org/10.1099/jmm.0.47497-0 cattamanchi a, dowdy d, davis j, et al. sensitivity of direct versus concentrated sputum smear microscopy in hiv – infected persons suspected of having pulmonary tuberculosis. bmc infect dis. 2009;9(1):53. https://doi.org/10.1186/1471-2334-9-53 xia h, song y, zhao b, et al. multicentre evaluation of ziehl-neelsen and light-emitting diode fluorescence microscopy in china. int j tuberc lung dis. 2013;17(1):107–112. https://doi.org/10.5588/ijtld.12.0184 chang ew, page a-l, bonnet m. light-emitting diode fluorescence microscopy for tuberculosis diagnosis: a meta-analysis. eur respir j. 2016;47(3):929–937. https://doi.org/0.1183/13993003.00978-2015 shenai s, minion j, vadwai v, et al. evaluation of light emitting diode-based fluorescence microscopy for the detection of mycobacteria in a tuberculosis-endemic region. int j tuberc lung dis. 2011;15(4):483–488. https://doi.org/10.5588/ijtld.10.0229 article information authors: jane y. carter1 orgenes e. lema1 magdaline w. wangai1 charles g. munafu2 †philip h. rees3 jackson a. nyamongo4 affiliations: 1african medical and research foundation, nairobi, kenya2african medical and research foundation, kampala, uganda 3nairobi hospital, kenya 4ministry of health, kenya correspondence to: jane carter postal address: po box 30125, 00100 gpo, nairobi, kenya dates: received: 29 nov. 2011 accepted: 21 sept. 2012 published: 30 oct. 2012 how to cite this article: carter jy, lema oe, wangai mw, munafu cg, rees ph, nyamongo ja. laboratory testing improves diagnosis and treatment outcomes in primary health care facilitiesafr j lab med. 2012;1(1), art. #8, 6 pages. http://dx.doi.org/10.4102/ ajlm.v1i1.8 note: †philip howell rees passed away on 22 march 2012 aged 76. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. laboratory testing improves diagnosis and treatment outcomes in primary health care facilities in this original research... open access • abstract • introduction • materials and methods • ethical considerations • results • case data    • change in diagnosis    • changes in drug use and cost • discussion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ objective: to determine if use of basic laboratory tests improves diagnosis and treatment outcomes in outpatients attending rural primary health care facilities.setting: six rural health centres in kenya. design: cross-sectional study to observe change in diagnosis and treatment made by clinical officers after laboratory testing in outpatients attending six rural health centres in kenya. subject: the diagnosis and treatment of 1134 patients attending outpatient services in six rural health centres were compared before and after basic laboratory testing. essential clinical diagnostic equipment and laboratory tests were established at each health centre. clinical officers and laboratory technicians received on-site refresher training in good diagnostic practices and laboratory procedures before the study began. results: laboratory tests were ordered on 704 (62.1%) patients. diagnosis and treatment were changed in 45% of tested patients who returned with laboratory results (21% of all patients attending the clinics). 166 (23.5%) patients did not return to the clinician for a final diagnosis and management decision after laboratory testing. blood slide examination for malaria parasites, wet preparations, urine microscopy and stool microscopy resulted in most changes to diagnosis. there was no significant change in drug costs after laboratory testing. the greatest changes in numbers of recorded diseases following laboratory testing was for intestinal worms (53%) and malaria (21%). conclusion: effective use of basic laboratory tests at primary health care level significantly improves diagnosis and patient treatment. use of laboratory testing can be readily incorporated into routine clinical practice. on-site refresher training is an effective means of improving the quality of patient care and communication between clinical and laboratory staff. introduction top ↑ primary health care (phc) is the first-line contact for medical care, service and advice. in kenya, phc is provided by community health workers, and at dispensaries, health centres, and outpatient departments of hospitals. the world health organization (who) advocates for basic laboratory services to support clinical and public health activities at phc level1 and the government of kenya has planned for appropriate diagnostic services at all levels of the health care system (levels 1–6); currently, peripheral laboratories are established mainly down to health centre level (level 3). most health centres are situated in rural areas, where the majority of the population lives; however, considerable constraints remain in establishing rural laboratory units and supporting their operation. basic laboratory tests may assist in better diagnosis and management of six out of ten of the most common diseases and conditions seen in outpatients presenting to health centres and primary level hospitals.2,3 more information is needed on how clinicians working in phc units utilise laboratory tests for patient management, and which tests are most useful for diagnosing and managing patients in different geographical areas. in an editorial in the british medical journal, garner et al.4 raise the questions: do laboratory tests at this level result in altered clinical decision-making, and does access to laboratory testing actually improve the quality of patient care? between 1992 and 1994, the african medical and research foundation (amref) laboratory programme, in collaboration with the national public health laboratory services, ministry of health, kenya, conducted an essential laboratory programme feasibility study in seven rural health centres to determine an approach to effective and sustainable diagnostic services at primary health care level. the method for the study has been previously described (3), and the results are presented in another paper.5 to address the complete diagnostic cycle, the study addressed both clinicians and laboratory staff. during the feasibility study, a sub-study examined how clinicians used laboratory tests, and compared preand post-test diagnosis and treatment to examine the effect of laboratory testing on outpatient management. materials and methods top ↑ this was a cross-sectional study observing the work of clinical officers in the outpatient departments of six health centres participating in the essential laboratory programme feasibility study. each health centre was situated in one rural province in kenya to reflect variation in climate and accessibility. these were: isibania (kuria district, nyanza province), katilu (turkana district, rift valley province), kimilili (bungoma district, western province), mariakani (kilifi district, coast province), matuu (machakos district, eastern province) and wanjohi (nyandarua district, central province). a seventh health centre was excluded because of the absence of a full-time clinical officer. the sites and geographical profile of the study health centres are detailed elsewhere.5before the start of the study, a baseline survey was conducted to determine clinical, laboratory and public health activities and review existing facilities and staffing. clinical diagnostic equipment and supplies were supplemented to ensure every clinician had access to the following: stethoscope, otoscope, sphygmomanometer, torch, vaginal specula, thermometer, patella hammer, tongue depressors, examination gloves, weighing scales. basic laboratory tests were selected according to their potential usefulness in diagnosing and managing the most common diseases and conditions seen in outpatient practice; their operability in resource-limited settings; their rapidity and cost; and the technical skills of clinical and laboratory staff at health centre level. laboratory tests established at each health centre were: haemoglobin estimation to detect anaemia (haemoglobinometer/haemiglobincyanide method); blood slide for malaria and other blood parasites (field stain); total white blood cell count (manual, improved neubauer chamber) to support fever investigation; blood film examination for blood cell morphology and differential white blood cell count (reverse field stain) mainly to support anaemia and fever investigation; urine microscopy (examination of sediment) to detect urinary tract, sexually transmitted and s. haematobium infections; urine chemistry (dipsticks) for urine protein and glucose; stool microscopy (direct, eosin, iodine) to distinguish bacterial and parasitic infection; wet preparations of genital and skin specimens (direct, potassium hydroxide) to detect sexually transmitted infections and fungal skin infections; gram stain to identify bacterial and fungal infections; ziehl neelsen stain (standard and modified) to diagnose tuberculosis and leprosy; dark field illumination to detect spirochaetes in genital ulcers; rapid plasma reagin kit for syphilis screening. the study physician and laboratory technologist, accompanied by the district clinical officer and district laboratory technologist for each district, visited each health centre for 5 days at the study start to introduce and establish project activities. on-site refresher training addressed improved diagnostic practices through one-to-one training of clinical officers and laboratory technicians during routine outpatient clinics. flow sheets were developed outlining history taking, physical examination, and selection and interpretation of laboratory tests for the major clinical syndromes seen at primary health care level (fever, pallor, diarrhoea, cough, skin diseases, sexually transmitted infections), based on standard treatment guidelines produced by the ministry of health. amref designed and produced a poster, ‘use of essential laboratory tests’,6 and clinicians were provided with the following amref publications: ‘communicable diseases’, ‘child health’ and ‘medicine’. the study was conducted during 2–4-day support supervisory visits carried out 3–4 times at each site over the two-year period. subjects were patients attending general outpatient curative clinics with a new condition. clinical officers were requested to take a brief directed history and perform a targeted physical examination on every patient and to request laboratory tests in every case where results could contribute to diagnosis and/or management. a basic laboratory request form including name, age, sex, patient number, brief clinical notes, tests required, signature of clinician and date was completed for every patient. patients carried the request form to the laboratory, waited for the results and returned to the clinical officer for a management decision. laboratory staff collected all specimens except high vaginal swabs, endocervical swabs and some pus swabs, which were collected by the clinical officer. for patients requiring laboratory tests, clinical officers were asked to make a preliminary clinical diagnosis and treatment decision, which were entered in the study record sheet, before referring the patient for laboratory testing. after receiving laboratory results, diagnosis and treatment were amended as required. the study physician sat with each clinical officer and collected data from all consecutive patients attending with a new condition, whether laboratory tests were ordered or not. due to few clinical officers in the health centres, most children under five years were treated in the maternal and child health clinic and only seriously ill children were referred to the clinical officer. time taken for laboratory testing and overall patient time in the health facility were not recorded. the data were analysed by comparing preand post-test diagnosis and treatment. confidence intervals were computed at the 95% confidence level. ethical considerations top ↑ ethical approval for the study was provided by the ministry of health, kenya. results top ↑ 1134 new patients were recruited into the study. patients were examined by eight clinical officers in a total of 58 days over two years (ranging from 9 to 11 days at each health centre). the average number of patients seen at each health centre was 189 (range 108 to 339); 849 (75%) patients were more than 12 years of age. female patients predominated at all health centres except two (wanjohi 49%, isibania 46%) (table 1). table 1: patient characteristics, profile of laboratory testing and change in patient management. case data top ↑ laboratory tests were ordered on 704 (62.1%) patients (range 46.1% – 68.5% at each health centre); 971 tests were ordered (range 1–6 tests per patient, average 1.4); one test was ordered for 513 (72.9%: range 60% 82%) patients (table 1). while the study physician was present, 166 (23.5%) tested patients did not return to the clinical officer for a final diagnosis and management decision; 20 (2.8%) patients returned with incomplete laboratory results. of the 538 tested patients who returned with all or incomplete laboratory results, diagnosis and/or treatment were changed in 242 patients (45% of tested patients; 21% of all patients). table 1 shows the number of patients at each health centre with all results, complete results, or who did not return to the clinician. of the 971 tests ordered, there were no results in 250 (25.7%). from the 721 test results, 264 (36.6%) resulted in a change in patient diagnosis, drug treatment or both. table 2 shows the types of tests ordered and numbers of tests contributing to a change in diagnosis or treatment. table 2: laboratory tests and their effect on diagnosis and treatment. tests were grouped according to the purpose of making a diagnosis or defining a clinical syndrome. table 3 shows the change in diagnosis and treatment made by each group of tests. table 3: change in diagnosis or treatment after laboratory testing. change in diagnosis data on diagnosis were grouped according to major diseases and clinical syndromes. the number of diagnoses recorded before and after laboratory testing were compared. figure 1 shows the 10 most common diagnoses in all health centres, including patients that were not tested in the laboratory. figure 1: ten most common diagnoses in all health centres. changes in drug use and cost drug use indicators were applied to measure the effect of laboratory testing on drug prescription practices.7 these were: total number of medicines prescribed; percentage of patients with an antibiotic prescribed; and percentage of patients with an injection prescribed. table 4 compares drug use indicators before and after laboratory testing. drugs were grouped into major treatment categories. the number of drug courses prescribed and costs were compared before and after laboratory testing (figure 2). table 4: change in drug use indicators after laboratory testing. figure 2: number of drug courses prescribed and costs before and after laboratory testing. discussion top ↑ many clinics lack essential diagnostic equipment for patient examination. vaginal specula and swabs were provided in sterile packs in each consultation room to allow immediate vaginal examination. laboratory investigations took approximately 1 to 3 hours per patient and were usually completed by the end of the morning or afternoon clinic sessions. clinical officers worked efficiently with all equipment ready to hand and incorporated laboratory testing into their procedures without difficulty. a basic laboratory request form for all tests was appropriate at this level. the clinical impression or provisional diagnosis recorded in the patient’s notes before referring the patient to the laboratory facilitated the final management decision after the laboratory results were received.nearly a quarter of tested patients (23.6%) did not return with laboratory results, and 2.8% of patients returned with incomplete results. although the study supervisors worked in each health centre for 1 to 3 full days at a time, some patients may have returned with results after the supervisors had left. since the tests are rapidly performed and are intended to provide results before patients leave the facility, investigating causes of delay would be useful. many health centres were crowded and patients may have become discouraged by the waiting time. improved organisation of patient flow and adequate numbers of clinical and laboratory staff should increase the numbers of patients who complete investigations. for some tests, such as stool, urine and sputum examination, patients may not have produced specimens; syphilis screening was often processed in batches and patients were asked to return for results on another day. as nearly half the patients referred to the laboratory had a change in diagnosis and or treatment as a result of laboratory testing, it is important to ensure that more patients return to the clinician with completed results. clinical officers were advised to interpret laboratory results in the light of patients’ symptoms and signs. in general, the presence of pathology in specimens from symptomatic patients was reported as a positive diagnosis and patients were treated appropriately; however, symptomatic patients with negative test results were also sometimes treated based on the clinicians’ judgement for the following conditions: malaria, intestinal helminthic and protozoal infection, fungal skin infection, pelvic inflammatory disease, or fungal vaginal infection. a change in diagnosis usually resulted in a change of treatment; a few test results altered the diagnosis but not the treatment, e.g. change from e. histolytica to g. lamblia infection; however, these data may have public health implications. malaria diagnosis was reduced as a result of laboratory testing; all other diagnoses were increased, due to better assessment of other causes of fever, and the ability of the laboratory to confirm alternative diagnoses. in our study, 222 patients were given the diagnosis of malaria on the basis of 192 positive blood slides. the over-diagnosis of malaria despite use of diagnostic testing has been demonstrated in other studies 8,9 and is a major limitation to improved case management and cost savings on treatment.10 using a laboratory confirmed diagnosis of malaria as the reference standard, the sensitivity of clinical malaria diagnosis in this study was 78% and the specificity 39%. malaria infection lacks specific symptoms and signs, and clinical diagnosis is not improved with better history taking.11 there was no difference in blood-slide positivity rate in the presence (48.6%) or absence (47.8%) of another condition causing fever (p = 0.872), based on physical examination or basic laboratory testing. this finding has been reported elsewhere12 but requires further study. symptomatic patients with negative endocervical swabs were treated for pelvic inflammatory disease, as endocervical smear microscopy is not a sensitive predictor of disease.13 based on the sum of results from a group of tests, the sensitivity of clinical diagnosis of sexually transmitted infections was 65%. the test that made the most difference to diagnosis was examination of wet preparations of high vaginal swabs (53% change in diagnosis); examination of sediment of first part of morning urine to rule out urethritis changed the diagnosis in a single symptomatic male patient. these findings have major implications for continued use of syndromic approaches as practised by some disease control programmes. testing to determine specific diagnoses is recommended to reduce cost of treatment and improve compliance.13 syndromic management would be made more effective if tailored to the skills of clinical staff and available laboratory investigations at different health facility levels. the ability of a laboratory test to change diagnosis is an important measure of usefulness, but is not the only factor. in general, tests were used to confirm a suspected diagnosis, but tests performed to rule out conditions are also of value. based on the number of patients for whom laboratory tests altered diagnosis and the clinical and public health importance of the diseases confirmed, a selection of core tests can be drawn up for an outpatient health service. in this study, total and differential white cell count and thin blood film examination made little difference to diagnosis or patient management, although they were helpful in providing a more complete clinical picture. potassium hydroxide preparation and dark field examination, although useful, address few cases and may be technically more demanding. ziehl neelsen stain and syphilis screening addressed few cases but are important in diagnosing serious and treatable diseases, and should be retained. in this study setting, patients with suspected tuberculosis or syphilis would have been referred to another facility for laboratory confirmation, so the diagnosis in the health facility records was recorded as ‘changed’. the change in diagnostic data resulting from improved laboratory use in outpatient services could have a major impact on national health estimates and planning if applied country-wide. for example, in this study malaria diagnosis was reduced by 21%. change in drug costs before and after laboratory testing was less than 1%, as change in diagnosis generally resulted in the first proposed treatment being changed to another. selected drug use indicators for primary health care facilities7 reflected increased rational use of medicines with laboratory use. all injections (four) cancelled after laboratory testing were for antibiotic treatment of sti in adults. laboratory diagnosis may reduce drug costs if return visits for second or third treatments are avoided. the presence of the study physician may have impacted on clinician practice in this study (hawthorne effect).14 however, data collected during the overall 2-year study period showed clinical officers retained the level of patient referral to the laboratory in two health centres5 in the absence of the study physician. the proportion of patients referred for laboratory testing may therefore be used as an indicator of effective laboratory use.5 although these data were collected some years ago, the findings are particularly relevant given the increasing recognition of the importance of more accurate patient diagnosis, rational drug use, quality health services and cost-effectiveness; and attempts by governments to develop effective diagnostic services at peripheral health care levels. further studies are required to determine clinicians’ use of laboratory services in different health care settings and how diagnostic services can be designed for maximal utility and effectiveness. acknowledgements top ↑ the authors would like to thank the ministry of health, kenya, and the participating district administrations for their help and cooperation in allowing us to undertake this study in their health facilities. we would like to thank the following clinical officers in the selected health centres for their participation in the study: patrick akwatta, francis dosho, fredrick mutua, nehemiah ndirangu, dan ochieng, emmanuel shungu, rose sumaili, catherine wasike. tom omurwa and anthony kamau of amref assisted with data analysis. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this paper. authors’ contributions j.c. was the project leader, designed the study and collected the data. o.l. and m.w. contributed to the study design, supervised the laboratory testing during the study and assisted with data analysis. c.m. and p.r. and j.n. provided technical advice. j.c. wrote the manuscript; o.l. and j.n. critically revised the paper. references top ↑ 1. world health assembly. thirty-second world health assembly, geneva, 22 may 1979. wha32.16. health laboratory technology, hbk res: vol. 11, 3rd ed., 1.6.2.1. wha, geneva; 1979.2. carter jy, lema oe, rees ph. health laboratory services system of mainland tanzania: an evaluation. ministry of health, amref, swiss development co-operation, nairobi, kenya; 1989. 3. carter jy, lema oe, munafu cg, wangai mw, rees, ph. laboratory services in primary health care. east afr med j. 1993;70 (4 suppl):12–13. 4. garner p, kiani a supachutikul a. diagnostics in developing countries. br med j. 1997; 315:760–761. http://dx.doi.org/10.1136/bmj.315.7111.760 5. lema oe, carter jy, wangai mw, rees ph, nyamongo ja. key indicators can measure improved laboratory utilisation after interventions to strengthen diagnostic services in primary health care. in prep. 6. carter jy, materu sm, lema oe. basic laboratory services. in: seear md, editor. manual of tropical paediatrics. cambridge university press, cambridge, 2000; p. 426–427. 7. world health organization. promoting rational use of medicines: core components. who policy perspectives on medicines;september 2002. 8. reyburn h, mbakilwa h, mwangi r, mwerinde o, olomi r, drakeley c, whitty cjm. rapid diagnostic tests compared with malaria microscopy for guiding outpatient treatment of febrile illness in tanzania: randomised trial. br med j. 2007;334:403. http://dx.doi.org/10.1136/bmj.39073.496829.ae 9. barat l, chipipa j, kolczak m, sukwa t. does the availability of blood slide microscopy for malaria at health centres improve the management of person with fever in zambia? am j trop med hyg. 1999;60(6):1024-1030. 10. lubell y, reyburn h, mbakilwa h, mwangi r, chonya s, whitty cjm, mills a. the impact of response to the results of diagnostic tests for malaria: cost-benefit analysis. br med j. 2008;336:202-205. http://dx.doi.org/10.1136/bmj.39395.696065.47 11. bassett mt, taylor p, bvirakare j, chiteka f, govere e. clinical diagnosis of malaria: can we improve? j trop med hyg. 1991;94:65–69. 12. ouma p, skarbinski j, zurovac d, akhwale w, laserson k, slutsker l, hamel m. clinical diagnosis of uncomplicated malaria in older children and adults in kenya: an evidence base for newly introduced guidelines. am j trop med hyg. 2008;77(5). american society of tropical medicine and hygiene 56th annual meeting. abstract 351. 13. centers for disease control and prevention. 1998 guidelines for treatment of sexually transmitted diseases. morbidity and mortality weekly report. 1997; 47, no. rr-1:49–79. 14. rowe ak, lama m, onikpo f, deming ms. health worker perceptions of how being observed influences their practices during consultations with ill children. trop doct. 2002;32:166-167. abstract background description of the intervention lessons learnt recommendations acknowledgements references about the author(s) denis omali infectious diseases institute, makerere university college of health sciences, kampala, uganda allan buzibye infectious diseases institute, makerere university college of health sciences, kampala, uganda richard kwizera infectious diseases institute, makerere university college of health sciences, kampala, uganda pauline byakika-kibwika infectious diseases institute, makerere university college of health sciences, kampala, uganda department of medicine, school of medicine, makerere university college of health sciences, kampala, uganda rhoda namakula infectious diseases institute, makerere university college of health sciences, kampala, uganda joshua matovu infectious diseases institute, makerere university college of health sciences, kampala, uganda olive mbabazi infectious diseases institute, makerere university college of health sciences, kampala, uganda emmanuel mande infectious diseases institute, makerere university college of health sciences, kampala, uganda christine sekaggya-wiltshire infectious diseases institute, makerere university college of health sciences, kampala, uganda damalie nakanjako infectious diseases institute, makerere university college of health sciences, kampala, uganda department of medicine, school of medicine, makerere university college of health sciences, kampala, uganda ursula gutteck department of clinical chemistry, university hospital zurich, university of zurich, zurich, switzerland keith mcadam department of clinical research, london school of hygiene and tropical medicine, london, united kingdom philippa easterbrook department of human immunodeficiency virus, world health organization, geneva, switzerland andrew kambugu infectious diseases institute, makerere university college of health sciences, kampala, uganda jan fehr department of clinical chemistry, university hospital zurich, university of zurich, zurich, switzerland barbara castelnuovo infectious diseases institute, makerere university college of health sciences, kampala, uganda yukari c. manabe infectious diseases institute, makerere university college of health sciences, kampala, uganda division of infectious diseases, johns hopkins school of medicine, baltimore, maryland, united states mohammed lamorde infectious diseases institute, makerere university college of health sciences, kampala, uganda daniel mueller department of clinical chemistry, university hospital zurich, university of zurich, zurich, switzerland concepta merry department of pharmacology and therapeutics, trinity college dublin, dublin, ireland citation omali d, buzibye a, kwizera r, et al. building clinical pharmacology laboratory capacity in lowand middle-income countries: experience from uganda. afr j lab med. 2023;12(1), a1956. https://doi.org/10.4102/ajlm.v12i1.1956 lessons from the field building clinical pharmacology laboratory capacity in lowand middle-income countries: experience from uganda denis omali, allan buzibye, richard kwizera, pauline byakika-kibwika, rhoda namakula, joshua matovu, olive mbabazi, emmanuel mande, christine sekaggya-wiltshire, damalie nakanjako, ursula gutteck, keith mcadam, philippa easterbrook, andrew kambugu, jan fehr, barbara castelnuovo, yukari c. manabe, mohammed lamorde, daniel mueller, concepta merry received: 17 may 2022; accepted: 30 nov. 2022; published: 07 feb. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: research and clinical use of clinical pharmacology laboratories are limited in lowand middle-income countries. we describe our experience in building and sustaining laboratory capacity for clinical pharmacology at the infectious diseases institute, kampala, uganda. intervention: existing laboratory infrastructure was repurposed, and new equipment was acquired. laboratory personnel were hired and trained to optimise, validate, and develop in-house methods for testing antiretroviral, anti-tuberculosis and other drugs, including 10 high-performance liquid chromatography methods and four mass spectrometry methods. we reviewed all research collaborations and projects for which samples were assayed in the laboratory from january 2006 to november 2020. we assessed laboratory staff mentorship from collaborative relationships and the contribution of research projects towards human resource development, assay development, and equipment and maintenance costs. we further assessed the quality of testing and use of the laboratory for research and clinical care. lessons learnt: fourteen years post inception, the clinical pharmacology laboratory had contributed significantly to the overall research output at the institute by supporting 26 pharmacokinetic studies. the laboratory has actively participated in an international external quality assurance programme for the last four years. for clinical care, a therapeutic drug monitoring service is accessible to patients living with hiv at the adult infectious diseases clinic in kampala, uganda. recommendations: driven primarily by research projects, clinical pharmacology laboratory capacity was successfully established in uganda, resulting in sustained research output and clinical support. strategies implemented in building capacity for this laboratory may guide similar processes in other lowand middle-income countries. keywords: therapeutic drug monitoring; building laboratory capacity; resource-limited setting; hiv; uganda. background clinical pharmacology is the study of drugs in humans.1 the central dogma of clinical pharmacology is ‘drug concentration determines drug actions’.2 therapeutic drug monitoring (tdm) is the laboratory measurement of the drug concentration in a sample matrix.3 one of the aims of measuring drug concentrations in tdm is to adjust drug dose to optimise clinical outcomes and minimise adverse events in hard-to-manage diseases like hiv infection and other infectious diseases.3,4,5,6,7 therapeutic drug monitoring is routinely utilised in developed countries but is used only infrequently in lowand middle-income countries (lmics).5,8,9 laboratories can measure drug concentrations in different sample matrices using immunoassay platforms10,11,12 and chromatographic methods like high-performance liquid chromatography (hplc) and liquid chromatography-mass spectrometry (lc-ms).3,9 because of its high specificity, hplc is preferred over immunoassays, and its lower cost and local availability make it the preferred choice over lc-ms in many lmic laboratories.13 however, both hplc and lc-ms platforms require high technical expertise that may not be available in lmics, and they have weak supply chains for equipment and spare parts.12 staff training and retention are also more challenging in lmics due to limited pre-service training opportunities and limited career options. as such, despite evidence of its relevance for specialised patient care in other settings,5 tdm is not included in the standard care package within the treatment guidelines outlined by the uganda ministry of health.14 the infectious diseases institute (idi), makerere university college of health sciences, is an hiv centre of excellence located in mulago national referral hospital complex in kampala, uganda.15 the institute, established through international partnerships, is an academic research centre that commenced operations in 2002. by 2019, through its clinic in mulago and partners, idi was supporting 329 335 hiv-positive patients actively receiving antiretrovirals. laboratory units in idi included a college of american pathologists-certified idi clinical core laboratory and a smaller translational research laboratory that was created in 2007 to develop laboratory research capacity for immunology, molecular biology, microbiology, and clinical pharmacology. generally, the need for a functional clinical pharmacology laboratory cannot be overlooked, especially in settings with a high disease burden like lmics. efforts to enhance clinical pharmacology laboratory capacity must consider multifaceted needs, including human resources, knowledge building, and infrastructure.16,17,18 across its programming, idi uses a systematic approach (capacity pyramid) to highlight gaps in interdependent types of capacity – both personal (e.g. skills) and institutional (e.g. systems) – and inform comprehensive interventions.19 furthermore, collaborative partnerships between institutions in developing and developed countries have been used as a key strategy to address challenges in strengthening laboratory capacity.9,16,18 this article describes idi’s experience with developing capacity by establishing and sustaining a clinical pharmacology laboratory in uganda. description of the intervention ethical considerations ethics committee approval was not required for this research. this research involved no human or animal subjects. repurposing of existing laboratory infrastructure in a clinical pharmacology laboratory for tdm, the process workflow is critical for assay accuracy and should be considered in the design of the laboratory facility.20 between january 2007 and december 2008, 292.53 square feet of space in the translational research laboratory was repurposed to host the clinical pharmacology laboratory. two fume extractors (to eliminate toxic chemical fumes for personnel health and safety) and one fume hood (for specific sample processing and storage) were already existent before the laboratory was repurposed. a lighting system akin to natural light was installed to ease visibility. strong workbenches made of non-porous material were already available in the acquired translational research laboratory, and these were arranged for smooth workflow and to support the instruments. air conditioning and air filtration systems were also already installed in the acquired laboratory space; these helped minimise dust exposure and ensure ambient temperature for the instruments and laboratory personnel. constant laboratory operation was supported by both the national electric power supply and a backup generator installed at the idi in 2004. laboratory equipment acquisition two hplc-ultraviolet (hplc-uv) machines with inbuilt detectors, an autosampler, and pumps (shimadzu lc-2010cht, shimadzu, kyoto, japan) controlled by class-vp software version 6.1 (shimadzu, kyoto, japan) were installed in the laboratory (figure 1). the machines were obtained as generous donations from trinity college dublin in 2007 and the university of zurich in 2013. with internally generated funds, the laboratory acquired other primary instruments like an analytical balance, a ph meter, and a magnetic stirrer. the laboratory also received a generous donation of a centrifuge from the united states national institutes of health. figure 1: high-performance liquid chromatography-ultraviolet detection machines installed in the clinical pharmacology laboratory at the infectious diseases institute, makerere university college of health sciences, kampala, uganda between 2007 and 2013. in 2018, leveraging its relationship with the university of zurich, idi received a donation of an lc-ms machine (thermo scientific lcq fleet ion trap lc/msn model, thermo fisher scientific, san jose, california, united states), the first of its kind in uganda for tdm and pharmacological research (figure 2). subsequently, in 2019, a nitrogen generator was purchased (figure 3) to support the operations of the lc-ms. a technical service engineer (originally from south africa, but subsequently from within uganda) authorised by the manufacturer services the hplc-uv instruments biannually. trained idi laboratory staff service the lc-ms with expert guidance from the university of zurich. also, idi engineers based at the site oversee the periodic servicing of other laboratory equipment, including the analytical balance, centrifuge, and ph meter. figure 2: a liquid chromatography-mass spectrometry instrument installed in the clinical pharmacology laboratory at the infectious diseases institute, makerere university college of health sciences, kampala, uganda in 2018. figure 3: a nitrogen generator installed to support continuous nitrogen gas supply to the mass spectrometer in the clinical pharmacology laboratory at the infectious diseases institute, makerere university college of health sciences, kampala, uganda in 2019. laboratory human resource development human resource establishment quality human resource, which is an important aspect of local capacity, is the centre of most laboratory capacity-building programmes.16,17,21,22,23 establishing quality human resource reduces both appraisal and failure costs, thereby reducing the overall cost of quality, which is a burden in lmics.24 building on the quality of the makerere university-johns hopkins university collaboration that led to the establishment of a college of american pathologists-certified laboratory, two laboratory technologists that had rotated through the idi clinical core laboratory were recruited. human resource knowledge building during the establishment phase, two laboratory technologists were trained in methods for measuring drug concentrations using the hplc instrument, one at the university of cape town and the other at both radboud university and the university of zurich. in 2018, through the strong idi-university of zurich research collaboration, one staff member was further trained in the use of the lc-ms platform, leading to the donation of the equipment to idi. the training activities were conducted both physically and via virtual media platforms to expand knowledge and skills in the areas of sample processing and measurement of drug concentrations in different human sample matrices, results analysis, equipment operation and maintenance, method development and validation, and the supply chain process. using cascade training (table 1), the formally trained staff member trained five other laboratory technologists to expand the laboratory’s human resource capacity. continuing medical education was encouraged for the laboratory staff throughout the capacity-building programme at the idi. currently, the laboratory has a strong human resource capacity composed of three laboratory technologists and continues to get technical and mentorship support from the university of zurich. table 1: human resource training programmes conducted between 2006 and 2019. lessons learnt current capacity for drug concentration measurements pharmacokinetic assays a clinical pharmacology laboratory was developed at idi and currently has the capacity to measure drug concentrations to guide tdm and clinical pharmacology studies using 10 analytical methods for hplc-uv and six methods for lc-ms either simultaneously or singly. these methods are used to measure the concentrations of anti-tuberculosis drugs such as ethambutol, rifampicin, isoniazid, pyrazinamide, rifapentine, rifabutin and moxifloxacin. antiretroviral drugs analysed in the laboratory include nevirapine, efavirenz, atazanavir, lopinavir, tenofovir, saquinavir, darunavir, etravirine, dolutegravir and raltegravir. antiepileptics (phenytoin and carbamazepine), antibiotics (vancomycin, gentamicin, kanamycin and amikacin), and antimalarial drugs (lumefantrine, artemether and its metabolites and halofantrine) are also analysed in the laboratory. currently, the lc-ms methods are used to determine the concentrations of tenofovir, dolutegravir, amikacin, and antimalarial drugs, while the concentrations of other drugs are measured using hplc-uv. the laboratory staff can also execute in-house innovations to develop, validate, and optimise methods for measuring the concentration of several drugs using the hplc-uv and lc-ms. drug concentration data from the laboratory has enabled researchers working on pharmacokinetic studies to detect drug interactions and sub-therapeutic concentrations and monitor patient antiretroviral therapy adherence (table 2). table 2: pharmacokinetic studies formally supported by the clinical pharmacology laboratory at the infectious diseases institute in kampala, uganda between 2009 and 2015. laboratory quality assurance programme in 2017, the laboratory commenced its participation in an external quality assurance (eqa) programme for nevirapine, efavirenz, atazanavir, and lopinavir through the stichting kwaliteitsbewaking klinische geneesmiddelanalyse en toxicologie (kkgt) (association for quality assessment in therapeutic drug monitoring and clinical toxicology, amstelveen, the netherlands).28 the eqa samples analysed in 2017 and 2019 were within the kkgt acceptance range in the four annual rounds. in 2018, eqa test results were within the kkgt acceptance range except for atazanavir in round one, lopinavir in rounds two and three, and efavirenz in round four. in 2019, anti-tuberculosis drugs, including ethambutol, rifampicin, isoniazid, pyrazinamide, rifapentine, and rifabutin, were included in two annual rounds of the eqa programme. the first attempt yielded results within the kkgt acceptance range in both rounds for all anti-tuberculosis drugs except isoniazid in round two. the second attempt in 2020 was not conducted for anti-tuberculosis drugs due to interruptions in the shipment of the eqa samples to the laboratory. antibiotics (only amikacin and vancomycin) and antiepileptics (only phenytoin and carbamazepine) were included in the 2017 eqa subscription, with four annual rounds. attempts to analyse the antiepileptics in all four rounds yielded results that were outside the kkgt acceptable range. after laboratory preparations, vancomycin was included in the eqa programme in 2020 and yielded results within the kkgt acceptance range in rounds one and four. however, the vancomycin eqa results for rounds two and three were out of the kkgt acceptance range. amikacin was not tested together with vancomycin in the eqa programme because the method to measure amikacin concentrations in the laboratory was not developed until 2020. with mutual interdependence with collaborators, the laboratory continues to develop capacity for other kkgt programmes, with continuous optimisation of existing assays for the measurement of drugs like the antiepileptics for which measurement was unsuccessful in the previous attempts. the lack of research studies requiring the measurement of carbamazepine and phenytoin concentrations may have contributed to the lesser focus on the antiepileptics programme. the eqa helped laboratory staff to identify pitfalls in routine laboratory analyses. interference of other analytes with the kkgt eqa samples was found to be the major cause of out-of-range low scores in the antiretroviral programme. this challenge was corrected by optimising the methods for simultaneous measurement of efavirenz, lopinavir, and atazanavir and using a different analytical method that had no interference between analytes. generally, participation in the eqa scheme improved staff confidence in supporting research studies and clinicians seeking tdm services. laboratory support for therapeutic drug monitoring and the need for a clinical pharmacology laboratory clinicians and clinical researchers at the idi clinic and external institutions have successfully used drug concentration results from the laboratory for tdm, specifically to assess non-adherence to regimens or to switch or discontinue patient therapy because of suspected drug resistance and toxicities. clinicians obtain patients’ blood samples and send the harvested plasma to the clinical pharmacology laboratory for testing with a corresponding request form attached. results from the laboratory are returned to clinicians to inform clinical management (counselling or dose adjustment, where appropriate). cumulatively, the laboratory tested 181 clinical care samples from the adult infectious diseases clinic at the idi between 2013 and 2019. with this support for clinical management, the need for a clinical pharmacology laboratory cannot be overemphasised, especially in lmics where there is a high disease burden. challenges and solutions encountered during laboratory capacity-building processes the instruments used for drug concentration measurements are costly and were acquired free of charge to idi through international collaborations (grants and donations). nevertheless, the costs of equipment preventive maintenance remained a challenge because of the high costs of service vendors and spare parts. the laboratory was able to incorporate these costs within research project budgets over the years, including from competitive grants. uganda has only a few authorised vendors that supply genuine high-purity reagents of hplc and lc-ms grade, and these are also costly to procure. before the acquisition of a nitrogen gas generator, obtaining high-purity gases like nitrogen and helium was difficult. initial training of laboratory technologists was limited to two staff members since this had to be conducted overseas. fortunately, efforts to cascade training to other laboratory staff were successful at minimal costs. notably, these challenges raise the cost of measuring the concentration of a drug in a single sample to approximately $40 united states dollars, which is outside the reach of many patients. from inception, the laboratory’s business case has thus focussed on funded research and clinic projects. for example, the tdm services at the idi clinic had to be paused in 2019, after analysing 181 samples from clinic patients, due to funding constraints. however, the clinical pharmacology laboratory remained operational since no such laboratory supporting clinical care was established in the country. recommendations the strategies employed in our laboratory yielded tangible results similar to those observed in related laboratory capacity-building programmes.16,17,18,22,23,29,30 clinical pharmacology laboratory capacity was developed to measure concentrations of antiretrovirals, anti-seizures, antibiotics, antimalarials, and anti-tuberculosis drugs for tdm. the capacity to develop, validate, and optimise analytical methods for other drugs was also developed. the success recorded for this laboratory capacity-building process reflects the gradual progress to strengthen capacity from inception to date with collaborative support. the focus of the institution’s research programme in the field of pharmacokinetics was sustained over the years, enabling continuous cash flow to the laboratory in the form of research costs covering sample analysis. however, with short-term project support as the main source of laboratory resources, factors beyond the laboratory’s direct control such as grant success or availability of collaborators with aligned goals could lead to shocks in the near term and impair long-term sustainability. we therefore recommend that laboratories in lmics (and their parent institutions) must be prepared to strengthen and sustain results from collaborative programmes when external support ends. laboratories in lmics intending to build and sustain long-term capacity for clinical pharmacology should build local capacity through training and infrastructure development.16,17,18,31 as with other capacity-building programmes, staff training in our setting was challenging,16,31 requiring international travel and time off work. we further recommend the introduction of practical coursework for clinical pharmacology sessions in pre-service laboratory education (bachelor’s degree-level laboratory technology training) to ease future on-the-job staff training. the african field epidemiology track programme developed and adopted this strategy, positively changing the laboratory profession in its programme member countries.17,32 using established centres like idi for postgraduate training in pharmacology or for placements to support new laboratories could reduce costs associated with expensive international training. further, pharmacology postgraduates can be included in laboratory mentorship programmes in lmics. investment in appropriate infrastructure at inception not only promotes staff safety but can also prolong the lifespan of the equipment. high-level air conditioning mitigates challenges of hot and humid climates that may affect laboratory instruments. the use of fume extractors to eliminate environmental dust and toxic fumes from laboratory reagents and processes is essential for staff health and safety. laboratory fume hoods should be used together with proper reagent segregation to minimise the chances of explosions resulting from flammable reagent fume reactions. the outcomes presented in this article represent success at a site where clinical research and laboratory infrastructure were already present and operating to international standards at baseline. our experience may thus not apply to all lmics due to variations in local guidelines and socio-economic factors. a clinical pharmacology laboratory was established, and laboratory capacity was developed and sustained at the idi through strong capacity-building collaborative relationships. the laboratory significantly contributed to research capacity development at idi and other external institutions by providing answers to questions from several pharmacology research studies. building clinical pharmacology laboratory capacity in lmics is feasible and necessary to support the global goal of managing hiv infection and other diseases. acknowledgements we acknowledge peter smith, university of cape town, lance heinle, pfizer global health fellows program, and david m. burger, nijmegen, radboud university, for organising staff training. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions d.o. and a.b. contributed equally. c.m. and dm. contributed equally. d.o. and a.b. conceived and designed the concept. d.o. wrote the initial manuscript. d.o., a.b., r.k., p.b.-k., r.n., j.m., o.m., e.m., d.n., p.e., b.c., y.c.m., m.l., d.m., and c.m. participated in critical revisions of the manuscript for intellectual content. c.s.-w., p.b.-k., u.g., m.l., d.m., and c.m. gave mentorship and technical support. o.m., j.f., d.n., b.c., y.c.m., p.e., k.m., and a.k. participated in administrative support. all named authors approved the final manuscript for publication. sources of support we are grateful to realta foundation, the irish department for foreign affairs, and the health research board of ireland (ghra06/02, ghra07/09) for initial support to establish the laboratory. we acknowledge the support given by the university of zurich in the provision of equipment and staff training. we acknowledge funding support for equipment, supplies, and maintenance from the european and developing countries clinical trials partnership and world health organization tropical diseases research (csa-ebola-2015-353). d.o. is supported by the fogarty international center of the national institutes of health under award number d43tw009771. a.b. is currently a phd scholar supported through the fogarty international centre, national institute of health grant #2d43tw009771-06 ‘hiv and co-infections in uganda’. the authors also acknowledge the deltas africa-funded makerere uganda virus research institute infection and immunity (muii) programme (grant #107743/z/15/z) that has collaborated with idi in building capacity of the translational research laboratory. we thank the united states national institutes of health for the generous donation of a centrifuge to the laboratory. r.k. is currently a phd scholar supported through the deltas africa initiative grant #del-15-011 to thrive-2, from wellcome trust grant #107742/z/15/z and the united kingdom government. e.m. is currently a msc fellow supported through the united states national institutes of health – fogarty international center, breca grant #1u2rtw010672. m.l. is supported by european and developing countries clinical trials partnership (ria2018ef-2083) which is part of the european and developing countries clinical trials partnership programme supported by the european union’s horizon 2020 research and innovation program. data availability all data underlying the findings are fully available without restriction in the manuscript from the corresponding author (d.o.). disclaimer the authors alone are responsible for the views expressed in this publication and they do not necessarily represent the views, decisions, or policies of the institutions with which they are affiliated. the views and opinions of authors expressed herein do not necessarily state or reflect those of european and developing countries clinical trials partnership. references huang s-m, lertora jj, markey sp, atkinson aj, jr. principles of clinical pharmacology. san diego, california: academic press; 2012. holford nh, atkinson aj, jr. time course of drug response. in: aj atkins, dr abernethy, ce daniels, rl dedrick, sp markey, editors. principles of clinical pharmacology. bethesda, maryland: elsevier, 2007; pp. 301–311. https://doi.org/10.1016/b978-012369417-1/50059-6 kang js, lee mh. overview of therapeutic drug monitoring. korean j intern med. 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antiretroviral drug concentrations in uganda: design, methods and patient characteristics of the south study. bmj open. 2017;7(9):e014679. https://doi.org/10.1136/bmjopen-2016-014679 droste j, aarnoutse re, koopmans pp, hekster ya, burger dmj. evaluation of antiretroviral drug measurements by an interlaboratory quality control program. j acquir immune defic syndr. 2003;32(3):287–291. https://doi.org/10.1097/00126334-200303010-00007 niyonzima n, wannume h, kadhumbula s, et al. strengthening laboratory diagnostic capacity to support cancer care in uganda. am j clin pathol. 2021;156(2):205–213. https://doi.org/10.1093/ajcp/aqaa218 paglia mg, bevilacqua n, haji hs, et al. improvement of tuberculosis laboratory capacity on pemba island, zanzibar: a health cooperation project. plos one. 2012;7(8):e44109. https://doi.org/10.1371/journal.pone.0044109 petti ca, polage cr, quinn tc, ronald ar, sande ma. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006;42(3):377–382. https://doi.org/10.1086/499363 gitta sn, mukanga d, babirye r, dahlke m, tshimanga m, nsubuga p. the african field epidemiology network-networking for effective field epidemiology capacity building and service delivery. pan afr med j. 2011;10(suppl. 1):3. abstract introduction methods results discussion acknowledgements references about the author(s) teena s.m. thomas infection control services laboratory, national health laboratory services, johannesburg, south africa department of clinical microbiology and infectious disease, school of pathology, university of the witwatersrand, johannesburg, south africa juno thomas centre for enteric diseases, national institute of communicable diseases, johannesburg, south africa karren le roux infection control services laboratory, national health laboratory services, johannesburg, south africa department of clinical microbiology and infectious disease, school of pathology, university of the witwatersrand, johannesburg, south africa sanelisiwe t. duze department of clinical microbiology and infectious disease, school of pathology, university of the witwatersrand, johannesburg, south africa faith mkhwanazi infection control services laboratory, national health laboratory services, johannesburg, south africa adriano duse infection control services laboratory, national health laboratory services, johannesburg, south africa department of clinical microbiology and infectious disease, school of pathology, university of the witwatersrand, johannesburg, south africa citation thomas tsm, thomas j, le roux k, duze st, mkhwanazi f, duse a. diagnostic challenges with accurate identification of listeria monocytogenes isolates from food and environmental samples in south africa. afr j lab med. 2022;11(1), a1482. https://doi.org/10.4102/ajlm.v11i1.1482 note: additional supporting information may be found in the online version of this article as supplementary documents 1, 2 and 3. original research diagnostic challenges with accurate identification of listeria monocytogenes isolates from food and environmental samples in south africa teena s.m. thomas, juno thomas, karren le roux, sanelisiwe t. duze, faith mkhwanazi, adriano duse received: 02 dec. 2020; accepted: 11 feb. 2022; published: 23 may 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the 2017–2018 listeriosis outbreak in south africa warranted testing for listeria monocytogenes in food products and processing environments. diagnostic tests are needed to accurately differentiate l. monocytogenes from other listeria species. objective: the study assessed the performance of the commonly used tests in our setting to accurately identify l. monocytogenes. methods: the study was conducted in a public health laboratory in south africa. cultured isolates from food and environmental samples were tested both prospectively and retrospectively between august 2018 and december 2018. isolates were phenotypically identified using tests for detecting β-haemolysis, christie-atkins-munch-peterson, alanine arylamidase (alaa), mannosidase, and xylose fermentation. listeria monocytogenes isolates were identified using automated systems, microscan walkaway plus 96, vitek® ms, vitek® 2 and surefast listeria monocytogenes plus pcr. all results were compared to whole-genome sequencing results. results: β-haemolysis and christie-atkins-munch-peterson tests gave delayed positivity or were negative for l. monocytogenes and falsely positive for one strain of listeria innocua. the alaa enzyme and colorex listeria agar lacked specificity for l. monocytogenes identification. based on a few phenotypic test results, an aberrant l. monocytogenes strain and listeria seeligeri strain were reported. all automated platforms overcalled l. monocytogenes in place of other listeria species. conclusion: no test was ideal in differentiating listeria species. this is an issue in resource-limited settings where these tests are currently used. newer technologies based on enzyme-linked immunosorbent assay and other molecular techniques specific to l. monocytogenes detection need to be investigated. keywords: listeria monocytogenes; food; environmental samples; diagnostic challenges; africa. introduction an extensive outbreak of listeriosis occurred in south africa in 2017–2018. to date, it is the largest laboratory-confirmed listeria monocytogenes foodborne outbreak described globally.1 the l. monocytogenes strain responsible for the outbreak was characterised on whole-genome sequencing (wgs) analysis as multi-locus sequence type 6.2 consequently, wide-scale testing for l. monocytogenes in various food products and processing environments commenced. for appropriate food safety and public health interventions, the utilised diagnostic tests should reliably differentiate l. monocytogenes, the outbreak pathogen, from other listeria species. listeria species and l. monocytogenes share the same growth requirements and often coexist in the same environment; therefore, l. monocytogenes should be accurately discriminated from other co-occurring listeria species.3 six listeria species (listeria marthii, listeria ivanovii, listeria seeligeri, listeria innocua, listeria grayi and listeria welshimeri) are closely related to l. monocytogenes. this close-relatedness challenges species differentiation.4 although uncommon, ‘atypical’ strains, which do not exhibit typical phenotypic characteristics, of l. monocytogenes and other listeria species have also been described.5 several test methodologies are utilised to discriminate between the listeria species; however, each has its pitfalls. l. monocytogenes is positive for the christie-atkins-munch-peterson (camp) test on sheep blood agar within 24 h of incubation.4 however, weakly haemolytic (showing haemolysis beyond 24 h of incubation) or non-haemolytic strains are frequently detected. weak or no haemolysis is due to deletion of the hyl gene or its regulatory protein prfa, which regulates the expression of virulence factors required for l. monocytogenes pathogenesis.4 other listeria species, such as l. ivanovii (particularly with the camp test utilising rhodococcus equi), l. seeligeri, and some l. innocua strains also show haemolytic capabilities which can make the utility of this test pointless.5,6,7 listeria agar by ottaviani and agosti (agar listeria ottaviani & agosti medium, biorad, berkeley, california, united states) is recommended in the international organization for standardization 11290–1, 2017 standard for the isolation and differentiation of l. monocytogenes from other listeria species.8 all listeria species are selected for growth on the medium and produce blue-green colonies due to substrate degradation by β-d-glucosidase activity. l. monocytogenes and l. ivanovii can be differentiated from the other species due to the production of an opaque halo around the colonies as a result of phosphatidylinositol-specific phospholipase (pi-plc) activity.9 the timing of the appearance of the opaque halo is also indicative of the species type. the halo is produced after 24 h incubation by l. monocytogenes and after 48 h of incubation by l. ivanovii.3,10 other strains, such as l. seeligeri, l. welshimeri, and a few strains of l. innocua, may also possess the plca gene, which codes for phospholipase activity that is responsible for creating the opaque halo around its colonies.4,5 in addition, other bacterial species, like bacillus species, cellulosimicrobium funkei, enterococci, kochuria kristinae, marinilactibacillus psychrotolerans, rothia terrae, and coagulase-negative staphylococci, may also grow as blue-green colonies on agar listeria ottaviani & agosti medium.11 bacillus circulans, bacillus licheniformis, enterococcus faecalis, enterococcus faecium/durans, and staphylococcus sciuri can produce a halo as well, which can make differentiation of l. monocytogenes from l. ivanovii difficult.11 the analytical profile index listeria test (biomerieux, marcy d’etoile, france) fails in 10% – 15% of identification cases. the main reason for this failure is due to the weak colour determinations. this is particularly applicable to the arylamidase test. the arylamidase enzyme, tested for in the popular differentiation innocua monocytogenes (dim) test, is supposed to be negative in l. monocytogenes and positive in other listeria species.12 often a weak positive dim result was considered a negative result, increasing the false positive l. monocytogenes determinations.4 this might be due to the doubtfulness of the colour determinations by the reader of the test. furthermore, false negative identification in atypical l. monocytogenes strains is also frequent.4 matrix-assisted laser desorption time of flight (maldi-tof) mass spectrometry is a quick and easy methodology gaining popularity in several microbiology laboratories. however, maldi-tof reportedly misidentifies l. innocua as l. monocytogenes american type culture collection (atcc) strain and the l. seeligeri atcc strain as l. monocytogenes or l. innocua.4 rychert et al. reported that vitek® mass spectrometer (ms) version 2.0 system (biomerieux, marcy d’etoile, france) correctly identified only 76% (34/45) of l. monocytogenes to the species level and 9% (4/45) to the genus level, while in 15% (7/45) identification could not be finalised because split identification and re-testing were not performed.13 the vitek® 2 system has also been reported to misidentify l. monocytogenes as l. innocua based on a negative reaction for phospholipase c in 1.4% (4/288) of a collection of isolates tested.14 the instrument could not identify an l. monocytogenes strain and gave a species error in another study.15 in a previous evaluation of the vitek system, when genus level identification of various listeria species was sought, the instrument had a sensitivity of 97.5%.16 the microscan walkaway si system (siemens healthcare diagnostics, west sacramento, california, united states) could not identify one l. monocytogenes atcc strain baa–751 during a comparative study with the vitek® 2 compact system.17 the reason for this was potentially attributed to the limited number of listeria species strains on the database. during the investigation of the south african listeriosis outbreak in 2017–2018, four listeria species (l. monocytogenes, l. innocua, l. welshimeri, and l. seeligeri) were detected from food samples and environmental swabs tested at the infection control services public health laboratory in johannesburg, south africa. this is similar to what has been described elsewhere in outbreak settings.5 as a result, accurate discrimination of l. monocytogenes from other species is of critical importance. whole-genome sequencing is a useful tool for confirmatory identification of l. monocytogenes and can be used as the reference standard test for comparing other tests.18 subsequent to the reported limitations of listeria tests commonly utilised in most public health laboratories, particularly in lowand middle-income countries, the infection control services public health laboratory evaluated the performance of the commonly utilised phenotypic tests (conventional phenotypic tests and chromogenic media) for the identification of listeria species in comparison to wgs results. the infection control services public health laboratory also compared the performance of the different automated diagnostic systems available in the institution for the identification of l. monocytogenes utilising known listeria isolates characterised by wgs. the study results will inform whether current tests are acceptable for future use and, if not, it will justify the evaluation of other technologies for accurate identification of l. monocytogenes. methods ethical considerations only cultured isolates from food and environmental samples were utilised in this research. no isolates from animals or animal-derived samples were used. therefore, no ethical clearance was required. study design and samples used data for this analysis were collected prospectively from august 2018 to december 2018 at the infection control services public health laboratory in johannesburg. isolates were cultured from food and environmental swabs of several food processing facilities across all of the provinces in south africa during the listeriosis outbreak period. all listeria isolates were identified on vitek® 2 (biomerieux, marcy-i’etoile, france). the phenotypic tests were performed either to (1) confirm the initial identification from vitek® 2 or (2) to discriminate between listeria species if two species identifications were given by vitek® 2. as a result, not all phenotypic tests were performed on all isolates. the accuracy of the conventional phenotypic tests to discriminate the four listeria species (l. monocytogenes, l. innocua, l. welshimeri, and l. seeligeri) was assessed (table 1).3 the isolate identity (vitek® 2 and phenotypic testing) was confirmed by wgs. table 1: phenotypic tests used to discriminate listeria monocytogenes from l. innocua, l. welshimeri and l. seeligeri. these isolates included 39 l. monocytogenes and 36 listeria non-monocytogenes species, including 28 l. innocua, seven l. welshimeri, and one l. seeligeri. laboratory analyses beta (β)-haemolysis was performed on sheep blood agar. plates were checked daily for up to 72 h. the camp test was performed using the staphylococcus aureus atcc strain 25923. the positive controls used for this test were streptococcus agalactiae atcc 13813 and l. monocytogenes atcc 19115. the plates were examined daily for up to 72 h. the presence of arylamidase, mannosidase enzymes and acid production from d-xylose (dxyl) fermentation was assessed on the vitek® 2 gram-positive card based on the results of alanine arylamidase (alaa), α-mannosidase (aman), and dxyl. the vitek® 2 gram-positive card was inoculated with the isolates as per the vitek® 2 instrument training manual.19 the colorex listeria agar (e&o laboratories ltd, bonnybridge, united kingdom) has the same constituents as the agar listeria ottaviani & agosti medium. this medium was assessed and analysis was performed retrospectively using 59 of the 75 banked listeria isolates from the analysis of the phenotypic tests. the isolates included 36 l. monocytogenes, 16 l. innocua, and seven l. welshimeri. the laboratory also verified the performance of the automated platforms available. analysis was performed retrospectively in december 2018 using 50 known listeria isolates from the laboratory repository. the listeria species included 20 l. monocytogenes strains and 30 listeria species. the 30 listeria species included 27 l. innocua, two l. seeligeri, and one l. welshimeri. these isolates were tested on four platforms, namely (1) microscan walkaway plus 96 (beckman coulter life sciences, indianapolis, indiana, united states), (2) vitek® ms version 3.0 system (biomerieux, marcy-i’etoile, france), (3) vitek® 2, and (4) surefast listeria monocytogenes plus polymerase chain reaction kit (congen, berlin, germany), run on the roche light cycler 2.0 (roche, basel, switzerland) instrument. the surefast kit identifies l. monocytogenes by amplifying a fragment of prfa and the detection limit of this assay is 10 cfu/ml as per our laboratory verification. staff were blinded to the confirmatory wgs results of the isolates during the colorex listeria agar and automated systems assessments. data analysis the sensitivity, specificity, positive predictive value, and negative predictive value of the test methods for detecting l. monocytogenes were calculated. data were collected on excel spreadsheets (microsoft, redmond, washington, united states) and analysis was performed using two-by-two tables.20 calculations were done as follows: sensitivity = true positive l. monocytogenes isolates (test and wgs positive) / total wgs-confirmed l. monocytogenes isolates (true positive + false negative) × 100 specificity = true negative l. monocytogenes isolates (test and wgs negative) / total wgs-confirmed non-l. monocytogenes isolates (true negative + false positive) × 100 positive predictive value = true positive l. monocytogenes isolates (test and wgs positive) / total positive test results (true positive + false positive) × 100 negative predictive value = true negative l. monocytogenes isolates (test and wgs negative) / total negative test results (true negative + false negative) × 100 results the phenotypic results of the listeria species in comparison to the wgs results are summarised in the online supplementary table 1. performance of the phenotypic tests for l. monocytogenes identification the three phenotypic tests used to confirm the identification of l. monocytogenes by vitek® 2 and discriminate it from the other listeria species were β-haemolysis, the camp test, and alaa activity (table 2). table 2: performance characteristics of β-haemolysis, christie, atkins and munch-peterson test and differentiation innocua monocytogenes test in comparison to whole-genome sequencing in the identification of listeria monocytogenes, infection control services public health laboratory, south africa, august 2018 – december 2018. of the 39 l. monocytogenes isolates identified by wgs, all three phenotypic tests corroborated the wgs findings in 82% (32/39) of the isolates. β-haemolysis and the camp test were absent in 18% (7/39) of the isolates. delayed positivity to both of these tests occurred at 72 h in 5.1% (2/39) of isolates. one l. innocua isolate was falsely positive to both of these tests. of the l. monocytogenes isolates, 18% (7/39) were falsely positive for alaa on vitek® 2. all l. innocua isolates and the one l. seeligeri isolate were falsely negative for alaa. performance of the phenotypic tests for l. innocua identification of note, one out of the 28 l. innocua isolates was positive for both β-haemolysis and the camp test. performance of the phenotypic tests for l. welshimeri identification the phenotypic tests used to identify l. welshimeri identified all seven of the isolates correctly. however, there was one isolate that had two identification options on vitek® 2, namely l. monocytogenes and l. welshimeri. to discriminate between the two listeria species, β-haemolysis, camp, and dxyl fermentation tests were assessed. the isolate was negative for β-haemolysis and the camp test but positive for dxyl fermentation, which suggested that the isolate is l. welshimeri. however, wgs results identified the isolate as l. monocytogenes. performance indicators, such as sensitivity, specificity, positive predictive value, and negative predictive value, for the phenotypic tests (β-haemolysis, camp test and dxyl fermentation) differentiating l. welshimeri from l. monocytogenes were not done due to the low number of l. welshimeri isolates identified during the study period. performance of the phenotypic tests for l. seeligeri identification one isolate was previously identified as l. monocytogenes based on vitek® 2 identification (99% probability), positive β-haemolysis (at 24 h incubation), positive camp test (at 24 h incubation), negative alaa enzyme activity, negative dxyl fermentation, and positive aman activity. however, wgs identified this isolate as l. seeligeri. since there was only one l. seeligeri isolate, the performance of the tests (alaa activity, aman activity, and dxyl fermentation) to discriminate this species from l. monocytogenes was not done. performance of the colorex listeria agar in the identification of l. monocytogenes all 59 isolates representing the three listeria species produced blue-green colonies on colorex listeria agar and 73% (43/59) of these isolates produced an opaque halo around the colonies (figure 1).21 of the 43 isolates that produced a halo, 84% (36/43) were identified as l. monocytogenes on wgs, while the remaining 16% (7/43) of isolates were identified as l. innocua (n = 5) and l. welshimeri (n = 2) on wgs (online supplementary table 2). the sensitivity and specificity of the medium for accurate l. monocytogenes identification were 100% and 69%. the positive predictive value was 83.7% and negative predictive value was 100%. figure 1: listeria monocytogenes colonies on colorex listeria agar. performance of the automated systems in the identification of l. monocytogenes all the systems overcalled l. monocytogenes in place of other species (table 3). all listeria species results on the various automated systems are provided in online supplementary table 3. table 3: performance characteristics of the automated systems in comparison to whole-genome sequencing in the identification of listeria monocytogenes, infection control services public health laboratory, south africa, december 2018. discussion from the evaluation, there was no ideal test for differentiating the listeria species: all had limitations. β-haemolysis and the camp test are recommended to differentiate l. monocytogenes from l. innocua. however, these tests can give delayed positivity (up to three days later) or be negative for l. monocytogenes. in addition, they may be falsely positive for certain l. innocua strains. the dim test for alaa enzyme activity lacks specificity for l. monocytogenes detection. all of the other listeria species also tested negative for this enzymatic activity, disproving its utility. we report the first aberrant l. monocytogenes strain that fermented dxyl and an aberrant l. seeligeri strain that was negative for alaa activity and dxyl fermentation and positive for aman activity. this further illustrates atypical strains that may potentially exist in our setting, complicating identification. unfortunately, wgs could not be repeated on both of these isolates again to reconfirm the results and rule out the possibility of isolate mix-up. the colorex listeria agar was able to correctly identify all l. monocytogenes isolates; however, it lacked specificity in discriminating other listeria species from l. monocytogenes. limitations there were also several shortcomings associated with the four automated diagnostic platforms tested. all platforms overcalled l. monocytogenes in place of the other listeria species. this could be because these instruments were validated for clinical samples in which l. monocytogenes is the predominant species isolated. the vitek® ms misidentified l. monocytogenes for other listeria species and the vitek® 2 gave both l. monocytogenes and l. innocua options for a few l. innocua isolates. the worst-performing platform for l. monocytogenes identification was microscan and the best performer was the surefast polymerase chain reaction kit. based on the above results, alternate testing platforms for l. monocytogenes identification need to be investigated. several other diagnostic methodologies are available to detect l. monocytogenes. these include (1) detection of l. monocytogenes by antibody-based assays, (2) molecular test methods such as loop-mediated isothermal amplification (lamp), dna hybridisation or polymerase chain reaction utilising l. monocytogenes–specific gene targets that have been identified, and (3) the use of genetically engineered bacteriophages.3,7,9,12 many of these assays are available as commercial automated kits approved by regulatory authorities. these technologies have been reported to perform well in the detection of l. monocytogenes. in addition, the automated systems are high throughput and significantly shortens the time to results of the traditional methods. the possible disadvantages of the above technologies would be: cost, staff expertise to perform the tests, and inhibition of tests (antibody-based and molecular) by the sample matrix. the molecular assays may also detect non-viable organisms. conclusion we have demonstrated that the commonly used methodologies in most public health laboratories, particularly in lowand middle-income settings, are limited in differentiating l. monocytogenes from the other listeria species. the accurate identification of l. monocytogenes is critical since it is the most predominant listeria species causing human disease and, therefore, must not be missed by diagnostic tests. the large scale of this outbreak required upscaling laboratory support for public health sample testing. however, if the available systems in routine microbiology laboratories cannot discriminate between l. monocytogenes and the other listeria species, overcalling or underreporting of l. monocytogenes can occur. underreporting l. monocytogenes will prevent or delay identifying an outbreak source and promote its continuity with huge public health impact. overcalling l. monocytogenes leads to the unnecessary closure of food production lines, which has huge financial implications for the company involved. depending on the company’s distribution level, halting production can also impact the community. in the outbreak setting, where l. monocytogenes prevalence in samples was comparatively higher, the positive predictive value of most of the tests assessed was unacceptable. hence, in a non-outbreak setting, the performance of these tests will be worse. therefore, other technologies must be investigated for their discriminatory capabilities and accurate identification of l. monocytogenes in microbiology laboratories in lowand middle-income countries. acknowledgements the authors would like to acknowledge the following for their role in the study: (1) the molecular and public health staff of the infection control services laboratory, national health, laboratory services, for conducting the tests that were required, (2) the microbiology laboratory at charlotte maxeke johannesburg academic hospital for granting permission to use the vitek® 2 and vitek® ms instruments, and (3) the national institute of communicable diseases for providing whole-genome sequencing results for all the isolates used for this research. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions t.s.m.t. and a.d. conceived the original idea for the study. t.s.m.t. supervised the study. f.m., s.t.d. and k.l.r. carried out the laboratory work and data collection. t.s.m.t. analysed the data. j.t. assisted with the whole-genome sequencing data and provided critical input to the article. all authors provided critical inputs to the manuscript. sources of support this work was supported by the national health laboratory services, south africa, by providing all consumables used for this research. data availability derived data supporting the findings of this study are available 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https://www.eolabs.com/product/pp7021-colorex-listeria-iso/. abstract introduction methods results discussion acknowledgements references about the author(s) njabulo khumalo department of chemical pathology, national health laboratory service, cape town, south africa division of chemical pathology, faculty of health sciences, university of cape town, cape town, south africa cameron a. francis department of chemical pathology, national health laboratory service, cape town, south africa division of chemical pathology, faculty of health sciences, university of cape town, cape town, south africa siphiwe m. baloyi department of haematology, national health laboratory service, cape town, south africa division of clinical haematology, faculty of health sciences, university of cape town, cape town, south africa jody a. rusch department of chemical pathology, national health laboratory service, cape town, south africa division of chemical pathology, faculty of health sciences, university of cape town, cape town, south africa citation khumalo n, francis ca, baloyi sm, rusch ja. gamma gap and albumin-globulin ratio show poor sensitivity for monoclonal gammopathy screening in south africa. afr j lab med. 2025;14(1), a2505. https://doi.org/10.4102/ajlm.v14i1.2505 note: additional supporting information is available for download alongside the online version of this article as online supplementary document 1. original research gamma gap and albumin-globulin ratio show poor sensitivity for monoclonal gammopathy screening in south africa njabulo khumalo, cameron a. francis, siphiwe m. baloyi, jody a. rusch received: 22 may 2024; accepted: 10 dec. 2024; published: 26 mar. 2025 copyright: © 2025. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: monoclonal gammopathies, including multiple myeloma, present significant challenges in sub-saharan africa. diagnosis is often missed because of limited screening tools. the gamma gap and albumin-globulin ratio (agr) have been proposed as simple, cost-effective screening methods; however, their utility in settings with prevalent infectious and inflammatory diseases is unclear. objective: this study evaluated the diagnostic accuracy of gamma gap and agr in identifying patients who require further investigation for monoclonal gammopathies in south africa. methods: a retrospective analysis of 7946 patients who underwent investigations for monoclonal gammopathies at groote schuur hospital, south africa, between september 2015 and september 2022 was conducted. patients were classified based on monoclonal protein detection, and the gamma gap, agr, and multivariable models were evaluated for diagnostic performance. results: among the patients (median age: 61 years, 58% female [4632/7946] and 42% [3314/7946] male patients), 1231 had monoclonal proteins. a gamma gap cutoff of 46 g/l identified 35% of monoclonal cases (sensitivity), with 91% specificity and an area under the curve (auc) of 0.60. the agr showed a slightly better auc of 0.63, with 44% sensitivity and 80% specificity at a 0.85 cutoff. multivariable models incorporating age, sex, and hypogammaglobulinaemia improved performance, with the gamma gap model achieving an auc of 0.73, improving the sensitivity to 58%, with a specificity of 78%. conclusion: the gamma gap and agr showed low sensitivity and moderate specificity in screening for monoclonal gammopathies, highlighting the need for integrated diagnostic approaches combining clinical, demographic, and laboratory data to improve early detection in resource-limited settings. what this study adds: although cost-effective and widely available, gamma gap and agr have limited accuracy for screening monoclonal gammopathies when used alone in settings with prevalent infectious and inflammatory diseases. although the tests are good at ruling out monoclonality, they risk missing many true cases, delaying diagnosis and treatment. keywords: gamma gap; globulins; paraproteins; paraproteinaemias; albumin/globulin ratio; inflammation. introduction monoclonal gammopathies include both malignant conditions, such as multiple myeloma, plasmacytoma, and waldenström macroglobulinaemia, as well as the pre-malignant condition, monoclonal gammopathy of undetermined significance (mgus).1 these disorders are characterised by the abnormal proliferation of clonal plasma cells producing monoclonal immunoglobulins. multiple myeloma, the most serious and well-known form, often progresses from mgus and leads to complications such as anaemia, renal dysfunction, osteolytic lesions, and increased susceptibility to infections. despite advances in treatment, multiple myeloma remains incurable but manageable.2 multiple myeloma accounts for 1.3% of all malignancies and 15% of haematological malignancies, with an estimated 138 000 new cases diagnosed globally each year.3 however, the incidence and mortality rates are significantly higher in affluent regions like north america, western europe, and australia compared to less affluent regions such as sub-saharan africa, asia, and oceania.3 cowan et al.3 and rebbeck et al.4 suggest that this disparity may result from limited access to screening and diagnostic tools in resource-constrained settings. consequently, many cases of multiple myeloma go undetected or are diagnosed at advanced stages, leading to an underestimation of the true prevalence in regions such as south africa.3,4,5 screening for monoclonal gammopathies is critical for early detection, enabling timely intervention and reducing the risk of complications.6,7 this is particularly important in low-resource settings, where restricted access to advanced diagnostic tools contributes to delayed diagnoses and poorer outcomes.3,4 current screening and diagnostic methods, such as serum protein electrophoresis (spe), immunofixation, serum-free light chains (sflc), urine protein electrophoresis, and bone marrow biopsy, while effective, are costly and complex,8 limiting their accessibility in resource-poor environments.1 this underscores the need for simple, affordable, and reliable screening tools to determine the need for further investigation. the gamma gap and albumin-to-globulin ratio (agr), derived from routinely measured total protein and albumin concentrations, have been used as clinical screening tools for monoclonal gammopathies.9,10,11 their low cost and ease of calculation make them appealing options. however, their diagnostic performance, particularly across diverse populations, remains inadequately established. additionally, chronic infections, inflammatory conditions, and malnutrition can influence the gamma gap and agr, leading to inaccurate results.12,13,14 in regions such as sub-saharan africa, where the burden of infectious and inflammatory diseases is high, the gamma gap and agr may perform poorly as screening tools. south africa faces a significant burden of infectious and inflammatory diseases, including high rates of hiv, tuberculosis, viral hepatitis, and other chronic infections.15,16 this can result in missed true cases, delayed diagnoses, or wasted healthcare resources resulting from unnecessary investigations and follow-up for false positives, limiting the tools’ effectiveness. understanding these limitations is crucial to avoiding misdiagnoses and to optimising healthcare resources in already overburdened systems. assessing the performance of screening and diagnostic tools is crucial to guiding their clinical use and to support the development of more reliable methods. this study aimed to evaluate the diagnostic accuracy of the gamma gap and agr in identifying patients who required further investigation for monoclonal gammopathies in a population burdened by inflammatory and infectious diseases. we hypothesised that the two metrics would perform poorly within this population. methods ethical considerations ethical approval was obtained from the human research ethics committee of the university of cape town (reference no. hrec: 589/2023). data for this study were managed in accordance with the data management plan, adhering to data protection and privacy regulations. only data required for analysis were extracted and securely stored, and access was limited to authors. informed consent for individual inclusion was waived because of the use of data for secondary research purposes.17 the study adhered to international ethical standards, including the declaration of helsinki. study design this retrospective, observational study evaluated the diagnostic accuracy of the gamma gap and agr in classifying patients with suspected monoclonal gammopathies, with the goal of determining their effectiveness as screening tools in a real-world clinical setting. setting groote schuur hospital is an 893-bed tertiary teaching hospital in cape town, south africa. the hospital’s national health laboratory service laboratory is accredited to the iso 15189 standard, affiliated with the university of cape town, and provides extensive testing for monoclonal gammopathies for the public sector. data collection the data used in this study were extracted from the monoclonal gammopathy research group – university of cape town database (hrec: r042/2022) for subsequent analysis. study population the database comprised de-identified retrospective data for adult patients (≥ 18 years) who underwent investigations for monoclonal gammopathies in our laboratory. the data, sourced from the laboratory information system and analysers, included basic demographics (age and sex) and results from spe, immunofixation, total protein, and albumin conducted from 01 september 2015 to 30 september 2022. all patients with complete biochemical data were considered for inclusion. exclusion criteria included missing data or equivocal immunofixation results, necessary to classify patients into two outcome groups. cancelled tests, duplicate tests, and follow-up visits were also excluded to prevent verification or spectrum bias. hypogammaglobulinaemic patients underwent routine immunofixation to rule out small but clinically significant monoclonal proteins or interference. these samples were included in primary analysis but were excluded in sub-group analysis for comparison. laboratory analyses data were generated using routine clinical laboratory analysers and methods. the spe was performed using capillary zone electrophoresis on the minicap, and immunofixation was performed using agarose gel electrophoresis on the hydrasys 2 (both sebia, lisses, france). albumin and total protein were measured on the roche cobas© 6000 analyser (roche diagnostics gmbh, mannheim, germany) with a coefficient of variation of 5.02% for albumin and 3.95% for total protein. the laboratory processes spe requests from clinicians, and immunofixation is reflexively added by the pathologist based on clinical and biochemical information, detection of suspicious peaks, or the presence of hypogammaglobulinaemia. data analysis the monoclonal gammopathy research group – university of cape town database underwent a curation pipeline for checking and cleaning, with quality control checks to ensure accuracy and reliability for this study. statistical analyses were performed using python-based packages (python software foundation, beaverton, oregon, united states) in pycharm (jetbrains, prague, czech republic). figure 1 shows the flow of patients, and the algorithm used to categorise patients into two groups (‘group 0’ [no monoclonal protein] and ‘group 1’ [monoclonal protein detected]). patients were initially classified into three categories based on their spe results and local policy. those with ‘unremarkable’ spes, showing no suspicious peaks or irregularities (< 1 g/l), were placed in group 0. patients whose spe showed a suspicious peak required immunofixation and, based on the results, were placed into either group 0 or group 1. patients with equivocal immunofixation results were excluded to prevent misclassification. figure 1: flowchart depicting the stepwise categorisation of patients evaluated for monoclonal gammopathy at a tertiary hospital laboratory in south africa from 01 september 2015 to 30 september 2022. the patients were categorised into ‘group 0’ (no monoclonal protein detected), ‘group 1’ (monoclonal protein detected), and the ‘excluded group’ (exclusion criterion met). a total of 13 931 spes were assessed for eligibility. after excluding 5985 tests, 7946 unique patients were included. patients were then classified by immunofixation. 5675 patients did not require immunofixation and were assigned to group 0. from immunofixation, 1040 were negative for monoclonality and thus also categorised into group 0. positive immunofixation results categorised patients into group 1. the gamma gap was calculated as the difference between total protein and albumin concentrations (g/l). the agr was calculated as albumin divided by globulin, where globulin was equivalent to the gamma gap. normality of continuous variables was assessed using histograms, quantile-quantile plots, and the anderson-darling test. categorical variables were described as numbers, proportions, and percentages, and visualised using pie charts (online supplementary figure 1). continuous variables were expressed as median (interquartile range), and illustrated with histograms (online supplementary figure 2). chi-squared tests were used for categorical variables, and wilcoxon rank-sum for continuous variables. a p-value < 0.05 was considered significant. figure 2: receiver operating characteristic curves illustrating the ability of the gamma gap and albumin-to-globulin ratio (agr) to predict monoclonality in patients being evaluated for monoclonal gammopathy at a tertiary hospital laboratory in south africa from 01 september 2015 to 30 september 2022. (a) in the whole population; (b) by albumin (< 35 g/l) and protein (> 78 g/l) categories in the whole population; (c) by albumin and protein categories excluding hypogammaglobulinaemic patients; (d) illustrates the performance of the multivariable logistic regression models. continuous variables were z-score normalised, and categorical variables were encoded. receiver operating characteristic (roc) curves were constructed to evaluate diagnostic performance of gamma gap, agr, and logistic regression models, with area under the curve (auc) used as a measure of accuracy (online supplementary text 1). the optimal cut-offs were determined using the youden index, and sensitivity and specificity were calculated at these cut-offs. univariable logistic regression was performed to identify predictors of the outcome. multivariable models were then developed and optimised. akaike information criterion (aic) values were calculated to compare univariable and multivariable models. confusion matrices and classification reports were generated to assess model performance. subgroup analyses were performed to further evaluate factors that may influence metric performance. patients with hypogammaglobulinaemia were excluded, and subgroups based on albumin (< 35 g/l vs ≥ 35 g/l) and total protein (≤ 78 g/l vs > 78 g/l) categories, based on their respective reference intervals, were also examined. results patient characteristics a total of 13 931 records from 8705 unique patients were evaluated for eligibility (figure 1). after applying the inclusion and exclusion criteria, 5985 records were removed, leaving 7946 unique patients. these patients were categorised into ‘group 0’ (no monoclonal detected, n = 6715) and ‘group 1’ (monoclonal detected, n = 1231). continuous variables were distributed non-parametrically. a total of 58% of patients were female and 42% were male patients (table 1). sex distribution was more balanced (54% women and 46% men) among patients with monoclonality. the median (p25, p75) age was 61 (50, 71) years. table 1: population characteristics of patients evaluated for monoclonal gammopathy at a tertiary hospital laboratory in south africa from 01 september 2015 to 30 september 2022. total protein was higher in the monoclonal compared to no monoclonal detected group (75 [66, 89] vs 73 [67, 78] g/l, p < 0.001), while albumin was lower (36 [28, 41] vs 40 [34, 44] g/l, p < 0.001). the gamma gap was higher in the monoclonal group (36 [28, 55] vs 32 [28, 38], p < 0.001), while the agr was lower (0.97 [0.54, 1.39] vs 1.23 [0.92, 1.52], p < 0.001). among 615 hypogammaglobulinaemic patients, 41% had a monoclonal protein, while 59% did not (p < 0.001) (online supplementary table 1). approximately one-third of patients had low albumin (< 35 g/l; reference interval: 35 g/l – 52 g/l) and one-third had high total protein (> 78 g/l; reference interval: 60 g/l – 78 g/l). receiver operating characteristic curves the gamma gap demonstrated an auc of 0.60 (95% ci: 0.58–0.62), while agr (0.63 [95% ci: 0.61–0.65]) was slightly higher (table 2; figure 2a). at an optimal cutoff of 46 g/l for the gamma gap, sensitivity was 35% (95% ci: 33–38) and specificity was 91% (95% ci: 90–92). for agr, an optimal cutoff of 0.85 had a sensitivity of 44% (95% ci: 41–47) and specificity of 80% (95% ci: 79–81). table 2: diagnostic accuracy of gamma gap, albumin-to-globulin ratio, and multivariable logistic regression models for predicting monoclonality in patients evaluated for monoclonal gammopathy at a tertiary hospital laboratory in south africa from 01 september 2015 to 30 september 2022. including hypogammaglobulinaemic patients detracted from the predictive power of both metrics. excluding these patients improved the auc to 0.68 for both. performance was further influenced by clinically relevant albumin and total protein categories (table 2; figure 2b and 2c). the gamma gap and agr performed better in patients with low albumin (auc both 0.67) and even better when hypogammaglobulinaemic patients were excluded (auc 0.75 and 0.73). both metrics showed improved performance in patients with high total protein (auc 0.81 and 0.80). excluding hypogammaglobulinaemic patients did not affect performance in this subgroup. conversely, the metrics performed more poorly in subgroups with normal/raised albumin and low/normal total protein. excluding hypogammaglobulinaemic patients from these analyses improved the performance in the normal/raised albumin group (auc 0.61 and 0.62). logistic regression models two multivariable models were developed for the gamma gap and agr using an iterative process. univariable logistic regression indicated that gamma gap, agr, age, albumin, and total protein, as well as the categories sex, albumin, protein, and hypogammaglobulinaemia, were all predictors of the outcome (all p < 0.001; online supplementary table 2). during development, high correlation between gamma gap, agr, total protein, and albumin prompted development of two separate multivariable models: the ‘gap’ model and the agr model. initially, the gap model included gamma gap, age, sex, and categories for albumin, total protein, and hypogammaglobulinaemia. during optimisation, albumin and protein categories were excluded – adding complexity without improving predictive power (online supplementary table 3). similarly, the albumin category was excluded from the agr model. the final gap model, based on 5578 observations, converged successfully and explained 18% of the variance (pseudo r2 = 0.180), while log-likelihood was −1984.5, outperforming the null model (−2419.9). the likelihood ratio test confirmed the joint significance of predictors (p < 0.0001). the gap model achieved auc 0.73, sensitivity 58% and specificity 78% (table 2; figure 2d). the agr model performed similarly, with auc 0.71 (sensitivity 55% and specificity 81%). we then assessed how well the gamma gap and agr could predict hypogammaglobulinaemia (online supplementary table 4). at an optimal cutoff of 27 g/l, the gamma gap performed well, with auc 0.86, sensitivity 80% and specificity 83%. the agr was considerably less sensitive (57%) and specific (75%), with auc 0.71. model comparison the performance of the two multivariable models was compared with the univariable models for gamma gap and agr. the aic values were 3979 for the multivariable gap model and 4211 for the agr model, suggesting that the gap model provided a better balance between goodness-of-fit and complexity. in contrast, the aic values for the univariable models were much higher (6320 for the gap model, and 6655 for the agr model), indicating that the multivariable models offered superior performance despite their complexity. confusion matrices and classification reports showed that multivariable models achieved higher accuracy, with gap and agr models achieving 75% and 78% accuracy, compared to 69% and 75% for the univariable models, respectively (online supplementary figure 3; online supplementary table 5). discussion our findings highlight that the gamma gap and agr provide limited diagnostic accuracy for identifying monoclonal gammopathies in real-world clinical settings with a high burden of inflammatory and infectious diseases, such as in south africa. the low sensitivity of these tests indicates a significant number of positive cases may go undetected, delaying diagnosis and treatment. however, their high specificity ensures accurate identification of most patients without the condition, reducing false positives and unnecessary further investigations. while this enhances their utility for ruling out cases, sensitivity should be prioritised for screening purposes to ensure all cases are captured. the sub-group analyses we performed offer further insights. the diagnostic performance of the gamma gap and agr improved when albumin and total protein concentration categories were considered. including hypogammaglobulinaemic patients in the analysis confounded the results, as 41% of these patients had monoclonal proteins despite significantly lower total protein and gamma gap values. notably, a low gamma gap (< 27 g/l) was predictive of hypogammaglobulinaemia, indicating that both high and low gamma gap values should raise suspicion for monoclonality in appropriate clinical contexts. previous studies have shown disparities in diagnostic performance across different contexts. in an australian study, thakkinstian et al. reported higher accuracy for the gamma gap, with an odds ratio of 4.5 (95% ci: 3.8–5.4) at a threshold of 41 g/l.10 similarly, in a study performed at duke university medical center in the united states, dupuis et al. demonstrated a strong correlation between the gamma gap and monoclonal protein concentrations, emphasising its potential as a rapid, simple test in resource-limited settings.11 unlike this present study, both studies were conducted in well-resourced settings and did not address the influence of inflammatory and infectious diseases on test performance. in contrast, liu et al.13 observed limited sensitivity but high specificity for the gamma gap (auc of 0.64 [95% ci: 0.60–0.69], sensitivity of 15.4%, specificity of 95.4%, threshold of 40 g/l) in a united states community-based study that included patients with mgus, hiv, and hepatitis c. these findings align with our results, suggesting that diagnostic performance is compromised in settings with a high burden of infectious and inflammatory conditions. while we found that the gamma gap and agr perform similarly, few studies have specifically evaluated the agr in this context. a study by tarín-arzaga et al.18 in mexico included all spe results (n = 1578) from 255 individuals and found that the agr (auc = 0.74, sensitivity = 59%, specificity = 79%) performed better than the gamma gap (auc = 0.71, sensitivity = 52%, specificity = 80%) overall.18 however, when predicting a monoclonal protein ≥ 10 g/l, both metrics showed excellent diagnostic performance, with an auc of 0.94, and comparable sensitivity (84% vs 87%) and specificity (87% vs 84%) values, respectively. in south africa, where infectious and inflammatory diseases are prevalent,15,16 conditions such as hiv, tuberculosis, chronic hepatitis (b and c), rheumatoid arthritis, and systemic lupus erythematosus compete for attention in the differential diagnosis of monoclonal gammopathy. these conditions are often associated with abnormal protein levels and polyclonal hypergammaglobulinaemia. these changes increase the gamma gap and lower the agr, complicating clinical decisions on diagnostic pathways and additional testing, in resource-limited settings. careful clinical evaluation is essential to differentiate monoclonal gammopathy from these prevalent conditions. studies have reported inconsistent gamma gap thresholds. similar to our findings, a canadian study by suleman et al. found a low sensitivity (47.7%) but moderate specificity (77.1%) at a threshold of 40 g/l.19 when they evaluated the threshold of 44 g/l, the specificity improved to 89.2%, at the expense of sensitivity (33.7%).19 in contrast, pawar and hedge in india observed high sensitivity (82.1%) and specificity (85.4%) at a lower threshold of 32.5 g/l, concluding that the gamma gap reliably distinguishes monoclonality.20 however, their findings are limited by a small sample size (n = 147), despite being conducted in a similar low-to middle-income country setting with a high burden of infectious and inflammatory diseases (n = 147). furthermore, hassan and hameed in iraq found that, at a threshold of 29.1 g/l, the gamma gap distinguished treated from untreated multiple myeloma patients with an auc of 0.735 (95% ci: 0.66–0.904, p < 0.001), achieving 76.5% sensitivity and 70.6% specificity.21 in our study, multivariable models outperformed univariable models, although both demonstrated low precision and recall in detecting monoclonal cases, with the agr model performing slightly better. the gap model, however, showed a significantly lower aic value, indicating a better balance between model fit and simplicity. despite this, both models have limited diagnostic utility in isolation and would benefit from further optimisation by incorporating additional predictive factors. this highlights the importance of a comprehensive approach to accurately predict monoclonal gammopathies,13 as demonstrated by thakkinstian et al., who used five variables (gamma gap, age, sex, haemoglobin, and egfr) in their optimised model, achieving an auc of 0.80.10 this study was strengthened by confirmation of their findings in a validation data set (n = 4312). recommendations universal screening for monoclonal gammopathy in asymptomatic individuals is not recommended.20 this study aimed to evaluate the gamma gap and agr as supplementary tools to identify patients at risk of monoclonal gammopathy, rather than replacing specialised tests such as spe, immunofixation, sflc, or urine protein electrophoresis. these commonly requested and cost-effective tests may aid in risk stratification. evidence has consistently shown that combining tests enhances diagnostic sensitivity in monoclonal detection, with a combination of spe, immunofixation, and sflc providing the most accurate diagnostic outcomes.21,22,23,24,25 strengths this study leveraged a large real-world sample from a high-burden setting, with tests conducted on standard equipment and reviewed by pathologists, ensuring high-quality data. limitations several limitations may affect the generalisability of the findings, including the focus on patients already under investigation for monoclonal gammopathy, retrospective design, and absence of clinical details like comorbidities or medication use. excluding patients with equivocal immunofixation results, while reducing misclassification, further limits applicability in cases of inflammation or unclear findings. conclusion neither the gamma gap nor the agr demonstrates sufficient reliability as standalone screening tools for monoclonal gammopathy in settings with a high burden of infectious and inflammatory diseases. while both metrics showed moderate specificity, their low sensitivity highlights the risk of missed cases. these findings emphasise the need for a more integrated approach that combines clinical, demographic, and laboratory data, particularly in resource-limited settings. further research is essential to develop more effective screening strategies and explore the potential of combining these markers with other clinical parameters to improve diagnostic performance. acknowledgements the authors would like to extend their gratitude to vida van dyk and all the routine staff at the c17, chemical pathology laboratory of the national health laboratory service at groote schuur hospital, cape town, south africa, for conducting the laboratory tests used in this study. we would also like to thank the chemical pathologists and registrars for their work in reporting these tests. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions j.a.r. and s.m.b. conceived the research idea. n.k., j.a.r., and s.m.b. assisted in producing the protocol and applying for ethical approval. c.a.f. handled the data collection and data cleaning. j.a.r., c.a.f., and n.k. performed data analysis. j.a.r., s.m.b., c.a.f., and n.k. wrote and revised the article. sources of support this research did not receive any form of grant from any funding agency, in the government, commercial or non-profit sectors. data availability data are not available publicly but may be requested from the corresponding author, n.k., upon reasonable request and after consultation with the human research ethics committee of the university of cape town. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. the article does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the authors are responsible for this article’s results, findings, and content. references rajkumar sv, dimopoulos ma, palumbo a, et al. international myeloma working group updated criteria for the diagnosis of multiple myeloma. lancet oncol. 2014;15(12):e538–e548. https://doi.org/10.1016/s1470-2045(14)70442-5 palumbo a, anderson k. multiple myeloma. n engl j med. 2011;364(11):1046–1060. https://doi.org/10.1056/nejmra1011442 cowan aj, allen c, barac a, et al. global burden of multiple myeloma: a systematic analysis for the global burden of disease study 2016. jama oncol. 2018;4(9):1221–1227. https://doi.org/10.1001/jamaoncol.2018.2128 rebbeck tr. cancer in sub-saharan africa. science. 2020;367(6473):27–28. https://doi.org/10.1126/science.aay4743 finestone e, wishnia j. estimating the burden of cancer in south 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practice. cancers. 2022;14(12):1–11. https://doi.org/10.3390/cancers14122930 abstract introduction methods results discussion acknowledgements references about the author(s) faithful makita-chingombe international pharmacology specialty laboratory, school of pharmacy, university of zimbabwe college of health sciences, harare, zimbabwe anthony t. podany antiviral pharmacology laboratory, university of nebraska medical center, omaha, nebraska, united states timothy mykris antiviral pharmacology laboratory, university of nebraska medical center, omaha, nebraska, united states farai muzambi international pharmacology specialty laboratory, school of pharmacy, university of zimbabwe college of health sciences, harare, zimbabwe richard w. browne translational pharmacology research core, center for integrated global biomedical sciences, school of pharmacy and pharmaceutical sciences, university at buffalo, buffalo, new york, united states andrew j. ocque translational pharmacology research core, center for integrated global biomedical sciences, school of pharmacy and pharmaceutical sciences, university at buffalo, buffalo, new york, united states robin difrancesco translational pharmacology research core, center for integrated global biomedical sciences, school of pharmacy and pharmaceutical sciences, university at buffalo, buffalo, new york, united states lee c. winchester antiviral pharmacology laboratory, university of nebraska medical center, omaha, nebraska, united states courtney v. fletcher antiviral pharmacology laboratory, university of nebraska medical center, omaha, nebraska, united states tinashe mudzviti international pharmacology specialty laboratory, school of pharmacy, university of zimbabwe college of health sciences, harare, zimbabwe charles c. maponga international pharmacology specialty laboratory, school of pharmacy, university of zimbabwe college of health sciences, harare, zimbabwe translational pharmacology research core, center for integrated global biomedical sciences, school of pharmacy and pharmaceutical sciences, university at buffalo, buffalo, new york, united states gene d. morse translational pharmacology research core, center for integrated global biomedical sciences, school of pharmacy and pharmaceutical sciences, university at buffalo, buffalo, new york, united states citation makita-chingombe f, podany at, mykris t, et al. cross-validation of a high-performance liquid chromatography nevirapine plasma assay in a resource-limited setting in zimbabwe. afr j lab med. 2021;10(1), a1264. https://doi.org/10.4102/ajlm.v10i1.1264 brief report cross-validation of a high-performance liquid chromatography nevirapine plasma assay in a resource-limited setting in zimbabwe faithful makita-chingombe, anthony t. podany, timothy mykris, farai muzambi, richard w. browne, andrew j. ocque, robin difrancesco, lee c. winchester, courtney v. fletcher, tinashe mudzviti, charles c. maponga, gene d. morse received: 08 may 2020; accepted: 24 feb. 2021; published: 08 july 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract an international hiv pharmacology specialty laboratory (psl) was established at the university of zimbabwe to increase bioanalytical and investigator capacities. quantitation of plasma nevirapine in samples from the aids clinical trials group protocol 5279 was compared between the university of nebraska medical center psl and the university of zimbabwe psl. both psls employed internally developed methods utilising reverse-phase high-performance liquid chromatography with ultraviolet detection. eighty-seven percent of the cross-validation results exhibited ± 20% difference. keywords: cross-validation; hiv; root-cause analysis; high-performance liquid chromatography. introduction the university of zimbabwe (uz) international hiv pharmacology speciality laboratory (ipsl) was established to increase hiv pharmacology-related research and support research investigator capacity in africa.1 the uz-ipsl performs drug development assay and clinical research specimens analyses, specifically drug-drug interactions, pharmacokinetic and pharmacodynamic data analyses. the clinical pharmacology quality assurance programme, which monitors all psls within the aids clinical trials group, provided technical guidance to and developed a comparative nevirapine validation study for the uz-ipsl to analyse replicate hiv clinical samples. validation of the nevirapine bioanalytics method2 was essential at the time because of nevirapine use in the sub-saharan region and results were generated by a reverse-phase high-performance liquid chromatography (hplc) system using ultraviolet detection with gradient-elution separation.3,4,5 this report compares nevirapine bioanalysis at two laboratories: the united states-based, university of nebraska medical center pharmacology specialty laboratory (unmc-psl) and the uz-ipsl. the unmc-psl served as the aids clinical trials group 5279 (a5279) protocol designated laboratory. the unmc-psl, during the time of the a5279 study, participated in the clinical pharmacology quality assurance proficiency testing (pt) programme to assure nevirapine assay performance and accuracy. although each psl validated the nevirapine assay separately, the lack of data comparing the bioanalytical capacity of pharmacology laboratories in low-middle income countries using clinical samples (rather than spiked plasma samples) was the justification for this project. therefore, implementation of the project was the last stage in determining uz-ipsl’s readiness to begin assaying human samples for clinical trials. methods ethical considerations data files used in this comparison study were de-identified and clinical pharmacology quality assurance was blinded to any data associated with samples. this article followed all ethical standards for research without direct contact with human or animal subjects. project design the uz-ipsl utilised the a5279 replicate samples (n = 95) that were available at the uz college of health sciences clinical trial research center (uzchs-ctrc) for cross-validation of nevirapine assays between the unmc-psl and the uz-ipsl. a5279 was a multi-centre, phase iii clinical trial investigating the use of short-course rifapentine or isoniazid for the prevention of active tuberculosis in hiv-positive adults.6 in a5279, nevirapine pharmacokinetics when co-administered with rifapentine and isoniazid was one of the exploratory objectives. all a5279 primary aliquots collected from the uz aids clinical trials group ctrc were analysed for nevirapine by the protocol designated psl at the unmc-psl. a5279 sample collection and processing plasma samples for the quantitation of nevirapine concentrations were obtained from a5279 study participants. blood was collected into potassium ethylenediaminetetraacetic acid collection tubes and transported on ice to the processing laboratory within 1 h where the plasma was separated from cells by centrifugation (1200 × g, 10 min, 4 °c). the plasma aliquots were frozen (–70 °c) and shipped to the unmc-psl. the uzchs-ctrc retained secondary aliquots for the uz-ipsl cross-validation assay. university of nebraska medical center psl nevirapine assay a5279 primary plasma aliquots were shipped from uzchs-ctrc on dry ice to the unmc-psl for determination of the nevirapine. the unmc-psl nevirapine determination assay had a quantitation range of 25 ng/ml to 10 000 ng/ml. samples measuring above the quantitation range were diluted and reanalysed using a validated dilution protocol. solid-phase extraction was utilised to prepare samples for analysis. the unmc-psl utilised a waters e2695 hplc coupled to a waters 2489 ultraviolet (waters, milford, massachusetts, united states) detector, which was controlled with empower 2 (waters, milford, massachusetts, united states) software as the analytical platform. the performance of the assay was compliant with the united states food and drug administration bioanalytical guidelines for method validation.7 university of zimbabwe international hiv psl nevirapine assay replicate plasma aliquots (n = 95) from a5279 available at the uzchs-ctrc were shipped to the uz-ipsl for determination of the nevirapine concentration for cross-validation. the uz-ipsl measured nevirapine using a validated hplc-ultraviolet assay detailed previously.2 the uz-ipsl chromatographic system consisted of a shimadzu lc20a hplc using ultraviolet photodiode array detection (model spd-m20a) and labsolutions software (version 5.8; kyoto, japan). the assay quantitation range was 500 ng/ml to 15 000 ng/ml. samples that were above the quantitation range were diluted and reanalysed. the performance of the assay was compliant with united states food and drug administration bioanalytical guidelines. peak purity for nevirapine was assessed to confirm the absence of interferences from potential impurities also extracted from the patient’s sample. a5279 samples were assayed over five runs; the two last runs included samples that were above the upper limit of quantification and were diluted (1:8) according to the validated dilution protocol.2 data management and analysis the uz-ipsl nevirapine results were submitted to the aids clinical trials group data management center portal using the data submission utility (frontier science foundation inc., brookline, massachusetts, united states). the a5279 study team approved the release of the unmc a5279 nevirapine concentration results for comparison with the uz-ipsl results. the original nevirapine concentration data from the unmc-psl were used in the specified a5279 protocol pharmacokinetics analysis; no uz-ipsl derived pharmacokinetics data were used in the per-protocol study analysis. the data management center compiled the data and provided the de-identified data files to the clinical pharmacology quality assurance pt unit to perform blinded, comparative statistical analyses of the nevirapine results. after examination (omnibus 2) and finalisation of the data, the analyses of the paired t-test, deming regression and bland-altman analyses were completed using prism graphpad (version 8.3; san diego, california, united states) software. results ninety-five replicate plasma aliquots were bioanalysed. the nevirapine concentration range in the analysed samples was ~2000 ng/ml – 34 000 ng/ml (~17-fold). of this, 97% (n = 92) ranged between 2000 ng/ml and 20 000 ng/ml and the remaining 3% (n = 3) were above 20 000 ng/ml and not contiguous (table 1, figure 1a)9. the nevirapine concentration values from the laboratories were tested for normality and only data ≤ 15 000 ng/ml or less were normally distributed. data 15 000 ng/ml reflected the upper limit of the uz-ipsl assay and was appropriate because it more closely represented the previously reported nevirapine steady state concentrations (5086 ng/ml – 13 368 ng/ml or 19.1 um to 50.2 um).3,4,5 of the data, 93% (n = 88) were 15 000 ng/ml or less (table 1)9 while 7% (n = 7) were above this value. therefore, the seven sample pairs were excluded from the method comparison. after exclusion, an additional sample pair was identified as a significant outlier using the grubbs test and was also excluded. figure 1: differences in nevirapine concentrations assayed at unmc-psl (omaha, nebraska, united states, october 2017) and uz-ipsl (harare, zimbabwe, may 2018); (a) scatter plot (n = 95 data points). (b) deming regression plot of 87 data points (outliers excluded). boxes in (a) indicate unmc upper limit of quantitation (dashed) and uz-ipls upper limit of quantitation (solid lines). table 1: a comparative study of nevirapine assay results conducted at the university of zimbabwe international hiv pharmacology specialty laboratory (harare, zimbabwe, may 2018) and the university of nebraska medical center pharmacology specialty laboratory (omaha, nebraska, united states, october, 2017). the two-tailed paired t-test of the final data set (n = 87) (table 1)9 showed uz-ipsl nevirapine values were significantly higher with a constant error of 3% – 4% more than unmc-psl values. the deming regression analysis (figure 1b)9 and the bland-altman plots (figure 2)9 further confirmed the higher values from the uz-ipsl. as shown in figure 2a9, regression analysis using the difference plot indicated an increasing proportional difference between the clinical pharmacology laboratories as the concentration of nevirapine increased. however, when the data were normalised by concentration, using the % difference analysis (figure 2b)9, the correlation was no longer significant. figure 2: differences in nevirapine samples (n = 87) assayed at unmc-psl (omaha, nebraska, united states, october 2017) and uz-ipsl (harare, zimbabwe, may 2018). (a) bland-altman difference plot indicating proportionally higher difference at high concentrations (mean difference = −430). (b) bland-altman percentage difference on normalisation by concentration, correlation not significant (mean % deviation = −4.49). shaded areas present confidence interval limits for mean (green shade) and agreement limits (red shade). discussion drug concentrations for nevirapine reported in the literature for hiv-positive individuals indicate a steady state maximum of 5740 ng/ml (5000–7440) and minimum of 3730 ng/ml (3200–5080)8. both nevirapine assays from the two laboratories can detect concentrations that might be below the target concentration for virologic suppression or above the upper limit of the therapeutic range. although the uz-ipsl nevirapine concentrations were higher than those of unmc-psl and this difference was statistically significant, it is unlikely to have clinical significance. the united states food and drug administration does not provide a window of acceptance for cross-validation studies but allows laboratories to determine a priori the acceptance criteria. using the established acceptance window for clinical pharmacology quality assurance pt samples, about 20% of the final target value, and expected concentrations of nevirapine, this difference was acceptable. the outcomes of the cross-validation also presented an opportunity to perform a root-cause analysis. the uz-ipsl’s trend of pt outcomes before and during the a5279 sample analysis period was explored. while all pt results were satisfactory, agreement of initial pt sample analyses showed unbiased accuracy, while during subsequent analyses which coincided with a5279 sample analysis, the uz-ipsl values exhibited a biased, high trend (4% – 19%) as compared to the final target values of that pt round. this shift in bias and accuracy was provided within the pt report and the uz-ipsl was able to complete a root-cause analysis and remediate as needed. limitations one limitation to consider is that the calibration ranges for the two equipment sets used for the nevirapine analysis were not identical. future comparisons of this nature may benefit from evaluations where the calibration range is the same for both laboratories. in addition, limited data points above 20 000 ng/ml entailed difficulties in quantitation of proportional error at that level. conclusion the cross-validation study provided evidence that the uz-ipsl performance using a nevirapine assay that was validated in their laboratory was acceptable and correlated with the results of an experienced psl. this study afforded the uz-ipsl a valuable opportunity to implement operations using its validated nevirapine assay for the analysis of samples obtained from a clinical research protocol and adopt procedures for handling of protocol specimens based on experience. furthermore, the outcomes of the cross-validation emphasised the value of pt and provided an occasion to perform root-cause analysis. acknowledgements the uz-ipsl acknowledges the contribution of dr marshall munjoma for his support during the laboratory work that led to the development of this manuscript. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions f.m.-c. was the project leader and f.m. was the laboratory technologist. t.mudzviti and c.c.m. provided manuscript review and supervised the uz-ipsl activities for the project. a.t.p. was clinical pharmacologist and c.v.f. was the psl director and a5279 pharmacology investigator. t.mykris and l.c.w. were laboratory technologists. a.j.o., r.w.b., r.d. and g.d.m. provided clinical pharmacology quality assurance project design and data analysis. all authors critically revised intellectual content and approved the final version to be published. sources of support university of zimbabwe ipsl: project was funded by the united states national institute of allergy and infectious diseases, national institutes of health (aids clinical trials group grant 5um1ai106701-04). university of nebraska psl: project was funded by the united states national institute of allergy and infectious diseases, national institutes of health, department of health and human services awards atp (k23 ai134307), and cvf (um ai106701). clinical pharmacology quality assurance program: this project has been funded in whole or in part with federal funds from the united states national institute of allergy and infectious diseases, national institutes of health and the department of health and human services, under contract numbers hhsn272201500006c and hhsn272200800019c. center for aids research: this publication was made possible through core services and support from the university of rochester center for aids research, a united states national institutes of health-funded programme (p30 ai078498). data availability data are available from (1) clinical pharmacolog quality assurance program translational pharmacology research core, center for integrated global biomedical sciences, school of pharmacy and pharmaceutical sciences, university at buffalo, state university of new york, buffalo, new york, united states, (2) international psl, school of pharmacy, university of zimbabwe college of health sciences, po box a178, avondale, harare, zimbabwe and (3) antiviral pharmacology laboratory, university of nebraska medical center, omaha, nebraska, united states. disclaimer the content is solely the responsibility of the authors and does not necessarily represent the official views of the united states national institutes of health. references mtisi tj, maponga c, monera-penduka tg, et al. strategic establishment of an international pharmacology specialty laboratory in a resource-limited setting. afr j lab med. 2018;7(1):a659. https://doi.org/10.4102/ajlm.v7i1.659 makita-chingombe f, ocque aj, difrancesco r, et al. development and validation of a high performance liquid chromatography method to determine nevirapine in plasma in a resource-limited 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httrps://doi.org/10.1056/nejmoa1806808 united states food and drug administration. guidance for industry: bioanalytical method validation. rockville, md: fda; 2001. nevirapine pk fact sheet [homepage on the internet]. 2016 [cited 2020 mar 19]. https://liverpool-hiv-hep.s3.amazonaws.com/prescribing_resources/pdfs/000/000/059/original/hiv_factsheet_nvp_2016_mar.pdf?1520612265 clinical pharmacology quality assurance, proficiency testing unit. 2019. blind nevirapine comparison report [not published]. article information author: edward m. kamau1 affiliation: 1special programme for research and training in tropical diseases, world health organization correspondence to: edward kamau postal address: c/o oms/tdr, avenue appia 20, geneva, 1211, switzerland dates: received: 26 apr. 2012 accepted: 09 apr. 2013 published: 27 aug. 2013 how to cite this article: kamau em. navigating laboratory services quality in challenging environments: a perspective for implementation in small, low-income countries and post-conflict settings. afr j lab med. 2013;2(1), art. #48, 4 pages. http://dx.doi.org/10.4102/ ajlm.v2i1.48 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. navigating laboratory services quality in challenging environments: a perspective for implementation in small, low-income countries and post-conflict settings in this original research... open access • abstract • introduction    • background • rationale for investing in constrained settings • the quality systems approach    • initiating a national laboratory quality system • basic characteristics in challenging environments    • organisation    • equipment    • purchasing and inventory    • records and process management    • information and occurrence (non-conformance) management    • assessment, process improvement and customer focus    • personnel and work environment • challenges • conclusion • acknowledgements    • competing interest • references abstract top ↑ the need to establish and maintain good laboratory practices is recognised universally. however, due to differences in resources available for health services in different countries, allocation of financial and human resources in poor countries is severely constrained. the constraints faced by poor countries call for innovative approaches that would guarantee the minimum acceptable quality while striving to meet the highest standards. in resource-limited setting, it may be justifiable to develop and use ‘fit for purpose’ quality standards based on internationally-recognised laboratory quality management frameworks or protocols. introduction top ↑ background under ideal conditions, medical practitioners rely on the use of quality laboratory data for evidence-based medical decision-making. public health programme officials also rely upon laboratory data in order to detect outbreaks of disease through laboratory surveillance, determine policy for the implementation of disease-control measures, monitor disease, and determine the impact of control programmes. furthermore, there is a heightened awareness of the importance of public health and clinical laboratories in ensuring that society is protected from re-emerging infectious agents and recurrent epidemics and pandemics.1 although the existence of laboratory infrastructure is a prerequisite for the generation of specific laboratory parameters, quality outcomes result from adherence to national laws and guidelines; and of course, appropriate leadership and management are critical. in addition to laws and regulations, the medical device industry, governments, world health organization, non-governmental organisations and professional societies should work together to develop quality standards leading to an improvement of testing and assurance with regard to the quality of laboratory data.2 these laws and systems help ensure that all laboratory testing is of the highest quality. rationale for investing in constrained settings top ↑ the key driving force to strengthen laboratory capacity in resource-constrained countries is to increase accuracy and reliability of data for diagnosis, treatment and control of diseases. in most, if not all, of the small, low-income countries in africa,3 there is an urgent and overwhelming need to address not only laboratory quality standards but also the inadequate technical and human capacity to deliver services, weak integration amongst disease control and prevention programmes and low appreciation of contemporary developments in laboratory diagnosis. furthermore, some countries face an additional challenge with respect to their official and/or national language, and greater support for francophone and lusophone countries by providing training and documentation in the major languages of the continent will help to address inequities in health research, as well as creating a unique area of work for the african society for laboratory medicine (aslm). supporting african nations should achieve the objectives of equal treatment and comparison of countries in similar situations.4 low-income countries and post-conflict settings present the greatest challenges as well as opportunities in laboratory medicine. with the appropriate tools and implementation of standards, laboratory professionals can demonstrate the contribution that high quality laboratory services make in improving the health status of the population. the evidence for improvement would come from data on the detection, management, prevention and research for neglected diseases. in the area of neglected tropical disease, a modest investment is likely to have maximum impact. there may be no ‘one size fits all’ solution to challenges in laboratory service quality, and each country or situation needs thorough scrutiny, as routine approaches that have worked well elsewhere may not be appropriate. at the same time, major challenges remain: overcoming the main infectious diseases; inadequate research capacity; the increasing burden of non-communicable diseases; and a constantly changing economic situation. countries or regions emerging from armed conflict are in dire need of quality laboratory services. there is also a need to motivate countries to prioritise laboratory services in their national health plans, to take advantage of new funding mechanisms and to commit more domestic resources to health care services. the implementation process is based on the following quality systems approach: 1. acknowledgement of the need to improve the laboratory services at all decision-making levels within countries and to articulate potential health and economic benefits. 2. assessment of capacity, infrastructure and training needs. 3. national consensus meeting of all stakeholders, including the development of guidelines and policy documents. 4. identification and designation of a national quality assurance laboratory or office and leadership structure. 5. allocation of resources to the maintenance of quality requirements. 6. development and provision of technical training and supervision. 7. participation in national and regional external quality control programmes that promote the implementation of corrective and preventative action plans that can be validated. 8. participation in an accreditation programme. this step-wise process is not specific to addressing laboratory services in the target settings only; it would be applicable wherever there are efforts to improve the quality of laboratory testing for any disease. the quality systems approach top ↑ the implementation of quality practices requires a systematic approach in a comprehensive and coordinated effort to meet quality objectives. quality assurance (qa) is focused on providing confidence that quality requirements will be fulfilled. in most resource-constrained countries, there has been little coordination towards objectives and almost no resources are available to implement activities that would ensure quality laboratory services.to achieve quality results, it is crucial that everyone who is involved in the process of laboratory testing is part of the quality process, from the person collecting specimens to the person making use of the test results. the purpose of a quality system is to avoid errors, provide consistent performance, ensure the integrity of data, increase efficiency and cost-effectiveness, provide customer satisfaction, training opportunities, and build credibility for the laboratory service on offer. in addition, all aspects of the testing process, covering the entire quality assurance cycle of pre-analysis, analysis and post-analysis (see figure 1), must be addressed. figure 1: model quality assurance cycle. initiating a national laboratory quality system the basis for addressing laboratory quality issues should be to develop a specific team with responsibility for implementing activities that will strengthen the capacity of the laboratory infrastructure. based on information gathered from questionnaires or country-specific national laboratory services reports submitted by disease control units, and at the invitation of the ministries of health (moh), stakeholders in laboratory services could initiate an assessment of laboratories by holding a meeting to review the country’s laboratory diagnosis action plan (if any is available) or developing such a plan for quality system implementation.5 this first step in implementing support for a quality laboratory system ensures a commitment on the part of the moh toward strengthening laboratory capacity. this commitment is essential for effective and relevant change and to begin the process of addressing organisational structures that will contribute to the laboratory’s ability to provide quality services.subsequent to obtaining the appropriate commitment, a detailed assessment of the current laboratory system and practices in the country, which should give special attention to identifying the weakest link(s) in the quality system, will determine the most critical gaps and enable priorities to be established for addressing those gaps. the assessment should not only take into account the national, provincial and peripheral laboratory infrastructure, but should also determine the management and communication practices between the laboratory personnel and medical practitioners. furthermore, relationships with the formal higher education sector (universities and medical training colleges) will need to be established, in order to influence and adopt curricula to meet the country’s needs and to engage these institutions in refresher and on-the-job training programmes. the assessment report should indicate clearly what actions are necessary and the time frame required for implementation of the quality system. more often than not, the first tangible activity would be to provide training on quality systems development to a core team of potential trainers. this is based on the realisation that most laboratory managers and technologists endeavour to provide quality results but have limited information regarding what steps need to be taken in order or to what benchmarks activities are to be performed. in parallel with the above activities, it would be necessary to address other specific technical training needs such as the selection and validation of tests, training of those likely to perform laboratory testing and preparation for the establishment of a national integrated laboratory service. a major requirement would be to ensure that there are competent personnel who have the mandate to implement quality systems. another critical component would be the creation or promotion of a central national laboratory to be the reference centre for the country or region with the responsibility of developing and reviewing standardised operating procedures (sops), selecting laboratory equipment and providing qa support, supervision, monitoring and evaluation. basic characteristics in challenging environments top ↑ the following are essential features for the organisation and management of a quality laboratory system. organisation the organisation component of quality systems implementation involves the planning and organising of the quality programme, defining the scope of authority and responsibility of staff and the allocation of resources to sustain quality requirements. in addition, job descriptions, training and orientation, continuing professional education, and competence and performance appraisal would provide confidence that quality standards will be met and upheld. equipment quality control (qc) of equipment includes selection of appropriate instruments and ensuring correct operation, providing for installation and initial calibration, and establishing maintenance mechanisms, including service contracts. routine servicing, repairing and provision of information for troubleshooting and regular review of documentation are essential with regard to maximising the useful life of laboratory equipment. purchasing and inventory it is essential to define criteria for products and services to be purchased, as well as to establish a system for the receiving, inspecting, accepting or rejecting, storing and recording of all incoming and outgoing materials. the ability to assess and maintain the inventory, in addition to establishing a system to link supplies to their users, activities or specific records, would enhance the development of an efficient procurement management system. records and process management establishing records and process management requires developing standardised document formats, systematic revision, approval and distribution, as well as managing patient test records, storage, retrieval and destruction systems. providing an oversight on all laboratory operations such as methodology evaluation, validation procedures, sops, qc and external quality assessment are essential for process control. information and occurrence (non-conformance) management information and occurenace management requires the management of incoming and outgoing information, standardisation of information capture, maintaining the confidentiality of patient information and ensuring competency in the appropriate information technology skills. timely and effective resolution of laboratory errors minimises the negative effects that such occurrences can have on the integrity of laboratory services. assessment, process improvement and customer focus regular evaluation of the entire qa cycle and the systematic evaluation of all laboratory procedures must be performed in order to ensure continued improvement in the quality of laboratory services. it also involves proactive gathering of information on customer satisfaction through surveys and feedback channels, as well as the use of said information to improve, recognise and reward the staff who provide a quality service. personnel and work environment the provision of adequate facilities, working and storage areas enhances reliable testing and ensures a safe working environment. challenges top ↑ one of the main constraints associated with laboratory quality systems in challenging circumstances is the failure to realise that, as a public good, laboratory services should be achieved equitably and to the highest attainable level, making the case for the hard-to-reach populations a daunting task with regard to the provision of patient services that are comparable in quality to those offered elsewhere. furthermore, there is often the reluctance to hold a routine laboratory to the same or higher standards as the reference laboratories, coupled with the common qa weaknesses that are found even in well-resourced settings. it is worth noting that a laboratory service system is only as strong as its weakest link and identifying the link(s) where there is potential for maximum impact of limited resources is not always straightforward. conclusion top ↑ the strengthening or establishment of a laboratory services quality system in challenging environments and, in particular, in small, low-income countries or regions and those emerging from armed conflicts, is clearly an important goal and the activities outlined above are achievable. this approach is in line with the promotion of the point-of-care platform by aslm for low income and lower-to-middle income countries, as it will require taking a step further up the ladder of hard-to-reach populations. for some of these settings, a modest improvement can help to address some of the complaints that may have precipitated the conflict whilst restoring devastated services and achieving the objective of equal treatment and comparison with countries or areas in similar situations. however, the implementation and improvements in laboratory services cannot be addressed in isolation. the use of evidence-based decision making in both clinical practice and in public health will require a change in attitude to one that values laboratory data. finally, an improvement in laboratory infrastructure alone will not be beneficial unless similar or greater attention is given to the broader healthcare system. acknowledgements top ↑ the author is grateful for comments received from reviewers and journal editors. all activities leading to the publication of this article did not require any financial support. competing interest the author declares that he has no financial or personal relationship(s) that may have inappropriately influenced him in writing this article. the views expressed by the author in this article do not reflect the views of his affiliated institutions. references top ↑ 1. world health organization. laboratory biosafety manual. 3rd ed. geneva: who; 2004.2. world health organization south-east asia region and western pacific region. laboratory quality standards and their implementation. geneva: who; 2011. 3. economic and social council. committee for development policy: report on the 7th session (14–18 march 2005). supplement no. 13. new york: un publications; 2005. 4. whitman g. who’s fooling who? the world health organization’s problematic ranking of health care systems. cato institute briefing papers no. 101, february 28, 2008. [page on internet]. [c2008] [cited 2011 apr 13]. available from: www.cato.org/publications/briefing-paper/whos-fooling-who-world-health-organizations-problematic-ranking-health-care-systems 5. world health organization south-east asia region and western pacific region. development of national health laboratory policy and plan. geneva: who; 2011. 6. centers for disease control and prevention. a modified pictorial representation: laboratory quality assurance and standardization programs. [page on internet]. [n.d.] [cited 2013 july 20]. available from: htttp://www.cdc.gov/labstandards article information authors: julia driessen1 henry limula2 oliver j. gadabu3 gervase gamadzi2 edwin chitandale2 anne ben-smith4 noor alide2 gerald p. douglas5 affiliations: 1department of health policy and management, university of pittsburgh, pittsburgh, united states 2kamuzu central hospital, ministry of health, lilongwe, malawi 3baobab health trust, lilongwe, malawi 4department of biomedical informatics, university of pittsburgh, united states 5center for health informatics for the underserved, university of pittsburgh, united states correspondence to: julia driessen email: driessen@pitt.edu postal address: 130 de soto street, a614 crabtree hall, pittsburgh, united states dates: received: 10 mar. 2014 accepted: 13 apr. 2015 published: 11 june 2015 how to cite this article: driessen j, limula h, gadabu oj. informatics solutions for bridging the gap between clinical and laboratory services in a low-resource setting. afr j lab med. 2015;4(1), art. #176, 7 pages. http://dx.doi.org/10.4102/ajlm.v4i1.176 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. informatics solutions for bridging the gap between clinical and laboratory services in a low-resource setting in this original research... open access • abstract • introduction    • problem statement       • key focus       • contribution to field • research method and design    • setting    • procedure    • ethical considerations • results • discussion    • limitations of the study    • recommendations    • conclusion • trustworthiness    • reliability and validity • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: there has been little formal analysis of laboratory systems in resource-limited settings, despite widespread consensus around the importance of a strong laboratory infrastructure. objectives: this study details the informational challenges faced by the laboratory at kamuzu central hospital, a tertiary health facility in malawi; and proposes ways in which informatics can bolster the efficiency and role of low-resource laboratory systems. methods: we evaluated previously-collected data on three different aspects of laboratory use. a four-week quality audit of laboratory test orders quantified challenges associated with collecting viable specimens for testing. data on tests run by the laboratory over a one-year period described the magnitude of the demand for laboratory services. descriptive information about the laboratory workflow identified informational process breakdowns in the pre-analytical and post-analytical phases and was paired with a 24-hour sample of laboratory data on results reporting. results: the laboratory conducted 242 242 tests over a 12-month period. the four-week quality audit identified 54% of samples as untestable. prohibitive paperwork errors were identified in 16% of samples. laboratory service workflows indicated a potential process breakdown in sample transport and results reporting resulting from the lack of assignment of these tasks to any specific employee cadre. the study of result reporting time showed a mean of almost six hours, with significant variation. conclusions: this analysis identified challenges in each phase of laboratory testing. informatics could improve the management of this information by streamlining test ordering and the communication of test orders to the laboratory and results back to the ordering physician. introduction top ↑ problem statement the healthcare challenges faced by resource-limited countries require an efficient and accessible laboratory infrastructure. laboratory testing plays an irreplaceable role in the diagnosis and treatment of diseases such as hiv, tuberculosis, and malaria.1 monitoring the progression of hiv through laboratory measures such as viral load and cd4 counts is important for managing patients on antiretroviral therapy (art) and identifying treatment failure. the serious public health threat of drug-resistant tuberculosis also requires laboratory testing for drug sensitivity.2 whilst rapid diagnostic tests are available for malaria and are in use in many health facilities, many countries still consider microscopy performed in the laboratory as the gold standard because it detects a wider array of species.3 if laboratory tests are not available or used, there is a substantial risk of inappropriate treatment, which harms the patient, wastes already limited resources and contributes to increased drug resistance.4,5 despite the need for laboratory testing in addressing the infectious disease burden, many laboratories are ill-equipped to play a central role in the diagnostic and care delivery process in resource-constrained countries.6 as a result of poor laboratory services and staffing, there is a diagnostic culture amongst physicians of circumventing laboratory testing and using other, less reliable signals to diagnose diseases such as malaria, or treating for malaria despite a negative laboratory result.7,8,9,10 in many low-income countries, there is reluctance to order tests such as a sputum microscopy for tuberculosis, since the patient often dies before the results are received.11 in malawi, for example, 40% of hospitals have only one trained laboratory technician.12,13 malawi's national art programme has relied primarily on clinical criteria for art treatment initiation.14 whilst compelling, these anecdotal insights and macro-level measures offer an incomplete picture of laboratory testing in low-resource settings and do not indicate how these shortages impact healthcare delivery and health outcomes. for example, investing in additional microscopes may have a limited effect if the necessary reagents are unavailable or physicians rarely order tests that require microscopes. a better understanding of the micro-level dynamics of laboratory testing, as well as the role of the laboratory within a low-resource health system, can identify high-value steps to laboratory strengthening. key focus laboratory testing is typically described as a three-stage process: pre-analytical, analytical and post-analytical.15,16 most studies of laboratory testing, both in lowand high-resource settings, focus on the analytical phase, or the analysis step within the laboratory; it has also been noted that the preand post-analytical phases are common sources of delays and errors.17 understanding the dynamics of a low-resource laboratory will also highlight the potential role of informatics in bolstering the efficiency of the laboratory system. laboratory testing is both materially and informationally intensive. whereas most coverage of laboratory systems in low-resource settings focuses on the material needs, such as sufficient quantities of reagents and working centrifuges, the information burden is just as demanding, but far less well-understood. informatics streamlines the management and transfer of data and thus may be appropriate for addressing these information barriers. however, better evidence is needed around the informational demands and barriers experienced in laboratories in low-resource settings. one problem where informatics solutions have been employed in low-resource settings is in improving clinic access to centralised laboratory testing results.18,19 other systems have been implemented in facilities with on-site laboratories in order to improve management of laboratory histories. in most cases, these systems involve data entry of paper forms, with the goal of maintaining accurate patient histories.19 contribution to field in this article we present detailed data on the role and workflow of clinical laboratory testing at kamuzu central hospital (kch), an 800+ bed tertiary health facility in lilongwe, malawi's capital city, with the goal of understanding other ways that informatics can be used to address laboratory challenges. kch has been an incubator for developing informatics interventions in the clinical setting since 2001. the hospital has an extensive local area network connecting more than 60 computers across the hospital campus. this is, in turn, connected to a dedicated power backup system and linked to a wireless metropolitan area network spanning greater lilongwe. aspects of this work have been described elsewhere.20,21,22 laboratory systems strengthening and the promotion of good laboratory practices are supported by a number of technical partners and the laboratory is currently in the process of preparing for an audit under the framework of strengthening laboratory management toward accreditation (slmta). four different aspects of the laboratory are assessed: (1) the demand for laboratory testing in the hospital; (2) the burden of untestable samples; (3) the process of laboratory testing in a typical patient visit; and (4) the time required for reporting of results. research method and design top ↑ setting kch is a tertiary care facility in the capital city of malawi. it serves an area of over four million people, with approximately 50 000 admissions and 245 000 outpatient visits per year.23 the laboratory at kch houses nine different departments: haematology, parasitology, microbiology, molecular biology, serology, flow cytometry, biochemistry, histology and blood bank. the laboratory operates continuously with 27 professional staff, although staffing levels are reduced at nights and on weekends. procedure we analysed previously-collected data from laboratory records and the results of studies done as part of a quality improvement effort by the kch laboratory. in 2009, a quality audit was performed on the samples sent for testing to the kch laboratory over a four-week period.24 a total of 3549 samples were evaluated for completeness of the test orders and viability of the samples. if a test order or sample was deemed untestable, the reason for this classification was noted. issues with test orders included incorrect or incomplete forms and unlabeled samples. samples were classified as non-viable if the sample quantity was insufficient, or if the samples were clotted, haemolysed, too old, or in the wrong container. results were stratified by department. an additional step as part of this quality improvement effort was to analyse the time required for results reporting. this consisted of tracking the amount of time that results were waiting in the laboratory for pick-up over a 24-hour period. a total of 25 patients had test orders sent to the laboratory during this observation period. no patient details were captured for these samples. information about test volume was obtained from the kch laboratory for the time period july 01, 2010 to june 30, 2011. this information is routinely collected by the laboratory and included, for each assay conducted, the total number of tests performed per month. overall test volume indicates a conservative estimate of the demand for testing in this setting, since some tests could not be performed because of equipment malfunctions and/or reagent shortages. the demand for certain assays is evidence of the types of pressures faced by the laboratory, since different assays have different time sensitivities and testing demands. we describe the critical steps of the laboratory testing workflow that involve information transfer. the three phases of testing are commonly broken down into nine discrete steps (order, collection, identification, transportation, preparation, analysis, reporting, interpretation, action); and we define the stages within kch, specifying for each step the location and staff members involved, as well as other pertinent details.15 the goal of this exercise is to identify the informational demands and potential process breakdowns in clinical laboratory testing at kch to better understand the potential role of informatics. ethical considerations this article describes a rationale for introducing informatics interventions to improve the quality of laboratory services in low-resource settings. the motivation is supported by results from previously-conducted quality improvement audits. no primary research was conducted, thus the work described here does not meet the criteria for requiring institutional review board approval. results top ↑ of the 3549 samples evaluated as part of the quality audit, 54% (n = 1923) were not testable (table 1). there was variation in this rate across the departments within the laboratory, ranging from 5% of samples for microbiology to 70% of samples sent to the blood bank department. an insufficient sample volume was the most common reason for a sample being deemed untestable (n = 1606). this characterised over 80% of untestable samples (n = 1923) and 45% of all audited samples (n = 3549). of the high number of samples that were considered untestable because of insufficient blood volume, the vast majority came from the paediatric department, where it is challenging to get sufficient blood from a sick and frequently dehydrated infant. test order forms were filled out either incorrectly or incompletely in just over 16% (n = 591) of audited samples. the least frequent problems identified were samples that were mixed up, too old, or in the wrong container. table 1: results of 2009 quality audit. between july 01, 2010 and june 30, 2011 the kch laboratory conducted 242 242 tests (table 2). the number of tests carried out for the parasitology and blood bank departments accounted for over half of the laboratory's total test workload during this time. the most common tests were: malaria parasites; full blood count; blood grouping and cross matches; and cd4 count. there was considerable monthly variation in the number of tests conducted, reflecting the seasonality of diseases such as malaria. the average monthly test load was 20 187 tests, with a standard deviation of 3263 tests. there were several classes of tests, including blood lipids and hormones, which were not conducted at all during this time period because of inoperative equipment and/or lack of reagents. table 2: kamuzu central hospital laboratory test workload, july 01, 2010 – june 30, 2011. figure 1 defines the total testing process workflow at kch according to the commonly-specified nine stages of laboratory testing, and indicates the staff responsible for the task.15,25 the process begins and ends on the patient ward with the physician, who makes the initial request for a laboratory test and also determines a course of action based on the interpretation of the test results. the pre-analytical phase is initiated with the ordering of a test and also includes the sample collection and identification or matching of the sample with the patient. these two tasks are both completed by a clinician or nurse and are associated with some of the issues identified in the quality audit, such as incomplete or incorrect labeling and sample mix-ups. incomplete or illegible labeling result from a number of factors, including insufficient label space and lack of necessary information at time of ordering. the pre-analytical phase then extends beyond the patient ward to include transport of the sample to the laboratory and preparation of the sample for testing by a laboratory technician. the transportation phase represents the transfer of the specimen and associated information from one department to another within the hospital; this task is not assigned to a specific job title in the hospital. it is most likely to be carried out by a nurse, a patient attendant, or a janitor, but there is no established routine for sample transport. samples waiting for transport to the laboratory are typically stored in a treatment room or at a nursing station, and are taken with varying frequency to the laboratory. it is thus clear that one challenge is a lack of awareness of the time required for sample transport, so issues such as delays or misplaced samples are not proactively addressed. in addition, time is spent during this stage transcribing various information from the test order form, so information is being duplicated. this phase concludes with the preparation phase, in which the laboratory technician uses the test order information to prepare the sample for analysis. informational gaps may cause delays at this point in the process because, just as the sample transport does not have a systematic workflow, the reporting of sample and test order errors back to the wards is similarly unstructured. figure 1: total testing process workflow at kch.1 description of staff involved in total test process at kch. steps shaded in grey indicate that information transfer is occurring. the analytical phase includes a single step, namely, the analysis of the laboratory sample. information is generated as part of this process and is combined with information supplied during the pre-analytical phase (patient age, gender, etc.) to generate a result. the manual nature of matching the test results with the corresponding patient based on patient name is another informational step that can cause delays and potentially lead to reporting errors. the process of reporting the result back to the physician is the start of the post-analytical phase and, again, is not a formalised process. it could be performed by a variety of personnel at unspecified frequencies. results are left in the laboratory entryway in cubby-style pigeon holes for pick-up. often, when someone is sent from a ward to drop off laboratory samples, they will also pick up and deliver any results that are available for that ward. this means that critical results may not be reported to the wards in an expedited manner. table 3 presents the duration of the reporting stage for laboratory results from a 24-hour observation of the laboratory. results were processed for all 25 patients who had test orders sent to the laboratory during the observation period. test orders were reduced as the haematology instrument was not operational at that time and polymerase chain reaction results for outpatients were delivered through a different mechanism. additionally, the hospital census was unusually low that day. of the 25 results, 18 were collected during the 24-hour observation window, whilst seven remained in the pigeon holes. the average duration of the reporting stage was just under six hours, with significant variation. two of the results were collected immediately because they were related to a critical patient and the laboratory called the ward when the results were available. on the other hand, over one-fifth of results spent more than 16 hours in the laboratory before being collected. table 3: result reporting turnaround time from a 24-hour quality audit (n = 18).† discussion top ↑ the results present a multi-faceted depiction of laboratory testing in a hospital in a low-resource setting, focusing on both the demand for laboratory testing and the informational challenges in meeting that demand. with more than 240 000 tests conducted at the kch laboratory during a one-year period, it is clear that laboratory services are very much in demand within the hospital, matching the rhetoric around the importance of accessible laboratory services in low-resource settings. however, the diagnostic process is information-intensive; and the quality audit and workflow analysis suggest that the capture, management, and transfer of this information are a significant barrier to maximising the laboratory's role at kch. the quality audit identified informational barriers in the collection and identification of samples. almost one-sixth of samples were untestable because of incomplete or incorrect paperwork. the quality audit was conducted as a quality improvement effort and, as motivation for the study, laboratory employees articulated several challenges associated with incorrect or incomplete test orders. firstly, patient details, such as age and gender, affect the interpretation of the results; and test orders that omit these data increase the likelihood of an interpretation error. secondly, the time and date of the sample is particularly important for tests sent to either the microbiology or the biochemistry departments, so the absence of this information compromises the accuracy of the test. finally, the current system for results reporting relies on ward information from the test order, so when this information is not included it is common for results to never reach the patient. these pre-analytical errors and delays are similar to those found in other low-resource laboratory environments.26 post-analytical delays were also evident from the analysis of results reporting, although the sample size was small and further examination is warranted. these inaccuracies have implications for both the hospital and the patients. untestable samples equate to wasted resources, including the physical supplies for the sample, such as the syringe and collection vessels, as well as employee time involved in collecting the sample and communicating the error. for patients, these errors amount to delays in care; surgical patients may face delays in scheduled surgeries if laboratory results are not ready, whilst for others it may mean an extra night in the hospital. efficient laboratory testing is particularly important for patients in critical condition, namely, those who face delays in life-saving care and/or empirical treatments prescribed in the absence of confirmatory laboratory results. the findings of the quality audit also informed the interpretation of the data around the frequency of laboratory testing. the information about test volume reflected tests conducted by the kch laboratory and therefore serves as a conservative measure of the demand for laboratory services at kch. there are at least two reasons that demand for laboratory services may be greater than that shown in table 1. first, samples deemed untestable may not always be corrected and re-sent to the laboratory, as the process for informing clinicians of untestable samples is unstructured and thus possibly lengthy; and clinicians may choose to pursue diagnosis and treatment without confirmatory laboratory results. second, material shortages, such as reagents and properly functioning equipment, may prohibit the performance of certain kinds of tests. the workflow analysis mapped the laboratory testing process at kch to the standard stages of testing and identified the informational components of each stage. this exercise identified a lack of formalised workflow around the transport of samples from the wards to the laboratory and the reporting of results from the laboratory to the wards. the reporting delays were confirmed in the study of reporting times, although a limitation of this component of the results is the small sample size. these gaps suggest that one unique aspect of the informational challenges faced by laboratory systems is the geographic scale of the testing process. unlike the paperwork issues identified in the quality audit, the workflow issue involves interactions amongst multiple agents across different departments. the standard approach to laboratory testing does not necessarily reconcile these multiple players and environments. information systems tend to have a departmental focus. for example, electronic medical records are patient-centric systems focusing primarily on managing clinical patient data. whilst this often includes laboratory test results, electronic medical records functionality does not extend into the laboratory. laboratory information systems, on the other hand, are typically specimen-centric systems, focusing on processes and workflows within the laboratory. in this scenario the generation of test orders and reporting of test results often falls within the gap between the electronic medical records and the laboratory information system. we propose a system for supporting specimen management, increasing visibility into the status of all orders for both clinical as well as laboratory staff and following a model embraced by courier companies (dhl/federal express/ups) to track and manage packages. this simple model has three main components: (1) the generation of a test order and associated paperwork; (2) the ability to monitor the status of the order, complete with exception alerts when applicable; and (3) electronic results reporting back to the ward. the approach of real-time monitoring of specimens as they move through each stage of the process is novel and is likely to have a higher impact in a low-resource setting, where challenges are arguably greater. in table 4, we summarise problems identified in the kch workflow and present proposed informatics interventions for each. table 4: problems identified and proposed informatics solutions. the proposed informatics interventions address problems identified at kch that would not generally be considered as being within the scope of a traditional laboratory information system implementation.27 this application of informatics to the pre-analytical and post-analytical phases is novel and these stages are natural targets for informatics interventions because they are information-intensive and often where errors arise.17 such an approach, which recognises that much of the total testing process takes place outside the laboratory, may also improve the perception of laboratory testing amongst clinicians. whilst clinicians in many of these settings have a tendency to circumvent laboratory testing in favour of more superficial, less accurate diagnostic signals, they may be more likely to opt for laboratory testing if they perceive it to be quick and accurate.7,8 we recognise that informatics interventions cannot solve all problems. whilst our proposed informatics intervention does not prevent insufficient samples from being sent to the laboratory, it provides a formal mechanism for reporting and correcting those errors, potentially saving time in the laboratory testing process and improving the timely delivery of care. a variation of the proposed intervention could include decision support tools that remind the nurse preparing the order of the sample requirements (sample amount, container type, etc.). the need for investment in laboratory infrastructure for disease prevention and control is recognised in the literature.28 resource shortages, such as laboratory technicians, microscopes and access to electricity, are commonly cited as limiting factors in improving laboratory services.29 the informational challenges of laboratory testing in low-resource settings, whilst more challenging to identify than the resource limitations, will limit the impact of additional resources if unaddressed. this article synthesised an array of data about laboratory operations in a low-resource hospital setting, calling attention to these informational challenges. next steps will include a larger-scale effort to document the pre-analytical and post-analytical phases at kch and other low-resource hospital laboratories. in addition, whilst the proposed interventions are hypothetical at this point, we are in the early stages of modeling such systems. limitations of the study this study focuses on the informational challenges associated with laboratory testing at kch and does not consider other challenges that may serve as limiting factors, such as the availability of reagents and other physical resources required for testing. resolving informational challenges in the laboratory workflow may have a limited impact on overall laboratory performance if resource-based constraints are present. furthermore, the findings from the results reporting study must be interpreted with caution, as the sample size was small. recommendations future research will attempt to further quantify the workflows and challenges within the laboratory at kch as well as those in other low-resource settings. in addition, evaluation of informatics solutions targeting the laboratory will speak to the extent that informational challenges are limiting the stature and role of the laboratory at kch. conclusion in this article, we present a multi-faceted depiction of the laboratory testing process and informational challenges in a low-resource setting. one criticism of laboratory process analyses, even in more advanced settings, is the focus on the analytical phase.27 we presented evidence that encompassed all three stages of testing and identified two specific informational challenges: (1) complete testing paperwork; and (2) efficient, timely communication between the wards and laboratory. indeed, these issues were identified because of the wider focus on the preand post-analytical phases, which capture the multiple players and locations involved in the complete laboratory testing process. informational barriers and inefficiencies arose at the transition points, the transfer of responsibility from one role or location to another during the testing process. whilst information such as test orders and results should support workflow and decision making, in this case it appears that challenges in information management are undermining these processes. the plight of laboratory services in low-resource settings is at once loudly decried and woefully under-investigated. here we presented examples of informational barriers in the preand post-analytical phases of the total testing process in a hospital in a low-resource setting – challenges which, by their nature, are predisposed to be mitigated or potentially even eliminated by informatics interventions. future work will attempt to design, implement, and evaluate informatics solutions to these and other barriers to more efficient and integral laboratory systems in low-resource settings. trustworthiness top ↑ the results presented represent the actual findings of the analyses described in the research method and design section, without alteration. reliability and validity the quality audit and turnaround time study reflect standard methods of measuring laboratory performance; and test volume is a standard measure of laboratory workload. acknowledgements top ↑ the authors would like to acknowledge the contributions of kch staff who conducted the data collection. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions j.d. (university of pittsburgh) led the analysis and manuscript writing; n.a., h.l., g.g. and e.c. (kamuzu central hospital) assisted with study concept and data acquisition; o.j.g. (baobab health) contributed to study design; a.b.-s. (university of pittsburgh) assisted with study concept, data analysis, and manuscript drafting; and g.p.d. 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touchscreen electronic medical record systems to support and monitor national scale-up of antiretroviral therapy in malawi. plos med. 2010;7(8):e1000319. http://dx.doi.org/10.1371/journal.pmed.1000319 hoffman m, mofolo i, salima c, et al. utilization of family members to provide hospital care in malawi: the role of hospital guardians. malawi med j. 2013;24(4):74–78. gamadzi g, chitandale e, yohane i. kch lab quality assurance programme update (internal report). lilongwe, malawi: kamuzu central hospital; 2009. plebani m, lippi g. closing the brain-to-brain loop in laboratory testing. clin chem lab med. 2011;49(7):1131–1133. http://dx.doi.org/10.1515/cclm.2011.617 mbah ha. phlebotomy and quality in the african laboratory. afr j lab med. 2014;3(1), art. #132, 4 pages. http://dx.doi.org/10.4102/ajlm.v3i1.132 lober wb, revere d, hills r. a lab-emr interoperability profile as an ehealth architecture component for resource-constrained settings. stud health technol inform. 2010;160(pt 1):257–261. nkengasong jn. a shifting paradigm in strengthening laboratory health systems for global health: acting now, acting collectively, but acting differently. am j clin pathol. 2010;134(3):359–360. http://dx.doi.org/10.1309/ajcpy5asuejyq5rk petti ca, polage cr, quinn tc, et al. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006;42(3):377–382. http://dx.doi.org/10.1086/499363 references about the author(s) glenda m. davison south african medical research council/cape peninsula university of technology (samrc/cput) cardiometabolic health research unit, faculty of health and wellness, cape peninsula university of technology, cape town, south africa department of biomedical sciences, faculty of health and wellness, cape peninsula university of technology, cape town, south africa division of haematology, faculty of health, university of cape town, cape town, south africa citation davison gm. the silent epidemic: tackling the burden of anaemia in africa. afr j lab med. 2025;14(1), a2788. https://doi.org/10.4102/ajlm.v14i1.2788 editorial the silent epidemic: tackling the burden of anaemia in africa glenda m. davison copyright: © 2025. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. anaemia is described as a condition in which the level of haemoglobin drops below the normal value for an individual’s age, sex and physical state. it most often develops as a symptom of an underlying disease or condition and leads to decreased oxygen delivery to tissues resulting in weakness and chronic fatigue.1 the world health organization (who) has identified anaemia as a global health problem which has extensive consequences, including poor health, lack of economic productivity, and increased morbidity and mortality. in 2021, it was predicted that the global prevalence of anaemia was 24%, which translates to 1.9 billion affected individuals, with women and children making up the majority.2 the causes of anaemia vary significantly by geographical region, sex and age, with developing countries in sub-saharan africa and asia carrying the highest burden. a recent assessment by the who reported that in africa, 106 million women and 103 million children under the age of 5 years suffered from anaemia, with the primary cause being iron deficiency.3 besides nutritional deficiencies including iron, vitamin b12 and folate, the underlying causes of anaemia are complex and include haemoglobinopathies, parasitic infections, viruses, drugs, and non-communicable diseases such as malignancy and chronic renal disease. in women of childbearing age, heavy menstrual loss, post-partum bleeding and pregnancy can cause anaemia and, if not treated, can lead to complications for both the newborn baby and the mother.3 in africa, the causes are highly complex and interrelated. for example, parasitic infections such as malaria and helminths can result in haemolysis and blood loss, leading to iron deficiency. dual infection with plasmodium falciparum and hookworm (ancylostoma duodenale and necator americanus) is well described and has been identified as a leading cause of anaemia, particularly among school children.4 to further complicate the problem, africa has a high burden of genetic haemoglobin disorders such as thalassemia and sickle cell disease. these inherited anaemias, which predominate in central, east and west africa, result in reduced red cell lifespans as well as chronic inflammation and haemolysis.1 although haemoglobinopathies are not prominent in southern africa, the prevalence of hiv and tuberculosis remains high. hiv is closely associated with tuberculosis and both result in chronic anaemia resulting from abnormal iron metabolism and a suppressed bone marrow.5 in addition, although the administration of antiretroviral therapy can reduce anaemia in hiv patients, some classes of antiviral drugs can cause it to occur.6 besides infectious disease, non-communicable disorders such as chronic kidney disease are rising rapidly in africa.7 chronic kidney disease leads to a reduction in the production of erythropoietin, a hormone essential for red cell production and erythropoiesis in bone marrow. consequently, chronic kidney disease has been identified as an increasingly important cause of anaemia, particularly in the elderly, and is associated with hypertension, hiv, diabetes and autoimmunity. despite the high prevalence of anaemia in africa, it is often seen as a neglected condition, particularly in young children under 5 years and women. although by 2030 the who aims to reduce anaemia in women and children by half, progress is slow.8 most strategies have focused on improving nutrition with iron fortified foods and, while this is important, iron deficiency is only one cause of anaemia; to effectively tackle the problem, research is needed to examine the interplay between all the different causes of the condition. this is particularly important on the african continent, which has a high burden of infectious, nutritional and non-communicable diseases. the continent of africa is diverse, and one barrier to achieving these goals is that regional and population-specific reference ranges for haemoglobin and full blood count parameters are lacking. red blood cell parameters can vary significantly according to geographic region, sex as well as age and, if not interpreted correctly, could result in an overestimation or underestimation of the problem.2 population-specific reference ranges must therefore be developed to remove uncertainty and create a true and relevant definition of anaemia. recently, the who published a guideline for haemoglobin cutoffs; however further clarity, particularly in africa, is required.9 african scientists have a vital role to play in this task. the causes of anaemia are multifactorial, and early detection can lead to prompt treatment, prevention of severe anaemia, and the timely identification of an underlying disorder which requires further investigation. it has therefore been proposed that haemoglobin screening should be included in all antenatal clinic visits for women. this can be difficult in rural areas where there is no access to a laboratory and, therefore, the incorporation of point-of-care haemoglobin screening may provide a solution. in kenya, point-of-care haemoglobin testing was incorporated into a panel of tests which included syphilis, hiv and malaria. this was offered to all pregnant women attending seven sites in rural western kenya. after 8 months, the results demonstrated a significant increase in testing with early identification of anaemia and a significant improvement in clinical management.10 it has also been proposed that haemoglobin or haematocrit testing, at the bedside, be performed for all paediatric and emergency hospital patients, with the goal of implementing effective blood management and the prevention of unnecessary blood transfusions which are still an essential treatment for severe acute anaemia.11 haemoglobin screening using point of care testing has been utilised in several african regions; however, an analysis of these reports indicated that the haemoglobin values were often inaccurate and clinically unusable. the major challenges were errors in sampling, inadequate training and the stability of reagents.12 these obstacles must be addressed before point-of-care anaemia screening can be used effectively. one solution is to offer focused and follow-up training for the healthcare professionals performing these tests. anaemia remains a global health challenge, particularly in lowand middle-income countries. to support the united nations in achieving its sustainable development goals,13 particularly goal 2 (zero hunger) and goal 3 (good health and well-being) by 2030, urgent attention must be given to the complex and multifactorial nature of anaemia. african scientists and healthcare professionals are essential in tackling this burden, and the building of strong partnerships among african and regional organisations is critical for effective progress. references weatherall dj, hatten c. anaemia: pathophysiology, classification, and clinical features. in: warrellda, cox tm, firth jd, editors. oxford textbook of medicine. 5th ed. oxford: oxford university press; 2010. p. 5359–5366. gardner wm, vilchis-tella vm, perumal n, et al. prevalence, years lived with disability, and trends in anaemia by severity and cause, 1990–2021: findings from the global burden of disease study 2021. lancet haematol. 2023;10(9):e713–e734. world health organization. anaemia [homepage on the internet]. 2025 [cited 2025 feb 10]. available from: https://www.who.int/news-room/fact-sheets/detail/anaemia brooker s, akhwale w, pullan r, et al. epidemiology of plasmodium-helminth co-infection in africa: populations at risk, potential impact on anemia, and prospects for combining control. am j trop med hyg. 2007;77(6 suppl):88–98. https://doi.org/10.4269/ajtmh.2007.77.88 cao g, wang y, wu y, jing w, liu j, liu m. prevalence of anemia among people living with hiv: a systematic review and meta-analysis. eclinicalmedicine. 2022;44:101283. https://doi.org/10.1016/j.eclinm.2022.101283 harding bn, whitney bm, nance rm, et al. antiretroviral drug class and anaemia risk in the current treatment era among people living with hiv in the usa: a clinical cohort study. bmj open. 2020;10:e031487. https://doi.org/10.1136/bmjopen-2019-031487 kaze ad, ilori t, jaar bg, et al. burden of chronic kidney disease on the african continent: a systematic review and meta-analysis. bmc nephrol. 2018;19:125. https://doi.org/10.1186/s12882-018-0930-5 world health organization, united nations children’s fund. the extension of the 2025 maternal, infant and young child nutrition targets to 2030: discussion paper [homepage on the internet]. geneva: world health organization and unicef; 2019 [cited 2025 feb 13]. available from: https://data.unicef.org/resources/who-unicef-discussion-paper-nutrition-targets/ world health organization. guideline on haemoglobin cutoffs to define anaemia in individuals and populations [homepage on the internet]. geneva: world health organization; 2023 [cited 2025 feb 13]. available from: https://www.who.int/publications/i/item/9789240088542 young n, taegtmeyer m, aol g et al. integrated point-of-care testing (poct) of hiv, syphilis, malaria and anaemia in antenatal clinics in western kenya: a longitudinal implementation study. plos one. 2018;13(7):e0198784. https://doi.org/10.1371/journal.pone.0198784 uyoga s, george ec, bates i, et al. point-of-care haemoglobin testing in african hospitals: a neglected essential diagnostic test. br j haematol. 2021;193(5):894–901. https://doi.org/10.1111/bjh.17431 brehm r, south a, george ec. use of point-of-care haemoglobin tests to diagnose childhood anaemia in lowand middle-income countries: a systematic review. trop med int health. 2024;29(2):73–87. https://doi.org/10.1111/tmi.13957 united nations. transforming our world: the 2030 agenda for sustainable development [homepage on the internet]. united nations; 2015 [cited 2025 feb 14]. available from: https://sdgs.un.org/2030agenda introduction covid-19 and cytokine storm covid-19 and t cell impairment covid-19 and immunological investigations acknowledgements references about the author(s) brahim admou center of clinical research, faculty of medicine and pharmacy, mohamed vi university hospital, cadi ayyad university, marrakech, morocco abdelhamid hachimi department of intensive care, faculty of medicine and pharmacy, mohamed vi university hospital, cadi ayyad university, marrakech, morocco mohamed abdenasser samkaoui department of anesthesiology and intensive care, faculty of medicine and pharmacy, mohamed vi university hospital, cadi ayyad university, marrakech, morocco citation admou b, hachimi a, samkaoui ma. how can clinical immunology laboratories contribute to the management of severe covid-19 cases in limited resource contexts? afr j lab med. 2020;9(1), a1282. https://doi.org/10.4102/ajlm.v9i1.1282 opinion paper how can clinical immunology laboratories contribute to the management of severe covid-19 cases in limited resource contexts? brahim admou, abdelhamid hachimi, mohamed abdenasser samkaoui received: 27 may 2020; accepted: 03 sept. 2020; published: 02 nov. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction since its appearance, the coronavirus disease 2019 (covid-19) that is caused by severe acute respiratory disease coronavirus 2 (sars-cov-2), has been responsible for severe respiratory disease similar to diseases commonly associated with the coronaviruses such as severe acute respiratory syndrome and middle east respiratory syndrome; covid-19 frequently requires hospitalisation in the intensive care unit and has had a high mortality rate.1 the severity of covid-19 is generally stratified as asymptomatic, mild (without signs of severe or critical illness), severe (with signs of breathing difficulties) or critical (with signs of respiratory failure, shock and multiple organ failure).2 the contribution of the medical laboratory to the categorisation of covid-19 clinical forms is not yet well defined.3 after confirmation of infection by real-time polymerase chain reaction, biochemical and haematological analyses provide fundamental biological parameters for determining disease severity. similarly, the clinical immunology laboratory could play an important role in elucidating diverse immunological abnormalities associated with the disease. in particular, immunological testing could better categorise the severe and critical forms of covid-19, and subsequently assist treating physicians during the entire course of therapy. the purpose of this opinion article is to highlight the position of the immunology laboratory in the management of severe and critical covid-19 cases, specifically in countries with limited resources. covid-19 and cytokine storm covid-19 is marked by an overproduction of pro-inflammatory cytokines and chemokines, mainly interleukin (il)-6, il-10, tumor necrosis factor (tnf)-α, il-1β, il-2, il-10, interferon-γ and monocyte chemoattractant protein-1.4 other biological abnormalities may predict the severity or progression of the disease, such as abnormal coagulation activation, leukopenia, high levels of c-reactive protein, ferritin, d-dimers, aminotransferase, lactate dehydrogenase and creatin kinase.3,5 elevated levels of pro-inflammatory cytokines have been shown to characterise severe lung infection, which manifests as respiratory distress, multiple organ failure and adverse outcomes in sars-cov-2 infection.6,7 in addition, il-6, il-10, and tnf-α levels significantly increase during infection and drop during recovery,8 suggesting that the intensity of the cytokine release correlates with disease activity. interestingly, il-6 may be a marker for monitoring serious covid-19 cases.9 moreover, simultaneous high levels of il-6 and d-dimers have been shown to be narrowly associated with severe forms of the disease in adult patients. therefore, measuring them in combination allows for greater specificity and sensitivity for predicting severe cases of covid-19.3 covid-19 and t cell impairment deregulation of the immune response, particularly t lymphocytes, appears to be strongly linked to the pathology of covid-1910; direct infection of lymphocytes by sars-cov-2 has been proposed as a cause for acute lymphocyte decline.3,11 lymphocytes express the receptor angiotensin-converting enzyme 2, which has been suggested to be the primary receptor targeted by sars-cov-2.12 moreover, altered t lymphocytes could be an important factor in worsening symptoms in patients, which makes lymphopenia a relevant marker of disease severity, hence the need for intensive care unit admission.11 it has been demonstrated that the severe respiratory syndrome that manifests due to covid-19 is characterised by cluster of differentiation (cd)4 and cd8 t lymphopenia, correlating with disease severity.4,13 on the other hand, covid-19 patients who require intensive care unit hospitalisation have significantly higher levels of il-6, il-10 and tnf-α, with lower levels of cd4 and cd8 t cell counts,8 with which levels of cytokines and t cells are inversely correlated.6,8 moreover, because t lymphocytes are generally crucial for amortising exaggerated natural immune reactions against viral infection, t cell defects can lead to worsening inflammatory responses during covid-19, whereas restoring the number of these lymphocytes can improve them.6 in accordance with this hypothesis, it has been shown that 4–6 days following the onset of infection, t lymphocyte counts drop to their lowest level, whereas cytokine levels reach their maximum. conversely, the restoration of the number of t lymphocytes is associated with a decrease in serum levels of various cytokines, such as il-6, il-10 and tnf-α.6 owing to the cytokine storm, and other immunological predictors of severity of sars-cov2 infection, these may also be helpful in choosing anti-inflammatory drugs, especially corticosteroids for their potential benefit in the reduction of inflammation-induced lung damage in severe covid-19 patients.1 covid-19 and immunological investigations having shown that the most relevant immunological parameters include inflammatory cytokines and t lymphocyte subpopulations, the clinical immunology laboratory is positioned to be important for assessing covid-19 patients, especially for categorising, or even predicting severe cases.2,10 cytokine levels, especially that of il-6, can be individually measured on immunoassay analysers, whereas other cytokine profiles can be explored using either enzyme-linked immunosorbent assays or other specific biotechnologies like luminex®, 200™ (or multiplex) (luminex®, austin, texas, united states) and flow cytometry systems, which allow for a large-scale quantitative measurement.14 the assessment of the main t cell subsets, such as cd3+, cd4+ and cd8+, requires only a simple phenotyping procedure conductible on a mini-cytometer, which is largely available worldwide even in limited-resource context laboratories, thanks to hiv management programs. more developed flow cytometry platforms allow for comprehensive phenotyping assays, enabling the investigation of naive, memory and regulatory t cells; in severe covid-19 patients, early data on t cell subpopulation abnormalities show increased naive helper t cells and decreased memory helper t cells, associated with a lower number of regulatory t cells.10 using a receiver operating characteristic curve to compare fatal and recovered covid-19 cases, xu et al.2 considered 559 cells/µl, 235 cells/µl, and 104 cells/µl of cd3+, cd4+t, and cd8+ t cell subsets as warning values, below which there was a significantly higher risk of in-hospital death.2 indeed, there is a need for regional data analyses before determining cut-off values of these t cell subsets. however, t cell lymphopenia might be accentuated by possible primary or acquired immune deficiency conditions, potentially revealed by covid-19, and must then be considered first when interpreting cell phenotyping values and when managing patients as well.15 conclusion for sars-cov-2 infection, alongside other clinical and biological parameters, the measurement of inflammatory cytokines, mainly il-6, as well as cd4+ and cd8+ t cell assessment, should be systematically planned during management of the disease. these markers could be useful for identifying severe cases requiring prompt admission to intensive care units, and for monitoring patient disease progression. these investigations are within the reach of almost all clinical immunology laboratories in the world. close collaboration between immunologists and physicians is essential for effective global efforts against this highly threatening pandemic. acknowledgements we would like to express our sincere gratitude and deepest appreciation to yacine berka for his highly valuable contribution in the editing of this manuscript. competing interests the authors have declared that no competing interests exist. authors’ contributions all authors contributed equally to this work. b.a. conceptualised and wrote the major parts of the manuscript. a.h. co-wrote the clinical aspects of the manuscript. m.a.s. contributed to the conceptualisation and validated a writing review of the manuscript. ethical considerations ethical clearance was not required for this study. sources of support this research received no specific grant from any funding agency in the public commercial or not-for-profit sectors. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references huang c, wang y, li x, et al. clinical features of patients infected with 2019 novel coronavirus in wuhan, china. lancet. 2020;395(10223):497–506. https://doi.org/10.1016/s0140-6736(20)30183-5 xu b, fan c-y, wang a-l, et al. miao. suppressed t cell-mediated immunity in patients with covid-19: a clinical retrospective study in wuhan, china. j infect. 81(1):e51–e60. gao y, li t, han m, et al. diagnostic utility of clinical laboratory data determinations for patients with the severe covid-19. j med virol. 2020;92(7):791–796. https://doi.org/10.1002/jmv.25770 pedersen s-f, ho y-c. sars-cov-2: a storm is raging. j clin invest. 2020;130(5):2202–2205. https://doi.org/10.1172/jci137647 henry b-m, de oliveira m-h-s, benoit s, et al. hematologic, biochemical and immune biomarker abnormalities associated with severe illness and mortality in coronavirus disease 2019 (covid-19): a meta-analysis. clin chem lab med. 2020;58(7):1021–1028. https://doi.org/10.1515/cclm-2020-0369 liu j, li s, liu j, et al. longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of sars-cov-2 infected patients. ebio med. 2020;55:102763. https://doi.org/10.1016/j.ebiom.2020.102763 zhou j, chu h., li c, et al. active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis. j infect dis 2014;209(9):1331–1342. https://doi.org/10.1093/infdis/jit504 diao b, wang c, tan y, et al. reduction and functional exhaustion of t cells in patients with coronavirus disease. front. immunol. 2020;11:827. https://doi.org/10.3389/fimmu.2020.00827 zhu z, cai t, fan l, et al. clinical value of immune-inflammatory parameters to assess the severity of coronavirus disease 2019. int j infect dis. 2020;95:332–339. https://doi.org/10.1016/j.ijid.2020.04.041 qin c, zhou l, hu z, et al. dysregulation of immune response in patients with covid-19 in wuhan, china. clin infect dis. 2020. https://doi.org/10.1093/cid/ciaa248 tan l, wang q, zhang d, et al. lymphopenia predicts disease severity of covid-19: a descriptive and predictive study. signal transduct target ther. 2020;5(1):33. https://doi.org/10.1038/s41392-020-0159-1 xu h, zhong l, deng j, et al. high expression of ace2 receptor of 2019-ncov on the epithelial cells of oral mucosa. int j oral sci. 2020;12:8. https://doi.org/10.1038/s41368-020-0074-x chen g, wu d, guo w, et al. clinical and immunologic features in severe and moderate coronavirus disease 2019. j clin invest. 2020;130(5):2620–2629. https://doi.org/10.1172/jci137244 medeiros ni, gomes jas. cytometric bead array [cba] for measuring cytokine levels in chagas disease patients. methods mol biol. 2019;1955:309–314. https://doi.org/10.1007/978-1-4939-9148-8_23 brough h-a, kalayci o, sediva a, et al. managing childhood allergies and immunodeficiencies during respiratory virus epidemics – the 2020 covid-19 pandemic. pediatr allergy immunol. 2020;31(5):442–448. https://doi.org/10.1111/pai.13262 abstract introduction methods results discussion acknowledgements references about the author(s) naseem cassim department of molecular medicine and haematology, university of the witwatersrand, johannesburg, south africa national priority programme, national health laboratory service, johannesburg, south africa lindi m. coetzee department of molecular medicine and haematology, university of the witwatersrand, johannesburg, south africa national priority programme, national health laboratory service, johannesburg, south africa wendy s. stevens department of molecular medicine and haematology, university of the witwatersrand, johannesburg, south africa national priority programme, national health laboratory service, johannesburg, south africa deborah k. glencross department of molecular medicine and haematology, university of the witwatersrand, johannesburg, south africa national priority programme, national health laboratory service, johannesburg, south africa citation cassim n, coetzee lm, stevens ws, glencross dk. addressing antiretroviral therapy-related diagnostic coverage gaps across south africa using a programmatic approach. afr j lab med. 2018;7(1), a681. https://doi.org/10.4102/ajlm.v7i1.681 original research addressing antiretroviral therapy-related diagnostic coverage gaps across south africa using a programmatic approach naseem cassim, lindi m. coetzee, wendy s. stevens, deborah k. glencross received: 18 sept. 2017; accepted: 02 may 2018; published: 12 nov. 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: a major challenge facing south africa is the concomitant hiv and tuberculosis epidemics. the national health laboratory service provides testing for staging hiv-positive patients, monitoring patients on antiretroviral therapy (art) and diagnosing tuberculosis. not all health districts have equivalent art-related coverage in particular for cd4 and hiv viral load testing. objectives: the integrated tiered service delivery model coverage precinct approach was used to address art-related testing service coverage gaps in a manner that balances cost, quality and equity. methods: an algorithm was developed to identify and address art-related diagnostic coverage gaps. data was extracted from the corporate data warehouse and oracle systems for the period of april 2015 to march 2016. daily test volumes were based on 21.73 working days per month. data were analysed using ms excel and mapped using arccatalog and arcmap. capacity analysis was informed by the available testing-platforms. results: health district daily hiv viral load volumes ranged from 2 to 1308 samples. nineteen candidate laboratories were identified to address the coverage gaps. following the proximity analysis, testing was consolidated at four candidate laboratories, resulting in 13 revised candidate laboratories. the revised candidate laboratory daily hiv viral load referrals ranged between 5 and 205 samples, with cd4 volumes between 6 and 85 samples. remaining coverage gaps were identified in seven municipalities. conclusions: the study demonstrated that the service coverage precinct approach could be used to identify coverage gaps for a defined art-related testing repertoire. introduction a major challenge facing south africa is the concomitant hiv and tuberculosis epidemics.1,2 it is estimated that 36.7 million people globally were living with hiv in 2016, with a prevalence of 0.8%, of which 25.5 million people live in sub-saharan africa (~70% of the global hiv burden).3,4 in south africa, it is estimated that seven million people are living with hiv, with an adult prevalence rate of 19.2% in 2015.4,5 despite many obstacles faced by south africa between 2010 and 2014, the response to the aids epidemic resulted in the largest antiretroviral treatment (art) programme in the world, with over two million hiv-infected people receiving treatment by 2010.6 in 2006, the south african government approved the ambitious national strategic plan for hiv and aids and sexually-transmitted infections (2007–2011) and committed the government to providing art to 80% of those eligible.7,8,9 in 2010, with ~900 000 people on art, south africa launched the national hiv counselling and testing campaign that aimed to test 20 million people over 20 months.7,8 the required art scale up in south africa necessary to meet the hiv counselling and testing campaign targets led to the announcement that accreditation would be abandoned and that all public healthcare facilities would be geared up to provide art.7,8 with the accreditation requirement removed, art services were decentralised to the majority of health facilities over the next few years (n = ~3000).7 this change removed the need for a national team to accredit health facilities for art provision. district coordinators could use a checklist to speed up the expansion of art services to the community level. the introduction of nurse initiated management of antiretroviral treatment at primary health care facilities facilitated further decentralisation of art, strengthened retention of patients in care and reduced the burden of managing uncomplicated cases at referral hospitals.10,11 currently, the joint united nations programme on hiv/aids (unaids) estimated that seven million people are living with hiv in south africa with a prevalence rate of 19.2% for adults (>15 years).12 unaids also reported 380 000 new hiv infections in 2015, with 180 000 deaths due to aids.12 by 2015, 3.3 million individuals were on art, resulting in 48% coverage of all hiv-positive individuals.5 in 2015, six countries accounted for 60% of the global tuberculosis burden, with the highest burden in india, followed by indonesia, china, nigeria, pakistan and south africa.13,14 tuberculosis is also a leading killer of hiv-positive people (35% of deaths were due to tuberculosis).13 by 2015, a tuberculosis incidence of 834 cases per 100 000 population was reported for south africa (includes hiv-positive tuberculosis cases).13 the incidence of multidrug-resistant tuberculosis was 37 cases per 100 000 population.13 tuberculosis treatment coverage was estimated to be 64% overall, increasing to 97% for patients with a known hiv status.13 eighty-five per cent of patients in hiv/ tuberculosis care were on art. south africa developed the national tuberculosis programme that aimed to find, treat and prevent tuberculosis in order to avoid tuberculosis deaths and reduce transmission.14 the national tuberculosis programme has substantially strengthened the national tuberculosis control programme.14,15 significant milestones for the national tuberculosis programme include the implementation of directly-observed treatment short course,15 introduction of fixed-dose combination drugs, conducting the national drug-resistance survey, introduction of hain mtbdrplus (multidrug-resistant tuberculosis rapid test) and xpert mtb/rif (replacement for sputum smear microscopy).14 significant challenges, however, remain to reduce the tuberculosis burden in south africa. the laboratory service plays a critical role in diagnosing tuberculosis and monitoring treatment.16,17 similarly, the laboratory service is required to evaluate patients and monitor virological suppression once on art.18 in south africa, the national health laboratory service (nhls) provides access to diagnostics to 80% of the population attending public sector health care facilities through a network of laboratories across nine provinces.19 the nhls aims to provide quality, affordable and sustainable health laboratory medicine, provide training for health science education and undertake innovative and relevant research.19 routine clinical pathology services are offered at all 288 laboratories providing routine haematology, chemical pathology and microbiology testing. for the hiv programme, the nhls provides cd4, hiv viral load, xpert mtb/rif and other routine testing to support art initiation, as defined in the national guidelines.18 cd4 testing within the nhls is standardised using flow cytometers (beckman coulter, miami, florida, united states) and the panleucogating method.20,21 cd4 laboratories use either the mpl/cellmek platform at busier laboratories or the aquois system for lower throughput laboratories.22,23,24 there are currently 49 laboratories offering cd4 testing. hiv viral load testing is offered using the roche (roche diagnostics, basel, switzerland) and abbott (abbott molecular, abbott park, illinois, united states) platforms. laboratories allocated to the roche diagnostic platform use the hiv-1 test (v2.0) assay on either the cobas ampliprep/cobas taqman, cobas 6800 or cobas 8800 analysers.25 additionally, some laboratories allocated to the abbott molecular platform use the realtime hiv-1 assay with the abbott m2000 realtime system (m2000sp and m2000rt) analyser.26 hiv viral load testing is currently offered at 17 laboratories using both the roche and abbott assays. xpert mtb/rif testing is based on the cepheid (sunnyvale, california, united states) genexpert platform.27,28 the genexpert technology is a modular system and supplied as a gx4 (i.e., four modules), gx16, gx48 and gx80.29 there are 214 nhls laboratories offering xpert mtb/rif testing, using a decentralised approach across all 52 health districts.30 however, not all health districts have equivalent coverage for cd4 and hiv viral load testing. the integrated tiered service delivery model (itsdm) described for cd4 testing across south africa to achieve coverage, proposed 104 cd4 testing sites consisting of 60 laboratories (tier-3 to tier-5), 22 point-of-care (poc) hubs (tier-2), and 22 decentralised poc testing sites (tier-1) that would only service one health facility.31 in particular, tiers two and three were identified as potential operational platforms to enable extending hiv testing services to existing nhls laboratories. the aim of the latter was to deliver equitable access to laboratory services by providing technology in the respectively identified laboratory that appropriately matched service delivery requirements within the defined service coverage precinct of the laboratory in question.31,32 the efforts to date have been test-specific (cd4-linked) and as such, a new analysis that optimally addresses art-related testing coverage gaps is needed. for example, adding only hiv viral load testing to a rural district laboratory repertoire is not sufficient to meet the needs for basic art-related test repertoire. to improve diagnostic coverage, repertoire testing demands include concurrent access to cd4, cryptococcal antigen (crag), xpert mtb/rif, creatinine (including estimated calculation of glomerular filtration rate), alanine transaminase (alt), full blood count (fbc), and hepatitis b surface antigen tests.18 a national burden of disease study conducted between 1997 and 2010 by the south african medical research council reported that the four major broad causes of death in south africa were hiv and tuberculosis, non-communicable diseases, injuries, and other type one conditions such as nutritional deficiencies.33,34,35 the burden of these non-communicable diseases will probably increase as the roll-out of art scales up further and reduces mortality from hiv.34 the scale of the challenge posed by the combined and growing burden of hiv, tuberculosis and non-communicable diseases demands an extraordinary response from the health care system.34 the lessons learnt from implementation of the cd4 itsdm31 and other related work36,37 to address coverage will be used to investigate coverage gaps for a defined art-related test repertoire. the aim of this study is to use the existing nhls laboratory capacity to address art-related coverage gaps and deliver services where some testing gaps exist in a sustainable manner that balances cost, quality and coverage. methods ethical considerations ethics clearance for this work was obtained from the university of the witwatersrand (study number: m1706108). this study involved the secondary analysis of laboratory test volumes data that does not contain any patient identifiers. no patient recruitment was necessary as routine laboratory data was used for the study. study design this study is based on the lessons learnt from the itsdm and extends these concepts to develop an algorithm that addresses service coverage for a defined test repertoire. the algorithm developed was used to identify existing nhls laboratories (candidates) that are not currently offering hiv viral load and cd4 testing that could be used to offer these diagnostic services and address art-related testing coverage gaps (figure 1). laboratories were assigned an alias in each province, e.g. ec1 for the first laboratory alphabetically in the eastern cape province. as the hiv viral load test currently has the smallest national service footprint, this test will be used as the baseline test from which to assess coverage gaps. figure 1: flow diagram steps used to identify candidate laboratories to address antiretroviral therapy-related testing coverage gaps, south africa. the seven steps used in the algorithm are described below: step 1: display service coverage precinct for the hiv viral load test a list of hiv viral load laboratories was compiled, including latitude and longitude coordinates. these data were collected using a spreadsheet and converted to a shapefile using arccatalog (redlands, california, united states) and loaded as a new layer on arcmap (redlands, california, united states)38 (displayed as a blue circle). the analysis toolbox was used to access the proximity toolset to display a 250 km buffer (equivalent to 2.5–3 h travel time depending on road infrastructure) around each hiv viral load laboratory to represent the current service coverage precinct. health facilities within the current hiv viral load service coverage precinct were assumed to have adequate access to testing.31 areas outside the service coverage precinct were assumed to lack coverage, requiring additional testing sites to address gaps. step 2: display health district daily volumes hiv viral load samples tested within the nhls are recorded in the laboratory information system through a data interface to the analyser. specimen-level laboratory information system data are then replicated to the corporate data warehouse for national programmatic reporting. health district hiv viral load test volumes for the period april 2015 to march 2016 (fiscal year [fy]2015/2016) were extracted from the corporate data warehouse. the data extract included the health district name and total hiv viral load test volumes, that is, the number of samples tested in the fy2015/2016. daily health district hiv viral load test volumes were calculated assuming an average of 21.73 working days per month and 12 months per year. in south africa, the municipal demarcation board is responsible for determining municipal boundaries.39 it also provides spatial data files at the provincial, district, municipal and ward levels.39 health district shape files were downloaded from the municipal demarcation board website39 and edited using arccatalog to add a new data field to capture district daily hiv viral load volumes (integer data type).38 the shapefile was added as a new layer on arcmap38 and edited to manually capture daily hiv viral load test volumes for 52 health districts. using the symbology functionality in arcmap,38 the daily hiv viral load volumes were displayed using quantiles with graduated colours (across six classes) for easy visual interpretation. each class was allocated a different colour, namely: 8–16, dark green; 17–64, green; 65–150, light green; 151–300, orange; 301–600, light red; and 601–1380, dark red. the cut-off values were adjusted manually for the first two classes to reflect the daily capacity of the genexpert gx4 (16 samples per day) and the gx16 (64 samples per day). step 3: identify a candidate laboratory in the hiv viral load service coverage gap areas a microsoft excel list of nhls laboratories that do not currently provide hiv viral load testing, with their respective latitude and longitude coordinates, were loaded as a new layer on arcmap. the maps were printed on an a3 paper in colour and given to three individuals to identify candidate laboratories in the current hiv viral load coverage gaps. the three lists were reviewed to generate the approved list of candidate laboratories. this list was converted to a shapefile using arccatalog.38 this shapefile was then added as a new layer of the map and visualised as red circles using the symbology function in arcmap.38 step 4: assess whether candidate laboratories offer antiretroviral therapy-related repertoire testing for each test performed within the nhls network, the laboratory information system generates single or multiple tariff code or codes to generate expenditure data (e.g. tariff code 3020 is allocated to the glucose test). these data are used to generate accounts on the oracle enterprise resource planning system in the accounts receivable module,40 which generated the volume and revenue report that details test volumes by tariff code. the tariff code volumes provide an easy analysis of test volumes where a one-to-one relationship exists between a test and its respective tariff code. this is the case for the art-related test repertoire. year-to-date volumes and revenue data from the march report, namely fy2015/16 volumes,41 were used. the report includes cost centre numbers, laboratory name, tariff code, tariff description, year-to-date test volumes and referred test volumes. referred test volumes indicate the number of tests referred to another laboratory when this test is not offered by the receiving laboratory. the data extract was analysed using microsoft excel to determine: (1) financial year 2015/16 cd4, creatinine, alt, fbc and xpert mtb/rif test volumes for each laboratory; and (2) where these tests are not offered, the referral test volumes. the aim of this analysis was to identify whether the candidate laboratories currently provide the required test repertoire. tariff codes were identified for the hiv viral load, cd4, crag, alt, fbc and xpert mtb/rif tests to assess year-to-date testing and referred volumes. the testing status of each candidate laboratory was to be reported as a table, meaning that if no test volumes were found for a specific test (tariff code) in fy2015/16, it was assumed the laboratory did not offer testing. step 5: assess candidate laboratories in close proximity (≤ 150 km) to avoid over-capacitation in a geographic area within a 150 km circumferential service coverage precinct, one laboratory would be able to provide sufficient coverage, removing the need to over-capacitate multiple laboratories. the aim of this step was to prevent over-capacitation and provide optimal testing at a candidate laboratory with the required test repertoire. an independent visual inspection was again conducted by three single individuals. they were requested to identify laboratory clusters on the printed map for candidate laboratories in close proximity. the responses were consolidated to identify clusters. for each cluster identified, google maps was used to determine inter-laboratory distances (kilometres) and drive times (minutes). the google maps drive time is not able to factor aspects such as stop or go sections due to road construction, adverse weather, traffic congestion and terrain and road condition. where laboratories in a cluster were >150 km from each other, they were added to the list of revised candidate laboratories, as the laboratories were too far apart for consolidated testing. where the inter-laboratory distance was ≤ 150 km for laboratories in a cluster, the laboratory currently offering cd4 testing and reporting the highest test volumes was the preferred choice for consolidation in the cluster. where consolidation was indicated, the combined hiv viral load and cd4 volumes were used to determine the required testing capacity. where a candidate laboratory did not currently offer hiv viral load or cd4 testing based on volumes and revenue year-to-date data, referred test volumes were used. all daily volume calculations assumed 21.73 working days per month and 12 months per year. this assumption is based on small district (rural) laboratories that offer an 8-hour service, but may not be applicable to larger laboratories that offer a 24-hour service impacting on testing capacity. the capacity analysis was done in consultation with three individuals from the national priority programme unit, with expertise in either cd4 or hiv viral load testing. one hiv viral load and cd4 platform was then allocated to each candidate laboratory based on the daily volumes or referrals (consolidated volumes where applicable). for the purpose of this paper, daily cd4 volumes of less than ten samples were allocated to the facspresto (becton dickinson, san diego, california, united states) platform. step 6: display service coverage precinct for the revised candidate laboratories in the identified clusters where consolidation was proposed, excluded laboratories were removed to generate a list of revised candidate laboratories. the list of revised candidate laboratories, with latitude and longitude values, was converted to a shapefile using arccatalog and added as a new layer (reported as red circles) on arcmap.38 a service coverage precinct buffer (250 km) was drawn around the laboratory on arcmap using the proximity tool to assess the additional service coverage the precinct provided (yellow circle).38 step 7: assess whether antiretroviral therapy-related testing service coverage gaps have been fully addressed for municipal areas without adequate coverage, town names were added to arcmap. for these areas, either poc testing or additional laboratory sites are proposed. results daily district hiv viral load test volumes only 5 out of 52 districts reported daily hiv viral load test volumes between 601 and 1308, namely city of cape town metro, city of johannesburg metro, ehlanzeni, ekurhuleni metro and ethekwini metro (figure 2). these health districts requested 33% of the national hiv viral load test volumes. figure 2: current daily hiv viral load test volumes per day per health district with the laboratories offering testing (n=17), south africa. nine health districts reported daily hiv viral load test volumes between 301 and 600; 22 health districts reported a daily hiv viral load test volume between 151 and 300 samples; only eight health districts had daily hiv viral load volume between 1515 and 300 while just 6 districts reported between 16 and 64 tests per day. there were two districts that reported daily volumes ≤ 16 samples per day, namely central karoo and namakwa health districts. candidate laboratories in the hiv viral load service coverage gaps the arcmap analysis reported adequate hiv viral load coverage in the gauteng, kwazulu-natal and mpumalanga provinces (figure 3). poor coverage was identified across all districts in the northern cape province. selected health districts with poor coverage in the western cape, eastern cape, north west, free state and limpopo provinces were also identified. nineteen candidate nhls laboratories were identified to scale up services (table 1). of these candidate laboratories, six were identified in the western cape province (wc1–wc6). four candidate laboratories were identified in the northern cape province (nc1–nc4) and three in the eastern cape province (ec1–ec3). only two candidate laboratories were identified in each of the limpopo (lp1–lp2), free state (fs1–fs2) and north west (nw1–nw2) provinces. figure 3: current hiv viral load coverage provided using a service coverage precinct of 150 km circumference and the identification of candidate laboratories in the coverage gaps (n = 19), south africa. table 1: current testing offered by the candidate laboratories to address the antiretroviral therapy-related testing coverage gaps, south africa. antiretroviral therapy-related testing currently provided by candidate laboratories all candidate laboratories (n = 19) provide xpert mtb/rif, creatinine (including a calculated estimated glomerular filtration rate), alt and fbc testing at present (table 1). none of the candidate laboratories, however, offer hiv viral load or hepatitis b surface antigen testing. five candidate laboratories already offer cd4 testing, namely fs2, nw1, nc1, nc4 and wc2. candidate laboratories in close proximity four clusters were identified following visual inspection. inter-laboratory distances ranged from 52.6 to 83.1 kilometres. similarly, travel times ranged from 44 to 62 min (table 2). consolidation of testing was proposed at the nw1, wc2 and fs2 laboratories as they currently offer cd4 testing. the ec2 laboratory performed 28 000 xpert mtb/rif, creatinine, alt and fbc tests, compared to 10 000 for the ec3 laboratory in fy2015/16. therefore, the recommendation for testing consolidation at the ec2 laboratory for this cluster. as such, the list of identified candidate laboratories (n = 19) was reduced to 13, with six laboratories removed due to close proximity to the proposed site, namely nw2, wc3, wc4, wc5, fs1 and ec3. the remaining 13 laboratories were referred to as revised candidate laboratories (figure 4). figure 4: analysis of additional coverage provided by the 13 candidate laboratories that would provide the required diagnostic support for antiretroviral therapy services, south africa. table 2: google maps distances and travel times between candidate laboratories in close proximity across the four clusters identified, south africa. analysis of test volumes and capacity required for cd4 and hiv viral load testing for the revised candidate laboratories daily hiv viral load referrals for the revised candidate laboratories (n = 13) ranged from 5 (nc2) to 205 (fs2) samples per day (table 3). the fs2, nw1 and wc2 laboratories reported daily volumes of 205 (fs2), 138 (nw1) and 80 (wc2). the nc2, wc1 and wc6 laboratories all reported lower daily volumes, ranging from five to seven samples per day. table 3: daily hiv viral load and cd4 test volumes, capacity required and testing tier for candidate laboratories, south africa. for cd4 testing, four laboratories with lower daily volumes (6–9 samples) would cope with the low throughput of a bd facspresto platform. the remaining laboratories (n = 9) would have sufficient capacity with the bc aquios platform to perform up to 120 samples per day (daily volumes ranged between 18 and 85). identify remaining antiretroviral therapy-related testing coverage gaps following the introduction of the antiretroviral therapy-related test repertoire at the revised candidate laboratories the majority of the art-related diagnostic coverage gaps would be addressed with the exception of some rural municipal areas that include brandvlei, bredasdorp, pofadder, pomfret, prieska, swellendam and williston, with daily hiv viral load test volumes of ≤ 6 samples per day (figure 4). discussion this article describes an approach to establishing where service deficiencies lie across a national programme, using a step-by-step approach. each step provides detail of the analysis undertaken to make the final decision to identify and address coverage gaps. the work is by no means fully comprehensive but should be viewed as a guideline of how to approach the mammoth task of where to start rolling out laboratory services across a national programme, irrespective of whether the programme is a vast network of laboratories that may be required for extensive art support services or a smaller network that involves other clinical laboratory testing. the adequacy of art-related diagnostic coverage is best addressed across a defined test repertoire that provides access to all testing required for art initiation and monitoring of hiv-positive patients enrolled on art. addressing the coverage gaps for a single test would not have the desired impact on art initiation, as patients would be required to wait for those tests results that are not included in the local test repertoire. the 250 km service coverage precinct was based on the hub-and-spoke logistics model used within the nhls.42 on average, samples are in transit with the courier for up to 3.5 hours before reaching the local nhls laboratory. to meet the minimum six-hour requirement for plasma hiv viral load samples, a service coverage precinct of 250 km was allocated (~2.5 h travel time). an advantage of providing art-related coverage is the ability to support integrated patient care envisaged by the ideal clinic initiative.43 the ideal clinic initiative is a national department of health programme that aims to systematically improve and correct deficiencies in primary health care facilities in the public sector as a mechanism to promote health and to prevent illnesses and further complications through health promotion, early detection, treatment and appropriate referral.43 an ideal clinic is an integrated primary health care facility with good infrastructure, adequate staff, adequate medicine and supplies, good administrative processes, and sufficient adequate bulk supplies.43 the success of the planned national health insurance will depend on a well-functioning primary health care system.43 an ideal clinic should use clinical policies, protocols and guidelines to deliver integrated health care services. this includes the integrated clinical services management approach that builds on the strengths of the hiv programme to deliver integrated care to patients by taking a patient-centric view.43 integrated care removes the requirement for patients to present for care on multiple visits (e.g. well baby clinic [immunisation], prevention of mother-to-child transmission of hiv check-ups and treatment for non-communicable diseases), rather allowing for a patient with multiple linked conditions to be treated in a single visit.44 integrated care could be supported by providing decentralised art-related diagnostics. improving the capacity of local haematology and chemical pathology testing would have a broader impact for the diagnosis of chronic diseases such as diabetes and cardiovascular diseases.44 many of the routine platforms used by the laboratories could easily be extended to include additional tests such as fasting glucose and troponin-t. additionally, some of the polyvalent platforms could be used for multiple tests. for example, the xpert platform is suitable for hiv viral load testing at the candidate laboratories due to the lower daily volumes. as laboratory personnel have already been exposed to this platform for tuberculosis testing, they have acquired the skills to competently use this platform, so that adding the additional test should be relatively easy to implement. should laboratory test volumes increase dramatically in future, either additional xpert platforms could be used or the laboratory could migrate to a higher-throughput roche or abbott hiv viral load platform.27,45 for cd4 testing, the aquios and bd facspresto platforms24,46 are proposed, given the current daily volumes. both platforms are relatively easy to use and have been tested at remote laboratories.46 for hepatitis b surface antigen testing, the vikia lateral flow test (biomerieux, marcy l’etoile, france) is proposed as an option. in south africa, crag screening using the lateral flow assay (immy, norman, oklahoma, united states) is currently offered by all cd4 laboratories as a reflex test following a cd4 ≤100 cells/µl. irrespective of the cd4 technology used, the simplicity of the crag lateral flow test will facilitate easy implementation into a candidate laboratory as the test was primarily designed for use at the poc. for additional art monitoring tests, such as fasting cholesterol and triglycerides, it would be beneficial to perform these tests at larger referral nhls laboratories, where both the required platforms and mono-specialist trained medical technologists are available. these test results are not required for initiation onto art, but can be available for the subsequent clinic visit. if a decision is made to implement fasting cholesterol and triglyceride testing in a candidate laboratory, the alere cholestech ldx analyser (alameda, california, united states) has been evaluated and found to provide clinically-equivalent results when compared to conventional laboratory based platforms.47 however, this platform does not provide a triglycerides result. suitable low throughput platforms for triglyceride testing are not currently available, affirming the need to refer these tests to a centralised facility. outcomes from the cd4 itsdm suggest that using existing nhls laboratories first to extend diagnostic coverage before investigating poc hubs or decentralised poc (true-poc) could reduce costs related to fit-for-implementation site development, training and competency costs and improve capacity.31,48 this approach is similar to public health initiatives such as extending hiv counselling and testing to the clicks® chain of stores in south africa to improve access to hiv counselling and testing services.49 additionally, the itsdm balances high-throughput testing in urban areas with decentralisation in hard-to-reach rural areas. this study has demonstrated that the itsdm could be used to both identify and address coverage gaps for national programs other than cd4, such as hiv viral load, tuberculosis and non-communicable diseases. once approval has been obtained to implement art-related testing at the proposed revised candidate laboratories, extensive planning is required, including site visits, gap analysis and identifying and addressing staffing requirements. in the implementation phase, activities include laboratory preparation, staff recruitment, analyser procurement followed with delivery and set-up, user training and finally, competency assessment and verification (fit-for-purpose). these activities need to be synchronised through a harmonising coordinating body, described as tier-6 in the itsdm, which will coordinate, streamline and standardise all processes relating to implementation, training and quality. in the south african context, this harmonising tier is provided for by the national priority programme unit of the nhls. additional cadres of staff, such as the phlebotomist technicians or medical technicians,50 could be considered to extend testing by recruiting and training community members for long-term sustainability where poc facilities may be needed to extend laboratory services in hard-to-reach areas.51 improved access to art-related testing should improve the overall access to laboratory service delivery to support clinical services in these remote areas. furthermore, building capacity for art-related testing could be an important stepping stone to improving access for the diagnosis of chronic diseases such as cardiovascular diseases, diabetes, chronic respiratory conditions and cancer.52 the algorithm is intended to be an iterative process. once a solution has been implemented, the algorithm could be repeated until all coverage gaps are addressed. if the step-by-step methodology is automated on the corporate data warehouse, this approach could be applied to multiple tests in real time, enabling key coverage decisions within the nhls. limitations this study was a desktop exercise and is based only on data extracted for fy2015/16. it does not reflect nhls plans for expansion of services but is merely an exercise to demonstrate the processes followed in the development of the itsdm and how the model can be applied to inform on other laboratory services. the concepts and methodology described here can be developed in any organisation to provide real time analysis of coverage gaps in a national network of laboratories as well as extend the concept to other laboratory test services with minimal effort. the visual assessment of candidate laboratories in proximity could be further replaced by an automated proximity analysis, as described elsewhere.36,37,53 finally, the model outcomes presented here need vigorous review and testing by senior area managers using their local insights to identify constraints such as space availability, logistics routing and staffing availability to assess the viability of each proposed testing site. costing of the various tiers, including decentralised itsdm, has been undertaken and published elsewhere31,50 with evidence of improved service delivery provided by extending and decentralising testing.48 further health economic studies would be of value to determine both the implementation and incremental cost of providing decentralised art-related testing as described in this work. both a bottom-up and top-down costing analysis would be required to assess costs using a provider perspective, especially in the context of recent local data which suggests a large advanced hiv disease burden in south africa,54 with the ambitious unaids 90-90-90 hiv treatment goals proposed for the third world.55 conclusion this article has demonstrated that a service coverage precinct approach could be used to identify coverage gaps for an art-defined test or -relevant repertoire depending on the programmatic requirements that need to be addressed. the itsdm approach could be used as a starting point to define best practices to introduce sustainable testing as part of a national service extending laboratory coverage to other chronic disease testing. this approach addresses coverage gaps in a cost effective manner. acknowledgements the authors thank the staff of the nhls corporate data warehouse and finance department, as well as pedro da silva, natasha gous, somayya sarang and puleng sheila morokane of the nhls national priority programme for their assistance. we would like to thank dr sergio carmona for his expert advice on hiv viral load testing within the nhls. competing interests the authors declare that they have no financial or personal 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cassim n, coetzee lm, schnippel k, glencross dk. estimating implementation and operational costs of an integrated tiered cd4 service including laboratory and point of care testing in a remote health district in south africa. plos one. 2014;9(12):e115420. https://doi.org/10.1371/journal.pone.0115420 gous nm, berrie l, dabula p, stevens w. south africa’s experience with provision of quality hiv diagnostic services. afr j lab med. 2016;5(2):436. https://doi.org/10.4102/ajlm.v5i2.436 world health organisation (who). noncommunicable diseases [homepage on the internet]. c2017 [cited 2017 jul 06]. available from: http://www.who.int/mediacentre/factsheets/fs355/en/ smith j, smith h, cassim n, et al., editors. geographic location and capacity optimisation modelling to plan effective and efficient diagnostics service placement. first international conference of the african society for laboratory medicine (aslm); cape town, south africa, 1–7 december 2012. coetzee lm, cassim n, glencross dk. analysis of hiv disease burden by calculating the percentages of patients with cd4 counts <100 cells/µl across 52 districts reveals hot spots for intensified commitment to programmatic support. s afr med j. 2017;107(6):507–513. https://doi.org/10.7196/samj.2017.v107i6.1131 joint united nations programme on hiv/aids (unaids). 90-90-90. an ambitious treatment target to help end the aids epidemic [homepage on the internet]. c2014 [cited 2017 mar 12]. available from: http://www.unaids.org/sites/default/files/media_asset/90-90-90_en_0.pdf abstract introduction methods results discussion acknowledgements references about the author(s) odilon d. kaboré laboratory of bacteriology and virology, sourô sanou university teaching hospital, bobo-dioulasso, burkina faso burkina faso national reference laboratory for antimicrobial resistance, bobo-dioulasso, burkina faso laboratory of emerging and re-emerging pathogens, school of health sciences, nazi boni university, bobo-dioulasso, burkina faso higher institute of health sciences, nazi boni university, bobo-dioulasso, burkina faso mélissa j. gonfouli laboratory of emerging and re-emerging pathogens, school of health sciences, nazi boni university, bobo-dioulasso, burkina faso merci muhigwa laboratory of emerging and re-emerging pathogens, school of health sciences, nazi boni university, bobo-dioulasso, burkina faso abdourahmane sow region wide laboratory services, west african health organization, bobo-dioulasso, burkina faso fernand michodigni laboratory of emerging and re-emerging pathogens, school of health sciences, nazi boni university, bobo-dioulasso, burkina faso andré nagalo laboratory of emerging and re-emerging pathogens, school of health sciences, nazi boni university, bobo-dioulasso, burkina faso jacques zoungrana laboratory of emerging and re-emerging pathogens, school of health sciences, nazi boni university, bobo-dioulasso, burkina faso higher institute of health sciences, nazi boni university, bobo-dioulasso, burkina faso department of infectious and tropical diseases, sourô sanou university teaching hospital, bobo-dioulasso, burkina faso arsène hema department of quality, sourô sanou university teaching hospital, bobo-dioulasso, burkina faso augustin konkobo laboratory of emerging and re-emerging pathogens, school of health sciences, nazi boni university, bobo-dioulasso, burkina faso centre muraz, bobo-dioulasso, burkina faso hervé kafando laboratory of emerging and re-emerging pathogens, school of health sciences, nazi boni university, bobo-dioulasso, burkina faso adama ouattara higher institute of health sciences, nazi boni university, bobo-dioulasso, burkina faso division of urology, sourô sanou university teaching hospital, bobo-dioulasso, burkina faso armel poda laboratory of emerging and re-emerging pathogens, school of health sciences, nazi boni university, bobo-dioulasso, burkina faso higher institute of health sciences, nazi boni university, bobo-dioulasso, burkina faso department of infectious and tropical diseases, sourô sanou university teaching hospital, bobo-dioulasso, burkina faso sylvain godreuil joint research unit, research institute for development, national center for scientific research, inserm, faculty of infectious diseases and vectors: ecology, genetics, evolution and control, university of montpellier, montpellier, france abdoul-salam ouedraogo laboratory of bacteriology and virology, sourô sanou university teaching hospital, bobo-dioulasso, burkina faso burkina faso national reference laboratory for antimicrobial resistance, bobo-dioulasso, burkina faso laboratory of emerging and re-emerging pathogens, school of health sciences, nazi boni university, bobo-dioulasso, burkina faso higher institute of health sciences, nazi boni university, bobo-dioulasso, burkina faso centre muraz, bobo-dioulasso, burkina faso citation kaboré od, gonfouli mj, muhigwa m, et al. in vitro antibacterial activity of tigecycline against multidrug-resistant bacteria isolated at the sourô sanou university teaching hospital in bobo-dioulasso, burkina faso. afr j lab med. 2024;14(1), a2895. https://doi.org/10.4102/ajlm.v14i1.2895 original research in vitro antibacterial activity of tigecycline against multidrug-resistant bacteria isolated at the sourô sanou university teaching hospital in bobo-dioulasso, burkina faso odilon d. kaboré, mélissa j. gonfouli, merci muhigwa, abdourahmane sow, fernand michodigni, andré nagalo, jacques zoungrana, arsène hema, augustin konkobo, hervé kafando, adama ouattara, armel poda, sylvain godreuil, abdoul-salam ouedraogo received: 07 june 2025; accepted: 14 oct. 2025; published: 18 dec. 2025 copyright: © 2025. the author(s). licensee: aosis. this work is licensed under the creative commons attribution 4.0 international (cc by 4.0) license (https://creativecommons.org/licenses/by/4.0/). abstract background: tigecycline, an antibiotic effective against multidrug-resistant bacteria (mdrb), remains inaccessible in burkina faso’s hospitals for urgent care. given the resulting therapeutic challenges and mortality in emergency services, evidence-based study of tigecycline’s efficacy on local bacterial clinical isolates is necessary before recommending its use. objective: this study aimed to evaluate the activity of tigecycline on mdrb isolates at the sourô sanou university teaching hospital laboratory, burkina faso. methods: this was a cross-sectional study with prospective and consecutive sampling of mdrbs. the latter included extended-spectrum β-lactamase-producing enterobacterales (esbl-e), carbapenem-resistant strains (crs), and methicillin-resistant staphylococcus aureus (mrsa), which were isolated from urine, blood, pus and puncture fluids between 01 june 2022 and 31 august 2022. antimicrobial susceptibility testing was conducted using the modified kirby-bauer method, and the results were interpreted according to the standard set by the european committee on antimicrobial susceptibility testing in 2021. results: a total of 117 mdrbs, including 93 enterobacterales, 15 carbapenem-resistant acinetobacter baumannii (crab), and 9 mrsas were collected. the proportion of esbl-e was 58% (68/117), followed by crs (34%, 40/117) and mrsa (8%, 9/117). the activity of tigecycline was 95.5% (43/45) on esbl-e, 72.5% (29/40) on crs (including 10/15 crab), and 89% (8/9) on mrsa. conclusion: the activity of tigecycline was highly effective on esbl-e, carbapenem resistant enterobacterales and mrsa, and moderate on crab. what this study adds: this was the first report on the evaluation of tigecycline activity on mdrbs in burkina faso. this non-marketed antibiotic in burkina faso could represent an alternative to spare carbapenems in the treatment of esbl-e infections, and a last resort antibiotic against susceptible crs infections in burkina faso’s hospitals. keywords: acinetobacter baumannii; burkina faso; enterobacterales; extended-spectrum β-lactamase-producing enterobacterales; in vitro; methicillin-resistant staphylococcus aureus; multidrug-resistant bacteria; tigecycline. introduction antimicrobial resistance (amr) has emerged as one of the leading public health threats of the 21st century, and poses an economic burden to the healthcare system because of long hospitalisations and higher morbidity and mortality.1 in 2019, there were approximately 4.95 million deaths associated with antimicrobial resistance, including 1.27 million attributable to multidrug-resistant bacteria (mdrb). the low-income countries of sub-saharan africa are the most affected, with an estimated mortality of 27.3 deaths per 100 000 inhabitants.2 the most problematic mdrbs encompass extended-spectrum β-lactamases (esbl)-producing enterobacterales (esbl-e), carbapenemase-producing enterobacterales (known as carbapenem-resistant enterobacterales, carbapenem-resistant acinetobacter baumannii (crab), and pseudomonas aeruginosa. in addition to vancomycin-resistant enterococcus and methicillin-resistant staphylococcus aureus (mrsa), they constitute a class of ‘superbugs’ called eskape, an acronym with dual significance reflecting both the first letter of the bacterial name and their ability to evade the bactericidal effects of antibiotics.3,4 given the global surge in esbl-e and carbapenem-resistant organisms, it is imperative to find alternative treatments to carbapenems and develop novel therapeutic agents to combat infections caused by these resistant bacteria. new antibiotics have been introduced into the market of developed countries over the last two decades to improve the treatment regimens of mdrb infections,5,6,7 but remain inaccessible in lowand middle-income countries affected by high rates of therapeutic deadlocks linked to superbug infections.2,8 one of these new antibiotics is tigecycline, a semisynthetic 9-tertbutyl-glycylamido derivative of minocycline, which acts by higher binding affinity to the 30s ribosomal subunit of bacteria and by blocking entry of amino-acyl trna molecules into the a site of ribosomes. amino acid residues are prevented from becoming incorporated into elongating peptide chains, thus leading to inhibition of protein synthesis. in addition, the glycylamido side chain at the 9th position on the d-ring results in enhanced activity and allows the molecule to resist efflux pumps compared to classical tetracyclines.9 this antibiotic inhibits the growth of multiple resistant gram-positive, gram-negative and anaerobic bacteria, and atypical bacteria, including mrsa and esbl-e, but it remains vulnerable to the chromosomally-encoded multidrug efflux pumps of pseudomonas aeruginosa and proteae sub-species.10,11 in 2005, tigecycline was approved by the us food and drug administration (fda),12,13 and the european commission granted a valid marketing authorisation throughout the european union for tigecycline (tygacil®) on 24 april 2006.14 despite being available in developed countries and proven effective against mdrb in many studies, this life-saving antibiotic remains inaccessible for urgent treatments in the hospitals of burkina faso, resulting in preventable deaths caused by therapeutic deadlocks.5,12,15 in this regard, the current priority for the hospitals in burkina faso is to find alternatives to reduce the overuse of carbapenems in esbl-e infections, while exploring effective antibiotic regimens to treat carbapenem-resistant organisms in therapeutic deadlock contexts. this study aimed to evaluate the activity of tigecycline on mdrb isolated from clinical samples at the referral healthcare centre in bobo-dioulasso. the expected results will allow us to authorise the supply of tigecycline in the reserve antibiotics for the treatment of patients in the therapeutic deadlocks. methods ethical considerations the study was conducted as part of the routine diagnosis of bacterial infections under the authorisation of the ethics committee for research at the national reference laboratory for antimicrobial resistance (approval number: n°2022-06-053mshp/sg/chuss/dg/dl/sbv/lnr-ram), in line with the declaration of helsinki. the anonymity of the participants involved in the study was maintained in this study. only the laboratory number was mentioned to guarantee confidentiality. no specific declaration of informed consent was required from the patients, as the experiments were part of the routine diagnosis supervised by the burkina faso ministry of health and public hygiene, which has the authority to process patient data for public health surveillance purposes. the outcomes of this work will be used to improve antibiotic prescriptions in patients with mdrb in burkina faso’s hospitals. study design, period, population and site this was a cross-sectional study with a prospective design that used consecutive sampling of mdrb clinical isolates esbl-e, carbapenem-resistant strains (crs) and mrsa from patients between 01 june 2022 and 31 august 2022. bacteriological analysis was carried out at the bacteriology laboratory of sourô sanou university teaching hospital, bobo-dioulasso. this laboratory hosts the burkina faso national reference laboratory for antimicrobial resistance. data and clinical samples collection the participants’ demographic, clinical service and sample types data were collected. all patients whose samples were positive for esbl, crs, and mrsa phenotypes, except p. aeruginosa and members of the proteae tribe (proteus spp., providencia spp. and morganella morganii) which are intrinsically resistant to tigecycline,13 were enrolled. the samples consisted of urine, pus, puncture fluids, and blood. isolation, identification and antimicrobial susceptibility testing media used for bacterial isolation regarding the isolation of mdrbs from urine samples, the culture media employed in this process were cystine lactose electrolyte deficient agar, and eosin methylene blue agar. considering bacterial isolation from blood culture, pus, and serous fluid cultures, appropriate agar plates (enriched chocolate, eosin methylene blue or chapman agar) were selected based on the presumed bacterial characteristics from a gram stain. bacterial identification bacterial presumptive identification was based on their morphological and cultural characteristics. enterobacterales and non-fermenting gram-negative bacilli were identified using commercialised kits, analytical profile index (api) 20e and api 20 ne strips (biomérieux, marcy-l’étoile, france), respectively.16 for gram-positive cocci, catalase and coagulase tests were used. briefly, the catalase test was conducted using the slide method with 1 drop of 3% hydrogen peroxide to observe an immediate bubble formation, indicating the presence of the catalase enzyme. regarding the coagulase test, a lump of bacterial cells was mixed into a drop of ethylenediaminetetraacetic acid-treated rabbit plasma on a microscope slide to observe the clumping of bacterial cells within 10 seconds. when a sample of gram-positive cocci responded positively to the catalase and coagulase tests, the microorganism was confirmed as s. aureus. antimicrobial susceptibility testing susceptibility of each isolate to various antimicrobial agents was determined through the kirby-bauer (kb) method (disc-diffusion method)17 on mueller-hinton agar (mha) plates (biomaxima, lublin, poland). briefly, single colonies of each clinical isolate were suspended in 5 ml sterile distilled water. the turbidity of the suspension was adjusted to 0.5 mcfarland’s standard and subsequently spread onto the surface of the mha plate using a sterile cotton swab. afterwards, antibiotic-impregnated disks were carefully placed onto an mha plate using forceps, and the plates were then incubated at 37 °c overnight. the inhibition diameters of 15 µg tigecycline were measured and interpreted as resistant or susceptible using standard reference values according to ca-sfm-eucast 2021.18 phenotypic antibiotic-resistance analysis extended-spectrum β-lactamase detection using the double synergy test the double synergy test was used for the detection of esbl production. briefly, it consisted of placing a 30 µg disk of third-generation cephalosporin (cefotaxime, ceftriaxone, ceftazidime) and/or other (aztreonam [30 µg] or cefepime [30 µg] at a distance of 30 mm, centre to centre, of an amoxicillin/clavulanic acid disk (20 µg/10 µg). the production of esbl resulted in very characteristic synergistic images between the third-generation cephalosporin disk or aztreonam (30 µg) and amoxicillin/clavulanic acid disk (20 µg/10 µg): a so-called champagne cork image.18 carbapenem-resistant strains detection regarding the detection of carbapenem-resistant enterobacterales, any isolate with decreased susceptibility to ertapenem (inhibition diameter < 25 mm; 10 µg) through agar diffusion test was considered resistant. for a. baumannii, a decreased susceptibility to imipenem 10 µg (inhibition diameter < 21 mm) or meropenem 10 µg (inhibition diameter < 15 mm) through agar diffusion test was considered resistant.18 methicillin-resistant s. aureus detection for mrsa identification, strains with a cefoxitin (30 µg disk) zone diameter < 22 mm were considered as mrsa phenotype.18 activity of tigecycline on multidrug-resistant bacteria detection we tested the activity of tigecycline (15 µg) on all mdr strains of escherichia coli in accordance with the eucast 2021 guidelines, and an inhibition diameter ≥ 18 mm was considered sensitive to tigecycline. for the other species of enterobacterales, the inhibition diameter ≥ 19 mm has been considered for susceptible strains as suggested by the fda (https://www.accessdata.fda.gov/drugsatfda_docs/label/2013/021821s026s031lbl.pdf). all mrsa strains having an inhibition diameter ≥ 19 mm were considered susceptible to tigecycline.18 considering crab, susceptibility to tigecycline was inferred using the clear zone diameters suggested by the fda criteria.19 susceptibility to tigecycline was defined based on clear zone diameters as susceptible (≥ 19 mm), whereas clear zone diameters < 19 mm were considered as resistant, although fda guidelines suggest intermediate for 15 mm – 18 mm, or ≤ 14 mm for resistant.19 quality controls quality control of antimicrobial susceptibility testing was assessed by using reference strains such as e. coli atcc 25922, s. aureus atcc 25929, and klebsiella pneumoniae atcc 700603, in accordance with eucast guidelines.18 data processing and analysis data were recorded in a microsoft excel 2016 worksheet, which was used to create graphs and tables. statistical analysis was performed with epi info 7™ software. the qualitative variables were expressed in proportions, whereas the quantitative variables were expressed in means with their 95% confidence intervals. proportions of mdrbs sensitive or resistant to tigecycline were compared with the pearson chi-square test when there was a normal distribution; otherwise, the fisher’s exact test was used. the significance threshold of 5% was considered for all statistical tests. results a total of 986 non-duplicate samples were included during the study period (01 june 2022 to 31 august 2022). the bacteriological yield was 44.52% (439/986) positive cultures. amongst the 439 positive cultures, 117 strains were mdrbs (26.65%), isolated from 117 patients (figure 1). figure 1: distribution of non-duplicate clinical samples and multidrug-resistant bacteria (n = 117) isolated, sourô sanou university teaching hospital in bobo-dioulasso, burkina faso, 01 june 2022 – 31 august 2022. patient sociodemographic characteristics out of the 117 patients observed with mdrbs, the male gender was predominant (53.8%). the average age of the 117 patients from whom we isolated mdrbs was 41.27 ± 17.9 years, ranging from 1 month to 85 years. the most represented age group was 40–50 years (17.9%), followed by 20–30 years (14.5%). regarding the distribution of mdrbs by patient origin, 80% came from various hospitalisation wards of the sourô sanou university teaching hospital, and 20% from other health facilities in the city of bobo-dioulasso. considering the hospitalisation wards, the urology department was most represented with 14.5%, followed by the intensive care unit (12%). distribution of multidrug-resistant bacteria (n = 117) according to bacterial species out of the 117 mdr isolates, e. coli was the most represented bacterial species (p < 0.018) with a frequency of 53% (62/117), followed by k. pneumoniae accounting for 17% (20/117). as described in figure 2, crab was isolated with a frequency of 13%, mrsa at 9%, and k. oxytoca at 5%. figure 2: distribution of multidrug-resistant bacteria according to bacterial species, sourô sanou university teaching hospital in bobo-dioulasso, burkina faso, 01 june 2022 – 31 august 2022 (n = 117). distribution of multidrug-resistant bacteria according to resistance phenotypes out of the 117 mdrbs isolated, the most predominant phenotype was esbl-producing enterobacterales (58%), including e. coli (72.58%), followed by k. pneumoniae with 67%. the resistance phenotypes to carbapenems (crs) represented 37% (p < 0.001; esbl-e versus crs). all strains of a. baumannii (n = 15) were resistant to carbapenems. for mrsa, nine strains were isolated, as detailed in table 1. table 1: identification and distribution of multidrug-resistant bacteria according to resistance phenotypes, sourô sanou university teaching hospital in bobo-dioulasso, burkina faso, 01 june 2022 – 31 august 2022 (n = 117). in vitro activity of tigecycline on multidrug-resistant bacteria species nine strains of enterobacterales, including 6 (66.67%) e. coli (2 esbls and 4 crs) and 3 (33.33%) k. pneumoniae (1 esbl and 2 crs), exhibited resistance to tigecycline. other enterobacteria, k. oxytoca, raoultella terrigena, and serratia marcescens, were all susceptible to tigecycline. considering the strains of a. baumannii, 66.67% (10/15) were susceptible to tigecycline. there was no statistically significant difference in the distribution of in vitro activity of tigecycline on mdrbs species (p > 0.32), as detailed in table 2. table 2: tigecycline activity according to multidrug-resistant bacteria species, sourô sanou university teaching hospital in bobo-dioulasso, burkina faso, 01 june 2022 – 31 august 2022. tigecycline activity against resistance phenotypes out of 117 antibiotic-resistant bacterial isolates, the overall activity of tigecycline on all mdrbs was 95.33% (102/117) with p-value < 0.002. out of the 68 esbl-e strains, tigecycline was active against 65 (95.59%) of total strains. considering the 40 crs strains, 29 (72.50%) were susceptible to tigecycline. of the nine mrsa isolated, only one strain was resistant. the activity of tigecycline against resistance phenotypes were described in table 3. table 3: activity of tigecycline against resistance phenotypes, sourô sanou university teaching hospital in bobo-dioulasso, burkina faso, 01 june 2022 – 31 august 2022 (n = 117). tigecycline activity on the two predominant isolates of enterobacterales (e. coli and klebsiella spp.) resistance phenotypes regarding the esbl-producing e. coli clinical isolates (45 strains) and the 17 carbapenem-resistant e. coli strains, tigecycline was active in 96% esbl strains (43/45), while 76% crs were found to be susceptible to tigecycline (13/17), p < 0.04 (figure 3). figure 3: tigecycline activity against (a) escherichia coli resistance phenotypes (n = 62; p < 0.04) and (b) klebsiella spp. resistance phenotypes (n = 27; p < 0.1), sourô sanou university teaching hospital in bobo-dioulasso, burkina faso, 01 june 2022 – 31 august 2022. considering the klebsiella spp. clinical isolates (27 strains), and the 7 carbapenem-resistant of k. strains, tigecycline was active on 20 esbl klebsiella spp. (14 k. pneumoniae and 6 k. oxytoca) while 71% carbapenem-resistant k. pneumoniae have been found to be susceptible to tigecycline (5/7), p < 0.1 (figure 3). discussion limited treatment options for mdrbs represent a serious global health issue, particularly in lowand middle-income countries that struggle to access new antibiotics to overcome emerging crs, esbl-e, and mrsa infections.8 in this context, we carried out this study to investigate the in vitro activity of tigecycline against mdrbs and to provide local data to advocate for the availability of tigecycline in the reserve antibiotics for the management of therapeutic impasses. regarding the esbl-e, a high rate of tigecycline activity (96%, 65/68) was observed in this study. this is similar to that reported in a systematic review in which tigecycline was microbiologically active against the majority of esbl-e, including more than 99% of 1936 resistant e. coli isolates.20 in addition, our results were consistent with those of zhang et al.21 in a global study on antimicrobial susceptibility between 2012 and 2016. the authors observed good activity of tigecycline on 98.4% of e. coli and 90.4% of serratia spp.21 furthermore, our observation was comparable with the finding reported by morosini et al. in spain (93.7%),22 but higher than the one mentioned by bazi et al. for the esbl-e (76%).23 globally, tigecycline has excellent in vitro activity against almost all esbl-e.20 this property allows it to evade many common resistance mechanisms, making it a suitable alternative for the treatment of many serious and life-threatening infections for which other antibiotics are no longer appropriate.24 as a result, tigecycline might circumvent the overuse of carbapenems during the era of mdr in esbl-e in burkina faso’s hospitals, as demonstrated in a previous study in thailand.25 considering carbapenem-resistant gram-negative bacteria, we observed a susceptibility of 72.5% (29/40) to tigecycline. this finding is higher than that reported by bazi et al. in morocco,23 and others in réunion island.26 the authors in morocco found an overall susceptibility rate of 57% of carbapenem-resistant gram-negative bacteria to tigecycline, and those in réunion, 67%. these differences could be explained by the long-term routine use of tigecycline in these countries compared to burkina faso, where it has not yet been marketed. indeed, carbapenem-resistant gram-negative bacteria employ several resistance mechanisms, including decreased expression of outer membrane porins, modification of the tigecycline’s target site, and overexpression of efflux pumps to inactivate tigecycline.27 therefore, the strength of the selection pressure for tigecycline would be an important parameter contributing to the complexity of resistance evolution in localities where it is widely used.28 regarding carbapenem-resistant enterobacterales, tigecycline activity was higher with carbapenem-resistant e. coli (13/17, 76%) than that observed with carbapenem-resistant k. pneumoniae (crkp; 5/7, 71%), and this observation is supported by numerous studies.29,30,31 for example, fatima et al. in pakistan reported a better activity of tigecycline on 97% (34/35) for carbapenem-resistant e. coli versus 86.3% (88/102) for crkp.30 the same trends were observed by wang et al. in china, with significantly higher susceptibility rates of 97.73% for carbapenem-resistant e. coli compared to 64.86% for crkp.32 likewise, an increasing prevalence of crkp isolates was reported from china to cause decreased tigecycline susceptibility.33 however, the lower susceptibility rates observed in our study compared to others may be because of our small sample size.32 regarding the efflux-mediated mechanisms in crkp, including high expression of the acrab-tolc and oqxab efflux pumps, appears to play a key role in tigecycline resistance.34,35 in our study, the susceptibility rate for crab with tigecycline was 66%. this is similar to those reported by bazi et al. (66%) in morocco23 and by navon-venezia, leavitt, and carmeli (66%) in israel.36 however, our observation was lower than that of nahid et al. in south africa. the authors reported a susceptibility of 75.8% to tigecycline on crab.37 the lower rate of the susceptibility that we found must be counterbalanced by the influence of culture media and testing technique, which often shows false-intermediate or false-resistant results in tigecycline susceptibility testing.38 previous studies found some discrepancies between different antimicrobial susceptibility testing methods39 and the mha composition (mha from oxoid versus mha from hopebio).40 accordingly, broth microdilution is the reference method for testing tigecycline susceptibility, and a recent study reported that broth microdilution confirmed 91% susceptible crab strains to tigecycline yielded categorical agreement of 87.3% for crab with kb, suggesting that the kb method often shows false-resistant results.38 in addition, it has been reported that disk diffusion (used in our study) and e-test have a poor correlation with broth microdilution as the reference method, especially for a. baumannii. the content of manganese, zinc, and magnesium in different mha may also explain these discrepancies in tigecycline inhibition diameters.40,41 however, caution should be taken in interpreting false resistance phenotypes through the disk diffusion assay in our study, which may exclude wrong inhibitory activity of tigecycline as the only therapeutic option in a field where multi-resistance is predominant.42,43,44 in cases of resistance, it would be advisable to carry out the antimicrobial susceptibility testing in a liquid medium using the vitek-2 compact (biomérieux, marcy-l’étoile, france)45 to provide reliable results to the clinician. regarding the mrsa strains isolated in our study, we observed that one (1/9) strain was resistant to tigecycline (11%). this observation was consistent with the finding of che hamzah et al. in terengganu, malaysia, who found a sensitivity of 94% (85/90) to tigecycline on mrsa.46 by contrast, the susceptibility rate that our study found (89%) is lower than that observed by other authors who reported a high susceptibility of mrsa to tigecycline (> 99.9%) amongst 7098 s. aureus isolates.47,48 similarly, khalili et al. reported a susceptibility of 100% on 99 mrsa tested with the kb method in tehran, iran.49 however, the lower number of mrsa isolates (< 30) and the kb method used in our study may reduce the susceptibility rate, which does not allow us to draw a definitive conclusion about the activity of tigecycline on mrsa. despite the relatively low rate of tigecycline susceptibility on mrsa in our study compared to others, resistance to tigecycline is barely found in s. aureus worldwide,47,49,50 and this molecule could be considered as an alternative antibiotic for most mrsa infections to decrease the risk of resistance to vancomycin, which remains the last-resort antibiotic for the treatment of mrsa infections. strengths and limitations this was the first study documented in burkina faso, that investigated tigecycline activity on mdrbs and could guide public health policy. this was a laboratory-based study with a limited sample size and locations. it should be supported by further clinical evaluations of the efficacy and adverse effects of tigecycline with multiple doses. recommendations the authors recommend that the direction of sourô sanou university teaching hospital should make tigecycline available for treatment of infections caused by esbl-e, carbapenem-resistant bacteria, or mrsa strains. we also request the hospital practitioners to use tigecycline as a reserve antibiotic to treat mdrb strains. we urge the other hospitals in the country to conduct their own studies to evaluate the effectiveness of tigecycline against clinical resistant bacteria (esbl-e, crs and mrsa) before considering it as a reserve antibiotic. conclusion in this current study, the activity of tigecycline was highly effective in vitro against esbl-e and moderate against carbapenem-resistant enterobacterales and crab and on mrsa. therefore, tigecycline was recommended as an option for the treatment of mdrb infections in the hospitals of burkina faso. however, it should be used in combination therapies to mitigate resistance, a common drawback of monotherapy against crs. furthermore, the presence of resistance phenotypes amongst the mdrbs reported in this study, even before the official introduction of tigecycline in burkina faso’s hospitals, necessitates its usage as a reserve antibiotic. finally, despite the fda’s warning of an increased mortality risk associated with the use of tigecycline,51 this non-marketed antibiotic in burkina faso could represent a suitable therapeutic alternative to spare carbapenems in the treatment of esbl-e infections. acknowledgements we are grateful to madam salimata ouattara, djamilatou diéssongo and adama kaboré for their technical assistance. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. credit authorship contribution odilon d. kaboré and abdoul-salam ouedraogo: conceptualisation. abdourahmane sow, sylvain godreuil: methodology and data curation. mélissa j. gonfouli, merci muhigwa, odilon d. kaboré: writing original draft. odilon d. kaboré, mélissa j. gonfouli, merci muhigwa, abdourahmane sow, adama ouattara, andré nagalo, hervé kafando, augustin konkobo, jacques zoungrana, arsène hema, armel poda, abdoul-salam ouedraogo and fernand michodigni: writing review & editing. all authors reviewed the article, contributed to the discussion of results, approved the final version for submission and publication, and take responsibility for the integrity of its findings. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability all relevant data that support the findings are available in this article. additional supporting information may be found upon reasonable request from the corresponding author, odilon d. kaboré. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. the article does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the authors are responsible for this article’s results, findings, and content. references ventola cl. the antibiotic resistance crisis. pharmacol ther. 2015;40(4):277–283. murray cj, ikuta ks, sharara f, et al. global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. lancet 2022;399(10325):629–655. https://doi.org/10.1016/s0140-6736(21)02724-0 pendleton jn, gorman sp, gilmore bf. clinical relevance of the eskape pathogens. expert rev anti-infect ther. 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2021;40(6):1149–1154. https://doi.org/10.1007/s10096-020-04123-z zarkotou o, pournaras s, altouvas g, et al. comparative evaluation of tigecycline susceptibility testing methods for expanded-spectrum cephalosporinand carbapenem-resistant gram-negative pathogens. j clin microbiol. 2020;50(11):3747–3750. https://doi.org/10.1128/jcm.02037-12 qu j, zong z, yu r, hu t, ye h, lü x. impact of test methodology, media type and ion content on the susceptibility of acinetobacter spp. to tigecycline. j antimicrob chemother. 2014;69(6):1710–1711. https://doi.org/10.1093/jac/dkt546 fernández-mazarrasa c, mazarrasa o, calvo j, arco a del, martínez-martínez l. high concentrations of manganese in mueller-hinton agar increase mics of tigecycline determined by etest. j clin microbiol. 2009;47(3):827–829. https://doi.org/10.1128/jcm.02464-08 jones rn, ferraro mj, reller lb, schreckenberger pc, swenson jm, sader hs. multicenter studies of tigecycline disk diffusion susceptibility results for acinetobacter spp. j clin microbiol. 2007;45(1):227–230. https://doi.org/10.1128/jcm.01588-06 casal m, rodríguez f, johnson b, et al. influence of testing methodology on the tigecycline activity profile against presumably tigecycline-non-susceptible acinetobacter spp. j antimicrob chemother. 2009;64(1):69–72. https://doi.org/10.1093/jac/dkp169 hope r, mushtaq s, james d, et al. tigecycline activity: low resistance rates but problematic disc breakpoints revealed by a multicentre sentinel survey in the uk. j antimicrob chemother. 2010;65(12):2602–2609. https://doi.org/10.1093/jac/dkq370 lall s, bhat v, biswas s, khattry n. comparative evaluation of in vitro activity of tigecycline using the disc diffusion method and the vitek-2 compact in clinical isolates at a tertiary care cancer center. med lab j. 2024;18(4):17–21. che hamzah am, yeo cc, puah sm, et al. tigecycline and inducible clindamycin resistance in clinical isolates of methicillin-resistant staphylococcus aureus from terengganu, malaysia. j med microbiol. 2019;68(9):1299–1305. https://doi.org/10.1099/jmm.0.000993 pfaller ma, huband md, streit jm, flamm rk, sader hs. surveillance of tigecycline activity tested against clinical isolates from a global (north america, europe, latin america and asia-pacific) collection (2016). int j antimicrob agents. 2018;51(6):848–853. https://doi.org/10.1016/j.ijantimicag.2018.01.006 yang q, xu y-c, kiratisin p, dowzicky mj. antimicrobial activity among gram-positive and gram-negative organisms collected from the asia-pacific region as part of the tigecycline evaluation and surveillance trial: comparison of 2015 results with previous years. diagn microbiol infect dis. 2017;89(4):314–323. https://doi.org/10.1016/j.diagmicrobio.2017.08.014 khalili h, dashti-khavidaki s, khaleghi s, maleki z, rasoolinejad m. evaluation of tigecycline activity against methicillin-resistante staphylococcus aureus isolated from biological samples. iran j pharm res. 2010;9(1):61–65. putnam sd, sader hs, farrell dj, jones rn. sustained antimicrobial activity of tigecycline against methicillin-resistant staphylococcus aureus (mrsa) from united states medical centers from 2004 through 2008. j chemother. 2010;22(1):13–16. https://doi.org/10.1179/joc.2010.22.1.13 prasad p, sun j, danner rl, natanson c. excess deaths associated with tigecycline after approval based on noninferiority trials. clin infect dis. 2012;54(12):1699–1709. https://doi.org/10.1093/cid/cis270 abstract introduction methods results discussion acknowledgements references about the author(s) patrick orikiriza epicentre mbarara research centre, mbarara, uganda department of microbiology, faculty of medicine, mbarara university of science and technology, mbarara, uganda dan nyehangane epicentre mbarara research centre, mbarara, uganda daniel atwine epicentre mbarara research centre, mbarara, uganda john j. kisakye department of biological sciences, college of natural sciences, makerere university, kampala, uganda kennedy kassaza epicentre mbarara research centre, mbarara, uganda juliet-mwanga amumpaire epicentre mbarara research centre, mbarara, uganda yap boum ii epicentre mbarara research centre, mbarara, uganda citation orikiriza p, nyehangane d, atwine d, et al. evaluation of the sd bioline tb ag mpt64 test for identification of mycobacterium tuberculosis complex from liquid cultures in southwestern uganda. afr j lab med. 2017;6(2), a383. https://doi.org/10.4102/ajlm.v6i2.383 original research evaluation of the sd bioline tb ag mpt64 test for identification of mycobacterium tuberculosis complex from liquid cultures in southwestern uganda patrick orikiriza, dan nyehangane, daniel atwine, john j. kisakye, kennedy kassaza, juliet-mwanga amumpaire, yap boum ii received: 20 oct. 2015; accepted: 23 may 2016; published: 31 mar. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: to confirm presence of mycobacterium tuberculosis complex, some tuberculosis culture laboratories still rely on para-nitrobenzoic acid (pnb), a traditional technique that requires sub-culturing of clinical isolates and two to three weeks to give results. rapid identification tests have improved turnaround times for mycobacterial culture results. considering the challenges of the pnb method, we assessed the performance of the sd bioline tb ag mpt64 assay by using pnb as gold standard to detect m. tuberculosis complex from acid-fast bacilli (afb) positive cultures. objectives: the aim of this study was to determine the sensitivity, specificity and turnaround time of the sd mpt64 assay for identification of m. tuberculosis complex, in a setting with high prevalence of tuberculosis and hiv. methods: a convenience sample of 690 patients, with tuberculosis symptoms, was enrolled at epicentre mbarara research centre between april 2010 and june 2011. the samples were decontaminated using nalc-naoh and re-suspended sediments inoculated in mycobacterium growth indicator tubes (mgit) media, then incubated at 37 °c for a maximum of eight weeks. a random sample of 50 known negative cultures and 50 non-tuberculous mycobacteria isolates were tested for specificity, while sensitivity was based on afb positivity. the time required from positive culture to reporting of results was also assessed with pnb used as the gold standard. results: of the 138 cultures that were afb-positive, the sensitivity of the sd mpt64 assay was 100.0% [95% ci: 97.3 – 100] and specificity was 100.0% (95% ci, 96.4 – 100). the median time from a specimen receipt to confirmation of strain was 10 days [iqr: 8–12] with sd mpt64 and 24 days [iqr: 22–26] with pnb. conclusion: the sd mpt64 assay is comparable to pnb for identification of m. tuberculosis complex and reduces the time to detection. introduction proper diagnosis is the first step toward better management and prevention of tuberculosis transmission. the stop tb partnership has recently described the actions and resources needed to end tuberculosis in the world by 20301. the world is now focusing on a ‘paradigm shift’ that will see countries improve case finding and decrease tuberculosis incidence rates by at least 10% annually. in order to achieve this global plan to end tuberculosis, all healthcare practitioners will be required to ensure that 90% of vulnerable groups are screened, 90% of those diagnosed are started on treatment and 90% are successfully treated. as such, one of the global priorities is on diagnosis.1 discovery, development and rapid uptake of new tools and interventions have been highlighted as major requirements for the success of this plan. currently, significant success has been noted with the integration of the xpert® mtb/rif (cepheid, united states) assay into clinical practice.2 while this tool has the advantage of detecting mycobacterium tuberculosis complex and mutations associated with rifampicin resistance, within approximately two hours2, challenges exist in low-resource settings related to instrument breakdown, inconsistent electric power supply, delayed maintenance, irregular supply of cartridges, limited machine capacity (four tests every two hours) and errors.2 despite the new tool, approximately three million tuberculosis cases annually are not diagnosed,1 thus causing a public health challenge. tuberculosis and hiv form a deadly synergy,3 yet the number of bacilli in the sputum samples of co-infected patients is usually low. as a result, countries including uganda have adopted national tuberculosis programme guidelines that prioritise the use of the xpert® mtb/rif assay on sputum from vulnerable groups such as children and on acid-fast bacilli (afb)-smear-negative, hiv-positive patients with signs and symptoms of tuberculosis.4 however, the majority of persons with presumptive tuberculosis do not necessarily know their hiv status and may not be eligible for the xpert® mtb/rif assay. bacteriological confirmation of tuberculosis by culture isolation currently remains the diagnostic reference standard recommended by the world health organization.5 however, even with a positive liquid culture there is need to differentiate m. tuberculosis complex and non-tuberculous mycobacteria (ntm). although this can easily be achieved by the current molecular methods such as line probe assays, these are complex and bear high infrastructural and human resource requirements.6 traditional methods, such as the use of para-nitrobenzoic acid (pnb) on ziehl-neelsen (zn) positive cultures, are simple, but require pure isolates, which delays results (2–3 weeks).6, 7 this delay impacts clinical management of the patient and potentially prolongs transmission among contacts of tuberculosis patients. there are additional costs associated with incubation requirements, such as staff time, space in the incubator, electricity and other factors that are not always considered.7 recently, rapid methods of identifying m. tuberculosis complex from afb-positive cultures have been developed. these rely on chromatographic detection of mpt64, a protein that is produced by m. tuberculosis complex during its metabolism in cultures.8 among these, the two commonly-available methods include the capilia tb-neo assay (tauns laboratories, inc., numazu, japan) and the sd bioline tb ag mpt64 assay (standard diagnostics, yongin-si, gyeonggi-do, republic of korea; hereafter, sd mpt64 [assay]). these assays have the advantage of being inexpensive, easy to use and readily available, even in low-resource settings.9 they are easily stored at room temperature and allow for results from positive cultures within 15 minutes.10 available data from evaluation of the capilia tb-neo assay indicate that it has excellent sensitivity and specificity.9,11,12 few studies have been done on the sd mpt64 assay in field settings.13,14,15 nevertheless, from the results of these studies, there is general agreement that these methods are suitable replacements for the traditional methods. the aim of this study was to determine the sensitivity, specificity and turnaround time of the sd mpt64 assay for identification of m. tuberculosis complex, in a setting with high prevalence of tuberculosis and hiv. methods ethical considerations the samples were obtained from patients enrolled at the epicentre mbarara research centre with approval from the faculty research and ethics committee and the institutional review board at mbarara university of science and technology, as well as the uganda national council for sciences and technology. all patients signed an informed consent form to participate in the main study and allow further testing on the samples and isolates. study design samples for this cross-sectional study were obtained from patients with signs and symptoms of pulmonary tuberculosis according to world health organization guidelines.16 we enrolled 690 patients at the epicentre mbarara research centre, mbarara, uganda, between april 2010 and june 2011 in a separate study to assess the utility of colorimetric methods to detect m. tuberculosis complex in patients with suspected tuberculosis.17 patients were eligible if they reported a cough for more than two weeks, were at least 15 years of age and signed an informed consent. after consenting, patients’ samples were collected, stored in a cool box and then transported immediately to the laboratory. they were decontaminated using the n-acetyl l-cysteine (nalc)-naoh procedure.18 the concentrated sediments were homogenised with phosphate buffer, inoculated in mycobacterium growth indicator tubes (mgit) (bd, franklin lakes, new jersey, united states) medium and then incubated at 37 °c for up to eight weeks. mgit cultures were read daily on a manual reader until growth was detected. to check for the presence of afb all positive cultures were tested by ziehl-neelsen (zn) smear microscopy; to check for contamination, positive cultures were tested by blood agar. the epicentre laboratory participates in an external quality assurance scheme for culture with the national health laboratory services, south africa. for this study, a convenience sample of 138 afb-positive cultures was considered for testing with sd mpt64 and pnb. the two tests were performed immediately on the isolate according to the manufacturer’s recommendations and laboratory protocol. if afb was present together with contamination, the isolate was re-decontaminated with naoh, sub-cultured in a fresh culture tube and monitored for pure growth before repeating pnb. to determine specificity, we randomly selected 50 known-negative mgit samples from the study, which were tested independently with sd mpt64 and pnb. to confirm ability of the test to rule out ntms, an additional sample of 50 known isolates of mycobacterium fortuitum were sub-cultured into mgit using a standard inoculum of mcfarland 0.5. these were monitored daily until positivity was detected by the mgit instrument. we performed the sd mpt64 assay and pnb on each of them as described above for the mgit-negative cultures (figure 1). figure 1: study profile. the pnb media was prepared locally, stored between 2 °c – 8 °c and quality controlled according to our standard operating procedures. the test required two lowenstein jensen slants one with and another without pnb reagent, the latter serving as negative control. both slants were inoculated with 500 µl of standard inoculum, incubated at 37 °c and read weekly until colonial growth was observed. identification of m. tuberculosis complex was made based on presence of growth in the control tube (without pnb), with no growth in the tube with pnb. manufacturer instructions were followed in performing the sd mpt64 assay. in brief, 100 µl of mixed mgit culture was added directly to the test cartridge and allowed to flow chromatographically for 15 minutes. a positive result was indicated by a red band on the test window in addition to the control band. data were collected using case report forms and double entered using voozanoo software version 2 (epiconcept, paris, france). turnaround time was calculated by measuring the time taken from specimen receipt to reporting of a positive sd mpt64 or pnb assay. ease of use of the techniques was determined through a questionnaire that was filled out by all technicians performing the tests. statistical analyses statistical analysis was performed using stata se v.11 software (college station, texas, united states, 2009). we considered a sample positive with m. tuberculosis complex when confirmed by the pnb gold standard and negative if not confirmed positive for m. tuberculosis complex by the gold standard. for sd mpt64, performance was calculated by estimating the sensitivity, specificity and 95% confidence interval. results the median age (years) and interquartile range (iqr) was 38 (30–48) with a gender ratio (male/female) of 49/51 and hiv positivity of 58.6%. of the 138 afb-positive mgit cultures among the 690 patients included in the study, 136 cultures (98.6%) were confirmed positive for m. tuberculosis complex by both sd mpt64 and pnb, and two as ntm (table 1). this gave a sensitivity of 100% [95% ci: 97.3–100]. all the known negative cultures and ntm were reported as negative for m. tuberculosis complex, giving a specificity of 100.0% [95% ci: 96.4–100], respectively. table 1: cross tabulation of sd mpt64 assay and para-nitrobenzoic acid. the median time from specimen receipt to confirmed identification of m. tuberculosis complex was 10 days [iqr: 8–12 days] with sd mpt64 and 24 days [iqr: 22–26] with pnb. all the technicians who performed the laboratory tests reported that the sd mpt64 assay was easy to use and did not require additional training other than the standard operating procedure. this was not the case with the pnb method. discussion few studies have evaluated the performance of the sd mpt64 assay in field settings with high tuberculosis and hiv burdens. this study confirms prior evaluations and increases the evidence that the test has excellent sensitivity and specificity in identifying m. tuberculosis complex using pnb as a gold standard, in a ugandan field setting where there is high tuberculosis and hiv co-infection. there have been previous evaluations in various countries using different gold standards. all the investigations have shown that the technique has high sensitivity and specificity. one study used reference bacterial strains and mycobacterium bovis field isolates from animals and found a sensitivity of 96.5% [95% ci: 91.2–99.0] and specificity of 100% [95% ci: 96.7–100].19 the same group also found a high positive predictive value of 100% [95% ci: 96.7–100], and a negative predictive value of 92.9% [95% ci: 82.7–98.0]. the sd mpt64 assay was evaluated on a large number of clinical isolates in india and performed with 100.0% sensitivity and specificity.9 another recent study in india used isolates from extra-pulmonary and ntm samples and found sd mpt64 to have a 100.0% sensitivity and specificity compared with conventional tests such as niacin, nitrate reduction and pnb.13 although the sample size was small, they demonstrated the accuracy, cost effectiveness and early identification of m. tuberculosis complex in patients with paucibacillary extra-pulmonary tuberculosis. the performance of the capilia tb-neo assay has been evaluated extensively. one study evaluated its performance using reference strains of m. tuberculosis complex, ntm and other non-related bacteria. they used nucleic acid assays in addition to the capilia tb-neo and sd mpt64 assays. the capilia tb-neo assay had 99.6% sensitivity and 100% specificity, unlike the sd mpt64 assay which had several false positive results with the ntm that were attributed to a high concentration of the bacterial antigen20. this poor specificity, however, was not observed in our study possibly due to only one ntm species evaluated. in uganda, a study done in kampala city provided evidence that the capilia tb-neo assay can be used to identify m. tuberculosis complex successfully from blood cultures.12 they found an overall sensitivity of 98.4% and overall specificity of 97.6% compared to polymerase chain reaction, which supports its use for blood culture isolates. limitations this study was nested within a colorimetric study and thus relied on study design, population selection and sample size of the primary study. the lack of other ntm strains limited exploration of false positivity as reported in other studies. recommendations basing on our findings, we recommend that the sd bioline tb ag mpt64 test be used in place of pnb for identification of m. tuberculosis complex from liquid cultures. conclusion the sd bioline mpt64 assay has good sensitivity and specificity for rapid discrimination between m. tuberculosis complex and ntm in clinical isolates. it also has a short turnaround time in confirming m. tuberculosis complex in afb-positive cultures. in addition, the technicians reported that the test is easy to perform. acknowledgements we would like to express our gratitude and thanks to the study participants. we also thank the epicentre nurses and laboratory personnel, especially john mary ngattu, henry munyambabazi and esther turyashemererwa, who participated in the data collection. we also acknowledge médecins sans frontières for funding the study that generated this data. special thanks to christopher c. moore for a thorough review of this manuscript. finally, we are grateful to the uganda research student support fund for linking the study team. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support this work was made possible with funding from médecins sans frontières. additional support was received from the uganda research student support fund, an initiative that was conceived to fund global students’ research and to link programmes between ugandan universities and several international partners to conduct research on global health issues. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. authors’ contributions p.o., d.n., j.j.k. and y.b. conceived the idea and designed the experiments. p.o., d.n., j.j.k., k.k. and y.b. performed the laboratory experiments. p.o., d.n., j.m.a. and d.a. analysed the data. p.o., d.n., j.j.k., k.k., j.-m.a. and y.b. wrote and reviewed the manuscript. all authors approved the final version of the manuscript. p.o. was the project leader, while y.b. and j.m.a. were project supervisors. all authors made conceptual contributions and were responsible for experimental and project design. p.o., 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kit. indian j med microbiol. 2015;33(5):122–125. http://dx.doi.org/10.4103/0255-0857.150912 kumar vg, urs ta, ranganath rr. mpt 64 antigen detection for rapid confirmation of m. tuberculosis isolates. bmc res notes. 2011;24:79. http://dx.doi.org/10.1186/1756-0500-4-79 kanade s, nataraj g, suryawanshi r, mehta p. utility of mpt 64 antigen detection assay for rapid characterization of mycobacteria in a resource constrained setting. indian j tuberc. 2012;59:92–96. world health organization. treatment of tuberculosis: guidelines. 4th ed. geneva, switzerland: who; 2010. boum y, orikiriza p, rojas-ponce g, et al. use of colorimetric culture methods for detection of mycobacterium tuberculosis complex isolates from sputum samples in resource-limited settings. j clin microbiol. 2013;51:2273–2279. http://dx.doi.org/10.1128/jcm.00749-13 kent pt, kubica gp. 1985. public health mycobacteriology: a guide for the level iii laboratory. u.s. department of health and human services, centers for disease control and prevention, atlanta, ga. byeon hs, ji mj, kang ss, et al. performance of the sd bioline tb ag mpt64 rapid test for rapid confirmation of mycobacterium bovis isolates from animals. j vet sci. 2014;16:31–35. http://dx.doi.org/10.4142/jvs.2015.16.1.31 chikamatsu k, aono a, yamada h, et al. comparative evaluation of three immunochromatographic identification tests for culture confirmation of mycobacterium tuberculosis complex. bmc infect dis. 2014;1:54. abstract introduction methodology results discussion acknowledgements references about the author(s) nse o. umoh department of medical laboratory science, faculty of health sciences and technology, ebonyi state university, abakaliki, nigeria chimezie f. nwamini department of medical laboratory science, faculty of health sciences and technology, ebonyi state university, abakaliki, nigeria nyoho j. inyang department of medical laboratory science, faculty of basic medical sciences, ambrose alli university, ekpoma, nigeria anthony n. umo department of medical microbiology and parasitology, college of health sciences, university of uyo, uyo, nigeria victor u. usanga department of medical laboratory science, faculty of health sciences and technology, ebonyi state university, abakaliki, nigeria amos nworie department of medical laboratory science, faculty of health sciences and technology, ebonyi state university, abakaliki, nigeria michael o. elom department of medical laboratory science, faculty of health sciences and technology, ebonyi state university, abakaliki, nigeria boniface n. ukwah department of medical laboratory science, faculty of health sciences and technology, ebonyi state university, abakaliki, nigeria citation umoh no, nwamini cf, inyang nj, et al. prevalence of urinary schistosomiasis amongst primary school children in ikwo and ohaukwu communities of ebonyi state, nigeria. afr j lab med. 2020;9(1), a812. https://doi.org/10.4102/ajlm.v9i1.812 original research prevalence of urinary schistosomiasis amongst primary school children in ikwo and ohaukwu communities of ebonyi state, nigeria nse o. umoh, chimezie f. nwamini, nyoho j. inyang, anthony n. umo, victor u. usanga, amos nworie, michael o. elom, boniface n. ukwah received: 09 apr. 2018; accepted: 12 may 2020; published: 24 aug. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: urinary schistosomiasis is a serious public health challenge in some communities of ebonyi state, south-east nigeria, partly resulting from a lack of adequate epidemiological data for the institution of effective control strategies. objective: this study evaluated the prevalence and risk factors of urinary schistosomiasis in rural communities of ebonyi state, south-east nigeria. methods: a total of 300 students, comprising 185 boys and 115 girls, were randomly selected for the study between july and december 2016. a questionnaire was administered to all participants to determine the risk factors for the disease in the area. urine specimens collected from the participants were processed by sedimentation and examined microscopically for the eggs of schistosoma haematobium. results: the overall prevalence rate for urinary schistosomiasis was 8.0%. students aged 6–10 years had the highest prevalence of infection (10.3%). the prevalence was significantly higher amongst male students (10.3%; p = 0.038) compared with female students (4.4%). logistic regression analysis showed a significant association between schistosomiasis infection and freshwater contact activities (p = 0.007; odds ratio = 1.89; 95% confidence interval: 4.33–16.17). contact with stream, pond, river and well water were associated with infection rates of 25%, 14%, 5.3%, and 4.4%, respectively. conclusion: a relatively low prevalence of urinary schistosomiasis was found in the area. participants’ socio-economic status and dependence on contaminated water sources were core modifiable risk factors. health education and development of potable water infrastructure, amongst other interventions, would likely reduce the burden and transmission of urinary schistosomiasis in this locality. keywords: urinary schistosomiasis; transmission; prevalence; ebonyi state; nigeria. introduction urinary schistosomiasis is a parasitic disease of the tropics and sub-tropics caused by infection of humans with the trematode (parasitic flatworm) known as schistosoma haematobium. although highly preventable, the disease ranks second only to malaria in terms of prevalence and socio-economic importance of parasitic diseases in endemic tropical and subtropical countries.1,2,3 schistosomiasis remains a challenging disease of public health importance, with approximately 779 million people estimated in 2008 to be at risk globally.4 worldwide, nigeria has the highest prevalence of urinary schistosomiasis, with about 29 million cases and about 101 million people at risk of infection in 2010.5,6,7 the high prevalence of urinary schistosomiasis in nigeria has been ascribed mainly to the wide distribution of bulinus spp., the snail host of s. haematobium, and the indiscriminate passage of urine harbouring s. haematobium eggs by infected individuals into water lodging the snail host.8,9 several factors, including poor sanitation, poverty, ignorance, limited access and availability of health facilities and social amenities, also account for the high prevalence of urinary schistosomiasis in developing countries.10 human infection results when man comes into contact with water harbouring the infective stage of the parasites, the free-swimming cercariae, which have the capability of directly penetrating the water-softened, intact skin of humans who are carrying out water-related activities, such as fishing, laundry, bathing and swimming.11,12 the presence of the intermediate snail hosts of the parasite and increased human contact with contaminated water bodies are the key determining factors that favour the transmission of the disease. people at maximum risk are those who live in, or travel to, endemic areas and make contact with water containing the intermediate host.8 children are usually prone to acquiring urinary schistosomiasis, because of a strong tendency for playing in water, which predisposes them to infection.10 early detection of the parasite in infected persons is key to the control and prevention of the disease, particularly with praziquantel therapy.13 parasitological diagnosis involving microscopic examination of urine for the parasite eggs is the most practical and widely-used method for detecting infected individuals;14 however, immunoserological diagnosis involving detection of the parasite antigens and antibodies with techniques such as an enzyme-linked immunosorbent assay have been reported to be very efficient, especially in early infections.15 school-age children constitute an ideal target group for investigation of urinary schistosomiasis in endemic communities, because of their known habits of poor hygiene and playing in water, which enhance the chances of infection with the parasites. the data generated from this age group have proven valuable, not only for justifying their inclusion in mass treatment programmes, but also for determining the need for such interventions.10,16 urinary schistosomiasis is a serious public health challenge in some communities of ebonyi state, south-east nigeria.17 in order to provide a contemporary roadmap for establishing suitable prevention and control strategies, this study evaluated the prevalence and risk factors of the disease in some rural communities of the area. methodology ethical considerations ethical approval for this study (fhst/ec/28) was obtained from the ethical committee, faculty of health sciences and technology, ebonyi state university, abakaliki (ebsu/2016/51032). study population the participants of this cross-sectional study, conducted between july 2016 and december 2016, were selected by simple random sampling (with the aid of a random number table) from amongst students of six primary schools in the ikwo and ohaukwu local government areas (lgas), following official authorisation by both councils. out of 348 pupils initially recruited from both lgas (ikwo, n = 186, ohaukwu, n = 162), a total of 300 students, comprising 185 boys and 115 girls, aged between 5 and 15 years, participated in this study based on the inclusion requirements of written consent, completed questionnaire, and submission of urine specimens. a signed or thumb-printed written consent for voluntary participation of each student was obtained from a parent or guardian. parents and guardians were informed that participants’ information would be treated with utmost confidentiality and used for the purpose of the research only before giving consent. they were also informed of the potential health and social benefits of the study. to protect the participants’ information, personal and quasi identifiers (such as occupation of the parents) were masked with numbers and letters, respectively. the sample size was determined using the formula described by charan and biswas18 for cross-sectional studies. study area this study was carried out in ohaukwu and ikwo lgas of ebonyi state, south-east nigeria. ohaukwu lga is located at latitude 6°31’58.3”n and longitude 8°1’22.9”e (central part) of ebonyi state and has an area of about 517 km2, with an estimated population of about 195 337, whereas ikwo lga is located at latitude 6°4’59”n and longitude 8°5’59”e (northern part) of ebonyi state and has an area of about 500 km2, with a population of about 214 969.19 the two lgas are separated from each other by a distance of about 30 km. this area has a typical tropical climate, comprising dry and wet seasons, with an annual average rainfall of 1300 mm and an atmospheric temperature of about 30 °c; the vegetation characteristics are predominantly guinea savannah. the area is made up of several rural communities traversed by streams and rivers, which constitute the inhabitants’ major source of water for domestic use, recreation and economic activities, particularly in the absence of pipe-borne water and other social amenities. it is mostly inhabited by peasant and subsistence farmers, whose activities have an important bearing on the ecology of the area. rice farming, mainly in swampy terrains, is the major occupation in the area. administration of questionnaire and collection of samples a questionnaire titled ‘investigation of risk factors associated with the transmission of urinary schistiosomiasis in ikwo and ohaukwu communities’ was administered verbally to each participant, parent, or guardian by the research assistants with the generous support of school tutors, who helped in communicating effectively in the local dialect. participants’ information sought for in the questionnaire included age, residence, source(s) of water for domestic use, water contact activities, history of diseases with symptoms of haematuria, access to healthcare facilities, and the occupation and educational status of parents or guardian, amongst others. each participant was given a sterile dry plastic universal container with a screw lid, in which they were asked to take a terminal urine sample between 10:00 and 12:00, when the ova load is maximal.20 each container was labeled with the sex, age and number of the participant as provided in the questionnaire form. fresh urine samples collected were examined macroscopically for presence of blood (haematuria). the samples were then preserved by adding 5 ml of dilute (0.3%) carbol-fuchsin solution to each 10 ml of urine,21 and transported to the laboratory in an ice-pack. variables definition categorical variables were used to assess the risks factors and prevalence of urinary schistosomiasis in this study. there were six categories, namely: lga, sex, age, occupation, water sources, and water contact activities. processing of samples microscopic examination of the urine samples for urinary schistosomiasis detection was based on detection of terminal-spine eggs of s. haematobium using a sedimentation concentration technique.18 ten millilitres of each urine sample was collected from each sample container into a centrifuge tube and spun for 5 minutes at 3000 revolutions per minute to concentrate the eggs. thereafter, the supernatant fluid was discarded into a petri dish. a drop of the sediments was transferred to a clean and grease-free glass slide, covered with a coverslip and examined microscopically using x100 and x400 magnification for eggs of s. haematobium, recorded as eggs/10 ml of urine. statistical analysis cronbach’s alpha was used to verify the reliability of the data obtained in this study. the prevalence values were calculated by finding the percentage of the factors. associations between demographic characteristics (age, sex, and occupation) and the prevalence of infection were analysed using pearson’s chi-square test. multivariate logistic regression analysis was used to evaluate the geographical and behavioural risks associated with s. haematobium infection. the geographical variables used in the analysis model included borehole, well, pond, stream and river water sources, whilst the behavioural variables comprised swimming, fishing, washing and other domestic uses. p-values of less than 0.05 were considered significant, and 95% confidence intervals were used to locate outliers. results prevalence of urinary schistosomiasis in ohaukwu and ikwo schistosoma haematobium infection was detected in 24 (8.0%) of the 300 students examined. the prevalence of infection was 10.0% (n = 150) in ikwo lga and 6.0% (n = 150) in ohaukwu lga. the difference between the prevalence of infection in ohaukwu and ikwo lgas was not statistically significant (p = 0.24; table 1). table 1: prevalence of urinary schistosomiasis in ohaukwu and ikwo, nigeria, december 2016. demographic distribution of students with schistosoma haematobium infection the highest prevalence of infection by age was 9.4% (n = 160) amongst students aged 6–10 years. students aged 11–15 years had a lower prevalence rate of 7.0% (n = 72), followed by those aged 1–5 years (n = 50; 6.0%), whereas students aged more than 15 years had the lowest (n = 18; 5.5%). the association between prevalence of infection and age of the participants was not statistically significant (p = 0.84; table 2). table 2: demographic distribution of subjects with schistosoma haematobium infection in ohaukwu and ikwo, nigeria, december 2016. of the 185 male students, 19 (10.3%) were infected with s. haematobium compared with 5 (4.3%) of the 115 female students. the impact of sex on the prevalence of infection was statistically significant (p = 0.038; table 2). the highest prevalence of infection by occupation (n = 128; 11.7%) was found amongst students whose parents or guardians were farmers, whilst children of public servants had the least (n = 61; 3.2%) (table 2). a prevalence of 7.0% (n = 85) was found amongst children whose parents or guardians were traders, and 3.8% (n = 26) where the parents or guardians were fishermen. the relationship between the prevalence of infection and the occupation of parents was not statistically significant (p = 0.23; table 2). association between sources of water/contact and schistosoma haematobium infection the highest rate of infection (n = 48; 25%) was found amongst students who utilised stream water mainly for domestic purposes. infection rates of 14.0% (n = 50) were found amongst those that used pond water, 5.3% (n = 57) for river water, and 4.4% (n = 45) for well water. there was no case of infection amongst students who utilised water from borehole facilities; the odds of infection were about three times higher with participants who utilised freshwater sources within the locality compared with those who used borehole water, but this association was not statistically significant (p = 0.55; odds ratio = 2.77; 95% confidence interval: 7.23–22.77; table 3). table 3: association between sources of water and schistosoma haematobium infection (n = 300), nigeria, december 2016. a high infection rate of 10.5% (n = 95) was found amongst participants who engaged in swimming, compared with other activities that involved contact with freshwater sources (table 4). multivariate analysis showed a statistically significant association between participants’ water contact activities and the prevalence of infection (p = 0.007; odds ratio = 1.89; 95% confidence interval: 4.33–16.17). table 4: distribution of schistosoma haematobium infection by freshwater contact activities (n = 270), nigeria, december 2016. discussion urinary schistosomiasis is reported to be endemic in virtually all rural regions of nigeria, because of a widespread occurrence of ecological and socio-economic factors associated with the disease.8,9,22,23 this study confirmed the existence of urinary schistosomiasis in ebonyi state, south-east nigeria with a prevalence of 8.0% in the study area. this prevalence rate was low compared with previous studies in the area, which reported higher rates, 49.7% for ohaukwu lga and 11.0% for onicha lga.17 the low prevalence rate of the disease found in the present study could, firstly, be attributed to the impact of a preventive praziquantel-treatment programme, initiated by the world health organization in 2014, for school-aged children and special risk groups in the area.24 secondly, development of private borehole water facilities often for commercial purposes is trending in some communities of this locality. although not accessible or affordable to a large segment of this rural population for patronage, access to this source of water by some residents, mainly for domestic uses, may have drastically reduced the risk of contact with parasite-infested water sources. this study found a higher prevalence of infection in communities without such facilities. based on such a finding, it would be rather surprising that the disease prevalence was lower in ohaukwu than ikwo lga, which has a few communities in close proximity to infrastructural and healthcare facilities in the state capital city, abakaliki. access to such amenities by the inhabitants of these communities should essentially impact positively on their living conditions, with a likelihood of reducing the prevalence level of the disease. this was, however, not the case, perhaps because of a combination of factors, including poor perception of the disease transmission dynamics, and a high probability of occupation-related infections in these agrarian communities, where parents often go to rice farms in the company of their children. nevertheless, the association between parents’ occupation and the prevalence of urinary schistosomiasis in this study was not statistically significant (p = 0.235). previous studies in some parts of nigeria had reported high prevalence rates of 48.8% (bauchi state), 44% (adamawa state), 55.7% (cross river state), and 21.5% (ebonyi state), which were associated mainly with the predominant occupation of the indigenes, such as fishing and farming.25,26,27,28,29 in concordance with reports from many parts of nigeria,28,30,31 this study found the influence of sex to be an important epidemiological factor in the transmission of urinary schistosomiasis, with a significantly higher prevalence of infection amongst male students compared with female students (p = 0.038). this agrees with the finding of okoli and odaibo32 in a previous study that attributed higher infection rates of urinary schistosomasis amongst school boys in ibadanto to a greater involvement in outdoor activities, such as swimming, washing, paddling of canoes, and irrigation. however, persons who have greater contact with the snail breeding loci are more likely to acquire the infection, regardless of their sex.28 most students in primary school classes are in the 6–10-year age bracket, and therefore it was not surprising that this age group had the highest prevalence of infection, perhaps on account of greater involvement in water-related activities compared with other groups, and not necessarily due to increased vulnerability. children are generally known to be vulnerable to urinary schistosomiasis, because of their strong tendency to play in water, which predisposes them to the infection.10 limitations regardless of the age or sex of students, contact with freshwater sources was a crucial factor for disease transmission, based on the pattern of infection found in this study. the prevalence of the disease was particularly high amongst children who utilised stream water for domestic and other uses, compared with other sources of water, including ponds and river. no case of infection was found amongst students who had unlimited access to borehole water. this finding is consistent with the fact that s. haematobium infection occurs only where there is contact between the population and freshwater sources harbouring the snail vector as well as the infective stage of the parasite.30 thus, this study may have been considerably limited by an inability to examine the water sources for the snail host, and quantify the participants’ water contact activities. nonetheless, a significant association was found between infection and students’ water contact activities. the zero-prevalence rate of the disease found amongst students who utilised borehole water facilities in this locality may underscore the development of infrastructure in rural communities as an important control approach for urinary schistosomiasis. conclusion this study reports a low prevalence of 8.0% for urinary schistosomiasis amongst children in the study area, with a likelihood of further expansion in the disease rate and foci, if appropriate control measures are not initiated urgently by the local health authorities. transmission of the disease in these communities is enhanced mainly by residents’ dependence on unsafe sources of water for domestic use, as well as behavioural and socio-economic tendencies that promote risky contacts with potentially parasite-infested water bodies. continued disease surveillance and selective praziquantel chemotherapy for infected persons, health education of the residents for improved perception of the behavioural and socio-economic activities associated with the disease, and development of water sources would likely improve the living conditions of this rural population with a consequent reduction in the burden and transmission of the disease. future studies on urinary schistosomiasis in these communities should include specific analysis of the water bodies, including malacological evaluation, as well as quantification of the water contact activities of the participants. acknowledgements we are grateful to the school teachers and parents of participants who helped generously in filling communication gaps during administration of our questionnaire and specimen collection. competing interests the authors have declared that no competing interest exists. authors’ contributions n.o.u. designed the study, wrote the protocol and part of the manuscript, managed the analysis, and vetted the manuscript. c.f.n. wrote the first draft of the manuscript, managed specimen collection and carried out the laboratory analysis. m.o.e. and b.n.u. co-designed the study and supervised the laboratory analysis. n.j.i. and a.n.u. managed the statistical analyses. v.u.u. and a.n. managed the literature search and administration of the questionnaire. all authors read and approved the final manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial or 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1999;4(4):308–315. https://doi.org/10.1046/j.1365-3156.1999.00388.x abstract introduction methods results discussion acknowledgements references about the author(s) naseem cassim national health laboratory service (nhls), national priority programmes, johannesburg, south africa department of molecular medicine and haematology, faculty of health sciences, university of witwatersrand, johannesburg, south africa honora smith department of mathematical sciences, university of southampton, southampton, united kingdom lindi m. coetzee national health laboratory service (nhls), national priority programmes, johannesburg, south africa department of molecular medicine and haematology, faculty of health sciences, university of witwatersrand, johannesburg, south africa deborah k. glencross national health laboratory service (nhls), national priority programmes, johannesburg, south africa department of molecular medicine and haematology, faculty of health sciences, university of witwatersrand, johannesburg, south africa citation cassim n, smith h, coetzee lm, et al. programmatic implications of implementing the relational algebraic capacitated location (racl) algorithm outcomes on the allocation of laboratory sites, test volumes, platform distribution and space requirements. afr j lab med. 2017;6(1), a545, https://doi.org/10.4102/ajlm.v6i1.545 original research programmatic implications of implementing the relational algebraic capacitated location (racl) algorithm outcomes on the allocation of laboratory sites, test volumes, platform distribution and space requirements naseem cassim, honora smith, lindi m. coetzee, deborah k. glencross received: 18 aug. 2016; accepted: 16 sept. 2016; published: 28 feb. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract introduction: cd4 testing in south africa is based on an integrated tiered service delivery model that matches testing demand with capacity. the national health laboratory service has predominantly implemented laboratory-based cd4 testing. coverage gaps, over-/under-capacitation and optimal placement of point-of-care (poc) testing sites need investigation. objectives: we assessed the impact of relational algebraic capacitated location (racl) algorithm outcomes on the allocation of laboratory and poc testing sites. methods: the racl algorithm was developed to allocate laboratories and poc sites to ensure coverage using a set coverage approach for a defined travel time (t). the algorithm was repeated for three scenarios (a: t = 4; b: t = 3; c: t = 2 hours). drive times for a representative sample of health facility clusters were used to approximate t. outcomes included allocation of testing sites, euclidian distances and test volumes. additional analysis included platform distribution and space requirement assessment. scenarios were reported as fusion table maps. results: scenario a would offer a fully-centralised approach with 15 cd4 laboratories without any poc testing. a significant increase in volumes would result in a four-fold increase at busier laboratories. cd4 laboratories would increase to 41 in scenario b and 61 in scenario c. poc testing would be offered at two sites in scenario b and 20 sites in scenario c. conclusion: the racl algorithm provides an objective methodology to address coverage gaps through the allocation of cd4 laboratories and poc sites for a given t. the algorithm outcomes need to be assessed in the context of local conditions. introduction the national health laboratory service (nhls) of south africa provides national coordination for the laboratory service, staff training, quality control and quality assurance, as well as managing the overall quality management system. within the national network of 266 laboratories, cd4 testing is currently offered at 59 laboratories to facilitate the staging and monitoring hiv-infected patients. these laboratories operate all levels of pathology service, including routine diagnostic chemical pathology, haematology and microbiology services. cd4 testing is offered using an integrated tiered service delivery model (itsdm) that matches daily testing demand with appropriate testing capacity. this is required to manage laboratory workload, turn-around-time (tat), instrument capacity utilisation and cost.1,2 cd4 testing is standardised using beckman coulter (beckman coulter, miami, florida, united states) equipment and the panleucogating platform.3,4 in 2014, the nhls performed 3.9 million cd4 tests. the itsdm model aims to ensure efficient, cost-effective provision of quality testing across all health districts.1 this ‘full coverage’ model strives toward equitable access to cd4 testing by providing technology that appropriately matches service delivery requirements, based on factors such as test volumes, distances from referring clinics to cd4 laboratories, and the package of clinical services offered by health facilities.1 five testing tiers, along with a sixth coordinating tier, are defined in the itsdm model: (1) true point-of-care (poc) services (tier 1), reserved for hard-to-reach areas, where nursing staff attending to patients operate the testing system (< 3 tests per system per day) and initiate patients onto antiretroviral therapy; (2) poc hubs (tier 2), namely, ‘mini-laboratories’ using only poc equipment for all relevant hiv and tuberculosis tests at a rate of < 10 samples per day in rural health districts; (3) community laboratories (tier 3) processing less than 100 samples per day; (4) district laboratories (tier 4) processing between 100 and 299 samples per day; (5) high volume centralised laboratories (tier 5) processing in excess of 300 samples per day; and (6) tier 6, representing a national reference/‘monitoring and evaluation’ centre responsible for coordination, harmonisation and standardisation of testing, as well as coordination of training and quality control across a national network of laboratories and related testing sites.1 it would also be cost effective to consolidate tier 4 and/or tier 5 laboratories into larger centralised laboratories (‘super-laboratory’/tier 6 level) that could process in excess of 600 samples per day, depending on efficiency of local transport systems.1 this would, however, create coverage gaps that would need to be addressed with poc in remote ‘hard to reach’ areas.1 an analysis of test volumes identified that the majority of cd4 samples were received from primary healthcare clinics (67%), with a further 8% from larger community healthcare centres; this tells us that hospitals only account for 25% of test volumes.1 this confirms the decentralisation of cd4 test requests. additionally, 14 health districts in south africa do not have a local in-district cd4 services.1 this requires cd4 samples to be referred to distant testing laboratories, which affects both tat and specimen integrity. as a result, extended tats were observed for several of these districts, for example, vhembe and thabo mofutsanyane health districts.1 by using a relative euclidian radius of 100 km around each cd4 laboratory (‘service precincts’), both over-and under-subscribed areas were identified.1 the under-subscribed areas were predominantly in health districts in the northern cape and eastern cape provinces,1 whereas significant over-subscription of cd4 testing was noted in the kwazulu-natal province, specifically in the ethekwini health district.1 the world health organization guidelines for a successful health laboratory network state that it is vital to provide equitable access to quality laboratory services, with specific attention focused on rural, semi-urban and underserved areas.5 the flow diagram (figure 1) describes this process, detailing all the steps required to deliver a cd4 result to a health facility. cd4 samples are collected by healthcare workers and delivered to the local laboratory by either courier and/or messenger. where the local laboratory offers cd4 testing, results are delivered following sample analysis. if cd4 testing is not offered at the local laboratory the procedure is as follows: (1) sample registered as a referral on the laboratory information system; (2) sample packaged for delivery to the testing laboratory; (3) sample transported by inter-laboratory courier service; (4) samples registered at the cd4 laboratory’s laboratory information system; (5) sample tested; (6) result printed at the local laboratory; and (vii) result delivered to the health facility. the nhls strategic plan (2010–2015) aimed to deliver quality, timely, accessible and customer-focused services.6 figure 1: end to end process for cd4 samples from sample collection to using the result during the patient consultation. the current cd4 service has predominantly implemented tiers 4 and 5 of the itsdm model. tier 4 laboratories use the epics xl mcltm flow cytometers with the prepplus iitm workstation from beckman coulter. tier 5 laboratories use higher throughput platforms with greater automation that could process up to 800 samples per day, depending on the number and configuration of the platforms implemented, for example, the 2 + 1 configuration that consists of two cellmektm cell preparation systems and one mpltm flow cytometer (also from beckman coulter). the nhls is currently replacing the epics xl mcltm flow cytometers with the aquois™ load and go system (beckman coulter). the different flow cytometer platforms offer daily throughput of up to 386 samples per day and per system, suitable for busy laboratories (cellmektm and mpltm). in contrast, the epics xl mcltm offers a daily capacity of up to 150 samples per day per system, which is appropriate for smaller laboratories at district hospitals. poc testing was based on the alere pimatm platform (alere technologies gmbh, jena, germany). a pilot tier 3 laboratory was implemented at the rural de aar laboratory7 which demonstrated improved access to cd4 testing for the district. the nhls has a large pool of laboratories currently not performing cd4 testing that could potentially be utilised to rapidly expand services while maintaining quality in a cost-effective approach.8 the current challenge the nhls faces is over-capacitation in urban areas and under-capacitation in selected rural areas. therefore, the objective of this study was to identify and address coverage gaps, identify over-subscribed areas and assess the impact the relational algebraic capacitated location (racl) outcomes would have on placement of laboratory sites, test volumes, platform choice and space requirements. methods spatial representation of racl algorithm output for each scenario the racl algorithm was developed to allocate cd4 laboratories and poc sites to ensure coverage for all health facilities to cd4 testing within a pre-determined travel time (t) using a set coverage approach.9,10 health facilities were clustered within a radius of 5 km to speed up the algorithm; for example, multiple health facilities in the town of colesberg were clustered due to their proximity. latitude and longitude data for all health facilities and nhls laboratories were used to determine euclidean distances between health facility clusters and nhls laboratories.9,10 due to financial constraints, google® maps directions api web service was used to obtain drive time estimations for a representative sample of euclidean distances.9,10 this dataset was used to generate a linear regression analysis between drive time (hours) and euclidean distance (km).9,10 based on this analysis, the euclidean distance dataset for each health facility cluster was converted to travel times.9 the algorithm allocates the lowest number of cd4 laboratories and/or poc sites to ensure coverage within the defined t.9,10 health facility clusters outside t are then allocated as poc sites.9,10 the logic applied for the racl algorithm is depicted in figure 2. figure 2: flow chart of the steps followed by the racl algorithm. for the purposes of this study, data included 3200 health facilities tested at 62 cd4 testing sites (part of the 266 nhls laboratories) with a daily capacity of 3600 tests. provincial and/or nhls boundaries were not considered for modelling. the racl model was repeated for the following travel times:9,10 scenario a: t = 4 hours scenario b: t = 3 hours scenario c: t = 2 hours. the algorithm outcomes for the scenarios above were visualised using google® fusion tables as follows:9,10 red point marker icon (letter a): existing cd4 laboratories. red point marker icon (letter e): introduction of cd4 testing at an existing nhls laboratory (that does not currently offer cd4 testing). small blue circle: poc sites. lines represent the euclidian distance (km) from the health facility cluster to the cd4 laboratory as follows: (1) green, where t = 2 hours; (2) purple, where t = 3 hours; and (3) orange, where t = 4 hours. the algorithm outcomes for each scenario included:9,10 number and placement of cd4 laboratories and poc sites. euclidian distances from the health facility cluster to the allocated cd4 laboratory. daily test volumes for cd4 laboratories. distribution of laboratory test volumes for each scenario, daily laboratory testing volumes were reported using a line chart. the dataset was sorted from the highest to lowest test volumes, and current daily volumes for 2015 were also reported. analysis of platform and space requirements for the 10 busiest laboratories for each scenario the top 10 laboratories with the highest daily volumes for each scenario were used to assess the appropriate: platform to be deployed, i.e. xl mcl or cellmek/mpl number of platforms required to provide sufficient instrument capacity bench space required to accommodate platform(s) based on the supplier width recommendations: for example, the cellmek requires a bench depth of 1.2 m, which includes the computer, monitor, bottles, cables and tubing. additionally, the percentage increase in daily testing volumes from current annual volumes (2015) was assessed. results spatial representation of racl algorithm output for each scenario for scenario a, (t = 4), testing would be offered using a fully-centralised approach with only 15 cd4 laboratories (figure 3 and table 1). additionally, cd4 testing would be introduced at the springbok, upington and vredenburg laboratories to meet a t of four hours in the northern cape, western cape and north west provinces.9 figure 3: spatial reporting of (a) scenario a, (b) scenario b, and (c) scenario c using google® fusion tables (copyright of selective analytics). table 1: number of cd4 laboratories and point-of-care sites for scenarios a, b and c. in scenario b (t = 3), the majority of the testing would be performed by tier 4 (n = 24) and tier 5 (n = 6) laboratories with poc reserved for hard-to-reach areas.1 additionally, 11 tier 3 laboratories were proposed. for this scenario, testing would be retained at 30 of the existing cd4 laboratories. additionally, cd4 testing would be established at 11 laboratories including springbok, beaufort west, vredendal, de aar, upington, george, graaf-reinet, tshwaragano and thabazimbi laboratories to improve accessibility.9 the decentralised scenario c (t = 2) would increase cd4 laboratories to 61, with 20 poc sites. distribution of cd4 laboratories for scenario a (t = 4), only 15 cd4 testing laboratories are required to ensure equitable access with no poc sites required.9 this would imply the closure of 44 cd4 testing laboratories (74.5% reduction in current platform). in scenario b (t = 3), 41 cd4 laboratories were allocated with only two poc sites (cd4 testing will cease at 16 laboratories to be closed). with a halved t value in scenario c (2 hours), 61 cd4 laboratories and 20 poc sites were required to ensure equitable access.9 this would require cd4 testing to be stopped at two laboratories and the establishment of 20 new poc sites. distribution of laboratory daily volumes for scenario a, there would be a significant increase in daily test volumes, with one laboratory required to increase capacity four-fold (figure 4).9 the majority of the cd4 testing would be performed using the cellmek and mpl platform (nine laboratories would perform between 346 and 3541 tests per day). for high-throughput laboratories, two cellmek/mpl systems would be able to produce 1080 cd4 results in a 10-hour work day. even for scenario a, this would require at least four cellmek/mpl systems to cope with testing demand (capacity of 4320 samples per day). figure 4: daily laboratory test volumes for scenarios a, b and c compared to current 2015 volumes. daily laboratory test volumes peak at 1142 in scenarios b and 992 in scenario c. these would necessitate two cellmek/mpl systems. an additional requirement for scenario c is lower-throughput cd4 platforms for laboratories performing between 11 and 50 tests per day (n = 8 tier 3 laboratories). analysis of platform and space impact for the 10 busiest laboratories for each scenario in scenario a, four laboratories would be required to increase throughput between 87% and 305% (table 2). at the busiest laboratory, this equates to allocating up to 27 metres of bench space to cope with a daily volume of 3541. to ensure adequate coverage, centralisation is paired with low-volume decentralised laboratories, leaving five laboratories to perform less than 40 samples per day (tier 2). table 2: daily laboratory volumes for the 10 busiest laboratories for scenarios a, b and c with the platform, space requirements and percentage increase in volumes compared to 2015 volumes. for scenario b, the top 10 laboratories are required to make minor changes to their throughput ranging between 5% and 38%. additionally, the required bench space varies from 5.27 to 13.56 metres. in scenario c, similar increases in throughput are reported at busier laboratories. however, decentralised poc testing reduces volume daily volumes for laboratories 9 and 10. discussion the racl algorithm outcomes reported an inverse relationship between t and the numbers of cd4 laboratories/poc sites required to provide coverage. this indicates that the number of cd4 laboratories required to ensure coverage would increase with a decreasing t. as t is increased, it would be possible to ensure coverage with fewer laboratories. the key would be to establish a t that could deliver a clinically-acceptable tat in line with the standard of care. the current south african guidelines require a cd4 count within seven days to fast-track patients ≤ 200 cells/µl.11 the racl algorithm was repeated for each of three travel times (4, 3 and 2 hours), based on a required tat of 24 hours. each scenario provides a solution to address coverage based on a clinically-accepted tat. scenario a represents a highly-centralised model that requires efficient logistics to achieve a t of four hours. the benefits of centralised testing include improved cost efficiency as well as improved testing quality with fewer laboratories.8 challenges include staff recruitment, space availability and infrastructural costs to prepare laboratories for cd4 testing. additionally, pre-analytical capacity would have to be upgraded to handle increased sample volumes. a key challenge would be higher logistics costs due to increased inter-laboratory referrals that would also affect specimen integrity. scenario b offers a mix of predominantly laboratory-based cd4 testing with limited poc testing in ‘hard-to-reach’ areas. several new cd4 testing sites at existing nhls laboratories identified have already been implemented by the nhls. many of these rural laboratories require limited testing capacity and were therefore identified as tier 3 laboratories by the itsdm. a good example is the de aar laboratory that was implemented in 2012. this laboratory was able to cope with testing demand and reduce tat substantially from 20.5 to 8.2 hours.7 additionally, with only two staff members, they performed satisfactorily on 10 external quality assessment trials (n = 20 samples).7 this demonstrates that a small, rural tier 3 laboratory can integrate cd4 testing for a small incremental cost.8 additional analysis was performed for the national health insurance pilot districts (n = 11) to assess service gaps.12 this study identified that four new tier 3 cd4 testing laboratories would be required to improve access to testing.12 these testing sites coincide with the new cd4 testing sites proposed in scenario b. in scenario c, testing is extended to 61 laboratories with 20 poc sites. many of the poc sites proposed would be within an acceptable t if a tier 2 site (poc hub) were established in calvinia. furthermore, a tier 2 site would serve multiple health facilities and offer access to multiple tests (cd4, xpert mtb/riftm, creatinine, alanine transaminase and haemoglobin/full blood count).1 a tier 2 site would be more cost-effective than multiple tier 1 sites.8 across the three scenarios, laboratory-based cd4 testing varies between 15 and 61 sites. for scenario a and b, between 18 to 44 existing cd4 laboratories would have to be consolidated, resulting in significant changes to sample logistics, equipment and staff allocation. reducing a platform of 62 laboratories to 15 would require a massive scale-up to cope with increased test volumes. this would include a scale-up in the pre-analytical section as well and could incur both renovation and verification costs. consolidation would require staff to relocate to urban areas; this would need to be handled using a change management approach with additional costs. additionally, samples would have to travel further, increasing per kilometre logistics costs. in summary, the racl algorithm identified the optimal placement of nhls laboratories for a range of t to enable the delivery of a cd4 service that balances the need for equitable access and cost-effectiveness. however, evidence from the pilot tier 3 laboratory demonstrates that additional tier 3 laboratories or tier 2 poc hubs could increase coverage in a more cost-effective manner than poc sites. limitations travel times reported are based on a representative sample of google® maps directions drive times. the drive time for all health facility clusters could not be obtained due to funding availability. therefore, travel times could be underestimated in areas with poor road infrastructure resulting in additional coverage gaps requiring additional testing sites and/or poc. the racl algorithm would have to be rerun should any of the assumptions change, namely, tat, number of health facilities and test volumes. it would be difficult to provide a statistical analysis of the data presented by the racl algorithm, such as a list of proposed laboratories/poc sites. additionally, an assessment of local conditions must accompany the findings of the racl algorithm to ensure optimal coverage. recommendations based on the results of this study, it is recommended that the racl algorithm be implemented on the corporate data warehouse to enable real-time analysis of coverage gaps. the limitations of using google’s drive times should be addressed. additionally, the algorithm should be extended to include other priority tests, such as xpert mtb/rif®, hiv viral load, hiv dna pcr and pap smear. conclusion the racl algorithm provides laboratory management with an objective methodology to identify and address coverage gaps. however, the algorithm outcomes must be assessed in conjunction with local knowledge to address coverage gaps using a sustainable approach. acknowledgements the authors thank wendy stevens, sergio carmona, jon smith, sherry drury and sithembile mojalefa. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support none. authors’ contributions h.s. developed the mathematical model, conceived the racl algorithm and developed the computer systems to display the model outcome spatially. d.k.g., n.c. and l.m.c. assisted with input parameters for the model and evaluation of model output. d.k.g., n.c. and l.m.c. conceived the analysis of the impact of implementing the algorithm outcomes on test volumes, platform distribution and space requirements. all authors contributed to the manuscript development. references glencross dk, coetzee lm, cassim n. an integrated tiered service delivery model (itsdm) based on local cd4 testing demands can improve turn-around times and save costs whilst ensuring accessible and scalable cd4 services across a national programme. plos one 9(12): e114727. http://doi.org/10.1371/journal.pone.0114727 coetzee lm, cassim n, glencross dk. a cost analyses of mobile laboratory cd4 testing in a national health insurance (nhi) pilot site. poster presented at the first international conference of the african society for laboratory medicine, cape town, south africa; 2012. glencross dk, janossy g, coetzee lm, et al. large-scale affordable panleucogated cd4+ testing with proactive internal and external quality assessment: in support of the south african national comprehensive care, treatment and management programme for hiv and aids. cytometry b clin cytom. 2008;74(suppl 1):s40–s51. glencross dk, scott le, jani iv, et al. cd45-assisted panleucogating for accurate, cost-effective dual-platform cd4+ t-cell enumeration. cytometry. 2002;50(2):69–77. world health organization. guide for national public health laboratory networking to strengthen integrated disease surveillance and response (idsr) [document on the internet]. c2008 [cited 2016 july]. available from: http://www.cdc.gov/globalhealth/healthprotection/idsr/pdf/htl-afro-cdc-lab-guidelines-for-nln.pdf national health laboratory service. nhls strategic plan 2010–2015 [document on the internet]. c2010 [cited 2016 july]. available from: http://www.nhls.ac.za/assets/files/nhls_strategic_plan_2010-15%20for_doh.pdf coetzee l, cassim n, glencross d. implementation of a new ‘community’ laboratory cd4 service in a rural health district in south africa extends laboratory services and substantially improves local reporting turnaround time. s afr med j. 2015;106(1):82–87. http://doi.org/10.7196/samj.2016.v106i1.10081 cassim n, coetzee lm, schnippel k, et al. estimating implementation and operational costs of an integrated tiered cd4 service including laboratory and point of care testing in a remote health district in south africa. plos one. 2014;9(12):e115420. http://doi.org/10.1371/journal.pone.0115420 smith j, smith h, cassim n, et al. geographic location and capacity optimisation modelling to plan effective and efficient diagnostics service placement. paper presented at the first international conference of the african society for laboratory medicine, cape town, south africa; 2012. smith hk, smith jp, glencross dk, et al. siting of hiv/aids diagnostic equipment in south africa: a case study in locational analysis. int t oper res. 2017;1–35. national department of health. national consolidated guidelines for the prevention of mother-to-child transmission of hiv (pmtct) and the management of hiv in children, adolescents and adults. pretoria, south africa; 2015. coetzee lm, cassim n, glencross dk. using the tiered service approach to assess gaps in laboratory service delivery in the eleven pilot districts of the national health insurance (nhi). poster presented at the first international conference of the african society for laboratory medicine, cape town, south africa; 2012. abstract introduction methods results discussion acknowledgements references about the author(s) nireshni mitchev department of medical microbiology, school of laboratory medicine and medical sciences, university of kwazulu-natal, durban, south africa ravesh singh department of medical microbiology, school of laboratory medicine and medical sciences, university of kwazulu-natal, durban, south africadepartment of medical microbiology, national health laboratory service, durban, south africa nigel garrett centre for the aids programme of research in south africa, durban, south africa discipline of public health medicine, school of nursing and public health, university of kwazulu-natal, durban, south africa veron ramsuran kwazulu-natal research innovation and sequencing platform, school of laboratory medicine and medical sciences, university of kwazulu-natal, durban, south africa abraham j. niehaus department of medical microbiology, school of laboratory medicine and medical sciences, university of kwazulu-natal, durban, south africadepartment of medical microbiology, national health laboratory service, durban, south africa koleka p. mlisana department of medical microbiology, school of laboratory medicine and medical sciences, university of kwazulu-natal, durban, south africacentre for the aids programme of research in south africa, durban, south africadepartment of academic affairs, research and quality assurance, national health laboratory service, durban, south africa citation mitchev n, singh r, garrett n, ramsuran v, niehaus aj, mlisana kp. performance of taqman probes for the detection of sexually transmitted infections in south african women. afr j lab med. 2021;10(1), a1124. https://doi.org/10.4102/ajlm.v10i1.1124 brief report performance of taqman probes for the detection of sexually transmitted infections in south african women nireshni mitchev, ravesh singh, nigel garrett, veron ramsuran, abraham j. niehaus, koleka p. mlisana received: 07 nov. 2019; accepted: 08 jan. 2021; published: 31 mar. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract neisseria gonorrhoeae, chlamydia trachomatis, trichomonas vaginalis and mycoplasma genitalium are the four main aetiologies of sexually transmitted infections responsible for vaginal discharge syndrome (vds). commercially available multiplex polymerase chain reaction (pcr) assays are expensive and generally not customisable. we evaluated a highly customisable singleplex pcr approach by testing it in parallel with the anyplex™ ii sti-7 detection assay in a cohort of south african women that presented with vds between may 2016 and january 2017. our multiple singleplex pcr strategy proved to be a simple, accurate, rapid, affordable and scalable option for diagnosing vds. keywords: sexually transmitted infections; vaginal discharge syndrome; molecular diagnostics; validation; taqman. introduction the world health organization estimates that approximately one million curable sexually transmitted infections (stis) occur globally each day.1 neisseria gonorrhoeae, chlamydia trachomatis and trichomonas vaginalis are the three most common pathogens causing stis worldwide, with co-infection also common.1,2 globally, as of 2016, there were approximately 86 million cases of gonorrhoea, 127 million cases of chlamydia and 156 million cases of trichomoniasis among 15–49 year-olds, with estimated prevalence rates of 0.9%, 3.8% and 5.3% in women specifically.1,3 such estimates are important for the effective prevention and control of stis but are generally lacking for most lowand middle-income countries.4 torrone et al. recently published a meta-analysis of data from african countries that highlights this point.4 prevalence figures varied widely between studies and ranged from 1.4% to 15.2% for n. gonorrhoeae, 1.2% to 20.6% for c. trachomatis and 6.6% to 29.7% for t. vaginalis.4 the prevalence of most stis is generally greater in certain high-risk populations such as those with a high prevalence of hiv co-infection and women aged 15–24 years.4 sexually transmitted infections and their complications are one of the top five reasons why females in lowand middle-income countries attend healthcare facilities.1,2 although the majority of stis are asymptomatic, asymptomatic stis can increase the probability of transmission of hiv and other stis, as well as have adverse effects on reproduction potential and maternal and newborn health.1,2,5,6 syndromic management of stis is widely practised and was introduced in south africa in 1995.7,8 unfortunately, due to its inability to effectively identify and treat a large number of individuals with asymptomatic infections, syndromic management has failed to decrease the prevalence of gonorrhoea and chlamydia in south africa.7,8 diagnosing the aetiology of most stis using culture methods is notoriously difficult and can take several days to complete. multiplex polymerase chain reaction (pcr) assays are currently the preferred diagnostic method used for identifying and subsequently managing both symptomatic and asymptomatic stis in many well-resourced countries.9 since pcr assays are not dependent on organism viability, they are often up to 20% – 30% more sensitive than conventional phenotypic methods and they can simultaneously detect multiple pathogens and also serve as point-of-care tests.9,10,11,12,13 polymerase chain reaction also performs well on noninvasively obtained specimens like urine and self-collected vaginal swabs.10 our study aimed to evaluate the suitability of a highly customisable singleplex real-time pcr approach using commercially available taqman® probes (thermo fisher scientific, waltham, massachusetts, united states) for the identification of n. gonorrhoeae, c. trachomatis, t. vaginalis and myocoplasma genitalium in vaginal samples from our local population. the anyplex™ ii sti-7 detection assay (seegene, seoul, south korea) was used as the comparator method. this widely used multiplex real-time pcr assay displays excellent sensitivity and specificity characteristics compared with other diagnostic tools.10 methods ethical considerations women attending the prince cyril zulu communicable disease centre (durban, south africa) for sti care were approached and, if willing to participate, provided written informed consent for the study. study data were collected, anonymised and managed using password-protected redcap electronic data capture tools (vanderbilt university, nashville, tennessee, united states), and stored on a secure server. this study was approved by the biomedical research ethics committee of the university of kwazulu-natal (study approval number: be534/16). samples as part of the centre for the aids programme of research in south africa 083 cohort study between may 2016 and january 2017, which was previously described in detail, women aged 18–40 presenting for sti care at a clinic in durban were assessed for enrolment and participation.7,14 hiv-positive women, pregnant women and those engaging in sex work were excluded due to predetermined eligibility criteria.15 participants consented to vaginal swab specimen collection for molecular testing. eswab™ collection and transport kits (copan diagnostics, brescia, italy) were used as per the manufacturer’s instructions. dna extraction and polymerase chain reaction collection swabs were vortexed for 30 s while inside their transport tubes, whereafter 500 µl of the suspension was added to a 1.5 ml eppendorf tube. this was followed by centrifugation and resuspension of the sediment in 200 µl distilled water. after heating and sonication steps, 5 µl of the resultant sample was added to 15 µl pre-aliquoted master mix. for the taqman® probe assays, master mixes were prepared separately for each single target reaction in a 96-well plate. the anyplex™ ii sti-7 detection assay was used according to the manufacturer’s specifications and was performed using a cfx96 real-time pcr system (biorad, hercules, california, united states).10 taqman® probes were used in a singleplex format on the abi® 7500 real-time pcr instrument from applied biosystems (thermo fisher scientific, waltham, massachusetts, united states). data management and analysis anyplex™ ii sti-7 detection assay results were analysed and interpreted with seegene viewer software (seegene, seoul, south korea). contingency 2 × 2 tables were used to determine the sensitivity, specificity, positive predictive value and negative predictive value for each of the taqman® probes with anyplex™ ii sti-7 detection assay as the gold standard.10,16,17 results a total of 267 women were screened for stis at an urban clinic in durban, south africa. vaginal discharge (n = 106; 39.7%) was the most common symptom reported.15 vaginal swabs were available for molecular investigation from 250 (93.6%) women. two molecular assays were used in parallel to detect the presence of four sexually transmitted microorganisms implicated in vaginal discharge syndrome. the yield obtained from 250 samples by each of the two methods ranged between 3.6% and 13.6% (table 1). at least one of n. gonorrhoeae, c. trachomatis, t. vaginalis or m. genitalium was identified in 22.8% (57/250) of the study population. c. trachomatis was the most common organism found in co-infections and was present in 42% (5/12) of the n. gonorrhoeae, 25% (3/12) of the m. genitalium and 11% (1/9) of the t. vaginalis infections. table 1: yield obtained with anyplex™ ii sti-7 detection assay and taqman® probes, from 250 vaginal swab specimens collected from patients attending prince cyril zulu communicable disease centre (durban, south africa) between may 2016 and january 2017. taqman® probe sensitivity ranged from 91.67% to 100%, and specificity ranged from 98.74% to 100.00% (table 2). negative predictive values ranged from 99.08% to 100.00% and positive predictive values ranged from 90.00% to 100.00%, except for m. genitalium (78.57%). table 2: performance characteristics of taqman® probes for 250 vaginal swab specimens collected from patients attending prince cyril zulu communicable disease centre (durban, south africa) between may 2016 and january 2017. final results were available after 130 min and 180 min with the taqman® probes and anyplex™ ii sti-7 detection assay, respectively. this includes 45 min for dna extraction and 15 min for results analysis. discussion the prevalence of any of the main curable stis, including syphilis and those caused by n. gonorrhoeae, c. trachomatis and t. vaginalis, in women in kwazulu-natal, south africa was previously reported to be as high as 13%.18 in our study, we observed the overall prevalence of stis to be 22.8%. our prevalence data is not a true representation of the general population, because we recruited participants from a sexually active cohort of women that presented with symptoms to an sti clinic. the xpert® ct/ng assay (cepheid, sunnyvale, california, united states) was previously used to investigate this cohort of samples.7 for the present study, we employed the anyplex™ ii sti-7 detection assay as the comparator, because the xpert® ct/ng assay can identify c. trachomatis and n. gonorrhoeae, but not t. vaginalis and m. genitalium. sensitivity, specificity, positive predictive value and negative predictive value characteristics of the taqman® probes for the four investigated organisms compared well to the chosen reference method. the only exception was the low positive predictive value observed for m. genitalium. this deviation may be due to the small sample size and requires further investigation. except for certain antibiotic-resistant organisms, the stis detected in our study can usually be treated with a course of antibiotics; however, if they remain undiagnosed and subsequently untreated, a range of serious health issues may follow. complications include elevated hiv transmission rates, infertility as well as life-threatening ectopic pregnancy and stillbirths.19 a rapid, accurate and affordable sti diagnostic tool is therefore essential to identify and treat affected individuals.13 molecular testing methods are advantageous over phenotypic testing methods due to the rapid availability of results. polymerase chain reaction methodologies can also detect various predefined targets at the same time, including drug-resistance determinants. singleplex pcr methods offer some important advantages over multiplex methods, principally in terms of ease of optimisation, customisability and target quantification. new gene targets of interest can be added in a flexible way without the requirement of a full revalidation process of the existing targets. our multiple singleplex pcr system can easily be adapted to diagnose various other clinical syndromes caused by bacterial, viral, fungal and parasitic microorganisms, including meningitis, blood stream infections, respiratory infections, diarrhoea, and urinary tract infections. singleplex pcr systems allow target quantification, which has potential diagnostic, prognostic and clinical monitoring functions. turnaround times with our multiple singleplex pcr were 50 min shorter than with the multiplex method. the clinical impact of this time difference, when non-immediately life-threatening infections are concerned, is probably insignificant. multiplex pcr methodologies are generally significantly more cost-effective than singleplex pcrs. however, this does not hold true when small numbers of molecular targets are considered.20 costing of the four targets used in our testing system indicates a considerable cost saving of $15.27 (united states dollars [usd]) per test, over the comparator method that is priced at $24.67 usd per test. affordability can be increased even further by using a 384-well reaction plate, making it an attractive option for high-throughput environments. with our system, we can easily adopt a workflow that is suitable for both research and clinical laboratories. limitations limitations of this study include the relatively small sample size and the low positivity rate obtained by both methodologies. multiple other microorganisms and dual infections may also have been responsible for vaginal discharge syndrome in some women, but were not considered in this investigation. conclusion the performance of four taqman® probes (thermo scientific) was assessed by comparison with the anyplex ii sti-7 detection assay. our multiple singleplex real-time taqman® pcr approach proved to be highly sensitive and specific for the detection of c. trachomatis, n. gonorrhoeae, t. vaginalis and m. genitalium. this approach offers an accurate, cost-effective and scalable option for identifying these pathogens in our patient population. acknowledgements the authors would like to acknowledge the aids programme of research in south africa 083 sti study participants and staff at the national health laboratory services (inkosi albert luthuli central hospital, durban, south africa) and centre for the aids programme of research in south africa. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions n.m., r.s. and k.p.m. conceptualised the study. k.p.m. was responsible for the overall supervision of the project and the acquisition of funding. n.m. and n.g. were responsible for project administration. n.m., r.s. and v.r. decided on the methodology and performed the laboratory investigations. n.m. curated all the data and the data analysis was done together with a.j.n. all authors participated in the writing of the manuscript. sources of support this project was funded through a grant by the department of science and technology and the national research foundation centre of excellence in hiv prevention as well as a grant from the united states – south african program for collaborative biomedical research, through the south african medical research council and the united states national institutes of health (grant number: ai116759). data availability the authors confirm that the data supporting the findings of this study are available within the article or its supplementary materials. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references world health organization. report on global sexually transmitted infection surveillance 2018 [homepage on the internet]. 2019 [cited 2019 jul 13]. available from: https://www.who.int/reproductivehealth/publications/stis-surveillance-2018/en/ tayoun ana, burchard pr, caliendo am, scherer a, tsongalis gj. a multiplex pcr assay for the simultaneous detection of chlamydia trachomatis, neisseria gonorrhoeae, and trichomonas vaginalis. exp mol pathol. 2015;98(2):214–218. https://doi.org/10.1016/j.yexmp.2015.01.011 rowley j, van der hoorn s, korenromp e, et al. chlamydia, gonorrhoea, trichomoniasis and syphilis: global prevalence and incidence estimates, 2016. bull world health organ. 2019;97:548–562. torrone ea, et al. prevalence of sexually transmitted infections and bacterial vaginosis among women in sub-saharan africa: an individual participant data meta-analysis of 18 hiv prevention studies. plos med. 2018;15(6):e1002511. https://doi.org/10.1371/journal.pmed.1002511 kaida a, dietrich jj, laher f, et al. a high burden of asymptomatic genital tract infections undermines the syndromic management approach among adolescents and young adults in south africa: implications for hiv prevention efforts. bmc infect dis. 2018;18:499. https://doi.org/10.1186/s12879-018-3380-6 workowski ka, bolan ga, centers for disease control and prevention. sexually transmitted diseases treatment guidelines, 2015. mmwr recomm rep. 2015;64(rr-03):1–137. garrett nj, osman f, maharaj b, et al. beyond syndromic management: opportunities for diagnosis-based treatment of sexually transmitted infections in lowand middle-income countries. plos one. 2018;13(4):e0196209. https://doi.org/10.1371/journal.pone.0196209 garrett nj, mcgrath n, mindel a. advancing sti care in low/middle-income countries: has sti syndromic management reached its use-by date? sex transm infect. 2016;93(1):4–5. https://doi.org/10.1136/sextrans-2016-052581 lee sj, park dc, lee ds, choe hs, cho yh. evaluation of seeplex std6 ace detection kit for the diagnosis of six bacterial sexually transmitted infections. j infect chemother. 2012;18(4):494–500. https://doi.org/10.1007/s10156-011-0362-7 choe hs, lee ds, lee sj, et al. performance of anyplex ii multiplex real-time pcr for the diagnosis of seven sexually transmitted infections: comparison with currently available methods. int j infect dis. 2013;17(12):e1134–e1140. https://doi.org/10.1016/j.ijid.2013.07.011 trembizki e, costa amg, tabrizi sn, whiley dm, twin j. opportunities and pitfalls of molecular testing for detecting sexually transmitted pathogens. pathology. 2015;47(3):219–226. https://doi.org/10.1097/pat.0000000000000239 papp jr, schachter j, gaydos ca, van der pol b. recommendations for the laboratory-based detection of chlamydia trachomatis and neisseria gonorrhoeae – 2014. mmwr recomm rep. 2014;63(rr-02):1–19. unemo m. current and future antimicrobial treatment of gonorrhoea – the rapidly evolving neisseria gonorrhoeae continues to challenge. bmc infect dis. 2015;15:364. https://doi.org/10.1186/s12879-015-1029-2 garrett n, mitchev n, osman f, et al. diagnostic accuracy of the xpert ct/ng and osom trichomonas rapid assays for point-of-care sti testing among young women in south africa: a cross-sectional study. bmj open. 2019;9(2):e026888. https://doi.org/10.1136/bmjopen-2018-026888 garrett n, maharaj b, osman f, et al. p4.115 high uptake of effective expedited partner therapy among young women with stis and their partners in south africa. sex transm infect. 2017;93(suppl 2):a233. https://doi.org/10.1136/sextrans-2017-053264.610 naaktgeboren ca, bertens lcm, van smeden m, et al. value of composite reference standards in diagnostic research. bmj. 2013;347:f5605. https://doi.org/10.1136/bmj.f5605 alonzo ta, pepe ms. using a combination of reference tests to assess the accuracy of a new diagnostic test. stat med. 1999;18(22):2987–3003. https://doi.org/10.1002/(sici)1097-0258(19991130)18:22%3c2987::aid-sim205%3e3.0.co;2-b naidoo s, wand h, abbai ns, ramjee g. high prevalence and incidence of sexually transmitted infections among women living in kwazulu-natal, south africa. aids res ther. 2014;11:1. https://doi.org/10.1186/1742-6405-11-31 centers for disease control and prevention. sexually transmitted disease surveillance report [homepage on the internet]. 2016 [cited 2019 may 28]. available from: https://www.cdc.gov/std/stats16/cdc_2016_stds_report-for508websep21_2017_1644.pdf deshpande a, white ps. multiplexed nucleic acid-based assays for molecular diagnostics of human disease. expert rev mol diagn. 2012;12(6):645–659. https://doi.org/10.1586/erm.12.60 article information authors: ernest p. makokha1 samuel mwalili1 frank l. basiye1 clement zeh1 wilfred i. emonyi2 raphael langat3 elizabeth t. luman4 jane mwangi1 affiliations: 1division of global hiv/aids, us centers for disease control and prevention, kenya2academic model providing access to healthcare (ampath), moi university school of medicine, kenya 3henry jackson foundation, kenya 4international laboratory branch, division of global hiv/aids, us centers for disease control and prevention, united states correspondence to: ernest makokha postal address: po box 606-00621, nairobi, kenya dates: received: 06 aug. 2014 accepted: 15 sept. 2014 published: 03 nov. 2014 how to cite this article: makokha ep, mwalili s, basiye fl, et al. using standard and institutional mentorship models to implement slmta in kenya. afr j lab med. 2014;3(2), art. #220, 8 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.220 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. using standard and institutional mentorship models to implement slmta in kenya in this original research... open access • abstract • introduction • research methods and design    • slmta laboratories    • mentorship models    • ethical considerations    • baseline, mid-term and exit audits    • data analysis • results • discussion    • conclusion • acknowledgements    • competing interests    • authors’ contributions    • cdc disclaimer • references abstract top ↑ background: kenya is home to several high-performing internationally-accredited research laboratories, whilst most public sector laboratories have historically lacked functioning quality management systems. in 2010, kenya enrolled an initial eight regional and four national laboratories into the strengthening laboratory management toward accreditation (slmta) programme. to address the challenge of a lack of mentors for the regional laboratories, three were paired, or ‘twinned’, with nearby accredited research laboratories to provide institutional mentorship, whilst the other five received standard mentorship.objectives: this study examines results from the eight regional laboratories in the initial slmta group, with a focus on mentorship models. methods: three slmta workshops were interspersed with three-month periods of improvement project implementation and mentorship. progress was evaluated at baseline, mid-term, and exit using the stepwise laboratory quality improvement process towards accreditation (slipta) audit checklist and scores were converted into a zeroto five-star scale. results: at baseline, the mean score for the eight laboratories was 32%; all laboratories were below the one-star level. at mid-term, all laboratories had measured improvements. however, the three twinned laboratories had increased an average of 32 percentage points and reached one to three stars; whilst the five non-twinned laboratories increased an average of 10 percentage points and remained at zero stars. at exit, twinned laboratories had increased an average 12 additional percentage points (44 total), reaching two to four stars; non-twinned laboratories increased an average of 28 additional percentage points (38 total), reaching one to three stars. conclusion: the partnership used by the twinning model holds promise for future collaborations between ministries of health and state-of-the-art research laboratories in their regions for laboratory quality improvement. where they exist, such laboratories may be valuable resources to be used judiciously so as to accelerate sustainable quality improvement initiated through slmta. introduction top ↑ medical research laboratories are scattered globally in order to support drug testing and development, epidemiologic and clinical studies, and emergency needs. these laboratories are typically well-funded state-of-the-art facilities with expert staff and well-maintained equipment. in kenya, there are five high-performing research laboratories accredited to international standards. funded by various international organisations, these laboratories are located in different regions of the country where they conduct donor-driven research. by contrast, as of the end of 2012, none of kenya’s 300 public sector clinical laboratories were accredited to international standards.1 kenya’s picture is representative of most of the developing world where public sector laboratories have suffered many years of neglect and operate without functioning quality management systems.2,3 as a result, a majority of these laboratories are persistent underperformers with regard to disease surveillance and outbreak investigation.4 in some clinical settings, unreliable and inaccurate laboratory services continue to discourage healthcare providers from using laboratory testing to support disease diagnosis.5 this practice may delay the realisation of the united nation’s millennium development goals, as quality laboratory testing is critical for medical diagnostics, care and treatment of diseases.6in the last several years, because of resources provided primarily by the us president’s emergency plan for aids relief (pepfar) and its strategic partners, public sector laboratories in kenya are on the mend. for instance, kenya was amongst the first african countries to develop national laboratory policy guidelines and a five-year national strategic plan in an effort to improve the overall quality of laboratory services to meet the rising demand for hiv testing and treatment monitoring.7 developed with support from pepfar, this strategic plan addressed a wide range of laboratory strengthening needs, including establishment of quality systems and local and international external quality assessment (eqa) programmes.8 these developments built the foundation for laboratory quality system improvements and enabled the country to roll out the strengthening laboratory management toward accreditation (slmta) programme in april 2010, nine months after its launch in kigali, rwanda in 2009.3 since the launch of slmta, observational studies have suggested that the mentorship component, especially when aligned to laboratory accreditation goals and overall plans of the ministry of health (moh), provides substantial impact on laboratory quality improvement.9 slmta mentorship approaches have ranged from standard short-term visits10,11 to newer models of peer or embedded mentoring.11,12 in the standard short-term model, a mentor visits the mentee laboratory and stays in the laboratory for a short stint, often a week or less. during this period, the laboratory quality officer works under the guidance of the mentor to address laboratory quality gaps identified during prior assessment. this standard approach is used by most countries because it requires few staff and is thus low in cost, especially if in-country mentors are used. however, short visits may limit the amount of knowledge and skills transferred from the mentor to the mentee, as well as behavioural changes, both of which are critical requirements for successful transformation of quality systems practices in the laboratory. on the other hand, peer or embedded mentoring involves a mentor being in a laboratory for an extended duration, often weeks or months at a time. this enables the mentor to better understand the practices and personalities of the mentee laboratory and promotes positive changes in processes and behaviours.9 both of these approaches are dependent on continuous availability of laboratory experts to serve as mentors. in much of sub-saharan africa, including kenya, there is a generalised shortage of laboratory workforce;13 thus, maintaining a cohort of mentors to cover the ever-increasing number of laboratories in the region enrolling in slmta is a challenge. this challenge was brought to the fore in kenya where, through pepfar support, the first cohort of 12 high-volume laboratories – four national level and eight regional-level – began slmta implementation in 2010. with only six trained laboratory mentors available to cover the wide geographical spread of laboratories and to make repeated visits to the slmta laboratories, kenya devised a multi-pronged mentorship approach for the 12 laboratories. the four national laboratories in nairobi received comprehensive multi-partner technical support in an effort to achieve international standards of accreditation. for the eight regional laboratories, five were assisted by the six available mentors through standard short-term visitations as per the slmta guidelines. the remaining three regional laboratories received institutional mentorship (‘twinning’) through pairing with internationally-accredited high-performing research laboratories in their regions. in this article, we focus on the eight regional laboratories where standard and institutional mentorship approaches were employed. we describe the institutional mentorship process and the improvements in laboratory quality systems in both twinned and non-twinned laboratories. research methods and design top ↑ the kenya moh purposively selected the eight regional laboratories to ensure geographical and regional balance (figure 1). the selected laboratories are categorised as level v within kenya’s national medical laboratory services policy,7 comprising seven provincial general hospital laboratories and one high-volume district hospital laboratory, all of which serve as regional hubs for eqa networks and back-up testing. the laboratories are functionally divided into seven testing departments: haematology, clinical chemistry, bacteriology/tuberculosis (tb) microscopy, blood transfusion, parasitology, histopathology/cytology and serology/virology/cd4. these departments are equipped with high-throughput equipment to support high-volume testing. each testing bench has a technical head, typically a specialised laboratory technologist with a higher national diploma qualification. figure 1: map of kenya showing the location of regional strengthening laboratory management toward accreditation (slmta) cohort i laboratories. all eight regional laboratories provide 24-hour service, seven days a week for specialised testing and other referral services to their regional catchment area, including cd4, clinical chemistries, hiv viral load and hiv early infant diagnosis. in addition, these laboratories serve as nodal points for split-sample quality assurance testing for district laboratories within their catchment region. all eight laboratories have infrastructure to allow for efficient workflow, such as running water and back-up power source. at the onset of the slmta programme, all laboratories were participating in the following eqa schemes: the united kingdom national eqa scheme (neqas) for cd4 testing; the human quality assessment scheme (huqas) for clinical chemistry and haematology testing; national hiv proficiency testing for rapid hiv testing; malaria microscopy eqa provided by the east african regional eqa scheme (reqas); and tb sputum microscopy provided by the kenya national tb laboratory. in addition, five of the laboratories were participating in the quality assessment and standardization for immunological measures (qasi) cd4 testing scheme, with plans to cascade this service to the peripheral laboratories. all eight laboratories were using a paper-based laboratory information system at the beginning of the programme. slmta laboratories in may 2010, the first four-day slmta workshop was conducted for representatives from each of the laboratories. after this workshop, the laboratories went through a three-month period of improvement project implementation and on-site mentorship. this was followed by a second slmta workshop in september 2010, followed by another round of improvement project implementation and on-site mentorship. the final slmta workshop was held in january 2011. mentorship models two mentorship models were employed for the eight regional laboratories: the standard mentorship approach in five laboratories; and institutional twinning in three. the standard mentorship model is described in detail elsewhere14 and is based on mentors spending periods of time in laboratories to guide and oversee the quality systems improvement process. six in-country, practising laboratory professionals were trained as mentors and completed the slmta training-of-trainers curriculum. five were assigned to one laboratory each, whilst the sixth was tasked with overall coordination of mentorship activities. as an initial assignment, each mentor assisted their target laboratory to develop a three-month work plan, outlining slmta workshop topics, potential improvement projects and a plan for documentation of laboratory performance. following each workshop, mentors visited their assigned laboratory once a month, with each visit lasting five days. at each visit, the mentor used the world health organization regional office for africa (who afro) stepwise laboratory quality improvement process towards accreditation (slipta) checklist in order to measure and review improvements. in addition, the mentor worked with laboratory staff to accomplish a set of improvement projects identified during the slmta workshops.for the institutional mentorship approach, the laboratory branch of the us centers for disease control and prevention (cdc), kenya office, contacted research laboratories to solicit their support for facility-based stepwise improvements of quality systems in targeted moh laboratories. these laboratories are accredited to either international organization for standardization (iso) or the college of american pathologists (cap) standards. based on this agreement, each of the three regional moh laboratories in western kenya was twinned after the first slmta workshop with an accredited medical research laboratory operating in the same region (table 1). this twinning relationship lasted for the duration of slmta programme. through this partnership and under the guidance of the slmta coordinating mentor, research laboratories worked with the slmta laboratories to support the implementation of quality improvements through initial engagement and development of an action plan, documentation review and equipment assessment, training and on-site mentorship, exchange visits and performance review (table 2). table 1: accredited research laboratories and the public laboratories twinned with them through the strengthening laboratory management toward accreditation (slmta) programme in western kenya. table 2: institutional mentorship process. ethical considerations the multi-country slmta programme was approved by the cdc as non-human research. under this approval, cdc staff provided technical support for work that did not involve possession or analysis of identifiable data or interaction with participants’ data. baseline, mid-term and exit audits slmta progress is evaluated using the slipta checklist.15 the slipta framework provides stepwise recognition toward fulfilment of the iso 15189/17025 standard.3 as per slipta guidelines, scores for every laboratory are converted into a zeroto five-star scale, with minimums set at 55% for one star, 65% for two stars, 75% for three stars, 85% for four stars and 95% for five stars.the slipta checklist covers the 12 quality system essentials (qses) as defined by the clinical and laboratory standards institute,16 which can be grouped into three stages of the quality cycle.17 resource management (pre-examination) tasks include organisation and personnel, equipment management, purchasing and inventory, and facilities and safety. process management (examination) tasks include documents and records, client management, information management and process control. improvement management (post-examination) tasks include management reviews, internal audit, corrective action and occurrence/incidence management. in march to april 2010, three weeks after enrolment and before the first slmta workshop, baseline audits were conducted in all eight laboratories, using the slipta checklist. the audits were conducted by a team of in-country auditors led by a visiting auditor from the american society for clinical pathology (ascp). in addition to the baseline audits, a rapid assessment of the services provided by these laboratories was conducted. results from the baseline audit and rapid assessment provided a basis for developing facility-specific work plans. mid-term audits were conducted six months after enrolment, after the second slmta workshop and corresponding improvement projects, also using the slipta checklist; these audits were conducted by an independent institution, the kenya accreditation service (kenas). following the final slmta workshop and improvement project period, about 13 months after the baseline audit, exit audits were conducted by kenas auditors. data analysis data from the baseline, mid-term and exit audits were collected and analysed using sas software version 9.3 (sas institute inc., cary, nc 2012). analysis focused on describing the results of the two mentorship approaches. slipta scores and star ratings were calculated by summing all points across the 12 qses. in addition, we examined results for each qse separately and computed an average score by quality cycle stage, weighing all qses in the cycle equally. exploratory data analysis was conducted by computing descriptive statistics (i.e., percentages, means and medians); graphical representation of the qse scores was created for the twinned and non-twinned laboratories. results top ↑ at baseline, the eight laboratories had a mean score of 32% on the slipta checklist.15 scores ranged from 16% to 44%, with all laboratories below the one-star level (table 3). the mean baseline score for twinned laboratories was 36% and for non-twinned laboratories, 30%. table 3: summary of audit results at baseline, mid-term, and exit of the strengthening laboratory management toward accreditation (slmta) programme, kenya 2010. there were considerable improvements in all but one laboratory at mid-term audit, with twinned laboratories improving substantially more than non-twinned laboratories (mean 32 percentage points vs. 10 percentage points). all three twinned laboratories had reached at least one star whilst all non-twinned laboratories were still at zero stars. at exit audit, twinned laboratories improved an additional average 12 percentage points to 80%, with one laboratory each at 2, 3 and 4 stars. non-twinned laboratories improved an additional average 28 percentage points to 68%, with three laboratories at one star, one at two stars and one at three stars. figure 2 shows qse results for the two sets of laboratories over the project period. for eight of the 12 qses, mean scores increased more amongst the twinned than non-twinned laboratories. the greatest improvements amongst the twinned laboratories were in the areas of occurrence/incidence management (from 0% to 80%), documents and records (from 9% to 79%) and internal audit (from 0% to 67%). greatest improvements for non-twinned laboratories were in the areas of documents and records (from 7% to 71%), facilities and safety (from 31% to 87%) and client management (from 28% to 78%). overall, laboratories scored highest in the resource management category of qses (73% for non-twinned and 82% for twinned laboratories at exit) and lowest in improvement management (53% and 71%, respectively) (figure 3). non-twinned laboratories improved slightly more than twinned laboratories in the resource management stage (41 percentage points vs. 38 percentage points), whereas twinned laboratories improved slightly more in the process management stage (44 percentage points vs. 45 percentage points) and substantially more in the improvement management stage (30 percentage points vs. 56 percentage points). figure 2: performance on 12 quality system essentials for twinned and non-twinned laboratories at baseline and exit of the strengthening laboratory management toward accreditation (slmta) programme. figure 3: average performance of twinned and non-twinned laboratories by quality cycle stages at baseline and exit of the strengthening laboratory management toward accreditation (slmta) programme. discussion top ↑ the eight regional-level laboratories in kenya’s first slmta cohort all made substantial quality improvements, moving from zero slipta stars to anywhere from one to four stars in a period of one year. laboratories twinned with research institutions started slightly higher and improved nearly twice as much as non-twinned laboratories during the first half of the programme. however, after mid-term, non-twinned laboratories increased their efforts and nearly caught up with their twinned counterparts. by the exit audit, twinned laboratories had improved by 44 percentage points, whilst non-twinned laboratories had improved by 38 percentage points.twinning slmta laboratories with research institutions addressed several observed needs. previous site assessment reports had documented a large disparity between government-funded and research-sponsored laboratories with regard to available equipment, supplies, quality assurance, safety practices and overall management. a consultancy team from the association of public health laboratories observed that there had previously been little to no sharing of resources and knowledge between the two groups, even where they were physically close in location;18 such was the case with the kenya medical research institute (kemri) hiv research laboratory, with state-of-the-art facilities and iso accreditation since 2007, and the nyanza provincial general hospital, which are located on the same campus. the twinning approach helped to bring the laboratories together to bridge the quality differential gap between the research laboratories and public laboratories that serve the majority of the population. twinning moh laboratories with high-performing laboratories also helped to alleviate the shortage of slmta mentors. the approach gave the government-sponsored laboratories access to highly-skilled personnel with technical expertise from the research laboratories, who were able to both mentor and monitor their quality performance. in addition, institutional mentorship provided extended contact time and engaged the entire laboratory workforce in the twinned moh laboratory, facilitating measurable qse improvements across the laboratory testing system. in the context of ongoing laboratory strengthening efforts19 and pepfar’s recent approaches to promoting public–private partnerships to enhance african countries’ laboratory systems and service delivery,20 kenya’s twinning model is the first of its kind to create in-country laboratory partnerships in order to address not only quality systems, but also sustainable strategies for capacity building. review of previous laboratory partnerships reveals two scenarios: the majority have been international partnerships focusing largely on vertical disease programmes and emerging global disease surveillance programmes,21 whilst the remainder focused on transfer of technologies in health and support of national scientists to apply for grants from local and international funding agencies.22 the results presented here provide evidence that institutional twinning of in-country high-performing laboratories with moh counterparts through the slmta programme is a practical means of achieving quality improvements for public diagnostic laboratories in low-resource settings. the exchange visits between the twinned laboratories provided the mentee laboratory staff additional motivation to adopt and implement quality systems in their laboratories. through exchange visits, the mentee laboratories had opportunities to experience first-hand good laboratory practices and thereafter found them easier to implement at their own facilities. in addition to the rapid improvements achieved through twinning, periodic visits to the research laboratories by the mentee laboratory staff promoted an integrated understanding of laboratory quality systems and further enhanced quality services provided by the mentee laboratories. in our study, twinned laboratories had the most advances in the improvement management category, which focuses mainly on post-examination tasks. these twinned laboratories did particularly well in occurrence and/or incidence management, internal audit and corrective action, all of which are critical for reducing preventable laboratory errors and hence ensuring that laboratories produce accurate results for quality patient care. in contrast, non-twinned laboratories improved more in the resource management and process management areas, which focus on pre-examination and examination tasks. these tasks require hospital management decisions and activities that not only support, but also enhance technical work performed by the laboratory. within one year of mentorship, several improvements were recorded in the non-twinned laboratories, including the establishment of laboratory personnel files with job descriptions, an accessible system for laboratory records and documents, management review calendars and a clinician handbook. the two sets of laboratories recorded comparable improvements in the equipment management and management review qses. however, twinning was associated with better outcomes in the qses that typically require close communication between the laboratory and upper management for approval of decisions and budgetary needs. several factors may limit the generalisation of our observations. firstly, the number of laboratories was very small, prohibiting statistical assessment of the difference in results between the twinned and non-twinned laboratories. secondly, laboratories were not assigned randomly to their respective mentorship model, so any effects could have resulted from factors that also influenced assignment; however, the two sets of laboratories had identical slmta training and comparable mean baseline scores, both overall and in most categories. thirdly, mentorship works through a positive rapport established between the mentor and mentee; despite use of a standardised checklist, personality differences amongst mentors and slmta laboratory staff members, as well as the greater contact time and frequent benchmarking visits for the twinned laboratories, may have affected the results. finally, one of the moh laboratories is located on the same campus as its twinned research laboratory; whilst this proximity has the advantage of very close monitoring and review of quality improvements, results may not be typical of what would be expected with more distant twinned laboratories. kenya’s institutional mentorship approach went beyond standard slmta training to include on-site provision of good clinical laboratory practice training. through benchmarking and exchange visits, the moh laboratories were able to observe, practise and develop personnel files, competency assessment tools, levey-jennings charts, quality control runs and eqa data tools. this institutional mentorship approach appears to have contributed to the rapid improvement in the twinned laboratories during the first half of the programme, as the moh laboratory staff quickly put into practice what they had observed. the limited progress made during the second half of the programme amongst twinned laboratories suggests that further improvements may rely on external factors, such as additional funding and staff; and that institutional mentorship may be most efficient when used for short durations. public–private partnership models have been gaining popularity in recent years as a potential source of funding and technical support for improvement in public sector activities. the world bank suggests that these partnerships may be a good way to build skills in the public sector.23 balancing the need for support with the host government’s responsibility for providing services to the public is critical. whilst the potential benefits are encouraging, it must be kept in mind that supporting public health laboratories is not the long-term responsibility of these research facilities; thus, care should be taken to identify ways to streamline the mentoring process and to use the programme as a catalyst for sustainable development of a permanent cadre of well-trained leaders within the public sector in order to ensure that the generosity of these research laboratories is maximised. judicious use of this valuable resource and appropriate acknowledgement will help to ensure a mutually beneficial outcome, encouraging private laboratories to include partnership in their mandate as a means of fulfilling corporate social responsibility. conclusion the institutional twinning model holds promise for future collaborations between ministries of health and state-of-the-art local laboratories accredited to international standards. this model may be used to foster long-standing and sustainable partnerships between public health and research laboratories. acknowledgements top ↑ this research has been supported by pepfar through cooperative agreement number 5 u2g ps001285-02 from pepfar phase ii through the cdc, division of global hiv/aids (dgha). competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions e.p.m. (cdc, kenya) was the project leader, responsible for implementing the supervisory mentorship approach described and drafting the manuscript. s.m. (cdc, kenya) helped with the data analysis. f.l.b. (cdc, kenya), w.i.e. (ampath) and r.l. (henry jackson foundation) assisted in implementing the twinning mentorship described. c.z. (cdc, kenya) edited the manuscript and e.t.l. (cdc, atlanta) reviewed the manuscript and made conceptual contributions. j.m. (cdc, kenya) designed the twinning mentorship described and edited the manuscript. cdc disclaimer the findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the cdc and the government of kenya. references top ↑ 1. schroeder lf, amukele t. medical laboratories in sub-saharan africa that meet international quality standards. am j clin pathol. 2014;141(6):791–795. http://dx.doi.org/10.1309/ajcpq5ktkagsscfn2. world health organization. joint who–cdc conference on health laboratory quality systems. who/hse/ihr/lyo/2008.3 [document on the internet]. c2008 [cited 2014 mar 15]. available from: http://www.who.int.ihr/lyon/report20080409.pdf 3.gershy-damet gm, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. http://dx.doi.org/10.1309/ajcptuuc2v1wjqbm 4.nkengasong j, 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laboratory quality improvement process towards accreditation (slipta). afr j lab med. 2012;1(1), art. #6, 8 pages. http://dx.doi.org/10.4102/ajlm.v1i1.6 10. nzabahimana i, sebasirimu s, gatabazi jb, et al. innovative strategies for a successful slmta country programme: the rwanda story. afr j lab med. 2014;3(2), art. #217, 6 pages. http://dx.doi.org/10.4102/ajlm.v3i2.217 11.nzombe p, luman et, shumba e, et al. maximising mentorship: variations in laboratory mentorship models implemented in zimbabwe. afr j lab med. 2014;3(2), art. #241, 8 pages. http://dx.doi.org/10.4102/ajlm.v3i2.241 12. audu ra, onubogu cc, nwokoye nn, et al. improving quality in national reference laboratories: the role of slmta and mentorship. afr j lab med. 2014;3(2), art. #200, 7 pages. http://dx.doi.org/10.4102/ajlm.v3i2.200 13. chankova s, muchiri s, kombe g. health workforce attrition in public sector in kenya: a look at the reasons. hum resour health. 2009;7:58. http://dx.doi.org/10.1186/1478-4491-7-58 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meili rc, et al. strengthening laboratory systems in resource-limited settings. am j clin pathol. 2010;134(3):374–380. http://dx.doi.org/10.1309/ajcpdqosb7qr5glr 20.pepfar. new public–private partnership to strengthen laboratory systems [page on the internet]. c2007 [cited 2014 aug 08]. available from: http://www.pepfar.gov/documents/organization/94561.pdf 21.gambel jm, hibbs rg jr. u.s. military overseas medical research laboratories. mil med. 1996;161(11):638–645. 22.chandiwana s, ornbjerg n. review of north-south and south-south cooperation and conditions necessary to sustain research capability in developing countries. j health popul nutr. 2003;21(3):288–297. 23.yu d, souteyrand y, banda ma, et al. investment in hiv/aids programs: does it help strengthen health systems in developing countries? global health. 2008;4:8. http://dx.doi.org/10.1186/1744-8603-4-8 abstract introduction methods results discussion acknowledgements references about the author(s) shaheed v. omar centre for tuberculosis, world health organization tb supranational reference laboratory network, national institute for communicable diseases, national health laboratory service, johannesburg, south africa lavania joseph centre for tuberculosis, world health organization tb supranational reference laboratory network, national institute for communicable diseases, national health laboratory service, johannesburg, south africa halima m. said centre for tuberculosis, world health organization tb supranational reference laboratory network, national institute for communicable diseases, national health laboratory service, johannesburg, south africa department of medical microbiology, university of the free state, bloemfontein, south africa farzana ismail centre for tuberculosis, world health organization tb supranational reference laboratory network, national institute for communicable diseases, national health laboratory service, johannesburg, south africa department of medical microbiology, university of pretoria, pretoria, south africa nabila ismail centre for tuberculosis, world health organization tb supranational reference laboratory network, national institute for communicable diseases, national health laboratory service, johannesburg, south africa thabisile l. gwala centre for tuberculosis, world health organization tb supranational reference laboratory network, national institute for communicable diseases, national health laboratory service, johannesburg, south africa nazir a. ismail centre for tuberculosis, world health organization tb supranational reference laboratory network, national institute for communicable diseases, national health laboratory service, johannesburg, south africa department of medical microbiology, university of pretoria, pretoria, south africa citation omar sv, joseph l, said hm, et al. whole genome sequencing for drug resistance determination in mycobacterium tuberculosis. afr j lab med. 2019;8(1), a801. https://doi.org/10.4102/ajlm.v8i1.801 brief report whole genome sequencing for drug resistance determination in mycobacterium tuberculosis shaheed v. omar, lavania joseph, halima m. said, farzana ismail, nabila ismail, thabisile l. gwala, nazir a. ismail received: 07 mar. 2018; accepted: 28 sept. 2018; published: 21 feb. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract south africa remains challenged with a high tuberculosis burden accompanied by an increase in drug resistant cases. we assessed the use of the illumina miseq, a next-generation sequencing platform for whole genome sequencing, followed by bioinformatic analysis using a commercial software package to determine resistance to selected drugs used for mycobacterium tuberculosis treatment in our setting. whole genome sequencing shows potential as a diagnostic platform for the detection of drug resistance in mycobacterium tuberculosis with the provision of information for several drugs simultaneously. introduction drug-resistant tuberculosis poses a significant challenge to tuberculosis control programmes in high burden settings.1 undiagnosed drug resistance leads to further transmission, poor patient outcomes and potential for amplification of drug resistance, impeding the world health organization’s (who) strategy to end tuberculosis by 2035. the drug-resistant tuberculosis outbreaks in tugela ferry2 and other regions of south africa3 highlight the need for early and accurate diagnosis of drug resistance. often, comprehensive phenotypic baseline testing is not available nor is a robust surveillance programme in place to inform regimen changes appropriate to local resistance profiles.4 a paradigm shift is needed in the approach to diagnosis and surveillance of drug-resistant tuberculosis to ensure that new drug potential is not lost due to the evolution and spread of resistant strains. molecular testing such as the line probe assay and xpert mtb/rif assay (cepheid, sunnyvale, california, united states) show potential superiority in overall performance over phenotypic drug susceptibility testing (dst).5,6 a targeted sequencing approach for resistance detection in mycobacterium tuberculosis by application of next-generation sequencing benchtop platforms showed good performance in terms of sensitivity.7 with the decreasing cost of next-generation sequencing, whole genome sequencing (wgs) could be applied for this purpose as an alternative to conventional phenotypic methods.8,9 the direct benefit of wgs is its ability to provide organism identification, strain relatedness and a drug resistance profile for characterised resistance-conferring mutations. in addition, wgs may be useful for resistance determination for newer drugs lacking validated dst such as bedaquiline and delamanid, utilising information available for the genetic basis associated with resistance in vitro to these novel drugs.10,11 we assessed the use of the illumina miseq® sequencing,12 followed by bioinformatic analysis using a commercial software (clc genomics workbench, qiagen, venlo, the netherlands) for drug resistance determination at the national tuberculosis reference laboratory in south africa. methods ethical considerations ethical approval was not required for this laboratory-based study as only anonymised isolates were used. sample selection twenty geographically diverse clinically isolated m. tuberculosis strains, with varying resistance profiles and spoligotype patterns, isolated between june 2012 and january 2013 were selected for this pilot evaluation (table 1). laboratory processing for culture, smear microscopy and dst were performed according to who guidelines.13 six of the 20 isolates had discordant phenotypic results between initial and repeat testing to either the fluoroquinolones or pyrazinamide. table 1: summary of performance for drug resistance determination using the mgit960, mtbdrplus assay and whole genome sequencing. routine laboratory phenotypic testing phenotypic dst was performed on the bactec mycobacterial growth indicator tube (mgit) 960 system (becton dickinson diagnostic systems, sparks, maryland, united states) following the manufacturer’s recommendation. first and second-line anti-mycobacterial drugs (rifampicin, isoniazid, ofloxacin, moxifloxacin, ptyrazinamide, amikacin, and kanamycin) were tested following the who 2012 policy guidelines.14 replicate testing was performed on any isolate resistant to pyrazinamide or second-line drugs on initial testing. next-generation sequencing wgs was performed using the miseq version 2 kit (illumina, san diego, california, united states). in brief, dna was extracted using the nuclisens easymag system (biomérieux, marcy-l’étoile, france) from a 200 µl aliquot of heatinactivated, mgit-cultured isolate and concentrations quantified using the qubit dsdna hs (high sensitivity) assay (life technologies, carlsbad, california, united states). libraries were prepared using nextera xt kit (illumina, san diego, california, united states) following the manufacturers’ protocol with one modification (figure 1). the modification deviated at the normalisation step, where the indexed dna libraries concentrations were quantified as described above and normalised to 4 nm by addition of tris-cl (10 nm, ph8.5 with 0.1% tween20). thereafter, the indexed libraries of all 20 isolates were pooled to a final concentration of 12 pm and loaded onto the miseq for sequencing. figure 1: operational workflow for phenotypic drug resistance determination versus resistance prediction by whole genome sequencing. the batch size is calculated at 20 isolates [estimated hands-on time]. bioinformatic analysis of wgs data clc genomics workbench version 6.0.1 (qiagen, venlo, the netherlands) was used for bioinformatic analysis. variant tables for genetic targets associated with resistance to rifampicin, isoniazid, fluoroquinolones (ofloxacin and moxifloxacin), pyrazinamide, aminoglycosides (amikacin and kanamycin), bedaquiline and delamanid (table 2) were generated using the map reads to reference tool and quality-based variant detection algorithm on clc genomics workbench using the h37rv sanger reference genome (genbank nc000962.3). the following cut-offs were applied to call a single nucleotide polymorphism or insertion/deletion: a minimum paired coverage depth of five times (5×), frequency of > 70% and a phil’s read editor, or phred, quality score of ≥ q20 (≥ 99% accuracy) at the variant position and neighbouring nucleotides within a radius of five base pairs. to ensure that an isolate was truly wild-type for a specific gene target, we further ran the create statistics for target regions on clc genomics workbench to ensure that the entire length of the gene investigated was completely sequenced. since no thresholds have been formally established for bioinformatic analysis, we utilised less stringent parameters than those previously described.15 table 2: table detailing first-line, second-line and novel tuberculosis drugs, their resistance-associated genes and their length. association of mutations as resistance predictors were identified using the tb drug resistance mutation database (tbdreamdb) database16 primarily. if a mutation was not listed, literature, including newer published databases such as tbprofiler and phyresse, was surveyed to identify the association.17,18 putative mutations associated with the novel drugs bedaquiline and delamanid were exclusively identified using published literature.11,19 the rpob-associated mutations were converted to the widely used escherichia coli nomenclature (addition of 81 codon positions).20 resolving discordant phenotypic and wgs results discordant results were resolved using the minimum inhibitory concentration broth microdilution method (trek sensititre, thermofisher, waltham, massachusetts, united states) and interpreted using the critical concentrations established by hall et al. (2012).21 in the case of pyrazinamide, the modified wayne’s test22 was used to resolve discordance. additionally, the genotype mtbdrplus assay version 2 (mtbdrplus) (hain lifesciences, nehren, germany) line probe assay was performed according to the manufacturer’s instruction for the first-line drugs rifampicin and isoniazid on all isolates. figure 1 provides an overview of the operational workflow for this study. results concordance between wgs and the phenotypic dst method for resistance determination was noted for all isolates except one phenotypically susceptible isolate for all targets explored (table 1 and table 2). the phenotypically susceptible isolate harboured a known resistance associated mutation in the fabg1/maba (inha promoter) region (inha promoter c-15t) detected by wgs. this finding was confirmed by the mtbdrplus assay displaying an inhamut1 mutation, and resistance was confirmed by the broth microdilution assay (minimum inhibitory concentration of 0.25 µg/ml) (table 2). interestingly, we found that a multidrug resistant isolate was incorrectly classified as susceptible to rifampicin by the mtbdrplus assay and resistant by both mgit dst and wgs; the latter detected the presence of the rpobl511p mutation, a known rifampicin resistance determinant. three phenotypic discordant pyrazinamide isolates included were susceptible by wgs and susceptibility was confirmed by the modified wayne’s test. of note was the finding that one isolate had a pncathr114met mutation by wgs that was not listed in the tbdreamdb database, and literature confirmed this not to be associated with resistance.24 resolution testing using the modified wayne’s test confirmed susceptibility. the phenotypically discordant fluoroquinolone isolates (n = 3) were predicted to be susceptible by wgs, displaying a wild-type gyra and gyrb gene. repeat dst was in agreement with wgs for moxifloxacin; however, two of the three isolates remained resistant to ofloxacin. resolution testing using the broth microdilution assay confirmed susceptibility for both isolates, showing a minimum inhibitory concentration of 1 µg/ml. wgs for novel drugs bedaquiline and delamanid showed no resistance-associated mutations. discussion the application of whole genome next-generation sequencing technology for drug resistance determination in m. tuberculosis has been shown to be a valuable tool in this study. despite the small sample size, the performance of wgs for predicting resistance was consistent with published studies containing subsets of south african isolates.25,26 the use of the miseq® offers reduced hands-on preparation time (~6 h per batch of isolates) compared to other next-generation sequencing technologies.27 bioinformatic analysis using the commercial software was relatively straightforward, particularly the use of workflows for automation. once a workflow is saved, imported data are automatically analysed, producing a final output table displaying mutations. however, sequence analysis requires an understanding of the associated genetic targets and drug resistance mutations. concordance of wgs with initial mgit dst was lacking for isoniazid, pyrazinamide and fluoroquinolones; however, resolution testing improved agreement between wgs and phenotypic drug susceptibility profiles for the discordant isolates, even at a coverage of 5× (paired) with acceptable quality scores. the use of this technology for resistance determination in m. tuberculosis is currently limited due to the lack of a comprehensive tuberculosis mutation catalogue predicting susceptibility, as seen in the case of the pncathr114met mutation in this study, which was not associated with resistance. furthermore, sequence data can only be generated from cultured isolates, creating a lag between specimen receipt and a positive culture. despite these limitations, wgs could benefit the majority of patients by enabling them to be placed on optimum regimens sooner in comparison to phenotypic methods. limitations the small sample size was inadequate for assessing diagnostic performance statistically. in addition, the data are based on a first attempt without any optimisation for sequence output. conclusion wgs correctly predicted resistance or susceptibility using commercial bioinformatics software based on already identified resistance-determining mutations. our findings suggest the system shows promise as a tool for predicting drug resistance in a short time frame for multiple drugs and multiple samples simultaneously, provided the genetic basis for resistance is well described. acknowledgements detailed wgs results and statistics are available from the corresponding author. we would like to thank professor jasper rees and jonathan feathers (agricultural research council, biotechnology platform, pretoria, south africa) for their valuable input and assistance. we further would like to thank the technical staff at the centre for tuberculosis. competing interests the authors declare 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warren rm, donald pr, et al. frequency and implications of pyrazinamide resistance in managing previously treated tuberculosis patients. int j tuberc lung dis. 2006;10(7):802–807. huitric e, verhasselt p, koul a, andries k, hoffner s, andersson di. rates and mechanisms of resistance development in mycobacterium tuberculosis to a novel diarylquinoline atp synthase inhibitor. antimicrob agents chemother. 2010;54(3):1022–1028. https://doi.org/10.1128/aac.01611-09 zignol m, cabibbe am, dean as, et al. genetic sequencing for surveillance of drug resistance in tuberculosis in highly endemic countries: a multi-country population-based surveillance study. lancet infect dis. 2018;18(6):675–683. https://doi.org/10.1016/s1473-3099(18)30073-2 loman nj, constantinidou c, chan jz, et al. high-throughput bacterial genome sequencing: an embarrassment of choice, a world of opportunity. nat rev microbiol. 2012;10(9):599–606. https://doi.org/10.1038/nrmicro2850 abstract background description of the intervention lessons learnt recommendations acknowledgements references about the author(s) patrick a. njukeng global health systems solutions, douala, cameroon charles njumkeng global health systems solutions, douala, cameroon callistus ntongowa global health systems solutions, douala, cameroon mohammed abdulaziz africa centres for disease control and prevention, addis ababa, ethiopia citation njukeng pa, njumkeng c, ntongowa c, abdulaziz m. strengthening laboratory networks in the central africa region: a milestone for epidemic preparedness and response. afr j lab med. 2022;11(1), a1492. https://doi.org/10.4102/ajlm.v11i1.1492 lessons from the field strengthening laboratory networks in the central africa region: a milestone for epidemic preparedness and response patrick a. njukeng, charles njumkeng, callistus ntongowa, mohammed abdulaziz received: 08 dec. 2020; accepted: 11 feb. 2022; published: 19 may 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: health systems in the central africa region are among the weakest and least funded in the world. the lack of laboratory networks and adequately trained personnel with clearly defined responsibilities has hampered the implementation of laboratory quality improvement programmes. global health systems solutions (ghss) obtained a grant from the africa centres for disease control and prevention to develop laboratory networks for disease surveillance and strengthen the quality of laboratory testing in the central africa region. intervention: one year after the grant was awarded on 01 october 2018, ghss has launched a regional integrated surveillance and laboratory network (rislnet) for central africa and developed national laboratory strategic plans and policies for member states, eight frameworks and guideline documents, as well as a website for rislnet central africa. ghss has also launched an extension for community health outcomes platform to supervise laboratories enrolled for accreditation, installed a basic laboratory information system (blis) in four laboratories in four member states, and trained 247 laboratory personnel and laboratory experts on blis, quality assurance, external quality assurance, strengthening laboratory management towards accreditation (slmta), quality management systems, and equipment maintenance and calibration. lessons learnt: participating laboratories now serve as reference laboratories for covid-19 testing in various countries. point-of-care testing, using the genexpert platform, has been the central strategy for the scale-up of covid-19 testing in the central africa region. recommendations: expanding slmta to other laboratories within central africa will significantly improve the quality management of laboratories for a better healthcare system. keywords: covid 19; laboratory; health system; strengthening; network; quality; management; response. background clinical laboratories form an essential component of the health system. in addition to providing test results for disease diagnosis and guiding treatment by detecting drug resistance, they form the backbone of disease surveillance and public health response to outbreaks and epidemics.1,2,3 efforts to strengthen health systems should therefore focus on laboratory services and systems as they provide primary information that informs decision making for best healthcare outcomes.4 the role of the laboratory in responding to epidemics is undoubtedly a vital one judging from the lessons learned in recent years. the 2014 ebola outbreak in west africa revealed the need to organise and maintain laboratory systems or networks that can rapidly and effectively adjust to carry out new diagnostic testing or laboratory services in response to large-scale epidemics.5 another lesson learned from the ebola outbreak was the need to set up a platform that enables planning and preparedness activities to be rapidly adapted to emerging pathogens or outbreaks.6 the current coronavirus disease 2019 (covid-19) pandemic has further emphasised the overarching role of quality laboratory systems in disease outbreak preparedness and response in africa and, specifically, in the central africa region. health systems in the central africa region are among the weakest in the world and have received the least funding support when compared to other regions.7,8 however, funding sources and advanced testing technologies are increasingly being made available to laboratories in the region from bodies such as the african union/africa centres for disease control and prevention (cdc), african society for laboratory medicine, the world health organization’s regional office for africa and the united states cdc, among others. nonetheless, laboratory quality improvement is still hindered by the lack of adequately trained personnel with clearly defined responsibilities, as well as the lack of well-established organisations and laboratory networks. to promote the strengthening of laboratory systems, the network of national public health institutes needs to be strengthened through continuous advocacy and staff capacity development. global health systems solutions (ghss), an international non-governmental organisation based in cameroon, obtained a grant from africa cdc to establish and strengthen public health laboratory systems and networks in the central africa region. ghss worked in close collaboration with africa cdc and the regional collaboration center to achieve this strategic vision of the africa cdc. the principal goal of the african union/africa cdc grant was to develop laboratory networks for disease surveillance and strengthen the quality of laboratory testing across nine countries in the central africa region, namely gabon, cameroon, central african republic, chad, congo brazzaville, equatorial guinea, burundi, democratic republic of congo, and sao tome and principe. this goal was predicated on three key objectives. the first objective was to develop a framework and statute for the central africa regional integrated surveillance and laboratory network (rislnet) that defines its function and operations, permitting the establishment of a website to share framework documents and experiences between the different countries. the second objective was to implement a quality management system (qms) towards the accreditation of laboratories in the region with emphasis on the quality of point-of-care (poc) testing, biosafety and biosecurity, equipment maintenance, sample referral systems and antimicrobial resistance surveillance. the final objective was to support selected member states to develop the laboratory components of regional proposals to address antimicrobial resistance. this article discusses the activities in the first year of the project implementation and the role of the implementation in the fight against covid-19, which is currently the greatest global challenge. description of the intervention ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. development of a framework and statute for the rislnet two workshops were organised in july 2018 and march 2019 to accomplish this task. the first workshop brought countries together within the circumscription of the central africa regional collaboration centre of africa cdc, and this was a key step towards setting up a network of health institutes and laboratories within the region. in this forum, experts from the human, animal and environmental health sectors of the nine countries came together in a workshop in brazzaville, gabon, and began to share contacts, create linkages and share information and experiences within the region. the workshop, which ended with the launching of rislnet for the central africa region under the patronage of the minister of health of the republic of congo, also saw the inauguration of bureau members for the central africa rislnet. a second workshop was organised in malabo, equatorial guinea, to guide the development of the national laboratory strategic plan and policies for member states. to foster the development of a framework and statute for the central africa rislnet, frameworks and guidelines were developed that included: a framework and statute for rislnet, a sample transport system framework for the central africa rislnet, a quality manual template for laboratory testing, and guidelines for laboratory biosafety and biosecurity. a website was also developed for the central africa rislnet to enhance the sharing of public health information, laboratory quality best practices and laboratory quality documents, as well as to encourage networking among public health laboratories in the central africa region. mapping and linkage of centres of excellence and laboratories in the region using the extension for community health outcomes platform the extension for community health outcomes (echo) was launched at ghss’ head office in douala, cameroon, to support the mentorship and supervision of seven laboratories enrolled for accreditation. these laboratories were linked and coordinated together with centres of excellence and public health laboratories for event-based and laboratory-based disease surveillance. they include laboratoire l’hôpital general de reference national, n’djamena, chad; laboratoire de l’hôpital prince regent charles, bujumbura, burundi; laboratoire national de sante publique, brazzaville, congo brazzaville; laboratorio national de referencia para tuberculose sao tome, sao tome and principe; international centre of medical research of franceville, franceville, gabon; laboratorio castroverde, malabo, equatorial guinea; and laboratoire national de biologie clinique et de sante publique, bangui, central african republic. the sample transport and information sharing systems between these laboratories and countries’ centres of excellence and national reference laboratories have been linked to echo. implementation of qms and biosafety in the region a training workshop was held in n’djamena, chad, from 22 july 2019 to 26 july 2019. biologists, laboratory managers, and laboratory technicians were trained on laboratory qms and the implementation of immediate improvement projects to speed up the world health organization’s regional office for africa accreditation process. the training served as a springboard for international organization for standardization-15189 accreditation, as 21 participants were trained on productivity management, quality assurance, documents and records management, and the use of the strengthening laboratory management towards accreditation (slmta) toolbox. development of a framework for implementing qms for poc testing this activity was designed to increase the accuracy and reliability of poc testing, which is useful for the rapid detection of endemic and outbreak diseases. a 5-day training of trainers was conducted from 28 october 2019 to 01 november 2019 in libreville, gabon, to build the capacity of 26 healthcare personnel to support the institutionalisation and sustainability of poc testing. this activity aimed to ensure that quality assurance officers are well equipped with the skills, knowledge and abilities necessary to perform assigned tasks. the world health organization/cdc-approved poc comprehensive quality assurance training package was used, with emphasis on the stepwise process for improving the quality of hiv-related point-of-care testing checklist.9 implementation of the computing for good (c4g) blis eight hospital laboratories, one public health laboratory and one reference laboratory each in burundi, congo, chad, and sao tome and principe, benefited from the computing for good (c4g) basic laboratory information systems (blis) network. after a baseline survey, gaps identified in each of the laboratories were addressed to allow the implementation of the blis network, and the ghss team embarked on the implementation phase to install and implement a c4g blis network in these laboratories to improve turn-around times and to support clinical decision making. the ghss team also purchased and installed information technology materials and network connectivity to support the management of c4g blis in the laboratories. the laboratory staff were then trained on the use of blis for the proper management of laboratory data and the day-to-day activities of the laboratory. lessons learnt the covid-19 disease is entirely new, and the characteristics of its causative virus, the severe acute respiratory syndrome coronavirus-2, are not fully understood.10 drug development typically takes years of scientific studies, and unfortunately, the new coronavirus has not given the world that length of time. at the start of the pandemic, there was no defined guideline available for the management of the disease. the echo platform provides practitioners with the opportunity to come together weekly to share experiences from the treatment centres in different countries. this will enhance patient management as expertise on better patient management strategies will easily be shared among different countries. this platform will also permit various treatment centres to discuss treatment challenges and receive feedback and contributions from colleagues. there is a great need to boost fragile health systems like those in the central africa region for better disease surveillance and outbreak response. a body such as ghss with vast experience in supporting health systems in central africa can utilise the echo platform established in the region to support the respective countries as they respond to this pandemic. it can guide infection prevention and control and virtual training on biosafety practices and sample collection, packaging and transportation for healthcare workers via the echo platform, all of which are crucial in the fight against covid-19. this platform is now used regularly by ghss to strengthen the quality of testing in cameroon and the democratic republic of congo. the rislnet initiative, after one year of its implementation, has been very helpful in the fight against the covid-19 outbreak. at a moment when health systems across the globe are facing significant challenges, it is the product of the rislnet initiative (the elected centres of excellence, referral network, trained personnel, and developed guidance documents) that has formed the basis of the sub-region’s strategy in the fight against covid-19. as covid-19 testing is not entirely decentralised within member states, the sample transport system framework for rislnet central africa can easily be adapted to the covid-19 sample transport network within member states. furthermore, the laboratory testing quality manual template put in place will guide good laboratory practice for covid-19 testing and ensure timely, accurate and reliable results. the guidelines for laboratory biosafety and biosecurity should provide an appropriate foundation for the implementation of safety practices for the protection of frontline healthcare workers who are most at risk. most importantly, the rislnet website11 has been essential in the sharing of information of public health interest, laboratory quality best practices, and laboratory quality documents. the website contains the emergency numbers of member states, provides updates on the covid-19 situation and enhances the sharing of covid-19 strategic documents, recommendations, guidelines, feedback from meetings, etc. the slmta programme was established because the united states cdc, world health organization’s regional office for africa and stakeholders recognised the poor state of medical laboratory systems in africa and prioritised the need to strengthen them to fight multiple diseases.4 few laboratories in central africa recognise the importance of developing qmss, laboratory strategic plans, and protocols that facilitate continuous laboratory quality improvement for patient care. the implementation of slmta in africa has improved laboratory quality, biosafety, standardisation, maintenance, record keeping, and reporting in africa, all of which are crucial to the optimal function of laboratories.12,13 the laboratories that participated in the rislnet initiative were mentored for accreditation using the slmta toolkit. it is therefore not surprising that these laboratories now serve as reference laboratories for covid-19 testing in their respective countries. the countries would have found it challenging to quickly respond to the covid-19 outbreak if the laboratories had not received training on qms, quality assurance, biosafety and biosecurity, and equipment maintenance and calibration. also, poc testing has been at the centre of the strategy to scale-up covid-19 testing in central africa. notably, genexpert, which is currently being used in scaling up covid-19 testing in central africa, was the focus of this implementation. the participants trained in the implementation of qms for poc testing are now adequately equipped to lead their countries in the scale-up of covid-19 testing. participants were also trained on capacity development for healthcare personnel to support the institutionalisation and sustainability of poc testing. thus, they can easily initiate, implement, evaluate and provide corrective action to the various poc sites carrying out covid-19 diagnoses. the use of a c4g blis network in these laboratories will improve turn-around times for covid-19 testing, which is very critical for clinical decision making, surveillance efforts, and the enforcement of preventive measures. the provision of information technology materials and network connectivity to support the management of c4g blis will facilitate data sharing and communication between the testing laboratories and collection sites. laboratory staff trained on the proper use of blis can properly manage testing data and rapidly generate and report testing statistics to health authorities for quick decision making. the impact of these project implementation activities on the regional response to covid-19 cannot be understated but may be difficult to quantify. nonetheless, lessons learned in the past have shown that strengthening laboratory systems is the best strategy for outbreak preparedness and response. during the ebola epidemic in west africa between 2013 and 2016, laboratory systems were overwhelmed, and the medical world saw the need to rethink the future of global laboratory medicine. in west africa, the laboratory system could not diagnose haemorrhagic fevers and other diseases and, as a result, the public health experts failed to notice the outbreak in time.5 resultant from weak laboratory systems, public health authorities in the west africa region continues to miss ebola cases, leading to ebola illnesses and deaths erroneously attributed to other infections.14 similarly, the world health organization was only notified of the ebola outbreak characterised by fever, severe diarrhoea, vomiting, and a high fatality rate in guinea in march 2014; however, epidemiological investigations linked laboratory-confirmed cases with a presumed first fatal case of the outbreak in december 2013.15,16 thus, weak laboratory systems frustrate surveillance systems and decrease the quality of patient care, which are integral parts of the preventive and therapeutic interventions needed in disease control. recommendations expanding slmta to other laboratories within member states in the central africa region will significantly improve the quality management in these laboratories and result in a better healthcare system. the laboratories enrolled in this programme can be further supported through supportive site visits or coaching through the echo platform; this will improve their effectiveness in responding to covid-19 testing challenges. outlining a clear plan to support these sites will help sustain efforts towards achieving accreditation and boost their current role in the fight against covid-19. more funding is required for these efforts and the improvement of health systems in central africa. acknowledgements the authors express sincere gratitude to the funding bodies (africa cdc and african union) and all the authorities of central africa. we acknowledge the various laboratories involved and the personnel who led the implementation at different levels. we equally applaud the diverse team of experts at ghss for their relentless efforts and commitment. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions p.a.n. conceived, designed and supervised the project implementation. c. njumkeng participated in the field implementation of the project and drafted the paper. c. ntongowa coordinated the project implementation. m.a. directed the project implementation and reviewed and corrected the final manuscript. all authors read and approved the final manuscript. sources of support this project was funded by the african union commission and the africa centres for disease control and prevention. however, the results and conclusions made in this publication are made by the authors and do not represent the official position of the african union commission and africa centres for disease control and prevention. data availability data sharing is not applicable to this article. disclaimer all authors were involved in year one activity implementation of laboratory networks for disease surveillance and strengthening of the quality of laboratory testing in central africa. references panteghini m. the future of laboratory 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https://www.nytimes.com/2014/12/30/health/how-ebola-roared-back.html abstract introduction methods results discussion acknowledgements references about the author(s) anura david wits diagnostic innovation hub, faculty of health sciences, university of the witwatersrand, johannesburg, south africa lesley e. scott wits diagnostic innovation hub, faculty of health sciences, university of the witwatersrand, johannesburg, south africa pedro da silva national priority program, national health laboratory services, johannesburg, south africa elizabeth mayne national priority program, national health laboratory services, johannesburg, south africa division of immunology, university of cape town, cape town, south africa wendy s. stevens wits diagnostic innovation hub, faculty of health sciences, university of the witwatersrand, johannesburg, south africa national priority program, national health laboratory services, johannesburg, south africa citation david a, scott le, da silva p, mayne e, stevens ws. storage of mycobacterium tuberculosis culture isolates in microbanktm beads at a south african laboratory. afr j lab med. 2023;12(1), a2172. https://doi.org/10.4102/ajlm.v12i1.2172 note: additional supporting information may be found in the online version of this article as online supplementary document 1. original research storage of mycobacterium tuberculosis culture isolates in microbanktm beads at a south african laboratory anura david, lesley e. scott, pedro da silva, elizabeth mayne, wendy s. stevens received: 28 jan. 2023; accepted: 23 aug. 2023; published: 25 oct. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: mycobacterium tuberculosis complex (mtbc) isolates are typically stored at −70 °c in cryovials containing 1 ml aliquots of a liquid medium, with or without 50% glycerol. multiple uses of the culture stock may decrease the strain viability while increasing the risk of culture contamination. small culture aliquots may be more practical; however, storage capacity remains challenging. microbanktm beads (25 beads/vial) for the long-term storage of fungal cultures is well documented, but their use for storing mtbc isolates is uninvestigated. objective: the study aimed to determine the feasibility of using microbanktm beads for long-term storage of mtbc isolates at a laboratory in south africa. methods: in february 2020, 20 isolates in liquid culture were stored in microbanktm beads, following an in-house developed protocol, at −70 °c. at defined time points (16 months [15 june 2021] and 21 months [18 november 2021]), two beads were retrieved from each storage vial and assessed for viability and level of contamination. results: stored liquid isolates demonstrated mtbc growth within an average time-to-detection of 18 days following retrieval, even at 21 months post storage. contaminating organisms were detected in 2 of 80 (2.5%) culture isolates. conclusion: microbanktm beads will allow for the reculture of up to 25 culture isolates using a reduced culture volume compared to current storage methods. microbanktm beads represent a storage solution for the medium-term storage of mtbc isolates. what this study adds: this study evaluated the use of microbanktm beads as an alternate method for storing mtbc culture isolates at −70 °c and provided a suitable option for medium-term storage of mtbc. keywords: mycobacterium tuberculosis; culture isolates; storage; liquid culture; microbank beads. introduction tuberculosis diagnosis is a challenge in many health centres. the introduction and subsequent endorsement by the world health organization of the genotype mtbdrplus (hain lifesciences, nehren, germany),1 xpert mtb/rif and xpert mtb/rif ultra assays’ (cepheid, sunnyvale, california, united states),2,3 and the truenat mtb, mtb plus and mtb-rif dx assays (molbio diagnostics, goa, india)4 highlighted the advantages of using molecular technologies for diagnosis. although there is a lag in developing new tuberculosis diagnostic assays, in july 2021, another group of technologies received the world health organization’s conditional recommendation for use in tuberculosis diagnosis.5 these are cobas mtb and cobas mtb-rif/inh (roche, basel, switzerland),6 fluorotype® mtbdr (bruker, nehren, germany),7 realtime mtb (abbott, illinois, united states),8,9 and the bd max mdr-tb assay (becton dickinson, franklin, new jersey, united states).10 the treatment action group report provides information on current and new screening and triage tools.11 the expanding tuberculosis diagnostics pipeline and other non-molecular, culture-based, microscopy and radiology innovations can increase testing and earlier disease detection, even in difficult-to-detect cases, such as in hiv co-infected individuals; expand the spectrum of drug-susceptibility testing; and reduce costs.12 once a technology becomes available, it must be validated to ensure conformity to the manufacturer’s claims and/or target product profiles and verified to assess suitability within the intended settings.13,14,15,16,17,18 both validation and verification require suitable reference material that is stable and can be safely and efficiently transported. mycobacterial species pose a potential biosafety risk, especially for staff responsible for storing and transporting or aliquoting isolates. culture isolates should also be stored in an appropriate medium and at the correct temperature to maintain viability. these requirements have both cost and space implications. one of the earliest studies on long-term storage and preservation of mycobacteria was performed in 1972.19 mycobacterium tuberculosis complex (mtbc) (cultured on proskauer and beck medium) and mycobacterium bovis (cultured on sauton’s medium) were stored in nine different diluents. these included sterile skim milk; 1% gelatin buffered at ph 6.8; 1:5 dilution of sauton’s medium; 0.25% triton wr 1339 in sauton’s medium; a solution containing 8.3% dextran, 7.5% glucose, and 0.025% triton wvr 1339; 15% aqueous solution of lactose at ph 5.0; 5% sodium glutamate; tween-albumin medium; and distilled water. archive stocks were stored at two different temperatures (−20 °c and −70 °c) for 3 years. there was a significant reduction in mtbc isolate viability when stored at −20 °c versus −70 °c. in addition, the methods applied had safety concerns, such as the bottling procedures, appropriate container selection, transportation, and the risk of aerosolisation (once the bottle was opened). although most of these issues were resolved, the storage media and safety considerations were not ideal.19 in 2005, huang et al. demonstrated that mtbc isolates from solid media could be stored at −70 °c in 7h9 broth without significant loss in viability (> 90%) for up 7 years, while the viability of strains preserved directly from mycobacteria growth indicator tubes (mgits) was acceptable for up to 3 years.20 in 2014, the world health organization released a laboratory manual recommending mtbc isolate storage in 7h9 broth with glycerol,21 media preparation, specific equipment, and sterile work areas. although the world health organization-recommended technique efficiently stores and maintains mtbc strain viability, metcalfe et al. and hanekom et al. reported a possible loss of strain fitness due to the subsequent need for sub-culture to maintain stock culture isolates.22,23 furthermore, subculturing is prone to contamination, which may further risk the loss of valuable isolates. kremer et al. reported an alternative option to collect samples from the surface of a frozen medium by scraping the top layer without defrosting the remaining suspension.24 however, the use of a frozen stock may reduce the viability of isolates if the entire tube thaws during the subculturing process. glass beads have demonstrated medium-term storage for mtbc with strain recovery rates of > 94% within 30 days of incubation by giampaglia et al.25 however, both the beads and storage vial require pre-sterilisation and preparation of storage medium, which increases the process cost and is labour intensive. the microbanktm platform (www.https://pro-lab.com/products/clinical-microbiology/bacteriology/microbank/) has approximately 25 sterile beads per storage vial and a specifically formulated cryopreservative for low-temperature storage. it is potentially a suitable alternative storage solution for mtbc isolates. the microbanktm beads are porous, allowing microorganisms to adhere to the bead surface. a new culture can be grown by inoculating culture media with a single bead from the storage. thus, the process can be repeated for the 25 beads of each isolate compared to the current method, where the number of possible new cultures depends on how much of the stock is used for each reculture. some studies26,27,28 have reported reasonable fungal isolate recovery rates (96% – 100%) from the microbanktm beads; however, as far as we know, the microbanktm storage solution has not been assessed for mtbc isolate recovery. nevertheless, the microbanktm beads may maintain longer mtbc viability and allow maximum usage with 25 beads, reducing process cost and preserving precious culture isolates. this study aimed to determine the feasibility of using the microbanktm beads to store mtbc isolates from a laboratory in south africa. methods ethical considerations ethics approval to store mtbc isolates from residual mtbc-positive liquid culture on the microbanktm system was obtained from the human research ethics committee at the university of the witwatersrand in south africa (ethics approval number: m1511110). the mgit culture isolates were allocated a study number to maintain participant confidentiality. informed consent was, therefore, not required. isolates were described based solely on their laboratory characteristics, including mgit ziehl neelsen, blood agar, line probe assay and drug-susceptibility testing. preparation of culture isolates for microbanktm bead storage twenty residual mtbc-positive culture isolates from the department of molecular medicine and hematology research laboratory at a university in south africa, containing a heavy initial inoculum (approximately 105 to 106 colony-forming units per millilitre [cfu/ml]) from mgits were selected. the time-to-detection (ttd) for each culture isolate was determined and provided by the bactec™ mgit 960 mycobacteria culture system (becton dickinson, sparks, maryland, united states) up to 42 days after incubation. isolates comprised rifampicinand isoniazid-susceptible, multidrug-resistant, and rifampicin-only or isoniazid-only resistant strains. an atcc 25177 strain (davies diagnostics, johannesburg, south africa) was included as a positive control. before their storage on the microbanktm beads (pro lab diagnostics, texas, united states) in february 2020, the liquid culture isolates were stored in their original tubes at room temperature for ~5 months following removal from the bactec™ mgit 960 system. the mgits were vortexed for 30 s to break up clumps before removing 1 ml of liquid culture. the bead inoculum was prepared by centrifuging 1 ml of liquid culture at 3000 g for 15 min at room temperature. a volume of 500 μl of supernatant was removed, and the remaining 500 μl of the re-suspended pellet (using a pipette) was added to one microbanktm bead vial. the resulting suspension was then inverted 10 times and incubated at room temperature for 60 min allowing the mycobacteria to adhere to the bead surface. the liquid was then aspirated, and the vial was stored at −70 °c per manufacturer’s instructions.29 at defined time points (16 months [15 june 2021] and 21 months [18 november 2021]), two beads were aseptically removed with a pipette tip from each isolate vial. the beads were then added to two separate mgits containing 0.8 ml of polymyxin b, amphotericin b, nalidixic acid, trimethoprim and azlocillin antibiotic mixture (becton dickinson, sparks, maryland, united states) and incubated in a bactec™ mgit 960 system at 37 °c for a maximum of 42 days. for those culture isolates that flagged positive, the presence of acid-fast bacilli was confirmed by smear microscopy (ziehl neelsen stain).30 the presence of contaminating organisms were confirmed by visual inspection of any ‘abnormal’ growth in the mgits (where possible) and/or plating an aliquot of the culture onto blood agar and then analysing the plate for any growth after 48 h of incubation at 37 °c. a mgit tbc identification mpt64 antigen test (becton dickinson, sparks, maryland, united states) test was used to confirm the presence of mtbc in the mgit isolates. staff performing the storage and retrieval procedure were asked to compare the microbanktm procedure (in terms of ease to use) to the glycerol procedure currently used in the laboratory. data analysis the mgit ttd for each bead was obtained from the bactec™ mgit 960 printout. the ttds were captured on a microsoft excel spreadsheet (microsoft corporation, redmond, washington, united states). a one-way analysis of variance was performed on the culture isolate ttd using sas (sas institute inc 2013. sas/access® 7.1 interface to adabas, cary, north carolina, united states) to determine if there were significant differences at each time point. in addition, the number of archive beads that successfully grew mtbc, the number contaminated, and the number that failed to grow were reported. results after a storage period of 16 and 21 months, 34 of 40 (85%) archive beads successfully grew mtbc, 5 of 40 (12.5%) did not demonstrate growth after the 42-day incubation, and 1 of 40 (2.5%) demonstrated contamination (figure 1). the baseline ttd for the positive control culture isolate, atcc 25177, was 15.9 days (table 1). the positive control demonstrated a mean ttd of 8.4 days and 20.5 days after 16 months and 21 months. for isolates, where both beads retrieved at the same time point, demonstrated growth of mtbc, 15 of 16 and 13 of 15 after 16 and 21 months showed similar ttds (figure 1). isolates 4 and 17 demonstrated > 8-day difference in ttd between the two beads retrieved simultaneously. the box and whisker plot (figure 1) displays the distribution of means representing the ttd for the original culture, month 16 and month 21. the one-way analysis of variance indicated a significant difference among the means of the original, month 16 and month 21 ttd (f (2, 57) = 9.17, p < 0.001) (online supplementary file table 1). post-hoc analysis using tukey’s honestly significant difference test revealed that the original ttd (mean = 8.06 days) was significantly lower than both month 16 (mean = 19.18 days) and month 21 (mean = 17.10 days) ttd. however, no significant difference was observed between the mean culture isolate ttd at month 16 and month 21. figure 1: distribution of means for the original, month 16 (15 june 2021), and month 21 (18 november 2021) time to detection at a laboratory in south africa. the upper and lower ends of the box are the upper and lower quartiles. the box covers the interquartile interval, where 50% of the ttds are found. the diamond represents the mean ttd at each timepoint and the horizontal line in each box represents the median ttd at each timepoint. table 1: time to detection for culture isolates retrieved from microbanktm beads at two sampling points, 16 (15 june 2021) and 21 months (18 november 2021) at a laboratory in south africa. regarding usability, laboratory staff reported that the microbanktm beads were technically undemanding (requiring only routine laboratory procedures such as centrifugation) and easy to use. furthermore, no additional preparation of reagents was required. discussion storage of mtbc culture isolates on the microbanktm beads was performed at −70 °c as recommended19,21,31 and mtbc isolates were recovered within 39 days of incubation in the bactec™ mgit™ 960 instrument with minimal contamination. for archive cultures where contamination was seen, culture isolates grown from the second bead from the same vial did not show contamination suggesting that contamination occurred during the mgit reculture process rather than during processing for storage. since the microbanktm bead platform doesn’t address the possible contamination problem due to multiple openings and closing of the vials, sterility during the reculture process is critical. although the statistical analysis showed a significant difference in the ttd between the original and post-storage ttd, the ttd between the 16-month and 21-month time points was not statistically significant. this increased ttd could be attributed to decreased metabolic activities of the mtbc bacilli due to their storage at low temperatures.31 there was variability in ttd at both time points post storage, even in beads from the same vial, which may suggest that growth depends on the number of bacilli adhering to the bead. rather than a loss in viability, a decreased number of bacilli on some beads may also be the reason for some culture isolates demonstrating a lack of growth after the 42-day incubation period and highlighting the importance of the uniform distribution of bacteria. it is therefore essential to rigorously invert the microbanktm vial at the time of inoculation to assist with the uniform distribution of the bacteria across the beads. the ttd has been described as a time-to-event variable, which represents an indirect measurement of the bacterial load,32 and it is possible to reduce the ttd by increasing the colony-forming units of the initial inoculum. increasing colony-forming units may be achieved by incubating the culture isolates at 37 °c for a more extended period to allow for increased growth or increasing the starting volume of the inoculum. the susceptibility profile of the culture isolates also did not appear to impact the viability of the culture isolate or the ttd. recovery rates of mtbc preserved from culture media (stored at −70 °c for 7 years) can vary from 97.4% to 100%.20 room temperature storage of culture isolates has also been investigated.33 although the bacteria remained viable for up to 6 years, recovery rates were low (54.0% to 66.4% depending on storage conditions, such as the availability of an air conditioner) with a contamination rate of ~7%. recovery rates of mtbc using the microbead method after a 21-month storage period were demonstrated to be 85%. although this lower recovery rate appears to be a limitation of this method, further investigation is required using an increased inoculum volume or a higher culture concentration and a longer storage period. a key advantage of the microbank™ beads is their safety profile. the minimal liquid volume reduces the risk of leakage and subsequent aerosolisation. the system also does not require the preparation and autoclaving of media. due to the increased risk of defrosting the entire vial and potential loss of the stock culture isolate, current practice recommends storing two vials of stock material in two separate freezers, which already has cost and space implications.34 with the microbank™ bead storage, 25 individual isolate recoveries can be made from a single vial, allowing efficient storage, which is ideal for space-constrained environments. with routine subcultures, it has been reported that frequent subcultures may distort drug-susceptibility levels and patterns of strains,35 but with the microbank™ system, since the primary culture isolate is technically not sub-cultured, this risk is minimal. for ease of identification within biorepository settings, microbanktm beads are available in a variety of colours. although not investigated in this study, microbanktm beads provide an attractive alternative for transporting viable isolates and for applications such as proficiency testing. the current transport of infectious mtbc culture isolates for further confirmatory reflex testing, such as sequencing, requires strict safety adherence criteria and is costly. typically a secondary watertight container with absorbent material, together with outer packaging,36 is employed for shipping isolates and limitations are placed on the total allowable volume of liquid material in a single shipping container.37 the microbanktm bead platform will require the same safety considerations as conventional storage but offers a reduced risk of aerosolisation since the beads are not transported with any liquid. limitations this study has some limitations, the most important being the absence of long-term storage data and a small sample size. investigation into the use of microbanktm beads for transporting mtbc was not performed. conclusion the use of microbanktm beads for medium-term storage of mtbc isolates at −70 °c shows promise, with growth observed within ~5 weeks of resuscitation, even at 21 months sampling post storage, and demonstrates advantages in ease of use. acknowledgements the authors would like to thank vidya keshav for assistance with editing of the manuscript, caitlin dixon and jonathan tsoka for data analysis, lyndel singh and the clinical laboratory services tuberculosis laboratory staff for assistance with laboratory testing. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions a.d. carried out the experiment. l.e.s. and a.d. conceived the original idea. l.e.s. supervised the project. a.d. took the lead in writing the manuscript. a.d., l.e.s., p.d.s., e.m. and w.s.s. provided critical feedback and helped shape the research, analysis and manuscript. sources of support united kingdom medical research council, with funds received from the united kingdom government’s newton fund under the united kingdom/south africa newton fund grant 015newton tb (w.s.s. and l.e.s.) and the bill & melinda gates foundation grant opp1171455 (w.s.s., l.e.s. and a.d.). data availability the authors confirm that the data supporting the findings of this study are available within the article. disclaimer the views expressed in the submitted article are those of the authors and not an official position of the institution or funder. references molecular line probe assays for rapid screening of patients at risk of multi-drug resistant tuberculosis (mdr-tb) [homepage on the internet]. world health organization; 2008 [cited 2022 may 14]. available from: https://stoptb.org/assets/documents/about/cb/meetings/15/2.08-11%20rolling%20out%20diagnostics%20in%20the%20field/2.08-11.2%20line%20probe%20assays.pdf world health organization (who) endorses new rapid tuberculosis test [homepage on the internet]. world health organization; 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[cited 2022 nov 26]. available from: https://stoptb.org/wg/gli/assets/documents/srt/msh%20rwanda%20-%20isolate%20storage%20packaging%20and%20transportation.pdf international air transport assoication (iata). dangerous goods regulation. iata guidelines. 60th ed. 2019. montreal: iata. references about the author(s) tumelo satekge department of chemical pathology, faculty of health sciences, university of limpopo, polokwane, south africa adekunle okesina department of chemical pathology and immunology, faculty of health sciences, university of ilorin, illorin, nigeria john anetor department of chemical pathology, faculty of health sciences, university of ibadan, ibadan, nigeria rajiv erasmus department of chemical pathology, faculty of health sciences, stellenbosch university, cape town, south africa citation satekge t, okesina a, anetor j, erasmus r. the status of newborn screening in africa: situation analysis, future plans and call to action. afr j lab med. 2025;14(1), a2973. https://doi.org/10.4102/ajlm.v14i1.2973 editorial the status of newborn screening in africa: situation analysis, future plans and call to action tumelo satekge, adekunle okesina, john anetor, rajiv erasmus copyright: © 2025. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. newborn screening (nbs) is a pivotal public health service with the aim of early detection of potential health issues in neonates shortly after birth.1 specifically, biochemical nbs involves the systematic screening of newborns for a range of inherited or congenital disorders that are mostly asymptomatic at birth. timely diagnosis and immediate treatment of these genetic disorders are essential to avert irreversible health complications resulting in lifelong morbidity and/or mortality in the paediatric population.2 the world health organization (who) has recommended that a policy of universal nbs be adopted in all countries and communities which have established rehabilitation services.3 newborn screening has a direct linkage to the united nations sustainable development goal 3 which seeks to eliminate preventable deaths of newborns and children under 5 years of age by 2030.4 newborn screening was first conceived in 1961 when dr robert guthrie (the ‘father’ of nbs) introduced the newborn bloodspot screening for phenylketonuria with a microbiological inhibition assay now known as the guthrie test.5 by late 1963, 400 000 infants were screened for phenylketonuria in 29 states in the united states, detecting 39 cases at an estimated incidence 1:10 000. the success story of the nbs for phenylketonuria has led to the legislation of universal nbs being passed in 1963 in massachusetts, followed by many other developed countries, including the united kingdom. the legislative endorsement paved the way for the development of more assays to expand nbs and include more congenital disorders including inborn errors of metabolism (iems).6 despite the positive impact of nbs to achieve desirable outcomes in the screened infants, lowand middle-income-countries, particularly in africa, have not accomplished the implementation of universal nbs. newborn screening took a backseat for a long time in africa as a result of prioritisation of communicable diseases, particularly hiv/aids and tuberculosis. the prevention of mother to child transmission and immunisation programmes have successfully reduced the infectious disease burden on the continent. recent reports have revealed that there is now an increasing contribution of non-communicable diseases, especially congenital disorders, to infant morbidity and mortality.2 the existing infrastructure and resources of the impactful hiv/aids and immunisation programmes should provide the platform to launch and integrate nbs initiatives in african countries. a good example is dried blood spot sampling for hiv screening performed during the immunisation schedules, which can be expanded to screen for other congenital disorders.7 the challenges of implementing a sustainable nbs programme are well documented, and an important one of these is lack of strong political will to enable government’s endorsement and prioritisation. in most of these countries, the limited financial support from governments has led to the over-reliance on external funding sourced from non-government and humanitarian organisations. this threatens the long-term sustainability of nbs programmes, because funding from these external sources is unlikely to be a permanent guarantee.8,9 the consortium of newborn screening in africa initiative, in partnership with the american society of hematology and other organisations, have established sickle cell disease (scd)-targeted nbs programmes in sub-saharan african countries.10 the extremely high incidence of scd in sub-saharan africa and its devastating clinical outcomes, as evidenced by high morbidity and mortality in the region, has largely informed the intervention of the consortium of newborn screening in africa and other non-governmental organisations, and has also attracted the attention of the who. the who regional office for africa has highlighted the need for developing national policy on nbs of scd directed at primary prevention, early detection, advocacy, surveillance and raising public awareness through community education.11 at the last workshop held on 04 june 2025 in abuja, jointly organised by revvity and the newborn screening consortium nigeria, an nbs entity based in nigeria appealed to the consortium of newborn screening in africa to add congenital hypothyroidism to scd screening. the workshop was a side event to the 5th global congress on scd, which was held in abuja on 03 june 2025–06 june 2025. the success of the nbs is dependent on the programmes being fully incorporated into the routine health system processes, which may be hard to achieve. the nbs initiatives need to be structured to enable maximal follow-up of the neonates and families for confirmatory testing and initiation of treatment. digital applications were introduced in ghana to conveniently manage the scd nbs data on the healthcare workers’ mobile phones, which also facilitates follow-ups. it is worth noting that ghana and morocco have been successful in sustaining one of the longest running nbs programmes in africa.9,12 this success has been attributed to the considerable commitment and support from the local government in the building of local capacity. a notable challenge in the public health facilities is the early hospital discharges (a few hours after birth) as a result of limited maternity resources. the established reference cut-off values of biomarkers and metabolites to screen endocrinopathies and iems, respectively, are validated for nbs after 24 h of age. the expanded nbs in the setting of africa will rely on research studies to derive appropriate reference cut-off values at 6 h after birth, to enable nbs prior to discharges. the literature emerging from several african nations emphasises the point that iems are not exceedingly rare as often perceived. this underscores the magnitude of the problem and the need for africa to rise to the challenge.2,13 early treatment of some iems is often simple and extremely effective and is often by modifying the infant’s diet; for example, early withdrawal of galactose in galactosaemia, as well as the well-known low phenylalanine diet for phenylketonuria patients, can be lifesaving. however, because of a lack of awareness and paucity of sophisticated equipment such as the tandem mass spectrometry in most african states, nbs for iems is close to non-existent in africa, unlike in the developed nations. an article by anetor, orimadegun, and anetor14 is insightful in raising awareness of affordable and readily available techniques to pragmatically screen for iems in resource-limited settings. additionally, the dried blood spot should be encouraged, as it can be stored and revisited as the nbs programme expands. likewise, point-of-care testing in the forms of paper chromatography and haemoglobin isoform-specific antibodies in a lateral flow platform are cost-effective and user-friendly alternative methods of screening of scd in remote areas.15 as demonstrated by nbs pilots in south africa and other african countries, nbs of congenital hypothyroidism mandates prioritisation in africa to promote neurodevelopment and avert long-term intellectual disability cost effectively through nbs.16 community engagement with endorsement from traditional leaders has been demonstrated to yield success in some nbs initiatives by sensitively addressing the cultural beliefs that are often accompanied by stigmatisation resulting from misconceptions.9,10 the international federation of clinical chemistry and laboratory medicine task force on global nbs was tasked to develop nbs initiatives in lowand middle-income countries and to address the challenges that hinder the implementation of systematic nbs. one of the notable accomplishments of the task force is the successful development of a well-organised national nbs programme in the philippines, which serves as a model to many lowand middle-income countries and from which africa can gain insights.17 in the same vein, the african federation of clinical chemistry nbs committee was formed to raise awareness of nbs on the continent, and to encourage as many african countries as possible to develop a policy on nbs that is implementable and sustainable. in the spirit of commemorating the international nbs day, the committee organised a first-of-its-kind webinar on the dire need for nbs, representing all regions of africa. with panellists carefully selected to represent the whole of africa, the panel took the opportunity to call on all african countries to embrace universal nbs and to comply with the world health assembly resolutions 77 of 2024, and 78 of 2025, calling on all lowand middle-income countries to embrace and implement universal nbs. in conclusion, it is evident that there is an urgent need for african countries to heed the call to action, drawing inspirations from developed nations that are already screening for up to 60 conditions. we can also draw lessons from other developing countries, such as the philippines, to gain the encouragement required to implement a sustainable nbs programme. the implementation of universal nbs in africa will require partnerships and collaborative efforts from key stakeholders to overcome the challenges and for the governments to legislate the nbs policy to combat infant morbidity and mortality on the african continent. references anderson r, rothwell e, botkin jr. newborn screening: ethical, legal, and social implications. annu rev nurs res. 2011;29(1):113–132. https://doi.org/10.1891/0739-6686.29.113 malherbe hl, bonham j, carrihill m, et al. newborn screening in south africa: the past, present, and plans for the future. rare dis orphan drugs j. 2024;3:7. https://doi.org/10.20517/rdodj.2023.49 world health organization. newborn and infant hearing screening: current issues and guiding principles for action [homepage on the internet]. 2010 [cited 2023 sep 15]. available from: https://www.who.int/publications/i/item/9789241599496 conroy n, gilmore b. child mortality and the sustainable development goals: a challenge and an opportunity. ir j med sci. 2017;186:357–358. https://doi.org/10.1007/s11845-016-1463-1 therrell bl, padilla cd, borrajo gj, et al. current status of newborn bloodspot screening worldwide 2024: a comprehensive review of recent activities (2020–2023). int j neonatal screen. 2024;10(2):38. https://doi.org/10.3390/ijns10020038 levy hl. robert guthrie and the trials and tribulations of newborn screening. int j neonatal screen. 2021;7(1):5. https://doi.org/10.3390/ijns7010005 hsu l, nnodu oe, brown bj, et al. white paper: pathways to progress in newborn screening for sickle cell disease in sub-saharan africa. j trop dis public health. 2018;6(2):260. https://doi.org/10.4172/2329-891x.1000260 wesonga rm, awe oi. an assessment of traditional and genomic screening in newborns and their applicability for africa. inform med unlocked. 2022;32:101050. https://doi.org/10.1016/j.imu.2022.101050 archer nm, inusa b, makani j, et al. enablers and barriers to newborn screening for sickle cell disease in africa: results from a qualitative study involving programmes in six countries. bmj open. 2022;12(3):e057623. https://doi.org/10.1136/bmjopen-2021-057623 green ns, zapfel a, nnodu oe, et al. the consortium on newborn screening in africa for sickle cell disease: study rationale and methodology. blood adv. 2022;6(24):6187–6197. https://doi.org/10.1182/bloodadvances.2022007698 world health organization regional office for africa. sickle-cell disease: a strategy for the who african region [homepage on the internet]. 2011 [cited 2024 may 20]. available from: https://apps.who.int/iris/handle/10665/1682 ohene-frempong k, oduro j, tetteh h, nkrumah f. screening newborns for sickle cell disease in ghana. pediatrics. 2008;121(suppl_2):s120–s121. https://doi.org/10.1542/peds.2007-2022uuu saadallah aa, rashed ms. newborn screening: experiences in the middle east and north africa. j inherit metab dis. 2007;30(4):482–489. https://doi.org/10.1007/s10545-007-0660-5 anetor ji, orimadegun be, anetor go. a pragmatic approach to the diagnosis of inborn errors of metabolism in developing countries. afr j lab med. 2023;12(1): 1946. https://doi.org/10.4102/ajlm.v12i1.1946 twum s, fosu k, felder ra, sarpong ka. bridging the gaps in newborn screening programmes: challenges and opportunities to detect haemoglobinopathies in africa. afr j lab med. 2023;12(1): 2225. https://doi.org/10.4102/ajlm.v12i1.2225 arrigoni m, zwaveling-soonawala n, lafranchi sh, van trotsenburg ap, mooij cf. newborn screening for congenital hypothyroidism: worldwide coverage 50 years after its start. eur thyroid j. 2025;14(1):e240327. https://doi.org/10.1530/etj-24-0327 padilla cd, therrell jr bl, alcausin mmlb, et al. successful implementation of expanded newborn screening in the philippines using tandem mass spectrometry. int j neonatal screen. 2022;8(1):8. https://doi.org/10.3390/ijns8010008 abstract introduction methods results discussion acknowledgements references about the author(s) lindi-marie coetzee national health laboratory service, national priority programme, johannesburg, south africa department of molecular medicine and haematology, university of the witwatersrand, johannesburg, south africa naseem cassim national health laboratory service, national priority programme, johannesburg, south africa department of molecular medicine and haematology, university of the witwatersrand, johannesburg, south africa deborah k. glencross national health laboratory service, national priority programme, johannesburg, south africa department of molecular medicine and haematology, university of the witwatersrand, johannesburg, south africa citation coetzee l-m, cassim n, glencross dk. using laboratory data to categorise cd4 laboratory turn-around-time performance across a national programme. afr j lab med. 2018;7(1), a665. https://doi.org/10.4102/ajlm.v7i1.665 original research using laboratory data to categorise cd4 laboratory turn-around-time performance across a national programme lindi-marie coetzee, naseem cassim, deborah k. glencross received: 04 aug. 2017; accepted: 19 dec. 2017; published: 28 junw 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background and objective: the national health laboratory service provides cd4 testing through an integrated tiered service delivery model with a target laboratory turn-around time (tat) of 48 h. mean tat provides insight into national cd4 laboratory performance. however, it is not sensitive enough to identify inefficiencies of outlying laboratories or predict the percentage of samples meeting the tat target. the aim of this study was to describe the use of the median, 75th percentile and percentage within target of laboratory tat data to categorise laboratory performance. methods: retrospective cd4 laboratory data for 2015–2016 fiscal year were extracted from the corporate data warehouse. the laboratory tat distribution and percentage of samples within the 48 h target were assessed. a scatter plot was used to categorise laboratory performance into four quadrants using both the percentage within target and 75th percentile tat. the laboratory performance was labelled good, satisfactory or poor. results: tat data reported a positive skew with a mode of 13 h and a median of 17 h and 75th percentile of 25 h. overall, 93.2% of cd4 samples had a laboratory tat of less than 48 h. 48 out of 52 laboratories reported good tat performance, i.e. percentage within target > 85% and 75th percentile ≤ 48 h, with two categorised as satisfactory (one parameter met), and two as poor performing laboratories (failed both parameters). conclusion: this study demonstrated the feasibility of utilising laboratory data to categorise laboratory performance. using the quadrant approach for tat data, laboratories that need interventions can be highlighted for root cause analysis assessment. introduction the national health laboratory service (nhls) provides cd4 testing for staging hiv-positive persons and monitoring for people living with hiv on antiretroviral therapy (art) within the public health sector in south africa. cd4 testing is currently offered at 52 laboratories using beckman coulter equipment and the panleucogating method.1,2 while most cd4 testing takes place at district and regional hospitals, samples predominantly originate from primary healthcare facilities.3 an integrated tiered service delivery model for cd4 testing has been implemented within the nhls in south africa3 to facilitate widespread universal coverage of cd4 services across the cd4 laboratories. the model strives to provide cd4 testing technology that appropriately matches service delivery requirements,3 based on the daily test volumes in any given laboratory and the laboratory’s respective demarcated service coverage precinct.3 five testing tiers are defined for cd4 services ranging from tier-5 (high volume, centralised testing sites) to tier-1 (point-of-care sites used to extend laboratory services into hard-to-reach areas).3 most cd4 laboratories process more than 100 samples per day, with some high volume sites processing in excess of 500 samples per day.1,3 to ensure that all cd4 results are received in a timely fashion and expedite local antiretroviral (art) treatment according to guidelines at the clinical interface, cd4 laboratories are mandated to report all cd4 results within a total laboratory turn-around time (tat) of 48 h. the south african 2015 art guidelines4 prescribe a six-month cd4 testing schedule for individuals diagnosed with hiv infection but not eligible for art.4 despite adjustment of these guidelines in 2016 to incorporate a universal ‘test and treat’ strategy, where all hiv-positive persons are eligible for treatment irrespective of their cd4 counts, the cd4 guideline5 testing and treatment requirements have been retained. emphasis is put on baseline cd4 testing for assessing immune status and fast tracking individuals into care, especially < 200 cells/ul, or screening for opportunistic infection.6,7 adults on art thus undergo cd4 testing at 12 months and then annually when indicated clinically.4 the overall mandate for care is to ensure timely art initiation for all eligible persons within two weeks of cd4 testing and within seven days for fast tracking.4,5 mean tat has been used to report service efficiency and laboratory performance.8,9 the distribution of mean laboratory tat is typically a non-gaussian distribution;10 use of mean tat is thus not an accurate reflection of overall tat performance in laboratories. hawkins describes tat distributions as demonstrating a positive skew typically to the right, highlighting the need to assess tail size.10 therefore, to better understand tat performance, an analysis of median tat is a more useful measure to provide information about the performance norm, such as the median performance of laboratories. the analysis also provides information about the exception to the median, that is, the outliers (the tail size),11 allowing for a more balanced assessment of tat performance and removal of attention on a single indicator: mean or median tat. cd4 results need to be available to meet the sevento 14-day standard of care for art services. the aim of this study was to investigate a method to highlight tat performance to close the gap on service deficiencies. cd4 tat efficiencies were assessed using the median and 75th percentile tat, as well as the percentage of samples within the tat target of 48 h. methods ethical considerations ethics clearance for this work was obtained from the university of the witwatersrand (study approval number: m1706108). turn-around time data extraction retrospective data were extracted from the corporate data warehouse for the 2015–2016 fiscal year (01 april 2015 to 31 march 2016). fields included the episode number, registered date, reviewed date, tat in hours, flow cytometer serial number and the name of the cd4 laboratory. both the registered and reviewed dates are generated automatically by the laboratory information management system (lims) and used to calculate the tat. tat data were analysed using microsoft access 2013 and excel 2013 (albuquerque, new mexico, united states) and stata, version 12 (college station, texas, united states). data anomalies such as unreviewed cd4 results, samples reviewed before they had been registered due to incorrect date and time settings, for example were removed using stata. the cd4 laboratory tat was calculated from registration on the lims at the referring laboratory to results reviewed or authorised by a senior medical technologist at a cd4 testing laboratory (figure 1). the cd4 tat measure was used in this study as an indicator of the overall efficiency of the laboratory network to transport cd4 samples between laboratories and conduct testing. although it would be optimal to assess facility-to-facility tat, this was not possible as an adequate sample tracking system is not in place. figure 1: a logical flow of samples from the referring to the testing laboratory, indicating the individual components of total laboratory turn-around time (green). this consists of lab-to-lab (time from registration at referral laboratory to registration at testing laboratory); reg-to-tst (time to capture cd4 result from registration at testing laboratory) and tst-to-rvw (time from cd4 result to authorisation on lims). purple elements represent sample collection at the health facility and transport to the referring laboratory, as well as result delivery back to the health facility, which are not included in the laboratory turn-around time calculations. turn-around time distribution analysis graphpad prism version 6 software (la jolla, california, united states) was used to assess tat distribution on a histogram indicating the 25th, 50th, 75th, 95th and 99th percentiles across all cd4 samples tested within the 2015–2016 period. stata was used to generate descriptive statistics, including the mean, median, inter-quartile range and percentiles. national turn-around time range distribution analysis specimen-level laboratory tat data (hours) of all 52 cd4 testing laboratories were analysed and categorised as satisfactory (≤ 48 h) or unsatisfactory (> 48 h) using stata software. the annual performance plan target is for 85% of samples to meet the 48 h tat target.8 individual laboratory turn-around time performance categorisation a scatter plot was created using microsoft excel reporting the respective laboratory’s percentage of samples within the target tat (x-axis) and the 75th percentile tat (y-axis) to categorise individual laboratory performance into four quadrants. laboratories in quadrant 1 (q1) had both ≥ 85% of samples within the target tat and a 75th percentile tat ≤ 48 h (both parameters within target), and were categorised as having ‘good’ performance. laboratories in quadrant 2 (q2) were defined as laboratories with ≥ 85% of samples within the target tat but a 75th percentile tat is > 48 h (only the 75th percentile did not meet the target), and were categorised as having ‘satisfactory’ performance. laboratories in quadrant 3 (q3) had < 85% of samples within the target tat and a 75th percentile tat > 48 h (both parameters out of target), and were categorised as having ‘poor’ performance. finally, laboratories in quadrant 4 (q4) had < 85% of samples within the target tat (out of target) and a 75th percentile tat ≤ 48 h (within target), and were categorised as having ‘satisfactory’ performance. quadrants were colour coded and labelled. analysis of contributing factors for turn-around time performance all laboratories from q1 with good performance were selected for workflow analysis and tat component analysis. in addition, two laboratories with satisfactory performance (q4) and two laboratories categorised with poor performance (q3) were assessed for workflow and laboratory tat components. tat components included: (1) laboratory-to-laboratory: time from registration at the referral laboratory to registration at the cd4 testing laboratory; (2) registration-to-testing: time from registration at the testing laboratory to capture of the cd4 result on lims; and (3) testing-to-review: time from capture of cd4 result on lims to authorisation (review and confirmation of physical result printout from flow cytometer platform) by a senior medical technologist (figure 1). for the workflow analysis, the hour of the day (1–24) when samples were registered, tested and reviewed were analysed to assess hourly frequencies to determine the synchronisation of these laboratory activities. the data were analysed and reported using microsoft excel, graphpad prism and stata, with both the median and 75th percentile components of the tat reported. pre-analytical (facility to referring laboratory) and post-analytical (review to result delivery to a service provider) times were outside the scope of this study. results the extracted data contained 3 618 856 rows of tat data for the 2015–2016 fiscal year. exclusion of data anomalies removed 3.6% (131 629 rows) of data. data anomalies included invalid dates, for example ‘1800/01/01’ caused by an incorrect computer clock and tat calculation errors. the final tat analysis was conducted on a dataset of 3 487 227 cd4 samples tested across the nhls network between april 2015 and march 2016. for the period reported, the mean tat was 22 h, with a median of 17 h and an inter-quartile range of 11 h–25 h. national turn-around time distribution the 2015–2016 fiscal year cd4 tat data was skewed to the right with a mode of 13 h. figure 2 depicts a right-tailed distribution with a long tail resulting in a skewness of 4.6. a 25th percentile tat of 11 h and a median of 17 h were reported, both within the 48 h target. a 75th percentile tat of 25 h was reported. the tat target of 48 h was exceeded at the 95th (58 h) and 99th percentile (117 h), representing 6.8% of all samples tested. figure 2: histogram displaying turn-around time distribution across a national cd4 programme with the blue, red, orange, green and yellow lines representing the 25th, 50th (median), 75th, 95th and 99th percentiles, respectively, and the bold black line indicating the target turn-around time of 48 h. national turn-around time range distribution figure 3 reveals that 93.2% of cd4 samples had a tat ≤ 48 h, that is satisfactory tat. there were 236 555 (6.8%) samples with a tat > 48 h, meaning unsatisfactory tat. figure 3: a pie graph representing sample volumes and percentage of samples with satisfactory (≤ 48 h) and unsatisfactory (> 48 h) cd4 turn-around time performance in the 2015–2016 fiscal period overall. individual laboratory turn-around time performance analysis of individual laboratory tat performance (figure 4) showed that 48/52 (93.2%) testing laboratories were represented in q1, meeting both the 85% of samples within the target tat and a 75th percentile target ≤ 48 h. overall, 50 out of 52 (96.2%) laboratories achieved the 75th percentile target of a ≤ 48 h tat (q1 and q4). there were no representative laboratories in q2 (satisfactory performance as defined in the methodology), and q4 had two laboratories with satisfactory performance. there were two laboratories in q3 that showed poor performance, with a < 85% of samples within the target tat and a 75th percentile tat of > 48 h. these laboratories reported 63% and 65% of samples with the target tat, and their 75th percentile values were 67 h and 71 h, respectively. figure 4: scatter plot of cd4 laboratories reporting the percentage of samples within the tat target (x-axis) and the 75th percentile (y-axis) with tat performance categorised into four quadrants (n = 52). quadrant 1 (q1, green) represents good performance with both > 85% of samples within the tat target and the 75th percentile < 48 h. quadrant 2 (q2, blue) identifies satisfactory performance with > 85% of samples within the tat target, but with a long tail at the 75th percentile (> 48 h). quadrant 3 (q3, red) reports poor performance where both the percentage of samples within the tat target and the 75th percentile values are not meeting their targets. quadrant 4 (q4, purple) represents satisfactory laboratories with < 85% of samples within the tat target, but within the 75th percentile target of 48 h. laboratories represented in q1 as having good performance included 31 using the high-throughput fully-automated mpl/cellmek testing platform and 17 using the low-throughput automated aquios testing platform with varying test volumes. no correlation between test volumes and tat was indicated in this study (data not shown, p > 0.1). platform and test volumes for q3 and q4 also included both aquios and mpl systems with low to high test volumes. analysis of contributing factors for turn-around time performance workflow analysis was done for data of all laboratories by quadrant. among laboratories with good performance (q1; n = 48), sample testing and review was done throughout the day, with a decline in testing and reviewing from 16:00 to midnight (figure 5a). registration peaked between 12:00 and 16:00, with another peak at 20:00–22:00. testing and review followed each other closely during the 24 h testing period, meaning that there was no significant time lapse between testing and review. figure 5: (a) represents the workflow over a 24 h period of all good performing laboratories (n = 48) versus, (b) a satisfactory performing laboratory (as identified in figure 4, q4), (c) represents two poor performing laboratories, (d) indicates the breakdown of tat into registration time in green, testing time in red and review time in blue. among laboratories with poor performance (q3; n = 2), registration peaked around 15:00–16:00 and again at 20:00–22:00, with testing occurring between 07:00 and 18:00 (daytime) (figure 5b). a significant lapse was noted between testing and review, with a peak in review at 14:00–18:00. the two laboratories with satisfactory performance (q4) showed testing throughout the 24 h, with review following testing during the day, peaking at 06:00–10:00, with very little reviewing taking place between midnight and 06:00 (figure 5c). when comparing tat components for the three quadrants, registration took the longest for q4 laboratories (9 h). testing times were about equal for q4 and q3 (25 h–27 h), but were significantly longer than testing time for q1 (17 h) (figure 5d). review times were also significantly longer for both q3 (5 h) and q4 (8 h) compared with q1 (1.3 h on average). discussion turn-around time is one of the most perceptible aspects noted by clinicians with respect to laboratory services10,12 and is frequently used to gauge laboratory service performance.9 it can also be used by the laboratory service provider to appraise efficiencies across a network of testing facilities. laboratory test tat depends on the nature of test, the clinical significance and time to clinical intervention.10,12 for example for cd4 services, determining an acceptable local tat target is based on the standard of care for enrolling patients onto art. in south africa, this equates to art initiation within 14 days of cd4 testing or seven days where a cd4 is < 200 cells/µl.4,5 using these guidelines, national and individual laboratory cd4 tat performance was assessed against the target of 48 h. currently within the nhls, the average or mean tat of non-normally distributed data is routinely reported to assess the performance of cd4 laboratories. this study, however, indicated that the median tat is a better measure of central tendency, given the right tailed distribution. in addition to using the median, two more measures, namely the 75th percentile and percentage of samples within the target tat, were introduced to assess tail size and the overall proportion of samples meeting the 48 h tat target. the intersection of these three variables provide a more accurate assessment of cd4 tat performance, as laboratories can report a median and 75th percentile within tat, but fail to meet the 85% threshold requirement for samples within the target tat. for the study period overall, 93.2% of samples were reported within the nhls 48 h tat target. the median or 50th percentile cd4 tat of 17 h reported here further revealed that a large number of samples were reported in less than a third of the time of the tat target. however, a small proportion of samples failed to meet the target tat. the national cd4 tat distribution demonstrated a long tail to the right. this may be due in part to local tat challenges causing delays, as reflected at the 95th tat percentile of 58 h. even at the 99th percentile, cd4 results were released within 5 days (117 h); that should be well within the local national department of health guidelines for art treatment initiation standard of care.4,5 time delays from facility to referral laboratory and review to result in hand were not included, as these times are not currently captured. plotting the percentage of samples within the target tat versus the 75th percentile as a scatter plot provides important visual insights about how laboratories perform in relation to their peers and against set targets. this information can potentially be incorporated into a dashboard with stop lighting for individual laboratories, or regions for management interventions to understand at a glance, and to hone in on sites at risk for bottlenecks in service delivery. good overall laboratory tat performance was noted for 48 out of 52 laboratories, only two laboratories had satisfactory performance and only two had poor performance (both percentage of samples within target and 75th percentile ≤ 48 h not met). it is to be expected that centralised testing will result in longer pre-analytical laboratory-to-laboratory tat as samples are brought into the testing facility from multiple distant referral sites (figure 1). differences in performance (quadrant placements) were, however, not related to the location of laboratories, testing platform, test volumes, staff component or hours worked (i.e. 24 h service vs 8 or 12 h). tat is affected by a multitude of factors or combinations thereof, that may or may not be related to test volumes. these may include synchronisation of registration, testing and review activities, pre-analytical delays, frequency of courier logistics, instrument capacity and downtime, staffing availability and competency, lims bandwidth and optimal workflow. the deficiency of the current lims system in use in the nhls is that it does not offer detailed end-to-end tracking capability to identify all components of pre-analytical and analytical tat. auto review, using delta check rules, to automatically release cd4 results is currently not in use and may dramatically reduce testing-to-review tat. total laboratory automation (for larger core laboratories) and auto verification showed a marked decrease in tat for various laboratory tests.13,14 in the absence of a fully optimised lims system, on-site audits are indicated for laboratories performing outside the acceptable target, to at least ensure that the laboratory testing or reviewing phase is optimised. audits as a tool for continued improvement of laboratory tat was recently reported for chemistry results in a typical south african teaching hospital, confirming its application in tat root cause analysis of outlying performers.15 in addition, a system for long-term monitoring of performance should also be in place to ensure continued acceptable performance by all testing laboratories. the workflow analysis of laboratories depicted in figure 5 showed the impact of delayed testing and reviewing on meeting tat targets. the main difference between laboratories was workflow synchronisation as evidenced by the time lapse between sample registration and testing, and testing and reviewing. laboratories with poor performance were assisted through on-site visits to assist with workflow. root cause analysis identified that the unavailability of qualified staff may lead to bottlenecks in testing or authorisation. the desktop analysis of tat described in this article shows that it is possible to categorise tat performance and identify the in-laboratory component contributing to delays in tat. limitations this study used predominantly data from the corporate data warehouse to assess laboratory tat data. the data presented here cannot differentiate all the various components of pre-analytical, analytical and post-analytical delays for cd4 testing tat. time delays from facility to referral laboratory and review to result in hand are not currently monitored, so in the absence of a local sample tracking system, it is not possible to assess end-to-end cd4 service delivery. recommendations the following recommendations are proposed to improve tat monitoring: develop a weekly tat monitoring report incorporating the aspects described above namely, scatter plot, component analysis and, where required, workflow analysis. develop an operational corporate data warehouse tat dashboard to facilitate real-time access to tat data. conclusion this study demonstrated the feasibility of establishing laboratory tat performance using secondary data from the corporate data warehouse. additionally, the shortcomings of using only a mean or median to assess tat performance are highlighted. once a monitoring system has been developed to provide real-time tail size data, the national cd4 programme would be able to plan focused interventions to proactively resolve poor service delivery levels. acknowledgements the authors thank cd4 trainers sherry drury, sithembile mojalefa, and neo mokone who assisted with site visits, cd4 laboratory managers for their assistance and support and mr manfred tepper from the nhls corporate data warehouse. the authors also thank dr denise lawrie who shared her knowledge and expertise in laboratory quality management. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support d.k.g. thanks the south african national research foundation for funding for rated researchers. author contributions d.k.g. supervised the study by providing leadership and oversight. d.k.g. was also the project leader. n.c and l.-m.c. designed the study, developed the methodology and conducted the research. l.-m.c. provided overall validation of data analysis as data curation. n.c. and l.-m.c. assisted with data interpretation and formal analysis of data. all authors contributed to writing the initial draft. d.k.g. was responsible for reviewing the article and overall project administration. references glencross dk, janossy g, coetzee lm, et al. large-scale affordable panleucogated cd4+ testing with proactive internal and external quality assessment: in support of the south african national comprehensive care, treatment and management programme for hiv and aids. cytometry b clin cytom. 2008;74(suppl 1):s40–s51. https://doi.org/10.1002/cyto.b.20384 glencross dk, 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http://intranet.nhls.ac.za/assets/files/policy/nhls_annual_report_2015.pdf goswami b, singh b, chawla r, et al. turn around time (tat) as a benchmark of laboratory performance. indian j clin biochem. 2010;25(4):376–379. https://doi.org/10.1007/s12291-010-0056-4 hawkins rc. laboratory turnaround time. clin biochem rev. 2007;28(4):179–194. glencross dk, coetzee lm, cassim n, et al, editors. development of a ‘full coverage’ tiered plan to ensure optimized laboratory and point-of-care cd4 service delivery. first international conference of the african society for laboratory medicine (aslm); 1–7 december, 2012, cape town, south africa. pati hp, singh g. turnaround time (tat): difference in concept for laboratory and clinician. indian j hematol blood transfus. 2014;30(2):81–84. https://doi.org/10.1007/s12288-012-0214-3 lou ah, elnenaei mo, sadek i, et al. multiple preand post-analytical lean approaches to the improvement of the laboratory turnaround time in a large core laboratory. clin biochem. 2017;50(15):864–889. https://doi.org/10.1016/j.clinbiochem.2017.04.019 archetti c, montanelli a, finazzi d, et al. clinical laboratory automation: a case study. j public health res. 2017;6(1):881. https://doi.org/10.4081/jphr.2017.881 imoh lc, mutale m, parker ct, erasmus rt, zemlin ae. laboratory-based clinical audit as a tool for continual improvement: an example from csf chemistry turnaround time audit in a south-african teaching hospital. biochem med. 2016;26(2):194–201. https://doi.org/10.11613/bm.2016.021 introduction medical laboratory waste laboratory waste management status quo in lowand middle-income countries causes of poor waste management potential health and environmental impact of improper waste management tackling poor waste management conclusion references about the author(s) pasipanodya i. machingura ruredzo department of laboratory diagnostic and investigative sciences, faculty of medicine and health sciences, university of zimbabwe, harare, zimbabwe bettina chale-matsau department of chemical pathology, faculty of health sciences, university of pretoria, pretoria, south africa national health laboratory service, pretoria, south africa citation machingura ruredzo pi, chale-matsau b. mainstreaming of biomedical waste management: best practices for clinical laboratories in africa. afr j lab med. 2025;14(1), a2881. https://doi.org/10.4102/ajlm.v14i1.2881 editorial mainstreaming of biomedical waste management: best practices for clinical laboratories in africa pasipanodya i. machingura ruredzo, bettina chale-matsau copyright: © 2025. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction biomedical waste comprises liquid and solid material generated during provision of healthcare services encompassing diagnostics and patient management. as these toxic, radioactive, or infectious products are potentially hazardous to human and environmental health, proper management of biomedical waste is of concern.1 modes of exposure to biomedical risks include cuts or pricks, intimate contact with skin or mucous membranes, inhalation or ingestion of hazardous substances.2 the world health organization reports that an estimated 15% of waste generated by healthcare activities is hazardous material which may be infectious, toxic, carcinogenic, flammable, corrosive, explosive, or radioactive.3 the open burning and low temperature incineration of such waste may cause emission of dioxins, furans, and particulate matter.4,5 as patient care is mostly associated with submission of samples (e.g. fluid or biopsy specimens) to a pathology laboratory to aid in diagnosis, clinical laboratories are among the major sources of healthcare waste. in lowand middle-income countries (lmics), healthcare waste is often not separated into hazardous or non-hazardous wastes, causing the quantity of hazardous waste to be much higher.3,6 the objective of this editorial is to highlight the health and environmental risks associated with biomedical waste management in clinical laboratories and to point out reasonable measures to best address these challenges. medical laboratory waste medical laboratory wastes carry a higher potential for infection and cause of injury by accidental needle sticks. it is reported in some countries that hazardous medical waste is handled and disposed together with household waste, which causes a health risk to municipal works, the public, and the environment.7 ideally, the management of the laboratory waste that includes biological agents should include a validated decontamination determined by the risk assessment process as stipulated in international and local waste disposal guidelines. the decontamination process can be conducted by a combination of chemical disinfectants and autoclaving, and incineration can be used to supplement in some cases. thus, it is essential that laboratory personnel have basic knowledge on cleaning, disinfection, and sterilisation.3 laboratory waste management status quo in lowand middle-income countries healthcare services in lmics continue to grow, and increased availability and use of diagnostic services is leading to growing rates of biomedical waste generation. large quantities of biomedical waste are generated in medical laboratories. most of these originate from wards, clinics, and surgical theatres as fluid (e.g. blood, urine, saliva, cerebrospinal fluid) and tissue obtained from patients during processes associated with diagnosis and treatment monitoring.8,9 waste in the laboratory is handled by medical laboratory professionals, who in some cases have limited training in waste management. there are items that are not harmful, such as packaging, but that are often collected and stored with hazardous material. because of a lack of training in waste management, medical laboratory professionals have limited knowledge of proper waste segregation. most clinical laboratories entrust the responsibility of waste removal and disposal to a registered service provider, with the expectation that proper measures are followed. following pick-up, biomedical waste is taken to disposal sites and manually sorted prior being subjected to respective mechanisms that include decontamination prior to being taken for recycling, landfills, or incineration, the products of which are then disposed in landfills. because of resource restraints in lmics, waste handlers may not have adequate protective clothing or training in waste management. in many instances, landfills are shallow and incineration is conducted in open facilities at suboptimal temperatures,10 further worsening the hazardous nature of biomedical waste. causes of poor waste management poor waste management is caused by limited legal frameworks, lack of awareness of the health hazards of the waste, inadequate training in appropriate waste management, lack of proper waste management and disposal systems, inadequate financial and human resources, and lack of prioritisation of waste management.11 furthermore, many countries lack appropriate regulations, or do not monitor and enforce them.3 the prevailing unpleasant economic climate in most lmics further compromises biomedical waste management, thus pushing the issue further down on the agenda of policymakers. it is important even when biomedical waste management policies are available to renew them periodically, in order to incorporate new research and system development points.2 the world health organization provides material that can be utilised to develop both standard and more specific procedures for dealing with pathogenic agents in the laboratory.3,12 potential health and environmental impact of improper waste management healthcare activities generate different kinds of waste from non-hazardous to hazardous. while harmful waste constitutes a small fraction of total waste, pooling all biomedical waste together increases the amount that must be disposed of. hospitals and laboratories in lmics lack appropriate waste segregation, storage, collection, and disposal practices, increasing the health risk to health professionals, waste handlers, and the rest of the community.13,14 potential harms include exposure to infectious pathogen-bearing waste material, toxins, and carcinogenic chemicals that may be inhaled, absorbed through skin, or introduced to the body from needle sticks.13 certain medical waste such as noxious chemicals and persistent spores from pathogens may remain airborne for an extended period of time. open burning and incinerators produce outdoor air pollution. products of partially incinerated waste disposed of in low-lying fields can easily seep into water systems. biological, chemical, and radioactive materials all have the potential to contaminate water bodies.4 bacteria in the waste has the potential to infiltrate ground water and surface water. liquid laboratory waste from cleaning work benches and glassware and effluent from analysers are incorporated into municipal wastewater. municipal wastewater is partially treated and released into rivers. this inappropriate handling of biomedical waste can have long-lasting effects on humans, animals, and plants, affecting the food chain and future generations. regulated, sustainable waste management practices can reduce the harmful effects of biomedical wastes.7 tackling poor waste management pre-service training and refresher courses for laboratory personnel must encompass this important aspect of biosafety. studies have shown that 85% of laboratory waste is non-harmful and is generally combined with the 15% of hazardous waste that is harmful, thus materially increasing the load of potentially harmful waste.3 proper segregation of waste and appropriate disposal is required to safeguard human health and minimise environmental impact.8 laboratory personnel require skills training in inventory management, including regular checking of expiration dates and avoiding overstocking. training institutions must continually update their training on safety to ensure appropriate pre-service training on laboratory waste management and support laboratories though continuing professional development on biosafety. laboratory personnel must take due consideration of the safety of themselves and the population at large, with governments taking leadership to ensure there are appropriate regulations, monitoring, and enforcement to ensure compliance.2 laboratory personnel have an ethical obligation to ensure the safety of the laboratory and community at large. laboratory waste audits can be conducted to guide planning and decision-making for laboratories. such audits can provide data on the amount, type, and laboratory areas where waste is generated to provide a baseline for waste management initiatives.11 laboratory management personnel are expected to adhere to and encourage implementation of national and international guidelines for biomedical waste management, and to ensure that laboratory personnel are familiar with these regulatory frameworks.15 conclusion research in waste management is also essential to help provide evidence for policy review and strengthening. clinical laboratories may benefit from conducting audits that will provide perspective on the quantities of each category of waste generated, so as to streamline management strategies. in order to realise improvements in biomedical waste management, laboratories should consider inclusion of training and regular refresher courses for medical laboratory professionals that can be incentivised as part of continuous professional development. this is a call for laboratory professionals to keep waste management on the radar as they practise. references balekouzou a, pamatika cm, nambei sw, et al. management of biomedical waste in two medical laboratories in bangui, central african republic. pan afr med j. 2016;23(1):237. https://doi.org/10.11604/pamj.2016.23.237.8786 singh h, kamal yt, mishra ak, et al. harnessing the foundation of biomedical waste management for fostering public health: strategies and policies for a clean and safer environment. discov appl sci. 2024;6(3):89. https://doi.org/10.1007/s42452-024-05735-2 world health organization. laboratory biosafety manual. geneva: world health organization; 2004. liu l, gong y, miao y, et al. new trends in pollution prevention and control technology for healthcare and medical waste disposal in china. processes. 2023;12(1):7. https://doi.org/10.3390/pr12010007 zhao l, zhang f-s, chen m, liu z, wu dbj. typical pollutants in bottom ashes from a typical medical waste incinerator. j hazard mater. 2010;173(1–3):181–185. https://doi.org/10.1016/j.jhazmat.2009.08.066 yazie td, tebeje mg, chufa ka. healthcare waste management current status and potential challenges in ethiopia: a systematic review. bmc res notes. 2019;12:1–7. https://doi.org/10.1186/s13104-019-4316-y agbere s, melila m, dorkenoo a, et al. state of the art of the management of medical and biological laboratory solid wastes in togo. heliyon. 2021;7(2):e06197. https://doi.org/10.1016/j.heliyon.2021.e06197 chitnis v, vaidya k, chitnis d. biomedical waste in laboratory medicine: audit and management. indian j med microbiol. 2005;23(1):6–13. https://doi.org/10.1016/s0255-0857(21)02704-3 pandey a, ahuja s, madan m, asthana ak. bio-medical waste managment in a tertiary care hospital: an overview. j clin diagn res. 2016;10(11):dc01. https://doi.org/10.7860/jcdr/2016/22595.8822 singh n, ogunseitan oa, tang y. medical waste: current challenges and future opportunities for sustainable management. crit rev environ sci technol. 2022;52(11):2000–2022. https://doi.org/10.1080/10643389.2021.1885325 opperman cj, singh s, barton f. appropriate disposal of waste in the laboratory: neglected but not forgotten. afr j lab med. 2022;11(1):1–2. https://doi.org/10.4102/ajlm.v11i1.1786 rowe dr, abdel-magid im. handbook of wastewater reclamation and reuse. london: crc press; 2020. jansen k, kocks d, roberts h. hazardous waste management solutions in south africa – still a challenge. j soc occup med. 2020;24(4):1–5. chisholm jm, zamani r, negm am, et al. sustainable waste management of medical waste in african developing countries: a narrative review. waste manag res. 2021;39(9):1149–1163. https://doi.org/10.1177/0734242x211029175 singhal l, tuli ak, gautam v. biomedical waste management guidelines 2016: what’s done and what needs to be done. indian j med microbiol. 2017;35(2):194–198. https://doi.org/10.4103/ijmm.ijmm_17_105 abstract introduction viable but non-culturable state: what exactly does this mean? bacteria known to enter the viable but non-culturable state cell changes in the viable but non-culturable state cellular repair mechanisms detection of pathogens in the viable but non-culturable state escherichia coli in the viable but non-culturable state induction into and in vitro resuscitation of e. coli in the viable but non-culturable state potential virulence of e. coli in the viable but non-culturable state acknowledgements references about the author(s) jennifer a. pienaar faculty of health sciences, department of biomedical technology, university of johannesburg, johannesburg, south africa water and health research centre, faculty of health sciences, university of johannesburg, johannesburg, south africa atheesha singh water and health research centre, faculty of health sciences, university of johannesburg, johannesburg, south africa tobias g. barnard water and health research centre, faculty of health sciences, university of johannesburg, johannesburg, south africa citation pienaar ja, singh a, barnard tg. the viable but non-culturable state in pathogenic escherichia coli: a general review. afr j lab med. 2016;5(1), a368. http://dx.doi.org/10.4102/ajlm.v5i1.368 review article the viable but non-culturable state in pathogenic escherichia coli: a general review jennifer a. pienaar, atheesha singh, tobias g. barnard received: 29 sept. 2015; accepted: 14 jan. 2016; published: 04 may 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the persistence and pathogenicity of pathogenic bacteria are dependent on the ability of the species to survive in adverse conditions. during the infectious process, the organism may need to pass through certain hostile anatomical sites, such as the stomach. under various environmental stresses, many bacteria enter into the viable but non-culturable (vbnc) state, where they are ‘alive’ or metabolically active, but will not grow on conventional media. escherichia coli bacteria encounter several diverse stress factors during their growth, survival and infection and thus may enter into the vbnc state. objectives: this review discusses various general aspects of the vbnc state, the mechanisms and possible public health impact of indicator and pathogenic e. coli entering into the vbnc state. method: a literature review was conducted to ascertain the possible impact of e. coli entering into the vbnc state. results: escherichia coli enter into the vbnc state by means of several induction mechanisms. various authors have found that e. coli can be resuscitated post-vbnc. certain strains of pathogenic e. coli are still able to produce toxins in the vbnc state, whilst others are avirulent during the vbnc state but are able to regain virulence after resuscitation. conclusion: pathogenic and indicator e. coli entering into the vbnc state could have an adverse effect on public health if conventional detection methods are used, where the number of viable cells could be underestimated and the vbnc cells still produce toxins or could, at any time, be resuscitated and become virulent again. introduction the survival of a microbial species largely depends upon its ability to subsist in hostile environments.1,2 when environmental conditions become unfavourable, bacteria must be able to withstand stress and assume strategies which permit them to endure until suitable conditions for growth are re-established.1 certain bacterial genera achieve this by developing resistant structures, for example, endospores, whereas many others enter a state of very low metabolic activity, usually referred to as the viable but non-culturable (vbnc) state.1,2 this suggests that this state is a unique adaptation strategy used by many bacterial species for long-term survival when exposed to hostile environmental stresses.3,4 vbnc cells are characterised by culturability loss on growth agar, which hinders their detection by conventional culture-based techniques. this leads to an underestimation of total viable cells in clinical or environmental specimens and could therefore pose a danger to public health.3 escherichia coli (e. coli) is a genetically diverse species that contains both commensal and pathogenic variants. strains of e. coli and related gram-negative coliform bacteria predominate amongst the aerobic commensal flora in the gut of humans and animals.5,6 e. coli is a non-sporulating, facultative anaerobe7 that typically colonises the infant gastrointestinal tract within hours of life, from which time on the host and e. coli derive mutual benefit.8 in the digestive tract, commensal strains are situated in the large intestine, particularly in the caecum and colon.7 there are various pathogenic strains of e. coli that possess a variety of disease-causing mechanisms such as diverse toxin, secretion, adhesion and siderophore systems, as well as many others.6 the benign e. coli strains have the potential to acquire genetic elements encoding for virulence factors and could themselves become pathogenic.6 both non-pathogenic and pathogenic species of e. coli have been shown to survive sub-lethal environmental stress conditions by entering into the vbnc state.9,10,11,12,13 virulent bacteria, such as pathogenic e. coli that are able to enter into a vbnc state, are a major public health concern, because they are able to return to the infectious state after transport in animal hosts.14 reports indicate that many species of pathogenic bacteria survive treatment and persist in pasteurised milk, processed food and drinking water, as well as in the environment.14 water is routinely tested for indicator bacteria (such as e. coli and enterococcus faecalis) and if they are not detected at a concentration below the guidelines, the water is considered to be safe for consumption. thus, in circumstances where there is a chance of vbnc pathogens, additional molecular tests would be required in order to reduce potential outbreaks of infectious disease.15 many studies conducted over the past 30 years have shown that the vbnc state is a significant strategy for bacterial survival.3 the presence of cells in the vbnc state in water (and food) have various significant consequences, an example being that e. coli cannot be used as an indicator of faecal contamination when the cells are in the vbnc state.16 the purpose of this review was to highlight the possible impact on public health of e. coli entering into the vbnc state and may be of interest to environmental scientists and medical clinicians. we systematically searched scientific literature databases (science direct, springer, google scholar, wiley, directory of open access journals, plos, ncbi) using keyword combinations of terms such as ‘viable but non-culturable’, ‘escherichia coli’, ‘virulence’, ‘resuscitation’, ‘induction’ and ‘pathogens’. articles older than 10 years were avoided, when possible, in order to ensure that the latest information on the topic was obtained. all studies involving the vbnc state were included, particularly those relating to e. coli. viable but non-culturable state: what exactly does this mean? the vbnc state is defined as a state of dormancy that certain species of bacteria can enter into when confronted with adverse environmental conditions15 such as extreme temperature changes,2,13,15,17 starvation,13,15,17 high osmotic pressure,2,13,15,17 exposure to chlorine,13,15 sharp changes in ph,15 oxygen availability, heavy metals, or exposure to white light.15,17 these conditions could be of a fatal nature if the organism did not enter into a vbnc state.17 when bacterial cells are able to grow and form colonies on conventional culture media, they are said to be ‘culturable’, whereas they are ‘viable’ if they are metabolically or physiologically active.15 bacteria in the vbnc state cannot be cultured on routine media; however, they retain metabolic activity, respiration, membrane integrity and slow gene transcription.17,18 in spite of their low metabolic rate in this state, after specific resuscitation protocols they may again become culturable.19 in an archetypal vbnc response, when the vbnc cells are exposed to environmental stresses a regular decline in colony-forming units is seen. however, total cell counts remain fairly constant. the viable count is very important in determining the vbnc state, as this will show whether the unculturable colonies are dormant but alive, or dead. there are several methods that can demonstrate viability, but all show some aspect of intact bacterial membranes and/or cellular metabolic activity.17 bacteria known to enter the viable but non-culturable state to date, the list of bacteria that have been shown to enter the vbnc state constantly increases; some of the notable human pathogens are pathogenic e. coli,13,15,17,19,20 mycobacterium tuberculosis, campylobacter spp., klebsiella pneumoniae, helicobacter pylori, listeria monocytogenes,15,17,19,20 pseudomonas aeruginosa, several salmonella and shigella spp.,13,15,17,19,20 vibrio cholerae and vibrio parahaemolyticus.15,17,19,20 universal indicators of faecal contamination, namely, commensal e. coli and e. faecalis, also enter into the vbnc state.15 cell changes in the viable but non-culturable state bacteria in the vbnc state generally exhibit dwarfing (decrease in cell size),17,19,21 acquire a coccal morphology,21 show pronounced metabolic changes such as decrease in respiration rates and nutrient transport17,19 and macromolecular synthesis.17,19,21 plasmids are retained and adenosine triphosphate levels and membrane potential remain high.19 there can be variation in nucleic acid content (particularly rna) when bacteria enter the vbnc state.21 day and oliver22 found that there were significant changes in membrane fatty acid composition of vibrio vulnificus after incubation at 5 °c in seawater. the results indicated that changes in the fatty acid composition occurred prior to entry into the vbnc state, suggesting that the capability to maintain fluidity of the membrane may be an aspect in this physiological response. cell death occurred in bacteria where there was inhibition of fatty acid synthesis, indicating that fatty acid synthesis is essential for cells entering the vbnc state. cellular repair mechanisms microbial recovery is characterised by the ability to revert to a functionally ‘normal’ state after enduring damage to essential components. this may be achieved by a resuscitation period in a favourable environment.23 after exposure to potentially harmful environmental factors, microorganisms may be classified as either ‘dead’ (irreversibly or lethally injured) or ‘alive’. live cells can be further classified as ‘uninjured’ (normal) or ‘injured’ (stressed, reversibly or sub-lethally injured).23 prolonged exposure of bacterial cells to a principal stress may ultimately lead to irreversible cell death. however, if the injured cells are removed from the environment that is causing the stress, cell recovery may then occur. the ability of the microorganism to once again be culturable on selective media is interpreted as a recovery from the initial injury and points to a repair of the particular metabolic and synthetic functions that were damaged whilst under the particular stress.24 depending on the type and degree of stress, specific biochemical events will be required for repair to occur. the synthesis of dna, rna, adenosine triphosphate and proteins, as well as the reorganisation of existing macromolecules, are some of the metabolic processes that occur during repair. bacteria also amass intracellular compounds that protect membranes and macromolecules from injury.25 these repair mechanisms are achieved via signal transduction systems that sense environmental stresses and regulate synchronised expression of various genes involved in mechanisms of cellular defence.26 in e. coli, trehalose has been recognised as the primary protective osmolyte; trehalose biosynthesis is induced by osmotic shock, extreme heat and cold, desiccation and entry into stationary phase.25 trehalose synthesis is under rna polymerase, sigma s (rpos) regulation. as with other polyols, trehalose protects proteins from denaturation by heat and may also protect against pressure.27 in enteric bacteria such as e. coli, rpos is the main regulator of the general stress response. various stress factors affect rpos transcription in different ways with or without the assistance of other cellular regulatory proteins. under normal circumstances, rpos levels are relatively low as rpos messenger rna forms a stable secondary structure, which results in reduced transcription; clpxp protease also repeatedly degrades the protein.26 detection of pathogens in the viable but non-culturable state because bacteria in the vbnc state are no longer culturable, alternate methods must be employed to demonstrate whether or not these cells are alive.19 generally, these assess viability by testing any of the following criteria: presence of an intact cellular membrane, demonstration of metabolic activity,4,19,20 expression of specific genes (e.g. 16s ribosomal rna) and differential expression of specific proteins (e.g. beta-d-glucuronidase in e. coli).4 16s ribosomal rna has been used in several vbnc studies, because these cells uphold levels of ribosomal rna as high as normal cells and retain reductase activity (essential for living cells).4 bacteriophages have also been revealed to be useful in detection of vbnc cells, as phage replication properties of live cells can be shown.28 table 1 shows some of the methods employed to detect vbnc bacteria. table 1: methods of detection of viable but non-culturable bacteria. escherichia coli in the viable but non-culturable state because of both biotic and abiotic ecological factors, aquatic ecosystems such as rivers and oceans represent a hostile environment for allochthonous bacteria such as e. coli;31,32 thus, these bacteria have developed the vbnc state.32 the survival and growth of e. coli in foods depends on the interaction between extrinsic or environmental factors (such as ph, temperature and water activity) and intrinsic factors (i.e. those related to food).26 the non-culturability linked with the vbnc state poses a potential public health problem, because the methods that are generally employed for detection and counting of e. coli depend on culturing.32 under altered environmental conditions, porins are vital for the survival of bacteria.33 the major outer membrane proteins (omp) in e. coli are ompf and ompc, whose gene expression is regulated by envz (an osmolarity sensor protein) and ompr, which responds primarily to changes in osmolarity.33 figure 1 illustrates the envz/ompr osmoregulatory system in e. coli.34 a study by darcan et al.33 found that wild-type and porin mutant (ompc or ompf deficient) e. coli populations entered vbnc under stress conditions of ph, osmolarity and starvation, whereas envz mutants (deficient of envz) were not able to enter a vbnc state. the researchers concluded that because the envz mutant population could not sense the environmental changes, they did not enter into a vbnc state when exposed to the tested adverse conditions. the analysis of the outer membrane subproteomes in vbnc e. coli was studied by muela et al.,32 who discovered sets of proteins that were modulated during vbnc induction. antigen 43 beta-subunit, outer membrane protein tolc and ompt were modulated by starvation, ompa and nlpa (lipoprotein 28) by photo-oxidation, and fiu (catecholate sidephore receptor fiu), fepa (ferrienterobactin receptor), antigen 43 alpha-subunit and adenosine triphosphatase by seawater exposure. only four identified proteins (elongation factor-tu, δ-3 phosphoglycerate dehydrogenase, threonine synthase and enolase) showed an alteration in their expression regardless of the stress factor employed. ompa maintains the structural integrity of the outer membrane and is an essential protein in e. coli; decreases in its expression have been associated with culturability loss in aquatic environments. elongation factor-tu has an essential role in biosynthesis and becomes membrane bound when e. coli is starved of certain nutrients.32 another study10 reported that enterohaemorrhagic e. coli (ehec) o157:h7 displayed markedly increased levels of ompw and a prevalence of elongation factor-tu protein after induction into the vbnc state using hydrogen peroxide. in 2008, a study35 showed that after passage in food through a mouse, the ompw stress response of ehec o157:h7 increased (increase in ompw expression) when induced into the vbnc state. the authors suggested that the different stress response of ompw was introduced by means of in vivo passage genetic alteration.35 figure 1: diagrammatical representation of the envz/ompr osmoregulatory system in escherichia coli. pathogenic ehec attach and efface intestinal mucosal cells and secrete cytotoxic shiga toxins (stx) 1 and 2 through the outer membrane.36 consequently, membrane fluidity may play a vital role in the secretion of these toxins. in an attempt to cope with environmental stresses, bacteria induce an alteration in the composition of membrane lipids.36 yuk and marshall36 investigated the relationships between acid resistance and composition of membrane lipids, as well as membrane lipid composition and stx secretion in three e. coli strains: e. coli o157:h7; an rpos-deficient mutant of ehec (ehec-rm); and non-pathogenic e. coli (npec). they found that decimal reduction times (d-values) in simulated gastric fluid of cells adapted to acid were higher than those of non-acid-adapted cells, regardless of the strain. in microbiology, the decimal reduction time is used when assessing thermal death time and thermal resistance. it is the exposure time required to cause death in 90% of the initial population under constant temperature and under specified conditions.37 acid adaptation levels decreased cis-vaccenic acid (c18:1_7c) and increased palmitic acid (c16:0) in the membrane lipids of all strains. the ratio of cis-vaccenic acid to palmitic acid increased at acidic ph, which caused a decrease in the fluidity of the membrane. the greatest stx concentrations of 2470 ng/ml (ehec) and 1460 ng/ml (ehec-rm) were seen in ehec adapted to ph 8.3 and ehec-rm adapted to ph 7.3. furthermore, the ratio of extracellular to intracellular stx concentration decreased at acidic ph, possibly as a result of the reduction of membrane fluidity. the results may suggest that whilst the rpos gene does not directly affect acid resistance in acid-adapted cells it causes a decrease in membrane fluidity which may, in turn, cause decreased stx secretion and increase acid resistance.36 despite extensive investigation of vbnc state, relatively little is known about its genetic control.3 nonetheless, two regulators, rpos and oxyr (dna-binding transcription dual regulator), appear to be important for induction of the vbnc state.3,38 rpos is a sigma factor essential for general stress response and survival in the stationary phase and has been shown to mediate expression of 10% of the genome in e. coli upon exposure to conditions that cause stress.3,38 various studies have suggested rpos involvement in the survival of e. coli in the vbnc state.18,38,39 kusumoto, asakura and kawamoto18 demonstrated that rpos mutants (inactivated rpos gene) resulted in faster induction into the vbnc state in e. coli and salmonella spp. boaretti et al.38 established that lack of rpos resulted in diminished ability of e. coli cells to remain in a vbnc state for long periods of time, which led to more rapid cell death. after 33 days in an artificial oligotrophic medium incubated at 4 °c, the parental strain of e. coli became non-culturable, whereas the rpos mutant lost culturability in only 21 days. a study conducted in 200539 proposed that reactive oxygen species play a role in the formation of vbnc cells. desnues et al.40 showed that vbnc e. coli display reduced superoxide dismutase activity, which resulted in an increase in oxidative damage. arana et al.41 subjected e. coli to hydrogen peroxide treatment and showed that some of the cells entered into the vbnc state. stationary phase e. coli were inoculated into filter-sterilised river water samples, then exposed to varying concentrations of hydrogen peroxide for 15 min at 20 °c. the total number of cells (control group), as determined with acridine orange direct count, was higher than the colony-forming unit counts (culturable count) of e. coli exposed to hydrogen peroxide. these results suggest that oxidative stress response regulation may be involved in the initiation of the vbnc state. table 2 summarises some of the proteomic changes seen in e. coli that have entered into the vbnc state, as well as which of these are under rpos and/or oxyr control. table 2: reported proteomic changes in the viable but non-culturable state in escherichia coli. induction into and in vitro resuscitation of e. coli in the viable but non-culturable state numerous environmental and chemical factors have been reported to induce the vbnc state.19 entry into the vbnc state is usually as a result of a natural stress, for example starvation; however, cells may also enter into this state during processes that are normally thought of as bactericidal, such as wastewater chlorination and pasteurisation of milk.17 this has implications for public health, as some of the established methods for food and water sanitation may, in fact, induce the vbnc state. table 3 summarises a number of methods that have been reported to induce the vbnc state in e. coli. table 3: inducers of viable but non-culturable state in escherichia coli. it is important to note that the vbnc phenotype is reversible; that is, bacteria that enter this state may again become culturable.3 the transition of bacterial cells from the vbnc state back to a culturable state is known as resuscitation.46 various authors theorise that the vbnc state forms part of the bacterial life cycle and hence constitutes a survival strategy in the face of unfavourable conditions. conversely, other studies have reported that non-culturable bacteria cannot be resuscitated.1 the underlying idea is that the vbnc phenotype is only of interest if cells in this state can resuscitate back to a state of culturability.2 although rich media was the first stimulus found to induce resuscitation, subsequent studies have identified a variety of stimuli that can trigger resuscitation.3 the following are some of the mechanisms that have been used to resuscitate bacterial cells from a vbnc state: use of host cells (e.g. embryonated chicken eggs); presence of specific compounds, such as amino acids, resuscitation promotion factor and autoinducers;47 and the removal of environmental stress (e.g. addition of nutrients to starved cells, return to darkness, change to a suitable temperature).3 it is important to note that for vbnc detection and resuscitation, a rich medium should generally be used, since some cells that may have been injured (but have not become vbnc) during exposure to the vbnc-inducing stress might not be capable of growing on differential or selective media containing antibiotics and/or other restrictive compounds.3 these injured cells have a greater sensitivity to components of the growth medium that are not customarily inhibitory, but they are not considered vbnc cells since they can be cultured on non-selective media.3 ohtomo and saito48 demonstrated that resuscitation from the vbnc state to the culturable state occurs in e. coli with the removal of environmental stress. exposure of these cells to high saline stress caused a significant decrease in the number of colony-forming units, but when the culture was relieved of the stressful condition the number of colony-forming units returned, within two hours, to the same level as before the stress. in 2011, pinto et al.46 tested the ability of vbnc e. coli to resuscitate under a minimal medium supplemented with different amino acids, discovering that a combination of glutamine, threonine, leucine and methionine would be adequate to trigger resuscitation of one of the tested strains (eco3). it was proposed that in order to initiate resuscitation, these amino acids may bind to receptors on the cell surface or be transported into the cells. additionally, they found that increased resuscitation was observed at 37 °c as opposed to 25 °c. bacteria communicate with each other by means of a process known as quorum sensing and can modulate population density-dependent behaviour such as biofilm formation and pathogenicity. population size estimation is accomplished by producing and responding to certain chemical signalling molecules.49 it has been shown that e. coli use the s-ribosylhomocysteinase (luxs)/autoinducer (ai)-2 quorum sensing system for population communication and were found to produce at least two autoinducers, ai-2 and ai-3.50 one study47 showed that ehec o157:h7 can be resuscitated using its als, such as ai-2, that are produced during biofilm formation. that fact that ais support resuscitation of bacteria in the vbnc state has a significant implications, in that commensal e. coli in the intestine can produce ais similar to those produced by ehec o157:h7.47 thus, the human intestine could prove a suitable environment for these pathogenic vbnc cells to undergo restoration of culturability. figure 2 summarises the vbnc state and the connection to repair mechanisms, induction and resuscitation. figure 2: the viable but non-culturable state.19, 23, 24, 25, 47 potential virulence of e. coli in the viable but non-culturable state for those species of bacteria that cause human infections, the underestimation or even non-detection of viable cells in quality control samples from clinical samples, water distribution systems and the food industry, may present a grave public health risk.3 quite a number of cases of bacterial infection are not linked with the isolation of the causative agent.51 it has been theorised that this may be as a result of a viral cause, low bacterial concentrations or non-culturability of the bacteria. during infection, these pathogens may reach sites in the body where they are exposed to molecules that hinder their growth (e.g. exogenous antibacterial drugs, substances made by the host, or the host’s resident bacterial population). stress conditions found in vivo may induce the bacteria into a vbnc state.51 bacterial populations in the vbnc state have consequences in their own right, because they maintain activity and thus participate in the functioning of the ecosystems, in energy production and in the carbon cycle. furthermore, pathogenic bacteria in the vbnc state are still able to produce toxins, thereby having a negative effect on their host.1 pathogenic e. coli are classified into groups based on their mechanisms of pathogenicity, clinical syndromes, virulence factors and distinctive o:h serotypes. the overall pathogenesis of these strains consist of mucosal site adhesion/colonisation, host defence evasion, multiplication and host damage.52 the groups include enterotoxigenic e. coli, enteropathogenic e. coli, enteroinvasive e. coli, enteroaggregative e. coli and enterohaemorrhagic e. coli.26 a summary of the individual virulence mechanisms of these strains is outlined in table 4. table 4: mechanisms of virulence in pathogenic escherichia coli. viable but non-culturable pathogenic e. coli have been implicated in a variety of diseases. pathogenic bacteria, whilst in the vbnc state, can be avirulent. the problem arises when some of the organisms can regain their virulence after resuscitation into culturable cells under suitable conditions.3 table 5 summarises the pathogenic e. coli that have been shown to enter the vbnc state. table 5: pathogenic escherichia coli known to enter the viable but non-culturable state. liu et al.11 showed that ehec o157:h7, when exposed to various environmental stresses, entered into the vbnc state and was still able to produce potent shiga toxins (both stx1 and stx2), which are responsible for the chief symptoms of haemolytic uremic syndrome and haemorrhagic colitis. in 1998, an ehec o157:h7 outbreak in salted salmon roe occurred in japan. it was found that patient samples containing o157:h7 would not grow on culture media after incubation in 13% nacl, but after being resuscitated in yeast extract broth, 90% of the cells were shown to be viable. these findings suggested that almost all cells are capable of entering into the vbnc state when exposed to salt water.41 zhao et al.13 induced e. coli o157:h7 into the vbnc state by exposure to high-pressure co2 treatment (one of the non-thermal techniques used for pasteurisation). they were able then to resuscitate the organism using tryptic soy broth at 37 °c. their results demonstrated that high-pressure co2 treatment could induce e. coli into the vbnc state, which has future public health implications since this technique is operational and soon to be employed for pasteurisation of liquid foods (such as fruit juice and milk) on a commercial scale.57 they concluded that all products treated by high-pressure co2 treatment should be checked for vbnc populations of bacteria using molecular-based methods in order to ensure the safety of the product. waste water forms the chief reservoir of human enteric bacteria such as e. coli. enteric bacteria are potential sources of disease; thus, it is imperative to properly disinfect waste water before it reaches the intakes for water treatment plants. chlorine, in the form of hypochlorous acid, is generally used to this end as it is an exceptionally powerful antibactericidal agent.16 in 2005, a study16 found that when e. coli k12 and s. typhimurium cells were exposed to a mixture of free and combined chlorine (as is used in waste water disinfection), a small proportion of the cells survived in the vbnc state. the researchers were not successful in resuscitating the cells, but concluded that the presence of the non-culturable cells still posed a possible threat to public health as evidence from other studies indicate that the cells of e. coli and s. typhimurium are capable of resuscitation. another study45 tested the effects of sunlight and seawater on e. coli h10407 and found that the bacteria entered into a vbnc state after exposure to both solar irradiation and seawater. the researchers established that e. coli retained its pathogenicity in the vbnc state, as enterotoxins were still produced. lothigius et al.44 determined that enterotoxigenic e. coli may still be metabolically active and viable after incubation in both freshand salt water for extended periods of time because of the vbnc state. it was found that the cell wall remained intact and there was expression of both metabolic and virulence genes after three months of incubation in water.43 another significant health implication of the vbnc state in pathogenic e. coli is possible antibiotic resistance in such cells.3,4 vbnc cells have a low metabolic rate and thus antibiotics targeting components or activities in active cells may prove less effective in quiescent cells.3,4 the vbnc bacterial populations may develop resistance to antibiotics, then resuscitate and initiate infection.19 an additional possibility is that the antibiotic acts an inducer for the vbnc state, as suggested by a study by mason et al.,58 where e. coli was exposed to 10 or 100 times the minimum inhibitory concentration of ciprofloxacin. the results showed that the colony-forming units decreased over time; however, there was no decrease in total cell numbers as seen by means of flow cytometry and light microscopy.58 conclusion vbnc pathogenic e. coli pose a public health risk, particularly those that may be present in water or food, because they still display metabolic activity, but cannot be detected via standard laboratory techniques, such as culturability.12 the existence of the vbnc state raises some serious questions about quality procedures that were previously thought to be relatively fool-proof, such as antibiotics testing, the sterility of medicinal drugs and the interpretation of routine food and water testing. it masks the actual number of viable cells that may, at any time, be resuscitated, emphasising the need to be aware that this state exists and take it into account when doing quality testing.1 acknowledgements competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions j.a.p. was responsible for conception, design and manuscript writing. a.s. and t.g.b. were responsible for critically reviewing the 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http://www.foodtech-portal.eu/index.php?title=supercritical_carbon_dioxide_pasteurization_of_liquid_food#aboutitp mason dj, power eg, talsania h, et al. antibacterial action of ciprofloxacin. antimicrob agents chemother. 1995;39(12):2752–2758. http://dx.doi.org/10.1128/aac.39.12.2752 abstract introduction ethical considerations case presentation management and outcomes discussion acknowledgements references about the author(s) kabir abdullahi department of morbid anatomy and forensic medicine, faculty of basic medical sciences, college of health sciences, usmanu danfodiyo university, sokoto, nigeria yahaya mohammed department of medical microbiology and parasitology, faculty of basic medical sciences, college of health sciences, usmanu danfodiyo university, sokoto, nigeria saddiku a. sahabi department of medical microbiology and parasitology, faculty of basic medical sciences, college of health sciences, usmanu danfodiyo university, sokoto, nigeria mahmood m. dalhat nigerian field epidemiology and laboratory training program, abuja, nigeria citation abdullahi k, mohammed y, sahabi sa, dalhat mm. coexistence of kaposi sarcoma and molluscum contagiosum on the same site in a hiv-aids patient: a very rare occurrence. afr j lab med. 2019;8(1), a747. https://doi.org/10.4102/ajlm.v8i1.747 case study coexistence of kaposi sarcoma and molluscum contagiosum on the same site in a hiv-aids patient: a very rare occurrence kabir abdullahi, yahaya mohammed, saddiku a. sahabi, mahmood m. dalhat received: 26 dec. 2017; accepted: 05 oct. 2018; published: 29 apr. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract introduction: there have been numerous reported opportunistic infections among hiv/aids patients. however, coexistence of kaposi sarcoma and molluscum contagiosum on the same site is a rare finding. case presentation: a 37-year-old man poorly adherent to antiretroviral therapy presented with molluscum contagiosum and kaposi sarcoma occurring simultaneously on numerous skin lesions around mid-2017 at usmanu danfodiyo university teaching hospital, sokoto state, nigeria. management and outcome: the patient was counselled and re-initiated on a second-line highly active antiretroviral therapy regimen. the patient’s lesions resolved three months later. discussion: the case is presented to improve the index of suspicion among clinicians and pathologists on such rare occurrences. keywords: kaposi sarcoma; molluscum contagiosum; hiv; aids. introduction molluscum contagiosum has been classified as an aids-defining illness; it usually causes a self-limiting skin lesion, but can become widely disseminated. it has a predilection for the head and neck area of individuals with aids.1 lesions range in size from 0.2 cm to 0.6 cm, although giant forms have been reported. they classically have an umbilication. on the other hand, kaposi sarcoma has a more generalised distribution, affecting more organs and systems. the lesions of kaposi sarcoma are usually purple in colour, flattened or raised and they are more difficult to manage and contribute more to mortality.1,2 coexistence of kaposi sarcoma and molluscum contagiosum in the same patient is rare and more difficult to diagnose and manage. we present a case report of a patient living with hiv, with multiple skin lesions, for which both diseases were diagnosed. ethical considerations ethical approval to conduct the study was sought and obtained from the health, research and ethics committee (hrec) of usmanu danfodiyo university teaching hospital, sokoto, nigeria, with approval number uduth/hrec/2018/no. 658. consent and permission were obtained from the patient to use his picture and details for the study. case presentation a 37-year-old man who had been hiv-positive for 2 years later became poorly adherent to first-line antiretrovirals (defaulted for more than 6 months). he re-presented at our facility again with low cd4 count (98 cells per µl) and high viral load (> 10 000.00 copies/ml) and had developed progressive, generalized, asymmetrical, non-scaly, maculo-papular, hyperpigmented focally nodular cutaneous lesions involving the head, neck, trunk (anteriorly and posteriorly), and the proximal upper and lower limbs, especially on the medial surfaces with the largest nodule reaching 2.5 cm in diameter, over a period of 3 months (figure 1 and figure 2). he was on a first-line highly active antiretroviral therapy regimen (zidovudine/lamivudine/nevirapine) before defaulting. we conducted a skin biopsy for histopathology. figure 1: anterior view of the skin lesion diagnosed as kaposi sarcoma and molluscum contagiosum. figure 2: posterior view of the skin lesion diagnosed as kaposi sarcoma and molluscum contagiosum. histopathological findings the laboratory received a small tissue fragment measuring 3 cm × 2 cm × 2 cm fixed in 10% buffered formalin. tissue was sectioned following processing and embedding in paraffin wax. light microscopy conducted on the haematoxylin and eosin stained tissue revealed a cellular nodular tumor composed of slit and sieve-like spaces containing red blood cells. these spaces were lined by plump dark cells with eosinophilic cytoplasm. in another focus within the lesion was a lobular lesion composed of enlarged keratinocytes whose nuclei were distended by eosinophilic amorphous bodies, consistent with molluscum bodies (figure 3 and figure 4). these findings are pathognomonic of both kaposi sarcoma and molluscum contagiosum (coexisting). figure 3: low power view of the coexisting kaposi sarcoma (golden arrow) and molluscum contagiosum (black arrow). haematoxylin and eosin staining x 40. figure 4: (a) section showing a lobular lesion composed of enlarged keratinocytes with central eosinophilic molluscum bodies. (b) section showing plump spindle cells with bland nuclei delimiting slit-like vascular spaces, consistent with kaposi sarcoma. haematoxylin and eosin staining x 200. management and outcomes the patient was counselled and re-initiated on a second-line highly active antiretroviral therapy regimen (tenofovir/lamivudine/lopinavir). he was re-evaluated three months after re-initiation. he has since been compliant (current cd4 count of 450 cells per µl and an undetectable viral load of < 20 copies/ml). the patient’s lesions resolved, even though no dermatological procedures or creams were used. discussion kaposi sarcoma and molluscum contagiosum both have viral infectious aetiologies, and commonly occur when the cd4 cell count is less than 150 cells per µl.2 the former is caused by a herpes virus, and the latter by a pox virus.2 our patient most likely had the infection either as reactivation or new infection during his period of non-adherence when his cd4 count and viral load deteriorated. we initially had a clinical suspicion of lepromatous leprosy due to the widespread nature of the lesions but the absence of nerve involvement and loss of sensation ruled it out. the hallmark of aids is increased susceptibility to opportunistic infections.3 kaposi sarcoma and molluscum contagiosum are categorized as aids-defining illnesses. even though their coexistence4 in hiv/aids patients has been widely described, the occurrence of the two diseases side by side within the same lesion is a rare occurrence.5 this case highlights why it is necessary to have a high index of suspicion when dealing with immunocompromised patients from clinical examination to sampling, during biopsy and ultimately in interpretation for the coexistence of skin diseases. conclusion clinicians and pathologists should be mindful of unusual presentations of opportunistic aids-defining illnesses in hiv/aids patients. our index patient was poorly adherent to treatment resulting in low cd4 counts and high viral loads. consequently, all efforts should be made to ensure adherence to treatment by hiv patients to optimise outcomes. the case report also highlights the importance of laboratory investigations and the evidence they provide in making accurate diagnoses in a patient population that is known to be at risk for multiple opportunistic infections affecting the same organ at the same time. we present this case, because of its unusual occurrence and also the need to counsel patients on compliance to highly active antiretroviral therapy medication once diagnosed with hiv/aids. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions k.a. and y.m. conceptualized the project. k.a. and s.a.s. were responsible for experimental and project design and performed additional experiments. m.m.d. made conceptual contributions. y.m. prepared the final manuscript that was reviewed by k.a., s.a.s. and m.m.d. source of support this work was conducted as part of authors routine work, no funds were received for this work. references grayson w. the hiv-positive skin biopsy. j clinpathol. 2008;61(7):802–817. https://doi.org/10.1136/jcp.2007.054015 elder de, elenitsas r, johnson bl, murphy gf lever’s, editors. histopathology of the skin. 9th ed. philadelphia, pa: lippincott williams & wilkins; 2005. wayne g. recognition of dual or multiple pathology in skin biopsies from patients with hiv/aids. pathol res int. 2011;2011:398546. singh vr, singh s, pandey ss. numerous giant molluscacontagiosa and kaposi’s sarcomas with hiv disease. indian j dermatol venereol leprol. 1996;62:173–174. prasad busarla sv, sayed s, nazarian rm, gimbel dc, moloo z, sohani ar. kaposi sarcoma in association with molluscumcontagiosum: an uncommon diagnosis in a single biopsy and potential diagnostic pitfall. am j dermatopathol. 2012;34(1):7–9. https://doi.org/10.1097/dad.0b013e31822438c6 acknowledgements references about the author(s) cristina gutierrez independent consultant, vigo, spain akos somoskovi global health technologies, global good fund, intellectual ventures laboratory, bellevue, washington, united states kris natarajan global health technologies, global good fund, intellectual ventures laboratory, bellevue, washington, united states david bell global health technologies, global good fund, intellectual ventures laboratory, bellevue, washington, united states citation gutierrez c, somoskovi a, natarajan k, bell d. need for better adherence to optimal incubation temperature for quality laboratory diagnostics and antibiotic resistance monitoring. afr j lab med. 2018;7(2), a789. https://doi.org/10.4102/ajlm.v7i2.789 scientific letter need for better adherence to optimal incubation temperature for quality laboratory diagnostics and antibiotic resistance monitoring cristina gutierrez, akos somoskovi, kris natarajan, david bell received: 13 feb. 2018; accepted: 26 july 2018; published: 06 dec. 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. in the omics era, incubation of human specimens and bacterial cultures continues to be the cornerstone for detection, identification and drug susceptibility testing (dst) of bacterial pathogens. accurate results require bacterial incubation under optimal physicochemical conditions. temperature is a key physicochemical factor that affects the bacterial environment, making incubators indispensable in clinical laboratories. human pathogens generally multiply best at temperatures similar to those of the human host (35°c – 37°c). biochemical tests for identification in pure isolates are recommended to be performed at 36°c ± 2°c, and dst at 35°c± 1°c.1,2,3,4 for mycobacterium tuberculosis complex and most nontuberculous mycobacteria, the recommended temperature is 37°c in both cases. in a recent survey to determine the incubator requirements for clinical microbiology laboratories in resource-limited countries, we analysed several parameters including the temperatures used to incubate clinical specimens for primary bacterial isolation, replicating bacterial cultures and performing dst. the data were reported in a self-administered questionnaire conducted from april 2017 to june 2017 by 12 laboratories from three countries in africa (cameroon, ivory coast and madagascar), three countries in the americas (haiti, guyana and bolivia) and one country in asia (bangladesh). the clinical specimens considered were sputum, pharyngeal or nasopharyngeal swabs, stools, urethral or vaginal swabs, skin, pus, urine, lymph nodes, blood, bone marrow, cerebrospinal fluid and other normally sterile body fluids. the analysed incubation temperatures referred to 26 pathogenic bacteria or pathogenic bacteria groups frequently isolated from clinical specimens (figure 1). figure 1: incubation temperature in 12 laboratories from seven countries for isolation, subculture and dst of 26 pathogenic bacteria or pathogenic bacteria groups frequently isolated from clinical specimens. laboratories working with mycobacteria incubated both specimens and isolates at the appropriate temperature of 37°c for m. tuberculosis complex and nontuberculous mycobacteria, or at 30°c in the case of mycobacterium ulcerans. to target the other 24 bacteria or groups of bacteria, specimens were incubated for primary isolation at either 35°c or 37°c with a tendency towards 37°c. laboratories employed the same distribution of temperatures for sub-culturing isolates and performing dst, with the exception of one laboratory sub-culturing acinetobacter baumanii, salmonella typhi and salmonella paratyphi at 42°c. the reported temperature fluctuation during incubation was ± 2°c for 55% of the incubators, ± 1°c for 29%, and ± 3°c for 16%. clinical laboratories are often faced with the need to grow a priori unidentified bacteria and potentially polybacterial specimens from non-sterile sites. to best support recovery of bacterial pathogens, as well as biochemical testing and dst incubation requirements, a temperature of 35°c is likely ideal. at 35°c, most human bacterial pathogens with differing optimum growth temperature will grow reliably, although colonies may appear small or require additional incubation due to their slower growth rate.5 dst is recommended to be performed at 35°c ± 1°c.3,4 the use of 37°c by most laboratories puts the reliability of dst results in question. incubation at 37°c also risks fluctuations to dying off temperatures. our field survey revealed fundamental failures regarding basic and easy-to-control requirements of incubation that could negatively impact dst, and therefore the battle against antimicrobial resistance. it is difficult to assess the real magnitude of the problem, because this study relied on self-reported laboratory procedures, which may be subject to bias. with the growing importance of identifying isolates and trends of antibiotic-resistant human pathogens, it is alarming that the simplest global recommendations fail to reach the workplace. more rigorous attention must be paid to bacterial incubation, including dst requirements, through the institution of evidence-based protocols and use of quality instruments that can provide accurate temperature levels, as well as operating under appropriate quality assurance practices and with stricter adherence to existing guidelines. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. sources of support none. authors’ contributions c.g., a.s. and k.n. designed the project. d.b. made conceptual contributions. c.g. collected and analysed the data. c.g., a.s. and d.b. co-wrote the manuscript. references forbes ba, sahm df, weissfeld as. traditional cultivation and incubation. in: tille p, editor. bailey & scott’s diagnostic microbiology. 12th ed. philadelphia, pa: elsevier health sciences, 2007; p. 93–119. jorgensen jh, turnidge jd. susceptibility test methods: dilution and disk diffusion methods. in: murray pr, editor. manual of clinical microbiology. 9th ed. washington dc: asm press, 2007; p. 1152–1172. the european committee on antimicrobial susceptibility testing. disk diffusion method for antimicrobial susceptibility testing – version 6.0. eucast, european society of clinical microbiology and infectious diseases: basel, switzerland; 2017. matuschek e, brown dfj, kahlmeter g. development of the eucast disk diffusion antimicrobial susceptibility testing method and its implementation in routine microbiology laboratories. clin microbiol infect. 2014;20:o255–o266. https://doi.org/10.1111/1469-0691.12373 winn w, allen s, janda w, et al. phases of the diagnostic cycle: analytical phase. in: koneman ew, editor. color atlas and textbook of diagnostic microbiology. 6th ed. baltimore, md: lippincott williams & wilkins, 2006; p. 15–42. abstract introduction purpose methods outcomes lessons learned acknowledgements references about the author(s) zelda r. moran earth institute, columbia university, new york, new york, united states atta b. frimpong millenium promise alliance, accra, ghana pablo castañeda-casado glaxosmithkline, brentford, united kingdom francis k. frimpong millenium promise alliance, accra, ghana manuela b. de lorenzo glaxosmithkline, brentford, united kingdom yanis ben amor center for sustainable development, earth institute, columbia university, new york, new york, united states citation moran zr, frimpong ab, castañeda-casado p, frimpong fk, de lorenzo mb, ben amor y. tropical laboratory initiative: an innovative model for laboratory medicine in rural areas. afr j lab med. 2019;8(1), a922. https://doi.org/10.4102/ajlm.v8i1.922 lessons from the field tropical laboratory initiative: an innovative model for laboratory medicine in rural areas zelda r. moran, atta b. frimpong, pablo castañeda-casado, francis k. frimpong, manuela b. de lorenzo, yanis ben amor received: 15 oct. 2018; accepted: 12 feb. 2019; published: 26 sept. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: communities in rural, low-resource settings often lack access to reliable diagnostics. this leads to missed and misdiagnosed cases of disease and contributes to morbidity and mortality. objective: this paper describes a model for providing local laboratory services to rural areas of ghana, and provides suggestions on how it could be adapted and expanded to serve populations in a range of rural communities. methods: the tropical laboratory initiative (tli) system in ghana comprises one central laboratory where samples delivered from clinics by motorbike riders are analysed. test requests and results are communicated on a mhealth application, and the patient does not have to visit the laboratory or travel beyond the clinic to receive a test. the tli also serves as a research base. the laboratory is accredited by the national health insurance authority, and accepts the national health insurance. the tli serves several communities in amansie west, ashanti region, and currently works with 10 clinics. the nearest hospital is a one-hour drive away and is the only other nearby facility for diagnostics beyond basic rapid tests. results: demand for services has increased yearly since the launch in 2010, and the tli currently provides over 1000 tests to approximately 350 patients monthly. the majority of patients are female, and the most common tests are for antenatal care. our experience demonstrates that laboratory services can be affordable and most components already exist, even in rural areas. conclusion: ministries of health in low-resource settings should consider this model to complement the rapid tests available in clinics. integrating with an insurance system promotes financial sustainability. keywords: laboratory; burden of disease; community health; data quality; disease reporting; epidemiology; global health; health services; maternal and child health; rural health. introduction access to reliable diagnostics can be a barrier to appropriate, timely medical treatment in resource-limited settings. under-diagnosis and misdiagnosis of diseases contribute to morbidity and mortality, often despite the existence of effective treatments. primary care laboratory medicine can help address this gap, and should be a priority for health system strengthening programmes.1,2,3 access to diagnostics in rural, low-resource settings is often either nonexistent or limited to basic rapid diagnostic tests, forcing healthcare providers to rely on referral or presumptive treatment. here, we present results and reflection on a rural laboratory network in ghana called the tropical laboratory initiative (tli), a collaboration between columbia university, ghana health service, millennium promise alliance, and ghana’s national health insurance authority. our model could be used as a template for increasing access to laboratory services in low-resource rural communities worldwide. several examples of rural diagnostic laboratory and specimen referral systems have been described in sub-saharan africa, the caribbean, and vietnam.4 in uganda, a specimen referral system consisting of ‘hub’ laboratories around the country to serve rural ‘spoke’ health facilities was first initiated in 2013,5 and samples are transported to each hub by motorbike – a model quite similar to the one described here.4,5,6,7 in haiti, a similar hub and spoke model has been implemented since 2011 but with a focus on improving access to hiv testing and monitoring at laboratories with cd4+ count and viral load testing services.8 in ethiopia, a referral system for blood samples and dried blood spots utilises both couriers and the national postal system for transporting specimens from lower-tiered to higher-tiered health facilities with more advanced laboratory services.4,9 publications on these initiatives are scarce, but the results observed include reduced turn-around time in ethiopia and uganda, and reduced cost of sample processing in uganda.4,7 the aim of the tli is to establish a model of specimen referral and increased access to diagnostic testing for rural populations in ghana, with an added element of same-day delivery of electronic test results to healthcare providers, and integration with the national health insurance scheme (nhis) to support financial sustainability. purpose the tli was established in the south-western ashanti region in 2010 with the goal of improving the quality of and access to diagnostics for a network of neighbouring clinics. prior to the inception of the tli, none of the clinics offered diagnostics beyond basic rapid diagnostic tests, which do not exist for many common diseases and health conditions affecting the population. as a result, most patients were referred to the nearest district hospital for testing, about a one-hour drive from the referring clinics. people were often unable to make this journey, especially if they were ill or had trouble accessing transportation. in addition, it is possible that the community may have lost confidence in local clinics and bypassed them completely, going directly to the district hospital for treatment (authors’ observations). this would result in unnecessary demand for basic testing services at the hospital, and does not promote a strong community-level primary care system. the tli was created to prevent unnecessary referrals, increase the speed of urgent referrals, and to provide insight into the health needs and disease burdens of rural populations. as of 2019, the tli provides analysis of blood, urine, stool, and sputum samples from 10 health centres that are 14–45 km away from the nearest hospital (average 30 km), and serve about 31 000 people in several villages. test results are produced on the same day that samples are received at the laboratory, and often on the same day that the patient presents to the clinic. ethical considerations this work was approved by the columbia university institutional review board, number irb-aaap3007, as a non-human subjects study. methods the tropical laboratory initiative model the tli was designed to serve a network of partner clinics. the model consists of one central rural laboratory, composed of three rooms – the main laboratory, a phlebotomy room, and an office – and staffed with two laboratory technologists and two motorbike riders who deliver samples from nearby clinics. the motorbike riders, who are not laboratory scientists, were trained in safe blood collection and sample storage as part of a special training module developed by the president’s emergency plan for aids relief (pepfar). they support nurses by collecting blood or other specimens from patients as requested by clinic staff. the riders have daily routes during which they visit each clinic three times per week, and remain at each clinic to collect samples for approximately 2 hours. the timing of each route is designed such that the riders are at the clinics on busy days and at times when most patients normally present to the clinic. the samples are stored at 4 degrees celsius in refrigerators at the clinics, if they are collected before motorbike riders arrive or on a day when they are not scheduled to come, and are transported in cold chain in insulated styrofoam to the laboratory. at the beginning of the project in 2010, the tli served six clinics that received test results on paper forms, but the model has since evolved: paper records were replaced by sms in 2012, and updated in 2015 to the current tablet-based platform, which works using a mhealth application called commcare (dimagi, cambridge, massachusetts, united states; https://www.dimagi.com/commcare/). the mhealth system provides forms for test requests, results, and secure data storage, and allows health workers to order tests and view results instantly, while laboratory technologists can view requests and input results onto a tablet. test results are normally available within 6–12 h of sample collection, or within 48 hours in the case of samples collected when there was no motorbike pickup that day. results can be viewed by the referring nurse or midwife immediately after they are entered (figure 1). figure 1: the tropical laboratory initiative model. apart from providing community diagnostic services, the laboratory also serves as a research centre for epidemiologic studies and validation of diagnostic devices. an external quality assurance system for haemoglobin, tuberculosis and malaria testing is in place with the affiliated district hospital; samples are tested at the tli, and sent to the hospital for the same tests. the results are compared to ensure that the tli test results are of high quality. administrative status in 2017, the laboratory was accredited as an independent facility by the national health insurance authority, becoming – to our knowledge – the first non-hospital affiliated laboratory serving a rural area in ghana. normally, a rural laboratory would be accredited as part of a healthcare facility, which means that payments to the laboratory would go directly to the affiliated clinic or hospital. independent status enables the laboratory to easily serve patients from multiple facilities, making it easier to provide diagnostics to a wider catchment area. as a result, the tli receives insurance reimbursements, so that any ghanaian enrolled in the nhis can receive tli services for free. this supports the sustainability of the business model and promotes universal access to diagnostic services. uninsured patients pay at rates determined by the nhis for private laboratories. the tli will pursue accreditation through stepwise laboratory quality improvement process towards accreditation (http://www.aslm.org/what-we-do/slipta/) as recommended by the ghanaian government. diagnostic tests package as the first laboratory of its kind in ghana, accreditation involved negotiations with ghana health services and the national health insurance authority on what diagnostics package to offer, and what tests would be inapplicable or impractical at the rural level. a list of services was finalised (table 1), with the goal of providing diagnostics well beyond what is normally available at the clinic level. tests for both infectious and chronic diseases, as well as blood and urine analysis for antenatal and postnatal care, are available, allowing clinic health workers to order tests previously unavailable to them, and to request confirmation if there is doubt about any clinic test. while the services offered are greatly expanded compared to the status quo, they are limited to those tests that local clinic health workers are qualified to order and interpret, and that do not require advanced equipment or specialised reagents. as a result, the tli model could be adapted or replicated in many rural settings, without the need for expensive materials or additional training for health workers. table 1: diagnostic tests offered at the tropical laboratory initiative site in tontokrom, ghana, july 2019. technical and scientific constraints operating a rural laboratory implies technological and operational limitations, and the tli is often affected by power outages, water shortages, and severe seasonal dust. equipment repairs are more difficult due to the remote location, and certain laboratory materials and reagents can be a challenge to replace during stock-outs. therefore, all services are provided using low-tech, affordable instruments and accessible reagents, and a power generator is available during outages. all tests are carried out with basic equipment: mainly microscope, centrifuge, water bath, colorimeter, solar-powered refrigerator, and generator (table 1). one of the main advantages of the model is that centralisation allows for easier supervision: equipment in need of repair is identified immediately and repaired within days, whereas it would be more difficult to address these issues at multiple individual clinics. outcomes sample volume the tli laboratory was opened in 2010, and the number of tests and patients has increased steadily since 2012 (figure 2). on average, the number of patient visits has increased by 43% each year, with a 144% increase between 2014 and 2015. in 2017, the tli served 4171 patients, providing an average of 1019 tests to 350 patients per month. the number of tests requested is highest on days when women normally come for antenatal care, suggesting that this model may be particularly suitable for supporting maternal and child health efforts. increasing demand suggests that the laboratory is not yet operating at capacity, and has the potential to serve a greater number of people in the surrounding community. figure 2: tests performed at the tropical laboratory initiative by year. patient population between 2010 and 2017, the tli returned test results for 15 247 patient visits and approximately 46 000 individual tests (most visits result in multiple tests), many of which were for pregnant women. indeed, 82% of all patient visits between 2011 and 2017 were by female patients, and 57% of female patients (47% of all patients) were pregnant. about 42% of visits from male patients were boys under 5 years old, compared to 9% of visits from female patients (table 2). the majority of female patients (75%) were between 15 and 35 years old. data on sex was missing for 73 patients (0.48%), and age was missing for 216 (1.42%) patients. (since the tli now accepts the national health insurance, it would be possible to determine how often patients have multiple sets of tests ordered by a clinic, if the nhis number was used as a unique identifier. this could be particularly useful for monitoring how often pregnant women return for antenatal care, and how many women receive the recommended tests, an important indicator for research in maternal and reproductive health. table 2: demographic data of patients served at the tropical laboratory initiative, tontokrom, ghana, 2010–2017. tests performed diagnostic services include tests for malaria, typhoid, hiv, and tuberculosis, along with antenatal care bloodwork and stool and urine examination (table 1, table 3). haemoglobin, blood group and rhesus, g6pd deficiency, hepatitis b, and sickle cell (all antenatal tests) compose 67% of tests performed at the tli. since 2010, 22% of tests were for haemoglobin, 15% for blood group and rhesus factor, 15% for hepatitis b, 15% for sickle cell, and 7% for syphilis. about 14% of all tests were malaria films, which nurses may request if they suspect malaria but the rapid diagnostic test at the clinic was negative. the remaining tests were for typhoid, parasites (urine and stool), and a limited number of hiv tests (usually performed at the clinics). overall, 57% of female patients and 8% of male patients were tested for sickle cell disease or its trait: 12% of female patients and 17% of male patients tested positive. these results provide new insights into the disease burdens of the local community, and long-term surveillance could be useful in early detection of outbreaks (in the case of infectious diseases) and for monitoring the success of public health programmes. table 3: summary of test types performed at the tropical laboratory initiative facility, tontokrom, ghana, 2010–2017. infectious disease test results many of the infectious disease diagnostics performed at the tli were also associated with antenatal care, and included syphilis, hepatitis b, and malaria. among female patients, 8.5% of tests for syphilis and 10% of tests for hepatitis b were positive (table 4), which is low compared to the estimated 13.1% regional prevalence in ashanti.10 table 4: results of common tests for infectious diseases, tontokrom, ghana 2010–2017. hiv tests are performed at the tli only if clinics run out of rapid diagnostic tests, or for confirmation when requested. hiv test results are not recorded in commcare due to patient confidentiality and are only communicated in person to the clinic nurse or hiv counselor. national health insurance scheme enrolment since the tli began accepting nhis reimbursements in 2017, payment using nhis was used for 77% of female patients receiving tests, 87% of pregnant women, and 42% of male patients (mostly under the age of 5). in ghana overall, national enrolment in the nhis was estimated at 40% in 2016, with 60% of the enrolled population being pregnant, under 18, over 70, or in the poorest income bracket, and therefore exempt from paying premiums.11 in 2017, close to 70% of all patients at the tli paid using the nhis. lessons learned potential for clinical, epidemiological and research benefits the tli experience demonstrates how basic laboratory services can be offered in rural, low-resource communities. access to diagnostics can promote efficient treatment and facilitate appropriate linkage to care. additionally, it provides valuable data, revealing disease burdens and health care-seeking trends, which could be critical insights for public health interventions. for example, it is estimated that 2% of babies born in ghana have sickle cell disease, and that sickle cell trait prevalence may be around 30% nationally.12 at the tli, 12.3% of female patients and 16.97% of male patients tested positive for sickle cell disease or sickle cell trait (the oxidation-reduction method does not distinguish between trait and disease). it is possible that this area of ashanti has lower rates of sickle cell trait and sickle cell disease, and this demonstrates how local laboratory services can reveal specific trends and inform public health efforts. secondly, the age demographic served by the tli is highly skewed toward reproductive age among female patients, and under 5 years among male patients. this demonstrates that mothers bring young boys to the clinic regularly, but adult men visit less commonly. conversely, female patients are far more likely receive diagnostics after the age of 5, and often during pregnancy. integration with the nhis system reveals which demographics are currently best served by national insurance, and provides insight into healthcare-seeking behaviours among these groups. for example, 87% of pregnant women used the nhis to pay for tli services, and further analysis could reveal whether they are getting the antenatal tests at the recommended frequency throughout pregnancy. data from a larger network of local centralised laboratories following the tli model in rural settings could support research into healthcare needs and disease burdens that are currently very difficult to investigate, and the value of this knowledge to the development of public health programmes has great potential. human resources and technology the tli model relies on well-trained laboratory technologists, managers, nurses, and motorbike riders, but the model does not pay for nurses who are government staff. the system fully depends on the dedication and enthusiasm of clinic healthcare workers who, in addition to collecting samples and ordering the tests, must use commcare to request those tests and view results. since clinics also retain paper records, using commcare results in additional, and often redundant, record keeping for already busy clinics. if this model is to be expanded, extra support staff and streamlined record keeping is needed. the tli laboratory technicians currently on the staff are from a large urban area, and relocating to a rural area was challenging; the tli provided higher salaries as an incentive. if the model is replicated, staff retention of laboratory technicians in rural areas could be difficult, and staff motivation would need to be a priority. besides workload, one factor impacting the services offered at the tli is that clinic healthcare workers are trained and qualified to order a limited scope of tests, meaning that the tli can perform tests that clinics do not perform and are not equipped to interpret. these include liver and kidney function, lipid testing, and others. increased laboratory services at the local level could inform governments on what additional training nurses and midwives could be given in order to take advantage of more tests, expanding the services available at local levels. it is also possible that the tli could offer testing requested by hospitals for patient monitoring at a local level. context for implementation various factors influence when and if building new laboratory systems is appropriate in rural areas of sub-saharan africa or other low-resource settings. first, the laboratory system should fill a void in the healthcare system, and not detract from or be redundant with any services already offered locally. for example, if laboratory services are (or are expected to be) offered within clinic facilities, establishing a parallel laboratory would reduce the need for these services to be prioritised. rural laboratories should be established in locations with no other diagnostic facilities available within a reasonable distance. the tli laboratory in ghana works only with facilities where no laboratory services are offered at the clinic, and that do not have alternative facilities within a reasonable distance given the terrain and access to public transport. second, laboratories should be established in rural locations with a number of well-attended health facilities. the ideal location for a laboratory is one where enough samples can be collected to result in full-time or nearly full-time utilisation of laboratory staff and equipment – efficiency and cost-effectiveness of the system decreases if only a small number of samples are delivered each day. the location of the tli was established in a populated rural area where the nearest reliable diagnostic facility was at the district hospital, in most cases tens of kilometres away – ensuring steady demand from the referring health centres. third, the methods for transporting and storing samples should be considered carefully when designing any rural laboratory system or specimen referral programme. there must be an inexpensive way to transport samples, ideally one that does not require any equipment or technical skill beyond what is already locally available. finally, the laboratory system must be enthusiastically supported by local government health departments. locations of laboratories and the scope of diagnostic services should ultimately be determined by them, and the easy communication of reports and data between facilities and other levels of the health system should be part of the programme’s design. sustainability the tli was established using a grant from becton dickinson and company and in-kind donations from glaxosmithkline, and is now sustained largely through internally generated funds with continued support from becton dickinson. in areas like ghana with national or health insurance systems, the model has potential to be fully or partially financially sustainable through insurance reimbursements. in 2017, nhis reimbursements were sufficient to cover 130% of all basic laboratory operation costs (reagents, consumables, office supplies, electric bills, and similar), excluding salaries. many tests require only low-cost reagents, so services can be inexpensive for patients paying out of pocket. demand for services at the tli have consistently increased since the project was launched, and continues to increase, suggesting that the laboratory is not yet meeting the community demand. encouragement to healthcare workers from tli management and ghana health service, as well as adding additional nearby clinics to the tli network, could substantially increase revenue to the laboratory, bringing it closer to true financial sustainability. further research should be conducted on business models for laboratories operating in countries without national health insurance, and the tli model will continue to work toward a business model that can cover all staff salaries. conclusion lessons learned are summarised in box 1. the tli model demonstrates that a strong national laboratory system covering rural areas has potential for both epidemiologic surveillance and research on new or improved diagnostics. all or many components of a successful laboratory system already exist in rural areas, and there is often no need to invest in costly and complicated infrastructure, or in extensive training if services fit within what health workers are equipped to offer and interpret. lastly, there is potential for laboratory systems in rural areas to be wholly or partially financially sustainable, and this should be considered a priority for public health programmes worldwide. acknowledgements the work of the tropical laboratory initiative was supported by a charitable donation from becton dickinson and company. the supporting source had no involvement in any part of this research or the publication. competing interests all authors declare no conflict of interest. authors’ contributions z.r.m. analysed all data and drafted the original manuscript. a.b.f., p.c.-c., f.k.f. and m.b.d.l. contributed to data collection and reviewed the final manuscript. y.b.a. developed the concept, initiated the programme with m.b.d.l., supervised the work, and drafted the manuscript with z.r.m. sources of support the work of the tropical laboratory initiative was supported by a charitable donation from becton dickinson and company. the supporting source had no involvement in any part of the research or manuscript. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the discussion and perspectives offered in this article are those of the authors, and do not represent the view of any affiliated institution or funding organisation. references sayed s, cherniak w, lawler m, et al. improving pathology and laboratory medicine in low-income and middle-income countries: roadmap to solutions. lancet (london, england). 2018;391(10133):1939–1952. https://doi.org/10.1016/s0140-6736(18)30459-8 wilson ml, fleming ka, kuti ma, looi lm, lago n, ru k. access to pathology and laboratory medicine services: a crucial gap. lancet (london, england). 2018;391(10133):1927–1938. https://doi.org/10.1016/s0140-6736(18)30458-6 horton s, sullivan r, flanigan j, et al. delivering modern, high-quality, affordable pathology and laboratory medicine to low-income and middle-income countries: a call to action. lancet (london, england). 2018;391(10133):1953–1964. https://doi.org/10.1016/s0140-6736(18)30460-4 fonjungo pn, alemnji ga, kebede y, et al. combatting global infectious diseases: a network effect of specimen referral systems. clin infect dis. 2017;64(6):796–803. https://doi.org/10.1093/cid/ciw817 kiyaga c, sendagire h, joseph e, et al. uganda’s new national laboratory sample transport system: a successful model for improving access to diagnostic services for early infant hiv diagnosis and other programs. plos one. 2013;8(11):e78609. https://doi.org/10.1371/journal.pone.0078609 kagimba m, agaba j. government sets up 77 laboratory hubs. new vision; 2015; 9 january. donnenberg s. the results are in: how a national sample and results transport network is improving patient care in uganda [news article]. addis ababa: african society for laboratory medicine; 2015. louis fj, osborne aj, elias vj, et al. specimen referral network to rapidly scale-up cd4 testing: the hub and spoke model for haiti. j aids clin res. 2015;6(8). https://doi.org/10.4172/2155-6113.1000488 kebede y, fonjungo pn, tibesso g, et al. improved specimen-referral system and increased access to quality laboratory services in ethiopia: the role of the public-private partnership. j infect dis. 2016;213(suppl 2):s59–s64. https://doi.org/10.1093/infdis/jiv576 ofori-asenso r, agyeman aa. hepatitis b in ghana: a systematic review & meta-analysis of prevalence studies (1995–2015). bmc infect dis. 2016;16(130). https://doi.org/10.1186/s12879-016-1467-5 alhassan rk, nketiah-amponsah e, arhinful dk. a review of the national health insurance scheme in ghana: what are the sustainability threats and prospects? plos one. 2016;11(11):e0165151. https://doi.org/10.1371/journal.pone.0165151 kyerewaa edwin a, edwin f, etwire v. controlling sickle cell disease in ghana – ethics and options. pan afr med j. 2011;10(14). abstract background description of the intervention lessons learnt recommendations acknowledgements references about the author(s) lance d. presser global engagement program, mriglobal, gaithersburg, maryland, united states jeanette coffin global engagement program, mriglobal, gaithersburg, maryland, united states lamine koivogui centre de recherche et de formation en infectiologie de guinée, université gamal abdel nasser de conakry, conakry, guinea allan campbell central public health reference laboratory, freetown, sierra leone julian campbell central public health reference laboratory, freetown, sierra leone fatmata barrie central public health reference laboratory, freetown, sierra leone jone ngobeh central public health reference laboratory, freetown, sierra leone zein souma central public health reference laboratory, freetown, sierra leone samuel sorie central public health reference laboratory, freetown, sierra leone doris harding central public health reference laboratory, freetown, sierra leone alimou camara institut national de santé publique, conakry, guinea pepe tohonamou institut national de santé publique, conakry, guinea basala traore institut national de santé publique, conakry, guinea frank a. hamill global engagement program, mriglobal, gaithersburg, maryland, united states joe bogan global engagement program, mriglobal, gaithersburg, maryland, united states sharon altmann global engagement program, mriglobal, gaithersburg, maryland, united states casey ross global engagement program, mriglobal, gaithersburg, maryland, united states jay mansheim global engagement program, mriglobal, gaithersburg, maryland, united states robert hegerty global engagement program, mriglobal, gaithersburg, maryland, united states scott poynter global engagement program, mriglobal, gaithersburg, maryland, united states scott shearrer global engagement program, mriglobal, gaithersburg, maryland, united states carmen asbun global engagement program, mriglobal, gaithersburg, maryland, united states brendan karlstrand global engagement program, mriglobal, gaithersburg, maryland, united states phil davis global engagement program, mriglobal, gaithersburg, maryland, united states jane alam global engagement program, mriglobal, gaithersburg, maryland, united states david roberts global engagement program, mriglobal, gaithersburg, maryland, united states paul d. stamper global engagement program, mriglobal, gaithersburg, maryland, united states jean ndjomou global engagement program, mriglobal, gaithersburg, maryland, united states nadia wauquier global engagement program, mriglobal, gaithersburg, maryland, united states mohamed koroma global engagement program, mriglobal, gaithersburg, maryland, united states alhaji munu global engagement program, mriglobal, gaithersburg, maryland, united states jason mcclintock global engagement program, mriglobal, gaithersburg, maryland, united states mar mar global engagement program, mriglobal, gaithersburg, maryland, united states true burns global engagement program, mriglobal, gaithersburg, maryland, united states stephen krcha global engagement program, mriglobal, gaithersburg, maryland, united states citation presser ld, coffin j, koivogui l, et al. the deployment of mobile diagnostic laboratories for ebola virus disease diagnostics in sierra leone and guinea. afr j lab med. 2021;10(1), a1414. https://doi.org/10.4102/ajlm.v10i1.1414 lessons from the field the deployment of mobile diagnostic laboratories for ebola virus disease diagnostics in sierra leone and guinea lance d. presser, jeanette coffin, lamine koivogui, allan campbell, julian campbell, fatmata barrie, jone ngobeh, zein souma, samuel sorie, doris harding, alimou camara, pepe tohonamou, basala traore, frank a. hamill, joe bogan, sharon altmann, casey ross, jay mansheim, robert hegerty, scott poynter, scott shearrer, carmen asbun, brendan karlstrand, phil davis, jane alam, david roberts, paul d. stamper, jean ndjomou, nadia wauquier, mohamed koroma, alhaji munu, jason mcclintock, mar mar, true burns, stephen krcha received: 29 sept. 2020; accepted: 18 mar. 2021; published: 22 oct. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: ebola virus emerged in west africa in december 2013. the ease of mobility, porous borders, and lack of public health infrastructure led to the largest ebola virus disease (evd) outbreak to date. intervention: the 2013 evd outbreak signalled the need for laboratory diagnostic capabilities in areas without strong public health systems. as part of the united states’ department of defense response, mriglobal was contracted to design, fabricate, equip, deploy, and operate two mobile diagnostic laboratories (mdls). the first laboratory analysed blood samples from patients in an adjacent ebola treatment centre (etc) and buccal swabs from the deceased in the community in moyamba, sierra leone. the second laboratory was deployed to support an etc in conakry, guinea. the department of defense provided real-time quantitative reverse transcription polymerase chain reaction assays that were deployed and validated on-site. lessons learnt: prompt and accurate molecular diagnostics reduced sample turn-around times from over 24 h to under 4 h. experienced laboratory staff tested up to 110 samples per day and on-site engineering proved necessary for mdl setup and operation. as the ebola response slowed, the sustainment of the mdls’ operations was prioritised, including staff training and the transition of the mdls to local governments. training programmes for local staff were prepared in sierra leone and guinea. recommendations: the mriglobal mdl team significantly contributed to establishing increased laboratory capacity during the evd outbreak in west africa. using the mdls for molecular diagnosis is highly recommended until more sustainable solutions can be provided. keywords: ebola; ebola virus; sierra leone; guinea; diagnostics; laboratory capacity; service expansion; epidemic; outbreak; outbreak response; west africa; mobile diagnostic laboratories. background in march 2014, the world health organization was notified regarding a cluster of disease in guinea characterised by fever, severe diarrhoea, vomiting, and high fatality rate. eventually, the disease was identified as a novel strain of the ebola virus.1 further investigation suggested the first fatality of the outbreak had occurred in december 2013 in guinea.1 when the outbreak was declared over, 28 616 ebola virus disease (evd) cases including 11 310 total deaths had been reported.2 the unprecedented scale of this evd outbreak resulted in sustained human-to-human transmission, the consequences of which are still being elucidated. ebola virus is a biosafety level 4 (bsl-4) agent. specimen inactivation should be performed in at least a bsl-3 laboratory, after which routine diagnostic specimen testing can be performed in a bsl-2 laboratory. when the outbreak began, the closest bsl-3 laboratory was in nigeria and was being used for tuberculosis diagnostics. it was not capable of evd testing, in that it lacked polymerase chain reaction (pcr) machines and had no validated assay and reagents. similarly, the closest bsl-4 laboratory was situated in gabon, 3000 km away from freetown, and unable to assist with evd diagnostics timeously. none of the west african countries hit hardest by the evd outbreak (guinea, sierra leone, or liberia) had adequate evd diagnostic facilities; this necessitated the development of mobile diagnostic laboratory (mdl) units, and improvement of other laboratory capacities in the region to help control the outbreak. the united states department of defense initiated the cooperative biological engagement program in west africa through the defense threat reduction agency to contain the biological agent (ebola virus), enhance biosafety and biosecurity, and strengthen the region’s ability to detect, diagnose, and report public health emergencies of international concern to the world health organization.3 mriglobal was awarded the contract to design, assemble, equip, and deploy rapid response mdls for molecular detection of ebola virus in patient samples. at the invitation of the sierra leonean and guinean governments, as well as the department of defense, the non-profit organisation mriglobal designed, built, delivered, and operated mdls during the evd outbreak starting in december 2014. by 2016, mriglobal had shifted its focus from emergency response to a smooth transition of management, which included staff training and support to the central public health reference laboratory in sierra leone and the national institute of public health in guinea. we describe here the deployment of the mdls for the evd outbreak response and discuss the successes and challenges experienced. description of the intervention ethical considerations the evd outbreak response was declared a public health emergency of international concern by the world health organization on 08 august 2014. the standard operating procedures used for diagnostic testing were approved by the world health organization, the department of defense, and the ministry of health in guinea and sierra leone. diagnostic results were released as quickly as possible following specimen analyses. neither mriglobal nor the department of defense retained any samples as they were either destroyed or turned over to the host country. mobile diagnostic laboratory design from an engineering standpoint, the project’s goal was to build a mobile, self-sustained, self-contained (safe) laboratory ready for delivery in less than six weeks. mriglobal engineers selected 20-foot (~6.1 m) intermodal containers because they provide a rugged, watertight, customisable shell that can be easily transported. mriglobal has over 15 years of experience in designing, building, maintaining, and deploying similar containerised laboratories around the world. mriglobal engineers had to consider customisation such as including surfaces that were easy to decontaminate and providing attachment and stabilisation points for all pieces of equipment within the labs, including lighting, heating, ventilation, and air conditioning systems. mriglobal’s engineers and scientists worked together to design the laboratory to ensure safe handling and testing of samples and the accommodation of equipment required for operations in both countries. the laboratories were to include three separate areas – sample inactivation and extraction, reagent preparation, and quantitative reverse-transcription polymerase chain reaction (qrt-pcr) areas inside the two laboratory containers. the first container housed the sample inactivation and extraction area where infectious samples would be processed and inactivated inside of a class ii type b2 biological safety cabinet (bsc). four bscs were placed in the inactivation laboratory to handle the anticipated sample volume. after delivery, modifications to the container were necessary to allow for sample pass-through between the two containers and to properly exhaust the bscs (figure 1c). figure 1: mriglobal mobile diagnostic laboratory that was deployed in response to the west africa ebola virus outbreak. (a) interior of polymerase chain reaction laboratory unit. (b) interior of master mix laboratory. (c) interior of inactivation and extraction laboratory unit. (d) interior of locker room. a partition was built inside the second laboratory container to make two separate work areas for the reagent preparation area and the qrt-pcr area (figure 1a and 1b). due to concerns about possible contamination in the reagent preparation area, additional air handling equipment was used to provide positive pressure to the reagent preparation area, thus ensuring it would remain clean. office space, supplies store, personal protective equipment (ppe) locker rooms, restroom, shower room, and a tool warehouse were also deemed necessary. a standard container was used to provide both office space and supply storage. mriglobal worked with a mobile restroom manufacturer to design a mobile trailer that would provide restroom and shower facilities as well as a locker room (figure 1d). an additional small container was used as the tool warehouse and office space for the on-site engineer. mobile diagnostic laboratory transportation and installation mriglobal was responsible for arranging transportation of these laboratories to guinea and sierra leone. to best satisfy the schedule requirements, air cargo was used. the aviastar-sp antonov an-124 ruslan (figure 2) was the only aeroplane option due to some issues including the mdl size and cargo weight and the runway length in guinea and sierra leone. figure 2: transport of mriglobal mdl from the united states to guinea and sierra leone. (a) antonov an-124 aircraft used to transport the mriglobal mdl from kansas city, missouri, united states, to guinea and sierra leone sites. (b) interior of antonov an-124 aircraft, loaded with mdl units. (c) truck unloading mdl units in moyamba, sierra leone. (d) hydraulic system used to unload mdl units in moyamba, sierra leone. once the equipment arrived at the sites, laboratory equipment was unpacked. the bscs were certified by the on-site engineers and the electrical lines were run. however, the engineering team was faced with technical challenges relating to the electrical power supply, safe water and sewer connections, laundry facilities, biohazard waste disposal, and internet connectivity. the mdls were built with the united states electrical standards of 120 volts and 60 hertz. these electrical standards are not shared in guinea or sierra leone and this resulted in difficulties replacing or servicing equipment. also, there was the issue of an unstable power supply. to address the unstable power supply, two diesel generators were purchased, and fuel contracts were established. each generator could provide light for the entire laboratory system on its own. however, the generators were significantly larger than necessary, resulting in reduced efficiency of the generators and increased fuel expenses. an automatic transfer switch was used to continuously monitor the power produced by the generator. to address water availability and to provide a sewer connection and a laundry facility, a small container was used to house the water equipment for the laboratory, including the two safety showers which were located directly outside of the laboratory exit. the container housed a water pump, pressure bladder tanks, and the laundry facility. the system was designed so it could accept water from a supplied water line or a tank stored on top of the water container, depending on what was available when the laboratory reached its final destination. the restroom and shower trailers were built with black water storage tanks but were also capable of being connected to a septic or sewer system. to ensure proper disposal of biohazard waste, a medical incinerator (elastec mediburn, carmi, illinois, united states) capable of temperatures above 1000 °c was installed. this ensured all infectious and pathological waste generated by the laboratory was safely disposed of. the environment in guinea and sierra leone also presented challenges. the mdls were consistently exposed to high temperatures, high humidity, and heavy rains, which resulted in the rapid decomposition of many elements of the mdls. these were addressed using guidelines and assessment tools provided in the ‘report on the status of emerging and dangerous pathogen laboratory network bsl-3 in select countries in the african region’.4 when possible, repairs and parts were sourced locally. when local repairs or parts were not available, they were included in quarterly laboratory supply shipments that originated in the united states. staff composition and worksite layout in sierra leone, the mdl arrived on 18 december 2014 and sample testing started on 12 january 2015 (25 days). in guinea, the time between the arrival of the mdl arrival and the start of sample testing was shortened to 13 days (21 april 2015 to 04 may 2015). the composition of the mdl team was a rotation of four scientists, two engineers, and multiple drivers at each site. the initial site layout for both sierra leone and guinea is shown in figure 3. figure 3: worksite layout for the mriglobal mobile diagnostic laboratory, moyamba, sierra leone. each laboratory unit is labelled appropriately. specimen collection blood and swab specimens were delivered to the mdl sites in conakry, guinea and moyamba, sierra leone, primarily by motorbike couriers or from the adjacent ebola treatment centre. when receiving the specimens, the staff wore coveralls, sleeves, and double gloves. ppe was stored and donned in the locker room unit (figure 1d). the surface of the specimen bucket and the sample packaging bag were disinfected by spraying with 0.5% hypochlorite solution. specimen inactivation and nucleic acid extraction the mriglobal ebola response team inactivated samples in the inactivation and extraction laboratory unit (figure 1c) wearing appropriate ppe. the ppe included coveralls, sleeves, triple gloves, and a powered air-purifying respirator system. the sample transport container was disinfected with 0.5% hypochlorite solution and opened within the bsc. for samples requiring malaria testing, the malaria ag p.f. test (sd bioline, chicago, illinois, united states) was performed. whole blood or plasma sample inactivation was performed using trizol liquid sample (thermofisher, waltham, massachusetts, united states). following inactivation, sample rna was extracted using the ez1 virus mini kit version 2.0 in the ez1 advanced xl hardware platform (qiagen, hilden, germany). extracted rna from samples was immediately sent for qrt-pcr amplification or stored at –20 °c. quantitative reverse transcription polymerase chain reaction assays the qrt-pcr assays were performed using the ebola zaire taqman and ebola zaire taqman-mgb assays provided by the department of defense and joint program manager-medical countermeasure systems critical reagents program (figure 1a). the assays were authorised for use on the 7500 fast dx real-time pcr instrument (applied biosystems, foster city, california, united states).5 the multiplex pcr steps were programmed as follows: stage 1 – 15 min at 50 °c, stage 2 – 5 min at 95 °c, stage 3 – 1 s at 95 °c, 26 s at 60 °c, stage 4 – 30 s at 40 °c.5 specimen storage the mdl was equipped with –20 °c freezers. as a result, only short-term (< 14 days) storage of patient samples was maintained. the specimens were well packed and the surface disinfected by 0.5% hypochlorite solution before storage. the mdl sites were guarded round the clock and all freezers and containers were locked. test result release samples from patients were assigned an individual, unique identification number by the emergency operations, established by each country’s ministry of health. when a sample was collected, the clinician was asked to complete a ‘viral haemorrhagic fever case investigation form’. the sample and the investigation form were marked with the case identification number and patient name and promptly transported to the laboratory. the case identification number provided a unique number for tracking the specimen from the patient, and the results of the specimen. the information in the test report included the case identification number, the ct value determined by qrt-pcr, and the confirmed result (yes, no, or suspect). according to the agreement with the health authorities, the mriglobal ebola response team did not contact hospitals directly. instead, mriglobal submitted the test report to the world health organization and a local emergency operations centre, which delivered consistent and timely results to each hospital and treatment centre. transfer of ownership the united states government supported the transfer of ownership of the supplies, materials, and equipment to the ministry of health of guinea on 23 september 2016 and to the ministry of health of sierra leone on 16 march 2017. personnel from each recipient country were trained before the transfer of ownership to allow for long-term independent maintenance of the improved diagnostic capabilities. in preparation for the transfer of ownership to the ministry of health of guinea, mriglobal developed and provided a series of trainings, each followed by a test and evaluation. training included tabletop exercise and a field operational demonstration. the tabletop exercise was designed to engage ministries of health and local and international partners in a collaborative effort to define how these new facilities and capabilities can best be merged into the existing national laboratory response systems and to capture stakeholder recommendations for improving long-term sustainability. the field training exercise was observed by evaluators and referees to gauge the capability of the newly trained staff in performing essential laboratory functions safely and effectively. the transfer of laboratory capacity to sierra leone and guinea is aimed at helping both countries fulfil the ‘core capacity requirements for surveillance and response’ as outlined in the international health regulations 2005 annex 1.6 other partner capabilities many other international partners played a role in the evd response in west africa, and there were numerous types of laboratories and laboratory diagnostics deployed. the dutch government deployed three mdl units, the chinese government deployed one mdl unit and built a bsl-3 laboratory and hospital outside of freetown, sierra leone, and the united states centers for disease control and prevention established a field laboratory in bo, sierra leone, alongside a host of other partner activities.7,8,9,10 the two mdl laboratories that were deployed by mriglobal tested 18 624 total samples without a safety incident. having adequate laboratory capacity, provided almost entirely by international partners, was key to meeting sample turn-around time criteria, proper diagnosis, contact tracing, and ultimately containment of the evd outbreak. lessons learnt the challenge assigned to mriglobal by the department of defense was to quickly deploy safe and effective laboratory diagnostic capabilities to sierra leone and guinea to address the evd outbreak. numerous international partners were or became involved in guinea and sierra leone, including but not limited to the united states centers for disease control and prevention, the chinese centre for disease control and prevention, médecins sans frontières international, public health england, world health organization, partners in health, the dutch government,8 and a consortium of nigerian scientists with support from the european union and african union. mriglobal operated the mdls commissioned by department of defense in guinea and sierra leone and tested thousands of samples safely. there were numerous challenges and lessons learned while establishing the mdls in guinea and sierra leone. many of the challenges were resolved by collaborating with the host government and other international partners. the primary goal of the mdls was to provide a biologically safe laboratory to perform timely and quality diagnostics. however, without the dedicated support of engineering and logistics staff, the project would not have achieved a high level of success. recommendations the mriglobal mdls in both conakry and freetown are still in use and will continue to be utilised by both countries, as well as international partners in the future. the diagnostic testing that is being performed in both laboratories has expanded over the past few years to include assays for influenza, severe acute respiratory syndrome coronavirus 2, dengue, chikungunya, zika, and more. using the mdls for their molecular diagnostics is highly recommended until more sustainable solutions can be provided. since their initial deployment, the mdls in sierra leone and guinea have increased both countries’ integrated disease surveillance and response systems, and adherence to international health regulations.6 in both sierra leone and guinea, molecular testing for severe acute respiratory syndrome coronavirus 2 was performed using the capacity provided by the mdl. the mriglobal mdl provides a reproducible, strategic solution for the rapid deployment of molecular diagnostics in resource-limited settings. the strength of the mriglobal mdl is the ability to rapidly build and deploy it to almost anywhere in the world. however, the mriglobal mdl is expensive (other organisations deployed significantly cheaper laboratory operations that were of greater or equal sample testing efficiency and safety) and in resource-limited settings the mdls are extremely challenging to maintain. therefore, the deployment of mdls should be carefully considered, given the cost and context. acknowledgements the authors express their deep gratitude to many partners, including the sierra leone and guinea health authorities, various donors, and local and international organisations whose contributions have helped support efforts to build quality clinical and public health laboratory systems. the authors would like to thank the support staff at mriglobal, and the staff at the us embassy in guinea and sierra leone. the authors deeply appreciate the translation services provided by david tolno and michel haba in conakry. the views expressed in the submitted article are the authors’ own, not an official position of mriglobal or the funding agency responsible, and no official endorsement should be inferred. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions l.d.p., j.c., l.k., a.c., j.c., f.b., j.n., z.s., s. sorie, d.h., a.c., p.t., b.t., f.a.h., j.b., s.a., c.r., j.m., r.h., s.p., s. shearrer, c.a., b.k., p.d., j.a., d.r., p.s., j.n., n.w., m.k., a.m., j.m., m.m. and t.b. contributed to the design and implementation of the research, to the analysis of the results, and to the writing of the manuscript. sources of support funding for the study was received from the united states defense threat reduction agency cooperative biological engagement program contracts hdtra1-08-d-0008 and hdtra1-15-c-0007. data availability data sharing is not applicable to this article, as no new data were created or analysed in this study. disclaimer the views expressed in the submitted article are the authors’ own, not an official position of mriglobal or the funding agency responsible, and no official endorsement should be inferred. references baize s, pannetier d, oestereich l, et al. emergence of zaire ebola virus in guinea. n engl j med. 2014;371:1418–1425. https://doi.org/10.1056/nejmoa1404505 world health organization. who ebola situation report [homepage on the internet]. 2016 [cited 2019 mar 06]. available from: 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a mobile biosafety level-3 laboratory in sierra leone during the 2014 ebola virus epidemic. plos negl trop dis. 2017;11(5):e0005622. https://doi.org/10.1371/journal.pntd.0005622 reusken c, smit p, pas s, et al. ebola virus laboratory response: the three dutch mobile laboratories in liberia and sierra leone. clin microbiol infect dis. 2016;1(4):1–7. https://doi.org/10.15761/cmid.1000s1003 nigeria mobile lab in sierra leone: bringing skills learned in one outbreak to another [homepage on the internet]. 2015 [cited 2020 aug 11]. available from: https://www.afro.who.int/news/nigerian-mobile-lab-sierra-leone-bringing-skills-learned-one-outbreak-another sealy tk, erickson br, taboy ch, et al. laboratory response to ebola – west africa and united states. mmwr suppl. 2016;65(suppl 3):44–49. https://doi.org/10.15585/mmwr.su6503a7 abstract introduction methods results discussion acknowledgements references about the author(s) kyle degruy centers for disease control and prevention, division of global hiv and tb, international laboratory branch, atlanta, georgia, united states katherine klein centers for disease control and prevention, division of tb elimination, laboratory branch, atlanta, georgia, united states zilma rey centers for disease control and prevention, division of global hiv and tb, international laboratory branch, atlanta, georgia, united states patricia hall centers for disease control and prevention, division of global hiv and tb, international laboratory branch, atlanta, georgia, united states andrea kim centers for disease control and prevention, division of global hiv and tb, international laboratory branch, atlanta, georgia, united states heather alexander centers for disease control and prevention, division of global hiv and tb, international laboratory branch, atlanta, georgia, united states citation degruy k, klein k, rey z, hall p, kim a, alexander h. development of dried tube specimens for xpert mtb/rif proficiency testing. afr j lab med. 2020;9(1), a1166. https://doi.org/10.4102/ajlm.v9i1.1166 original research development of dried tube specimens for xpert mtb/rif proficiency testing kyle degruy, katherine klein, zilma rey, patricia hall, andrea kim, heather alexander received: 14 jan. 2020; accepted: 24 june 2020; published: 29 sept. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: proficiency testing (pt) is part of a comprehensive quality assurance programme, which is critical to ensuring patients receive accurate and reliable diagnostic testing. implementation of the cepheid xpert® mtb/rif assay to aid in the diagnosis of tuberculosis has expanded rapidly in recent years; however, pt material for xpert mtb/rif is not readily available in many resource-limited settings. objective: to develop an accurate and precise pt material based on the dried tube specimen (dts) method, using supplies and reagents available in most tuberculosis culture laboratories. methods: dried tube specimens were produced at the united states centers for disease control and prevention from 2013 to 2015 by inactivating liquid cultures of well-characterised mycobacterial strains. ten percent of dts produced were tested with xpert mtb/rif and evaluated for accuracy and precision. results: validation testing across eight rounds of pt demonstrated that dts are highly accurate, achieving an average of 96.8% concordance with the xpert mtb/rif results from the original mycobacterial strains. dried tube specimen testing was also precise, with cycle threshold standard deviations below two cycles when inherent test cartridge variability was low. conclusion: dried tube specimens can be produced using equipment already present in tuberculosis culture laboratories, making xpert mtb/rif pt scale-up more feasible in resource-limited settings. use of dts may fill the gap in tuberculosis laboratory access to external quality assessment, which is an essential component of a comprehensive continuous quality improvement programme. keywords: external quality assessment; eqa; xpert mtb/rif; proficiency testing; dried tube specimen; dts. introduction the world health organization (who) estimated that 10.0 million people developed tuberculosis and 1.5 million died from tuberculosis disease worldwide in 2018.1 human immunodeficiency virus contributes to this epidemic, with 860 000 (8.6%) of the 10.0 million tuberculosis patients also being co-infected with hiv. approximately 251 000 people living with human immunodeficiency virus died from tuberculosis in 2018.1 rapid tuberculosis diagnosis and treatment initiation are therefore critical to reducing tuberculosis-related deaths and ongoing transmission in communities. access to rapid, quality diagnostic testing is a significant barrier to global tuberculosis elimination.2 the who estimated that approximately 3 million cases of tuberculosis disease in 2018 went undiagnosed or unreported, and only 186 772 of the estimated 500 000 people who developed either multidrug-resistant or rifampicin-resistant tuberculosis were diagnosed.1 the implementation of liquid culture and molecular methods have shortened the time-to-detection of mycobacterium tuberculosis and time-to-determination of the m. tuberculosis drug susceptibility profile.3,4 the cepheid xpert® mtb/rif assay (cepheid, sunnyvale, california, united states) is a cartridge-based, hemi-nested, real-time polymerase chain reaction assay detecting m. tuberculosis and genetic mutations associated with resistance to rifampicin.5 this assay provides test results within two hours of beginning the test when compared to conventional culture and phenotypic drug susceptibility testing, which may take eight weeks or longer.5,6 the who recommends xpert mtb/rif as one of the initial diagnostic tests for all individuals evaluated for tuberculosis.7 quality assurance (qa) is vital in the clinical laboratory for accuracy and reliability of diagnostic testing. clinicians depend on accurate and rapid results for appropriate patient diagnosis and treatment.8 rapid implementation of the xpert mtb/rif assay occurred in many resource-limited settings to quickly increase capacity for tuberculosis diagnosis and treatment initiation; unfortunately, qa activities have not kept pace with the expansion of xpert mtb/rif testing. when we initiated development of the dried tube specimen (dts) for the xpert mtb/rif external quality assessment (eqa) procedure in 2012, only 386 of 907 (43%) global xpert mtb/rif testing sites participated in eqa, and only 77 of 318 (24%) african testing sites, participated in eqa.9 quality assessment programmes for xpert mtb/rif are needed to ensure patients receive accurate tuberculosis diagnostics, rifampicin resistance testing, and follow-up test results. a comprehensive qa plan for xpert mtb/rif includes documented training and competency assessment for all operators, verification of the genexpert instrument or modules, new lot testing, temperature monitoring for testing site and kit storage, routine monitoring of quality indicators, and eqa, including onsite supervisory visits and participation in proficiency testing (pt).10,11 proficiency testing provides opportunities for the laboratory to identify errors in the testing process and implement systems to detect and prevent those errors.12 although a limited number of xpert mtb/rif pt panels are currently available to testing sites in resource-limited settings, many must be procured from commercial international providers.13 this may be cost prohibitive, and transport of infectious material can be complicated.14 because of these constraints, as well as the rapidly expanding number of testing sites, there is a critical need for countries to implement a simple and sustainable national pt programme in order to ensure the accuracy of xpert mtb/rif results. we modified the dts method originally developed for hiv rapid test pt to produce pt panels for the xpert mtb/rif assay.15 the novel dts-based xpert mtb/rif pt panel methodology described here was developed to address the need for countries to sustainably produce their own pt panels for the xpert mtb/rif assay. methods ethical considerations no patient specimen collection was required, and no patient identifiers were recorded on quality assurance tools. participation in the xpert mtb/rif quality assurance and pt programme was voluntary and free of charge. no incentives were provided. this study was approved by the cdc center for global health, division of human research protection (cgh hsr tracking # 2014-082). dried tube specimen panel preparation dried tube specimens for this study were prepared at the united states centers for disease control and prevention (cdc; atlanta, georgia, united states), international laboratory branch between 2013 and 2015. the xpert mtb/rif cartridge captures and lyses intact m. tuberculosis bacilli.5 dried tube specimens were developed using whole cell inactivation in order to preserve the cell structure of the selected mycobacterial strains. this more closely simulates dna extraction from a patient specimen. all dts preparation steps were performed in a biosafety level 3 containment laboratory with personal protective equipment, including respiratory protection. dried tube specimens were prepared using well-characterised rifampicin-resistant and rifampicin-susceptible m. tuberculosis strains and non-tuberculous mycobacteria strains obtained from the american type culture collection, who proficiency testing challenges, and the cdc’s collection of laboratory-derived m. tuberculosis strains. m. tuberculosis strains were characterised using phenotypic and genotypic methods, including the middlebrook 7h10 method of proportion drug susceptibility testing and sanger sequencing of the rpob gene.6,16 strains were inoculated into panta-supplemented bactec® mgit (mycobacteria growth indicator tube) 7 ml tubes (modified middlebrook 7h9 broth base) (becton, dickinson and company; sparks, maryland, united states) and incubated in the bactec® mgit 960® instrument (becton, dickinson and company; sparks, maryland, united states) until flagged positive for culture growth. cultures were then incubated an additional 4–6 days at 37 °c in an auxiliary incubator and inoculated onto middlebrook 7h10 agar and blood agar (remel, lenexa, kansas, united states) for enumeration of mycobacteria and contamination checks. mycobacteria were inactivated using a 2:1 ratio of xpert mtb/rif sample reagent (sr) (cepheid; sunnyvale, california, united states) to mgit culture in sterile 50 ml conical tubes (figure 1). the suspensions were incubated at 15 °c – 30 °c, and vortexed for 30 seconds every 15 minutes for a total of two hours. mgit culture/sr suspensions were neutralised with 20 ml – 25 ml of in-house prepared, sterile phosphate buffer ph 6.8 and centrifuged at 3000 × g for 15 min. the pellets were washed with an additional 45 ml phosphate buffer, centrifuged, and the supernatants were discarded. pellets were then resuspended in 5 ml of phosphate buffer, transferred to sterile glass tubes with 5–10 3 mm glass beads (thermo fisher scientific, waltham, massachusetts, united states), vortexed for 5 min, and allowed to settle for 15 min. supernatants above the beads were transferred to new sterile glass tubes, labelled as stock suspensions, and stored at 2 °c – 8 °c. inactivation verification was performed by inoculating 0.5 ml of stock suspension into mgit tubes supplemented with panta and incubating for two cycles for a total of 84 days. only stocks testing negative for growth were considered for panel preparation and distribution. figure 1: overview of dried tube specimens for xpert mtb/rif preparation procedure, 2013–2015, united states centers for disease control and prevention (atlanta, georgia, united states). pre-testing xpert mtb/rif pre-testing of five dts from each stock was conducted to evaluate dts accuracy and precision on a limited scale prior to preparing large numbers of pt samples. dried tube specimens were prepared by diluting an aliquot of each stock suspension 1:10 with in-house prepared, sterile saline, adding food grade dye at a concentration of 0.1% (volume/volume) to each dilution, and transferring 100 µl of each stock dilution into five 4 ml cryovials. cryovials were left uncapped in a class ii biosafety cabinet (bsc) until all liquid evaporated and aliquots were visibly dry. dried tube specimen samples were then capped and stored in the dark at room temperature until tested. dried tube specimens were rehydrated with 2.5 ml of sr, shaken vigorously 20 times and incubated at room temperature for 15 min with additional shaking repeated after 10 min (figure 2). the xpert mtb/rif cartridges were inoculated with approximately 2.0 ml of rehydrated samples using the transfer pipette provided with the kit, and tested immediately on a genexpert iv or genexpert viii using genexpert dx software version 4.0 (cepheid, sunnyvale, california, united states), according to manufacturer’s instructions.17 dried tube specimens prepared in 2013 were tested with xpert mtb/rif assay g4 research use only cartridges. dried tube specimens prepared in 2014 and 2015 were tested with food and drug administration-cleared xpert mtb/rif us in vitro diagnostic (ivd) cartridges. figure 2: testing dried tube specimens using xpert mtb/rif, 2013–2015, united states centers for disease control and prevention (atlanta, georgia, united states). to ensure that dts yielded accurate xpert mtb/rif results, m. tuberculosis detection and rifampicin resistance results from dts pre-testing were verified against the expected xpert mtb/rif results for the parent strain (as determined by prior phenotypic and genotypic testing). results were considered acceptable if the qualitative m. tuberculosis detection and rifampicin resistance results matched the expected results for the parent strain for all five pre-tested dts from the stock. precision was evaluated to verify uniform suspension of the organism in the stock using the cycle threshold (ct) standard deviation (sd). in line with previous studies, probe a was selected for most analysis as it was the first probe to reach the detection threshold.18 the sds of probe a ct values were calculated for all dts and compared between different samples within each panel. probe c ct values were used when probe a did not bind. the sd for the cartridge internal specimen processing control (spc) ct was also calculated for all samples and panels to investigate whether inherent xpert mtb/rif cartridge variability contributed to variation in dts ct values. to ensure that adequate amounts of inactivated organism were present in the dts and that the integrity of dna was not compromised during inactivation, the average xpert mtb/rif semi-quantitative result (i.e. the category assigned to the amount of m. tuberculosis dna based on the ct of the first probe detected) was required to fall in the medium (ct = 16–22) to low (ct = 22–28) range. stock selection and dried tube specimen panel validation the five most precise and accurate stocks (i.e. the stocks with the lowest ct sd and lowest mean ct with dts all yielding expected test results during pre-testing) were selected to aliquot dts for use in the pt programme. each xpert mtb/rif pt panel included one dts per stock, for a total of five samples. eight unique panels were prepared for use in the pt programme from 2013 (2013-a, 2013-b, 2013-c, and 2013-d) to 2015 (2014-a, 2015-a, 2015-b, and 2015-c) as described above. a strict bsc cleaning protocol (1% sodium hypochlorite followed by 70% ethanol rinse of all inner walls, sash, work surface and under work surface) was employed before aliquoting each panel. in most cases, only one sample was dried at a time. in situations where more than one sample was dried in the same bsc at the same time, all dts had the same expected result. an aliquoting order was routinely followed: (1) ‘tuberculosis not detected’ (non-tuberculous mycobacteria) samples were first aliquoted, then dried, capped, and removed; (2) ‘tuberculosis detected, rifampicin resistance not detected’ samples followed; and finally (3) ‘tuberculosis detected, rifampicin resistance detected’ samples were aliquoted last. no culture manipulation was performed in the bsc while dts samples were drying. once prepared, panels were validated by confirming accurate and precise test results for 10% of the dts produced using the same methodologies outlined above. ten dts from each stock underwent validation for panel 2013-a (50 total), 18 for panel 2013-b (90 total), 25 for panels 2013-c, 2013-d, and 2014-a (125 total per panel), 50 for panels 2015-a and 2015-b (250 total per panel), and 55 for panel 2015-c (275 total). results across all eight panels, the mean ct values for probe a (or probe c when probe a did not bind) ranged from 19.3 to 26.2, which were within the acceptable xpert mtb/rif semi-quantitative range of medium to low (data not shown). the spc ct ranged from 0.0 to 40.8 across all eight panels with a mean ct range of 23.8–27.8. when panel validation results were compared with the expected xpert mtb/rif results for the parent strain, dts tested from panels 2013-a through 2015-c ranged from 96.0% to 98.5% concordant (table 1). only three (0.2%) of the 1290 dts tested were discordant, and of those, two were false negative and one was false positive. a total of eight (0.6%) dts samples, including four from 2013-b, three from 2013-c, and one from 2013-d, yielded indeterminate rifampicin results. when examining cts for all discordant and rifampicin-indeterminate results, we found that the cts for the test cartridge spc were consistently high (35.3–38.2) with the exception of one discordant result from 2015-b (25.7). table 1: accuracy of dried tube specimen xpert mtb/rif test results collected during panel validation, 2013–2015, united states centers for disease control and prevention (atlanta, georgia, united states). a total of 30 errors were encountered while testing the 1290 dts, which resulted in an overall error rate of 2.3% (table 2). this rate ranged from 0.0% to 4.4% across panels (table 1). errors encompassed six different error codes (table 2). no ‘invalid’ or ‘no result’ results were observed. table 2: errors received during validation of dried tube specimens for 2013–2015 proficiency testing panels, 2013–2015, united states centers for disease control and prevention (atlanta, georgia, united states). while dts samples were found to be accurate (96.8% concordance overall), sample precision varied, with ct sd values ranging from 0.9 to 6.2 (figure 3). the sd of the cartridge spc also varied among dts pt samples, ranging from 0.7 to 10.5. we observed higher variability in probe a ct (sd: 1.0–6.2) and spc ct values (sd: 1.4–10.5) for panels produced in 2013 and tested with xpert mtb/rif assay g4 cartridges than for those produced in 2014 (1.0–1.9; 0.9–3.3) and 2015 (0.9–1.7; 0.7–2.9), which were tested with food and drug administration-cleared xpert mtb/rif us ivd cartridges. figure 3: standard deviations of probe a and specimen processing control cycle threshold values for dried tube specimens containing mycobacterium tuberculosis complex tested during panel validation, 2013–2015, united states centers for disease control and prevention (atlanta, georgia, united states). discussion the dts method was originally developed for hiv rapid test pt and subsequently used to produce pt panels for other assays such as syphilis, hiv viral load and malaria rapid test, with good results.15,19,20,21 this study demonstrates that an accurate and precise m. tuberculosis pt panel for the xpert mtb/rif assay can also be produced using the dts technique. the panel validation process verifies the accuracy and precision of each dts panel produced and adheres to clinical and laboratory standards institute recommendations for characterising molecular pt materials.12 the dts panels produced according to this methodology were accurate, achieving 96.8% test result concordance with parental stocks for the detection of m. tuberculosis and rifampicin resistance. interestingly, the discordant and indeterminate samples, with the exception of the 2015-b false negative discordant sample, had spc ct values above 35. a previous quantitative assessment by blakemore et al. found that xpert mtb/rif assay results with spc ct values above 34 could be quantified inaccurately.22 thus, it is possible that the majority (2 of 3) of the discordant results and all 8 of the rif indeterminate results in the dts panel evaluations could be due to factors inherent to the xpert mtb/rif assay cartridges. the overall error rate was relatively low (2.3%), and no trend in error rates was observed over the study period. eighty percent of the errors observed were due to error codes 5006 and 5007 (probe check failure). these errors often are associated with the incorrect sample volume added to the cartridge, incorrect filling or bubbles in the cartridge reaction tube, or probe integrity issues.10 the remaining errors (codes 2005, 2014, 2037 and 5011) are associated with instrument or cartridge performance. additionally, the dts method delivers a consistent amount of m. tuberculosis to each sample, such that testing of dts prepared from the same stock yielded probe a ct values with an sd similar to the sd of the spc internal control ct values in most cases. there is no current standard for molecular pt variability. however, the standard deviations observed for dts produced in 2014 and 2015 and tested with food and drug administration-cleared xpert mtb/rif us ivd cartridges are in line with those seen by scott et al. using a similar inactivation technique.18 xpert mtb/rif sr has been shown to effectively inactivate mycobacteria while leaving the cells intact.18 although this evaluation did not assess the cell structure post-inactivation, other investigators confirmed the presence of whole bacilli following sr-mediated inactivation of m. tuberculosis using flow cytometry.18 inoculation of dts stock suspensions into mgit culture provides confirmation of inactivation prior to distribution of dts. furthermore, the incubation of mgit culture for 4–6 days post-positivity did not adversely affect inactivation with sr and was found to consistently yield dts samples with the desired m. tuberculosis dna concentration for dts preparation and testing. while the accuracy and precision of dts are similar to those reported for other pt panels, the simplicity of matrix preparation and feasibility for transfer to low-resource settings set the dts xpert mtb/rif pt panel apart.13 additionally, since the procedure is similar to specimen processing for tb culture, much of the laboratory expertise, technical skill, equipment (e.g., class ii bsc, safety centrifuge, bactec mgit 960 or 320 instrument, vortex, and an auxiliary incubator), and consumables necessary for dts preparation already exist in tuberculosis culture laboratories in resource-limited countries. many national tuberculosis reference laboratories in these settings have implemented bactec mgit 960 culture and drug susceptibility testing in recent years, making mgit 7 ml tube media the most common liquid media for m. tuberculosis culture and detection.23 lastly, since dts are prepared within the tubes utilised for sample rehydration, testing of dts does not require additional laboratory supplies. not only are expertise, equipment, and supplies readily available, but dts are also estimated to be low cost. regarding transport, parekh et al. reported that the use of dts for hiv pt eliminates the need for cold chain and results in a significant reduction in shipping costs.15 reagent costs are also low, as approximately 500 dts can be made from a single inactivated mgit culture. it is therefore possible for one culture laboratory, such as a national tuberculosis reference laboratory, to produce and validate enough dts to provide pt material for all testing sites in a country. the dts method can thus provide resource-limited countries with a suitable pt material to create and manage their own sustainable xpert mtb/rif pt programme. external quality assessment using dts can serve as an important component of a laboratory qa programme, which is necessary to ensure that patients receive accurate and reliable diagnostic testing services.11 challenges and limitations the primary challenge associated with producing dts was variability between ct values from separate aliquots of the same dilution. however, the inherent cartridge-to-cartridge variability in ct values that was observed when comparing the sd of probe a to the sd of the spc likely plays a role in the variability observed in dts ct values. after the xpert mtb/rif assay gained food and drug administration clearance in 2013, we transitioned from using xpert mtb/rif assay g4 research use only cartridges in 2013 to xpert mtb/rif us ivd cartridges in 2014 and 2015. the range of sds of probe a ct values decreased from 1.0–6.2 in 2013 to 0.9–1.9 in 2014–2015. this decrease further suggests that the ct value variability and high sd values observed were likely due to inconsistencies between cartridges. however, the variability of dts could also be due to mycobacteria (particularly m. tuberculosis) clumping in mgit cultures, leading to an uneven dts stock suspension and difficulty in achieving equal numbers of bacilli in each aliquot. additional evaluations are underway to improve both the accuracy and precision of dts. another limitation of using the dts technique for pt is that the dts sample type (inactivated, dried mycobacteria) does not resemble the most commonly tested clinical sample type, sputum. while the use of dts for pt does not allow for the complete evaluation of all the procedural steps or potential pitfalls involved in xpert mtb/rif testing of sputum, it could be an adequate substitute. for example, creating lyophilised, m. tuberculosis-spiked sputum samples would involve the purchase and maintenance of expensive equipment, such as lyophilisers, and increase biosafety risk for laboratorians. alternately, the use of liquid samples increases biohazard risk to both shipping and laboratory personnel and increases transportation costs. therefore, the safety and cost-saving benefits of dts may outweigh potential disadvantages associated with assessing proficiency of xpert mtb/rif testing using dried, inactivated m. tuberculosis in resource-limited settings. recommendations and next steps we are continuing to study and refine the technique for producing dts, including the investigation of heat inactivation of m. tuberculosis for improved biosafety, stability and sample accuracy and precision. since development of this novel pt technology, a voluntary xpert mtb/rif dts-based pt programme has been rolled out to 26 countries and united states-affiliated territories, as described in ‘a global proficiency testing program for xpert mtb/rif using dried tube specimens’ by klein et al.24 in addition, in 2016 we began the transfer of dts technology to countries as part of a pt package that includes feedback and corrective actions to sustainably improve the quality of international diagnostic testing services. in 2019, the scope of the pt programme was increased to include the xpert mrb/rif ultra assay (cepheid, sunnyvale, california, united states) where the same panel prepared for xpert mtb/rif pt was also successfully validated for use as pt for the xpert mtb/rif ultra assay. conclusion proficiency testing for the xpert mtb/rif assay, as part of a comprehensive qa programme, should be a priority for every national tuberculosis programme. the dts technique creates accurate, precise, and safe pt panels. designing this preparation procedure around the availability of equipment and reagents in national tuberculosis reference laboratories in resource-limited countries promotes the transfer of dts technology to both national and regional programmes and assists countries in implementing consistent, sustainable eqa programmes. acknowledgements we thank bharat parekh for his inspiration and guidance, and adeboye adelekan, elizabeth prentince and wendy stevens for assistance accessing laboratories willing to pre-test dts panels. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions k.d. and h.a. conceived and designed the experiments, k.d. and k.k. performed the experiments and analysed the data, and k.d., k.k., z.r. a.k. and p.h. wrote the paper. k.d., k.k., z.r., p.h., a.k. and h.a. reviewed and approved the manuscript. sources of support this research has been supported by the president’s emergency plan for aids relief (pepfar) through the centers for disease control and prevention, atlanta georgia, united states. data availability statement data access level is public without identifying information of proficiency testers, testing sites and country. disclaimer the findings and conclusions in this paper are those of the authors and do not necessarily represent the official position of the centers for disease control and prevention or other funding agencies. references world health organization. global tuberculosis report 2019 [homepage on the internet]. c2019 [cited 2020 may 20]. geneva: who press. available from: https://www.who.int/tb/publications/global_report/en/ world health organization. the end tb strategy [homepage on the internet]. c2014 [cited 2020 may 20]. geneva: who press. available from: 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aidoo m. field assessment of dried plasmodium falciparum samples for malaria rapid diagnostic test quality control and proficiency testing in ethiopia. malar j. 2015;14:11. https://doi.org/10.1186/s12936-014-0524-z blakemore r, nabeta p, davidow al, et al. a multisite assessment of the quantitative capabilities of the xpert mtb/rif assay. am j respir crit care med. 2011;184(9):1076–1084. https://doi.org/10.1164/rccm.201103-0536oc foundation for innovative new diagnostics. liquid culture and drug susceptibility testing [homepage on the internet]. switzerland: foundation for innovative new diagnostics; c2016 [cited 2020 may 20]. available from: https://www.finddx.org/wp-content/uploads/2016/02/mgit_manual_nov2006.pdf klein k, degruy k, rey z, et al. a global proficiency testing program for xpert mtb/rif using dried tube specimens, 2013–2015. afr j lab med. 2020. forthcoming. article information authors: adino d. lulie1 tilahun m. hiwotu1 achamyeleh mulugeta1 adisu kebede1 habtamu asrat1 abnet abebe1 dereje yenealem1 ebise abose1 wondwossen kassa1 amha kebede1 mary k. linde2,3 gonfa ayana1 affiliations: 1ethiopian public health institute (ephi), ethiopia2shawnee state university, portsmouth, ohio, united states 3american society for clinical pathology (ascp), united states correspondence to: adino lulie postal address: ethiopian public health institute, patriot street, po box 1242, ethiopia dates: received: 06 aug. 2014 accepted: 15 sept. 2014 published: 03 nov. 2014 how to cite this article: lulie ad, hiwotu tm, mulugeta a, et al. perceptions and attitudes toward slmta amongst laboratory and hospital professionals in ethiopia. afr j lab med. 2014;3(2), art. #233, 6 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.233 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. perceptions and attitudes toward slmta amongst laboratory and hospital professionals in ethiopia in this original research... open access • abstract • introduction • research methods and design    • data analysis procedures • results    • survey       • regional-level laboratory managers’ perceptions of slmta       • facility-level laboratory quality officers’ and managers’ perceptions of slmta       • hospital chief executive officers’ perceptions of slmta    • focus groups       • commitment, integration, output and impact of slmta on healthcare systems       • cost and sustainability       • the role of mentorship and partnership       • challenges of slmta implementation • discussion    • conclusion and recommendations • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: strengthening laboratory management toward accreditation (slmta) is a competency-based management training programme. assessing health professionals’ views of slmta provides feedback to inform program planning, implementation and evaluation of slmta's training, communication and mentorship components.objectives: to assess laboratory professionals’ and hospital chief executive officers’ (ceos) perceptions and attitudes toward the slmta programme in ethiopia. methods: a cross-sectional descriptive survey was conducted in march 2013 using a structured questionnaire to collect qualitative data from 72 laboratory professionals and hospital ceos from 17 health facilities, representing all regions and two city administrations in ethiopia. focus groups were conducted with laboratory professionals and hospital administration to gain insight into the strengths and challenges of the slmta programme so as to guide future planning and implementation. results: ethiopian laboratory professionals at all levels had a supportive attitude toward the slmta programme. they believed that slmta substantially improved laboratory services and acted as a catalyst for total healthcare reform and improvement. they also noted that the slmta programme achieved marked progress in laboratory supply chain, sample referral, instrument maintenance and data management systems. in contrast, nearly half of the participating hospital ceos, especially those associated with low-scoring laboratories, were sceptical about the slmta programme, believing that the benefits of slmta were outweighed by the level of human resources and time commitment required. they also voiced concerns about the cost and sustainability of slmta. conclusion: this study highlights the need for stronger engagement and advocacy with hospital administration and the importance of addressing concerns about the cost and sustainability of the slmta programme. introduction top ↑ laboratory services are an integral part of clinical decision-making and contribute to various aspects of health services, including the making of diagnostic and therapeutic decisions for patients, as well as disease monitoring and prevention.11 historically, laboratories in developing countries have been under-resourced and marked by poor performance. these issues have fostered distrust in laboratory data amongst clinicians, reinforcing cycles of inadequate investment in laboratory systems. however, with recent emphasis on improving access to testing so as to meet the needs of expanded treatment and prevention programmes for hiv and other major diseases, the demand for diagnostics in resource-limited settings has increased substantially.12,3laboratory accreditation is used widely in developed countries to encourage and document improvements in the quality and reliability of test results. however, for laboratories in developing countries accreditation is a daunting challenge that only a handful of public laboratories in africa have surmounted.14 as a result, the world health organization’s regional office for africa has suggested that member countries improve the performance standards of their laboratories by implementing laboratory quality management systems (lqms), establishing intermediate quality level goals and working toward accreditation in a stepwise manner.15 the laboratory network in ethiopia is organised according to size and scope: health centre laboratories, hospital laboratories, regional reference laboratories and national reference laboratories. the ethiopian public health institute (ephi), formerly known as the ethiopian health and nutrition research institute, developed its first laboratory master plan in 2005 with a focus on building laboratory systems in the country. one of the strategic objectives was to expand and strengthen the national lqms. to accelerate the implementation of the lqms, ethiopia adopted the strengthening laboratory management toward accreditation (slmta) programme, a competency-based management training programme that uses a series of short didactic courses and work-based learning projects to bring about immediate and measurable laboratory improvements and to prepare laboratories for accreditation.16,7 in 2010–2012, ethiopia implemented the slmta programme in two cohorts with a total of 45 laboratories, comprising national and regional reference laboratories, referral hospital laboratories and district hospital laboratories throughout the country.18 hiwotu et al. detail slmta implementation in ethiopia, documenting substantial improvements to laboratory quality.19 since its introduction in 2009, slmta has been implemented in 617 laboratories in 47 countries.110 however, no study has yet been published assessing the attitudes of healthcare professionals toward the programme. this study aimed to assess laboratory professionals’ and hospital chief executive officers’ (ceos) perceptions and attitudes toward slmta programme implementation in ethiopia in order to provide feedback for programme planning, implementation, communication and mentorship activities. research methods and design top ↑ a cross-sectional descriptive study was conducted in march 2013 using structured surveys and focus group discussions. seventeen facilities in total were selected purposively so as to ensure representation by geographic region and laboratory type, with one laboratory selected for each of the nine regions and two city administrations in the country, two national specialised hospitals in the capital city (addis ababa) and four additional hospitals in larger regions. all hospitals included in the survey had participated in the ethiopian hospital management initiative and hospital ceos had received master of hospital administration degrees through the ministry of public health as part of this programme.111 a total of 72 people – 55 laboratory professionals (three to four per facility) and 17 hospital ceos (one per facility) – were surveyed using questionnaires tailored to each group of respondents (table 1). hard and electronic copies of the questionnaires were distributed and all questionnaires were completed and returned to study investigators. table 1: profiles of laboratory professionals focus group participants. two focus groups representing 12 of the 17 facilities were conducted, each with five laboratory quality officers and three laboratory managers (table 2). in two-hour long interviews, investigators collected detailed data through open-ended questions and group discussions on the importance and sustainability of the slmta programme, the role of partners and the challenges facing the programme. participants also shared their insights for future plans and implementation. of the 16 focus group participants, three quality officers and one laboratory manager had also participated in the survey. the principal investigator, who had experience with qualitative data collection methods, facilitated the discussion, whilst the co-investigator, experienced in public health research methodology, captured the discussions through note-taking and audio recording. table 2: types of evaluation questions asked in surveys of laboratory professionals and hospital chief executive officers. data analysis procedures quantitative data were entered and analysed using microsoft1® excel, after which descriptive statistics were used to present the findings. qualitative data were analysed by transcribing and categorising responses. results top ↑ survey regional-level laboratory managers’ perceptions of slmta data collected from the eight regional laboratory managers showed an overwhelming agreement on the importance of the slmta programme. all of the participants agreed that the programme had brought substantial improvements to the quality of laboratory services, one saying that slmta was ‘a catalyst for healthcare reform’ in ethiopia. respondents reported that stock outs of reagents were reduced, data management systems were improved and interruption of service resulting from equipment problems was minimised.respondents cited the commitment and participation of trained laboratory personnel, the national implementing unit and other implementing partners as major driving forces in the successful implementation of the slmta programme. all participating laboratory managers agreed that slmta has helped to accelerate improvements in laboratory networking, sample referral systems, competency of laboratory professionals through need-based trainings, laboratory equipment maintenance and electronic or paper-based data management. despite the significant benefits of the slmta programme, two of the eight respondents indicated that laboratory quality improvements were not as large as they had expected. the main challenges they faced in implementing the programme included high turnover of trained staff, inconsistent and inadequately-trained mentors and a lack of awareness regarding the importance of slmta at some facilities. the respondents recommended further training for laboratory staff and sensitisation workshops for medical directors and hospital ceos. for sustainable slmta implementation throughout the country, respondents suggested national strategic planning and empowerment of regional laboratories to oversee the programme in their respective regions. facility-level laboratory quality officers’ and managers’ perceptions of slmta of the 47 facility laboratory managers and quality officers surveyed, 40 (85%) viewed the slmta programme as being a critical step in the laboratory quality improvement process. all participants reported that slmta impacted every laboratory process and believed that positive and sustainable changes had occurred at all levels of the laboratory. they reported more confidence about the procedures to follow as well as a better understanding of the tests they performed, and felt that they were given more responsibilities for laboratory quality improvement processes.thirty-five (74%) of the participants reported satisfaction with the slmta training methods and that they gained important knowledge and experience. the remaining 12 (26%) respondents had various complaints about the training methods. for example, one respondent (participant 03) reported that ‘the programme was launched without availing computers and providing basic computer skill training’ as there were no computers available at the training facility. half of the participating quality officers and laboratory managers related that, even though slmta demanded more resources than were currently available, improvements and changes were vital for their laboratories. they reported that the ministry planned to provide the resources necessary for accreditation preparation. the other half indicated that slmta’s value for accreditation preparation was low in comparison to the resources and time required to implement. one said, ‘[w]e were spending so much of our time preparing different documents that had no potential impact on the quality of laboratory services’ (participant 01). thirty-five (74%) respondents indicated that they faced challenges resulting from a lack of commitment on the part of laboratory staff and management. the remaining 12 (26%) respondents said that staff and management commitment was not a problem in their facilities and that slmta implementation was facilitated by post-training orientation, close communication with management, scheduling of regular staff meetings and motivation of the staff and management. all participants agreed that slmta had improved communication between laboratory staff and management and had led to measurable quality improvements. they reported that the most dramatic improvements were seen in reduced turnaround times, decreased equipment down times, new and functional data management systems and minimised supply lead times. additionally, laboratory logistics information systems had been implemented and storage conditions improved. twenty-eight (60%) quality officers and laboratory managers indicated that there had been no regular mentor visits in their laboratories, either from ephi or from implementing partners. all participants cited a lack of consistency amongst mentors and limited time for mentorship as being critical barriers to slmta implementation. hospital chief executive officers’ perceptions of slmta from the survey of 17 hospital ceos, 10 (59%) understood the importance, requirements and desired outcomes of the slmta programme, whilst seven (41%) were uncertain. the notable difference between the two groups was that the former worked more closely with laboratory management.all 17 hospital ceos agreed that the programme was resource-demanding and focused more on documentation than on actual laboratory testing. eight (47%) believed that slmta was of insufficient value in their facilities given the significant amount of precious human resources consumed. seven of these eight ceos were from facilities that had scored zero stars at the exit audit. on the other hand, six (35%) ceos whose facilities had improved and had scored one to three stars at the exit audit noticed the laboratory improvements and felt that the programme was valuable. nine of the 17 hospital ceos (52.9%) reported that they were so impressed with the programme that they were using the laboratory as a model for transforming their entire hospital system. focus groups commitment, integration, output and impact of slmta on healthcare systems focus group members raised several issues during their discussions regarding outputs and impacts of slmta. they noted that they were proud of the results, which measurably improved quality of patient care. one participant shared his opinion:‘slmta brings an irreversible laboratory revolution in our country.’ (participant 07) he continued by saying that laboratory professionals are the key to quality health services, as acknowledged by the ministry of public health. other respondents noted that the slmta programme promotes the standardisation of laboratory services, which contributes to customer satisfaction and confidence. they reported that there were now stronger and clearer laboratory policies and referral systems and that communication between laboratories had improved. additional reported benefits of slmta included continuous improvement of services, opportunities for laboratories to conduct self-audits and movement toward compliance with national and international regulatory requirements. participants noted that slmta has been a catalyst for total health care reform and improvement in ethiopia. one focus group participant noted the new integrated pharmaceutical logistics information system, which ‘improved the supply chain system of all pharmaceutical drugs in the country’ (participant 05), as well as hospital reform initiatives aimed at improving the quality of the healthcare system, both of which were inspired by the slmta programme. in addition, focus group participants generally agreed that slmta sparked an institution-wide revolution in safety and infection prevention practices throughout their hospitals. all laboratory staff were trained in basic laboratory bio-safety and post-exposure prophylaxis principles in facilities that implemented slmta. as one participant said: ‘vaccination and safe waste disposal are coming true in our facility, [a reality that seemed] beyond our ability and capacity before.’ (participant 16) cost and sustainability a majority of the focus group participants agreed that the programme required more resources and time than they had anticipated. one participant said:‘even though i have no doubt of the importance of slmta for my country, the programme requires more resources which will hinder implementation in resource-limited situations.’ (participant 04) on the other hand, some participants did not notice the cost of slmta, since the programme is mainly funded by support from the government and implementing partners. participants expressed great concern about sustainability. all agreed on its importance, but noted that neither ephi nor the implementing partners who initiated slmta in ethiopian laboratories had designed strategies for sustaining the programme. health facility management and the regional health bureau were not made aware of the need and cost of implementing and sustaining slmta. furthermore, the roles of facility management and of the regional health bureau have not been clearly defined; this complicates the issue, since participants believe that sustaining the slmta programme is the responsibility of the government. participants agreed that sufficient action had not been taken to decentralise responsibility from the government to the management at their facilities, primarily because of a lack of regular communication between management at the facilities and ephi. they recommended that all stakeholders, including laboratory management, facility management, ephi and implementing partners, maintain close communication in order to share the responsibility of sustaining the slmta programme. the role of mentorship and partnership participants agreed that regular mentorship as part of the slmta programme was important, but noted that this component was absent in some of their facilities. major concerns included deficiencies in the number of trained mentors, uncoordinated planning between ephi and regional facilities and lack of awareness of the importance of mentorship for quality management systems. participants pointed out that the time allocated for mentorship was insufficient and that there were inconsistent skill levels amongst mentors. one shared her experience:‘even though we have limited mentorship services, we have tremendous knowledge and experience from mentors that improves our facility performance.’ (participant 06) the majority of the discussants agreed that implementing partners have played an important role in slmta execution. they have worked in collaboration with ephi on capacity-building activities, such as renovation of laboratories, donation of modern laboratory equipment and provision of mentorship and basic training to laboratory staff on safety and quality management. one regional hospital representative shared: ‘the role of partners in the slmta programme was an essential piece for slmta implementation in our facility.’ (participant 15) however, some participants argued that the role of partners should be to build capacity and focus on short-term solutions, rather than to provide long-term support for slmta. one participant expressed concerns that partners have provided ‘a lot of support, but there was no joint planning between facilities and partners’ (participant 10). other participants wanted to take on more of the responsibility, saying: ‘we want the help of partners based on our plans and requirements.’ (participant 09) challenges of slmta implementation common challenges identified included lack of commitment from facility management and laboratory staff, turnover of trained staff, insufficient regular mentorship, inadequate laboratory infrastructure, lack of awareness of the importance of the slmta programme and scarce resources. other impediments included frequent equipment malfunction due to power interruption and lack of coordination between implementing partners and ephi. discussion top ↑ the attitudes of laboratory personnel in this study were generally supportive of the slmta programme. they recognised that slmta fundamentally restructured laboratory operations, heightened the confidence of laboratory professionals, improved data management systems and reduced turnaround times, equipment down times and supply lead times. study participants agreed that the programme resulted in substantial improvements to the quality of laboratory services, laid the foundation for the implementation of an integrated pharmaceutical logistics information system and served as a catalyst for total healthcare reform and improvement in ethiopia. laboratory staff concerns focused on the issues of retention of trained staff, lack of regular mentor visits and resource requirements with regard to slmta implementation.hospital ceos were more sceptical of slmta and raised concerns regarding programme costs and the prolonged process associated with implementation. in addition, many hospital ceos did not have a clear understanding of the benefits of the slmta programme and most of those in hospitals whose laboratories remained at the zero-star level at the exit audit did not believe that the value of the improvements merited the human resources and time consumed. the roles of hospital management and regional health bureaus should be afforded greater attention in the implementation of slmta. overall, the greatest concern was the cost and sustainability of slmta. previous studies have found similar results. alkhenizan and shaw conducted a systematic review of the global literature on the attitude of healthcare professionals toward accreditation.112 two of the 17 studies identified focused on the laboratory personnel’s perceptions towards accreditation. both studies found that most of the respondents preferred to work in an accredited laboratory because accreditation increased their confidence in the procedures they followed. however, the majority had concerns about the cost of accreditation and whether it had an effect on the quality of laboratory results. the interface between laboratories and clinicians is important, as clinicians play a critical role in the use of laboratory services and test results; however, physicians and nurses were not interviewed in this study. an evaluation of their attitudes toward laboratory services before and after slmta implementation and their perspective on slmta in general would provide additional relevant information. conclusion and recommendations laboratory professionals had a positive attitude toward slmta implementation in ethiopia, seeing it as a driving force for substantial improvements in sample referral linkage, laboratory commodity management and laboratory data management systems. half of the hospital ceos were positive about the programme, whilst the other half, mainly those with low-scoring laboratories, were sceptical, highlighting the need for stronger engagement with hospital administration in order to address concerns about cost and sustainability. future studies focusing on the cost-benefit of the slmta programme and long-term sustainability of results within slmta laboratories, as well as of the programme as a whole, would be beneficial. acknowledgements top ↑ this work was sponsored and completed by ephi, ethiopia. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions a.d.l. (ephi) was the primary author of the manuscript, designed the methodology and questionnaire, supervised all the procedures and finalised the manuscript. t.m.h. (ephi) was responsible for the design of the focus group discussion sessions, as well as the qualitative part of the project, and jointly conceived the study with the primary author. a.m. (ephi) conducted the literature review. adisu k. (ephi) collected and interpreted the data and made conceptual contributions. h.a. (ephi) participated in the writing up of the results and advised on data analysis. a.a. (ephi) collected and interpreted the data and made conceptual contributions. d.y. (ephi) reviewed the flow of information and participated in the writing of the discussion. e.a. (ephi), w.k. (ephi) and amha k. (ephi) made conceptual contributions. m.k.l. (shawnee state university; ascp) was responsible for reviewing and structuring the manuscript. g.a. (ephi) is the who stepwise laboratory quality improvement process towards accreditation (slipta) focal point for ethiopia, proving critical insight and follow up on the project. references top ↑ 1. nkengasong n. strengthening laboratory services and systems in resource-poor countries [editorial]. am j clinpathol. 2009;131(6):774. http://dx.doi.org/10.1309/ajcp8gyx8ktkdatz2. abimiku ag. building laboratory infrastructure to support scale-up of hiv/aids treatment, care, and prevention: in-country experience. am j clinpathol. 2009;131(6):875–886. http://dx.doi.org/10.1309/ajcpelmg6gx6rqsm 3. massambu c, mwangi c. the tanzania experience: clinical laboratory testing harmonization and equipment standardization at different levels of a tiered health laboratory system. am j clinpathol. 2009;131(6):861–866. http://dx.doi.org/10.1309/ajcp3zaafupcixig 4. schroeder lf, amukele t. medical laboratories in sub-saharan africa that meet international quality standards. am j clin pathol.2014;141(6):791–795. http://dx.doi.org/10.1309/ajcpq5ktkagsscfn 5. gershy-damet gm, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clinpathol. 2010;134(3):393–400. http://dx.doi.org/10.1309/ajcptuuc2v1wjqbm 6. yao k, maruta t, luman et, nkengasong jn. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. 2014;3(2), art. #194, 8 pages. 7. yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clinpathol. 2010;134(3):401–409. http://dx.doi.org/10.1309/ajcpnbbl53fwuiqj 8. ephi, federal democratic republic of ethiopia. laboratories upgrade towards who-afro laboratory accreditation performance report. addis ababa, ethiopia; 2013. 9. hiwotu tm, ayana g, mulugeta a, et al. laboratory system strengthening and quality improvement in ethiopia. afr j lab med. 2014;3(2), art. #228, 6 pages. http//dx.doi.org/10.4102/ajlmv3i2.228 10. yao k, luman et, slmta collaborating authors. evidence from 617 laboratories in 47 countries for slmta-driven improvement in quality management systems. afr j lab med. 2014;3(2), art. #262, 11 pages. 11. kebede s, abebe y, wolde m, et al. educating leaders in hospital management: a new model in sub-saharan africa. int j qual health care. 2010;22(1):39–43. http://dx.doi.org/10.1093/intqhc/mzp051 12. alkhenizan a, shaw c. the attitude of health care professionals towards accreditation: a systematic review of the literature. j famcommunity med.2012;19(2):74–80. abstract introduction methods results discussion acknowledgements references about the author(s) nada a. abdelrahim department of medical microbiology, faculty of medical laboratory sciences, nile university, khartoum, sudan nahla mohamed department of virology, faculty of clinical microbiology, umeå university, umeå, sweden magnus evander department of virology, faculty of clinical microbiology, umeå university, umeå, sweden clas ahlm department of infection and immunology, faculty of clinical microbiology, umeå university, umeå, sweden imad m. fadl-elmula department of pathology and clinical genetics, faculty of medicine, al-neelain university, khartoum, sudan assafa academy, kartoum, sudan citation abdelrahim na, mohamed n, evander m, ahlm c, fadl-elmula im. human herpes virus type-6 is associated with central nervous system infections in children in sudan. afr j lab med. 2022;11(1), a1718. https://doi.org/10.4102/ajlm.v11i1.1718 original research human herpes virus type-6 is associated with central nervous system infections in children in sudan nada a. abdelrahim, nahla mohamed, magnus evander, clas ahlm, imad m. fadl-elmula received: 29 aug. 2021; accepted: 25 may 2022; published: 22 sept. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: human herpes virus type-6 (hhv-6) is increasingly recognised as a febrile agent in children. however, less is known in sub-saharan african countries, including sudan. objective: we investigated the involvement of hhv-6 in paediatric central nervous system (cns) infections in khartoum, sudan. methods: febrile patients aged up to 15 years with suspected cns infections at omdurman hospital for children from 01 december 2009 to 01 august 2010 were included. viral dna was extracted from leftover cerebrospinal fluid (csf) specimens and quantitatively amplified by real-time polymerase chain reaction (pcr) at umeå university in sweden. results: of 503 csf specimens, 13 (2.6%) were positive for hhv-6 (33.0% [13/40 of cases with proven infectious meningitis]). the median thermal cycle threshold for all hhv-6-positive specimens was 38 (range: 31.9–40.8). the median number of virus copies was 281.3/pcr run (1 × 105 copies/ml csf; range: 30–44 × 103 copies/pcr run [12 × 103 – 18 × 106 copies/ml csf]). all positive patients presented with fever and vomiting; 86.0% had seizures. the male-to-female ratio was 1:1; 50.0% were toddlers, 42.0% infants and 8.0% teenagers. most (83.0%) were admitted in the dry season and 17.0% in the rainy season. cerebrospinal fluid leukocytosis was seen in 33.0%, csf glucose levels were normal in 86.0% and low in 14.0%, and csf protein levels were low in 14.0% and high in 43.0%. conclusion: among children in sudan with cns infections, hhv-6 is common. studies on the existence and spread of hhv-6 chromosomal integration in this population are needed. keywords: hhv-6; viral neuroinfections; viral meningitis; aseptic meningitis; real-time pcr. introduction human herpes virus type-6 (hhv-6) is a major cause of acute febrile illnesses in young children1 where most are infected by the age of three.2 the virus remains latent in white blood cells (i.e. monocytes and macrophages) following a primary infection and persistence in salivary glands.3 reactivation of hhv-6 can occur in the case of an immunosuppression which may result in complications affecting various systems including the central nervous system (cns).3 young immunocompetent children who suffer from fever and seizures can develop cns disease in primary hhv-6 infections.4,5 in this matter, several authors have described the major role that could be played by the virus as a cause of paediatric neuroinfections.6,7,8,9,10,11 detecting hhv-6 nucleic acids in the cerebrospinal fluid (csf) by molecular assays indicates active virus replication, hence cns infection. this interpretation is complicated by the phenomenon of hhv-6 chromosomal integration.12,13 the only human herpes virus that is found integrated in the human genome and can be passed on vertically from parent to child is hhv-6.14 this occurs occasionally, and is claimed to be detected by simply identifying persistently high concentrations of hhv-6 nucleic acids in blood because of chromosomal integration in white blood cells.12,13 in contrast, integrated hhv-6 dna is highly unexpected in normal cell-free body compartments, including the csf.11,13,15 ward16 stated that viral load should be high to identify a condition as chromosomal integration and low virus copies would indicate an infection. normal csf glucose with normal or elevated proteins is the usual finding in viral infections of the cns.17 increased numbers of white blood cells (˃ 5 cell/mm3) in csf indicates inflammation; however, normal csf cellular counts in patients with proven cns infections have been frequently reported.17,18,19,20 it is therefore recommended not to limit hhv-6 testing to patients with increased csf cellular counts.21 most patients recover fully from an hhv-6 infection, but rare complications leading to neurological impairments or death can also occur.22,23 little is known on hhv-6 infections in saharan and sub-saharan africa, and the infection has never been investigated in sudan. we, therefore, intended to identify the involvement of hhv-6 in cns infections in a large group of children in khartoum, sudan. this report is a part of a larger study on the microbial aetiologies of cns infections in this population. methods ethical considerations the ethical clearance for conducting this study was obtained from the ethical review board of the national center for neurological sciences in september 2009. patient consent was determined to be unnecessary and was waived. patients were not contacted directly; data were obtained from hospital files and were kept anonymous at all stages of the study. excess specimens were obtained from the hospitals main laboratory after all officially requested tests were applied. permission to collect data and specimens was granted from hospital authorities verbally (from hospital general director; authors’ attestation on file) and in writing (from laboratories administration, federal ministry of health). study materials a total of 503 csf specimens were obtained from febrile (˃ 37 °c) meningitis-suspected attendees of the omdurman hospital for children in khartoum, sudan. patients aged 0 to 15 years who were admitted between 01 december 2009 and 01 august 2010 were included. clinical and demographic data were obtained from hospital records retrospectively. routine csf analyses were performed at the microbiology laboratory of omdurman hospital. an aliquot of csf was frozen at −80 °c for further analysis for hhv-6 dna at the department of clinical microbiology, umeå university, umeå, sweden. human herpes virus type-6 dna was extracted then quantitatively amplified by real-time polymerase chain reaction (pcr) using taqman® (eurogentec®, seraing, liège, belgium) method. viral dna extraction the qiaamp ultrasens virus technology kit (qiagen®, hilden, germany) was used for viral dna extraction in csf. one ml specimen was used to concentrate viral dna: buffer ac (qiagen®, hilden, germany) was added, shortly incubated, then sediment by low g-force (i.e. 1000–1200 × g) centrifugation (eppindorf centrifuge 5415d®, hamburg, germany) to pellet nucleic acid complexes. the supernatant was discarded; the pellet was re-suspended in buffer ar (qiagen®, hilden, germany) and proteinase k and incubated for 10 min at 40°c. binding conditions were adjusted by adding buffer ab (qiagen®, hilden, germany); the lysate was applied to the silica gel membrane spin column. during brief centrifugation (i.e. 3000–5000 × g), dna selectively binds to the membrane. remaining contaminants and enzyme inhibitors were removed by centrifugation (i.e. 3000–5000 × g) in two wash steps using buffer aw1 then buffer aw2. pure viral nucleic acids were eluted in 30 µl low-salt buffer ave (qiagen®, hilden, germany) twice. each elute (60 µl) was divided into two aliquots (30 µl each) and preserved at −80 °c. taqman® real-time polymerase chain reaction viral gdna amplification and detection was performed by real-time analysis using the applied biosystems® 7900ht fast real-time pcr system (foster city, california, united states) and the taqman® (eurogentec®, seraing, liège, belgium) probe (reporter dye famtm on 5´ end and quencher dye tamratm on 3´ end). the lower assay detection limit is one virus copy per ml specimen. forward and reverse primers (figure 1) and probes (dna technology®, aarhus, denmark) were diluted to reach final working concentration of 25 µm (standardised concentration by umeå university hospital). commercially provided oligonucleotide products were diluted to the suitable working solution and the recommendation of quantitect® (qiagen®, hilden, germany) q pcr protocol was followed. a pcr master mix solution was prepared for harry (hhv-6) primers (figure 1) as follow: volumes of 12.5 µl of 2 × quantitecttm probe pcr master mix (qiagen®, hilden, germany), 1.0 µl forward primer (final concentration 25 µm), 1.0 µl reverse primer (final concentration 25 µm), 0.8 µl probe (final concentration 20 µm) and 7.2 µl rnase-free water (ambion®, waltham, massachusetts, united states) were added into 2.5 µl template gdna to complete a total reaction volume of 25.0 µl per single pcr reaction. the mixture was pulse-vortexed (vortex-genie pulse®, bohemia, new york, united states) and centrifuged briefly (i.e. 3000 × g). then, 22.5 µl single reaction mix was transferred into a well of a microamptm optical 96-well reaction plate (applied biosystems®, foster city, california, united states) to which 2.5 µl template gdna was added to reach total volume of 25.0 µl per one reaction. figure 1: specifications of human herpes virus type 6 primers and probe in khartoum, sudan, december 2009 – august 2010. the pcr plates were covered by microamptm optical adhesive film (applied biosystems®), concentrated at the bottom of the plate wells by spinning at low speed in a cold centrifuge (i.e. 1200 × g) using allegratm x-12r centrifuge (beckman coulter®, brea, california, united states). each pcr reaction plate was designed for carrying eight standard dilutions, one negative reverse transcriptase preparation, one non-template control containing ddh2o and duplicates or triplicates of each experimental dna template. the standard used for pcr had 5 × 106 (5e6) virus copies (obtained from the virology department, umeå university hospital). the first standard preparation was used without dilution, having a concentration of 5e6 copies, followed by 1/10 serial dilutions for the subsequent seven preparations to reach final concentrations of 5e5, 5e4, 5e3, 5e2, 5e1, 5 and 1 virus copy. real-time cycler thermal condition was as follows: heating at 50 °c for 2 min, initial activation of hotstartaq® dna polymerase (qiagen®, hilden, germany) at 95 °c for 10 min, denaturation at 94 °c for 15 s and combined annealing and extension at 60 °c for 60 s. the cycle was repeated 45 times taking an approximate duration of 90 min including generation of amplification curve. data were analysed using the software abi 7900ht sds plate utility version 2.4 (applied biosystems®). statistical analysis statistical package program statistical package for social sciences version 21 (ibm corp., chicago, illinois, united states) was used. categorical variables were expressed in frequencies and percentages and cross-tabulated and pearson chi-square tested for statistically significant differences under the 0.05 level. numerical variables were described using measures of central tendency and of dispersion; pearson’s correlation and its 95% confidence intervals were calculated. results clinical, demographic and conventional laboratory findings all of the following describe patients with positive csf hhv-6 dna: all patients with clinical data (100%; 7/7; six cases out of a total of 13 had missing clinical data) presented with fever (˃ 37 °c) and vomiting and six (86%) with seizures (figure 2). for the 12 patients with available demographic data, the male-to-female ratio was 1:1 (figure 2). figure 2: demographic, clinical and conventional laboratory data for human herpes virus type-6 positive patients, khartoum, sudan, december 2009 – august 2010. half of the patients were toddlers, followed by 42% (5/12; 1 missing case) infants and 8% (1/12) a 15-year-old teenager. most patients (83%; 10/12) were admitted to the hospital in the dry season (december to june) and the remaining 17% (2/12) admitted in the rainy season (july to august). cerebrospinal fluid specimens from 42% (5/12) were traumatic; cytological and chemical analyses were not performed. the remaining 58% (7/12) were clear; 33% (1/7) showed increased csf white blood cells (50 cells/mm3) with 60% lymphocytosis and the remaining 67% (6/7) showed normal csf white blood cell count (< 5 cells/mm3). cerebrospinal fluid glucose levels for most specimens (86%; 6/7) were normal (45 mg/dl – 100 mg/dl) and one specimen (14%) was low (30 mg/dl). cerebrospinal fluid proteins levels were high (> 45 mg/dl) in 43% (3/7), normal (14 mg/dl – 45 mg/dl ) in 43% (3/7) and low (11 mg/dl) in 14% (1/7) (figure 2). all 13 hhv-6 positive cases did not show evidence of rapid-growing-bacteria coexisting in csf on gram’s stain and in vitro bacterial culture. however, 23% (3/13) were positive for other non-cultivable microbes. all patients (100%; 7/7) recovered and were discharged. real-time polymerase chain reaction findings polymerase chain reaction testing for hhv-6 dna in csf revealed 13 (2.6%) positive specimens out of a total of 503. median cycle threshold (ct) for all hhv-6 positive specimens was 38 with a range of 31.9 to 40.8. median virus copy was 281.3 per pcr run (1 × 105 virus copies/ml csf) with a range of 30 to 44 × 103 copies per pcr run (12 × 103 and 18 × 106 virus copies/ml csf). individual pcr data for all 13 positive specimens are shown in table 1. standard dilutions with viral copies of 5e6 to 5e2 produced amplification curves between ct 24 and ct 36. the generated standard curve plot showed perfect negative association between ct and viral quantity. this was repeatable in all pcr runs. table 1: thermal cycle and virus load for human herpes virus type-6 positive individual cerebrospinal fluid specimens using quantitative taqman real-time polymerase chain reaction, khartoum, sudan, december 2009 – august 2010. the final classification of cases based on clinical and molecular findings is shown in table 2. table 2: classification of cases based on clinical and molecular findings, khartoum, sudan, december 2009 – august 2010. discussion human herpes virus type-6 is becoming increasingly recognised as an emerging cns pathogen; nevertheless, it has never been investigated in sudan and very little is known about it in sub-saharan african countries or those of the meningitis belt. in the present study, hhv-6 dna was identified in the csf of 2.6% of paediatric patients with suspected meningitis. this result is close to the findings of hosseininasab24 who identified csf hhv-6 dna in 1.5% (1/65) of children with aseptic meningitis in southern iran. tavakoli21 reported similar findings of 1.8% (27/1482) in new york, united states. another study in new york by yao11 revealed a significantly higher prevalence of 40.0% (14/35) in patients with cns infections who tested negative for other cns pathogens. among well-defined groups in our study, the prevalence was also high, accounting for 33.0% out of 40 cases with proven infectious meningitis (table 2). in the present study, unlike yao’s approach, cases with other detectable cns pathogens were not excluded because of possible co-infections, as frequently reported.21,25,26,27 it has been reported that primary infection with hhv-6 always occurs in young children (age 0 to 2 years).5,28,29 human herpes virus type-6 infections, especially to the cns, can also occur in healthy older children and adults, but it is thought to be due to virus reactivation.22 among 24 hhv-6 positive patients in tavakoli’s study,21 42% were infants ≤ 3 years and 12.5% were teenagers 11–17 years old. out of our 13 hhv-6 positive cases, 92% were infants ≤ 2.3 years and one (8%) was a 15-year-old teenager. the ratio of boys to girls in this study was 1:1 which agrees with tavakoli’s findings of 1.3:1.1. clinical signs and symptoms in hhv-6 meningitis are not specific.30 out of the 24 examined patients, tavakoli reported fever in 71.0%, altered mental status in 67.0%, headache in 29.0% and seizures in 33.0%. other reported symptoms were muscle weakness, muscle pain and stiff neck, which are general symptoms for meningitis or encephalitis.21 in the present study, all hhv-6 positive cases presented with fever and vomiting, 86.0% presented with seizures, 14.3% with chills and 14.3% with a stiff neck. none of our patients developed skin rash, which is the only specific – but rare – symptom in hhv-6 meningitis.30 findings of both tavakoli and hosseininasab concur. normal csf glucose with normal or elevated proteins is the usual finding in viral infections of the cns,17 as found in this study, where all specimens showed normal csf glucose concentration, 33% normal proteins and 67% high proteins. unfortunately, chemical analysis of the csf is ineffective in case of viral infections; however, its significance is in distinguishing bacterial from aseptic aetiologies which is a crucial preliminary step in deciding adequate treatment. cerebrospinal fluid leukocytosis was seen in 33%. increased csf white blood cell numbers indicate inflammation; however, normal csf cellular counts do not rule out viral aetiologies. in fact, normal csf cellular counts in patients with proven cns infections were frequently reported.17,18,19,20 normal csf profile was reported in 25% of hhv-6 positive cases in tavakoli’s study21; accordingly, hhv-6 testing should not be limited to patients with abnormal csf profiles. thermal ct is defined as the number of cycles required for the fluorescent signal to cross the threshold (i.e. exceed background level). median ct for our positive cases was 38 with a range of 31.9 to 40.8. in tavakoli’s study, ct values ranged from 25.03 to 39.92. in quantitative real-time pcr, ct values inversely correlate with viral loads; therefore, a low ct value indicates a high viral load and vice versa. our ct values and viral loads were found to be significantly (p = 0.029) inversely correlated (r = −0.6, 95% confidence interval: −0.1 to −0.9) indicating significant variation of viral loads among our patients. substantial variation among viral loads in patients was also reported by tavakoli.21 the phenomenon of hhv-6 chromosomal integration is in debate; while some11,12,13 consider it an easily identifiable condition based on the presence or absence of nucleus containing blood cells in different body compartments, ward16 believes the few leukocytes that are usually present in normal csf can reveal positive viral dna in the case of hhv-6 integration. in the present study, the csf was clear with no observed cells in most cases (86%), while a single case (14%) showed an increased csf cell count. ward16 elaborated that in order to identify a condition as chromosomal integration, viral load should be high while low virus copies would indicate an infection. they16 reported a significantly lower (i.e. 2.4 log10 copies/ml) csf hhv-6 dna concentration in 9 children with primary infection in comparison with 21 patients with viral chromosomal integration (i.e. 4.0 log10 copies/ml). in the present study, the median csf virus concentration was 1 × 105 copies per ml with a minimum virus concentration of 12 × 103 copies per ml and a maximum of 18 × 106 copies per ml. while ward16 recommended identifying low virus copies (≤ 103) as an acute hhv-6 infection and high virus copies (≥ 104) as chromosomal integration, collot31 identified viral integration in approximate concentrations of 103 to 106 copies per ml. in the present study, we identified viral concentrations as low as 104 and as high as 107. several other studies reported high csf viral loads in patients with hhv-6 cns infections.11,21 in addition, other authors12,13 insist that chromosomal integration is a rare condition. accordingly, we assume the detected hhv-6 dna in our mostly cell-free csf specimens is more likely to be from free replicating virus than from chromosomally integrated virus. in infants, primary hhv-6 infection is an important cause of febrile seizures with an incidence of 13% in the united states.4 knowing that febrile seizures and vomiting were dominant symptoms among our population and the most frequent age group was children up to the age of 2.3 years, therefore, further supported our assumption. despite this, and for the sake of scientific relevance, we are not ruling out the possibility of integration among our identified cases. for this reason, studies to identify the prevalence of hhv-6 integration among healthy sudanese population are warranted. alongside hhv-6, mixed microbial infections were identified in three cases in this study. several authors also reported mixed viral infections to the cns.21,25,26,27 despite moderate neurological sequel, and less likely death, patients usually recover fully from an hhv-6 infection22,23, as fortunately observed in this population. limitations a major limitation in this study, however, is that we were unable to further genotype our identified hhv-6 viral dna because of a limited csf volume (i.e. all available csf was consumed in testing and identifying multiple microbes – reported in other publications). conclusion human herpes virus type-6 cns infection is frequent in this population (i.e. identified in one-third of cases with proven infectious meningitis). we recommend studying the existence and spread of hhv-6 chromosomal integration in the healthy sudanese population. acknowledgements we acknowledge all laboratory technologists in the bacteriology departments of omdurman hospital for children, the national health laboratory and the central public laboratories at the ministry of health for providing clinical data and specimens. we also thank the administrations of sudan medical and scientific research institute (sumasri) of the university of medical sciences and technology (umst) and that of the faculty of medical laboratory sciences of al-neelain university in khartoum, sudan, for providing working space and allowing use of laboratory ware. we are indebted for the kind gesture of a complete bench fee waiver that was provided by the department of clinical microbiology, umeå university in umeå, sweden, to perform all molecular investigations. we thank professor hassan mohammed ali for his great efforts in revising the manuscript. competing interests we, the authors, declare that we have no competing interests with respect to the research, authorship or publication of this article. authors’ contributions n.a.a. developed research questions and design, collected and managed all data, performed all laboratory work, statistical analysis and interpretation, wrote and edited the text. n.m. advised on the approach and methodology and guided and supervised the molecular laboratory work. m.e. and c.a. contributed to the approach and methodology, supervised the molecular laboratory work, edited and proofread the manuscript and were major contributors in the overall study. i.m.f.-e. supervised the research process throughout, contributed in the development of research questions, design and methodology, and managed all logistics and clinic-based activities. all authors have read and approved the final manuscript. sources of support molecular laboratory investigations were supported by the department of clinical microbiology at umeå university in umeå, sweden. all remaining project expenses 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https://doi.org/10.1128/jcm.40.7.2445-2451.2002 article information how to cite this article: how to cite: the supplement coordinators and guest editors. information for action: ajlm’s special issue on transforming the quality of laboratory medicine through the strengthening laboratory management toward accreditation programme. afr j lab med. 2014;3(2), art. #279, 1 page. http://dx.doi.org/10.4102/ ajlm.v3i2.279 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. information for action: ajlm’s special issue on transforming the quality of laboratory medicine through the strengthening laboratory management toward accreditation programme in this foreword... open access this inaugural special issue of the african journal of laboratory medicine spotlights the critical need for continuous quality improvement in laboratories and the impressive global expansion and impact of one programme aimed to make such improvements a reality: the strengthening laboratory management toward accreditation (slmta) programme. slmta that teaches laboratory managers how to implement practical quality management systems in resource-limited settings using available resources. with a series of short courses and work-based improvement projects supported by site visits and mentoring, slmta is designed to achieve rapid, measureable improvement in laboratories. slmta was launched in 2009, and has since been implemented in some 617 laboratories in 47 countries worldwide. this special issue provides a comprehensive collection of programme results, including a detailed description of the slmta methodology and variations thereof, analyses of global programme data, and a summary of the progress made in developing a cadre of indigenous slmta trainers to facilitate global scale-up. a two-part comprehensive review of the literature summarizes the qualitative and quantitative results, and identifies strategic directions for the future of the programme. equally, or perhaps more important, are the numerous reports of countryand laboratory-level experiences providing in-depth lessons learned from ground-level implementers. from these papers we learn about factors contributing to success in implementing a quality management system, as well as pitfalls to beware of, and glean practical insights from the slmta pioneers who have gained first-hand experience striving for continuous quality improvement. these articles focus on navigating the balance between country ownership and effective partnership, using innovative incentives to accelerate improvements, building local human resources, and developing a culture of quality in laboratories that is sustainable. other articles relate experiences, such as continuing on to the goal of international accreditation, and addressing the need to extend the slmta programme beyond the laboratory to all parts of the health system. the remaining articles focus on specific facets of the programme, including the impact of mentorship, whether establishing in-country training facilitators is less expensive than using international trainers, and the pros and cons of conducting centralized versus facility-based training. others explore how to engage local resources, such as research laboratories and partners, and the benefit of using electronic tools to streamline the audit process. until now, little has been published on this groundbreaking programme, as efforts thus far have been focused on implementation rather than evaluation. this collection contains a vast wealth of information from a programmatic and observational point of view. the more complicated work of rigorous programme evaluation – for example, studies using randomized interventions and control groups; formal calculations of cost-effectiveness; and assessment of the health impact of laboratory quality improvement – remains to be done. by combining these studies into a single collection, we hope to assist readers with assimilation of the results into a meaningful understanding of the slmta programme and its implementation. whether used by individual laboratories to improve quality of services, by country-level managers and partners to guide slmta implementation, or by ministry of health leaders and other decision-makers as a source of evidence for large-scale planning, the data and insights of those who have successfully implemented the programme provide a wealth of knowledge and information for evidence-based decision-making to ensure continuous quality improvement for better patient care and public health outcomes. the supplement coordinators and guest editors article information authors: oladipo a. aboderin1,2 olufemi adefehinti3 babatunde w. odetoyin1 amadin a. olotu2 iruka n. okeke4 olugbenga o. adeodu3,5 affiliations: 1department of medical microbiology and parasitology, obafemi awolowo university, ile-ife, nigeria2department of medical microbiology and parasitology, obafemi awolowo university teaching hospitals complex, ile-ife, nigeria 3department of paediatrics, obafemi awolowo university teaching hospitals complex, ile-ife, nigeria 4department of biology, haverford college, haverford, united states 5department of paediatrics and child health, obafemi awolowo university, ile-ife, nigeria correspondence to: oladipo abo postal address: department of medical microbiology and parasitology, college of health sciences, obafemi awolowo university, 220005, ile-ife, nigeria dates: received: 25 june 2011 accepted: 19 jan. 2012 published: 04 june 2012 how to cite this article: aboderin oa, adefehinti o, odetoyin bw, olotu aa, okeke in, adeodu oo. prolonged febrile illness due to ctx-m-15 extended-spectrum β-lactamase-producing klebsiella pneumoniae infection in nigeria. afr j lab med. 2012;1(1), art. #16, 4 pages. http://dx.doi.org/10.4102/ ajlm.v1i1.16 note: this report was presented as a poster (abstract c-119) at the 111th general meeting, american society for microbiology, may 21–24, new orleans, louisiana, united states. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. prolonged febrile illness due to ctx-m-15 extended-spectrum β-lactamase-producing klebsiella pneumoniae infection in nigeria in this case studies... open access • abstract • introduction • case report • discussion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ we report on an 8-year-old patient with septicaemia unresponsive to therapy for five weeks. undetected, extended-spectrum β-lactamase (esbl) production by the infecting klebsiella strain was regarded as responsible for treatment failure. intravenously administered imipenem during the sixth week led to sustained resolution of fever. resource-limited hospitals can incur prohibitive costs from esbl-producer infections because of diagnostic limitations and consequent treatment failure involving prolonged supportive therapy. introduction top ↑ the first report of plasmid-encoded β-lactamase capable of hydrolysing the extended-spectrum cephalosporins was published in 1983.1 since then, extended-spectrum β-lactamases (esbls) have become an increasingly important resistance mechanism among enterobacteriaceae worldwide. a 2006 report of the infectious diseases society of america listed esbl-producing klebsiella pneumoniae and escherichia coli among drug-resistant microbes for which new therapies are urgently needed.2 reports show that the esbl problem is rapidly evolving and increasing in severity and scope with the discovery of new esbls, particularly the ctx-m types, which have become the most prevalent.3 the β-lactamases are the greatest threat to the usefulness of β-lactam antibiotics such as the penicillins and cephalosporins. of all the different types of β-lactamases, esbls currently have the greatest clinical impact in terms of diversity and distribution as well as the ability to hydrolyse expanded-spectrum third generation cephalosporins. the earliest variants of esbls originated as a result of point mutations in the genes for broad-spectrum β-lactamases whilst newer ones, including the most successful such as ctx-m, arose by acquisition from the environmental metagenome through horizontal gene transfer.4 cephalosporins as bactericidal, cell wall-active β-lactam agents were introduced in the 1980s and as a result of effectiveness against broad-spectrum β-lactamases became standard for treatment of severe conditions such as bloodstream infections, pneumonia and intra-abdominal infections, until esbls started compromising usefulness in response to overuse and selective pressure. organisms that produce esbls are an important reason for therapy failure with cephalosporins and have serious consequences for infection control. furthermore, ctx-m esbl enzymes have been associated with coresistance to other agents including trimethoprim-sulphamethoxazole, tetracycline, gentamicin, tobramycin and ciprofloxacin.5 it is essential that clinical microbiology laboratories rapidly and reliably detect and report esbl-producing organisms. case report top ↑ an eight-year-old girl presented with a two-week history of fever, abdominal pain, passage of watery stool and recurrent vomiting. there was also a history of frequent micturition with occasional dysuria but neither haematuria nor passage of dark-coloured urine. prior to presentation in the teaching hospital, she had been admitted to a distant private hospital for five days, where she was treated with amoxicillin, ciprofloxacin and artesunate (doses and duration of treatment are unknown). she was discharged from that hospital because her state of health was not improving significantly and also because of the need for better family support. there was no other history of hospital admission or blood transfusion and no history suggestive of haemoglobinopathy. immunisation and nutritional history were essentially normal.general physical examination revealed a conscious but ill-looking, somewhat pale, febrile (temperature 38.5 °c) girl. she was moderately dehydrated and jaundiced. her weight was 22 kg (86% of expected weight for age). there was no facial or pedal oedema. the respiratory rate was 32 cycles per min and breathing was regular; pulse regular at 120 beats per min and with good volume. her blood pressure was 90/50 mmhg. the significant systemic findings on examination at admission were severe suprapubic tenderness, moderate hepatosplenomegaly (firm, not tender) and negative renal angle tenderness. all other systems were normal. conventional blood cultures were done at five different times after admission. there was a growth of klebsiella sp. on three occassions. once, the isolate was sensitive to gentamicin and ceftriaxone but resistant to all available antibiotics tested on the other two occasions. urine and stool cultures did not yield growth of any pathogens. screening for human immunodeficiency virus (hiv), hepatitis c virus (hcv) and hepatitis b surface antigen (hbsag) was negative. full blood counts showed a haematocrit ranged between 14% and 30%, white cell counts of 8 x 109/l – 9.2 x 109/l and an essentially normal platelet count (260 x 109/l). the erythrocyte sedimentation rate was 80 mm/hr (westergreen method) and the haemoglobin phenotype (by electrophoresis) was as. serum biochemistry parameters were all normal except for conjugated hyperbilirubinaemia. repeated abdominal ultrasonography showed findings that are consistent with hepatosplenomegaly in a septicaemic patient. whilst in hospital and when fever was uncontrolled and persistent, the patient was given fresh whole blood transfusions thrice and exchange blood transfusions twice, amongst other forms of treatment. fever remained persistent for five weeks following admission, despite different courses of antibiotics involving ciprofloxacin, gentamicin, ceftazidime, ceftriaxone and amoxicillin-clavulanic acid (table 1). it was only at this point that the possibility of infection with an esbl-producing organism was considered. esbl-producers are not routinely sought in the diagnostic laboratory. during the sixth week, the klebsiella sp. isolate from the patient was tested and confirmed to be producing an esbl. immediately following this test result, treatment was commenced with imipenem (not routinely available in the hospital) and there was dramatic resolution of fever. the patient remained free of fever for one week after receiving imipenem and was subsequently discharged. two weeks later, when she reported for follow-up, she was still fever-free and healthy. table 1: treatment interventions. the klebsiella sp. isolate was identified as klebsiella pneumoniae subspecies pneumoniae using the api 20e identification strips for enterobacteriaceae (biomérieux, marcy-l’étoile, france). presumptive esbl phenotypic testing and confirmation in the organism was performed by disc diffusion tests on mueller hinton agar by employing ceftazidime (30 µg) and cefpodoxime (10 µg) alone and in combination with clavulanic acid as ceftazidime-clavulanic acid (30/10 µg) and cefpodoxime-clavulanic acid (10/1 µg) respectively. results were interpreted using the clinical and laboratory standards institute (clsi) criteria for disc diffusion.6 antimicrobial susceptibility testing for the organism was carried out by the disc diffusion technique according to the guidelines and recommendations of clsi.6 the isolate was resistant to streptomycin, gentamicin, chloramphenicol, tetracycline, nalidixic acid, ciprofloxacin, ampicillin, trimethoprim, sulphamethoxazole, ceftriaxone, cefepime and amoxicillin-clavulanic acid, but susceptible to imipenem.genomic dna was extracted from the isolate using the wizard genomic extraction kit (promega) according to the manufacturer’s directions and used as template for pcr reactions targeting resistance elements and genes. platinum pcr supermix (invitrogen) was used for all reactions, and pcr cycle conditions were as recorded in the original articles describing the primers (table 2). we employed oligonucleotides that prime the conserved ends of the cassette regions of class 1 and 2 integrons respectively to screen for these elements (table 2).7,8 as shown in figure 1, we were able to determine that the strain harboured a class 1, but not a class 2 integron. sequencing of the 1.6 kb amplified class 1 cassette region revealed that it was identical to the cassette region of plasmid pip1206 (genbank accession number nc_010558), containing two integrated cassettes: a dfra17 cassette encoding resistance to trimethoprim, and an aada4 aminoglycoside resistance cassette.9 since the integron did not contain an esbl cassette, we screened the isolate for blactx-m type genes, employing primers that amplify an internal fragment from multiple blactx-m alleles (table 2)10. the resulting 550 bp product shown in figure 2 was sequenced and found to be identical to the corresponding region of blactx-m-15. table 2: oligonucleotides for pcr reactions. figure 1: pcr amplification of the variable regions of class 1 and class 2 integrons. (a) class 1 integron-variable regions amplified using lev5’cs and lev3’cs primers. lane 1: no template; lane 2: e. coli strain 042 bearing an aada cassette within a class 1 integron; lane 3: e. coli strain 17-2 bearing the dfra1-sat-aada cassette sequence within a class 2 integron; lane 4: k. pneumoniae subsp. pneumoniae isolate k01 from this study; lane 5: 1 kb ladder plus (invitrogen). marker size fragments are indicated to the right of the gel in kilobase pairs. (b) class 2 integron-variable regions amplified using hep51 and hep74 primers with samples shown in (a) loaded on to the gel. (c) cassette content and orientation of the k01 integron amplified in (a), as predicted from the dna sequence. figure 2: pcr amplification of a ctx allele from k. pneumoniae subsp. pneumoniae isolate k01 from this study. lane 1: 1 kb ladder plus (invitrogen). lane 2: k01. lane 3: esbl-negative e. coli strain 042. lane 4: no template. marker size fragments are indicated to the left of the gel in kilobase pairs. in the absence of a positive control, the identity of the band amplified from strain k01 was determined by sequencing. the cost of repeated investigations (table 3) was n14 000.00 ($90.92), which is more than a tenfold increase on projected diagnostic expenses, had a diagnosis estimate been made immediately on admission. antibiotics and fresh whole blood transfusions (thrice) as well as exchange blood transfusions (twice) cost n89 900.00 ($583.77). these and other treatment interventions effectively doubled the cost of treatment as compared to that for what would normally have been appropriate therapy after admission. finally, prolonged hospital accommodation, feeding and nursing care over 48 days amounted to n20 400.00 ($132.46) in contrast to n2975.00 ($19.32) for admission for one week, an almost tenfold increase. table 3: investigations. discussion top ↑ the occurrence and spread of infections resulting from esbl-producing organisms have been well documented in countries of europe, asia and north america.11 in contrast, data on the epidemiology of esbl enzymes is very limited in nigeria. molecular analysis of eight nigerian esbl-producing enterobacter species in 2001 detected only tem and shv-like esbls and no ctx-m types.12 in a study of klebsiella pneumoniae isolates associated with community-acquired urinary tract infections between 2002 and 2003 in ibadan, nigeria, ctx-m group 1, -like enzymes were found in 17 (57%), but ctx-m-15 was identified in only two isolates.13 olowe et al. investigated the occurrence of ctx-m esbl-producing e. coli and found nine of 79 ampicillin-resistant hospital isolates to be esbl producers.14 more recently, a case of necrotising fasciitis was reported in a nigerian patient in the uk.15 morganella morganii and citrobacter freundii carrying the ctx-m-15 esbl gene were isolated from the patient, highlighting the presence of ctx-m genes in africa even though there is a scarcity of reports in the literature. here, we describe a case of prolonged, uncontrolled fever found to be due to esbl-producing k. pneumoniae. to the best of our knowledge, this is the first documented clinical course and outcome of esbl-producing bacterial infection in nigeria. failure to recognise and initially diagnose the presence of an esbl-producing organism resulted in considerable expense in the management of the infection. this includes the cost of different courses of ineffective antibiotics for five weeks, exchange blood transfusions and whole blood transfusions, as well as hospital charges resulting from prolonged stay in hospital. the estimated avoidable cost of supportive therapy and investigation related to possible alternative diagnoses was almost $600 in a country where the average annual per capita income is $2300 and health care resources are severely limited. the clinical diagnostic microbiology laboratory plays a crucial part in the detection and reporting of esbl-producing bacteria, and it is important that laboratories be fully aware of the significance of esbl-producing organisms and the best methods for detecting them, as in our case. resource-limited hospitals can incur prohibitive costs associated with esbl-producer infections because of prolonged supportive therapy and treatment failure following the use of readily available antibiotics. diagnostic improvements to allow routine detection and reporting of esbl production in enterobacteriaceae will help greatly in avoiding these costs. acknowledgements top ↑ this work was supported by a branco weiss fellowship (awarded to ino) from the society-in-science, swiss federal institute of technology (eth), zurich, switzerland. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions o.a.a. (obafemi awolowo university) coordinated the research. o.a. (obafemi awolowo university teaching hospitals complex) a.a.o. (obafemi awolowo university teaching hospitals complex), o.a.a. and o.o.a. (obafemi awolowo university teaching hospitals complex) managed the patient (case) clinically. a.a.o., o.a.a. and b.w.o. (obafemi awolowo university) performed microbiological testing whilst i.n.o. carried out molecular experiments. a.o.a. and i.n.o. (haverford college) drafted the paper, to which o.a., b.w.o., o.o.a. and a.a.o. contributed. o.a.a. and i.n.o. undertook revision of the manuscript. all authors approved the final version. references top ↑ 1. knothe h, shah p, krcmery v, antal m, mitsuhashi s. transferable resistance to cefotaxime, cefoxitin, cefamandole and cefuroxime in clinical isolates of klebsiella pneumoniae and serratia marcescens. infection. 1983;11:315–317. http://dx.doi.org/10.1007/bf01641355, pmid:6321357 2. talbot gh, bradley j, edwards je, gilbert d, scheld m, bartlett jg. bad bugs need drugs: an update on the development pipeline from the antimicrobial availability task force of the infectious diseases society of america. clin infect dis. 2006;42:657–668. http://dx.doi.org/10.1086/499819, pmid:16447111 3. cantón r, coque tm. the ctx-m β-lactamase pandemic. curr opin microbiol. 2006;9:466–475. http://dx.doi.org/10.1016/j.mib.2006.08.011, pmid:16942899 4. poirel l, kāmpfer p, nordmann p. chromosome-encoded ambler class a β-lactamase of kluyvera georgiana, a probable progenitor of a subgroup of ctx-m extended-spectrum β-lactamases. antimicrob agents chemother. 2002;46:4038–4040. http://dx.doi.org/10.1128/aac.46.12.4038-4040.2002, pmid:12435721, pmcid:132763 5. pitout jdd. multiresistant enterobacteriaceae: new threat of an old problem. expert rev anti-infect ther. 2008;6:657–669. http://dx.doi.org/10.1586/14787210.6.5.657, pmid:18847404 6. clinical and laboratory standards institute. performance standards for antimicrobial susceptibility testing: twentieth informational supplement. clsi document m100-s20. wayne, pa: clinical and laboratory standards institute; 2010. 7. lévesque c, piché l, larose c, roy ph. pcr mapping of integrons reveals several novel combinations of resistance genes. antimicrob agents chemother. 1995;39:185–191. http://dx.doi.org/10.1128/aac.39.1.185, pmid:7695304, pmcid:162507 8. white pa, mciver cj, rawlinson wd. integrons and gene cassettes in the enterobacteriaceae. antimicrob agents chemother. 2001;45:2658–2661. http://dx.doi.org/10.1128/aac.45.9.2658-2661.2001, pmid:11502548, pmcid:90711 9. périchon b, bogaerts p, lambert t, frangeul l, courvalin p, galimand m. sequence of conjugative plasmid pip1206 mediating resistance to aminoglycosides by 16s rrna methylation and to hydrophilic fluoroquinolones by efflux. antimicrob agents chemother. 2008;52:2581–2592. http://dx.doi.org/10.1128/aac.01540-07, pmid:18458128, pmcid:2443923 10. bonnet r, recule c, baraduc r, et al. effect of d240g substitution in a novel esbl ctx-m-27. j antimicrob chemother. 2003;52:29–35. http://dx.doi.org/10.1093/jac/dkg256, pmid:12775683 11. pitout jdd, laupland kb. extended-spectrum β-lactamase-producing enterobacteriaceae: an emerging public-health concern. lancet infect dis. 2008;8:159–166. http://dx.doi.org/10.1016/s1473-3099(08)70041-0, pmid:18291338 12. aibinu ie, ohaegbulam vc, adenipekun ea, ogunsola ft, odugbemi to, mee bj. extended-spectrum β-lactamase enzymes in clinical isolates of enterobacter species from lagos, nigeria. j clin microbiol. 2003;41:2197–2200. http://dx.doi.org/10.1128/jcm.41.5.2197-2200.2003, pmid:12734278, pmcid:154721 13. soge oo, queenan am, ojo kk, adeniyi ba, roberts mc. ctx-m-15 extended-spectrum β-lactamase from nigerian klebsiella pneumoniae. j antimicrob chemother. 2006;57:24–30. http://dx.doi.org/10.1093/jac/dki429, pmid:16319181 14. olowe oa, grobbel m, büchter b, lübke-becker a, fruth a, wieler lh. detection of blactx-m-15 extended-spectrum β-lactamase genes in escherichia coli from hospital patients in nigeria. int j antimicrob agents. 2010;35:206–207. http://dx.doi.org/10.1016/j.ijantimicag.2009.10.004, pmid:20022221 15. soleimanian s, gordon nc, wareham dw. polymicrobial necrotizing fasciitis involving enterobacteria producing ctx-m-15 extended-spectrum β-lactamases. j med microbiol. 2011;60:135–137. http://dx.doi.org/10.1099/jmm.0.021998-0, pmid:20813849 abstract introduction methods results discussion acknowledgements references about the author(s) mulalo molaudzi department of oral biological sciences, university of the witwatersrand, johannesburg, south africa julitha molepo department of oral biological sciences, university of the witwatersrand, johannesburg, south africa citation molaudzi m, molepo j. the use of real-time polymerase chain reaction and an adenosine deaminase assay for diagnosing pleural tuberculosis. afr j lab med. 2019;8(1), a731. https://doi.org/10.4102/ajlm.v8i1.731 original research the use of real-time polymerase chain reaction and an adenosine deaminase assay for diagnosing pleural tuberculosis mulalo molaudzi, julitha molepo received: 30 nov. 2017; accepted: 19 feb. 2019; published: 28 aug. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the diagnosis of pleural tuberculosis remains a challenge, because the most widely used conventional diagnostic tools are unable to rapidly detect mycobacterium tuberculosis in pleural fluid with sufficient sensitivity. objectives: the aim of this study was to evaluate the usefulness of an adenosine deaminase assay and real-time polymerase chain reaction (qpcr) in diagnosing pleural tuberculosis. methods: one hundred and five consecutive pleural fluid specimens collected between august 2008 and march 2009 were assessed. among the 105 specimens, 50 (48%) were unconfirmed tuberculosis cases, 21 (20%) were confirmed tuberculosis cases and 34 (32%) were non-tuberculosis cases (controls). real-time pcr was performed using the light cycler mycobacterium detection kit according to the manufacturer‘s instructions (roche diagnostics). an adenosine deaminase assay was carried out using a commercial colorimetric assay kit as a user-defined method on a beckman dxc 600 synchron analyser. results: the sensitivity of the qpcr was 67% and specificity was 100%. the sensitivity of the adenosine deaminase assay was 80% and specificity was 94%. conclusion: the findings show that the adenosine deaminase assay had higher sensitivity than qpcr. real-time pcr had 100% specificity, thus a combination of the two methods may be useful for the diagnosis of pleural tuberculosis. keywords: pleural tuberculosis; adenosine deaminase assay; qpcr; real-time polymerase chain reaction. introduction extrapulmonary tuberculosis is common in south africa and is exacerbated by the high rate of hiv infection in the country. in 2014 a high incidence of extrapulmonary tuberculosis (88.6 per 100 000 population) was reported in south africa.1 pleural tuberculosis is the most common form of extrapulmonary tuberculosis after lymph node tuberculosis.2 early diagnosis is essential in order to effectively treat and control the disease. due to the paucibacillary nature of pleural tuberculosis, the sensitivity of pleural fluid microscopy (about 10%) and culture (about 20%) in pleural tuberculosis cases is low.3,4,5 culture also has a longer turn-around time, ranging from 4 to 8 weeks.6 the poor sensitivity and specificity of the conventional methods of diagnosis suggest the need for more effective methods, such as an adenosine deaminase assay (ada) and real-time polymerase chain reaction (qpcr). pleural fluid ada has been shown to be a valuable biochemical marker that has a high sensitivity and specificity for pleural tuberculosis diagnosis.7 the use of ada as a diagnostic marker has additional merits, since results can be produced rapidly.8 molecular diagnostic tests such as qpcr have been used in the diagnosis of tuberculosis. however, the sensitivity of qpcr varies, because different methods target different genes such as rpob, 16s rrna and is6110.6,9 advantages of qpcr over conventional pcr (polymerase chain reaction)testing include fast availability of results, decreased risk of contamination and quantification of bacterial load.10 it is important to find a rapid and reliable test for the diagnosis of pleural tuberculosis, particularly in developing countries such as south africa where there is high incidence of tuberculosis and hiv. although the utility of ada and qpcr for the diagnosis of pleural tuberculosis has been reported worldwide, there is a need to evaluate these tests in developing countries with a high tuberculosis burden.11 this study evaluated the use of ada and qpcr for the diagnosis of pleural tuberculosis. methods ethical considerations the study was approved by the university of limpopo (medunsa campus) research and ethics committee (study approval number: mrec/p/177/2008:pg). study population one hundred and five consecutive pleural fluid specimens submitted to the dr george mukhari tertiary chemical pathology laboratory in pretoria, south africa, between august 2008 and march 2009 were included in the study. the specimens were from 71 suspected tuberculosis cases (patients with suggestive symptoms of tuberculosis; symptoms included coughing for more than 2 weeks, night sweats, fever and loss of weight) and 34 non-tuberculosis cases/controls (patients with other clinical causes of pleural effusion). pleural fluid of all suspected tuberculosis cases (n = 71) and 32 of the control patients were exudates and only 2 of the control specimens were transudates. among the two patients with transudates, one was diagnosed with malignancy and the other one was diagnosed with diabetes. patient demographic data, including age, sex and clinical history, were collected from the national health laboratory services data intensive system and applications database. permission to use samples and access the database was granted by the head of chemical pathology. individual informed consent was not sought, because the study was conducted on routine samples only and it did not involve additional samples or change in the treatment of patients. clinical and laboratory diagnosis all pleural fluid specimens were stained using auramine o and cultured on lowenstein-jensen agar slants and mgit bactec tubes containing middlebrook medium (becton dickinson, sparks, maryland, united states). all the culture positive samples were confirmed by ziehl-neelsen staining. a diagnosis of pleural tuberculosis was made when the pleural fluid was culture positive and acid fast bacilli (afb)-positive for mycobacterium tuberculosis. the unconfirmed pleural tuberculosis cases were diagnosed by the clinical presentation, such as history of tuberculosis infection, chronic cough, night sweats and loss of weight. the non-tuberculosis pleural effusion cases were patients with pleural effusion caused by other medical conditions (table 1). table 1: demographic characteristics of cases included in the study, pretoria, south africa, 2008-2009. dna extraction pleural fluid samples were concentrated by centrifugation at 3000 g for 15 min, the supernatant was discarded and the pellet resuspended in 2 ml of phosphate buffered saline buffer ph 6.8. the samples were mixed by vortexing for 5 seconds. the extraction of dna was performed on concentrated pleural fluid specimens using the amplicor respiratory sample preparation kit according to the manufacturer’s instructions (roche diagnostics, mannheim, germany). briefly, 500 µl wash solution was added to 100 µl of concentrated pleural fluid sample in a 1.5 ml tube. the mixture was vortexed for 5 s, centrifuged at 12 500 g for 10 minutes, and the supernatant discarded. about 100 µl of lysis buffer was added to the pellet and the tubes were vortexed for 5 s to resuspend the pellet. the samples were then incubated at 60 °c for 45 min. following incubation, the samples were spun down for 5 s, 100 µl of neutralising buffer was added and the mixture was vortexed for 5 s. the lysate was stored at 2 °c – 8 °c for later use. real-time polymerase chain reaction real-time pcr was performed using the light cycler mycobacterium detection kit according to the manufacturer’s instructions (roche diagnostics, mannheim, germany). a total of 20 µl pcr mixture containing 0.25 µl of 8.5 uracil dna glycosylase, 0.75 µl internal control, 4 µl master mix, 11 µl detection mix and 4 µl dna sample was prepared in a capillary tube. an internal control was used to control for the presence of inhibitors. the master mix contained the enzyme hot start taq polymerase, mgcl2, deoxynucleoside triphosphate (dttp, dctp, datp, dgtp) and pcr buffer. the detection mix contained the primers, hybridising probes, fluorescein and the acceptor probe lightcycler red. the primers targeted the 16s rrna sequence of the m. tuberculosis complex. real-time pcr was performed on the light cycler 2.0 (roche diagnostics, mannheim, germany) real-time instrument according to the following protocol: incubation at 40 °c for 10 min, denaturation at 95 °c for 10 min; amplification consisted of 45 cycles of: 95 °c for 10 s, 50 °c for 10 s with a single acquisition mode and 72 °c for 20 s. this was followed by a melting curve analysis to determine melting temperatures for the sequences targeted by the hybridisation probes. this comprised three stages: 95 °c for 1 min, 40 °c for 2 min and 70 °c for 1 min, ramp rate 0.1 and a continuous acquisition mode, followed by cooling at 40 °c for 30 s. analysis of the samples was done in two steps: pcr amplification, where the target amplicon for each sample was detected between the annealing and elongation steps as sigmoid curves at 640 nm. the melting temperature calling, where the melting temperature specific for each subtype in the sample, was determined using the light cycler 2.0 software (roche diagnostics, mannheim, germany). samples were regarded positive for mycobacteria if they had exponential amplification with a crossing point value of less than 35 with a signal intensity of more than 0.02 and negative with no amplification on the 640/back 530 nm channel and exponential amplification for the internal control in the 705/back 530 nm channel having a signal intensity of more than 0.02. a melting temperature of 53.5 °c – 56.5 °c indicated m. tuberculosis, 48 °c – 51 °c m. avium and 57 °c – 60 °c m. kansasii. adenosine deaminase assay the ada was analysed using a commercial colorimetric assay kit according to manufacturer’s instructions (diazyme general atomics, poway, california, united states). the activity in the patient’s specimen was calculated with the formula: activity in specimen = (optical density specimen – optical density specimen blank)/(optical density standard – optical density reagent blank) × 50 with the result expressed in units/l. an ada value of 30 units/l or higher was considered to be positive. the assay was done on a dxc 600 synchron analyser (beckman coulter, brea, california, united states) using user-defined methods. data analysis the results of the ada assay and qpcr against the culture, the gold standard for diagnosis of tuberculosis, were entered into 2 × 2 tables, wherein the sensitivity was calculated by using the formula tp/(tp+fn), specificity by using the formula tn/(fp+tn), positive predictive value by using the formula tp/(tp+fp) and negative predictive value by using the formula tn/(tn+fn), where tp = true positives, fn = false negatives, fp = false positives and tn = true negatives. the mean and standard deviation of ada and the mean age were calculated using epi info version 3.3 (centers for disease control and prevention, atlanta, georgia, united states). results among the 105 specimens, 50 (48%) were unconfirmed tuberculosis cases, 21 (20%) were confirmed tuberculosis cases and 34 (32%) were non-tuberculosis cases (controls) (table 1). all of the 34 (100%) controls were afb negative, and none showed visible growth on culture medium. of the 71 suspected tuberculosis cases, 50 (70%) were culture-negative and 21 (30%) were culture positive (i.e. confirmed tuberculosis cases). of the 21 culture positive samples, 3 (14%) were afb positive. of the 21 confirmed tuberculosis (pleural tuberculosis) cases, 52% were men and 48% were women, with age ranging from 20 to 65 years and a mean age of 38.5 years (table 1). of the 50 unconfirmed tuberculosis cases, 50% were men and 50% were women, with age ranging from 19 to 85 years and a mean age of 39 years. of the 34 non-tuberculosis pleural effusion cases, 38% (13/34) had malignancy, 26% (9/34) had congestive cardiac failure, 15% (5/34) had chronic renal failure, 6% (2/34) were diabetic, 6% (2/34) had pneumonia, 6% (2/34) had polyarthritis and 3% (1/34) had anaemia. among the 34 control patients, 56% (19/34) were women and 44% (15/34) were men, and their ages ranged from 23 to 78 years, with a mean age of 49 years. real-time polymerase chain reaction of the 21 confirmed cases, 14 (67%) were qpcr and culture positive, while 7 (33%) were qpcr negative. none of the specimens from the control group and the unconfirmed tuberculosis cases were positive by qpcr. the sensitivity of the qpcr was 67% and specificity was 100% (table 2). the positive predictive value was 78% and negative predictive value was 86%. table 2: operating characteristics of real-time polymerase chain reaction and adenosine deaminase assay in comparison with the gold standard culture in pleural tuberculosis, pretoria, south africa, 2008-2009. adenosine deaminase assay of the 21 confirmed cases, 17 (81%) were ada and culture positive, and 4 (19%) were ada negative. of the 50 unconfirmed cases, 39 (78%) were ada positive and 11 (22%) were ada negative. the ada levels among the confirmed pleural tuberculosis cases ranged from 8 units/l to 134.3 units/l with a mean ada value of 52.2 ± 21.66 units/l. the ada levels among the unconfirmed pleural tuberculosis cases ranged from 5.68 units/l to 200 units/l with a mean ada value of 50.12.2 ± 21.77 units/l. the ada levels among the control group ranged between 2.4 units/l and 98 units/l, with a mean ada value of 12.7 ± 8.64 units/l. only two (6%) of the 34 non-tuberculosis pleural effusion cases were ada positive with values of 39 units/l and 98 units/l. both patients with elevated levels of ada had pneumonia. the mean ada level among the pleural tuberculosis cases was significantly higher than that of the control group, p < 0.0001. the sensitivity of ada was 80% and specificity was 94% (table 2). the positive predictive value was 89% and negative predictive value was 88%. discussion the diagnosis of pleural tuberculosis is challenging due to the low number of bacilli in the pleural fluid. the detection of pleural tuberculosis using afb microscopy and culture methods is not effective as both of these methods have low sensitivity, and the culture method has a longer turn-around time of up to 8 weeks.2,12 this study evaluated the use of qpcr and ada for the diagnosis of pleural tuberculosis. the sensitivity and specificity of qpcr varies depending on the type of test used.13,14,15 in this study, the sensitivity of qpcr against culture was found to be 67%. our findings are similar to those of previous studies.13,14,16 rosso et al.17 reported even lower sensitivity of 42.8%. a high number of low sensitivities for pcr were reported in a meta-analysis study.13 the low sensitivity of qpcr may be explained by the low number of bacilli or the presence of inhibitors in the pleural fluid.18 a study by casallas-rivera et al.19 reported qpcr hybridisation probe sensitivity of 66.7%. this low sensitivity makes it difficult to use pcr as a method for ruling out tuberculosis. in contrast to our findings, higher sensitivity of qpcr has been reported. a study by kalantri et al.16 reported 80% sensitivity for qpcr. a high qpcr specificity of 100% was found in our study, and other authors have reported similar results. a meta-analysis conducted by pai et al.13 reported commercial and in-house pcr methods to have high specificities (98% and 93%), which suggests a potential role of pcr in confirming the diagnosis of pleural tuberculosis. other studies reported similar results. recently, casallas-rivera et al.19 used qpcr to detect pleural tuberculosis in 40 patients and specificity was found to be 93.5%. in another study, a pcr specificity of 93.8% in 87 patients was reported.20 it is noteworthy that some of these comparisons were made between qpcr, the method used in this study which detects amplification during the early phases of the reaction, and pcr, which is a conventional/traditional method detecting amplification at the final phase or end-point of the reaction. pleural fluid ada activity has been shown to be a valuable biochemical marker that has a high sensitivity and specificity for pleural tuberculosis diagnosis.7 the mean ada values among the confirmed pleural tuberculosis and unconfirmed cases in our study were 52.2 ± 21.66 units/l and 50.12 ± 21.77 units/l respectively, which were both significantly higher than that of the control group (12.70 ± 8.64 units/l), with a p value of less than 0.0001. these results are in accordance with several other studies.21,22,23 the current study showed high ada sensitivity (80%) and specificity (94%) for the diagnosis of pleural tuberculosis. several previous studies reported contrasting results to ours. in a study by mo-lung et al.,7 210 patients with pleural effusion were studied, and higher ada sensitivity (87.3%) and lower specificity (91.8%) were observed. a study by zaric et al.24 evaluated the diagnostic value of the ada assay in 121 patients and found a higher sensitivity (89.2%) and a lower specificity (70.4%). another study evaluated the ada assay in more than 2000 patients and reported higher sensitivity (93%) and lower specificity (90%).25 kashyapi et al.26 showed higher ada sensitivity (82%) and lower specificity (83%). ada levels in non-tuberculous lymphocytic effusions rarely exceed the diagnostic cut-off for tuberculosis, and various ada cut-off values have been used, ranging from 30 units/l to 50 units/l. in our study, ada levels were above the cut-off of 30 units/l value in two (5.8%) patients for whom the diagnosis of pleural tuberculosis was ruled out; both the patients had pneumonia with ada levels of 98 units/l and 39.8 units/l. this is in agreement with one study where high levels of ada were reported in some cases of parapneumonic effusions and adenocarcinoma.27 lee et al.28 measured ada levels in non-tuberculous lymphocytic effusion and found that ada values were above the cut-off of 40 units/l in one complicated parapneumonic effusion and two cases of lymphoma. a study by porcel and vives29 also showed that it is rare that ada in non-tuberculous lymphocytic effusions is above the cut-off value. in that study, only two of eight patients with high ada levels had uncomplicated parapneumonic effusion with ada levels of 58.9 units/l and 40.5 units/l. jiménez et al.30 studied 410 patients with non-tuberculous lymphocytic effusions and ada levels reached the diagnostic cut-off for tuberculosis (40 units/l) in seven of the 410 cases. two patients had bronchogenic carcinomas, two had complicated parapneumonic effusions, one had a diagnosis of lymphoma, one had a mesothelioma and one case was idiopathic. roughly one-third of parapneumonic effusions had ada levels above 40 units/l.31 differences in ada activity between tuberculosis and malignancy may be due to differences in t-helper phenotypes or the presence of memory cd4+ cells in tuberculosis.23 conclusion the ada assay has a high sensitivity (80%) and specificity (94%) and hence it is still a useful tool for the diagnosis of pleural tuberculosis. the sensitivity of qpcr was 67% and specificity was 100%. the low sensitivity of qpcr suggests that this test should not be used for excluding a diagnosis of pleural tuberculosis. however, the high specificity of qpcr suggests that a combination of the two methods may be useful for the diagnosis of pleural tuberculosis. adenosine deaminase assay (ada) activity in pleural fluid can differentiate between pleural disease due to tuberculosis and effusion due to non-tuberculous lymphocytic effusion. in addition, the ada result is available on the same day compared to culture, which takes about two weeks. more studies on the diagnostic value of qpcr are needed. acknowledgements the authors would like to acknowledge the staff of chemical pathology, national health laboratory services, dr george mukhari hospital for collection of samples. we acknowledge the late prof. h.f. joubert, who co-supervised the project. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions m.m. conceived the idea. m.m. carried out experiments. j.m. supervised the project. both j.m. and m.m. contributed to the final version of the manuscript. sources of support national health laboratory services research trust: 9/10/2007; prof h.f. joubert. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the author(s) and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references karstaedt as. extrapulmonary tuberculosis among adults: experience at chris hani baragwanath academic hospital, johannesburg, south africa. s afr med j. 2013;104(1):22–24. https://doi.org/10.7196/samj.6374 sharma sk, mohan a. extrapulmonary tuberculosis. indian j med res. 2004;120:316–353. diacon ah, van de wal bw, wyser c, et al. diagnostic tools in tuberculous pleurisy: a direct comparative study. eur respir j. 2003;22:589–591. https://doi.org/10.1183/09031936.03.00017103a porcel jm, esquerda a, vives m, et al. etiology of pleural effusions: analysis of more than 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milovancev a, et al. differential diagnosis of tuberculous and malignant pleural effusions: what is the role of adenosine deaminase? lung. 2008;186:233–240. https://doi.org/10.1007/s00408-008-9085-7 porcel jm, esqueda a, bielsa s. diagnostic performance of adenosine deaminase activity in pleural fluid: a single-center experience with over 2100 consecutive patients. eur j intern med. 2010;21:419–423. https://doi.org/10.1016/j.ejim.2010.03.011 kashyap rs, kainthla rp, mudaliar av, purohit hj, taori gm, daginawala hf. cerebrospinal fluid adenosine deaminase activity: a complimentary tool in the early diagnosis of tuberculous meningitis. cerebrospinal fluid res. 2006;3:1–6. https://doi.org/10.1186/1743-8454-3-5 dikensoy o, namiduru m, hokaoglu s, ikidag b, filiz a. increased pleural fluid adenosine deaminase in brucellosis is difficult to differentiate from tuberculosis. respiration. 2002;69:556–559. https://doi.org/10.1159/000066465 lee yc, rogers jt, rodriguez rm, miller kd, light rw. adenosine deaminase levels in nontuberculous lymphocytic pleural effusions. chest. 2001;120:356–361. https://doi.org/10.1378/chest.120.2.356 porcel jm, vives m. etiology and pleural fluid characteristics of large and massive effusions. chest. 2003;124:978–983. https://doi.org/10.1378/chest.124.3.978 jiménez castro d, díaz nuevo g, pérez-rodríguez e & light rw. diagnostic value of adenosine deaminase in nontuberculous lymphocytic pleural effusions. eur respir j. 2003;21:220–224. https://doi.org/10.1183/09031936.03.00051603 porcel jm, vives m, esquerda a, ruiz a. usefulness of the british thoracic society and the american college of chest physicians guidelines in predicting pleural drainage of nonpurulent parapneumonic effusions. respir med. 2006;100:933–937. https://doi.org/10.1016/j.rmed.2005.06.017 acknowledgements references about the author(s) awadia a. ahmeidi department hematology, faculty of medical laboratory science, university of science and technology, khartoum, sudan ashraf musa abeer hospital, muscat, oman hend s. ahmed department hematology and blood transfusion, faculty of medical laboratory science, omdurman ahlia university, khartoum, sudan adel a. elahmar communicable disease center, harmad medical corporation, doha, qatar ryhana b. goota ministry of health, khartoum, sudan ibtihal a. ahmed faculty of medical laboratory science, ibn sina university, khartoum, sudan abdelhakam h. ali department microbiology, faculty of medical laboratory science, university of al butana, rufaa, sudan mushal allam national institute for communicable diseases, national health laboratory service, johannesburg, south africa mozan o. hassan department hematology and blood transfusion, faculty of medical laboratory science, omdurman ahlia university, khartoum, sudan citation ahmeidi a.a, musa a, ahmed h.s, et al. inflammatory markers as predictors of mortality in covid-19 infection. afr j lab med. 2020;9(1), a1298. https://doi.org/10.4102/ajlm.v9i1.1298 scientific letter inflammatory markers as predictors of mortality in covid-19 infection awadia a. ahmeidi, ashraf musa, hend s. ahmed, adel a. elahmar, ryhana b. goota, ibtihal a. ahmed, abdelhakam h. ali, mushal allam, mozan o. hassan received: 12 june 2020; accepted: 07 oct. 2020; published: 21 dec. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. since the origination of the severe acute respiratory syndrome coronavirus-2 (sars-cov-2) in china in december 2019 and its subsequent global spread, it became critical to understand the inflammatory process associated with this virus and the coronavirus disease 2019 (covid-19) and to understand the changes associated with the different inflammatory parameters. the clinical presentation of covid-19 cases varies greatly from mild symptoms among the majority of patients, to severe disease leading to death in some patients. many covid-19 cases had increased levels of inflammatory cytokines and other infection-related biomarkers.1 the inflammatory markers, including interleukin-6, d-dimer, neutrophil-to-lymphocyte ratio and high-sensitivity c-reactive protein (hs-crp) levels, were found to be indicative of severe covid-19 in reports that emerged from china.2 however, their association with mortality among covid-19 patients has not been reviewed. in this article, we identify inflammatory markers (interleukin-6, d-dimer, neutrophil-to-lymphocyte ratio and hs-crp) as predictors of covid-19 mortality. laboratory tests for these markers are simple, cheap and available and can be performed in outbreak areas with limited resources. according to zhou et al., d-dimer levels exceeding 1.0 µg/ml at hospital admission correlated significantly with death among hospitalised covid-19 patients in china, with a p-value of less than 0.001.3 another study in wuhan, china, which included 343 in-patients with confirmed covid-19, showed that elevated d-dimer levels over 2 µg/ml at an early stage of hospitalisation correlated significantly with a high risk of death.4 also, according to another study from china that included 1099 confirmed covid-19 patients, results revealed that median d-dimer and c-reactive protein levels were higher among severe cases compared to non-severe cases, demonstrating that high d-dimer and c-reactive protein levels were significantly associated with covid-19 severity.5 additionally, in wuhan jinyintan hospital, china, a study showed that high levels of interleukin-6 were significantly associated with a high mortality rate.6 this finding was corroborated by silberstein in this study.7 liu y et al. in zhongnan hospital of wuhan university identified an elevation in the neutrophil-to-lymphocyte ratio as an independent and significant predictor of mortality among 245 hospitalised covid-19 patients, with an 8% increase in mortality with each unit increase in neutrophil-to-lymphocyte ratio.8 many studies reported a correlation between high levels of hs-crp and mortality rate in covid-19 patients. a study done among 375 patients with confirmed sars-cov-2 infection revealed that elevated hs-crp levels were significantly associated with a high mortality risk.9 in another study conducted to evaluate fatal outcomes among covid-19 patients, 187 patients from china were included among whom 43 died, and results revealed that high levels of hs-crp was significantly associated with mortality.10 we conclude that elevation in levels of these four inflammatory markers may be indicative of covid-19 infection severity and mortality. we suggest that these parameters may be helpful predictors of covid-19 severity and could be used as early predictors for case management before deterioration. on the other hand, these parameters cannot be used independently for initial diagnosis and physicians need to monitor the presence of other infections that may interfere with the elevation of these markers. finally, we recommend that similar studies on inflammatory markers should be conducted in african populations to demonstrate the levels of these markers among african covid-19 patients and their association with covid-19 severity and mortality. acknowledgements competing interests the authors have declared that no competing interests exist. authors’ contributions a.a.a., a.m., a.a.e. and i.a.a. developed the concepts and performed the literature search. m.o.h. and m.a. helped to supervise the project. a.h.a and r.b.g. edited the manuscript. a.a.a., a.h.a. and a.a.e. prepared and reviewed the manuscript. h.s.a. helped in writing the literature review. m.a. and m.o.h. were responsible for final approval of the version to be published. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references qin c, zhou l, hu z, et al. dysregulation of immune response in patients with covid-19 in wuhan, china. clin infect dis. 2020;71(15):762–768. https://doi.org/10.1093/cid/ciaa248 chen n, zhou m, dong x, et al. epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study. lancet. 2020 feb 15;395(10223):507–513. https://doi.org/10.1016/s0140-6736(20)30211-7 zhou f, yu t, du r, et al. clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study. lancet. 2020;395(10229):1054–1062. https://doi.org/10.1016/s0140-6736(20)30566-3 zhang l, yan x, fan q, et al. d-dimer levels on admission to predict in hospital mortality in patients with covid-19. j thromb haemost. 2020 jun;18(6):1324–1329. https://doi.org/10.1111/jth.14859 guan wj, ni zy, hu y, et al. clinical characteristics of coronavirus disease 2019 in china. n engl j med. 2020 apr 30;382(18):1708–1720. wu c, chen x, cai y, et al. risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease 2019 pneumonia in wuhan, china. jama intern med. 2020;180(7):934–943. https://doi.org/10.1001/jamainternmed.2020.0994 silberstein m. correlation between premorbid il-6 levels and covid-19 mortality: potential role for vitamin d. int immunopharmacol. 2020 nov;88:106995. https://doi.org/10.1016/j.intimp.2020.106995 liu y, du x, chen j, et al. neutrophil-to-lymphocyte ratio as an independent risk factor for mortality in hospitalized patients with covid-19. j infect. 2020;81(1):e6–e12. https://doi.org/10.1016/j.jinf.2020.04.002 yan l, zhang ht, goncalves j, et al. an interpretable mortality prediction model for covid-19 patients. nat mach intell. 2020;2(5):283–288. https://doi.org/10.1038/s42256-020-0180-7 guo t, fan y, chen m, et al. cardiovascular implications of fatal outcomes of patients with coronavirus disease 2019 (covid-19). jama cardiol. 2020;5(7):811–818. https://doi.org/10.1001/jamacardio.2020.1017 response from makgopa et al. acknowledgements references about the author(s) josef finsterer neurology and neurophysiology centre, vienna, austria citation finsterer j. carriers of the m.3243a>g variant should not be labelled with an acronym before they have been systematically screened for multisystem disease. afr j lab med. 2024;13(1), a2527. https://doi.org/10.4102/ajlm.v13i1.2527 scientific letter carriers of the m.3243a>g variant should not be labelled with an acronym before they have been systematically screened for multisystem disease josef finsterer received: 08 june 2024; accepted: 09 aug. 2024; published: 30 sept. 2024 copyright: © 2024. the author licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. we read with interest the article by makgopa et al. about a 45-year-old male with maternally inherited diabetes and deafness (midd) syndrome due to the variant m.3243a>g.1 family history was positive for hearing loss (mother, two sisters, nephew), diabetes (two sisters, nephew), cardiac disease (mother, two sisters), and kidney failure (niece).1 two other family members (third sister, nephew) tested positive for the m.3243a>g variant.1 the study is compelling but raises concerns that should be discussed. the first point is that neither heteroplasmy rates nor mitochondrial dna (mtdna) copy number have been determined. in order to assess the rate of progression and outcome, it would have been imperative to at least determine heteroplasmy rates in various affected and unaffected tissues. since heteroplasmy rates can influence phenotypic presentation and can explain the phenotypic heterogeneity between family members, it would have been helpful to know these values. the second point is that the cause of heart failure in the index patient and one of his sisters was not specified. was the heart failure due to coronary artery disease, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, takotsubo syndrome, left ventricular hypertrabeculation, or malignant ventricular arrhythmias? knowledge of cardiac disease that causes death in at least four family members (mother, index patient, two sisters) is important for the care of other living family members and for genetic counselling. what were the results of long-term electrocardiogram recordings, what were the pro-brain natriuretic peptide values in the clinically affected living family members, and did cardiac magnetic resonance imaging show hypertrophy of the left ventricular myocardium or left ventricular hypertrabeculation? did the mother have signs of heart failure such as leg oedema, exertional dyspnoea, or neck vein distension before death? the third point is that we disagree with the diagnosis of midd in the index patient. the index patient suffered not only from diabetes and hearing impairment, but also from congestive heart failure, myopathy and renal failure.1 therefore, this patient cannot be diagnosed with midd but rather should be classified as midd plus.2 the fourth point is that the index patient did not undergo autopsy. a patient with a mitochondrial disorder should undergo an autopsy, especially if he or she has not been systematically screened for multisystem involvement throughout his or her lifetime. the m.3243a>g variant may have manifested itself not only in the ears, heart, muscles, and pancreas, but generally in all organs.3 since pulmonary embolism was suspected, it should have been confirmed by autopsy. since the m.3243a>g commonly manifests in the brain,4 we should know whether any of the clinically affected family members had cognitive decline, seizures, ataxia, movement disorder, or stroke-like episodes. surprisingly, the index patient’s mother was depicted as living in the pedigree shown in figure 2 in makgopa et al. it should be clarified whether she was deceased as described in the case description or is actually alive as stated in the pedigree. in conclusion, this interesting study has limitations that put the results and their interpretation into perspective. removing these limitations could strengthen the conclusions and reinforce the study’s message. all unresolved questions must be clarified before readers can uncritically accept the study’s message. carriers of the m.3243a>g variant should not be labelled with an acronym before they have been systematically screened for multisystem disease. response from makgopa et al. we thank the author of this scientific letter for their interest in our article. they have raised some interesting and compelling arguments, and we appreciate their perspective. however, the title of the letter may be a bit misleading, as it appears to imply that erroneous diagnoses were made in the makgopa et al. article. our article sought to highlight the importance of recognising the midd pattern and ensuring that maternal relatives of a patient with midd are screened and followed up, as they are at risk of developing the condition. maternal relatives should be tested for the mtdna variant, as they are likely to be obligate carriers. as stated in our article, genetic testing was considered for available maternal relatives. the article clearly indicates that the proband was not available, as he was deceased (table 2 in makgopa et al.) at the time when the genetic testing was conducted. while testing heteroplasmy may be desirable, the frequency may change with age across tissues, even within the same individual, and the impact of heteroplasmy still remains unresolved. although not accurate, heteroplasmy levels can be broadly estimated on the restriction fragment length polymorphism band intensities. there is no doubt that these are positive cases. we think it is important to point out that the unavailability of heteroplasmy determination in the local clinical setting cannot be an excuse not to diagnose cases. capability of an assay to accurately detect mtdna variants is very important. the polymerase chain reaction–restriction fragment length polymorphism method used in this study has appreciable sensitivity to provide correct diagnosis. it is interesting that the author suggested the diagnosis of midd plus. indeed, patients described by makgopa et al. had variable presentations. however, midd plus, as suggested in this letter, is not a formal diagnosis. it is noteworthy that none of the patients described by makgopa et al. were found to have mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (melas) syndrome. in conclusion, we hope that concerns raised in the letter do not in any way dilute the important message of the article by makgopa et al. there is no doubt that the letter further indicates the importance of actively screening patients with midd and their maternal relatives, as previously reported. it is worth emphasising that families need to be followed up, as they may be at risk of developing either midd, melas or other forms of primary mitochondrial disease related to this variant. acknowledgements competing interests the author declares that they have no financial or personal relationships that may have inappropriately influenced them in writing this scientific letter. author’s contribution j.f. is the sole author of this scientific letter. ethical considerations this scientific letter followed all ethical standards for research without direct contact with human or animal subjects. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability data sharing is not applicable to this scientific letter as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this scientific letter are those of the author and are the product of professional research. it does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the author is responsible for this scientific letter’s results, findings, and content. references makgopa h, kemp t, meldau s, honey em, chale-matsau b. maternally inherited diabetes and deafness with a variable presentation across three generations within a pedigree, south africa. afr j lab med. 2024;13(1):2384. https://doi.org/10.4102/ajlm.v13i1.2384 finsterer j, frank m, mishra a. genetic background and phenotypic heterogeneity of melas and maternally inherited diabetes and deafness. int j clin exp pathol. 2015;8(11):15439–15441. el-hattab aw, adesina am, jones j, scaglia f. melas syndrome: clinical manifestations, pathogenesis, and treatment options. mol genet metab. 2015;116(1–2):4–12. https://doi.org/10.1016/j.ymgme.2015.06.004 oyama m, iizuka t, nakahara j, izawa y. neuroimaging pattern and pathophysiology of cerebellar stroke-like lesions in melas with m.3243a>g mutation: a case report. bmc neurol. 2020;20(1):167. https://doi.org/10.1186/s12883-020-01748-7 abstract introduction methods results discussion acknowledgements references about the author(s) armand m. mambote department of disease control, faculty of veterinary medicine, university of zambia, lusaka, zambia ruth l. mfune department of public health, faculty of medicine, copperbelt university, ndola, zambia victor daka department of public health, faculty of medicine, copperbelt university, ndola, zambia melai mubanga department of environmental health, school of medicine, eden university, lusaka, zambia mary mubiana department of disease control, faculty of veterinary medicine, university of zambia, lusaka, zambia flavien n. bumbangi department of medicine and clinical sciences, faculty of medicine, eden university, lusaka, zambia chitalu chanda ministry of health, lusaka, zambia john b. muma department of disease control, faculty of veterinary medicine, university of zambia, lusaka, zambia citation mambote am, mfune rl, daka v, et al. seroprevalence and risk factors of brucella antibodies among patients seeking medical attention at health facilities in selected districts of western province in zambia. afr j lab med. 2025;14(1), a2885. https://doi.org/10.4102/ajlm.v14i1.2885 original research seroprevalence and risk factors of brucella antibodies among patients seeking medical attention at health facilities in selected districts of western province in zambia armand m. mambote, ruth l. mfune, victor daka, melai mubanga, mary mubiana, flavien n. bumbangi, chitalu chanda, john b. muma received: 31 may 2025; accepted: 09 sept. 2025; published: 11 dec. 2025 copyright: © 2025. the author(s). licensee: aosis. this work is licensed under the creative commons attribution 4.0 international (cc by 4.0) license (https://creativecommons.org/licenses/by/4.0/). abstract background: brucellosis is a neglected zoonosis that impacts public health and livestock productivity. it is endemic in zambia, particularly in regions with extensive livestock farming, such as the western province. objective: this study aimed to determine the seroprevalence of brucella antibodies and identify risk factors for brucella exposure among healthcare-seeking patients at health facilities in western province, zambia. methods: a cross-sectional study was conducted from 16 december 2022 to 31 december 2023 among 197 patients at health facilities in mongu, senanga, and limulunga districts of western province. serum samples were collected and tested for brucella antibodies using the rose bengal test (rbt) and competitive-enzyme linked immunosorbent assay. according to the serial interpretation of results, only sera that tested positive on rbt and competitive-enzyme linked immunosorbent assay were considered positive. a structured questionnaire was used to gather epidemiological data. results: most study participants were women (55.8%), married (54.31%), and had a primary level of education (55.33%). there was a 4.57% seroprevalence of brucella antibodies. most participants (67.01%) were unaware that brucella affects humans, and 91.88% lacked knowledge about its mode of transmission to humans. multivariable logistic regression model showed that the number of animals kept was a statistically significant risk factor (p < 0.039) associated with human brucella seropositivity. conclusion: brucella antibodies were detected among patients visiting health facilities in western province, zambia. the number of animals kept was significantly associated with brucella antibodies. what this study adds: this is the first study on the seroprevalence of brucella antibodies in patients at health facilities in zambia, addressing a research gap that has largely centred on livestock. this information will help to develop public health strategies for preventing and controlling human brucellosis intervention. keywords: brucella antibodies; human brucellosis intervention; risk factors; seroprevalence; western province; zambia. introduction brucellosis is an infectious zoonotic infection caused by intracellular gram-negative coccobacilli bacteria of the genus brucella, and is among the seven neglected zoonotic diseases.1,2 the disease is widespread, affecting humans, a wide range of wild animals, and economically viable domestic livestock such as cattle, goats, sheep, donkeys, camels, swine, and dogs.3 currently, 12 brucella species have been identified; however, only b. melitensis, b. abortus, b. suis, and, on rare occasions, b. canis, are classified as causing human infection.4 humans can become infected by consuming unpasteurised dairy products, or through direct contact with secretions from infected animals.4 typical symptoms observed in most high-risk occupational groups affected, such as veterinarians, laboratory workers, abattoir workers, slaughterhouse workers, livestock workers, and farmers, include fever, headaches, physical weakness, sweats, and back pain.5 the disease profoundly impacts developing countries with weak public and animal health systems.2 according to the world health organization (who), more than 500 000 new human cases are reported yearly worldwide.5 human brucellosis studies have reported varying seroprevalences across africa: 31.3% in egypt,6 24.1% in nigeria,7 5.6% in cameroon,8 5.7% and 31.8% in kenya,9 18.7% in uganda,10 6.1 % in rwanda,11 and 1.41% in tanzania.12 however, there is scarce information on human brucellosis in zambia, as more studies have focused on animals than humans.13,14 the findings are intended to improve stakeholder awareness and to strengthen brucellosis diagnosis, surveillance, and prevention efforts in endemic regions. methods ethical considerations ethical approval was obtained from excellence in research ethics and science (reference number: 2018-dec-004), the national health research authority, and the ministry of health prior to the study. written informed consent was obtained from all participants. for participants below 18 years, written informed consent was obtained from their parents or legal guardians, followed by their assent. the participants were informed of the purpose of the study, procedures, risk of minimal pain and discomfort at the injection site during blood sample collection, their benefits, and the right to withdraw at any time during the study. all data were restricted to the investigators and treated confidentially; no participant identifiers were used. to ensure participant data confidentiality, all personal identifiers were deleted from the data set and replaced with unique codes. the data, which was stored on password-protected computers, could only be accessed by the research team. any published results are given in aggregate form, ensuring that individual individuals are not recognised. study design and setting a cross-sectional study was conducted between 16 december 2022 and 31 december 2023 among patients seeking care at health facilities in three districts of western province, zambia: senanga, limulunga, and mongu. the study involved collecting blood samples from consenting symptomatic patients and epidemiological data using a pretested structured questionnaire. the province and districts were selected based on the fact that western province is one of the major livestock-producing provinces, where bovine brucellosis has been reported.15 western province covers an area of 126 386 km2 and lies between the latitudes 14 °s and 17 °s and longitudes 22 °e and 25 °e.16 it is characterised by the barotse floodplains of the zambezi river and the sandy soils. the main economic activity in the province is livestock production, followed by fishing and crop production. three health facilities were selected in senanga district: ngundi, sikumbi, and lui wanya health centres. two facilities were included from mongu district (lealui mini hospital and sefula rural health centre) and two from limulunga district (limulunga mini hospital and ikwichi rural health centre). study population and sampling strategy the sample size was estimated using the ausvetepitool software (http://epitools.ausvet.com.au/), assuming an expected prevalence of 20%,17 a desired absolute precision of 5% and a confidence level of 95%. a sample size of 225 participants was considered after increasing the required sample size (n = 195) by 15% to account for sample haemolysis and data incompleteness. sample haemolysis was accounted for because of the distance (approximately two days) between the study area where samples were collected and the laboratory in lusaka. the 15% was considered based on previous literature.18 the sample size was distributed in the three districts according to the weight index (table 1). table 1: distribution of sample size of humans weighted by district populations. included in the study were all consenting patients at the health facilities during the study period who presented any of the following signs and symptoms: intermittent or persistent fever, headache, weakness, profuse sweating, chills, arthralgia, weight loss and joint pain, with a negative result for. the patients with another confirmed diagnosis, such as smear-positive tuberculosis, malaria, salmonella, febrile disease, and those who did not consent, were excluded from the study. patients who presented with different signs and symptoms, or were unwilling to participate, were excluded from the study. sample and data collection a clinical officer or nurse collected 4 ml of blood from each consenting participant into a labelled sterile plain tube and kept at +4 °c for 24 h. the serum samples were separated using a portable field centrifuge (tomy digital mx-300, japan) and stored in labelled cryovial tubes at −20 °c until transportation to the university of zambia school of veterinary medicine (unzavet) for laboratory analysis. epidemiological data were collected from participants using a structured questionnaire adopted from a similar study.17 the questionnaire had four parts: (1) socio-demographic characteristics, (2) knowledge and attitude of participants regarding brucellosis, (3) practices of participants towards brucellosis, and (4) risk factors. laboratory analysis serum samples were screened for brucella antibodies using the rose bengal test (rbt, id. vet, and innovative diagnostics, france) as described elsewhere. only rbt-positive serum samples were subjected to competitive-enzyme linked immunosorbent assay (c-elisa). briefly, for the rbt, 30 µl of serum was mixed with 30 µl of rose bengal antigen on a slide. the mixture was gently rocked for 4 min and observed for agglutination. the presence of agglutination was considered positive. the c-elisa analysis was done according to the manufacturer’s guidelines and reagent kit manual (svanovirr brucella–ab c-elisa, boehringer ingelheim svanova, sweden). briefly, 50 μl of mab-solution was added into all wells used for controls and samples (time difference between controls: a positive, weak positive, and negative control were included in a 96-well plate; samples and mab-solution addition did not exceed 10 min). the plate was sealed, and the reagents were thoroughly rinsed for 5 min using a plate shaker. the plate was then incubated at room temperature (25 °c) for 30 min, rinsed four times with phosphate-buffered saline–tween buffer. the wells were filled up at each rinse. the plate was then emptied and tapped hard to remove all remaining fluid. then, 100 μl of the conjugate solution was added to each well, and the plate was sealed and then incubated at room temperature for 30 min. rinsing was repeated, as explained above. then, 100 µl substrate solution was added to each well and incubated for 10 min at room temperature (timing began after the first well was filled). the reaction was stopped by mixing 50 µl of stop solution in the same order as the substrate solution. then, the optic density (od) of controls and samples was measured at 450 nm in a microplate photometer (the air was used as a blank). the od was measured within 15 min after the addition of the stop solution to prevent fluctuation in od values. the od of the positive control was the one with which the od of each test serum was compared to establish the final result (negative or positive). determination of the positive and negative tests using the cut-off was provided in the c-elisa kit guide. negative results were determined by a percent inhibition of < 30%, while positive results were ≥ 30%. in this study, serial interpretation of the results was used. therefore, only sera testing positive on both rbt and c-elisa were regarded as positive. data analysis the data were entered, coded and cleaned using microsoft excel 2016® (microsoft, redmond, washington, united states) and analysed using stata version 17 (statacorp llc, college station, texas, united states). categorical data were expressed as a percentage, and seroprevalence was calculated by dividing the number of positive serum samples by the total samples examined. descriptive statistics were used to summarise participant characteristics and seroprevalence. categorical variables were presented as frequencies and proportions (%), and continuous variables were summarised using medians (interquartile range) based on their distribution. the seroprevalence of brucella antibodies, defined as a sample positive on both the rbt and confirmatory c-elisa, was calculated as a proportion with a 95% confidence interval (ci) derived from the wilson score method to ensure accuracy for proportions near zero. the odds ratio, 95% ci, and fisher’s exact tests were computed to see the degree of association of the risk factors with brucella seropositivity. using the cut-off, negative results were determined by a percent inhibition of < 30%, while positive results were ≥ 30% for c-elisa. the independent effects of risk factors on anti-brucella spp. seropositivity were assessed using univariable analysis. variables with a p-value ≤ 0.25 were used as variables in the multivariable logistic model, which was built using a backwards selection strategy. the odds ratio was calculated at 95% ci to see the degree of association between brucella antibodies and the risk factors. the model’s predictability was estimated by the receiver operating characteristic (roc) curve analysis, while the hosmer and lemeshow test was used to test model fit. results socio-demographic characteristics of study participants out of the 225 samples collected, 197 were acceptable for analysis and 28 were haemolysed, hence could not be included in the study. out of the 197 participants, the study had more women (55.8%, n = 110) than men (44.2%, n = 87). the participants’ mean age was 36 years, ranging from 10 to 81 years. about (54.31%, n = 107) were married, (55.33%, n = 109) had a primary education, and (68.02%, n = 134) were unemployed. senanga district had more participant (48.23%, n = 93), than mongu and limulunga districts (table 2). table 2: socio-demographic variables of study participants (n = 197). seroprevalence of human brucella antibodies from the 197 serum samples that were acceptable for analysis, the estimated seroprevalence of brucella antibodies among the patients attending the health facilities in western province was 4.57% (n = 9, 95% ci: 2.34–8.49). the seroprevalence was higher in senanga and mongu (2.03%) than limulunga district (table 3). table 3: distribution of brucella antibodies per study district. knowledge and attitudes of participants regarding brucellosis most of the participants, 114 (57.87%) had obtained their information on brucellosis from veterinary officers, while only a few, 13 (6.60%), were aware that brucellosis can affect humans. most participants, 132 (67.01%), were ignorant about the mode of transmission to humans, while only 16 (8.12%) had the symptoms of brucellosis, as shown in table 4. table 4: knowledge and attitude of participants about brucellosis (n = 197). risk factors associated with brucella antibodies initially, the univariable analysis looked at the relationship between possible risk factors and the dichotomous outcome variable, brucella antibodies presence (table 5). some characteristics, including age, marital status, occupation, education level, and gender, did not correlate with brucella antibodies presence (table 5). the number of animals kept (p < 0.049) and exhibiting brucellosis symptoms (p < 0.004) were statistically significantly associated with brucella antibodies. table 5: univariable analysis of risk factors associated with brucella antibodies. as demonstrated by the multivariable logistic regression model (table 6), the number of animals retained was a predictor of brucella spp. seropositivity (p < 0.039). the odds of one keeping several animals being seropositive were 6.49 (95% ci: 1.10–38.13). the predictability of the model is indicated by the roc curve (area under the curve = 0.760) (figure 1). the area under the roc indicated that the model can correctly predict up to 76% of the outcome. the hosmer and lemeshow test for goodness-of-fit (χ2 = 0.76; with a p-value of 0.785) indicated that the model fit the data adequately. figure 1: receiver operating characteristic curve demonstrating the predictability of the model (area under the curve = 0.760). table 6: multivariable logistic regression analysis for risk factors associated with human brucella antibodies. discussion seroprevalence of human brucella antibodies the study found a 4.57% seroprevalence of brucella antibodies among the participants, which is close to the 6.1% reported among patients attending a district hospital in rwanda.11 brucella antibodies were higher in patients from senanga district than mongu and limulunga districts. this can be attributed to the fact that most of the samples collected in senanga were from rural health centres, while most samples in mongu and limulunga districts were collected from mini-hospitals. our 4.57% finding is lower than the 20.2% reported in the southern province of zambia.17 however, our study focused on febrile patients seeking medical attention, while the former targeted occupationally exposed humans (herdsmen and abattoir workers). the study findings are also lower than the 14.9% prevalence observed among community hospital patients in southwestern uganda.19 southwestern uganda has a high milk production and cultural practices of raw milk consumption, which increases brucellosis exposure. a similar study in saudi arabia reported a seroprevalence of 12.8% among febrile patients at a referral hospital.20 the variations seen in results can be because of the various serodiagnostic methods used. the higher rbt positivity compared to c-elisa may reflect cross-reactivity or past exposures; the use of c-elisa improved specificity. brucellosis serology showed a significant difference between the results of the rbt (18.27%) and confirmatory c-elisa (4.57%). because of its high sensitivity, the rbt can produce false positives by cross-reacting with antibodies against other gram-negative bacteria with similar o-polysaccharide antigens, such as escherichia coli o:157 or yersinia enterocolitica o:9. moreover, it may detect subclinical or low-level exposures that do not necessarily indicate active illness. in this high-risk pastoralist community, rbt-positive/c-elisa-negative results likely represent the background immunological stimulation, despite our serial testing approach (rbt screening followed by c-elisa confirmation), which is the recommended standard to maximise diagnostic specificity. therefore, the 4.57% seroprevalence is a precise and cautious estimate of actual brucella exposure. knowledge and attitude of participants regarding brucellosis about half of the respondents (57.87%) were knowledgeable about brucellosis, similar to findings in uganda.21 low knowledge levels increase the risk of brucella infection, weaken control measures and contribute to disease underreporting in the country. only 6.60% of participants were aware that brucella can affect humans, while the majority (67.01%) were ignorant about the mode of transmission to humans. the low awareness levels in our study may be attributed to the limited formal education received by participants and the lack of sensitisation on zoonotic diseases. risk factors associated with brucella antibodies the number of animals kept was a risk factor that was significantly associated with human brucella seropositivity. the odds of someone keeping animals being seropositive were 6.49. a similar study conducted in kenya found that herd size was a significant risk factor for exposure to brucella spp.22 the model roc curve demonstrated the predictability of the model (area under the curve = 0.760). the hosmer and lemeshow test showed that the model fitted the data, thus increasing its reliability in predicting, with roc areas around 0.760, as confirmed by a p > 0.78. the reduced model predictability may be because of our small sample size, which reduces the ability to detect smaller effect sizes, or it could suggest that the level of livestock exposure is the main factor driving brucellosis seropositivity in this community, with other factors acting as mediators or being associated. study limitations the sample size for the study was less than targeted, but enough to carry out the planned analysis without affecting its validity. a small sample size and few positive cases are reflected in some wide cis, suggesting caution in interpreting associations. however, some samples were haemolysed and excluded from analysis; hence, other positive cases were possibly not detected. our study did not present the bacteriological or molecular-based test results; hence, the seropositivity results might be caused by previous exposure to infection or cross-reactivity. the c-elisa test used in this study is highly specific. serial interpretation of rbt and c-elisa increases the specificity of the test regime. therefore, our reported 4.57% seroprevalence offers a precise and cautious estimate of actual brucella exposure. recommendations in rural livestock communities with endemic brucellosis, increasing routine testing and upholding a high index of clinical suspicion may enhance case discovery and lessen the burden of the disease. furthermore, health authorities should organise public health awareness campaigns to educate healthcare workers and the communities about brucellosis, its risk factors, modes of transmission, and preventive measures. the practice of raw milk consumption and its products should be discouraged. lastly, there is a need for further bacteriological and molecular studies to understand the epidemiology of brucellosis. controlling brucellosis in animals will be the most efficient method of controlling it in humans. conclusion a total of 4.57% of febrile patients seeking care at health facilities in western province tested positive for brucella antibodies. most participants were unaware that humans can contract brucella or how the disease is transmitted. the number of animals kept was statistically associated with brucella seropositivity. these findings underscore the need for public health interventions to enhance awareness, promote regular screening, and strengthen preventive measures for brucellosis in health facilities in western province, zambia. acknowledgements this article includes content that overlaps with research originally conducted as part of armand m. mambote’s master’s thesis titled ‘seroprevalence of human brucella antibodies and associated risk factors among patients seeking medical attention in selected health facilities in western province of zambia’, submitted to the department of disease control, university of zambia, in 2024. the thesis was supervised by j.b. muma and r.l. mfune. portions of the data, analysis, and discussion have been revised, updated, and adapted for journal publication. the original thesis is publicly available at: https://dspace.unza.zm/handle/123456789/9050. the author affirms that this submission complies with ethical standards for secondary publication, and appropriate acknowledgement has been made of the original work. the authors would like to thank the provincial health officer, district health officers, clinical officers and nurses for their support during sample collection, and the laboratory staff at the department of disease control, school of veterinary medicine, university of zambia, for their assistance during laboratory analysis. while their contribution was important, it did not meet the criteria for authorship. a preprint version of this work was previously published on medrxiv (https://doi.org/10.1101/2024.07.25.24311011) and we acknowledge its role in shaping the final manuscript. competing interests the authors declare that no financial or personal relationships inappropriately influenced the writing of this article. authors’ contributions r.l.m and j.b.m participated in the conception of the idea, investigation and the writing, reviewing and editing of the manuscript. a.m.m. took part in data curation, formal analysis, investigation, methodology and prepared the original draft manuscript. v.d., m. mubanga, m. mubiana and c.c. participated in reviewing and editing of the manuscript. f.n.b. and a.m.m. validated the software, while r.l.m. and j.b.m. supervised the research activities. funding information this work was supported by the international foundation sciences (grant number 13-b-6519-1), which funded the brucellosis project, and the africa centre of excellence for infectious disease of humans and animals world bank-funded project (grant number p151847). data availability the data that support the findings of this study are not openly available because of reasons of sensitivity and are available from the corresponding author, r.l.m., upon reasonable request disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. the article does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the authors are responsible for this article’s results, findings, and content. references pappas g, papadimitriou p, akritidis n, christou l, tsianos ev. the new global map of human 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university of zambia, 2017; p. 260. nyirenda ss, sakala m, moonde l, et al. prevalence of bovine fascioliasis and economic impact associated with liver condemnation in abattoirs in mongu district of zambia. bmc vet res. 2019;15(1):33. https://doi.org/10.1186/s12917-019-1777-0 mubanga m, mfune rl, kothowa j, et al. brucella seroprevalence and associated risk factors in occupationally exposed humans in selected districts of southern province, zambia. front public health. 2021;9:754244. https://doi.org/10.3389/fpubh.2021.745244 browner ws, newman tb, cummings sr, et al. designing clinical research. 5th ed. philadelphia, pa: lippincott williams & wilkins; 2022. migisha r, nyehangane d, boum y, et al. prevalence and risk factors of brucellosis among febrile patients attending a community hospital in south western uganda. sci rep. 2018;8(1):1–8. https://doi.org/10.1038/s41598-018-33915-9 alkahtani am, assiry mm, chandramoorthy hc, al-hakami am, hamid me. sero-prevalence and risk factors of brucellosis among suspected febrile patients attending a referral hospital in southern saudi arabia (2014–2018). bmc infect dis. 2020;20:26. https://doi.org/10.1186/s12879-020-4763-z kansiime c, mugisha a, makumbi f, et al. knowledge and perceptions of brucellosis in the pastoral communities adjacent to lake mburo national park, uganda. bmc public health. 2014;14(1):242. https://doi.org/10.1186/1471-2458-14-242 kairu-wanyoike s, nyamwaya d, wainaina m, et al. positive association between brucella spp. seroprevalences in livestock and humans from a cross-sectional study in garissa and tana river counties, kenya. plos negl trop dis. 2019;13(10):e0007506. https://doi.org/10.1371/journal.pntd.0007506 abstract introduction methods results discussion acknowledgements references about the author(s) foluke a. fasola department of haematology, college of medicine, university of ibadan, ibadan, nigeria department of haematology, university college hospital, ibadan, nigeria adeola a. fowotade department of medical microbiology & parasitology, college of medicine, university of ibadan, ibadan, nigeria adedayo o. faneye department of virology, college of medicine, university of ibadan, ibadan, nigeria adeyeni adeleke department of haematology, college of medicine, university of ibadan, ibadan, nigeria citation fasola fa, fowotade aa, faneye ao, adeleke a. prevalence of hepatitis b virus core antibodies among blood donors in nigeria: implications for blood safety. afr j lab med. 2022;11(1), a1434. https://doi.org/10.4102/ajlm.v11i1.1434 original research prevalence of hepatitis b virus core antibodies among blood donors in nigeria: implications for blood safety foluke a. fasola, adeola a. fowotade, adedayo o. faneye, adeyeni adeleke received: 23 oct. 2020; accepted: 20 apr. 2022; published: 26 july 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: anti-hepatitis b core antibody (anti-hbc) testing improves transfusion safety by detecting past and current hepatitis b virus (hbv) infection while detecting hepatitis b surface antigen (hbsag) in serology-negative hbv infection. however, occult hbv infection (obi) (serum or liver hbv dna-positive but hbsag-negative) remains unaddressed among replacement blood donors – family members or friends who donate to replace blood transfused to a relative. objective: this study assessed risk factors for a positive anti-hbc test among donors with obi and determined the anti-hbc-positive status of replacement donors. methods: the study was conducted at the university college hospital blood bank, ibadan, nigeria, using blood samples collected from blood donors between april 2019 and may 2019. donors were screened for hbsag by rapid diagnostic test (rdt) and enzyme-linked immunosorbent assay (elisa) and anti-hbc by elisa, while hbv dna was detected using a semi-nested polymerase chain reaction. results: of the 274 participants, 15 (5.5%) were hbsag-positive by rdt and 36 (13.1%) by elisa, while 133 (48.5%) were anti-hbc positive. out of 232 hbsag-negative donors, 107 (46.1%) were anti-hbc positive. of the 107 hbsag-negative but anti-hbc-positive samples, only one (0.93%) was hbv dna-positive. the hbv dna-positive donor was hbsag-negative by both rdt and elisa tests. conclusion: this study establishes a potential risk for hbv transmission from isolated anti-hbc-positive donors to blood recipients. hbc immunoglobulin (antibody) m testing to identify blood units requiring further screening with polymerase chain reaction to detect obi can prevent hbv transmission through blood transfusion. keywords: anti-hbc antibodies; donors; blood safety; hbv dna; occult hbv. introduction blood transfusion is an indispensable life-saving therapy for patient management; however, one of its adverse effects is the potential for transmitting infections. hepatitis b virus (hbv) is the most frequent transfusion-transmitted viral infection.1 in sub-saharan africa, the median risk of hbv infection following blood transfusion is 4.3/1000 units2 and the risk from blood screened by enzyme immunoassay for recipients younger than ten years old is 1:326.3 due to its public health burden, tackling hbv is a global health strategy for achieving the 2030 agenda for sustainable development.4 when an individual has an acute-phase infection, the first viral antigen to appear in the blood is hepatitis b surface antigen (hbsag), which persists even in the chronic phase but is undetectable when the virus is cleared from the blood. thus, hbsag is the hbv infection detection marker. the hbv genome is enclosed within a ‘core particle’ made of hepatitis b core protein or antigen (hbc). while the host is clearing the hbsag by developing hbsag antibodies (anti-hbs), the host also produces hbc immunoglobulin (antibody) m. during this period, known as the ‘window period’, the host is infectious, and the serological marker for infection is anti-hbcigm,5 as the period is associated with undetectable levels of both hbsag and anti-hbsag. blood donors at this infection stage (tail end carriers) can transmit hbv.6 progression of hbv infection to the chronic state is associated with the presence of both immunoglobin g (igg) and immunoglobin m (igm) anti-hbc against hbc. the natural course of infection in persons with chronic hbv infection could terminate as ‘inactive carriers’ with occult hbv infection (obi), defined as the existence of low-level hbv dna in the serum (< 200 iu/ml), cells of the lymphatic (immune) system, or hepatic tissue of patients with serological markers of the previous infection (anti-hbc or anti-hbs positive) and the absence of serum hbsag.7,8 hence, positive anti-hbc is considered a key obi marker.9 most blood bank screening panels consist of hbsag and anti-hbc total (igm + igg).10 the anti-hbc total (igm + igg) is used to detect both previous and current hbv infection. the hbsag, anti-hbc, and hbv nucleic acid tests (nat) are often used in blood banks in high-income countries to detect infection, during the window period, obi, and genetic and antigenic viral variants, thus ensuring optimal blood safety levels. a substantial percentage of blood recipients are likely to be the immunosuppressed, women, and children.11 transfusion of blood from obi donors to pregnant women can increase vertical transmission of viral hepatitis. vertically infected babies are prone to long-term complications of hbv infection, such as liver disease. sadly, the university college hospital (uch) blood bank in ibadan, nigeria, cannot detect hbv infection in blood donors with undetectable hbsag. in the uch blood bank, as in many developing countries, most blood units are from replacement donors, against the recommended voluntary blood donors for safe blood supply. replacement blood donors are family members or friends who donate blood to replace blood transfused relatives, in our setting pregnant relatives. thus, the inability to detect hbv infection in blood donors with undetectable hbsag implies a possible risk of post-transfusion hepatitis from anti-hbc-positive individuals. therefore, it is imperative to determine the anti-hbc positivity status of replacement donors who constitute the most significant proportion of our blood donors5,10 as with nigerian hospital-based blood banks.12 anti-hepatitis b core antibody tests and nat are excluded in our blood donor algorithm; therefore, this study aimed to assess the risk factors for positive anti-hbc in donors and the anti-hbc status of replacement donors to improve blood safety. methods ethical considerations the university of ibadan/university college hospital (ui/uch) ethics review team approved this research (19/0204). all recruited participants gave written informed consent. the confidentiality of participants was maintained by coding the samples, and only authorised personnel had access to participants’ identities. study location and participants the research was conducted at the blood bank of uch in ibadan, nigeria, an 850-bed hospital with 163 examination couches, excluding private suites. this blood bank collects, stores, and processes blood. the target population were replacement blood donors whom the patient or patient’s relatives recruited to replace blood used. only donors who presented blood donation forms indicating the patient on whose behalf blood was being donated were eligible. all donors were recruited from april 2019 to may 2019. in our blood bank, donors were allowed to donate blood if: donors were between 18 and 65 years, weighed 50 kg and above, passed the copper sulphate test and the verbal questioning stage, and were negative for transfusion-transmitted infections, including syphilis, hiv, hbv and hepatitis c virus. donors were excluded from this study if they: voluntarily came to donate blood, had a tattoo (exclusive of tribal marks obtained from infancy), failed the copper sulphate screening test or failed routine eligibility questioning by blood bank staff (which includes questions about previous blood donations, breastfeeding and menstruation status for female donors, presence of any health condition such as hypertension or diabetes, and hiv and hepatitis c virus status). in this study, a questionnaire was administered to include consecutive potential replacement blood donors who had passed the copper sulphate test and blood bank staff eligibility oral questioning stage. the questionnaire was translated into the native language (yoruba) for non-english-speaking participants and collected the patient’s demographic characteristics and knowledge of and risk factors of hbv. four questions tested hbv knowledge with ‘yes’ or ‘no’ questions. each ‘yes’ response scored 1, while each ‘no’ response scored 0. knowledge was graded from 0 to 4 based on increasing correct responses, that is, 1 = only one yes, 2 = two yeses, 3 = three yeses and 4 = four yeses. sample collection six millilitres of venous blood were collected from each donor into sterile plain bottles labelled with the donor’s identity number. the blood was allowed to stand for 45 min, and then the serum was separated by centrifugation daily. the serum was then stored in two aliquots at –80 °c. the samples were thawed to room temperature for each laboratory analysis. the serological tests were performed a week after collecting samples, and the polymerase chain reaction (pcr) test was carried out in july 2019, two months after the collection of samples. serology the blood bank’s screening algorithm identifies a blood donor as hbv-positive using hbsag rapid diagnostic test (rdt) kit and a conventional semi-automated enzyme-linked immunosorbent assay (elisa). the bio-check rapid kit test (bio-check, san francisco, california, united states) employs lateral chromatographic flow, while the monolisa™ hbs ag ultra elisa kit (bio-rad, marnes-la-coquette, france) employs a qualitative one-step solid-phase enzyme-linked immunoassay technique of the ‘sandwich’ type. each test kit came with positive and negative controls used in each assay. first, the rapid test was administered to all donors pre-donation. donors who were hbsag-negative by the rapid test were further tested with the semi-automated elisa test. then, using one of the two aliquots of sera stored, all samples were tested further for anti-hbc using the hbcab elisa kit (melsin medical co., ltd, changchun, china). the anti-hbc assay measures the total anti-hbc, including the igg and igm hbc antibodies. hepatitis b virus dna detection sera from blood donors that were anti-hbc positive were tested for hbv dna with primers targeting the hbv pre-s gene in a semi-nested pcr protocol as described below. briefly, the total hbv dna was extracted from the samples using a dna extraction method previously described by wang et al.12 the hbv pre-s gene was amplified in a semi-nested pcr using three primers. the 979 (5ʹcaaaagacccacaattctttgacatactttccaa3ʹ) and sf (5ʹgtgtcttggccaaaattcgcagt3ʹ) primers were used in the first pcr run, while the primers 979 and mc-f (5ʹtcggatccggtatgttgcccgtttgtc3ʹ) were used in the second pcr round. the cycling conditions used are as follows: 95 °c for 5 min; 40 cycles of 95 °c for 30 s, 60 °c for 45 s, and 72 °c for 45 s; and a final extension of 72 °c for 7 min. the amplicon of ~550 base pairs was analysed by gel electrophoresis using 2% (weight/volume) agarose gel and stained with sybr stain (jena biosciences, jena, germany). amplicons were visualised in an ultraviolet illuminator. the negative control was a previously known hbv-negative sample, while the positive control was a previously confirmed hbv dna-positive sample. occult hbv infection was defined as hbv dna-positive, and serology hbsag-negative and anti-hbc positive. data analysis statistical package for social sciences version 20 (ibm corp., chicago, illinois, united states) was used. frequencies and percentages in tables or charts were used to present the demographic characteristics, risk factors for hbv infection among donors, and hepatitis b screening results for participants. means and standard deviations were used to summarise continuous variables such as the age of participants. odds ratios (ors) and 95% confidence intervals (cis) were calculated to test for the association between risk factors and anti-hbc positivity. the chi-square was also used to test the association between anti-hbc, socio-demographic features and risk factors of the participants. the statistical significance was set at p < 0.05. results socio-demographic and risk factors for acquisition of hepatitis b virus infection from blood donors the 274 participants included in the study ranged from 18 to 62 years (mean age of 32.0 ± 8.86 years). approximately half of the participants, 157 (57.3%), were married, and 25 (9.1%) had multiple sexual partners, while 14 (5.1%) had been previously treated for a sexually transmitted disease (table 1). twenty-eight (10.2%) participants had scarification or tribal marks, 87 (31.8%) shared sharp objects (pedicure, manicure and use of razor blade), and 9 (3.3%) had previously used sex performance enhancement recreational drugs. furthermore, 18 (6.6%) participants had been previously transfused with blood, 7 (2.2%) had jaundice in the last year, and 57 (20.8%) had been asked to do a test for hepatitis b before coming to donate blood. participants’ hbv knowledge scores were: zero, 120 (46.9%); one, 29 (11.3%); two, 21 (8.1%); three, 57 (22.3%); and four, 29 (11.3%). table 1: demographic characteristics of 274 blood donors in ibadan, nigeria, between april 2019 and may 2019. risk factors for hepatitis b virus and anti-hepatitis b core protein or antigen positivity a total of 133 blood donors were anti-hbc positive. the proportion of participants aged 18–35 years with a positive anti-hbc (42.2%) was lower than the proportion of participants above 36 years with a positive anti-hbc (61.8%). donors showed a declining prevalence of anti-hbc with an increasing hbv knowledge score (p = 0.046). the odds of being hbcab positive were 0.35 times less likely among blood donors who had a knowledge score of 4 than blood donors who had a knowledge score of 0 (95% ci: 0.14; 0.84). all 9 participants who used recreational drugs had a positive anti-hbc compared to 117/252 (46.8%) of participants who did not use (p = 0.001). in contrast, there was a lower proportion of participants who shared sharp objects with positive anti-hbc, 34 (39.1%), compared to 96 (52.9%) for those who did not share (table 2). the sharp objects shared included blades and sharps used for a pedicure, manicure, beauty treatment, and shaving hair. the age, gender, educational level, number of sexual partners, history of sexually transmitted disease transfusion and jaundice did not show any significant statistical relationship to anti-hbc positivity. thirteen donors had prior hbv vaccination. five (33.3%) of the hbv vaccinated donors were positive for anti-hbc, while 10 (66.7%) were negative. however, the difference was not statistically significant. the odds of being hbcab positive were 2.22 times more likely among blood donors over 35 years old than those aged 18–35 years (95% ci: 1.32; 3.72). the odds of being hbcab positive were 0.35 times less likely among blood donors who had a knowledge score of 4 than blood donors who had a knowledge score of 0 (95% ci: 0.14; 0.84). the relative risk (rr) of blood donors being hbcab positive was 2.14 times higher among blood donors who had ever used recreational drugs before or during sex than in blood donors who never used recreational drugs before or during sex (95% ci: 1.88; 2.43). there is a 2.23 times higher likelihood of anti-hbc positivity for blood donors who had ever been transfused with blood. blood donors who shared sharp objects were 0.57 times less likely to be hbcab positive compared to donors who do not share sharp objects (95% ci of or: 0.34; 0.96). the comparison of the different risk factors with hbcab is shown in figure 1. figure 1: risk factors for hepatitis b virus infection among 133 donors with positive anti-hepatitis b core protein or antigen in ibadan, nigeria, between april 2019 and may 2019. table 2: comparison of hepatitis b virus risk factors among anti-hepatitis b core-positive and -negative blood donors in ibadan, nigeria, between april and may 2019. occult hepatitis b virus infection of the 274 blood donors, 42 (15.3%) cases were hbsag-positive by both or either rdt or elisa: 9 (3.28%) by both rdt and elisa, 6 (2.19%) by rdt only, and 27 (9.85%) by elisa only; 232 (84.7%) were hbsag-negative (rdt and elisa negative). almost half (133/274; 48.5%) of the donors were anti-hbc positive, while 26/274 (9.5%) donors were hbsag-positive and anti-hbc positive. among the 259 participants who tested negative for hepatitis b by rdt, 121 (46.7%) had a positive anti-hbc result, while 110 (46.2%) of the 238 participants who tested negative for hbsag by elisa test had a positive anti-hbc (table 3). table 3: patterns of anti-hepatitis b core results for rapid and enzyme-linked immunosorbent assay tests for hepatitis b surface antigen and relationship of the two hepatitis b surface antigen detection assays in ibadan, nigeria, between april 2019 and may 2019. of the 232 blood donors that were hbsag-negative by any method, 107 (46.1%) were positive for anti-hbc. however, only one case was anti-hbc positive, hbsag-negative and hbv dna-positive (0.93%). discussion this study on hbc antibodies among replacement blood donors, who form a significant proportion of blood donors in nigeria, showed that 48.8% were positive for total anti-hbc (igg and igm), and over 60.0% of the donors had a tertiary education level, with 47.0% of the donors having an hbv infection knowledge score of 0. in addition, the risk factors for hbv acquisition among anti-hbc-positive donors were being older than 35 years, having poor knowledge of the hbv transmission route, and the use of sex enhancement recreational drugs. an inconsistent positivity rate was observed when rdt and elisa were used to screen for hbsag among blood donors. this incongruity in the results suggests that it is better to use both methods in screening blood for transfusion to reduce the escape of hbv-positive blood into the transfusion pool, especially when hbv dna screening is not in use. testing for anti-hbc has been used in blood transfusion in low hbv-endemic regions to minimise the incidence of post-transfusion hepatitis following transfusion of hbsag-negative blood. the assay identifies chronically infected low-level hbv donors.13 the prevalence of anti-hbc (35.7%) among our hbsag-negative blood donors is high. other studies have reported lower anti-hbc prevalence in low and intermediate hbv-endemic regions and in some high hbv-endemic regions like india14 and north africa.15 the higher prevalence of anti-hbc (35.7%) among the blood donors may be attributed to a higher number of blood donors who had current or past exposure to hbv since anti-hbc persists for life even though the hbv infection is resolved. the possibility of obi should be considered in hbsag-negative but anti-hbc-positive blood donors. occult hbv is more frequently diagnosed in anti-hbc-positive individuals than in anti-hbc-negative individuals.16,17 occult hbv has been reported to range from 3.0% to 17.0% in nigeria.18,19 the prevalence of anti-hbc antibody positivity among blood donors varies between and within countries, depending on the assay method. the anti-hbc test lacks specificity as test reagents reactivity varies between manufacturers, which may affect between-study comparisons.20 depending on the assay type and screening algorithms, false-reactivity rates ranged between 16.0% and 75.0%.21 the confirmatory algorithm is complex. however, a positive anti-hbc in a high hbv-endemic region establishes a history of hbv infection and portends a high risk of obi with the possibility of deferring the blood donor. japhet et al. from nigeria reported a lower anti-hbc prevalence of 13.0% and a higher prevalence of 19.6% hbsag.22 the lower anti-hbc prevalence was probably caused by the igm anti-hbc test used in that study, which detects acute infections but misses chronic hbv carriers, whereas the current study detected total anti-hbc (igm and igg). the shortcoming of the total anti-hbc study is that it includes all blood donors who had been exposed to hbv including those with resolved infection. this shortcoming may exaggerate the prevalence of hbv infection in high-prevalence regions, thereby shrinking the size of the eligible blood donor pool. dhawan et al. observed a significantly higher anti-hbc (8.4% vs 6.9%) among replacement donors than among voluntary donors in india. however, a wide range of prevalence of anti-hbc positivity has been reported among indian donors, and this has been attributed to the use of assays with varying sensitivities in different studies for screening the donors and the nature of the study population.23 this study suggests that donors who had ever used recreational drugs are likely to be positive for anti-hbc. therefore, stringent blood donor screening criteria using a standard questionnaire that provides confidentiality for donors in addition to educational materials to eliminate donors who had ever used recreational drugs may significantly reduce hbv-positive donors irrespective of whether they are voluntary or replacement blood donors. comparing this study with other studies that investigated total anti-hbc in nigeria, the prevalence of 48.5% is higher than 32.5% by ogunfemi et al. in ilorin,24 but lower than 60.7% and 90.0% from the studies by akinbami et al. in lagos18 and ojo et al. in ife.25 the 90.0% prevalence was reported pre-hbv vaccination. levels of anti-hbc have been shown to decrease in the donor population; this could be attributed to acquired immunity from vaccination and increased awareness.26 similarly, in germany, the trend for anti-hbc-positive status was 1.17% in 2007 but decreased to 0.72% in 2015.27 summarily, the variations in prevalence have been associated with the different assays used, study design and hbv endemicity. some studies investigated anti-hbc in hbsag-negative donors, while some studies investigated in both hbsag-negative and hbsag-positive donors.23,25,26,27 total anti-hbc+/hbsagdonors of 107 (46.1%) in our study is higher than igm anti-hbc+/hbsagdonors of 49 (18.1%) and 20 (4.4%) for maiduguri28 and ile-ife,29 nigeria. the higher anti-hbc+/hbsagdonors in our study could suggest a higher potential for hbv blood transmission from donors. however, this may not be the case because high anti-hbc prevalence does not always imply high obi frequency.27 although other studies reported a lower prevalence of anti-hbc in some nigerian blood centres, the anti-hbc prevalence in those blood centres is still significant to cause an increase in donor deferral rate and reduce blood supply if anti-hbc testing is added to the blood donor screening algorithm. the anti-hbc test included in screening blood donors in hbv non-endemic countries is not routinely used in hbv-endemic countries because many blood products would be discarded due to positive result even though most of the blood would be safe for transfusion.20 total anti-hbc-positivity was significantly higher among donors older than 35 years compared to younger donors, which is contrary to the statistically non-significant difference observed in ilorin, nigeria.23,24 studies from bangladesh, italy, and korea also reported high anti-hbc-positive status in older donors, which was attributed to the acquisition of hbv infections in earlier years when hbv was highly endemic.30,31,32 although the prevalence of hbv infection is still high in nigeria, declining rates have been reported.33 this study suggests that donors younger than 35 years may be less likely to have hbv infection or be an occult hbv carrier. occupation and educational level did not significantly affect the anti-hbc status of the donors. however, a good knowledge of the virus transmission was associated with a lower anti-hbc-positive rate, which suggests that ignorance about the virus increases vulnerability to infection. therefore, health education to increase awareness of hbv transmission and prevention could reduce infection among the future donor population. the finding of lower anti-hbc antibodies among those who share sharp objects in salons may not have any obvious explanation, but olayinka et al. reported in a study in nigeria that public barbing salon clipper cuts, manicure and pedicure cuts, and scarification were not significantly associated with the presence of hbsag.34 all blood donors who used recreational drugs were anti-hbc positive. blood donors who use recreational drugs and are positive should be identified and permanently deferred from donating blood. surprisingly, five blood donors who had hbv vaccination were anti-hbc positive. vaccine recipients who develop anti-hbc might not have responded to the vaccine. it may also be indicative of hbv infection with escape mutants.20,33 the anti-hbsag (anti-hbs) titre of the vaccinated donors was not determined, so we could not confirm if the vaccination provoked an immune response in them. sharing of sharp objects, being sexually active, the number of sex partners and unprotected sex evidenced by those with an sexually transmitted disease are risk factors for hbv infection35,36 but were not evident in our study. this might be due to the targeted study population and the study’s small sample size. the finding in this study does not rule out the danger associated with collecting blood from donors with risk factors. recruiting donors with risk factors is strongly discouraged, especially where exhaustive screening tests are not available. the screening of the donors with anti-hbc-positive and hbsag-negative sera for hbv dna showed that occult infection was present in one of the 107 donors (0.93%). the presence of hbv dna in 0.93% of our serologically screened donors suggests that there is a potential for transmission of hepatitis b to blood recipients. this is higher than 0.56% of blood donors in cameroon35 and 0.5% in ghana,36,37 but less than 11.54% – 14.5% reported in blood donors in egypt.37,38 this study establishes that hbv infection may occur among recipients of blood from donors that are isolated anti-hbc positive. it might be difficult to sustain the blood supply if all the 48.5% of the blood donors who have anti-hbc in their blood are deferred or rejected. implementing anti-hbc testing could make blood safer. since exclusion of all anti-hbc-positive donors may reduce the availability of safe blood in the blood banks of countries with high hbv prevalence, and screening all blood donors with nat to determine the hbv dna status may be too expensive in resource-limited regions, an algorithm that attempts to detect occult hbv is being suggested. the complementary use of both rdt and elisa for hbsag screening of blood donors should continue for maximum identification of hbsag-positive donors. the recommended algorithm includes testing all hbsag-negative blood (by both rdt and elisa) for antibodies to hbc and subjecting anti-hbc-positive blood to nat to identify potential infectious blood units. this will improve the safety of the blood supply and reduce costs while capturing samples that are likely to have undetected threats. limitations the confirmatory algorithms for true positivity of anti-hbc include secondary testing with an alternative elisa, testing for anti-hbs, anti-hbe or hbeag. the most frequently used is anti-hbs. however, this study did not investigate the anti-hbs titre of the participants. this might affect the significance of the observed anti-hbc and hbv dna positivity in the donor population. conclusion almost half of the donors in this study were anti-hbc positive, suggesting that a large proportion of the donors had been exposed to hbv. moreover, donors who were anti-hbc positive and hbsag-negative could have hbv dna. we propose a testing algorithm that can be utilised in hbv-endemic, resource-limited settings. all donors should be tested for hbsag (by both elisa and rdt); those who are hbsag-negative should be further tested for anti-hbc, while only those who are both hbsag-negative and anti-hbc positive should undergo mini-pool nat. this algorithm optimises blood safety and prevents hbv transmission from infected blood units. acknowledgements we acknowledge the cooperation and support of the blood bank donor section staff in carrying out this study. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions f.a.f. was involved in the conception and design of the study, analysis and interpretation of data, drafting of the article, and final manuscript review. f.a.f., a.a.f., a.o.f. and a.a. were involved in laboratory analysis. a.a.f. was involved in the design of the study. a.o.f. was involved in the analysis of data and review of the manuscript. a.a. was involved in the administration of questionnaires and sample collection. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability data that support the findings of this study are available from the corresponding author, f.f., upon reasonable request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any 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anti-hbc and hbv-dna among blood donors in north africa; western libya. international blood research & reviews. 2015;3:152–159. https://doi.org/10.9734/ibrr/2015/18364 arora s, doda v, kirtania t. sensitivity of individual donor nucleic acid testing (nat) for the detection of hepatitis b infection by studying diluted nat yield samples. blood transfus. 2015;13(2):227–232. de la fuente ra, gutiérrez ml, garcia-samaniego j, fernández-rodriguez c, lledó jl, castellano g. pathogenesis of occult chronic hepatitis b virus infection. world j gastroenterol. 2011;17(12):1543–1548. https://doi.org/10.3748/wjg.v17.i12.1543 akinbami aa, oshinaike oo, dosunmu oa, et al. seroprevalence of hepatitis b e antigen (hbe antigen) and b core antibodies (igg anti-hbcore and igm antihbcore) among hepatitis b surface antigen positive blood donors at a tertiary centre in nigeria. bmc res notes. 2012;5(1):167. https://doi.org/10.1186/1756-0500-5-167 oluyinka oo, tong hv, bui tien s, et al. occult hepatitis b virus infection in nigerian blood donors and hepatitis b virus transmission risks. plos one. 2015;10(7):e0131912. https://doi.org/10.1371/journal.pone.0131912 seo dh, whang dh, song ey, han ks. occult hepatitis b virus infection and blood transfusion. world j hepatol. 2015;7(3):600–606. https://doi.org/10.4254/wjh.v7.i3.600 candotti d, laperche s. hepatitis b virus blood screening: need for reappraisal of blood safety measures? front med (lausanne). 2018;5:29. https://doi.org/10.3389/fmed.2018.00029 japhet mo, adesina oa, donbraye e, adewumi mo. hepatitis b core igm antibody (anti-hbcigm) among hepatitis b surface antigen (hbsag) negative blood donors in nigeria. virol j. 2011;8(1):513. https://doi.org/10.1186/1743-422x-8-513 dhawan h-k, marwaha n, sharma r-r, et al. anti-hbc screening in indian blood donors: still an unresolved issue. world j gastroenterol. 2008;14(34):5327–5330. https://doi.org/10.3748/wjg.14.5327 ogunfemi mk, olawumi ho, olokoba ab, et al. prevalence of antibody to 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https://doi.org/10.3233/hab-2011-0242 salawu l, adegoke ao, aboderin ao, huraina ha. hepatitis b viral markers in surface antigen negative blood donors: the need to look beyond antibody negativity. west afr j med. 2011;30(4):292–295. shil n, biswas j, khatun a, et al. incidence of anti-hbc antibody (igg and igm) among hbsag negative apparently healthy blood donors. bangabandhu sheikh mujib med univ j. 2016;9(4):201–204. https://doi.org/10.3329/bsmmuj.v9i4.30241 romanò l, velati c, cambiè g, fomiatti l, galli c, zanetti ar. hepatitis b virus infection among first-time blood donors in italy: prevalence and correlates between serological patterns and occult infection. blood transfus. 2013;11(2): 281–288. lim ya, yoon s. an experience of use of anti-hbc and anti hbs for blood donor screening tests at a tertiary hospital blood center in korea. korean j lab med. 2009;(1):59–65. https://doi.org/10.3343/kjlm.2009.29.1.59 olayinka at, oyemakinde a, balogun ms, et al. seroprevalence of hepatitis b infection in nigeria: a national survey. am j trop med hyg. 2016;95(4):902–907. https://doi.org/10.4269/ajtmh.15-0874 musa bm, bussell s, borodo mm, samaila aa, femi ol. prevalence of hepatitis b virus infection in nigeria, 2000–2013: a systematic review and meta-analysis. niger j clin pr. 2015;18(2):163–172. https://doi.org/10.4103/1119-3077.151035 fopa d, candotti d, tagny ct, et al. occult hepatitis b infection among blood donors from yaoundé, cameroon. blood transfus. 2019;17(6):403–408. allain j-p, candotti d, soldan k, et al. the risk of hepatitis b virus infection by transfusion in kumasi, ghana. blood. 2003;101(6):2419–2425. https://doi.org/10.1182/blood-2002-04-1084 el-zayadi ar, ibrahim eh, badran hm, et al. anti-hbc screening in egyptian blood donors reduces the risk of hepatitis b virus transmission. transfus med. 2008;18(1):55–61. https://doi.org/10.1111/j.1365-3148.2007.00806.x mahmoud ai, elsherbiny nm, afifi na, ahmed bm, yasin as. occult hepatitis b infection among blood donors in al azhar university hospital, upper egypt: the current status after 25 years of vaccine introduction. egypt j immunol. 2018;25(1):45–56. abstract introduction methods results discussion acknowledgements references about the author(s) cheikh fall department of virology, institut pasteur de dakar, dakar, senegal aurélie cappuyns praesens foundation, brussels, belgium oumar faye department of virology, institut pasteur de dakar, dakar, senegal steven pauwels praesens foundation, brussels, belgium gamou fall department of virology, institut pasteur de dakar, dakar, senegal ndongo dia department of virology, institut pasteur de dakar, dakar, senegal moussa m. diagne department of virology, institut pasteur de dakar, dakar, senegal cheikh t. diagne department of virology, institut pasteur de dakar, dakar, senegal makhtar niang department of virology, institut pasteur de dakar, dakar, senegal alassane mbengue department of virology, institut pasteur de dakar, dakar, senegal martin faye department of virology, institut pasteur de dakar, dakar, senegal idrissa dieng department of virology, institut pasteur de dakar, dakar, senegal babacar gningue quality department, institut pasteur de dakar, dakar, senegal abdoulaye bousso senegalese health emergency operation center, ministry of health, dakar, senegal ousmane faye department of virology, institut pasteur de dakar, dakar, senegal rudi pauwels praesens foundation, brussels, belgium amadou a. sall department of virology, institut pasteur de dakar, dakar, senegal citation fall c, cappuyns a, faye o, et al. field evaluation of a mobile biosafety laboratory in senegal to strengthen rapid disease outbreak response and monitoring. afr j lab med. 2020;9(2), a1041 https://doi.org/10.4102/ajlm.v9i2.1041 original research field evaluation of a mobile biosafety laboratory in senegal to strengthen rapid disease outbreak response and monitoring cheikh fall, aurélie cappuyns, oumar faye, steven pauwels, gamou fall, ndongo dia, moussa m. diagne, cheikh t. diagne, makhtar niang, alassane mbengue, martin faye, idrissa dieng, babacar gningue, abdoulaye bousso, ousmane faye, rudi pauwels, amadou a. sall received: 25 apr. 2019; accepted: 29 may 2020; published: 20 aug. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: past and recent outbreaks have highlighted the vulnerability of humans to infectious diseases, which represent serious economic and health security threats. a paradigm shift in the management of sanitary crises is urgently needed. based on lessons from the 2014 ebola outbreak, the praesens foundation has developed an all-terrain mobile biosafety laboratory (mbs-lab) for effective field diagnostics capabilities. objective: the aim of the study was to train african teams and run a field evaluation of the mbs-lab, including robustness, technical and operational sustainability, biosafety, connectivity, turn-around times for testing and result delivery. methods: the mbs-lab was deployed in senegal in october 2017 for a six-month field assessment under various ecological conditions and was mobilised during the dengue outbreaks in 2017 and 2018. results: the mbs-lab can be considered an off-grid solution that addresses field challenges with regard to working conditions, mobility, deployment, environment and personnel safety. blood (n = 398) and nasal swab (n = 113) samples were collected from 460 study participants for molecular screening for acute febrile illnesses and respiratory infections. the results showed that malaria (particularly in kédougou) and upper respiratory tract infections remain problematic. suspected dengue samples were tested on board during the dengue outbreaks in 2017 (882 tests; 128 confirmed cases) and 2018 (1736 tests; 202 confirmed cases). conclusion: the mbs-lab is an innovative solution for outbreak response, even in remote areas. the study demonstrated successful local ownership and community engagement. the mbs-lab can also be considered an open mobile healthcare platform that offers various opportunities for field-deployable, point-of-care technologies for surveillance programmes. keywords: mobile biosafety laboratory; mbs-lab; field deployment; outbreak; senegal; point-of-care. introduction past and recent disease outbreaks (e.g. severe acute respiratory syndrome, middle east respiratory syndrome-related coronavirus, severe acute respiratory syndrome coronavirus, ebola, zika, dengue) have shown that infectious diseases continue to affect the lives of people, while also representing social, economic and national security threats that can quickly evolve into global health crises.1 for example, the 2014–2016 ebola outbreak in west africa cost $32.6 billion and resulted in the loss of 11 000 lives.2 furthermore, dengue fever has become endemic in africa with recurrent outbreaks in different countries.3,4,5 a parallel threat is the rise of antimicrobial resistance with an estimated 4.1 million deaths per year, expected to lead to a $42 trillion loss to the african economy by 2050.2 it is therefore urgent to further strengthen infectious disease surveillance and outbreak preparedness. compared to the resources devoted to dealing with other global threats such as terrorism, climate change or war, little investment is dedicated to infectious disease outbreak preparedness.6,7,8 in recent years, health professionals and decision-makers have identified several strategies needed to mitigate risk.9 however, up to now, preventive initiatives have underestimated threats. in view of this, there is an increasing international community alliance, not only to raise funding, but more importantly to develop adequate and rapid deployment of task forces to prevent or contain declared outbreaks.10 in fact, the typical pattern of infectious disease preparedness today can be characterised by international mobilisation during outbreaks, followed by relaxation and diminishing investments. the dependence on crisis response is both costly and ineffective (especially in preventing future outbreaks).8 whereas ignorance and lack of technology may have been an excuse in the past, more can and should be done for the development of strategies to achieve global health security, including a commitment from public authorities, the availability of appropriate healthcare infrastructure and qualified staff, and international operating ‘disease-fighting forces’ that can be deployed rapidly when needed.11,12 according to the world bank, africa needs between $2 billion and $3.5 billion a year for epidemic preparedness. in addition, this requires political leadership, financial commitment, partnerships and innovation.11 based on their field observations in west africa during the 2014–2016 ebola epidemic and driven by the ambition to bring advanced technologies to communities that need it most, the praesens foundation developed a truck-based mobile biosafety laboratory (mbs-lab) that included an isolator for safe handling of samples and a fully automated molecular diagnostic platform. the goal was to develop an additional tool for better preparedness and faster response to outbreaks and epidemics. the purpose was to fill the gap between the so-called suitcase-based or boxed field mobile laboratories and the much larger container-based laboratories. this combines rapidity, biosafety and advanced diagnostic technology for the rapid detection and identification of pathogens, even in hard-to-reach regions with limited to non-existent healthcare infrastructure. the key objective of this study was to evaluate the mbs-lab in an african context and to prepare local staff for its use, especially during outbreaks. the mbs-lab was challenged under various field conditions to test its operational readiness regarding the following indicators: robustness, technical and operational sustainability, autonomy, connectivity, maintenance, biosafety procedures, logistics, turn-around times and communication of test results. with its current ecosystem of fixed laboratories, field stations, national surveillance network and experience in responding to various outbreaks, the institut pasteur de dakar (ipd), which hosts a world health organization collaborating center for arboviruses and haemorrhagic fever viruses, was chosen as a partner to evaluate this mbs-lab. methods ethical considerations ethical approval to conduct the study was provided by the ministry of health, republic of senegal (0154/msas/dprs/cners; protocol sen17/48). biosafety considerations a closed biosafety isolator equipped with a high-efficiency particulate air filter was integrated in the mbs-lab. a negative pressure cascade (−25 pascal [pa], −50 pa and −25 pa inside the isolator) provided a safe environment for handling different classes of pathogen, thereby eliminating the need for personal protective equipment required for high-containment laboratory facilities. in addition, regular decontaminations were conducted by fumigation with hydrogen peroxide (h2o2) both within the isolator and the workspace of the mbs-lab (nocospray, oxy’pharm, paris, france). to prevent external contamination and ensure cleanliness inside the mbs-lab, operators were required to wear disposable shoe covers. study setting the mbs-lab was built in belgium and shipped to dakar, senegal, in september 2017. this prospective study was carried out in five different localities in senegal based on their climatological and ecological differences (from the dry northern to the humid southern regions) for six months (from october 2017 to march 2018). work was conducted in various healthcare settings ranging from district hospitals and laboratories to primary healthcare settings and outreach initiatives in remote areas. after a week of training of ipd staff, the mbs-lab was first deployed in the kédougou area (south-eastern senegal) from 08 to 23 october 2017, before being mobilised unexpectedly for dengue outbreak management in louga city (north-western senegal) from 25 october to 23 november 2017. following this event, extensive field evaluation continued in the following sites: (1) barkedji, linguère and dahra localities, which are not far from louga (03–29 december 2017), (2) bandafassi and angoussaka villages near kédougou (13–27 january 2018), (3) saint-louis city and debi-tiguette village next to the bird sanctuary of djoudj in the north (11–23 february 2018) and (4) sokone and karang in the fatick region in the central part of country, near the gambia (11–24 march 2018) (figure 1). the mbs-lab travelled over 7000 kilometres during the pilot study. figure 1: map indicating the study areas in senegal, 2017–2018. prior to deployment, ipd laboratory staff received appropriate training to work in the mbs-lab, which covered (1) necessary precautions to prevent exposures, (2) biosafety practices and procedures and (3) data and material management. in addition, the training covered realistic scientific, medical, technical and operational challenges that could be encountered in a field situation. drivers and maintenance technicians were also trained extensively for their jobs. overall, a team of more than 15 people have been trained and have participated in one or more field deployments. study population and sampling the study population included people visiting local healthcare facilities and presenting acute febrile illness or respiratory symptoms. they consisted of male and female patients of all ages who fully consented to participate in the study. for children and patients under 18 years of age, the parents or legal guardians signed the consent forms. a syndromic approach was adopted for diagnostics. therefore, blood (n = 398) and nasopharyngeal swab (n = 113) samples were collected and analysed using a multiplex strategy for the detection of plasmodium genus, arboviruses (including main flavivirus species, chikungunya and rift valley fever virus), salmonella genus and respiratory viruses (such as influenza, respiratory syncytial virus and human metapneumovirus). staff composition one scientist was in charge of coordination between local healthcare settings and the laboratory, such as specimen reception and the release of analysis results. two laboratory technicians were in charge of operations, including molecular diagnostic activities, cleaning of the laboratory and daily reporting. the dedicated driver of the mbs-lab assisted in setting up the laboratory and stabilising the vehicle. during operations, he ensured security around the laboratory and monitored its energy supply. he was also trained to resolve any minor technical issues related to the vehicle and equipment. maintenance and biosafety personnel were on call in dakar and assisted in the field on request. mobile biosafety laboratory workflow sample reception samples and clinical information forms were collected daily on-site and from neighbouring healthcare facilities for analysis in the mbs-lab, respecting cold-chain protocols during transportation. samples were then introduced from the outside directly into the isolator using the secured exterior sample hatch. the access to the sample hatch and entrance to the mbs-lab were for authorised personnel only, using a magnetic badge or key. handling inside the isolator for the protection of laboratory staff and the environment, all samples were unpacked only inside the isolator, in order to minimise the risk of exposure (figure 2). the surfaces of packaging and sample tubes were decontaminated with aniospray disinfectant (laboratoires anios, lille, france). easy and safe handling was the main objective for the design of the isolator with a negative internal pressure of up to −50 pa. the entrance and exit pass boxes flanking the isolator were set at −25 pa. therefore, specimens containing any class of infectious pathogen can be handled within the mbs-lab. figure 2: features of the mobile biosafety laboratory and platforms, senegal, 2017–2018. (1) closed under-pressured biosafety isolator; (2) idylla platform; (3) idylla cartridge; (4) smartcycler instrument; (5) telecommunication system; (6) external view of the mbs-lab; (7) biohazard waste container; (8) laboratory refrigerator. molecular platforms the modularity and flexibility of the mbs-lab enable rapid detection and identification of various pathogens using the on-board multiplexing molecular platforms. the idylla™ system (biocartis, mechelen, belgium) is a fully automated, real-time polymerase chain reaction (real-time transcriptase–polymerase chain reaction) molecular testing system designed to offer results in the minimum amount of time. all components required for nucleic acid extraction, purification, real-time transcriptase–polymerase chain reaction amplification and detection are integrated in a single cartridge that was further loaded into the idyllatm system. handling time was less than five min per sample and the liquid-tight, disposable cartridges greatly reduce the risk of contamination.13 the idylla™ ebola virus triage test, which was approved by the united states food and drug administration for emergency use authorisation, and tropical fever panel cartridges (prototype assay) were tested and compared with reference methods (manuscript in progress).14 in addition, real-time multiplex transcriptase–polymerase chain reaction tests were performed on the smartcycler device (cepheid, sunnyvale, california, united states), using a set of lightmix kits containing pre-mixed primers and probes (tib molbiol, berlin, germany) for the simultaneous detection and differentiation of up to three different pathogens in less than 1 h. results were analysed according to the manufacturer’s recommendations. runs were valid if results generated for all controls (positive and negative) were correct. samples were considered positive if there was an amplification curve with a crossing point value within the defined cut-off (crossing point < 39), equivocal if the crossing point value was higher than the cut-off and negative if there was no amplification. specimen and reagents storage the mbs-lab was equipped with a 4 °c refrigerator for short-term storage of inactivated products and reagents (figure 2). in addition, there was a portable ultra-low mini −80 °c freezer (shuttle™ ult-25ne, stirling, athens, ohio, united states) with capacity to store up to 1000 specimens, which were first stowed in cryoboxes. testing report, data processing and analysis prior to testing, samples were codified with unique anonymous numbers for traceability purposes, linking the sample number, patient identification, case definition and consent form. data were reported in a microsoft excel database (microsoft corporation, redmond, washington, united states), cleaned, and analysed using r software (r foundation, vienna, austria). the diagnostic results were delivered to physicians as early as possible for improved patient management. results overall, 460 participants, consisting of 224 women and 236 men (sex ratio = 1.05), were recruited. the median age was 18 years (range: 2 months to 70 years). a total of 398 blood samples and 113 nasal swabs were screened using the commercial lightmix kits (tib molbiol, berlin, germany) based on a syndromic approach for the diagnosis of febrile infections, including malaria, those caused by salmonella or arboviruses (chikungunya and rift valley fever virus, and flaviviruses such as dengue, zika, west nile and yellow fever virus) and respiratory diseases associated with influenza, parainfluenza, human metapneumovirus, adenovirus and respiratory syncytial viruses. the results showed that malaria remains problematic in senegal, particularly in the southern areas, where 60% (98/162) of the blood samples collected in kédougou were positive (table 1). the other malaria cases (15%, 29/195) were reported during the deployment in the north-western areas (barkedji and dahra-linguère) in december 2017. table 1: distribution of blood samples collected and pathogens identified in kédougou, barkedji, dahra-linguère, saint-louis and karang-sokone areas, october 2017–march 2018. the other major health problem was respiratory tract infections, particularly among young children. indeed, 21 metapneumovirus, 15 influenza, 3 parainfluenza and 4 picornavirus cases were diagnosed from nasal swab samples. co-infection was found in two patients with metapneumovirus and influenza virus, one with malaria and metapneumovirus, one with malaria and picornavirus and one with metapneumovirus and picornavirus (tables 1 and 2). table 2: distribution of nasopharyngeal swab samples collected and pathogens identified in kédougou, barkedji, dahra-linguère, saint-louis and karang-sokone areas, october 2017–march 2018. otherwise, the mbs-lab was mobilised during the 2017 and 2018 dengue outbreaks at the request of the ministry of health. consequently, 882 and 1736 suspected samples have been tested on-board, with 128 and 202 confirmed cases (manuscript in progress). performance of the mbs-lab the features and performance of the mbs-lab are detailed below. all-terrain truck a mercedes sprinter was converted with a six-wheel driveline and increased gross vehicle mass to 7 tons. a fixed laboratory cabin was then installed on the chassis. this combination offers a good balance between optimised mobility, size and robustness. the mbs-lab fills the gap between the extreme flexibility and mobility of suitcase-based mobile laboratories and fixed container-based solutions. the all-terrain vehicle can cover most of africa’s diverse terrain and poor roads with a driving range of about 550 km. a support vehicle (toyota hilux) accompanied the mbs-lab during deployments for personnel transportation and for carrying additional laboratory materials (figure 3). figure 3: deployment and implementation of the mobile biosafety laboratory in the field, kédougou, senegal, october 2017. power system power for the mbs-lab was supplied by lithium-ion batteries, which can be charged by the on-board diesel generator, the local electrical grid (with an inline voltage regulator) or by the vehicle’s alternator. this system guaranteed energy autonomy for the proper functioning of the fully equipped laboratory, including the air conditioning, refrigerator, lighting, telecommunication system and diagnostic equipment (idylla™ and smartcycler™ instruments) for at least one working day relying on batteries alone. the whole laboratory was self-reliant for at least a week; the air conditioning system had the largest impact on energy consumption. the vehicle was equipped with a 100-litre fuel tank and as long as diesel fuel is available, the laboratory can be operational. in the event of a total power cut-off, the isolator had a dedicated uninterruptible power supply to ensure safe shut down and decontamination. the entire power management system can be remotely monitored and controlled using the on-board communication capabilities. geolocation system a real-time satellite geo-positioning system was installed that allows for remote tracking of the vehicle. the system sent alerts when the mbs-lab left or entered a predefined geofenced zone. the cabin was equipped with an emergency distress signal and badge identification of drivers for security and traceability. communication system the mbs-lab was equipped with 3g/4g cellular routers and worldwide-secured satellite networks, which guaranteed permanent connectivity and secure communication channels between the on-field mobile laboratory and the external world. both systems also had the ability to create a local wi-fi network. these allowed for the remote monitoring of connected instruments and control computers that together form a data acquisition and monitoring system. the status and data of the mbs-lab, including the isolator pressures, local time, temperature, humidity, power system monitoring and control, test reports and error logs, were displayed on several dedicated screens in the mbs-lab. laboratory air conditioning the mbs-lab interior was kept at a controlled temperature and humidity even in hot (up to 54 °c) and humid environments. this not only created a comfortable working environment for personnel during long shifts in the laboratory, but also ensured that all equipment could function within optimal operating conditions. laboratory refrigerator a laboratory refrigerator (4 °c) provided a cold chain for the short storage of reagents as well as (inactivated) samples for further confirmation testing. the safe storage of vaccines and medicines and their transportation to and from faraway health clinics until the point of administration is another potential use. hydraulic levelling system this system automatically stabilises and levels the mbs-lab in less than five minutes on uneven terrain. once deployed, the mbs-lab is no longer susceptible to any motion induced by people entering the laboratory or by wind shear, and complies with the requirements of certain equipment to only be operated when levelled. maintenance because the base vehicle was kept standard and is based on a widely used light commercial vehicle, the manufacturer’s recommended engine maintenance intervals are not affected and regular spare parts can be used. a selection of spare parts for both the vehicle and the power supply equipment is always on board for field repairs. special spare parts were shipped to dakar when needed and installed when the mbs-lab was between deployments. in one instance, a maintenance technician was sent to the field to perform an urgent repair. the power supply system can be monitored and controlled remotely, which allows the system to send out alerts in case of issues that can also be resolved remotely, sometimes in combination with the installation of spare parts carried on board such as fuses. systematic technical checks were performed in dakar after each mission to keep the mbs-lab ready for its next (unexpected) deployment. turn-around times prior to the availability of the mbs-lab, samples had to be sent to ipd for specific diagnostic testing, causing slow turn-around times, which have a considerable effect on the quality of samples, the reliability of tests and the reporting of results. the decentralisation of testing with the mbs-lab has dramatically cut turn-around times for samples processing and results delivery from at least a week to hours (average: 4 h). biosafety risk management the mbs-lab was designed for the safe handling of all types of pathogens and reduced human and environmental exposure to potentially harmful agents. the three key elements of biological containment are laboratory practices, safety equipment and facility design. some characteristics are detailed below. personnel flow usually the mbs-lab’s occupancy does not exceed two laboratory technicians. before and after manipulation, the surface and materials used were decontaminated with aniospray disinfectant (laboratoires anios, lille, france). samples and materials flow samples and materials were brought into the isolator through the first pass box with a negative pressure of −25 pa. this pass box has two entry doors, one inside the laboratory for materials and one connected to the external sample hatch. accordingly, samples were deposited from outside into the mbs-lab via this sample hatch and from there were handled safely within the isolator, avoiding any contact between the sample and the laboratory technician. a unidirectional workflow was respected. waste management all potentially infectious waste materials were disposed of in a leak-proof biohazard waste container (jce biotechnology, za bioparc, lyon, france) (compliant with the normative requirements nf x 30-511, un 3291 and un 3249). this was safely connected to the inside of the isolator via a leak-proof hatch system (according to the recommendations of the international organisation for standardization [iso] 10648-2), preventing contamination risks.15,16 any materials which had to be taken out of the isolator, including rna samples and test cartridges, were disinfected with aniospray (laboratoires anios, lille, france) and placed through the sterile pass box before being carried out for further operations. another trash bin was dedicated to non-infectious materials. full containers were securely closed and awaited transport in the accompanying vehicle before being sent to the ipd for incineration. discussion the rising frequency of emerging infectious diseases, their increasing geographic spread and their expanding impact should make overcoming pandemic diseases an international priority. it is widely recognised that infectious diseases constitute social, economic and global security threats. outbreak control that relies exclusively on international response, mostly mounted in crisis mode, leaves little time or opportunity to train local teams and is not sustainable, as after the crisis response very little infrastructure or expertise is left behind. if time is of the essence, it seems logical to have local, regional and continental rapid response teams that are well trained and equipped with the appropriate knowledge and tools. diagnostic needs for pathogens with epidemic potential need to be addressed ahead of the next epidemic. by doing so, we can create a preparedness ecosystem that will allow the shift from a cumbersome, costly emergency response to rapid, cost-effective action for both known and unknown pathogens. key issues seem to be support for public health emergency preparedness, surveillance and response management, workforce development and knowledge at the regional and country level to withstand emerging diseases and other unexpected health events. different models of truck-based laboratories have already been developed for outbreak management or bioterrorism response17,18,19; some were deployed during the 2014−2016 ebola outbreak in west africa.19 however, mobility and flexibility to move from one hotspot to another and energy management, especially in remote areas, turned out to be the biggest challenges. in this study, the performance of the mbs-lab was evaluated against field conditions over a six-month period. the mbs-lab can be considered an off-grid solution that addresses field challenges with regard to mobility, deployment, environment and personnel safety, and operator working conditions. permanent connectivity using cellular and satellite network connections makes the transmission of data and provision of real-time information possible. the design of the mbs-lab offers safe and comfortable working conditions for operators. designed for and tested by african experts, it has demonstrated successful local ownership and management. placing patients, their healthcare providers and local communities at the centre of these activities has contributed to local support from both political and medical authorities, which proved to be key factors for a successful initiative. acting under the auspices of the senegalese ministry of health and local health authorities, the mbs-lab was fully integrated and embedded into the local health system to reinforce capacity building. the mbs-lab can be seen as an extension of a strong local reference laboratory that serves as a base station, providing trained operators with logistical support in terms of laboratory consumables and supplies, waste management and storage of biological samples collected during missions. the pilot study met the overall objective of cutting down turn-around times for diagnostic testing from (at least) a week to hours, through the decentralisation of testing at the most remote level and on-site multiplex testing using molecular platforms at the sentinel sites that served as satellite health posts. no extensive set-up or installation time is required. once on-site, the mbs-lab can be operational within the hour and moves easily between sites, which is useful during an epidemic investigation. by avoiding the need for the transportation of infectious clinical samples to centralised laboratories, the time to generate actionable results and logistical burdens are dramatically reduced. this approach led to better-informed decision-making and improved case management, even of highly mobile populations. because they relied on accurate diagnosis, treatments were more targeted and not based on clinical symptoms only. more than 1300 samples have been safely handled inside the mbs-lab, including blood samples and nasopharyngeal swabs for the detection of pathogens associated with the main tropical infectious diseases. overall, tests were correctly conducted and the results reported on average within 4 h upon receipt. prior to the deployment, no local laboratory capacity was available in selected sites and samples had to be sent to the ipd for analysis, resulting in a turn-around time of at least a week. in other words, a functional mechanism was in place but might be improved by mobile testing capacity. during the six-month period (from october 2017 to march 2018), acute respiratory infections were the common causes of febrile illnesses in practically all the settings in which the mbs-lab operated. human metapneumovirus influenza viruses, parainfluenza virus and picornavirus were the most commonly detected pathogens and were found mostly in children under 5 years, as previously reported.20 these results highlighted once again the pivotal role of respiratory viruses in acute respiratory infections. similar results were found in other countries.20,21,22 co-infection cases have become more apparent since the introduction of multiplex molecular assays; however, the impact on disease severity with arboviruses seems less well defined.23 besides the acute respiratory infections, malaria was also observed in a significant proportion. malaria is endemic in senegal, with a stratified transmission pattern characterised by a low incidence in the dry and northern regions and a relatively high incidence in the southern and humid areas. the high transmission season starts from july and continues until october, corresponding to the rainy season.24 the majority of malaria cases were found in kédougou with a peak of transmission in october during the rainy season. these findings are in agreement with other malaria reports, which shows the accuracy of the results provided by the mbs-lab and indicates that this platform could also be used to monitor the most deadly human parasite.24,25 however, no arbovirus cases were observed during the investigation at kédougou, an area where arbovirus infection is reputed to be endemic.25 whereas in asia and the americas human-to-human transmission by mosquitoes is the current form of arbovirus circulation, in west africa sylvatic circulation is predominant.26 with entomological and virological surveillance programmes, several epidemic events have been observed in kédougou after sylvatic amplification in mosquitoes.26 with climate change, urbanisation and population mobility, sporadic cases or small outbreaks are at risk of turning into large epidemics.27,28 indeed, ancestral sylvatic dengue transmission, initially carried by non-human primates and aedes mosquitoes in the forests of west africa, is now characterised by larger outbreaks, as exemplified by the recent epidemics in senegal in 2017 and 2018.29,30 in that framework, the mbs-lab was deployed, and 882 (in 2017) and 1736 (in 2018) sera samples were handled (unpublished data). the mbs-lab played a key role in managing the outbreaks with proximity, rapid response and early patient management. the response to the dengue virus outbreak has shown that the surveillance network (senegalese syndromic sentinel surveillance network) can mobilise targeted diagnostic efforts to assist in controlling disease outbreaks. as such, the mbs-lab complements this system by adding mobile testing capacity. on top of acute disease surveillance with a direct impact on public health, the mbs-lab can act in a rapid response capacity to address epidemics, ensure preventive disease surveillance and serve as a health monitoring platform and a provider of primary healthcare. as an open mobile healthcare platform, it offers various opportunities for field-deployable point-of-care technologies (e.g. molecular detection platform with multiplexing or syndromic panel testing, lateral flow, sequencing, etc.). moreover, this platform generates high-quality data that can be turned into new insights and knowledge (disease intelligence) and offers great potential for the development of disease surveillance software and epidemiological tools for integrated public health surveillance. conclusion in summary, we describe the set-up and operations of the mbs-lab, first deployed in senegal for extensive field evaluation resulting from a strong partnership between the praesens foundation and the ipd. the trained teams and mbs-lab stationed in dakar now act as a standby epidemic task force. overall, the mbs-lab is a forward-looking solution for outbreak response in remote areas with a high risk for emerging infectious diseases. however, it can also be considered an open mobile healthcare platform that offers various opportunities for field-deployable point-of-care technologies for surveillance programmes. it offers rapidly deployable, connected and state-of-the-art technology for effective field diagnostics capabilities. with extensive training and knowledge sharing, this experience perfectly illustrates that by investing in local capacity building efforts that engage communities and establish the necessary trust before a crisis hits, a country will be able to take local ownership of potential future outbreak responses and to address regional laboratory testing needs autonomously. this innovative solution has the potential to be scaled across the african continent. other domains such as public health emergencies, research, refugee camps, armies, clinical trials and vaccination campaigns also need to be explored for intervention. acknowledgements we thank the hospital staff of all partner healthcare settings and local populations for their involvement and efforts in the project. we thank the drivers, and any other staff involved in the project at the institut pasteur de dakar for their help in logistics maintenance, quality management and laboratory activities. many thanks to our partners and donors such as the praesens foundation, the senegalese ministry of health (via the prevention branch) and the global health security agenda programme for their continuous support and efforts in the project. competing interests the authors have declared that no competing interests exist. authors’ contributions r.p., s.p., a.c., ousmane f. and a.a.s. conceived the project. oumar f., b.g., a.c., c.f. and c.t.d. conceived and planned the experiments. a.b. coordinated activities of dengue outbreak responses. c.f., c.t.d., oumar f., b.g., a.c., s.p., m.f., m.m.d., i.d., m.n., g.f., a.m. and n.d. were involved in the field study. c.f. and a.c. took the lead in writing the manuscript in consultation with all the authors. sources of support the praesens foundation supported the mobile laboratory development and part of the field missions. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references nii-trebi ni. emerging and neglected infectious diseases: insights, advances, and challenges. biomed res int. 2017;2017:5245021. https://doi.org/10.1155/2017/5245021 african union. peace and security council discuss public health threats to the continent urge integration of effective public 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https://doi.org/10.1016/j.tmaid.2018.11.002 article information authors: jessina masamha1 beth skaggs2 isabel pinto3 ana paula mandlaze4 carolina simbine4 patrina chongo4 leonardo de sousa1 solon kidane5 katy yao6 elizabeth t. luman6 eduardo samogudo4 affiliations: 1center for global health (cgh), division of global hiv/aids (dgha), us centers for disease control and prevention (cdc), maputo, mozambique2cgh, division of global disease detection and emergency response, cdc, atlanta, united states 3central laboratory department, ministry of health, maputo, mozambique 4national institute of health, ministry of health, maputo, mozambique 5association of public health laboratories, silver spring, united states 6cgh, dgha, cdc, atlanta, united states correspondence to: jessina masamha postal address: cdc, 7th floor jat complex, 267 zedequias manganhela avenue, maputo, mozambique dates: received: 10 sept. 2014 accepted: 21 sept. 2014 published: 03 nov. 2014 how to cite this article: masamha j, skaggs b, pinto i, et al. working toward a sustainable laboratory quality improvement programme through country ownership: mozambique’s slmta story. afr j lab med. 2014;3(2), art. #253, 6 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.253 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. working toward a sustainable laboratory quality improvement programme through country ownership: mozambique’s slmta story in this original research... open access • abstract • introduction • research methods and design    • institutionalisation of the slmta programme    • implementation • results • discussion    • challenges    • conclusion • acknowledgements    • competing interests    • authors’ contributions    • cdc disclaimer • references abstract top ↑ background: launched in 2009, the strengthening laboratory management toward accreditation (slmta) programme has emerged as an innovative approach for the improvement of laboratory quality. in order to ensure sustainability, mozambique embedded the slmta programme within the existing ministry of health (moh) laboratory structure.objective: this article outlines the steps followed to establish a national framework for quality improvement and embedding the slmta programme within existing moh laboratory systems. methods: the moh adopted slmta as the national laboratory quality improvement strategy, hired a dedicated coordinator and established a national laboratory quality technical working group comprising mostly personnel from key moh departments. the working group developed an implementation framework for advocacy, training, mentorship, supervision and audits. emphasis was placed on building local capacity for programme activities. after receiving training, a team of 25 implementers (18 from the moh and seven from partner organisations) conducted baseline audits (using the stepwise laboratory quality improvement process towards accreditation [slipta] checklist), workshops and site visits in six reference and two central hospital laboratories. exit audits were conducted in six of the eight laboratories and their results are presented. results: the six laboratories demonstrated substantial improvement in audit scores; median scores increased from 35% at baseline to 57% at exit. it has been recommended that the national tuberculosis reference laboratory apply for international accreditation. conclusion: successful implementation of slmta requires partnership between programme implementers, whilst effectiveness and long-term viability depend on country leadership, ownership and commitment. integration of slmta into the existing moh laboratory system will ensure durability beyond initial investments. the mozambican model holds great promise that country leadership, ownership and institutionalisation can set the stage for programme success and sustainability. introduction top ↑ reliable laboratory services and networks are fundamental components of well-functioning health systems and are essential for patient management, disease detection and control, surveillance and outbreak investigations.1 however, in most resource-poor countries, mozambique included, access to quality laboratory services is limited,2 resulting in delayed detection of outbreaks and lengthy or inaccurate diagnoses that may compromise patient care and outcomes. laboratory quality improvement efforts are intended to strengthen laboratory services, leading to advances in the overall health system and the health of the nation.the national laboratory network in mozambique consists of reference laboratories and clinical laboratories. clinical laboratory services are integrated into a tiered national health service (nhs) that comprises central, provincial and district hospitals, as well as health centres (figure 1). of the more than 1300 health facilities in the nhs, only 314 (24%) have laboratories; approximately 30% of these offer only microscopy and rapid test services. whilst strides have been made to improve laboratory infrastructure, a great number of health facilities and laboratories remain under-resourced in areas such as electricity, water supply, physical environment and equipment. in addition to constrained infrastructure, laboratories are affected by frequent stock outs of reagents and consumables, equipment breakdowns, insufficient numbers of qualified laboratory staff and a lack of laboratory quality systems. figure 1: tiered ministry of health laboratory network in mozambique. addressing gaps in the laboratory network is a critical objective for laboratory systems strengthening and a top priority for the government of mozambique.3 consequently, the ministry of health (moh) drafted a national laboratory policy that defines the governance structure for the national network in order to ensure consistent provision of quality laboratory services for clinical diagnosis, research and surveillance.3 the national laboratory policy outlines the ministry’s commitment to the goals of implementing quality management systems (qms) and pursuing national or international accreditation for its reference, central and provincial hospital laboratories.3 with these goals in mind, in 2010 the moh in mozambique implemented strengthening laboratory management toward accreditation (slmta), an innovative training and mentorship programme for continuous quality improvement aimed at improving laboratory qms and preparation for accreditation.4 in addition to slmta, the moh adopted the world health organization regional office for africa's (who afro) stepwise laboratory quality improvement process towards accreditation (slipta), a framework for measuring and recognising quality levels in public health laboratories in developing countries.5 slipta incorporates a checklist to audit laboratory performance and a scoring system to determine a laboratory’s level on the pathway toward achieving accreditation to the international organization for standardization (iso) 15189 standard. the moh incorporated slmta and slipta into the existing health system infrastructure in order to overcome the challenges met by some externally-supported programmes that failed to last after support ended.6 often, externally-supported programmes are viewed as short-term projects and therefore change is either resisted or temporary; desirable outcomes are either not achieved or not maintained. institutionalisation, careful planning, local capacity building, country ownership and strong leadership have been described as being key ingredients for sustainable programmes.7 this article outlines the steps followed by the mozambican moh to establish a national framework for quality improvement and embed the slmta programme within existing moh laboratory systems. results from the initial cohort of participating laboratories are presented along with insights into strategies to establish programme sustainability. research methods and design top ↑ institutionalisation of the slmta programme in 2011, a national laboratory technical working group (twg) consisting of key moh personnel and partners was established to build a framework for a national laboratory quality improvement programme. the heads of reference laboratory services and clinical laboratory services within the moh were appointed as co-chairs. the twg developed a slmta implementation plan, which included training, mentorship, supervision and audits. roles and responsibilities for each stakeholder were defined. the moh created a logistics and administrative position under which the slmta coordinator was hired and a slipta focal person was appointed. the moh coordinated financing for programme implementation from its various partners. the twg met weekly to coordinate and monitor the implementation of slmta. to ensure maximum programme integration and success, the twg felt it was critical for slmta to be accepted as a national moh programme within the laboratory network rather than as an externally-implemented project. to localise the programme, the slmta training tool kit was translated into portuguese, locally-relevant implementation and advocacy strategies were developed and a portuguese acronym, fogela (fortalecer a gestão de laboratórios para acreditação), was created. moh twg members were assigned leading roles in advocacy, planning and implementation. prior to implementation, the slmta programme was introduced by twg co-chairs through presentations at an annual national health directors meeting hosted by the moh. various members of the twg introduced the programme at other meetings with key health leaders, including provincial health directors and medical directors in the provinces.the slmta programme in mozambique was designed to be implemented in a phased, hierarchical approach in which top-tier laboratories (national reference laboratories and central hospital laboratories) were prioritised for enrolment in the first cohort. after gaining experience implementing the programme in their own laboratories, trained managers and quality officers would themselves become a resource for training, mentoring and supervising laboratories enrolled in succeeding cohorts. after demonstrating competence in slmta implementation, provincial quality focal points would lead implementation at lower laboratory tiers within their provinces. to ensure feasibility, a small number of laboratories would be enrolled in successive years, prioritising provincial hospital laboratories. this phased approach was seen as essential for the development of slmta into a sustainable moh programme that could keep pace with human and financial resource needs and maintain programme results. country ownership requires sufficient institutional capacity to define and implement a strategy.7 the need for trainers, mentors, supervisors and auditors was identified, and training was conducted in order to build the various skills required by moh staff to successfully roll out and sustain the slmta programme. in december 2010, in collaboration with a global public healthcare foundation, mozambique trained 15 auditors using the who afro auditor training curriculum. the following year, six of the auditors who were also twg members were trained as slmta trainers, four from the moh and two from the us centers for disease control and prevention (cdc) office in mozambique. these slmta trainers also received training in mentorship and supervision and, as part of the twg, oversaw programme implementation in the first cohort of laboratories. there was little in-country experience in implementing qms at the inception of slmta. thus, three expatriate laboratory professionals with experience in qms development were contracted as mentors and trained by an experienced slmta mentor in the provision of guidance and support. in 2012, in preparation for programme decentralisation and expansion, mozambique conducted the first portuguese-language slmta training-of-trainers (tot) course, which included 18 laboratory professionals from mozambique and three from angola. mozambique now has a team of 25 slmta trainers, including four master trainers (two from the moh) capable of conducting tots. implementation eight laboratories were enrolled in the first slmta cohort, including six reference laboratories and two central hospital laboratories. in january 2011, newly-trained auditors were paired with the experienced consultant auditors to conduct baseline audits for the enrolled laboratories using the slipta checklist. the checklist is divided into 12 sections. laboratory quality is benchmarked on a zeroto five-star scale, awarded based on a percentage of the total score: < 55% assigned zero stars, 55% – 64% one star, 65% – 74% two stars, 75% – 84% three stars, 85% – 94% four stars and ≥ 95% five stars.5the slmta curriculum was then presented during three 3–5-day workshops conducted four months apart. three key staff members from each laboratory who were mandated to lead the implementation of quality improvement in their laboratory participated in the workshops: the laboratory manager, the quality officer and one head of section. throughout the programme, mentors worked with the laboratory staff, spending sixto eight-week periods embedded in a laboratory, followed by an eight-week absence. selected improvement projects such as documentation, equipment management, inventory management, quality control and safety were implemented following each workshop. two follow-up supervisory visits were conducted in each laboratory during these periods in order to provide technical assistance and to monitor the implementation process. the first visit was to support data collection for the selected improvement projects and to review laboratory action plans; the second visit six to eight weeks later was to monitor progress. supervision reports were shared with participating laboratories and feedback on progress and challenges was shared with laboratory and hospital management. in march 2012, after completion of the slmta curriculum, locally-trained auditors, supported by expert international auditors, conducted exit audits of the laboratories. summaries from audit findings, corresponding scores and laboratory star ratings were shared with laboratory management and staff, hospital directors and provincial health department officials. results top ↑ all eight laboratories completed the three slmta workshops and the assigned improvement projects within the implementation period. of the eight laboratories, six had complete exit audit data; the remaining two reference laboratories had some missing data and were excluded from the analyses. the six laboratories with complete scores demonstrated overall improvement after 12 months of implementation (figure 2). at baseline, all laboratories began with zero stars (median score 35%, range 14% – 50%). at exit, scores for all laboratories improved (median exit score 57%, range 44% – 76%; improvements 7–53 percentage points). one laboratory remained at zero stars (though its score increased from 14% to 44%), three laboratories were at one star, one laboratory was at two stars and one laboratory had reached three stars. the areas of greatest average improvement (> 40 percentage points) were client management and customer service, corrective action, purchasing and inventory and management reviews (figure 3). the areas of least improvement (< 15 percentage points) were information management, equipment, facilities and safety and internal audit. figure 2: baseline, exit and official slipta audit results and star ratings for the first cohort* of laboratories based on the slipta checklist. mozambique slmta programme, january 2011 to june 2013. figure 3: average performance of six laboratories enrolled in first slmta cohort in mozambique across the 12 sections of the slipta checklist before and after slmta implementation as measured by baseline and exit audits (january 2011 to march 2012). the best-performing laboratory at the exit audit was the national tuberculosis (tb) reference laboratory, which attained three stars (76%). this laboratory had an overall improvement of 53 percentage points across all 12 checklist sections, with improvements of ≥ 80 percentage points in corrective action and purchasing and inventory. at the end of the programme, three of the six laboratories were officially enrolled into the who afro slipta programme for review by auditors from the african society for laboratory medicine. the scores from the official audits were slightly higher than exit audit results, at 58% for nampula central hospital, 72% for the cellular immunology reference laboratory and 79% for the national tb reference laboratory (figure 2). the national tb reference laboratory was recommended to apply for international accreditation. a work plan was developed to address gaps identified and a target set for the laboratory to have the accreditation audit in august 2014 with the portuguese accreditation institute based in portugal. discussion top ↑ through slmta, mozambique has begun to make substantial progress in laboratory quality improvement and has developed a plan to ensure that continued progress is both feasible and sustainable. the progress made would be short-lived without country ownership and leadership, both of which are fostered by institutionalisation of the programme. the moh made a sustained effort to take ownership of the slmta programme as part of their overarching strategy to improve laboratory quality systems, embedding it within the moh structure in order to ensure that planning, advocacy and implementation were led and championed by moh personnel. as a result, laboratory management and staff viewed slmta as a permanent moh programme, rather than as a ‘one-off’ event that would soon disappear.moh leadership not only influenced perception of the programme but also staff commitment. laboratory personnel were wholly committed to the programme, working creatively and beyond designated work hours in order to implement assigned improvement projects. in one laboratory, the quality manager and laboratory staff contributed money to purchase file folders for laboratory documents. in another, laboratory personnel paid for tokens to reward staff who made outstanding contributions to quality improvement projects. in fact, so much excitement was generated over the programme that managers of laboratories yet to be enrolled in slmta wanted to know when their laboratories would be considered for future cohorts. the moh led advocacy efforts, such as introduction of the programme and presentation of results at key strategic forums, which were aimed at promoting senior-level ownership, support and commitment crucial for the success of the programme. these efforts led to widespread buy-in and interest in the programme amongst provincial health and hospital directors as well as key moh decision makers. the provincial and hospital directors received regular updates during routine slmta supervisory visits and showed a keen interest in knowing how their laboratories were progressing and what they could do to support the laboratory staff. hospital directors began to visit the laboratories to follow up on projects and to ask ‘how many stars’ their laboratories had now achieved. these hospital directors commented on the increased interaction with the laboratories and improved understanding of their institutions’ quality indicators; and became involved in resolving problems that were beyond the laboratories' control. for example, when one laboratory showed decreased customer satisfaction as a result of staff shortages, the hospital director engaged the provincial health director to request additional laboratory staff at her facility. as guided by the implementation plan, expansion of the slmta programme is underway. in october 2012, a second slmta cohort was launched. in keeping with the tiered strategy, laboratories from central, provincial, general and rural hospitals were eligible to apply and 10 laboratories were selected. as slmta roll-out continues, the laboratory twg has set programme implementation goals based on the country’s capacity and available financial, human and material resources. the moh continues to lead all aspects of programme planning and implementation and, since 2012, approximately 30% of funding for the programme has been incorporated into annual moh budgets. mozambique has built a foundation for a sustainable laboratory quality improvement programme; however, to guarantee longevity, critical aspects of the national laboratory system must be strengthened. establishing a national laboratory policy is essential with regard to sustaining the improvements achieved through the slmta programme.1 whilst mozambique has drafted such a policy, it has not yet been given official approval, limiting the moh’s ability to drive the implementation of qms and enforce adherence to quality standards across the laboratory network. without such a formal policy, continued and sustained progress cannot be mandated systematically. mozambique has set a goal of accreditation for its six reference laboratories and 10 central and provincial hospital laboratories. however, international accreditation is not a goal that is achievable for all laboratories in the network. thailand has successfully implemented a model to accredit laboratories according to national standards that vary by laboratory level.8 to ensure continuous quality improvement through expansion of slmta to lower-tier laboratories, mozambique may consider developing a similar framework that outlines national standards for quality laboratory services, against which rural hospital and health centre laboratories could be monitored and evaluated. once a policy is in place, clear measures must be established to assess progress and effectiveness, as well as to sustain laboratory strengthening efforts. nkengasong et al. suggest using the indicator ‘number of laboratories accredited in the public health sector’ in order to track the progress of quality systems implementation for a national laboratory network.9additional indicators could include: number of provinces with dedicated quality management officers; percentage of laboratories audited in the previous 12 months; percentage of audited laboratories demonstrating improvement as measured by the slipta checklist; and percentage of laboratories implementing external proficiency testing for select services. beyond system structures and policy, human resources are a critical requirement for the slmta programme. yao et al. recommend an investment in human resources and allowance of time for dedicated personnel to successfully implement and sustain slmta activities so as to achieve the goal of laboratory accreditation.4 as mozambique continues to work toward increasing coverage of slmta, it is critical to develop local training and mentoring capacity. significant upfront investment in local capacity building will keep long-term costs down and ensure sufficient reach and uptake of the national programme. for example, in zimbabwe, shumba et al. found that it cost less over the long term to engage and train local quality managers as auditors, trainers and mentors than to import international consultants to do the job.10 minimizing costs is essential because programmes that require unacceptable levels of resource commitment are unsustainable.6 challenges mozambique has invested in developing a large implementation team that has been trained adequately for the task. in fact, in addition to conducting in-country training, mozambican-based trainers have since assisted with slmta training in angola and peru; whilst its master trainers have supported tots in kenya, ethiopia and the dominican republic. however, with the exception of the slmta logistics coordinator, none of these programme implementers work in an official capacity for the slmta programme. because of competing priorities, slmta activities may not always be completed adequately, as evidenced by missing exit audit data for two laboratories in the first cohort. within the laboratories, quality managers have bench responsibilities that compete with improvement projects and continued maintenance of the qms. to be successful, the terms of reference for quality managers must be revised to include adequate time for implementation of the laboratory’s qms. although the initial plan was to groom local mentors from the first cohort of laboratories for subsequent cohorts, it has been a challenge to secure time for these now ‘more experienced’ staff members to leave their day-to-day work and provide mentorship to newly-enrolled laboratories. staffing moh quality departments with personnel dedicated to maintaining the daily operations of the quality improvement programme may address some of these challenges. conclusion successful implementation of the slmta programme requires partnership; however effectiveness and long-term viability depend on country leadership, ownership and commitment. the moh has collaborated with cdc and various partners to implement the slmta programme, whilst at the same time assuming leadership for the process, including establishment of the structures necessary for long-term programme success. the mozambican model of institutionalisation of slmta into existing moh laboratory systems demonstrates that country leadership, commitment and ownership can provide a strong platform for sustainability. by taking advantage of the current influx of laboratory resources to strengthen existing structures, build essential human resource capabilities and establish guiding strategies and policies, the mozambique laboratory quality improvement programme is designed to be sustainable and is positioned to achieve the goal of providing quality laboratory services across the country. acknowledgements top ↑ this programme has been supported by the us president’s emergency plan for aids relief (pepfar) through the cdc under the terms of cooperative agreement number u2gps12985. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions j.m. (cdc, mozambique) supported the planning and implementation of the programme and wrote the manuscript. b.s. (cdc, atlanta) provided oversight for the programme and substantial input into the writing of the manuscript. i.p. (central laboratory department, moh), a.p.m., c.s. and p.c. (all national institute of health, moh), l.s. (cdc, mozambique) and s.k. (association of public health laboratories) supported the planning and implementation of the programme. k.y. and e.t.l. (cdc, atlanta) substantially revised the manuscript. e.s. (national institute of health, moh) championed the institutionalisation of the programme, led its implementation and reviewed the manuscript. cdc disclaimer the findings and conclusions in this report are those of the author and do not necessarily represent the official position of the cdc. references top ↑ 1. gershy-damet gm, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2011;134(3):393–400. http://dx.doi.org/10.1309/ajcptuuc2v1wjqbm2. olmsted ss, moore m, meili rc, et al. strengthening laboratory systems in resource-limited settings. am j clin pathol. 2011;134(3):374–380. http://dx.doi.org/10.1309/ajcpdqosb7qr5glr 3. ministry of health mozambique. national laboratory policy; 2012 (unpublished). 4. yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin pathol. 2011;134(3):401–409. http://dx.doi.org/10.1309/ajcpnbbl53fwuiqj 5. world health organization’s regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation in the african region (with checklist) [document on the internet]. c2011 [cited 2013 may]. http://www.afro.who.int/en/clusters-a-programmes/hss/blood-safety-laboratories-a-health-technology/blt-highlights/3859-who-guide-for-the-stepwise-laboratory-improvement-process-towards-accreditation-in-the-african-region-with-checklist.html 6. swerrison h. understanding the sustainability of health programs and organisational change. a paper for the victorian quality council [document on the internet]. c2007 [cited 2013 may]. available from: http://www.health.vic.gov.au/qualitycouncil/downloads/sustainability_paper.pdf [url no longer available]. 7. world bank. country ownership [page on the internet]. c2008 [cited 2013 may]. available from: http://web.worldbank.org/archive/website01013/web/0__co-89.htm 8. wattanasri n, manoroma w, viriyayudhagorn s. laboratory accreditation in thailand: a systemic approach. am j clin pathol. 2011;134(4):534–540. http://dx.doi.org/10.1309/ajcpzyy19wmkmazt 9.nkengasong j. a shifting paradigm in strengthening laboratory health systems for global health: acting now, acting collectively, but acting differently. am j clin pathol. 2011;134(3):359–360. http://dx.doi.org/10.1309/ajcpy5asuejyq5rk 10.shumba e, nzombe p, mbinda a, et al. weighing the costs: implementing the slmta programme in zimbabwe using internal versus external facilitators. afr j lab med. 2014;3(2), art. #248, 6 pages. http://dx.doi.org/10.4102/ajlm.v3i2.248 abstract introduction methods results discussion acknowledgements references about the author(s) oytip nathalang graduate program in medical technology, faculty of allied health sciences, thammasat university, pathumthani, thailand panasya rassuree graduate program in medical technology, faculty of allied health sciences, thammasat university, pathumthani, thailand kamphon intharanut graduate program in medical technology, faculty of allied health sciences, thammasat university, pathumthani, thailand wanlapa chaibangyang graduate program in medical technology, faculty of allied health sciences, thammasat university, pathumthani, thailand núria nogués laboratori d’immunohematologia banc de sang i teixits, barcelona, spain citation nathalang o, rassuree p, intharanut k, chaibangyang w, nogués n. genomic analysis of kel*03 and kel*04 alleles among thai blood donors. afr j lab med. 2024;13(1), a2294. https://doi.org/10.4102/ajlm.v13i1.2294 original research genomic analysis of kel*03 and kel*04 alleles among thai blood donors oytip nathalang, panasya rassuree, kamphon intharanut, wanlapa chaibangyang, núria nogués received: 16 aug. 2023; accepted: 17 jan. 2024; published: 19 mar. 2024 copyright: © 2024. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the kell blood group system is clinically important in transfusion medicine, particularly in patients with antibodies specific to kell antigens. to date, genetic variations of the kell metallo-endopeptidase (kel) gene among thai populations remain unknown. objective: this study aimed to determine the frequencies of kel*03 and kel*04 alleles among thai blood donors using an in-house polymerase chain reaction-sequence-specific primer (pcr-ssp) method. methods: blood samples obtained from 805 unrelated central thai blood donors at a blood bank in pathumthani, thailand, from march 2023 to june 2023, were typed for kpa and kpb antigens using the column agglutination test, and the results for 400 samples were confirmed using dna sequencing. a pcr-ssp method was developed to detect the kel*03 and kel*04 alleles, and genotyping results were validated using known dna controls. dna samples obtained from thai donors in central (n = 2529), northern (n = 300), and southern (n = 427) thailand were also genotyped using pcr-ssp for comparison. results: all 805 (100%) donors had the kp(a−b+) phenotype. the pcr-ssp genotyping results agreed with the column agglutination test and dna sequencing. all 3256 thai blood donors had the homozygous kel*04/kel*04 genotype. frequencies of the kel*03 and kel*04 alleles among thai donors differed significantly from those of japanese, native american, south african, brazilian, swiss, and german populations. conclusion: this study found a 100% kel*04 allele frequency in three thai populations. these data could provide information on kel*03 and kel*04 allele frequencies to estimate the risk of alloimmunisation in thai populations. what this study adds: this study demonstrates that in-house pcr-ssp can be used to determine kel*03 and kel*04 alleles to predict kpa and kpb antigens. even though only homozygous kel*04/kel*04 genotypes were found among thai donor populations, the established pcr-ssp method may be useful for estimating the risk of alloimmunisation in other populations. keywords: kell blood group system; phenotype; allele; genotype; thai population. introduction the high immunogenicity of antigens in the kell blood group system (international society of blood transfusion symbol, kel, and number, 006), particularly the k antigen, is clinically significant in transfusion medicine.1 kell antigens are detectable as early as the 10th week of pregnancy and reach full maturity after birth. immunoglobulin g antibodies against kell antigens may develop during transfusion or pregnancy, leading to haemolytic transfusion reactions and haemolytic disease of the foetus and newborn.2,3 currently, 37 antigens have been identified in this system. individuals without kell antigens are referred to as having the k0 phenotype (kell null), which is very rare across all populations. patients with antibodies against kell antigens, such as k/k (kel1/kel2), kpa/kpb (kel3/kel4) and jsa/jsb (kel6/kel7), frequently experience haemolytic transfusion reactions and haemolytic disease of the foetus and newborn issues.2,4 in general, kell antigen frequencies differ among populations. for example, the frequency of the k+ phenotype is high among caucasians (7% – 9%) but less than 0.1% among asians.1,3 in a related study of 1522 thai blood donors from the national blood centre, thai red cross society, the k−k+ phenotype was the most common (98.16%), followed by k+k+ (1.78%) and k+k− (0.07%).4 low-incidence antigens like the kpa antigen are present in less than 1% of the general population, whereas high-incidence antigens like the kpb antigen are found in more than 99% of the population.1,2,3 kpa and kpb antigens are not included in screening or panel cells. hence, additional panel cells are needed to identify antibody specificity among patients with antibodies against these antigens (anti-kpa or anti-kpb).2,5 anti-kpa and anti-kpb are typically immunoglobulin g antibodies and exhibit reactivity in the indirect antiglobulin test, especially when using enzyme-treated red blood cells. the conventional tube test and column agglutination test (cat) are routinely used to identify kpa and kpb antigens and to determine kp(a+b−), kp(a+b+), and kp(a−b+) phenotypes.1,6 the limitations of typing for these antigens using conventional tube test and cat methods include the high cost of commercial antiserum, as well as the observation of positive direct antiglobulin test and positive autocontrol results in chronically transfused patients.1,2,3 genotyping of the kell metallo-endopeptidase (kel) gene for the kel*03 and kel*04 alleles is an alternative to serological testing for predicting kpa and kpb antigens. it is useful for selecting appropriate blood for patients with anti-kpa and anti-kpb antibodies, as well as in anthropology studies to determine allele frequencies within populations.7,8,9 various polymerase chain reaction (pcr)-based techniques have been used to identify the kel*03 and kel*04 alleles. the pcr-restriction fragment length polymorphism and polymerase chain reaction-sequence-specific primer (pcr-ssp) methods have been used to genotype these alleles among iranian patients with alloimmunised multi-transfused thalassaemia7,8 and to determine allele frequencies in the multi-ethnic brazilian population.9 a multiplex pcr assay was also developed to genotype 35 red blood cell antigens among austrian blood donors and provided reliable results compared with serological testing.10 subsequently, luminex-based genotyping platforms have also been implemented to predict red blood cell phenotypes among patient and donor populations.11,12,13,14,15 furthermore, matrix-assisted laser desorption/ionisation time of flight mass spectrometry was used to determine blood group alleles in blood donors.16,17,18 this method offered high throughput and proved to be less time-consuming than routine serological testing.16,17,18 however, genotyping using luminex-based platforms and matrix-assisted laser desorption/ionisation time of flight mass spectrometry techniques requires costly reagents and equipment, thus making them unsuitable for laboratories with established budgets. since the kel*03 and kel*04 allele frequencies in thai populations remain unknown, this study aimed to detect kel*03 and kel*04 alleles among blood donors in central, northern, and southern thailand using in-house pcr-ssp and to compare the observed allele frequencies with those of other donor populations. methods ethical considerations ethical clearance to conduct this study was obtained from the human research ethics committee of thammasat university, thailand (no. coe no. 007/2566), according to the declaration of helsinki. written informed consent was obtained from all individuals. study population leftover ethylenediaminetetraacetic acid-anticoagulated blood samples were collected from 805 unrelated healthy thai blood donors at the blood bank, thammasat university hospital, pathumthani, thailand, from march 2023 to june 2023. all the donors who qualified for inclusion had already passed the questionnaire and physical screening procedures for blood donation provided by the national blood centre, thai red cross society. blood samples positive for hepatitis b virus, hepatitis c virus, hiv, or syphilis were excluded from the study. in addition, the study included 1724 leftover dna samples from the blood bank, thammasat university hospital, pathumthani; 300 samples from the blood bank section, faculty of medicine, chiang mai university, chiang mai; and 427 samples from the regional blood centre, 12th songkhla, thai red cross society, songkhla, thailand. dna controls for the kp(a+b−) and kp(a+b+) phenotypes were obtained from laboratori d’immunohematologia banc de sang i teixits, barcelona, spain. dna extraction genomic dna was extracted from the samples using the dneasy blood & tissue kit (qiagen, valencia, california, united states) according to the manufacturer’s instructions and stored at −20 °c before further analysis. serological testing for kpa and kpb antigen typing altogether, 805 blood samples were analysed to detect the kpa and kpb antigens, using the cat method with an anti-kpa/kpb id-card (bio-rad laboratories, cressier, switzerland) made up of six microtubes containing either anti-kpa or anti-kpb of human origin within the gel matrix. briefly, 10 µl of 5% red cell suspension in id-diluent 1 (bio-rad laboratories, cressier, switzerland) was added to the appropriate microtubes of the id-card. the id card was then centrifuged, and the results were recorded. sequence-based genotyping genomic dna from 400 of the samples tested using cat were randomly selected and sequenced to identify kel*03 and kel*04 alleles. primers were designed for each target gene (table 1) using national center for biotechnology information primer-blast (https://www.ncbi.nlm.nih.gov/tools/primer-blast/).19 polymerase chain reaction amplification for the kel gene exon 8 containing single nucleotide variant c.841 c > t [rs8176059], p.arg281trp, was performed using seq-kp-f forward and seq-kp-r reverse primers, with an expected fragment size of 358 base pairs (bp). each pcr reaction mix (total volume: 40 µl) was prepared using 3 µl of genomic dna (50 ng/l), 3 µl each of the forward and reverse primers (10 µm), 20 µl of 2x pcr reaction mixture (phusion high-fidelity pcr master mix, new england biolabs, ipswich, massachusetts, united states), and 11 µl of sterile distilled water. the pcr reactions were carried out in a t100 thermal cycler (bio-rad laboratories, inc., hercules, california, united states). pcr reactions included 30 s of initial denaturation at 98 °c, followed by 10 cycles of 10 s at 98 °c and 60 s at 69 °c, then 20 cycles of 10 s at 98 °c, 50 s at 60 °c, and 30 s at 72 °c, and a final extension step for 5 min at 72 °c. the pcr products were separated on a 1.5% agarose gel with a 100 bp dna molecular weight marker (fermentas, carlsbad, california, united states), stained using sybr safe dna gel stain (invitrogen, paisley, united kingdom), electrophoresed in 1x tris-borate-edta solution at 100 volts for 30 min, and then visualised with a blue-light transilluminator. the pcr products were then purified using a gel extraction kit (genejet gel extraction kit, fisher scientific baltics uab, vilnius, lithuania), and the eluted fragments were sequenced directly using sanger sequencing (u2bio sequencing service, bangkok, thailand) with the aforementioned pcr primers. table 1: primer sequences designed for the detection of kel*03 and kel*04 alleles from samples of healthy thai blood donors at thammasat university hospital, thailand, march 2023 – june 2023. genotyping using polymerase chain reaction-sequence-specific primer kel*03 and kel*04 alleles were identified using an in-house pcr-ssp technique with custom primers and amplification conditions (table 1). the kp-t-a-f and kp-rev primers were used for kel*03 amplification, while the kp-c-b-f and kp-rev primers were used for kel*04 amplification. two sets of pcr reaction mixtures, set a for identifying kel*03 alleles and set b for kel*04 alleles, were performed for each sample. each pcr reaction mixture (total volume: 10 µl) was prepared using 1 µl of template genomic dna (50 ng/l), 1 µl each of the forward and reverse primers (10 µm), and 5 µl of 2 × pcr reaction mixture (green hot start pcr master mix, biotechrabbit gmbh, hennigsdorf, germany). co-amplification of the human growth hormone (hgh) gene using 1 µl of hgh-f and hgh-r primers (6 µm) was conducted as the internal control in each set. the pcr reactions were carried out in a t100 thermal cycler (bio-rad laboratories, inc., hercules, california, united states) and included 30 s of initial denaturation at 95 °c, followed by 10 cycles of 30 s at 95 °c and 60 s at 69 °c, then 20 cycles of 10 s at 95 °c, 50 s at 62 °c and 30 s at 72 °c, and a final extension step for 5 min at 72 °c. after electrophoresis, pcr products were examined with a blue-light transilluminator. the expected pcr product size for both the kel*03 and kel*04 amplification reactions was 158 bp, while that of the internal control, the hgh gene, was 434 bp. known dna controls of kel*03/kel*03, kel*03/kel*04 and kel*04/kel*04 genotypes were used as positive and negative controls in parallel with the test samples. the kel*03/kel*03 genotype was indicated if amplification was observed with only the kel*03 reaction (set a), while the kel*04/kel*04 genotype was indicated if amplification was observed with only the kel*04 reaction (set b). if amplification occurred in both sets of reactions, the kel*03/kel*04 genotype was indicated. the specificity of pcr-ssp was examined by cross-amplifying the kel*03-specific primers with the kel*04/kel*04 genotype and vice versa. the pcr-ssp method was conducted on a total of 3256 dna samples (including the 400 samples tested using dna sequencing) to identify kel*03 and kel*04 alleles. repeatability to evaluate the repeatability of the newly developed pcr-ssp, 100 randomly selected samples with known genotypes by dna sequencing were retested by pcr-ssp. upon completion of pcr-ssp genotyping in all 3256 samples, 100 randomly selected dna samples from among the 400 blood donors with genotyping obtained by standard dna sequencing were retested using pcr-ssp under the same conditions as previously described to test the repeatability of our in-house pcr-ssp technique for kel*03 and kel*04 genotyping. to improve the validity and reliability of the evaluation, technicians were blinded to the pcr-ssp results. dna sequencing was performed in the event of any discrepant genotyping results obtained using the pcr-ssp and serological assays. comparison of kel*03 and kel*04 allele frequencies between populations kel*03 and kel*04 allele frequencies among thai blood donors were compared with those reported in filipino, southeast asian, chinese, japanese, korean, south asian, native american, alaska native/aleut, hawaiian/pacific islander, south african, brazilian, swiss, and german populations.9,11,15,16,17 statistical analysis gene and allele frequencies were simply estimated by counting the number of times each gene and allele was observed in samples from each population. the calculation of allele frequency for kel*03 is based on the gene counting method; every kel*03/kel*03 individual has two kel*03 alleles, and a kel*03/kel*04 individual has one. this is relative to all alleles in the population, that is, two times the total number of samples. this was also applied to the kel*04 allele frequency calculation: the chi-square (χ2) test was used to evaluate whether the observed genotype frequencies agreed with the expected frequencies under the hardy-weinberg equilibrium. the two-tailed fisher’s exact test with bonferroni adjustment of homogeneity was used to determine the differences in allele frequencies between central, northern, and southern thai populations and to compare these differences with those reported in other populations.9,11,15,16,17 an adjusted p-value of less than 0.004 was considered statistically significant. all statistical analyses were conducted using spss® version 16.0 (spss inc., chicago, illinois, united states). results serological testing and dna sequencing a total of 805 blood samples were tested for kpa and kpb antigens using cat. all donors (100%) had the kp(a−b+) phenotype. kp(a+b−) and kp(a+b+) phenotypes were not found in this donor population. the sequencing results of the 400 randomly selected samples revealed that these donors were homozygous at the kel gene locus, having the kel*04/kel*04 genotype (figure 1). these results agreed with the serological testing results using cat. figure 1: electropherogram of the sequenced kel*03 and kel*04 polymorphism region of the kel gene in a thai blood donor, thammasat university hospital, thailand, march 2023 – june 2023. the homozygous state of the c.841c position at exon 8 identified in kel*04/kel*04 genotypes is shown. genotyping using polymerase chain reaction-sequence-specific primer according to the pcr-ssp results, among 3256 examined samples, a 158 bp product was only detected using the particular primer sets for kel*04 allele detection, thus indicative of the kel*04/kel*04 genotype. the hgh internal control was also amplified in all reactions (figure 2). three samples with the kp(a−b+), kp(a+b−), and kp(a+b+) phenotypes as determined using dna sequencing were used as known dna controls to validate the genotyping results for both alleles. for all three control samples, there was no cross-amplification detected between kel*03 and kel*04 primer sets. figure 2: genotyping of kel*03 and kel*04 alleles using known dna controls by polymerase chain reaction sequence-specific primers at thammasat university hospital, thailand, march 2023 – june 2023. in total, to detect kel*03 and kel*04 alleles, we genotyped 2529 samples obtained from central thai blood donors, 300 samples from northern thai donors, and 427 samples from southern thai donors using the newly developed pcr-ssp. all 3256 thai blood donors were homozygous at the kel gene locus, having the kel*04/kel*04 genotype. the kel*03/kel*03 and kel*03/kel*04 genotypes were not detected (table 2). the retesting results for the sub-group of 100 blood sample donors with known genotype agreed with the first round of pcr-ssp testing for all samples. the frequencies of the three genotypes, including kel*03/kel*03, kel*03/kel*04, and kel*04/kel*04 genotypes, were consistent between the central thai, northern thai, and southern thai populations according to the hardy-weinberg equilibrium (χ2 = 0.00, degree of freedom = 2, p = 1.00). table 2: kel*03 and kel*04 genotype and allele frequencies among thai blood donors at thammasat university hospital, thailand, march 2023 – june 2023. kel*03 and kel*04 allele frequencies in thai and other populations the kel*03 and kel*04 frequencies among the thai donors in this study were similar to those reported in filipino (p = 0.29),11 southeast asian (p = 1.00),11 chinese (p = 0.12),11 korean (p = 1.00),11 south asian (p = 0.05),11 alaska native/aleut (p = 0.008),11 and hawaiian/pacific islander (p = 1.00) populations11 (table 3). there were, however, significant differences in kel*03 and kel*04 allele frequencies between the thai donors in this study and individuals in a south african population15 (kel*03: 0.0000 vs 0.0067; and kel*04: 1.0000 vs 0.9933; p = 0.002). in addition, there were significant differences in allele frequencies between thai donors and individuals in japanese (p < 0.001),11 native american (p < 0.001),11 brazilian (p < 0.001),9 swiss (p < 0.001),16 and german populations (p < 0.001).17 table 3: comparison of the kel*03 and kel*04 allele frequencies between thai blood donors (thammasat university hospital, thailand, march 2023 – june 2023) and blood donors from other populations. discussion in this study, 3256 dna samples from three thai blood donor populations were tested using pcr-ssp. the kel*03 allele predicting the kpa antigen was not observed. among the 805 samples from central thai blood donors tested using cat in this study, the kp(a−b+) phenotype was the only phenotype detected (100%). the sequencing results of 400 of these 805 samples confirmed the occurrence of the kel*04/kel*04 genotype. the pcr-ssp genotyping results obtained in this study were accurate as demonstrated by the absence of cross-amplification between the two primer sets used to identify the kel*03 and kel*04 alleles. moreover, the results of the pcr-ssp repeatability testing on 100 randomly selected samples from 400 known genotypes by pcr-ssp and dna sequencing were consistent with the first round of testing. genotyping techniques such as pcr-ssp are more applicable and cost-effective than serological techniques for determining the prevalence of antigens in extended blood group systems. while reference laboratories do have high-throughput luminex-based genotyping technologies available, they require expensive reagents and complicated equipment. the developed pcr-ssp method makes it more affordable to screen a large number of samples for kpa prediction. using the two-tailed fisher’s exact test with an uncorrected p-value of 0.05, there were significant differences in kel*03 and kel*04 allele frequencies between individuals in thai populations and those in south asian, japanese, native american, alaska native/aleut, south african, brazilian, swiss, and german populations.9,11,15,16,17 however, using the bonferroni-adjusted p-value cutoff of 0.004, the significant differences observed between the thai populations and the south asian and alaska native/aleut populations were considered false positives. therefore, there were only significant differences in kel*03 and kel*04 allele frequencies between thai donors and donors from japanese, native american, south african, brazilian, swiss, and german populations.9,11,15,16,17 this means that anti-kpa antibodies may be more common among patients in these populations than among thai patients. the majority (approximately 95%) of kp(a−b+) donors in japanese, native american, brazilian, swiss, and german populations would be easily selected to transfuse among patients producing anti-kpa. this is because the kp(a+) antigen is a low-frequency antigen, and the probability of finding compatible blood units is high.1,2 in contrast, only a few donors with the kp(a+b−) phenotype can serve as donors to patients with the kp(a+b−) phenotype who have anti-kpb antibodies.2 in this study, the prevalence of the predicted kp(a−b+) phenotype was similar among thai donors and individuals from filipino, southeast asian, chinese, korean, south asian, alaska native/aleut, and hawaiian/pacific islander populations.11 this finding is consistent with existing data on kpa alloimmunisation:1,2 so far, anti-kpa has not been reported in the aforementioned populations. given the low prevalence of kp(a+b−) observed in this study, autologous donation or compatible blood recruitment from family members will be necessary to provide safe blood transfusion in thai patients with anti-kpb antibodies. a related study on genetic distance analysis of thai blood donors based on rh, mns, kidd diego, and kell blood group genotypes including 10 alleles – rhce*e, rhce*e, gypb*03, gypb*04, jk*01, jk*02, di*01, di*02, kel*01, and kel*02 – revealed a close cluster pattern formed by highly related populations, such as those from thai, filipino, southeast asian, chinese, and hawaiian/pacific islander populations.20 in our study, however, the kel*03 and kel*04 frequencies among the thai donors were similar to those observed in the filipino, southeast asian, chinese, korean, south asian, hawaiian/pacific islander, and alaska native/aleut populations. a large proportion of the current thai, filipino, southeast asian, chinese, korean, south asian, and hawaiian/pacific islanders populations possibly descended from a common ancestor.20 however, the observed differentiation of blood group polymorphisms may have arisen a long time ago in different sub-populations, and there may not have been any significant genetic admixture thereafter. a prior study conducted in thailand in 2023 to investigate the distribution of the 10 blood group alleles revealed that the alaska native/aleut population differed from the thai population with regard to pairwise genetic distances.20 this differentiation may have been influenced by a combination of cultural practices, particularly within-group marriages, and the consequences of group isolation.21 nevertheless, more research using larger sample sizes on additional relevant blood group alleles may support the findings of genetic similarities within populations and can be used in anthropological studies. limitations one limiting factor of the pcr-ssp assay stems from the need for samples with known and relatively rare phenotypes, particularly kp(a+b−) and kp(a+b+), to ensure the test’s validity and reliability. in addition, an erroneous prediction of the k0, kmod, kel: 1 weak, and other variants of the kel gene – which are exceedingly rare in thai and other populations – may occur when using pcr-ssp to determine kel*03 and kel*04 alleles. the confirmation of these phenotypes using serological techniques and kel gene sequencing is thus suggested.2,22 to obtain a more representative picture of the prevalent kel gene variants among thai blood donors, the genotypic results reported in this study need to be verified in large-scale studies involving all regions in thailand. conclusion this study found a 100% kel*04 allele frequency in three thai populations. this data could provide information on kel*03 and kel*04 allele frequencies to estimate the risk of alloimmunisation in thai populations. acknowledgements the authors would like to thank the blood bank, thammasat university hospital for support during this study. competing interests the authors declare that they have no financial or personal relationships that may have appropriately influenced them in writing this article. authors’ contributions o.n. designed and conceived the study. p.r. and n.n. collected samples. o.n., k.i., and p.r. performed laboratory tests. p.r., w.c., and o.n. prepared and analysed the data. k.i. and p.r. performed the statistical analysis. o.n. and w.c. wrote the manuscript. all authors discussed the results and contributed to the final manuscript. sources of support the study was funded in part by the national research council of thailand. data availability the authors confirm that the data supporting the findings of this study are available within the article. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. it does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the authors are responsible for this article’s results, findings, and content. references fung mk, eder af, spitalnik sl, westhoff cm. technical manual. 19th ed. bethesda, md: aabb; 2017. daniels g. human blood groups. 3rd ed. west sussex: blackwell science; 2013. cohn cs, delaney m, johnson st, katz lm. technical manual. 20th ed. bethesda, md: aabb; 2020. fongsarun j, nuchprayoon i, yod-in s, kupatawintu p, kidprasirt c. blood groups in thai blood donors [in thai]. thai j hematol transfus med [serial online]. 2002 [cited 2023 jun 23]; 12(4). available from: http://tsh.or.th/file_upload/files/vol12-404bloodgroupsinthaiblooddonors.pdf marsh wl, redman cm. the kell blood group system: a review. transfusion. 1990;30(2):158–167. 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https://doi.org/10.9790/3008-09229097 ncbi/primer-blast: finding primers specific to your pcr template (using primer3 and blast) [homepage on the internet]. [cited 2022 oct 22] available from: www.ncbi.nlm.nih.gov/tools/primer-blast/ intharanut k, chaibangyang w, nimnuch p, nathalang o. genetic variation of blood group polymorphisms among thai blood donors [in thai]. j hematol transfus med [serial online]. 2023 [cited 2023 oct 18]; 73(1). available from: https://he01.tci-thaijo.org/index.php/jhematoltransfusmed/article/view/262367/176706 delfin f, min-shan ko, a, li, m, et al. complete mtdna genomes of filipino ethnolinguistic groups: a melting pot of recent and ancient lineages in the asia-pacific region. eur j hum genet. 2014;22(2):228–237. https://doi.org/10.1038/ejhg.2013.122 bejrachandra s, saipin j, udee s, et al. transfusion in a rare blood type, k0 with anti-ku: a case report [in thai]. thai j hematol transfus med [serial online]. 1998 [cited 2023 jun 23];8(3). available from: http://www.tsh.or.th/file_upload/files/v8%20n3%20255.pdf abstract introduction methods results discussion acknowledgements references about the author(s) felicity gopolang department of global health, schools of public health and medicine, international training and education center for health (i-tech), university of washington, seattle, washington, united states fales zulu-mwamba laboratory services unit, directorate of clinical care and diagnostic services, ministry of health zambia, lusaka, zambia davy nsama laboratory services unit, directorate of clinical care and diagnostic services, ministry of health zambia, lusaka, zambia annika kruuner zambart, lusaka, zambia dailes nsofwa laboratory quality management systems, centers for disease control and prevention (cdc) zambia, lusaka, zambia ishmael kasvosve faculty of health sciences, university of botswana, gaborone, botswana royce gomo immunogene labs, ruwa, zimbabwe tiny motlhabane medical laboratory technology department, institute of health sciences, gaborone, botswana bhavna chohan department of global health, schools of public health and medicine, international training and education center for health (i-tech), university of washington, seattle, washington, united states olusegun soge department of global health, schools of public health and medicine, international training and education center for health (i-tech), university of washington, seattle, washington, united states daniel osterhage department of global health, schools of public health and medicine, international training and education center for health (i-tech), university of washington, seattle, washington, united states nancy campbell department of global health, schools of public health and medicine, international training and education center for health (i-tech), university of washington, seattle, washington, united states michael noble department of pathology and laboratory medicine, university of british columbia, vancouver, british columbia, canada ann downer department of global health, schools of public health and medicine, international training and education center for health (i-tech), university of washington, seattle, washington, united states jean-frederic flandin department of global health, schools of public health and medicine, international training and education center for health (i-tech), university of washington, seattle, washington, united states anya nartker department of global health, schools of public health and medicine, international training and education center for health (i-tech), university of washington, seattle, washington, united states catherine koehn department of global health, schools of public health and medicine, international training and education center for health (i-tech), university of washington, seattle, washington, united states linda k. nonde hiv and aids twinning center program, american international health alliance (aiha), lusaka, zambia aaron shibemba laboratory services unit, directorate of clinical care and diagnostic services, ministry of health zambia, lusaka, zambia clement b. ndongmo center for disease control and prevention (cdc) zambia, lusaka, zambia martin steinau center for disease control and prevention (cdc) zambia, lusaka, zambia lucy a. perrone department of global health, schools of public health and medicine, international training and education center for health (i-tech), university of washington, seattle, washington, united states citation gopolang f, zulu-mwamba f, nsama d, et al. improving laboratory quality and capacity through leadership and management training: lessons from zambia 2016–2018. afr j lab med. 2021;10(1), a1225. https://doi.org/10.4102/ajlm.v10i1.1225 original research improving laboratory quality and capacity through leadership and management training: lessons from zambia 2016–2018 felicity gopolang, fales zulu-mwamba, davy nsama, annika kruuner, dailes nsofwa, ishmael kasvosve, royce gomo, tiny motlhabane, bhavna chohan, olusegun soge, daniel osterhage, nancy campbell, michael noble, ann downer, jean-frederic flandin, anya nartker, catherine koehn, linda k. nonde, aaron shibemba, clement b. ndongmo, martin steinau, lucy a. perrone received: 17 mar. 2020; accepted: 22 feb. 2021; published: 30 apr. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: competent leadership and management are imperative for delivering quality laboratory services; however, few laboratory managers receive job-specific training in organisational management and leadership. objective: to develop and evaluate participants’ competencies in organisational leadership and management as measured through learner and laboratory quality improvement assessments. methods: this professional development programme employed a mentored, blended learning approach, utilising in-person didactic and online training, with the practical application of a capstone project in the laboratories. programme impact was evaluated through a series of preand post-laboartory assessments using the stepwise laboratory improvement process towards accreditation checklist, as well as learner-competency assessments through online quizzes and discussions. results: from 2016 to 2018, 31 managers and quality officers from 16 individual laboratories graduated from the programme having completed capstone projects addressing areas in the entire laboratory testing process. laboratories increased their compliance with the international organization for standardization 15189 standard and all but two laboratories significantly increased their accreditation scores. two laboratories gained three stars, two laboratories gained two stars, and five laboratories gained one star. five laboratories subsequently achieved international organization for standardization 15189 accreditation in 2019. conclusion: this programme taught leadership theory to laboratory managers and allowed them to implement leadership and management practices in the laboratory setting. programmes such as this complement existing laboratory quality management training programmes such as strengthening laboratory management toward accreditation. keywords: leadership; quality management; workforce development. introduction medical laboratories are a critical component of healthcare because they provide essential data for effective patient care, pathogen detection, disease surveillance and response. enabling access to quality laboratory services is a challenge in low-resource settings1 and many laboratories in resource-constrained countries provide poor quality diagnostic testing with incorrect, unreliable, or significantly delayed test results. competent laboratory management and leadership are vital for delivering quality laboratory services and laboratories need leaders who can utilise their resources effectively in a variable healthcare environment.2 these leadership skills are required to effect beneficial change in complex healthcare settings3 and work effectively across disciplines; however, they are not skills that laboratory managers (lms) commonly cultivate during conventional academic programmes.4 few laboratory supervisors ever receive formal laboratory management and leadership training for their roles.5,6,7 effective laboratory quality management requires that laboratory supervisors have not only the technical knowledge of quality management systems (qms) and national and international standards for medical laboratory quality such as the international organization for standardization (iso)15189 but also the strong leadership and managerial skills to lead their staff and drive accreditation efforts.8,9,10,11,12 the strengthening laboratory management toward accreditation (slmta) programme was launched in 2009,13,14 and provides iterative quality management training to hundreds of laboratory personnel. slmta programme addresses common workforce knowledge gaps in resource-constrained settings via a multi-workshop implementation model. the guide for the stepwise laboratory improvement process towards accreditation15 checklist was endorsed by the world health organization regional office for africa in 2011. it serves as a benchmarking tool to monitor laboratory conformity to the iso15189 quality standard and the slmta programme. as of 2019, slmta has been implemented in 1368 laboratories globally and of these 191 (7.16%)16 have been iso15189 accredited. while significant progress has been made in laboratory quality through qms training programmes such as slmta in the last decade,17 strengthening the impact of these programmes across the continent and increasing representation of the laboratory sector in the upper levels of healthcare governance requires further investments in leadership and management training for lms and directors.18 public health leadership training is evidently beneficial to clinical practitioners and policymakers19,20 and there is a need for wider access to similar programmes for laboratory professionals.21 however, there are limited formal leadership programmes available.22,23,24,25,26,27 to address this gap, the certificate program in laboratory leadership and management28 was developed in 2013 at the university of washington in consultation with global laboratory practice experts. the goal of the certificate program is to build a scalable professional development programme aimed at building the leadership and management skills of laboratory staff in supervisory positions. the participant criteria ultimately ensured that participants were in the leadership position to make substantive and impactful improvements in their laboratory’s testing quality and operations. the programme was implemented in zambia for two years starting in 2016 to strengthen leadership and management competencies of lms and quality assurance officers from key tertiary public and military hospital laboratories. also, this programme aimed to improve the laboratories’ quality of diagnostic services and their compliance with the stepwise laboratory improvement process towards accreditation (slipta) checklist towards achieving iso15189 accreditation. we aim to describe the effectiveness of this laboratory leadership programme in two zambian cohorts, using the laboratories’ compliance with the slipta checklist as the main outcome measure. methods ethical considerations approval to conduct the study was received from the human research ethics committee, university of new england (approval number he13-240). program design and implementation the certificate program was implemented in two cohorts from 2016–2018; each programme cohort completed course and project works in 9 months. this culturally appropriate and effective25 programme employed a mentored, competency-based,29 blended learning approach. it was designed for adult learners and courses were delivered in-person and online. participants delivered a capstone project, which is an individualised, practical application of a quality improvement (qi) project (figure 1). in each cohort, two in-person sessions bookended the online coursework. the in-person sessions served as the programme orientation and finale sessions. figure 1: structural overview for the laboratory leadership and management programme in zambia, 2016–2018. two cohorts of participants from 16 laboratories across zambia participated from 2016–2018, each cohort taking 9 months to complete the programme work. both programme years utilised a similar approach to adult experiential learning, utilising a blended solution of online and face-to-face instruction, a robust online discussion board as well as close faculty and mentorship support for individual capstone projects conducted at participant’s home laboratories. orientation and finale sessions were conducted in lusaka. seventeen laboratory managers from 16 laboratories completed the 2016–2017 programme and completed 16 unique capstone projects. for the second cohort, 16 laboratory managers and 15 quality managers completed the programme and conducted 15 unique capstone projects. the orientation session introduced participants to the programme structure, content, learning goals and expectations, mentor-participant guidelines, the online learning management system (lms; canvas™ learning management system, london),30 and the laboratory assessment and audit tools to be used (e.g. slipta).15 following the orientation, participants returned to their worksites where they discussed the programme with their staff and chief medical superintendent before starting the baseline audit process and online coursework. the results of the baseline audits identified the cp focus area and provided a guideline for the development of cp work plans. the curriculum for the 2016 cohort included five courses from the university of washington delivered sequentially, the first on laboratory quality and systems (delivered in-person), followed by laboratory leadership, laboratory management, communicating laboratory information, and implementing diagnostic technology. the latter topics were delivered online via the canvas lms. each online course was four weeks long and included 20 h – 25 h (~5–6 h/week) of mixed media instruction and a weekly discussion. each course was followed by a 2–3-week instruction intermission during which participants submitted their cp-related assignments. the cp was a customisable qi project designed and implemented by participants at their laboratories with close support from mentors and faculty. the cp process began after the orientation session, with a baseline laboratory audit conducted over a period of 1 week using the slipta checklist.15 through their cps, participants were to exemplify team leadership and improve teamwork through delegation and a system of accountability. the curriculum for the second cohort (2017–2018) included a university of british columbia quality assurance online curriculum for quality assurance officers. this online course was delivered in seven online modules via the blackboard lms system (blackboard inc., washington, district of columbia, united states) and conveyed traditional qms principles of shewhart,31,32 deming,33,34,35,36 crosby,37 and juran38,39 with additional perspectives by faculty. curriculum courses delivered instructions necessary for compliance with the iso quality and competence (iso15189) requirements and expectations for medical laboratories. the lms in the second cohort undertook advanced training based on kouzes and posner’s textbook and workbook ‘the leadership challenge’.40 the lms participated in the online coursework concurrently with the quality assurance officers and conducted joint cps in their home laboratories. both programme cohorts ended with an in-person meeting where participants presented their cps to their peers, mentors and faculty and received a programme completion certificate. participant and mentor selection the programme was specially designed for quality assurance officers and lms who are currently working in a managerial role in a health laboratory; participants and mentors were selected by the programme’s selection committee following specific eligibility criteria. mentors had an average of 20 years’ experience in the clinical laboratory field and were paired with up to seven participants. mentors provided on-site and remote coaching using various communication channels, including the lms discussion board, email, skype (microsoft corp., redmond, washington, united states), and whatsapp™ (facebook inc., menlo park, california, united states) calls. mentors provided step-by-step support and motivated participants to apply knowledge gained from the global classroom to address management challenges such as staff resistance to change, particularly from some long-serving staff members. mentors also reinforced messages of individual leadership and accountability by encouraging laboratory staff at all service levels to implement smaller qi projects. the staff were to identify gaps related to the lm’s cp and led efforts to find solutions. mentors also coached participants to organise and conduct meetings with the laboratory staff, the quality team, and the senior hospital administrators. these proposed meetings were aimed at engaging all laboratory staff and the hospital administration with the implementation and review of the laboratory improvement program. learner and programme evaluation the programme was evaluated based on both learner and facility impact. learner outcome metrics included self-rated competency and graded assessments including graded participation in the weekly online discussion board accompanying each course, course exams and cp-related assignments (analysis of laboratory audit result, cp project proposal, work plan development, implementation update, final report and project presentation).41 course surveys, exit interviews, and facility pre-programme and post-programme slipta checklist audits conducted by the ministry of health were also used to evaluate the programme. at the end of each programme year, via an online programme survey, qualitative programme feedbacks were received from the participants on various aspects of the programme. participants identified the most valued aspect of the programme and evaluated the curriculum quality, the cp process, and the mentor’s support. also, a post-programme evaluation survey was conducted by an independent organisation in 2018.42 the survey utilised a likert scale rating system to collect anonymised data from both cohorts on how participants felt their abilities had changed since they graduated from the programme. all quantitative and qualitative evaluation data were collected from survey responders and analysed using excel software (microsoft corp., redmond, washington, united states). results demographics and graduation rate participants of both programmes were selected using established eligibility criteria from key laboratory facilities as indicated by the zambia ministry of health (table 1). overall, 31 individuals completed the programme with 16/17 (94%) graduating in 2016 and 26/31 (84%) in 2018. these graduates (25 men and 6 women) conducted their programme work at 16 individual hospital laboratories from all nine provinces in zambia (table 2). nine lms completed both cohorts. eight mentors from zambia, botswana, and zimbabwe (three men, five women) supported each paired programme participant for an average of 3 h per week. table 1: participant and mentor selection criteria, zambia, 2016–2018. table 2: programme demographics, zambia, 2016–2018. capstone project scope and success thirty-one cps were completed by graduates in these two years and the cp topics addressed a range of issues on the total laboratory testing process (table 3). in addition to these formal projects, supplemental qi projects were undertaken by other staff in the laboratory adjacent to the cp’s topical area. these supplementary projects which were undertaken by the general laboratory staff also contributed to the improved laboratory performance and addressed issues such as updating standard operating procedures to minimise specimen cross-contamination, implementing new duty rosters for daily equipment maintenance activities during public holidays and weekends and phlebotomy service task-shifting. the smaller qi projects strengthened both the internal and external laboratory communication channels and improved laboratory safety via the introduction of hand-washing facilities, controlled laboratory access, and routine class ii biological safety cabinet smoke tests. table 3: capstone project topic areas, zambia, 2016–2018.† quality improvement progress the ministry of health conducted baseline (the beginning of each programme year) and exit (the end of the 9-month programme) slipta audits. both audits were utilised as benchmarking tools to measure the impact of the cp. after the programme period in 2018, the slipta checklist audit scores of 14 out of the 16 participating laboratories (87.5%) increased (figure 2), with nine laboratories also improving their slipta star rating. two laboratories gained three slipta stars, another two gained two slipta stars, and five gained one slipta star. of the seven other laboratories, six maintained their star rating while one laboratory lost a star rating. three laboratories achieved five slipta stars by the end of the programme and five of these participating laboratories have achieved iso15189 accreditation16 at the time of this writing. figure 2: changes in laboratory audit scores before (2016) and after (2018) the laboratory leadership and management programme in zambia, 2016-2018. participants and representatives from the ministry of health conducted baseline stepwise laboratory improvement program towards accreditation audits of each laboratory at the beginning (2016) and end of the programme (2018). audit scores are shown as whole numbers with a maximum score of 275 points. learner satisfaction participants self-reported significant improvements of key competencies as a result of the programme as indicated by internal (table 4) and external surveys (figure 3). all participants reported improvement in their leadership and management knowledge and skills as well as laboratory practice compliance. more than 95% reported improved competencies in various other laboratory abilities such as critical analyses and interpretation of laboratory data, communication, collaboration with clinicians on result utilisation, improvement of laboratory practice compliance and accountability in line with national and international standards, implementation of essential quality assurance practices (timeliness, reliability and accuracy of testing), and application of leadership and management skills. participants also involved other laboratory staff in learning by downloading recorded lectures for others to watch offline as a team and discussed weekly topics as a group. the programme was also highly rated by mentors as indicated from both internally and externally conducted surveys. figure 3: participants’ self-perceived changes in key abilities after laboratory leadership and management programme in zambia, 2016–2018. a likert scale-based survey conducted of all programme graduates was conducted in 2018 by an external organisation. graduates of the programme self-reported key changes in abilities as a result of the programme (n = 24 respondants) and percentage of each response were calculated and shown here. table 4: qualitative feedback from participants about the programme, zambia, 2016–2018. discussion this programme set out to improve the leadership and quality management skills of a cohort of laboratory supervisors in zambia including improving their competencies in management, communication, policy development, laboratory data analysis, and international quality management principles to improve the laboratories’ ability to deliver quality clinical and public health services. the blended learning programme was successful in achieving a > 80% graduation target rate for both cohorts. participants indicated in surveys that the programme improved their leadership and management skills and subsequently their laboratory’s performance. all respondents reported that they thought the programme applied to their work and that they would recommend the programme to their peers. the continuous support and motivation from faculty and mentors43,44,45 ensured participants were supported during the entire programme period. also, the employment of an effective and reliable online lms to deliver high-quality asynchronous online courses and support a robust real-time discussion board to foster the cultivation of a strong community of practice among each cohort contributed to the high retention rate observed. the online discussion board was utilised daily for communication and enabled participants to share best laboratory leadership, management and advocacy practices with their peers and receive valuable feedback. importantly, the programme was valued by participants because it delivered both theoretical and practical applications of effective laboratory leadership and management. the cp was a unique component of the programme, unifying the entire laboratory around a common goal and fostering a strong working relationship between the management team and the technical staff. this process resonated the importance of strong teams in furthering an organisation’s mission. this blended learning programme is therefore unique in that the modular online curriculum is adaptable to any environment, allowing for customisation with location-specific needs and inclusion of a global audience and experts, regardless of the time zone. the potential for local ownership and expansion of this programme is immense as evidenced by the breadth of project topics participants undertook as well as the adjacent qi projects. the projects improved participant’s leadership and management skills as well as their laboratory’s qms in line with iso15189 (as measured by the preand post-programme slipta audits). the projects also addressed internal indicators of laboratory quality such as specimen rejection rate, turnaround time and client satisfaction. all participating laboratories demonstrated qi; however, not all these improvements are captured by the slipta audits. notably, the structured programme content and sustained faculty and mentor engagement are implicated in these observed laboratory qms improvements and contributed to the international recognition of three participating laboratories. as of the time of this writing, five of these participating laboratories have now achieved iso15189 accreditation. some challenges were encountered in the two-and-a-half-year programme. specifically, management staff changes in some facilities within the two programme periods challenged the continuum of qi. particularly, staff turnover in 2017 correlated with lower qi in many of the participating laboratories. also, other implementing partners at times were simultaneously present on-site with programme mentors which reduced available contact time with mentees. mentors expressed challenges such as mentees not responding to communications and availing themselves during distant mentorship. personal time management was the only participant self-reported challenge; concurrently meeting programme and work responsibilities was demanding for participants. as such, future efforts will be made by the programme developers to condense the programme length based on feedback, offer all of the coursework online to minimise on-site time, and include content pertaining to personnel time management and motivation particularly when there are competing interests. distance learning programmes that include significant components of field or work-based training are proving to be highly effective in fostering practical competency development and behaviour change in learners46 and the results of this programme for lms is no exception. importantly, the modular online curriculum and blended format of the programme is permissive of adoption and adaptation by local institutions such as universities and professional associations. adoption and implementation by local organisations will have added benefit to laboratory professionals either by contributing to university degrees such as diplomas or continuing education credits as part of an annual licensure programme or career advancement points for leadership positions. cost elements of the programme include faculty time, mentor honorarium, data plans for participants, lms maintenance and logistical costs for on-site coaching and in-person meetings. programme implementation costs could likely be reduced should the programme be converted to a completely online programme including mentorship. however, the impact and quality of an entirely online programme are yet to be evaluated. although massive open online courses offer exciting opportunities to distribute knowledge on a massive and global scale, a full understanding of their effectiveness to deliver competency-based training to healthcare professionals remains limited and further research is warranted.47 the value of laboratory leadership programmes such as the one we describe here are starting to receive greater attention from the public health practice community48 and should be supported alongside other efforts to strengthen national laboratory systems.49 limitations this programme was limited to a selected group of participants from zambia who were selected based on their position in their organisation or their occupation. as such, success in this programme was dependent on staff continuity in the programme and vulnerable to disruptions caused by staff reassignment. should the programme become more financially sustainable through user fees, the global audience could be expanded and no longer tied to priority facilities as determined by external donors. recommendations leadership and management training, such as this training programme, is highly recommended as it can lead to measurable impacts in the laboratory. leadership and management training programmes such as this programme are highly recommended to complement existing qms training programmes such as slmta. professional development programmes for healthcare practitioners delivered through online learning platforms should also include an applied project where learned theory from the global classroom can be applied to the job. conclusion this programme affirms the impact of formal leadership and management training on laboratory capacity and builds on previous investments to improve quality, system operability, and preparedness. the programme emphasised the functional practices of organisational leadership and effectively supplemented quality management training programmes; it can be implemented alongside other efforts to strengthen national laboratory systems. acknowledgements we express our appreciation to the hospitals, laboratory leadership, and staff at the 16 laboratories that participated in this programme over the two years. we also thank carl henn, audra stark, dickson kaoma, naomi mwanza, justina mthoniswa, and esther pandawe at aiha for assistance with local logistics support for course delivery and mentoring visits. we are grateful to alec mcgee, debbie confer, elizabeth scott, laura livingston, tom furtwangler, caitlyn bradburn, chris joss, lindsay mumm, leah klug, candice moss, solmaz shotorbani, jennifer hubber, jessica mcpherson, jennifer antilla, ali mokdad, olivier defawe, ellen wilcox, carlyn collins, patricia sadate-ngatchou, and robert martin for their contributions in programme development. we thank cardea services for the programme impact evaluation. we also thank subject matter experts from the university of washington, united states centers for disease control and prevention, sandia national laboratories, association of public health laboratories, washington state department of health, world health organization, university of british columbia, george washington university, and path. we thank anne fox for assistance with graphic design. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions f.g. developed the manuscript draft as the lead author, as well as reviewing and editing towards finalisation of the article. t.m. contributed to the review and editing of the manuscript draft. b.c. was a regional mentor and capstone project. d.o. was responsible for the background literature review and initial identification of target journals. j.-f.f. contributed to editing the manuscript. r.g. contributed (technical review and editing) to the final version of the manuscript. f.z.-m. participated as a mentor and provided input on the manuscript. d. nsama assisted to obtain access to resources and monitored the progress. c.k. performed programmatic, fiscal and operational management, and provided review of the final manuscript. n.c. was lead teacher in the leadership course, coached managers in developing a vision, mission and values for their teams as well as understanding how to motivate team members to engage in the changes needed to a more an effective and efficient laboratory. m.s. coordinated and oversaw the programme and edited the manuscript. a.n. reviewed the manuscript. o.s. contributed to the development and review of the manuscript. c.b.n. contributed to the manuscript writing, reading and approval to the final version. a.d. reviewed and approved the final version. i.k. reviewed and edited the manuscript draft. a.k. reviewed the manuscript draft. l.k.n. reviewed the manuscript draft. d. nsofwa reviewed the manuscript draft. m.n. was the course developer and lead faculty for the university of british columbia quality management course and mentored all quality managers in the programme in 2017. a.s. wrote the manuscript together with f.g. with input from co-authors. sources of support funding for this work was provided by the president’s emergency plan for aids relief through the united states department of health and human services, health resources and services administration under the terms of a cooperative agreement (u97ha04128) awarded to aiha. data availability data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the findings and conclusions of this report are those of the authors and do not necessarily represent the official position of the funding agencies. references martin r, barnhart s. global laboratory systems development: 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[cited 2020 jun 20]. london international development centre (ldic); 2011. available from: https://lidc.ac.uk/_assets/dl4h%20main%20report.pdf rowe m, osadnik cr, pritchard s, maloney s. these may not be the courses you are seeking: a systematic review of open online courses in health professions education. bmc med educ. 2019 sep 14;19(1):356. https://doi.org/10.1186/s12909-019-1774-9 albetkova a, isadore j, ridderhof j, et al. critical gaps in laboratory leadership to meet global health security goals. bull world health organ. 2017;95(8):547–547a. https://doi.org/10.2471/blt.17.195883 opio a, wafula w, amone j, kajumbula h, nkengasong jn. country leadership and policy are critical factors for implementing laboratory accreditation in developing countries: a study on uganda. am j clin pathol. 2010;134(3):381–387. https://doi.org/10.1309/ajcp6kmotclisgj3 abstract introduction methods results discussion acknowledgements references about the author(s) terver m. akindigh infectious diseases unit, jos university teaching hospital, jos, nigeria abba o. joseph infectious diseases unit, jos university teaching hospital, jos, nigeria christiana o. robert department of microbiology, faculty of natural sciences, university of jos, jos, nigeria ocheme j. okojokwu department of microbiology, faculty of natural sciences, university of jos, jos, nigeria juliet n. okechalu department of microbiology, faculty of natural sciences, university of jos, jos, nigeria joseph a. anejo-okopi department of microbiology, faculty of natural sciences, university of jos, jos, nigeria citation akindigh tm, joseph ao, robert co, okojokwu oj, okechalu jn, anejo-okopi ja. seroprevalence of hepatitis b virus co-infection among hiv-1-positive patients in north-central nigeria: the urgent need for surveillance. afr j lab med. 2019;8(1), a622. https://doi.org/10.4102/ajlm.v8i1.622 brief report seroprevalence of hepatitis b virus co-infection among hiv-1-positive patients in north-central nigeria: the urgent need for surveillance terver m. akindigh, abba o. joseph, christiana o. robert, ocheme j. okojokwu, juliet n. okechalu, joseph a. anejo-okopi received: 09 mar. 2017; accepted: 04 dec. 2018; published: 27 june 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract we report the seroprevalence of hepatitis b surface antigen among hiv-positive patients at a clinic in north-central nigeria. screening for hepatitis b virus was based on serological markers. alanine aminotransferase levels and cd4+ t-lymphocyte counts were compared between patients. the study showed that 9.2% were positive for hepatitis b surface antigen with significant differences between alanine aminotransferase levels of patients with and without hepatitis b virus. we recommend a national surveillance system to monitor control efforts. keywords: antiretroviral therapy; alanine aminotransferase; cd4+ t-lymphocytes; hepatitis b virus; hiv; serological markers. introduction hepatitis b virus (hbv), a circular dna virus with humans serving as the only reservoir1, remains a worldwide public health problem. transmission of the virus occurs via exposure to infected blood and body fluids.1 in areas where the prevalence of hepatitis b surface antigen (hbsag) is more than 8%, there are two broad possibilities for how transmission can occur: either it happens vertically at birth from chronically infected mothers to their newborns or horizontally during childhood or in adulthood.2,3 the infection caused by the virus is among the leading causes of liver disease, with liver-related deaths occurring in an estimated 25% – 30% of the population of chronic carriers, principally cirrhosis and hepatocellular carcinoma.4 a fifth of the current global population is estimated to be seropositive for hbsag.5 as at 2013, an estimated 13.6% of the nigerian population had been reported to be chronic carriers of hbsag.6 consequently, the burden of disease is considered to be high, as evidenced by the high incidence of morbidity and mortality associated with the virus.7 the similarity in transmission routes for hbv and hiv makes co-infection very common.8 hbv/hiv co-infection has been shown by some studies to increase morbidity and mortality when compared to hiv mono-infection.9,10 it has been reported that co-infection facilitates replication and decreases clearance of both viruses, as a result of impaired innate and adaptive immune responses.11,12 the hiv-positive sub-population deserves attention, considering the enormous public health consequences that co-infection with hbv can have. this cross-sectional study reports the prevalence of hbv infection among hiv-positive patients receiving treatment and care from the jos university teaching hospital (juth), infectious diseases unit. methods ethical considerations approval of the study protocol was obtained from the institutional ethics committee at juth (study approval number, juth/dcs/adm/127/xix/5967). study population this laboratory-based study was carried out by examining 120 hiv patients older than 18 years on highly active antiretroviral therapy accessing care at juth who provided written informed consent. the sample size was determined using the following formula:13 where z = alpha at 95% confidence level = 1.96 p = prevalence of hepatitis b virus infection14 = 7.2% = 0.072 q = 0.928 e = margin of error = 0.05 n = minimum sample size = 10.2 with 10% attrition = 10.2, thus n = 20 collection of patients’ blood whole blood samples were obtained from each patient and used for screening for hbv serological markers as well as for performing cluster of differentiation cd4+ t-lymphocyte enumeration and clinical chemistry analysis of alanine aminotransferase (alt). the process involved collection of blood using a bd precision glide™ into a 4 ml edta bd vacutainer® (becton dickinson, plymouth, united kingdom) for cd4+ t-lymphocyte enumeration and into a 6 ml bd vacutainer® clot activator tube (becton dickinson, plymouth, united kingdom). serological marker assay plasma was obtained by centrifuging collected whole blood at 2500 rpm for 1 min and testing for hepatitis b serologic markers, including the surface antigen (hbsag), surface antibody (anti hbs), envelope antigen (hbeag), envelope antibody (anti hbe) and core antibody (anti hbc) of viral particles as performed using the rapid hbv combo test kit (acumen diagnostics incorporated, china) according to the manufacturer’s instructions. cd4+ t-lymphocyte enumeration cd4+ t-lymphocyte counts for each patient were obtained by adding 20 µl of the patient’s whole blood to 20 µl of cd4+ easy count kit monoclonal antibody, then incubating in the dark for 15 min. afterwards, 800 µl of cd4+ easy count kit no lyse buffer was added to the incubated mixture and analysed using a partec cyflow cytometer (sysmex-partec, munster, germany). evaluation of alanine aminotransferase alt levels were obtained using a cobas c311 (roche diagnostics, mannheim, germany) chemistry auto-analyser. the method involved using an eppendorf 5708 centrifuge to spin the blood sample and obtain an aliquot of serum, which was then placed in a sample cup and processed using the cobas c311 auto-analyser to determine the serum alt levels of patients. data analysis data were analysed using spss version 17 (ibm corporation, armonk, new york, united states) to obtain descriptive statistics, evaluate associations between socio-demographic variables and positivity for hbv. we also determined whether there was a difference between alt and cd4+ counts using an unpaired t-test for the mean values of both groups. decisions on statistical significance were made at a 5% level of significance. results out of the 120 patients on highly active antiretroviral treatment who participated in the study, 88 (73.3%) were women and 32 (26.7 %) were men (table 1). participants in this study had a mean age of 40.4 ± 10.6 years with male participants significantly older than their female counterparts in the study. similarly, the alt levels of male participants were significantly higher than female participants. table 1: patient characteristics by sex, jos university teaching hospital hiv clinic, august 2015. among the hiv-positive patients in the study, 11 were positive for hbsag (9.2%) (table 2). for the other serological markers in the study, 15 patients (12.5%) were positive for anti hbs 1 patient (0.8%) was positive for hbeag, 27 patients (22.5%) were positive for anti hbe, and 32 patients (26.7%) were positive for anti hbc. table 2: seroprevalence of hepatitis b serological markers? among hiv-positive adult patients attending the jos university teaching hospital hiv clinic, august 2015. being in the 40–49-year age group was significantly associated with hbv/hiv co-infection (p = 0.019) (table 3). our study reported a higher rate of hbv infection among the male hiv patients (12.0% versus 7.9% in female patients), but this observation was not significant (p = 0.494). the associations of hbv infection with age, educational level and marital status were also not statistically significant. no association between infection and marital status (p = 0.875) when they were compared to patients who were negative for hbsag. table 3: socio-demographic characteristics and laboratory parameters of hbv infection among hiv-positive adult patients, jos university teaching hospital hiv clinic, august 2015. the mean cd4+ counts of hiv/hbv co-infected patients were lower than patients who were only positive for hiv; this difference was not statistically significant (p = 0.938) (table 3). however, the mean alt values for patients positive for hbsag were significantly higher than for patients negative for hbsag (p = 0.033). discussion this present study observed a 9.2% sero-prevalence of hbsag among hiv-positive patients attending the hiv treatment clinic at juth. this is slightly lower than the 12.5% prevalence reported from north-western nigeria in 201315 and the 11.5% observed in 2012 another health facility in north-central nigeria.16 however, another study conducted in southwestern nigeria reported a much lower prevalence of 5.7% among hiv-positive patients in 2009.17 these reports which show differences in regional prevalence may point to different epidemiological factors at work in different parts of the country and reveal a gap in our current understanding of the epidemiology of hiv/hbv co-infection. national surveillance systems to provide a full picture of the co-infection landscape are urgently needed to identify epidemiological trends over time to make sure suitable prevention measures are deployed. our results agree with the conclusions of a recent meta-analysis by musa and colleagues6 that showed that in nigeria, there is a continued decline in prevalence of hepatitis b infection; they estimated that the rate of decline has been approximately 0.8% annually. while our finding provides significant feedback regarding the impact of the hbv vaccination programme in nigeria, the result remains higher than the 8% threshold that the world health organization considers endemic among hiv-positive individuals. we made an observation similar to other studies regarding men having higher rates of infection than women.17,18 the report of a recent study by akhtar et al.19 in a tertiary hospital in malaysia and another report from north-east nigeria20 for hbv co-infection in hiv patients agree with our observation of an age-specific association in patients aged 40–49 years. a global age-specific prevalence, which is highest in sub-saharan africa, has been reported from a systematic review of data on worldwide prevalence collected over a 27-year period.21 the association between increasing age and being positive for hbsag by several workers reinforces the current paradigm that the epidemiology of hbv in nigeria is primarily via horizontal transmission.22 in the present study, serum alt levels in patients who were positive for hbsag were significantly higher than alt levels in patients who were hbsag-negative, which is a comparable observation to that made by otegbayo et al.23 it is possible that the patients in our study population were in the late reactivation phase where high serum alt levels accompany progressive liver damage.24 there is a growing interest in understanding serum hbsag levels across the different phases of hbv infection.25 however, our inability to quantify hbsag titers and ascertain the different phases of infection are limitations of our study. although two studies documenting the natural history of the infection in asian and european cohorts reported no significant association between alt activity and serum hbsag titers,26,27 an african perspective for the different phases may reveal a different picture in the course of infection. conclusion despite the overall decline in hbv prevalence, it remains endemic in hiv-positive populations and requires effective surveillance to fully understand the epidemiology of hiv/hbv co-infection in nigeria. increased uptake of a vaccination programme among hiv-positive patients who are susceptible to hbv infection, along with the identification of patients with hbv infection for treatment, are critical strategies in prevention and control activities. acknowledgements we are grateful to the management of the juth infectious diseases unit for their support and also to the entire staff of the centre for their cooperation and technical contributions to this study. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions this work was carried out in collaboration between all authors. author t.m.a. performed data analysis and result interpretation, prepared the tables, wrote the final manuscript and reviewed the final draft. c.o.r. & a.o.j. designed and performed the experiments, and wrote and reviewed manuscript. j.a.a.-o. conceptualised the experiment protocol, supervised the experiments and reviewed the final draft of the manuscript. j.n.o. and o.j.o. reviewed the manuscript and made corrections to manuscript. all authors read and approved the final manuscript. source of support none. references hwang ew, cheung r. global epidemiology of hepatitis b virus (hbv) infection. n am j med sci. 2011;4(1):7–13. https://doi.org/10.7156/v4i1p007 kiire cf. the epidemiology and prophylaxis of hepatitis b in sub-saharan africa: a view from tropical and subtropical africa. gut. 1996;38(suppl 2):s5–s12. https://doi.org/10.1136/gut.38.suppl_2.s5 hoffmann cj, thio cl. clinical implications of hiv and hepatitis b co-infection in asia and africa. lancet infect dis. 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https://doi.org/10.21276/apjhs.2014.1.4.35 akhtar a, khan ah, sulaiman sa, soo ct, khan k. hbv and hiv co-infection: prevalence and clinical outcomes in tertiary care hospital malaysia. j med virol. 2016;88:455–460. https://doi.org/10.1002/jmv.24347 mustapha sk, jibrin yb. the prevalence of hepatitis b surface antigenaemia in patients with human immunodeficiency virus (hiv) infection in gombe, nigeria. ann afr med. 2004;3(1):10–12. ott jj, stevens ga, groeger j, wiersma s. global epidemiology of hepatitis b virus infection: new estimates of age-specific hbsag seroprevalence and endemicity. vaccine 2012;30(12):2212–2219. https://doi.org/10.1016/j.vaccine.2011.12.116 ikobah j, okpara h, elemi i, ogarepe y, udoh e, ekanem e. the prevalence of hepatitis b virus infection in nigerian children prior to vaccine introduction into the national programme on immunization schedule. pan afr med j. 2016;23:128, 1–9. https://doi.org/10.11604/pamj.2016.23.128.8756 otegbayo ja, taiwo bo, akingbola ts, et al. prevalence of hepatitis b and c positivity in a nigerian cohort of hiv-infected patients. ann hepatol. 2008;7(2):152–156. vigano m, lampertico p. clinical implications of hbsag quantification in patients with chronic hepatitis b. saud j gastroenterol. 2012;18(2):81–86. https://doi.org/10.4103/1319-3767.93805 chan hl, wong vw, tse am, et al. serum hepatitis b surface antigen quantitation can reflect hepatitis b virus in the liver and predict treatment response. clin enterol hepatol. 2007;5:1462–1468. https://doi.org/10.1016/j.cgh.2007.09.005 jaroszewicz j, serrano bc, wursthorn k, et al. hepatitis b surface antigen levels in the natural history of hepatitis b virus (hbv) infection: a european perspective. j hepatol. 2010;52:514–522. https://doi.org/10.1016/j.jhep.2010.01.014 nguyen t, thompson ajv, bowden s, et al. hepatitis b surface antigen levels during the natural history of chronic hepatitis: a perspective on asia. j hepatol. 2010;52:508–513. https://doi.org/10.1016/j.jhep.2010.01.007 abstract introduction methods results discussion acknowledgements references about the author(s) jessica s. hurwitz department of pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa mae newton-foot department of pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa department of medical microbiology, tygerberg hospital, national health laboratory service, cape town, south africa kristien nel van zyl department of pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa pieter nel department of pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa department of medical microbiology, tygerberg hospital, national health laboratory service, cape town, south africa citation hurwitz js, newton-foot m, nel van zyl k, nel p. fosfomycin susceptibility testing and resistance mechanisms in enterobacterales in south africa. afr j lab med. 2024;13(1), a2252. https://doi.org/10.4102/ajlm.v13i1.2252 original research fosfomycin susceptibility testing and resistance mechanisms in enterobacterales in south africa jessica s. hurwitz, mae newton-foot, kristien nel van zyl, pieter nel received: 05 july 2023; accepted: 18 dec. 2023; published: 19 mar. 2024 copyright: © 2024. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: fosfomycin treatment of urinary tract infections is increasingly attractive due to escalating antibiotic resistance rates among urinary pathogens. standard antibiotic susceptibility testing methods perform poorly for fosfomycin as there is poor correlation between susceptibility results and clinical outcomes in urinary pathogens other than escherichia coli. objective: we evaluated the performance of fosfomycin susceptibility testing in e. coli and klebsiella pneumoniae to determine whether fosfomycin susceptibility is associated with molecular resistance mechanisms. methods: forty-six each of e. coli and k. pneumoniae clinical isolates were obtained from a tertiary hospital in south africa, from 01 june 2017 to 31 january 2018. agar dilution, disk diffusion, and gradient diffusion were performed and interpreted using the clinical laboratory standards institute and european committee on antimicrobial susceptibility testing guidelines. molecular resistance mechanisms were identified by whole genome sequence analysis. results: disk diffusion and gradient diffusion were accurate alternatives for fosfomycin susceptibility testing in e. coli (98% categorical agreement), but not in k. pneumoniae (47% categorical agreement). all e. coli isolates contained at least one resistance mechanism, but only one isolate with a fosa gene was resistant. in k. pneumoniae, 63% (29/46) and 70% (32/46) of isolates were susceptible to fosfomycin, using clinical laboratory standards institute and european committee on antimicrobial susceptibility testing breakpoints, respectively, despite all isolates containing a fosa gene and a uhpt mutation. conclusion: a better understanding of fosfomycin susceptibility and improved antibiotic susceptibility testing tools could improve diagnostic capability and clinical guidelines for fosfomycin treatment of urinary tract infections. what this study adds: this study highlights the importance of adhering to interpretive guidelines when performing antimicrobial susceptibility testing and the need for simplified, accurate and standardised susceptibility testing methodology and interpretation for fosfomycin in enterobacterales organisms. keywords: fosfomycin; phenotypic susceptibility testing; molecular resistance mechanisms; escherichia coli; klebsiella pneumoniae; enterobacterales. introduction urinary tract infections (utis) caused by gram-negative and gram-positive bacteria, including those of the enterobacterales order and enterococcus species, pose a global burden, with more than 404 million individuals being affected in 2019.1,2 the most common causes of lower uncomplicated utis are escherichia coli and klebsiella pneumoniae,3 with e. coli causing 70% – 95% of upper and lower utis.4,5 due to increased antibiotic resistance among urinary pathogens to more commonly used antibiotics and concerns with the side-effect profiles, alternative treatment options for utis are being assessed.6,7 fosfomycin, an old antibiotic with broad-spectrum bactericidal activity against most urinary pathogens, is an excellent empiric choice for the treatment of lower uncomplicated utis.8,9,10 fosfomycin acts by binding to uridine-diphosphate-n-acetylglucosamine enolpyruvyl transferase (mura)11 to prevent the production of n-acetylmuramic acid, a peptidoglycan precursor.12 this inhibits peptidoglycan formation, leading to bacterial cell lysis and cell death.13 fosfomycin resistance is mediated by modification of the target mura protein, direct modification of fosfomycin by fos enzymes,14 or by mutations in the glycerol-3-phosphate transport and the hexose phosphate uptake transport systems and their regulators, which are responsible for fosfomycin uptake.8 there is limited data in south africa on fosfomycin susceptibility in e. coli and other enterobacterales due to a lack of consensus in the interpretation of susceptibility results by the clinical and laboratory standards institute (clsi) and the european committee on antimicrobial susceptibility testing (eucast), limited research, and a lack of information regarding associations between susceptibility test results, molecular mechanisms of resistance, and clinical outcomes.9 nevertheless, e. coli fosfomycin resistance rates were reported to be as low as 3.2% in europe, asia and the united states between 2004 and 2009, with studies done after 2010 only suggesting a slight increase in resistance rates.15 in south africa, two studies in 2020 and 2022 described fosfomycin resistance rates of 4.3% – 4.5% in uti isolates and 2% – 8% in enterobacterales.3,16 the clsi and eucast guidelines provide fosfomycin susceptibility breakpoints for e. coli but the only breakpoints available for other enterobacterales are agar dilution minimum inhibitory concentration (mic) breakpoints from eucast, which are the same as those for e. coli. this is due to insufficient clinical and mic data for establishing both clinical breakpoints and an epidemiological cut-off value. due to the absence of breakpoints for other enterobacterales, e. coli breakpoints have been used for interpretation of fosfomycin susceptibility.3,15,16 the inappropriate interpretation of fosfomycin susceptibility for k. pneumoniae may, however, lead to unfavourable clinical outcomes when the agent is used therapeutically.8,17,18 this study aimed to describe the association between phenotypic susceptibility to fosfomycin and molecular mechanisms of resistance to fosfomycin in both e. coli and k. pneumoniae. methods ethical considerations ethical approval was obtained from the stellenbosch university health research ethics committee (n14/06/069, u22/05/180). a consent waiver was received since the isolates used were anonymised, the patients would neither be harmed nor benefit from the study directly, and no patient information was included in this study. the research data were stored electronically on a password-protected device accessible by the principal investigator only. isolate collection forty-six each of e. coli and k. pneumoniae previously isolated from patient blood cultures (bact/alert® culture media, biomérieux, marcy-l’étoile, france) at a tertiary hospital in south africa between 01 june 2017 and 31 january 2018 as part of routine patient management were collected. species identification was done as part of routine diagnostic procedures using the vitek®2 automated system (biomérieux, marcy-l’étoile, france). fosfomycin susceptibility testing agar dilution was performed by spotting 1 μl of a 1:4 dilution of a 0.5 macfarland suspension of each bacterial isolate on mueller-hinton agar plates (sigma aldrich, st. louis, missouri, united states) containing glucose-6-phosphate and fosfomycin (fosfomycin sodium vetranal®, sigma aldrich, st. louis, missouri, united states) at a range of concentrations (1 μg/ml – 256 μg/ml), in two-fold increments.17 agar dilution was performed in triplicate, and the mic was determined as the lowest antibiotic concentration at which bacterial growth was not observed in at least two replicates following incubation at 37 °c for 16 h in ambient air.19 gradient diffusion was performed using fosfomycin mic strips (0.064 μg/ml – 1024 μg/ml) (liofilchem, roseto degli abruzzi, italy) on mueller-hinton agar plates (green point media, cape town, south africa) inoculated with a 0.5 mcfarland bacterial suspension and incubated at 37 °c for 16 h in ambient air. the mic was interpreted at the concentration where the zone of the ellipse intercepted with the strip.20 kirby bauer disk diffusion was performed using fosfomycin/glucose-6-phosphate discs (200 μg) (mast group, bootle, united kingdom) on mueller-hinton agar plates inoculated with a 0.5 mcfarland bacterial suspension. the zone of inhibition (zoi) was measured following incubation at 37 °c for 16 h in ambient air. quality control was performed using the fosfomycin susceptible e. coli american type culture collection 25922 strain (atcc, manassas, virginia, united states), with an mic range of 0.5 μg/ml – 2 μg/ml and a zoi range of 22 mm – 30 mm, as specified by clsi.17 the antibiotic susceptibility testing results were analysed using the interpretive categories and breakpoints established by the clsi (2020) and eucast (2020).17,18 for e. coli, clsi interprets zoi ≥ 16 mm/mic ≤ 64 μg/ml as susceptible, zoi = 13 mm/mic – 15 mm/mic = 125 μg/ml as intermediate, and zoi ≤ 1 mm/mic ≥ 256 μg/ml as resistant; due to the absence of breakpoints for k. pneumoniae, e. coli breakpoints were used for interpretation. for e. coli, eucast interprets zoi ≥ 24 mm/mic ≤ 32 μg/ml, as susceptible and zoi < 24 mm/mic > 32 μg/ml as resistant, but for k. pneumoniae, there are only agar dilution breakpoints; therefore e. coli disk diffusion breakpoints were used for interpretation. for disk diffusion, eucast recommends disregarding isolated colonies that are within the zoi, while clsi advises taking them into account.17,18 for gradient diffusion, the manufacturer’s guidelines suggest different approaches for e. coli and k. pneumoniae: for e. coli, single colonies in the zone of the ellipse should be disregarded, while for k. pneumoniae, colonies within 3 mm of the strip should be considered for both clsi and eucast interpretations.20 fosfomycin resistance mechanisms whole genome sequencing analysis whole genome sequencing and genome assembly were previously performed on the e. coli and k. pneumoniae isolates using the illumina miseq platform (illumina, inc., san diego, california, united states).21 resistance gene identifier version 5.2.1 (mcmaster university, south hamilton, ontario, canada) was used to identify fosfomycin resistance mechanisms in the assemblies, including fosa genes and the multidrug transporter mdtg, and mutations in the target gene mura, the uhpt and glpt transporter genes, and the cyaa and ptsi regulator genes, using the comprehensive antibiotic resistance database (mcmaster university, south hamilton, ontario, canada).22 outputs were generated for 95% identity. the artemis genome browser version 18.2.0 (wellcome sanger institute, hinxton, cambridgeshire, united kingdom)23 was used to manually view and curate the gene sequences to confirm the presence of the fosfomycin resistance mechanisms in a subset of assemblies. data analysis the performance of disk diffusion and gradient diffusion relative to the gold standard method, agar dilution, was described using categorical agreement, essential agreement (for gradient diffusion), and minor, major, and very major error rates. categorical or essential agreement of greater than 90% and minor, major, and very major error rates of less than 3% were considered acceptable.24 the combinations of mutations and genes detected by whole genome sequencing analysis were compared to gold standard agar dilution mics. data were stored, and statistical calculations were done, in microsoft® excel® for microsoft 365, 2022 (microsoft®, redmond, washington, united states). results fosfomycin susceptibility according to the gold standard method, agar dilution, 98% (45/46) of the e. coli isolates were susceptible to fosfomycin, according to both clsi and eucast guidelines. of the k. pneumoniae isolates, 70% (32/46) were susceptible, 22% (10/46) resistant, and 9% (4/46) intermediate, according to clsi breakpoints, while 63% (29/46) of the k. pneumoniae isolates were susceptible and 37% (17/46) were resistant according to eucast breakpoints. for e. coli, gradient diffusion showed 100% categorical agreement with agar dilution using eucast breakpoints, with one major error (2%) when interpreting the mics according to clsi breakpoints (table 1). the essential agreement between gradient diffusion and agar dilution for e. coli was only 11%. gradient diffusion did not perform well against agar dilution for k. pneumoniae, with a categorical agreement of around 75% depending on the guidelines used and an essential agreement of only 65%. disk diffusion showed a categorical agreement of 100% against agar dilution using both clsi and eucast breakpoints for e. coli. disk diffusion did not perform well for k. pneumoniae, with a categorical agreement of 46% using eucast and 82% using clsi breakpoints. table 1: performance of fosfomycin gradient diffusion and disk diffusion susceptibility testing in comparison to agar dilution for 92 clinical isolates from south africa (2017–2018) interpreted using the clinical laboratory standards institute (2020) and european committee on antimicrobial susceptibility testing (2020) breakpoints. mechanisms of fosfomycin resistance various fosfomycin resistance mechanisms were detected in e. coli, with all the isolates carrying the mdtg gene and 93% (43/46) having the e448k mutation in the glpt gene (table 2). mutations in uhpt and the regulator genes, cyaa and ptsi, were also detected. a fosa gene was only detected in one e. coli isolate; this isolate had the highest fosfomycin mic at greater than 256 μg/ml and was the only e. coli isolate classified as fosfomycin resistant. in the absence of fosa, mics ranged from 1 μg/ml to 16 μg/ml regardless of the presence of different combinations of glpt, uhpt, and cyaa mutations, and the presence or absence of mdtg genes (table 3). table 2: fosfomycin resistance mechanisms in 92 clinical isolates from south africa (2017–2018). table 3: combinations of fosfomycin resistance mechanisms and minimum inhibitory concentration distributions for 92 clinical isolates from south africa (2017–2018). all of the k. pneumoniae isolates had a fosa gene, either fosa5 (6/46, 13%) or fosa6 (40/46, 87%), and the e350q mutation in uhpt (table 2). both combinations were present in susceptible and resistant isolates with mics that ranged from 1 μg/ml to 256 μg/ml (table 3). discussion in this study, disk diffusion and gradient diffusion performed well in comparison to the gold standard agar dilution25 for e. coli using both clsi and eucast guidelines. however, in the absence of interpretative criteria for other enterobacterales species, neither method performed adequately for k. pneumoniae. fosfomycin resistance mechanisms were identified in all e. coli and k. pneumoniae isolates, regardless of mic, highlighting further challenges associated with fosfomycin susceptibility testing. regardless of the interpretive guideline used, all the tests in this study, except gradient diffusion, identified a single fosfomycin-resistant e. coli isolate. this low fosfomycin resistance rate (2%) is in keeping with the findings of previous studies.3,15,16,26 gradient diffusion interpreted using clsi breakpoints failed to identify the one resistant isolate, and the essential agreement between agar dilution and gradient diffusion for e. coli was low. the manufacturers’ guidelines for interpreting single colonies within the zone of ellipse of gradient diffusion strips vary. liofilchem recommends that for e. coli, single colonies should be ignored when measuring the zone of the ellipse, regardless of whether the clsi or eucast guidelines are used for interpretation.20 also, the clsi and eucast breakpoints differ,17,18 influencing the interpretation. the different interpretations of mic values did not greatly influence the susceptibility interpretation in e. coli, resulting in an acceptable categorical agreement of 98% according to clsi (with a very major error rate of 2%) and 100% according to eucast guidelines. for e. coli, disk diffusion showed 100% categorical agreement with agar dilution using both clsi and eucast guidelines, although eucast advises ignoring isolated colonies in the zoi, and clsi advises considering them.17,18 for k. pneumoniae, there were conflicting susceptibility results when interpreted using the two guidelines, clsi and eucast, even for the gold standard agar dilution method. although the variation in mic values was lower when comparing gradient diffusion to agar dilution in k. pneumoniae than in e. coli, neither essential nor categorical agreement was acceptable. similarly, the categorical agreement between agar dilution and disk diffusion, interpreted using both clsi (82%) and eucast (46%) guidelines, was unacceptable. this is suspected to be due to the extrapolation of the guidelines provided by clsi and eucast for e. coli, to k. pneumoniae.17,18 there were more single colonies within the inhibition zones in k. pneumoniae than e. coli, making it challenging to interpret the susceptibility results. liofilchem advises that for k. pneumoniae, isolated colonies within the zone of the ellipse of the mic strips should be considered irrespective of the interpretation guideline,20 and this contributed to the higher essential agreement for k. pneumoniae compared to e. coli. the lower categorical agreement for disk diffusion was interpreted using eucast guidelines, disregarding isolated colonies within the zoi. based on these findings, neither gradient nor disk diffusion should be used as alternatives to agar dilution for fosfomycin susceptibility testing in k. pneumoniae. furthermore, in agreement with this study, bijllaardt et al. concluded that the breakpoints for e. coli should not be used for k. pneumoniae.27 although various mutations associated with fosfomycin resistance in e. coli were detected in the e. coli isolates, no mura mutations were detected, and only one isolate containing a fosa gene was fosfomycin resistant. raised fosfomycin mics were not detected in e. coli isolates with various combinations of fosfomycin resistance-associated mutations. a study by doesschate et al. also found that e. coli isolates that did not have fosa genes were susceptible to fosfomycin.28 all of the k. pneumoniae isolates had a fosa gene (fosa5 or fosa6) and the e350q mutation in the uhpt gene. the presence of these resistance mechanisms however had no correlation with the fosfomycin mics of the the different isolates. it is, therefore, unclear whether these mechanisms contribute to fosfomycin resistance in this organism. the differential expression of fosa may account for the single colonies observed within the zoi in k. pneumoniae isolates.29 susceptibility tests for fosfomycin in k. pneumoniae are unreliable, with no clear association between phenotypic susceptibility and the presence of resistance mechanisms. the gold standard agar dilution test produced different results depending on the interpretive guidelines used, and there was low agreement between gradient diffusion or disk diffusion and agar dilution. limitations although both gradient diffusion and disk diffusion, interpreted using clsi and eucast guidelines, produced an acceptable categorical agreement for e. coli, a more extensive sample set of isolates containing more fosfomycin-resistant isolates, and with mics around the breakpoints should be analysed to confirm these findings. the aim of the study was to correlate the fosfomycin resistance mechanisms with the phenotypic susceptibility results. however, due to the very low prevalence of fosfomycin resistance amongst e. coli isolates, with only one resistant isolate, we were unable to test the significance of any association. similarly, it was clear that there was no association between fosfomycin mic and resistance mechanisms in k. pneumoniae. not all fosfomycin resistance genes or mutations may be included in the comprehensive antibiotic resistance database22, and therefore, mutations or genes that confer resistance could have been missed. thus, whole genome sequence data should be manually searched to identify potential novel fosfomycin resistance mutations, which could be further validated to improve our understanding of fosfomycin susceptibility. the expression of the resistance mechanisms, specifically the fosa genes, was not evaluated in this study. differential expression may account for the variable mics and susceptibility results despite the presence of these genes in all k. pneumoniae isolates. conclusion fosfomycin molecular resistance mechanisms were not associated with phenotypic susceptibility to fosfomycin in e. coli or k. pneumoniae, except fosa3 in one resistant e. coli isolate. disk diffusion and gradient diffusion perform well for fosfomycin susceptibility testing for e. coli but not for k. pneumoniae. these findings support the guidelines given by clsi and eucast in that disk diffusion and gradient diffusion susceptibility testing should not be performed for k. pneumoniae and that the e. coli breakpoints should not be used for interpreting k. pneumoniae susceptibility. acknowledgements the authors would like to acknowledge prof. a. whitelaw and prof. a. dramowski for the isolate collection, drs s. reuter and y. snyman for whole genome sequencing, and the national health laboratory service microbiology laboratory of tygerberg hospital for sample processing. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions p.n. and m.n.f. designed the study. j.s.h. performed all experiments and data analysis. k.n.v.z. assisted with data analysis and methodology. j.s.h., m.n.f., k.n.v.z. and p.n. collectively interpreted the results. j.s.h. wrote the draft article, with the support and supervision of p.n. and m.n.f. all authors reviewed and approved the final article. sources of support this work was supported by the stellenbosch university undergraduate and honours research project fund (rp-22-04-006). data availability the data that support the findings of this study are available from the corresponding author, p.n., upon reasonable request. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. it does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the authors are responsible for this article’s results, findings, and content. references stamm we, norrby sr. urinary tract infections: disease panorama and challenges. j infect dis. 2001;183 suppl 1:s1–s4. https://doi.org/10.1086/318850 zeng z, zhan j, zhang k, chen h, cheng s. global, regional, and national burden of urinary tract infections from 1990 to 2019: 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[fosfomycin, co-trimoxazole and nitrofurantoin in the treatment of recurrent uncomplicated urinary tract infections in type 2 diabetes mellitus]. wiad lek [serial online]. 2007;60(5–6):235–240. available from: https://europepmc.org/article/med/17966887 brown ed, vivas ei, walsh ct, kolter r. mura (murz), the enzyme that catalyses the first committed step in peptidoglycan biosynthesis, is essential in escherichia coli. j bacteriol. 1995;177(14):4194–4197. https://doi.org/10.1128/jb.177.14.4194-4197.1995 eschenburg s, priestman m, schönbrunn e. evidence that the fosfomycin target cys115 in udp-n-acetylglucosamine enolpyruvyl transferase (mura) is essential for product release. j biol chem. 2005;280(5):3757–3763. https://doi.org/10.1074/jbc.m411325200 castañeda-garcía a, blázquez j, rodríguez-rojas a. molecular mechanisms and clinical impact of acquired and intrinsic fosfomycin resistance. antibiotics. 2013;2(2):217–236. https://doi.org/10.1074/jbc.m411325200 suarez je, mendoza mc. plasmid-encoded fosfomycin resistance. antimicrob agents chemother. 1991;35(5):791–795. https://doi.org/10.1128/aac.35.5.791 mueller l, cimen c, poirel l, descombes mc, nordmann p. prevalence of fosfomycin resistance among esbl-producing escherichia coli isolates in the community, switzerland. eur j clin microbiol infect dis. 2019;38(5):945–949. https://doi.org/10.1007/s10096-019-03531-0 mothibi lm, bosman nn, nana t. fosfomycin susceptibility of uropathogens at charlotte maxeke johannesburg academic hospital. s afr j infect dis. 2020;35(1):a173. https://doi.org/10.4102/sajid.v35i1.173 clsi. performance standards for antimicrobial susceptibility testing. 30th ed. clsi guideline m100. wayne, pa: clinical and laboratory standards institute; 2020. european committee on antimicrobial susceptibility testing. breakpoint tables for interpretation of mics and zone diameters, version 9.0 [homepage on the internet]. 2020 [cited 2023 nov 09]. available from: http://www.eucast.org/clinical_breakpoints/ lu cl, liu cy, huang yt, et al. antimicrobial susceptibilities of commonly encountered bacterial isolates to fosfomycin determined by agar dilution and disk diffusion methods. antimicrob agents chemother. 2011;55(9):4295–4301. https://doi.org/10.1128/aac.00349-11 fosfomycin mic test strip technical sheet mts45 – rev. 1. liofilchem 09.11.2017. [cited 2023 nov 09]. available from: http://www.liofilchem.net/login.area.mic/technical_sheets/mts45.pdf dramowski a, aiken am, rehman am, et al. mortality associated with third-generation cephalosporin resistance in enterobacteriaceae bloodstream infections at one south african hospital. j glob antimicrob resist. 2022;29:176–184. https://doi.org/10.1016/j.jgar.2022.03.001 alcock bp, raphenya ar, lau tty, et al. card 2020: antibiotic resistome surveillance with the comprehensive antibiotic resistance database. nucleic acids res. 2020;48(d1):d517–d525. https://doi.org/10.1093/nar/gkz935 carver t, harris sr, berriman m, parkhill j, mcquillan ja. artemis: an integrated platform for visualisation and analysis of high-throughput sequence-based experimental data. bioinformatics. 2012;28(4):464–469. https://doi.org/10.1093/bioinformatics/btr703 tenover fc. mechanisms of antimicrobial resistance in bacteria. am j med. 2006;119(6):s3–s10. https://doi.org/10.1016/j.amjmed.2006.03.011 mezzatesta ml, la rosa g, maugeri g, et al. in vitro activity of fosfomycin trometamol and other oral antibiotics against multidrug-resistant uropathogens. int j antimicrob agents. 2017;49(6):763–766. https://doi.org/10.1016/j.ijantimicag.2017.01.020 ou lb, nadeau l. fosfomycin susceptibility in multidrug-resistant enterobacteriaceae species and vancomycin-resistant enterococci urinary isolates. can j hosp pharm. 2017;70(5):368–374. https://doi.org/10.4212/cjhp.v70i5.1698 van den bijllaardt w, schijffelen mj, bosboom rw, et al. susceptibility of esbl escherichia coli and klebsiella pneumoniae to fosfomycin in the netherlands and comparison of several testing methods including etest, mic test strip, vitek2, phoenix and disc diffusion. j antimicrob chemother. 2018;73(9):2380–2387. https://doi.org/10.1093/jac/dky214 ten doesschate t, abbott ij, willems rjl, et al. in vivo acquisition of fosfomycin resistance in escherichia coli by fosa transmission from commensal flora. j antimicrob chemother. 2019;74(12):3630–3632. https://doi.org/10.1093/jac/dkz380 ortiz-padilla m, portillo-calderón i, de gregorio-iaria b, et al. interplay among different fosfomycin resistance mechanisms in klebsiella pneumoniae. antimicrob agents chemother. 2021;65(3):e01911-20. https://doi.org/10.1128/aac.01911-20 abstract introduction methods results discussion acknowledgements references about the author(s) kerri-lee a. francois discipline of virology, faculty of health sciences, school of laboratory medicine and medical sciences, college of health sciences, university of kwazulu-natal, durban, south africa national health laboratory services, inkosi albert luthuli central hospital, durban, south africa nokukhanya msomi discipline of virology, faculty of health sciences, school of laboratory medicine and medical sciences, college of health sciences, university of kwazulu-natal, durban, south africa national health laboratory services, inkosi albert luthuli central hospital, durban, south africa kerusha govender discipline of virology, faculty of health sciences, school of laboratory medicine and medical sciences, college of health sciences, university of kwazulu-natal, durban, south africa national health laboratory services, inkosi albert luthuli central hospital, durban, south africa lilishia gounder discipline of virology, faculty of health sciences, school of laboratory medicine and medical sciences, college of health sciences, university of kwazulu-natal, durban, south africa national health laboratory services, inkosi albert luthuli central hospital, durban, south africa pravi moodley discipline of virology, faculty of health sciences, school of laboratory medicine and medical sciences, college of health sciences, university of kwazulu-natal, durban, south africa national health laboratory services, inkosi albert luthuli central hospital, durban, south africa raveen parboosing discipline of virology, faculty of health sciences, school of laboratory medicine and medical sciences, college of health sciences, university of kwazulu-natal, durban, south africa national health laboratory services, inkosi albert luthuli central hospital, durban, south africa department of medical virology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa indrani chetty discipline of virology and national health laboratory service, inkosi albert luthuli central hospital, durban, south africa lunga xaba discipline of virology and national health laboratory service, inkosi albert luthuli central hospital, durban, south africa aabida khan discipline of virology, faculty of health sciences, school of laboratory medicine and medical sciences, college of health sciences, university of kwazulu-natal, durban, south africa national health laboratory services, inkosi albert luthuli central hospital, durban, south africa citation francois k-la, msomi n, govender k, et al. seroprevalence of sars-cov-2 immunoglobulin g in hiv-positive and hiv-negative individuals in kwazulu-natal, south africa. afr j lab med. 2023;12(1), a2065. https://doi.org/10.4102/ajlm.v12i1.2065 original research seroprevalence of sars-cov-2 immunoglobulin g in hiv-positive and hiv-negative individuals in kwazulu-natal, south africa kerri-lee a. francois, nokukhanya msomi, kerusha govender, lilishia gounder, pravi moodley, raveen parboosing, indrani chetty, lunga xaba, aabida khan received: 26 aug. 2022; accepted: 29 mar. 2023; published: 29 june 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: kwazulu-natal ranked second highest among south african provinces for the number of laboratory-confirmed cases during the second wave of the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) pandemic. the seroprevalence of sars-cov-2 among certain vulnerable groups, such as people living with hiv in kwazulu-natal, is unknown. objective: the study aimed to determine the prevalence of sars-cov-2 immunoglobulin g (igg) in hiv-positive versus hiv-negative patients. methods: this was a retrospective analysis of residual clinical blood specimens unrelated to coronavirus disease 2019 (covid-19) submitted for diagnostic testing at inkosi albert luthuli central hospital, durban, from 10 november 2020 to 09 february 2021. specimens were tested for sars-cov-2 immunoglobulin g on the abbott architect analyser. results: a total of 1977/8829 (22.4%) specimens were positive for sars-cov-2 antibodies. seroprevalence varied between health districts from 16.4% to 37.3%, and was 19% in hiv-positive and 35.3% in hiv-negative specimens. seroprevalence was higher among female patients (23.6% vs 19.8%; p < 0.0001) and increased with increasing age, with a statistically significant difference between the farthest age groups (< 10 years and > 79 years; p < 0.0001). the seroprevalence increased from 17% on 10 november 2020 to 43% on 09 february 2021 during the second wave. conclusion: our results highlight that during the second covid-19 wave in kwazulu-natal a large proportion of people living with hiv were still immunologically susceptible. the reduced seropositivity in people with virological failure further emphasises the importance of targeted vaccination and vaccine response monitoring in these individuals. what the study adds: this study contributes to data on sars-cov-2 seroprevalence before and during the second wave in kwazulu-natal, south africa, which has the highest hiv prevalence globally. reduced seropositivity was found among people living with hiv with virological failure, highlighting the importance of targeted booster vaccination and vaccine response monitoring. keywords: sars-cov-2 igg prevalence; seroprevalence hiv-positive and hiv-negative; anti-sars-cov-2 igg hiv-positive and negative; seroprevalence sars-cov-2 igg; kwazulu-natal. introduction coronavirus disease 2019 (covid-19), caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), is one of the most significant pandemics in history. in africa, south africa has the highest number of confirmed cases (africa cdc covid-19 dashboard, 2021 – https://africacdc.org/covid-19/). kwazulu-natal province ranked second-highest nationally for the number of laboratory-confirmed cases during the second wave of the sars-cov-2 epidemic in south africa.1 the first wave in kwazulu-natal was around june 2020 – july 2020. the wave was followed by lower but sustained viral circulation between august 2020 and october 2020 and a more explosive second wave from november 2020 to february 2021. the second wave was of greater magnitude than the first, with the emergence of the more transmissible beta variant.2,3 the third wave of the epidemic in south africa started in may 2021, with a resurgence in kwazulu-natal in june 2021. the more transmissible delta variant rapidly displaced the beta variant between may 2021 and august 2021, while the even more highly transmissible omicron variant overtook the delta variant between november 2021 and january 2022.2,4 polymerase chain reaction (pcr) is most accurate for the diagnosis of acute sars-cov-2 infection.5 however, the use of pcr-confirmed cases for reporting may underestimate the true extent of the sars-cov-2 pandemic. although not indicated for the diagnosis of acute infection, serology may give a more accurate reflection of the true prevalence of infection in people who may not have had a pcr test during acute infection or who were asymptomatic.6 even though south africa has passed the third, fourth and fifth waves of the sars-cov-2 epidemic and is currently experiencing a transition phase, serological surveillance remains a vital tool to understand the true extent of sars-cov-2 exposure and inform public health responses.7 seroprevalence in 2020 was estimated to be 5–10 times higher than reported cases identified by pcr.8,9,10 a population-based seroepidemiological survey in gauteng province in south africa, that started 8 weeks into the first wave and ended at the peak of the second wave, estimated that there were 2.89 million sars-cov-2 infections, which is 7–8 times higher than the reported 332 000 pcr cases.10 local sars-cov-2 seroprevalence rates were over 60% in black south african blood donors11 and 30% – 40% among patients accessing care during the first wave in the cape town metropolitan sub-districts.12 similarly, seroprevalence studies in spain, geneva and new york reported seroprevalence higher than the number of reported cases.13,14,15 ideally, population-based serosurveys should be used to estimate seroprevalence nationally.16 however, serosurveys require extensive resources, and involve active recruitment and prospective follow-up of individuals. using residual clinical specimens submitted for either routine screening or diagnostic testing could provide a snapshot16,17 and is a convenient way to conduct passive surveillance. in south africa, seroprevalence studies using convenience specimens have been conducted by the south african blood bank services (sanbs) in the western cape and gauteng.11,12,18 kwazulu-natal has the world’s highest hiv prevalence19 and has the second highest number of reported sars-cov-2 infections nationally. second-wave infections in south africa caused by the beta variant were associated with increased disease severity, especially in people living with hiv20 when no vaccines were available. thus, an accurate estimate of sars-cov-2 seroprevalence among people living with hiv was important in the early phase of the sars-cov-2 epidemic for outbreak surveillance, implementation of vaccination strategies, and to assess the effectiveness of non-pharmaceutical interventions in kwazulu-natal. we therefore determined the sars-cov-2 immunoglobulin g (igg) seroprevalence among people with hiv infection in kwazulu-natal. methods ethical considerations this study was approved by university of kwazulu-natal biomedical research ethics committee (ref. bca256/010). informed patient consent was not required as this was a retrospective study using residual diagnostic specimens. no additional specimens were collected. all residual clinical specimens were de-identified and labelled with a unique study number; patient privacy and confidentiality data were protected in accordance with the declaration of helsinki. data were collected in a password-protected excel spreadsheet (microsoft corp., redmond, washington, united states) on a dedicated computer behind firewall-protected servers, which was only accessible to the primary investigator. study design the authors retrospectively tested residual clinical sera and plasma specimens submitted to the department of virology at inkosi albert luthuli central hospital, durban, for diagnostic testing unrelated to covid-19 from 10 november 2020 to 09 february 2021, representing the period before and during the second wave (22 november 2020 to 27 march 2021) of the sars-cov-2 epidemic in kwazulu-natal. sample size a minimum sample size of 7294 was estimated using openepi (version 3.1; http://www.openepi.com/)21 based on a sars-cov-2 prevalence of 5% to 10% before the second wave peak with a maximum 1% margin error and using published seroprevalence data from studies done globally (5%,10 1% – 6.9%22 and 1% – 10%8), the assumed point prevalence was calculated. using the stata® (statacorp, college station, texas, united staes) sampsi function, the sample size was calculated to compare sars-cov-2 seropositivity in hiv-positive and hiv-negative individuals with a power of 80%, two-sided alpha of 0.05 and equal size in each group. a sample size of 3682 would be sufficient to detect a 1% difference if the prevalence is low (1%) and a 3% difference if the prevalence is high (13%).8,13,22 specimen selection sera and plasma specimens with sufficient volumes from the viral serology (hiv and hepatitis b enzyme-linked immunosorbent assay and hiv viral load laboratory sections) were selected for sars-cov-2 igg testing. specimens with inadequate volumes (< 100 µl) were excluded. the selected specimen results were linked to demographic information extracted from the laboratory information system. specimens from the same patient were tested once only. laboratory methods testing for sars-cov-2 igg was performed on the abbott architect using the abbott architect sars-cov-2 igg assay (abbott diagnostics, abbott park, illinois, united states).23 sera and plasma specimens were stored at 2 °c – 8 °c for not more than 7 days and −20 °c for long-term (> 7 days) storage. the assay is an automated two-step chemiluminescent microparticle immunoassay for qualitative detection of sars-cov-2 igg antibodies against the sars-cov-2 nucleocapsid protein. the assay was performed and interpreted according to the manufacturer’s instructions.24 statistical analysis statistical analysis was done using stata® version 15 (statacorp, college station, texas, united states). we performed a logistic regression analysis to determine whether age, gender, hiv seropositivity, and hiv viral load were associated with sars-cov-2 seropositivity, with significance level set to 0.05. results a total of 9083 specimens were tested. however, 254 specimens were excluded from the analysis due to existing sars-cov-2 igg test requests (n = 248) or mismatched laboratory information (n = 6). a total of 8829 (table 1) specimen results were analysed: 5724 (64.8%) of the specimens were from female and 2865 (32.5 %) from male patients, while 240 (2.7%) had no specified gender; 1309 (14.8%) of sera specimens had hiv enzyme-linked immunosorbent assay results, and 5522 (62.5%) of plasma specimens had hiv viral load results. table 1: demographic characteristics of specimens collected in kwazulu-natal, south africa, from 10 november 2020 to 09 february 2021. the average sars-cov-2 seropositivity across 11 health districts (figure 1) during the second wave was 22.4%; the ilembe district had the highest seropositivity (37.3%), followed by king cetshwayo (35.1%), and umkhanyakude (32%). weekly seropositivity increased with increasing sars-cov-2 cases (figure 2), reaching 43% during the second wave in kwazulu-natal. figure 1: sars-cov-2 immunoglobulin g seropositivity per district in kwazulu-natal, south africa, from 10 november 2020 to 09 february 2021. figure 2: temporal relationship between seropositivity and polymerase chain reaction positivity of specimens collected in kwazulu-natal, south africa, from 10 november 2020 to 09 february 2021. seropositivity among female patients was 23.6% (odds ratio: 0.705, 95% confidence interval: 0.617–0.806) while male patients had lower odds of seropositivity (p < 0.0001) (table 1). seropositivity increased with increasing age, with a 1.012 increase in odds for every year increase in age (p < 0.0001) (figure 3). seropositivity was highest in the 70–79 year age group and lowest in those under 9 years of age. seropositivity (table 1) was higher in the hiv-negative compared to the hiv-positive (35.3% vs 19%; odds ratio: 0.402; 95% confidence interval: 0.349–0.463). hiv positivity with a viral load greater than 1000 copies/ml was associated with lower odds of sars-cov-2 seropositivity (odds ratio: 0.518; 95% confidence interval: 0.401–0.669). no statistically significant interaction (at a 10% level) was noted between hiv status and age or gender. figure 3: age and seropositivity of specimens collected in kwazulu-natal, south africa, from 10 november 2020 to 09 february 2021. discussion we assessed the sars-cov-2 igg seropositivity in convenience specimens submitted to the department of virology at inkosi albert luthuli central hospital, durban, before and during the peak of the second covid-19 wave caused by the beta variant. we observed an increase in seropositivity from 17% before to 43% during the second wave in kwazulu-natal, reflective of the increase in the number of infected individuals during the second wave, with a mean sars-cov-2 seropositivity of 22.4% across the study period. there were statistically significant associations between sars-cov-2 seropositivity, age, gender, and hiv status. our results indicate that the true extent of the sars-cov-2 epidemic in kwazulu-natal may have been underestimated due to reporting of pcr results alone. this is similar to other local seroprevalence studies that report higher seroprevalence compared to the number of reported pcr cases.10,11,12,25 the seropositivity in our study was lower than the seroprevalence reported by surveillance studies in cape town (40%) and gauteng (27.8%).12,18 the lower prevalence may be because the specimens were not collected immediately after the first wave in kwazulu-natal; they were collected 4 months later, thus affecting the detection of igg, which wanes over time. the sentinel surveillance studies were started directly after the first wave in both cape town and gauteng. furthermore, the majority (63%) of the specimens in our study were from hiv-positive individuals; 10% of these specimens had an hiv viral load greater than 1000 copies/ml and reduced sars-cov-2 seropositivity. hiv viral load greater than 1000 copies/ml had a statistically significant association with lower odds of sars-cov-2 igg positivity. hiv infection is associated with a reduced immune response to infectious pathogens.26 however, in individuals on antiretroviral therapy, immune responses to sars-cov-2 are reported to be similar to that in hiv-negative individuals.27,28 people on antiretroviral therapy may display immune reconstitution;26 however, chronic immune activation and impaired b-cell responses in individuals on antiretroviral therapy with virologic failure may result in reduced antibody responses26,28 and lower igg concentrations,29 accounting for the lower seropositivity in those with hiv infection. lower sars-cov-2 igg concentrations may affect the detectability of igg,30 which may be lower than the detectable limit of the assay. based on a local evaluation of the performance of the abbott architect sars-cov-2 igg assay, sensitivity is highest 30–41 days after a positive pcr, following moderate to severe infection.23 however in our study reduced seropositivity was most likely due to reduced immunity in people with uncontrolled hiv and the timing of sampling in relation to the epidemic waves.23 we have demonstrated that sars-cov-2 seropositivity increased with age, with children under 10 years having the lowest seropositivity (11.1%). similar patterns have been described locally25 and internationally.15 our reported seropositivity (10%) in children under 10 years during the second wave in kwazulu-natal indicated that these children may have been more vulnerable to sars-cov-2 infection. however, the rate of infections in south africa among these children was 1.4%,31 suggesting that pcr testing alone may have underestimated infection rates or that these children were asymptomatic. however, a seroprevalence in children under 9 years during the first wave in gauteng was reported to be about 20%,18 most likely due to gauteng having the highest infection rate nationally during the entire epidemic.1 we found no discernible pattern in the district seroprevalence during the second wave. however, the rate of infections in the ethekwini, ilembe, ugu, and uthukela districts were subsequently lower in the third wave.1 limitations firstly, the specimens tested were from individuals accessing public healthcare and thus may not represent the general population. however, it potentially represents people living with hiv accessing antiretroviral therapy in our province. secondly, surveillance using residual clinical specimens without exposure time data, as done in this study, may underestimate seropositivity.32 thirdly, seropositivity may be underestimated since sars-cov-2 igg detection and durability is directly proportional to disease severity.33,34 although antibody positivity was still high in another study at 8 months after mild and asymptomatic infection,35 overall detectability and durability may be reduced in hiv-positive persons.36 fourthly, the abbott architect sars-cov-2 igg assay detects igg to nucleocapsid antigens only and at the time was the only approved commercial serological assay by the south african health products regulatory authority.37 the use of nucleocapsid igg solely to determine seroprevalence may underestimate the true rate of past infections.32 nucleocapsid igg declines more rapidly than spike igg. however, nucleocapsid igg is more sensitive within the first 14 days of sars-cov-2 infection as compared to spike igg.34 lastly, antibody cross-reactivity with other betacoronaviruses may lead to false positives.23,38 however, the abbott architect sars-cov-2 igg has a specificity of 99%.23,38 conclusion our results highlight that after the second wave in kwazulu-natal, many people accessing public healthcare were immunologically susceptible. furthermore, reduced seropositivity in hiv-positive individuals with hiv viral loads greater than 1000 copies/ml further emphasised the importance of targeted vaccination of people living with hiv and monitoring response to vaccination in these individuals.39,40 acknowledgements the authors would like to acknowledge and thank the staff from the department of virology at inkosi albert luthuli central hospital for their assistance and dedication to the research project, as well as the national health laboratory service for providing the kits used for this research. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions k.-l.a.f., n.m., k.g., l.g., p.m., r.p., i.c., l.x. and a.k. contributed to the conceptualisation, ethics application, writing of the manuscript, data collection and analysis, and have read and agreed to the published version of the manuscript. sources of support this research did not receive a grant from any funding agency in the public, commercial, or non-profit sectors. we would like to express gratitude to the national health laboratory service for providing kits used for this research. data availability the authors confirm that the data supporting the findings of this study are available within the article. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any of the affiliated institutions of the authors. references bapela mp, ngcobo a, sosibo c, mhlongo b. kwazulu-natal department of health covid-19 situation report. 9 june 2022. durban: doh; 2022. abdool karim ss, baxter c. impact of sars-cov-2 variants of 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faulkner n, cornish gh, et al. preexisting and de novo humoral immunity to sars-cov-2 in humans. science. 2020;370(6522):1339–1343. https://doi.org/10.1126/science.abe1107 hassold n, brichler s, ouedraogo e, et al. impaired antibody response to covid-19 vaccination in advanced hiv infection. aids. 2022;36(4):f1–f5. https://doi.org/10.1097/qad.0000000000003166 xu x, vesterbacka j, aleman s, nowak p. high seroconversion rate after vaccination with mrna bnt162b2 vaccine against sars-cov-2 among people with hiv – but hiv viremia matters? aids. 2022;36(3):479–481. https://doi.org/10.1097/qad.0000000000003135 abstract background description of the intervention lessons learnt recommendations acknowledgements references about the author(s) seth attoh jm wadhwani department of anatomical pathology, pathology division laboratory, 37 military hospital, accra, ghana francis k.m. tetteh department of microbiology, pathology division laboratory, 37 military hospital, accra, ghana mary mcaddy jm wadhwani department of anatomical pathology, pathology division laboratory, 37 military hospital, accra, ghana kingsley ackah department of microbiology, pathology division laboratory, 37 military hospital, accra, ghana richmond kyei jm wadhwani department of anatomical pathology, pathology division laboratory, 37 military hospital, accra, ghana marcus moroti department of microbiology, pathology division laboratory, 37 military hospital, accra, ghana cynthia boateng department of chemical pathology, pathology division laboratory, 37 military hospital, accra, ghana laurinda adusu-donkor department of chemical pathology, pathology division laboratory, 37 military hospital, accra, ghana joseph boafo department of haematology, pathology division laboratory, 37 military hospital, accra, ghana alhassan yakubu department of quality assurance, pathology division laboratory, 37 military hospital, accra, ghana sarah kwao department of quality assurance, pathology division laboratory, 37 military hospital, accra, ghana emmanuel sarkodie department of haematology, pathology division laboratory, 37 military hospital, accra, ghana nana-banyin koranteng department of chemical pathology, pathology division laboratory, 37 military hospital, accra, ghana monica a. addo department of chemical pathology, pathology division laboratory, 37 military hospital, accra, ghana frederick hobenu jm wadhwani department of anatomical pathology, pathology division laboratory, 37 military hospital, accra, ghana kwasi agyeman-bediako jm wadhwani department of anatomical pathology, pathology division laboratory, 37 military hospital, accra, ghana raymond d. fatchu department of quality assurance, pathology division laboratory, 37 military hospital, accra, ghana citation attoh s, tetteh fkm, mcaddy m, et al. challenges with the pursuit of iso 15189 accreditation in a public health laboratory in ghana. afr j lab med. 2022;11(1), a1448. https://doi.org/10.4102/ajlm.v11i1.1448 lessons from the field challenges with the pursuit of iso 15189 accreditation in a public health laboratory in ghana seth attoh, francis k.m. tetteh, mary mcaddy, kingsley ackah, richmond kyei, marcus moroti, cynthia boateng, laurinda adusu-donkor, joseph boafo, alhassan yakubu, sarah kwao, emmanuel sarkodie, nana-banyin koranteng, monica a. addo, frederick hobenu, kwasi agyeman-bediako, raymond d. fatchu received: 10 nov. 2020; accepted: 04 apr. 2022; published: 19 july 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: accreditation is important for all medical laboratories, particularly public health laboratories in developing countries. several laboratories in ghana implemented the requirements of the international organization for standardization (iso) 15189 but were unable to proceed to accreditation. this article describes the challenges faced by the pathology division laboratory of the 37 military hospital, accra, ghana, during the acquisition of iso 15189 accreditation and suggests solutions for a better approach. intervention: following iso 15189 accreditation in 2017, an online survey was conducted between 01 and 30 march 2020 among the laboratory staff. respondents were required to grade, on a scale of 0 (least) to 5 (most), the extent to which 16 key challenges influenced the process of obtaining accreditation. key informant interviews were also held with laboratory personnel who were directly involved in the establishment of the quality management system in the laboratory and the accreditation acquisition process. lessons learnt: documentation, laboratory safety measures, laboratory management support, and reagent unavailability were estimated as the challenges that most affected the acquisition of laboratory accreditation. challenges such as poor communication, staff apathy and workload had the least effect on the accreditation process. there was no difference in challenges identified between persons who worked in the laboratory before or after accreditation (p = 0.11). recommendations: to surmount the anticipated challenges, there is the need for national strategic direction for laboratory accreditation, hospital and laboratory management support for the accreditation acquisition and maintenance processes, and sufficient technical assistance in the form of training and mentorship. keywords: iso 15189:2012; accreditation; strengthening laboratory management towards accreditation; laboratory; challenges. background the quest to meet international standards, gain international recognition and acceptability, win the trust and confidence of clinicians and patients, improve quality of care, and improve the capacity to measure performance and streamline operations is driving medical facilities to pursue international accreditation.1 accreditation and the pursuance of quality health systems in lowand middle-income countries have become critical in achieving universal health coverage.2 a well-structured and well-implemented healthcare system can result in better health outcomes and reduce health inequalities, hospitalisation rates, and hospitalisation costs.3,4 according to the world health organization, despite the huge investments made in the health sector globally, including the medical laboratory, health systems and laboratory practices still face significant challenges.5 in sub-saharan africa, several initiatives focused on medical laboratory improvement have resulted in few practical and sustainable outcomes.6,7 key factors that negatively influence the accreditation process include the lack of prioritisation of accreditation, inadequate allocation of resources for attaining and maintaining accreditation, poor understanding of the importance of accreditation by both laboratory personnel and health authorities, and the high cost of the process.8 between the years 2010 and 2013, ghana enrolled 15 public sector medical laboratories in the strengthening laboratory management towards accreditation programme to develop laboratory systems towards accreditation readiness and subsequent accreditation.9 at the close of the programme, three laboratories were awarded a four-star rating according to the strengthening laboratory management towards accreditation grading system.9 however, despite the quality improvement within the respective laboratories, none has proceeded to accreditation due to major gaps threatening the acquisition of accreditation. even for laboratories that have attained accreditation in other countries, weak hospital management support, inadequate documentation, inefficient equipment, irregular supply of reagents, little mentorship, and high staff turnover have been identified as bottlenecks to attaining and maintaining accreditation.10,11 as several african countries within the sub-region, including ghana, pursue healthcare reforms to achieve universal health coverage, the need for national accreditation policies and systems has become a priority for several ministries of health.12 the pathology division laboratory of the 37 military hospital became the first public sector laboratory to be accredited for methods in chemical pathology and haematology in accra, ghana, in 2017.8 learning from its experiences and challenges, the laboratory has maintained its accreditation status over the years while gradually expanding its scope to include other departments in the division. the microbiology department also subsequently obtained international organization for standardization (iso) 15189:2012 accreditation in 2019 from the southern african development community accreditation service. this article describes the challenges faced by the pathology division laboratory of the 37 military hospital in accra, ghana, in the pursuit of iso 15189:2012 accreditation and suggests solutions to the identified challenges. description of the intervention ethical considerations ethical clearance, with reference number 37mh-irb/ds/ipn/417/2020, was received from the institutional review board of the 37 military hospital, accra, ghana. participants were required to respond to a consent section before voluntarily completing the online questionnaire. to protect the privacy of participants, no personal information was collected. data collection the study was conducted in the pathology division laboratory of the 37 military hospital in accra, the capital of ghana, west africa, which received iso 15189 accreditation for haematology, chemical pathology, and microbiology test methods between 2017 and 2019. the laboratory also operates other departments, including histopathology, serology, and a blood transfusion service. key informant (ki) interviews were conducted with personnel who were directly involved in the establishment of the quality management system (qms) of the laboratory and the accreditation acquisition process. these kis included the officer-in-charge of the division, laboratory manager, quality manager, and heads of department for the haematology, chemical pathology, and microbiology departments. the kis were coded (ki-1, ki-2, etc.) to maintain confidentiality. interviews were digitally voice recorded after securing interviewee consent. interviews were conducted in english, transcribed verbatim immediately thereafter, and then reviewed by members of the research team to ensure their validity. the main themes explored covered personnel, documentation, and management. an electronic questionnaire was sent to all laboratory staff between 01 and 30 march 2020 to obtain staff opinions on the challenges with the laboratory’s accreditation acquisition process. the key challenges were selected based on other studies10 and the laboratory’s observed experiences during the iso 15189 implementation phase. these covered challenges related to documentation, laboratory management support, laboratory safety, reagent availability, equipment availability, mentorship, support from doctors, hospital management support, staff attrition, staff qualification, staff strength, laboratory information management system, support from nurses, workload, staff apathy, and communication. participation in the study was voluntary. participants were required to grade, on a scale of 0 (least challenging) to 5 (most challenging), the extent to which 16 key challenges impacted the accreditation acquisition process of the pathology division laboratory. the three most challenging and three least challenging aspects of the accreditation process were identified. the maximum score attributable to each challenge is 520 (total number of respondents [n = 104] multiplied by the maximum score applicable [n = 5]). data analysis data collected were processed and analysed in microsoft excel 2016 (microsoft corporation, redmond, washington, united states) and stata ic/16 (statacorp, college station, texas, united states). summary descriptive statistics were used to describe the characteristics of the data set obtained from survey respondents. the number of responses on each challenge to accreditation was also represented as percentages for each score category. a multivariate regression model was used to compare the scores of key challenges between personnel who joined the pathology division laboratory before accreditation versus after accreditation, adjusting for employee type, education, gender, and awareness of the accreditation status of the pathology division. p-values less than 0.0025 were considered statistically significant. lessons learnt a total of 106 of the 110 personnel working in the laboratory completed the structured questionnaire, representing a 96.4% response rate. there were two non-responses on time of joining the laboratory (before or after accreditation) and five non-responses on job title. more respondents (67/104, 64.4%) were present throughout the laboratory’s accreditation acquisition process compared to respondents (37/104, 35.6%) who joined the laboratory after the accreditation in 2017 (table 1). table 1: characteristics of study respondents (n = 106) at the pathology division laboratory, 37 military hospital, accra, ghana, november 2020. the pathway to iso 15189 accreditation is marked with several challenges, especially in resource-limited environments. this, however, does not make it an improbable cause. notable among the challenges to the accreditation process were laboratory personnel attrition, personnel attitude to change, service interruptions and logistic constraints, documentation, continuous external assessment outcomes, and size and complexity of the laboratory (figure 1). figure 1: scoring of key challenges to accreditation by laboratory staff at the pathology division laboratory, 37 military hospital, accra, ghana, november 2020. documentation, availability of reagents, workload, and staff strength were among the highest-rated challenges. these challenges are consistent across different laboratories.10 challenges such as nurses’ support, doctors’ support and staff qualification were rated among the least challenging. the role of laboratory management in ensuring that the requirements of the international standards are understood by laboratory personnel is important to ensure collective buy-in and conformance. where such inadequacies exist, mentorship would be needed to complement management roles in the application of the relevant standards.8 when controlled for employee type, education, gender, and awareness of the accreditation status of the pathology division, there was no statistically significant difference in responses between respondents who joined the laboratory before or after accreditation (table 2, figure 2). this is a clear indication that best practices implemented in the build-up to any accreditation process do not significantly change the laboratory’s processes, especially in laboratories where qms may be seen as a burden. this ease of transition may be associated with the conscious effort to involve personnel in the accreditation process, thus increasing their level of satisfaction and making them more committed to the process.13 when staff are committed and involved in the accreditation process from the beginning, good commitment is more likely to persist throughout the life cycle of the laboratory. figure 2: comparison of the scores of key challenges to accreditation between personnel who joined the pathology division laboratory, 37 military hospital, accra, ghana, before versus after accreditation, november 2020. table 2: multivariate regression model comparing the scores of key challenges between personnel who joined the pathology division laboratory, 37 military hospital, accra, ghana, before versus after accreditation, november 2020. key informant interviews staff attrition critical among the challenges to the accreditation process was laboratory personnel attrition. ki-1 acknowledged: ‘as a military facility, military personnel are moved every now and then on military and allied duties resulting in significant reduction in staff numbers and consequent increase in workload on the remaining personnel.’ (k1-1, male, laboratory scientist) as a result, persons trained and deemed competent in the quality system were lost and new persons introduced. this can be described as a ‘brain drain’ of laboratory personnel. according to ki-2 (female, laboratory manager), ‘between 2014 and 2017, five out of eight members of the laboratory quality steering committee members had been changed’. this certainly increased the amount of work the few remaining personnel had to do to support the quality system. to address this situation, the laboratory developed and deeply integrated a staff orientation and training programme to consistently update old and new staff with the status quo and requirements of the laboratory qmss. additionally, for every key appointment (e.g. quality manager, safety officer) there were two assigned deputies. although these deputies had technical roles, they also understudied the key officer in preparation for taking over when one was moved. the attrition of personnel may constitute a significant form of brain drain in a laboratory that has built the capacity of its personnel to implement the requirements of the qms. the effect of attrition is felt when the quality system is established around personalities and not as a functional system. establishing a functional system is to ensure that developed policies and procedures are followed as required. however, there is the need to maintain a good level of stability of key personnel such as the quality manager and the quality team players to maximise the human resource capacity. personnel attitude to changing work routines the attitude of personnel to changing work routines was another challenge that was apparent during the application process. according to ki-4: ‘laboratory personnel considered the accreditation process and its requirement as additional work burden which added very little immediate financial benefits to one’s pocket hence the lack of enthusiasm towards the process.’ (k1-4, female, laboratory scientist supervisor) we opine that the general perception among personnel is that the implementation of a quality system towards accreditation is entirely different from the ‘regular’ laboratory work. hence, staff are more likely to go about their routine duties and only implement quality system requirements if they remember to do so. both ki-3 and ki-4 emphasised: ‘the same personnel who are deployed on technical bench duties and processing of samples were the very staff involved in the numerous administrative and documentation tasks. they are simply overwhelmed with tasks and had the right to complain.’ (k1-3 & k1-4, laboratory scientist supervisors) the routine duties of laboratory staff and the quality requirements associated with iso accreditation are more interconnected than mutually exclusive as perceived. the perception of the two being separate stems from the situation where too much emphasis is placed on getting documentation done to the detriment of patient care or vice versa. additionally, lack of motivation coupled with the extra duties increases that perception gap. personnel are originally employed by their managers to run patient samples and churn out results. therefore, any additional duty is expected to come with some extra, usually financial, remuneration. where this fails to occur, as is the case in most public sector laboratories because they do not manage their own budget, enforcing such a change is met with firm resistance or indifference. this situation is however contrary to a study done in lebanon where accreditation was found to be an impetus for better performance.2 it took several one-on-one mentorship and coaching sessions and in-house continuous education on iso 15189 accreditation, the improvement process, the requirements of the stepwise laboratory improvement process towards accreditation checklist, good clinical laboratory practices, and laboratory qms to get a lot more personnel on board with the process. according to ki-2: ‘the concerns of laboratory personnel regardless of how often or how much they complain is good feedback. though we knew that indeed they were overwhelmed, everyone was overwhelmed. however, we initiated a staff of the month award scheme to motivate personnel.’ (ki-2, female, laboratory manager) this ‘staff of the month’ award included a lunch package for the winner and a crate of drinks for the entire department, as well as the display of the winner’s photograph at the front desk. service interruptions and logistic constraints interruptions in the continuity of laboratory service was considered one of the challenges to the accreditation process. ki-1 explained: ‘service interruptions usually occurred as a result of delays by suppliers to deliver reagents and other consumables, as well as bureaucratic procurement processes. these mostly resulted in reagent stock-outs. most of the reagents and consumables required for testing are imported and are very capital intensive. due to the amount involved and the foreign exchange transaction policies of the government of ghana, it became even more difficult to carry out such procurement.’ (k1-1, male, laboratory scientist) ki-1 acknowledged as a solution: ‘to ensure a more regular and continuous supply of reagents and consumables, some level of financial autonomy was given to the laboratory. an accountable imprest was also set aside for the laboratory. this brought to the fore the level of support offered by the hospital management.’ (k1-1, male, laboratory scientist) the level of support and trust provided sufficient drive for the staff and management of the laboratory to improve their interest in the whole process and secure the coveted accreditation. all kis agreed that the absence of a dedicated budget for procurement of laboratory logistics was the main reason for the service interruptions occurring in the laboratory. continuous external assessment outcomes in preparation for accreditation, self-checks and external assessments are needed to ascertain the readiness of the laboratory for the final accreditation assessment. according to ki-1: ‘we were subjected to several external assessments; however, it was as if the outcomes did us ‘more harm than good’. despite the efforts put into preparing for external assessments, the scores were low and staff were demoralised. different assessors almost reported different findings for the same requirement.’ (k1-1, male, laboratory scientist) all kis agreed that assessments (internal and external) are critical as required by the standard but that it is important to focus on managing the quality system rather than on the numbers or scores of an assessment. documentation constant documentation of activities is one of the key requirements of accreditations in general. the iso 15189 standard requires the generation of evidence of the implementation of laboratory policies, processes, and procedures. ki-3 said: ‘it is like you have to document everything you do or do not do, this is hard, this is not something we are or were used to, hence the massive resistance.’ (k1-3, laboratory scientist supervisor) in a time of evidence-based laboratory medicine, it is only imperative to document all activities as proof that they were carried out as required. the key role of conformance to documentation in any quality system cannot be overemphasised. ki-1 commented: ‘it is not possible for any laboratory to implement a quality system when the personnel who are the direct implementers of the system are not committed to the process. no matter how good the policies and processes are, if the people do not conform to it, it is useless.’ (k1-1, male, laboratory scientist) this represented one of the biggest challenges to manage in an environment where documentation and conformance are not part of the culture. group training and consistent one-on-one coaching with staff on the importance and benefits of documentation were conducted. most importantly, results of documentation (e.g. occurrence, quality indicators, temperature monitoring, etc.) were periodically reviewed and communicated to staff to help them appreciate the need for and benefits of keeping quality records. size and complexity of the laboratory ki-5 (laboratory scientist) stated that ‘the medical laboratory, to the ordinary person, may be perceived as a small space within which simple tests are performed’, and further opined that the converse rather is true, stating that ‘the laboratory is a large complex network of several units and departments involved in an interconnected series of test activities’. this was a major challenge. the laboratory has five testing departments, collectively performing over 100 test panels and generating results for over 300 analytes. ‘at a point we were confused, not knowing exactly what was going on because they were just too many’, ki-2 (laboratory manager) said. the initial attempt to get all methods accredited at once was not working. the burden of work was overwhelming due to the size and complexity of the laboratory. therefore, the laboratory adopted a stepwise approach to attaining its much-desired accreditation. this phased approach implied prioritising the methods to be accredited and working at accreditation in smaller chunks. chemical pathology and haematology were therefore chosen for the initial accreditation process, with the subsequent addition of microbiology a year later. no historical precedence all kis acknowledged that the accreditation of a public sector laboratory in ghana was considered highly improbable. this made it even more difficult for laboratory staff to be convinced that it was possible to be accredited as a public sector laboratory. as explained by ki-2: ‘staff always made reference to other bigger hospitals in ghana and in the west african sub-region that if those facilities were not accredited though better placed to be, how was it going to be possible for the military hospital which is resource-stricken be accredited.’ (k1-2, female, laboratory manager) however, it was clear that although there was no historical precedence, with the needed guidance and mentorship, it was possible to meet the requirements for accreditation. according to ki-1 and ki-5: ‘effective mentorship is a pivotal element for any public sector laboratory seeking accreditation. in the absence of a historical precedence, our mentorship program guided the development of policies and process and further assisted in the implementation, conformance and the review process.’ (k1-1 & k1-5, laboratory scientists) in a country where the motivation of the government towards laboratory accreditation is weak, securing accreditation for any public sector laboratory is usually a huge challenge.14 the inadequacies in the national support for accreditation result in the inadequate dedication of resources (human, capital and infrastructural) towards attaining and maintaining accreditation. while a national focus is required, hospital management support, including support from other healthcare workers such as doctors and nurses, cannot be overemphasised.8 management support plays a key role in the accreditation process in resource-limited settings, and the engagement of hospital management with laboratory management accelerates the accreditation acquisition process. perspectives about the challenges with securing accreditation vary between laboratory personnel and management staff. while laboratory personnel identified excessive documentation, weak laboratory management support, and inadequate safety as the main challenges to accreditation, management personnel identified staff attrition, personnel attitudes, and the size and complexity of the laboratory facility as the main challenges. however, both groups of personnel identified excessive documentation and service interruption as key challenges. there is a need for constant feedback across levels of hierarchy to get everyone on the same page in the quest for accreditation. that way, the resources, although limited, can be better apportioned to address common challenges. limitations the study was limited to staff of the pathology division laboratory so the perspectives of hospital management, other hospital staff and clients were not solicited. the ki interviews did not include personnel whose testing activities were not within the scope of accreditation. some bias may therefore be inherent in the responses. recommendations to make laboratory accreditation more relevant and, consequently, surmount the anticipated challenges, there is the need for national strategic direction for laboratory accreditation, hospital and laboratory management support for the accreditation acquisition and maintenance processes, and sufficient technical assistance in the form of training and mentorship. acknowledgements we are grateful to the management and staff of the entire pathology division of the 37 military hospital for their contribution and support in the conduct of this study. special thanks go to dr edward asumanu of the postgraduate college of the 37 military hospital for providing professional guidance. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions s.a. conceived the idea, l.a.-d. started the initial draft and the manuscript and was completed by r.d.f., f.k.m.t., k.a., r.k., m. mcaddy, m. moroti, c.b., j.b., a.y., s.k., e.s., n.-b.k., m.a.a., f.h., and k.a.-b. all authors reviewed the manuscript and provided feedback. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability data sharing is not applicable to this article. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references alemnji ga, zeh c, yao k, fonjungo pn, regional c, office k. strengthening national health laboratories in sub-saharan africa: a decade of remarkable progress. trop med int health. 2014;19(4):450–458. https://doi.org/10.1111/tmi.12269 el-jardali f, hemadeh r, jaafar m, et al. the impact of accreditation of primary healthcare centers: successes, challenges and policy implications as perceived by healthcare providers and directors in lebanon. bmc health serv res. 2014;14:86. https://doi.org/10.1186/1472-6963-14-86 schoen c, osborn r, doty mm, squires d, peugh j, applebaum s. a survey of primary care physicians in eleven countries, 2009: perspectives on care, costs, and experiences. health aff. 2009;28(6):w1171–w1183. https://doi.org/10.1377/hlthaff.28.6.w1171 department of health and ageing. primary health care reform in australia: report to support australia’s first national primary health care strategy. 2009; canberra: department of health and ageing. who. the world health report 2008 – primary healthcare: how wide is the gap between its agenda and implementation in 12 high-income health systems? health policy. 2012;7(3):38–58. https://doi.org/10.12927/hcpol.2013.22778 masanza mm, nqobile n, mukanga d, gitta sn. laboratory capacity building for the international health regulations (ihr[2005]) in resource-poor countries: the experience of the african field epidemiology network (afenet). bmc public health. 2010;10 suppl 1(suppl 1):1–7. https://doi.org/10.1186/1471-2458-10-s1-s8 gershy-damet gm, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. https://doi.org/10.1309/ajcptuuc2v1wjqbm attoh s, kodjoe it, boateng c, et al. lessons learnt of the first public sector iso 15189 accredited laboratory in ghana. postgrad med j ghana. 2020;9(1):1–5. nkrumah b, van der puije b, bekoe v, et al. building local human resources to implement slmta with limited donor funding: the ghana experience. afr j lab med. 2014;3(2):1–7. https://doi.org/10.4102/ajlm.v3i2.214 girma m, desale a, hassen f, sisay a, tsegaye a. survey-defined and interview-elicited challenges that faced ethiopian government hospital laboratories as they applied iso 15189 accreditation standards in resource-constrained settings in 2017. am j clin pathol. 2018;150(4):303–309. world health organization. regional office for the eastern mediterranean. (2004). quality improvement in primary health care: a practical guide. who regional publications, eastern mediterranean series (26). https://apps.who.int/iris/handle/10665/119694 mate ks, rooney al, supachutikul a, gyani g. accreditation as a path to achieving universal quality health coverage. global health. 2014;10(1):1–8. https://doi.org/10.1186/s12992-014-0068-6 paccioni a, sicotte c, champagne f. accreditation: a cultural control strategy. int j health care qual assur. 2008;21(2):146–158. https://doi.org/10.1108/09526860810859012 grochau ih, ten caten cs, de camargo forte mm. motivations, benefits and challenges on iso/iec 17025 accreditation of higher education institution laboratories. accredit qual assur. 2018;23(3):183–188. https://doi.org/10.1007/s00769-018-1317-9 abstract introduction methods results discussion acknowledgements references about the author(s) edward asumanu postgraduate college, 37 military hospital, accra, ghana seth attoh department of anatomical pathology, 37 military hospital, accra, ghana raymond x. servor department of anatomical pathology, 37 military hospital, accra, ghana clement laryea department of medicine, 37 military hospital, accra, ghana mary mcaddy department of anatomical pathology, 37 military hospital, accra, ghana fred hobenu department of anatomical pathology, 37 military hospital, accra, ghana raymond factchu department of anatomical pathology, 37 military hospital, accra, ghana kwesi agyemang-bediako department of anatomical pathology, 37 military hospital, accra, ghana edward o. nyarko public health division, 37 military hospital, accra, ghana godwin k. nyarko department of anatomical pathology, 37 military hospital, accra, ghana marcus k. moroti department of anatomical pathology, 37 military hospital, accra, ghana lawrence edusei department of anatomical pathology, 37 military hospital, accra, ghana citation asumanu e, attoh s, servor rx, et al. causes of death and post-mortem testing for sars-cov-2 in a tertiary hospital during the covid-19 pandemic in ghana. afr j lab med. 2022;11(1), a1766. https://doi.org/10.4102/ajlm.v11i1.1766 original research causes of death and post-mortem testing for sars-cov-2 in a tertiary hospital during the covid-19 pandemic in ghana edward asumanu, seth attoh, raymond x. servor, clement laryea, mary mcaddy, fred hobenu, raymond factchu, kwesi agyemang-bediako, edward o. nyarko, godwin k. nyarko, marcus k. moroti, lawrence edusei received: 19 oct. 2021; accepted: 23 aug. 2022; published: 23 nov. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: causes of death during the coronavirus disease 2019 (covid-19) pandemic ranhttp://crossmark.crossref.org/dialog/?doi=10.4102/ajlm.v11i1.1766=pdf&date_stamp=2022-11-23ge from direct consequences of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection to deaths unrelated to sars-cov-2. another feature of the pandemic is the post-mortem testing for sars-cov-2. understanding these aspects of covid-19 are essential in planning and limiting the impact of sars-cov-2 virus on healthcare systems. objective: this study investigated the underlying causes of death and the presence of sars-cov-2 in bodies received at the 37 military hospital, accra, ghana, during the covid-19 pandemic. methods: the study was conducted from 4–27 may 2020. deceased patients that met the inclusion criteria were prospectively selected during the expanded surveillance period for sars-cov-2 testing, autopsy and determination of underlying and immediate cause of death. results: a total of 161 deceased patients were analysed with 53 autopsies. the overall positive test rate for sars-cov-2 was 14.9% (24/161 patients), with a positive rate of 5.0% (8/161 patients) for nasopharyngeal samples and 30.2% (16/161 patients) for bronchopulmonary samples. the underlying causes of death were not related to sars-cov-2 infection in 85.1% (137/161) of patients, sars-cov-2-associated 12.4% (20/161) and sars-cov-2-induced in 2.5% (4/161). cardiovascular complications formed the most common cause of death in patients with or without sars-cov-2. conclusion: there was a high positive rate of sars-cov-2 in post-mortem cases. however, most deaths were not caused by sars-cov-2 but by cardiovascular complications. the high rate of bronchopulmonary positive results for sars-cov-2 requires that autopsies be done in suspicious cases with negative nasopharyngeal sampling. keywords: post-mortem; severe acute respiratory syndrome coronavirus 2; coronavirus disease 2019; surveillance; pathology. introduction in 2019, the world was struck by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) which caused the coronavirus disease 2019 (covid-19).1,2 the resulting covid-19 pandemic continues to pose challenges to healthcare systems. compared to previous pandemics, the covid-19 pandemic has exhibited a cyclical nature and unique manifestation in different geographical areas.2 various aspects of the disease have been reported from epidemiology to mortality.3,4,5,6,7 studies on mortality do not report on other causes of death within the pandemic,1,6,7 although these are equally important in the fight against the sars-cov-2. autopsies are indispensable in clarifying the cause of death, as well as for quality control and audit in medical practice. it is an established fact that a clinically determined cause of death is not as reliable as an autopsy-determined cause of death.8,9 real-time reverse transcription polymerase chain reaction (rrt-pcr) is the standard laboratory technique for testing for sars-cov-2 and can be used for samples taken at autopsy.10 identifying pathological consequence of sars-cov-2 at autopsy can be a useful addition to rrt-pcr. surveillance has remained a cornerstone in the management of the covid-19 pandemic. different types of surveillance systems have been used during the covid-19 pandemic, such as the hospital-based mortality surveillance and routine surveillance.11 as part of its expanded surveillance protocol, the 37 military hospital in accra, ghana, screened all deceased patients received at the department of anatomical pathology morgue for sars-cov-2 and cause of death in may 2020. the aim of the study was to determine the underlying causes of death and the presence of sars-cov-2 using rrt-pcr for all cases admitted to the department of anatomical pathology during the study period. methods ethical considerations permission was granted by the 37 military hospital’s institutional review board for the study with ethical approval number 37mh-irb ipn/mast/2020. no consent was required of the next of kin for the autopsies. to ensure privacy and confidentiality of the deceased patients, data was de-identified, coded and stored with passwords on electronic media. study design this prospective study was conducted over a three-week period from 4 to 27 may 2020. this was the first institutional surveillance study in ghana on deceased patients at an anatomical pathology department. the study was conducted at the j.m. wadhwani department of anatomical pathology at the 37 military hospital in accra, ghana. the department receives about 4000 deceased individuals of all categories of death from the hospital and its environs annually and conducts approximately 1500 autopsies. the department conducts autopsies for patients who have died from unknown cause for medico-legal or coroner’s inquiry, homicide, suicide, accidents, misadventure and routine hospital cases for autopsy (where the cause of death is not known by the attending physician). deceased patients with a known cause of death, where a medical certificate of cause of death has been provided by the attending physician, are stored by the department prior to burial.12 two categories of deceased patients, in-hospital deaths (ihds) and brought-in-dead cases (bids), formed the study population. in-hospital deaths are deaths that occurred while patients were being managed in the hospital. brought-in-dead deaths are those that occurred outside the hospital or before the individual had been seen by a physician in the hospital. all deceased individuals received 4–27 may 2020 were included in the study, except for individuals less than one year old, unidentified individuals and patients diagnosed as having covid-19 in the hospital. study procedures were conducted in three phases. first, samples were collected for sars-cov-2 testing. second, an autopsy was completed for a subset of patients when requested by the coroner or physician. finally, the underlying and immediate cause of death for all individuals was determined. sample collection for sars-cov-2 nasopharyngeal samples were collected from all deceased patients who met the inclusion criteria by the anatomical pathology team using sterile swab sticks. additional bronchopulmonary samples were collected from patients who underwent autopsy as part of their post-mortem examinations. for the bronchopulmonary specimens, one sample was taken from swabs of secretions in the tertiary bronchi and another from fluid squeezed from the lungs, after sectioning, into an ordinary sterile specimen container. these samples were sent for rrt-pcr test at a national reference laboratory, noguchi memorial institute for medical research in accra, ghana. autopsy a complete autopsy was done by certified department pathologists on individuals only when directed by the coroner or referred by the attending physician as per department policy. all available case notes and records were reviewed by the pathologists. selected organs, including the lungs, heart, kidney, liver, spleen, intestines and brain, were sampled for histopathological examination and some photographs taken. determination of underlying cause of death cause of death was considered to be the disease or injury which initiated the train of events leading directly to death or the circumstances of an accident or violence which produced the fatal injury. at least two certified pathologists determined the underlying cause of death for all individuals studied and reported on the histological findings. the pathologists analysed the clinical notes, as well as the cause of death provided by the attending physician, to determine the underlying cause of death for cases that did not undergo autopsy. in cases that underwent autopsy, the underlying cause of death was determined by evidence after histopathological examination and using the clinical notes, if any. data analysis the data collection form captured the biodata and the clinical cause of death for all patients who did not undergo autopsy. the cause of death for those who underwent autopsy was also recorded. the rrt-pcr test results were received in batches from the noguchi memorial institute for medical research and matched to the individual patient to complete the data collection process. underlying causes of death were categorised into three groups. the first group comprised deaths not related to sars-cov-2 infection. this group included patients who tested negative for sars-cov-2 and who had various underlying causes of death (e.g. massive cerebral haemorrhage due to hypertension); these are referred to as ‘non-sars-cov-2-related deaths’. the second group comprised deaths associated with, but not caused directly by, sars-cov-2 infection. this group included patients with a positive sars-cov-2 test but whose underlying cause of death was not directly attributable to sars-cov-2 (e.g., perforated duodenal ulcer); these are referred to as ‘sars-cov-2-associated deaths’. the last group comprised of deaths directly induced by sars-cov-2 infection. this group included patients with a positive sars-cov-2 test and whose underlying cause of death was attributable to sars-cov-2 (e.g., acute respiratory distress syndrome with or without pneumonia); these are referred to as ‘sars-cov-2-induced deaths’. collected data were managed in microsoft excel (2016; norristown, pennsylvania, united states), which was used to calculate frequencies and proportions. results study population a total of 161 deceased patients, including 96 ihds and 65 bids, were examined over the study period (table 1). the mean age of the study population was 53.3 years (range: 18 months – 90 years) with a male-to-female ratio of 1:1. among the age groups above 40 years, there was an increasing ratio of sars-cov-2 infection compared to non-sars-cov-2 infection. a total of 53 autopsies were performed. all deceased patients had nasopharyngeal samples collected for testing, and those that underwent full autopsy (n = 53) also had a bronchopulmonary sample collected. there was a total of 24 rrt-pcr tests positive for sars-cov-2 from both nasopharyngeal and bronchopulmonary samples. the overall positive test rate from the study population was 14.9% (24/161), with a positive rate of 5.0% (8/161) for nasopharyngeal samples and 30.2% (16/53) bronchopulmonary samples. of patients who had no autopsy, eight had sars-cov-2-associated causes of death and a positive nasopharyngeal test and 100 had a negative nasopharyngeal test and a cause of death that was not associated with sars-cov-2. eight patients with a negative nasopharyngeal test had a bronchopulmonary specimen that tested positive for sars-cov-2. of the 161 patients included in the study, four patients’ (2.5%) deaths were directly caused by sars-cov-2, 20 patients (12.4%) tested positive for sars-cov-2 but had another cause of death; for the majority of patients, the cause of death was not related to sars-cov-2 infection (n = 137, 85.1%). table 1: characteristics of deceased individuals examined for sars-cov-2 infection, 37 military hospital, accra, ghana, 4–27 may 2020. sars-cov-2-induced deaths of the four sars-cov-2-induced deaths, three were ihds and one was bid (table 2). all cases had severe pulmonary oedema and diffuse alveolar damage (dad) with prominent hyaline membrane formation (figure 1). there was severe congestion with intra-alveolar deposition of fibrinous tissue in case 1 (figure 1a). case 2 also showed large pneumocytes with enlarged nuclei and granular amphophilic cytoplasm and microthrombi in the smaller pulmonary arteries (figures 1a and figure 1c) as well as edema, congestion, proliferation of fibroblasts and lymphocytic infiltrates in the interstitium (figure 1d). case 3 showed very prominent dad and hyaline membrane formation, interstitial fibrosis, infiltration of the pulmonary interstitium and alveolar spaces by macrophages and scattered multinucleated giant cells. microthrombi were also noted in smaller pulmonary arteries (figure 1c). case 4 revealed thrombi in branches of the coronary and renal arteries (figure 1e and figure 1f). figure 1: haematoxylin and eosin staining of various organs in sars-cov-2-induced deaths at the 37 military hospital, accra, ghana, may 2020. (a) severe congestion with intra-alveolar deposition of fibrinous tissue (black arrow) ×400. (b) type 2 pneumocyte hyperplasia (black arrows), diffuse alveolar damage (dad) with early hyaline membrane (hm) formation (green arrows) ×400. (c) dad with prominent hm formation (yellow arrow), interstitial fibrosis (green arrow) and a thrombus in a small pulmonary artery (black arrow) ×100. (d) dad with prominent hm (red arrow), oedema (black arrows), congestion (green arrow), interstitial fibrosis and lymphocytic infiltrates (yellow arrow) ×100. (e) thrombus formation (black arrow) in a branch of a coronary artery (green arrow) in the heart muscle (red arrow) ×400. (f) micro-thrombus (black arrow) in a renal arteriole ×100. table 2: summary of sars-cov-2 induced deaths during post-mortem at the 37 military hospital, accra, ghana, may 2020. sars-cov-2-associated deaths of the 20 patients with a positive sars-cov-2 test and an unrelated cause of death, 13 were ihd and seven were bid (table 3). the mean age was 54.7 with a range of 24–90 years. the male:female ratio was 1:1. cardiovascular disorders constituted almost half of the underlying causes of death in this category (n = 9). hypertension was the most common cardiovascular disorder among both ihds and bids. other causes of death included tumours (n = 1), accidents and trauma (n = 4), infections (n = 1), and others (n = 5). table 3: summary of sars-cov-2 associated deaths during post-mortem at the 37 military hospital, accra, ghana, may 2020. non-sars-cov-2-related deaths of the 137 non-sars-cov-2-related deaths, 80 were ihd and 57 were bid. the age range was 2–88 years with a male: female ratio of 1:1 for patients who had an autopsy, the clinical diagnoses were confirmed, except for one case of primary liver cell carcinoma, which had been diagnosed clinically as cirrhosis of the liver. broad categories of causes of death identified were: cardiovascular disorders (n = 49), tumours (n = 23), accident and trauma (n = 13), diabetes mellitus-related complications (n = 11), infections (n = 18), liver disorders (n = 6), renal disorders (n = 1), and others (n = 16). hypertension and its related complications formed the most common cause of death in patients without sars-cov-2 (table 4). two patients who tested negative for sars-cov-2 on rrt-pcr for both the nasopharyngeal and bronchoalveolar samples had lung tissue histopathology consistent with acute respiratory distress syndrome or dad. there were five ihd and two bid with ‘miscellaneous’ causes of death. the bid deaths were from viral hepatitis (n = 1) and severe asthma (n = 1), whereas the ihd deaths were from intravascular haemolysis (n = 1), viral meningitis (n = 1) and malaria (n = 3). table 4: summary of non-sars-cov-2 related deaths at the 37 military hospital, accra, ghana, may 2020. discussion the study identified three categories of deaths that occurred during the pandemic. there were a significant number of undiagnosed sars-cov-2 infections among deceased individuals received by the anatomical pathology department of the 37 military hospital with unique histopathological features of sars-cov-2 induced deaths. the potential for community spread of the virus from handling of unsuspected sars-cov-2 deceased individuals without personal protective equipment, as well as the sars-cov-2 cases found during autopsy, are a missing link in efforts to control the pandemic. there are other documented missing links affecting overall sars-cov-2 control such as non-uniform data collection, vaccine hesitancy and non-reliable energy infrastructure in low-resource environments.13,14,15 in an italian multicentre study conducted in 2020, the presence of sars-cov-2 in bronchoalveolar samples was 32.7% of suspected cases with 76% having a double negative nasopharyngeal test.16 our study also found sars-cov-2 in the bronchopulmonary specimens of cases with negative nasopharyngeal tests and in cases who died from other conditions. these findings need to be factored into the current health policy on sars-cov-2. the underlying cause of death is critical in accurate reporting and documentation for better epidemic surveillance and effective planning of control measures.17 this is the first reported post-mortem study data for ghana that has categorised underlying causes of death into three groups: non-sars-cov-2 related (85.1%), sars-cov-2 associated (12.4%) and sars-cov-2 induced (2.5%). the case fatality rate in ghana for sars-cov-2 infection was reported to be an average of 0.5% in july 2020.18 this did not include deaths due to sars-cov-2 as identified by post-mortem examinations. the presence of post-mortem-diagnosed cases of sars-cov-2-induced deaths in this study implies that the overall case count is higher than reported for ghana. the need to include cases identified by pathology departments during post-mortem examinations in defining all case mortality has been emphasised.19 this highlights the importance of post-mortem examination in understanding the natural history of diseases and defining a true mortality rate. accurate reporting is a requirement in the documentation of cause of death. in its guidelines for certification and classification of covid-19 as a cause of death, the who requires a distinction between underlying cause of death and immediate cause of death in the completion of part i of the international form of the medical certificate of cause of death.20 this was to encourage uniformity of data collection to guide health policy, but differences in classification are emerging.20,21 the evolution of the pandemic and the understanding of the disease requires a simple categorisation of underlying of causes of death. the categorisation adopted in this study is useful for determining accurate case counts during clinical audits of deaths. identifying subgroups of covid-19 case mortality is useful in managing the pandemic.22 of particular interest were the 20 sars-cov-2-associated deaths in our study. these cases were categorised as having died from their underlying medical conditions, rather than from sars-cov-2. the prioritisation of care for sars-cov-2 infections by the health system, rather than the underlying condition, might have contributed to their mortality. most deaths during the pandemic have been reported as being related to underlying conditions.8,23 this raises the question as to whether sars-cov-2 has the potential to delay care among patients with underlying medical conditions. the presence of sars-cov-2 among the bids patients in our study suggests a high community transmission of the disease. such cases may be asymptomatic and likely to result in an increased disease burden in the absence of strict protocols. interestingly, our study found a high proportion (85.1%) of deaths not attributable to sars-cov-2. the impact of the pandemic on health systems and the lack of provision of optimum care are attributable causes to the phenomenon of high proportions of non-sars-cov-2-related deaths.24 among the bid cases in our study not attributable to sars-cov-2, hypertension, infection, liver cirrhosis, diabetes mellitus and pulmonary embolism-related complications were the leading causes of death. causes of death not attributable to sars-cov-2 for cases from the hospitals (in descending order) included hypertension, tumours, accident and trauma, diabetes mellitus and infections. in ghana, the prevalence of major non-communicable diseases, especially hypertension, and their risk factors has increased and contributes significantly to the disease burden.25 hypertension and its related complications were the leading causes of death in both groups of patients. non-communicable metabolic diseases, tumours and trauma-related deaths rank high in the study. this emphasises the need for aggressive clinical care for non-communicable diseases even in the face of a positive sars-cov-2 test. our study found an overall sars-cov-2 case positivity rate of 14.90% among deceased patients received by the anatomic pathology department; these are not included in ghana’s national data on sars-cov-2. the study found a mean age of 55 years for male patients and 53 years female patients with an equal gender ratio. this is a different demographic pattern from the reported increased susceptibility to covid-19 among the elderly, those with severe comorbidities and among male patients.26 while our study found a high proportion of comorbidities among the elderly, the deaths were not directly from sars-cov-2, but from the associated cormorbidities.23 it can be extrapolated that, in the context of an underlying medical condition, the primary disease possess more risk than the sars-cov-2 infection. severe acute respiratory syndrome corona virus 2 has been known to survive longer in cold temperatures and on fomites, with guidelines emerging for the handling of an autopsy in suspected cases.27,28 in our study, the pathology department discovered 24 sars-cov-2-infected deceased patients, of which 20 were previously undiagnosed. the absence of appropriate personal protective equipment and standard protocols predisposed staff to a high risk of infection. it is recommended that there should be a national protocol for the safe performance of autopsies during the pandemic. the site of sample collection has a direct correlation with the sensitivity of rrt-pcr tests, and bronchopulmonary samples give the highest viral yield.29,30,31 in our study, bronchopulmonary specimens gave a higher positivity rate than nasopharyngeal specimens, implying that our bronchopulmonary specimens also had a higher viral load. the higher level of virus in bronchial secretions should be a recognised risk for medical staff during oropharyngeal endoscopic procedures and anaesthesia. the necessary awareness and preventive practices to avoid contracting the virus should be in place. the analysis of the histology of specimens from covid-19 cases provides useful information about the disease. our study found inflammatory features in the lungs and thrombus formation in the arterial systems of major organs. this combination of pulmonary congestion with peri-vascular lymphocytic infiltrates, interstitial/intra-alveolar fibrosis, type 2 pneumocyte hyperplasia, dad with hyaline membrane formation, as well as arterial thrombosis, are likely to result in the rapid and severe hypoxemia noticed among covid-19 patients. an ensuing cellular response in the presence of an underlying medical disease could contribute to the high mortality in these patients. a higher proportion of such patients in our study had cardiovascular disease. the role of the thrombus formation and subsequent hypoxia on vascular endothelium need to be investigated. in our study, there were two cases with the clinical and histopathological features of acute respiratory distress syndrome/dad that had negative sars-cov-2 rrt-pcr test results. there were no definitive diagnoses at the time of autopsy. even though both patients had comorbid conditions, evidence at post-mortem did not attribute their deaths to the comorbid conditions. a recent case series report ascribed such situations to a ‘probable’ case of covid-19 as the cause of death.30 such cases, who test negative for sars-cov-2 but have clinical features suggestive of covid-19, should be considered as a subgroup of covid-19 to help in case management and prevention protocols, considering the high mortality associated with the disease. the negative pcr results of these two cases are most likely to be false-negative results. such false-negative errors may be the result of pre-analytical issues,32 such as poorly collected samples, mislabelling, improper storage or transport, or interference from medications. that notwithstanding, there are other causes of acute respiratory distress syndrome/dad that should be considered, and an autopsy should therefore be encouraged to distinguish between sars-cov-2-induced deaths and sars-cov-2-associated deaths. the study showed that a significant number of clinically diagnosed cases of covid-19 may be false. deaths could have been wrongly labelled as due to covid-19, because of a positive sars-cov-2 test by the nasopharyngeal route and presence of respiratory difficulty. such patients would have been denied dignified burials by their relatives, instead of the practice of mass burial as mandated by national policy at the beginning of the pandemic. these findings are a useful addition to ghana’s national data to determine which deceased patients can be safely released to families and which require supervised burials. the importance of routine screening in high-risk clinical practice among asymptomatic patients and guidelines to protect staff must be emphasised. limitations it would have been ideal to conduct autopsies for all 161 patients in this study, in order to determine the pathological changes present, presence of sars-cov-2 in bronchopulmonary specimens, as well as the autopsy cause of death. unfortunately, this was not possible, because in some cases a medical certificate of cause of death had been issued by the attending physician so no autopsy was requested. this study was also limited to one institution and may not reflect the nationwide pattern. conclusion there was a high positive rate of sars-cov-2 infection among the patients in this study who underwent post-mortem examination. however, the majority of those deaths were not caused by sars-cov-2 but by cardiovascular complications. thus, there should be aggressive clinical care for non-communicable diseases. the high sars-cov-2 infection rate among the deceased may reflect a high rate in the population. the high rate of bronchopulmonary positive results for sars-cov-2 requires that autopsies be conducted in suspicious cases with negative nasopharyngeal sample test results. false-negative rrt-pcr test results for nasopharyngeal samples were common enough that there is a need to intensify preventive protocols. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions e.a., s.a., r.x.s., c.l., m.m. and f.h. contributed toward the conceptualisation of the study. e.a. and s.a. did analyses and wrote the article. r.x.s. and c.l. assisted with the conceptualisation and investigation of the study. m.m. assisted with conceptualisation and data curation and f.h. assisted with conceptualisation, investigation, and resources. r.f. and l.e. assisted with analysis. r.f., k.a.-b., e.o.n., m.k.m. and l.e. assisted with resources. k.a.-b. and m.k.m. contributed toward investigations. m.k.m. assisted with data curation, g.k.n. data validation and l.e. data analyses. sources of support the research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability data are available on request from the corresponding author, e.a. disclaimer the views expressed are those of the authors and do not reflect that of the institution. references rahimi pordanjani s, hasanpour a, askarpour h, et al. aspects of epidemiology, pathology, virology, immunology, transmission, prevention, prognosis, diagnosis, and treatment of covid-19 pandemic: a narrative review. int j prev med. 2021 may 15;12:38. https://doi.org/10.4103/ijpvm.ijpvm_469_20 sohrabi c, alsafi z, o’neill n, et al. world health organization declares global emergency: a review of the 2019 novel coronavirus (covid-19). int j surg. 2020 may;76:71–76. 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future directions in research, practice and policy. ghana med j. 2012;46(2 suppl):1–3. jin jm, bai p, he w, et al. gender differences in patients with covid-19: focus on severity and mortality. front public health. 2020 apr 29;8:152. https://doi.org/10.3389/fpubh.2020.00152 xue y, lai l, liu c, niu y, zhao j. perspectives on the death investigation during the covid-19 pandemic. forensic sci int synerg. 2020 apr 9;2:126–128. https://doi.org/10.1016/j.fsisyn.2020.04.001 santurro a, scopetti m, d’errico s, fineschi v. a technical report from the italian sars-cov-2 outbreak. postmortem sampling and autopsy investigation in cases of suspected or probable covid-19. forensic sci med pathol. 2020 sep;16(3):471–476. https://doi.org/10.1007/s12024-020-00258-9 wang x, tan l, wang x, et al. comparison of nasopharyngeal and oropharyngeal swabs for sars-cov-2 detection in 353 patients received tests with both specimens simultaneously. int j infect dis. 2020 may;94:107–109. https://doi.org/10.1016/j.ijid.2020.04.023 attoh sa, hobenu f, edusei l, et al. postmortem diagnosis of covid-19: antemortem challenges of three cases at the 37 military hospital, accra, ghana. afr j lab med. 2020;9(1):1290. https://doi.org/10.4102/ajlm.v9i1.1290 edler c, schröder as, aepfelbacher m, et al. dying with sars-cov-2 infection – an autopsy study of the first consecutive 80 cases in hamburg, germany. int j legal med. 2020 jul;134(4):1275–1284. https://doi.org/10.1007/s00414-020-02317-w payne d, newton d, evans p, et al. preanalytical issues affecting the diagnosis of covid-19. j clin pathol. 2021;74:207–208. https://doi.org/10.1136/jclinpath-2020-206751 http://www.ajlmonline.org open access page 1 of 1 reviewer acknowledgement acknowledgement to reviewers in an effort to facilitate the selection of appropriate peer reviewers for the african journal of laboratory medicine, we ask that you take a moment to update your electronic portfolio on https://ajlmonline.org for our 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https://ajlmonline.org https://ajlmonline.org https://ajlmonline.org/index.php/ajlm/user https://ajlmonline.org/index.php/ajlm/user mailto:publishing@aosis.co.za article information authors: phidelis m. maruti1 ekesa a. mulianga1 lorna n. wambani1 melda n. wafula1 fidelis a. mambo2 shadrack m. mutisya3 eric n. wakaria4 erick m. mbati5 angela a. amayo4 jonathan m. majani1 bryan nyary6 kilian a. songwe3 affiliation: 1ministry of health-kenya, kenya2department of health sciences, masinde muliro university, kenya 3a global healthcare public foundation, kenya 4management sciences for health-kenya, kenya 5aids, population and health integrated assistance plus (aphia plus) western, kenya 6international healthcare and development, nigeria correspondence to: phidelis maruti postal address: bungoma district hospital, 14-50200 bungoma, kenya dates: received: 22 may 2014 accepted: 02 july 2014 published: 16 sept. 2014 republished: 07 nov. 2014 how to cite this article: maruti pm, mulianga ea, wambani ln, et al. creating a sustainable culture of quality through the slmta programme in a district hospital laboratory in kenya. afr j lab med. 2014;3(2), art. #201, x pages. http://dx.doi.org/10.4102/ ajlm.v3i2.201 note: article republished with updated references relating the special issue. copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. creating a sustainable culture of quality through the slmta programme in a district hospital laboratory in kenya in this original research... open access • abstract • introduction • research method and design • results and discussion    • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: bungoma district hospital laboratory (bdhl), which supports a 200-bed referral facility, began its strengthening laboratory management toward accreditation (slmta) journey in 2011 together with eight other laboratories in the second round of slmta rollout in kenya.objectives: to describe how the slmta programme and enhanced quality interventions changed the culture and management style at bdhl and instilled a quality system designed to sustain progress for years to come. methods: slmta implementation followed the standard three-workshop series, mentorship site visits and audits. in order to build sustainability of progress, bdhl integrated quality improvement processes into its daily operations. the lab undertook a process of changing its internal culture to align all hospital stakeholders – including upper management, clinicians, laboratory staff and maintenance staff – to the mission of sustainable quality practices at bdhl. results: after 16 months in the slmta programme, bdhl improved from zero stars (38%) to four stars (89%). over a period of two to three years, external quality assessment results improved from 47% to 87%; staff punctuality increased from 49% to 82%; clinician complaints decreased from 83% to 16; rejection rates decreased from 12% to 3%; and annual equipment repairs decreased from 40 to 15. twelve months later the laboratory scored three stars (81%) in an external surveillance audit conducted by kenya accreditation service (kenas). conclusion: management buy-in, staff participation, use of progress-monitoring tools and feedback systems, as well as incorporation of improvement processes into routine daily activities, were vital in developing and sustaining a culture of quality improvement. introduction top ↑ laboratory systems are one of the core capacities that countries must develop in order to comply with world health organization (who) international health regulations, since they play a major role in the key processes of detection, assessment, response, notification and monitoring of events.1 as laboratory results influence up to 70% of medical diagnoses,2 reliable laboratory services are essential for the provision of safe and effective treatment to patients. the quality of laboratory services is a major factor that directly affects the quality of healthcare in a country.2the strengthening laboratory management toward accreditation (slmta) programme promotes rapid, measurable improvement in laboratories of developing countries. slmta is implemented through multiple workshops with intervening site visits to support improvement projects. kenya began the slmta implementation process with 12 laboratories in april 2010. bungoma district hospital laboratory (bdhl) was enrolled in the second slmta round in february 2011, along with eight other laboratories. after the first three months of slmta implementation, bdhl management noted, from both the internal audit report and general observations, that little progress had been made. as a result, radical changes were phased in to encourage all laboratory staff to participate in improvement activities, adopt more disciplined and stringent work duties and schedules, engage in laboratory planning and include hospital management and other stakeholders in the process. this article describes how the slmta programme and enhanced interventions changed the culture and management style at bdhl, instilling a system designed to sustain progress for years to come. research method and design top ↑ bungoma district hospital, a primary care facility with very limited resources, started operating in 1952 as a chief’s native health centre. located in bungoma town, it now serves as a referral hospital for the north-western region of kenya. with 200 in-patient beds, it offers both out-patient and in-patient services and provides laboratory services for haematology, serology, clinical chemistry, immunology, microbiology, parasitology and blood banking.consistent with the slmta protocol,3 a baseline audit was conducted in february 2011, followed by three workshops, two one-week mentorship site visits after each workshop, a mid-term audit and an exit audit in march 2012. to determine the impact of changes made at bdhl and the sustainability of the new systems, an external surveillance audit was conducted by the kenya accreditation service (kenas) in february 2013, 12 months after slmta concluded. the non-profit charity a global healthcare public foundation played an important role in conducting the three workshops, on-site mentorship and conference call follow up. in addition, the foundation provided funding for laboratory facility and equipment upgrades. efforts were made to engage all hospital management in the process, as well as other stakeholders, including the hospital maintenance unit, the procurement and/or supplies unit and clinicians. slmta was integrated by laboratory staff into routine work processes and a succession plan was developed for laboratory management to ensure the sustainability of the quality improvements. this included appointing a deputy for each key function, performing on-the-job mentorship of staff on slmta, as well as regular internal reviews of progress. further engaging stakeholders and ensuring continued progress, the laboratory staff conducted weekly customer surveys (for patients/clients), which informed improvement projects. slmta uses the who’s regional office for africa’s (who afro) stepwise laboratory quality improvement process towards accreditation (slipta) framework to both guide improvement activities and evaluate programme effectiveness.4unlike traditional pass/fail accreditation schemes, slipta uses a zeroto five-star scale to recognise the evolving fulfilment of international organization for standardization (iso) 15189 requirements. laboratories that fail to achieve at least 55% on their audit score receive a zero-star rating; 55% − 64% yields one star, 65% − 74% yields two stars, 75% − 84% yields three stars, 85% − 94% yields four stars and laboratories that achieve 95% or more receive five stars.4 this stepwise approach acknowledges laboratories where they stand, supports them with a series of evaluations to demonstrate improvement and both recognises and rewards their progress. the slipta process is not intended to replace established accreditation schemes, but rather to provide an interim pathway to the realisation of international laboratory standards.5 several indicators were measured to assess the impact of slmta implementation. firstly, results from routine external quality assessment (eqa) panels from human quality assessment services (huqas), conducted three times per year for 22 analytes, were compared; the average annual percentage of correct responses from 2010 to 2013 are presented. secondly, staff punctuality in 2011–2013 was assessed based on data from an employee time clock, defined as the average overall percentage of person-days that staff arrived on time for their shift. thirdly, clinician and customer satisfaction were assessed by means of a ‘how do you rate us’ form that was made available to all patients in 2012–2013 and clinicians in 2011–2013; the proportion of forms submitted with complaints was calculated. fourthly, annual average sample rejection rates for all laboratory tests were calculated for 2011–2013; and finally, equipment repairs and the proportion carried out by external engineers versus internal staff from the hospital’s biomedical engineering department were assessed for 2011–2013. results and discussion top ↑ bdhl’s baseline audit score was 38% (0 stars). after slmta implementation, the laboratory scored 89% (4 stars) at the exit audit. the surveillance audit carried out 12 months afterward yielded a score of 81% (3 stars) (figure 1). figure 1: results for baseline, mid-term, exit and surveillance audits, bungoma district hospital laboratory, kenya. without hospital management support, sustainable changes are difficult to achieve. poor eqa data for chemistry and haematology, as well as an increased mortality rate in medical wards from 3% in 2009 to 9% in 2011, were presented to management to demonstrate that laboratory failures could be contributing to deaths, especially amongst hiv patients for whom treatment depends heavily on chemistry results. hospital management approved the purchase of a new fully-automated analyser to replace the old semi-automated analyser and control materials, and convinced partners to donate air conditioners for the laboratory. as a result, erratic temperatures no longer interfered with the quality of results or turnaround time and overall eqa results improved from 47% in 2010 to 87% in 2013 – above the set target of 80% (table 1). because staff buy-in is also crucial, further efforts were made to encourage participation throughout the hospital. an annual award scheme for the entire hospital was established in order to motivate staff to improve patient care. in addition, a ‘wall of fame’ and a ‘wall of shame’ were instituted in order to further inculcate a culture of friendly competition amongst staff and to ensure conformity to the set standards. these activities led to the prompt release of test results, thereby improving turnaround time and the efficiency and quality of patient care throughout the hospital. however, the laboratory experienced substantial staff turn-over in 2013, with four laboratory personnel being transferred, including the medical superintendent and laboratory manager. this left the laboratory with nine staff members, thus causing acute staff shortages and gaps in laboratory management, with the result being that customer complaints increased from 3% in 2012 to 22% in 2013. a new policy for clocking-in and -out for laboratory staff and the introduction of a leave request form resulted in an increase in staff punctuality from 49% in 2011 to 82% in 2013 (table 2), as well as an increase in staff availability to perform assigned tasks. this temporarily addressed staff shortages. quarterly meetings for one-on-one mentorship with each laboratory staff member were introduced. during these meetings, laboratory management reminded staff members of strategic objectives, thanked them for their hard work, provided feedback on their performance and suggested areas of improvement. in response to positive feedback and this collaborative approach, laboratory staff members reported feeling appreciated, more engaged and willing to be part of a team to improve healthcare quality in the hospital. the laboratory also began holding quarterly meetings with clinicians to discuss sample rejection rates, clinicians’ perception of the laboratory and suggestions for improvement. the proportion of clinicians who reported complaints on the feedback form decreased from 83% in 2011 to 16% in 2013, whilst the total number of form submissions increased from 76 to 252. sample rejection rates declined from 12% in 2011 to 3% in 2013 and clinicians reported in meetings that their confidence in laboratory results had improved. the laboratory also met regularly with stakeholders from the maintenance and procurement departments in order to advocate for prompt routine preventive equipment maintenance. consequently, the number of equipment repairs decreased from 40 in 2011 to 15 in 2013 and the proportion of repairs conducted by an external engineer versus the hospital biomedical engineering department decreased from 80% to 20% (table 1). table 1: summary of quality indicators before and after slmta implementation. in order to sustain the gains achieved, slmta was integrated into daily routines, building a foundation for continuous improvement. discussions of improvement projects are now included in regular laboratory staff meetings; the laboratory conducts weekly hands-on continuous medical education sessions; and all staff members are now involved in budget and planning discussions. these changes were designed in order to improve the laboratory staff’s customs, beliefs and attitudes in the workplace, leading to widespread and lasting staff support of laboratory quality improvement activities. sustainability is further enhanced by quarterly internal audits, conducted by the laboratory quality officer. to monitor processes, staff members identify causes of problems and suggest possible solutions. a root cause analysis is conducted and corrective action is identified, following a two-step procedure. step 1 involves the development of a cause-and-effect diagram (figure 2) in order to categorise probable causes of non-conformity under ‘the 6 ms’ of machinery, methods, measurement, manpower, materials and milieu (environment).6 using objective evidence, the quality officer then examines each probable cause and, based on the evaluation, the staff then works by process of elimination to identify those items which were most likely associated with the non-conformity. figure 2: cause-and-effect diagram. step 2 is the root cause investigation. using the problems identified in step 1, an investigation into their root cause is performed. for example, the quality officer may recognise that laboratory personnel do not have proper competency records. to find the source of this problem, the officer may ask ‘why?’ and then receive a variety of answers, including: • staff are not aware of the need for competency assessment. • staff were not trained on the procedure for competency assessment. • the quality officer thought the procedure was covered during training. • no records of training are kept. • the training procedure does not mention the need to keep records. after this questioning, the root cause of the problem may become clear: for example, perhaps the training procedure does not fully address the need for record keeping. the quality officer may then recommend that, in order to improve the system, the training procedure must be revised. this process of identifying problems and selecting improvement activities allows for a clear understanding of what is hindering efficient and reliable work in the laboratory and provides appropriate solutions for improvement (table 2). table 2: summary of improvement activities. conclusion bdhl successfully used slmta to progress from zero to four stars within a 16-month period and to maintain a three-star rating for 12 months thereafter. this quality improvement required substantial effort and a collaborative approach. fundamental steps were necessary in order to create and maintain a culture that supports continuous quality improvement. firstly, universal rules were established and enforced, such as adopting written protocols and practices that prescribe clear policies, procedures, values and behaviours. secondly, the principles and techniques of quality improvement and their associated behaviours were taught so that staff members could learn both the concepts and how to apply them. finally, it was critical to reinforce these principles and behaviours on a continual basis. bdhl employees were recognised and rewarded when they demonstrated adherence and consequences were made clear for noncompliance. leaders and managers did not allow laboratory staff to become complacent, simply meeting basic or minimum requirements. everyone was pressed continually for professional excellence, growth and improvement.for quality management systems to be implemented effectively and sustained, the hospital’s management and staff must be involved and willing to participate. bdhl can attest that sustainable improvement is achieved by engaging all hospital stakeholders, leading to increased confidence in the laboratory on the part of clinicians, nurses and patients. long-term sustainability will rely on continued vigilance, training of new staff and participation of all stakeholders. acknowledgements top ↑ the authors wish to acknowledge the commitment of the bungoma district hospital management for nursing the implementation to reality, including the entire bdhl staff, procurement unit staff and hospital maintenance unit staff who all worked tirelessly to achieve the implementation. gratitude goes to a global public health foundation for its mentorship and support; to the us centers for disease control and prevention (cdc) kenya office for its support and coordination; to the us president’s emergency plan for aids relief (pepfar) for providing funds to implement slmta; and to global scientific solutions for health for their manuscript support. finally, we wish to acknowledge talkmore maruta for his training and encouragement. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. authors’ contributions p.m.m. (ministry of health-kenya) was responsible for experimental and project design. e.a.m., l.n.w. and m.n.w. (all ministry of health-kenya) performed most of the experiments. f.a.m. (masinde muliro university), s.m.m. (a global healthcare public foundation), e.m.m. (aphia plus), a.a.a. (management sciences for health-kenya), j.m.m. (ministry of health-kenya) and b.n. (international healthcare and development) all made conceptual contributions. e.n.w. (management sciences for health-kenya) was responsible for the calculations; and k.a.s. (a global healthcare public foundation) was the project leader. references top ↑ 1.masanza mm, ngobile n, mukanga d, et al. laboratory capacity building for the international health regulations (ihr[2005]) in resource-poor countries: the experience of the african epidemiology network (afenet). bmc public health. 2010;10(suppl 1):s8. http://dx.doi.org/10.1186/1471-2458-10-s1-s82.guzel o, guner ei. iso 15189 accreditation: requirements for quality and competence of medical laboratories, experience of a laboratory i. clin biochem. 2009;42(4–5):274–278. http://dx.doi.org/10.1016/j.clinbiochem.2008.09.011 3.yao k, maruta t, luman e, et al. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. in press. 4.who afro. who guide for the stepwise laboratory improvement process towards accreditation in the african region (with checklist) [document on the internet]. c2012 [cited 2014 jul 20]. available from: http://www.afro.who.int/en/clusters-a-programmes/hss/blood-safety-laboratories-a-health-technology/blt-highlights/ 3859-who-guide-for-the-stepwise-laboratory-improvement-process-towards-accreditation-in-the-african-region-with-checklist.html 5.gershy-damet gm, rotz p, cross d, et al. world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. http://dx.doi.org/10.1309/ajcptuuc2v1wjqbm 6.wikipedia. ishikawa diagram [page on the internet]. n.d. [cited 20 jul 2014]. available from: http/en.wikipedia.org/wiki/ishikawa_diagram abstract introduction methods coordination and alignment of activities awareness creation surveillance infection prevention and control antibiotic stewardship innovation and research perspectives on the implementation of ghana’s national action plan conclusions acknowledgements references about the author(s) japheth a. opintan department of medical microbiology, university of ghana, accra, ghana citation opintan ja. leveraging donor support to develop a national antimicrobial resistance policy and action plan: ghana’s success story. afr j lab med. 2018;7(2), a825. https://doi.org/10.4102/ajlm.v7i2.825 lessons from the field leveraging donor support to develop a national antimicrobial resistance policy and action plan: ghana’s success story japheth a. opintan received: 01 may 2018; accepted: 22 aug. 2018; published: 06 dec. 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: to mitigate the increasing trend of antimicrobial drug resistance (amr), the global action plan (gap) on amr was adopted at the 68th world health assembly in may 2015. subsequently, member countries were encouraged to mirror the five key strategic objectives of gap to develop their respective national action plans (naps) by 2017. country-specific data on amr is, however, critical for a comprehensive nap that will inform policy and also anchor all the objectives of gap. systematic reviews have been suggested by some authors to generate relevant data to inform nap development. objectives: this article highlights ghana’s success story in the development of its amr policy documents and how it could further be implemented through donor support. methods: literature and desk review of the activities of ghana’s national platform on antimicrobial resistance leading to the development of the nap and amr policy was done. results: ghana launched its nap together with the accompanying policy document in april 2018. country-specific data, which guided these documents, were obtained by leveraging donor support activities through the national platform on antimicrobial resistance. conclusion: ghana’s success story on the development of amr policy documents is pivoted on a strong political will and the leveraging of donor support for specific activities. introduction antimicrobials are important medicines that revolutionised modern medicine and prevented needless morbidity and mortality associated with infectious diseases. antimicrobials also paved the way for complex surgical procedures like hipbone replacement to be more possible. inappropriate use and substandard antimicrobials in humans, animals and aquaculture have, however, contributed to the current global threat of antimicrobial resistance (amr). to address amr at the global level, different stakeholders were tasked to put a global action plan (gap) together. in may 2015, the world health assembly adopted gap’s five key strategic plans to be mirrored by member countries. the tripartite body, consisting of world health organization (who), food and agriculture organization (fao) and international organization for animal health, commissioned and continues to assist several countries to generate the necessary data for individual country national action plans (naps). funding from these agencies has led to the development of several assessment toolkits that target laboratory needs, capacities and data generation on amr.1 some of these toolkits can be applied at the local and country levels. the african union, through the africa centres for disease control and prevention (africa cdc), recently met in addis ababa to discuss a framework for amr containment within the sub-region. with its audience, authority and access to varied resources, the african union can better leverage resources for amr containment in the sub-region. for example, the african union can effectively complement strategies to control and contain antimicrobial use (amu) and amr by formulating the necessary regulatory and legislative frameworks for adaptation by member states. inevitably, individual country naps, which outline surveillance systems to feed data into global amu or amr databases, must start at local and regional levels. at each of these levels, both human and material resources must be adequately leveraged through partnerships. ghana has been successful at leveraging both internal and donor partners’ resources to develop its nap and its accompanying policy document.2,3 on 11 april 2018, his excellency, president nana addo dankwa akufo-addo, launched both documents which received massive media coverage. this article highlights ghana’s success story on the development of both documents and also provides some impetus on how further resources can be leveraged for implementation, using the one health concept. methods this article is based on literature and desk review of the activities of ghana’s national platform on antimicrobial resistance (npar). the ministry of health leads the npar which operates on a one health framework. npar works in consultation with public and private agencies, academic and research institutions, professional bodies and civil society organisations. smaller technical working groups are occasionally tasked to perform various activities at specific time periods and report back to the larger platform. financial and technical support for npar were provided by the government of ghana, the swedish international development agency, united kingdom department for international development, the antibiotic drug use, monitoring and evaluation of resistance (admer) project, who and fao. coordination and alignment of activities leadership is key to the coordination and alignment of all activities that generate data to inform amr policy. ghana’s success story began with the formation of the npar in 2011. the director of pharmacy who led several amr advocacies4 chaired the quarterly meetings of npar. the leadership role played by the ministry of health and donor support from swedish international development agency through action on antibiotic resistance (react) paved the way for ghana’s amr policy. the npar brought together different stakeholders from specialties such as veterinary, research institutions, academia, media and civil society organisations. bringing different players from human, animal and environmental sectors together meant that individuals who used to work in ‘silos’ began talking to each other and sharing ideas. sooner, the magnitude and complexities of amu or amr became evident to all stakeholders. ministries like the ministry of food and agriculture, ministry of environment, science, technology and innovation, ministry of fisheries and aquaculture development and the regional fao eventually joined the crusade. thus, awareness creation, which forms part of the strategic objectives of gap, started with discussions around the table. through react, the ministry of health leveraged support for stakeholder analysis for amr policy in ghana. ministry of health also worked with civil society organsations to educate the community on issues related to amu or amr. donor support was also leveraged from the admer project, a research project supported by the danish international development agency. the overarching aim of the admer project was ‘prudent use of antibiotics for improved human health’. to inform policy, the admer project constituted an advisory board with membership from key stakeholders, including the ministry of health. the chairperson of npar leveraged support from the admer project for research activities that fed into ghana’s amr policy development.5,6,7,8 ghana-dutch research activities through the moh in mid-2005 also provided useful data that covered multiple disciplines. findings from these research activities provided data for amr surveillance in humans.9 the newman et al. study was one of the first large-scale studies on antimicrobial drug resistance in humans in ghana. the five key strategic pillars of the gap are anchored on awareness creation, surveillance, infection prevention and control, antibiotic stewardship, and research and innovation. ghana’s success story is highlighted under these broad strategic objectives below. awareness creation a knowledge, attitudes, practices and perceptions study was carried out by some members of ghana’s npar in 2014.10 findings from this study showed that there is a gap in the knowledge and perception of optimal antibiotic prescription practices among prescribers. this knowledge gap is not so different from surveys done by who in other sub-regions.11 policy and regulatory studies were also leveraged through the admer project.12 importantly, findings from most of the above studies were shared with multiple stakeholders, including sector ministries, directors, policymakers and the general public. react trained and used civil society organisations to create awareness on amu or amr in the communities. several groups, including opinion leaders, farmers groups and associations, were targeted and sensitised on issues related to amu or amr. since the inception of ‘antibiotics awareness week’, ghana has been participating, with the ministry of health leading various activities including media engagements during these celebrations. in 2015, admer hosted the first ever african conference on amr in ghana with the theme, ‘who is winning the antibiotic resistance war: bacteria or man?’13 this conference may have created awareness on the largest scale in ghana and probably the sub-region. the conference was organised in partnership with ministry of health, who, the university of ghana and other key stakeholders. over 120 participants, including health professionals, journalists and the general public, attended the conference. surveillance local and regional baseline data is needed to inform amr policy at country levels. through ghana-dutch collaboration, the first national surveillance of amr in humans was done in 2007.9 in 2015, donor support from react and admer was leveraged for a second nation-wide amr surveillance in ghana.8 the 2015 nation-wide study used whonet to store and to analyse surveillance data. in addition to the baseline data generated to inform policy, this study identified gaps that needed to be addressed before ghana’s nap implementation. for example, human resources, laboratory supplies and quality control checks would be crucial during implementation of a nap. compared to human studies, there is a paucity of surveillance data in the veterinary and aquaculture sectors in ghana. information on the role of the environment is also lacking. a one health concept will require building capacities in these other important sectors. empirically, it had been suspected that the quality of antimicrobials in the country may not be good, but there were no scientific data to support this fact. however, an important subsequent scientific study by one of the admer doctoral students provided some baseline data on the quality of antibiotics in the country. the study showed that most antibiotics purchased from the open market were of poor quality. infection prevention and control ghana has a national policy on infection prevention and control (ipc).14 however, in practice, the dictates of this policy are not strictly followed. studies conducted at both tertiary and secondary levels of health care facilities in ghana, indicate that uptake of ipc practices are not optimal.12,15 small-scale studies in ghana have shown that compliance to hand hygiene recommendations before and after patient contact are low.16 donor support to improve water, sanitation, and hygiene (wash) programmes, running water infrastructure and hospital waste disposal are much needed to control infections in ghana. in many health care facilities in ghana, the tasks of ipc have been handed to quality assurance committees. this does not augur well for the appropriate implementation of ipc practices. donor support can be leveraged to train many more full-time ipc personnel. in 2000, newman et al., funded by the neo pharmacy centre, conducted a point prevalence survey at the korle-bu teaching hospital.17 this survey gave a prevalence rate of healthcare-associated infections of approximately 7%. further to this, a team of researchers with funding from the danish international development agency recently conducted a multi-centre point prevalence survey of health care associated infections under the healthcare associated infections ghana project.18 this study was conducted in 10 different hospitals within ghana, including teaching, regional and district hospitals. the overall prevalence rate was 8% with levels varying between 3.5% and 14.4% among different hospitals.19 in addition to this study, doctoral and post-doctoral students on the project are carrying out several other studies on infection control related topics. these include surgical site infections, neonatal and puerperal sepsis, cost associated with health care associated infections as well as ethnography studies.18 antibiotic stewardship there is a systematic challenge with antibiotic stewardship in ghana. there have been reports of high prevalence of unwarranted antibiotic use in health care facilities at all levels of health care.5,15 there is often a challenge between excess and access.20 the admer project generated some baseline data on antibiotic prescription practices and what informs prescription.5,12 innovation and research local capacity for manufacturing of antibiotics and diagnostics is limited. for example, over 70% of the essential medicine needs of ghana are imported.21 this gives room for the importation of counterfeit and substandard medicines. donor support and investment is needed to improve local capacity in research, development and production of medicines in ghana. studies have shown that several exotic plants in ghana have medicinal properties. partnerships with the centre for scientific research into plant medicine, mampong, akwapim, ghana could serve a good purpose. there is also the need to leverage donor support for diagnostics, especially point of care tools.22 perspectives on the implementation of ghana’s national action plan the president of ghana, his excellency, nana addo dankwa akufo-addo, launched both ghana’s nap and policy documents on 11 april 2018. honorable ministers of state – ministry of health, ministry of food and agriculture, ministry of environment, science and innovation, ministry of fisheries and aquaculture development – were present to show their commitment. also present to show their commitment were the tripartite body (who, international organization for animal health, fao), legislators and diplomatic corps. great leadership from these important statesmen will be required for an effective implementation of the nap. the implementation target, outlined in the nap, is between 2017 and 2021. this means ghana may be late in some targets, although some activities have already commenced. it is estimated that 21 million dollars will be needed to implement the five-year activities outlined in ghana’s nap. identified lead implementing government ministries and departments would have to factor these activities into their annual budgets. donor support must also be leveraged for some key activities outlined in the nap. fortunately, key activities of the nap have been costed and lead implementing agencies also identified. this should make it easier for developmental donors to partner with government for the naps implementation. the united kingdom government’s support through the fleming fund is perhaps a low-hanging-fruit that ghana can fall on, especially for amu or amr surveillance in humans, animals, aquaculture and the environment. ghana can also leverage support from the fleming fund to build capacity and infrastructure for the other key strategic objectives including ipc and antibiotic stewardship. the concept of ghana’s nap is one health. however, greater infrastructure would be required in the animal, aquaculture and environmental sectors. great political will and amr champions in these sectors would be required for a successful implementation. conclusions through great leadership and donor support leveraging, requisite data was generated to inform ghana’s nap and policy documents. a much greater coordination and alignment of activities is required for full and smooth implementation. donor support can be harnessed through bodies such as the who, fao and the international organization for animal health, the african union and the fleming fund to facilitate implementation. acknowledgements dr appiah-korang labi, prof mercy j. newman, mr george hedidor and mrs martha gyansa-lutterodt are acknowledged for proofreading and ensuring that the facts are correct. competing interests the author declares that he has no financial or personal relationships which may have inappropriately influenced him in writing this article. sources of support none. references atlas. pfizer’s atlas surveillance database: a key tool in the fight against amr [homepage on the internet]. 2017. available from: https://www.amrindustryalliance.org/case-study/antimicrobial-testing-leadership-and-surveillance-atlas/ ghana nap. ghana national action plan on antimicrobial resistance, 2017–2021. 1st ed. accra: ministry of health, ministry of food and agriculture, ministry of environment, science, technology and innovation, ministry of fisheries and aquaculture development, 2017; pp. 1–103. republic of ghana. policy on antimicrobial use and resistance. 2017. 1st ed.; pp. 1–36. gyansa-lutterodt m. antibiotic resistance in ghana. lancet infect dis. 2013;13:1006–1007. https://doi.org/10.1016/s1473-3099(13)70196-8 ahiabu ma, tersbøl bp, biritwum r, et al. a retrospective audit of antibiotic prescriptions in primary health-care facilities in eastern region, ghana. health policy plan. 2016;31:250–258. https://doi.org/10.1093/heapol/czv048 andoh la, dalsgaard a, obiri-danso k, et al. prevalence and antimicrobial resistance of salmonella serovars isolated from poultry in ghana. epidemiol infect. 2016;144:3288–3299. https://doi.org/10.1017/s0950268816001126 obeng-nkrumah n, twum-danso k, krogfelt ka, newman mj. high levels of extended-spectrum beta-lactamases in a major teaching hospital in ghana: the need for regular monitoring and evaluation of antibiotic resistance. am j trop med hyg. 2013;89:960–964. https://doi.org/10.4269/ajtmh.12-0642 opintan ja, newman mj, arhin re, et al. laboratory-based nationwide surveillance of antimicrobial resistance in ghana. infect drug resist. 2015;8:379–389. https://doi.org/10.2147/idr.s88725 newman mj, frimpong e, donkor es, et al. resistance to antimicrobial drugs in ghana. infect drug resist. 2011;4:215–220. asante kp, boamah ea, abdulai ma, et al. knowledge of antibiotic resistance and antibiotic prescription practices among prescribers in the brong ahafo region of ghana: a cross-sectional study. bmc health serv res. 2017;17:422. https://doi.org/10.1186/s12913-017-2365-2 who. antibiotic resistance: multi-country public awareness survey. 2015 [homepage on the internet]. available from: http://www.who.int/drugresistance/documents/baselinesurveynov2015/en/ yevutsey sk, buabeng ko, aikins m, et al. situational analysis of antibiotic use and resistance in ghana: policy and regulation. bmc public health. 2017;17:896. https://doi.org/10.1186/s12889-017-4910-7 admer project. antibiotic drug use monitoring and evaluation of resistance project. 2015 [homepage on the internet]. available from: http://admerproject.org/ republic of ghana. national policy and guidelines for infection prevention and control in health care settings. 2015, pp. 1–146. labi ak, obeng-nkrumah n, nartey et, et al. antibiotic use in a tertiary healthcare facility in ghana: a point prevalence survey. antimicrob resist infect control. 2018;7:15. https://doi.org/10.1186/s13756-018-0299-z asare a, enweronu-laryea cc, newman mj. hand hygiene practices in a neonatal intensive care unit in ghana. j infect dev ctries. 2009;3:352–356. newman mj. nosocomial and community acquired infections in korle bu teaching hospital, accra. west afr j med. 2009;28:300–303. ghana hai. healthcare associated infections in ghana project [homepage on the internet]. 2015. available from: https://haiproject.org/ labi ak, obeng-nkrumah n, owusu e, et al. multi-centre point-prevalence survey of hospital-acquired infections in ghana. j hosp infect. in press. https://doi.org/10.1016/j.jhin.2018.04.019 cars o. securing access to effective antibiotics for current and future generations. whose responsibility. ups j med sci. 2014;119:209–214. https://doi.org/10.3109/03009734.2014.912700 seiter a, gyansa-lutterodt m. policy note: the pharmaceutical sector in ghana. 2009. okeke in. laboratory systems as an antibacterial resistance containment tool in africa. afr j lab med. 2016;5:497. https://doi.org/10.4102/ajlm.v5i3.497 abstract introduction research method and design results discussion acknowledgements references about the author(s) jenifer l. vaughan department of molecular medicine and haematology, university of the witwatersrand, johannesburg, south africa department of haematology at the chris hani baragwanath academic hospital, national health laboratory services, johannesburg, south africa sakina loonat department of haematology at the chris hani baragwanath academic hospital, national health laboratory services, johannesburg, south africa nazeer alli department of molecular medicine and haematology, university of the witwatersrand, johannesburg, south africa department of haematology at the chris hani baragwanath academic hospital, national health laboratory services, johannesburg, south africa citation vaughan jl, loonat s, alli n. evaluation of the accuracy of the cellavision™ dm96 in a high hiv-prevalence population in south africa. afr j lab med. 2016;5(1), art. #313, 5 pages. http://dx.doi.org/10.4102/ajlm.v5i1.313 original research evaluation of the accuracy of the cellavision™ dm96 in a high hiv-prevalence population in south africa jenifer l. vaughan, sakina loonat, nazeer alli received: 30 mar. 2015; accepted: 10 dec. 2015; published: 09 mar. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract introduction: the cellavision™ dm96 (dm96) is a digital microscopy system which performs well in developed countries. however, to date it has not been evaluated in africa, where the pathology spectrum encountered is very different. in particular, its utility in a setting with high hiv prevalence has not been assessed, which is of interest because of the morphological aberrations often seen in hiv-positive patients. objectives: this study aimed to evaluate the accuracy of the dm96 in a south african laboratory, with emphasis on its performance in samples collected from hiv-positive patients. methods: a total of 149 samples submitted for a routine differential white cell count in 2012 and 2013 at the chris hani baragwanath academic hospital in johannesburg, south africa were included, of which 79 (53.0%) were collected from hiv-positive patients. results of dm96 analysis preand post-classification were compared with a manual differential white cell count and the impact of hiv infection and other variables of interest were assessed. results: preand post-classification accuracies were similar to those reported in developed countries. reclassification was required in 16% of cells, with particularly high misclassification rates for eosinophils (31.7%), blasts (33.7%) and basophils (93.5%). multivariate analysis revealed a significant relationship between the number of misclassified cells and both the white cell count (p = 0.035) and the presence of malignant cells in the blood (p = 0.049), but not with any other variables analysed, including hiv status. conclusion: the dm96 exhibited acceptable accuracy in this south african laboratory, which was not impacted by hiv infection. however, as it does not eliminate the need for experienced morphologists, its cost may be unjustifiable in a resource-constrained setting. introduction the differential white cell count (dwcc) is a frequently requested laboratory investigation and is, historically, a labour-intensive test. although the guidelines established by the international consensus group for haematology to identify samples where peripheral smear review may be omitted1 have considerably reduced the workload on the morphology bench, manual smear review remains necessary in a substantial proportion of cases. with the number of skilled medical technologists on the decline worldwide, morphology skills are becoming ever scarcer, particularly in africa where laboratory resources are, in general, grossly strained.2,3 novis et al. reported the smear review rate to be proportional to the number of occupied beds in the hospital served,4 which they speculated to reflect a higher pathology burden in larger hospitals. in sub-saharan africa, the pathology burden (and hence the need for smear review) is compounded by the hiv epidemic, which places an extra strain on haematology services because of the many haematological complications of hiv infection. there is thus a dire need for analysers that can improve laboratory efficiency in this setting. the cellavision™ dm96 (cellavision ab, lund, sweden; hereafter, ‘dm96’) is a digital microscopy system that has the potential to minimise the time required by a morphologist to perform a manual dwcc. it comprises: an automated microscope that scans the blood smear; a digital camera that captures images of all the cellular and particulate material on the slide; and a computer that classifies each image by means of complex algorithms. it has been demonstrated to have good performance characteristics in developed countries,5,6,7,8,9 but to date has not been evaluated in africa, where the spectrum of pathology encountered is very different. most notable in sub-saharan africa is the extremely high prevalence of hiv infection. more than 25.5 million people live with hiv in this region, compared with less than 2.5 million in western/central europe and north america combined.10 the effect of this epidemic is greatest in communities with a poor socio-economic background. in south africa, the most substantial impact is on state-sector hospitals. because hiv infection is often associated with a number of morphological peculiarities of the white cells, including the frequent presence of atypical activated lymphocytes and abnormal nucleation of the neutrophils,11 its impact on the performance of the dm96 is of interest for laboratories operating in areas with high hiv prevalence. the chris hani baragwanath academic hospital (chbah) is a large referral centre in johannesburg, south africa, which serves a community with a poor socio-economic background and high hiv prevalence. the aim of this study was to assess the accuracy of the dwcc generated by the dm96 in the chbah laboratory, with emphasis on its performance in samples collected from hiv-positive patients. research method and design ethical considerations ethical clearance was obtained from the human research ethics committee of the university of the witwatersrand, johannesburg, south africa (clearance number: m090688). sample selection and analysis the study was performed at the national health laboratory service (nhls) haematology laboratory at chbah in soweto, johannesburg, south africa. a total of 149 peripheral blood samples were selected from edta-anticoagulated specimens submitted for a dwcc over the course of 2012 and 2013. samples were selected to cover a wide range of white cell counts and were included only if the patient had an hiv test result available in the laboratory information system (lis) (disalab version 04.16.04.373, laboratory system technologies, boksburg, gauteng, south africa). slides were made and stained with may-grünwald/giemsa by an automated slide maker and stainer (sp-100, sysmex, kobe, japan) as per standard operating procedure for the performance of a dwcc. each smear was examined by an experienced morphologist and analysed with the dm96 within a 24-hour period. the manual dwcc was performed on 100 cells. the dm96 was set to analyse 110 images per sample. each image was pre-classified as an unidentified cell, a neutrophil, a lymphocyte, a monocyte, a granulocyte precursor (i.e., a promyelocyte, myelocyte or metamyelocyte), a blast, an eosinophil, a basophil, a nucleated red cell, a giant platelet, a platelet aggregate, a smear cell or an artefact. cells which the instrument identified as being band cells were classified as neutrophils. the images were then viewed by the same morphologist who performed the manual dwcc. the morphologist either verified the dm96 pre-classification or reclassified the cells. hereafter, the initial dwcc performed by the dm96 will be referred to as the ‘pre-classification dwcc’ and the final dwcc following review by the morphologist will be referred to as the ‘post-classification dwcc’. ‘misclassification’ will refer to cells requiring reclassification by the morphologist. for each sample, the data available in the lis were reviewed and pertinent information recorded. this included the clinical information provided by the attending clinician, demographic details, evidence of infection (including c-reactive protein levels and culture results), recent hiv viral load and cd4 counts (where appropriate), exposure to anti-retroviral therapy, as well as bone marrow aspirate/trephine biopsy and other histology findings. the data were recorded in excel™ spreadsheets (microsoft office excel™ 2007, redmond, washington, united states). no identifying patient information was recorded. all data collection and analysis were performed by a haematopathologist working in the haematology laboratory of the chbah. statistical analysis demographic data are presented as medians (interquartile range [iqr]), mean (± standard deviation [sd]) and proportions, as appropriate. the accuracy of the dm96 dwcc was evaluated by linear regression (preand post-classification) and bland-altman (post-classification) analyses comparing the absolute count for neutrophils, monocytes, lymphocytes, eosinophils, basophils and blasts with those obtained by manual counting. the misclassification rate was determined as the proportion of all counted cells requiring reclassification. a multivariate linear regression analysis was performed to assess the impact of variables of interest, including hiv infection, on misclassification rates. any data point with a standard residual of greater than 2.5 was excluded from analysis and p-values less than 0.05 were considered statistically significant. statistical analysis was performed using statistica software, version 12.5 (stat soft [pty] ltd; tulsa, oklahoma, united states). results patient demographic and clinical data are summarised in table 1. the median white cell count (wcc) was 6.76 × 109/l (range 0.28–262). the wcc was < 1.5 × 109/l in 14 patients (9.4%) and > 50 × 109/l in 12 patients (8.1%). slightly over half (n = 79; 53%) of patients were hiv-positive, whilst ~40% (n = 61) had a history of malignancy. the prevalence of malignancy was similar between hiv-positive and hiv-negative patients, although the spectrum of malignant disease varied substantially between these two groups. the dominant malignancy amongst hiv-positive patients was high-grade lymphomas, whereas leukaemias were more common amongst hiv-negative patients. despite this difference, the proportion of patients with abnormal cells present in the peripheral blood was similar between the two groups. not surprisingly, evidence of infection was substantially more common amongst hiv-positive patients. table 1: patient demographic and clinical data for samples included in study, chris hani baragwanath academic hospital haematology laboratory, johannesburg, south africa, 2012–2013 (n = 149). analysis of accuracy overall, pre-classification dwcc accuracy was very poor, with good correlation occurring only for neutrophils (table 2). correlation co-efficient (cc) values improved substantially post-classification, but remained below 0.9 for all cell types except neutrophils and blasts, and was poor for eosinophils, basophils and monocytes. however, the bland-altman analysis showed that agreement was within acceptable limits of bias for neutrophils, lymphocytes and blasts, with borderline acceptable agreement for monocytes. a substantial negative bias was evident for both eosinophils and basophils, but because of the low levels of these cell types, this translated into small differences in absolute values, the clinical significance of which was negligible. table 2: accuracy of cellavision™ dm96 analyser, chris hani baragwanath academic hospital haematology laboratory, johannesburg, south africa, 2012–2013. analysis of misclassification rates although only 3.5% of cells were classified as ‘unidentified’ in the pre-classification dwcc, overall 16% required reclassification (table 3). as the most common cell type, neutrophils were the most frequently misclassified, but overall, only 7.6% of all neutrophils required reclassification. in contrast, close to 30% of monocytes, eosinophils and blasts, as well as > 90% of basophils, were misclassified. multivariate analysis revealed a significant relationship between the number of misclassified cells and both the wcc and the presence of malignant cells in the blood (table 4). no other variables analysed, including hiv status, had a significant association with the number of misclassified cells. table 3: misclassification of cells by cellavision™ dm96 analyser, chris hani baragwanath academic hospital haematology laboratory, johannesburg, south africa, 2012–2013 (n = 149). table 4: associations of variables of interest with misclassification rates of cells by cellavision™ dm96 analyser, chris hani baragwanath academic hospital haematology laboratory, johannesburg, south africa, 2012–2013 (n = 149).† discussion in this study, we assessed the performance of the dm96 as compared to a manual dwcc in 149 samples collected from patients with a wide range of infections and haematological pathologies in a large south african state hospital serving a population with high hiv prevalence. results from similar studies performed in developed countries have varied. park et al. showed excellent correlation (cc > 0.9) for all cell types except promyelocytes and basophils.13 in contrast, although most other studies have also shown good correlations for neutrophils, lymphocytes and blasts, correlations were generally poorer for monocytes (cc 0.67–0.83), eosinophils (cc 0.73–0.88), and basophils (0.05–0.76).6,7,8,9 similarly in our study, there was excellent correlation (cc > 0.95) for both neutrophils and blasts and substantially weaker correlation (cc < 0.75) for eosinophils and basophils. interestingly, the correlations for both lymphocytes (cc = 0.86) and monocytes (cc = 0.51) were poorer in our study than those reported previously. nonetheless, we found that agreement was within acceptable limits of bias for both the monocyte and lymphocyte values, and accuracy was thus judged to be adequate for these parameters. the diversity of results between studies may be attributable, in part, to the proportion of pathological samples included in each study. in addition, park et al. found that correlations improved in samples with low wccs when the dm96 was preset to analyse a higher number of white cells (300–500), which may have impacted their overall correlation results.13 the relatively poorer correlation consistently seen for monocytes, basophils and/or eosinophils is likely a result of the predictably poor precision expected when cells present in small numbers are assessed by a limited cell count. reassuringly, where discrepancies between the methods were evident in our study, the differences translated into small changes in absolute cell counts, the clinical significance of which was negligible. although hiv infection has well documented effects on white cell morphology,11 misclassification rates in our study were not associated with hiv status. misclassification rates were significantly associated with wcc and the presence of malignant cells in the peripheral blood, whereas chemotherapy exposure, a history of prior malignancy and the presence of infection were not. ‘unidentified cells’ made up 3.5% of cells in our study, but overall, 16% required reclassification. our misclassification rate was higher than that described previously by rollins-raval, raval and contis, who assessed the performance of the dm96 over a six-month period at three separate sites.14 the number of unidentified cells in that study was ~1% and the number of misclassified cells ranged from 4.6% – 12.7%. the higher misclassification rate in our study is likely a product of the large number of specimens containing malignant cells and selection of a large number of samples with extreme wccs for the purpose of evaluating the analyser. thus, samples with abnormal wccs were disproportionately prevalent in comparison to the routine laboratory workload. the overall accuracy of the dm96 is therefore likely to be better than shown in our study, particularly when the wcc is near normal and malignant cells are not present. that being said, in the current era of sophisticated full blood count analysers able to perform accurate dwccs in a timely manner, the necessity of performing manual dwccs is restricted to samples in which the analyser fails to perform an accurate dwcc because of the presence of abnormal white cells. consequently, the accuracy of results for samples with abnormal cells present is of greatest interest, as these specimens would potentially be eligible for analysis with the dm96 because of the need for a manual dwcc. the time required to perform a dm96 dwcc has been demonstrated to be less than that required for a manual dwcc,6,8,9 which raises hopes for greater laboratory efficiency when a manual dwcc is needed. however, the increased misclassification rate in the presence of abnormal cells shown in our study necessitates reclassification of a greater number of cells, which prolongs the time required to perform a dm96 dwcc in this setting. this could conceivably negate the marginal reduction in the time it takes for experienced morphologists to perform a dwcc using the dm96 as compared to a manual count. cornet, perol and troussard suggested that the dm96 would prove to be time efficient, as the few unidentified cells could be quickly and easily classified and validated, thus reducing the time spent on microscopy by technical staff.5 however, we found that the number of unidentified cells comprised only about 20% of the misclassified cells; thus, re-assignment of only the unidentified cells would compromise accuracy to an unacceptable extent. time efficiency is also undermined in samples with very low wccs, where accuracy is reportedly increased by pre-setting the analyser to count 300–500 cells. however, this improvement comes at the expense of a longer analysis time.13 moreover, the improvements in cell recognition and flagging technology in automated analysers mean that the vast majority of samples can be reviewed by scanning the peripheral smear without the need for a manual dwcc. thus, in samples without a substantial number of abnormal cells present, the potential analytical time advantage expected from the dm96 is eliminated by the performance of a ‘smear scan’ in lieu of a manual dwcc. given the benefits of a smear scan over a manual dwcc in the majority of samples, as well as the poorer time efficiency of the dm96 anticipated in samples with leukopenia or abnormal cells present, the utility of the dm96 is placed in question. this is emphasised by the user-dependent nature of correlation studies shown by briggs et al., where the accuracy of the dm96 was noticeably poorer when the analysis was performed by less-experienced microscopists.6 clearly, although the dm96 is a brilliant piece of innovative technology, it does not eliminate the need for skilled morphologists. limitations there are some limitations to our study which should be considered when interpreting the results. a large proportion of our hiv-negative patients had a malignancy, so they cannot be regarded as being representative of the normal population. no normal control group was included, so the accuracy of the dm96 was therefore not assessed in the normal population. our sample size is relatively small and the number of samples included with extreme wccs and abnormal cells present most likely skewed our results to some extent. conclusion this study showed that the performance of the dm96 in an african laboratory serving a population with a high hiv-prevalence was similar to that described in developed countries. however, significant intervention from experienced morphologists remains necessary for the validation of results, particularly when malignant cells are present. given the substantial cost of this sophisticated instrument, it would be difficult to justify its routine use in a resource-constrained setting. acknowledgements the authors would like to acknowledge and thank all of the technical staff members of the national health laboratory service haematology laboratory at the chris hani baragwanath academic hospital. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. sources of support this project was performed on samples submitted for routine testing, and therefore required no funding. authors’ contributions j.l.v. (university of the witwatersrand and national health laboratory services) was responsible for peripheral smear and dm96 analysis, data analysis and writing the paper. s.l. (national health laboratory services) assisted with peripheral smear and dm96 analysis. n.a. (university of the witwatersrand and national health laboratory services) co-wrote the paper and provided editorial oversight. references barnes pw, mcfadden sl, machin sj, et al. the international consensus group for hematology review: suggested criteria for action following automated cbc and wbc differential analysis. lab hematol. 2005;11(2):83–90. http://dx.doi.org/10.1532/lh96.05019 beck s, & doig k. laboratory managers’ views on attrition and retention of laboratory personnel. clin lab sci. 2005 fall;18(4):238–247. petti ca, polage cr, quinn tc, et al. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006 feb 1;42(3):377–382. http://dx.doi.org/10.1086/499363 novis da, walsh m, wilkinson d, et al. laboratory productivity and the rate of manual peripheral blood smear review: a college of american pathologists q-probes study of 95,141 complete blood count determinations performed in 263 institutions. arch pathol lab med. 2006 may;130(5):596–601. cornet e, perol jp, troussard x. performance evaluation and relevance of the cellavision dm96 system in routine analysis and in patients with malignant hematological diseases. int j lab hematol. 2008 dec;30(6):536–542. http://dx.doi.org/10.1111/j.1751-553x.2007.00996.x briggs c, longair i, slavik m, et al. can automated blood film analysis replace the manual differential? an evaluation of the cellavision dm96 automated image analysis system. int j lab hematol. 2009 feb;31(1):48–60. http://dx.doi.org/10.1111/j.1751-553x.2007.01002.x lee lh, mansoor a, wood b, et al. performance of cellavision dm96 in leukocyte classification. j pathol inform. 2013;4:14. http://dx.doi.org/10.4103/2153-3539.114205 ceelie h, dinkelaar rb, van gelder w. examination of peripheral blood films using automated microscopy; evaluation of diffmaster octavia and cellavision dm96. j clin pathol. 2007 jan;60(1):72–79. http://dx.doi.org/10.1136/jcp.2005.035402 kratz a, bengtsson hi, casey je, et al. performance evaluation of the cellavision dm96 system: wbc differentials by automated digital image analysis supported by an artificial neural network. am j clin pathol. 2005 nov;124(5):770–781. http://dx.doi.org/10.1309/xmb9k0j41lhlatay unaids. fact sheet: 2014 global statistics [document on the internet]. c2015 [cited 2015 sep 04]. available from: http://www.unaids.org/sites/default/files/media_asset/20150714_fs_mdg6_report_en.pdf. bain b. the haematological features of hiv infection. br j haematol. 1997 oct;99(1):1–8. http://dx.doi.org/10.1046/j.1365-2141.1997.2943111.x westgard qc. quality requirements: desirable biological variation database specifications [page on the internet]. c2014 [cited 2015 oct 24]. available from: https://www.westgard.com/biodatabase1.htm. park sh, park cj, choi mo, et al. automated digital cell morphology identification system (cellavision dm96) is very useful for leukocyte differentials in specimens with qualitative or quantitative abnormalities. int j lab hematol. 2013 oct;35(5):517–527. http://dx.doi.org/10.1111/ijlh.12044 rollins-raval ma, raval js, contis l. experience with cellavision dm96 for peripheral blood differentials in a large multi-center academic hospital system. j pathol inform. 2012;3:29. http://dx.doi.org/10.4103/2153-3539.100154 ajlm 8(1)_2019_contents.indd http://www.ajlmonline.org open access table of contents i original research morphological patterns of anaemia among pregnant women from sudan abuobieda b. abusharib african journal of laboratory medicine | vol 8, no 1 | a743 | 14 october 2019 original research haematological values in a healthy adult population in yaoundé, cameroon martin e. oloume, abas mouliom, bernard f. melingui, suzanne belinga, julie s. nana, mathurin tejiokem, francoise n. sack, jeanne manga, annie r. epote african journal of laboratory medicine | vol 8, no 1 | a852 | 31 october 2019 original research onsite healthcare worker acceptability and performance of the point-of-care pima cd4 assay in dar es salaam, tanzania mary e. schmitz, karen chang, nichole arnett, luciana kohatsu, ruth lemwayi, michael mwasekaga, john nkengasong, omotayo bolu, fausta mosha, larry westerman african journal of laboratory medicine | vol 8, no 1 | a740 | 21 november 2019 original research assessment of the aquios flow cytometer – an automated sample preparation system for cd4 lymphocyte panleucogating enumeration daniel rhodes, guislaine carcelain, mike keeney, christophe parizot, dominika benjamins, laurine genesta, jin zhang, justin rohrbach, denise lawrie, deborah k. glencross african journal of laboratory medicine | vol 8, no 1 | a804 | 05 december 2019 original research resazurin microtitre plate assay and sensititre® mycotb for detection of mycobacterium tuberculosis resistance in a high tuberculosis resistance setting prenika jaglal, melendhran pillay, koleka mlisana african journal of laboratory medicine | vol 8, no 1 | a840 | 13 december 2019 lessons from the field development and validation of a high performance liquid chromatography method to determine nevirapine in plasma in a resource-limited setting faithful makita-chingombe, andrew j. ocque, robin difrancesco, charles maponga, farai muzambi, tsitsi g. monera-penduka, tinashe mudzviti, takudzwa j. mtisi, gene d. morse african journal of laboratory medicine | vol 8, no 1 | a880 | 16 may 2019 lessons from the field strengthening laboratory capacity for detection of respiratory viral pathogens through the global health security agenda (ghsa) framework brett whitaker, karen a. alroy, erica guthrie, sarah schildecker, susan hiers, jill woodard, s. arunmozhi balajee african journal of laboratory medicine | vol 8, no 1 | a861 | 18 july 2019 48 55 61 69 79 88 95 page i of ii table of contents i editorial extending the breadth of african laboratory medicine iruka n. okeke african journal of laboratory medicine | vol 8, no 1 | a1128 | 05 december 2019 review article review of molecular subtyping methodologies used to investigate outbreaks due to multidrug-resistant enteric bacterial pathogens in sub-saharan africa anthony m. smith african journal of laboratory medicine | vol 8, no 1 | a760 | 14 march 2019 original research implementation and evaluation of the presto combined qualitative real-time assay for chlamydia trachomatis and neisseria gonorrhoeae in rwanda vicky cuylaerts, irith de baetselier, claude m. muvunyi, lambert mwambarange, hilde smet, john rusine, viateur musengamana, janneke van de wijgert, tania crucitti african journal of laboratory medicine | vol 8, no 1 | a739 | 18 april 2019 original research molecular detection of enterovirus d68 among children with acute respiratory tract infection in ghana joyce a. kubi, mohamed mutocheluh, joseph h.k. bonney, william k. ampofo, john k. odoom african journal of laboratory medicine | vol 8, no 1 | a732 | 26 june 2019 original research molecular confirmation and phylogeny of lassa fever virus in benin republic 2014–2016 olumuyiwa b. salu, ayorinde b. james, honoré s. bankolé, jijoho m. agbla, magloire da silva, fernand gbaguidi, christian f. loko, sunday a. omilabu african journal of laboratory medicine | vol 8, no 1 | a803 | 22 august 2019 original research organisational management of hospital blood transfusion services in nairobi county, kenya: evidence of implementation anne n. mbuthia, eunice m. mwangi, musa o. ong’ombe african journal of laboratory medicine | vol 8, no 1 | a676 | 26 august 2019 original research the use of real-time polymerase chain reaction and an adenosine deaminase assay for diagnosing pleural tuberculosis mulalo molaudzi, julitha molepo african journal of laboratory medicine | vol 8, no 1 | a731 | 28 august 2019 original research xpert® mtb/rif assay on formalin-fixed paraffin-embedded tissues in the diagnosis of extrapulmonary tuberculosis allan n. njau, samuel m. gakinya, shahin sayed, zahir moloo african journal of laboratory medicine | vol 8, no 1 | a748 | 18 september 2019 1 3 13 19 24 30 38 43 vol 8, no 1 (2019) issn: 2225-2002 (print) | issn: 2225-2010 (online)african journal of laboratory medicine http://www.ajlmonline.org open access table of contents ii lessons from the field blueprint for building a biorepository in a resource-limited setting that follows international best practices alash’le g. abimiku, talishea croxton, petronilla j. ozumba, ndidi agala, olasinbo balogun, emmanuel jonathan, enzenwa onyemata, kachimi ndifon, sunji nadoma, thankgod anazodo, sam peters, christine m. beiswanger african journal of laboratory medicine | vol 8, no 1 | a722 | 28 august 2019 lessons from the field medical laboratory accreditation in a resource-limited district health centre laboratory, addis ababa, ethiopia daniel m. desalegn, boja d. taddese, nebiyou yemanebrhane, mulye s. getahun, kumera t. kitila, tariku t. dinku, kassahun d. asferie, elizabeth a. wolde, gemechis b. tura, tilahun b. mersha, alemayhu w. rorissa, daniel d. wondimagegnehu, tinsae k. hailu, abrham t. bika african journal of laboratory medicine | vol 8, no 1 | a793 | 19 september 2019 lessons from the field tropical laboratory initiative: an innovative model for laboratory medicine in rural areas zelda r. moran, atta b. frimpong, pablo castañeda-casado, francis k. frimpong, manuela b. de lorenzo, yanis ben amor african journal of laboratory medicine | vol 8, no 1 | a922 | 26 september 2019 lessons from the field a comprehensive district-level laboratory intervention after the ebola epidemic in sierra leone annelies w. mesman, musa bangura, sahr m. kanawa, joseph s. gassimu, kerry l. dierberg, mohamed m. sheku, j. daniel orozco, regan h. marsh african journal of laboratory medicine | vol 8, no 1 | a885 | 22 october 2019 lessons from the field design and implementation of a clinical laboratory information system in a low-resource setting timothy m. mtonga, faheema e. choonara, jeremy u. espino, chimwemwe kachaje, kenneth kapundi, takondwa e. mengezi, soyapi l. mumba, gerald p. douglas african journal of laboratory medicine | vol 8, no 1 | a841 | 28 october 2019 case study coexistence of kaposi sarcoma and molluscum contagiosum on the same site in a hiv-aids patient: a very rare occurrence kabir abdullahi, yahaya mohammed, saddiku a. sahabi, mahmood m. dalhat african journal of laboratory medicine | vol 8, no 1 | a747 | 29 april 2019 case study late diagnosis of multidrug-resistant tuberculosis in a child at dr george mukhari academic hospital, ga-rankuwa, south africa: a case report bakani a. siwele, ndivhuho a. makhado, matodzi t. mariba african journal of laboratory medicine | vol 8, no 1 | a783 | 29 july 2019 99 111 116 123 130 137 140 case study atypical presentation of herpes simplex virus type 1 infection in paediatric burns patients in a large tertiary hospital, south africa mpho l. sikhosana, asma salloo, monica birkhead, kerrigan mccarthy african journal of laboratory medicine | vol 8, no 1 | a916 | 23 october 2019 brief report whole genome sequencing for drug resistance determination in mycobacterium tuberculosis shaheed v. omar, lavania joseph, halima m. said, farzana ismail, nabila ismail, thabisile l. gwala, nazir a. ismail african journal of laboratory medicine | vol 8, no 1 | a801 | 21 february 2019 brief report utilisation of fine needle aspiration cytology and biopsy in sokoto, nigeria: a five-year review kabir abdullahi, mohammed umar, saddiku m. sahabi african journal of laboratory medicine | vol 8, no 1 | a809 | 30 may 2019 brief report seroprevalence of hepatitis b virus co-infection among hiv-1-positive patients in north-central nigeria: the urgent need for surveillance terver m. akindigh, abba o. joseph, chrisitiana o. robert, ocheme j. okojokwu, juliet n. okechalu, aje j. anejo-okopi african journal of laboratory medicine | vol 8, no 1 | a622 | 27 june 2019 brief report potential role of lu/bcam in hiv-related atherosclerosis modisa s. motswaledi, ishmael kasvosve, oluwafemi o. oguntibeju african journal of laboratory medicine | vol 8, no 1 | a792 | 30 september 2019 brief report hyperuricaemia is associated with dyslipidemia but not hba1c among type 2 diabetes mellitus patients in botswana ellen gobusamang, naledi g. nyepetsi, modisa s. motswaledi, ishmael kasvosve african journal of laboratory medicine | vol 8, no 1 | a786 | 07 november 2019 correction corrigendum: antimicrobial resistance surveillance in ethiopia: implementation experiences and lessons learned rajiha a. ibrahim, amete m. teshale, surafel f. dinku, negga a. abera, abebe a. negeri, feven g. desta, eyasu t. seyum, adugna w. gemeda, wubshet m. keficho african journal of laboratory medicine | vol 8, no 1 | a1109 | 06 december 2019 reviewer acknowledgement african journal of laboratory medicine | vol 8, no 1 | a1136 | 12 december 2019 145 149 154 157 161 165 169 170 page ii of ii about the author(s) passoret vounba economic community of central african states (eccas) commission/fourth phase of the regional disease surveillance systems enhancement project (redisse iv), libreville, gabon severin loul economic community of central african states (eccas) commission/fourth phase of the regional disease surveillance systems enhancement project (redisse iv), libreville, gabon ludovic f. tamadea economic community of central african states (eccas) commission/fourth phase of the regional disease surveillance systems enhancement project (redisse iv), libreville, gabon joël f.d. siawaya department of laboratory services, chu mère-enfant fondation jeanne ebori, libreville, gabon regional integrated surveillance and laboratory network (rislnet) for central africa, libreville, gabon citation vounba p, loul s, tamadea lf, siawaya jfd. corrigendum: microbiology laboratories involved in disease and antimicrobial resistance surveillance: strengths and challenges of the central african states. afr j lab med. 2023;12(1), a1913. https://doi.org/10.4102/ajlm.v12i1.1913 note: doi of original article published: https://doi.org/10.4102/ajlm.v11i1.1570 correction corrigendum: microbiology laboratories involved in disease and antimicrobial resistance surveillance: strengths and challenges of the central african states passoret vounba, severin loul, ludovic f. tamadea, joël f.d. siawaya published: 09 may 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. in the published article, vounba p, loul s, tamadea lf, siawaya jfd. microbiology laboratories involved in disease and antimicrobial resistance surveillance: strengths and challenges of the central african states. afr j lab med. 2022;11(1), a1570. https://doi.org/10.4102/ajlm.v11i1.1570, there is an omission in the acknowledgements statement. the corrected acknowledgements statement appears below. the original incorrect wording: we sincerely thank dr skander hathroubi (from humboldt university of berlin, germany) for his contribution by reading and correcting the first draft of the manuscript. the revised and updated wording: we sincerely thank dr skander hathroubi (from humboldt university of berlin, germany) for his contribution by reading and correcting the first draft of the manuscript. this study was commissioned and supported by the regional disease surveillance systems enhancement (redisse) project in central africa, which is financially supported by the world bank and managed by the economic community of central african states (eccas). we would like to thank eccas and the world bank for supporting our efforts through redisse. the authors apologise for this error. the correction does not change the study’s findings of significance or overall interpretation of the study’s results or the scientific conclusions of the article in any way. acknowledgements references about the author(s) christoffel j. opperman green point tb laboratory, national health laboratory service, cape town, south africa sarishna singh green point tb laboratory, national health laboratory service, cape town, south africa francois barton green point tb laboratory, national health laboratory service, cape town, south africa citation opperman cj, singh s, barton f. appropriate disposal of waste in the laboratory: neglected but not forgotten. afr j lab med. 2022;11(1), a1786. https://doi.org/10.4102/ajlm.v11i1.1786 scientific letter appropriate disposal of waste in the laboratory: neglected but not forgotten christoffel j. opperman, sarishna singh, francois barton received: 12 nov. 2021; accepted: 04 apr. 2022; published: 14 july 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. laboratory waste management should focus on environmental and worker safety in a cost-effective manner to ensure ongoing diagnostic testing in accredited laboratories, especially in lowand middle-income countries with limited resources.1 for example, facilities focusing on mycobacterium tuberculosis generate biosafety level three infectious material that must be decontaminated and disposed of correctly to maintain good laboratory practice within a legislative framework.2 therefore, a holistic outline to support sustainable waste management in the laboratory is essential. this may include various components, such as waste disposal awareness campaigns, keeping abreast of technological advances,3 or implementing managerial policies. in this letter, we discuss practical suggestions for appropriately disposing of different waste types generated in most laboratories, with specific reference to a high-throughput, public, m. tuberculosis diagnostic laboratory. a technical brief published in 2020 by the global fund on sustainable healthcare management highlighted strategic waste management and best practice principles to limit hazardous infectious waste.4 their recommendations include: waste avoidance, reduction, and minimisation.4 in our laboratory setting, the disposal of hazardous biological material is not weight dependent. therefore, switching from single-use items to reusable equipment in low-risk laboratory areas that are not dedicated to processing or culturing and reducing ‘space-occupying’ objects, such as disposable laboratory coats (figure 1, number 1), effectively reduces waste and cost. digital platforms can limit paperwork and, thereby, paper waste. it is often noticed that forms and labels are discarded in biological waste containers within a busy laboratory. disposal of these and other reusable materials in bins designated for recyclables is not only a cost-saving initiative but should be a moral obligation on our journey to a ‘green’ and sustainable environment. implementing local guidelines with testing algorithms is essential to limit unnecessary investigations that generate extensive ‘routine diagnostic’ waste (figure 1, number 2). waste created by high sample rejection rates secondary to leaked specimens, insufficient volumes of poor quality, inappropriately submitted sample types, unlabelled containers, mismatched samples with the laboratory request forms, samples unsuitable due to contamination, et cetera, and should form part of quality control procedures. gatekeeping (reducing tests that can be avoided without negatively impacting patient management) and letting local healthcare facilities know the reason when they have a sample rejected from a laboratory can positively reinforce national guidelines and reduce sample rejection.5 figure 1: open-lid image from a biological infectious waste disposal container in a reference tuberculosis laboratory at the end of a specimen processing shift in cape town, south africa, 2022. 1, disposable laboratory coat; 2, appears to be a rejected specimen; 3, sterile cepheid xpert® mtb/rif ultra packaging material; 4, extensive plastic packaging from a tuberculosis specimen; 5, large amount of absorbable cleaning paper; 6, disposable latex gloves. laboratory waste should be classified according to the category that will dictate the waste management approach.6 laboratory products and kits containing nontoxic materials that can be discarded in general waste should be sought during the procurement process. for example, sterile cepheid xpert® mtb/rif ultra (solna, stockholm, sweden) packaging material (figure 1, number 3) does not require a biological infectious material container for discard. in addition, large amounts of packaging (figure 1, number 4) and large containers should be avoided during transportation from a local healthcare facility to the laboratory. such packaging creates a financial burden and consumes space in waste bins. although it is tempting to use recyclable products in the laboratory (plastics), laboratory professionals should be careful not to contaminate new products when using recycled instruments, as partial decontamination could cause erroneous results and impact patient management. unless being used to absorb spilled liquids, surface cleaning materials, such as paper towels, should be kept to a minimum (figure 1, number 5). care should be taken to maximise the use of personal protective equipment in the laboratory to preserve the supply chain, particularly during the coronavirus disease 2019 pandemic.7,8 to our knowledge, no guideline has been published on how many times gloves should be changed without obvious contamination between laboratory samples; discretion must be used in this regard (figure 1, number 6). a ‘just in time’ approach should be utilised when purchasing inventory. this could limit waste generated by reagents or diagnostic tests lost to expiration. however, we acknowledge that many african laboratories have constrained resource allocations and challenges maintaining sustainable budgets. this correspondence is not intended to be an all-inclusive guideline on the management of waste in the laboratory. instead, we looked critically into the waste bins to remind all laboratory workers about their responsibilities and opportunities for disposing of waste diligently and correctly. staff should be trained and updated regularly on correct waste disposal procedures. in addition, waste auditing systems should be implemented to gather robust data, guide planning, and assist laboratory managers with decision-making.4 auditing reviews on the amount, type, and laboratory area of waste generated ought to form the baseline for waste management initiatives. after all, correct waste disposal remains a ‘low-hanging fruit’ for saving money in every laboratory. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions c.j.o. conceptualised and wrote the original draft, then reviewed and edited the manuscript. s.s. and f.b. reviewed and edited the manuscript. ethical considerations this article does not contain any studies involving human participants performed by any of the authors. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability data sharing is not applicable to this article, as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect official policy or position of any affiliated agency of the authors. this article does not contain any studies involving human participants performed by any of the authors. references tuberculosis laboratory biosafety manual. in: essential biosafety measures for tb laboratories. geneva: world health organization; 2012. available from: https://www.ncbi.nlm.nih.gov/books/nbk179129/ johnson kr, braden cr, cairns kl, et al. transmission of mycobacterium tuberculosis from medical waste. jama. 2000;284(13):1683–1688. https://doi.org/10.1001/jama.284.13.1683 myneedu vp, aggarwal a. disposal of the large volume of sputum positive for mycobacterium tuberculosis by using microwave sterilisation technology as an alternative to traditional autoclaving in a tertiary respiratory care hospital in delhi, india. infect prev pract. 2020;2(3):100072. https://doi.org/10.1016/j.infpip.2020.100072 technical brief: sustainable health care waste management. geneva: the global fund; 2020. rooper l, carter j, hargrove j, hoffmann s, riedel s. targeting rejection: analysis of specimen acceptability and rejection, and framework for identifying interventions in a single tertiary healthcare facility. j clin lab anal. 2017;31(3):e22060. https://doi.org/10.1002/jcla.22060 ferronato n, torretta v. waste mismanagement in developing countries: a review of global issues. int j environ res public health. 2019;16(6):1060. https://doi.org/10.3390/ijerph16061060 yuan x, wang x, sarkar b, sik ok y. the covid-19 pandemic necessitates a shift to a plastic circular economy. nat rev earth environ. 2021;2:659–660. https://doi.org/10.1038/s43017-021-00223-2 bauchner h, fontanarosa pb, livingston eh. conserving supply of personal protective equipment-a call for ideas. jama. 2020;323(19):1911. https://doi.org/10.1001/jama.2020.4770 article information authors: wendy arneson1 cathy robinson1 bryan nyary2 affiliation: 1american society for clinical pathology institute, global outreach, usa2independent consultant correspondence to: wendy arneson postal address: 33 w monroe street, suite 1600, chicago, il 60603 usa dates: received: 30 apr.2012 accepted: 02 aug.2012 published: 18 june 2013 how to cite this article: arneson w, robinson c, nyary b. biomedical laboratory science education: standardising teaching content in resource-limited countries. afr j lab med. 2013;2(1), art. #56, 6 pages. http://dx.doi.org/10.4102/ ajlm.v2i1.56 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. biomedical laboratory science education: standardising teaching content in resource-limited countries in this original research... open access • abstract • introduction • research method and design • results • ethical considerations    • potential benefits and hazards    • recruitment procedures    • informed consent    • data protection • trustworthiness    • reliability • discussion • limitations of the study    • recommendations • conclusion • acknowledgements    • competing interests    • authors' contributions • references abstract top ↑ background: there is a worldwide shortage of qualified laboratory personnel to provide adequate testing for the detection and monitoring of diseases. in an effort to increase laboratory capacity in developing countries, new skills have been introduced into laboratory services. curriculum revision with a focus on good laboratory practice is an important aspect of supplying entry-level graduates with the competencies needed to meet the current needs. objectives: gaps in application and problem-solving competencies of newly graduated laboratory personnel were discovered in ethiopia, tanzania and kenya. new medical laboratory teaching content was developed in ethiopia, tanzania and kenya using national instructors, tutors, and experts and consulting medical laboratory educators from the united states of america (usa). method: workshops were held in ethiopia to create standardised biomedical laboratory science (bmls) lessons based on recently-revised course objectives with an emphasis on application of skills. in tanzania, course-module teaching guides with objectives were developed based on established competency outcomes and tasks. in kenya, example interactive presentations and lesson plans were developed by the usa medical laboratory educators prior to the workshop to serve as resources and templates for the development of lessons within the country itself. results: the new teaching materials were implemented and faculty, students and other stakeholders reported successful outcomes. conclusions: these approaches to updating curricula may be helpful as biomedical laboratory schools in other countries address gaps in the competencies of entry-level graduates. introduction top ↑ laboratory capacity has been diminished in resource-limited countries, but efforts to improve capacity are ongoing at present.1 new technologies are being introduced into the laboratory service of developing countries, including automation, flow cytometry and molecular diagnostics.2 there is also a focus on developing good laboratory practices through quality systems.3 the american society for clinical pathology (ascp) has facilitated training-of-trainers (tot) in-service training workshops for working laboratory professionals in the areas of haematology, biochemistry, cd4 testing and management in many developing countries.successful implementation and maintenance of these technologies in public health laboratories has required both in-service training and the improvement of education in medical laboratory schools in order to invest in workforce development.4,5 during the time of new methodand instrument roll-out, in-service training has effectively helped increase laboratory capacity to meet immediate needs, but training organisations in developing countries find that other approaches are required for maintaining the momentum achieved following any training activities.6 likewise, input from laboratory supervisors and faculty of medical laboratory schools gathered at stakeholders’ meetings by the centers for disease control and prevention (cdc) and its partners revealed that new biomedical laboratory technician (bmlt) graduates do not have the knowledge or competencies needed in order to meet the needs of these new technologies in medical laboratories.7 recognised experts in science curriculum revision for global health settings have advised that education for students entering healthcare settings should be focused on useful skills, knowledge and attitudes for the workforce based on job descriptions and responsibilities.8,9 in order to make the new skills sustainable in the laboratory setting, the usa head office of the cdc directed its partner organisations to also provide a focus on medical laboratory school education to include revision of training materials to meet new competencies.7 education and technical training of individuals in vocational schools and colleges prior to entry into the workforce is termed pre-service training. this has been a major focus of the global outreach mission of the ascp.10,11,12 the ascp developed a pre-service training programme which takes place in each country over an 18 month to 24 month timeframe, involving revision of curricula and teaching materials and providing training to teaching staff of biomedical laboratory science (bmls) schools. a successful aspect of eventually increasing laboratory capacity in developing countries is through training of students in medical laboratory schools and, specifically, through curriculum revision. curricula were revised and aligned so that both knowledge and practical skills were taught and assessed using a variety of educational resources, based on recognised educational principles.13 in addition, each curriculum was written to include a focus on applications and problem-solving outcomes, rather than focusing on the recall of information. countries requesting assistance in building laboratory capacity, with support from usa cdc, called for technical assistance in curriculum revision in certain bmls programmes. the ascp and the cdc instituted curriculum revisions and development of new teaching content in several african countries, including ethiopia, kenya and tanzania following their request for assistance. this process began in 2007 when the ascp assembled a team of usa -based consultants. these consultants are usa bmls educators with proficiency in assessment, curriculum development, and teaching techniques as well as expertise in one or more disciplines within bmls. this consultancy team created lessons for each discipline (e.g. haematology, biochemistry, immunohematology, parasitology, microbiology, histology, cytology and management). using a model for effective curriculum revision and implementation similar to one recommended by an internationally-recognised trainer,8 these consultants met in each country with bmls educators from their respective schools and with stakeholders (including experts from national reference laboratories, national cdc staff and laboratory staff from the ministry of health) for on-site assessments, assistance in curriculum review and revision, and mentorship as part of the pre-service training programme.10,14,15 following pedagogical training of the teaching staff and curriculum revision, the general process for developing outcomes-based teaching content was to conduct workshops in order to create lesson plans and teaching lessons. biomedical laboratory leadership from each country selected a different lesson plan template in consideration of their own needs based on stakeholder and faculty input. l. cuban, a recognised leader in education, has noted that there are often difficulties in implementing official curricula, which may arise as a result of personal choices or due to level of teaching experience.16 in order to minimise these difficulties and discrepancies in what is actually taught in the classroom, a harmonised curriculum and standardised teaching content were developed in ethiopia, tanzania and kenya, so that all students graduated with equivalent knowledge and competencies. this stage of curriculum revision involved the formation of teaching materials, lectures, exercises, and hands-on activities for laboratory students to learn. oneto three-week workshops were held prior to the start of the new teaching year to provide the opportunity to develop standardised teaching content in order to meet the newly-revised learning objectives. these lesson plans were shared with participants in the workshop setting and offered an opportunity to practise teaching a new topic with evaluation and input from facilitators and stakeholders. research method and design top ↑ can lesson plans and teaching lessons developed in-country with national teaching staff and international consultants be implemented effectively in order to teach newly-revised curricula? this case study describes the outcomes of these workshops held to develop teaching content with bmls educators from three different countries. observations and anecdotal evidence are provided in this manuscript so as to answer this question.in ethiopia, tanzania and kenya, following needs assessments of representative laboratory schools and stakeholders’ meetings to identify the scope of work and competencies needed for entry-level graduates, the medical laboratory science curriculum was revised with input from both national bmls educators and usa bmls educator experts. following approval of the curriculum, workshops were held with the same educators and usa consultant educators to develop standardised medical laboratory teaching content. whilst differences existed in the outcomes and steps taken with the medical laboratory schools in the three african countries, there were also similarities in the overall process. in two workshop settings, biomedical laboratory science faculty members from five universities in ethiopia, along with the usa consultants, developed standardised lessons for each of the 26 professional medical laboratory courses in the four-year bachelor of science (bsc) curriculum. interactive presentations and lesson plans, developed independently by both usa-based consultants and the in-country medical laboratory educators, served as resources to assist in developing country-specific lectures. the lesson content in each of the 26 medical laboratory courses was developed to meet current course-learning objectives with an emphasis on application of skills. in the first workshop held in ethiopia, faculty experts created lessons collaboratively in small groups with input from larger groups represented by the five universities. the lessons were designed to fit a 1 to 2 hour didactic teaching session or a 3 hour practical session. these lessons contained references, instructor notes, appropriate illustrations, pedagogically-interactive study questions and activities. the electronic slide-presentation lessons emphasised an application of cognitive skills. approximately half of the teaching content was developed for the entire professional curriculum in the first workshop. following this workshop, the national educators continued to develop the lectures and other teaching aids for their courses. additionally, the standardised electronic presentation lectures were reviewed and gaps were identified. the same national medical laboratory faculty and usa consultants participated in a second workshop to complete the task of developing standardised teaching content (including didactic and practical sessions for all courses) and addressing gaps in the first lesson plans identified during implementation based on anecdotal evidence from the national bmls educators. in tanzania, the usa consultants met in a workshop with tutors and experts from 10 government and private medical laboratory schools to develop standardised teaching content. this process was similar to that described for ethiopia; however, the newly-developed content was unique in that it was developed for an articulated competency-based curriculum. in competency-based curricula, modules are used rather than courses. the module content was created from various series of hierarchical outcomes following a detailed analysis of competencies required in the workplace so that specific entry-level workplace tasks could be taught and assessed. in addition, the format of developing the standardised teaching content was unique when compared with the other examples described because a detailed lesson plan in the form of a teaching-session guide was created, rather than lectures with notes. module teaching-session guides, based on established competency outcomes and tasks, were developed in small groups by both the faculty experts and usa educator consultants. each teaching-session guide included, (1) the module and session title, (2) learning objectives, (3) total session time, (4) resources needed, (5) content outline, (6) more detailed content listed in order with illustrations, (7) pedagogically-interactive activities, (8) at least one assessment item and (9) references. in kenya, the usa consultant educators developed and delivered approximately 80 lesson plans with accompanying slide presentation lessons that addressed theory and practical skills for new technologies and topics as identified by the needs assessment. this was provided at a workshop in which the kenyan medical laboratory lecturers from 11 campuses worked collaboratively with the usa consultants and stakeholders. in discipline-specific groups, the lesson plans were adapted to kenyan-specific terminology and laboratory applications. this teaching content served as a resource and foundation for the lectures to be adapted as needed at any time after the workshop concluded.15 for kenya, the decision to include all medical laboratory lecturers from all 11 campuses was made to facilitate consensus for use of the didactic and laboratory lesson plans when teaching the courses.15 results top ↑ the faculty spokespersons from the five universities in ethiopia were given a short survey via email, which elicited a 20% return rate. the faculty spokespersons from six laboratory schools in tanzania were given a short survey via email and there was a 16.7% return rate. in addition, direct observations were made by technical staff from the ascp in seven out of eight laboratory schools. there was a 100% return rate of the direct surveys given to faculty spokespersons from the 11 campuses of the kenyan laboratory schools.the five universities in ethiopia implemented standardised electronic slide presentation lectures for 26 medical laboratory professional courses. anecdotal comments from the survey of biomedical laboratory schools’ spokespersons (sp) provided the following information regarding the use of standardised teaching lesson plans in the first year: ‘the materials become uniform throughout the country, which benefits both faculty and students.’ (sp1, department chair) ‘students say they are better able to understand and learn the subject matter because the lectures and labs are more organised and more interactive.’ (sp1, department chair) ‘students report that they know exactly what is expected of them and that they are better prepared for exams from the wording of the objectives.’ (sp1, department chair) ‘the laboratory schedule for each lecture/course helps avoid ambiguity and ensures lab space is available as needed.’ (sp1, department chair) ‘the only reported drawback so far comes when faculty only use the outline materials on the powerpoint® slides and do not provide supplementary information. this makes more in-depth learning difficult for students without access to textbooks or the internet.’(sp1, department chair) eight of the 10 medical laboratory programmes in tanzania implemented the teaching-session guides for the year-one course modules and implementation was verified by direct observation by the ascp technical staff. anecdotal comments from a survey of bmls spokespersons regarding the use of the lessons during the first year were as follows: ‘the general feeling among students taking this courses is that there is good understanding, although we did not give a questionnaire to see how many had a feeling of good understanding or not good understanding.’ (sp2, head of school) ‘we enrolled 91 students in the nta level-four, semester one, for academic year 2010–2011, but [the number that] remain [are] 77 students (and, therefore, 14 failed to progress).’ (sp2, head of school) ‘what we did is that we compared the [number of students who] failed [with those who] passed, [and the] majority passed.’ (sp2, head of school) results from a survey conducted by ascp one year post-implementation of the standardised teaching content with medical laboratory lecturers from kenya revealed the following: ‘faculty like using the same lesson plan template for all lectures and labs as the materials are all in the same place and easy to find and use. they also report that following the lesson plan and objectives keeps them on task. at the end of the lesson, they know they have covered all the materials related to the topic. previously, lecturers reported they wandered around and got off-topic and weren’t sure what materials they had or had not covered.’ (sp3, head of school) ‘lecturers reported that they liked the fact that students on every campus are receiving the same information in each class and are all prepared similarly at graduation with regard to knowledge and skills’. (sp3, head of school) ‘students reported that they liked the lessons better because the lecturers stayed on topic and the material was easier to learn. students like having objectives because objectives help them know how in-depth they need to focus on specific information and because objectives help them study for exams.’ (sp3, head of school) ‘students appreciate (powerpoint®) handouts so they can look over the information before class. they can also follow along with the lecturer more easily and, because they aren’t consumed with taking copious notes, they are able to use critical thinking skills to ask questions about the materials during the lecture or lab class.’ (sp3, head of school) ethical considerations top ↑ this study did not involve experimentation with human subjects. potential benefits and hazards there were no risks to the subjects of this study. recruitment procedures all participants in the development and assessment of the curricula, did so as part of their work responsibilities and were invited to participate with formal letters of invitation from their ministries of health. informed consent no informed consent was needed or received. data protection anecdotal data was received via email from a secured website. trustworthiness top ↑ anecdotal data in the form of comments was listed directly in the study and not altered. reliability no follow up to the results has been provided at this time. discussion top ↑ lesson plans and lessons can be developed in-country with national teaching staff and international consultants in a workshop setting and can be implemented effectively in order to teach newly-revised curricula based on this evidence from three countries and 26 laboratory schools. needs assessments prior to these curriculum workshops indicated a lack of standardised teaching content, especially in new technologies or with quality assurance. different formats for lesson plans can be used based on the type of curriculum and the level of expertise of the teaching staff but it is important to create formal lesson plans or teaching-session guides when implementing a new curriculum that includes many new theoretical or practical skills.limitations included the need for alternative methods of delivering the electronic lessons when power supplies were interrupted or when computer equipment needed to be shared amongst multiple instructors. having printers to print out lessons bridged some gaps when instructors had to revert to the blackboard or whiteboard to deliver visual aspects of the lessons. in addition, not all faculty members were able to attend the teaching content development workshops and, consequently, other teaching staff may have developed lessons for them. if proper mentoring was not provided for the use of these lesson plans, the new lesson plans became a limiting step for novice faculty members. turnover in teaching staff sometimes accelerated this limitation. another limitation was in planning realistic lesson plans for hands-on practice that matched the current or expected available resources. sometimes the new curriculum called for teaching practical skills that were not fully resourced as hands-on lessons. lesson plans that contained the critical-thinking skills but had simulations of missing resources were developed in the workshops in order to bridge gaps with respect to immediate resource limitations. lessons learned regarding the implementation of new content to meet the revised bmls curriculum include the following main points: • it is critical to the overall success of pre-service activities to conduct a pre-assessment of the university’s resources and inventory in order to circumvent challenges when the implementation of teaching content begins. for example, if lessons are developed in powerpoint®, the sustainability of the resources and the lecturers’ skills must be maintained or the lessons will not be possible to implement effectively. • prior to the development and implementation of teaching content, it is important to the success of the pre-service programme to provide teaching-methods training to the instructors, including interactive teaching strategies, objective-writing practice, and effective practices in the development and use of electronic teaching media (such as powerpoint® and word®). an emphasis on the mechanics of good visual appearance and animation was relevant. • it was found to be important to plan for periodic meetings and reviews between faculty, campuses and universities after implementation in order to build a support system for sustainability of the curriculum and retention of the faculty. similarities in developing teaching content for these three african countries included the following: • teaching content was developed to address a standard set of medical laboratory competencies in all three countries. • this was accomplished by a systematic approach to identifying the competencies and by including both stakeholders and usa educator consultants. • in all three countries, the common elements of teaching content and standard format were determined in large-group settings; specific discipline content was developed in small-group settings. • all three countries developed standardised teaching content for use in bmls programmes throughout their country. • all three countries implemented the standardised teaching content for the new curriculum successfully. differences in the implementation of content in these three countries included the following: • teaching content for tanzania was developed as teaching-session guides and organised in teaching guides for competency-based modules, whilst teaching content used in ethiopia and kenya was developed in lesson plans and electronic presentation format and organised within the framework of traditional courses. • teaching content for tanzania was organised in a prescriptive teaching guide with nine mandatory components, as opposed to teaching content organisation in ethiopia and kenya, which was less prescriptive, allowing for more flexibility in delivery style. • standardised teaching content for ethiopia and tanzania was completed with direct usa consultant involvement in a workshop setting, whilst the standardised teaching content for kenya was completed by all campus lecturers following the workshop without direct involvement from the usa consultants. limitations of the study top ↑ due to the limitations of funding for the study, stakeholders have not been surveyed to determine if the newly-revised and implemented curricula meet the gaps in competencies that previous curricula did not fill. the number of new graduates trained with this new curricula have been quantified. recommendations in order to ascertain whether the new curricula provide the necessary competencies to meet the needs of each country and increase laboratory capacity in quantity and quality, surveys of employers and other stakeholders in these countries should be implemented, analysed and reported as part of future research studies. conclusion top ↑ teaching content should be revised to match the needs of the workforce and can effectively be developed by collaboration of national bmls instructors with international bmls educator consultants. new teaching methods, including the use of electronic slide presentations, can be used to implement content that will address new skills and emerging technologies. providing new technologies and methods for teaching content, however, should not eliminate the need for interactive strategies to involve students in active learning. in other words, the immediate appeal of animation and images that can be provided by electronic slide presentations should be used to enhance active (rather than passive) learning. the implementation of curriculum teaching content must be in conjunction with review and revision of assessments in order to ascertain that they address directly the knowledge, skills and attitude objectives specified in each course or module. the authors anticipate that by sharing this information, it may be helpful to biomedical laboratory faculty and administrators in other countries as they consider how to revise and implement standardised curriculum content. acknowledgements top ↑ the authors wish to acknowledge mr mistire wolde (addis ababa university), mr peter kariuki (kenya medical training college) and mr. manase nsunza (singida, tanzania) for their contributions to the evaluation phase of this project. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. authors’ contributions w.a. and c.r. 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[c1995] [cited 2013 may 03] available from: http://larrycuban.files.wordpress.com/2011/08/1476538.pdf abstract background conception, construction and assembly of the modular biosafety level 3 laboratory impact: system strengthening and drug-resistant tuberculosis diagnostic capacity barriers encountered in the process of establishing and maintaining biosafety level 3 laboratory services in nigeria discussion conclusion acknowledgements references about the author(s) gambo aliyu institute of human virology, university of maryland school of medicine, baltimore, maryland, united states nicholas ezati institute of human virology, abuja, fct, nigeria mosunmola iwakun institute of human virology, abuja, fct, nigeria sam peters institute of human virology, abuja, fct, nigeria alash’le abimiku institute of human virology, university of maryland school of medicine, baltimore, maryland, united states citation aliyu g, ezati n, iwakun m, et al. diagnostic system strengthening for drug resistant tuberculosis in nigeria: impact and challenges. afr j lab med. 2017;6(2), a502. https://doi.org/10.4102/ajlm.v6i2.502 lessons from the field diagnostic system strengthening for drug resistant tuberculosis in nigeria: impact and challenges gambo aliyu, nicholas ezati, mosunmola iwakun, sam peters, alash’le abimiku received: 10 june 2016; accepted: 17 nov. 2016; published: 31 mar. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the increasing prevalence of drug-resistant tuberculosis and the threat of extensively-drug-resistant tuberculosis in hiv hotspots have made the detection and treatment of drug-resistant tuberculosis in the sub-saharan africa setting a global public health priority. objective: we sought to examine the impact and challenges of tuberculosis diagnostic capacity development for the detection of drug-resistant tuberculosis and bio-surveillance using a modular biosafety level 3 (bsl-3) laboratory in nigeria. method: in 2010, the united states president’s emergency plan for aids relief (pepfar) programme, through the institute of human virology at the university of maryland in baltimore, maryland, united states, deployed a modular, bsl-3 laboratory to support the national tuberculosis programme in drug-resistant tuberculosis detection and bio-surveillance for effective tuberculosis prevention and control. results: from 2010 until present, sputum samples from 11 606 suspected cases in 33 states were screened for drug-resistant tuberculosis. of those, 1500 (12.9%) had mono-resistant tuberculosis strains, and 459 (4.0%) cases had multidrug-resistant tuberculosis. over the last four years, 133 scientists were trained in a train-the-trainer programme on advanced tuberculosis culture, drug susceptibility testing, line-probe assays and xpert® mtb/rif, in addition to safety operations for biosafety facilities. power instability, running cost and seasonal dust are notable challenges to optimal performance and scale up. conclusion: movable bsl-3 containment laboratories can be deployed to improve diagnostic capacity for drug-resistant tuberculosis and bio-surveillance in settings with limited resources. background nigeria has a limited high-level certified laboratory infrastructure and trained human resources to support a comprehensive public health response to the tuberculosis pandemic.1 until recently, the conventional culture-based drug susceptibility testing (dst) platforms for detecting drug-resistant tuberculosis were available only in select public and private laboratories.2 presumptive cases of multi-drug resistant (mdr) tuberculosis from different parts of the country went to the distant coastal city of lagos for diagnosis and at the nigerian institute for medical research, which housed the only tuberculosis reference laboratory for a population of over 170 million. the number of culture reference laboratories has risen to seven, yet they covered only 4% to 8% of the world health organization-recommended population target of one functioning culture laboratory per 500 000 to one million population.3 according to a 2011 who report on global tuberculosis control, two in 100 of the newly detected tuberculosis cases and nine in 100 of re-treated cases in nigeria were mdr tuberculosis.4 the national tuberculosis and leprosy training centre in zaria has the largest tuberculosis referral center in northern nigeria, with an average of 25–30 new smear-positive tuberculosis cases enrolled in treatment and care monthly; about 27% of the enrolled tuberculosis cases are co-infected with hiv.5 in addition to the reference laboratory, the centre has a large outpatient clinic for the management of tuberculosis, hiv and leprosy. it also has training facilities as the national training centre for tuberculosis and leprosy. it has a modest inpatient facility, mainly for the treatment of mdr tuberculosis, with a 20-bed capacity and two isolation rooms. with over 80% of estimated tuberculosis cases currently undetected,6 the steady rise in case notification from 40 000 cases in 1999 to about 140 000 in 2010,7 coupled with increasing prevalence of drug-resistant tuberculosis and the potential threat of co-infection with hiv in patients with drug-resistant tuberculosis, underscore the need to strengthen diagnostic capacity for detection of tuberculosis and mdr tuberculosis in nigeria.8,9,10 the scale-up of cepheid genexpert® for mycobacterium tuberculosis and rifampicin resistance detection within a two-hour time frame requires complementary capacity for confirmatory dst with culture or line-probe assays. to enhance detection of tuberculosis, mdr tuberculosis and extensively-drug-resistant tuberculosis, as well as treatment monitoring across nigeria, the establishment of biosafety level 3 (bsl-3) laboratories with capacity for culture and dst is a priority. however, the few existing containment laboratories in nigeria function suboptimally, with frequent breakdowns linked to design, structural complexities, adoptability and a harsh operational environment. to guarantee the optimal performance required of biosafety facilities to meet the unmet needs of the national tuberculosis programme, the institute of human virology at the university of maryland in baltimore, maryland, united states, developed a modular pre-constructed bsl-3 laboratory for the national tuberculosis and leprosy training centre in zaria, kaduna state, nigeria, with a five-year contractual agreement for service and local staff training in bsl-3 laboratory operation and maintenance. in this article, we describe the establishment of the prototype of the modular bsl-3 laboratory as a platform for mdr tuberculosis surveillance in nigeria and its impact on system strengthening and the challenges encountered. conception, construction and assembly of the modular biosafety level 3 laboratory following several failed attempts to upgrade or renovate existing structures and uncertainties about the structural building requirements to withstand the negative pressure required for such laboratories, germfree laboratories, inc., based in ormond beach, florida, united states, was contracted by the institute of human virology at the university of maryland to design, construct, deliver and assemble the movable bsl-3 laboratory with input from the institute’s staff. following several modeling sessions, the laboratory was designed using two 40-foot containers with all the equipment required to make it functional. it was then disassembled and the components and accessories were shipped to lagos, nigeria, and delivered to zaria by oversized haulage truck to the national tuberculosis and leprosy training centre, where the laboratory is currently located (figure 1). figure 1: delivery and assembly of the modular bsl-3 laboratory, zaria, kaduna state, nigeria, 2010. the laboratory has the following sections: (a) an alcove; (b) a specimen receipt area with an autoclave and a pass-through-interlocked window where samples are received from outside, checked and registered; (c) a processing and inoculation section containing three biological hoods and a centrifuge; (d) a culture manipulation room with two bactec mgit 960 (becton dickinson, franklin lakes, new jersey, united states) instruments; and (e) a mechanical room. in the culture manipulation room, growth tubes with inoculated samples are grown in one bactec mgit 960 instrument and dst is done in the other. the mechanical room houses all regulatory mechanisms, a heating, ventilation, air-conditioning, cooling control system and control panels for the operation of the laboratory, which can be viewed remotely from germfree laboratories in florida (figure 2). this is used for monitoring the functionality of the laboratory, and for the training and mentoring of nigerian biotech engineers. laboratory scientists from different parts of country receive trainings on good laboratory practices, assays, advanced culture with dst, and handling of hazardous materials. figure 2: sections of the modular bsl-3 laboratory, zaria, kaduna state, nigeria, 2010. two backup generators, a 250 and a 100 kva, are used to supplement the public power supply to provide a 24-hour seven-day supply of electricity. the heating, ventilation, air-conditioning, cooling system and all equipment are further supported through a 60 kw and 30 kva three-phase online power inverter system. to keep rodents out, a mesh fence was built around the laboratory and fumigation was performed. the estimated cost for the modular bsl-3 laboratory, including equipment, delivery and the concrete platform upon which the lab rests, was $720 000. the two power generators cost $70 105, while the walkways, overhead water tanks, emergency showers, closed-circuit television system and other structural work cost $80 749. the sum of the estimated expense for the establishment of the modular bsl-3 laboratory was $870 854. impact: system strengthening and drug-resistant tuberculosis diagnostic capacity the modular laboratory serves as one of the two tuberculosis national reference laboratories in nigeria. services were mostly limited to the northern states until 2013, but by 2015 both northern and southern states were served (figure 3). the laboratory performs and builds in-country capacity to perform solid and liquid cultures, dst, line-probe assays and genexpert tests. the line-probe assay and genexpert tests are not performed in the bsl-3 laboratory, but in a separate laboratory in the same complex by the same laboratory personnel. figure 3: sample referral trends in different parts of nigeria for drug-resistant tuberculosis testing at the modular bsl-3 laboratory, zaria, kaduna state, nigeria, 2010. a total of 89 scientists have received training on advanced tuberculosis culture, drug susceptibility testing and line-probe assays, and 44 scientists from nine states have been trained on genexpert. these include state external quality assurance officers and programme staff responsible for activating new genexpert sites, monitoring and supervision. the trained scientists from zonal tuberculosis reference laboratories now perform these tests in their respective laboratories and train their colleagues. the bsl-3 laboratory is also used for the preparation of external quality assurance panels for genexpert tests, line-probe assays and tuberculosis culture and dst tests across the country. over the period from january 2010 to april 2016, sputum samples from 11 606 presumptive drug-resistant tuberculosis cases in 33 states were processed using solid and liquid cultures, then dst was performed using line-probe assays or solid proportion and bactec mgit 960 methods. of those, 1500 (12.9%) had mono-resistant strains, while 459 (4.0%) cases had mdr tuberculosis. the laboratory currently collaborates with the san rafael world health organization supranational reference laboratory in milan, italy, the national institute of communicable diseases in south africa, and the uganda supranational tuberculosis reference laboratory on external quality assessment, and is enrolled in the world health organization regional office for africa’s strengthening laboratory management towards accreditation step-wise accreditation programme with support from the african society for laboratory medicine and the medical laboratory science council of nigeria. the laboratory supported the 2010–2011 national prevalence survey for mdr tuberculosis and the 2012 tuberculosis prevalence survey in nigeria. barriers encountered in the process of establishing and maintaining biosafety level 3 laboratory services in nigeria as in most developing countries, an erratic power supply is a major challenge in nigeria. the modular bsl-3 laboratory was operated on stand-by generators supported with a high capacity inverter system to ensure a steady supply of electricity. this added to the cost of operationalising the laboratory. the dry harmattan wind from the sahara desert generates a lot of dust in winter in the north of nigeria, where the laboratory is located. germfree industries, inc. designed a pre-filtration unit using easily replaceable filters to filter the air supply before it reaches the more costly high-efficiency particulate air supply filters of the heating, ventilation and air conditioning system. the public water supply at the site was infrequent and insufficient for laboratory use at the time of installation. an overhead tank was designed specifically to supplement the public water supply for the laboratory. a water filter was attached to ensure that clean water reached the distiller. waste disposal was a concern and was addressed by passing liquid waste through a pre-treatment system before it is deposited into a septic tank. spillage incidents at the operational training sessions necessitated the provision of an additional emergency shower to enhance personal protection. access to the shower had to be fabricated to link it to the two emergency doors of the laboratory. discussion there are few bsl-3 tuberculosis laboratory prototypes in the sub-saharan african setting. the modular bsl-3 laboratory offers a useful alternative for construction of such a facility. lack of indigenous firms with professional expertise in the construction and maintenance of a bsl-3 containment laboratory to international standards – which is a critical first step in establishing a biosafety laboratory11 – informed the decision to buy a pre-constructed model. a modular laboratory was deployed by zamstar in 2009 in zambia to conduct a national prevalence survey. like our model, it was constructed in a 40-foot container with all equipment and accessories.12 however, compared with the zamstar model, our model cost more, because of additional accessories including power backups, cameras, walk ways, and overhead water tanks required for optimal functioning of the laboratory. although the establishment of this laboratory appears to be expensive in the beginning, given the unmet need for expertise in biosafety containment and development of local capacity, it is an investment worth the cost. during the five years after its instalment, operations have been optimised to meet the local needs of the national tuberculosis programme without a break in service delivery. with support from our modular bsl-3 laboratory, nigeria was able to conduct national tuberculosis surveillance on a representative sample with best estimates of tuberculosis and drug-resistant tuberculosis prevalence. the scientists trained in tuberculosis detection and dst from various zonal laboratories that form the country’s tuberculosis laboratory network are now supporting tuberculosis diagnosis and treatment monitoring of patients in their respective zones. this capacity development programme has helped expand the number of laboratories supporting the tuberculosis control programme from three in 2010 to seven in 2016. with the increasing demand for services resulting from the scale-up of innovative strategies for intensified case finding, this investment is expected to contribute to epidemic control by improving early detection and monitoring of treatment of drug-resistant tuberculosis in nigeria. elsewhere in africa, locally constructed bsl-3 laboratories are able to operate optimally.13,14 however, in many settings within sub-saharan africa, the experience and expertise of local construction workers may not guarantee internationally-accepted standards for biosafety containment facilities.11,13 to further guarantee standards and prevent failures experienced on previous attempt to integrate such services in locally constructed laboratories, the pepfar programme entered into a service contract with germfree laboratories, inc. this allowed the local team of biomedical engineers, who would be shouldered with the responsibility of maintaining the unit for the long term, to work closely with the germfree team for a brief period after the installation. now that the unit is locally maintained, germfree only performs annual certification and preventive maintenance. the laboratory is linked to suprareference laboratories for external quality assessment. our experience provides evidence that it is feasible to establish and maintain modular containment laboratories in settings with limited resources. international donor agencies and policy makers should consider replicating this model in high tuberculosis-burden settings, especially in places where expertise in construction and supervision of bsl-3 facilities are not locally available, as demonstrated in the case of nigeria. these laboratories are easy to integrate into national tuberculosis programs and serve as training facilities for capacity development. upgrading an existing tuberculosis laboratory to a bsl-3 standard for detection of drug-resistant tuberculosis, as was successfully done in the kingdom of lesotho, is another alternative way of providing the needed diagnostic services.14 if that is considered, the experience and expertise of the construction firm is necessary to guarantee internationally-acceptable standards. conclusion a modular bsl-3 laboratory was deployed to strengthen detection of tuberculosis and drug-resistant tuberculosis in nigeria. it operates optimally and collaborates effectively with established international laboratories to strengthen the national tuberculosis programme. priority should be given to modular laboratories in settings with limited expertise in building facilities for the isolation and characterisation of dangerous biological agents such as drug-resistant and mdr tuberculosis. since the establishment of this first prototype model in a developing country setting, germfree laboratories, inc. has used the knowledge acquired to establish a modified and more affordable biosafety level 2 system that has the capacity for bsl-3 practices with the addition of a class iii biosafety cabinet, also referred to as glove box. the addition of the class iii biosafety cabinet with a direct sample entry pass-box makes it possible to safely handle highly-infectious samples. box 1: lessons learned. acknowledgements competing interests the interpretation and presentation of the data presented in this report were not influenced by any of the authors’ personal or financial relationships with other people or organisations. sources of support establishment of the modular bsl-3 laboratory to support nigeria’s national tuberculosis control programme was funded by the cdc pepfar programme through the institute of human virology, university of maryland school of medicine. authors’ contributions g.a. conceived the idea, analysed data and wrote the first draft. n.e. and m.i. collected the data and contributed to writing of the first draft. m.i., s.p. and a.a. critically review the draft for intellectual content. references parsons lm, somoskövi a, gutierrez c, et al. laboratory diagnosis of tuberculosis in resource-poor countries: challenges and opportunities. clin microbiol rev. 2011;24(2):314–350. https://doi.org/10.1128/cmr.00059-10 lawn sd, mwaba p, bates m, et al. advances in tuberculosis diagnostics: the xpert mtb/rif assay and future prospects for a point-of-care test. lancet infect dis. 2013;13(4):349–361. https://doi.org/10.1016/s1473-3099(13)70008-2 world health organization. policy framework for implementing new tuberculosis diagnostics. who: geneva, switzerland; 2010. world health organization. global tuberculosis control report (2011) [document on the internet]. c2011 [cited 2017 jan 16]. available from: https://apps.who.int/iris/bitstream/10665/44728/1/9789241564380_eng.pdf aliyu g, el-kamary ss, abimiku a, et al. prevalence of non-tuberculous mycobacterial infections among tuberculosis suspects in nigeria. plos one. 2013;8(5):e63170. https://doi.org/10.1371/journal.pone.0063170 world health organization. global tuberculosis report. 2015. who: geneva, switzerland; 2015. united states embassy in nigeria. nigeria tuberculosis fact sheet [document on the internet]. c2012 [cited 2016 oct 29]. available from: https://photos.state.gov/libraries/nigeria/487468/pdfs/january%20tuberculosis%20fact%20sheet.pdf aliyu g, el-kamary ss, abimiku a, et al. mycobacterial etiology of pulmonary tuberculosis and association with hiv infection and multidrug resistance in northern nigeria. tuberc res treat. 2013;2013:650561. https://doi.org/10.1155/2013/650561 dinic l, akande p, idigbe eo, et al. genetic determinants of drug-resistant tuberculosis among hiv-infected patients in nigeria. j clin microbiol. 2012;50(9):2905–2909. https://doi.org/10.1128/jcm.00982-12 lawson l, yassin ma, abdurrahman st, et al. resistance to first-line tuberculosis drugs in three cities of nigeria. trop med int health. 2011;16(8):974–980. https://doi.org/10.1111/j.1365-3156.2011.02792.x mourya dt, yadav pd, majumdar td, et al. establishment of biosafety level-3 (bsl-3) laboratory: important criteria to consider while designing, constructing, commissioning & operating the facility in indian setting. indian j med res. 2014;140(2):171–183. kosloff br, de haas p. muyoyeta m, et al. use of containerized laboratories in the zamstar tb prevalence survey in zambia: a progress report [document on the internet]. c2010 [cited 2017 jan 16]. available from: https://www.zambart.org.zm/wp-content/uploads/2015/04/zamlab-ctl-poster-iuatld-2010-ps-101361-14.pdf ssengooba w, gelderbloem sj, mboowa g, et al. feasibility of establishing a biosafety level 3 tuberculosis culture laboratory of acceptable quality standards in a resource-limited setting: an experience from uganda. health res policy syst. 2015;13:4. https://doi.org/10.1186/1478-4505-13-4 paramasivan cn, lee e, kao k, et al. experience establishing tuberculosis laboratory capacity in a developing country setting. int j tuberc lung dis. 2010;14(1):59–64. abstract introduction materials and methods results discussion acknowledgements references about the author(s) henry a. mbah labtrail global llc, smyrna, delaware, united states feyisayo e. jegede biological science department, bayero university kano, gwarzo road kano, nigeria family health international-360, garki,federal capital territory, abuja, nigeria surajudeen a. abdulrahman department of public health, penang medical college, penang, malaysia tinuade i. oyeyi biological science department, bayero university kano, gwarzo road kano, nigeria citation mbah ha, jegede fe, abdulrahman sa, oyeyi ti. evaluation of standard diagnostic rapid test kits for malaria diagnosis among hiv patients in kano, nigeria. afr j lab med. 2018;7(1), a698. https://doi.org/10.4102/ajlm.v7i1.698 original research evaluation of standard diagnostic rapid test kits for malaria diagnosis among hiv patients in kano, nigeria henry a. mbah, feyisayo e. jegede, surajudeen a. abdulrahman, tinuade i. oyeyi received: 06 oct. 2017; accepted: 13 aug. 2018; published: 05 dec. 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: malaria diagnosis among hiv-positive patients is uncommon in nigeria despite the high burden of both diseases. objectives: we evaluated the performance of a malaria rapid diagnostic test (mrdt) against blood smear microscopy (bsm) among hiv-positive patients in relation to anti-retroviral treatment (art) status, cd4+ count, fever, cotrimoxazole prophylaxis and malaria density count. method: a cross-sectional study involving 1521 consenting randomly selected hiv-positive adults attending two art clinics in kano, nigeria, between june 2015 and may 2016. venous blood samples were collected for testing with mrdt, bsm, and cd4+ t cells count by cytometry. biodata and other clinical details were extracted from patient folders into an excel file, cleaned, validated, and exported for analysis into spss version 23.0. sensitivity, specificity, predictive values of mrdt were compared with bsm with a 95% confidence interval. results: malaria parasites were detected in 25.4% of enrollees by bsm and 16.4% by mrdt. overall sensitivity of mrdt was 58% and specificity was 97%. cotrimoxazole prophylaxis and fever status did not affect mrdt sensitivity and specificity. unexpectedly, the sensitivity was highest at parasite density count of less than 500 cells/µl. at cd4+ t cells count over 500 cells/µl the sensitivity was higher (62.4%) compared to 56% at less than 500 cells/µl. in the non-art group sensitivity was higher (65%) compared to those on art (56%) but the specificity was similar. all differences were significant for all variables (p < 0.05). conclusion: although the mrdt specificity was good, the sensitivity was poor, requiring further evaluation for use in malaria diagnosis among hiv-malaria co-infected persons in these settings. introduction malaria remains a major public health problem in nigeria with 97% of the population at risk.1 in 2016 nigeria had about 57.3 million cases of malaria with over 112 000 deaths reported.2 although malaria prevalence in nigeria varied between states, a 60.6% prevalence has been reported in a population-based study across five local government areas of kano state.3 hiv prevalence among adults in nigeria was about 2.9% with about 3.2 million persons living with hiv in 2016.4 the greatest burden of disease due to both hiv and malaria (predominantly plasmodium falciparum) occurs commonly in sub-saharan africa5 and the rates of co-infection make interaction between both diseases a global health focus.5,6,7 a key factor for effective management of malaria is early and accurate diagnosis. the global impact of malaria has prompted an increase in the development of diagnostic strategies. the updated world health organization (who) recommendations indicate that all suspected cases of malaria must be confirmed with a laboratory diagnostic test before treatment.8 this enables differentiation of malarial and non-malarial fevers, to prevent unnecessary use of antimalarial drugs.9,10 this is very crucial among hiv-positive patients, where febrile illnesses other than malaria are common. opportunistic infections and other febrile illnesses mimic malaria in hiv patients.5 although microscopy is the current gold standard for the diagnosis of malaria, it is time consuming and labour intensive.11,12,13 in many malaria-endemic areas like nigeria, it is difficult to maintain the required technical skills, equipment and infrastructure particularly in rural areas,14,15 where the disease is more prevalent. hence, the use of rapid diagnostic tests (rdt) in such endemic areas is recommended.16,17 furthermore, part of the national malaria control strategy plan for 2014 to 2020, with a vision of having a malaria-free nigeria, is to test all care-seeking persons suspected of suffering from malaria using rdt or microscopy.1 data from reviewed literature showed that very few studies have evaluated the validity of rdt for malaria diagnosis in the general population in nigeria.14,15,18 thus far, only one study has evaluated the performance of a malaria rdt (mrdt) among 387 hiv-positive persons in nigeria.19 overall mrdt sensitivity, specificity, positive and negative predictive value have not yet been properly evaluated especially among hiv-positive individuals.20,21 the objective of the study was to evaluate the performance of a commercially available mrdt (based on p. falciparum histidine-rich protein type-2 and plasmodium lactate dehydrogenase), the standard diagnostics (sd) bioline mrdt, compared to the gold standard, microscopy, in the diagnosis of malaria among hiv-positive participants with respect to malaria parasite density, cd4+ t cells count, cotrimoxazole prophylaxis use, anti-retroviral treatment (art) status and occurrence of fever. the choice of sd bioline was on the premise that this rapid test kit is the most commonly used mrdt for routine malaria diagnosis in kano state. materials and methods ethical considerations the protocol was reviewed by kano state hospitals management board local ethical committee and written approval was given (study approval number: hmb/gen/488/1 of 17/04/2015). participant consent was obtained from all respondents. study setting and population this was a cross-sectional study carried out from june 2015 to may 2016 at two health facilities in kano city, nigeria, a malaria endemic area.3 the facilities are infectious diseases hospital kano (idh) and murtala mohammed specialist hospital kano. the population of interest in this study were hiv-positive adult participants who routinely visit the two health facilities to access art services. they consisted of male and female (non-pregnant) patients (on art and non-art) aged 18 years and older enrolled in an art programme who had not been on an antimalarial drug in the past 14 days and who willfully consented to participate in the study. data collection and sampling technique a total of 1521 consenting participants were randomly selected and blood specimens were collected and analysed for the presence of malaria parasite using both rdt and microscopy techniques and density of malaria parasites was determined. additionally, relevant socio-demographic and clinical data including clients’ code, age, gender, use of co-trimoxazole prophylaxis, art status (receiving or not receiving treatment), and episodes of fever were retrieved from both patients’ folders and lafiya management information system (lamis®) software with the support of trained data entry clerks. we ensured data confidentiality through participant coding and restricting access to the data to only research team members. laboratory analysis (rapid diagnostic test, blood microscopy and parasite density determination) from 1521 consenting hiv-positive participants, 4 ml venous blood sample was aseptically collected using sterile vacutainer needle and holder into an ethylenediaminetetraacetic acid (edta) anticoagulant tube. the blood samples were mixed properly to avoid clotting before laboratory investigations. we used the mrdt kit (code 05fk30) as described by the manufacturers (standard diagnostics bioline korea, 2013). for microscopy slide method, both thin and thick smear slides were prepared and examined. the microscopy procedure adopted was described by who.23 to ensure the quality of testing, mrtd and microscopy were performed by independent laboratory personnel and mrtd results were blinded to microscopists. all mrtd kits were stored according to manufacturer recommendation and used before the expiry date. the two microscopists responsible for slide examinations in both facilities have attended who training on malaria microscopy. additionally, 10% of the slides were randomly selected for re-testing. about 2% of the results were discordant between the first and second readers and were subjected to further evaluation by a third reader before a final decision was made. malaria parasite density for each positive smear was calculated using individual white blood cell count according to this formula: data processing and analysis data were reviewed, cleaned and validated in a microsoft 2013 excel file (microsoft corp., redmond, washington, united states) and analysed using statistical package for social scientists (spss version 23.0; ibm, armonk, new york, united states). the sensitivity, specificity, positive predictive value and negative predictive value of mrdt were estimated against blood smear microscopy (bsm), the gold standard, at 95% confidence interval in relation to (1) malaria parasite density count, (2) fever (presence or absence), (3) cd4+ t cells count, (4) co-trimoxazole prophylaxis use, and (5) art status (currently on art or not taking art). in addition, differences in performance of mrdt against bsm were analysed (overall and for sub-groups of the five variables mentioned above) based on level of ‘equality of marginal positives’ in disease classification between the two using mcnemar tests and significance level was set at p < 0.05. results study population this study included a total of 1521 hiv-positive participants consisting of 1074 (70.6%) women and 447 (29.4%) men. the mean age was 37.20 with a standard deviation (sd) of 10.41 years. data on clinical information showed that the majority (84.7%) were on art. an almost equal proportion of the participants (n = 772, 50.8%) were on daily dose co-trimoxazole prophylaxis for prevention against opportunistic infection including malaria parasite infection while 749 (49.2%) were not on co-trimoxazole prophylaxis. about (7%) complained of fever. the mean current cd4+t cells count of the participants was 401.35 cells/µl ± 239.24 cells/µl, and ranged from 6 cells/µl to 1736 cells/µl. the majority (n = 1038, 68.2%) of the respondents had a cd4+t cells count lower than 500 cells/µl, while 31.8% (n = 483) had cd4+ t cell counts of 500 cells/µl or higher. the mean malaria parasite density was 265 cells/µl ± 31.8 (sd) cells/µl with a range of 20 cells/µl to 2500 cells/µl. a majority (87.8%) had malaria parasite density less than 500 cells/µl, while the rest (12.2%) had parasite density 500 cells/µl or higher. prevalence of malaria parasite among hiv-positive patients based on microscopy versus malaria rapid diagnosis test the prevalence of malaria was 25.4% with bsm technique (gold standard method) and only 16.4% with mrdt. it is worth nothing that participants were recruited at routine art follow-up visits, with or without symptoms of malaria. almost all (99.2%) malaria infections were plasmodium falciparum species while dual infection of plasmodium falciparum and plasmodium vivax species occurred in only three (0.8%) participants (table 1). the overall sensitivity of the mrdt method was 58% and specificity 97%. the positive predictive value was 89% and the negative predictive value was 87% (table 2). a 2.5% (28/1135) false positive result and 27.6% (55/200) false negative result were observed. table 1: prevalence of malaria parasite among hiv-positive patients determined by malaria rapid diagnosis test and standard blood smear microscopy. table 2: overall performance of malaria rapid diagnosis test in comparison with standard blood smear microscopy among hiv positive participants. overall, there was significant difference in mrdt performance compared to bsm (p < 0.001), with the mrdt demonstrating a ‘fair’ performance and discriminatory capacity of 0.775 (area under the curve = 0.775, 95% ci: 0.743–0.804) of the studied population. performance of malaria rapid diagnostic test in comparison with standard microscopy different malaria parasite densities the mrdt showed a sensitivity of about 72% among hiv-positive patients with malaria density count between 0 and 499 cells/µl and greater than 1000 cells/µl. unexpectedly, a low sensitivity of about 34% was observed when density was between 500 and 999 cells/µl (table 3). table 3: performance of malaria rapid diagnostic test in comparison with standard blood smear microscopy at different malaria parasite density, fever status, cotrimoxazole prophalaxis,cd4+ count and art status mrdt performance was significantly different from bsm findings, regardless of malaria parasite density count of participants (p < 0.005). presence or absence of fever the mrdt showed slightly lower but similar sensitivity in cases with and without fever, but similar specificity for both groups of participants. however, positive predictive value was higher (95.6%) among clients that complained of fever compared to those without (88.1%) fever (table 3). in those with or without fever, mrdt performance was significantly lower compared to bsm findings (p < 0.0) co-trimoxazole prophylaxis mrdt performed similarly in sensitivity and specificity in both groups, although higher positive predictive value was observed among participants who were not on co-trimoxazole (table 3). regardless of participant’s co-trimoxazole status, the performance of mrdt was significantly lower in sensitivity and specificity compared to bsm findings (p < 0.001) in both groups. current cd4+t cells count/µl mrdt revealed higher (sensitivity 62%, specificity 99%, positive predictive value 91% and negative predictive value 91%) diagnostic accuracy among participants with cd4+t cells count greater than 500 cells/µl. this was lower (sensitivity 56%, specificity 97%, positive predictive value 88% and negative predictive value 85%) among participants with cd4+t cells count under 500 cells/µl (table 3). the observed differences in performance were statistically significant (p < 0.001). anti-retroviral treatment status the mrdt revealed a higher (65%) sensitivity among hiv participants who were not on art compared (56%) to those on art. a similar pattern of higher positive predictive value (98%) was observed among the non-art group compared to 87% among the art group (table 3). for both groups of participants (art vs non-art), the observed differences in performance of mrdt were significantly lower in sensitivity compared to bsm findings among the non-art group (p < 0.001). discussion we detected malaria parasites in 25.4% of participants by bsm and 16.4% by mrdt. overall sensitivity of mrdt was 58% and specificity was 97%. the sensitivity and specificity were similar irrespective of co-trimoxazole and fever status. at malaria parasite density count of under 500 cells/µl, sensitivity was 73% and between 500 and 999 cells/µl, sensitivity was 34%. at cd4+ t cell count over 500 cells/µl the sensitivity was higher (62.4%) compared to 56% at under 500 cells/µl. in the non-art group sensitivity was higher (65%) compared to those on art (56%) but the specificity was similar. although blood smear microcopy still remains the gold standard for malaria diagnosis, several limitations ranging from infrastructure and equipment to competence still exist in endemic areas like nigeria. these justify the need for an alternate method like mrdt.15 since who recommended the use of mrdt, several test kits have flooded the market, hence the need for constant field validation to verify manufacturers’ claims. this is critical, particularly among hiv-positive patients in whom other febrile illnesses and infections mimic malaria.5 the prevalence of malaria in hiv-positive patients in this study was 25.4% based on microscopy compared to 16.4% based on mrdt method. this was similar to a tanzanian study reporting a malaria prevalence of 23.8% using microscopy method among hiv-positive patients compared to 17.5% with mrtd24. however, our findings varied from a similar study in lagos, nigeria, involving a smaller sample size of 387 hiv patients where a malaria prevalence of about 19% was reported for both bsm and mrdt of another brand19. another study in burkina faso among 114 hiv-positive participants showed a malaria prevalence of about 45% for mrdt and 42% for bsm.21 it is worth noting that in this study, participants were recruited at routine follow-up visits, regardless of suspicion of malaria unlike the other studies comparing performance of bsm and mrdt in hiv-positive patients suspected of suffering from or having symptoms of malaria. for example, the tanzania24 and burkina faso21 studies looked at hiv-positive children suspected of malaria, while those from lagos,19 uganda,10,20 and malawi25 looked at adults with hiv attending outpatient clinics suspected of having malaria. thus, the direct comparison of proportions positive for malaria in our studies with the others must be considered with caution. mrdt overall sensitivity and specificity reported in this study was similar to a 55.4% sensitivity reported in lagos, nigeria, but varied in specificity (90.3%).19 contrary to our results, a 100% sensitivity and similar specificity of about 95.4% was documented in a burkina faso study among hiv-positive participants.21 other studies on mrdt performance among hiv-positive population in malawi,25 and two different studies in uganda10,20 have shown higher sensitivity of over 94%. in these studies, the specificities averaged over 97% like in ours except for the malawi study that reported about 51% specificity.25 the 2.5% false positive results observed with the use of mrdt may be an indicator of residual antigenemia. the average time that histidine rich protein-2 (pfhrp-2) remains positive after resolution of parasitaemia is about 2 weeks, although it has been shown that the protein can take over a month to clear.26 using mrdt, a false positivity rate of 5.9% had been reported in burkina faso among hiv-positive patients. the 27.55% false negative results observed might be partly due to deletion or mutation of hrp2 gene in the malaria parasite, which is the most common target for mrdt.27,28 it has been reported to exhibit a high level of polymorphism.29,30 this is an important factor that may affect the performance of mrdt based on antigen detection.31,32 in addition, false negative results have been associated with blood samples taken beyond the period of malaria fever paroxysms.33 p. falciparum isolates without the hrp2 gene have been shown to be important contributors to false negative hrp2-based mrdt testing.34,35 moreover, only plasmodium falciparum releases hrp2; therefore, the presence of non-falciparum infection as another malaria species co-infection in plasmodium falciparum may also give a false negative result. other factors that might possibly affect mrdt field performance are shelf life and heat stability.36,37 in this study, the test kits were stored based on manufacturers’ recommendations and used before the expiry date on the mrdt kits. unexpectedly, with the mrdt, we observed a lower sensitivity of 34% among hiv-positive participants with a malaria density count between 500 cells/µl and 999 cells/µl compared to a 73% sensitivity when the malaria density count was less than 500 cells/µl. however, the specificity at between 500 cells/µl and 999 cells/µl was 97.5%. this was contrary to a previous study in lagos, nigeria, that observed an increase in sensitivity from 90.9% at parasite densities greater than 200 cells/µl to 97.6% at densities greater than 500 cells/µl.19 generally, at lower parasitaemia, variability in sensitivity is more common.20,38,39 similar observation of false negative mrdt results at parasite counts even higher than 1000 cells/µl have been reported in other studies19,25,40,41 and may be attributed to prozone effect observed with immunochromatographic tests such as mrdt.42 all the same, many mrdts today can achieve excellent sensitivity and specificity for p. falciparum at parasite density lower than 500 cells/µl.11 besides this study, and to the best of our knowledge, only one other study has assessed performance of mrdt among hiv-positive patients based on febrile status in africa.10 mrdt performance in our study showed similar sensitivity and specificity as reported by mills et al.,10 in febrile or non-febrile cases in rural uganda. apparently, mrdt reliability is independent of febrile status. mrdt performance was similar in terms of sensitivity, specificity and positive predictive value regardless of whether a participant was on co-trimoxazole prophylaxis or not. information on similar studies is not available; however, with microscopy a relatively higher prevalence has been reported on those without co-trimoxazole prophylaxis compared with those on it.19,22,43 mrdt performance revealed a higher sensitivity among participants with a cd4+ t cell count greater than 500 cells/µl compared to a lower sensitivity among participants with a cd4+ t cell count under 500 cells/µl. although information on similar studies is not available, it has been reported in a similar setting that hiv-positive participants with cd4+ t cell counts 250 cells/µl or higher are significantly less likely to have patent parasitaemia.19 the mrdt revealed a higher sensitivity among hiv-positive participants who were non-art compared to those on art. lower incidence of malaria among hiv-positive adults on art have been reported in different settings10,43,44 as well as reports of antimalarial properties of some antiretroviral drugs.45,46 based on other reports in the general population, the use of mrdt method as an alternative to bsm in malaria endemic areas is recommended for epidemiological studies. however, the performance varied depending on species of the malaria parasite, level of parasitaemia, and immunity.47 for some of these reasons, such recommendation must be taken with caution in hiv-malaria co-infected persons, even though no differential effect of hiv infection on mrdt performance (specifically the sd bioline antigen-based method) has been reported in literature.25 strengths and limitations this study was sufficiently powered having a large sample size of 1521 hiv-positive participants across the two health facilities. apparently, this may be the first study to assess the performance of mrdt in an african setting of significantly high hiv and malaria co-morbidity in association with variables like immune status, parasitaemia and some clinical (co-trimoxazole prophylaxis, fever and art status) factors. the cross-sectional design of this study was a limitation as it was difficult to determine how the potential antimalarial effect of co-trimoxazole prophylaxis and art could have affected mrdt performance over time. note that we observed better diagnostic accuracy of mrdt among participants who were neither on co-trimoxazole prophylaxis nor on art. secondly, we did not measure individual body temperature to correlate it with participants’ reports of fever. conclusion as observed in this study, the sensitivity of sd bioline, which is based on p. falciparum histidine-rich protein type-2 and plasmodium lactate dehydrogenase, is lower than who recommendation for mrdt.48 thus, further evaluation is required to determine its suitability in malaria diagnosis among hiv-malaria co-infected patients in these settings. it is worth speculating that a similar observation with the mrdt could have been observed in non–hiv-positive persons. trustworthiness, reliability and validity the data reported in this current article reflect findings of work done on evaluation of standard diagnostic bioline rapid test kits for malaria diagnosis among hiv patients in kano, nigeria, by our research team members who all participated in the study design, execution, collection, analysis of data and report writing. the research outcomes presented in this report were from the diagnostic evaluation of standard diagnostic bioline rapid test kits for malaria diagnosis among hiv-positive participants in kano, nigeria, at infectious diseases hospital and murtala mohammed specialist hospital kano. although previous studies have reported on the evaluation of rapid malaria diagnosis against gold standard microscopy methods with a focus on the general population, it is worth noting that data among hiv-positive persons under different conditions are not readily available. hence, this study sought to address paucity of data on performance evaluation of standard diagnostic bioline among hiv-positive patients in relation to various factors such as fever status, cd4+ cell count, art status, co-trimoxazole prophylaxis and malaria density. the outcomes of this study and proposed recommendations could be useful in public health programme planning in such settings where hiv and malaria burden is high. apart from disease prevalence data reported, malaria density count and performance of rapid test kits was also evaluated in addition to other clinical and useful variables. this may serve as a baseline upon which comparison may be made in future. acknowledgements the authors wish to express their sincere appreciation to all the health care workers of both facilities especially the laboratory staffs for various support provided during the study and ensuring a conducive working atmosphere for success. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article and its content is solely the authors’ responsibility and views expressed do not necessarily represent the official views of the study facilities and any affiliated institution listed. sources of support none. authors’ contributions h.a.m and t.i.o were the project leaders. f.e.j, t.i.o and s.a.a were responsible for the experimental and project design. f.e.j and s.a.a performed most of the experiments. h.a.m, f.e.j and t.i.o made conceptual contributions. f.e.j and s.a.a performed some of the experiments. f.e.j and t.i.o prepared the samples and data analysis. data interpretation was performed by s.a.a, f.e.j, h.a.m and t.i.o. f.e.j and s.a.a drafted the manuscript. h.a.m and t.i.o reviewed the manuscript for scientific soundness. all authors reviewed and approved the final version of the manuscript. references united states agency for international development. president’s malaria initiative nigeria: malaria operational plan fy 2013 [homepage on the internet]. president’s malaria initiative; 2015. p. 1–74. available from: https://www.pmi.gov/docs/default-source/default-document-library/malaria-operational-plans/fy-15/fy-2015-nigeria-malaria-operational-plan.pdf?sfvrsn=6 world health organization. world malaria report 2017 [homepage on the internet]. 2017. 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http://www.who.int/malaria/publications/atoz/rdt2.pdf abstract background description of the intervention results lessons learnt recommendations acknowledgements references about the author(s) emma e. kploanyi school of public health, university of ghana, legon, accra, ghana joseph kenu school of public health, university of ghana, legon, accra, ghana benedicta k. atsu school of public health, university of ghana, legon, accra, ghana david a. opare national public health and reference laboratory, ghana health service, accra, ghana franklin asiedu-bekoe public health division, ghana health service, accra, ghana lee f. schroeder department of pathology and clinical laboratories, university of michigan, ann arbor, michigan, united states david w. dowdy department of epidemiology, johns hopkins bloomberg school of public health, baltimore, maryland, united states alfred e. yawson department of community health, university of ghana medical school, accra, ghana ernest kenu school of public health, university of ghana, legon, accra, ghana citation kploanyi ee, kenu j, atsu bk, et al. an assessment of the laboratory network in ghana: a national-level atlas survey (2019–2020). afr j lab med. 2023;12(1), a1844. https://doi.org/10.4102/ajlm.v12i1.1844 note: additional supporting information may be found in the online version of this article as online supplementary document 1. lessons from the field an assessment of the laboratory network in ghana: a national-level atlas survey (2019–2020) emma e. kploanyi, joseph kenu, benedicta k. atsu, david a. opare, franklin asiedu-bekoe, lee f. schroeder, david w. dowdy, alfred e. yawson, ernest kenu received: 11 feb. 2022; accepted: 29 sept. 2022; published: 08 feb. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: integrated health systems with strong laboratory networks are critical in improving public health. the current study assessed the laboratory network in ghana and its functionality using the assessment tool for laboratory services (atlas). intervention: a national-level laboratory network survey was conducted among stakeholders of the ghanaian laboratory network in accra. face-to-face interviews were conducted from december 2019 to january 2020, with follow-up phone interviews between june and july 2020. also, we reviewed supporting documents provided by stakeholders for supplementary information and transcribed these to identify themes. where possible, we completed the laboratory network scorecard using data obtained from the atlas. lessons learnt: the laboratory network (labnet) scorecard assessment was a valuable addition to the atlas survey as it quantified the functionality of the laboratory network and its overall advancement toward achieving international health regulations (2005) and global health security agenda targets. two significant challenges indicated by respondents were laboratory financing and delayed implementation of the ghana national health laboratory policy. recommendations: stakeholders recommended a review of the country’s funding landscape, such as funding laboratory services from the country’s internally generated funds. also, they recommended laboratory policy implementation to ensure adequate laboratory workforce and standards. keywords: laboratory systems; laboratory network; atlas; epidemic-prone diseases; labnet scorecard; laboratory capacity; laboratory readiness; laboratory strengthening. background integrated health systems with effective and efficient laboratory networks1,2 are gaining wide recognition for their critical role in managing high-burden endemic infections, particularly hiv, tuberculosis and malaria,3,4 and accelerating disease outbreak response. the importance of laboratory outbreak response has been highlighted by the severe acute respiratory syndrome (sars, 2003), h1n1 (2009), meningitis (2009), cholera (2010), middle east respiratory syndrome (2012), ebola virus disease (2014), zika virus disease (2015), and yellow fever (2016–2017) outbreaks, as well as the current coronavirus disease 2019 (covid-19) pandemic.5,6,7,8,9 there are considerable delays in outbreak detection and communication for most infectious disease outbreaks originating from africa,10,11 as exemplified in the delayed response to the west african ebola outbreak, which was reported four months after the index infection and, more recently, the delayed diagnosis of suspected covid-19 cases in africa.6,12,13 also, the covid-19 pandemic has underscored the diagnostic capacity challenges facing african countries. as of january 2022, ghanaian sars coronavirus 2 (sars-cov-2) daily testing rates was around 70 per 1000 people, in contrast to over 2300 per 1000 people in the united states.14 improving laboratory networks is a critical step toward controlling health emergencies.15 the global health security agenda (ghsa) requires countries to establish tiered national laboratory systems and ‘determine an appropriate level of diagnostic capability at each level of the public health hierarchy from national to the district’.16 the laboratory network in ghana was assessed in 2006, focusing on hiv diagnostic resources, using the assessment tool for laboratory services (atlas).17 the assessment identified the need for guidelines in biosafety, inventory control and logistics management information systems, and the application of policies and procedures.18 however, there have been no publicly available assessments of the laboratory network in ghana since then. in this study, we conducted the atlas survey with a wider scope, including a laboratory network (labnet) scorecard evaluation.19 the atlas survey was conducted using the central-level questionnaire to describe the national laboratory network in terms of its organisational structure, management of laboratory services and logistics, as well as quality regulation. a portion of the labnet evaluation was added to provide a quantitative measure of functionality to corroborate the qualitative findings from the atlas survey on core capabilities. the assessment was conducted to identify strengths and weaknesses of the laboratory network and provide recommendations for its improvement. description of the intervention ethical considerations the study obtained ethical approval from the ghana health service ethical review committee (ghs-erc 005/05/19) and noguchi memorial institute for medical research institutional review board (fwa 00001824). in addition, the director-general of the ghana health service (ghs) granted permission before the study was conducted. written consent was obtained from every participant before conducting an interview. study design and setting this was a qualitative study conducted among key stakeholders in the laboratory network in accra, ghana. it is part of a more extensive, ongoing study to map and model the laboratory network in ghana for three epidemic-prone diseases (epds) – bacterial meningitis, measles and yellow fever – and three diseases of public health importance (dphi): hiv, tuberculosis and hepatitis c virus (hcv). these diseases guided our choice of stakeholder interviewed. data collection we had a consultation with stakeholders from the national public health & reference laboratory (nphrl) and disease surveillance department of ghs in november 2019 to adapt the national-level united states agency for international development atlas20 to suit the ghanaian context, as recommended in the atlas guidelines. we identified stakeholders to be interviewed with the atlas during the consultation session. the atlas comprises nine sections: organisation, policy, forecasting and procurement, financing, storage and distribution, inventory control system, laboratory information management system, supervision, and general questions. face-to-face interviews were conducted from december 2019 to january 2020, with follow-up phone interviews conducted between june 2020 and july 2020. also, supporting documents provided by stakeholders were reviewed for supplementary information, and responses were transcribed to identify themes. where possible, data were used to complete the labnet scorecard.19 the labnet scorecard has been validated for assessing a country’s laboratory network functionality in implementing the international health regulations (2005) and attaining the ghsa targets.19 our labnet scorecard assessment was based on five of the nine core capabilities closely related to aspects of the laboratory network assessed by the atlas: political, legal, regulatory and financial framework; structure and organisation of the laboratory networks; laboratory information (management system); infrastructure, equipment and supplies; and quality of laboratory services. the scores were converted to percentages reflecting overall advancement toward standards or targets, and interpretations were drawn on each component’s functionality stage based on the weakest score (supplementary table 1). survey implementation a total of 12 respondents were interviewed: two representatives from clinical laboratories unit (clu) under the institutional care division, ghs; deputy directors at supplies, stores and drugs management (ssdm) and the finance division, ghs; the head and quality manager for nphrl; and a representative each from the health facilities regulatory agency and allied health professionals council. relevant stakeholders from disease control programmes including the expanded programme of immunisation (epi) with focus on epds, national tuberculosis control programme (ntp), national aids and sexually transmitted infections control programme (nacp) and national viral hepatitis control programme (nvhcp) were also interviewed. results organisational structure of clinical and public health laboratories the ghs tiers into the national, regional, district and sub-district levels with the clu coordinating vertical laboratory activities (figure 1). respondents reported over 800 public clinical laboratories, including 10 regional, 112 district and about 692 sub-district health facility laboratories. based on data from the national database district health information management system ii (dhims2) (ghana health service with technical assistance from the university of oslo health informatics department, accra, ghana), the ghs laboratories conduct biochemistry (n = 342), haematology (n = 397) and microbiology tests (n = 365). the public health division of the ghs is responsible for the public health laboratories (phls) that surveil measles, rubella, yellow fever, tuberculosis, hiv and other diseases. the nphrl in accra has oversight responsibility for three other phls situated in tamale (northern zone), kumasi (middle zone), and sekondi (western zone). figure 1: organisational structure of clinical and public health laboratories in ghana as of january 2020. the atlas assessment provided the number of laboratory facilities in each category in january, 2020. laboratories in the ghs organisational structure are tiered into three levels: tertiary (nphrl and teaching hospital laboratories), secondary (regional hospital laboratories and zonal phls) and primary laboratories (laboratories in district, sub-district facilities and health centres). the relationship between tiers is based on referrals, flowing from lower-tier to higher-tier laboratories, with higher-tier facilities assigned some oversight responsibilities on lower-tier laboratories (e.g., outreach training and supportive supervision [otss]). the four teaching hospitals, although directly under ministry of health (moh) but not ghs, only play a key role in the referral system. the regional laboratory scientists also serve as laboratory coordinators in various regions. however, the districts lack such coordinators due to inadequate staff capacity. the epi, nacp, ntp and nvhcp collaborate with clinical laboratories and phls to conduct laboratory testing. the epi has outsourced all laboratory services and logistics management for epds to nphrl. while the nacp has art sites at 488 facilities, including laboratories, the ntp works with about 337 laboratories in the health system. the nphrl coordinates national-level activities of nvhcp, whereas the health directorates coordinate activities at the regional and district levels. there are no specific laboratories designated for hcv testing under the current programme. policies and other guidelines the moh had no dedicated laboratory policy development unit at the time of assessment. thus, the clu represents the ghs on clinical laboratory policy issues. the laboratory technical committee comprising members from all agencies of moh and ghs developed the ghana national health laboratory policy21 that was approved in 2013; however, the ghana national health laboratory policy is still not operational. the policy describes the laboratory organisation, services and test menu by level, staffing norms, logistics management, quality management and laboratory information system. the primary level test menu covers basic parasitology and bacteriology, cytology, histopathology, serology, clinical chemistry and haematology tests. in contrast, the secondary level test menu covers more sophisticated testing such as immunohistochemistry and nucleic acid testing. finally, the tertiary level test menu covers more specialised tests and the phls focus on epd-dphis in support of public health surveillance (figure 2). figure 2: test menu for primary, secondary and tertiary tiers in the ghanaian laboratory network, 2020. the policy document on infection prevention and control22 is fully operational. it contains guidelines that cover infection prevention and biosafety. the phls perform tests using standard operating procedures (sops). also, the clinical laboratories conform to international and funding programme standards. however, harmonised general clinical testing is lacking, particularly the use of different automated equipment at the facility level for haematology, immunology and chemistry. financing funding for laboratory services in the country is fragmented for infrastructure, supplies and equipment. there are different funding sources for target programmes and phls; general clinical testing relies on internally generated funds and national health insurance system (nhis) reimbursement on diagnostic services and tests listed on the nhis benefits package.23 the government of ghana only provides funding for infrastructure, some equipment and workers’ salaries. the primary funding donors were the global fund (figure 3), which provided the most funding for hiv and tuberculosis, and the world health organization (who), which funds epds. currently, no donor funding is available for hcv laboratory services, nhis covers only hcv screening and remaining costs are borne by patients. stakeholders estimated a 20% – 50% gap in funding for the six epd-dphis. figure 3: financing of laboratory services for priority disease conditions in ghana, 2020. different units and divisions coordinate programme-specific testing for epd-dphis. most programmes procure laboratory supplies at the central level rather than allocate funds to laboratories. the nphrl receives most supplies and funds from donors, allocating these to the zonal phls through their divisional heads. outbreaks also determine the allocation of financial resources. target programmes and the nphrl have separate budgetary line items for laboratory services, supplies and equipment. however, the finance unit of the ghs works with a highly aggregated budget, the medium term expenditure framework plan for moh24; hence, there is no specific budget line for laboratory services. since ghana attained lower-middle-income status, a transition plan was implemented in 2015, requiring the government to increase funding each year with a full transition scheduled in 2022. forecasting and procurement the target programmes, nphrl and health facilities consistently forecast and procure needed laboratory supplies; however, there is no standardised forecasting method. the epi does not prepare forecasts for epds because they have outsourced this role to nphrl. the situation is quite peculiar for nvhcp as they also prepare forecasts but do not receive funding for laboratory supplies. unless otherwise requested by funders, the public procurement act 663 guides procurement as amended in 2016. for example, at ntp, a procurement request to ssdm initiates an open tender with the selection of vendors by the central tender committee. a diverse stakeholder team monitors the procurement process. the average lead time is programme-specific, being six months for nacp and inconsistent for ntp due to time lags between contract award and item supply. although the programmes operate independently, there are instances when they carry out joint activities, especially between the ntp and nacp. their procurement units lead procurement and monitoring for general clinical testing of their target disease conditions at the facilities. all programmes indicated adequate laboratory supplies, except the nvhcp, which has no funding for laboratories. the nacp supplies reagents and haematology and chemistry analysers to the art sites, whereas the ntp supplies reagents to all tuberculosis testing laboratories. however, there was little information on the adequacy of supplies at the phls and clinical laboratories, as this assessment was conducted only at the national level. although multiple systems manage laboratory supplies, respondents reported that duplicating efforts had been minimised. storage and distribution the integrated scheduled delivery system implements the last mile distribution strategy of laboratory supplies and equipment to all levels other than phls. currently, there are four storage facilities for laboratory supplies and cold chain reagents at the central level, and although these storage facilities have adequate cold chain capacity, the regional stores do not. thus, a central medical store is being built for future storage needs. a third-party logistics firm is contracted for distribution as ghs delivery vehicles are insufficient. the nphrl handles supplies at the national level for onward distribution to the zonal phls. the programmes intervene when there is a need for redistribution or emergency distribution. inventory control the international organization for standardization (iso) 15189 requires establishing an inventory system with preset minimum and maximum stock levels. hence, sops have been developed by the ssdm indicating the minimum, reorder points and maximum stock levels for facilities. the minimum and maximum stock levels are two and three months for the central level and three and six months for the regional medical stores. the laboratory facilities determine order quantities for general clinical testing supplies, higher-level authorities at the central level for programmes, and the head of nphrl for all phls. the laboratory scientists at the facilities collaborate with their procurement units to conduct stock-taking twice annually, using various mechanisms, such as whatsapp platforms (meta platforms, menlo park, california, united states), microsoft excel spreadsheets (microsoft, redmond, washington, united states), and gx-alerts (cepheid, sunnyvale, california, united states), aside from the monthly stocks reports. the who monitors stock for measles, rubella and yellow fever through kit management reports regularly submitted by the nphrl, while the nphrl monitors activities, including stock balances, at the zonal phls. specimen transport system based on the current testing algorithm, sputum samples from all new suspected cases are tested using genexpert® machines (cepheid, sunnyvale, california, united states). thus, the available 127 genexperts® are strategically placed within a 2-h drive to most facilities, easing specimen referral. the ghana postal courier service is contracted to transport samples on specific pickup days, ensuring collection within two days. tuberculosis drug resistance testing is performed at five laboratories, with specimen referral arranged by the requesting facility through public transport or courier service. hiv viral load testing and early infant diagnosis are performed at 10 regional and four teaching hospitals. as with tuberculosis diagnosis, the courier service transports hiv specimens on facility-specific pickup days. the requesting facility arranges specimen transport for all other specimen referrals. laboratory information system management the health information management system used in all health facilities is the dhims2. this database is an upgrade from the dhims, which incorporates tracking by target programmes and improves cause-of-death statistics. like most clinical databases, the dhims2 captures laboratory service data at an aggregate level down to facility, but not patient level. the basic laboratory information system also reports into dhims2 periodically. similarly, e-tracker data for hiv viral load are captured in the dhims2. two other databases do not directly report data into dhims2: gx-alert for genexpert, and epiinfo (epi info™, centers for disease control and prevention, atlanta, georgia, united states) or data on epds. gx-alert data are aggregated at the facility level and entered manually into dhims2 whereas epiinfo captures data on the who disease network that are reported directly to the who. no entries are made into dhims2 (figure 4). figure 4: reporting system for laboratory services management information in ghanaas of 2020. the phls prepare weekly yellow fever reports and communicate positive results via phone. other reports are submitted either electronically or as hard copies or by both methods. standard national forms used to collect and report data into dhims2 include case-based forms and other national forms developed by the clu for haematology, chemistry and microbiology testing. they also provide demographic data crucial for planning immunisation activities. there are also logbooks at the laboratories that provide information on the laboratory tests requested and conducted. however, only aggregated data are available at the national level. as described, reports from these databases are useful for quantifying commodities during forecasting, procurement, inventory control, targeted screening, planning public health interventions, notifying international organisations on disease burden and outbreaks, and seeking donor support. however, not all facilities submit data as and when due. the nphrl monitors reporting rates and supports supervision exercises organised by phls. supervision the clu does not supervise private laboratories. instead, it carries out supportive supervision for general clinical testing at the regional level. another regional-level team supervises the district and sub-district levels. in addition, the clu performs supervisory visits every six months using the otss checklist for malaria diagnosis and the integrated monitoring and supervision checklist for general supervision. for the phls, a national-level supervisory team monitors reagent availability, storage and shelf life, and staff competencies and credentials using checklists. supervision is quarterly at the nphrl but less frequent at the zonal phls due to insufficient logistics; however, the laboratory managers monitor daily activities. target programmes, mainly nacp and ntp, organise supervisory visits to laboratories every quarter or twice annually, depending on resources. these programmes are building local capacity so that representatives can supervise activities at the district level. the epi only provides supervision and support for the national and regional cold rooms. quality regulation and accreditation at the time of assessment, no laboratory had been iso accredited. however, the clu has assessed some laboratories preparing for accreditation using the stepwise laboratory improvement process towards accreditation checklist.25 the clu also carries out external quality assurance through otss (supplementary figure 1), supporting the implementation of iso 15189. the nphrl and zonal phls have implemented quality management systems based on this standard with self-regulation. the health facilities regulatory agency under the moh regulates laboratory quality independently of the clu. they issue laboratory operation licenses based on an intensive checklist inspection, with tiered accreditation: 50% (6-month license) or 90% (3-year renewal) of items. unannounced laboratory monitoring is carried out twice annually and could lead to a reassignment of the tier, influencing the laboratory’s nhis reimbursement. the allied health professionals council regulates laboratory professionals through licensing examinations and sanctions. although the council is enjoined by act 857 to supervise practitioners, this is lacking due to fund constraints. instead, their interventions are responses to malpractice brought to their attention. the assessment of laboratory network functionality using the laboratory network scorecard the self-report of stakeholders on five of the nine core capabilities of the laboratory network using the labnet scorecard indicated that the laboratory information management system advanced the most (74%) toward achieving the international health regulations (ihr) and ghsa targets (figure 5; supplementary table 1). three capabilities were similarly rated: structure and organisation of the laboratory networks (58%), infrastructure, equipment and supplies (62%), and quality of the laboratory services (63%). however, the political, legal, regulatory and financial framework rated the lowest (33%), with the finance component at the least functional stage as it lacked vital attributes. figure 5: country results for assessment of the laboratory network in ghana using the labnet scorecard, 2020. (a) overall advancement of core capabilities towards the standards taking all scores into consideration; (b) stages of functionality highlighting weak scores. lessons learnt experiences although atlas guidelines recommend group discussions for administering the tools, we adopted key informant interviews due to stakeholders’ conflicting and limited availability for a group discussion. multiple stakeholders with in-depth knowledge and experience in one or more tool sections were interviewed to obtain a holistic picture of the current laboratory network. this mode of administering the tool offered the added advantage of receiving more detailed responses than obtained in a time-limited group discussion. moreover, the key informant interviews served as a basis to receive further information from respondents to consolidate findings. some respondents recommended other stakeholders that could better respond to some sections of the tool; hence, the earlier list of stakeholders generated from the consultative meeting was updated a few times. the labnet scorecard assessment was a valuable addition to the atlas survey as it provided quantitative responses that served as a reflection of the overall advancement toward standards and targets set by ihr (2005) and ghsa. based on the experiences described, we drew lessons, and identified some strengths and challenges: lessons the atlas was a useful baseline assessment of the entire laboratory network because multiple stakeholders with in-depth knowledge and experience in one or more tool sections were interviewed. the labnet scorecard assessment was a useful addition to the atlas survey as it assessed the functionality of the laboratory network through quantitative responses that served as a reflection of the overall advancement toward targets set by ihr (2005) and ghsa. the lowest labnet score was obtained in the ‘legal and regulatory framework’ assessment; the ghana national health laboratory policy although approved has not yet been implemented, driving the lack of harmonised test menu, reagents and equipment. thus, the laboratory policy needs to be implemented to ensure an adequate workforce and standardisation of laboratories. a weak legal and regulatory framework impacts all other labnet core components and must be addressed first. vertical programmes rely heavily on external funding, whereas health facilities solely rely on internally generated funds for laboratory supply procurement and service delivery. this presents the opportunity for a better collaboration with vertical programmes to access external funding or adapt the funding mechanisms of health facilities through targeted policy and administrative interventions. the supervision and quality regulation organised by the administrative units and vertical programmes align with iso standards and could be leveraged for the accreditation of laboratories. strengths the assessment found a collaboration between disease control programmes, clinical and phls in the forecasting, procurement and distribution of laboratory supplies, testing for priority target disease conditions, and supportive supervision for laboratory testing. the assessment also found nhis reimbursement on diagnostic services and tests listed on the benefits package as an opportunity to increase local financing of laboratory services. some gaps identified by the first atlas assessment18 have been addressed over the years through the development and implementation of the policy for infection prevention and control, and sops guiding inventory control and laboratory testing. it is commendable that the country has a national database into which data from clinical and phls are reported and easily accessed for decision-making. the labnet assessment indicated that the laboratory information management system advanced the most toward achieving ihr and ghsa targets. the specimen transport arrangements between some target programmes (ntp and nacp) and ghana postal courier service serve as a good foundation to developing a specimen referral and transport system for all priority conditions. challenges laboratory funding was a major challenge indicated by most respondents. target programmes rely heavily on external funding; the clu reported that the internally generated funds and the often-delayed nhis reimbursements were insufficient to maintain testing capacity (figure 3). funding from the government of ghana was insufficient for laboratory services, and there was no specific budget line for these services at the central level. inadequate funds at the central level affect holistic and integrated supervision. currently, the clu takes advantage of the otss for malaria diagnosis to conduct general supervision. three out of five stakeholders from clu, nphrl and epi indicated that some laboratories run out of reagents and other supplies due to insufficient funds. the nvhcp has especially suffered from inadequate funding for its activities. programmes that receive external funding do not have the liberty of discretionary expending as funders dictate spending. another challenge is the delayed implementation of the laboratory policy approved in 2013. as a result, test menu harmonisation and reagents and equipment standardisation are a challenge, affecting specimen and patient referral to higher tiers. in addition, there is no organised system at the national level for sample transportation except for hiv and tuberculosis diagnoses. study limitations this assessment was carried out to characterise the laboratory network in ghana using the atlas, a qualitative tool. in addition, some aspects of the labnet scorecard were used to measure functionality quantitatively in support of findings on five out of the nine core capabilities that were related to some aspects of the atlas administered. ideally, the entire qualitative and quantitative labnet evaluation should be applied as part of a multisectoral workshop led by the moh, with results based on consensus. thus, future assessments should consider administering the entire labnet tool and convene workshops to validate findings. conclusion laboratories under the ghana health service are tiered into three levels with the clinical laboratories vertically coordinated by the clu whereas the public health unit coordinates the phls. the laboratories collaborated with target programmes in the forecasting, procurement and distribution of laboratory supplies, supportive supervision and testing for priority conditions. no laboratory had yet received iso accreditation at the time of assessment although some had been assessed using the stepwise laboratory improvement process towards accreditation checklist. in terms of functionality, the laboratory information management system advanced the most toward achieving ihr and ghsa targets whereas the political, legal, regulatory and financial framework lagged behind with the finance component at the lowest stage of functionality. major gaps were identified in laboratory financing and the implementation of the national health laboratory policy. these should represent the focus of future initiatives to strengthen the laboratory network in ghana. recommendations stakeholders recommended that the funding landscape for the country be reviewed. considerations should be made to fund laboratory services from the country’s internally generated funds and expand the scope of nhis. also, the moh should create a laboratory unit to collaborate with administrative units for laboratories in all their agencies to update and implement the national health laboratory policy to guide improvement of the laboratory network towards achieving the ghsa targets. moreover, plans for a national sample referral and transportation system that accommodate a wide range of clinical testing needs should be operationalised. the collaboration between administrative units and target programmes could be improved through joint supervision to optimise the resources available. also, the quality regulation in laboratories align with iso standards and could be leveraged for the accreditation of laboratories. a follow up labnet assessment could be conducted to help document the laboratory network’s progress towards attaining the ghsa targets on all nine core capabilities. acknowledgements the authors are grateful to all stakeholders in the ghanaian laboratory network who provided their inputs during data collection and validation. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions e.k., l.f.s., d.w.d. and a.e.y. designed the study; all authors contributed to the implementation of the research. e.e.k. analysed the results and wrote the manuscript with support from j.k., b.k.a., l.f.s., d.a.o. and f.a.-b. all authors provided critical feedback and contributed to the final manuscript. sources of support the study was funded by the united states national institutes of health (1 r01 ai136977-01a1) under the terms of the agreement awarded to the regents of the university of michigan. data availability the data that support the findings of this study are available on request from the corresponding author, e.k. disclaimer the views expressed in this article are the authors’ own, not an official position of the collaborating institutions or the funding agency responsible and no official endorsement should be inferred. references olmsted ss, moore m, meili rc, et al. strengthening laboratory systems in resource-limited settings. am j clin pathol. 2010;134(3):374–380. https://doi.org/10.1309/ajcpdqosb7qr5glr world health organization. the maputo declaration on strengthening of laboratory systems. in: consensus meeting on clinical laboratory testing harmonization and standardization [homepage on the internet]. maputo: world health organization – regional office for africa; 2008 [cited 2021 nov 10]. 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https://www.mofep.gov.gh/sites/default/files/pbb-estimates/2018/2018-pbb-moh.pdf world health organization regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation in the african region (with checklist). brazzaville: who afro; 2012. abstract introduction methods results discussion acknowledgements references about the author(s) heidi albert find, cape town, western cape, south africa jean de dieu iragena world health organisation, african region country office, brazzaville, congo kekeletso kao find, campus biotech, geneva, switzerland donatelle erni find, campus biotech, geneva, switzerland teferi mekonen african society for laboratory medicine, addis ababa, ethiopia philip c. onyebujoh world health organisation, african region country office, harare, zimbabwe citation albert h, de dieu iragena j, kao k, et al. implementation of quality management systems and progress toward accreditation of national tuberculosis reference laboratories in africa. afr j lab med.2017;6(2), a490.https://doi.org/10.4102/ajlm.v6i2.490 original research implementation of quality management systems and progress towards accreditation of national tuberculosis reference laboratories in africa heidi albert, jean de dieu iragena, kekeletso kao, donatelle erni, teferi mekonen, philip c. onyebujoh received: 13 may 2016; accepted: 07 oct. 2016; published: 31 mar. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: laboratory services are essential at all stages of the tuberculosis care cascade, from diagnosis and drug resistance testing to monitoring response to treatment. enabling access to quality services is a challenge in low-resource settings. implementation of a strong quality management system (qms) and laboratory accreditation are key to improving patient care. objectives: the study objective was to determine the status of qms implementation and progress towards accreditation of national tuberculosis reference laboratories (ntrls) in the african region. method: an online questionnaire was administered to ntrl managers in 47 world health organization regional office for africa member states in the region, between february and april 2015, regarding the knowledge of qms tools and progress toward implementation to inform strategies for tuberculosis diagnostic services strengthening in the region. results: a total of 21 laboratories (43.0%) had received slmta/tb-slmta training, of which 10 had also used the global laboratory initiative accreditation tool. however, only 36.7% of ntrls had received a laboratory audit, a first step in quality improvement. most ntrls participated in acid-fast bacilli microscopy external quality assurance (95.8%), although external quality assurance for other techniques was lower (60.4% for first-line drug susceptibility testing, 25.0% for second-line drug susceptibility testing, and 22.9% for molecular testing). barriers to accreditation included lack of training and accreditation programmes. only 28.6% of ntrls had developed strategic plans and budgets which included accreditation. conclusion: good foundations are in place on the continent from which to scale up accreditation efforts. laboratory audits should be conducted as a first step in developing quality improvement action plans. political commitment and strong leadership are needed to drive accreditation efforts; advocacy will require clear evidence of patient impact and cost-benefit. introduction the burden of tuberculosis in africa remains high. the world health organization (who) reported 1 342 000 tuberculosis cases in the region in 2014; 28% of the global caseload. the region suffers from the highest per-capita burden; 281 cases per 100 000 population, more than double the global average. despite meeting the millennium development goal target of a falling tuberculosis incidence rate, the african region failed to meet the targets for 50% reduction in tuberculosis prevalence and mortality. furthermore, the attainment of these targets varied among countries, with 40 countries achieving the millennium development goal tuberculosis incidence target and only 18 countries achieving the 50% reduction in tuberculosis mortality target. rates of multi-drug resistant tuberculosis vary across the continent, with average rates of 2.1% (0.5–3.7) among new cases and 11% (6.7–16) among previously-treated cases.1 high-quality laboratory services are an essential component of all stages of the tuberculosis care cascade, from diagnosis and drug resistance testing to monitoring response to treatment.2,3 however enabling access to quality tuberculosis diagnostic services for populations in need is a major challenge in low-resource settings. the end tb strategy calls for universal access to drug susceptibility testing (dst).4 however, the who reported that only 6.4% of new bacteriologically-confirmed tuberculosis cases and 33% of previously-treated cases received dst in 2014.1 laboratory services on the continent are known to suffer many challenges, including poor infrastructure, inadequate human resource capacity, and weak underlying health systems.3 a number of recent regional initiatives have emphasised the need for strengthening quality systems of laboratories in the region, including the world health organization (who) regional office for africa 58th session (afr/rc58/r6, yaoundé, september 2008),5 the maputo declaration (january 2008),6 the kigali declaration on strengthening laboratory management towards accreditation (july 2009),2 and the african society for laboratory medicine’s ministerial call for action (december 2012).7 a strong laboratory quality management system (qms) is critical to ensuring the quality of testing. a weak qms may result in laboratory errors that can lead to both overand under-diagnosis of tuberculosis, interruptions in service, and delayed reporting of results, leading to a negative impact on patient care. failure to meet minimum safety standards may put laboratory workers, patients and the community at risk.8 such requirements can only be consistently met by concerted efforts to develop and maintain quality management systems within the tuberculosis laboratories. accreditation is a formal process of recognition that appropriate and sufficient quality systems have been implemented. accredited laboratories are recognised as meeting certain quality standards and having the necessary technical processes in place, as well as administrative systems needed to ensure quality results. evidence from various highand low-income settings8,9,10,11 demonstrates that the implementation of qms and accreditation leads to a measurable improvement in the quality of services and increased patient safety due to a reduction in laboratory errors. national tuberculosis reference laboratories (ntrls) play a critical role in ensuring quality tuberculosis diagnostic services throughout the whole diagnostic testing network, conducting advanced testing (such as liquid culture, firstand second-line dst), supervision of peripheral laboratories, external quality assurance, and conducting drug surveillance surveys and operational research. quality improvement of ntrls is a critical first step in building strong diagnostic networks to deliver quality laboratory services for patient care. accreditation of ntrls was a specific goal in the global plan to stop tb (2006–2015).12 the plan estimated that less than 5% of ntrls globally were accredited by 2006, and a target of more than 50% ntrls being accredited by 2015 was established. despite some progress, the goal of 50% ntrls being accredited remains unmet and, especially in the african region, only a minority of ntrls, or any other tuberculosis laboratories, have reached this standard. at the time of writing, ntrls in south africa, botswana, mozambique and uganda (8% of ntrls in the african region) had achieved accreditation according to the iso 15189 standard (south africa national accreditation service, www.sanas.co.za and instituto portugues de acreditacao, www.ipac.pt). fifty-four per cent of countries in the african region reported having a formal qms toward achieving laboratory accreditation implemented at the ntrl in 2014.1 however, the extent of progress toward accreditation of individual laboratories remains unclear. furthermore, only six ntrls have received a formal external audit conducted by the african society for laboratory medicine using the who’s stepwise laboratory quality improvement process towards accreditation (slipta) programme (mekonen t, personal communication). there are a number of key resources available to assist tuberculosis laboratories in developing and maintaining a qms, which are reviewed in the global laboratory initiative’s (gli) recently-published guide for providing technical support to tb laboratories in lowand middle-income countries.13 adherence to the iso 15189:2012 international standard (www.iso.org) is used by regulating authorities and accreditation bodies for recognising the competence of medical laboratories. the who’s laboratory quality management system provides a comprehensive overview of laboratory qms and comes in the form of a modular training package and manual.14 gli developed an online tuberculosis-specific tool to assist laboratories with accreditation preparedness (www.gliquality.org). slipta is a stepwise monitoring and auditing framework developed by the who regional office for africa, based on iso 15189. assessment using the slipta checklist is scored and rated on a scale of one to five stars, with five stars being considered an indicator of readiness for international accreditation. strengthening laboratory management toward accreditation (slmta) is a structured quality improvement programme, which teaches laboratory managers how to implement practical qms in resource-limited settings (www.slmta.org). the programme includes a series of workshops and work-based improvement projects supported by site visits and mentoring, and impact of the programme is measured using the slipta checklist.15 the foundation for innovative new diagnostics found has developed a tuberculosis-specific version of slmta (tb-slmta), including a harmonised checklist based on slipta and the gli tool.16 gli africa, a partnership focused on strengthening tuberculosis diagnostic networks in the african region,17 has established a programme aimed at supporting quality improvement and accreditation of ntrls on the continent. as a first step in this initiative, a survey was developed to determine the current status of implementation of qms at ntrls, and the knowledge and use of various tools to assist in accreditation preparedness. methods survey development and dissemination the aim of the survey was to gather baseline data on ntrl status regarding qms and accreditation, to inform planning for support to countries working towards accreditation. the questionnaire was developed between december 2014 and february 2015 by a gli africa taskforce, which comprised key stakeholders, including the african society for laboratory medicine, who regional office for africa, gli, the who supranational reference laboratory (srl) uganda, the us centers for disease control and prevention, and find. following finalisation of the questionnaire content in english, translations were made into french and portuguese. the survey was disseminated via email from the who regional office for africa to the who representatives/who liaison officers in 47 who african region member states, containing links to the survey in the three languages. the survey was disseminated on 4 february 2015, along with an explanation of the purpose of the survey, and the closing date for responses was 17 february 2015. who country representatives were responsible for dissemination of the survey to the national tuberculosis control programme (ntp) and ntrl managers. organisations represented on the gli africa task force also followed up with ntp/ntrl managers in countries to encourage survey participation. countries that did not respond by the deadline were contacted, either by one of the authors (j.i.) or by in-country consultants, to request a response. respondees were given the option to respond via the online survey tool, or via email or fax. all responses were received by 25 april 2015. survey questions the survey consisted of questions in several key areas: (a) familiarity with various tools and approaches that can be used when implementing a qms; (b) progress with implementing qms in their laboratory; (c) data from internal or external qms assessments; (d) programmes and partners supporting strengthening of qms in their laboratory; and (e) challenges faced in strengthening qms. survey questions and possible responses are provided in full in table 1. table 1: national tuberculosis reference laboratory accreditation preparedness survey questions data management data from the online survey were managed via the who extranet data system. survey responses were exported from the who system in .csv format, and data analysis was conducted in microsoft excel (2013 version; microsoft, redmond, washington, united states). results responses were received from all 47 who african region member states. a total of 49 responses from ntrls were received; one per country except for zimbabwe and nigeria where each has two ntrls which responded individually. all responses were submitted via the online survey tool. knowledge of quality management system tools a total of 93.9% (46/49) of ntrls reported awareness of at least one of the qms tools listed (gli stepwise process towards tb laboratory accreditation, who laboratory quality management system handbook, slipta, slmta, and/or tb-slmta. five ntrls reported knowing all of the qms tools mentioned, all of which had either achieved accreditation or were known to be actively working toward accreditation. three laboratories reported having no knowledge of any of the tools; all three laboratories were from non-anglophone countries. overall, 69.2% of english-speaking ntrls had knowledge of the gli tool, compared with 60.0% for portuguese-speaking ntrls and 55.6% for french-speaking ntrls. progress with implementation of quality management system twenty-one laboratories (42.9%) reported having staff trained by either slmta or tb-slmta; 13 (26.5%) by slmta, 6 (12.2%) had staff trained by both slmta and tb-slmta, while two (4.1%) reported staff receiving only tb-slmta training (figure 1). figure 1: implementation of gli and slmta/tb-slmta approaches by national tuberculosis reference laboratories in the world health organization’s african region† ten laboratories reported actively implementing the gli tool; two of which had already achieved accreditation and one laboratory was close to achieving accreditation at the time of the survey. four additional laboratories reported partially implementing the gli tool, all of which had been audited in 2013 or 2014 and received two, three, three and four slipta stars levels. twenty-one countries reported having a slipta focal point appointed within the ministry of health. all laboratories that reported using the gli tool also reported having staff trained on slmta or tb-slmta. two laboratories who reported actively implementing gli and tb-slmta were from who srls that were already accredited but whose staff received tb-slmta training to build capacity for providing qms support to other countries in the region. a higher proportion of anglophone countries reported implementing qms using the gli tool and had received training by slmta or tb-slmta. 65.4% of countries that responded to the survey in english had staff who had participated in slmta and/or tb-slmta training, as opposed to 40.0% of portuguese-speaking ntrls and only 11.1% of french-speaking ntrls. implementation of a qms using the gli tool varied according to the language of the ntrls, with 23.1% of english-speaking ntrls, 20.0% of portuguese-speaking ntrls and 5.6% of french-speaking ntrls reporting full use, and 30.8% of english-speaking ntrls, 20.0% of portuguese-speaking ntrls and 22.2% of french-speaking ntrls reporting partial use of the gli tool (figure 2). figure 2: national tuberculosis reference laboratories in the world health organization’s africa region† whose staff participated in slmta/tb-slmta training, according to country survey response language. audits eighteen ntrls (36.7%) reported having had an audit conducted using the slipta checklist. the date of the most recent audit and the audit score are shown in figure 3. it was not documented whether these audits were internal or external and, if external, the identity of the auditing body. an additional four laboratories reported having planned audits that had not yet been conducted. figure 3: auditing of national tuberculosis reference laboratories in the world health organization’s african region† using the stepwise laboratory quality improvement process towards accreditation (slipta) checklist, date of last audit and slipta star grading achieved at last audit. external quality assurance a total of 98.0% (48/49) of the ntrls reported participation in at least one eqa programme, with 46 of these 48 sites (95.8%) being enrolled in acid-fast bacilli smear microscopy eqa. first-line dst was the next most common eqa in which ntrls participated (60.4%), followed by second-line dst (25.0%) and molecular tuberculosis testing (22.9%). the most commonly-reported eqa providers were the who srl network and the national institute for communicable diseases (south africa). of the 20 ntrls not enrolled in first-line dst eqa, 13 of these laboratories had links with an srl. national tuberculosis reference laboratories’ formal collaboration agreements with supranational reference laboratories forty (40/49; 81.8%) ntrls reported having a formal agreement with an srl. the majority of srl support came from belgium (30%), south africa (18%), uganda (18%) and italy (12%) (figure 4). the most common form of support provided by srls was provision of eqa panels, technical support, training and supervision visits. figure 4: collaboration agreements established between world health organization supranational reference laboratories and national tuberculosis reference laboratories (in the world health organization african region†) barriers to implementation of a quality management system of those laboratories not actively implementing qms, the reasons for not having done so a qms in their laboratory are shown in figure 5. a lack of training, poor staff motivation, lack of an accreditation programme and insufficient on-site supervision were cited as the major reasons for non-implementation of a qms. interestingly, lack of funds was the lowest-rated response among the options provided in answer to this question. however, when laboratories were asked a separate question about the barriers to implementing qms, lack of funds was cited as one of the main reasons. furthermore, only 28.6% of respondees reported that they had included a budget and workplan for laboratory accreditation in their strategic plans or global fund concept notes. figure 5: reasons reported by national tuberculosis reference laboratories in the world health organization african region† for not currently working towards accreditation. discussion recommendations although the majority of laboratories had knowledge of qms tools, and almost half reported receiving slmta training or using the gli tool, only a third of the laboratories reported having conducted an audit, an essential first step in establishing a baseline and developing an action plan to address recommendations and corrective actions resulting from the audit. furthermore, responses from a number of countries suggested that there may be some confusion between the different tools mentioned in the survey. since this survey did not provide a detailed measure of qms implementation in individual laboratories and only a small proportion of laboratories were able to provide audit data, a full audit of laboratories using the slipta checklist is an urgent priority in order to develop a detailed action plan and budget for achieving accreditation. it is also recommended that information on accreditation is disseminated to countries in the region explaining the process of accreditation, the tools available and an implementation roadmap is provided to assist countries with planning. additionally, a regional database of suitably-qualified organisations and individuals who are able to provide technical assistance to countries for accreditation should be developed. participation in eqa for acid-fast bacilli smear microscopy was very high, with 95.8% of ntrls participating in an eqa scheme. however, eqa for other diagnostic techniques was less frequently conducted, with 60.4% of ntrls not participating in first-line dst eqa and very low participation in eqa for second-line dst and molecular methods. it is not known what proportion of the ntrls conduct second-line dst, and therefore the low participation in eqa may, in some cases, reflect the fact the second-line dst is not currently conducted in the country. the same may apply to the low enrolment of ntrls in molecular testing. furthermore, the extent to which ntrls perform eqa of laboratories within their network was not reported; it is these laboratories that conduct the majority of microscopy testing for patient care. participation in eqa for all ntrls should be urgently expanded for all diagnostic technologies performed. this can be achieved by expanding access to eqa programmes through the srl network or other recognised providers. the majority of countries reported a formal agreement with an srl. however, capturing of detailed information on the extent of support and to what extent the srl is fulfilling the country’s need for technical support was outside the scope of the current survey. establishment of formal relationships between the nine countries not currently supported by srls should be prioritised. provision of eqa panels for all diagnostic technologies to all supported countries should be an essential component of the support provided by the srl network or via other providers. there was a disparity in qms implementation and accreditation between anglophone and non-anglophone countries on the continent. this is anticipated to be due to the greater availability of tools and support in anglophone countries in the region. translation of any training materials or tools found to be lacking should be performed as an urgent priority, as well as building greater capacity for technical support in francophone and lusophone countries. a lack of planning and budgeting were highlighted as major gaps, with almost three quarters of laboratories not having included plans for accreditation in their strategic planning processes. gli africa can play a role in providing guidance on planning and budgeting for accreditation, as well as advocating for the importance of quality improvement and accreditation as being key contributors toward countries meeting sustainable development goals for health. an assessment of country capacity and availability of partners and donors to support countries in planning, funding and implementing accreditation programmes should be conducted. laboratories need to elicit senior management support within the ministry of health and include funding for quality improvement and accreditation of tuberculosis laboratories in their country strategic plans.18 equally, donors need to allocate adequate funding for quality improvement and accreditation programmes. to ensure local political commitment, as well as continued and adequate donor funding, need more evidence on the benefit of accreditation. such evidence should focus on the patient impact of improved quality and specifically the benefit of accreditation per se as opposed to quality improvement in the absence of accreditation, as well as including a costing and cost-benefit analysis. limitations the scope of this survey was to obtain a general overview of the knowledge and status of qms among ntrls on the continent, and not to provide detailed information about particular laboratories. therefore, a clear indication as to the status of qms implementation in individual laboratories was not possible as approximately two-thirds of laboratories had not been assessed using a recognised qms checklist. this survey focused on ntrls and did not determine the status of qms and progress toward accreditation for other laboratories in the network. in most countries, it would be anticipated that the ntrl is likely to be the most advanced in terms of qms. it would be important to obtain information on the status of qms in other laboratories, particularly those performing culture, dst and line-probe assay. the survey was administered in three main lingua franca used in the african region; namely, english, french and portuguese. however, the who recognises other languages, including spanish, as being the official language used in equatorial guinea. there were some conflicting responses given to several questions, implying that the questions were not clearly understood by all respondents. finally, this survey was conducted among the 47 who african region member states and not the 54 countries comprising the whole african region; therefore, generalisation of our conclusions to the entire african region should be made with caution. conclusions progress has been made in implementing qms in laboratories in the african region. however, more than 90% of ntrls in the region are not accredited. nonetheless, a good foundation is in place on the continent from which to scale up laboratory accreditation efforts. the high level of participation in eqa for smear microscopy can be leveraged to expand the range of diagnostic tests covered by eqa. the majority of ntrls reported having established a formal agreement with an srl, although the scope and quality of support was not determined in this study. building capacity for auditing is urgently needed, for both internal and external audit. auditing of ntrls is needed as a first step to inform individual country action plans and budgeting to achieve accreditation. political commitment and strong leadership are needed to drive accreditation efforts; advocacy will require clear evidence of patient impact and cost-benefit to enable funding mobilisation from donors or governments among the many competing priorities for healthcare budgets. dissemination of information on accreditation, coordination of technical support for planning, budgeting and implementing quality improvement and accreditation programmes is an urgent priority. support for non-anglophone countries should be strengthened, including translation of documents and tools, and provision of technical support in appropriate languages. acknowledgements the authors would like to thank the following members of the gli africa task force who contributed to review of the questionnaire: heather alexander (us centers for disease control and prevention), talkmore maruta, ndlovu nqobile, diallo samba (african society for laboratory medicine), moses joloba (who srl uganda), as well as christopher gilpin (who geneva). we are grateful to maria alice telles who assisted with translation of the survey into portuguese. we are thankful to the ntrl managers who took the time to complete the survey, as well as the who country representatives who assisted with dissemination of the survey. competing interests the authors are involved in implementing programmes in the african region to support laboratories towards accreditation. h.a., k.k., j.d.d.i., t.m. and p.c.o are members of the global laboratory initiative for africa, whose mission is to support strengthening of tb diagnostic networks in the region. sources of support none. authors contributions h.a., j.d.d.i., k.k., t.m. and p.c.o. contributed to the conception of the project and development and critical review of the questionnaire. j.d.d.i. conducted the data collection. h.a. and d.e. conducted the data analysis. h.a. drafted the manuscript. all authors contributed to the critical review of the manuscript and agreed with the manuscript content. references world health organization. global tuberculosis report 2015. geneva, switzerland: who; 2015. gershy-damet gm, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. https://doi.org/10.1309/ajcptuuc2v1wjqbm birx d, de souza m, nkengasong jn. laboratory challenges in the scaling up of hiv, tb, and malaria programs: the interaction of health and laboratory systems, clinical research, and service delivery. am j clin pathol. 2009;131(6):849–851. https://doi.org/10.1309/ajcpgh89qdswfons world health organization. the end tb strategy. geneva, switzerland: who; 2015. world health organization regional office for africa. strengthening public health laboratories in the who african region: a critical need for disease control (document afr/rc58/6) [document on the internet]. c2008 [cited 2016 dec 13]. available from: http://apps.who.int/iris/bitstream/10665/1777/1/afr-rc58-r2.pdf world health organization regional office for africa. the maputo declaration on strengthening laboratory systems [document on the internet]. c2008 [cited 2016 dec 19]. available from: http://www.who.int/diagnostics_laboratory/maputo-declaration_2008.pdf african society for laboratory medicine. ministerial call for action: strengthening laboratory services in africa. international conference of the african society for laboratory medicine, 2012, cape town, south africa [document on the internet]. c2012 [cited 2016 dec 19]. available from: http://aslm.org/?wpdmdl=3 allen lc. role of a quality management system in improving patient safety – laboratory aspects. clin biochem. 2013;46(13–14):1187–1193. https://doi.org/10.1016/j.clinbiochem.2013.04.028 wattanasri n, manoroma w, viriyayudhagorn s. laboratory accreditation in thailand: a systemic approach. am j clin pathol. 2010;134(4):534–540. https://doi.org/10.1309/ajcpzyy19wmkmazt peter tf, rotz pd, blair dh, et al. impact of laboratory accreditation on patient care and the health system. am j clin pathol. 2010;134(4):550–555. https://doi.org/10.1309/ajcph1skq1hnwghf kibet e, moloo z, ojwang pj, et al. measurement of improvement achieved by participation in international laboratory accreditation in sub-saharan africa: the aga khan university hospital nairobi experience. am j clin pathol. 2014;141(2):188–195. https://doi.org/10.1309/ajcpv8a9mrwhgxef stop tb partnership. the global plan to stop tb, 2006–2015. actions for life: towards a world free of tuberculosis. int j tuberc lung dis. 2006;10(3):240–241. global laboratory initiative. guide for providing technical support to tb laboratories in lowand middle-income countries. geneva, switzerland; 2015. world health organization. laboratory quality management system handbook [document on the internet]. c2011 [cited 2016 may 01]. available from: http://www.who.int/ihr/publications/lqms_en.pdf yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin pathol. 2010;134(3):401–409. https://doi.org/10.1309/ajcpnbbl53fwuiqj foundation for innovative new diagnostics. tb laboratory quality management systems towards accreditation harmonized checklist. geneva, switzerland: find; 2014. messele t. why gli africa: the objectives/conceptual framework, planning and future role for gli africa. african society for laboratory medicine conference, cape town, south africa; 2014. nkengasong jn, mesele t, orloff s, et al. critical role of developing national strategic plans as a guide to strengthen laboratory health systems in resource-poor settings. am j clin pathol. 2009;131(6):852–857. https://doi.org/10.1309/ajcpc51blobbpakc acknowledgements references about the author(s) john nkengsong us centers for disease control and prevention, atlanta, georgia, united states debrah i. boeras international laboratory branch, us centers for disease control and prevention, atlanta, georgia, united states alash’le abimiku institute of human virology, university of maryland school of medicine, baltimore, maryland, united states rosanna w. peeling international laboratory branch, us centers for disease control and prevention, atlanta, georgia, united states citation nkengsong j, boeras di, abimiku a, peeling rw. assuring the quality of diagnostic testing: the future is now. afr j lab med. 2016;5(2), a558. http://dx.doi.org/10.4102/ajlm.v5i2.558 opinion paper assuring the quality of diagnostic testing: the future is now john nkengsong, debrah i. boeras, alash’le abimiku, rosanna w. peeling received: 31 aug. 2016; accepted: 31 aug. 2016; published: 17 oct. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. in the 1 february 2006 issue of clinical infectious diseases, petti et al.1 described laboratory medicine in africa as a barrier to effective healthcare and highlighted the need for increased investment in laboratory services in africa. in response to that article, berkelman et al.2 voiced their concern that laboratory services have become the ‘achilles heel’ of global efforts to combat infectious diseases and antimicrobial resistance. now, 10 years later, the future of laboratory medicine in africa has never been brighter and many have played a part. countries are committed to using innovative technologies and connectivity solutions to ensure the quality of diagnostic testing, not only in laboratories but at point-of-care (poc) sites across the continent. the world health organization (who) has provided leadership through several landmark events, starting with the maputo declaration for strengthening laboratory health systems in january 2008,3 followed by the lyon statement on the need for developing countries to establish practical quality management systems in april 2008.4 specifically in africa, the yaoundé resolution was issued by the who regional office for africa (who afro) in 2008 to strengthen public health laboratories in the who african region.5 since july 2009, several african countries, donors, the who and implementing partners launched a programme for strengthening laboratory management, with the aim of accelerating national laboratory services toward iso 15189 accreditation in the african region. the who afro accreditation scheme adopts the stepwise laboratory improvement program towards accreditation (slipta) programme. the purpose of slipta is not to replace existing accreditation schemes such as those offered by the college of american pathologists, international organization for standardization, south african national accreditation system or south african development community accreditation service, but to serve as a scheme to assist the laboratories in obtaining these internationally-recognised accreditation standards. in 2011, the african society for laboratory medicine (aslm) was launched as a pan-african professional body working to advocate for the critical role and needs of laboratory medicine and networks throughout africa as a means of improving the quality of health services. aslm has become the implementing partner for slipta.6 at the same time, another set of driving forces to improve the quality of laboratories and increase access to quality-assured testing came from disease control and elimination programmes for hiv, tuberculosis and malaria. in particular, the president’s emergency plan for aids relief, the bill & melinda gates foundation, unitaid and others have galvanised industry to develop innovative poc technology platforms. however, implementation of poc tests has been fraught with problems as the health systems in which testing must occur are often weak, fragmented and, experience a critical shortage of healthcare personnel in high-burden countries, ill-prepared for any additional testing. poc testing has now become a double-edged sword in that, while poc tests can increase access to testing and allow patients to be linked to evidenced-based care, its introduction has put tremendous stresses on fragile healthcare systems. decentralising testing requires programmes for training and monitoring effectiveness to be amplified a hundredto a thousand-fold in magnitude, while ensuring quality test results. the introduction of cd4 count testing has shown that managing a network of poc sites has proven to be challenging for ministries of health, with significant operator and instrument errors, frequent instrument breakdowns and stock-outs of reagents and other supplies. technological innovation is not sufficient; innovation in health service delivery is urgently needed. as part of its unitaid grant to facilitate the evaluation and implementation of poc tests for cd4, hiv viral load and early infant diagnosis, the london school of hygiene & tropical medicine conducted consultations in nine countries with an interest in quality assurance. these countries have shown commitment and enthusiasm for embracing quality assurance, in particular a system of external quality assessment (eqa). as shown in this supplement, these countries have developed policies for ensuring the quality of diagnostics performed at both laboratories and at the poc. their commitment has not stopped at developing a laboratory or poc policy – the implementation plans include setting up a national system of quality assurance. to overcome health system constraints, the london school of hygiene & tropical medicine collaborated with the ministry of health in zimbabwe to pilot middleware solutions to show that connectivity allows ministries of health to monitor instrument and operator performance across the country. connectivity solutions can also be used to facilitate improvements in supply chain management to avoid stock-outs and wastage, making health systems more efficient, and to improve patient outcomes. quality comes at a cost but, compared with the human and economic costs of not having quality assurance, that cost is insignificant. the london school of hygiene & tropical medicine, in collaboration with the us centers for disease control and prevention, have developed models and tools for integration of poc and laboratory systems, including costing an overall eqa programme. the model, soon to be posted on the international diagnostics centre/london school of hygiene & tropical medicine website, allows countries to estimate and budget for an eqa programme as part of the cost of an overall diagnostic programme. it is now possible to include this cost in the overall cost of introducing a new diagnostic test. these innovations and tools can be applied to eqa programmes for other diseases. in the last 10 years, who afro has established networks of laboratories for malaria, tuberculosis, enterics and meningitis in the regions that are linked by eqa programmes. who and the us centers for disease control and prevention published an eqa policy manual for hiv poc testing in 2015. aslm is committed to sustaining these quality assurance efforts in the region with implementing partners such as the us centers for disease control and prevention, the clinton health access initiative and the elizabeth glaser pediatric aids foundation. aslm as a pan-african organisation can leverage competencies, expertise and the comparative advantage of each organisation, as well as academia to build capacity, mobilise funds and advocate for the value of quality-assured diagnostics to procurement agencies such as unitaid and the global fund. the outlook for laboratory medicine in the region has never been brighter. the future of diagnostics is now. no new diagnostics should be introduced without first establishing a quality assurance programme and no diagnostic should be procured without including a budget for quality assurance. acknowledgements the authors would like to acknowledge all of the countries and partners that participated in regional quality assurance consultations and contributed to this supplement. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. sources of support none. author contributions j.n., d.i.b., a.a., and r.w.p all contributed equally to the conception, writing, and revision of the manuscript. all authors approved the final version for publication. references petti ca, polage cr, quinn tc, et al. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006;42(3): 377–382. http://dx.doi.org/10.1086/499363. epub 2005 dec 20. berkelman r, cassell g, specter s, et al. the “achilles heel” of global efforts to combat infectious diseases. clin infect dis. 2006;42(10):1503–1504. http://dx.doi.org/10.1086/504494 world health organization’s regional office for africa. the maputo declaration on strengthening of laboratory systems [document on the internet]. c2008 [cited 2013 jan 02]. available from: http://www.who.int/diagnostics_laboratory/maputo-declaration_2008.pdf world health organization. joint who–cdc conference on health laboratory quality systems. who/hse/ihr/lyo/2008.3 [document on the internet]. c2008 [cited 2013 jan 03]. available from: http://www.who.int/ihr/lyon/report20080409.pdf gershy-damet gm, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. http://dx.doi.org/10.1309/ajcptuuc2v1wjqbm african society for laboratory medicine. http://aslm.org/ abstract introduction methods results discussion acknowledgements references about the author(s) razia b. banderker department of pathology, division of chemical pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa department of pathology, division of chemical pathology, national health laboratory service, cape town, south africa fatima b. fazel department of internal medicine, division of clinical haematology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa annalise e. zemlin department of pathology, division of chemical pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa department of pathology, division of chemical pathology, national health laboratory service, cape town, south africa aye-aye khine division of chemical pathology, national health laboratory service, green point, cape town, south africa thumeka p. jalavu department of pathology, division of chemical pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa department of pathology, division of chemical pathology, national health laboratory service, cape town, south africa citation banderker rb, fazel fb, zemlin ae, khine a-a, jalavu tp. serum-free light chain test utilisation at a south african academic laboratory and comparison with serum protein electrophoresis results. afr j lab med. 2023;12(1), a2201. https://doi.org/10.4102/ajlm.v12i1.2201 original research serum-free light chain test utilisation at a south african academic laboratory and comparison with serum protein electrophoresis results razia b. banderker, fatima b. fazel, annalise e. zemlin, aye-aye khine, thumeka p. jalavu received: 24 mar. 2023; accepted: 05 sept. 2023; published: 24 nov. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: serum protein electrophoresis (spe), urine protein electrophoresis and immunofixation electrophoresis were traditionally utilised for the diagnosis of monoclonal gammopathies. the quantitative serum-free light chain (sflc) assay is reportedly more sensitive and has been introduced to recent clinical guidelines. objective: this study aimed to investigate sflc test utilisation and describe spe findings in patients with abnormal sflc ratios. methods: a retrospective audit of sflc analyses was conducted in cape town, south africa, from may 2018 to april 2020. agreement between abnormal sflc ratios and spe results was determined in a sub-group of patients screened for monoclonal gammopathies. serum-free light chains were analysed using freelite® kappa and lambda assays. results: of the 1425 patients included in the audit, 741 (52%) had abnormal sflc ratios; 636 (45%) had increased and 105 (7%) had decreased sflc ratios. in a sub-group analysis of 117 new patients with an abnormal sflc ratio, 57 had a monoclonal protein (m-protein) on spe (49%), and 60 (51%) did not. four out of 60 patients without m-protein had a plasma cell dyscrasia, while renal impairment or inflammatory response accounted for the rest. of the 57 patients with a m-protein and abnormal sflc ratio, 41 (72%) had a plasma cell dyscrasia, seven (12%) had lymphomas and nine patients (16%) were unclassifiable. conclusion: serum-free light chains should be requested when there is a high index of clinical suspicion. neither sflc nor spe should be performed in isolation when screening patients for monoclonal gammopathy, to ensure that no patient is missed. what this study adds: the study adds to the evidence on sflc test utilisation. serum protein electrophoresis alone may miss cases of light chain myeloma, while sflc performed in isolation may produce false positive results in the setting of inflammatory disorders or renal impairment, leading to unnecessary further investigation. keywords: monoclonal gammopathy; multiple myeloma; m-protein; paraprotein; plasma cell dyscrasia; polyclonal gammopathy; free light chains. introduction monoclonal gammopathies (mg) comprise a spectrum of disorders, from the pre-malignant monoclonal gammopathy of undetermined significance (mgus) to multiple myeloma (mm), which is associated with significant morbidity and mortality.1 a hallmark of mg is the production of a monoclonal protein (m-protein) (except in cases of non-secretory mm).2 some synonyms include ‘paraprotein’, ‘m-component’, and ‘monoclonal immunoglobulin’. the m-protein may consist of intact immunoglobulins (heavy and light chains bound together), free light chains (not bound to a heavy chain) or fragments of either one. the m-protein is produced more commonly by a malignant clone of plasma cells but may also be produced by clonal lymphoplasmacytic cells (as seen in waldenström’s macroglobulinaemia).3 according to the 2019 cancer association of south africa cancer registry statistics, mm accounted for 0.5% of all histologically diagnosed cancers. the most common age group at diagnosis was 60–64 years among both male and female patients.4 for decades, serum protein electrophoresis (spe) and urine protein electrophoresis (upe) and immunofixation electrophoresis (ife) were the initial tests for diagnosing mg. a limitation of spe is that it can only identify m-proteins with a concentration greater than 500 mg/l – 2000 mg/l, and patients with lower concentrations of m-protein, such as those with oligosecretory myeloma, may be missed, especially if the m-protein is a free light chain.5,6 also, for accurate quantification of the m-protein in urine, a 24-h urine collection is preferred over a random specimen. collection of urine for 24 h is inconvenient for patients and often incorrectly collected. however, a recent local study suggested random urine specimens may be used to estimate the 24-h urine m-protein concentration with equations in patients previously diagnosed with mm.7 the quantitative serum-free light chain (sflc) assay was developed in 2001 by the binding site group5 and can detect light chains at concentrations less than 1 mg/l, making it a more sensitive method than spe and upe.8 the international myeloma working group released a consensus guideline for investigating patients suspected of having mm in 20119 and guidelines on the use of the binding site sflc assay in screening, diagnosis and monitoring of mg in 2009.10 the two recommended screening profiles are either spe with ife and the sflc assay, or spe with ife and upe with urine immunofixation electrophoresis (uife).11 the sflc assay was added to our laboratory test repertoire in 2018. although spe and sflc have been recommended, we have subjectively noted that clinicians at our centre frequently request a single test (either spe or sflc) when screening patients for mg. this practice may be an attempt to reduce hospital expenditure; however, it may lead to false-negative screening results and poorer patient outcomes. this study aimed to investigate sflc test utilisation and to describe the spe findings in those with abnormal sflc ratios in the screening of patients for mg at tygerberg hospital (tbh) national health laboratory service (nhls). methods ethical considerations this study was approved by the health research ethics committee of stellenbosch university (s19/10/270, 05 december 2019) and was performed according to the declaration of helsinki. informed consent was not required as the study was a laboratory-based, retrospective audit. all results were de-identified to maintain patient confidentiality and only shared with members of the study team when deemed necessary. data were stored on password-protected devices. study design the study was a retrospective audit of laboratory data and clinical folders of select adult patients on whom sflc assays were requested at tbh nhls for 2 years, from may 2018 to april 2020. setting tygerberg hospital is a 1380-bed tertiary academic hospital in cape town, western cape, south africa, affiliated with stellenbosch university’s faculty of medicine and health sciences. the nhls is the sole laboratory service provider for public healthcare in south africa. the nhls laboratory at tbh participates in internal and external quality control programmes and is an international organization for standardization 15189 accredited laboratory. serum-free light chain samples are referred for analysis from regional hospitals and clinics surrounding tbh. data collection data of all sflc performed in the above period were extracted and anonymised from the nhls laboratory information system (trakcare lab enterprise, intersystems corporation, cambridge, massachusetts, united states) by the corporate data warehouse. serum-free light chain results were classified into five groups: 1 – normal kappa (κ) and lambda (λ) free light chains with normal κ/λ ratio, 2 – raised κ/λ ratio, 3 – decreased κ/λ ratio, 4 – raised κ and λ with normal κ/λ ratio (inflammatory pattern), and 5 – other (normal κ/λ ratio with raised or decreased κ or λ free light chains). stepwise exclusions were performed (figure 1). repeat sflc requests in individual patients were excluded as patients with a mg would have multiple requests over time, skewing demographic statistics. after the initial demographic analysis, groups 1, 4 and 5, and all results from sites other than tbh, were excluded to allow comparison of abnormal sflc ratio with electrophoresis results. results of tbh patients were selected as the electronic medical records were accessible for these patients. medical records of patients seen at other facilities were not readily available. in the final step, patients known with mg and those without an spe performed within 3 months (90 days) of sflc testing were excluded. the relationship between abnormal κ/λ ratio and spe was investigated in tbh patients. figure 1: stepwise exclusion of laboratory data in the study conducted at tygerberg hospital national health laboratory service, south africa, from may 2018 to april 2020. laboratory analyses serum-free light chain serum samples were analysed using the immunoturbidimetric freelite® kappa and lambda assays (the binding site group ltd, birmingham, united kingdom) on the roche cobas® 6000 c601 instrument (roche diagnostics gmbh, mannheim, germany). the coefficient of variation for the freelite® normal internal quality controls was κ 3.98% and λ 3.79%, and κ 2.52% and λ 4.14% for the high internal quality controls (may 2019). the manufacturer’s reference intervals were previously verified in a local study12 and applied as follows: κ free light chain (flc): 3.3 mg/l – 19.4 mg/l, λ flc: 5.7 mg/l ‒ 26.3 mg/l, and κ/λ ratio: 0.26‒1.65. both increased and decreased ratios were considered abnormal.5 the reference interval of 0.37‒3.1 for κ/λ ratio in renal impairment was applied in a select group of patients with abnormal ratio and undetectable m-protein on spe.13 serum protein electrophoresis and ife were performed on the sebia hydrasys 2 (sebia, lisses, france) semi-automated agarose gel electrophoresis system. clinicians at our centre requested spe, while ife was reflexively added by the reporting pathologist if necessary, based on the clinical context or spe appearance (presence of suspected m-protein or hypogammaglobulinaemia). data analysis laboratory data were captured in microsoft excel spreadsheets (microsoft corporation, redmond, washington, united states). demographic statistics of sflc requests and the frequency of abnormal sflc results were determined using statistical package for social scieces, version 27 (ibm corp., armonk, new york, united states). descriptive statistics were determined, where appropriate. results after the data extraction was performed, 1425 individual test results were identified (figure 1) and included in the retrospective audit. sixty percent (n = 860/1425) of patients were female, with a mean age of 59 years (standard deviation ± 13) (table 1). the most requests were received for patients in the 51–70-year age group (figure 2). a minority of requests were received from primary (n = 19, 1.3%) and secondary healthcare institutions (n = 228, 16%). most requests were received from tertiary healthcare institutions (n = 1178; 82.7%). just over half (n = 741, 52%) of κ/λ ratios were abnormal; 44.6% (n = 636) had an increased ratio, and 7.4% (n = 105) had a decreased ratio. figure 2: age distribution of patients for whom serum-free light chain assays were requested at tygerberg hospital national health laboratory services, south africa, from may 2018 to april 2020. table 1: demographics of 1425 patients with serum-free light chain requests received at tygerberg hospital national health laboratory services, south africa, from may 2018 to april 2020. six patients out of 199 (3%) were excluded due to the absence of an spe within 3 months of the sflc request. after stepwise exclusion was performed (figure 1), 117 new patients from tbh with sflc patterns 2 and 3 (abnormal κ/λ ratio) were identified and had sflc findings compared with spe/ife. of those, 57 (49%) had an m-protein present on spe/ife, and 60 (51%) did not. forty-one patients were diagnosed with a plasma cell dyscrasia (72%), seven patients had lymphoma (12%), and nine patients were unable to be classified due to the absence of a bone marrow biopsy and incomplete investigation (16%) (table 2). out of 28 patients diagnosed with mm, 15 were female, with a female-to-male ratio of 1.2:1. of the four patients who were hiv-positive, two had mm, one had mgus, and one had plasmablastic lymphoma. table 2: clinical features of 57 patients identified with monoclonal gammopathy at tygerberg hospital, south africa, between may 2018 and april 2020. of the 57 patients with mg, two had an immunoglobulin g λ m-protein with an unexpectedly raised κ/λ ratio (figure 3). both cases had mildly increased ratios of 1.99 (patient 1, figure 3a) and 2.09 (patient 2, figure 3b), respectively (reference interval: 0.26–1.65). renal impairment could not account for the mildly raised ratios as both patients had normal renal function. bone marrow biopsy revealed a diagnosis of mm with λ restriction in patient 2, while bone marrow findings were normal in patient 1, suggestive of mgus. sixty patients had an abnormal κ/λ ratio without detectable m-protein on spe and their diagnoses were investigated (figure 4). in 38 patients with renal impairment, 2 patients (5.3%) had κ/λ ratios outside of the renal κ/λ ratio reference interval (0.37‒3.1). one patient had a κ/λ ratio of 0.13 and was diagnosed with a mg, while the other had a κ/λ ratio of 4.3 but the diagnosis could not be determined as bone marrow biopsy had not been performed. four patients out of 60 had been diagnosed with a mg (6.7%) based on bone marrow or tissue biopsy findings (table 3). of the 19 patients with a presumed inflammatory response, 17 had polyclonal hypergammaglobulinaemia on spe (89.5%). most of these were from specialities within internal medicine, namely pulmonology (n = 4), neurology (n = 4), rheumatology (n = 2) and gastroenterology (n = 2). figure 3: serum immunofixation gels in two patients with raised κ/λ ratios and λ monoclonal proteins at tygerberg hospital, south africa, between may 2018 and april 2020. a, patient 1. b, patient 2. the black arrows indicate positions of the monoclonal proteins in each immunofixation gel. figure 4: classification of patients with abnormal ratio and absent m-protein on serum protein electrophoresis at tygerberg hospital, south africa, from may 2018 to april 2020. table 3: findings in four patients diagnosed with monoclonal gammopathies, without detectable m-protein on immunofixation electrophoresis at tygerberg hospital, south africa, from may 2018 to april 2020. discussion the study is one of the first african studies investigating sflc test utilisation and correlating abnormal sflc ratios with spe findings. we found that most patients who underwent mg screening were over 50 and were primarily female. most sflc requests came from tertiary healthcare institutions. the most frequently observed sflc pattern was that of a raised κ/λ ratio. few patients were excluded for lack of a recent spe, suggesting that most patients were screened with both spe and sflc, with good adherence to clinical guidelines. in the sub-group analysis of patients with abnormal κ/λ ratio, over half did not have a detectable m-protein on spe. a minority of patients in the sub-group diagnosed with mg were hiv-positive. as mgs are more frequently suspected and diagnosed in older populations, our finding of more frequent screening in older populations is expected.14 the gender distribution in our study was unexpected as higher incidences of mg have been reported internationally among male patients.15 a previous study performed at a tertiary hospital in the western cape included 100 myeloma patients diagnosed between 2008 and 2015. the authors found a female predominance, with a female-to-male ratio of 1.78:1 (unpublished data). an older study performed at a tertiary hospital in gauteng (2004–2009), which included 34 myeloma patients, also found a female predominance (2.4:1).16 a possible reason for this discrepancy could be a difference in healthcare-seeking behaviour among genders in our population. national census data from 2011 obtained by statistics south africa reported that 24.4% of men (compared to 21.1% of women) did not consult a health worker during recent episodes of illness or injury.17 this poor healthcare-seeking behaviour among men could also explain why more female patients than male patients were diagnosed with mg in our study cohort. the low number of sflc requests from primary and secondary healthcare institutions is concerning as many patients could present to their local clinics for non-specific symptoms of mm, such as fatigue or back pain.14 a lack of awareness about mg among health workers at the primary care-level facilities may explain the low screening rate. alternatively, these facilities may not have the budget to request the relatively expensive tests required for screening, necessitating patient referral for further investigation. the relatively few requests received from primary and secondary healthcare institutions may also explain why patients in our setting often present late and with overt signs of end-organ damage. many patients newly diagnosed with mg were not thoroughly investigated as they did not have a bone marrow biopsy performed. one patient did not return for the scheduled biopsy, and one had demised before the biopsy could be performed. it is unclear why the rest were not investigated further, although possible reasons could be that the patients demised at home, clinicians did not view or understand the implications of the screening results, or the patients did not return for other reasons (lack of funds to pay for transport or misunderstanding their diagnosis). additionally, clinicians may have made a preliminary diagnosis of low-risk mgus in certain patients, thus deferring bone marrow biopsy.18 in an audit of ife and sflc results between january 2018 and may 2019 performed in india, authors also noted poor follow-up of patients which they attributed to financial constraints, ignorance or patient death.19 an unexpected finding in our study was the presence of a raised κ/λ ratio and λ m-protein on spe in two patients. with a λ m-protein, the ratio should be decreased.8 patient 1’s serum was sent to another laboratory for repeat sflc testing, and a similar pattern was seen, confirming the result. patient 2’s serum had not been referred for confirmation. we postulate that this discordance between the sflc ratio and spe may be explained by the tendency of λ flc to form dimers, which could obscure target epitopes necessary for detection by the sflc assay antibodies. as described in a previous study, the sflc assay had a relatively high false-negative rate compared to spe and ife in identifying patients with a λ m-protein.20 in the presence of an inflammatory disease process, it would then be possible to see a mildly increased ratio. it is important to note that an abnormal sflc ratio is not entirely specific to the presence of a mg. with decreases in renal glomerular filtration rate, there is a corresponding decrease in renal clearance of flc, leading to a mild increase in the sflc ratio in the absence of mg.13 a large number of patients with an abnormal sflc ratio and no m-protein on spe in our study cohort could be explained by renal impairment. applying the proposed renal reference interval would then allow them to be classified as having a normal sflc ratio. it is not uncommon for myeloma patients to present with renal impairment; one patient with renal failure and absent m-protein on spe was determined to have mm on bone marrow biopsy. in this case, the ratio was 0.13, falling outside of the renal reference interval (0.37–3.1).13 in addition to renal impairment, inflammatory disorders such as autoimmune diseases have been identified as causes for elevated sflc concentrations and a normal to mildly raised κ/λ ratio due to polyclonal increases in light chain production.21,22 in our study, many patients with normal renal function, abnormal sflc ratio and absent m-protein had a polyclonal increase in gamma globulins on spe. this could imply the presence of an inflammatory process. most of these requests were from sub-specialities of internal medicine, in particular from pulmonology, neurology and rheumatology. other studies have reported on the finding of a mildly raised κ/λ ratio with inflammatory disorders, and alternative reference intervals with higher upper limits have been suggested to reduce the rates of false positive ratios while maintaining diagnostic sensitivity. hill et al. (2006) showed that the positive predictive value for an increased ratio improved from 34% to 58% when a cut-off of > 3.0 was used, and further improvement to 78% when a cut-off of > 5.0 was applied.23 similarly, sandfeld-paulsen et al. (2022) found that increasing the ratio cut-off to 4.32 allowed a reduction in false positive results from 19% to 6%.24 as noted in previous guidelines, sflcs are especially useful in identifying and monitoring light-chain mm and light-chain amyloidosis.3,10 in the current study, four patients with negative spe and abnormal ratios had the diagnoses of light-chain mm and solitary plasmacytoma. this finding demonstrates the utility of requesting both spe and sflc in the initial screening of patients. studies have shown that this combination of tests allows identification of virtually all cases of mg,25,26 although upe and uife is still recommended when screening for light-chain amyloidosis.10 the current study focused on new patients not previously identified with a mg as there is ongoing discussion regarding the ideal mg screening test combination: spe/ife and sflc or spe/ife and upe/uife.1,27 in a recently published clinical update article, rajkumar recommended that the initial investigation for a patient suspected of having mm should include spe, serum ife and sflc.1 this is based on a study by katzmann et al., who identified 428 patients with positive m-protein on urine ife and determined the diagnostic sensitivity of different screening strategies. they determined that spe with ife alone would have missed 6.5% of patients (primarily light chain amyloidosis), while sflc alone would have missed 14% of patients. the combination of spe, ife and sflc identified 99.5% of patients. the two cases missed by this combination were diagnosed with mgus and did not require medical intervention.25 in practice, collecting a single serum specimen rather than collecting both serum and urine from patients for screening purposes is also more convenient. in a review article, singh described spe and upe with ife as the gold standard for diagnosing mg.27 when a m-protein is identified on upe, it is diagnostic of mg, while an abnormal sflc ratio is not. therefore, the test combination of spe/ife and upe/uife does not produce false positive results as with sflc testing.27 false positive results may lead to unnecessary further investigation and patient anxiety.28 while the above is true, it is crucial to consider that upe is typically a semi-automated method and, therefore, more labour-intensive than fully automated sflc analysis. automation allows higher throughput and shorter turnaround time for sflc testing. urine protein electrophoresis and uife also require visual inspection and manual interpretation, leaving room for human error. limitations a limitation of our study is the small number of patients included in the comparison of κ/λ ratio with spe. samples for sflc analysis are received from multiple sites around the country; however, only a limited number of these sites use electronic medical record-keeping. it was not feasible to trace the physical medical records of these patients, and we were, therefore, limited to patients assessed at our centre. a further limitation is that we did not determine the diagnostic sensitivity and specificity of the available screening strategies due to the small numbers of patients with biopsy-confirmed mm included in the study. due to the study’s retrospective nature, we did not look at other markers of inflammation, which may be helpful in patients with a mildly increased κ/λ ratio and absent m-protein on spe. conclusion this study is one of the first to report on sflc findings in an african context. it highlights challenges unique to a lowerto middle-income setting, such as the high rates of patients lost to follow-up or incomplete investigation and low volumes of tests received from primary and secondary care levels. the authors have also demonstrated some of the strengths and limitations of sflc and spe methods in screening patients for mg. the findings showed that many sflc results were in keeping with an inflammatory pattern, and hence, in patients suspected to have mg, sflc requests should be based on a high index of clinical suspicion and performed with spe. as per the literature, the most critical finding is that neither sflc nor spe should be performed in isolation to ensure no patient is missed. acknowledgements the authors would like to acknowledge prof. tonya esterhuizen from the biostatistics unit, division of epidemiology and biostatistics, for assistance with data organisation and analysis through the support from the stellenbosch university faculty of medicine and health sciences dean’s fund. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions r.b.b. was responsible for study design, data collection and curation, data analysis, data visualisation, original draft and review of the article. f.b.f. contributed to study design, data analysis, original draft and review of the article. a.e.z., a-a.k. and t.p.j. contributed to conceptualisation, study design, original draft and review of the article. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability data are not publicly available but may be requested from the corresponding author, t.p.j., upon reasonable request and after consultation with the health research ethics committee of stellenbosch university. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references rajkumar sv. multiple myeloma: 2022 update on diagnosis, risk stratification, and management. am j hematol. 2022;97(8):1086–1107. https://doi.org/10.1002/ajh.26590 jenner e. serum free light chains in clinical laboratory diagnostics. clin chim acta. 2014;427:15–20. https://doi.org/10.1016/j.cca.2013.08.018 tate j. the paraprotein – an enduring biomarker. clin biochem rev. 2019;40(1):5–23. 2019 cancer association of south africa (cansa) cancer registry [homepage on the internet]. 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however, there is a dearth of information on nosocomial candidaemia in nigeria, despite the increasing use of more invasive therapeutic modalities, immunosuppressive agents and increasing incidence of immunosuppression as a result of malignancies and hiv.objectives: to determine the hospital-based frequency of candidaemia in a tertiary hospital in ibadan, nigeria. method: this was a prospective descriptive study which included 230 immunosuppressed patients. all isolates were identified to the species level using both conventional and automated methods. thereafter, all candida species isolated were tested for antifungal susceptibility using the broth microdilution method. results: candidaemia occurred in 12 (5.21%) of the 230 study patients, with c. tropicalis accounting for 50% of the infections. four patients (33.3%) presented with c. parapsilosis, one (8.3%) with c. albicans and one (8.3%) with a mixed infection of c. albicans and c. tropicalis. all 12 isolates were sensitive to fluconazole (minimal inhibitory concentration < 8 mg/ml). univariate analysis revealed that old age, multiple surgeries and long-term hospitalisation were significant contributing factors for the occurrence of candidaemia. eleven (91.7%) of the 12 patients with candidaemia had candida colonisation of other sterile sites including the bladder, peritoneum and trachea. furthermore, bivariate analysis revealed that mucositis (p = 0.019) and diarrhoea (p = 0.017) were significantly associated with an increased risk of candidaemia. the crude mortality rate of candidaemia was 91.7%. conclusion: this study highlights the significance of nosocomial candidaemia and the need for proactive laboratory investigation and clinical management of this life-threatening disease. introduction top ↑ since the 1980s, candida infections have become a growing problem.1,2,3,4 martin et al. reported a 207% increase in incidence between 1979 and 2000 in the united states.5 morbidity rates in immunocompromised patients are as high as 50%.6,7 patients with underlying clinical conditions, such as haematological malignancies, long-term intensive care unit (icu) stays and prematurity, are at high risk for candidaemia. independent risk factors for infection include treatment with broad-spectrum antibacterial agents, immunosuppressive therapy and/or parenteral nutrition; prior haemodialysis; and having implanted prosthetic devices.8,9,10,1112candidaemia poses a complex clinical puzzle. firstly, blood culture, the gold standard for diagnosis and treatment of candidaemia, is positive in less than 50% of infected patients and takes several days for a diagnosis.13 second, trends in incidence and species distribution differ between geographical zones and healthcare facilities.14 thirdly, some species of candida are resistant to fluconazole. without rapid diagnosis and treatment, the consequences are clear: a 20% increase in mortality was found in cases where antifungal therapy was delayed for more than 12 hours.15,16 as there is insufficient data on nosocomial candidaemia in nigeria, we conducted a prospective study to establish the frequency of candidaemia, predisposing factors and the susceptibility of candida spp. to fluconazole. research method and design top ↑ conducted from 01 september 2008 to 30 august 2009 at university college hospital, a 960-bed, tertiary referral centre in ibadan, nigeria, this study enrolled 230 immunosuppressed patients who had been admitted at least 10 days prior to the start of the study and had persistent pyrexia (temperature > 38 °c). study subjects included patients presenting with haematological malignancies; icu patients; very low birth weight (vlbw) premature neonates (temperature > 38 °c or < 35 °c); patients who had undergone multiple abdominal surgical procedures during their current admission; diabetic patients with persistent pyrexia; and hiv patients. the patients’ underlying diseases were classified broadly, as can be seen in table 1. exclusion criteria included non-consenting patients, patients without clinical signs of infection and patients already receiving empirical antifungal therapy. probability proportionate to size was used to estimate the number of patients selected per targeted study population group.17 table 1: demographic characteristics of patients studied and underlying diseases. participation in the study was voluntary and, after receiving written consent from the patients or their immediate relatives, the investigators explained the study in both english and yoruba and then administered a semi-structured questionnaire, adapted from a european confederation of medical mycology intensive care unit (ecmm icu) form. the questionnaire was divided into three sections: socio-demographic data; medical and surgical history (based on known risk factors for candidaemia); and laboratory findings. candidaemia was defined as being the isolation of candida species from at least one positive blood culture sample in patients with clinical signs of bloodstream infection. two venous blood samples of five ml each were collected from each patient. ten millilitres of blood was added to each bactec blood culture bottle and incubated at 37 °c using a bactec 9050 blood culture system (becton dickinson, inc., sparks, md, usa), an automated system that contains antibiotic-absolving resins, allowing for the culturing of blood from patients on antibiotics without discontinuing treatment. additional advantages of this system are its rapid turnaround time and increased yield compared with the conventional, manual blood culture system. positive samples were examined microscopically using direct gram staining; those showing yeast were cultured onto sabouraud’s dextrose agar (oxoid, uk) and chromagar candida (chromagar, france). the germ tube test was used for presumptive diagnosis of c. albicans, but all isolates were identified to the species level using api 20c aux and/or id 32c automated strip detection (biomerieux vitek, inc., st louis, mo, usa). susceptibility testing to fluconazole was performed using the broth microdilution method in accordance with the clinical and laboratory standards institute (clsi) recommendations (m27-a2).18 stock inocula were prepared by adding 24–48-hour-old test candida to roswell park memorial institute (rpmi) broth and adjusting to a 0.5 mcfarland standard. the stock inocula were diluted 1:1000 and 100 μl of each was added to two-fold 100 μl dilutions (ranging from 0.12 to 64 μg/ml) of fluconazole on the microtitre plate as well as to a drug-free medium. after incubating the microtitre plates at 35 °c for 24–48 hours, the amount of growth in a well containing the antifungal agent was compared with the amount of growth in an antifungal-free, growth-control well. the minimum inhibitory concentration (mic) was the lowest concentration of antifungal agent that visibly inhibited 50% growth of the organism. isolates with an mic < 8 mg/ml were considered to be susceptible to fluconazole; isolates with an mic > 64 mg/ml were resistant.18 those with an mic from 16–32 mg/ml were fluconazole susceptible dose-dependent (s-dd).18 quality control was ensured by the inclusion of clsi-recommended quality control strains candida parapsilopsis atcc 22019 (mic range 2–8 mg/ml) and candida krusei atcc 6258 (mic range 16–64 mg/ml). quantitative data from the questionnaire were entered into microsoft excel 2003 (v11.0) (microsoft, redmond, wa) and spss 15.0 for windows (spss inc., chicago, 2006) was used for the analysis. the univariate analysis used descriptive statistics and tables, including frequency distribution; and the bivariate analysis used the chi-square test with statistical significance set at p < 0.05. results top ↑ of the 230 study patients, the median age was 18 years, with 27.5% (n = 63) being less than one year old and 10.9% (n = 25) older than 60. the male-to-female ratio was 1.12:1. all patients were admitted for hospitalisation: 39.7% (n = 89) for 11–20 days and 17.8% (n = 42) for more than 30 days (table 1). seven percent (n = 16) of the patients underwent more than one operation (table 2). most of the study population (97.4%; n = 224) were treated with broad-spectrum antibiotics; 45.7% (n = 105) of these received 11–20 days of different combination of broad-spectrum antibiotics. the median duration of antibiotic use was 15.5 days, with only 11.7% (n = 27) of patients receiving antibiotic treatment for more than 21 days (table 2). most patients had at least one invasive procedure during their admission, with 92.2% (n = 212) undergoing intravenous cannulation and 8.7% (n = 7) undergoing venous cutdown (table 2). other treatments used in this population included antifungal therapy for non-invasive candidiasis, anti-cancer chemotherapy, radiotherapy, steroids and highly-active antiretroviral therapy (haart). table 2: treatment history of patients in the study. the distribution pattern of isolates from blood culture samples showed that staphylococcus aureus (42.9%; n = 30) and klebsiella spp. (20.0%; n = 14) were the most common pathogens, followed by candida spp. (17.1%; n = 12) (figure 1). of the enrolled patients, 12 (5.2%) had candidaemia. c. tropicalis (n = 6; 50%) was the most frequently-identified candida spp. (figure 2). based on chromagar candida phenotypes, one patient had a polymicrobial infection with both c. albicans and c. tropicalis. candida spp. was also isolated from non-venous, invasive sites in 11 (91.7%) of the 12 patients (figure 3). all candida spp. isolates were susceptible to fluconazole (mic < 8 mg/ml). in addition, the api 20c aux and id 32c identification of the candida spp. were in concordance with one another. figure 1: bar chart showing distribution of pathogens causing bloodstream infections. figure 2: bar chart showing distribution of candida spp. causing bloodstream infections. figure 3: of the 12 (5.2%) patients with candidaemia in this study, age (p = 0.8) and sex (p = 0.16) distributions were not of statistical significance. these patients’ underlying diseases included solid tumours, diabetes, hiv, infective endocarditis, haematological malignancy, vlbw, severe head injury and sepsis. there was no significant relationship (p = 0.18) between underlying disease and the various candida spp. detected. using chi-square analysis, diarrhoea (p = 0.017) and oral thrush (mucositis) (p = 0.019) were the only statistically-significant symptoms (table 3). the crude mortality rate was 91.7% (11 out of 12 patients). table 3: association of symptoms of immunosuppression in patients who developed candidaemia. ethical considerations top ↑ ethical approval for the study was obtained from the national health research ethics committee (nhrec/ 05/01/2008a) and, in keeping with ethical procedures, the patients or their immediate relatives provided informed written consent. potential benefits and hazards the sample collection is a minimum risk procedure (venipuncture under standard precautions). benefits to the participants included free blood culture and feedback of results to the managing clinicians. trustworthiness top ↑ confidentiality was ensured by the identification of participants with code numbers. other personal details of the participants were kept in safe keeping with the lead researcher for 5 years in a secured file. discussion top ↑ candidaemia is a nosocomial infection commonly seen in critically-ill patients and those with haematological malignancies.1,2,4,6,19 in this study, candida spp. ranked third among pathogens causing blood stream infections (bsis) in hospitalised patients admitted to the study centre. findings from north american and european surveillance programmes of hospital-acquired infections showed that isolates of candida spp. were the fourth most common cause, accounting for 8% – 10% of nosocomial bsis.19 this difference could exist because all of the participants in this study were immunosuppressed; not all presented with bsis; and the developed world has established protocols and guidelines for the diagnosis, management and prevention of invasive fungal infections.the hospital-based frequency of candidaemia in this study was 5.2%, which could possibly be a result of nigeria’s progressive changes in the medical and surgical management of patients over the last five years. these improvements have reduced the vulnerability of critically-ill patients to haematogenous dissemination of candida spp. whereas previously, bacterial and/or viral infections often resulted in fatalities. conversely, antifungal drugs are not administered routinely, either empirically or as prophylaxis, in the management of high-risk patients in nigeria, which could possibly explain the high mortality rates from candidaemia. several risk factors for candidaemia have been shown in previous studies and confirmed in this study. in this study, patients under the age of one (n = 63) accounted for 27.5% of the study population and 25% of patients with candidaemia. this is consistent with findings by saiman et al. which showed that infants, especially premature, vlbw babies, have a higher risk of candidaemia because of their immature immune systems and frequent intubation.20 patients over the age of 60 (n = 25) accounted for 10.9% of the studied population and 16.7% of the cases of candidaemia, which is relatively high, but not of statistical significance; however, schelenz found that old age was an independent risk factor for candidaemia.21 two of the patients had infective endocarditis, one of whom had undergone cardiac surgery, a high-risk factor for fungal endocarditis, with an infection rate of 0.23% – 1.0%.22,23 another known risk factor for candidaemia is long-term hospital stays, especially in icu settings.22 there was a correlation between length of hospital stay and frequency of candidaemia, further confirming the nosocomial source of the infection and that prolonged hospital stays are risk factors.2,3,19 broad-spectrum antibiotic use is a major risk factor for the development of candidaemia because these medications eliminate the bacterial gut flora and encourage the overgrowth and dissemination of gastrointestinal fungal commensals.1921 almost all of the enrolled patients with candidaemia were taking several antibiotics, for a median period of 15.5 days. most patients had intravenous lines in situ, but only 13.6% (n = 7) underwent intravenous cutdown (femoral vein). 11 of the 12 patients with candidaemia had the same candida spp. isolated from other sites. symptoms associated with candidaemia were mucositis (p = 0.019) and diarrhoea (p = 0.017). these findings are consistent with those of other reports showing mucosal colonisation to be an independent risk factor for candidaemia;21,24 however, there are no previous studies citing any relationship between candidaemia and diarrhoea. the high proportion of c. tropicalis (50%) reported here is consistent with a study in india by verma et al. where the proportion was 46%.25 the crude mortality rate in our study was 91.7% (11 out of 12 patients) compared with the 35% – 75% crude mortality rates reported in other studies.23 this could be attributed to small sample sizes, as well as to the fact that the clinicians at the study site did not administer antifungal drugs routinely, either on an empirical or a prophylactic basis, for the management or prevention of candidaemia. clinicians may not consider candidaemia as a cause of infection because of limited training in nosocomial diseases, the absence of knowledge of disease prevalence (ncbi pubmed searches reveal few publications in the field from nigeria) and the lack of an established infrastructure for high-quality laboratory testing. limitations of the study top ↑ limitations of this study include the small sample size and generalised study population. conclusion top ↑ this study highlights the clinical significance of nosocomial candidaemia and the need for proactive investigation for possible invasive candidiasis. this is particularly needed in nigeria where patients have to pay for every laboratory investigation carried out in the hospital; as a result, clinicians cannot proactively and/or repeatedly request tests or cultures. this study highlights the need for affordable laboratory tests and an improved understanding of the dangers of untreated candidaemia. finally, improved communication and collaboration between clinicians and clinical microbiologists is essential for the development of measures to control the emerging threat of nosocomial candidaemia. acknowledgements top ↑ the authors would like to acknowledge prof. marianna viviani for her contribution with the eccm icu form and her advice on specimen storage and transportation, as well as drs olubunmi olubanwo and victor uwadike for their help in sample collection and data collation. competing interests prof. malcolm richardson has received research funding and speaker fees from gilead science, pfizer, msd and astellas. the other authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions r.o.o. (university college hospital, nigeria) was involved in the conception and design of the study, as well as with the acquisition, analysis and interpretation of the data. she also wrote the article. r.a.b. (university college hospital, nigeria) was involved in the conception and design of the study, as well as the write up. m.r. (university of manchester) had substantial involvement in the revision of the article and performed the expert review prior to submission. o.o.o. (university of lagos, nigeria) was involved in the conception of the article, as well as the data collection and analysis. m.a.p. 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children. eur j pediatr. 1999;158(4):275–280. http://dx.doi.org/10.1007/s004310051071 23.dean da, burchard kw. fungal infection in surgical patients. am j surg. 1996;171(3): 374–382. http://dx.doi.org/10.1016/s0002-9610(97)89647-x 24.jordà-marcos r, alvarez-lerma f, jurado m, et al. risk factors for candidaemia in critically ill patients: a prospective surveillance study. mycoses. 2007;50(4):302–310. http://dx.doi.org/10.1111/j.1439-0507.2007.01366.x 25.verma ak, prasad kn, singh m, et al. candidaemia in patients of a tertiary health care hospital from north india. indian j med res. 2003;117:122–128. abstract introduction methods results discussion acknowledgements references about the author(s) mmachuene i. hlahla department of forensic pathology, faculty of health sciences, university of limpopo, polokwane, south africa moshibudi j. selatole department of forensic pathology, faculty of health sciences, university of limpopo, polokwane, south africa citation hlahla mi, selatole mj. could ante-mortem computed tomography be useful in forensic pathology of traumatic intracranial haemorrhage? afr j lab med. 2021;10(1), a1040. https://doi.org/10.4102/ajlm.v10i1.1040 original research could ante-mortem computed tomography be useful in forensic pathology of traumatic intracranial haemorrhage? mmachuene i. hlahla, moshibudi j. selatole received: 24 apr. 2019; accepted: 25 feb. 2021; published: 29 july 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: imaging techniques have proven valuable in forensic pathology practice, with computed tomography being preferred for forensic use. in the era of virtual autopsy and a lowto middle-income, resource-constrained country, a question arises as to whether ante-mortem computed tomography (act) could be cost-effective by reducing the number of invasive autopsies performed. objective: the objective of this study was to assess the usefulness of act in forensic pathology by examining discrepancy rates between act scans and autopsy findings in cases of deceased individuals with traumatic intracranial haemorrhages and assess factors associated with discrepancies. methods: eighty-five cases of act and autopsy reports from 01 january 2014 to 31 december 2016 from the polokwane forensic pathology laboratory, south africa, were analysed retrospectively. using cohen’s kappa statistics, measures of agreement and resultant discrepancy rates were determined. also, the discrepancy patterns for each identified factor was also analysed. results: the discrepancy rate between act and autopsy detection of haemorrhage was 24.71% while diagnostic categorisation of haemorrhage was 55.3%. classification discrepancy was most observed in subarachnoid haemorrhages and least observed in extradural haemorrhages. a markedly reduced level of consciousness, hospital stay beyond two weeks and three or fewer years of doctors’ experience contributed to classification discrepancies. conclusion: ante-mortem computed tomography should be used only as an adjunct to autopsy findings. however, the low discrepancy rate seen for extradural haemorrhages implies that act may be useful in the forensic diagnosis of extradural haemorrhages. keywords: forensic imaging; ante-mortem computed tomography; traumatic intracranial hemorrhage; forensic autopsy. introduction imaging techniques have in recent years been found to be greatly useful in forensic pathology.1,2 these modalities not only serve to augment but lower the invasive autopsies performed.3 the latter is convenient for various reasons, including religious and economic reasons. computed tomography (ct) has been used as the preferred imaging modality in forensic imaging.4 though in the era of virtual autopsies, the usefulness of ante-mortem computed tomography (act) compared to the inexpensive invasive autopsy in a middle-income country, such as south africa, must be justified. thus, it is necessary to determine whether act will be an augmentation or a cost-effective alternative to autopsy. head injuries are common, usually carry a high mortality rate, and are therefore important in forensic pathology practice. while post-mortem ct has been found useful in determining the cause of death in cases of traumatic intracranial haematomas,2,5 there is limited empirical evidence for the use of act scan findings to this effect. discrepancies between clinical or ante-mortem ct scan and autopsy findings exist and are common.6 according to bruno et al.,7 radiologic interpretations cannot be programmed because interpretation is subject to a variety of factors such as doctors’ (radiologists’ and pathologists’) expertise and complex psychophysiological and cognitive level of the patient (e.g. level of consciousness). thus, act interpretation errors are inevitable. this study assessed the rate and pattern of discrepancies between act scan and conventional autopsy findings of intracranial haemorrhages in cases of traumatic head injury submitted for autopsy at a facility in a rural south africa province. methods ethical considerations ethical clearance for the study was secured from turfloop research and ethics committee (trec/268/2017: pg). consent was not applicable as we were not working on human subjects but case reports, permission for which was obtained from the limpopo provincial department of health. the case files were coded with numbers and names of the deceased were not recorded. study design this quantitative descriptive study retrospectively analysed 85 cases of deceased individuals across three years from 01 january 2014 to 31 december 2016. cases sustained head injuries, underwent act imaging and had no surgical intervention after being referred to an academic hospital. deceased cases were subjected to autopsy procedure as per legal requirement at the attached forensic pathology facility in limpopo, a rural province of south africa. data collection data were obtained from post-mortem reports from the forensic pathology laboratory while ct scan reports, clinical data and human resource records were obtained from the pietersburg hospital records section and radiology department. autopsy findings served as a reference point because studies have demonstrated that it remains superior to act scan for the detection of brain injuries.1,8 the majority (n = 80) of the cases had only one act scan done and the rest a second; only the first scan reports were therefore used in the study. the following intracranial haemorrhages were assessed from these reports: epidural, subdural and subarachnoid haemorrhages. data analysis data were captured and analysed using microsoft excel (microsoft office professional plus 2013; microsoft corp., redmond, washington, united states) and international business machines corporation statistical package for the social sciences version 23 (armonk, new york, united states) running under microsoft windows (microsoft corp., redmond, washington, united states). cross-tabulations were used to establish the percentage agreement between act scan and autopsy findings of extradural haemorrhage (edh), subdural haemorrhage (sdh) and subarachnoid haemorrhage (sah) single or combination occurrence. if there is agreement between the act scan and autopsy findings the individual case scored 1, but if there is a disagreement between the act scan and autopsy findings, the case scored 0. levels of agreement and resultant discrepancy rates were determined using cohen’s kappa statistics. the pattern of discrepancies for identified factors such as the level of consciousness by glasgow coma scale, the length of hospital stay, the experience of the clinician (radiologist and pathologist) and the site of haemorrhage was also evaluated. results were considered statistically significant when p was less than 0.05. results agreement in the detection and diagnosis of haemorrhages in 75.29% (64/85) of cases, the act scan and autopsy agreed on the presence or absence of haemorrhage with a kappa coefficient of 0.3834 (table 1). the remaining 24.71% represents the overall discrepancy rate between the act scan and autopsy detection of haemorrhage. table 1: agreement between autopsy and ante-mortem computed tomography findings in the detection of intracranial haemorrhages, south africa, 2014–2016. the highest agreement between the act scan and autopsy finding was recorded in the diagnosis of edh (agreement 90.59%, kappa coefficient 0.5823 and discrepancy rate 9.41%) (table 1). with regard to sdh, the agreement was 74.12% with a kappa coefficient of 0.4857 and a discrepancy rate of 25.88% (table 1). the lowest agreement of 65.88% with a kappa coefficient of 0.3219 and the highest discrepancy rate of 34.12% was found for sah (table 1). in 38 of the 85 cases (44.7%; 25 with and 13 without haemorrhage), both methods agreed in the diagnostic intracranial haemorrhage category (i.e. single, binary or ternary combinations of haemorrhages), denoting a diagnostic category discrepancy of 55.3% (table 2 and table 3). act agreed in 7 of the 11 cases categorised as singular sah by autopsy (bolded) but reclassified three cases as having binary haemorrhage (sah and sdh) and one case as absent (no haemorrhage). for 25 cases classified as ‘absent’ (having no haemorrhage) by autopsy, act agreed in 13 (bolded) cases but categorised the remaining 12 as having different haemorrhages. table 2: summary of diagnostic category for intracranial haemorrhage, south africa, 2014–2016. table 3: summary of misclassification of detection category agreement and discrepancies observed for haemorrhages, south africa, 2014–2016. pattern of discrepancies with regard to identified factors the analysis revealed that most discrepant intracranial haemorrhage diagnoses (5 out of 8 cases for edh; 15 out of 22 cases for sdh; 19 out of 29 cases for sah) were seen in patients with markedly low levels of consciousness, denoting severe traumatic head injury (figure 1). figure 1: discrepancies and level of consciousness, south africa, 2014–2016. 1 = mild (gcs 15–13), 2 = moderate (gcs 12–9), 3 = severe (gcs ≤ 8), 0 = unspecified. half (4/8) of the discrepant cases for edh were found to have been admitted on average for a day or less (figure 2). on the other hand, most discrepancies for sah were seen in cases that were admitted for two weeks or less (9/29) and four weeks or more (10/29) on average, while for sdh the majority (9/22) stayed in the hospital for more than a month. figure 2: discrepancies and length of admission, south africa, 2014–2016. in addition, more discrepant case diagnoses were seen with a radiologist or pathologist who had less than three years of working experience (figure 3). figure 3: discrepancies and level of experience (in years), south africa, 2014–2016. discussion the current study found a significant difference in haemorrhage detection between act and autopsy. individual intracranial haemorrhage detection discrepancies ranged from 9.41% to 34.12%, with sah carrying the highest rate. this is consistent with what has been reported by a number of previous studies that collectively reveal discrepancy rates ranging from 9% to 39%.5,9 there are varying findings concerning discrepancy rates for sah with the majority of previous studies showing high discrepancy10,11,12 in keeping with the current study. the study also found that in general act had a diagnostic accuracy of 44.7%. as such, the high level of discrepancy for sah was attributable to misclassification, which may mean misdiagnosis, and to a combination of haemorrhages that could have masked the sah. also, a high sah discrepancy rate (79% of sah cases) was noted with prolonged length of hospital stay, probably due to haemorrhage resorption with time. a progressive clearance of red blood cells in the cerebrospinal fluid results in approximately 50% of sahs not being visualised after one week of occurrence,13,14 and this process can be shorter or longer.15 this implies that the majority of the sah previously diagnosed under an act scan may after approximately three weeks not be seen in an autopsy if the patient demises. this is dependent on the amount of sah in the leptomeninges. this was evident from the current study where the act scan superseded autopsy in the diagnosis of sah, which was consistent with previous studies.11,14 the high level of agreement of agreement (90.59%) observed for edh diagnosis (with high true negative rate) was corroborated in a similar study comparing act and autopsy findings11 and when comparing post-mortem ct diagnoses to autopsy results.9 we opine that the high level of discrepancy in cases admitted for a day or less may be because it takes more than a day for the typical shape of the haemorrhage to appear. more discrepancies were noted in cases with markedly low levels of consciousness, denoting severe injuries. this may be because critically ill patients may not assume specific positions required to visualise the haemorrhage, particularly when the haemorrhage is small in size, a notion also suggested by liisanantti and ala-kokko.5 practitioners (radiologists and pathologists) with a low level of experience made more discrepant diagnoses. although most studies support that experience appears to decrease discrepancies,16,17 they also agree that there are multifactorial and complex factors that can result in those discrepancies. some stipulate that the main reason for the discrepancies in lower postgraduate trainees has been identified as a lack of knowledge.18,19 doctors with low levels of experience are typically registrars and junior medical officers. lee et al.20 report that discrepancies tend to be higher among registrars, owing to ‘physical discomfort, eye strain and lack of motivation’, which intensify by the end of the workday. moreover, registrars and medical officers work overtime, during which they have to work overnight. this may result in focusing difficulty and hence reduced diagnostic or detection accuracy.21,22 length of admission has also been shown to affect agreement for sdh. the study showed that the greater part of the discrepant sdh cases were seen in patients who were admitted for more than a month on average. a possible reason could be the effects of the healing processes, which can happen in thinner blood collection cases such that after a month the haemorrhage may be enclosed or completely absorbed.23,24 brain slicing intervals during autopsies are usually not the same as the intervals used during act imaging and this may also account for the general discrepancy rate. delayed intracranial haemorrhage could occur after the act scan is obtained and could also account for the discrepancies as most of the cases did not undergo a subsequent act scan. a useful scenario to study this would have been to have the act and autopsy on the same day. limitations the major limitation of this study is the small sample size. moreover, the study addressed only three types of brain haemorrhages (edh, sdh and sah); therefore, the findings cannot be generalised to forensic pathology practice, or any other type of intracranial haemorrhage. further research with a larger sample size and broader scope is recommended. conclusion the overall detection discrepancy rate of 24.74% and ct diagnostic accuracy of 44.7% implies that act scans may not be used as an altervative to reduce the number of autopsies performed at the mentioned facility but can only be used as an adjunct to autopsy. however, the low discrepancy rate in edh, especially after a day of admission, implies that act may be useful for the diagnosis of this haemorrhage in the forensic setting. a markedly reduced level of consciousness, length of hospital stay depending on the type of haemorrhage and three or fewer years of doctors’ experience all contributed to discrepancies observed between act and autopsy findings. the study employed a limited sample and thus calls for more similar studies in both high and lowand middle-income countries. acknowledgements we would like to thank mr p. mphekgwana (university of limpopo) and mr a. poopedi (pietersburg provincial hospital) for statistical analyses. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions m.i.h. conducted the research and produced a mini-dissertation. m.j.s. supervised m.i.h. and prepared the publication manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability statement data sharing is not applicable to this article as no new data were 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accuracy. j am coll radiol. 2010;7(9):698–704. https://doi.org/10.1016/j.jacr.2010.03.004 waite s, scott j, gale b, fuchs t, kolla s, reede d. interpretive error in radiology. ajr am j roentgenol. 2017;208(4):739–749. https://doi.org/10.2214/ajr.16.16963 itabashi hh, andrews jm, tomiyasu u, erlich ss, sathyavagiswaran l. forensic neuropathology – a practical review of the fundamentals. burlington, vt: academic press; 2007. saukko p, knight b. knight’s forensic pathology. 4th ed. new york, ny: crc press; 2016. abstract introduction methods results discussion acknowledgements references about the author(s) susan k. musau department of laboratory, maryland global initiatives corporation (mgic), nairobi, kenya christina mwachari department of laboratory, maryland global initiatives corporation (mgic), nairobi, kenya elvis kirui department of strategic information, maryland global initiatives corporation (mgic), nairobi, kenya junghae muthoni laboratory department, centers for disease control, nairobi, kenya taylor lascko center for international health, education, and biosecurity, university of maryland, baltimore, maryland, united states natalia blanco center for international health, education, and biosecurity, university of maryland, baltimore, maryland, united states alash’le abimiku school of medicine, university of maryland, baltimore, maryland, united states emily koech department of laboratory, maryland global initiatives corporation (mgic), nairobi, kenyacenter for international health, education, and biosecurity (ciheb), nairobi, kenya citation musau sk, mwachari c, kirui e, et al. implementing an intensified mentorship approach towards accelerated medical laboratory accreditation in 10 counties in kenya. afr j lab med. 2022;11(1), a1814. https://doi.org/10.4102/ajlm.v11i1.1814 original research implementing an intensified mentorship approach towards accelerated medical laboratory accreditation in 10 counties in kenya susan k. musau, christina mwachari, elvis kirui, junghae muthoni, taylor lascko, natalia blanco, alash’le abimiku, emily koech received: 14 dec. 2021; accepted: 18 mar. 2022; published: 01 july 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: despite kenya’s roll-out of the strengthening laboratory management towards accreditation programme in 2010, most laboratories had not made significant or tangible improvements towards accreditation by 2016. in april 2016, the university of maryland, baltimore enrolled 27 facilities in the standard strengthening laboratory management towards accreditation programme. objective: this study aimed to describe and evaluate the implementation of an intensified mentorship strategy on laboratory accreditation. methods: in october 2017, the university of maryland, baltimore implemented intensive mentorship in 27 hospital laboratories in nairobi, kiambu, meru, embu, muranga, nyeri, laikipia, nyandarua, tharaka-nithi, and kirinyaga counties in kenya. laboratories were paired with competent mentors whose skills were matched to facility gaps. baseline and follow-up assessments were done between april 2016 and march 2019 using the world health organization’s stepwise laboratory quality improvement process towards accreditation checklist and overall scores of the 12 quality system essentials and star ratings (from zero to five, based on scores) used to evaluate the effectiveness of the intensified mentorship. results: in september 2017, 14 laboratories scored zero stars, three scored one star, eight scored two stars, one scored three stars, and one laboratory was accredited. by march 2019, eight laboratories were accredited, five scored four stars, 10 scored three stars, three scored two stars, and only one scored one star. the average score change with the intensified approach was 81.5 versus 53.9 for the standard approach. conclusion: the intensified mentorship strategy resulted in fast-tracked progress towards laboratory accreditation and can be adopted in similar resource-limited settings. keywords: accreditation; slmta; slipta; qms; intensified mentorship; kenya. introduction implementing a laboratory quality management system (qms) is critical to providing reliable results for clinical decision-making.1 inaccurate laboratory results can lead to inappropriate interventions, adversely affecting the credibility of the laboratory, and may invite legal action.2 laboratory accreditation formally recognises both the qms and the technical competence of a laboratory to perform specific tests.1,3 laboratory accreditation is a system of standard procedures that aims to improve laboratory services’ quality, results’ accuracy, and patients’ safety.4,5 most laboratories rely on international quality standards for medical laboratories (international organization for standardization 15189), which specify the requirements of quality and competence to medical laboratories. this set of standards is comprehensive but also resource-intensive.5 resource-limited settings face several challenges when implementing quality standards and practices in laboratories, including financial constraints, human resource capacity, limited physical infrastructure, lack of equipment and equipment maintenance, failure to create and/or implement national laboratory policies, and substandard performance in laboratory quality indicators due to a lack of quality standards.6,7 the strengthening laboratory management towards accreditation (slmta) programme was launched in 2009,8 and adopted in kenya in 2010. strengthening laboratory management toward accreditation is a competency-based programme that uses a series of short courses and work-based learning projects to effect immediate and measurable laboratory improvements while empowering laboratory managers to implement a practical qms to ensure better patient care. a standard slmta training programme spans from 12 to 18 months. it includes three workshops spaced between qms’ application and improvement projects’ implementation. the process is supported by regular supervisory visits or on-site mentoring. audits are conducted at the beginning, midterm, and end of the training using the world health organization’s stepwise laboratory quality improvement process towards accreditation (slipta) checklist to assess strengths, weaknesses, and progress made.9 laboratories scoring three stars and above are eligible to apply to the kenya accreditation service, the sole national accreditation body mandated to offer accreditation services for laboratories in kenya. kenya has progressed tremendously in using the slmta programme to accredit medical laboratories, however, only 13 laboratories had been accredited by 2016 through the slmta programme.10,11 with more than 3000 medical laboratories in kenya offering basic diagnostic services, a considerable gap remains in implementing qms. the university of maryland, baltimore (umb), with funding from the united states president’s emergency plan for aids relief through the united states centers for disease control and prevention (cdc), has worked with kenya’s national and county governments to enhance laboratory systems to support hiv/tuberculosis services. as part of this scope, umb, through the boresha maabara programme, supported and mentored 27 hospital laboratories from 10 selected counties (nairobi, kiambu, meru, embu, muranga, nyeri, laikipia, nyandarua, tharaka and kirinyanga) in implementing continuous quality improvement initiatives, and laboratory qms mentorship towards accreditation. the hospitals laboratories included all cadres, such as county, districts and low-level health centres. the objective of this paper is to describe and evaluate an intensified laboratory qms mentorship strategy, which was introduced by umb to move public health laboratories in kenya to accreditation. methods ethical considerations the implementation protocol was approved by the university of maryland, baltimore institutional review board (hp-00094192) and amref ethics and scientific review committee (p 815/2020). this project was also reviewed in accordance with the cdc human research protection procedures. the institutional review boards gave this protocol a “not human research” determination. only aggregated non-human and non-identifiable data were used for this analysis. progression of mentorship strategy at the boresha maabara programme’s inception in april 2016, 27 facilities were enrolled in the slmta programme. baseline audits using the slipta checklists were conducted in april 2016. key healthcare workers, including laboratory managers and quality assurance officers, were enrolled in three consecutive qms training workshops. identified gaps were addressed through improvement projects, and their progress was evaluated during midterm audits. the standard slmta mentorship approach, as described by makokha et al.,12 was used to support the facilities towards achieving accreditation, including embedding qms mentors and supervisory mentorships in the laboratories for 360 days. using this approach, 15 mentors were assigned to at least two slmta facilities. in addition to laboratory qms mentorship, mentors were involved in supporting continuous quality improvement initiatives for hiv and tuberculosis testing-related services in other facilities. this broad scope of work stretched their workload and time (figure 1). figure 1: comparison between the standard and intensified mentorship structures in university of maryland, baltimore from april 2016 to march 2019, kenya. the boresha maabara programme implemented an intensified mentorship approach, starting october 2017, to accelerate the accreditation process. in this approach, seven mentors were identified and dedicated purely to mentoring the 27 laboratories towards accreditation (figure 1). the approach was overseen by a intensively trained qms coordinator who had technical expertise and laboratory accreditation experience. the qms coordinator led planning, supervising, and training facility teams on qms initiatives. mentorship involved conducting initial audits, reviewing non-conformities for each laboratory, and planning meetings for target setting and mentor pairing to support facilities based on non-conformities. mentor competencies and strengths were matched with the laboratory needs, based on the 12 quality system essentials, and mentorship addressed the 12 quality system essentials for each laboratory to resolve all non-conformities. mentorship was complemented with international organization for standardization 15189:2012, method verification, root cause analysis/corrective action and preventive action, and safety trainings based on the laboratory’s needs. monthly virtual meetings were held to check on the progress of all 27 laboratories, which involved re-strategising and re-identifying the skillsets required in different facilities, including re-assigning mentors to different laboratories, if needed. internal audits were conducted by umb mentors, and external audits by the african society for laboratory medicine and kenya accreditation service. the cdc engaged african society for laboratory medicine to conduct external audits to map slmta laboratories in africa, which allowed additional audits to be conducted before kenya accreditation service assessments for accreditation. data collection and analysis baseline and quarterly audits using the slipta checklist (figure 2) were conducted across the 27 laboratories, starting from april 2016 to march 2019, and scores were assigned based on standard scores allocated to the 12 slipta sections. data were analysed using descriptive statistics and graphical representations of the star ratings. accredited laboratories were allocated the highest slipta score (275) of a five-star rating during the computation. we calculated means, standard deviations, medians, and interquartile ranges for scores. the statistical analysis was performed using stata statistical software release 17 (college station, texas, united states: statacorp llc). figure 2: timeline of the evaluation of 27 laboratories starting april 2016 to march 2019, kenya. results at baseline in april 2016, 23 laboratories scored zero stars, three scored one star, and one was already accredited. after 18 months of implementation of the standard mentorship strategy (april 2016 – september 2017), 14 laboratories scored zero stars, three scored one star, eight scored two stars, one scored three stars and one laboratory was accredited, showing slow improvement. in september 2017, the midterm audit demonstrated the common non-conformities emanated from three slipta sections: management review meetings (section 2), evaluation and audits (section 6), and identification and control of non-conformities (section 10) (figure 3). these non-conformities were addressed throughout the intensified mentorship through targeted trainings on how to convene and conduct management review meetings, root cause analysis, corrective actions, and internal audits. figure 3: spider chart showing mean scores for 10 university of maryland, baltimore-supported laboratories for stepwise laboratory quality improvement process towards accreditation sections audited in september 2017, kenya. eighteen months after implementing the intensified mentorship strategy (october 2017 – march 2019), eight laboratories were accredited, with five scoring four stars, 10 scoring three stars, three scoring two stars and only one laboratory scoring one star (figure 4). seven laboratories were newly accredited following the intensified mentorship, while the number of laboratories with ≥ 3 stars improved by 91%. figure 4: audit star ratings comparison between the standard and intensified mentorship approach for university of maryland, baltimore-supported laboratories from april 2016 to march 2019, kenya. across the evaluation period, the median slipta checklist scores increased. with the standard mentorship approach, the median increased from 79 (interquartile range: 55–117) in april–june 2016 to 148 (interquartile range: 116–191) in july–september 2017. with the intensified mentorship, the median score increased to 225 (interquartile range: 207–275) by january–march 2019. the average change of audit scores while using the standard approach was 53.9. the average change with the intensified approach was 81.5, showing greater score improvement after implementing the intensified approach (table 1). table 1: median scores against the standard mentorship (april 2016 – sep 2017) and the intensified mentorship approaches (oct 2017 – mar 2019) for university of maryland, baltimore-supported laboratories in kenya. discussion the boresha maabara programme focused on intensified mentorship of facilities towards reaching and maintaining accreditation. this mentorship approach allowed facilities to be mentored by mentors with the skills necessary to close identified gaps. mentors were allowed to focus on their areas of expertise, which led to a rapid improvement in laboratory audit scores and fast-tracked the laboratories towards accreditation. the standard mentorship approach, as recommended by the slmta programme, assumes that knowledgeable and competent mentors capable of implementing all 12 quality system essentials independently are available.13 makokha et al. noted that the standard mentorship approach that has embedded mentors handling all quality system essentials did not yield results in kenya as expected, as the majority of the laboratories stagnated with minimal improvements.12 these findings suggest the need to re-strategise effective strategies towards laboratory accreditation.12 each country may need to modify the standard approach and develop a country-specific strategy. quality management system mentors may have varying skill levels across the different slipta sections; therefore, they are naturally more effective mentoring in those areas of expertise. our model of pairing qms mentors based on their skills brought together a well-rounded team to provide mentorship to facilities and to resolve non-conformities quickly. other challenges of the standard approach have been documented, including unstructured and often unscheduled mentorship visits that lacked practical work plans and had inconsistent mentorship strengths; these circumstances led to an inability to implement some quality essentials as required.13 the intensified strategy adopted by umb, led by an experienced qms coordinator, provided structured mentorship visits by qualified mentors with monthly reviews of progress. constant interactions between the qms coordinator and mentors allowed opportunities to learn mentors individual strengths and weaknesses, thereby enabling suitable mentor pairs for a well-rounded team. assigning mentors to facilities based on their skillsets and gaps identified in these facilities resulted in tasks being completed more quickly. eventually, this model not only resulted in strengthened qms in the facilities, but also in improved mentors’ technical capacities as mentors learned from each other. monthly meetings between mentors and the qms coordinator through virtual systems provided a forum for sharing progress and discussing laboratory-related challenges and allowed for follow-up of action items. university of maryland, baltimore’s strategy also emphasised dedicating mentors solely to qms, which allowed them to focus their time and effort towards mentoring assigned facilities towards accreditation. while this approach may appear resource-intensive, it allowed for shorter periods of intense interaction with facilities than the standard mentorship approach, as it enabled non-conformities to be efficiently resolved; the model is actually more beneficial for resource-limited countries.14 in our model, accrediting all facilities was in the best interest of all mentors; these mentors functioned together as a network and depended on each other rather than competing against each other. some challenges experienced in the field included inadequate laboratory staffing in the supported facilities; additional mentor-time spent on training lab personnel on qms, as it is not part of the medical laboratory science training curriculum; lack of sufficient budget allocations for qms activities; and use of substandard equipment failing the verification process. these challenges could be overcome by top management embracing qms, building it as part of the medical laboratory science curriculum in the training institutions, and allocating portions of the budget specifically to qms. these factors could also aid in maintaining the achieved gains over time. limitations the paper is restricted to umb experience, staff, and mentorship structure in 10 kenyan counties (nairobi, kiambu, meru, embu, muranga, nyeri, laikipia, nyandarua, tharaka, and kirinyanga) and is not necessarily representative of the whole country. the audits were also conducted by different people and are subject to inter-personnel variations. conclusion the intensified mentorship strategy, led by an experienced qms coordinator working with qms-dedicated mentor teams, resulted in accelerated progress towards accreditation. this strategy could be adopted to accelerate the process of accreditation in similar countries with proper planning and supervision to ensure success. including top-level management, clinicians, and all laboratory staff in the qms process is also essential for enhancing sustainability. additionally, incorporating qms as part of the medical laboratory science curriculum in the training institutions and allocating portions of the budget specifically for qms could help maintain the achieved gains over time. acknowledgements we wish to acknowledge the support from the university of maryland under which this manuscript was written. we thank all the mentors including andrew mboche, sebastian muoki, kola mbangula, vincent mutava, james njogu, beryl mukhwana and lawrence gitonga who worked tirelessly to ensure that the qms was implemented in the laboratories. we also appreciate the contribution of our program managers alan logendo and patricia rarriw. our gratitude goes to both national and county governments that supported this process. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions s.k.m. was the quality management system coordinator and wrote the article; c.m. was the project director and reviewed the article; e. kirui performed the statistical analysis; j.m. was the cdc activity manager for the grant and reviewed the article; t.l. reviewed the article; n.b. reviewed the article and monitored key timelines; a.a. was a principal investigator to the grant and reviewed the article; and e. koech was the country director overseeing implementation of the grant and critically reviewed the article. sources of support this article has been supported in part by the united states president’s emergency plan for aids relief through the united states centers for disease control and prevention, under the terms of cdc-rfa-gh15-1544. data availability data not available due to ethical/ownership restrictions. disclaimer the findings and conclusions in this article are those of the authors and do not necessarily represent the official position of the funding agencies. references guevara g, gordon f, irving y, et al. the impact of slmta in improving laboratory quality systems in the caribbean region. afr j lab med. 2014;3(2):199. https://doi.org/10.4102/ajlm.v3i2.199 hollensead sc, lockwood wb, elin rj. errors in pathology and laboratory medicine: consequences and prevention. j surg oncol. 2004;88(3):161–181. https://doi.org/10.1002/jso.20125 international organization for standardization (iso): iso 15189:2012 medical laboratories – requirements for quality and competence. 2012. geneva, switzerland. zima t. accreditation of medical laboratories – system, process, benefits for labs. j med biochem. 2017;36(3):231–237. https://doi.org/10.1515/jomb-2017-0025 world health organization. laboratory quality management system: handbook, version 1.1. france: world health organization; 2011. guindo ma, shott jp, saye r, et al. promoting good clinical laboratory practices and laboratory accreditation to support clinical trials in sub-saharan africa. am j trop med hyg. 2012;86(4):573–579. https://doi.org/10.4269/ajtmh.2012.11-0691 elbireer am, opio aa, brough rl, jackson jb, manabe yc. strengthening public laboratory service in sub-saharan africa: uganda case study. lab med. 2011;42(12):719–725. https://doi.org/10.1309/lm2obnyy9d0uxzjo yao k, luman e, authors s, moyo s. evidence from 617 laboratories in 47 countries for slmta-driven improvement in quality management systems. afr j lab med. 2014;3(2):362. https://doi.org/10.4102/ajlm.v3i2.262 yao k, maruta t, luman et, nkengasong jn. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. 2014;3(3):194. https://doi.org/10.4102/ajlm.v3i2.194 slmta. slmta laboratories that have achieved accreditation. 2021. https://slmta.org/accredited-labs/ african society for laboratory medicine (aslm). internationally accredited laboratories in africa. 2017. https://aslm.org/resource/gmap/ makokha ep, mwalili s, basiye fl, et al. using standard and institutional mentorship models to implement slmta in kenya. 2014. afr j lab med. 2014;3(2):220. https://doi.org/10.4102/ajlm.v3i2.220 maruta t, rotz p, peter t. setting up a structured laboratory mentoring programme. afr j lab med. 2013;2(1):77. https://doi.org/10.4102/ajlm.v2i1.77 adane k, girma m. iso 15189 laboratory support … how does iso 15189 laboratory accreditation support the delivery of healthcare in ethiopia? a systematic review. ethiopian j health sci. 2018;29(2):259–264. https://doi.org/10.4314/ejhs.v29i2.13 abstract introduction ethical considerations case presentation management and outcome discussion acknowledgements references about the author(s) emmanuel o. irek department of medical microbiology and parasitology, obafemi awolowo university teaching hospitals complex, ile-ife, osun state, nigeria temitope o. obadare department of medical microbiology and parasitology, obafemi awolowo university teaching hospitals complex, ile-ife, osun state, nigeria patrick a. udonwa department of ophthalmology, obafemi awolowo university teaching hospitals complex, ile-ife, osun state, nigeria olajumoke laoye department of ophthalmology, obafemi awolowo university teaching hospitals complex, ile-ife, osun state, nigeria oyekola v. abiri department of medical microbiology and parasitology, obafemi awolowo university teaching hospitals complex, ile-ife, osun state, nigeria adenike o. adeoye department of ophthalmology, obafemi awolowo university teaching hospitals complex, ile-ife, osun state, nigeria aaron o. aboderin department of medical microbiology and parasitology, obafemi awolowo university teaching hospitals complex, ile-ife, osun state, nigeria citation irek eo, obadare to, udonwa pa, et al. cylindrocarpon lichenicola keratomycosis in nigeria: the challenge of limited access to effective antimicrobials. afr j lab med. 2017;6(1), a612. https://doi.org/10.4102/ajlm.v6i1.612 case studies cylindrocarpon lichenicola keratomycosis in nigeria: the challenge of limited access to effective antimicrobials emmanuel o. irek, temitope o. obadare, patrick a. udonwa, olajumoke laoye, oyekola v. abiri, adenike o. adeoye, aaron o. aboderin received: 06 feb. 2017; accepted: 18 apr. 2017; published: 11 july 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract introduction: we report a rare cause of keratitis, due to cylindrocarpon lichenicola, in a farmer with keratomycosis. despite the acknowledged virulence of this fungus, a suitable antifungal for its management was not accessible. case presentation: a 67-year-old farmer presented with a two-week history of pain, mucopurulent discharge, redness and a corneal ulcer with a visual acuity of hand movement in the right eye. with a working diagnosis of infective keratitis, corneal scrapings were taken under a slit lamp biomicroscope for microbiological testing. direct lactophenol cotton blue mounts revealed septate fungal hyphae, while fungal culture on sabouraud dextrose agar at room temperature grew woolly mould phenotypically consistent with c. lichenicola. management and outcome: the patient was started on hourly topical natamycin (5%), ciprofloxacin (0.3%), two-hourly instillation of tobramycin (0.3%) and atropine (1%) twice daily for three months following the isolation of the fungus. the eye healed with a corneal scar and no improvements in visual acuity. discussion: this infection was difficult to manage due to the inaccessibility of a suitable antifungal, namely, voriconazole in our setting. hence, there is a need for prompt identification and early institution of suitable antifungals in any patient with suspected keratomycosis. introduction filamentous fungi are an emerging cause of keratitis worldwide,1 including occurrences of some rarer fungi. only a few cases of keratitis due to cylindrocarpon lichenicola have been reported in published literature in the world, none of these in sub-saharan africa. the occurrence of keratomycosis is, however, partly associated with farming,2 which is a predominant occupation in the tropics. hence, keratomycosis from plant or soil particles laden with fungal materials is not uncommon in these areas.3 c. lichenicola is a hyaline filamentous fungus which is also called fusarium lichenicola and has been reclassified as a member of the fusarium solani species complex.1 however, it rarely causes keratitis,4 although it has been implicated in cutaneous mycosis in immunocompetent patients.5,6 it has devastating effects on the eye following infection, despite use of suitable antifungals.3 in this article, we describe fungal keratitis caused by c. lichenicola in a man living in a semi-urban region in nigeria. ethical considerations informed consent written informed consent was obtained from the patient after adequate explanation was provided and confidentiality was assured. data protection the patient’s identification data were stored on a password protected computer accessible only to the authors and the managing team. case presentation a 67-year-old male farmer presented to the ophthalmology unit of obafemi awolowo university teaching hospitals complex in ile-ife, nigeria, with a two-week history of pain, mucopurulent discharge and redness in the right eye. there was no history of foreign body entry into the right eye, nor was there ocular trauma or instillation of traditional eye medication. the patient had earlier used chloramphenicol eye drops which he obtained over the counter. his fasting blood sugar, complete blood count and electrolyte urea and creatinine were essentially normal for his age. the patient’s hiv status was negative on serology testing. further, ocular examination revealed a visual acuity of hand movement in the right eye, unaided and aided. slit lamp biomicroscopic examination of the right eye showed a diffuse conjunctival hyperemia and a 5.5 x 4 mm corneal ulcer with raised and irregular margins (figure 1) which stained with fluorescein (figure 2). whitish stromal infiltrates were present in the ulcer bed and around the ulcer margins with associated stromal oedema and folds in the descemet’s membrane. anterior chamber examination revealed hypopyon of about one-eighth of the anterior chamber height. pupil was round but sluggishly reactive to light and there was early lens opacity and no glow on fundoscopy in the right eye. ocular findings in the left eye were essentially normal. a presumptive diagnosis of infective keratitis was made. during the slit lamp biomicroscopy, corneal scrapings were taken from the margins and the base of the ulcer and were sent to the microbiology and parasitology laboratory for bacterial and fungal tests. direct lactophenol cotton blue mounts revealed septate fungal hyphae, while direct gram stain showed cellular debris but no microorganisms. culture on chocolate agar yielded scanty growth of cottony white colonies after 48 hours of incubation at 37°c. however, culture on sabouraud dextrose agar at room temperature supported growth of woolly mould with reddish brown pigmentation on the agar after 48 hours (figure 3). lactophenol cotton blue staining of the mould under light microscope (x400 magnification) revealed conidiophores consisting of phialides, arranged in brush-like structures. moreover, the phialides were cylindrical with small collarettes producing hyaline, smooth-walled conidia, which were arranged in masses. the macroconidia were septate, cylindrical with rounded apex and flat base7 (figure 4). no bacteria were seen. the morphology of the mould identified was consistent with c. lichenicola.7 figure 1: right eye examination showing hypopyon, corneal ulcer measuring 5.5 x 4 mm with irregular margins. figure 2: right eye examination under slit lamp with fluorescein dye highlighting the corneal ulcer. figure 3: woolly mould with reddish brown pigmentation after culture of corneal scraping on sabouraud dextrose agar at room temperature for 48 hours. figure 4: cylindrocarpon lichenicola chlamydospores, conidiophores and conidia stained with lactophenol phenol cotton blue (x400 magnification). management and outcome this patient was started on hourly topical natamycin (5%), ciprofloxacin (0.3%), two-hourly instillation of tobramycin (0.3%) and atropine (1%) twice daily. he was also placed on oral fluconazole (200 mg) daily, 250 mg of acetazolamide daily and oral analgesics, all for three months following the isolation of the fungus. despite the duration of use of the medication, the patient’s vision did not improve, as the visual acuity remained hand movement in the right eye (unaided and aided) and healed with a corneal scar. discussion keratomycosis caused by c. lichenicola is rare in humans and challenging to manage. although voriconazole has been used with success in some reports in managing c. lichenicola,8 its accessibility in our setting is limited. generally, there is lack of access to some specific antimicrobial agents needed for possible successful treatment of infections (such as this) in lowand middle-income countries such as nigeria.9 although the circumstance following the occurrence of the fungus in this patient could not be fully ascertained, farming has been associated with keratomycosis.2 prompt diagnosis with early institution of medication for keratomycosis caused by c. lichenicola is therefore needed to avert debilitating effects on the eye, such as were seen in this patient, or visual loss as reported in literature.3 moreover, appropriate access to effective antimicrobials, in this circumstance voriconazole, in lowand middle-income countries is essential while restricting inappropriate use (which drives antimicrobial resistance). although keratomycosis caused by c. lichenicola has been reported in different parts of the world,4,8,10 this appears to be the first report of such in published literature from sub-saharan africa. acknowledgements we wish to thank our families for their encouragement during this research. we also appreciate the support provided by all resident doctors of the medical microbiology and ophthalmology units of obafemi awolowo university teaching hospitals complex during this case report. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support none authors’ contributions e.o.i. was the research coordinator. t.o.o. and o.v.a. performed most of the microbiological and fungal studies. p.a.u. and o.l. conducted the ophthalmology review of the patient. a.o.adeoye and a.o.aboderin undertook the revision of the manuscript. references summerbell rc, schroers hj. analysis of phylogenetic relationship of cylindrocarpon lichenicola and acremonium falciforme to the fusarium solani species complex and a review of similarities in the spectrum of opportunistic infections caused by these fungi. j clin microbiol. 2002;40(8):2866–2875. thomas pa, kaliamurthy j. mycotic keratitis: epidemiology, diagnosis and management. clin microbiol infect. 2013;19(3):210–220. https://doi.org/10.1111/1469-0691.12126 mangiaterra m, giusiano g, smilasky g, et al. keratomycosis caused by cylindrocarpon lichenicola. med mycol. 2001;39(1):143–145. kaliamurthy j, jesudasan ca, prasanth da, et al. keratitis due to cylindrocarpon lichenicola. j postgrad med. 2006;52(2):155–157. champa h, sreeshma p, yegneswaran prakash p, et al. cutaneous infection with cylindrocarpon lichenicola. med mycol case rep. 2013;2(1):55–58. https://doi.org/10.1016/j.mmcr.2013.01.006 diongue k, diallo ma, seck mc, et al. tinea pedis due to cylindrocarpon lichenicola beginning onycholysis. med mycol case rep. 2016;11:13–15. https://doi.org/10.1016/j.mmcr.2016.02.002 ellis d, davis s, alexiou h, et al. descriptions of medical fungi. 2nd ed. adelaide, south australia; 2007. mitra a, savant v, aralikatti a, et al. the use of voriconazole in the treatment of cylindrocarpon keratomycosis. cornea. 2009;28(2):217–218. https://doi.org/10.1097/ico.0b013e3181870315 laxminarayan r, matsoso p, pant s, et al. access to effective antimicrobials: a worldwide challenge. lancet. 2016;387(10014):168–175. https://doi.org/10.1016/s0140-6736(15)00474-2 goel insan n, mane v, chaudhary bl, et al. a review of fungal keratitis: etiology and laboratory diagnosis. int j curr microbiol app sci. 2013;2(6): 307–314. article information authors: madisa mine1,2 sikhulile moyo2 penny stevens3 kurt michael3 vladimir novitsky2,6 kgomotso makhaola1,2 aida asmelash2 s’khatele molefhabangwe2 elias woldegabriel2 gaseboloke mothowaeng2 talkmore maruta2 charity kamhukamwe1 phibeon m. mangwendeza2 molly holmes-pretorius6 isaac mtoni1 modisa motswaledi4 rosemary musonda2 ndwapi ndwapi4 joseph makhema2 richard marlink2,6 khumo seipone5 tendani gaolathe2 max essex2,6 affiliations: 1national health laboratory, gaborone, botswana2botswana-harvard aids institute partnership (bhp), princess marina hospital, gaborone, botswana 3safety monitoring in international laboratories (smile), johns hopkins university, baltimore, maryland, united states 4department of clinical services, ministry of health, gaborone, botswana 5department of hiv/aids prevention and care, ministry of health, gaborone, botswana 6harvard school of public health, boston, massachusetts, united states correspondence to: madisa mine postal address: 5353 church road, extension 10, gaborone, botswana dates: received: 15 june 2011 accepted: 11 nov. 2011 published: 15 dec. 2011 how to cite this article: how to cite this article: mine m, moyo s, stevens p, et al. immunohaematological reference values for hiv-negative healthy adults in botswana. afr j lab med. 2011;1(1), art. #5, 7 pages. http://dx.doi.org/10.4102/ ajlm.v1i1.5 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. immunohaematological reference values for hiv-negative healthy adults in botswana in this original research... open access • abstract • introduction • materials and methods    • study design, setting and population    • study procedures • laboratory procedures    • blood collection and hiv serology    • flow cytometric analysis    • haematological analysis • ethical considerations    • statistical analysis • results • discussion    • limitations of the study • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: clinical laboratories in botswana have relied entirely on the reference intervals for normal immunohaematological values provided by manufacturers’ kits and textbooks.objectives: the aim of this study was to determine the means, medians, 2.5th and 97.5th percentile reference intervals, for normal immunohaematological values in healthy adults in botswana. method: a total of 261 healthy participants comprising 126 men (48%) and 135 (52%) women were enrolled in the southern part of botswana, and immunological and haematological laboratory parameters were measured. results: the mean age was 28.8 (95% confidence interval [ci] 27.7–29.8) years, with a median of 27 years and a range 18–66 years. the mean haemoglobin level was significantly lower for women (12.4 g/dl; 95% ci 12.1% – 12.7%) than men (15.1 g/dl; 95% ci 14.9% – 15.3%). the women’s haemoglobin reference values (9.0 g/dl – 15.0 g/dl) levels were lower than observed in predominantly white populations (12.0 g/dl – 16.0 g/dl), but comparable with regional consensus reference intervals (9.5 g/dl – 15.8 g/dl) recently defined for east and southern africa. conclusion: the established values provide an important tool for patient management and could influence decisions on inclusion of participants and adverse events in clinical trials conducted locally. introduction top ↑ the human immunodeficiency virus (hiv) constitutes a major public health problem in botswana with a prevalence of 17.6% 1 in the general population and 31.8% amongst pregnant woman.2,3 botswana is one of the countries in africa that has led a very high response to the epidemic through a range of multilevel interventions, including the widespread access to antiretrovirals (arvs),4,5 and has built capacity to conduct international prevention, treatment and vaccine trials.6 a valid scientific safety evaluation of arvs and hiv vaccines relies on the availability of locally defined reference values for parameters of clinical interest.7reference intervals are essential for monitoring patho-physiological changes after infection or disease states, or following the administration of drugs in therapeutic or clinical interventions and vaccine studies. several multicenter trials, including phase i and/or phase ii vaccine studies and global use of arvs, have increased the need for regional and locally established reference values.8 reference values vary considerably in different populations, geographical regions, climate, race and dietary habits.9,10,11,12,13,14,15 reference intervals studies conducted in african countries have shown differences within populations and sometimes within sub-groups, and marked differences with values from white populations.8,13,16,17,18 these studies have also observed significant differences with values from developed countries, in haematologic parameters such as haemoglobin and absolute neutrophil, which are normally used for toxicity grading in clinical trials, and consequently have implications in patient management and the conduct of clinical trials.18,19,20,21 a significant number of potential african volunteers are excluded from participating in vaccine trials because screening, enrollment and follow-up monitoring is mostly based on values derived from predominantly white populations. a study in uganda19 documented an exclusion rate of as high as 69% of vaccine clinical trial participants because of haematologic abnormalities, 83% of whom could have been included in the studies if local reference ranges were used. kibiya et al.8 observed that the lower limit of neutrophil counts and haemoglobin reference ranges qualify to be graded as moderate and severe adverse events, respectively, according to the division of aids (daids) toxicity grading table.22 determination of the cd4+ t-lymphocyte counts play a pivotal role on the initiation and monitoring of patients on highly active antiretroviral therapy (haart); hence the urgent need for establishing cd4+ lymphocytes reference intervals for local populations. some studies have observed that black people and afro–caribbean people have lower total white cell counts, and neutrophil and platelet counts, than white people.9,23,24 immunohaematological reference values for a healthy botswanan population have never been established formally, whereas in many african countries, it is common practice to use reference values established in predominantly white populations, which may not be representative of the botswanan population (which comprises mostly black people). in order to conduct investigation drug (ind) and vaccine trials successfully, an assessment of the local reference intervals is required.25,26,27 the aim of this study was to establish the immunohaematological reference values in healthy adults in botswana following the clinical laboratory standards institute (clsi) (formerly nccls) guidelines28 in order to improve patient care, the participation of individuals in clinical trials and the evaluation of adverse events. materials and methods top ↑ study design, setting and population a cross sectional study was conducted amongst healthy adult volunteers that were recruited at the voluntary counselling and testing center (vct) known as tebelopele (a setswana name that means ‘foresight’) from around gaborone, the capital city of botswana. potential volunteers who tested hiv-negative in a parallel testing algorithm that used determine hiv-1/2 (abbott laboratories, il, usa) and unigold hiv (trinity biotech, ireland) rapid test kits, were recruited into the study. the rapid hiv results were confirmed in the laboratory by using two enzyme-linked immunosorbent assays as per national guidelines. study procedures potential apparently healthy hiv-negative volunteers who consented to participate in the study were included. a brief screening questionnaire was administered by experienced study nurses. the questionnaire was designed using the clinical laboratory standards institute (clsi) guidelines28 (formerly nccls) and the following categories were excluded: • pregnant • breastfeeding • patients who had been inpatients in a hospital or who had been subjectively ill during the last month • patients receiving medical treatment • patients who had a recent or recurrent infection, including hiv and malaria • patients who had smoked in the hour before blood was drawn • patients who had donated blood in the preceding month. participants had access to their hiv-testing results through voluntary counselling and testing protocol, and obtained all other results through a registered physician for review and referral to appropriate care. participants with underlying clinical conditions were excluded from the analysis as well. laboratory procedures top ↑ blood collection and hiv serology approximately 15 ml whole blood was collected from the cubital vein with a vacutainer system in k3edta. samples were analysed on the day of collection. hiv status was further confirmed in plasma from the samples by using an enzyme-linked immunosorbent assays vironostika hiv uniform ii plus o (biomérieux france, marcy l’etoile, france) and murex hiv-1.2.o murex (biotech, dartford, uk) according to manufacturer’s instructions. a parallel algorithm was applied and the tests were performed at the botswana harvard hiv reference laboratory, following national guidelines that included confirmation with western blot or dna pcr for all discordant results. flow cytometric analysis t-lymphocyte subsets were analysed on a facscalibur flow cytometer (becton dickinson immunocytometry systems, san jose, california) by using a four-colour immunofluorescence reagent (bd multitest cd3 fluorescein isothiocyanate [fitc]/cd8, phycoerythrin [pe]/cd45, peridinin chlorophyll protein [percp]/cd4, allophycocyanin [apc]), and trucount tubes according to the manufacturer’s instructions. the list-mode data was acquired and analysed with multiset software (becton dickinson immunocytometry systems). the instrument used was validated prior to use and it was monitored daily with trucount controls materials; it was also enrolled in an external quality assurance scheme by the united kingdom external quality assessment scheme (ukneqas). analysis was conducted by trained and competent personnel. haematological analysis haematology parameters were determined from whole blood by using the sysmex xe-2100 (sysmex, kobe, japan), according to the manufacturer’s instructions. the xe-2100 is capable of measuring 32 parameters, including the white blood cell (wbc) 5-part differential into lymphocytes, monocytes, eosinophils, neutrophils and basophils. it also provides a 14-parameter haemogram, as well as an integral reticulocyte analysis that includes an immature reticulocyte fraction, a nucleated red blood cell (nrbc) count and calculated parameters. these calculated parameters are, the mean corpuscular volume (mcv) (fl), the corpuscular haemoglobin (mch) (pg), the mean corpuscular haemoglobin concentration (mchc) (g/dl), and the rbc distribution width by standard deviation (rdw-sd) (fl). the accuracy and precision of the instrument was monitored daily by using commercial quality control materials, as well as quarterly through enrollment in an external quality assurance scheme by the college of american pathologists (cap). trained and competent personnel performed the analysis. ethical considerations top ↑ the study was approved by the health research and development committee (hrdc) of the ministry of health in botswana. written informed consent was obtained from participants prior to their enrollment. those people found to be hiv-positive were excluded from the study, and referred for hiv care and treatment clinics for further management. cd4 and haematology results that were obtained, were offered free of charge as per the botswanan guidelines. statistical analysis reference intervals were calculated according to the clsi guidelines document c28-a2 28 by applying non-parametric methods. the medians were calculated and reference values were determined as the 2.5th and 97.5th percentiles, respectively, of the distribution of reference values. the mean, median, and standard deviation values were calculated for each immunohaematological parameter. we used confidence ratio, which is the ratio of (average confidence interval width) to (reference interval), as described by rhoads,29 to evaluate the impact of the sample size. clsi recommends a minimum of 120 healthy patients per group. the non-parametric mann-whitney u test was used to determine any statistically differences between laboratory values for men and women. p-values < 0.05 were considered significant. all statistical analyses were carried out with ep evaluator release 8 (david g. rhoads and associates, kennett square, pennsylvania, usa) and stata 11.0 (satacorp, college park, texas, usa). results top ↑ screening and enrollment started in may 2008 and ended in june 2008. a total of 294 individuals were screened: 145 men (49%) and 149 women (51%). a total of 33 (11.2%) of screened volunteers were excluded, following enrollment based on defined exclusion criteria. the remaining 261 participants’ samples were included in the study, and comprised 135 women (51%) and 126 men (49%). the mean age was 28.8 (95% ci, 27.7–29.8) years, with a median of 27 and a range 18–66 years.in our results (table 1 and table 2) we show the means, medians and 95th percentile reference values, according to gender, for cd4+ and cd8+ t-lymphocytes absolute values and percentages. the median cd4 cell count in the women (924 cells/mm3) was significant higher than in the men (744 cells/mm3), with p < 0.05. the calculated combined reference intervals for cd4+ and cd8+ t-lymphocytes were 261 cells/mm3 – 1667 cells/mm3 and 261 cells/mm3 – 1538 cells/mm3 for men, and 268 cells/mm3 – 1667 cells/mm3 for women (table 1). the reference intervals for cd8+ t-lymphocytes (table 2) were comparable to those obtained from a blood donor population. the study furthermore shows that the cd4% and cd8% reference intervals for women were 27–63, for men 27–60, and for both sexes 27–63 (table 3). the cd8% reference intervals were 12–46 for women, 11–45 for men, and 11–46 for both sexes. the absolute cd4 and cd8 values for botswana were lower than for most african countries (table 4). table 1: cd4+ cell counts for healthy hiv-negative adults in botswana. table 2: cd8+ cells counts for healthy hiv-negative adults in botswana. table 3: cd4% and cd8% cells counts for healthy hiv-negative adults in botswana. table 4: comparison of ratio of cd4+ to cd8+ and absolute cd4+, cd8+ and lymphocyte counts between different populations. our research shows the reference intervals for the haematological parameters (mean; mean s.d; and 95% ci for mean, median, range 2.5th to 97.5th percentile) partitioned by gender (table 5 and table 6). the red blood cell (rbc) parameters (median haemoglobin, haematocrit, and rbc) and wbc were statistically different according to gender (p < 0.05). there were; however, no gender-specific differences observed for some white blood cell (wbc) subsets (neutrophils, eosinophils, lymphocytes, reticulocytes and basophils), except for monocytes (p = 0.0089). our research also shows haematological reference intervals from this study and those from certain african countries14,16,18,19 and non-african populations (table 7).30 the haemoglobin reference intervals were generally higher than in east africa, but lower than those from ethiopia and us-based comparison populations. table 5: means, medians and 95th percentile reference intervals for haematological parameters for 261 hiv-negative adults in botswana. table 6: means, medians and 95th percentile reference intervals of white blood cell subset percentages for 261 hiv-negative adults in botswana. table 7: comparison of haematological parameters between different populations. discussion top ↑ the reference intervals for immunohaematological and clinical chemistry parameters, which may serve as botswanan standards for the interpretation of laboratory results, were established from 260 hiv-negative participants (134 women [52%] and 126 men [48%]) aged 18–66 years, from around gaborone. as expected, the percentage cd4+ and cd8+ t-lymphocytes varied less between women and men. malone et al.31 reported large fluctuations in repeated cd4+ cell counts in hiv-positive patients and can be explained, in part, by cd4+ cell count diurnal cycle, and also by high variability in total lymphocyte counts. hiv-negative women have a higher average cd4+ t-lymphocytes count than men, which confirms the findings of bussmann et al.32 the cd4 cell counts are lower in botswana than those observed in east africa and usa. this has obvious implications on the clinical staging of aids and the assessment for disease progression.the significant gender difference in red blood cell count, haemoglobin and haematocrit, agrees with other studies16 and with the well-established fact that men have higher values for the red blood cell parameters than women, partly because of the influence of the hormone androgen on erythropoiesis, and partly because of menstrual loss. the red blood cell parameters for the botswanan population were within the intervals for the ethiopian population, even though the expectation was that altitude induces erythropoiesis, which has accounted for the red blood cell parameters of ethiopia that were consistently higher than those of many other african countries.16 the median haemoglobin level was significantly lower for women than for men, which is consistent with previous findings.8,9,21,27 the female haemoglobin reference value (9.0 g/dl to 15.0 g/dl) levels were lower than observed in predominantly white populations (12.0 g/dl to 16.0 g/dl), but comparable with east african and southern african regional consensus reference intervals (9.5 g/dl to 15.8 g/dl) recently defined for east and southern africa. the division of aids (daids) has haematological criteria for grading the severity of potential vaccine-related adverse events.22 white blood cell and platelet counts, and haemoglobin levels are used as inclusion and/or exclusion criteria in many clinical trials. the lower limit of botswana’s normal female reference range for haemoglobin qualifies as a moderate adverse event and a significant proportion of the woman may not qualify to be enrolled for phase i and/or phase ii vaccine trials. the methodologies in this study are commonly used, and should serve well as guidelines for the represented population. laboratories that wish to adopt these reference intervals should explore any differences in methodology, and should verify the transference and appropriateness of the reference intervals to their laboratory. limitations of the study although this study meets the minimum clsi requirements for establishing valid reference intervals, it is be important to explore further geographic and ethic differences that may exist within the botswanan population. one limitation of our study is that, despite our efforts to include only healthy subjects, the use of a questionnaire, as well as hiv screening and a review by a physician, it was not feasible to screen for all medical conditions that might have influenced the laboratory results. for instance, participants were not examined for other infections such as respiratory infections, recent transient gastroenteritis and sexually transmitted infections. all participants were adults drawn from voluntary counselling and testing facilities. when reporting reference values, the actual representativeness of the data is an important concern for the underlying population. as in most studies, we cannot totally exclude the possibility that there is some sampling bias because of use of a self-selecting population. our analysis excluded participants who showed any signs or symptoms of disease, however, and it is thus likely that our data are representative of healthy adults from the southern region of botswana. gaborone is the capital city of botswana and has a diversity of ethnic groups; consequently, reference values from this region are not likely to make a significant clinical difference. because of access to a reference laboratory working under iso and gclp standards, it was not easy to include regions that are further away from the capital city, but this data is still considered to be a very important baseline for similar future studies. the data are also immediately applicable in studies and clinical care that is an improvement over the current practice of using reference values derived from elsewhere. conclusion top ↑ this is the first study in botswana to document haematological reference intervals for healthy adults. our study shows that clinical reference values developed within the region are more appropriate for the botswanan population than those adopted from developed countries. the established values provide an important tool for patient management and could influence decisions on the inclusion of participants, as well as the management of adverse events. it could also improve scientific validity in clinical trials that are conducted locally and regionally. acknowledgements top ↑ the authors would like to thank the participants of this study for their time and patience. the authors also extend their gratitude to the voluntary counselling and testing (vct) management for availing the use of their facilities for the recruitment of the study participants. in addition, the authors would like to thank the ministry of health and botswana harvard partnership for supporting the study. the project was also supported by grant number u01ai069456 from the national institute of allergy and infectious diseases. the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institute of allergy and infectious diseases or the national institutes of health. competing interests the authors declare that they have no financial or personal relationship(s) which may have influenced them inappropriately in writing this article. authors’ contributions m. mine (national health laboratory), v.n. (harvard school of public health), r. marlink (harvard school of public health) and m.e. (harvard school of public health) conceived and designed the study, and m. mine, s. moyo (bhp princess marina hospital), p.s. (johns hopkins university), k. michael (johns hopkins university) and m.h.-p. (harvard school of public health) wrote the article. s. moyo and m. mine analysed the data. clinical support was provided by a.a. (bhp princess marina hospital), n.n. (ministry of health), j.m. (bhp princess marina hospital), k.s. (ministry of health), t.g. (bhp princess marina hospital), e.w. (bhp princess marina hospital) and i.m. (national health laboratory) (medical doctors), and by s. molefhabangwe (deceased) (bhp princess marina hospital), as well as by g.m. (bhp princess marina hospital) (study nurses and recruiters). k. makhaola (national health laboratory), t.m. (princess marina hospital), c.k. (national health laboratory), p.m.m. (bhp princess marina hospital), r. musonda (bhp princess marina hospital) and m. motswaledi (ministry of health) provided laboratory support. references top ↑ 1. central statistics office/national aids coordinating agency (cso/naca). botswana behavioural aids impact survey iii. gaborone, botswana: central statistics office/national aids coordinating agency; 2010.2. ministry of health botswana. 2007 botswana anc second generation hiv/aids sentinel surveillance. technical report. de la hoz gomez f, anderson mg, bodika s, et al., editors. gaborone, botswana: botswana government print; 2009. 3. plosmedicineeditors. a new vision for clinical trials in africa. plos med. 2004;1:e71. http://dx.doi.org/10.1371/journal.pmed.0010071, pmid:15630472, pmcid:539056 4. ojikutu b, makadzange at, gaolathe t. scaling up art treatment capacity: lessons learned from south africa, zimbabwe, and botswana. curr infect dis rep. 2008;10:69–73. http://dx.doi.org/10.1007/s11908-008-0012-0, pmid:18377818 5. 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local reference ranges for full blood count and cd4 lymphocyte count testing. s afr med j. 2009;99:243–248. pmid:19588777 15. otahbachi m, simoni j, simoni g, et al. gender differences in platelet aggregation in healthy individuals. j thromb thrombolysis. 2010;30:184–191. http://dx.doi.org/10.1007/s11239-009-0436-x, pmid:20039102 16. tsegaye a, wolday d, otto s, et al. immunophenotyping of blood lymphocytes at birth, during childhood, and during adulthood in hiv-1-uninfected ethiopians. clin immunol. 2003;109:338–346. http://dx.doi.org/10.1016/j.clim.2003.08.008, pmid:14697749 17. menard d, mandeng mj, tothy mb, kelembho ek, gresenguet g, talarmin a. immunohematological reference ranges for adults from the central african republic. clin diagn lab immunol. 2003;10:443–445. pmid:12738646, pmcid:154963 18. karita e, ketter n, price ma, et al. clsi-derived hematology and biochemistry reference intervals for healthy adults in eastern and southern africa. plos one. 2009;4:e4401. http://dx.doi.org/10.1371/journal.pone.0004401, pmid:19197365, pmcid:2632744 19. eller la, eller ma, ouma b, et al. reference intervals in healthy adult ugandan blood donors and their impact on conducting international vaccine trials. plos one. 2008;3:e3919. http://dx.doi.org/10.1371/journal.pone.0003919, pmid:19079547, pmcid:2593783 20. wells j, shetty ak, stranix l, et al. range of normal neutrophil counts in healthy zimbabwean infants: implications for monitoring antiretroviral drug toxicity. j acquir immune defic syndr. 2006;42:460–463. http://dx.doi.org/10.1097/01.qai.0000224975.45091.a5, pmid:16810112 21. lubega ir, fowler mg, musoke pm, et al. considerations in using us-based laboratory toxicity tables to evaluate laboratory toxicities among healthy malawian and ugandan infants. j acquir immune defic syndr. 2010;55:58–64. http://dx.doi.org/10.1097/qai.0b013e3181db059d, pmid:20588184, pmcid:3033212 22. division of aids (daids). the division of aids table for grading the severity of adult and pediatric adverse events (daids ae grading table). usa: national institutes of health; 2004. 23. bussmann h, wester cw, masupu kv, et al. low cd4+ t-lymphocyte values in human immunodeficiency virus-negative adults in botswana. clin diagn lab immunol. 2004;11:930–935. pmid:15358655, pmcid:515279 24. lawrie d, coetzee lm, becker p, mahlangu j, stevens w, glencross dk. local reference ranges for full blood count and cd4 lymphocyte count testing. s afr med j. 2010;99. pmid:20459912 25. mills e, cooper c, guyatt g, et al. barriers to participating in an hiv vaccine trial: a systematic review. aids. 2004;18:2235. http://dx.doi.org/10.1097/00002030-200411190-00003, pmid:15577535 26. gilmour jw, stevens ws, gray c, de souza m. laboratory expansion to large-scale international hiv preventive vaccine trials. curr opin hiv aids. 2007;2:201. http://dx.doi.org/10.1097/coh.0b013e3280eec77a, pmid:19372887 27. stevens w, kamali a, karita e, et al. baseline morbidity in 2,990 adult african volunteers recruited to characterize laboratory reference intervals for future hiv vaccine clinical trials. plos one. 2008;3:e2043. http://dx.doi.org/10.1371/journal.pone.0002043, pmid:18446196, pmcid:2312327 28. national committee for clinical laboratory standards (nccls). how to define and determine reference intervals in the clinical laboratory: approved guideline. 2nd ed. wayne, pa: national committee for clinical laboratory standards; 2000. 29. rhoads dg. understading reference intervals. in: rhoads dg, editor. ep evaluator release 8 for evaluating clinical laboratory methods. kennett square, pennsylavania: david g. rhoads and associates, inc.; 2007. 30. cabot rc, harris nl, shepard j-ao, et al. normal reference laboratory values. n engl j med. 2004;351:1548–1563. http://dx.doi.org/10.1056/nejmcpc049016, pmid:15470219 31. malone jl, simms te, gray gc, wagner kf, burge jr, burke ds. sources of variability in repeated t-helper lymphocyte counts from human immunodeficiency virus type 1-infected patients: total lymphocyte count fluctuations and diurnal cycle are important. j acquir immune defic syndr. 1990;3:144–151. pmcid:1967309 32. bussmann h, wester cw, masupu kv, et al. low cd4+ t-lymphocyte values in human immunodeficiency virus-negative adults in botswana. clin diagn lab immunol. 2004;11:930–935. pmid:15358655, pmcid:515279 abstract introduction methods results discussion acknowledgements references about the author(s) hammad ali division of global hiv and tuberculosis, centers for disease control and prevention, atlanta, georgia, united states peter minchella division of global hiv and tuberculosis, centers for disease control and prevention, atlanta, georgia, united states geoffrey chipungu division of global hiv and tuberculosis, centers for disease control and prevention, lilongwe, malawi evelyn kim division of global hiv and tuberculosis, centers for disease control and prevention, lilongwe, malawi james kandulu ministry of health, lilongwe, malawi dalitso midiani ministry of health, lilongwe, malawi andrea kim division of global hiv and tuberculosis, centers for disease control and prevention, atlanta, georgia, united states mahesh swaminathan division of global hiv and tuberculosis, centers for disease control and prevention, atlanta, georgia, united states steve gutreuter division of global hiv and tuberculosis, centers for disease control and prevention, atlanta, georgia, united states john nkengasong division of global hiv and tuberculosis, centers for disease control and prevention, atlanta, georgia, united states daniel singer division of global hiv and tuberculosis, centers for disease control and prevention, lilongwe, malawi citation ali h, minchella p, chipungu g, et al. infant hiv diagnosis and turn-around time for testing in malawi, 2015. afr j lab med. 2020;9(1), a904. https://doi.org/10.4102/ajlm.v9i1.904 original research infant hiv diagnosis and turn-around time for testing in malawi, 2015 hammad ali, peter minchella, geoffrey chipungu, evelyn kim, james kandulu, dalitso midiani, andrea kim, mahesh swaminathan, steve gutreuter, john nkengasong, daniel singer received: 30 aug. 2018; accepted: 27 aug. 2020; published: 26 nov. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: for hiv-exposed infants in malawi, there are missed opportunities at each step of the testing and treatment cascade. objective: this study assessed factors associated with hiv positivity among infants in malawi and turn-around times for infant hiv testing. methods: hiv testing data for infants aged 0–18 months from 2012 to 2015 were extracted from the malawi hiv laboratory information management system and analysed using logistic regression. turn-around time was defined as time between collection of samples to results dispatch from the laboratory. results: a total of 106 997 tests were included in the analyses. a subset of 76 006 observations with complete dates were included in the turn-around time analysis. overall positivity was 4.2%. factors associated with positivity were increasing age (infants aged 3–6 months: adjusted odds ratio [aor] = 2.24; infants aged 6–9 months: aor = 3.42; infants aged > 9 months: aor = 4.24), female sex (aor = 1.08) and whether the mother was alive and not on antiretroviral therapy at time of the infant’s test (aor = 1.57). provision of hiv prophylaxis to the infant after birth (aor = 0.38) was found to be protective against hiv positivity. the median turn-around time was 24 days (increased from 19 to 34 days between 2012 and 2015). conclusion: infant hiv positivity has decreased in malawi, whereas turn-around time has increased. factors associated with positivity include increasing age, female sex, and whether the mother was alive and not on antiretroviral therapy at the time of the infant’s test. keywords: hiv; malawi; infant hiv testing; turn-around time. introduction malawi has a high prevalence of hiv; in 2016, 9.2% of persons aged 15–49 years, 920 000 people (550 000 women, 60%) aged 15 years and older, and 110 000 children (0–14 years of age) were living with hiv.1 among all people living with hiv, 680 000 were on antiretroviral therapy (art) as of 2016.1 currently, there are 717 sites that offer art to hiv-positive patients in malawi and there are nine laboratories nationwide that provide infant hiv virologic testing.2 like many countries in sub-saharan africa, malawi is striving towards achieving the joint united nations programme on hiv/aids 90-90-90 targets. these targets include 90% of people living with hiv diagnosed, 90% of the people living with hiv with known status accessing treatment, and 90% of those who are on treatment virally suppressed.3 in malawi, an estimated (antiretroviral adjusted) 76.8% of 15–64-year-old people living with hiv know their hiv status, 91.4% who know their status self-report current use of art and 91.3% of those who self-report art use were virally suppressed.4 the second 90 requires ‘test and start’, which is defined as provision of art for all people living with hiv irrespective of their cluster of differentiation 4 counts.5 national guidelines recommend that hiv dna should be detected using polymerase chain reaction (pcr) in infants who are exposed to hiv. when resources and facilities are available, this should happen at the first opportunity after the infant is older than 6 weeks.6 blood is collected by facility nurses and dried blood spot specimens are created and sent to one of the nine laboratories nationwide that provide infant hiv virologic testing. if the pcr test is negative, a rapid antibody test is done from age 12 months onwards. if the rapid antibody test done at age 12 months returns negative, another rapid antibody test is done at age 24 months, if breastfeeding stopped at least 6 weeks before. if still breastfeeding, then the test is done six weeks after stopping breastfeeding. in spite of these clear guidelines, hiv diagnosis in hiv-exposed infants has remained a challenge in the country. the malawi population-based hiv impact assessment 2015–2016 showed that around half (49.4%) of infants born to hiv-positive women received a virologic hiv test within 2 months of birth, and 29.3% received a test between 2 and 12 months.4 these malawi population-based hiv impact assessment estimates are based on a self-report of the mother and are higher than the estimates from a 2014 cohort study, which showed that only 39% – 41% of hiv-exposed infants older than age 24 months had documented evidence of an hiv test result, with 3% of those tested being positive.2 about 82% – 89% of those who had a positive result or who were diagnosed with severe hiv disease were initiated on art.2 these data highlight the potential opportunities missed at each step of the testing and treatment cascade for hiv-exposed infants, that is, more infants need to be tested and 10% – 20% of those tested positive were not started on art. malawi started universal treatment (i.e., ‘test and start’) for hiv-positive children younger than age 12 months in 2011.7 in 2014, this was scaled up to cover all children younger than age 5 years,6 and in 2016 malawi implemented ‘test and start’ to cover all people living with hiv nationally.8 one component of ‘test and start’ implementation is scaling up of hiv virologic testing among infants born to hiv-positive mothers, and this requires a rapid expansion of laboratory services in malawi. there is a paucity of adequate paediatric hiv data generally in malawi and specifically on factors associated with infant hiv positivity. in addition to difficulties in testing all infants born to hiv-positive mothers, there have been significant challenges in turn-around times for infant hiv test results, thus affecting early diagnosis in malawi. turn-around time is a metric used to assess the length of time between steps in the laboratory testing process. longer turn-around times lead to delays in getting test results, which lead to delayed diagnoses. any delay in diagnosis invariably leads to delays in the initiation of treatment, leading to poorer health outcomes that can result in higher mortality rates among untreated infants. the objectives of this study were to look at hiv positivity in infants in malawi over time, and assess factors associated with positivity. in addition, we looked at the turn-around time for infant hiv testing to understand the timeliness of diagnoses. methods ethical considerations this study was approved as exempt 45 cfr 46.101(b)(4) by the centers for disease control and prevention institutional review board (#6890) and the national health science research committee, ministry of health, malawi (#1675). no extra data were collected for the purpose of these analyses. the malawi hiv laboratory information management system (lims) database does not include patient names but does include unique patient identification numbers, which were scrambled prior to analysis, so that they may be used to clean or de-duplicate the data set but cannot be linked back to a patient. study design a cross-sectional analysis was conducted on data from the lims. the united states president’s emergency plan for aids relief supported the development of a lims in malawi, which captures basic information on each infant hiv and viral load sample sent for testing. although originally developed to project reagent needs for laboratories conducting virologic testing and thus keep track of laboratory functions, the database offers important information both on the utilisation of laboratory services as well as on the effectiveness of hiv services. data included in the lims are routinely collected as part of regular provision of hiv services. these data include patient demographics, records of sample transportation and laboratory test outcomes. all data are entered into encrypted databases that are centrally stored and are password protected. data analysis data between 2012 and 2015 were extracted from the infant hiv testing database of the malawi lims. records for infants aged 0 to 18 months at the time of specimen collection were included in the analysis. tests were excluded from the analysis if the test result was missing. there were two patient identification numbers available in the system that were used. data were excluded if both patient identification numbers were missing to avoid duplication. based on the patient identification numbers, if there was more than one record found in the database for a patient identification number, the record with the latest test date was kept and the others dropped before analysis. infant hiv positivity was defined as detection of hiv dna using a pcr test. in malawi, specimens with positive or indeterminate results are re-tested for confirmation. table 1 shows the variables from the lims database that were included in the analysis. overall turn-around time was defined as the time between collection of the sample at the healthcare facility to the dispatch of the results from the laboratory back to the facility. data were dropped from the turn-around time analysis if the date values were implausible (e.g., if the date of the test was before the date of the collection or date of birth). mean and median turn-around times were calculated between all major steps of the process. table 1: variables extracted from the laboratory information management system to review infant hiv diagnosis and turn-around time for testing in malawi, 2012–2015. since the malawi lims includes routinely collected data, a number of variables had missing values. observations were missing from the records for infant feeding option (60%), reason for pcr testing (37%), infant sex (26%), art and life status of the mother (alive and on art, alive but not on art, deceased; 12%), multiple-birth indicator (8%), region (1.2%) and age (0.2%). we presumed that observations in all these variables were missing at random and used fully conditional chained equations9 to impute 50 completed data sets for analysis. bivariate and multivariable logistic regression analyses were undertaken on each of the multiply imputed data sets, and combined using rubin’s rules,10 to identify factors associated with infant hiv positivity. the initial multivariable model included all variables approaching significance (p < 0.25) in the bivariate model, plus all plausible two-way interactions provisionally detected by changes in odds ratios between the bivariate analysis and a full main-effects model. we eliminated covariates one at a time – beginning with the interactions – based on p-values from wald tests to identify the simplest plausible multivariable model, retaining only covariates and interactions for which p ≤ 0.05. our inferences were based upon the adjusted odds ratios (aor) from the final multivariable model and their 95% confidence intervals (ci). stata version 15 (statacorp lp, college station, texas, united states) was used to conduct analyses. results the 2012–2015 data set included 125 713 tests, and 106 997 tests (85.1%) from unique infants aged 0–18 months which were included in the analysis of associations with hiv positivity. of those, only 76 006 tests had complete date recordings and those were used for the analysis of turn-around times. a total of 23 954 tests were conducted in 2012, 27 437 in 2013, 29 880 in 2014 and 25 726 in 2015. most infants tested were younger than 3 months old (66.6%); there were almost equal numbers of male (39 628) and female (39 525) infants and most infants were from southern malawi (69.1%; table 2). table 2: population characteristics and factors associated with a positive hiv result in infants in malawi, 2012–2015. hiv positivity the overall infant hiv positivity between 2012 and 2015 was 4.2% (n = 4448), which reduced from 5.7% in 2012 to 3.1% in 2015. positivity was 4.3% in male infants (n = 1701) and 4.7% in female infants (n = 1840) and increased by age (2.3% in infants aged < 3 months old, 5.8% in infants aged 3–6 months old, 9.6% in infants aged 6–9 months old, and 12.4% in infants aged > 9 months old; table 2). positivity was higher if the mother was not on art during pregnancy or labour (22.6%) or if the mother’s art status was unknown (4.4%), compared to women who were on art during pregnancy or labour (2.5%). positivity was highest for infants of deceased mothers (5.6%), compared to those whose mothers were alive and on art (2.3%). positivity was higher if the infant was not given hiv prophylaxis at birth (18.1%) compared to those who were given prophylaxis at birth (2.5%). on multivariable analyses, the odds of testing positive for hiv decreased by 18% per year (aor 0.82, 95% ci: 0.70–0.84). other factors significantly associated with testing positive for hiv were increasing age of the infant (3–6 months: aor 2.24, 95% ci: 2.07–2.42; 6–9 months: aor 3.42, 95% ci: 3.13–3.74; > 9 months: aor 4.24, 95% ci: 3.84–4.67), female sex (aor 1.08, 95% ci 1.01–1.16), whether the mother was alive but not on art at the time of the infant hiv test (aor 1.57, 95% ci: 1.36–1.82), and if pcr was conducted because the infant was presumed to have severe hiv disease (aor 2.37, 95% ci: 1.65–3.39) (table 2). immediate postpartum provision of hiv prophylaxis to infants was protective against testing positive for hiv (aor 0.38, 95% ci: 0.33–0.44). the effects of art during pregnancy or labour varied among regions, as manifested by an interaction between region and art provision during pregnancy or labour. infants born to mothers who received art during pregnancy or labour were equally likely to test positive regardless of region. conversely, the aors for infants born to mothers who did not receive art during either pregnancy or labour varied among regions, and infants born in the central region had the lowest aor (2.80, 95% ci 2.06–3.81) relative to baseline (infants born in the northern region to mothers who received art during pregnancy or labour) (table 3). table 3: interaction between antiretroviral therapy in labour or pregnancy and region as factors associated with positive hiv results in infants in malawi, 2012–2015. turn-around time the overall median turn-around time between hiv-related sample collection at the healthcare facility to the dispatch of results from the laboratory back to the facility was 24 days (interquartile range: 15–36 days), whereas the mean turn-around time was 29.1 days (standard deviation: 22.5 days) (table 4). the median turn-around time was 19 days in 2012 but increased by 79% to 34 days in 2015. median turn-around time was longest in the northern region (28 days, interquartile range: 20–40 days), followed by central (27 days, interquartile range: 17–40 days) and southern region (23 days, interquartile range: 15–35 days). median times increased for all steps from 2012 to 2015: median turn-around time between sample drawn at the health facility and being received at the laboratory increased from 11 days in 2012 to 16 days in 2015; the median turn-around time between the sample being received and the sample testing increased from 4 to 6 days; median turn-around time between sample testing and the dispatch of the result from the laboratory back to the facility increased from 0 to 3 days. table 4: mean and median turn-around time by year for hiv tests in infants in malawi, 2012–2015. discussion this study used data from malawi lims, a routinely collected hiv data source, and showed that the overall hiv positivity in hiv-exposed infants reduced from 5.7% in 2012 to 3.1% in 2015; however, the overall median turn-around time between collection of the sample at the healthcare facility to the dispatch of the results from the laboratory back to the facility increased from 19 days in 2012 to 34 days in 2015. in addition, lims data were analysed to identify factors associated with hiv positivity in infants, including infant age and maternal art uptake. these results highlight the point that routine testing data from lims can be used not only to inform service delivery but to conduct epidemiological analyses to fill gaps in knowledge about hiv infection. the positivity results from this study (3.1% in 2015) are similar to the results reported by the ministry of health (3% in 2014).2 infant hiv positivity reduced between 2012 and 2015 and prevention of mother-to-child-transmission (pmtct) initiatives are likely the main reason for this. specifically, implementation of option b+ (where all pregnant women living with hiv are started on lifelong art irrespective of their cluster of differentiation 4 count)11 – as opposed to option a (in which pregnant women receive art prophylaxis antenatal and intrapartum, along with a postpartum ‘tail’ regimen) and option b (in which all women receive art starting in the antenatal period with continued treatment throughout the breastfeeding duration) – has reduced the mother-to-child-transmission (mtct) of hiv. the malawi population-based hiv impact assessment found that 97.9% of self-reported hiv-positive women aged 15–49 years who gave birth in the last 12 months before the survey reported having received art.4 it is important to note that malawi introduced option b+ in 2011, before it became a recommendation by the world health organization.12 since then, the coverage of pregnant women who received art to prevent mtct increased from 62% in 2012 to 79% in 2015 (and further increased to > 95% in 2018).13 owing to the increase in the number of women who received art to prevent mtct, the number of new hiv infections averted have increased, from 8100 in 2012 to 8600 in 2015 (and further increased to 9600 in 2018), and the number of new infections among children aged 0–14 years has decreased from 9300 in 2012 to 6400 in 2015 (and further decreased to 3500 in 2018).13 in addition to the increase in pmtct, the proportion of infants born to hiv-positive women who received an hiv virological test within the first 2 months after birth increased from 5% in 2012 to 22% in 2015 (and further increased to > 95% in 2018).13 thus, the reduction in positivity rates is probably led by the reducing mtct rates due to option b+. another possible factor contributing to decreased positivity is the increase in testing in healthy infants due to the expansion of early infant diagnosis testing. this study identified a number of factors found to be associated with hiv positivity in infants. increasing age was associated with the risk of testing positive. this could be due to a delay in infant testing, duration of breastfeeding, or lack of access or adherence to care and treatment. most mothers breastfeed in malawi and the longer a mother who is not on art breastfeeds, the greater the risk of transmission of hiv to the infant.14 current malawi guidelines dictate that hiv-exposed infants should be tested between age 6 and 8 weeks. however, this finding shows that at least some of the infants are not getting tested by 6–8 weeks which may be because mothers are not accessing care and treatment for their hiv. this could be due to non-disclosure issues which can pose a risk for sexual transmission of hiv to the partner, and impact the implementation of pmtct.15 also, since the latest test result was selected from the lims for this study, some results may reflect a repeat or confirmatory test and thus could have contributed to the observed late testing. infants born to mothers who received art during pregnancy or labour experienced indistinguishable risks of hiv infection across all regions. however, infants born to mothers who did not receive art during either pregnancy or labour were more likely to have been infected, and their odds ratio varied among regions. infants born in the central region to mothers who did not receive art had a slightly lower aor relative to baseline than their counterparts born in the northern and southern regions; however, the confidence intervals overlapped for all three regions. infants were at higher risk of being tested positive if mothers were alive and not on art at the time of the test compared to those whose mothers were alive and on art. this shows the importance of providing art during pregnancy to women living with hiv14,16,17,18,19 and during labour,18 which is well established as a pmtct measure. human immunodeficiency virus-exposed infants not provided with hiv prophylaxis at the time of birth were at an increased risk of being hiv-positive as opposed to those who were provided with prophylaxis at birth. in addition, hiv-exposed infants with presumed severe hiv disease at the time of the test were at a higher risk of being hiv-positive. various studies have reported that suboptimal use of art and inadequate skilled care during the delivery are factors that increase mtct.14,16 human immunodeficiency virus prophylaxis reduces the concentration of hiv virus in the infants’ blood.20,21 thus, hiv prophylaxis should be provided to all exposed infants to reduce perinatal transmission of hiv and those who test positive should be started on art immediately. lastly, female infants were at a higher risk of testing hiv-positive compared to male infants. there is some evidence that female infants, compared to male infants, have higher susceptibility to hiv infection before birth. one study reported a significant association between sex and hiv infection, with female infants being at a much higher risk for hiv acquisition than male infants at birth, suggesting in utero acquisition.22 currently, infant hiv virologic testing in malawi depends on the availability of equipment, laboratory space and trained technicians.23,24 out of the 28 districts in malawi, only 7 have molecular laboratories that can perform infant hiv virologic testing. therefore, samples need to be transported to these laboratories for testing from the districts with no molecular laboratories. this can increase the turn-around time for testing, resulting in delays in the return of the result back to the patient and start of treatment if the result is hiv-positive. multiple factors can prolong turn-around time, including delays with sample transport due to inclement weather, shortage of fuel and holidays.25 laboratory factors can also delay turn-around time, including staff shortages, work overload, stock-out of reagents, issues with equipment and other administrative delays.26 the increase in turn-around time documented in this study occurred during a period of rapid expansion of option b+ for pmtct. the pmtct programme uses the same mechanisms for transport of samples and art and the same laboratory facilities for hiv viral load testing. the increased turn-around time likely represents stresses on the system across all levels as scale-up occurred. a sample tracking and online result reporting system – so samples can be tracked during delivery and results can arrive at the facility as soon as tested – can help with improving turn-around time and providing results as soon as possible to clinicians. another option could be the use of hiv point-of-care testing which can deliver results at the test site. although point-of-care testing is currently not available on a routine basis in malawi, a point-of-care early infant diagnosis implementation pilot conducted in malawi reduced the turn-around time from collection of sample to art initiation and increased the proportion of patients initiating art.27 the findings in this study are strengthened by the fact that the data set used in this study is the national laboratory data set which includes data from all tests conducted at the national laboratories in the country. this includes tests conducted at the public health facilities and the externally managed facilities and there is a minimal chance of testing happening at a laboratory outside the national laboratories. additionally, the data are available from 2012 onwards which allows for interpretation of the trends over time. the availability of turn-around time data provides a rare opportunity to monitor the delivery of laboratory services on a national scale. findings from this study may be applicable throughout sub-saharan africa, especially in countries that are scaling up virologic testing. limitations like all studies, this study has limitations. firstly, as the data set only included routinely collected variables, only a limited set of variables could be included in the logistic regression. some other important variables that could provide insight into other demographic, behavioural and clinical factors associated with hiv positivity in infants could not be included, as they are not collected routinely. secondly, multiple patient identification numbers were available in the data set. however, none of the identification numbers were completely available for the majority of the patients. therefore, the data had to be de-duplicated using various patient identification numbers, which, in spite of all efforts, may have led to some duplication in the data set. thirdly, a number of variables had missing data. this was due to incomplete data recording and changes in the hiv test requisition form over time. we attempted to address this by doing multiple imputations and using the imputed data to conduct the regression analyses. for the turn-around time analysis, nearly 40% of tests had to be excluded from analysis, if the dates were missing or if the dates were implausible. better data management and quality control is required to resolve issues of missing data, which will improve the measurement of indicators. fourthly, the variable ‘mother’s life and art status’ at the time of the infant’s hiv test may not have been correctly entered. it seems that when the mother’s life and art status were missing, it was recorded as mother not on art. this is important to note, because in the option b+ era in malawi, it is unlikely that so many women were hiv-positive and not on art. lastly, lims is not connected to the clinical systems; thus, we were unable to ascertain outcomes of positive tests, that is, what proportion of infants were put on treatment and how quickly. as lims is a routinely collected data source, it can potentially be used for surveillance purposes. however, as described, currently there are a number of data quality and completeness issues that may limit the ability of lims to be used as a surveillance system. attention to detail is required to improve the quality and completeness of lims data, and routine data cleaning and validation procedures should be implemented before lims can be considered as a surveillance system. in addition, a formal comparison of data generated by lims and those generated by other data systems (e.g., electronic medical records) is required to validate the use of lims as a surveillance system in malawi. conclusion although infant hiv positivity has decreased over time in malawi, the turn-around time has increased over the same period of time. active engagement from all stakeholders is required to reduce the turn-around time to assist with implementation of ‘test and start’ among infants in the country. the earlier hiv is detected in infants, and treatment started by the clinician, the better the outcomes among these infants. therefore, it is imperative to test early and provide the results to clinicians as soon as possible so that appropriate care and treatment are provided to infants. although this study shows that lims can provide important information about infant hiv virologic testing, we do not know what proportion of patients actually received these results and what proportion of results were actually acted upon by clinicians. although lims in malawi was designed to provide information for the laboratories, it can provide other important epidemiological pieces of information. this evidence-based information can guide development and restructuring of policy and programmes in the country. acknowledgements the authors would like to acknowledge all the implementing partners in malawi for their support of this study and the mapping exercise. thanks to ermias amene, statistician at the centers for disease control and prevention, for his assistance. competing interests no conflicts of interest are declared by authors. authors’ contributions h.a., a.k. and d.s. conceptualised the study; h.a. and s.g. conducted the analysis; h.a. wrote the first draft; p.m., g.c. and a.k. provided technical input; all authors provided input on the initial and final drafts. sources of support this analysis was supported by the president’s emergency plan for aids relief through the centers for disease control and prevention. data availability statement data sharing is not applicable to this article as no new data were collected in this study. disclaimer the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the funding agencies. references joint united nations programme on hiv/aids. malawi hiv and aids estimates [homepage on the internet]. 2017 [cited 2020 nov 2]. available from: http://www.unaids.org/en/regionscountries/countries/malawi/ government of malawi. malawi aids response progress report 2015. lilongwe: government of malawi; 2015. joint united nations programme on hiv/aids. 90-90-90 an ambitious treatment target to help end the aids epidemic [homepage on the internet]. 2014 [cited 2020 nov 2]. available from: http://www.unaids.org/en/resources/documents/2017/90-90-90 ministry of health malawi. malawi population-based hiv impact assessment (mphia) 2015–16: final report. lilongwe: ministry of health malawi; 2018. world health organization. guideline on when to start antiretroviral therapy and on pre-exposure prophylaxis for hiv. geneva: world health organization; 2015. ministry of health malawi. clinical management of hiv in children and adults. lilongwe: ministry of health malawi; 2014. ministry of health malawi. clinical management of hiv in children and adults. lilongwe: ministry of health malawi; 2011. chinele j. malawi government says determined on new art regime. the times malawi. 2016 feb 13 [cited 2020 nov 2]. available from: https://times.mw/malawi-government-says-determined-on-new-art-regime van buuren s. multiple imputation of discrete and continuous data by fully conditional specification. stat methods med res. 2007;16(3):219–242. https://doi.org/10.1177/0962280206074463 rubin d. multiple imputation for nonresponse in surveys. new york, ny: wiley; 1987. united nations children’s fund. options b and b+: key considerations for countries to implement an equity-focused approach. new york, ny: unicef; 2012. kieffer mp, mattingly m, giphart a, et al. lessons learned from early implementation of option b+: the elizabeth glaser pediatric aids foundation experience in 11 african countries. j acqui immune defic syndr. 2014;67(suppl 4):s188. https://doi.org/10.1097/qai.0000000000000372 joint united nations programme on hiv/aids [homepage on the internet]. aidsinfo. 2019 [cited 2020 nov 2]. available from: http://aidsinfo.unaids.org/ berhan z, abebe f, gedefaw m, tesfa m, assefa m, tafere y. risk of hiv and associated factors among infants born to hiv positive women in amhara region, ethiopia: a facility based retrospective study. bmc res notes. 2014;7(1):876. https://doi.org/10.1186/1756-0500-7-876 hatcher am, woollett n, pallitto cc, et al. bidirectional links between hiv and intimate partner violence in pregnancy: implications for prevention of mother-to-child transmission. j int aids soc. 2014;17(1):19233. https://doi.org/10.7448/ias.17.1.19233 anígilájé e, dabit o, ageda b, hwande s, bitto 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translating research into policy and practice. jama. 2000;283(9):1175–1182. https://doi.org/10.1001/jama.283.9.1175 volmink j, siegfried n, van der merwe l, brocklehurst p. antiretrovirals for reducing the risk of mother-to-child transmission of hiv infection. cochrane database syst rev. 2007;1:cd003510. https://doi.org/10.1002/14651858.cd003510.pub2 taha te, nour s, kumwenda ni, et al. gender differences in perinatal hiv acquisition among african infants. pediatrics. 2005;115(2):e167–e172. https://doi.org/10.1542/peds.2004-1590 fiscus sa, cheng b, crowe sm, et al. hiv-1 viral load assays for resource-limited settings. plos med. 2006;3(10):e417. https://doi.org/10.1371/journal.pmed.0030417 roberts t, bygrave h, fajardo e, ford n. challenges and opportunities for the implementation of virological testing in resource-limited settings. j int aids soc. 2012;15(2):17324. https://doi.org/10.7448/ias.15.2.17324 minchella pa, chipungu g, kim aa, et al. specimen origin, type and testing laboratory are linked to longer turnaround times for hiv viral load testing in malawi. plos one. 2017;12(2):e0173009. https://doi.org/10.1371/journal.pone.0173009 thiha s, shewade hd, philip s, et al. factors associated with long turnaround time for early infant diagnosis of hiv in myanmar. glob health action. 2017;10(1):1395657. https://doi.org/10.1080/16549716.2017.1395657 mwenda r, vojnov l, saka e, et al. significantly improved antiretroviral therapy initiation rates after the implementation of point of care early infant diagnosis. african society for laboratory medicine conference cape town, south africa. 3–8 december 2016. abstract introduction methodology results discussion acknowledgements references about the author(s) naseem cassim national health laboratory service, johannesburg, south africa lindi m. coetzee national health laboratory service, johannesburg, south africa department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa deborah k. glencross national health laboratory service, johannesburg, south africa department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa citation cassim n, coetzee lm, glencross dk. categorising specimen referral delays for cd4 testing: how inter-laboratory distances and travel times impact turn-around time across a national laboratory service in south africa. afr j lab med. 2020;9(1), a1120. https://doi.org/10.4102/ajlm.v9i1.1120 original research categorising specimen referral delays for cd4 testing: how inter-laboratory distances and travel times impact turn-around time across a national laboratory service in south africa naseem cassim, lindi m. coetzee, deborah k. glencross received: 06 nov. 2019; accepted: 16 sept. 2020; published: 21 dec. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the south african national health laboratory service provides laboratory services for public sector health facilities, utilising a tiered laboratory model to refer samples for cd4 testing from 255 source laboratories into 43 testing laboratories. objective: the aim of this study was to determine the impact of distance on inter-laboratory referral time for public sector testing in south africa in 2018. methods: a retrospective cross-sectional study design analysed cd4 testing inter-laboratory turn-around time (tat) data for 2018, that is laboratory-to-laboratory tat from registration at the source to referral receipt at the testing laboratory. google maps was used to calculate inter-laboratory distances and travel times. distances were categorised into four buckets, with the median and 75th percentile reported. wilcoxon scores were used to assess significant differences in laboratory-to-laboratory tat across the four distance categories. results: cd4 referrals from off-site source laboratories comprised 49% (n = 1 390 510) of national reporting. a positively skewed distribution of laboratory-to-laboratory tat was noted, with a median travel time of 11 h (interquartile range: 7–17), within the stipulated 12 h target. inter-laboratory distance categories of less than 100 km, 101–200 km, 201–300 km and more than 300 km (p < 0.0001) had 75th percentiles of 8 h, 17 h, 14 h and 27 h. conclusion: variability in inter-laboratory tat was noted for all inter-laboratory distances, especially those exceeding 300 km. the correlation between distance and laboratory-to-laboratory tat suggests that interventions are required for distant laboratories. keywords: hiv; cluster of differentiation 4; cd4; immune status; inter-laboratory referral; distance; travel time. introduction laboratory services in south africa are provided within the public sector by the national health laboratory service (nhls).1 this mandate is delivered through a national network of 255 laboratories ranging from large high-throughput laboratories located in metropolitan areas to smaller laboratories placed in rural district hospitals.1 testing is typically offered using a tiered laboratory service approach.2 in the lower tiers, a basket of eight standard pathology tests are performed locally to provide emergency services and include full blood count, urea and electrolytes, cardiac enzymes, glucose, liver enzymes, among others. specialised assays and tests supporting local hiv/aids and tuberculosis testing regarded as high-volume workloads are transferred through the laboratory referral network to appropriate specialist and dedicated testing laboratories, depending on the nature of the test. tertiary centres and academic laboratories have high workloads that are facilitated by using more sophisticated, high-throughput analysers.2 cd4 testing, for example, is centralised into 43 laboratories located strategically across the country to ensure widespread full-service coverage.2,3 in south africa, samples are collected daily from health facilities through a network of couriers and transported to the local source laboratory. these laboratories perform a basic repertoire of tests. more specialised tests are referred to the nearest testing laboratory with an expanded test repertoire. a cd4 count is used to assess the patient’s immune status (level of immune suppression) at presentation and during therapy.2 additionally, it assesses the risk of opportunistic disease, including co-infections such as cryptococcal meningitis in patients with cd4 counts lower than 100 cells/µl.4,5 global estimates of advanced hiv disease have remained largely unchanged over the last 5 years despite increased antiretroviral therapy (art) coverage in low-and middle-income countries.6 between 30% and 50% of hiv-positive individuals in lowand middle-income countries are estimated to have cd4 counts under 200 cells/µl.6 in south africa, coetzee et al. reported that the percentage of samples with a cd4 count under 100 cells/µl ranged between 7.8% and 11.5% for the 2014/2015 financial period across south africa’s nine provinces,7 while the burden of advanced disease (cd4 count ≤ 200 cells/µl), reported by carmona et al., approaches nearly 40% among those entering care for the first time in 2016.8 approximately 3.5 million cd4 tests are typically processed per annum throughout the nhls. the burden of advanced disease thus weighs heavily on this laboratory service. patients with advanced hiv disease are expedited into care and art initiation, usually within 7 days of diagnosis.6 to meet the requirements of ‘fast-tracking’ patients into care and onto art, cd4 results need to be delivered timeously to referring health facilities. this, in turn, requires that samples be delivered in good time to the testing laboratories, within a 12-h window (as per internal guideline), to allow laboratory testing to commence and be completed within the organisational stipulated testing inter-laboratory turn-around time (tat) of 40 h. within the nhls, there are 255 source laboratories that refer cd4 samples to the 43 testing laboratories (5.9:1). the organisational tat cut-off for the inter-laboratory referral of cd4 samples (or ‘lab-to-lab’ time) is set at 12 h. previous work revealed that the placement of cd4 testing equipment and testing capacity, within an area identified with longer tat attributable to pre-analytical causes, led to a marked shortening of the associated pre-analytical tat and noticeably shorter overall tat.9 glencross et al. reported using a service radius around existing cd4 testing laboratories to determine healthcare facilities and clinics that lay outside of existing service precincts.2 this study facilitated the identification of additional testing sites required to improve local tat.2 to achieve prompt sample referral within the cd4 network to address pre-analytic tat, the nhls employs a ‘hub and spoke’ approach whereby each testing cd4 laboratory receives referred samples from multiple source laboratories where the samples are first accepted into the laboratory network (but where there are no facilities to perform the testing).10 at the referring (source) laboratory, the cd4 sample is registered (as a referral) onto the laboratory information system (lis). thereafter, samples are transported to the centralised testing site; the inter-laboratory timeframe is completed when the samples are received and receipted on the lis at the testing site. the lis automatically generates the relevant dates and times of sample dispatch and receipt that are used to determine the inter-laboratory tat in hours.10 for the purpose of this study, this inter-laboratory time period (component), from registration at the source laboratory to referral receipt at the testing laboratory, is termed the ‘lab-to-lab’ tat. it is anticipated that the distance and travel time for inter-laboratory referral could potentially impact lab-to-lab tat performance. laboratories located more than 300 km from a testing laboratory are expected to have longer lab-to-lab tat. conversely, laboratories within a 100 km radius are expected to have shorter travel times and lab-to-lab tat. with a decentralised laboratory service model, it would be assumed that an inter-laboratory referral distance would not exceed 250 km. the 250 km radius is based on the study by cassim et al. that used the integrated tiered service delivery model (itsdm) coverage precinct approach to address art-related testing service coverage gaps.2,10 the cassim et al. study assumed that a 250 km buffer around each laboratory would adequately address art-related test coverage.10 the relationship between travel time, distance and lab-to-lab tat performance would identify gaps in the current service model, where additional decentralisation would deliver better inter-laboratory tat performance. the objective of this study was to determine the impact of distance on inter-laboratory referral time in 2018 for public sector testing in south africa. methodology ethical considerations ethical clearance was obtained from the university of the witwatersrand (m1706108). this study was a retrospective analysis of nhls laboratory data, did not use any patient identifiers and did not involve any direct patient contact. study design and data analysis this study used a cross-sectional study design. microsoft access and excel (microsoft corp., redmond, california, united states) were used to prepare data11 and analysis was performed using sas 9.4 (sas, cary, north carolina, united states).12 laboratory data for all cd4 samples referred for centralised testing were extracted from the corporate data warehouse for the 2018 calendar year across south africa. the corporate data warehouse is an entity of the nhls and houses all lis data generated by its laboratories. a referral cd4 outcome was assigned where both the source (referring) laboratory and testing laboratories fields were populated in the lis data. google maps (alphabet, inc., mountain view, california, united states) was used to calculate both inter-laboratory distances and travel times using confirmed coordinates of laboratories (latitude and longitude).13 data were summarised in a worksheet for later analysis. the lis date and time stamps were used to calculate the lab-to-lab (inter-laboratory referral) time in hours (time from registration at the source laboratory to the time of referral receipt at the testing laboratory). due to the limited data points, the following delays could not be determined and are accounted for in the lab-to-lab tat: (1) between registration at the source laboratory to pick up by the courier, (2) transport to the testing laboratory and (3) delivery at the testing laboratory for referral receipt. skewness can be quantified as a representation of the extent to which a given distribution varies from a normal distribution.14 a normal distribution reports a skewness of zero compared to a positively skewed distribution with a value of greater than zero.14 for a normal distribution, descriptive statistics such as the mean and standard deviation can be reported. however, for a skewed distribution, the median and interquartile range are reported. hence, skewness dictates what descriptive statistics may be reported. for each cd4 sample, the requesting health facility is captured to facilitate the delivery of patient reports. in the lis, each health facility has a designated location code. this code is captured in the lis based on health facility details provided on the laboratory request form. unfortunately, very few health facilities had latitude and longitude data making it difficult to calculate the distance to the local source laboratory. cd4 samples that were not referred (i.e. that were received directly at testing laboratories) were excluded for the purpose of this study. data were reported as descriptive statistics, and visualised using histograms. a scatter plot was used to demonstrate the relationship between referral times (hours) and distances (km), with descriptive statistics reported. the inter-laboratory distances between source and testing sites were categorised into four buckets; (1) ≤ 100 km, (2) 101–200 km (3) 201–300 km and (4) > 300 km. the aim of the analysis was to determine whether lab-to-lab tat was influenced by distance. the analysis could also reveal whether decentralisation within a 200 km radius would decrease lab-to-lab tat. the number of health facilities associated with local source laboratories in each of the four inter-laboratory distance buckets was reported. the lab-to-lab tat component and descriptive statistics (median and 75th percentile) were reported, using the non-parametric wilcoxon scores (rank sum) test to determine differences in tat reported among the distance categories. in addition, inter-laboratory referral routes longer than 300 km that referred 1000 or more samples in 2018 are reported as a separate table reporting: (1) source laboratory, (2) testing laboratory, (3) distance, (4) travel time (hours), (5) percentage within 12-h cut-off and (6) 75th percentile, using source and testing laboratory aliases. the projected daily referral volumes were calculated based on the assumption of 21.73 working days per month. results there were 2 844 242 cd4 samples performed in 2018, of which 1 390 510 (49%) were referred for centralised testing within the nhls laboratory network. distribution of inter-laboratory referral times and distances a linear relationship exists between referral times and distances. lab-to-lab data reported a positively skewed distribution (skewness = 2.83). a lab-to-lab median of 11 h with an interquartile range of 7–17 h was reported. the mode was 8 h, with a range of 96 h (figure 1). figure 1: distribution of inter-laboratory cd4 testing referral laboratory-to-laboratory turn-around time in the 2018 calendar year for national health laboratory service laboratories in south africa. median, interquartile range, mode and range are indicated (inset). the inter-laboratory cd4 testing referral distance data also reported a positively skewed distribution (skewness = 1.66) (figure 2). the median distance travelled by referred samples was 67.1 km (interquartile range = 20.1–123.0 km; mode = 14.4 km; range = 559 km). the distribution of inter-laboratory referral travel times was also positively skewed (skewness = 1.33). the median was 0.9 h, with an interquartile range of 0.4–1.63 h, a mode of 0.35 h, and a range of 6.15 h. figure 2: relationship between inter-laboratory cd4 testing referral distance and travel time between source and cd4 testing laboratories in the 2018 calendar year across the national health laboratory services in south africa. inter-laboratory cd4 testing referral performance by distance category overall, 67% of samples were referred to testing laboratories within a 100 km inter-laboratory referral distance, increasing to approximately 93% of samples within a 200 km radial precinct. approximately 7% of samples were transported 201 km or more from their source laboratory to their sister centralised testing laboratory. overall, for referral distances under 300 km, all inter-laboratory precincts (both the mode and median lab-to-lab tat) met the organisational inter-laboratory tat cut-off of 12 h. longer lab-to-lab tats were noted from referral or source laboratories further than 300 km from testing laboratories. analysis of the 75th percentile lab-to-lab tat, unlike the mode and median parameters, revealed that only inter-laboratory referral distances of under 100 km met the 12-h cut-off (table 1). the wilcoxon scores (rank sum) test indicated a significant difference in lab-to-lab tat across the four distance categories. table 1: inter-laboratory cd4 testing referral laboratory-to-laboratory performance by distance categories in the 2018 calendar year for national health laboratory services laboratories in south africa. laboratories serviced the majority of health facilities (80%) with an inter-laboratory referral distance within 200 km. there were 12% and 7.5% of health facilities that were serviced by laboratories with an inter-laboratory referral distance of 201 km – 300 km and more than 300 km. distant inter-laboratory routes performance analysis for five source laboratories, distance to the nearest testing facility exceeded 300 km (table 2). distances ranged from 304 km to 560 km (mean distance of 387 km). the percentage of samples transferred to the centralised testing site within the cut-off tat of 12 h was 36%, 37% 6%, 83% and 8% for source laboratories s1, s2, s3, s4 and s5. the median lab-to-lab tat across each of the five sites exceeded the stipulated tat cut-off for all five sites. the 75th percentile lab-to-lab tat revealed further variation of pre-analytical inter-laboratory tat efficiency, that is, 10 h – 29 h for individual laboratories. daily cd4 referral volumes from these laboratories ranged from 6 to 24 samples. table 2: laboratotry-to-laboratory performance of inter-laboratory referral routes over 300 km and involving at least 1000 referred samples in the 2018 calendar year for national health laboratory service laboratories in south africa. discussion in this study, the impact of distance and travel time on lab-to-lab tat as a pre-analytical component of total tat was assessed to categorise laboratory service efficiency and, in national service, understand where bottlenecks of logistics may occur. we observed moderate correlation between distance travelled and the overall recorded inter-laboratory lab-to-lab tat. however, across all service precincts, especially those exceeding a radius of 300 km, variable inter-laboratory tat was recorded. generally, median lab-to-lab tat of referrals within a 200 km radius of a testing facility (n = 210 laboratories; 93% of total referrals) was 11 h and fell within the organisational stipulated inter-laboratory tat cut-off of 12 h. this finding confirms that previous implementation of decentralised cd4 testing facilities into lower service tiers has positively impacted the efficiency of local service delivery.2,9 earlier work identified that well-planned decentralisation in remote areas has been successfully implemented in the northern cape (de aar, upington and tshwaragano laboratories).2 despite previous decentralisation drives and a national median inter-laboratory tat of 11 h for at least 93% of referred samples, there are still outliers noted. for example, where distances travelled to a centralised testing facility was between 100 km and 200 km, a 75th percentile inter-laboratory tat of 17 h was noted. therefore, although most of the referring times reported here were within the inter-laboratory transfer cut-off of 12 h, service gaps are evident (where referral laboratories failed to deliver their samples to the testing laboratory within the stipulated time frame). glencross et al. developed the itsdm that consists of five tiers to facilitate sustainable ‘full-service coverage’ across south africa.2 this model could be used to extend cd4 services into rural and remote areas with tier 3 (community) or tier 2 (point-of-care and hub) services that complement more centralised testing offered by tiers 1 and 2.2 the tier 3 sites would be installed in existing laboratories with existing infrastructure.2 candidate laboratories are identified using the itsdm,2 to service health facilities outside the current service precinct.2,9 the implementation of new tier 3 community laboratories in this context has a proven role in reducing pre-analytical tat, particularly where samples are required to travel distances in excess of 300 km.9 coetzee et al. have previously reported the successful impact of the placement of an itsdm-identified community cd4 laboratory in an area experiencing long overall tat; a dramatically reduced tat was realised in this rural district.9 this was achieved by adding cd4 testing capacity to the existing test repertoire in the local laboratory (de aar) which substantially reduced the pre-analytical component of total tat of testing in the area. in the current study, based on the number of samples referred and the proportion of samples within cut-off tat, sites 1 and 2 should be eligible for local capacity placement. in some cases (i.e. sites 3, 4 and 5), review of local logistics could improve the pre-analytical component, especially as it was revealed that one of the five sites, which was 304 km from its sister testing site, achieved a remarkable 75th percentile lab-to-lab tat of around 10 h. this suggests that distance alone is not the primary determining factor influencing lab-to-lab tat. this study demonstrated a median lab-to-lab tat of 15 h, with a right skewed distribution and a 75th percentile of 27 h for referrals outside a 300 km service precinct to the nearest testing laboratory. this finding, again, reiterates the need to apply the itsdm approach described above to assist with identifying sites for capacity development and alleviation of the longer inter-laboratory tats identified in this work. at least four sites with low test volumes and exceeding 300 km to the nearest testing facility warrant the introduction of a local service that could be accomplished with lower throughput platforms (daily capacity of 40 samples).15,16,17 testing systems such as the beckman coulter aquios cytometer or becton dickinson facspresto are easy to use and suitable for existing smaller laboratories requiring minimal refurbishment. costing assessments done by cassim et al. have demonstrated that the incremental cost for decentralising cd4 testing to community laboratories is a mere $2.05.18 this low incremental cost is due to the placement agreement model where the cost of the analysers for new sites is included in the reagent costs. whether this is a high or low volume site requiring different platforms, the cost to offer one test remains the same. furthermore, the proposed assays have been validated locally and have world health organization pre-qualification,19 reporting acceptable precision to predicate methods for both venous or capillary blood sampling.15 the challenges for the decentralisation model include the availability of adequately trained staff, the need to increase resupply points of reagents and consumables at lower-level facilities, and the ease of access to suppliers for maintenance and repairs. irrespective of costs, the impact on patient care is invaluable.20 with only about 50% of tested cd4 samples referred between laboratories, the nhls strives to have adequate testing facilities for maximum coverage especially in high-burden and remote areas,2 and, as mentioned above, has set a maximum target time of 12 h for samples to be transported from source laboratories to the centralised testing facilities. it would make sense for inter-laboratory tat (referral time) to be directly proportional to distance. this assumption is based on the provision of adequate and reliable delivery vehicles and appropriately maintained roads. the road conditions and how the collection and transportation routes are designed would also affect tat performance. the operating times of referral sites may also impact delays in tat as not all offer a 24-h service. human resource factors can also lead to delays and longer tat, including the work hours, work ethos and diligence of both drivers and receiving office staff at the centralised facility.21 a limitation of this analysis is that it focuses on the inter-laboratory referral after samples are received within the laboratory network. the laboratory pre-analytical phase of tat is measured from the first registration in an nhls laboratory to receipt in the testing facility; it does not include the time from venepuncture to first registration on the lis system. however, as the latter time periods are poorly recorded, it is difficult to determine if a longer lab-to-lab tat is due to a delay in time from sample collection to registration at the source laboratory. challenges in the pre-analytical and analytical arms would have to be addressed separately due to differences in the nature of challenges and the involvement of different personnel. therefore, separate corrective action interventions need to be set in motion. this may include redesigning the specimen referral network which could include additional routes or potentially identifying some sites for point-to-point routing instead of a multi-stop route, or vice versa.2 to adequately address all delays of inter-laboratory referral tat, all aspects of this phase should be addressed. not all of the processes undertaken during the transfer of samples are documented and traceable, that is, not all steps generate a time and date stamp on the lis or are date-stamped at the point of venesection. the introduction of electronic monitoring of all total tat would facilitate a more complete view of possible points of delay across the entire sample journey and identify aspects for corrective action. vertical audits can be used to identify sources of delay on different days of the week to calculate average (delay) times for each process.22 in addition, workflow analyses could assist in mapping out (and eliminating) processes or steps that do not add value.22 through this process, a standard operating procedure for all pre-analytical processes could be introduced across facilities. limitations the study used predominantly laboratory data to assess the performance of cd4 test referrals across the nhls. this study did not report data for other tests as the inter-laboratory referral network is integrated. all the concepts reported in this study for cd4 testing apply to other referred tests as well. no information was available to review time from sample collection to registration onto the laboratory network, as well as time after sample registration at the source laboratory to collection by the courier. conclusion this study demonstrates that most referrals for cd4 testing reach their testing facilities within the expected 12-h window, with some outliers identified. here, differences in inter-laboratory referral distances and lab-to-lab tat performance suggest that there are inconsistent systems and practices in use to transfer samples between centres. further investigation to understand the root causes would assist in aligning efficient delivery of all samples between facilities. there is a need for additional data collection for the inter-laboratory referral process to better understand where service bottlenecks exist. this study identified the need for electronic data recording at multiple stages of sample inter-laboratory referral such that bottlenecks can easily be identified and resolved to optimise timely referrals. for distances exceeding 300 km, the establishment of additional community cd4 laboratories is recommended. acknowledgements the authors would like to thank the corporate data warehouse, and staff at the national priority programme unit for their assistance. competing interests the authors have declared that no competing interests exist. authors’ contributions d.k.g. supervised the study by providing leadership and oversight and was also the project leader. n.c. and l.m.c. designed the study, developed the methodology, conducted the research, conducted the data analysis and prepared the maps. d.k.g. provided editorial comments and technical input. all authors contributed to the manuscript development. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references national health laboratory service (nhls). annual report 2017/18 [homepage on the internet]. johannesburg: national health laboratory service (nhls); 2018 [cited 2018 jul 25]. available from: http://www.nhls.ac.za/assets/files/an_report/nhls_ar_2018.pdf glencross dk, coetzee lm, cassim n. an integrated tiered service delivery model (itsdm) based on local cd4 testing demands can improve turn-around times and save costs whilst ensuring accessible and scalable cd4 services across a national programme. plos one. 2014;9(12):e114727. https://doi.org/10.1371/journal.pone.0114727 glencross dk, janossy g, coetzee lm, et al. large-scale affordable panleucogated cd4+ testing with proactive internal and external quality assessment: in support of the south african national comprehensive care, treatment and management programme for hiv and aids. cytometry b clin cytom. 2008;74(suppl 1):s40–s451. https://doi.org/10.1002/cyto.b.20384 govender np, glencross dk. national coverage of reflex cryptococcal antigen screening: a milestone achievement in the care of persons with advanced hiv disease. s afr med j. 2018;108(7):534–535. https://doi.org/10.7196/samj.2018.v108i7.13094 coetzee lm, cassim n, sriruttan c, mhlanga m, govender np, glencross dk. cryptococcal antigen positivity combined with the percentage of hiv-seropositive samples with cd4 counts <100 cells/mul identifies districts in south africa with advanced burden of disease. plos one. 2018;13(6):e0198993. https://doi.org/10.1371/journal.pone.0198993 world health organisation (who). prevent hiv, test and treat all – who support for country impact: progress report [homepage on the internet]. geneva: world health organisation (who); 2016 [cited 2019 jul 25]. available from: http://apps.who.int/iris/bitstream/10665/251713/1/who-hiv-2016.24-eng.pdf?ua=1 coetzee lm, cassim n, glencross dk. analysis of hiv disease burden by calculating the percentages of patients with cd4 counts <100 cells/microl across 52 districts reveals hot spots for intensified commitment to programmatic support. s afr med j. 2017;107(6):507–513. https://doi.org/10.7196/samj.2017.v107i6.11311 carmona s, bor j, nattey c, et al. persistent high burden of advanced hiv disease among patients seeking care in south africa’s national hiv program: data from a nationwide laboratory cohort. clin infect dis. 2018;66(suppl_2):s111–s117. https://doi.org/10.1093/cid/ciy045 coetzee lm, cassim n, glencross dk. implementation of a new ‘community’ laboratory cd4 service in a rural health district in south africa extends laboratory services and substantially improves local reporting turnaround time. s afr med j. 2015;106(1):82–87. https://doi.org/10.7196/samj.2016.v106i1.10081 cassim n, coetzee lm, stevens ws, glencross dk. addressing antiretroviral therapy-related diagnostic coverage gaps across south africa using a programmatic approach. afr j lab med. 2018;7(1):681. https://doi.org/10.4102/ajlm.v7i1.681 microsoft corporation. microsoft office 2013 albuquerque [homepage on the internet]. new mexico: microsoft corporation; 2019 [cited 2019 jul 01]. available from: https://products.office.com/en/home sas institute. sas 9.3 [homepage on the internet]. cary, nc: sas institute; 2019 [cited 2019 jul 01]. available from: https://www.sas.com/en_us/software/sas9.html google. google maps: distance and travel time beween two laboratories mountain view [homepage on the internet]. ca: google; 2019 [cited 2019 aug 12]. available from: https://www.google.com/maps investopedia. skewness [homepage on the internet]. new york, ny: investopedia; 2019 [cited 2020 jun 12]. available from: https://www.investopedia.com/terms/s/skewness.asp coetzee lm, glencross dk. performance verification of the new fully automated aquios flow cytometer panleucogate (plg) platform for cd4-t-lymphocyte enumeration in south africa. plos one. 2017;12(11):e0187456. https://doi.org/10.1371/journal.pone.0187456 coetzee lm, lawrie d, stevens w, venter f, osih r, glencross dk, editors. performance evaluation of pimatm cd4 point-of-care (poc) analyzers in a controlled laboratory setting, johannesburg, south africa. xviii international aids conference; 2010 jul 18–23; vienna. coetzee lm, moodley k, glencross dk. performance evaluation of the becton dickinson facspresto near-patient cd4 instrument in a laboratory and typical field clinic setting in south africa. plos one. 2016;11(5):e0156266. https://doi.org/10.1371/journal.pone.0156266 cassim n, coetzee lm, schnippel k, glencross dk. estimating implementation and operational costs of an integrated tiered cd4 service including laboratory and point of care testing in a remote health district in south africa. plos one. 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2018;32(1):e22180. https://doi.org/10.1002/jcla.22180 abstract introduction methods results discussion acknowledgements references about the author(s) sarah mcalpine department of chemical pathology, national health laboratory service, pretoria, south africa bettina chale-matsau department of chemical pathology, national health laboratory service, pretoria, south africa department of chemical pathology, faculty of health sciences, university of pretoria, pretoria, south africa citation mcalpine s, chale-matsau b. impact of potassium test sample rejections on routine laboratory service, south africa. afr j lab med. 2023;12(1), a2239. https://doi.org/10.4102/ajlm.v12i1.2239 original research impact of potassium test sample rejections on routine laboratory service, south africa sarah mcalpine, bettina chale-matsau received: 19 june 2023; accepted: 01 nov. 2023; published: 22 dec. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: accurate potassium measurements are necessary for effective clinical management of hyperkalaemia. pre-analytical factors may affect laboratory measurements, leading to erroneous results and inappropriate patient management and negatively impact the efficiency and finances of laboratories and hospitals. objective: this study evaluated the impact of rejected potassium test requests on laboratory service. methods: we conducted a retrospective descriptive study to assess potassium test data at a public laboratory in pretoria, gauteng, south africa, using samples collected from an academic hospital, peripheral hospitals, and outpatient clinics between january 2018 to december 2018. we assessed the relationship between reasons for rejection and health facility type, as well as financial implications for the laboratory. results: the potassium result rejection rate was 15.1% (29 806 samples), out of the 197 405 requests received. the most common reasons for rejection were old sample (> 1 day old) (41.4%; 12 348 rejections) and haemolysis (38.2%; 11 398 rejections). the most frequent reason for rejections at the central, academic hospital was haemolysis (42.0%), while old sample was the most common reason for rejection at peripheral hospitals (43.4%; 4119/9493 requests) and outpatient health facilities (57.2%; 7208/12 605 requests) (p = 0.022). the total cost of potassium sample rejection over the study period was substantial, given the resource constraints in this setting. conclusion: peripheral hospitals and outpatient departments accounted for the majority of rejected potassium testing results, possibly resulting from delays in transportation; causing substantial financial impact on the laboratory. improved sample collection, handling, and expedited transportation are recommended. what this study adds: this study highlights the importance of appropriate sample collection and handling and the undesirable consequences of non-adherence to these pre-analytical considerations. keywords: hyperkalaemia; pseudohyperkalaemia; pre-analytical factors; result rejection; sample rejection. introduction hyperkalaemia is defined as potassium levels greater than 5.5 mmol/l.1 it can occur following dysfunction of the renin-angiotensin-aldosterone system due to kidney failure, adrenal hypofunction, or the use of certain drugs, thus resulting in decreased potassium elimination.2,3 other common causes of hyperkalaemia include increased release of potassium from cells, as seen in diabetic ketoacidosis due to decreased insulin levels, as well as cellular breakdown or necrosis, such as rhabdomyolysis, tumour lysis syndrome, haemolysis, trauma, and burns.4,5 hyperkalaemia is also not unusual in patients with thrombocytosis or leucocytosis.5,6 persistent hyperkalaemia can be life-threatening, causing cardiac arrhythmias with possible cardiac arrest or paralysis of the respiratory muscles even with slight deviations from normal levels (3.5 mmol/l – 5.0 mmol/l).1,6 thus, accurate laboratory potassium measurements are necessary for clinicians to make appropriate medical decisions. within the laboratory, the quality and confidence of a test result depends on the quality and functioning of the total testing process, which encompasses the pre-analytical, analytical and post-analytical stages.7,8 this is important as 60% – 70% of medical decisions rely on laboratory investigations.9 due to advancements in laboratory methods, instruments, and monitoring through internal quality control and external quality assurance programmes, fewer errors are typically attributed to the analytical phase.8,10 the pre-analytical phase involves test requisition and sample labelling, collection, handling, transport, and processing. this phase accounts for up to 70% – 75% of all laboratory errors reported, most of which are due to human errors.8,9 these errors can lead to samples that are unsuitable for analysis or unreportable results requiring rejection. pre-analytical factors that may affect sample quality include haemolysed, clotted, or icteric samples, as well as samples that are mislabelled, unlabelled, or collected in inappropriate tubes. insufficient sample volume not only limits the number of tests that can be done from the received sample but can also affect sample quality, as the blood-to-additive ratio may interfere with analysis and produce erroneous results.8,9 mild contamination by ethylenediaminetetraacetic acid (edta-k), a potassium-containing anticoagulant, may also cause subtle analyte changes that can be missed.11 prolonged centrifugation of samples during the pre-analytical phase can also interfere with sample quality where, for example, platelets can lyse, resulting in falsely elevated potassium levels.12,13,14 falsely elevated potassium levels or pseudohyperkalaemia occurs when generated results are not consistent with the clinical features of the patient.4,12 not only can pseudohyperkalaemia result in inappropriate patient management, but it can also mask hypokalaemia, leading to a missed diagnosis as concentrations may falsely present within a ‘normal’ reference interval.12 the most commonly occurring source of pseudohyperkalaemia is haemolysis, which is reported to cause up to fivefold more rejections than any other reason.7,13 a subset of haemolysed samples may result from endogenous causes such as haemolytic anaemia, immune reactions, toxin exposure, and haemodialysis treatment, or due to direct damage to red blood cells.3,5 in vitro or exogenous haemolysis accounts for most haemolysed cases. causes include poor sample acquisition technique, substandard handling and transport, prolonged storage time, and delayed processing.15,16 sample acquisition techniques that specifically affect potassium include prolonged tourniquet application and patient fist clenching, which can cause a 1 mmol/l – 2 mmol/l increase in potassium levels due to potassium efflux from cells during muscle contraction.12,13,14 the use of needles with unsuitable diameters during sample collection can also result in haemolysis. in addition, cold temperatures can decrease the function of the sodium/potassium-adenosine triphosphase pump, resulting in the passive movement of potassium down the concentration gradient and out of red blood cells.12 rejection of samples or test results inconveniences both patients and healthcare personnel as it delays urgent clinical decision-making. specimen recollection may be required, leading to prolonged admission for the patient and unnecessary costs to the laboratory and the hospital or clinic.10,17 pre-analytical errors are estimated to cost approximately 0.23% – 1.02% of a hospital’s total operating budget in the united states.9,10 while there are at present no local studies on the financial implications of hyperkalaemia on the health sector in south africa, the prevalence of hyperkalaemia18 and its contribution to prolonged hospital stay have been previously described.19 magwai and colleagues recently described a rejection rate of 1.4% in an academic laboratory in south africa.20 although sample and result rejection may appear to be a small and insignificant factor in the total testing process, it is an important quality indicator for laboratories and can have great implications for the patient and health facility. this study thus aimed to assess the frequency and reasons for potassium test request rejection at the national health laboratory service (nhls), tshwane academic division (tad), south africa, during the period from 01 january 2018 to 31 december 2018 and the financial impact of these rejections on this laboratory. methods ethical considerations this study was approved by the university of pretoria research ethics committee (number 578/2020). patient consent was waived by the ethics committee as the study was based on retrospective data. patient details such as name, address, hospital number or unique laboratory identification numbers were not used to ensure patient confidentiality. study design and setting a retrospective descriptive observational study was conducted using data for potassium test requests, including both resulted and rejected tests, at the nhls tad chemistry laboratory from 01 january 2018 to 31 december 2018. these data were requested from the nhls data warehouse and were on samples from facilities in pretoria, gauteng, south africa. variables considered were reasons for rejection and the facility where the sample was obtained. rejected potassium test requests were either for unreported results or samples not tested for potassium, each of which was replaced by a reason for rejection. potassium requests generally form part of a urea and electrolytes profile. the nhls tad is a tertiary laboratory located in the 832-bed, steve biko academic hospital. the laboratory also receives samples from six peripheral hospitals (ph) and 264 outpatient clinics located at average distances of 60 km and 30 km from the laboratory. the nhls has small laboratories based at phs. these small laboratories are mostly closed after work hours, during which test requests are referred to tad. the laboratory receives approximately 350 000 all-inclusive test requests per month, tested using the abbott architect c8000 chemistry analyser (abbott laboratories, chicago, illinois, united states). we excluded all data for which patient location was not recorded as well as samples registered at peripheral laboratories and referred to tad. data analysis we evaluated data on rejected potassium test results from three categories of health facilities to determine the frequency of result rejection and reasons for rejection, and to estimate the financial loss incurred by the laboratory as a result of these rejections. financial loss was estimated based on the nhls cost-per-test charge (potassium cost at r30.35 south african rand [zar] per test) for 2018 (study period). data analysis and descriptive statistics were computed using microsoft excel 2016 (microsoft corporation, redmond, washington, united states) spreadsheets. the r statistical software version 3.6.0 (r foundation, indianapolis, indiana, united states) was used for statistical calculations.21 categorical data were expressed as frequencies and percentages. we compared the distribution of rejection reasons between steve biko academic hospital (a central academic hospital), phs and outpatient health facilities using the chi-square test. p-values less than 0.05 were considered statistically significant. results potassium test result rejection rate and reasons for rejection the total number of potassium tests requested during the study period was 197 405 (table 1), of which 167 599 were resulted and 29 806 were rejected (15.1%). the most common reasons for rejection were old samples (1 day or older) (41.4%; n = 12 348 rejections), haemolysed samples (38.2%; n = 11 398 rejections), edta-k contamination (which was based on decreased magnesium, calcium, and alkaline phosphatase results) (6.3%; n = 1864 rejections), and labelling errors (3.5%; n = 1046 rejections), which included missing or mismatched information on the sample and the request form (figure 1). other rejection reasons, accounting for 10.6% (n = 3150) of all rejections, were grouped as miscellaneous reasons and included ‘leaked’, ‘wrong tube’, ‘lost in transit’, ‘insufficient specimen’, ‘specimen container broken’, ‘duplicate registration’, and ‘specimen not received’. figure 1: reasons for rejection of potassium test requests at the national health laboratory service, tshwane academic division, gauteng, south africa, january 2018 – december 2018. table 1: number of potassium test requests and rejections by source facility category. national health laboratory service, tshwane academic division, gauteng, south africa, january 2018 – december 2018. result rejections based on facility type rejected potassium test requests were from a total of 271 facilities, which were divided into three categories. these were steve biko academic hospital, which is the central academic hospital, phs (n = 6), and outpatient health facilities (n = 264). outpatient health facilities made up 42.3% of the total number of rejected results, phs made up 31.9%, and steve biko academic hospital alone made up 25.9% of all potassium result rejections in the specified study period. haemolysed sample, which caused 42.0% of rejections, was the most reported reason for rejection from steve biko academic hospital (figure 2). this was followed by miscellaneous reasons for rejection (19.8%) and edta-k contamination (15.5%). this differed from the phs and the outpatient health facilities where old sample was the most common cause of rejection (43.4% and 57.2%), followed by haemolysed samples (40.78% and 34.0%). there was a statistically significant difference (p = 0.022) between the reasons for potassium result rejection at the different facility types (table 2). figure 2: reasons for rejection of potassium test requests at the national health laboratory service, tshwane academic division, gauteng, south africa, january 2018 – december 2018. table 2: observed and expected values for number of potassium test rejections by source facility category. national health laboratory service, tshwane academic division, gauteng, south africa, january 2018 – december 2018. financial impact of potassium test results rejection the nhls cost per test for potassium testing as per the 2018 price list was approximately $1.90 united states dollars (usd; equivalent to r30.35 zar). with a total of 29 806 rejected potassium test requests, the total cost to the laboratory was approximately $56 000.00 usd (r986 876.00 zar) in 2018 (figure 3). figure 3: the estimated financial loss incurred by the national health laboratory service, tshwane academic division, gauteng, south africa due to potassium test request rejections from steve biko academic hospital, peripheral hospitals, and outpatient health fa cilities, january 2018 – december 2018. discussion the potassium result rejection rate for the nhls tad chemistry laboratory in 2018 was 15.1%, and there was a statistically significant difference between the reasons for potassium result rejection at the different facility types. old sample was the main reason for rejections, comprising 41.4% of total rejections. this was the most common reason for rejection for both phs and outpatient facilities. owing to the location of some of these facilities, it is likely that the reasons for delayed analysis might be multifactorial. these are likely to include distance, long time lapse following collection, storage, and transportation conditions. other potential reasons for delay in testing may include batch testing or factors related to the testing laboratory itself such as excessive workload, instrument issues, and sample sharing between departments, which may delay sample arrival to the chemistry laboratory as previously described in studies from china and the united states.16,22 old samples are rejected because of biochemical changes that occur as red blood cells break down during long storage, thus leading to erroneous results.15 delays in sample analysis can affect accuracy and quality, and, as indicated in a study from the united states, samples stored at temperatures of 2 °c – 8 °c for longer than 24 h result in spuriously elevated potassium levels due to cessation of the sodium/potassium-adenosine triphosphatase pump.12 a study from france has indicated that although potassium levels are stable at room temperature, samples should be analysed within 4 h – 6 h for accurate measurements.23 moreover, increased potassium levels have been reported in samples stored at room temperature within 1 h of collection in a study done in china in 2017.16 another factor to consider when samples are sent to external or distant laboratories for testing is whether the samples were centrifuged at the collection site prior to transportation or only after they arrived at the testing laboratory. delayed serum separation may cause potassium to leak out of the red blood cells, yielding hyperkalaemia. ideally, according to a united kingdom study done in 2003, samples should be centrifuged within 1 h of collection to prevent these falsely increased potassium levels.24 additionally, an important aspect to be aware of for potassium measurements is susceptibility to seasonal pseudohyperkalaemia, where potassium levels are elevated in the cooler winter months owing to the inhibition of the sodium/potassium-adenosine triphosphatase pump activity.12,24 interestingly, the main reason for result rejection in this study, old sample, is not the most common cause of rejection reported in previous studies done in ethiopia, the united states, and turkey between 2015 and 2016.10,11,25,26 the major reasons for rejection reported in these studies include haemolysis, insufficient sample, incorrect sample or tube, clotted samples, contamination, and mislabelled or unlabelled samples.26,27 this difference may, however, be explained by the fact that about 74% of all samples received at this laboratory were referred from off-site facilities, thus making delayed sample analysis a major factor causing rejection. the second most common reason for potassium result rejection in this study was haemolysed samples (38.2%). this was the main reason for potassium result rejection at the steve biko academic hospital (42.02%), a facility that makes up a quarter of all samples received by the laboratory. at the peripheral facilities, approximately 50% of rejections were due to haemolysed samples, which is lower than previous reports from australia and turkey in 2010, where haemolysed samples account for about 60% of all rejections.6,28 findings from previous studies done in italy in 2008 and malaysia in 2019 suggest that haemolysis is the main cause of pseudohyperkalaemia.7,13 making medical decisions based on these erroneous results can lead to inappropriate patient care. this may occur when there is pseudohyperkalaemia or in instances when hypokalaemia is missed due to false potassium elevation in the sample.12 a malaysian study done in 2019 demonstrated that although haemolysis may occur in vivo, this only occurs in about 2% of cases.7 when in vivo haemolysis is suspected, additional information such as patient history, haptoglobin measurements, bilirubin levels, and red blood cell count are required to arrive at the diagnosis.7 in 2020, a study in italy reported that in vitro or exogenous causes are more common and include poor sample collection techniques, poor sample handling and transport, prolonged storage time and inadequate storage temperature.29 personnel collecting samples should be trained and educated on the importance of correct collection techniques. a study investigating the impact of educational training among nurses in a hospital in oman in 2017 showed about 75.9% improvement in sample quality after re-training.30 the availability of specially trained phlebotomy personnel within a facility is important not just for proper blood sample collection, but also to ease the workload on the already overworked nurses and doctors, who may tend to rush sample collection due to the increased workload.30 haemolysed samples certainly pose a serious challenge for clinical laboratories.7 contamination by edta-k anticoagulant as a cause of pseudohyperkalaemia accounted for 6.3% of the potassium rejections in this study and is common in clinical chemistry laboratories.31 as reported in studies from thailand in 201932 and india in 2020,33 edta-k contamination may potentially lead to patient mismanagement. thus, technique and order of draw need to be cautiously considered during sample collection to avoid contamination.34 rejection of test results can negatively impact patient care at the health facility and laboratory. importantly, rejected results can delay critical and potentially life-saving clinical decisions. based on our calculations, the nhls tad incurred a financial loss of almost r1 000 000.00 zar (approximately $56 000.00 usd) in a single year due to potassium test request rejections. an additional aspect to consider is the person-hours wasted due to these rejections. rejection of 29 806 potassium test requests over a 12-month period equates to approximately 2500 rejections per month. if the processing time of each of these samples is 10 min, 25 000 min or 416 h per month are lost on wasted labour. this equates to approximately 17 days per month. also, additional time is wasted as laboratory staff remove erroneous results and assign reasons for rejection. to solve this, the nhls tad could implement automatic cancellation by the laboratory information system of potassium requests that are older than 1 day. this would prevent the sample from being analysed and eliminate the subsequent wastage of financial resources and labour. limitations access to data was mainly limited to nhls archived data with no knowledge how the samples were collected or stored. the rejection data comprised mostly potassium requests that were either of unreported results or samples not tested for potassium, each of which was replaced by a reason for rejection and could therefore not be reported. the cost of these rejections to the hospitals and the effect on patient management could not be quantified as no clinical information was available for this estimation. the cost could only be estimated for the laboratory and could not be done for the health facilities. conclusion findings from this study have shown a high rate of rejection of potassium test requests across all facilities, with phs and outpatients accounting for most of the rejected potassium test requests. old sample was the most common reason in facilities located off site due to delays in sample processing. as indicated in the study, test rejections can have significant financial implications for the laboratory. acknowledgements the authors acknowledge the head of department of chemical pathology prof. tahir pillay for support. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions s.m. drafted the original manuscript, b.c.-m. was involved in conceptualisation and supervision of the study as well as editing of the manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability the study is based on retrospective data. authors confirm that data supporting the findings of the study are available within the manuscript. disclaimer the views and opinions 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https://doi.org/10.15415/jmrh.2015.21001 lippi g, guidi gc, mattiuzzi c, plebani m. preanalytical variability: the dark side of the moon in laboratory testing. clin chem lab med. 2006;44(4):358–365. https://doi.org/10.1515/cclm.2006.073 koseoglu m, hur a, atay a, cuhadar s. effects of hemolysis interference on routine biochemistry parameters. biochem med. 2011;21(1):79–85. https://doi.org/10.11613/bm.2011.015 barbato l, campelo md, pigozzo s, et al. rejection of hemolyzed samples can jeopardize patient safety. ejifcc. 2020;31(1):15. al-ghaithi h, pathare a, al-mamari s, villacrucis r, fawaz n, alkindi s. impact of educational activities in reducing pre-analytical laboratory errors: a quality initiative. sultan qaboos univ med j. 2017;17(3):e309. https://doi.org/10.18295/squmj.2017.17.03.008 schlüter k, cadamuro j. erroneous potassium results: preanalytical causes, detection, and corrective actions. crit rev clin lab sci. 2023;60(6):1–24. https://doi.org/10.1080/10408363.2023.2195936 laiwejpithaya s, piyophirapong s. pseudohyperkalemia caused by edta contamination: a not uncommon pre-analytical error. siriraj med j. 2019;71(4):322–323. https://doi.org/10.33192/smj.2019.49 borkotokey m, dutta k. spurious hyperkalemia due to edta contamination: commonly overlooked and potentially dangerous. jmscr. 2020;8(11):150–151. https://dx.doi.org/10.18535/jmscr/v8i11.26 ercan ş, ramadan b, gerenli o. order of draw of blood samples affect potassium results without k-edta contamination during routine workflow. biochem med. 2021;31(2):267–275. https://doi.org/10.11613/bm.2021.020704 introduction factors contributing to the rise of antimicrobial resistance economic burden of antimicrobial resistance limited availability of new antimicrobials laboratories’ role in antimicrobial resistance conquering antimicrobial resistance: from global priority to your priority conclusion references about the author(s) rajiv t. erasmus department of biomedical sciences, cape peninsula university, cape town, south africa division of chemical pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa chikwelu l. obi school of science and technology, sefako magkatho health sciences university, pretoria, south africa sajini souda department of pathology, university of botswana, gaborone, botswana citation erasmus rt, obi cl, souda s. the scourge of antimicrobial resistance: containing a global crisis. afr j lab med. 2024;13(1), a2645. https://doi.org/10.4102/ajlm.v13i1.2645 editorial the scourge of antimicrobial resistance: containing a global crisis rajiv t. erasmus, chikwelu l. obi, sajini souda copyright: © 2024. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction with the introduction of antibiotics in the 1940s, infectious diseases, which had dominated the field of healthcare for centuries, became treatable and deaths preventable. but today, antibiotic use is being threatened by antimicrobial resistance (amr) – bacteria, viruses, fungi, and parasites that are difficult to defeat using available antibiotics. antimicrobial resistance is a normal process that occurs progressively due to genetic changes in pathogens, driven primarily by the misuse and overuse of antimicrobials in humans, animals and plants.1 antimicrobial resistance has become a global threat. in the first comprehensive assessment of the global burden of amr from 1990 to 2021, it was estimated that 4.71 million (95% uncertainty interval: 4.23–5.19) deaths were associated with bacterial amr, including 1.14 million (95% uncertainty interval: 1.00–1.28) deaths attributable to bacterial amr.2 worldwide, antibiotic use increased by 65% between 2000 and 2015, largely associated with overconsumption among developing countries with rising incomes. while the highest antibiotic consumption rates in 2000 were in the united states, france, spain, new zealand, and hong kong, by 2015, the four top consumers of antibiotics were in lowto middle-income countries, such as turkey, tunisia, algeria, and romania.3 in the global antimicrobial resistance and use surveillance system (glass) 2022 report that highlighted bacterial pathogen resistance rates, the median stated rates in 76 countries were 42% for third-generation cephalosporin-resistant escherichia coli and 35% for methicillin-resistant staphylococcus aureus.4 this is concerning illustratively, when considering that in urinary tract infections caused by e. coli, 1 in 5 cases exhibited reduced susceptibility to standard antibiotics such as ampicillin, co-trimoxazole, and fluoroquinolones in 2020.4 africa will be home to 2.5 billion people by 2050.5 the united nations environment program6 estimated that 10 million people will die of amr-related causes across the world, and the world health organization reported that 4.1 million of these people will reside in africa.7 according to abubakar and salman,8 the prevalence of antibiotic use among hospitalised patients in africa varied from 27.6% to 83.5%, with the greater prevalence across west africa (51.4% – 83.5%) and north africa (79.1%) when compared to east africa (27.6% – 73.7%) and south africa (33.6% – 49.7%). factors contributing to the rise of antimicrobial resistance antibiotics are not just used to counter infections in humans. they are also used to maintain the health of animals and crops. the primary cause of amr in humans is the inappropriate use of antimicrobials. research shows that 30% of all antibiotics prescribed in united states acute-care hospitals are either unnecessary or inappropriate.9 additionally, antibiotics are prescribed in doctor’s offices and emergency departments for infections that do not need them (e.g. colds and influenza). antibiotic resistance also spreads easily across the globe today, due to the ease and affordability of travel. the united states centers for disease control prevention reports that 1 billion people travel across international borders each year, and 350 million travellers arrive in the united states through more than 300 ports of entry.10 a resistance threat from anywhere could spread quickly simply by people travelling across borders. the increase in amr worldwide is also caused by animal and environmental contributors. antibiotics are regularly added to animal feed and drinking water to prevent sickness. increases in amr will make treating infections in animals more difficult and cause animal infections to be more severe,3 with ripple effects on zoonotic diseases. economic burden of antimicrobial resistance the world bank group’s final report on drug-resistant infections of 2017 indicated that amr was more damaging in low-income countries, where the gross domestic product may be eroded by more than 5% of the base case.11 in an optimistic low-amr setting, the modelled losses of global output may exceed $1 trillion per year and, after 2030, may reach $2 trillion annually by 2050.11 the report also highlights that this may imply a reduction in other aspects of population well-being, because resources that could have been devoted to reducing poverty or other health-related challenges may need to be re-routed to financing the additional costs of the increased burden of disease. antimicrobial resistance doesn’t just contribute to healthcare costs – it also impacts levels of productivity. the united states centers for disease control prevention estimates that annual losses in productivity will cost the united states $35 billion a year.3 in low-income countries, amr not only reduces productivity levels but also shrinks workforces because of associated sickness and premature death.3 challenges, including regular access to clean water and good sanitation, exacerbated by poverty, coupled with the endemicity of hiv/aids, enhance the risk of infection and subsequent amr,12 with coronavirus disease 2019 further compromising healthcare infrastructures. the high rates of resistance to commonly prescribed and dispensed antibiotics across sub-saharan africa are further worsened by high rates of inappropriate prescribing and dispensing of antimicrobials, weak diagnostic capabilities, variable implementation of regulations concerning the dispensing of antimicrobials without a prescription, and variable access to effective healthcare.12 other compounding factors that add to the challenges of rising amr rates in sub-saharan africa include the availability of substandard or falsified or fake antibiotics. this arises from currently weak regulatory systems, limited local manufacturing, and inadequate quality assurance testing of antimicrobials, as well as concerns with available professionals and co-operation between professional groups.12 some traditional diagnostics, such as growing cultures to identify infectious pathogens, can take too much time. as a result, clinicians may be forced to prescribe antimicrobials before test results are completed, which can lead to overuse and misuse of antibiotics. limited availability of new antimicrobials the development of new antimicrobial treatments has dropped dramatically since the 1990s and is reaching a crisis stage today. according to the world health organization, there were only 27 new antibiotics for priority pathogens in clinical development in 2021, down from 31 in 2017.13 in 2019, the pew charitable trusts14 documented worldwide efforts to develop new antibiotics. they identified 43 antibiotics in development: four with new applications, 19 that could treat infections caused by some gram-negative bacteria, 10 each that could address urgent threats from gonorrhoea or clostridium difficile; only one in four of these was a novel drug.14 new drug development faces daunting hurdles, including lengthy pathways to approval, high costs of development, and low success rates. to address the current amr crisis, developing a more robust pipeline of new antimicrobials, especially from medicinal plants and animals, must become a priority. laboratories’ role in antimicrobial resistance microbiology laboratories provide reliable systems, equipment, and technologies to obtain meaningful data that can be used to detect and monitor infections, amr, and the efficacy of different antibiotic treatment options. microbiology and virology laboratories play critical roles in reporting susceptibility studies, which are important in assessing the susceptibility of pathogens to specific antibiotics. additionally, they support the detection of changes in antibiotic resistance over time. microbiologists can assist clinicians in providing diagnostic stewardship through identifying specific infections, interpreting test results, and guiding antibiotic treatment. they also assist in identifying susceptibility patterns and conducting research to expand advanced and rapid diagnostic testing methods, as well as contributing to data collection and examining new evidence for the appropriate use of antibiotics. by researching novel antimicrobial compounds, laboratory professionals assist in expanding the antibiotic pipeline conquering antimicrobial resistance: from global priority to your priority stopping the spread of amr demands aggressive actions to prevent or minimise infections worldwide. measures may include improved antibiotic use, antibiotic prescription, monitoring of amr genes, uncovering new amr genes, and the use of antimicrobials in farm animals. efforts over the past decade have shown that the disciplined, multidisciplinary approach that characterises antimicrobial stewardship can drive measurable improvements. infection prevention and control, vaccinations, and environmental interventions also contribute to tackling amr. but success depends on: a relentless commitment to action from healthcare leadership high levels of engagement by a wide variety of healthcare clinicians and professionals data collection and sharing on a local, national, and international basis continuous creation and refinement of evidence-based best practices collaboration between healthcare providers and public health officials extensive education and training within healthcare, across the community timely development of novel diagnostic tools and new antibiotic options. monitoring of antibiotic use, surveillance, awareness and education, community efforts, developing tools for detection and monitoring, as well as developing dashboards, are strategies that can be used to contain amr. rapid diagnostics offer quicker turn-around times and greater accuracy, but as new technologies, they are more expensive and require training and mastery for the effective interpretation of results. vaccines are also a key preventative measure to limit future infectious diseases and any subsequent inappropriate antimicrobial use with implications for the development of amr. vaccines are also less likely to induce resistance.12 each healthcare professional must accept responsibility, underpinned by commitment, to tackle the menace of amr. conclusion tackling the challenges posed by amr requires a multi-pronged approach, including commitment of healthcare professionals, education, and awareness campaigns on the drawbacks of the indiscriminate use of antibiotics in humans, animals and plants, inappropriate antimicrobial prescribing by healthcare workers, and monitoring of the genes coding for amr. the concept of ‘one health’ may be threatened by the ripple effects of amr. adequate budgetary allocation for the clinical management of amr, research on development of new antimicrobials, and laboratory diagnostics or investigations of the array of factors responsible for amr, including sentinel surveillance, are critical imperatives in addressing the conundrum of amr. references world health organization. anti-microbial resistance, key facts [homepage on the internet]. 2023 [cited 2024 aug 31]. available from: https://www.who.int/news-room/fact-sheets/detail/antimicrobial-resistance#:~:text=amr%20is%20a%20natural%20process,in%20humans%2c%20animals%20and%20plants antimicrobial resistance collaborators. global burden of bacterial antimicrobial resistance 1990–2021: a systematic analysis with forecasts to 2050. lancet. 2024;404(10459):1199–1226. https://doi.org/10.1016/s0140-6736(24)01867-1 dadgostar p. antimicrobial resistance: implications and costs. infect drug resist. 2019;12:3902–3910. https://doi.org/10.2147/idr.s234610 world health organization. global antimicrobial resistance and use surveillance system (glass) report: 2022. geneva: world health organization; 2022. statista. forecast of the total population of africa from 2020 to 2050 [homepage on the internet]. 2024 [cited 2024 aug 30]. available from: https://www.statista.com/statistics/1224205/forecast-of-the-total-population-of-africa/ united nations environmental program. bracing for superbugs: strengthening environmental action in the one health response to antimicrobial resistance [homepage on the internet]. 2023 [cited 2024 aug 30]. available from: https://www.unep.org/resources/superbugs/environmental-action world health organization. african region, health topics [homepage on the internet]. 2024 [cited 2024 aug 30]. available from: https://www.afro.who.int/resistamr abubakar u, salman m. antibiotic use among hospitalised patients in africa: a systematic review of point prevalence studies. j rac ethn health disparities. 2024;11(3):1309–1329. https://doi.org/10.1007/s40165-023-01610-9 centre for disease control prevention. core elements of hospital antibiotics stewardship programs. atlanta, ga: us department of health and human services, cdc; 2019. available at https://www.cdc.gov/antibiotic-use/core-elements/hospital.html. centre for disease control prevention. antibiotic resistance threats in the united states, 2019. atlanta, ga: us department of health and human services, cdc; 2019. the world bank. final report – drug-resistant infections. a threat to our economic future [homepage on the internet]. 2017 [cited 2022 jun 05]. available from: https://documents1.worldbank.org/curated/pt/323311493396993758/pdf/final-report.pdf godman b, egwuenu a, wesangula e, et al. tackling antimicrobial resistance across sub-saharan africa: current challenges and implications for the future. exp opin drug saf. 2022;21(8):1089–1111. https://doi.org/10.1080/14740338.2022.2106368 world bank. by 2050, drug-resistant infections could cause global economic damage on par with 2008 financial crisis. press release. 2016; sept 20. pew charitable trusts. tracking the global pipeline of antibiotics in development. 2019 [cited n.d.]. available from: https://www.pewtrusts.org/en/research-and-analysis/issue-briefs/2019/09/tracking-the-global-pipeline-of-antibiotics-in-development#:~:text=as%20of%20december%202019%3a,phase%203%20will%20be%20approved. abstract introduction methods results discussion acknowledgements references about the author(s) oluwatoyin a. babalola institute for advanced medical research and training, college of medicine, university of ibadan, ibadan, nigeria ayodele ogunkeyede department of surgery, university of ilorin teaching hospital, ilorin, kwara state, nigeria abayomi b. odetunde institute for advanced medical research and training, college of medicine, university of ibadan, ibadan, nigeria foluke fasola department of haematology, college of medicine, university of ibadan, ibadan, nigeria anthony a. oni † department of medical microbiology, college of medicine, university of ibadan, ibadan, nigeria chinedum p. babalola institute for advanced medical research and training, college of medicine, university of ibadan, ibadan, nigeria sickle cell hope alive foundation (schaf), nigeria adeyinka g. falusi institute for advanced medical research and training, college of medicine, university of ibadan, ibadan, nigeria sickle cell hope alive foundation (schaf), nigeria citation babalola oa, ogunkeyede a, odetunde ab, et al. haematological indices of sickle cell patients with chronic leg ulcers on compression therapy. afr j lab med. 2020;9(1), a1037. https://doi.org/10.4102/ajlm.v9i1.1037 note: †1955–2015. original research haematological indices of sickle cell patients with chronic leg ulcers on compression therapy oluwatoyin a. babalola, ayodele ogunkeyede, abayomi b. odetunde, foluke fasola, anthony a. oni, chinedum p. babalola, adeyinka g. falusi received: 23 apr. 2019; accepted: 23 sept. 2020; published: 21 dec. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: recurrent chronic leg ulcers and its are morbidities associated with sickle cell anaemia (sca). compression therapy increases the rate of healing of these ulcers and also decreases the rate of recurrence. objective: this study evaluated the haematological parameters of patients with sca and chronic leg ulcers placed on high compression bandaging to provide data for improved ulcer management and prevention. methods: eighteen patients with sca and chronic leg ulcers were recruited for treatment by compression therapy in ibadan, nigeria, from march to june 2015. eighteen sca patients with no history of chronic leg ulcers were age and sex matched and recruited as controls. blood samples, wound biopsies and swabs were collected at different time points for full blood count, microbiology, culture and antimicrobial susceptibility tests. haemoglobin variants were quantified by high performance liquid chromatography. fasting blood sugar was tested for leg ulcer patients to determine diabetic status. results: ulcers ranged from 0.5 cm2 to 416 cm2 (median: 38.4 cm2). post-intervention ulcer size ranged from 0.0 cm2 to 272 cm2 (median: 18.6 cm2, p < 0.001); four ulcers completely healed. compared to the control group, haematological indices at commencement of treatment were more severe in leg ulcer patients (p = 0.02). no patients with chronic leg ulcer was diabetic. microorganisms isolated from the leg ulcers include pseudomonas aeruginosa, staphylococcus aureus, proteus sp., escherichia coli and klebsiella oxytoca. conclusion: measures to improve haematological parameters during leg ulcer treatment in sca patients should be taken to aid wound healing. keywords: chronic leg ulcer; sickle cell anaemia; wound healing; haematological parameters; compression therapy. introduction chronic leg ulcer is one of the morbidities associated with sickle cell anaemia (sca). chronic leg ulcer is a wound on the skin that occurs between the knee and the foot, showing no signs of healing after 3 months of proper treatment.1 in sca patients, the polymerisation of haemoglobin s (hbs) leads to the formation of sickled red blood cells (rbcs) which become trapped in the microcirculatory system. this leads to microvascular vaso-occlusion causing tissue ischaemia and associated pain. occlusion of the veins in the legs leads to venous hypertension, which may cause varicose eczema, deposition of scar tissue and iron pigments in the skin, and oedema in the lower leg. this may result in the breakdown of the skin or delay healing in the case of a leg injury, leading to chronicity.2 any leg ulcer in the presence of a venous disorder is described as a venous ulcer. other considerations for venous ulcer diagnosis are an ankle brachial pressure index greater than 0.8 with absence of features of rheumatoid vasculitis, malignancies and diabetic mellitus. the gold standard in the management of chronic venous leg ulcers is the four-layered compression therapy bandaging system. sickle cell associated ulcers (scus) can be precipitated by many factors such as trauma, infection, severe anaemia and usually occur with venous stasis disease, which requires compression therapy.2 the microorganisms invading the tissues of the skin contribute to the delayed healing observed in chronic leg ulcers.3 bacteria produce and also stimulate the production of substances (from activated neutrophils) that are detrimental to the body’s wound healing processes.4,5 these substances include proteases such as metalloproteases, elastases and collagenases that destroy the building blocks needed for normal wound healing. in this proteolytic environment, bacteria can continue to multiply and use up local resources such as oxygen and nutrients that are also needed for the wound healing.4 diabetes mellitus increases the risk for the development of infection and impaired wound healing in individuals due to a decreased cell and growth factor response.6 the effect of diabetes mellitus in scus is yet to be assessed. ulcer incidence in sickle cell disease (scd) patients in the united states ranges from 2.5% to 25% and 75% in jamaica.7,8 in nigeria and ghana, it is about 10%,9,10 and the rate of recurrence of ulcers is high (> 70%).9,11,12 chronic leg ulcer thus contributes to recurrent and prolonged misery in these patients. compression therapy increases the rate of healing of scus and also reduces the rate of recurrence.7,8,9 there is inadequate education on the prevention and management of leg ulcers among people living with scd and their caregivers. the therapeutic strategy for scus is complex and multidisciplinary efforts to offer holistic care is poor. this study is therefore designed to address the paucity of information about the laboratory markers of scus among people living with scd and also serves as a basis for health promotion interventions for the prevention of scus and improvement in clinical interventions in nigeria and other african countries. methods ethical considerations this study was approved by the university of ibadan/university college hospital joint ethics review committee, college of medicine, university of ibadan; protocol identification number ui/ec/15/0133. sickle cell disease patients in need of leg ulcer treatment at the haematological clinics of the university college hospital and ring road state hospital, ibadan, voluntarily participated in this study. oral informed consent was obtained from each of the participants using a format explaining the details of the procedure, possible complications that may arise using the compression therapy technique for wound care and the importance of the biopsies and periodic blood sample collection. a code number was assigned to each patient and their samples to protect participants’ privacy. study design in a pilot hospital-based study, 18 sca patients with chronic leg ulcers were recruited from haematology clinics at university college hospital and ring road state hospital, ibadan, for treatment by compression therapy (first use case in nigeria) for a period of 3 months (13 weeks) between march 2015 and june 2015. there were 6 men and 12 women with ages ranging from 19 to 44 years. eighteen age and sex matched sca patients with no history of chronic leg ulcers were recruited as controls. sickle cell anaemia patients below 18 years as well as adult sca patients attending the named facilities who did not give informed consent were excluded from this study. those with chronic leg ulcers with an ankle brachial pressure index less than 0.8 were also excluded. a pre-tested semi-structured questionnaire was administered to each of the patients for a history of leg ulcer, family history and socio-demographic details. a four-layer high compression bandaging system described previously.13 was used in this study. dressing was done once a week for 13 weeks. sample collection and processing blood samples were collected before commencement of treatment (t0) from both groups. for the leg ulcer group, blood samples, wound swabs and tissue biopsies (in normal saline and in peptone) were also collected on the 5th week (t1) and 10th week (t2) of treatment. microscopy, culture and sensitivity tests were carried out on the wound biopsies and swabs. full blood count was carried out using a swelab haematology analyser (boule diagnostics, stockholm, sweden) and the level of haemoglobin variants was determined by high performance liquid chromatography using the biorad variant ii hemoglobin testing system (clinical diagnostics, hercules, california, united states). the patients were prescribed antibiotics based on the result of antibiotic sensitivity tests. a fasting blood sugar test was performed for the leg ulcer patients to determine their diabetic status. statistical analysis statistical analyses were done using ibm statistical package for social sciences version 23 (ibm corporation, armonk, new york, united states). the differences in haematological parameters between the control and test groups were determined by the student’s t-test. the paired t-test was used to evaluate changes in haematological parameters within chronic leg ulcer patients during the therapy. spearman’s rank correlation was used to determine relationships between different haematological parameters and between haematological indices and size of ulcers at t0. the level of statistical significance was set at p < 0.05. results description of patients, ulcers and healing rate eighteen patients with leg-ulcers and a similar number of ulcer-free controls were included in the study. there were 25 uclers in all as seven patients had bilateral ulcers. the ulcers ranged from 0.5 cm2 to 416 cm2 with a median ulcer size of 38.4 cm2. the ages of the ulcers were 1–22 years (with recurrence). within the 3 months of compression therapy, four of the patients had complete close-up of the ulcer wound, 16 of them had more than a 50% healing rate (the percentage of the size of wound closure to the initial wound size) and the remaining five had a healing rate of 35% – 50%. the post-intervention median ulcer size ranged from 0.0 cm2 to 272 cm2 with a median of 18.6 cm2 (p < 0.001). the full details of patients, ulcers and healing rates have been described elsewhere.12 full blood count analysis haematocrit (hct) at commencement of treatment was lower in the leg ulcer patients compared to the control group (19.11% vs 26.17%, p < 0.001). mean corpuscular volume (95.33 femtolitre vs 87.73 femtolitre, p = 0.02), mean corpuscular haemoglobin (mch) (30.82 picogram vs 28.46 picogram, p = 0.05), red cell distribution width (27.78% vs 22.68%, p = 0.004), white blood cell count (wbc) (13.90 vs 8.70, p < 0.001) and platelet count (plt) (487.42 vs 299.08, p < 0.001) were higher in the leg ulcer patients (table 1). in the sickle cell patients, hct at t2 was significantly higher than at t0 (p = 0.04). there was an improvement in most of the haematological indices from t0 to t2, but that between t0 and t1 was more profound than t1 to t2 (table 1). elevated wbc was more prevalent in leg ulcer patients (66.7%) at t0 compared to the control group (11.1%) p < 0.001. table 1: mean values for full blood count parameters across the different time periods in adult sickle cell anaemia patients with (cases) and without (controls) chronic leg ulcers at the university college hospital and ring road state hospital, ibadan, nigeria, march–june 2015. there was a positive correlation between wound size and wbc (0.538, p = 0.01, figure 1a) and also between wound size and plt (0.418, p = 0.04, figure 1b). there was a poor correlation between wound size and haemoglobin (hgb) (0.024, p = 0.46, figure 1c). the leg ulcer patient with the highest hgb (8.2 g/dl) in this study test group had the biggest ulcer (416 cm2). on exclusion of this patient, the positive correlation between wound size and wbc became r = 0.473, p = 0.03 (figure 1d) and that between wound size and plt became more significant at r = 0.618, p = 0.004 (figure 1e). however, that between wound size and hgb became r = -0.216, p = 0.20 (figure 1f). figure 1: correlation between wound size and some haematological parameters at t0 in adult sickle cell anaemia patients with chronic leg ulcers at the university college hospital and ring road state hospital, ibadan, nigeria, march–june 2015. correlation between wound size (cm2) and (a) wound size versus white blood cell count (103/µl), (b) wound size versus platelet count (103/µl) and (c) wound size versus haemoglobin concentration (g/dl) in all the leg ulcer patients. correlation between wound size (cm2) and (d) wound size versus white blood cell count (103/µl), (e) wound size versus platelet count (103/µl) and (f) wound size versus haemoglobin concentration (g/dl) excluding one leg ulcer patient with the highest haemoglobin concentration and biggest wound size. in the patients with chronic leg ulcers, there was a positive correlation between wbc and plt and negative correlations between wbc and hgb as well as between hgb and plt with and without exclusion of the patient with the biggest ulcer at t0. there was no correlation between any of these three parameters in the patients who had not experienced a chronic leg ulcer in their lifetime (i.e. the control group) (table 2). table 2: correlation between white blood cell count, platelet count and haemoglobin concentration at t0 in adult sickle cell anaemia patients with (cases) and without (controls) chronic leg ulcers at the university college hospital and ring road state hospital, ibadan, nigeria, march 2015 – june 2015. high performance liquid chromatography analysis the concentration of hbs was higher in the leg ulcer group (86.25% vs 82.87%, p = 0.03). the difference in the mean foetal haemoglobin (hbf) level between the leg ulcer group (6.01% ± 3.10%) and control group (7.49% ± 5.89%) was not statistically significant (p = 0.19) but four of the control patients and only one of the leg ulcer patients had a hbf level greater than 10%. microbial culture test bacteria isolated from the wound swabs and biopsies include pseudomonas aeruginosa, staphylococcus aureus, proteus mirabilis, escherichia coli and klebsiella oxytoca, with pseudomonas aeruginosa being the most prevalent (55% – 70%) across the different culture tests. fasting blood sugar test none of the leg ulcer patients was diabetic. results of the fasting blood sugar test for all the participants in the test group ranged from 64 mg/dl to 82 mg/dl. discussion this study was carried out to evaluate the haematological parameters in patients with sca and chronic leg ulcers, and also to assess the haematological changes during healing of these ulcers while being managed with high compression bandaging, an as yet unemployed method of chronic leg ulcer treatment in nigeria. there were marked differences in some haematological parameters between sca patients with and without chronic leg ulcers and haematological parameters improved with healing of the ulcers. monitoring of haematological parameters for improvement during treatment of chronic leg ulcers and after wound closure in sca patients will aid healing and might also reduce the rate of recurrence. the mean hgb and hct of the case patients (those with chronic leg ulcers) at t0 were significantly lower than those of the control patients (those who have never had a chronic leg ulcer). a high rate of haemolysis is one of the major causes of a reduced steady state haemoglobin in sickle cell patients. sickle cell anaemia patients with a lower steady state haemoglobin concentration are at a higher risk of developing chronic leg ulcer.14,15 according to ladizinski et al.,16 the inability of a wound to heal could be a result of an inadequate supply of blood or a very low haemoglobin level (< 8 g/dl). one of the causes of scus is lack of oxygen which may be attributable to overall anaemia and reduced oxygen-carrying capacity of sickled haemoglobin.8 the addition of recombinant human erythropoietin (a glycoprotein hormone that stimulates red blood cell production) subcutaneously at weekly intervals resulted in a rapid and complete healing of a chronic leg ulcer.17 hence, treatment of chronic leg ulcers should aim at anaemia correction. however, this should be done with caution and within a threshold that will be beneficial to the patient as a much higher hct may also lead to hyperviscosity and increase the risk of vaso-occlusion. a wider red cell distribution width as compared to the control group, with a mean corpuscular volume within normal haematological range, in the test patients alludes to anaemia caused by bleeding from regular treatments, folate deficiency or vitamin b12 deficiency. there was a slight improvement in the hct level from t0 to t2 mainly due to constant education of the patients on the need to improve the quality of their diet and better compliance with routine haematinics. nolan et al.14 and hassan et al.18 found that patients with leg ulcers had higher wbcs as is the case in this study. the majority of the patients with leg ulcers had elevated wbc counts at the beginning of the study. elevated wbc, which is due to recurrent inflammation and vasculopathy, is one of the hallmarks of disease severity.19,20,21 patients with elevated wbc may be more susceptible to developing chronic leg ulcer because leukocytes stick to other blood cells and the endothelium due to an upregulation of adhesion molecules, thereby promoting vaso-occlusion.22 vaso-occlusion of the veins in the leg may cause tissue ischaemia with a resultant breakdown of the skin or delayed healing of wounds, leading to chronicity.2 higher wbc may also be due to microbial colonisation or infection of the ulcers. a lower wbc count was observed at t2 and this may be due in part to the use of antibiotics prescribed (based on the result of an antibiotic sensitivity test) a week before t1 sampling. infections that are not resolved may lead to the non-healing of wounds.16 there is a the need to control infections to promote wound closure and healing. in this study, wound size was positively correlated with wbc. therefore, efforts to reduce elevated wbc in leg ulcer treatment may aid wound closure. there was a significantly higher number of platelets in the leg ulcer patients than in the control group of this study. in jamaica, cumming et al.23 found that leg ulceration was associated with higher plt. hypercoagulability can trigger leg ulcer formation as it may cause ischaemia to the skin, resulting in friability and ulceration.24 scus are characterised partly by an increase in clotting ability as a result of increased plt, hypercoagulability and a measured increase in clotting factors at the wound itself.7 in this study, plt was also positively correlated with wound size. wirth et al.25 asserted that an efficient decrease in plt may lead to healing of skin ulcers. there was a negative correlation between hgb and wbc as well as between hgb and plt. this means a higher rate of haemolysis may result in elevated wbc and plt counts. the observation that these three parameters were not significantly correlated in patients without chronic leg ulcers lends credence to the notion that lower steady state hgb and elevated wbc and plt counts may predispose sickle cell patients to chronic leg ulcers. koshy et al.15 noted that there was a lower incidence of leg ulcers in scd patients with a hbf concentration higher than 10%. in jamaica, cumming et al.23 also found that ulceration was negatively associated with foetal haemoglobin levels. in this study however, the difference in hbf levels between the two study groups was not statistically significant, probably due to the small sample size. the presence of hbf may help to reduce the rate of haemolysis by reducing sickling potential. having hbf within the cell reduces its ability to polymerise due to the formation, in hbs and hbf mixtures, of asymmetrical hybrid tetramers of the type α2βsγ. the contact sites of hb βs for polymerisation is abolished, and hence prevents sickling26 and this may also reduce subsequent inflammatory reactions. however, elevated hbf levels may not always be protective as the pancellular distribution, rather than the concentration of hbf in the haemolysate is more effective in inhibiting polymerisation.27 the level of hbs was lower in the control patients. a lower hbs level is usually due to higher hbf levels or the presence of alpha or beta thalassemia which was not assayed in this study. reduced hbs concentration leads to a lower rate of sickling and, consequently, a reduced rate of haemolysis. haemolysis of sickled rbcs leads to the release of activated molecules (plasma haemoglobin) which react with nitric oxide to form inert nitrate. nitric oxide maintains vascular function and normal blood flow28,29 and has also been shown to be vital to cutaneous wound healing.30,31 nitric oxide suppresses aggregation of platelets, secretion of procoagulant proteins and the expression of cell adhesion molecules on endothelial cells, thereby promoting blood flow.28 hence, improvement in leg ulcer management may be achieved by the introduction of agents that have the capacity to reduce sickle erythrocyte density as well as those that can make nitric oxide more available in tissues. none of the chronic leg ulcer patients in this study was diabetic. diabetes is quite rare in sickle cell patients6,32,33 and does not have anything to do with the non-healing ulcers of patients in this study. venous incompetence leads to reduced blood flow, and may cause increased blood vessel permeability with the attendant leaking of fluid and proteins into tissue spaces, a condition known as oedema.34 localised oedema traps leukocytes in the tissues, and oxygen-free radicals and harmful substances released by the leukocytes are capable of causing tissue damage, which may eventually lead to the development of ulceration. blood clotting factors are also triggered in reduced blood flow.35,36 according to clare et al.,37 ‘hypoxia-induced sickling, the rheological effects of white cell counts, and activation of components of the coagulation system may all contribute’ to venous incompetence. this explains why reduced hct, elevated plt and wbc may increase the risk of leg ulceration in individuals with scd. compression therapy aids healing of leg ulcers by increasing venous blood flow,38 thereby making more readily available the supply of adequate nutrients and other factors necessary for wound healing. on the other hand, the risk of blood clot formation and oedema is reduced, thereby promoting healing. however, there may be a recurrence of ulcers if haematological conditions are not monitored and addressed. this was the case for 3 of the previously healed participants in this study. management and prevention of chronic leg ulcer in scd patients should address underlying haematological indices as no one method of dressing may totally prevent recurrence.39,40 the wearing of compression stockings after wound healing may help to maintain normal venous pressure and prevent recurrence due to skin tissue breakdown.41,42 recommendations chronic leg ulcer is multifactorial and hence, in order to make the correct diagnosis and give the most effective treatment, health practitioners should adopt a multidisciplinary approach to a systematic patient evaluation to determine the ulcer pathogenesis.34 laboratory investigations to monitor haematological indices and to reveal any underlying infection should be incorporated in the treatment and management modalities of chronic leg ulcer in nigeria. agents that will reduce the rate of sickle red blood cell destruction and cell adherence to endothelial venules should be explored by health practitioners in the prevention of the formation and recurrence of ulcers and also in its management. several chemical agents have been suggested and some are under clinical trials.43 some investigators44,45 have also shown that ascorbic acid favours nitric oxide production in endothelial cells by stabilising nitric oxide synthase cofactor tetrahydrobiopterin. it is therefore suggested that the inclusion of vitamin c at recommended doses be explored in the daily routine drugs for individuals living with scd for the prevention and management of chronic leg ulcers and other haemolysis-related sickle cell phenotypes. limitations one limitation of this study is the small sample size. the high cost of compression therapy was the major determinant of the sample size. laboratory markers such as elevated serum lactose dehydrogenase, reticulocyte count, bilirubin level and some genetic factors which were not assayed in this study have also been associated with a risk of chronic leg ulcers in patients with scd,7,14 assaying for all these factors in a larger cohort of patients may help to identify other laboratory markers of chronic leg ulcer susceptibility. conclusion sickle cell patients with a lower level of hgb and elevated wbc and plt counts at steady state may be at a higher risk of non-healing leg ulcers. most of the haematological indices improved with healing of the leg ulcers placed on high compression bandaging. an interdisciplinary approach to provide a favourable microenvironment for healing of ulcers, and the monitoring of haematological parameters for the prevention of recurrence, is recommended. acknowledgements sickle cell hope alive foundation (schaf) – or the recruitment of patients and provision of funds for this study. the members of staff of haematology department, ring road state hospital, ibadan, who cooperated and contributed their time to make this study possible. the patients who cooperated and endured the pain of biopsies and treatment. medical education partnership initiative in nigeria (national institute of health) – for providing guidance in the data analysis and write-up of this work. competing interests the authors have declared that no competing interests exist. authors’ contributions o.a.b. was involved in the design, planning, investigation and data analysis of the study and the manuscript write-up. a.o. was involved in the design, planning and investigation. a.b.o. and c.p.b. assisted with the planning and implementation of the study. f.f. revised the manuscript. a.a.o. assisted with the microbiology investigation. a.g.f. was involved in the study design and planning, and assisted with secondary level facility access, project coordination and manuscript revision. sources of support funding was provided by the sickle cell hope alive foundation (schaf). data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do 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schneider jm, kraaus ap, kitabchi ae. the prevalence of diabetes mellitus in sickle haemoglobinopathies. j clin endocrinol metab. 1979;48(2):192–195. https://doi.org/10.1210/jcem-48-2-192 jarrett oo, olorundare ei. type 1 diabetes mellitus in a known sickle cell anaemia patient: a rare combination in nigeria. afr j med sci. 2014;43(2):177–181. partsch h. understanding the pathophysiological effects of compression. in: s calne (ed). ewma position document; understanding compression therapy. london: mep ltd; 2003. p. 2. agale sv. chronic leg ulcers: epidemiology, aetiopathogenesis, and management. ulcers. 2013;2013:413604. https://doi.org/10.1155/2013/413604 shami sk, shields da, scurr jh, coleridge smith pd. leg ulceration in venous disease. postgrad med j. 1992;68(804):779–785. https://doi.org/10.1136/pgmj.68.804.779 clare a, fitzhenley m, harris j, serjeant gr. chronic leg ulceration in homozygous sickle cell disease: the role of venous incompetence. br j haematol. 2002;119(2):567–571. https://doi.org/10.1046/j.1365-2141.2002.03833.x cullum na, nelson ea, fletcher a, sheldon t. compression for venous leg ulcers. cochraine database syst rev. 2001;(2):cd000265. https://doi.org/10.1002/14651858.cd000265 el khatib am, hayek sn. leg ulcers in sickle cell patients: management challenges. chronic wound care manage res. 2016;(3):157–161. https://doi.org/10.2147/cwcmr.s85455 o’donnell tf, passman ma, marston wa, et al. management of venous leg ulcers: clinical practice guidelines of the society for vascular surgery and the american venous forum. j vasc surg. 2014;60(2):3s–59s. https://doi.org/10.1016/j.jvs.2014.04.049 nelson ea, bell-syer sem. compression for preventing recurrence of venous ulcers. cochrane database syst rev. 2014;(9):cd002303. https://doi.org/10.1002/14651858.cd002303.pub3 anderson i. compression hosiery to reduce leg ulcer recurrence. nurs times. 2013;109(6):18–20. kapoor s, little ja, pecker lh. advances in the treatment of sickle cell disease. mayo clin proc. 2018;93(12):1810–1824. https://doi.org/10.1016/j.mayocp.2018.08.001 mortensen a, lykkesfeldt j. does vitamin c enhance nitric oxide bioavailability in a tetrahydrobiopterin-dependent manner? in vitro, in vivo and clinical studies. nitric oxide. 2014;36:51–57. https://doi.org/10.1016/j.niox.2013.12.001 parker wm, qu z, may jm. intracellular ascorbate prevents endothelial barrier permeabilization by thrombin. j biol chem. 2015;290(35):21486–21497. https://doi.org/10.1074/jbc.m115.662098 abstract background description of the intervention lessons learnt recommendations acknowledgements references about the author(s) augustine o. mpamugo center for international health, education and biosecurity, maryland global initiatives corporation, university of maryland, baltimore (umb), abuja, nigeria nnaemeka c. iriemenam division of global hiv and tb, united states centers for disease control and prevention, abuja, nigeria adebobola bashorun national aids, viral hepatitis, and stis control programme, federal ministry of health, abuja, nigeria olumide o. okunoye division of global hiv and tb, united states centers for disease control and prevention, abuja, nigeria orji o. bassey division of global hiv and tb, united states centers for disease control and prevention, abuja, nigeria edewede onokevbagbe center for international health, education and biosecurity, maryland global initiatives corporation, university of maryland, baltimore (umb), abuja, nigeria tapdiyel jelpe division of global hiv and tb, united states centers for disease control and prevention, abuja, nigeria matthias a. alagi division of global hiv and tb, united states centers for disease control and prevention, abuja, nigeria chidozie meribe division of global hiv and tb, united states centers for disease control and prevention, abuja, nigeria rose e. aguolu department of research monitoring and evaluation, national agency for the control of aids, abuja, nigeria charles e. nzelu department of planning, research and statistics, federal ministry of health, abuja, nigeria segun bello center for international health, education and biosecurity, maryland global initiatives corporation, university of maryland, baltimore (umb), abuja, nigeria department of epidemiology and medical statistics, faculty of public health, college of medicine, university of ibadan, ibadan, nigeria babatunde ezra center for international health, education and biosecurity, maryland global initiatives corporation, university of maryland, baltimore (umb), abuja, nigeria christine a. obioha center for international health, education and biosecurity, maryland global initiatives corporation, university of maryland, baltimore (umb), abuja, nigeria baffa s. ibrahim center for international health, education and biosecurity, maryland global initiatives corporation, university of maryland, baltimore (umb), abuja, nigeria oluwasanmi adedokun center for international health, education and biosecurity, maryland global initiatives corporation, university of maryland, baltimore (umb), abuja, nigeria akudo ikpeazu national aids, viral hepatitis, and stis control programme, federal ministry of health, abuja, nigeria chikwe ihekweazu nigeria centers for disease control, abuja, nigeria talishiea croxton institute of human virology, university of maryland school of medicine, baltimore, maryland, united states sylvia b. adebajo institute of human virology, university of maryland school of medicine, baltimore, maryland, united states mcpaul i.j. okoye division of global hiv and tb, united states centers for disease control and prevention, abuja, nigeria alash’le abimiku institute of human virology, university of maryland school of medicine, baltimore, maryland, united states citation mpamugo ao, iriemenam nc, bashorun a, et al. lessons learnt from assessing and improving accuracy and positive predictive value of the national hiv testing algorithm in nigeria. afr j lab med. 2024;13(1), a2339. https://doi.org/10.4102/ajlm.v13i1.2339 note: additional supporting information may be found in the online version of this article as online supplementary document 1. lessons from the field lessons learnt from assessing and improving accuracy and positive predictive value of the national hiv testing algorithm in nigeria augustine o. mpamugo, nnaemeka c. iriemenam, adebobola bashorun, olumide o. okunoye, orji o. bassey, edewede onokevbagbe, tapdiyel jelpe, matthias a. alagi, chidozie meribe, rose e. aguolu, charles e. nzelu, segun bello, babatunde ezra, christine a. obioha, baffa s. ibrahim, oluwasanmi adedokun, akudo ikpeazu, chikwe ihekweazu, talishiea croxton, sylvia b. adebajo, mcpaul i.j. okoye, alash’le abimiku received: 16 oct. 2023; accepted: 22 may 2024; published: 28 aug. 2024 copyright: © 2024. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: hiv testing remains an entry point into hiv care and treatment services. in 2007, nigeria adopted and implemented a two-test rapid hiv testing algorithm of three hiv rapid test kits, following the sequence: alere determine (first test), unigoldtm (second test), and stat-pak® as the tie-breaker. sub-analysis of the 2018 nigeria hiv/aids indicator and impact survey data showed significant discordance between the first and second tests, necessitating an evaluation of the algorithm. this manuscript highlights lessons learnt from that evaluation. intervention: a two-phased evaluation method was employed, including abstraction and analysis of retrospective hiv testing data from january 2017 to december 2019 from 24 selected sites supported by the united states president’s emergency plan for aids relief programme. a prospective evaluation of hiv testing was done among 2895 consecutively enrolled and consented adults, aged 15–64 years, accessing hiv testing services from three selected sites per state across the six geopolitical zones of nigeria between july 2020 and september 2020. the prospective evaluation was performed both in the field and at the national reference laboratory under controlled laboratory conditions. stakeholder engagements, strategic selection and training of study personnel, and integrated supportive supervision were employed to assure the quality of evaluation procedures and outcomes. lessons learnt: the algorithm showed higher sensitivity and specificity in the national reference laboratory compared with the field. the approaches to quality assurance were integral to the high-quality study outcomes. recommendations: we recommend comparison of testing algorithms under evaluation against a gold standard. what this study adds: this study provides context-specific considerations in using world health organization recommendations to evaluate the nigerian national hiv rapid testing algorithm. keywords: lessons learnt; hiv/aids; hiv rapid test; testing algorithm; nigeria. background hiv testing services (hts) remain an entry point into hiv care and treatment.1 hiv testing is the first pillar of the joint united nations program on hiv/aids (unaids) 95-95-95 cascade,2 which helps people living with hiv to access hiv prevention and treatment services. in addition, hiv testing supports programmatic efforts to reach the unaids target of 95% of people living with hiv becoming aware of their hiv status by 2030.3 as of 2020, nigeria had about 1.8 million people living with hiv, representing about 5% of the global burden and 7% of the african burden by the end of 2020.4 with the support of donors and implementing partners, in 2020, the government of nigeria made substantial progress towards attaining the unaids 95-95-95 targets needed to achieve hiv epidemic control by 2030. thus, by the end of 2020 in nigeria, 73% of people living with hiv were diagnosed, 89% of those diagnosed accessed hiv treatment, and 78% of those who accessed hiv treatment were virally suppressed.4 the adoption of the test and treat strategy5 for anti-retroviral therapy initiation underscores the importance of accurately diagnosing true positives. false positivity due to misdiagnosis will result in erroneously initiating such wrongfully diagnosed persons on lifelong anti-retroviral therapy, which would have significant emotional, financial, and psychosocial implications for the affected individuals.6,7,8 this places enormous responsibility on the country’s government to identify, adopt, and implement an appropriate hiv testing algorithm that complies with world health organization guidelines for hiv algorithm selection.9 validating the national hiv testing algorithm is crucial to determining its optimal performance across testing streams.10 before 2005, there was no formal evaluation of hiv rapid test kits for developing a national algorithm in nigeria.11 in 2006, in collaboration with the united states president’s emergency plan for aids relief (pepfar) programme, nigeria implemented and completed two phases of evaluation; the first was a laboratory-based evaluation that rated nine hiv rapid test kits with a specific focus on non-cold chain-dependent rapid test kits to suit both infrastructure and the varied national skilled level.11 the second was a field evaluation of the interim algorithm to assess the test performance in a non-controlled environment and interpretations of the test by non-laboratory personnel such as hiv counsellors. the world health organization had recommended using rapid hiv tests, since the conventional enzyme immunoassay and western blot constituted a challenge and reduced access to testing, especially in resource-limited nations.9 thus, the phased evaluations identified suitable algorithms based on combining two or more rapid tests with a diagnostic accuracy comparable to enzyme immunoassay and western blot strategies.11 based on the initial evaluation report in nigeria, three serial hiv testing algorithms were recommended for use in three combinations: (1) determinetm hiv 1/2 (abbott molecular inc., des plaines, illinois, united states), stat-pak® hiv 1/2 assay (chembio diagnostic systems inc., medford, new york, united states) and bunditm (bundi international diagnostics ltd, aba, abia state, nigeria); (2) unigoldtm (trinity biotech, plc., wicklow, ireland), stat-pak® (chembio diagnostic systems, inc. medford, new york, united states) and bunditm; and (3) determinetm, unigoldtm and stat-pak®.11 consequently, nigeria adopted a serial algorithm in 2007, which was a two-test algorithm comprising of alere determine hiv rapid test as the first test, unigoldtm hiv rapid test as the second test, and stat-pak® hiv rapid test as the tie-breaker.11,12 the two-test national hiv rapid testing algorithm was used during the 2018 nigeria aids indicator and impact survey to estimate the national hiv prevalence and incidence. the success of this survey gave a more accurate status of the national hiv prevalence in nigeria compared to previous surveys.10,13,14 however, a sub-analysis of 2018 nigeria aids indicator and impact survey data revealed a concordance rate of 56.6% between the first and second tests for hiv tests performed in the field, indicating that the nigeria hiv rapid testing algorithm may be performing suboptimally in these settings.10 this article presents lessons learnt from evaluating the performance of the national hiv testing algorithm in nigeria using a two-phase approach that (1) retrospectively analysed hiv testing programme data collected from january 2017 to december 2019 from routine hiv testing sites across the six geopolitical zones of nigeria, and (2) prospectively analysed collected samples from six geopolitical zones of nigeria. description of the intervention ethical considerations ethical clearance for this evaluation was obtained from the national health research ethics committee of nigeria (approval number 01/01/2007-22/05/20208) and the university of maryland baltimore institutional review board (approval number hm-hp-00091258-1). this project was reviewed in accordance with united states centers for disease control and prevention (cdc) (project id: 0900f3eb81b4a2a1, accession number cgc-ngra-5/14/20-4a2a1) human research protection procedures and was determined to be research, but united states cdc investigators did not interact with human subjects or have access to identifiable data or specimens for research purposes. setting nigeria has an estimated population of 220 544 164, based on the united nations world population prospects (2022 revision) estimate, and covers a land mass of 923 768 square kilometers.15 nigeria has 36 states, and a federal capital territory grouped into six geopolitical zones. study design a cross-sectional design was implemented. the evaluation was conducted in two phases: a prospective and a retrospective study. phase 1: retrospective evaluation the performance of the national hiv rapid testing algorithm was evaluated retrospectively by reviewing and analysing hiv testing programme data of de-identified patients from 24 purposively selected pepfar-supported sites in 12 states across the six geopolitical zones of nigeria. data were abstracted from the paper-based national daily hiv testing register by trained data clerks. the criteria for site selection included hiv prevalence levels of the states and hiv testing volume of at least 1000 tests per year (table 1). the period covered was january 2017 to december 2019. the abstracted data were entered directly into a custom-built electronic data collection platform. table 1: states selected for the retrospective phase of evaluation of hiv rapid testing algorithm in nigeria, july 2020 – september 2020. phase 2: prospective evaluation sampling – state and site selection: sampling for this evaluation was done in six states across the six geopolitical zones, consisting of one state per zone. the six states were purposively selected based on their prevalence levels (low, medium, and high) according to the 2018 nigeria aids indicator and impact survey report,16 as well as security and safety considerations. three hts sites per state per zone were selected based on-site selection criteria: availability of hts, low, medium, and high positive case yields, ease of transportation and shipments to the national reference laboratory (nrl), and security and safety consideration. in total, 18 hts sites were used for the evaluation (table 2). table 2: purposively selected states for the prospective phase of the evaluation of hiv rapid testing algorithm in nigeria, july 2020 – september 2020. sample size: the estimated sample size was 3000 (1800 negatives and 1200 positives), comprising 500 samples (300 negatives and 200 positives) per geopolitical zone. the actual sample collected was 2895, comprising 1817 negatives and 1078 positives, from participants aged 15 years – 64 years, who consented to and accessed hts services from july to september 2020. a 10 ml ethylenediaminetetraacetic acid vacutainer was used to collect blood samples from all participants. survey procedures: consenting survey participants were recruited, and capillary blood was drawn by finger sticks to perform the first test in the algorithm. when the first test was non-reactive, the participant was given a negative hiv result and escorted to the site’s laboratory for a venous whole blood collection into a 10 ml ethylenediaminetetraacetic acid tube. the same process was done for all hiv-negative results across the three sites until the targeted 300 hiv-negative samples were achieved for that state/geopolitical region. subsequent negative results from the consenting participants were excluded from the study. similarly, when the first test was reactive, the participant was also taken to the site’s laboratory, where whole venous blood was collected into a 10 ml ethylenediaminetetraacetic acid tube, and part of this sample was used to perform the second test. if the second was also reactive, the participant was confirmed hiv positive according to the national hiv testing algorithm. however, if the result of the second test was non-reactive, then a tie-breaker test was performed, and the result of the tie-breaker was taken as the final hiv test status of the participant. this process was conducted across the study sites for all participants who were reactive at the first test until the targeted 200 hiv positives per state/geopolitical zone were achieved. the remnants of the 10 ml blood samples were then separated by centrifugation (1500 × g for 10 min or ~3000 rpm) into plasma suspension, and the plasma was aliquoted into two cryo-tubes of about 1.5 ml each, and labelled with a pre-printed sticker containing de-identified patient identity codes comprising state, site, and alphanumeric codes. the codes were pre-printed as barcode labels to avoid documentation errors and improve tracking of sample shipments from the field to the nrl. the aliquots were packaged in cryo-boxes and shipped to the nrl in abuja in −20 °c credo boxes. all plasma samples received at nrl were stored at −86 °c ultra-low freezers, dw-hl 678s zhongke meiling (zhongke meiling cryogenic company limited, 1862 zishi road, hefei city, anhui, china) in the national biorepository unit until they were used for further testing according to the evaluation protocol. repeat testing using the same national hiv rapid testing algorithm was performed in a controlled laboratory environment by skilled laboratorians who were given refresher training before the testing using validated test kits. also, a confirmatory assay using the geenius™ hiv-1/2 supplemental assay (bio-rad laboratories. redmond, washington, united states) was done for all the hiv positives in the field and at the nrl; all discordant hiv results between the field (hiv-positive) and nrl (hiv-negative); as well as all discordant results between the first and second tests done at nrl. the accuracy and reproducibility of the national hiv rapid testing algorithm performed at the nrl were confirmed by a higher platform multiplex hiv-1/2 assay, luminex® magpix® (luminex corporation, austin, texas, united states). all the evaluation samples were further characterised using the multiplex bead assay technology (luminex corporation, austin, texas, united states). two laboratorians tested the samples (in duplicate) using the multiplex bead assay platform, and discordant results (between the testers) were also retested (in triplicate) for concurrent hiv diagnosis and serotyping. survey participants’ inclusion and exclusion criteria: clients aged 15 years – 64 years who sought hts services at the selected health facilities and consented to participate in the evaluation were included, while clients aged less than 15 years and above 64 years, as well as those who did not consent to participate, were excluded from the evaluation. quality assurance measures selection, training, and competency assessment of recruited personnel (quality assurance measure) proper selection and training of the field staff was one of the key measures to ensure the quality of the algorithm evaluation process. a hundred and three field staff were recruited for retrospective data abstraction, while eighteen experienced laboratory scientists were engaged as prospective phase data collectors and sample processing and shipment officers. due to the high impact of the coronavirus disease 2019 pandemic at the time of the study, nigeria imposed a national policy on physical meeting restrictions. this restriction resulted in an innovative cost-saving alternative model: a 5-day, remote virtual training, which did not compromise the quality of normal physical training. a total of 169 study personnel, including representatives from the government of nigeria, were trained. at the nrl, three different groups of competent testers were recruited and given separate refresher trainings: seventeen (17) laboratory scientists who had earlier participated in the hiv/aids national impact survey of 2018 and had obtained technical experience on the functionality of the geenius™ hiv-1/2 supplemental assay, five (5) laboratory professionals who were highly experienced in hiv serology rapid test, and two (2) laboratory professionals who were experienced with the multiplex assay. they were all required to achieve a 100% pass score in the competency assessment administered after the training to qualify as testers in the prospective phase of the survey at nrl. standardisation of laboratory processes in the field and at the national reference laboratory to further assure the quality of laboratory activities, a laboratory operational manual was developed, which contained all the processes and procedures required from pre-examination to post-examination phases, from sample management to result transmission into the evaluation database. this document was printed and disseminated to laboratory staff involved in the prospective phase of the evaluation across the evaluation sites to ensure standardisation of processes towards a quality output. supervision field supervision ensured that best practices were followed and the study protocol was strictly adhered to at all project implementation phases. two rounds of supervisory visits were conducted within 3 months during the field evaluation phase. all field supervisors comprising representatives from the implementing partners, national aids, viral hepatitis, and stis control programme (nascp), and national agency for the control of aids, were trained on field assessment before the visit. a checklist was developed and administered to assess compliance and adherence to the evaluation protocol. the site laboratory supervisor supervised on-site specimen collection across the selected sites for the prospective evaluation, per the protocol. the central technical supervisors (nascp, national agency for the control of aids, united states cdc, and university of maryland baltimore) monitored the field sites to ensure that consent forms were duly completed, to assure quality and volume of specimens collected, and to confirm storage conditions. specimen information was clearly and anonymously unlinked from patients’ identifiers. issues observed in the first visit, such as irregularities in data entries, overshooting of data entry targets for negative samples, and deficiencies in field sample storage facilities, were addressed before the second visit. the united states cdc team and/or other vital stakeholders provided daily and scheduled technical and operational oversight on testing quality to ensure the laboratory-controlled environment was met and the associated evaluation protocol-oriented guidelines were followed throughout the testing at the nrl. data collection test data for the retrospective and prospective evaluations were collected on mobile tablet devices using custom-built software (online supplementary figure 1, online supplementary figure 2, and online supplementary figure 3). each study participant was randomly assigned a unique identifier. all test data across the 12 states were transmitted and synchronised into a central server and dashboard using real-time monitoring and support systems. only designated personnel had access to the data. all personnel involved in data abstraction signed a data confidentiality agreement. data security (storage and ownership) the collected data were stored on password-protected tablets provided only to the data entry clerks throughout the evaluation. the tablet information was backed up daily. the final data set was maintained and stored in a secure server at the university of maryland baltimore central office. all data and reports belonged to the federal ministry of health. data analysis results from the field testing using the national rapid testing algorithm were compared with those from the laboratory testing at the nrl using the same algorithm. the positive predictive value and negative predictive value were calculated based on the reference testing results, using geenius™ as the confirmatory test and the test performed at the nrl as the reference or standard (table 3). table 3: a two-by-two table showing the level of agreement between test results and geenius™ in nigeria, july 2020 – september 2020. dissemination the evaluation outcome with a detailed report was shared with the government of nigeria (nascp, and national agency for the control of aids), pepfar, global fund, the national laboratory technical working group, and other critical in-country stakeholders, including multilateral organisations and implementing partners. appropriate approvals were obtained, and consent was sought from the co-investigators and the government of nigeria before study data were published. study findings have been published in a peer-reviewed journal.17 the evaluation findings were further disseminated to stakeholders (including multilateral partners, bilateral partners, and implementing partners) at a forum made available by nascp, to support a consensus with partners and stakeholders for a future policy decision. the evaluation complied with the pepfar evaluation standards of practice requirements on final report accessibility. lessons learnt the findings of the main evaluation study, which have been published in another study,17 show higher positive predictive value and better concordance rates between the first and second tester when the indices at the nrl under controlled conditions were compared against the field evaluations. this evaluation method has some distinguishing features from similar studies across sub-saharan africa. first, while similar evaluations focused commonly on the performance characteristics of individual test kits of an algorithm,11,18 this evaluation method focused on assessing the quality of testing in the entire testing strategy of the current algorithm used in nigeria. the benchmark for comparison was the algorithm performance at the nrl under controlled settings, such as using trained, competent personnel, controlled environmental temperature, quality-assured test kits, and adherence to testing protocols. the positive predictive values, algorithm agreement rates, and first/second test concordance rates across testing points were compared between the field and nrl. this study method aligns with methods used in other similar studies where performance characteristics of test kits were compared across different testing points, prevalence settings, and regional, environmental, and personnel factors in algorithm selection.19,20,21,22 this evaluation followed the national hiv rapid testing algorithm strictly. the two-test serial rapid testing algorithm was used at the field level and in a controlled laboratory environment to compare performance, resolve discordances between the two settings, and discordance between the firstand second-line tests. the use of geenius™ hiv-1/2 supplementary assay and hiv multiplex assay technology further confirmed the testing quality in both settings.17 cameroon used a similar method to evaluate its two-test national hiv testing algorithm at the field level and in a controlled laboratory setting with quality management systems. the cameroon national hiv testing algorithm also confirmed their field and laboratory testing using the geenius™ hiv-1/2 supplementary assay.23 stakeholder engagement proper stakeholder engagement is a foundational stone in any project planning and implementation success.24 before the commencement of the algorithm evaluation, key stakeholders were line-listed and consulted for their ownership and support toward the activity’s success. the united states cdc funded the project and provided technical and oversight functions; the government of nigeria, through its agencies (nascp and national agency for the control of aids), took ownership and provided leadership and control of project activities which gave the project unrestricted access to the government facilities through their chief medical directors and laboratory directors. involving the pepfar implementing partners overseeing each site ensured adequate technical personnel, laboratory infrastructures, and unrestricted access to the selected sites across the states. engaging the nigerian centers for disease control as an implementing partner in the evaluation provided unrestricted access to facilities and personnel at the nrl, as well as facilitating long-term storage of post-evaluation samples at the biorepository laboratory. the cumulative efforts of all stakeholders provided the synergy that produced a very successful evaluation. constitution of the team of field supervisors a well-composed field supervisory team that included government representatives from the national and state levels provided an acceptable and result-oriented supportive visit. a second supervisory visit to review the status of the recommendations on the non-conformities of the first visit was a quality improvement initiative that further impacted the quality of study processes and outcomes. also, the regular visits of the united states cdc technical team to the nrl to ensure compliance with the expected controlled conditions for testing at the nrl gave rise to a high-quality outcome in laboratory results from the three groups of testers. competency assessment: criteria for selection of testers competent and qualified personnel are critical to the laboratory quality management system.25 using competency assessment instead of preand post-tests in the selection process ensured that only competent personnel were recruited to conduct testing at nrl. this factor also significantly contributed to the quality of the evaluation process, such as a reduced number of repeat tests, minimal supervisor attention, and a high concordance rate between first and second tests. innovative approaches to training and study design furthermore, this study innovatively employed virtual training and concurrent competency assessment methodology in addition to regular virtual and/or supportive physical supervision, where possible, due to coronavirus disease 2019 -associated restrictions, to ensure high-quality and optimal testing service delivery at the field and laboratory levels during the survey-implementation period. the retrospective component of the study enabled the team to review the paper-based national daily hiv testing register for concurrence. in line with world health organization guidance on the country selection of suitable algorithm, this evaluation underscored the importance of periodic assessment of an entire algorithm under different settings and conditions beyond evaluating individual test kit performance.9 real-time data transmission and dashboard review a custom-built data-capturing mobile app was installed on tablet devices and distributed to field staff for real-time data capture and transmission to a central server. a dashboard was created that provided vital analytics on the daily efforts in the field, and access was given to the critical project management team members to review, track, and coordinate field performance remotely on a daily basis across the 12 states. this strategy provided motivation, proper direction, and redirection of field activities across the states, which resulted in achieving a 96.5% (2895) sample collection within the set time frame of less than 3 months.17 limitations the study methods did not include confirmatory testing for all the negative samples derived from the field and nrl using the geenius™ hiv-1/2 supplemental assay. due to cost constraints, only 10% of the negatives were subjected to confirmatory testing. testing all negatives will increase the probability of finding more false negatives and accuracy in determining the true specificity of the algorithm under evaluation. recommendations this study methods provide a reference study design for periodic assessment of the national hiv rapid testing algorithm’s performance under different settings and conditions, beyond evaluating the performance of individual test kits. in addition, we recommend that all samples tested in the field and the reference laboratory be confirmed using a gold standard to identify all true positives and negatives. acknowledgements the evaluation working group includes jerry gwamna, nwando mba, gabriel ikwulono, obinna nnadozie, ayodele fagbemi, and grace bassey. the authors thank all participants that consented to the study. in addition, the authors extend their gratitude to ado g. abubakar, mary okoli, chimaoge c. achugbu, andrew n. thomas, mudiaga k. esiekpe, abubakar iliyasu bichi, tamunonengiyeofori israel, chinwe n. ugwu, and erasogie evbuomwan for their technical assistance on the hiv multiplex assay. the authors also extend their gratitude, especially to ndidi agala, for coordinating the sample receipt, accessioning, and storage at the biorepository of the national reference laboratory. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions a.o.m., o.a., n.c.i., o.o.b., o.o.o., a.a., m.i.j.o., and s.b.a. were responsible for conceptualisation of the study. a.o.m., s.b., b.e., c.a.o., b.s.i, o.a., r.e.a, m.a.a., c.m., e.o., n.c.i., o.o.b., o.o.o., t.c., a.a., m.i.j.o., and s.b.a. were involved in the investigation, and n.c.i., o.o.b., o.o.o., t.j., s.b., b.e., c.a.o., b.s.i., o.a., r.e.a., m.a.a., c.m., and e.o. defined the methodology. o.a., a.a., m.i.j.o., a.b., a.i., n.c.i., c.e.n., and s.b.a. supervised the project. a.o.m., o.o.b., o.o.o., n.c.i., s.b., and o.a. wrote the original draft, and a.o.m., n.c.i., a.b., o.o.o., o.o.b., e.o., t.j., m.a.a., c.m., r.e.a, c.e.n., s.b., b.e., c.a.o, b.s.i., o.a., a.i., c.i., t.c., s.b.a., m.i.j.o., and a.a. were involved in review and editing of the submission. sources of support this project was supported by the united states president’s emergency plan for aids relief through the united states centers for disease control and prevention under the grant number nu2gghoo2108 noa 02-03 to the university of maryland, baltimore. data availability data sharing is not applicable to this article, as no new data were created or associated with this manuscript. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. it does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the authors are responsible for this article’s results, findings, and content. references am k, pradeep k. hiv counseling and testing – entry point for prevention and care: gujarat experience. nat j 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al. evaluation of simple rapid hiv assays and development of national rapid hiv test algorithms in dar es salaam, tanzania. bmc infect dis. 2009:9;9:1–7. https://doi.org/10.1186/1471-2334-9-19 klarkowski d, glass k, o’brien d, lokuge k, piriou e, shanks l. variation in specificity of hiv rapid diagnostic tests over place and time: an analysis of discordancy data using a bayesian approach. plos one. 2013;8(11), e81656. https://doi.org/10.1371/journal.pone.0081656 kosack cs, page al, beelaert g, et al. towards more accurate hiv testing in sub-saharan africa: a multi-site evaluation of hiv rdts and risk factors for false positives. j int aids soc. 2017;20(1), 21345. https://doi.org/10.7448/ias.20.1.21345 kosack cs, shanks l, beelaert g, et al. hiv misdiagnosis in sub-saharan africa: performance of diagnostic algorithms at six testing sites: performance. j int aids soc. 2017;20(1), 21419. https://doi.org/10.7448/ias.20.1.21419 tegbaru b, wolday d, messele t, et al. assessment of the implementation of hiv-rapid test kits at different levels of health institutions in ethiopia. ethiop med j. 2007;45(3):293–299. tagny ct, bissim m, djeumen r, et al. the use of the geeniustm hiv-1/2 rapid confirmatory test for the enrolment of patients and blood donors in the who universal test and treat strategy in cameroon, africa. vox sang. 2020;115(8):686–694. https://doi.org/10.1111/vox.12942 alqaisi if. the effects of stakeholder’s engagement and communication management on projects success. in: matec web conf; 2018. p. 162. 3rd international conference on buildings, construction and environment engineering (bcee3-2017). 23–25 october 2017, sharm el-shiekh, egypt. campbell n. improving laboratory quality and capacity through leadership and management training: lessons from zambia 2016–2018. afr j lab med. 2021;10(1):1225. https://doi.org/10.4102/ajlm.v10i1.1225 http://www.ajlmonline.org open access african journal of laboratory medicine issn: (online) 2225-2010, (print) 2225-2002 page 1 of 1 corrigendum read online: scan this qr code with your smart phone or mobile device to read online. authors: john nkengasong1 debrah i. boeras2 alash’le abimiku3 rosanna w. peeling2 affiliations: 1us centers for disease control and prevention, atlanta, georgia, united states 2international diagnostic centre, london school of hygiene and tropical medicine, london, united kingdom 3institute of human virology, university of maryland school of medicine, baltimore, maryland, united states corresponding author: debrah boeras, dboeras@globalhealthig.com date: published: 19 july 2017 how to cite this article: nkengasong j, boeras di, abimiku a, peeling rw. corrigendum: assuring the quality of diagnostic testing: the future is now. afr j lab med.2017;5(2), a661. https://doi.org/10.4102/ajlm. v5i2.661 copyright: © 2017. the authors. licensee: aosis. this work is licensed under the creative commons attribution license. in the version of this article initially published, the first author’s surname was misspelled and the affiliations for the second and last authors were misstated. the author list and affiliations are hereby corrected to: john nkengasong us centers for disease control and prevention, atlanta, georgia, united states debrah i. boeras international diagnostic centre, london school of hygiene and tropical medicine, london, united kingdom alash’le abimiku institute of human virology, university of maryland school of medicine, baltimore, maryland, united states rosanna w. peeling international diagnostic centre, london school of hygiene and tropical medicine, london, united kingdom the errors have been corrected in the pdf version of the article. we apologise for any inconvenience that this may have caused. corrigendum: assuring the quality of diagnostic testing: the future is now read online: scan this qr code with your smart phone or mobile device to read online. note: doi of original article: http://dx.doi.org/10.4102/ajlm.v5i2.558 http://www.ajlmonline.org http://orcid.org/0000-0002-9850-3479 http://orcid.org/0000-0003-2213-0204 http://orcid.org/0000-0001-7404-8873 mailto:dboeras@globalhealthig.com https://doi.org/10.4102/ajlm.v5i2.661 https://doi.org/10.4102/ajlm.v5i2.661 http://crossmark.crossref.org/dialog/?doi=10.4102/ajlm.v5i2.661=pdf&date_stamp=2017-07-19 http://dx.doi.org/10.4102/ajlm.v5i2.558 abstract introduction methods results discussion acknowledgements references about the author(s) brian r. kullin department of molecular and cell biology, faculty of science, university of cape town, cape town, south africa sharon reid department of molecular and cell biology, faculty of science, university of cape town, cape town, south africa valerie abratt department of molecular and cell biology, faculty of science, university of cape town, cape town, south africa citation kullin br, reid s, abratt v. clostridium difficile in patients attending tuberculosis hospitals in cape town, south africa, 2014–2015. afr j lab med. 2018;7(2), a846. https://doi.org/10.4102/ajlm.v7i2.846 original research clostridium difficile in patients attending tuberculosis hospitals in cape town, south africa, 2014–2015 brian r. kullin, sharon reid, valerie abratt received: 04 june 2018; accepted: 28 sept. 2018; published: 06 dec. 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: diarrhoea due to clostridium difficile infection (cdi) poses a significant burden on healthcare systems around the world. however, there are few reports on the current status of the disease in sub-saharan africa. objectives: this study examined the occurrence of cdi in a south african population of tuberculosis patients, as well as the molecular epidemiology and antibiotic susceptibility profiles of c. difficile strains responsible for disease. methods: toxigenic c. difficile in patients with suspected cdi attending two specialist tuberculosis hospitals in the cape town area were detected using a pcr-based diagnostic assay (xpert® c. difficile). c. difficile strains isolated from pcr-positive specimens were characterised by ribotyping, multilocus variable-number tandem-repeat analysis and antibiotic susceptibility testing. results: the period prevalence of cdi was approximately 70.07 cases per 1000 patient admissions. strains belonging to ribotype 017 (rt017) made up over 95% of the patient isolates and all of them were multi-drug resistant. multilocus variable-number tandem-repeat analysis revealed several clusters of highly related c. difficile rt017 strains present in tuberculosis patients in several wards at each hospital. conclusion: tuberculosis patients represent a population that may be at an increased risk of developing cdi and, in addition, may constitute a multi-drug resistant reservoir of this bacterium. this warrants further investigation and surveillance of the disease in this patient group and other high-risk patient groups in sub-saharan africa. introduction clostridium difficile infection (cdi) is the most common cause of nosocomial diarrhoea in the developed world, with complications of the disease, including potentially life-threatening pseudomembranous colitis and toxic megacolon.1 the total costs of cdi treatment are estimated to be as high as $2871.00 (united states dollars) to $4846.00 per patient in the united states and between $5243.00 and $8570.00 per patient in european countries,2 and there is evidence to suggest that treatment failure and disease relapse rates are increasing.3 since the turn of the century, several significant outbreaks of cdi have occurred and have been documented in north america and europe.4,5,6 toxigenic c. difficile strains produce two large clostridial toxins, tcda and tcdb, both of which show cytotoxic activity and are responsible for the majority of disease symptoms.7 a third binary toxin, cdtab is also produced by some strains, such as those belonging to ribotype 027 (rt027) and rt078, and may be associated with increased disease severity in some settings.8 several toxin variant strains have also been identified. the most common of these is a subset of strains that produce only one functional toxin (tcdb) and is mainly comprised of members of the rt017 group. this group is widespread in asia9 and is capable of causing severe disease across diverse populations.6 the most common risk factor for the development of cdi is previous exposure to antibiotics, particularly clindamycin and cephalosporins and, less frequently, fluoroquinolones.10 the resulting dysbiosis allows proliferation of c. difficile and the progression of disease symptoms. additional risk factors include advanced age,11 gastric acid suppression therapy12 (although this is sometimes debated13), co-morbidities such as hiv14 and exposure to long-term healthcare facilities.15 there have also been several case reports of cdi occurring in patients with tuberculosis,16,17,18,19 possibly related to the long-term intensive antibiotic therapy that they typically receive. however, there have been very few studies to date specifically looking at cdi in this patient group. chang et al.20 examined a potential link between fluoroquinolone use and cdi in tuberculosis patients in hong kong and concluded that the risk of cdi was moderate for these patients, while lee et al.21 detected a relatively low incidence of cdi in korean tuberculosis patients (2.83 cases per 1000 adults). more recently, however, legenza et al.22 identified tuberculosis as an independent risk factor for cdi in patients in cape town, south africa, and there has been at least one report of a cdi outbreak occurring in a tuberculosis hospital in the eastern cape province,23 suggesting the need for further research in this area. while the overall burden of cdi in sub-saharan africa is largely unknown, data from the limited number of available studies suggest that the prevalence of cdi in the region is comparable to that of high income countries in europe and north america.24,25 in south africa, studies carried out in the vhembe district of limpopo province and cape town in the western cape report a prevalence of toxigenic c. difficile of between 10% and 20% in patients with diarrhoea.26,27,28 however, at least two of the previous studies undertaken in the country relied on diagnostic testing that detected toxin a alone, meaning that the rate of cdi in the country may be underestimated.27,29 additionally, epidemiological information regarding cdi and the strains responsible for disease is currently lacking, particularly for hiv-positive/tuberculosis-positive patients, who may be at an increased risk of developing cdi. therefore, the aim of the current study was to examine cdi in patients attending two specialist tuberculosis hospitals in cape town and to determine the molecular epidemiology of strains isolated from these patients. methods ethical considerations all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and national research committees and with the helsinki declaration and its later amendments. this study was approved by the ethics committee of the university of cape town (hrec number: 310/2008). for this type of observational and retrospective study formal consent is not required. study setting and sample collection adult patients with tuberculosis attending two specialist tuberculosis hospitals (designated hospital a and hospital b) in cape town between september 2014 and september 2015 were included as part of a larger surveillance study for cdi that has been published more fully elsewhere.30 both hospitals house patients undergoing tuberculosis therapy with adult wards divided into long-stay multi-drug resistant (mdr) and extensively drug resistant (xdr) wards and short-stay separate male and female wards for drug sensitive patients. inclusion criteria were clinical suspicion of cdi (based on a c. difficile diagnostic test request from the attending clinician) and an unformed stool sample, collected in sterile specimen containers. during the study period, stool samples submitted to the national health laboratory service diagnostic laboratory at groote schuur hospital that tested positive for c. difficile using the xpert® c. difficile platform (cepheid, sunnyvale, california, united states) were retained at −70 °c for further culture analysis. recorded patient data were limited to patient identification number (necessary to identify repeat specimens), age, gender as well as the hospital and ward the patient was in at the time of sample submission. c. difficile isolation and characterisation c. difficile was isolated from stool samples using cycloserine cefoxitin egg yolk agar.31 briefly, stool was homogenised in phosphate buffered saline and heat shocked at 60 °c for 10 minutes, before being inoculated onto cycloserine cefoxitin egg yolk agar. cultures were incubated for 24–48 hours at 37 °c in an anaerobic chamber under an atmosphere of 5% h2, 10% co2 and 85% n2 (forma scientific, model 1024, marietta, ohio, united states). putative c. difficile isolates were confirmed by pcr targeting the c. difficile tpi gene, as well as the various toxin-encoding genes (tcda, tcdb, cdta, cdtb).32,33 isolates were typed by capillary gel electrophoresis-based ribotyping34 and matched against a local strain database, which included a selection of profiles from the cardiff anaerobe reference unit (named according to the standard nomenclature) and the swedish institute for communicable disease control (names preceded by ‘se’ in the designation).30 further characterisation by multilocus variable-number tandem-repeat analysis (mlva) was carried out as previously described.35 briefly, the calculated numbers of repeats at each locus were used to calculate a dissimilarity matrix based on the manhattan distance between isolates. this dissimilarity matrix was used to construct minimum spanning trees using kruskal’s algorithm implemented in the mstgold program (version 2.4)36 with a final consensus tree generated from 2000 bootstrap replicates. final tree editing was carried out using the gephi graph visualiser (version 0.9.2).37 strains with a total summed tandem-repeat difference of two or less across all loci were considered clonally related based on approaches used in previous studies.35,38,39 antibiotic susceptibility testing minimum inhibitory concentrations (mics) for various antibiotics were determined using the gradient diffusion strip method. testing was carried out using either etest® strips (biomérieux, johannesburg, south africa) (metronidazole, vancomycin, moxifloxacin and erythromycin) or mic test strips (liofilchem, roseto degli abruzzi, italy) (rifampicin) and isolates were cultured on brucella agar (supplemented with 5% horse blood, 5 μg/ml haemin and 10 μg/ml vitamin k). clinical breakpoints were obtained from clinical & laboratory standards institute tables40 (metronidazole, moxifloxacin, erythromycin), from european committee on antimicrobial susceptibility testing tables41 (vancomycin), and published data (rifampicin).42 c. difficile atcc 700057 and c. difficile 11/11 were used as control strains showing full susceptibility and reduced susceptibility to metronidazole respectively.43 results xpert data between september 2014 and september 2015, initially, a total of 212 c. difficile test requests were received from the two hospitals. slightly more than half (120; 56.6%) of the submitted samples were from female patients. repeat specimens were obtained from a total of seven patients. two of these were classified as duplicate specimens (received from the same patient within a two-week period) and were removed from subsequent analyses, while the remaining five were classified as recurrent disease, defined as including both relapse and reinfection cases. the minimum, maximum and median durations between tests for patients with recurrent disease were 60, 161 and 90 days, respectively. samples were submitted from patients housed in nine different wards across the two hospitals. a total of 117 test requests (55.7%) were received from patients in drug sensitive (ds) tuberculosis wards, 77 (36.7%) from patients in mdr tuberculosis wards and four (1.9%) from patients in pre-xdr tuberculosis and xdr tuberculosis wards. a further 12 samples (5.7%) did not have ward information. overall, xpert c. difficile-positive results were obtained for 152 samples (72.4%) in the non-repeat dataset (figure 1). a total of three samples (1.4%) yielded indeterminate results and were not repeated. the period prevalence for hospital a was approximately 84.05 cases per 1000 patient admissions and for hospital b was approximately 60.03 cases per 1000 admissions (70.07 cases per 1000 admissions overall). most samples (145; 69%) were submitted by patients between the ages of 24 and 45 and the median age for xpert c. difficile positive patients (median 38, interquartile range 31–45.25) was slightly higher than for xpert c. difficile negative patients (median 34, interquartile range 30–40). figure 1: detection of c. difficile in samples provided by patients attending hospital a and hospital b stratified by patient age. the relative proportions of samples provided by patients in hospital a and hospital b testing positive (+ve) and negative (-ve) by the xpert® c. difficile test (xpert), along with those that yielded an indeterminate or invalid result (ind). numbers above the columns represent total number of tests performed for each category. frequency of clostridium difficile infection cases stratified by ward the cumulative frequency of xpert c. difficile-positive samples received from each ward in the two hospitals was analysed in 28-day windows for the entire study period, to allow the identification of periods of increased cdi prevalence (figure 2). hospital a experienced several periods of increased incidence (five or more samples per 28-day window). ward 2 (mdr tuberculosis) showed four peaks, occurring throughout the study period. ward 3 (ds tuberculosis) showed two peaks, the first of which occurred during the november–december 2014 period with the second occurring near the end of the study. hospital b experienced three periods of increased incidence across two wards. both ward 1 (ds tuberculosis) and ward 3 (ds tuberculosis) showed peaks during the november–december 2014 period, with an additional peak during january 2015 observed for ward 1. figure 2: frequency of cdi cases per ward. the number of cdi cases in 28-day windows for each ward in hospital a (a) and hospital b (b) over the study period. windows move along the x-axis in one-day steps. wards are designated as housing patients undergoing standard tuberculosis treatment (ds) or mdr/xdr treatment regimens (mdr/xdr). isolation and molecular epidemiology of c. difficile strains of the 152 xpert c. difficile positive samples, only 119 (78.3%) were retained for culture analysis mostly due to insufficient left over sample. toxigenic c. difficile (n = 110) were isolated from 110 of these samples (92.4%). rt017, toxin a-b+ isolates accounted for 105 (95.5%) of the total number of strains isolated from patients in both ds tuberculosis and mdr tuberculosis wards. the remaining five toxin a+b+ isolates were typed as rt002 (one isolate), rt046 (one isolate) and rt(se)108 (three isolates) and were all isolated from samples submitted by patients in ds tuberculosis wards from hospital b. mlva revealed close relationships between many of the rt017 strains from the two hospitals. just over half (53.4%) of all isolates from hospital a clustered in one large group of clonally related strains (summed tandem-repeat difference ≤ 2) in a minimum spanning tree (figure 3a). strains in this group were isolated throughout the course of the study from patients in all four wards that experienced cases of cdi. a second smaller group of eight strains was also evident and comprised of strains isolated from three of the four wards between january 2015 and november 2015. hospital b showed several groups of three or more clonally related strains (figure 3b), which together accounted for 76.9% of all isolates. as for hospital a, the clusters contained strains that were isolated from patients in different wards throughout the study period. it was noted that, in both hospitals, sets of three or four clonally identical strains were isolated from different patients in a single ward at different time points, suggesting possible patient-to-patient transfer events. figure 3: minimum spanning tree of mlva data for c. difficile rt017 isolates showing their clonal relationships and isolation sites. strains isolated from hospital a (a) and hospital b (b) are represented by circles with the date of isolation included in the circle and the circle colour representing the ward that the patient was in at the time of sample submission. larger circles represent multiple identical isolates, with the size of the circle proportional to the number of isolates. the total summed tandem-repeat difference between strains is given by the numbers between each circle. clonally related isolates (summed tandem-repeat difference ≤ 2) are grouped within the shaded areas – () pairs, () clusters. the trees have been redrawn for ease of viewing and are not to scale. figure 3 (continues...): minimum spanning tree of mlva data for c. difficile rt017 isolates showing their clonal relationships and isolation sites. strains isolated from hospital a (a) and hospital b (b) are represented by circles with the date of isolation included in the circle and the circle colour representing the ward that the patient was in at the time of sample submission. larger circles represent multiple identical isolates, with the size of the circle proportional to the number of isolates. the total summed tandem-repeat difference between strains is given by the numbers between each circle. clonally related isolates (summed tandem-repeat difference ≤ 2) are grouped within the shaded areas – () pairs, () clusters. the trees have been redrawn for ease of viewing and are not to scale. antibiotic susceptibility of isolates complete antibiotic susceptibility testing data (metronidazole, vancomycin, erythromycin and moxifloxacin) were available for a total of 77 isolates, 43 from patients attending hospital a and 34 from patients attending hospital b. additional antibiotic susceptibility testing data were available for some strains and these were included in the analysis. all isolates from both hospitals were susceptible to vancomycin in vitro (table 1). similarly, none of the isolates was resistant to metronidazole, although four isolates from hospital a showed reduced susceptibility to the antibiotic (mic > 2 mg/l). all but one of the tested isolates (a rt002 isolate) were resistant to rifampicin. all tested rt017 isolates (n=72) were resistant to moxifloxacin, while the remaining non-rt017 isolates were susceptible to the antibiotic. erythromycin resistance was observed in over two-thirds of the isolates (72.6%). multi-drug resistance was observed for all tested rt017 strains (table 2), with 73.6% of isolates resistant to erythromycin, moxifloxacin and rifampicin and a further 26.4% of isolates co-resistant to moxifloxacin and rifampicin. co-resistance was also observed for rt(se)108 strains, with two isolates resistant to erythromycin and rifampicin. there were no significant differences in antibiotic resistance patterns between wards and between hospitals. table 1: antibiotic susceptibility data for isolates. table 2: multi-drug resistance by ribotype. discussion there is a clear need for further study and increased surveillance of cdi in sub-saharan africa. both hospital and community-acquired cdi cases have the potential to pose significant challenges to healthcare systems and there are currently no data on the economic burden of cdi outbreaks in the region. an inclusive ‘one-health’ approach involving researchers at multiple levels in the community and hospital environment is important. a first step in this direction is the establishment of systematic sentinel surveillance of cdi in high-risk populations to monitor the incidence of cdi, as well as the predominant strain types and circulating antibiotic resistance phenotypes. similar programmes in high income nations have been successful in reducing both the incidence of infection and mortality due to cdi44 and would be of benefit to countries in sub-saharan africa. in the current study, cdi cases were examined in at-risk populations of patients undergoing long-term anti-tuberculosis therapy. cdi was common at both tuberculosis institutions with at least four wards experiencing spikes in the infection rates during the study period. three of these wards housed patients undergoing ds tuberculosis treatment regimens, while one housed patients receiving mdr tuberculosis therapy. although c. difficile outbreak definitions vary across countries, a rate of five or more cases in a single ward with at least 20 beds over a four-week period is generally used as a threshold to initiate outbreak investigations. that this occurred in four different wards during the study is concerning and suggests the need for enhanced surveillance of c. difficile in these hospitals to help to prevent further infections. toxin a-negative, toxin b-positive rt017 strains made up the majority of tuberculosis patient isolates. strains belonging to this ribotype have been implicated in outbreaks in canada,45 china,46 korea,47 argentina,48 israel,49 japan50 and europe6,51 and they are often resistant to multiple drugs.42,52 moreover, in previous studies rt017 strains have been shown to have a similar 30-day mortality rate of the so-called ‘hypervirulent’ rt027 strains.5,53 rt017 strains were also commonly isolated from other hospitals in cape town during the same time period, suggesting that these strains are circulating more broadly in the cape town patient population.30,54 it is possible that the previous use of standalone enzyme immunoassay diagnostic tests that target toxin a alone allowed rt017 strains to proliferate undetected in the region, providing a reservoir of strains in the healthcare environment with the potential to cause outbreaks in susceptible patient populations. several rt017 strains isolated from both hospitals were highly related by mlva, suggesting possible patient-to-patient strain transfer. in a large scale epidemiological analysis of c. difficile strains infecting patients in england, eyre et al.55 defined several possible transmission relationships for patients with clonally related c. difficile strains. for hospital-acquired cases, these included ‘ward contacts’ (two or more patients occupying the same ward over a concurrent time interval that allowed for direct patient-to-patient transmission), ‘ward contamination’ (patients occupying the same ward but with an interval of 1–28 days separating the discharge or end of infectivity of the initial case and the admission of patients who subsequently developed cdi) and ‘hospital contacts’ (patients in different wards at the same hospital over a similar time interval to ward contacts). unfortunately, detailed ward occupation data and the duration of disease symptoms were not available for patients in this study and it is, therefore, not possible to differentiate between the three different transmission relationships in the current analysis. nevertheless, the isolation of several clonally related strains from samples submitted by patients in the same ward within a 28-day time interval suggests that ward contact or ward contamination occurred along with possible hospital contact for samples from patients occupying different wards. interestingly, strains with identical mlva profiles were also isolated from different patients at longer time intervals of three to seven months. this has been observed previously55 and may be due to the formation of spores that persist in a genetically quiescent state on surfaces in the hospital environment. metronidazole is the recommended antibiotic for initial episodes of cdi, with vancomycin reserved for cases that do not respond to initial treatment and for cases of recurrent disease. no resistance to either of these antibiotics was observed for local isolates. however, a small number of strains exhibited a slightly elevated metronidazole mic of 4 mg/l. this may be clinically relevant as the maximum level of metronidazole that can be maintained in the gut during oral therapy ranges between 0.25 mg/l and 9.5 mg/l.1 fidaxomicin, an alternative treatment for cdi with a narrow spectrum of activity, is not yet widely available for use in south africa. apart from one non-rt017 isolate, all c. difficile strains isolated from patients attending specialist tuberculosis hospitals were resistant to rifampicin. this included samples from both ds tuberculosis and mdr tuberculosis patients and contrasts with a concurrent set of isolates obtained from patients attending other hospitals in the cape town area (37% of strains resistant to rifampicin).30 it is also higher than that observed for strains isolated from the general hospital population in europe (0% – 63.64%).42 rifampicin is included as part of the standard ds tuberculosis treatment regimen and exposure to antibiotics belonging to this class has been associated with the development of resistance in c. difficile.56 similar results have been observed for rt046 strains isolated from a tuberculosis hospital population in poland.16 resistance to rifamycin in c. difficile is usually associated with mutations in the rpob gene, which typically confer cross-resistance to multiple members of the antibiotic class and, importantly, are associated with a low fitness burden, allowing the phenotype to be stably maintained in the bacterial population.57 fluoroquinolone resistance was also common among c. difficile strains isolated from tuberculosis patients. previous exposure to fluoroquinolones is a recognised risk factor for the development of cdi, and it is thought that acquisition of resistance to third and fourth generation fluoroquinolones by rt027 strains was a contributing factor to several hospital outbreaks in north america during the early 2000s.4,58 in south africa, moxifloxacin is included as part of the mdr tuberculosis treatment regimen, and all c. difficile strains isolated from mdr tuberculosis patients were resistant to the antibiotic. resistance to fluoroquinolones in c. difficile is chromosomally encoded,59 readily develops following exposure to this class of antibiotics60 and is associated with a low fitness cost to the bacterium.61 therefore, the development of resistance to fluoroquinolones, as well as rifampicin, is likely to be stably maintained in the c. difficile population circulating among tuberculosis patients. one reason proposed for the relatively low rates of cdi in patients undergoing anti-tuberculosis therapy in previous studies is that rifampicin is often effective against c. difficile and may help to protect against cdi in these patients.20 since this protection would be lacking in a background of circulating rifampicin-resistant strains, the presence of rifampicin resistance among the rt017 isolates in the current study is significant. moreover, co-resistance to fourth generation fluoroquinolones would allow the same strains to persist in patients undergoing mdr tuberculosis treatment regimens. together, these results may help to explain the increased risk of cdi in tuberculosis patients in cape town22 compared to previous studies in hong kong20 and korea21 and confirm the need to monitor this patient population more carefully. limitations there are several limitations to the current study. as mentioned, detailed ward occupation data were not available, and this will need to be collected in future analyses to identify various potential strain transmission routes. additionally, while mlva has previously been shown to be as sensitive as whole genome sequencing in tracking c. difficile transmission,38 it has been noted that rt017 strains show very little variation for several of the loci included in the scheme,30,35 suggesting that the method may not be suitable for examining rt017 transmission dynamics. therefore, we are currently performing whole genome sequencing on selected isolates to determine the fine-scale molecular epidemiology of local rt017 strains in order to complement the mlva results. conclusion the presence of a relatively large number of mdr rt017 c. difficile strains in patients attending specialist tuberculosis hospitals in cape town is noteworthy, especially given their potential to cause outbreaks. many of the isolates were closely related by mlva, and there is some evidence to suggest that patient-to-patient transfer of strains took place during the study period. tuberculosis patients, many of whom are co-infected with hiv, may represent a population that is vulnerable to cdi and further studies should be undertaken to evaluate the risk in this patient group. additionally, the propensity of c. difficile to form highly resistant spores that are continually shed by individuals with cdi warrants additional studies to investigate potential contamination of surfaces present in the hospital environment. tuberculosis patients who do not go on to develop active cdi may still be colonised and subsequently become carriers of the organism to the community or other healthcare facilities. finally, information regarding c. difficile in other sub-saharan countries is currently lacking and further research is needed to understand the epidemiology of the organism in the region more thoroughly. acknowledgements media preparation: elzane cronje and the staff at the national health laboratory service media laboratory. sample collection and plating: dr justyna wojno, dr lourens robberts, dr diane rip, zubeida salaam-dreyer and the staff at national health laboratory service microbiology groote schuur hospital capillary gel electrophoresis: alvera vorster. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support this study was funded by the national research foundation of south africa and the south african medical research council. b.r.k. acknowledges the claude leon foundation and the carnegie corporation of new york for postdoctoral fellowships. authors’ contributions s.r. and v.a. were the project leaders. b.r.k. performed the experiments. b.r.k., s.r. and 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of clostridium difficile in vitro. antimicrob agents chemother. 2015;59:1794–1796. https://doi.org/10.1128/aac.04503-14 ajlm 7(1)_contents.indd http://www.ajlmonline.org open access table of contents i original research evaluation of standard diagnostic rapid test kits for malaria diagnosis among hiv patients in kano, nigeria henry a. mbah, feyisayo e. jegede, surajudeen a. abdulrahman, tinuade i. oyeyi african journal of laboratory medicine | vol 7, no 1 | a698 | 05 december 2018 original research metabolic effects occurring in irradiated and non-irradiated red blood cellular components for clinical transfusion practice: an in vitro comparison faieqa adams, gregory r.m. bellairs, arthur r. bird, oluwafemi o. oguntibeju african journal of laboratory medicine | vol 7, no 1 | a606 | 06 december 2018 case study ecthyma gangrenosum on the face of a malnourished child with pseudomonas sepsis: simulating cancrum oris khadijat o. isezuo, usman m. sani, usman m. waziri, bilkisu i. garba, yahaya mohammed, joy f. legbo, nazish p. aquil, fatima i. abubakar, memuna omar african journal of laboratory medicine | vol 7, no 1 | a756 | 05 december 2018 brief report detection of the janus kinase 2 v617f mutation using a locked nucleic-acid, real-time polymerase chain reaction assay tshiphiri senamela, marleen kock, piet becker, joachim j.c. potgieter african journal of laboratory medicine | vol 7, no 1 | a658 | 31 january 2018 brief report common uropathogens among diabetic patients with urinary tract infection at jinja regional referral hospital, uganda barbara i. nabaigwa, bashir mwambi, john okiria, caesar oyet african journal of laboratory medicine | vol 7, no 1 | a621 | 09 february 2018 brief report human parvovirus b19-induced anaemia in pre-school children in ilorin, nigeria oluwaseyi s. ashaka, olajide o. agbede, adesuyi a. omoare, samuel k. ernest african journal of laboratory medicine | vol 7, no 1 | a615 | 10 may 2018 brief report external quality assessment programme for early infant diagnosis of hiv-1, mozambique, 2011–2014 nalia ismael, orvalho augusto, adolfo vubil, sofia o. viegas, fernanda miambo, patrina chongo, nédio mabunda african journal of laboratory medicine | vol 7, no 1 | a664 | 11 october 2018 brief report assessment of international consensus group for haematology smear review rules among patients with plasmodium falciparum malaria in johannesburg, south africa jenifer l. vaughan, nazeer alli, sandra havyarimana, estee benade african journal of laboratory medicine | vol 7, no 1 | a715 | 29 november 2018 reviewer acknowledgement african journal of laboratory medicine | vol 7, no 1 | a934 | 05 december 2018 56 64 73 77 81 84 88 93 97 page i of i table of contents i editorial building resources to meet evolving laboratory medicine challenges in africa iruka n. okeke african journal of laboratory medicine | vol 7, no 1 | a915 | 29 november 2018 lessons from the field strategic establishment of an international pharmacology specialty laboratory in a resource-limited setting takudzwa j. mtisi, charles maponga, tsitsi g. monera-penduka, tinashe mudzviti, dexter chagwena, faithful makita-chingombe, robin difranchesco, gene d. morse african journal of laboratory medicine | vol 7, no 1 | a659 | 12 february 2018 lessons from the field implementation of the laboratory quality management system (iso 15189): experience from bugando medical centre clinical laboratory – mwanza, tanzania medard beyanga, lisa gerwing-adima, kahima jackson, benjamin majaliwa, henrico shimba, simon ezekiel, charles massambu, dickson majige, michael mwasegaka, wilson mtotela, patrick mateta, christa kasang african journal of laboratory medicine | vol 7, no 1 | a657 | 31 july 2018 original research detection of minority drug resistant mutations in malawian hiv-1 subtype c-positive patients initiating and on first-line antiretroviral therapy zhiyong zhou, kevin tang, guoqing zhang, nellie wadonda-kabondo, kundai moyo, lori a. rowe, joshua r. devos, nick wagar, du-ping zheng, hongxiong guo, john nkengasong, mike frace, scott sammons, chunfu yang african journal of laboratory medicine | vol 7, no 1 | a708 | 30 may 2018 original research low-cost diagnostic test for susceptible and drug-resistant tuberculosis in rural malawi annie zhang, enoch jumbe, robert krysiak, sabeen sidiki, holden v. kelley, elly k. chemey, chancy kamba, victor mwapasa, juan i. garcía, alison norris, xueliang j. pan, carlton evans, shu-hua wang, jesse j. kwiek, jordi b. torrelles african journal of laboratory medicine | vol 7, no 1 | a690 | 04 june 2018 original research using laboratory data to categorise cd4 laboratory turn-around-time performance across a national programme lindi-marie coetzee, naseem cassim, deborah k. glencross african journal of laboratory medicine | vol 7, no 1 | a665 | 28 june 2018 original research district and sub-district analysis of cryptococcal antigenaemia prevalence and specimen positivity in kwazulu-natal, south africa naseem cassim, lindi m. coetzee, nelesh p. govender, deborah k. glencross african journal of laboratory medicine | vol 7, no 1 | a757 | 11 october 2018 original research addressing antiretroviral therapy-related diagnostic coverage gaps across south africa using a programmatic approach naseem cassim, lindi m. coetzee, wendy s. stevens, deborah k. glencross african journal of laboratory medicine | vol 7, no 1 | a681 | 12 november 2018 1 3 9 15 24 30 37 45 vol 7, no 1 (2018) issn: 2225-2002 (print) | issn: 2225-2010 (online)african journal of laboratory medicine background complexities of naat selection challenges of robust and rapid naat evaluations conclusion acknowledgements references about the author(s) lesley e. scott department of molecular medicine and haematology, school of pathology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa lara d. noble department of molecular medicine and haematology, school of pathology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa ashika singh-moodley department of molecular medicine and haematology, school of pathology, faculty of health sciences, university of the witwatersrand, johannesburg, south africanational priority programme, national laboratory services, johannesburg, south africa trish kahamba department of molecular medicine and haematology, school of pathology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa diana r. hardie division of medical virology, faculty of health sciences, university of cape town, cape town, south africa wolfgang preiser division of medical virology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa national health laboratory service, cape town, south africa wendy s. stevens department of molecular medicine and haematology, school of pathology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa national priority programme, national laboratory services, johannesburg, south africa citation scott le, noble ld, singh-moodley a, et al. challenges and complexities in evaluating severe acute respiratory syndrome coronavirus 2 molecular diagnostics during the covid-19 pandemic. afr j lab med. 2022;11(1), a1429. https://doi.org/10.4102/ajlm.v11i1.1429 opinion paper challenges and complexities in evaluating severe acute respiratory syndrome coronavirus 2 molecular diagnostics during the covid-19 pandemic lesley e. scott, lara d. noble, ashika singh-moodley, trish kahamba, diana r. hardie, wolfgang preiser, wendy s. stevens received: 15 oct. 2021; accepted: 07 feb. 2022; published: 26 apr. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. background severe acute respiratory syndrome coronavirus 2 (sars-cov-2), the causative agent of coronavirus disease 2019 (covid-19), was first identified in wuhan, china, in december 2019.1 since the world health organization (who) declared covid-19 a global pandemic on 11 march 2020,2 more than 280 million cases of infection and over 5 million deaths had been reported globally by 31 december 2021.3 the primary method of diagnosing infection with sars-cov-2 is by nucleic acid amplification technology (naat). diagnostic assays have become available at an impressive rate, with a total of 514 molecular assays listed by december 2021.4 regulatory bodies in australia, brazil, canada, europe, japan and the united states, and organisations such as the who, the united states food and drug administration, the foundation for innovative new diagnostics (find), and the united states centers for disease control and prevention (cdc) provide ongoing updates on assay performance. many assays, however, remain unavailable in certain geographic regions; in-house assays are in use that are not accessible to these evaluation bodies, and the ongoing emergency use need has meant that national programmes have had to perform their own evaluations. furthermore, in mid-2020, limited guidance was available for performing such evaluations and equally limited guidance existed on acceptance criteria or patient use cases.5 the find had verified 21 naat assay manufacturer claims6 (< 8% of available assays by july 2020), and the who target product profile (tpp) version 0.1 was only released in august 2020.7 through emergency validations in south africa, numerous complexities of molecular testing were quickly realised. many of these align with those described elsewhere,8,9 but additional difficulties arose due to drastic local lockdown measures, and the experience of using assays during lockdown yielded several relevant insights. complexities of naat selection the entire pathology value chain needs to be considered when selecting suitable naats. each step contributes to meaningful and valid test results: patient sampling, specimen type, specimen collection and handling (stability), viral rna purification, amplification and detection, and result interpretation (figure 1). specimen collection and handling challenges include swab type, collection site, collection and pre-treatment methods, and transport. swabs are manufactured from different materials (cotton, nylon, polyester, rayon and foam) and may be spun or flocked.10,11 thus, swabs differ in absorption properties, viral capture and release. swab shafts may be made from wood, plastic or aluminium, and some of these materials are incompatible with certain collection and laboratory techniques (e.g. wooden shafts are unsuitable for paediatric sampling and may inhibit naat).10 commonly used swab collection sites are the nasopharynx, mid-turbinate region, nasal cavity or oropharynx, but the method used for a particular specimen is often not documented or made known to a testing laboratory. alternative specimen types include saliva, gargle, sputum and faecal material. furthermore, multiple swabs may be combined in a single testing vial. after collection, the swabs may be transported dry or in plain saline, phosphate-buffered saline, or various preservation media (e.g. viral transport media). the medium may vary in volume (1 ml – 3 ml), impacting viral concentration. specimens are transported at 2 °c – 8°c or at ambient temperature, which, in some settings, means high humidity and temperature. certain testing laboratories insist on inactivation of viral swabs for biosafety, which could include chemical or heat pre-treatment. figure 1: considerations for severe acute respiratory syndrome coronavirus 2 molecular assays across the pathology value chain. viral rna extraction (or purification) is prone to variability. the majority of rna purification methods rely on automated magnetic bead extraction performed on automated platforms (e.g. nuclisens® easymag® [biomérieux, johannesburg, south africa]). these differ in processing capacity, input volume, purified rna elution yield and turnaround time (tat). manual methods range from spin columns (e.g. rneasy mini spin [qiagen, hilden, germany]), which rely on centrifugation, to crude approaches that are used where no extraction instrumentation is available. the latter include direct lysis-to-polymerase chain reaction (pcr) kits (e.g. lyra® direct sars-cov-2 assay [quidel®, san diego, california, united states]), proprietary lysis buffers (e.g. bosphore ex-tract dry swab rna [anatolia geneworks, istanbul, turkey]) or simply heat (where rapid sample preparation is needed).12 these procedures aim to disrupt viral particles to release rna and inactivate inhibitory enzymes. added variables with the lysis approach could be a specimen’s exposure time to the buffer and buffer compatibility with pcr. the primary specimen input testing volume also differs between technologies: 300 µl added directly to an xpert® xpress® sars-cov-2 cartridge (cepheid, sunnyvale, california, united states), 400 µl to the cobas® sars-cov-2 assay (roche, basel, switzer;and) and 500 µl to the realtime sars-cov-2 assay (abbott, chicago, illinois, united states). similarly, most rna extraction platforms require input of anywhere between 100 µl and 300 µl of raw specimen. furthermore, for a direct lysis-to-pcr assay, input volume can range from 10 µl preservation buffer (nagene [diagnóstica longwood, zaragoza, spain]) to 30 µl raw specimen (smartchek®), and ideally the patient swab should be added directly to the proprietary medium. the naats used are also variable. they are performed on a variety of platforms that may be closed (dedicated) (e.g. m2000® [abbott, chicago, illinois, united states]) or open (‘plug-and-play’ approach subsequent to extraction using different systems and instruments). the latter requires compatibility with real-time thermocycler instruments (e.g. cfx96 touch™ [biorad, hercules, california, united states], quantstudio [thermo fisher scientific, waltham, massachusetts, united states]). throughput ranges from a single test at a time (e.g. genexpert® [cepheid, sunnyvale, california, united states]) to 1000–1500/day (e.g. cobas® [roche, basel, switzerland]). many of the open-platform thermocyclers use 96-well plates and may require specimen batching. as with specimen input volume, the rna input volume also varies between assays (e.g. 8 µl for the allplex sars-cov-2 assay [seegene, seoul, south korea], 10 µl for the taqpath™ sars-cov-2 assay [thermo fisher scientific, waltham, massachusetts, united states], and 14 µl for the perkinelmer™ sars-cov-2 rt-qpcr reagent kit ce-ivd [perkinelmer™, waltham, massachusetts, united states]. a number of novel assays have rna input volumes as low as 5 µl. the differences in volume for extraction or pcr may influence theoretical limits of detection. in addition to these complexities, the sars-cov-2 genes (envelope [e], nucleocapsid [n], spike [s], membrane [m], open reading frames 1a and 1b [orf1ab], rnase-dependent rna polymerase [rdrp]) or combination of genes targeted by naat are inconsistent. these have been reported to affect overall assay sensitivity and specificity,13,14 and may also be impacted by viral genetic evolution that can affect gene targets.15 furthermore, there is the complex layer of different diagnostic algorithms implemented within countries that often change over time. for example, some settings report specimen results based on a single gene target, while others require two or more gene targets or result confirmation by a second naat method using a different gene target. automated software algorithms are not always available for all pcr platforms, and result analysis may require skilled user input (e.g. visual interpretation of amplification curves or adjustment of threshold settings and thus cycle thresholds [ct]). this adds another, potentially subjective, layer of variability. challenges of robust and rapid naat evaluations the complex variables described above come to the fore when clear, comparative standardised evaluations are conducted. such evaluations generally had to be performed in parallel with managing covid-19 testing emergencies. in our case, the most challenging issue experienced early in the pandemic was access to sufficient numbers of relevant clinical specimens available for inclusion in evaluation challenge panels. the volume of extracted rna per specimen was often only sufficient to test a limited number of naats. thus, constant panel ‘manufacture’ was required, with the potential to introduce variability (e.g. different specimens) across evaluations. the sample size, the numbers of positive and negative panel specimens, and the range in viral concentration (high, medium or low) of challenge specimens may influence sensitivity scores. the last addresses the need for assays to correctly identify differences in patient risk stratification: while there does not appear to be a difference in median ct between symptomatic and asymptomatic patients,16 a lower ct (higher viral load [vl]) in real-time pcr assays may indicate increased virus transmissibility16,17 and may impact patient outcomes.17,18 the impact of sars-cov-2 variants on technologies15 should also be considered, as specimens selected from waves of infection driven by different variants could affect the technology (e.g. s gene target failure of the taqpath sars-cov-2 assay [thermo fisher scientific, waltham, massachusetts, united states] with the alpha19 and omicron20 variants). these challenges contribute to generating variability in the assay performance outcome and may make the difference between accepting different assays and criteria. false negative results can arise from low levels of virus due to patient or sample characteristics (i.e. either biologically or artefactually), degraded viral rna, viral genetic variation or presence of inhibitors. false positive results can arise from inaccurate ct threshold settings, and contamination during processing. the correct placement of naat technologies should also be considered. a less sensitive assay may be valuable in settings that receive specimens from patients with high vl concentrations (low ct values), that is, emergency settings or referral centres. in contrast, community or mobile screening and testing sites will require assays with greater sensitivity. in addition to clinical specimens, several types of reference materials (plasmids, biomimetic standards, or viable, inactivated or lysed virus) have become commercially available (e.g. accuplex™ sars-cov-2 reference material kit [seracare, milford, massachusetts, united states]), although these may not always be compatible with naat primer or probe sequences. access to locally manufactured sars-cov-2 viral culture supernatant is an asset to expanding an evaluation panel, independent of naat target genes. culture dilutions may be used to measure assay precision, linearity and, potentially, the limit of detection. another limitation is often kit size, where only a limited number of specimens can be tested per kit supplied or where single-plex pcr assays limit the number of specimens in a test run (e.g. single-plex with 3 gene targets can only assess 32 specimens per 96-well rna extraction plate including controls). other considerations determined during performance evaluation are assay ease of use, time to reportable result and throughput. general considerations, often listed in a tpp are: testing footprint, number of testing steps, operator skills required to perform the test, minimum pipetting volume, biosafety requirements, internal controls, positive and negative batch controls, reagent storage stability, reagent reconstitution required, training needs, maintenance, additional consumables and connectivity options, to name a few. moreover, it is important to select suitable comparator technology, a limitation also described by axell-house et al.21 this may be based on who recommendations, or, under emergency use, current in-county methodology already approved for standard of care (soc). in our case, at least seven soc technologies are in use to report patient results across south africa, and residual specimens from these assays were available for challenge panels. the goal of performance evaluation is to determine whether a new method or technology is as good as soc and use thereof will not alter patient care. however, this does pose a challenge if soc specimens are in a different format or rna degrades during specimen storage. our group obtained institutional review board approval to use residual patient specimens for evaluations with an informed consent waiver. where fresh or paired specimens were needed, a full clinical trial was required with informed consent, which increased the time needed and cost of evaluations. once an evaluation is completed, method comparison involves determining accuracy (sensitivity and specificity) or agreement, precision (reproducibility), linearity and limit of detection. recommendations are made based on analytical performance, as well as ease of use. in the absence of acceptance criteria guidelines, we investigated these parameters for an initial 24 assays using a standardised evaluation protocol approach as described in table 1. over and above the already mentioned complexities and challenges, it became apparent that single method comparison acceptance criteria might not be applicable to all assays. we identified four types or categories of assays submitted for evaluation: (1) closed systems (incorporating rna extraction and naat), (2) standalone pcr kits to be performed off already purified rna, (3) direct lyse-to-pcr, where no front-end rna purification or extraction is available or where there is a need for rapid testing, and (4) standalone rna extraction or purification kits. the 24 assays were among groups 1 (n = 9), 2 (n = 12) and 3 (n = 3), with the predominant viral gene targets being n (35.0%), followed by orf1ab (25.0%), e (20%), s (10.0%) and rdrp (10.0%). a sensitivity score of > 90% was achieved by 62.5% (15/24) of assays and > 95% sensitivity achieved by 37.5% (9/24) compared to soc. those that employ direct lyse-to-pcr (group 3) were below the acceptance criteria by at least a 20% drop in median sensitivity (72.0%) compared to the median sensitivity in group 1 (95%) and group 2 (92.0%). the clinical specimens used in these assays’ evaluation panels covered a range in sars-cov-2 vl, with a median target ct = 27. group 3 performed well on specimens with high vl (ct < 30), and hence another challenge is not only in the choice of range in clinical specimens being evaluated, but potentially the need to apply different acceptance criteria, bearing in mind that group 3 assays are designed for use where no front-end rna extraction (or rapid) system is available. the reduced sensitivity needs to be weighed against the rapid availability of a result which would likely identify patients who are newly symptomatic and at the height of being infectious. therefore, the diagnostic dilemma facing regulators and end users could be ‘no available test’ or ‘specimens routed a further distance (with ensuing longer tat) to a testing laboratory with access to front-end rna extraction technology’, or a ‘less sensitive test with potentially shorter tat and greater clinical relevance’. table 1: key evaluation features of a protocol applied under emergency use and acceptance criteria. conclusion selecting clinically relevant, laboratory-compatible and well-performing sars-cov-2 naat assays or systems for patient care is complex and subject to several challenges, as also highlighted elsewhere.9,22 current evaluation protocols are not robustly performed under emergency circumstances,21 and performance acceptance criteria and technology placement may require flexibility.23 assays themselves have also improved with time. added layers of complexity that we experienced were donated tests that did not always follow required regulatory pathways prior to implementation, and suppliers requesting evaluation from multiple laboratories in the hope of improving their performance score. however, having a new test ready for use is only half the battle, and still requires rapid implementation and scale-up, with further factors requiring investigation such as cost, supply chain management, training, quality assessment, interfacing to existing laboratory information systems, compatibility to existing testing landscapes, continuous quality monitoring and post-market surveillance, to name a few. acknowledgements network of evaluating laboratories: national health laboratory service (lucia hans, kim steegen, irene ketseoglou, puleng marokane, pedro da silva, somayya sarang, koleka mlisana), national institute of communicable diseases (olga perovic, mignon du plessis), clinical laboratory service (blessing kadira, lyndel singh, timothy moshema). developers of viral culture stock: universities of stellenbosch and the western cape (tasnim suliman), national health laboratory service (bavesh kana, bhavna gordhan) and harsha desai and mohapi mokone (programme managers engaging with suppliers and regulators). competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions l.e.s., l.d.n., a.s.-m., t.k., d.r.h., w.p. and w.s.s. contributed equally to the design and implementation of the research, to the analysis of the results and to the writing of the article. ethical considerations the use of residual blood, swab and universal transport medium specimens for research and development of molecular and serology assays for sars-cov-2 was approved by the university of the witwatersrand human research ethics committee (medical): m1911201. sources of support w.s.s., l.e.s., l.d.n. and t.k. are supported by funding received from the bill and melinda gates foundation through the innovation in laboratory engineered accelerated diagnostics investment (grant number opp1171455). data availability data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references zhu n, zhang d, wang w, et al. a novel coronavirus from patients with pneumonia in china, 2019. new engl j med. 2020;382(8):727–733. https://doi.org/10.1056/nejmoa2001017 world health organization. who director-general’s opening remarks at the media briefing on covid-19 – 11 march 2020 [homepage on the internet]. 2020 [cited 2020 apr 2]. available from: 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[cited 2021 dec 22]. available from: https://www.gov.uk/government/publications/wuhan-novel-coronavirus-guidance-for-clinical-diagnostic-laboratories/covid-19-guidance-for-alternative-swab-types-and-transport-media kahamba tr, noble l, stevens w, scott l. comparison of three nasopharyngeal swab types and the impact of physiochemical properties for optimal sars-cov-2 detection. j vaccines vaccin. 2020;s6:005. https://doi.org/10.1101/2020.10.21.20206078 marais g, naidoo m, hsiao n-y, valley-omar z, smuts h, hardie d. the implementation of a rapid sample preparation method for the detection of sars-cov-2 in a diagnostic laboratory in south africa 2020. plos one. 2020 oct 20;15(10):e0241029. https://doi.org/10.1371/journal.pone.0241029. ecollection 2020 li d, zhang j, li j. primer design for quantitative real-time pcr for the emerging coronavirus sars-cov-2. theranostics. 2020;10(16):7150–7162. https://doi.org/10.7150/thno.47649 gand m, vanneste k, thomas i, et al. use of whole genome sequencing data for a first in silico specificity evaluation of the rt-qpcr assays used for sars-cov-2 detection. int j mol sci. 2020;21(15):5585. https://doi.org/10.3390/ijms21155585 world health organization. tracking sars-cov-2 variants [homepage on the internet]. 2020 [cited 2021 dec 17]. available from: https://www.who.int/en/activities/tracking-sars-cov-2-variants/ singanayagam a, patel m, charlett a, et al. duration of infectiousness and correlation with rt-pcr cycle threshold values in cases of covid-19, england, january to may 2020. euro surveill. 2020;25(32):pii=2001483. https://doi.org/10.2807/1560-7917.es.2020.25.32.2001483 rao sn, manissero d, steele vr, pareja j. a systematic review of the clinical utility of cycle threshold values in the context of covid-19. infect dis ther. 2020;9(3):573–586. https://doi.org/10.1007/s40121-020-00324-3 prebensen c, hre plm, jonassen c, et al. sars-cov-2 rna in plasma is associated with icu admission and mortality in patients hospitalized with covid-19. clin infect dis. 2020;73(3):e799–e802. https://doi.org/10.1093/cid/ciaa1338 european centres for disease prevention and control. rapid increase of a sars-cov-2 variant with multiple spike protein mutations observed in the united kingdom (20 december 2020) [homepage on the internet]. 2020 [cited 2021 jan 12]. available from: https://www.ecdc.europa.eu/sites/default/files/documents/sars-cov-2-variant-multiple-spike-protein-mutations-united-kingdom.pdf national institute for communicable diseases. sars-cov-2 sequencing update 26 november 2021. prepared by the national institute for communicable diseases (nicd) of the national health laboratory (nhls) on behalf of the network for genomics surveillance in south africa (ngs-sa) [homepage on the internet]. 2021 [cited 2021 nov 27]. availalbe from: https://www.nicd.ac.za/wp-content/uploads/2021/11/update-of-sa-sequencing-data-from-gisaid-26-nov_final.pdf axell-house db, lavingia r, rafferty m, clark e, amirian es, chiao ey. the estimation of diagnostic accuracy of tests for covid-19: a scoping review. j infect. 2020;81(5):681–697. https://doi.org/10.1016/j.jinf.2020.08.043 yuzhong xu mc, xinchun chen, jialou zhu. current approach in laboratory testing for sars-cov-2. int j infect dis. 2020;100:7–9. https://doi.org/10.1016/j.ijid.2020.08.041 mina mj, parker r, larremore db. rethinking covid-19 test sensitivity – a strategy for containment. n engl j med. 2020;383:e120. https://doi.org/10.1056/nejmp2025631 south african health products regulatory authority. md018: specification criteria for covid-19 molecular test kits [homepage on the internet]. [cited 2020 sep 14]. available from: http://www.sahpra.org.za/wp-content/uploads/2020/07/md018-specifications-molecular-test-kits-v1-22072020.pdf abstract introduction methods results discussion acknowledgements references about the author(s) adeyemi t. adeyemo department of medical microbiology and parasitology, obafemi awolowo university teaching hospitals, ile-ife, nigeria babatope kolawole department of medicine, faculty of clinical science, obafemi awolowo university, ile-ife, nigeria vincent o. rotimi department of microbiology, faculty of medicine, kuwait university, kuwait city, kuwait aaron o. aboderin department of medical microbiology and parasitology, obafemi awolowo university teaching hospitals, ile-ife, nigeria department of medical microbiology and parasitology, faculty of basic medical science, obafemi awolowo university, ile-ife, nigeria citation adeyemo at, kolawole b, rotimi vo, aboderin ao. multicentre study of the burden of multidrug-resistant bacteria in the aetiology of infected diabetic foot ulcers. afr j lab med. 2021;10(1), a1261. https://doi.org/10.4102/ajlm.v10i1.1261 original research multicentre study of the burden of multidrug-resistant bacteria in the aetiology of infected diabetic foot ulcers adeyemi t. adeyemo, babatope kolawole, vincent o. rotimi, aaron o. aboderin received: 03 may 2020; accepted: 22 oct. 2020; published: 23 mar. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: infected diabetic foot ulcer (idfu) is a public health issue and the leading cause of non-traumatic limb amputation. very few published data on idfu exist in most west african countries. objective: the study investigated the aetiology and antibacterial drug resistance burden of idfu in tertiary hospitals in osun state, nigeria, between july 2016 and april 2017. methods: isolates were cultured from tissue biopsies or aspirates collected from patients with idfu. bacterial identification, antibiotic susceptibility testing and phenotypic detection of extended-spectrum beta-lactamase and carbapenemase production were done by established protocols. specific resistance genes were detected by polymerase chain reaction. results: there were 218 microorganisms isolated from 93 idfus, comprising 129 (59.2%) gram-negative bacilli (gnb), 59 (27.1%) gram-positive cocci and 29 (13.3%) anaerobic bacteria. the top five facultative anaerobic bacteria isolated were: staphylococcus aureus (34; 15.6%), escherichia coli (23; 10.6%), pseudomonas aeruginosa (20; 9.2%), klebsiella pneumoniae (19; 8.7%) and citrobacter spp. (19; 8.7%). the most common anaerobes were bacteroides spp. (7; 3.2%) and peptostreptococcus anaerobius (6; 2.8%). seventy-four idfus (80%) were infected by multidrug-resistant bacteria, predominantly methicillin-resistant s. aureus and gnb producing extended-spectrum β-lactamases, mainly of the ctx-m variety. only 4 (3.1%) gnb produced carbapenemases encoded predominantly by blavim. factors associated with presence of multidrug-resistant bacteria were peripheral neuropathy (adjusted odds ratio [aor] = 4.05, p = 0.04) and duration of foot infection of more than 1 month (aor = 7.63, p = 0.02). conclusion: multidrug-resistant facultative anaerobic bacteria are overrepresented as agents of idfu. a relatively low proportion of the aetiological agents were anaerobic bacteria. keywords: infection; diabetic foot; ulcers; multidrug-resistance; bacteria; antibiotic; anaerobic culture; samples. introduction infected diabetic foot ulcer (idfu) is associated with inlammation or purulence occurring in sites below the ankle in persons with diabetes mellitus.1 it is a major global public health issue with a substantial medical, socio-economic and psychological burden. infected diabetic foot ulcer is one of the most common diabetes-related infections in clinical practice, and a common indication for hospital admission.1 ulceration often precedes foot infection in diabetic patients, with peripheral vascular disease, peripheral neuropathy and visual impairment and immunological disturbances also playing contributory roles. infection impairs the healing process and aggravates the condition of patients with diabetic foot ulcer (dfu) and could lead to great disability, septicaemia and death if not promptly and properly managed. at 7.2% (95% confidence interval [ci]: 5.1–9.3%) and higher than the global prevalence of 6.3% (95% ci: 5.4–7.3%), africa has the second highest global prevalence of dfu, a precursor of idfu.2 foot infections are more common and lethal in africa than elsewhere globally.3 between 25% and 60% of diabetic patients with a background foot ulcer will develop idfu which remains a major reason for non-traumatic amputation of the lower limbs.4 the foot infection can progress to irreversible septic gangrene which often necessitates life-saving amputation of the lower limb.5 patients with idfu have 15–46 times higher risk of limb amputation than those with non-diabetic related ulcers.6 more than 1 million diabetic patients may require limb amputation worldwide yearly, and a greater percentage of them are in developing countries.7 wide varieties of organisms, including anaerobic bacteria, have been implicated in the aetiology of idfu depending on the severity of infection and time from onset to presentation at healthcare facilities. advanced idfus with features of sepsis at admission usually harbour anaerobic pathogens.8 the emergence and current global threat of antimicrobial resistance in the face of dwindling antibiotics in the development pipeline has added a new twist to the burden of idfu.9 increasing involvement of multidrug-resistant organisms (organisms resistant to at least three different antimicrobial classes)10 in diabetic patients with infected foot ulcers has significantly reduced antibiotic treatment options, thus posing a serious challenge particularly in resource-constrained lowand middle-income countries where access to antimicrobial drugs is of grave concern.11 it has also increased the length of hospital stay and cost of treatment, and caused additional morbidity and mortality.12 these situations have assumed worrisome trends in which resistance is building up to antibiotics of last resort; pathogens showing considerable resistance to vancomycin and carbapenems are particularly becoming more common as agents of foot infection in diabetic patients.13 various studies have reported many independent risk factors and predictors of multidrug-resistant idfu including previous hospitalisation for the same wound, prolonged antibiotic therapy, ulcer type and increased ulcer size, presence of osteomyelitis, poor glycaemic control, prolonged duration of foot ulcer infection as well as proliferative retinopathy.14,15,16,17,18 according to bakele et al., predictors of lower limb amputation by multivariate logistic regression analysis were advanced ulcer grade, inappropriate antibiotic use, overweight, obesity, poor blood glucose control and neuropathy.19 furthermore, albuminuria, diabetic nephropathy and charcot arthropathy were noted as predictors of poor healing of diabetic foot ulcer.20 a recent systematic review and meta-analysis on the global burden of diabetic foot ulceration in cameroon, west africa, concluded that paucity of data impedes strategies for treatment and prevention of foot infections in diabetic patients.2 thus, our study was designed to determine the prevalent bacteria involved in idfus, assess the burden of multidrug-resistance (mdr) among the isolates and evaluate the associated risk factors. methods ethical considerations ethical approval for this study was granted by the ethics and research committees of the obafemi awolowo university teaching hospitals complex and ladoke akintola university college of technology with protocol numbers erc/2015/11/02 and lth/er/2016/01/254. information about the study and participant involvement was fully explained to patients, and properly signed and dated written informed consent forms were obtained from patients before their recruitment into the study. results of wound biopsy microscopy, culture and sensitivity were made available for patients’ management. study population the prospective, cross-sectional, multicentre, hospital-based study was carried out in osun state, southwest nigeria, between july 2016 and april 2017. it included three existing tertiary healthcare facilities in the state: obafemi awolowo university teaching hospitals complex, wesley guild hospital, ilesa, and ladoke akintola university of technology teaching hospital, osogbo. all consecutive diabetic patients (both hospitalised and outpatients) with foot infections meeting the criteria for diagnosis of idfu seen and managed at these hospitals were recruited into the study. they were clinically assessed and foot lesions graded according to the diabetic foot infection severity classification system issued by the infectious disease society of america.8 only non-duplicate patients and samples were included in the study. all inpatients were followed up with regular check-ups physically in the wards until they either died or were discharged. sample collection and bacterial identification aspirates were obtained from deep-seated abscesses, and tissue samples were collected after washing the wound vigorously with sterile saline and debridement of the slough to exclude mere colonisers. necrotic tissues were curetted into anaerobic basal broth (oxoid, basingstoke, hants, united kingdom) for anaerobic culture. the samples were immediately transported to the laboratory and processed within 2 h of sample collection by inoculating them onto a set of selective and non-selective media which were: 5% (volume/volume) sheep blood agar (ba; oxoid, basingstoke, hants, united kingdom), macconkey agar (oxoid, basingstoke, hants, united kingdom), chocolate agar and anaerobic basal agar (oxoid, basingstoke, hants, united kingdom) supplemented with 5% (abscesses) laked sheep blood, vitamin k1 (1 µg/ml), l-cysteine hydrochloride (5 µg/ml) and gentamicin (100 µg/ml) (gentamicin blood agar). inoculated plain ba and macconkey agar plates were incubated in air and chocolate agar plates in co2 at 37 °c for 24 h. inoculated plain gentamicin ba plates, as well as gentamicin ba with kanamycin (75 µg/l) and vancomycin (5 µg/l) supplements, were incubated under anaerobic conditions made up of 80% h2, 10% co2 and 10% n2 for 48 h and extended for 5 days if necessary; anaerobiosis was achieved using a bactron anaerobic chamber (sheldon manufacturing, inc., cornelius, oregon, united states). representative colonies were identified by colonial morphology, gram staining characteristics and conventional biochemical tests including catalase and oxidase tests. facultative anaerobic gram-negative bacilli (gnb) and streptococcus spp. were further identified with microbact™ gnb 24e (oxoid, basingstoke, hants, united kingdom) and rapid™ str (thermo fisher scientific, remel products, lexena, kansas, united states), while staphylococcus spp. were further identified with a coagulase test, characteristic growth appearance on mannitol salt agar and a dnase test. the obligate anaerobes were identified by rapid™ ana ii (thermo fisher scientific, remel products, lexena, kansas, united states). yeast isolates were identified by gram staining and germ tube tests. quality control strains, staphylococcus aureus atcc 25923, escherichia coli atcc 25922, bacteroides fragilis atcc 25285 and peptostreptococcus anaerobius atcc 27337, were used to assess the quality of the media and identification systems. the quality of our bacterial identification system and procedures (for aerobes, facultative anaerobes and obligate anaerobes) were assured by ensuring that the control bacterial strains were identified to their names. antibiotic susceptibility test antibiotic susceptibility testing for aerobes and facultative anaerobes was performed using the modified kirby-bauer disc diffusion technique as recommended by the clinical and laboratory standards institute (clsi).21 gram-positive isolates were tested with the following antibiotic discs (oxoid, basingstoke, hants, united kingdom): penicillin (10 µg), cefuroxime (30 µg), ceftriaxone (30 µg), cefepime (30 µg), co-amoxiclav (20/10 µg), ciprofloxacin (5 µg), chloramphenicol (30 µg), gentamicin (10 µg), amikacin (10 µg), trimethoprim-sulfamethoxazole (1.25 µg/23.75 µg), cefoxitin (30 µg), erythromycin (15 µg), ampicillin-sulbactam (10 µg/10 µg), piperacillin-tazobactam (100 µg/10 µg) and vancomycin (30 µg). vancomycin (256–0.015 µg/ml) minimum inhibitory concentration (mic) strip (oxoid, basingstoke, hants, united kingdom) was used to test for vancomycin resistance among methicillin-resistant s. aureus (mrsa). gram-negative isolates were tested with the following antibiotic discs (oxoid, basingstoke, hants, united kingdom): ceftriaxone (30 µg), ceftazidime (30 µg), cefotaxime (30 µg), cefepime (30 µg), co-amoxiclav (20/10 µg), ciprofloxacin (5 µg), gentamicin (10 µg), amikacin (10 µg), chloramphenicol (30 µg), aztreonam (30 µg), trimethoprim-sulfamethoxazole (1.25 µg/23.75 µg), ampicillin-sulbactam (10 µg/10 µg), piperacillin-tazobactam (100 µg/10 µg), cefoxitin (30 µg), meropenem (10 µg) and ertapenem (10 µg). discrete colonies were emulsified in sterile saline to match 0.5 mcfarland turbidity standards from where confluence inocula were made on mueller-hinton agar (mha) plates with sterile cotton swabs. the swabbed mha plates were allowed to dry at room temperature and a set of six antibiotic discs were placed evenly spaced on each of the plates. vancomycin resistance was tested in the methicillin-resistant s. aureus isolates by placing a mic evaluation strip (oxoid, basingstoke, hants, united kingdom) on inoculated mha. after 18–24 h of incubation, the diameter of the zone of inhibition around each antibiotic disc was measured and recorded. vancomycin mic values were also recorded for the s. aureus. zones of inhibition of each antibiotic as well as vancomycin mic values were interpreted as ‘sensitive’, ‘intermediate’ or ‘resistant’ in accordance with clsi guidelines.21 isolates with intermediate sensitivity were regarded as ‘resistant’. the quality of antibiotic susceptibility testing consumables (including antibiotic discs and mha) and procedures were assured with bacterial control strains (e. coli atcc 25922, s. aureus atcc 25923 and s. aureus atcc 43300). zones of inhibition of tested antibiotics on the bacterial control strains fell within their quality control ranges according to clsi.21 extended-spectrum β-lactamase production was confirmed among enterobacteriaceae and other gnb that showed reduced susceptibility to at least one of the tested third-generation cephalosporins (cefotaxime 30 µg, ceftazidime 30 µg and ceftriaxone 30 µg) or aztreonam (30 µg) by a combination disc diffusion test (cddt).21 cddt was done using both single discs of cefotaxime (30 µg) and ceftazidime (30 µg) and their respective clavulanic acid containing discs (cetotaxime-clavulanate 30/10 µg, ceftazidime-clavulanate 30/10 µg). a 5 mm or more increase in zone of inhibition of one or more combination discs as compared with their respective single discs was taken as confirmatory evidence of extended-spectrum beta-lactamase (esbl) production.21 ampc beta-lactamase production was detected by ampc disc test as described by anjali et al. on isolates which show resistance to at least one third-generation cephalosporin and a β-lactamase inhibitor.22 a broth culture of e. coli atcc 25922 was adjusted to 0.5 mcfarland turbidity standard and inoculated onto mha plates. sterile filter paper discs (6 mm) were moistened with distilled water (about 20 µl) and up to five colonies of the test organism was transferred onto the filter paper. afterwards, a cefoxitin (30 µg) disc and the inoculated filter paper disc were placed next to each other and almost touching on inoculated media. this setup was incubated overnight at 37 °c. a flattening or indentation of the zone of inhibition of cefoxitin in the vicinity of the test disc (inoculated filter paper) indicated a phenotypic confirmatory evidence of ampc β-lactamase production.22 gram-negative bacilli with intermediate sensitivity or resistance to one or more carbapenems were tested for production of carbapenemases by the modified hodge test and interpreted by clsi guidelines.21 organisms that were phenotypically mdr, including esbl-producing gnb, carbapenem-resistant gnb and mrsa, were further tested for resistance-determining genes using polymerase chain reaction (pcr)-based protocols with specific oligonucleotide primers23–27 (table 1); template bacterial dna was extracted by the boiling method.28 electrophoresis of each pcr product (5 µl) was carried out in 1.5% (weight/volume) agarose gel (biomatik, kitchener, ontario, canada) in 1x tris-acetate-edta (tae) buffer for 45 min. the size of amplified products was estimated using 100 base pairs molecular weight marker (100–1200 base pairs). table 1: oligonucleotides primers and amplification reactions for targeted resistance genes. statistical analysis data analysis was performed with statistical package for social sciences version 20 (spss inc., chicago, illinois, united states). comparison of mean values was done using the student’s t-test for continuous variables and the chi-square test for categorical variables. risk factors for infection of diabetic foot by mdr organisms were identified by logistic regression analysis. logistic regression was used to determine predictive associations of variables that showed statistical significance by bivariate analysis. a p-value of less than 0.05 was considered to be statistically significant. results sociodemographic and clinical characteristics of patients ninety patients (53 male and 37 female) presented with 93 idfus during the 11-month study period. the patients ranged between 18 and 85 years (mean 54.7 ± 12.8 years) of age. of the 93 cases of foot infections, 56 (60.2%) had lasted for at least 1 month, and 70 (75.3%) were in-hospital patients. sixty-six (71.0%) of the ulcers were categorised as wagner’s grade 3 and above. most (n = 74; 82.2%) of the patients used antibiotics in the month preceding presentation at the healthcare facilities, while 93.3% (n = 84) had commenced antibiotics before collection of wound samples (table 2). table 2: association between clinical and sociodemographic variables of infected diabetic foot ulcer patients from tertiary healthcare facilities in osun state, nigeria, july 2016 to april 2017. predictors and treatment outcomes of multidrug-resistant bacterial infections in infected diabetic foot ulcer significant factors associated with the presence of mdr organisms in diabetic foot infections included peripheral sensory neuropathy, a foot infection duration of more than a month and admission duration of more than a month (table 2). further analysis with logistic regression however identified only peripheral neuropathy (adjusted odds ratio = 4.05, 95% ci: 1.08–15.13) and foot infection duration of more than a month (adjusted odds ratio = 7.63, ci: 1.64–35.39) as the predisposing factors for acquisition of multidrug-resistant bacteria among patients with idfu. a substantial proportion (33/70; 47.1%) of the inpatients had poor treatment outcomes; poor outcomes noted in 53.4% (31/58) of patients with mdr infections included major limb amputation (below and above knee amputation) (18/58; 31.0%) and death (13/58; 22.4%). infections by these mdr bacteria have significant association with poor treatment outcomes (adjusted odds ratio = 5.11, 95% ci: 1.23–29.67). distribution of isolates among diabetic foot cases all 93 wound specimens obtained from the 90 patients (three patients had bilateral foot ulcers) in the three healthcare facilities were positive on bacterial culture with only 10 (10.8%) of them yielding a single organism each. among the polymicrobial cultures, 45 (48.4%) yielded two organisms per culture, 34 (36.6%) yielded three organisms per culture while 4 (4.3%) yielded four organisms per culture. results further showed that there was a total of 218 organisms isolated from the 93 specimens cultured with an average of 2.34 organisms per sample. of the organisms, 129 (59.2%) were gram-negative aerobic and facultative anaerobic bacilli, 59 (27.1%) were gram-positive aerobic cocci and 29 (13.3%) were anaerobic bacteria; only 1 (0.5%) was yeast. s. aureus (34; 15.6%) was the single most common organism followed by e. coli (23; 10.6%) and pseudomonas aeruginosa (20; 9.2%). others included klebsiella spp. (19; 8.7%), citrobacter spp. (19; 8.7%), enterococcus spp. (14; 6.4%), enterobacter spp. (11; 5.0%), proteus mirabilis (10; 4.6%) and acinetobacter spp (9; 4.1%). on the other hand, the predominant anaerobic bacteria were bacteroides spp. (7; 3.2%) and p. anaerobius (6; 2.8%) as shown in table 3. table 3: bacterial aetiological agents of infected diabetic foot ulcers in tertiary healthcare facilities in osun state, nigeria, july 2016 to april 2017. antibiotic resistance pattern of aerobic isolates gram-positive bacteria were highly resistant to trimethoprim-sulfamethoxazole (69.5%), penicillin g (66.1%) and gentamicin (40.1%) but demonstrated low-level resistance to piperacillin/tazobactam (6.8%) and amikacin (10.2%). on the other hand, gram-negative bacteria were highly resistant to the third-generation cephalosporins which included ceftriaxone (56%), cefotaxime (55%) and ceftazidime (48.1%), as well as trimethoprim-sulfamethoxazole (89%), gentamicin (54.3%) and ciprofloxacin (54.3%). low rates of resistance were however shown to ertapenem (6.4%), piperacillin/tazobactam (9.3%) and amikacin (12.4%). prevalence and pattern of multidrug resistance among bacterial isolates in infected diabetic foot ulcer further analysis of resistance profiles in the organisms showed that of the 188 aerobic isolates, 121 (64.4%) were mdr, being resistant to one or more agents in at least three antibiotic classes (table 4). the prevalence rates of mdr among gpc and gnb were 55.9% and 68.2%. multidrug resistance rates were generally high among the isolated bacteria especially acinetobacter species (88.9%), enterococcus species (84.6%), enterobacter species (81.8%) and citrobacter species (73.7%). overall prevalence of mdr bacteria among the idfu cases was 80% (n = 74) with rates among in-patient and outpatient cases being 82.9% (n = 58) and 69.6% (n = 16). twelve (35.3%) s. aureus were methicillin-resistant, while of the 129 gnb, 43 (33.3%) were esbl-producing and 10 (7.8%) were carbapenem-resistant (table 5). high esbl production rates were seen among enterobacter species (54.6%), klebsiella species (52.6%), citrobacter species (52.6%) and e. coli (43.5%). other esbl-producing species found were hafnia alvei (1; 20.0%), providencia spp. (1; 33.0%) and morganella morganii (2; 28.6%). ampc β-lactamase production was detected among citrobacter spp. (1; 5.3%), e. coli (2; 8.7%) and m. morganii (1; 14.3%). carbapenem resistance was seen among acinetobacter baumannii (4; 44.4%), h. alvei (2; 40.0%), p. aeruginosa (3; 15.0%) and m. morganii (1; 14.3%). further tests showed that among the 10 carbapenem-resistant isolates, only four, h. alvei (2/4) and a. baumannii (2/4), were carbapenemase-producing. table 4: prevalence of multidrug resistance among bacterial isolates from infected diabetic foot ulcer patients from tertiary healthcare facilities in osun state, nigeria, july 2016 to april 2017. table 5: types and mechanisms of antibiotic resistance among bacterial agents of infected diabetic foot ulcer from tertiary healthcare facilities in osun state, nigeria, july 2016 to april 2017. detection of resistance genes ten (83.3%) of the 12 mrsa isolates harboured the meca gene. at least one of the esbl genes investigated was detected in 86.0% (n = 37) of the 43 esbl-producing organisms. the most common esbl gene detected was blactx-m, harboured by 30 (69.8%) of the phenotypically confirmed esbl-producing isolates. others were blatem (27; 62.8%) and blashv (8; 18.6%) (table 6). thirty-one (72.1%) of the esbl-producing gnb had at least two esbl genes. four of the 10 carbapenem-resistant species were phenotypically confirmed to be carbapenemase-producers. carbapenemase and metallo-beta-lactamase genes were detected in all of the four phenotypically confirmed carbapenemase-producers; they were blavim (n = 3), blakpc (n = 2) and blandm (n = 1). these genes were detected in all of the carbapenemase-producing h. alvei (2/2) and a. baumannii (2/2). table 6: detection of extended-spectrum beta-lactamase genes among phenotypically confirmed extended-spectrum beta-lactamase producing isolates from tertiary healthcare facilities in osun state, nigeria, july 2016 to april 2017. discussion this study shows that a wide range of bacteria are agents of infection of diabetic foot ulcers; it also reveals the high level of antibiotic resistance among the aerobic and facultative anaerobic bacteria with a large proportion of patients having multidrug-resistant infection leading to poor treatment outcomes. infected diabetic ulcers continue to be polymicrobial infections involving aerobic as well as obligate anaerobic organisms. infected diabetic foot ulcers in this study have an average of two different bacteria implicated in the disease, and this is typical of diabetic foot infections across sub-saharan africa and asia.29 gram-negative aerobic bacteria including e. coli, p. aeruginosa, klebsiella species and enterobacter species predominate, reflecting the long-standing nature of these infections as a consequence of poor health-seeking behaviour in low-resourced developing countries.8,30 gram-negative bacteria are more commonly implicated in infected diabetic ulcers in developing countries where most patients present late to healthcare facilities with advanced diseases.29 furthermore, a wide range of anaerobic bacteria primarily bacteroides species and p. anaerobius are important agents of the infections and were isolated from a third of the cases in this study. this suggests infections that are chronic and below the superficial layers of the skin.31 anaerobic bacteria account for 13.3% of the organisms isolated in this study, a higher prevalence than previously reported in the institution32 and may be attributable to deployment of a better anaerobic culture method in which specimens were processed and incubated in a bactron anaerobic chamber. higher prevalence rates of obligate anaerobes were however reported by ikeh et al. in jos, nigeria (17%),33 and al-benwan in kuwait (15.3%).34 low rates noted by richard et al. (1%)35 in france and yates et al. (1%)36 in australia may be due to the fact that most patients tend to seek medical care early enough in countries with good health insurance coverage which will enable a higher proportion to present with low-grade foot infection.37 antibiotic resistance remains a huge problem among diabetic foot ulcer infections; it worsens prognosis and makes treatment outcomes poor.38 multidrug-resistant bacteria were common (74/93; 80%) among idfu cases in this study, and this is possibly due to inappropriate antibiotics use and unrestricted access to antimicrobial drugs in many lowand middle-income countries.39 this is similar to findings in studies conducted in other developing countries40,41 but contrasts findings of several studies in high-income countries including france which reported low prevalence of mdr bacteria among patients with idfu.38,42 a wide spectrum of aerobic bacteria isolated in this study were found to be multidrug-resistant, comparable to findings elsewhere in africa and asia with high mdr rates involving mainly s. aureus, enterobacteriaceae, and p. aeruginosa.41,43 one in every three isolates of s. aureus in this study was mrsa. although prevalence of mrsa appears to be rising in africa, most of the countries have rates lower than 50%.44 our study also revealed that meca, the most common determinant that confers methicillin resistance on s. aureus, was detected in 83.3% of the mrsa strains and this is similar to the observation of chaudhry et al. who detected the gene in 20 (84%) of the 25 phenotypically confirmed mrsa isolates.45 mrsa strains that lack the meca gene may demonstrate methicillin resistance on account of alternate mechanisms of penicillin resistance such as the possession of mecc, a variant of meca discovered in 2011, or other mutations in genes encoding penicillin-binding proteins.46 extended-spectrum β-lactamases, which confer resistance to expanded-spectrum cephalosporins, were produced by 33.3% of all gnb isolated; all but two of the esbl-producing gnb belonged to the family enterobacteriaceae and included e. coli, klebsiella and citrobacter species. the burden of esbl-producing gnb is enormous among patients with idfu especially in poor-resourced countries with prevalence rates being reported to range from 23% to 49% across africa and asia.22,43,45,47,48 the most prevalent esbl gene was the ctx-m which has been reported as the most predominant variant worldwide.49 in the present study, only 10 (7.8%) of the gram-negative bacteria were resistant to the carbapenems. carbapenem resistance-determining genes were present in a. baumannii, h. alvei and m. morganii. carbapenems as drugs of last resort in the treatment of resistant gnb infections have variable but increasing rates of resistance.13 independent risk factors for acquisition of mdr bacteria found in our study are peripheral sensory neuropathy and foot infection duration longer than a month. peripheral neuropathy does not only make diabetics susceptible to foot ulceration but also makes insensate (neuropathic) foot ulcers become more extensive due to continuous painless trauma. loss of protective pains could cause patients not to present to healthcare facilities early enough. in developing countries, such patients with more chronic infections (> 1 month duration) would have engaged in self-prescribed antibiotic use for a prolonged period of time leading to selective pressure and emergence of mdr foot infection.39,50 this is similar to reports among idfu cases from india.40,51,52 other authors also documented the prolonged duration of wound infection as a predictor of infection of diabetic foot ulcers with mdr bacteria.53,54 contrary findings have however been documented from other studies in china, iran and portugal.41,42,55 our finding is also discordant with the report of noor et al. which established that ulcer size is a risk factor for infection by mdr organisms.54 this study also observed a significant association between presence of mdr bacteria in idfu and long duration of hospitalisation (> 1 month) similar to previously documented reports by another author in turkey.14 we did not find any sociodemographic factors that were significantly associated with the occurrence of mdr idfu in our study in agreement with other reports.40,52,53 in contrast, trivedi et al. in the united states noted smoking as an independent risk factor for multidrug-resistant foot wound infection.56 furthermore, in this study, infection of diabetic foot ulcers by mdr pathogens was found to have a significant association with poor treatment outcomes including major limb amputation and mortality. in agreement with our findings, the adverse effects of mdr diabetic foot infection on treatment had been underscored in a systematic review and meta-analysis of data from 28 studies reporting a treatment failure rate of 22.7% and significant association between mdr foot infections and treatment failure.57 limitations the limitation of the study was that the number of patients recruited was limited to 90 and this was because the study was time-bound. also, outpatients could not be followed up because of the multicentre nature of the study. resistance profiles of obligate anaerobic bacteria could not be determined and whole genome sequencing (for strain relatedness) was also not done due to lack of financial support for the study. conclusion the spectrum of agents causing idfu is wide and includes numerous species of aerobic and anaerobic bacteria. there is a high prevalence of mdr aerobic bacteria among them which poses a great limitation to the effective treatment of cases. acknowledgements the authors are grateful to faith bankole and ayo isinkaye for assistance in data management and babatunde odetoyin for technical assistance in the central science laboratory, obafemi awolowo university, ile-ife, nigeria. competing interests the authors have declared that no competing interests exist. authors’ contributions a.t.a. and a.o.a. conceived and designed the study. b.k. and v.o.r. contributed to the design of the study. a.t.a. and a.o.a. conducted laboratory experiments. a.t.a. and a.o.a. analysed the data. a.t.a., a.o.a. and v.o.r. wrote the final report. all authors reviewed and approved the final report. sources of support this study was supported by the obafemi awolowo university teaching hospitals complex. data availability the data sets used and analysed during the current study are available from the corresponding author on reasonable request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references lipsky ba, senneville e, abbas zg, et al. guidelines on the diagnosis and treatment of foot infection in persons with diabetes (iwgdf 2019 update). diabetes metab res rev. 2020;36(s1):e3280. https://doi.org/10.1002/dmrr.3280 zhang p, lu j, jing y, tang s, zhu d, bi y. 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smd, kamal ma. antibiotic resistance and extended spectrum beta-lactamases: types, epidemiology and treatment. saudi j biol sci. 2015;22(1):90–101. https://doi.org/10.1016/j.sjbs.2014.08.002 levin me. an overview of the diabetic foot: pathogenesis, management and prevention of lesions. inter j. diab dev countr. 1994;14:39–47. valappil rk, krishnan s, bai sdk. multidrug resistant organisms in diabetic foot ulcers-analysis of risk factors and clinical outcome. j med sci clin res. 2017;9(2):18138–18143. https://doi.org/10.18535/jmscr/v5i2.144 zubair m, abida m, jamal a. clinicobacteriology and risk factors for the diabetic foot infection with multidrug resistant microorganisms in north india. biol med. 2010;2(4):22–34. hartemann-heurtier a, robert j, jacqueminet s, et al. diabetic foot ulcer and multidrug-resistant organisms: risk factors and impact. diabet med. 2004;21(7):710–715. https://doi.org/10.1111/j.1464-5491.2004.01237.x noor s, borse ag, ozair m, raghav a, parwez i, ahmad j. inflammatory markers as risk factors for infection with multidrug-resistant microbes in diabetic foot subjects. foot (edinb). 2017;32:44–48. https://doi.org/10.1016/j.foot.2017.05.001 ji x, jin p, chu y, feng s, wang p. clinical characteristics and risk factors of diabetic foot ulcer with multidrug-resistant organism infection. inter j lower extrem wounds. 2014;13(1):64–71. https://doi.org/10.1177/1534734614521236 trivedi u, parameswaran s, armstrong a, et al. prevalence of multiple antibiotic resistant infections in diabetic versus nondiabetic wounds. j pathog. 2014;2014:173053. http://doi.org/10.1155/2014/173053 vardakas kz, horianopoulou m, falagas me. factors associated with treatment failure in patients with diabetic foot infections: an analysis of data from randomized controlled trials. diabetes res clin pract. 2008;80:344–351. https://doi.org/10.1016/j.diabres.2008.01.009 abstract introduction methods results discussion acknowledgements references about the author(s) timothy m. mtonga department of biomedical informatics, school of medicine, university of pittsburgh, pittsburgh, pennsylvania, united states faheema e. choonara kamuzu central hospital, lilongwe, malawi jeremy u. espino department of biomedical informatics, school of medicine, university of pittsburgh, pittsburgh, pennsylvania, united states chimwemwe kachaje baobab health trust, lilongwe, malawi kenneth kapundi baobab health trust, lilongwe, malawi takondwa e. mengezi baobab health trust, lilongwe, malawi soyapi l. mumba baobab health trust, lilongwe, malawi gerald p. douglas department of biomedical informatics, school of medicine, university of pittsburgh, pittsburgh, pennsylvania, united states citation mtonga tm, choonara fe, espino ju, et al. design and implementation of a clinical laboratory information system in a low-resource setting. afr j lab med. 2019;8(1), a841. https://doi.org/10.4102/ajlm.v8i1.841 note: additional supporting information may be found in the online version of this article as online appendix 1. lessons from the field design and implementation of a clinical laboratory information system in a low-resource setting timothy m. mtonga, faheema e. choonara, jeremy u. espino, chimwemwe kachaje, kenneth kapundi, takondwa e. mengezi, soyapi l. mumba, gerald p. douglas received: 30 may 2018; accepted: 28 june 2019; published: 28 oct. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: reducing laboratory errors presents a significant opportunity for both cost reduction and healthcare quality improvement. this is particularly true in low-resource settings where laboratory errors are further exacerbated by poor infrastructure and shortages in a trained workforce. informatics interventions can be used to address some of the sources of laboratory errors. objectives: this article describes the development process for a clinical laboratory information system (lis) that leverages informatics interventions to address problems in the laboratory testing process at a hospital in a low-resource setting. methods: we designed interventions using informatics methods for previously identified problems in the laboratory testing process at a clinical laboratory in a low-resource setting. first, we reviewed a pre-existing lis functionality assessment toolkit and consulted with laboratory personnel. this provided requirements that were developed into a lis with interventions designed to address the problems that had been identified. we piloted the lis at the kamuzu central hospital in lilongwe, malawi. results: we implemented a series of informatics interventions in the form of a lis to address sources of laboratory errors and support the entire laboratory testing process. custom hardware was built to support the ordering of laboratory tests and review of laboratory test results. conclusion: our experience highlights the potential of using informatics interventions to address systemic problems in the laboratory testing process in low-resource settings. implementing these interventions may require innovation of new hardware to address various contextual issues. we strongly encourage thorough testing of such innovations to reduce the risk of failure when implemented. keywords: low-resource setting; laboratory testing; laboratory information system; malawi; informatics interventions. introduction laboratory testing plays a vital role in clinical decision-making. it is estimated that up to 70% of medical decisions in high-resource healthcare settings are made based on clinical laboratory test results.1,2 even though access to clinical laboratory services is comparatively lower in low-resource settings, studies show that clinicians in low-resource settings also make most decisions based on laboratory testing.3,4 despite the importance of laboratory test results in clinical decision-making, little effort has been made in low-resource settings to improve the entire laboratory testing process, which starts when the test is first ordered and ends when the results are interpreted and a clinical decision is made.5 laboratory errors include a wide variety of mistakes in the testing process and have no universally accepted definition. we define a laboratory error as any event or mistake that leads to failure to perform a laboratory test, misdiagnosis of a laboratory test, or delayed reporting of laboratory test results. in 2001, it was estimated that laboratory errors accounted for $200 million – $400 million in american healthcare expenditures per annum.6 since then, the rate of utilisation of laboratory services has increased, making the reduction of laboratory errors a significant opportunity for cost reduction and healthcare quality improvement. recent studies have tried to categorise errors using phases of the total testing process, which comprises pre-analytical, analytical and post-analytical phases.7 the pre-analytical phase covers all activities from when the test is ordered to when the specimen is delivered to the laboratory for testing. the analytical phase covers the activities involved in the actual testing of the specimen and the post-analytical phase involves the reporting and interpretation of the laboratory result. among the phases of the total testing process, it has been observed that most laboratory errors happen outside of the analytical phase.8 an example of an error outside the analytical phase is the mislabelling of a specimen, which could happen during the drawing of a sample in the pre-analytical phase. while error rates vary between health facilities, it is estimated that 32% – 75% of all laboratory errors happen in the pre-analytical phase.9 error rates in the analytical phase are estimated in the range of 13% – 32% and in the post-analytical phase in the range of 9% – 31%.9 informatics interventions may be useful in reducing such laboratory errors. examples of such interventions are computer-aided ordering of laboratory tests, barcode labelling of specimen tubes, and automating the reporting of laboratory test results. these interventions are often provided using computer systems that allow physicians to order diagnostic tests, medications, and other procedures, commonly referred to as computerised provider order entry.10 computerised provider order entry is often a part of a larger electronic health record system. however, such comprehensive electronic health record systems have low penetration in low-resource settings where the burden of disease is high and laboratory errors are further exacerbated by poor infrastructure, shortages in trained workforce and informational challenges.1,11 although laboratory information systems (lis) have been shown to help reduce laboratory errors, little information is available on the implementation of these in low-resource settings. furthermore, most descriptions of lis implementations in low-resource settings focus on the analytical phase of the total testing process.12 in this article, we describe preliminary work in developing a lis that addresses problems using informatics interventions to support all phases of the total testing process in a low-resource setting with no pre-existing computerised provider order entry system. methods ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. setting we implemented a lis at the kamuzu central hospital (kch) in lilongwe, malawi, between january and march 2015. the kamuzu central hospital is a 750-bed government-operated referral hospital. the laboratory at kch comprises eight departments: microbiology, parasitology, serology, haematology, molecular biology, blood bank, flow cytometry and biochemistry. these departments perform laboratory tests for both outpatients and inpatients at the hospital and conducted 242 242 tests between 01 july 2010 and 30 june 2011.13 the system described in this article was piloted in the outpatient tuberculosis clinic of the hospital and the microbiology department of the laboratory starting 31 march 2015. user requirements and system capabilities requirements for the lis were provided by laboratory technicians in the form of user stories. a user story is a succinct way of representing a task that a user will want to perform using an information resource.14 it includes the role of the user, the task or action and the benefit, goal or achievement. an example of a user story in this context is: as a laboratory technician, i want to know which specimen was drawn first so that i can prioritise it for analysis to reduce the number of non-viable specimens. we compiled a consolidated list of user stories for each phase of the total testing process. we used that list to define a set of functionality requirements from the laboratory technicians. to ensure that no core functionality was omitted from the specifications, we leveraged the laboratory information system functionality assessment toolkit (lis-fat) developed by the association of pathology informatics. this assessment toolkit provides 850 declarative statements that describe the functions that a lis should possess.15 an example of a functionality statement from lis-fat is: a laboratory information system should provide intelligent sample labelling that groups samples based on the test to be done and prints them out. the lis-fat was originally intended for use as a lis evaluation checklist. however, in our implementation, we repurposed it to define capabilities for the proposed system. furthermore, we recognised that the lis-fat was primarily developed for use in a setting with adequate resources and some aspects of it may not be well suited for a low-resource setting. we therefore assessed the lis-fat functionality statements and selected those that focused on direct user needs and were most applicable in a low-resource setting. special effort was made to ensure that major functional categories of the lis-fat were not overlooked. this resulted in a customised lis-fat applicable to a low-resource setting, with the declarative statements describing the core requirements for lis in this setting. to elucidate the dependencies that could drive the design phase, all functionality statements created in this step were grouped into high, medium, and low priority categories by a group of laboratory management personnel. this helped determine the order in which the functionality would be implemented to ensure that the most important functionality was implemented first. system design and development laboratory information system software can be commercial, open-source, or home-grown. we chose to build on existing open-source lis software and customise it based on our functional requirements. before any functionality was implemented, we conducted a design validation study of two open-source lis software systems to determine the extent to which they implemented the required functionality for the kch laboratory.16 these systems were open enterprise laboratory information system (openelis) and basic laboratory information system (blis).12,17 we assessed and ranked the systems based on the number of functionality requirements that they satisfied for the lis implementation at kch. a functionality requirement was considered satisfied if the lis had a feature that could be used to achieve the goal of that requirement. the choice between the systems was based on the total number of required functions that each of the systems possessed. the system with the most functionality requirements was selected as the foundation upon which the lis implementation at kch would be built, and a comprehensive design was made around it to ensure that all functional requirements were met. we also realised that information systems frequently emphasise the collection and use of data for reporting purposes and streamlining workflow. however, the use of such systems does not necessarily result in improved outcomes. to maximise the value of the lis, we considered problems identified in the laboratory testing process and described in previous publications.11,13 targeted informatics interventions were developed and incorporated into the system’s design to address each of these problems. upon completion of the system design, a team of three developers (c.k., k.k., t.m.m.) iteratively implemented and integrated the remaining functionality over eight weeks from mid-january to mid-march 2015. during this time, clinicians and laboratory personnel provided initial feedback which we used to refine the user interfaces for the new features. results user requirements and system capabilities a list of 34 user stories was compiled and mapped into functionality statements for the kch lis implementation. an additional 41 statements were added from our review of the lis-fat statements. the selected lis-fat statements had direct user benefits in keeping with the user stories provided by laboratory technicians and were most suitable for lis implementation in low-resource settings. these 75 statements formed the core functionality requirements for the lis implementation at kch. system design and development in our design validation study, we independently assessed blis and openelis against our set of 75 functionality requirements for the kch lis. the basic laboratory information system met 25 (33%) of the functionality requirements and openelis met 22 (29.3%). a detailed breakdown of the functionality that each system had is presented in table 1. table 1: functionality assessment of two open-source laboratory information systems for the kamuzu central hospital laboratory testing process, malawi, 2015. following the design validation study, blis was selected as the base software for the lis implementation at kch. to support the pre-analytical and post-analytical phases, we built clinician-facing laboratory order entry and results reporting software modules. since functionality was already delineated by phases, each phase could be easily conceptualised as an independent component in a larger system. the decision to adopt a modular approach was further driven by the understanding that modules are easier to maintain than a single monolithic system. with the modular approach, any software module can be easily replaced should a more suitable alternative be identified. for example, if another type of lis software was chosen to replace the customised blis at kch, it could be easily integrated because of the modular approach. this provides flexibility for future improvements of the system. each module also addressed specific challenges with targeted informatics interventions. a summary of these is provided in table 2. table 2: problems in the laboratory testing process and interventions implemented in the laboratory information system to address them at the kamuzu central hospital, malawi, 2015. developing bedside solutions to facilitate the bedside use of the lis, we designed custom hardware in the form of a mobile workstation that clinicians could use for ordering laboratory tests and reviewing laboratory results. the mobile workstation is equipped with a low-cost 9-inch tablet computer, a barcode scanner, and a thermal label printer as shown in figure 1. to provide complete mobility, the workstation is powered by batteries and does not need to be plugged in to a power outlet during use. the batteries are charged between ward rounds when the mobile workstation is not in use. the mobile workstation also provides room for the clinicians and nurses to easily carry around all medical supplies and consumables required for specimen collection during ward rounds. the provision of space for medical supplies was made as a value addition for the medical personnel and eliminated the need for a separate cart for medical supplies. figure 1: a mobile workstation equipped with a tablet computer, label printer and barcode scanner, kamuzu central hospital, malawi, 2015. to provide visibility into the status of laboratory tests and results at each stage of the testing process, we also built a dashboard application. this application runs on raspberry pi mini-computers connected to 23-inch screens that are mounted in relevant work areas both in the laboratory as well as in the hospital wards. on the screen, we display context-specific work lists such that each user only sees the processes in which they are involved and on which they must act. for example, the dashboard in the microbiology department only shows specimens that require microbiological tests and not any other specimens. a screenshot of the dashboard is provided in figure 2. figure 3 depicts how the dashboard application, blis and the laboratory order entry and results reporting modules are integrated. figure 2: an example of a nursing station dashboard at the kamuzu central hospital, malawi, 2015. figure 3: architecture of laboratory information system implementation at the kamuzu central hospital, malawi, 2015. laboratory testing workflow with the laboratory information system the testing process begins with the clinical decision to order a laboratory test to assess the patient’s condition. to initiate an order, the clinician uses a unique national patient identifier in the form of a barcode to open the patient’s record in the order entry module. patient identifiers are issued to patients on arrival at the hospital after completion of a one-time patient registration process, which generates an adhesive label to be affixed to a patient’s personal health passport. a health passport is a paper-based continuity of care document kept by the patient. the framework for uniquely identifying patients at kch has been described in detail elsewhere.18 scanning the patient’s barcode opens the patient’s summary in the order entry module displaying the patient’s past test orders and their status, including test results when available. from this page, the clinician can place new test orders and initiate the pre-analytical phase of the total testing process. in addition to test ordering, the order entry module also maintains and displays an up-to-date catalogue of all the tests that are currently being offered at the facility. this intervention helps prevent ordering of tests that are unavailable in the laboratory. once the test order has been placed through the order entry module, a health level 7 message is sent to the blis server via a health level 7 message router to record the test order. the blis server responds by issuing an accession number for the specimen, which is printed on a label in barcode and human readable form together with other test order details during specimen collection. the label is then manually affixed to the specimen container. this accession number is used to track the specimen throughout the testing process. the time at which the specimen label is printed is used in the system as the approximate time when the specimen was collected. once the order has been placed, it appears on the relevant nursing station dashboard as well as the laboratory reception dashboard. this was done to provide a visual cue for the laboratory receptionist to anticipate incoming specimens from the ward while at the same time reminding nursing station staff that they need to collect and transport the specimens to the laboratory. once a specimen has been collected, the dashboard displays its viability based on how long it has been since it was drawn. this serves as a reminder for the nursing staff to bring the specimens to the laboratory on time. the specimen will continue to show on the nursing station dashboard until it has been received at the laboratory reception. when the specimen arrives at the laboratory, the laboratory receptionist scans the barcode and performs a visual inspection of the specimen container and test order documentation. based on this, the receptionist determines whether the specimen should be accepted or rejected. for example, a specimen can be rejected if it is no longer viable depending on when it was drawn and the type of test that was ordered. when a specimen is rejected, a notification appears on the nursing station dashboard informing the nursing staff to redraw the specimen. if the specimen is accepted at laboratory reception, it is sent to the appropriate laboratory department for analysis and an entry is added to that department’s dashboard. this is the beginning of the analytical phase. the department dashboard acts as a dynamic worklist informing laboratory technicians of tests that need to be run and results that have yet to be recorded. once a specimen has been analysed, the laboratory technician enters the result using a touchscreen workstation in the laboratory department to complete the analytical phase of the testing process. the nursing staff are notified of the new result through the nursing station dashboard and can now print out the test result and affix it to the patient’s health passport or medical chart for review by the clinician. discussion in this article, we have described the process used to implement a lis in a low-resource setting, specifically at kch in lilongwe, malawi. we aimed to demonstrate a problem-driven approach that implements individual informatics interventions to support the laboratory testing process in a low-resource setting. we demonstrated this by piloting a system that supported the entire testing process for outpatient tuberculosis screening tests at kch. an example of a problem that was addressed in this implementation is the incorrect use of specimen containers for various tests. to address this problem, a picture of the correct container and required specimen volume for each test type was presented to the users during specimen drawing as an electronic job aid. these two interventions addressed the cause of 84% of all untestable specimens at kch that we reported in a previous article.13 while the pilot implementation in the outpatient tuberculosis clinic achieved our main goal, it also limited our ability to measure the impact of the interventions. for instance, sputum is the only specimen type collected in the outpatient tuberculosis clinic at kch. therefore, we could not measure the impact of the specimen container decision support on specimen viability. furthermore, since this ward serves outpatients, the laboratory turnaround time for these tests is not an accurate measure of process efficiency as the review of the results depends on when the patient returns to the hospital, and not when the actual test result itself becomes available. despite these limitations, there are several points worth noting from this work. a set of 75 functionality requirements (online appendix 1) for lis in low-resource settings was produced as part of this project. this contains significantly fewer requirements than the 850 statements found in the lis-fat document, which are more appropriate for high-resource settings. these 75 requirements can be used by other implementers in low-resource settings to design and evaluate their own lis. a further point can be made about the actual design of the implementation. the use of distinct modules separated by the phases of the total testing process offers several benefits. not only can the modules be more easily maintained, they can also be easily replaced should better alternatives be identified. this offers significant benefits going forward as the implementation is not tightly coupled to any single piece of software. this implementation further highlights the benefits that open-source software provides with regard to systems implementation in low-resource settings. software development takes time and is expensive. however, using existing open-source software has the potential to vastly reduce both effort and cost. for instance, design and development took only 10 weeks because existing software was reused for some parts of our system. this could have been significantly longer if everything was built from scratch. therefore, we recommend that other implementers in low-resource settings find ways of making use of the many open-source products in the health informatics community as this can reduce their effort and expenditure. using cheaper alternatives is a common approach to cost reduction. in this implementation, we did this by using tablets that cost $60.00 for the workstations instead of the $650.00 touchscreen clinical workstations that we have used in the past. the tablets presented a significant price reduction and other desirable qualities like the ability to easily be mounted on the mobile workstation. there seemed to be no significant problems with the tablets during the testing phase. however, when we deployed them in the hospital, the tablets often stopped responding and would occasionally power down during use without warning. this led to the loss of all current work that clinical staff had done related to the current patient or specimen and was very disruptive to the workflow. our experience with the tablets emphasises the need for rigour in the testing of new hardware before deployment. in the next iteration, we will address this by replacing the tablets with raspberry pi mini-computers and 10.1-inch touchscreen displays. we have comprehensively tested this solution and believe that these new computers will perform more reliably than the tablets and will not significantly inflate the expenditure on the project as they cost less than $200.00 each.19 the main limitation of this implementation was our inability to measure the impact that the interventions had on various laboratory key performance indicators such as turnaround times for laboratory tests. in addition, we did not deploy the mobile workstation in the inpatient wards for use during ward rounds. this was mainly due to dependencies that had to be met before deploying the system to inpatient wards at kch. in the future, we intend to perform field usability evaluations for the mobile workstations and problem impact studies to quantify the effect of the various interventions on laboratory key performance indicators. lessons learnt from this pilot have informed the continuing scale-up of lis implementations in malawi. a revised version of this system has now been deployed in three central hospitals and four district hospitals.20 revisions have focused on improving operations in the analytical phase by interfacing instruments to the lis. future efforts will focus on maximising the benefits in the pre-analytical and post-analytical phases where most laboratory errors occur. acknowledgements the authors would like to thank the clinical and laboratory staff at kamuzu central hospital (kch), and the laboratory information management systems program secretariat. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions t.m.m. drafted the manuscript and assisted in the design and development of all the required software components. f.e.c. coordinated the implementation in the kamuzu central hospital (kch) laboratory and influenced the design through original contributions and critical feedback. j.u.e. and s.l.m. analysed open-source laboratory information system software for potential use at kch. c.k. and k.k. assisted in the design and development of all the required software components. t.e.m. coordinated project activities and oversaw implementation. g.p.d. conceived the original idea and designed custom hardware for the implementation. all authors critically revised the manuscript. sources of support this project was supported by the president’s emergency plan for aids relief through the centers for disease control and prevention under the terms of 3u2ggh000729-02s1. the authors acknowledge the united states centers for disease control and prevention which funded the development of the lis at kch through their cooperative agreements with the malawi ministry of health and baobab health trust. data availability statement all software described in this article has been made available on github. the list of functionality statements for laboratory information systems in low-resource settings has been included as supplemental material (online appendix 1). no further data were created or analysed in this study. disclaimer the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the funding agencies. references becich mj. information management: moving from test results to clinical information. clin leadersh manag rev. 2000;14(6):296–300. hallworth mj. the ‘70% claim’: what is the evidence base? ann clin biochem. 2011;48(6):487–488. https://doi.org/10.1258/acb.2011.011177 wilson ml, fleming ka, kuti ma, et al. access to pathology and laboratory medicine services: a crucial gap. lancet. 2018;391(10133):1927–1938. https://doi.org/10.1016/s0140-6736(18)30458-6 moyo k, porter c, chilima b, et al. use of laboratory test results in patient management by clinicians in malawi. afr j lab med. 2015;4(1):8. https://doi.org/10.4102/ajlm.v4i1.277 price cp, john as, christenson r, et al. leveraging the real value of laboratory medicine with the value proposition. clinica chimica acta. 2016;462:183–186. https://doi.org/10.1016/j.cca.2016.09.006 bologna l, hardy g, mutter m. reducing specimen and medication error with handheld technology. annual conference and exhibition; chicago, il: healthcare information and management society; 2001. plebani m. exploring the iceberg of errors in laboratory medicine. clinica chimica acta. 2009;404(1):16–23. 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https://doi.org/10.1145/2909609.2909657 driessen j, limula h, gadabu oj, et al. informatics solutions for bridging the gap between clinical and laboratory services in a low-resource setting. afr j lab med. 2015;4(1), 1426–1431. https://doi.org/10.4102/ajlm.v4i1.176 cohn m. user stories applied: for agile software development. massachusetts, us: addison-wesley professional; 2004. tuthill jm, friedman ba, balis uj, et al. the laboratory information system functionality assessment tool: ensuring optimal software support for your laboratory. j pathol inform. 2014;5. https://doi.org/10.4103/2153-3539.127819 evaluation methods in biomedical informatics [homepage on the internet]. charles p. friedman, springer [cited 16 december 2016]; available from: http://www.springer.com/us/book/9780387258898. monu r. design and implementation of a basic laboratory information system for resource-limited settings. [master’s thesis]. atalanta, ga: georgia institute of technology;2010. douglas gp, gadabu oj, joukes s, et al. using touchscreen electronic medical record systems to support and monitor national scale-up of antiretroviral therapy in malawi. plos med. 2010;7(8). https://doi.org/10.1371/journal.pmed.1000319 mtonga t, abaye m, rosko sc, et al. a comparative usability study of two touchscreen clinical workstations for use in low resource settings. j health informat afr. 2018;(2). https://doi.org/10.12856/jhia-2018-v5-i2-209 baobab health [cited 2019 may 7]. available from: http://baobabhealth.org/wherewework.php. abstract introduction methods results discussion acknowledgements references about the author(s) ivy j. mutai phage biology laboratory, institute of primate research, nairobi, kenya department of biochemistry, biotechnology and microbiology, faculty of pure and applied sciences, kenyatta university, nairobi, kenya angela a. juma phage biology laboratory, institute of primate research, nairobi, kenya martin i. inyimili department of human anatomy, university of nairobi, nairobi, kenya atunga nyachieo phage biology laboratory, institute of primate research, nairobi, kenya anthony k. nyamache department of biochemistry, biotechnology and microbiology, faculty of pure and applied sciences, kenyatta university, nairobi, kenya citation mutai ij, juma aa, inyimili mi, nyachieo a, nyamache ak. efficacy of diversely isolated lytic phages against multi-drug resistant enterobacter cloacae isolates in kenya. afr j lab med. 2022;11(1), a1673. https://doi.org/10.4102/ajlm.v11i1.1673 original research efficacy of diversely isolated lytic phages against multi-drug resistant enterobacter cloacae isolates in kenya ivy j. mutai, angela a. juma, martin i. inyimili, atunga nyachieo, anthony k. nyamache received: 13 july 2021; accepted: 04 may 2022; published: 11 aug. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: enterobacter cloacae causes nosocomial infections in 15% of patients in lowand middle-income countries with emergence of carbapenem resistance. the utilisation of bacteriophages for therapeutic purposes is crucial for eradicating these resistant bacterial strains. objective: this study evaluated the efficacy of lytic phages on bacterial isolates of e. cloacae and determined their stability in various physicochemical conditions. methods: twenty-nine lytic phages were isolated from the waste water of six informal settlements in nairobi county, kenya, from july 2019 to december 2020 and cross-reacted with 30 anonymised clinical isolates of e. cloacae. six phages were then selected for physicochemical property studies. phages were described as potent upon lysing any bacterial strain in the panel. results: selected phages were stable at 4 °c – 50 °c with a 5.1% decrease in titre in four of six phages and a 1.8% increase in titre in two of six phages at 50 °c. the phages were efficient following two weeks incubation at 4 °c with optimal activity at human body temperature (37 °c) and an optimal ph of 7.5. phages were active at 0.002 m and 0.015 m concentrations of ca2+ ions. the efficiency of all phages decreased with increased exposure to ultraviolet light. all phages (n = 29) showed cross-reactivity against anonymised clinical isolates of e. cloacae strains (n = 30). the most potent phage lysed 67.0% of bacterial strains; the least potent phage lysed 27.0%. conclusion: this study reveals the existence of therapeutic phages in kenya that are potent enough for treatment of multi-drug resistant e. cloacae. keywords: lytic phages; enterobacter cloacae; multi-drug resistance; nosocomial infections; nairobi county; kenya. introduction enterobacter cloacae is a gram-negative rod-shaped bacteria that is associated with nosocomial infections.1 this organism is naturally resistant to ampicillin, amoxicillin/clavulanic, cephamycin and the firstand second-generation cephalosporins due to chromosomally encoded ampc β-lactamase.1 enterobacter cloacae is a normal flora of the human gastrointestinal tract, which can cause opportunistic infections among immunocompromised individuals, the elderly and newborns.1,2 this organism causes septicemia, endocarditis, urinary tract infections, wound infections, and meningitis in newborns.1,2 these infections can lead to prolonged hospitalisation and higher cost of treatments, increased antibiotic use and pressure leading to the development of antimicrobial resistance in the hospital. in addition, the rates of morbidity and mortality among critical patients can also increase.1,2 enterobacter cloacae causes nosocomial infections that affect at least 7% of patients in high-income countries; such infections are twice as high (15%) in the lowand middle-income countries.3 enterobacter cloacae is ranked as the third most predominant organism causing nosocomial infections and the second most predominant carbapenem-resistant organism, according to studies conducted in the united states.1,4,5,6 according to nyangacha et al., e. cloacae resistance was observed among patients suffering from tungiasis, or jigger disease, a secondary infection in a study done in the western part of kenya with the following resistance profiles: ampicillin (75.0%), amoxicillin-clavulanic acid (25.0%), tetracycline (50.0%), ceftazidime (25.0%) and cefuroxime (25.0%).7 globally, the emergence and spread of carbapenemase producers e. cloacae has been reported with the prevalence rate of 59.5% being reported in japan and china in three tertiary hospitals.8,9,10 this record of a high rate of drug resistance is a worrying trend for this organism.10 there is a growing need to conduct active surveillance of e. cloacae, in order to control and prevent further spread of drug resistance to other low-prevalence countries.8,10,11 in order to control the current rise in antimicrobial resistance, it is clear that an alternative to the use of antibiotics is urgently needed. bacteriophages (phages) have been proposed as an alternative to antibiotics based on reported evidence.12,13 bacteriophages are obligate parasites; hence, they infect bacteria and form potential antimicrobial agents capable of killing bacteria, including the drug resistant strains.11,13,14 phages naturally multiply through feeding on the bacteria leading to their lysis which in the process regulates bacterial populations within the ecosystems.13,14,15 since phages are very specific to bacteria, their lysis process could be exploited for the development and production of new therapeutic agents.11 given this revelation, phage hunting is now pursued to aid in disease management, as an alternative to elimination and prevention of multi-drug resistant (mdr) strains.10,11 however, e. cloacae is a bacteria of medical importance, but there is limited information on the application of bacteriophages as alternative antimicrobial agents against this bacteria.1,2,5 additionally, there is scarce information on studies of lytic phages against e. cloacae in kenya. the efficacy of phages in treatment of disease-causing bacteria has been exhibited by numerous reports. on the other hand, there is scarce information about their physicochemical properties. furthermore, the stability of kenyan phages under various extreme conditions of alkalinity, acidity, high and low temperatures, ultraviolet exposure, salinity and storage has not been fully studied. we argue that determining the effect of external factors that could influence the yield and potency of phage preparations is important as one prepares phages as an alternative to antibiotics. this study, therefore, evaluated the efficacy of lytic phages in vitro on a panel of bacterial isolates of e. cloacae and their stability under different physicochemical environments.. methods ethical considerations ethical clearance was not required for this study, since there were no human subjects or animals models used in this research; however, this study was registered with the institutional research and ethics committee (iserc) of the institute of primate research (ipr/irc/2014). the bacterial isolates used in this study were anonymised clinical isolates obtained from the kenya medical research institute (kemri) center for microbiology (seru#2767), in nairobi, kenya. for environmental waste water collection, an approval was issued by nairobi water and sewerage company (nawasco#ncwsc/trg14/109). sample collection environmental waste water collection was done by the institute of primate research phage biology group from july to august 2019. samples were collected from six informal settlements: kibera, dandora, kariobangi, huruma, mathare and korogocho, all in nairobi county, kenya. three samples per settlement were collected making a total of 18 samples. dark screw cap containers (to prevent direct light) were used to collect the samples which were transported to the institute of primate research phage laboratory using cooler boxes as the secondary containment followed by storage at 4 °c and processed within three weeks. the three samples per settlement were named using number designations in the following order: 1, 2 and 3. bacterial isolation and identification an mdr isolate of e. cloacae isolated from environmental waste water was used for phage isolation. this bacterium was identified using culture media, microscopic and biochemical examination using vitek ii machine (biomérieux, marcy-l’etoile, france) for identification and antimicrobial susceptibility of bacteria.16 a panel of anonymised clinical bacterial isolates (not evaluated for antimicrobial susceptility test) of e. cloacae (n = 30) were subjected to the phages to assess cross infectivity. phage isolation phages were isolated through an enrichment method according to the methods described by akhtar et al. and clokie et al. with slight modifications.17,18 briefly, 30 ml of each environmental waste water was centrifuged at 10 000 × gravitation for 10 min (centrific™, centrifuge fisher scientific, waltham, massachusetts, united states). the supernatant was mixed with an equivalent volume of tryptose soy broth (tsb) (himedia, mumbai, india), and inoculated with 1 ml of 18 h-old mdr e. cloacae culture. the mixture was incubated overnight at 37 °c in a shaker incubator at 120 rotations per minute (lab-line® incubator-shaker, waltham, massachusetts, united states). the cultures were then centrifuged at 10 000 × gravitation for 10 min, and the supernatant sterilised using a 0.22 µm syringe filter (millipore, merck, darmstadt, germany) and stored at 4 °c for use in the spot test. in vitro screening for phages (spot testing) phages were screened through spot test procedures according to clokie and kropinski (2010) with slight modifications.18 briefly, a lawn of 24 h-old mdr e. cloacae isolate (100 µl) was prepared in soft agar (0.7%) with tsb on a tryptose soy agar (tsa) plate (himedia, mumbai, india). ten-fold serial dilutions of the phage filtrate were prepared and 5 µl of each dilution was spotted on a well-labelled plate with dilutions ranging from 10−1 to 10−8 followed by overnight incubation at 37 °c. observation of plaques (clear-patched regions) on the bacterial lawn was recorded as positive for the phage. for plates with distinct plaques, the well-isolated plaques were harvested using a sterile pasteur pipette and suspended in 200 µl sterile saline magnesium (sm) buffer (100 mm sodium chloride, 10 mm magnesium sulphate, 50 mm tris-hcl, ph 7.5 and 0.01% weight by volume gelatin), vortexed and incubated at room temperature for 1 h before centrifuging at 4000 gravitation for 5 min to remove any remaining debris, labelled and stored at 4 °c. preparation and titration of phage lysate (plaque assay) for plates without well-isolated plaques, the double agar overlay method was employed with slight modifications.19 briefly, 100 µl of the 10-fold serial dilution of the lysate (from the spot with the least number of plaques) was mixed with 100 µl of 18 h-old e. cloacae in 6 ml molten agar (0.7% agar with tsb) and dispensed on tsa plate (1.5% agar with tsb) medium and allowed to solidify before incubation at 37 °c for 18 h. well-isolated plaques were identified and marked before harvesting each plaque using a sterile pasteur pipette, vortexed and incubated at room temperature for 1 h before centrifuging at 4000 gravitation for 5 min to remove any remaining debris, labelled well and stored at 4 °c. calculation of phage titre in plates with distinct plaques for the spot test and plaque assay, the plaques were counted respective to their dilution factors employing the following formula: effect of temperature on phage titre phage adsorption rates on the host bacterium were recorded at the temperatures 4 °c, 10 °c, 20 °c, 37 °c and 50 °c. then 100 µl of actively growing host strain cultures in tsb to an optical density of 600 nm (od600) of 0.6 was used to make an overlay on tsa plates. the selected phages were then incubated at these different temperatures for 1 h and then placed at room temperature (20 °c – 27 °c) for 30 min prior to performing spot tests. the outcomes were given as a log10 of phage titre.20,21,22 effect of ph on phage titre the effect of ph 2, 5, 7, 9, 11 and 13 on phage titre and viability of phages was studied in tsa plates by the spot test method.18 the sm buffer was adjusted to the desired ph by the use of naoh and hcl. the actual ph of the sm was determined with a ph meter (hanna instruments inc. woonsocket, rhode island, united states) and this was used as a control or standard. the adjusted sm buffers were used for serial dilutions of the phages under study. dilutions of the phage stocks were done to get the working dilution factors. roughly, 106 pfu/ml of 20 µl phages (individual phages) was added to 180 µl of sm buffer, after an earlier adjustment of ph (2–11), in eppendorf tubes, followed by 30 min of incubation at 37 °c. the remaining phages were determined by spotting the phages in different ph and the outcomes indicated as pfu/ml.23 influence of ca2+ ions on phage titre and phage stability the effect of the divalent cations on bacterial lysis and phage adsorption was investigated by varying the concentration of cacl2: 0.000 m, 0.005 m, 0.010 m and 0.015 m in soft agar during preparation. plaque assays were conducted in duplicate, followed by overnight incubation at 37 °c. phage titre was determined at the different salt concentrations as log10 and comparisons made with the increase in salt concentrations.21 storage stability of enterobacter cloacae phages stability of the phages during storage was investigated using a previously described method with slight modifications.24 briefly, 3 ml of the selected phages with known phage titre were aliquoted into 15 ml centrifuge tubes and wrapped with aluminium foil to prevent direct light and kept at –20 °c, 4 °c, and 37 °c for two weeks. the spot test method was used to determine the effectiveness and the efficiency of the phages after storage. effect of ultraviolet light on phage titre the spot test method was used to determine the effect of ultraviolet light on phage irradiation with various modifications.25 briefly, 10 µl of each phage of known titre from each site was aliquoted into five sterile pcr tubes and labelled as 0, 5, 10, 15 and 20 min of ultraviolet light exposure. the phages for each time period were placed in a biosafety cabinet level 2 (290 nm – 320 nm, bsc-2, haier, tokyo, japan) and the ultraviolet light turned on for the required time; the phages were then removed simultaneously. polymerase chain reaction (pcr) tubes have been found to permeate ultraviolet light rays and affect the integrity of dna; hence, the tubes were capped during this study. host range determination of phages to ensure the specificity of bacteriophages, their effect on other bacterial genera and species was investigated. the anti-bactericidal efficacy of individual phages (n = 29) was evaluated through the spot test method against each individual e. cloacae isolate (n = 30) as described by kutter (2009).23 staphylococcus aureus, a bacteria from another genera, was used as negative control. a volume of 5 µl of individual phage stock was spotted on a tsa plate with a lawn of 100 µl overnight cultured host bacteria in soft agar, which was examined for bacterial lysis after 18 h – 24 h. the spot tests were performed in duplicate. a clear zone was considered as a positive infection in the tests and negative with no cross-reactivity.26 a phage was termed ‘potent’ upon lysing any bacterial strain in the host range panel. a phage with the widest spectrum of lysis activity on the tested bacterial strains was termed the ‘most potent’, while a phage with the lowest spectrum of activity on the tested bacterial strains was termed the ‘least potent’. a bacteria that was sensitive to a phage infection was termed ‘susceptible’. data analysis all the experiments were performed in triplicate and the mean values obtained. statistical entry was carried out and given treatment in microsoft excel 20 for windows (microsoft, redmond, washington, united states) and epidemiological information (epi info7tm, centers for disease control and prevention, atlanta, georgia, united states). one-way analysis of variance using statistical package for social sciences version 20 (spss inc., chicago, illinois, united states) was used to determine significant differences at p < 0.05. the surviving phage population obtained in each study were converted to log10 pfu/ml. data presentation was performed using graphpad prism version 5.00 for windows (graphpad software, san diego, california, united states). results antimicrobial susceptibility profile of the host bacterium (enterobacter cloacae) this isolate was resistant to five classes of antibiotics namely: beta-lactams, penicillins (ticarcillin or clavulanic acid, and piperacillin), cephalosporins (cefuroxime, cefuroxime axetil, ceftriaxone, and cefepime), monobactams (aztreonam), chloramphenicols (amphenicol), tetracycline (tetracycline, minocycline), quinolones (levofloxacin) and sulphonamides (trimethoprim). it was susceptible only to: meropenem (carbapenem) and tigelcycline (glycylcycline). isolated phages a total of 29 phage strains were isolated, with four phages from dandora, five phages from huruma, five phages from kibera, five phages from kariobangi, seven phages from korogocho and three phages from mathare. these phages were named using number-letter designations according to the sample source. for example, from kibera 1 settlement: the phages were named kibera 1a, 1b, and 1c. all the phages were subjected to host range studies. from the 29 phages isolated from the six sources, one phage from each source that had complete lytic properties and a lytic zone diameter of ≥ 3 mm was selected for study of physicochemical properties. effect of temperature on phage titre the isolated phages were stable from 4 °c to 50 °c (figure 1). there was a slight decrease in phage titre in four out of six phages at 50 °c, and two out of six phages had a slight increase in phage titre at 50 °c. in addition, one out of five phages had a constant titre from 4 °c to 30 °c. figure 1: effect of temperature on phages isolated in nairobi county, kenya, july 2019 to december 2020. phages were named based on source and phage strain number for example huruma 1b (p12) means huruma 1b source; phage strain number 12. effect of ph on phage titre no phages had any lytic activity at ph 2. all the phages were stable from ph 5 to 11 ph (slightly acidic to strong base). no phages had activity at ph 13 (figure 2). figure 2: effect of ph on phages isolated in nairobi county, kenya, july 2019 to december 2020. phages were named based on source and phage strain number for example huruma 1b (p12) means huruma 1b source; phage strain number 12. influence of ca2+ ions on phage titre and phage stability the addition of calcium chloride (0.002 m – 0.015 m) salt increased the adsorption rate and phage titre of phages in three out of five of the phages and a decrease in two out of five phages (figure 3). figure 3: effect of salt on phages isolated in nairobi county, kenya, july 2019 to december 2020. phages were named based on source and phage strain number, for example huruma 1b (p12) means huruma 1b source; phage strain number 12. storage stability of enterobacter cloacae phages all the phages were stable and efficacious at 4 °c and 37 °c. there was minimal or no activity at −20 °c following two weeks of storage (figure 4). figure 4: effect of storage on phage effectiveness and stability isolated in nairobi county, kenya, july 2019 to december 2020. the plates show the stability and lysis activity of: (a) huruma 1b phage, (b) dandora 3a phage, (c) kibera 1b phage, (d) korogocho 3a phage, (e) kariobangi 3b3 phage, and (f) mathare 1a phage at 4 °c, 37 °c and −20 °c following 2 weeks of storage. these phages were isolated in nairobi county, kenya, between july 2019 and december 2020. the phages were stable and efficacious at 4 °c and 37 °c with minimal or no activity at −20 °c. effect of ultraviolet light on phage titre exposure to ultraviolet light (290 nm – 320 nm) resulted in decreased phage titre from the 5th min to the 15th min. at the 20th min, there was no activity at all, which indicated that all the phages had been sterilised (figure 5). figure 5: effect of ultraviolet light exposure on phage survivability isolated in nairobi county, kenya, july 2019 to december 2020. the plates show the effect of ultraviolet light exposure from 0 min, 5 min, 10 min, 15 min and 20 min on: (a) huruma 1b phage, (b) dandora 3a phage, (c) kibera 1b phage, (d) korogocho 3a phage, (e) kariobangi 3b3 phage, and (f) mathare 1a phage isolated in nairobi county, kenya, between july 2019 and december 2020. the selected phages had decreased activity with increase in time from the 5th min to the 15th min, and no activity at all at the 20th min because all the phages had been destroyed. host range spot test analysis on enterobacter cloacae bacterial isolates all the isolated e. cloacae phages (n = 29) showed cross-reactivity against e. cloacae strains (n = 30) (figure 6). in order of individual phage potency from the highest to the least, the reactivity levels were: 67% (1 phage), 63% (3 phages), 60% (4 phages), 57% (5 phages), 53% (12 phages), 50% (1 phage), 47% (1 phage) and 27% (2 phages). according to the most susceptible bacteria to phages from the highest to the least, the following data were obtained: 100% (8), 97% (1), 93% (6), 86% (1), 79% (1), 31% (1), 21% (1), 14% (1), 10% (2), 7% (2) and 0% (7) (figure 7). figure 6: percentage potency of isolated phages on enterobacter cloacae strains isolated in nairobi county, kenya, july 2019 to december 2020. figure 7: susceptibility of enterobacter cloacae bacteria to isolated phages in nairobi county, kenya, july 2019 to december 2020. discussion this study found that the e. cloacae isolates from environmental waste water were resistant to five classes of antibiotics and hence termed as a mdr organisms. this is an indication that our environment habours mdr isolates that could be pathogenic to human and animal health. the antimicrobial profile of this isolate made it a good candidate for phage isolation. all of the isolated e. cloacae phages showed cross-reactivity against e. cloacae strains with the most potent phage lysing 67% of the bacterial strains and the least potent phage lysing 27% of the bacterial strains. a bacteria that was sensitive to a phage infection was termed as susceptible, with the highest susceptibility rate at 100% and the least susceptible at 0%. these results have similarities with previous studies done in india in 2019, on the host range of e. cloacae phage, which was able to lyse four species of the bacteria.27 additionally, in a study done in portugal in 2015, the use of three phages of e. cloacae as a cocktail for inactivation of urinary tract infections increased the potency of the phages in killing mdr e. cloacae.28 this specificity of the phages to the host bacteria is being exploited for therapeutic purposes in the treatment of various mdr bacteria.29 the specificity of the lytic activity is a characteristic that has been utilised for the development and production of novel therapeutic agents.11 host-range specificity in phage therapy is one of the major advantages for its success while it spares the commensal microbes from destruction during remedy.30 the specificity of the phages to their host bacteria is attributed to the phage host receptors involved in recognition, interaction and adsorption during attachment.31 additionally, the receptors are recognised by the ends of the virion’s long tail fibres of the phage towards the host bacteria.32 the stability of the e. cloacae phages obtained from this study varied from 4 °c to 50 °c. this stability concurs with e. cloacae phages previously isolated in lahore, pakistan.33 additionally, the phage titre fluctuated with different temperature conditions: there was a slight decrease in phage titre in four of six phages at 50 °c while two of six phages had a slight increase in phage titre at 50 °c. in addition, one of five of the phages had a constant titre from 4 °c to 30°c. the observed variation was also observed in a study done in iowa, united states, with the yield of phages being highly dependent on temperature.34,35 the elucidation of phage stability at different temperatures is needed to establish phage effectiveness as alternative therapeutic agents. from our ph stability studies, it is evident that the isolated e. cloacae phages were not stable in very acidic environments such as ph 2. this could be associated with the denaturation of the phage protein coat at low ph and stability being attained at basic ph above 5, with optimal activity in ph-neutral conditions (ph 7.5).36 however, the inactivation and reduction of the lytic activity of the phages decreased in high alkaline conditions (ph 11–13). this could be attributed to dissociation of the capsid protein due to high concentrations of hydrogen and hydroxyl ion in the solution.36 these findings concur with findings from previous studies done in portugal and poland with optimal phage stability at neutral ph (7.5).24,37 the ability of these phages to survive in the neutral ph (7.5) could be exploited or utilised in various applications such as sterilisation of hospital equipment, industrial mass production and for therapeutic purposes in patients with mdr infections of e. cloacae. in our study, there was increased activity of the isolated phages in 0.002 m and 0.015 m concentrations of ca2+ ions. but there was a slight drop of phage activity (1.79% drop) at 0.05 m ca2+ concentration in four out of six6 phages. in a study done in ireland in 2015, calcium was found to accelerate the phage lytic cycle with an impact on dairy fermentations.38 some phages require the cation for nucleic acid injection, efficient adsorption to cell wall binding sites and enhanced stability.38,39 the addition of the salts in our study corresponded with the above findings. salt availability also aids in the penetration processes of the phage genome into the host cytoplasm.39 a slight drop in phage titre of some phages might have been caused by the increase in growth of phage aggregates that might have resulted from neutralisation of the negatively charged moieties on the phage surface by cation binding with an increase in calcium salt concentration.40 all the phages in the current study were stable and efficacious at refrigerated temperature (4 °c) with optimal activity at human body temperature (37 °c) and minimal or no activity after being frozen (−20 °c) for two weeks. the crystal structure of ice destroys phages at −20 °c; hence, this storage is highly discouraged.41,42 the viability of phages at 4 °c has also been reported in other studies.24,37,43 a 5% – 10% glycerol addition to the phage suspension possibly warrants safe viability and infectivity for 30 days or longer-term storage at −20 °c or −70 °c.44 ultraviolet light is known to kill viruses and bacteria cells by disrupting their dna by damaging the thymine bases through creating a reaction between molecules or creating dimers.45 in our study, phages had decreased activity after exposure to ultraviolet light, and efficiency and efficacy decreased upon continuous sterilisation up to the 15th min and no activity at the 20th min. this effect of irradiation has also been reported in previous studies done in the united states in 2002 and 1947.25,46 in addition, a study conducted in china in 2020 reported that ultraviolet light potentially reduced phage titres in pathogen reduction quality.47 limitations in host range determination, a panel of 30 anonymised clinical isolates of e. cloacae and one staphylococcus aureus (control) were used for cross-reactivity studies. however, no antimicrobial susceptibility studies for these bacterial isolates were carried out. conclusion this study reveals the existence of the most potent lytic phages that are effective on mdr e. cloacae isolates found in kenya. the existence of diverse phage strains from the sampled areas provided an effective cocktail of phages that could be used as antimicrobial agents. findings from this study demonstrate that physicochemical properties influence the efficacy of phages in their antimicrobial activities and are worth considering. acknowledgements this work was inspired and partly funded by dr elizabeth (betty) kutter (the evergreen state college, united states). we appreciate ipr for the laboratory space and partial funding support. we acknowledge dr lilian musila (kemri, center for microbiology) for laboratory technical support. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions all authors approved the study and submission. i.j.m., a.k.n. and a.n. conceived the idea. i.j.m. carried out the experiment supervised by a.k.n. and a.n., a.a.j. and m.i.i. offered technical support. i.j.m., a.k.n. and a.n. wrote the manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability the authors confirm that the data supporting the findings of this study are available within the article. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any 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assurance programmes outcomes challenges and lessons learnt acknowledgement references about the author(s) matilu mwau kenya medical research institute, nairobi, kenya mamo umuro national public health laboratory services, nairobi, kenya collins o. odhiambo kenya medical research institute, nairobi, kenya citation mwau m, umuro m, odhiambo co. experience from a pilot point-of-care cd4 enumeration programme in kenya. afr j lab med. 2016;5(2), a439. http://dx.doi.org/10.4102/ajlm.v5i2.439 country profile experience from a pilot point-of-care cd4 enumeration programme in kenya matilu mwau, mamo umuro, collins o. odhiambo received: 15 mar. 2016; accepted: 11 aug. 2016; published: 17 oct. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. hiv situation in kenya kenya is a hiv ‘high burden’ county in africa with both a generalised and a concentrated epidemic within its population of 47 million.1 hiv prevalence reached a peak of 10.5% in 1995–1996, but declined to approximately 6.0% in 2013.2 the number of people living with hiv is estimated to have increased from about 1.2 million in 2009 to 1.4 million in 2013.3 this number is projected to continue increasing due to improved survival attributable to antiretroviral therapy programmes. the total number of new hiv infections is estimated to have declined by about 15.0% over the last five years from about 116 000 in 2009 to around 100 000 in 2013.3 over the last five years, the number of annual aids-related deaths has declined, from about 85 000 in 2009 to 58 000 in 2013. the prevalence of hiv in infants has also been declining, with numbers dropping from around 14.4% in 2007 to 8.8% in 2015. this decline is partly due to a robust prevention of mother-to-child transmission programme. it is estimated that 100 000 infants are born to hiv-positive mothers every year. approximately 60 000 children are on treatment for hiv. however, only 50.0% of infants receive a timely virologic test and antiretroviral therapy coverage is suboptimal, which can be attributed to limited access to diagnosis.4 laboratory infrastructure in support of hiv care and treatment in kenya for adults, counselling services and hiv testing using rapid test strips are widely available.3 up to 83% of women and 71% of men report having tested at least once in their lifetime. access to testing is not uniform across the country, with the northern parts (mainly arid, underpopulated and low hiv-prevalence areas) reporting less than 10% testing.3 following the ‘test and treat’ guidelines released by the world health organization in 2014,5 cd4 testing is on the decline. there are approximately 170 bd facscount™ machines in kenya, and fewer than 30 bd facscalibur™ machines (personal communication, nphls). these are concentrated mainly in highly-populated parts of the country. the total number of tests for cd4 done in 2015 was only 439 443, down from more than 800 000 a year earlier. it is likely that some cd4 testing demand will persist even after ‘test and treat’ is adopted, mainly driven by demand from clinicians and for management of cryptococcal meningitis and other opportunistic infections. early infant diagnosis (eid) testing began in 2006 and is offered at seven national laboratories.4 the laboratories conduct 95% of all the testing on a referral basis. the monthly workload is 5000 tests, and has been steady. this volume represents only 50% of the infants who should be tested, due to suboptimal access, partly as a result of the centralised laboratory referral system. the abbot m2000sp™ and roche cobas® ampliprep/cobas® taqman® systems are in widespread use, running on medium throughput. viral load testing was available as a private service in kenya until october 2014, when the national programme decided to roll out the service free of charge in the public sector. there are seven laboratories that form part of the viral load testing network, with a combined total of 17 roche and 16 abbott instruments. between them, up to 900 000 viral load tests will be done in 2016. the demand, however, is estimated to be approximately 3.4 million viral load tests over the next three years. quality assurance framework and policy for hiv lab and point-of-care testing to help improve access to viral load and eid testing, the ministry of health encouraged evaluations of new technologies as they become available. it has also developed a point-of-care (poc) testing policy to guide the implementation of poc testing to support laboratory practice and bring services closer to patients. a poc technical working group comprising several stakeholders, including the elizabeth glaser pediatric aids foundation, the clinton health access initiative and the united nations children’s fund, is overseeing the implementation. currently, the national public health laboratories services, in collaboration with key stakeholders, is putting together a national poc roadmap that addresses the introduction and scale-up of poc testing in the existing landscape. the kenya medical research institute has been evaluating technologies as they become available in order to determine their suitability for use in kenya.6,7,8 currently, eid and viral load poc testing devices are under evaluation, with results expected in late 2016. guidelines for poc technology evaluation and adoption were developed and launched in 2014. the national coordinator for poc evaluations is the national public health laboratories services, but there are sub-coordinators in all 47 counties of kenya (figure 1). the coordinators are mainly involved in supportive supervision. a national laboratory technical advisory committee develops policies and guidelines for poc testing. other components of the guideline address training, competency assessment and certification of end-users. the guideline also addresses the selection and assessment criteria for poc sites. the ministry of health and implementing partners have started discussions on mapping of viral load and eid to identify gaps that poc devices could help fill. figure 1: kenya point-of-care quality assurance programme management structure. existing quality assurance programmes kenya has a national quality assurance programme that has been pursuing laboratory accreditation through the strengthening laboratory management towards accreditation approach.6 the programme also supports external quality assessment (eqa), training and registration of users, and harmonisation of protocols and standards. eqa occurs three times a year in kenya through an intermediate model involving both local and international eqa providers. international panels are sourced from human quality assessment services, public health agency of canada (qasi), the us centers for disease control and prevention, and the world health organization, depending on the test. the eqa panels are distributed to county hubs for redistribution to the testing sites. national panels, including dried tube specimens, split samples, rechecking and internal controls, are procured centrally. the national programme also supports validation of new tests/equipment, certification of personnel and routine maintenance of equipment. routine supervision is decentralised. all seven central molecular testing laboratories participate in the us centers for disease control and prevention, atlanta, proficiency testing for viral load, eid and hiv drug resistance testing. additionally, a subset (4/7) of the central testing labs participate in a quarterly inter-laboratory eqa programme, with all expected to participate during 2016. samples are processed between the participating laboratories and results are discussed every quarter to enhance quality. it is conceivable that there will be three cycles of eqa annually for eid and viral load when the appropriate poc testing devices become available. outcomes the poc testing pipeline has only had a few technologies that have reached clinical evaluation, and of those, the alere pima™ and bd facspresto™ have shown the biggest potential for use. following satisfactory evaluation findings,8 kenya deployed 48 alere pima devices across 34 of its 47 counties in 2014, including four devices at sites supported by médecins sans frontières (figure 2). training for an additional 62 sites was completed in march 2015, which increased the coverage of poc cd4 devices in the country to 44 out of 47 counties. overall, 96 sites have a total of 119 pima devices. at least three personnel from each testing site were trained on the use of the poc testing device. figure 2: cd4 device mapping in kenya. a total of 298 functioning cd4 instruments were clustered across 47 counties. site selection was based on a national map of existing cd4 sites and extensive discussion between regional implementing partners and individual county health management teams who oversee all health-related issues in the county. this process led to the generation of a list of 150 facilities that fit the site selection criteria agreed upon by the poc technical working group, such as total patient volumes per health facility, distance travelled to central testing hubs, and the total throughput of the pima device. these criteria were used to generate a score for each site that was eligible for pima placement. in addition, qualitative criteria, such as security and availability of personnel, were considered when selecting placements for the initial 44 devices from the 150 sites generated. the same site selection methodology was applied for the 62 additional pima devices that were deployed in early 2015. the ministry of health has enacted a law passed by parliament that recommends device leasing (as opposed to purchase) for all medical diagnostic services in the country. in this model, the device belongs to the manufacturer during its usage period. the manufacturer is responsible for the maintenance of the device. the cost of the cartridge, service and maintenance is bundled and packaged together with the cost per test. in 2014, trainings were conducted using the super-user training model and included the training of county laboratory coordinators, implementing partners, and a team from the national hiv reference laboratory. however, the quality of training that cascaded down through training-of-trainers was poor and therefore required follow-up for on-site re-training by the ministry of health, supported by implementation partners. in 2015, trainings focused on an end-user training model. while most facilities were able to begin testing within one to two weeks after the super-user training, there was delayed end-user/facility-based training in some facilities due to conflicting commitments by either the county laboratory coordinator or the implementing partner. the lessons learned from 2014 have been applied to the 2015 training curriculum, such as the switch from super-user training to end-user training. follow-up visits after the initial training and implementation of the 44 pima devices showed that most poc testing sites adapted their clinical workflow around the availability of onsite cd4 testing. nearby satellite sites are now redirecting their samples to pima poc testing sites. using this approach, the 119 pima devices performed an average of 2432 tests per month, or 6.5% of all cd4 tests done in the country. the bd facspresto was rolled out at 13 sites, for an average of 818 tests a month. at poc testing sites, 71.5% of patients received same day results, while post-implementation assessments have shown a reduction in time to initiation on antiretroviral therapy from two months8 to 27.5 days at poc testing sites.6 additionally, 11.7% of all eligible patients were initiated on antiretroviral therapy on the same day they were tested. challenges and lessons learnt the first phase of implementation of poc cd4 testing devices in kenya revealed a number of challenges: testing errors resulted in wastage of a significant number of test cartridges for the pima device. in 2014, the error rate was 7.6% of all tests done, while in 2015, this rate increased to 13.6% in the first two quarters of the year. the maximum pass rate for the eqa system over 40 rounds was only 82.0%. lack of reporting on commodity consumption resulted in stock-outs at some sites and the delay in issuing new control beads for the pima machines. machine downtimes reduced overall access to the service. movement of staff within counties while organisational structures were finalised meant that some of the staff trained as super-users were transferred to new regions and could no longer provide support to the pima poc testing sites in the region in which they were trained. other competing engagements of some county super-users meant that they were unable to provide consistent mentorship or support to the users they trained. lack of internet in remote regions meant data transmission to the national database was erratic. lessons learnt poc technologies for hiv have the potential to improve access to diagnosis and to reduce time to result or to initiation of treatment. kenya, being a high hiv burden country, is keen to adopt technologies that offer the most promise and is therefore constantly evaluating them. during the rollout of the alere pima and bd facspresto systems, some lessons have been learnt that can guide future adoption of poc testing for viral load and eid. the rate of testing errors, and the causes, does not differ markedly from what others have reported.10 errors attributed to the user can be mitigated by supportive supervision and trainings; device errors can be reduced through preventative maintenance, while errors attributed to the sample can be managed through training of end-users. the poc cd4 testing programme was hampered by poor reporting on commodity consumption. the net effect was a lack of supplies, leading to avoidable machine downtime. in our experience, supportive supervision improves reporting. an even more effective solution is automating the reporting by means of digital transmission systems.4,10,11 this requires that poc devices come with preinstalled global system for mobile communications or general packet radio service capability. after each eqa cycle, the national hiv reference laboratory and supporting partners need to conduct intervention meetings with participating sites with unsatisfactory reports to support improvement of performance and enhance quality service delivery (figure 3). kenya is undertaking a costing exercise for quality assurance for poc testing, (wherein 3 members of the kenya medical research institute have been undergoing a series of trainings, facilitated by the london school of hygiene and tropical medicine and the international diagnostics centre, to build their capacity to cost quality assurance models), but regardless of the findings, the ministry of health will need to identify additional support from implementing partners to establish local production of eqa panels. figure 3: (a) cd4 pima™ poc testing trends 2014–2015 error rates; (b) drill down to sites with high error rates: top 36 sites in kenya contributing to 75% of the total error rates. conclusion kenya has been very engaged with the evaluation and implementation of poc testing and pursues the highest quality of care for kenyans through quality diagnostics. the moh is continuously expanding quality assurance programmes with an emphasis on the accreditation of laboratories and plans for local production of external quality assurance panels. implementing partners can continue to work with the ministry of health to mitigate further risk and to address the various challenges raised above in some of the following ways: including different end-users in trainings to address the turnover of staff and support trained super-users; and leveraging resources across relevant partners and organisations to ensure that an internet connection is available at every facility to enable online reporting of monthly consumption. the lessons learned through the implementation of pima are guiding the planning for eid and viral load poc; primarily site selection and continuous quality assurance for monitoring and evaluation. the collaboration between implementing partners and ministry of health is an important aspect to planning for sustainable quality poc testing. acknowledgement the authors are grateful to poc testing implementing partners, including the clinton health access initiative, who supported pima implementation in kenya. we also thank the london school of hygiene and tropical medicine for invitation and support towards the development of a quality assurance framework for kenya. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support none. references tanser f, de oliveira t, maheu-giroux m, et al. concentrated hiv subepidemics in generalized epidemic settings. curr opin hiv aids. mar 2014;9(2):115–125. http://dx.doi.org/10.1097/coh.0000000000000034 unaids. hiv and aids estimates [page on the internet]. c2013 [cited 2016 sep 14]. available from: http://www.unaids.org/en/regionscountries/countries/kenya national aids control council. kenya aids response progress report. nairobi, kenya: nacc; 2014. khamadi s, okoth v, lihana r, et al. rapid identification of infants for antiretroviral therapy in a resource poor setting: the kenya experience. j trop pediatr. dec 2008;54(6):370–374. http://dx.doi.org/10.1093/tropej/fmn036 epub 2008 may 29. world health organization. consolidated guidelines on hiv prevention, diagnosis, treatment and care for key populations. geneva, switzerland: who; 2014. yao k, maruta t, luman et, et al. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. 2014;3(2), 8 pages. http://dx.doi.org/10.4102/ajlm.v3i2.194 mwau m, adungo f, kadima s, et al. evaluation of pima™® point of care technology for cd4 t cell enumeration in kenya. plos one. 2013;8(6):e67612. http://dx.doi.org/10.1371/journal.pone.0067612 odeny ta, decenso b, dansereau e, et al. the clock is ticking: the rate and timeliness of antiretroviral therapy initiation from the time of treatment eligibility in kenya. j int aids soc. 2015;18:20019. http://dx.doi.org/10.7448/ias.18.1.20019 ecollection 2015. fajardo e, metcalf c, piriou e, et al. errors generated by a point-of-care cd4+ t-lymphocyte analyser: a retrospective observational study in nine countries. bull world health organ. sep 1 2015;93(9):623–630. http://dx.doi.org/10.2471/blt.14.146480 peeling rw. diagnostics in a digital age: an opportunity to strengthen health systems and improve health outcomes. int health. nov 2015;7(6):384–389. http://dx.doi.org/10.1093/inthealth/ihv062 stevens w, gous n, ford n, et al. feasibility of hiv point-of-care tests for resource-limited settings: challenges and solutions. bmc med. 2014;12:173. http://dx.doi.org/10.1186/s12916-014-0173-7 abstract introduction methods results discussion acknowledgements references about the author(s) adeyinka a. davies department of medical microbiology and parasitology, olabisi onabanjo university teaching hospital, sagamu, nigeria bram spruijtenburg department of medical microbiology and immunology, canisius-wilhelmina hospital (cwz)/dicoon, nijmegen, the netherlands radboudumc-cwz center of expertise for mycology, nijmegen, the netherlands eelco f.j. meijer department of medical microbiology and immunology, canisius-wilhelmina hospital (cwz)/dicoon, nijmegen, the netherlands radboudumc-cwz center of expertise for mycology, nijmegen, the netherlands iriagbonse i. osaigbovo department of medical microbiology, school of medicine, university of benin, benin city, nigeria oluwaseyi balogun department of biomedical engineering, university of lagos, idi-araba, surulere, nigeria abiola adekoya department of radiology, olabisi onabanjo university teaching hospital, sagamu, nigeria titilola gbaja-biamila nigeria institute of medical research, yaba, lagos, nigeria saint louis university college of public health and social justice, st. louis, missouri, united states of america jacques f. meis radboudumc-cwz center of expertise for mycology, nijmegen, the netherlands institute of translational research, cologne excellence cluster on cellular stress responses in aging-associated diseases (cecad) and excellence center for medical mycology, university of cologne, cologne, germany rita oladele department of medical microbiology and parasitology, lagos university teaching hospital, idi-araba, lagos, nigeria citation davies aa, spruijtenburg b, meijer efj, et al. molecular identification and antifungal susceptibility testing of aspergillus species among patients with chronic pulmonary aspergillosis in nigeria. afr j lab med. 2025;14(1), a2674. https://doi.org/10.4102/ajlm.v14i1.2674 original research molecular identification and antifungal susceptibility testing of aspergillus species among patients with chronic pulmonary aspergillosis in nigeria adeyinka a. davies, bram spruijtenburg, eelco f.j. meijer, iriagbonse i. osaigbovo, oluwaseyi balogun, abiola adekoya, titilola gbaja-biamila, jacques f. meis, rita oladele received: 29 oct. 2024; accepted: 11 apr. 2025; published: 11 july 2025 copyright: © 2025. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: triazole resistance in aspergillus spp. has therapeutic implications for managing chronic pulmonary aspergillosis (cpa) worldwide. however, antifungal susceptibility testing (afst) is not routinely performed in nigeria, a country with a high cpa burden. objective: this study aimed to confirm the identity of aspergillus spp. isolated from patients with cpa using molecular methods, determine their antifungal susceptibility profile, and ascertain phylogenetic relatedness. methods: this study examined 47 aspergillus isolates from sputum samples obtained in a prospective longitudinal study of cpa prevalence among 141 consenting symptomatic tuberculosis patients in lagos, nigeria, between june 2021 and may 2022. the preliminary phenotypically identified aspergillus spp. were further identified by amplifying the calmodulin gene and performing afst against seven antifungal agents using the clinical laboratory standard institute (clsi) micro-dilution method, as well as determining their phylogenetic relatedness. results: the 51 patients who met the diagnostic criteria for cpa included 30 (59.0%) male and 21 (41.0%) female patients (age range: 17–68 years). thirty-six (71.0%) had positive aspergillus cultures. an isolate, initially identified phenotypically as a. fumigatus, was reidentified as a. pseudonomiae. phylogenetic analysis on a. fumigatus and a. flavus isolates suggested the absence of clonal transmission. all isolates were susceptible to the tested antifungals. conclusion: clinical aspergillus isolates from azole-naïve patients with cpa did not demonstrate triazole resistance. nonetheless, afst is required for patients on long-term azole therapy and systematic surveillance of clinical and environmental isolates is recommended to detect the emergence of azole-resistant phenotypes. what this study adds: this study underscores the importance of routine surveillance for antifungal resistance to detect the occurrence of resistance strains early in clinical settings, as this has therapeutic implications for patients harbouring resistant phenotypes. keywords: chronic pulmonary aspergillosis; antifungal susceptibility test; phylogenetic analysis; tuberculosis; aspergillus spp.; nigeria. introduction in 2022, the world health organization released the first fungal priority pathogens list, with aspergillus fumigatus categorised as a critical pathogen.1 aspergillus fumigatus accounts for many allergic and invasive lung conditions, principal amongst those with chronic pulmonary aspergillosis (cpa). outside europe, specifically in asia and africa, non-fumigatus species such as a. flavus and a. terreus are also increasingly implicated.2,3,4 regardless of the aspergillus spp., cpa is a slowly progressing but debilitating disease which affects individuals with structural lung defects. the annual incidence is 1 837 272 cases, of which about 340 000 (18.5%) die.5 the prevalence of cpa in tuberculosis-treated patients has been reported as 49.7% in nigeria,6 50% in ghana,3 and 57% in india.7 a combination of symptomatology persisting over 3 months, radiological findings, positive cultures, and serological tests is required to diagnose. the disease often requires long-term treatment with systemic antifungal agents, preferably triazoles such as itraconazole or voriconazole administered orally. antifungal therapy improves symptoms and reduces morbidity and mortality of the disease in most patients. identifying clinical and microbiological resistance during treatment is of utmost importance to limit relapse and improve the long-term survival rate. besides being useful for treating all forms of aspergillosis,8 triazoles, which have a broad spectrum of antifungal activity, are also used as environmental fungicides to prevent fungal spoilage of crops by phytopathogens.9 cross-resistance between medical and environmental triazoles has been documented.10 azole resistance in a. fumigatus was first reported in clinical isolates of azole-experienced cpa patients in 199711 and azole-naïve invasive aspergillosis patients in 2014.12 since then, several countries have reported resistance to triazoles in clinical and environmental isolates,8,13 including africa, where documented resistance to environmental aspergillus isolates has been reported from the eastern and western regions.14 also, tr34/l98h was described from a clinical aspergillus isolate in kenya.15 this emphasised that azole resistance in aspergillus spp. is present, although the lack of proficiency and resources to routinely perform antifungal susceptibility testing (afst), underestimation of azole resistance in a. fumigatus prevalence persists. however, the rising trends in azole-resistant a. fumigatus globally made the panel of experts recommend subjecting all clinical aspergillus spp. isolates to afst,16 which has not been feasible in africa. additionally, surveillance data on triazole resistance in clinical aspergillus spp. from africa has been sparse. therefore, this study aimed to determine the antifungal susceptibility profile of clinical isolates of aspergillus after identifying them to the species level using molecular methods. methods ethical considerations this study was approved by the institutional ethical committees of lagos university teaching hospital in december 2020 (reference number: adm/dcst/hrec/app/3868) and by the nigeria institute of medical research in january 2021 (reference number: irb/20/096). written informed consent was obtained from all participants, with data entered into a private, secure computer with access limited to investigators. test results were kept confidential and disclosed only to the patient and their healthcare provider. study design this was a descriptive study of aspergillus isolates obtained from a previously reported prospective longitudinal study conducted to determine the cpa incidence in two tuberculosis treatment clinics in lagos, nigeria (lagos university teaching hospital and nigeria institute of medical research, yaba), between 01 june 2021 and 31 may 2022.6 in the previous study, 141 consenting adult patients who were previously treated for tuberculosis (1–4 years earlier) and were clinically classified as retreatment (n = 79; 56.0%) and post-tuberculosis treatment (n = 62; 44.0%) patients were recruited. fifty-one patients were diagnosed with cpa based on a combination of positive bordier aspergillus-specific igg assays (bordier affinity products, crissier, switzerland) in patient serum, typical chest x-ray features of cpa, and isolation of aspergillus spp. from high-volume sputum cultures,17 of these, 47 aspergillus spp. were cultured from the sputum of 36 patients. the demographic characteristic and aspergillus spp. distributions were analysed with ibm statistical package for social science software version 25 (ibm corp., armonk, new york, united states). isolate culture and dna extraction as part of the current study, phenotypic identification of isolates recovered from sputum was carried out at the mycology laboratory of lagos university teaching hospital. identification was based on colonial morphology and microscopic features as previously described by davies et al.6 thereafter, isolates were transported to canisius-wilhelmina ziekenhuis hospital (nijmegen, the netherlands) on sterile filter paper impregnated with tween 20 for molecular identification, performed between 04 december 2022 and 15 february 2023. at canisius-wilhelmina ziekenhuis hospital, the isolates were stored at -80 °c, according to standard procedures, until use. the isolates were grown on sabouraud dextrose agar plates (oxoid, hampshire, united kingdom) for 7 days at 30 °c. for the dna extraction, isolates were resuspended in 400 µl magna pure bacteria lysis buffer (roche diagnostics gmbh, mannheim, germany) and magna lyser green beads (roche diagnostics gmbh, mannheim, germany).18 the conidia were lysed by a magna lyser instrument (roche diagnostics gmbh, mannheim, germany) for 30 s at 6500 rpm. all tubes were consecutively inactivated at 100 °c for 10 min. dna was extracted and purified using the magna lyser system (roche diagnostics gmbh, mannheim, germany). the magna pure and vial na small volume kit was used following the manufacturer’s instructions, as previously described by de groot et al.19 molecular species identification as part of the current study, a polymerase chain reaction amplifying the calmodulin (cam) gene was used for species identification using primers cmd5 5’-ccgagtacaaggargccttc-3’ and cmd6 5’-ccgatrgaggtcatracgtgg-3’, as previously described.20 generated amplicons were purified according to the ampliclean protocol (nimagen, nijmegen, the netherlands) and the sequencing polymerase chain reaction was performed using 0.5 µl brilliantdye premix, 1.75 µl brilliantdye 5x sequencing buffer (nimagen, nijmegen, the netherlands), 1 µl cmd6 primer (5.0 µm), 5.75 µl water and 1 µl purified dna. ensuing amplicons were purified using the d-pure purification method (nimagen, nijmegen, the netherlands) and sequenced on a 3500 xl genetic analyzer (applied biosystems, foster city, california, united states). calmodulin sequences of a. fumigatus cbs 487.65 (ab259965.1), a. niger cbs 101700 (gu195633.1), a. flavus cbs 117622 (ef202068.1), and a. pseudonomiae dto:267-i4 (kp330066.1) were extracted from the national center for biotechnology nucleotide database. alignment and tree building were done with the clustal omega multiple alignment algorithm.21 visualisation and editing were made with itol v6 (https://itol.embl.de).22 calmodulin sequences generated in the present study were deposited under genbank accession numbers oq915062–oq915108. antifungal susceptibility testing as part of the current study, afst against amphotericin b (bristol myers squib, woerden, the netherlands), itraconazole (janssen cilag, beerse, belgium), voriconazole (pfizer central research, sandwich, united kingdom), posaconazole (merck, haarlem, the netherlands), isavuconazole (basilea pharmaceutica, basel, switzerland), anidulafungin (merck, haarlem, the netherlands) and micafungin (merck, haarlem, the netherlands) was performed by broth microdilution following the clinical laboratory standard institute (clsi) m38-a2 protocol.23 the spores were diluted in rpmi 1640 medium and adjusted with a genesys 20 spectrophotometer (thermo fisher scientific, waltham, massachusetts, united states)19 to obtain final concentrations of 1 × 105 to 5 × 105 cfu/ml suspension and subsequently transferred into microtiter plates with the appropriate antifungal concentrations. the plates were incubated at 35 °c for 48 h and visually interpreted. minimal inhibitory concentrations (mics) were read as the lowest antifungal concentration with a 100% growth reduction when compared to the growth control.18 for the echinocandins, minimal effective concentrations were determined microscopically as the lowest drug concentration with the growth of small, rounded, compact hyphal forms compared to the hyphal growth in the control. tentative antifungal breakpoints based on expert opinion and epidemiological cutoff values were implemented for different aspergillus spp. according to clsi, the epidemiological cutoff value for a. fumigatus is 2 µg/ml for testing amphotericin b, in comparison, 4 µg/ml for a. flavus, whereas, for voriconazole, ≤ 0.5 µg/ml was interpreted as susceptible, 1 µg/ml as intermediate, and ≥ 2 µg/ml as resistant.24 short tandem repeat genotyping as part of the current study, a. fumigatus and a. flavus, isolates were genotyped as previously described by de valk et al.25 and rudramurthy et al.26 in short, multiplex polymerase chain reaction reactions amplifying nine short tandem repeat markers were performed using 1× faststart taq polymerase buffer without mgcl2, deoxynucleoside triphosphates (0.2 mm), mgcl2 (3 mm), forward and reverse primers (5 µm), 1 u faststart taq polymerase (roche diagnostics gmbh, mannheim, germany), and isolated dna on a thermocycler (biometra, westburg, göttingen, germany).18 products were analysed on a 3500 xl genetic analyser (applied biosystems, foster city, california, united states), and corresponding copy numbers were determined and analysed using genemapper software (applied biosystems, foster city, california, united states), as previously described by spruijtenburg et al.27 data analysis the demographic data were entered directly into microsoft excel (microsoft, redmond, washington, united states), while the laboratory results were first entered into a book dedicated solely to this study and later transferred into microsoft excel. the participants were categorised into re-treatment and post-tuberculosis treatment groups. descriptive analysis was done with ibm statistical package for social science software version 25 (ibm corp., armonk, new york, united states), with data presented in frequency and percentages. these data were then displayed in tabular form. a multistate categorical coefficient was used to determine genetic relatedness between isolates (bionumerics v7.6.1 software; applied mathd nv, sint-martens-latem, belgium). results of the 51 patients who met the case definition for cpa, 47 aspergillus spp. were recovered from the sputum cultures of 36 (70.6%) patients. participants included 19 (37.3%) male patients and 17 (33.3%) female patients, with ages ranging from 21 to 75 years. aspergillus flavus (n = 18; 35.3%) was the most common species isolated. however, multiple aspergillus spp. were also isolated from 10 patients, with nine consisting of a combination of two different species, while the remaining patient had a mixture of a. flavus, a. fumigatus, and a. niger isolated (table 1). table 1: demographic, clinical characteristics, and culture findings of patients recruited from lagos university teaching hospital and nigeria institute of medical research, yaba, nigeria, between 01 june 2021 and 31 may 2022. molecular identification genotypic species determination was based on calmodulin sequencing using clustal omega multiple sequence alignment, which resulted in the identification of 28 a. flavus sensu stricto strains, 11 a. fumigatus sensu stricto strains, seven a. niger sensu stricto strains, and one a. pseudonomiae (figure 1, table 2). figure 1: phylogenetic tree of aspergillus species from nigeria, between 01 june 2021 and 31 may 2022. table 2: isolate overview of aspergillus species, including minimal inhibitory concentrations in mg/l and host clinical information, all isolated from sputum. analysis was performed at canisius-wilhelmina ziekenhuis hospital nijmegen, the netherlands between 04 december 2022 and 15 february 2023. antifungal susceptibility forty-seven aspergillus isolates were tested using the clsi m38-a microdilution method; anidulafungin and micafungin mics were ≤ 0.008 µg/ml for all strains. for the a. pseudonomiae isolate, mics were 0.5 µg/ml for amphotericin b, 0.25 µg/ml for itraconazole, 1 µg/ml for voriconazole, 0.125 µg/ml for posaconazole, and 1 µg/ml for isavuconazole. using tentative breakpoints based on clsi, no resistant isolates were found. out of the four azoles, posaconazole showed the highest in vitro activity based on the lowest mic50 of 0.063 µg/ml for a. flavus, a. fumigatus and a. niger. itraconazole, voriconazole, and isavuconazole demonstrated the lowest in vitro activity, with a mic50 value of 0.5 µg/ml against all three species. notable mic differences between species were observed for amphotericin b, with a. flavus having an mic50 of ≥ 2 two-fold dilutions higher than a. fumigatus and a. niger (table 2). short tandem repeat genotyping using a multiplex polymerase chain reaction, nine markers in a. fumigatus and a. flavus strains were amplified, and then analysed to investigate the genotype relatedness with bionumerics v7.6.1 software (applied mathd nv, sint-martens-latem, belgium). for both species, all isolates displayed unique genotypes (figure 2 and figure 3), and isolates did not cluster based on retreatment tuberculosis or post-tuberculosis treatment (figure 2a and figure 3a). all 28 a. flavus isolates differed from each other in at least three out of nine markers (figure 2b), while a. fumigatus isolates differed in six or more short tandem repeat markers (figure 3b). figure 2: cluster analysis of genotyped aspergillus flavus isolates (n = 28) from nigeria, between 01 june 2021 and 31 may 2022: (a) a minimum spanning tree with branch lengths indicating the similarity between isolates. thin solid lines display variation in two markers, thin dashed lines variation in three markers and thin dotted lines in four or more markers (b) an unweighted pair-group method with arithmetic mean dendrogram displays copy numbers, isolate identifications (ids), and genotype numbers. figure 3: cluster analysis of genotyped aspergillus fumigatus isolates (n = 28) from nigeria, between 01 june 2021 and 31 may 2022: (a) a minimum spanning tree with branch lengths indicating the similarity between isolates. thin dotted lines display variation in four or more markers (b) an unweighted pair-group method with arithmetic mean dendrogram displays copy numbers, isolate identifications (ids) and genotype numbers. discussion this study demonstrated that cpa among the participants in this current study is more commonly associated with a. flavus than a. fumigatus, which is concordant with other studies from tropical countries. all clinical isolates were fully susceptible to azoles, echinocandins and amphotericin b. this is the first report of afst in clinical isolates of aspergillus in nigeria. the species distribution of aspergillus isolates in this study (a. flavus n = 18, 35.3%; a. fumigatus n = 5, 9.8%; a. niger, n = 2, 3.9%) differs from studies conducted in nigeria, where a. fumigatus recovered from sputum cultures of patients with hiv-tuberculosis co-infection accounted for 42.1% in the northern part in 2019 and 57.14% in the southwestern part in 2016.28,29 cultures of 10 participants in the index study contained more than one aspergillus spp., and it is not certain if these represented co-infections or merely colonisation. however, there were no significant differences in the aspergillus spp. and the afst between retreatment tuberculosis and post-tuberculosis patients. only one aspergillus isolate from a retreatment tuberculosis was identified as a. pseudonomiae. phenotypic techniques can misidentify aspergillus spp. with similar morphological features, and complementing with genotypic methods is expected to improve identification accuracy. besides the common pathogenic aspergillus spp., an isolate of a. pseudonomiae, which belongs to the section flavi, was isolated. this isolate was initially identified as a. fumigatus based on colonial morphology and microscopic appearance. aspergillus pseudonomiae was first isolated from soil and insects in the united states,30 and has only been isolated once so far from the environment in nigeria; clinical infections are uncommon except for one report from a patient diagnosed with cpa.31 it is difficult to ascertain if the isolate from our study represents colonisation or was an actual cause of cpa, since only a single sputum specimen with a. pseudonomiae was collected from this patient who declined further participation. besides this isolate, there was a high correlation between phenotypic identification and molecular methods in this study. while this may imply the reliability of phenotypic methods, especially for use in low-resource settings, the study’s relatively small sample size must be considered; therefore, further studies are needed before making definite recommendations. based on available clinical breakpoints and epidemiological cut-off values from the clsi, all the aspergillus strains in this study were susceptible to the tested antifungals, including azoles.22,23 overall, the mics for all the aspergillus spp. were the same, except for amphotericin b against a. flavus. several other studies have reported higher amphotericin b mics against a. flavus when compared to other aspergillus spp.31 it is thought that a. flavus displays higher ergosterol levels with increased activity of peroxidase and superoxide dismutase, resulting in reduced susceptibility.32 since high mics to amphotericin b are associated with therapeutic failure, voriconazole is the preferred drug for treating all forms of invasive aspergillosis.31 the absence of triazole resistance in aspergillus spp. in this study was not surprising, because the study participants had no prior exposure to azole drugs and no reports of environmental or cross-resistance to triazoles have been reported in the region to date; a study conducted on soil samples in lagos nigeria reported that all the aspergillus spp. isolated were susceptible to voriconazole and itraconazole.33 in contrast, another study reported 2.0 to 2.2% prevalence from soil samples collected in west africa, including burkina faso and nigeria.14 however, the study site of the reported azole resistance aspergillus spp. in nigeria was outside lagos. in east africa, triazole-resistant aspergillus strains have been reported, from agricultural settings and clinical isolates in tanzania and kenya.15,34,35,36 outside africa, the prevalence of azole-resistant clinical isolates in europe steadily increased from 1.7% in 1997 to 9% in 2009,37 and 15% as of 2018,38 while it was 4.3% in a single centre study from china,39 and 3.5% in the united states.40 the exponential increase in agricultural azole fungicides used for crop protection and material preservation against phytopathogens may be responsible for observed rates of azole-resistant aspergillus spp. in these regions.10,41,42 meanwhile, information about azole fungicides in africa is scarce and only the study conducted in kenya described azole resistance from azole-naïve and azole-experienced soil samples.15 thus, the lack of surveillance studies may have resulted in the low prevalence reported in africa.14 therefore, coordinated data collection with a standardised protocol and continuous surveillance of environmental and clinical aspergillus spp. is warranted in nigeria. the absence of a. fumigatus and a. flavus isolates with identical or highly related short tandem repeat genotypes suggests no transmission of these fungal species between patients within or between hospitals. for a. fumigatus, microsatellite typing has extensively been applied and suggests a high genetic diversity.28,40 limitations the results of this study may not be generalisable to other locations within nigeria. other limitations include the relatively small number of isolates and the high frequency of mixed cultures, as it was not possible to determine if these represented co-infections or colonisation. the limitations notwithstanding, the data generated support using azoles as first-line therapy for cpa treatment in the study location. conclusion molecular species identification helped to identify the aspergillus strains accurately. antifungal susceptibility results showed susceptible strains in this study, suggesting that de novo or environmentally mediated resistance is unlikely to be an issue in managing cpa patients. however, given the long-term treatment required, surveillance for resistance development during therapy is warranted. systematic surveillance of clinical and environmental aspergillus spp. is needed in nigeria to monitor for the emergence of azole-resistant phenotypes. acknowledgements we thank the entire staff of the department of medical microbiology and parasitology, lagos university teaching hospital, idi-araba, lagos, and radboudumc-canisius-wilhelmina hospital center of expertise for mycology, nijmegen, the netherlands, for their immense support during the study. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions a.a.d., b.s., e.f.j.m., i.i.o., o.b., a.a., t.g.-b., j.f.m. and r.o. contributed equally to the study. a.a.d. was involved in the conception and design of the study, literature review, data collection, analysis, and the first draft of the article. b.s., j.f.m., and e.f.j.m. contributed to the analysis and writing of the methods, and reviewed the first draft of the manuscript. i.i.o., r.o., j.f.m. and t.g.-b. reviewed the first draft of the article. a.a. reviewed the chest x-ray, and o.b. was involved in the statistical analysis. a.a.d., b.s., e.f.j.m., i.i.o., o.b., a.a., t.g.-b., j.f.m. and r.o. read and approved the final article. funding information this research received no specific grant from any funding agency in the public, commercial, or not for profit sectors. data availability calmodulin sequences of the aspergillus spp. were deposited under genbank accession number oq915062-oq915108. the data that support the findings of this study are available on request from the corresponding author, a.a.d. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. the article does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the authors are responsible for this article’s results, findings, and content. references world health organization. world health organization fungal priority pathogens list to guide research, development, and public health action [homepage on the internet]. 2022 [cited 2023 nov 28]. available from: https://www.who.int denning dw, riniotis k, dobrashian r, sambatakou h. chronic cavitary and fibrosing pulmonary and pleural aspergillosis: case series, proposed nomenclature change, and review. clin infect dis. 2003:37(suppl 3):s265–s280. https://doi.org/10.1086/376526 ocansey bk, otoo b, adjei a, et al. chronic pulmonary aspergillosis is common among patients with presumed tuberculosis relapse in 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fumigatus: a side-effect of environmental fungicide use? lancet infect dis. 2009:9(12):789–795. https://doi.org/10.1016/s1473-3099(09)70265-8 chen y, dong f, zhao j, et al. high azole resistance in aspergillus fumigatus isolates from strawberry fields, china, 2018. emerg infect dis. 2020;26(1):81–89. https://doi.org/10.3201/eid2601.190885 abstract introduction methods results discussion acknowledgements references about the author(s) shahin sayed department of pathology and laboratory medicine, aga khan university hospital, nairobi, kenya susan c. lester department of pathology, brigham and women’s hospital, boston, massachusetts, united states department of pathology, harvard medical school, harvard university, boston, massachusetts, united states michael wilson department of pathology and laboratory services, denver health, denver, colorado, united states university of colorado school of medicine, aurora, colorado, united states daniel berney barts cancer institute at queen mary university of london, london, united kingdom ricard masia department of pathology, harvard medical school, harvard university, boston, massachusetts, united states department of pathology, massachusetts general hospital, boston, massachusetts, united states zahir moloo department of pathology and laboratory medicine, aga khan university hospital, nairobi, kenya department of pathology, university of ottawa, ottawa, ontario, canada jennifer stall department of pathology, harvard medical school, harvard university, boston, massachusetts, united states department of pathology, massachusetts general hospital, boston, massachusetts, united states alexia eslan african strategies for advancing pathology, denver, colorado, united states stephanie ayers african strategies for advancing pathology, denver, colorado, united states angela mutuku college of pathologists of east, central and southern africa, nairobi, kenya jeannette guarner department of pathology and laboratory medicine, emory university school of medicine, atlanta, georgia, united states citation sayed s, lester sc, wilson m, et al. creation and pilot testing of cases for case-based learning: a pedagogical approach for pathology cancer diagnosis. afr j lab med. 2017;6(1), a637. https://doi.org/10.4102/ajlm.v6i1.637 original research creation and pilot testing of cases for case-based learning: a pedagogical approach for pathology cancer diagnosis shahin sayed, susan c. lester, michael wilson, daniel berney, ricard masia, zahir moloo, jennifer stall, alexia eslan, stephanie ayers, angela mutuku, jeannette guarner received: 13 apr. 2017; accepted: 21 june 2017; published: 25 oct. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: case-based learning (cbl) is an established pedagogical active learning method used in various disciplines and defined based on the field of study and type of case. the utility of cbl for teaching specific aspects of cancer diagnosis to practising pathologists has not been previously studied in sub-saharan africa. objectives: we aimed to pilot test standardised cancer cases on a group of practising pathologists in sub-saharan africa to evaluate case content, clarity of questions and delivery of content. methods: expert faculty created cases for the four most commonly diagnosed cancers. the format included mini-cases and bullet cases which were all open-ended. the questions dealt with interpretation of clinical information, gross specimen examination, morphologic characteristics of tumours, ancillary testing, reporting and appropriate communication to clinicians. results: cases on breast, cervical, prostate and colorectal cancers were tested on seven practising pathologists. each case took an average of 45–90 min to complete. questions that were particularly challenging to testers were on: specimens they should have been but for some reason were not exposed to in routine practice. ancillary testing and appropriate tumour staging. new knowledge gained included tumour grading and assessment of radial margins. revisions to cases were made based on testers’ feedback, which included rewording of questions to reduce ambiguity and adding of tables to clarify concepts. conclusion: cases were created for cbl in kenya, but these are applicable elsewhere in africa and beyond to teach cancer diagnosis. the pilot testing of cases prepared faculty for the actual cbl course and feedback provided by the testers assisted in improving the questions and impact on day-to-day practice. introduction the use of standardised cases to teach different levels of trainees in the medical professions is a common practice. the use of case-based learning (cbl), a well-established pedagogical active learning method with divergent definitions depending on the discipline and type of ‘case’ employed,1 to teach specific aspects of a cancer diagnosis to practising pathologists and pathology fellows or trainees is not frequently employed. pathologists use daily sign-out cases on their desks to teach trainee residents. this approach has served many but is trainer dependent and based on the local practice rather than standard practice. as an example, if synoptic pathology reporting of cancers is not routine practice in a particular country, this will not be part of what is taught to resident trainees. case-based learning is a process by which trainees actively learn through a clinical presentation that serves as a stimulus to acquire additional knowledge on the specific clinical entity to solve problems. williams2 emphasised that cbl is an educational paradigm posing contextualised questions that allow students to develop a collaborative approach to their education by fostering integrated learning and promoting self-assessment, reflection and life-long learning. in the medical setting, cases provide the student with the background of a patient or other clinical situation.3 the description can be vague but have adequate content to facilitate evaluation.4,5 in addition, supporting information that helps trainees acquire knowledge may include: vital signs, clinical signs and symptoms, laboratory results, book chapters and even the latest research articles. the instructor facilitates knowledge construction and directs students away from a predominantly passive, lecture-driven mode.6 case-based learning is often contrasted with problem-based learning (pbl) and the differences between pbl and cbl are not always clear. barrows and tamblyn7 defined pbl as the learning that results from the process of working towards the understanding of a resolution of a problem where the problem is encountered first in the learning process. problem-based learning is more self-directed and allows students to explore various domains of the problem based on their prior knowledge but there is no guidance provided by facilitator even if learners deviate from the problem.8,9 in contrast, cbl is a guided inquiry method, with defined learning outcomes, and the teaching builds on prior knowledge, integrates data and considers application to future situations. this in turn encourages teamwork and accountability, as well as engages participants in their learning to think about plausible answers instead of passively receiving the information.10 thus both cbl and pbl are used to stimulate and underpin the active acquisition of knowledge, skills and attitudes,11 although cbl is more structured. this paper focuses on the process of creating standardised cancer cases and the pilot testing of the same. the pilot testing determined the validity of the questions and their acceptability. we describe the outcome and learning points from the pilot exercise and the improvements made on the cases based on the feedback received. methods ethical considerations the research ethics committee, aga khan university, approved this study (approval number: 2017/rec-39). a total of two senior and five mid-career faculty members provided written informed consent to participate in pilot testing of the cases in two sessions over an interval of three weeks. testers were practising pathologists from a sub-saharan africa teaching institution with more than five years postgraduate teaching experience in surgical pathology. study design we elected to create cases that included the four most commonly diagnosed cancers by pathologists in the east, central and southern africa regions: breast, cervical, prostate and colorectal cancers.12,13 the format of the cases included mini-cases (consisting of a narrative with tightly focused questions that assisted learners to apply a variety of concepts) and bullet cases (consisting of two to three sentences with one or two directed teaching points). all questions were open-ended so that the participants would write down an answer; there were no multiple-choice questions and only one correct answer. the questions in each of the cases were aimed at emphasising the pathological features of the cancers that have clinical impact on diagnosis, treatment and prognosis for the four selected cancers. the cases included questions that dealt with interpretation of clinical information (e.g. previous treatment, tumour marker data, other relevant history), aspects regarding description and processing of gross specimens (e.g. dissection and inking), morphologic characteristics of tumours (e.g. mitoses, grading, amount of tumour in biopsies), further ancillary testing (e.g. immunohistochemistry) and how tumours should be reported (including synoptic reporting) and the appropriate relevant communication to clinicians. answers to the questions were provided in powerpoint format as reference material to the testers. the questions were formatted in a cascading style with an increasing level of complexity. the faculty created 10 mini cases (2 breast cases, 2 cervical cases, 3 colorectal cases and 3 prostate cases) in 14 to 50 questions and 4 bullet cases (all prostate) with 1–4 questions for each bullet case. after the cases were created, the expert faculty and facilitators reviewed and discussed the cases and questions prior to the pilot testing. the aim of the testing was to evaluate case content and the clarity of the questions, and define the best manner of delivery (e.g. the best method to present microscopic images using glass slides and microscopes, printed photographs, or whole-slide digitised images on screens). we estimated the time each case would take to solve, so that testers would have an idea of the amount of uninterrupted time they would need to devote to testing of these cases. the process of creation of the cases and the pilot testing exercise is illustrated in supplementary figure 1. supplementary figure 1: flow chart of pilot testing process. pilot testing session the main objectives of pilot testing the cases were to: scrutinise the need for each question and document feedback from testing group participants. review and improve upon the questions that were not clear. determine the approximate length of time it took to complete each one of the cases. a faculty member who had gone through the cases with the experts who created them facilitated the pilot testing session. testers were practising pathologists from a kenyan teaching institution with more than five years of postgraduate teaching experience in surgical pathology. the testers were given an overview of the capacity building project for which the cases were being created. they were informed about the objectives of the pilot testing and were asked to provide specific feedback regarding: finding the answers, and the ease with which they found the answers, to the questions in the printed powerpoint material provided. information that was new to them after having solved the cases. the total time it took to answer each one of the cases and which questions took the most time to answer. other instructions given to testers included: they were encouraged to work in groups and advised to give themselves a block of 2–3 h to solve the cases for each cancer. however, they were asked to spend no more than 10 min per question on a case. they were told to go over the cases and answer the questions in each case in the sequence presented and to work without interruptions. they were provided with hard copies of the powerpoint lectures and synoptic reporting templates and encouraged to refer to these so as to answer the questions. the participants were instructed to write down issues they saw with questions (e.g. responses could vary, could not find answer, too difficult, spent too much time). lastly, we (faculty and support staff) conducted a survey of testers to gain knowledge of acceptability of the standardised cases. results a total of two senior and five mid-career faculty members agreed to participate in pilot testing of the cases in two sessions over an interval of three weeks. two cancers were pilot tested in each session. table 1 provides details of the topics that were challenging to testers and the new concepts learned after the cbl exercise. table 1: case type, challenges encountered and new knowledge gained. depending on the complexity of the case and the number of questions, each case took approximately 45 to 90 min to complete. the questions that were found to be challenging related to those cases and specimen types that the participants were not exposed to in routine practice within their local context. these included handling of breast specimens post neoadjuvant therapy, handling of rectal resection specimens and radical prostatectomy specimens. other challenging questions included those related to molecular testing and use of complementary immunohistochemistry testing (e.g. microsatellite instability testing for colorectal cancer) and appropriate staging according to the tumour-node-metastasis system (e.g. determining whether serosal penetration is present in colorectal cancer). in addition, participants felt they had acquired new knowledge with regard to grading of tumours (assessing breast mitotic counts and the new grade group system for prostatic cancers), appropriately assessing radial margins for colorectal resection specimens and gained clarity on the indications and interpretation of the microsatellite instability testing for colorectal cancers. based on the feedback given by the testers, changes were made to the cases. these changes included rewording of questions felt to be ambiguous or confusing and adding tables to some cases to clarify concepts (e.g. the distinction between tumour size and tumour stage). regarding the reference material given to the testers (i.e. the printed powerpoint presentations), important feedback was provided. participants noted that they should be instructed to review the presentations prior to attempting to answer the questions. in addition, participants suggested that it would be helpful to label the images in the presentations with the type of neoplasia or teaching point being demonstrated, such that the presentations could ‘stand alone’ as educational material (i.e. a live lecture was not necessary). there was debate among the expert faculty and facilitators regarding the presentation of the reference powerpoint material to the testers (i.e. electronically rather than printed). the printed version on the a4-sized paper used for the testing had very small font size and was difficult to read (supplementary figure 2). therefore, participants suggested that if printed material is used for the cases, the powerpoint slides should be printed using a larger font size. supplementary figure 2: examples of powerpoint slides printed for pilot testing exercise. an electronic post-testing feedback survey was administered to the seven pilot testers three months after the exercise. four of the testers who had taken part in the pilot testing exercise responded to the survey. they found the cases very useful and stated that the knowledge gained was applicable in their day-to-day practice. all of them commented that they have implemented changes in their practice based on what they learned from the cbl. some of the testers, however, would have preferred video microscopy sessions with actual glass slides, longer duration of training sessions and prior access to reference or reading material in preparation for the cases. discussion the pilot testing of standard cancer cases provided information regarding acceptability, time it may take for pathologists in kenya to solve these cases and knowledge gained. moreover, at least one of the topics for each cancer in which the testers struggled were the same as the ones for which they gained knowledge. these cases were used one month later in a study that compared knowledge gained after a lecture-based course versus a cbl course for pathologists practising in east, central and southern africa. the rationale of using the cbl type of instruction is that by ‘doing’ there is better retention of concepts with emphasis on critical thinking and comprehension of the defined problem and arriving at a solution in the appropriate context.14,15 in any residency training, review of cases with faculty is how residents learn. this system is faculty dependent and the content of instruction will vary from one faculty member to the next reviewing a case. in most instances, especially for pathology, the system provides opportunity for one-on-one teaching. the training content in this context is subject to the local practice. in addition, if a case of a rare entity is unavailable during the trainee’s residency years, the resident misses out on the opportunity to see such a case, thus limiting their exposure and learning. creating cases for cbl therefore allows for standardised instruction to be provided across the residency years. testers of our cases felt that the material was easy to relate to and that the amount of content and the complexity included was appropriate to their needs and daily practice. we included mini cases and bullet cases, but the two different format types and lengths of questions did not make a difference to the participants’ level of engagement. according to milliard et al.,16 the success of a case is highly dependent upon the interest it elicits among the students in the synthesis and applicability of the information provided for the learning process.16 in order to create student engagement and promote in-depth discussions, case length, realism and level of intrigue should be considered.17,18 our cases had all the essential facts to facilitate integration of the information provided so as to arrive at a predetermined definitive solution. we did not have questions that allowed multiple possible solutions.19 the answers to the questions were present in the powerpoint presentations that were given to the testers at the same time they were given the cases, but the majority of the testers found it distracting to refer to the materials. this could be explained by the fact that the powerpoint presentations were printed with two slides per page using letter-size paper. this meant that the font size was very small and may have discouraged participants from utilising the materials. faculty therefore agreed that when using these cases an electronic version of the reference material needs to be provided or the presentations should be printed as one slide per page. we established a set of guidelines for the testers which they were required to adhere to when solving the cases. two factors may be key to the success of a cbl platform: group activation and accompanying peer instruction.15,20,21 as an example, it may be useful to illustrate the benefits of teamwork15 or in our cases the need to answer the questions in order as each built upon the previous answer. introducing cbl pedagogies to students for the first time may be bit of a challenge as it may result in resistance; therefore, one should take into consideration that the instructor must be skilled in facilitating group learning activities. clear instructions on the cbl template, goals and timelines and provision of the case prior to the learning activity can mitigate this challenge.15,22 failure of cbl implementation is mainly caused by poor instructional planning as previously reported by struyven et al.23 our seven testers worked together to solve the cases. some have stated that six participants in a group should be the maximum group size permitted as it allows for closer interaction and participation of each group member.22 the discussion should be paced to ensure that trainees and instructors alike become familiarised with the cbl style.15 as cited by kulak and newton,15 in order to strengthen instructor–student engagement, the facilitator should familiarise themselves with student names. the amount of time that it took for questions to be answered by the testers was between 45 and 90, minutes depending upon the case. this was very similar to what was expected by the faculty, but testing the cases was nonetheless instrumental in confirming the amount of time required for case completion in this particular context. after the testing, the faculty decided to include references in the cases to guide participants to the location of the pertinent information in the presentations, as it was deemed to be too time-consuming to locate the information otherwise. to optimise time utilisation and sustain a focused discussion on the case in hand, the instructor ideally should be familiar with all aspects of the case.15,24 we found that very minor changes were needed for the cases and questions; however, the expert faculty that created the cases found the feedback from the testers very informative. it allowed the expert faculty who practise in a western context to become familiar with african realities and tailor the instructional delivery of the cases accordingly. having a faculty familiar with the local environment to conduct the pilot testing also permitted the group members a level of comfort in expressing their views, providing honest feedback and remaining engaged throughout the process. in summary, we created standardised cancer cases for cbl in east, central and southern africa, but these same cases could be used elsewhere to teach cancer diagnosis. our pilot testing prepared us better for the actual cbl course and gave us a sense of possible problems to encounter. in the survey performed three months later, testers stated that they had changed their practice to incorporate the concepts learned. as an example, testers indicated that they had adopted the new prostate grading system into practice. this emphasises that this active pedagogical approach to learning pathological concepts for cancer diagnosis was beneficial to our testers who seemed to have learned as much as if they had participated in the course. acknowledgements the authors would like to thank drs zuriel daniel, joseph r. ndungu, miinda pramenas okemwa, emily adhiambo rogena, edwin walong and wairimu waweru of the department of human pathology university of nairobi and dr mary mungania of kenyatta national hospital for participating in the pilot testing of cases. the authors would like to also thank the aga khan university hospital, nairobi, for their administrative support. competing interest the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. sources of support funded by: administrative supplements to promote cancer prevention and control research in low and middle income countries department of health and human services public health services. grant number: ncipar-15-155; principal investigator: dr michael wilson. daniel berney was supported by orchid cancer (http://www.orchid-cancer.org.uk). author contributions s.s. was the lowand middle-income country (lmic) lead and wrote the manuscript; s.c.l., d.b., r.m., j.s. were the high-income country (hic) faculty who prepared the cases and reviewed the manuscript; m.w. was the hic project leader and reviewed the manuscript, z.m. was the lmic faculty and reviewed the manuscript; a.e. was the hic project administrator and reviewed the manuscript; s.a. was the hic research analyst; a.m. was the lmic project coordinator and assisted with manuscript preparation according to journal guidelines; j.g. was the hic project consultant and faculty and contributed to writing and review of the manuscript. references sturdy s. scientific method for medical practitioners: the case method of teaching pathology in early twentieth-century edinburgh. bull hist med. 2007;81(4):760–792. https://doi.org/10.1353/bhm.2007.0093 williams b. case based learning – a review of the literature: is there scope for this educational paradigm in prehospital education? emerg med j. 2005;22(8):577–581. https://doi.org/10.1136/emj.2004.022707 wetherell j, mullins g. the use of student journals in problem-based learning. med educ. 1996;30(2):105–111. https://doi.org/10.1111/j.1365-2923.1996.tb00727.x kahn p, o’rourke, k. understanding enquiry-based learning. in: barrett t, labhrainn i, fallon h, editors. handbook of enquiry and problem-based learning: irish case studies and international perspectives. galway: centre for excellence in learning and teaching, nui; 2005. p. 1–12. [cited 2017 jan 3]. available from: http://www.aishe.org/readings/2005-2/chapter1.pdf shulman ls. toward a pedagogy of cases. in: shulman jh, editor. case methods in teacher education. new york: teachers college press. 1992; p. 1–30. fernández-santander a. cooperative learning combined with short periods of lecturing: a good alternative in teaching biochemistry. biochem mol biol educ. 2008;36(1):34–38. https://doi.org/10.1002/bmb.20141 barrows hs, tamblyn rm, barrows. problem-based learning: an approach to medical education. new york springer publishing company. 1980;1:224. [cited 2017 jan 3]. available from: http://books.google.com/books?id=9u-5djuqq2uc srinivasan m, wilkes m, stevenson f, nguyen t, slavin s. comparing problem-based learning with case-based learning: effects of a major curricular shift at two institutions. acad med. 2007;82(1):74–82. https://doi.org/10.1097/01.acm.0000249963.93776.aa schneider dk. case-based learning.– edutech wiki. [cited 2017 jan 10]. available from: http://edutechwiki.unige.ch/en/case-based_learning bastable sb. nurse as educator: nurse as educator principles of teaching and learning for nursing practice, 4th ed. london: jones and bartlett. 2014. aamodt a. case-based reasoning: foundational issues, methodological variations, and system approaches. ai commun. 1994;7:39–59. korir a, okerosi n, ronoh v, mutuma g, parkin m. incidence of cancer in nairobi, kenya (2004–2008). int j cancer. 2015;2059(137):2053–2059. https://doi.org/10.1002/ijc.29674 wabinga hr, nambooze s, amulen pm, okello c, mbus l, parkin dm. trends in the incidence of cancer in kampala, uganda 1991–2010. int j cancer. 2014;135(2):432–439. https://doi.org/10.1002/ijc.28661 richardson jte. instruments for obtaining student feedback: a review of the literature. assess eval high educ. 2005;30(4):387–415. https://doi.org/10.1080/02602930500099193 kulak v, newton g. a guide to using case-based learning in biochemistry education. biochem mol biol educ. 2014;42(6):457–473. https://doi.org/10.1002/bmb.20823 millard jt. television medical dramas as case studies in biochemistry. j chem educ. 2009;86(10):1216–1218. https://doi.org/10.1021/ed086p1216 cornely k. content and conflict: the use of current events to teach content in a biochemistry course. biochem mol bio ed. 2003;31(3):173–6. gijbels d, dochy f, van den bossche p, segers m. effects of problem-based leaming : a meta-analysis from the angle of assessment. rev educ res. 2005;75(1):27–61. https://doi.org/10.2307/3516079 herreid c.f. what makes a good case? j coll sci teaching. 1997/98;27:163–165. hussain rmr, mamat whw, salleh n, saat rm, harland t. problem-based learning in asian universities. stud high educ. 2007;32(6):761–772. https://doi.org/10.1080/03075070701685171 moust jhc, van berkel hjm, schmidt hg, et al. case based learning – a review of the literature: is there scope for this educational paradigm in prehospital education? biochem mol biol educ. 2005;3(4):577–581. https://doi.org/10.1136/emj.2004.022707 mostert mp, sudzina mr. undergraduate case method teaching: pedagogical assumptions vs the real world. in: proceedings of the annual meeting of the association of teacher educators; 1996 february; st louis (mo). [cited 2017 jan 10]. available from: http://files.eric.ed.gov/fulltext/ed395900.pdf struyven k, dochy f, janssens s. students’ perceptions about evaluation and assessment in higher education: a review. assess eval high educ. 2005;30(4):325–341. https://doi.org/10.1080/02602930500099102 cliff wh, wright aw. directed case study method for teaching human anatomy and physiology. am j physiol. 1996;270(6 pt 3):s19–s28. abstract introduction methods results discussion acknowledgements references about the author(s) malcolm t. ellapen department of virology, albert luthuli hospital, durban, south africa terry j. ellapen department of sport, rehabilitation and dental sciences, faculty of health, tshwane university of technology, pretoria, south africa yvonne paul department of sport, rehabilitation and dental sciences, faculty of health, tshwane university of technology, pretoria, south africa citation ellapen mt, ellapen tj, paul y. the growth patterns of the medical technology profession in south africa. afr j lab med. 2021;10(1), a1164. https://doi.org/10.4102/ajlm.v10i1.1164 original research the growth patterns of the medical technology profession in south africa malcolm t. ellapen, terry j. ellapen, yvonne paul received: 10 jan. 2020; accepted: 22 oct. 2020; published: 23 apr. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: constant appraisal of healthcare workforce trends is vital; this measure determines the adequacy of the workforce in meeting its society’s healthcare demands. this includes determining the number of the incoming workforce (students, interns) and the active or practising workforce relevant registries. objective: this study aimed to examine patterns of workforce growth in the medical technology profession (students, interns and practitioners) from 2008 to 2018 in south africa. methods: student, intern and practitioner medical technology registries, from the 2012/2013, 2016/2017 and 2017/2018 health professions council of south africa (hpcsa) annual reports, were analysed. the number of national health laboratory service and private laboratory posts were secured from the national health laboratory service plan performance report. a comparison between the total number of south african medical technology (private and public) posts occupied versus the hpcsa practitioner 2016 register was completed, to determine the saturation status. results: annual student, intern, and practitioner registries indicated a mean growth of 6.8%, 28.9%, and 0.7% from 2008 to 2018. the transition of interns to practitioners is progressively dwindling (2015–2018). the practitioner register showed a 1.2% decline in registration from 2013 until 2018. in 2016, only 55.9% of the hpcsa registered medical technologists were employed (p < 0.001). conclusion: there are more medical technologists than available public and private sector posts. the progressively growing student register compared to the dwindling practitioner register indicates attrition in the profession. an investigation identifying the reasons why graduates neglect to register as practising medical technologists should be undertaken. keywords: medical technology; attrition; growth; interns; practitioner. introduction sub-saharan african countries have fewer employed healthcare staff compared to australia, the united kingdom and the united states.1 in 2013, these developed countries had an estimated 59.4 healthcare practitioners per 10 000 of their populations, compared to an estimated 22.8 healthcare practitioners per 10 000 obtainable in sub-saharan african countries.1,2 this deficiency in the healthcare staff to population ratio adversely influences the quality of healthcare provision in sub-saharan african countries. lopes et al. described the attrition in a profession as the premature voluntary resignation of an employee from their vocation due to illness, death, emigration or expected superannuation.3 common reasons cited for premature exit from healthcare professions are poor salary packages, transition to more lucrative employment opportunities, job dissatisfaction, lack of upward job mobility, poor working conditions and family responsibilities.2,4 the primary reason for job dissatisfaction is unrealistic employment expectations.5 in many healthcare professions, interns are expected to register as practising healthcare professionals immediately after completing their mandatory residency in order to secure employment.6 a new scenario is, however, emerging where graduate laboratory scientists refrain from registering and practising as healthcare scientists or technologists after completing their residency.2,7,8 this contributes to the poor profession transition which adversely affects the pool of available scientists.2 most research pertaining to career transition on brain drain has concentrated on doctors, professional nurses and pharmacists who prematurely leave their professions.2,9,10 a literature search on the sabinet search engine (january to july 2019), employing the keywords ‘attrition of medical technologists’ revealed 15 records. following title and abstract analyses, only a single study relating to this research interest remained, which is suggestive of the paucity of literature on the aforementioned topic in africa.2 anecdotal reports also suggest that the south african medical technology profession may be experiencing attrition. medical technologists analyse human tissue, as well as body fluids or excretions; laboratory findings are then utilised by the medical practitioner or dentist to make a clinical diagnosis or institute medical or dental treatment.6 the constant scarcity of medical technologists and laboratory scientists in the united kingdom and the united states inspired research aimed at uncovering the reasons for their premature attrition from the medical profession. data have informed the development and implementation of some laboratory staff retention strategies.11,12,13 despite the important role that medical technologists play concerning clinical diagnoses, there is little research on this workforce’s growth in south africa. this preliminary investigation aimed to identify the medical technology profession’s growth patterns by reviewing the student, intern and practitioner medical technology registries from 2008 to 2018 on the health professions council of south africa (hpcsa) database. it is intended that the preliminary findings of this article will encourage further research to uncover the predisposing factors influencing the intern medical technologists’ attrition that will in turn inform retention strategies. methods ethical considerations these data are in the public domain (hpcsa website); therefore, no ethical approval for the study was needed. study design and data source the study was a retrospective descriptive analysis of the medical technology profession growth in south africa. the period observed was from 2008 to 2018. the student, intern and practitioner registries of the medical technology profession were obtained from the hpcsa annual reports.14,15,16 a student medical technologist-in-training is currently completing a medical technology degree and is registered with the hpcsa as a student-in-training. an intern medical technologist is a student who has completed their theoretical requirements and has partially fulfilled the practical requirements for the medical technology degree but is currently in the final year of mandatory clinical residency. being a student, the intern medical technologist is therefore registered with the hpcsa. a practitioner is a medical technologist who has completed the theoretical requirements for the academic degree and the mandatory clinical residency of both the training institution and the hpcsa. the hpcsa register of medical technologists serves to identify all eligible practitioners. without hpcsa registration a medical technologist cannot practise. statistical analysis the percentage change in registration between each successive year among the students, interns and practitioners was calculated as shown in equation 117: to determine the growth or attrition of the medical technology professionals in a given year, the number of registered interns was compared to the number of newly registered practitioners in the subsequent year. growth or attrition was calculated as: in equation 2, nrp is the number of newly registered practitioners and imt is the number of intern medical technologists. the comparison of the total number of medical technologists employed in 2016 according to the competition commission health market inquiry versus the number of hpcsa registered medical technologists was made to determine existence of over-saturation or under-saturation in the profession.14,15,18 the descriptive statistics adopted were mean, standard deviation and percentages. a chi-square analysis, between the numbers of national health laboratory service and private laboratory posts in comparison to the number of practitioners, was completed for the year 2016. an independent student t-test was used to compare annual intern to practitioner medical technologist transition rate, as well as the annual growth rate of interns and practitioners (alpha was set at p < 0.05). results overall, there was a positive increase in the number of student, intern and practitioner medical technologists (table 1).14,15 the mean growth in the number of student medical technologists was 6.8% while that of the interns was 28.9%, despite observed declines in 2015 and 2018. the annual registration of practitioners showed a mean growth of 0.7% over the period of 2008 to 2018 (table 1).14,15 table 2 shows the intern to practitioner rate; an increase was recorded from 2008 to 2012, while from 2013 to 2018 the number of interns registering as practitioners following their residency dwindled. the annual practitioner registry growth rate from 2008 until 2012 was 2.3%, as compared to the diminishing growth rate of −1.2% from 2013 until 2018. a comparative review of the growth of the intern medical technologists and practitioner registries from 2013 until 2018 significantly differed (p = 0.04). intern medical technologist registries grew at an annual mean rate of 12.7% (±15.3), while practitioner registries dwindled by an annual mean rate of −1.2% (±6.7%). table 3 indicates a significant difference between the number of newly registered interns compared to the registered practitioners (p < 0.047). table 1: growth rate patterns of student, intern and practitioner registries of the south african medical technology profession (2008–2018). table 2: the transition rate of change of south african medical technologists interns to practitioners (2008–2018). table 3: comparison between the transitional number of south african interns, practitioners and newly registered practitioners from 2008 to 2018. there are both public and private pathological laboratories and depots in south africa. the public laboratories and depots fall within the ambit of the south african national health laboratory service. the largest south african private pathological laboratories include pathcare, ampath and lancet; these saturate approximately 90% of the private market.18 in 2016, the competition commission health market inquiry reported that the south african national pathology group had a total number of 10 295 employees, which comprised 3000 medical technologists. of the 3000 medical technologists, national health laboratory service employed 1473 practitioners (49.1%).18 table 3 indicates that the number of previously registered practitioners significantly differed from the newly registered practitioners (p < 0.0001). discussion the hpcsa student register is composed of students pursuing a vocation of medical technology, who are registered with one of the accredited south african tertiary training institutions. students-in-training cannot complete clinical practice, if they are not registered with the hpcsa.6 however the student register does not distinguish among first-year, second-year and third-year students-in-training. the register does distinguish student-in-training, intern medical technologist and practitioner.6 the student register increased from 2008 to 2018. these statistics are reassuring, and they epitomise the ongoing interest in the medical technology profession. anagnostopoulou et al. and ossai et al. reported that undergraduates who are pleased with the quality of education and residency they receive continue with the academic process.19,20 as such the steady growth in the student registration can be seen as arising out of the satisfaction of both students and their benefactors with the quality of education and training provided by south african tertiary education institutions. however, to ascertain whether the growth in the student registry is a direct result of the satisfaction that students and their benefactors feel, regarding the quality of their training and education, a more direct investigation of the perceptions of students-in-training should be undertaken. the progression of student-in-training medical technologists to intern medical technologists is marked by positive average growth. this is reminiscent of the fact that undergraduates are ardent to earn their degree and these statistics furthermore reflect the work-integrated learning stage of the clinical internship, which falls under the purview of both academic training institutions and clinical laboratories. clinical internships are offered by both public and private laboratories, allowing students the opportunity to complete their clinical training. the practitioner register showed a meagre positive growth rate from 2008 to 2018 of 0.7%. this rate is below the global growth rate of the medical technology profession which has fluctuated between 2% and 8% from 2010 to 2018, with an average growth rate of 3.65%.21 in 2016 the hpcsa had 5362 registered medical technologists (table 2), but according to the competition commission health market inquiry only 3000 (55.9%) were employed in both public and private healthcare sectors; 2362 (44.1%) were unemployed or not practising.14,15,18 this statistic shows that there were 2362 medical technologists unaccounted for, which could be seen as over-saturation of the profession. there was a steady decline in the intern to practitioner transition rate from 2013 to 2018, stressing the attrition of intern medical technologists to practitioners. these findings concur with those of noden et al. who reported that many intern and graduate medical technologists are actively leaving the profession while some seek further postgraduate education.2 the precise rationale for exist from the south african medical technology profession is unknown as yet. therefore, further investigation is warranted. while there are several possible reasons for this premature exodus from the profession, mcclure postulated that the primary reason many healthcare laboratory graduates and interns do not practise the profession is due to poor career advancement opportunities. thus, interns are upskilling themselves to be more marketable in other alternative, more financially lucrative, professions.7 the annual medical technologist salary packages offered by the various south african pathological laboratories range as follows: ampath (south african rand [zar] r247 000.00), lancet laboratories (r249 000.00), national health laboratory service (r287 000.00) and pathcare (r314 000.00).22 the united states annual salary packages for medical technologists range from $53 576.00 united states dollars (usd) to $52 712.00 usd, which is between r931 150.88 to r916 134.56 (conversion rate of $1.00 usd:r17.38 on 02 june 2020 [using conversion rate http://www.payscale.com]). in hong kong, the average medical technologist salary package is hong kong dollars (hkd) $264 906.00, which is equivalent to r593 389.44 (conversion rate of $1.00 hkd:r2.24 on 02 june 2020). a common alternative career path is academia. the average annual south african academic salary is r309 281.00, which is almost equivalent to the highest salary package in pathcare.21 beck and doig reported that poor financial remuneration packages are fundamental stimuli for premature graduate and young healthcare laboratory staff resignation.12 another reason is that many older medical scientists continue working until retirement age, thereby limiting the employment opportunities of graduates.8 young graduates are thus driven to change their professions to secure employment. presently there are more registered medical technologists than there are available practitioner posts in both the private and public sectors.17 the initial problem the authors identified from the preliminary data is that there is a lack of jobs for approximately 40% of the practitioner registry; where are these practitioners going to find an occupation? this information needs to be relayed to the training institutions to reduce the student entrance numbers, which can help prevent further over-saturation. a pragmatic solution would be to create more medical technologist posts, because most laboratories are understaffed, resulting in the staff being overworked. recommendations for prospective studies the study confirms the attrition of the profession at the point of a career transition from intern medical technologist to practitioner. the possible reasons for this remain unknown. it is recommended that an investigation identifying the reasons why graduates neglect to register as practising medical technologists be undertaken. the attrition of healthcare laboratory staff in developed countries has encouraged intensive research, which uncovered the rationale behind their decisions.11,12,13 it is suggested that similar investigations be undertaken in south africa. the study should also try to determine the factors predisposing the change in student-in-training, intern medical technologist and practitioner perceptions and attitudes from initial desire to pursue a career in medical technology to leaving the profession. limitations this preliminary study has the following limitations, which warrant further investigation: how many intern medical technologists complete their residency, what are the potential career options of those interns who complete their residency, how many interns who complete their residency continue to stay in south africa, how many travel aboard to secure jobs and how many leave the profession? conclusion the south african medical technology profession has steadily grown its student and intern medical technologist registers. the evidence indicates that there are more registered practitioners available than public and private sector posts (suggesting potential over-saturation). also, many intern medical technologists are refraining from registering as practitioners, concurring with anecdotal reports of the ongoing attrition of the profession in south africa. it is postulated that the lack of employment opportunities in south africa is driving the attrition of intern to practitioner medical technologist transition. the concern lies in a not-so-distant future scenario, where there may be under-saturation of medical technologists in south africa, created by the present attrition of intern to practitioner transition. however, the underlying factors predisposing this attrition need to be determined in prospective investigations. future investigations determining the perceptions and attitudes of students-in-training, intern medical technologists and practitioners towards continuing in the profession is essential. the evidence gleaned from these investigations should be employed by the medical technology profession in conjunction with the south african department of health to draft and implement strategies to prevent attrition and over-saturation. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions m.t.e. was involved in the conceptualisation, drafting and reviewing of the manuscript. y.p. was involved in drafting and reviewing the manuscript. t.j.e. was involved in conceptualisation, gathering data, drafting and reviewing the manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability the authors confirm that the data supporting the findings of this study are available within the article. disclaimer the views and opinions expressed in this article are those of the 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[cited 2018 jul 05]. available from: http://www.labtestingmatters.org/responding-to-the-continuing-personnel-shortages-in-laboratory-medicine/ health professions council of south africa. annual report (2012/2013). pretoria: hpcsa. 2013; p. 21. health professions council of south africa. annual report (2016/2017). pretoria: hpcsa. 2017; p. 35. health professions council of south africa. annual report (2017/2018). pretoria: hpcsa. 2018; p. 33. ellapen tj, barnard m, swanepoel m, hammill hv, paul y, strydom gl. emerging trends in the profession of biokinetics: a 10 year perspective (2007–2017). int j med med sci. 2018;10(6):71–78. https://doi.org/10.5897/ijmms2018.1364 national health laboratory service (nhls). annual performance plan fiscal year: 2019/20 [homepage on the internet]. 2019 [cited 2020 jun 20]. available from: http://pmg-assets.s3-website-eu-west1.amazonaws.com/nhls_app_2019_2020.pdf anagnostopoulou e, mavroidis i, giossos y, koutsouba m. student satisfaction in the context of a postgraduate programme of the hellenic open university. turk online j distance educ. 2015;16(2):40–55. https://doi.org/10.17718/tojde.52944 ossai en, alo at, onwe bc, okoro do, ezeagu ne, ogbonnaya lu. prevalence and predictors of perceived stress: a study among medical students of ebonyi state university abakaliki, nigeria. j exp res. 2019;7(1):57–66. https://doi.org/10.9734/ajarr/2019/v3i129779 statista. total global medical technology growth per year from 2010 to 2022 [homepage on the internet]. 2019 [cited 2020 jun 20]. available from: https://www.statista.com/statistics/329048/worldwide-medical-technology-growth-per-year/ payscale.com. the average salary of a south african physiotherapist [homepage on the internet]. 2020 [cited 2020 jun 20]. available from: http://www.payscale.com abstract introduction methods results discussion acknowledgements references about the author(s) joseph n. enuma department of biological sciences, university of abuja, abuja, nigeria felix o. sanni department of research and development, fescosof data solution, ota, ogun state, nigeria malau b. matur department of zoology, university of jos, jos, plateau state, nigeria njab e. jean laboratory department, total laboratory project for africa, abudja, nigeria tosan erhabor department of haematology, medical laboratory science council of nigeria, abuja, nigeria iheukwumere i. egbulefu laboratory service, labhub medical & diagnostic centre-nigeria, abuja, nigeria citation enuma jn, sanni fo, matur mb, et al. malaria an opportunistic infection in hiv/aids patients? – a nigerian experience. afr j lab med. 2022;11(1), a1842. https://doi.org/10.4102/ajlm.v11i1.1842 original research malaria an opportunistic infection in hiv/aids patients? – a nigerian experience joseph n. enuma, felix o. sanni, malau b. matur, njab e. jean, tosan erhabor, iheukwumere i. egbulefu received: 28 jan. 2022; accepted: 03 aug. 2022; published: 24 nov. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: hiv and malaria interact at the level of the host’s susceptibility to infection, but little is known about the effect of hiv on malaria infection in nigeria. objective: this study estimated the prevalence of malaria parasitaemia and its relationship with hiv immunodeficiency. methods: this cross-sectional study was conducted in two hospitals in abuja, nigeria between october 2012 and march 2013 among 600 respondents, comprising 200 hiv-negative controls, 200 hiv-positive patients on antiretroviral therapy (art), and 200 hiv-positive patients not on art. malaria parasites, malaria density and absolute cd4 counts were carried out on all three groups. participants with cd4 counts below 350 cells/mm3 were considered immunocompromised and likely to develop opportunistic infections. results: most study participants were aged 21–40 years (65.2%). the mean cd4 counts of hiv-positive patients not on art (300 ± 211 cells/mm3) and those on art (354 cells/mm3) were significantly lower than among controls (834 cells/mm3) (p < 0.001). malaria prevalence was not statistically different between the controls (44.5%), patients on art (40.5%), and those not on art (39.5%) (p = 0.562). compared to 7% immunodeficiency among controls, 56% of patients on art and 65.5% of those not on art had a cd4 count < 350 cells/mm3 (p < 0.001). the prevalence of malaria parasitaemia among immunodeficient individuals (42.4%) was similar to prevalence among those with cd4 counts > 350 cells/mm3 (40.8%; p = 0.695). conclusion: these findings suggest that malaria parasitaemia is not an opportunistic infection among hiv-positive individuals in nigeria. keywords: malaria; infection; hiv; opportunistic; cd4. introduction with about 14% of the world’s population, sub-saharan africa is home to roughly 67% of all people living with hiv/aids.1,2 on the other hand, the world health organization estimates that sub-saharan africa accounts for more than 90% of the worldwide burden of malaria.3 as a result, any interaction between the two infections is expected to have a considerable impact on health in the region, particularly in countries like nigeria, which now has the largest malaria burden in sub-saharan africa and ranks first among african countries affected by hiv/aids.3 opportunistic infections occur more frequently and with greater severity in individuals with compromised immune systems, such as those positive for hiv.4 opportunistic infections are less prevalent today than during the early stages of hiv and aids, owing to improved medications that reduce hiv viral load and strengthen the immune system.5 however, many people living with hiv continue to develop opportunistic infections, because they are unaware of their hiv status, are not on treatment, or their treatment does not keep their viral load low enough for their immune system to fight infections.6 the possible repercussions and public health impact of hiv and malaria’s geographical overlap have been identified and explored in recent years.5,7,8 the two diseases interact at the level of the host’s susceptibility to infection. hiv infection increases the probability of clinical and severe malaria, while plasmodium falciparum infection increases hiv viral load.8,9 additionally, dual infection has been found to contribute to the spread of both illnesses in sub-saharan africa, raising concerns about treating coinfected patients and the possibility of therapeutic interactions.10 because of the significant geographic and social overlap between people infected with hiv and malaria, complex interactions at the individual and group levels are possible.11 notably, any relationship between the two infections has the potential to have considerable public health implications due to their respective frequencies.12 hiv infection may exacerbate malaria’s burden by increasing susceptibility to infection or impairing antimalarial medications’ preventative and therapeutic efficiency, as both are immune-mediated.13 however, early 1990s investigations failed to reveal substantial interactions between malaria and hiv in coinfected children and adults who had acquired semi-immunity to malaria, probably due to research design problems and a lack of information about immunosuppression.14 in the early 2000s, it was shown that hiv infection has a clinical effect on malaria infection and disease and that this effect appears to be reliant on the dynamics of malaria transmission and the degree of hiv-associated immunosuppression.15,16 hiv-positive individuals who have not acquired immunity to malaria exhibit a significant increase in the severity of infection, in contrast to those who have naturally acquired immunity to malaria, for whom hiv infection is associated with just a mild rise in clinical disease.17 malaria has been identified as a risk factor for concurrent hiv infection at the population level in the recent past.13 studies have addressed the prevalence of asymptomatic malaria,18,19 association between clinical malaria and immunosuppression among hiv-positive patients.20 and effects of malaria infection on hiv-positive individuals in nigeria,4,18,19,21,22 but information on malaria as an opportunistic infection in hiv-positive individuals is scanty. as such, this study sought to estimate the prevalence of malaria parasitaemia and its relationship with hiv immunodeficiency among hiv-positive patients in abuja, nigeria. methods ethical considerations this study received approval from the federal capital territory health research ethics committee (fhrec) with approval number fhrec/2012/11/34/05–11-12 the study protocol was made clear to the participants, and their informed consent was obtained, both orally and in written forms, before recruiting them to the study. information that could be traced to any of the respondents was not collected. data obtained from the participants were treated confidentially and for research purposes only. study design this prospective, randomised, cross-sectional study assessed malaria as an opportunistic infection in hiv-positive patients in abuja, nigeria, between october 2012 and march 2013. abuja is home to all of the country’s ethnic groups and six geographical regions. the study investigated two main groups: hiv-positive patients and hiv-negative individuals attending the hiv/aids clinics in either the wuse general hospital or asokoro district hospital. these hospitals are central to the federal capital territory and represent nigeria’s abuja metropolis. a total of 600 individuals were recruited to the study by using a convenience method to select participants by asking consented respondents if they were on antiretroviral therapy (art) or not until the required number of participants was completed, to ensure equal numbers of participants in each of three groups: hiv-negative, hiv-positive on art, hiv-positive not on art. hiv-negative individuals served as controls for the study. only hiv-positive and hiv-negative patients attending the two hospitals were included in the study; those who did not attend these hospitals were excluded. data collection after consent was given and before being enrolled into the study, hiv tests were done for the control patients to determine that they were truly hiv-negative and for the hiv-positive patients not on art to confirm they were truly hiv-positive. hiv tests were not conducted on hiv-positive patients on art, because we confirmed their status from the treatment case files retrieved from the hospitals. all participants were tested for the presence and density of malaria parasites and for cd4 count to assess their immune status. age and gender were collected from patients’ medical records. experimental procedures collection of blood samples for hiv test a 5 ml blood sample was collected into an ethylenediaminetetraacetic acid tube by the staff of the laboratory departments at both hospitals for all three tests: hiv test, malaria parasite examination and cd4 count. samples that could not be tested immediately were stored in a refrigerator at 2 °c – 6 °c and tested within 48 h. blood samples were tested at the same facility in which they were collected and were not transported to another facility. hiv testing three test kits, namely, determine hiv 1/2 (abbott japan co., ltd., tokyo, japan), uni-gold hiv 1/2 (chembio diagnostic systems, inc. hauppauge, new york, united states) and stat pak hiv 1/2 (trinity biotech plc., bray, co. wicklow, ireland), which are the national hiv testing algorithm screening tests23 (first-line test: determine hiv 1/2; confirmatory test: uni-gold hiv 1/2; tie breaker test: stat pak hiv 1/2) used for the hiv testing. whole blood was used for the testing. using the determine kit, one drop of whole blood was put onto the sample compartment of the kit, and two drops of chase buffer were added and allowed to run for 10 min before the reading was taken. the appearance of two pink lines on the strip indicates that the sample is positive; only one line indicates the test is negative. if there is no line at all, the test is considered invalid. positive results were confirmed with the confirmatory test kit (uni-gold). since we need hiv-negative participants in this group to serve as inherent controls, patients with hiv-positive results were excluded from the study. malaria parasite examination one or two drops of venous blood were spread on a clean, dry slide to make a thick blood film. the film was allowed to air dry, and the slide was then fixed in 100% alcohol for approximately 30 min. the slide was then flooded with a fresh solution of 10% giemsa stain (merck kgaa, darmstadt, germany), freshly prepared in distilled water. the stain was allowed to remain for approximately 30 min. after 30 min, the slide was rinsed in tap water and allowed to air dry thoroughly. when the thick film was completely dried, a drop of oil immersion was applied to an area of the film which appeared mauve coloured (usually around the edges). the oil was spread to cover about 10 mm in diameter to allow the film to be examined first at low magnification (olympus microscope, new york microscope company, hicksville, new york, united states). the area that was well stained but not too thick was selected. a 100x objective lens was used to examine malaria parasites and malaria pigments. at least 100 high power microscope fields were examined for parasites. the approximate numbers of parasites (trophozoites, schizonts and gametocytes) and whether malaria pigment was present in the white cells were reported, with the result being graded as either + (mild or moderate parasitaemia), ++ (severe parasitaemia) or +++ (very severe parasitaemia). when no malaria parasites were seen, the sample was reported as ‘none seen’.24 estimating malaria parasites numbers (density) a drop of immersion oil was placed on the stained slide and viewed under a 100× objective lens. two hundred (200) leucocytes were counted while noting the number of malaria parasites (trophozoites and schizonts) present during the count. the number of parasites per microlitre was calculated using the formula: where n = number of parasites counted; t = assumed total white blood cell (leucocyte) count (usually 8000) in cells/µl, and l = number of leucocytes counted.24 estimation of cd4 the flow cytometer (sysmex partec’s cyflow™ counter system, sysmex, kobe, japan) was prepared for analysis, and the operating software was prepared for measurement. the sample tube was filled with prepared sample suspension (20 µl of cyflow™ cd4 pe antibody plus 20 µl thoroughly mixed whole blood). the mixture was allowed to incubate in the dark for 15 min, mixing the sample every 5 min during incubation. then 800 µl of cd4 buffer was added and mixed gently. the tube was filled not to more than 2/3 of its volume. the sample was inserted onto the sample port until a ‘click’ sound was heard. the sample was fully mounted within one second. the measurement (acquisition) started automatically; the operating software indicated the prerun, run and count status. in the prerun phase, cells were quickly transported to the flow cuvette’s analysis position; they were analysed and classified into histograms on real-time display in the run phase and counted for a given volume (in a cubic millilitre of blood) in the final stage. after the count phase, the acquisition finished automatically. the button end was clicked to complete the acquisition manually, or the sample tube was removed from the sample port. after each day run, 1.6 ml of cleaning fluid and 1.6 ml of sheath fluid were used for cleaning.25 data analysis data were collected and cleaned in microsoft excel (microsoft corp., redmond, washington, united states) before they were exported to ibm-spss statistics for windows (version 25.0., released 2017; ibm, armonk, new york, united states) for analysis. in the analyses, hiv-positive participants were categorised as being on art or not on art according to their self-reported status at the time of recruitment. participants were categorised as immunodeficient if they had a cd4 count of less than 350 cells/mm3 and were hiv-positive. the chi-square test was used to compare malaria and immune suppression proportions in the study groups. analysis of variance was used to compare the mean cd4 counts in the study groups, using a 95% confidence level, and a p-value of less than 0.05 was considered significant. results most study participants were aged 21–40 years (65.2%) (table 1). this age group was predominant across all study groups, constituting 49.5% of controls, 75% of hiv-positive patients on art and 71.0% of patients not on art. similarly, most study participants were female (69.0%), constituting 61.5% of controls, 74.0% of hiv-positive patients on art and 71.5% of hiv-positive patients not on art. table 1: sociodemographic characteristics of controls, hiv-positive patients with and without antiretroviral therapy, abuja, nigeria in 2012–2013. the mean cd4 counts of hiv patients not on art (300 ± 211 cells/mm3, i.e., immunodeficient) and those on art (mean: 354 cells/mm3) were significantly lower than the control group (mean: 834 cells/mm3) (p < 0.001) (table 2). on the other hand, malaria prevalence was not significantly different across all study groups (controls, 44.5%; patients on art, 40.5%; those not on art, 39.5%; (p > 0.562). compared to 7% immunodeficiency recorded among the control group, more than half of hiv-positive patients on art (56.0%) and 65.5% of those not on art were immunodeficient (p = 0.001). table 2: prevalence of malaria and immunodeficiency status among controls and hiv-positive patients with and without antiretroviral therapy, abuja, nigeria in 2012–2013. the prevalence of malaria parasitaemia among immunodeficient individuals was 42.4%, which was not significantly different (p = 0.695) from the 40.8% among those with a cd4 count of 350 cells/mm3 and above (table 3). table 3: prevalence of malaria parasitaemia across immunodeficiency status of all participants in abuja, nigeria in 2012–2013. most malaria cases across all groups were found within the group aged 21–40 years, including 52.8% of controls, 79.0% of hiv patients on art, 75.9% of hiv-positive patients not on art and 77.5% of all hiv-positive patients (p = 0.001) (table 4). similarly, the group aged 21–40 years also comprised the majority of immunodeficient participants (controls, 64.3%, hiv-positive patients on art, 73.3%, hiv-positive patients not on art, 74.8%, and all hiv-positive patients, 74.1%). table 4: comparison of malaria and immunodeficiency with age of hiv-positive and hiv-negative individuals in abuja, nigeria 2012–2013. discussion the mean cd4 count among our study controls was 834 cells/mm3; it was 354 cells/mm3 among hiv-positive patients on art, and 300 cells/mm3 among hiv-positive patients not on art, which shows that those hiv-positive patients not on art were immunodeficient. several studies have supported this finding, reporting how a cd4 count of less than 350 cells/mm3 makes a patient immunodeficient, elevating their chances of being susceptible to different diseases.20,26 a study conducted in india reported that a lower cd4 count increases the chance of infection among hiv-positive children below the age of 14 years.27 they found the prevalence of opportunistic infections in 90.91% of the hiv-positive patients with a cd4 count of less than 200 cells/mm3, 21.21% among those with a cd4 count between 200 and 349 cells/mm3, and 23.81% among those with a cd4 count of 350 cells/mm3 – 499 cells/mm3, while patients with cd4 counts < 500 cells/mm3 had the least amount of infection (11.43%).27 this finding may also explain why hiv clients not on art with a cd4 count of less than 350 cells/mm3 had a higher percentage of immunodeficiency. however, with a lowered immunity (lower mean cd4 count) among hiv-positive patients on art and those not on art, one would ordinarily expect a higher prevalence of malaria parasitaemia in these groups, but this was not the case in our study. the study found that malaria prevalence was higher among controls and hiv-positive patients on art, which might be explained by various factors, including the number of respondents in each group. additionally, genotype may play a role, as it has previously been reported that individuals with aa genotype are more susceptible to malaria.28 this claim was further substantiated by stating that malaria parasites do not thrive in individuals with sickle cell trait, since haemoglobin ‘s’ is known to inhibit the growth and reproduction of malaria parasites, which may explain the finding of a higher percentage of controls have the aa genotype.28 art may also potentially impact an individual’s vulnerability, as studies have shown that it is less effective against the asexual ring stages of p. falciparum in the blood.29,30 the prevalence of parasitaemia was not significantly higher among the group that was both hiv-positive and immunodeficient than in the negative group, which shows malaria is not an opportunistic infection in hiv-positive individuals. the study also found that most respondents who tested positive for malaria in all groups were between 21 and 40 years. a study conducted in southwest ethiopia also reported that malaria was more prevalent among hiv-positive individuals within a similar age range (25–34 years).31 studies in jos, nigeria,4 europe,6 and beira, mozambique22 have similarly reported that the prevalence of parasitaemia among hiv patients is usually among this age group. on the other hand, studies conducted on the mainland of tanzania32 and in nankoma, uganda33 dispute this finding, indicating that the occurrence is typically highest among those under the age of 20, while another in cross river state, nigeria discovered that age has no bearing on this.18 limitations abuja is the central area of study, which may not fully represent nigeria. notwithstanding, the findings of the study are relevant to the nigeria situation since the study areas is the capital city of nigeria where people from different geopolitical regions reside. conclusion this study reported immunodeficiency as a cd4 count of less than 350 cells/mm3. however, this did not have a statistically significant influence on the presence of malaria parasites in the blood of hiv-positive individuals. hiv-positive patients on art and controls still recorded high rates of malaria. therefore, these findings suggest that malaria parasitaemia is not an opportunistic infection among hiv-positive patients in abuja, nor is malaria parasitaemia more common in hiv infection in nigeria. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions each author proofread the work and contributed considerably to the writing. f.o.s. and n.j.e. conceptualised the project, m.b.m. and n.e.j. were heavily involved in data curation, while t.e. and i.i.e. oversaw the article’s data analysis. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability data supporting the findings of this study are available from the corresponding author, f.o.s., on request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references unaids. global aids update – confronting inequalities [homepage on the internet]. 2021 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opportunistic and other intestinal parasitic infections in aids patients, hiv seropositive healthy carriers and hiv seronegative individuals in southwest ethiopia. east afr j public health. 2008;5(3):169–173. https://doi.org/10.4314/eajph.v5i3.38998 ezeamama ae, spiegelman d, hertzmark e, et al. hiv infection and the incidence of malaria among hiv-exposed children from tanzania. j infect dis. 2012;205(10):1486–1494. https://doi.org/10.1093/infdis/jis234 katrak s, day n, ssemmondo e, et al. community-wide prevalence of malaria parasitemia in hiv-infected and uninfected populations in a high-transmission setting in uganda. j infect dis. 2016;213(12):1971–1978. https://doi.org/10.1093/infdis/jiw057 building resources to meet evolving laboratory medicine challenges in africa references about the author(s) iruka n. okeke department of pharmaceutical microbiology, university of ibadan, ibadan, nigeria citation okeke in. building resources to meet evolving laboratory medicine challenges in africa. afr j lab med. 2018;7(1), a915. https://doi.org/10.4102/ajlm.v7i1.915 editorial building resources to meet evolving laboratory medicine challenges in africa iruka n. okeke copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. building resources to meet evolving laboratory medicine challenges in africa two brief reports in this issue spotlight intersections between communicable and non-communicable disease. nabaigwa et al.1 report on urinary tract infections in diabetic patients at a ugandan hospital, where they found hyperglycaemia associated with urinary tract infections, and ashaka et al.2 describe anaemia of likely infectious origin: human parvovirus b19. as these reports illustrate, disease, as we know it, has become more complicated. each patient will likely be best served by multiple tests performed in different laboratory medicine departments in order to get the most informative description of his or her disease. at no time before now has laboratory medicine been so central to healthcare delivery.3 three decades ago, a yes or no response to the question: ‘is this patient infected with hiv?’ might have been sufficient to initiate state-of-the-art treatment in africa and was well above the standard of care.4,5 since then, the use of cd4+ counts and hiv viral load information to guide care has come from gains in knowledge and the appreciation that appropriate diagnosis and treatment access is central to containing the disease.6 however, diagnostic needs have advanced even beyond that standard for reasons that are actually determined by the virus: hiv drug resistance is a new challenge, brought about by viral evolution and the selective pressure from antiretrovirals. thus, new knowledge and disease evolution will continue to press us to change. in this issue, zhou et al.’s7 response to the need for drug resistance information was to evaluate the application of viral population deep sequencing for people living with hiv in malawi. they demonstrate that deep sequencing can identify drug-resistant minority variants, making informed drug choices that eliminate rather than select resistant hiv lineages possible. the question that their study raises, because the study used roche 454 sequencing via a research collaboration, is how to apply strategies like it routinely at the point of care in malawian and other african clinics. this is a ‘when?’ and not a ‘whether?’ question of course: once next generation sequencing is available for diagnosis of one disease, it can be used for a range of diagnostics with very little additional infrastructural and human resource development. some of the technologies that will aid laboratory contributions to better care are relatively new ones, operating on new platforms and taking advantage of what we now know about the molecular basis of disease. zhou et al.’s7 next generation sequencing approach is a case in point. but still others will be extensions of tried and true technologies that may not require new skills and infrastructure. for example, zhang et al.8 (this issue) described the results of field testing a simple thin layer agar culture-based method that can detect drug-resistant tuberculosis in under 2 weeks using chromogenic plates that can be cultured in remote or resource-limited settings. while not as fast, they found it to be almost as accurate as xpert® mtb/rif testing and much cheaper. both the zhang8 and zhou7 articles showcase techniques that have already been deployed in other parts of the world but had not previously been piloted in african healthcare settings. in the end, an evidence-based selected suite of essential phenotypic and genotypic tests, including sequence-based ones, must be available to all patients and the more rapidly we can assess test utility in local settings, the sooner african patients will have access to faster and more precise diagnoses. for this reason, this journal will continue to value reports of local testing and adaptation and other forms of implementation science. alongside bench technique improvements are the quality management systems that will ensure that each patient sample is appropriately processed in time to influence care. innovation is needed even here and this volume includes some interesting new initiatives. coetzee et al.9 (this issue) demonstrate a simple mechanism for using laboratory data to measure performance, turn-around time in that specific instance, and ismael et al.10 describe an external quality assessment programme for early infant hiv diagnosis. to process laboratory specimens locally, and to the highest standards, it will often be necessary to upgrade or entirely refurbish existing laboratories or plant new ones. while this prospect may be seen as difficult or even daunting, the experiences of others, such as beyanga et al.11 and mtisi et al.12 (both this issue) are essential for replicating success and avoiding costly pitfalls. beyanga et al.11 outline the path of a tanzanian diagnostic laboratory to accreditation. in their description of the construction of a specialised pharmacology laboratory capable of performing pharmacokinetic analyses, mtisi et al.12 outline the process from the stage of initiating public-private international partnerships, through human resource development to preparations for iso 15189 accreditation. ultimately, as mtisi et al.’s article12 emphasises, quality must be an initial goal and not an afterthought. it is not sufficient for laboratories to claim to have performance capabilities; these must be assured. and while the majority of african diagnostic laboratories remain to be accredited, step-wise progression towards this goal is essential for ensuring diagnostic sufficiency in the near future. references nabaigwa bi, mwambi b, okiria j, oyet c. common uropathogens among diabetic patients with urinary tract infection at jinja regional referral hospital, uganda. 2018. afr j lab med. 2018;7(1), a621. ashaka os, agbede oo, omoare aa, ernest sk. human parvovirus b19-induced anaemia in pre-school children in ilorin, nigeria. 2018. afr j lab med. 2018;7(1), a615. milner da jr, holladay eb. laboratories as the core for health systems building. clin lab med. 2018;38(1):1–9. https://doi.org/10.1016/j.cll.2017.10.001 who. aquired immune deficiency syndrome (aids), 1987 case definition for the cdc/who case definition for aids. wkly epidemiolo rec. 1988;63:1–8. okeke in. divining without seeds: the case for strengthening laboratory medicine in africa. ithaca, ny: ilr/cornell university press; 2011. p. 222. who. consolidated guidelines on the use of antiretroviral drugs for treating and preventing hiv infection: recommendations for a public health approach. geneva: world health organization; 2016. zhou z, tang k, zhang g, et al. detection of minority drug resistant mutations in malawian hiv-1 subtype c-positive patients initiating and on first-line antiretroviral therapy. 2018. afr j lab med. 2018;7(1), a708. zhang a, jumbe e, krysiak r, et al. low-cost diagnostic test for susceptible and drug-resistant tuberculosis in rural malawi. 2018. afr j lab med. 2018;7(1), a690. coetzee l-m, cassim n, glencross dk. using laboratory data to categorise cd4 laboratory turn-around-time performance across a national programme. 2018. afr j lab med. 2018;7(1), a665. ismael n, augusto o, vubil a, et al. external quality assessment programme for early infant diagnosis of hiv-1, mozambique, 2011–2014. afr j lab med. 2018;7(1), a664. https://doi.org/10.4102/ajlm.v7i1.664 beyanga m, gerwing-adima l, jackson k, et al. implementation of the laboratory quality management system (iso 15189): experience from bugando medical centre clinical laboratory – mwanza, tanzania. 2018. afr j lab med. 2018;7(1), a657. mtisi tj, maponga c, monera-penduka tg, et al. strategic establishment of an international pharmacology specialty laboratory in a resource-limited setting. 2018. afr j lab med. 2018;7(1), a659. article information authors: olufemi s. amoo1 idowu a. taiwo2 olumuyiwa o. salu1 azuka p. okwuraiwe1 chika k. onwuamah1 morenike a. awe1 osaga o. oforomeh1 daniel i. onwujekwe3 oliver c. ezechi3 audu r. ajuma1 affiliations: 1human virology laboratory, nigerian institute of medical research, yaba, lagos, nigeria2department of cell biology & genetics, university of lagos, akoka, lagos, nigeria 3clinical sciences division, nigerian institute of medical research, yaba, lagos, nigeria correspondence to: olufemi samuel amoo postal address: pmb 2013, yaba, lagos dates: received: 25 may 2012 accepted: 19 sept. 2012 published: 15 may 2013 how to cite this article: amoo os, taiwo ia , salu oo, et al. comparison of cobas/ampliprep taqman and amplicor hiv-1 monitor test in lagos, nigeria. afr j lab med. 2013;2(1), art. #68, 4 pages. http://dx.doi.org/10.4102/ ajlm.v2i1.68 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. comparison of the cobas/ampliprep taqman and amplicor hiv-1 monitor tests in lagos, nigeria in this original research... open access • abstract • introduction • materials and method • viral load assay • ethical considerations • results • discussion • acknowledgement    • competing interests    • authors’ contributions • references abstract top ↑ background: the use of real-time polymerase chain reaction (pcr) technology options is increasing in resource-limited settings because they are faster, improve assay sensitivity, have higher throughput, larger dynamic ranges and reduced rates of contamination. in 2010, unaids ranked nigeria as the second highest population of people living with hiv and aids (2.98 million people) in the world. objective: the objective of this study was to compare the analytical performances of the amplicor hiv-1 monitor (version 1.5) and the cobas ampliprep/taqman (version 2.0) used in monitoring hiv disease progression in hiv-infected individuals. method: in a cross-sectional study, hiv-1 rna values obtained with the amplicor hiv-1 monitor version 1.5 were compared with those of the cobas/ampliprep taqman hiv-1 version 2.0 in a routine clinical setting. between may and november 2011, 176 plasma samples collected were analysed in parallel using both techniques. data analysis was done using statgraphics centurion xvi and medcalc version 12.0. result: the correlation coefficient for the two assays was 0.83 and the level of agreement using a bland–altman plot was 94.2%. conclusion: these findings suggest that the results from the two methods were comparable, hence the cobas/ampliprep taqman version 2.0 is recommended for high-volume laboratories. introduction top ↑ the world health organisation guidelines for the treatment of hiv-1-infected patients recommend viral load as a major marker in disease prognosis.1in conjunction with other immunological tests, hiv viral load is used to assess the efficacy of antiretroviral drugs. therefore, accurate measurement of hiv-1 viral load is essential to provide clinicians with valuable information to determine treatment decisions. recently, new quantitative hiv-1 assays have been designed to cope with increasing molecular diversity of the virus, to overcome the issue of turnaround time and the challenges of viral load estimation encountered with manual methods.2 however, there have been reports of plasma viral load discrepancies between the amplicor monitor test and one of the technologically improved methods, the cobas ampliprep/taqman.3 in a study in south africa, both assays have been reported to have a good agreement,4 and it is important that a similar study is repeated because of the different subtypes found in this region. therefore, there is need to establish a relationship between these two assays.manual methods for nucleic acid extraction are the most time-consuming and challenging aspect of viral load measurement. in addition, they require skilled technical personnel and extended ‘hands-on’ time. automation of the extraction process, on the other hand, has the potential to significantly increase reliability, sample throughput and efficiency. globally, nigeria has the second highest number of people infected with hiv.5 the widespread use of antiretroviral drugs and their availability in low-resource countries has not only brought a form of relief by improving the health of the individuals infected with hiv but has also led to more people living with hiv and aids seeking care and treatment.6 in nigeria, assays are being expanded to manage more patients because of the evidence of their use in early detection of drug resistance.7 therefore, there is need for testing laboratories to prepare to meet with the high demand as it has to do with meeting turnaround time and providing the quality of results needed for efficient patient management. due to the superior technology and ease of use of the cobas ampliprep/taqman, it is recommended that it replace the manual amplicor as a monitoring tool for hiv-1 rna. however, it is good laboratory practice that these monitoring tools are validated before use, especially in places where various hiv-1 subtypes exist.8,9,10 the aim of this study was therefore to compare hiv-1 rna values obtained with the amplicor hiv-1 monitor version 1.5 with those of the cobas taqman hiv-1 assay in a routine clinical setting. materials and method top ↑ in a retrospective study between may and november 2011, 176 archived plasma samples previously tested with the amplicor monitor test and stored at −80 °c in the human virology laboratory were assayed for hiv-1 viral load using the amplicor monitor version 1.5 hiv-1 viral load technique. samples within the detection range of 400 copies/ml and 750 000 copies/ml were selected and assayed with the cobas ampliprep/taqman version 2.0. the subjects’ informed consent was obtained before inclusion in the study. data were analysed using epi info 2008 (version 3.5.1), statgraphics centurion xvi (version 16.0.3) and microsoft office excel 2007. the results are presented as mean and standard deviation (s.d.). agreement between the two methods being compared was also assessed using correlation coefficient, linear regression and bland-altman analysis. differences between means were considered significant when p ≤ 0.05. viral load assay top ↑ amplicor hiv-1 monitor test (version 1.5): this assay targets only the gag p24 region using conventional polymerase chain reaction (pcr) method. the lower limit of quantitation is 2.60 log10 copies/ml and upper limit of quantitation is 5.87 log10 copies/ml. the standard specimen volume is 200 μl. nucleic acid were extracted, amplified and hybridised as recommended by the kit manufacturer. the amplicor hiv‑1 monitor test is based on five major processes, namely specimen preparation; reverse transcription of target rna to generate complimentary dna (cdna); pcr amplification of target cdna using hiv-1 specific complementary primers; hybridisation of the amplified products to oligonucleotide probes specific to the targets; and detection of the probe linked with an enzyme so as to give color reaction later on.cobas ampliprep/cobas taqman hiv-1 test, version 2.0: this assay simultaneously targets the gag and the ltr region with two dually labelled hybridisation probes. the lower limit of quantitation is 1.30 log10 copies/ml and upper limit of quantitation is 7.0 log10 copies/ml. the specimen volume required for this method is 1000 μl. upon loading the sample in appropriate racks, nucleic acid extraction, amplification and detection are performed using the cobas taqman hiv-1 test, v2.0 software on the cobas ampliprep/cobas taqman instrument as specified by equipment manual. the cobas ampliprep/cobas taqman hiv-1 test is based on three major processes: specimen preparation to isolate hiv-1 rna; reverse transcription of the target rna to generate cdna; and simultaneous pcr amplification of target cdna and detection of cleaved dual-labeled oligonucleotide probes specific to the target. sequencing and subtyping: the viroseq kit and abi 3130xl genetic analyser were used to sequence the samples. sequences for reference subtypes and crfs were downloaded from los alamos sites; they were aligned and bootstrapped with patients’ sequences in clustalx and visualised nj plots. ethical considerations top ↑ ethical approval has been reviewed; the protocol and safety guidelines satisfied the conditions of the nigerian institute of medical research (nimr) institutional review board (irb), policies regarding experiments that use human subjects. results top ↑ we examined 176 stored plasma specimens of hiv positive patients obtained between may and november, 2011. analysis of the 176 samples in which viral load was determined revealed that discrepancies of more than 0.5 log10 copies/ml existed for 44 (25%) samples. of these 44 samples with discrepancies, 29 (66%) had lower values with the amplicor monitor test while 15 (34%) had higher values. twenty samples were sequenced from the 176 samples and their subtypes were obtained. the correlation coefficient between the cobas amplicor and the ampliprep/taqman for these samples are shown according to subtypes obtained in table 1. they all had good correlation coefficient between the assays, with the exception of subtype crf 43_02g (r = 0.42). there was a strong correlation coefficient between the viral load values obtained with the 176 samples for the two assays (r = 0.83 p value < 0.21 figure 1). the overall performance between the two assays also indicates a strong relationship, with 94.2% degree of agreement as revealed by the bland–altman graph (figure 2), which represents the number of samples ranging within the mean ± 2 s.d. interval. table 1: correlation coefficient between assays for hiv-1 subtypes. figure 1: the correlation coefficient between the cobas ampliprep/taqman hiv-1 assay and the cobas amplicor assay. figure 2: a bland-altman plot showing the degree of agreement between the cobas ampliprep/taqman and the cobas amplicor monitor assays. the numbers of samples ranging within the mean ±2 s.d. interval is 163 of 176 (94.2%). discussion top ↑ generally, there was a good correlation coefficient in viral loads between the assays for all subtypes except crf 43_02g. however, in nigeria, the subtypes ag and g are most prevalent15 and the viral loads for these subtypes were well correlated in this study.8 of the 44 samples with discrepancies of more than 0.5 log10 copies/ml, the amplicor monitor assay had more samples with lower viral load titre values compared to the cobas ampliprep/taqman. the cobas ampliprep/taqman targets both the gag and ltr region, whereas the amplicor monitor assay targets only the gag region.overall, these data indicate that the two assays have similar performances for the quantitation of hiv-1 rna amongst the samples tested. the high level of agreement (94.2%) observed between the two assays in this study was also reported by previous authors12,13 who demonstrated good overall agreement of the test results using absolute bias plot.14recently, in a study to investigate the impact of genetically diverse hiv samples from china on performance of cobas ampliprep/taqman and amplicor monitor assays, it was demonstrated that the viral loads of different hiv genotypes measured by the use of the two assays were comparable.1 the cobas ampliprep/taqman hiv-1 version 2.0 assay is a fully automated system with continuous sample loading and thereby has higher throughput, shorter turnaround time and minimum risk of contamination throughout sample processing. this has a major advantage for clinical laboratories in efficient patient management, especially for a laboratory such as ours that assays over 17 000 hiv-1 rna viral load samples annually. using the cobas system, the laboratory could assay 36 000 viral load samples annually, with fewer skilled personnel. moreover, the nigerian government plans to expand access to hiv-1 viral load testing in the current art programme, so the use of this automated system will make it easier to cope with the increased demand. in conclusion, the study shows that there is no significant average bias between the two assays compared. however, laboratory personnel and physicians should be aware that good laboratory practice and other factors could influence the outcome of laboratory reports. acknowledgement top ↑ we would like to acknowledge roche diagnostics for donating the cobas ampliprep/taqman equipment to the nimr and for providing the kits for its evaluation. aids preventive initiative in nigeria (apin) is also acknowledged for supporting hiv and aids clinical and laboratory management in the nimr. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. authors’ contributions a. o.s. (human virology, nimr), was the project leader and wrote the manuscript. t. i. a. (university of lagos) and a.r.a. (human virology, nimr) assisted in project design, supervised the project and proof read the manuscript. s.o.b. (human virology, nimr), and o.a. (human virology, nimr), performed some of the experiment. o.c.k. (human virology, nimr), performed some of the experiment and assisted in data analysis. a.m.a. (human virology, nimr), assist in sample preparation and utility wash up. o.o.o. (human virology, nimr), performed the data analysis. o.d.i. and e.o.c. (clinical sciences, nimr), were the clinicians that attended to the patients on visit days. references top ↑ 1. department of health and human services (dhhs). guidelines for the use of antiretroviral agents in hiv-infected adults and adolescents. washington dc, us government printing office; 2011, p. 174.2. de bel a, marissens d, debaisieux l et al. correction of underquantification of human immunodeficiency virus type 1 load with the second version of the roche cobas ampliprep/cobas taqman assay. j. clin. microbiol. 2010;48(4):1337–1342. http://dx.doi.org/10.1128/jcm.01226-09 3. damond f, roquebert b, bénard a et al. human immunodeficiency virus type 1 (hiv-1) plasma load discrepancies between the roche cobas amplicor hiv-1 monitor version 1.5 and the roche cobas ampliprep/cobas taqman hiv-1 assays. j. clin. microbiol. 2007;45(10):3436–3438. http://dx.doi.org/10.1128/jcm.00973-07 4. scott l, carmona s, stevens w. performance of the new roche cobas ampliprep-cobas taqman version 2.0 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http://dx.doi.org/10.1016/j.jcv.2004.09.025 13. foulongne v, montes b, didelot-rousseau m, segondy m. comparison of the lcx human immunodeficiency virus (hiv) rna quantitative, realtime hiv, and cobas ampliprep-cobas taqman assays for quantitation of hiv type 1 rna in plasma. j. clin. microbiol. 2006;44(8):2963–2966. http://dx.doi.org/10.1128/jcm.00341-06 14. bland jm, altman dg. measuring agreement in method comparison studies. stat. methods med. res. 1999;8:135–160. http://dx.doi.org/10.1191/096228099673819272 15. hawkins c, chaplin b, idoko j, ekong e, adewole i, gashau w, murphy rl, kanki ip, apin plus/harvard pepfar team. clinical and genotypic findings in hiv-infected patients with the k65r mutation failing first line antiretroviral therapy in nigeria. j acquir immune defic syndr. 2009;52(2):228. http://dx.doi.org/10.1097/qai.0b013e3181b06125 abstract background description of the intervention lessons learnt recommendations acknowledgements references about the author(s) abdourahamane yacouba faculté des sciences de la santé, université abdou moumouni, niamey, nigerlaboratory team covid-19, niamey, niger adamou lagaré laboratory team covid-19, niamey, nigercentre de recherche médicale et sanitaire, niamey, niger daouada alhousseini maiga faculté des sciences de la santé, université abdou moumouni, niamey, nigerlaboratory team covid-19, niamey, niger halimatou moumouni sambo laboratory team covid-19, niamey, niger direction des laboratoires de santé, ministère de la santé publique, niamey, niger sani ousmane laboratory team covid-19, niamey, niger centre de recherche médicale et sanitaire, niamey, niger zelika hamidou harouna laboratory team covid-19, niamey, niger hôpital national amirou boubacar diallo, niamey, niger boubacar marou faculté des sciences de la santé, université abdou moumouni, niamey, niger maman k. sanoussi laboratory team covid-19, niamey, niger balki aoula laboratory team covid-19, niamey, niger ali amadou laboratory team covid-19, niamey, niger hôpital de l’amitié niger-turquie, niamey, niger hassane boureima laboratory team covid-19, niamey, niger hôpital général de référence de maradi, maradi, niger saidou amatagas laboratory team covid-19, niamey, niger hôpital national de zinder, zinder, niger abdoulaye ousmane faculté des sciences de la santé, université dan dicko dankoulodo de maradi, maradi, niger eric adehossi faculté des sciences de la santé, université abdou moumouni, niamey, niger covid-19 experts group, niamey, niger saidou mamadou faculté des sciences de la santé, université abdou moumouni, niamey, niger covid-19 experts group, niamey, niger citation yacouba a, lagaré a, alhousseini maiga d, et al. laboratory organisation and management of sars-cov-2 infection in niger, west africa. afr j lab med. 2020;9(1), a1308. https://doi.org/10.4102/ajlm.v9i1.1308 lessons from the field laboratory organisation and management of sars-cov-2 infection in niger, west africa abdourahamane yacouba, adamou lagaré, daouada alhousseini maiga, halimatou moumouni sambo, sani ousmane, zelika hamidou harouna, boubacar marou, maman k. sanoussi, balki aoula, ali amadou, hassane boureima, saidou amatagas, abdoulaye ousmane, eric adehossi, saidou mamadou received: 20 june 2020; accepted: 01 oct. 2020; published: 21 dec. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: as the coronavirus disease 2019 (covid-19) pandemic unfolds, laboratory services have been identified as key to its containment. this article outlines the laboratory organisation and management and control interventions in niger. intervention: the capitol city of niger, niamey, adopted a ‘national covid-19 emergency preparedness and response plan’ to strengthen the preparedness of the country for the detection of severe acute respiratory syndrome coronavirus-2. laboratory training and diagnostic capacity building were supported by existing active clinical and research laboratories for more rapid and practicable responses. the national reference laboratory for respiratory viruses located at the centre de recherche médicale et sanitaire was designated as the reference centre for covid-19 testing. the national plan for covid-19 testing is being gradually adopted in other regions of the country in response to the rapidly evolving covid-19 emergency and to ensure a more rapid turn-around time. lessons learnt: after the decentralisation of covid-19 testing to other regions of the country, turn-around times were reduced from 48–72 h to 12–24 h. reducing turn-around times allowed niger to reduce the length of patients’ stays in hospitals and isolation facilities. shortages in testing capacity must be anticipated and addressed. in an effort to reduce risk of shortages and increase availability of reagents and consumables, niamey diversified real-time reverse transcriptase–polymerase chain reaction kits for severe acute respiratory syndrome coronavirus-2 detection. recommendations: continued investment in training programmes and laboratory strategy is needed in order to strengthen niger’s laboratory capacity against the outbreak. keywords: severe acute respiratory syndrome coronavirus-2; sars-cov-2; coronavirus disease 2019; covid-19; laboratory diagnosis; west africa; niger. background in december 2019, a new viral respiratory infection emerged. the infection was first detected and reported in the chinese province of hubei and has since spread globally, affecting people and socio-economic development in both developing and developed countries. the disease, later named the coronavirus disease 2019 (covid-19) and declared a global pandemic by the world health organization, is caused by a species of coronavirus known as severe acute respiratory syndrome coronavirus-2 (sars-cov-2), which belongs to the family coronaviridae and the genus betacoronavirus.1,2,3 worldwide, real-time reverse transcriptase–polymerase chain reaction (rrt-pcr) is the gold standard method used to detect the presence of sars-cov-2 rna in clinical specimens. as of 15 june 2020, 175 503 laboratory-confirmed covid-19 cases were reported in africa, with 4111 deaths.4 these figures make africa the continent least affected by this pandemic, with the most significantly affected countries in the region being south africa (70 038 cases), nigeria (16 085 cases), ghana (11 422 cases), and algeria (10 919 cases).4 the index case in the republic of niger was detected on 19 march 2020. this was an imported case involving a 36-year old man who arrived by road from burkina faso. since the official declaration of the index case, a total of 980 cases have been confirmed as of 15 june 2020.5 as this pandemic unfolds, laboratory services have been identified as key to containment efforts. this article outlines the laboratory organisation and management and control interventions in niger. description of the intervention ethical consideration this article followed all ethical standards for research without direct contact with human or animal subjects. organisational response severe acute respiratory syndrome coronavirus-2 is an emerging respiratory pathogen spreading rapidly through the general population. niamey, the capitol city of niger, adopted the ‘national covid-19 emergency preparedness and response plan’ (available from https://tinyurl.com/y2x3vpmt) to strengthen niger’s preparedness for the detection of sars-cov-2. this plan details the procedures for containment, contact tracing and screening based on the risk of spread. the plan is structured around five major strategic axes including reinforcement of coordination, strengthening of epidemiological surveillance, strengthening of health services capacities, reinforcement of risk communication and community engagement, and, lastly, the creation of isolation facilities. eight committees have been set up to implement the national covid-19 emergency preparedness and response plan, one of which is the laboratory and research team. the national covid-19 emergency preparedness and response plan provides $16 484 884.48 (united states dollars) to enable expedited building and implementation of sars-cov-2 testing capacity in the eight regions of the country in tune with the expansion of the pandemic. the laboratory and research team, which is charged with the responsibility of improving laboratory capacity and capability, is structured into three groups comprising a pre-analytical group (sample collection, inactivation, identification numbers), an analytical group (rrt-pcr testing) and a post-analytical group (validation and reporting test results). for more rapid and practicable responses, laboratory diagnostic capacity building is being supported by existing active clinical and research laboratories. the national reference laboratory for respiratory viruses located at the centre de recherche médicale et sanitaire (cermes) was designated as the reference centre for covid-19 testing. the veterinary research laboratory ‘laboratoire central de l’elevage’, the national reference laboratory for hiv and tuberculosis, the national hospital of niamey and the research institute for sustainable development supported the laboratory response by providing equipment for pcr techniques. moreover, seven laboratory technicians from these centres, who have had extensive training on pcr techniques, were mobilised by the ministry of health, in addition to the cermes technicians, to support the response by performing the rrt-pcr testing at cermes, which received and analysed samples collected from across the country. as part of covid-19 response, niamey and its partners provided logistical air support for sample transport from remote regions such as diffa, zinder, maradi, tahoua and agadez. gradually, the national plan on covid-19 testing is being adopted in other regions of the country in response to the rapidly evolving covid-19 emergency and to ensure a more rapid turn-around time (tat). this adoption involves gradual decentralisation of the sars-cov-2 rna rrt-pcr assays to three regions, tahoua, maradi and zinder, located 550, 662 and 891 kilometres from niamey (figure 1). the choice of these regions was based on several factors, including the distance from the capital city, the number of close contacts in these regions, the number of confirmed cases at the time of the decentralisation and the capacity of the laboratory facilities on site. figure 1: laboratory organisation and adaptation for covid-19 pandemic in niger. the 68 cases (in red) recorded on 11 april 2020 denote the peak between 19 march 2020 (index case) and 15 june 2020. the tahoua laboratory analysed samples collected from the tahoua and agadez regions. samples collected from the zinder and diffa regions were analysed in zinder, whereas the maradi laboratory analysed samples collected in the maradi region. samples collected from the niamey, dosso and tillabery regions were analysed at cermes. logistics and testing capacity are handled by cermes, which reported the availability of the reagents and materials needed to sample patients and to perform the rrt-pcr on a weekly basis to the ministry of health. laboratory diagnosis severe acute respiratory syndrome coronavirus-2 is classified as a risk group 3 human pathogen, similar to middle east respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus.6 the interim guidance from the world health organization suggests that non-propagative processing and handling of specimens containing sars-cov-2 must be performed in standard biosafety level-2 laboratories.7,8 consequently, niamey leveraged the safe practices and techniques, safety equipment and appropriate facility design of cermes to reduce or eliminate exposure of laboratory workers to sars-cov-2-containing materials according to the world health organization requirements.7 before the decentralisation of sars-cov-2 rna rrt-pcr assays to other regions, samples collected from across the country were sent to cermes. samples from remote regions such as diffa, zinder and agadez were transported using flights in accordance with category b transportation regulations.9 in an effort to help close contact management, sample collection was performed during home visits. ribonucleic acid extraction from samples was performed manually using qiaamp® viral rna mini kit (250) (qiagen gmbh, hilden, germany) or nucleic acid isolation or purification reagent (daan gene co., ltd, guangzhou, china) according to the manufacturer’s instructions. two teams of four laboratory technicians were dedicated to rna extraction at cermes. these teams were often overworked due to the large volume of samples handled. qualitative rrt-pcr assays were performed using the nucleic acid testing kit (daan gene co., ltd, guangzhou, china).10 two target genes, the open reading frame1ab (orf1ab) and nucleocapsid protein (n), were simultaneously amplified. a cycle threshold value lower than 40 was defined as a positive test result, and a cycle threshold value of 40 or more was defined as a negative outcome according to the manufacturer’s protocol. alternatively, rrt-pcr targeting the rdrp gene of the sars-cov-2 or sars-like coronavirus was performed using the lightmix® modular sars-cov-2 rdrp (tib molbiol, berlin, germany). positive samples for rdrp gene were confirmed by performance of rrt-pcr for the detection of the e gene using lightmix® sarbecov e gene plus equine arteritis virus control (tib molbiol, berlin, germany). given the extraordinary demand for reagents and consumables, risk of supply shortages became the main issue. niamey experienced a shortage of rna extraction kits and this led to the prioritisation of the testing of vulnerable people, health professionals and patients requiring hospitalisation. as of 15 june 2020, a total of 5386 samples had been tested for sars-cov-2 in niger. of these, 980 (18.2%) were confirmed positive (figure 2). all test results, positive or negative, were immediately reported to the national committee, which is responsible for communication on the pandemic. the highest number of new daily confirmed cases (68 cases) was detected on 11 april 2020. figure 2: covid-19 situation in niger as of 15 june 2020. (a) numbers of sars-cov-2 rna-positive patients. (b) map of niger showing the distribution of sars-cov-2-positive patients according to region. in march and april 2020, before the decentralisation of sars-cov-2 rna rrt-pcr assays to other regions, the number of samples received exceeded the capacity of the single centralised laboratory earmarked for the testing. consequently, tats were between 48 h and 72 h. this tat length often created a disconnect between clinicians and laboratory staff. indeed, according to the clinicians, the tat of 48–72 h delayed treatment and increased patients’ length of stay, particularly in isolation facilities where patients are often asymptomatic. after decentralisation, on 15 june, 2020, the tat had decreased significantly to 12 h at maradi and between 12 h and 24 h at tahoua, zinder and cermes, niamey. lessons learnt experience gained in the management of previous outbreaks (rift valley fever and meningitis) helped niamey to build a quick response to the covid-19 pandemic. however, additional control efforts are needed to improve the laboratory strategy and response against covid-19 in the country. firstly, a well-coordinated laboratory strategy and operational plan is needed in order to address the shortcomings of the existing plan. moreover, considering the importance of improved sars-cov-2 laboratory capacity, the country should provide extensive training to laboratory technicians in preparation for rapid expansion of laboratory diagnostic capacity. secondly, laboratory tat is critical in determining the success of both the laboratory response programme and the management of patients. reducing the tat allowed reductions in patients’ length of stay in hospitals and isolation facilities. thirdly, communication between clinical and laboratory staff needs to be improved in order to ensure that laboratory results are understandable to clinicians. fourthly, the shortages in testing capacity need to be anticipated and addressed. if capacity is exceeded, priority should be given to the testing of vulnerable patients, health professionals and patients requiring hospitalisation. niamey diversified rrt-pcr kits for sars-cov-2 detection to reduce the risk of shortages and increase the availability of reagents. it has been demonstrated that rna extraction kits from different manufacturers are interchangeable.11 however, diversifying the type of rrt-pcr kits for sars-cov-2 detection can lead to some variation in the detection rate between kits.12 it has been well documented that the limit of detection of different rrt-pcr kits can differ substantially.13,14 therefore, care must be taken in the interpretation of the results when using different kits for the monitoring of patients. fifthly, the overwhelming number of specimens to test highlighted the need for fast methods to extract viral rna. ribonucleic acid extraction from samples was performed manually in niger. in addition to the high risk of contamination, manual extraction of viral rna is time consuming and requires a large number of laboratory professionals. lastly, amplification of multiple target genes (e.g. n, e, rprd genes) could be used to avoid invalid results and increase sensitivity for detection of new genomic variants.15,16 recommendations despite tat improvement in the laboratory management of covid-19 testing, several additional control efforts are needed to improve the laboratory response against covid-19 in niger. considering the importance of improved sars-cov-2 laboratory capacity, continued investment in training programmes and laboratory strategy is needed to systematically guide the laboratory response against the outbreak. acknowledgements authors would like to acknowledge the contributions of the national covid-19 emergency preparedness and response committee for the technical, resource and logistical contributions, the government of niger, the ministry of health and all its partners for the good support that formed the basis of a favourable working environment for the response against covid-19 pandemic in niger. we would also like to thank dr ahmed olowo-okere of usmanu danfodiyo university, sokoto-nigeria, for language editing. competing interests the authors have declared that no competing interests exist. authors’ contributions a.y. and s.m. conceived and designed the study. data acquisition was done by a.l. the draft manuscript was written by a.y., while b.m., d.a.m., h.m.s., h.b., s.o., e.a., s.m., m.k.s, z.h.h., h.b., s.a., a.o., a.a. and b.a. critically reviewed the manuscript. all authors have read and agreed to the final version of this manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability statement the data are available from the corresponding author upon reasonable request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated 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rt-pcr in covid-19 detection: issues affecting the results. expert rev mol diagn. 2020 may 3;20(5):453–454. https://doi.org/10.1080/14737159.2020.1757437 penarrubia al, ruiz m, porco r, et al. multiple assays in a real-time rt-pcr sars-cov-2 panel can mitigate the risk of loss of sensitivity by new genomic variants during the covid-19 outbreak. int j infect dis. 2020;97:225–229. https://doi.org/10.1016/j.ijid.2020.06.027 abstract introduction methods results discussion acknowledgements references about the author(s) atang bulane department of medical microbiology & virology, university of the free state, bloemfontein, south africa anwar hoosen department of medical microbiology & virology, university of the free state, bloemfontein, south africa citation bulane a, hoosen a. use of matrix-assisted laser desorption/ionisation-time of flight mass spectrometry analyser in a diagnostic microbiology laboratory in a developing country. afr j lab med. 2017;6(1), a598. https://doi.org/10.4102/ajlm.v6i1.598 original research use of matrix-assisted laser desorption/ionisation-time of flight mass spectrometry analyser in a diagnostic microbiology laboratory in a developing country atang bulane, anwar hoosen received: 07 dec. 2016; accepted: 30 june 2017; published: 08 dec. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: rapid and accurate identification of pathogens is of utmost importance for management of patients. current identification relies on conventional phenotypic methods which are time consuming. matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (maldi-tof ms) is based on proteomic profiling and allows for rapid identification of pathogens. objective: we compared maldi-tof ms against two commercial systems, microscan walkaway and vitek 2 ms. methods: over a three-month period from july 2013 to september 2013, a total of 227 bacteria and yeasts were collected from an academic microbiology laboratory (n = 121; 87 gram-negatives, seven gram-positives, 27 yeasts) and other laboratories (n = 106; 35 gram-negatives, 34 gram-positives, 37 yeasts). sixty-five positive blood cultures were initially processed with bruker sepsityper kit for direct identification. results: from the 65 blood culture bottles, four grew more than one bacterial pathogen and maldi-tof ms identified only one isolate. the blood cultures yielded 21 gram-negatives, 43 gram-positives and one candida. there were 21 escherirchia coli isolates which were reported by the maldi-tof ms as e. coli/shigella. of the total 292 isolates, discrepant results were found for one bacterial and three yeast isolates. discrepant results were resolved by testing with the api system with maldi-tof ms showing 100% correlation. conclusion: the maldi-tof ms proved to be very useful for rapid and reliable identification of bacteria and yeasts directly from blood cultures and after culture of other specimens. the difference in time to identification was significant for all isolates. however, for positive blood cultures with minimal sample preparation time there was a massive difference in turn-around time with great appreciation by clinicians. introduction in clinical microbiology laboratories, the identification of microorganisms in patient specimens has historically been based on the detection of pathogen-specific phenotypic characteristics.1 these include microscopic and colony morphology features and biochemical phenotypes that can be detected with either manual or automated methods.1 the most commonly used automated systems in south africa are the vitek 2 ms and microscan walkaway systems. although, these systems allow for the identification of most bacterial isolates with great accuracy, they are costly and time consuming. they rely on the active metabolic processes of the pathogen and as a result long incubation periods are required. the introduction of the matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (maldi-tof ms) method, which is based on proteomic profiling, has provided fast, reliable and cost-effective identification of bacteria and yeast.2,3,4 the maldi-tof ms assay has also shown the ability to directly identify bacteria or fungi from positive blood cultures.5,6,7 this greatly reduces the turn-around time for patients with suspected sepsis. at present, blood culture analysis takes at least 24 h or more before a definitive diagnosis is achieved.8 maldi-tof ms has been routinely used in clinical laboratories in european countries from 2009 but is not widely used in south africa. several reports show that it is reproducible, and produces results that are comparable to genome sequencing.9,10,11 in this study, the maldi-tof ms was evaluated for diagnostic microbiology in a developing country. the first part of the study compared maldi-tof ms identification results with identification by the microscan walkaway system (siemens healthcare diagnostics, sacramento, california, united states) at an academic laboratory. the second part of study compared isolates from a regional hospital and two private pathology laboratories that used the vitek 2 ms (biomérieux, marcy l’etoile, france). the last part of study compared organisms from blood culture vials when a positive signal was emitted from the bact/alert 3d system (biomérieux, marcy l’etoile, france). methods collection and analysis of clinical isolates samples were collected from 15 july 2013 to 30 september 2013 (three-month period). bacteria and yeast isolates retrieved from clinical samples from an academic diagnostic microbiology laboratory (national health laboratory service [nhls], universitas hospital, bloemfontein, free state, south africa) and three other laboratories, one at kimberly hospital (northern cape, south africa) and two private pathology laboratories (bloemfontein, free state, south africa), were analysed. the laboratories identified isolates to the species level with standard biochemical methods using either the microscan walkaway system (siemens healthcare diagnostics, sacramento, california, united states) or the vitek 2 ms system (biomérieux, marcy l’etoile, france). isolates were tested in parallel with the maldi-tof ms assay (bruker daltonics, bremen, germany). the technician who carried out the maldi-tof ms assay was blinded to the identity of the isolates. the analysis of the isolates from the academic diagnostic microbiology laboratory was done in real time, whereas the samples from regional and private laboratories were assayed in batches. blood culture analysis this analysis was only performed for the samples from the academic diagnostic microbiology laboratory. only the initial positive culture from each patient was used to avoid duplicate analyses of samples from the same septic episode. charcoal-free, positive blood culture bottles were gram-stained and protein was extracted according to manufacturer’s instructions using the bruker sepsityper protein extraction kit (bruker daltonics, bremen, germany). in brief, 200 μl of lysis buffer was added to 1.0-ml aliquot of positive blood culture (bact/alert 3d ms aerobic, anaerobic bottles). this mixture was centrifuged for 1 min at 13 000 rpm. the supernatant was removed and the pellet was re‑suspended in 1 ml of washing buffer. the supernatant was discarded and the pellet was further re-suspended in 75% ethanol. ethanol-formic acid extraction was then performed as per the manufacturer’s instructions. after extraction 1 μl of protein supernatant was spotted on a 96-spot maldi-tof ms target plate, overlaid with matrix and analysed using biotyper version 3.0 according to the manufacturer’s instructions. the only modification to this procedure was that instead of a 1.0 ml aliquot, a 3 ml aliquot was used for protein extraction from positive paediatric blood culture bottles. the escherirchia coli atcc 25922 reference strain was used as a positive control, and matrix with no organism was used as a negative control in the analysis. matrix-assisted laser desorption/ionisation-time of flight mass spectrometry analysis clinical isolate identification by the maldi-tof ms assay was performed according to the manufacturer’s instructions. in brief, the isolated colonies were directly applied onto steel msp 96 maldi ground steel target plates (bruker daltonics, bremen, germany). the yeast isolates were overlaid with 2 μl formic acid for protein extraction and 2 μl of alpha-cyano-4-hydroxy cinamic acid matrix, while the bacterial isolates were directly overlaid with 2 μl alpha-cyano-4-hydroxy cinamic acid matrix. the microflex lt instrument was calibrated twice a week by using the bruker daltonics bacterial test standard. when scores greater than 2.0 were generated, they were classified as ‘high-confidence’ (secure species), whereas scores between 1.7 and 1.99 were classified as ‘intermediate confidence’ (genus only) and scores of less than 1.7 were classified as ‘unacceptable’. the results obtained by maldi-tof ms were compared to the results obtained by the conventional methods using the microscan walkaway system and vitek 2 ms after analysis by the respective laboratories. the api coryne v 2.0 (biomérieux, marcy l’etoile, france) was used to resolve discrepancies between maldi-tof ms and vitek 2 ms results. the isolated colonies were directly applied onto steel msp 96 maldi ground steel target plates (bruker daltonics, bremen, germany). the yeast isolates were overlaid with 2 μl formic acid for protein extraction and 2 μl of alpha-cyano-4-hydroxy cinamic acid matrix, while the bacterial isolates were directly overlaid with 2 μl alpha-cyano-4-hydroxy cinamic acid matrix. the plates were analysed by the maldi-tof ms machine. turn-around time for identification of blood culture isolates the turn-around time for identification from blood cultures was determined by calculating the time elapsed between the incubation of the sample in the automated instrument and identification by standard laboratory methods. the maldi-tof ms assay turn-around time was calculated from the instrument flagging a positive blood culture to the time when the maldi-tof ms machine completed the interpretation of the spectra. results a total of 227 isolates were evaluated over the three-month study period (academic diagnostic microbiology laboratory n = 121; other laboratories n = 106). of the 227 isolates, 122 were gram-negative bacteria, 41 gram-positive bacteria and 64 yeast isolates. among the isolates from the academic diagnostic microbiology laboratory, 87 were gram-negative, seven gram-positive and 27 yeast isolates, whereas 35 gram-negatives, 34 gram-positives and 37 yeast isolates were from the other laboratories. the 122 gram-negative isolates analysed by maldi-tof ms were 100% concordant at the species level with the vitek 2 ms and microscan walkaway systems (table 1). a total of 98 (80.3%) isolates generated a high confidence score, 22 (18.0%) had an intermediate score and two (1.6%) had a very low confidence score (proteus mirabilis and hafnia alvei). despite their low score, identification by both the microscan walkaway system and the vitek 2 ms system was in concordance with the maldi-tof ms identification. table 1: gram-negative bacterial isolates identified (n = 122), bloemfontein, south africa, 15 july 2013 to 30 september 2013. for the 41 gram-positive isolates identified by maldi-tof ms (table 2), 26 (63.4%) generated a high confidence score, 14 (34.1%) had an intermediate score and 1 (2.4%) had a poor score. forty out of the 41 gram-positive isolates showed concordance at the species level while one was discrepant. of the 40 cpncordant isolates, seven were from the microscan walkaway system and 33 from the vitek 2 ms system. despite the poor score for the streptococcus pneumoniae isolate, there was concordance with the vitek 2 ms. the discrepant isolate was identified as arthrobacter spp by maldi-tof ms with an intermediate score value (1.7–1.9), but was identified by the vitek 2 ms as staphlococcus aureus. however, the isolate was confirmed as arthrobacter spp by the api coryne (biomérieux, marcy l’etoile, france) with a score value of 64.5%. table 2: gram-positive bacterial isolates identified (n = 41), bloemfontein, south africa, 15 july 2013 to 30 september 2013. among the 64 yeast isolates analysed by maldi-tof ms (table 3), 48 (75.0%) showed an acceptable identification score of 1.7 or higher. of the 64 isolates, 16 (25.0%) generated a high score, 32 (50.0%) had an intermediate score and 16 (25.0%) had a poor identification score. for the 16 yeast isolates with a poor identification score by maldi-tof ms, their identities were found to be in agreement with the microscan walkaway system for the isolates from academic laboratory and vitek 2 ms for the isolates from other laboratories. among the isolates with an intermediate score, two (4.2%) isolates (1.7–1.99) were identified as candida parapsilosis and candida dubliniensis by maldi-tof ms but were identified as candida albicans by the microscan walkaway. however, vitek 2 ms also identified them as c. parapsilosis and c. dubliniensis. table 3: yeast isolates identified by maldi-tof ms (n = 64), bloemfontein, south africa, 15 july 2013 to 30 september 2013. there was 100% concordance for bacterial identification between the maldi-tof ms assay and the blood cultures (tables 4 and 5). in total, 65 blood culture bottles were first gram stained and then analysed by maldi-tof ms; 60 (92.0%) were monomicrobial, four (6.0%) were polymicrobial and one (2.0%) was cultured a yeast. ultimately 21 (32.0%) blood culture isolates were classified as gram-negative bacteria, 43 (66.0%) as gram-positive bacteria and 1 (2.0%) as candida spp. table 4: gram-negative bacteria identified directly from blood cultures by maldi-tof ms (n = 21), bloemfontein, south africa, 15 july 2013 to 30 september 2013. table 5: gram-positive bacteria identified directly from blood cultures by maldi-tof ms (n = 43), bloemfontein, south africa, 15 july 2013 to 30 september 2013. of the four polymicrobial blood bottles, the maldi-tof ms assay correctly identified only one organism (table 6). candida spp (score 1.84) was identified as the candida guiliermondii (ana) by the maldi-tof ms, while the microscan walkaway system identified it as candida zeylanoides. the yeast was further tested by the vitek 2 ms and was identified as c. guiliermondii. table 6: four polymicrobial blood cultures and direct identification by maldi-tof ms (n = 4), bloemfontein, south africa, 15 july 2013 to 30 september 2013. the average identification time by maldi-tof ms for 65 positive blood culture bottles from the time a signal was generated by the blood culture machine was 35 min. the conventional methods required 48 h. discussion despite the common use of maldi-tof ms in laboratories based in developed countries, this system is not frequently used in developing countries. for this reason, few data are available on the efficacy of maldi-tof ms for routine diagnosis of bacterial and yeast identification in microbiology laboratories in developing countries such as south africa. in this study, the utility of maldi-tof ms for the identification of bacterial and yeast species either from clinical specimens or directly from blood culture broth was evaluated and compared with routinely used accepted methods (microscan walkaway system and vitek 2 ms) in a resource-limited country. current procedures, such as conventional and automated methods that are used in blood culture identification, delay pathogen identification for hours, or even days when fastidious bacteria are involved. in this study, an average of 48 h was required for pathogen identification from blood cultures, whereas it took a mere 35 min on average for extraction and identification of both bacteria and yeast directly from the same blood cultures by the maldi-tof ms. this system has shown the potential to reduce delays that currently exist between blood culture sampling and the availability of the results to clinicians and hence allows initiation of early speciesor genus-oriented empirical treatment. the rapid identification of the causative agent is not only important in treatment selection; according to other studies based on rapid identification techniques, it also reduces therapeutic costs.12 although it has been reported that the reliability for maldi-tof ms in identification of gram-positive bacteria is lower when compared to gram-negative bacteria in blood cultures,12 in the current study maldi-tof ms was able to correctly identify all gram-positive isolates that were supplied and the majority had good score values. polymicrobial blood cultures were found in 4 of the 65 (6.2%) positive samples, and maldi-tof ms could only identify 1 of the pathogens among these mixed blood cultures. the identification score values ranged between 1.45 and 2. this is a limitation for the use of the maldi-tof ms system for identification of pathogens directly from polymicrobial blood samples. one candida spp was identified directly from a positive blood culture sample. the candida spp was identified as c. guiliermondii (ana) by the maldi-tof ms with a score value of 1.84, whereas the microscan walkway system identified it as c. zeylanoides. the yeast isolate was further tested by the vitek 2 ms and identified as c. guiliermondii. when maldi-tof ms was evaluated for the identification of bacterial isolates, results showed that maldi-tof ms allows excellent identification at the species level for all the gram-positive and gram-negative bacteria, as a significant proportion of bacterial isolates identified showed high correlation with the microscan walkaway and vitek 2 ms results (99.4%). according to literature on the use of maldi-tof ms, species identification can be attained at a threshold of 2.0.13 however, among the 292 samples analysed in the current study, 107 had an identification score of less than 2 by maldi-tof ms. interestingly, the maldi-tof ms identities of these 107 isolates correlated with those given by microscan walkaway system and vitek 2 ms. similar results have also been reported in other studies where a score value lower than 2 still resulted in reliable species identification.3,14,15,16 it was hypothesised that the low identification score by maldi-tof ms may be due to the low analyte concentration rather than a low degree of relatedness to the information in the database. the inclusion of low-scoring samples in the current study increased the sensitivity of the maldi-tof ms. bacterial isolates that were identified as s. pneumoniae by the maldi-tof ms were confirmed as such by microscan walkaway system. for identification of yeast isolates, maldi-tof ms was able to reliably identify 62 of the 64 (96.9%) isolates that were included in the current study. a total of four different yeast species were identified in correlation with the vitek 2 ms and microscan walkaway systems. there were two discrepant yeast isolates from a swab and catheter tip which were identified as c. dubliniensis (score 1.85) and c. parapsilosis (score 1.91) by maldi-tof ms. the two isolates were both identified as c. albicans by the microscan walkaway system and the maldi-tof ms identification was confirmed by the vitek 2 ms. the correct identification of the candida spp is important, since the infection and clinical impact of other candida spp other than c. albicans seem to be increasing among hiv-positive patients, which leads to challenges in empirical antifungal treatment.19 candida spp, including candida glabrata and c. parapsilosis, are known to be resistant to fluconazole,20 which is one of the drugs most widely used in the treatment of systemic fungal infections. rapid identification of this species is essential for proper treatment and management. limitations the sample size for direct identification from blood cultures was relatively small and there was only one yeast isolate. a larger sample including more yeast isolates would have provided useful information. conclusion the maldi-tof ms assay proved to be very useful for rapid and reliable identification of bacterial and yeast pathogens directly from blood cultures and isolates from other specimens at academic and private pathology laboratories in a developing country. the difference in time to identification for all isolates was significant between the maldi-tof ms and the two other automated systems (microscan walkaway and vitek 2 ms). however, for positive blood cultures with their minimal sample preparation time, there was a massive difference in turn-around time, contributing to great laboratory efficiency. acknowledgements our thanks and sincere appreciation to the staff of the academic and private diagnostic microbiology laboratories who kindly provided isolates for testing and bruker daltonics, south africa, for providing us with some of the reagents and for allowing us to perform the maldi-tof ms on their machine. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support the department of medical microbiology and virology, university of the free state, provided financial support for reagents and accommodated the bruker maldi-tof ms in the laboratory. authors’ contributions a. b. was responsible for the data collection, analysis and write up of the manuscript. a.h. was responsible for building up of proposal, data analysis and write-up of the manuscript. references koneman ew, editor. color atlas and textbook of diagnostic microbiology. 6th ed. 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https://doi.org/10.1128/jcm.02485-09 prod’hom g, bizzini a, durussel c, bille j, greub g. matrix-assisted laser desorption ionization-time of flight mass spectrometry for direct bacterial identification from positive blood culture pellets. j clin microbiol. 2010;48(4):1481–1483. https://doi.org/10.1128/jcm.01780-09 mestas j, felsenstein s, bard jd. direct identification of bacteria from positive bact/alert blood culture bottles using matrix-assisted laser desorption ionization–time-of-flight mass spectrometry. diagn microbiol infect dis. 2014;80(3):193–196. https://doi.org/10.1016/j.diagmicrobio.2014.07.008 endimiani a, tamborini a, luzzaro f, lombardi g, toniolo a. epidemiology of bloodstream infections and time to detection of positive blood cultures: an evaluation of the automated bact/alert and bactec 9240 systems. new microbiol. 2002;25(1):9–16. mellmann a, cloud j, maier t, et al. evaluation of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry in comparison to 16s rrna gene sequencing for species identification of nonfermenting bacteria. j clin microbiol. 2008;46(6):1946–1954. https://doi.org/10.1128/jcm.00157-08 mellmann a, bimet f, bizet c, et al. high interlaboratory reproducibility of matrix-assisted laser desorption ionization-time of flight mass spectrometry-based species identification of nonfermenting bacteria. j clin microbiol. 2009;47(11):3732–3734. https://doi.org/10.1128/jcm.00921-09 almuzara m, barberis c, traglia g, et al. evaluation of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry for species identification of nonfermenting gram-negative bacilli. j microbiol methods. 2015;112:24–27. https://doi.org/10.1016/j.mimet.2015.03.004 stevenson l, drake s, et al. rapid identification of bacteria in positive blood culture broths by matrix-assisted laser desorption ionization-time of flight mass spectrometry. j clin microbiol. 2010;48(2):444–447. https://doi.org/10.1128/jcm.01541-09 khot p, couturier m, wilson a, croft a, fisher ma. optimization of matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis for bacterial identification. j clin microbiol. 2012;50(12):3845–3852. https://doi.org/10.1128/jcm.00626-12 bizzini a, durussel c, bille j, greub g, prod’hom g. performance of matrix-assisted laser desorption ionization-time of flight mass spectrometry for identification of bacterial strains routinely isolated in a clinical microbiology laboratory. j clin microbiol. 2010;48(5):1549–1554. https://doi.org/10.1128/jcm.01794-09 christner m, rohde h, wolters m, et al. rapid identification of bacteria from positive blood culture bottles by use of matrix-assisted laser desorption-ionization time of flight mass spectrometry fingerprinting. j clin microbiol. 2010;48(5):1584–1591. https://doi.org/10.1128/jcm.01831-09 van veen s, claas e, kuijper ej. high-throughput identification of bacteria and yeast by matrix-assisted laser desorption ionization-time of flight mass spectrometry in conventional medical microbiology laboratories. j clin microbiol. 2010;48(3):900–907. https://doi.org/10.1128/jcm.02071-09 he y, li h, lu x, stratton cw, tang yw. mass spectrometry biotyper system identifies enteric bacterial pathogens directly from colonies grown on selective stool culture media. j clin microbiol. 2010;48(11):3888–3892. https://doi.org/10.1128/jcm.01290-10 werno am, christner m, anderson tp, murdoch dr. differentiation of streptococcus pneumoniae from nonpneumococcal streptococci of the streptococcus mitis group by matrix-assisted laser desorption ionization-time of flight mass spectrometry. j clin microbiol. 2012;50(9):2863–2867. https://doi.org/10.1128/jcm.00508-12 pappas p, kauffman c, andes d, et al. clinical practice guidelines for the management of candidiasis: 2009 update by the infectious diseases society of america. clin infect dis. 2009;48(5):503–535. https://doi.org/10.1086/596757 hughes c, beggs w. action of fluconazole (uk-49, 858) in relation to other systemic antifungal azoles. j antimicrob chemother. 1987;19(2):171–174. https://doi.org/10.1093/jac/19.2.171 abstract introduction methods results discussion acknowledgements references about the author(s) innocent uwimana school of public health, national university of rwanda, field epidemiology and laboratory program (feltp), kigali, rwanda. rwanda biomedical centre/biomedical services, national reference laboratory division, kigali, rwanda. nestor bizimungu rwanda biomedical centre/biomedical services, national reference laboratory division, kigali, rwanda. fabrice ingabire rwanda biomedical centre/biomedical services, national reference laboratory division, kigali, rwanda. elyse mukamukwiye rwanda biomedical centre/biomedical services, national reference laboratory division, kigali, rwanda. odette sharangabo rwanda biomedical centre/biomedical services, national reference laboratory division, kigali, rwanda. semuto c. ngabonziza rwanda biomedical centre/biomedical services, national reference laboratory division, kigali, rwanda. elaine kamanzi rwanda biomedical centre/biomedical services, national reference laboratory division, kigali, rwanda. citation uwimana i, bizimungu n, ingabire f, et al. trends in leprosy case detection in rwanda, 1995–2011: analysis of 17 years of laboratory data. afr j lab med. 2017;6(1), a426. https://doi.org/10.4102/ajlm.v6i1.426 original research trends in leprosy case detection in rwanda, 1995–2011: analysis of 17 years of laboratory data innocent uwimana, nestor bizimungu, fabrice ingabire, elyse mukamukwiye, odette sharangabo, semuto c. ngabonziza, elaine kamanzi received: 31 jan. 2016; accepted: 14 oct. 2016; published: 28 feb. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: leprosy, or hansen’s disease, is a chronic, infectious disease caused by mycobacterium leprae. it remains one of the leading causes of deformity and physical disability. objective: we analysed laboratory records to assess trends in prevalence rates and case detection rates (cdrs) in rwanda. methods: a retrospective review of detected leprosy cases from the records of the rwanda national reference laboratory over a 17-year period (1995–2011) was conducted. skin biopsy samples were analysed microscopically using ziehl-neelsen staining technique to identify m. leprae. results: cumulatively, 266 suspected cases were reported between 1995 and 2011. of the suspected cases, 77 (28.9%) were laboratory confirmed as having leprosy. among detected cases, 59 (76.6%) were men and 18 (23.4%) women. the male:female ratio was 3:1. there were 77 registered leprosy cases over the 17-year period of the study, and the prevalence rate was 0.005 per 10 000 population. a gradual decrease in the prevalence rate was observed from 0.015 per 10 000 population in 2003 to 0.003 per 10 000 population in 2010. from 1995 to 2011, the cdr did not exceed one per 10 000 population. conclusion: this laboratory review demonstrates a declining trend in prevalence rates and cdr during the period of the study. early case detection and a sustainable leprosy control programme remain the cornerstones of reducing the physical and socio-economic burden of leprosy in rwanda. introduction despite the availability of powerful, multi-drug therapies, leprosy remains one of the world’s most infectious diseases and is a leading cause of deformity and physical disability. leprosy, or hansen’s disease, is a chronic infection caused by mycobacterium leprae. for affected patients, leprosy carries a significant stigma and contributes to their isolation from the rest of the world. the global burden of leprosy in 1993 was estimated at 2.4 million of leprosy cases worldwide against 10 to 12 million in 1980, and 5.4 million in 1985.1 in 2010, the registered prevalence of leprosy worldwide was 211 903 cases, with the world health organization (who) reporting 244 796 new cases detected during 2009.2 the who reported that the highest number of cases occurred in south east asia (n = 166 115 new cases), followed by the african region (n = 28 934 new cases).2 in 1991, the who issued a resolution to achieve global elimination of leprosy by 2000, with ‘elimination’ defined as a reduction in prevalence of the disease to less than 1 case per 10 000 population.3 although this target was achieved in 2000, many countries continue to experience transmission of the disease. currently, leprosy remains endemic in several countries, including india, indonesia and brazil, which have the highest global burden of leprosy worldwide.4 in the who americas region, brazil ranks first with 19.2%, followed by suriname with 7.2% and paraguay with 6.3%. in the who’s eastern mediterranean region, sudan had 4.9% and qatar 2.8%. in the southeast asia region, nepal detected 14.8%, east timor 14.5% and india 11.0%. in the western pacific region, kiribati had 95.3%, marshall island had a detection rate of 80.8% and nauru 30.0%. in the who african region, comoros represented the highest detection rate, with 44.3% cases per 100 000 inhabitants in 2009, followed by liberia with 10.8% and sierra leone with 8.0%.5 the who’s introduction of multi-drug therapy, a combination of three drugs – dapsone, rifampicin and clofazimine – has contributed spectacularly to the improvement of leprosy case management and disease control. use of this multi-drug therapy in association with political commitment has decreased the prevalence of leprosy worldwide in several countries, strengthening disease surveillance by coordinating available internal and external resources for leprosy control and striving for integration of leprosy control into general health services for an inclusive and better disease surveillance.6,7 in 1995, the rwanda national tuberculosis and leprosy control programme initiated efforts to increase detection and treatment of leprosy cases by engagement of community health workers and education for awareness of leprosy to the community. despite strong measures put in place to eliminate leprosy, rwanda is still recording new cases. in 2010, eight new cases with grade-2 disabilities were recorded in rwanda. the present study aimed to assess the trends in the prevalence rates and case detection rates (cdrs) in rwanda to evaluate whether the country meets the who leprosy elimination target. methods ethical considerations this study was conducted after obtaining authorisation from the ethical committee of the school of public health, university of rwanda. the authorisation for using laboratory records was sought from the rwanda biomedical centre, national reference laboratory division. no authorisation numbers were issued. no patients’ names were used during data collection or analysis, as patients were delinked from their names and codes were used during data collection. setting, study period and study population this retrospective analysis of leprosy cases detected over a 17-year period was conducted using data from the laboratory records of the rwanda national reference laboratory division in kigali, rwanda. the national reference laboratory is a referral laboratory dedicated to performing specialised laboratory tests from various health facilities, including intermediate district hospitals and health centres at the peripheral level. the national reference laboratory receives and performs laboratory tests for all suspected cases of leprosy countrywide. data were retrieved from laboratory registers for smear microscopy examinations performed from january 1995 to december 2011. examinations were conducted for all suspected cases of leprosy on samples collected from patients who attended any health facility in rwanda. laboratory tests and interpretation exudates from slit skin were collected and sent immediately to the national reference laboratory division. smears were stained by using the hot ziehl-neelsen staining technique to detect acid alcohol-resistant bacilli in skin smears collected from skin lesions, ear lobes, elbows and/or other exudates. the ziehl-neelsen test was used to assess bacillus morphology and the bacterial index. the bacterial index represents the quantitative bacillary load (number of bacilli) and is expressed according to a logarithmic scale ranging from 0 to 4+. a positive smear was classified as ‘multibacillary’ if its bacterial index was evaluated to be 1+ to 4+ and as ‘paucibacillary’ if its bacterial index was either negative or scanty.8,9 leprosy smear microscopy interpretation was done by a well-trained laboratory technologist to identify m. leprae. the who recommends definitive identification and confirmation of leprosy if one of the two criteria are found: (1) skin biopsy smear positive; or (2) characteristic anaesthetic leprosy skin lesions, with or without nerve thickening or enlargement, with sensory or motor loss.10 data collection and analysis data collected from the laboratory registers were entered in epi-info (version 3.5.3). distribution of variables, including age, sex and quantitative bacillary load, was collected and analysed by describing simple frequencies. the case detection rate (cdr), an important indicator being used under the national leprosy eradication programme, and prevalence rate per 10 000 inhabitants were calculated according to who guidelines, as described elsewhere. briefly, cdr was calculated based on the number of cases detected in a year multiplied by 100 000 and divided by the total population in that year.11,12,13 results a total of 266 suspected cases were reported between 1995 and 2011 (figure 1). of the suspected cases, 77 (28.9%) were laboratory confirmed as being infected with m. leprae. the remaining cases (n = 189; 71.1%) were microscopically negative for acid alcohol-resistant bacilli or leprosy bacilli. among the detected cases, 76.6% (n = 59) were men and 23.4% (n = 18) women. the male:female ratio was 3:1. the most affected age group was the group of patients over age 45 years. multibacillary patients with a bacteriological index ranging from 1+ to 4+ were detected in 73.0% of men and 21.0% of women. paucibacillary cases were less common, at 3.9% in men and 2.6% in women (figure 1). figure 1: results of enrolled patients for laboratory detection of mycobacterium leprae, rwanda, 1995–2011. the highest number of cases was recorded in 2010, with a cdr of 0.33 per 100 000 population. in 2005, more suspected cases (23.0%; 61/266) were recorded, but the cdr was lower (0.03 per 100 000 inhabitants) (figure 2). figure 2: suspected cases compared to laboratory-confirmed cases, rwanda, 1995–2011. there were 77 registered leprosy cases over the 17-year study period, with a prevalence rate of 0.005 per 10 000 population. the prevalence rate decreased from 0.015 per 10 000 population in 2003 to 0.003 per 10 000 population in 2010 (figure 3). from 1995 to 2011, the cdr did not exceed one per 10 000 inhabitants. figure 3: prevalence rate of leprosy in rwanda from 2003–2010. discussion the present study shows that over a period of 17 years, the prevalence of leprosy in rwanda has decreased and remained below the who’s elimination target of less than 1 case per 10 000 population. this might be due to increased awareness amongst community health workers, as well as a community sensitisation effort for case detection and treatment. annually, the who describes and provides regional cdrs from individual national cdrs in a listing of the top 33 endemic countries and top 14 individual countries. in 1998, the who found that in seven of the top 14 countries, including india and brazil, the cdr was above two per 10 000. india contributed 79% to global case detection in the same year. the african, american and southeast asian who regions each accounted for about 30%, once india was excluded. between 1994 and 2000, case detection did not decrease in these three who regions, according to the same estimates.14 our study shows that rwanda can be classified among the countries that have achieved the who target phase of leprosy elimination (prevalence rate < 1/10 000). the gradual decrease in leprosy prevalence rates shows that rwanda has reached the who target of leprosy elimination as it was defined in 1991.15 studies conducted elsewhere, such as in togo, lome, where a similar retrospective analysis was conducted from january 1990 to december 2005, found similar decreases in leprosy prevalence trends, with prevalence and cdr dropping remarkably from 1990 to 2005.16 in our analysis, leprosy case detection and the prevalence rates dropped noticeably over an eight-year period, from 0.015 per 10 000 population in 2003 to 0.003 per 10 000 population in 2010, and from 1995 to 2011 the cdr did not exceed one per 10 000 inhabitants. this gradual decrease was the result of the good surveillance system established countrywide. although the cdr in rwanda did not exceed one per 10 000 inhabitants from 1995 to 2011, multibacillary patients with a bacteriological index ranging from 1+ to 4+ were highly represented in our analysis. similar cdrs are found elsewhere in the world. an epidemiological survey on leprosy in metropolitan france found a cdr of 0.003 per 10 000 inhabitants.17 in french guyana, near the border with brazil, a study found a cdr of 0.53 cases/10 000 inhabitants/year; this area had the highest number of leprosy cases in the world and ranked second worldwide after india, which in 2009 detected the highest number of new cases.1,18 the low detection of leprosy cases among women in our study lead to a 3:1 male:female ratio, which could be explained by better adherence to drug regimens among women when compared to men, as has been found elsewhere.19 limitations there are two major limitations for the present study. first, we were not able to trace and study patient treatment outcomes. additionally, because this was a retrospective data analysis from routine laboratory records, some information was missing, which hampered our ability to analyse some important variables. recommendations we recommend early case detection through an active case detection strategy for a sustainable leprosy control programme. further studies and nationwide surveys are recommended for continuous monitoring of leprosy, patient treatment outcomes and its estimation for better disease surveillance and case management countrywide. conclusion our study shows that rwanda has achieved the who global leprosy elimination target of less than one case per 10 000 and has demonstrated a declining trend in leprosy prevalence and cdrs. the attainment of the who leprosy elimination target became a reality in rwanda by putting various strategies and interventions in place, including sensitisation of community health workers, active disease surveillance using active samples and data collection, and political will. these strategies are the cornerstone that will allow reduction of physical disabilities and socio-economic burden of leprosy in the country. acknowledgements the authors acknowledge the support of the rwanda biomedical centre, national reference laboratory division for permission to use laboratory data. the authors thank all laboratory staff at the national reference laboratory for laboratory testing, and all health facilities involved in the data collection. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. sources of support none. authors’ contributions all authors of this paper directly participated in the planning, execution, or analysis of this study. i.u. was the project leader, participated in planning, data analysis, and writing of the manuscript. i.u., f.i., e.m., o.s., s.c.n. and e.k. reviewed the manuscript for important intellectual content. n.b. performed the laboratory tests. s.c.n. performed critical appraisal of the manuscript. all authors read and approved the final version submitted. references noordeen sk. elimination of leprosy as a public health problem: progress and prospects. bull world health organ. 1995;73(1):1–6. world health organization. global leprosy situation, 2010. wkly epidemiol rec. 2010;85(35):337–348. world health organization. who expert committee on leprosy. world health organ tech rep ser. 1998;874:1–43. world health organization. leprosy update. wkly epidemiol rec. 2011;86(36):389–400. penna ml, penna go. leprosy frequency in the world, 1999–2010. mem inst oswaldo cruz. 2012;107(suppl. 1):3–12. noordeen sk, lopez bravo l, daumerie d. global review of multidrug therapy (mdt) in leprosy. world health stat q. 1991;44(1):2–15. daumerie d. leprosy in the who african region. world health stat q. 1991;44(1):16–22. lastória jc, de abreu mamm. leprosy: a review of laboratory and therapeutic aspects – part 2. an bras dermatol. 2014;89(3):389–401. https://doi.org/10.1590/abd1806-4841.20142460 shepard cc, mcrae dh. a method for counting acid-fast bacteria. int j lepr other mycobact dis. 1968;36(1):78–82. aftab h, nielsen sd, bygbjerg ic. leprosy in denmark 1980–2010: a review of 15 cases. bmc res notes. 2016;9:10. https://doi.org/10.1186/s13104-015-1768-6 mustapha g, olusegu oj, mustapha s, et al. leprosy elimination: progress and challenges in nigeria; kaduna state tb and leprosy control programme as a case study. afr j infect dis. 2012;6(1):5–9. ogbeiwi oi. progress towards the elimination of leprosy in nigeria: a review of the role of policy implementation and operational factors. lepr rev. 2005;76(1):65–76. kumar a. letter to editor: new case detection and prevalence rates in leprosy. lepr rev. 2003;74(2):177–179. meima a, richardus jh, habbema jd. trends in leprosy case detection worldwide since 1985. lepr rev. 2004;75(1):19–33. richardus jh, habbema jd. the impact of leprosy control on the transmission of m. leprae: is elimination being attained? lepr rev. 2007;78(4):330–337. saka b, kombate k, mouhari-toure a, et al. leprosy in lome, togo: retrospective study of 383 cases. med trop (mars). 2008;68(5):496–498. bret s, flageul b, girault py, et al. epidemiological survey of leprosy conducted in metropolitan france between 2009 and 2010. ann dermatol venereol. 2013;140(5):347–352. https://doi.org/10.1016/j.annder.2013.02.019 domergue v, clyti e, sainte-marie d, et al. leprosy in french guyana: a retrospective study from 1997 to 2006. med trop (mars). 2008;68(1):33–37. ranganadha rao pv, peri s, porichha d, et al. a review of trends in new case-detection in subarnapur district of orissa. indian j lepr. 2006;78(2):153–165. abstract introduction methods results discussion acknowledgements references about the author(s) shaun m. grobler department of haematology and cell biology, faculty of health sciences, university of the free state, bloemfontein, south africa department of haematology, national health laboratory service, bloemfontein, south africa anne-cecilia van marle department of haematology and cell biology, faculty of health sciences, university of the free state, bloemfontein, south africa department of haematology, national health laboratory service, bloemfontein, south africa citation grobler sm, van marle a-c. effects of sample age and storage temperature on the flow cytometric diagnosis of chronic lymphocytic leukaemia in south africa. afr j lab med. 2025;14(1), a2688. https://doi.org/10.4102/ajlm.v14i1.2688 original research effects of sample age and storage temperature on the flow cytometric diagnosis of chronic lymphocytic leukaemia in south africa shaun m. grobler, anne-cecilia van marle received: 09 nov. 2024; accepted: 28 feb. 2025; published: 31 may 2025 copyright: © 2025. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: chronic lymphocytic leukaemia (cll) is a haematological neoplasm with characteristic flow cytometric immunophenotyping. pre-analytical variables impact the quality and reproducibility of flow cytometric data, which could alter the diagnosis from cll to atypical cll (acll). objective: this study investigated the effects of pre-analytical variables, specifically sample age and storage temperature, on the stability of key antigens used in the diagnosis of cll. methods: serial flow cytometric analyses were performed from january 2022 to march 2023 on blood samples of 10 cll patients from the universitas academic hospital haematology clinic in bloemfontein, south africa. samples were stored at room and refrigerator temperatures and analysed at baseline, 24 h, 48 h, 72 h and 96 h. we recorded the percentage and intensity of antigen expression of cll makers, including cd5, cd20, cd23, cd79b, cd200 and sigm, and assessed whether these affected the adapted and modified matutes scores. results: statistically significant changes were observed in cd5 (p = 0.028), cd23 (p = 0.003) and cd200 (p = 0.005) expression, with better stability at refrigerator temperature. two samples showed changes in both matutes scores by 24 h, irrespective of storage temperature. by 48 h, scores changed to acll in six room-temperature and four refrigerated samples. a majority shift in diagnosis to acll (modified matutes: n = 8/10; adapted matutes: n = 7/10) was observed at 96 h for refrigerated samples. conclusion: these findings indicate that pre-analytical variables influence antigen stability in cll samples, with better preservation at refrigerator temperature, recommending analysis within 48 h of collection. what this study adds: this study highlights the impact of pre-analytical variables on the flow cytometric diagnosis of cll. extended room temperature storage alters antigen expression, shifting matutes scores and potentially affecting the final diagnosis. the findings emphasise optimised sample handling, for improved diagnostic accuracy in laboratory medicine. keywords: chronic lymphocytic leukaemia; atypical chronic lymphocytic leukaemia; flow cytometry; matutes score; pre-analytical variables; storage time; storage temperature. introduction chronic lymphocytic leukaemia (cll) is a clonal haematological disorder consisting of immunologically inept, small, monomorphous and well-differentiated b-lymphocytes that aberrantly express the antigens cd5 and cd23 on their surfaces.1 a sustained (≥ 3 months) monoclonal b-lymphocytosis of ≥ 5 × 109/l in the peripheral blood, with characteristic morphological and immunophenotypic features, is required to establish the diagnosis of cll.2 immunophenotyping not only confirms the diagnosis of a mature, clonal b-cell neoplasm but also helps to differentiate cll from other b-cell lymphoproliferative disorders (b-lpds). notwithstanding substantial advances in cytogenetics and molecular biology, morphology and immunophenotyping remain the benchmark for diagnosing mature b-lpds.3 the matutes score, developed in 1994, uses five surface markers (cd23, cd5, fmc7, surface immunoglobulin [sig], and cd22) to diagnose cll, with scores of 4 or 5 indicating cll, and scores below 4 suggesting non-cll; a score of 3 implies atypical cll (acll).4,5,6 in 1997, a modification known as the moreau score replaced cd22 with cd79b to better distinguish cll from other b-lpds.4,5,6 cooperative groups, including the euroflow consortium, now omit fmc7 and use cd20 to streamline testing without compromising diagnostic accuracy.5,7,8 cd200, identified in 2009, further improved the score’s accuracy, distinguishing cll from mantle cell lymphoma.9 an adapted matutes score that includes cd200 has been shown to achieve nearly 100% diagnostic accuracy for cll, underscoring the value of these refinements.10 the concept of acll, initially described by criel et al.,11 refers to a b-lpd with atypical morphology, including larger cll cells with pleomorphic nuclear contours. the revised 4th edition of the world health organization classification of tumours of hematopoietic and lymphoid tissues describes acll as having at least 10% circulating ‘cll cells’ with prominent nuclear clefts and folds.12 the incidence of acll is reported to be 7% among all b-lpds diagnosed by flow cytometry.13 atypical cll is described as having a lower matutes score owing to the unusually brighter expression of sig or cd79b, lack of cd5 or cd23, or positivity for fmc7/cd20.11,13,14 these immunophenotypic changes may, however, be secondary to trisomy 12, acting as a confounding variable.11,14 whether the distinction between cll and acll is clinically significant remains controversial, although acll has been associated with a more aggressive clinical course and a higher probability of disease progression.14 owing to the lack of a concrete standardised concept of acll,15 and ambiguous definitions in the successive world health organization classifications,3,16 acll is now used more generically to denote forms with an unusual immunophenotype, regardless of blood smear findings.16 the impact of pre-analytical variables on the quality and reproducibility of flow cytometry data is well described.17,18,19,20 parameters such as the type of anticoagulant in which samples are collected, the storage time and the storage temperature of samples before analysis, were found to affect absolute cell counts and antigen expression. however, these effects have primarily been assessed on healthy subjects’ blood samples. extensive comparative studies to determine the optimal pre-analytical flow conditions for b-lpd samples are lacking.17,19,20 universitas academic hospital national health laboratory service (nhls) is in the city of bloemfontein in the free state province of central south africa. universitas academic hospital nhls serves as the referral laboratory for the free state, northern cape and north west provinces, and provides diagnostic and laboratory services for a population of 8.41 million people, occupying a total geographic surface area of 607 596 km2.21 universitas academic hospital nhls often receives blood samples requiring immunophenotyping from distant peripheral clinics, which only reach the laboratory 3–5 days after collection. consequently, universitas academic hospital nhls frequently diagnoses immunophenotypically ‘atypical cll’ cases, where the morphological assessment of these blood samples supports ‘typical’ cll. this raises the question of whether we truly see more atypical cases or whether the sample age and storage conditions significantly affect the percentage antigen expression and intensity of expression on lymphoid cells in haematological malignancies. the aim of our study was to measure objectively the effects of sample age (from time of collection to time of analysis) and storage conditions (room versus refrigerator temperature) on the percentage antigen expression and the intensity of expression of surface antigens in cll. we wanted to determine the optimal storage conditions to produce quality results and establish a cut-off time after which samples are considered unsuitable for reliable diagnoses. methods ethical considerations the study was conducted in accordance with the declaration of helsinki and approved by the health sciences research ethics committee of the university of the free state (reference number ufs-hsd2021/1257/3011), free state department of health and the national health laboratory service. as per the institutional review board, no informed consent was required as there was no interaction between the principal investigator and the patients, nor did the study’s outcome affect patients’ well-being or medical care. the blood samples used in this study were the surplus blood of routine samples collected by trained nursing staff during the patient’s scheduled haematology clinic visits, independent of our research. the researchers did not have access to patient sociodemographic or clinical details. study design and patient selection whole blood samples in vacutainers containing ethylenediamine tetra-acetic acid (edta) (becton dickinson [bd], san jose, california, united states) were obtained by designated clinic personnel from patients with confirmed diagnoses of cll attending the universitas academic hospital haematology clinic, bloemfontein, south africa, between january 2022 and march 2023. the blood samples used in this study were the surplus blood of the routine blood samples collected by trained nursing personnel during the patients’ haematology clinic visit, independent of our research. full blood count and peripheral blood smear results were screened to identify eligible samples. these included samples with morphologically detectable disease, regardless of whether the patients were newly diagnosed or receiving treatment. the edta samples were immediately de-identified and assigned study numbers to ensure patient confidentiality, after which baseline (within 12 h of collection) flow cytometric analysis was performed. only samples with a baseline immunophenotype of typical cll were included in the study. following baseline flow cytometry, each sample was then divided into two separate specimen tubes: one stored at refrigerator temperature (4 °c) and the other at room temperature (rt), defined as 23 °c in nhls laboratories.22,23 these two aliquots per sample were then analysed after 24 h, 48 h, 72 h and 96 h of storage at the respective temperatures. all flow cytometric analyses were performed on the facscanto™ ii (bd, san jose, california, united states). the percentage antigen expression, median fluorescence intensity (mfi) and difference in mfi for each marker were recorded, as shown in figure 1, which illustrates the study design. figure 1: schematic representation of the study design, universitas academic hospital, bloemfontein, south africa, january 2022 to march 2023. sample processing sample preparation was done according to the euroflow protocols.22 because of the inclusion of surface membrane immunoglobulins (sigm, skappa, slambda), a wash-stain-lyse approach was followed. in brief, 3000 µl of phosphate-buffered saline was mixed with 300 µl of edta whole blood, followed by centrifugation at 540 g for 5 min in a heraeus megafuge 8 centrifuge (thermo scientific inc., waltham, massachusetts, united states). thereafter, the supernatant was discarded and these steps repeated once more. the cell pellet was then resuspended in 300 µl of phosphate-buffered saline. the correct volume of cells required per tube was calculated using equation 1: this calculated volume was incubated for 15 min at rt in the dark with 10 µl of each of the following monoclonal antibodies: cd5, cd19, cd20, cd23, cd43, cd45, cd79b, cd200, sigm, skappa, and slambda. the chosen antibody panel (table 1) was adapted from the euroflow lymphoid screening tube and b-lpd panels including those markers considered crucial in the diagnosis of cll.1,5,7 table 1: antibody panel design for flow cytometric analysis of patients attending the universitas academic hospital haematology clinic, bloemfontein, south africa, january 2022 to march 2023. the cells were then lysed with 2000 µl of diluted bd facs™ lysing solution (bd biosciences, san jose, california, united states) and incubated at rt in the dark for another 15 min. following centrifugation at 540 g for 5 min, the supernatant was discarded, and the cell pellet finally resuspended in 500 µl of phosphate-buffered saline for analysis on the facscanto™ ii (bd, san jose, california, united states). sample analysis and interpretation analysis was conducted using the bd facsdiva™ version 8 (bd, san jose, california, united states) and infinicyt™ version 2.0 (cytognos, santa marta de tormes, salamanca, spain) software. the lymphocyte gate (forward versus side light scatter), cd45 and cd19 against side scatter were employed for gating. normal t-lymphocytes were used as internal negative reference populations (nrp), and, where necessary, with markers such as cd5, typically expressed by t-cells, neutrophils were employed as the nrp. positive designation for markers were assigned if expression was present in > 20% of monotypic b cells24,25,26 or when the fluorescent signal for a particular marker was greater than that of the internal nrp. negative staining was assigned when the fluorescence of any given marker was similar to that of the nrp (qualitative assessment).22,26 median fluorescence intensity, and specifically mfi difference, that is, the intensity of expression in monotypic b cells compared to that of the internal reference populations in arbitrary units provided by the analysis software, and converted to logarithms, was used for quantitative assessment. table 2 and figure 2 represent an example of the mfi measurements for the different markers in one cll sample. table 2: an example of the median fluorescence intensity measurements for the different markers in one sample of a patient with chronic lymphocytic leukaemia at universitas academic hospital, bloemfontein, south africa, january 2022 to march 2023. figure 2: example of change in median fluorescence intensity of one subject from baseline (dotted histogram) compared to 96 h (solid histogram) at universitas academic hospital, bloemfontein, south africa, january 2022 to march 2023. in addition to the positive or negative designation, markers were further described according to the intensity of the positive signal as either dim, moderate, bright or variable by comparing the fluorescent signal, expressed in logarithms, of each marker to the nrp. dim staining was defined as mildly increased when compared to the nrp (less than one logarithm brighter than the nrp). moderate staining was at least one logarithm brighter than the nrp, and bright staining was defined as at least two logarithms brighter than the nrp (table 2).26 two cll scoring systems, the moreau score and the adapted matutes score, were applied to the flow cytometric data for the various intervals and temperatures to determine a change in the diagnosis from cll (table 3).1,4,6,10 the diagnosis of cll, employing the moreau and the adapted matutes scores, necessitates the observation of dim (1 point) or bright (0 points) expression of certain markers (sig and cd79b). consequently, instances of transition to moderate expression did not result in point deduction within the scoring framework. table 3: modified matutes/moreau and adapted matutes scores for the diagnosis of chronic lymphocytic leukemia at universitas academic hospital, bloemfontein, south africa, january 2022 to march 2023. data analysis to assess whether the mfi difference observed at 24 h, 48 h, 72 h or 96 h during storage of the blood samples at rt or 4 °c were different from their initial baseline values (at < 12 h, rt), a two-factor analysis of variance was used. when the two-factor analysis of variance test showed significant changes, a post-hoc two-tailed t-test was used to determine which pairwise comparison of means (storage time versus storage temperature) contributed to the overall significant difference. analysis was performed using microsoft® excel® (microsoft corporation, redmond, washington, united states). results samples included twelve samples were initially screened, although only 10 samples were included in the final analysis. two samples were excluded because of ‘atypical’ cll scores calculated at baseline. median fluorescence intensity and median fluorescence intensity difference irrespective of storage temperature, some markers, including cd23, cd200 and sigm, showed > 10% decrease in mfi difference (cd23: p = 0.003; cd200: p = 0.005; and sigm: p = 0.07) already at 24 h. the markers that did not show a significant change in mfi difference in relation to time or storage temperature were cd20, cd79b, skappa and slambda. at rt, the mfi difference for cd5 showed a statistically significant decrease only at 96 h (p = 0.028), whereas the samples kept at 4 °c showed no significant change in mfi difference over time (p = 0.08 at 96 h). cd23 showed statistically significant changes with storage time (p ≤ 0.001) and storage temperature (p = 0.025), with a decrease in the mfi difference of > 15% from baseline already at 24 h for both rt (p = 0.015) and 4 °c (p = 0.032). cd200 was more sensitive to time (p = 0.05) compared to temperature (p = 0.07), with > 25% decrease in mfi difference at 48 h (p = 0.013) in the rt samples. however, the samples kept at 4 °c showed no significant change in mfi difference over time (p = 0.08 at 96 h). storage temperature rather than time played a statistically significant role in sigm expression (p = 0.006). it was noteworthy that the sigm mfi difference was more stable at rt, while a decrease of > 90% was observed in the 4 °c samples at 48 h (p = 0.022). in addition, skappa and slambda were more stable at rt, compared to samples kept at 4 °c. there was, however, no clinically significant decrease in the 4 °c samples, even at 96 h (p = 0.11 for skappa and p = 0.13 for slambda) (figure 3). figure 3: changes in median fluorescence intensity difference over time for each marker, cd23 (a), cd200 (b), cd5 (c), slgm (d), cd20 (e), cd79b (f), kappa (g) and lambda (h), at the different storage temperatures at universitas academic hospital, bloemfontein, south africa, january 2022 to march 2023. the solid line represents the means of the median fluorescence intensity difference and the shaded area indicates the range of results at the various time points. chronic lymphocytic leukaemia scores to determine whether storage time or temperature would affect the cll diagnostic scores, the moreau and adapted matutes scores were calculated at various time points. a two-way analysis of variance was performed to compare the effect of storage time and temperature on the mfi for the markers included in the cll scores. both the moreau and the adapted matutes scores showed statistically significant variation with temperature (moreau: p = 0.007; and adapted matutes: p = 0.005) and time (moreau: p ≤ 0.001; and adapted matutes: p ≤ 0.001). a post-hoc analysis confirmed 48 h to be the most significant time point for both scoring systems in rt samples (moreau: p = 0.002; and adapted matutes: p = 0.011) as well as the 4 °c samples (moreau: p = 0.018; and adapted matutes: p = 0.040). the moreau score of most rt samples (n = 6) and four of the 4 °c samples changed to atypical cll at 48 h. a majority shift (n = 8 for the moreau score; n = 7 for the adapted matutes score) in diagnosis from typical cll to atypical cll for the 4 °c samples was only seen at 96 h (figure 4). figure 4: temporal evolution of the chronic lymphocytic leukaemia diagnoses (from baseline to 96 h) at universitas academic hospital, bloemfontein, south africa, january 2022 to march 2023. top: moreau score at room temperature (a) and 4 °c (b). bottom: adapted matutes score at room temperature (c) and 4 °c (d). discussion in this study, we observed statistically significant changes in cd5, cd23, and cd200 antigen expression, with antigen stability notably improved under refrigerated conditions. the majority of samples stored at rt (n = 6) and only four of the refrigerator samples displayed a shift in diagnosis towards acll at the 48 h mark. however, a majority shift in diagnosis from cll to acll/non-cll for the refrigerator samples was only evident after a 96 h storage duration. the international council for standardisation of haematology and the international clinical cytometry society emphasise the need for standardised pre-analytical procedures, with recommendations that samples be analysed within 48 h when stored at rt.20 however, bain et al. suggest that samples should ideally be analysed within 24 h if transported and stored at rt, particularly when referral to central laboratories is involved.27 other studies, like those by diks et al.,19 have highlighted the impact of pre-analytical handling on data quality, noting that sample processing should be tailored to the cell type and clinical context. notably, most of these investigations used samples from healthy donors, not individuals with haematological malignancies.17,18,19 comparative studies on optimal pre-analytical conditions for b-lpd specimens remain limited,19 underscoring a gap that our research set out to address. our findings align with the euroflow consortium, which examined pre-analytical variables on samples from patients with haematological malignancies and suggested that protocols may need adjustments based on sample type and markers analysed.28 notably, we found that cll antigen expression is better preserved at refrigerator temperatures than rt, which contrasts with previous reports.27 among the assessed markers, cd23 and cd200 showed statistically significant expression changes over time, with better stability at refrigerated temperatures. different definitions of rt worldwide could partially explain these discrepancies. for instance, rt standards range from 15 °c to 30 °c globally, depending on climate and regulatory guidelines.29,30 in south africa, where this study was conducted, rt typically falls between 21 °c and 26 °c,31 with 22 °c ± 2 °c advised for blood-handling facilities.31,32 additionally, structural similarities between cd23 and cd200 – both containing immunoglobulin-like domains – might influence their stability under varying conditions.33,34 our study suggests that maintaining samples at refrigerator temperature, rather than rt, may better preserve sample integrity and accuracy in flow cytometric analyses. this principle applies not only to flow cytometry samples but has also been shown to be true for full blood count parameters when processing is delayed.35 interestingly, sigm levels decreased significantly under refrigerated conditions, potentially because of its low baseline expression in cll cells, making it more susceptible to degradation. alternatively, guo et al. found that interleukin-4 (not assessed in this study) could increase sigm expression in cll,36 while jackson et al. reported lower interleukin-4 levels at 4 °c compared to rt,37 potentially explaining the observations. although the evaluation of cd200 is not a mandatory criterion in cll diagnosis as defined by the world health organization,3 its inclusion has been advocated in the euroflow guidelines, and previous research has attested to its high diagnostic utility, especially in distinguishing cll from mantle cell lymphoma.38 the adapted matutes score represents a four-marker scoring system, which incorporates cd200, streamlining the antibody requirements for cll diagnosis, and offering valuable advantages in resource-constrained settings.10 in the current research, the adapted matutes score exhibited greater diagnostic accuracy for cll than the moreau score when dealing with samples subjected to delayed analysis. arguably, the inclusion of fewer markers susceptible to time and temperature-dependent changes may translate to a more robust scoring system with heightened accuracy. the influence of storage conditions on the percentage antigen expression and mfi of markers employed in the cll scoring systems allowed a shift in the calculated scores to support a diagnosis of acll or even another b-lpd. notable deviations in the adapted matutes and moreau scores were discerned in two samples after only 24 h of storage, irrespective of the temperature setting. this may partly be explained by the elevated white cell counts (± 200 × 109/l) observed in these two samples. however, underlying genetic aberrations may also have contributed. the clinical significance of acll remains debatable, and in the absence of a standardised and universally accepted definition for acll,14 the term is often used more broadly to describe cases with an unconventional immunophenotype and is unlikely to represent a single biological entity.15 as such, the term ‘b-lpd other than cll’ may be more appropriate to prevent the inadvertent exclusion of other b-lpd subtypes. with treatment modalities revolutionising to more targeted therapies, the distinction between cll and other b-lpds invokes significant treatment implications. limitations budgetary constraints imposed limitations on the number of samples subjected to analysis. despite only including 10 patients in our study, each patient’s sample underwent flow cytometric analysis nine times, encompassing baseline assessments and subsequent evaluations at four different time points, both under refrigerator and rt conditions. we enrolled patients exhibiting morphologically detectable cll and a typical baseline immunophenotype, irrespective of their treatment status. it is important to note that the impact of treatment could potentially alter the expression of specific markers in cll. for our analysis, we adhered to specific fluorochromes according to the euroflow guidelines.7,22 these findings may not be universally applicable to products from alternative manufacturers. samples were collected in edta tubes. various studies have indicated that the stability of cell surface marker expression and population distribution is greater in sodium-heparin blood compared to edta blood.19,20 however, it should be noted that storing sodium-heparin samples is associated with a more rapid decline in leukocyte counts over time. the use of whole blood fixation strategies (cyto-chex®, transfix®) to preserve cell populations in stored samples intended for flow cytometric analysis was beyond the scope of this study. yet, these reagents have been shown to reduce signal intensity.19,20 future studies could involve larger cohorts, encompassing diverse haematological malignancies. in addition, correlations between disease progression, molecular abnormalities, or treatment interventions and alterations in antigen expression of cells could be investigated. conclusion in conclusion, this study showed that storage time and storage temperature impact the stability of surface markers required for the diagnosis of cll, which we can translate to our clinical setting. to ensure reliable results, referral samples expected to reach the laboratory more than 48 h after collection must be maintained at refrigerator temperatures during storage and transportation. while our study specifically focused on cll as a proof of concept, the implications of our findings likely extend beyond the markers employed in cll diagnosis and the pre-analytical phase may need to be customised to the clinical application. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions s.m.g. and a.-c.v.m. conceptualised and designed the study. s.m.g. wrote the draft protocol, wrote the draft manuscript, and carried out the flow cytometric analysis of the samples. s.m.g. and a.-c.v.m. coordinated all aspects of the implementation of the study and participated in the experimental phase of the study and contributed to the writing of the article and approval of the final version. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the data that support the findings of this study are available from the corresponding author, a.-c.v.m., upon reasonable request. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. the article does not necessarily reflect the 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of sample storage on the measurement of lymphocyte subpopulations from hiv-1-positive and control subjects. j immunol methods. 1992;151(1–2):217–225. https://doi.org/10.1016/0022-1759(92)90120-i correia rp, bortolucci aca, lopes acw, et al. recommendations for quality assurance in multiparametric flow cytometry: first consensus of the brazilian group of flow cytometry (gbcflux). j bras patol med lab. 2015;51(6):389–396. https://doi.org/10.5935/1676-2444.20150061 diks am, bonroy c, teodosio c, et al. impact of blood storage and sample handling on quality of high dimensional flow cytometric data in multicenter clinical research. j immunol methods. 2019;475:112616. https://doi.org/10.1016/j.jim.2019.06.007 davis bh, dasgupta a, kussick s, han jy, estrellado a. validation of cell-based fluorescence assays: practice guidelines from the icsh and iccs – part ii – preanalytical issues. cytometry b clin cytom. 2013;84(5):286–290. https://doi.org/10.1002/cyto.b.21105 government communications 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[homepage on the internet]. 11th ed. strasbourg: european directorate for the quality of medicines and healthcare; 2022 [cited 2023 nov 18]. available from: https://www.edqm.eu/en/european-pharmacopoeia-ph.-eur.-11th-edition steedman m. temperature standards for cold chain logistics [homepage on the internet]. 2022 [cited 2023 nov 01]. available from: https://www.cargo-wise.co.uk/blog/temperature-standards-for-cold-chain-logistics#:~:text=ambient%20temperature. boshoff t. managing office temperature. labour guide south africa [homepage on the internet]. 2015 [cited 2023 sep 04]. available from: https://labourguide.co.za/health-and-safety/managing-office-temperature hardwick j. blood storage and transportation. isbt sci ser. 2020;15(s1):232–254. https://doi.org/10.1111/voxs.12599 hatherley d, barclay an. the cd200 and cd200 receptor cell surface proteins interact through their n-terminal immunoglobulin-like domains. eur j immunol. 2004;34(6):1688–1694. https://doi.org/10.1002/eji.200425080 acharya m, borland g, edkins al, et al. cd23/fcεrii: molecular multi-tasking. clin exp immunol. 2010;162(1):12–23. https://doi.org/10.1111/j.1365-2249.2010.04210.x kaur a, lee h-k, del giudice d, singh i, eldibany m. effect of temperature fluctuation and transport time on complete blood count parameters. am j clin pathol. 2019;152(suppl 1):s112. https://doi.org/10.1093/ajcp/aqz121.019 guo b, zhang l, chiorazzi n, rothstein tl. il-4 rescues surface igm expression in chronic lymphocytic leukemia. blood. 2016;128(4):553–562. https://doi.org/10.1182/blood-2015-11-682997 jackson cj, reppe s, eidet jr, et al. optimization of storage temperature for retention of undifferentiated cell character of cultured human epidermal cell sheets. sci rep. 2017;7(1):8206. https://doi.org/10.1038/s41598-017-08586-7 sandes af, de lourdes chauffaille m, oliveira crmc, et al. cd200 has an important role in the differential diagnosis of mature b-cell neoplasms by multiparameter flow cytometry. cytometry b clin cytom. 2014;86(2):98–105. https://doi.org/10.1002/cytob.21128 what is the problem? blood donation shortages what are the causes of reduced blood supply and donations? what is the impact of decreased blood donations? how do we address blood shortages? acknowledgements references about the author(s) kenneth b. david faculty of pharmaceutical sciences, kaduna state university, kaduna, nigeriahull york medical school, university of hull, hull, united kingdom knovicks simfukwe school of veterinary medicine, university of zambia, lusaka, zambia mohamed b. musa faculty of pharmacy, omdurman islamic university, khartoum, sudan steven munharo training and research unit of excellence, college of medicine, university of malawi, blantyre, malawi don e. lucero-prisno iii department of global health and development, london school of hygiene and tropical medicine, london, united kingdom citation david kb, simfukwe k, musa mb, munharo s, lucero-prisno iii de. impact of covid-19 on blood donation and supply in africa. afr j lab med. 2021;10(1), a1408 https://doi.org/10.4102/ajlm.v10i1.1408 opinion paper impact of covid-19 on blood donation and supply in africa kenneth b. david, knovicks simfukwe, mohamed b. musa, steven munharo, don e. lucero-prisno iii received: 27 sept. 2020; accepted: 23 june 2021; published: 25 oct. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. what is the problem? blood donation shortages as the coronavirus disease 2019 (covid-19) continues to spread in africa, unprecedented disruptions at all levels of human endeavours including healthcare delivery systems have been recorded.1 one of the major areas of healthcare systems affected is blood supply – a commodity needed for the survival of many patients. blood and blood products such as cryoprecipitate, plasma, immune globulins, platelets, etc., are required for the management of medical conditions including but not limited to trauma, renal impairment, cancer, sickle cell anaemia, haemorrhagic shock, and other medical conditions related to acute or chronic loss of blood. despite the covid-19 pandemic, health problems and accidents occur including maternal morbidities, malnutrition, blood-transmitted infectious diseases (hiv and aids, hepatitis c virus infection, hepatitis b virus infection, syphilis and anaemia-inducing infectious diseases such as malaria, which is particularly worse in rainy seasons in countries such as malawi and nigeria,2 kidney disease, liver disease, and cancer, etc.). hence, blood donation shortages caused by the covid-19 pandemic are wrecking africa’s already overwhelmed blood transfusion services and are a guaranteed threat to a positive patient outcome, particularly for children under the age of 5, who are the recipients of 54% of the 118.5 million blood collected in low-income countries.3 in countries that rely on voluntary blood donations, particularly from students, the trends of blood supply and donation are likely to decline in many other countries if the covid-19 pandemic continues. this will consequently and adversely affect beneficiaries in various hospitals, particularly in countries whose sole blood donors are volunteers and students.4 what are the causes of reduced blood supply and donations? blood transfusions save lives, hence the need to maintain an adequate supply of blood. blood donors have made it possible for blood to be available for transfusion to other patients. these donors can either be family members, voluntary non-renumerated donors, or paid donors. the world health organization preferentially recommends voluntary non-renumerated donors as the major source of blood for transfusion purposes.5,6 the uncertainty in the pattern of blood demand and supply and the inadequacy of human resources (which can be reduced due to ill health) are some of the most pressing challenges posed by the covid-19 pandemic.7 the major contributors to low donations in africa are restricted movements, lockdowns, and closure of blood donation institutions.8 others include fear of contracting covid-19 by both donors and healthcare workers due to limited knowledge on severe acute respiratory syndrome coronavirus 2 (sars-cov-2) transmission. in zambia, for instance, there was a sharp decline in the number of blood donations because donors were afraid that by donating their blood, they could contract the virus. the lockdown imposed in the country was also a precipitating factor.5 however, the challenge might be more severe in countries where blood donation is not a culture. for instance, in kenya, less than 1% of the 47 million kenyans donated blood in the 2018/2019 year.9 in south africa, there was a decreased blood supply and increased blood demand when schools and colleges were shut down because over 30% of the below 1% daily blood donors are younger than 25 years old.10 what is the impact of decreased blood donations? covid-19 has caused some blood transfusion centres to miss their blood collection targets. for instance, the zambia national blood transfusion services target for the first quarter of 2020 was 18 750 units, but the institution barely collected 6516 units, representing 34.7% of the initial target.5 the botswana blood transfusion service had enjoyed an annual collection of 25 000 units, but due to covid-19 preventive measures, only 11 000 units had been collected as of may 2020. this implied an impending decrease in the units of blood obtainable in a pandemic-affected year.8 the national blood transfusion services in botswana was able to collect only about 23 000 units of blood and blood products against the 45 000 units set as the target for the year 2020.11 in kenya, covid-19 has also reduced blood collection manpower (staff) due to ill health, social distancing measures placed in all institutions, and the lack of sufficient effective protective equipment to ensure staff and client safety.9 a study conducted at a teaching hospital in nigeria revealed that 71.4% of patients with various types of malignancies such as acute myeloid leukaemia, bladder cancer, extra-orbital malignancy, prostate cancer, and lung cancer died due to the lockdown imposed in the country which disrupted the availability of blood donors.12 to this end, transfusion institutions – both hospital and independent blood transfusion units – have a key role to play in monitoring the demand and supply of blood and blood products to ensure that these products are adequately available. how do we address blood shortages? when considering issues related to the availability of blood and blood products for clinical management, the principle of demand and supply should be evaluated.13 decrease demand proactive measures aimed at reducing demand and increasing the supply of blood and blood products must be implemented, particularly, at this time of the covid-19 pandemic. these include patient blood management and strengthened cross-matching protocols for allogenic blood transfusion to reduce repeat transfusion.10 increase supply to maintain supply, intense mobilisation campaigns and safer donation protocols are required. mobilisation campaigns should be aimed at creating awareness: educating the populace on the importance of blood donations, debunking covid-19-related blood donation myths, and sharing extra safety and screening measures instituted in the facilities.14 unlike the recorded decline in the number of donated blood and blood products in africa, india for instance devised ways to boost the confidence level of donors for outdoor blood donation drives during the lockdown period. this allowed the blood bank to receive adequate supplies of blood amid the pandemic.14 some of the measures taken to boost the confidence of donors during the pandemic included the education of both staff and blood donors on covid-19 preventive measures, the observation of safety protocols while conveying the donors to the venue of the blood donation drive, the provision of face masks and other personal protective equipment to all donors and staff, the compulsory use of hand sanitisers, and the use of infrared thermometers to check the body temperature of all the donors. also, the use of ‘namaste’ as the mode of greetings to avoid handshakes was reinforced.14 like other coronaviruses, sars-cov-2 infects the upper and lower respiratory tract and its rna is shed into the serum or plasma of infected persons. thus, some studies reported that sars-cov-2 can be detected in either blood plasma or serum.15,16 theoretically, this implies that blood recipients can be infected with sars-cov-2 if they receive infected blood or blood products. however, wang and his co-authors in 202015 disagreed with this theoretical claim by saying: ‘to date, there are no reported cases of sars-cov-2 transmission by any blood product but transfusion transmission cannot yet be completely excluded’. also, the world health organization has stated that the role of blood-borne transmission of sars-cov-2 is uncertain as the very low viral titres in plasma suggest a low risk of blood-borne transmission of the virus.17 fortunately, there has not been any reported case of sars-cov-2 transmission via transfusion in any country.15 consequently, it is not mandated for blood donors to get tested for sars-cov-2.17 however, because of the high number of asymptomatic covid-19 infections, the safety of blood donors and staff needs to be ensured. aside from the routine screening for infectious diseases, the screening of potential donors for covid-19 exposure and symptoms should be done before donation, that is, at the time of donor clearance. the world health organization recommends that donor restrictions be modified according to the covid-19 community transmission status so as not to worsen the unavailability of donated blood. some of the guidelines include sensitising donors about self-deferring upon the onset of symptoms, and instructing donors to inform the blood collection centre if they develop a respiratory illness. deferring lasts for 14 days once a positive test is confirmed and delaying lasts for another 14 days after recovering from covid-19 symptoms.18,19 improve management finally, blood inventory management should be implemented to prevent stockouts in emergency cases, collected blood and blood products should be harnessed judiciously.14 conclusion covid-19 is a threat to blood transfusion services in many countries, particularly lower-income countries. with a recorded decline in blood collection rates and missed targets, the impact casts a shadow on health and health outcomes amid the covid-19 pandemic. covid-19 offers african governments a chance to intensify efforts to effectively tackle all the uncertainties posed by the pandemic,20 implement responsive systems, and strategise effective ways to reach yearly targets even in pandemics.21 with steps taken to judiciously utilise available blood and broaden the sources of blood donation amid the pandemic, the impact of covid-19 will be mitigated. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions k.b.d. and d.e.l-p. iii conceived the idea. k.b.d., k.s., m.b.m. and s.m. wrote the draft of the manuscript. d.e.l-p. iii reviewed and assisted with data collection and the language edit. all of the authors have read and agreed to the final manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. data availability data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references david kb, thomas n, solomon jk. epidemiology of covid-19 in africa: daily cumulative index and mortality rate. int j infect control. 2020;16(2). https://doi.org/10.3396/ijic.v16i2.008.20 cho hj, koo jw, roh sk, et al. covid-19 transmission and blood transfusion: a case report. j infect public health. 2020;13(11):1678–1679. https://doi.org/10.1016/j.jiph.2020.05.001 who. blood safety and availability [homepage on the internet]. 2020 [cited 2020 may 3]. available from: https://www.who.int/news-room/fact-sheets/detail/blood-safety-and-availability wang y, han w, pan l, et al. impact of covid-19 on blood centres in zhejiang province china. vox sang. 2020;115(6):502–506. https://doi.org/10.1111/vox.12931 who. blood transfusion safety [homepage on the internet]. 2021 [cited 2021 feb 14]. available from: https://www.who.int/bloodsafety/voluntary_donation/en/ allain jp, sarkodie f, boateng p, asenso k, kyeremateng e, owusu-ofori s. a pool of repeat blood donors can be generated with little expense to the blood center in sub-saharan africa. transfusion (paris). 2008;48:735–741. https://doi.org/10.1111/j.1537-2995.2007.01599.x kasanga m, mudenda s, gondwe t, chileshe m, solochi b, wu jian. impact of covid-19 on blood donation and transfusion services at lusaka provincial blood transfusion centre, zambia. pan afr med j. 2020;35(2):74. https://doi.org/10.11604/pamj.supp.2020.35.2.23975 ngwako, t. botswana blood donation decline amid covid-19 [homepage on the internet]. allafrica. [cited 2021 apr 24]. available from: https://allafrica.com/stories/202006020164.html mwai p. why has kenya been facing serious shortages of human blood? [homepage on the internet]. 2020 [cited 2020 may 3]. available from: https://www.bbc.com/news/world-africa-51458114 kaserer a, rössler j, braun j, et al. impact of a patient blood management monitoring and feedback programme on allogeneic blood transfusions and related costs. anaesthesia. 2019;74:1534–1541. https://doi.org/10.1111/anae.14816 swanka c. botswana’s blood bank half empty [homepage on the internet]. [cited 2021 apr 24]. available from: https://www.google.com/amp/s/www.sundaystandard.info/botswanas-blood-bank-half-empty/%3famp/ ugwu ao, madu aj, efobi cc, ibegbulam og. pattern of blood donation and characteristics of blood donors in enugu, southeast nigeria. niger. j clin pract. 2018;21:1438–1443. himan y, patidar gk, arora s. covid-19 pandemicresponse to challenges by blood transfusion services in india: a review report. isbt science series. 2020;15(4):365–373. https://doi.org/10.1111/voxs.12563 gupta am, ojha s, poojary m, sumathi sh, nagaraju p, dhokle r. organization of the outdoor blood donation drives amid novel coronavirus pandemic and national lockdown: an experience from a tertiary care oncology institution in india. transfus apher sci. 2020;59(5):102878. https://doi.org/10.1016/j.transci.2020.102878 wang w, xu y, gao r, et al. detection of sars-cov-2 in different types of clinical specimens. jama. 2020;323(18):1843–1844. https://doi.org/10.1001/jama.2020.3786 chang l, zhao l, gong h, wang l, wang l. severe acute respiratory syndrome coronavirus 2 rna detected in blood donations. emerg infect dis. 2020;26:1631–1633. https://doi.org/10.3201/eid2607.200839 who. transmission of sars-cov-2: implications for infection prevention precautions [homepage on the internet]. scientific brief. 2020 [cited 2020 may 3]. available from: https://apps.who.int/iris/rest/bitstreams/1286634/retrieve adebisi ya, oke gi, ademola ps, chinemelum ig, ogunkola io, lucero-prisno iii de. sars-cov-2 diagnostic testing in africa: needs and challenges. pan afr med j. 2020;35(2):4. https://doi.org/10.11604/pamj.2020.35.4.22703 aneke jc, okocha ce. blood transfusion safety; current status and challenges in nigeria. asian j transfus sci. 2017;11(1):1–5. https://doi.org/10.4103/0973-6247.200781 stanweth sj, new hv, apelseth to, et al. effects of the covid-19 pandemic on supply and use of blood for transfusion. lancet haematol. 2020. https://doi.org/10.1016/s2352-3026(20)30186-1 lucero-prisno de 3rd, adebisi ya, lin x. current efforts and challenges facing responses to 2019-ncov in africa. glob health res policy. 2020;5:21. https://doi.org/10.1186/s41256-020-00148-1 abstract introduction research method and design results discussion acknowledgements references about the author(s) pamela michelow department of anatomical pathology, university of the witwatersrand and national health laboratory service, johannesburg, south africa amanda sherrin department of anatomical pathology, university of the witwatersrand and national health laboratory service, johannesburg, south africa louise rossouw department of anatomical pathology, university of the witwatersrand and national health laboratory service, johannesburg, south africa samson mohaleamolla department of anatomical pathology, university of the witwatersrand and national health laboratory service, johannesburg, south africa denise evans department of internal medicine, university of the witwatersrand, johannesburg, south africa avril swarts department of medicine, university of the witwatersrand, johannesburg, south africa ntombiyenkosi rakhombe right to care, helen joseph hospital, johannesburg, south africa jennifer s. smith department of epidemiology, university of north carolina, chapel hill, north carolina, united states lineberger comprehensive cancer center, university of north carolina, chapel hill, north carolina, united states cynthia firnhaber department of medicine, university of the witwatersrand, johannesburg, south africa right to care, helen joseph hospital, johannesburg, south africa citation michelow p, sherrin a, rossouw l, et al. performance of the cellslide® automated liquid-based cytology system amongst hiv-positive women. afr j lab med. 2016;5(1), art. #278, 6 pages. http://dx.doi.org/10.4102/ajlm.v5i1.278 original research performance of the cellslide® automated liquid-based cytology system amongst hiv-positive women pamela michelow, amanda sherrin, louise rossouw, samson mohaleamolla, denise evans, avril swarts, ntombiyenkosi rakhombe, jennifer s. smith, cynthia firnhaber received: 03 nov. 2014; accepted: 13 nov. 2015; published: 01 feb. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: many women undergoing cervical screening as part of a national south african screening programme may be positive for hiv. the performance of liquid-based cytology (lbc) on samples from hiv-positive women needs to be determined. objectives: the performance of the cellslide® automated lbc system was evaluated as a possible alternative to conventional cytology in a national cervical cancer screening programme. methods: split samples from 348 hiv-positive women attending an hiv treatment clinic in johannesburg, south africa were examined by conventional cytology and monolayer lbc methods. all samples were stained, examined and reported in the same manner. cytotechnologists were blinded to the conventional smear diagnosis if the lbc smear was screened and vice versa. results: the same percentage of inadequate smears (1.4%) was obtained by conventional cytology and lbc. atypical squamous cells of undetermined significance were observed in 5.2% of conventional smears and 4.0% of lbc smears. low-grade squamous intraepithelial lesions were found in 35.6% of conventional smears and 32.7% of lbc smears. only one conventional smear was categorised as atypical squamous cells – cannot exclude a high-grade lesion, whereas five such cases were identified on lbc. high-grade squamous intraepithelial lesions were seen in 21.6% of conventional smears and 23.3% lbc smears. no invasive carcinoma was identified. conclusion: the performance of the cellslide® lbc system was similar to that of conventional cytology in this population of high-risk hiv-positive women, indicating that it may be introduced successfully as part of a cervical cancer screening programme. introduction cervical cancer is a significant cause of morbidity and mortality in south africa. in 2009, it was the second-most common cancer amongst south african women, with an age-standardised incidence rate of 22.33 per 100 000.1 amongst black south african women, cervical cancer was the most commonly histologically diagnosed cancer in 2009, with an age-standardised incidence rate of 26.19 per 100 000.1 unfortunately, more recent incidence data are not available. from a global perspective, an analysis of 187 countries showed that approximately 200 000 women died from cervical cancer in 2010, of which a significant proportion were women aged between 15 and 49 years in under-resourced nations.2 in south africa, 30.2% of women of reproductive age were hiv-positive in 2010, whereas the rate was 29.5% in 2012.3,4 the burden of cervical cancer and its precursor lesions is intensified amongst hiv-positive women. in a study from johannesburg, hiv-positive women had a higher prevalence of cervix lesions related to human papillomavirus (hpv) compared with hiv-negative women, even after controlling for confounding variables such as age and sexual behaviour.5 this finding reflects the high rate of co-infection of hiv and hpv. such co-infection is often associated with higher prevalence of high-risk hpv infections and increased rates of intraepithelial lesions such as high-grade squamous intraepithelial lesions (hsil), which are often large, multifocal, and have higher recurrence rates. invasive cervical cancer is also more common amongst hiv-positive women, occurring approximately 10 years earlier and with more rapid progression and poorer prognosis, than amongst hiv-negative women.6,7 the national south african guidelines for cervical cancer screening of hiv-positive women recommend screening upon diagnosis of hiv. if the results are negative, follow-up screening every one to three years is recommended. if the initial results show a low-grade lesion (i.e. atypical squamous cells of undetermined significance [ascus] or low-grade squamous intraepithelial lesion [lsil]), a repeat screening is required one year later.8 if a high-grade lesion is found (i.e. hsil or atypical squamous cells – cannot exclude a high-grade lesion [asch]), the patient is referred for further evaluation. suggested screening modalities include conventional and liquid-based cytology (lbc), hpv testing, visual inspection with acetic acid and visual inspection with lugol’s iodine. a number of studies to compare conventional cytology, hpv testing and visual inspection with acetic acid for screening of hiv-positive women in south africa are currently underway.9,10 it is estimated that 80% of south africans use public–sector healthcare facilities, including the national health laboratory service. approximately 1 million screenings using the conventional cervical cytology method were reported by the national health laboratory service in 2013.11 lbc is an alternative method of preparing cervical smears for microscopic examination and is widely used in well-resourced nations. however, although it was developed 15 years ago, it has not been adopted into the south african health sector. several meta-analyses comparing lbc with conventional cytology have reported conflicting results. abulafia et al.12 concluded that a commonly used lbc test was more sensitive and specific compared with conventional cytology for diagnosing cervical dysplasia, whereas arbyn et al.13 and davey et al.14 determined that lbc neither reduces the number of unsatisfactory smears nor improves detection of hsil. karnon et al. found that there is uncertainty regarding the ‘relative effectiveness (and cost-effectiveness) of the two main lbc techniques’.15 advantages of lbc include that screenings for hpv and other sexually transmitted infections, including chlamydial and gonococcal infections, can be performed on the lbc collection fluid, without the need for collecting a separate specimen. hpv testing can be performed on the fluid in the vial, and hpv testing or cytology can be performed on the same sample.16,17 a large percentage of south african women undergoing routine cervical screening may be hiv-positive.3,4 as there is a paucity of literature examining whether lbc can be successfully used to screen for cervical abnormalities in hiv-positive women, the benefit of introducing lbc as part of a national cervical cancer screening programme in south africa is unclear. the aim of this study was to determine whether cellslide® (audit diagnostics, cork, ireland), an automated lbc processing system for the preparation of thin-layer smears, can be used successfully as a screening modality in a high-risk hiv-positive population. research method and design ethical considerations the study protocol was reviewed and accepted by both the university of the witwatersrand (human ethics committee) and the university of north carolina. study design and setting the study was a non-randomised, prospective, observational evaluation. the study population comprised 348 hiv-positive women involved in a cervical cancer screening study in south africa.5 all participants were enrolled consecutively. women aged between 18 and 65 years were recruited from an hiv treatment clinic at a tertiary government hospital in johannesburg, south africa, between november 2009 and august 2011. participants were approached for the study whilst in the hiv clinic awaiting medications or an appointment with a healthcare practitioner. women were ineligible to participate if they were pregnant, had previously undergone a hysterectomy or treatment for cervical neoplasia or cancer, were severely ill or had signs or symptoms suggestive of a sexually transmitted disease. women who had completed treatment for a sexually transmitted disease were eligible. women who were menstruating at the time of study enrolment were asked to return within two weeks to participate. the main reasons that women declined to participate in the study were the fear of losing their place in the queue and time constraints. a cervical fluid sample was taken with a cervical broom. a split-sample method was then used, whereby a conventional cytology smear (pap smear) was prepared by spreading the collected material onto a glass slide and spray fixing immediately, followed by placing the tip of the brush in a vial containing cellslide® preservative solution (audit diagnostics, cork, ireland). both specimens were processed in the cytology unit of the department of anatomical pathology, university of the witwatersrand/national health laboratory service in johannesburg, south africa. the manufacturer’s instructions were used when preparing the cellslide® thin-layer slide. both types of sample were stained, coverslipped, examined under the microscope and reported using the bethesda system for reporting cervical cytology.18 the bethesda system is a widely used cytology reporting system that not only provides guidelines for specimen adequacy but also offers standardised reproducible criteria for cytologic lesions such as ascus, lsil, asch and hsil. the aim of the bethesda system is to minimise inter-observer variability and facilitate communication between the clinician and the laboratory.18 laboratory investigations the conventional and cellslide® cervical smears were examined by different cytotechnologists. the cytotechnologists reporting the cellslide® smears were blinded to the conventional smear diagnosis and vice versa. thirteen cytotechnologists reported out some of the conventional cytology and some of the cellslide® thin-layer slides. cytotechnologists participate in stringent internal quality assurance activities. some of these include all reportedly negative smears undergoing rapid review, all positive smears being evaluated by two technologists and evaluation of each technologist’s ascus : sil ratio. external quality assurance activities employed by this laboratory include the australian rcpa quality assurance proficiency programme and laboratory accreditation by external accreditation bodies. if quality assurance activities identify suboptimal performance by a cytotechnologist, re-training and intense monitoring of the work quality are undertaken. statistical analysis categories were compared using the chi-square test or fisher’s exact test for proportions. we determined the accuracy of cellslide® for identifying the diagnostic category correctly compared to the ‘gold standard’ of conventional cytology (pap smears). the number of positive and negative samples as tested using cellslide® was compared to the number of samples with and without each cervical abnormality of interest (e.g., hsil, asch, lsil, ascus) as determined by conventional cytology. we calculated the sensitivity, specificity, positive predictive value and negative predicative value for each diagnostic category, as well as the corresponding 95% confidence intervals (ci; binomial distribution assumed). the kappa coefficient was used to test for agreement between diagnostic categories for the split sample. kappa values < 0.4 were considered to indicate poor agreement, whereas values of 0.41–0.75 were considered to indicate moderate (fair to good) agreement and values > 0.75 were regarded as indicating excellent agreement.19 as both methods used the same categories within the rating scale, a weighted kappa coefficient was not required. all analyses were performed at a 5% significance level using sas version 9.1 (sas institute, cary, north carolina, united states). results very few inadequate smears were obtained by the respective methods (only five [1.4%] for both conventional cytology and cellslide®) (table 1). a high percentage of abnormal smears were identified with both methods, and a diagnosis of negative for intraepithelial lesion/malignancy (nilm) was found for only 125 (35.9%) by conventional cytology and 129 (37.1%) by cellslide®. lsil was the most frequently diagnosed epithelial abnormality (124 [35.6%] by conventional cytology and 114 [32.7%] by cellslide®). both preparation methods diagnosed a substantial number of hsil cases (75 [21.6%] by conventional cytology and 81 [23.3%] by cellslide®). the cellslide® method diagnosed an additional six cases of hsil (p < 0.001). ascus was diagnosed in 18 samples (5.2%) using conventional cytology and in 14 samples (4.0%) using cellslide® (p < 0.001), whereas asch was diagnosed in only one sample (0.3%) using conventional cytology and in five samples (1.4%) using cellslide® (p < 0.014). no cases of glandular lesions or invasive carcinoma were diagnosed by either method. table 1: results from conventional cytology and cellslide® automated liquid-based cytology amongst hiv-positive women (n = 348) in johannesburg, south africa (november 2009 to august 2011).† twenty cases diagnosed as lsil by conventional cytology were diagnosed as hsil by cellslide®, and 15 cases diagnosed as lsil by cellslide® were diagnosed as hsil by conventional cytology. four cases diagnosed as asch by cellslide® were diagnosed as lsil by conventional cytology. no cases diagnosed as asch by one method (either conventional cytology or cellslide®) were diagnosed as hsil by the other method. three cases diagnosed as ascus by cellslide® were diagnosed as hsil by conventional cytology, but no cases diagnosed as ascus by conventional cytology were diagnosed as hsil by cellslide®. the agreement between the two diagnostic methods was poor for asch (κ = 33.0, 95% ci: 1.4–33.0) (table 2). agreement was also considered poor for ascus, because the kappa value was close to the poor–moderate cut-off and the 95% ci was wide (κ = 41.1, 95% ci: 18.6–62.5). agreement was moderate for lsil and hsil, and excellent for nilm. table 2: level of agreement between conventional cytology and cellslide® for each diagnostic category amongst samples from hiv-positive women (n = 348) in johannesburg, south africa (november 2009 to august 2011). for hsil, cellslide® showed sensitivity of 76.0% (95% ci: 64.8–85.1) and specificity of 91.0% (95% ci: 87.0–94.2), with a false-omission rate < 7%, compared with conventional cytology (table 3). in addition, when compared with conventional cytology, cellslide® showed sensitivity of 89.6% (95% ci: 82.9–94.4) and specificity of 92.2% (95% ci: 87.8–95.4) for nilm, sensitivity of 70.2% (95% ci: 61.3–78.0) and specificity of 87.7% (95% ci: 82.6–91.7) for lsil, and sensitivity of 100% (95% ci: 2.5–100) and specificity of 98.8% (95% ci: 97.1–99.7) for asch. table 3: diagnostic accuracy of cellslide® compared with conventional cytology in samples from hiv-positive women in johannesburg, south africa (november 2009 to august 2011). discussion the results show excellent to moderate agreement between conventional cytology and the automated lbc system cellslide® for diagnosis of nilm, lsil and hsil in this population of hiv-positive women. poor agreement was observed between the two methods for diagnosis of ascus and asch. hpv testing is becoming increasingly popular as a primary cervical screening modality. in south africa, lbc can be efficacious, should hpv testing be implemented. hpv testing and co-testing with either cytology or reflex cytology, if certain high-risk hpv types are found, can be performed on the same vial, without the need for collecting two samples from the same patient. the lbc method may support faster microscope screening and therefore more samples could be screened daily, although not all studies have demonstrated this.12,13,14,15 the lbc method is reported to perform better when using computer-assisted screening devices,12,13,14,15 which is an advantage given the large number of pap smears performed in south african public-sector healthcare facilities. the cost associated with lbc has to be considered. results on the cost-effectiveness of lbc are conflicting, depending on whether studies found improvements in the adequacy or detection of abnormality rates. a study by taylor et al.20 found that a commonly used lbc product reduced the number of inadequate smears but did not improve detection of histology-confirmed disease (cervical intraepithelial neoplasia grade 1 or worse) and therefore concluded that the increased cost does not justify the implementation of lbc. in contrast, a review by cox21 showed that lbc was cost-effective. a study by de bekker-grob et al.22 similarly determined that lbc can be cost-effective as a cervical screening modality, specifically if the cost of lbc exceeded the cost of conventional cytology by less than €3.2, the sensitivity of lbc was at least 3% – 5% greater than conventional cytology and the rate of inadequate smears on conventional cytology was at least 16.2%. only one study has investigated the performance of lbc on samples from hiv-positive women.23 the findings need to be considered critically to inform a decision about introducing lbc in the south african public health sector, as a large percentage of south african women eligible for cervical screening may be hiv-positive.6,7,24 the study by swierczynski et al.23 concluded that conventional cytology and lbc detected the same number of squamous intraepithelial lesions. however, the diagnosis of ascus by lbc was more likely to be associated with a squamous intraepithelial lesion on follow-up compared with diagnoses of ascus by conventional cytology. they also determined that both methods could readily identify infectious organisms. in the current study, the conventional cytology and cellslide® methods detected a similar number of unsatisfactory smears and ascus and lsil diagnoses, but more cases of asch and hsil were diagnosed by cellslide®. furthermore, we found excellent agreement between the two methods for diagnosis of nilm and moderate agreement for diagnosis of lsil and hsil. our finding of poor agreement for diagnosis of ascus and asch highlights the well-described poor intraand inter-observer reproducibility for these epithelial abnormalities.25,26,27 however, it is important to note that the small number of samples in the ascus and asch categories could have compromised the accuracy of the statistical analysis. the results of the current study show that cellslide® has good sensitivity and specificity for nilm. as a screening test, a negative result is useful for determining that the patient does not have the disorder. at this initial screening, cellslide® correctly identified more than 90% of samples that showed no cervical abnormalities. similar to these results, a brazilian study found that the agreement between conventional cytology and lbc was highest in the nilm category.28 the authors further noted that this influenced the agreement rate, as the majority of cervical smears were negative. in comparison, the proportion of samples that showed abnormal cytology exceeded 60% for both preparation methods in the current study. other studies have also documented below-excellent agreement for epithelial abnormalities when comparing conventional cytology to lbc preparations.27,28 factors that influence agreement include the method employed to collect the smear, variations in cellular material between the conventional and lbc samples and the level of experience of the cytotechnologists in interpreting the smears.29,30,31,32,33,34 limitations one of the main limitations of the current study is the small number of samples in some of the epithelial abnormality categories (e.g., ascus, asch) as determined by the bethesda system; these results should be interpreted with caution. another limitation is that cellular morphology is somewhat different on lbc preparations compared with conventional cytology and cytotechnologists face a learning curve when moving from conventional cytology to lbc.30 in addition, training in lbc cytomorphology is recommended to facilitate accurate interpretation of an lbc smear.34 as this study was conducted over only a limited time period, cytotechnologists may not have mastered lbc cytomorphology completely. accurate costing of a cervical smear, whether for conventional cytology or lbc, is a complex problem and beyond the scope of the current study. however, cost is a critical factor and must be considered when deciding whether to move to lbc or continue using conventional cytology. conclusion results obtained with cellslide® were similar to those of conventional cytology in this population of high-risk hiv-positive women. the technique may therefore be used successfully should it be decided to move to lbc. acknowledgements we would like to thank the nurse clinicians at right to care for performing the cervical smears and the cytotechnologists at the national health laboratory service for screening the samples. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. sources of support the study was supported by the university of north carolina center for aids research (p30-ai50410), usaid pepfar (674-a-00-08-00007-00) and the south african research chairs initiative of the department of science and technology (phe za.09.0265). authors’ contributions p.m. (university of the witwatersrand and national health laboratory service) and c.f. (university of the witwatersrand and helen joseph hospital) were the main investigators, designed the study, analysed the results and wrote the manuscript. a.sh., l.r. and s.m. (university of the witwatersrand and national health laboratory service) prepared and examined the cervical smears. a.sw. (university of the witwatersrand) was the data manager. n.r. (helen joseph hospital) enrolled patients in the study, performed cervical smears and was responsible for patient follow-up. d.e. (university of the witwatersrand) and j.s.s. 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http://dx.doi.org/10.1002/(sici)1097-0339(199803)18:3<230::aid-dc14>3.0.co;2-l article information author: nicolas bouchet1 affiliation: 1quality assurance department, centre national de recherche et de formation sur le paludisme (cnrfp), burkina faso correspondence to: nicolas bouchet email: nikolas.bouchet@gmail.com postal address: 01 bp 2208, ouagadougou, burkina faso dates: received: 08 apr. 2014 accepted: 24 sept. 2014 published: 31 july 2015 how to cite this article: bouchet b. iso 15189:2012: what changes for african laboratories? afr j lab med. 2015;4(1), art. #325, 3 pages. http://dx.doi.org/10.4102/ajlm.v4i1.325 note: this article is the english translation of the french version published on 13 may 2015: bouchet b. iso 15189: 2012: quels changements pour les laboratoires africains? afr j lab med. 2015;4(1), art. #181, 4 pages. http://dx.doi.org/10.4102/ajlm.v4i1.181 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. iso 15189:2012: what changes for african laboratories? in this opinion paper... open access • introduction • iso 15189:2012    • process approach    • quality manual    • risk management    • laboratory information management    • minor changes • conclusion • acknowledgements    • competing interests • references introduction top ↑ the international organization for standardization's (iso) 15189 is the gold standard quality criteria for medical laboratories1 and accreditation is the recognition of a quality system in full compliance with this standard. aware of the current challenges in the fields of public health and biomedical research, african medical laboratories may need to develop quality management systems (qms) compliant with the requirements of iso 15189. launched by the world health organization's regional office for africa (who afro) and partners, the stepwise laboratory quality improvement process towards accreditation (slipta) and strengthening laboratory management toward accreditation (slmta) programmes have emerged in recent years in africa to support laboratories in the implementation of quality processes.2,3,4,5 as with any other quality system, iso standards evolve and are revised periodically. at the end of 2012, iso published the third edition of iso 15189,6 which replaces the 2007 version.7 the goal of this article is to highlight the major changes between the two versions of this standard, as well as the modifications these changes will require in quality systems already in place in laboratories, especially in african laboratories. iso 15189:2012 top ↑ in the new version, the standard is a clear document which is better organised than the 2007 version. the title was shortened and the organisation of the different sections has been adjusted to follow a more logical order, allowing users to gain a better understanding of the different requirements. during the presentation of the new version, iso spoke of a ‘technical revision’; however, a reading of this new version reveals changes that are actually more substantial. process approach a process is ‘a set of interrelated or interacting activities that transform inputs into output elements’.8 the process approach describes the organisation of work as a series of interconnected steps with the goal of meeting optimal customer satisfaction and achievement of objectives. the goal is to make the organisation more functional and less dependent on hierarchy, with the introduction and development of cross-functional management, a result-oriented culture and teamwork within the laboratory. the 2007 version already addressed this concept, but left the choice open to laboratories as to whether or not to follow this approach in their qms. the process approach already existed in the qms based on the general quality standard, iso 9001, which introduced the concept in its 2000 version9 and maintained it in the 2008 version.10 the 2012 version of iso 15189 is much more explicit: the process approach has become a requirement (subsection 4.2.1), albeit one that could be complicated to implement for african laboratories. in fact, this approach may be difficult to implement for any small medical laboratory with a small staff; such laboratories represent the majority of medical laboratories in sub-saharan africa. the process approach involves the mapping of the effect of processes, that is, to represent all the processes needed for the qms and their sequences by determining the interactions that bind them, and appointing process managers with clearly-delineated role definitions. in small organisations, the same person may have both an overall and a detailed view of operations, thus a precise mapping of different processes is often seen as unnecessary. for example, if in a laboratory of four employees each individual is involved in all processes, the establishment of a map may seem superfluous. however, this approach does have its advantages. firstly, it emphasises the ultimate goal of the qms – patient outcomes – by defining and clearly showing each staff member's role in the achievement of these outcomes. secondly, it allows analysis (and thus improvement) of the performance of the laboratory. medical laboratories have an advantage an implementing this approach, since their activities are centred around three main processes defined in the standard: the preanalytical process (section 5.4), the analytical process (section 5.5) and the postanalytical process (section 5.7). by focusing on these three major processes, a laboratory may be able to link to other processes (such as equipment and inventory management, human resources, customer satisfaction, continuous improvement, etc.) for a fairly simple mapping. quality manual for the quality manual, the 2007 version (subsection 4.2.4) proposed a plan in 23 points; the 2012 version focuses on describing six key points in a laboratory quality manual (subparagraph 4.2.2.2): the quality policy, a description of the scope of the qms, the organisation and the structure of the laboratory, the roles and responsibilities of laboratory management, the description of the document management system and the policies established for the qms, with reference to the activities that support them. this change will thus allow each laboratory to define the terms of its quality manual based on its own quality system, which will demonstrate mastery of the standard by the laboratory and the ability of the laboratory to adapt the standard to local realities, which often proves difficult in sub-saharan africa. it will also simplify the writing of this document, which is the cornerstone of the qms. risk management this is arguably the major change in the new version of the standard. it appears in subparagraph 4.1.1.4, where it is stated that the role of the laboratory director is to ‘design and implement a contingency plan’ and that these plans should be tested periodically. subsection 4.14.6 (risk management) also addresses this subject, focusing on the risks of possible failures on the test results; everything must be done to reduce and/or eliminate these risks. the concept is also found in the new section on the management of laboratory information (subsection 5.10.3); it is stated in this paragraph that ‘the laboratory shall have documented contingency plans […] in the event of failure or downtime’. this concept of risk management is an important introduction in this new version of the standard; it will require substantial work for its implementation, as all contingencies should be identified and evaluated (e.g. stock-outs of reagents caused by delivery delays, failure of an analyser, power failure, malfunction of a printer, etc.). each situation must be included in the ‘contingency plan’ with: a solution to the problem (correction). a method to inform both patients and physicians. a plan to communicate the problem internally. corrective actions to prevent the recurrence of the problem, and procedures/protocols to return to normal operations. these plans must then be ‘tested periodically’ and this process must be documented; these tests should then be used to improve the plan if necessary. these plans will also be audited as part of an internal audit of the qms. let us take as examples power failures and delays in supply of reagents. these are rare events in industrialised countries, but are common problems in sub-saharan africa. many african capitals still experience rolling blackouts during certain periods of the year and delivery delays are common for laboratory reagents. a risk with a very low probability of occurrence in industrialised countries may occur with a very high frequency in sub-saharan african countries. it could thus be difficult to implement risk management as described in the standard in african laboratories. contingency plans can be implemented periodically for some aspects, but some things that might otherwise be considered ‘exceptional’ may need to be considered routine [in a sub-saharan setting]. it should be noted that this introduction of the concept of risk management in the new version of iso 15189 is part of a more global trend. this concept, which comes from industry, has entered the regulatory world in recent years. in the process of revision of the iso 9001 standard, which is the basic reference for quality management systems (the new version is scheduled for 2015), the concept of a risk management approach was identified by the expert group in charge of the revision of iso 9001 during the 26th meeting of the iso technical committee in tokyo in 200911 and it will be integrated into the new version. risk management is clearly one of the objectives of this new version of iso 9001, requiring one to project into the future to anticipate all possible problems that could prevent customer satisfaction, which is the main objective of the standard. in the field of medical laboratories, this concept was introduced in the united states by the new regulations in quality control, with the internal quality control plan, following the clinical and laboratory standards institute (clsi) ep23-a guideline.12 this document introduced the concept of risk management in the field of internal quality control in medical laboratories.13,14,15 laboratory information management section 5.10 is not really new in the new version of the standard; it is rather annex b of the 2007 version, which has now become a normative, with the obligation to implement these requirements. this entire section focuses on data protection and the management of the information system. the laboratory must ensure that the software tools involved in managing laboratory information (collection, processing, recording, reporting, data retention) are validated by suppliers, regularly maintained and secure. laboratories must consider these new requirements and act accordingly, although they may be difficult to implement and to document in the african context. minor changes section 4.14, which was dedicated only to internal audits in the 2007 version, is more complete in the 2012 version, because it deals with all aspects of audits and evaluations: review of requests, procedures and requirements for samples, assessment of customer feedback, staff suggestions, internal and external audits, risk management and monitoring of quality indicators. the goal of this entire process of evaluation/audit is to improve the laboratory quality system, ensuring that all quality processes necessary to ensure the best quality for patients’ results have been implemented in order to meet customer requirements. a new subparagraph (4.1.1.3) introduces ethics rules about potential conflicts of interest, the integrity of staff, ethical considerations in handling samples of human origin and confidentiality of patients. this point is very important because the culture of business ethics is a concept seldom implemented in sub-saharan africa, contrary to the concept of bioethics, which was mentioned in the 2007 version (annex c), but has disappeared in the 2012 version. conclusion top ↑ iso 15189 remains the ‘gold standard’ for quality in medical laboratories. the cleaner organisation of the 2012 version will allow laboratories to better understand this standard and better meet the standard requirements. in fact, it is an obligation for laboratories to use the iso standard to move toward accreditation in order to ensure the trust of patients and to gain national and international respect. the challenge is huge for african laboratories, which must adapt to this standard whilst taking into account the specific conditions in which they are based. it is desirable that who afro adapts its slipta checklist to this new version of the standard, generally adapting all support programmes to include the changes of the 2012 version. acknowledgements top ↑ my thanks to dr issa nébié ouedraogo and dr. issiaka soulama (both cnrfp, burkina faso) for their valuable comments and discussions on the manuscript, as well as to dr sodiomon b. sirima, the executive director of the cnrfp for his support. competing interests the author declares that he has no financial or personal relationship(s) that may have inappropriately influenced him in writing this article. references top ↑ datema tam, oskam l, klatser pr. review and comparison of quality standards, guidelines and regulations for laboratories. afr j lab med. 2011;1(1), art.#3, 7 pages. gershy-damet g, rotz p, cross d. belabbes e, cham f, ndihokubwayo j, et al. the world health organization african region laboratory accreditation process. am j clin pathol. 2010; 134:393-400. http://dx.doi.org/10.1309/ajcptuuc2v1wjqbm maruta t, motebang d, wanyoike j, peter t, rotz pj. impact of mentorship on who-afro strengthening laboratory quality improvement process towards accreditation (slipta). afr j lab med. 2012;1(1), art. #6, 8 pages. yao k, mckinney b, murphy a, rotz p, wafula w, sendagire h, okui s, et al. improving quality management systems of laboratories in developing countries. am j clin pathol. 2010;134:401-409. http://dx.doi.org/10.1309/ajcpnbbl53fwuiqj mothabeng d, maruta t, lebina m, lewis k, wanyoike j, mengstu y. strengthening laboratory management towards accreditation: the lesotho experience. afr j lab med. 2012;1(1), art. #9, 7 pages. organisation internationale de normalisation (iso). iso 15189 : 2012. laboratoires de biologie médicale – exigences concernant la qualité et la compétence. troisième édition 2012-11-01, version corrigée 2013-03-01. organisation internationale de normalisation (iso). iso 15189 : 2007. laboratoires d’analyses de biologie médicale – exigences particulières concernant la qualité et la compétence. deuxième édition 2007-04-15, version corrigée 2007-09-15. site internet de l’organisation internationale de normalisation (iso). disponible à cette adresse: www.iso.org organisation internationale de normalisation (iso). iso 9000 : 2005 systèmes de management de la qualité – principes essentiels et vocabulaire. troisième édition 2005-09-15. organisation internationale de normalisation (iso). iso 9001 : 2000 systèmes de management de la qualité – exigences. troisième édition 2000-12-15. organisation internationale de normalisation (iso). iso 9001 : 2008 systèmes de management de la qualité – exigences. quatrième édition 2008-11-15, version corrigée 2009-07-15. clinical and laboratory standards institute. ep23-a: laboratory quality control based on risk management; approved guideline. 2011. person n. developing risk-based quality control plans: an overview of clsi ep23-a. clin lab med, 2013, 33, 15-26. http://dx.doi.org/10.1016/j.cll.2012.11.003 westgard j. perspectives on quality control, risk management, and analytical quality management. clin lab med, 2013, 33, 1-14. http://dx.doi.org/10.1016/j.cll.2012.10.003 nichols j. laboratory quality control based on risk management. ann saudi med. 2011 may-jun; 31(3): 223–228. http://dx.doi.org/10.4103/0256-4947.81526 ajlm 12(1)_2023_contents.indd http://www.ajlmonline.org open access table of contents i review article bridging the gaps in newborn screening programmes: challenges and opportunities to detect haemoglobinopathies in africa seth twum, kwadwo fosu, robin a. felder, kwabena a.n. sarpong african journal of laboratory medicine | vol 12, no 1 | a2225 | 14 december 2023 original research consequences of rpob mutations missed by the genotype mtbdrplus assay in a programmatic setting in south africa nomonde r. mvelase, lindiwe p. cele, ravesh singh, yeshnee naidoo, jennifer giandhari, eduan wilkinson, tulio de oliveira, khine swe swe-han, koleka p. mlisana african journal of laboratory medicine | vol 12, no 1 | a1975 | 06 february 2023 original research alternative methods for calculating percentage haemolysis of red cell concentrates in peripheral blood banks in sri lanka caroline a. fernando, deklanji t. dissanayake, uththara i. hewamana, shyamini rathnaweera, wickrama a. samanthilake, ranga tudugala, kithsiri b. jayasekara, kumudu kuruppu african journal of laboratory medicine | vol 12, no 1 | a1987 | 23 february 2023 original research antimicrobial susceptibility of bacterial uropathogens in a south african regional hospital alicia naidoo, afsana kajee, nomonde r. mvelase, khine swe swe-han african journal of laboratory medicine | vol 12, no 1 | a1920 | 03 march 2023 original research using the slipta checklist to assess laboratory readiness for joint commission international accreditation abdul k. el karaaoui, nada assaf african journal of laboratory medicine | vol 12, no 1 | a2044 | 15 march 2023 original research use of full blood count parameters and haematology cell ratios in screening for sepsis in south africa jason van rensburg, saarah davids, carine smuts, glenda m. davison african journal of laboratory medicine | vol 12, no 1 | a2104 | 11 april 2023 original research modelling cd4 reagent usage across a national hierarchal network of laboratories in south africa naseem cassim, lindi-marie coetzee, deborah k. glencross african journal of laboratory medicine | vol 12, no 1 | a2085 | 15 may 2023 original research performance of two multiplex flow cytometric assays for antibody detection in egyptian patients alshymaa a. ahmed, alia a. el shahaway, heba m. kadry, nora m. said african journal of laboratory medicine | vol 12, no 1 | a2099 | 18 may 2023 original research covid-19 positive cases among asymptomatic individuals during the second wave in ndola, zambia jonathan gwasupika, victor daka, justin chileshe, moses mukosha, steward mudenda, bright mukanga, ruth l. mfune, gershom chongwe african journal of laboratory medicine | vol 12, no 1 | a2119 | 31 may 2023 57 65 73 80 88 96 103 110 117 page i of iii table of contents editorial time to treat the climate and nature crisis as one indivisible global health emergency kamran abbasi, parveen ali, virginia barbour, thomas benfield, kirsten bibbins, stephen hancocks, richard horton, laurie laybourn-langton, robert mash, peush sahni, wadeia m. sharief, paul yonga, chris zielinski african journal of laboratory medicine | vol 12, no 1 | a2335 | 20 november 2023 editorial taking the train of digital health and artificial intelligence to improve medical laboratory service in africa: key considerations rajiv erasmus, pascale ondoa african journal of laboratory medicine | vol 12, no 1 | a2329 | 30 november 2023 opinion paper medical laboratory practice in malawi – current status symon f. nayupe, patrick mbulaje, steven munharo, parth patel, don e. lucero-prisno iii african journal of laboratory medicine | vol 12, no 1 | a1921 | 06 january 2023 opinion paper why pathogen genomics is crucial in africa’s public health lamech m. mwapagha african journal of laboratory medicine | vol 12, no 1 | a2166 | 22 september 2023 review article the covid-19 pandemic in sub-saharan africa: the significance of presumed immune sufficiency abel o. idowu, yusuf o. omosun, joseph u. igietseme, anthony a. azenabor african journal of laboratory medicine | vol 12, no 1 | a1964 | 30 january 2023 review article key success factors for the implementation of quality management systems in developing countries iryna tanasiichuk, olha karaman, larysa natrus african journal of laboratory medicine | vol 12, no 1 | a2058 | 31 january 2023 review article surveillance of antimicrobial resistance in human health in tanzania: 2016–2021 neema camara, nyambura moremi, janneth mghamba, eliudi eliakimu, edwin shumba, pascale ondoa, beverly egyir african journal of laboratory medicine | vol 12, no 1 | a2053 | 22 may 2023 review article a pragmatic approach to the diagnosis of inborn errors of metabolism in developing countries john i. anetor, bose e. orimadegun, gloria o. anetor african journal of laboratory medicine | vol 12, no 1 | a1946 | 30 may 2023 review article epidemic forecast and preparedness for explosive-cerebrospinal meningitis outbreak in nigeria using the preventive vaccination strategy iseimokumo c. peletiri, rosemary c. nwachukwu c. nwachukwu, diweni c. peletiri, esther q. onoja, charity t. tulagha, ikaprite i. igbalibo, grace m. ayanbimpe, eugene i. ikey african journal of laboratory medicine | vol 12, no 1 | a2086 | 17 november 2023 1 5 7 11 14 24 32 40 48 vol 12, no 1 (2023) issn: 2225-2002 (print) | issn: 2225-2010 (online)african journal of laboratory medicine http://www.ajlmonline.org open access table of contents ii original research selected micronutrient status of school-aged children at risk of schistosoma haematobium infection in suburban communities of nigeria samson e. olerimi, ehitare i. ekhoye, oriasotie s. enaiho, alexander olerimi african journal of laboratory medicine | vol 12, no 1 | a2034 | 31 may 2023 original research seroprevalence of sars-cov-2 immunoglobulin g in hiv-positive and hiv-negative individuals in kwazulu-natal, south africa kerri-lee a. francois, nokukhanya msomi, kerusha govender, lilishia gounder, pravi moodley, raveen parboosing, indrani chetty, lunga xaba, aabida khan african journal of laboratory medicine | vol 12, no 1 | a2065 | 29 june 2023 original research evaluation of tumour marker utilisation and impact of electronic gatekeeping in the province of kwazulu-natal, south africa immaculate s. dlamini, verena gounden, nareshni moodley african journal of laboratory medicine | vol 12, no 1 | a2027 | 30 june 2023 original research lipids and apolipoproteins c-iii and e among treatment-naïve and treatment-experienced persons with hiv in nigeria mercy n. okunorobo, nwakasi k. nnamah, ugomma a. ude, enyioma a. ude african journal of laboratory medicine | vol 12, no 1 | a2018 | 17 july 2023 original research performance of the automated sysmex xn-3000 analyser for detecting white blood cell abnormalities in south africa jasmine ramiah, dashini pillay, nadine rapiti african journal of laboratory medicine | vol 12, no 1 | a2140 | 19 july 2023 original research providing specimen transport through an online marketplace in the northern region of ghana abass abdul-karim, david opare, ulysses balis, lee f. schroeder african journal of laboratory medicine | vol 12, no 1 | a2062 | 20 july 2023 original research missed opportunities for integrated testing of hiv and tuberculosis on the genexpert platform in lesotho gamuchirai p. gwaza, monkoe leqheka, tsietso mots’oane, sabine dittrich, kekeletso kao african journal of laboratory medicine | vol 12, no 1 | a2132 | 28 august 2023 original research establishment of a stable proficiency testing matrix in transfusion microbiology in south africa xoliswa l. mpumlwana, winnie kruger, ute jentsch african journal of laboratory medicine | vol 12, no 1 | a2095 | 30 august 2023 original research storage of mycobacterium tuberculosis culture isolates in microbanktm beads at a south african laboratory anura david, lesley e. scott, pedro da silva, elizabeth mayne, wendy s. stevens african journal of laboratory medicine | vol 12, no 1 | a2172 | 25 october 2023 122 128 134 141 148 154 162 169 176 original research role of ctx-m-15 gene in spread of extended-spectrum beta-lactamases among immunocompetent patients in ghana noah obeng-nkrumah, gloria d. tawiah-abrokwa, enid owusu, francisca duah, daniel oduro-mensah, paul kwao, bako evariste, appiah-korang labi african journal of laboratory medicine | vol 12, no 1 | a2135 | 20 november 2023 original research serum-free light chain test utilisation at a south african academic laboratory and comparison with serum protein electrophoresis results razia b. banderker, fatima b. fazel, annalise e. zemlin, aye-aye khine, thumeka p. jalavu african journal of laboratory medicine | vol 12, no 1 | a2201 | 24 november 2023 original research impact of novel software on laboratory expenditure at an academic hospital in south africa zoliswa mayekiso, kelechi e. oladimeji, guillermo a. pulido estrada, charles hongoro, teke r. apalata african journal of laboratory medicine | vol 12, no 1 | a2159 | 30 november 2023 original research critical values notification: a nationwide survey of practices among clinical laboratories across nigeria lucius c. imoh, idris y. mohammed, ifeyinwa d. nnakenyi, ephraim u. egbuagha, tomisin m. adaja, chinelo p. onyenekwu african journal of laboratory medicine | vol 12, no 1 | a2249 | 15 december 2023 original research impact of potassium test sample rejections on routine laboratory service, south africa sarah mcalpine, bettina chale-matsau african journal of laboratory medicine | vol 12, no 1 | a2239 | 22 december 2023 lessons from the field building clinical pharmacology laboratory capacity in lowand middle-income countries: experience from uganda denis omali, allan buzibye, richard kwizera, pauline byakika-kibwika, rhoda namakula, joshua matovu, olive mbabazi, emmanuel mande, christine sekaggya-wiltshire, damalie nakanjako, ursula gutteck, keith mcadam, philippa easterbrook, andrew kambugu, jan fehr, barbara castelnuovo, yukari c. manabe, mohammed lamorde, daniel mueller, concepta merry african journal of laboratory medicine | vol 12, no 1 | a1956 | 07 february 2023 lessons from the field an assessment of the laboratory network in ghana: a national-level atlas survey (2019–2020) emma e. kploanyi, joseph kenu, benedicta k. atsu, david a. opare, franklin asiedu-bekoe, lee f. schroeder, david w. dowdy, alfred e. yawson, ernest kenu african journal of laboratory medicine | vol 12, no 1 | a1844 | 08 february 2023 case study challenges faced by the hiv testing system in lowand middle-income countries rachel s. kamgaing, yagai bouba, samuel m. sosso, jeremiah e. gabisa, aubin nanfack, joseph fokam, laure ngono, nadine fainguem, michel c.t. tommo, krystel n. zam, junie f. yimga, désiré k. takou, alexis ndjolo african journal of laboratory medicine | vol 12, no 1 | a1974 | 31 january 2023 182 192 199 207 214 220 227 236 page ii of iii http://www.ajlmonline.org open access table of contents iii case study neonatal presentation of a patient with liddle syndrome, south africa nicolene steyn, bettina chale-matsau, aron b. abera, gertruida van biljon, tahir s. pillay african journal of laboratory medicine | vol 12, no 1 | a1998 | 14 april 2023 brief report evaluation of xpert carba-r for detecting carbapenemase-producing organisms in south africa sanelisiwe t. duze, teena thomas, tshegofatso pelego, sabelle jallow, olga perovic, adriano duse african journal of laboratory medicine | vol 12, no 1 | a1898 | 05 january 2023 brief report effect of delayed sample draw after blood collection for haemoglobin test in south korea hyerim kim, jongmin kim african journal of laboratory medicine | vol 12, no 1 | a2008 | 28 march 2023 brief report characterisation of genes encoding for extended spectrum β-lactamase in gram-negative bacteria causing healthcareassociated infections in mwanza, tanzania jenipher g. mwakyabala, conjester i. mtemisika, stacy mshana, adam a. mwakyoma, vitus silago african journal of laboratory medicine | vol 12, no 1 | a2107 | 12 april 2023 brief report antibacterial activity of soil-isolated bacillus altitudinis/pumilus complex against methicillin-resistant staphylococcus aureus from mwanza, tanzania reuben n. abednego, vitus silago african journal of laboratory medicine | vol 12, no 1 | a2167 | 18 july 2023 240 246 251 255 260 correction corrigendum: microbiology laboratories involved in disease and antimicrobial resistance surveillance: strengths and challenges of the central african states passoret vounba, severin loul, ludovic f. tamadea, joël f.d. siawaya african journal of laboratory medicine | vol 12, no 1 | a1913 | 09 may 2023 correction corrigendum: formulation of phage cocktails and evaluation of their interaction with antibiotics in inhibiting carbapenemaseproducing klebsiella pneumoniae in vitro in kenya noutin f. michodigni, atunga nyachieo, juliah k. akhwale, gabriel magoma, abdoul-salam ouédraogo, andrew n. kimang’a african journal of laboratory medicine | vol 12, no 1 | a2028 | 09 may 2023 correction corrigendum: the application of sigma metrics in the laboratory to assess quality control processes in south africa marli van heerden, jaya a. george, siyabonga khoza african journal of laboratory medicine | vol 12, no 1 | a1996 | 15 may 2023 reviewer acknowledgement acknowledgement to reviewers editoial office african journal of laboratory medicine | vol 12, no 1 | a2325 | 19 december 2023 265 266 267 268 page iii of iii abstract introduction methods results discussion acknowledgements references about the author(s) erica-mari nell division of haematological pathology, department of pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa national health laboratory service, tygerberg hospital, cape town, south africa zivanai c. chapanduka division of haematological pathology, department of pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa national health laboratory service, tygerberg hospital, cape town, south africa citation nell e, chapanduka zc. aetiology of pancytopenia: experience of a south african tertiary academic centre. afr j lab med. 2022;11(1), a1645. https://doi.org/10.4102/ajlm.v11i1.1645 original research aetiology of pancytopenia: experience of a south african tertiary academic centre erica-mari nell, zivanai c. chapanduka received: 10 june 2021; accepted: 18 feb. 2022; published: 31 may 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: pancytopenia is a manifestation of numerous disease entities. the causes of pancytopenia differ with geographic region, socio-economic factors and hiv prevalence. awareness of the common causes of pancytopenia may aid timely diagnosis. objective: this study aimed to determine the aetiology of pancytopenia in a south african population. methods: a retrospective observational study of adult patients presenting with pancytopenia at tygerberg academic hospital, south africa, from january 2016 to december 2017 was performed. data on pancytopenia cases were obtained from the laboratory information system and utilised to determine the causes of pancytopenia. results: a total of 673 cases of pancytopenia were identified. the most common causes of pancytopenia were chemoradiation therapy (25%), sepsis (18%), haematological malignancy (9%), advanced hiv (7%), and megaloblastic anaemia (6%). the diagnostic yield of bone marrow examinations (bme) was 57% (n = 52/91). the aetiology of pancytopenia differed according to age, with malignancy being a more common cause of pancytopenia among the elderly. conclusion: several easily recognisable and treatable conditions can manifest as pancytopenia. prompt management of such conditions, notably sepsis and megaloblastic anaemia, can result in the resolution of the cytopenias and negate the need for a bme. however, haematological malignancy and unexplained pancytopenia strongly rely on a bme to establish a diagnosis. pancytopenia investigations, when guided by appropriate clinic-laboratory findings, can promptly identify the underlying aetiology, while also identifying cases where an expedited bme is required. this is valuable in resource-conscious medicine. keywords: pancytopenia; sepsis; hiv; haematological malignancy; nutritional deficiency; megaloblastic anaemia; aging. introduction the term pancytopenia is used to describe a reduction in all three haematopoietic cell lines in the peripheral blood, namely erythrocytes, leukocytes and platelets. pancytopenia is not a disease entity, but rather the manifestation of several diseases and signifies the need for investigation. chemotherapy and radiation therapy are predictable causes of pancytopenia.1 the occurrence of pancytopenia in the absence of chemotherapy or radiation therapy is a common diagnostic dilemma and a bone marrow examination (bme) is recommended if the cause of pancytopenia cannot be otherwise ascertained. pancytopenia can also be caused by central inadequate production or peripheral destruction/sequestration,1 and the incidence of the various causes of pancytopenia differs based on geographic, socio-economic, dietary and other factors. in india, numerous studies have highlighted megaloblastic anaemia and aplastic anaemia as the commonly encountered causes of pancytopenia.2,3,4,5,6,7,8,9,10,11,12,13,14,15 studies from neighbouring countries, including pakistan,16,17,18,19,20 bangladesh21,22 and nepal,23,24,25 show similar findings. climate also affects the aetiology of pancytopenia by affecting disease transmission.26 as such, malaria, which is endemic in bangladesh, was found to be the most common cause of pancytopenia in a bangladeshi study.22 in high-income countries, however, the disease profile is quite different. studies done in france, sweden, south korea, oman and the united states demonstrate that haematological malignancies (hms) are the most common cause of pancytopenia.27,28,29,30,31,32 a study in mexico revealed a mix of hm and megaloblastic anaemia as the most common causes,33 demonstrating the role that socio-economic status and access to healthcare has on the aetiology of pancytopenia. in africa, the causes of pancytopenia follow the pattern of poor nutrition and poor access to healthcare. studies performed in zimbabwe, djibouti, morocco and tunisia showed that megaloblastic anaemia was the most common cause of pancytopenia.34,35,36,37 furthermore, hiv was found to be the third most common cause of pancytopenia in both the zimbabwean and djiboutian studies.34,35 to our knowledge, a study by retief and heyns performed in bloemfontein in 1976 is the only previous study investigating the aetiology of pancytopenia in south africa.38 the most common cause of pancytopenia as identified in that study was bone marrow aplasia (49.0%), and over two-thirds of those cases were attributed to radiation or chemotherapy. infectious agents (9.7%) were the second most common cause, followed by megaloblastic anaemia (9.6%). the hiv pandemic started in 1981, thus the effect of hiv on pancytopenia was not seen in the retief and heyns study.39 there is currently a high burden of hiv infection in south africa, and by the middle of 2019, 13.5% of south africans, that is 7.97 million people, were living with hiv.40 cytopenias are commonly observed in patients with hiv infection and are often multifactorial in aetiology.41 while cytopenias in hiv are common, the prevalence of pancytopenia in hiv clinics in african countries is low: 0.7% in an ethiopian study and 0.5% in a ugandan study.42,43 the pancytopenia prevalence is much higher (8.7%) in hiv clinics in puerto rico.44 this study aimed to contribute to the body of knowledge regarding the aetiology of pancytopenia in a developing country with a high burden of hiv and to assess the most common causes of pancytopenia across different age groups. methods ethical considerations this study was approved by the human research ethics committee review board of stellenbosch university (study approval number: s18/08/171) and all research was performed in accordance with relevant regulations with anonymised data. the requirement for informed consent was waived by the ethics committee due to the retrospective nature of the study. setting, specimens and defining criteria a retrospective cross-sectional descriptive study was conducted over a two-year period. all adult patients with new-onset pancytopenia who were treated at tygerberg academic hospital, cape town, south africa, from 01 january 2016 to 31 december 2017 were identified and included in our study. pancytopenia was defined as leucocyte count < 4 × 109/l, haemoglobin < 10 g/dl, and a platelet count < 100 × 109/l. these parameters are comparable with other studies investigating the aetiology of pancytopenia.6,7,14,15,16,21,45,46 patients were excluded from the study if they received clinical care at another facility even though the bme was reported at tygerberg academic hospital. data collection and interpretation the data were obtained from the laboratory information system (lis) of the national health laboratory service. an lis search was conducted to identify cases meeting the study criteria for pancytopenia. the identified pancytopenia cases were reviewed on the national health laboratory service lis to identify the cause of the pancytopenia. information retrieved from the lis included the reports of peripheral blood smears, bmes, vitamin b12 and serum folate levels, iron studies, viral studies, mycobacterium tuberculosis culture and genexpert® tests (cepheid, sunnyvale, california, united states), malaria rapid tests (binaxnow malaria, abbott laboratories, chicago, illinois, united states) and thick and thin smears, autoimmune screens, sepsis markers, and blood cultures. in addition, information on patient age, gender, hiv status, cd4 count, and hiv viral load was obtained. advanced hiv was defined as a cd4 count < 200 cells/µl.47,48 since the aetiology of cytopenias in hiv is multifactorial, evidence of contributing factors such as folate deficiency, opportunistic infection or malignancy were sought in hiv-positive patients. in the absence of any other cause of cytopenias, and if the cd4 count was < 200 cells/µl, the pancytopenia was attributed to the advanced hiv. data analysis statistical analysis was done in conjunction with the biostatistics department of stellenbosch university. percentages were calculated for categorical variables. age was presented as mean and standard deviation. non-parametric data such as hiv viral load, cd4 count, time to bme and time to resolution of pancytopenia were presented as medians and interquartile ranges. pearson’s chi-square tests were used to assess the statistical differences in the frequencies of causes of pancytopenia between the hiv-positive and negative groups, as well as between different age groups. the data were analysed using statistical package for the social sciences version 25 (ibm corp., armonk, new york, united states). results a total of 695 cases of new-onset pancytopenia were identified within the two-year period. further investigation showed that seven cases had platelet clumping with a subsequent platelet count above 100 × 109/l, and 15 cases were due to sample dilution, having been collected from a cannulated infusion vein. thus the total number of true pancytopenia cases was 673 over the specified period (figure 1). figure 1: flow diagram of case selection and stratification to identify the causes of pancytopenia at tygerberg academic hospital, cape town, south africa, from january 2016 to december 2017. true pancytopenia was confirmed in 673 cases. chemoradiation therapy was found to be the cause in 166 cases, leaving 507 non-iatrogenic cases with unidentified underlying aetiologies. of those, 91 patients had a bme and the cause of pancytopenia was identified in 52 of these patients. in 16 patients whose bme showed non-specific findings, a cause for pancytopenia was found using information available on the lis. using information available on the lis, the cause of pancytopenia was identified in 340 of the remaining 416 patients who did not have a bme. of the 673 patients, 273 (41%) were male and 400 (59%) were female. the mean age at which pancytopenia was diagnosed was 44 ± 15 years (range: 18–87 years). most common causes of pancytopenia chemotherapy and/or radiation therapy was the most common (25%; 166/673) cause of pancytopenia (figure 2). the chemoradiation therapy was for the treatment of non-haematological malignancies (51%; 85/166) and hm (49%; 81/166) (figure 3). figure 2: frequency of different causes of pancytopenia among adult patients at tygerberg academic hospital, cape town, south africa, from january 2016 to december 2017. figure 3: pancytopenia cases at tygerberg academic hospital, cape town, south africa, from january 2016 to december 2017 for which chemoradiation therapy was the cause. (a) site of nhm, (b) haematological malignancy (hm) subtypes. the most common causes of pancytopenia among the remaining 507 (75%) cases were sepsis (18%; 122/673), hm (9%; 62/673), advanced hiv with no other identifiable cause (7%; 49/673) and megaloblastic anaemia (6%; 42/673). a cause for pancytopenia could not be established in 15% (98/673) of cases. haematological conditions were found to be common causes of pancytopenia. these included hm (9%; 62/673) and aplastic anaemia (3%; 20/673). six of the 20 patients with aplastic anaemia had a paroxysmal nocturnal haematuria clone. serum vitamin b12, folate and ferritin levels were measured in 280 of the 507 patients with non-iatrogenic pancytopenia. of these, 12% (33/280) had isolated folate deficiency and 3% (9/280) had vitamin b12 deficiency. infection contributed to a considerable proportion of pancytopenia cases. sepsis was the most common non-iatrogenic cause of pancytopenia. in 80% of sepsis cases (97/122), a blood culture was positive for an organism. gram-negative bacteria were more commonly cultured (61%; 74/122) compared to gram-positive bacteria (16%; 19/122). the remaining four cultures were positive for candida albicans. in 62% (76/122) of sepsis-associated pancytopenia cases, the pancytopenia resolved. the median time to resolution of pancytopenia was 2 days (interquartile range: 1–6). twenty-three (3%; 23/673) patients had positive mycobacterium tuberculosis cultures/genexpert®; the majority (19/23) of these were hiv-positive patients. nine (1%; 9/673) patients had malaria. description of pancytopenia in patients with hiv of the 507 non-iatrogenic pancytopenia cases, 41% (207/507) were hiv-positive, 47% (236/507) were hiv-negative, and the hiv status was unknown in 13% (64/507). the median cd4 count was 94 cells/µl (interquartile range: 33–202 cells/µl, n = 175 patients tested) and the median hiv viral load was 1096 copies/ml (interquartile range lower than detectable limit – 91 560 copies/ml, n = 113 patients tested). the viral load was undetectable in 14% (29/207) of the hiv-positive patients with pancytopenia, and advanced hiv was seen in 68% (141/207). the cd4 count was not performed in 27 hiv-positive patients with pancytopenia, thus the proportion of patients with advanced hiv may be underestimated. further investigation in patients with advanced hiv revealed additional contributing causes of pancytopenia, including sepsis (22%; 31/141), folate deficiency (13%; 18/141) and tuberculosis (10%; 14/141). moreover, advanced hiv was the only identifiable cause of pancytopenia in 24% (49/207) of the hiv-positive patients, accounting for 7% of all cases (49/673). the aetiology of pancytopenia differed between hiv-positive and hiv-negative patients (figure 4). haematological malignancies and aplastic anaemia were significantly more common in hiv-negative patients (p < 0.0001), while folate deficiency (p = 0.004), tuberculosis (p < 0.0001) and thrombotic microangiopathy (p = 0.011) were more common among hiv-positive patients. there was no significant difference in the proportion of sepsis cases between the hiv-positive and hiv-negative groups (p = 0.66). figure 4: proportions of various causes of pancytopenia among hiv-positive and hiv-negative patients at tygerberg academic hospital, cape town, south africa, from january 2016 to december 2017. bone marrow examination to determine pancytopenia aetiology bone marrow examination was performed to determine the cause of pancytopenia in 91 of the non-iatrogenic cases and this revealed the cause of pancytopenia in 52 cases (57%; 52/91). thirty-six of these had hm (40%; 36/91), nine had aplastic anaemia (10%; 9/91), and three had non-haematological malignancies (3%; 3/91). the most common hms were myelodysplastic syndrome (11%) and acute leukaemia (10%). notably, in four of the nine acute leukaemia cases, there were scanty or no blasts (< 1%) on the peripheral blood smear. in the remaining 39 bmes, no specific bone marrow pathology could be identified. causes of pancytopenia in different age groups the aetiology of pancytopenia also varied according to age (figure 5). while sepsis was the most common cause of pancytopenia among young (18–39 years old) and middle-aged (40–59 years old) patients, hm was the most common cause of pancytopenia in the oldest age group (60–89 years old). there was a significantly higher proportion of patients diagnosed with hm (p < 0.0001) and non-haematological malignancies (p = 0.012) among patients aged 60–89 years old. the increase in hm seen in the 60–89-year-old category was mainly due to an increase in the number of myelodysplastic syndrome cases. the number of cases of sepsis, megaloblastic anaemia and aplastic anaemia did not significantly differ between the age categories. figure 5: most common causes of non-treatment-related pancytopenia among adult patients at tygerberg academic hospital, cape town, south africa, from january 2016 to december 2017. (a) all patients (n = 507), (b) 18–39 years old (n = 250), (c) 40–59 years old (n = 172), (d) 60–89 years old (n = 85). discussion this study showed that chemoradiation therapy was the most common cause of pancytopenia in adult patients at a south african tertiary academic centre. the most common non-treatment-related causes of pancytopenia were sepsis, hm, advanced hiv, and megaloblastic anaemia. the aetiology of pancytopenia as found in our study is comparable to the findings of a previous south african tertiary institution study.38 their study showed chemoradiation therapy to be the most common cause of pancytopenia and revealed infection as another prominent cause.38 haematological malignancy, which was not a prominent cause of pancytopenia in their study, was the third most common cause of pancytopenia in this study. infection, megaloblastic anaemia and aplastic anaemia are common causes of pancytopenia in developing countries while hm is the most common cause in developed countries (table 1). in comparison to the aetiologies of pancytopenia described by studies across the world, our study shows features common to both developing and developed countries. this is similar to the findings of a study performed in mexico where the most common causes of pancytopenia were hm and megaloblastic anaemia.32 in south africa, there are considerable differences in access to health services between socio-economic groups, and the poor face many predisposing factors that are recognised as social determinants of ill health.49 hiv and other communicable diseases are concentrated among the lower socio-economic groups of south africa.50 this may explain why infections contributed considerably to the aetiology of pancytopenia in our study. table 1: most common causes of pancytopenia across different studies globally, 1976–2019. advanced hiv was found to be one of the common causes of pancytopenia in our study, similar to studies performed in zimbabwe, djibouti, india and the united states.12,32,34,35 in our study, 41% of patients were hiv-positive. the majority of these patients had advanced hiv, and in a quarter of these, advanced hiv was found to be the only identifiable cause of pancytopenia. additionally, our study showed that hiv status alters the aetiology of pancytopenia; folate deficiency, tuberculosis and thrombotic microangiopathy were significantly more common among hiv-positive patients, while aplastic anaemia and hm were significantly more common among hiv-negative patients. interestingly, despite the known risk of hiv-associated lymphomas in advanced hiv,51 we did not find an increase in hm-associated pancytopenia in hiv-positive patients. this may be because lymphomas do not typically cause pancytopenia even when there is bone marrow infiltration.52 haematological malignancies were a more common cause of pancytopenia in our study than in other studies performed in developing nations (table 1). the burden of cancer is highest in affluent regions and is thought to be associated with diet, lifestyle and environmental exposures. with increasing urbanisation and globalisation, developing nations are increasingly exposed to diet and lifestyle changes, including smoking, a sedentary lifestyle and obesity, which were previously mainly seen in developed societies.53 in addition, developing nations have an increased risk of viral infection, including infection with oncoviruses, which can lead to malignancy.53 expensive diagnostic approaches are also increasingly available in tertiary centres, allowing for easier diagnosis of malignancy, which may be missed in rural regions or countries with fewer resources. these factors are likely contributing to the changing landscape of pancytopenia aetiology in south africa. megaloblastic anaemia as a cause of pancytopenia was not as prevalent in our study as in studies performed in india3,4,5,7,8,9,10,11,13,15 and the rest of africa.34,35,36,37 this is likely because the south african government introduced legislation in 2003 for the mandatory folate fortification of the staple maize meal and wheat flour.57,58 nevertheless, folate deficiency was more common in hiv-positive patients than in hiv-negative patients in this study, likely reflecting poor folate absorption due to hiv-associated gastrointestinal disease. importantly, six of the 48 patients with megaloblastic anaemia in our study had an underlying pathology leading to depletion of folate reserve. this highlights the importance of follow-up in patients with haematinic deficiencies and the importance of further investigation if there is a poor response to the haematinic replacement or clinical suspicion of an underlying condition. the aetiology of pancytopenia also varied with age. there were significantly more malignancies, both haematological and non-haematological, in the age group 60–89 years. this is comparable to the findings of studies in turkey and iran.45,46 globally, malignancies are not a more common cause of pancytopenia in older patients. in studies conducted in india, megaloblastic anaemia was found to be the most common cause of pancytopenia, with no noticeably higher frequency of malignancy among older people.6,12,15 the substantial variation in the frequency of malignancy as a cause of pancytopenia in the elderly is most likely due to regional risk factors for megaloblastic anaemia, as well as variable availability of expensive diagnostic approaches such as flow cytometry, cytogenetics and next-generation sequencing for the diagnosis of malignancy. the findings of this study highlight that good clinical and laboratory correlation is required for prompt and cost-effective identification of the underlying cause of pancytopenia to guide management. chemoradiation is a predictable cause of pancytopenia; however, cell counts should also be monitored, and if the cell counts do not improve, investigation for vitamin b12/folate deficiency or marrow infiltration should be performed. sepsis was another easily identifiable cause of pancytopenia in our study. the median time to resolution of sepsis-induced pancytopenia was two days. therefore, when sepsis is treated empirically, cell counts should be monitored for recovery. should cell counts fail to recover, further investigation is required. vitamin b12 and folate deficiency are important to exclude in cases of pancytopenia. in cases where haematinic deficiency is confirmed based on serum levels, poor response to replacement therapy may indicate underlying marrow pathology requiring bme. knowledge of the vitamin b12 and folate serum levels are also important for the interpretation of bmes, especially if myelodysplastic syndrome is suspected. additionally, if there are features of myelodysplastic syndrome, acute leukaemia or bone marrow infiltration (such as unexplained leucoerythroblastic reaction) in peripheral blood or, importantly, if the pancytopenia is unexplained, a bme should be fast-tracked in the investigation of the pancytopenia. furthermore, in patients with hiv, vitamin b12 and folate deficiency should be excluded early, as folate deficiency was found to be common in the hiv patients in our study. moreover, in hiv-positive patients, there should be a thorough investigation for tuberculosis, and a bme is recommended to investigate marrow infiltration by opportunistic infections and malignancy. in older patients, there should be a high clinical suspicion of malignancy. ultimately, bme remains a valuable procedure for the investigation of unexplained pancytopenia. limitations one of the limitations of our study was that only retrospective information available on the lis was used to identify the cause of pancytopenia. as a result, the contribution of drugs and co-morbidities was unknown and a cause for pancytopenia could not be identified in 14% of patients. the world health organization defines advanced hiv as either cd4 count < 200/µl or clinical stage iii or stage iv disease. however, the clinical stage of hiv disease was not known in this study. thus the definition of advanced hiv was based only on cd4 count, and the patients with advanced hiv may be underestimated.48 clinicopathological correlation would also have improved the study. furthermore, it is noteworthy that our study was performed in a tertiary hospital, thus selection bias may have resulted in chemoradiation therapy being the most common cause of pancytopenia. socio-economic, geographical and ethnic factors are known to influence the aetiology of pancytopenia. it is noteworthy, however, that differences in study design also influence the described aetiology of pancytopenia reported by different studies. study designs differ in their definitions of pancytopenia and whether clinical or laboratory data or both were used. additionally, some studies only evaluate bme patients, while some include all patients with pancytopenia. some studies may also include children, and others may include patients that had chemotherapy or radiation therapy. these differences influence the top causes of pancytopenia between different studies. conclusion this study shows that the most common causes of new-onset pancytopenia in adults at a south african tertiary academic centre are chemoradiation therapy, sepsis, hm, advanced hiv and megaloblastic anaemia. these results demonstrate the need for the prompt recognition and treatment of sepsis and megaloblastic anaemia, early recognition of hiv and initiation of antiretroviral therapy, and a thorough investigation for malignancy. integration of clinical, laboratory and radiological findings to guide investigation of pancytopenia allows for prompt diagnosis, while also elucidating where bme should be expedited in the investigation of pancytopenia. acknowledgements we would like to acknowledge mr wessel kleinhans for his assistance with data extraction from the laboratory information system. competing interests the authors declare that the 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health nutr. 2008;11(3):307–313. https://doi.org/10.1017/s136898000700033x abstract introduction methods results discussion acknowledgements references about the author(s) adesola olalekan department of medical laboratory science, faculty of basic medical sciences, college of medicine, university of lagos, lagos, nigeria bamidele iwalokun department of molecular biology and biotechnology, nigerian institute of medical research, lagos, nigeria marcellinus aguwa department of clinical psychology, federal neuro-psychiatric hospital, lagos, nigeria abosede adegbite department of medicine, federal neuro-psychiatric hospital, lagos, nigeria barakat bello department of medical laboratory science, faculty of basic medical sciences, college of medicine, university of lagos, lagos, nigeria sunday adesola infectious diseases hospital, lagos, nigeria olusola ojurongbe center for emerging and re-emerging infectious diseases, ladoke akintola university of technology, oyo, nigeria olayinka ogunleye department of pharmacology, therapeutics and toxicology, college of medicine, lagos state university, lagos, nigeria taiwo ojurongbe department of statistics, osun state university osogbo, osun, nigeria citation olalekan a, iwalokun b, aguwa m, et al. infection prevention and control practices, policy adherence and knowledge of healthcare workers at covid-19 treatment centres in nigeria. afr j lab med. 2025;14(1), a2878. https://doi.org/10.4102/ajlm.v14i1.2878 original research infection prevention and control practices, policy adherence and knowledge of healthcare workers at covid-19 treatment centres in nigeria adesola olalekan, bamidele iwalokun, marcellinus aguwa, abosede adegbite, barakat bello, sunday adesola, olusola ojurongbe, olayinka ogunleye, taiwo ojurongbe received: 28 may 2025; accepted: 12 aug. 2025; published: 12 dec. 2025 copyright: © 2025. the authors. licensee: aosis. this work is licensed under the creative commons attribution 4.0 international (cc by 4.0) license (https://creativecommons.org/licenses/by/4.0/). abstract background: infection prevention and control (ipc) practices are crucial for protecting patients and healthcare workers (hcws), especially during the coronavirus disease 2019 (covid-19) pandemic. this crisis has underscored the importance of ipc strategies in understanding health system readiness and strengthening preparedness for future pandemics. objective: this study investigated healthcare personnel’s ipc knowledge, adherence to safety policies, and implementation of ipc procedures in covid-19 treatment centres across nigeria. methods: a cross-sectional, multicentre study was conducted among 113 respondents, that is, 57 hcws and 56 volunteers, from 23 june 2020 to 15 march 2021. an electronic questionnaire adapted from validated instruments was used. results: out of 113 respondents, 69 (61%) demonstrated good ipc practices, with high adherence (n = 105, 92.9%) to face mask usage and hand hygiene. only 50 (44.2%) reported receiving basic training on ipc. although personal protective equipment (ppe) was available, 25% did not consistently wear full ppe when attending to covid-19 patients. most hcws (105; 93%) opposed testing patients without consent, and 100 (88.5%) affirmed the availability of standardised ipc protocols. no significant association was observed between age, gender, years of experience, and ipc compliance (p-values: 0.097, 0.287, and 0.699). interestingly, 33 (29.2%) hcws with less than 10 years of experience exhibited better ipc practices. facility policies such as confidentiality and non-discrimination were mostly upheld, with 90 (79.6%) participants agreeing that discriminatory practices should have consequences. conclusion: while face mask use and hand hygiene compliance were high, gaps remained in ipc training and consistent use of full ppe. strengthening training, ipc knowledge, policy standardisation, and resource equity is important for stronger ipc compliance during health emergencies. what this study adds: the study identified key factors supporting future pandemic preparedness by examining the control and preventive strategies implemented at various ctcs in nigeria. it also emphasised the need for standardised policies, which are essential for building resilient healthcare systems during public health crises. keywords: infection prevention and control; healthcare workers; coronavirus disease 2019; facility policies; pandemic preparedness; knowledge assessment. introduction infection prevention and control (ipc) is a holistic approach aimed at curbing and halting the transmission of infectious agents and the diseases they cause among healthcare workers (hcws), patients, their relatives, and the broader community. when ipc measures are not implemented properly, they can lead to a decline in the quality of healthcare, increased harm, and the spread of diseases. healthcare workers and other staff in health institutions are key stakeholders in the implementation of ipc activities, such as hand hygiene, use of personal protective equipment (ppe), injection safety, and adherence to world health organization (who) standard guidelines,1 especially during public health emergencies such as the covid-19 pandemic. infection prevention and control practices were compromised during the covid-19 pandemic because of a shortage of ipc experts and volunteers to support personal hygiene and facility-level infection prevention efforts.2 the united states centers for disease control and prevention recommended strict ipc guidelines for covid-19 to help limit its transmission. these guidelines included the use of visible signage (e.g. signs and posters) at entrances and other strategic locations such as waiting areas, elevators and cafeterias. the united states centers for disease control and prevention also emphasised using respiratory protection, such as respirators, well-fitting face masks, or cloth masks to cover the mouth and nose, thereby reducing the spread of respiratory secretions during breathing, sneezing, talking and coughing. the universal use of ppe by healthcare personnel was encouraged strongly through distancing and avoiding crowded areas. in addition, the united states centers for disease control and prevention recommended optimising engineering controls to reduce exposure risks. for example, setting up physical barriers in reception and triage areas and establishing designated routes to direct symptomatic patients through waiting rooms and triage areas. dedicated medical equipment was advised to minimise cross-contamination when managing patients with confirmed or suspected severe acute respiratory syndrome coronavirus 2 infection.3 one of the improvisations of the covid-19 era in africa was the provision of veronica buckets in different locations in the covid-19 treatment centres (ctcs). this ensured access to running water for hand washing, aligning with hand hygiene principles in ipc protocols.4 furthermore, the who has underscored consistently the necessity for well-designed and effectively executed ipc programmes to curb the transmission of the virus both domestically and internationally.5 infection prevention and control encompasses a set of practices and procedures that must be implemented in healthcare settings to prevent the transmission of infections between patients and hcws.6 effective ipc also maintains a safe hospital environment and safeguards against community transmission of infectious illnesses during outbreaks.7 successful pandemic control, such as during covid-19, requires ipc strategies guided by well-established policies.8 however, poor compliance with ipc measures in lowand middle-income countries, especially in africa and asia, has been a significant challenge in managing the covid-19 pandemic.9,10,11,12 during the covid-19 pandemic, the implementation of ipc measures varied between lowand middle-income countries and high-income countries. differences were observed in compliance strategies, available infrastructure, and the level of facility engagement. nigeria, one of africa’s most populous countries, was globally recognised for its swift and effective containment of the ebola outbreak in 2014.13 however, its capacity and strategy to control the covid-19 pandemic through effective ipc implementation in treatment centres remains insufficiently documented. a key factor in strengthening future pandemic preparedness is the understanding of the infection control and prevention strategies that were employed in various ctcs in nigeria. this includes evaluating adherence to ipc policies and assessing the awareness of covid-19-related protocols among hcws and volunteers. such data can serve as a foundation for developing more robust preparedness plans for future emerging infectious diseases. therefore, this study aims to evaluate the infection prevention strategies used by hcws and volunteers in ctcs in nigeria, along with their knowledge of disease control, confidentiality practices and adherence to facility policies. methods ethical considerations ethical approval for the survey was obtained from the hospital research ethics committee of the college of medicine, university of lagos (ethics approval number: cmul/hrec/05/20/738). the purpose of the research was outlined clearly in the electronic questionnaire, which included a statement of consent for participation. no names or other identification information was collected, and a statement was provided on the respondents’ rights and privacy. participants were only able to complete the questionnaire after indicating their agreement to the ethical and consent declarations study design and population this cross-sectional, questionnaire-based multicentre study involved 113 workers, comprising 57 hcws and 56 volunteers from various ctcs across nigeria, with the majority based in lagos (82; 75%), the epicentre of the outbreak in the country. the study was conducted between 23 june 2020 and 15 march 2021. other states represented included ogun (7; 6.2%), oyo and the federal capital territory abuja (4; 3.5% each), bayelsa (3; 2.6%), katsina, kebbi, osun, and rivers (2; 1.8% each), and abia, borno, ebonyi, kwara, and zamfara (1; 0.9% each) (table 1). a variety of healthcare professionals and support personnel working in ctcs participated in the study, including nurses (31; 27.43%), health attendants (19; 16.81%), medical laboratory scientists (16; 14.16%), medical doctors (14; 12.39%), and pharmacists (3; 2.65%), along with others (30; 26.56%) (detailed in figure 1). all participants provided consent and completed the electronic questionnaire-based survey, which was disseminated via online platforms, including email and whatsapp group chats. nurses and ward attendants were involved in patient care; medical laboratory scientists performed patient investigations, including nasal swabbing for severe acute respiratory syndrome coronavirus 2 testing and confirmation; medical doctors also provided patient care; pharmacists were responsible for dispensing drugs; and other volunteers contributed their professional skills in various roles at the ctcs. in this study, a volunteer is defined as a respondent who is not employed full-time in a covid-19-related hospital, but who volunteered at a ctc (table 2). figure 1: distribution of healthcare workers and volunteers who participated in the study, nigeria, 23 june 2020 to 15 march 2021. table 1: frequency of the participants per state in nigeria, 23 june 2020 to 15 march 2021. table 2: socio-demographic analysis of the respondents, nigeria, 23 june 2020 to 15 march 2021. questionnaire the electronic questionnaire used to collect information from respondents was a modified version of the validated instrument for measuring hiv-related stigma and discrimination among health facility staff. this comprehensive questionnaire, originally developed by the united states agency for international development, the united states president’s emergency plan for aids relief, and the health policy project, was adapted for this study.14 in addition, excerpts from the who guidelines on ipc strategies for covid-195 were incorporated to assess that covid-19 ipc processes were carried out across various facilities. the self-administered electronic survey comprised two sections. the first section of the questionnaire collected demographic information, including gender, age (as of the respondent’s last birthday), profession, location of covid-19 service delivery, and years of experience. the second section evaluated respondents’ knowledge, facility policies, and infection control practices using a three-point likert scale with response options: yes, no, or not applicable. data analysis the database was automatically generated in an excel spreadsheet, then collated and exported to statistical package for the social science version 27 (ibm corp., armonk, new york, united states) for descriptive and comparative analyses. the chi-square test was employed to compare different categories of respondents involved in the study and to analyse prevention and control activities in the ctcs. those who had a score below the mean for the ipc practice variables (avoiding physical contact, wearing of face masks, wearing of gloves during all aspects of patient care, implementing any special ipc measures for patients with covid-19 that are not used for other patients, and wearing full ppe), when pooled together, were classified as having poor or inadequate use of protective measures; and those who scored greater than or equal to the mean score were classified as having good or adequate practices. results demographics and general characteristics of the respondents the total number of participants was 113, of whom 79 (69.9%) were between 31 and 50 years of age, and 51 (45.1%) had less than 10 years of experience in infectious disease-related work. approximately half (56; 49.6%) of the respondents who worked in the ctcs at the time of the study were volunteers (table 2). there was no significant association between the socio-demographic variables and the ipc practices by all the respondents (table 3). the distribution of the category of hcws and volunteers involved in this study is indicated in figure 1. cross-tabulation between the professional category of respondents and the category of training indicated that some volunteers had prior training in ipc, as they were healthcare professionals who volunteered to work in the ctcs during the pandemic. these individuals were not originally part of the full-time staff of ctc (table 4). table 3: association between socio-demographic and infection control practice among the respondents, nigeria, 23 june 2020 to 15 march 2021. table 4: classification of training by profession, nigeria, 23 june 2020 to 15 march 2021. knowledge of infection prevention and control strategies most (44.2%) of the respondents had received basic training on patient confidentiality, ipc of covid-19, privacy, patient informed consent, discrimination and key population stigma. the other category of respondents had received training in ipc for covid-19 (33.6%), as shown in table 4. table 5 highlights the different training categories to which respondents were exposed. key population stigma and discrimination, particularly in the context of covid-19 care, refer to the negative attitudes and beliefs directed towards individuals with covid-19, and discrimination as denial of care or unequal treatment. knowledge of this is essential to promote equity, trust and adherence to ethical standards in patient management.14 table 5: previous training by the participants, nigeria, 23 june 2020 to 15 march 2021. infection control practices by the healthcare workers in the covid-19 treatment centres infection control practices that most hcws could apply vary between good practices (61%) and those ipc methods that were poorly executed (39%) (figure 2). the application of alcohol-based hand sanitisers, the use of face masks, and hand washing with soap and water were the most common practices among the hcws in the ctcs (table 6). most hcws did not wear complete ppe during the care and management of patients at the centres. of the hcws in these centres, 88 (77.9%) use special infection-control measures for patients infected with covid-19, which they normally do not use with other patients not infected with covid-19 (table 7). in addition, 75% of healthcare workers reported wearing full ppe while working with covid-19-confirmed patients at the treatment centres. figure 2: infection control practices among the respondents, nigeria, 23 june 2020 to 15 march 2021. table 6: distribution of infection prevention and control practices (n = 113), nigeria, 23 june 2020 to 15 march 2021. table 7: infection prevention control practices at the covid-19 treatment centres, nigeria, 23 june 2020 to 15 march 2021. health policy and regulations in the facility basic policies and regulations such as informed consent, discrimination and confidentiality, supply of ipc materials in the facility, and standard hospital protocol in handling patients were assessed among hcws in these treatment centres. most of the hcws (93%) believe that testing individuals without their knowledge (informed consent) is not advisable. in addition, 81.4% believed that there are sufficient supplies of ipc materials and ppe in the facilities, while 88.5% feel that the standard operating procedures guiding their daily operations at the centres are accessible (table 8). interestingly, 15.9% of the hcws in this study are indifferent to issues of patient discrimination and disclosure of their patients’ status (confidentiality) while working at the facility (table 8). table 8: health policy and regulations in the facility, nigeria, 23 june 2020 to 15 march 2021. discussion our study observed that 44.2% of respondents had received basic training on ipc related to covid-19, including hand hygiene, use of ppe, patient informed consent, privacy, confidentiality, and issues surrounding key population stigma and discrimination. the most common ipc practices among hcws at the treatment centres were the use of face masks, handwashing with soap and water, and the application of alcohol-based hand sanitisers. in addition, 75% of respondents reported wearing full ppe while attending to patients confirmed to have covid-19. notably, 93% of hcws believed that testing individuals without their knowledge or consent was inappropriate. this study highlighted the knowledge and practices of ipc among hcws and volunteers who worked in ctcs in nigeria during the 2020–2022 pandemic, primarily in lagos state (table 1). the study also examined their adherence to established policies in these facilities. the who emphasises that ipc is vital for limiting disease transmission and safeguarding both hcws and the wider community. the attitudes, knowledge and practices of hcws regarding ipc play a crucial role in ensuring patient safety and improving treatment outcomes. in this study, years of work experience, age, and gender are not associated with the infection control practices among the respondents (p > 0.05). of the 113 participants, 61 (54%) were male and 52 (46%) were female. of the women, 40 (57.8%) demonstrated good ipc, which is lower than the 70.4% reported among 301 female participants in a similar study conducted in kaduna, nigeria.15 these differences may be due to variations in training, occupational roles, and hygiene practices. in a study from ethiopia, male hcws were three times more likely to demonstrate good ipc,16 a trend also seen in china, and attributed to their lighter workloads and differing occupational roles.17 our study observed that hcws with fewer years of work experience (≤ 10 years) demonstrated better ipc in 33 out of 69 (47.8%) cases, which may be attributed to recent updated training methods, adherence to protocols owing to fear of consequences, technological adaptability, supervisory pressure and heightened awareness. in contrast, a study conducted in ethiopia in 2021 found that hcws with longer years of experience had good ipc practices compared to those with less than 5 years of experience.16 similarly, in pakistan, logistic regression analysis showed that experienced (> 5 years) hcws were more likely to adhere to precautionary practices, probably because of their skills and experience in managing with public health emergencies.18 in our study, healthcare workers and volunteers aged between 31 and 50 demonstrated good ipc practices in 44 (38.9%) cases. this contrasts with findings from south africa, where younger hcws achieved higher knowledge scores than their older counterparts, possibly because of their broader use of digital information sources.19 however, another study suggests that older hcws may perform better because of their accumulated experience.18 only 50 (44.2%) of our study participants had basic knowledge of ipc, confidentiality, and consent, in contrast with studies in ethiopia (88.2%) and afghanistan (85%), which reported a higher level of ipc knowledge.20,21 similarly, a study in nepal reported 76% adequate ipc knowledge,22 while another nigerian study reported 56%.23 these discrepancies may be because of differences in sampling methods or to the scope of the questionnaire items. the relatively low knowledge level in our study may also reflect the small proportion (33%) of respondents who received covid-19-specific ipc training (table 4). comprehensive training and ongoing education should be prioritised. our study showed that 61% of hcws practised good ipc, including avoiding physical contact, wearing of face masks and gloves during all aspects of patient care, implementing special ipc measures for covid-19 patients, and wearing full ppe for covid-19, higher than the 41% reported in another nigerian study15 but lower than compliance rates observed in india, uganda, and ghana.24,25,26 the most common ipc practices included wearing face masks (92.9%), followed by wearing gloves during all aspects of patient care (90.3%) (table 7). these findings align with a ugandan study reporting 93% mask use.26 however, in ghana, ppe use was reported to be lower.27 in additional, some other studies highlighted poor adherence to mask and glove use, often because of discomfort and ppe shortages.28,29,30,31 most participants in our study reported that ipc supplies were adequately available, reducing their perceived risk of contracting covid-19. in contrast, a brazilian study noted significant ppe shortages.32 adequate ppe is essential to prevent hcw infections and reduce community transmission. other studies similarly reported good supply availability during covid-19 management.33,34 a large majority (93%) of our participants affirmed the availability of standard operating procedures in their facilities, which enhances both patient care and infection prevention. however, iyal and colleagues attributed low ipc compliance in their study to the lack of covid-19-specific guidelines.15 meanwhile, a study in ghana reported low protocol adherence despite the availability of guidelines.27 therefore, making protocols readily accessible and promoting adherence are critical to effective ipc in treatment centres. interestingly, 93% of our participants disagreed with testing patients for covid-19 without their consent. this finding, however, does not align with who guidance, which allows for exceptions during public health emergencies under specific legal frameworks.35 a united states study revealed public support for clear communication and consent, although mandatory testing was accepted in high-risk settings like schools or hospitals.36 the study also assessed perceptions of discrimination, with 79.6% of respondents indicating that there would be consequences for discriminating against patients with covid-19. this probably reflects awareness of professional ethical standards,37 and underscores the role of ethical compliance in building trust and improving patient outcomes. respect for confidentiality and consent remains fundamental to effective healthcare delivery. limitations of the study the small number of participants and geographical distribution limit the generalisation of the study. in addition, the study is based on self-reported data and information provided by the participants, which could not be verified. secondly, the potential influence of social desirability bias might have affected the responses on ipc compliance. understanding the context of the covid-19 response, participants may have felt compelled to indicate higher adherence to ipc protocols than what was done. this bias may have led to an exaggerated view of compliance rates. future studies must validate self-reported data using other methods to ascertain accurately the ipc practices carried out by the respondents. however, despite these limitations, this constraint did not undermine its overall significance and the findings can inform future research and policy development for pandemic preparedness. lagos state was the epicentre of the covid-19 pandemic in nigeria, which accounts for the higher number of respondents from treatment centres in the state. future research should include representation from all six geopolitical zones to generate findings that can be generalised and inform national policy. conclusion generally, our study highlights the infection control practices carried out in the ctcs. practices such as the use of alcohol-based hand rub, wearing of medical masks, and handwashing with soap and water were the predominant infection prevention measures implemented at the centres. in addition, hcws demonstrated notable knowledge of covid-19 and adhered to consent and confidentiality protocols in several treatment facilities across nigeria during the pandemic. these findings support improved preparedness and response strategies for future emerging or re-emerging infectious diseases in africa. continuous training and improved knowledge of ipc, particularly in the proper use of ppe, transmission modes, encouraging adherence, enforcement of sanctions for non-compliance and containment strategies, are essential for strengthening health systems and for protecting hcws. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article credit authorship contribution adesola olalekan: conceptualisation, methodology, investigation, writing – original draft, project administration, writing – review & editing, funding acquisition. bamidele iwalokun: formal analysis, investigation, writing – original draft, writing – review & editing. marcellinus aguwa: methodology, investigation, writing – original draft, writing – review & editing. abosede adegbite: writing – original draft, writing – review & editing. barakat bello: writing – original draft, validation, writing – review & editing. sunday adesola: methodology, writing – original draft, writing – review & editing. olusola ojurongbe: methodology, writing – original draft, writing – review & editing. olayinka ogunleye: methodology, writing – original draft, writing – review & editing. taiwo ojurongbe: methodology, formal analysis, writing – original draft, writing – review & editing, supervision. sources of support adesola olalekan received internet services and stationery funding from the da boyz covid-19 research support from the college of medicine, university of lagos. data availability the authors confirm that the data supporting the findings of this study are available in the article and its references. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. the article does not necessarily reflect the official policy or position of any 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activities in high schools utah, november 2020–march 2021. mmwr morb mortal wkly rep. 2021;70:785–791. https://dx.doi.org/10.15585/mmwr.mm7021e2 centers for disease control and prevention. national center for immunization and respiratory diseases (u.s.). division of viral diseases. interim guidance for antigen testing for sars-cov-2. 2022 [cited 2025 feb 02]. https://stacks.cdc.gov/view/cdc/113645 abstract background description of the intervention lessons learnt recommendations acknowledgements references about the author(s) fausta s. mosha department of communicable diseases, world health organization (eastern mediterranean regional office), cairo, egypt rana hajjeh director programme management, world health organization (eastern mediterranean regional office), cairo, egypt rachel ochola who health emergencies (whe) programme, (eastern mediterranean regional office), cairo, egypt amany ghoniem department of communicable diseases, world health organization (eastern mediterranean regional office), cairo, egypt yvan j.-f. hutin department of communicable diseases, world health organization (eastern mediterranean regional office), cairo, egypt citation mosha fs, hajjeh r, ochola r, ghoniem a, hutin yj-f. implementation of the laboratory strategic framework to strengthen health laboratory services in eastern mediterranean region, 2016–2023. afr j lab med. 2025;14(1), a2611. https://doi.org/10.4102/ajlm.v14i1.2611 lessons from the field implementation of the laboratory strategic framework to strengthen health laboratory services in eastern mediterranean region, 2016–2023 fausta s. mosha, rana hajjeh, rachel ochola, amany ghoniem, yvan j.-f. hutin received: 13 sept. 2024; accepted: 06 dec. 2024; published: 30 apr. 2025 copyright: © 2025. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: laboratories and diagnostics services are critical to universal health coverage and public health response. we assessed the extent of the implementation and functionality of the 2016–2023 eastern mediterranean region (emr) laboratory strategic framework. intervention: documents and reports from world health organization country offices were examined between september 2022 and november 2022, supplemented by stakeholder-provided documents, to enhance data collection and reporting across the framework’s five goals. an intervention using a performance evaluation scorecard assessed the progress of emr member states (mss) towards strengthening health laboratory services, with findings validated during the december 2022 regional public health laboratory directors’ meeting in egypt. lessons learnt: we analysed results from 21 of 22 mss. three (14%) mss, all high income, had the capacity to implement all indicators, while only one of five low-income (20%) mss could not demonstrate any capacity across all five goals evaluated. irrespective of income category, the least implemented domains were: (1) availability of either or both fully implemented laboratory policy, and (2) a fully implemented integrated national laboratory strategic plan, both of which were implemented in only 50% of mss. recommendations: addressing the identified gaps requires concerted efforts, collaboration, and sustained investment to ensure the delivery of high-quality laboratory services and advance public health outcomes across the emr. implementation of laboratory strategies should be coordinated through the specific laboratory department or unit at the ministry of health level, above the central public health laboratories and with the support of a national laboratory technical working group. what this study adds: this study revealed substantial gaps in implementing laboratory policies and strategic plans in the emr, with full implementation achieved by only 50% of mss. it underscores the necessity for coordinated efforts and sustained investment to enhance laboratory services and promote effective laboratory practices in emr. keywords: laboratory; strategic framework; diagnostic services; health coverage; public health response. background among major global health priorities, three, namely universal health coverage, antimicrobial resistance, and the global health security agenda, require better access to quality laboratory services.1 laboratories establish diagnosis, guide therapy, and monitor disease progression and disease surveillance by offering timely and accurate information.2,3,4 laboratories provide early diagnosis in cases of public health signals, thereby ensuring health security and national development, and fulfilling international obligations, such as those outlined in the international health regulations of 2005.5 in addition, laboratories contribute to sustainable core capacities and the development of programmes for diseases such as hiv, tuberculosis, and malaria.6 there were various initiatives to improve diagnostics in the region, including for 2005–2009,7 and for 2010–2011.8 building on previous diagnostic initiatives (2005–2011), the eastern mediterranean region (emr) focused on improving vaccination coverage in countries with low third dose of the diphtheria, pertussis and tetanus vaccine and district coverage. this included capacity building for healthcare workers and strengthening of immunisation systems. efforts were also focused on enhanced surveillance for emerging infections and support of high-risk populations. in public health emergencies, technical assistance was provided to meet core epidemiological and laboratory capacities, particularly in areas with high expatriate movement, ensuring timely response and improved disease monitoring. in 2018, the world health organization (who) formulated the essential diagnostic list.9 however, the pace of advancement has been insufficient. high-income countries have easily adapted their well-organised routine laboratory systems and services, but these are often neglected in intermediate and low-income settings,10,11,12 with gaps remaining. comprehensive national laboratory strategic plans and policies are needed to enhance the laboratory systems, and this is most important in lowand middle-income countries because of the limited resources.13 these plans should consider legal and regulatory frameworks, administrative and technical management structure, and effective human resources and retention strategies. implementation of laboratory quality management systems and the establishment of monitoring and evaluation systems are also required. prioritisation of procurement and maintenance of equipment, along with infrastructure enhancement, ensure sustainable improvement in national laboratory systems.14 finally, effective leadership, commitment, and coordinated efforts from host governments while fostering public–private partnerships can increase access to quality laboratory services.15,16,17 in october 2016, the 63rd session of the regional committee for the emr of the who endorsed a strategic framework for strengthening health laboratory services for 2016–2020 through resolution em/rc63/r.4.18,19 the aim was to enhance sustainable national health laboratory systems to improve clinical and public health services, focusing on epidemic-prone diseases, health security, and emergency preparedness. the framework established six strategic goals: strengthening leadership and governance, improving organisation and quality management, ensuring sustainable and competent human resources, ensuring safe and secure laboratory environments, promoting effective laboratory networking, and enhancing coordination and use of laboratory services. the coronavirus disease 2019 (covid-19) pandemic profoundly disrupted public health systems, but had mixed effects in terms of laboratory systems strengthening. on one hand, there was unprecedented leapfrogging with concurrent setup of new capacity. in 2020, a surge in polymerase chain reaction testing capacity occurred in response to the urgent need for expanded diagnostic capabilities in the emr. subsequently, in 2021, the emergence of viral variants spurred a substantial increase in genomic sequencing capacity to effectively monitor and characterise genetic variations of severe acute respiratory syndrome coronavirus 2.20 however, this new capacity failed to include a system to manage the diagnosis and surveillance of other communicable diseases, including antimicrobial resistance. in 2020, the emr member states (mss) temporarily extended the resolution to 2023 to promote a well-defined direction, and to set standards and decision-making on resource (capital and human) allocation. however, the eastern mediterranean regional office needed a re-assessment before re-designing a new framework. we needed to leverage lessons learnt since 2016, while capitalising on the progress achieved during the covid-19 response. this article describes overall progress towards the implementation of the emr strategic framework for strengthening health laboratory services, achievements, challenges, opportunities, and new priorities as the framework resolution came to an end in 2023. description of the intervention ethical considerations ethics committee approval was not required for this research. this research involved no human or animal subjects. review of documents we reviewed available grey literature focused on health system planning, performance and implementation, including documented health policy reports, planning dashboards, performance reports, country implementation plans, periodical reports, and other eligible documentation from the period 2016 to 2022. scorecards data collected the who eastern mediterranean regional office developed a performance evaluation indicator-based matrix (or scorecard) to assess the progress made by mss towards implementation of the regional framework for strengthening health laboratory services. the scorecard focused on the five key goals of the framework (2016 to 2023). we developed specific indicators for the five goals on leadership, legislation and governance, including tiered and integrated laboratory networks (10 indicators). the other indicators were on human resources for laboratory service delivery (8 indicators), and laboratory biosafety and biosecurity (7 indicators). we also developed indicators for the implementation of laboratory quality systems (6 indicators). as the outcome of all five goals will have a direct effect on the rational and evidence-based use of laboratory services, we did not develop indicators for the sixth goal on promotion of rational and evidence-based use of laboratory services. the scores ranged from 1 to 4 (no capacity, limited capacity, developed capacity, demonstrated capacity). initially, we looked at a high-level overview of the extent of implementation of the priority indicators. data collection procedure we sent the scorecards to all 22 who country offices in eastern mediterranean regional office in september 2022, along with instructions on how to fill in the scorecard, with a requested turn-around time of 6 weeks. we also listed the number of documents to be submitted as evidence of the mentioned capacity. the who country offices shared the information with the national reference laboratory management, who filled in the scorecard with their quality committee. the director of the national reference laboratory shared the filled-in scorecard together with the attached appendices as evidence of the capacity to the public health laboratory unit in the who emr. data analysis and validation we extracted a selection of indicators from each strategic area to plot graphs. the who emr held three laboratory manager’s meetings to validate the results and to plan for the next phase of the strategy to sustainably strengthen the public health laboratory services in the region. the first meeting, held on 05 december 2022 – 07 december 2022,21 involved directors of central public health laboratories and partners. member states reviewed progress and challenges on implementation of the regional framework for strengthening health laboratory services as recorded in the scorecard by the mss. the second meeting (06 march 2023 – 08 march 2023)22 was with technical officers at the central public health laboratories and regional laboratory partners, to deep dive and to agree on the new priorities for the next framework and alternative modalities to strengthen laboratory governance and implementation of laboratory quality management systems. the third meeting (14 august 2023 – 16 august 2023)23 was with the policymakers at the ministries of health and laboratory regional partners to discuss and deliberate on key policy shifts to attain sustainable delivery of quality laboratory services in the emr. the deliberation from this meeting provided information to shape the new who emr strategy to strengthen laboratory services. lessons learnt participation in the assessment we analysed results from 21 of the 22 emr mss, representing a range of low (five), lower-middle (seven), upper-middle (three) and high-income (six) economies with varying geographical size and development status. the missing data were from an upper-middle-income country that did not respond before the given deadline. overall scorecard laboratory system strengthening varied by ms and by defined goals. three (14%) mss, all high-income, had some capacity to implement all indicators, while only one low-income (5%) ms could not demonstrate any capacity across all indicators of the five goals evaluated. specifically, four (80%) mss in the low-income group, five (71%) lower-middle income, one (33%) upper-middle income, and one (17%) in the high-income group still had no capacity to implement 19% (6/31) or more indicators. none of the mss in both lowand lower-middle income brackets had implemented any national staffing plan for their respective networks based on workload forecasting. implementation of the national laboratory policy and national laboratory strategic plan was least implemented, irrespective of the ms’s income group. further challenges across the region included underfunding, insufficiently trained laboratory personnel, outdated and poorly maintained infrastructure and equipment, shortages of essential reagents and consumables, inadequate bio-risk management, and limited implementation of quality assurance and quality control measures, among other indicators. leadership and governance the scorecard indicated that only three mss (14%) have integrated all types of laboratories into a laboratory network, while four mss (19%) reportedly have a fully implemented laboratory policy in place. it was further noted that five mss (24%) have successfully implemented an integrated national laboratory strategic plan. meanwhile, seven mss (33%) were reported to have a dedicated national laboratory budget that fully addresses intersectoral functions with defined operational plans. additionally, 10 mss (48%) were identified as having a designated national reference laboratory responsible for coordinating public health functions across the national laboratory network. finally, 13 mss (62%) were reported to have dedicated laboratory budgets at all levels of their human health network (figure 1). figure 1: full implementation of selected leadership and governance indicators, 2022. effective, tiered, and integrated laboratory referral networks and enhanced coordination six mss (29%) had a well-established laboratory coordination mechanism at their ministry of health, including other multiple ministries and the private sector. ten mss (48%) had laboratory networks for all functions with clearly defined tier-specific roles, but only three (14%) integrated all types of laboratories into their networks. human resources for laboratory service delivery six mss (29%) had demonstrated capacity with regard to human resources for laboratory service delivery. additionally, seven (33%) mss each demonstrated capacity for quality and safety management through various topics in pre-service laboratory training curricula, had pre-service and in-service training programmes for laboratory management, as well as competency-based training curricula in line with national standards. an additional four (19%) had licensed laboratory workers. licensure was based on education, training and competency assessment, and a national staffing plan for the laboratory network based on workload forecasting (figure 2). figure 2: number of countries fully implemented selected human resources for laboratory indicators, 2022. laboratory biosafety and biosecurity eight mss (38%) had an up-to-date laboratory biosafety manual in all facilities, while 12 (57%) mss had all the basic safety equipment available to all laboratory workers and at all levels. furthermore, six (29%) mss had appointed safety officers in both public and private sector laboratories, while seven (33%) mss had certified entities regularly maintaining biosafety cabinets across all relevant tiers. none of the mss, however, had a regulated system of biobanking implemented at any level of their laboratory systems. additionally, eight (38%) mss had established waste management policies in accordance with level-specific biosafety and biosecurity requirements. finally, all laboratories in nine (43%) of the mss had access to incinerators that complied with national standards (figure 3). figure 3: the extent to which selected safety indicators from 2022 have been fully implemented in countries. quality of laboratory services there were variations on the level of implementation of the laboratory quality management systems (figure 4). eleven (52%) mss had fully implemented standardised procedures for internal quality control across their entire network for all tests carried out. five (24%) mss had established an external quality assessment programme for priority diseases across all tiers, facilitating feedback on results and implementing further actions for improvement. ten mss (48%) had reference laboratories participating in an international external quality assessment programme, while seven (33%) mss had filled laboratory quality officer positions in all public sector laboratories. four (19%) mss had implemented quality management activities in all laboratories, as well as implementing mandatory certification and accreditation standards for their specific laboratory settings. figure 4: number of countries that have effectively implemented selected quality systems indicators, 2022. recommendations results of this review point to the multifaceted landscape of laboratory system strengthening efforts and capacity levels across mss in the emr towards defined goals. few mss exhibited robust capacity on the implementation of the framework. however, we identified limited laboratory capacity in some mss that represents a barrier to implementation and sustainable diagnostic services. the gaps persisted, particularly concerning the implementation of national strategic planning frameworks. leadership and governance structures were heterogeneous among mss, pointing to variations in investment levels in laboratory budgets and stages of policy implementation. despite the availability of dedicated laboratory budgets in over 50% of the mss, and designated national reference laboratories, 76% (16) mss had yet to fully actualise developed policies and strategic plans. to realise the full implementation of related indicators that are still lagging, these still need to trickle down to all levels of leadership and governance. these challenges may be exacerbated by the lack of national-level monitoring of laboratory service performance. furthermore, laboratories often do not receive sufficient priority and recognition within national health systems. organisational bottlenecks, including insufficient leadership and governance, can influence the health system.24,25 in the absence of national laboratory strategies, it is difficult to provide comprehensive national laboratory services. the importance of laboratories was appreciated during the covid-19 pandemic, which exemplified the need for timely and quality laboratory services. in response to covid-19’s urgent laboratory needs, investments by governments and partners have strengthened laboratory services and systems in many countries. however, these have often been focused, mainly intended to address covid-19.26 to strengthen and sustain laboratory services, laboratory services must be integrated at various levels of the laboratory network. the evidence-based integrated and tiered laboratory network would form the basis for improved laboratory services.1 several mss showcased well-established coordination mechanisms and tiered networks. however, there are still existing challenges with integrating laboratory services. the implementation of a network approach has provided greater cost savings, efficiencies, increased coverage, and increased access,27 and hence improved network functionality and coordination mechanisms. a comprehensive array of human resources for health directed towards the promotion or improvement of human health is needed to achieve universal health coverage by 2030.28 however, in the emr, human resources capacities for laboratory service delivery presented a complex landscape, with fewer than half of the mss demonstrating proficiency. while there were commendable efforts in incorporating quality and safety management topics in training curricula, and in providing pre-service and in-service training programmes, challenges persisted in developing and implementing national staffing plans and competency-based training curricula. laboratory biosafety and biosecurity measures presented another area of concern, despite notable progress on the provision of personal protective equipment for staff. strides have been made in safety equipment availability and the appointment of safety officers, but critical deficiencies remained, including the absence of regulated biobanking systems and the imperative for enhanced waste management policies aligned with biosafety and biosecurity requirements. to ensure the safety of personnel and the environment, mss will need to develop and implement a national regulatory framework that addresses biosafety and biosecurity with the inclusion of other sectors, such as animal health and environment. overall, our analysis underscores the complex nature of laboratory system strengthening efforts within the emr, characterised by an array of achievements and challenges. addressing the identified gaps demands concerted efforts, collaboration, and sustained investment to ensure the delivery of high-quality laboratory services and advance public health outcomes across the emr. to sustain the gains from 2016 to 2023, with substantial investments during the covid-19 pandemic, mss will need to support laboratory systems as a priority, by developing a national laboratory policy within the national health development plan that will guide the implementation of a national integrated laboratory strategic plan. the implementation of laboratory strategy will be coordinated through the specific laboratory department or unit at the ministry of health level, above the central public health laboratories. this will be able to oversee both public health and clinical laboratory services. this can go hand-in-hand with the creation of the multisectoral and multi-programme national laboratory technical working group to better coordinate and integrate laboratory services in the mss. the new laboratory framework to strengthen laboratory services in the emr (2024–2029) therefore prioritises laboratory leadership and governance, laboratory quality management systems, and biosafety and biosecurity. the who emr will coordinate the efforts of mss through the establishment and technical support of a regional laboratory technical working group with representation from mss and partners. acknowledgements we acknowledge the invaluable contributions of laboratories and partners across mss in the eastern mediterranean region, whose tireless efforts and dedication have immensely advanced health laboratory services. their unwavering commitment has been instrumental in implementing the laboratory strategic framework and addressing regional health challenges. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions f.s.m. conceptualised the study, including the carrying out of data collection and the corresponding data analysis, and wrote the article. r.h. and a.g. performed validation of the data and contributed to the writing of the article. r.o. verified the methodology, performed data analysis and contributed to the writing of the article. y.j.-f.h. conducted the initial formal analysis and review of the methodology, and contributed to the writing of the article. f.s.m., r.h., r.o., a.g., and y.j.-f.h. approved the final version of the article. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the data that support the findings of this manuscript are available from the world health organization database, but restrictions apply to the availability of these data. data were used under licence for the current study and so are not publicly available. however, data may be made available by the corresponding author, r.o., upon reasonable request and with permission from the ministry of health of the eastern mediterranean mss. disclaimer the views and opinions expressed in this article are those of the authors and are the product 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for health collaborators. measuring the availability of human resources for health and its relationship to universal health coverage for 204 countries and territories from 1990 to 2019: a systematic analysis for the global burden of disease study 2019. lancet. 2022;399(10341):2129–2154. https://doi.org/10.1016/s0140-6736(22)00532-3 abstract introduction methods results discussion acknowledgements references about the author(s) jemal alemu department of medical laboratory sciences, college of health sciences, addis ababa university, addis ababa, ethiopia armauer hansen research institute, addis ababa, ethiopia aklilu lemma institute of pathobiology, addis ababa university, addis ababa, ethiopia balako gumi aklilu lemma institute of pathobiology, addis ababa university, addis ababa, ethiopia aster tsegaye department of medical laboratory sciences, college of health sciences, addis ababa university, addis ababa, ethiopia abdulaziz sherif department of internal medicine, college of health sciences, addis ababa university, addis ababa, ethiopia fisihatsion tadesse department of internal medicine, college of health sciences, addis ababa university, addis ababa, ethiopia amha gebremedhin department of internal medicine, college of health sciences, addis ababa university, addis ababa, ethiopia rawleigh howe armauer hansen research institute, addis ababa, ethiopia citation alemu j, gumi b, tsegaye a, et al. flow cytometric characterisation of acute leukaemia in adolescent and adult ethiopians. afr j lab med. 2025;14(1), a2394. https://doi.org/10.4102/ajlm.v14i1.2394 note: additional supporting information is available for download alongside the online version of this article as online supplementary document 1. original research flow cytometric characterisation of acute leukaemia in adolescent and adult ethiopians jemal alemu, balako gumi, aster tsegaye, abdulaziz sherif, fisihatsion tadesse, amha gebremedhin, rawleigh howe received: 28 dec. 2023; accepted: 28 oct. 2024; published: 23 jan. 2025 copyright: © 2025. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: flow cytometric characterisation of acute leukaemia is a key diagnostic approach for clinical management of patients, but is minimally practised in resource-constrained settings like ethiopia. objective: this study aimed to determine the immunophenotypes of acute leukaemia by flow cytometry at tikur anbessa specialised hospital, addis ababa, ethiopia. methods: a cross-sectional study was conducted on adolescent and adult inpatients consecutively admitted from april 2019 to june 2021. peripheral blood samples were stained for surface and cytoplasmic markers, and analysed by four-colour flow cytometry. results: of 140 cases aged 13 years to 76 years, 74 (53%) were men and 66 (47%) were women, 68 (49%) had acute lymphocytic leukaemia (all), 65 (46 %) had acute myelogenous leukaemia (aml), and 7 (5.0%) had acute leukaemia non-otherwise specified. acute lymphocytic leukaemia was more common among adolescent and male cases; aml was more common among adult and female cases. among all subtypes, b-cell acute lymphocytic leukaemia cases (73.5%) were more common than t-cell acute lymphocytic leukaemia (26.5%). a subset of acute leukaemia, cd19+/cd56+ aml was identified in 3 cases (6% of aml). of the b-cell all cases, 21 (42%) were cd34+/cd10+/cd66c+, 10% were cd34+/cd10+/cd66c–, 32% were cd34-/cd10+, and 6% were cd34+/cd10–. an unexpectedly high number of t-cell all cases that lacked surface cd3 were observed to have significantly higher levels of aberrantly expressed myeloid markers. conclusion: we observed multiple phenotypes identifying subtypes of acute leukaemia cases, extending our previous studies in ethiopia. what this study adds: this study extends previous studies by describing phenotypically defined subsets of all and aml which, in addition to diagnosis, may have useful prognostic value for clinicians. keywords: flow cytometry; acute leukaemia; ethiopia; phenotype; cell surface biomarker; cytoplasmic biomarker. introduction acute leukaemias are malignant disorders of precursor cells because of chromosomal rearrangements and multiple gene mutations, resulting in a high number of transformed cells within the bone marrow.1,2 leukaemia is diagnosed according to clinical features, laboratory tests including morphological examination of blood smears, flow cytometry immunophenotyping, and cytogenetic and molecular analysis.3,4 flow cytometry contributes important information to the assigning of leukaemic cell lineages, subtypes, and subsets in the diagnosis of and optimal therapy for acute leukaemia.5 flow cytometric analysis determines expression patterns for cell surface and cytoplasmic molecules defining maturation stages, lineage, and subtype differentiation, as well as aberrantly expressed markers.6 while numerous studies have been conducted in developed countries on flow cytometric phenotyping of acute leukaemias, far less is known in resource-limited settings in east africa, such as ethiopia. moreover, the method is not well established for routine care at the main tertiary care referral hospital of ethiopia. using this technology, we are continuing our efforts to integrate patient care with research and teaching. we previously conducted an initial research study7 showing the feasibility of this approach in this resource-limited setting.8 our current work was aimed to determine the immunophenotypes of acute leukaemia by flow cytometry at tikur anbessa specialised hospital, addis ababa, ethiopia. methods ethical considerations our study received approval from the institutional research board of aklilu lemma institute of pathobiology, addis ababa university (reference no.: alipb irb/009/2011/2018), the college of health science, addis ababa university (protocol no.: 001/19/im), and the armauer hansen research institute (ahri)/all africa leprosy, tuberculosis and rehabilitation training center (alert) ethics review committee (protocol no.: po34/18). written consent was obtained from adult participants, whereas written assent was obtained from adolescent cases or their families/caregiver. consent was also obtained from control participants. all personal information obtained from the study was coded to maintain complete confidentiality throughout the study period. study design and setting the present study was a cross-sectional study done at the haematology wards of tikur anbessa specialised hospital in addis ababa, ethiopia, between april 2019 and june 2021. tikur anbessa specialised hospital is the largest referral hospital in the country for the diagnosis and treatment of malignancies. study population and sampling strategy the study includes 140 adult and adolescent (aged 13–17 years) inpatients, consecutively diagnosed with acute leukaemia and admitted to the adult haematology ward, who had not yet started chemotherapy and who were recruited for the study. bone marrow samples from each patient had been obtained, and smears stained with haematoxylin and eosin were confirmed and classified as acute myelogenous leukaemia (aml) or acute lymphocytic leukaemia (all). the sample size of 140 was calculated by using the percentage of acute leukaemia cases (among total patients within a typical a 36-month period in the haematology ward) and the margin of error set at 5%, with level of confidence at 95% (zα/2 = 1.96). sample and data collection important socio-demographic as well as related clinical information was collected using a standardised form. cases/families/caregivers were interviewed for demographic characteristics and clinical data were extracted from patient cards by haematology fellows as well as internal medicine residents under the supervision of the principal investigator and senior advisers. in addition, we used test results from healthy individuals as positive controls for some of the markers as a quality control. over 80 tikur anbessa specialised hospital laboratory staff members, ahri research staff, or alert hospital laboratory staff volunteered a whole blood sample. we selected apparently healthy professionals by checking their complete blood count result with five differential count analysers and for the presence of abnormal results, including detection of immature cells. a total of three samples were rejected. a peripheral blood sample of about 2 ml was collected from each patient by experienced and trained nurses, and transferred to tripotassium ethylenediaminetetraacetic acid vacutainer tubes (becton dickinson, pont-da-claix, france). the blood samples were sent to tikur anbessa specialised hospital main laboratory for complete blood count testing on an automated haematological analyser (sysmex 2000i; sysmex corporation, kobe, japan) and retested for complete blood count (by sysmex kh 21n; sysmex corporation, kobe, japan, or coulter dxh 800; beckman coulter, shirley, new york, united states) at the alert hospital, and the average values were taken. samples were transported on the same day to the ahri immunology laboratory for flow cytometry analysis. laboratory analyses flow cytometry we used a previously published procedure for flow phenotyping.7 briefly, for cell surface staining, 100 ml of blood was added to 5 ml polystyrene tubes to which various combinations of fluorescent-conjugated antibodies were added.8 the antibody target molecules and fluorochromes included: cd8-fitc, cd4-pe, cd45-percp-cy5.5, cd3-apc, cd19-fitc, cd56-pe, cd33-fitc, cd34-pe, cd117-apc, cd13-fitc, cd10-pe, hla-dr-apc, isotype control igg1-fitc, isotype control igg1-pe, isotype control igg1-apc, cd66c-pe, cd15-fitc, cd14-fitc, cd7-pe, cd22-apc, and cd64-apc. mixtures containing cd8/cd4/cd3 and cd33/cd34/cd117 antibodies, and cd3, cd15, cd19, cd10, cd56 individual antibodies were obtained from agilent technologies (glostrup, denmark); the remaining monoclonal antibodies were received from becton dickinson (san jose, california, united states). antibodies were organised into 10 panels, typically using 4 antibody colours per panel. the same panel design was used for all leukaemia cases. this was done to assure uniformity among all subjects within the study as well as to ascertain potential aberrant expression of lineage-specific markers on some leukaemias, for example myeloid markers cd13, cd33, mpo, and cd66c on b-cell all (b-all).8,9 cytoplasmic staining was performed with the following antibodies: cd45-percp-cy5.5, terminal deoxynucleotidal transferase-fitc, cmyeloperoxidase (cmpo)-fitc, cd79a-pe, cd3-apc, igg1-fitc, igg1-pe, igg1-apc. the cmpo/cd79a/cd3 mixture and terminal deoxynucleotidal transferase antibodies were obtained from agilent technologies (glostrup, denmark); the remaining antibodies were from becton dickinson (san jose, california, united states). staining was performed as described for phenotyping.7 acquisition was performed with a facs calibur four-colour flow cytometer (becton-dickinson, durham, new hamshire, united states; an instrument that measures six parameters such as side scatter, forward scatter, and four types of fluorochrome-coated antibodies) using cell quest software (bd biosciences, san jose, california, united states), with typically 30 000 to 100 000 events acquired. data analysis data were imported into an ms excel (microsoft, redmond, washington, united states) spreadsheet and thereafter exported to an ms access (microsoft, redmond, washington, united states) database where the data were reviewed, cleaned, and analysed with spss version 25 statistical software (ibm corp, armonk, new york, united states). a given marker was considered positive if > 20% of the leukaemia gated cells were above the cutoff threshold established by isotype controls separately for surface and cytoplasmic staining11; a representative gating example is depicted in online supplementary figure 1. in some instances, marker-positive cells were expressed as a percentage of gated leukaemia cells. in addition, for selected samples, marker density was calculated by dividing the mean fluorescence of marker-positive cells by that of marker-negative cells. the chi-square test was used to determine associations between variables, and p < 0.05 indicates statistical significance; the mann-whitney u parametric test was used for continuous data. interpretation and grouping of acute leukaemia types and subtypes leukaemic cells were identified, typically (but not always) on the basis of reduced expression of cd45 and side scatter. gated cells were then evaluated for expression of different markers. the threshold for marker positivity was determined by parallel samples stained with control isotype markers as described.7 since markers typically were not entirely specific for a given lineage, we used world health organization (who)10 and associazone italiana ematologia oncologia pediatrica and the berlin-frankfurt-münster11 guidelines for aml, b-all, and t-cell all (t-all) classifications, and additional references for aml subtyping by flow cytometry.12 there are no generally accepted guidelines for the sub-classification of aml by flow cytometry aligned with the french-american-british categories, but we arbitrarily subdivided them into categories of aml ‘m0-2-like’ leukaemias (with phenotypes of either aml m0, m1 or m2), ‘m3-like’, ‘m4-like’ and ‘m5-like’, based on the percentage of cases with positive (> 20%) leukaemia cells and/or the percentage of leukaemia cells positive across those patients. moreover, we provisionally defined cases that did not exhibit a straightforward lineage commitment, as acute leukaemia non-otherwise specified.13 results demographic, clinical and flow cytometry characteristics out of a total of 140 patients, the ages ranged from 13 to 76 years, with a median of 25 (interquartile range: 18–35) (table 1). sixty-two per cent (46/74) of male patients and 54.5% (36/66) of female patients were within the age group of 18 years – 39 years (data not shown in table). no statistically significance associations were found between gender and acute leukaemia types or subtypes. the majority of patients clinically presented with anaemia (71.4%). based on flow cytometry analysis, out of 140 cases, 68 (49%) were identified as all (b-cell and t-cell), and 65 (46%) as aml. seven (5%) cases were acute leukaemia non-otherwise specified, although two cases appeared to meet requirements for mixed lineage (table 1). comparison of flow cytometry classification with independent classification into aml and all, using morphology of bone marrow smears, gave a concordance of 72%. leukaemia and age were significantly associated (p < 0.001). low haemoglobin and high white blood count levels were detected across all leukaemia types, without statistically significant differences. the highest platelet counts were found among t-all cases. table 1: demographic, complete blood counts (haemoglobin, white blood count, and platelet count) and acute leukaemia types among adolescent and adult acute leukaemia patients before chemotherapy at tikur anbessa specialised hospital, addis ababa, ethiopia, april 2019 to june 2021. expression of markers in acute leukaemia types, subtypes and subsets nearly all (96%) b-all patients were positive for all three b lineage markers (cd19, ccd79a, cd22). importantly, none of the ‘b-cell specific’ markers was uniquely specific to b-all. a number of cases of t-all expressed myeloid markers. acute myelogenous leukaemia was observed to be positive for cd117, cd34 and cmpo (table 2). among b-all, 42% were positive for the aberrantly expressed myeloid marker, cd66c, together with cd34 and cd10 (21/50), 10% with cd34+/cd10+/cd66c– (5/50), 6% cd34+/cd10– (3/50) and 32% cd34–/cd10+ (16/50). one of the cd34+/cd10–cases was cd66c positive, while of the cd34–/cd10+ subset, 29% were cd66c positive (table 3). among scd3 positive t-all, the average percentage of scd3 positive cells (79.4%) was less than the average ccd3 positive cells (99%), and scd3 was an average of 2.8-fold lower in density than that of ccd3; this difference was not observed among mature t-cells in the same samples (data not shown). notably, cmpo density among cmpo-positive t-all leukaemias was substantially 5.4 times less than that of cmpo-positive aml cases (p < 0.001) (table 2). table 2: percentage of patients who tested positive for indicated markers among acute leukaemia cases, tikur anbessa specialised hospital, addis ababa, ethiopia, april 2019 to june 2021. table 3: percentage of patients positive for indicated markers among b-cell acute lymphocytic leukaemia subsets at tikur anbessa specialised hospital, addis ababa, ethiopia, april 2019 to june 2021. distribution of b-cell acute lymphocytic leukaemia subsets in age when b-all subsets were stratified according to age, among b-all cases as characterised by cd34, cd10 and cd66 markers, 46% (6/13) of adolescents had a cd34+/cd10+/cd66+ phenotype and 8% (1/13) had a cd34+/cd10– phenotype (table 4). moreover, 41% (15/37) of adult cases had a cd34+/cd10+/cd66+ phenotype, while 5% (2/37) were cd34+/cd10–. importantly, there were no statistically significant differences in the distribution of b-all subsets according to age (p = 0.93). table 4: distribution of b-cell acute lymphocytic leukaemia subsets and acute myelogenous leukaemia subtypes among acute leukaemia patients by age group, tikur anbessa specialized hospital, addis ababa, ethiopia, april 2019 to june 2021. when comparing scd3 negative and t-all positive phenotypes, 20% of t-all cases without surface cd3 were positive for cd13, 30% were positive for cd33, and 42% were positive for mpo (table 5). among cases with scd3 expressed, 8% were positive for cd13, 0% were positive for cd33, and 20% were positive for mpo. table 5: differential expression of cell surface markers cd3 positive and negative t-cell acute lymphocytic leukaemia at tikur anbessa specialised hospital, addis ababa, ethiopia, april 2019 to june 2021. expression of markers in acute myelogenous leukaemia subtypes among the 65 aml-like cases, 46 were thus arbitrarily classified as aml m0-2-like (71%), 10 as m3-like (15%), 6 as m4-like (9%) and 3 as m5-like (5%). acute myelogenous leukaemia m0-2-like expressed markers of immaturity, 93% cd34, 100% cd117 and 93% hla-dr positive, as well as other myeloid markers. of note cd19 and cd56 were co-expressed in three cases (5%). acute myelogenous leukaemia m5-like were typified by the demonstration of markers of mature monocytes cd64 and/or cd14, and lack of immature markers. acute myelogenous leukaemia m4-like were notable for the presence of two distinct populations of leukaemia with differing densities of cd45 and side scatter, distinct from each other and from normal leukocytes, with one subpopulation bearing immature markers (cd34 and cd117), and the other monocyte markers cd14 and/or cd64. acute myelogenous leukaemia m3-like cells were those intermediate between extremes of m0-2-like and m4-like or m5-like. these were typically cd117 positive and hla-dr negative (figure 1). among aml subtypes, only the aml-m0-m2-like subtype was reported in adolescent cases (100% [5/5]), whereas all aml-like subtypes were reported in adult cases, including aml-m3-like, aml–m4-like and aml-m5-like (table 4). figure 1: immunophenotypes of arbitrary acute myelogenous leukaemia (aml) subtypes among acute leukaemia cases at tikur anbessa specialised hospital, addis ababa, ethiopia, april 2019 to june 2021. (a) immunophenotypes of aml-m3-like (n = 10), (b) aml-m0-m2-like (n = 46), (c) aml-m4-like (n = 6), and (d) aml-m5-like (n = 3) leukaemia cases. for (a), (b), and (d), markers are depicted either as percentage of cases with > 20% leukaemic cells (vertical bars) or percentage of leukaemia cells positive across all patients (indicated at the bottom row). for figure (c), aml-m4-like, percentage of leukaemia cells positive across all patients is depicted for each of two separable populations, an immature myeloblast-like cell, and a more mature monocyte-like cell. discussion we observed a similar distribution of aml, b-all, t-all, as well as age associations, to our previous 2017 study7 and another african study.14 secondly, we defined frequencies of subsets of b-all based on flow cytometric phenotypes having known associations with cytogenetic abnormalities, and find similarities consistent with previously reported prevalence. in particular, we observed a high prevalence of cd10+/cd34+/cd66+ b-all likely to be associated with t(9;22), and cd10+/cd34– b-all leukaemias, at least some of which have been associated with t(1:19) translocations.15 thirdly, myeloid markers were preferentially expressed on t-all leukaemias with the most immature phenotypes. fourthly, we report arbitrary flow cytometric classification of subtypes of aml based on a review of the study,16 and identify a subset expressing cd19 and cd56 consistent with the t(8;21) translocation. collectively, these findings underscore the importance of multi-dimensional approaches for leukaemia diagnosis and prognosis. we described subsets of b-all, using cd34, cd10 and, for some cases, cd66c, because these are commonly used markers and data exist describing their associations with clinical outcomes17 and with genetic aberrations.18 moreover, cd34 and cd10 are well-described markers whose expression aids in delineating steps in early b-cell development.19,20 thus, cd34+/cd10–, cd34+/cd10+ and cd34–/cd10+ represent successive stages of immature b-cell differentiation from pre-pro19,20 cells to, ultimately, transitional b-cells, although reports differ on the classification of intermediate pro-b and pre-b cells.19,20 of note, cd34+/cd10+ b-all has also been termed common b-all.21 these stages also appear to be similar to the b1, b2 and b3 subsets of b-all defined by the european group for the immunological characterization of leukemias classification,22 though reports differ on the classification of cd34+ positivity on pre-b cells.23 including cd66c in the analysis allowed us to identify four subsets of b-all. the cd34+/cd10+/cd66c+ subset was common in our study. a study in saudi arabia reported that the cd66c, cd34 and cd10 positivity phenotype were associated with, but not perfectly predictive of, the t(9;22) translocation, which is common in b-all, particularly among adults.8 of note, cd66c positivity in b-all is also common in cases with hyperdiploidy.24,25 among adults, it has a worse prognosis,26 but outcomes may be enhanced with the inclusion of tyrosine kinase inhibitors specific for the fusion gene bcr-abl.27 our finding that 40% of b-all in this cohort had the cd34+/cd10+/cd66c+ phenotype is consistent with another study.25 a second common phenotype in our study was cd10+/cd34–, consistent with another study.9 b-cell acute lymphocytic leukaemia with the t(1;19) translocation often has this phenotype, and reportedly with a favourable prognosis with intensive chemotherapy regimens.28 the prevalence of t(1;19) has been observed to be around 5% e,28 much lower than the associated phenotype that we report here. if the cd34–cd10+ phenotype is strongly predictive of t(1;19) aberrations, this implies that such aberrancies are exceptionally high in the ethiopian setting. however, we favour the possibility that whereas most t(1;19) may be cd10+cd34–,15 the converse is not true, and there may be many other rearrangements or mutations present in this subset apart from the t(1;19) translocation. further molecular work will be required to explore this possibility. less common in this study were two additional b-all phenotypes, the cd10+/cd34+/cd66c– phenotype, and the cd34+/cd10– subset (with or without cd66c). the cd10+/cd34+/cd66c– b-all subtype has been shown to be correlated with the t(12;21) translocation, which is the most common aberration in paediatric b-all, and which carries a good prognosis.29 in our cohort, the prevalence of this phenotype was low (10%), but our cohort was primarily composed of adult and adolescent leukaemia cases, so this lower frequency would be expected.17 the cd34+/cd10– subset has been shown to be associated with chromosome 11 mutations involving the mll gene.30 the most common of these translocations is t(4;11) (mll-af4), present in 5% of all.18 our finding of a 6% prevalence of the cd34+/cd10– phenotype is consistent with this. we defined two populations of cytoplasmic cd3 expressing t-all, those without and those with detectable surface cd3 (scd3). according to previous european group for the immunological characterization of leukemias classifications, the former would likely comprise the more immature t1 and t2, and the latter the less immature t3 and t4 phenotypes.31 since we did not evaluate cd1 expression, we were unable to distinguish the cortical t3 from the more mature t4 phenotype. in our t-all cases, scd3, when detectable, was variable in percentage, and typically of reduced intensity compared with peripheral normal t-cells. a notable difference between the scd3 negative and positive subsets was the greater expression of myeloid markers cd33, cmpo and cd13, among the most immature t-all. cmyeloperoxidase expression is uncommon among t-all, although it has been described previously.32 we observed expression of myeloid antigens in b-all as well, confirming an other study.33 it is possible that some of the t-all and b-all described here may better fit the diagnosis of mixed phenotype acute leukaemia. this would imply a much higher prevalence of this subtype in the ethiopian setting. however, weak expression of cmpo in particular is permitted within the classification criteria of associazone italiana ematologia oncologia pediatrica-berlin-frankfurt-münster11 for t-all, and we observed on average at least 4-fold lower levels of cmpo on those leukaemias we classified as t-all. from a mechanistic perspective, given that small fractions of early t-progenitor cells in normal individuals can express some myeloid markers,16,34 these leukaemias might persistently express incompletely repressed myeloid genes, as suggested for b-all.33 further characterisation will be necessary to distinguish between these possibilities. there are no generally accepted guidelines for the sub-classification of aml by flow cytometry. originally, the french-american-british classifications defined subgroups of aml-m0, -m1, -m2, -m3, -m4, -m5, -m6 and -m7, which represented, respectively, aml with minimally differentiated, aml without maturation, aml with maturation, acute promyelocytic leukaemia, acute myelomonocytic leukaemia, acute monocytic leukaemia, acute erythroid leukaemia and acute megakaryocytic leukaemia. the newer who classifications10 comprise mostly recurrent cytogenetic translocations in aml, and aml not otherwise categorised. the former category includes aml with t(8;21), aml with t(16;16), aml with t(15;17), as well as several other translocations and genetic anomalies or mutations which are less common. the t(8;21) translocation is commonly associated with some aml-m2, the t(16;16) with some aml-m4, and the t(15;17) with some aml-m3. however, many genetic anomalies do not neatly map to old fab categories, and, conversely, many aml do not have defined cytogenetic anomalies. the new who general category of aml not otherwise categorised thus includes subcategories which align with the previous fab system.10 because we were neither able to obtain cytogenetics for our samples, nor able to obtain fab subtypes by morphology on most of the samples, we opted for an ad hoc flow cytometric classification based on published literature.35 there are extensive published data available, both in relation to fab types as well as in relation to abnormalities.35 in the present study, we made arbitrary classifications based on these published data. with regard to aml-m0, -m1 and -m2, we made no attempt to differentiate between these types using flow cytometry. we further subdivided aml into m3-like, m4-like, m5-like categories. in comparing our distributions of aml subtypes with the reported literature our prevalence aml m0-2-like was higher than what would be predicted by other reports in the literature, and our estimated prevalence of m4-like and m5-like were lower.36 among the four aml subtypes, all identified in adults and aml-m0-m2-like subtypes were more identified in adolescent cases, and it is similar with a previous study.37 in this study, m0-2-like was largely defined on the basis of the simultaneous expression of immature markers, including cd34, cd117 and hla-dr. the majority of aml were of this arbitrary classification. however, cd34 and cd117 co-expression is not universal in aml-m0, -m1, and/or -m2.35 thus, this may be a principal explanation why our prevalence estimates for aml subtypes differ from other studies.38 a distinct subset of aml-m2 leukaemias include those with the t(8;21) translocation, which typically results in the aberrant expression of cd19 and cd56, a phenotype which has been reported in association with the t(8;21) translocation.39 we observed three such cases in this cohort within our ‘m0-2-like’ subtype, numbers in line with predictions based on a study evaluating t(8;21).39 notably, these three did not express other b-cell markers making a diagnosis of mixed phenotype or bi-lineage phenotype unlikely.40 a previous study described the t(8;21) translocations with lower density or lower percentage of positive cells expressing these two markers,41 and we did observe other ‘m0-2-like’ with similar phenotypes which could also conceivably express the t(8;21) translocation. further work will be required to assess this possibility. in addition, we observed that among the ‘m0-2-like’ leukaemias, a relatively high fraction expressed the cd7 and tdt markers, typically associated with early lymphocyte progenitors and cells of the t-cell lineage, respectively. this was also observed in other subtypes of aml. the observation that such markers are commonly expressed among aml leukaemia is well documented.42 acute myelogenous ‘m4-like’ leukaemia was easily visualised through the identification of immature myeloblasts and more mature monoblast-/monocyte-like populations.12 aml-m4 leukaemia is associated with the inv16 chromosome abnormality and also with bone marrow eosinophilia, leading to the unique who diagnostic classification. however, not all forms of aml-m4 express this abnormality,43 and other common abnormalities include ccaat enhancer binding protein alpha (cebpa) and nmp1 mutations,44,45 both with generally good prognosis.46 acute myelogenous ‘m5-like’ leukaemia was primarily based in this study on the detection of monocyte-like cell subpopulations expressing cd14 and cd64 and lack of immaturity markers such as cd34 and cd117, though the latter two markers among m5 leukaemias have been cited in the literature.40 a number of mutations have been associated with m5, notably the mll containing t(9;11)47 and also forms with npm1 mutations,44 although both mutations are also seen in other aml types.44 the definition we used for aml ‘m3-like’ leukaemias represented those with phenotypes more intermediate to those of the immature marker expressing aml ‘m0-2-like’ and those expressing monocyte markers. of note, the majority of ‘m3-like’ leukaemias in this study were cd34–/cd117+ and hla-dr–, a flow cytometry phenotype often described in association with aml-m3.48 importantly the aml-m3 phenotype is mostly associated with the t(15;17) rearrangement,12 but it is important to note that occasional t(15;17) translocations exist which are associated with other aml subtypes,49 and even markers such as ccd3.50 recommendations importantly, our study underscores the heterogeneity of leukaemia, and the need for future use of markers of molecular mutations or chromosomal arrangements, as well as evaluation of long-term patient outcomes. these are planned for future studies. limitations our study was limited by the numbers of markers we utilised. in particular, we did not look for subsets of aml requiring additional markers to define erythroblastic or megakaryocyte leukaemia, mast, natural killer, basophil or dendritic cell leukaemias, and did not characterise mixed lineage or phenotype leukaemias. in addition, since morphology results from smears can only clearly distinguish between myeloid and lymphoid lineages, clear-cut comparisons cannot be made between morphology approaches and flow cytometry. conclusion acute leukaemia diagnosis and prognosis represent a challenge in resource-limited settings. most of the more advanced approaches utilised in developed country settings are unavailable; moreover, no single approach, even among advanced methodologies, is alone sufficient for diagnosis and prognosis according to current who guidelines. in the present study we have attempted to come up with a compromise approach, utilising more affordable four-colour flow cytometry and engaging in more detailed phenotypic characterisation of patient and leukaemia subtypes. however, it is clear that this approach, while promising, will require considerably more standardisation and validation in the context of who classification as well as patient outcomes. acknowledgements we are grateful to addis ababa university, the armauer hansen research institute, tikur anbessa specialised hospital, the alert hospital, especially professionals at the medical laboratory department, the international clinical laboratories, addis ababa, ethiopia, for their technical support during the study, and to all patients who voluntarily participated in this study. this article is partially based on j.a.’s dissertation entitled ‘prevalence of viral infections and flow cytometric analysis among acute leukaemia patients at tikur anbessa specialised hospital, addis ababa, ethiopia’ towards the degree of phd, aklilu lemma institute of pathobiology, addis ababa university, ethiopia, with supervisors prof balako gumi, dr rawleigh howe, prof aster tsegaye and dr amha gebremedhin. competing interests the author reported that they received funding from addis ababa university and the armauer hansen research institute, addis ababa, ethiopia, which may be affected by the research reported in the enclosed publication. the author has disclosed those interests fully and has implemented an approved plan for managing any potential conflicts arising from their involvement. the terms of these funding arrangements have been reviewed and approved by the affiliated university in accordance with its policy on objectivity in research. authors’ contributions j.a. and r.h. conceptualised and designed the study, collected laboratory data, performed laboratory testing, and prepared the article. b.g. and a.t. reviewed and analysed the data. a.g., a.s. and f.t. collected and interpreted the clinical data. r.h., a.t., a.s. and a.g. revised the article and supervised the study. j.a., b.g., a.t., a.s., f.t., a.g., and r.h. approved the final article. sources of support our study was financially supported by addis ababa university and the armauer hansen research institute, addis ababa, ethiopia. data availability the datasets analysed and presented in this study are available from the corresponding author, j.a., on reasonable request and are not publicly available, because of either privacy or ethical restrictions. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. the article does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the authors are responsible for this article’s results, findings, and content. references shipley jl, butera jn. acute myelogenous leukemia. expert hematol. 2009;37(6):649–658. https://doi.org/10.1016/j.exphem.2009.04.002 tebbi ck. etiology of acute leukemia: a review. cancers. 2021;13(9):2256. https://doi.org/10.3390/cancers13092256 lee p-s, lin c-n, liu c, huang c-t, hwang w-s. acute leukemia with myeloid, b-, and natural killer cell differentiation: a novel form of multilineage leukemia. arch pathol lab med. 2003;127(2):e93–e95. https://doi.org/10.5858/2003-127-e93-alwman mason j, griffiths m. molecular diagnosis of leukemia. expert rev mol diagn. 2012;12(5):511–526. https://doi.org/10.1586/erm.12.44 weir eg, borowitz mj. flow cytometry in the 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haematol. 1999;105(1):198–207. https://doi.org/10.1111/j.1365-2141.1999.01312.x singh zn, duong vh, koka r, et al. high-risk acute promyelocytic leukemia with unusual t/myeloid immunophenotype successfully treated with atra and arsenic trioxide-based regimen. j hematopathol. 2018;11:67–74. https://doi.org/10.1007/s12308-018-0329-z article information authors: megan a. rensburg1 tandi matsha2 mariza hoffmann1 mogamat s. hassan2 rajiv t. erasmus1 affiliations: 1division of chemical pathology, national health laboratory service, tygerberg hospital, stellenbosch university, south africa2faculty of health and wellness sciences, cape peninsula university of technology, south africa correspondence to: megan rensburg postal address: po box 19113, tygerberg 7505, south africa dates: received: 14 mar. 2011 accepted: 19 jan. 2012 published: 04 june 2012 how to cite this article: rensburg ma, matsha t, hoffmann m, hassan ms, erasmus rt. distribution and association of hs-crp with cardiovascular risk variables of metabolic syndrome in adolescent learners. afr j lab med. 2012;1(1), art. #10, 6 pages. http://dx.doi.org/10.4102/ ajlm.v1i1.10 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. distribution and association of hs-crp with cardiovascular risk variables of metabolic syndrome in adolescent learners in this original research... open access • abstract • introduction • materials and methods    • subjects    • measurement of blood pressure, glucose and lipids    • measurement of hs-crp    • statistical analysis • ethical considerations • results • discussion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ objective: metabolic syndrome (mets) and its associated cardiovascular risk are on the increase in children. high-sensitivity c-reactive protein (hs-crp) has emerged as a useful marker for inflammation associated with atherosclerosis and cardiovascular disease. our aim was to determine the distribution of hs-crp in an effort to identify the mets variable that is critical in modulating plasma crp levels in a population of south african adolescents.design: a cross-sectional analytical study design was used for this investigation, where the dependent and independent variables were measured simultaneously. methods: anthropometric variables, blood pressure, fasting blood glucose and lipids were performed on 324 consenting learners aged 15–18 years from three different ethnic groups (black, white and coloured). the national cholesterol education program adult treatment panel iii (ncep atp iii) for ages 15–18 year olds was used to define mets. results: the prevalence of mets and obesity was 3.7% and 7.1%, respectively. the hs-crp levels were significantly higher in subjects with a waist-circumference greater than the 90th percentile (p < 0.01) and in obese learners with mets, but was lower in adolescents with normal weight and mets. median hs-crp levels increased with an increasing number of metabolic abnormalities and exceeded 3 mg/l in 19% of adolescents. gender and ethnic differences were observed. conclusion: our findings suggest that obesity and waist circumference appear to be major mediators of hs-crp levels in south african adolescents. introduction top ↑ globally, obesity and its accompanying metabolic complications are on the increase. childhood obesity has reached epidemic proportions and is one of the most serious public health problems facing the developed and, increasingly, the developing world.1 metabolic syndrome (mets), a constellation of interrelated conditions of insulin resistance, hyperlipidaemia, hypertension and obesity, is one of the recognised complications or associations of obesity. a pro-inflammatory and pro-thrombotic state contributing to endothelial dysfunction is a common feature of those with mets.2 it is well recognised that inflammation plays a key role in the pathophysiology of atherosclerosis and cardiovascular disease (cvd).3 markers of endothelial dysfunction and inflammation include tumour necrosis factor-alpha (tnf-α), the interleukins (il) and c-reactive protein (crp). tnf-α is a cytokine secreted from endothelial and smooth muscle cells, macrophages and fat cells. it enhances monocyte recruitment into developing atherosclerotic lesions and therefore links obesity with atherosclerosis.4 il-6 is the major pro-coagulant cytokine. it increases plasma levels of plasminogen activator inhibitor type 1, fibrinogen and crp. lifestyle modifications, including a mediterranean-style diet and weight loss, reduce serum concentrations of il-6, il-7, and il-18.5 patients with coronary heart disease have markedly elevated levels of il-1, especially those with unstable disease.6 pro-inflammatory cytokines, including il1-β, il-6 and tnf-α, are released from macrophages within the vessel wall during an inflammatory response.4 these cytokines mediate distant inflammatory effects, including activation of hepatic genes encoding acute-phase reactants, crp, fibrinogen and serum amyloid a. crp induces synthesis of other cytokines, cellular adhesion molecules (cams) and tissue factor in monocytes and endothelial cells.4,7 tissue factor activation provides the link between inflammation and coagulation by activating the coagulation cascade. crp was traditionally measured in patients to assess and monitor systemic inflammation during an acute infection. values exceeding 10 mg/l are accepted as evidence of probable infection and/or sepsis. the need for detection very low levels (< 3 mg/l) only became necessary when crp was discovered to be a useful marker of endothelial dysfunction and future cvd risk. high-sensitivity c-reactive protein (hs-crp) has emerged as a useful biomarker for vascular inflammation associated with atherosclerosis and it may directly promote atherosclerotic processes.8,9 as such, elevated hs-crp levels, as a response to the increase in the secretion of cytokines of adipose origin, have been used as a marker of cardiovascular risk and diabetes in adults.10 various hs-crp methods have been introduced, fulfilling the need of measuring crp levels at these low levels. in apparently healthy adults, hs-crp levels of more than 3 mg/l are associated with a high risk of future cvd. many studies have examined the clustering of metabolic abnormalities, but the metabolic phenotype continues to be less well understood in children in terms of criteria, prevalence and clinical implications.11 studies have reported high values of hs-crp amongst obese children and adolescents, indicating a certain level of inflammation.3,12 childhood crp values have also been shown to predict adult crp levels independently of other metabolic risk factors.13 the us national health and nutrition examination survey (usnhanes) reported that adolescents with more metabolic abnormalities had higher crp levels.11 the few studies conducted in developing countries have shown a relatively high prevalence of mets that is paralleled by the increasing obesity in children and adolescents.14 a recent study on the prevalence of mets amongst south african children reported an overall rate of 6.5% using the national cholesterol education program expert panel on detection, evaluation and treatment of high blood cholesterol – adult treatment panel iii (ncep atp iii) definition.15 in south africa, morbidity and mortality related to non-communicable disease remain prominent, despite the sustained increase in deaths from chronic infections such as hiv and aids and tuberculosis.16,17 this is particularly important when providing interventions that target cvd and their risk factors, as these chronic conditions place a huge financial and human resource burden on south africa’s health budget. the measurement of hs-crp concentrations may thus provide an indication of future risk of cvd, which may lead to earlier intervention. therefore, the aim of this study was to determine the distribution of hs-crp in a population of south african adolescent learners and to determine its association with cardiovascular risk factor variables of mets, in an effort to identify the variable that is critical in modulating plasma hs-crp levels in this population. materials and methods top ↑ subjects our study population formed part of a larger study involving 1683 learners aged 8–18 years, recruited through a proportionally stratified multistage random sampling technique from government-funded secondary schools in the western cape province of south africa. the inclusion criteria were, (1) male or female learners aged 15 years – 18 years, (2) fasted overnight (≥ 10 h) and (3) no current or recent history of infectious disease (on self-report). participants were excluded if the measured hs-crp values were > 10 mg/l, or if there were any missing data. recent and/or current infectious disease was excluded by self-report and brief medical examination on the day of sampling. measurement of blood pressure, glucose and lipids finger prick blood performed by trained nursing staff was used for the estimation of fasting blood glucose (fbg) and lipid levels using the accutrend gct glucometer and cardiochektm pa analyser (polymer technology systems, inc., indianapolis, usa), respectively. the glucometer coefficient of variation is stated as < 3% and accuracy is approximately 5%, when compared to a hexokinase method. the cardiochektm analyser showed reasonable compliance with ncep goals for coefficients of variation and bias measurements.18 blood pressure measurements were performed using a semi-automatic digital blood pressure monitor (rossmax int. ltd, taipei, taiwan) according to world health organization guidelines.19 the ncep atp iii for ages 15–18 years were used to define mets20 (table 1). individuals were classified as overweight or obese, according to age and gender, as described by cole et al.21 measurement of hs-crp different platforms exist to measure hs-crp, including turbidimetric, nephelometric and immunometric assays (two-site chemiluminescent). these different methodologies compare well in precision and comparative studies.22 venous blood was collected and the serum was separated and frozen at -20 °c before batch analysis. the hs-crp was measured by an hs-crp assay, based on the highly sensitive near infrared particle immunoassay rate methodology (immage® immunochemistry system, beckman coulter inc., brea, usa), with an analytical sensitivity of 0.2 mg/l and a functional sensitivity of 0.11 mg/l (measuring range 0.2 mg/l – 1440 mg/l). participants with hs-crp concentrations < 0.2 mg/l had a value assigned to them of 0.2 mg/l. statistical analysis the statistica 8 software package (stratsoft inc., tulsa, usa) was used to perform statistical analyses. descriptive data are represented as medians because hs-crp has a non-gaussian distribution and non-parametric statistics have been applied. other variables, such as glucose, triglycerides and cholesterol, are presented as means, as they follow a normal distribution. for categorical data, group comparisons were performed using the mann whitney u-test. a 5% significance level (p < 0.05) was used for determining significant differences. table 1: criteria for the metabolic syndrome are listed below and can be any three of more. ethical considerations top ↑ the study was approved by the faculty of health science ethics committee at the university of stellenbosch and consent to conduct the study was obtained from the western cape education department. ethical clearance was given for the collection of venous blood samples from the participants older than 12 years. finally, a parent or guardian gave consent for the adolescents’ participation in this study and the participants themselves assented. results top ↑ three-hundred-and-twenty-nine learners who satisfied the inclusion criteria were recruited for the study. five were then excluded from this initial study population as a result of hs-crp levels > 10 mg/l, leaving 324 learners for this study. of this total, 186 (57.4%) were female, whilst 138 (42%) were male. the final study population comprised 136 (42%) black learners, 62 (19%) white learners and 126 (39%) coloured learners.the distribution of hs-crp was skewed, with higher median hs-crp concentrations in female learners than male learners (1.77 mg/l vs 1.39 mg/l, respectively; p = 0.15). significant racial differences were observed with higher median hs-crp levels in non-white learners (white – 1.02 mg/l; black – 1.74 mg/l; coloured – 1.76 mg/l). the hs-crp levels correlated positively with the learners’ body mass index (bmi) and waist circumference, but not with high density lipoprotein cholesterol (hdl-c) or triglycerides. the prevalence of mets and obesity was 3.7% and 7.1%, respectively. obesity was higher in all female learners, whilst coloured learners (17%) had a nearly three times higher rate of obesity than white learners (5%) or black learners (4%). the median hs-crp levels in the mets group were 2.56 mg/l, compared to 1.67 mg/l in those without mets. the hs-crp values correlated positively with an increase in the number of mets components present (figure 1). four (33%) subjects with mets had a normal weight and their median hs-crp levels were significantly lower than the obese group (table 2). because low-grade systemic inflammation may underlie, at least in part, the clustering of metabolic risk factors, we examined the relationship between hs-crp concentrations and components of the mets. there was no association between the individual components, blood pressure, triglycerides, fasting blood glucose and hs-crp noted. more than 60% of the participants had hdl values below the cut-off of 1.04 mmol/l, with a mean value 0.97 mmol/l. there was a significant difference (p < 0.01) noted in the crp concentrations between those with a normal waist circumference, compared to those with a waist circumference greater than the 90th percentile (1.39 mg/l vs 3.22 mg/l, respectively). the hs-crp levels of > 3 mg/l were seen in 18.5% (n = 60) of learners. table 3 compares baseline demographic data and laboratory values for children with crp levels of ≥ 3 mg/l and those with crp levels of < 3 mg/l. these subjects were likely to have higher bmis, with a higher percentage of obese subjects. however, 13% (n = 33) of children with levels > 3 mg/l had normal bmi. discussion top ↑ inflammation is an important pathogenic factor in the development of atherosclerosis and coronary heart disease, especially in those with diabetes, obesity and mets. an important asset of our study is that it investigated the prevalence of mets in adolescents from three different racial groups in south africa. mets, as defined by the modified ncep atp iii classification, was observed in 3.7% of all children, with hs-crp values nearly three times higher in children with mets compared to those without it.20 previous studies have reported that hs-crp concentrations correlate with individual components of the mets.11 we observed a positive linear trend in hs-crp levels, with an increasing number of features of mets. we also observed no association between the individual components: blood pressure, triglycerides, fasting blood glucose and hs-crp. of the metabolic variables studied, adiposity, as measured by bmi and waist circumference, was the major predictor of hs-crp (similar to results obtained from international studies3,11), suggesting obesity as a mediator of excess crp seen in the mets. mets has been associated with higher crp levels even in the absence of obesity,3,11,23 but this was not observed in our study. considerably lower hs-crp levels were observed in children with mets but with normal weight (also termed metabolically obese, normal-weight children), with the median hs-crp in this group being 0.65 mg/l, compared to 3.9 mg/l in the obese group. previous studies have suggested that metabolically obese but normal-weight individuals are also at an increased risk of developing type 2 diabetes and cvd24; however, this needs to be explored further in our setting in the light of these observations.this community-based study has demonstrated that hs-crp levels in south african children vary by race and sex and relate adversely to risk variables of mets. the hs-crp values ranged from 0.2 mg/l to 9.9 mg/l, with higher median hs-crp concentrations in female learners (1.77 mg/l vs 1.39 mg/l). similar observations have been made by lambert et al.24 in 16-year-old children from quebec, canada, as well as in the 1999–2000 us nhanes11 and the bogalusa studies3. however, in the taipei children heart study of crp concentrations no gender differences were observed.25 sex differences have been attributed to the effect of estrogens, which have been implicated in the transcriptional control and clearance of hs-crp.26 as observed in adults,27 we found that there were also ethnic differences, with black and coloured children having higher hs-crp levels. ethnic differences in body fat patterning are thought to account for some of the observed differences.28 in the bogalusa study, hs-crp levels showed racial differences and related to measures of obesity.3 for a given waist circumference, black boys and girls from the usa were found to have higher abdominal subcutaneous adipose tissue and lower visceral adipose tissue than their white peers.28 similar observations have been reported in south african children, in whom relatively more subcutaneous fat and greater waist circumference have been observed in stunted girls, which may predispose them to obesity in adulthood.29 in the strong without anorexia nervosa (swan) study, which examined 3154 women from different ethnic groups, afro-american ethnicity was associated with higher hs-crp levels after controlling for bmi, age and other risk factors, suggesting that genetic variants of hs-crp and dietary and lifestyle factors could also be contributory factors.27,30 the implications of these elevated hs-crp levels need further study, as south african black people generally have a better cardiovascular risk profile than other ethnic groups. obesity rates were comparable to those observed in many developed countries and obese learners in our study had a significantly higher median hs-crp (4.00 ± 0.58 mg/l) compared to their normal-weight counterparts (1.28 ± 0.12 mg/l). because fat distribution appears to have a more important influence on cardiovascular risk factors in young subjects than overall adiposity, abdominal obesity has been identified as the main determinant of several metabolic disorders.31,32 the relationship of waist circumference as a measure of abdominal obesity appears to be explained by its strong association with visceral adipose tissue, therefore explaining the strong association with inflammatory biomarkers such as hs-crp.33 we compared the hs-crp concentration of those with a normal waist circumference to those individuals with a waist circumference greater than the 90th percentile and, as expected, found a significant difference (p < 0.01). this finding is consistent with international results and highlights that an increase in abdominal obesity mirrors visceral adiposity and increased inflammation.33 waist circumference, particularly in young south african women, has been shown to be a better predictor of insulin resistance than visceral adipose tissue.34 whilst these results re-affirm that high hs-crp values are most likely caused by increased fat deposition, we also observed that more than half of the children with hs-crp > 3 mg/l were of normal weight, suggesting that a sizable number of apparently healthy children may be at increased cardiovascular risk. despite these observations in this and other studies, it is not recommended for hs-crp to be a suitable target for therapeutic intervention as no causal association between hs-crp and mets phenotypes has been reported.35 one platform to measure crp, regardless of the clinical context, is ideal in the clinical laboratory as it could lead to decreased cost and turn-around-time of analysis, as most laboratories have dedicated instruments for measurement of hs-crp and batch-analysis is usually performed. there are several limitations to our findings. the primary limitation is the use of a single hs-crp measurement. in adults, repeat hs-crp measurements are recommended, as concentrations are affected by factors such as recent infection and inflammatory conditions. the measurement of other inflammatory biomarkers of endothelial dysfunction could have added more value to the study. other tests that could have excluded active infection, for example cytokines or white blood cell count, were not conducted. a second limitation is that this was not a national study and excluded learners from private schools. another limitation is that the study outcomes depended on the definition of mets. insulin resistance, the key component of the mets, was not determined. furthermore, direct measurement of body fat mass and distribution was not assessed and the study was observational and cross-sectional in nature, making it difficult to differentiate between cause and effect, and associations. in summary, we have found that hs-crp is increased in south african adolescents with metabolic abnormalities and the mets phenotype. markers of adiposity, such as bmi and waist circumference, appear to be strong mediators of hs-crp and many learners had hs-crp levels greater than 3 mg/l, a value that has been used in adults to indicate high cvd risk. in our setting, hs-crp may be useful marker to assess early atherosclerosis and cardiovascular risk, particularly in those children with normal bmi. figure 1: association between the number of metabolic components and high-sensitivity c-reactive protein (hs-crp). table 2: metabolic syndrome, body mass index (bmi) and high-sensitivity c-reactive protein (hs-crp) levels amongst the study participants (n = 324). table 3: baseline demographic data and laboratory results of the study participants (n = 324). acknowledgements top ↑ this research was supported by a grant from the national health and laboratory service research trust and the medical research council of south africa. we wish to thank the cape peninsula university of technology for their financial contribution, as well as professor martin kidd for performing the statistical analyses. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this paper. authors’ contributions r.t.e. (stellenbosch university) was the project leader; all authors contributed equally to experimental and project design and data collection. m.r. (stellenbosch university), t.m. (cape peninsula university of technology) and r.t.e. wrote the manuscript. references top ↑ 1. weiss r, dzuira j, burgert t, et al. obesity and metabolic syndrome in children and adolescents. n engl j med. 2004;350(23):2362–2374. http://dx.doi.org/10.1056/nejmoa031049, pmid:15175438 2. odrowaz-sypniewska g. markers of pro-inflammatory and pro-thrombotic state in the diagnosis of metabolic syndrome. adv med sci. 2007;52:246–250. pmid:18217426 3. patel da, srinivasan sr, xu j, li s, chen w, berenson gs. distribution and metabolic syndrome correlates of plasma crp in biracial (black-white) younger adults: the bogalusa heart study. metabolism. 2006;55:699–705. http://dx.doi.org/10.1016/j.metabol.2005.07.015, pmid:16713426 4. koh kk, han sh, quon mj. inflammatory markers and the metabolic syndrome. j am coll cardiol. 2005;11:1979–1985. 5. esposito k, marfella r, ciotola m, et al. effects of a meditereanean-style diet on endothelial dysfunction and markers of vascular inflammation in the 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al. progress and challenges in metabolic syndrome in children and adolescents. circulation. 2009;119:628–647. http://dx.doi.org/10.1161/circulationaha.108.191394, pmid:19139390 34. jennings cl, lambert ev, collins m, levitt ns, goedecke jh. the atypical presentation of the metabolic syndrome components in black african women: the relationship with insulin resistance and the influence of regional adipose tissue distribution. metabolism. 2009;58:149–157. http://dx.doi.org/10.1016/j.metabol.2008.09.007, pmid:19154946 35. timpson nj, lawlor da, harbord rm, et al. c-reactive protein and its role in metabolic syndrome: mendelian randomization study. lancet. 2005;366:1954–1959. http://dx.doi.org/10.1016/s0140-6736(05)67786-0 references about the author(s) rajiv erasmus department of chemical pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa department of pathology, faculty of medicine, university of botswana, gaborone, botswana department of biomedical sciences, faculty of health sciences and wellness, cape peninsula university of technology, cape town, south africa pascale ondoa african society for laboratory medicine, addis ababa, ethiopia department of global health, institute of global health and development, university of amsterdam, amsterdam, the netherlands citation erasmus r, ondoa p. taking the train of digital health and artificial intelligence to improve medical laboratory service in africa: key considerations. afr j lab med. 2023;12(1), a2329. https://doi.org/10.4102/ajlm.v12i1.2329 editorial taking the train of digital health and artificial intelligence to improve medical laboratory service in africa: key considerations rajiv erasmus, pascale ondoa copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. similar to other scientific fields, laboratory medicine is undergoing a digital revolution. software, algorithms and digital applications are increasingly incorporated into in-vitro diagnostics to enhance automated processes for the collection, storage, integration and analysis of data to inform patient management, to aid in population-based investigations, or to improve the quality and effectiveness of laboratory testing.1,2 some of the most popular examples of how digitisation can revolutionise the practice of laboratory medicine in africa include automation of specimen management and testing (e.g., cobas® system workflow from roche), connection of various instruments to monitor their functionality in real time and assist operators (e.g., genexpert® from cepheid), and laboratory information systems that support the swift reporting of test results and real-time diagnostics of the performance of entire laboratory networks. digital technologies are also being used to streamline processes for quality assurance and laboratory operations.3 digitised laboratory testing processes provide new opportunities for uncovering trends, increase the usability of state-of-the-art technology and may accelerate the utilisation of test results for improved patient and public health outcomes.3,4,5 the emergence of digital data in the field of laboratory medicine brings the possibility of using artificial intelligence (ai) to aid in clinical decision-making, disease monitoring and prediction of outcomes and patient safety. artificial intelligence is defined as the ability of computer systems to perform tasks that normally require human intelligence, such as learning, problem-solving, visual perception, decision-making and communication.4,5,6,7,8 artificial intelligence takes advantage of digital data to integrate health records, symptom profiles, demographic data and other patient information to better predict diagnoses and recommend treatments. machine learning is the type of ai most frequently used in laboratory medicine. one example of machine learning is a computer algorithm that can ‘digest’ historical information from thousands of pathology slides, identify patterns of interpretation and develop an algorithm for detecting cancer cells quickly through reducing human interpretation errors as well as the length of time required for visual inspection by a human operator.4,5,6,7,8 in another interesting use of ai, machine learning has been used in the prediction of the spread of coronavirus disease 2019, by analysis of viral variant data, hot spots and contact tracing.2 artificial intelligence may also be able to assist in the development of new diagnostics and treatment strategies, which would be an important advance.9 the integration of digitisation and ai into laboratory medicine certainly has the potential to improve the accuracy, efficiency, usability and productivity of laboratory services. indeed, the simultaneous scaling up of paper-based information, digitisation of information and processes, and implementation of ai are creating unique opportunities for leapfrogging from a conventional landline system to a digital diagnostic ecosystem. however, for african health systems that already face well-known chronic challenges, these changes also pose risks for overwhelming laboratory medicine with too many challenges or inadequate technology to meet them.10 undoubtedly, integrating digitisation and ai into laboratory and diagnostic services implementation in the lowand middle-income settings of africa is expected to pose some challenges and barriers. these include: (1) investment costs in required hardware, software, data storage and maintenance; (2) privacy, ethical and regulatory concerns around the sharing of sensitive personal data; and (3) human factors, including the building of computer and ai literacy among healthcare workers, as well as lack of trust lack of trust or confidence in digital technology and ai tools, or fear of losing jobs or skills.11,12 these challenges and barriers are amplified in many developing countries and may include issues around the standardisation of tools and the complexity of conducting quality assurance on digitised processes and the ai component of diagnostics. lessons learnt from disruptions in settings already embracing digital technologies can inform the way africa adopts and scales up ai into healthcare in general and diagnostics in particular.11,12 so how do we take it from there? as with other technology innovations, the implementation of new digital technologies and ai should clearly respond to a clinical need and potential patient applications should be clearly defined by users of diagnostic services. the implementation of infrastructure should include multiple stakeholders that tap experts both within and outside of the health sectors. this is particularly important with respect to data acquisition, data creation, model development and deployment. critical to the scale-up and democratisation of digital technologies and ai is addressing gaps in access to electricity and internet connections on the continent. as of september 2023, only 47% of africans have reliable access to electricity (https://data.worldbank.org), and there was only 43% penetration for internet access in 2021 (www.statista.com). additionally, a whole cadre of experts must be built to manage data collection, storage, analysis and interpretation. this gap has already been demonstrated by the gap in bioinformaticians observed during the scale-up of next-generation sequencing in the context of the africa centres for disease control and prevention pathogen genomic initiative.13 finally, a thorough monitoring and evaluation framework is warranted to assess where and how ai and digital technology are actually improving patient and public health and how these can be integrated and implemented in african healthcare settings. integrating digital technologies and ai into laboratory services has the potential to transform diagnostic services and contribute to the implementation of the may 2023 resolution of 76th world health assembly on strengthening diagnostics in africa and other countries to improve positive patient, public and global health outcomes.14 regional agencies such as the africa centres for disease control and prevention have already taken the lead in developing continental frameworks to guide the implementation of digital technologies in public health systems,15 as well as in launching initiatives such as healthconnekt to build the internet connectivity infrastructure in africa, to allow the continent to align with the world health organization’s deliberate strategy to use digital technology as a force to transform primary healthcare.16 moving forward, africa-led knowledge networks, such as the african society for laboratory medicine’s laboratory strengthening community of practice and laboratory directors forum17,18 can play a major role in identifying priorities, best practices, operational requirements and ethical considerations for the implementation of digital and ai technology in african laboratory medicine. the digital revolution is a game changer for africa, which can drive a parallel digital revolution in laboratory diagnostics. continental frameworks that have been developed now need to be made operational to benefit from this transformation. references jovičić sž, vitkus d. digital transformation towards the clinical laboratory of the future. perspectives for the next decade. clin chem lab med. 2023;61(4):567–569. https://doi.org/10.1515/cclm-2023-0001 forrester. the lab of the future is here [homepage on the internet]. 2021. [cited 2023 sept. 20]. available from: https://assets.thermofisher.com/tfs-assets/cmd/reference-materials/wp-95975-lab-future-wp95975-en.pdf gungoren ms. crossing the chasm: strategies for digital transformation in clinical laboratories. clin chem lab med. 2023;61(4):570–575. https://doi.org/10.1515/cclm-2022-1229 peschisolido s. lab 4.0: making digital transformation work for your laboratory [homepage on the internet]. technology networks informatics; 2022. [cited 2023 sept. 20]. avaliable from: https://www.technologynetworks.com/informatics/articles/lab-40-making-digital-transformation-work-for-your-laboratory-358583 naugler c, church d. automation and artificial intelligence in the clinical laboratory. crit rev clin lab sci. 2019;56(2):98–110. https://doi.org/10.1080/10408363.2018.1561640 khatab z, yousef gm. disruptive innovations in the clinical laboratory: catching the wave of precision diagnostics. crit rev clin lab sci. 2021;58(8):546–562. https://doi.org/10.1080/10408363.2021.1943302 ahmed z. ai and laboratory medicine: opportunities and challenges (linkedin). 2023. a guide to ai in the enterprise [homepage on the internet]. tech target. 2021, p. 2–25. [cited 2023 sept. 20]. available from: https://media.techtarget.com/digitalguide/images/misc/ea-marketing/eguides/a_guide_to_artificial_intelligence_in_the_enterprise.pdf al-antari ma. artificial intelligence for medical diagnostics-existing and future ai technology! diagnostics (basel). 2023;13(4):688. https://doi.org/10.3390/diagnostics13040688 oleribe oo, momoh j, uzochukwu b, et al. identifying key challenges facing healthcare systems in africa and potential solutions. int j gen med. 2020;12:395–403. https://doi.org/10.2147/ijgm.s223882 jiang l, wu z, xu x, et al. opportunities and challenges of artificial intelligence in the medical field: current application, emerging problems, and problem-solving strategies. j int med res. 2021;49(3):3000605211000157. https://doi.org/10.1177/03000605211000157 church dl, naugler c. using a systematic approach to strategic innovation in laboratory medicine to bring about change. crit rev clin lab sci. 2022;59(3):178–202. https://doi.org/10.1080/10408363.2021.1997899 onywera h, ondoa p, nfii f, et al. boosting pathogen genomics and bioinformatics workforce in africa. lancet. 2023. https://doi.org/10.1016/s1473-3099(23)00394-8 strengthening diagnostics capacity [homepage on the internet]. world health organization; 2023. [cited 2023 sept. 20]. available from: https://apps.who.int/gb/ebwha/pdf_files/wha76/a76_r5-en.pdf digital transformation strategy [homepage on the internet]. africa cdc; 2023. [cited 2023 sept. 20]. available from: https://africacdc.org/download/digital-transformation-strategy/ technical series on primary health care [homepage on the internet]. world health organization; 2018. [cited 2023 sept. 20]. available from: https://www.who.int/docs/default-source/primary-health-care-conference/digital-technologies.pdf laboratory systems strengthening community of practice (labcop) [homepage on the internet]. african society for laboratory medicine (ajlm). [cited 2023 sept. 20]. available from: https://aslm.org/what-we-do/labcop/ aslm launches the laboratory directors’ forum to transform laboratory best practice in africa [homepage on the internet]. african society for laboratory medicine (ajlm); 2022. [cited 2023 sept. 20]. available from: https://aslm.org/press-release/aslm-launches-the-laboratory-directors-forum-to-transform-laboratory-best-practice-in-africa/ abstract introduction ethical considerations case presentation management and outcome discussion acknowledgements references about the author(s) fatima b. jiya department of paediatrics, usmanu danfodiyo university teaching hospital, sokoto, nigeria paul k. ibitoye department of paediatrics, usmanu danfodiyo university teaching hospital, sokoto, nigeria nma m. jiya department of paediatrics, usmanu danfodiyo university teaching hospital, sokoto, nigeria maryam amodu-sanni department of paediatrics, usmanu danfodiyo university teaching hospital, sokoto, nigeria yahaya mohammed department of medical microbiology and parasitology, usmanu danfodiyo university teaching hospital, sokoto, nigeria dada m. aquib department of radiology, usmanu danfodiyo university teaching hospital, sokoto, nigeria lukman k. coker department of paediatrics, usmanu danfodiyo university teaching hospital, sokoto, nigeria citation jiya fb, ibitoye pk, jiya nm, et al. emphysematous pyelonephritis in an infant from sokoto, north-western nigeria. afr j lab med. 2021;10(1), a1181. https://doi.org/10.4102/ajlm.v10i1.1181 case study emphysematous pyelonephritis in an infant from sokoto, north-western nigeria fatima b. jiya, paul k. ibitoye, nma m. jiya, maryam amodu-sanni, yahaya mohammed, dada m. aquib, lukman k. coker received: 27 jan. 2020; accepted: 03 dec. 2020; published: 26 apr. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract introduction: emphysematous pyelonephritis is a life-threatening necrotising bacterial infection of the kidneys. it is rare among children and can be fatal if not promptly identified and treated. case presentation: a 7-month-old male infant presented to the emergency paediatric unit of usmanu danfodiyo university teaching hospital, sokoto, nigeria, on 12 november 2019 with a 5-day history of fever and vomiting, and a 3-day history of a progressively enlarging, left-side abdominal mass. there was associated excessive crying on micturition, refusal to feed and weight loss. he looked ill and was in respiratory distress, irritable, febrile (38.8 °c), moderately dehydrated and pale. his weight and length were 5.5 kg and 64 cm. he had a tender, firm and ballotable abdominal mass on the left flank measuring 8 cm × 10 cm. his pulse rate was 140 beats/min, blood pressure 60/40 millimetres of mercury and respiratory rate was 65 cycles/min. he had widespread coarse crepitations and normal heart sounds on chest auscultation. management and outcome: an initial diagnosis of sepsis was made. other considerations were nephroblastoma and neuroblastoma. ceftriaxone and blood transfusion were commenced with subsequent administration of intravenous fluids. further radiologic investigations revealed emphysematous pyelonephritis. the patient had percutaneous drainage and extended spectrum β-lactamase-producing escherichia coli (sensitive to meropenem) which was isolated from the aspirate culture after 48 h of incubation. meropenem could not be commenced because of non-availability and high cost. the patient subsequently deteriorated and died from septic shock. conclusion: emphysematous pyelonephritis has a fulminant course when not diagnosed promptly and treated adequately. keywords: emphysematous pyelonephritis; infection; kidney; infant; sokoto. introduction emphysematous pyelonephritis (epn) is a severe necrotising and progressive infection of the kidneys which is characterised by the formation of gas within the renal parenchyma, collecting system, or the perinephric tissue.1 it is rare in children, with the majority of cases occurring among adults with diabetes mellitus.2 gas-forming organisms are said to be the causative organisms of which escherichia coli is the most common.3 the pathogenesis is unclear but factors thought to increase predisposition to developing epn include decreased host immunity, increased levels of glucose in the tissue, impaired tissue perfusion, obstruction of the urinary system and presence of gas-forming organisms in the host tissue.1 the diagnosis of epn requires clinical features supported by radiologic investigations and isolation of the offending organisms. depending on the stage of the disease, treatment could be medical alone or a combination of medical and surgical interventions.1 ethical considerations ethical approval to conduct the study was obtained from uduth health research ethics committee with registration number nhrec/30/012/2019. authors obtained permission from the caregivers to publish the clinical details of the patient. case presentation we report the case of y.b (initials of infant used to retain anonymity), a 7-month-old male infant that was referred from a secondary health facility in sokoto, nigeria, to usmanu danfodiyo university teaching hospital (uduth), sokoto in november 2019. he was brought by his parents to the emergency paediatric unit of uduth on account of a 5-day history of fever and vomiting, and a 3-day history of progressively enlarging left-side abdominal mass which was noticed incidentally and said to be tender to touch. there was no preceding history of trauma and there were no masses on other body parts. there was associated excessive crying, crying on micturition, refusal to feed and weight loss. he was admitted at the referring hospital at the onset of illness where he had anti-malaria (artesunate), and antibiotic (cefuroxime) treatment, as well as a blood transfusion with no significant improvement, necessitating referral to uduth sokoto 72 h later. operating within a resource-constrained health system, uduth is the highest tertiary level healthcare facility within the state. both of his parents have no formal education and are ‘petty traders’ with a combined average earning of 40 000.00 nigerian naira ($132.00 united states dollars [usd]) per month. physical examination revealed an ill-looking child in respiratory distress, irritable, febrile (axillary temperature = 38.8 °c), moderately dehydrated, pale, anicteric acyanosed with no significant peripheral lymphadenopathy. his weight and length were 5.5 kg and 64 cm, while oxygen saturation (spo2) was 89% in room air. he had a tender, firm and ballotable abdominal mass extending from the left lumbar region to the left iliac region, measuring 8 cm × 10 cm. the right kidney, liver and spleen were not palpable. he had normal male external genitalia and was not circumcised. his pulse rate was 140 beats/min, blood pressure 60/40 millimetres of mercury and respiratory rate was 65 cycles/min. chest auscultation revealed vesicular breath sounds with widespread coarse crepitations and normal heart sounds. the neurologic examination was also normal. a clinical diagnosis of sepsis with focus on the chest and urinary tract with malaria was made. other considerations were nephroblastoma and neuroblastoma. management and outcome broad spectrum empirical antibiotic (intravenous ceftriaxone) treatment was commenced empirically, and the patient was transfused with blood (haematocrit was 22%) and subsequently placed on intravenous fluids. blood film for malaria parasite was negative, and metabolic panel and complete blood count were normal except for anaemia (table 1). urinalysis showed proteinuria, glycosuria and leucocyturia but urine microscopy and culture yielded no significant growth (table 1). human immunodeficiency virus dna polymerase chain reaction was negative. chest radiograph revealed multiple patchy perihilar opacities. abdominal ultrasound demonstrated relative renomegaly of the left kidney with a bipolar length of 99 mm, turbid collection with multiple pockets of air within it and marked thinning of the parenchyma. the right kidney was normal in outline, position and size (72 mm in bipolar length) and other organs were normal in appearance (figure 1). computerised tomography (ct) scan of the abdomen revealed an enlarged left kidney with bipolar length and transverse diameters of 97 mm and 68 mm, reduced renal parenchyma enhancement (hounsfield units = 36–42), while multiple oval and tubular negative density areas (hounsfield units = 543–723) were noted centrally and in subcapsular regions suggestive of air in the collecting system and renal parenchyma with overall features in keeping with epn. the right kidney was normal in position, outline and size (bipolar diameter 63.8 mm and transverse diameter 40.8 mm), no mass lesions or calculus were seen, and other organs were normal (figure 2 and figure 3). the patient’s diagnosis was changed to septicaemia complicated by unilateral (left) emphysematous pyelonephritis class ii. ceftriaxone treatment was continued while awaiting the culture result, and an ultrasound guided percutaneous drain was inserted, which drained about 60 ml of purulent, blood-stained material containing air bubbles. the result of aspirate microscopy, culture and sensitivity revealed numerous pus cells on microscopy, and growth of e. coli confirmed to be extended spectrum β-lactamase-positive via phenotypic method, which was sensitive only to meropenem. antibiotic therapy was changed to intravenous meropenem but could not be commenced due to unavailability, as well as the high cost of the medication where available. although there was reduction in his temperature to 37.9 °c, he subsequently developed shock and deteriorated rapidly. he did not respond to resuscitation and died 24 h later. although not the recommended treatment for this case with stage ii epn, nephrectomy was considered but the rapidity in deterioration in the patient’s condition (shock and death within few hours) did not allow for adequate counselling and preparation for the procedure. figure 1: sonogram of the left kidney of an emphysematous pyelonephritis patient at the usmanu danfodiyo university teaching hospital, sokoto, nigeria, november 2019. figure shows renomegaly, reduced renal parenchyma echogenicity and multiple irregular mixed echoes with dirty shadowing (blue arrow). figure 2: contrast enhanced computed tomogram of an emphysematous pyelonephritis patient at the usmanu danfodiyo university teaching hospital, sokoto, nigeria, november 2019. figure shows left renomegaly, contrast enhancement and multiple air densities within the calyceal system (orange arrow). figure 3: non–contrast-enhanced computed tomogram of an emphysematous pyelonephritis patient at the usmanu danfodiyo university teaching hospital, sokoto, nigeria, november 2019. figure shows left renomegaly with multiple air density area within the parenchyma and subcapsular region (orange arrows). table 1: laboratory data of the emphysematous pyelonephritis patient on the second day of admission at the usmanu danfodiyo university teaching hospital, sokoto, nigeria, november 2019. discussion the 7-month-old male infant in this study had acute onset of fever, vomiting and a left-side ballotable abdominal mass with radiologic features of stage ii epn confirmed to be caused by extended spectrum β-lactamase e. coli on aspirate microscopy, culture and sensitivity. emphysematous pyelonephritis is a severe necrotising infection of the renal parenchyma that causes gas accumulation within the renal tissues, with or without the involvement of the peri-renal spaces.4,5 reports of epn has been documented in adults, especially among diabetics, hypertensives and renal transplant recipients following end-stage renal disease.6 women are said to be more at risk than men.1,5 emphysematous pyelonephritis is rare among children with the first known paediatric case reported in a 10-year-old female in 1985.7 there are few reports of epn among children in studies from south africa,8 texas9 and saudi arabia,10 with none of the patients having diabetes mellitus nor was there significant gender preponderance in the occurrence of epn. however, one of the reported cases was a transplant recipient.10 the index patient is, to the best of our knowledge, the first documented case in a child in sokoto state, and possibly in nigeria. unlike in previous reports9,10 in which cases had underlying medical conditions (chromosomal abnormality with ectopic right ureter, and end-stage renal disease from neurogenic bladder), our case was apparently healthy with no identifiable risk factors prior to the development of epn. possibly, our case is an indication that epn can in very rare instances occur in previously apparently healthy children. the most common causative bacterial organism is e. coli, which is also the organism that was isolated from the aspirate specimen of our patient. other pathogenic agents of epn include klebsiella, proteus, pseudomonas, citrobacter, enterococcus and streptococcus species. rare organisms such as coagulase negative staphylococcus, clostridium, candida species and aspergillus fumigates have also been reported.1,3,9,10 unlike in our case, the study by siddique et al.9 reported enterobacter cloacae as the causative agents of epn in their patient. the pathogenesis of epn is not yet known; however, the formation of carbon dioxide from the fermentation of glucose in urine and kidney tissues is thought to be the main mechanism.1 the predisposing factors that have been implicated include the presence of a gas-forming bacterial organisms, high glucose levels in tissues, immunosuppression (e.g. diabetes and immunosuppressive therapy), urinary tract obstruction, and poor tissue perfusion.1,11,12 local tissue ischaemia in the presence of a gas-producing pathogen is thought to exacerbate tissue destruction, encourage the production of pus, and inhibit the removal of locally produced gas, leading to epn.11 other speculated mechanisms are that the increased levels of glucose in the tissues together with decreased blood supply to the kidneys contributes to the anaerobic metabolism of glucose and lactate by the organisms and thereafter the production of gases like carbon dioxide, hydrogen, nitrogen, oxygen and methane by the gas-forming organisms.13 the clinical manifestation of epn in children is similar to that of adults with the main features being fever, anorexia, nausea, vomiting, flank pain with or without palpable mass, and dysuria.7,13 our patient presented with some of the aforementioned symptoms. the classification of epn is via radiologic imaging, and the most commonly used classification system is by huang and seng using ct scan to classify epn into five (1–4b) classes.1 our patient’s ct scan findings (figure 2 and figure 3) placed him at epn class ii because the demonstrated air went beyond the left collecting system to involve the renal parenchyma. although earlier studies9,10 reported renal ultrasonograms suggesting air collection in the renal parenchyma of their patients, epn was not staged using a impossible ct scan in the studies making it impossible to compare the stage of epn in our study with theirs. the clinical presentation, radiologic imaging and isolation of causative organisms confirms the diagnosis of epn.1 modalities of treating patients with epn are said to have changed over time, with intensive conservative management assuming a prominent position, depending on the class of epn and other co-morbidities. broad spectrum antibiotic therapy with percutaneous drainage is the standard recommended treatment protocol for epn classes i and ii. the treatment options for patients with epn classes iii and iv depend on the presence and number of risk factors such as shock, acute kidney injury, thrombocytopaenia and coma. the choice of treatment for patients with fewer than two risk factors is antibiotics and percutaneous drainage. in the presence of two or more risk factors, nephrectomy is the recommended treatment.1 our patient had percutaneous drainage but could not commence the appropriate antibiotic (meropenem) therapy due to non-availability and high cost. although uduth is the highest tertiary level option within the state, meropenem is not indigenously produced to the best of our knowledge. although available, his parents could not afford the supply of ten 500 mg powder for the injection, which cost 18 000.00 nigerian naira ($59.00 usd).14 unlike in our patient, other studies have reported good response to antibiotics like third-generation cephalosporin, fluoroquinolones and vancomycin.6,8,9 good outcomes for epn have been reported with prompt initiation of recommended treatment.9 however, epn may run a fatal course if not identified and treated early.4,5,9 there was a delay in diagnosing the index case due to late presentation. additionally, the isolated organism (extended spectrum β-lactamase e. coli) was resistant to the empirical antibiotic (ceftriaxone) therapy. the recommended antibiotic (meropenem) could not be commenced because of the aforementioned reasons. it is, therefore, not surprising that the patient succumbed to the illness. conclusion our experience brings to the fore the occurrence of epn in an infant and the fulminant course it can take when not diagnosed promptly and treated adequately. our case highlights the importance of a high index of suspicion, the resistance of extended spectrum β-lactamase e. coli to the empirical antibiotic (ceftriaxone), and the need to ensure availability as well as cost effectiveness of recommended antibiotics in the study location. these measures will go a long way in the timely identification of cases and will improve the outcome of management of initial classes (1–3) of epn. acknowledgements competing interests the authors have declared that no competing interests exist. authors’ contributions f.b.j. conceptualised, designed and wrote the original draft. p.k.i., n.m.j. and y.m. critically revised and supervised the manuscript. m.a.-s., d.m.a. and l.k.c. acquired the data. all authors approved the final version to be published. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references huang jj, tseng cc. emphysematous pyelonephritis: clinicoradiological classification, management, prognosis, and pathogenesis. arch intern med. 2000;160(6):797–805. https://doi.org/10.1001/archinte.160.6.797 pontin ar, barnes rd, joffe j, kahn d. emphysematous pyelonephritis in diabetic patients. br j urol. 1995;75(1):71–74. https://doi.org/10.1111/j.1464-410x.1995.tb07237.x mohsin n, budruddin m, lala s, al-taie s. emphysematous pyelonephritis: a case report series of four patients with review of literature. ren fail. 2009;31(7):597–601. https://doi.org/10.1080/08860220903003396 lu yc, chiang bj, pong yh, et al. emphysematous pyelonephritis: clinical characteristics and prognostic factors. int j urol. 2014;21(3):277–282. https://doi.org/10.1111/iju.12244 schicho a, stroszczynski c, wiggermann p. emphysematous cystitis: mortality, risk factors, and pathogens of a rare disease. clin pract. 2017;7(2):930. https://doi.org/10.4081/cp.2017.930 camelia a, abhijit s, shankar a, et al. a case series of emphysematous pyelonephritis. case rep med. 2014;2014:587926. https://doi.org/10.1155/2014/587926 pode d, perlberg s, fine h. emphysematous renal and perirenal infection in nondiabetic patient. urology. 1985;26:313–315. https://doi.org/10.1016/0090-4295(85)90139-6 ambaram pr, petersen kl. emphysematous pyelonephritis in children. pediatr infect dis j. 2016;35(10):1159–1161. https://doi.org/10.1097/inf.0000000000001254 siddique k, seikaly mg. emphysematous pyelonephritis in an infant. pediatr infect dis j. 2013;32(10):1157–1158. https://doi.org/10.1097/inf.0b013e31829aaae3 al-makadma as, al-akash si. an unusual case of pyelonephritis in a pediatric renal transplant recipient. pediatr transplant. 2005;9(2):258–260. https://doi.org/10.1111/j.1399-3046.2004.00276.x turney jh. renal conservation for gas-forming infections. lancet. 2000;355(9206):770–771. https://doi.org/10.1016/s0140-6736(99)00351-7 yang wh, shen nc. gas-forming infection of the urinary tract: an investigation of fermentation as a mechanism. j urol. 1990;143(5):960–964. https://doi.org/10.1016/s0022-5347(17)40151-0 singh su, mcglynn l, fordham m. emphysematous pyelonephritis. bju int. 2010;107(9):1474–1478. https://doi.org/10.1111/j.1464-410x.2010.09660.x drugs.com. meropenem prices, coupons and patient assistance programs [homepage on the internet]. [cited 2019 nov 10]. available from: http://www.drugs.com abstract introduction methods results discussion acknowledgements references about the author(s) koumpingnin nebie national blood center of ouagadougou, ouagadougou, burkina faso laboratory of haematology, department of fundamental sciences, health sciences research and training unit, university joseph ki-zerbo, ouagadougou, burkina faso salam sawadogo national blood center of ouagadougou, ouagadougou, burkina faso laboratory of haematology, department of fundamental sciences, health sciences research and training unit, university joseph ki-zerbo, ouagadougou, burkina faso salifo sawadogo national institute for medical sciences, university nazi boni, bobo-dioulasso, burkina faso souro sanou teaching hospital, bobo-dioulasso, burkina faso jérôme koulidiati laboratory of haematology, department of fundamental sciences, health sciences research and training unit, university joseph ki-zerbo, ouagadougou, burkina faso yalgado ouedraogo teaching hospital, ouagadougou, burkina faso habi y.a. lengani yalgado ouedraogo teaching hospital, ouagadougou, burkina faso abdoul g. sawadogo national blood center of ouagadougou, ouagadougou, burkina faso jérôme babinet centre national de référence pour les groupes sanguins (cnrgs), national institute for blood transfusion, paris, france mohammed khalloufi french establishment of blood, bobigny, france saliou diop department of haematology, university cheikh anta diop, dakar, senegal eléonore kafando laboratory of haematology, department of fundamental sciences, health sciences research and training unit, university joseph ki-zerbo, ouagadougou, burkina faso laboratory of haematology, paediatric teaching hospital charles de gaulle, ouagadougou, burkina faso citation nebie k, sawadogo s, sawadogo s, et al. red blood cell alloimmunisation in multi-transfused patients from an haemodialysis service in burkina faso. afr j lab med. 2022;11(1), a1625. https://doi.org/10.4102/ajlm.v11i1.1625 original research red blood cell alloimmunisation in multi-transfused patients from an haemodialysis service in burkina faso koumpingnin nebie, salam sawadogo, salifo sawadogo, jérôme koulidiati, habi y.a. lengani, abdoul g. sawadogo, jérôme babinet, mohammed khalloufi, saliou diop, eléonore kafando received: 12 may 2021; accepted: 26 may 2022; published: 26 sept. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: in burkina faso, red blood cell (rbc) transfusion remains the crucial anaemia treatment following chronic renal failure (crf) as erythropoietin and its analogues are unavailable. however, blood group matching beyond the abo and rhesus is not common in burkina faso. thus, alloimmunisation is a potential issue for transfused patients. objective: our study aimed to identify anti-erythrocyte antibodies in multi-transfused crf patients at the yalgado ouedraogo teaching hospital, ouagadougou, burkina faso. methods: this cross-sectional study, conducted from october 2018 to november 2019, included crf patients who had received at least two rbc units. we screened patients for the presence of rbc antibodies using three commercial cells panels and identified antibody specificities for positive screenings using 11 cells panels for an indirect antiglobulin test (iat) in a low ionic strength microcolumn gel-card system. results: two hundred and thirty-five patients (45.1% female; average age: 41.5 years) were included. the median number of blood units received per patient was 10 (interquartile range: 5–20). the overall alloimmunisation rate was 5.9% (14/235). antibodies identified included: anti-d (1 case), anti-c (1 case), anti-d+c (4 cases), anti-cw (1 case), anti-e (1 case), anti-s (1 case) and anti-lea (1 case). in four positive patients, the specificity of the antibodies was indeterminate. no risk factors were associated with alloimmunisation. conclusion: in burkina faso, screening for rbc alloantibodies should be mandated for patients at risk. the high rate of indeterminate antibodies suggests the need to develop a local rbc antibody panel adapted to the local population. keywords: blood transfusion; alloimmunisation; rbc antibody; crf; burkina faso. introduction blood transfusion, specifically the transfusion of red blood cells (rbc), significantly contributes to the modern healthcare system. every day, blood transfusion saves lives in developing countries where acute anaemia caused by malaria, sickle cell disease (scd), pregnancy-related events and other trauma remains high or is on the rise. for example, in burkina faso, around 103 731 rbc units were used in 2017.1 however, this did not meet the transfusion needs of the country. moreover, this number is far lower than the theoretical needs of around 196 000–580 000 per the world health organization estimation method iii (i.e. 1% – 3% of the 19.5 million inhabitants).2 besides the chronic blood shortage, developing countries also face poor quality of blood products and their unsafe use.3 indeed, residual risks of transfusion-transmitted infections remain high4,5 due to inadequacies in blood donor selection and retention and laboratory screening of blood donations. furthermore, blood transfusion adverse events are underestimated due to the weakness or nonexistence of haemovigilance and quality management systems.6,7 finally, although transfusion-transmitted infections and major blood groups matching errors are worrying, blood transfusion safety issues, alloimmunisation and the occurrence of alloantibody are also pressing issues, especially in multi-transfused patients such as those undergoing haemodialysis for chronic renal failure (crf).4,8,9,10 anaemia is highly prevalent in end-stage renal disease patients, often non-regenerative normochromic normocytic anaemia caused by inadequate renal erythropoietin production. erythropoietin infusions or other erythropoietin-stimulating agents manage anaemia in end-stage renal disease patients. the united states food and drug administration recommends an erythropoietin haemoglobin target range of 100 g/l – 120 g/l11 and expressly states that erythropoietin-stimulating agents should be used to increase haemoglobin only to the level necessary to avoid transfusion.11,12,13 in 2016, an expert committee advocated for including erythropoietin-stimulating agents in the world health organization model list of essential medicines to reduce the need for transfusions in patients with end-stage chronic kidney disease. erythropoietin-stimulating agents prevent transfusion-related risks, facility requirements, and risk management costs in the event of possible harm (infections, haemosiderosis).14 however, erythropoietin-stimulating agents treatments are out of reach for most patients in our context. therefore, rbc transfusion is used to manage crf-related anaemia and scd patients. meanwhile, our country faces poor pre-transfusion compatibility practices; abo and rhd matching is the only mandatory screening for rbc transfusions. no other blood group is considered, and no alloantibody screening or compatibility test is performed.15 given this context, high rbc alloantibodies frequency is expected among transfused patients; however, there is a paucity of data on this. thus, this study determined the frequency of anti-erythrocyte alloimmunisation and identified alloantibody specificities among crf multi-transfused patients in yalgado ouedraogo teaching hospital, ouagadougou, burkina faso. methods ethical considerations both the yalgado ouedraogo teaching hospital direction and the internal ethical committee of the national blood transfusion centre (authorisation no. 015/cnts/dg/cirs, 03/23/2018) approved the study. the nurses and the medical doctor in charge of the interview and other data collection obtained verbal informed consent. also, the data were password protected and accessible only by the first author. results were shared with staff and patients and used to influence patients’ future transfusions. study setting this study was conducted in the nephrology and haemodialysis unit of the teaching hospital yalgado ouedraogo of ouagadougou, burkina faso, where about 400 patients with chronic kidney failure undergo haemodialysis yearly. we conducted this cross-sectional study from january 2018 to december 2019 and included haemodialysis end-stage chronic kidney failure patients who had ever received rbc transfusions at least twice. socio-demographic information, clinical data, and medical history of each included patient were recorded on a standardised survey form during an in-person interview with the medical doctor responsible for the study or trained nurses. data collected include gender, age, date of the first transfusion, date of the last transfusion received, number of transfusions, the total number of blood units received since crf started, and number and type of adverse reactions related to transfusions reported. additionally, the number of pregnancies, live and still births, abortions, and anti-d injection use were also reported for female patients. five mililitres of blood was drawn from each patient into ethylenediaminetetraacetic acid tubes. the sample was centrifuged, and the obtained plasma was used for alloantibodies screening and identification. testing methods we used the indirect antiglobulin test method with the gel column agglutination card technique (invitrogel ahg, mtc invitro diagnostics ag, bensheim, germany). in this technique, the gel column contains an anti-human antibody that traps irregular antibodies present in a patient’s plasma and fixed on rbc. agglutinated rbcs are trapped in the gel column, making the agglutination easy to read.16,17 in the first step, a panel of three rbc reagents (invitrocell screen i-ii-ii, mtc invitro diagnostics ag, bensheim, germany), that targets antigens d, c, c, e, e, v, cw, k, k, kpa, kpb, jsa, jsb, fya, fyb, jka, jkb, lea, leb, p1, m, n, s, s, lua, lub and xg*a antibodies, were used for screening. samples positive for any antibody were further tested to identify antibody specificity using an 11 rbc panel (invitrocell ident 11, mtc invitro diagnostics ag, bensheim, germany) that targets the same antigens in the screening stage. an enzyme-treated rbc panel was not used. statistical analyses we used epi-infotm software (version 7.2.2.2, centers for disease control and prevention, atlanta, georgia, united states) for data analysis. the frequencies and percentages are given with a 95% confidence interval. we used the chi-square test to compare proportions, and differences were considered significant for p < 0.05. results baseline characteristics during the study period, 235 patients with crf were included, comprising 45.1% (106/235) female patients. the mean age was 41.9 (standard deviation 14.5 years; median 41 years; range 15–86 years). the mean number of received rbc units was 18 units ranging from two to 160 rbc, while the median number of received blood units per patient was 10 (interquartile range: 5–20). about 55.2% (128/232) had received more than 10 rbc units (table 1). table 1: social and demographic characteristics of patients with chronic renal failure, yalgado ouedraogo teaching hospital of ouagadougou, burkina faso, 2018. red blood cell alloimmunisation prevalence of the 235 patients included, 14 had alloantibodies, representing an overall positivity rate of 5.9%. four of the 14 patients (28.6%) had indeterminate antibody specificity. in 10 patients, 14 antibodies were identified: 5 anti-d, 5 anti-c, 1 anti-e, 1 anti-cw, 1 anti s, 1 anti-lea; four patients were positive for both anti-d and anti-c (table 2). table 2: characteristics of patients with red blood cell alloimmunisation, yalgado ouedraogo teaching hospital of ouagadougou, burkina faso, 2018.† most antibodies (12 of 14; 85.7%) were of the anti-rh blood group antigens, with anti-d and anti-c being the most prevalent, each accounting for 35.7% (table 3). table 3: specificity and frequency of alloantibodies found among multi-transfused patients of the yalgado ouedraogo teaching hospital of ouagadougou, burkina faso, 2018.† red blood cell alloimmunisation risk factors there were no differences in the mean age (43.3 vs 41.8 years, p = 0.21) and the mean number of blood units received (13.0 vs 15.7 rbc units, p = 0.36) between immunised and non-immunised patients. the alloimmunisation rate was higher in patients who had received more than 10 rbc units (7.4% vs 2.3%, p = 0.12), but this difference was statistically insignificant. there were no other factors associated with alloantibodies (table 4). table 4: factors associated with alloimmunisation in multi-transfused patients with chronic renal failure, yalgado ouedraogo teaching hospital, ouagadougou, burkina faso, 2018. discussion our study aimed at determining the frequency and the specificity of alloantibodies among the multi-transfused haemodialysis crf patients at the teaching hospital yalgado ouedraogo of ouagadougou (burkina faso). we found an alloimmunisation rate of 5.9% with antibodies mainly of the anti-rh blood group antigens specificity. this study overviews of rbc immunological risks among patients with chronic diseases who are lifelong blood transfusion patients. although there have been recent changes in the blood transfusion system in burkina faso, including the replacement of multiple hospital-based blood banks with a centralised system, standardisation and harmonisation of practices,6,18,19,20 improved blood collection and infectious disease screening, some improvements towards managing blood recipients are necessary. for example, compactibility screening is still limited to the abo and rhd antigens, contrary to obtainable standards in high-income countries, where rare groups, at least rh-kell major antigens, are screened for before transfusion. moreover, in burkina faso, alloantibody screening tests and laboratory compatibility tests using at least an indirect antibody test as recommended is not implemented: the patient’s plasma and a sample of the rbc units are tested for agglutination on a glass surface. this study was the first in the country to use the gel column card method, one of the current best methods for alloantibody screening. nevertheless, the study presents some limitations as complementary antibody identification techniques, such as enzyme-treated red cells reagents panels (papain, bromelain or other) or wide-range panels, were not used. the lack of complementary identification can explain the high rate (4 of 14; 28.7%) of alloantibody undetermined specificity (inconclusive antibody identification).21 the overall alloimmunisation rate of 5.9% among crf patients undergoing haemodialysis on our study is consistent with the findings of two systematic reviews and meta-analysis studies conducted by ngoma et al. in 2015 and boateng et al. in 2019. these studies reported an overall alloimmunisation rate of 6.95% and 7.5% in sub-saharan africa.22,23 our results are similar to those of kafando et al., who found an alloimmunisation rate of 4.2% among children transfused with rh-kell unmatched blood units.24 alloimmunisation rates in the same range were reported in uganda (6.1%), rwanda (6.4%), sudan (4.0%) and tanzania (4.1%). however, some reported higher rates: uganda in 2010 (10.2%), mali in 2013 (10.3%) and nigeria in 2015 (9.3%).22,25,26,27,28 these results reflect the poor immunological safety of blood transfusions in sub-saharan africa, where blood transfusion is performed based only on the blood donor and recipient abo and rhd antigens matching. alloimmunisation rates observed in our study and other studies from sub-saharan africa are lower than those observed in europe and north america when they only screened for abo and rhd. prevalences ranged from 18% to 76% in the united kingdom and united states.29,30,31,32,33,34 in france, the rate was about 30%.35,36 despite the mandatory donor and recipient rh-kell antigens matching before transfusion in these developed countries, alloimmunisation rates in those settings are higher than ours.34,37,38 this serves as a reminder that the risk of alloimmunisation is multiparametric, depending on the population’s subgrouping or prevalent diseases.30,36 also, high rates of alloimmunisation could be due to antigen discrepancies between transfused rbc concentrates collected from donors with european ancestry and scd recipients who are often of sub-saharan african descent.36,37 a similar hypothesis was assumed in some other countries with multi-ethnic groups, such as iran.39 in burkina faso, blood group antigen distribution is established for abo and rhd within blood donors and patients.40,41 there is no data about rh subgroups or other important rbc antigens. it is known that significant differences in the distribution of blood group antigens within the country’s natural ethnic groups could exist. sawadogo et al. found that the phenotype o was more frequent in the central-west, central and east regions corresponding to ‘mossi’, ‘gourounsi’, and ‘gourmantché’ areas, whereas the phenotype a and ab were more prevalent in ‘boucle du mouhoun’ and ‘hauts-bassins’ regions and the ‘bwaba’ and ‘bobo’ areas. the phenotype o negative was infrequent in ‘bwaba’.40 these studies suggest that in burkina faso, with more than 50 ethnic groups, dominant blood groups vary between or are specific to particular ethnic groups. thus, new studies should be conducted to establish blood subgroup frequencies and rbc matching strategies in the country. in our study, the antibody specificity of four participants of 14 (28.6%) was indeterminate. this impairment could be due to the discrepancy between the european-sourced red cell reagent panel and our population. this situation highlights the need to implement local panels for rbc alloantibodies testing as with some other lowand middle-income countries.42,43,44 furthermore, boateng et al. claim that creating and maintaining a database of phenotyped blood donors will facilitate the selection of matched blood components for emergency transfusions as seen in sub-saharan africa and help locally manufacture rbc reagents. thus, rbc alloantibodies screening may become more economical and sustainable for multi-transfused patients, particularly patients with scd in this zone.22 the majority (85.7%) of the alloantibodies found in this study were anti-rh group antigens. anti-d and anti-c antibodies accounted for 35.7%, followed by anti-e and anti-cw. in a previous study of children who received transfusions in burkina faso, anti-c and anti-e were the most frequent.24 in our study, the two mainly represented antibodies were co-associated (anti-d+c) in 4 of 14 patients. the predominance of rh group antibodies was also reported in some other west african countries, as well as in côte d’ivoire,45 mali,26 senegal46 and nigeria,27,47 but in these studies, anti-e was the most often encountered when compared to anti-d and anti-c. surprisingly, we found rh anti-d antibodies, which could be due to errors occurring during patients’ blood typing. our hospital has reported as many as 46 blood typing errors yearly (unpublished data). another hypothesis is that partial rhd antigen carriage is frequent in individuals with african ancestry. in this case, an rhd-positive patient can develop alloantibodies after receiving rhd-positive rbc, as reported by chou et al.30,48 the same hypothesis applies to partial c carriers.49 this study tried to identify the risk factors associated with alloimmunisation. neither gender, age, nor the number of blood units received was associated with alloimmunisation in our study. however, ifeoma et al.47 in nigeria, senghor et al.46 in senegal and natukunda et al.50 in uganda have associated these factors with alloimmunisation; the small size of our sample might have prevented the observation of such associations. limitations one limitation of this study was that we could not screen for antibodies within a reasonable time after each transfusion. for many patients, screening occurred months or years after the last transfusion event. this delay may have impacted the alloimmunisation rate. conclusion this study showed that rbc alloimmunisation is a reality in multi-transfused patients in burkina faso. therefore, exhaustive donor-patient blood matching beyond abo and rhd is necessary for lifelong transfused patients, such as crf and scd patients. further investigations are needed to efficiently establish the distribution of rbc antigens and phenotypes among blood donors and patients in the country, which may facilitate rbc reagent manufacturing. acknowledgements salfo kellé for patients interview, data and sample collection. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions k.n. designed the study, collected data, participated in samples testing, contributed to data analysis and drafted the manuscript. salam sawadogo, salifo sawadogo, j.k., j.b., h.y.a.l. and a.g.s. contributed to designing the study, data analysis and interpretation. m.k., s.d. and e.k. critically reviewed and revised the manuscript. all of the authors approved the final version of the manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability data are available from the corresponding author, k.n., upon request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy 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https://doi.org/10.1111/j.1537-2995.2009.02382.x natukunda b, mugyenyi g, brand a, schonewille h. maternal red blood cell alloimmunisation in south western uganda. transfus med. 2011;21(4):262–266. https://doi.org/10.1111/j.1365-3148.2011.01073.x abstract introduction methods results discussion acknowledgements references about the author(s) oluwaseyi s. ashaka department of medical microbiology and parasitology, college of health sciences, university of ilorin, ilorin, kwara state, nigeria olajide o. agbede department of medical microbiology and parasitology, college of health sciences, university of ilorin, ilorin, kwara state, nigeria adesuyi a. omoare department of medical microbiology and parasitology, college of health sciences, university of ilorin, ilorin, kwara state, nigeria samuel k. ernest department of paediatrics and child health, college of health sciences, university of ilorin, ilorin, kwara state, nigeria citation ashaka os, agbede oo, omoare aa, ernest sk. human parvovirus b19-induced anaemia in pre-school children in ilorin, nigeria. afr j lab med. 2018;7(1), a615. https://doi.org/10.4102/ajlm.v7i1.615 brief report human parvovirus b19-induced anaemia in pre-school children in ilorin, nigeria oluwaseyi s. ashaka, olajide o. agbede, adesuyi a. omoare, samuel k. ernest received: 09 feb. 2017; accepted: 02 dec. 2017; published: 10 may 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract sera collected from 57 anaemic and 115 non-anaemic age-matched pre-school children in ilorin, nigeria, between november 2014 and december 2015 were assayed for human parvovirus b19-specific igm antibodies by using the enzyme linked immunosorbent assay technique. a total of 17 (29.8%) anaemic children and 18 (15.7%) non-anaemic children were positive for parvovirus b19 infection. infection with parvovirus b19 is common in this population, and screening for the virus during differential diagnosis is recommended. introduction parvovirus b19 is a small, non-enveloped, single-stranded dna virus.1 the nucleocapsid of the virus has icosahedral symmetry. parvovirus b19 is transmitted through the respiratory routes and through contact with infected blood and blood products to susceptible individuals, including children with underdeveloped immune systems. risk factors associated with parvovirus b19 infection include blood transfusion, circumcision, tribal marks and scarification.2 interest in parvovirus b19 has increased because of the burden of anaemia among children in developing countries where multiple other causes of anaemia abound.3,4 the cause of anaemia among pre-school children living in sub-saharan africa has been associated with malaria, haemoglobinopathy, iron deficiency, folic acid deficiency, vitamin b12 deficiency, vitamin a deficiency, hiv infection, helminthic infestation, sickle cell disease and autoimmune haemolytic anaemia3. in nigeria, anaemia may also be due to iron deficiency resulting from insufficient dietary intake in children. the majority of hospital admissions for children under the age of five years have been attributed to malaria.5 in addition, sickle cell disease contributes significantly to the burden of anaemia in this population.6 some reports on the co-infection of plasmodium sp. with parvovirus b19 exists and it is known that both agents affect red blood cells.7 the association of human parvovirus b19 with anaemia, especially among pre-school children, has been established by other studies, which have reported levels between 7% and 14%.4 parvovirus b19 preferentially targets young erythrocytes and temporarily suppresses red blood cell production.8 parvovirus infection in anaemic pre-school children contributes to anaemia and could also be responsible for increased childhood morbidity and mortality among children with other underlying causes of anaemia.9 this raises the index of suspicion that parvovirus b19 may be involved in increasing the burden of anaemia in pre-school children. this study was initiated at the university of ilorin teaching hospital and the children’s specialist hospital to determine the status of pre-school children with anaemia regarding possible infection with human parvovirus b19. methods ethical considerations this study was conducted in compliance with the helsinki declaration and was approved by the health research and ethics committee of the university of ilorin teaching hospital and the hospital management bureau at the children’s specialist hospital (approval number erc pan/2014/09/1314). the parents or guardians of participants gave written informed consent before their children were enrolled in the study. all data were analysed anonymously throughout the study. a semi-structured questionnaire was administered to obtain relevant information on the children’s sociodemographic characteristics; evidence of rash and laboratory findings were documented. study design and participants this was an observational, prospective, cross-sectional, hospital-based case control study. patients were consecutively recruited from among pre-school children who visited the university of ilorin teaching hospital or the children’s specialist hospital between november 2014 and december 2015. all consecutive patients visiting either hospital were recruited. all patients recruited resided in ilorin, a city characterised by communal living, an open sewage system, and challenges for water utilisation and sanitation in its underdeveloped urban areas. all patients presenting with anaemia at either hospital were identified using a protocol set up by the paediatric clinics that classified patients with haematocrit values of ≤ 30% as anaemic (cases) and patients with haematocrit values of > 30% as non-anaemic (controls).10 controls with a significant fever (temperature ≥ 38 °c) were excluded from the study. it should be noted that the type of anaemia examined in this study was based on reticulocyte count with normal range value of 0.8% – 2.2% (haematocrit value: 31.7% – 39.6%). patients with sickle cell disease were deliberately excluded from this study. serologic testing serum samples were collected from all participants. three millilitres of blood was drawn from each participant by a paediatrician. aliquots were decanted into tubes without anticoagulant for parvovirus b19 immunoglobulin m (igm) serology and into edta anticoagulant tubes for haematocrit analysis to confirm anaemia. blood sample bottles were labelled with a unique sample code starting with ‘a’ for cases (children with anaemia) and a code starting with ‘c’ for controls. determination of haematocrit was done by centrifugation using standard procedures at a fixed speed of 11 000 revolutions per minute (rpm) for 10 minutes (hawksley & sons ltd, sussex, united kingdom). serum samples were separated by centrifugation at 400 revolutions per minute and stored at -20°c for three months after which the enzyme linked immunosorbent assay (elisa) was performed. the elisas used a recombinant vp1 protein to capture igm antibody response to viral capsid protein-1 antigen of human parvovirus b19 (vircell, granada, spain). all assays were performed according to the manufacturer’s written instructions (vircell, granada, spain). parvovirus antigen that contained inactivated parvovirus b19 antigen (vp2 obtained in baculovirus) was used. positive control which contained serum with parvovirus antigen and negative control contained serum without parvovirus antigen were tested with each batch of patient samples. statistical analysis the generated data were systematically analysed as appropriate for mean, proportion and chi-square test using spss for windows statistical software version 18.0 (spss inc., chicago, illinois, united states; 2009). the chi-square was used to determine differences between groups and a one-sided probability of < 0.05 was considered statistically significant. every child meeting the case definition was enrolled in the study. age-matched children (±5 months from the age of a case) were selected as controls from among children meeting the inclusion criteria who attended the health facility during the study period. results a total of 172 pre-school children younger than age five participated in the study; 92 were boys, 80 were girls and the mean age for all children was 1.66 years. a total of 21 (22.82%) boys and 14 (17.5%) girls were positive for human parvovirus b19 infection. a total of 57 participants were anaemic, of whom 17 (29.8%) were positive for parvovirus b19 infection; 115 participants were non-anaemic, of whom 18 (15.7%) were positive for parvovirus b19 infection (table 1). a large percentage of anaemic participants (n = 40; 70.2%) were negative for parvovirus b19 infection. overall, 97 (84.4%) participants had no anaemia and no parvovirus b19 infection. only two of the 17 anaemic children positive for parvovirus b19 infection had visible rash. table 1: comparison of human parvovirus b19 infection among anaemic and non-anaemic children, ilorin, nigeria, november 2014–december 2015 (n = 172) prevalence of parvovirus b19 infection among anaemic participants was highest among the 13–24-month age group (n = 7) (figure 1). prevalence of parvovirus b19 infection among non-anaemic participants was highest among the 49–60 month age group (n = 6). figure 1: distribution of human parvovirus b19 infection among age groups of anaemic and non-anaemic pre-school children in ilorin, nigeria, november 2014–december 2015 (n = 172). discussion our study found that both anaemic and non-anaemic pre-school children living in ilorin, nigeria tested positive for human parvovirus b19 infection. of the 57 anaemic children, 17 (29.8%) were parvovirus b19-positive, and of the 115 non-anaemic children, 18 (15.7%) were parvovirus b19-positive. few studies have reported on prevalence of parvovirus b19 infection in children. the prevalence of parvovirus b19 infection in such studies ranges between 5% and 14.3% among children with sickle cell anaemia.11,12 this is lower than the prevalence of 29.8% we found among pre-school children with anaemia in ilorin. our study also found that parvovirus b19-infection prevalence was higher in children with anaemia when compared to non-anaemic controls and that the difference was statistically significant. this difference may be as a result of recent infection with human parvovirus b19 in this study population. a study in italy reported that parvovirus b19 can persist in immunocompetent symptomatic and non-symptomatic individuals by the presence of viral dna in different tissue but this was observed in the absence of viraemia and anti-b19 igm.13 these results have implications for children under the age of five years, because such children are vulnerable to the detrimental effects of anaemia. the presence of infection due to human parvovirus b19 may exacerbate anaemia among children who had already have anaemia due to other causes. that a majority of anaemic pre-school children tested negative for human parvovirus b19 infection points to other aetiologies as the cause of anaemia in these children. in the tropics, anaemia may be due to iron deficiency resulting from insufficient dietary intake in children. in nigeria, the majority of hospital admissions among children younger than the age of five years have been attributed to malaria.14 malaria is thought to be the primary cause of severe anaemia in at least 50% of people living in malaria-endemic areas.5 it has also been shown that parvovirus b19 infection can play a critical role in the aetiology of severe anaemia in areas that are highly endemic for malaria.15 our results also have implications for children living in the sickle cell belt of the tropics. nigeria has a carrier rate for the sickle cell gene of 20%–30%, and sickle cell disease affects 2%–3% of the nigerian population of more than 160 million people.6 sickle cell anaemia may well become a life-threatening complication in patients infected with parvovirus b19. the presence of parvovirus b19 infection may exacerbate anaemia in children that have sickle cell disease.16 among the 115 non-anaemic controls in this study, 18 (15.7%) tested positive for human parvovirus b19 infection. it is possible that these children had been exposed to parvovirus b19 recently and that they had recovered from infection-related anaemia prior to enrolment in the study. it could also be that the children who tested positive were truly anaemic but their haematocrit values had not risen to the cut-off value used in this study. limitations there are some limitations to our study, which should be noted. we were unable to perform pcr, qpcr or other genotypic testing for parvovirus b19 due to limited resources. additionally, the dynamics of recovery from anaemia as a result of rapid erythropoiesis in healthy infected children and the impact of other anaemia-causing factors may have influenced results obtained in this study. recommendations there is an urgent need to initiate specific public health interventions to prevent anaemia and its attendant consequences in children. appropriate screening for parvovirus b19 with igm antibody detection and nucleic acid detection would exclude this viral agent during differential diagnosis of anaemia common in this age group. conclusions the high prevalence rate found in our study shows that human parvovirus b19 infection is common in this population of pre-school children. acknowledgements competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support none. authors’ contributions o.s.a. was responsible for writing the publication, designing the experiment, data analysis, laboratory investigation and sample collection. o.o.a. was responsible for designing the experiment and writing the publication. a.a.o. was responsible for laboratory investigation and data analysis and s.k.e. was responsible for designing the experiment and sample collection. references rogo ld, mokhtari-azad t, kabir mh, rezaei f. human parvovirus b19: a review. actavirologica. 2014;58(3):199–213. lefrere j, servant-delmas a, candotti d, et al. persistent b19 infection in immunocompetent individuals: implications for transfusion safety. blood. 2005;106:2890–2895. tolentino k, friedman j. an update on anaemia in less developed countries. american journal of tropical medicine and hygiene. 2007;77(1):44–51. frickhofen n, chen zj, young ns, cohen bj, heimpel h, abkowitz jl. parvovirus b19 as a cause of acquired chronic pure red cell aplasia. br j haematol. 1994;87:818–824. doi: 10.1111/j.1365-2141.1994.tb06743.x. jasbir ks, kiersten bj. reduction in the burden of malaria anaemia in benin. confirmation of an anti-vector approach at national level. int j trop med. 2009;4(3):104–111. ademola sa. management of sickle cell disease: a review for physician education in nigeria (sub-saharan africa). anaemia. 2015:21 pages. duedu ko, sagoe kw, ayeh-kumi pf, affrim rb, adiku t, huat lb. the effects of co-infection with human parvovirus b19 and plasmodium falciparum on type and degree of anaemia in ghanaian children. asian pac j trop biomed. 2013;3(2):129–139. heegaard ed, brown ke. human parvovirus b19. clinmicrobiol rev. 2002;15(3):485–505. wildig j, michon p, siba p, et al. parvovirus b19 infection contributes to severe anemia in young children in papua new guinea. j infect dis. 2006;194:146–153. emodi i. the anaemias. in: azubuike jc, nkangineme ke, editors. paediatrics and child health in a tropical region. 2nd ed. owerri, nigeria: african educational services; 2007. pp. 355–363. girei ai, alao oo, joseph de, damulak do, orkuma j, banwat eb. haematological profile of sickle cell anaemia in children with human parvovirus b19 infection in jos, north central nigeria. jclin med res. 2010;2(9):152–155. iwalokun ba, iwalokun so, hodonu so. seroprevalence of parvovirus b19 antibodies and evidence of viremia among nigerian patients with sickle cell anaemia. j biomed res. 2013;27(4):272–282. corcioli f, zakrzewska k, rinieri a, et al. tissue persistence of parvovirus b19 genotypes in asymptomatic persons. j med virol. 2008;80(11):2005–11. orimadegun ae, fawole o, okereke jo, akinbami fo, sodeinde o. increasing burden of childhood severe malaria in a nigerian tertiary hospital: implications for control. j troppediatr. 2007;53(3):185–189. manning l, laman m, rosanas-urgell a, et al. severe anemia in papua new guinean children froma malaria-endemic area: a case–control etiologic study. plosnegl trop dis. 2012;6(12):e1972. diallo da, guindo a, dorie a, et al. human parvovirus b19 infection in sickle cell anaemia patients in mali: a case-control study. archives de pediatrie. 2011;18:962–965. article information authors: ahoefa vovor 1 ameyo dorkenoo1 yao layibo2 affiliations: 1faculté mixte de médecine et de pharmacie, université de lomé, togo 2institut national d’hygiène lomé, togo correspondence to: ahoefa vovor postal address: faculté mixte de médecine et de pharmacie université de lomé bp 1515 lomé togo dates: received: 24 feb. 2013 accepted: 27 aug. 2013 published: 18 dec. 2013 how to cite this article: vovor a, dorkenoo a, layibo, y. performance d’hemocue hb 201+ dans le diagnostic de l’anémie de l’enfant dans les structures sanitaires du niveau périphérique au togo. afr j lab med. 2013;2(1), art. #28, 5 pages. http://dx.doi.org/10.4102/ ajlm.v2i1.28 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. performance d’hemocue hb 201+ dans le diagnostic de l’anémie de l’enfant dans les structures sanitaires du niveau périphérique au togo in this original research... open access • abstrait • abstract • introduction • matériel et méthodes • population étudiée    • l’échantillonnage    • critères d’inclusion • méthodes d’étude    • aspects éthiques • analyse des données • résultats    • population d’étude    • caractéristiques générales des enfants inclus dans l’étude    • étude comparative des deux méthodes       • comparaison des deux méthodes selon les valeurs du taux d’hémoglobine       • comparaison des deux méthodes pour le diagnostic de l’anémie • discussion    • contraintes de l’étude    • population étudiée    • analyse comparative des valeurs du taux d’hémoglobine    • évaluation du test hemocue 201+® dans le diagnostic de l’anémie • conclusion • remerciements    • intérêts concurrents    • contributions des auteurs • références abstrait top ↑ contexte: l’anémie est un problème de santé publique dans le monde entier, et notamment dans les pays en développement. elle a des répercussions majeures sur la santé et sur le développement économique et social d’un pays. la prise en charge des patients anémiés étant nécessaire, il faut un diagnostic biologique précis, et donc un dosage du taux d’hémoglobine par des méthodes fiables.objectif: évaluer les performances diagnostiques du test hemocue hb201+®. méthodes: étude comparative de la mesure du taux d’hémoglobine à partir du photomètre hemocue hb 201+® et d’analyseurs d’hématologie chez 213 enfants de 6 à 59 mois souffrant d’un paludisme simple; la détermination du taux d’hémoglobine par les analyseurs est retenue comme méthode de référence pour évaluer hemocue hb201+®. résultats: 72.8% des valeurs obtenues par hemocue hb201+® étaient à ±1 g/dl de celles de la méthode de référence. le coefficient de corrélation de pearson était de 0.80. la prévalence de l’anémie était de 79.3% pour la méthode de référence et de 77.9% pour hemocue hb201+®. la sensibilité et la spécificité de l’analyseur hemocue hb201+® étaient respectivement de 95.1% et de 65.3%. conclusion: les résultats de l’étude ont montré que le test hemocue hb201+® présentait une bonne sensibilité, une spécificité moyenne et une exactitude moyenne dans le diagnostic de l’anémie et dans le dosage de l’hémoglobine. son utilisation peut être recommandée dans les structures périphériques afin de faciliter le diagnostic biologique de l’anémie et sa prise en charge dans les populations vivant dans les zones difficiles d’accès. abstract top ↑ performance of hemocue hb201+ in the diagnosis of anaemia in children in health facilities at the peripheral level in togobackground: anaemia is a global public health problem, especially in developing countries. anaemia has major repercussions on health status, as well as on the economic and social development of a country. effective care of anaemic patients requires a reliable and precise diagnostic test that can determine haemoglobin levels. objective: to evaluate the diagnostic performance of the hemocue test hb201+®. method: this study compared haemoglobin levels measured using the photometer hemocue hb201+® with those measured by analysers of haematology. children aged 6 to 59 months who suffered from uncomplicated malaria were eligible for inclusion. haemoglobin levels determined by the analysers were considered the reference for evaluation of the levels measured using hemocue hb201+®. results: 72.8% of the values obtained by hemocue hb201® were within ±1g/dl of the reference value. the pearson correlation coefficient was 0.80. the prevalence of anaemia was 79.3% using the reference method and 77.9% using hemocue 201+®. the sensitivity and the specificity of hemocue hb201+® were 95.1% and 65.3% respectively. conclusion: the study results showed that the hemocue hb201 test+® provided good sensitivity, average specificity and average precision, both for the diagnosis of anaemia and for the determination of haemoglobin levels. it may be used in peripheral centres to facilitate the laboratory diagnosis of anaemia and its management in populations that live in areas with difficult accessibility. introduction top ↑ l’anémie est un problème mondial de santé publique elle touche, selon les estimations de l’organisation mondiale de la santé (oms), deux milliards d’individus dans le monde. elle est nettement plus fréquente dans les pays en voie de développement chez les enfants et les femmes où la principale étiologie est la carence en fer aggravée essentiellement par les parasitoses intestinales et le paludisme; ses conséquences sont néfastes pour le développement de l’enfant.1 le diagnostic de l’anémie est déterminé par des signes cliniques et confirmé par la biologie; il repose sur le dosage de l’hémoglobine qui doit être réalisé par des méthodes et équipements fiables (spectrophotomètre de biochimie ou analyseur d’hématologie). au togo, le système sanitaire est organisé de façon pyramidale selon trois niveaux: un niveau central, un niveau intermédiaire (régional) et un niveau périphérique. si les laboratoires d’analyses médicales et biologiques des niveaux central et régional de la pyramide de santé du togo disposent de ces équipements, beaucoup de laboratoires de biologie médicale, notamment du niveau périphérique, utilisent des méthodes de dosage du taux d’hémoglobine (méthode de tallquist2 et méthode de sahli et gowers3) désuètes dont le niveau de fiabilité des résultats fournis est sujet à caution.4 l’hémoglobinomètre portable, hemocue 201+® (hemocue ab, angelholm, suède), un appareil mesurant par photométrie le taux d’hémoglobine dans le sang, a le mérite d’être accessible et d’utilisation relativement plus facile et donc plus adapté aux réalités des laboratoires du niveau périphérique. l’objectif de cette étude était d’évaluer les performances de l’hémoglobinomètre hemocue hb201+® pour le diagnostic de l’anémie de l’enfant afin de le rendre disponible dans les structures sanitaires périphériques en remplacement des techniques actuellement utilisées; plus spécifiquement, il s’agissait de: • comparer les résultats obtenus par hemocue hb201+® et les analyseurs d’hématologie, syxmex kx-21® et mindray bc 3000®. • définir la sensibilité, la spécificité et l’exactitude d’hemocue hb201+® matériel et méthodes top ↑ il s’agit d’une étude analytique comparant la mesure du taux d’hémoglobine à partir du photomètre hemocue hb 201+® à celle effectuée par des analyseurs d’hématologie sysmex kx 21® et mindray bc 3000®.cette étude a été réalisée du 13 août au 14 décembre 2007. le prélèvement et la réalisation des dosages par hemocue hb201+® ont été réalisés sur les sites de surveillance de l’efficacité des antipaludiques à lomé (au centre médico-social d’adakpamé et au centre médico-social jérusalem d’agbalépédogan) et à sokodé (au centre médico-social bon secours et à la polyclinique de sokodé). les patients y ont été recrutés et le dosage de l’hémoglobine à l’aide de l’hémoglobinomètre hemocue hb 201+® y a été effectué; les dosages à l’aide des analyseurs ont été réalisés dans les laboratoires du chu-campus à lomé (sysmex kx 21®) et du chr de sokodé (mindray bc 3000®). population étudiée top ↑ les sujets ayant participé à la présente étude étaient des enfants âgés de 6 à 59 mois admis en consultation externe dans les structures sanitaires précitées. l’échantillonnage la taille minimale de l’échantillon était de 174 enfants, calculée à partir de la formule de schwartz: n = ε2pq/i2, la prévalence p de l’anémie chez l’enfant étant estimée à 87 %5, q = 1 – p, ε = 1.96 et la précision i = 5%. critères d’inclusion ont été inclus les enfants âgés de 6 à 59 mois admis en consultation externe dans les centres de santé précités les jours ouvrables de 7h30 à 11h00. ces enfants doivent répondre aux critères d’inclusion et de non inclusion définis pour l’évaluation de l’efficacité thérapeutique des combinaisons à base d’artémisinine sur sites sentinelles au togo.6 les critères d’inclusion étaient: = être âgé de 6 mois à 6 ans = avoir de la fièvre (température axillaire supérieure ou égale à 37.5 °c ou température rectale supérieure à 38 °c) ou rapporter un épisode de fièvre dans les 72 dernières heures = avoir une goutte épaisse et un frottis sanguin positif. les critères de non inclusion étaient: = présence d’un ou de plusieurs signes de danger ou de paludisme grave défini par l’oms = présence d’une malnutrition sévère (périmètre brachial < 11 cm; rapport poids/taille < 70%; œdèmes bilatéraux aux membres inférieurs) = existence d’une autre pathologie fébrile évidente = prise d’une quelconque médication à action antipaludique au cours des deux dernières semaines. méthodes d’étude top ↑ l’échantillon biologique était constitué de sang total. deux types de prélèvements ont été effectués: du sang capillaire prélevé au niveau de la pulpe du doigt pour le dosage sur hemocue et 2 ml de sang veineux prélevés dans un tube contenant de l’anticoagulant edta pour le dosage sur les analyseurs d’hématologie. les prélèvements sur edta ont été acheminés dans des glacières au centre hospitalier universitaire (chu) campus et au centre hospitalier régional (chr) de sokodé, structures situées à moins de 3 km des sites de prélèvement, respectivement pour les prélèvements effectués à lomé et sokodé.le dosage sur hemocue hb 201+® a été effectué dans ces quatre centres; le dosage par l’analyseur sysmex kx-21® a été effectué au chu campus de lomé pour les prélèvements provenant des deux structures sanitaires de lomé et le dosage par l’analyseur mindray bc 3000® a été réalisé au chr de sokodé pour les prélèvements provenant des deux structures sanitaires de sokodé. les techniciens de laboratoire ont été formés afin d’assurer la standardisation des méthodes de diagnostic (la reproductibilité interindividuelle). un contrôle de qualité inter laboratoire a été systématiquement effectué entre le laboratoire d’hématologie du chu campus et celui du chr de sokodé pour les deux automates. aucune différence n’a été observée entre les résultats des deux analyseurs. l’anémie a été définie par un taux d’hémoglobine inférieur à 110 g/l. aspects éthiques la clairance éthique, délivrée par le ministère de la santé pour la conduite des tests d’efficacité et du profil de l’hémogramme au cours de l’accès palustre simple, a couvert cette étude. analyse des données top ↑ l’analyse statistique des résultats a été réalisée sur les logiciels excel 2003 et spss12. les paramètres statistiques étudiés étaient:• le coefficient de corrélation de pearson pour la détermination du lien entre les valeurs obtenues par hemocue hb 201+® et l’automate. • les limites de dispersion des différences entre les valeurs du taux d’hémoglobine obtenues par hemocue 201+® et celles obtenues par les analyseurs, déterminées par la méthode de bland et altman.7 les limites de variation acceptables ont été fixées de façon arbitraire à ±10 g/l autour de la moyenne des différences entre les valeurs du taux d’hémoglobine obtenues à partir des deux méthodes. la méthode de dosage sur hemocue hb201+® sera dite exacte lorsque les limites de dispersion des différences des valeurs des deux méthodes (moyenne ±2 sd) définies par le graphique de bland et altman ne dépasseront pas les limites de variation acceptables préalablement fixées. les performances intrinsèques (sensibilité et spécificité) et extrinsèques (valeur prédictive positive et valeur prédictive négative) du test hemocue hb201+® par rapport à l’anémie ont été déterminées. résultats top ↑ population d’étude huit cent seize enfants ont été reçus en consultation durant la période de l’étude; 340 étaient éligibles et 213 ont été inclus dans l’étude. caractéristiques générales des enfants inclus dans l’étude l’âge moyen était de 35.3 ± 14.8 mois, avec des valeurs extrêmes de 7 mois à 59 mois. le sex-ratio était de 1.39, avec une prédominance masculine. le poids moyen était de 12.8 ± 3 kg et la température moyenne de 38.7 ± 0.9 °c (tableau 1). étude comparative des deux méthodes comparaison des deux méthodes selon les valeurs du taux d’hémoglobine le taux d’hémoglobine moyen était respectivement de 95.0 g/l (±18,1) et de 94.1 g/l (±18.1) pour les deux analyseurs d’hématologie et pour hemocue hb201+® (figure 1). la moyenne des différences entre les valeurs des deux méthodes était de 0.9 ± 11.5 g/l. les limites considérant 95% des valeurs (± 2 sd) étaient de -22.1 g/l pour la limite inférieure et +23.9 g/l pour la limite supérieure. le coefficient de corrélation de pearson était de 0.80. le tableau 2 montre la répartition du nombre d’échantillons selon les différences de taux d’hémoglobine entre les deux méthodes. soixante-douze virgule soixante-dix-sept pour cent des valeurs d’hemocue hb 201®+ étaient à ±10 g/l de celles de la méthode de référence et 90.6% à ± 20 g/l des mesures de référence (tableau 2). figure 1: dispersion des différences entre les valeurs du taux d’hémoglobine par la méthode hemocue hb201+® et la méthode analyseurs selon la méthode de bland et altman. comparaison des deux méthodes pour le diagnostic de l’anémie la prévalence de l’anémie était de 81.2% (tableau 3). autant à lomé qu’à sokodé, l’hemocue hb 201+® présentait une bonne sensibilité et une spécificité moyenne; 90.2% des patients étaient effectivement anémiés lorsque hemocue hb 201+® donnait un taux d’hémoglobine inférieur à 110 g/l (tableau 4). discussion top ↑ contraintes de l’étude notre étude étant couplée à une étude déjà réalisée à d’autres fins, certaines informations qui permettaient de faire une évaluation plus complète de la performance d’hemocue hb 201+® ont fait défaut. la répétabilité d’hemocue hb201+® et des deux analyseurs pour le dosage de l’hémoglobine n’a pas été préalablement évaluée; les limites de variations acceptables ont donc été fixées de façon arbitraire à ±10 g/l.on aurait également pu disposer d’un groupe témoin d’enfants apparemment sains et non anémiés pour faciliter la comparaison. population étudiée la population d’étude a regroupé 213 enfants des deux sexes âgés de 6 à 59 mois avec une légère prédominance masculine, le poids moyen était de 12.8 kg et la température moyenne de 38.7 °c. le sex-ratio retrouvé dans notre étude était peut être dû au hasard car avant l’âge de 5 ans tous les enfants, indépendamment du sexe sont prédisposés au paludisme étant donné qu’ils ne sont pas encore pré-immunisés.bien que la population d’étude soit inégalement répartie sur les deux sites, le nombre d’enfants participant à l’étude étant plus élevé à lomé qu’à sokodé, les caractéristiques sociodémographiques des enfants enrôlés sur les deux sites étaient similaires. analyse comparative des valeurs du taux d’hémoglobine il n’existait pas de différence significative entre le taux moyen d’hémoglobine obtenu avec les analyseurs (95 g/l ±18.1) et celui obtenu par hemocue hb201+® (94.1 g/l ±18.1). le coefficient de corrélation de pearson entre les deux méthodes était de 0.80, démontrant ainsi la proximité des valeurs obtenues avec les deux méthodes. les limites de dispersion des différences selon la méthode statistique de bland et altman, limites entre lesquelles 95% des valeurs des différences se trouvaient, étaient de -22.1 g/l et +23.9 g/l. l’exactitude du test hemocue hb201+® était de 88.3%. ces résultats sont similaires à ceux trouvés par tayou et al. au cameroun en 2004 pour la détermination de l’hémoglobine chez les donneurs de sang.8 ceux-ci ont trouvé des limites de dispersion de -20.6 g/l à + 13.8 g/l, une exactitude de 93.6% et un coefficient de corrélation de 0.91. nos résultats diffèrent en revanche de ceux trouvés par paddle et al. en angleterre en 20024, qui ont trouvé des limites de dispersion plus étroites que les nôtres (-11.6 g/l et + 1.6 g/l). la prévalence de l’anémie était de 79.3% pour la méthode de référence et de 77.9% pour hemocue hb201+®. les prévalences de l’anémie par les deux méthodes ne présentent aucune différence significative. soixante-douze virgule soixante-dix-sept pour cent des valeurs d’hemocue hb 201+® étaient à ±10 g/l de celles de la méthode de référence. ces résultats diffèrent de ceux trouvés par paddle et al.4: 95.3% de leurs résultats se trouvaient à ±10 g/l de celles de la méthode de référence. il faut noter que ces derniers ont dû évaluer l’échelle de couleurs développée par l’oms pour le dosage de l’hémoglobine et comparer les résultats du taux d’hémoglobine obtenus avec hemocue à ceux obtenus par la méthode des couleurs et ceux déterminés par une méthode de référence, l’analyseur technicon h3. table 1: caractéristiques générales des enfants inclus dans l’étude. table 2: nombre d’échantillons selon les différences du taux d’hémoglobine entre les deux méthodes. table 3: prévalence de l’anémie à partir des deux méthodes de dosage. table 4: évaluation de la performance intrinsèque et extrinsèque de l’hemocue hb 201+®. évaluation du test hemocue 201+® dans le diagnostic de l’anémie hemocue hb 201+® a montré une sensibilité de 96.8% et de 93%, et une spécificité de 59.1% et de 70.4% respectivement sur les sites de lomé et de sokodé. sur l’ensemble des deux sites d’étude, hemocue hb 201+® présentait une sensibilité de 95.1% et une spécificité de 65.3%. ces résultats sont similaires à ceux obtenus par neville à dundee (en écosse) en 19879, qui a obtenu une sensibilité et une spécificité respective de 88.5% et 77.6% dans une étude comparative de la méthode hemocue avec l’analyseur automatique elt 80ws (ortho diagnostic systems ltd) chez 235 patients recrutés dans un centre de santé. d’autres études ont rapporté une très bonne spécificité de la méthode hemocue hb201+®: 97.1% pour tayou et al. en 2004 au cameroun8 et 94.2% pour sari et al. en 1998 en indonésie.10 au cameroun, l’automate celly 70 a été utilisé comme méthode de référence et en indonésie, hemocue hb201+® a été comparé à la méthode directe de la cyanméthémoglobine. cette différence avec nos résultats peut être due au grand nombre de faux positifs (17) trouvés par rapport à la référence choisie. ceci peut également être dû au fait que l’utilisation de la 3e ou 4e goutte lors de la manipulation n’a pu être rigoureusement respectée. de plus, les conditions d’utilisation d’hemocue hb 201+® assez restrictives peuvent également être incriminées: en effet, les microcuvettes d’hemocue hb 201+®, qui contiennent des réactifs, ne peuvent pas résister à une température supérieure à 30 °c et doivent être utilisées dans un court délai après ouverture. la nouvelle génération de cet équipement, hemocue hb 301+®11 serait davantage adaptée aux réalités des laboratoires de niveau périphérique, puisque ses microcuvettes, qui ne contiennent pas de réactif, résistent à une température pouvant aller jusqu’à 40 °c, et sont relativement moins coûteuses et donc plus abordables. d’autres études doivent être réalisées en vue d’évaluer hemocue hb 301+® pour le dosage de l’hémoglobine dans nos structures sanitaires. conclusion top ↑ les résultats de l’étude ont montré que le test hemocue hb201+® présentait une bonne sensibilité, une spécificité moyenne et une exactitude moyenne tant dans le diagnostic de l’anémie que dans le dosage de l’hémoglobine. son utilisation peut être recommandée dans les structures périphériques afin de faciliter le diagnostic biologique de l’anémie et sa prise en charge dans les populations vivant dans les zones difficiles d’accès. remerciements top ↑ nous adressons nos remerciements aux parents et tuteurs qui ont accepté que leurs enfants participent à l’étude, aux enfants et aux membres des équipes des sites où le recrutement des enfants a été fait. intérêts concurrents aucun conflit d’intérêt: les auteurs déclarent n’avoir aucun lien financier ou personnel les ayant influencé de façon inappropriée pendant la rédaction de l’article. contributions des auteurs a.v. (université de lomé) conception, conduite de l’étude, analyse des données, rédaction de l’article. a.d (université de lomé) conception, conduite de l’étude, rédaction de l’article. y.l. (institut national d’hygiène) conduite de l’étude et analyse des données. références top ↑ 1. oms, unicef. joint statement: focusing on anaemia, towards an integrated approach for effective anaemia control. genève: organisation mondiale de la santé; 2004.2. oms hémoglobine: utilisation d’un comparateur in manuel des techniques de base pour le laboratoire médical, p 487, oms; 1982 3. oms – hémoglobine: dosage par la méthode de sahli in manuel des techniques de base pour le laboratoire médical, p. 487, genève: organisation mondiale de la santé; 1982. 4. paddle jj. evaluation of the haemoglobin colour scale and comparison with the haemocue haemoglobin assay. bull organ mond santé; 80: 813-6; 2002. 5. ministère de la santé. enquête sur l'anémie et les facteurs associés dans les ménages du togo. lomé: ministère de la santé/togo; 2000.oms. protocole de suivi de l'efficacité des antipaludiques. genève: organisation mondiale de la santé; 2003. 6. bland jm, altman dg. statistical methods for assessing agreement between two methods of clinical measurement. lancet; 327: 307–10; 1986 http://dx.doi.org/10.1016/s0140-6736(86)90837-8 7. tayou c, monny l, mbanya d. évaluation de deux techniques de dosage de l’hémoglobine chez les donneurs de sang. transfus clin biol.; 13: 331-4; 2006. 8. neville r. evaluation of portable haemoglobinometer in general practices. bmj; 294:1263-5; 1987. http://dx.doi.org/10.1136/bmj.294.6582.1263, pmid:3109609, pmcid:pmc1246433 9. sari m, de pee s, martini e, et al. estimating the prevalence of anaemia: a comparison of three methods. bulletin of the world health organization; 79 (6): 506-11; 2001. pmid:11436471, pmcid:pmc2566437 10. tayou tagny c, kouam l, mbanya d. évaluation du nouvel appareil hemocue hb 301 dans le dosage de l’hémoglobine chez des femmes enceintes camerounaises. ann biol clin; 66 (1): 90-4; 2008 article information authors: souleymane sanon1 adama gansane1 lamoussa paul ouattara1 abdoulaye traore1 issa nebie ouedraogo1 alfred tiono1 donatella taramelli2 nicoletta basilico 3 sodiomon bienvenu sirima1 affiliations: 1centre national de recherche et de formation sur le paludisme, ouagadougou, burkina faso2dipartimento di scienze farmacologiche e biomolecolari (disfeb), università di milano, italy 3dipartimento di scienze biomediche, chirurgiche e odontoiatriche, università di milano, italy correspondence to: souleymane sanon postal address: 01 bp 2208, ouagadougou 01, burkina faso dates: received: 26 july 2012 accepted: 15 oct. 2012 published: 08 mar. 2013 how to cite this article: sanon s, gansana a, ouattara lp, et al. in vitro antiplasmodial and cytotoxic properties of some medicinal plants from western burkina faso. afr j lab med. 2013;2(1), art. #81, 7 pages. http://dx.doi.org/10.4102/ ajlm.v2i1.81 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. in vitro antiplasmodial and cytotoxic properties of some medicinal plants from western burkina faso in this original research... open access • abstract • introduction • material and methods    • study site and plants    • crude extracts preparation    • in vitro antiplasmodial assays       • plasmodium falciparum culture       • evaluation of antiplasmodial effect of extracts    • in vitro cytotoxicity assay of extracts on human cells • results    • antimalarial effects    • toxicity • discussion • acknowledgments    • competing interests    • authors' contributions • references abstract top ↑ background: resistance of malaria parasites to existing drugs complicates treatment, but an antimalarial vaccine that could protect against this disease is not yet available. it is therefore necessary to find new effective and affordable medicines. medicinal plants could be a potential source of antimalarial agents. some medicinal plants from burkina faso were evaluated for their antiplasmodial and cytotoxic properties in vitro.methods: crude dichloromethane, methanol, water-methanol, aqueous and alkaloids extracts were prepared for 12 parts of 10 plants. chloroquine-resistant malaria strain k1 was used for the in vitro sensibility assay. the plasmodium lactacte dehydrogenase technique was used to determine the 50% inhibitory concentration of parasites activity (ic50). the cytotoxic effects were determined with hepg2 cells, using the tetrazolium-based colorimetric technique, and the selectivity index (si) was calculated. results: sixty crude extracts were prepared. seven extracts from terminalia avicenoides showed ic50 < 5 µg/ml. the ic50 of dichloromethane, methanol, aqueous and alkaloids extracts ranged between 1.6 µg/ml and 4.5 µg/ml. three crude extracts from combretum collinum and three from ficus capraefolia had an ic50 ranging between 0.2 µg/ml and 2.5 µg/ml. crude extracts from these three plants had no cytotoxic effect, with si > 1. the other plants have mostly moderate or no antimalarial effects. some extracts from cordia myxa, ficus capraefolia and opilia celtidifolia showed cytotoxicity, with an si ranging between 0.4 and 0.9. conclusion: our study showed a good antiplasmodial in vitro activity of terminalia avicenoides, combretum collinum and ficus capraefolia. these three plants may contain antiplasmodial molecules that could be isolated by bio-guided phytochemical studies. introduction top ↑ malaria remains a serious worldwide health problem due to the emergence and spread of parasite resistance to well-established antimalarial drugs and mosquito vectors resistant to insecticides.1 in sub-saharan african populations, malaria is one of the diseases causing most morbidity and mortality. it is estimated that each year malaria causes nearly 800 000 deaths,2,3 mostly of african children aged below 5 years. in burkina faso, malaria is the leading cause of hospitalisation, with 2 million cases per year, and it remains also the principal cause of death in local health centers, with a rate of 50.7% in 2009.4 although continued attempts to develop a vaccine for malaria are ongoing, distribution of mosquito nets, household spraying, and prophylaxis remain the primary prevention methods and antimalarial drugs remain the only treatment option.5 in order to decrease the risk of chemo-resistance to most of the antimalarial drugs, the world health organization (who) has recommended artemisinin-based combination therapies (acts) for the management of uncomplicated p. falciparum malaria cases. unfortunately, act treatment failures have been reported in some countries.6,7,8,9 in addition, these drugs are expensive, limiting their use in a population with average annual income around $100. thus the use of traditional and less expensive preparations is common.10 historically, many drugs effective against parasitic diseases stem from traditional medicine, such as quinine and artemisinin.11,12,13 today, 30% of drugs on the pharmaceutical market come from nature14 and medicinal plants constitute a popular source of potential antimalarial agents. in the in western area of burkina faso, ten medicinal plants are widely used by traditional healers,15 although little scientific data exist on their effectiveness in treating malaria. the aim of this study is to determine which of these plants show promising antimalarial effects as well as low toxicity in vitro, paving the way for potential pharmaceutical development. material and methods top ↑ this study was approved by the national ethical committee to be conducted with the traditional healers on the study site. study site and plants the study was conducted in the province of comoe, where malaria transmission is continuous and which covers 15 871 km2 with 277 384 inhabitants. with an average annual rainfall of 900 mm, this area is one of the wetter regions of the country, and has a large plant biodiversity. the herbal pharmacopoeia has expanded rapidly in this province, and traditional healers collaborate with researchers in the field of herbal medicine. twelve samples consisting of leaves and bark from ten plants (terminalia avicenoides, combretum collinum, ficus capraefolia, anthocleista nobilis, celtis integrifolia, cordia myxa, lophira lanceolata, oppilia celtidifolia, securinega virosa and tapinanthus dodoneifolius) were identified. the scientific identification and the herbarium were made by botanical taxonomists from the centre national de recherche scientifique et technologique (cnrst) of burkina faso. the specimen voucher number from this herbarium was deposited in centre national de recherche et de formation sur le paludisme (cnrfp). these parts of plants were used by traditional healers to treat malaria-like symptoms. samples were harvested during the rainy season, washed, dried and pulverised for phytochemical extractions. crude extracts preparation extraction based on maceration, decoction and preferential depletion methods were used to obtain five types of extract for each of the 12 plant parts. we obtained three organic extracts, one aqueous with water, and one crude alkaloid. crude organic extracts were prepared by maceration for 16 hours successively with dichloromethane (ch2cl2), methanol (ch3oh) and water-methanol (ch3oh/h2o) solvents. plant powder (20 g) was used for these organic extraction methods with 500 ml of each solvent. ch2cl2 extract was air dried at room temperature. ch3oh and ch3oh/h2o extracts were freeze-dried with lyophilisator (brand) after total evaporation of solvents. aqueous extracts were prepared by boiling 10 g of plant powder in 500 ml of purified water for 30 minutes. after cooling, solutions were filtered on cotton wool and freeze-dried. crude alkaloid extracts were obtained by alkalinisation with nh4oh of the plant powder and extraction with ch2cl2 for 24 hours. plant powder (20 g) was used by applying the classical alkaloids extraction method.16 after 16 hours of maceration with ammoniac and ch2cl2, a percolation was made with ch2cl2 solvent. then 500 ml of dichloromethane layer was concentrated under vacuum and then extracted with a 2% solution of h2so4. the aqueous acid solution was alkalinised again with nh4oh and extracted with ch2cl2, and a crude alkaloids extract was obtained by concentration. each of the 60 extracts was dissolved in appropriate solvent of dimethyl sulfoxide (dmso) and purified water (dmso/water) to give a homogeneous solution at an initial concentration of 1 mg/ml. samples were then serially diluted with complete culture media (rpmi 1640 with albumax) to achieve the required concentration with dmso concentration < 0.5%. in vitro antiplasmodial assays plasmodium falciparum culture sixty extracts were tested for their effectiveness in preventing growth of the most common chloroquine-resistant strain of malaria found in burkina faso, plasmodium falciparum k1. the malaria strains were provided by the london school of tropical medicine and hygiene (england) and were grown under standard conditions as previously described.17 they were maintained in continuous culture at the cnrfp, in a sterile atmosphere under a laminar flow hood in 200 µl blood group o+ using rpmi 1640 (megacell, sigma aldrich, usa) medium (5 ml) supplemented with 2-hydroxyethlpiperazine-n-2ethanesulfonic acid (hepes acid) (25 mm; gibco-brl, paisley, scotland), nahco3 (25 mm), 1% albumax and washed erythrocytes to yield a final haematocrit of 4%. parasitaemia was maintained between 1% and 6% by dilution with non-infected o+ erythrocytes. blood was obtained from subjects who had not received antimalarial treatment during the previous two weeks and had an aa electrophoresis (no sickle cell trait). the 75 cm3 culture flasks were incubated for 24 hours at 37 °c in a co2 incubator (heracell 150, forma scientific), with 2% o2, 5% co2, 93% n2 and 90% humidity. every 24 hours, supplemented media was renewed and parasitemia was checked by blood smear with optical microscope. evaluation of antiplasmodial effect of extracts antimalarial effects were quantified with respect to inhibition of parasite growth, as measured by the production of plasmodium lactate dehydrogenase (pldh). testing was performed in three steps in duplicate in 96-well flat bottom plates (tpp, switzerland).malaria parasites were cultured with seven different concentrations of each extract and parasite growth was assessed by the production of pldh. each extract was applied in a series of seven duplicate dilutions (final concentrations ranging from 0.78 µg/ml to 50 µg/ml) on two rows. 100 µl of asynchronous parasitised erythrocytes at a hematocrit of 2% with parasitemia between 1.5% and 2% were prepared in 100 µl of each extract. the plates were then placed at 37 °c in a modular incubator chamber with a humidified atmosphere of the same gas mixture as above for 72 hours. dihydroartemisinin was used to validate the malaria test and chloroquine diphosphate salt (sigma aldrich) was used to validate the real chloroquine resistance of malaria strain k1. infected and uninfected erythrocytes o+ were used as positive and negative controls, respectively. parasite growth was determined by measuring the content of parasite lactate dehydrogenase18 using malstat, ntb/pes reagents. the microplates were read with a spectrophotometer (biotek el x 808) at a wavelength of 650 nm. absorbance data were entered into microsoft excel to calculate the percent inhibition relative to positive control from the mean of raw data for each concentration. table curve version 5.0 software was used to plot inhibition curves and calculate the inhibition concentration of drug that reduced the pldh activity by 50% (ic50). the results were categorised following deharo’s et al classification:19 good antimalarial effect ic50 < 5 µg/ml, moderate antimalarial effect 5 µg/ml ≤ ic50 < 10 µg/ml and inactive extract ic50 ≥ 10 µg/ml. in vitro cytotoxicity assay of extracts on human cells human hepatoma cells atcc # hb-8065 (hepg2) were obtained from the american type culture collection (atcc, manassas, usa). the cells were grown in rpmi-1640 (megacell, sigma aldrich, usa) supplemented with 10% fetal bovine serum (v/v), 1% mix (v/v) of 200 mm l-glutamine, 10 000 iu/ml penicillin and 10 mg/ml streptomycine, (gibcobrl) within a humidified atmosphere of 6% co2 and 94% de n2 at 37 °c over 5 days. cultures were maintained by sub-culturing flasks every 4 days at 5 × 104 cells/25 cm2 flask by trypsination. the cytotoxicity of the extracts was assessed using a tetrazolium salt mtt (3-[4.5-dimethylthiazol-2-yl]-2.5-diphenyltetrazolium bromide) (sigma) colorimetric method, based on reagent cleavage by mitochondrial dehydrogenase in viable cells.20 growing cells were placed at a density of 5 × 104 cells per well in microplates in 100 µl of culture medium. the microplates were incubated in a humidified atmosphere of 6% co2 and 94% n2 at 37 °c for 24 hours. for each extract, seven duplicate dilutions were prepared with concentrations ranging from 0.78 µg/ml to 50 µg/ml in dmso and then in medium culture. for each test, we used a growth (cells plus medium only), positive (with doxorubicin) and a negative control (with dmso) respectively. cytotoxicity was scored as the percentage reduction in absorbance at 570 nm versus that of the untreated control culture. a selectivity index (si), corresponding to the ratio between antimalarial and cytotoxic properties, was calculated according to the following formula: siextract = ic50cytotoxic/ic50malaria. the extract was considered to have negligible cytotoxicity if si > 1.21 results top ↑ antimalarial effects among the 60 extracts tested, 21 were identified as having good antimalarial effects (ic50 < 5 µg/ml), 14 with moderate effects (5 µg/ml≤ ic50 < 10 µg/ml), and 25 as inactive (ic50 ≥ 10 µg/ml) (table 1). terminalia avicenoides produced the most effective antimalarial extracts, with four coming from its leaves and three from its stem bark (table 2). three extracts from combretum collinum and three from ficus capraefolia also had good antimalarial effects (table 2). extracts from the seven other plants had mostly moderate to little effect. table 1: in vitro antimalarial and cytotoxic effects of indigenous plant extracts. table 2: promising crude extracts from medicinal plants according to antiplasmodial activity level and cytotoxicity. toxicity the extracts with antiplasmodial effects from terminalia avicenoides, combretum collinum and ficus capraefolia, had low risk of cytotoxicity (si > 1), with si ranging from 4 to 140 (table 1). higher risk of cytotoxicity (si < 1) was found with five extracts from four plants: a water-methanol extract from celtis integrifolia (si > 0.5), methanol and water-methanol extracts from cordial myxa (si = 0.9 and si = 0.5, respectively), aqueous extract from ficus capraefolia (si = 0.4) and water-methanol extract from opilia celtidifolia (si = 0.4). discussion top ↑ the best antimalarial effects were obtained with extracts of three plants, namely, terminalia avicenoides, combretum collinum and ficus capraefolia. the four crude extracts from the leaves and three from the stem bark of terminalia avicenoides showed good antimalarial effects against the cq-resistant strain k1, with ic50 values ranging between 1.16 µg/ml to 4.53 µg/ml. in nigeria, a similar study showed that methanol extracts of leaves of terminalia avicennoides had an ic50 = 14.09 µg/ml with the k1 malaria strain.22 based on deharo’s efficiency criteria, results from nigeria are different from our findings. these differences may be related to many parameters, including the local environment and the collection periods, which contribute to the variation of plant chemical components as shown in a previous study on seasonal effects on bioactive compounds.23 the biological results could be linked to the laboratory techniques used. in phytochemical studies, extraction methods are often different because the ability of extracting a solvent of chemical groups is related to its polarity. concerning biological assessment, the results are often based on the sensitivity of techniques used. a previous study done in australia pointed to the difference of sensitivity of the flow cytometry assay, the sybr green plate reader assay, the plasmodium falciparum lactate dehydrogenase assay and light microscopy used in in vitro antiplasmodial activity assessments.24 despite the difference of ic50 values ​​obtained, which could be related to laboratory sensitivity techniques and environmental conditions, the antiplasmodial properties of terminalia avicenoides were promising. our study confirms the pharmacological properties of this plant species shown by its antifungal activity against candida albicans25 and parasitogical activity on trypanosoma mali.26 another study showed good antidiarrheal properties for an aqueous extract of the roots of terminalia avicennoides.27 it is recommended that future studies should consider this plant as a potential source of antiplasmodial molecules. our study appears to be the first to demonstrate the antimalarial effects of this plant. with combretum collinum, dichloromethane, water-methanol and alkaloid extracts of leaves had good antimalarial activity, with ic50 values ranging from 0.2 µg/ml to 2.14 µg/ml. it appears that our findings are the first report of extracts exhibiting in vitro antiplasmodial activities. however, studies have shown the pharmacological property of some species of the same botanical family (combretaceae). acetone extracts from combretum molle, which belongs to the same botanical family, have shown good antimalarial effects, with ic50 values from 2.2 µg/ml28 to 8.2 µg/ml28 and selective inhibition effect of hiv-1 replication.29 the plant also has antifungal, anti-inflammatory and antilarvicidal properties.30,31,32 according to these results, the antimalarial effect obtained with combretum collinum can be explained by the presence of chemical groups specific to this botanical family. ficus capraefolia is the third plant which has good antimalarial effect, with dichloromethane, methanol and crude alkaloid extracts (ic50 < 5 µg/ml ranging between 0.95 µg/ml and 2.85 µg/ml). our study appears to be the first report to show the antimalarial activity of this plant. other studies have shown pharmacological properties of other species of the same botanic family (moraceae). thus, ficus sycomorus (l) had a moderate antimalarial effect with ic50 < 10 µg/ml,16 and ficus sur (forssk) was inactive, with ic50 = 27.4 µg/ml and ic50 > 100 µg/ml using chloroquine resistant strains ent 30 and vi/s respectively.33 in the other species of moraceae, which is ficus fistulosa reinw, molecules such as the flavonoid artonin f, flavonoid 7-demethylartonol and flavonoid cycloartobiloxanthone have been isolated.34,35 the pharmacological effects that our study and others show could be attributed to this chemical group, but a bioguided phytochemical study would be necessary to verify whether it has the same antimalarial molecules as the ones found in our species. besides the three previous plant species, anthocleista nobilis, opilia celtidifolia, lophira lanceolata, securinega virosa and tapinanthus dodoneifolius all showed moderate antimalarial activity. a literature search on the antimalarial activities of the aforementioned plants has shown no data. these pharmacological properties could be attributed to alkaloids which could be contained in this plant.36,37 analgesic properties38 and antibacterial activity39 of ethanol and methanol extracts have been shown in cordia mixa. in kenya, aqueous and methanol extracts from leaves of securinega virosa showed antimalarial activity using the cq-resistant strain d6 with ic50 of 25.52 µg/ml and 2.28 µg/ml respectively.40 our study confirms the antimalarial property shown by the kenyan study, using another cq-resistant strain k1. otherwise, this antimalarial effect could be attributed to virosecurinine, an alkaloid isolated from its leaves.41 our study appears to be the first report showing a moderate antimalarial effect for lophira lanceolata but its antimicrobial activity against candida albicans has been described previously in cameroun.42 in nigeria, a study showed that a methanol extract of the leaves of tapinanthus dodoneifolius has very good antimicrobial properties.43 the promising antimalarial extracts from terminalia avicenoides, combretum collinum and ficus capraefolia showed no cytotoxic effect, with a high selectivity index > 10. this finding is in concordance with some cytotoxicity studies of extracts from african medicinal plants.44,45 otherwise, among the extracts which have moderate and no antimalarial effects, the risk of cytotoxicity for human cells has been reported, as shown by water-methanol extracts from celtis integrifolia (si > 0.5), methanol and water-methanol extracts from cordia myxa (respectively si = 0.9 and si = 0.5), aqueous extracts from ficus capraefolia (si = 0.4) and water-methanol extracts from opilia celtidifolia (si = 0.4). this shows that the toxicity of a drug depends on effective dose used. furthermore, it is also important that even though they are effective in treating malaria, the cytotoxicity of to these plants must be taken into account especially by traditional healers for whom the major issue is giving the correct dosage. as shown in previous studies, in traditional medicine practice, medicinal plants can be used but the dosage must be taken into consideration.46,47 the results of this study can improve the traditional use of plants and protect people from risks following their administration. it appears for the first time from scientific investigation that terminalia avicennoides, ficus capraefolia and combretum collinum may be potential sources of antimalarial agents due to their good antimalarial effects and the lack of cytotoxic effects of their extracts. the seven other plants have mostly moderate antimalarial effects but some extracts from celtis integrifolia, cordial mixa and opilia celtidifolia showed cytotoxic effects. these plants can be used in traditional medicine by paying close attention to the dosage. the promising results obtained can be a starting point to seek bioactive compounds by bioguided fractionation and biological studies, for the development of new drugs. acknowledgments top ↑ we thank the european commission, who fully funded the work through project lshp-ct-2005-018834 of european commission’s 6th framework programme (fp6). we thank who/tdr, who funded the study site characterisation and the ethnobotanical study to identify traditional healers and medicinal plants through project a30930. we thank all antimal consortium members and all staff of cnrfp, where the project was implemented. special thanks to professor steven ward, susan jones and tracy seddon from lstm (england), prof. henri vial from university of montpelier (france), and prof. christian doerig from monash university (melbourne, australia) for their valuable contributions during the project implementation. thanks to the entire population of the province of comoe, especially the traditional healers, for their involvement in the plant identification process. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this paper. authors’ contributions s.s. (centre national de recherche et de formation sur le paludisme) was the project coordinator, the person in charge of overseeing all research activities in the field and the laboratory, the data analysis, the writing of the paper. g.a. (centre national de recherche et de formation sur le paludisme) contributed to the project implementation by supervising laboratory activities and the paper review. o.l.p. (centre national de recherche et de formation sur le paludisme) was a student who contributed to the plant harvesting and implementation of laboratory activities. s.s.b., t.a. (centre national de recherche et de formation sur le paludisme) were in charge of monitoring the research activities, quality control activities, data validation and the paper review. t.d. and n.b. (university of milan) are responsible for the laboratory in milan where they trained on pldh technique and technology transfer the cnrfp staff, and contributed to the monitoring of susceptibility tests, the scientific data validation and the paper review. references top ↑ 1. wellems te, plowe cv. chloroquine-resistant malaria. j infect dis. 2001;184(6): 770–776. http://dx.doi.org/10.1086/3228582. who. world malaria report, geneva; 2010. 3. roselyne nt, leonardo l, cinzia cn, geme ud, serge ry, giulio l, et al. potential of a khaya ivorensis-alstonia boonei extract combination as antimalarial prophylactic remedy. j ethnopharmacol. 2011;137:743–751. http://dx.doi.org/10.1016/j.jep.2011.06.036 4. dgiss – mission ministère de la santé. utilisation des données statistiques www.insd.bf/fr/img/arc-ssn_rapport_final_sommaire_sante.pdf; 2010. 5. sirima sb, gansane a. artesunate–amodiaquine for the treatment of uncomplicated malaria. expert opin investig drugs. 2007;16:1079–1085. 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accepted: 11 feb. 2022; published: 29 apr. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: haemoglobinopathies are inherited haemoglobin disorders that result in anaemia characterised by erythrocyte anisopoikilocytosis. red cell distribution width (rdw) measures anisopoikiloytosis and is readily reported by haematology analysers as a complete blood count parameter. the utility of rdw as a diagnostic marker of haemoglobinopathies in kenya remains undetermined and undocumented. objective: this study aimed to determine the diagnostic efficacy of rdw in discriminating haemoglobinopathy and haemoglobinopathy-free cases in kenya. methods: the case-control study used randomly selected haematology analyser outputs for haemoglobinopathy-free (241, 49.4%) and haemoglobinopathy cases (247, 50.1%) aged 1 month to 66 years old tested in the aga khan hospital, kisumu, and its satellite centres in western kenya from 01 january 2015 to 31 december 2020. results were verified using high performance liquid chromatography. the receiver operating characteristic (roc) curve was used to evaluate the diagnostic power of rdw as a biomarker for sickle cell disease (scd) and sickle cell trait phenotypes and β-thalassaemia. results: the rdw showed diagnostic significance in scd phenotypes at 21.1 roc curve coordinate with 67.7% sensitivity, 90.0% specificity, 0.789 accuracy, 70.5% positive predictive validity, 88.8% negative predictive validity, 6.77 positive likelihood ratio, 0.36 negative likelihood ratio and 18.94 (11.4–31.4) odds ratio. conclusion: an rdw of 21.1% is potentially a predictor of scd haemoglobin phenotypes and should be included in the haematology screening algorithm as a critical value, above which suspected cases qualify to be investigated for scd. keywords: red cell distribution width; surrogate marker; biomarker; haemoglobinopathies; patients; western kenya. introduction haemoglobinopathies, thalassaemia syndromes, and structural haemoglobinopathy variants – haemoglobin s, haemoglobin e, haemoglobin c – are hereditary haemoglobin disorders resulting from mutations in genes encoding the haemoglobin polypeptide chain. these structural haemoglobin disorders result in functionally impaired molecules; impairment includes inefficient oxygen supply and susceptibility to destruction by the victim’s reticuloendothelial system, with consequential fatal or life-threatening severe anaemia and hypoxia.1,2 the world health organization reports that approximately 7% of the global population carry an inherited haemoglobin disorder gene and that about 300 000 infants are born with severe haemoglobin disorders annually, with over 200 000 being born in the sub-saharan african countries.1,3 if proper measures are not put in place, it is estimated that between 2010 and 2050, 14 282 000 babies will be born with sickle cell disease (scd), of which 82% will be born in sub-saharan african countries.4 haemoglobinopathies are neglected but increasing global health problems; children with scd who live in sub-saharan africa have an estimated high mortality rate of 50% – 80% by age 5.1,5,6,7,8,9,10,11 parents who are carriers of haemoglobinopathies have a 25% risk of genetically transferring potentially severe disorders to offspring, thus making prevention and control of these disorders difficult. therefore, due to the recessive character of haemoglobinopathy inheritance, researchers have recommended screening for the carrier state in potentially susceptible populations.12,13 studies have shown that the kenyan population has a significant burden of haemoglobin disorders that remains undocumented as with many other sub-saharan african countries. non-surveillance is due to the high financial cost of haemoglobinopathy laboratory tests. these tests include the world health organization recommended haemoglobin electrophoresis and genotyping tests for population and newborn screening.11,14 consequently, a simple model to unmask cases needs to be established. laboratory testing is the surest way to diagnose blood disorders; however, most affected children in african countries continue to die in early childhood, usually undiagnosed, due to the lack of effective programmes for early detection and treatment. a prospective cohort study in the kilifi area of kenya documented a 50% – 90% mortality rate in children under five years with scd, which was consistent with the 50% – 80% mortality rate recorded among undiagnosed and untreated children across africa. the authors recommended prioritising scd diagnosis and management health research in africa. the current study sought to determine the potency of red cell distribution width (rdw) as a surrogate marker for haemoglobinopathies circulating in a vulnerable population in resource-poor settings of western kenya.4,5,7,6 the kenyan regions served by the aga khan hospital kisumu, being within the malaria holoendemic region of western kenya, lie in the lake victoria economic block region which is known to have a high burden of haemoglobinopathies, particularly sickle cell haemoglobinopathy.11,15,16 the need for a less costly haemoglobinopathies laboratory testing method in western kenya is imperative. previous studies have demonstrated the utility of rdw as a discriminatory marker of iron deficiency anaemia from other microcytic anaemias while other studies have demonstrated the ability of this haematological parameter to discriminate iron deficiency anaemia and thalassaemias. the haematological index or parameter, rdw, seems able to discriminate haemoglobinopathies generally from other erythrocyte disorders associated with anaemia.17,18,19,20,21,22 this makes rdw a potentially simpler, cheaper, faster, and potentially dependable laboratory assay method for haemoglobinopathy detection suitable in low-income settings of western kenya. a sickling test was recommended for screening scd in children since it proved to have high sensitivity and specificity compared to solubility test and peripheral blood film in a study done in uganda. however, unknown adult haemoglobinopathy carriers were left out by the same study; thus, the present study sought to unmask these carriers from the general population.23 the rdw measures variation in red blood cell sizes (anisocytosis) and shapes (poikilocytosis) in cell volume within the red cell population. these are erythrocyte phenotypic features that are commonly abnormal in the presence of haemoglobinopathy.24 the rdw is one of the haematological indices routinely generated by automated haematology analysers in clinical laboratory assays; therefore, it is a simple, faster, cheaper, and widely used test in routine practice as part of a full haemogram report. the rdw has been studied as a significant entity in various disease pathogeneses involving erythrocyte size and shape variations24,25,26 it is derived and presented as a coefficient of variation. studies have shown the potential utility of the rdw as a marker for laboratory detection of haemoglobinopathies, but there is a paucity of data on its use in kenya. genetic variation and environmentally and socioculturally imposed epigenetic changes have been shown to influence gene-phenotype.27 it is uncertain if the rdw values and their relationship with haemoglobin phenotypes on populations in other geographical settings can apply to the kenyan scenario, especially the malaria holoendemic lake victoria basin. the overall goal of the study was to contribute to improving the chances of survival of infants and children with haemoglobinopathies by enabling financial access to timely laboratory testing through a dependable but affordable assay method. its main objective was to establish the overall accuracy of the rdw as a surrogate marker of haemoglobinopathies among age-mixed patients. methods ethical considerations ethical approval was granted by the masinde muliro university ethical review committee (reference mmu/cor:403012 vol 3(03) and national commission of science and technology (nacosti) (ref. 407653). permission to collect data was approved by aga khan hospital, kisumu ethics committee (reference adm/007/089) thus patients’ consent was not needed. the raw data was stored by the principal investigator in restricted rooms and electronic data was coded to maintain anonymity in password proof computers. sample size determination sample size calculation was performed using cochrans’s formula for sample size determination in case-control and other comparative studies.28 assuming 19% prevalence of α-thalassaemia and sickle cell among children enrolled in a malaria vaccine clinical trial study done at kombewa in lake victoria basin, western kenya, a confidence level of 95% and a precision level of 5%, sample size of 237 was obtained; since this was a two-arm study (case-control), an equal control of 237 was needed, giving a minimum required sample size of 474.16 study design this was a hospital-based cross-sectional retrospective comparative study of 488 randomly selected high performance liquid chromatography confirmed haemoglobinopathy, but non-iron deficiency, subjects (cases) (n = 247, 50.1%) and haemoglobinopathy-free and non-anaemic results (control group) (n = 241, 49.4%) with corresponding complete blood counts from hospital databases for the aga khan hospital, kisumu, and its western kenya satellites. data collection data were obtained from the laboratory database on patients examined at the hospital’s haematology laboratory for the past five years from 01 january 2015 to 31 december 2020. complete blood count reports were performed using various sysmex analysers (kx-21n, xp 300, sysmex xnl 330, symex xs 500i and sysmex xs1000i; sysmex corporations, kobe, japan). the cases were individuals who were confirmed for various haemoglobinopathies using a high performance liquid chromatography (bio-rad d10) machine (bio-rad laboratories, hercules, california, united states). excluded cases included those without their respective complete blood counts reports, those with confirmed leukaemia, and those cases that had received transfusion in the past three months. the control group consisted of individuals presumed free from disorders normally associated with abnormality of red erythrocyte shape and size (including haemolytic, macrocytic or iron deficiency anaemia) and haemoglobinopathy-free. these were age-mixed people electrophoretically confirmed to have normal haemoglobin (haemoglobin aa genotype) and had haemoglobin concentrations of ≥ 9.5 g/dl for ≤ 5-year-olds, ≥ 10.5 g/dl for ≤ 12-year-olds and ≥ 11 g/dl for ≥ 13-year-olds.29 all participants had rdw results as part of the complete blood counts from automated haematology analysers, but cases had additional haemoglobin profiles. data analysis statistical package for social sciences version 20 (spss inc., chicago, illinois, united states) was used to analyse data with kolmogorov-smirnov and shapiro-wilks tests. these tests revealed that the control group was a skewed (non-normal) distribution (p < 0.05); thus, a non-parametric statistics test, the kruskal-wallis h-test, was used to assess the rdw variation within haemoglobinopathy variants while the mann whitney u-test was used to compare the rdw variations between groups as recommended by nahm, 2016.30 accordingly, the normal rdw reference values were derived from the control group as the upper limit of 95% confidence interval (ci) of the median. data were summarised as a median and interquartile range for the rdw and percentage for haemoglobinopathy presence. the clinical utility of the rdw was studied through receiver operating characteristic (roc) curves analysis to assess its diagnostic efficacy in differentiating diseased (haemoglobinopathy) from non-diseased (haemoglobinopathy-free) population. the sensitivity and specificity at optimal points by use of youden index, plus predictive values, likelihood ratio (lr), and odds ratio (or) at the 5% significance level (p = 0.05) were determined.31 results demographic characteristics of study participants the proportions of the control group (49.4%, n = 241) and the case group (50.6%, n = 247) did not differ significantly (p = 0.740) in a total of 488 individuals (table 1). there was no significant difference (p = 0.502) between the number of male (43.9%, n = 214) and female (56.1%, n = 274) participants. the majority of the participants were from the kisumu station (49.0%, n = 239), followed by busia (15.4%, n = 75) and homabay (12.3%, n = 60; p < 0.001). the rest of the participants were distributed among the rest of the locations. there was a statistically significant variation in scd frequencies across the three age groups: ≤ 5-year-olds (42.4%, n = 207), ≤ 12-year-olds (19.9%, n = 97), and ≥ 13-year-olds (37.7%, n = 184; p < 0.001). table 1: demographic characteristics of study participants and rdw in control and case (haemoglobinopathies) groups in western kenya, 01 january 2015 – 31 december 2020. rdw in haemoglobinopathy phenotyping the median rdw difference was 6.2 between the case (20.7, interquartile range [iqr] = 8.3) and the control groups (14.5, iqr = 2.7; 95%, ci = 9.1–19.9; p < 0.001). the rdw median for scd phenotypes were: haemoglobin ss genotype, 25.4 (iqr = 5.5); haemoglobin ss genotype + β-thalassemia, 23.3 (iqr = 7.9); and haemoglobin ss genotype + haemoglobin f, 20.9 (iqr = 5.5) which were significantly higher compared to the control group (14.5, iqr = 2.7; 95% ci = 9.1–19.9; p < 0.001). individuals with pure haemoglobin as genotype had a significantly higher (p < 0.001) rdw median of 16.4 (iqr = 6.5) compared to the control group (14.5, iqr = 2.7; 95% ci = 9.1–19.9). the rdw was high in haemoglobin as genotype + haemoglobin f (24.2, p = 0.449), haemoglobin as genotype + β-thalassemia (20.9, iqr = 10.5; p = 0.791) and β-thalassaemia (19.9, iqr = 8.6; p = 1.00) patients but the difference was not statistically significant when compared to the control group (14.5, iqr = 2.7; 95% ci = 9.1–19.9). diagnostic efficacy of the rdw in haemoglobinopathies at given optimal points, the rdw demonstrated its diagnostic efficacy by marking some haemoglobinopathies with a high sensitivity, specifity, youden index and asymptotic significance (p < 0.001) with their roc curves flowing upwards on the left side of the curve (table 2; figure 1). on the other hand, the diagnostic utility of rdw for some haemoglobinopathies was marked by low sensitivity, specifity and youden index and did not have asymptotic significance with their roc curve flowing along the diagonal line (figure 2 and figure 3). table 2: summary of rdw predictive ability in haemoglobin disorders in western kenya, 01 january 2015 – 31 december 2020. figure 1: red cell distribution width, roc curve in scd phenotypes (haemoglobin ss genotype, haemoglobin ss genotype + haemoglobin f, haemoglobin ss genotype + β-thalassaemia) in western kenya, 01 january 2015 – 31 december 2020. (a) red cell distribution width roc curve in diagnosis of homozygous scd. (b) red cell distribution width roc curve in diagnosis of sickle cell disease with haemoglobin f. (c) red cell distribution width roc curve in diagnosis of scd with β-thalassaemia. figure 2: red cell distribution width roc curve in sickle cell triat phenotypes (haemoglobin as genotype and β-thalassaemia) in western kenya, 01 january 2015 – 31 december 2020. (a) red cell distribution width, receiver operating characteristic curve for sickle cell trait flowing along diagonal line of the roc curve. (b) red cell distribution width, receiver operating characteristic curve for sickle cell trait+β-thalassaemia also flowing along the diagonal line of the roc curve. figure 3: red cell distribution width roc curve in β-thalassaemia in western kenya from 01 january 2015 – 31 december 2020. red cell distribution roc curve in sct phenotyping the rdw proportion for pure haemoglobin as genotype was 21.1% (n = 103; roc curve flowed along the diagonal line with a youden index = 0.501; p = 0.976) for diagnosis of pure sickle cell trait (sct) phenotype (table 2, figure 2). similarly, the rdw roc curve coordinates at the optimal point of 19.8 did not have diagnostic significance (p = 0.399, sensitivity, 50%, specificity 70%; curve flowed along the diagonal line with a low youden index = 0.600) for the diagnosis of haemoglobin as genotype + β-thalassaemia. red cell distribution roc curve in scd phenotyping the rdw proportion for haemoglobin ss genotype was 9.2% (n = 45; optimal point = 21.1; p < 0.001; accuracy = 0.892; sensitivity = 86.7%; specificity = 80.0%) (table 2, figure 1) pushing the curve to the left upper side of the roc curve. the proportion for haemoglobin ss genotype and haemoglobin f was 4.1% (n = 20; optimal point = 21.8; p < 0.001; sensitivity = 70.0%; specificity = 67.6%; youden index = 0.766), with the roc curve flowing above the diagonal line. a haemoglobin ss genotype + β-thalassaemia prevalence of 12.7% (n = 62) was obtained and at 17.7 the rdw optimal point (p < 0.001; sensitivity = 78.0%; specifity = 64.5%; youden index = 0.805). roc curve in β-thalassaemia the rdw optimal point of 16.8 for β-thalassaemia was not significant (p = 0.706). the roc curve flowed along the diagonal line area under curve = 0.539 with sensitivity = 63% and specificity = 60%) (table 2; figure 3). sensitivity and specificity of rdw in scd phenotype diagnosis the roc curves grouped haemoglobinopathies into two groups serving as an excellent significant (p < 0.001) biomarker in scd phenotypes diagnosis; but it was poor in the diagnosis of sct phenotypes and β-thalassemia (low youden index, sensitivity, and specificity) (table 2). therefore, the efficacy of rdw as a biomarker for use in scd phenotype diagnosis was evaluated in a single roc curve (figure 4) giving a sensitivity of 67.7%, specificity of 90.0% and an accuracy of 0.789 at an optimal point of 21.1 (table 2). figure 4: red cell distribution width roc curve in haemoglobin ss phenotypes in western kenya, 01 janauary 2015 – 31 december 2020. this roc curve demonstrates the efficacy of rdw at 21.1% optimal point in scd (haemoglobin ss) phenotypes diagnosis. predictive validity, likelihood and or of rdw in scd phenotyping the rdw at 21.1 optimal value, recorded 70.5% positive predictive validity and 88.8% negative predictive validity in scd phenotypes diagnosis (table 2). similarly, the same optimal value had a 6.77 positive likelihood ratio (lr+), 0.36 negative likelihood ratio (lr-) and 18.94 or in the diagnosis of haemoglobin ss phenotypes. discussion the rdw was able to diagnose scd phenotypes significantly (p < 0.001), but could not diagnose sct phenotypes and β-thalassaemia (p > 0.05; low youden index, sensitivity, and specificity). the overall accuracy of the rdw proved to be an excellent biomarker for scd haemoglobinoathies; thus, unknown (seemingly haemoglobinopathy-free) cases with rdw above 21.1 need to be confirmed using advanced technology. therefore, countries with limited financial resources who have not implemented newborn and population screening can use this potential biomarker as a cost-effective approach. a worthless test has a youden index of 0.5, poor sensitivity of 50%, a specificity of about 50% and a roc curve that flows along the diagonal line and thus is unable to distinguish diseased from non-diseased individuals.31 the rdw could not serve as a biomarker for pure sct (p = 0.976) and sct+β-thalassaemia (p = 0.399) diagnoses; both had low youden index, sensitivity and specifity. in homozygous scd, the rdw had a sensitivity of 86.7%, specificity of 80% and an accuracy of 0.892 which are features of a significant (p < 0.001) biomarker at 21.1 roc curve coordinate.31 similar findings of elevated rdw were documented by webster and castro32 with homozygous scd having the highest rdw, followed by heterozygous scd+β-thalassaemia and then scd and haemoglobin f. the rdw proved to be an excellent diagnostic marker for scd and haemoglobin f at 20.8 roc curve coordinate where a sensitivity of 70.0% and a specificity of 67.6% were obtained with a high accuracy of 0.766. the roc curve demonstrated that the rdw was an excellent diagnostic marker (p < 0.001) in detecting scd+β-thalassaemia with an accuracy of 0.805. the β-thalassaemia roc curve flowed along the diagonal line with poor diagnostic significance (p = 0.706) similar to qurtom et al’s report33 of β-thalassaemia having a normal or mildly elevated rdw at a mean of 15.4 ± 1.21, meaning it is not possible to use the rdw to differentiate β-thalassemia from the healthy population. the rdw roc curves were able to discriminate (p < 0.001) scd phenotypes from the healthy population but could not diagnose sct phenotypes and β-thalassaemia. the optimal rdw value for the overall efficacy in diagnosing scd phenotypes was 21.1 (sensitivity = 67.7%; specificity = 90.0%; accuracy = 0.789). this means that in a population, an rdw > 21.1 can identify 67.7% of the individuals as having scd while an rdw of < 21.1 will identify 90% of individuals who would test negative for scd. this implies that at 21.1, the rdw can be used as an optimal diagnostic biomarker for scd phenotypes in western kenya. a positive predictive value tells how likely an individual will test positive for a given disease. in regard to the present study, patients with rdw > 21.1 were 70.5% likely to test positive for scd indicating 29.5% would still test negative for scd even when the rdw was > 21.1. negative predictive value tells how likely an individual will be to test negative for a particular disease meaning 88.1% of patients with rdw < 21.1 would be free from scd while 11.9% would still test positive. therefore, the rdw is proving to be a good diagnostic biomarker for scd phenotypes among haemoglobinopathies listed in the present study. to this end, this is the first-ever attempt to determine the likelihood of using the rdw value to diagnose scd phenotype in western kenya. likelihood ratios are clinically more useful than sensitivity and specificity in determining the usefulness of diagnostic tests. the positive likelihood ratio (lr+) expresses how likely a test is going to correctly diagnose the presence of the condition where the greater the lr+, the more likely the test is going to give a true positive diagnosis. the rdw > 21.1 had a lr+ of 6.77, meaning any individual having the rdw > 21.1 is 6.77 times more likely to test positive for scd. negative likelihood ratio (lr–) is defined by akobeng34 as the ratio of how likely a test will correctly diagnose the absence of a condition whose value is usually < 1; the closer the value gets to zero, the better the test is correctly indicating the absence of the condition. regarding the present study, lr– was 0.36, meaning the probability of a person with the rdw < 21.1, is 0.36 times less likely to be free from scd. it is important to note a test having both lr+ and lr– close to 1 has little influence to predict the presence or absence of a disease and is, therefore, worthless in clinical practice. on the same optimal value, an or of 18.9 was obtained meaning that individuals with the rdw > 21.1 were 18.9 times at greater risk of having scd haemoglobinopathy compared to those with the rdw < 21.1. limitations sickle cell traits +haemoglobin f and +β-thalassaemia recorded abnormally elevated rdw that did not have statistical significance due to the small sample size. conclusion these findings indicate that the rdw is a promising diagnostic marker for scd phenotypes; thus, rdw of 21.1 should be included in the haematology policy screening algorithm as a critical value above which the unknown cases qualify to be investigated for sickle cell haemoglobinopathy. however, the data used were retrospective and hence the diagnostic utility of this haematological index for haemoglobinopathy should be explored further using prospective data. a confirmatory test would still be needed and therefore provision and use of powered mini-electrophoretic equipment will be appropriate in low-resource settings. acknowledgements we are grateful to mr. raphael kitonga for allowing us to use his home-based library and its accessories in addition to steadfast moral support. we appreciate the aga khan hospital, kisumu laboratory manager lucy mathenge and dr simon onsongo head of the pathology department for supporting us during data collection. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions b.m.m. conceptualised the study, analysed data, wrote the original draft of the manuscript and critical reviews. g.s. conceptualised the study, and contributed to the validation and critical reviews. p.o. conceptualised the study, gave critical technical expert advice on the study, validation and gave critical reviews on the manuscript. sources of support the authors received no financial support for the research, authorship, or publication of this article. data availability data supporting the findings of this study is available upon request from the corresponding author, b.m.m. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references weatherall d. the inherited disorders of haemoglobin: an increasingly neglected global health burden. 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their use in clinical practice. acta paediatr. 2007;96(4):487–491. https://doi.org/10.1111/j.1651-2227.2006.00179.x abstract introduction methods results discussion acknowledgements references about the author(s) danielle martin department of clinical microbiology and infectious diseases, national health laboratory service and faculty of health sciences, university of the witwatersrand, johannesburg, south africa rispah chomba department of clinical microbiology and infectious diseases, national health laboratory service and faculty of health sciences, university of the witwatersrand, johannesburg, south africa tshegofatso pelego national institute of communicable diseases, centre for tuberculosis, johannesburg, south africa sanelisiwe t. duze department of clinical microbiology and infectious diseases, national health laboratory service and faculty of health sciences, university of the witwatersrand, johannesburg, south africa citation martin d, chomba r, pelego t, duze st. in silico and in vitro validation of a duplex polymerase chain reaction assay for detecting cronobacter sakazakii in powdered infant milk. afr j lab med. 2025;14(1), a2902. https://doi.org/10.4102/ajlm.v14i1.2902 original research in silico and in vitro validation of a duplex polymerase chain reaction assay for detecting cronobacter sakazakii in powdered infant milk danielle martin, rispah chomba, tshegofatso pelego, sanelisiwe t. duze received: 13 june 2025; accepted: 16 sept. 2025; published: 30 nov. 2025 copyright: © 2025. the authors. licensee: aosis. this work is licensed under the creative commons attribution 4.0 international (cc by 4.0) license (https://creativecommons.org/licenses/by/4.0/). abstract background: cronobacter sakazakii causes life-threatening infections in neonates, primarily transmitted through contaminated powdered infant formula (pif). in lowand middle-income countries, limited surveillance and diagnostic capacity hinder accurate detection of c. sakazakii, highlighting the need for rapid and affordable testing methods. objective: to validate a duplex polymerase chain reaction (pcr) assay for rapidly detecting cronobacter spp. and c. sakazakii in pif using both in silico and in vitro approaches. methods: this study was conducted in south africa between march and august 2022. seven gene targets and their primers were selected from published literature. to assess sensitivity and specificity, in silico pcr was performed using genome sequences of cronobacter and non-cronobacter species from the national center for biotechnology information database. the best-performing primers were selected for an in vitro analysis using bacterial isolates and pif samples from the infection control service laboratory. the specificity of the assay was assessed using eight foodborne pathogens, and further evaluated using pif samples artificially contaminated with cronobacter spp., bacillus cereus, and salmonella typhi. results: the best-performing primers, lpfa_1, fimg, and fimp1, showed 100% sensitivity and specificity. duplex pcr assay successfully detected both cronobacter spp. and c. sakazakii with no cross-reactivity with non-cronobacter pathogens, and remained effective in the presence of contaminants such as b. cereus and salmonella typhi. conclusion: the validated duplex pcr assay offers a rapid, specific, and affordable assay for detecting cronobacter spp. and c. sakazakii in pif. what this study adds: this assay combined in silico and in vitro validation of a rapid and affordable pcr assay for pif screening in resource-limited settings. keywords: cronobacter spp.; cronobacter sakazakii; powdered infant formula; polymerase chain reaction; specificity; sensitivity. introduction cronobacter sakazakii is a gram-negative, opportunistic, motile, food-borne bacterium within the enterobacterales family.1 originally described as ‘yellow-pigmented enterobacter cloacae’, it was renamed enterobacter sakazakii in 1980, and later reclassified under the genus cronobacter.2,3 the genus currently comprises seven species: cronobacter condimenti, c. dublinensis, c. malonaticus, c. muytjensii, c. sakazakii, c. turicensis, and c. universalis.3 among these, only c. sakazakii, c. turicensis, and c. malonaticus are associated with human diseases, particularly c. sakazakii, the most clinically significant species, often associated with neonatal meningitis.4,5,6,7 the majority of c. malonaticus infections occur in adults and are less severe.6 cronobacter sakazakii is a clinically significant pathogen, particularly in neonates and infants, where infections can lead to meningitis, necrotising enterocolitis, septicaemia, and even death.8,9 premature infants, or those with low birthweight or a compromised immune system, are at an even greater risk of infection, and outbreaks of disease have been reported in hospital units for newborns.10,11 cronobacter infections are often attributed to contaminated powdered infant formula (pif), which is a nonsterile product.1,11,12 cronobacter sakazakii has also been isolated from breast milk expressed with contaminated pump equipment,13,14 as well as other environmental sources, including pacifiers, bottles, feeding utensils, and kitchen sink surfaces, all of which serve as potential points of contamination for pif.14 despite its severity, the public health impact of c. sakazakii is often underestimated, particularly in lowand middle-income countries because of the absence of active surveillance systems, underreporting of cases, and inadequate diagnostic infrastructure.15 although c. sakazakii has been detected in various food products and environments across africa, including south africa,16,17,18 egypt,19,20,21 côte d’ivoire,22 and nigeria,23 epidemiological data remain scarce. this diagnostic gap underscores the need for rapid, accurate, and affordable detection methods to ensure the safety of pif and enable timely public health interventions, as c. sakazakii infections are associated with high morbidity and mortality.7 moreover, survivors often suffer irreversible neurological sequelae, such as brain abscesses, quadriplegia, developmental delays, hydrocephalus, and motor impairment.5,24,25,26 traditional culture-based methods, such as those recommended by the us food and drug administration, the international organization for standardization, and the international dairy federation (iso/ts 22964:2006), are widely used for detecting c. sakazakii in pif and are considered reliable.27 however, these methods are labour-intensive, require a pre-enrichment step, and can take up to six days to yield results. additionally, traditional biochemical tests and chromogenic media used for confirmation are limited by phenotypic overlap between cronobacter spp. and other enterobacteriaceae, which can lead to misidentification.27,28 furthermore, competition from faster-growing organisms and interference from background microflora can reduce recovery rates and hinder accurate detection.29 molecular methods, such as polymerase chain reaction (pcr), offer higher sensitivity and specificity, along with faster turnaround times. existing pcr assays often lack species-level specificity and may require multiple reactions to differentiate closely related cronobacter spp.4,30,31 moreover, the limited availability of rapid and accurate detection methods is compounded by the high cost and technical complexity of the existing assays, making routine testing especially challenging in resource-limited settings. this study, therefore, aimed to optimise and validate an accurate and affordable duplex pcr assay for the rapid and simultaneous detection of cronobacter spp. and c. sakazakii in pif, ideal for use in resource-limited settings. methods ethical considerations an ethics waiver (reference number: w-cbp-220422-03) was obtained from the witwatersrand human research ethics committee (medical), university of the witwatersrand, johannesburg, south africa. the study was conducted between march 2022 and august 2022, and involved no human participants or animal research. bacterial isolates and pif samples were sourced from the infection control service laboratory of the national health laboratory service. the pif samples were tested for contaminants during routine food testing at the infection control service laboratory before use in artificial contamination experiments. all of the experiments complied with institutional and laboratory biosafety and ethical guidelines. the data generated contained no personal identifiers and were managed following institutional data protection policies. study design this study was a laboratory-based validation combining in silico and in vitro approaches to evaluate the performance of primers for a duplex pcr assay that can rapidly detect cronobacter spp. and c. sakazakii in pif. the study was conducted at the infection control service laboratory of the national health laboratory service at the university of the witwatersrand in johannesburg, south africa, between march 2022 and august 2022. sample collection this study used a collection of 27 complete cronobacter genome sequences (20 c. sakazakii, 2 c. malonaticus, 1 c. turicensis, 1 c. dublinensis, 1 c. universalis, 1 c. muytjensii, and 1 c. condiment), and 20 complete genome sequences of non-cronobacter foodborne pathogens, including three salmonella typhi, two shigella sonnei, two listeria monocytogenes, three e. cloacae, two escherichia coli, three bacillus cereus, two klebsiella pneumoniae, and three staphylococcus aureus. the genome sequences were downloaded as fasta files from the national center for biotechnology information database (ncbi: https://www.ncbi.nlm.nih.gov) and were used in the in silico pcr analysis. in addition, 11 previously characterised foodborne pathogens and four pif samples (pif7069, pif6748, pif7070, and pif6749) from two brands were obtained from the infection control service laboratory through routine diagnostics; these were used for the in vitro pcr analysis. the foodborne pathogens included three cronobacter isolates (c. turicensis, c. sakazakii m0020, and c. sakazakii m0022) and eight non-cronobacter isolates (salmonella typhi, s. sonnei, l. monocytogenes, e. cloacae, e. coli, b. cereus, k. pneumoniae, and s. aureus). routine food testing confirmed that pif7069, pif6748, and pif7070 were free of bacterial contamination, whereas pif6749 was positive for b. cereus. gene targets and primer selection a literature search through pubmed, google scholar, and scopus was performed to identify gene targets for the molecular detection of cronobacter spp. and c. sakazakii. the search was performed using the following terms: ‘cronobacter sakazakii’, ‘cronobacter spp.’, ‘pcr’, ‘gene target’, ‘species-specific primers’, ‘genus-specific primers’. seven gene targets were selected based on published studies that reported their potential diagnostic value in differentiating cronobacter spp., specificity, sensitivity, and availability of the primer sequences. these seven gene targets included six species-specific genes (cgca, gyrb, rpob, fimp1, fimg, lpfa_1) and one genus-specific gene (grxb). the corresponding primer sequences for each gene target were downloaded and used in the in silico pcr analysis (table 1). table 1: gene targets and primer sequences for detecting cronobacter spp. and cronobacter sakazakii. in silico pcr analysis genome sequences of the 27 cronobacter species and 20 non-cronobacter foodborne pathogens were used in the in silico pcr analysis to determine the sensitivity and specificity of the primer sequences for each gene target. the in silico pcr was performed using an online platform available at http://insilico.ehu.es/pcr/, which simulates theoretical pcr amplification based on primer sequences and genome sequences.35 each primer pair was tested against each of the 47 genome sequences, using default settings to determine the presence or absence of an amplification product matching the expected amplicon size. the in silico pcr analysis provides the predicted start position and size of the amplicon. results were recorded as positive (+) if the expected product size was detected, and negative (-) if no product was observed. thereafter, the sensitivity and specificity of each primer set were calculated using equations 1 and 2. the species-specific primers with the highest sensitivity and specificity for c. sakazakii detection were selected for further validation in vitro. pcr optimisation and specificity cronobacter turicensis, c. sakazakii m0020, and c. sakazakii m0022, and eight non-cronobacter foodborne pathogens were cultured on nutrient agar (thermo fischer scientific, waltham, massachusetts, united states) and incubated overnight at 36 °c. crude genomic dna was extracted by suspending two loopfuls of bacterial colonies in 200 µl of tris-ethylenediaminetetraacetic acid (te) buffer, followed by incubation at 95 °c for 25 min, vortexing, and centrifugation at 12 000 rpm for 3 min. the supernatant was collected and stored at 8 °c until further use. two singleplex pcr assays targeting the lpfa_1 and grxb genes, were optimised in a 25 µl reaction volume containing 12.5 µl of 2x pcr master mix (thermo fischer scientific, waltham, massachusetts, united states), 2 µl of dna, 0.2 µm primers, and nuclease-free water. the pcr cycling conditions were 95 °c for 3 min, 35 cycles of 95 °c for 30 s, 51 °c for 30 s, 72 °c for 60 s, followed by a final extension at 72 °c for 10 min. amplicons were resolved by gel electrophoresis on a 2% agarose gel stained with gelred™ (anatech, johannesburg, south africa), using 1x tris-borate-edta buffer at 110 v, and visualised using the geldoc system (bio-rad laboratories, hercules, california, united states). the duplex pcr assay targeting both lpfa_1 and grxb was optimised under the same conditions, with each primer added at a final concentration of 0.2 µm. in all pcr reactions, c. turicensis and c. sakazakii m0020 were used as positive controls for the grxb and lpfa_1, and nuclease-free water was used for the negative control. the specificity of the duplex pcr assay was evaluated using genomic dna from c. sakazakii m0022, c. turicensis, and eight non-cronobacter foodborne pathogens listed above. the specificity of the assay was calculated using equation 2. artificial contamination to artificially contaminate the pif samples with c. sakazakii, 5 µl of a suspension (108 colony-forming units/ml) was added to 10 g of each sample in a sterile glass bottle with 100 ml buffered peptone water, followed by incubation for 6 h at 36 °c. this resulted in an estimated final concentration of approximately 5.0 × 106 colony-forming units/ml in the pre-enrichment. the pif7070 sample was also inoculated with 5 µl of salmonella typhi (108 colony-forming units/ml). after incubation, 40 ml of the pre-enrichment broth was centrifuged at 3000×g for 10 min. the pellet was resuspended in 200 µl of supernatant, and genomic dna was extracted using the high pure pcr template preparation kit (roche diagnostics gmbh, mannheim, germany), followed by pcr amplification using the duplex pcr assay. powdered infant formula samples pif7070 and pif6749 were specifically used to evaluate assay interference. the experiment was performed in duplicate. data analysis all data were collected and recorded using microsoft excel® 2010. for both in silico and in vitro pcr assays, amplification results were recorded based on the presence or absence of the expected amplicon size. similarly, results from the artificially contaminated pif samples were classified as positive or negative according to band visualisation on a 2% gel electrophoresis. a true positive was defined as a c. sakazakii or cronobacter spp. yielding a positive amplification with the expected amplicon size. a true negative was defined as a non-cronobacter foodborne pathogen (or genome sequence) with no amplification, confirming assay specificity. false positives were recorded when amplification occurred in non-cronobacter spp., and false negatives when the assay failed to amplify the target genes for cronobacter spp. the results were tabulated, and the sensitivity and specificity were calculated using equation 1 and equation 2: results in silico pcr performance in total, seven primer pairs corresponding to the seven gene targets for cronobacter spp. and c. sakazakii detection were evaluated against 47 genome sequences using in silico pcr. species-specific primers targeting the lpfa_1, fimg, and fimp1 genes demonstrated 100% sensitivity and specificity for c. sakazakii detection during in silico pcr. the cgca and gyrb primers had 100% specificity, with sensitivities of 94.7% and 89.5%, each missing one or two c. sakazakii sequences during the in silico pcr (table 2). the rpob primers exhibited the lowest performance, with sensitivity and specificity of 78.9% and 92.6%, whereas the genus-specific grxb primers detected all cronobacter spp. except c. dublinensis and c. condiment, resulting in 92.3% sensitivity and 100% specificity (table 2). based on these results, lpfa_1 and grxb primers were selected for in vitro testing. table 2: summary of the sensitivity and specificity of each primer pair based on in silico polymerase chain reaction analysis. in vitro pcr performance two singleplex pcr assays targeting the lpfa_1 and grxb genes were successfully optimised and effectively detected the respective genes in c. sakazakii m0022, c. sakazakii m0020, and c. turicensis (figure 1a). additionally, a duplex pcr assay targeting both genes was optimised, demonstrating simultaneous detection of both the lpfa_1 and grxb genes in c. sakazakii m0022, c. sakazakii m0020, while only detecting the grxb gene in c. turicensis (figure 1b). figure 1: (a) gel electrophoresis of the singleplex polymerase chain reaction (pcr) assays targeting the lpfa_1 (881 bp; lanes 2–5) and grxb (378 bp; lanes 6–9) genes: lane 1, dna ladder (100 bp, thermo fischer scientific, waltham, massachusetts, united states); lane 2, cronobacter sakazakii m0022; lane 3, c. turicensis; lane 4, c. sakazakii m0020; lane 5, negative control; lane 6, c. sakazakii m0022; lane 7, c. sakazakii m0020; lane 8, c. turicensis; and lane 9, negative control. (b) duplex pcr assay targeting c. sakazakii and cronobacter spp. in a single reaction: lane 1, dna ladder (fastruler, thermo fischer scientific, waltham, massachusetts, united states); lane 2, c. sakazakii m0022; lane 3, c. sakazakii m0020; lane 4, c. turicensis; and lane 5, negative control. the duplex pcr assay was 100% specific for cronobacter spp. and c. sakazakii, with successful amplification of the grxb gene in c. sakazakii m0022, c. sakazakii m0020, and c. turicensis, as well as the amplification of the lpfa_1 gene in c. sakazakii m0022 and c. sakazakii m0020, and no amplification in any of the eight non-cronobacter foodborne pathogens (figure 2a). moreover, the lpfa_1 and grxb genes were successfully detected in the pif samples artificially contaminated with c. sakazakii m0022 (figure 2b, table 3), with no interference from the non-cronobacter foodborne pathogens in pif samples pif6749 (containing b. cereus) and pif7070 (containing salmonella typhi). these results demonstrate the assay’s reliability and suitability for detecting cronobacter spp. and c. sakazakii in pif, even in the presence of other foodborne pathogens. figure 2: (a) gel electrophoresis showing the specificity of the polymerase chain reaction (pcr) assay: lane 1, dna ladder (100 bp, promega, madison, wisconsin, united states); lanes 2–9, non-cronobacter species (escherichia coli, listeria monocytogenes, bacillus cereus, salmonella typhi, klebsiella pneumoniae, staphylococcus aureus, enterobacter cloacae, shigella sonnei); lane 10, cronobacter turicensis; lane 11, c. sakazakii m0022; lane 12, c. sakazakii m0020; and lane 13, negative control. (b) polymerase chain reaction results from the artificially contaminated powdered infant formula (pif) samples: lane 1, dna ladder (fastruler, thermo fischer scientific, waltham, massachusetts, united states); lane 2 and lane 5, pif contaminated with c. sakazakii m0022; lane 3, pif with b. cereus and c. sakazakii m0022; lane 4, pif with salmonella typhi and c. sakazakii m0022; lane 6, c. sakazakii m0020; lane 7, c. turicensis; and lane 8, negative control. table 3: cronobacter sakazakii detection in artificially contaminated powdered infant formula samples. discussion in silico pcr is a valuable tool used to predict amplification efficiency, specificity, and amplicon size by aligning primers against target genomes, facilitating the selection of optimal primer pairs for pcr assays.36 in our study, primers targeting the rpob, cgca, and gyrb genes exhibited suboptimal sensitivity and specificity for c. sakazakii detection. the rpob gene requires two separate pcr reactions to differentiate between c. sakazakii and c. malonaticus, limiting its diagnostic utility.31 our findings are consistent with previous studies reporting limited species-level resolution of the cgca and gyrb primers.31,33 in contrast, primers targeting the fimp1, fimg, and lpfa_1 showed 100% specificity and sensitivity in silico. however, the short amplicon size of fimp1 (103 bp) reduces its suitability for conventional pcr. prior studies investigating the suitability of fimg and lpfa_1 genes for c. sakazakii detection using bioinformatics and in vitro approaches demonstrated superior sensitivity for lpfa_1 over fimg, supporting the selection of the lpfa_1 gene for further validation.4 the genus-specific grxb gene, while slightly less sensitive, showed broad detection across cronobacter genomes and 100% specificity within our study sample, reinforcing its value for genus-level identification. our findings are consistent with previous studies that have successfully employed the grxb gene for the detection of cronobacter spp.34,36,37,38 in vitro validation confirmed the efficacy of the selected primers in detecting cronobacter spp. and c. sakazakii. singleplex and duplex pcr assays targeting lpfa_1 and grxb successfully detected c. sakazakii and c. turicensis with high specificity. the duplex assay enabled simultaneous detection of both targets in artificially contaminated pif, even in the presence of other foodborne pathogens such as b. cereus and salmonella typhi. these results corroborate the in silico findings and demonstrate the assay’s robustness and reliability. importantly, the duplex pcr assay reduced turnaround time to approximately 12 h, compared to the 3–6-day protocol required by the conventional culture-based method recommended by the united states food and drug administration.27,39 this time efficiency, combined with high specificity and affordability, makes the assay particularly suitable for resource-limited settings where rapid screening is essential for public health interventions and outbreak response. although culture-based methods remain the cornerstone of microbiological diagnostics, particularly for strain isolation, antimicrobial susceptibility testing, and whole-genome sequencing, which are essential for surveillance and outbreak investigations. the duplex pcr assay reported in this study will complement culture-based methods by serving as a rapid screening tool for c. sakazakii in pif, enabling early detection of contaminated pif products. the assay will facilitate timely interventions and support targeted use of culture-based confirmation, especially in resource-limited settings where laboratory capacity and turnaround time are constrained. by identifying positives quickly, this pcr assay will enable resource-limited laboratories to prioritise samples for culture, thereby optimising resource use and improving overall diagnostic turnaround time. limitations this study had a few limitations. the in vitro sensitivity of the duplex pcr assay could not be fully assessed because of the limited availability of c. sakazakii and other cronobacter spp. isolates, despite efforts to source additional strains from public and private laboratories. however, incorporating in silico validation of the primers using publicly available genome sequences from multiple cronobacter species allowed us to evaluate primer specificity across diverse species. in silico analysis thus provided a valuable alternative for assay validation. this approach also supports the growing utility of bioinformatics tools in enhancing assay design. another limitation of this study is that the sensitivity of the assay was determined using high bacterial concentrations, which may not fully represent the variable contamination levels of c. sakazakii typically found in pif, as contamination may occur at low levels, and accurate detection under such conditions is critical for food safety. future work should therefore include the determination of the assay’s limit of detection using serial dilutions of c. sakazakii spiked into pif. while this study focused on conventional pcr to ensure affordability and accessibility in basic laboratory settings, it may not fully meet the throughput and automation needs of modern food safety laboratories. adapting the assay into a real-time multiplex pcr assay will allow for quantitative detection, improved sensitivity, and reduced turnaround time. additionally, integrating automated dna extraction workflows could streamline sample processing and minimise human error, making the assay more suitable for high-throughput environments and outbreak response scenarios. these enhancements would support broader implementation in both resource-limited and well-equipped laboratories, contributing to more efficient monitoring of cronobacter contamination in infant formula. conclusion this assay combined in silico and in vitro validation of a rapid and affordable duplex pcr assay for the simultaneous detection of cronobacter spp. and c. sakazakii in pif. the performance of the assay was not affected by the presence of non-cronobacter species such as b. cereus and salmonella typhi. this duplex pcr assay can facilitate the diagnosis of c. sakazakii in pif, particularly in resource-limited settings, contributing to improved epidemiological surveillance and screening of cronobacter spp. in pif. its adaptability for routine diagnostics and outbreak investigations further underscores its potential for integration into national food safety monitoring programmes. acknowledgements the authors would like to acknowledge the staff of the infection control service laboratory of the national health laboratory service for providing us with the bacterial isolates and powdered infant formula samples used in this study. this article is partially based on danielle martin’s thesis entitled, ‘optimizing a pcr assay for rapid detection of cronobacter sakazakii in powdered infant formula’, towards the degree of bhsc honours in the department of clinical microbiology and infectious diseases, university of the witwatersrand, south africa, submitted in december 2022, with supervisors rispah chomba, tshegofatso pelego and sanelisiwe t. duze. the thesis is not publicly available. competing interests the author reported that they received funding from national research foundation which may be affected by the research reported in the enclosed publication. the author has disclosed those interests fully and has implemented an approved plan for managing any potential conflicts arising from their involvement. the terms of these funding arrangements have been reviewed and approved by the affiliated university in accordance with its policy on objectivity in research. authors’ contributions all of the authors contributed to this manuscript. s.t.d. and r.c. conceptualised the original idea for the study and supervised this study. d.m. and t.p. carried out the laboratory work, data collection, and analysis; d.m. and s.t.d. wrote the manuscript; and r.c., d.m., t.p., and s.t.d. reviewed the final draft of the article. funding information this work was supported by the national research foundation (nrf) under sanelisiwe t. duze (grant number 138279). data availability the authors confirm that the data supporting the findings of this study are available within the article. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. the article does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the authors are responsible for this article’s results, findings, and content. references jason j. prevention of invasive cronobacter infections in young infants fed powdered infant formulas. pediatrics. 2012;130(5):e1076–e1084. https://doi.org/10.1542/peds.2011-3855 farmer jj, 3rd, asbury ma, hickman fw, brenner dj, enterobacteriaceae study group. 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opportunistic pathogen of concern to neonatal health. front pediatr. 2015;3:38. https://doi.org/10.3389/fped.2015.00038 abstract introduction methods results discussion acknowledgements references about the author(s) yogandree ramsamy department of medical microbiology, school of laboratory medicine and medical sciences, antimicrobial research unit, university of kwazulu-natal, national health laboratory services, durban, south africa sabiha y. essack antimicrobial research unit, college of health sciences, university of kwazulu-natal, durban, south africa benn sartorius school of nursing and public health, university of kwazulu-natal, durban, south africa miriam patel antimicrobial research unit, college of health sciences, university of kwazulu-natal, durban, south africa koleka p. mlisana national health laboratory services, university of kwazulu-natal, durban, south africa citation ramsamy y, essack sy, sartorius b, patel m, mlisana kp. antibiotic resistance trends of eskape pathogens in kwazulu-natal, south africa: a five-year retrospective analysis. afr j lab med. 2018;7(2), a887. https://doi.org/10.4102/ajlm.v7i2.887 original research antibiotic resistance trends of eskape pathogens in kwazulu-natal, south africa: a five-year retrospective analysis yogandree ramsamy, sabiha y. essack, benn sartorius, miriam patel, koleka p. mlisana received: 07 aug. 2018; accepted: 28 sept. 2018; published: 06 dec. 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: to combat antimicrobial resistance, the world health organization developed a global priority pathogen list of antibiotic-resistant bacteria for prioritisation of research and development of new, effective antibiotics. objective: this study describes a five-year resistance trend analysis of the eskape pathogens: enterococcus faecium, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa and enterobacter spp., from kwazulu-natal, south africa. methods: this retrospective study used national health laboratory services data on 64 502 eskape organisms isolated between 2011 and 2015. susceptibility trends were ascertained from minimum inhibitory concentrations and interpreted using clinical and laboratory standards institute guidelines. results: s. aureus was most frequently isolated (n = 24, 495, 38%), followed by k. pneumoniae (n = 14, 282, 22%). decreasing rates of methicillin-resistant s. aureus (28% to 18%, p < 0.001) and increasing rates of extended spectrum beta-lactamase producing k. pneumoniae (54% to 65% p < 0.001) were observed. carbapenem resistance among k. pneumoniae and enterobacter spp. was less than 6% during 2011–2014, but increased from 4% in 2014 to 16% in 2015 (p < 0.001) among k. pneumoniae. p. aeruginosa increased (p = 0.002), but resistance to anti-pseudomonal antimicrobials decreased from 2013 to 2015. high rates of multi-drug resistance were observed in a. baumanni (> 70%). conclusion: this study describes the magnitude of antimicrobial resistance in kwazulu-natal and provides a south african perspective on antimicrobial resistance in the global priority pathogen list, signalling the need for initiation or enhancement of antimicrobial stewardship and infection control measures locally. introduction the burden of multi-drug resistant (mdr) bacteria has increased substantially worldwide1 and infections caused by these organisms continue to pose a great challenge to public health systems and populations at large. mdr is defined as non-susceptibility to at least one agent in three or more antimicrobial categories.2 mdr bacterial infections are among the top three threats to global public health. due to the lack of surveillance programmes, the true extent of antimicrobial resistance in the african region is unknown or underestimated. additionally there is no formal framework for collaboration among surveillance programmes in the region.1 a request was made to the world health organization (who) by member states in 2017 to develop a global priority pathogen list (ppl) of antibiotic-resistant bacteria to help in prioritising the research and development of new and effective antibiotic treatments.3 due to the high prevalence of multi-drug resistance among eskape bacteria, defined by the infectious diseases society of america as enterococcus faecium, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa and enterobacter spp., these pathogens feature prominently in the global ppl of antibiotic-resistant bacteria and are the focus of this article. members of the eskape group of bacteria ‘escape’ the biocidal action of antibiotics and present new paradigms in pathogenesis, transmission and resistance of infectious diseases.4 eskape pathogens with multiple drug resistance mechanisms have been implicated in life-threatening nosocomial infections, especially among critically ill individuals,5 presenting clinicians with serious therapeutic challenges.5 the global ppl stratifies bacterial pathogens into three priority tiers: critical, high and medium.3 carbapenem-resistant a. baumannii, p. aeruginosa and enterobacteriaceae spp., which includes k. pneumoniae, feature in the critical priority tier. methicillin-resistant, vancomycin intermediate and resistant s. aureus, in addition to e. faecium, are featured in the high priority tier. the eskape group of bacteria deserve special attention due to their association with antibiotic resistance,6 as infections caused by this particular group of pathogens result in high mortality and morbidity rates, increased healthcare costs, diagnostic dilemma and difficulty in the initiation of empirical treatment.7 as a control measure to decrease the incidence of infections due to eskape pathogens, site-by-site surveillance studies and antibiograms are necessary to inform effective empiric therapy.8 this study assessed trends in annual resistance rates for all eskape pathogens processed over a five-year period from 2011 through to 2015 in kwazulu-natal, south africa. evidence before this study pathogen surveillance and antimicrobial resistance surveillance informs effective antimicrobial prescribing, in addition to controlling the spread of resistant pathogens within the local environment. it is well known that dedicated surveillance systems form an integral strategy to combat antibiotic resistance; however, surveillance of antibiotic-resistant bacteria is severely lacking in most african countries. the largest antimicrobial resistance surveillance data set from sub-saharan africa was published in 2017 and reflected antimicrobial resistance trends from isolates implicated in bloodstream infections from adults and children in malawi over a 19-year period were described.9 there have been no long-term surveillance data reports describing eskape pathogens and their antimicrobial resistance patterns from kwazulu-natal, south africa, except for national sentinel site surveillance describing antimicrobial resistance among k. pneumoniae isolates from bloodstream infections from 2009–2012. hence, this study aimed at providing a description of eskape pathogens and their antibiotic resistance trends over a five-year period in the province of kwazulu-natal, south africa. added value of this study the eskape group of bacteria are among the critical and high priority pathogens on the global ppl. this study provides the largest data set of the eskape pathogens gathered from adults and children who presented to the largest public service hospitals in kwazulu-natal, south africa, over a five-year period. the surveillance data provided in this manuscript provide a good baseline surveillance intervention to combat antimicrobial resistance. implications of all the available evidence in this study the who global ppl identifies priority pathogens for the research and development of new antibiotics. knowledge of these pathogens and their resistance patterns in local contexts is pivotal in our fight against antimicrobial resistance. despite a decreasing trend in methicillin-resistant s. aureus (mrsa), there is emergence and rapid expansion of extended spectrum beta-lactamase (esbl)-producing k. pneumoniae coupled with fluoroquinolone resistance observed in this study. additionally, increasing resistance to third-generation cephalosporins and amoxicillin-clavulanate is a growing concern, as there is now a greater reliance on the carbapenems to treat infections caused by k. pneumoniae. increasing resistance to carbapenems observed in k. pneumoniae is slowly becoming a reality in kwazulu-natal and may spell the beginning of the end for carbapenems against enterobacteriaceae such as k. pneumoniae. continued surveillance will provide valuable information on antimicrobial resistance patterns for eskape pathogens, which will assist in informing empiric antimicrobial therapy. methods ethical considerations this study was approved by the biomedical research ethics committee of the university of kwazulu-natal (study approval number: be085/12). study sites data collected from august 2011 to december 2015 at nine participating public sector hospitals from two districts across the province of kwazulu-natal were utilised in this study. the nine participating hospitals are the largest in the province and represent all levels of healthcare as described in tables 1 and 2.10 table 1: public hospitals and bed numbers in kwazulu-natal, south africa. table 2: levels of healthcare in kwazulu-natal, south africa. bacterial samples each participating centre extracted data of all isolates categorised as eskape pathogens. all body sites were considered acceptable sources. samples submitted for microbiological analysis included blood, urine, catheters (central venous catheter + hemodialysis) and respiratory specimens, in addition to samples from other body sites. not every patient who presented to a healthcare facility with an infection submitted a sample for microbiological analysis. additionally, the data presented included organisms from all specimen types; hence, colonisation was not distinguished from infection. antimicrobial susceptibility testing all participating laboratories in this study subscribed to the national health laboratories services (nhls) proficiency testing scheme. evaluations are carried out quarterly and samples for the scheme are prepared with assistance from the national institute for communicable diseases – centre for opportunistic, tropical and hospital infections, a division of the nhls. thus, all laboratories participating in this study were proficient in testing bacterial samples. all participating laboratories were responsible for sample processing, including isolate identification and susceptibility testing. pathogen identification was determined using the vitek 2 (biomerieux, marcy l’étoile, france) platform. antimicrobial susceptibility testing and minimum inhibitory concentration determinations were performed using the vitek 2 (biomerieux, marcy l’étoile, france) platform. the results were interpreted according to the criteria of the clinical laboratory standards institute.11 the antimicrobial test panel included: penicillin, ampicillin, amoxicillin-clavulanate, ceftriaxone, cefepime, cefuroxime, cefoxitin, ceftazadime, imipenem, meropenem, ertapenem, piperacillin-tazobactam, amikacin, gentamicin, erythromycin, clindamycin, linezolid, teicoplanin, vancomycin, fusidic acid, mupirocin, tetracycline, oxacillin, rifampicin, nitrofurantoin, colistin, trimethoprim/sulfamethoxazole, ciprofloxacin and tigecycline. methicillin resistance in s aureus was inferred by oxacillin resistance.11 the vitek 2 ast-n255 card was used to perform antimicrobial susceptibility testing on gram-negative, whereas the ast-p603 card was used for gram-positive organisms. presumptive esbl production in k. pneumoniae was determined using the minimum inhibitory concentration for ceftriaxone and ceftazidime as per the clinical laboratory standards institute guidelines.11 isolates were characterised as susceptible or resistant using clinical laboratory standards institute-approved breakpoints.11 data extraction and analysis all information regarding specimen collection, sampling and patient laboratory results is deposited on the nhls laboratory database. the use of this database and access to patient information is restricted to laboratory staff working within the nhls. thus, data collection was done at the nhls and entailed extraction of isolate information from this computerised laboratory database. the data received were de-duplicated, anonymised and patient confidentiality was maintained at all times. isolate information, specimen type and results of antimicrobial susceptibility testing, including minimum inhibitory concentration data, were also extracted. once the data were extracted, trends in the total number of eskape pathogens and their antimicrobial resistance patterns were determined. these trends were compared over a five-year period. statistical analysis all data processing and analyses were performed using stata 13.0 software se (statacorp. lp, 2013, college station, texas, united states). categorical data were presented using stratified frequency tables (n and %). trends or associations were assessed using the standard pearson’s chi-square (χ2) test. if expected cell count in the cross tabulation contained fewer than five observations (sparse numbers), then the fisher’s exact test was utilised instead. a p-value of less than 0.05 was considered statistically significant. results distribution of species by clinical specimens for the period 2011–2015, a total of 64 502 eskape clinical isolates were recovered from clinical specimens (figure 1). the distribution of the isolated eskape organisms varied during this period. the clinical specimens included respiratory specimens, blood, urine, catheters, which included central venous and hemodialysis catheters, as well as samples from other sites, including wounds. respiratory samples included sputum, bronchi alveolar lavage and endotracheal aspirates. overall, s. aureus (24 495, 38.0%) was the most frequently isolated pathogen followed by k. pneumoniae (14 282, 22.2%), p. aeruginosa (11 231, 17.4%), a. baumannii (8010, 12.4%), enterobacter spp. (4267, 6.6%), e. faecium (2217, 3.4%) (table 3 and figure 1). similarly, s. aureus (3787, 35.8%) and k. pneumoniae (3059, 28.9%) were the most frequently isolated species from blood cultures. gram-negative organisms namely k. pneumoniae (2469, 29.2%), a. baumannii (2036, 24.1%) and p. aeruginosa (2015, 23.8%) were the most common isolates from respiratory samples, followed by gram-positive species, of which s. aureus was the most frequently isolated (1353, 16%). k. pneumoniae, a member of the enterobacteriaceae group, was the predominant species isolated from urine (4997, 55.5%). catheters included a combination of central venous, arterial and hemodialysis catheters. a. baumannii (31.5%), s. aureus (23.4%) and k. pneumoniae (23.1%) were the most common isolates cultured. other sample types included wound swabs, pus swabs and aspirates. the most frequently isolated species from other sample types was s. aureus (18 397, 53.4%). figure 1: trends in the number of eskape pathogens isolated from clinical samples collected in public sector healthcare facilities in kwazulu-natal from 2011 to 2015. table 3: distribution of eskape pathogens isolated from clinical samples collected in public sector healthcare facilities in kwazulu-natal from 2011–2015. antibiotic resistance during the five-year period, high levels of antibiotic resistance were detected among the eskape pathogens. overall, the most common resistance pattern in gram-negative eskape pathogens was resistance to amoxicillin-clavulanate and ceftriaxone. the majority of the a. baumannii isolates were resistant to multiple drugs, with over 70% of isolates resistant to all tested antimicrobial agents except colistin, amikacin and gentamicin. isolates of p. aeruginosa remained fairly susceptible to agents like amikacin, piperacillin-tazobactam, meropenem imipenem and colistin. overall, colistin resistance was less than 10% over the five-year period for the gram-negative eskape pathogens. between 2011 and 2015, 7788 (54.5%) k. pneumoniae isolates were resistant to amoxicillin-clavulanate. more than 50% of all isolates were resistant to third-generation cephalosporins, including ceftriaxone (8326, 58.3%) and ceftazadime (7412, 51.9%), indicative of esbl production; 5317 (37.2%) isolates were resistant to ciprofloxacin. of the 14 282 k. pneumoniae isolates, 922 (6.4%) were resistant to meropenem (table 4). increasing antimicrobial resistance trends were observed in k. pneumoniae (figure 2). resistance to ceftriaxone increased from 54.6% in 2011 to 65.5% in 2015 (p = 0.018). for the period 2011–2014 average resistance to ciprofloxacin was 35%, which increased to 42% in 2015 (p = 0.015). resistance to amoxicillin-clavulanate increased from 50.6% in 2011 and to 62.7% in 2015 (p = 0.009). between 2011 and 2014, resistance to meropenem was 5% or less. this was followed by a significant increase to 16% in 2015 (p < 0.001). colistin resistance in k. pneumoniae was approximately 2%, but this was not confirmed by reference or molecular methods. both european committee on antimicrobial susceptibility testing and the clinical laboratory standards institute recommend broth microdilution for antimicrobial susceptibility testing of colistin, but broth microdilution is rarely used in routine microbiology laboratories.12 broth microdilution is not routinely performed at these kwazulu-natal public sector microbiology laboratories and as a result they may have failed to detect heteroresistance. figure 2: resistance profile of k. pneumoniae (2011–2015). table 4: resistance profiles of gram-negative eskape pathogens (2011–2015). high mdr rates were observed for a. baumannii with over 70% of isolates resistant to meropenem, imipenem, ciprofloxacin, piperacillin-tazobactam and ceftazidime (table 4). accumulative average resistance to piperacillin-tazobactam, ceftazadime, ciprofloxacin, meropenem, and amikacin observed in p. aeruginosa was 1706 (15.2%), 1601 (14.2%), 1501 (13.4%), 1090 (9.7%) and 716 (6.3%), (p < 0.001) (table 4). although 840 (7.5%) samples of p. aeruginosa, were resistant to colistin, this was not confirmed by reference or molecular methods. over the five-year period, an increase in the total number of p. aeruginosa was noted (p = 0.002) (figure 1). during 2013–2015, decreasing trends in resistance patterns were observed in p. aeruginosa to ceftazadime (from 17% to 13%, p = 0.004), piperacillin-tazobactam (from 27% to 21%, p < 0.001), meropenem (from 18% to 10%, p < 0.001), ciprofloxacin (from 22% to 18%, p = 0.002), and amikacin (from 10% to 8%, p < 0.001) (figure 3). figure 3: resistance profile of p. aeruginosa (2011–2015). during the five-year period, the average accumulative resistance rate to available carbapenems observed in enterobacter spp. was 5%. resistance to ciprofloxacin was greater in e. cloacae (16%) compared to e. aerogenes (8%) (data not shown). as illustrated in figure 4, rates of mrsa varied between 18% and 31% per year. overall, a decreasing trend in the proportion of mrsa was observed between 2011 and 2014, 28% to 18% (p < 0.001). decreasing resistance rates or trends across several antibiotic classes, namely gentamicin, clindamycin, ciprofloxacin, erythromycin and rifampicin, were also noted. no resistance to vancomycin, teicoplanin and linezolid was observed. of the 24 495 isolates of s. aureus isolated over the study period, 22 596 (92%) were resistant to penicillin, 12 168 (49%) were resistant to co-trimoxazole, 6034 (24%) were resistant to cloxacillin, 5352 (21.8%) were resistant to ciprofloxacin, 4916 (20%) were resistant to erythromycin, 4702 (19%) were resistant to rifampicin, and 917 (3.7%) were resistant to clindamycin. all isolates of e. faecium (2217) were susceptible to vancomycin and linezolid. figure 4: resistance profile of s. aureus in the eskape group (2011–2015). esbl-producing k. pneumoniae, increased from 54.9 in 2011 to 65.5% in 2015 (p = 0.018). mrsa decreased from 31.4% in 2012 to 19.1% in 2015 (p < 0.001). mdr a. baumanni decreased from 85% in 2012 to 70% in 2015 (figure 5). overall, during the five-year period 6351 (79.2%) of all a. baumannii were mdr, 6034 (24.6%) of all s. aureus were mrsa and 8511 (59.5%) of k. pneumoniae were esbl-producers. figure 5: distribution of highly resistant eskape pathogens (2011–2015). discussion antimicrobial resistance is now a global pandemic that threatens the health, economic and social well-being of every individual.13 antimicrobial resistance among bacterial pathogens is increasing globally at a rapid rate, including south africa.14 data from the centers for disease control and prevention indicate that the six eskape bacteria are responsible for two-thirds of all healthcare-associated infections.15 the eskape group of pathogens feature among the critical and high priority pathogens on the who global ppl. in this study, we report on five-year surveillance of eskape pathogens and describe the incidence and resistance profiles of the eskape pathogens in kwazulu-natal, south africa. effective infection prevention and control bundles, comprising hand hygiene, contact precautions and a change in antibiotic prescription culture, advocated locally and internationally may have contributed to the possible decline in mrsa rate;16 however, the number of esbl-producing k. pneumoniae increased. infection prevention and control interventions should impact on decreasing both pathogens, but a notable increase in the number of esbl-producing k. pneumoniae was observed. the use of third-generation cephalosporins, as part of the integrated management of childhood illness guidelines17 and sexually transmitted infections management guidelines in south africa,18 may have contributed to the increase in esbl-producing k. pneumoniae. third-generation cephalosporins are administered empirically at the discretion of a community centre healthcare professional on suspicion of meningitis, sepsis or as part of syndromic management for sexually transmitted infections. in our study, the percentage resistance of 24% to methicillin observed in s. aureus was lower than the 46% that was described by perovic et al., in 2015.16 mrsa proportions exceeded 20% in all who regions, and in some countries it exceeded 80%.1 the epidemiology of mrsa in africa was highlighted in a systematic review of 263 articles. prevalence of mrsa in africa varied from 12% in tunisia to 82% in egypt.16 our findings confirm that vancomycin resistance among s. aureus is yet to be encountered in kwazulu-natal. decreasing trends in antimicrobial resistance to erythromycin, clindamycin, rifampicin and ciprofloxacin were observed in s. aureus during the study period. nhls public sector susceptibility data (january–december 2009) presented by crowther et al. showed varying rates of mrsa.19 the data obtained were from eight laboratories located in academic hospitals in south africa, excluding kwazulu-natal. rates of cloxacillin resistance in s. aureus varied between 24% and 84%. additionally, clindamycin resistance reported in that study varied between 15% and 70% in comparison to less than 3% observed in our analysis. k. pneumoniae was the most common gram-negative organism isolated from the eskape group and during the study period k. pneumoniae accounted for almost one-third of all eskape organisms isolated from blood. in a 2015 global surveillance report by healthcare-associated infections surveillance network and united states national healthcare safety network 8.7% and 9.9% of all hospital acquired infections were attributable to k. pneumoniae20. a cause for concern in this study is the steady increase in antimicrobial resistance to amoxicillin-clavulanate and third-generation cephalosporins reflecting inhibitor-resistance and esbl production in k. pneumoniae. perovic et al. reported similar proportions of esbl-producing k. pneumoniae (68.3%) from bloodstream infections over a four-year period.21 in another south african study, dramowski et al. reported that 75.7% and 78.3% of all k. pneumoniae isolated from paediatric community-acquired and hospital-acquired bloodstream infections were esbl producers.22 according to a who report in 2014, resistance rates of k. pneumoniae to third-generation cephalosporins varied between 8% and 77% in africa.1 extended spectrum β-lactamase (esbl) production in escherichia coli and k. pneumoniae have been reported in thailand (rate between 3.0% and 23.1%), china (65% and 31.9%), and india (67.0% and 55.0%).23 who reported that resistance to third-generation cephalosporins observed in k. pneumoniae was greater than 30% worldwide and greater than 60% in some countries.1 the rise in ciprofloxacin resistant k. pneumoniae in 2015 could be due to increased use of ciprofloxacin for treatment of esbl-producing k. pneumoniae infections. more significantly, ciprofloxacin is used as a first-line therapy for uncomplicated urinary tract infections in the south african standard treatment guidelines.10 during the period 2010–2012, national sentinel site surveillance for antimicrobial resistance in k. pneumoniae from south africa was performed and 46.5% of all isolates were resistant to ciprofloxacin.21 due to the large number of quinolone-resistant and esbl-producing k. pneumoniae, treatment for severe k. pneumoniae infections rely on carbapenems.24 perovic et al. noted less than 6% resistance rate to carbapenems in k. pneumoniae during 2010–2012.21 with reference to our study, although resistance to carbapenems was generally low between 2011 and 2014, a significant increase of 16% was noted in 2015, indicative of an emerging problem. massive surveillance gaps in documenting carbapenem-resistant k. pneumoniae from africa exist and this is a great challenge. in 2014, the who reported that 0% – 4% of k. pneumoniae from africa were resistant to carbapenems; however, these data were obtained from only four countries1 on the african continent. increasing carbapenem resistance in k. pneumoniae during 2015 could very well represent the global trends in increasing resistance of enterobacteriacae to carbapenems. the who has highlighted alarming rates of carbapenem resistance in k. pneumoniae, exceeding 50% in some countries of the eastern mediterranean and europe.1 limited therapeutic options to treat infections due to carbapenem-resistant k. pneumoniae are among the greatest challenges facing clinicians. therapeutic options include colistin, but a recent italian study showed that among 178 carbapenemase-producing k. pneumoniae isolates from different hospitals, 43% were also resistant to colistin.25 tigecycline is not available for use in the public healthcare sector in kwazulu-natal; therefore, susceptibility and resistance data for this drug were not analysed in this study. the only remaining intervention to prevent dissemination of mdr organisms, such as carbapenemase-producing k. pneumoniae, is infection control.24 the significant decrease in resistance observed in p. aeruginosa to many of the anti-pseudomonal antimicrobial agents may be attributed to the fact that the majority of p. aeruginosa was isolated from non-sterile sites. it is well known that p. aeruginosa is a long-term coloniser and can persist for prolonged periods of time; hence, many clinicians opt not to administer systemic antimicrobials in a patient who is otherwise clinically well. the emergence and spread of antibiotic-resistant gram-negative pathogens can be attributed to antibiotic selective pressure;26 therefore, the likelihood of developing mdr p. aeruginosa is reduced when no antimicrobials are administered for colonising bacteria. greater than 70% rates of resistance were observed in a. baumanni for most antimicrobials tested, except gentamicin, amikacin and colistin, where resistance was 68%, 18% and 4%, respectively. susceptibility to amikacin and colistin remained stable for a. baumannii during the five-year period. the propensity for development of resistance to a drug is lower when it is used in combination than with monotherapy. this could explain the stable susceptibility to amikacin as it is only used as part of a dual therapy and never alone in south africa. colistin susceptibily could be due to its lack of availability or restricted use in the public health sector in south africa. a motivation and prior approval is mandatory prior to drug administration; hence, most clinicians are unable to access the drug. additionally, in the majority of patients with a. baumannii, treatment is not offered as most acinetobacter species are considered long-term colonisers of low virulence potential and are intrinsically resistant to all but a few antimicrobial agents.27,28 therapy for these colonisers would only lead to the emergence of other mdr organisms. even in the face of directed therapy, acinetobacter has the ability to develop resistance,27 and the isolation of colistin-resistant subpopulations of acinetobacter is of great concern.28 drawing a comparison to the global burden of mdr a. baumannii, a recent systemic review and meta-analysis by bialvaei et al. (2017) highlighted that the pooled prevalence of mdr a. baumannii was 72% annually, with frequencies of between 22% and 100%,29 similar to the findings in the present study. limitations there are a number of limitations of this study. the total number of samples received by all the laboratories during the study period was not available. there were deficiencies in data collection for 2014 due to an upgrade of microbiological diagnostic systems, resulting in unrecoverable surveillance data. additionally, although this surveillance reflects nine hospitals across two districts in kwazulu-natal, the private health sector was not included, and the results reflect only the public health sector in kwazulu-natal, south africa. conclusion this is the first study describing resistance trends in who global priority pathogens within kwazulu-natal. the high prevalence rates of antimicrobial resistance observed in eskape pathogens signal the need to improve antimicrobial stewardship and infection prevention and control programmes in the region. acknowledgements we acknowledge all contributions from the following individuals: dr p. bhola, dr n. dlamini, dr s. haffejee, dr k. lutchminarian, dr p. mahabeer, dr y. mahabeer, dr a. niehaus, dr p. ramjathan, dr l. sookan, dr k. swe swe han. national health laboratory services – microbiology staff in kwazulu-natal. competing interests s.y.e. reports personal fees and non-financial support as a member of the global respiratory infection partnership sponsored by an unconditional educational grant from reckitt & benckiser (pty) ltd, uk, outside the submitted work. all other authors declare that they have no competing interests. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. no funding was received for the design of the study, collection, analysis, and interpretation of data and in writing the manuscript. authors’ contributions k.p.m and s.y.e. conceived the study. k.p.m, s.y.e and y.r designed the study. y.r, s.y.e and k.p.m. performed the literature search. y.r. and m.p. prepared the data. the statistical analysis was performed by b.s. b.s., y.r. and m.p. performed the data analysis. all authors interpreted data, wrote the manuscript, and approved the final version. references world health organization. antimicrobial resistance: global report on surveillance. april 2014. available from: http://www.who.int/drugresistance/documents/surveillancereport/en magiorakos ap, srinivasan a, carey rb, et al. multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. clin microbiol infect. 2012;18(3):268–281. https://doi.org/10.1111/j.1469-0691.2011.03570.x world health organization. global priority list of antibiotic-resistant bacteria to guide research, discover, and development of new antibiotics; 2017. 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2006;43(supplement 2):s62–s69. https://doi.org/10.1086/504481 murray ck, hospenthal dr. acinetobacter infection in the icu. crit care clin. 2008;24(2):237–248, vii. https://doi.org/10.1016/j.ccc.2007.12.005 ramsamy y, muckart dj, han ks. microbiological surveillance and antimicrobial stewardship minimise the need for ultrabroad-spectrum combination therapy for treatment of nosocomial infections in a trauma intensive care unit: an audit of an evidence-based empiric antimicrobial policy. s afr med j. 2013;103(6):371–376. https://doi.org/10.7196/samj.6459 bialvaei az, kouhsari e, salehi-abargouei a, et al. epidemiology of multidrug-resistant acinetobacter baumannii strains in iran: a systematic review and meta-analysis. j chemother. 2017;29(6):327–337. https://doi.org/10.1080/1120009x.2017.1338377 abstract introduction development and implementation process success parameters discussion future goals and challenges conclusion acknowledgements references about the author(s) takudzwa j. mtisi international pharmacology specialty laboratory, school of pharmacy, university of zimbabwe college of health sciences, harare, zimbabawe charles maponga international pharmacology specialty laboratory, school of pharmacy, university of zimbabwe college of health sciences, harare, zimbabawe center for integrated global biomedical sciences, university at buffalo, buffalo, new york, united states tsitsi g. monera-penduka international pharmacology specialty laboratory, school of pharmacy, university of zimbabwe college of health sciences, harare, zimbabawe tinashe mudzviti international pharmacology specialty laboratory, school of pharmacy, university of zimbabwe college of health sciences, harare, zimbabawe dexter chagwena international pharmacology specialty laboratory, school of pharmacy, university of zimbabwe college of health sciences, harare, zimbabawe faithful makita-chingombe international pharmacology specialty laboratory, school of pharmacy, university of zimbabwe college of health sciences, harare, zimbabawe robin difranchesco center for integrated global biomedical sciences, university at buffalo, buffalo, new york, united states gene d. morse center for integrated global biomedical sciences, university at buffalo, buffalo, new york, united states citation mtisi tj, maponga c, monera-penduka tg, et al. strategic establishment of an international pharmacology specialty laboratory in a resource-limited setting. afr j lab med. 2018;7(1), a659. https://doi.org/10.4102/ajlm.v7i1.659 lessons from the field strategic establishment of an international pharmacology specialty laboratory in a resource-limited setting takudzwa j. mtisi, charles maponga, tsitsi g. monera-penduka, tinashe mudzviti, dexter chagwena, faithful makita-chingombe, robin difranchesco, gene d. morse received: 07 july 2017; accepted: 17 oct. 2017; published: 12 feb. 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: a growing number of drug development studies that include pharmacokinetic evaluations are conducted in regions lacking a specialised pharmacology laboratory. this necessitated the development of an international pharmacology specialty laboratory (ipsl) in zimbabwe. objectives: the aim of this article is to describe the development of an ipsl in zimbabwe. methods: the ipsl was developed collaboratively by the university of zimbabwe and the university at buffalo center for integrated global biomedical sciences. key stages included infrastructure development, establishment of quality management systems and collaborative mentorship in clinical pharmacology study design and chromatographic assay development and validation. results: two high performance liquid chromatography instruments were donated by an instrument manufacturer and a contract research organisation. laboratory space was acquired through association with the zimbabwe national drug regulatory authority. operational policies, standard operating procedures and a document control system were established. scientists and technicians were trained in aspects relevant to ipsl operations. a high-performance liquid chromatography method for nevirapine was developed with the guidance of the clinical pharmacology quality assurance programme and approved by the assay method review programme. the university of zimbabwe ipsl is engaged with the united states national institute of allergy and infectious diseases division of aids research networks and is poised to begin drug assays and pharmacokinetic analyses. conclusions: an ipsl has been successfully established in a resource-limited setting through the efforts of an external partnership providing technical guidance and motivated internal faculty and staff. strategic partnerships were beneficial in navigating challenges leading to laboratory development and training new investigators. the ipsl is now engaged in clinical pharmacology research. introduction ideally, research institutes should have access to a full complement of analytical laboratories within their jurisdiction. the laboratories should be well equipped and staffed with competent analytical scientists. since many clinical studies involve drug interventions and pharmacokinetic evaluations, accessible assays should include analytical methods for common approved and investigational therapeutic interventions. this enables the laboratory to serve a wide range of clinical trial protocols. there has been a growing number of clinical studies involving prevention and therapeutic drug interventions conducted in zimbabwe. most of the studies are conducted by the university of zimbabwe (uz) college of health sciences’ clinical trials unit (uzchs-ctu) through research networks sponsored by the united states’ national institutes of health (nih). currently the uzchs-ctu collaborates with five hiv research networks, including the aids clinical trials group (actg), hiv prevention trials network, international maternal pediatric adolescent aids clinical trials, microbial trials network and hiv vaccines trials network.1 within the actg structure, specialties in immunology, virology, pharmacology, tuberculosis and genomics form the actg laboratory center network structure. prior to establishment of the uz international pharmacology specialty laboratory (ipsl), the uz-university of california, san francisco ctu had capacities in the other actg hiv laboratory specialties except pharmacology. pharmacology specialty laboratories (psls) lead drug development research projects that include bioavailability, drug-drug interactions and determining pharmacokinetic parameters for drugs. the uzchs-ctu implements phase ii and iii protocols involving numerous licensed antiretroviral and other new investigational drugs. in addition, the use of traditional medicines in zimbabwe and their potential drug interactions2 as well as other culture-specific issues, for example dietary habits,3 create a need for building pharmacology research capacities. while the laboratory is currently developing expertise in the assaying of drug concentrations in biological matrices, current research protocol requires that these samples be transported to other countries for analysis. shipping research biological specimens across national borders impacts clinical research processes negatively. the shipment approval procedures to assure ethical analysis of the samples are laborious, time-consuming, costly and have resulted in abortion of clinical studies in the past.4 national institutional review boards are also more rigorous when reviewing such protocols, as there is need to assure conformance on the part of laws of different countries. the ethical review process, which has to be as explicit as possible on the plans for the samples, is consequently more complicated and time-consuming. in addition, there is often a hesitation from local participants to enrol in studies that involve shipment of samples to other countries. the time taken with sample shipment procedures and transportation delays research timelines. this is usually accompanied by high shipping charges, which increase the cost of research. in addition, the integrity of samples may be compromised, if shipping procedures are not followed correctly as they are moved long distances across borders. lastly, ensuring that all analytical procedures conform to what has been established during the informed consent process becomes more complicated across jurisdictions. the major challenge is that it is often more difficult for originating institutional review boards to conduct inspections of the recipient laboratories against their intended expectations.5 in 2007, uz and the university at buffalo (ub), state university of new york, united states, entered into a memorandum of understanding; its scope included teaching, research, exchange of faculty and students, as well as staff development. this formed on the backbone of the initial ub-uz initiative that has been active since 2002. in 2005, a formal agreement to address clinical pharmacology research and training needs was adopted. consequently, a progressive series of awards and milestones enabled ub to guide uz during the establishment of the ipsl in zimbabwe. this manuscript describes the collaborative development process, including challenges and strategies. development and implementation process competitive research supporting awards as a foundation for the international pharmacology specialty laboratory multiple funding sources were utilised to develop and implement the uz-ipsl. in 1998, the actg sponsored a fellowship in clinical pharmacology at ub that was completed by the current uz-ipsl principal investigator. in 2009, the actg principal investigator and the co-principal investigator visited uz to discuss and assess the potential for infrastructure and subsequent capacity building to develop, implement and operate an ipsl. shortly after this visit, the university of california, berkley aids international research and training program provided a sub-award to ub. this award supported two master of philosophy candidates from the uz school of pharmacy and department of pharmacology and created a seed that would lead to an aids international research and training program (aitrp) focused on clinical pharmacology research between uz and ub (2009) and begin the uz-ipsl investigator training. the nih division of aids (daids) increased the scope of the hiv research network’s pharmacology laboratory network to include the uzchs ipsl, enabling the daids clinical pharmacology quality assurance program (cpqa) to assist in this process.6 finally, in december 2011, the uz-ipsl successfully competed for a developmental psl that would interact with each of the hiv research networks. infrastructure and equipment initially, laboratory space was allocated within the university of zimbabwe. laboratory equipment donations were solicited from manufacturers through ub. donations from waters associates, inc., research triangle international and merck laboratories included two high-performance liquid chromatography (hplc) systems, basic laboratory equipment, laboratory supplies and computer software for pharmacokinetic analysis. additionally, basic equipment such as refrigerators, freezers, centrifuges, ph meters and evaporators were purchased through the nih uz-ipsl developmental specialty laboratory grant award. the scientific and industrial research and development centre provided the required certification and calibration of ancillary equipment. with the addition of two hplc systems, an agreement was made in 2012 with the ministry of health and child care through a partnership with the national drug regulatory authority and the medicines control authority of zimbabwe (mcaz), to house the laboratory at the mcaz. to ensure completion of assays without interruption, integrity of stored specimens and protection of sensitive laboratory equipment, a backup generator and uninterrupted power supply systems were installed. human resources development the roles within the uz-ipsl were distributed among a team of laboratory scientists, technologists and aitrp fellows. table 1 provides an overview of the team’s established positions, the diverse professional backgrounds, and training received to support the development of this laboratory research team. training focused on the development of chromatographic methods, laboratory quality management systems (qms) and clinical pharmacology research and most was done at the translational pharmacology research core (tprc) at ub, new york, united states. additional technical training was done in-house through the cpqa programme.6,7 in the process, individual training records were documented to provide evidence to support the appropriateness of appointed responsibilities in the various areas of research shown in table 1. table 1: uz-ipsl positions, professional qualifications and training received. one laboratory scientist, the qms implementation manager, was tasked with developing the standard operating procedures (sops) and overseeing the implementation of the qms. the role involved conducting onsite monitoring and mentoring in clinical laboratory sciences. a second laboratory scientist, the laboratory supervisor, was tasked with implementing the qms system and assay methods, as well as technician training. both were trained extensively on chromatographic assays at the national institute of allergy and infectious diseases laboratory data management system. one laboratory technician was tasked with ensuring efficient resource utilisation, facilities improvement and maintenance. two technicians worked under the direct supervision of the laboratory supervisor. when the uz-ipsl received initial designation as a developmental psl in 2011, it was utilised as a core laboratory to support concurrently running capacity building programmes. in this role, the uz-ipsl provided bioanalytical training support to aitrp fellow research projects and daids hiv research network protocols, through assay planning, mentoring and seminars in clinical pharmacokinetics and pharmacodynamics for research protocol design and data analysis. high-performance liquid chromatography method development and validation hplc methods for the determination of antiretroviral drugs in human plasma were developed under the technical guidance of cpqa. initial assay development and validation focused on the determination of nevirapine in plasma. nevirapine was considered highly relevant to the uz-ipsl development agenda, because it was widely used in the setting. subsequently, an hplc method for efavirenz was developed and validated. policies and standard operating procedures using the iso 15189:2009 standard for medical laboratories, sops were developed for all laboratory processes.8 more than 30 sops were developed. the sops were used to validate all the equipment used to develop and validate hplc methods, to ensure equipment performance in the zimbabwean setting. iso 15189 was specifically chosen as it covers requirements that would need to be addressed to achieve the laboratory’s vision of enlarging the scope of the laboratory to include performing pharmacology tests for clinical decision-making. the aim was to target accreditation with southern african development community accreditation service (sadcas), a regional, non-profit, multi-economy accreditation body whose mission is to provide credible, cost-effective accreditation services. under the twinning partnership agreement between sadcas and the south african nation accreditation system, it is now mandatory that all medical laboratories performing clinical assays in zimbabwe seeking to obtain iso 15189 accreditation go through the sadcas.9 oversight for quality assurance of both standard and novel assays was provided by the cpqa proficiency testing programme.6 most clinical studies conducted in zimbabwe are nih funded so meeting cpqa requirements was strategic. the process was guided by the cpqa and included individual staff development, mentored laboratory training, continuous technical guidance and communications and site assessments to monitor progress. an initial site development assessment was done in 2011 from which the cpqa made specific infrastructure and qms development recommendations. to meet cpqa requirements, the uz-ipsl performed assays on proficiency testing samples, generated assay validation report reviews, and participated in several daids cross-network clinical trial and clinical pharmacology laboratory group networking activities. progress was tracked electronically and through continued interaction between cpqa and uz-ipsl personnel. the most recent assessment, an implementation assessment, was done in 2016 and was the basis for the recommendation for iso accreditation. success parameters we set out to develop a laboratory that would be acceptable in zimbabwe with the initial primary focus to support the actg scientific agenda by participating in actg protocols. as this involved bringing together professionals from different scientific disciplines, different levels of training were required and tailored to each individual. essentially, investigators obtained training in the order of basic laboratory, pharmacology, then chromatography training. once these fundamentals were achieved, the laboratory then embarked on and successfully completed the method development and validation required to analyse actg protocol samples following cpqa approval. evidence of productivity associated with the uz-ipsl can be highlighted by the increasing number of funded grants, manuscripts and collaborations throughout the development process. the ub-uz aitrp was recently funded for another five years; it is now known as the ub-uz hiv research training program and has since been awarded additional supplemental grants in oncology and behavioral sciences. one investigator also received a center for aids research award through the university of rochester to support doctoral research. other fellows have also received scholarship and fellowship awards from various sponsors including the letten foundation, world health organization special programme for research and training in tropical diseases career development fellowships and the uz promoting excellence in research and faculty enhanced career training programme. in addition, from 2008 to date, 20 manuscripts have been written and numerous abstracts and conference presentations made by uz-ipsl investigators and fellows at various international conferences including those organised by the african society for laboratory medicine, the international aids society and the society of quality assurance. discussion overall, implementation of the strategy was successful. however, it was not without challenges. first, the cpqa requirements and sadcas accreditation requirements had to be met. in contrast to laboratories conducting routine clinical chemistry tests, cpqa developmental and implementation assessments are necessary for psls seeking to offer pharmacology assay services to actg-funded studies. once the laboratory was operational, methods required cpqa approval and then proficiency testing participation to maintain assurance of continuous competency. in order to expand the scope to offer accredited clinical laboratory services, local regulations require all clinical medical laboratories to receive relevant accreditation (in this case iso 15189) through sadcas. aiming to achieve both simultaneously demanded more time, effort and resources. as a strategy, development of sops focused on meeting the rigorous iso 15189 requirements. however, iso 15189 standards are particular for medical laboratories and do not address some of the intricacies of pharmacology. therefore, the detail of the cpqa-recommended policies and procedures were then incorporated. cpqa approval for the first uz-ipsl hplc method, determination of nevirapine in human plasma, was officially attained in december 2016. this gave the uz-ipsl an opportunity to contribute to pharmacology assay services for international protocols, and secure additional funding to proceed with addressing the outstanding iso 15189 requirements. another ongoing challenge has been the recruitment and retention of laboratory scientists with adequate training to perform drug assays. two important criteria need to be fulfilled by suitable scientists to work in a psl: the ability to work with biological matrices and an understanding of clinical pharmacology assays. there are currently only two programmes in the country training medical laboratory scientists and neither programme has a strong clinical pharmacology component. as such, extra training in clinical pharmacology is required. while the uz-psl development process has successfully trained two medical laboratory scientists, retaining them before the laboratory secures substantial assay contracts has been a challenge. as a retention strategy, only one medical laboratory scientist was contracted on a full-time basis with a competitive salary. the other was contracted on a part-time basis and prioritised for postgraduate training support from the aitrp. the aitrp also provided mentorship for laboratory-based studies, enabling the second laboratory scientist to work and study in the same environment while applying the skills to a specific aitrp project. this arrangement was a very effective retention strategy and has served to enhance the outcomes for both the funded programmes. securing service contracts for the hplc instrumentation was also a challenge. there are currently limited technical support and supplies available in zimbabwe due to the prevailing socioeconomic situation. this lack of expedient supply chains for instrument engineers threatened to slow down laboratory development. the uz-ipsl had to rely on regional vendors, further constraining the limited laboratory funding. close liaison with an established regional ipsl and the cpqa programme has assisted the uz-ipsl in locating cost-effective regional vendors. in addition, a new hplc system was purchased through actg support to sustain current analytical capacity. future goals and challenges the collaboration of the uz-ipsl with the uzchs-ctu will ensure that investigators within the network, who themselves may have other research or clinical studies, will continue to utilise and hence sustain the uz-ipsl’s research agenda. in addition, a newly funded hiv research and training program will continue to utilise the uz-ipsl as a foundational resource to support research projects for its doctoral students and postdoctoral fellows. these fellows have assumed scientific leadership responsibility in multiple research areas including bioequivalence, nanomedicine, infectious diseases, cancer, translational pharmacology, pharmacovigilance, nutrition pharmacology, phytopharmacology and pharmacogenomics within the uz-ipsl. this will lead to growth in the types of drug assays that are available in the uz-ipsl to support daids network clinical trials, as well as the growing pharmaceutical, regulatory and research industry. in addition, the planned inclusion of clinical pharmacology research from multiple networks will positively impact the laboratory’s sustainability. however, it is important to note that the aforementioned will require greater capacity to accommodate an increasing number of protocols. this results in an increased number of sample analyses for more drug analytes and requires significant assay development and validation efforts. technologies may also change with corresponding technology advancements. thus, continual funding, capacity building and training are crucial to keep abreast with relevant platforms. given the advancements in drug delivery systems such as nanoparticles and cell and tissue targeted drugs, assays in other matrices such as hair, foetal tissue and cerebrospinal fluid are desirable. such assays require analytical systems with higher specificity and sensitivity, that is, mass spectrometry. the cost associated with acquisition, installation and training for such instrumentation is another challenge for the uz-ipsl. to address this challenge, several strategies have been considered and are progressing. these include negotiating for allocation through new or current funding sources while pursuing collaborations within the university and other local research institutions where access to mass spectrometers might be available. possibilities of sponsorship through philanthropic agencies are also being investigated. conclusion the uz-ipsl provides a reproducible strategic approach for the development and implementation of an accredited psl in a resource-limited setting. the strength of the uz-ipsl lies in its ability to overcome several challenges through strategic partnerships, and in its diverse inter-professional human resources. these strengths should serve the uz-ipsl well in the planned development of assays for more drugs investigated in studies. lessons learned acknowledgements the authors acknowledge the administrative, operational and technical laboratory support provided by kelly tooley, farzia kaufman, primrose jaravani, charlene taylor, jill hochreiter, jill lapham and alfred tarumbwa. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. sources of support uz-ipsl: grant number: 5um1ai106701-04 aitrp and hrtp: the project described was supported by grant numbers d43tw010313, d43tw007991 and d43tw007991 01a2s1 from the fogarty international center. the content is solely the responsibility of the authors and does not necessarily represent the official views of the fogarty international center or the national institutes of health. clinical pharmacology quality assurance program: this project has been funded in whole or in part with federal funds from the national institute of allergy and infectious diseases, national institutes of health, department of health and human services, under contract numbers hhsn272201500006c and hhsn272200800019c. center for aids research: this publication was made possible through core services and support from the university of rochester center for aids research, an nih-funded programme (p30 ai078498). the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institutes of health. authors’ contributions c.m. and g.d.m. provided leadership for the project. r.d., t.j.m. and f.m.-c. provided technical expertise. t.g.m.-p., t.m. and d.c. provided clinical expertise for the project. all authors contributed to writing the manuscript and approved the final version for publication. references uzchs-ctu. university of zimbabwe college of health sciences clinical trials unit; 2017 [our research]. available from: http://www.uz-ucsf.co.zw/research/ mudzviti t, maponga cc, khoza s, et al. the impact of herbal drug use on adverse drug reaction profiles of patients on antiretroviral therapy in zimbabwe. aids res treat. 2012;2012:4. https://doi.org/10.1155/2012/434171 bepe n, madanhi n, mudzviti t, et al. the impact of herbal remedies on adverse effects and quality of life in hiv-infected individuals on antiretroviral therapy. j infect dev ctries. 2011;5(1):48–53. monera-penduka tg, maponga cc, morse gd, et al. capacity for ethical and regulatory review of herbal trials in developing countries: a case study of moringa oleifera research in hiv-infected patients. j pharm policy pract. 2017;10(1):9. https://doi.org/10.1186/s40545-017-0099-5 national research council panel on collecting storing, accessing, protecting biological specimens, biodata in social surveys. the national academies collection: reports funded by national institutes of health. in: hauser rm, weinstein m, pool r, et al, editors. conducting biosocial surveys: collecting, storing, accessing, and protecting biospecimens and biodata. washington, dc: national academies press (us) national academy of sciences; 2010:1–2 difrancesco r, rosenkranz sl, taylor cr, et al. clinical pharmacology quality assurance program: models for longitudinal analysis of antiretroviral proficiency testing for international laboratories. ther drug monit. 2013;35(5):631–642. https://doi.org/10.1097/ftd.0b013e31828f5088 difrancesco r, tooley k, rosenkranz sl, et al. clinical pharmacology quality assurance for hiv and related infectious diseases research. clin pharmacol ther. 2013;93(6):479–482. https://doi.org/10.1038/clpt.2013.62 thelen mh, vanstapel fj, kroupis c, et al. flexible scope for iso 15189 accreditation: a guidance prepared by the european federation of clinical chemistry and laboratory medicine (eflm) working group accreditation and iso/cen standards (wg-a/iso). clin chem lab med. 2015;53(8):1173–1180. https://doi.org/10.1515/cclm-2015-0257 sadcas-sanas. joint communiqué on sadcas accreditation services. tpaj 01-04, guidelines for sadcas/sanas joint assessments under the twinning partnership arrangement (2014). approved and implemented 28/10/2014. abstract introduction methods results discussion acknowledgements references about the author(s) kamela l. mahlakwane division of medical virology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa division of medical virology, tygerberg hospital, national health laboratory service, cape town, south africa wolfgang preiser division of medical virology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa division of medical virology, tygerberg hospital, national health laboratory service, cape town, south africa nokwazi nkosi division of medical virology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa division of medical virology, tygerberg hospital, national health laboratory service, cape town, south africa nasheen naidoo division of clinical pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africadivision of clinical pathology, tygerberg hospital, national health laboratory service, cape town, south africa gert van zyl division of medical virology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa division of medical virology, tygerberg hospital, national health laboratory service, cape town, south africa citation mahlakwane kl, preiser w, nkosi n, naidoo n, van zyl g. delays in hiv-1 infant polymerase chain reaction testing may leave children without confirmed diagnoses in the western cape province, south africa. afr j lab med. 2022;11(1), a1485. https://doi.org/10.4102/ajlm.v11i1.1485 original research delays in hiv-1 infant polymerase chain reaction testing may leave children without confirmed diagnoses in the western cape province, south africa kamela l. mahlakwane, wolfgang preiser, nokwazi nkosi, nasheen naidoo, gert van zyl received: 04 dec. 2020; accepted: 24 mar. 2022; published: 23 june 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: early diagnosis and confirmation of hiv infection in newborns is crucial for expedited initiation of antiretroviral therapy. confirmatory testing must be done for all children with a reactive hiv pcr result. there is no comprehensive data on confirmatory testing and hiv pcr test request rejections at national health laboratory service laboratories in south africa. objective: this study assessed the metrics of routine infant hiv pcr testing at the tygerberg hospital virology laboratory, cape town, western cape, south africa, including the proportion of rejected test requests, turn-around time (tat), and rate of confirmatory testing. methods: we retrospectively reviewed laboratory-based data on all hiv pcr tests performed on children ≤ 24 months old (n = 43 346) and data on rejected hiv pcr requests (n = 1479) at the tygerberg virology laboratory over two years (2017–2019). data from sample collection to release of results were analysed to assess the tat and follow-up patterns. results: the proportion of rejected hiv pcr requests was 3.3%; 83.9% of these were rejected for various pre-analytical reasons. most of the test results (89.2%) met the required 96-h tat. of the reactive initial test results, 53.5% had a follow-up sample tested, of which 93.1% were positive. of the initial indeterminate results, 74.7% were negative on follow-up testing. conclusion: a high proportion of hiv pcr requests were rejected for pre-analytical reasons. the high number of initial reactive tests without evidence of follow-up suggests that a shorter tat is required to allow confirmatory testing before children are discharged. keywords: infant hiv pcr; confirmatory testing; early infant diagnosis; eid; laboratory diagnosis; antiretroviral therapy; turn-around time; follow-up testing. introduction the early diagnosis of hiv infection in infants is very important.1,2 infants who are initiated on antiretroviral therapy (art) within seven days of life are four times more likely to achieve early viral suppression than those who commence art later.3,4 the south african national guidelines on the prevention of mother-to-child transmission of hiv recommend that hiv polymerase chain reaction (pcr) tests be conducted within 3–6 days of birth and that art should be immediately initiated in all children with detectable hiv nucleic acid while awaiting the follow-up hiv pcr results.5 early initiation of art is associated with improved virological, immunological, and clinical outcomes.6,7,8,9,10 early diagnosis of hiv in infants is done by testing for viral nucleic acid (dna and/or rna) in blood samples, usually by pcr.11 current guidelines, including those issued by the western cape department of health and south african national department of health, recommend the testing of all hiv-exposed infants by pcr within seven days of birth (i.e., birth pcr), at approximately 10 weeks of age for those who tested negative at birth, at 18 weeks for high-risk infants who received extended prophylactic art for 12 weeks, at six months, and after cessation of breastfeeding.5,12,13 the post-breastfeeding hiv test may either be by pcr if the child is younger than 18 months of age or by serology if the child is older.5,11,12 the world health organization recommends that hiv antibody testing be done after at least three months post-breastfeeding to allow for hiv antibody development and to prevent missing hiv infection in the last few days of breastfeeding.13 a negative antibody test in an infant older than 18 months is confirmation that the infant is not infected.13 although the world health organization recommends hiv pcr testing for infants ≤ 18 months old, the age cut-off for hiv pcr testing at national health laboratory service (nhls) laboratories is 24 months rather than 18 months. this is based on studies that showed that maternal hiv antibody clearance took longer than 18 months in some perinatally exposed infants, with seroreversion rates of 89.3%, 94.2%, and 100.0% at ages 12, 18, and 24 months, respectively.14,15 nevertheless, this policy has not been formalised in the laboratory diagnostic booklet, the western cape provincial testing guidelines, or the national department of health hiv testing guidelines. given the consequences of hiv infection (such as lifelong art), a follow-up sample must be collected as soon as possible from all children with a reactive infant pcr result to confirm the diagnosis or detect false-positive initial results.11,16 however, a negative test result following an initial reactive result does not necessarily constitute false positivity, particularly in infants who are on prophylaxis or art. clinical actions taken based on false-positive results may have dire biomedical (exposure to drug toxicity and side effects), psychosocial (possible stigma and negative impact on life outlook and future relationships), financial (art costs), and medico-legal implications.16 once initiated on art, it may become impossible to distinguish a virally suppressed child from one who was never infected.4 in south africa, the nhls provides laboratory testing for the public health sector, providing healthcare for approximately 85% of the population who do not have medical insurance.17 the nhls aims to issue results for at least 80% of infant pcr tests within a 96-h turn-around time (tat).18 this tat is defined by the nhls19 as the time interval between sample reception and the release of results by a virologist (i.e., laboratory tat). this tat does not consider the periods between sample collection and reception, or between the review of results and the receipt of results by the patient. for this study, we refer to the period between sample collection and authorisation of results as ‘clinical tat’. the nhls performs all hiv early infant diagnosis testing using the roche® cobas® ampliprep/cobas® taqman® system (roche® molecular systems, inc., branchburg, new jersey, united states) in a network of centralised laboratories.17 results of this assay could be positive, negative, or indeterminate. indeterminate qualitative hiv pcr results on the roche® cobas® ampliprep/cobas® taqman® system were first described in the tygerberg hospital virology laboratory in 2012.20,21 during the period covered by this study, indeterminate results were defined as a cycle threshold of ≥ 33, or a relative fluorescence intensity below five.22 these diagnostic criteria have since been revised,23,24,25,26 mostly based on collated data, which showed cycle threshold value cut-offs to be the best predictor of reproducible positive results. the cycle threshold value of 33 was shown to most precisely differentiate clear positive from irreproducible cases.23 in the tygerberg virology laboratory, hiv pcr testing is done only once on each sample unless the results are invalid, in which case a single retest is done on the remnant sample. samples that produce invalid results for the second time are rejected with this reason attached: ‘failed after repeated attempts’. all other results (i.e., negative, positive, or indeterminate) are released as such, with no repeat testing of the remnant sample. the world health organization reports that most women and their newborn infants are likely to be discharged within one to two days following uncomplicated vaginal delivery, and within two to four days following uncomplicated caesarean delivery.27 thus, infant birth hiv pcr results should, ideally, be available before the child gets discharged to allow immediate optimal management (prophylaxis, art, or confirmatory testing). hiv pcr tat strategies should thus seek to address this challenge. in this study, a laboratory-based retrospective review was conducted to determine the proportion of rejected infant hiv pcr requests, assess laboratory conformance with tat requirements, and assess whether there were confirmatory tests done following reactive infant hiv pcr results performed on the roche® cobas® ampliprep/cobas® taqman® system. methods ethical considerations this study was approved by the health research ethics committee (hrec) of stellenbosch university (reference number: s19/03/053). the hrec does not require patient consent when residual clinical samples are used for research if the research findings will not impact the patient or change their management in any way. access to the extracted laboratory data was limited to the researchers. study design and setting this is a retrospective review of data generated at the tygerberg hospital virology laboratory in cape town, western cape, south africa, between july 2017 and june 2019. the data was downloaded from the laboratory information system (lis) of the tygerberg laboratory (trakcare lab, intersystems corporation, cambridge, massachusetts, united states). the tygerberg virology laboratory renders diagnostic virological pathology services to tygerberg hospital and other health facilities from its drainage areas. all samples received in the laboratory go through the routine process of sample reception, registration and analysis, as well as review of results. all hiv-1 qualitative pcr results from ethylenediaminetetraacetic acid-anticoagulated whole blood and dried blood spot samples from children aged ≤ 24 months that were tested at the tygerberg virology laboratory between 01 july 2017 and 30 june 2019 were included in this study. samples from patients older than 24 months (n = 534) or with unknown age (n = 287), quality control samples (n = 29), and samples with coded names or surnames (n = 15) were excluded. a total of 43 346 tests were included in this study. data collection rejection of test requests a raw data set of rejected hiv pcr requests for children aged ≤ 24 months was extracted from the lis (n = 1479) and assessed to determine the pattern of hiv pcr request rejections by the laboratory. infant pcr test requests may be rejected for various reasons, as included in the lis and nhls standard operating procedure for sample rejection. these reasons may be pre-analytical (e.g. ‘unsuitable age’ for children aged > 24 months) or analytical (e.g. ‘laboratory error’). samples from other peripheral laboratories are referred both digitally and physically. digital referrals that are not accompanied by the sample are rejected as ‘lost in transit’ or ‘specimen not received’ after consultation with the referring laboratory or the requesting clinician. all ethylenediaminetetraacetic acid-anticoagulated samples are discarded after seven days from the date of collection. samples that are not tested within this period are rejected as ‘sample too old’. the term ‘insufficient specimen’ refers to remnant samples with initial invalid results and insufficient sample volume for repeat testing. the classification of these rejections as pre-analytical is, therefore, arbitrary. turn-around time laboratory tat was calculated based on the nhls definition,19 while clinical tat was analysed as earlier defined. the time points were extracted from the lis as recorded during each step of the testing process. the results were then stratified using a 96-h tat cut-off. follow-up testing all results were grouped using the unique laboratory code, that is, the medical record number, which is assigned to each patient and remains the same for all subsequent test requests. the time-to-follow-up was calculated as the number of days between the first and second sample collection dates as recorded on the lis. in line with the guidelines, all positive and indeterminate results were expected to have a follow-up sample sent for confirmatory testing as soon as possible, while all the negative samples were expected to be followed up as per schedule. negative follow-up test results were further checked to determine if a third follow-up sample was tested. data analysis data were analysed using microsoft excel 2010 version 14 (microsoft corporation, redmond, washington, united states). rejected test requests were stratified by reason for rejection and further summarised into pre-analytical and analytical reasons for rejection. results rejected test sets out of the 1479 rejected samples, 1241 (83.9%) were rejected for pre-analytical reasons, and 238 (16.1%) for analytical reasons (figure 1). ‘duplicate requests’ (21.3%), ‘insufficient specimen’ (21.1%), and ‘specimen not received’ (16.1%) were the most common pre-analytical reasons for sample rejections (table 1). other reasons that each constituted less than 1% of the reasons for test rejection were grouped as ‘other various reasons’. figure 1: pre-analytical and analytical reasons for hiv polymerase chain reaction test rejection at tygerberg hospital, cape town, south africa, between july 2017 and june 2019. table 1: reasons for rejection of hiv polymerase chain reaction test requests at tygerberg hospital, cape town, south africa, between july 2017 and june 2019. demographics of the 43 346 samples that were tested for infant hiv pcr, 27 978 (64.5%) were the patients’ first or initial hiv pcr samples, while 18 393 (42.4%) samples were collected at birth. among the 9585 patients who were tested for the first time after seven days of life (i.e. beyond birth pcr), 5715 (59.6%) were tested between day eight and week 12 of life (median = 10 weeks; interquartile range [iqr] = 8–11 weeks). the proportions of male (49.9%) and female patients (49.8%) were similar, with the sex of 149 (0.3%) patients being unknown. turn-around time in total 38 653 (89.2%) test results were within the 96-h laboratory tat cut-off (median = 44 h; iqr = 31–64), while the remaining 4693 (10.8%) were not (median = 116 h; iqr = 106–137) (figure 2). figure 2: numbers of hiv pcr requests, rejected samples, and follow-up tests conducted at tygerberg hospital, cape town, south africa, between july 2017 and june 2019. however, for the clinical tat, only 33 245 (76.7%) samples were signed out within 96 h (median = 57 h; iqr = 47–73), while 10 101 (23.3%) test results were released after 96 h (median = 119 h; iqr = 105–140). the median tat from sample collection to sample registration was 17 h (iqr = 8–24). follow-up hiv pcr test results of the 520 (1.9%) reactive initial results, approximately half were followed up by testing of a subsequent sample. similarly, 9409 (50.3%) of the initial negative results had subsequent samples tested, with only 1264 (13.4%) of these tested at 10 weeks of age (6–12 weeks of life) as per the national hiv testing guidelines. nine (0.7%) of these 10-week samples had positive or indeterminate results. it should be noted that some of the follow-up results may have not been linked to the initial samples and may have been missed in this analysis. of the 520 initial reactive tests, 251 (48.3%) were tested at birth, with 62.9% (n = 158) of those subjected to confirmatory testing. the remaining 269 (51.7%) were tested after seven days of life, and 44.6% (n = 120) of those had confirmatory testing conducted. the reactive results were further analysed to assess the agreement between initial and confirmatory results. one hundred and eighty-nine (93.1%) patients with initial positive results were also positive following confirmatory testing, while five (2.5%) were indeterminate, and nine (4.4%) had discordant results (negative on follow-up testing) (table 2). further scrutiny of these nine results revealed that all of them had a third follow-up hiv pcr test done, with four testing positive and five testing negative. of the 75 patients with initial indeterminate results who had a confirmatory sample tested, 56 (74.7%) had negative results. of these, 30 (53.6%) had a third hiv pcr follow-up test done, 24 (42.9%) had no third follow-up hiv pcr or hiv viral load test conducted, while two patients had hiv viral load follow-up tests conducted, rather than an hiv pcr test (table 3). among the 30 patients who had a third hiv pcr test conducted, 29 (96.7%) were negative. one of two patients who had hiv viral load follow-up tests done had detectable hiv viral load (862 328 copies per millilitre). the presence of detectable hiv nucleic acid in this quantitative molecular test is confirmatory of hiv infection. table 2: comparison of results of the initial reactive infant polymerase chain reaction tests and confirmatory tests conducted at tygerberg hospital, cape town, south africa, between july 2017 and june 2019. table 3: follow-up tests for samples that initially tested indeterminate at tygerberg hospital, cape town, south africa, between july 2017 and june 2019. urgency of follow-up samples of the 278 follow-up tests conducted, 147 (52.9%) were performed more than seven days after the initial test (mean: 82 days; range: 8–606 days). among the 131 (47.1%) follow-up samples tested within the first seven days of life, only 52 (39.7%) were tested within three days of the initial test. discussion in this study, we determined that 3.3% of infant hiv pcr requests were rejected, and that the majority of the test results met the tat requirements. infant hiv pcr requests were rejected for various reasons, mainly due to duplicated requests, insufficient sample volumes, and loss of specimen in transit. these rejections may occur when more than one clinician requests the same test for the same patient, when a sample is received in the laboratory with a volume lower than that required for the test, or when test requests are not accompanied by the patient’s sample. the proportion of rejected samples is concerningly high and negatively impacts the early diagnosis and management of hiv-positive infants. comparative data from other settings is limited, thus making it difficult to determine the rejection threshold across laboratories. an overall appraisal of the tat showed that the tygerberg virology laboratory meets the standards set by the nhls national strategic plan. however, the average laboratory tat was longer when only positive and indeterminate results, which were the focus of our study, were considered; 14.9% of these results were released after the required 96 h. the clinical tat was also longer, with only 76.7% of results released within 96 h. the clinical tat may thus require some revisions to enable the expedition of early infant diagnosis and art initiation. point-of-care hiv pcr testing has been shown to accomplish same-day diagnosis for infants,28 with a significantly higher number of infants accessing confirmatory testing,29 thereby assisting with rapid art initiation. this study also found that only 39.7% of the reactive confirmatory tests were done within three days after the initial test. on average, most newborns would have been discharged from the hospital within three days of birth. if hiv birth pcr test results are received within this period, newborns with positive results are likely to be initiated on art before being discharged, thus reducing the number of patients lost to follow-up.28 although new mothers are required to visit the clinic within 6 days after birth, the 6-day maternal postnatal clinic visit was reported to be at 58.0% in the western cape between 2017 and 2019.30 the delay in follow-up testing (over seven days after initial testing) in 59.3% of patients in this study may be as a result of this low uptake or because our 96-h tat only accounts for the processes within the laboratory. while 1.9% of initial test results were reactive, only 53.5% of these had the prescribed confirmatory test done. it is unknown whether the other reactive results (46.5%) had confirmatory tests done, and this may mean that many hiv-positive patients may not have been initiated on art, or that a substantial proportion would have been started on art without confirmatory test results, effectively exposing some uninfected children to lifelong art. the 53.5% follow-up test rate is similar to the 53.0% confirmatory test rate reported in a study in tshwane, south africa.31 this proportion may be different from what is seen in clinical practice31 as some of the patients, particularly those aged > 18 months, may have had either hiv viral load or serological follow-up tests. these patients would have been missed in our study, as we focused specifically on hiv pcr results. the discordant negative results in 4.4% of patients with an initial reactive result indicate that these initial reactive results may have been false reactive or may be due to a rapid decline in hiv nucleic acid concentration following exposure to highly active art in infants receiving antiretroviral drugs (as prevention of mother-to-child transmission or combination art), making it difficult to make a definitive diagnosis of hiv infection. most children with indeterminate results tested negative on subsequent follow-up tests. again, this could either indicate that a large proportion of these indeterminate results were initially false reactive, or may indicate rapid hiv nucleic acid decay in children who are receiving art for prophylaxis, which may be two or three drugs in high-risk cases.4,32 it should, however, be noted that the indeterminate cut-offs used in this study were based on the reproducibility of a particular assay, with a particular chemistry and software, and within a different prevention of mother-to-child transmission context. these were later improved. recently, hiv diagnostic criteria have been revised to address this diagnostic dilemma.23,24,25,26 only 13.4% of patients with negative birth pcr results received a follow-up test within 10 weeks as per schedule; about half (50.3%) were later followed up after 10 weeks. this is consistent with other similar local studies on 10-week hiv pcr follow-up tests.33 at ten weeks, 0.7% of the infants in this study tested positive or indeterminate on hiv pcr, which is lower than the national hiv prevalence of 0.9% and the national strategic plan target of 1.3% at 10 weeks.30 it is, however, higher than the reported western cape rate of 0.5%.30 it should be noted that while these patients could have been missed at birth following intrauterine infection, they also could have been infected either perinatally or postnatally. just over half of the infants who required confirmatory pcr testing received such tests. it remains unclear whether the patients who were not followed up were hiv positive or not. a small proportion of babies received the 10-week follow-up test. the patients who did not receive this test may only be seen in the hospital later in life when they are sick. clinician education on hiv testing guidelines may help to reduce this number, and strengthening systems to reduce the time between first and confirmatory hiv pcr tests could also be beneficial. most of the indeterminate results were negative on the second and third follow-up tests. it was not known if patients were receiving prevention of mother-to-child transmission regimens or combination art during the periods of observation, as that may have resulted in undetectable hiv nucleic acid. the new early infant diagnosis criteria, as well as a separate and independent hiv pcr testing platform, may help resolve the challenges presented by indeterminate hiv pcr results. limitations our interpretation of the tat data did not account for the current workflow practice, in which hiv pcr testing is processed only during weekdays (monday to friday). the fact that no tests are done on weekends may result in tat variability. this study is thus not indicative of laboratories that operate a shift system (i.e., a 24-h service). occasionally, hiv pcr results also fail to transmit to the lis, further delaying the tat. another limitation of the study was the classification of indeterminate results using outdated criteria that have since been revised. some of these results may now be re-classified as positive. follow-up tests were reconciled using a unique laboratory code (medical record number) for each patient. however, we noted that some patients may have erroneously had more than one medical record number. this could have occurred in instances where samples from babies were labelled with the mother’s name (i.e., baby of xyz) and then later labelled with the baby’s name, resulting in non-reconciled patient profiles. this might have contributed to the under-reporting of follow-up tests, as these would have been missed. we, however, compared data provided on actual request forms with those captured by the lis and found them to match. also, some patients could have moved to other health facilities outside of our testing site. it must be noted that this study was limited to the tygerberg hospital virology laboratory, and thus may not be representative of the whole western cape region (i.e. groote schuur hospital and green point complex). the magnitude of this data concern is unknown due to our inability to assess the number of follow-up results that were not linked with the initial patient results. conclusion a high proportion of infant hiv pcr requests were rejected for various reasons. hiv pcr testing tat at the tygerberg laboratory is at par with the national requirements. however, this 96-h laboratory tat may need to be revised as it may negatively impact the number of children who receive confirmatory testing after a positive result. a larger study is recommended for a clearer appreciation of the hiv pcr testing challenges. acknowledgements the authors of this manuscript would like to extend their gratitude to the national health laboratory service for allowing them to use its data. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions k.l.m. conceptualised the research project, carried out the investigation and analytical calculations, and wrote the original draft. w.p. supervised the project and edited and reviewed the final version of the manuscript. n. nkosi provided critical feedback and helped shape the research, analyse the data, and edit the manuscript. n. naidoo helped with the analytical calculations and the drafting and editing of the manuscript. g.v.z. supervised the project and helped with the editing and review of the final version of the manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the data that support the findings of this study are available on request from the corresponding author, k.l.m. the data are not publicly available due to their containing information that could compromise the privacy of patients whose samples were used in this research project. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the national health laboratory service or university of stellenbosch. references mofenson lm, cohn j, sacks e. challenges in the early infant hiv diagnosis and treatment cascade. j acquir immune defic syndr. 2020;(84 suppl 1):s1–s4. https://doi.org/10.1097/qai.0000000000002366 webb k, chitiyo v, mahachi n, et al. brief report: improving early infant diagnosis observations: estimates of timely hiv testing and mortality among hiv-exposed infants. j acquir immune defic syndr. 2020;83(3):235–239. https://doi.org/10.1097/qai.0000000000002263 dominguez-rodriguez s, tagarro a, palma p, et al. reduced time to suppression among neonates with hiv initiating antiretroviral therapy within 7 days after birth. j acquir immune defic syndr. 2019;82(5):483–490. https://doi.org/10.1097/qai.0000000000002188 veldsman ka, maritz j, isaacs s, et al. rapid decline of hiv-1 dna and rna in infants starting very early antiretroviral therapy may pose a diagnostic challenge. aids. 2018;32(5):629–634. https://doi.org/10.1097/qad.0000000000001739 department of health. guideline for the prevention of mother to child transmission of communicable infections. health, editor. pretoria: doh; 2019. lilian rr, kalk e, technau kg, sherman gg. birth diagnosis of hiv infection in infants to reduce infant mortality and monitor for elimination of mother-to-child transmission. pediatr infect dis 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zhang x, et al. delayed seroreversion of specifical antibody against hiv in hiv-exposed infants: a retrospective cohort study. hiv med. 2020;21(11):718–721. https://doi.org/10.1111/hiv.13026 dunning l, francke ja, mallampati d, et al. the value of confirmatory testing in early infant hiv diagnosis programmes in south africa: a cost-effectiveness analysis. plos med. 2017;14(11):e1002446. sherman gg, lilian rr, bhardwaj s, candy s, barron p. laboratory information system data demonstrate successful implementation of the prevention of mother-to-child transmission programme in south africa. s afr med j. 2014;104(3 suppl 1):235–238. https://doi.org/10.7196/samj.7598 national health laboratory service. national laboratory health service strategicplan for the fiscal years 2015/16–2019/20. johannesburg: nhls; 2016. national health laboratory service. strategic plan for the fiscal years 2015/16–2019/20. johannesburg: nhls; 2016. maritz j, preiser w, van zyl gu. establishing diagnostic cut-off criteria for the cobas ampliprep/cobas taqman hiv-1 qualitative test through validation against the amplicor dna test v1.5 for infant diagnosis using dried blood spots. j clin virol. 2012;53(2):106–109. https://doi.org/10.1016/j.jcv.2011.12.002 maritz j, van zyl gu, preiser w. irreproducible positive results on the cobas ampliprep/cobas taqman hiv-1 qual test are different qualitatively from confirmed positive results. j med virol. 2014;86(1):82–87. https://doi.org/10.1002/jmv.23811 haeri mazanderani a, moyo f, sherman gg. missed diagnostic opportunities within south africa’s early infant diagnosis program, 2010–2015. plos one. 2017;12(5):e0177173. https://doi.org/10.1371/journal.pone.0177173 haeri mazanderani a, moyo f, kufa t, maritz j, sherman gg. differentiating clearly positive from indeterminate results: a review of irreproducible hiv-1 pcr positive samples from south africa’s early infant diagnosis program, 2010–2015. diagn microbiol infect dis. 2018;91(3):248–255. https://doi.org/10.1016/j.diagmicrobio.2018.02.019 haeri mazanderani a, sherman gg. evolving complexities of infant hiv diagnosis within prevention of mother-to-child transmission programs. f1000res. 2019;8:1637. https://doi.org/10.12688/f1000research.19637.1 luo r, boeras d, broyles ln, et al. use of an indeterminate range in hiv early infant diagnosis: a systematic review and meta-analysis. j acquir immune defic syndr. 2019;82(3):281–286. https://doi.org/10.1097/qai.0000000000002104 vojnov l, penazzato m, sherman g, et al. implementing an indeterminate range for more accurate early infant diagnosis. j acquir immune defic syndr. 2019;82(3):e44–e46. https://doi.org/10.1097/qai.0000000000002081 world health organization. who recommendation on postnatal discharge following uncomplicated vaginal birth. the who reproductive health library. geneva: world health organization; 2018. boeke ce, joseph j, wang m, et al. point-of-care testing can achieve same-day diagnosis for infants and rapid art initiation: results from government programmes across six african countries. j int aids soc. 2021;24(3):e25677. odhiambo co, githuka g, bowen n, et al. point-of-care early infant diagnosis improves adherence to the testing algorithm in kenya. j int assoc provid aids care. 2020;19:2325958220906030. https://doi.org/10.1177/2325958220906030 massyn n, pillay y, padarath a. district health barometer 2017/18. durban: health systems trust; 2019. moyo f, haeri mazanderani a, feucht ud, et al. monitoring diagnosis, retention in care and viral load suppression in children testing hiv polymerase chain reaction-positive in two districts in south africa. s afr med j. 2019;109(9):686–692. https://doi.org/10.7196/samj.2019.v109i9.13765 veldsman ka, janse van rensburg a, isaacs s, et al. hiv-1 dna decay is faster in children who initiate art shortly after birth than later. j int aids soc. 2019;22(8):e25368. https://doi.org/10.1002/jia2.25368 kalk e, kroon m, boulle a, et al. neonatal and infant diagnostic hiv-pcr uptake and associations during three sequential policy periods in cape town, south africa: a longitudinal analysis. j int aids soc. 2018;21(11):e25212. https://doi.org/10.1002/jia2.25212 abstract introduction methods results discussion acknowledgements references about the author(s) jurette s. grove department of chemical pathology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa national health laboratory service, johannesburg, south africa siyabonga khoza department of chemical pathology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa national health laboratory service, johannesburg, south africa dineo v. mabuza department of chemical pathology, faculty of pathology, pathcare laboratories, johannesburg, south africa shaida b. khan department of chemical pathology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa national health laboratory service, johannesburg, south africa citation grove js, khoza s, mabuza dv, khan sb. an audit of the iron status of patients at chris hani baragwanath academic hospital, in johannesburg, south africa. afr j lab med. 2024;13(1), a2509. https://doi.org/10.4102/ajlm.v13i1.2509 note: additional supporting information may be found in the online version of this article as online supplementary document 1. original research an audit of the iron status of patients at chris hani baragwanath academic hospital, in johannesburg, south africa jurette s. grove, siyabonga khoza, dineo v. mabuza, shaida b. khan received: 24 may 2024; accepted: 12 aug. 2024; published: 18 oct. 2024 copyright: © 2024. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: iron deficiency is a common disorder, especially in developing countries. accurately assessing iron status remains challenging, particularly for patients with chronic diseases such as hiv and chronic kidney disease, prevalent in south africa. objective: this study aimed to determine how ferritin cut-offs affect iron status classification in adult patients treated at a tertiary hospital in south africa. additionally, it assessed the frequency of these conditions and the impact of age and gender on iron status. methods: this retrospective study analysed iron profiles in adult patients from 01 october 2020 to 31 march 2021. iron status was categorised into five groups: iron deficiency anaemia (ida), anaemia of chronic disease, ida with anaemia of chronic disease, iron deficiency without anaemia, and iron replete based on haemoglobin, transferrin saturation, and ferritin levels. the impact of using two different ferritin cut-off values (15 µg/l and 30 µg/l) was investigated. results: the study included 3221 complete iron profiles. there was a predominance of female patients (2.2:1 ratio). anaemia of chronic disease was the most prevalent iron disorder (39%), regardless of ferritin cut-off. using a higher ferritin cut-off of 30 µg/l significantly increased the detection rates of both ida and iron deficiency without anaemia (p < 0.001). conclusion: this study suggests that a higher ferritin threshold (30 µg/l) might improve diagnosis of iron disorders in settings with high inflammatory diseases. further studies are needed to refine thresholds. local guidelines should be adjusted to consider higher ferritin cut-offs, and longitudinal studies are recommended to evaluate long-term outcomes. what this study adds: this study confirms the use of higher ferritin cut-offs for enhanced detection of iron deficiency states. the findings also emphasise the ongoing need for establishing simple, standardised, and accurate methods for iron status classification. keywords: iron; iron status; ferritin; ferritin diagnostic threshold; iron deficiency; iron deficiency anaemia; anaemia of chronic disease. introduction iron deficiency is the most common nutritional disorder worldwide, and more prevalent in developing countries.1,2,3 pregnant women, young children, and women of reproductive age are at higher risk.4,5 iron deficiency refers to decreased or depleted iron stores, which precedes the development of ineffective haematopoiesis resulting in iron deficiency anaemia (ida).4 iron deficiency can also manifest as part of complex pathological processes seen in chronic diseases, where it is called functional iron deficiency. without treatment, iron deficiency can cause significant morbidity and mortality, making an accurate and timely diagnosis important.6 accurate determination of iron status is often difficult and complex. the gold standard for diagnosis of iron deficiency is a bone marrow biopsy demonstrating the absence of stainable iron.2,7 however, since this approach is costly and invasive, biochemical tests are more frequently utilised for diagnosis. a working group from the world health organization has recommended five biomarkers for the work-up of iron deficiency, namely haemoglobin, mean cell volume (mcv), zinc protoporphyrin, soluble transferrin receptor, and serum ferritin.8 although haemoglobin concentration, mcv and ferritin are readily available in south africa, zinc protoporphyrin and soluble transferrin receptor are not commonly measured. specifically, in our setting at chris hani baragwanath academic hospital, the markers available to assess iron status are haemoglobin concentration, mcv, serum iron, transferrin with transferrin saturation (tsat), and serum ferritin. the diagnosis of the various pathological processes rests on the integrated interpretation of these tests.2,7,9,10 interpretation of these biochemical parameters with a subsequent diagnosis is not always simple, as the effect of inflammation is a confounding factor that needs to be considered. chronic inflammation is associated with production of cytokines, which blunts the erythropoietin response. this results in a disruption of normal iron homeostasis and causes increased uptake of iron, with subsequent retention thereof in the reticuloendothelial system. the outcome is iron-restricted erythropoiesis, followed by the development of anaemia of chronic disease (acd) with an elevated ferritin, low haemoglobin, and decreased tsat.2,3,11 furthermore, inflammation induces pro-inflammatory cytokines such as interleukin 6, which increases hepcidin production. hepcidin acts as a regulator of iron homeostasis by mediating the internalisation of iron and degradation of ferroportin, the iron exporter. this results in decreased iron release from enterocytes and macrophages and, subsequently, to lower plasma iron levels than required to meet the body’s needs. in addition, serum ferritin is significantly elevated during inflammation.12,13,14,15 in the south african setting, a national health and demographic survey found that as much as a third of south african women of reproductive age are anaemic based on point-of-care haemoglobin measurement, although iron status was not assessed.6 a meta-analysis found that the prevalence of anaemia in women of reproductive age ranged from 22.0% to 44.0%, compared to iron deficiency which ranged from 7.7% to 19.0%, and ida, specifically, which ranged from 9.7% to 10.5%, utilising a ferritin cut-off of < 15 µg/l.16 furthermore, it was found that ida in pregnant patients resulted in an increased risk for premature birth, thus emphasising the importance of a correct and timely diagnosis.17 in the general south african population, 12.6% of participants had anaemia, compared to the high prevalence of 39.8% for iron deficiency, in this case specifically using the higher ferritin cut-off of < 30 µg/l for improved sensitivity.4 the distribution of anaemia between the sexes indicated a much higher prevalence of 18.3% in women compared to 2.9% in men.4 despite these assessments, the iron status of hospital-attending patients in the south african context has not been studied. a 2016 study in cape town, south africa, found acd to be the predominant cause of anaemia in patients with hiv and tuberculosis, seen in over 95% of their study population, also utilising the higher ferritin cut-off of < 30 µg/l.18 importantly, this study was more extensive in the usage of other biochemistry of iron status such as soluble transferrin receptor, as well as infective markers such as c-reactive protein, which likely improved the sensitivity. the high prevalence of chronic inflammatory conditions like hiv, as well as chronic kidney disease, together with the presence of inflammation in hospitalised patients, increases the prevalence of acd.19,20 this raises the question on the iron status of an inpatient population, and on the diagnostic impact of coexisting acd and iron deficiency. evidently, the cut-off used for ferritin for the diagnosis of iron deficiency is not harmonised, with most studies suggesting using < 30 µg/l, and others, < 15 µg/l.2,10 clinicians at our site frequently rely on a ferritin cut-off value of < 15 µg/l for the diagnosis of ida, based on the world health organization guidelines,8 and this is applied universally, regardless of sex. the question remains whether this international guideline is applicable to the south african population and, more specifically, to a hospital-attending cohort. to our knowledge, there are no local studies that have investigated the effect on the classification of iron status using these different cut-offs to date. given the aforementioned context, there is a need to investigate the diagnostic utility of these available biochemical parameters for iron status evaluation in the south african milieu. thus, the aims of this retrospective study were to analyse and categorise the iron profiles in a south african adult population treated at a tertiary hospital setting, and to evaluate how various ferritin thresholds influence the identification of iron deficiency. methods ethical considerations ethical approval to conduct the study was obtained from the human research ethics committee of the university of the witwatersrand (reference no.: m210512). patients’ informed consent was not required due to the retrospective nature of the study. patient data were de-identified to maintain patient privacy and anonymity. data were securely stored on a password-protected excel spreadsheet (office 2016, microsoft, redmond, washington, united states), accessible only to the investigators of the study. the research was conducted adhering to the ethical principles of the declaration of helsinki (2013). study design and setting this study employed a retrospective cross-sectional design analysing laboratory data from patients attending chris hani baragwanath academic hospital in soweto, south africa. pathology services are provided primarily by the national health laboratory services, which serves 80% of the population in the public sector health system. data collection data for the period from 01 october 2020 to 31 march 2021 were obtained from the national health laboratory services corporate data warehouse after mining from the laboratory information system (trackcare lab, intersystems corporation, cambridge, massachusetts, united states). the study included results from adult patients (> 18 years) attending chris hani baragwanath academic hospital who had the following parameters: sex, haemoglobin, mcv, iron, transferrin, tsat, and ferritin. patient results with missing data for age, sex, haemoglobin, ferritin, or tsat were excluded. laboratory analysis the iron profile analyses (serum iron, transferrin and ferritin) were conducted on the roche cobas® 8000 automated chemistry analyser (roche diagnostics, mannheim, germany). iron was measured using the ferrozine colourimetric method without deproteinisation. iron reacts with ferrozine to form a coloured complex which is measured photometrically. transferrin and ferritin were measured using immunoturbidimetric methods utilising anti-human transferrin and anti-human ferritin antibodies, respectively. transferrin saturation was calculated according to the formula: % saturation = (iron / [transferrin x 25.6]) × 100%. the haematological parameters (haemoglobin, mcv) were measured using the sysmex xn10/xn20 series automated haematology analyser (sysmex corporation, kobe, japan). haemoglobin was measured spectrophotometrically using a non-cyanide sodium lauryl sulphate. mean cell volume was calculated as follows: haematocrit % × 10 ÷ red blood cell count. the national health laboratory services laboratory at chris hani baragwanath academic hospital maintains accreditation status with the relevant governing bodies and participates in external quality assurance schemes for ongoing independent quality assessment. data processing data included in the study were classified into ida, acd, iron deficiency with acd (iron deficiency + acd), iron deficiency without anaemia (idwa), and iron replete using pre-defined criteria and two different cut-offs for ferritin (table 1). haemoglobin and ferritin levels were used as hard criteria for classification, while tsat was used primarily as an adjunct. results that did not fall into these categories using the criteria were classified as indeterminate. table 1: criteria for iron status classification, johannesburg, south africa, 01 october 2020 to 31 march 2021. statistical analysis statistical analyses were performed using spss software 29.0.0.0 (ibm corp. armonk, new york, united states). all the data were non-parametric when the normality was evaluated using the shapiro-wilk test. categorical data were presented using frequencies and percentages. non-parametric data were presented as median (interquartile range). the chi-squared test was used to compare categorical variables, while the mann-whitney u test was used to compare the medians of two groups of continuous data. for continuous data with three or more groups, the kruskal-wallis test with bonferroni correction was employed to assess the differences in medians among the groups. a p-value of < 0.05 was considered statistically significant. results demographics following the removal of duplicate episodes (n = 2249), results showing an age below 18 years (n = 248), results with missing demographic data (n = 48), and incomplete profiles (n = 3891), 3221 episodes from a total of 9657 episodes were included in the analysis (table 2). there was a predominance of women with a ratio of 2.2:1. women were also younger overall, with a median age of 42 years, compared to 50 years for men. this difference was statistically significant in the ida (p < 0.001) and iron deficiency + acd (p < 0.001) groups, but not for the acd (p = 0.430), idwa (p = 0.260), iron replete (p = 0.212) and indeterminate (p = 0.345) groups (figure 1). the majority of requests originated from the inpatient population (62%), primarily from the medical (82%) and surgical departments (17%) for male patients, and from the medical (68%) and maternity (23%) departments for female patients (online supplementary figure 1). figure 1: age differences between male and female patients in different iron status groups defined using a ferritin 30 µg/l, johannesburg, south africa, 01 october 2020 to 31 march 2021. table 2: iron status of patients in johannesburg, south africa, 01 october 2020 to 31 march 2021. frequencies of iron disorders based on ferritin cut-off irrespective of the ferritin cut-off used for iron status classification, acd was the most prevalent iron disorder (overall = 39%) in this study. in contrast, the frequency of patients with ida, iron deficiency + acd, idwa and iron replete varied depending on the ferritin cut-off employed for the classification (table 2). the use of the higher ferritin cut-off of 30 µg/l results in a statistically significant increase in the frequency of ida (p < 0.001) and idwa (p < 0.001). utilising this higher ferritin cut-off in combination with a tsat of <16% improved the differentiation of idwa from the indeterminate group, reducing the frequency of the latter from 11% to 5% (online supplementary figure 2). the lower ferritin cut-off also precludes the identification of idwa in the male group. the iron deficient states, ida, idwa, and iron deficiency + acd were more frequent in female patients. less than 15% of the study group could not be classified into any iron status category as per the criteria in table 1 and were therefore classified as indeterminate. distributions of parameters across different iron status groups the pattern of distribution of parameters in the ida group remained consistent across the two different ferritin cut-offs employed (table 3). lower levels of iron, tsat and mcv were found in the ida and iron deficiency + acd groups compared to other groups. the highest ferritin levels were present in the acd (median = 554 µg/l [258–1156]) and indeterminate (median = 170 µg/l [58–361]) groups. the iron replete and indeterminate groups were statistically similar in the distribution of parameters, except for transferrin and ferritin (table 3). table 3: distribution of parameters according to diagnosis using two different ferritin cut-offs, johannesburg, south africa, 01 october 2020 to 31 march 2021. discussion employing a multi-marker approach for the assessment of iron status, we classified 3221 iron profiles using haemoglobin, serum ferritin with two different cut-offs, and tsat. consistent with another study conducted in 2020 in patients attending a hospital in italy,21 acd (39%) was the most common iron disorder in our setting, irrespective of the ferritin threshold (15 µg/l vs 30 µg/l). using different thresholds did not affect the frequency, as the diagnostic criterion employed was a serum ferritin concentration > 100 µg/l, and the observed median ferritin levels (554 µg/l, interquartile range: 258–1156) were substantially elevated. the predominance of acd in our setting can be attributed to the high burden of pro-inflammatory communicable and non-communicable disorders such as hiv, chronic kidney disease, type 2 diabetes, and metabolic syndrome. anaemia of chronic disease results from a cytokine-mediated disruption of iron homeostasis, affecting red blood cell survival, erythropoiesis, and iron efflux from macrophages. the elevations in serum ferritin seen in these patients stem from stimulation of the divalent metal transporter 1 with increased intracellular iron shifts.11,22 the risk of developing acd increases with advancing age, which corresponds with our observation that acd was the most frequent iron disorder in the elderly (38.%). among all the categories in our study, patients in the acd group had the lowest levels of transferrin (median = 1.65 µg/l, interquartile range: 1.29–2.01). this observation can be explained by the hepatic inhibition of transferrin production as a consequence of the ongoing inflammatory process.23 an examination of the impact of applying the two distinct ferritin cut-offs on the distribution of patients between iron status categories revealed a noticeable shift in all groups except for acd. when using a higher cut-off of 30 µg/l, patients initially categorised as iron deficiency + acd, iron replete, or indeterminate were re-classified as iron deficiency and idwa, with a 1.7-fold increase for iron deficiency, and a 5.6-fold increase for idwa. the cut-off of 30 µg/l has been shown to have significantly higher sensitivity (98%) when compared to a serum ferritin of 15 µg/l, which is highly specific but lacks sensitivity for recognising iron deficiency, as shown in studies conducted in 1998 in america and 1993 in sweden.24,25 this may explain the higher prevalence of profiles identified as indicative of iron deficient states when using 30 µg/l as a serum ferritin threshold. the use of a higher threshold could inadvertently lead to an increase in false positives, but the primary advantage is the increased detection rate of cases that might otherwise go undetected when utilising the lower cut-off of 15 µg/l, which may include up to 50% of true cases of ida.26 within our clinical setting, there is widespread adoption of the world health organization-recommended ferritin cut-off value of 15 µg/l for the diagnosis of ida. however, potential confusion may arise due to the presence of method-specific (and potentially sex-specific) reference intervals for ferritin reported by the national health laboratory services laboratory information system. it is crucial to recognise that these reference intervals are not directly applicable to the diagnosis of all iron status disorders. instead, they represent the range of ferritin values observed in a healthy population. it is important to identify and treat iron deficiency, even in the absence of anaemia (normal haemoglobin levels), in order to minimise any potential adverse consequences, such as reduced cognitive function, poor performance, and poorer outcomes across a range of medical conditions that may occur in idwa.27,28,29 although previous research indicates that the frequency of idwa may be twice as high as that of ida,30 our study population produced a lower than expected frequency (< 5% using either ferritin cut-off), likely because our group was primarily made up of patients attending a hospital or hospitalised, where ida and acd are expectedly more common. despite our low frequency of idwa (3%) being in sharp contrast to that identified in a healthy outpatient population in lebanon in 2020 (57.5% using ferritin < 30 µg/l),31 it remains consistent with the established sex differences, where female patients exhibit a higher tendency for iron deficiency.31 the differences in prevalence estimates may also be due to lack of consistency in definitions and parameters, making it challenging to accurately identify idwa.28 the common practice is relying on a normal haemoglobin combined with a low ferritin in a patient with suggestive clinical symptoms, but there is a lack of guidance for diagnosing idwa in patients with disorders known to affect ferritin levels. as recommended by al-naseem et al. from england in 2021,28 when using a low tsat as a diagnostic criterion in patients where ferritin levels are elevated, our study showed the prevalence of idwa increases to 8% (ferritin cut-off 15 µg/l) and 9% (ferritin cut-off 30 µg/l). these results imply that the inclusion of tsat may reclassify patients who, according to traditional parameters, would otherwise be classified as indeterminate using our criteria. it is important to acknowledge that tsat is a ratio of serum iron to transferrin or total iron binding capacity, and is therefore susceptible to variations in either of these parameters.32 serum iron levels are strongly influenced by dietary consumption and show pronounced diurnal fluctuations, while conditions such as chronic illness and malnutrition suppress the synthesis of transferrin.33 this is supported by evidence from research conducted in 2022 on kenyan patients that tsat (< 20%) shows a relatively low predictive value (31.5%) in identifying idwa.34 mixed aetiology anaemia, iron deficiency + acd, is characterised by the co-occurrence of true iron deficiency in patients with acd. the underlying mechanism is thought to be chronic blood loss associated with a disorder with persistent inflammatory activity.35 it is important to accurately diagnose true iron deficiency in acd patients in order to prevent delayed iron replacement and failure to identify the underlying cause of blood loss.36,37 the substantial overlap in conventional blood parameters makes it difficult to distinguish between ida, iron deficiency + acd, and acd. a limit of < 100 µg/l has been suggested as a diagnostic criterion for iron deficiency + acd to account for the inflammation-induced elevation in ferritin.14 in our study, using this cut-off, iron deficiency + acd ranked second behind acd (defined by ferritin < 15 µg/l) as the most common iron disorder. it is interesting to note however, that a 2021 study conducted in gastroenterology patients in the united kingdom35 has shown that non-traditional markers such as hepcidin, soluble transferrin receptor (stfr), stfr/log ferritin index, and reticulocyte haemoglobin equivalent, can be used to correctly reclassify a substantial proportion of patients in this group as either ida or acd alone, indicating that using only traditional markers may overstate the true prevalence of iron deficiency + acd.37 however, these markers are not without limitations. for instance, the soluble transferrin receptor exhibits an inverse relationship with tissue iron availability and thus is elevated in iron deficiency states. however, due to its correlation with erythropoiesis, its levels also increase in response to enhanced erythropoietic activity, which poses a limitation.32 while the stfr/log ferritin index has been proposed to improve diagnostic accuracy in anaemia classification compared to stfr alone, the routine use of both soluble stfr and the stfr/log ferritin index remains limited due to cost considerations and significant analytical challenges. these challenges include the lack of standardisation in assay methodologies as well as diagnostic thresholds.37,38 in our study, the observed parameters in patients with ida form a classic picture of microcytic anaemia, hypoferraemia, low ferritin levels, decreased tsat percentages, and elevated transferrin levels. interestingly, regardless of the cut-off, median ida ferritin levels remained less than 15 µg/l. mean cell volume was not used in this study as a diagnostic criterion for classification, but its analysis showed that subjects with iron deficiency (ida, iron deficiency + acd, and idwa) tended to have lower median values than subjects with acd, iron replete, and indeterminate categories. notably, the median mcv for only the ida group (72.8 fl, interquartile range: 67.1–79.1) fell below the reference range in our study. studies show that up to 40% of ida patients may have a normal mcv, thus its usefulness as a stand-alone diagnostic tool for iron deficiency is very limited.39 lastly, there was a subset of profiles in our study that could not be categorised into any of the pre-defined groups according to our specified criteria and were deemed indeterminate. this unclassifiable group was predominantly characterised by profiles with normal haemoglobin, elevated serum ferritin, and borderline low tsat levels, and is suggestive of underlying inflammation potentially affecting these parameters. it is noteworthy that a sizeable number of the profiles would be re-classified as idwa had the tsat been used in cases of elevated ferritin levels, decreasing the frequency of this group from 13% to 5% (ferritin < 15 µg/l) and 11% to 5% (ferritin < 30 µg/l). limitations as a retrospective study, this analysis of laboratory data is inherently limited by the lack of information on clinical diagnoses and potentially incomplete data, which may impact the interpretability of the findings. the lack of a comparison to the gold standard diagnostic test, a bone marrow biopsy, made it impossible to verify the accuracy of the ida classification using ferritin. the lack of inflammatory marker data in this study restricts our ability to incorporate them directly into the analysis of inflammatory conditions groups and the impact on ferritin levels. specifically, this data cohort was obtained during the coronavirus 2019 pandemic, which could have affected results due to the effect of inflammation on ferritin and transferrin concentration. while a range of ferritin cut-off values have been proposed for the diagnosis of acd, we opted to utilise a cut-off of 100 µg/l. this specific value aligns with the established threshold for initiating iron replacement therapy in patients with confirmed ida.11 our selection of a tsat < 16% for iron status classification differs from the < 20% cut-off employed by some authors, potentially affecting direct comparability of prevalence to other studies.33 conclusion in the absence of an acceptable non-invasive gold standard marker for iron deficiency, a ferritin cut-off of 30 µg/l might be a suitable threshold for iron deficiency assessment. while this approach is likely to result in a small increase in false-positive diagnoses, more true iron deficiency cases will be identified compared to a lower ferritin cut-off of 15 µg/l. furthermore, considering the potential underdiagnosis of idwa based on existing literature, we recommend screening for iron deficiency using ferritin in patients with normal haemoglobin who present with suggestive clinical symptoms. our findings highlight the difficulties in accurately assessing iron status using traditional laboratory tests. non-standardised ferritin cut-offs, suboptimal test performance in inflammatory settings, and considerable overlap in marker profiles between iron deficiency and other conditions contribute to these challenges. further investigation into the diagnostic accuracy, as well as the feasibility of employing novel markers in our setting, is warranted. acknowledgements we express our gratitude to the national health laboratory service corporate data warehouse for their assistance in data mining and for granting us permission to utilise their data for our investigation. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions the conceptualisation of the study and methodology was developed by j.s.g., d.v.m., s.b.k., and s.k. j.s.g. was involved in data collection, and s.b.k. conducted the data analysis. j.s.g., d.v.m., s.b.k., and s.k. reviewed the findings and contributed to the final article. j.s.g. and s.b.k. created the first draft, and d.v.m. and s.k. edited it. sources of support this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. data availability all data generated from this study are available upon reasonable request from the corresponding author, j.s.g. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. it does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the authors are responsible for this article’s results, findings, and content. references thomas c, thomas l. biochemical markers and hematologic indices in the diagnosis of functional iron deficiency. clin chem. 2002;48(7):1066–1076. https://doi.org/10.1093/clinchem/48.7.1066 brugnara c. iron deficiency and erythropoiesis: new diagnostic approaches. clin chem. 2003;49(10):1573–1578. 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https://doi.org/10.1182/blood-2010-05-286260 skikne bs, punnonen k, caldron ph, et al. improved differential diagnosis of anemia of chronic disease and iron deficiency anemia: a prospective multicenter evaluation of soluble transferrin receptor and the stfr/log ferritin index. am j hematol. 2011;86(11):923–927. https://doi.org/10.1002/ajh.22108 lyle an, budd jr, kennerley vm, et al. assessment of who 07/202 reference material and human serum pools for commutability and for the potential to reduce variability among soluble transferrin receptor assays. clin chem lab med. 2023;61(10):1719–1729. https://doi.org/10.1515/cclm-2022-1198 bermejo f, garcía-lópez s. a guide to diagnosis of iron deficiency and iron deficiency anemia in digestive diseases. world j gastroenterol. 2009;15(37):4638–4643. https://doi.org/10.3748/wjg.15.4638 hiv situation in nigeria laboratory infrastructure and hiv-related testing in nigeria quality assurance framework and policy for hiv laboratory and point-of-care testing quality assurance framework for hiv early infant diagnosis and viral load existing quality assurance programmes lessons learnt conclusion and way forward acknowledgements references about the author(s) ado abubakar national external quality assessment laboratory, saye zaria, kaduna state, nigeria institute of human virology, maina court, plot 252, herbert macaulay way cbd, abuja, nigeria samuel peters institute of human virology, maina court, plot 252, herbert macaulay way cbd, abuja, nigeria oyebimpe balogun institute of human virology, maina court, plot 252, herbert macaulay way cbd, abuja, nigeria sophia osawe institute of human virology, maina court, plot 252, herbert macaulay way cbd, abuja, nigeria plateau state human virology research center, plateau hospital, jos, nigeria ille mamman institute of human virology, maina court, plot 252, herbert macaulay way cbd, abuja, nigeria plateau state human virology research center, plateau hospital, jos, nigeria joshua barde national external quality assessment laboratory, saye zaria, kaduna state, nigeria university of maryland school of medicine, baltimore, maryland, united states emmanuel ojo family health international (fhi 360), garki abuja, nigeria nicholas ezati institute of human virology, maina court, plot 252, herbert macaulay way cbd, abuja, nigeria national tuberculosis and leprosy training center, saye, zaria, kaduna state, nigeria jide bango hiv/aids division, federal ministry of health, edo house abuja, nigeria evelyn ngige hiv/aids division, federal ministry of health, edo house abuja, nigeria anthony emeribe medical laboratory science council of nigeria, durumi, abuja, nigeria alash’le abimiku institute of human virology, maina court, plot 252, herbert macaulay way cbd, abuja, nigeria university of maryland school of medicine, baltimore, maryland, united states citation abubakar a, peters s, balogun o, et al. implementing quality assurance for laboratory-based and point-of-care hiv testing in nigeria. afr j lab med. 2016;5(2), a455. http://dx.doi.org/10.4102/ajlm.v5i2.455 country profile implementing quality assurance for laboratory-based and point-of-care hiv testing in nigeria ado abubakar, samuel peters, oyebimpe balogun, sophia osawe, ille mamman, joshua barde, emmanuel ojo, nicholas ezati, jide bango, evelyn ngige, anthony emeribe, alash’le abimiku received: 03 apr. 2016; accepted: 11 aug. 2016; published: 17 oct. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. hiv situation in nigeria the african national congress sentinel survey in nigeria has shown a steady decrease in hiv prevalence since 2001, with the following trend: 5.8% in 2001, 5.0% in 2003, 4.4% in 2005, 4.6% in 2008, 4.1% in 2010, and 3.4% in 2013.1,2 despite this favourable picture, nigeria has a mixed hiv epidemic and has populations and states where the prevalence is significantly higher than the national estimates (figure 1). nigeria has a population of 173.6 million.3 it is estimated that about 3.2 million people live with hiv in nigeria and about 220 393 new hiv infections occurred in 2013, with 210 031 deaths from aids-related cases.2 only about 42% of those who need antiretroviral therapy are covered. only 4.1% of hiv-exposed infants and 17% of women who tested positive for hiv received their virological and serological test results at the appropriate time, respectively (table 1).2,4 prevention of mother-to-child transmission of hiv coverage is about 20.2%, with only 4.6 % hiv-infected pregnant women assessed for antiretroviral therapy eligibility, through either clinical staging or immune monitoring tests during the period.2 viral load coverage is only 10% (institute of human virology nigeria, unpublished data), with just 419 reported laboratory-based cd4 instruments and 27 cd4 point-of-care (poc) instruments5 (table1). according to the 2010 integrated biological and behavioural surveillance survey, hiv prevalence among the most at-risk populations in nigeria is much higher than among the general population. prevalence is estimated at 27.4% among brothel-based female sex workers and 21.7% among non-brothel-based female sex workers. men who have sex with men had a prevalence of 17.2% (table 1).2,6 figure 1: hiv prevalence in nigeria by state. table 1: key characteristics of the hiv epidemic in nigeria. laboratory infrastructure and hiv-related testing in nigeria high-quality laboratory service is essential in any hiv prevention, treatment, care, and support programme, as it ensures that clients are reliably diagnosed and treated for hiv and related opportunistic infections, such as cryptococcus spp. laboratory services are also essential for monitoring the quality of care and patient’s adherence and response to therapy. in 2015, with the help of the us presidents emergency program for aids relief (pepfar), nigeria upgraded its laboratory infrastructure for hiv diagnosis and monitoring, including cd4, hiv serology, early infant diagnosis (eid) and viral load testing, for a total of 566 laboratories. in 2016, 428 of these laboratories, of which 76 are in tertiary and 352 in secondary facilities, are still being supported by pepfar. the remaining 138 primary facility-based laboratories have been transitioned to the government of nigeria.4 in addition, 61 laboratories have been supported for genexpert® (cepheid) external quality assessment (eqa) through proficiency testing panels from the united states centers for disease control and prevention (cdc) in atlanta. laboratory services have been provided to over 500 000 tuberculosis patients in care and treatment.5 in addition, the improved national hiv/aids laboratory guideline has been distributed to all 20 universities and 47 colleges in nigeria offering medical laboratory science programmes. finally, close to 3000 laboratory personnel have benefitted from in-service laboratory trainings over a three-year period. transitioning of molecular testing in pcr-supported laboratories to the government of nigeria is taking place to minimise the disruption of eid and viral load services, and to ensure that government of nigeria staff are trained on and proficient in the performance of pcr-related activities. in 2016, five pcr laboratories were being transitioned to the government of nigeria. as pepfar scales up viral load for patient monitoring, cd4 testing may be limited to staging and monitoring where viral load testing is not yet available. the current plan of the government of nigeria, supported by pepfar, is to scale up viral load services in the 32 high burden local government areas in nigeria and to establish a network of laboratories that can eventually support viral load testing nationally. obviously, a robust and dependable quality assurance strategy must accompany such expansions. quality assurance framework and policy for hiv laboratory and point-of-care testing the national eqa framework in nigeria is conducted through a centralised system, adopting last-mile distribution through a courier company (figure 2). the medical laboratory science council of nigeria, in partnership with the axios foundation, developed the national eqa centre at the national tb and leprosy training center, zaria, between 2008 and 2013, with support from the division of global hiv and tb of the cdc. the institute of human virology, nigeria, currently supports the medical laboratory science council of nigeria to execute this programme. while the centre produces hiv serology panels for distribution using dried tube specimens,7 cd4 panels are obtained from oneworld accuracy (canada). in addition, the digital proficiency testing’s oneworld accuracy platform is used for bioinformatics and statistical analyses for both hiv serology and cd4 for the 428 laboratories still supported by pepfar (figure 3). the anticipated plan is that laboratories supported by the government of nigeria and those that are privately owned will also join the quality assurance programme to ensure high-quality practices in all laboratories supporting patient care. figure 2: nigeria national external quality assessment structure. figure 3: nigeria centralised national external quality assessment model. quality assurance framework for hiv early infant diagnosis and viral load cdc-nigeria established a cost-free proficiency testing programme in nigeria for molecular laboratory tests used for eid in 2006, and hiv viral load tests in 2010. there are currently 18 advanced laboratories participating in proficiency testing for viral load dried tube specimens and 23 laboratories participating in eid proficiency testing – a significant increase from the single laboratory, plateau state human virology research center in jos, where the programme was initiated in 2006. the plateau state human virology research center now serves as a distribution centre to all participating laboratories in the country (figure 4). the cdc has also transitioned the responsibility of the production of these proficiency testing panels to the bacteriology and virology laboratory at the university cheikh anta diop in dakar, senegal, thereby building regional capacity to support countries in west africa. the molecular laboratories in nigeria currently participate in two proficiency testing events per year for eid and viral load. this proficiency testing is an essential component in a comprehensive laboratory quality assurance programme for the care and treatment of hiv-infected patients for any country. figure 4: early infant diagnosis and viral load external quality assessment structure. existing quality assurance programmes in addition to the provision of proficiency testing panels by the national external quality assessment laboratory, the national quality assurance team conducts an on-site assessment by strengthening laboratory management toward accreditation8-certified trainers, mentors and auditors. rapid test kit evaluations and post-market validation of all hiv rapid test kits supplied by pepfar or the government of nigeria for use in nigeria are also an integral part of quality assurance. the implementing partners assist the state quality assurance teams in conducting hiv rapid-test-kit post-market validation in the states. in a recent drive to achieve accreditation through the world health organization regional office for africa’s stepwise laboratory quality improvement process towards accreditation9 and strengthening laboratory management toward accreditation programmes, with support from the cdc, eight laboratories in nigeria that have attained 4to 5-star strengthening laboratory management toward accreditation grades have been enrolled for iso 15189 accreditation through the south african national accreditation system. sites that have yet to attain a 4-star grade receive continuous quality improvement activities that include training and on-site mentorship and will be presented for stepwise laboratory quality improvement process towards accreditation audit after being confirmed ready by the in-country team. lessons learnt the success achieved in implementing a quality assurance programme in nigeria can be attributed to: ownership and commitment on the part of the host institutions;10 technical support provided by trained nigerian personnel in the different organisations and institutions functioning as pepfar and global health implementing partners; and leadership and commitment from the government of nigeria. in our experience, an important lesson for health facilities is that quality assurance requires sustained expenditure, human and material resources, and dedication. however, it is highly rewarding for both the health institutions and their clients in the end. the support provided by implementing partners to the government has been invaluable in developing structures, strengthening referral networks, improving antiretroviral therapy uptake, access to viral load testing, shipment, data exchange, and commodity logistics. hiv rapid-test-kit quality improvement implementation will ensure that all hiv testing points use standardised log books, run controls, participate in a proficiency testing programme and ensure that testers are certified. provision of viral load poc testing will help to meet testing targets, as the number of pcr laboratories cannot meet the demand, especially in rural settings. it will also reduce the challenge of the logistics involved in maintaining cold chain during specimen referrals. conclusion and way forward over a period of 10 years, the pepfar programme has provided the foundation in nigeria for eqa support for cd4, hiv serology, blood chemistry, haematology, hiv viral load and eid testing to over 566 sites in nigeria. in line with the country operational plan,11 the government of nigeria has already taken significant steps to support the 138 laboratories that have been transitioned to it and provide eqa support for hiv serology and cd4. the remaining 428 laboratories are still supported by pefpar, together with an additional 18 viral load laboratories and 23 eid laboratories, under the coordination of the government of nigeria as part of the transition plan. similarly, for molecular diagnostics (i.e., viral load and eid), the plateau state human virology research center handles the enrollment of these laboratories in eqa proficiency testing from the cdc and dakar, senegal. the transitioning of this activity to the university cheikh anta diop in senegal will ensure reduction in costs and will foster ownership in the african sub-region. with the current expansion of viral load testing to high hiv-burden states and local governments in nigeria, the need for a substantial number of viral load poc testing platforms backed by a robust eqa programme in nigeria is important for both the public and private sectors. the post-market validation of imported rapid test kits before distribution will assure that rapid test kits used in the country are of the highest quality. with pepfar-supported laboratories being integrated into the mainstream healthcare facility laboratories under the oversight and commitment of the federal ministry of health and medical laboratory science council of nigeria, quality assurance uptake and ownership is on the increase, including among non-pepfar supported laboratories. the establishment of the national quality assurance team, which drives the post-market validation of rapid test kits, will ensure that quality assurance is a priority in both public and private healthcare facilities in nigeria. it also sends a strong message that quality assurance standards are important to the nation and must be adhered to. further steps should be taken to involve state quality assurance officers in the preparation of dried tube specimens in all 36 states in nigeria. as the country struggles to comply with the aggressive unaids targets of 90-90-90, the need to increase of poc testing needs to be addressed and appropriate quality assurance put in place to ensure that the increased volume does not compromise quality. the low coverage in antiretroviral therapy, eid testing, prevention of mother-to-child transmission, cd4 poc testing, and hiv viral load testing can be improved through integrated tiered referral networks, public–private partnerships, and programme ownership by the government of nigeria. acknowledgements competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. sources of support none. references federal ministry of health, nigeria. national hiv seroprevalence sentinel survey among pregnant women attending antenatal clinics in nigeria: technical report 2010. nigeria: department of public health, national aids/sti control program; 2010. national agency for the control of aids, nigeria. national hiv/aids prevention plan 2014–2015. abuja, nigeria: naca; 2014. the world bank. population, total [page on the internet]. c2013 [cited 2016 sep 28]. available from: http://data.worldbank.org/indicator/sp.pop.totl national agency for the control of aids, nigeria. global aids response country progress report [document on the internet]. c2015 [cited 2016 sep 27]. available from: http://www.unaids.org/sites/default/files/country/documents/nga_narrative_report_2015.pdf orloff s. standardization and harmonization of laboratory equipment: selected country case studies [document on the internet]. c2010 [cited 2010 mar 22]. available from: http://www.who.int/hiv/amds/17_cdc_case_studies_orloff.pdf federal ministry of health, nigeria. hiv integrated biological and behavioural surveillance survey (ibbss) [document on the internet]. c2010 [cited 2016 sep 28]. available from: http://www.popcouncil.org/uploads/pdfs/2011hiv_ibbss2010.pdf parekh bs, anyanwu j, patel h, et al. dried tube specimens: a simple and cost-effective method for preparation of hiv proficiency testing panels and quality control materials for use in resource-limited settings. j virol methods. 2010;163(2):295–300. http://dx.doi.org/10.1016/j.jviromet.2009.10.013 epub 2009 oct 28. yao k, maruta t, luman et, et al. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. 2014;3(2), 8 pages. http://dx.doi.org/10.4102/ajlm.v3i2.194 world health organization regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation (slipta) in the african region (with checklist). brazzaville, republic of congo: who afro; 2015. abimiku ag. building laboratory infrastructure to support scale-up of hiv/aids treatment, care and prevention: in-country experience. am j clin pathol. 2009;131(6):875–886. http://dx.doi.org/10.1309/ajcpelmg6gx6rqsm united states department of state. fy 2015 nigeria country operational plan (cop) [document on the internet]. c2015 [cited 2016 sep 28]. available from: http://www.pepfar.gov/documents/organization/250298.pdf article information authors: nestor bangoura1 abou a.m. diouara2 mohamed cissé1 halimatou d. ndiaye2 souleymame mboup2 ahidjo ayouba3 coumba t. kane2 affiliations: 1service de dermatologie chu donka, cta, conakry, guinée 2laboratoire de bactériologie virologie chu aristide le dantec, université cheikh anta diop de dakar, sénégal 3umi 233 ird de montpellier, france correspondence to: coumba kane email: ctourekane@yahoo.co.uk postal address: 30 avenue pasteur, bp: 7325 dakar, sénégal dates: received: 24 jan. 2014 accepted: 12 dec. 2014 published: 26 june 2015 how to cite this article: bangoura n, diouara aam, cissé m. quantification de la charge virale et tests de résistance du vih-1 aux arv à partir d’échantillons dbs (dried blood spots) chez des patients guinéens sous traitement antiretroviral. afr j lab med. 2015;4(1), art. #168, 7 pages. http://dx.doi.org/10.4102/ajlm.v4i1.168 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. quantification de la charge virale et tests de résistance du vih-1 aux arv à partir d’échantillons dbs (dried blood spots) chez des patients guinéens sous traitement antirétroviral in this original research... open access • abstrait • abstract • introduction • conception et méthode d’étude    • patients et sites de collecte des échantillons    • echantillonnage et conservation des prélèvements dbs    • l’extraction des acides nucléiques    • la quantification de la charge virale (cv)    • génotypage et analyse phylogénétique • considérations éthiques • résultats    • caractéristiques de la population d’étude    • charge virale (cv) et tests de résistance    • phylogénie et caractérisation moléculaires des souches virale • discussion • remerciements    • conflit d’intérêt    • contributions des auteurs • références abstrait top ↑ problématique: comme dans plusieurs pays du sud, le suivi virologique des patients sous traitement antirétroviral (tarv) en guinée est timide voire inexistant dans certaines localités. le but de cette étude était d’évaluer la faisabilité technique et logistique de l’utilisation des dbs dans les tests de charge virale (cv) et de génotypage. méthode: de septembre à octobre 2010, les dbs ont été préparés à partir de prélèvements sanguins de patients adultes sous tarv. le délai d’envoi des échantillons au laboratoire de référence était de 30 jours maximum après le prélèvement et se faisait à température ambiante. la cv a été quantifiée et les échantillons de patients en échec virologique (cv ≥ 3 log10 copies/ml) ont été génotypés selon le protocole de l’anrs. l’algorithme de stanford version 6.0.8 a été utilisé pour l’analyse et l’interprétation des mutations de résistance. résultats: parmi les 136 patients inclus, 129 et 7 étaient respectivement sous première et deuxième ligne de traitement avec une médiane de suivi de 35 mois [iqr: 6-108]. l’échec virologique a été noté chez 33 patients. parmi eux, 84.8% (n = 28/33) ont bénéficié d’un génotypage. le taux de résistance global était de 14% (n = 19/136). le crf02_ag était le sous type viral le plus prévalent (82%; n = 23) conclusion: en plus de montrer la faisabilité technique et logistique des tests de cv et de génotypage à partir des dbs, ces résultats montrent l’intérêt de leurs utilisations dans le suivi virologique des patients sous tarv. cette étude a permis également de documenter l’échec virologique, la résistance aux arv et la diversité génétique du vih-1 en guinée. mots clés: vih-1, résistance aux arv, dbs (dried blood spots), guinée conakry, génotypage, charge virale. abstract top ↑ quantification of viral load and resistance tests of hiv-1 to arvs from dried blood spots samples in guinean patients undergoing antiretroviral treatment. problem: as in several countries of the south, the virological monitoring of patients undergoing antiretroviral treatment (arvt) in guinea is low or non-existent in some locations. the aim of this study was to assess the technical and logistical feasibility of the use of (dried blood spots) dbss in viral load (vl) and genotyping tests. method: from september 2010 to october 2010, dbs were prepared from blood samples of adult patients under arvt. the samples had to be sent to the reference laboratory within 30 days after the sample had been done at ambient temperature. the vl was quantified and the samples of patients with virological failure (cv ≥ 3 log10 copies/ml) were genotyped according to the anrs protocol. the stanford algorithm, version 6.0.8, was used to analyse and interpret the resistance mutations. results: amongst the 136 included patients, 129 and 7 were under first and second line treatment respectively, and monitored for an average of 35 months [iqr: 6-108]. virological failure was noticed among 33 patients. among them, 84.8% (n = 28/33) benefited from genotyping. the global resistance rate was 14% (n = 19/136). crf02_ag was the most prevalent viral subtype (82%; n = 23) conclusion: in addition to demonstrating the technical and logistic feasibility of vl and genotyping tests from dbss, these results show the relevance of their use in the virological monitoring of patients under arvt. also, this study made it possible to provide information on virological failure, arv resistance and the hiv-1 genetic diversity in guinea. introduction top ↑ en guinée, la prévalence du vih chez les adultes (15-49ans) est estimée à 1.4%.1 la mise sous traitement antirétroviral (tarv) des patients vivant avec le vih/sida a débuté en 1999. depuis, le nombre de sites de prise en charge (pec) opérationnels des patients répartis dans le pays a augmenté et est passé de 46 en 2012 à 51 en fin 2013. la couverture nationale en centres de conseils et dépistage volontaire (cdv) était de 66 sites et 131 sites de prévention de la transmission mère-enfant (ptme) pour 464 structures offrant les services de consultations prénatales (cpn) en 2013.2 de plus, la couverture en tarv a connu une hausse au cours de ces années et est passée de 22.50% en 2007 à 56.91% en fin 2011, pour 40 258 personnes éligibles au tarv selon les lignes directrices de l’oms.3 a l’image de plusieurs pays à ressources limitées, du fait d’un manque d’infrastructures, d’équipements biomédicaux et de personnels qualifiés, les tests moléculaires, notamment la charge virale (cv) et le génotypage, ne sont pas tout le temps disponibles. par conséquent, le suivi des patients sous tarv en guinée se fait essentiellement en se basant sur des critères clinico-immunologiques.4 l’utilisation du papier buvard comme support de prélèvement sanguin alternatif au plasma permettrait la collecte et le transport des échantillons des sites périphériques vers le centre de référence national ou international. le papier buvard (dbs) a été largement utilisé dans le diagnostic sérologique et moléculaire de l’infection à vih.5,6 de plus, les dbs ont été utilisés dans la détermination de la cv et des tests de résistance du vih-1 aux arv.7,8,9 plusieurs études ont évalué et validé le dbs en le comparant au plasma10,11,12,13, qui est le type d’échantillon de référence pour les tests de cv et de génotypage. le dbs est un support facile d’utilisation, moins exigeant que le plasma et le sérum car ne nécessitant pas une chaine de froid pour la conservation, le stockage et le transport des échantillons. c’est dans ce contexte que s’inscrit cette étude pionnière en guinée dont l’objectif était d’évaluer la faisabilité technique et logistique des tests de cv et de génotypage du vih-1 à partir d’échantillons dbs de patients sous tarv collectés dans des conditions de terrain. conception et méthode d’étude top ↑ patients et sites de collecte des échantillons cette étude a porté sur des patients adultes (≥ 18 ans) sous tarv depuis au moins 6 mois, suivis dans le cadre du programme national. les patients inclus dans cette étude ont été recrutés consécutivement sur la période allant de septembre à octobre 2010 au niveau de 4 sites de prise en charge (pec). les individus infectés par le vih-2 ou coinfectés par le vih-1 et vih-2, de même que les femmes ayant bénéficiés d’une prévention de la transmission mère-enfant du vih (ptme), n’étaient pas inclus. le choix de ces sites a été fait de façon aléatoire et sur la base de l’existence d’association de personnes vivant avec le vih pouvant faciliter la collecte des échantillons. un des sites est situé dans la capitale, le centre de traitement ambulatoire de conakry, et les 3 autres sont localisés dans les régions de boké, mamou et labé, distants respectivement de 300, 350 et 600 km de la capitale (figure 1). figure 1: répartition des sites de collecte des échantillons. echantillonnage et conservation des prélèvements dbs pour chaque patient, 5 ml de sang total ont été recueillis dans un tube edta par ponction veineuse au niveau du pli du coude pour servir à la préparation de 2 cartes de papier filtre whatman 903®, conformément aux règles d’hygiène et de sécurité , et 50 µl de sang total ont été déposés sur chacun des 5 spots à raison d’une carte dbs, préalablement identifiée et datée. les échantillons ont été séchés pendant la nuit à température ambiante (30 à 37 °c). chaque échantillon dbs a été placé dans un sachet plastique individuel hermétiquement fermé en présence de dessiccateurs. puis, ils ont été conservés et stockés sur site à température ambiante. l’envoi des dbs vers le laboratoire de bactériologie virologie de l’hôpital aristide le dantec de dakar au sénégal pour les tests de cv et de génotypage s’est fait par voie terrestre dans les 30 jours suivant le prélèvement. l’extraction des acides nucléiques a partir de 2 spots de chaque échantillon dbs/patients et à l’aide de l’appareil nuclisens minimag (biomérieux, craponne, france), les acides nucléiques totaux ont été obtenus par extraction magnétique et manuelle selon la chimie de boom.14 en effet, les spots découpés à l’aide d’un « puncher » dédié ont été trempés dans un tube contenant 2 ml de tampon de lyse et agité pendant 30 minutes à température ambiante. les acides nucléiques ont été extraits et élués dans 25 µl de tampon d’élution après une série de centrifugation et des lavages avec des tampons 1, 2 et 3 comme précédemment décrit.10 la quantification de la charge virale (cv) la détermination de la cv a été effectuée à partir de l’extrait obtenu et ceci en utilisant kit nuclisens easyq hiv-1 v2.0 (biomérieux, craponne,france) conformément aux instructions du fabriquant. la plateforme utilisée était nuclisens® easyq (biomérieux, lyon, france) et le principe est une amplification de type nasba. le seuil de détectabilité de la technique est de 800 copies/ml.15 dans cette présente étude, le seuil de l’échec virologique a été fixé à 3 log10 copies/ml génotypage et analyse phylogénétique le génotypage a été effectué selon le protocole de l’anrs (http://www.hivfrenchresistance.org/) qui consiste à faire des amplifications séparées par pcr des fragments de la protéase en entier et des 240 premiers codons de la reverse transcriptase (rt) du gène pol en utilisant respectivement les couples d’amorces 5’prot1/3’prot1 et mj3/mj4 comme amorces externes et 5’prot2/3’prot2 et a35/ne35 comme amorces internes. les produits de pcr de 2ème tour ont été purifiés avec le kit qiaquick gel extraction kit®, (qiagen, courtaboeuf, france) conformément aux indications du fabriquant. l’adn purifié a été directement séquencé sur la plateforme abi prism 3100 avant (genetic analyzer applied biosystem) selon la technologie du big dye terminator technology®v3.1 (applied biosystems, courtaboeuf, france). les séquences obtenues ont été assemblées et manuellement éditées sur le logiciel seqmantm ii 5.08 de la suite de dnastar® software (lasergene, konstanz, germany). l’analyse et l’interprétation des mutations de résistance ont été réalisées sur l’algorithme de l’université de stanford version 6.0.8 (http://hivdb.stanford.edu/). les séquences générées ont été alignées avec des séquences références du vih-1 groupe m et l’ensemble des formes recombinant les crfs disponibles sur los alamos hiv database (http://www.hiv.lanl.gov/content/index). trois séquences de chaque sous-type pur ont été incluses dans l’alignement. et les séquences ont été alignées avec l’algorithme de muscle puis l’alignement dégapé obtenu a été avec le programme de gblocks du logiciel seaview v4.4.1. l’arbre phylogénétique de maximun de vraisemblance (phyml) a été également généré sur seaview v4.4.1 avec comme paramètres supports de branche déterminés par la méthode approximate likelihood ratio test (alrt), option sh-like. l’analyse de similarité et de bootscanning pour la confirmation des formes recombinantes (crfs, urfs) a été effectuée sur le logiciel simplot v3.5.1.16,17 l’analyse et les calculs statistiques des données ont été réalisés avec les logiciels epi info v3.5.4 et microsoft excel. considérations éthiques top ↑ cette étude est une sous-étude d’un projet multicentrique impliquant 3 pays de l’afrique de l’ouest (sénégal, mali et la république de guinée) et a été approuvée par les comités éthiques nationaux de ces pays. les patients ont été recrutés consécutivement sur base volontaire, sur une période allant de septembre à octobre 2010 après signature d’un formulaire de consentement libre et éclairé. pour garder confidentielles les données des participants, un code unique a été attribué à chaque prélèvement. résultats top ↑ caractéristiques de la population d’étude au total 136 patients infectés par le vih-1 sous tarv ont participé à cette étude. parmi eux, 129 étaient sous traitement de première ligne (2inti + 1innrt) et 7 sous deuxième ligne (2inti + 1ip/r), avec une médiane de suivi thérapeutique de 35 mois [iqr: 6-108 mois]. le sexe ratio homme/femme était de 0,64 et l’âge médian était de 38 ans [iqr: 18-61 ans] boîte 1. charge virale (cv) et tests de résistance l’échec virologique (cv ≥ 3 log10 copies/ml) a été observé chez 33 patients soit un taux de 24.26% (n = 33/136). parmi eux, 4 étaient sous deuxième ligne de tarv et 13/29 patients ont eu des changements de molécules de première ligne (exemple: d4t par azt) pour des raisons cliniques ou de tolérance. dans le tableau 1, figurent entre autre l’historique du traitement et les données virologiques des patients en échec virologique. selon la durée du traitement, 4/13, 7/31 et 22/92 patients étaient en échec virologiques respectivement à des intervalles 6-12, 13-24 et > 24 mois. tableau 1: données liées aux patients en échec virologique (cv ≥ 3 log10 copies/ml). boîte 1: caractéristiques de la population d’étude et nombres de participants par sites. au total, 28/33 échantillons de patients en échec virologique ont pu être génotypés soit un taux d’amplification réussi de 84.84%. la médiane de cv de ces échantillons de patients en échec virologique génotypés (n = 28) était de 4 log10 copies/ml [iqr: 3-6.7] et celui des échantillons non amplifiés (n = 5) était de 3.1 log10 copies/ml [iqr: 3-3.6] pour une p-value = 0.02. au moins une mutation conférant une résistance à une molécule antirétrovirale a été observée chez 19 patients, soit un taux de résistance globale de 14%. la mutation m184v (n = 13) et les tams (thymidine analogue-associated mutations) (n = 32) étaient les plus fréquemment observées pour les inti et la k103n (n = 11) et y181c (n = 7) pour les innti. leurs survenues étaient d’autant plus marquées que la durée du traitement était élevée (figure 2). l’insertion t69 a également été observée chez 5 patients dont un en deuxième ligne. d’autres mutations comme la v198i, g190a/g et y188a/l ont été également notées chez 4, 3 et 2 patients respectivement (tableau 1). par ailleurs, aucune mutation de résistance majeure aux ip (inhibiteur de protéase) n’a été observée et ceci qu’il s’agisse de patients avec une virémie supérieure à 3 log10 copies/ml ou pas, de patients sous première ligne ou deuxième ligne de traitement. figure 2: fréquences des mutations observées en fonction de la durée du traitement. phylogénie et caractérisation moléculaires des souches virale l’analyse phylogénétique des séquences nucléotides a permis de montrer la prédominance du crf02_ag, 82% (n = 23/28). les sous types d et crf06_cpx ont été observés dans les proportions respectives 7% (n = 2/28) et 11% (n = 3/28) (figure 3). figure 3: arbre phyml montrant la relation phylogénétique entre les séquences requêtes (n=28, en traits pointillés) et les références (en traits pleins) dans la région du gène pol (pr+rt) du vih-1. discussion top ↑ cette étude décrit pour la première fois en guinée les données relatives à l’échec virologique et le taux de résistance du vih-1 chez des patients sous tarv. précédemment, dans des conditions relativement similaires à celles décrites dans cette étude, du fait que le plasma soit le type d’échantillon de référence pour la quantification de la cv et la réalisation des tests de résistance, nous avons effectué des études d’évaluation et de faisabilité des tests virologiques à partir d’échantillons dbs. ce fut, d’une part, des comparaisons de valeurs de cv et de profils de résistance entre échantillons pairs plasma et dbs8 et, d’autres part, la faisabilité des tests de résistance à partir de dbs collectés et acheminés dans des conditions réelles de vie.10 le but de cette présente étude était d’évaluer la faisabilité technique et logistique des tests de cv et de génotypage à partir d’échantillons dbs de patients sous tarv collectés dans des conditions de terrain. l’échec virologique a été observé dans 24.26% (n = 33/136) des cas et parmi eux 2/3 des patients étaient à plus de 24 mois de traitement (tableau 1). le taux de suppression virologique (75.7%) semble être satisfaisant pour une médiane de suivi de 35 mois.18 garrido et al en 2008 ont rapporté un taux de suppression virologique similaire mais avec une médiane de suivi thérapeutique de 12 mois.7 dans cette étude, nous avons obtenu un taux d’amplification réussi de 84.84% comparable à ceux obtenus en tanzanie,19 en espagne20 et légèrement inférieur à ceux obtenus précédemment au sénégal10 et en guinée conakry (94%).21 cinq échantillons de patients en rebond virologique, dont la médiane de cv était faible (3.13 log10 copies/ml [3.06-3.63]), n’ont pas pu être génotypés, malgré plusieurs tentatives. ceci pourrait également être dû à une dégradation des acides nucléiques durant les processus de conservation et de stockage des prélèvements dbs à température ambiante. d’ailleurs, plusieurs études ont rapporté un faible taux d’amplification réussi pour des échantillons à cv < 5000 (3.69 log10) copies/ml contrairement à ceux ayant des cv élevées (> 10000 [4 log10] copies/ml).20,22,23,24 les mutations de résistances sélectionnées chez les patients en échec virologique dans cette étude (tableau 1) étaient en accord avec les schémas thérapeutiques en cours ou antérieurs. la proportion de patients ayant ces mutations augmente avec la durée du traitement (14/19 ; supérieure à 24 mois). par ailleurs, la mutation m184v conférant une résistance au 3tc/ftc et les tams pour les inti et les mutations k103n et y181c pour les innti (figure 2), les plus prédominantes ici, ont été également celles observées dans plusieurs études conduites en afrique subsaharienne.7,25,26 l’insertion t69, conférant une multi-résistance aux inti27 et retrouvée chez certains patients, reflète la composition de leur régime thérapeutique, qui inclut soit la didanosine (ddi), soit la stavudine (d4t) ou encore la zidovudine (azt). ces molécules sont connues pour sélectionner la mutation t69.28,29,30 par ailleurs, les résultats de génotypage ont montré que 27.2% (n = 9/28) des patients en échec virologique étaient porteurs de virus sauvages (i.e. encore sensibles aux arv). cet échec virologique serait probablement lié à une mauvaise observance ou encore aux variants minoritaires que le ‘bulk sequencing’ utilisé dans cette étude ne peut pas détecter. des observations similaires ont été rapportées à abidjan (côte d’ivoire) et à bangui (république centrafricaine).31,32 une virémie élevée sous tarv est associée à un risque d’émergence de la résistance du vih aux médicaments. ainsi, cette étude met en évidence la nécessité d’améliorer l’observance au traitement. l’analyse phylogénétique des souches virales étudiées montre une forte prédominance du crf02_ag (81%, n = 26), comme précédemment rapporté dans une étude de résistance primaire du vih-1 chez des patients nouvellement infectés à conakry.21 ces résultats montrent la faisabilité technique et logistique des tests de cv et de génotypage à partir de prélèvements dbs. d’un autre côté, cette étude montre l’intérêt de l’utilisation des dbs comme support de prélèvement dans le monitoring virologique des patients sous tarv en guinée, pays où le suivi virologique est encore peu structuré. de plus elle a permis de documenter le taux de patients en échec virologique, la résistance du vih-1 aux arv et la diversité génétique en guinée. remerciements top ↑ nous remercions l’organisation ouest africaine de la santé (ooas) pour avoir financé cette étude, solthis guinée (solidarité thérapeutique et initiative contre le sida) pour son assistance, toutes les équipes de recherches ayant contribué à ce travail, l’ensemble du personnel des sites de pec du guinée, les différentes organisation des pvvih/sida et les patients qui ont bien voulu participer à cette étude. conflit d’intérêt les auteurs déclarent qu’ils n’ont aucun lien financier ou personnel qui pourrait les influencer de façon inappropriée en écrivant cet article. contributions des auteurs c.t.k. (université cheikh anta diop) était le chef de projet. c.t.k, h.d.n., a.a.m.d. (université cheikh anta diop) et m.c. (service de dermatologie chu donka, cta, conakry) étaient responsables de la conception expérimentale et de celle du projet. a.a.m.d. (université cheikh anta diop) et n.b. (service de dermatologie chu donka, cta, conakry) ont effectué les analyses virologiques et écrit le manuscrit initial. tous les auteurs ont participé à sa rédaction et édition finales. tous les auteurs ont lu et approuvé le manuscrit final. références top ↑ unaids. unaids report on the global aids epidemic. 2012:pp:1–212 [unaids global aids report web site]. disponible sur: http://www.unaids.org/en/media/unaids/contentassets/documents/epidemiology/2012/gr2012jc2434_worldaidsday_results_en.pdf. [consulté le 16 mai 2013]. cnls-guinée. revue des progrès vers la réalisation des cibles de la déclaration 2011 de l’onu sur le vih et le sida. rapport narratif. 2014:pp:1–36. http://www.unaids.org/fr/regionscountries/countries/guinea/ [consulté le 05 juin 2014]. cnls-guinée. rapport ungass 2012 _guinée. 2012:pp:1-71. [unaids global aids report web site]. disponible sur http://www.unaids.org/en/dataanalysis/knowyourresponse/countryprogressreports/2012countries/ce_gn_narrative_report[2011].pdf. [consulté le 26 juin 2013] pnpcsp-ist/sida. normes et protocoles de prise en charge de l’infection par le vih chez l’adulte et l’enfant en guinee. 2012:pp:1–103. [who web site]. disponible sur http://www.who.int/hiv/pub/guidelines/guinea_art.pdf. 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oct;176(4):899–903. http://dx.doi.org/10.1086/516511 winters ma, coolley kl, girard ya, et al. a 6-basepair insert in the reverse transcriptase gene of human immunodeficiency virus type 1 confers resistance to multiple nucleoside inhibitors. j clin invest. 1998 nov 15;102(10):1769–1775. http://dx.doi.org/10.1172/jci4948 scherrer au, von wyl v, joos b, et al. predictors for the emergence of the 2 multi-nucleoside/nucleotide resistance mutations 69 insertion and q151m and their impact on clinical outcome in the swiss hiv cohort study. j infect dis. 2011 mar 15;203(6):791–797. http://dx.doi.org/10.1093/infdis/jiq130 messou e, chaix ml, gabillard d, et al. increasing rate of tams and etravirine resistance in hiv-1-infected adults between 12 and 24 months of treatment: the voltart cohort study in cote d’ivoire, west africa. j acquir immune defic syndr. 2013 jun 21. http://dx.doi.org/10.1097/qai.0b013e3182a009e4 pere h, charpentier c, mbelesso p, et al. virological response and resistance profiles after 24 months of first-line antiretroviral treatment in adults living in bangui, central african republic. aids res hum retroviruses. 2012 apr;28(4): 315–323. http://dx.doi.org/10.1089/aid.2011.0127 abstract introduction methods results discussion acknowledgements references about the author(s) ayanda g.p. jali department of haematology, health king edward viii hospital, university of kwa-zulu natal, durban, south africa department of haematology, national health laboratory service, inkosi albert luthuli academic hospital, durban, south africa bongani b. nkambule department of human physiology, school of laboratory medicine and medical sciences, university of kwazulu-natal, durban, south africa citation jali agp, nkambule bb. prevalence and aetiology of moderate and severe thrombocytopenia in a tertiary and quaternary centre in kwazulu-natal. afr j lab med. 2020;9(1), a799. https://doi.org/10.4102/ajlm.v9i1.799 original research prevalence and aetiology of moderate and severe thrombocytopenia in a tertiary and quaternary centre in kwazulu-natal ayanda g.p. jali, bongani b. nkambule received: 12 mar. 2018; accepted: 02 june 2020; published: 24 aug. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: thrombocytopenia is a common haematological disorder, characterised by platelet counts below 150 × 109/l. the aetiology of thrombocytopenia is multifactorial; notably, in a misdiagnosis this condition may be due to pre-analytical laboratory artefacts. knowledge about the common aetiology of thrombocytopenia will assist clinicians in decision-making and interpretation of laboratory tests and this may lead to prompt, adequate patient management and cost-saving measures. objective: this study determined the prevalence and aetiology of moderate and severe thrombocytopenia in a tertiary or quaternary laboratory in durban, kwazulu-natal, south africa. methods: we conducted a retrospective study at the inkosi albert luthuli central hospital haematology laboratory between october 2015 and april 2016. a total of 2076 full blood count results with a platelet count of less than 100 × 109/l were retrieved from the inkosi albert luthuli academic hospital database. laboratory data were extracted and matched with clinical data and used to identify the potential aetiology of thrombocytopenia. results: the prevalence of thrombocytopenia was 14.9% within the selected study period. the haematology or oncology wards and clinic accounted for 55.2% of thrombocytopenia cases, whereas the adult and paediatric intensive care units accounted for 29.3%. notably, 15.5% of thrombocytopenia cases were reported in non-haematology wards and clinics. the most common cause of thrombocytopenia was chemotherapy which accounted for 38.5% of all causes. conclusion: in our tertiary and quaternary setting, thrombocytopenia in adults was most common in patients admitted to haematology and oncology wards. moreover, chemotherapy-induced thrombocytopenia accounted for more than a third of all these cases. keywords: thrombocytopenia; prevalence; aetiology; south africa; haematology. introduction thrombocytopenia is a common clinical condition that is associated with multiple systemic diseases. thrombocytopenia is characterised by platelet counts below 150 × 109/l. however, only platelet counts of less than 100 × 109/l are considered clinically significant.1 moderate thrombocytopenia is defined as a platelet count range of 50–100 × 109/l, while severe thrombocytopenia is classified by a platelet count of less than 50 × 109/l.2 the aetiology of thrombocytopenia varies and may be caused by mild to life-threatening clinical conditions. however misdiagnosis of thrombocytopenia also occurs, and could be the result of artefactual laboratory errors.3,4 a full blood count and peripheral blood smear are mandatory as an initial test in patients with thrombocytopenia.1,5 although there have been advances in the automation of haematology analysers, the peripheral blood smear still remains an important diagnostic tool.1 it provides an informed interpretation of the full blood count results, as there are morphological abnormalities that an automated analyser cannot detect.3,6 in particular, it helps to distinguish between thrombocytopenia due to laboratory errors and true thrombocytopenia.3 thrombocytopenia can be caused by decreased thrombopoiesis in the bone marrow and increased sequestration of platelets by the spleen as a result of malignancy, aplastic anaemia, myelodysplastic syndrome and opportunistic infections.1,7 another aetiology of thrombocytopenia is increased peripheral destruction of platelets that may occur following disseminated intravascular coagulation, thrombotic microangiopathy and increased platelet sequestration due to hypersplenism.4,8 for prompt and adequate management of patients with thrombocytopenia, a comprehensive review of the patients’ medical records should be performed by the treating clinician, and appropriate physical examination and investigations need to be performed.1 previous studies have shown an increased prevalence of thrombocytopenia in hospitalised patients in developing and developed countries.4,9,10 in patients with haematological malignancies receiving chemotherapy, thrombocytopenia as a consequence of drug toxicity levels has been reported.12 understanding the aetiology of thrombocytopenia in hospitalised patients is pivotal, as thrombocytopenia may lead to complications in patients with a variety of conditions.9,10,11 in a previous study conducted in johannesburg, south africa in 2012, the authors reported on chemotherapy and sepsis as the common causes of thrombocytopenia, irrespective of hiv status.4 however, the prevalence and aetiology of thrombocytopenia in a tertiary or quaternary hospital setting remains unknown as data describing this remain scarce. the aim of this study was to determine the prevalence and clinical diagnosis associated with thrombocytopenia in patients presenting at inkosi albert luthuli central hospital in kwazulu-natal between october 2015 and april 2016. methods ethical considerations the study received ethical approval from the university of kwazulu-natal biomedical research and ethics committee (approval number: be 297/16). sample selection and data extraction this retrospective study was conducted at the department of haematology at inkosi albert luthuli central hospital (ialch), durban, kwazulu-natal, south africa. the department offers diagnostic haematology services to the entire province of kwazulu-natal. the ialch is an 846-bed referral hospital that provides tertiary level services to the entire kwazulu-natal province and parts of the eastern cape. data collection patients’ diagnostic test results and clinical notes were retrieved from the ialch trak laboratory data management system, version 6.10.56 (intersystems corporation, cambridge, massachusetts, united states). the extracted laboratory data items included the peripheral blood film report, diagnosis, platelet count, ward number and hospitalisation status. clinical data items were retrieved from meditech version 615 (meditech, westwood, massachusetts, united states). the extracted patient information included age, sex, type of ward, hospitalisation status and the cause of thrombocytopenia in these patients. a total of 2076 full blood count results were extracted from the trak database. these results were from samples received at ialch haematology laboratory on non-consecutive weekdays from october 2015 to april 2016. this was in an effort to minimise the inclusion of repeat samples in our analysis. all samples were analysed using the same sysmex xe5000 analyser (sysmex corporation, chuo-ku, kobe, japan). the study included patient reports that fulfilled the study inclusion criteria of a platelet count of < 100 × 109/l and had a corresponding peripheral blood smear report (figure 1). figure 1: study design. this study included full blood count reports retrieved from the department of haematology for patients presenting at the inkosi albert luthuli central hospital, durban, kwazulu-natal, south africa (october 2015 – april 2016). sample size determination in order to determine the prevalence of thrombocytopenia, we computed a minimum required sample size (n) of 304 based on the thrombocytopenia prevalence of 20% and an appropriate precision (d) of 0.05 and a 95% confidence interval width of 15.5% – 24.5%.13 furthermore, a sub-sample size of 170 was required to estimate the aetiology of thrombocytopenia with a 5% probability of error and assuming that 50% of cases are due to acquired factors. statistical analysis the kolmogorov-smirnov and lilliefors tests for normality were used to assess data distribution. parametric data such as platelet count and cluster of differentiation 4 t-cell counts were reported as mean and standard deviations. platelet counts were ordered into two categories (i.e. < 50 and 51–100). the fisher’s exact test was used for comparisons between ordinal data (age, platelet count, diagnosis and race). the prevalence of thrombocytopenia was reported as a proportion of patients with the disease. furthermore, associations between patient characteristics (age, sex and hospital ward) and aetiology of thrombocytopenia were assessed. a p-value of < 0.05 was regarded as statistically significant. all data analyses were performed using the stata version 13.1 statistical software (statacorp lp, college station, texas, united states). results a total of 2076 full blood count reports were retrieved from the ialch database. among these, 174 reports that met the inclusion criteria were included in the study (figure 1). the extracted participant data included age, gender, peripheral blood film report, diagnosis, platelet count, ward number and hospitalisation status. patient characteristics the study comprised 174 thrombocytopenia patients with a mean age of 24.4 ± 2.9 years. this consisted of patients receiving both inpatient and outpatient care (table 1). the hiv status of the thrombocytopenia patients was reported in 146 (83.9%) of the included cases, while it was not determined in 28 (16.1%) (table 1). thirty-one (21.2%) of the thrombocytopenia patients were hiv-positive with a mean cluster of differentiation 4 t-cell count of 261.3 ± 199.3 cells/µl. table 1: characteristics of patients presenting at the department of haematology inkosi albert luthuli central hospital, durban, kwazulu-natal, south africa (october 2015 – april 2016). prevalence and aetiology of moderate to severe thrombocytopenia the overall prevalence of thrombocytopenia was 14.9%. the paediatric population (0–12 years) had the highest prevalence of thrombocytopenia (37.4%) compared with those aged 13–24 years (21.8%), 25–36 years (14.9%), 37–48 years (9.8%), 49–60 years (9.8%) and > 60 years (6.3%) (table 1). in the overall cohort, the prevalence of thrombocytopenia was higher in female patients (51.1%) compared to male patients (48.9%), p < 0.001. thrombocytopenia was most common in inpatients (85.6%) compared to outpatients (14.4%), p < 0.001. the adult and paediatric haematology and oncology wards and clinic had the highest prevalence of thrombocytopenia (55%), whereas the adult and paediatric intensive care units (29.3%) and the non-haematology wards and clinic (15.5%) had a lower prevalence, p < 0.001. overall, severe thrombocytopenia (platelet count ≤ 50 × 109/l) was twofold higher (64.9%) than moderate thrombocytopenia (35.1%), p = 0.004 (table 2). table 2: aetiology of thrombocytopenia in patients presenting at the department of haematology inkosi albert luthuli central hospital, durban, kwazulu-natal, south africa (october 2015 – april 2016). chemotherapy-induced thrombocytopenia (38.5%) and sepsis (27.6%) accounted for the majority of thrombocytopenia cases (table 2). malignancies accounted for 11.5%, immune thrombocytopenic purpura 4.6% and aplastic anaemia 4.6%. aetiologies with a prevalence less than 4% were grouped into one category and these comprised trauma as well as pre-eclampsia, storage disorders, autoimmune disorders, hereditary thrombocytopenia and human leukocyte antigen antibodies to platelets, which accounted for between 0.6 and 1.7%. discussion in this study, we report a thrombocytopenia prevalence of 14.9%, which is higher than the 8.6% prevalence reported in a similar study conducted at an academic state hospital in johannesburg, south africa.4 this higher prevalence may be due to the fact that the current study was conducted at a tertiary and quaternary hospital, to which only specific cases are referred and this could result in an overestimation of the prevalence of thrombocytopenia. in addition, other studies have reported on seasonal and genetic variations in platelet counts.14 taken together these factors may account for the differences in the prevalence of thrombocytopenia. in fact, seasonal variations in platelet counts have been reported with lower platelet counts observed in spring and summer, while slightly elevated platelet counts have been observed in autumn and winter.14,15 notably our study period fell within a low thrombocytopenia season (summer and autumn); this may suggest that the reported platelet counts in patients with severe thrombocytopenia were not influenced by seasonal variations but may have led to an underestimation of thrombocytopenia in our setting. notably, only 17.8% of the patients included in our study were hiv-positive compared to 36% reported in the previous retrospective study by vaughan et al. reporting on patient full blood count reports authorised in 2012, at the chris hani baragwanath academic hospital, south africa.4 we further report on a higher frequency of thrombocytopenia in hospitalised patients compared to outpatients, which is similar to that previously described.4 this may be due to the differences in diagnosis and disease severity between admitted patients and outpatients. moreover, in our study thrombocytopenia was particularly common among patients admitted to haematology and oncology wards, intensive care units, neonatal intensive care units and medical wards, while a minority of thrombocytopenia cases (1.7%) were from non-haematology clinics. these findings are consistent with those previously reported. interestingly, in our study thrombocytopenia was common particularly among children below the age of 12 years and the majority of the cases were due to sepsis. more than a third of cases referred to the haematology department with thrombocytopenia were classified as chemotherapy-induced. the mechanism of thrombocytopenia in chemotherapy-induced thrombocytopenia involves reduced platelet production, an increased rate of platelet destruction and enhanced platelet clearance by immune mechanisms.7 bone marrow infiltration by malignancy can cause suppression of megakaryopoiesis, resulting in thrombocytopenia.1,7 in our study, malignancies accounted for more thrombocytopenia cases when compared to bone marrow failure. contrary to our findings, a study conducted in johannesburg, south africa in 2012 showed that bone marrow failure accounted for 9.6% of cases while malignancies accounted for 7.4% of thrombocytopenia cases.4 thrombocytopenia due to sepsis accounted for 27.6% of the cases, a majority of which were in the intensive care unit. the majority of cases presenting with sepsis had severe thrombocytopenia; these findings were similar to those previously described.16 studies have shown that the prevalence of thrombocytopenia in sepsis ranges from 33.8% to 60%.5,6,17 immune thrombocytopenic purpura, an isolated thrombocytopenia with no associated clinical conditions,18 was seen in 4.6% of the participants and most participants were between 13 and 24 years; the majority were in the obstetrics and labour ward (87.5%). in adults, immune thrombocytopenic purpura is a chronic disease resulting from an autoimmune disorder mediated by platelet antibodies, increased platelet destruction and impaired platelet production.18,19 contrary to literature, immune thrombocytopenic purpura was not common among the paediatric group (12.5%).19 neonatal thrombocytopenia commonly occurs as a result of increased platelet destruction or sequestration resulting from infections, respiratory distress syndrome or infants whose mothers had pre-eclampsia.20 thrombocytopenia can also be the result of decreased platelet production in congenital abnormalities of the newborn such as thrombocytopenia absent radii.20 thrombocytopenia can also be seen in children who are well and commonly as an isolated thrombocytopenia resulting from immune causes such as in infants born to mothers with immune thrombocytopenic purpura and in those with neonatal alloimmune thrombocytopenia. in our study, the prevalence of pregnancy-associated thrombocytopenia was 5.2%, which is lower in comparison to other studies.8,9,21 pregnancy-associated thrombocytopenia occurs in 6% – 10% of pregnant patients; however, its prevalence depends on its association with other medical conditions.22 the diagnosis of gestational thrombocytopenia is a diagnosis of exclusion and accounts for the majority of all cases of thrombocytopenia during pregnancy.23,24 limitations care should be taken in generalising the findings of the study because of the small numbers included in the study. the patient characteristics of those included may also differ from those seen at other levels of the healthcare system. other limitations encountered were a lack of family history and other medications patients were taking, as this would have allowed for the classification and consideration of familial and drug-induced thrombocytopenia. conclusion in our study, thrombocytopenia was common in hospitalised patients compared to outpatients. chemotherapy, sepsis and malignancies were the most common causes of thrombocytopenia. focused investigations are warranted for prompt patient management. knowledge of the prevalence and common aetiology of thrombocytopenia in a healthcare facility will assist clinicians in decision-making and interpretation of laboratory test results, leading to prompt and adequate patient management and potentially offsetting patient costs that may be incurred due to unwarranted laboratory investigations. further research in this field is required at different levels of healthcare as differences in the reported prevalence of thrombocytopenia may be the result of differences in patient demographics and clinical presentation of patients, which may differ between these facilities. acknowledgements the authors would like to thank the department of haematology, national health laboratory service laboratory, inkosi albert luthuli central hospital and inkosi albert luthuli central hospital for granting access to the patient database. competing interests the authors declare that there are no financial, personal or professional competing interests that may interfere with this work. authors’ contributions a.g.p.j. performed the experiments, designed the study and wrote the paper; b.b.n. contributed to the revision of the intellectual content. all authors gave their approval of the final version to be submitted for publication. sources of support b.b.n. is a university of kwazulu-natal developing research innovation, localisation and leadership in south africa fellow. developing research innovation, localisation and leadership in south africa is a united states national institutes of health d43 grant (d43tw010131) awarded to the university of kwazulu-natal in 2015 to support a research training and induction programme for early-career academics. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references stasi r. how to approach thrombocytopenia. hematology am soc hematol educ program. 2012;2012(1):191–197. https://doi.org/10.1182/asheducation-2012.1.191 gernsheimer t, james ah, stasi r. how i treat thrombocytopenia in pregnancy. blood. 2013;121(1):38–47. https://doi.org/10.1182/blood-2012-08-448944 zhang l, xu j, gao l, pan s. spurious thrombocytopenia in automated platelet count. lab med. 2018;49(2):130–133. https://doi.org/10.1093/labmed/lmx081 vaughan jl, fourie j, naidoo s, subramony n, wiggill t, alli n. prevalence and causes of thrombocytopenia in an academic state sector laboratory in soweto, johannesburg, south africa. s afr med j. 2015;105(3):215–219. 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fountain em, arepally gm. etiology and complications of thrombocytopenia in hospitalized medical patients. j thromb thrombolysis. 2017;43(4):429–436. https://doi.org/10.1007/s11239-016-1467-8 chaturvedi s, kohli r, mccrae k. over-testing for heparin induced thrombocytopenia in hospitalized patients. j thromb thrombolysis. 2015;40(1):12–16. https://doi.org/10.1007/s11239-014-1123-0 soff ga, shaw j, kilpatrick k, marongiu a, park j. burden of thrombocytopenia in adult cancer patients receiving chemotherapy. j clin oncol. 2019;37(15):1555–1555. https://ascopubs.org/doi/abs/10.1200/jco.2019.37.15_suppl.1555 louie ks, micallef jm, pimenta jm, forssen um. prevalence of thrombocytopenia among patients with chronic hepatitis c: a systematic review. j viral hepat. 2011;18(1):1–7. https://doi.org/10.1111/j.1365-2893.2010.01366.x buckley mf, james jw, brown de, et al. a novel approach to the assessment of variations in the human platelet count. thromb haemost. 2000;83(03):480–484. https://doi.org/10.1055/s-0037-1613840 liu b, taioli e. seasonal variations of complete blood count and inflammatory biomarkers in the us population-analysis of nhanes data. plos one. 2015;10(11):e0142382. https://doi.org/10.1371/journal.pone.0142382 venkata c, kashyap r, farmer jc, afessa b. thrombocytopenia in adult patients with sepsis: incidence, risk factors, and its association with clinical outcome. j intensive care. 2013;1(1):9. https://doi.org/10.1186/2052-0492-1-9 boechat tde o, silveira mf, faviere w, macedo gl. thrombocitopenia in sepsis: an important prognosis factor. rev bras ter intensiva. 2012;24(1):35–42. thota s, kistangari g, daw h, spiro t. immune thrombocytopenia in adults: an update. cleve clin j med. 2012;79(9):641–650. https://doi.org/10.3949/ccjm.79a.11027 grimaldi-bensouda l, nordon c, michel m, et al. immune thrombocytopenia in adults: a prospective cohort study of clinical features and predictors of outcome. haematologica. 2016;101(9):1039–1045. https://doi.org/10.3324/haematol.2016.146373 arora m, goyal l, khutan h. prevalence of thrombocytopenia during pregnancy and its effect on pregnancy and neonatal outcome. ann int med dent res. 2019;3(2):4. https://doi.org/10.4103/ijh.ijh_17_18 sebitloane hm. thrombocytopenia during pregnancy in women with hiv infection receiving no treatment. s afr med j. 2016;106(2):210–213. https://doi.org/10.7196/samj.2016.v106i2.9903 dwivedi p, puri m, nigam a, agarwal k. fetomaternal outcome in pregnancy with severe thrombocytopenia. eur rev med pharmacol sci. 2012;16(11):1563–1566. mccrae kr. thrombocytopenia in pregnancy: differential diagnosis, pathogenesis, and management. blood rev. 2003;17(1):7–14. https://doi.org/10.1016/s0268-960x(02)00056-5 ciobanu am, colibaba s, cimpoca b, peltecu g, panaitescu am. thrombocytopenia in pregnancy. maedica (buchar). 2016;11(1):55. abstract introduction methods results discussion acknowledgements references about the author(s) abera a. kitaba national clinical bacteriology and mycology reference laboratory, ethiopian public health institute, addis ababa, ethiopia zelalem t. bonger national clinical bacteriology and mycology reference laboratory, ethiopian public health institute, addis ababa, ethiopia degefu beyene national clinical bacteriology and mycology reference laboratory, ethiopian public health institute, addis ababa, ethiopia zeleke ayenew national clinical bacteriology and mycology reference laboratory, ethiopian public health institute, addis ababa, ethiopia estifanos tsige national clinical bacteriology and mycology reference laboratory, ethiopian public health institute, addis ababa, ethiopia tesfa addis kefale national clinical bacteriology and mycology reference laboratory, ethiopian public health institute, addis ababa, ethiopia yonas mekonnen national clinical bacteriology and mycology reference laboratory, ethiopian public health institute, addis ababa, ethiopia dejenie s. teklu national clinical bacteriology and mycology reference laboratory, ethiopian public health institute, addis ababa, ethiopia elias seyoum national clinical bacteriology and mycology reference laboratory, ethiopian public health institute, addis ababa, ethiopia abebe a. negeri national clinical bacteriology and mycology reference laboratory, ethiopian public health institute, addis ababa, ethiopia citation kitaba aa, bonger zt, beyene d, et al. antimicrobial resistance trends in clinical escherichia coli and klebsiella pneumoniae in ethiopia. afr j lab med. 2024;13(1), a2268. https://doi.org/10.4102/ajlm.v13i1.2268 original research antimicrobial resistance trends in clinical escherichia coli and klebsiella pneumoniae in ethiopia abera a. kitaba, zelalem t. bonger, degefu beyene, zeleke ayenew, estifanos tsige, tesfa addis kefale, yonas mekonnen, dejenie s. teklu, elias seyoum, abebe a. negeri received: 17 july 2023; accepted: 11 jan. 2024; published: 27 mar. 2024 copyright: © 2024. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: clinicians rely on local antimicrobial resistance pattern data to guide empiric treatment for seriously ill patients when culture and antimicrobial susceptibility testing results are not immediately available. objective: this study aimed to analyse 5-year trends in antimicrobial resistance profiles of escherichia coli and klebsiella pneumoniae isolates. methods: bacteriology reports from 2017 to 2021 at the ethiopian public health institute were analysed retrospectively. isolates were identified using either the vitek 2 compact system, the bd phoenix m50 instrument, or conventional biochemical tests. antimicrobial susceptibility testing was conducted using either the kirby-bauer disk diffusion method or the vitek 2 compact system and bd phoenix m50 systems available at the time of testing. the cochran armitage trend test was employed to test the significance of antimicrobial resistance trends over time. p-values less than 0.05 were considered statistically significant. results: of the 5382 bacteriology reports examined, 458 (9%) were on e. coli and 266 (5%) were on k. pneumoniae. both k. pneumoniae (88%) and e. coli (65%) demonstrated high resistance to extended-spectrum cephalosporins. however, both k. pneumoniae (14%) and e. coli (5%) showed lower rates of resistance to carbapenems compared to other antimicrobials. in k. pneumoniae, resistance to carbapenems (from 0% to 38%; p < 0.001) and ciprofloxacin (from 41% to 90%; p < 0.001) increased significantly between 2017 and 2021. conclusion: both organisms showed very high resistance to broad-spectrum antibiotics. additionally, k. pneumoniae demonstrated a statistically significant rise in ciprofloxacin and carbapenem resistance. what this study adds: this study emphasises the significance of regular reporting of local antimicrobial resistance patterns as this information can guide appropriate empiric therapy and efforts to address antimicrobial resistance issues. keywords: antimicrobial resistance; retrospective analysis; trend analysis; escherichia coli; klebsiella pneumoniae; ethiopian public health institute; ethiopia. introduction the emergence and spread of antimicrobial-resistant enterobacteriaceae, particularly escherichia coli and klebsiella pneumoniae, is a critical health problem and threatens the effective prevention and treatment of serious infections.1 antimicrobial-resistant e. coli and k. pneumoniae are responsible for a high proportion of serious nosocomial infections, including urinary tract, bloodstream, abdominal, and respiratory tract infections, and are a serious concern for global health and development.2,3 e. coli and k. pneumoniae develop resistance to beta-lactam antibiotics primarily by producing carbapenemase and extended-spectrum beta-lactamase enzymes.4,5,6,7 klebsiella pneumoniae tends to be significantly more resistant than e. coli, with carbapenem resistance rates exceeding 25% reported in several southern european countries.7 infections caused by carbapenem-resistant, extended-spectrum beta-lactamase-producing, and multidrug-resistant e. coli and k. pneumoniae are more difficult to treat and occur at higher frequencies.2,3,7 the united states centers for disease control and prevention has classified carbapenem-resistant e. coli and k. pneumoniae, as urgent threats and extended-spectrum beta-lactamase-producing e. coli and k. pneumoniae, as serious threats, whereas the world health organization classified them as critical priority pathogens requiring the development of new active antimicrobial agents.2,3 globally, several studies and antimicrobial resistance surveillance systems have reported increasing rates of antimicrobial resistance among e. coli and k. pneumoniae.8,9,10,11,12,13 for example, a 10-year retrospective study in china found that the rate of carbapenem-resistant k. pneumoniae increased significantly, from 6.7% in 2010 to 56.7% in 2019.12 similarly, a surveillance report from a hospital in malawi revealed a substantial increase in ciprofloxacin-resistant e. coli (from 0.0% in 1998 to 31.1% in 2016) and ciprofloxacin-resistant klebsiella species (from 1.7% in 1998 to 70.2% in 2016).14 several scientific reports out of africa have emphasised the fast-growing threat of antimicrobial resistance.8,11,14,15,16,17,18 the threat appears to be receiving more attention in the region than ever before, as evidenced by an increase in the number of publications on the subject, as well as the initiatives by the african society for laboratory medicine and the african centers for disease control and prevention.19 even though studies in ethiopia have reported a high burden of antimicrobial resistance among e. coli and k. pneumoniae,8,17,18 there is limited information on the trends of antimicrobial resistance among these pathogens in ethiopia. therefore, the current study was designed to analyse 5-year antimicrobial resistance trends among e. coli and k. pneumoniae recovered from specimens referred to the ethiopian public health institute. the findings of our study may provide local antimicrobial resistance patterns of e. coli and k. pneumoniae to clinicians to guide empiric treatment for seriously ill patients when culture and antimicrobial susceptibility testing results are not immediately available. moreover, the findings may provide information for initiatives aimed at combating the problem of antimicrobial resistance, including infection prevention and control practices and antimicrobial stewardship. methods ethical considerations due to the retrospective nature of this study, we were unable to obtain consent from the patients who provided specimens. therefore, a waiver of informed consent to conduct the study was requested and approved by the institutional review board of the ethiopian public health institute with approval number ephi-irb-413-2021. to maintain confidentiality, patient names and other personal identifiers were encrypted, and unique identification numbers were utilised to identify data. therefore, the study was conducted in accordance with helsinki declaration as revised in 2013. data collection this retrospective study included routine bacteriology culture reports of e. coli and k. pneumoniae isolates obtained from various clinical specimens at the ethiopian public health institute between january 2017 and december 2021. the laboratory provides diagnostic services as well as a range of research activities. it was accredited by the ethiopian accreditation service following the requirements of the international organization for standardization 15189:2012. isolate identification and antimicrobial susceptibility testing isolation and identification of the bacteria were achieved by culturing the specimens onto appropriate culture media and incubating them at 35 °c – 37 °c following laboratory standard operating procedures. the isolated bacteria were identified using one of the following laboratory methods available at the time of testing: vitek 2 compact system (biomérieux, marcy-l’étoile, france), bd phoenix m50 (becton, dickinson and company, franklin lakes, new jersey, united states), and standard biochemical tests. for standard biochemical tests, the following biochemical tests were used along with gram staining for isolate identification: triple sugar iron agar (oxoid ltd., basingstoke, hampshire, england), lysine iron agar (biomark, pune, maharashtra, india), sulfide indole motility (himedia, mumbai, maharashtra, india), simmons citrate agar (biomark, pune, maharashtra, india), urea agar (himedia, mumbai, maharashtra, india), and oxidase (liofilchem, roseto degli abruzzi, italy). antimicrobial susceptibility testing was carried out using either the kirby-bauer disk diffusion method on muller hinton agar (oxoid ltd., basingstoke, hampshire, england), the vitek 2 compact system, or the bd phoenix m50 system. the results of antimicrobial susceptibility tests (susceptible, intermediate, and resistant) were interpreted using the latest clinical and laboratory standards institute m100 criteria available at the time of testing.20 quality control for antimicrobial susceptibility testing and biochemical tests and culture was carried out as per the clinical and laboratory standards institute guidelines20,21, manufacturer instructions, and laboratory standard operating procedures using different american-type culture collection strains. e. coli american-type culture collection 25922, staphylococcus aureus american-type culture collection 25923, and pseudomonas aeruginosa american-type culture collection 27853 strains were used for quality control during antimicrobial susceptibility testing. grouping antimicrobial agents for trend analysis for trend analysis, we grouped related antimicrobials according to drug classes using the clinical and laboratory standards institute m02 guidelines.21 antimicrobial agents were categorised as extended-spectrum cephalosporins, aminoglycosides, firstand second-generation cephalosporins, carbapenems, β-lactam-combination agents, ciprofloxacin, and trimethoprim/sulfamethoxazole. resistance to at least one of the several agents within each antimicrobial class was used to define resistance at the antimicrobial class level. isolates were considered extended-spectrum cephalosporin-resistant if they were resistant to at least one of the extended-spectrum cephalosporins: ceftazidime, cefepime, and ceftriaxone. aminoglycoside-resistant isolates were those that were resistant to at least one of the following aminoglycosides: amikacin, tobramycin, and gentamycin. strains that were resistant to amoxicillin/clavulanic acid or piperacillin/tazobactam were deemed resistant to a β-lactam-combination agent. carbapenem resistance was defined as resistance to meropenem or imipenem. strains resistant to cefazolin or cefuroxime were considered resistant to firstand second-generation cephalosporins. data extraction the bacteriology records from the laboratory logbook were entered into whonet software (who collaborating centre for surveillance of antimicrobial resistance; https://whonet.org/software.html). the following information was obtained: type of specimen, final culture results, identity of the organism (for positive cultures), susceptibility testing results, date of specimen collection and receipt at the laboratory, and patients’ demographics (age and gender). to avoid bias from repetitive culture, only data on the first isolate from each patient were included. statistical analysis the whonet software was used to analyse resistance rates.22 for each species, the resistance rates were calculated by dividing the number of resistant isolates by the total number of isolates tested. this was calculated for each year, and the yearly trends over the 5 years were assessed. we also determined the distribution of pathogens according to the age and gender of patients, as well as specimen types and year of isolation. statistical analysis was performed using r software,23 and the cochran armitage trend test was employed to test the statistical significance of antimicrobial resistance trends over time. p-values less than 0.05 were considered statistically significant. results a total of 7199 clinical specimens were collected from 2017 to 2021 (figure 1). specimens other than blood, urine, and pus (n = 1745) were excluded from this study; isolates from stool, throat swabs, nasal swabs, and other specimens were not considered pathogens. additionally, specimens with missing information on patient age and gender (n = 72) were excluded from this study. in total, 5382 records of blood, pus, and urine specimens with complete information were obtained, from which 458 (9%) e. coli and 266 (5%) k. pneumoniae isolates were recovered. the combined prevalence of e. coli and k. pneumoniae was 13% (724/5382). figure 1: flow diagram of applied criteria for selection of bacteriology culture data for this study. ethiopian public health institute, addis ababa, ethiopia, january 2017 – december 2021. distribution of e. coli and k. pneumoniae by age, gender, and specimen type there were more male patients (n = 2890; 54%) than female patients (n = 2492; 46%) (table 1). the majority of the patients (n = 2459; 46%) were aged between 19 and 45 years. of the 5382 specimens, 42% were urine specimens, 40% were blood samples, and 18% were pus samples. out of the 724 isolates, 458 (63%) were e. coli, the majority (n = 356; 78%) of which were recovered from urine. the remaining 266 (37%) isolates were k. pneumoniae, of which 129 (50%) were recovered from blood specimens. about 82% of all e. coli and k. pneumoniae isolates were recovered from blood and urine specimens. e. coli (n = 261; 57%) was more prevalent among female patients, while k. pneumoniae (n = 153, 58%) was more common in male patients. table 1: distribution of escherichia coli and klebsiella pneumoniae by age, gender, and specimen types. ethiopian public health institute, addis ababa, ethiopia, january 2017 – december 2021. five-year resistance patterns of e. coli and k. pneumoniae isolates among the e. coli isolates, the highest rates of resistance were recorded against ampicillin (88%), piperacillin (84%), and tetracycline (80%), and the lowest resistance rates were recorded against amikacin (2%), meropenem (3%), and imipenem (5%) (figure 2). among the k. pneumoniae isolates, the highest resistance rates were observed against cefazolin (91%), cefuroxime (91%), ceftriaxone (92%), and piperacillin (97%), while the lowest resistance rates were recorded against nitrofurantoin (9%), amikacin (13%), meropenem (13%), and imipenem (17%). figure 2: resistance rates of escherichia coli and klebsiella pneumoniae to tested antimicrobials at the ethiopian public health institute, addis ababa, ethiopia, january 2017 – december 2021. antimicrobial resistance trends of e. coli despite the observed high rates of resistance to common antimicrobial agents among the e. coli isolates in this study, we found no statistically significant increasing trends in resistance to any of the tested antimicrobials (figure 3). the resistance rates of e. coli to ciprofloxacin ranged from 60% to 82% and from 71% to 80% for trimethoprim/sulfamethoxazole between 2017 and 2021. the proportion of ciprofloxacin-resistant e. coli was 70% in 2017 but fell to 68% in 2018 and 60% in 2019, before rising to 72% in 2020 and 80% in 2021. figure 3: antimicrobial resistance trends among escherichia coli at the ethiopian public health institute, addis ababa, ethiopia, january 2017 – december 2021. carbapenem resistance trends in e. coli fluctuated during the study period. in 2017, the proportion of carbapenem-resistant e. coli was 4%. this proportion rose to 10% in 2018, then fell to 2% in 2019 and 0% in 2020 before rising again to 9% in 2021. resistance rates to the extended-spectrum cephalosporins (range: 57% – 71%) and firstand second-generation cephalosporins (range: 54% – 72%) were continuously high throughout the study period. in 2017, the proportion of extended-spectrum cephalosporin-resistant e. coli was 71%. this proportion dropped slightly to 65% in 2018, 63% in 2019, and 57% in 2020 before rising again to 69% in 2021. for the aminoglycosides, the proportion of resistant e. coli isolates ranged from 23% to 31% between 2017 and 2021, while for the β-lactam combinations, the proportion of resistant e. coli isolates dropped from 41% to 20%. antimicrobial resistance trends of k. pneumoniae overall, k. pneumoniae exhibited high levels of antimicrobial resistance, with 88% of the isolates resistant to extended-spectrum cephalosporins, 51% resistant to aminoglycosides, 44% resistant to ciprofloxacin, and 14% resistant to carbapenems (figure 4). there was a statistically significant increase in ciprofloxacin resistance and carbapenem resistance rates among the k. pneumoniae isolates (p < 0.0001). over the 5 years, the proportion of carbapenem-resistant k. pneumoniae increased significantly from 0% in 2017 to 8% in 2018 and 2019, and from 19% in 2020 to 39% in 2021 (p < 0.001). from 2017 to 2018, the proportion of ciprofloxacin-resistant k. pneumoniae decreased from 41% to 24%. however, by 2019, this proportion increased to 39%, before surging to 71% in 2020 and 90% in 2021 (p < 0.001). over the first 3 years of the study period, the prevalence of trimethoprim/sulfamethoxazole-resistant k. pneumoniae ranged between 81% and 85%. however, this proportion rose to 100% in 2020 before decreasing to 91% in 2021. figure 4: antimicrobial resistance trends among klebsiella pneumoniae at the ethiopian public health institute, addis ababa, ethiopia, january 2017 – december 2021. among the k. pneumoniae isolates, resistance rates to the extended-spectrum cephalosporins remained above 80% throughout the study period. in 2017, 57% of k. pneumoniae isolates were resistant to aminoglycosides. however, the resistance rate decreased to 46% in 2018 and 42% in 2019, before rising to 71% in 2020 and 67% in 2021. the proportion of k. pneumoniae isolates that were resistant to the β-lactam combinations ranged from 33% to 79% between 2017 and 2021. in the first 2 years of the study period, the proportion of k. pneumoniae isolates resistant to the β-lactam combinations increased from 50% to 79%, before decreasing to 54% in 2019. subsequently, in both 2020 and 2021, the resistance levels dropped below 50%, reaching 33% in 2020 and 42% in 2021. discussion in this study, e. coli displayed high rates of resistance against ampicillin, piperacillin, and tetracycline, and low rates of resistance against amikacin and meropenem. k. pneumoniae exhibited high rates of resistance to cefazolin, cefuroxime, ceftazidime, trimethoprim-sulphamethoxazole, ceftriaxone, and piperacillin, and low rates of resistance against amikacin and meropenem. regarding the trends in resistance observed over 5 years, our findings indicate that there were no statistically significant increases in resistance to the tested antimicrobials among e. coli isolates. however, we did observe a statistically significant rise in the rates of ciprofloxacin and carbapenem resistance among the k. pneumoniae isolates. the ethiopian standard treatment guidelines for general hospitals provide comprehensive recommendations on the use of various antibiotics for empiric treatment.24 these guidelines consider the anatomical location and severity of different infections. for instance, in the case of sepsis, the guidelines recommend a combination of ampicillin and gentamicin, penicillin g, or a combination of an aminoglycoside or ciprofloxacin with ceftazidime.24 the guidelines suggest various medications, such as amoxicillin, cephalexin, ciprofloxacin, nitrofurantoin, ampicillin, gentamicin, trimethoprim-sulphamethoxazole, cefuroxime, ceftriaxone, and additional options, for the treatment of urinary tract infections.24 in the case of wound infections, the guidelines recommend piperacillin-tazobactam, ampicillin-sulbactam, cefazolin, amoxicillin-clavulanate, doxycycline, trimethoprim-sulphamethoxazole, and cefuroxime. these guidelines aim to ensure the effective and responsible use of antibiotics in ethiopian hospitals.24 regarding local prescribing practices, a study conducted in 2021 at dilchora referral hospital in ethiopia revealed that ciprofloxacin was the most frequently prescribed antibiotic for treating patients with urinary tract infections, followed by norfloxacin and amoxicillin/clavulanic acid.25 another study at ambo university referral hospital, ethiopia, from 2019, reported that ceftriaxone was the most commonly prescribed single antibiotic, accounting for 21.7% of prescriptions.26 in that study, the combination of ceftriaxone and azithromycin was the most popular choice for empiric treatment of community-acquired pneumonia, accounting for 50.7% of prescriptions. the authors also highlighted a non-adherence rate of 36.4% to the national guidelines for antibiotic use. furthermore, a study conducted at four governmental hospitals in eastern ethiopia in 2017 identified amoxicillin, ceftriaxone, and ciprofloxacin as the top three prescribed antibacterial drugs.27 lastly, research from addis ababa, ethiopia, from 2016, revealed that amoxicillin was the most frequently prescribed antibiotic, accounting for 44.8% of prescriptions, followed by ciprofloxacin at 13.6%, and trimethoprim-sulphamethoxazole at 11.2%.28 the observed high resistance patterns of both e. coli and k. pneumoniae isolates to commonly used antibiotics may be attributed to these factors. given that these pathogens have demonstrated high resistance to the antibiotics recommended in the ethiopian standard treatment guidelines, it is necessary to regularly revise the guidelines in accordance with local antimicrobial resistance levels. in this study, we observed a significant increase in carbapenem resistance rates among k. pneumoniae, reaching 38% by the end of 2021. in agreement with our findings, china’s antimicrobial surveillance network reported a significant rise in the prevalence of meropenem-resistant k. pneumoniae from 2.9% in 2005 to 26.3% in 2018 (p < 0.001).29 furthermore, a 20-year (1997–2016) report from the sentry antimicrobial surveillance program revealed a significant increase in carbapenem resistance among k. pneumoniae, rising from 0.7% in 1997 to 14.2% in 2016 in europe (p < 0.001).30 this increase in carbapenem resistance might be explained by poor hospital infection control and prevention practices and inappropriate prescription practices.31 inappropriate prescription practices, in turn, may be related to the scarcity of bacteriology laboratories capable of promptly detecting resistant bacteria and providing antibiograms to clinicians in health facilities in developing countries.31 in contrast to our findings, however, earlier studies at 14 new york city hospitals reported a decline in carbapenem-resistant k. pneumoniae at 6 of the 14 hospitals (carbapenem-resistant k. pneumoniae decreased from 38% in 2006 to 29% in 2009; p < 0.001).32 another study from the united states reported that the occurrence of carbapenem-resistant k. pneumoniae isolates declined significantly in a public health system in new york, united states, from 2016 to 2020, but increased between january 2021 and june 2022.33 these differences in observed trends could be attributed to strong infection control and prevention efforts done to decrease and prevent carbapenem-resistant k. pneumoniae infections in both hospital and community settings in new york city.32 ciprofloxacin resistance rates increased significantly among k. pneumoniae from 2017 (41%) to 2021 (90%), consistent with findings of a study conducted in sichuan, china, from 2017 to 2020 that reported increasing resistance of k. pneumoniae to ciprofloxacin from 14.7% in 2017 to 26.5% in 2020.34 similarly, the sentry antimicrobial surveillance program reported increasing rates of ciprofloxacin resistance among k. pneumoniae from 7.3% in 1997 to 27.9% in 2016 in europe.30 furthermore, the taiwan surveillance of antimicrobial resistance programme (2002–2012)35 reported a significant decrease in the proportion of ciprofloxacin-susceptible k. pneumoniae (average of 89.9% from 2002 to 2006 to an average of 81.6% from 2008 to 2012). conversely, however, the korean antimicrobial resistance monitoring system reported an almost constant trend for ciprofloxacin-resistant k. pneumoniae from 2013 to 2015.13 geographical variations may explain these observed differences. high resistance rates to both first-line and last-resort antibiotics were observed in our study among e. coli and k. pneumoniae isolates. overall, the rates of resistance to trimethoprim/sulfamethoxazole (e. coli 75% and k. pneumoniae 85%), ciprofloxacin (e. coli 69% and k. pneumoniae 44%), the first-generation to fourth-generation cephalosporins (e. coli 65% and k. pneumoniae 88%), and aminoglycosides (e. coli 28% and k. pneumoniae 51%) among both species during the 5-year study period were very high. this is consistent with the findings of previous studies in ethiopia,8,17,18 kenya,36 and uganda.37 these high rates of resistance could be related to the high prevalence of irrational antibiotic use and self-prescription practices in ethiopia.38,39,40,41,42 as a result, these antimicrobials may no longer be considered effective treatment options for infections caused by e. coli and k. pneumoniae. this, in turn, underlines the importance of developing bacteriology laboratory capacity in healthcare facilities to ensure that isolate identification and antimicrobial susceptibility testing results are made available for clinicians. furthermore, antimicrobial stewardship programmes are required to monitor and regulate antibiotic use. our findings are inconsistent with those of a retrospective analysis of data from a national surveillance network in switzerland over 8 years (2009–2016) that showed lower resistance among e. coli and k. pneumoniae to commonly used antibiotics such as thirdand fourth-generation cephalosporins (e. coli < 6%, k. pneumoniae < 5%), ciprofloxacin (e. coli > 14%, k. pneumoniae > 12%), and trimethoprim/sulfamethoxazole (e. coli < 23%, k. pneumoniae < 11%).43 this may be explained by the low use of antibiotics in both community and hospital settings in switzerland.43 limitations the lack of molecular identification for k. pneumoniae could have led to the misidentification of various species within the k. pneumoniae species complex. due to the retrospective nature of this study, we were unable to confirm resistance genes using polymerase chain reaction. this would have provided valuable additional information regarding the presence or absence of specific resistance genes in the organisms studied, enhanced our understanding of the mechanisms underlying antibiotic resistance, and potentially allowed a more comprehensive analysis of the genetic determinants contributing to resistance patterns. conclusion based on the findings of this study, both k. pneumoniae and e. coli showed very high resistance to both first-line and last-resort antibiotics recommended in the ethiopian standard treatment guidelines. however, the antimicrobial resistance trends for the majority of the antimicrobial agents fluctuated throughout the study period. furthermore, k. pneumoniae showed a statistically significant increasing trend of resistance to carbapenems and ciprofloxacin. this is concerning, since it may compromise the treatment of critically ill patients. this necessitates continued infection control efforts, together with diagnostic and antimicrobial stewardship programmes in healthcare facilities. additionally, it is necessary to regularly revise the guidelines in accordance with local antimicrobial resistance levels. acknowledgements we would like to thank the ethiopian public health institute for allowing us to extract antimicrobial resistance data and conduct this research. we also extend our appreciation to ms mulushewa g/egziabeher for her contribution in preparing the required culture media for this study. in addition,we would also express our gratitude ms shishig masresha and tigist basheda for their valuable assistance in cleaning and sterilising the required petri dishes and other equipment essential for the study. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions a.a.k. played a substantial role in the study conception, whonet software configuration, data curation and analysis, writing the original draft, review and editing. z.t.b. participated in study whonet software configuration and data curation and analysis. d.b., z.a., e.t., t.a.k., d.s.t., y.m., and e.s. participated in writing the original draft, review and editing. a.a.n. participated in supervising and writing, reviewing and editing. all authors read and approved the final manuscript. sources of support the authors received no financial support for the research, authorship, and/or publication of this article. data availability raw data were extracted from the ethiopian public health institute antimicrobial resistance surveillance data storage. raw data are available upon reasonable request from the corresponding author, a.a.k. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. it does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the authors are responsible for this 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improve surveillance delay emergence limit transmission mitigate harm enablers of africa centres for disease control and prevention’s framework implementation of africa centres for disease control and prevention’s framework acknowledgements references about the author(s) jay k. varma africa centres for disease control and prevention, addis ababa, ethiopia john oppong-otoo african union, inter-african bureau for animal resources, nairobi, kenya pascale ondoa african society for laboratory medicine, addis ababa, ethiopia olga perovic national institute for communicable diseases, johannesburg, south africa university of witwatersrand, johannesburg, south africa benjamin j. park united states centers for disease control and prevention, atlanta, georgia, united states ramanan laxminarayan center for disease dynamics, economics and policy, washington, d.c., united states princeton university, princeton, new jersey, united states rosanna w. peeling london school of tropical medicine and hygiene, london, england, united kingdom constance schultsz amsterdam institute for global health and development, amsterdam, the netherlands han li china center for disease control and prevention, beijing, china china pla institute for disease control and prevention, beijing, china chikwe ihekweazu nigeria center for disease control, abuja, nigeria amadou a. sall institut pasteur, dakar, senegal baboucarr jaw african union, inter-african bureau for animal resources, nairobi, kenya john n. nkengasong africa centres for disease control and prevention, addis ababa, ethiopia citation varma jk, oppong-otoo j, ondoa p, et al. africa centres for disease control and prevention’s framework for antimicrobial resistance control in africa. afr j lab med. 2018;7(2), a830. https://doi.org/10.4102/ajlm.v7i2.830 opinion paper africa centres for disease control and prevention’s framework for antimicrobial resistance control in africa jay k. varma, john oppong-otoo, pascale ondoa, olga perovic, benjamin j. park, ramanan laxminarayan, rosanna w. peeling, constance schultsz, han li, chikwe ihekweazu, amadou a. sall, baboucarr jaw, john n. nkengasong received: 09 may 2018; accepted: 26 july 2018; published: 06 dec. 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. antimicrobial resistant (amr) organisms are increasing globally, threatening to render existing treatments ineffective against many infectious diseases.1,2 amr strains of bacteria, fungi, parasites, and viruses prolong illness, increase case fatality, facilitate transmission, and increase treatment costs.3,4 in africa where many health systems are weak, the likelihood of amr increasing and the consequences of amr infections are particularly high, and drug resistance has already been documented for hiv and the pathogens that cause malaria, tuberculosis, typhoid, cholera, meningitis, gonorrhoea, and dysentery.5 patients in these countries have limited access to accurate diagnosis and adequate antimicrobial treatment, which can lead to sepsis and other life-threatening complications.6,7 many factors contribute to the emergence, persistence, and transmission of amr. although amr strains arise naturally due to genetic changes in replicating microorganisms, their emergence is accelerated by inappropriate use of antimicrobial agents in humans, animals, and the environment.8 amr emergence may be amplified by substandard or counterfeit antimicrobials, which impair treatment of existing infections and may select for amr strains.9 transmission of amr is accelerated by inadequate infection prevention and control in health care facilities, by contamination of the food supply with amr bacteria, by impaired access to potable water, and by limitations in public health prevention programmes, including immunisation, sanitation, and sexual health.10 globally, drug resistance causes an estimated 700 000 deaths each year, and if current trends continue, amr could result in over 10 million deaths per year and over $100 trillion (united states dollars) in lost output globally by 2050.11 recognising the urgent need for action, the world health assembly adopted the global action plan on antimicrobial resistance in may 2015, and the world health organization established the global antimicrobial resistance surveillance system.12 in 2016, african countries committed to address amr as part of the united nations general assembly special meeting and, in june 2017, committed to accelerate implementation of the international health regulations and promote africa’s health security, of which amr surveillance and control is a core component.13,14 to implement these commitments, the africa centres for disease control and prevention (africa cdc) officially launched its ‘framework for antimicrobial resistance control, 2018–2023’ in october 2017.15 established in january 2017, africa cdc is a ‘specialised technical institution’ of the african union (au) responsible for strengthening capacity for surveillance and disease intelligence, emergency preparedness and response, laboratory systems, information systems, and public health research and workforce development in africa.16 africa cdc developed its framework with four principles in mind. firstly, the strategy should help advance the global action plan. secondly, while more information is needed about incidence, prevalence, and interventions, africa cdc should follow the ‘precautionary principle’ and take action against resistance now. thirdly, policy and practice are as important as diagnostic or treatment technologies in mitigating amr. finally, africa cdc has an opportunity to leverage its unique position in the au to raise awareness, secure commitments, and influence policy at the head of state level. after consultation with subject matter experts in africa and globally, africa cdc developed a framework that has four objectives: (1) improve surveillance of amr organisms among humans and animals, (2) delay emergence of amr, (3) limit transmission of amr and (4) mitigate harm among patients infected with amr organisms. table 1 lists the high priority activities africa cdc will pursue for each of these areas. table 1: priority areas for africa centres for disease control and prevention framework for antimicrobial resistance control, 2018–2023. improve surveillance understanding the full extent of amr and its impact in africa is challenged by a lack of continent-wide amr surveillance data, especially for pathogens that require complex testing methods.3 although gains have been made in collecting data on resistance to some pathogens, such as hiv, mycobacterium tuberculosis, and plasmodium spp., several challenges remain, including inadequate demand by clinicians for diagnostic testing, laboratory infrastructure, resources to continuously collect, transport, and test specimens for amr surveillance, use of standardised protocols, quality assurance, systematic surveillance of amr in animals and their products in africa, and collaboration between the human and animal health sectors.17,18 africa cdc’s framework identified several areas in which it can strengthen laboratory capacity for surveillance in humans and animals. delay emergence reducing the need for and inappropriate use of antimicrobials can help delay emergence of amr. in human health, extensive studies have been conducted in the united states, europe, and other high-income settings on approaches to promote prudent antimicrobial prescribing – an approach known as antimicrobial stewardship.19 success has been achieved when stewardship programmes include structural changes and behavioral modification techniques.20,21,22 economic incentives for prescribing vary by country but can be particularly problematic when physicians or health care facilities rely on antimicrobial sales to fund operations. in many settings, persons obtain antimicrobials directly from pharmacies with no evaluation by or prescription from a physician.23 additionally, substandard and counterfeit antimicrobials are widely available in africa; substandard antimicrobials can promote amr by containing levels of an agent sufficient to exert selective pressure for resistant organisms but insufficient levels to kill those organisms.9 there are limited studies about the effectiveness or essential components of antimicrobial stewardship programmes in africa and other resource-limited settings, particularly involving the engagement of non-physician prescribers.24 in animals, antimicrobials are used for prophylaxis, treatment, and growth promotion, and it is reasonably assumed that most antimicrobial consumption occurs during food production, not through prescriptions to treat illness. prudent use in these settings is a critical risk management approach to delaying emergence, but is challenging, because food producers have a strong economic incentive to use antimicrobials and weak incentive to use them prudently.25,26 the au has made a high-level commitment to food safety, engaging partners from multiple sectors, including the industry. africa cdc can leverage this commitment by making policymakers more aware of the link between food production, amr emergence, and food-borne illness and by promoting prudent antimicrobial use in collaboration with animal health, agriculture, and other related authorities. limit transmission transmission of amr occurs frequently in health care facilities.27 such transmission can have severe consequences, because pathogens in health care facilities are more likely to be multi-drug resistant. hospitalised people are more susceptible to these severe illness, and these pathogens can also be spread outside of the hospital. the basic components of all programmes include: strong political commitment and dedicated resources for infection control, strict adherence to protocols for hand hygiene and for identification, isolation, and management of potentially infectious patients, adequate supplies and equipment for patient care, systems for infectious waste management, building design, and maintenance to reduce transmission, and continuous monitoring of process, outcome, and impact indicators.28 the west africa ebola response demonstrated that effective hospital-based infection prevention and control programmes require intensive support to initiate and sustain. africa cdc’s framework, therefore focuses primarily on advocacy, policy, and guidance to mobilise sufficient resources for infection prevention and control. in animal health, implementation of risk-based food safety systems – such as hazard analysis of critical control points, good handling practices, and good agricultural practices – can reduce the flow of amr pathogens in food to humans, especially when combined with proper nutrition to reduce the need for antimicrobials.29 mitigate harm strong antimicrobial stewardship programmes promote adherence to clinical treatment guidelines, helping delay emergence of amr and improve outcomes among patients already infected with amr organisms. diagnostic stewardship will also be essential to guide such treatment.30 substandard diagnostic tests and laboratory supplies remain common problems across africa and can lead to amr when tests lead to unnecessary antibiotic treatment. limiting their availability will be challenging, given the large number of health care delivery sites that may be using such products and the insufficient supply of quality diagnostic tests. a major ethical dilemma for africa cdc’s framework will be to balance antibiotic access versus excess.31 although antibiotic usage is excessive at a population level, many vulnerable groups lack access to effective antimicrobial treatment. africa cdc’s framework focuses on promoting antimicrobial and diagnostic stewardship, while seeking ways to maintain access to life-saving diagnostics and treatments. enablers of africa centres for disease control and prevention’s framework laws and policies play a critical role in framing, enabling, and protecting public health. africa cdc will leverage its stature and authority as a specialised agency of the au to advocate for laws and policies to monitor, prevent, and mitigate amr. such policy initiatives, however, can only succeed with robust involvement of civil society. to date, engagement of civil society for amr has been challenging, because the science can be complex to explain, the threat often characterised as distant, patients’ stories of illness and death often not told because of under-diagnosis and because they occur in marginalised groups, interventions not readily distilled into high-impact slogans, and public health agencies investing too little in civil society engagement. to effectively control amr, each country will need leadership and staff that have the education and skills to implement amr control programmes in both human and animal health sectors. the most pronounced human resources gaps are likely in laboratory services – from bench microbiologists to managers – and in health care infection prevention and control. africa cdc and partners are working together on a public health workforce strategy for the continent and mobilising resources to support hiring, training, and retaining of staff. by highlighting the urgency and severity of the amr threat, africa cdc will advocate for both donors and government ministries to allocate more funding to laboratory services and infection prevention and control personnel. implementation of africa centres for disease control and prevention’s framework from 4–5 april 2018, africa cdc convened amr focal points from african countries and partners to review the africa cdc amr framework and prioritise activities for the next year. one major conclusion of the meeting was that africa cdc should establish a formal structure to coordinate amr activities within the au, particularly with the au’s animal health agency (the inter-african bureau for animal resources), across member states, and with multilateral and non-governmental partners in africa. participants proposed that this entity’s first task should be to transform the africa cdc amr framework into an au-wide framework and ensure that it more fully addresses amr issues in animal and environmental health. in addition to governance and coordination, participants proposed that africa cdc focus activities in the next year on developing and harmonising tools to map and assess human and animal diagnostic laboratory capacity for amr, developing guidelines for prudent antimicrobial use in humans and in animals that reflect what is currently known about amr in africa, and developing minimum standards for infection prevention and control in health care facilities to limit amr transmission. conclusion africa cdc’s framework for amr is broad and aspirational, reflecting the urgency and complexity of this public health problem and the potential for africa cdc as an institution to help galvanise action and provide guidance to member states. progress in implementing this framework will require close coordination across and high-level commitment from member states and partners in africa. acknowledgements the conclusions, findings and opinions do not necessarily reflect the official position of the united states centers for disease control and prevention or the authors’ affiliated institutions. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support none. authors’ contributions j.k.v. drafted the manuscript. all authors contributed to the design and analysis of this project and all authors reviewed, approved and substantively revised the manuscript. references world bank. drug-resistant infections: a threat to our economic future [homepage on the internet]. washington, dc; 2016. available from: http://pubdocs.worldbank.org/en/689381474641399486/1701381-amr-lab-report-web.pdf laxminarayan r, duse a, wattal c, et al. antibiotic resistance-the need for global solutions. lancet infect dis. 2013;13:1057–1098. https://doi.org/10.1016/s1473-3099(13)70318-9 ndihokubwayo jb, yahaya aa, desta at, et al. antimicrobial resistance in the african region: issues, challenges and actions proposed. key determinants for health in the african region. african health monitor. 2013;16. world health organization. drug resistant tuberculosis is now at record levels [homepage on the internet]. geneva; 2010. available from: http://www.who.int/mediacentre/news/releases/2010/drug_resistant_tb_20100318/en/ essack sy, desta at, abotsi re, agoba ee. antimicrobial resistance in the who african region: current status and roadmap for action. j pub health. 2016;39:8–13. https://doi.org/10.1093/pubmed/fdw015 le doare k, bielicki j, heath pt, sharland m. systematic review of antibiotic resistance rates among gram-negative bacteria in children with sepsis in resource-limited countries. j pediatric infect dis soc. 2015;4:11–20. https://doi.org/10.1093/jpids/piu014 musicha p, cornick je, bar-zeev n, et al. trends in antimicrobial resistance in bloodstream infection isolates at a large urban hospital in malawi (1998–2016): a surveillance study. lancet infect dis. 2017;17(10):1042–1052. https://doi.org/10.1016/s1473–3099(17)30394-8 levy sb, marshall b. antibacterial resistance worldwide: causes, challenges and responses. nat med. 2004;10:s122–s129. https://doi.org/10.1038/nm1145 newton pn, green md, fernández fm, day np, white nj. counterfeit anti-infective drugs. lancet infect dis. 2006;6:602–613. https://doi.org/10.1016/s1473-3099(06)70581-3 okeke in, lamikanra a, edelman r. socioeconomic and behavioral factors leading to acquired 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bjerrum l. antimicrobial resistance: risk associated with antibiotic overuse and initiatives to reduce the problem. ther adv drug saf. 2014;5(6):229–241. https://doi.org/10.1177/2042098614554919 arnold sr, straus se. interventions to improve antibiotic prescribing practices in ambulatory care. cochrane database syst rev. 2005;(4):cd003539. https://doi.org/10.1002/14651858.cd003539.pub2 davey p, marwick ca, scott cl, et al. interventions to improve antibiotic prescribing practices for hospital inpatients. cochrane database syst rev. 2017;(2):cd003543. https://doi.org/10.1002/14651858.cd003543.pub4 hulscher me, grol rp, van der meer jw. antibiotic prescribing in hospitals: a social and behavioural scientific approach. lancet infect dis. 2010;10:167–175. https://doi.org/10.1016/s1473-3099(10)70027-x morgan dj, okeke in, laxminarayan r, et al. non-prescription antimicrobial use worldwide: a systematic review. lancet infect dis. 2011;11:692–701. https://doi.org/10.1016/s1473-3099(11)70054-8 cox ja, vlieghe e, mendelson m, et al. antibiotic stewardship in low-and middle-income countries: ‘same, but different’? clin microbiol infect. 2017;23(11):812–818. https://doi.org/10.1016/s1198-743x(17)30365-8 world animal health organization (oie). terrestrial animal health code – chapter 6.7. in: harmonization of national antimicrobial resistance surveillance and monitoring programmes [homepage on the internet]. 2017. available from: http://www.oie.int/fileadmin/home/eng/health_standards/tahc/current/chapitre_antibio_harmonisation.pdf landers tf, cohen b, wittum te, larson el. a review of antibiotic use in food animals: perspective, policy, and potential. public health rep. 2012;127:4–22. https://doi.org/10.1177/003335491212700103 weinstein ra. controlling antimicrobial resistance in hospitals: infection control and use of antibiotics. emerg infect dis. 2001;7:188–192. https://doi.org/10.3201/eid0702.010206 world health organization. guidelines on core components of infection prevention and control [homepage on the internet]. 2016. available from: http://www.who.int/gpsc/ipc-components/en/ food animal organization. impact of animal nutrition on animal welfare [homepage on the internet]. 2011. available from: www.fao.org/3/a-i3148e.pdf morgan dj, malani p, diekema dj. diagnostic stewardship – leveraging the laboratory to improve antimicrobial use. j am med assoc. 2017;318:607–608. https://doi.org/10.1001/jama.2017.8531 das p, horton r. antibiotics: achieving the balance between access and excess. lancet. 2016;387(10014):102–104. https://doi.org/10.1016/s0140-6736(15)00729-1 http://www.ajlmonline.org open access page 1 of 1 reviewer acknowledgement acknowledgement to reviewers in an effort to facilitate the selection of appropriate peer reviewers for the african journal of laboratory medicine, we ask that you take a moment to update your electronic portfolio on https://ajlmonline.org for our files, allowing us better access to your areas of interest and 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maruta patrick mateta genevieve mezoh james miller a.h. mohamed meade morgan fausta s. mosha jacob s. mwebi prisha naidoo kameko nichols patrick njukeng johannes nossent ifeanyi nsofor jacinta nwogu roseangela nwuba stephen obaro celestina obiekea michael ofori olusegun s. ojo jude n. okoyeh ayobami olaniyi gisele oliveira geoffrey omuse cyprian onyeji jose ortiz de la rosa philip o. oshun judith owen maria pardos de la gándara dimitri poddighe nira pollock raymond pranata jackson a. roberts vincent rotimi celal satici nadeem shaikh andre st. aubyn bowers yogesh kumar swami vu thi thom ralph timperi oyewale tomori cristina touriño noemi r. tousignant lara vojnov larry westerman p. yagupsky http://www.ajlmonline.org� https://ajlmonline.org https://ajlmonline.org https://ajlmonline.org https://ajlmonline.org/index.php/ajlm/user https://ajlmonline.org/index.php/ajlm/user mailto:publishing@aosis.co.za abstract background methodology results discussion acknowledgements references about the author(s) medard beyanga department of clinical laboratory services, bugando medical center, mwanza, tanzania lisa gerwing-adima department of clinical laboratory services, bugando medical center, mwanza, tanzania kahima jackson department of clinical laboratory services, bugando medical center, mwanza, tanzania benjamin majaliwa department of clinical laboratory services, bugando medical center, mwanza, tanzania henrico shimba department of clinical laboratory services, bugando medical center, mwanza, tanzania simon ezekiel department of clinical laboratory services, bugando medical center, mwanza, tanzania charles massambu tanzania ministry of health community development, gender, elderly and children, dodoma, tanzania dickson majige tanzania ministry of health community development, gender, elderly and children, dodoma, tanzania michael mwasegaka us centers for disease control and prevention, dar es salaam, tanzania wilson mtotela clinical and laboratory standards institute, wayne, new jersey, united states patrick mateta clinical and laboratory standards institute, wayne, new jersey, united states christa kasang medical mission institute, wuerzburg, bavaria, germany citation beyanga m, gerwing-adima l, jackson k, et al. implementation of the laboratory quality management system (iso 15189): experience from bugando medical centre clinical laboratory – mwanza, tanzania. afr j lab med. 2018;7(1), a657. https://doi.org/10.4102/ajlm.v7i1.657 lessons from the field implementation of the laboratory quality management system (iso 15189): experience from bugando medical centre clinical laboratory – mwanza, tanzania medard beyanga, lisa gerwing-adima, kahima jackson, benjamin majaliwa, henrico shimba, simon ezekiel, charles massambu, dickson majige, michael mwasegaka, wilson mtotela, patrick mateta, christa kasang received: 21 june 2017; accepted: 13 feb. 2018; published: 31 july 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: use of laboratory evidence-based patient health care in tanzania remains a complex problem, as with many other countries in sub-saharan africa. as at 2010, 39 african countries, including tanzania, had no clinical laboratories that met the minimum requirements for international laboratory standards (international organization for standardization [iso] 15189). objective: the aim of this article is to share experience from bugando medical centre laboratory’s milestones in reaching iso 15189 accreditation. methods: mentors to address the laboratory management and technical requirements performed a gap analysis using the southern african development community accreditation system checklist. several non-conformances were detected. system and technical procedures were developed, approved and communicated. quality indicators were established to measure laboratory improvement and to identify issues which require immediate and preventive actions. results: the departments’ external quality assessment performance increased after iso 15189 implementation (e.g. parasitology from 45% to 100%, molecular biology from no records to 100%, biochemistry 50% to 95%, tuberculosis microscopy 60% to 100%, and microbiology from 48.1% to 100%). there was a reduction in complaints, from eight to two per week. rejected samples were reduced from 7.2% to 1.2%. turn-around time was not recorded before implementation but reached 92% (1644/1786) of the defined targets, and the proportion of contamination in blood cultures decreased from 16% to 4%. conclusion: our experience suggests that the implementation of a quality management system is possible in resource-limited countries like tanzania. mentorship is necessary and should be done by professional laboratory mentors trained in quality management systems. financial resources and motivated staff are key to achieving iso 15189 accreditation. background the use of laboratory evidence-based patient healthcare in tanzania remains a complex problem, as in many other countries in sub-saharan africa.1 clinicians often believe that laboratory tests are additional health costs, because diagnosis and treatment are often done using empirical clinical judgement. laboratory results are perceived to be unreliable, especially when they are found to be discordant with clinical indications.2 it has been emphasised that healthcare professionals should make clinical decisions based on the best available evidence.3 access to unreliable diagnostic services and misdiagnosis causes confusion during patient management, and can result in unnecessary expenditure and in some cases death.4 there are recent global calls to provide more resources for the diagnosis, treatment and prevention of infectious diseases affecting the african population. in contrast, clinicians, financial controllers and public policy makers in resource-limited settings may be unaware of the importance of a laboratory-proven diagnosis for appropriate disease management.4 there is sufficient evidence that laboratory services are essential for guiding patient care. however, their central role has been neglected in resource-limited countries for decades.5 laboratory results should guide about 70% of clinical decisions and promote excellence in providing the best patient care. provision of accurate and reliable laboratory test results is only possible if the laboratory meets a minimum standard that provides credibility of the reported test results.6 until 2010 there were 380 laboratories which met international standards for quality in africa.7 tanzania, along with 38 of the 49 african countries assessed, had no clinical laboratories that met the minimum requirements for international quality standards to ensure reliability of reported patient results and competence of staff.7 the maputo declaration of 2008 pointed out that strengthening of laboratory systems requires collaboration between laboratories, governments and supporting partners.8 this collaboration can only be met by creating and implementing individual country plans and strategies which utilise the laboratory for diagnosis of diseases.9 before the maputo declaration, the ministry of health community development, gender, elderly and children, in collaboration with the clinical & laboratory standards institute (clsi), and with support from the president’s emergency plan for aids relief, had been working together to implement quality management systems (qms) in five zonal referral hospital laboratories. bugando medical centre laboratory was among the five referral hospital laboratories.10,11 the aim of this article is to share experience from the bugando medical centre laboratory’s journey to international organization for standardization (iso) 15189 accreditation. the experience will inform most laboratories currently implementing qms to refamiliarise themselves with the actual steps required before inviting assessors to assess laboratories and give recommendations for iso accreditation. methodology ethical considerations we used data from routine laboratory qms operations. no patient information was used; thus there was no ethical review required for this article. gap analysis the laboratory gap analysis was done for both management and technical areas of the laboratory to establish baseline data. the approach was to evaluate current laboratory practices and compare it with laboratory standards as stipulated by iso. the gap analysis revealed numerous non-conformances after comparing actual performance with the standard. the gaps identified included lack of knowledge about the laboratory standard, insufficient technical skills among staff and the use of unverified technical methods. other gaps were: absence of established policies and technical procedures, samples tested by staff who were deemed incompetent, and lack of staff training and supervision to carry out a test (see box 1). box 1: list of gaps that were identified during the gap analysis. implementation of the quality management system implementation of the qms started with management: the hospital management appointed the laboratory director, laboratory manager and quality manager, whereafter the laboratory manager appointed section heads to form the laboratory management team as recommended by iso. the laboratory organisational structure was set primarily to create lines of authority, reporting and communication, and to avoid overlapping of tasks during the qms implementation process. the laboratory manager and quality manager attended several qms training sessions which were organised by the ministry of health community development, gender, elderly and children in collaboration with clsi. these trainings were followed by awareness meetings that involved hospital management to make sure that they understood the processes and to ensure the availability of the resources required to enable the qms implementation process. hospital management became supportive after gaining a clear understanding of the advantages that a proper and functional qms offers to both patients and the hospital itself. laboratory mentorship and document development a laboratory mentor and an advisor were identified to guide laboratory management during the development of laboratory policies and procedures. the mentor was an employee of clsi, the organisation that was supporting the system implementation. he was a laboratory professional who had a master’s degree in business administration, and was trained in qms mentorship and laboratory audits. the advisor was a biomedical laboratory scientist with a master’s degree in public health and infectious disease. available laboratory policies and procedures were formalised; those from outside the laboratory were adapted and contextualised to the laboratory setting to form the laboratory manual. the established policy manual formed the basis of other procedures, including the laboratory’s qms procedures. the laboratory system procedures (box 2) were developed to cover all quality system essentials, as directed by iso,12 while documentation of technical procedures provided standardisation of all laboratory tests. developed documents were approved by the laboratory director and communicated to all laboratory staff. box 2: laboratory quality management procedures. apart from implementation of laboratory quality essentials (all processes in the laboratory workflow that provide the building blocks of quality, e.g. document control),13 the laboratory prioritised four components, which included staff training and competency, methods validation and verification, practising internal quality control in each testing procedure performed within the laboratory, as well as enrolment in external quality control programme schemes. laboratory management established quality indicators to measure the laboratory’s improvement and to identify corrective and preventive actions. staff training and competency assessment the laboratory developed a procedure to guide management in conducting training and assessing staff competencies. during the policies training session, the quality officer would first address the clause within iso, then would make sure it had been properly interpreted among laboratory staff and adequately documented within the laboratory policy manual. system procedures were introduced to explain step-by-step approaches to ensure implementation among laboratory staff. quality management system forms were designed to provide evidence of implementation of procedures, as documented in the laboratory policy manual. training sessions were concluded with ‘quizzes’ to check understanding of the discussed procedures. staff that scored below 80% on the quizzes given after training were retrained and reassessed for competencies. the responsibility for training on technical procedures was assigned to heads of sections, who provided step-by-step guidance on technical procedures. before staff were allowed to carry out a test, they were assessed by testing known samples, internal controls and external quality assessment materials for reproducibility of results, as well as being checked for all related activities for the respective procedures before being deemed competent. staff competence procedures also included examination related to scores of 80% on tests. heads of sections and the laboratory manager declared staff to be competent and any deviation of the expected requirements prompted retraining. the quality officer took responsibility for adequate implementation and filing of competence forms in personnel files. method validation and verification the laboratory developed procedures to guide staff on how to validate or verify technical methods; the procedures clearly clarified the difference between verification and validation. verification was defined as processes that are carried out to prove that the method can provide intended use only when capability data are available; it referred to standard methods used without modification. validation14 was defined as processes that are carried out to prove that the method is fit for the laboratory intended use when no capability data are available; it referred to non-standard laboratory designed methods, which are subjected to subsequent modification. therefore for validation, the laboratory has to set criteria to accept or reject the test method. qualitative tests samples from previously reported results were used to verify methods. samples were selected randomly (diseased and non-diseased samples) and assigned, in a blinded study, to two different staff members (verifier 1 and verifier 2) deemed competent to carry out the test under verification. staff carrying out the test were not aware of the results and were supervised, to avoid sharing of results. they were subjected to a similar testing set-up and provided with the method to conduct samples testing. the results produced were again compared to the expected results, also the sensitivity and specificity of the test methods were calculated. the verification results which met the set criteria or manufacturer claims provided sufficient evidence to prove the suitability of the test methods. the verification results which did not meet the criteria demonstrated unfitness of the method for testing of patients. validation or verification of quantitative tests accuracy, precision and linearity were determined for individual test methods. manufactured quality control material was used during the validation or verification process, and control samples were analysed in duplicate three times a day for five days. precision was calculated to determine coefficient of variation (%), and accuracy was determined to obtain ranges at 95% confidence. the ranges calculated were compared to that of the manufacturer. linearity was performed by doing serial dilutions on control samples; the diluted samples were tested using the method under verification process, and the results obtained were analysed to obtain the linear line, which was then compared to that of the manufacturer. the laboratory management agreed that the method was fit for testing patients if an acceptable linear line could be visualised after analysis. internal and external quality assessments the laboratory, with assistance from funding provided by the president’s emergency plan for aids relief, procured internal quality control samples and enrolled in external quality assessment (eqa) schemes. internal quality controls were performed as documented in the established internal quality control procedure. laboratory staff were trained to evaluate the results of internal controls before they report a patient’s results. the laboratory received eqa samples from thistle (south africa), one world accuracy (canada), the national quality assessment scheme (united kingdom), the national health laboratory quality assurance & training centre (tanzania) and the united states centers for disease control and prevention (united states).15 samples were received and processed within the deadline, and results from eqa providers were reviewed to identify opportunities for improvement. eqa scores were set to be 80%; when this score was not achieved, it was documented as a non-conformance event, which required root cause analysis and implementation of an identified corrective action. quality indicators quality indicators were defined as any measure of the system whereby data collected in a specified period are analysed to determine the improvement of the established system.16 examples of quality indicators which were established to measure laboratory improvement included: external quality assurance performance; number of customer complaints; sample turn-around time; number of rejected samples; equipment downtime; and blood culture sample contamination rate. the laboratory set targets were: (1) eqa performance above 80%, (2) five customer complaints per week, and (3) 90% of samples received to be tested and reported on within the established turn-around time. turn-around time was defined as time taken from receiving a sample in the laboratory until dispatch of results to a patient. neither rejected samples nor blood culture contamination should exceed 3% of the total samples received. quality indicators were reviewed by section members, chaired by the heads of sections and the quality officer, and then in quarterly intervals by the quality assurance committee. deviations from set targets prompted improvement actions. results external quality assessment (percentage score) for the parasitology section increased from 45% before implementation of iso to 100% after implementation, the biochemistry section performance increased from 50% to 95%, the molecular biology section performance increased from no record to 100%, the tuberculosis microscopy section increased from 60% to 100%, and the microbiology section increased from 48.1% to 100% (table 1). the number of complaints decreased from eight to two complaints per week. rejected samples decreased from 7.2% (18/250) to 1.2% (3/240). the overall turn-around time performance was not recorded before implementation, but reached 92% (1644/1786) of the defined targets, and contamination of blood culture samples decreased from 16% (25/160) to 4% (5/116). table 1: quality indicators before and after implementation of iso 15189, bugando medical centre clinical laboratory, tanzania. the gap analysis was conducted on 23 january 2012, the implementation started immediately to address the gaps obtained and accreditation was awarded on 26 march 2014. after qms implemented laboratory quality improvement activities and monitoring of indicators were possible as displayed in figures 1–3. figure 1: parasitology turn-around times from january to december 2016. gap analyses were conducted in january 2012, and implementation of laboratory quality improvement activities started immediately to address the gaps. accreditation was awarded in march 2014 and indicators have been monitored since. figure 2: chemistry sample rejection from january to december 2016. figure 3: microbiology external quality assessment january–december 2016. accreditation after 24 months of intensive work, including mentoring activities, the laboratory was iso 15189 accredited under a southern african development community accreditation services/south african national accreditation system (sadcas/sanas)17 partnership, with registration number md002. tests in five sections were accredited, including microbiology, parasitology, molecular biology, biochemistry and tuberculosis microscopy. the laboratory witnessed visible advantages of implementing qms. for example, work done being appreciated by customers, passing eqas, decreased turn-around times and fewer complaints (table 1), through monthly monitoring and review of established quality improvement indicators. discussion implementation of iso 15189-based qms in a routine clinical laboratory at the tertiary hospital, bugando medical centre, was successful in preparing the laboratory for accreditation after 24 months of intense effort. the initial anxiety of staff was counteracted by supportive visits by the ministry of health and mentors and constructive advice. the newly-established heads of sections, quality officer, laboratory manager, laboratory director and top hospital management were informed to support the programme. however, the implementation procedures required close follow-up of staff to ensure adherence to the established system, which was not always easy. the laboratory management realised that implementation of qms also provided regular objective assessment through internal and external audits, ensuring continous improvement in operations. regular mentorship and a quality officer with strong leadership skills transformed the laboratory into a quality-driven organisation. a customer survey, conducted to measure what clinicians were saying about the laboratory services, revealed that clinicians of the hospital were able to trust and make clinical decisions supported by laboratory results.10 this was also observed in botswana, where laboratories with mentorship, support from hospital management and ‘strong lab staff camaraderie’ supported the implementation process.18 similar findings were observed in laboratories in zimbabwe and kenya, where mentorship played an important role in the successful implementation of the iso standard.19,20 the laboratory experienced a marked increase of intrinsic motivation during the presence of external clsi mentors, and the team started to demonstrate a high level of commitment, working beyond official hours without any financial expectations. following successful accreditation, the quality officer observed a sudden decline in commitment of the team, as described in the life cycle of an organisation.21 staff relaxed after their initial hard work, and the laboratory manager and quality officer were challenged to ensure timely intervention to avoid losing the achieved goals. the laboratory management ensured translation of the achieved goals into daily laboratory operations with its advantages to patient care. there may be a time where a short-term mentor must come in again, as was done in zimbabwe, when trying to identify an optimal model for mentorship.22 our observations are similar to that reported by a tuberculosis laboratory in kisumu, kenya, where culture contamination rate decreased from 15.4% to 5.3% compared to the reduction from 16% to 4% observed in our laboratory before and after implementation of the qms. the only difference is that the laboratory in kenya was implementing the standard in a tuberculosis laboratory and was on the implementation process, thus was not accredited as yet.23,24 similar results were achieved by the strengthening laboratory management towards accreditation programme in 47 african countries, where the programme is implementing its activities. it was observed in five years of operation that the strengthening laboratory management towards accreditation approach formed a unique capacity to assist laboratories to make progress in improving quality of services as a way to achieve accreditation. however, our implementation of similar activities took 24 months of intensive work.17,25 conclusions our experience suggests that implementation of a qms is possible in resource-limited countries like tanzania, if adequately supported with human and financial resources. to achieve this level of standard requires trained and well-motivated laboratory staff to implement the system. mentorship is necessary and should be done by laboratory professionals trained in qms, who have implemented the system. financial resources and motivated staff are key to achieving iso accreditation. acknowledgements competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support none. authors’ contributions m.b., l.g.-a., c.k., k.j., b.m., h.s., s.e., c.m., d.m., m.m., w.m. and p.m. conceived the plan and structure of the laboratory quality management system in their respective departments and collected the data and indicators. m.b. and c.k. drafted the article, did the analysis and interpreted the data. all authors read and approved the final article. references niessen lw, grijseels ew, rutten ff. the evidence-based approach in health policy and health care delivery. soc sci med. 2000;51(6):859–869. https://doi.org/10.1016/s0277-9536(00)00066-6 andiric lr, massambu cg. laboratory quality improvement in tanzania. am j clin pathol. 2015;143(4):566–572. https://doi.org/10.1309/ajcpab4a6wwpyien akobeng ak. principles of evidence based medicine. arch dis childhood. 2005;90(8):837–840. https://doi.org/10.1136/adc.2005.071761 petti ca, polage cr, quinn tc, ronald ar, sande ma. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006;42(3):377–382. https://doi.org/10.1086/499363 muula as, maseko fc. medical laboratory services in africa deserve more. clin infect dis. 2006;42(10):1503. https://doi.org/10.1086/503678 guzel o, guner ei. iso 15189 accreditation: requirements for quality and competence of medical laboratories, experience of a laboratory i. clin biochem. 2009;42(4):274–278. https://doi.org/10.1016/j.clinbiochem.2008.09.011 schroeder lf, amukele t. medical laboratories in sub-saharan africa that meet international quality standards. am j clin pathol. 2014;141(6):791–795. https://doi.org/10.1309/ajcpq5ktkagsscfn nkengasong jn, nsubuga p, nwanyanwu o, et al. laboratory systems and services are critical in global health: time to end the neglect? am j clin pathol. 2010;134(3):368–373. https://doi.org/10.1309/ajcpmpsinq9brmu6 nkengasong jn, mesele t, orloff s, et al. critical role of developing national strategic plans as a guide to strengthen laboratory health systems in resource-poor settings. am j clin pathol. 2009;131(6):852–857. https://doi.org/10.1309/ajcpc51blobbpakc woodcock s, fine g, mcclure k, unger b, rizzo-price p. the role of standards and training in preparing for accreditation. am j clin pathol. 2010;134(3):388–392. https://doi.org/10.1309/ajcp03tfpbkeyynt mcclure k, arbique j, rendell a, rizzo-price p. clsi: building laboratory capacity in africa. clin microbiol newslett. 2009;31(13):95–99. https://doi.org/10.1016/j.clinmicnews.2009.06.001 gershy-damet g-m, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. https://doi.org/10.1309/ajcptuuc2v1wjqbm soin ss. total quality essentials: using quality tools and systems to improve and manage your business. new york, ny: mcgraw hill professional; 1999. whiting p, rutjes a, dinnes j, et al. development and validation of methods for assessing the quality of diagnostic accuracy studies. health technol assess winch engl. 2004;8(25):iii, 1–234. libeer j-c. role of external quality assurance schemes in assessing and improving quality in medical laboratories. clin chim acta. 2001;309(2):173–177. https://doi.org/10.1016/s0009-8981(01)00518-6 sciacovelli l, o’kane m, skaik ya, et al. quality indicators in laboratory medicine: from theory to practice. clin chem lab med. 2011;49(5):835–844. https://doi.org/10.1515/cclm.2011.128 sadcas. accreditation process [homepage on the internet]. n.d. [cited 2017 sept 23]. available from: http://www.sadcas.org/accreditation-process mokobela k, moatshe m, modukanele m. accelerating the spread of laboratory quality improvement efforts in botswana. afr j lab med. 2014;3(2):art.# 207, 1–6. ntshambiwa k, ntabe-jagwer w, kefilwe c, samuel f, moyo s. translating a national laboratory strategic plan into action through slmta in a district hospital laboratory in botswana: original research. afr j lab med. 2016;3(2):1–5. makokha ep, mwalili s, basiye fl, et al. using standard and institutional mentorship models to implement slmta in kenya: original research. afr j lab med. 2016;3(2):1–8. lester dl, parnell ja, carraher s. organizational life cycle: a five-stage empirical scale. int j organ anal. 2003;11(4):339–354. https://doi.org/10.1108/eb028979 nzombe p, shumba e, zimuto sn, et al. maximising mentorship: variations in laboratory mentorship models implemented in zimbabwe: original research. afr j lab med. 2016;3(2):1–8. musau s, mccarthy k, okumu a, et al. experience in implementing a quality management system in a tuberculosis laboratory, kisumu, kenya. int j tuberc lung dis. 2015;19(6):693–5. https://doi.org/10.5588/ijtld.14.0886 audu ra, sylvester-ikondu u, onwuamah ck, et al. experience of quality management system in a clinical laboratory in nigeria: lessons from the field. afr j lab med. 2012;1(1):1–5. yao k, luman et, nkengasong jn, et al. the slmta programme: transforming the laboratory landscape in developing countries: lessons from the field. afr j lab med. 2016;3(2):1–8. article information authors: robert n. maina1 doris m. mengo1 abdikher d. mohamud1 susan m. ochieng1 sammy k. milgo1 connie j. sexton2 sikhulile moyo3 elizabeth t. luman2 affiliations: 1kenya accreditation service (kenas), kenya 2us centers for disease control and prevention, atlanta, united states 3botswana-harvard aids institute partnership, princes marina hospital, botswana correspondence to: robert maina postal address: po box 47400-00100, nairobi, kenya dates: received: 11 aug. 2014 accepted: 12 sept. 2014 published: 03 nov. 2014 how to cite this article: maina rn, mengo dm, mohamud ad, et al. progressing beyond slmta: are internal audits and corrective action the key drivers of quality improvement?. afr j lab med. 2014;3(2), art. #222, 7 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.222 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. progressing beyond slmta: are internal audits and corrective action the key drivers of quality improvement? in this original research... open access • abstract • introduction • research methods and design    • site selection    • slmta training    • audits    • mentorship    • data analysis • results • discussion    • limitations    • conclusion • acknowledgements    • competing interests    • authors’ contributions    • cdc disclaimer • references abstract top ↑ background: kenya has implemented the strengthening laboratory management toward accreditation (slmta) programme to facilitate quality improvement in medical laboratories and to support national accreditation goals. continuous quality improvement after slmta completion is needed to ensure sustainability and continue progress toward accreditation. methods: audits were conducted by qualified, independent auditors to assess the performance of five enrolled laboratories using the stepwise laboratory quality improvement process towards accreditation (slipta) checklist. end-of-programme (exit) and one year post-programme (surveillance) audits were compared for overall score, star level (from zero to five, based on scores) and scores for each of the 12 quality system essential (qse) areas that make up the slipta checklist. results: all laboratories improved from exit to surveillance audit (median improvement 38 percentage points, range 5–45 percentage points). two laboratories improved from zero to one star, two improved from zero to three stars and one laboratory improved from three to four stars. the lowest median qse scores at exit were: internal audit; corrective action; and occurrence management and process improvement (< 20%). each of the 12 qses improved substantially at surveillance audit, with the greatest improvement in client management and customer service, internal audit and information management (≥ 50 percentage points). the two laboratories with the greatest overall improvement focused heavily on the internal audit and corrective action qses. conclusion: whilst all laboratories improved from exit to surveillance audit, those that focused on the internal audit and corrective action qses improved substantially more than those that did not; internal audits and corrective actions may have acted as catalysts, leading to improvements in other qses. systematic identification of core areas and best practices to address them is a critical step toward strengthening public medical laboratories. introduction top ↑ accurate, timely and affordable medical diagnosis to support patient care and management remains a challenge in developing countries. many laboratories are ill prepared to respond to health emergencies, yet their services are critical for the detection of new pathogens and containment of disease outbreaks.1 establishing a quality management system (qms) to support accreditation is a demanding process requiring great organisational skills, motivation and huge investment that may be both overwhelming and unfeasible for public medical laboratories with limited resources. in addition to the cost of developing these systems, barriers are raised by the lack of awareness amongst healthcare workers regarding the benefits of accreditation on health services. several publications have highlighted the advantages of a comprehensive qms in a laboratory setting along with recognition of areas of qms that were at risk of failing.2,3, 4,5 accreditation is now widely recognised as being an essential element of strengthening the qms in public health laboratories and may also have a positive effect on other sectors of the healthcare system.6,7 the past few years have seen increased support for health systems in africa through funding from initiatives such as the us president’s emergency plan for aids relief (pepfar); the global fund to fight aids, tuberculosis and malaria; the global health initiative; and organisations such as the world bank.8 strengthening integrated laboratory services within public health laboratories, as opposed to focusing on disease-specific programmes, has been encouraged as a means of establishing a cost-effective laboratory system.9 the growing recognition of the importance of laboratory services has resulted in the launch of several important initiatives including the strengthening laboratory management toward accreditation (slmta) programme. slmta was developed by the us centers for disease control and prevention (cdc) in collaboration with the american society for clinical pathology (ascp), the clinton health access initiative and the world health organization’s regional office for africa (who afro), with the aim of promoting immediate measurable improvement in the laboratories of developing countries.10,11,12 the kenya health service delivers medical laboratory services through a network of 958 laboratories, of which 70% belong to the government, 20% to nongovernmental organisations and 10% to the private sector.13 as of the end of 2012, eight (< 1%) of the 958 laboratories in kenya had been accredited, none of which were in the government sector.4 as accreditation of health facilities is a key goal of the country’s 2008–2012 strategic plan,13 kenya’s ministry of health (moh) established a national accreditation steering committee to coordinate laboratory accreditation activities. after slmta’s launch in july 2009, the moh, with support from cdc’s kenya office, adopted the programme and enrolled 53 laboratories in six cohorts between 2010 and 2013 from amongst national reference, provincial, and district-level laboratories. the moh set a target to accredit at least five laboratories from the first cohort of 13 to the international organization for standardization (iso) 15189 standard by the end of 2014. six main partners have helped implement slmta in kenya: ascp, a global health care public foundation (aghpf), management sciences for health (msh), the kenya aids vaccine initiative (kavi), the african field epidemiology network (afenet) and the world bank. twenty laboratories so far have graduated from the slmta programme. after completing the slmta programme, laboratories still had substantial quality gaps to fill before seeking accreditation. this paper reviews the performance of five laboratories from the first slmta cohort in kenya, highlighting the progress made in the year after completion of the programme and identifying areas critical to implementation and sustainability of a sound qms. research methods and design top ↑ site selection five laboratories from the first slmta cohort in kenya were selected for this evaluation based on two primary factors: they had the same group of mentors and mentorship schedules after the exit audit; and all were audited during the same timeframe by the same team of auditors. for the purpose of confidentiality these laboratories are presented as a–e. laboratory b is a private clinical research laboratory, whilst the other four are government-run provincial-level laboratories with significant automation in haematology and flow cytometry but only semi-automated equipment in clinical chemistry. slmta training the slmta training for the first cohort took place in 2010 and consisted of three workshops of four days each. module one and cross-cutting activities were taught in workshop one, modules two to six were taught in workshop two and modules seven to 10 were taught in workshop three. the trainers were slmta training-of-trainers (tot) graduates employed by cdc’s kenya office. two staff members from each of the five laboratories participated in the workshops. these participants were quality managers and laboratory managers, whose responsibilities included overseeing the implementation of qms, and training and supervising the laboratory personnel. these workshops were interspersed with periods of three months, during which participants implemented improvement projects based on what was learned at the previous workshop and laboratory-specific quality gaps. audits baseline audits were conducted in april 2010, one month before the first slmta workshop. since they were conducted by non-certified auditors, their findings are not included in this analysis. exit audits were conducted in august 2011, eight months after the third workshop. surveillance audits were conducted one year later, in august 2012. audits were conducted using who afro’s stepwise laboratory quality improvement process towards accreditation (slipta) checklist. the slipta checklist comprises 111 questions subdivided into 12 sections that represent the quality systems essentials (qses) from the clinical and laboratory standards institute.14 for each positively answered question, points were allocated (two, three or five); partially positive answers were awarded one point. a star rating was assigned as follows: five stars (244–258 points, ≥ 95% compliance), four stars (219–243 points, 85% – 94% compliance), three stars (193–218 points, 75% – 84% compliance), two stars (167–192 points, 65% – 74% compliance) and one star (142–166 points, 55% – 64% compliance). a score of 141 points or less (< 55%) received zero stars.15 both exit and surveillance audits were conducted by auditors certified by the kenya accreditation service (kenas), the sole national accreditation body in kenya. auditors were selected on the basis of training and technical expertise, as well as having had no previous engagement with the laboratory being audited to safeguard impartiality. all auditors had completed core training on slipta as well as training on iso requirements for quality and competency in medical laboratories (iso 15189) and guidelines for qms auditing (iso 19011), had attended an annual assessor refresher course and had conducted at least one slipta audit. the composition of each audit team was based on the scope of the laboratory being audited. methods of audit included: (1) review of documents; (2) review of records (procedures, minutes of the meetings, quality control data, corrective actions, work plans); (3) staff interviews; and (4) observation and witnessing of testing procedures. the interviews were open-ended questions used to obtain information required by the slipta checklist. the laboratory management and staff received prior notification of the audit dates and auditors’ names. the auditors selected the staff and the procedure to be observed, using sampling techniques that ensured objectivity and impartiality of the audit process. this ensured that staff did not have prior knowledge of who was to be observed. audits were conducted by two auditors per laboratory over a two-day period. each auditor recorded their findings in their checklist before compiling the report in a consensus checklist that included the final score and the star rating for each laboratory. a summary report of all findings (both positive and negative) and a corrective action request form (for each nonconformity) were completed by the auditors on site and submitted to the laboratory management at the end of the second day of audit. a senior laboratory staff member was required to sign each form so as to acknowledge receipt of the findings. any divergent opinions between the auditors and the laboratories were resolved on site before writing of the final report. mentorship mentorship during slmta implementation was unstructured, with mentors visiting laboratories for unspecified periods of time without concrete work plans. this deficiency was partly because of a lack of training for mentors, as well as the lack of a proper mentorship programme in the country by 2010. in contrast, after the exit audit new mentors were embedded in the five facilities for two weeks per month for four months. these mentors followed comprehensive work plans in the eight weeks they were in the facilities, which covered the entire slipta checklist. the mentors were consultants in qms implementation in medical laboratories contracted by the implementing partner. their qualifications included diploma or a bachelor’s degree in medical laboratory sciences, quality management training based on iso 15189 and the slipta checklist, and experience working in an accredited laboratory or one undergoing the accreditation process. data analysis retrospective data mining and analysis were conducted using slipta audit data comprehensive reports and corrective action forms for each laboratory at the kenas offices. we compared the exit and surveillance audit scores, evaluated performance in the individual qses and identified key areas of progress and challenges in implementation of a sound qms. we also examined nonconformities at the exit and surveillance audits with a focus on identification of common issues. any nonconformity identified at both the exit and surveillance audits in the same laboratory was classified as a recurring nonconformity. statistical analysis was done using graphpad prism software version 6.02 (graphpad software, la jolla, california, usa, www.graphpad.com) and microsoft® excel. results top ↑ exit audit scores were below the one-star level for all laboratories except b, which scored three stars at exit. all of the laboratories increased their scores from exit to surveillance audit; two achieved one star, two achieved three stars and the laboratory that began at three stars achieved four stars (figure 1). median scores increased from 37% at exit to 75% at surveillance audit (improvement range 5–45 percentage points). figure 1: performance of the five laboratories based on stepwise laboratory quality improvement process towards accreditation (slipta) checklist scores and star ratings. the two laboratories with the highest per-person workload (a and e) had the most improvement from exit to surveillance audit, whilst the laboratory with the lowest volume (b) and highest exit score had the least improvement (figure 1). the lowest median qse scores at exit were internal audit, corrective action and occurrence management, and process improvement (all < 20%) (figure 2). the highest medians were in purchasing and inventory, facilities and safety, and organisation and personnel (all > 45%). each of the 12 qses improved substantially at the surveillance audit (range 24 – 75 percentage points), with the greatest improvement in client management and customer service, internal audit and information management (≥ 50 percentage points). the smallest improvements were in purchasing and inventory, management reviews, organisation and personnel, and occurrence management and process improvement (≤ 25 percentage points). by the surveillance audit, the areas of greatest challenge were occurrence management and process improvement, corrective action and management reviews (all ≤ 55%), whilst the strongest areas were client management and customer service, facilities and safety, purchasing and inventory, and information management (all ≥ 78%) (figure 2). figure 2: median scores of the five laboratories in the 12 quality system essentials (qses). in order to examine more closely the relationship between specific areas of improvement and overall success, we compared results for laboratories a and e, which started with zero stars at exit (median 34%) and reached three stars at surveillance (median 76%), to those for laboratories c and d, which also started at zero stars (median 42%) and reached one star at surveillance (median 59%). we excluded laboratory b in this sub-analysis because it was markedly smaller than the others and started with a substantially higher score at exit. most striking were the results for the internal audit and corrective action qses. all four of these laboratories scored 0% in internal audit at exit. laboratories a and e both excelled in this area post-slmta, increasing their score to 60% by the surveillance audit. laboratories c and d, on the other hand put little focus on this area, scoring only 10% at the surveillance audit (figure 3). moreover, laboratories a and e both excelled in corrective action, increasing from 0% to 50% and from 8% and 67%, respectively, whilst laboratories c and d decreased from 8% to 0% and from 63% to 25%, respectively. when excluding internal audit and corrective action, the other qses for laboratory a and e improved by a median of 48 percentage points from exit to surveillance audit, whilst those from c and d improved by 15 percentage points. no other qses had such consistent results. figure 3: performance of laboratories a, c, d and e in the 12 quality system essentials (qses). ten nonconformities were common to all five laboratories: lack of critical procedures, lack of or incomplete management review records, incomplete personnel files, lack of equipment or method validation, lack of equipment calibration records, deficient internal audit, inconsistent internal quality control monitoring, unacceptable proficiency testing results, ineffective corrective action and deficient quality indicator monitoring (table 1). at the exit audits, laboratories a and e had the most nonconformities. however, these laboratories developed corrective action plans that covered most of the nonconformities, so that by the surveillance audit their nonconformities were reduced by more than half. laboratories c and d, on the other hand, reduced their nonconformities by only 12% – 40% at the surveillance audit, with many recurring issues. table 1: number of nonconformities at exit and surveillance audits and implementation of the corrective action plan by the laboratory. discussion top ↑ all five laboratories in this study made notable improvements from exit to surveillance audit, displaying sustained and even increased efforts after completion of the slmta programme. whilst laboratory b outperformed the other laboratories both at its exit and surveillance audits, this may in part reflect the smaller size, lower workload and less complicated management system of this small private research laboratory compared with the four hospital laboratories. laboratories c and d reached one star at their surveillance audits, recording more modest improvements than laboratories a and e, which reached three stars despite having a higher number of tests per technician. overall, results suggest that intensive mentorship provided post-slmta may be helpful with regard to accelerating quality improvement. in an attempt to identify the factors associated with the relatively greater success of laboratories a and e, we examined closely their improvements by qse. for the most part, all of the laboratories tended to struggle in the same areas (primarily internal audit, corrective action, and occurrence/management and process improvement) and excelled in the same areas (organisation and personnel, facilities and safety, and purchasing and inventory). however, laboratories a and e improved substantially in the internal audit qse, increasing their scores from 0% to 60% in this area by the surveillance audit; laboratories c and d did not. they also improved well in corrective action, whilst the scores for this qse decreased in laboratories c and d. furthermore, improvements to these scores alone do not account for the greater overall improvements of laboratories a and e, as their scores in other areas also improved substantially more than laboratories c and d (48% versus 15%). the qse ‘internal audit’ involves checking whether internal audits have been conducted by trained auditors and examining the corrective actions carried out to rectify the audit findings. iso 15189 mandates that internal audits be conducted at least annually.16 conducting internal audits can help a laboratory stay focused and provides concrete information with which to better understand its own areas of weakness and make decisions for improvement.17 the american association for laboratory accreditation suggests that internal audits are critical because they allow laboratories to identify the root and potential causes of problems that need to be eliminated so that the problems are prevented from occurring the first time or recurring after correction. subsequently, corrective or preventative action is taken to address a problem and eliminate the root cause of the problem.18 thus, in laboratories a and e, internal audits and corrective actions may have acted as catalysts, facilitating improvement in other areas. in addition, discussion of internal audit results within the laboratory may have helped to develop an overall quality culture, not only amongst laboratory staff but also amongst hospital management, as has been identified elsewhere as a key factor in slmta success.19 whilst this hypothesis is generated using a very small number of laboratories and various findings have been reported in other small studies,20,21,22 our results suggest that further evaluation of the benefits of conducting regular internal audits followed by corrective action is warranted. the 12 qses can be divided into three quality stages: resource management (pre-analytical), process management (analytical) and improvement management (post-analytical).23 the three qses with the lowest scores for these laboratories at exit audit (internal audit, corrective action, and occurrence management and process improvement) are all part of the improvement management stage. datema et al.24 point out that this stage is given the lowest scoring weight of the three quality stages in the slipta system (16% of the total score, compared with 48% for resource management and 36% for process management in the original scoring structure; and 20%, 48% and 33%, respectively, in the current scoring structure).15 they argue that ‘this stage is of prime importance for sustaining the continuous improvement cycle’24 and that by awarding it fewer points, laboratories may be ‘less stimulated to invest effort in improvement management’.24 overall, data on nonconformities obtained from the exit and surveillance audit reports showed that the actions taken to address deficiencies were often inadequate and, in most instances, root causes were not established and eliminated. the completeness and implementation of the corrective action work plan varied between laboratories. laboratories a and e developed focused corrective action work plans based on the findings of the exit audit and implemented them thoroughly. in contrast, laboratories c and d made less progress in implementing corrective action plans. of note is that, despite attaining four stars, laboratory b had 19 nonconformities at the surveillance audit, including 12 recurring nonconformities from the exit audit. it may be that this laboratory had already covered the easier parts of the checklist during slmta implementation and the remaining weak areas, such as occurrence management and internal audit, were hardest to implement and sustain post-slmta. recurring nonconformities are graded as major events and could potentially lead to a serious breakdown of the qms if not addressed in a timely fashion. thus, even laboratories with high slipta scores may still have major nonconformities that could pose a threat to sustainability of the qms and prevent accreditation. limitations the circumstances in each country and in every laboratory within a country are unique, each facing multiple interconnected challenges. whilst our analysis of five laboratories identified some interesting findings, it will be critical to expand this analysis using data from a large number of laboratories in multiple settings. this will give a better picture of performance trends across the 12 qses both during and after slmta implementation, as well as allowing for generalisation of the important issues facing laboratories today and the critical challenges impeding success and sustainability. our study was limited by the lack of comparable baseline data from which to evaluate the common issues that existed before slmta implementation, as baseline data were excluded in order to minimise variability in auditor scoring. finally, additional surveillance audits will be needed for the assessment of long-term sustainability of programme results. conclusion this analysis reveals common gaps in laboratory qses that can be addressed during and after slmta implementation. in particular, internal audits and subsequent corrective actions may play key roles in catalysing improvements in other areas, although a more systematic global evaluation is needed in order to generalise common problems and determine the best practices to address them. acknowledgements top ↑ this publication was made possible by support from pepfar phase ii through cooperative agreement number 5 u2g ps001285-02 from the cdc, division of global hiv/aids (dgha). we thank dr. jane mwangi from cdc’s kenya office for her valuable suggestions and the mentors of the first cohort laboratories, especially lucy otieno, moses oyaya, florence mawere and hanington ahenda, as well as the staff from laboratories a–e for providing valuable information. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions r.n.m. (kenas) collected, analysed and interpreted the data and wrote the manuscript. d.m.m. (kenas) and a.d.m. (kenas) assisted in data collection. s.m.o. (kenas) was involved in both auditor selection and data collection. s.k.m. (kenas) provided guidance on selection of facilities to include in this analysis. c.j.s. (cdc, atlanta) was involved in data analysis and interpretation. s.m. (botswana-harvard aids institute partnership) assisted in data analysis. e.t.l. (cdc, atlanta) assisted in data analysis and interpretation and significantly shaped the focus of the article. cdc disclaimer the findings and conclusions in this paper are those of the author(s) and do not necessarily represent the official position of the cdc or the government of kenya. references top ↑ 1.nkengasong jn, nsubuga p, nwanyanwu o, et al. laboratory systems and services are critical in global health: time to end the neglect? am j clin pathol. 2010;134(3):368−373. http://dx.doi.org/10.1309/ajcpmpsinq9brmu6 2.nicklin w, dickson s. the value and impact of accreditation in health care: a review of the literature. canada: accreditation canada; 2009. 3.harper j.c, sengupta s, vesela k, et al. accreditation of the pdg laboratory. hum reprod. 2010; 25(4) 1051−1065. http://dx.doi.org/10.1093/humrep/dep450 4.schroeder lf, amukele t. medical laboratories in sub-saharan africa that meet international quality standards. am j clin pathol. 2013;141(6):791–795. http://dx.doi.org/10.1309/ajcpq5ktkagsscfn 5.zeh ce, inzaule sc, magero vo, et al. field experience in implementing iso 15189 in kisumu, kenya. am j clin pathol. 2010;134(3):410–418. http://dx.doi.org/10.1309/ajcpzirkdus5lk2d 6.peter tf, rotz pd, blair, dh, et al. impact of laboratory accreditation on patient care and the health system. am j clin pathol. 2010;134(4):550–555 http://dx.doi.org/10.1309/ajcph1skq1hnwghf 7.alkhenizan a, shaw c. impact of accreditation on the quality of healthcare services: a systematic review of the literature. ann saudi med. 2011;31(4):407–416. http://dx.doi.org/10.4103/0256-4947.83204 8.mccoy d, chand s, sridhar d .global health funding: how much, where it comes from and where it goes. health policy plan. 2009;24(6):407–417. http://dx.doi.org/10.1093/heapol/czp026 9.parsons lm, somoskovi a, lee e, et al. global health: integrating national laboratory health system and services in resource-limited settings. afr j lab med.2012;1(1), 5 pages. 10.yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin pathol. 2010;134(3):401–409. http://dx.doi.org/10.1309/ajcpnbbl53fwuiqj 11.luman et, yao k, nkengasong jn. a comprehensive review of the slmta literature part 1: content analysis and future priorities. afr j lab med. 2014;3(2), art. #265, 11 pages. http://dx.doi.org/10.4102/ajlm.v3i2.265 12.gershy-damet gm, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. http://dx.doi.org/10.1309/ajcptuuc2v1wjqbm 13.ministry of public health and sanitation. strategic plan. the second national health sector strategic plan of kenya. reversing the trends [document on the internet]. c2008 [cited 2014 oct 07]. available from: http://www.internationalhealthpartnership.net/ fileadmin/uploads/ihp/documents/country_pages/kenya/kenya%20ministy_of_public_health_and_sanitation_strategic_plan%202008-2012.pdf 14.clinical and laboratory standards institute. application of a quality management system model for laboratory services; approved guidelines – third edition. clsi document gp26-a3 [isbn 1-56238-553-4]. clinical and laboratory standards institute; 2004. 15.world health organization’s regional office for africa. who-afro stepwise laboratory quality improvement process towards accreditation (slipta) checklist [document on the internet]. c2012 [cited 2013 may 31]. available from: http://www.afro.who.int/en/clusters-a-programmes/hss/blood-safety-laboratories-ahealth-technology/blt-highlights/3859-who-guide-for-thestepwise-laboratory-improvement-process-towards-accreditation-in-the-africanregion-with-checklist.html 16.international organization for standardization. iso 15189 medical laboratories – requirements for quality and competence. geneva: international organization for standardization; 2005. 17.clinical and laboratory standards institute. assessments: laboratory internal audit program; approved guideline – vol: 33 no. 15. clsi document qms15-a [isbn 1-56238-895-9]. clinical and laboratory standards institute; 2013. 18.american association for laboratory accreditation. internal auditing course training manual [document on the internet]. c2012 [cited 2014 jul 03]. available from: http://www.aavld.org/assets/accreditation/accreditation_documents/internal%20auditor%20course%20handbook.pdf 19.maruti pm, mulianga ea, wambani ln, et al. creating a sustainable culture of quality through the slmta programme in a district hospital laboratory in kenya. afr j lab med. 2014;3(2), art. #201, 5 pages. http://dx.doi.org/10.4102/ajlm.v3i2.201 20.mothabeng d, maruta t, lebina m, et al. strengthening laboratory management towards accreditation: the lesotho experience. afr j lab med. 2012;1(1), art. #9, 7 pages. 21.makokha ep, mwalili s, basiye fl, et al. using standard and institutional mentorship models to implement slmta in kenya. afr j lab med. 2014;3(2), art. #220, 8 pages. http://dx.doi.org/10.4102/ajlm.v3i2.220 22.mokobela ko, moatshe mt, modukanele m. accelerating the spread of laboratory quality improvement efforts in botswana. afr j lab med. 2014;3(2), art. #207, 6 pages. http://dx.doi.org/10.4102/ajlm.v3i2.207 23.international organization for standardization. iso 9001:2008 quality management systems – requirements. geneva: international organization for standardization; 2008. 24.datema ta, oskam l, van beers sm, et al. critical review of the stepwise laboratory improvement process towards accreditation (slipta): suggestions for harmonization, implementation and improvement. trop med int health. 2012;17(3):361–367. article information authors: michael l. kasusse1,2 nazarius m. tumwesigye1 steven aisu3 joseph k.b. matovu1,2 rhoda wanyenze1,2 affiliations: 1makerere university school of public health, kampala, uganda 2maksph-cdc fellowship program, makerere university school of public health, kampala, uganda 3central public health laboratories, ministry of health uganda, kampala, uganda correspondence to: michael kasusse email: lkasussem@yahoo.com postal address: po box 32060, kampala, uganda dates: received: 27 aug. 2014 accepted: 15 aug. 2015 published: 30 nov. 2015 how to cite this article: kasusse ml, tumwesigye nm, aisu s, matovu jkb, wanyenze r. effectiveness of the credit-line approach for support of cd4 equipment functionality in northern uganda. afr j lab med. 2015;4(1), art. #234, 5 pages. http://dx.doi.org/10.4102/ajlm.v4i1.234 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. effectiveness of the credit-line approach for support of cd4 equipment functionality in northern uganda in this original research... open access • abstract • introduction • methods • results • discussion    • limitations    • recommendations    • conclusion • acknowledgements    • funding sources    • competing interests    • authors’ contributions • references abstract top ↑ background: improving laboratory service delivery requires a functioning logistics and supply system. uganda’s ministry of health uses the credit-line approach to provide laboratory supplies including commodities for cd4 test equipment. objectives: we examined the effectiveness of the credit-line approach in improving laboratory service delivery by using the functionality of cd4 test equipment as a proxy indicator. method: a cross-sectional survey was conducted at 7 level-three health centres (hc iiis), 18 level-four health centres (hc ivs), and 10 hospitals in 15 districts of mid-northern uganda, including the lango (17 facilities) and acholi sub-regions (18 facilities), between july 2013 and august 2013. functionality, was determined through selfand interviewer-administered questionnaires. the chi-squared test was used to assess differences in functionality by sub-region, facility type, and equipment type. results: a total of 38 cd4 test analysers were assessed. of these, 26 (68%) were functional. in hospitals, 85% of cd4 analysers were functional, in hc ivs, 67% were functional and in hc iiis, 43% were functional. the differences did not reach statistical significance. in the lango sub-region, 72% of analysers were functional; in the acholi sub-region, 65% were functional. non-functionality was mainly due to lack of reagents and cartridges, as well as low staffing levels of laboratory technicians with the skills necessary to operate the equipment. conclusion: the credit-line approach supported the functionality of cd4 equipment in the surveyed facilities. however, there is a need to address issues of staffing and availability of reagents to enhance the functionality of cd4 equipment and improve patient care, especially at hc iiis. introduction top ↑ until 2003, the ministry of health (moh) of uganda used a ‘push’ system to guarantee supply of medical commodities, especially in areas affected by natural disasters or areas such as northern uganda, which was affected by a 20-year-long civil war. in the push system, the authorised supplier determines the types and quantities of supplies to be issued to health facilities.1 inconsistencies remained between the needs of the user areas and the medical items supplied. in many health facilities, there were frequent stock outs of supplies (including reagents for cd4 equipment) and large quantities of expired items (including cartridges for cd4 equipment).2 as a consequence, this led to non-functioning equipment and patients missing cd4 monitoring. the moh introduced a ‘pull’ system in 2003 to overcome the challenges of frequent stock outs and expired items. this system required health facilities to determine the types and quantities of medical items that they needed.3,4 an assessment of the performance of the pull system to improve the availability of equipment and reduce expiration of medical supplies showed that the pull system improved the availability of medical supplies, but did not address challenges such as inadequate training of staff, lack of transport and inadequate funding.5 in order to address the challenges of funding and transport, the moh introduced the ‘credit-line’ approach in 2009 at the government-owned national medical stores supported by development partners, including the united states centers for disease control and prevention, uganda; the clinton foundation health access initiative, uganda; unitaid; and global fund, amongst others. of the funding available, 80% was allocated to public health facilities and 20% to private not-for-profit health facilities.6 this approach requires that, during every cycle of two or four months, each health facility is allocated a financial vote, known as a credit line, from which to draw an equivalent of equipment and supplies. the moh’s central public health laboratories (cphl), the laboratory activities unit, identifies a list of supplies, recommends specifications, quantifies and makes forecasts that cater to the credit line for each facility. laboratory commodities and supplies, including cd4 equipment commodities, are procured and distributed or transported to the districts by the national medical stores for public facilities and by a private, not-for-profit store, the joint medical store, for public, not-for-profit facilities. each health facility is tasked with placing orders, picking up the supplies from the districts, maintaining consumption data and sending the data to cphl for use in making other forecasts.6 however, the role of this approach in supporting the functionality of facilities’ cd4 equipment has not been evaluated in northern uganda. laboratory monitoring of hiv patients determines eligibility for anti-retroviral treatment (art), which slows down the progression of hiv to aids, in addition to monitoring the efficacy of art after initiation.7 it has been shown that, compared with viral load, cd4 count is a better predictor of clinical progression of hiv to aids and is a better guide for initiation of art.8,9 in low-income countries, laboratory monitoring of patients on art remains controversial because of ongoing resource limitations.10 in addition, unreliable or inaccurate testing leads to unnecessary costs in areas that already experience shortages. this, in turn, leads to the perception that laboratory testing is unhelpful or that it could compromise patient care.11 in developing countries, cd4 testing is common in urban areas, where patients can undertake multiple visits to clinics. the expansion of art programmes into rural areas created a need for point-of-care (poc) cd4 testing in order to overcome logistical barriers in the timely dissemination of test results and initiation to care programmes.12 rapid, reliable and affordable poc cd4 tests are not yet widely available.13,14 some of the poc cd4 test technologies on the market or in development include: pointcare® now; cyflow® minipoc; pima™ cd4; coulter cd4 count kit; dynal® t4 quant kit; daktari cd4; mbio® cd4 system; visitect® cd4; and zyomyx®. some of these are being used in the northern region of uganda.15 poc cd4 testing is an efficient intervention to reduce pre-treatment loss to follow up, because it enables clinics to stage patients rapidly on site, so that more patients are determined to be eligible and initiated on art.16 the aim of diagnostic poc testing is to minimise the wait time to obtain a test result, allowing clinicians and patients to make a quick clinical decision. in resource-limited settings, the benefits of poc testing outweigh its costs by focusing on the relevant clinical outcomes.17 there is also a need for laboratory follow-up of hiv patients in resource-limited settings, if appropriate cd4 test equipment is used. the auto 40 system, which uses thermoresistant reagents, is one example of a cd analyser that is appropriate for such settings.18 the moh’s credit-line approach is intended to ensure adequate supply of such equipment and associated reagents for clinical monitoring of patients with hiv. we evaluated the effectiveness of the credit-line approach for improving laboratory service delivery to healthcare facilities in northern uganda. facilities were surveyed to determine the functionality of their cd4 equipment as a proxy indicator of the effectiveness of the approach, including reasons for non-functionality. various factors, such as facility type and staffing levels, were examined to suggest ways of improving laboratory service delivery in the country. methods top ↑ as part of the national laboratory supplies quantification and verification exercise, between 01 july 2013 and 02 august 2013, the moh’s cphl conducted a cross-sectional survey at health facilities in the lango and acholi sub-regions of mid-northern uganda. the lango sub-region includes eight districts and the acholi sub-region, seven districts. facilities in the study area included the following types ordered by complexity of service delivery: private for-profit; private not-for-profit and government or public clinics, including level-two health centres, which are closest to communities and offer basic health services; level-three health centres (hc iiis); level-four health centres (hc ivs) which offer more complex services similar to hospitals; general hospitals; and regional referral hospitals. at least one government laboratory facility was included for each of the 15 districts of mid-northern uganda (figure 1). laboratories in an hc iv facility were selected preferentially, because the moh plans to transform hc ivs into laboratory hubs. figure 1: area of survey: acholi and lango sub-regions of uganda. depending on the availability of the laboratory managers (or ‘in charges’), semi-structured questionnaires were either self-administered by the laboratory managers or interviewer-administered to the available laboratory staff by the research team. the research team, which was composed of three cphl technical staff, including a team leader and a driver, travelled to each facility to distribute and collect the questionnaires. the questionnaires gathered the following information: the type and location of cd4 equipment and functionality; reasons for non-functionality; and staffing levels for each cadre, including laboratory technologists, laboratory technicians, laboratory assistants and microscopists, by facility. completed questionnaires were first reviewed in the field by the research team to ensure completeness and accuracy. the questionnaires had an optional telephone contact field and collectors contacted the respondents to validate information that was not clear. the statistical package for the social sciences statistical software (version 17.0; spss inc., chicago, il 2008) was used for data entry and analysis. the chi-squared method was used to evaluate whether there was a statistically-significant difference between the functionality of cd4 equipment by sub-region, facility level or equipment type. the outcome variable for determining the effectiveness of the credit-line approach was whether the cd4 equipment at each facility was functional. functional was defined as cd4 equipment that was capable of carrying out cd4 tests. the credit-line approach was considered to be effective for facilities with functional cd4 test equipment. we also evaluated whether facility level, distribution and type of cd4 equipment or the staffing levels by facility affected the presence of functional cd4 equipment. p-values below the conventionally accepted significance level of 0.05 (or 5%) were considered to be statistically significant. results top ↑ a total of 16 self-administered questionnaires were completed by the managers of laboratory facilities. laboratory managers were absent at 19 laboratory facilities, at which the research team conducted interviewer-administered questionnaires with available staff. thus, of the 68 public facilities in the lango and acholi sub-regions, a total of 35 facilities were assessed (table 1), including 7 hc iiis, 18 hc ivs and 10 hospitals in the lango (17 facilities) and acholi (18 facilities) sub-regions. these 35 facilities included 38 cd4 analysers – 18 in the lango sub-region and 20 in the acholi sub-region. pima was a predominant type of cd4 equipment and the majority of cd4 analysers were in hc iv facilities (18 of 38; 48%). overall, 26 of the 38 (68%) cd4 analysers were functional (table 2). although the lango sub-region had fewer cd4 analysers than the acholi region, a higher percentage were functional (72% in lango vs. 65% in acholi). similarly, although there were more cd4 analysers at hc iv facilities, hospitals had a higher percentage of functional cd4 equipment (67% in hc iv facilities vs. 85% in hospitals); hc iiis had the lowest percentage of functional cd4 analysers (43%). there were no significant differences in the functionality of cd4 equipment by sub-region, facility level or equipment type. non-functionality of cd4 equipment was mainly because of the lack of reagents or cartridges (table 3). other reasons given included expired cartridges, unpredictable power sources and lack of controls. low staffing levels were reported amongst the various laboratory cadres with the skills necessary to operate the equipment (table 4). the lowest levels were observed amongst laboratory technologists at hospitals (6 of 31 positions filled; 19%) and laboratory assistants at both hc iii (4 of 12 positions filled; 33%) and hc iv facilities (19 of 70 positions filled; 27%). table 1: distribution of cd4 analysers at healthcare facilities surveyed in northern uganda, july–august 2013. discussion top ↑ this assessment of the credit-line approach for providing laboratory supplies (including commodities for the functionality of cd4 equipment) to health facilities found that 68% of cd4 equipment was functional, despite the low staffing levels of laboratory cadres by facility level. staffing issues related to provision of commodities and supplies still play a role in the effectiveness of the credit-line approach, especially in areas with a history of war and natural disasters.3,4,5 despite persistent resource constraints,10 poc cd4 testing is being implemented successfully in northern uganda to overcome logistical barriers to the timely dissemination of test results and initiation to care programmes.12 table 2: functionality of cd4 equipment by region, facility level and equipment type. limitations the credit-line approach in uganda is being supported by developing partners to provide commodities and supplies to public and private not-for-profit health laboratories for the functioning of cd4 equipment.6 this approach excludes specialised testing at high-level reference laboratories, which play a very important role in outbreaks and disease surveillance in the country. when interpreting our results, readers should also consider the lack of baseline data to use as a comparison between the push and pull systems with the credit-line approach; the fact that the selection of laboratories included in the analysis was not random; and that the intended respondents (laboratory managers) were not available for more than half of the surveyed laboratories where interviewer-administered questionnaires were instead conducted with laboratory staff members. recommendations there is a need to address issues of staffing and availability of reagents to enhance the functionality of cd4 equipment and improve patient care, especially at hc iiis. staffing issues related to the functionality of cd4 equipment used in the clinical monitoring of hiv patients, such as provision of commodities and supplies, should be regarded as highly important in the effectiveness of the credit-line approach. such issues may include recruitment, as well as retention and training. there is also a need to include specialised facilities in the credit-line approach systems for meaningful laboratory services in uganda. conclusion the credit-line approach supports the functionality of cd4 equipment used in the clinical monitoring of hiv patients in areas with a history of war and natural disasters, such as northern uganda. staffing issues related to provision of commodities and supplies play a role in the effectiveness of the credit-line approach. table 3: reasons for non-functionality of cd4 equipment. table 4: staffing levels of facilities by cadre. acknowledgements top ↑ the authors appreciate the support provided by mr wilson nyegenye (laboratory logistics coordinator of the moh’s cphl) in conceiving, designing and carrying out the survey. the authors also acknowledge dr olico okui of monitoring and evaluation technical assistance at the makerere university school of public health, for reviewing the drafts, as well as mr. william lali (coordinator quality assurance, cphl) for providing technical input on issues concerning laboratory work during the manuscript’s correction stage. funding sources the authors would also like to acknowledge the moh’s cphl and the maksph-cdc fellowship program for providing the financial and material support to conduct the survey. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions m.l.k. (makerere university school of public health, maksph-cdc fellowship program) was responsible for the project design, survey, statistical calculations and writing the manuscript. n.m.t. (makerere university school of public health) was responsible for scientific and conceptual contributions to the manuscript. s.a. (cphl, moh) was the project leader. j.k.b.m. (makerere university school of public health, maksph-cdc fellowship program) provided contextual contributions on the subject of the article and r.w. 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primary health clinics: an observational cohort study. lancet. 2011;378(9802):1572–1579. http://dx.doi.org/10.1016/s0140-6736(11)61052-0 drain pk, hyle ep, noubary f, et al. diagnostic point-of-care tests in resource-limited settings. lancet infect dis. 2014;14(3):239–249. pmid: 24332389, http://dx.doi.org/10.1016/s1473-3099(13)70250-0 dieye tn, diaw pa, daneau g, et al. evaluation of a flow cytometry method for cd4 t cell enumeration based on volumetric primary cd4 gating using thermoresistant reagents. j immunol methods. 2011;372(1–2):7–13. pmid: 21835181, http://dx.doi.org/10.1016/j.jim.2011.07.012 abstract introduction methods results discussion acknowledgements references about the author(s) franklin s. azebaze agueguia center for the study and control of communicable diseases (csccd), faculty of medicine and biological sciences (fmbs), university of yaoundé 1 (uy1), yaoundé, cameroon paul talla department of internal medicine and specialties, faculty of medicine and biomedical sciences (fmbs), yaoundé general hospital, university of yaoundé 1 (uy1), yaoundé, cameroon marie c. okomo assoumou department of microbiology, haematology, parasitology and infectious diseases, faculty of medicine and biomedical sciences (fmbs), university of yaoundé 1 (uy1), yaoundé, cameroon graeme b. jacobs division of medical virology, department of pathology, faculty of medicine and health sciences, stellenbosch university, tygerberg, south africa cedric h. mbakam center for the study and control of communicable diseases (csccd), faculty of medicine and biological sciences (fmbs), university of yaoundé 1 (uy1), yaoundé, cameroon elise guiedem center for the study and control of communicable diseases (csccd), faculty of medicine and biological sciences (fmbs), university of yaoundé 1 (uy1), yaoundé, cameroon martha tongo mesembe center for the study and control of communicable diseases (csccd), faculty of medicine and biological sciences (fmbs), university of yaoundé 1 (uy1), yaoundé, cameroon emilia lyonga center for the study and control of communicable diseases (csccd), faculty of medicine and biological sciences (fmbs), university of yaoundé 1 (uy1), yaoundé, cameroon george mondinde ikomey center for the study and control of communicable diseases (csccd), faculty of medicine and biological sciences (fmbs), university of yaoundé 1 (uy1), yaoundé, cameroon citation azebaze agueguia fs, talla p, okomo assoumou mc, et al. involvement of cd95 and ligand in cd4+ t-cell and cd8+ t-cell depletion and hepatic cytolysis in patients with chronic viral hepatitis b. afr j lab med. 2021;10(1), a1224. https://doi.org/10.4102/ajlm.v10i1.1224 original research involvement of cd95 and ligand in cd4+ t-cell and cd8+ t-cell depletion and hepatic cytolysis in patients with chronic viral hepatitis b franklin s. azebaze agueguia, paul talla, marie c. okomo assoumou, graeme b. jacobs, cedric h. mbakam, elise guiedem, martha tongo mesembe, emilia lyonga, george mondinde ikomey received: 16 mar. 2020; accepted: 20 oct. 2020; published: 15 mar. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: chronic viral hepatitis b (hbv) is characterised by progressive hepatocyte destruction and t-cell depletion. the mechanisms of the cd95-cd95 ligand (cd95l) signalling pathway during this chronic disease and the cirrhotic process remains unclear. objective: we evaluated the involvement of the cd95-cd95l receptor-ligand system in t-cell depletion and hepatic cytolysis in patients with chronic hbv. methods: this was a cross-sectional study conducted from september to december 2018 at the yaoundé general hospital, cameroon. four ml of whole blood was collected and analysed. the cd95 and cd95l levels, as well as the cd4+ t-cell and cd8+ t-cell counts, were performed by enzyme-linked immunosorbent assay and flow cytometry. results: of the 130 hbv-positive patients, 36 (27.7%) were cirrhotic and 94 (72.3%) were non-cirrhotic. the cirrhotic patients had significantly elevated cd95 (p < 0.001) and cd95l (p = 0.001) plasma levels, compared with non-cirrhotic patients. the cd4/cd8 ratios were lower in cirrhotic patients, compared to non-cirrhotic patients (p < 0.001). there were statistically significant correlations between cd95 level and cd4+ t-cell counts, between cd95 level and cd8+ t-cell counts, between cd95 level and the cd4/cd8 ratio, between cd95 level and fibrosis score, and between cd95l level and fibrosis score. conclusion: cd95 and cd95l could be involved in t-cell depletion and hepatic cytolysis during the pathogenesis of chronic hbv and could potentially be used as biomarkers for immunological and hepatic monitoring in patients with chronic hbv. keywords: hbv; cd95-cd95l; cd4+ t-cells; cd8+ t-cells; cirrhosis. introduction the hepatitis b virus (hbv) is the leading cause of chronic liver disease and remains a major global health burden.1 chronic hbv infection is denoted by the persistence of the hbv surface antigen in the host for at least 6 months.1 viral hepatitis b is characterised by the progressive onset of fibrotic hepatic lesions; the pathogenesis remains unclear.2 counts of cd4+ and cd8+ t-cells, which play an indispensable role in the resolution of hbv infection,2 are significantly lower in chronic hbv, but the mechanisms resulting in their depletion remain unclear.3 the severity of liver lesions caused by infection varies according to the immune status of the patient.2 an appropriate immune response leads to the necrosis of infected hepatocytes and the elimination of the virus.1 an excessive immune response induces massive hepatocyte destruction, while immunotolerance is marked by abundant but asymptomatic viral multiplication without destruction of hepatocytes.3 in chronic carriers of hbv, this immune response exists, but it is insufficient to eliminate the virus.3 the persistence of hbv in hepatocytes leads to repeated attempts to eliminate them by t-cells.2 this is mediated by several immune mechanisms, including apoptosis, which is a physiological process of cell death, implementing an actual cell lysis programme.3 the triggering of this ‘programmed death’ is done through the activation of specialised signalling pathways, namely the cd95-cd95 ligand (cd95l) pathway.3 cd95 (fas or apo-1) is the receptor of cd95l (fas ligand, fasl); cd95 belongs to the tumor necrosis factor-receptor/nerve growth factor-receptor (tnf-r/ngf-r) family.3 members of this family are characterised by the presence of cysteine-rich domains in their extracytoplasmic portion; cd95 has three cysteine-rich domains.3 the ‘death receptor’ subfamily is distinguished at the intracytoplasmic portion that contains a domain of about 80 amino acids called the ‘death domain’.3 the induction of apoptosis by cd95 follows its oligomerisation by an agonist monoclonal antibody or by its natural ligand, cd95l.3 the latter belongs to the superfamily of tnf/ngf.3 the cd95 molecule is expressed in many tissues including the liver, the heart and the hematopoietic tissues.3 however, a total absence of expression of the cd95 protein in humans has no direct repercussions on the lymphoid system.4 cd95 is expressed on the surface membrane of activated t-lymphocytes or b-lymphocytes, during the first activation.5 after a second activation, the t-lymphocyte expresses cd95l and kills the activated lymphocytes (death in ‘trans’), including itself (death in ‘cis’), which is called ‘activation-induced cell death’ (aicd).5 the cd95-cd95l interaction, therefore, represents a route of control of the immune response, and the regulation of autoimmune proliferations.5 cd95-mediated t-cell apoptosis is a well-known mechanism for the prevention of immunopathogenesis and the maintenance of immune tolerance through the contraction of t-cell responses. this is described as an immune checkpoint mechanism.5 three mechanisms maintain peripheral t-cells homeostasis: t-cells anergy, suppression by regulatory t-cells and aicd.6 the population of effector t-cells is controlled by aicd, which is initiated by repeated stimulation of the t-cell receptor via apoptosis mediated by cd95.7 thus, aicd mediated by cd95 plays a very important role within the peripheral immune system.6 the mechanisms of the cd95-cd95l apoptotic signalling pathway during chronic hbv remains to be elucidated.6 our objective was to evaluate the involvement of cd95-cd95l receptor-ligands in t-cell depletion and hepatic cytolysis in patients with chronic hbv. methods ethical considerations ethical approval to conduct the study was obtained from the institutional ethics and research committee for human health, of the catholic university of central africa (no. 2019/0803/ceirsh/ess/mim). written and verbal informed consent was given by all participants. the study was conducted according to the ethical principles and guidelines of the international declaration of helsinki 2013. all procedures were standard and presented a minimal risk to participants. the names of the participants were replaced by alphanumeric codes and kept in a safe place, accessible only by the principal investigator. the physical documents were kept in secure locked drawers within the processing site. the digital documents were encrypted and secured by passwords and stored on secure hard disks. study design we performed a cross-sectional study from september to december 2018 of patients with chronic hbv infection. participants received consultation at the hepato-gastroenterology department of the yaoundé general hospital, cameroon. patient recruitment was consecutive and non-probabilistic. the results of the biological analysis were returned to the patients and incorporated into their medical records. study population from the fibro test examination, patients were divided based on their metavir fibrosis score into cirrhotic and non-cirrhotic hbv-infected patients. recruited patients were between 18 and 60 years of age. patients with liver diseases of other aetiologies or those having hbv infection and a history of autoimmunity, drug-dependence or co-infection with other viruses, including hepatitis c virus (hcv), hepatitis d virus (hdv), hiv and human t-cell leukemia virus type 1, were excluded from the study. the selection criteria for the control participants were: not having a medical history of hbv, hcv, hdv, human t-cell leukemia virus type 1 or hiv infection, autoimmunity and not having consumed alcohol in the past 7 years. these control participants were selected from a population of blood donors at the yaoundé general hospital and screened for these viral infections and diseases. social characteristics (age and sex) and clinical information (cirrhosis statute, levels of cd95 and cd95l, counts of cd4+ t-cells, cd8+ t-cells and cd4/cd8 ratio) were collected for each participant using a standard questionnaire. sample collection and analysis site four millilitres of whole blood was collected into ethylene diamine tetra acetate acid anticoagulant tubes (greiner bio-one international gmbh, kremsmünster, austria), and transported at room temperature to the immuno-virology laboratory of the center for the study and control of communicable diseases of the faculty of medicine and biomedical sciences of the university of yaoundé 1. these samples were analysed within 24 h for cd4+ t-cells, cd8+ t-cells and the cd4/cd8 ratio. plasma was obtained by centrifugation of whole blood at a speed of 5000 revolutions per minute for 5 min and aliquoted in cryovials. plasma samples were frozen at −20 °c, for the subsequent determination of cd95 and cd95l levels. metavir scores of fibrosis were collected from patients’ medical records. determination of cd4+ t-cell and cd8+ t-cell count and the cd4/cd8 ratio we used the whole blood collected for the determination of cd4+ t-cells, cd8+ t-cells and the cd4/cd8 ratio. this was performed by flow cytometry, which is a technology that provides rapid multi-parametric analysis of single cells in solution, with specific fluorochrome-conjugated antibodies, using the becton dickenson fluorescent activated cells sorting system (biosource, san jose, california, united states). results were obtained automatically for the absolute cd4+ t-cells and cd8+ t-cells count, including the cd4/cd8 ratio. this was done according to the manufacturer’s recommendations. determination of cd95 and cd95l plasma level the determination of the cd95 and cd95l plasma levels were performed by the sandwich enzyme-linked immunosorbent assay (elisa) technique (quantikine®, r&d systems, abingdon, united kingdom), following the manufacturer’s instruction. optical densities were measured at 450 nm using an elisa reader (sunrise tecan, tecan gmbh, grödig/salzburg, austria), and all samples were assayed in duplicate. the cd95 and cd95l levels in the samples were determined by extrapolating the results from a standard curve. statistical analysis data from this study were recorded in microsoft office excel 2016, and statistical analysis was performed using epiinfo® 7.0 (division of public health surveillance and informatics epidemiology program office, ms k74 centers for disease control and prevention, atlanta, georgia, united states) and graph pad prism 5.0 (graph pad software inc., la jolla, california, united states). comparisons between cd4+ t-cells, cd8+ t-cells, cd4/cd8 ratio, cd95 and cd95l between different groups were performed using the non-parametric test of mann-whitney and kruskal-wallis. the correlations between cd95, cd95l, cd4+ t-cells, cd8+ t-cells, cd4/cd8 ratio and fibrosis score were established using the spearman’s correlation coefficient (r). all values of p under 0.05 were considered statistically significant at a confidence interval of 95%. results for 190 patients recruited, 130 (68.42%) were hbv-infected patients, and 60 (31.58%) were control participants. among the control participants, 36 (60.00%) were male and 24 (40.00%) were female, with an average age of 35 ± 13 years. of the 130 hbv-infected patients, 36 (27.70%) were cirrhotic and 94 (72.30%) were non-cirrhotic. among the cirrhotic patients, 23 (64.00%) were male and 13 (36.00%) were female, with an average age of 38 ± 16 years. among non-cirrhotic patients, 52 (55.3%) were male and 42 (44.70%) were female, with an average age of 31.54 ± 9 years (table 1). table 1: social and clinical characteristics of hepatitis b virus-positive patients at yaoundé general hospital, cameroon, september–december 2018. the level of cd95 in cirrhotic patients ranged from 0.6 picograms per millilitre (pg/ml) to 6.5 pg/ml, with a median of 5.3 pg/ml. the level of cd95 in non-cirrhotic patients ranged from 0.7 pg/ml to 6.3 pg/ml, with a median of 3.2 pg/ml. the difference between the cd95 levels of the cirrhotic and non-cirrhotic patients was statistically significant, with a p-value of less than 0.001 (figure 1a). the level of cd95l in cirrhotic patients ranged from 1.8 pg/ml to 5.9 pg/ml, with a median of 4.2 pg/ml. the level of cd95l in non-cirrhotic patients ranged from 1.3 pg/ml to 5.3 pg/ml, with a median of 3.6 pg/ml. the difference between the cd95l levels of the cirrhotic and non-cirrhotic patients was statistically significant, with a p-value of 0.001 (figure 1b). figure 1: level of cd95 and cd95l in cirrhotic and non-cirrhotic hepatitis b virus-positive patients at yaoundé general hospital, cameroon, september–december 2018. the cd4+ t-cell values in cirrhotic patients ranged from 608 cells/µl to 1024 cells/µl, with a median of 784.5 cells/µl. the cd4+ t-cell values in non-cirrhotic patients ranged from 688 to 1116 cells/µl, with a median of 901.3 cells/µl. the difference between the cd4+ t-cell values of the cirrhotic and non-cirrhotic patients was statistically significant, with a p-value of 0.003 (figure 2a). figure 2: value of cd4+ t-cells, cd8+ t-cells and the cd4/cd8 ratio in cirrhotic and non-cirrhotic hepatitis b virus-positive patients at yaoundé general hospital, cameroon, september–december 2018. the cd8+ t-cell values in cirrhotic patients ranged from 508 cells/µl to 1003 cells/µl, with a median of 774.5 cells/µl. the cd8+ t-cell values in non-cirrhotic patients ranged from 585 cells/µl to 1013 cells/µl, with a median of 786 cells/µl. the difference between the cd8+ t-cell values of the cirrhotic and non-cirrhotic patients was not statistically significant, with a p-value of 0.417 (figure 2b). the cd4/cd8 ratio in cirrhotic patients ranged from 1.0 to 1.2, with a median of 1.0. the cd4/cd8 ratio in non-cirrhotic patients ranged from 1.0 to 1.4 with a median of 1.2. the difference between the cd4/cd8 ratios of the cirrhotic and non-cirrhotic patients was statistically significant, with a p-value of less than 0.001 (figure 2c). within the group of non-cirrhotic and cirrhotic patients infected with hbv, there was a statistically significant direct correlation between cd95 and cd4+ t-cells (r = 0.042, p = 0.016), and between cd95 and the cd4/cd8 ratio (r = 0.123, p = 0.01). within the group of cirrhotic patients infected with hbv, there was a statistically significant direct correlation between cd95 and fibrosis score (r = 0.021, p = 0.003). in this same group, there was a statistically significant inverse correlation between cd95 and cd8+ t-cells (r = −0.029, p = 0.016) (table 2). we also found, within this same group, a statistically significant direct correlation between cd95l and the fibrosis score (r = 0.099, p = 0.003). there were statistically significant inverse correlations between the fibrosis score and the cd4+ t-cells (r = −0.280, p < 0.001), and between the fibrosis score and cd4/cd8 ratio (r = −0.455, p < 0.001) within the group of cirrhotic patients infected with hbv (table 2). table 2: correlation between cd95, cd95l, fibrosis score, cd4+ t-cells, cd8+ t-cells and cd4/cd8 ratio of hepatitis b virus-positive patients at yaoundé general hospital, cameroon, september–december 2018. discussion we sought to assess the implications of cd95-cd95l receptor-ligands in t-cell depletion and hepatic cytolysis, in patients with chronic hbv. we found that the plasma levels of cd95 and cd95l were significantly elevated in cirrhotic patients, compared to non-cirrhotic patients. the cd4/cd8 ratios were lower in cirrhotic patients, compared to non-cirrhotic patients. there were statistically significant correlations between the cd95 level and cd4+ t-cell count, between the cd95 level and the cd8+ t-cell count, between the cd95 level and cd4/cd8 ratio, between the cd95 level and fibrosis score, and between the cd95l level and fibrosis score. the concentrations of cd95 and cd95l were higher in cirrhotic patients than in non-cirrhotic patients, with a statistically significant difference between the two groups. these results corroborate those of peter et al.: they observed low constitutive expression of cd95 in non-cirrhotic patients with chronic hbv, compared to patients with cirrhosis related to hbv.8 the results obtained in this study agree with literature that cd95 is overexpressed during hbv infection.9 the expression of cd95 increases in response to a primary stimulus and this makes hepatocytes more susceptible to stimulation by cd95l. this hypothesis is supported by the observation that induction of cd95 expression occurs following chronic lymphatic histiocytic inflammation in different epithelial cells.6 these results suggest that liver damage in hbv-infected patients may primarily involve the destruction of hepatocytes by t-cells using the cd95-cd95l receptor-ligand system. immune system abnormalities are associated with t-cell depletion.10 we found statistically significant differences in cd4+ t-cell values between cirrhotic and non-cirrhotic patients. mean values of cd4+ t-cells in cirrhotic patients were lower than those observed in non-cirrhotic patients. also, the mean values of cd8+ t-cells in cirrhotic patients were higher than in non-cirrhotic patients. these abnormalities can be caused by abnormal regulation of the activation gene for the antigen 4 of cytotoxic t-cells, the lymphocytes 3, the immunoglobulin t domain and the mucin 3 domain, the cell death receptor 1, the cd244/2b4, the cd160 and by the t-cells immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif domains.11,12 the average cd4/cd8 ratio in cirrhotic patients was similar to that found in 2016 by yang et al.13 the cd4/cd8 mean ratio, however, was lower than that found in non-cirrhotic patients. this could reflect a possible increased immune depletion in cirrhotic patients, compared to non-cirrhotic patients. we highlighted the existence of a statistically significant direct correlation between cd95 and cd4+ t-cells in hbv-infected patients, cirrhotic and non-cirrhotic. at the same time, there was a statistically significant, but inverse, correlation between cd95 and cd8. these results could be explained by the fact that the increase in activated cd8 t-cells is followed by their differentiation into cytotoxic t-cells. cytotoxic t-cells are effector lymphocytes that cause the destruction of neighbouring cells having cd98 receptors on their surfaces, thus causing a decrease in the cd98 receptor. the statistically significant direct correlation between cd95 and the cd4/cd8 ratio observed in cirrhotic hbv-infected individuals could be due to the influence of the number of activated cd4+ t-cells carrying cd95 receptors, in the cd4/cd8 ratios. the increase in the number of activated cd4+ t-cells directly involves an increase in the production of cd95 receptors, which in turn increases the plasma concentration of cd95 receptors. although the correlation observed between cd95l and cd4+ t-cells and between cd8+ t-cells and the cd4/cd8 ratio was not statistically significant, both had inverse relationships in cirrhotic hbv-infected individuals. the cd95l level evolved in the opposite direction to the cd4+ t-cells, cd8+ t-cells and the cd4/cd8 ratio. this could be explained by the maintenance of the peripheral tolerance of t-cells activated by aicd mediated by the interaction between cd95 and cd95l.6 indeed, over-activated effector t-cells are harmful to the immune system and must be removed from body tissues. also, there was a statistically significant direct correlation between the metavir fibrosis score and the cd95 and cd95l concentrations in these cirrhotic hbv-infected patients. this reflected an association between liver injury and activation of the cd95-cd95l apoptosis pathway. these results corroborate those obtained by peter et al. in a study of cd95 receptor and ligand involvement in hepatic injury,8 where cd95 receptor expression was very high in hepatocytes in hbv-related cirrhosis.12 these results are also parallel to those obtained in the case of infections with hcv by hayashi and mita,14 where the expression of cd95 was upregulated according to the severity of inflammation of the liver. there was a statistically significant inverse correlation between the fibrosis score and the cd4+ t-cell count as well as the cd4/cd8 ratio in cirrhotic hbv-infected patients. these results reflected an association between the specific cellular immune response via the activation of the cd4+ t-cells who are the ‘coordinators’ and the liver lesions that occurred; this therefore suggests that the destruction of hepatocytes in hbv infection would be induced by t-cells, using cd95-cd95l mediation.12 there was no statistically significant inverse correlation between the fibrosis score and cd8+ t-cell concentration in cirrhotic hbv-infected patients. these results show that expression levels of the constitutive receptor cd95 are functionally sufficient to mediate apoptosis of hepatocytes and t-cells. limitations our study did not evaluate the activation of the cd95-cd95l signalling pathway during the evolution of hbv infection. we, therefore, propose a longitudinal study, which will evaluate the activation of the cd95-cd95l signalling pathway during the progression of the viral infection, from cultures of hepatocytes infected with hbv, and which will evaluate the inhibition of cd95 receptors as an immunotherapeutic method in patients with chronic hbv. conclusion the levels of cd95 and cd95l were higher in cirrhotic patients compared to non-cirrhotic patients. the cd4+ t-cell counts, cd8+ t-cell counts and cd4/cd8 ratios were respectively lower, higher, and lower in cirrhotic patients compared to non-cirrhotic patients. thus, cd95-cd95l could be involved in t-cell depletion and hepatic cytolysis during the pathogenesis of chronic hbv and could potentially be used as biomarkers for immunological and hepatic monitoring in patients with chronic hbv. acknowledgements we thank the staff of the yaoundé general hospital, cameroon, and the participants of this study. competing interests the authors have declared that no competing interests exist. authors’ contributions f.s.a.a., p.t. and g.m.i. designed the project and the main conceptual ideas. g.m.i., p.t., c.h.m. and g.b.j. worked on the technical and methodological details. f.s.a.a. and p.t. recruited participants and collected blood samples. g.m.i., m.t.m., g.b.j., c.h.m., e.g. and e.l. participated in the planning and supervision of the work. f.s.a.a. performed the biological analysis of the blood samples. f.s.a.a. and c.h.m. carried out the statistical analysis of the data and designed the figures and tables. f.s.a.a., g.m.i., c.h.m., g.b.j., m.c.o.a. and m.t.m. contributed to the interpretation of the results and the writing of the manuscript. all the authors discussed the results and commented on the manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability statement the data supporting the results of this study are available on request from the corresponding author, f.s.a.a. the data are not publicly available because they contain information that could compromise the confidentiality of research participants. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references dane ds, cameron ch, briggs m. virus-like particles in serum of patients with australia-antigen-associated hepatitis. lancet. 1970;1(7649):695–698. https://doi.org/10.1016/s0140-6736(70)90926-8 klenerman p, thimme r. t cell responses in hepatitis c: the good, the bad and the unconventional. gut. 2012 aug;61(8):1226–1234. https://doi.org/10.1136/gutjnl-2011-300620 maryam k, seyed ma, heidar s. comparison of serum 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the ratios of peripheral blood t lymphocytes for patients with hepatitis b virus-related acute-on-chronic liver failure. biomed res. 2016;27(2):517–520. hayashi n, mita e. fas system and apoptosis in viral hepatitis. j gastroenterol hepatol. 1997 oct;12(9–10):s223–s226. https://doi.org/10.1111/j.1440-1746.1997.tb00504.x article information author: henry a. mbah1 affiliation: 1senior technical advisor lab, fhi-360, nigeria correspondence to: henry mbah postal address: fhi-360, plot 1073-a1, j.s. tarka street, godab plaza, area 3 garki-abuja, nigeria dates: received: 06 aug. 2013 accepted: 03 apr. 2014 published: 18 aug. 2014 how to cite this article: mbah ha. phlebotomy and quality in the african laboratory. afr j lab med. 2014;3(1), art. #132, 4 pages. http://dx.doi.org/10.4102/ ajlm.v3i1.132 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. phlebotomy and quality in the african laboratory in this opinion paper... open access • abstract • introduction    • phlebotomy and the error-prone pre-analytical stage    • phlebotomy has been undervalued worldwide    • current practice in africa       • staff       • space       • quality of specimen       • logistics       • identification       • safety       • documentation       • training    • existing complexity in the setup    • recommendations • conclusion • acknowledgements    • competing interest • references abstract top ↑ phlebotomy, the act of drawing blood through venepuncture, is one of the most common medical procedures in healthcare, as well as being a basis for diagnosis and treatment. a review of the available research has highlighted the dearth of information on the phlebotomy practice in africa. several studies elsewhere have shown that the pre-analytical phase (patient preparation, specimen collection and identification, transportation, preparation for analysis and storage) is the most error-prone process in laboratory medicine. the validity of any laboratory test result hinges on specimen quality; thus, as the push for laboratory quality improvement in africa gathers momentum, the practice of phlebotomy should be subjected to critical appraisal. this article offers several suggestions for the improvement of phlebotomy in africa. introduction top ↑ medical laboratory services, despite playing a pivotal role in africa’s healthcare system, have suffered gross neglect for decades.1,2 government investment in laboratories is often inadequate, resulting in substandard laboratory infrastructure; poorly-trained and unmotivated staff; and a limited scope of testing services. furthermore, it is widely understood that insufficient investment in the laboratory can have a negative effect on the quality of testing services, impacting the overall quality of healthcare. over the past five years, efforts have been made to improve laboratory services in africa. in january 2008, the consensus meeting on clinical laboratory testing harmonization and standardization convened governments, agencies and development partners in maputo, mozambique.3 the meeting established the maputo declaration on strengthening of laboratory systems, aimed at improving clinical laboratory services in developing countries.3 subsequent meetings held in 2008 on african laboratory medicine in lyon, france, yaoundé, cameroon and dakar, senegal formulated strategies for laboratory strengthening and arrived at several landmark achievements.3 in 2009, the world health organization, regional office for africa (who-afro) and partners launched the stepwise laboratory quality improvement process towards accreditation (slipta) to help laboratories in resource-limited settings strive toward international accreditation.3,4 the african society for laboratory medicine (aslm) was launched in 2011 and aslm’s first international conference for laboratory medicine was held in 2012. phlebotomy and the error-prone pre-analytical stage phlebotomy, also called venesection or venotomy, is the incision into a vein for the purpose of drawing blood5 that is used for laboratory analysis, diagnosis, transfusions and research. the person who performs phlebotomy is called a phlebotomist. phlebotomists in africa are also commonly responsible for collecting and properly packaging specimens (blood, sputum, urine, other body fluids, tissues, etc.), accepting incoming specimens and routing specimens to the proper section for analysis. the significant role phlebotomists play in the delivery of essential healthcare services has been described numerous times in existing literature.6,7,8,9phlebotomists are often the only laboratory professional a patient will meet during a hospital stay.accurate and precise laboratory test results depend on properly-performed phlebotomy in order to obtain high-quality specimens. the most well-trained testing staff, using the most sophisticated instruments, cannot produce accurate results from a poorly-collected specimen. traditionally, the laboratory testing process is divided into three phases: pre-analytical, analytical, and post-analytical. there have also been suggestions that the pre-analytical phase be divided into a ‘pre-pre-analytical phase’ and a ‘true pre-analytical phase’.10 phlebotomy falls within the realm of the pre-pre-analytical phase, which includes steps (test requesting, patient and sample identification, sample collection and sample transportation) that may neither be performed in the laboratory nor undertaken by laboratory personnel. the post/pre-analytical phase, which is carried out in the laboratory after specimen reception, involves the steps required to prepare samples for analysis (centrifugation, aliquoting and sorting). several studies suggest that in laboratory diagnosis, most errors occur within the pre-analytical phase (46% – 70%), followed by the post-analytical phases (18% – 47%), with the fewest errors occurring in the analytical phase (7% – 13%).11,12,13 the most frequent pre-analytical errors in laboratory medicine include: missing sample and/or test request; incorrect or missing identification; in vitro haemolysis; undue clotting; use of the wrong container; contamination from infusion route; insufficient sample; inappropriate blood-to-anticoagulant ratio; insufficient mixing of the sample; inappropriate transport; and incorrect storage conditions.12 phlebotomy has been undervalued worldwide historically, the critical role of phlebotomy has been overlooked,14 having been suggested as the most underestimated procedure in healthcare.6,14,15 for example, although most employers in the united states (us) require valid certification or a licence issued by an accredited phlebotomy training programme or a professional body such as the american society of phlebotomy technicians (aspt), only five us states mandate phlebotomy certification for practise.16 according to a recent survey on phlebotomy practice in 28 european countries, 21% of the countries do not require specific training for phlebotomy; national phlebotomy guidelines are available in only 25% of the countries; and only 36% have specific training available as a continuous educational resource.17 in many countries (and most countries in europe), there are no professional phlebotomists.13 phlebotomy is performed by doctors, nurses, laboratory staff and other healthcare professionals. current practice in africa anecdotal evidence (i.e., accounts from individual healthcare workers) from countries such as cameroon, chad, côte d’ivoire, kenya and nigeria indicates a high prevalence of suboptimal phlebotomy practices. unfortunately, because of the paucity of published information on phlebotomy practice in africa, this paper refers to information from europe and the us, where causes of phlebotomy service issues have been well researched and may be similar to those faced in resource-limited settings. staff in many facilities, laboratory phlebotomy staff draw blood from outpatients whereas doctors and nurses usually draw blood from inpatients. less oversight from the laboratory could contribute to service quality issues if medical staff have multiple tasks to perform simultaneously. use of trained phlebotomy-specific personnel may greatly reduce pre-analytical error rates.in many facilities across africa, resource constraints require that staff be cross-trained for multiple tasks, possibly eroding specific skills and resulting in excessive workloads. furthermore, the practice of rotating staff through different facilities likely decreases institutional expertise, especially if adequate planning and training are not performed well in advance. a study in europe found that the rates of pre-analytical errors are higher for inpatients than outpatients, for whom procedures are performed by personnel under direct laboratory control.18 a publication from the us concluded that phlebotomists are preferred over nurses and physicians for blood draws.19 increasingly, phlebotomy skills are being diluted in african healthcare settings through the implementation of task shifting and multi-skilled staffing strategies. thus, a decrease in phlebotomy expertise is exposing an increasing number of facilities to serious underlying safety problems and the likelihood of liabilities. space in most cases, dedicated space is not available for specimen collection; blood is drawn in patient waiting areas, laboratory result-collecting areas, in the heart of the laboratory, or in corridors and passageways, without demarcation. thus, confidentiality may be compromised. quality of specimen the correct order of blood draw is not well understood and recommended volumes are not always considered.20 blood may be drawn from intravenous infusion devices and needles may be withdrawn with the tourniquet still in place. there may also be inadequate quality checks by supervisors. logistics materials and supplies are often inadequate; blood is frequently drawn into an ordinary syringe and moved, exposed, from the wards to the laboratory. using ordinary syringes, the volume and proportion of mixture with anticoagulant and other additives relative to the intended test may be ignored. in the wards in particular, piercing the skin with ordinary needles and failure to put pressure on the puncture site, have most likely caused risky situations in which blood leaks down the patient’s arm onto the bed and clothing. identification poor labelling is a major source of concern.20 labelling is done locally, by matching the patient name with a number written with wax marker, coloured pencil, or marker onto the tubes and, in some cases, on the rubber tube caps only. this may lead to a mix-up of specimens during processing when, in some cases, the tops are removed before centrifugation. some technicians may claim they still know which tube belongs to which patient, which is highly unlikely. transfusion-related deaths21 and undue surgeries22 have been traced back to patientor specimen-identification errors. one laboratory even reported pregnancy in a male patient. safety some staff members may not wear or change gloves23 between patients, either because of limited supply or lack of training and supervision. in some cases, staff members have expressed concern that they would not be able to feel a vein with gloves on. touching the incision site to find the vein after alcohol swabbing is common23 and can expose the patient to risk of infection. sharps containers and colour-coded waste bins are often absent; it is not uncommon to find only a single trash bin without lining for general use. such practices have exposed healthcare workers to needle-stick injuries and sharps injuries.24,25 documentation standard operating procedures (sops) are not readily available. where they exist, sops are too often locked up and not easily accessible to all staff members who perform phlebotomy. in the wards, clinical sops are severely lacking. policies are rarely available and job aids are uncommon, or there is no appropriate place to affix them. training special training, continuous and refresher training and certification or competency assessment in phlebotomy are rarely mandatory. it is worth noting that in south africa, continuous phlebotomy training and certification is required. in donor-funded antiretroviral therapy (art) programmes, the drive to put as many persons as possible on art may overshadow plans for phlebotomy training. ironically, the quality of an art programme hinges on the entry point of sample collection and testing. existing complexity in the setup although the importance and vulnerability of the preand post-analytical phases have been acknowledged for many years, current quality management programmes still tend to focus on the performance and efficiency of analytical processes and activities within the direct control of the laboratory. there are concerns on the thoroughness of coverage of the pre-analytical phase in the major accreditation schemes. the current tiered quality improvement scheme, slipta, focuses on resource management covering nine of the 14 requirements considered by iso 15189/2007 on pre-examination procedures.26 despite the coverage by iso 15189/2007, pre-analytical errors remain, even in an accredited laboratory.27 to date, the pre-analytical variables that lie outside the direct control or supervision of laboratory personnel are difficult to monitor, as tools and policies are not fully standardised or harmonised worldwide. recommendations some suggestions are proposed to stimulate thoughts and actions that will help to improve phlebotomy practice and reduce pre-analytical errors in laboratory medicine. firstly, clear written procedures from existing guidelines should be developed.28,29 phlebotomy techniques should be standardised and sops, operative guidelines and preventive and reporting policies widely disseminated. secondly, a dedicated phlebotomist should be appointed, if possible, and/or specific healthcare professional training, continuous education and routine competency assessment should be enhanced. quality indicators focusing on the pre-analytical phase should be adapted, implemented and monitored in quality improvement projects.30 external quality assurance programmes should be modified to check the entire examination process, including pre-analytical and post-analytical procedures. phlebotomy services in health facilities should be centralised and communication amongst healthcare professionals and interdepartmental cooperation should be improved. finally, field studies should be undertaken in order to address the dearth of research in the area of phlebotomy practice and pre-analytical errors in africa. conclusion top ↑ in some african countries, the quality of phlebotomy, the entry point to laboratory testing, is inadequate. this important period in the struggle for laboratory quality improvement in africa provides a window of opportunity to enhance strategies to improve phlebotomy practices. acknowledgements top ↑ special thanks are extended to professor julius n. anyu of the department of public administration at the university of the district of columbia, usa and miss ashley mbah, a high school junior at st. thomas more academy, magnolia, delaware, usa, for reviewing this article. the views expressed herein are those of the author and do not necessarily reflect those of fhi-360. competing interest the author declares that he has no financial or personal relationship(s) which may have inappropriately influenced him in writing this article. references top ↑ 1.petti ca, polage cr, quinn tc, et al. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006;42(3):377–382. 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bonini p, et al. haemolysis: an overview of the leading cause of unsuitable specimens in clinical laboratories. clin chem lab med. 2008;46(6):764–772. http://dx.doi.org/10.1515/cclm.2008.170 19.caruana c. the laboratory test begins with phlebotomy. lab med. 2003;34(8): 566–576. http://dx.doi.org/10.1309/095mc7vc1hycl1cj 20.makubi an, meda c, magesa a, et al. audit of clinical-laboratory practices in haematology and blood transfusion at muhimbili national hospital in tanzania. tanzan j health res. 2012;14(4):1–8. 21.myhre ba, mcruer d. human error − a significant cause of transfusion mortality. transfusion. 2000;40(7):879–885. http://dx.doi.org/10.1046/j.1537-2995.2000.40070879.x 22.chassin mr, becher ec. the wrong patient. ann intern med. 2002; 136(11):826–833 [comment in rice f. the wrong patient. ann intern med. 2003;138(6):518, author reply 518–519]. http://dx.doi.org/10.7326/0003-4819-136-11-200206040-00012 23.wondimu h, addis z, moges f, et al. bacteriological safety of blood collected for transfusion at university of gondar hospital blood bank, northwest ethiopia. isrn hematol. 2013; 308204:7 pages. 24.nsubuga fm, jaakkola ms. needle stick injuries among nurses in sub-saharan africa. trop med int health. 2005;10(8):773–781. http://dx.doi.org/10.1111/j.1365-3156.2005.01453.x 25.kebede g, molla m, sharma hr. needle stick and sharps injuries among health care workers in gondar city, ethiopia. saf sci. 2012;50(4):1093–1097. http://dx.doi.org/10.1016/j.ssci.2011.11.017 26.datema ta, oskam l, van beers sm, et al. critical review of the stepwise laboratory improvement process towards accreditation (slipta): suggestions for harmonization, implementation and improvement. trop med int health. 2011;17(3):361–367. 27.simundic am, nikolac n, vukasovic i, et al. the prevalence of preanalytical errors in a croatian iso 15189 accredited laboratory. clin chem lab med. 2010;48(7): 1009–1014. http://dx.doi.org/10.1515/cclm.2010.221 28.world health organization. who guidelines on drawing blood : best practices in phlebotomy. geneva: world health organization; 2010. 29.ernst d. procedures for the collection of diagnostic blood specimens by venipuncture. wayne, pa: clinical and laboratory standards institute; 2007. 30.plebani m, chiozza ml, sciacovelli l. towards harmonization of quality indicators in laboratory medicine. clin chem lab med. 2013;51(1):187–195. http://dx.doi.org/10.1515/cclm-2012-0582 abstract introduction methods results discussion acknowledgements references about the author(s) diana r. hardie department of pathology, faculty of health sciences, university of cape town, cape town, south africa national health laboratory service, cape town, south africa howard newman national health laboratory service, port elizabeth, south africa joanna reid national health laboratory service, cape town, south africa division of medical virology, faculty of health sciences, stellenbosch university, cape town, south africa nei-yuan hsiao department of pathology, faculty of health sciences, university of cape town, cape town, south africa national health laboratory service, cape town, south africa gert van zyl division of medical virology, faculty of health sciences, stellenbosch university, cape town, south africa lucia hans national health priority programme, national health laboratory service, johannesburg, south africa department of molecular medicine, faculty of health sciences, university of the witwatersrand, johannesburg, south africa jasantha odayar division of epidemiology and biostatistics and the centre for infectious disease epidemiology and research, faculty of health sciences, university of cape town, cape town, south africa stephen korsman department of pathology, faculty of health sciences, university of cape town, cape town, south africa national health laboratory service, cape town, south africa citation hardie dr, newman h, reid j, et al. field study to determine the reliability of hiv viral load results shows minimal impact of delayed testing in south africa. afr j lab med. 2024;13(1), a2364. https://doi.org/10.4102/ajlm.v13i1.2364 note: additional supporting information may be found in the online version of this article as online supplementary document 1. original research field study to determine the reliability of hiv viral load results shows minimal impact of delayed testing in south africa diana r. hardie, howard newman, joanna reid, nei-yuan hsiao, gert van zyl, lucia hans, jasantha odayar, stephen korsman received: 09 nov. 2023; accepted: 20 mar. 2024; published: 28 may 2024 copyright: © 2024. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: understanding factors that impact hiv viral load (vl) accuracy in resource-limited settings is key to quality improvement. objective: we evaluated whether testing delay and specimen storage between 25 °c and 30 °c before testing affected results. methods: between november 2019 and june 2023, 249 individuals on antiretroviral therapy, or with newly diagnosed hiv, were recruited from clinics in cape town and gqeberha, south africa, and three plasma preparation tubes were collected. one tube was tested within 24 h, while the others were stored uncentrifuged at ambient temperatures before testing. centrifugation and testing of matched samples were performed on day 4 and day 7 after collection. results: time delay and ambient storage had minimal impact in specimens with a day 1 vl of > 100 copies/ml. when grouped by day 1 vl range, 96% – 100% of specimens at day 4 and 93% – 100% at day 7 had vls within 0.5 log copies/ml of the first result. the greatest variability at days 4 and 7 was observed when the day 1 vl was < 100 copies/ml. however, there was no trend of increasing difference over time. of day 1 specimens with undetectable vl, or vl < 50 copies/ml, 80% had concordant results at day 4 and 78% at day 7. conclusion: these results show that vl is stable in plasma preparation tubes for 7 days when stored at room temperature. there is significant variability in specimens with low vl, but variability is not affected by testing delay. what this study adds: ideal hiv vl testing conditions are frequently unachievable in resource-limited settings. data are needed on whether this impacts on the validity of test results. our results provide reassurance that storage at ambient temperature for up to 7 days before testing does not substantially affect the vl result. keywords: hiv viral load stability; delayed testing; plasma preparation tubes; high-throughput viral load testing; diagnostic accuracy. introduction viral load (vl) testing is an essential tool for monitoring patients on anti-retroviral therapy (art) and, at a programmatic level, provides data to assess the joint united nations programme on hiv/aids 95:95:95 targets,1 specifically the third target which defines the proportion of patients on art that are virologically suppressed. south africa has the largest art treatment programme in the world, with > 6 million people on therapy.2 in 2022, more than 6 m vl tests were performed as part of this programme.3 south africa’s vl testing model involves centralised testing in high-throughput laboratories. major challenges that have the potential to compromise test accuracy relate to logistics and pre-analytical problems, which include sub-optimal specimen collection, transport, storage, handling, and testing delay. currently, vl platform manufacturers advise that plasma should be separated within 6 h to 24 h of collection, then stored at 4 °c or frozen at −80 °c if not tested within 5 days.4,5 in resource-constrained environments, specimens frequently reach testing sites beyond the recommended time after collection and may be subjected to further delays before testing takes place. the extent to which these pre-analytical factors compromise the accuracy of results has been evaluated in laboratory simulation studies,6,7 and also a systematic review.8 findings suggest that viral rna is preserved beyond the currently recommended testing time. good preservation of viral rna was also found in a local study performed at groote schuur hospital (gsh) using routine diagnostic specimens collected in ethylenediaminetetraacetic acid tubes or ethylenediaminetetraacetic acid-plasma preparation tubes (ppt). in this study, diagnostic specimens were stored after initial hiv vl testing at a range of times and storage temperatures and then re-tested.9 the vl in specimens stored for up to 1 week had very good concordance with the first result.9 however, tubes had already been centrifuged prior to storage and the study was performed entirely in the laboratory environment. in addition, it was only possible to follow trends in vl from the same patient over 2 time points due to specimen volume constraints. more studies are therefore needed to gain a better understanding of the factors that affect vl test reliability, as this knowledge is critical for improving the standard of testing in resource constrained environments, like south africa. to expand on our previous findings, a field study was undertaken where participants were recruited to provide additional specimens for vl testing. by means of serial testing of specimens from the same participants we evaluated the extent to which testing delay and adverse storage (including storage temperature and centrifugation) affects the reliability of vl results. methods ethical considerations ethical approval for the study was obtained from the human subjects research ethics committee of the faculty of health sciences at the university of cape town (hrec ref 159/2019). written informed consent was obtained from all participants prior to enrolment. only individuals of 18 years or older were recruited. study-related data were stored on a password-protected device to which only the principal investigator had access. study objectives the study objectives were as follows: to evaluate the impact of delayed testing on the reliability of hiv rna quantification in diagnostic specimens, collected and stored in un-centrifuged ppt tubes and to describe the impact of storage at a warmer ambient temperature (storage between 25 °c and 30 °c) in diagnostic specimens in un-centrifuged ppt tubes. we also evaluated whether centrifugation (enabling physical separation of plasma from cells) prior to storage improves the concordance of results where testing is delayed (evaluated at one site, namely gsh). study design the study was performed at three sites within the national health laboratory service network of hiv vl testing laboratories. patients were recruited from four art clinics, three in the cape metro and one in the eastern cape province of south africa. enrolment for two sites (gqeberha/port elizabeth [pe] and tygerberg hospital [tbh]) ran from 21 november 2019 to 24 january 2020 for pe, and 05 december 2019 to 23 january 2020 for tbh. enrolment for the third site (gsh) began on 28 august 2022 and ended on 06 june 2023 (following normalisation of laboratory services after the severe acute respiratory virus-coronavirus 2 pandemic). patients who were due to have a vl test or who were newly diagnosed with hiv and not yet on therapy, were approached, and consented to give two extra blood tubes (5 ml – 7 ml per tube) for hiv vl testing. agnostic patient recruitment was not done as we were not trying to collect a sample set reflective of the hiv-positive population in south africa, but rather wanted to evaluate a technical issue in stored specimens. thus, we aimed to collect roughly half of the specimens from patients with low-level or undetectable viral loads (on art) and half from patients who were expected to have unsuppressed vls (not yet started on antiretroviral drugs). three ppt tubes were collected during the same blood draw and delivered to the regional hiv vl testing laboratory (figure 1). on arrival, one tube was centrifuged (at approximately 3000 g for 10 min) and sent for immediate hiv vl testing. testing was completed within 24 h of sample collection. the result from this test was issued for routine patient management. the other two tubes were stored uncentrifuged in routine field conditions where the temperature varied between 25 °c and 30 °c. four days after collection, one of the stored tubes was centrifuged (at 3000 g for 10 min) and a vl test was performed. similarly, 7 days after collection, the third tube was centrifuged and a vl test was performed. viral load testing was performed using the routine assay in each of the three participating laboratories at the time of study, namely roche cap/ctm (tbh laboratory), roche 6800 (pe laboratory) (both roche molecular systems, pleasanton, california, united states) and abbott alinity m (gsh laboratory; abbott laboratories, chicago, illinois, united states). on a subset of patients (103 specimens, done at gsh laboratory only), hiv vl was repeated at day 7 on the tube that had been centrifuged and tested on day 1. the day 1 centrifuged tubes were stored under the same conditions as the yet untested ones, prior to repeat testing. the tubes were not re-centrifuged prior to repeat testing on day 7. they were used to assess whether storage in an already centrifuged state improved stability of vl in the sample. figure 1: study plan for testing factors affecting accuracy of viral load samples, south africa, 2019–2023. data analysis data analysis was performed using microsoft excel (microsoft corporation, redmond, washington, united states). bland-altman plots were used to evaluate differences in day 1 versus day 4 vl results and between day 1 and day 7.10 viral load values were log transformed as this is the standard way of expressing these values in clinical practice. for analysis, unless otherwise specified, samples with a vl that was lower than the limit of detection (ldl) were assigned a value of ‘1’ (0 log) copies/ml. results with a vl value of < 20 copies/ml were assigned a value of ‘19’ (1.28 log) copies/ml, otherwise the log value of the reading in copies/ml was used for analysis. we considered a difference in vl of < 0.3 log (or 2-fold difference) to reflect ‘no change’ as this is within the expected variability of this technology. a change of > 0.5 log copies/ml (> 3-fold difference) was considered likely to reflect a change that was caused by factors other than random expected variation.11 in addition, the results were evaluated in terms of whether the results obtained at day 4 or day 7 resulted in a reclassification of a patients’ vl control status. for this analysis: a viral load of ≤ 0 copies/ml was considered to reflect viral suppression;12,13 a viral load of 50 copies/ml–1000 copies/ml was considered to reflect low-level viraemia (llv);12,13 and a vl value of > 1000 copies/ml was considered to reflect unsuppressed vl or viral failure.12,13 results a total of 249 participants were consented and provided 3 ppt tubes for testing (51 from tbh, 60 from gqeberha (pe) and 138 from gsh. details from each study site are given in table 1. specimens from 6 patients arrived at the laboratory more than 24 h after collection and these were excluded from the study. altogether, 123 participants were already on art and 120 were newly diagnosed as hiv-positive, but not yet on art. table 1: characteristics of patients and testing sites used for viral load test accuracy study, south africa, 2019–2023. day 1 vls were segregated into the following vl categories: 55 (22.6%) were ldl; 46 (18.9%) were detectable, but < 50 copies/ml; 7 (2.9%) were 50 copies/ml – 100 copies/ml; 28 (11.5%) were 2–3 log copies/ml; 30 (12.3%) were 3–4 log copies/ml; 36 (14.8%) were 4–5 log copies/ml; 30 (12.3%) were 5–6 log copies/ml; and 11 (4.5%) were > 6 log copies/ml (table 2). table 2: proportion of samples tested on day 4 and day 7 within 0.3 log copies/ml and 0.5 log copies/ml of day 1 viral load value, by day 1 vl category, south africa, 2019–2023. applying current guidelines to define the participants’ hiv control status, as determined by the day 1 vl value, 101 participants would have been classified as virally suppressed, 35 had llv and 107 had unsuppressed vls (table 3). for reference, the full data set containing the matched day 1, day 4, and day 7 vl results for each patient is available as online supplementary document 1. table 3: viral load concordance on day 4 and day 7 according to viral control status as determined by the day 1 test, south africa, 2019–2023. viral load concordance at days 1, 4 and 7 overall, there was very little difference in paired readings at both day 4 and day 7 compared with day 1. the mean bias at day 4 was 0.15 and for day 7 was 0.12 log copies per ml, indicating a higher vl reading in the samples where testing was delayed (figure 2). for samples with an initial vl of > 2 log copies/ml, the paired vl value at day 4 and day 7 was within 0.5 log copies/ml in 96% – 100% of instances for day 4 and 93% – 100% of instances for day 7 (table 2), signifying minimal impact of the testing delay on the quantification. the greatest variation was observed for specimens with day 1 vls less than 2 log copies/ml. figure 2: variability of all paired viral load results at days 4 and 7, south africa, 2019–2023. bland-altman plots of day 1 versus day 4 vl (a) and day 1 versus day 7 (b) show that viral load measurements tended to be systematically higher by an average of 0.12 log at day 4 and 0.14 log at day 7 as compared to day 1. the bias tended to be greater in samples with low rna levels (< 2 log copies/ml); (c) shows variability of paired viral loads of day 1 samples that were re-run (r) on day 7, with the bland altman plot revealing minimal bias and even lower variance in day 1 versus day 7 results. green lines represent +2 and −2 standard deviations and the red dotted line represents the mean difference in hiv viral load on the bland altman plots. when differences in paired vls were evaluated using important thresholds that define clinical response, namely viral suppression (vl undetectable or < 50 copies/ml), llv (vl 50 copies/ml – 1000 copies/ml) and viral failure/unsuppressed vl (> 1000 copies/ml) (table 3), similarly good concordance was observed, with most specimens at days 4 and 7 having vl in the same clinical range as day 1. lowest agreement was in the 101 samples with a day 1 vl below 50 copies/ml, but nonetheless 81% remained < 50 copies/ml at the day 4 test and 79% at the day 7 test. most of the discordant results, 17 specimens at day 4 and 19 specimens at day 7, shifted into the llv category, while for 2 specimens at day 4 and 2 specimens at day 7, the vl was > 1000 copies/ml. concordance was higher, at 86% in specimens that were llv on day 1 testing at both days 4 and 7, and even higher, at 96% in specimens where the day 1 vl was > 1000 copies/ml. patients with a day 1 vl that was detectable, but < 50 copies/ml, were 2.6 times more likely to have a detectable vl at day 4 and 3.4 times more likely to have a detectable vl at day 7 than patients with an undetectable day 1 vl (ldl). overall, detection in a later specimen appeared to be stochastic. there was no trend to suggest that this phenomenon worsened with time. its frequency appeared to be affected by testing site and technology used, as it varied from 0% (gsh, abbott alinity), 11% and 22% at days 4 and 7 (tbh, roche cap/ctm) to 50% (pe, roche cobas 6800). viral load concordance in samples centrifuged on day 1 storage of specimens already centrifuged appeared to improve concordance. the day 7 vl values in specimens that were stored in a centrifuged condition (plasma separated from the cells) were very similar to the day 1 value (figure 2c). the mean difference in vl between day 1 and day 7 was 0.03 log copies/ml and standard deviation was narrower, at 0.42, than for the specimens that were first centrifuged and tested on day 7. only one result had a clinically significant difference (–1.1 log copies/ml) in vl between day 1 (4.95 log copies/ml) and day 7 (3.85 log copies/ml). discussion in this field study conducted in cape town and gqeberha in south africa between 2019 to 2023, we found that delay of hiv vl testing for up to 7 days after specimen collection and storage at ambient temperature had minimal effect on test results. these findings are compatible with previous work done in south africa in 2017, using stored laboratory specimens,9 and also support the findings of a systematic review by bonner et al.8 the study conditions were designed to match more closely the real-world situation in lowand middle-income countries with high hiv disease burdens and large art programmes, where testing is often delayed and storage temperatures are higher than recommended due to logistical issues. these results provide reassurance that time delay and ambient storage is not a major cause of result inaccuracies. most of the variability that resulted in misclassification of a patient’s status was observed in specimens with day 1 vls of < 2 log copies/ml (from virally suppressed patients and those with llv). in particular, a proportion of results in the viral suppression range shifted into the llv range at one or both later time points, and some that were llv shifted into the viral suppression range. the most probable explanation for this is the inherent variability of real-time polymerase chain reaction technology in specimens with target levels close to the limit of detection.14 in such specimens, target may be only intermittently detected in test replicates, and greater variability can be expected in the quantified value.15 of note is that specimens from patients that were virally suppressed, but had a detectable vl that was < 50 copies/ml at day 1, were 2 times to 3 times more likely to have a detectable vl in a stored specimen than if the vl was undetectable at day 1. low-level viraemia reflects a state of incomplete viral suppression and has been associated with increased risk of vl failure in the future.16,17,18 because of this, there is a move to set the vl indicative of viral failure (currently defined as > 1000 copies/ml) at a lower vl threshold, such as at 200 or 50 copies/ml. as can be seen in this study, this can be expected to increase the chance of misclassification of patients due to the inherent variability of real-time polymerase chain reaction in this range. confirmation of viraemia on a second specimen would help to mitigate this and is recommended in current guidelines.12,13 occasional specimens displayed a higher-than-expected variability, namely 4 that were virally suppressed on day 1, but > 1000 copies/ml at either day 4 or day 7. it is probable that factors other than time delay were responsible. potential explanations for this stochastic variability could be specimen contamination with hiv rna during processing, inadequate centrifugation prior to testing or some other undefined laboratory factor.19,20,21 technical factors clearly played a role in this study, as the proportion of specimens that were ldl on day 1, but detectable later, varied markedly at the different testing sites. one additional measure to improve test concordance that was evaluated was centrifugation of specimens prior to storage. in a subset of specimens, the same tube that was centrifuged and tested on day 1, was rerun on day 7. these results showed very low variability and no specimen had a clinically actionable change in vl when comparing the day 1 and day 7 results. a caveat is that this experiment was only done at the site where there was very low variability in the stored specimens anyway. limitations only three specimens were collected from each patient, and this limited the number of factors we could evaluate. testing occurred across three sites and different testing platforms could have accounted for some of the differences that were seen, for example a much lower rate of detectable viral rna in later specimens with day 1 suppressed vl at one site. these will need to be evaluated at a later stage. specimens were only tested once on day 1. a repeat test on day 1 would have provided key data on the inherent variability of vl results on our platforms and given context to the variability observed at the later time points. conclusion our field study provides further evidence that time delay has minimal impact on vl quantification when specimens are stored at room temperature for up to 7 days. this extends the time allowable before vl testing needs to be done. most of the discordant results were in the llv range and likely reflect inconsistent detection at the limit of detection of the assay. centrifugation of specimens prior to storage improves the concordance of results at later time points and early centrifugation after collection should be prioritised for hiv testing programmes. the contribution of other patient-related and technical issues requires further investigation. acknowledgements we thank widaad zemanay and nchimunya hapepa for help with project management and sr dyonase for assistance with patient recruitment. we also thank staff at national health laboratory service laboratories with help processing study samples. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions d.r.h., s.k., n.-y.h., h.n., l.h. and g.v.z. all contributed to designing the study. h.n. recruited patients and oversaw testing at the port elizabeth (gqeberha) laboratory. j.r. recruited patients and oversaw testing at the tygerberg laboratory. j.o. recruited patients and d.r.h. oversaw testing at the groote schuur hospital laboratory. d.r.h. wrote the manuscript and all authors contributed to data analysis and reviewing of the final manuscript. sources of support this research received no specific grant from any funding agency. the national health laboratory service provided support for testing of study samples. no other formal funding was received for this study. data availability the authors confirm that the data supporting the findings of this study are available within its supplementary materials. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. it does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the authors are responsible for this article’s results, findings, and content. references robert b, brown eb. fast-track ending the aids epidemic by 2030. unaids publ [serial online]. 2014;(1):1–14. 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2021;12(1):2919–2931. https://doi.org/10.1080/21505594.2021.2004743 ellman tm, alemayehu b, abrams ej, arpadi s, howard aa, el-sadr wm. selecting a viral load threshold for routine monitoring in resource-limited settings: optimizing individual health and population impact. j int aids soc. 2017;20:16–18. https://doi.org/10.1002/jia2.25007 procop gw, taege aj, starkey c, et al. preanalytic process linked to spuriously elevated hiv viral loads: improvement on an fda-approved process. diagn microbiol infect dis. 2017;89(1):44–46. https://doi.org/10.1016/j.diagmicrobio.2016.09.003 kran amb, jonassen tø, sannes m, et al. overestimation of human immunodeficiency virus type 1 load caused by the presence of cells in plasma from plasma preparation tubes. j clin microbiol. 2009;47(7):2170–2174. https://doi.org/10.1128/jcm.00519-09 salimnia h, moore ec, crane lr, macarthur rd, fairfax mr. discordance between viral loads determined by roche cobas amplicor human immunodeficiency virus type 1 monitor (version 1.5) standard and ultrasensitive assays caused by freezing patient plasma in centrifuged becton-dickinson vacutainer brand plasma prepa. j clin microbiol. 2005;43(9):4635–4639. https://doi.org/10.1128/jcm.43.9.4635-4639.2005 abstract introduction description of laboratory development building blocks building on the foundation discussion acknowledgements references about the author(s) kirtika patel department of immunology, moi university, eldoret, kenya r. matthew strother oncology department, university of otago, christchurch, new zealand francis ndiangui department of pathology, moi teaching and referral hospital, eldoret, kenya david chumba department of pathology, moi university college of health sciences, eldoret, kenya william jacobson department of pathology, indiana university school of medicine, indianapolis, indiana, united states cecelia dodson histology laboratory, indiana university health, indianapolis, indiana, united states murray b. resnic department of pathology, brown university, providence, rhode island, united states randall w. strate department of pathology, indiana university school of medicine, indianapolis, indiana, united states james w. smith department of pathology, indiana university school of medicine, indianapolis, indiana, united states citation patel k, strother rm, ndiangui f, et al. development of immunohistochemistry services for cancer care in western kenya: implications for lowand middle-income countries. afr j lab med. 2016;5(1), a187. http://dx.doi.org/10.4102/ajlm.v5i1.187 lessons from the field development of immunohistochemistry services for cancer care in western kenya: implications for lowand middle-income countries kirtika patel, r. matthew strother, francis ndiangui, david chumba, william jacobson, cecelia dodson, murray b. resnic, randall w. strate, james w. smith received: 07 may 2014; accepted: 03 dec. 2015; published: 04 may 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: cancer is becoming a major cause of mortality in lowand middle-income countries. unlike infectious disease, malignancy and other chronic conditions require significant supportive infrastructure for diagnostics, staging and treatment. in addition to morphologic diagnosis, diagnostic pathways in oncology frequently require immunohistochemistry (ihc) for confirmation. we present the experience of a tertiary-care hospital serving rural western kenya, which developed and validated an ihc laboratory in support of a growing cancer care service. objectives, methods and outcomes: over the past decade, in an academic north-south collaboration, cancer services were developed for the catchment area of moi teaching and referral hospital in western kenya. a major hurdle to treatment of cancer in a resource-limited setting has been the lack of adequate diagnostic services. building upon the foundations of a histology laboratory, strategic investment and training were used to develop ihc services. key elements of success in this endeavour included: translation of resource-rich practices to a resource-limited setting, such as using manual, small-batch ihc instead of disposableand maintenance-intensive automated machinery, engagement of outside expertise to develop reagent-efficient protocols and supporting all levels of staff to meet the requirements of an external quality assurance programme. conclusion: development of lowand middle-income country models of services, such as the ihc laboratory presented in this paper, is critical for the infrastructure in resource-limited settings to address the growing cancer burden. we provide a low-cost model that effectively develops these necessary services in a challenging laboratory environment. introduction cancer is a leading cause of mortality in lowand middle-income countries (lmics), already accounting for more deaths than tuberculosis, hiv and/or aids and malaria combined.1,2 according to the international agency for research on cancer there were more than 600 000 new cancer cases and more than half a million cancer deaths in africa in 2008, with a projected doubling by 2030.3 the need to develop an infrastructure for cancer research and care in lmics has been recognised by various researchers.4,5,6 a core component of cancer infrastructure is adequate pathology services. the breast health global initiative provides excellent resource-stratified guidelines for the development of pathology services.7 however, development of pathology services in lmics faces a number of challenges, including insufficient funds, lack of skilled personnel at all levels (technicians to pathologists), unavailable equipment and unreliable supply chains for consumables.8,9,10 despite these challenges, there have been a number of approaches to solving the deficits in lmic pathology services. three primary themes characterise these efforts: the development of systems to export specimens, either preor post-processing, for reading and diagnosis by out-of-country pathologists; services offered in-country through volunteer pathologists from other countries, and the use of telepathology.11 however, there are shortfalls with each of these approaches. specimen export faces challenges with acquisition of export permits, postage costs and risk of specimen loss during transit. in addition, long turnaround times hamper appropriate patient care.12 volunteer pathology services have difficulty identifying sufficient pathologists who have time available for travel and service provision, loss of income for the pathologist and the cost of airfare, visas, and room and board. once arrived, these experts often face a learning curve as they work with older and frequently ill-maintained equipment.12 telepathology, although promising, is presently slowed by inadequate bandwidth and the cost of required equipment.12 each of these approaches provides key elements to the successful development of pathology services for cancer care in resource-limited settings. these approaches depend on the development of sufficient local infrastructure and expertise. in the absence of development of local expertise, inadequate sample preservation and preparation can render a well-resourced pathology laboratory inadequate for diagnosis. in this paper, we describe our effort to combine all three of these approaches, along with investment in local resources and extensive local training, to develop pathology services in a resource-limited setting for immunohistochemistry (ihc), which is essential for reliable cancer diagnosis, prognosis and therapeutic decision-making.13 this process required extensive collaboration, investments in training and the local infrastructure, as well as coordination with telepathology resources and volunteer pathologists. description of laboratory development the academic model providing access to healthcare programme the academic model providing access to healthcare (ampath) programme links the resources of the moi teaching and referral hospital (mtrh), kenya’s second referral hospital, with the expertise and creativity of the moi university school of medicine (musom) and a consortium of north american academic medical centres (indianna university, brown university, duke university, lehigh valley hospital allentown, pennsylvania, university of notre dame, providence portland medical center, purdue university, university of massachusetts, medical school, university of utah, school of medicine, university of toronto, faculty of medicine and university of california, san francisco). the collective goal is to find sustainable solutions to many aspects of health and disease in western kenya.14,15 ampath-oncology evolved to deliver appropriate care to cancer patients presenting to mtrh and found that a major impediment to a successful cancer care programme was that pathology was limited only to morphologic diagnosis. building blocks according to world bank data, in 2014, the population of kenya was 44.86 million people (http://data.worldbank.org/country/kenya#; accessed: 21 april 2016.). at present, there are only two tertiary-care facilities in kenya, one serving nairobi and eastern kenya, and mtrh, which serves the rift valley and western kenya. mtrh serves a catchment area that includes half of kenya’s population – approximately 20 million people. to start building an ihc programme, a first step was an assessment of the existing resources at mtrh, musom and ampath. there were four general pathologists and six laboratory technicians serving in the department of pathology who provided diagnostic surgical pathology services to this population. pathologists have a masters in medicine (mmed) qualification and are employed by mtrh and musom, whereas technicians are registered members of the kenya technicians and technologist board and hold a certificate, diploma or degree. the surgical pathology laboratory at mtrh performs grossing services and processes tissue with a semi-automated tissue processor. final diagnosis is based on morphologic assessment with haematoxylin and eosin staining. clinical personnel at mtrh frequently have co-appointments at musom. the university also has non-clinical faculty involved in extensive laboratory and research endeavours. relevant to ihc services, the department of immunology had 10 staff members, was responsible for the training of medical, dental, nursing, physical therapy and environmental health students, and had 100 m2 of laboratory space for training. within the department of immunology, one faculty member with extensive prior experience with ihc was identified, having trained and worked in the united kingdom, and was a certified assessor for the united kingdom national external quality assurance scheme. ampath, which had been operational since 2001, had also put some infrastructure in place. prior investment in mtrh pathology included a tissue processor, tissue embedding station, convection oven, microtome, tissue section flotation water bath and microscopes. a critical contribution was maintaining service contracts and reagent agreements with a local company representative (dako, nairobi, kenya), which services ihc equipment and stocks reagents. this allowed for functional equipment and easy access to reagents, a frequent issue in resource-limited settings. in addition, ampath had extensive connections to pathologists through its large number of research projects and linked university programmes in north america. several pathologists had travelled to kenya to establish digital pathology training resources for musom. the combined resources available for development of ihc are presented in table 1. table 1: building blocks: existing resources within musom, mtrh, and ampath, western kenya, before 2009. building on the foundation the development of the ihc service required several key components to be coordinated, purchased or developed: the physical housing of an ihc laboratory, the coordination of equipment, the organisation of the service, training of personnel and ongoing quality assessments. physical housing: discussions between all partners (mtrh, musom and ampath) identified space in the existing histopathology laboratory for ihc services. renovations were required for temperature control (to ensure reagent stability and process reproducibility), air flow (to ensure safety for technicians using solvents for slide preparation) and back-up power supplies (for both the air conditioning unit and the reagent storage refrigerators). coordination of equipment: the equipment of the ihc laboratory itself was procured through repurposing of local equipment, donations and direct purchases through grants and donations. table 2 outlines the equipment and consumables required for the ihc laboratory, along with the sources of these items. table 2: strategic investments to develop immunohistochemistry laboratory services, western kenya, 2009–2012. organisation of service: administrative structures had to be established between partners to facilitate personnel management, establish processes for requesting ihc and reporting results, and perform administrative tasks such as ordering reagents. a laboratory director was assigned from the musom department of immunology and charged with overseeing daily operations and reporting to the head pathologist within mtrh’s histopathology department. management of the laboratory’s finances (both expenditures and billing) was routed through an office for research and sponsored projects maintained by all three partners. training of personnel: four histopathologists, four technicians and two doctoral students in immunology were trained both through short group workshops conducted by the local ihc expert and representatives from dako, the company providing ihc reagents (nairobi, kenya), and through one-on-one instruction taught by the consortium of north american universities. personnel were selected for training from amongst available staff at mtrh who were willing to take on the extra ihc training duties. skills developed included manual staining and use of the autostainer. pathologists were also trained on the importance of ihc in diagnosis and prognosis, and sessions with surgeons emphasised the importance of quick fixation for good ihc results. protocols were developed to ensure that specimen transport from the operating theatre to the histopathology laboratory occurred in a timely manner. quality assessment: following initial competency assessments by north american experts, protocols were developed to ensure ongoing quality and reproducibility. methods used to maintain high quality included: written standard operating procedures, inclusion of positive and negative controls in all staining batches and use of electronic image transfer for consultation with north american pathologists. in addition, during routine site visits by the north american pathologists, a selection of ihc slides were assessed and stained slides were sent to reference laboratories at indiana university and the university of california, san francisco. further, the ihc laboratory was enrolled in the united kingdom national external quality assessment scheme, an external quality assessment scheme, for validation and quality assurance/quality control of staining and reporting. the current immunohistochemistry programme: operations and outcomes operations establishing a functioning ihc service started at the level of tissue acquisition. standard operating procedures were designed to ensure that specimens are immediately placed in formalin, that the time of collection is recorded and that the attending surgeons make radial cuts across larger specimens to ensure adequate penetration of formalin. schedules were developed for specimen transport to the laboratory and rapid grossing, followed by processing and embedding into paraffin within 24 hours of resection. a major quality improvement effort was required to ensure 3–5-µm sectioning and ultimately required gross analysis is performed by a pathologist rather than a technologist. the lead pathologist must request specific ihc stains following haematoxylin and eosin staining and reporting. for the ihc laboratory, one pathologist is assigned for all aspects of ihc analysis, including reporting and dispatch of results. antigen retrieval for ihc is performed with a pt link machine (pre treatment link, dako north america, inc., carpentaria, california, united states). antigen retrieval is achieved by using high-ph tris-edta (ph 8.0) (dako k8004) and a low-ph citrate buffer (ph 6.0) (dako k8005). for example, high-ph antigen retrieval is used for oestrogen and progesterone receptors, whereas a low-ph antigen retrieval buffer is used for her2. the dako envision flex (dako, k8000 denmark) kit is used for immunostaining. endogenous peroxide is blocked by dako hydrogen peroxide (dako sm801 k8002). ready-to-use primary antibodies are used and staining is performed as per the manufacturer’s (dako ir/is series) instructions. envision flex mouse linker (k 8021) is used as a secondary antibody. slides are than labelled with envision flex linked with horseradish peroxidase (hrp) (dako sm802) and the staining is visualised by using the substrate dab chromogen mix (dako sm 803). slides are counterstained with envision flex heamatoxyline (dako k8008), dehydrated and cover slipped, and examined under the microscope. two tris buffer washes of 5 min each are performed between all steps. known positive and negative controls are also stained for all runs for quality assurance. other immunostains used to report other tumours are shown in table 3. the allred scoring system is used to score the slides for the hormones.16 the other immunostains are reported as percentage positive or negative in the tumours. most stains are performed manually, although used infrequently at this time, a donated autostainer (dako autostainer plus, dako north america, inc., carpentaria, california) is maintained for large-batch staining (48 slides). table 3: current productivity of the mu/mtrh/ampath immunohistochemistry laboratory, western kenya, 2009–2012. once the ihc staining is complete, all staff pathologists have an opportunity to review the slides for interpretation. three pathologists from indiana university visited the laboratory for a two-week period twice a year and one pathologist from brown university visited the laboratory once during the inception period. these visits provided continuous training. as per protocol, ihc slides are read together with the pathologist, ihc laboratory director and technologists. all ihc results are recorded in a logbook for technical purposes and in a file dedicated to ihc, with the matched control slide results for future reference. all slides are catalogued, dated and stored for easy reference. paraffin blocks are also catalogued and stored, according to standard laboratory practices. owing to constrained resources, scanning slides for electronic storage and subsequent distribution was not practical. consequently, a photomicroscope was put in place to photograph slides in the mtrh ihc laboratory. we are adapting this approach to capture images using a mobile phone, likely the best approach in a resource-poor laboratory. outcomes from 2006 to 2012, there was increased demand for basic histopathology – 1200 cases in 2006 rising to 6500 cases by 2012 – largely related to the clinical oncology programme. along with this increased service demand, immunostaining provided an invaluable adjunct to diagnosis of cancer. to date, the ihc laboratory performed 406 immunostains. before 2009, the turnaround time for results was at least three weeks, whereas after 2012 the turnaround time decreased to one week. patients were treated as per the results obtained. the types of other immunostains performed and the method for external validation are shown in table 3. box 1: lessons learned. discussion recent studies in sub-saharan africa have suggested improving pathology services to be key in cancer control, treatment and research.17,18 building local capacity for ihc in lmics has several tangible benefits: rapid turnaround time, improved pre-treatment diagnostic accuracy and capability for more rapid treatment initiation. further, it avoids the risk of specimen loss associated with the use of remote laboratory facilities. currently, the mtrh ihc laboratory is the only laboratory of its kind in a public institution in kenya. however, similar efforts are ongoing in malawi, which has already proven to be a robust platform for providing cancer care to its patients and for research.19 currently our laboratory performs oestrogen, progesterone and her2 staining for breast cancer treatment following guidelines,20,21 differentiation of lymphomas using a basic panel of antibodies,22,23 research-based diagnostics for kaposi’s sarcoma (ks) using latency-associated nuclear antigen (lana-1) and wilm’s tumour. this work helps to facilitate treatment of patients, avoid treatment of clinically ambiguous presentations of disease as cancer24 and more efficiently utilise limited treatment resources. however, in developing ihc capacity several issues were highlighted in pathology in kenya. it quickly became clear that reliable ihc results were dependent upon good morphology, which, in turn, relied upon proper handling and fixation of tissues.25 identifying simple issues, such as replacing non-standardised fixatives with neutral buffered formalin and standardising fixation, avoided issues of masked antigen, which were found to be important to allow interpretation of stains.19 in addition, investment in skills development of technologists was required to allow thin (3−5 μm), reproducible sections of paraffin-embedded tissues to be cut for ihc and with regard to the use and repair of the microtome. the ihc staining technique is a new technique and can be performed manually or with the use of an autostainer and the pt link machines for antigen retrieval at our institution. several technologists had to be trained on the principles of the ihc technique, methodology and the use of equipment for ihc. retention of highly qualified personnel after training is an ongoing concern, owing both to personnel leaving for better employment and to internal procedures whereby technologists are rotated regularly across clinical laboratories within mtrh, a mechanism designed to ensure equity amongst technologists. this approach, common in resource-constrained settings, required specific negotiation with hospital administration and allowed ongoing skills development. pathologists and technical staff should be trained locally, where the resources and equipment are relevant. training should be continuous and immunostaining should be performed at least once a week for maintaining knowledge, skills and technical ability. training of local pathologists and technologists by experts from other institutions (locally-based and internationally-based) should be encouraged. under ideal circumstances, there should be ongoing support from experts for troubleshooting, quality assurance and external review of slide evaluation and reporting. this process required extensive collaboration, investments in training and the local infrastructure and coordination with telepathology resources and volunteer pathologists. however, a notable lesson learnt is that not all resource-rich practices can translate to the low-resource environment. ihc performed manually was less costly and equally effective compared with the use of an autostainer. autostaining used considerable amounts of reagents and solvents and frequent mechanical failures were experienced owing to power fluctuations. similarly, we found that a microwave or a pressure cooker instead of expensive antigen retrieval equipment was adequate for good ihc staining in western kenya. given the high cost of servicing, we recommend manual batch processing of tissues for staining as the standard methodology in a resource-poor setting. small amounts of manual staining can be performed in slide storing boxes instead of having to use expensive humidifying chambers. diaminobenzidine (dab), which is a potential carcinogen, is used as a chromogen during ihc staining. gloves and a face mask have to be worn when handling dab. the dab waste at our institution was diluted with water to a concentration of 0.9 mg/ml before being discarded in the hazardous waste container. the ihc laboratory is air-conditioned so there is a constant exchange of air and the temperature is maintained at 20 °c. all the stages that involved solvents such as alcohol and xylene (i.e. dewaxing, dehydrating and cover slipping) are performed in the histology laboratory, which is equipped with a fume hood.26 consumables and reagents (e.g. alcohol, xylene, disposable plastic pipettes, pipette tips, cover slips and charged slides) remain a challenge to acquire and, relative to developed settings, are quite expensive. further, there can be substantial delays in receiving purchased quality reagents. we are continuing to negotiate local purchase versus importing supplies from our international partner institutions, where competitive pricing, quality assurance and reliable delivery may outweigh the drawbacks of international importation. despite our obvious cost constraints, we have selected more expensive ready-to-use polymer-based reagents for the following reasons: procedural simplification as a result of bound secondary antibody and a detection system which minimises errors, no reliance on avidin and biotin for conjugation and the potential for high background readings, and longer shelf stability.20 our experience has been that the additional cost of buying ready-to-use reagents, which are more expensive ($200 united states dollars [usd] for 200 primary antibody tests) than the undiluted reagents ($200 usd for 300 tests), is outweighed by the benefits of obtaining consistent results, with fewer chances of repetition, in a lmic setting. conclusion researchers working in sub-saharan africa have stressed the urgent need for improvement of cancer therapy by improving pathological diagnostic capabilities as part of capacity building efforts in low-resource settings.27 the global task force on expanded access to cancer care and control in developing countries has recommended improving cancer diagnosis and pathology expertise.28 local capacity in pathology and laboratory services therefore has to be improved. opportunities for collaborative models involving regional and global partners should be employed as pathology services develop in lmics.29 ultimately, this multi-modal approach resulted in an efficient lmic pathology service performing basic immunostains with the resources and expertise to facilitate expansion. our experience illustrates that it is possible to establish extended pathology services efficiently with the help of partnership and collaboration in kenya, a lmic. development of lmic models of services such as this is critical to resource-constrained settings seeking to address the growing burden in cancer prevention, care and research. acknowledgements competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. sources of support this work was supported in part by the national council of science and technology ncst/5/003/call2/09 and pfizer grant. an ihc autostainer and a pt link machine for antigen retrieval were donated by indiana university. authors’ contributions k.p. carried out the work described and conceived and wrote the manuscript. r.m.s. had oversight of ampath-oncology, helped coordinate the international expertise and resources, and contributed to conception and writing of the manuscript, f.n. was the pathologist in charge of breast cancers and d.c. was the pathologist in charge of other tumours. w.j. was a visiting pathologist and c.d. helped in setting up the ihc laboratory and provided technical support. as visiting pathologists, m.b.r. provided advice for ihc diagnosis and r.w.s. provided technical support and advice in diagnoses. j.w.s. helped with coordination and acquiring instrumentation and reagents for the ihc laboratory. references jemal a, bray f, center mm, ferlay j, ward e, forman d. global cancer statistics. ca cancer j clin. 2011;61(2):69–90. http://dx.doi.org/10.3322/caac.20107 kanavos p. the rising burden of cancer in the developing world. ann oncol. 2006;17(suppl 8):viii15–viii23. ferlay j, shin h-r, bray f, forman d, mathers c, parkin dm. estimates of worldwide burden of cancer 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middleand low-income countries: the experience of a case-control study of inflammatory breast cancer in north africa in the past 10 years. cancer med. 2012;1(3):338–349. http://dx.doi.org/10.1002/cam4.36 knaul fm, frenk j, shulman l for the global taskforce on expanded access to cancer care and control in developing countries. closing the cancer divide: a blueprint to expand access in low and middle income countries [report on the internet]. boston ma: harvard global equity initiative; c2011 [cited 2015 oct 24]. available from: http://gtfccc.harvard.edu/fs/docs/icb.topic1063570.files/ccd_report_111027.pdf. roberts dj, wilson ml, nelson am, adesina am, fleming ka, milner d, et al. the good news about cancer in developing countries – pathology answers the call. lancet. 2012;379(9817):712. http://dx.doi.org/10.1016/s0140-6736(12)60306-7 abstract introduction methods results discussion acknowledgements references about the author(s) mo se kwon division of medical virology, department of pathology, faculty of health sciences, university of cape town, cape town, south africa national health laboratory service, cape town, south africa khumo o. sematle division of medical virology, department of pathology, faculty of health sciences, university of cape town, cape town, south africa national health laboratory service, cape town, south africa lucia hans department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa national priority programme, national health laboratory service, johannesburg, south africa stephen korsman division of medical virology, department of pathology, faculty of health sciences, university of cape town, cape town, south africa national health laboratory service, cape town, south africa nei-yuan hsiao division of medical virology, department of pathology, faculty of health sciences, university of cape town, cape town, south africa national health laboratory service, cape town, south africa diana r. hardie division of medical virology, department of pathology, faculty of health sciences, university of cape town, cape town, south africa national health laboratory service, cape town, south africa citation kwon ms, sematle ko, hans l, et al. using data science to investigate rising hiv low-level viraemia results at groote schuur laboratory in south africa. afr j lab med. 2025;14(1), a2953. https://doi.org/10.4102/ajlm.v14i1.2953 note: additional supporting information is available for download alongside the online version of this article as online supplementary document 1. original research using data science to investigate rising hiv low-level viraemia results at groote schuur laboratory in south africa mo se kwon, khumo o. sematle, lucia hans, stephen korsman, nei-yuan hsiao, diana r. hardie received: 25 july 2025; accepted: 05 nov. 2025; published: 20 dec. 2025 copyright: © 2025. the author(s). licensee: aosis. this work is licensed under the creative commons attribution 4.0 international (cc by 4.0) license (https://creativecommons.org/licenses/by/4.0/). abstract background: following a major service disruption across the national health laboratory service, backlogged hiv viral load (vl) specimens from limpopo province were rerouted to groote schuur hospital (gsh) laboratory in the western cape province. during this time, an increase in low-level viraemia (llv; 50 copies/ml – 1000 copies/ml) was observed at the gsh laboratory, raising concerns about possible pre-analytical and analytical issues, including compromised specimen quality and possible contamination. objective: to determine whether the observed increased llv was due to analytical errors (e.g. contamination), pre-analytical factors such as prolonged turnaround times (tat), or underlying epidemiological differences. methods: hiv vl data from 2023–2024 for limpopo and the western cape were analysed using python. viral load results were grouped into predefined categories and compared. turnaround times were plotted and thembisa model estimates were used to assess hiv prevalence and antiretroviral therapy (art) coverage. longitudinal patient-level analysis evaluated vl trends as a proxy for adherence. in addition, quality control data were evaluated at testing sites. results: limpopo specimens showed higher llv (20%) and lower viral suppression (< 50 copies/ml) at 70%, compared to western cape (13% llv, 81% suppression), where follow-up and suppression outcomes were also higher. no clear evidence indicated that extended tat or potential instrument contamination affected vl results significantly. conclusion: the increase in llv at gsh was linked primarily to processing specimens from limpopo, highlighting regional differences in hiv vl result distributions. these differences probably reflect variations in art access and adherence, rather than laboratory-related issues such as delayed tat, sample quality, or contamination. what this study adds: this study shows how province-specific hiv result patterns correlate with art adherence, using a python script to assess serial vls and follow-up suppression. it demonstrates the value of routine data analysis in monitoring high-throughput hiv vl tests, which are often auto-released without pathologist oversight. keywords: laboratory testing; hiv; viral load; data science; low-level viraemia; pre-analytical factors; art programme; contamination. introduction viral load (vl) testing is the cornerstone of monitoring hiv-1 viraemia in people living with hiv who have been initiated on antiretroviral therapy (art). viral load testing does not only monitor treatment success but also informs clinical decisions regarding adherence interventions and regimen changes in cases of virologic failure. the national health laboratory service (nhls) in south africa provides hiv vl testing at 17 centralised laboratories across the country,1 with smaller local laboratories referring samples to these reference facilities. in october 2024, during an internal quality assessment at the groote schuur hospital (gsh) laboratory, we noted an increase in low-level viraemia (llv) results, defined as hiv vls between 50 copies/ml and 1000 copies/ml, as well as a rise in values below the limit of quantification. the number of results reported as lower than detectable (ldl) decreased, while hiv vls > 1000 copies/ml remained stable. the mechanisms underlying llv remain poorly understood. patients with llv are often maintained on failing regimens, which can promote the accumulation of drug-resistant mutants, virologic rebound, increased risk of virological failure, poorer clinical outcomes, and ongoing transmission.2 it is essential to distinguish sustained llv from intermittent llv, also known as ‘blips’, which are typically short-lived and carry a lower risk of resistance or disease progression. the recent rise in llv rates in south africa is particularly concerning, especially following the nationwide rollout of dolutegravir by 2022, which was expected to enhance viral suppression.3 this trend mirrors observations from other southern african development community countries, such as botswana,4 where dolutegravir scale-up has similarly not led to the anticipated reductions in virologic failure and llv prevalence. upon further enquiry, it was noted that the gsh laboratory had started receiving a large volume of specimens from another central laboratory in the limpopo province, outside of the usual catchment area of our laboratory, citing operational difficulties and testing backlogs. it is worth noting that most hiv samples in limpopo are processed at one centralised laboratory in the province, located approximately 1739 kilometres from gsh, which is located in the western cape province.5 the transit times for these specimens were inevitably prolonged as they were transported by road to gsh, and the specific conditions under which specimens were transported could not be confirmed (i.e. to what degree the cold chain was maintained). this study aims to evaluate hiv vl data from both laboratories and to investigate whether the increase in llv observed at gsh is associated with an increase in referred specimens from outside the province, and whether the extended transit times affected sample quality and vl results. an accompanying increase in reactive negative controls also raised concerns regarding possible contamination and false positive results. the study further considers whether the current national virological failure threshold (> 1000 copies/ml) remains appropriate, and explores how art coverage and adherence may influence the distribution of vl results across provinces. methods ethical considerations this study was conducted in accordance with the ethical principles outlined in the declaration of helsinki. ethical approval was granted by the human research ethics committee of the university of cape town (reference number: 233/2025). approval to access and analyse data from the nhls database was obtained through the nhls academic affairs and research management system. informed consent was waived, as all published data is aggregated and contains no personal identifiers. data were analysed and stored on a password-locked computer and only accessible to the study co-investigators. hiv viral load testing hiv-1 vl testing was performed using the abbott alinity m hiv-1 assay (abbott molecular, des plaines, illinois, united states), a fully automated real-time, reverse transcriptase polymerase chain reaction system used in south african laboratories affiliated with the nhls. prior to testing, specimen tubes were centrifuged at 3000 g for 10 min to separate plasma or serum. the assay processes 0.2 ml to 1.0 ml of plasma or serum, extracting hiv-1 rna via magnetic microparticle technology and amplifying two highly conserved genomic regions alongside an internal rna control to monitor assay performance. for specimens with volumes less than 0.2 ml, a 1:5 dilution is prepared and analysed using the 1 ml protocol. the assay demonstrates a limit of detection of 20 copies/ml and a quantification range of 20 copies/ml – 10 000 000 copies/ml, with the lower limit of quantification dependent on input volume (50 copies/ml for 0.2 ml protocol; 20 copies/ml for 1.0 ml protocol). it is validated for hiv-1 groups m (subtypes a–h and circulating recombinant forms), o, and n, ensuring broad subtype inclusivity for accurate clinical monitoring.6 data collection access to patient-level hiv vl data stored in the nhls corporate data warehouse in johannesburg was obtained through the academic affairs and research management system. following institutional approval, hiv vl data from the trakcare laboratory information system were extracted for the limpopo and western cape provinces, covering the period from 2023 to 2024. the data were provided in comma-separated values (.csv) format. to investigate the potential impact of laboratory contamination on negative control results, relevant maintenance records were requested from an abbott representative for both laboratories. in addition, contextual information on hiv prevalence and art coverage was sourced from the thembisa model7 — a comprehensive, publicly available hiv modelling tool developed for south africa. the model integrates demographic, behavioural and treatment-related data to estimate hiv trends and assess intervention impacts, and it is updated routinely at both national and provincial levels. data/statistical analysis data were analysed using a custom python script developed with the pandas library (python software foundation, wilmington, delaware, united states), an open-source data analysis toolkit used widely in data science for efficient manipulation, cleaning and transformation of structured data. there were two main datasets: one extracted for limpopo province and the other for the western cape province. the initial step of the script involved a data clean-up process, during which duplicate records, records falling outside the specified time period, entries with incorrect data types or formats, and records with missing information in key fields were removed. after the clean-up process, the number of hiv vl records from the western cape between 01 january 2023 and 31 december 2024 was 818 989, while the limpopo dataset contained 1 169 903 records. the script then aggregated individual vl results into predefined ranges: ldl, < 50 copies/ml, 50–199 copies/ml, 200–1000 copies/ml, and > 1000 copies/ml. results below the limit of quantification (< 20 copies/ml or < 50 copies/ml depending on volume protocol), as well as absolute values < 50 copies/ml, were grouped into the ‘< 50’ category. monthly counts were grouped based on specimen ‘collection date’ for both laboratories (online supplementary tables 1–5). for comparative purposes, absolute monthly counts were converted into percentages of total vl tests per month. these data were visualised using clustered bar graphs in microsoft excel (microsoft corporation, redmond, washington, united states) to facilitate interpretation (online supplementary figures 1–4). monthly turnaround time (tat) was plotted on the same graph as a secondary line chart, measured in days. turnaround time was calculated as the difference between ‘registration date’ (when specimens were logged into the laboratory information system) and ‘entered date’ (when test results were entered automatically post-processing on the alinity m platform). a separate chart was created to assess operations in limpopo by grouping the number of specimens by month using the ‘collection date’ field and again with the ‘entered date’ field. this helped to evaluate workflow efficiency, identify potential backlogs, and determine whether delayed processing had contributed to increased tats and the referral of specimens to reference laboratories outside the province. if the number of specimens collected in a given month exceeded the number processed, it suggested the presence of a backlog. to quantify objectively the deviations observed in vl distributions, 2024 monthly results were compared with corresponding 2023 data within each vl range. z-scores were calculated as standardised deviations (z = [result – mean]/standard deviation) based on 2023 means and standard deviations, allowing consistent comparison across categories (online supplementary tables 1–5). heatmaps generated using the seaborn python library visually represented these standardised deviations over time. this study also examined potential epidemiological factors contributing to vl distribution changes. longitudinal patient-level vl data were analysed to assess art adherence and virological suppression by examining serial vl results, follow-up testing rates, mean intervals between tests, and suppression rates. in the western cape, where a unique medical record number is assigned consistently to each patient, vl results were aggregated using this identifier. in contrast, for limpopo and other provinces that do not use a standardised medical record number system, a probabilistic matching approach was used. a record linkage algorithm was developed to generate a synthetic ‘unique_id’, based on a weighted scoring system incorporating patient attributes such as name, surname, date of birth, sex, folder number, and hospital attended. pairs of records exceeding a predefined similarity threshold were grouped as belonging to the same individual. given the computational intensity of large-scale pairwise comparisons, records were first pre-clustered by the first letter of the surname to enhance processing efficiency. no patient identifiers are present in the output data and only aggregated results were analysed. use of artificial intelligence artificial intelligence tools such as chatgpt (openai), claude ai (anthropic), and deepseek were used to assist in the development and refinement of the python scripts used for data processing and analysis. these tools helped to correct syntax errors, troubleshoot logic issues, and optimise script functionality. in addition, they were used to improve grammar, clarity and consistency in the manuscript. at no point was any patient-identifiable information or unprocessed data uploaded into any artificial intelligence platform. all analyses were conducted locally in full compliance with institutional data governance policies and ethical guidelines. the use of artificial intelligence was limited to supportive roles in coding and editorial assistance. all data interpretation, analysis, and conclusions were independently conducted and verified by the authors. results groote schuur hospital laboratory between february 2024 and august 2024, the distribution of hiv vl results at gsh remained consistent. on average, 53% of results were classified as ldl, 31% were < 50 copies/ml, 4.9% fell within 50–199 copies/ml, 2.7% within 200–1000 copies/ml, and 8.1% were > 1000 copies/ml (figure 1). notable increases in vls above 1000 copies/ml were seen in january 2024 and december 2024, probably reflecting a subset of newly diagnosed individuals who had not yet started treatment and were not yet engaged in routine art clinic follow-up. figure 1: hiv viral load result distribution for groote schuur hospital laboratory grouped monthly by ‘collection date’ from january 2024 to december 2024 (n = 204 966, average of 17 081 specimens collected per month). from september 2024 onwards, a shift was observed in the vl distribution. lower than detectable results began to decline, while the proportions in the < 50, 50–200, and 200–1000 categories increased (> 1000 copies/ml category remained unchanged). the most significant changes occurred in november 2024, with ldl dropping to 42.85%, < 50 rising to 36.18%, and 50–199 increasing to 9.17%. figure 3 shows that these changes exceeded two standard deviations, especially in october 2024 and november 2024. these months coincided with a surge in sample referrals from limpopo: 5213 in september 2024, 11 925 in october 2024, and 12 998 in november 2024 (figure 2). figure 2: hiv viral load specimen volume at groote schuur hospital laboratory grouped monthly by ‘collection date’ and by ‘episode region’, from january 2024 to december 2024. figure 3: heatmap illustrating how hiv viral load distribution at groote schuur hospital from january 2024 to december 2024 deviated from its reference data. to determine whether this influx affected overall patterns, the analysis was repeated excluding limpopo specimens. when only western cape (western cape northern cape catchment area) data were included, the vl distributions remained steady after august 2024, mirroring the earlier part of the year. z-scores also remained mostly below 2 (figure 3 and figure 4), indicating no significant deviations. while tats appeared longer in june 2024 and july 2024, as a result of retrospective data entry after the cyberattack, the actual vl distributions during this period remained stable (z-score < 3), despite reduced specimen numbers (figure 2). figure 4: hiv viral load result distribution for groote schuur hospital laboratory grouped monthly by ‘collection date’ from january 2024 to december 2024 (excluding samples from the limpopo region). contamination event in 2024 groote schuur hospital laboratory observed an increase in reactive negative controls during november 2024 and december 2024, with seven reactive cases reported in november 2024 and six in december 2024, compared to only zero or one per month in the preceding months. there was also an increase in positive environmental controls, with vls ranging between < 20 copies/ml up to log 3 copies. this increase coincided with a surge in testing volume driven by external referrals, which may have elevated the risk of contamination events. compounding the situation, the limpopo laboratory was concurrently managing its own contamination issue, evidenced by a spike of 13 reactive negative controls in november 2024 (average of 1.4 reactive negative controls in preceding months), which prompted a temporary shutdown of one of its instruments to carry out a full decontamination procedure. no notable deviation in the hiv vl distribution was observed in the gsh data in 2024 after excluding results from limpopo specimens during this period, as illustrated in the heatmap and clustered bar graph (figure 3 and figure 4). the contamination event was resolved following standard maintenance and decontamination procedures, including ethanol-based cleaning of the amplification area, with no further spikes in reactive negative controls observed in the couple of months that followed (online supplementary table 6). specimens collected in limpopo province figure 5 shows the overall distribution of vl results for limpopo specimens, grouped by vl range. from september 2024, some specimens were sent to external laboratories such as gsh owing to local backlogs, although most were still tested within limpopo (online supplementary figure 5). interestingly, the vl distribution for limpopo differed noticeably from those seen at gsh and across the western cape (online supplementary figures 1–4). figure 5: hiv viral load result distribution for limpopo specimens grouped monthly by ‘collection date’ from january 2024 to december 2024 (n = 614 840, average of 51 237 specimens collected per month). between january 2024 and july 2024, limpopo’s vl results were fairly stable. the < 50 copies/ml category showed minor fluctuations in march, june, and july 2024, averaging about 33% of results. on average, vl results during this period were distributed as follows: 41% ldl, 31% below 50 copies/ml, 13% between 50 and 199, 6.3% between 200 and1000, and 9.7% above 1000 copies/ml. from september 2024 onwards, the ldl proportion dropped to about 34%, while < 50 copies/ml increased to about 37%. the other categories remained largely unchanged (online supplementary figure 6). turnaround times peaked at about 10 days in march 2024, probably because of high specimen volumes that overwhelmed local processing capacity. another peak occurred during the june 2024 to july 2024 cyberattack period, although this was partly because of delays in retrospective data capture (online supplementary figure 5). despite these operational issues, vl distributions remained stable up to july 2024, with only slight increases in the < 50 category (z-score > 3) in march, june, and july 2024 (online supplementary figure 6). as lab operations improved after august 2024, tat decreased steadily, reaching just two days by december 2024. strangely, this coincided with a drop in ldl results and a rise in the < 50 copies/ml category. a comparison of results by testing site (limpopo laboratory vs gsh) showed no major differences in vl patterns or any consistent link between tat and vl distribution (figure 6, online supplementary figure 7, and online supplementary figure 8). figure 6: hiv viral load result distribution for limpopo specimens by test site from september 2024 to november 2024. hiv prevalence, antiretroviral therapy coverage, and adherence data according to the 2024 estimates of the thembisa model, hiv prevalence in the western cape was approximately 6.9%, with art coverage about 74%. in comparison, limpopo had an estimated hiv prevalence of 9.8% and an art coverage rate of 72.4%, of which the national hiv prevalence is 12.8% and art coverage is 77.7%.7 by using a custom record linkage algorithm on python, an analysis of the 2023–2024 limpopo data identified 590 136 unique patients, excluding those with only one vl test conducted after 31 august 2024 (as these patients are most likely newly diagnosed and unlikely to have had follow-up vls by the end of the dataset on 31 december 2024). among these patients, 301 953 instances of vl ≥ 50 copies/ml were recorded, of which more than one instance can belong to a single patient. of these, only 151 340 instances (50.12%) had a follow-up vl result, with a mean time to follow-up of 211 days. among those with follow-up tests, 74 281 (49.08%) achieved virological suppression (< 50 copies/ml). in the western cape, 402 537 unique patients were identified after applying the same exclusion criteria. there were 164 025 instances of vl ≥ 50 copies/ml, with 89 732 (54.71%) followed up. the average time to follow-up was 225 days, and 48 711 (54.28%) of those followed up were virologically suppressed (table 1). table 1: distribution of unique patients by initial hiv viral load category, with corresponding follow-up testing percentages, average number of days to follow-up, and follow-up viral load outcomes categorised by the same predefined ranges, in limpopo and western cape provinces, 01 january 2023 to 31 december 2024. discussion with the recent expansion of the national art programme and increased hiv vl testing, concerns have arisen about the impact of pre-analytical factors on vl results.1,8 this study explored additional factors including analytical and population-level programme dynamics, and how all these factors may play a collective role in vl result interpretation. pre-analytical factors operational disruptions, specimen backlogs, and longer tats were evident in limpopo from early 2024. while the cyberattack later that year most likely worsened these issues, it probably did not affect vl results directly in subsequent months. although many specimens were lost during june 2024 and july 2024, those processed showed no major shifts in vl distribution (figure 4 and figure 5). a notable change in limpopo’s vl profile emerged only after the laboratory information system was restored in august 2024, characterised by a decline in ldl results and a rise in vls < 50 copies/ml (figure 5). given the stability of other vl categories, this may reflect specimen degradation from factors such as viral particle leakage, causing ldl samples to be misclassified as < 50 copies/ml. however, previous studies have shown that hiv-1 rna remains stable at temperatures up to 37 °c and during transit times of up to seven days, reliably differentiating suppression, llv, and virological failure.8,9,10,11 in resource-limited settings, testing is usually confined to reference laboratories distant from sampling sites. for example, a northern cape study highlighted a 250 km radius between nhls district laboratories, with centralised cd4 testing limited to three facilities.12 such distances represent significant pre-analytical challenges that affect access and tats.13,14 although increased tats were suspected to affect vl distribution, including for limpopo specimens processed at gsh, no consistent correlation was found between prolonged tat and rising llv results (figure 5, online supplementary figure 7, and online supplementary figure 8). analytical factors in light of increased reactive negative controls at gsh laboratory in november 2024 and december 2024, machine contamination was initially considered as a possible explanation for the rise in llv results (online supplementary table 6). however, when limpopo specimen results were excluded from the dataset for gsh, the hiv vl distribution remained largely consistent with previous months. a separate analysis comparing results of limpopo specimens processed at either gsh or limpopo during the same time period revealed similar distribution patterns unique to the limpopo province, regardless of the testing site. according to the alinity m hiv-1 assay package insert,6 several integrated mechanisms minimise the risk of contamination during testing. these include the use of aerosol-barrier pipette tips, which are disposed after each use to prevent cross-sample carryover, and the performance of amplification and detection within sealed reaction vessels, which are discarded following analysis. the assay also employs deoxyuridine triphosphate in place of deoxythymidine triphosphate during dna synthesis, enabling the enzymatic degradation of any carryover amplicons by uracil-dna glycosylase prior to the next amplification cycle. the heat-labile uracil-dna glycosylase enzyme is inactivated during the rt-pcr process, preventing interference with the generation of new double-stranded dna products.15 the carryover rate for the alinity m hiv-1 assay was evaluated in the package insert by alternating high positive (1 000 000 copies/ml) and hiv-1-negative plasma samples across multiple runs. of 720 negative replicates, one tested positive, yielding an overall carryover rate of 0.1% (95% confidence interval: 0.0% – 0.8%). this might explain why, despite having contamination issues during this period, the hiv vl proportions remained mostly unaffected during november 2024 and december 2024. for reference purposes, an unrelated contamination event, which occurred at gsh laboratory in 2025, where leakage of amplicons from an uncapped reaction vessel led to a significant decline in the ldl vl results, is mentioned in online supplementary text 1. up to this point, our findings indicate that the rise in llv results observed at gsh was driven primarily by an increase in referred specimens from limpopo, which exhibited a distinct hiv viral load distribution compared to specimens from the laboratory’s usual catchment areas. this provincial variation in hiv vl distribution appears to be specific to region and did not correlate with pre-analytical factors such as extended transit times or compromised sample integrity as assumed previously. to explore potential reasons for these inter-provincial differences, the study was extended to examine broader population-level programme indicators, including hiv prevalence, art coverage and adherence to treatment. population-level programme factors comparing limpopo and western cape provinces reveals marked differences in vl distribution, despite similar hiv prevalence and art coverage (about 72% – 74%). in limpopo, about 20% of vl results fall within the llv ranges (50–200 copies/ml and 200–1000 copies/ml), with 70% suppressed below 50 copies/ml (including ldl). in contrast, western cape reports about 13% llv and 81% suppression below 50 copies/ml (including ldl). table 1 highlights that higher initial vl predicts a lower likelihood of suppression on follow-up, consistent with studies linking persistent llv to virological failure and drug resistance.16,17,18,19 these findings raise questions about the adequacy of the current virological failure threshold of > 1000 copies/ml, which was established by the world health organization for dried blood spot testing in decentralised settings.20 lowering it to 400 copies/ml could allow earlier detection of treatment failure, reduce transmission risk and accommodate for viral blips, while enabling resistance testing when needed. follow-up rates among unsuppressed patients are suboptimal in both provinces—50.12% in limpopo and 54.71% in western cape. interestingly, the vl > 1000 category shows the highest follow-up rate and the shortest time to follow-up, yet it also had the lowest rate of virological suppression. observing the general trend, western cape province has higher follow-up and suppression rates, alongside lower llv proportions on follow-up (table 1), suggesting that adherence challenges can, at least in part, explain the differences observed in vl result distributions between the two provinces. barriers to art adherence are multifactorial, including side effects, nondisclosure, and stigma.21,22 structural and socioeconomic factors, such as limited healthcare access and long travel distances, are especially severe in provinces such as limpopo. these systemic inequities affect rural and under-resourced communities disproportionately, necessitating targeted interventions. limitations information regarding disruptions to services, such as art access, or other epidemiological factors that could have influenced hiv vl results during the study period is uncertain. the exact pre-analytical handling of specimens in limpopo is also unclear, including whether samples were centrifuged upon arrival or only just prior to processing on the alinity m platform. in addition, details about specimen transport conditions are lacking, particularly as temperature loggers were not used. regarding the python-based record linkage script, the records were first pre-clustered according to the first letter of the patient surname before any pairwise comparisons were done to match similar records. as records in different clusters are never compared against each other, patients with typographical errors in the first letter of their surname, or use of a different surname (e.g., married women) or instances where name and surname are switched around were not linked to each other and were treated as separate patients. other relevant patient clinical information, such as treatment interruption, was not available for this study. conclusion this study highlights the value of routine data analysis in high-throughput laboratory settings, where hiv vl results are frequently auto-released without direct oversight by a pathologist. aggregating vl data helps to identify distribution trends, which tend to remain relatively consistent over time. establishing a baseline for these patterns allows for the early identification of anomalies and reduces the risk of inaccurate results being reported unintentionally. differences in hiv vl distribution between provinces appear to be driven more by epidemiological factors, such as art adherence, than by laboratory-related pre-analytical variables. even in real-world conditions where tats reached up to 10 days, no significant effect was observed on vl outcomes (figure 5). similarly, the cyberattack on the information technology system did not appear to alter hiv vl result distributions, although it seemed to have a chain effect later on, disrupting normal laboratory operations and referral pathways, leading to specimens being rerouted to external reference laboratories outside the province. large volumes of specimens sent to gsh from limpopo owing to local backlogs still showed a distinct and consistent vl distribution pattern, different from that of the western cape, regardless of transit duration or test location (figure 6). this event highlights the potential for future multi-centre verification and validation, particularly in cases where significant and unexplained deviations in hiv vl results occur together with other confounding factors like contamination, which can further complicate the interpretation of results. to understand better the broader factors influencing vl distribution, further research is needed, specifically exploring population-level hiv prevalence, art coverage, and adherence. one major obstacle is the lack of a consistent unique identifier or medical record number across provinces; currently, only the western cape captures these data systematically. analyses like this can provide objective insights into art adherence and can highlight areas where policy changes — such as expanding access to vl testing, including point-of-care options23 — could help to improve adherence and move closer to achieving the remaining targets of the joint united nations programme on hiv/aids 95–95–95 strategy. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. credit authorship contribution mo se kwon: conceptualisation, methodology, formal analysis, investigation, writing – original draft, visualisation, project administration, software validation, data curation, writing – review & editing, supervision. khumo o. sematle: conceptualisation, methodology, investigation, writing – original draft, visualisation, project administration, writing – review & editing, supervision. lucia hans: writing – review & editing, supervision. stephen korsman: writing – review & editing, supervision. nei-yuan hsiao: writing – review & editing, supervision. diana r. hardie: conceptualisation, methodology, investigation, writing – original draft, visualisation, project administration, writing – review & editing, supervision. all authors reviewed the article, contributed to the discussion of results, approved the final version for submission and publication, and take responsibility for the integrity of its findings. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the data that support the findings of this study are owned by the nhls and are not publicly available owing to patient confidentiality and data protection regulations. access to the data may be granted upon reasonable request from the corresponding author, mo se kwon, subject to institutional approvals and the nhls data access policy. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. the article does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the authors are responsible for the results, findings, and content of this article. references national health laboratory service. annual report 2022–2023 [homepage on the internet]. johannesburg: national health laboratory service; 2023. [cited 2024 nov 20]. available from: https://www.nhls.ac.za/key-documents/annual-reports/ bangalee a, hans l, steegen k. feasibility and clinical relevance of hiv-1 drug resistance testing in patients with low-level viraemia in south africa. j antimicrob chemother. 2021;76(10):2659–2665. https://doi.org/10.1093/jac/dkab220 hans l, cassim n, sarang s, et al. hiv viral load testing in the south african public health setting in the context of evolving art guidelines and advances in technology, 2013–2022. diagnostics (basel). 2023;13(17): 2731. https://doi.org/10.3390/diagnostics13172731 choga ot, lemogang gm, choga wt, et al. high prevalence of reverse transcriptase inhibitors associated resistance mutations among people living with hiv on dolutegravir-based antiretroviral therapy in francistown, botswana. j antimicrob chemother. 2025;80(3):767–776. https://doi.org/10.1093/jac/dkae472 google maps. distance from mankweng hospital to groote schuur hospital [homepage on the internet]. 2025. 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[cited 2024 jun 23]. available from: https://thembisa.org/downloads hardie d, korsman s, ameer s, vojnov l, hsiao ny. reliability of plasma hiv viral load testing beyond 24 hours: insights gained from a study in a routine diagnostic laboratory. plos one. 2019;14(7):e0219381. https://doi.org/10.1371/journal.pone.0219381 amellal b, murphy r, maiga a, et al. stability of hiv rna in plasma specimens stored at different temperatures. hiv med. 2008;9(9):790–793. https://doi.org/10.1111/j.1468-1293.2008.00632.x vandamme am, van laethem k, schmit jc, et al. long-term stability of human immunodeficiency virus viral load and infectivity in whole blood. eur j clin invest. 1999;29(5):445–452. https://doi.org/10.1046/j.1365-2362.1999.00462.x bonner k, siemieniuk ra, boozary a, roberts t, fajardo e, cohn j. expanding access to hiv viral load testing: a systematic review of rna stability in edta tubes and ppt beyond current time and temperature thresholds. plos one. 2014;9(12):e113813. https://doi.org/10.1371/journal.pone.0113813 coetzee lm, cassim n, glencross dk. newly implemented community cd4 service in tshwaragano, northern cape province, south africa, positively impacts result turn-around time. afr j lab med. 2022;11(1): 1376. https://doi.org/10.4102/ajlm.v11i1.1376 dawande pp, wankhade rs, akhtar fi, noman o. turnaround time: an efficacy measure for medical laboratories. cureus. 2022;14(9):e28824. https://doi.org/10.7759/cureus.28824 girdwood sj, crompton t, cassim n, et al. optimising courier specimen collection time improves patient access to hiv viral load testing in south africa. afr j lab med. 2022;11(1): 1725. https://doi.org/10.4102/ajlm.v11i1.1725 kleiboeker sb. quantitative assessment of the effect of uracil-dna glycosylase on amplicon dna degradation and rna amplification in reverse transcription-pcr. virol j. 2005;2:29. https://doi.org/10.1186/1743-422x-2-29 li jz, gallien s, do td, et al. prevalence and significance of hiv-1 drug resistance mutations among patients on antiretroviral therapy with detectable low-level viremia. antimicrob agents chemother 2012;56:5998–6000. https://doi.org/10.1128/aac.01217-12 hermans le, moorhouse m, carmona s, et al. effect of hiv-1 low-level viraemia during antiretroviral therapy on treatment outcomes in who-guided south african treatment programmes: a multicentre cohort study. lancet infect dis. 2018;18(2):188–197. https://doi.org/10.1016/s1473-3099(17)30681-3 ryscavage p, kelly s, li jz, harrigan pr, taiwo b. significance and clinical management of persistent low-level viremia and very-low-level viremia in hiv-1infected patients. antimicrob agents chemother. 2014;58(7):3585–3598. https://doi.org/10.1128/aac.00076-14 vardhanabhuti s, taiwo b, kuritzkes dr, eron jj jr, bosch rj. phylogenetic evidence of hiv-1 sequence evolution in subjects with persistent low-level viraemia. antivir ther. 2015;20:73–76. https://doi.org/10.3851/imp2772 world health organization. who recommendations [homepage on the internet]. 2013. [cited 2025 feb 12]. available from: https://www.who.int/publications mashele v, marincowitz gjo, marincowitz c. factors influencing adherence to antiretroviral therapy among young adults in limpopo province. s afr fam pract. 2024;66(1):e1–e6. https://doi.org/10.4102/safp.v66i1.5973 nhlongolwane n, shonisani t. predictors and barriers associated with non-adherence to art by people living with hiv and aids in a selected local municipality of limpopo province, south africa. open aids j. 2023;17(1):1–8. https://doi.org/10.2174/18746136-v17-230726-2023-2 drain pk, dorward j, bender a, et al. point-of-care hiv viral load testing: an essential tool for a sustainable global hiv/aids response. clin microbiol rev. 2019;32(3):e00097–e000918. https://doi.org/10.1128/cmr.00097-18 page 1 of 1 reviewer acknowledgement http://www.ajlmonline.org open access read online: scan this qr code with your smart phone or mobile device to read online. acknowledgement to reviewers in an effort to facilitate the 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reviewed were finally published. we apologise for any names that have been inadvertently left out. these individuals provided their services to the journal as a reviewer from 01 october 2021 to 30 september 2022. moses adoga moses o. akiibinu hazim a. alhiti george asare ojor r. ayemoba emmanuel o. babafemi marinus barnard ebi c. bile naseem cassim macdonald m. chaava bettina chale-matsau modibo coulibaly anicet dahourou shewaneh damite tjeerd datema glenda m. davison alpha a. diallo mustapha dibbasey yakhya dieye titilope m. dokunmu peter drotman beverly egyir adetoun ejilemele okechukwu ekenna mathias emokpae kathleen england nicolas epie folorunso o. fasina foluke a. fasola tusabe fred lisa gerwing-adima verena gounden eshetu l. haile samson m. haumba rena hoffman fatih hunç prenika jaglal thumeka jalavu frantz jean louis feyisayo e. jegede sudhakar kalvatala celso khosa byung s. kim daniel kimani jean kloppers winny koster elsie c. kruger george b. kyei bashir laway raphael lihana 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http://www.ajlmonline.org� https://ajlmonline.org mailto:submissions@ajlmonline.org mailto:submissions@ajlmonline.org https://ajlmonline.org https://ajlmonline.org https://ajlmonline.org/index.php/ajlm/user https://ajlmonline.org/index.php/ajlm/user mailto:publishing@aosis.co.za acknowledgement to reviewers abstract introduction methods results discussion acknowledgements references about the author(s) ashandree reddy department of chemical pathology, faculty of health sciences, university of kwazulu-natal, durban, south africanational health laboratory service, durban, south africa nadine rapiti national health laboratory service, durban, south africadepartment of haematology, faculty of health sciences, university of kwazulu-natal, durban, south africa verena gounden department of chemical pathology, faculty of health sciences, university of kwazulu-natal, durban, south africanational health laboratory service, durban, south africa citation reddy a, rapiti n, gounden, v. comparison of 24-hour versus random urine samples for determination and quantification of bence jones protein in a south african population. afr j lab med. 2021;10(1), a1228. https://doi.org/10.4102/ajlm.v10i1.1228 original research comparison of 24-hour versus random urine samples for determination and quantification of bence jones protein in a south african population ashandree reddy, nadine rapiti, verena gounden received: 20 mar. 2020; accepted: 04 mar. 2021; published: 04 aug. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the international myeloma working group and college of american pathologists recommend a 24-h urine collection to determine the bence jones protein (bjp) excretion level for monitoring treatment response in patients with multiple myeloma (mm). there are several issues related to sample collection and the method is prone to inaccuracy. objective: this study compared measured 24-h to random urine collections for the quantitation of bjp in a south african population. methods: sixty-six patients with mm submitted random urine samples with their routine 24-h urine collection from april 2016 – march 2018. measured 24-h urine bjp was compared to two estimated 24-h bjp excretions calculated as follows: estimation 1 (e1): estimated 24-h bjp (mg/24 h) = urine bjp/creatinine ratio (mg/mmol) × 10. estimation 2 (e2): estimated 24-h bjp (mg/24 h) = urine bjp/creatinine ratio (mg/mmol) × 15 mg/kg for women or × 20 mg/kg for men. results: correlation of estimation equations e1 and e2 to the measured 24-h urine bjp was 0.893. patients showed no difference in classification of treatment response using either the e1 or e2 estimation equations when compared to the measured 24-h urine bjp results. conclusion: this study demonstrates that the estimated 24-h bjp shows a high degree of correlation with the measured 24-h bjp and can likely be used to monitor treatment response in south african patients with mm. keywords: multiple myeloma; bence jones protein; random urine; estimated 24-h bence jones protein. introduction plasma cell neoplasms are a group of disorders which include multiple myeloma (mm), where a clone of plasma cells can secrete a homogenous immunoglobulin or its components. these may be identified as a monoclonal peak on analysis by serum or urine protein electrophoresis (upep).1 the presence, level and type of monoclonal immunoglobulin have important implications in diagnosis, staging and treatment of these disease states.2 the core diagnostic features of mm include the presence of neoplastic plasma cells on bone marrow aspirate, radiological evidence of osteolytic lesions and detection of monoclonal immunoglobulin in serum or urine.1 it is the second most common haematological cancer accounting for 1% of all malignancies worldwide. multiple myeloma was responsible for 0.43% of newly diagnosed cases of malignancies in south africa in 1999 with the incidence being reported at approximately 0.00054%.3 while the incidence is highly variable among countries, studies indicate that the incidence of mm has increased uniformly since 1990 with the largest increase in middle and low-middle income countries.4,5 the prevalence of mm is higher in hiv-positive compared to uninfected individuals.6 this increases the disease burden of mm in south africa, a country that has a high hiv epidemic prevalence with 7.5 million people living with hiv.7 monoclonal free light chains (flcs) appearing in the urine are referred to as bence jones proteins (bjps). detection and measurement of bjps are utilised to diagnose and monitor monoclonal gammopathies.8,9 once renal tubular reabsorption is saturated, bjp is present in urine. in approximately 20% of mm cases, bjps may occur in the absence of a monoclonal band in the serum, making their detection a valuable test for this malignancy.8,10 levels of bjps may be quantified by upep. following electrophoresis of the urine specimen and staining of the gel, the size of the bjp peak is measured using a densitometry scan of the peak. the percentage area of the peak is then multiplied by the total urine protein concentration of the sample to provide a semi-quantitative value for the bjp. confirmation of the presence of bjp following upep is performed via immunofixation. the international myeloma working group (imwg) and the college of american pathology recommend a 24-h urine collection for quantification of urine bjp.9,11 although 24-h urine is a definitive means to determine renal protein excretion, it has several issues especially those related to sample collection (table 1)12,13,14,15. in particular, the impracticality of a 24-h collection together with the high likelihood of incomplete collections hinder the accuracy of the test. hence the use of random or early morning urine collections has been suggested to avoid the problems associated with 24-h collections. the clinical utility of measured urine protein is improved when expressed as a ratio to urine creatinine.12,13,14 as creatinine excretion in urine is fairly constant throughout the 24 h, measurement of protein creatinine ratios allows correction for variations in urine concentration. the use of protein creatinine ratios has become widespread for routine urine protein analysis and several studies have demonstrated good correlation with the 24-h collection.11,12,13 table 1: comparison of the advantages and disadvantages of random and 24-h urine collection for bence jones protein. while the use of bjp to creatinine ratio has emerged as an alternative to the 24-h collection, few studies have examined its correlation with the 24-h collection and no reported study to the authors’ knowledge has reviewed its utility in an african population.14,15,16,17 the haematology clinic at king edward viii hospital is the referral centre for the management of patients with mm and other plasma cell neoplasms from the entire province of kwazulu-natal, south africa. many of these patients carry their 24-h collections, travelling several hundred kilometres using public transport to reach the haematology clinic. this is not ideal for maintaining sample stability while also being inconvenient and embarrassing for the patient.18 despite new therapy options, mm is largely incurable, and most patients relapse and require a change in management. laboratory testing plays a vital role in monitoring response to treatment as well as detecting a relapsed inpatient on treatment.9 this, together with the previously described issues related to 24-h urine collections, prompted us to examine the utility and validity of measured 24-h urine compared to random urine collections for the quantitation of bjp in a south african population. methods ethical considerations ethical approval to conduct the study was acquired from the biomedical research ethics committee, university of kwazulu-natal (ref. no. be509/15). written informed consent was taken from each participant in english or isizulu depending on their requirement. the raw data were securely stored by the principal investigator and the compiled electronic data were anonymised and password protected for use only by those involved in the study. participants study participants were recruited from the haematology clinic at king edward viii hospital, durban, south africa. samples were collected over a period of two years (april 2016 – march 2018). all participants had the diagnosis of mm (per imwg criteria) and were at different stages of the disease and treatment. sample collection each participant collected a 24-h urine sample for bjp following a standard protocol as part of the routine clinical assessment. the 24-h collection was started the day before the clinic visit. on submission of the 24-h collection, the participants immediately collected a random urine sample as per instructions provided. thymol was used as the preservative for the 24-h urine sample and no preservative was utilised for the random sample. both samples were submitted to the laboratory immediately. the 24-h collections were analysed as per routine by the chemical pathology laboratory. the random urine samples were analysed for urine total protein (utp) and creatinine. aliquots of the random urine samples were then frozen at –70 °c and stored for a maximum of one month (stability as per manufacturer) until the upep was performed.18 laboratory analysis for both random and 24-h urine collections, upep was performed using the sebia hydragel 7 high resolution kit run on the sebia hydrasys (sebia, norcross, georgia, united states). quantitation of upep fractions was performed using the sebia hydrasys densitometer system and phoresis software (sebia, norcross, georgia, united states). acid violet staining was used, and the sensitivity of this method allows bjp to be detected at concentrations of 15 mg/l – 20 mg/l of the original urine. urine samples for immunofixation electrophoresis (ife) analysis were concentrated using bjp concentrators from the sebia hydrasys kit for all utp samples measuring less than 0.7 g/l.18 urine total protein and urine creatinine were measured using standard spectrophotometric methods on the siemens advia 1800 chemistry analyser (siemens diagnostics, tarrytown, new york, united states). a dye-binding method using pyrogallol red was used to quantify utp.19 urine creatinine was measured using the modified kinetic jaffe method.20 only those 24-h urine samples that were positive for bjp had their respective random samples analysed to determine comparability. the measured 24–h bjp excretion was calculated as follows: %bjp peak on densitometer × utp (g/l) × 24-h urine volume (litre [l]) and multiplied by 1000 mg/24 h. the estimated 24 bjp using the random urine values were calculated as per the two formulae below: for e1, a factor of 10 was utilised because while daily excretion of creatinine is dependent on muscle mass, an average daily loss of 10 mmol of creatinine can be expected.13 e2 was based on the average urinary creatinine excretion which is higher in men (14 mg/kg/day – 26 mg/kg/day) than women (11 mg/kg/day – 20 mg/kg/day).13 of the included participants, three had paired before and after treatment samples. for the purpose of this study, they were classified according to imwg response criteria21 using only the urine and serum electrophoresis data. a complete response is a negative serum and urine m-protein immunofixation; a very good partial response is serum and urine m-protein detectable by immunofixation but not by electrophoresis or a greater than 90% reduction in serum m-protein plus urine m-protein level under 100 mg/24 h; a partial response is a greater than 50% reduction in serum m-protein and a greater than 90% or to under 200 mg/24 h reduction in 24 h urine m-protein; progressive disease is described as an increase of greater than 25% from the lowest response value in any one or more of the following: (1) serum m-component or the absolute increase must be over 0.5 g/dl, (2) urine m-component and/or the absolute increase must be > 200 mg/24 h. stable disease is not meeting criteria for complete response, very good partial response, partial response or progressive disease.21 demographic details and clinical histories were collected from the patients’ clinical records. the body mass index was calculated as weight/height2 (kg/m2) and categorised according to the world health organization. statistical analysis statistical analyses were performed using microsoft® excel (microsoft® office 2016, microsoft, redmond, washington, united states) and medcalc for windows, version 10.0 (medcalc software, ostend, belgium). the shapiro-wilk test was used to assess normality. for non-parametric data, the spearman rank correlation and passing-bablock regression analysis was utilised for comparison of different estimated 24-h bjp equations to the measured 24-h bjp. categorical data were compared using the kruskal wallis test. wilcoxon paired-sample analysis was used to compare continuous variables. a p-value of less than 0.05 was deemed to be statistically significant. results a total of 66 paired 24-h and random urine samples were collected. twenty-two samples (33%) had detectable bjp on 24-h upep and 19 (29%) had a quantifiable bjp in g/24 h. three patients had faint bands below the detectable limit (< 15 mg/l) on the measured 24-h urine with percentage bjp calculated, but did have a quantifiable bjp peak on their paired random urine sample. the urine total protein (tp) for those three 24-h samples were 0.1 g/l, 0.07 g/l and 0.05 g/l which was much lower than their random paired samples of 1.6 g/l, 1.1 g/l and 1.8 g/l. one sample had a quantifiable 24-h bjp peak (tp 0.1 g/l) with no peak on the random urine sample (tp 4.2 g/l) but the monoclonal band was present on urine immunofixation. the 22 samples with detectable bjp were from 19 patients as three patients had repeat collections within the study period. for the three patients, only their first collection was included when analysing data except when categorising the responses. there were 10 women and 9 men with 18 of the 19 patients being black african. the remaining patient was of indian descent. only 10 patients were tested for hiv with only 1 being positive. serum free light chains (sflcs) were also only measured in seven of these patients. of the 19 patients, two had no urine immunofixation request by a pathologist or analysis despite having detectable bjp on upep (table 2). the mean age was 55.8 years old (standard deviation ± 6.6) and the mean body mass index was 27.5 m2/kg (standard deviation ± 5.4) (table 3). table 2: patient characteristics including demographics, immunotyping and relevant laboratory results obtained from king edward hospital, durban, south africa, april 2016 – march 2018. table 3: summary data of the patient characteristics, the mean or median for the measured 24-h bence jones protein and the estimated bence jones protein for the 19 patients, between april 2016 and march 2018, durban, south africa. the correlation between e2 and the 24-h bjp is 0.88 for women and 0.988 for men. the e2 equation was based on the average urinary creatinine excretion which is higher in men (14 mg/kg – 26 mg/kg per day) than women (11 mg/kg – 20 mg/kg per day). the correlation between e1 which is based on creatinine excretion and 24-h bjp is 0.82 for women and 0.975 for men (table 4). table 4: estimated equations (e1 and e2) and 24-h bence jones protein categorised according to gender between april 2016 and march 2018, durban, south africa. analysis following categorisation of the three patients with paired samples per imwg bjp response criteria (table 5) showed no significant difference in classification of treatment response using either the e1 or e2 estimation equations when compared to the measured 24-h urine bjp results. absolute or percentage difference from sample a (before treatment) and sample b (after treatment) did not show pr which is characterised by a greater than 50% reduction of serum m-protein and reduction in 24-h urinary m-protein by over 90% or to under 200 mg/24 h. nor did it show progressive disease with an increase of more than 25% from the lowest response value urine m-component (the absolute increase must be > 200 mg/24 h). the average period between sample a and sample b was nine months. the treatment comprised supportive and specific individualised therapy. the specific therapy included the use of alkylating agents such as cyclophosphamide or melphalan, immunomodulatory agents like thalidomide and steroids. all three patients were classified as stable disease as per the response criteria. the revised international staging system for mm is not affected by the difference between the measured and estimate equations for these three patients as bjp is not included in its criteria. table 5: comparison of 24-h bence jones protein with the estimate equations (e1 and e2) in three paired patient samples before and after treatment over the april 2016 – march 2018 period in durban, south africa, together with their response classification. the spearman rank correlation for both estimation equations e1 and e2 was 0.893 when compared to the measured 24-h bjp. passing-bablock regression analysis showed that the e2 estimation equation had a smaller proportional bias with a slope of 0.968 compared to the e1 estimation equation slope of 0.671 when compared to the measured 24-h bjp (figure 1 and figure 2). figure 1: regression analysis of measured 24hr bence jones protein excretion versus e1 estimation for 24-h bence jones protein during the period april 2016 – march 2018 in durban, south africa. figure 2: regression analysis of measured 24-h bence jones protein excretion versus e2 estimation for 24-h bence jones protein, during the period april 2016 – march 2018 in durban, south africa. discussion the spearman rank correlation of 0.893 signifies a high degree of correlation between the estimate equations and 24-h bjp. this indicates that although the estimate equations do not precisely approximate the 24-h bjp, they are comparable and hence can be useful. the estimate equations also did not consistently overestimate or underestimate the 24-h measurement. the e2 estimation demonstrates a closer correlation and smaller proportional bias with the measured 24-h bjp compared to the e1 estimation equation and may be preferred (table 4, figure 1 and figure 2). importantly, when the three paired samples’ estimated bjps were used to classify patients according to imwg treatment response, there was no significant difference with the performance of the measured 24-h bjp and the estimated bjp using the e1 and e2 equations (table 5). this is key with regard to being able to use the random specimens for monitoring of disease. although there was a limited number of samples, this study indicates the potential use of both 24-h bjp estimates to monitor response in patients with mm and this is in keeping with prior findings in other studies performed in different population groups.16,17 the average age of mm patients in this study was 55.8 years old, which is much younger when compared to that reported in developed countries with the average age at diagnosis ranging from 65 to 70 years.22 due to the high prevalence of hiv infection in the 15–49-year-old age group (19%) in south africa, further research with the appropriate clinical and laboratory information will be key in determining if hiv is a significant risk factor for mm in the younger population.7 unfortunately this information was not available to us at the time. other findings like translocation (11;14) have been reported to be more prevalent in younger myeloma patients.23 a previous study demonstrated that it may be possible to use the protein/creatinine ratio from random urine samples to estimate the 24-h bjp excretion.16 another study concluded that protein concentrations in the same individual are relatively constant. this group also demonstrated that early morning spot specimens had a linear relation with measured 24-h bjp collections and were preferred over random urine collection.17 because patients travelled long distances and arrived at the haematology clinic at varying times, early morning specimens were a challenge to collect. despite this, our study was still able to demonstrate that a random sample can be comparable to a 24-h bjp and can be used to monitor disease response to treatment. light chains are more challenging to detect than complete igs.24 the sflc assay has increasingly been used and tracks well with proteinuria in individual patients.25,26 the greater sensitivity when compared to urine analysis has brought forth the widespread use and incorporation of sflc measurements into multiple guidelines for the management of myeloma; most recently it is a myeloma defining event in asymptomatic patients.9,27 all the study participants had a serum protein electrophoresis and a upep but surprisingly only a few had sflcs. we found only seven patients had sflcs and one of the seven samples had leaked during transit. the sflc assay is not readily available in our province (kwazulu-natal). additionally, due to inter-patient variation in the renal metabolism of light chains, quantification of proteinuria cannot be predicted by the sflc concentration.28,29,30 fifteen of the 19 patients had glomerular infiltration rates under 60 ml/min per 1.73 m2 which may affect the renal metabolism of sfl’s. the imwg states that once a diagnosis of mm is made, a 24-h upep and immunofixation should be done for patient monitoring and these measures are not replaceable with sflc.21,30 multiple myeloma is associated with significant mortality and morbidity and is considered largely incurable and fatal without treatment. with the introduction of new classes of effective drugs for the treatment of mm, improved frequencies and the degree of patient response have been observed. many treatments have been shown to significantly prolong survival and simultaneously improve the quality of life. unfortunately, all patients will ultimately relapse after treatment and will require a change to a more responsive therapy. this necessitates regular periodic monitoring of disease to detect relapse. laboratory testing plays a vital role in monitoring response to treatment as well as detecting relapse in a patient on treatment.9,21 to further elucidate the findings in this study, future studies should include a larger cohort of patients that have measurements before and after treatment enabling a more robust comparison for response criteria. this study is the first to use and to demonstrate the potential utility of the estimated 24-h urine bjp in an african population group. both the e1 and e2 calculations are simple to perform while utp and urine creatinine measurements are easily available on routine chemistry analysers. together with other studies, this study adds to the body of evidence available for use of random urine in estimating 24-h bjp for patient monitoring.16,17 limitations as a result of only including patients with densitometrically quantifiable bjp on the measured 24-h bjp, the small sample size was a limitation. however, this was also a limitation noted in other studies reviewing the use of random urine collections for bjp estimation.16,17 measuring the creatinine on the 24-h urine collections to verify the accuracy of the collection would have been beneficial.17 another limitation is the challenges associated with the method to quantify bjp. different proteins have varying affinities for the dyes used to stain electrophoretic gels and thus a lack of linearity of the densitometry response may be seen. bence jones protein may also co-migrate with other proteins or present with several bands making it complex to define the bjp peak correctly by densitometry. the measurement of bjp is not standardised and to minimise the mentioned analytical variability, it is suggested that patients should be followed up at the same laboratory, which was adhered to in this study.31 we suggest using the random bjp to monitor known patients with mm who already have confirmed bjp on immunofixation to minimise the above-mentioned limitations associated with measuring bjp on electrophoresis. conclusion the random urine bjp estimates e1 and e2 are simple, rapid, easily available and an inexpensive method that is comparable to a 24-h bjp. it can potentially be used for monitoring known patients with mm including light-chain disease. we have demonstrated that when using the imwg response classification, both e1 and e2 equations did not differ from the measured 24-h bjp. although we had a few paired samples, their encouraging comparison and utility will promote further studies with larger cohorts. acknowledgements the authors are grateful to sunitha sathabridg, a technologist at the national health laboratory service based at inkosi albert luthuli central hospital, for assistance in analysis of the urine protein electrophoresis samples. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions v.g. conceived the research idea and assisted with literature review, research protocol development, statistical analysis, interpretation of data and review of the first draft write-up. a.r. compiled the literature review and assisted in research protocol development, ethical and hospital management approval, data collection and processing, statistical analysis, interpretation of data and the journal article first draft write-up. n.r. contributed by reviewing the research protocol and coordinating data collection. sources of support the authors would like to acknowledge the south african national health laboratory service for the funding of this study. data availability raw and derived data supporting the findings of this study are available from the corresponding author, a.r., on request. disclaimer the views expressed in the submitted article are those of the authors and not an official position of the institution. references burtis ca, ashwood er, bruns de. tietz textbook of clinical chemistry and molecular diagnostics. volume 25. london: elsevier health sciences, 2012; p. 547–548. tate j, caldwell g, daly j, et al. recommendations for standardized reporting of protein electrophoresis in australia and new zealand. ann clin biochem. 2012;49(3):242–256. https://doi.org/10.1258/acb.2011.011158 visser hf, visser a, snyckers ch, goller r, pool r, myburgh jg. retrospective review of multiple 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[cited 2018 jan 11]. available from: http://seer.cancer.gov/statfacts/html/mulmy.html duek a, trakhtenbrot l, avigdor a, nagler a, leiba m. multiple myeloma presenting in patients younger than 50 years of age: a single institution experience. acta haematol. 2020;144(1):58–65. https://doi.org/10.1159/000507414 abraham rs, clark rj, bryant sc, et al. correlation of serum immunoglobulin free light chain quantification with urinary bence jones protein in light chain myeloma. clin chem. 2002;48(4):655–657. https://doi.org/10.1093/clinchem/48.4.655 alyanakian ma, abbas a, delarue r, arnulf b, aucouturier p. free immunoglobulin light-chain serum levels in the follow-up of patients with monoclonal gammopathies: correlation with 24-hour urinary light-chain excretion. am j hematol. 2004;75(4):246–248. https://doi.org/10.1002/ajh.20007 bradwell ar, carr-smith hd, mead gp, harvey tc, drayson mt. serum test for assessment of patients with bence jones myeloma. lancet. 2003;361(9356):489–491. 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protein. clin chem lab med. 2005;41(3):338–346. https://doi.org/10.1515/cclm.2003.054 abstract introduction methods results discussion acknowledgements references about the author(s) mokshanand fhooblall school of laboratory medicine and medical sciences, university of kwazulu-natal, durban, south africa fikile nkwanyana howard college, university of kwazulu-natal, durban, south africa koleka p. mlisana department of medical microbiology, national health laboratory services, durban, south africa citation fhooblall m, nkwanyana f, mlisana kp. evaluation of the biofire® filmarray® blood culture identification panel on positive blood cultures in a regional hospital laboratory in kwazulu-natal. afr j lab med. 2016;5(1), a411. http://dx.doi.org/10.4102/ajlm.v5i1.411 original research evaluation of the biofire® filmarray® blood culture identification panel on positive blood cultures in a regional hospital laboratory in kwazulu-natal mokshanand fhooblall, fikile nkwanyana, koleka p. mlisana received: 07 jan. 2016; accepted: 27 july 2016; published: 30 sept. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: there are presently many non-culture-based methods commercially available to identify organisms and antimicrobial susceptibility from blood culture bottles. each platform has its benefits and limitations. however, there is a need for an improved system with minimal hands-on requirements and short run times. objectives: in this study, the performance characteristics of the filmarray® bcid panel kit were evaluated to assess the efficiency of the kit against an existing system used for identification and antimicrobial susceptibility of organisms from blood cultures. methods: positive blood cultures that had initially been received from hospitalised patients of a large quaternary referral hospital in durban, south africa were processed as per routine protocol at its medical microbiology laboratory. positive blood cultures were processed on the filmarray bcid panel kit in parallel with the routine sample processing. inferences were then drawn from results obtained. results: organism detection by the filmarray bcid panel was accurate at 92.6% when organisms that were on the repertoire of the kit were considered, compared to the combination methods (reference method used in the study laboratory). detection of the antimicrobial resistance markers provided by the panel and reference method demonstrated 100% consistency. blood cultures with a single organism were accurately identified at 93.8% by filmarray, while blood cultures with more than one organism were identified at 85.7%. conclusion: the filmarray bcid panel kit is valuable for detection of organisms and markers of antibiotic resistance for an extensive range of organisms. introduction blood cultures are a crucial element in the diagnostic workup of newly-admitted patients and for monitoring suspected bloodstream infection in inpatients.1 identification of organisms responsible for bacteraemia and/or fungaemia, as well as antimicrobial susceptibility patterns of these organisms, are currently done from blood cultures via various methods. many systems (phenotypic and genotypic methods) exist for workup of blood cultures.1,2 however, shortcomings in identification of microorganism(s) from blood cultures do still exist.1 there is a need for a system or combination of systems that offer a compromise between accuracy, precision and timing of reporting of organisms from positive blood cultures. timely and appropriate institution of antibiotics following the susceptibility report of isolated organisms from the laboratory should be also made possible.1 this would be in line with present recommendations of antibiotic stewardship programmes and control of transmission of multidrug-resistant organisms. ideally, for organism identification and susceptibility pattern derivation from blood cultures, a system should have a turnaround time of less than two hours and minimal hands-on time.1 the available genotypic and phenotypic systems, both nucleic acidand non-pcr-based, are attractive options, although each has its specific limitations.3,4 current systems do not offer a comprehensive range of organisms, with some detecting only bacteria but not detecting yeasts. not all systems offer antimicrobial susceptibility information and not every laboratory has adequate and/or skilled personnel to prepare samples for running on molecular kits.1 hands-on time with samples refers to the actual time spent in preparing the run for the samples to be tested.1 run time refers to the interval between setting up the assay and obtaining a result fit for reporting.3 resistance genes refer to gene sequences present in the organisms that may or may not be expressed. expression of resistance phenotypes may be seen as lack of susceptibility to certain antimicrobials.3 the filmarray® bcid panel (biomérieux clinical diagnostics, salt lake city, utah, united states) is a pcr-based molecular platform,5 and high analytical sensitivities of over 90% have been reported by other studies.6,7,8,9,10 it can potentially identify 19 bacteria, five yeasts and three antibiotic resistance markers from positive blood culture bottles. the filmarray panel has been studied by blaschke et al.,6 altun et al.,7 rand and delano,8 bhatti et al.,9 southern et al.10 and ward et al.11 in the different studies, filmarray was compared to various methods for organism detection rate and time taken for organism identification, in view of improving current identification and susceptibility testing.6,7,8,9,10,11 it would be highly beneficial to have identification and susceptibility reports within 60 minutes of a bottle flagging positive, as proposed by the filmarray package insert. with our present combination methods, a minimum of 24 hours would be needed from the time a blood culture becomes positive to reporting identification and resistance patterns. this study was undertaken to assess filmarray, as an option to improve on blood culture reporting. the plethora of bacteria and yeasts covered by the filmarray panel is frequently recovered in blood cultures. the genetic mutations that were available as antimicrobial resistance genes are also of interest. in a quaternary reference hospital, organisms recovered were from patients exposed to a variety, and possibly suboptimal doses, of antibiotics as they are referred from various clinics across kwazulu-natal. this forms a perfect niche for multidrug-resistant organisms. carbapenems, amongst others, are extensively used in private and public sectors in kwazulu-natal, and estimates of klebsiella pneumoniae-type carbapenemase (kpc) resistance gene are needed. the kpc gene, amongst others, offered by filmarray would be interesting to consider from that perspective. methods ethical considerations ethics approval was obtained from the biomedical research ethics committee of the university of kwazulu-natal (be456/14) and the study was done on blood cultures received by the laboratory. consent was obtained from the patients whose samples were used as per the existing agreement between the durban national health laboratory services medical microbiology laboratory, the university of kwazulu-natal and the hospital where the study was undertaken. study setting and design the study was conducted at the quaternary-level facility for the province of kwazulu-natal, south africa, a regional referral hospital with a bed capacity of 846. becton dickinson bactec (becton dickinson, united states) blood cultures were received as per routine work from all wards and intensive care units of the hospital covered by the medical microbiology laboratory of the national health laboratory services, durban, kwazulu-natal. per routine processes, the blood cultures are loaded into the automated blood culture continuous monitoring system bactec™ fx system (bd diagnostics, franklin lakes, new jersey, united states). they remain incubated in the instrument pending organism growth and detection. after five days of no signal received from a blood culture bottle, the bottle is retrieved and resulted as ‘no growth after five days’. blood cultures that become positive are retrieved and worked up by gram stain and subculture. plates from subculture are interpreted by microbiologists and further biochemical or automated tests are utilised. ultimately, positive blood cultures are reported with organism identity and susceptibility. in this study, blood cultures received between february and april 2015 that became positive were evaluated using a prospective analytical approach. during the study, there was no request made to wards to send more blood cultures or to restrict the number of blood cultures made available for the study. any blood culture that became positive was included in the study. the number of positive blood cultures from the same patient, patient name, patient clinical diagnosis or antibiotic received by patient with positive blood culture were not used as exclusion criteria. data on positive blood culture results were drawn from our laboratory information system (lis) – trakcare lab, version 6.10, intersystems corporation (cambridge, massachusetts, united states). the bd bactec fx system used for blood cultures and the vitek® 2 microbial id/ast testing system (biomérieux clinical diagnostics, united states) used by our combination methods were interfaced to the lis. data on identification and susceptibility of organisms recovered by filmarray were recorded into microsoft excel 2013 (microsoft, redmond, washington, united states). identification and susceptibility patterns of organisms recovered from positive blood cultures by combination methods were drawn from the lis and corresponding blood cultures run on filmarray were drawn and compared. reproducibility was achieved by retesting five random positive blood cultures once each on filmarray. the results from the first run on the instrument were compared to the second run. four external controls were used as external standards to confirm the good running of the filmarray panel for this study. external controls american type culture collection (atcc) strains were used as external controls and set up by both subculture and filmarray. strains used (all obtained from davies diagnostics, randburg, south africa) included: listeria monocytogenes atcc 13932, klebsiella pneumoniae atcc 1705, candida kruzei atcc 6758, pseudomonas aeruginosa atcc 27853, candida tropicalis atcc 66029, hemophilus influenzae type b atcc 33533 and candida glabrata atcc mya 2956. these atcc strains were used for quality control in the laboratory as internal quality control for bench controls and were performed weekly. pure colonies of these strains were inoculated into brain heart infusion broth and incubated for 30 minutes. using an optical densitometer, a 0.5 mcfarland standard suspension was made from this brain heart infusion broth and injected into sterile blood cultures. these blood cultures were loaded into the bd bactec fx system and retrieved when positive. one hundred microlitres of the contents of the positive blood culture were aspirated and mixed with sample buffer (provided in the kit). the pouch was loaded onto the filmarray instrument and the result read later. the controls – in the form of atcc strains spiked in blood cultures – were run randomly in between the runs of the positive blood cultures from patients on the filmarray instrument. four positive blood cultures (with a total of seven atcc strains) were run on filmarray, dispersed between 101 positive blood cultures that were received from hospital. this represented an average of 0.04 controls per assay run. combination methods the usual protocol of blood culture workup in the study’s laboratory constituted the combination methods. these were used collectively in this study as the reference method. after a blood culture bottle became positive, drops from its contents were smeared on glass slides, air-dried and gram stained. this was followed by microscopy of the smear. based on the morphology of the organism seen on the gram stain, different culture media were inoculated and incubated under appropriate temperature and atmospheric conditions for at least 18 hours. for gram-positive cocci in clusters, blood agar, mannitol-salt agar and dna plates were set up. for gram-positive cocci in chains, blood agar with optochin disc, macconkey agar and bile esculin agar were set up. for small gram-positive bacilli, blood agar and bile esculin agar were set up. for large gram-positive bacilli, blood agar and egg yolk agar plates were set up. for gram-negative bacilli, chocolate agar and macconkey agar were set up. for gram-negative cocci, chocolate agar and macconkey agar were set up. for yeasts, blood agar and sabouraud’s dextrose agar were set up. interpretation and further testing of recovered organism(s) were done the next day. colonies of organisms were tested by: catalase, indole and oxidase tests; staphylococcal latex agglutination tests; and streptococcal grouping assays. germ tube tests were done on suspected yeasts colonies. suspensions were made of the colonies and set up for api 20e, api 20ne, api 20 strep, api nh, api coryne, api 20 c aux (biomérieux clinical diagnostics, united states) and the vitek 2 microbial id/ast testing system (biomérieux clinical diagnostics, united states). antibiotic susceptibility testing was done using the kirby-bauer disk-diffusion testing system for oxacillin resistance screening, screening of extended-spectrum beta-lactamases (esbls) and modified-hodge test screening. esbls are enzymes that confer resistance to many beta-lactam antibiotics.12 antimicrobial gradient test (etest) was also used for minimum inhibitory concentration evaluation. minimum inhibitory concentration is the lowest concentration of antibiotic that inhibits discernable growth of an organism following overnight incubation. it permits broad differentiation into categories of susceptibility and nonsusceptibility.5 the identification and susceptibility reports were read the next day. the date and time each blood culture became positive and the actual date and time report of blood culture was reported were recorded in microsoft excel for all blood cultures included in the study. the different times taken for reporting positive blood cultures were compared and the minimum turnaround time determined for positive cultures by combination methods. filmarray bcid panel each positive blood culture was run on a single filmarray kit within eight hours of becoming positive. the positive blood culture was gram-stained, subcultured and simultaneously run on filmarray. one hundred microlitres of the contents of the blood culture bottle were aspirated, mixed with sample buffer (provided) and loaded into the filmarray pouch. the pouch was loaded onto the filmarray instrument, which was connected to a computer system. using the filmarray software interface, the different steps of dna extraction, nested and multiplex pcrs and post-amplification analysis may be visualised and timed. at the end of the run, a report was automatically generated which documented it as having any detectable organism(s) as well as any antimicrobial resistance gene(s) – meca, vana/b and kpc. once a positive blood culture was setup on filmarray, a timer was started and time until report generation was noted and compared to the time recorded in the printout report from the filmarray instrument. validity of results was also included in the report, with invalid results reported if either of the two in-built internal controls failed. statistical analyses in this study, identification and antimicrobial susceptibility of organisms obtained by the filmarray panel were compared to those obtained by combination methods. identification that was brought down to the nearest precision possible (genus/species/complex/subspecies level achievable) by filmarray with respect to the results seen with combination methods was labelled as ‘precisely identified’; organisms that were missed or misidentified (genus/species/complex/subspecies level), despite being on the panel, were labelled as either ‘missed’ or ‘misidentified’. the number of organisms, rather than the number of blood cultures, was used to evaluate the performance of filmarray in the statistical analyses. sensitivity, specificity, positive predictive value and negative predictive value as well as agreement were analysed. calculations of sensitivities, specificities, positive predictive values and negative predictive values were adjusted according to results obtained. ‘true positive’ was defined as an organism on the filmarray repertoire, identified by combination methods and also accurately identified by filmarray. ‘true negative’ was defined as any microorganism not on the filmarray repertoire and not detected by filmarray. ‘false positive’ describes any microorganism identified by the filmarray that was not mentioned in the filmarray kit specifications because of design limitations. ‘false negative’ was any microorganism on the filmarray repertoire that was identified by combination methods but was reported by filmarray as any result other than the correct identification (missed/misidentified). organisms detected by filmarray while not identified by combination methods were excluded from the number of organisms detected by filmarray in our calculations. this was due to the assumption that the combination methods was the gold standard method. the combination methods made use of trusted and proven phenotypic methods; and would be (in theory) superior to the filmarray panel for organism detection. calculations were done using ibm spss statistics for windows (version 22.0; ibm corp, armonk, new york, united states). results over the study period, 2119 blood cultures were received by the laboratory and 22.3% (472/2119) were positive. a total of 113 positive blood cultures were worked up both by combination methods and filmarray kits. three blood culture bottles were reported as invalid by the filmarray instrument. four blood culture bottles were used as external controls on filmarray by inoculation with known atcc organisms. all four external controls yielded the desired identification. five blood culture bottles were randomly repeated on filmarray to evaluate reproducibility, and all five gave the same results each time they were run. this permitted the actual evaluation of 101 positive blood culture bottles containing clinical isolates, comparing filmarray to combination methods. overall, 101 positive blood cultures were tested; 92.1% (93/101) with one organism and 7.9% (8/101) with more than one organism by the combination methods. all positive blood cultures included in the study had organisms detectable on initial gram stain. in addition, all 101 positive bottles were identified down to the presence of one or more organisms by combination methods. in total, 109 organisms were detected by combination methods from 101 positive cultures. this is because some blood cultures had one organism, and other blood cultures had more than one organism detected. of the organisms, 86.2% (94/109) were on the repertoire of the filmarray panel for potential detection. the performance of filmarray on positive blood cultures is described (blood culture-wise) in figure 1. with 91.6% (76/83) organisms correctly identified as part of the filmarray repertoire we found equally acceptable accuracy of the filmarray bcid panel, organism wise: 92.6% (87/94) organisms identified that were identifiable (as per the repertoire claimed by the manufacturer) compared to combination methods. performance of filmarray on blood cultures with one type of organism and blood cultures with more than one organism is further elaborated in table 1. organisms and resistance genes recovered by filmarray are in tables 2 and 3 – where, notably, the majority of organisms recovered were gram-positive organisms. conflicting results between filmarray and combination methods are shown in table 4. filmarray missed and/or misidentified 7.5% (7/94) of the organisms. table 1: performance characteristics of filmarray compared to combination methods. table 2: performance of filmarray on blood cultures with one type of organism – by microorganism and antibiotic resistance marker table 3: performance of filmarray on blood cultures with more than one type of organism – by microorganism and antibiotic resistance marker. table 4: breakdown of conflicting results. figure 1: comparison of blood culture results using combination methods against filmarray. a minimum of 24 hours was needed by combination methods to obtain results from positive blood cultures. a maximum of 65 minutes was needed when using filmarray for obtaining identification and potential susceptibility information of an organism from a positive blood culture. once the kit was manually set up and loaded on the filmarray instrument, the run, taking 55–60 minutes preceding report generation, was all automated, needing no operator intervention. organisms recovered that had resistance genes undetectable by filmarray included: esbl-producing klebsiella pneumoniae, esbl-producing enterobacter cloacae, esbl-producing escherichia coli, esbl-producing proteus mirabilis and carbapenemase-producing acinetobacter baumannii (also producing metallobetalactamases and oxacillinases). in certain blood cultures run, there was another form of discrepancy – where more organisms were detected by filmarray than the combination methods. these included 14 isolates comprising these organisms: enterobacter cloacae complex, proteus spp., serratia marcescens, acinetobacter baumannii, haemophilus influenzae, escherichia coli, enterococcus spp., streptococcus spp., candida parapsilosis and staphylococcus aureus. discussion good correlation was seen between the filmarray and combination methods for identification of organisms and resistance genes. an overall accurate identification rate of 92.6% was achieved for organisms identifiable by filmarray from all blood cultures. this was slightly superior to the 91% sensitivity achieved overall in a separate study by blaschke et al., which used a developmental version of the filmarray,6 but slightly below sensitivities found by other studies.7,8,9,10,11 higher sensitivity of filmarray was seen with positive cultures with one type of organism (93.8% [75/80] organisms detected) than with cultures with more than one organism (85.7% [12/14] detected) in our study. this corroborated with a higher proportion of organisms not accurately identified (either missed or misidentified) from cultures with more than one organism tested by filmarray. this was in line with previous work done on the filmarray bcid, which also found lower sensitivity for cultures that detected more than one organism.6,7,8,10 nonetheless, while detection of organisms from blood cultures with one type of organism appears better overall, organisms that were missed by filmarray in our study were more from blood cultures with one type of organism. three out of four false negatives reported were seen from blood cultures with one type of organism. that was in correlation with blaschke et al., who also had a higher number of false negatives from blood cultures with one type of organism as compared to blood cultures with more than one organism (37.5%).6 of concern were organisms that were on the repertoire of the filmarray but not detected and still picked up by combination methods. if filmarray were applied, these results would have been erroneously interpreted as negative. overall, over 7% of organisms (7/94) were falsely classified as negative in our study, although it did include enterococcus spp., which was also missed in two blood cultures. this false-negative rate was higher than findings from other studies which reported lower rates.6,8,10,11 enterococcus spp. was not detected by filmarray from two blood cultures in our study. this is significant, as some enterococcus spp. bloodstream infections carry a high mortality rate, necessitating early and appropriate antibiotic infusion, especially in those with suspected infective endocarditis. enterococcus spp. was also amongst those missed by filmarray in both blaschke et al. (n = 1)6 and altun et al. (n = 2).7 misidentification of methicillin-sensitive staphylococcus spp. as methicillin-sensitive staphylococcus aureus was observed on two blood cultures (blood cultures with one type of organism) in our analysis. this was a species classification mistake by filmarray and delivery of such results might lead to overtreatment with cloxacillin. similar mislabelling has been observed in other studies.9,10 there was negative misclassification of staphylococcus (at genus level) by filmarray in our study, which was in contrast to findings by blaschke et al. and altun et al., where staphylococcus was missed by filmarray while recovered by culture.6,7 streptococcus spp. was erroneously identified as enterococcus spp. by filmarray on two instances in blood cultures in our analysis. this type of misclassification could lead to the wrong choice of antibiotic, as therapy choices for these two are dichotomous. during our investigation, candida albicans was the only yeast recovered from patient blood culture in two instances and was also accurately identified by the filmarray. congruous findings of 100% identification rate of yeasts were also seen by altun et al.7 and southern et al.10 this was as opposed to findings by blaschke et al. where only 75% sensitivity was achieved for candida albicans.6 antibiotic resistance marker detection by filmarray was not an issue in our study. all meca and vana/b genes detected corresponded with culture and vitek results for the respective samples. the kpc gene was not detectable in the clinical samples tested and hence could not be commented on. meca gene detection was inaccurate in previous studies, with bhatti et al. detecting it by filmarray, when not actually present, in four specimens.9 ward et al. also reported six spurious instances of meca detection from both methicillin-sensitive staphylococcus spp. and methicillin-sensitive staphylococcus aureus.11 however, lack of detection of meca was also observed from nine coagulase-negative methicillin-resistant staphylococcus species in their study. minimal laboratory personnel training would be needed for incorporation of filmarray into the routine daily workflow of the laboratory. the stand-alone component of filmarray during its 55–60 minute run time is beneficial. if implemented, this would permit attention to other laboratory work pending the report on positive blood culture. the filmarray instrument software interface also enabled quick troubleshooting and can be coupled to a laboratory information system for more efficient updates of results. in addition, when the filmarray pouch is poorly rehydrated, the system immediately informs laboratory personnel so that an entire hour is not wasted. finally, the 65 minutes required for running filmarray on a positive blood culture was substantially shorter than the time required to run the combination methods (a minimum of 24 hours). limitations filmarray covered only three types of genetic markers (meca, vana/b and kpc). other mechanisms of resistance detected by combination methods, such as esbls, were not provided by filmarray and were therefore missed. as such, an efficient comprehensive antibiotic susceptibility system may be needed with this system. in certain blood cultures, detection of organisms by filmarray was in excess of that of combination methods. these discrepant results were not included in calculations but may have been due to poor performance of combination methods or detection of nucleic acid from non-viable organisms by filmarray in the blood culture.1 our sample size with regard to assessing performance characteristics of filmarray was small at 101. increasing the pool of positive blood cultures tested, with a more diverse range of isolates, would have been beneficial. recommendations one positive blood culture can be run at a time on the filmarray instrument. based on the aforementioned rapid reporting times of this instrument, filmarray would be appropriate to reduce turnaround time in a microbiology laboratory of a hospital. the unavoidable delays in testing more than one positive blood culture could be overcome by using at least two units of the filmarray instrument. the identity of the organism, along with clinical information, would help clinical microbiologists and clinicians rule out whether a positive blood culture contains a contaminant or an actual pathogen, necessitating start or change of therapy.2,4 conclusion our assessment of the filmarray panel on positive blood cultures demonstrated reasonable accuracy and practical benefits. our results were in agreement with the reference method used in this study in the majority of positive blood cultures tested. yet, for the time being, it should be used coupled to an existing identification and antibiotic susceptibility determination system. this, if utilised for rapid communication of results to the physician in the ward, would ameliorate the choice of initial antimicrobial to patients and also drastically affect patient management. acknowledgements the authors are grateful to dr raveen parboosing for his contribution to the statistical analysis. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. sources of support the biofire filmarray instrument and kits were provided by biomérieux. this paper was supported by research project number brec ref: be456/14. authors’ contributions m.f. was the principal investigator, and was responsible for the running of tests and write-up of the analysis. f.n. was the statistician in charge of statistical data handling. k.p.m. was the supervisor, providing advice and assisting with the write-up of the analysis. references paolucci m, landini mp, sambri v. conventional and molecular techniques for the early diagnosis of bacteraemia. int j antimicrob agents. 2010;36(suppl 2):s6–s16. 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zelalem t. bonger, degefu beyene, zeleke ayenew, estifanos tsige, tesfa addis kefale, yonas mekonnen, dejenie s. teklu, elias seyoum, abebe a. negeri african journal of laboratory medicine | vol 13, no 1 | a2268 | 27 march 2024 original research effect of covid-19 lockdown on glycated haemoglobin testing and utilisation in kwazulu-natal, south africa rucita severaj, verena gounden african journal of laboratory medicine | vol 13, no 1 | a2302 | 13 may 2024 original research field study to determine the reliability of hiv viral load results shows minimal impact of delayed testing in south africa diana r. hardie, howard newman, joanna reid, nei-yuan hsiao, gert van zyl, lucia hans, jasantha odayar, stephen korsman african journal of laboratory medicine | vol 13, no 1 | a2364 | 28 may 2024 original research invisible and undervalued: a qualitative study of laboratory workers’ experiences and perceptions of laboratory strengthening in sierra leone mohamed b. jalloh, eva vernooij, alice street african journal of laboratory medicine | vol 13, no 1 | a2292 | 31 may 2024 original research association between dyslipidemia and cervical intraepithelial neoplasia: a case-control study in south-western uganda frank ssedyabane, thomas c. randall, joseph ngonzi, rogers kajabwangu, alexcer namuli, joy muhumuza, josephine n. najjuma, deusdedit tusubira african journal of laboratory medicine | vol 13, no 1 | a2374 | 16 july 2024 original research lecithin-cholesterol acyltransferase and paraoxonase-1 levels in atherosclerotic cardiovascular disease patients in nigeria promise c. nwaejigh, maria o. ebesunun, oluwaseye m. oladimeji african journal of laboratory medicine | vol 13, no 1 | a2286 | 17 july 2024 original research performance comparison of three commercial multiplex molecular panels for respiratory viruses at a south african academic hospital clinton van der westhuizen, mae newton-foot, pieter nel african journal of laboratory medicine | vol 13, no 1 | a2415 | 20 august 2024 original research antibiotic susceptibility pattern among children admitted to a hospital in nigeria: a retrospective study aderonke o. oluwo, mary a. lawal, cecilia a. mabogunje, olubunmi t. okurame african journal of laboratory medicine | vol 13, no 1 | a2362 | 20 august 2024 42 49 58 64 75 83 90 97 page i of ii table of contents editorial the scourge of antimicrobial resistance: containing a global crisis rajiv t. erasmus, chikwelu l. obi, sajini souda african journal of laboratory medicine | vol 13, no 1 | a2645 | 29 november 2024 opinion paper fighting cervical cancer in africa: taking a closer look at human papillomavirus 35 sophia u. okeke african journal of laboratory medicine | vol 13, no 1 | a2243 | 29 january 2024 opinion paper implementation of antimicrobial resistance surveillance in ghana using the integrated disease surveillance and response strategy beverly egyir, alfred bortey, kwabena o. duedu, gifty boateng, franklin a. bekoe, george hedidor, michael adjabeng, nicholas t.k.d. dayie, noah obeng-nkrumah, japheth a. opintan african journal of laboratory medicine | vol 13, no 1 | a2404 | 13 august 2024 opinion paper the human cell atlas: promises, recent developments, and bridging the african single-cell data gap enahoro s. abhulimen african journal of laboratory medicine | vol 13, no 1 | a2583 | 19 december 2024 lessons from the field lessons learnt from assessing and improving accuracy and positive predictive value of the national hiv testing algorithm in nigeria augustine o. mpamugo, nnaemeka c. iriemenam, adebobola bashorun, olumide o. okunoye, orji o. bassey, edewede onokevbagbe, tapdiyel jelpe, matthias a. alagi, chidozie meribe, rose e. aguolu, charles e. nzelu, segun bello, babatunde ezra, christine a. obioha, baffa s. ibrahim, oluwasanmi adedokun, akudo ikpeazu, chikwe ihekweazu, talishiea croxton, sylvia b. adebajo, mcpaul i.j. okoye, alash’le abimiku african journal of laboratory medicine | vol 13, no 1 | a2339 | 28 august 2024 original research sentinel laboratory compliance with best practices in burkina faso’s antimicrobial resistance surveillance network dame yenyetou, emmanuel zongo, emilie dama, merci muhigwa, issouf sanou, charles sawadogo, soumaya ouangraoua, ibrahim sangare, abdoulaye nikiema, anicet g. dahourou, abdoul s. ouedraogo african journal of laboratory medicine | vol 13, no 1 | a2259 | 30 january 2024 original research fosfomycin susceptibility testing and resistance mechanisms in enterobacterales in south africa jessica s. hurwitz, mae newton-foot, kristien nel van zyl, pieter nel african journal of laboratory medicine | vol 13, no 1 | a2252 | 19 march 2024 original research genomic analysis of kel*03 and kel*04 alleles among thai blood donors oytip nathalang, panasya rassuree, kamphon intharanut, wanlapa chaibangyang, núria nogués african journal of laboratory medicine | vol 13, no 1 | a2294 | 19 march 2024 1 4 7 12 16 23 29 35 vol 13, no 1 (2024) issn: 2225-2002 (print) | issn: 2225-2010 (online)african journal of laboratory medicine http://www.ajlmonline.org open access table of contents original research role of ace2 and tmprss2 polymorphisms in clinical severity and outcomes of covid-19 in egypt walaa samy, osama a. gaber, rania m. amer, nahawand a. el-deeb, ahmed a. abdelmoaty, ahmed l. sharaf, ahmed m. el-gebaly, rasha mosbah, maha e. alsadik, amal fawzy, alshymaa a. ahmed african journal of laboratory medicine | vol 13, no 1 | a2375 | 27 august 2024 original research association between coagulation indicators and menorrhagia among women in kenya phidelis m. marabi, stanslaus k. musyoki, fred monari, paul m. kosiyo, collins ouma african journal of laboratory medicine | vol 13, no 1 | a2438 | 17 september 2024 original research challenges with communication of critical laboratory results in a resourcelimited setting in south africa ameerah davids, annalise e. zemlin, elsie c. kruger african journal of laboratory medicine | vol 13, no 1 | a2457 | 23 september 2024 original research an audit of the iron status of patients at chris hani baragwanath academic hospital, in johannesburg, south africa jurette s. grove, siyabonga khoza, dineo v. mabuza, shaida b. khan african journal of laboratory medicine | vol 13, no 1 | a2509 | 18 october 2024 original research eskape pathogen incidence and antibiotic resistance in patients with bloodstream infections at a referral hospital in limpopo, south africa, 2014–2019: a cross-sectional study tiyani c. mthombeni, johanita r. burger, martha s. lubbe, marlene julyan, molebogeng r. lekalakala-mokaba african journal of laboratory medicine | vol 13, no 1 | a2519 | 29 november 2024 104 112 120 126 134 original research healthcare waste management knowledge, attitudes and practices of laboratory workers at a regional hospital, lesotho ts’aletseng m. siimane, motlatsi e. nts’ihlele african journal of laboratory medicine | vol 13, no 1 | a2485 | 06 december 2024 scientific letter carriers of the m.3243a>g variant should not be labelled with an acronym before they have been systematically screened for multisystem disease josef finsterer african journal of laboratory medicine | vol 13, no 1 | a2527 | 30 september 2024 scientific letter pathobiology of hiv-related metabolic and cardiovascular comorbidities: towards a unifying mechanism zohreh jadali african journal of laboratory medicine | vol 13, no 1 | a2582 | 25 october 2024 case study maternally inherited diabetes and deafness with a variable presentation across three generations within a pedigree, south africa herbert makgopa, tanja kemp, surita meldau, engela m. honey, bettina chale-matsau african journal of laboratory medicine | vol 13, no 1 | a2384 | 29 may 2024 brief report diagnostic cut-off value of haemoglobin a1c for diabetes mellitus in harare, zimbabwe chido w. bvumbi, vinie kouamou, ngalulawa kone, trust zaranyika, lloyd bowora, hilda t. matarira, raylton p. chikwati african journal of laboratory medicine | vol 13, no 1 | a2373 | 23 april 2024 142 150 152 154 159 page ii of ii vol 13, no 1 (2024): special collection: transforming african laboratory diagnostic systems through digital technology and artificial intelligence guest edited by prof. rajiv t. erasmus vol 13, no 1 (2024) review article advances in estimating plasma cells in bone marrow: a comprehensive method review ethan j. gantana, ernest musekwa, zivanai c. chapanduka african journal of laboratory medicine | vol 13, no 1 | a2381 | 11 july 2024 164 review article impact of artificial intelligence and digital technology-based diagnostic tools for communicable and non-communicable diseases in africa chikwelu l. obi, joshua o. olowoyo, thembinkosi d. malevu, liziwe l. mugivhisa, taurai hungwe, modupe o. ogunrombi, nqobile m. mkolo african journal of laboratory medicine | vol 13, no 1 | a2516 | 21 november 2024 174 reviewer acknowledgement african journal of laboratory medicine | vol 13, no 1 | a2652 | 21 november 2024 188 about the author(s) rajiha a. ibrahim ethiopian public health institute, addis ababa, ethiopia amete m. teshale ethiopian public health institute, addis ababa, ethiopia surafel f. dinku ethiopian public health institute, addis ababa, ethiopia negga a. abera ethiopian public health institute, addis ababa, ethiopia abebe a. negeri ethiopian public health institute, addis ababa, ethiopia feven g. desta ethiopian public health institute, addis ababa, ethiopia eyasu t. seyum ethiopian public health institute, addis ababa, ethiopia adugna w. gemeda ethiopian public health institute, addis ababa, ethiopia wubshet m. keficho american society for microbiology, addis ababa, ethiopia citation ibrahim ra, teshale am, dinku sf, et al. corrigendum: antimicrobial resistance surveillance in ethiopia: implementation experiences and lessons learned. afr j lab med. 2019;8(1), a1109. https://doi.org/10.4102/ajlm.v8i1.1109 note: doi of original article: https://doi.org/10.4102/ajlm.v7i2.770 corrigendum corrigendum: antimicrobial resistance surveillance in ethiopia: implementation experiences and lessons learned rajiha a. ibrahim, amete m. teshale, surafel f. dinku, negga a. abera, abebe a. negeri, feven g. desta, eyasu t. seyum, adugna w. gemeda, wubshet m. keficho published: 06 dec. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. in the version of this article published earlier, the surname of the second author, amete m. teshale, was inadvertently misspelt as ‘teshal’. the author’s surname should have appeared as ‘teshale’ throughout the author list and ‘how to cite’ information section. this correction does not alter the study’s findings of significance or overall interpretation of the study results. the authors apologise for any inconvenience caused. abstract introduction methods discussion acknowledgements references about the author(s) naseem cassim national health laboratory service (nhls), johannesburg, south africa department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa lindi m. coetzee national health laboratory service (nhls), johannesburg, south africa department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa manfred e.e. tepper national health laboratory service (nhls), johannesburg, south africa louella perelson national health laboratory service (nhls), johannesburg, south africa deborah k. glencross national health laboratory service (nhls), johannesburg, south africa department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa citation cassim n, coetzee lm, tepper mee, perelson l, glencross dk. timely delivery of laboratory efficiency information, part ii: assessing the impact of a turn-around time dashboard at a high-volume laboratory. afr j lab med. 2020;9(2), a948. https://doi.org/10.4102/ajlm.v9i2.948 lessons from the field timely delivery of laboratory efficiency information, part ii: assessing the impact of a turn-around time dashboard at a high-volume laboratory naseem cassim, lindi m. coetzee, manfred e.e. tepper, louella perelson, deborah k. glencross received: 18 dec. 2018; accepted: 04 nov. 2019; published: 29 apr. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: in south africa’s national health laboratory service, ad hoc mean turn-around time (tat) reporting is an important indicator of performance. however, historic static tat reporting did not assess very long or very short times. an interactive tat dashboard was developed using the following tat measures; (1) median, (2) 75th percentile and (3) percentage of within cut-off tat to allow for improved differentiation of tat performance. objectives: the objective of our study was to demonstrate increased efficiency achieved by using an interactive tat dashboard. methods: a retrospective descriptive study design was used. creatinine tat outcomes were reported over 122 weeks from a high-volume laboratory in gauteng, south africa. the percentage of within cut-off and 75th percentile tat were analysed and reported using microsoft excel. a focus group session was used to populate a cause and effect diagram. results: the percentage of within cut-off tat increased from 10% in week 4 to 90% and higher from week 81. the 75th percentile decreased from 10 hours in week 4 to under 5 h from week 71. component tat analysis revealed that the 75th percentile testing was 5 h or longer for weeks 4, 5 and 48. the 75th percentile review tat ranged from 1 h to 15 h. from week 41, the review tat was under 1 h. conclusion: our study demonstrated that the use of an interactive tat dashboard coupled with good management can dramatically improve tat and efficiency in a high-volume laboratory. keywords: turn-around-time; laboratory efficiency; pathology; laboratory medicine. introduction turn-around time (tat) is an important performance indicator of laboratory efficiency to deliver patient results.1 in the south african national health laboratory services, ad hoc mean tat reports were previously produced for laboratory managers. these tat reports assessed performance based on the national health laboratory service global annual performance plan (app) tat cut-offs specific for individual tests.2 reports were provided intermittently in a static form that assessed central tendency only (i.e. the tail size was not reported) and did not allow for drilling functionality to access additional, more detailed, information to direct meaningful corrective action (i.e. laboratory or sample-level tat breakdown). to improve on these tat reporting systems, coetzee et al. used three additional measures to assess tat efficiency: (1) median tat, (2) 75th percentile tat (tail size) and (3) percentage of within cut-off tat.3 these measures accurately assessed outliers as tail size and could be used by laboratories to address workflow issues and identify testing delays for intervention. tail size refers to the volume of samples in a positively skewed data distribution that has a long tail to the right. these samples often have a much higher tat value than the central tendency (median) for this data distribution. tail size can be measured as the percentage of samples that exceed a defined tat cut-off in hours or as a percentile. initially, the three measures described above were reported in microsoft excel (redmond, washington, united states) worksheet format from august 2016 to june 2017.4 thereafter, from july 2017, an interactive dashboard was developed that reported tat data for a basket of tests using the microstrategy desktop (tysons, virginia, united states) analytics tool.5 previously, the static reports and, more recently, the interactive dashboard reports are distributed to area (province), business (district) and laboratory managers. data can be reviewed in the interactive dashboard reports across the provincial, district or laboratory levels through drilling functionality, which makes it possible to slice through a data hierarchy to reveal additional details6 contained within the aggregated data. in this way, tat data presented can be visualised at the national, provincial and laboratory level on the same dashboard page. the approach allows various levels of manager to drill down from a ‘bird’s-eye’ view of tat performance nationally to the provincial or individual laboratory level. within the dashboard, tat can be viewed for a basket of tests including routine haematology full blood count with platelet and differential testing, international normalised ratio, activated prothrombin testing and d-dimers, chemical pathology testing including urea and electrolytes, liver function testing, glucose, cholesterol, among others, as well as microbiology testing for hiv (hiv viral load, hiv dna polymerase chain reaction), tuberculosis (xpert mtb/rif [mycobacterium tuberculosis dna/resistance to rifampicin) and syphilis (rapid plasma reagin and treponema pallidum antibodies) and, lastly, cluster of differentiation 4 (cd4) testing. proxy marker analytes are used to assess performance of the respective matched assay panel, for example creatinine is used as the proxy test to review the urea and electrolytes performance. each test has its own predetermined tat determined at the local level according to the level of care, with absolute national app cut-offs noted. global tat outcomes for each test are reported according to specifically stipulated, organisation-determined tat app at the national level and are described elsewhere.2,7 national app cut-offs are set bearing in mind the multi-tiered service that accommodates reporting from primary health care referral to large tertiary centres that may offer emergency services, and do not necessarily reflect the respective individual, laboratory-stipulated tat, which may be self-determined by laboratories based on their local clinical needs. armed with the knowledge of tat and which tests are identified as poor performers in the interactive dashboard, laboratory managers can identify and address areas of concern through review of the contributing causes.8 this is achieved through root cause analysis, a method of problem-solving used to identify the root causes (faults or problems) and determine the most probable underlying causes of error.8 the ultimate aim of root cause analysis in tat monitoring is to formulate corrective actions that either mitigate or eliminate the identified causes to return tat efficiency and performance to acceptable levels. the aim of this study was to report on the impact of an interactive dashboard that provides weekly information about tat and enables laboratory and senior managers to monitor tat and identify problematic areas for corrective action. the hypothesis was that an interactive tat dashboard delivering week-by-week information about laboratory tat provides the impetus for continuous service review and implementation of appropriate corrective action, where required, to ensure the timeliness of laboratory reporting. data are presented from a single, busy, routine automated clinical pathology laboratory at a large regional hospital to reveal how the described tat dashboard served to continually highlight ongoing tat delays for urea and electrolyte (creatinine) result reporting and, ultimately, facilitated sustained corrective action. methods ethical considerations ethics clearance was obtained from the university of the witwatersrand (study approval number: m1706108). no patient identifiers were extracted with data. study design and samples used a retrospective descriptive study design was used to analyse laboratory data and highlight the impact of interventions by observing trends. qualitative focus group sessions were used to unpack the root causes of poor performance. convenience sampling was used. for the purpose of this study, the tat performance for creatinine testing, which had poor tat at the start of the study, was used to demonstrate how dashboard monitoring of tat could highlight and impact the tat. creatinine testing outcomes were reported with an app cut-off of 90% within 5 hours.2 weekly tat data, from the week ending 07 august 2016 (01 august 2016 to 07 august 2016) to the week ending 02 december 2018 (26 november 2018 to 02 december 2018) was reviewed (122 weeks). data extraction and turnaround time definition the data extract contained the following variables: (1) report week ending date, for example 23 october 2016 (monday to sunday), (2) laboratory name, (3) test method name, (4) tat cut-off, (5) test volumes, (6) percentage of within cut-off tat, (7) median tat, (8) 75th percentile tat, (9) inter-laboratory referral 75th percentile tat, (10) testing 75th percentile tat and (11) review 75th percentile tat. all tat 75th percentile values were reported in hours. each week was numbered, that is, 1–122. tat data refer to total tat (i.e. time of first registration to time of result release after review) if not otherwise specified for tat components. all data were prepared and analysed using microsoft excel (redmond, washington, united states).4 the testing tat time interval was calculated from time of registration in the testing laboratory to time of result generation on the analyser interface. review tat (tst-to-rvw [test-to-review]) is the time taken by a senior technologist to review the patients’ results on the laboratory information system, making sure all quality checks were adequately performed before releasing (authorising) the patients report. the recorded time interval, that is, the review tat, was calculated afterwards for each individual sample outcome from the time of result generation to the time of authorisation or review. percentage within cut-off turnaround time analysis the percentage of within cut-off tat was calculated as the total number of samples meeting the organisation’s tat cut-off criteria of 5 h for urea and electrolytes testing divided by the total number of tests performed, expressed as a percentage, per week. the results were reported as a line chart (indicating the week number and app cut-off of 90%). data were segmented into three phases: (1) baseline: week 1 to 44 (week ending 04 june 2017), (2) dashboard intervention: week 45 to 63 (week ending 15 october 2017) and (3) post-intervention from week 64 to 122 (week ending 02 december 2018). the dashboard intervention period indicates the switch from using an excel worksheet to the interactive dashboard. 75th percentile turnaround time analysis the 75th percentile was calculated for total tat per week, as well as for tat components, that is, testing and review. as tests were local hospital-based and not referred from surrounding laboratories, the pre-analytical tat component was not applicable. when samples are referred, the pre-analytical tat measures the interval (time taken to transport the sample between laboratories) from registration at the source (the laboratory where the sample was received) to the testing laboratory. results from this analysis were plotted as 75th percentile, per testing week, for both total and component tat. root cause analysis the root cause analysis diagram was used to identify potential factors causing poor tat performance.9 causes were grouped into the following headings: (1) equipment and supplies, (2) environmental, (3) rules, policies or procedures and (4) staff or personnel. focus group meetings were arranged with the laboratory manager and section supervisors to identify causes and to populate the cause and effect diagram. a voice recorder was used to create the cause and effect diagram using microsoft visio (redmond, washington, united states).5 results this laboratory performed 326 081 tests for the financial period 2016/2017, 341 760 tests for 2017/2018 and 399 538 tests for 2018/2019. assuming 24/7 operations, this equates to between 894 and 1095 tests per day (booplal n 2019, personal communication). prior to the implementation of the interactive dashboard, weekly tat data were extracted from the corporate data warehouse that houses laboratory information system data within the national health laboratory service. weekly microsoft excel worksheets were prepared manually and distributed via email prior to the implementation of the interactive dashboard at week 45. percentage of within cut-off turnaround time analysis for the baseline phase, the percentage of tat within the cut-off fluctuated (range: 10% to 79%) (figure 1). during the intervention, the tat range again fluctuated from 59% to 97%. for the post-intervention phase, the percentage of tat with the cut-off ranged from 89% to 98%. the 90% cut-off was met for 42 consecutive weeks from week 81 to the end of the study period. figure 1: the percentage of within cut-off turn-around times for creatinine testing at a high-volume laboratory across 122 weeks after implementation of a weekly dashboard, gauteng, south africa, 2017. 75th percentile turnaround time analysis during the baseline phase, the total tat 75th percentile ranged from 4 h to 20 h, changing to 2–10 h for the intervention phase (figure 2). for the post-intervention phase, the 75th percentile range was 2–3 h. for testing tat, the 75th percentile for the baseline phase ranged from 2 h to 11 h and changed during the intervention phase to 1–6 h. in the post-intervention phase, the range was 1–2 h. in the baseline phase, the 75th percentile review tat ranged from 1 h to 15 h compared to 1–3 h for the intervention phase. the post-intervention phase reported a 75th percentile review tat of 1 h or less. figure 2: 75th percentile total turn-around time for creatinine testing at a high-volume laboratory across 122 weeks after implementation of an interactive weekly dashboard, gauteng, south africa, 2017. root cause analysis four major clusters of contributing causes were identified in the root cause analysis including: equipment and supplies, environmental causes, rules, policies and procedural causes and, lastly, staff and personnel factors (figure 3). with respect to equipment and supplies, the following causes were shown to have negatively impacted tat: (1) migration to the new platform (phased approach), (2) difficulties with procurement of laptops to facilitate after-hours off-site authorisation, (3) power outages, (4) bandwidth challenges (laboratory information system [lis]), (5) lis upgrade and (6) reagent or stock procurement. for rules, policies and procedures, the following problems were identified: (1) middleware had to be configured, tested and amended due to the changes brought about by a phased approach and (2) a substantial workload was transferred from a nearby laboratory without provision made for additional testing or staffing capacity. insufficient air conditioning and water leaks from the ceiling were highlighted as causative environmental factors. for staff and personnel considerations, the following were identified: (1) after-hours authorisation delay (by pathologists), (2) industrial action leading to delays, (3) paediatric and low-volume samples requiring manual processing caused bottlenecks in the workflow, (4) staff constraints (in terms of insufficient staff to manage the benches), (5) additional training of staff was required for new procedures and processes for the platform testing changes implemented, (6) prior to full automation, first-line manual sample preparation was needed to enable sample testing and, finally, (7) training for the new platforms provided occurred on site, but staff were also required to attend training off site leaving benches poorly staffed during training periods. figure 3: root cause analysis diagram developed in conjunction with the laboratory manager at a high-volume laboratory, gauteng, south africa, 2017. discussion in this study, it was hypothesised that the application of appropriate corrective action guided by an interactive tat dashboard indicating the proportion of samples within stipulated tat cut-offs and tail size (outliers) would result in improved performance.1,10 this was based on the assumption that delivering an interactive tat dashboard indicating outlier performance on its own would not result in improvement. good laboratory management and response with appropriate corrective action is the key catalyst to deliver a sustained quality of reporting and ensure the continual tat improvement of performance over time.11 all laboratories typically adhere to a quality management system (qms) that is used to assess laboratory quality from the pre-analytic phase through the testing and reviewing processes. a qms is defined as a set of coordinated activities that direct and control a laboratory with regard to quality.12 all aspects of the laboratory operation, including the organisational structure, processes and procedures, need to be addressed by the qms to assure quality.12 laboratory quality can be defined in terms of accuracy, reliability and timeliness (i.e. tat).12 one of the key practices for continuous improvement is the management review meeting, allowing the laboratory an opportunity to review annual performance as set out in the qms. the management review cycle involves planning, implementing, checking and acting on a quarterly basis to address shortfalls identified to effect continual improvement.12 in this study, we reveal how the introduction of a tat dashboard enabled senior management of the laboratory in question to assess tat performance for a particular battery of tests that had not met the stipulated tat cut-off (greater than 65% of results were outside of the stipulated tat cut-off). upon introduction of the dashboard, several areas of concern were immediately identified including pre-analytical, analytical and post-analytical factors. with respect to component tat, assessing specifically the timeframes of registration to testing and testing to release of the report, tat delays were attributable to delays of review during weeks 1 to 41, with further delays caused by testing (instrument) interruptions during weeks 4 and 48. the root cause analysis revealed several contributing factors categorised as equipment and supplies, rules, policies and procedures, environmental and personnel or staffing issues. specifically, the introduction of an auto-review rule process played an important role in improving tat cut-off. a similarly placed high-volume core laboratory in canada also reported that the implementation of a series of lean approaches in their busy laboratory, including automation and auto-review rules, were effective to more efficiently manage substantial volumes of samples while meeting tat cut-offs.13 several important lessons learned and documented by the study laboratory could serve as a template for outreach training to help other public sector laboratories achieve similar tat performance improvements and establish the practices adopted at this site. key learning outcomes emerged: firstly, the importance of the need to collate and actively review real-time information about tat, including components of tat, in ensuring overall timely reporting in laboratories was understood and confirmed. secondly, the value of vertical audits was demonstrated. vertical audits assisted in understanding what contributed to delayed tat, and specific focus on outlier samples and vertical audits directed subsequent meaningful corrective action. in line with the requirement of ongoing improvement of service delivery, a weekly ‘results for action’ statement was developed and found to be useful to deliver specific information at the sample or episode level. such reports, while getting the attention of senior management, could be directed to relevant managers to highlight specific problematic areas and guide the focus of managers’ attention to the investigation of true tat outliers or exceptions. such investigation (with solutions) of specifically identified problem areas could yield practical and advantageous outcomes, not only solving the issues at hand but more widely having a positive impact on overall service delivery improvement. the final lesson learned revolved around the importance of documenting and following through on corrective actions as part of the qms. this ensures that corrective actions taken have consequences and are sustained. in the services review presented here, the week-by-week reminders of outlying tat were a constant cue that solutions implemented had not been effective. re-evaluation and re-assessment allowed for streamlined processes to be considered when initial corrective actions had failed. also highlighted was the importance of conducting a root cause analysis, as cause and effect diagrams, to tease out and understand all aspects of errors and any contributing factors that may lead to delays in tat. it is also important to point out that although a corrective action may be resolved with a single intervention, more frequently corrective action is a multi-step process to identify possible solutions and alternatives. once implemented, these corrective steps require consistent monitoring and evaluation for sustained impact. here, the information provided by the dashboard offered objective evidence of identified issues that could be documented and presented to senior managers month to month and at the annual management meeting; that, in due course, enabled corrective action planning and the facilitated, necessary, mandates to effect better service. past approaches to improving turnaround times over the years there have been multiple approaches to monitoring tat reporting with the aim of improving tat and, in turn, patient care. approaches range from identifying outliers to implementing lean sigma six to process mapping.1,14,15,16,17 one of the earliest approaches described was the identification of tat outliers.1 holland et al. reported that the average length of stay in the emergency department across 11 hospitals correlated significantly with the percentage of outliers.15 in 2007, hawkins defined outliers as the tat of samples that exceeded the institution’s agreed tat cut-offs.1 outliers could also be defined as the tail size given the skewed tat distribution. therefore, the mean and confidence interval are not appropriate measures for assessing tat performance. in 2018, coetzee et al. used the 75th percentile and the percentage of samples within the cut-off tat to identify outliers at the laboratory and test levels.10 the development of routine monitoring systems to identify laboratories with a long tail size enabled focused interventions to proactively resolve poor service delivery.10 many laboratories have implemented lean six sigma approaches to improve tat performance.13 lean is defined as a continuous improvement system consisting of technical tools and management methods.16 one of the aims of a ‘lean’ approach is to find and eliminate waste.16 for example, waste may be introduced by the layout of a laboratory or by poor process design.16 long distances between the receiving office and testing laboratory could also, for example, encourage staff to move samples in batches resulting in tat delays.16 padgett et al. reported that the introduction of lean approaches for troponin testing resulted in substantial tat improvements and averted a proposed point-of-care testing implementation.16 stapleton et al. reported how a lean approach implemented over a three-year period in the laboratory resulted in tat reductions for all emergency tests17 by implementing both workflow improvements and a dedicated emergency bench.17 another approach to improve tat reported by barakauskas et al. involved using lis time stamps, direct observations and discussions with staff to construct various value stream and process maps for immunosuppressant drug level testing.14 the value stream map identified process bottlenecks that were addressed.14 the process map was reported in columns to represent the major groups of personnel and locations from the health care worker to the reference testing laboratory14 with the sequence of events and steps involved illustrated in a vertical direction.14 bottlenecks were also identified in the process map to plan improvement initiatives, for example emergency bag usage.14 ultimately, the aim of any of the approaches described above is to improve tat performance and, thus, patient care. although tat is especially important for emergency tests that have very short tat cut-offs, it is equally important to set cut-offs for other, less urgent tests to ensure that respective test results are received by attending physicians in a timely fashion to effect appropriate patient care, an important factor in assuring both the quality of care and the cost-effective use of hospital services.18 aside from patient care, tat delays also have the potential to waste valuable health resources caused by duplicate test requests, thereby increasing public health expenditure.18 in summary, the multiple approaches to improving tat performance across all laboratory tests play an important role in improving the quality of patient care. application to african contexts figure 4 describes an approach that would make it possible for laboratories in lowand middle-income countries to collate the data required to develop the tat reporting described in our study. in the first instance, laboratories would require a basic lis that could generate weekly data extracts as described in figure 4, using open source database software such as microsoft sql server (express, redmond, california, united states).19 equally, ‘mysql’,20 ‘firebird’21 or ‘cubrid’22 could be deployed to generate the aggregate data described. training is freely accessible via the internet for these software packages. there are multiple free online courses by providers such as ‘edx’, for example, where one can learn how to both develop and query a structured query language (sql) database.23,24 any of these software packages could be implemented on a local desktop or in a server environment, depending on the data volume, with very basic query tools using sql commands making it possible to develop dashboard tools using the step-by-step building blocks approach described in figure 4. figure 4: integration of laboratory information system specimen-level turnaround time data from multiple laboratories into a single, aggregated structured query language database for development of an interactive dashboard, gauteng, south africa, 2017. cross country collaboration and sharing of resources could play an important role in securing already developed dashboard tools for other african countries. a multi-country approach could reduce overall costs and effort. for example, a single tat dashboard could be developed for the southern african development community to ensure accessibility and provide scalability. to secure the system and provide confidentiality, each country could have access to their own data using data access privileges. the benefit of this approach is that after the methods, systems and dashboards are developed, it is easy to extend these developments to other countries with minimal additional cost. the only additional effort required at the country level would be to collate and share the data extracts with the umbrella organisers. limitations only lis data were used for our study. without a laboratory specimen tracking system, it is not possible to report end-to-end tat. the implementation of an end-to-end tracking system from the time of venesection to delivery into a laboratory, additionally integrated into a tat dashboard, could provide valuable supplementary date and time values to allow for an extended tat efficiency review. conclusion in summary, this study demonstrated that an interactive tat dashboard, reporting appropriate tat parameters, applied in the context of a qms, coupled with proactive and diligent management, can accurately identify outliers and lead to appropriate corrective action and sustained timely laboratory reporting. lessons learned acknowledgements the authors thank area, business and laboratory managers in gauteng for their participation and input. competing interests the authors declare no conflict of interest. the authors further declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions d.k.g. supervised the study by providing leadership and oversight as the project leader. n.c., m.e.e.t. and l.m.c. designed the study, developed the methodology and conducted the research. m.e.e.t. extracted the data. n.c. conducted the data analysis. l.p. provided content for the cause and effect diagram and provided details on the interventions implemented. d.k.g. provided editorial comments and technical input. all authors reviewed the results and contributed to the manuscript development. data availability statement we are not able to share the data. disclaimer the authors declare that the views expressed in the submitted article are their own and not the official position of any institution or funder. references hawkins rc. laboratory turnaround time. clin biochem rev. 2007;28(4):179–194. national health laboratory service (nhls). annual report 2017/18 [homepage on the internet]. johannesburg: national health laboratory service; 2018 [cited 2018 dec 10]. available from: http://www.nhls.ac.za/assets/files/an_report/nhls_ar_2018.pdf coetzee l, cassim n, tepper m, glencross dk. the importance of reporting individual weekly laboratory turn-around-time (tat) to identify outliers and underperformance masked during global annual tat review. african society for laboratory medicine (aslm) conference, abuja, nigeria. 2018; 2018 dec 10–13; abuja. 2018. poster: id ps-2.3b-070. microsoft corporation. microsoft office [homepage on the internet]. redmond, wa: microsoft corporation; 2018 [cited 2019 mar 12]. available from: https://products.office.com/en-za/products microstrategy. microstrategy desktop [homepage on the internet]. va: microstrategy; 2018 [cited 2018 dec 03]. available from: https://www.microstrategy.com/us/get-started/desktop corporation m. drill mode in a visualization in power bi [homepage on the internet]. redmont, wa: microsoft corporation; 2018 [cited 2018 dec 12]. available from: https://docs.microsoft.com/en-us/power-bi/consumer/end-user-drill cassim n, coetzee lm, tepper eem, glencross dk. facilitating timely delivery of information about laboratory efficiency, part i: developing an interactive dashboard for a basket of tests to assess turnaround time (tat) efficiency performance across a national laboratory service. afr j lab med. in press 2019. khan k. root cause analysis (rca) of prolonged laboratory turnaround time in a tertiary care set up. j clin diagn res. 2014;8(4):fc05–fc08. https://doi.org/10.7860/jcdr/2014/7269.4255 wikipedia. ishikawa diagram [homepage on the internet]. wikipedia; 2018 [updated 2018 october 26; cited 2019 jan 05]. available from: https://en.wikipedia.org/wiki/ishikawa_diagram coetzee lm, cassim n, glencross dk. using laboratory data to categorise cd4 laboratory turn-around-time performance across a national programme. afr j lab med. 2018;7(1)1–7. https://doi.org/10.4102/ajlm.v7i1.665 ezzelle j, rodriguez-chavez ir, darden jm, et al. guidelines on good clinical laboratory practice: bridging operations between research and clinical research laboratories. j pharm biomed anal. 2008;46(1):18–29. https://doi.org/10.1016/j.jpba.2007.10.010 world health organization (who), centers for disease control (cdc), clinical and laboratory standards institute (clsi). laboratory quality management system handbook [homepage on the internet]. geneva: world health organization; 2011. 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improved laboratory stat test turnaround time using lean six sigma. am j clin pathol. 2012;138(suppl_2):a184-a. https://doi.org/10.1093/ajcp/138.suppl2.36 wybenga dr, winkelman jw, tanasijevic mj, otten j. how fast is fast enough for clinical laboratory turnaround time?: measurement of the interval between result entry and inquiries for reports. am j clin pathol. 1997;108(4):400–405. https://doi.org/10.1093/ajcp/108.4.400 mysql. mysql community edition [homepage on the internet]. redwood city, ca: oracle corporation; 2019 [cited 2019 feb 03]. available from: https://www.mysql.com/products/community/ microsoft corporation. sql server 2017 express edition [homepage on the internet]. redmond, wa: microsoft corporation; 2019 [cited 2019 apr 10]. available from: https://www.microsoft.com/en-us/sql-server/sql-server-editions-express firebird foundation incorporated. firebird rdbms [homepage on the internet]. firebird foundation incorporated; 2019 [cited 2019 apr 10]. available from: https://firebirdsql.org/en/firebird-rdbms/ cubrid. cubrid 10.1 now live: better, faster, stronger [homepage on the internet]. cubrid; 2019 [cited 2019 apr 10]. available from: https://www.cubrid.org/ edx. querying data with transact-sql. cambridge, ma: edx; 2019 [cited 2019 apr 03]. founded by harvard university and mit in 2012, edx is an online learning destination and mooc provider, offering high-quality courses from the world’s best universities and institutions to learners everywhere. available from: https://www.edx.org/course/querying-data-with-transact-sql-2 edx. developing sql databases. cambridge, ma: edx; 2019 [cited 2019 apr 03]. founded by harvard university and mit in 2012, edx is an online learning destination and mooc provider, offering high-quality courses from the world’s best universities and institutions to learners everywhere. available from: https://www.edx.org/course/developing-sql-databases-0 abstract introduction methods results discussion acknowledgements references about the author(s) sam l. nsobya department of pathology, school of biomedical science, makerere university, kampala, uganda paul c. hewett population council, washington, district of columbia, united states sam kalibala population council, washington, district of columbia, united states barbara s. mensch population council, new york, new york, united statesphewett@popcouncil.org citation nsobya sl, hewett pc, kalibala s, mensch bs. performance of kalon herpes simplex virus 2 assay using dried blood spots among young women in uganda. afr j lab med. 2016;5(1), a429. http://dx.doi.org/10.4102/ajlm.v5i1.429 brief report performance of kalon herpes simplex virus 2 assay using dried blood spots among young women in uganda sam l. nsobya, paul c. hewett, sam kalibala, barbara s. mensch received: 24 feb. 2016; accepted: 25 may 2016; published: 16 aug. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract this study evaluated the performance of the kalon biological hsv2 igg enzyme-linked immunosorbent assay (kalon biological ltd, surrey, united kingdom) on dried blood spots (dbs) of various dilutions compared with plasma from young women aged 18–24 years in uganda. we estimated the sensitivity and specificity of three dbs dilutions using plasma as the reference. all three evaluated dbs dilutions yielded low sensitivities and specificities, with dbs 1:2 yielding the highest concurrence. other herpes simplex virus type 2 assays should be examined with regard to their utility for testing dbs. introduction herpes simplex virus type 2 (hsv-2) causes lifelong infections in exposed individuals and is a useful cumulative marker for unprotected sex. hsv-2 infection has also been shown to be an important co-infection for hiv, facilitating hiv acquisition and transmission, and accelerating disease progression.1 in uganda, it is estimated that about half of adults aged 15–59 years are infected with hsv-2.2 numerous studies have outlined the usefulness of dried blood spots (dbs) for the serologic diagnosis of infectious diseases, as well as for large-scale seroprevalence studies.3,4,5,6,7 since dbs do not require immediate refrigeration, occupy little space and are easily transported, they are an attractive means for biomarker-based studies, particularly in geographic settings with limited laboratory resources. recently-developed type-specific hsv antibody tests are based on the detection of antibodies to glycoprotein g1 (gg1), a marker for hsv-1 infection, and glycoprotein g2 (gg2), a marker for hsv-2 infection.8,9 validation of dbs for immunoglobulin g (igg)-based tests has been conducted using hsv-1-positive antibody samples from 22 healthy volunteers.10 hogrefe, ernst and su reported that testing data using a single dilution of dbs eluates (1:4) with the hsv type-specific enzyme-linked immunosorbent assay (elisa) method were similar to those of sera diluted to 1:101 using the standard herpeselect® elisa (focus diagnostics, california, united states).10 the efficiency of using igg eluted from dbs samples was found to be consistent with measurements of igg concentrations in most corresponding serum samples. however, results have been inconsistent when using the kalon biological hsv2 igg elisa assay (kalon biological ltd, surrey, united kingdom; hereafter kalon elisa hsv2 assay) when measuring antibodies to gg2 with the same testing protocol.10 in a household survey conducted in 2010, sexual behavior data and dbs specimens were collected from young women aged 18–24 years in kampala.11 when a 1:4 dilution was applied to our first 277 dbs specimens, the estimated hsv-2 prevalence was 2%. this prevalence was significantly lower than that reported previously in the 2004–2005 uganda hiv/aids sero-behavioural survey (uhsbs), which found a prevalence of 21% among women aged 15–19 years and 38% among women aged 20–24 years.2 when an additional 10 dbs specimens from our sample of young women were further analysed using a dilution of 1:2, the prevalence of hsv-2 increased three-fold.11 in the study reported here, we examined the performance of the kalon elisa hsv2 assay using dbs and plasma samples from stored specimens previously collected during the 2004–2005 uhsbs. the dbs laboratory assessment was conducted in late 2010 and early 2011. the study goal was to examine whether hsv-2 testing results based on dbs at various dilution levels were concordant with plasma-based results obtained from the same participants. methods ethical considerations participants who provided specimens to the 2004–2005 uhsbs consented to the long-term storage and future testing of their delinked blood specimens for which they would not receive results. we obtained additional permission for this study from the uganda ministry of health. the study was also reviewed and approved by the uganda virus research institute’s institutional review board (gc/127/11/10/15) and the population council institutional review board (protocol 433). study population this study was conducted using existing stored dbs and plasma specimens from the 2004–2005 uhsbs. the uhsbs was a nationally-representative household-based survey that sampled 19 656 adult respondents. the main objective of the uhsbs was to obtain national and sub-national estimates of hiv prevalence and selected indicators of hiv-related risk behaviours, programme coverage and hiv knowledge and attitudes. one of its specific objectives was to determine the magnitude and distribution of hiv and other sexually-transmitted infections, such as hsv-2, in uganda.2 the uhsbs estimated the national adult prevalence of hiv at 6.4% and that of hsv-2 at 44%.2 survey specimens and testing survey participants had venous blood samples drawn into 4.5 ml edta vacutainer tubes, from which dbs were produced using whatman ss903 specimen collection paper, air-dried overnight in plastic boxes and stored in lots of 20 separated by glassine paper in ziploc bags containing desiccants. in the field, blood was centrifuged and the plasma was transferred to microvials. plasma and dbs were transported periodically to a central laboratory in entebbe for processing and storage at -80 °c (plasma) and -20 °c (dbs). the plasma and dbs samples for the uhsbs were tested for hsv-2 antibodies using the kalon elisa hsv2 assay within several weeks of collection. results were classified as positive or negative using cutoffs as specified by the manufacturer. dbs validation study design for the purposes of this study, we randomly selected 110 stored dbs specimens from women aged 18–24 years whose plasma-based equivalents tested positive and 110 stored dbs specimens whose plasma-based equivalents tested negative using the kalon elisa hsv2 assay. laboratory testing and analysis was conducted at the molecular research laboratory in kampala as part of the makerere university-university of california, san francisco research collaboration on 2 may 2011. from each of these 220 dbs, a 6 mm-diameter (28 mm2) disk, containing approximately 50–75 µl of blood per spot, was punched out from the filter paper and soaked overnight at 4 °c in 150 µl of phosphate-buffered saline (ph 7.4). after the overnight elution step, the eluates were diluted at three different levels, 1:4, 1:3 and 1:2. each specimen was tested in triplicate with the kalon elisa hsv2 assay, as directed by the manufacturer’s instructions. data analysis frequencies of reactivity, non-reactivity, sensitivity and specificity with 95% confidence intervals were generated using sas® software, version 9.3 (sas institute inc., cary, north carolina, united states, 2011). dbs-based estimates of sensitivity and specificity were obtained using the plasma-based results as the reference. we determined the dilution level for dbs-based hsv-2 testing that yielded the highest (relative) sensitivity and specificity. results the final sample size included 210 individuals due to missing test results for 10 dbs. of the 210 plasma specimens, 104 (49.5%) were reactive and 106 (50.5%) were non-reactive for hsv-2 antibodies (table 1). the dbs 1:2 dilution yielded 116 (55.2%) reactive and 94 (44.8%) non-reactive results. dbs 1:3 produced 124 (59.0%) reactive and 86 (41.0%) non-reactive results, whereas dbs 1:4 yielded 110 (52.4%) reactive and 100 (47.6%) non-reactive results. table 1: hsv-2 seropositivity for plasma-based and dbs dilution-based assays using the kalon elisa hsv2 assay among young women in uganda, 2004–2005.† the 1:2 ratio of buffer and eluate yielded the highest sensitivity (84.6%) and specificity (73.6%) (table 2). the 1:3 dilution had the next-highest sensitivity (82.7%), but had the lowest specificity (64.2%). the 1:4 dilution showed the lowest sensitivity (73.1%) and a specificity of 67.9%. overall, 127 (60.5%) of the 210 specimens had concordant results between plasma and all three dbs dilutions (not shown in the tables). of the 127 concordant results, 69 (54.3%) were concordant positive and 58 (45.7%) were concordant negative. a total of 83 (39.5%) cases had a discordant result between plasma and at least one of the dbs dilutions. of the 83 discordant results, 35 (42.2%) were discordant positive and 48 (57.8%) were discordant negative. table 2: relative sensitivity, specificity and 95% confidence intervals of dried bloodspot dilutions tested with the kalon elisa hsv2 assay compared with plasma among young women in uganda, 2004–2005.† discussion the kalon elisa hsv2 assay has been shown to be sensitive and specific for hsv-2 diagnosis using plasma.2,12 in our study, a dilution of 1:2 showed the highest sensitivity and specificity compared with the 1:3 and 1:4 dilution levels. this estimated sensitivity and specificity is low relative to the plasma-based reference results. specifically, our findings differ from the estimated higher sensitivity and specificity found in a south african population which used the herpeselect elisa serological assay.13 however, earlier studies evaluating the plasma-based herpeselect elisa test in sub-saharan populations suggested a lower specificity than the plasma-based kalon elisa hsv2 assay.14 in addition, patterns of reactivity varied by dilution level. although not the optimal dilution based on sensitivity or specificity, dbs 1:3 had the highest proportional reactivity to hsv-2 antibodies (59.0%). all three dilutions yielded higher frequencies of reactivity than plasma (49.5%), specifically dbs 1:2 (55.2%), dbs 1:3 (59.0%) and dbs 1:4 (52.4%). we found low concordance between plasma-based and dbs-based results, which contrasts with the high concordance reported by hogrefe et al. using the herpeselect assay.10 limitations there are several limitations in our study. the sampling frame was limited to young women aged 18–24 years in uganda; thus, our results are not generalisable to men or older adults. additionally, the sample size was relatively small. further, we did not compare the quantity of igg in the dbs punch specimens to that in plasma specimens. finally, this study only used the kalon elisa hsv2 assay to evaluate dbs for hsv-2 diagnosis. other hsv-2 assays, such as the herpeselect 2 elisa igg and biokit hsv2 rapid assay are used in african countries and need to be further evaluated to compare their utility with dbs. conclusion in summary, our study examined the performance of the kalon elisa hsv2 assay, using dbs in a population of young women in uganda. dbs testing with the kalon elisa hsv2 assay revealed relatively low sensitivity and specificity compared with plasma-based results on the same individuals. while dbs would be an appealing and relatively simple means for biomarker-based studies, particularly in resource-constrained settings, the accuracy of this testing format would need to be substantially improved before its use could be recommended for epidemiological studies. acknowledgements the authors would like to thank angele marandet and wolfgang hladik at the united states centers for disease control for their support and encouragement in the development of the manuscript. we also thank joshua musinguzi of the ministry of health, aids control programme in uganda for his assistance in design and execution of the study. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. sources of support the study was funded by the national institutes of health grant number r01 hd 047764 s1, barbara s. mensch principal investigator, paul c. hewett, co-principal investigator. author contributions s.l.n. drafted the manuscript, which was reviewed and contributed to by p.c.h., s.k. and b.s.m. s.l.n. oversaw the testing of specimens in the laboratory and the reporting of the test results. s.l.n. and p.c.h. contributed to the analysis of the results. b.s.m. and p.c.h. conceived of the original study, with s.l.n. and s.k. assisting with its design and execution. references laeyendecker o, henson c, gray rh, et al. performance of a commercial, type-specific enzyme-linked immunosorbent assay for detection of herpes simplex virus type 2-specific antibodies in ugandans. j clin microbiol. 2004;42(4): 1794–1796. http://dx.doi.org/10.1128/jcm.42.4.1794-1796.2004 macro o. uganda hiv/aids sero-behavioural survey: 2004–2005. ministry of health; 2006. bailey nm, cunningham mp, kimber cd. the indirect fluorescent antibody technique applied to dried blood, for use as a screening test in the diagnosis of human trypanosomiasis in africa. trans r soc trop med hyg. 1967;61(5):696–700. http://dx.doi.org/10.1016/0035-9203(67)90135-6 beebe jl, briggs lc. evaluation of enzyme-linked immunoassay systems for detection of human immunodeficiency virus type 1 antibody from filter paper disks impregnated with whole blood. j clin microbiol. 1990;28(4):808–810. brody ja, mcalister r, haseley r, lee p. use of dried whole blood collected on filter paper disks in adenovirus complement fixation and measles hemagglutination inhibition tests. j immunol. 1964;92(6):854–857. jafri hs, torrico f, noh jc, et al. application of the enzyme-linked immunoelectrotransfer blot to filter paper blood spots to estimate seroprevalence of cysticercosis in bolivia. am j trop med hyg. 1998;58(3):313–315. takkouche b, iglesias j, alonso-fernandez jr, fernandez-gonzalez c, gestal-otero jj. detection of brucella antibodies in eluted dried blood: a validation study. immunol lett. 1995;45(1–2):107–108. http://dx.doi.org/10.1016/0165-2478(94)00247-o ashley rl, wald a. genital herpes: review of the epidemic and potential use of type-specific serology. clin microbiol rev. 1999;12(1):1–8. ashley r. type-specific antibodies to hsv-1 and -2: review of methodology. herpes. 1998;5:33–38. hogrefe wr, ernst c, su x. efficiency of reconstitution of immunoglobulin g from blood specimens dried on filter paper and utility in herpes simplex virus type-specific serology screening. clin diagn lab immunol. 2002;9(6):1338–1342. http://dx.doi.org/10.1128/cdli.9.6.1338-1342.2002 kelly ca, hewett pc, mensch bs, et al. using biomarkers to assess the validity of sexual behavior reporting across interview modes among young women in kampala, uganda. stud fam plann. 2014;45(1):43–58. http://dx.doi.org/10.1111/j.1728-4465.2014.00375.x lingappa j, nakku-joloba e, magaret a, et al. sensitivity and specificity of herpes simplex virus-2 serological assays among hiv-infected and uninfected urban ugandans. int j std aids. 2010;21(9):611–616. http://dx.doi.org/10.1258/ijsa.2009.008477 delany-moretlwe s, jentsch u, weiss h, et al. comparison of focus herpesselect and kalon hsv-2 gg2 elisa serological assays to detect herpes simplex virus type 2 antibodies in a south african population. sex transm infect. 2010;86(1):46–50. http://dx.doi.org/10.1136/sti.2009.036541 smith js, bailey rc, westreich dj, et al. herpes simplex virus type 2 antibody detection performance in kisumu, kenya, using the herpeselect elisa, kalon elisa, western blot and inhibition testing. sex transm infect. 2009;85(2):92–96. http://dx.doi.org/10.1136/sti.2008.031815 abstract introduction antiretroviral therapy monitoring low-level viraemia conclusion acknowledgements references about the author(s) nicholus nanyeenya department of epidemiology and biostatistics, school of public health, makerere university college of health sciences, kampala, uganda ministry of health central public health laboratories, kampala, uganda noah kiwanuka department of epidemiology and biostatistics, school of public health, makerere university college of health sciences, kampala, uganda damalie nakanjako department of medicine, school of medicine, makerere university college of health sciences, kampala, uganda gertrude nakigozi rakai health sciences project, masaka, uganda simon p.s. kibira department of community health and behavioral sciences, school of public health, makerere university college of health sciences, kampala, uganda susan nabadda ministry of health central public health laboratories, kampala, uganda charles kiyaga ministry of health central public health laboratories, kampala, uganda isaac sewanyana ministry of health central public health laboratories, kampala, uganda esther nasuuna infectious diseases institute, makerere university college of health sciences, kampala, uganda fredrick makumbi department of epidemiology and biostatistics, school of public health, makerere university college of health sciences, kampala, uganda citation nanyeenya n, kiwanuka n, nakanjako d, et al. low-level viraemia: an emerging concern among people living with hiv in uganda and across sub-saharan africa. afr j lab med. 2022;11(1), a1899. https://doi.org/10.4102/ajlm.v11i1.1899 review article low-level viraemia: an emerging concern among people living with hiv in uganda and across sub-saharan africa nicholus nanyeenya, noah kiwanuka, damalie nakanjako, gertrude nakigozi, simon p.s. kibira, susan nabadda, charles kiyaga, isaac sewanyana, esther nasuuna, fredrick makumbi received: 23 mar. 2022; accepted: 29 june 2022; published: 20 oct. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract attaining viral load (vl) suppression for over 95% of the people living with hiv on antiretroviral therapy is a fundamental step in enabling uganda and other sub-saharan african countries to achieve global sustainable development goal targets to end the hiv/aids epidemic by 2030. in line with the 2013 world health organization recommendations, several sub-saharan african countries, including uganda, use a threshold of 1000 hiv viral rna copies/ml to determine hiv viral non-suppression. the united states centers for disease control and prevention and the international association of providers of aids care deem this threshold very high, and hence recommend using 200 copies/ml to determine viral non-suppression. using 1000 copies/ml as a threshold ignores people living with hiv who have low-level viraemia (llv; hiv vl of at least 50 copies/ml but less than 1000 copies/ml). despite the 2021 world health organization recommendations of using intensive adherence counselling for people living with hiv with llv, several sub-saharan african countries have no interventions to address llv. however, recent studies have associated llv with increased risks of hiv drug resistance, virologic failure and transmission. the purpose of this narrative review is to provide insights on the emerging concern of llv among people living with hiv receiving antiretroviral therapy in sub-saharan africa. the review also provides guidance for uganda and other sub-saharan african countries to implement immediate appropriate interventions like intensive adherence counselling, reducing vl thresholds for non-suppression and conducting more research to manage llv which threatens progress towards ending hiv by 2030. keywords: hiv/aids; low-level viraemia; viral load testing; non-suppression; virologic failure. introduction by the end of 2020, an estimated 37.7 million people were living with hiv/aids; two-thirds (67%) were living in sub-saharan africa, and 27.5 million were accessing antiretroviral therapy (art).1 the increased access to art has necessited the monitoring of its efficacy among people living with hiv, hence the increased scale-up of hiv viral load (vl) monitoring. as the world strives to control the hiv epidemic, attaining vl suppression for over 95% of people living with hiv on art is a fundamental step in enabling uganda, and other sub-saharan african countries, to achieve the global targets of ending the hiv/aids epidemic by 2030, as stipulated in target 3.3 of the sustainable development goal 3.1,2,3 hiv viral non-suppression is defined as hiv rna viral copies equal to or greater than 1000 copies/ml as recommended by the world health organization (who).4 this is also an indication of hiv virologic treatment failure in uganda, and several other sub-saharan african countries like kenya, zambia and sierra leone, among others.5 low-level viraemia (llv) defined as having an hiv vl of at least 50 copies/ml but less than 1000 copies/ml (≥ 50 copies/ml to < 1000 copies/ml) is considered as being virally suppressed. despite the recent 2021 who recommendation to offer intensive adherence counselling (iac) to people living with hiv on art with llv,6 no special intervention is given to people living with hiv with llv in uganda and several other sub-saharan african countries; yet, llv has been associated with various clinically poor outcomes like hiv drug resistance and virologic failure.7,8 this narrative review was completed in january 2022, and the reviewed articles were published from march 2000 to june 2021. several databases including google scholar, pubmed, and science direct were searched using search terms like ‘low-level viraemia’, ‘viral load testing’, ‘non-suppression threshold’ and ‘virologic failure’. this review aims at guiding uganda and other sub-saharan african countries to take immediate appropriate interventions to manage llv. antiretroviral therapy monitoring highly active art has been the springboard in the management and control of hiv/aids over the years; the goal of highly active art is to lead to hiv viral suppression with an increase in the body immunity function.4 globally, access to art has increased, thereby improving the hiv treatment outcomes,1 and this has been a great milestone in achieving epidemic control. in uganda, an estimated 1.2 million (out of 1.4 million) people living with hiv were on art by 2019.9 as the efforts to scale-up people living with hiv on art continue, there is an inevitable need to monitor the efficacy of art among people living with hiv10 and several methods of art monitoring are recommended by who. these include patient monitoring involving clinical events and adherence monitoring, immunological monitoring involving cd4 cell count, and virologic monitoring comprising hiv vl testing.11, 12 virologic monitoring virologic monitoring which comprises hiv vl testing is the preferred way of monitoring treatment outcomes in people living with hiv on art, and who recommends use of a threshold of 1000 copies/ml to determine vl non-suppression, indicative of either poor drug adherence or virologic failure.6 however, the united states centers for disease control and prevention and international association of providers of aids care recommend a threshold of 200 copies/ml for vl non-suppression.13, 14 a number of sub-saharan african countries initiated the scale-up of vl testing from 2014 to 2015, following the who 2013 recommendation.15, 16 uganda began scaling up hiv vl testing for all eligible people living with hiv on art in 2014.16 upon initiation of art, the first vl test is done for people living with hiv at six months, and then at 12 months, after which it is repeated annually for suppressed adults, and every six months for suppressed children and adolescents. people living with hiv with a vl result below 1000 copies/ml have a suppressed vl and are routinely given adherence counselling in which they are encouraged to continue with art, and no other intervention is given.5 a decreased vl is associated with better clinical outcomes and slowed disease progression,17 and is also associated with reduced hiv incidence at community level.17, 18 however, there is limited data about the cut-off of viral copies at which the slowed disease progression and reduced hiv incidence at community level happens. people living with hiv/aids who have been on art for at least six months with a vl of 1000 copies/ml or more are non-suppressed. these people are offered monthly iac sessions for three months. after the iac sessions, a vl test is repeated in the fourth month to determine whether they have achieved vl suppression.5, 6, 12 if the repeat vl result after iac is still non-suppressed, this is considered as virologic failure provided non-adherence to art is ruled out. a switch committee is then convened to discuss switching the patient to another art line.5 several predisposing factors like poor art adherence, unawareness about the art benefits and other existing chronic illness, among others, have been associated with vl non-suppression.19, 20, 21 people living with hiv on art with non-suppressed vl are at increased risk of faster progression to aids, which is also associated with poor clinical outcomes.21, 22 tremendous progress has been achieved by several sub-saharan african countries in establishing comprehensive vl testing programmes23; for instance, in uganda, the number of annual vl tests has steadily increased annually from 16 411 vl tests (2% of people living with hiv on art) in 2014 to 1 332 335 vl tests (95% of people living with hiv on art) in 2020.24 challenges affecting the scale-up of vl testing, like suboptimal sample transportation and results delivery, limited human resource in health facilities, unawareness about vl testing among hiv healthcare providers and people living with hiv, protracted procurement processes, and poor adherence to national and who vl testing guidelines, have limited full coverage of vl testing in the country.23, 25 the other key important issue to note with the introduction of vl testing is llv. low-level viraemia definition and risk factors the who defines llv as a low but detectable vl that is, viraemia (≥ 50 copies/ml to < 1000 copies/ml).26 people living with hiv/aids on art with viraemia ≥ 50 copies/ml to < 1000 copies/ml are virally suppressed, but have llv.5, 27 previous studies have associated llv with baseline cd4 + t cell counts below 200 cells/mm3, art duration longer than 60 months, art regimens comprising of zidovudine, lamivudine and nevirapine (aor: 2.26, p < 0.001) or didanosine-based regimen, and existing subtype b′ infection.27, 28 justification for using a threshold of 1000 copies/ml for viral load non-suppression the use of 1000 copies/ml as a threshold for determining vl non-suppression in sub-saharan african countries including uganda, has generated enormous debate over the years, since it can lead to the accumulation of people living with hiv on art categorised as suppressed vl, but having llv.29 the recommendation by who to use this threshold of 1000 copies/ml30 was based on a public health perspective where dried blood spot samples were used instead of plasma as a specimen type for hiv-1 vl testing, facilitate the decentralisation of specimen collection and can increase access to vl testing in resource-limited settings like uganda due to their cost-effectiveness, as compared to plasma.31 unfortunately, the performance of dried blood spot samples for vl testing is lower, when compared to the gold standard sample type plasma, with a low sensitivity and specificity to detect treatment failure, and favours using a threshold of 1000 copies/ml.29, 32 slowed disease progression has been demonstrated in people living with hiv on art with a vl of less than 1000 copies/ml,17, 33 though the exact threshold at which llv predicts disease progression varies, and remains debatable.34 furthermore, a reduced risk of hiv transmission for people living with hiv on art with vl results less than; 1000 copies/ml,35 1500 copies/ml,36 and less than 1700 copies/ml,37 has been shown. no hiv transmission risks have been demonstrated below 400 copies/ml,38 but the range of viraemia between 400 copies/ml and 1000 copies/ml at which hiv transmission occurs, still remains unclear.39 prevalence and effects sustained vl suppression has been shown to reduce the occurrence of hiv drug resistance among people living with hiv on art and also leads to improved treatment outcomes.40, 41, 42 however new emerging resistance mutations have been demonstrated in people living with hiv on art with llv,7,8 where persistent llv has been associated with increased risk of virologic failure43; which is also associated with increased risk of adverse treatment outcomes.44 for the case of the sub-sahara african region, 19.3% of the participants had llv while 7.8% of the participants had persistent llv in a study, which aimed to evaluate virologic failure and its predictors in four african countries including uganda, kenya, tanzania and nigeria.45 furthermore, 57.5% of participants with persistent llv (plasma hiv rna > 50 copies/ml at two consecutive visits) in this study would later have confirmed virologic failure. however this study did not examine hiv drug resistance, which could be a key driver of virologic failure (failure of people living with hiv with a non-suppressed vl to suppress after three sessions of iac, and poor drug adherence has been ruled out), and also assessment of drug adherence was mainly through self reports which could have been affected by social desirability and recall bias.45 furthermore, in south africa, llv occurred in 23% of people living with hiv on art with an incidence of 11.5 per 100 person-years of follow-up (95% confidence interval: 11.4–11.7), during first-line art. in this study, llv was associated with increased hazards ratios of virological failure and the subsequent switch to second-line art, as compared with a non-dectectable vl of less than 50 copies/ml; and the risk of virological failure was increased more with increased ranges of llv.7 this study did not look at hiv drug resistance testing results in llv, because they were not available. in another south african study looking at hiv viraemia and mother-to-child transmission risk after art initiation in pregnancy in cape town, the risk of early hiv vertical transmission was greatly associated with vl at delivery, with noted risks of 0.25%, 2.0% and 8.5% among women with vl < 50 copies/ml, 50 copies/ml – 1000 copies/ml and > 1000 copies/ml at delivery, respectively (p < 0.001). this implies that women with llv at delivery had eight times the risk of hiv vertical transmission, as compared to women with a non-detectable vl < 50.46 in malawi, a study was conducted that comprised 1274 mothers and described vl suppression among hiv-positive mothers at 4–26 weeks postpartum, the factors associated with vl suppression, and the impact of vl suppression levels on mother-to-child transmission. this study indicated that 8.7% of these hiv-positive mothers had llv and were more likely to be adolescents, who had been on art for less than six months, with suboptimal adherence. llv was associated with 7.0% risk of mother-to-child transmission, as compared to 0.9% risk for mothers with a non-detectable vl.47 elsewhere, hiv drug resistance was detected in about 30.0% of people living with hiv with a vl test having the first episode of llv in british columbia; and these patients had an increased risk of developing virologic failure, compared to those without hiv drug resistance.48 another study indicated that there were 44.0% accumulated resistance mutations in 47 people living with hiv on art with two or more episodes of llv, and surprisingly the median viraemia was 267 copies/ml; and virologic failure followed in 16.0% of these people living with hiv.49 in the united kingdom, 30.0% of people living with hiv with llv acquired at least one drug resistant mutation50; and major resistant mutations were detected in 12.7% of people living with hiv with llv.51 proposed interventions reduced drug adherence has been associated with residual llv,52 and this implies that interventions to enhance treatment adherence could be offered to people living with hiv on art with llv to achieve a non-detectable vl, which is the recommended target of art in various international guidelines.53, 54 in the recent 2021 who consolidated guidelines, iac has been recommended to be offered to people living with hiv with llv,6 though most sub-sahara african countries have not yet started offering this intervention. furthermore, there is no data in uganda and other sub-sahara african countries to determine whether iac can be effective in achieving a non-detectable vl among people living with hiv on art with llv. few sub-sahara african countries like south africa7, 46 and malawi47 have undertaken research to understand llv and its implications, which has created a knowledge gap about the subject, thereby affecting key policy decisions in the region. conclusion there is an increasing frequency of llv among people living with hiv on art in uganda and across other sub-sahara african countries. this llv is associated with increased risks of hiv drug resistance and virologic failure which can affect the efficacy of art, and lead to accelerated hiv disease progression. with the advancing global targets to end the hiv epidemic, reduce transmission rates, and improve the clinical outcomes of the people living with hiv on art, there is an inevitable need to re-assess the use of the threshold of 1000 copies/ml to determine vl non-suppression, and to establish strategies to address the rising proportions of people living with hiv on art with unmanaged llv in the region. more research about the association between llv and drug resistance and virologic failure in sub-sahara africa also needs to be done. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions n.n., n.k., d.n., g.n. and f.m. designed the concept of the article. n.n., e.n. and i.s. drafted the article with guidance and inputs from n.k., d.n., g.n., f.m., s.p.s.k., s.n. and c.k. all of the authors read through the final article and approved it. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors references joint united nations programme on hiv/aids. global hiv & aids statistics – fact sheet [homepage on the internet]. 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[cited 2021 jun 25]. available from: https://www.eacsociety.org/guidelines/eacs-guidelines/ abstract introduction methods results discussion acknowledgements references about the author(s) victor williams unit of epidemiology and biostatistics, school of public health, faculty of health sciences, university of the witwatersrand, johannesburg, south africa marianne calnan university research co. llc, manila, philippines bassey edem vaccines and immunity theme, mrc unit the gambia, london school of hygiene and tropical medicine, fajara, gambia chukwuemeka onwuchekwa barcelona institute of global health (isglobal), barcelona, spain chika okoro universal health coverage and communicable and non-communicable diseases cluster, world health organization, owerri, imo state, nigeria christine candari university research co. llc, manila, philippines rhodora cruz university research co. llc, manila, philippines kennedy otwombe unit of epidemiology and biostatistics, school of public health, faculty of health sciences, university of the witwatersrand, johannesburg, south africa perinatal hiv research unit, faculty of health sciences, university of the witwatersrand, johannesburg, south africa citation williams v, calnan m, edem b, et al. genexpert rollout in three high-burden tuberculosis countries in africa: a review of pulmonary tuberculosis diagnosis and outcomes from 2001 to 2019. afr j lab med. 2022;11(1), a1811. https://doi.org/10.4102/ajlm.v11i1.1811 note: additional supporting information may be found in the online version of this article as online supplementary document 1. original research genexpert rollout in three high-burden tuberculosis countries in africa: a review of pulmonary tuberculosis diagnosis and outcomes from 2001 to 2019 victor williams, marianne calnan, bassey edem, chukwuemeka onwuchekwa, chika okoro, christine candari, rhodora cruz, kennedy otwombe received: 16 dec. 2021; accepted: 24 may 2022; published: 30 aug. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the rollout of genexpert aimed at increasing early diagnosis of tuberculosis to improve treatment outcomes and global tuberculosis targets. objective: this study evaluated trends in tuberculosis diagnosis and outcomes preand post-introduction of genexpert in three african countries – the democratic republic of the congo (drc), nigeria and south africa. methods: data from 2001 to 2019 were extracted from the world health organization’s data repository. descriptive analysis, paired t-tests and interrupted time series models were used. results: estimated tuberculosis incidence decreased from 327/100 000 to 324/100 000 in the drc, and from 1220/100 000 to 988/100 000 in south africa. incidence remained at 219/100 000 in nigeria. the tuberculosis case notification rate did not change significantly. increases in the new case treatment success rates were statistically significant (drc: p = 0.0201; nigeria: p = 0.0001; south africa: p = 0.0017); decreases in mortality were also statistically significant (drc: p = 0.0264; nigeria: p = 0.0001; south africa: p < 0.0001). time series models showed insignificant increases in new tuberculosis cases in drc (n = 1856, p = 0.085) and nigeria (n = 785, p = 0.555) from 2011 to 2019; and a statistically significant decrease in south africa (n = 15 269, p = 0.006). conclusion: improvements in tuberculosis treatment outcomes were achieved, but little progress has been made in new case notification due to varied implementation and scale-up of genexpert across the three countries. implementation barriers need to be addressed to achieve the required tuberculosis targets. keywords: tuberculosis; genexpert; xpert mtb/rif; africa; interrupted time series. introduction in december 2010, the world health organization (who) approved the xpert mtb/rif assay for the early detection of pulmonary tuberculosis (ptb), particularly in people living with hiv and presumptive multidrug-resistant tuberculosis (mdr-tb) clients.1 previously, sputum smear microscopy was the most used method for the diagnosis of ptb in lowand middle-income countries. based on the type and method of microscopy, the accuracy of detection varies between 20% and 80%,2,3,4 and depends on the technologist’s reading, the quality of the sample, and the smear staining technique. mycobacterium tuberculosis culture and drug susceptibility testing, which are the gold standard for tuberculosis diagnosis and detection of mdr-tb, are time-consuming, expensive, and requires sophisticated equipment which is not available in many facilities. therefore, active tuberculosis disease among people living with hiv who tend to have a paucibacillary disease2 or active mdr-tb disease is detected later, resulting in poorer tuberculosis treatment outcomes; hence, the need for a more reliable and timely means of diagnosis. genexpert for tuberculosis diagnosis although the genexpert platform (cepheid, sunnyvale, california, united states) was available as early as the 1990s, it was originally only used to detect anthrax.3 from 2006, the platform was more widely used to diagnose tuberculosis4,5 and, in 2010, received an endorsement from the who as a first-line tuberculosis diagnostic test among people living with hiv and those presumed to have mdr-tb.1 the xpert mtb/rif assay (cepheid, sunnyvale, california, united states) is an automated molecular (nucleic acid amplification) test for the diagnosis of tuberculosis with comparable accuracy to culture.4,6 it can also detect the m. tuberculosis complex dna and mutations linked to rifampicin resistance (a proxy for mdr-tb) from specimens in under two hours and reduces handling of the specimen by staff. the capability of the xpert mtb/rif assay to detect smear-negative tuberculosis offers an important advantage over smear microscopy, particularly for people living with hiv and children. its ability to detect rifampicin resistance within a short period improves the prospects of timely and appropriate treatment for mdr-tb. culture and drug susceptibility testing are, however, mandatory to complete the drug-resistance profile and to monitor treatment. the operational characteristics of the system which combines user safety, ease of use, and the potential for use as a point-of-care tool, make it an ideal system for quick diagnosis of tuberculosis. the higher cost per sample, the need for a constant power supply, and the need for regular validation are some of the challenges associated with using the system in resource-limited settings.7,8,9 the improved xpert mtb/rif ultra (cepheid, sunnyvale, california, united states), which has enhanced performance in detecting m. tuberculosis and resistant tuberculosis, has a higher sensitivity for smear-negative tuberculosis cases resulting from hiv and tuberculosis coinfection as well as improved rifampicin resistance results. it improves case finding among smear-negative tuberculosis patients who would otherwise have been missed and potentially have transmitted tuberculosis to other people. between 2010 and the end of 2015, 21 549 instrument modules and about 16.2 million xpert mtb/rif cartridges were procured by the public sector and non-governmental organisations from 122 high burden developing countries at concessionary prices.8 country adoption of genexpert the end tb strategy of 2016–2035, defined lists of countries with three types of high-burden tuberculosis infection – tuberculosis alone, mdr-tb, and tuberculosis/hiv coinfection – and identified 30 countries with a high burden of tuberculosis infection.10 fourteen countries, eight of which are in sub-saharan africa, belong to all three lists. xpert mtb/rif rollout in the 14 countries with high burdens of tuberculosis, tuberculosis/hiv coinfection, and mdr-tb infection has been variable. nonetheless, evidence of the impact of the introduction of xpert mtb on tuberculosis diagnosis and detection of drug-resistance at the population level has been demonstrated.11,12,13 in africa, the three highest burden countries are the democratic republic of the congo (drc), nigeria and south africa. the drc installed their first xpert mtb/rif assay in 2011 but has had variable scale-up due to recurrent conflict and the ebola epidemics. by 2016, the drc had only 226 modules with a smear-to-xpert ratio (defined as the market penetration of xpert within public sector organisations) of 18 and a 6% utilisation rate.14,15 a 2019 study indicated there was an increase in tuberculosis case-finding and rifampicin resistance among diagnosed cases; however, treatment gaps had a significant impact on the treatment outcomes for drug-resistant tuberculosis patients.16 nigeria installed the first xpert mtb/rif machine in 2011; by 2019, it had 1560 modules, a smear-to-xpert ratio of 2.3, and a 27% utilisation rate.14 an early implementation assessment conducted by mustapha et al. in 2015 reported minimal improvements in tuberculosis case-finding due to a multitude of challenges that affected accessibility;17 a follow-up assessment in 2018 found the same challenges.18 south africa issued national guidance recommending xpert as the first-line diagnostic test for tuberculosis in 2011–2012, because of the high hiv burden. the first xpert mtb/rif was installed in 2011 and by march 2019 there were 4228 installed modules, and the country had achieved a smear-to-xpert ratio of 0 and a 40% utilisation rate.19 however, xpert was only rolled out to the rural areas between 2013 and 2015. due to the variable uptake of the xpert mtb/rif assay since its endorsement in 2010, we set out to evaluate trends in tuberculosis incidence, case notification, and tuberculosis treatment outcomes preand post-introduction of genexpert in three high ptb-burdened african countries – the drc, nigeria, and south africa. the outcomes were reported between the years 2001 and 2019. methods ethical considerations this study used publicly available aggregated data from the world health organization’s website. identifiable data were not used and there was no direct contact with individuals, hence ethical approval was not required for this study. study design we used a trend design strategy20 to describe tuberculosis incidence, case notification and treatment outcome patterns before and after the introduction of genexpert in three selected tuberculosis high-burden countries in africa between 2001 and 2019. the trend design uses cross-sections at two or more points in time to assess specific changes in a population over a defined period.20 we identified countries with a high burden of tuberculosis, tuberculosis/hiv coinfection, and mdr-tb that introduced xpert within two years of who’s 2010 endorsement and were among the top 10 global high-burden lists based on the absolute number of reported tuberculosis cases21. data were extracted from the updated 2020 who tuberculosis burden estimates22, which are annual estimates from the who, as well as data submitted to the who by different countries. this included 12 tuberculosis indicators that describe annual estimates of tuberculosis incidence, diagnosed tuberculosis cases and notifications, and tuberculosis treatment outcomes. these 12 indicators were selected based on the experience of the researchers that these were adequate to describe tuberculosis incidence, case notification, and tuberculosis treatment outcomes. data analysis data were extracted in an excel file (microsoft corp., redmond, washington, united states) and imported into stata 15 (stata statistical software: release 15. statacorp lp, college station, texas, united states) for cleaning and analysis on 26 june 2021. the years 2001–2010 were selected to study trends before, and 2011–2019 after, rollout of genexpert as a routine tuberculosis diagnostic test. different indicators that describe tuberculosis incidence, tuberculosis treatment outcome, and mortality were retained for analysis22. tuberculosis incidence data per 100 000 population, treatment outcomes and mortality rates for preand post-genexpert periods were summarised by medians and interquartile ranges. additionally, these were presented in line graphs to illustrate trends in the two periods. a paired t-test was used to compare the values of each indicator to determine if there were significant differences in the values of the different indicators between the period before and after the introduction of genexpert within each country. a single group interrupted time series analysis using newey-west standard errors assessed the impact of the introduction of genexpert on the number of confirmed ptb cases diagnosed from 2001 to 2019 in the three countries, using 2011 as the year the programmatic implementation of xpert began. missing data for diagnosed ptb cases for nigeria in 2003 were inputted using the mean value from 2002 and 2004. statistical significance was placed at p < 0.05 and output from the three models are presented as a summary table and charts. the variable ‘t’ represents the time since the start of the study, ‘x2011’ represents the intervention period, ‘x_t2011’ represents the interaction of ‘_x’ and ‘_t’, and ‘treated’ represents the post-intervention linear trend. the ‘_cons’ represent the starting level of laboratory-confirmed ptb cases. a separate sensitivity model including tuberculosis/hiv coinfection rate was done for south africa to ascertain if the initial model may have been confounded by hiv coinfection. results descriptive summary new confirmed ptb cases, the estimated number of cases, new extrapulmonary cases, and tuberculosis notification rates were higher in the drc and nigeria but lower in south africa in the period after, compared to the period before, the introduction of genexpert (table 1)22. table 1: summary statistics of various tuberculosis indicators in three african countries 2001–2010, and 2011– 2019. the estimated tuberculosis incidence per 100 000 population was lower in the drc (324/100 000) and south africa (988/100 000) post-introduction of genexpert, but remained the same in nigeria (219/100 000). data on laboratory-confirmed rifampicin-resistant/mdr-tb was not available before the introduction of genexpert. however, there was an increase in diagnosed mdr-tb cases across the three countries after the introduction of genexpert (137 for the drc, 656 for nigeria, and 8981 for south africa). the tuberculosis case detection rate was comparable across the two periods in all three countries, while a decrease was observed in tuberculosis/hiv coinfection incidence post the introduction of genexpert (16% for the drc, 17% for nigeria, and 60% for south africa). the estimated tuberculosis mortality showed a downward trend in all three countries with the largest decline in south africa, from 395/100 000 to 116/100 000, after the introduction of genexpert. the treatment success rate in all three countries increased after the introduction of genexpert compared to the period before. trends in select tuberculosis indicators confirmed ptb cases (figure 1a)22 increased gradually from less than 50 000 cases in 2002 to slightly above 100 000 in the drc and about 80 000 in nigeria in 2019. however, in south africa, tuberculosis cases peaked at about 200 000 in 2011, dropping to about 120 000 in 2019. new extrapulmonary cases (figure 1b)22 followed a similar pattern to confirmed pulmonary cases but with much lower numbers; nigeria showed a slight decline in the number of cases reported between 2017 and 2019. figure 1: tuberculosis cases in three countries in africa 2001–2019 (a) new laboratory-confirmed cases 2001–2019; (b) new extrapulmonary cases 2001–2019. the case detection rate (figure 2a)22 and treatment success rate (figure 2b)22 showed a slow increase from 2001 to 2019 in all three countries. nigeria had the lowest case detection rate with the least increase. the drc showed a sharp decrease in treatment success rate in 2006, as did south africa in 2010, with south africa showing another decline after 2017. there was a marked increase in tuberculosis case detection rate in the drc and south africa from 2016, with a leveling off by 2019. figure 2: key tuberculosis indicators in three african countries 2001–2019 (a) case detection rate (%) 2001–2019; (b) treatment success rate (%) 2001–2019. south africa showed the most substantial decline in tuberculosis mortality per 100 000 population (figure 3)22 from 2001 to 2019, with the steepest decline occurring between 2011 and 2012. figure 3: tuberculosis-related mortality in three high-burden african countries 2001–2019. comparison between the two periods data for rifampicin-resistant tuberculosis and mdr-tb were not available before the introduction of genexpert; hence no comparison was done. a statistically significant increase was seen in the new pulmonary confirmed tuberculosis cases and the estimated number of all cases in drc (p = 0.0002, p < 0.0001) and nigeria (p = 0.0011, p < 0.0001) but not in south africa (p = 0.7413, p = 0.5081) (table 2)22. estimated tuberculosis incidence per 100 000 population decreased in the drc (p = 0.0067) and south africa (p = 0.1066), but there was no change in nigeria. the percentage of tuberculosis/hiv incidence and mortality per 100 000 population showed a statistically significant decrease in all three countries (drc, p = 0.0264; nigeria and south africa, p < 0.0001), while the percentage treatment success rate for new cases showed a statistically significant increase in the three countries (drc, p = 0.0201; nigeria, p < 0.0001; south africa, p = 0.0017). table 2: comparison of tuberculosis indicators pre(2001–2010) and post-introduction (2011–2019) of genexpert in three high tuberculosis burden countries in africa. interrupted time series models in the drc, the initial number of laboratory-confirmed cases of ptb was 50 747, and the number of cases increased significantly every year before 2011 by 3178 (p < 0.0001) (table 3, supplementary figures 1-3). in the first year of the intervention, cases of ptb decreased by 15 521 (p = 0.009), followed by a marginal increase in the yearly number of cases of diagnosed ptb (relative to the pre-intervention period) of 1856 (p = 0.085). after introducing the intervention, the post-trend estimate shows the cases of diagnosed ptb increased annually by 5034 (p ≤ 0.001). a similar trend was seen in nigeria except for the reduction in the number of cases in the year the intervention commenced, and the increase afterwards was not statistically significant. however, the post-trend estimate showed a statistically significant increase in cases of diagnosed ptb annually by 4295 (p ≤ 0.001). south africa showed a similar trend of a statistically significant annual increase of 7996 (p = 0.013) before 2011. there was a decline in diagnosed ptb cases by 157 in the first year of the intervention period (p = 0.994), however, this decrease was statistically significant annually after the intervention period by 15 269 cases (p = 0.006). the post-intervention linear trend also showed a significant decrease in cases of 7273 (p = 0.028). table 3: summary of interrupted time series models output for the three highest burden tuberculosis countries in africa 2001–2019. the models were not influenced by seasonality or any time-varying confounder. there was no over-dispersion as our outcome variable was continuous. an autocorrelation test was done for each of the three models up to seven lags, and there was no autocorrelation in any of the models. a separate model to ascertain the impact of hiv coinfection, which could have influenced the model output in south africa, indicated similar findings (online supplementary figure 4). discussion our analysis showed that an increase in the number of diagnosed tuberculosis cases in the drc and nigeria was statistically significant, but the number of cases diagnosed in south africa after the introduction of genexpert had decreased. tuberculosis case notification rates and case detection rates did not show major changes. there was, however, a significant reduction in tuberculosis/hiv coinfection and mortality per 100 000 population, with an increase in treatment success rate for new cases in all three countries. while all three countries installed their first xpert mtb/rif in 2011, there was variable adoption of xpert as a first-line diagnostic test, inconsistent scale-up and inadequate utilisation of xpert testing in each country.11,15 these factors may have considerably affected the case notification achievements of the three countries as shown in our study. in the drc, initial evaluation post rollout indicated genexpert improved detection of tuberculosis cases and rifampicin resistance in patients with smear-negative sputum.9 however, later evaluation reports indicated the coverage was low, with one xpert instrument serving 3.3 zones in the country.16 this was further complicated by recurrent stockouts and expiry of instrument cartridges as of 2018, although measures had been instituted to address these with additional staff complements and training.23 this study showed case notification and detection rates did not change considerably in nigeria, with no difference in incidence rate between the two periods. this was corroborated by a 2019 assessment of genexpert performance, which reported low case findings (about 25% of estimated cases) despite the increase in xpert modules and use of xpert as the main diagnostic tool in 2016 (xpert4all initiative).18 this may be because many health workers do not follow the standard protocol when a patient presents with a cough, without considering tuberculosis or ordering genexpert testing. in addition, there is under-utilisation of genexpert because of modular failures, poor power supply, inefficient sample transport mechanisms, weak data management and inadequate human resources to staff the remote test sites. however, the national programme did report an increase in tuberculosis case finding in those areas with access to xpert.18 additional studies on the utility of xpert in nigeria found that it increased tuberculosis case detection among smear-negative ptb patients.24 implementation data from the south african national health laboratory services from 2011 to 2019 demonstrateed a declining trend in the number of cases detected.19 this is consistent with what we have reported. in a study to assess the impact of xpert rollout on tuberculosis diagnostic yield and time to initiate therapy, the researchers observed marginal increases in tuberculosis case detection and time to initiate treatment, suggesting the need to address the health system to improve xpert mtb/rif efficiency.25 in a similar study, xpert increased the proportion of rifampicin-resistant confirmed tuberculosis and significantly reduced the time to diagnosis, commencing therapy, and the pre-treatment mortality in patients with drug-resistant tuberculosis.26 however, on-treatment mortality and treatment outcomes did not change, reflecting a need to address treatment gaps and treatment outcomes.26 similar to our findings, the health systems trust (an organisation established to assist with the transformation of the healthcare system in south africa) showed there has been an annual decline in tuberculosis incidence in south africa from 2011 to 2015 and data from the university of kwazulu-natal suggest that the rapid drop in tuberculosis cases detected could be attributed partially to the scale-up of antiretroviral therapy that took place between 2009 and 2014.27,28,29 however, about 150 000 tuberculosis cases are missed annually in south africa – nearly 50% of the tuberculosis burden in the country.30 patient-side and health system-side factors may contribute to the missed cases and thus reduce the impact of xpert on tuberculosis case detection and treatment outcomes. major health system barriers include laboratory-running challenges such as intermittent electricity supply and limited number of xpert cartridges, insufficient human resources, poor provider knowledge and skills to adhere to diagnostic guidelines and inadequate information management.30,31,32 on the patient side, barriers include loss to follow-up due to delayed diagnosis or family and work commitments, negative public sector care perceptions, and using private-sector care with limited financial resources.33,34 in modelling studies to determine the long-term outcome of scaling up the xpert platform with an algorithm that used xpert as the first-line diagnostic test, it was determined that if implemented optimally, the number of tuberculosis cases diagnosed per year would increase by 30% – 37%, while diagnosed mdr-tb cases would increase by 69% – 71%. eighty-one percent of the cases would be diagnosed after the initial visit compared to 46% at the time.35 the data from each of the three countries, while showing a modest rise in the number of tuberculosis cases diagnosed, did not meet the expectations of the modelling studies. despite the observed increases in the drc and nigeria, there are numerous health system challenges to the optimal rollout and utilisation of genexpert in the diagnosis of tuberculosis cases. south africa registered a decline in the number of tuberculosis cases diagnosed. variable scale-up, low utilisation rates and unaddressed patient-side and health-system barriers have negatively impacted the expected increase in tuberculosis case detection and rifampicin resistance detection, as well as treatment outcomes. xpert performance in other tuberculosis high-burden countries reflects similar trends for case detection and treatment success.15,36 limitations this study was not without limitations. firstly, we used data extracted from the who website for which we had no control in terms of how data was collected. this study did not have a mechanism for identifying data reported to the who that was not updated. there were missing data for some outcomes and variables but where feasible, missing data were imputed. however, the mean imputation approach used may not have yielded the optimal value, although it provided an estimate to work with. the output of our interrupted time series analysis may have been influenced by unmeasured confounders, particularly in south africa where the rollout of the antiretroviral therapy program may have impacted diagnosed tuberculosis cases. to address this, we conducted a sensitivity analysis to ascertain if hiv coinfection impacted the south african model. noting that interrupted time series models are generally not affected by typical confounders such as age distribution or socioeconomic status, like in the modelling of individual patient data, we believe the modelling approach was appropriate.37 nevertheless, this study has highlighted the key successes since the introduction of genexpert for tuberculosis diagnosis and the areas requiring improvement to be on track to end tuberculosis by 2035. conclusion while the genexpert testing platform has been lauded as a revolutionary tool to enhance tuberculosis case finding in meeting the end tb targets,10 tuberculosis case notification remains low in the three highest tuberculosis burden countries in africa. if lowand middle-income countries’ health systems do not address implementation challenges related to access and utilisation, the advantages of the xpert diagnostic platform will not be realised and these targets will be missed. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions v.w. designed the study, extracted, and analysed the data, and coordinated the writing of the manuscript. m.c. co-designed the study, wrote sections of the first draft, and interpreted the analysis. b.e. and c. onwuchekwa participated in data analysis and interpretation and writing of sections of the manuscript. c. okoro, c.c. and r.c. interpreted the analysis findings, provided input on the structure of the research, and revised the manuscript. k.o. guided the analysis, interpretation of results and presentation of the findings. all authors read and approved the final manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the data that supports the findings of this research are openly available at the world health organization data repository (https://www.who.int/tb/country/data/download/en/). disclaimer the views expressed in this manuscript are those of the authors and not 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editors. district health barometer, 2019/2020 [homepage on the internet]. durban: health systems trust; dec 2020 [cited 2021 jun 27]. available from: https://www.hst.org.za/publications/pages/dhb2019-20.aspx tomita a, smith cm, lessells rj, et al. space-time clustering of recently-diagnosed tuberculosis and impact of art scale-up: evidence from an hiv hyper-endemic rural south african population. sci rep. 2019;9(1):1–9. https://doi.org/10.1038/s41598-019-46455-7 mclaren zm, sharp a, brouwer e, nanoo a. the impact of anti-retroviral therapy on tuberculosis detection at the national level in south africa. am j trop med hyg. 2018;99(6):1407. https://doi.org/10.4269/ajtmh.17-0530 walt van der m, moyo s. the first national tb prevalence survey south africa 2018. pretoria: national department of health; 2021. murdoch j, curran r, van rensburg aj, et al. identifying contextual determinants of problems in tuberculosis care provision in south africa: a theory-generating case study. infect dis poverty. 2021;10(1):1–13. https://doi.org/10.1186/s40249-021-00840-5 adu pa, yassi a, ehrlich r, spiegel jm. perceived health system barriers to tuberculosis control among health workers in south africa. ann global health. 2020;86(1):15. https://doi.org/10.5334/aogh.2692 marais f, kallon ii, dudley ld. continuity of care for tb patients at a south african hospital: a qualitative participatory study of the experiences of hospital staff. plos one. 20191;14(9):e0222421. https://doi.org/10.1371/journal.pone.0222421 dudley l, mukinda f, dyers r, marais f, sissolak d. mind the gap! risk factors for poor continuity of care of tb patients discharged from a hospital in the western cape, south africa. plos one. 2018;13(1):e0190258. https://doi.org/10.1371/journal.pone.0190258 meyer-rath g, schnippel k, long l, et al. the impact and cost of scaling up genexpert mtb/rif in south africa. plos one. 2012;7(5):e36966. https://doi.org/10.1371/journal.pone.0036966 haraka f, kakolwa m, schumacher sg, et al. impact of the diagnostic test xpert mtb/rif on patient outcomes for tuberculosis. cochrane database syst rev. 2021;2021(5):cd012972. https://doi.org/10.1002/14651858.cd012972.pub2 bernal jl, cummins s, gasparrini a. interrupted time series regression for the evaluation of public health interventions: a tutorial. int j epidemiol. 2017;46(1):348–355. acknowledgements references about the author(s) elias chipofya malawi association of medical laboratory scientists, lilongwe, malawi citation chipofya e. a comparative framework for regulatory oversight in malawi’s medical laboratory professionals: lessons from kenya and nigeria. afr j lab med. 2025;14(1), a2941. https://doi.org/10.4102/ajlm.v14i1.2941 opinion paper a comparative framework for regulatory oversight in malawi’s medical laboratory professionals: lessons from kenya and nigeria elias chipofya received: 10 july 2025; accepted: 11 oct. 2025; published: 29 nov. 2025 copyright: © 2025. the author licensee: aosis. this work is licensed under the creative commons attribution 4.0 international (cc by 4.0) license (https://creativecommons.org/licenses/by/4.0/). robust professional regulatory oversight is crucial for ensuring the competence of healthcare practitioners and maintaining high-quality health systems.1,2 this is particularly vital for medical laboratory professionals, whose work underpins most clinical decisions, directly impacting diagnostic accuracy, treatment efficacy, and, ultimately, patient safety.3 when professional oversight is weak or violated, patient safety is directly compromised, leading to inconsistent care, an erosion of public trust, and potentially catastrophic outcomes.4 in healthcare, regulation is a policy that sets external rules for medical practice and administration, representing a sustained form of control by a public agency. regulatory oversight, a related but distinct concept, is the policy-driven mandate that translates these regulations into practical application.5 its practical application is seen in the structured processes that govern the education and professional development of health professionals. the primary mandates of regulatory oversight, including accreditation, licensure, and certification, are the key mechanisms for ensuring the initial and ongoing competence of medical practitioners.6 until the late 1960s, comprehensive global regulation for healthcare professionals, specifically in laboratory medicine, was limited. today, however, countries such as canada and the united kingdom demonstrate the immense value of professional oversight.2,7 this trend has extended to many african lowand middle-income countries, which have established formal regulatory frameworks for medical laboratory practice. for instance, the kenya medical laboratory technicians and technologists act of 1999 established the kenya medical laboratory technicians and technologists board (kmlttb), responsible for the regulatory oversight of training, registration, and licensing to ensure professional standards and quality control.8 similarly, the medical laboratory science council of nigeria (mlscn), established by the 2003 medical laboratory science council of nigeria act, holds responsibility for the regulatory oversight of its professional community.9 while these formal frameworks are vital for public health, robust oversight is often lacking in many lowand middle-income countries, where regulators are frequently resource-constrained, and oversight may be perceived as weak.10 malawi’s foundational medical regulation dates to the registration of medical practitioners ordinance, no. 9 of 1906, which first governed medicine in the british protectorate of nyasaland (now malawi) and established the nyasaland medical council.11 this was later superseded by the medical practitioners and dentists act of 1987, which created the medical council of malawi (mcm).12 the act, outlined in part iv, section 10, gives the mcm a broad mandate for regulatory oversight, including registering practitioners, overseeing training, and exercising disciplinary control.12,13 despite these foundational regulations, malawi’s healthcare system faces a significant regulatory oversight gap that endangers patient safety. the medical practitioners and dentists act of 1987 in malawi is critically deficient, failing to establish a dedicated framework for the professionalisation, training, and continuing development of medical laboratory professionals. this oversight deficiency means this vital workforce lacks the robust, consistent oversight found in countries such as kenya and nigeria, which consequently compromises diagnostic accuracy and professional growth. this article addresses the gap by comparatively analysing the regulatory frameworks of malawi, kenya, and nigeria. the analysis identifies key weaknesses in malawi’s approach, demonstrating the 1987 act’s need for substantial revision to meet modern international standards. the resulting evidence-based recommendations aim to strengthen professional governance, ultimately improving patient safety and public health outcomes in malawi. the regulatory process for healthcare professionals is a cohesive system with six interconnected components. it begins with (1) the accreditation of pre-service education, ensuring training institutions meet established curriculum standards.14 this is followed by (2) a thorough competency assessment to officially certify that individuals can effectively apply their skills and knowledge, which is vital in a rapidly evolving field like laboratory medicine.7,15 these competencies are then formalised through (3) a comprehensive system of registration, certification, and licensing.15,16 concurrently, (4) a clear scope of practice is defined and enforced to clarify authorised tasks, prevent patient safety risks, and improve efficiency.16 to ensure competence is maintained, (5) mandatory continuous professional development (cpd) requires lifelong learning in response to rapid scientific advances.2,17 finally, (6) the system upholds professionalism, which includes a commitment to public service and ethical behaviour. unprofessional conduct is the most common reason for disciplinary measures, and can significantly undermine public trust, directly affecting public health.18 malawi has a long and notable history of training medical laboratory professionals, with its first reputable programme established in 1968 at malamulo college of health sciences. this programme was so well regarded that it attracted students from across africa, including zambia, lesotho, uganda, zimbabwe, botswana, the democratic republic of congo, eswatini, sierra leone, ghana, sudan, ethiopia, and rwanda.19,20 this legacy is further evidenced by the mcm having registered 65 diplomas (3 years of training) and 79 certificates (2 years of training) from both malamulo and malawi college of health sciences between 1988 and 1999.13 despite this rich history, the medical laboratory profession’s growth is now hindered by a legislative gap in the medical practitioners and dentists act of 1987, a stark contrast to the dedicated statutory frameworks found in neighbouring countries such as kenya, nigeria, and south africa. this gap in the primary regulation is, to some extent, mitigated but not eliminated by the mcm’s active regulatory oversight (a policy-driven mandate). this oversight is evidenced by recent formal policy tools, such as the comprehensive undergraduate and postgraduate curriculum standards, the corresponding assessment checklists, the initial inspection checklist for training institutions, and the standard operating procedures for curriculum approval.21,22,23,24,25,26 the persistent challenge confronting medical laboratory professionals in malawi is a regulatory misalignment that originated in 2004. at that time, key academic institutions (the malawi college of health sciences and malamulo college of health sciences) sought to modernise their training and align it with global benchmarks by proposing to rename the qualification from medical laboratory technology to biomedical sciences. crucially, this academic modernisation shift was not formally recognised by the mcm. instead, the mcm maintained the outdated, traditional professional registration titles: medical laboratory assistants (for certificate holders) and medical laboratory technicians (for diploma holders). this initial regulatory discrepancy continues to pose a significant problem. for example, the kamuzu university of health sciences (kuhes) later renamed its undergraduate programme (introduced in 2006) from medical laboratory technology to medical laboratory sciences. despite the curriculum having undergone multiple important reviews in 2009, 2014, and 2016, kuhes graduates face the identical regulatory hurdle: the mcm still mandates the use of the older title, medical laboratory technologists, for official professional registration. furthermore, kuhes introduced a similar, separate bachelor of science in biomedical sciences programme in 2021. this creates a further complication, as the existence of different curricula for related disciplines at the same institutions, which can lead to internal competition for limited resources and negatively affect educational quality. these educational challenges are supported by a survey of african laboratory professionals by maruta, mataka and moyo, which provides empirical evidence of core training issues. the most common problems identified were a significant lack of resources and equipment (37.5%), limited clinical exposure (19.0%), insufficient curriculum coverage (9.5%), and inadequate faculty (7.7%).27 the issue is further complicated by the mcm’s public notice of 23 september 2024, formally acknowledging medical laboratory sciences at the kuhes as a regulated profession. however, the same notice concurrently indicated that the similar bachelor of science in biomedical sciences falls outside its regulatory scope. this disparity is starkly contrasted by the programme’s establishment timeline: the bachelor of science in biomedical sciences was established by mzuzu university in 2007, while kuhes introduced its similar, separate programme in 2021. graduates of the mzuzu university programme can register as medical laboratory technologists under the statutory regulatory body. this demonstrates a critical inconsistency: while the mcm regulates one title, the similar biomedical sciences title is not consistently protected across institutions, leading to varying registration eligibility. since the mcm regulates practice, not the universities, these inconsistencies in the programme curricula under the mcm’s purview cause significant confusion with regard to employment criteria. the root cause of these regulatory disparities is not the absence of a framework, but rather the mcm’s use of a principles-based standards framework. this approach, adopted in the revised standards for undergraduate and postgraduate education, prioritises programme rationale, learning outcomes, and format compliance over mandated, uniform content and titling across equivalent allied health disciplines, including the medical laboratory profession. this regulatory flexibility unintentionally permits the non-uniformity observed in equivalent titles across institutions, despite the mcm’s mandate to regulate practice.21,22 this failure in regulatory clarity is mirrored by the varied landscape of professional training across the african region. a previous expert study, drawn from an analysis of the region, demonstrated that although most professionals were trained at public institutions (82%), qualifications lacked uniformity. the bachelor’s degree (53%) was the most common first qualification, but qualification names were non-uniform, with medical laboratory sciences (43%) being the most prevalent title, followed by medical laboratory technologist (20%) and medical technology (19%). furthermore, while most qualifications included curriculum content such as genetics, molecular biology, cytology, laboratory management, histology, blood transfusion, haematology, immunology, chemistry, and microbiology, a standardised framework is still necessary to ensure consistency across all training institutions.27 the consequences of this regulatory confusion are starkly evident in the workforce. a literature review on standard occupation classifications by koo confirmed a wide range of global titles for the same roles, including medical laboratory scientist, medical technologist, clinical laboratory scientist, biomedical scientist, biomedical analyst, and medical laboratory technician.28 the absence of a clear, national title in malawi allows these different terms to be used interchangeably for the same roles. this lack of a defined professional identity and consistent regulation directly jeopardises diagnostic quality and patient safety by permitting unqualified individuals to assume critical roles.12,29 it also hinders career progression by creating confusion in employment criteria and contributing to the paradox of a high number of unemployed professionals, despite a significant national personnel gap. ultimately, the absence of a defined identity and consistent regulation for these vital medical laboratory professions directly jeopardises diagnostic quality and patient safety, while also hindering career progression. the governance structure for medical laboratory professionals in malawi, defined by the medical practitioners and dentists act of 1987, embodies a model of subordination and indirect oversight. while the mcm attempts to define allied health professions, including medical laboratory professionals, via its code of ethics, the primary legislation fails to formally recognise them.12,13,29 crucially, the profession suffers a significant systemic disadvantage because of its limited and indirect representation on the mcm.20 as stipulated in section 4 of the act, the council’s membership is dominated by medical practitioners and dentists, explicitly excluding a dedicated seat for the medical laboratory profession. the act reserves positions for: the chief of health services, a medical practitioner and a dentist from the public service, a medical practitioner from the christian health association of malawi, five members from the medical association of malawi, a medical practitioner representing kuhes, the secretary for justice and solicitor general, and at least three members from the general public (one being a medical practitioner or dentist in private practice).12,13 this lack of direct involvement of medical laboratory professionals inherently impedes their capacity to effectively shape their own professional regulation, ethical oversight, quality assurance, and accreditation.20,30 furthermore, malawi’s broad, centralised framework is vulnerable to political influence because the mcm’s council members and chairman are appointed directly by the minister.12,13 this political entanglement frequently undermines effective regulatory oversight, leading to deflected accountability and the bypassing of professional protocols.10,31 this malawian model stands in stark contrast to the frameworks established in regional counterparts such as kenya and nigeria, which operate under a paradigm of dedicated professional self-regulation. establishment of separate, dedicated statutory councils is done by both the kenya medical laboratory technicians and technologists act (cap 253a, 1999; the kmlttb) and the medical laboratory science council of nigeria act (2003; the mlscn). these bodies are explicitly empowered as independent entities to supervise, control, and regulate all facets of the profession, from prescribing training courses and approving institutions to professional licensing and ethical conduct. significantly, their governing boards are composed predominantly of practising medical laboratory professionals, often elected by their own associations. this model ensures that regulatory standards and policy development are driven by specialised expertise and professional ownership.8,9 this structure directly addresses malawi’s challenge of political entanglement by creating independent, profession-specific corporate bodies. the operational deficiencies confronting the malawian model, where the mcm faces a critical challenge rooted in a widespread misunderstanding of its roles among both the public and practitioners, further emphasise the benefits of autonomous regulatory frameworks.13,32 this internal confusion is severely compounded by persistent underfunding, as insufficient government budgetary allocations have consistently failed to support the mcm’s rigorous oversight and disciplinary actions for over a decade.13 this financial constraint aligns with broader concerns that limited resources hinder regulators’ capacity for high-quality, policy-relevant research,33 forcing the mcm to rely on fragmented, small-scale studies that impede evidence-informed policy development.17,34 by contrast, the dedicated statutory bodies in kenya (kmlttb) and nigeria (mlscn) are structurally better positioned, by virtue of their focused mandate and professional governance, to minimise operational confusion, manage public awareness, and advocate for sustainable, profession-specific funding models.8,9 the critical divergence concerns professional standardisation and quality assurance. malawi’s regulatory framework for medical laboratory staff, from assistants to post-graduate scientists, suffers from a fundamental lack of clearly defined roles and a structured scope of practice. this deficiency is in sharp contrast to established models, such as that for laboratory medicine consultants, which mandate a precise definition of competencies and job descriptions.35 indeed, since the scope of practice defines the necessary tasks, titles, and qualifications associated with medical laboratory science,15 its absence critically compromises professional clarity and quality control. compounding this issue is the absence of requirements for external verification of qualifications, statutory professional accreditation, and competency assessments.17 this collectively results in a severe lack of professional consistency, where numerous titles can be applied to identical roles, compromising the profession’s identity and mirroring issues found in unregulated systems that lack a protected title for medical laboratory professionals.36 these systemic failures significantly degrade service quality, lead to poorer patient outcomes, impede professional development, and heighten the threat of brain drain.37 conversely, models in kenya (kmlttb) and nigeria (mlscn) successfully mitigate these problems by establishing independent, profession-specific corporate bodies with governing boards primarily composed of practising professionals. this self-governing mandate ensures rigorous control over entry and quality, while structurally positioning them to minimise political interference, manage public awareness, and effectively enforce the precise competency requirements that malawi’s multi-disciplinary, politically entangled system fails to maintain. professionalism encompasses a high level of intellectual and technical expertise, the ability to practise independently, and a strong commitment to public service.38 regulatory oversight requires that practitioners consistently demonstrate a high standard of professional conduct, including probity and ethical behaviour. unprofessional or unethical conduct is the most common reason for disciplinary action taken by regulatory agencies and professional organisations.18 the mcm frequently receives allegations regarding issues such as illegal drug possession, operating unregistered premises, professional negligence, and practitioners providing services they are not competent to deliver.13 the mcm’s code of ethics and professional conduct explicitly outlines these as professional misconduct for which practitioners can face disciplinary action.13,29 beyond tracking these punitive actions, a more objective measure of whether regulatory oversight delivers a competent workforce and enhances patient safety is to compare data on pathology-related errors.15,39 however, these data are unavailable in malawi. the lack of a reliable error repository stems from systemic failures: frequent pre-analytical errors occur because of extreme delivery delays that deteriorate samples, and severe supply shortages (reagents, correct tubes, stationery) that force inadequate data recording.39 together, these factors create operational instability, making the collection of systematic errors impractical for directly assessing the regulatory impact on patient safety. the medical mcm operates with a policy-driven mandate for regulatory oversight of its cpd programme, a core component of its overall regulatory framework. this programme, initiated in 2008 under the medical practitioners and dentists act of 1987, serves to ensure practitioners maintain competence and uphold ethical obligations, a requirement reinforced by the mcm’s code of ethics and professional conduct.17,29,34 however, several critical issues undermine the programme’s practical implementation. first, the required annual point total saw an adjustment: the initial mandate of 50 cpd points for license renewal was later deemed unrealistic and revised to 30 points annually, which is the minimum explicitly stated in the official malawi cpd diary that practitioners must present for certification renewal.34 second, while the cpd diary explicitly provides detailed cpd codes (a through i) and rules for point calculation across nine distinct categories (e.g., specific caps on clinical meetings and conferences, or a point range for publications), this intricate, manual system is criticised in the academic literature for its inherent complexity and administrative burden, leading to a lack of operational clarity and practical consistency in its application and verification.17,34 finally, this issue of consistency is compounded by the fact that cpd activities are often confined to medical officers in the urban areas, thus creating a problem of professional isolation for practitioners in rural settings who lack access to necessary resources and electronic services.17 this is compounded by limited published information on the programme and generally low compliance rates in the country. a major barrier is the association of cpd activities with financial incentives such as per diems (daily allowances or payments provided to individuals, specifically healthcare workers, for attending training sessions, workshops, or seminars).17,40 this system prioritises monetary gain over genuine learning, leading to a lack of motivation when payments are absent, and has even resulted in training boycotts and pressure to share allowances with supervisors.40 additionally, the mcm’s regulations create a significant loophole, as cpd is not mandatory for all healthcare cadres, particularly the medical laboratory profession. this regulatory gap can lead to outdated skills, directly impacting diagnostic accuracy and delaying the adoption of new technologies.41 furthermore, many medical professionals, especially those in rural areas, face limited access to cpd opportunities because of a lack of essential resources such as computers and internet access.34 the programme is also perceived as being primarily for senior urban medical personnel, and some staff believe that skills are best updated through long service rather than formal training. by contrast, countries such as nigeria and kenya offer more effective models for regulatory oversight, particularly for specialised cadres. nigeria (mlscn) and kenya (kmlttb) have both implemented specific, targeted cpd frameworks for medical laboratory professionals.42,43 these frameworks detail how points are awarded for activities, from degrees to publications, ensuring clarity and accountability, which addresses a key challenge in malawi. while nigeria’s system has faced issues with sponsorship,41,44 kenya stands out with strict sanctions for non-compliance, including licence suspension.43 this directly contrasts with malawi’s problem of financial incentives (per diems) driving cpd participation, often causing training boycotts when payments are absent.17,40 the structured, profession-specific regulations in nigeria and kenya thus offer concrete models for malawi. by improving its administration, mitigating the reliance on financial incentives, and ensuring equitable access, malawi could align its policy-driven mandate with more effective practical implementation. based on the current landscape and international best practices, the following key recommendations are proposed to strengthen the medical laboratory profession: enact a dedicated medical laboratory professionals act (specialisation) action: legislate a new, profession-specific act to replace the current broad, multidisciplinary council model. goal: establish an independent or highly autonomous regulatory body to define and regulate all cadres, including postgraduates.20,30 this model must be consistent with kenya’s kmlttb and nigeria’s mlscn, ensuring professional duties align with their education and authority.35 reform the medical practitioners and dentists act (chapter 36:01 of the laws of malawi) action: if a dedicated act is not feasible, the 1987 act requires significant reform. goal: the act’s governance must be reformed to mitigate political influence.20 this requires a merit-based appointment process and mandatory dedicated council representation for medical laboratory and paramedical staff to counter dominant professional bias.45 this revised structure should align with multi-professional bodies such as the health and care professions council (united kingdom) and the health professions council of south africa.46,47 implement mandatory, competency-based continuous professional development action: make mandatory, competency-based cpd a prerequisite for the annual re-registration of all regulated professionals. goal: ensure consistent standards and diagnostic accuracy through mandatory, verifiable competency assessments.41,46 continuous professional development must cover gaps in pre-service training, specifically laboratory accreditation and quality management systems.41 the mcm must ensure that the cpd is well-structured and tailored.27 competency frameworks should be developed by the mcm, training institutions, or professional working groups, including the malawi association of medical laboratory scientists.16,48 establish a rigorous accreditation and licensing system for training action: develop and strictly enforce a rigorous, independent accreditation system for all medical laboratory training programmes and institutions. goal: address major challenges: lack of resources/equipment, limited clinical exposure, and inadequate faculty.27 a standardised national curriculum and classification framework is crucial to end the discrepancy between academic and regulatory titles, which causes inconsistent registration eligibility. licensing and inspection must be robust, and accreditation should be transparent and independently evaluated.14 accreditation must mandate minimum content, including microbiology, biosafety/biosecurity, and laboratory management.27 enhance transparency and accountability mechanisms action: significantly enhance the transparency and accountability of regulatory operations. goal: enhance enforcement and public trust by publishing regular, publicly accessible reports on regulatory activities, finances, and performance. clear, robust, and independent processes for public complaints and professional appeals are essential. foster professional engagement and ownership action: strengthen professional engagement and foster a sense of shared ownership amongst healthcare practitioners. goal: counter political influence by establishing formal dialogue platforms between the mcm and professional bodies (e.g. the malawi association of medical laboratory scientists), to ensure shared regulatory responsibility. prioritise empirical research to ensure regulations are feasible and effective. supporting local regulation empowers professionals and fosters ownership.10 delineate scopes of practice and establish career progression pathways action: develop and implement clearly delineated scopes of practice for each distinct professional grade. goal: address ill-defined scopes of practice and limited career progression.20,30 the mcm, training institutions, and the malawi association of medical laboratory scientists must formally define the scopes of practice, aligning them with competency principles.35 establish attractive career pathways, including formal designation of managerial/non-managerial posts and laboratory-specific post-graduate training (up to phd level) with defined, compensated career paths.27,41 this is crucial for professional retention and improving job satisfaction. in conclusion, malawi’s laboratory services are severely compromised by a confluence of deeply entrenched and interconnected challenges. by addressing these issues, malawi can not only safeguard its own public health but also serve as a crucial case study for how a targeted and profession-specific regulatory approach can fundamentally transform healthcare governance and outcomes in similar resource-constrained environments. acknowledgements the author thanks dr graham beastall and dr mike hallworth for their valuable insights and guidance during the conceptualisation and development of this opinion paper. competing interests the author declares that no financial or personal relationships inappropriately influenced the writing of this article. credit authorship contribution elias chipofya is the sole author of this article. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability no new data were generated or analysed for this opinion paper. all information synthesised is derived from publicly available literature and reports, as detailed in the references. disclaimer the views and opinions expressed in this article are those of the author and are the product of professional research. the article does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the author is responsible for this article’s results, findings, and content. references chiu p, leslie k, jang g, adams tl, thiessen n, kung jy. performance measurement and evaluation of health practitioner regulation: a scoping review protocol. plos 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https://web.mlscn.gov.ng/wp-content/uploads/filr/997/revised%20policy%20for%20continuing%20professional%20development%20(cpd)%20program.pdf kenya medical laboratory technicians and technologists board. the medical laboratory technicians and technologists (continuing professional development guideline) regulations 2024 [homepage on the internet]. nairobi: kenya medical laboratory technicians and technologists board; 2024 [cited 2025 oct 13]. available from: https://kmlttb.org/kmlttbregulations/documents/continuing%20professional%20development%20regulation.pdf ifeoma e, rebecca e, ezekiel o, et al. a cross-sectional study of the knowledge and attitude of medical laboratory personnel regarding continuing professional development. niger j med. 2015;56(6):425. https://doi.org/10.4103/0300-1652.171617 goldman ah. professional values and the problem of regulation. bus prof ethics j. 1986;5(2):47–59. https://doi.org/10.5840/bpej19865228 willson c, badrick t. a comparison of some certification/registration schemes available for medical laboratory scientists. aust j med sci. 2017;38(3):84–89. sims d, zingela z, mokhachane m, et al. medical education, reflections and perspectives from south africa: a review. bmc med educ. 2025;25:370. https://doi.org/10.1186/s12909-025-06910-8 willson c, healthineers s, badrick t. pathology laboratory scientific scope of practice and competence-based standards for a certified workforce. aust j med sci. 2018;39(1 & 2):32–37. article information authors: talkmore maruta1 philip rotz1 trevor peter1 affiliations: 1clinton health access initiative, lesotho correspondence to: talkmore maruta postal address: po box 354, harare, zimbabwe dates: received: 03 july 2012 accepted: 26 sept. 2012 published: 26 mar. 2013 how to cite this article: maruta t, rotz p, trevor p. setting up a structured laboratory mentoring programme. afr j lab med. 2013;2(1), art. #77, 7 pages. http://dx.doi.org/10.4102/ ajlm.v2i1.77 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. setting up a structured laboratory mentoring programme in this lessons from the field... open access • abstract • introduction • facility-based laboratory mentorship • identifying and training mentors • time: an essential resource commitment • optimal environment for mentoring success • structured mentoring model    • sufficient duration    • sufficient frequency of mentor engagement    • number of laboratories to be mentored    • size of laboratories to be mentored    • number of mentors and their available time    • skill level and experience of mentor    • funding    • logistics • mentor support and integration • supervision and accountability • measuring laboratory progress and mentoring effectiveness • structured reporting • summary • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ introduction: laboratory mentoring programmes can be an important vehicle to establish and solidify quality management systems and help laboratories achieve accreditation goals. different mentoring approaches have been used with varying levels of success. the authors provide a guide to implementing a structured laboratory mentorship programme based on their practical field experience. method: the study is based on experience in lesotho as well as subsequent roll out of a similar approach in the other african countries of zimbabwe, mozambique, swaziland and cameroon between 2009 and 2011. summary: we highlight critical elements to consider when setting up a long-term, sustainable and well-structured mentorship programme. these elements include: well-defined goals; sufficient length of mentor engagement on site; standardised approach across laboratories; measurement of progress using standardised tools; well-structured reporting mechanisms; alignment of the programme with overall ministry of health plans; and selection and training of the mentors. these elements will differ in application, depending on countries’ needs and available resources. a structured approach allows for scalability, comparison across laboratories and countries and an easier approach to budgeting and planning for countries intending to set up similar programmes. introduction top ↑ in recent years, attention has increasingly been turning towards improving overall healthcare in order to ensure higher quality, greater access and better value for money.1 to achieve this, countries have used various approaches, mainly training health professionals.1 one approach has been introducing quality improvement and quality management principles and concepts into pre-service or in-service training curriculum. quality-related curriculum in most medical trainings has been under-emphasised, despite frequent recommendations.2 in a survey conducted by the health foundation in 2012,1 quality improvement was found to be mandatory for pre-service medical training only in the usa. this leaves much of the training in quality to the in-service period for most other countries. in-service training ensures that health professionals who are already providing services have the opportunity to update their knowledge and skills according to the latest standardised practices. many different approaches have been used in quality related in-service training of health professionals, including classroom-based training, online training, workshops, and single session continued medical education courses. an interactive training approach that provides competency-based training has been shown to be effective in transferring skills as well as knowledge and attitudes.3 a culture of practicing quality routinely is a behaviour change issue and calls for a different approach to training.4 the foundation of health survey indicated that approaches that utilise active learning strategies, where participants put quality improvement into practice through close coaching and mentoring, are thought to be more effective than didactic classroom styles alone.1 however, most of the documented in-service training models are largely for clinical practice. anecdotal reports suggest that the laboratory side of healthcare has also been active in this area, but that most of the work remains undocumented. one successful model that has been documented is the lesotho mentorship model4, where a facility based approach by an experienced mentor resulted in considerable transfer of quality management skills to the laboratory staff. based on practical field experience from the lesotho mentorship model1 and subsequent roll out of similar models in five other african countries (mozambique, zimbabwe, swaziland, cameroon, and nigeria) as well as selected laboratories under the east african public health laboratory networking project (eaphlnp), we give guidance on how to set up a structured laboratory mentorship programme. the facility-based approach, with mentors spending extended periods of time on site, was also documented by gershy-damet et al.5 who recommended structured periods of mentor engagement times embedded in the daily life of a laboratory as valuable means for successful implementation of quality management systems. facility-based laboratory mentorship top ↑ laboratory mentoring programmes can be an important vehicle to establish and solidify quality management systems (qms) and help laboratories achieve accreditation goals.1 there are other methods that have been used to assist laboratories in attaining accreditation. the method described here is an initiative where experienced mentors were embedded in a laboratory for extended periods of time to develop an in-depth understanding of the laboratory and provide day-to-day assistance in the implementation of qms and preparation for accreditation. this facility-based and embedded approach to mentorship has been observed to have high impact in different settings.4,5the authors recognise that mentoring can be utilised for a variety of laboratory support purposes. for example, mentoring has proven to be a useful means of establishing new tests, like dna-pcr, viral load and tb culture. in these initiatives, an experienced mentor works with a laboratory for several months to help set up and problem-solve through the initial rollout of a given test. the mentoring approach discussed in this document engages the whole laboratory, targets qms implementation throughout the entire testing system and is staged as a series of engagements that aid laboratories as they progress toward accreditation. there are several training tools available in qms implementation.6 mentoring can be used independently or in conjunction with these tools, one of the tools that has been shown to have demonstrable, documented evidence of improvements is the strengthening laboratory management toward accreditation (slmta) training initiative, a task-based laboratory management training developed jointly by the united states centers for disease control and prevention, the american society for clinical pathology, and the clinton health access initiative.7 in some settings, slmta and mentoring have been shown to have better impact than each implemented independently.8 whether using mentoring as a means to deliver slmta at the laboratory level or as second-level support for laboratories that have completed slmta, these two efforts complement one another and can strengthen the drive to realise national accreditation goals. described below are some of the critical building blocks to consider when setting up a facility-based laboratory mentorship programme. the authors recognise that these suggested building blocks may not always apply in the same way in every setting, but believe that each of these elements should be considered. for example, although time spent on site is a critical element, the amount of time suggested in this guide may not be feasible in all cases. identifying and training mentors top ↑ special attention must be taken when selecting mentors, as they are a critical element in the success of the mentorship program.4 mentors can be drawn from experienced laboratory personnel available internationally, regionally or nationally. international mentors with experience in preparing laboratories for accreditation may provide valuable laboratory mentoring support. however, these persons can be difficult to identify, costly to employ and may not be available for extended periods of time. building a mentoring programme around regional and national mentors should be seriously considered. when compared to the cost of international mentors, the same level of funding may employ a greater number of regional or national mentors and therefore provide more total contact time with laboratories.in addition, while carefully selected international mentors may have experience that enables them to contribute particular expertise, it should also be recognised that experience with qms implementation under conditions common to many african laboratory systems is also an expertise – one well-suited to mentoring laboratories for accreditation. in addition, the in-depth familiarity with regional laboratory practice and national laboratory systems possessed by regional and national mentors can also contribute significantly to mentoring efficiency and effectiveness. further, investing resources in the development of regional or national mentors is a means of building capacity and strengthening long-term sustainability. one of the criteria to consider when selecting mentors is whether they have broad experience as a bench technician. this is important, as the mentor works in an embedded fashion. some management experience, at least at the bench section level, is also an important element to be considered, as the mentor would deal with varying management related issues. some background knowledge of qms and implementation of iso laboratory standards is vital to ensure that whatever is implemented and the resolving of non-conformities remain aligned with international standards. experience in conducting training would be useful, as training is one of the techniques used in delivering this facility-based mentorship.4 mentoring involves behaviour change. hence it is desirable to have a mentor who is persistent, patient and has a positive attitude, coupled with passion for laboratory quality improvement. the ability to work well with others and the ability to provide instruction and firm constructive correction is essential. not all the elements described above might be present in all potential candidates for mentoring. some elements, such as knowledge of standards, can be attained after recruitment through training and attachments at accredited laboratories or those working towards accreditation. this can provide the much needed hands-on experience in implementation of qms. once mentors have been selected, preparatory training will aid in readying new mentors for deployment. experienced mentors should be sought to provide training for new recruits. their training should include some didactic training, job-shadowing, working under the observation of an experienced mentor, work plan sharing and feedback with experienced mentors or amongst a pool if all are new. on-site follow-up reviews, especially during the early phases of the mentorship, are one means of training new recruits. in addition to this training, there are several additional training courses or experiences which may prove beneficial for mentors, either as part of their initial preparation or as ongoing professional development. examples include slmta training-of-trainers workshops and iso 15189 laboratory standard training. training in conducting laboratory assessments should be considered, as assessments are an integral component of the mentoring described. in countries where resources allow, international and/or regional mentors are used. in such cases, developing local capacity must be prioritised from the beginning of the programme. several methods are suggested in ensuring building of local mentoring capacity within the mentorship programme. local experienced laboratory staff can be twinned with an experienced internal or regional mentor. shortto long-term attachments at mentored laboratories or accredited laboratories where available, are one way of building local mentoring capacity. structured exchange programmes between laboratories, where peer-to-peer training and skills exchange can groom potential local mentors, are a cost-effective means of training and grooming local mentors. time: an essential resource commitment top ↑ mentoring programmes require resource commitment. the resource most critical to effective mentoring is time.4 mentors spend extended periods of dedicated time working with laboratories in an embedded fashion – an investment of weeks to months of in-laboratory presence. side-by-side instruction and guidance, provided through sustained time in the laboratory, are essential to create a culture that understands and values quality and to effect behaviour changes within the laboratory that ensure routine implementation of quality measures. it is worth noting that the side-by-side mentorship approach does not target technical skills training. emphasis is on assisting the mentees to build quality into the testing that they are already competent on. with adequate resource commitment, a well-structured mentoring programme can equip and empower laboratory staff and bolster laboratory performance. however, not all countries or initiatives are able to commit the in-lab staff time necessary for an effective mentoring programme. where resource commitment to mentoring is not currently feasible, consideration should be given to what can be accomplished through a programme of shorter duration coaching and/or site support visits. optimal environment for mentoring success top ↑ every effort should be made to ensure that mentoring programmes are implemented within a context conducive to the realisation of meaningful and sustainable laboratory improvement. mentoring efforts that are established without attention to the environmental conditions of the laboratory system are likely to find their success limited and short-lived.ministry of health (moh) support and involvement is crucial to mentoring success. mohs should be clear in their communication of accreditation goals, and mentoring as a way of building the knowledge, capacity and experience necessary for success. public identification of mentoring as an moh priority can increase the acceptance and standing of mentors in the eyes of laboratory staff and hasten buy-in and cooperation. it is advisable to detail programme expectations and responsibilities in a document shared with all parties. this encourages clarity, transparency, commitment and accountability. in some countries, this has even taken the form of a ‘contract’ between the moh, the laboratories, the mentors and the organisation they represent. programme design and site selection should be done in consultation with moh laboratory services leadership and in line with national accreditation goals detailed in laboratory strategic and operational plans. further, mentoring programmes should be designed with direct links to national laboratory quality assurance (qa) programmes. mentors should report and route information to the qa office and act as an extension of the qa office at the laboratory level, assisting with in-lab implementation of national qa initiatives. an added advantage for mentoring programme implementation is the presence of a national laboratory technical working group or a subcommittee dedicated to implementation of qms or accreditation. mentoring programmes should be represented in such a group to encourage sharing of on-the-ground challenges with a wider group of stakeholders and collective solution seeking. lastly, the optimal context for mentoring will also include active parallel national strengthening initiatives for service and maintenance of instruments, supply chain, proficiency testing, and routine assessment of laboratory performance. in-lab mentoring can yield many service-level benefits, but a number of critical areas may require action at the management level, far removed from any individual laboratory. combining laboratory mentoring efforts with initiatives that strengthen the national laboratory system provides the best opportunity for long-term success. structured mentoring model top ↑ mentoring programme design should be based upon well-defined goals and the model developed should reflect the following key commitments: • mentors spend extended periods of time in laboratories. • mentors are embedded in the life of the laboratory. • mentoring is delivered through a series of engagements over time. • laboratory progress is measured at specific points with standard tools (e.g., who-afro strengthening laboratory quality improvement process towards accreditation [slipta] checklist). • reporting is structural. the mentoring programme design will reflect the conditions and resources particular to each context. while mentoring programmes will differ, care should be taken to maintain the commitments detailed above. mentoring is about embedding someone in the life of a laboratory to fully understand its culture, processes and people. mentors work from within and alongside to help raise a laboratory’s level of performance. the authors have observed that mentorships of longer than four to eight weeks can be more effective than shorter periods.4 though special mentoring emphasis may be given to laboratory staff with greater levels of responsibility, facility-level mentoring should involve all laboratory staff and not centre solely on the manager or quality officer. mentors should emphasise a team approach to quality. the entire facility should have a stake in the improvement of the laboratory. several common variables should be considered in designing a mentoring model that will help laboratories achieve accreditation goals. sufficient duration mentoring requires a sustained time commitment. significant periods of contact time with the laboratory are necessary to change behaviour and practice. the initial engagement is crucial to success. it is during this time that mentors develop a comprehensive understanding of the rhythms, patterns, practices, and personalities within the laboratory. this period is also essential for relationship building, as many positive changes are realised as much through common cause as particular expertise. initial mentoring engagements of less than four weeks may not achieve this. some programmes dedicate a mentor to a laboratory for six to twelve consecutive weeks for an initial engagement. sufficient frequency of mentor engagement we view laboratory mentoring as a series of mentoring engagements. this approach enables the mentor to assist in implementation and contains periods where the laboratory functions on its own initiative. a model that prioritises several engagements with a given laboratory, in our view, gives the mentor and moh leadership the opportunity to gauge how well a laboratory is able to sustain, or even extend, quality improvement without the on-site presence of a mentor. these intervals provide an opportunity to evaluate the laboratory’s independent operations and reinforce positive practice and re-address areas of continuing concern. mentoring programmes should be designed to have a minimum of two engagements per laboratory with a period of four to eight weeks of independent operations between engagements. number of laboratories to be mentored the number of laboratories to be mentored should be in line with national accreditation priorities. however, this determination should also take into account available human, financial and logistical resources and the guidance above related to sufficient duration and frequency. size of laboratories to be mentored this can pertain to the number of sections in a laboratory or the size of its staff. large laboratories with many sections and staff may benefit from a mentoring approach that works on a section-by-section basis, spending several consecutive weeks focusing on each section. in smaller laboratories, a mentor should be able to work across all sections simultaneously, dealing with the laboratory as a whole. number of mentors and their available time the number of mentors and their availability for deployment is a common limitation confronted when designing mentoring programmes. this can result in tough choices about the mentoring model. when faced with such decisions, it is our view that mentoring programmes should focus on doing more for fewer laboratories.this may strike some observers as unfair or inadequate to their needs. the argument can be raised that greater reach would enable more laboratories to benefit by receiving at least a little mentoring. while sympathetic to the gap between needs and resources that this view attempts to address, it nonetheless mistakes mentoring to be a commodity that can maintain its value and return on investment while being sub-divided into smaller and smaller units. that is not the case. it has been argued above that the benefits of mentoring accrue through accretion – working side-by-side with the laboratory over an extended time. the benefits realised through mentoring are not accumulated in a linear fashion – spending one week in five labs is not equal to spending five weeks in one lab. on the surface, the ‘equation’ above may seem like it should be reflexive. but mentoring is about understanding a laboratory’s working culture and helping its primary members re-shape that culture to include the routine implementation of qms. it relies upon correction, repetition, repeated correction and correct repetition. because implementation of qms involves behaviour change, it is often better to provide concentrated mentoring to a few laboratories (along the lines described above) rather than stretch too far, provide diluted mentoring and realise few long-term gains. moreover, making the choice to engage a few laboratories more substantively provides the opportunity for greater experiential learning and capacity building of staff working in those laboratories. laboratory staff who have received in-depth mentoring and subsequently proven their ability to maintain and extend their laboratory’s qms are prime candidates to implement these same improvements in other laboratories, either on transfer or by themselves serving as mentors. a mentorship design that stretches too wide and has a thin in-lab presence is unlikely to have the same impact in terms of formation of a cadre of valuable managers and/or second-generation mentors. skill level and experience of mentor not all mentors will exhibit the same ‘work rate’. this is attributable to differences in skill level, experience, style, acceptability, and the particularities of the individual laboratories. inexperienced mentors may initially need to spend more time working in laboratories than experienced mentors. national mentors familiar with the intricacies of a country’s laboratory system may find it easier to address certain problems than external mentors who are unsure how or with whom they should work. mentoring programme design should consider whether inexperienced mentors or mentors unfamiliar with the national laboratory system may need more in-laboratory time.in addition, mentoring programmes should be attentive to the training needs of the mentors themselves. regular experience-sharing with other mentors, qms training, assessing methods of training, iso 15189 training, qms implementation study visits – these are some ways in which mentors can continue to build knowledge and skills that can contribute to their in-lab work. funding funding considerations contribute significantly to the mentoring model, sometimes necessitating difficult decisions. when faced with limited funds, consider demonstrating success and seeking additional monies to expand, rather than stretching too far and diluting programme impact, thus weakening the case for programme expansion. logistics mentors live and move within the areas surrounding the laboratories they work in. considerations around driving distances and frequencies of flight schedules must also be factored into mentoring design and budgets. mentor support and integration top ↑ technical and logistical support for mentors should also be considered. while the latter can often be addressed through office administrative staff, technical support – as well as integration – may require intentional networking and structure.technical support will invariably be needed, as mentors engage the laboratory system’s most deeply rooted problems. mentors should access technical support from leadership in the moh and other national experts. for this reason, mentor orientation should include introductions to all relevant resource persons. organisations that have outside laboratory expertise should link mentors with laboratory specialists and/or experienced mentors working in other settings with whom they can consult as the need arises. logistical support ensures that a mentor’s time and energy are primarily directed to laboratories. because mentors most often work in laboratories rather than an organisation’s office – and may travel extensively – assistance with transportation, accommodation, communication and stationery can be very helpful. the cost of transport, accommodation, meals, phone calls, internet, photocopying, faxing, etc. should be budgeted. integration of mentors is important internally, with the moh, with relevant laboratory working groups or subcommittees and with other laboratory stakeholders. because mentors often work independently for extended periods, they often operate outside the office or organisational work culture. specific efforts may be necessary to ensure that mentors are involved and recognised as valuable members of the organisation – for example, scheduling recurrent organisational meetings at times when they can participate. good mentors can be difficult to replace. once hired, inclusion and job satisfaction should not be ignored. if multiple mentors are working in a country, they should meet together routinely to share experiences and synchronise approaches. mentors should be integrated with the moh’s national quality department and a representative should participate in national working groups or subcommittees related to qms implementation or accreditation. this integration will encourage mentor observations from the laboratory level to inform system-level laboratory activities and vice versa. supervision and accountability top ↑ creating an environment that addresses common mentor needs and monitors programme progress enables mentors to focus on in-lab implementation of qms and accreditation preparedness.if employing a single mentor, supervision of the mentor may fall to a non-laboratory member of the management team. in-country supervision can assist with the immediate problem-solving needs of both parties. when non-laboratory personnel conduct mentor supervision, it is important to identify outside laboratory technical support to discuss laboratory issues that may arise. if a programme is designed for a team of mentors, someone should be tasked with coordinating the overall programme and providing supervision. this could be done by a non-lab programme manager or by designating a senior mentor who has reduced in-lab duties and coordinates and supervises the rest of the mentor team. mentor teams should pursue common objectives in a coordinated manner, within standard structures and with standardised moh-endorsed tools. while each mentor will have their favoured micro-approaches to working with a laboratory, a structured model, standard tools, and integration will help ensure that laboratories implement qms in a standard fashion that demonstrates coherence. accountability is another crucial element of programme design. mentors should be clear about to whom and how frequently they report their activities, in what format, what feedback they can expect and when they will receive it. reporting may include written reports, routine phone calls, regular presentations to the moh or a relevant working group, etc. just as competency assessments are required of all laboratory staff, mentors should also receive field observation as part of their supervision. in addition, mentored laboratories should provide evaluation feedback on mentoring engagements. follow-up contact with mentored laboratories and/or relevant moh staff should be regularised to identify any emerging concerns. ultimately, laboratory mentoring programmes are accountable to the moh’s laboratory leadership. as previously stated, the moh should be included in reporting structures, either directly or through the representation of a mentor coordinator or senior mentor. measuring laboratory progress and mentoring effectiveness top ↑ in addition to clearly stated goals, mentoring programmes should also have standard measures of performance in order to gauge laboratory progress and mentoring effectiveness. the regular collection of laboratory performance data using standard tools will indicate how well a laboratory is implementing qms and where the laboratory stands with regard to their accreditation goals. the who-afro slipta program can help in setting clear overall goals and objectives. for example, one mentoring objective may be to realise an improvement of two stars on the five-star slipta scoring scale. or it may be that all laboratories will operate at the level of at least three stars by the end of the project. or that a given laboratory will reach five stars and be ready to successfully achieve iso-15189 accreditation. the presence of the who-afro slipta checklist can serve as an important measuring tool since it is standardised and accepted for use across multiple countries. data collection intervals should be structured to capture information about laboratory performance during mentoring engagements as well as between mentoring engagements. it is recommended that assessments be conducted before mentorship begins, at the end of each mentor engagement, when the mentor returns to the laboratory after a period of absence and at the conclusion of mentoring. the importance of the ‘return’ assessment lies in the valuable information it can provide for understanding how well the laboratory is able to sustain qms implementation under its own strength and initiative. as with general accreditation preparation, independent assessments conducted by persons familiar with the who-afro slipta checklist can be an important means of validating progress. involving independent assessors at baseline and at regular intervals thereafter (e.g., semi-annually or annually) can help identify or confirm general trends and laboratories ready to apply for assessment by who-afro. independent assessment at the conclusion of the mentoring programme can aid in measuring overall success. these on-going assessments serve more than one purpose during the mentorship engagement period. as described above, the assessments at specific time points within the mentorship period provide a means of gauging the laboratory progress, mentoring effectiveness, how well a laboratory is implementing qms and where the laboratory stands with regard to their accreditation goals. as part of mentorship, these assessments are also a means of building capacity to conduct internal audits within the laboratory as they are jointly done with the quality manager or any of the persons designated to internal audits. collection of other performance data is strongly encouraged. creating a balanced scorecard of key indicators to monitor in mentored laboratories will provide data about the status of service level operations. the granularity and quantitative nature of this monitoring complements the broader evaluation of the who-afro slipta checklist and ensures that the subjectivity in scoring the slipta assessment is balanced with the presence of performance data. mentors arriving at a laboratory for the first time may need to develop a baseline for these indicators by reviewing performance over the last full month or four weeks. this may be difficult in situations where these data may not have been collected or recorded. while collection of these baseline data may be mentor driven, it is important to build this tracking into the monthly operations of the laboratory so that this monitoring becomes a routine laboratory activity. the collection of these data should be standardised across mentored laboratories and could include performance indicators such as turn-around time, service interruptions, stock-outs, equipment downtime, external quality assurance performance, customer complaints and specimen rejection rate. for ongoing monitoring, laboratory assessments with the who-afro slipta checklist should continue to be conducted even after the full mentoring engagement has finished. these assessments could be conducted twice a year. if slippage is noted, further mentoring engagement may be prescribed. incorporating these assessments into the moh’s evaluation of its laboratories should be strongly considered. data summaries should be reported and shared with the moh and key stakeholders. efforts should be made to include independent evaluation of mentored laboratories. if a mentoring team is in place, mentors can conduct assessments for laboratories they are not mentoring. another option is to have the senior mentor, a lab specialist or supervisor assess the laboratories, perhaps teamed with an moh quality officer. the crucial issue is to include assessments of laboratory performance by independent evaluators to promote objectivity and accountability. structured reporting top ↑ reporting mechanisms should allow full engagement of the laboratory staff, laboratory management and upper management, which may include hospital management and the moh. any findings and opportunities for improvement must be discussed with the laboratory staff and management and action plans jointly formulated.4 besides guarding against the mentor doing the work for the laboratory, it is one means of building capacity within the laboratory and ensuring sustainability. commitment from management is a requirement of iso 15189,9 and constant and persistent engagement of management through proactive reporting is one means of gaining support from them. management support is critical to the success of implementation of quality management system as significant issues require their direct support, e.g., funds for purchase of reagents and supplies, staffing and physical changes, among many. summary top ↑ laboratory mentorship has been provided over the years in many different formats with varying results.10,11 some achievements have been reported where use was made of short visits, with technical assistance through telephone and skype. the authors observed that there is lack of a documented and standardised or harmonised approach to mentorship. this guide seeks to highlight elements that the authors feel should be considered when setting up a long-term, sustainable mentorship programme. considerations of these guiding principles may be a step towards harmonising approaches to mentorship. harmonising the approach may allow scalability and easy comparison across countries. with a standardised approach, budgeting and planning for countries intending to set up mentorship may be easier, as they are able to single out the activities expected. in conclusion, the success of a mentorship programme depends on several factors: well-defined goals, sufficient length of mentor engagement on site, standardised approach across laboratories, measurement of progress using standardised tools, well-structured reporting mechanisms, alignment of the programme with overall moh plans, and selection and training of the mentors. these elements will differ in application, depending on countries’ needs and available resources. one to two weeks of mentorship engagements may be sufficient for laboratories that are far advanced in implementation of qms while six to eight weeks may be required for laboratories that are beginning the process. in some countries where resources to recruit international mentors are not available and local experienced and self-motivated staff may not have the other prerequisites described here, local mentors can be recruited and taken through a grooming phase until they are ready to mentor independently. acknowledgements top ↑ competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this paper. authors’ contributions t.m. wrote the manuscript, designed and implemented the model of mentorship described, p.r. assisted in designing the model described and edited the manuscript, and t.p. assisted in designing the model described and edited the manuscript. references top ↑ 1. the health foundation; 2012. evidence scan: quality improvement training for healthcare professionals. accessed 12 september 2012. http://www.health.org.uk/public/cms/75/76/313/3731/quality improvement training for healthcare professionals.pdf?realname=rqcuap.pdf 2. henley e. a quality improvement curriculum for medical students. jt comm j qual improv. 2002 jan;28(1):42–48. 3. sullivan rl, ann b, noel m et al. 1995. clinical training skills for reproductive health professionals. baltimore, maryland, jhpiego corporation. 4. maruta t, motebang d, wanyoike j, peter t, rotz pj. impact of mentorship on who-afro strengthening laboratory quality improvement process towards accreditation (slipta). afr j lab med. 2012;1(1), art. #6, 8 pages. http://dx.doi.org/10.4102/ajlm.v1i1.6 5. gershy-damet g-m, rotz p, cross d et al. the world health organization african region laboratory accreditation process improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134:393–400 393 doi: 10.1309/ajcptuuc2v1wjqbm 393 6. the world health organization quality management system training toolkit. accessed september 2012. http://www.who.int/ihr/training/laboratory_quality/en/index.html 7. yao k, mckinney b, murphy a et al. improving quality management systems of laboratories in developing countries. an innovative training approach to accelerate laboratory accreditation. am j clin path. 2010;134:401–409. http:// dx.doi.org/10.1309/ajcpnbbl53fwuiqj, pmid:20716796 8. mothabeng d, maruta t, lebina m, lewis k, wanyoike j, mengstu y. strengthening laboratory management towards accreditation: the lesotho experience. afr j lab med. 2012;1(1), art. #9, 7 pages. http://dx.doi.org/10.4102/ajlm.v1i1.9 9. iso 15189:2007(e): medical laboratories – particular requirements for quality and competence. 2nd edn. 10. buddeberg-fischer b, herta kd. formal mentoring programmes for medical students and doctors – a review of the medline literature. med teach. 2006;28(3):248–257. http://dx.doi.org/10.1080/01421590500313043 11. frei e, stamm m, buddeberg-fischer b. mentoring programs for medical students – a review of the pubmed literature 2000–2008. bmc med educ. 2010;10:32. http://dx.doi.org/10.1186/1472-6920-10-32, pmid:20433727 abstract introduction methods results discussion acknowledgements references about the author(s) gamuchirai p. gwaza department of impact, foundation for innovative new diagnostics (find), geneva, switzerland department of continuing education, university of oxford, oxford, united kingdom monkoe leqheka department of research and laboratory services, ministry of health, maseru, lesotho tsietso mots’oane department of research and laboratory services, ministry of health, maseru, lesotho sabine dittrich department of global public health, deggendorf institute of technology, deggendorf, germany kekeletso kao department of access, foundation for innovative new diagnostics (find), geneva, switzerland citation gwaza gp, leqheka m, mots’oane t, dittrich s, kao k. missed opportunities for integrated testing of hiv and tuberculosis on the genexpert platform in lesotho. afr j lab med. 2023;12(1), a2132. https://doi.org/10.4102/ajlm.v12i1.2132 original research missed opportunities for integrated testing of hiv and tuberculosis on the genexpert platform in lesotho gamuchirai p. gwaza, monkoe leqheka, tsietso mots’oane, sabine dittrich, kekeletso kao received: 29 nov. 2022; accepted: 15 june 2023; published: 28 aug. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: integrated testing, treatment and care are key strategies for addressing the dual burdens of tuberculosis and hiv. the genexpert instrument allows simultaneous hiv and tuberculosis testing, but its utilisation for integrated testing remains suboptimal. objective: the study determined the extent to which tuberculosis testing and hiv early infant detection (eid) were integrated on the genexpert platform, or the potential for integration at selected health facilities. methods: a mixed methods evaluation was conducted using retrospective secondary data analysis of laboratory records from 2017 to 2019, and semi-structured interviews. data were collected between january 2020 and march 2020 in lesotho. results: forty-four health staff were interviewed across 13 health facilities: one regional, nine district, and three clinic level. six were government facilities, six were mission hospitals, and one was a non-profit clinic. all facilities selected had at least one genexpert instrument used for tuberculosis or hiv testing; none included simultaneous testing for tuberculosis and hiv. in 2017, the average utilisation rate for the genexpert instrument for tuberculosis and eid testing was 63% and 24%, while in 2019, the average utilisation rate was 61% for tuberculosis testing and 27% for eid. conclusion: except for three sites where the testing rates were high, utilisation rates were sufficiently low that all the hiv eid and tuberculosis tests undertaken in 2017 and 2019 could have been performed using only the instruments currently dedicated to tuberculosis testing. there is a missed opportunity for the integration of testing for tuberculosis and hiv on the genexpert instrument. what this study adds: this study adds to the body of evidence on the need for integration of testing and highlights some practical and technical considerations for successful implementation of integrated tuberculosis and hiv testing. keywords: tuberculosis; hiv; tb; genexpert; testing; integrated diagnosis; lesotho. introduction integrated testing can improve health outcomes, save costs, and improve patient experiences.1,2,3,4 tuberculosis is the leading cause of death among people living with hiv, and people living with hiv are 18 times more likely to fall ill with tuberculosis.5 in 2020, 9% of the people diagnosed with tuberculosis lived with hiv.6 implementing integrated testing, treatment, and care activities has been identified as a key strategy for addressing the dual burdens of tuberculosis and hiv.7,8 however, integrating testing for hiv and tuberculosis is still suboptimal due to various factors, including limited resources, a lack of clarity on effective integration models, and a lack of coordination among disease programmes.9 these factors are common challenges in integrating testing for other disease conditions, such as febrile illnesses.10 in 2010, the world health organization (who) recommended using the xpert® mtb/rif assay based on the genexpert multidisease platform for diagnosing tuberculosis. the xpert® mtb/rif is a simple and robust test making it feasible for use by people with limited or no laboratory training in peripheral laboratories and clinics.11,12,13 in 2016, who also approved several point-of-care (poc) assays for hiv diagnostics, including the xpert hiv-1 qual for hiv qualitative testing for early infant diagnosis (eid) using whole blood or dried blood spots, which used the same genexpert platform.14 point-of-care testing provides the opportunity to reduce turnaround times (tat), limiting patient loss along the hiv testing cascade, reducing infant mortality, and allowing for task shifting to lower cadres of health workers at decentralised facilities.15,16,17 the genexpert instrument can simultaneously test multiple pathogens, such as tuberculosis and hiv, using different cartridges on the same instrument. the platform has also recently been used for coronavirus disease 2019 testing.18 as such, the genexpert platform can potentially improve service delivery efficiencies and reduce costs to the patient by integrating testing for tuberculosis and hiv.19 despite significant progress, lesotho still grapples with the dual burden of tuberculosis and hiv.18 progress on mother-to-child transmission of hiv was bolstered by introducing the poc eid tests,20 including the genexpert hiv-1 qual and m-pima hiv-1/2 detect/alere q.16 lesotho is among the 30 countries with a high tuberculosis burden and has the highest estimated incidence rate of 654 per 100 000 people.18 the foundation for new innovative diagnostics has supported the ministry of health in lesotho in rolling out tuberculosis testing on the genexpert platform since 2016. lesotho’s investment in molecular tuberculosis and hiv testing has progressively increased since this time, and the country has a total of 42 genexpert instruments with four modules (gxiv) in 21 health facilities that could be used for tuberculosis and hiv testing and 15 alere q instruments in 15 health facilities for hiv poc testing.21 due to the high tuberculosis and hiv co-infection rates, lesotho adopted a strategy of using xpert® mtb/rif as the primary diagnostic test for presumptive tuberculosis patients to increase the chances of tuberculosis detection.21 the country provided hiv viral load testing for all hiv-positive individuals per the who hiv treatment guidelines.20 it also offered hiv eid testing at poc to minimise loss of follow-up. the lesotho ministry of health guidelines for hiv eid testing are consistent with the who guidelines, recommending that the first virological test for infants exposed to hiv should be conducted at or around 6 weeks following birth and again after they are weaned from breastfeeding and all infants diagnosed with hiv should be started on anti-retroviral therapy immediately, irrespective of cd4 count.22 the who also strongly recommended that xpert® mtb/rif be used as the initial diagnostic test in individuals suspected of multidrug-resistant tuberculosis or hiv-associated tuberculosis.23 the foundation for new innovative diagnostics has been supporting efforts to promote testing integration where possible through conducting various diagnostic network optimisation activities in lesotho.24,25 this evaluation aimed to determine the implementation rate or potential for integrated tuberculosis and hiv testing on the genexpert platform in lesotho from 2017 to 2019. the evaluation focused on hiv eid and tuberculosis testing, as these were the only tests being performed on the genexpert in the health facilities at the time of the evaluation in january 2020. methods ethical considerations no formal ethical approval was required as this was part of a routine programme evaluation. we received permission from the ministry of health, department of research and laboratory services, and a representative from the department was part of the evaluation team. several ethical measures were taken to protect the data and interviewees, including anonymising their responses and providing aggregate numbers of tests. the interviewees provided informed consent and the interviews were conducted in a confidential space to ensure privacy. no patients were interviewed in this evaluation. study design integrated testing was defined in the evaluation to mean that different tests, that is, tuberculosis and hiv, could be conducted at the same time or consecutively on the same genexpert instrument. a mixed methods evaluation design was developed, integrating different methods to collect data on defined processes and outcome indicators. data were collected simultaneously with one researcher conducting interviews while the other reviewed the laboratory records at the health facilities. this data collection was conducted between 13 january 2020 and 24 january 2020. further data requests for missing or data unavailable in the field were shared between february 2020 and march 2020. the focus was on the cepheid xpert® mtb/rif, referred to as the genexpert instrument. secondary data analysis data were collected from laboratory records or files with data for tuberculosis and hiv eid tests conducted at the facility for the most recent period from 2017, 2018 and 2019. data for 2017 and 2019 were used in the evaluation as they were more complete and could be used for comparisons across the health facilities. complete data meant that the data included the day the test was taken, time started, time finished, result of the state, and error rates. semi-structured interviews these were conducted with the guide of a survey instrument consisting of two questionnaires. the ‘laboratory questionnaire’ covered questions related to genexpert instrument installation, staff training, instrument maintenance, quality management, data reporting, testing rates, costs and challenges faced in implementing tuberculosis and hiv testing. the ‘clinical questionnaire’ focused on patients’ general profile, economic costs, staff training, testing rates, linkage to treatment, health outcomes, and challenges related to tuberculosis and hiv patient management. the clinical questionnaire was also meant to provide more details on the health workers’ experience and how they perceived the patients’ experiences. the inclusion criteria for the interviews were that those interviewed had to be responsible for conducting the tuberculosis or the hiv test on the genexpert instrument or were working directly on the tuberculosis or hiv programme on a day-to-day basis. at each site, two people were interviewed working in the lab for tuberculosis or where the genexpert for tuberculosis was located and two people working in the maternal health division where the genexpert was located for hiv eid testing. the questionnaires were developed based on the metrics that the foundation for new innovative diagnostics had been using to monitor the genexpert implementation within the facilities prior to 2016 where they had provided funding for the activities. as such, they had been piloted and tested within the same contexts. setting lesotho employs a hub and spoke model for sample referrals. the hubs are laboratories often located in hospitals, either at the district or regional level, where the genexpert instrument is located.21 samples are referred from collection sites’ ‘spokes’ to testing sites’ ‘hubs’. the spokes are clinics or health centres where samples are collected and transported to the hubs for testing.26 generally, the hubs in peri-urban areas cover a large catchment area and receive samples from multiple spokes. sampling strategy genexpert testing sites in rural, urban, and peri-urban areas across the country were selected based on discussions with the ministry of health staff to ensure diversity in region and type of facility. the health facilities were purposely sampled to represent the geographic breadth and size of the facilities, that is, peri-urban, rural and urban sites and regional, district, and local hospitals and clinics.3 the facilities selected had to have at least one genexpert instrument used for tuberculosis or hiv testing. in some cases, the facility used genexpert for tuberculosis testing and the alere q for eid testing. these were still included in the evaluation and the data were later excluded from the analysis to allow for better comparison. data analysis the data were analysed in microsoft excel (microsoft corp., redmond, washington, united states). the utilisation rate for the genexpert instrument was calculated by dividing the actual number of tests performed each year (for a given site and disease) by the maximum possible number of tests that could be performed. the who guidelines recommend that the maximum capacity of a single, four-module genexpert instrument is 20 specimens per day.23 however, the recent global laboratory initiative guide recommends that one technician could perform more than 12 xpert® mtb/rif tests per day.27 for practical consideration, a maximum capacity of 12 specimens per day was used in the analyses, for a single gxiv, given three 2-h runs in a day. an assumption of 264 days of testing per annum, based on 22 days per month recorded during data collection at the sites, was used. based on these calculations, the maximum capacity of a single, four-module genexpert instrument is 3168 specimens each year (12 specimens/day × 264 days). we defined the standard tat in two distinct ways: (1) the time from sample collection to results being received in the laboratory and (2) the time from sample collection to when the patient receives results. these vary by countries and sites and are usually determined for each based on infrastructure capacity. the tat was collected through interviews. this is because laboratory records showed the time when the sample was received and when the test was completed and did not always show when the patient collected the results. during the interviews the staff were asked, ‘approximately how long does it take from the time the sample is collected to when the patient receives their results?’ if they could, they were asked to break down the times into the two stages defined above. results sample in total, 44 people were interviewed (24 tuberculosis interviews, 18 hiv interviews, and two ministry of health interviews), with an average of two people per site and two in the central ministry of health offices. we completed 42 questionnaires; 24 for tuberculosis and 18 for hiv. of the tuberculosis and hiv questionnaires, half focused on the clinical aspects, while the other half focused on the laboratory questionnaire. thirteen health facilities were included in the evaluation (table 1). twelve health facilities conducted tuberculosis testing on the genexpert instrument, (one site only had no tuberculosis testing: thamae health centre) and they served a combined total of 137 spoke sites. nearly half (46%; 6/13) of the hospitals had more than one gxiv, such as mafeteng (n = 3), berea (n = 2), motebang (n = 2), ntshekhe (n = 2), partners in health clinic (n = 2) and scott hospital (n = 2). nine health facilities had a gxiv for hiv eid testing and 73 spoke sites. table 1: location of health facilities visited in lesotho in january 2020. none of the 13 sites had integrated tuberculosis and hiv eid testing on the same instrument, instead, tuberculosis and hiv testing were conducted separately in different rooms by different people on different instruments. in 2017, there were 32 037 tuberculosis tests conducted on 16 gxiv machines (table 2). seven genexpert instruments for eid testing were installed in 2017. there were data available from three of the sites; a total of 2297 eid tests were conducted. overall utilisation rates for the three sites ranged from 54% to 72%. in 2017, the average utilisation rate for the genexpert instrument for tuberculosis and eid testing was 63% and 24%. table 2: number of tests conducted on the genexpert instrument, based on the data recorded in lab registers and hospital files. in 2019, there were 19 gxiv instruments used for tuberculosis testing across 12 sites, with a total of 37 347 tuberculosis tests conducted. nine gxiv instruments were used for eid testing across nine sites, and 6977 eid tests were conducted. overall utilisation rates ranged from 54% to 148%. in 2019, the average utilisation rate was 61% for tuberculosis testing and 27% for eid. turn-around time the tat for performing the tests and receiving the results in the laboratory is 2 h on the genexpert instrument. the time to get results to the patients at the hub site ranged from 2 h to 24 h across the sites for both tuberculosis and hiv testing (table 3). the average time to get the results to the patients in the spoke sites varied across the sites and ranged from 2 to 7 days. table 3: turn-around time for return of tuberculosis and early infant detection test results to patients at hub and spoke sites, lesotho, january 2020 – march 2020.† staff capacity the number of staff trained to operate the genexpert instrument varied across sites and diseases (table 4). on average, there were four trained staff for tuberculosis testing and three for hiv testing on the genexpert instrument. for the tuberculosis testing, staff trained were usually laboratory technicians, microscopists, or health technologists. tuberculosis testing was conducted within the central laboratory at the health facility. on the other hand, for hiv eid testing, nurses, professionals, and lay counsellors were trained to operate the genexpert instrument. the eid testing was done at the poc within the maternal wing of the health facility. table 4: staff trained to operate the genexpert instrument for testing, lesotho, january 2020 – march 2020.† linkage to treatment in all of the hiv eid sites, there was same-day initiation of treatment, with 100% of those testing positive being initiated on anti-retroviral therapy. the proportion of infants staying on treatment for at least 12 months was very high (100%) across six sites. for hiv satellite sites, five of the sites had same-day initiation of treatment (seboches, ntsheke, scott, motebang, thamae), while two (paray and mafeteng) had 2 days, one site (berea) had 4 days, and one site (maluti hospital) could be up to 10 days (table 5). care cascade and linkage to treatment data for tuberculosis were not complete or available for more than half of the facilities (7 out of 12) and were therefore excluded from the results. table 5: time for patient linkage to treatment for hiv early infant detection, lesotho, january 2020 – march 2020†. barriers to integrating tuberculosis and hiv testing on the same platform resources donor alignment: one barrier to integration noted during the interviews was the lack of coordination among the different international funding partners for tuberculosis and hiv. in many cases, each funder purchased their instrument and had different reporting requirements, making it challenging to integrate the workflow and testing even if the devices were housed in adjacent rooms and could have been used by both programmes. time: another barrier to integration was potential time constraints if testing was increased, especially in sites with a higher utilisation rate. training another concern raised in the interviews was that staff conducting hiv training had little training in tuberculosis testing and vice versa. some had received the training in the past but had yet to have a chance to put it into practice and would need more training before they could conduct the tests. information technology connectivity another issue mentioned was the need for a functional data connectivity system to record information and transmit results electronically to clinicians, which would have made integration easier. some sites had limited data availability as they still relied on a paper-based system or were transitioning to a digital system. most of the genexpert instruments were no longer linked or could use e-reporting. as a result, staff entered results manually, placing an extra burden on them. discussion in this evaluation of integrated testing of tuberculosis and hiv on the genexpert platform, none of the sites visited tested for tuberculosis and hiv on the same instrument, and most instruments were not operating at maximum capacity. this has been observed not just in africa but also in europe, because although the who european region has moved towards integration, this has had a limited effect on the programmatic organisation for tuberculosis and hiv nationally, which is partly dependent on donor funding that is often project-related and focused on a particular disease.13 the utilisation rates in our study were consistent with results from a previous diagnostic network mapping exercise in 2017 across the 10 districts of lesotho, which had shown low utilisation of genexpert instruments, with 19 out of 24 facilities sampled using instruments at less than 50% capacity.24 this underutilisation of genexpert instruments for tuberculosis has been reported in a trend analysis of 22 tuberculosis high-burden countries with just seven countries (37%) (south africa, tanzania, ethiopia, nigeria, pakistan, uganda, and zimbabwe) utilising the instruments for multiple tests, including tuberculosis and other diseases, such as hiv and hepatitis-c virus.28 similarly, for hiv eid, underutilisation was observed in another study in zimbabwe in 2019, where the average daily utilisation of poc eid instruments was 1.51 tests/day.29 a later study (2020) in lesotho confirmed that the genexpert instruments are largely underutilised, and relocation of instruments will deliver equivalent access to services compared to procuring new instruments.25 the relatively low utilisation rate at many of the sites evaluated in this study suggests there is potential for integrated testing in future, purely based on capacity. for example, in 2017, the three sites that installed new genexpert instruments could have integrated the eid tests on the available genexpert machines. at mafeteng, with the two machines available for tuberculosis, there would have been a total of 5668 tests (4347 for tuberculosis and 1321 for eid) out of a possible maximum of 6336 tests, which would have been an 89% utilisation rate. similarly, in maluti, 2291 tests (1849 tuberculosis and 442 eid) could have been conducted on one instrument with a 72% utilisation rate. in paray, 1699 (1165 tuberculosis; 534 eid) tests could have been undertaken at a 53.6% utilisation rate. in 2019, had all the eid tests been integrated or conducted on the genexpert instruments used for tuberculosis testing, five sites would still not have used the maximum capacity. only three of the sites would not have been able to handle both tests without an additional machine (table 2). integrating testing across major diseases can help leverage resources in developing countries.30 the feasibility of integrated testing was successfully demonstrated in 2018 in a study in zimbabwe on the genexpert platform for tuberculosis testing, detecting resistance to the antibiotic rifampicin, eid, and hiv viral load testing in the treatment monitoring of patients on anti-retroviral therapy.31 fortunately, this integration is already part of lesotho’s us president’s emergency plan for aids relief (pepfar) country support plan within the tiered laboratory network.20 however, there are still barriers to operationalising integrated testing in lesotho, which are common in other countries. for example, staff capacity was also identified as a barrier to tuberculosis and hiv integration in a 2009 paper in uganda. health workers had been trained on tuberculosis or hiv and not on tuberculosis-hiv integration. in addition, follow-up supervision and monitoring after the training were minimal.9 the genexpert instrument and many poc tests can improve patient experiences by reducing waiting times. in this evaluation, all the hub sites had a tat, that is, patients getting their results, of less than 4 h, consistent with similar studies that had shown same-day results return to a caregiver at testing sites and a tat of 5 days at spoke sites, a significant improvement over the conventional model of 61.7 days.26 the tat noted in this evaluation was a reduction from a 2019 in a study in eight african countries that had seen a tat of 9 days at spoke sites.16 this reduction could have responded to a country review of the joint tuberculosis and hiv programme, which noted that the tat in some spoke sites needed urgent attention as it could take up to 2 weeks.32 reducing tat can increase the testing rates for tuberculosis and hiv as it will cut down on travel time and costs, especially in rural areas, which may be a significant burden for some people. however, informal discussions with some laboratory workers during the evaluation showed that even though technically the tat for getting results to the patients at the hubs could be between 2 h and 3 h, which is the time it took to run the test, in practice the patient still had to wait at least a day to get their results. this was because the lab workers did not want the added pressure of patients waiting for their results as they had a lot of tests from their spoke sites to attend to. the time commitment by the patients constitutes an additional economic cost, precluding the patients and those accompanying them from doing other paid work or caring duties for several hours or days. interventions are needed to increase service coverage and utilisation of services in these areas. policy implications this evaluation highlights areas that need more focus and more detailed diagnostic network optimisation exercises to improve service delivery and allocate stocks, staff, equipment, and training. better coordination of donor support or increased flexibility in reporting of use will be essential to maximise the use of resources, where integration may be more effective than purchasing new equipment. monitoring and evaluation should be a routine part of the implementation and should be budgeted and demanded by policymakers and donors. health facilities may require additional resources and technical support to improve data collection, analysis, and dissemination. limitations access to data was a challenge in some sites, which affected the analysis. the evaluation focused on 2017 and 2019 data only, which was the most complete. due to a lack of variation in the cost data by site, a cost-effectiveness analysis could not be done. conclusion previous reviews and studies have shown that integrated testing is feasible and can improve health outcomes and patient experiences. the findings from this evaluation show that there are still operational challenges to implementing integrated testing. an understanding and further research of how and why integrated testing models work and in which contexts will allow for effective targeted interventions to improve testing coverage across several diseases especially taking advantage of the multidisease testing platforms. acknowledgements the authors would like to acknowledge the support from all the laboratory staff and health workers in the facilities visited. competing interests g.p.g., s.d. and k.k. were employed at the foundation for new innovative diagnostics at the time of the evaluation. authors’ contributions g.p.g. conceived of the presented idea, designed the evaluation, performed the data collection, and wrote the manuscript. m.l. helped in the data collection and review of the data. t.m. supervised the project and reviewed the data. s.d. contributed to the review of data collection instruments, manuscript review and final version of the manuscript. k.k. helped in the design of the project, data collection, supervision of project and review of manuscript. sources of support the evaluation was funded by the foundation for new innovative diagnostics, which receives support from several funding sources. data availability the authors confirm that the data supporting the findings from this study are available within this article. raw data supporting this evaluation’s findings are available from the corresponding author, g.p.g., upon reasonable request. disclaimer the views expressed in the submitted article are the authors’ own and not an official position of the ministry of health, lesotho or foundation for new innovative diagnostics. references wallrauch c, heller t, lessels r, kekana m, barnighusen t, newell m.-l. high uptake of hiv testing for tuberculosis patients in an integrated primary health care hiv/tb programme in rural kwazulu-natal. s afr med j. 2010;100(3):146–147. 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and optimization of laboratory services in lesotho. geneva: find; 2017. albert h, purcell r, wang yy, et al. designing an optimized diagnostic network to improve access to tb diagnosis and treatment in lesotho. plos one. 2020;15(6):e0233620. https://doi.org/10.1371/journal.pone.0233620 tiam a, gill mm, hoffman hj, et al. conventional early infant diagnosis in lesotho from specimen collection to results usage to manage patients: where are the bottlenecks? plos one. 2017;12(10):e0184769. https://doi.org/10.1371/journal.pone.0184769 stop tb partnership. gli practical guide to tb laboratory strengthening. geneva: global laboratory initiative; 2017. cazabon d, pande t, kik s, et al. market penetration of xpert mtb/rif in high tuberculosis burden countries: a trend analysis from 2014 – 2016. gates open res. 2018;2:35. https://doi.org/10.12688/gatesopenres.12842.1 frank sc, cohn j, dunning l, et al. clinical effect and cost-effectiveness of incorporation of point-of-care assays into early infant hiv diagnosis programmes in zimbabwe: a modelling study. lancet hiv. 2019;6(3):e182–e190. https://doi.org/10.1016/s2352-3018(18)30328-x birx d, de souza m, nkengasong jn. laboratory challenges in the scaling up of hiv, tb, and malaria programs: the interaction of health and laboratory systems, clinical research, and service delivery. am j clin pathol. 2009;131(6):849–851. https://doi.org/10.1309/ajcpgh89qdswfons ndlovu z, fajardo e, mbofana e, et al. multidisease testing for hiv and tb using the genexpert platform: a feasibility study in rural zimbabwe. plos one. 2018;13(3):e0193577. https://doi.org/10.1371/journal.pone.0193577 government of lesotho. final report for a joint review of hiv/tuberculosis and hepatitis programmes. maseru: government of lesotho; 2017. abstract introduction methods results discussion acknowledgements references about the author(s) john b. kalule department of biotechnical and diagnostic sciences, college of veterinary medicine, animal resources animal and biosecurity, kampala, uganda joseph tomusange department of biotechnical and diagnostic sciences, college of veterinary medicine, animal resources animal and biosecurity, kampala, uganda teddy namatovu department of biotechnical and diagnostic sciences, college of veterinary medicine, animal resources animal and biosecurity, kampala, uganda citation kalule jb, tomusange j, namatovu t. serological detection of brucellosis among febrile, malaria-negative children and domesticated dogs in an urban african setting. afr j lab med. 2020;9(1), a864. https://doi.org/10.4102/ajlm.v9i1.864 original research serological detection of brucellosis among febrile, malaria-negative children and domesticated dogs in an urban african setting john b. kalule, joseph tomusange, teddy namatovu received: 12 july 2018; accepted: 06 july 2020; published: 30 sept. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: childhood brucellosis and malaria are co-endemic febrile illnesses in some sub-saharan african countries. malaria and brucellosis co-infection or brucellosis sole infections are often missed due to an over emphasis on malaria and the lack of appropriate diagnostic infrastructure. brucellosis in dogs is usually overlooked and yet there is extensive contact between humans and their pets. objective: this study investigated brucellosis in children and dogs using a confirmatory serological testing series that screens for three brucella sp. methods: residual blood samples from malaria smear-negative febrile children were collected and tested for brucella sp and malaria parasite. during the same period, residual blood samples presented to a veterinary microbiology laboratory in the same area were tested for brucellosis using the same approach. results: a total of 105 human and 80 canine blood samples were tested for brucellosis antibodies. the seroprevalence of brucellosis was 22.86% (25/105) in children and 1.3% (1/80) in dogs using the card, buffered acidified plate antigen, and standard plate agglutination tests but was 0% using the rivanol precipitation plate agglutination test. conclusion: given that brucellosis can be caused by both smooth and rough colony strains, there is a need to modify the current serological surveillance strategy (targeted at only brucella abortus and other smooth colony brucella strains) to figure out the relative contribution of rough colony brucella strains (b. ovis and b. canis). since uganda is endemic for brucellosis there is a need to modify the brucellosis surveillance strategy. keywords: brucellosis; serology; malaria; febrile illness; diagnostics. introduction globally, human brucellosis remains the most common zoonotic disease with more than 500 000 new cases annually, most of which are in sub-saharan africa.1 generally, brucellosis has no age predilection; its transmission is linked to use of or contact with known sources of infection, such as consumption of poorly made dairy products,2,3 or contact with carcasses or aborted material of infected livestock. childhood brucellosis has previously been reported among children presenting with pyrexia of unknown origin.4 childhood brucellosis accounts for up to one-third of all cases of human brucellosis in endemic regions.5 uganda is one of the sub-saharan africa countries that are endemic for brucellosis and have, according to world organisation for animal health (oie) data, reported cases of human brucellosis.6 as per a 2006 study which estimated brucellosis globally, uganda had an estimated incidence of 1.8 new cases per 100 000 population.7 both the vaccine strain (brucella abortus s19) and the wild strains (b. abortus, b. melitensis, b. canis and b. suis) have been shown to be excreted in substantial amounts in livestock milk,8,9,10 a wholesome nutritious option for weaned children,11 thus milk is a vehicle of brucellosis transmission to humans.12 milk and/or milk products should be subjected to various physical sterilisation to kill potential pathogens such as brucella spp.13 however, due to various socio-economic factors, consumption of milk and milk products remains a risk factor for brucellosis in many regions in sub-saharan africa.10,14,15,16,17 nevertheless, other risk factors, such as environmental contamination with aborted foetal material, or contact with infected domesticated dogs or their body fluids, could potentially be associated with brucellosis,18 even among children. dogs have been proven to be reservoirs of highly pathogenic canine b. suis strains that can cause severe disease in humans19 and/or re-emergence of brucellosis on livestock farms.20 the clinical presentation of brucellosis is largely similar to that of malaria (high fever, joint pain, malaise, headache and chills)21; thus, there is a need to deploy sensitive, specific, rapid, and cost-effective laboratory diagnostic tools to differentiate them and avoid misdiagnosis.3 the situation is aggravated by the presence of the weak laboratory infrastructure in sub-saharan africa.22 moreover, the morbidity and mortality rates associated with malaria are high, yet co-infections with brucellosis in this setting are not uncommon.3 children presenting with high fevers, headaches, and joint pains are more likely to be treated empirically for malaria.23 the diagnosis of brucellosis requires the use of several clinical investigative techniques across haematology, biochemistry, radiology, bacteriology, serology and molecular biology.24 isolation of brucella spp. from the blood, bone marrow or other tissue fluids is the gold standard.5 unlike most high-income countries where brucellosis affects mostly animals, in sub-saharan africa it is arguably endemic in both humans and animals.25 in north america, where brucellosis affects both domestic (such as cattle and goats) and wild animals (such as elk and bison), highly efficient serological tests have been developed as part of surveillance diagnostic test schemes for both.26 seven such serological tests, the card (rose bengal) test, the complement fixation test, the rivanol precipitation plate agglutination (rppa) test, the standard plate agglutination (sp) test (spt), the buffered acidified plate antigen (bapa) test, the rapid automated presumptive test, and the fluorescence polarisation assay have been approved by the united states department of agriculture for the detection of b. abortus antibodies. brucella abortus is the most prevalent species in north america. the rapid slide agglutination test is often used to test for b. canis in dogs which can also be infected with b. suis.27 the b. abortus serological tests that are commonly used cannot detect antibodies to the rough colony variants b. canis and b. ovis. the north american serological tests have rarely been used in the endemic african regions; in fact, only the card test has been used frequently for serodiagnosis of brucellosis at the point of care for both humans and animals.28 the card test is highly efficient for diagnosis of human brucellosis, often outperforming tests that take longer to perform such as coombs, competitive enzyme-linked immunosorption assay, brucellacapt (immunocapture agglutination test; vircell, granada, spain),29 immunochromatography, and immunoprecipitation with brucella proteins.30 with the exception of the rppa and the fluorescence polarisation tests, the spt, card, rapid automated presumptive and bapa tests all have the limitation that immunoglobulin m antibodies against brucella spp. cross-react with those of other gram-negative bacteria such as salmonella spp. on the other hand, the immunoglobulin g antibodies that are detected using the rppa test do not cross-react and thus give fewer false positives. unlike seropositivity by rose bengal test or bapa, which in addition to indicating infection may also mean recovery from brucellosis, rppa test seropositivity can be used to differentiate between active infection or past infection.12 most studies of brucella spp. seroprevalence in uganda have used the rose bengal test (card test) without using a confirmatory serological test as categorised by the oie.31,32,33 the rppa test is commonly used as a confirmatory test, because the non-specific reactivity is reduced by precipitation of high molecular weight serum glycoproteins.12 the increased specificity means a reduction in the number of false positives. this study aimed to establish the seroprevalence of brucellosis due to b. abortus and b. suis in humans presenting with acute febrile illness and domesticated dogs in the same urban african setting. we hypothesised that the use of a confirmatory serological test (rppa) in addition to routinely used screening serological tests (bapa, card and spt) would enable us to rule out positive reactions due to vaccination of tested animals, previous infection history, or cross reactions34 and thus figure out the true seroprevalence of brucellosis due to b. abortus and b. suis in this setting. methods ethical considerations ethical clearance was obtained from the makerere university, college of veterinary medicine, animal resources and biosecurity (covab), school of biomedical and biotechnical laboratory sciences, research and ethics committee (sbls/rec/13/019). the ethical clearance was for use of residual blood samples from the paediatric ward at the clinical microbiology laboratory at mulago national referral hospital. the same clearance also permitted the use of residual blood samples from dogs at a small animal veterinary clinic at makerere university college of veterinary medicine animal resources and biosecurity. this study did not directly recruit or collect samples from either the febrile children or the febrile dogs. data on the ages and sex of the patients from whom the samples were collected were recorded. the samples were re-assigned a study number and all patient-identifying data were not accessed. the same was done for the residual blood samples from febrile dogs. study design and residual sample selection we opted to test residual blood samples from febrile children that were malaria smear-negative and residual blood samples from febrile dogs. the dogs, though owned by individual homes, closely interacted with the community in the study region; for instance, the pig abattoir in the community is potentially a common source of infection to both humans (via meat purchases) and dogs (either the abattoir waste was purchased by the dog owner as dog feed or the dogs accessed poorly disposed abattoir waste). all samples used were collected between january and june 2014 (figure 1). the malaria-negative residual paediatric blood samples were collected from the clinical microbiology laboratory of a regional referral hospital, while residual canine blood samples were from a local veterinary clinic which serves the central region. the residual blood samples were tested for brucellosis using three screening tests and one confirmatory test. figure 1: sample testing algorithm for blood samples from children and dogs in kampala, uganda (january and june 2014). re-testing human blood samples for malaria the included malaria-negative paediatric residual blood samples were re-tested for malaria to confirm that they were malaria-negative. the field’s stains a and b (himedia laboratories limited, mumbai, india) were used to stain malaria parasites and were viewed using light microscopy. briefly, a drop of blood was placed on a clean 25 mm x 75 mm glass slide (merck, darmstadt, germany) and a thick smear was made at the centre of the glass slide using the edge of another glass slide to spread to an area of 1 cm2. the smear was allowed to dry at room temperature for 1 min. it was then sequentially dipped into field’s stain a (himedia laboratories limited, mumbai, india) for 3 seconds and washed in de-ionised water for 3 s with gentle agitation, then it was dipped in field’s stain b for 3 s and washed in tap water for 5 s. the stained smear was then air dried and examined under a light microscope at x100 magnification under oil immersion for malaria parasites, as previously described.35 testing of human and canine sera samples for brucella abortus/suis antibodies human blood samples that were negative for malaria were processed by centrifugation and the serum obtained was tested for brucellosis using: the becton dickinson brucellosis card test kit 306® (national veterinary services laboratory [nvsl], ames, iowa, united states) – a screening test for antibodies against b. abortus/b. suis in serum and plasma; spt (nvsl, ames, iowa, united states); bapa (sl, ames, iowa, united states) and rppa (nvsl, ames, iowa, united states) – a precipitation and agglutination test. the bapa, rppa, card and sp kits screened for b. abortus/ b. suis antibodies in serum and plasma; tests were carried out according to the manufacturers’ instructions on all human and canine sera included in the study. except otherwise stated, the control sera used were the b. abortus complement fixation medium positive control serum (nvsl reagent 12-m) and the b. abortus complement fixation negative control serum (nvsl reagent 12-n). buffered acidified plate antigen test aseptically, 80 μl of each sample or control serum was pipetted onto separate squares (1.25 inch – 1.5 inch) etched on a 12.5-inch x 12.5-inch white ceramic bioassay glass plate. then 30 μl of buffered brucella plate antigen (nvsl, ames, iowa, united states) was added to each sample or control serum square. the serum-antigen suspension was then mixed using a glass stirrer to a diameter of approximately 27 mm and incubated for 4 minutes (±30 s) at room temperature (20 °c – 26 °c) in an enclosed space. after this incubation, it was stirred again and incubated a second time at room temperature for 4 min. the slides were then read in a minnesota box – an illuminator with an indirect source of light, a black background, and a cover to prevent evaporation of test reagents. rivanol precipitation plate agglutination test aseptically, 200 μl of each sample or control (positive or negative) was transferred into respective (well-labelled) tubes. thereafter, 200 μl of brucella rinavol solution was transferred into each tube and mixed by shaking for 1 min. the mixture was incubated at room temperature for 5 min for precipitation. the tubes were centrifuged at 3000 revolutions per minute to pellet the precipitates. aseptically, 80 μl, 40 μl, 20 μl and 10 μl of each sample or control supernatant were dispensed onto separate squares (1.25 inch – 1.5 inch) etched on a 12.5 inch x 12.5 inch white ceramic bioassay glass plate. then, 30 μl of b. abortus rivanol antigen was added. the serum-antigen mixture was stirred in circles (at least 8 circles) beginning from the 1:200 serum dilutions, then proceeding to the 1:25 dilutions of sample or control sera. the bioassay plate was then incubated at room temperature (25±2 °c) in an enclosed space for 6 min (±30 s) with rotation. the incubation with rotation was repeated for a further 6 min (±30 s). the reading was taken at the end of the second incubation under an illuminator according to the manufacturer’s instructions. card test aseptically, 30 µl of sample or control (positive or negative) were dispensed onto the reaction area on the card. subsequently, two drops of the b. abortus card test antigen (nvsl, ames, iowa, united states) were then dispensed onto the card adjacent to the sample. the antigen and serum were mixed using a clean wooden stirrer for approximately 15 s, followed by rocking movements for 4 min. standard plate agglutination tests aseptically, 80 µl, 40 µl, 20 µl and 10 µl of sample or control serum were dispensed onto separate squares on a white ceramic bioassay glass plate. following gentle mixing, 30 µl of antigen was dispensed to each square containing sample or control serum. the serum and antigen were mixed with a stirrer in circles. mixing was done beginning with the 1:200 dilution. twice, consecutively, the glass plate was rocked through four rotations, followed by incubation at 25±2 °c in an enclosed space for 4 min (±30 s). the white ceramic bioassay glass plate was rotated again and incubated for another 4 min (±30 s). the reaction was then read using a minnesota box (nvsl, ames, iowa, united states). statistical analysis data on the serological reactions were entered in microsoft office excel 2010 (microsoft corp., redmond, washington, united states) and then exported to epiinfotm 7.1.5.2 (centers for disease control and prevention, atlanta, georgia, united states) for analysis to determine proportions. the openepi diagnostic test evaluation calculator36 was used to estimate parameters for the performance of the different screening tests compared to the rppa test. results a total of 349 human residual blood samples were received at the clinical microbiology laboratory from the paediatric ward during the study period; 80 canine residual blood samples were received at the small animal veterinary clinic during the same time period. of the 349 human samples, 105 were smear-negative for malaria; 244 were smear-positive and excluded from further analysis. of the 105 malaria smear-negative samples, 44.8% (47/105) were from male patients and 55.2% (58/105) were from female patients. seroprevalence of brucellosis among the malaria smear-negative residual blood samples was 23.8% (25/105) by card, 23.8% (25/105) by bapa and 23.8% (25/105) by spt, but was 0% (0/105) using the rppa test (table 1). all the samples that tested positive using the card test were also positive using the bapa and the sp tests; therefore, there was a 100% agreement between card, bapa and sp tests. of the 25 samples that tested positive using the three screening tests, none (0%, 0/25) were confirmed as positive using the rppa test. of the 25 human samples that were positive, 40% (10/25) were from male patients, and 60% (15/25) were from female patients. there was no significant difference between sero-reactions for male and female children (p = 0.131). the mean age of the seropositive children was 12 (±0.8) years. table 1: sero-reactions to brucellosis tests for human and canine blood samples tested between january and june 2014 in kampala, uganda. eighty blood samples from dogs (one sample from each dog), all from households in the central region, were tested for b. suis antibodies, and only one was positive using the three screening serological screening tests – card, bapa and spt. thus, the seroprevalence of canine b. suis was 1.28% (1/80) using the screening tests. this sample was, however, negative using the rppa test (seroprevalence using the rppa test = 0%). there was no significant difference between the seropositivity for humans and dogs (p = 0.052). of the canine (1/80) and human (25/105) samples that tested positive by the sp test, all were positive at the two lowest dilutions (1:25 and 1:50). however, 60% (15/25) of the human samples tested positive at the third dilution (1:100) and 28% (7/25) tested positive at the fourth dilution (1:200). for the human samples, the specificity of the screening tests (card, bapa and spt) was 76.2% (67.2–83.3), sensitivity 0%, positive predictive value 0%, and the negative predictive value 100%. the cohen’s kappa statistic was 0. for the canine samples, the specificity of the screening tests (card, bapa and spt) was 76.2% (67.2–83.3), sensitivity 0%, positive predictive value 0%, and the negative predictive value 100%. the cohen’s kappa statistic was 0. discussion this study used three screening tests (card, bapa and sp) and an oie confirmatory test (rppa) to evaluate brucellosis as a possible aetiology of pyrexia of unknown origin among malaria smear-negative febrile children in a malaria-endemic african region. using routine serological screening tests, the seroprevalence of brucellosis in dogs was 1.3%, and 23.8% in humans. there was 100% agreement between card, bapa and sp tests, but all were negative using the confirmatory rppa test. the seroprevalence of brucellosis in both species using the oie-recommended confirmatory serological test – rppa test – was 0%. many serological studies conducted on brucellosis in endemic regions have reported a high prevalence of brucellosis.37,38,39,40 unlike earlier studies, this study deployed an oie confirmatory test in parallel with routinely used screening tests. the brucellosis prevalence reported without the use of a confirmatory test might be misleading, particularly when brucella culture, the gold standard for brucella spp. detection, is not carried out. brucella culture is difficult to execute and suitable laboratory facilities are often lacking in resource-limited brucellosis-endemic regions. the discrepancy between the screening and confirmatory tests found in the current study is similar to that reported in a study conducted in kenya.41 in this study, all the blood samples tested using the card and other screening tests were negative when tested using the rppa test. the difference in results can be explained by the fact that the most commonly used screening test in uganda, the card (rose bengal) test, deploys a b. abortus/suis antigen that would miss the detection of rough brucella spp. such as b. canis and b. ovis.42 also, cross-reaction is not uncommon when using card (rose bengal), leading to false positives. the smooth lipopolysaccharide o-chain of smooth b. abortus, b. melitensis and b. suis and other gram-negative bacteria expressing the lipopolysaccharide o-chain (such as escherichia coli o157:h7 and yersinia enterocolitica o:9) have been shown to cross-react with the card (rose bengal) test (b. abortus/suis antigen).43 also, the card test does not differentiate between vaccinated and infected animals, or of the recovered individuals (as would be expected in a brucellosis-endemic setting) from those with active infection.34 on the other hand, the rppa test is a quantitative and specific test which is able to rule out false-positive serological reactions.34 these factors could account for the difference in results between the card and the rppa test. additionally, even though there was agreement between the three serological screening tests (card, bapa and sp) the different screening serological tests target a different cluster of antibodies, some of which are not recognised by the oie. for instance, the sp test lacks oie recognition and detects immunoglobulin m, immunoglobulin g2, and immunoglobulin a (unpublished data, nvsl protocols, ames iowa, united states). while the bapa test has oie recognition, it detects mainly immunoglobulin g1 and immunoglobulin g2 and has a sensitivity ranging from 70% – 99%.44 therefore, the use of any one of the screening tests in isolation could infer non-detection of some of the antibody types. in this study, the screening tests were in total agreement, meaning that at least one of the antibody types they target was present, pointing to possible prior exposure or other cross-reactions, but not infection (as this would be confirmed using the rppa). the rppa test showed that all the test samples from humans and dogs were negative for brucellosis caused by the smooth brucella strains. specifically, the canine blood samples were negative for the highly pathogenic canine b. suis. the findings of this study contrasted with those of a similar study that showed a seroprevalence of 7.5% for human brucellosis in humans using the card test alone.3 the difference could be attributable to the fact that they did not use a quantitative confirmatory serological test (rppa) alongside the card test. since a positive brucellosis test would necessitate long-term use of antibiotics,45 an incorrect brucellosis diagnosis (false-positive serological reaction) means that patients are placed on unnecessary antibiotics, this in turn aids the development of antibiotic resistance.41 misdiagnosis of the causes of febrile illness in uganda is a common cause of the misuse of antibiotics.46 limitations the serological tests that were used in this study deployed antigens that could not aid the detection of rough colony brucella spp. such as b. canis and b. ovis. brucella canis is a species specific to dogs. therefore, even though the residual samples were negative for b. abortus and b. suis they may not be negative for b. canis. the flourescent polarisation assay is the recommended serological confirmatory test for brucellosis, but it was not used in this study; instead we used a supplemental confirmatory serological test. in this study we did not confirm infection by carrying out a culture for brucellosis. to the best of our knowledge, the findings of this study can be used as per the scope of the study and in light of its limitations as clearly pointed out. conclusion this study used the traditional brucellosis screening tests (bapa, card and sp) in addition to a quantitative serological confirmatory test (rppa). the additional use of rppa confirmed as negative the positive results obtained using the bapa, sp and card tests. this implies that the improved specificity on using rppa might help improve diagnostic accuracy by ruling out false-positive serological reactions. since this study did not set out to detect b. canis in dogs, but rather canine b. suis, other serological tests specific to b. canis would help to complete the brucellosis picture in dogs. acknowledgements we acknowledge the norman. e. borlaug international fellowship and the national veterinary services laboratory for providing the serological reagents. competing interests the authors declare that there are no financial or personal relationships that may have had an influence in the writing of this article. authors’ contributions j.b.k. participated in the conception of the idea, data analysis, and writing of the manuscript. t.n. and j.t. participated in the processing of the samples, and writing of the manuscript. sources of support funds for this study were provided by the norman. e. borlaug international fellowship in the east african region. data availability statement data is available from the corresponding author upon reasonable request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references godfroid j. brucellosis in livestock and wildlife: zoonotic diseases without pandemic potential in need of innovative one health approaches. arch public health. 2017;75:34. https://doi.org/10.1186/s13690-017-0207-7 nguna j, dione m, apamaku m, et al. seroprevalence of brucellosis and risk factors associated with its seropositivity in cattle, goats and 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https://doi.org/10.1371/journal.pone.0178780 hubbard k, wang m, smith dr. seroprevalence of brucellosis in mississippi shelter dogs. prev vet med. 2018;159:82–86. https://doi.org/10.1016/j.prevetmed.2018.09.002 ducrotoy mj, bardosh kl. how do you get the rose bengal test at the point-of-care to diagnose brucellosis in africa? the importance of a systems approach. acta trop. 2017;165:33–39. https://doi.org/10.1016/j.actatropica.2016.10.004 orduña a, almaraz a, prado a, et al. evaluation of an immunocapture-agglutination test (brucellacapt) for serodiagnosis of human brucellosis. j clin microbiol. 2000;38(11):4000–4005. https://doi.org/10.1128/jcm.38.11.4000-4005.2000 diaz r, casanova a, ariza j, moriyon i. the rose bengal test in human brucellosis: a neglected test for the diagnosis of a neglected disease. plos negl trop dis. 2011;5(4):e950. https://doi.org/10.1371/journal.pntd.0000950 muloki hn, erume j, owiny do, et al. prevalence and risk factors for brucellosis in prolonged fever patients in post-conflict northern uganda. afr health sci. 2018;18(1):22–28. https://doi.org/10.4314/ahs.v18i1.4 tumwine g, matovu e, kabasa jd, et al. human brucellosis: sero-prevalence and associated risk factors in agro-pastoral communities of kiboga district, central uganda. bmc publ health. 2015;15:900. erume j, roesel k, dione mm, et al. serological and molecular investigation for brucellosis in swine in selected districts of uganda. trop anim health prod. 2016;48(6):1147–1155. hall sm, confer aw, tabatabai lb, et al. detection of serum antibody to brucella abortus in cattle by use of a quantitative fluorometric immunoassay. j clin microbiol. 1984;20(6):1023–1027. bejon p, andrews l, hunt-cooke a, et al. thick blood film examination for plasmodium falciparum malaria has reduced sensitivity and underestimates parasite density. malar j. 2006;5:104. dean ag sk, soe mm. openepi: open source epidemiologic statistics for public health, version 3 [homepage on the internet]. n.d. [updated 2013 june 04; cited 2019 aug 15]. available from: www.openepi.com assenga ja, matemba le, muller sk, et al. epidemiology of brucella infection in the human, livestock and wildlife interface in the katavi-rukwa ecosystem, tanzania. bmc vet res. 2015;11:189. acosta-gonzález ri, gonzález-reyes i, flores-gutiérrez gh. prevalence of brucella abortus antibodies in equines of a tropical region of mexico. can j vet res. 2006;70(4):302–304. ducrotoy mj, bertu wj, ocholi ra, et al. brucellosis as an emerging threat in developing economies: lessons from nigeria. plos negl trop dis. 2014;8(7):e3008. njeru j, wareth g, melzer f, et al. systematic review of brucellosis in kenya: disease frequency in humans and animals and risk factors for human infection. bmc publ health. 2016;16(1):853. de glanville wa, conde-álvarez r, moriyón i, et al. poor performance of the rapid test for human brucellosis in health facilities in kenya. plos negl trop dis. 2017;11(4):e0005508. ebani vv, cerri d, fratini f, bey rf, andreani e. serological diagnosis of brucellosis caused by brucella canis. new microbiol. 2003;26(1):65–73. bonfini b, chiarenza g, paci v, et al. cross-reactivity in serological tests for brucellosis: a comparison of immune response of escherichia coli o157:h7 and yersinia enterocolitica o:9 vs brucella spp. vet ital. 2018;54(2):107–114. oie. manual of diagnostic tests and vaccines for terrestrial animals [homepage on the internet]. 2018 [cited 2019 oct 15]. available from: https://www.oie.int/international-standard-setting/terrestrial-manual yuan mj, li sh, huang y, et al. diagnosis and treatment of seven patients with brucellosis in non-pastoral areas. zhonghua nei ke za zhi. 2019;58(8):596–598. batwala v, magnussen p, nuwaha f. antibiotic use among patients with febrile illness in a low malaria endemicity setting in uganda. malar j. 2011;10:377. abstract introduction methods results discussion acknowledgements references about the author(s) lucas ampaire department of medical laboratory sciences, mbarara university of science and technology, mbarara, uganda abraham muhindo department of medical laboratory sciences, mbarara university of science and technology, mbarara, uganda patrick orikiriza epicentre mbarara research centre, mbarara, uganda faculty of medicine, mbarara university of science and technology, mbarara, uganda juliet mwanga-amumpaire epicentre mbarara research centre, mbarara, uganda lisa bebell massachusetts general hospital, boston, massachusetts, united states yap boum epicentre mbarara research centre, mbarara, uganda citation ampaire l, muhindo a, orikiriza p, et al. a review of antimicrobial resistance in east africa. afr j lab med. 2016;5(1), a432. http://dx.doi.org/10.4102/ajlm.v5i1.432 review article a review of antimicrobial resistance in east africa lucas ampaire, abraham muhindo, patrick orikiriza, juliet mwanga-amumpaire, lisa bebell, yap boum received: 06 mar. 2016; accepted: 24 june 2016; published: 15 sept. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background and objectives: knowledge of local and regional antimicrobial resistance (amr) is important for clinical decision making. however, surveillance capacity for amr is lacking throughout east africa, and current amr data are sparse. we sought to address this gap by summarising all available high-quality data on amr in the east africa region. method: we searched the pubmed database and african journals online archives in april and may 2015 using the search term ‘antimicrobial resistance and sub-saharan africa’ to find articles published from 2005 onwards. only full-text articles in english were included. results: we included 12 published articles in our analysis. most articles were on bloodstream infections, hospital-based and cross-sectional in design; a majority described either communityor hospital-acquired infections. high levels of amr to commonly-used antibiotics were reported, including 50% – 100% resistance to ampicillin and cotrimoxazole infections, emerging resistance to gentamicin (20% – 47%) and relatively high levels of resistance to ceftriaxone (46% – 69%) among gram-negative infections. much of the resistance was reported to be in klebsiella species and escherichia coli. among gram-positive infections, extensive resistance was reported to ampicillin (100%), gentamicin and ceftriaxone (50% – 100%), with methicillin-resistant staphylococcus aureus prevalence ranging from 2.6% – 4.0%. conclusion: overall, bacterial resistance was reported among commonly-used antibiotics (ampicillin, gentamicin and ceftriaxone), raising concern that these antibiotics may no longer be useful for treating moderate or severe bacterial infections in east africa. thus, empirical treatment of bacterial infections needs to be reconsidered and guided by local assessment of amr. improvements in the limited amount of quality data and lack of harmonisation in assessing the burden of amr are also needed. introduction without urgent, coordinated action by many stakeholders, the world is headed for a post-antibiotic era, in which common infections and minor injuries which have been treatable for decades can once again kill. (dr keiji fukuda, who assistant director-general for health security) the benefits of appropriate antibiotic use to treat bacterial infections are well established, although all antibiotic use carries a risk of inducing antimicrobial resistance (amr). throughout east africa there is a heavy burden of community-acquired infectious disease.1 unfortunately, the surveillance capacity for amr is minimal in most east african countries, and current data on amr patterns of common pathogenic bacteria are sparse.2 in addition, world health organization (who) surveillance reports indicate that there is inadequate coordination and harmonisation, compromising the ability to assess and monitor the situation.3 as a result, in these resource-constrained settings, the choice of antibiotic is often not based on known bacterial susceptibilities. limited capacity for microbiology testing in east africa coupled with a high burden of life-threatening bacterial infections reinforces a pattern of antibiotic prescription that is largely empirical, where amr is detected only by therapeutic failure. compounding the problem is the small repertoire of antimicrobials available in these settings, which are often of poor quality, when not counterfeit. in addition, in low-resource settings, antibiotics are often sold over the counter with minimal product regulation, oversight or quality control.4 the above, coupled with poor hygiene and infection control practices, may also spread community and/or hospital-acquired drug-resistant pathogens, further exacerbating the problem.5 the who global report3 on amr indicates that resistance of common bacteria has reached alarming levels in many parts of the world. furthermore, the report shows high proportions of resistance to third-generation cephalosporins and carbapenems: up to 54% among escherichia coli and klebsiella pneumoniae. unfortunately, in east africa, few good studies exist documenting the extent of amr. the global antibiotic resistance partnership conducted by the uganda national academy of sciences recently revealed worsening trends of resistance and diminishing effectiveness of antibiotics in uganda.6 some affordable drugs, such as penicillin g and cotrimoxazole, have been reported to have resistance at or near 100%.6 although such reports are concerning, the burden of amr in the east africa region is not well published. additionally, knowledge of the situation in many parts of the world further complicates the problem.3 better knowledge of the burden and proportion of infections caused by drug-resistant bacteria in low-resource settings would raise awareness of the need to prevent the rise and spread of drug resistance. understanding current levels of amr throughout east africa could improve clinical practice by guiding empirical antibiotic choice. toward this end, we reviewed the available evidence on the burden of amr among bacterial pathogens in east africa in order to inform current clinical practice and future research interventions to address antibiotic resistance. methods literature review we searched the pubmed database and african journals online archives in april and may 2015. we used the term ‘antimicrobial resistance and sub-saharan africa’ to find articles published from 2005 onwards. only articles in english were included. study selection criteria full-text articles were included if they reported the proportion of antibiotic resistance among clinical isolates of pathogenic bacteria collected from inpatients and outpatients in any of the following east african countries: uganda, kenya, tanzania, rwanda, ethiopia and democratic republic of congo. eligible studies were required to describe the patient population studied, organisms isolated, specific laboratory methods used for the determination of pathogen antimicrobial sensitivity patterns, and an interpretation of the specific minimum inhibitory concentration breakpoints or the diameter of the zone of inhibition of the antibiotics tested as described by the clinical and laboratory standards institute.7,8 both adult and paediatric patient populations were included, but case reports were excluded from the review as has been done previously.9 for overlapping studies reporting on the same clinical isolates, only the study with the largest sample size was included. in an effort to incorporate contemporary, relevant amr data, only studies published from 2005 onwards were included in the review. data extraction the extracted data included bacterial species isolated, the number of isolates tested for amr, specific antibiotics tested for resistance, and percentage of organisms resistant to each antibiotic. extracted data were grouped on the basis of whether they caused bloodstream infections or other infections. results we initially identified 150 articles: 140 from pubmed and 10 from african journals online. full-text articles were available for 34 papers identified by the search. of the remaining 116 manuscripts, only abstracts were freely available for 105 articles; 11 presented information on anti-tuberculosis drug resistance and lacked information on non-mycobacterial infections, which rendered them ineligible for inclusion in this review. of the 34 full-text articles available, 22 were excluded, because the data presented were from non-east african countries that were inseparable from data presented about east african countries (n = 5) or their laboratory methods were not well defined (n = 17) (figure 1). the remaining 12 articles were included in this review, six describing amr patterns in uganda,10,11,12,13,14,15 five in ethiopia16,17,18,19,20 and one in tanzania21 (table 1). neither studies from rwanda nor studies from the democratic republic of the congo met inclusion criteria for this review. all studies were hospital-based and cross-sectional in design, and the majority described both communityand hospital-acquired infections. four studies presented data on bloodstream infection, seven focused on other infections excluding bloodstream infections and one reported clinical specimens from multiple anatomical sites. figure 1: selection of publications for inclusion in this review. table 1: summary of east africa-based studies included in the analysis. antimicrobial resistance patterns bloodstream infections according to the disk diffusion methods used in studies included in this review, pathogens exhibited relatively high levels of resistance to antibiotics commonly used in east africa. for gram-negative organisms (table 2), 50% – 100% resistance was reported to ampicillin and cotrimoxazole, two of the most frequently-prescribed antibiotics in this region.16 high levels of resistance to ampicillin among children with bloodstream infections (75% – 100%)7,10,16 were also reported, as were lower, but significant, levels of resistance to gentamicin (20% – 47%).7,10,16 among gram-positive organisms isolated from bloodstream infections, overall relatively low levels of resistance were reported to gentamycin, ampicillin and chloramphenicol (4% – 12%).10,11 however, studies reporting specifically on hospital-acquired strains noted high-level resistance to ampicillin, gentamycin, chloramphenicol and trimethoprim-sulfamethoxazole (29%).11,19 table 2: antibiotic resistance patterns among patients with bloodstream infections. non-bloodstream infections surgical site infections among organisms isolated from surgical site infections in hospitalised patients, staphylococcus aureus and coagulase-negative staphylococcus were the most common gram-positive organisms, whereas klebsiella spp., proteus spp. and e. coli were the most common gram-negative organisms10,16,22 (table 3). multiple studies reported 100% resistance to ampicillin for surgical site infections among hospitalised adults.16,22 there was also notable resistance of gram-positive organisms to gentamicin and ceftriaxone in post-operative nosocomial isolates (50% – 100%), with methicillin-resistant staphylococcus aureus (mrsa) prevalence ranging from 2.6% – 4.0%.16 table 3: antibiotic resistance patterns among patients with other, non-bloodstream infections. urinary tract infections high-level resistance to ampicillin (50% – 100%) was seen in urinary tract infections, where e. coli was the most common pathogen.16,18 citrobacter freundii was a major cause of urinary tract infections in one study focused on obstetric fistula patients, with resistance to ampicillin, gentamicin, and ceftriaxone ranging from 46% – 69%.18 other causative organisms of urinary tract infections, such as klebsiella spp., enterobacter spp. and proteus spp., also showed significant resistance to ampicillin, gentamycin and ceftriaxone, some of the most commonly-used antibiotics in east africa.16,18 there was also high-level resistance reported of neisseria gonorrhoeae to ciprofloxacin (81%) among sex workers in uganda.21 gastrointestinal tract infections salmonella spp. in stool isolates from children under five years of age demonstrated complete resistance to ceftriaxone (100%). from the gastrointestinal tract, the commonly isolated bacteria were campylobacter spp. and shigella spp. campylobacter spp. showed moderate amounts of resistance to ampicillin (30%) and high-level resistance to gentamicin (70%). among shigella spp., relatively high numbers were resistant to ampicillin (63%), although resistance to gentamycin was lower at 27% in one ethiopian study.20 multiple body sites one study from uganda on phenotypic clindamycin resistance found that 109 (36%) of s. aureus isolates were resistant to clindamycin, of which 9 (3%) were constitutively resistant and 100 (33.3%) were inducibly resistant. in this study, s. aureus also showed significant resistance to trimethoprim-sulfamethoxazole (62%) and oxacilin (36%), with a demonstrated prevalence of mrsa equal to 36%.12 resistance to vancomycin, one of the last-line drugs for treating mrsa, was also found (7.3%).12 discussion in this review, we summarise the findings of 12 studies that demonstrate significant resistance across the east africa region to antibiotics important for everyday use. overall, although amr varies throughout the region, we found that most gram-negative organisms have limited susceptibility to ampicillin, ceftriaxone and gentamicin, which are commonly-used first-line empirical antibiotics in this region and recommended by the who integrated management of childhood illness for treatment of severely-ill infants. there was also significant resistance to cotrimoxazole, penicillins, quinolones, cephalosporins and aminoglycosides among gram-positive organisms. several published papers we reviewed reported single bacterial isolates resistant to multiple antibiotics. antibiotic resistance to multiple drugs was most common among gram-negative organisms isolated from hospital-acquired infections in post-operative patients and hospitalised adults.16,23 however, the susceptibility of gram-negative organisms to ciprofloxacin was generally reported to be good; thus, this may be the drug of choice for empirical use against post-operative nosocomial infections with gram-negative organisms. the finding of multi-drug resistance in this population suggests that efforts to promote appropriate antibiotic use and microbiological sampling of infected patients should be targeted to these groups in low-resource settings. the evidence presented here indicates that amr, especially to the widely-used antibiotics (ampicillin, tetracyclines and trimethoprim-sulfamethoxazole), is prevalent and common in east africa and may be a growing problem, especially among hospitalised and post-operative patients. however, resistance is likely under-reported in this region as noted by the who global report on amr in 2014,3 due to limited availability of diagnostic testing, microbiology support and limited comparability of laboratory standards. many of the same factors leading to the inability to test clinical isolates for antimicrobial susceptibility contribute to antibiotic overuse and misuse when laboratory data are lacking and can contribute to exacerbation of amr. even when information about amr is available, it may not be properly communicated to those prescribing medications in east africa, due to inadequate national laboratory strategic plans throughout the region. in addition, guidelines regarding appropriate selection of drugs are inadequate.11,18 compounding these problems is a lack of rigorous infection control procedures, all of which could lead to the development and spread of antibiotic resistant bacteria. these factors combine to support the spread of existing amr throughout east africa. preventing the development and spread of antimicrobial resistance amr is likely to become an even greater problem in east africa and may be exacerbated by overuse of antibiotics, the lack of oversight of antibiotic prescription, and the paucity of relevant local data on amr. to address these issues, existing antimicrobial stewardship programmes should be strengthened or, where they are not yet in place, they should be developed and implemented in all regional referral hospitals in response to these challenges. based on our findings, an area of particular focus should be hospitalised patients. existing but limited resources should be directed equally at discovering the causes and at appropriate treatment of infections in this population. additionally, there is a need to urgently scale-up training of both laboratory and pharmacy staff in antibiotic stewardship at health facilities where laboratory investigations are available. this is critical in communicating with clinicians, who are the cornerstone of proper management of patients with infections, especially with regard to the use of antibiotics. there is also need to regularly conduct antibiotic resistance surveys to establish evidence-based and locally-relevant antibiotic resistance information that would be helpful in creating guidelines to improve clinical practice. the implementation of the 2009 who global strategy for containment of amr through inter-continental-wide surveillance programmes as a health systems approach12,23,24,25 has met with a number of challenges. in lowand middle-income countries, implementing the strategy has proven difficult, because human and financial resources and microbiology expertise are insufficient. in addition, it is difficult to obtain appropriate sample sizes for an accurate representation of resistance patterns. novel approaches to antimicrobial surveillance are therefore needed for low-resource settings, which include the development of surveillance programmes utilising smaller sample sizes to provide locally-relevant amr patterns and to encourage appropriate empirical antimicrobial therapy.26 moreover, the development of new point-of-care diagnostic tools able to detect amr in a cost-effective way will improve patient management and limit the emergence of drug resistance. lastly, from among the total of 150 publications we identified, we considered only 12 due to the lack of standardisation and quality of the methodology and reporting. this highlights the scarcity of good quality data that could allow stakeholders to assess the real burden of amr. thus, better standardised research protocols are needed to evaluate the emergence of amr in different settings to obtain comparable results and implement tailor-made interventions. conclusion based on the findings in this review, resistance to commonly-used antibiotics is prevalent in east africa. multi-drug resistance has been noted as a rising threat in the region and threatens to further complicate the drug resistance burden, especially for non-bloodstream infections where a single isolate may be resistant to more than one antibiotic drug of choice in different or similar drug lines. data from interventional studies designed to reduce amr are particularly lacking in the east african context, where infectious disease prevalence is high. the profound lack of data on hospital-acquired infections and prevalence of amr in low-income east african countries calls for vigorous investigation and surveillance to better define the problem. there is a need for countries to promote acceptance of antimicrobial stewardship as a programmatic strategy, including pharmacy management, laboratory quality control, complete microbiology investigations and creation and dissemination of regional standard antibiograms. acknowledgements we are grateful to dan nyehangane and associate professor apecu o. richard for their critical review of the manuscript and the uganda research student support fund for their assistance. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions this work was carried out in collaboration between all authors. y.b. was the 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2005;43(2):745–749. http://dx.doi.org/10.1128/jcm.43.2.745-749.2005 nambatya jl, nyairo s, bironse m, et al. antibiotic use knowledge and behavior at a ugandan university. int j infect control. 2011;7(4):7 pages. duse ag. the global antibiotic resistance partnership (garp). s afr med j. 2011;101(8 pt 2):551. kimang’a an. a situational analysis of antimicrobial drug resistance in africa: are we losing the battle? ethiop j health sci. 2012;22(2):135–143. world health organization. country pharmaceutical situations. who press: geneva, switzerland; 2009. world health organization. who global strategy for containment of antibiotic resistance. who press: geneva, switzerland; 2001. abstract the need for quality assurance current point-of-care technologies for cd4, early infant diagnosis and viral load testing understanding quality assurance current external quality assessment schemes external quality assessment for point-of-care cd4, early infant diagnosis and viral load testing external quality assessment technology regional external quality assessment coordination centres benefits of external quality assessment programmes looking forward: sustainable, country-owned external quality assessment programmes in africa acknowledgements references about the author(s) adrienne f.a. meyers qasi, national hiv & retrovirology laboratories, public health agency of canada, jc wilt infectious diseases research centre, winnipeg, manitoba, canada department of medical microbiology and infectious diseases, university of manitoba, winnipeg, manitoba, canada department of medical microbiology, university of nairobi, nairobi, kenya paul sandstrom qasi, national hiv & retrovirology laboratories, public health agency of canada, jc wilt infectious diseases research centre, winnipeg, manitoba, canada department of medical microbiology and infectious diseases, university of manitoba, winnipeg, manitoba, canada thomas n. denny duke university, department of medicine, durham, north carolina, united states mackenzie hurlston centers for disease control and prevention, atlanta, georgia, united states terry b. ball qasi, national hiv & retrovirology laboratories, public health agency of canada, jc wilt infectious diseases research centre, winnipeg, manitoba, canada department of medical microbiology and infectious diseases, university of manitoba, winnipeg, manitoba, canada department of medical microbiology, university of nairobi, nairobi, kenya department of immunology, university of manitoba, winnipeg, manitoba, canada rosanna w. peeling london school of hygiene & tropical medicine, london, united kingdom debrah i. boeras london school of hygiene & tropical medicine, london, united kingdom citation meyers afa, sandstrom p, denny tn, et al. quality assurance for hiv point-of-care testing and treatment monitoring assays. afr j lab med. 2016;5(2), a557. http://dx.doi.org/10.4102/ajlm.v5i2.557 lessons from the field quality assurance for hiv point-of-care testing and treatment monitoring assays adrienne f.a. meyers, paul sandstrom, thomas n. denny, mackenzie hurlston, terry b. ball, rosanna w. peeling, debrah i. boeras received: 31 aug. 2016; accepted: 31 aug. 2016; published: 17 oct. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract in 2015, unaids launched the 90-90-90 targets aimed at increasing the number of people infected with hiv to become aware of their status, access antiretroviral therapies and ultimately be virally suppressed. to achieve these goals, countries may need to scale up point-of-care (poc) testing in addition to strengthening central laboratory services. while decentralising testing increases patient access to diagnostics, it presents many challenges with regard to training and assuring the quality of tests and testing. to ensure synergies, the london school of hygiene & tropical medicine held a series of consultations with countries with an interest in quality assurance and their implementing partners, and agreed on an external quality assessment (eqa) programme to ensure reliable results so that the results lead to the best possible care for hiv patients. as a result of the consultations, eqa international was established, bringing together eqa providers and implementers to develop a strategic plan for countries to establish national poc eqa programmes and to estimate the cost of setting up and maintaining the programme. with the dramatic increase in the number of proficiency testing panels required for thousands of poc testing sites across africa, it is important to facilitate technology transfer from global eqa providers to a network of regional eqa centres in africa for regional proficiency testing panel production. eqa international will continue to identify robust and cost-effective eqa technologies for quality poc testing, integrating novel technologies to support sustainable country-owned eqa programmes in africa. the need for quality assurance it is estimated that 36.9 million people globally are living with hiv, with more than two-thirds of these individuals located in resource-limited regions.1 the ambitious unaids 90-90-90 targets, aimed to end the hiv epidemic within the next 15 years, place diagnostics in a key role for achieving these goals: by 2020, 90% of all people living with hiv will know their hiv status; 90% of all hiv-positive people will receive sustained antiretroviral therapy; 90% of all people receiving antiretroviral therapy will achieve viral suppression.2 recent estimates suggest that 15.8 million people are receiving antiretroviral therapy. with the new world health organization guidelines recommending test and treat, it is expected that 30 million people living with hiv will have access to treatment by 2020.3 the achievement of the 90-90-90 targets depends on the availability of tests for hiv case detection, including early infant diagnosis (eid) and viral load for monitoring viral suppression. cd4 enumeration is still used for treatment initiation in many countries as they move toward the world health organization guidelines of test and treat, and plan to scale up viral load testing. however, access to these tests is reduced in resource-limited settings. recent efforts aimed at improving access to these assays have seen a large increase in novel technologies that can be performed at the point of care (poc). the rollout of rapid poc tests for hiv testing and treatment initiation has significantly reduced the time for patients to receive results and initiate therapy.4,5 as testing is decentralised to hundreds of sites outside of the laboratory, and being performed by non-laboratory staff, ensuring test quality and accuracy of results have become key considerations in hiv programmes.6 current point-of-care technologies for cd4, early infant diagnosis and viral load testing to date, there are two approved (world health organization-prequalified) poc platforms for cd4 and two for eid, whereas poc viral load platforms are in the evaluation pipeline.7 most of these poc technologies provide sample-in/answer-out diagnostic testing, making it possible for these tests to be performed with minimal training, outside of the traditional laboratory setting. most of the devices can withstand extreme transport and environmental conditions, provide a test result in less than one hour and have data transmission capabilities. some (and more are in development) can even operate on battery power.8 the rollout of cd4 poc devices in the last few years has provided us with lessons learnt in the implementation and quality management for poc testing. eid and viral load poc testing has not yet been fully implemented in the field, but will benefit from lessons learned from the cd4 experience to strengthen existing quality programmes and ensure integration of poc testing within these programmes. understanding quality assurance quality management is the entire process supporting the quality of testing, while quality assurance is the planned activities implemented to support the quality management system (figure 1).9,10 while it is highly recommended that a poc quality assurance plan address the entire quality assurance management cycle, in this report we focus on external quality assurance as a quality assurance activity relevant to poc testing, and the results serve as a strong indicator of laboratory quality.11 figure 1: world health organization/us centers for disease control and prevention quality assurance cycle to improve the quality of hiv-related point-of-care testing. poc devices have in-built internal quality controls to monitor in real time the performance of an instrument and, to some extent, the user. if any of the controls fail, the device should not provide a test result. in contrast, external quality assessment (eqa), often used interchangeably with proficiency testing, challenges the testing environment and allows for an external expert body to examine the processes and provide remedial action.9,12,13 the three components of an effective eqa laboratory programme are site supervision, retesting of specimens, and proficiency testing.14 supervision is integral to quality assurance and should be done routinely, but with minimal disruption to patient services. retesting is rarely done due to costs and time constraints, and is especially challenging in the poc environment. proficiency testing is the most widely-used approach to monitor the performance of the test used and the quality of testing. it involves blinded control material, meant to mimic patient specimens, sent from a reference laboratory to the testing site and results sent back for scoring. participation in eqa programmes is often mandated for clinical laboratories performing regular testing of patient samples for hiv care and management.15,16 a low proficiency testing score should alert the laboratory or testing site that there is a problem. often, identifying the exact nature of the problem, and then correcting the problem, is difficult, even for skilled technicians. the error can be either an instrument error or an operator error. investigation and remedial action should be taken immediately to correct the problem before patients’ results are adversely affected. current external quality assessment schemes a comprehensive external quality assurance programme consists of the following: proficiency testing panel design and production, eqa scheme/survey, results and reporting, customer service/technical support, and corrective action (table 1). table 1: considerations for external quality assessment programmes. an eqa survey or cycle consists of a panel of specimens intended to challenge or assess the testing event. eqa should, as far as possible, cover the entire range of tests, and the entire examination process, from sample reception, preparation and analysis to interpretation and reporting.17 the goal is to allow for regularity in testing that will highlight any changes or challenges a participant may encounter without having a long period of inaccurate testing going undetected. the reporting of results, as well as immediate corrective action to improve programmes, is the most critical component to any eqa programme. reporting results is a major challenge in resource-limited settings due to various reasons that result in wasted resources and systematic problems going undetected. the number of specimens or members in each panel and frequency of eqa often varies by type of test and the eqa provider. programmes often struggle to find an overall balance between ‘necessary’ and ‘sufficient’ in order to minimise costs and maximise resources. external quality assessment for point-of-care cd4, early infant diagnosis and viral load testing as new technologies are introduced, eqa needs to evolve to accommodate new testing strategies.18 in an effort to address the topic of quality of testing at hundreds, perhaps thousands of sites in a country, this section will consider what is necessary to ensure quality poc cd4, eid, and viral load testing. projected demands for point-of-care testing with ‘test and treat’ and the scale up of viral load testing for monitoring patients, cd4 volumes are projected to remain the same in the short term and eventually decline as programmes transition to viral load testing. while poc testing for eid and viral load is currently not offered in some countries, planning for the rollout of these tests is ongoing and presents a critical time to develop strategies for quality assurance of these tests. to ensure synergies, the london school of hygiene & tropical medicine established eqa international, bringing together eqa providers and implementers to develop a strategic plan for working with countries in africa to establish national poc eqa programmes and estimate the cost of setting up and maintaining the programme. members of eqa international include the public health agency of canada qasi programme, the us centers for disease control and prevention, duke university external quality assurance program oversight laboratory and immunology quality assessment, south africa national health laboratory service, zimbabwe national quality assurance programme and foundation mérieux. eqa international is working closely with funders and implementing partners to remain one step ahead of the phased rollout of poc testing for eid and viral load in africa. how to deliver on the demands of quality-assured point-of-care testing assuming hundreds and possibly thousands of devices will be placed in africa over the next five years, eqa international, in consultation with national hiv programme and laboratory managers from countries in africa with an interest in participating in eqa programmes for poc testing, agreed on a work plan for the standardisation, production and distribution of proficiency testing panels and reporting mechanisms, to cover cd4, eid and viral load poc testing in africa (table 2). table 2: proposed external quality assessment scheme for cd4, early infant diagnosis and viral load point-of-care testing. external quality assessment technology ideally, eqa proficiency testing specimens can be used for both poc and laboratory testing. while the use of fresh whole blood samples most closely resembles clinical samples tested by a participant, if an eqa programme has several hundred participants or a participant base located over a wide geographical area, it may not be feasible to access enough material and distribute to participants within the time-frame that the material is stable and effective for proper evaluation. commercially-stabilised whole blood products, while financially more challenging, are homogeneous and stable for weeks to months, which is beneficial for large eqa programmes. novel technologies are likely to be needed, given the volumes and potential logistical challenges, such as maintenance of sample integrity during transport. several innovative cost-effective applications are being explored to provide stabilised eqa specimens for cd4, eid and viral load testing. a major challenge with cd4 has been the necessity for whole blood and therefore stabilisers are required as a means of extending pre-analytic capacity for cd4 t-cell enumeration.19 considering poc sites, an ideal candidate would allow the eqa cd4 and eid specimens to remain stable for up to three weeks at ambient temperatures in-country. another innovative technology, the dried tube specimen (dts) has been successfully used for eqa for hiv and syphilis rapid diagnostic tests and for automated viral load laboratory tests.20,21,22,23,24 dts can likely be expanded for eqa of viral load poc tests as well as other tests. a study specifically looking at the relative impact of costs of implementing dts eqa with the rollout of a rapid syphilis testing programme found the incremental costs of eqa relatively small and further cost reductions were possible through programme redesign.25 eqa sample considerations for poc cd4, viral load and eid programmes are summarised in table 3. eqa international is working to identify robust and cost-effective eqa technologies for quality poc testing and integrating novel technologies to support sustainable eqa programmes. table 3: external quality assessment sample considerations. regional external quality assessment coordination centres currently, the majority of proficiency testing panels are procured from commercial eqa providers outside africa. shipment of proficiency testing panels to individual laboratories is costly and logistical challenges are frequently encountered. a regional approach would allow for distribution of a well-characterised panel of sufficient sample size to be used to compare performances across the region. the national health laboratory service in south africa is currently providing eqa for eid to more than 10 neighbouring countries. the afriqualab in senegal supports eqa for cd4 and eid to more than 40 countries. the zimbabwe national quality assurance program is establishing a regional eqa centre to support cd4, eid and viral load testing. finally, the african society for laboratory medicine has recently established a network of six collaborating centres across africa to strengthen laboratory medicine; these collaborating centres could also function as regional eqa centres. a network of regional eqa coordinating centres can be set up to produce or procure proficiency testing materials from global providers, and coordinate distributions (figure 2). these centres could also oversee the logistics and management of the eqa programme, providing timely corrective action and strengthening national quality assurance programmes and country capacity. through technology transfer, these centres could produce panels at minimal cost, using locally-sourced material. figure 2: global external quality assessment coverage through regional external quality assessment centres. regional eqa coordinating centres can also support other programmes and services. the world health organization regional office for africa has linked eqa programmes for their laboratory networks for influenza, malaria, meningitis, enterics and other diseases.26 the east african regional external quality assessment scheme supports eqa in five countries to improve the quality of essential diagnostic services in clinical laboratories at peripheral level. future capacities of regional eqa centres could serve as biobanking facilities that support test performance evaluations and validations, post-market surveillance, global health surveillance, and facilitate new test development. benefits of external quality assessment programmes there is strong evidence to indicate that participation in eqa programmes significantly improves testing performance.23,26,27 the us centers for disease control and prevention has developed eqa programmes to help participating laboratories monitor the quality of laboratory-based viral load and eid testing in lowand middle-income countries. in an analysis of the reported eqa data from more than 114 participating laboratories in 44 countries in the viral load programme, and 136 participating laboratories in 41 countries in the eid programme, they found that with increased participation came increased performance.23,27 the public health agency canada (qasi)-quality management system, designed to work globally, optimised its programme strategies in 2009 and immediately saw reduced error rates with their cd4 programme as well as significant reduction in inter-laboratory variability resulting from continuous participation in the qasi-quality management system.28 qasi provides detailed, extensive technology transfer workshops to programme participants using a ‘train-the-trainer’ format, which significantly improves participation and performance. with cd4, improvements in performance of programmes can be directly linked to improvements in patient care.4,11 apart from insights into the participants’ performance levels, eqa can also survey the overall national laboratory infrastructure and trace problems in processes. examples of this include the sample transport network and supply chain management system. these two systems often go hand-in-hand and contribute to reagents/kit stock-outs and poor quality of specimens. a comprehensive, coordinated eqa programme can identify these gaps and bring them to the attention of the country’s ministry of health and partners for immediate corrective action, strengthening the overall infrastructure of the country and thereby improving programmes and services. in addition, eqa programmes can also capture key indicators and document programme improvements over time. eqa is an important tool for providing the educational stimulus that will improve overall performance and standards.29 other quality assurance activities, such as training and site evaluations, can support a quality-assured environment.10,30 eqa also builds educational stimulus and promotes research capacity through technology transfer and trainings. the institute of public health in brussels, belgium looked at eqa for malaria rapid diagnostic tests and found that eqa prompted educational incentives that boosted self-confidence and credibility.31 eqa can also provide valuable information to programmes and manufacturers though post-market surveillance.32 post-market surveillance ensures that diagnostics continue to meet quality, safety and performance requirements. often failures go unreported, but though a proactive eqa programme, these failures can be captured and reported appropriately. participation in an external quality assessment program provides many benefits and valuable data to health authorities (figure 3):30,33 comparison of performance and results among different test sites. collection of data across different sites. early warning for systematic problems. objective evidence of testing quality. indicates areas that need improvement. survey the use of diagnostic algorithms. potential to build health security through surveillance and monitoring. figure 3: benefits of an external quality assurance programme to point-of-care sites, country programmes, and point-of-care industry. a key feature of many of these poc molecular devices is that they are equipped with data transmission capacities. apart from monitoring the quality of tests and testing to optimise supply chain management, data connectivity links data from diagnostic laboratories and poc test readers and devices to provide data on testing coverage, disease trends and timely information for early warning of infectious disease outbreaks.34 looking forward: sustainable, country-owned external quality assessment programmes in africa sustainability of eqa programmes requires country ownership. as countries in africa plan the scale-up of poc technologies for cd4, viral load and eid testing, political commitment toward quality should be reflected in country policies and implementation plans. putting in place eqa programmes that can identify gaps and challenges in the testing environment and address them immediately to increase the efficiency of healthcare systems and improve patient results, are urgent priorities. the role of implementing partners and industry in support of eqa and quality assurance programmes is also a strong consideration for the sustainability of quality-assured testing. partners and industry can bring numerous resources to these programmes. as an example, when zimbabwe was establishing their national quality assurance programme, the ministry of health brought together stakeholders as well as quality assurance and poc partners to share their vision for a coordinated national programme that would identify redundancies and gaps and leverage partner resources and commitment. this process secures a sustainable country-owned quality assurance national policy. acknowledgements cdc disclaimer: the findings and conclusions in this article are those of the authors and do not necessarily represent the official position of the u.s. centers for disease control and prevention. open review: this article underwent open peer review by ben cheng and david mabey. the editors and authors are grateful for their comments and suggestions. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support t.n. denny was supported by niaid daids iqa, contract no. hhsn272201400014c; niaid daids eqapol, contract no. hhsn272201000045c. authors’ contributions r.w.p and d.i.b drafted the manuscript. a.f.a.m., p.s., t.n.d., m.h., and t.b.b. reviewed the manuscript for important intellectual 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http://dx.doi.org/10.1186/1475-2875-9-359 world health organization. post-market surveillance of in vitro diagnostics [document on the internet]. c2015 [cited 2015]. available from: http://www.who.int/diagnostics_laboratory/postmarket/150819_pms_guidance_final_version.pdf?ua=1 kettelhut mm, chiodini pl, edwards h, et al. external quality assessment schemes raise standards: evidence from the ukneqas parasitology subschemes. j clin pathol. 2003;56(12):927–932. peeling rw. diagnostics in a digital age: an opportunity to strengthen health systems and improve health outcomes. int health. 2015;7(6):384–389. http://dx.doi.org/10.1093/inthealth/ihv062 abstract introduction methods results discussion acknowledgements references about the author(s) shanthie govender school of clinical medicine research laboratory, nelson mandela school of medicine, university of kwazulu-natal, durban, south africa lungile mbambo school of clinical medicine research laboratory, nelson mandela school of medicine, university of kwazulu-natal, durban, south africa makandwe nyirenda south african medical research council, hiv prevention research, durban, south africa motshedisi sebitloane department of obstetrics and gynecology, school of clinical medicine research laboratory, nelson mandela school of medicine, university of kwazulu-natal, durban, south africa nathlee abbai school of clinical medicine research laboratory, nelson mandela school of medicine, university of kwazulu-natal, durban, south africa citation govender s, mbambo l, nyirenda m, sebitloane m, abbai n. herpes simplex virus-2 infections in pregnant women from south africa: evaluation of the immunoflow rapid test. afr j lab med. 2020;9(1), a854. https://doi.org/10.4102/ajlm.v9i1.854 brief report herpes simplex virus-2 infections in pregnant women from south africa: evaluation of the immunoflow rapid test shanthie govender, lungile mbambo, makandwe nyirenda, motshedisi sebitloane, nathlee abbai received: 27 june 2018; accepted: 17 apr. 2020; published: 31 aug. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract the diagnostic performance of immunoflow, a rapid test for detecting herpes simplex virus type-2 (hsv-2) infections, was investigated in 248 antenatal women. approximately one hundred and seventy-seven (71%) of the enrolled women were infected with hsv-2. sero-positivity was associated with older age ([≥ 30 years] 104/177, 58%), having a secondary level of education but not tertiary level of education (125/177, 70.6%), and being unmarried (150/177, 84.7%). the sensitivity of the immunoflow test in relation to the herpeselect hsv-2 enzyme-linked immunosorbent assay was 89.7% and specificity was 96.2%. the immunoflow therefore can serve as a valuable test in screening for hsv-2 infections in pregnant women. keywords: hsv-2 infection; pregnant women; rapid test; south africa. introduction the south african hiv/aids and sexually transmitted infections (sti) strategic plan deems the prevention and early treatment of stis to be a major health priority for the country; emphasis has been placed on the delivery of quality services for testing and treating of stis.1 simple, rapid and affordable point-of-care tests for diagnosing stis may be advantageous, because patients can be tested and treatment commenced in a single visit.2 cassetteand strip-based point-of-care tests are highly applicable for use in resource-poor settings, because they do not require electricity, a laboratory or highly trained staff to perform the testing.3 additionally, such tests could have tremendous potential for use in community clinics in an sti-endemic country such as south africa. in highly endemic herpes simplex virus type-2 (hsv-2) regions such as south africa,4,5 routine screening for hsv-2 infections would pose a huge financial burden on the health system, because the only available diagnostic tests for hsv-2 are enzyme-linked immunosorbent assays (elisa), which are expensive and time consuming. due to the high turn-around time to receive test results, many infected individuals may be lost to follow up. in addition, elisa requires the use of specialised equipment and highly trained laboratory staff. to address these limitations, rapid point-of-care tests that: are easy to operate, provide results on the same day and are relatively inexpensive would result in a larger number of testing and subsequent treatment infected individuals. the immunoflow hsv test (genbio, san diego, california, united states) is a point-of-care cassette test that detects igg to hsv gg-2 (specific hsv type 2 and total hsv [type 1 + type 2]) for type-specific classification, which is not possible using whole virus lysate, and also provides epidemiological information on these diseases. currently, the diagnostic performance of this test has not been evaluated in a south african setting. additionally, there is a lack of published research studies on this point-of-care test, both locally and globally. this study compared the results of the immunoflow with the herpeselect hsv-2 elisa (focus diagnostics, cypress, california, united states). we used the herpeselect hsv-2 elisa from focus diagnostics as our reference test, because this test has been approved by the manufacturer and has a certification mark approval (gmbh, hannover, germany) for detection of hsv antibodies in pregnant women globally. methods ethical considerations the study and all study related materials were approved by the biomedical research ethics committee, university of kwazulu-natal (be392/17). study setting and population the study was conducted between april 2017 and august 2017 at the king edward viii hospital antenatal clinic in durban, kwazulu-natal, south africa. two hundred and forty-eight women participated in this study. during screening, an estimated 20% of the women approached refused study participation. the study criteria included: being pregnant and aged 18 years or older, willing to give written informed consent, willing to undergo a blood draw, and willing to allow the study team to document their hiv status from their clinic cards. data and specimen collection for this study, data on the women’s demographics and clinical information were recorded on a case report form. women who had symptoms of genital ulcers and sores were treated by syndromic management. for the syndromic management approach, patients presenting with a genital ulcer or sore were treated with aciclovir, oral, 400 mg 8-hourly for 7 days. venous blood (3.5 ml) was collected by a hospital professional nurse. the blood was collected into a serum separator gel tube. the blood was processed and tested at university of kwazulu-natal, school of clinical medicine research laboratory. immunoflow test the samples were tested according to the manufacturer’s instructions. test cassettes were placed on a dry level surface, and 100 µl of wash solution was added to the cassette. the patient samples were diluted in sample diluent and thereafter 200 µl of the dilution was added to the cassette. a second wash step was conducted before the addition of 100 µl of color g to the cassette. a final wash step was performed and the results were available within 2 min. each cassette included a reagent-positive control. the presence of a red or pink dot in the individual test and control windows was read as a positive result. herpeselect hsv-2 enzyme-linked immunosorbent assay the herpeselect hsv-2 elisa assay is a glycoprotein g-based type-specific elisa technique which produces qualitative results. two hundred ul of each serum sample was tested according to the manufacturer’s recommendations (focus diagnostics, cypress california, united states). controls that were provided with kits were included for all runs: igg high positive control (index value greater than 3.5); igg low positive control (index value between 1.5–3.5); and negative control (index value less than 0.8). the cut-off value used to determine a positive result was > 1.10 index value; 0.9–1.10 index values were considered equivocal and index values < 0.9 were considered as negative results. all samples that produced equivocal index values were re-tested. data analysis all analyses were performed using stata, version 14 (statacorp llc, college station, texas, united states). the diagnostic performance (i.e. sensitivity, specificity, positive predictive value, and negative predictive value) of the immunoflow test was compared to the gold standard herpeselect hsv-2 elisa. a p-value of < 0.05 was considered as significant. results a prevalence of 177/248 (71.4%) for hsv-2 was observed on the immunoflow test and prevalence was 195/248 (78.6%) on the herpeselect hsv-2 elisa. the prevalence of hiv in this population was 124/248 (50.0%). approximately (107/177) of the women were positive for hsv-2 and were also hiv-positive. the majority of the women who tested positive for hsv-2 were older than age 30 years (104/177, 58%, p = 0.001), had completed secondary education but not tertiary education (125/177, 70.6%, p = 0.05), were unemployed (107/177, 60.5%, p = 0.55), were unmarried (150/177, 84.7%, p = 0.02) and reported having more than one lifetime sexual partners (164/177, 92.6%, p = 0.0002) (table 1). table 1: description of pregnant women recruited from the antenatal clinic of the king edward viii hospital in durban, kwazulu-natal, south africa, 2017. the sensitivity of the immunoflow test was 89.7% and its specificity was 96.2%. of the 248 samples tested, 175 samples were correctly classified as positive by the immunoflow test. however, there were 2 samples that the immunoflow test classified as positive whereas the reference test classified these as negative. in addition, 20 samples were falsely classified as negative by the immunoflow (table 2). the positive predictive value of the immunoflow was 98.9% and its negative predictive value was 71.8%. the overall predicitive accuracy of the immunoflow test was 91.1% (95% confidence interval: 86.9% – 94.4%) (table 3). table 2: immunoflow rapid test results compared to herpeselect 2 enzyme-linked immunosorbent assay results in antenatal women, kwazulu-natal, south africa, 2017. table 3: performance of the immunoflow rapid test compared to the herpeselect 2 enzyme-linked immunosorbent assay in antenatal women, kwazulu-natal, south africa, 2017. discussion the 78.6% prevalence of hsv-2 reported in this study was found to be higher than other published studies on antenatal women.6,7,8,9,10 a high prevalence of hsv-2 was observed in women who had more partners, thereby emphasising the association between increased number of sex partners and risk of contracting stis. the performance of the immunoflow was comparable to other published reports on hsv-2 rapid tests, which reported sensitivities and specificities > 90%.11,12,13,14,15 other published studies on hsv-2 rapid tests have not reported on the performance of those tests in the presence of other viral infections, such as hiv, or genital symptoms relating to infection. in this study, the immunoflow rapid test performance was not shown to be negatively affected by hiv infection. the test yielded a sensitivity of 91.5% and specificity of 100% among hiv-positive women. in addition, the test was able to detect infection in women who were presenting with symptoms of genital ulcers or sores. among women who were symptomatic, the sensitivity of the test was 91% and its specificity was 97%. this study highlights that having a test such as the immunoflow test could greatly contribute to early detection and treatment of women with herpes simplex viruses for improved outcomes for pregnant woman and their babies. limitations the limitations of the study were as follows: western blotting could not be performed as a second confirmatory test due to the high cost of the tests. the testing was performed at a research laboratory by medical technicians and is not a reflection of how the test would be performed at a clinic. medical technicians are more experienced at laboratory procedures and quality checks. if the test had been conducted by a clinic nurse who has no laboratory experience, the results may have been different. this is yet to be confirmed. however, conducting evaluation studies at antenatal clinics will be a future research consideration. the test required the use of serum. with slight modifications, such as using blood collected by finger-prick instead of serum, this test could serve as a valuable test in screening for hsv-2 infections. however, this needs to be evaluated. conclusion overall, we have shown that the immunoflow rapid test performed well in relation to the elisa. rapid laboratory tests for diagnosis of stis will directly contribute to united nations sustainable development goals that focus on improvement in health.16 acknowledgements we gratefully acknowledge the contribution of the women who participated in this study. competing interests the authors declared no potential conflicts of interest with respect to the research, authorship or publication of this article. authors’ contributions n.a. designed and funded the study. m.n. performed all the statistical analysis. s.g. and l.m. performed all the laboratory testing and analysis. m.s. provided clinical assistance. all authors contributed to the writing of the final manuscript. sources of support we thank the national research foundation of south africa for funding this study. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references lewis da, maruma e. revision of the national guideline for first-line comprehensive management and control of sexually transmitted infections: what’s new and why? south afr j epidemiol infect. 2009;24(2):6–9. https://doi.org/10.1080/10158782.2009.11441341 hsieh y-h, hogan mt, barnes m, et al. perceptions of an ideal point-of-care test for sexually transmitted infections–a qualitative study of focus group discussions with medical providers. plos one. 2010;5(11):e14144. https://doi.org/10.1371/journal.pone.0014144 tucker jd, bien ch, peeling rw. point-of-care testing for sexually transmitted infections: recent advances and implications for disease control. curr opin infect dis. 2013;26(1):73. https://doi.org/10.1097/qco.0b013e32835c21b0 abbai ns, wand h, ramjee g. socio-demographic and behavioural characteristics associated with hsv-2 sero-prevalence in high risk women in kwazulu-natal. bmc rese note. 2015;8(1):1. https://doi.org/10.1186/s13104-015-1093-0 kenyon c, colebunders r, buve a, hens n. partner-concurrency associated with herpes simplex virus 2 infection in young south africans. int j std aids. 2013;24(10):804–812. https://doi.org/10.1177/0956462413482810 lima l, padalecki g, castro c, cordeiro j, de paula v. seroprevalence of human herpesvirus type 2 in a reference center for pregnant women in rio de janeiro, brazil. vrr. 2017;22:20–21. https://doi.org/10.17525/vrrjournal.v22i1.327 domercant jw, louis fj, hulland e, et al. seroprevalence of herpes simplex virus type-2 (hsv-2) among pregnant women who participated in a national hiv surveillance activity in haiti. bmc infect dis. 2017;17(1):577. https://doi.org/10.1186/s12879-017-2674-4 anjulo aa, abebe t, hailemichael f, mihret a. seroprevalence and risk factors of herpes simplex virus-2 among pregnant women attending antenatal care at health facilities in wolaita zone, ethiopia. virol j. 2016;13(1):43. https://doi.org/10.1186/s12985-016-0501-y nakubulwa s, kaye dk, bwanga f, tumwesigye nm, nakku-joloba e, mirembe fm. incidence and risk factors for herpes simplex virus type 2 seroconversion among pregnant women in uganda: a prospective study. j infect dev countr. 2016;10(10):1108–1115. https://doi.org/10.3855/jidc.6874 perti t, nyati m, gray g, et al. frequent genital hsv-2 shedding among women during labor in soweto, south africa. infect dis obstet gynecol. 2014;2014:article id 258291:8 pages. https://doi.org/10.1155/2014/258291 ashley rl, eagleton m, pfeiffer n. ability of a rapid serology test to detect seroconversion to herpes simplex virus type 2 glycoprotein g soon after infection. jcm. 1999;37(5):1632–1633. https://doi.org/10.1128/jcm.37.5.1632-1633.1999 wald a, ashley-morrow r. serological testing for herpes simplex virus (hsv)–1 and hsv-2 infection. clin infect dis. 2002;35(2):s173–s182. https://doi.org/10.1086/342104 philip ss, ahrens k, shayevich c, et al. evaluation of a new point-of-care serologic assay for herpes simplex virus type 2 infection. clin infect dis. 2008;47(10):e79–e82. https://doi.org/10.1086/592696 laderman ei, whitworth e, dumaual e, et al. rapid, sensitive, and specific lateral-flow immunochromatographic point-of-care device for detection of herpes simplex virus type 2-specific immunoglobulin g antibodies in serum and whole blood. cvi. 2008;15(1):159–163. https://doi.org/10.1128/cvi.00218-07 shevlin e, morrow ra. comparative performance of the uni-gold™ hsv-2 rapid: a point-of-care hsv-2 diagnostic test in unselected sera from a reference laboratory. j clin virol. 2014;61(3):378–381. https://doi.org/10.1016/j.jcv.2014.08.012 united nations general assembly. sustainable devlopment goals [homepage on the internet]. 2015 [cited 2018 jun 27]. available from: https://www.un.org/sustainabledevelopment/sustainable-development-goals/ abstract introduction methods results discussion conclusion acknowledgements references about the author(s) sarah j. girdwood health economics and epidemiology research office, department of internal medicine, school of clinical medicine, faculty of health sciences, university of the witwatersrand, johannesburg, south africa department of medical microbiology, amsterdam university medical center, amsterdam, the netherlands thomas crompton right to care, johannesburg, south africa naseem cassim department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa national health laboratory service, johannesburg, south africa floyd olsen national health laboratory service, johannesburg, south africa portia sejake national health laboratory service, johannesburg, south africa karidia diallo centers for disease control and prevention, pretoria, south africa leigh berrie centers for disease control and prevention, pretoria, south africa dorman chimhamhiwa right to care, johannesburg, south africa wendy stevens department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa national health laboratory service, johannesburg, south africa brooke nichols health economics and epidemiology research office, department of internal medicine, school of clinical medicine, faculty of health sciences, university of the witwatersrand, johannesburg, south africa department of medical microbiology, amsterdam university medical center, amsterdam, the netherlands department of global health and development, school of public health, boston university, boston, massachusetts, united states citation girdwood sj, crompton t, cassim n, et al. optimising courier specimen collection time improves patient access to hiv viral load testing in south africa. afr j lab med. 2022;11(1), a1725. https://doi.org/10.4102/ajlm.v11i1.1725 note: additional supporting information may be found in the online version of this article as online supplementary document 1 original research optimising courier specimen collection time improves patient access to hiv viral load testing in south africa sarah j. girdwood, thomas crompton, naseem cassim, floyd olsen, portia sejake, karidia diallo, leigh berrie, dorman chimhamhiwa, wendy stevens, brooke nichols received: 02 sept. 2021; accepted: 24 may 2022; published: 25 oct. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: south africa uses a courier network for transporting specimens to public laboratories. after the daily collection of specimens from the facility by the courier, patients not yet attended to are unlikely to receive same-day blood draws, potentially inhibiting access to viral load (vl) testing for hiv patients. objective: we aimed to design an optimised courier network and assess whether this improves vl testing access. methods: we optimised the specimen transport network in south africa for 4046 facilities (november 2019). for facilities with current specimen transport times (n = 356), we assessed the relationship between specimen transport time and vl testing access (number of annual vl tests per antiretroviral treatment patient) using regression analysis. we compared our optimised transport times with courier collection times to determine the change in access to same-day blood draws. results: the number of annual vl tests per antiretroviral treatment patient (1.14, standard deviation: 0.02) was higher at facilities that had courier collection after 13:36 (the average latest collection time) than those that had their last collection before 13:36 (1.06, standard deviation: 0.03), even when adjusted for facility size. through network optimisation, the average time for specimen transport was delayed to 14:35, resulting in a 6% – 13% increase in patient access to blood draws. conclusion: viral load testing access depends on the time of courier collection at healthcare facilities. simple solutions are frequently overlooked in the quest to improve healthcare. we demonstrate how simply changing specimen transportation timing could markedly improve access to vl testing. keywords: hiv viral load; scale-up; patient access; south africa; specimen transport. introduction the national health laboratory service (nhls) is the largest diagnostic pathology service provider in south africa, providing laboratory and related public health services to over 80% of the population.1 through a national network of laboratories, the nhls is responsible for supporting the national and provincial health departments in the delivery of healthcare and manages a courier network for transporting specimens from healthcare facilities to centralised testing laboratories. couriers collect specimens from all health facilities at least once per day and more frequently at high-volume facilities. however, anecdotal evidence suggests that after specimens have been collected by the courier from primary healthcare (phc) facilities for the day, patients that are still in the queue are unlikely to have their blood drawn that day, potentially inhibiting access to essential diagnostics. in the case of hiv patients specifically, if the patient is unable to return the following day, viral load (vl) testing could be delayed until the next clinical visit or when the patient is required to collect their next antiretroviral treatment (art) medication – potentially 2–6 months later – due to inadequate cold-storage facilities at phc facilities for blood samples. viral load testing is the recommended method for monitoring hiv patients on art. south africa, through the nhls, currently operates a highly centralised national network that conducted more than five million vl tests at 16 laboratories in 2018.2 despite this, currently, 16% – 31% of hiv-positive south africans have not had a vl test done within the guideline-recommended window (six months after art initiation and at 12-month intervals thereafter).3,4 patients with unsuppressed vl for extended periods without clinical action are at risk of increased morbidity, mortality, and/or onward transmission of hiv.5 improving access to vl testing would not only enhance patient-centred care but could result in higher levels of viral suppression.6 we aimed to design an optimised courier network that could provide expanded service access to all diagnostics available at phc facilities within the costs of the current system. we then aimed to assess the impact of the optimised courier network on vl testing access. methods ethical considerations this project was approved by the university of the witwatersrand human research ethics committee (medical) (hrec) as an additional project within the integrated laboratory data analysis for care programme (study approval number: hrec m160978). additional ethics approval was received from the center for global health, united states centers for disease control and prevention in atlanta (study approval number: cgh 2019-224). as this study involved the retrospective analyses of de-identified, aggregated data collected as part of routine care, no patient consent was sought. although sensitive information was not collected, all data extracted from the nhls were anonymised, aggregated by facility, and transferred to a secure server. patients or the public were not involved in the design, conduct, reporting, or dissemination plans of this research. study design we extended a previously described geospatial cost model to optimise the specimen transport network in south africa.7 courier routing was optimised for 4046 public facilities in south africa to improve turnaround times of specimen transport from the health facility to the laboratory. using data for a sample of phc facilities for which we had current sample transportation times (n = 356), we then used regression analysis to assess the relationship between current specimen transport time and vl testing access. finally, we compared our specimen transport times from the optimised model with current courier collection times for the subset of facilities (n = 356) to determine the percentage change in access to same-day blood draws. network optimisation for healthcare facilities data from all public sector healthcare facilities were extracted from nhls’s corporate data warehouse for a 12-month period (january 2018 to december 2018) and used in the model. extracted data included the number and type of tests (e.g. vl) requested per site, the location of the site, and the laboratory where specimens were sent to be tested. data from 4046 healthcare facilities from the nhls corporate data warehouse were included in the optimisation. these 4046 facilities accounted for 98% of the total test volumes that were couriered during the study period. facilities and laboratories in the corporate data warehouse were matched to the district health information system so that coordinate data from the district health information system could be used to locate the facilities and laboratories. data quality assessments were conducted on the coordinates to ensure accuracy, and missing coordinate data were sourced from internal implementing partner sources, as well as from google searches. the optimised system enhanced the functioning of the current specimen routing network by incorporating factors that would improve specimen validity and would be better aligned with patient-centred care (online supplementary text 1).7 the cost of the current system was calculated using nhls expenditure on contracted couriers. the contracted courier rate per kilometre was applied to the distance outputted by the routing optimisation model to estimate the cost of the optimised system. the optimisation of the specimen transport system was conducted using the arcgis pro network analyst tool (environmental systems research institute, redlands, california, united states) for vehicle routing problems, which optimises a set of transport routes taking into account the expected specimen volumes, distance from the laboratory to the facility, drive times, and other key constraints identified by stakeholders (online supplementary text 2).8 a 2019 tomtom (environmental systems research institute, redlands, california, united states) routable street data set was sourced from environmental systems research institute for use in the routing analyses. relevant outputs from the optimisation included facility name, corresponding route name, time of arrival at the facility by the courier, and the number of vl tests requested by that phc facility. using this output, it was possible to determine the average latest time that a courier would collect specimens at a facility per day. change in viral load access for a subset of facilities to assess whether the optimised specimen transport network improved vl access, we first determined whether facilities that historically had earlier courier collection times had lower vl test volumes compared to facilities with later courier collection times. we then quantified the additional number of patients that would be expected to access vl testing due to the expanded access brought about by delayed courier specimen collection times. to determine the current latest time at which a health facility is serviced by the courier network, we obtained data on current courier routes and the expected time of arrival at each facility by the courier. unfortunately, arrival time was not well documented and was only available for a subset of facilities in two provinces, gauteng and western cape (n = 497). from this sample, correctional service facilities and hospitals (n = 57), as well as facilities that did not have any patients on art or could not be linked to the district health information system (n = 80), were removed. data from the district health information system were used to determine the number of patients on art at the remaining 360 facilities (as of december 2018). finally, to determine the number of annual vl tests requested by each facility in 2018, the 360 facilities were matched to the nhls data from the corporate data warehouse. four facilities were not matched, resulting in a total sample of 356 phc facilities. data analysis using stata software (version 15.0, college station, texas, united states), we performed an unadjusted regression analysis to assess the relationship between current specimen transport time at the 356 phc facilities and vl testing access, defined as the number of annual vl tests performed per patient on art.9 the independent variable in the regression used the current average specimen transport time as a cut-off, with facilities categorised based on whether their average latest specimen transport time was before or after this cut-off. we also repeated the regression analysis, adjusting for the size of the phc facility in terms of the number of patients on art.10 a statistical significance cut-off of 5% was used. we estimated the time distribution of blood draws for patients at phc facilities based on data from an unpublished time-in-motion study on wait times conducted at an hiv outpatient facility in johannesburg in 2014.11 using this distribution, it was possible to estimate the number of additional patients that would be able to access a blood draw at different times in the day, depending on the arrival time of the courier. sensitivity a sensitivity analysis assessed the impact of changing the average latest courier time of the current system on the relationship between the time of the last courier collection for the day and the number of vl tests requested per patient on art. for each time point (representing the average latest time of courier pick-up at a facility), we determined whether there was a statistically significant difference in the average number of vl tests per patient on art between facilities whose last courier collection for the day occurred before that point versus those that had courier pick-ups after that point. in addition, we assessed how a different assumption regarding the distribution of patient flow (a more uniform distribution) at a phc facility would change our results in terms of patient access to same-day blood draws. results network optimisation the optimised network included 1179 courier routes, involved 136 000 km of driving per day, and serviced the 4046 facilities that are part of the nhls national network at least once per day. the cost to operate the current system is estimated to be $283 488 united states dollars (usd) per week, and the optimised system costs 1.3% less at $279 901 usd per week. through specimen network optimisation, the average latest time that patients can access blood draw for laboratory services was delayed to 14:48 from 14:15. in addition, with optimisation, an estimated 85% of specimens arrive at the laboratory by 16:00. impact of optimised network on viral load access in our sample of facilities for which we had complete current specimen transport times, the weighted average latest time an individual can access a blood draw (weighted by the vl volumes at the facility) is currently 13:36. under half (141/356; 40%) of the phc facilities had an average latest courier pick-up time before 13:36, while the average latest courier pick-up time at 215/356 (60%) facilities was after 13:36. the average number of annual vl tests conducted per patient on art was higher at facilities with courier pick-up after 13:36 (1.14; standard deviation: 0.02) compared to those with courier pick-up before 13:36 (1.06; standard deviation: 0.03). the number of vl tests conducted per patient on art was higher (by approximately 0.07) among facilities with courier collection after 13:36 compared to facilities with courier collection before 13:36, and the regression coefficient was significant (p < 5%), even when adjusted for phc facility size (table 1). table 1: relationship between average latest courier specimen collection time and average viral load tests per antiretroviral treatment patient† at public sector healthcare facilities in south africa, january 2018 to december 2018. data from an unpublished time-and-motion study on wait times show that the timing of blood draws was skewed to the right, with only 17% of patients getting a blood draw after 12:00 (figure 1).11 the median time for a blood draw was 09:42 (confidence interval: 08:50–11:15) and, on average, patients waited just under 3.5 h from the time that they arrived at the phc facility until a blood draw was conducted. for our sample of facilities in gauteng and the western cape, we compared the current average latest time for specimen pick-up of 13:36 to the optimised model transport time of 14:35. based on the distribution of the timing of blood draws, we estimated a 6% increase in patient access to same-day blood draws. with the assumption of a uniform patient distribution over time (i.e. a constant flow of patients for each hour that a health facility is open), the estimated patient access to same-day blood draw increased from 6% to 13%. figure 1: hourly distribution of patient blood draws at an outpatient clinic, johannesburg, south africa, 2014. in a sensitivity analysis, facilities that are serviced by a courier earlier in the day are more likely to have a significantly lower number of vl tests conducted per patient on art (figure 2). this was true until approximately 14:45, beyond which it was less likely that there would be a significant difference between the number of vl tests conducted per patient on art at the facilities. figure 2: relationship between time of courier collection and viral load access at public sector healthcare facilities in gauteng and western cape provinces, south africa, december 2019. discussion through the optimisation of the specimen transportation network, the estimated average time of specimen collection was delayed from 14:15 to 14:48 for the entire network of public facilities (n = 4046), and from 13:36 to 14:35 for the subset of facilities for which we had complete data (n = 356). this is an improvement over the current system as it alleviates the constraint faced by patients and facility staff to quickly collect blood samples before the courier arrives for the final time daily. earlier specimen arrival enables laboratory staff to meet the daily processing demands as laboratory spikes and workflow could be smoothed out and specimens processed as quickly as possible. based on our findings, delaying courier collection by an hour can increase patient access to same-day blood draws by 6% – 13% across the gauteng and western cape provinces, depending on the patient flow distribution at the phc facilities. this simple supply-side intervention to improve the logistics around specimen collection could improve access to vl testing and, consequently, increase vl test volumes and reduce the cost of specimen transportation. improving vl testing access in accordance with national guidelines is critical to attaining south africa’s goal of high levels of viral suppression among hiv patients to meet the last 95% of the hiv targets of the joint united nations programme on hiv and aids.12 one study conducted in four provinces in south africa found that only 69% of patients who were due for a vl test had a vl recorded in an electronic register, and only 24% of those who did not have a vl recorded had a blood draw recorded in their file.3 this suggests that a lack of blood draws, and not necessarily a failure of the result making it back to the phc facility, could be a major contributor to the lack of vl results. furthermore, another study5 observed a large number of missing vl test results for patients with a previously unsuppressed vl – patients at greatest risk for hiv morbidity, mortality and onward transmission.5 in another study conducted in the western cape, 84% had a vl test done when it was due.4 optimising the transportation for vl testing is only one mechanism to improve patient access to vl testing. there are alternative strategies that allow for blood to be drawn at any time of the day. if dried specimens (e.g. dried blood spots or dried plasma spots) were used for vl testing, for which they currently are not, specimens could be taken at any time during the day, even after the courier has picked up the specimens for the last time that day.13 further, should specimen stability be maintained beyond the current recommended limits,14 and should guidelines be adjusted to reflect this, this would also be an effective mechanism to increase access to vl testing. point-of-care tests could also be used, when it is cost-effective, to perform vl testing on demand.15,16 furthermore, where space and resources allow, additional cold-storage facilities could be made available at facilities where current cold storage is inadequate so that after the last courier collection for the day, blood samples can be collected and refrigerated until the next day. to our knowledge, this is the first paper to model the relationship between specimen transport collection times and vl access in a national vl programme. this analysis relies on innovative geospatial routing optimisation and primary data on laboratory and facility location matched to programmatic art data.17 although this paper has focused on the impact on vl access, these results are generalisable to other diagnostics where same-day transport is required for specimen integrity. limitations this study has several limitations. first, we sampled a subset of facilities in only two, predominately urban, provinces out of the nine provinces in south africa. nevertheless, while a more representative sample set may have produced different results, this would primarily have been driven by the current average latest time of specimen collection by couriers, which we varied in the sensitivity analysis. the relationship between specimen transport time and vl access would only become insignificant if the average latest time of specimen transport was later than 14:45, which is unlikely to be the case across the network. second, this project was conducted in south africa with an extensive well-functioning specimen referral network that ensures each facility is serviced at least once per day.18 extrapolation of these findings to other countries would require a consideration of the extent of their specimen referral networks. however, for less-developed specimen referral networks, the impact of optimising specimen collection or increasing the frequency of transport such that each facility is serviced at least once a day might result in larger patient access benefits. third, we relied on the patient flow distribution at one phc facility, where the median time of blood draw was 09:42 in the morning, to quantify the patient benefits of increased access. however, for phc facilities with later median times of blood draw (e.g., with a more uniform distribution of patients throughout the day), the benefits in terms of increased patient access of delaying the courier pick-up time would be greater. fourth, we did not quantify the patient benefits of receiving same-day blood draws. between 6% and 13% of patients requiring a blood draw for vl testing would not need to return to the phc facility another day, potentially reducing transport costs/opportunity costs of a missed day of work. patient scheduling and flow may also improve with later specimen transport as facility staff will be aware that there is more time to send patients to the blood draw queue and can thus book patients in for later times in the day, ultimately reducing the waiting time for patients. finally, while this project has primarily addressed phc facilities that predominantly operate during the week and close at 16:00, future research could look at the value of couriers collecting specimens after hours and on weekends at facilities that operate 24 h and/or on weekends. the benefits in terms of increased patient access to vl testing could even be larger as these facilities could potentially offer phlebotomy services to patients after hours and on weekends, where currently there are none. conclusion simple solutions are frequently overlooked in our quest to improve healthcare. delaying specimen pick-up times at phc facilities by an hour can improve patient access to vl testing at no additional cost. this has implications beyond vl testing and extends to all laboratory diagnostics, especially for specimens whose integrity can be compromised without refrigeration. acknowledgements the authors would like to thank the national health laboratory service for their valuable input with acquiring the data. the results from this manuscript have been presented at the conference on retroviruses and opportunistic infections 2020. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions w.s., k.d., l.b., and b.n. conceived the project. s.j.g., b.n. and, t.c. and n.c. acquired and analysed data for the model. s.j.g. and b.n. developed the model. s.j.g. and b.n. interpreted model results. s.j.g. and b.n. wrote the first draft of the manuscript. s.j.g., t.c., n.c, f.o., p.s., k.d., l.b., d.c., w.s. and b.n. read and approved the final manuscript. sources of support this work has been supported by the united states president’s emergency plan for aids relief (pepfar) through the united states centers for disease control and prevention under the terms of coag 5 nu2ggh001631-04-00: strengthening the delivery and expanding access to quality laboratory services and enhancing healthcare worker and laboratory safety in the republic of south africa under pepfar: protocol number 2019-224. the findings and conclusions in the report are those of the authors and do not necessarily represent the official position of the funding agencies. data availability there are restrictions to the availability of the data used to perform the analysis presented in this manuscript. requests for the underlying data used in this manuscript need to be directed to the national health laboratory service. (https://www.nhls.ac.za/; contact person: portia sejake portia.sejake@nhls.ac.za) disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references national health laboratory service. about us [cited 2022 feb 11]. available from: https://www.nhls.ac.za/about-us/ world health organization. chapter 4.1: clinical guidelines: antiretroviral therapy. in: consolidated guidelines on the use of antiretroviral drugs for treating and preventing hiv infection [homepage on the internet]. 2016 [cited 2022 sept 22]. available from: https://nationalgovernment.co.za/entity_annual/1714/2018-national-health-laboratory-service-(nhls)-annual-report.pdf pascoe s, huber a, murphy j, et al. identifying gaps in viral load monitoring: results from an evaluation of viral load reporting at primary health care facilities in south africa. in aids 2018 abstract book: aids 2018 22nd international aids conference – amsterdam. 2018. euvrard j, schulz t, hilderbrand k, et al. how accurately do routinely reported hiv viral load suppression proportions reflect progress towards the 90-90-90 target in the population on antiretroviral treatment in khayelitsha, south africa? s afr med j. 2019 mar 1;109(3):174–177. https://doi.org/10.7196/samj.2019.v109i3.13456 murphy ra, court r, maartens g, sunpath h. second-line antiretroviral therapy in sub-saharan africa: it is time to mind the gaps. aids res hum retroviruses. 2017;33(12):1181–1184. https://doi.org/10.1089/aid.2017.0134 drain pk, dorward j, violette lr, et al. point-of-care hiv viral load testing combined with task shifting to improve treatment outcomes (stream): findings from an open-label, non-inferiority, randomised controlled trial. lancet hiv. 2020;7(4):e229–e237. https://doi.org/10.1016/s2352-3018(19)30402-3 nichols be, girdwood sj, crompton t, et al. impact of a borderless sample transport network for scaling up viral load monitoring: results of a geospatial optimization model for zambia. j int aids soc. 2018;21(12):e25206. https://doi.org/10.1002/jia2.25206 esri. arcgis pro. network analyst tool. release 10.7. redlands, ca: environment systems research institute (esri); 2020. statacorp. stata statistical software: release 15. college station, tx: statacorp lp; 2017. fox mp, brennan at, nattey c, et al. delays in repeat hiv viral load testing for those with elevated viral loads: a national perspective from south africa. j int aids soc. 2020;23(7):e25542. https://doi.org/10.1002/jia2.25542 long l, batiancila r, girdwood s, majuba p, rosen s, lince-deroche n. booked appointments system: an evaluation of the acceptability and feasibility of booked appointments in a large hiv clinic in johannesburg, south africa [serial online]. 2022 [cited 2020 dec]. available from: https://www.medrxiv.org/content/10.1101/2022.06.30.22277110v1 unaids. fast-track. ending the aids epidemic by 2030 [homepage on the internet]. 2014 [cited 2020 nov 3]. available from: https://www.unaids.org/en/resources/documents/2014/jc2686_wad2014report nichols be, girdwood sj, shibemba a, et al. cost and impact of dried blood spot versus plasma separation card for scale-up of viral load testing in resource limited settings. clin infect dis. 2019;70(6):1014–1020. https://doi.org/10.1093/cid/ciz338 hardie d, korsman s, ameer s, vojnov l, hsiao n-y. reliability of plasma hiv viral load testing beyond 24 hours: insights gained from a study in a routine diagnostic laboratory. plos one. 2019;14(7):e0219381. https://doi.org/10.1371/journal.pone.0219381 girdwood s, crompton t, sharma m, et al. cost-effectiveness of adoption strategies for point of care hiv viral load monitoring in south africa. eclinicalmedicine. 2020;28:100607. https://doi.org/10.1016/j.eclinm.2020.100607 girdwood sj, nichols be, moyo c, crompton t, chimhamhiwa d, rosen s. optimizing viral load testing access for the last mile: geospatial cost model for point of care instrument placement. plos one. 2019;14(8):1–13. https://doi.org/10.1371/journal.pone.0221586 nichols k, girdwood sj, inglis a, et al. bringing data analytics to the design of optimized diagnostic networks in lowand middle-income countries: process, terms and definitions. diagnostics. 2021;11(1):22. https://doi.org/10.3390/diagnostics11010022 glencross dk, coetzee lm, cassim n. an integrated tiered service delivery model (itsdm) based on local cd4 testing demands can improve turn-around times and save costs whilst ensuring accessible and scalable cd4 services across a national programme. plos one. 2014;9(12):e114727. https://doi.org/10.1371/journal.pone.0114727 abstract introduction methods results discussion acknowledgements references about the author(s) naseem cassim department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa national health laboratory service, johannesburg, south africa neeshan ramdin department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa sadhaseevan moodly department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa national health laboratory service, johannesburg, south africa deborah k. glencross department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa national health laboratory service, johannesburg, south africa citation cassim n, ramdin n, moodly s, glencross dk. cost of running a full-service receiving office at a centralised testing laboratory in south africa. afr j lab med. 2022;11(1), a1504. https://doi.org/10.4102/ajlm.v11i1.1504 original research cost of running a full-service receiving office at a centralised testing laboratory in south africa naseem cassim, neeshan ramdin, sadhaseevan moodly, deborah k. glencross received: 29 dec. 2020; accepted: 19 apr. 2022; published: 13 july 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the national health laboratory service operates a platform of 226 laboratories across south africa, ranging from highly sophisticated central academic hospitals to distant rural hospitals. the core function of the national health laboratory service is to provide cost-effective and efficient health laboratory services in the public healthcare sector. objective: this study aimed to assess the comprehensive cost of running a full-service receiving office (ro) at the charlotte maxeke johannesburg academic hospital (cmjah) laboratory. methods: top-down costing was conducted, with the cost per registration as the main outcome of interest. the annual equivalent costs (aec) for the following categories were determined: registration materials, collection materials, staffing, laboratory equipment, building and electricity, and other operating costs. data for the period from 01 april 2019 to 31 march 2020 were included in the analyses. results: the aec was $1 657 483.00 united states dollars (usd) and the cost per registration was $0.766 usd. staff contributed 59.9% of the total cost per registration, while collection materials contributed 21.4%. the ro core staff (data clerks) contributed 50.8% of the total staffing costs, while messengers and drivers contributed 31.2%. the introduction of order entry at the cmjah and other primary healthcare facilities reduced the total aec by 20%. a single order entry application would serve both the cmjah and primary healthcare facilities hence we would prefer to not refer to order entries. conclusion: providing a comprehensive ro service costs approximately $1.00 usd per registration. the implementation of order entry at the cmjah would reduce aecs substantially and improve efficiency. keywords: receiving office; cost per registration; order entry; costing; pre-analytical. introduction the national health laboratory service (nhls) operates a platform of 226 laboratories across south africa, ranging from central academic to distant rural laboratories, with a mandate to provide cost-effective and efficient services in the public healthcare sector.1 to carry out this mandate, the nhls is premised on three pillars, namely diagnostics, research, and teaching and training.1 the charlotte maxeke johannesburg academic hospital (cmjah) is the largest nhls reference laboratory in south africa. the laboratory is international standards organization 15189-accredited and provides quality results that contribute to patient care.2 this laboratory houses the largest automated laboratory track system within the nhls network that allows third-party instrument connectivity, thus providing a wide repertoire of clinical pathology services.3 the automated track creates a harmonious flow of samples through the pre-analytical, analytical and post-analytical phases and ensures that the laboratory can provide state-of-the-art diagnostic services with efficiency, improved turn-around times, and minimal wastage.3 an estimated 4.8 million tests are performed annually at the cmjah laboratory, with steady year-on-year increases recorded. the receiving office (ro) plays a pivotal role in the correct and timeous capturing of samples into the laboratory information system (lis) to facilitate the delivery of patients’ reports and expenditure reporting. unfortunately, unlike the analytical phase at the cmjah laboratory, the pre-analytical processes at the ro are not automated. pre-analytics refer to the procedures carried out before actual sample testing and include patient identification, preparation, sample collection, sample packaging, sample transportation, and sample preparation for analysis and storage.4 currently, the ro receives completed paper-based request forms to start the data capture process, which is time-consuming as it requires the entry of details of the health facility, healthcare worker, patient, and requested test into the lis. details such as the time of sample receipt, sample sorting, data capture, and delivery to the laboratory are also manually entered into the lis. furthermore, in the present system, samples are handled multiple times in the ro in contrast to the analytical phase where the automated track uses a one-touch approach.3 in other settings, many of these manual processes can be automated through order entry (oe). for example, public sector laboratories such as the inkosi albert luthuli central hospital have adopted a paperless approach that uses oe to replace multiple manual ro tasks.5 order entry is an application that enables healthcare workers to create electronic orders within the facility,6 replacing traditional pen-and-paper methods for ordering laboratory investigations using request forms. a simplified linear workflow for oe would be as follows: at source, the healthcare worker identifies the need for a laboratory test to be performed and uses the oe application to select the tests required, with the patient and healthcare worker information populated by an electronic patient record system (eprc). the order is then electronically transcribed and sent to the national lis where it is accepted and the testing process commences. after results authorisation, data are translated back to the eprc and patient care is provided based on the results.7 with the oe workflow, all the required information is transmitted electronically and testing can commence with minimal ro processing, thus minimising transcription errors and improving order response,8 turn-around time, test ordering efficiency, laboratory utilisation, and, ultimately, patient care.9,10 as oe is part of the clinical workflow, it should ideally be implemented as a collaborative effort with the entire healthcare team.7 order entry can influence and control test ordering patterns through structured order screens, manipulation of order sets and the analysis of real-time data to assess the impact of such changes.10 this is especially important in the context of national treatment guidelines such as for the care of hiv-positive patients, in which tests are performed based on standardised patient workup and care.11 electronic gatekeeping, which is used to reduce unnecessary test requests, can also be programmed into the oe application to alert clinicians at the time of placing an order that their samples will likely not be processed.11 this study aimed to assess the comprehensive cost of running a full-service ro at a busy centralised academic laboratory that processes local samples and receives referred samples for specialist pathology testing and to assess the impact of the implementation of oe on ro costs. methods ethical considerations ethical clearance (approval number: m160978) was obtained from the university of the witwatersrand human research ethics committee (medical). no patient identifiers were collected and thus patient consent was not required. study setting this study was conducted at the full-service ro based at the cmjah laboratory, johannesburg, south africa. data are reported for the april 2019 to march 2020 financial period. workflow at the charlotte maxeke johannesburg academic hospital laboratory receiving office forty-two primary healthcare (phc) facilities and two national central hospitals are directly served by the cmjah laboratory; the laboratory offers all routine haematology, chemistry and microbiology testing, as well as specialist pathology testing (e.g. hiv viral load or cd4 testing, flow cytometry, cytogenetics, etc.). in addition, similar specialised tests that are referred from other phc facilities and hospitals outside the immediate precinct of the cmjah laboratory (n = 354) are also processed at the cmjah laboratory, including those based in the west rand, ekurhuleni, city of johannesburg, and sedibeng districts. samples collected at distant health facilities are delivered to the closest local nhls source laboratory using a hub-and-spoke courier network with multiple health facilities located along designated routes (figure 1). these source laboratories serve their respective surrounding health facilities and perform routine clinical pathology testing. all specialised tests such as hiv viral load are subsequently referred within the nhls network to academic testing facilities such as cmjah. an inter-laboratory referral courier network is used to transport the referred samples to the larger academic laboratories. locally sourced specimens are collected from hospital wards at cmjah (n = 64; samples transported every 2 h) and the nelson mandela children’s hospital (n = 6; samples transported every 4 h). the final source of specimens is the surrounding health facilities (e.g. alexandria community health centre) that deliver samples directly to the cmjah ro using a courier network. figure 1: sources of specimens and activities performed at the charlotte maxeke johannesburg academic hospital laboratory receiving office, south africa, april 2019 – march 2020. pre-analytical activities include sample receipt, sample sorting to identify where testing will take place, and registration on the lis. these activities take place at both the cmjah ro as well as at surrounding source laboratories. the registration process involves entering the details provided on the request form (patient demographics and health facility and healthcare worker details) into the lis and attaching barcodes with the episode numbers on all samples. samples not tested at the source laboratory are referred on the lis; this involves preparing a shipping list and packaging samples for courier pick-up. the pre-analytical activities performed at the cmjah ro are the same for surrounding health facilities. referral samples are accepted on the lis before testing. after testing, patient reports are printed and delivered to each health facility. costing methodology the costing analysis was done using microsoft excel (microsoft corporation, redmond, washington, united states). costs were obtained using historical cmjah expenditure data for the april 2019 to march 2020 financial period. the accounting stance was as a provider of the ro service, that is, all costs were obtained as the provider of laboratory services (costs incurred by the health facility were excluded). a top-down costing approach was used, with the main outcomes of interest being the annual equivalent cost (aec) and cost per registration. the cost per registration refers to the costs of all the activities conducted in the ro for each patient visit, during which one or more tests may be requested. therefore, it is not the cost per sample received as multi-disciplinary registration is performed. multi-disciplinary registration refers to a single registration on a single episode number of multiple tests requested for multiple pathology disciplines. costs were collected in south african rand (zar) and reported in united states dollars (usd) (using the monthly average exchange rate of r14.7835 zar to $1.00 usd for november 2019).12 the consolidated health economic evaluation reporting standards checklist was used in the preparation of this manuscript.13 costs were reported for the following categories: registration materials, collection materials, staff, laboratory equipment, building and electricity, and other operating costs. the consumer price index ranged from 3.6% (november 2019) to 4.6% (february 2020).14 furthermore, the world bank reported an ‘inflation, consumer prices (annual %)’ value of 4.12% for 2019.15 therefore, we assumed a 4% discount rate. organisational overhead costs were excluded; these included all corporate services such as human resources, finance, and information technology offered by the nhls corporate offices. registration materials included items such as disposable gloves, n95 masks, thermal barcode printer paper, paper (to print worklists for the laboratory), disposal boxes, etc. specimen collection materials included laboratory request forms, sharps disposal boxes, specimen collection kits and biohazard bags, which are all issued by the nhls to healthcare facilities for sample collection. receiving office staff included the business manager (grade d5), the ro manager (d1), supervisors (c3), and operational staff, consisting of data clerks (b2/4), messengers (a3), administrative officers (c1), drivers (b4) and cleaners (a1). for staff costs, we used the annual salary data that include medical aid, pension and other allowances.16 staff were categorised as ro core staff, messengers or drivers, ro management, cleaning, and overall management. the percentage of time spent on sample registration by each staff category was determined (table 1). table 1: percentage of time spent on registrations by staff categories and types at the charlotte maxeke johannesburg academic hospital laboratory receiving office, south africa, april 2019 – march 2020. the costs of the following laboratory equipment were included: a messenger monitoring system (used to track and log the times when messengers arrive and collect samples at each ward and deliver samples back to the ro), date and time stamp devices, multi-function printers, barcode scanners, operator chairs, workbenches, computers, network points, barcode printers, specimen tube racks, shopping baskets (used for sample delivery to each section), air conditioners, fridges, freezers (–20 °c), document scanners, and filing cabinets. to calculate the aec for laboratory equipment, working life was set at five years with a discount rate of 4%. working life is defined as the period that an asset is likely to remain in service. we used the microsoft excel ‘sum’ and ‘pmt’ functions (equation 1): studies have reported that the ‘pmt’ function in microsoft excel is easy to use for calculating the aec for equipment using assumptions of the discount rate, working life, and purchase price.17,18 to determine the total building costs, we multiplied the total area of the ro in square metres (m2) by the average building cost of r8163 zar per square metre.19 this average building cost is based on the statistics south africa estimate for building office spaces.19 to calculate the aec for building costs, we used a working life of 50 years and a discount rate of 4% and applied this to the total costs using the pmt formula. we determined the monthly cost of electricity at the cmjah laboratory and attributed 2% of the cost to the ro based on its size. we used the ro income statement to assign other operating costs. these included the aec for items such as cellular phones, computer consumables, printing, telephones, couriers, freight, postage, laundry, accreditation, waste disposal, uniforms, etc. the cost for lis licenses and other costs were also obtained from the income statement. costing analysis we reported the aec and the cost per registration. data were reported for each cost category. the percentage contribution of each cost category to the total cost per registration was reported. we assessed the impact of implementing oe at surrounding phc facilities only versus implementing oe at all surrounding phc facilities and all cmjah wards. to calculate costs for each oe scenario, the percentage reduction in manual ro activities was applied to the staff costs of ro clerks. a rejection rate of 6% was used to determine the reduction in data collection materials and registration materials (based on lis reports). samples are rejected because of electronic gatekeeping rules (such as minimum re-testing intervals) or for failing to meet essential criteria stipulated in the laboratory handbook (such as missing mandatory information or using the incorrect specimen type).20,21,22,23 it was assumed that oe would integrate electronic gatekeeping and rejection rules to reduce wastage.22,23 by integrating these rules, the oe application would alert the healthcare worker that mandatory information was missing and prevent the order from being placed. this would reduce costs by preventing the use of specimen collection materials, as well as other time-consuming activities, including sample transport, sample sorting, data capture, and sample rejection. currently, ro staff capture only 6% of samples that are rejected on the lis. the aec for the base case (as is) was compared to the two oe scenarios for each cost category. results data from a total of 2 163 421 registrations at the cmjah ro were included in the analyses. the cmjah ro consists of 68 employees (excluding the business manager and administrative assistant), including 34 data capturers, 22 messengers, four drivers, four cleaners, three managers or supervisors, and one administrative officer. assuming a 24/7 service for 365 days, this equates to a daily workload of 5927 registrations. on average, it takes 12 min per sample to complete sample receipt, sorting, registration, barcoding, and addition of test tubes to the track for delivery to each laboratory section. annual equivalent cost an aec of $1 657 482 usd was reported for the cmjah ro. staffing contributed $992 664 usd (59.9%), collection materials contributed $355 450 usd (21.4%) and other operating costs contributed $222 653 usd (13.4%) to the aec (table 2). registration materials ($54 622 usd), equipment ($20 444), and building and electricity ($11 650) collectively contributed $86 716 usd (5.2%) to the aec. table 2: annual equivalent costs and the cost per registration for each cost category at the charlotte maxeke johannesburg academic hospital laboratory receiving office, south africa, april 2019 – march 2020. cost per registration the total cost per registration was $0.766 usd. staffing contributed $0.459 usd, collection materials contributed $0.164 usd, other operating costs contributed $0.103 usd, registration materials contributed $0.025 usd, equipment contributed $0.009 usd, and building and electricity contributed $0.005 usd to the cost per registration. staffing costs the ro core staff (50.8%) and messengers and drivers (31.2%) contributed 82.0% of the total staffing costs, while the ro supervisor and ro manager contributed 14.4% of the total staffing costs. impact of order entry introducing oe at the surrounding phc facilities, which contribute 50% of registrations at cmjah, would reduce costs by $285 081 usd. by implementing oe at the surrounding phc facilities and the cmjah wards, the estimated cost savings increased to $335 514 usd (figure 2). this is a 20.2% cost reduction from the current costs of running the ro service. figure 2: impact of order entry implementation on the annual equivalent cost at the charlotte maxeke johannesburg academic hospital (cmjah) laboratory receiving office, south africa, april 2019 – march 2020. discussion the cost to register one sample at the cmjah ro was less than $1.00 usd. staffing was the major contributor to the overall cost, highlighting the manual nature of activities performed by ro staff. collection materials and other operating costs, on the other hand, contributed only one-third of the total ro costs. one of the current data challenges in healthcare services in south africa is that patient demographics and personal identifiers are captured on multiple data systems in different formats. this highlights the inefficiencies of the current paper-based system. at the cmjah, patients are assigned a unique patient identifier and their details are captured on the hospital information system when they first present for care. when they present to the pharmacy or radiology units, for example, their details are re-captured. also, when a laboratory test is requested, this information is transcribed onto the laboratory request form and then manually captured in the lis by data clerks. aside from the multiplied workload, this multipronged, multi-layered approach to patient data capture and sample tracking can lead to transcription errors and data field omissions. these manual transcription errors have been confirmed during random data audits of patient request forms where differences were observed between lis-contained information and information on the request forms (data not shown). inefficient use of existing information within the healthcare system thus increases the workflow complexity and workload in the ro, thereby increasing the need for a large staffing component to process and capture information on the lis. for the implementation of oe across the public health sector in south africa, the use of a national unique patient identifier is imperative to ensure consistency of information irrespective of the site of presentation. currently, no unique patient identifier is used within the public health system.24 in the present system, duplicate patient records are inadvertently created on the hospital information system or eprc systems as most phc facilities use alphanumeric codes that are not based on the national health identifier (nhid) to identify patients.25 the development of an electronic system that can generate a master patient index or nhid would prevent unnecessary data duplication.25 this nhid can be implemented using a centralised, semi-distributed or highly distributed model.25 the different models determine where the nhid is assigned, that is, at the national, regional or facility level.25 the aim would be to develop an nhid system that contains a master record of all patients that have accessed public healthcare services across south africa. a new nhid would only be assigned after searching the national master patient index list to prevent the duplication of patient information. this will facilitate the seamless data transfer of patient information to the oe application, enable longitudinal tracking of patients, and reduce healthcare costs by not repeating laboratory investigations that were already requested by another health facility. the cost to implement oe (capital expenditure and maintenance) and an electronic health record system is a function of bed size.26 the one-time capital cost to implement oe for a 720-bed facility was estimated at $16 026 676 usd (2012 equivalent),26,27 and the annual maintenance costs were estimated to be $2 015 807 usd.26,27 as this data were reported for four hospitals with an existing electronic health record system, we calculated the cost for a single hospital and used the interest rates reported by the international monetary fund to determine the equivalent value in 2019.28 between 2013 and 2019, the south african central bank policy rate reported a cumulative inflation rate of 6.63%.28 this equates to an annual cost of $4 809 449 usd per hospital in 2019.28 in contrast, a district hospital in kenya reported a cost of $2.1 million usd for the implementation of oe and $435 000 usd for annual maintenance.29 given that this hospital has 320 beds, this is not a reasonable cost estimate for oe implementation at cmjah (1088 beds).30 therefore, a local costing study is required to assess the implementation and maintenance costs of oe for all wards at cmjah, nelson mandela children’s hospital, and surrounding phc facilities. given the connectivity and information technology infrastructure challenges in the public health sector in south africa, extensive investments would have to be made to make oe accessible and to develop the necessary interfaces with the different hospital information system and eprc systems used by hospitals and phc facilities. to implement oe at phc facilities, some minimum infrastructures such as tablets, computers, internet connection and an eprc interface are required.9 these are required for the electronic transfer of orders to the lis and the return of results.9 for this electronic transfer, logical observation identifier names and codes could be used.31 implementing oe at the surrounding phc facilities and cmjah wards would result in an estimated annual cost saving of around 20%. should the cost of oe installation be similar to the cost of the cmjah ro, it would be possible to pay the capital cost of the system with the savings generated over a five-year period. there may also be additional cost savings generated by better adherence to treatment and pathology testing guidelines, reduced rejections and more appropriate ordering. another study reported that the implementation of oe at a large academic hospital resulted in an annual saving of $2.2 million usd compared to an investment of $11.8 million usd, albeit it took over five years for these savings to be realised.32 as indicated by birkmeyer et al., the primary motivation for introducing oe should be to improve the quality of care provided and not to reduce healthcare expenditure.33 it has been shown that oe has the potential to increase efficiency and effectiveness, thereby enhancing the quality of patient care.32 when implemented with clinical decision support systems, oe has the potential to reduce rejections and unnecessary test requests and alert clinicians of the required mandatory data fields and specimen criteria.34 in addition, as an electronic platform, oe could streamline the workflow in the health facility. the development of eprc systems in the united states has been challenging due to closed, proprietary and incompatible systems.35 likewise, for developing countries, purchasing proprietary oe systems from developed countries leads to higher implementation costs due to travel costs and exchange rate fluctuation. as a result, the use of locally developed or open-source oe could result in lower implementation costs and minimal expenditure required to extend the application across the public health sector. a good example of a locally developed information system is tier.net, which is used to collect data on patients receiving antiretroviral therapy.34 after being piloted in the western cape province, tier.net is now used across south africa. the implementation of oe across the public health system beyond cmjah would dramatically streamline the process of placing laboratory orders and receiving results. the major benefit of oe would be a move from paper-based to electronic systems, removing the requirement to re-capture information that exists in the eprc. in addition, the ro at laboratories would be dramatically scaled down given the electronic transfer of data. this would change the visibility of orders at both the health facility and laboratory, with better monitoring of samples and results that are outstanding. overall, the interface with the laboratory would become efficient and streamlined by bypassing multiple manual steps in the current workflow (figure 3). figure 3: typical workflow with order entry implemented at the charlotte maxeke johannesburg academic hospital laboratory receiving office, south africa, april 2019 – march 2020. once oe is implemented, the cmjah ro data clerks will not be retrenched. staff may be re-allocated, deployed to other duties, or transferred to other laboratories with staff shortages. in addition, clerks could be encouraged to consider training as laboratory technicians and migrating to the analytical phase where shortages exist. limitations this study excluded the cost of organisational overheads. should the organisational overheads be included, it would result in a minor change to the cost per registration as the organisational overheads would be divided by the number of registrations across the nhls. the costs reported would also vary depending on the size and complexity of the ro, as well as the distance of the ro from source laboratories. in a small province such as gauteng, health facilities are close (≤ 50 km) to testing laboratories. however, in more rural provinces such as the northern cape, facilities could be located over 300 km from their local laboratory, thus leading to higher logistics costs. another limitation is that this study did not consider the costs of training required to introduce oe. although the coronavirus disease 2019 pandemic has demonstrated that it is possible to conduct clinical training remotely,36 health facilities may not have access to the necessary bandwidth and computers present in a university environment.36 therefore, the cost of introducing oe should be assessed given the challenges with remote learning, especially in rural settings. conclusion providing a comprehensive ro service at a large referral laboratory in south africa costs less than $1.00 usd per registration; however, most of this expenditure can be attributed to the high ro staffing costs due to the manual nature of ro activities. the implementation of oe has the potential to reduce ro costs by as much as 20% while also improving efficiency, reducing turn-around times, and improving patient outcomes. acknowledgements the authors would like to thank the various suppliers for providing quotations. we would also like to thank the national health laboratory service for providing costs from the oracle erp system. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions n.c. developed and executed the research, conducted the costing analysis, prepared the first draft and made editorial input. s.m. and n.r. provided costing information, reviewed the data and reviewed the article. d.k.g. was the project leader and provided editorial input. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the data are not publicly available due to the authors not having permission to share the costing data. disclaimer the views expressed in this manuscript are those of the authors and not those of the university of witwatersrand or the nhls. references national health laboratory service (nhls). annual report 2018/19 [homepage on the internet]. johannesburg: national health laboratory service (nhls); 2019 [cited 2020 feb 19]. available from: https://www.nhls.ac.za/key-documents/annual-reports/ schneider f, maurer c, friedberg rc. international organization for standardization (iso) 15189. ann lab med. 2017;37(5):365–370. https://doi.org.10.3343/alm.2017.37.5.365 south african government. gauteng health launches automated pathology laboratory at charlotte maxeke [homepage on the internet]. johannesburg: south african government; 2017 [cited 2020 nov 03]. available from: https://www.gov.za/speeches/official-launch-nhls-charlotte-maxeke-johannesburg-academic-hospital%e2%80%99s-automated-pathology plebani m. quality indicators to detect 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[homepage on the internet]. statistics south africa (stats sa); 2019 [cited 2020 oct 20]. available from: http://www.statssa.gov.za/?p=7974 national department of health (ndoh), national health laboratory service (nhls). primary health care laboratory handbook: a step by step guide [homepage on the internet]. 2018 [cited 2021 nov 03]. available from: https://www.idealhealthfacility.org.za/app/document/download/20 pema ak, kiabilua o, pillay ts. demand management by electronic gatekeeping of test requests does not influence requesting behaviour or save costs dramatically. ann clin biochem. 2018;55(2):244–253. https://doi.org.10.1177/0004563217707980 elnenaei mo, campbell sg, thoni aj, lou a, crocker bd, nassar ba. an effective utilization management strategy by dual approach of influencing physician ordering and gate keeping. clin biochem. 2016;49(3):208–212. https://doi.org/10.1016/j.clinbiochem.2015.11.005 sripa p, hayhoe b, garg p, majeed a, greenfield g. impact of gp gatekeeping on 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precision medicine: extending an observables ontology unifying snomed ct and loinc. j am med inform assoc. 2018;25(3):259–266. https://doi.org.10.1093/jamia/ocx097 kaushal r, jha ak, franz c, et al. return on investment for a computerized physician order entry system. j am med inform assoc. 2006;13(3):261–266. https://doi.org.10.1197/jamia.m1984 birkmeyer cm, lee j, bates dw, birkmeyer jd. will electronic order entry reduce health care costs? eff clin pract. 2002;5(2):67–74. osler m, hilderbrand k, hennessey c, et al. a three-tier framework for monitoring antiretroviral therapy in high hiv burden settings. j int aids society. 2014;17(1):18908. https://doi.org.https://doi.org/10.7448/ias.17.1.18908 fraser h, biondich p, moodley d, choi s, mamlin b, szolovits p. implementing electronic medical record systems in developing countries. j innov health inform. 2005;13(2):83–95. https://doi.org.10.14236/jhi.v13i2.585 schmutz ams, jenkins ls, coetzee f, et al. re-imagining health professions education in the coronavirus disease 2019 era: perspectives from south africa. afr j prim health care fam med. 2021;13(1):e1–e5. https://doi.org.10.4102/phcfm.v13i1.2948 abstract introduction research method and design results discussion recommendations conclusion acknowledgements references about the author(s) ozayr h. mahomed discipline of public health medicine, university of kwazulu-natal, durban, south africa ruth lekalakala clinical microbiologist, university of limpopo, polokwane, south africa shaidah asmall senior technical advisor, national department of health, pretoria, gauteng, south africa naseem cassim data and logistics manager, national health laboratory service, johannesburg, gauteng, south africa citation mahomed oh, lekalakala r, asmall s, cassim n. implications of the introduction of laboratory demand management at primary care clinics in south africa on laboratory expenditure. afr j lab med. 2016;5(1), art. #339, 6 pages. http://dx.doi.org/10.4102/ajlm.v5i1.339 original research implications of the introduction of laboratory demand management at primary care clinics in south africa on laboratory expenditure ozayr h. mahomed, ruth lekalakala, shaidah asmall, naseem cassim received: 01 july 2015; accepted: 23 dec. 2015; published: 18 mar. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: diagnostic health laboratory services are regarded as an integral part of the national health infrastructure across all countries. clinical laboratory tests contribute substantially to health system goals of increasing quality of care and improving patient outcomes. objectives: this study aimed to analyse current laboratory expenditures at the primary healthcare (phc) level in south africa as processed by the national health laboratory service and to determine the potential cost savings of introducing laboratory demand management. methods: a retrospective cross-sectional analysis of laboratory expenditures for the 2013/2014 financial year across 11 pilot national health insurance health districts was conducted. laboratory expenditure tariff codes were cross-tabulated to the phc essential laboratory tests list (ell) to determine inappropriate testing. data were analysed using a microsoft access database and excel software. results: approximately r35 million south african rand (10%) of the estimated r339 million in expenditures was for tests that were not listed within the ell. approximately 47% of expenditure was for laboratory tests that were indicated in the algorithmic management of patients on antiretroviral treatment. the other main cost drivers for non-ell testing included full blood count and urea, as well as electrolyte profiles usually requested to support management of patients on antiretroviral treatment. conclusions: considerable annual savings of up to 10% in laboratory expenditure are possible at the phc level by implementing laboratory demand management. in addition, to achieve these savings, a standardised phc laboratory request form and some form of electronic gatekeeping system that must be supported by an educational component should be implemented. introduction diagnostic health laboratory services are regarded as an integral part of the national health system across all countries and have an important role in the continuum of care. laboratory testing provides access to screening of asymptomatic individuals at risk for developing disease, early detection of diseases and diagnostic confirmation; provides information on patients’ prognosis; assists with planning appropriate disease management strategies and monitoring patients’ response to treatment; and plays a pivotal role in ensuring patient safety by identifying hospital-acquired infections and other potential health related adverse events.1 the national health laboratory service (nhls) provides diagnostic laboratory services for the south african public health sector through over 300 laboratories across the nine provinces, thereby achieving 80% population coverage.2 the nhls is reimbursed by the provincial departments of health on a fee-for-service billing arrangement.2 through this payment mechanism, laboratory tests are itemised as tariff codes on an invoice, for example, tariff code 2210 denotes the haemoglobin test. these funds are obtained from the provincial equitable share portion of the national health budget. expenditures for laboratory services have increased by 45% from r3.1 billion south african rand in financial year (fy) 2010/2011 (01 april 2010 to 31 march 2011) to r4.5 billion by fy 2013/2014 (01 april 2013 to 31 march 2014).3 this was because of increases in laboratory test volumes from 80.2 million tests in fy 2011/2012 to 81.1 million in fy 2012/20134 and approximately 86 million tests in fy 2013/2014.2 the sharp increases in expenditures and test volumes in fy 2013/2014 can be attributed to growth in priority test volume. priority tests for patients living with hiv, tuberculosis and cervical cancer accounted for 16% of total test volume2 in fy 2013/2014. there was a 144% increase in the volume of genexpert mtb/rif assays, as well as a 27% increase in hiv viral load testing.3 the genexpert mtb/rif assay replaced the auramine smear for diagnosis of tuberculosis during this time period. there was a large difference in the price per test; auramine smears cost r24.34, whereas the genexpert mtb/rif assay cost r172.85 as of december 2013. the volume of laboratory testing is anticipated to increase with the introduction of general practitioners as care providers for patients at the primary healthcare (phc) level, because general practitioners are expected to order additional laboratory tests that were not previously requested under nurse-based phc services. furthermore, a renewed focus has been placed on integrated clinical services at the phc level5 and the introduction of algorithms to appropriately manage patient conditions.6 this is likely to further increase the volume of tests requested, thereby increasing the total expenditures of the public health system. demand management aims to improve the requesting of appropriate laboratory tests and results in reductions in public health expenditures without affecting clinical outcomes.7 the first step to implementing demand management involves defining what constitutes an ‘inappropriate’ request, based on some form of agreed-upon guidance.7 for example, this may involve standardising the repertoire of tests that may be requested by level of care, namely, phc and hospital services.7 similarly, evidence-based laboratory medicine involves eliminating laboratory tests with no clinical value and introducing new laboratory tests where evidence proves their efficacy and effectiveness.8 the implementation of this approach requires a pathologist-driven laboratory service that utilises context-appropriate evidence to guide testing and reduce public health expenditures. the south african national department of health has proposed a demand-management system in the form of an essential laboratory tests list (ell) to promote appropriate and cost effective usage of laboratory services at phc facilities without having a negative effect on patient outcomes. the ell includes the minimum set of tests that should be performed to offer comprehensive services at the phc level.9 in determining the ell for south africa, the world health organization criteria for the usefulness and clinical relevance of tests that influence diagnosis and patient management were considered.9 additionally, the ell requires that a single test be used, rather than multiple tests, if the single test provides adequate diagnostic information.9 for example, on the ell, the alanine transaminase test is substituted for the liver function panel test, as the alanine transaminase test provides the same diagnostic value without affecting patient outcomes. furthermore, the laboratory tests included on the ell were aligned to the national south african standard treatment guidelines,10 including the phc clinical algorithms that were introduced as a clinical supportive management component of the integrated chronic disease management mode.5 the aims of this study were to analyse current expenditures and the profile of laboratory tests currently requested at the phc level. in addition, we sought to determine the potential cost savings that could be achieved by the public health sector through the introduction of the demand-management-based ell. research method and design this was a retrospective cross-sectional analysis of phc laboratory expenditure for the fy 2013/2014 period across 11 national health insurance (nhi) pilot districts. data on district and facility level expenditures for the amajuba, city of tshwane metro, dr kenneth kaunda, eden, gert sibande, or tambo, pixley ka seme, thabo mofutsanyana, umgungundlovu, umzinyathi and vhembe districts were extracted from the nhls corporate data warehouse. the extracted data fields included customer account information, laboratory information, system location codes used to identify health facilities, tariff codes used for billing purposes (to identify the investigation(s) performed), annual test volumes and expenditures. all expenditure data were reported in south african rand. the ell defines the laboratory tests that could be requested at phc facilities by nursing staff or general practitioners. for example, nurses can request thyroid stimulating hormone tests; however, general practitioners can also request a free thyroxine 4 test. amongst other tests, the ell included haemoglobin, hiv viral load, genexpert for tuberculosis, cluster definition 4 (cd4) count, hiv dna pcr for infants, sputum and urine microscopy, smear, culture and sensitivity, total cholesterol and total triglycerides, prostatic specific antigens, cervical smears and glycated haemoglobin (hba1c). tests not on the ell for primary healthcare included full blood count, urea and electrolytes, hiv serology, liver function tests, drug levels for carbamezapine and tegretol, endocrine tests such as thyroid profiles and follicule stimulating hormones and luteinising hormone levels, arthritis screening, anti-nuclear factors and other immunological tests. each ell test was mapped to one or more nhls tariff code(s) from the expenditure data. a one-to-one or a one-to-many relationship exists between an ell test and the tariff code(s) used by the nhls. for example, the cd4 test represents a one-to-one relationship. however, the c-reactive protein test has a one-to-many relationship, as different tariff codes are used based on the laboratory methodology, for example, qualitative versus quantitative test. furthermore, some ell tests follow a diagnostic cascade, whereby based on an initial result, a subsequent investigation is performed, such as microscopy, culture and sensitivity. additionally, ell tests were grouped into logical test baskets, for example, lipogram for cholesterol and triglycerides. for the expenditure analysis, a microsoft access 2010 database (microsoft corporation, redmond, washington, united states) was used and the expenditures and ell mapping list were imported as tables. the expenditure tariff codes were reported with the ell test by creating a relationship between the two tables. queries were developed to report on tariff code expenditures based on the ell test. results test volumes and expenditures by health district approximately 4.5 million tests accounted for approximately r339 million in laboratory expenditures for diagnostic laboratory tests at the phc facilities in the 11 districts during the fy 2013/2014 study period (table 1). phc facilities within the city of tshwane (gauteng) accounted for the highest proportion of test volume and laboratory expenditures, followed by clinics within the or tambo district (eastern cape) and umgungundlovu district (kwazulu-natal). table 1: test volumes and laboratory expenditures for primary healthcare facilities within 11 national health insurance pilot districts, south africa, 2013–2014.† total expenditures on ell and non-ell laboratory tests of all laboratory expenditures for fy 2013/2014, 21 tests were responsible for ~92% (r310 million) of the total (table 2). laboratory tests for patients living with hiv were responsible for ~47% of all expenditures, of which hiv viral load accounted for ~32% of expenditures, cd4 for ~10% and hiv dna pcr for infants, ~6%. laboratory tests for tuberculosis diagnosis accounted for ~21% of the expenditure, including tuberculosis microscopy (~2%), genexpert (~18%) and tuberculosis culture (~1%). non-ell tests such as the full blood count and the urea and electrolyte tests were responsible for 5% of expenditure (r16 million). table 2: laboratory tests with the highest expenditures across the 11 national health insurance pilot districts, south africa, 2013–2014.† expenditure on non-ell tests by district across the 11 nhi pilot districts, 10% (r35 million) of laboratory expenditures at phc facilities were for tests that were not included on the ell (table 3). in five districts, including gert sibande, pixley ka seme, vhembe, city of tshwane and dr kenneth kaunda, the proportion of tests not included on the ell exceeded 10% of the phc’s laboratory expenditure. table 3: laboratory expenditures for essential laboratory list (ell) tests and non-ell tests within the 11 national health insurance pilot districts, south africa, 2013–2014.† profile of the non-ell tests processed of all the laboratory tests not included on the ell, 21 tests accounted for 91% (r31 million) of the total non-ell laboratory expenditures (r35 million) (figure 1). of these tests, the full blood count (28%) and urea and electrolyte (20%) tests were the main cost drivers for non-ell tests. the third main contributor was the different components of the liver function test, which together accounted for 17% of expenditures for non-ell tests (aspartate transaminase, 5%; total protein, 2%; albumin, 4%; total bilirubin, 3%; direct bilirubin, 2%; and lactate dehydrogenase, 1%). rhesus factor laboratory tests accounted for 10% of the non-ell laboratory expenditures. the remaining non-ell tests accounted for 9% of non-ell expenditure. figure 1: non-ell expenditures within the 11 national health insurance pilot districts, south africa, 2013−2014. analysis included data for the 21 most common tests not on the ell that were billed in financial year 2013/2014, which began on 01 april 2013 and ended 31 march 2014. these 21 tests represented 91% of ‘inappropriate’ (non-ell) laboratory expenditures for the phc facilities within the 11 nhi pilot districts. discussion this retrospective analysis of laboratory expenditure data for fy 2013/2014 indicated that facilities within the 11 nhi pilot districts accounted for approximately r339 million of all phc facility expenditures for diagnostic laboratory tests. the city of tshwane had the highest proportion for test volume and laboratory expenditure, followed by facilities within the or tambo and umgungundlovu districts. diagnostic tests for hiv and tuberculosis were the main cost drivers for laboratory expenditure. of the estimated r339 million total, approximately r35 million (10%) were for non-ell tests. full blood count, urea and electrolyte profiles, as well as liver function tests usually done to support the holistic management of patients on art, were the main cost drivers for non-ell tests. public health expenditures overall are expected to increase by an average of 7.9% between fy 2014/2015 and fy 2016/2017.6 laboratory expenditures are expected to increase by an average of 17.7% during the same time period. this is likely to increase budgetary pressures on an already cash-strapped public health sector and, in particular, on the nhls. however, despite these financial constraints, the nhls is expected to provide or maintain the same standard of service. laboratory, patient, healthcare provider and systemic factors are often cited as potential reasons for ‘inappropriate laboratory tests requests’.7 laboratory factors include prolonged turn-around-times, inability to access results due to the lack of information systems, laboratory request forms that enable the request of a panel test rather than an individual test and the availability of an open-ended, ‘other tests’ box.7 healthcare providers may inappropriately request tests because of inexperience, inadequate understanding or lack of awareness regarding the guidelines or protocols of management, a lack of information about the unit cost of each test, or routine practice.11 additionally, the poor filing systems within health facilities often result in duplicate test requests.7 in 2008, the carter review noted that 25% of pathology tests conducted in the united kingdom national health service were unnecessary7 and the implementation of demand management would conservatively result in a 20% savings in laboratory expenditures.12 our study found that an average of 10% of the laboratory test expenditures at the phc level were for non-ell tests. if use of the demand-management-based ell were extended to district, regional and tertiary hospitals, the savings would be in line with the carter estimate. a conservative estimate based on the 10% savings applied to all 3400 phc facilities across 52 nhls districts would result in an annual savings of r400 million to the health sector. recommendations currently, laboratories are required to perform all tests requested by the clinician and/or nursing staff, resulting in over-utilisation of services. to address inappropriate use of laboratory tests (i.e., non-ell tests) across south africa by phc facilities, three key initiatives are proposed. the first initiative is the development of a national ell. this would require the standardisation of typical clinical laboratory tests per level of healthcare, whilst taking into consideration local demographic and epidemiological factors.9 the second initiative would be to support the ell by developing a dedicated phc laboratory request form that lists only tests appropriate for the phc level. clinicians and nurses would thus be able to select only from amongst tests on the ell. an important aspect of the phc request form design would be to remove the ‘other tests’ box, which enables clinicians to request any investigation. the first two initiatives should, in turn, be supported by the third: electronic gatekeeping to reject tests that are inappropriately requested (not on the ell). however, in order to achieve this initiative, all phc health facilities would be required to use a health information system, including an order entry module with built-in rules to avoid inappropriate ordering. whilst the above measures may help to reduce inappropriate laboratory test requests, appropriate education initiatives directed at health service providers would also be required to support these interventions. these educational sessions should provide guidance on appropriate laboratory testing based on clinical guidelines and evidence-based laboratory medicine recommendations to ensure that specimens are collected in the correct manner.7 limitations this study was a retrospective cross-sectional study and used secondary data on expenditures; it was thus dependent on the accuracy of the data entered into the information systems. it was not possible to differentiate whether tests were coded accurately or combined when multiple individual tests were ordered. in addition, the study was limited to nhi health districts and the results may not be representative of other health districts in south africa. finally, the study focused on one aspect for potential savings. additional studies may be required to investigate other aspects of appropriate utilisation of the diagnostic laboratory services. conclusion this study demonstrated that considerable potential savings of up 10% in laboratory expenditure are possible following the introduction of an ell at the phc level, in addition to further laboratory demand management interventions. acknowledgements competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. sources of support the authors wish to thank sue candy and manfred tepper at the national health laboratory service corporate data warehouse for their assistance and support in extracting the health facility expenditure data. authors’ contributions o.h.m. (university of kwazulu-natal) drafted the article and conducted the analysis. r.l. (university of limpopo) provided technical input on the draft article. s.a. (national department of health) provided editorial comments and technical input on the essential laboratory list. n.c. (national health laboratory service) retrieved the data, conducted the preliminary analysis and drafted the article. references the lewin group, inc. the value of laboratory screening and diagnostic tests for prevention and health care improvement. washington: american clinical laboratory association and advanced medical technology association (advamed); 2009. national health laboratory service. annual report 2014/15. johannesburg: national health laboratory service; 2015. national treasury. estimates of provincial revenue and expenditure [document on the internet]. c2012 [cited 2015 march 11]. available from: http://www.treasury.gov.za/documents/provincial%20budget/2012/4.%20estimates%20of%20prov%20rev%20and%20exp/gt/gt%20-%20epre%20-%20full%20document.pdf. national health laboratory service. annual report-2012–13. johannesburg: national health laboratory service; 2013. mahomed o, asmall s, freeman m. an integrated chronic disease management model: a diagonal approach to health system strengthening in south africa. j healthcare poor underserved. 2014 nov;25(4):1723–1729. http://dx.doi.org/10.1353/hpu.2014.0176 national department of health. primary care 101. pretoria: knowledge translation unit, national department of health; 2008. fryer a, smellie w. managing demand for laboratory tests: a laboratory toolkit. j clin pathol. 2013 jan;66(1):62–72. http://dx.doi.org/10.1136/jclinpath-2011-200524 horvath a. from evidence to best practice in laboratory medicine. clin biochem rev. 2013 aug;34(2):47–60. world health organization. laboratory services for primary health care: requirements for essential clinical laboratory tests. geneva: world health organization, health laboratory technology unit; 1998. national department of health. standard treatment guidelines and essential medicines list for south africa, primary health care level. pretoria: national department of health; 2008. nardi r, berti f, fabbri l, et al. toward a sustainable and wise healthcare approach: potential contributions from hospital internal medicine departments to reducing inappropriate medical spending. ital j med. 2013;7(2):65–81. http://dx.doi.org/10.4081/itjm.2013.65 department of health. report of the second phase of the review of the nhs pathology services in england. london: department of health; 2008. acknowledgements references about the author(s) nqobile ndlovu african society for laboratory medicine, addis ababa, ethiopia yenew k. tebeje africa centres for disease control and prevention, addis ababa, ethiopia renuka gadde clinton health access initiative, boston, massachusetts, united states trevor peter clinton health access initiative, boston, massachusetts, united states citation ndlovu n, tebeje yk, gadde r, peter t. to break the cycle of covid-19 lockdowns and safely open up economies, we must ensure equitable access to diagnosis and treatment. afr j lab med. 2022;11(1), a1650. https://doi.org/10.4102/ajlm.v11i1.1650 opinion paper to break the cycle of covid-19 lockdowns and safely open up economies, we must ensure equitable access to diagnosis and treatment nqobile ndlovu, yenew k. tebeje, renuka gadde, trevor peter received: 17 june 2021; accepted: 10 feb. 2022; published: 27 may 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. in june 2021, sub-saharan africa was facing the worst crisis from coronavirus disease 2019 (covid-19) since the pandemic began, with cases and deaths rising amidst the emergence of new virulent strains of severe acute respiratory coronavirus 2.1 in the midst of this, countries need to ramp up effective prevention and treatment programmes in a way that permits economies to function. these measures include vaccination, physical distancing and wearing masks to protect others, testing and tracing to identify and isolate infected individuals and those who have been in contact with them, and treatment with emerging drug therapies in order to reduce transmission and save lives.2 there is optimism and hope in the global discussion on vaccination and novel therapies. however, effective covid-19 disease control is likely to require that countries jointly balance their investments in vaccines, personal protection, testing and treatment.3,4 testing programmes which have been a backbone of control efforts since the onset of the pandemic, now need to be integrated effectively with other interventions as well as continually improve their efficacy. for example, vaccine rollout must be supported by testing and surveillance data to improve outcomes. the use of routine testing data can help to identify transmission hot spots and to prioritize locations and populations within vaccination campaigns.5 testing data will be important for monitoring the occurrence of outbreaks within populations post-vaccination and to inform ongoing efforts to close residual gaps in coverage, including informing governments on how to develop targeteted approaches for emerging viral variants.5 testing is also needed for the effective use of emerging drug therapies and medical oxygen. these rely on diagnosis and treatment early in the course of disease to achieve the highest benefits in morbidity and mortality.4 in the near future, the combination of rapid tests and new drug regimens will allow the expansion of test and treat programmes that will make covid-19 a manageable infection. in the meantime, to operate schools and workplaces safely and sustainably, it is important to ensure that rapid testing is available where and when it is needed and that we have the tools in place to comprehensively monitor and track the disease.6 current testing efforts in many sub-saharan countries are well below the threshold of one test per 1000 population per week set by the world health organization.7 this means that not only is it not clear how many infections have occurred, but there is limited insight into the emergence and spread of new outbreaks. there are ongoing efforts to better understand vaccine variants through genomic sequencing but there are limitations. for example, sequencing technologies are still not widely available and they often do not provide real-time information on variant outbreaks to help limit spread.8 without the ability of individuals and public health programmes to understand their infection status and take actions limit disease spread, the current cycle of covid-19-related restrictions on work, school and travel will continue to threaten africa’s economies, increasing poverty and hunger, and reversing gains made in recent decades. that is why it is critical for the global health community, governments, and the private sector to come together to ensure that accurate, rapid antigen diagnostic tests (rdts) are accessible at community level, including self-tests. rapid antigen diagnostic tests must be available in all primary health care facilities and made available through public and private channels for self testing, and must be appropriately used to help reopen borders, schools, and places of work and worship.9,10 examples of this work were presented in a recent series of webinars organised by the african society for laboratory medicine and the africa centers for disease control and focused on expanding access to antigen rdts for covid-19.9 leading scientists and public health experts from africa, europe, the united states and asia presented the latest updates on testing and covid-19 control. in rwanda, covid-19 rdts are being used in public and private health facilities, at weddings, schools, prisons, churches, market places, as well as for refugees and others recommended by rwanda’s ministry of health. this has helped treble the number of testing locations and reduce testing costs. in south africa, rdts have been widely used in open border posts (land, sea, and air) and are being used within the provinces of the country. in nigeria, scaled-up use of rdts within national youth camps has made it possible to quickly detect cases and isolate youth as they entered the camps, thus preventing potential outbreaks.9 schools provide one of the best opportunities for testing and other strategies to prevent transmission. the most important lines of defence in preventing transmission of severe acute respiratory coronavirus 2 include making sure students do not come to school with symptoms, enforcing universal mask adherence and physical distancing, improving ventilation, and swiftly investigating any cases associated with schools, including contact tracing and quarantine and isolation.6 testing is another tool in the prevention arsenal for schools and can provide guidance on how to plan better prevention and increase layers of defense. testing also ensures early identification of cases among students and staff and helps with contact tracing efforts, and can also identify infection among students and staff at high-risk of developing severe disease due to underlying conditions and support investigations and studies in understanding the role children play in disease transmission. testing must reflect the purposes and resources of a community. sweeping guidance that schools should be closed if the positivity rate exceeds a certain threshold needs to be reviewed in the context of a country. for example, if a school is conducting all in-person classes and the prevalence in community increases, it may consider reducing class sizes and having students attend on alternating schedules. in other words, a deeper analysis should guide decision making. there is mounting evidence of the detrimental effects of school closures and the impact it is having on children from learning loss to emotional wellness.10 one can argue that schools should be in the same category as essential services, similar to healthcare facilities, in that they should only close when there is no other option. in september 2020, several organisations, including the world health organization, the global fund, the africa centres for disease control and prevention, find and others, announced an agreement to make available 120 million low-cost, high-quality antigen rdts for covid-19 for lowand middle-income countries.11 while this was necessary to ensure that tests were available, adoption and use of antigen testing was slow, and many countries implemented these professional-use tests widely within health facilities and at the community level (i.e., for symptomatic and high-risk individuals). high-performing self-tests are now more widely available and provide opportunities to expand access to testing, decongest health facilities, and limit the risk of transmission.12 it is important that countries identify the most effective use for both professional use rapid tests and self tests to be deployed within public testing programmes and for private use.13 now is not the time to debate whether we should be spending on vaccines, oxygen, personal protective equipment, drug treatment, or testing. effective programmes need to strategically deploy each of these interventions in an integrated way, just as integrated prevention, testing and treatment control programmes have been developed for other diseases. fortunately, antigen rdts with excellent performance are available and can be used at the point of care or in laboratory settings, and are increasingly available for self testing.12,14 even though much progress has been made in recent years, gaps in access to essential tests remain an issue throughout africa, including covid-19. while robust systems to deliver testing and surveillance for covid-19 are developed, it is important to ensure that they are sustainable into the future and also address other major health issues, including malaria, hiv, tuberculosis, cervical cancer and other diseases. maintaining a joint focus on prevention, testing and treatment will be necessary, building on partnerships between governments, donors, policymakers and industry to use the available tools to solve this global crisis. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions n.n. contributed to the planning and execution of the webinars that generated the basis of the paper. in addition, he contributed to the conceptualisation and the design of the write-up, reviewed and edited the paper. y.k.t. also contributed to the planning and execution of the webinars that generated the basis of the paper. in addition, he contributed in the conceptualisation and the design of the write-up, reviewed and edited the paper. r.g. contributed to the original conceptualisation, writing of the paper and execution of the webinars that generated the basis of the paper. in addition, he contributed to the design of the write-up, reviewed and edited the paper. t.p. contributed to the planning and execution of the webinars that generated the basis of the paper. in addition, he contributed to the design of the write-up, reviewed and edited the paper. ethical considerations this article followed all ethical standards for research. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for profit sectors. data availability data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references burke j. african nations fear more covid-19 deaths before vaccination begins [homepage on the internet]. the guardian, 04 february 2021. [cited 2021 mar 23]. available from: https://www.theguardian.com/global-development/2021/feb/04/african-nations-fear-more-covid-deaths-before-vaccination-begins noorbhai h. a mathematical model to guide the re-opening of economies during the covid-19 pandemic. ann med surg (london). 2020;57:5–6. https://doi.org/10.1016/j.amsu.2020.06.041 majumder j, minko t. recent developments on therapeutic and diagnostic approaches for covid-19. aaps j. 2021;23(1):14. https://doi.org/10.1208/s12248-020-00532-2 world health organization. who recommends two new drugs to treat covid-19 [homepage on the internet]. [cited 2020 jan 30]. available from: https://www.who.int/news/item/14-01-2022-who-recommends-two-new-drugs-to-treat-covid-19 koks s, williams rw, quinn j, et al. covid-19: time for precision epidemiology. exp biol med (maywood). 2020;245(8):677–679. https://doi.org/10.1177/1535370220919349 dzinamarira t, musuka g. the paradox of re-opening schools in zimbabwe in the covid-19 era. public health pract 2021;2:00070. issn 2666-5352. https://doi.org/10.1016/j.puhip.2020.100070 jani iv, peter tf. nucleic acid point-of-care testing to improve diagnostic preparedness. clin infect dis. 2022; ciac013. htttps://doi.org/10.1093/cid/ciac013 otu a, agogo e, ebenso b. africa needs more genome sequencing to tackle new variants of sars-cov-2. nat med. 2021;27:744–745. https://doi.org/10.1038/s41591-021-01327-4 african society for laboratory medicine. special covid-19 echo sessions 37 [homepage on the internet]. 2021 [cited 2021 mar 23]. available from: https://aslm.org/resource/special-covid-19-echo-session-37/ chen ih, chen cy, pakpour ah, griffiths md, lin cy. internet-related behaviors and psychological distress among schoolchildren during covid-19 school suspension. j am acad child adolesc psychiatry. 2020;59(10):1099–1102.e1. https://doi.org/10.1016/j.jaac.2020.06.007 world health organization. global partnership to make available 120 million affordable quality covid-19 rapid tests for low and middle income countries [homepage on the internet]. 2020 [cited 2022 jan 30]. available from: https://www.who.int/news/item/28-09-2020-global-partnership-to-make-available-120-million-affordable-quality-covid-19-rapid-tests-for-low--and-middle-income-countries boum y, eyangoh s, okomo mc. beyond covid-19 – will self-sampling and testing become the norm? lancet infect dis. 2021;21(9):1194–1195. https://doi.org/10.1016/s1473-3099(21)00197-3 africa cdc. new guidance to expand rapid antigen testing for covid-19 response in africa released [homepage on the internet]. 2020 [cited 2022 jan 30]. available from: https://africacdc.org/news-item/new-guidance-to-expand-rapid-antigen-testing-for-covid-19-response-in-africa-released/ peeling rw, olliaro pl, boeras di, fongwen n. scaling up covid-19 rapid antigen tests: promises and challenges. lancet infect dis. 2021;21(9):e290–e295. https://doi.org/10.1016/s1473-3099(21)00048-7 abstract introduction methods results discussion acknowledgements references about the author(s) faithful makita-chingombe international pharmacology specialty laboratory, school of pharmacy, university of zimbabwe college of health science, harare, zimbabwe andrew j. ocque center for integrated global biomedical sciences, school of pharmacy and pharmaceutical sciences, university at buffalo, translational pharmacology research core, new york state center of excellence in bioinformatics and life sciences, the state university of new york, buffalo, new york, united states robin difrancesco center for integrated global biomedical sciences, school of pharmacy and pharmaceutical sciences, university at buffalo, translational pharmacology research core, new york state center of excellence in bioinformatics and life sciences, the state university of new york, buffalo, new york, united states charles maponga international pharmacology specialty laboratory, school of pharmacy, university of zimbabwe college of health science, harare, zimbabwe center for integrated global biomedical sciences, school of pharmacy and pharmaceutical sciences, university at buffalo, translational pharmacology research core, new york state center of excellence in bioinformatics and life sciences, the state university of new york, buffalo, new york, united states farai muzambi international pharmacology specialty laboratory, school of pharmacy, university of zimbabwe college of health science, harare, zimbabwe tsitsi g. monera-penduka international pharmacology specialty laboratory, school of pharmacy, university of zimbabwe college of health science, harare, zimbabwe tinashe mudzviti international pharmacology specialty laboratory, school of pharmacy, university of zimbabwe college of health science, harare, zimbabwe takudzwa j. mtisi international pharmacology specialty laboratory, school of pharmacy, university of zimbabwe college of health science, harare, zimbabwe gene d. morse center for integrated global biomedical sciences, school of pharmacy and pharmaceutical sciences, university at buffalo, translational pharmacology research core, new york state center of excellence in bioinformatics and life sciences, the state university of new york, buffalo, new york, united states citation makita-chingombe f, ocque aj, difrancesco r, et al. development and validation of a high performance liquid chromatography method to determine nevirapine in plasma in a resource-limited setting. afr j lab med. 2019;8(1), a880. https://doi.org/10.4102/ajlm.v8i1.880 lessons from the field development and validation of a high performance liquid chromatography method to determine nevirapine in plasma in a resource-limited setting faithful makita-chingombe, andrew j. ocque, robin difrancesco, charles maponga, farai muzambi, tsitsi g. monera-penduka, tinashe mudzviti, takudzwa j. mtisi, gene d. morse received: 26 july 2018; accepted: 02 nov. 2018; published: 16 may 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: there are several instances where nevirapine pharmacokinetic monitoring may be useful, such as in special populations or pharmacokinetic drug interaction studies that require the ascertainment of nevirapine pharmacokinetics in the sub-saharan region. objectives: the main aim of this study was to produce a validated, sustainable and relevant nevirapine assay method that meets bio-analytical regulatory requirements. methods: the developed method utilised a waters 2795 alliance high performance liquid chromatography system with a 2996 photo diode array detector, an atlantis dc18 5 micron, 3.9 mm × 150 mm analytical column and a gradient flow rate of 1 ml/min. ultraviolet detection data were collected from 210 nm to 400 nm, extracted at 260 nm, and processed for nevirapine and internal standard peak height responses. results: the method proved to be linear (r2 0.995), precise (+1.92% – +9.69%) and accurate (-9.70% – 12.0%). recovery for the analyte and internal standard was between 98.8% and 114%. the method showed good specificity as no interferences were caused by common african traditional medicines, anti-tuberculosis medications or other concomitant antiretrovirals nor endogenous components. conclusion: the method is reproducible, relevant to our setting and uses considerably low plasma volumes with preservation of some consumables, a desirable key factor in a resource-limited setting. keywords: high performance liquid chromatography; nevirapine determination; method development and validation. introduction while nevirapine is no longer the preferred first-line non-nucleoside reverse transcriptase inhibitor for the management of hiv, it is still relevant as an option in zimbabwe. nevirapine is also available for use in hiv-exposed neonates for the prevention of mother-to-child transmission.1 in this regard, a validated method to determine nevirapine concentrations in plasma is desirable. research initiatives supported by several united states national institutes of health programmes, experienced mentors and commercial donations have contributed to the establishment of a clinical pharmacology laboratory to support hiv research in zimbabwe.2 however, method development and validation present resource and infrastructural challenges in resource-limited settings (rls). many limitations need to be considered during method development. the cost of equipment and availability of reagents and supplies are major determinants affecting the feasibility and sustainability of the method. patient factors such as concomitant medications and blood testing volumes must also be considered to assure method sensitivity and specificity challenges. there is evidence indicating that in african settings, people on antiretrovirals also utilise traditional herbal medication.3 high performance liquid chromatography (hplc) assays become compromised with introduction of diverse excipients, metabolites or active compounds eluting at the same retention time and absorbing at the same wavelength as the analyte.4 therefore, assessing for interference of concomitant antiretrovirals, traditional herbal medicines and medications for potential opportunistic diseases, such as anti-tuberculosis drugs, is relevant when developing assay specificity in rls. the sample matrix itself should also have minimal method interference. due to the need for frequent monitoring, hiv-positive patients will also have several tests that require drawing of blood.5 therefore, to minimise blood collection volumes, a sensitive assay method accommodating small sample volumes should be pursued. when preparing a sample for assay, extraction and concentrating steps can be optimised to improve method sensitivity. different techniques for extraction of analyte from the plasma matrix are employed. these vary from an expensive solid phase extraction (spe) such as cartridge extraction to a less expensive extraction approach such as protein precipitation using solvents. furthermore, a cost-effective but efficient sample concentration technique is an important factor. techniques to concentrate samples vary from nitrogen-dependent evaporation to centrifugal force applications. nitrogen evaporation is employed in some methods.6,7 however, this has limited application in rls due to the difficulty in accessing nitrogen gas. in addition, in rls reagents and materials are usually expensive due to the need for importation, which contributes to increased assay costs. sustainability of an assay also depends on the stability of these reagents. mass spectrometry-based methods have been employed in plasma assays for antiretrovirals in several pharmacology and pharmaceutical laboratories.8,9 these assays offer greater sensitivity and specificity when compared to ultraviolet-based detection.10,11,12 however, mass spectrometry-based analytical systems are expensive to acquire and maintain.13 furthermore, to avoid matrix effects frequently encountered with clinical plasma samples, costly internal standards are required. hplc with ultraviolet detection becomes a viable option when considering an assay for nevirapine in patients taking nevirapine as prescribed, because the plasma concentrations are fairly high and detectable by ultraviolet-based methods.14 despite limited resources, methods developed should still abide by regulations on method validation and be as effective (e.g., appropriate range of quantitation and specificity) as assaying the same analyte by mass spectrometry. bio-analytical method guidelines issued by national agencies accepting drug submissions as well as medical laboratory accreditation agencies provide key support for assuring laboratory performance measures. therefore, developing the capacity to monitor antiretroviral concentrations within local research institutions requires striking a balance between cost effectiveness and development of a robust method that ultimately does not compromise the integrity of sample results.10 the aim of this study was to develop and validate a suitable hplc method, using ultraviolet detection to determine nevirapine concentrations in human plasma in harare, zimbabwe, a resource-limited setting. the method was required to meet the standard requirements for sample analysis used to support clinical trials that incorporate nevirapine plasma concentration as an outcome measure.15 methods ethical considerations the method application was done using samples collected in 2013 at parirenyatwa hiv opportunistic infections clinic, harare, zimbabwe for a project trial approved by the joint research ethical committee 130/10 in harare, zimbabwe, and the medical research council of zimbabwe /b/255 in harare, zimbabwe (registration number nct01410058). method development chemicals, reagents, and equipment all antiretrovirals used (nevirapine, indinavir, efavirenz, atazanavir, ritonavir, lopinavir, zalcitabine, lamivudine, tenofovir, zidovudine, stavudine, abacavir and emtricitabine) were obtained from the national institutes of health aids research and reference reagent programme. nevirapine powder had a stated purity of 90% and indinavir powder, which was used as the internal standard (is), had a stated purity of 89%. human, heparinized plasma was donated by the national blood transfusion services (harare, zimbabwe). hplc-grade methanol and acetonitrile were purchased from microsep, romil pure chemistry (johannesburg, south africa). reverse osmosis water was produced from an elga purelab® water purifier (harare, zimbabwe). the hplc analyses were performed using a waters 2795 separation module (waters technologies corporation, milford, massachusetts, united states) equipped with waters 2998 pda detector and empower software (version 3; waters associates, milford, massachusetts, united states). instrumental and analytical conditions hplc, as described above, employing a waters atlantis dc18 3.9 mm × 20 mm, 5 µm preceded by a guard column was used. the final mobile phase consisted of (1) 10 mm ammonium acetate, acetonitrile and methanol (60:25:15) ph4 and mobile phase (2) 10 mm ammonium acetate, acetonitrile and methanol (20:50:30) ph4 and was delivered using the following gradient scheme: 0–3 min (100% 1), 4 min – 7 min (100% 2), and 7.01 min – 10 min (100% 1). the flow rate was 1 ml/min. the auto-sampler was set at ambient temperature and the column oven was maintained at 40 °c. the injection volume of sample was 65 µl. scanning was from 210 nm to 400 nm with data extracted at 260 nm for nevirapine and indinavir using empower software version 3. these conditions were used for method validation carried out based on the united states food and drug administration guidelines.15 sample treatment five mg of nevirapine and is reference grade powders were individually weighed on a calibrated analytical balance. both drugs were separately dissolved in 100% methanol and dilutions for their working standards were done in 50:50 methanol:water. is was spiked into calibration standards, quality controls or unknowns prior to extracting nevirapine by protein precipitation using cold acetonitrile. initial testing of plasma sample amounts ranged from 100 µl to 250 µl with various adjustments of precipitation solution. samples were then vortexed and centrifuged for 10 min at 7852 × g. a volume of the supernatant was transferred to a culture tube and evaporated in a centrivaptm (vwr labconco, kansas city, missouri, united states) benchtop centrifugal vacuum evaporator at 50 °c until dry. the dried sample was reconstituted with 200 µl mobile phase before being transferred to a polypropylene insert and placed in an auto-sampler vial. the inserts and vial tops were discarded after use while the vials were preserved for reuse. chromatography using various columns and different mobile phase conditions, optimisation of chromatographic separation of nevirapine and is was performed. sample carryover was ruled out by injecting mobile phase after injections of prepared plasma samples at the following nevirapine concentrations: 500 ng/ml, 1000 ng/ml, 5000 ng/ml and 10 000 ng/ml. mobile phase chromatograms of several sources of nevirapine-free plasma samples were inspected to ensure absence of peaks within the retention time window of both nevirapine and is. a concentration of 500 ng/ml was indicated as the lowest calibrator value with analyte response greater than 20% of the blank response. consequently, the lowest calibrator was the limit of quantification (loq) as per fda guidance. repeatability assessments were done at loq by carrying out six injections at an injection volume of 65 µl. method validation calibration curves and quality control preparation calibration concentrations were prepared from nevirapine stocks weighed separately from the stock used for quality controls (qc). calibration concentrations of 15 000 ng/ml, 13 000 ng/ml, 8000 ng/ml, 4000 ng/ml, 2000 ng/ml, 1000 ng/ml and 500 ng/ml were prepared based on the therapeutic range for nevirapine in plasma.16,17,18 the lower loq (lloq) was prepared at 500 ng/ml, lower quality control (lqc) at 1500 ng/ml, middle quality control (mqc) at 5000 ng/ml and highest quality control (hqc) at 12 000 ng/ml by dilution of stock solution into blank human plasma. intra and inter-day precision and accuracy of calibration curve, lower limit of quantification and quality controls a calibration curve was constructed for measuring every batch of validation samples with 1/x2 weighted linear regression. six samples were tested at each qc level including lloq on 3 days; a different analyst performed the analysis each day. accuracy and precision of dilution was measured using a prepared high out of quantitation quality control (hoq) at 45 000 ng/ml. dilution of the hoq was carried out in two different dilutions, 1:4 and 1:8 using blank plasma as diluent. stability freeze-thaw, reinjection and room temperature stability were determined at low and high qc concentrations in triplicate and compared to freshly prepared controls stored at (-70 °c). room temperature stability involved leaving the samples on the bench top for 6 h. freeze-thaw was performed over three freeze (-70 °c) and thaw (room temperatures) cycles. reinjection stability samples were assessed by re-injecting samples stored at 4 °c for 168 h. specificity and selectivity selectivity and specificity were investigated by assessing six different blank plasma samples and monitoring for appearance and potential interference by endogenous compounds. interference from other antiretrovirals (stavudine, tenofovir, abacavir, lamivudine, zidovudine, emtricitabine, atazanavir, ritonavir, lopinavir, efavirenz, dideoxycytidine), anti-tuberculosis drugs (isoniazid, rifapentine, pyrazinamide, desacetylrifapentine) and herbal supplements allium sativum (garlic) and moringa oleifera lam. were tested by spiking each component separately in blank plasma. twenty µl of 10 µg/ml for antiretrovirals and 50 µg/ml for anti-tuberculosis drugs were spiked into 180 µl of plasma and processed as described in the method. one whole syzygium aromaticum (clove) and one gram each of allium sativum (garlic) and moringa oleifera lam. were ground before extraction in methanol. after centrifugation for 15 min, 20 µl of the extract supernatant was spiked into 180 µl plasma before the sample was processed. individual component chromatograms were overlaid with lloq chromatograms. recovery recovery was performed at the hqc and lqc levels in six independent lots of human edta plasma (tests) and compared to the response observed in water (controls). to establish a control, plasma was replaced with water and the sample treated as described. method application the method has been routinely used in proficiency testing in an external quality assurance programme conducted by the united states national institute of allergies and infectious disease and the division of aids clinical pharmacology quality assurance programme, which conducts proficiency testing assessments for clinical pharmacology laboratories under the national institutes of health hiv research network.19 methodology utility was demonstrated by assessing reproducibility within ±20% by re-analysis of four patient samples from a previously reported investigation on effect of moringa oleifera lam. leaf powder on the pharmacokinetics of nevirapine in hiv‑positive adults.20 patients were on nevirapine 200 mg twice daily and samples were taken at timed intervals after consumption of moringa oleifera lam. (1.85 g). the trial registration number is nct01410058, jrec 130/10, mrcz/b/255. results method development sample treatment was successful using protein precipitation (400 µl acetonitrile), and a minimal amount of plasma sample (180 µl) was sufficient for the method. centrifugal vacuum evaporation, which improved sensitivity, was successfully employed using 550 µl of the sample supernatant (85% volume) using the centrivaptm. using auto-sampler vial inserts preserved the containment vials for reuse thus lowering consumable costs. gradient conditions at ph4 were ideal in improving sensitivity and selectivity. desirable retention times of 4.5 min for nevirapine and 5.9 min for indinavir were achieved within a run time of 10 min. chromatographic separation of nevirapine and is from each other and from endogenous plasma components was shown by an overlay of blank and lloq chromatograms (figure 1). absence of sample carry over and high repeatability (5.39%) indicated that the method was ready to move to the validation phase. figure 1: chromatograms of blank plasma (a) and blank plasma spiked with internal standard and nevirapine (b) showing no endogenous components interference at nevirapine and internal standard retention times. method validation the calibration curve was linear over the calibration range of 500 ng/ml – 15 000 ng/ml. an example of a calibration curve obtained for nevirapine is illustrated in figure 2. where a calibrator level other than the lloq was not within ±15%, the calibrator was omitted and the curve recalculated. the accuracy range was between −8.13 and +8.83% with a precision of < 12%. precision and accuracy of the qc samples were within acceptance criteria of united states food and drug administration and are summarised in table 1. precision was < 9.69% and accuracy ranged from −9.70 to +12.0% for lqc, mqc and hqc. lloq precision was < 7.53% and accuracy was +2.76% to +16.1%. hoq precision was < 2.84% and accuracy was +4.96% to +5.15% whether diluted 1:4 or 1:8. table 1: inter and intra day accuracy and precision of quality control samples at university of zimbabwe international pharmacology laboratory, 2016. figure 2: representative nevirapine calibration curve for inter-day precision and accuracy. the deviations from nominal concentrations for freeze-thaw, room temperature and reinjection stability were also within acceptable limits (table 2). none of the concomitant medications (figures 3, 4 and 5) interfered with the analyte or is, including additional antiretrovirals other than those taken in combination with nevirapine. achieved recovery for the analyte ranged from 103% to 114%, while the indinavir is recovery ranged from 98.8% to 113%. table 2: nevirapine stability over different conditions at university of zimbabwe international pharmacology laboratory. figure 3: chromatogram overlay of lloq with antiretrovirals, no interference was observed at nevirapine and internal standard retention times for all antiretrovirals. figure 4: chromatogram overlay of lloq with anti-tuberculosis drugs; no interference was observed at nevirapine and internal standard retention times. figure 5: chromatogram overlay of lloq with herbs, no interference at nevirapine and internal standard retention times was observed in all herbs. method application all samples analysed in the clinical pharmacology quality assurance programme external proficiency tests using this validated method were acceptable (accuracy from target values within 20%). the percentage bias for this method from the previously assayed patient samples was within 20% of reported values (-13% to +17% difference); an example of a patient chromatogram is shown in figure 6. figure 6: chromatogram of patient sample. (samples collected at parirenyatwa opportunistic infections clinic in 2013 and assayed at university of zimbabwe international pharmacology laboratory). discussion an optimised hplc-ultraviolet-based method for nevirapine determination in plasma was successfully developed and validated within a resource-limited setting. optimisations in assay development aimed at attaining a desirable chromatogram, ultraviolet detection wavelength, resolution and retention times for both the analyte and is in human plasma matrix. a minimal amount of 180 µl plasma was sufficient compared to other hplc-based nevirapine assay methods that utilised 500 µl or more.21,22,23 low sample amounts are desirable due to the need for frequent monitoring of hiv patients4 and the need for additional laboratory tests during antiretroviral treatment. while most sample treatment methods use spe cartridges, protein precipitation has been used with success by other researchers.24,25 even though spe may result in a cleaner sample, the technique requires consumable spe products and supporting interfaces such as manifolds and vacuum pumps that come at a greater cost than this method. employing centrifugal vacuum evaporation resulted in improved sensitivity and excluded the use of nitrogen gas, which is not locally produced. to reduce column burden and sustain longer column life, an inexpensive guard column was placed in line prior to the column. in addition, the method is designed to increase organic components in the gradient, helping to remove endogenous components and debris from the column. the analysis run time was shorter than that observed in other gradient or isocratic hplc-based nevirapine assay methods.21,22,23 the ph controlled mobile phase composition of the gradient method capitalised on the high solubility of nevirapine in organic solvents to achieve an early elution of nevirapine.26 the observed nevirapine and indinavir stability under different environmental subjections was reflective of analyte and is stability in plasma as proved by other scholars.27 to assure specificity of the method, several regional considerations were: national formularies for co-administered hiv antiretrovirals, as well as treatments for common co-infections and the local use of herbal supplements particularly moringa oleifera lam. and allium sativum (garlic).3 in that regard, and as achieved, other antiretrovirals, anti-tuberculosis drugs and indicated herbs should not interfere with or compromise the assay method. observed recoveries were comparable to those achieved by other extraction methods, for example by spe.28,29 the results during method application further confirmed the method’s specificity. conclusion a valid method to measure nevirapine in plasma was developed by using hplc-ultraviolet detection and resource-conserving techniques while maintaining the sensitivity, specificity, selectivity, accuracy and precision needed to monitor nevirapine at therapeutic ranges. the described method is evidence that despite limited resources, capitalisation of viable resources enables establishment of effective drug analysis methods relevant to rls. acknowledgements the authors thank waters technologies corporation, united states, for hplc donation and the translational pharmacology research core, buffalo, new york, united states, for laboratory equipment donations. the authors also thank laboratory technicians alfred tarumbwa and lorellie mungure for their support and efforts during the development and validation of this method. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. source of support research reported in this publication was supported by the university of zimbabwe international pharmacology specialty laboratory, funded by the united states national institute of allergy and infectious diseases of the national institutes of health under award numbers um1 ai068634, um1 ai068636 and um1 ai106701. the project described was supported by the university at buffalo – university of zimbabwe hiv research training program, funded by the fogarty international center under award number d43 tw010313. the content is solely the responsibility of the authors and does not necessarily represent the official views of the fogarty international center or the national institutes of health. authors’ contributions f.m-c. was the project leader and a.j.o. and r.d. were responsible for project design. f.m. performed most of the experiments. t.g.m-p., t.j.m. and t.m. made conceptual contributions to the manuscript. c.m. and g.d.m. critically revised intellectual content and approved the final version to be published. disclaimer the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institutes of health. references who. consolidated guidelines on the use of antiretroviral drugs for 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clinical pharmacology and quality assurance, stability of arvs in spiked plasma held at −70 °c. the research foundation on behalf of the state university of new york, university at buffalo, 2016. charbe n, baldelli s, cozzi v, castoldi s, cattaneo d, clementi e. development of an hplc–uv assay method for the simultaneous quantification of nine antiretroviral agents in the plasma of hiv-infected patients. j pharm ana. 2016;6(6):396–403. https://doi.org/10.1016/j.jpha.2016.05.008 fan b, stewart jt. determination of zidovudine/lamivudine/nevirapine in human plasma using ion-pair hplc. j pharm biomed anal. 2002;28(5):903–908. https://doi.org/10.1016/s0731-7085(01)00708-7 abstract introduction methods results discussion acknowledgements references about the author(s) erius tebuka department of pathology, weill bugando school of medicine, mwanza, united republic of tanzania bugando medical centre, mwanza, united republic of tanzania mwesige charles central pathology laboratory, bugando medical centre, department of hematology, mwanza, united republic of tanzania jeffer o. bhuko department of pathology, weill bugando school of medicine, mwanza, united republic of tanzania bugando medical centre, mwanza, united republic of tanzania mwanza region health center, mwanza, united republic of tanzania citation tebuka e, charles m, bhuko jo. prevalence and risk factors for red blood cell alloimmunisation among sickle cell patients in mwanza city, tanzania. afr j lab med. 2020;9(1), a823. https://doi.org/10.4102/ajlm.v9i1.823 original research prevalence and risk factors for red blood cell alloimmunisation among sickle cell patients in mwanza city, tanzania erius tebuka, mwesige charles, jeffer o. bhuko received: 01 may 2018; accepted: 05 mar. 2019; published: 10 sept. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: erythrocyte alloimmunisation can lead to complications such as delayed haemolytic transfusion reaction. objective: this study investigated the prevalence of and risk factors for red blood cell alloimmunisation among multiply transfused sickle cell disease (scd) patients in mwanza city, tanzania. methods: from may 2017 to july 2017, this descriptive, cross-sectional, hospital-based study enrolled 200 participants with scd who had received at least two units of blood in the previous year. blood count was performed using a sysmex haematology analyser. antibody screening was done by the tube method using a panel of three screening cells with known antigenicity. results: of the 200 patients enrolled, 108 (54%) were female. the median age was 4.5 years (interquartile range [iqr] = 6), the median number of transfusions was 3 (iqr = 1), and the median pre-transfusion haemoglobin level was 6.6 g/dl (iqr = 2.7). prevalence of alloimmunisation was 8.5% (17/200) with immunoglobulin g, and one patient developed cold immunoglobulin m antibodies. blood groups reported were rhesus c and e, kell, kidd and duffy. there was no statistically significant association between the number of transfusions and the risk of alloimmunisation. conclusion: the rate of alloimmunisation in multiply transfused scd patients was 8.5% and higher than other studies in east africa. thus, there is a need for extensive red blood cell screening and matching to minimize alloimmunisation and risk of delayed haemolytic transfusion reaction, particularly in scd and chronically transfused patients. keywords: sickle cell disease; alloimmunisation; alloantibody; screening cells; bugando medical centre; catholic university of health and allied sciences; red blood cells. introduction sickle cell disease (scd) is an inherited haemoglobin disorder; it is a genetic disease with high prevalence in the equatorial regions of africa, arabia, europe, and india. globally, approximately 300 000 people are born with scd annually.1 blood transfusions are necessary for the care and treatment of patients with scd.2,3 in africa, including tanzania, and the middle east, the abo and rhesus d blood grouping system are as the test parameters for blood transfusion, neglecting other red blood cell surface antigens, which are considered minority blood groups. however, this can cause serious complications, including alloimmunisation in scd patients, once a transfusion is done, without a thorough and complete screening of the antigenicity of both the donor and the recipient.4 alloimmunisation is the body’s response to foreign antigens after being subjected to cells or tissues with different antigenicity.5 complications of alloimmunisation include life-threatening conditions such as delayed hemolytic transfusion reactions, immediate agglutination, hyper hemolysis2 and autoimmune responses in individuals having blood transfusions, especially scd patients. studies have shown that antigenicity matching, for scd and chronically transfused patients, is important for decreasing the frequency of alloimmunisation and its associated complications6. the presence of alloantibodies is a major complication in scd, presenting challenges for medical management. antigen matching beyond the standard abo blood grouping system and rhesus typing has reduced complication rates.6 according to bashawri et al., race and ethnicity could be a risk factor for alloimmunisation in sickle cell anaemia patients.7 additionally, failure to screen for minor antigens such as kidd, duffy, and mns blood groups, might cause a mismatch and subsequent alloimmunisation in blood recipients.8. current practice in tanzania is forward grouping, which screens for blood groups abo and rhesus d. however, there are currently no guidelines for blood group screening at the national blood transfusion services. thus, patients are typically transfused without an extensive matching for compatibility. for example, no screening for kell antigens is conducted. additionally, little is known about the extent to which alloantibodies affect sickle cell patients locally. this study aimed to examine the prevalence of developing red blood cell alloimmunisation among multiply transfused scd patients and identify its associated risk factors, especially in mwanza (lake zone). this will, in turn, be valuable for the setting up of guidelines for extensive donor and recipient typing, especially for transfusion-dependent patients at bugando medical centre (bmc)/lake zone national blood transfusion services laboratories. methods ethical considerations ethical clearance was received from the catholic university of health and allied sciences and bmc joint research committee (ethical clearance number: 323/2017). permission to work in the bmc central pathology laboratory was also sought from the laboratory director and manager. written informed consent was sought from both inpatient and outpatient scd with multiple transfusions before including them in the study. study design this was a cross-sectional study conducted from may 2017 to july 2017 at the bmc, mwanza city (lake zone), tanzania. participants were inpatients and outpatients with scd who attended the bmc during the study period and consented to participate. patients were recruited until the estimated minimum sample size of 200 scd patients was reached; the sample size was estimated using the kishi lisle formula. the study included all scd patients attending bmc inpatient and outpatient departments for transfusion therapy purposes who had received at least two units of blood during the previous year the study excluded patients with no blood transfusion history and patients who did not consent to participate. a structured questionnaire was completed by the parent or guardian of the child and laboratory records confirmed scd, number of transfusions and interval of blood transfusion. the questionnaire was used to gather the demographic and medical history information of selected participants. sample collection one 4-ml blood sample was collected from each patient by phlebotomists at the central pathology laboratory at bmc into ethyldiammine tetraacetate tubes to avoid agglutination and preserve red blood cell (rbc) antigenicity. samples were stored at 2 °c – 6 °c refrigeration for not more than 3 days. laboratory analysis alloantiboy detection – indirect coombs test the presence of alloantibodies was determined by assessing antigen-antibody agglutination in patient serum where agglutination indicates incompatibility of patient’s serum antibodies with commercially purchased red cells. three commercially purchased red cell-antibody screening cell panels were used for screening the parted blood cells (screen cell 1, product code r1wr1; screen cell 2, product code r2r2; and screen cell 3, product code rr) (lorne laboratories, reading, berkshire, united kingdom). screening for the different rbcs antigens was done as described by the manufacturer. screen cell 1 had c, d, e, cw, m, n, s, p1, k, k, leb, fya, jka, and jkb red cells. screen cell 2 had d, e, c, m, s, p1, k, leb, fyb, and jkb red cells, and screen cell 3 had c, e, n, s, p1, k, kpa, lea, fyb, and jka red cells (table 1). a mixture of patient serum and the commercial red cell-antibody screening cells was centrifuged for 10 minutes at room temperature (25 °c). tubes were checked for agglutination confirming cold alloantibodies (immunoglobulin m). then low ionic strength solution was added to enhance antigen-antibody reaction, followed by incubation at 37 °c for 15 min to detect warm antibodies. after incubation, a cell wash was done by using a washing buffer to remove unbounded antibodies. finally, anti-human globulin (coombs reagent) was added to the tubes to detect the presence of alloantibodies. the tubes were then centrifuged at 1500 revolutions per minute for 3 min. the centrifuged mixture of patient serum, commercial red cells and anti-human globulin was read microscopically to assess the presence of agglutination. tubes with agglutination were considered ‘positive’, whereas tubes with no agglutination were considered ‘negative’. agglutination was classified as ‘strong’ when agglutination could be seen macroscopically or as ‘mild’ when agglutination could only be confirmed microscopically. alloantibodies were then classified as ‘warm’ alloantibodies (antibodies that react at or near 37 °c) or as ‘cold’ alloantibodies (antibodies that react below 37 °c). table 1: screening cells used and blood groups tested for alloantibodies, bugando medical centre, may 2017–june 2017.† autoantibody detection – direct antiglobulin test to rule out autoantibodies, which were not part of the study but can affect test results, a direct antiglobulin test was done. briefly, the patient’s red blood cells were washed three times with normal saline. two drops of anti-human globulin (coombs reagent) was added to the washed cells, mixed and centrifuged at 1500 revolutions per minute for 3 min. the centrifuged mixture of patient rbc and anti-human globulin was read microscopically for agglutination. tubes with agglutination were considered ‘positive’ for autoantibodies, whereas tubes with no agglutination were considered ‘negative’ for autoantibodies. to rule out anti-human globulin non-reactivity and validate negative results (tubes without agglutination), pre-sensitised commercial rbcs (lorne laboratories; reading, berkshire, united kingdom) coated with short-armed antibodies were added. if agglutination was observed, which was expected, then anti-human globulin was reactive and the patient’s result was considered valid. data management and statistical analysis after cross-checking, data were transferred directly to statistical package for social sciences software (international business machines corporation, chicago, illinois, united states) version 20 and analysed. continuous data were presented using medians and interquartile range, and the chi-square was used to test associations for categories available. p < 0.05 was considered statistically significant. results the median age of the study participants was 4.5 years (range: 0–26 years) (table 2). of the 200 patients enrolled, 17 patients were positive for alloantibodies. no autoantibodies were detected in any patient; all direct antiglobulin tests were negative. one patient developed immunoglobulin m cold antibodies and was excluded from downstream analysis, but did not experience the consequent nuisance hemolysis. table 2: demographic characteristics of sickle cell disease patients with multiple transfusions at bugando medical centre, may 2017–july 2017.† a total of 17 (8.5%) patients developed 23 warm alloantibodies and 183 (91.5%) tested negative for warm alloantibodies (table 3). of the 17 patients that developed warm alloantibodies, 11 had single alloantibodies and six had double alloantibodies. of the 23 identified alloantibodies, 87% (20/23) showed mild agglutination, and 13% (3/20) showed strong agglutination. among the alloimmunised patients, the number of transfusions ranged from 3 units to 6 units within the previous year. there was no significant statistical association between the number of transfusions and the risk of alloimmunisation [p = 0.07]. the median number of transfusions for patients without alloantibodies was 2 (median: 1.0) units of blood. all 183 patients who were negative for warm alloantibodies received ≤ 2 transfusions. table 3: demographic characteristics of sickle cell patients having warm alloantibodies at bugando medical centre, may 2017–july 2017. of the 23 identified alloantibodies, 12 were of the rhesus group (anti-e [2], anti-c [6], anti-c [3], anti-e [1]), 6 were of the kell group (anti-k), 2 were of the mns group (anti-m), 1 was of the kidd group (jka), 1 was of the duffy group (fya) and 1 was of the lewis group (lea) (table 4). table 4: warm alloantibodies in sickle cell patients with multiple blood transfusions who tested positive for alloantibody screening, bugando medical centre, may 2017–june 2017. discussion the alloimmunisation rate in the current study is higher than the 6.1% alloimmunisation rate reported by natukunda et al. in mulago kampala uganda and the 4.1% rate reported by meda et al. in muhimbili dar es salaam, tanzania.9,10 the differences in these rates could be attributed to differences in the median number of blood transfusions, which was lower (mean, 2(0) units) in the other two studies, whereas the mean number of transfusions at bmc was 3(1) units. the alloimmunisation rate of the current study was comparable to that reported by ugwe et al.3 of 9.3%. the study included 145 scd patients in nigeria; blood transfusion was found to be significantly associated with alloimmunisation (p = 0.027 where p < 0.05). as with our study, sex was not statistically significantly associated with the risk of alloimmunisation in all three studies. as of 2018, tanzania was the third leading country in africa for sickle cell anaemia after the democratic republic of the congo and nigeria.11 the detection of alloantibodies of the kell, mns, kidd, duffy and lewis blood groups is common12 and is corroborated by our study as anti-c, anti-c, anti-e, anti-e, anti-k were the most common alloantibodies detected. thus, these rbc antigens should be included in extended blood typing to detect alloantibodies to these groups. warm alloantibodies (often igg) are of clinical importance because they haemolyse red blood cells at body temperature whereas cold antibodies (igm) also known as nuisance antibodies rarely haemolyse red blood cells in vivo due to temperature inactivation.13 in tanzania, the lake zone area is leading with scd patients (bmc, mwanza) hence the numerous transfusions as part of their clinical management14. thus, the prevalence and risk factors for red blood cell alloimmunisation in this locale needs to be identified. this study found an alloimmunisation rate of 8.5% among scd patients at bmc in mwanza, tanzania, but found no significant association between alloimmunisation and the increased number of transfusions. limitations multiple transfusion is a risk factor for alloimmunisation, especially in sickle cell disease patients. however, in our study, multiply transfusion was not significantly associated with alloantibody development. this could be, in part, due to the small sample size, under-reporting and maybe because all participants had received blood transfusion at least once. more than half of the participants’ guardians or parents could not remember the actual number of blood transfusions a child had taken before attending bmc, thus influencing our final results. availability of commercial red cells is a problem because there are few haematological laboratories worldwide that can manufacture these red cells to cover the demands. our antibody screening/identification approach might be prohibitive for extensive antibody screening in institutions with larger samples requiring shorter turnaround time because it is labour intensive and time-consuming. other methods like gel, sprca, or even automated methods can be used as these are faster, thus reducing laboratory turnaround time. recommendations due to the common occurrence of alloantibodies and its detection within multiply transfused scd patients in bmc, the bmc blood bank and zonal national blood transfusion centre should obtain facilities and expertise that will allow for extensive phenotypic blood typing and matching to minimise the development of alloantibodies and effectively minimize risks of delayed haemolytic transfusion reaction in scd and chronically transfused patients. conclusion the rate of alloimmunisation in multiply transfused scd patients was 8.5% and higher than other studies from east and west africa. thus, there is the need for extensive red blood cell screening and matching to minimize alloimmunisation and risk of delayed haemolytic transfusion reaction, particularly in scd and chronically transfused patients. acknowledgements the authors send a special thanks to their families and sarah bhuko for their endurance and unyielding support during our research work. the authors acknowledge the support of lorne laboratories in the united kingdom for supplying reagents for this experimental paper and the technical support from bugando referral hospital-cpl department. the authors also offer their appreciation to the higher education students loans board for funding this research work. last but not least, the authors would like to extend their sincere gratitude and appreciation to all classmates who helped in one way or another during this research work. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this paper. authors’ contributions j.o.b. and e.t. conceived and designed the study. j.o.b, e.t. and m.c. contributed reagents/materials. j.o.b. and m.c. collected the data, and the data analysis was done by j.o.b. and e.t. j.o.b. wrote the manuscript. all authors read and approved the final manuscript. sources of support this study was funded by higher education students loans board, as a part of the degree awarded to jeffer bhuko by catholic university of health and allied sciences, mwanza, tanzania. reagents are commercial red cells which were purchased from lorne laboratories uk, and these were purchased with a fund from the higher education students loans board. data availability data sharing does not apply to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references kangiwa u, ibegbulam o, ocheni s, madu a, mohammed n. pattern and prevelence of alloimmunization in multiply transfused patients with sickle cell disease in nigeria. biomarker res. 2015;3(1):26. https://doi.org/10.1186/s40364-015-0050-3 yazdanbakhsh k, ware re, noizat-pirenne f. red blood cell alloimmunization in sickle cell disease: pathophysiology, risk factors, and transfusion management. blood. 2012;120(3):528–537. https://doi.org/10.1182/blood-2011-11-327361 ugwu n, awodu o, bazuaye g, okoye a. red cell alloimmunization in multi‑transfused patients with sickle cell anemia in benin city, nigeria. nigerian j clin pract. 2015;18(4):522–526. https://doi.org/10.4103/1119-3077.154204 heddle nm, soutar rl, o’hoski pl, et al. a prospective study to determine the frequency and clinical significance of alloimmunisation post-transfusion. br j haematol. 1995;91(4):1000–1005. https://doi.org/10.1111/j.1365-2141.1995.tb05425.x goding jw. monoclonal antibodies: principles and practice. london: academic press; 1996. https://books.google.co.tz/books?hl=en&lr=&id=jpk4zy4cnqqc&oi=fnd&pg=pp1&dq=goding+jw.+monoclonal+antibodies:+principles+and+practice.+academic+press&ots=4eztgtmgfr&sig=pwmoltkhu-af2qanx7pmtox3oy4&redir_esc=y#v=onepage&q=goding%20jw.%20monoclonal%20antibodies%3a%20principles%20and%20practice.%20academic%20press&f=false miller st, kimr d, styles la, dampier cd, roseff sd. red blood cell alloimmunization in sickle cell disease: prevalence in 2010. transfusion. 2013;53(4):704–709. https://doi.org/10.1111/j.1537-2995.2012.03796.x bashawri l. red cell alloimmunization in sickle-cell anaemia patients. eastern mediterranean health j. 2007;13(5):1181–1189. https://doi.org/10.26719/2007.13.5.1181 chou st, jackson t, vege s, smith-whitley k, friedman df, westhoff cm. high prevalence of red blood cell alloimmunization in sickle cell disease despite transfusion from rh-matched minority donors. blood. am soc hematol. 2013;122:1062–1071. https://doi.org/10.1182/blood-2013-03-490623 natukunda b, schonewille h, ndugwa c, brand a. red blood cell alloimmunization in sickle cell disease patients in uganda. transfusion. 2010;50(1):20–25. https://doi.org/10.1111/j.1537-2995.2009.02435 meda e, magesa p, marlow t, reid c, roberts d, makani j. red blood cell alloimmunization in sickle cell disease patients in tanzania. east afr j public health. 2014;11(2):775–780. https://doi.org/10.1016/j.ijans.2015.08.002 ambrose ee, makani j, chami n, et al. high birth prevalence of sickle cell disease in northwestern tanzania. pediatr blood cancer. 2018;65(1):e26735. https://doi.org/10.1002/pbc.26735 sihler kc, napolitano lm. complications of massive transfusion. vol. 137, chest. american college of chest physicians; 2010. p. 209–220. https://doi.org/10.1378/chest.09-0252 sokol rj, hewitt s, stamps bk. autoimmune haemolysis: mixed warm and cold antibody type. acta haematol [internet]. 1983;69(4):266–274. https://doi.org/10.1159/000206903 thakral b, saluja k, sharma rr, marwaha n. phenotype frequencies of blood group systems (rh, kell, kidd, duffy, mns, p, lewis, and lutheran) in north indian blood donors. transfus apher sci. 2010 aug 1;43(1):17–22. https://doi.org/10.1016/j.transci.2010.05.006 abstract introduction methods results discussion acknowledgements references about the author(s) immaculate s. dlamini department of chemical pathology, faculty of laboratory medicine, national health laboratory service, durban, south africa department of chemical pathology, faculty of laboratory medicine, university of kwazulu-natal, durban, south africa verena gounden department of chemical pathology, faculty of laboratory medicine, national health laboratory service, durban, south africa department of chemical pathology, faculty of laboratory medicine, university of kwazulu-natal, durban, south africa nareshni moodley department of chemical pathology, faculty of laboratory medicine, national health laboratory service, durban, south africa department of chemical pathology, faculty of laboratory medicine, university of kwazulu-natal, durban, south africa citation dlamini is, gounden v, moodley n. evaluation of tumour marker utilisation and impact of electronic gatekeeping in the province of kwazulu-natal, south africa. afr j lab med. 2023;12(1), a2027. https://doi.org/10.4102/ajlm.v12i1.2027 original research evaluation of tumour marker utilisation and impact of electronic gatekeeping in the province of kwazulu-natal, south africa immaculate s. dlamini, verena gounden, nareshni moodley received: 20 july 2022; accepted: 13 apr. 2023; published: 30 june 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: inappropriate testing remains a high healthcare cost driver. tumour marker tests are more expensive than routine chemistry testing. implementing test demand management systems like electronic gatekeeping (egk) has reportedly decreased test requests. objective: this study aimed to describe the appropriateness of tumour marker tests, carcinoembryonic antigen, alpha foetal protein, prostate-specific antigen, carbohydrate antigen 19-9, cancer antigen 15-3, cancer antigen 125, and human chorionic gonadotropin, and determine the effectiveness of the egk used in the public health sector in kwazulu-natal, south africa. methods: tumour marker test data for the kwazulu-natal province were extracted from the national health laboratory service central data warehouse for 01 january 2017 – 30 june 2017 (pre-egk) and 01 january 2018 – 30 june 2018 (post-egk implementation). questionnaires were sent to the clinicians in the regional hospitals ordering the most tumour marker tests to assess ordering practices. in addition, we assessed monthly rejection reports to determine the effect of the egk. results: the egk minimally reduced tumour marker requests or associated costs (1.4% average egk rejection rate). an overall 18% increase in the tumour marker tests occurred in 2018. the data suggest inappropriate tumour marker test utilisation, particularly for screening. conclusion: the introduction of egk as a test demand management had little impact on tumour marker test requests and costs. continuous education and reiteration of indications for tumour marker test use are required. what this study adds: this study demonstrates the ineffectiveness of egk in tumour marker orders, and provides some insight as to why these markers are being ordered, which is important in trying to decrease inappropriate ordering of these tests. keywords: tumour marker; demand management; electronic gatekeeping; minimum retesting interval; cost reduction. introduction serum tumour markers are biochemical markers released by tumour cells directly or indirectly as a source or effect of malignant development. tumour markers are a less invasive tool than a biopsy and are used to increase or decrease the clinical suspicion of a developing new or secondary cancer, detect the recurrence or progression of cancer, monitor response to treatment, and identify a specific therapeutic modality. ideally, requesting and testing a tumour marker should allow for effective patient management, when appropriately performed, and reduce unnecessary redundant costs.1 therefore, the measurement of non-invasive serum tumour markers has been pursued to expedite early diagnosis and detection of cancer aimed at reducing cancer morbidity and mortality. however, the diagnostic sensitivity and specificity of most currently available serum tumour markers are limited.2 the inappropriate use of serum tumour markers has been reported.3,4,5 the improved analytical sensitivity and specificity of high-output automated platforms have increased accessibility to the use of tumour markers and increased the use of serum tumour markers.6 however, the progression in instrumentation has been incongruent with the adoption of evidence-based guidelines to guide the appropriate use of tumour markers.7 the cost of inappropriate testing of tumour markers indirectly affects patient safety, depending on the management strategies initiated based on the results reported.8 increased healthcare costs with decreased healthcare budgets have forced laboratories to develop strategies to reduce and prevent inappropriate testing.9 demand management is one strategy that focuses on ensuring appropriate requests while ensuring quality care to the patient. laboratories have adopted several strategies to limit test demand, such as requiring requesting physicians to have a predetermined educational level, redesigning test request forms, and physical and electronic-based gatekeeping and reflex testing.9 most serum tumour markers are not recommended as first-line rule-in or rule-out tests for cancer, but rather for detecting tumour recurrence and monitoring treatment. thus, demand management strategies have been developed.9,10 some strategies have been validated for serum tumour marker testing,11 such as the minimum retesting interval (mri). the mri strategy stipulates the minimum time before repeating a test based on the test’s properties, such as clinical indication.12,13 the south african national department of health, in conjunction with the national health laboratory service, which serves the south africa public health sector facilities, have used the mri strategy, termed electronic gatekeeping (egk), to manage test demand since 2017. electronic gatekeeping was introduced to limit healthcare spending on ‘unnecessary’ laboratory investigations. the criteria for selecting the mri were based on a combination of literature and consensus agreement between expert clinicians representing the department of health in each region, and expert pathologists. electronic gatekeeping implementation studies have demonstrated that egk is an effective cost-saving tool for several laboratory tests. in 2010, tygerberg hospital management and the national health laboratory service conducted a pilot project in cape town, south africa, to identify the number of egk-rejected and egk-restored (i.e., approved for analysis) tests, the costs saved, and the impact test rejections. the study concluded that the egk was an effective and sustainable demand management tool. they found that most rejected tests were not restored, revealing the inappropriateness of those test requests. the use of egk did not appear to negatively impact patient care but was an effective cost-saving tool.14 however, an academic hospital in gauteng province, south africa, reported that egk test demand management does not dramatically influence requesting behaviour or save costs. they reported an unchanged monthly percentage of egk-held tests over a 22-month retrospective study period, suggesting that a solitary demand management strategy is not as effective as anticipated or as demonstrated in other studies.15 both the cape town and gauteng studies only reviewed the effect of routine chemistry testing demand, not tumour marker testing. to date, no study has reviewed the requesting nature of tumour markers in the south african public health sector. in the south african public healthcare sector, all laboratory and other diagnostic costs are borne by the state. tumour markers were chosen as they are one of the most highly requested tests in the chemistry laboratory, are more costly to process, and are thus charged at a higher rate than the more routine general chemistry testing. at the time of the study, the national health laboratory service chemical pathology laboratory at laboratory a provided tumour marker testing services for most patients in the public sector covering the entire province of kwazulu-natal, except a more northern region, where testing is provided by laboratory b, a national health laboratory service laboratory, which provides a smaller tumour marker test repertoire. the serum tumour markers that were assessed during this audit were carcinoembryonic antigen, alpha foetal protein (afp), prostate-specific antigen (psa), carbohydrate antigen 19-9 (ca 19-9), cancer antigen 15-3 (ca 15-3), cancer antigen 125 (ca 125), and human chorionic gonadotropin (hcg). disorders with high afp include hepatocellular carcinoma, hepatoblastoma, non-seminomatous testicular germ cell tumours of the embryonal carcinomas, cancers of the pancreas, lung, and gastric, and non-malignant processes such as acute viral hepatitis, liver cirrhosis, and obstructive jaundice.2,16,17 carcinoembryonic antigen is a tumour marker for gastrointestinal cancers, but it is also elevated in breast, lung and liver cancers, and non-malignant conditions like heavy smoking, bronchitis, gastritis, duodenal ulcer, liver diseases, pancreatitis, and colorectal polyposis.16,18 human chorionic gonadotropin is produced by embryonal tissue1 but is used as a tumour marker in seminomatous and non-seminomatous testicular tumours, ovarian germ cell tumours, the gestational hydatid form mole, choriocarcinoma, and non-testicular teratomas.19 carbohydrate antigen 19-9 is normally synthesised by the pancreas, biliary ductal cells, gastric, colon, and endometrial and salivary epithelia. it is mainly used to prognosticate and monitor response to interventions in patients with pancreatic and gastrointestinal cancer.1 increased ca 125 values most often are associated with epithelial ovarian cancer, although levels can also be increased in other malignancies, such as breast, endometrial, cervix, peritoneal, uterus, lung, pancreas, hepatocellular and non-hodgkin’s lymphoma and multiple benign disorders, which include pregnancy, endometriosis, uterine fibroids, pancreatitis, normal menstruation, pelvic inflammatory disease, and cirrhosis of the liver.1 cancer antigen 15-3 levels have been reported to be useful to prognosticate in breast cancer patients,20 but elevations of ca 15-3 levels are also seen in other malignancies, including pancreatic, lung, ovarian, colon, and liver cancer as well as benign breast and liver conditions.1 prostate-specific antigen aids in the diagnosis, risk assessment, and monitoring of prostate carcinoma, but it is also elevated in non-malignant conditions like acute urinary retention, benign prostatic hyperplasia, prostatitis, and urinary tract infection.1 this study aimed to describe the tumour marker requesting practices across different levels of healthcare in the province of kwazulu-natal, south africa, assess the effect of egk on these requesting practices and, lastly, determine via questionnaire the rationale for tumour marker requesting by the clinicians at the highest ordering facilities. methods ethical considerations ethics approval for this study was obtained from university of kwazulu-natal biomedical research ethics committee (number be035/18). written informed consent was received from the participating clinicians. data were collected on a password-protected computer and the primary investigator was the only person with access to it. patients were identified by hospital numbers and their identities were not revealed. questionnaires were anonymised and identified by numbers allocated to the specific sites. survey respondents were assured raw data would remain confidential and would not be shared. data collection the national health laboratory service central data warehouse reposits all test results generated by national health laboratory service laboratories. we extracted from the national health laboratory service central data warehouse all tumour marker tests performed in public healthcare facilities in kwazulu-natal province, south africa. the data extracted included requests made 6 months pre-egk (01 january 2017 to 30 june 2017) and 6 months post-egk implementation (01 january 2018 to 30 june 2018). the data consisted of results from laboratory a and b national health laboratory service chemical pathology laboratory. laboratory a offers the following tumour marker tests: carcinoembryonic antigen, afp, psa, ca 19-9, ca 15-3, ca 125, and hcg, whereas laboratory b offers all the above tests except ca 19-9. both laboratories analysed tumour markers on the siemens advia centaur xp (siemens, tarrytown, new york, united states). the mri rules that were in use for egk per tumour marker test were as follows: hcg: 1 day; afp: 1 month; psa: previous result abnormal = 1 month and previous result normal = 1 year; carcinoembryonic antigen: 1 month; ca 125: 1 month; ca 19-9: 1 month; and ca 15-3: 1 month; where: 1 day is 12 h, 1 month is 21 days, and 1 year is 322 days since daily tests are not performed at the same time each day and a repeat visit within a set interval (eg. week or month) may happen before the exact interval has passed. the monthly egk rejections reports were assessed from 01 january 2018 to 30 june 2018, to determine the number of requests rejected by gatekeeping in the included laboratories. the top five highest requesting units from all healthcare facilities over the period of the study were identified and chosen as the sites for questionnaire distribution. written informed consent and the questionnaire (adapted from mcginley and kilpatrick21) were hand delivered and obtained from clinicians from these selected facilities. the questionnaire identified who was ordering tumour markers and why. responses were collated in microsoft excel (microsoft corporation, redmond, washington, united states) for further evaluation. data analysis statistical analyses were performed using medcalc r version 18.11 (medcalc software, mariakerke, belgium) and microsoft excel 2016 (microsoft corporation, redmond, washington, united states). data were assessed for normality using the shapiro-wilk test. normal data were presented as mean ± standard deviation. the monthly average rejection rate was calculated. costing was done using on the national health laboratory service state price list 2017. results a total of 38 615 tumour marker tests for the specified analytes were processed during the 6-month pre-egk introduction period (01 january 2017 to 30 june 2017), while 45 567 tumour markers requests were processed in the post-egk implementation period (01 january 2018 to 30 june 2018). in 2018, there was an 18% increase in tumour marker tests processed. the most ordered tumour marker was psa (41.1% of tested tumour markers in 2017, and 38.4% in 2018), while the least ordered was ca 15-3 (< 3% of tested tumour markers in both 2017 and 2018) (table 1). table 1: summary of tumour markers processed in kwazulu-natal, south africa, 01 january 2017 to 30 june 2018. the majority of samples for psa, afp, carcinoembryonic antigen, ca 19-9, ca 15-3 and ca 125 had normal results (defined as results within the reference interval) in both years of the study period. in contrast, hcg had the most abnormal results (outside of reference interval) for both years in the study period, with 90.1% (2783/3088) abnormal results for 2017, and 92.1% (3771/4092) for 2018. clinical history data provided on the laboratory information system via the central data warehouse demonstrated that there were no clinical histories recorded for most samples (n = 21 299, 65%). a minority of samples (n = 1535, 5%) had a history of cancer documented on the request forms. nine percent (n = 2927) of requests indicated suspicion of malignancy or screening as request reason (figure 1). figure 1: clinical details for tumour marker test requests retrieved from laboratory information system in kwazulu-natal, south africa, 01 january 2018 – 30 june 2018. based on the national health laboratory service state price list 2017, the cost of normal results was markedly higher than abnormal results for both study periods. for example, from 01 january 2017 to 30 june 2017, normal results cost 3 995 553.00 south african rand (r) ($218 409.51 united states dollars [usd]), while abnormal results cost r1 210 461.00 ($66 167.61 usd). in the same period in 2018, normal results cost r4 764 052.00 ($260 418.09 usd) and abnormal results cost r1 181 010.00 ($64 557.73) (figure 2). figure 2: cost of resulted normal and abnormal tumour markers test requests in (a) 2017 and (b) 2018 in kwazulu-natal, south africa. all cost amounts are shown in south african rand (zar). most test requests were received from the outpatient departments or non-oncology clinics of the respective healthcare facilities. however, test requests from the oncology wards and clinics were the lowest. the tertiary academic hospitals made the fewest requests, followed by primary healthcare facilities. requests from district hospitals increased in 2018 by 58.7% to overtake regional hospitals as the main requestors (table 2). table 2: distribution of samples processed per location for tumour marker testing in kwazulu-natal, south africa, 01 january 2017 – 30 june 2018. the egk rejected an average of 95 tumour marker test requests per month from 01 january 2018 to 30 june 2018 with a total of 570 tests rejected over this period. additionally, during the same 6-month period, no egk-rejected tumour marker tests were restored. the total cost of tumour marker rejected test requests was r78 043.86 in 2018 (table 3). table 3: electronic gatekeeping rejection rate and costs saved in kwazulu-natal, south africa, 01 january 2018 – 30 june 2018. clinician questionnaire findings we reviewed 22 responses from the 37 questionnaires distributed (59% response rate). most respondents were from surgical departments (n = 24; 64%), followed by medical (n = 9; 24%), with the remainder (n = 3; 9%) being from general outpatient clinics or unspecified. participants consisted predominantly of junior staff (interns) and non-specialist medical officers. ninety-five percent (n = 21) of respondents indicated that their facility had no dedicated oncology unit or clinics. a further 91% (n = 20) of the participants were unaware of any local or international tumour marker test request guidelines for clinical practice. participants indicated the following as consequent actions to an abnormal tumour marker result: imaging studies (n = 20, 91%), 63% (n = 14) included biopsy, referral (n = 11, 50%), requesting another tumour marker test (n = 3, 14%), and 9% (n = 2) included repeating the tumour marker test. most respondents requested tumour markers to query suspected tumours; over 20% (n = 4) indicated their use to detect tumour sources (figure 3). figure 3: reasons for requesting tumour marker testing per questionnaire respondents, june 2018, in kwazulu-natal, south africa. discussion tumour markers are some of the more expensive clinical chemistry tests. based on the national health laboratory service test pricing for the period 2017/2018, the total cost of tumour marker testing for the two periods reviewed was more than r10 million ($546 631.50 usd). all rejected tumour marker tests were estimated to cost r78 043.86 ($4266.12 usd) in 2018. in the public sector in south africa, the cost of laboratory testing is paid by the department of health (state), with no cost to the patient. the egk rejects test requests before payment and laboratory testing, hence no refund is made on rejected requests. previous reports from 1997–2012 state that 20% – 50% of laboratory tests are inappropriate or are not evidence-based practices.22,23 pema, kiabilua and pillay (gauteng, south africa, in 2018),15 and smit, zemlin and erasmus (tygerberg, western cape, south africa, in 2015)14 reported significant cost reductions through egk of requests. however, the test requests reviewed were smaller-volume tests. our findings showed that the number of tumour marker tests rejected by the egk rules was minimal. fewer than 20 tests were rejected on average, per month, for each of the tumour markers apart from psa. this low rejection rate suggests that appropriate test ordering, per the test’s correct clinical requirement and guidelines, would be the most effective way of controlling inappropriate tumour marker test requests. appropriate test ordering practice requires education on and routine reiteration of appropriate request guidelines, and the development and implementation of national testing guidelines. the lack of continuous clinician education is a reported driver of inappropriate testing.24,25 education and continuous reiteration of best practices are especially important in the non-academic centres, where there are more generalists than specialists managing patients. the high request numbers from district health facilities support the fact that education regarding tumour marker utilisation is most needed in non-academic centres; however, district hospitals represent most hospitals servicing the population (n = 37) versus regional hospitals (n = 13).26 as evidenced by the respondents on the questionnaire, a lot of clinicians were requesting tumour markers to screen for malignancy. our findings may also be an indication that the egk rules require further review and are not strict enough to achieve sufficient demand management. these rules could include limiting requests to only two tumour marker tests on one visit or admission to dissuade panel screening. however, stricter rules may not be possible for hcg, as it is also a test for normal pregnancy and pregnancy-related disorders (for example ectopic pregnancy) and serial measurements are critical. human chorionic gonadotrophin was frequently requested for younger patients, probably because the laboratory information system could not distinguish between malignancy-related and pregnancy-related hcg testing. additionally, germ cell tumours in which hcg concentrations may be increased are more frequently seen in younger adults and adolescents. this is the first study, to the authors’ knowledge, that examines the use of tumour markers in sub-saharan healthcare facilities. the findings of this study suggest that repeat testing represents a small fraction of the cost associated with tumour marker requests and that inappropriate requests (use of all tumour markers as screening tests) are likely resulting in test overuse and associated increased healthcare costs. the introduction of egk has made little or no impact on the number and cost of tumour marker tests performed. while there are no national consensus guidelines for the utilisation of tumour markers in south africa, international guidelines or best practice documents are available to guide clinicians to order tests appropriately.27,28,29 we recommend developing local and national tumour marker ordering guidelines for all levels of healthcare. focused education at the undergraduate level and continuous professional development regarding appropriate utilisation of laboratory tests including tumour markers is also required. greater involvement of pathologists in spreading appropriate utilisation awareness and coaching of junior doctors is also essential. the increasing demands on limited healthcare resources and funding necessitate careful management of testing to ensure optimal patient care while managing costs. limitations one of the limitations of this study was that access to histology results was not available. thus, tumour marker test results could not be confirmed by tumour biopsy results. furthermore, the small number of questionnaires distributed may not be representative of the clinician cohort. additionally, we did not sample clinicians from different healthcare facility levels. furthermore, due to the geographical limitations, lack of internet availability, and other limited resources, we restricted questionnaire distribution to facilities within kwazulu-natal. in addition to that, hcg results were not separated into pregnant versus non-pregnant due to missing clinical records on many samples. lastly, the effects of interventions to improve clinician knowledge of tumour marker requests were not assessed. conclusion it appears that many clinicians do not appropriately request and utilise tumour marker tests and there is no guideline for tumour test ordering and result interpretation. the egk barely reduced tumour marker requests and costs as there was an increase in costs and testing numbers, despite egk implementation. education of doctors, stricter egk rules, and additional demand management measures may be required to make a noticeable demand difference. acknowledgements the authors would like to acknowledge the national health laboratory services laboratory information system (trak care) central data warehouse for the data retrieval, and the clinicians at the department of health hospitals who completed the study questionnaires. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article, except where otherwise indicated. authors’ contributions i.s.d., v.g. and n.m. confirmed they have contributed to the intellectual content of this paper and have met the following four requirements: (1) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (2) drafting or revising the article for intellectual content; (3) final approval of the published article; and (4) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability raw data were extracted from the national health laboratory service central data warehouse. costing was done using 2017 pricing at the national health laboratory service. figures with raw data include: figure 2, figure 3, table 1, table 2 and table 3. testing data may be made available on reasonable request from the corresponding author, n.m. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references sturgeon c. tumour markers. in: rifai n. tietz textbook of clinical chemistry and molecular diagnostics. chapter 31. st louis, missouri: elsevier, 2018; pp. 436–478. malati t. tumour markers: an overview. indian j clin biochem. 2007;22(2):17–31. https://doi.org/10.1007/bf02913308 ntaios g, hatzitolios a, chatzinikolaou a, et al. an audit of tumour marker utilisation in greece. eur j intern med. 2009;20(3):e66–e69. https://doi.org/10.1016/j.ejim.2008.07.026 ferraro s, mozzi r, panteghini m. tumour marker ordering: do not lose control: a prospective clinical trial. am j clin pathol. 2015;144(4):649–658. https://doi.org/10.1309/ajcpnzapjrb3t6kk al-mughales ja, alahwal ms. inappropriate practice in tumour marker 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geneva, switzerland citation emperado dm, mazzola lt, kelly-cirino c. an open-source molecular diagnostic platform approach for outbreak and epidemic preparedness. afr j lab med. 2020;9(2), a1017. https://doi.org/10.4102/ajlm.v9i2.1017 lessons from the field an open-source molecular diagnostic platform approach for outbreak and epidemic preparedness devy m. emperador, laura t. mazzola, cassandra kelly-cirino received: 29 mar. 2019; accepted: 08 july 2020; published: 28 sept. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: diagnostic development for outbreak pathogens has typically followed a disease-specific reactive rather than proactive response. given the diversity of outbreak pathogens, particularly those prioritised by the world health organization research and development blueprint, a more flexible and proactive approach to epidemic preparedness is needed to expand access to critical molecular diagnostic tests in peripheral and resource-constrained deployment settings. objective: new and more sustainable directives are needed to spur the development of high-quality products, particularly for epidemics more often found in lowand middle-income countries. to leverage and de-risk the development process, we present the benefits and challenges of an open-source business model for co-development of molecular diagnostic tests for decentralised settings. methods: we identify key outbreak pathogens that are available only for testing in high infrastructure laboratories and compare in-country installed base platforms that could be leveraged for menu expansion. key strengths and challenges for development are highlighted for both platform and assay developers, with discussion of how to leverage and de-risk the process through an open-source development model. results: depending on the specific partner strengths, options for partnership roles are presented. the proposed open-source business model addresses the particular challenges in the detection of outbreakand epidemic-prone pathogens in lowand middle-income countries, reduces development and deployment risks to support outbreak response, strengthens diagnostic capacity and creates a viable market for product developers. conclusion: we hope this model for a collaborative and open-source approach for molecular diagnostics serves to encourage stakeholders to consider co-development partnerships to improve outbreak preparedness and epidemic/pandemic response. keywords: diagnostics; development; outbreak; preparedness; infectious disease; test development. introduction diagnostics are a fundamental component of a successful outbreak response by enabling surveillance and early detection, rapid and appropriate patient management, and objective evaluation of outbreak containment, in addition to facilitating therapeutic and vaccine development.1,2,3,4 for outbreak and epidemic-prone pathogens, diagnostic development has typically followed a disease-specific reactive approach. faced with significant mortality and morbidity during the 2013–2016 ebola virus disease and 2015–2017 zika virus epidemics, substantial global funding was made available, resulting in the rapid development of new diagnostics, therapeutics and vaccines.5,6,7,8 however, only a handful of the diagnostic tests survived to become commercial products, and few found a sustainable market once the epidemics were declared contained and public funding was diverted elsewhere.5,9,10 a shift toward a more proactive and nuanced development strategy for outbreaks and epidemics is needed, especially to support diagnostic capacity in lowand middle-income countries (lmics). some epidemic-prone diseases are endemic in certain regions, requiring diagnostics that can differentiate active disease from background exposure. for example, lassa fever affects many countries in west africa, with resurging high rates of cases during the dry season.11,12,13 likewise, some emergent diseases share overlapping geographies and similar clinical symptoms (e.g. lassa fever and ebola virus disease, middle east respiratory syndrome coronavirus and influenza), an additional challenge for rapid and definitive diagnosis.2,14 rapid pathogen identification can be critical when early detection can direct lifesaving treatment and intervention, such as antibiotics for confirmed bacterial infections15 or vector control vaccination campaigns during a yellow fever outbreak.2 there are common challenges to the deployment of diagnostic tests during outbreaks.16 early detection and contact tracing at the first stages of an outbreak are critical to rapid containment. similarly, a rapid test turn-around time enables patients to be triaged both quickly and appropriately. in settings of endemic diseases with similar clinical indications, it will be invaluable to rapidly differentiate and identify the pathogen responsible for the outbreak. this kind of dynamic outbreak response demands access to diagnostics across a wide range of infrastructure, from reference laboratories to near-patient (npt) hospital laboratories to point-of-care (poc) clinics and community testing. in particular, the priority pathogens identified by the world health organization research and development blueprint17 present a challenge for diagnostic development for epidemic preparedness by any single developer, as the pathogens cover a wide range in clinical presentation, transmission mode, specimen type, lineage diversity, geography and outbreak frequency. a more flexible approach is needed to rapidly develop or adapt diagnostic tests for a wide variety of pathogens, which can be implemented in outbreak-appropriate deployment settings.18,19,20 molecular diagnostics for epidemic preparedness molecular diagnostics (mdx) plays an important role in disease diagnosis (detection and confirmation of active infection), case management and surveillance, as well as a supporting role in therapeutic and vaccine trials. due to the processing complexity and risks of contamination, mdx testing capacity is traditionally available only at reference-level or high-resourced laboratories, which can significantly delay patient diagnosis and intervention during an outbreak when specimen transport is a challenge. molecular diagnostics testing is particularly useful at peripheral health facilities, where sick patients first present, to allow for pathogen detection. however, peripheral settings typically have limited resources for laboratory testing: npt settings describe hospital and clinic-associated laboratories or mobile units, which support a minimal level of laboratory infrastructure and personnel; poc settings typically lack any laboratory infrastructure, including bedside, primary care and community-based testing.21 near-patient/point-of-care mdx platforms have been designed specifically to meet the requirements for rapid pathogen detection in low-infrastructure settings,22,23,24,25,26 with a compact and automated design for a simplified ‘sample in, result out’ processing.18,19,27,28 near-patient/point-of-care mdx target product profiles have been described in detail.29,30,31,32 successful implementation has been demonstrated in decentralised laboratories in lmics for diseases including hiv, tuberculosis, zika virus, ebola virus disease and influenza,22,23,24,33,34,35,36 suggesting that a broader menu of assays could be developed for these npt/poc platforms for outbreak detection. outbreak pathogen molecular diagnostics opportunities table 1 presents a list of outbreak pathogens identified as high priority pathogens for medical countermeasures due to their high epidemic and pandemic potential.37,38,39 most of these diseases have mdx tests (reagent kits) that are commercially available, but appropriate only for use in high infrastructure research laboratories. in some cases, the mdx test has not been validated clinically or approved for diagnostic use (i.e. research-use-only kit), while in other cases, particularly for newer pathogens, mdx tests are available only as published protocols from trusted reference laboratories.17,38,40 there is potential for available laboratory kits to be re-engineered for npt/poc testing, including kits for viral haemorrhagic pathogens41,42,43,44, arboviral diseases,45,46,47,48 respiratory pathogens49,50,51 and biothreat pathogens (including disease x).39,52,53 table 1: availability of molecular diagnostic tests for select outbreak pathogens. new diagnostic development approach traditional assay development model tests for novel diseases are typically developed in academic or national reference laboratories which have the expertise, infrastructure and resources for rapid development and validation of mdx tests. these ‘in-house’ or laboratory-developed mdx tests are published and distributed as protocols from an internationally trusted source.44,54,55,56 however, the test methods require a high level of expertise and are generally limited in deployment to other reference-level laboratories. commercial products can enable broader deployment, where the mdx test is manufactured as quality-assured kits that contain all of the necessary pre-measured test reagents. in the traditional development model, a commercial platform developer builds a portfolio of proprietary assays, using a cartridge specific to the developer’s instrument platform. the instrument, cartridge, reagents and consumables are manufactured by the platform developer and supported as a ‘sole source’ provision. commercial development requires significant financial resources from the developer, with the cost of test development, manufacturing, clinical validation and regulatory approval generally considered to be an investment toward sustainable sales. companies require some profit margin in order to sustain their business and further develop test menus. when the anticipated markets are small, such as for disease with limited or intermittent outbreaks, or where prices are constrained to the cost of goods (as is typical with global health/lmic markets), then there is little incentive for commercial development. in many resource-constrained settings, alternative test options are not affordable or simply not available. to support rapid menu expansion during new or repeated outbreaks, a more flexible concept for outbreak pathogen test development is needed. open-source diagnostic model in lmic settings, leveraging the existing diagnostic infrastructure is key to expanding the capacity for disease management and outbreak preparedness. rather than requiring repeated de novo investment, existing platforms could be utilised for test menu expansion, building on the existing human resources, laboratory testing capacity and supply chains. a similar approach has seen success in the integration of tuberculosisand hiv-testing at peripheral health centres.57 for outbreak response, more flexible initiatives are needed to support rapid test development and deployment scenarios. here we describe a new open-source business model for mdx test development using existing platforms for npt/poc diagnostic testing. as a key innovation, the open-source development model introduces an open-architecture cartridge designed to facilitate rapid development of tests by outside assay developers, where the platform developer provides an open, sterile cartridge that can be used by assay developers (academic or commercial) to rapidly prototype mdx tests for the existing platform. this development model leverages the strength of each developer, namely the available install base of a platform within relevant lmics and the availability of reagent kits for the detection of outbreak pathogens, albeit for high infrastructure laboratory use. as noted in table 2, this open-source development model can serve to decouple some of the risks for new test deployment. table 2: co-development leverage and risk reduction. this open-source model is intended to catalyse a more rapid approach to the development of diagnostic tests for decentralised or low-infrastructure settings, and to provide a more flexible approach to manufacturing tests for novel and endemic pathogens for lmics through co-development (table 3). variations on the co-development partnership include collaborative research, test kit sub-contract manufacturing, and licensing or acquisition. these variations each require specific research and development and supply agreements, as well as marketing and product support agreements between the partners. in some cases, a highly successful development partnership could lead to downstream acquisition. even in cases with limited sales, the ability to rapidly develop and deploy npt/poc tests for novel pathogens is valuable in improving local outbreak response. table 3: co-development roles for open-source model. model benefits and challenges an open-source model for diagnostics can provide a more flexible and cost-effective approach to mdx deployment in peripheral settings, provided that commercial developers can find mutual benefit in a strategic partnership rather than traditional competition. the co-development model outlined above is intended to leverage the differential strengths between assay and platform developers by decoupling the investment risk for test menu expansion to include pathogens that may have limited but critically important deployment. by leveraging existing assays and npt/poc infrastructure, new tests can be rapidly re-engineered with a more cost-effective and ‘on demand’ approach for manufacturing. for the platform developer, a comprehensive assay menu expands the user base, which may provide more resilient and sustainable market volumes for diagnostic manufacturing. for the assay developer, their existing assay portfolio is expanded to include testing across a broad range of settings.58,59,60,61 designed to utilise existing lmic resources, an acknowledged potential disadvantage of this business model is reduced competition in favour of entrenched player market stability and product availability. novel molecular platforms must be evaluated, both for their potential implementation in lmics, as well as their openness to work with partners in developing assays, particularly for outbreak-prone diseases. to support this new development model, innovative partnerships and finance solutions are needed, especially for proactive development and commercial viability. here, the role of product development partners can serve as both matchmaker and co-investor to incentivise co-development partnerships.62 the open-source model is currently being piloted by the foundation for innovative and new diagnostics to partner diagnostic assay and platform developers in support of outbreak preparedness in the near term (2–5 years).58 similarly, this development model would also benefit from support from the global health community toward sustainable market commitments, pooled procurement mechanisms, and funding for test stockpiling to establish more sustainable supply chains and support long term commitments to diagnostic manufacturers.16 some of these mechanisms are in place to address similar challenges in the vaccines sector and should be explored and adapted for diagnostics.63 conclusion despite the influx of interest at the onset of a new outbreak, it is more often the case that research and development costs for new diagnostics may be too high relative to market size to provide a sufficient incentive for product development, and sustained support can be particularly challenging, for pathogens that present seasonal or sporadic markets. alternative business models for diagnostic development could help reduce the high costs associated with bringing a new pathogen test to market. an open-source development partnership model can combine the strengths of commercial developers to reduce development the cost and time to market, while supporting existing supply chain infrastructure, training and proficiency.29,32 certainly, there are additional upstream and downstream challenges for pathogen test development. for new or poorly understood outbreaks, there are uncertainties in pathogen identification, epidemiology and disease kinetics. newly developed tests require additional resources for clinical validation, for which well-characterised specimens and calibration standards may be difficult to obtain.16 commercial products also require international and/or in-country regulatory approval, though this may be a lower hurdle for assays that were previously validated as laboratory tests. here, product development partnerships can also serve as matchmakers to allow for sample sharing between research sites and developers with promising technologies, as well as to leverage in-country experience to support evaluation studies in lmics that meet the requirements for regulatory approval. the availability of appropriate diagnostics is a key feature for outbreak and epidemic preparedness. early diagnosis of patients at initial point-of-care will enable timely patient and population interventions and support national surveillance systems for emerging and re-emerging diseases. collaborative efforts are needed to develop npt/poc diagnostics for all world health organization priority pathogens, to ensure access to high-quality diagnostics where testing is needed and to enable innovative funding mechanisms to support proactive development and market sustainability. we hope this article serves to encourage all diagnostic stakeholders (industry, academia, non-for-profit, non-governmental-organisations, governments) to foster partnerships in development and consider the open-source model early in the development of new diagnostics. acknowledgements competing interests d.m.e. and c.k.-c. are employees of find, while l.t.m. is a consultant at find; find is a non-profit organisation and product development partnership that works with industry partners to support diagnostic development. authors’ contributions d.m.e. and c.k.-c. were responsible for concept design. d.m.e. and l.t.m. performed literature reviews and co-wrote the manuscript. c.k.-c. reviewed and approved the final version of the manuscript. ethical consideration the authors confirm that ethical clearance was not required for this study. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors; c.k.-c., d.m.e. and l.t.m. are employed by find. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily 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innovation. health res policy syst. 2011;9:33. https://doi.org/10.1186/1478-4505-9-33 gavi. gavi ‘effective and fit for purpose’ [homepage on the internet]. c2017 [cited 2019 mar 29]. available from: https://www.gavi.org/library/news/statements/2017/gavi--effective-and-fit-for-purpose-/ abstract introduction methods results discussion acknowledgements references about the author(s) terese g. orum department of veterinary public health and preventive medicine, faculty of veterinary medicine, university of ibadan, ibadan, nigeria olayinka o. ishola department of veterinary public health and preventive medicine, faculty of veterinary medicine, university of ibadan, ibadan, nigeria oluwawemimo o. adebowale department of veterinary public health and preventive medicine, college of veterinary medicine, federal university of agriculture, abeokuta, ogun state, nigeria citation orum tg, ishola oo, adebowale oo. occurrence and antimicrobial susceptibility patterns of salmonella species from poultry farms in ibadan, nigeria. afr j lab med. 2022;11(1), a1606. https://doi.org/10.4102/ajlm.v11i1.1606 original research occurrence and antimicrobial susceptibility patterns of salmonella species from poultry farms in ibadan, nigeria terese g. orum, olayinka o. ishola, oluwawemimo o. adebowale received: 21 apr. 2021; accepted: 14 apr. 2022; published: 20 july 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: salmonella species are among the major foodborne pathogens causing diseases of economic and public health implications in poultry and humans globally. objective: this study aimed to determine the occurrence and antimicrobial susceptibility patterns of salmonella isolates from chickens in poultry farms in ibadan, southwest nigeria. methods: cloacal swab samples (n = 360) were obtained from chickens randomly selected from 10 poultry farms in five local government areas of ibadan, oyo state, from 04 april 2018 to 20 november 2018. bacterial identification and antimicrobial susceptibility testing were performed using established protocols. data were analysed using descriptive statistics and pearson’s chi-squared test at p ≤ 0.05 significance level. results: the overall prevalence of salmonella was 21.4%. there were statistically significant associations between salmonella prevalence and the farm location (p = 0.003), age of chickens (p < 0.001), and health status of chickens (p < 0.001). all salmonella isolates (n = 77; 100.0%) were resistant to cefuroxime. the isolates were also highly resistant to cotrimoxazole (n = 74; 96.1%), chloramphenicol (n = 73; 94.8%), meropenem (n = 72; 93.5%), gentamicin (n = 69; 89.6%), and tetracycline (n = 64; 83.1%). conclusion: the presence of drug-resistant salmonella in commercial layer chickens in ibadan is a potential threat to consumer health as it increases the risk of carcass contamination and pathogen propagation, and limits the options to control and treat infections in humans and animals. well-integrated national surveillance systems for monitoring salmonella and antimicrobial resistance in poultry are critical. keywords: salmonella species; antibiotics; antimicrobials; resistance; poultry and infection. introduction salmonella species is one of the primary causes of foodborne illnesses in humans globally.1 although most cases of salmonellosis are mild, some may lead to life-threatening enteric fever or an invasive form of the disease requiring antimicrobial treatment, especially among immunocompromised people, infants, and the elderly.2,3 the consumption of food animal products such as poultry meat has been implicated in human salmonellosis outbreaks worldwide.4 the salmonella genus comprises two species, salmonella bongori and salmonella enterica, and over 2500 different serotypes or serovars.5 in africa, non-typhoidal salmonellae are common causes of bacterial bloodstream infections in humans with clinical signs of fever6 and are associated with case fatality rates of about 20.0% – 25.0%.7 a report from nigeria showed that non-typhoidal salmonellae (43.5%) and typhoidal salmonellae (56.5%) were isolated from blood and stool samples collected from adults and children.8 poultry farming is a major source of livelihood for poor rural households and urban areas in many developing countries, including nigeria.8,9 the poultry sector has the potential to promote national economic growth and provide employment opportunities for nigerians.10 for instance, poultry farming serves as an additional occupation to supplement the income of small farm families.10 similarly, the poultry value food chain accounts for about a quarter of local meat production in nigeria and promotes the per capita animal protein consumption of the population.11 despite these advantages, small-scale poultry farmers in nigeria lose up to 18% of chicks in the first two weeks of rearing, often due to salmonellosis; this negatively impacts food security and the socio-economic status of farmers.12 the expansion of the poultry industry has increased the occurrence of salmonellosis, which has become an important public health threat in nigeria, causing heavy economic loss and higher human and animal morbidities and mortalities.13,14 the most prevalent human salmonella serovars are also common in poultry, suggesting a possible epidemiologic interface between human infections and poultry as a reservoir of salmonella.8 salmonella can contaminate poultry and propagate among breeding flocks and at all stages of the production cycle, including during hatching, transportation, slaughtering, processing and retailing, and subsequently spread to humans.15 antimicrobial resistance is an emerging global threat associated with the reduced efficacy of antimicrobials in the treatment of human and animal infections, prolonged morbidity, and death.16 multidrug-resistant organisms are an increasing challenge to human and livestock health worldwide, and nigeria is not excluded from the antimicrobial resistance burden. antimicrobials are used in veterinary practice as growth promoters, prophylactics or therapeutics. the indiscriminate use of antimicrobials has been linked to increased antimicrobial resistance among bacterial pathogens isolated from poultry and the poultry environment, including salmonella.17 therefore, poultry may harbour multidrug-resistant pathogens, which could be transmitted to human populations anywhere through direct contact or by consumption. hence, our study focused on determining the occurrence and antimicrobial susceptibility patterns of salmonella isolates from chickens in poultry farms in ibadan, oyo state, nigeria. methods ethical considerations the animal care and use research ethics committee (acurec), university of ibadan, nigeria, reviewed and approved the protocols of the study (ui-acurec/18/0105). verbal informed consent was obtained from farm owners before sampling chickens. study design and sampling method a cross-sectional study was conducted to determine the occurrence of multidrug-resistant salmonella in poultry farms in ibadan, oyo state, southwest nigeria. a multistage sampling technique was used to select the chickens for sampling. first, five local government areas (lgas) in ibadan metropolis, namely ibadan north, ibadan north east, ibadan north west, ibadan south west and ibadan south east were randomly selected for the study. next, a total of 10 commercial layer poultry farms (a–j), two from each of the five lgas, were purposively recruited based on the presence of commercial layer production and farmers’ consent. afterwards, a single pen from each farm was randomly selected, from which 36 chickens were sampled by systematic random sampling. the population of the poultry farms ranged from 500 to 2000 chickens aged between 15 and 36 weeks at the time of sampling; the breeds raised were isa brown and bovans nera black. the farms all operated an intensive management system using battery cages. sample size using a prevalence of 3.5% of salmonella species reported in layers from poultry farms in ilorin, kwara state,18 the sample size was calculated using the formula,19 where n = minimum sample number, z = confidence interval at 95% (1.96), p = prevalence level of subject (3.5%), and d = level of precision (2%). a sample size of 324 was determined, and a 10% non-contingency was added to make up for non-response, giving a minimum target sample size of 360. sample collection all sanitary and biosecurity protocols were observed by investigators to avoid the spread of pathogens between farms during sample collection. farm visits were conducted at about 2-week intervals over eight months from 4 april 2018 to 20 november 2018. each farm was visited once by the investigators and 36 samples were collected during each visit. the investigators visited each farm on dates specified by the farm managers to ensure that no biosecurity measures were compromised. a systematic random sampling was performed to collect samples from chickens within the pens irrespective of age, breed, and health status. a total of 360 cloacal swabs were collected from live chickens (layers) for the detection of salmonella. samples from farms a and c were taken from diseased chickens as the farmers refused access to the apparently healthy ones. cloacal swab samples were collected by gently rotating the cotton swab within the cloacae, with care taken to avoid contact with other areas to prevent contamination. all samples were transported in sterile 10 ml tubes on ice at 4 °c to the veterinary public health laboratory of the department of veterinary public health and preventive medicine, university of ibadan, for bacteriological analysis. no transport medium was used. samples were processed immediately on arrival at the laboratory. for each sampled chicken, information on the species, breed, age, health status, management system, and recent antibiotics administered was also obtained and documented. isolation of salmonella isolation of salmonella was carried out according to the international organization for standardization standard 6579 guidelines20 for isolation and characterisation of salmonella species. briefly, pre-enrichment was conducted by transferring each swab into 10 ml tubes containing buffered peptone water (oxoid, basingstoke, united kingdom). each tube was then thoroughly homogenised by vortexing and incubated at 37 °c for 18–20 h. subsequently, 0.1 ml of the pre-enrichment broth culture was inoculated into 10 ml of rappaport vassiliadis broth (oxoid, basingstoke, united kingdom) for the selective enrichment of salmonella and incubated at 42 °c for 24 h. a loopful from the overnight enrichment broth was then inoculated onto xylose lysine deoxycholate agar (oxoid, basingstoke, united kingdom) and brilliant green agar (oxoid, basingstoke, united kingdom), and the plates were incubated at 37 °c for 24 h. to obtain pure colonies, the presumptive salmonella colonies that appeared red with black centres on xylose lysine deoxycholate agar and pinkish-white or red surrounded by a red halo on brilliant green agar were subcultured on xylose lysine deoxycholate agar plates and incubated at 37 °c for 24 h. the pure cultures were subsequently inoculated on nutrient agar slants, incubated at 37 °c for 24 h and stored in the fridge at 4 °c. phenotypic characterisation of salmonella salmonella isolates were identified based on colony morphology, and further phenotypically confirmed and characterised using gram staining (negative) and standard laboratory biochemical tests such as indole (negative), methyl red (positive), voges-proskeur (negative), citrate (negative), oxidase (negative), catalase (positive), urease (negative), and triple sugar iron agar (acid/alkaline). also, sugar fermentation tests for glucose (positive), lactose (negative), mannitol (positive), sucrose (negative), and xylose (positive) were performed. antimicrobial susceptibility tests the antimicrobial susceptibility test was carried out using the agar disk diffusion method.21 the sensitivity patterns of the cultured isolates were reported for standard antibiotics according to the clinical laboratory standards institute.22 briefly, putative salmonella isolates on nutrient agar slants were subcultured onto nutrient agar plates and incubated for 24 h at 37 °c. subsequently, a colony from each plate was emulsified into sterile saline to achieve turbidity equivalent to 0.5 mcfarland standard (~ 108 cfu/ml). the inoculum was aseptically and uniformly spread on the surface of prepared muller hinton agar plates (oxoid, basingstoke, united kingdom). thereafter, the antibiotics disks (biomark laboratories, pune, maharashtra, india) were placed aseptically on the inoculated agar surface using sterile forceps and the plates were incubated at 37 °c for 16–18 h. the following antimicrobials were used: tetracycline (10 µg), cotrimoxazole (25 µg), gentamicin (10 µg), cefuroxime (30 µg), chloramphenicol (10 µg), ciprofloxacin (5 µg), meropenem (10 µg) and amikacin (30 µg). the zones of inhibition around each disk were measured and interpreted according to the clinical laboratory standards institute guidelines. the escherichia coli american type culture collection 25922 reference strain was used as the control. an isolate was considered multidrug-resistant if resistant to at least one antimicrobial in three or more antimicrobial classes. data analysis data were analysed using descriptive statistics such as proportions and percentages and presented as a table and figures. a pearson’s chi-squared (χ2) test and odds ratios were computed to test the association between the presence of salmonella and age, breed, farm location, and health status of the chickens. the level of significance was set at p ≤ 0.05. all statistical analyses were conducted using the statistical package for the social sciences version 21 (ibm corp., armonk, new york, united states, 2012). results a total of 77 (21.4%) of the 360 samples were positive for salmonella. farm c, one of the farms in ibadan north-west where only sick chickens were sampled, had the highest salmonella prevalence (21/36; 58.3%) (figure 1). overall, the prevalence of salmonella was higher among chickens of the isa brown breed (51/216; 23.6%) and chickens aged between 21 and 26 weeks (39/108; 36.1%) (table 1). table 1: association between salmonella prevalence and demographic variables of chickens from poultry farms in ibadan metropolis, nigeria, april 2018 – november 2018. figure 1: occurrence of salmonella species in chickens from poultry farms in ibadan metropolis, nigeria, april 2018 – november 2018. age, farm location, and health status of chickens were all associated with salmonella carriage. layer chickens aged between 21 and 26 weeks had four times the odds of salmonella carriage compared to those aged between 15 and 20 weeks (odds ratio = 4.52; 95% confidence interval: 1.97–10.4, p < 0.001). similarly, chickens in farms in ibadan north-west had three times higher odds of being salmonella-positive compared to those from ibadan north lga (odds ratio = 3.22; 95% confidence interval: 1.54–6.7; p = 0.003). farms in ibadan north-west lga operated under poor hygienic and low biosecurity conditions. interestingly, sick chickens had lower odds of harbouring salmonella than the healthy ones (odds ratio = 0.32; 95% confidence interval: 0.18–0.57; p < 0.001). antimicrobial resistance profiles of the salmonella isolates all of the isolates (n = 77; 100.0%) were resistant to cefuroxime. isolates were also highly resistant to cotrimoxazole (n = 74; 96.1%), chloramphenicol (n = 73; 94.8%), meropenem (n = 72; 93.5%), gentamicin (n = 69; 89.6%) and tetracycline (n = 64; 83.1%), but only moderately resistant to ciprofloxacin (n = 31; 40.3%) (figure 2). figure 2: antimicrobial resistance profile of salmonella isolates from chickens from poultry farms in ibadan metropolis, nigeria, april 2018 – november 2018. discussion food safety issues, including the presence of zoonotic and antibiotic-resistant pathogens in farm animals, are of increasing concern among consumers of animal food products globally. we reported a 21.4% prevalence of salmonella in commercial poultry in ibadan. previous studies have reported the prevalence of salmonella infection in poultry farms and chickens in nigeria to be between 2.0% and 43.0%,17,23,24,25,26,27,28,29,30,31,32 and this is similar to the prevalence reported in another study conducted in bangladesh.33 salmonella have been implicated in bacteraemia, septicaemia and gastroenteritis in humans, and their presence in poultry chickens may pose serious health risks to humans.34 the observed salmonella prevalence in the farms studied may be due to poor biosecurity measures, contamination from poultry premises or feed, or bacterial transmission via faecal matter by insects and rodents.35 chickens from poultry farms in ibadan north-west lga had three times the odds of being colonised by salmonella and this may be attributed to the unhygienic and poor sanitary measures observed on the farms during site visits, including the presence of heaps of faecal matter in the pens and absence of disinfectant foot dips. salmonella transmission in farms may also be promoted by inadequate knowledge and poor attitudes towards biosecurity and poultry management.36 similarly, as observed, leaving dead carcasses in the farm, lack of proper hygiene, and the presence of faeces in water could have accounted for the high prevalence of salmonella in farms in ibadan north-west. similar to past studies conducted in nigeria, colombia and baghdad,37,38,39 there was no association between chicken breed and salmonella colonisation in our study. the observed high rates of resistance to antimicrobials among the isolates is not surprising as most poultry farmers in the country have unlimited over-the-counter access to antimicrobials and indiscriminately use them as growth promoters and prophylactics40 without veterinary prescriptions.41,42 the ciprofloxacin resistance was lower in this study compared to another study conducted in nigeria,43 but similar to a report from the united states.44 all salmonella isolates were resistant to cefuroxime, which was the only cephalosporin tested in this study; this is similar to previous reports from nigeria, the united arab emirates, germany and india.17,45,46 this poses a serious threat to humans as cephalosporin-resistant salmonella can potentially be transmitted to humans via the consumption of such poultry or poultry products. moreover, there are limited treatment options for human infections caused by pathogens resistant to multiple classes of antimicrobials, particularly those involving the cephalosporins and fluoroquinolones.46 the high resistance to tetracycline documented in this study is not surprising. tetracycline is the most abused antibiotic for the treatment and prevention of poultry disease in nigeria.17,40,41 nevertheless, the tetracycline resistance rate in this study is higher than those reported in past studies in ghana47 and cote d’ivoire,48 possibly because of differences in geographical location, farm biosecurity levels, and national implementation of antimicrobial policies in these countries. limitations we were not able to identify the specific serovars of the salmonella isolates due to non-availability of antisera to perform serotyping. also, the findings of this study are not generalisable to farms in ibadan as only the five lgas in ibadan metropolis were sampled. further work, which should involve the detailed sampling of more lgas and serotyping of salmonella isolates, is needed to provide more insight into the prevalence of the various serovars circulating in the poultry population in ibadan, nigeria. conclusion we report a high prevalence of salmonella among chickens from poultry farms in ibadan, nigeria. apparently healthy and sick chickens may serve as potential shedders and propagators of drug-resistant salmonella, with possible implications for consumer health and the environment. data generated from this study could inform policies or guidelines for the improvement of on-farm hygiene and biosecurity best practices among poultry farmers to reduce the burden of salmonella in the industry. also, nigeria urgently needs well-coordinated or integrated national surveillance systems and networks to monitor antimicrobial use and resistance in animal and human populations and the environment. acknowledgements the authors appreciate the technical support provided by the technologists of the department of veterinary public health and preventive medicine, university of ibadan, ibadan, nigeria, particularly mr yemi okunlade. we are also grateful to the owners and managers of the poultry farms used in this study for their cooperation. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions o.o.i. conceived and designed the study. t.g.o., o.o.i. and o.o.a. undertook the methodology of the study. t.g.o. collected, analysed the samples and did the statistical analysis under the supervision of o.o.i. and o.o.a. furthermore, t.g.o. drafted the original manuscript, and o.o.i. and o.o.a. revised it. all of the authors approved the final manuscript. sources of support the research received no specific grant from any funding agency in the public, commercial or non-profit sector. data availability the data that support the findings of this study can be made available by the corresponding author, o.o.i., upon reasonable request. disclaimer the views and opinions expressed in 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hot, uhunmwangho a. the prevalence of salmonella species among poultry chickens in ekpoma edo-state, nigeria. int j community res. 2014;3(4):101–105. fashae k, ogunsola f, aarestrup fm, hendriksen rs. antimicrobial susceptibility and serovars of salmonella from chickens and humans in ibadan, nigeria. j infect dev ctries. 2010;4(8):484–494. https://doi.org/10.3855/jidc.909 mbuko ij, raj ma, ameh j, saidu l, musa wi, abdul pa. prevalence and seasonality of fowl typhoid disease in zaria-kaduna state, nigeria. j bacteriol res. 2009;1(1):001–005. aje anejo‑okopi j, ejiji isa s, audu o, fagbamila io, iornenge jc, smith is. isolation and polymerase chain reaction detection of virulence inva gene in salmonella spp. from poultry farms in jos, nigeria. j med tropics. 2016;18(2):98–102. https://doi.org/10.4103/2276-7096.192237 islam m, hassan j, khan mr. seroprevalence of mycoplasma gallisepticum infection in backyard and commercial layer chickens in bhola district, bangladesh. j adv vet anim res. 2014;1(1):11–15. https://doi.org/10.5455/javar.v1i1p11-15 manoj j, singh mk, singh yp. the role of poultry in food borne salmonellosis and its public health importance. adv anim vet sci. 2015;3(9):485–490. https://doi.org/10.14737/journal.aavs/2015/3.9.485.490 jones ft. a review of practical salmonella control measures in animal feed. j appl poult res. 2011;20(1):102–113. https://doi.org/10.3382/japr.2010-00281 raufu ia, ahmed oa, aremu a, odetokun ia, raji ma. salmonella transmission in poultry farms: the roles of rodents, lizards and formites. savannah vet j. 2019;2:1–4. ibrahim t, yakubu n, grace p, et al. antimicrobial resistance profile of salmonella typhimurium isolated from commercial poultry and poultry farm handlers in nasarawa state, nigeria. microbiol res j int. 2019;28(4):1–12. https://doi.org/10.9734/mrji/2019/v28i430136 rodriguez jm, rondón is, verjan n. serotypes of salmonella in broiler carcasses marketed at ibague, colombia. braz j poultry sci. 2015;17(4):545–552. https://doi.org/10.1590/1516-635x1704545-552 al-khayat l, khammas ej, ali rm, al-owaini ba, abdulrasool lms. rapid detection and identification of poultry salmonella serotypes using multiplex pcr assay. mrvsa. 2016;5(3):31–40. https://doi.org/10.22428/mrvsa.5.3-5.03102016 adebowale oo, adeyemo ok, awoyomi o, dada r, adebowale o. antibiotic use and practices in commercial poultry laying hens in ogun state nigeria. rev elev med vet pays trop. 2016;69(1):41–45. https://doi.org/10.19182/remvt.31170 awogbemi j, adeyeye m, akinkunmi eo. a survey of antimicrobial agents usage in poultry farms and antibiotic resistance in escherichia coli and staphylococci isolates from the poultry in ile-ife, nigeria. j infect dis epidemiol. 2018;3:47–55. adebowale oo, adeyemo fa, bankole n, et al. farmers’ perceptions and drivers of antimicrobial use and abuse in commercial pig production, ogun state, nigeria. int j environ res public health. 2020;17(10):3579–3599. https://doi.org/10.3390/ijerph17103579 salihu ae, onwuliri fc, mawak jo. antimicrobial resistance profiles of salmonella gallinarum isolates from free-range chickens in nasarawa state, nigeria. int j bacteriol res. 2014;2(1):19–27. malorny b, hoofa j, bunge c, helmuth r. multicenter validation of the analytical accuracy of salmonella pcr: towards an international standard. appl environ microbiol. 2013;69(1):290–296. https://doi.org/10.3390/ijerph17103579 rotimi vo, jamal w, pal t, sonnevend a, dimitrov ts, albert mj. emergence of multidrug-resistant salmonella spp. and isolates with reduced susceptibility to ciprofloxacin in kuwait and the united arab emirates. diagn microbiol infect dis. 2008;60(1):71–77. https://doi.org/10.1016/j.diagmicrobio.2007.07.007 sivakumar t, avinash saravanavel n, prabhu d, shankar t, vijayabaskar p. characterization of multidrug resistant patterns of salmonella sp. world j med sci. 2012;7(2):64–67. andoh la, dalsgaard a, obiri-danso k, newman mj, barco l, olsen je. prevalence and antimicrobial resistance of salmonella serovars isolated from poultry in ghana. epidemiol infect. 2016;144(15):3288–3299. https://doi.org/10.1017/s0950268816001126 bonny ac, karou atg, sanogo m, atobla k, ahonzo-niamke sl. prevalence of salmonella and their antibiotic susceptibility patterns in the district of abidjan, côte d’ivoire. int j biol chem sci. 2014;8(2):450–458. https://doi.org/10.4314/ijbcs.v8i2.5 abstract introduction methods results discussion acknowledgements references about the author(s) thumeka p. jalavu national health laboratory service, department of chemical pathology, faculty health sciences, stellenbosch university, stellenbosch, south africa megan rensburg national health laboratory service, department of chemical pathology, faculty health sciences, stellenbosch university, stellenbosch, south africa rajiv erasmus national health laboratory service, department of chemical pathology, faculty health sciences, stellenbosch university, stellenbosch, south africa citation jalavu tp, rensburg m, erasmus r. clinical staff knowledge and awareness of point-of-care-testing best practices at tygerberg hospital, south africa. afr j lab med. 2020;9(1), a853 https://doi.org/10.4102/ajlm.v9i1.853 note: additional supporting information may be found in the online version of this article as online supplementary document 1. original research clinical staff knowledge and awareness of point-of-care-testing best practices at tygerberg hospital, south africa thumeka p. jalavu, megan rensburg, rajiv erasmus received: 27 june 2018; accepted: 24 mar. 2020; published: 16 july 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: point-of-care testing (poct) is defined as testing done near or at the site of patient care with the goal of providing rapid information and improving patient outcomes. point-of-care testing has many advantages and some limitations which affect its use and implementation. objective: the aim of the audit was to determine the current practices, staff attitudes and training provided to hospital clinical staff. methods: the audit was conducted with the use of a questionnaire containing 30 questions. one hundred and sixty questionnaires were delivered to 55 sites at tygerberg academic hospital in cape town, south africa, from 21 june 2016 to 15 july 2016. a total of 68 questionnaires were completed and returned (42.5% response rate). results: most participants were nursing staff (62/68, 91%), and the rest were medical doctors (6/68, 9%). most participants (66/68, 97%) performed glucose testing, 16/68 (24%) performed blood gas testing and 17/68 (25%) performed urine dipstick testing. many participants (35/68, 51%) reported having had some formal training in one or more of the tests and 25/68 (37%) reported having never had any formal training in the respective tests. many participants (46/68, 68%) reported that they never had formal assessment of competency in performing the respective tests. conclusion: participants indicated a lack of adequate training in poct and, thus, limited knowledge of quality control measures. this audit gives an indication of the current state of the poct programme at a tertiary hospital and highlights areas where intervention is needed to improve patient care and management. keywords: poct; near-patient testing; iso 22870; pathology; chemical pathology. introduction point-of-care testing (poct) is defined as testing done at or near the site of patient care, with the aim of providing rapid information and improving patient outcomes.1 the goal of poct is to provide timely information regarding the patient’s condition, and to adjust management and improve the quality of care whilst avoiding medical errors.2 the ideal poct programme must meet several requirements; these include organisation, supervision, written procedures, operator training and competency testing, instrument evaluation, proficiency testing, quality control, and appropriate result recording and notification.1 the main guidelines used to design and implement poct programmes are the international organization for standardization (iso) 22870:2016 and clinical laboratory standards institute guidelines such as poct4,3,4 which provide comprehensive guidance on poct practice. the royal college of pathologists of australia has a framework for poct in the laboratory and at poct sites that can be adapted for use at independent sites offering poct.5 these are some of the goals for a poct programme applicable to the hospital environment. part of quality management may include internal quality control and external quality assurance (eqa), depending on the type of poct device in use. nursing staff form an essential part of the clinical team and routinely perform poct; therefore, a regular provision of training should be provided to ensure adequate knowledge and compliance with the above requirements.5 documentation, information management and record keeping are also an important part of the poct process. ideally, the systems should be linked to the laboratory information systems; if this is not available, the poct team should develop programmes to ensure that this is done.6,7 this process includes documentation of non-conformances, and protocols on how to identify, investigate and take appropriate corrective and preventative measures. indirect costs associated with poct may also include training of personnel, quality control, maintenance, external quality control/proficiency testing, etc.8,9 incidents of nosocomial infections related to sharing of devices between patients, as well as viral infections related to contaminated lancet holders, have been reported.10 these are some of the reasons why it is recommended to have a poct committee which ensures proper training and compliance with both local and international guidelines for the implementation and management of a poct programme. south africa currently has no formal national policy on poct and training of personnel for healthcare facilities. different hospitals implement their own programmes and it is not clear if the same protocols are followed everywhere. point-of-care testing is widely available for use in the general and some emergency hospital units. national guidelines are necessary to inform the planning of poct programmes, training and assessment of practitioners, or guidance on issues around quality management. hospital and facility managers are often the main stakeholders involved in poct programmes, without involvement of any laboratory representatives. it is against this background that different hospitals operate their poct programmes, without national guidelines; this is also applicable to tygerberg academic hospital (hereafter, tygerberg hospital). healthcare workers who use poct at tygerberg hospital in cape town, south africa, include approximately 550 medical staff (interns, officers, specialists, registrars, etc.) and nursing staff (approximately 2100 professional nurses, staff nurses, and nursing assistants). a variety of point-of-care tests are in use. for example, medical wards, such as the internal medicine and endocrine wards, use glucose meters and urine dipsticks on a regular basis, as do the diabetes and renal clinics. the intensive care and high care units use these poc tests as well as arterial blood gas analysers, which are benchtop poct devices that require more skill to operate than the other two tests and are often performed by medical staff, professional nurses or technicians, when available. this study audited current practices and training provided to hospital personnel who regularly use poct at tygerberg hospital, to determine whether collaboration is necessary between the laboratory and the hospital management team responsible for the current poct instruments used. the audit aimed to determine the current training provided to clinical staff about the use of poct devices, investigate staff practices and attitudes towards poct, and to ascertain the general level of training and knowledge of quality control in the practice of poct. the information gathered will help to give feedback to the stakeholders on areas that can be improved in the current poct programme. such areas include staff training, competency assessment, and regular refresher courses on both the theory and the practical aspects of poct. the stakeholders include hospital administration, nursing staff, doctors, clinical technologists, laboratory technologists and pathologists of the relevant disciplines. the audit was not requested by the hospital; it was laboratory initiated. methods ethical consideration ethics approval was obtained from the university of stellenbosch health research ethics committee (reference: s15/11/269). written permission was also obtained from the hospital management to conduct the audit in the hospital medical wards and outpatient clinics. participants were given information leaflets and signed informed consent forms after agreeing to participate in the study. study design this study is a descriptive, cross-sectional audit conducted with the use of a questionnaire containing 30 questions (supplementary document 1). a questionnaire was chosen as the most ideal and feasible format for conducting this qualitative audit. the questions were designed to cover the important aspects, based on the iso 22870 guideline. questionnaire the questionnaire was developed by the primary investigator, with the input and supervision of the co-investigators, based on a thorough literature review. this initial draft was further adapted to include the relevant sections from iso 22870 in order to incorporate quality management aspects. questions included general knowledge about poct at the different sites, theoretical knowledge around poct, formal training and competency assessment of poct operators, quality control measures and current perceptions of operators on their respective tests. formal training was defined as a lecture-type session lasting approximately 1 hour, including a practical demonstration and practice in the use of the specific poct device. questions were grouped in sections, with a few confirmatory questions which did not follow a specific order. routine performance of tests was assessed by asking about the frequency of tests performed per week; that is, participants were asked to indicate the average number of times they performed each test applicable to them (more than one test could be selected from a table). the questionnaire was validated at two locations and with colleagues within the department to ensure the questions were understandable prior to distribution of questionnaires. setting and tests included this study was conducted at tygerberg academic hospital, a tertiary hospital with an inpatient capacity of 1384 beds situated in the northern suburbs of cape town, western cape, south africa. the hospital serves a community of approximately 3.6 million from the public health system. three point-of-care tests – blood glucose, blood gases and urinalysis – were chosen for the audit, because they are performed routinely throughout the hospital and fall under the expertise of the study team. all poct devices used in the hospital are from roche accu-chek® (roche diabetes care gmbh, mannheim, germany). other non-chemistry point-of-care tests, such as those for hiv haemoglobin, were not included in the audit as they fall outside the scope of the investigators’ expertise. data collection and analysis an average of three questionnaires was delivered to 55 sites in the hospital, comprising wards, emergency units and outpatient clinics, between 21 june 2016 and 15 july 2016. a few sites only accepted one or two questionnaires, because the nursing managers could not identify anyone else who was suitable to participate, so the total number of questionnaires delivered was 160. site inclusion criteria included any hospital site that routinely uses poct as part of patient management. this included all general medical and surgical wards, emergency units, intensive care units and high care units. the psychiatric and orthopaedic wards and clinics were excluded, because of the low likelihood for use of poct at these sites. ward managers were approached to help with selection of suitable nursing staff to complete the questionnaires. we expected professional nurses, staff nurses and nursing assistants to form the majority of participants from the nursing side. we also expected interns, medical officers and registrars to form the majority of participants from the medical staff. this expectation was based both on their respective clinical duties and on their close involvement in daily patient care as medical doctors without the competing managerial duties applicable to senior and higher rank medical consultants and specialist doctors. students were excluded from the study because they were not employed by the hospital and were at different levels of study. participants were given an option to complete the questionnaire at the time of delivery or to complete it in their own time when not busy with core duties. all participants were encouraged to complete the questionnaire as comprehensively as possible and all those who delayed in completing the questionnaire were given a second or third chance to do so. information from the questionnaires was captured on microsoft excel 2016 version 16.0 (microsoft, redmond, washington, united states), which was used to calculate basic descriptive statistics for the data. in the analyses, each ward or clinic included in the study counted as a single site. missing data included questionnaires that were returned uncompleted and those which were completed only in part. the latter group was included in the data analysis as most of them had completed over 80% of the questions. missing data were also taken into account when specific questions were reported. responses are reported as percentages of the final sample size. each question analysed includes an indication of the percentages of those who did not answer the specific question (tables 1 and 2). no further adjustments were made to compensate for missing data. table 1: participant characteristics, ranks and clinical experience of tygerberg hospital, cape town south africa, 2016. table 2: awareness and knowledge of point-of-care best practices amongst healthcare workers at tygerberg hospital, cape town, south africa, june-july 2016. results study participants and tests administered out of the 160 questionnaires delivered, 68 were returned completed (42.5% response rate) (table 1). most participants (66/68, 97%) performed glucose monitoring, 16/68 (24%) performed blood gas testing, and 17/68 (25%) performed urine dipstick testing (table 2). a total of 52/68 (76%) performed one or more of the tests more than five times per week, mainly glucose, followed by urine dipsticks and blood gas analysis (table 1). knowledge and awareness of point-of-care-testing best practices although a majority (35/68, 51%) indicated that they had formal training, many indicated that they had not (25/68, 38%) (table 2). those participants who indicated that they had knowledge of device validation, reported that this was performed by either the clinical engineering department or the ward. most respondents (57/68, 78%) indicated that poct is necessary in their respective wards or clinics. most of the staff (42/68, 62%) indicated that they knew who was managing poct in their respective locations. the present study found that 31% (n = 21) of participants indicated they used a recording system in addition to patient files, 28% (n = 19) indicated they did not require use of a recording system, and 16% (n = 11) were unaware of a recording system in their ward or clinic. nineteen percent (n = 13) of participants indicated using other means of record control, mostly involving duplicating of result entries in the nursing notes as well as the designated charts on patient files; one participant indicated that doctors enter poct results on their computer for future reference. six percent (n = 4) of participants selected more than one of the four answer options, thus providing conflicting responses. when asked about the important step(s) before performing a point-of-care test, 47/68 (69%) correctly indicated that patient preparation was vital, whilst 9/68 (13%) indicated that confirmation of results was an important first step. a large proportion (25/68, 37%) indicated that the laboratory method was more accurate for glucose measurement in a patient with dehydration or shock, whilst 23/68 (34%) felt the glucose meter was as accurate as the laboratory; a further 11/68 (16%) indicated that the blood gas measurement is the most reliable if a patient is dehydrated. record keeping of test results was another parameter used as a marker of quality management in poct; 21/68 (31%) of participants said they used a recording system, 19/68 (28%) felt there was no need for it, and 24/68 (35%) either did not know if there was a recording system or selected other forms of a recording system. more than half of the participants (38/68, 56%) viewed poct as being an important part of patient management (table 2). discussion most respondents to this audit of the use of poct by clinical staff at a south african tertiary hospital found that poct was a vital part of patient care; this is important, as it is likely to ensure that the staff is open to learning and keeping up to date with new information and practices. the second main observation was that there is a lack of formal training of hospital staff in the practice of poct, and most of the participants indicated that they needed formal training in poct. this is an important issue which requires consideration by stakeholders as it may impact patient outcomes and improve staff confidence in performing the tests.11 staff confidence requires formal skills training and competency testing in order to minimise the risk of errors.12 errors can be attributed to several underlying reasons, including poor technique, abnormal haematocrit, failure to adhere to the correct procedure, and presence of interfering substances. for example, the poct devices used in the hospital are roche accu-chek® devices, which are known to be prone to galactose, ascorbic acid and ceftriaxone interference, and which may deliver false high or -low glucose results in the presence of interference.13 the package insert also states that the use of the glucose meter is not advised in patients with peripheral vascular disease or with dehydration from several causes. without theoretical knowledge relevant to the test performed, the clinical personnel are at a disadvantage and are not fully equipped to perform these tests. all persons involved in poct should be aware of potential interferences, why patient preparation is important, and the concepts of accuracy and precision. such knowledge requires training by experts in the field, such as laboratory professionals who would provide valuable input in the training of clinical personnel. the participants showed a limited awareness of quality control procedures, such as poct device validation and eqa. this was indicated by the high number of participants (54%, n = 37) who were not aware of any eqa involvement in their ward or clinic and the 53% (n = 36) who did not know if any device validation or verification was performed prior to the use of new poct devices in their wards or clinics. this is related to the lack of training in poct basics and principles. the purpose of poct device validation, internal quality control and eqa is to ensure that the results obtained are of a good quality and give confidence to the clinician who will initiate or change the treatment of the patient based on the result obtained from a poct device. laboratories are required to participate in internal quality control and eqa activities in order to be accredited to international standards. point-of-care testing programmes also benefit from such quality control measures, as this would allow them to compare with other poct sites and allow early identification of non-conformances. international guidelines, such as iso 22870:2016 and (clia) poct04, recommend operator training in both the theory and the practice of internal quality control of poct devices.3 some countries, such as australia and new zealand, have local guidelines on the use and implementation of poct based on both national and international recommendations.6,14 when testing for glucose, theoretical knowledge is required in order for the tester to be aware of factors such as haematocrit levels, systemic shock oxygenation status and exposure of strips to humidity, which can reduce the shelf-life of the strips.15,16 a low haematocrit level (< 30% – 35%) may lead to overestimation of glucose, whilst a haematocrit above 45% may lead to underestimation of glucose results by some poct devices. some of the above factors have predictable effects, such as overestimation or underestimation of tests such as glucose, or to the delivery of false-positive dipstick results because of exposure of the strips to humidity. exposure of glucose meter reagent strips to humidity does not have a predictable effect of overor underestimation of glucose results, unlike the aforementioned examples. some poct devices for glucose have been shown to have poor accuracy at critical glucose levels (critically high: > 33.3 mmol/l; critically low: < 2.2 mmol/l) and to have significant bias compared to other devices and the central laboratory method.17 it is therefore important for poct operators to know when to question a poct device result and to confirm with the central laboratory method. some studies have also shown that poct in high-risk patient groups, such as those patients in either adult or paediatric intensive care units, can lead to misdiagnosis.16,18 this requires staff to be very knowledgeable about potential sources of error, dealing with critical values and the use of protocols to guide poc test use in these settings. point-of-care testing has become an integral part of healthcare in both the primary care and the hospital setting.1 with the increasing use of poct, there is also an increasing need to adopt and practise global principles to avoid medical errors and ensure patient safety.2 potential disadvantages of poct include insufficient validation of trained and certified operators, insufficient supervision, limited understanding of quality control testing, little or no security of patient test results and quality control data and limited connectivity of poct devices.2,12 this audit sought to evaluate the current state of poct practice at tygerberg hospital by focusing on the most widely-available and commonly-used tests in the hospital. in agreement with international practices in the hospital setting,5,12 nurses form the bulk of the poct operators in this study. however, nnakenyi et al. showed different findings in their audit of 5 hospitals in nigeria. their study had 40% physicians, 32% nurses and 27% technologists, with a total of 98 participants across all five hospitals.19 the study above is comparable to the present study in terms of sample size and recorded a good response rate from doctors. our study was targeted at clinical staff, namely, nurses and doctors only. great effort was made to recruit doctors in this study; the poor response rate from the doctors in this study may indicate the low level of interest of doctors in poct. this finding supports the recommendation by some to keep poct programmes under the control of the laboratory. this would mean that the head of chemical pathology or the principal chemical pathologist becomes the chair of the poct committee in the hospital, and she or he would be directly involved in the decision making and running of the programme as recommended by international guidelines. in many hospitals in sub-saharan africa, poct is performed by clinical staff because of the limited availability of medical technologists, who are primarily employed in core laboratories with limited numbers, if any, involved in hospital poct. clinical staff are therefore at the forefront of hospital poct in sub-saharan africa and a good source of information about the practices, successes, and limitations of hospital poct in this setting. a study conducted in nigeria included only doctors, which provided a different perspective but limits a direct comparison between the different african studies.20 this study used an interviewer-administered questionnaire on doctors at two different hospitals in nigeria. the sample selection method was not explained clearly; the response from the two hospitals seems to have been 22% for one and 32% for the other. there is no specific mention of how the selection process was conducted and the rationale behind the exclusion of nursing staff in their study. our study, by contrast, mainly included nursing staff who are the main operators of poct in the hospital setting. the questionnaire was designed to obtain information, to identify current gaps in the system and to find solutions that may be easy to implement. some questions were asked to elicit information about record keeping and maintaining a clear paper trail. this has been shown to be a limitation of many poct programmes where devices do not have connectivity to the laboratory system and rely only on the manual transcription of results.21 there were varied responses to these questions in this study, and this may point to the lack of a formalised system for recording poct results separate from the entries in patient files. focus group discussions have been conducted amongst personnel to determine their perception and operational impact of poct on clinical duties in parts of rural australia and uganda (23,24).22,23 most participants of these focus groups indicated that they valued poct, but were dissatisfied with the implementation and their exclusion from the planning process. in our setting, the use of focus groups was not feasible, because of staff shortages and the limited time available to engage with the nursing staff whilst they concentrated on their clinical duties. the present study found that the majority of participants (76%, n = 52) also valued poct and regarded it as being an important component of patient care in their environment. many (66%, n = 45) indicated that they would want formal training in poct, 25% (n = 17) indicated no desire to have training in poct, and 9% (n = 6) did not answer the question. the questionnaire did not specifically include questions about views on implementation or involvement of participants in the planning of the poct programme in the hospital. the focus was primarily on the practice of poct, knowledge of poct theory, and perceptions of participants regarding poct in the wards and clinics where poct programmes are already implemented. the findings of the study are mainly applicable to tygerberg hospital and to other tertiary hospitals that do not currently have a poct training programme, and where the central laboratory is not involved in poct. these findings may not apply to other hospitals in south africa who have a different poct management system. the training of nurses should be explored in other institutions in south africa to give a comprehensive picture of the poct programmes in local hospitals. this should be followed by the development of training programmes and regular re-training to ensure that clinical personnel keep their knowledge and skills up to date. at the time this audit was conducted, there were no national guidelines or policies guiding the practice of poct in south african hospitals. there is limited published information on current practices in poct in south africa and within the rest of the african continent. many of the studies available in africa have looked at implementation of specific poct instruments and clinical outcomes. these studies do not primarily look at the availability of local guidelines and training of personnel on poct and therefore cannot be compared directly with the current study. compared with other poct programmes, such as hiv-poct, general poc biochemistry tests have been around for much longer. a collaborative study of zambia and south africa on hiv-poct found that intensive training, supervision and robust quality assurance mechanisms were required to optimise community hiv-poct.24 a similar approach can be applied to other poct programmes to improve their quality. this audit is the first of its nature to be conducted and reported in south africa. it will provide a basis for the laboratory and hospital to determine the need for collaborative training of clinical staff. limitations the limitations of the study include the small number of questionnaires sent, which was estimated based on the knowledge that not all staff in the ward perform poct and that those who do are usually busy with clinical duties and we did not want to distract them from service delivery. the response rate was quite low overall; in some sites, available staff members were busy when questionnaires were distributed and even upon follow-up, they still did not have time to complete them. this applied to both nursing staff and the mid-level/junior medical staff. the questionnaire did not focus on the views of participants regarding planning and implementation of poct programmes in the hospital, this information would have been valuable and used to gauge the general attitude of participants in being directly involved in the planning and implementation of poct in the hospital. some questions may not have been clear or explicit enough for participants to provide accurate feedback; even though the questionnaire was piloted with nursing staff and medical doctors, some participants may still have found some questions unclear. recommendations we recommend the introduction of training and certification programmes for point-of-care test operators in keeping with international guidelines. we also recommend that a poct coordinator be appointed to lead the current programmes with the assistance of a dedicated poct team in the hospital, as well as the involvement of the clinical laboratory for the continuous evaluation and improvement of the current programme. conclusion this audit found that a significant percentage of the participants did not receive adequate training in poct and had very limited knowledge of quality control measures. this audit gives an indication of the current state of the poct programme in the hospital and highlights areas where intervention is most needed to improve patient care. current guidelines recommend that hospital personnel have basic knowledge and skills to perform routine poct. appropriate implementation of a poct service requires focus on all aspects, including staff training and quality assurance. this information is also important to inform the department of health of the need to consider implementing guidelines and policies on poct in all health facilities in south africa. acknowledgements we would like to thank all of the nurses and doctors who agreed to take part in the study and all colleagues who gave advice and guidance during the development of the study protocol. we also thank the tygerberg academic hospital management for allowing us to conduct the audit in the hospital. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced the writing of this article. authors’ contributions all three listed authors were responsible for the study design and protocol development until submission to ethics. t.p.j. collected the data, through distribution and collection of questionnaires, and entry of data onto the microsoft excel spreadsheet for data analysis, and drafted the first manuscript. r.e. and m.r. further contributed by critical revision of the manuscript in preparation for submission. all listed authors approved of the final version of the manuscript for publication. source of support none. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references dyhdalo ks, howanitz pj, wilkinson ds, souers rj, jones ba. documentation of quality control and operator training at point-of-care testing. arch pathol lab med. 2014;138(11):1444–1448. 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biochemistry: metabolic and clinical aspects. 3rd ed. edinburgh: churchill livingstone, 2014; 932 p. dahm mr, mccaughey e, li l, et al. point-of-care testing across rural and remote emergency departments in australia: staff perceptions of operational impact. stud health technol inform. 2017;239:28–34. rasti r, nanjebe d, karlström j, et al. health care workers’ perceptions of point-of-care testing in a low-income country – a qualitative study in southwestern uganda. plos one. 2017 jul 27;12(7):e0182005. https://doi.org/10.1371/journal.pone.0182005 bock p, phiri c, piwowar-manning e, kosloff b, mandla n, young a, et al. understanding low sensitivity of community-based hiv rapid testing: experiences from the hptn 071 (popart) trial in zambia and south africa. j int aids soc. 2017 aug;20(suppl 6):21780. https://doi.org/10.7448/ias.20.7.21780 reviewer acknowledgement open accesshttp://www.ajlmonline.org page 1 of 1 the editors and guest editors would like to take this opportunity to thank all of those who provided scientific and logistical support for this issue of the african journal of laboratory medicine: should names have inadvertently been excluded from this list, the publisher apologises and undertakes to amend the exclusion in the next issue. ajlm african journal of laboratory medicine in an effort to facilitate the selection of appropriate peer reviewers for the african journal of laboratory medicine, we ask that you take a moment to update your electronic portfolio on www. ajlmonline.org for our files, allowing us better access to your areas of interest and expertise, in order to match reviewers with submitted manuscripts. if you would like to become a reviewer, please visit the journal website to register. to access your details on the website, you will need to follow these steps: 1. log into the online journal at http://www. ajlmonline.org 2. in your ‘user home’ [http://www.ajlmonline. org/index.php/ajlm/ user] select ‘edit my profile’ under the heading ‘my 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http://www.ajlmonline.org http://www.ajlmonline.org http://www.ajlmonline.org/index.php/ajlm/user http://www.ajlmonline.org/index.php/ajlm/user http://www.ajlmonline.org/index.php/ajlm/user article information authors: rosemary a. audu1 catherine c. onubogu2 rosemary n. okoye3 nkiru n. nwokoye2 chika k. onwuamah1 adesola z. musa4 toyosi y. raheem2 maureen n. aniedobe1 samuel j. nduaga3 ini-obong essien3 emmanuel o. idigbe2 affiliations: 1human virology laboratory, nigerian institute of medical research, nigeria2national tuberculosis reference laboratory, nigerian institute of medical research, nigeria 3clinical diagnostic laboratory, nigerian institute of medical research, nigeria 4monitoring and evaluation unit, nigerian institute of medical research, nigeria correspondence to: rosemary audu postal address: nigerian institute of medical research, pmb 2013, yaba, nigeria. dates: received: 25 mar. 2013 accepted: 09 july 2014 published: 24 oct. 2014 how to cite this article: audu ra, onubogu cc, okoye rn, et al. proficiency testing for hiv, tuberculosis and malaria diagnosis in clinical laboratories in nigeria. afr j lab med. 2014;3(1), art. #102, 9 pages. http://dx.doi.org/10.4102/ ajlm.v3i1.102 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. proficiency testing for hiv, tuberculosis and malaria diagnosis in clinical laboratories in nigeria in this original research... open access • abstract • introduction • research method and design    • phase 1: questionnaire survey    • phase 2: provision of proficiency testing service    • characterisation of panels       • hiv       • tuberculosis       • malaria    • panel distribution    • mentoring component    • data analysis    • ethical considerations • results    • questionnaire survey    • participation and response rate of laboratories    • proficiency testing panel testing results    • feedback from participating laboratories • discussion    • limitations of the study    • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: proficiency testing (pt) is a means of verifying the reliability of laboratory results, but such programmes are not readily available to laboratories in developing countries. this project provided pt to laboratories in nigeria.objectives: to assess the proficiency of laboratories in the diagnosis of hiv, tuberculosis and malaria. methods: this was a prospective study carried out between 2009 and 2011. a structured questionnaire was administered to 106 randomly-selected laboratories. forty-four indicated their interest in participation and were enrolled. four rounds of pre-characterised plasma panels for hiv, sputum films for tuberculosis and blood films for malaria were distributed quarterly by courier over the course of one year. the results were returned within two weeks and scores of ≥ 80% were reported as satisfactory. mentoring was offered after the first and second pt rounds. results: average hiv pt scores increased from 74% to 95% from the first round to the third round, but decreased in the fourth round. for diagnosis of tuberculosis, average scores increased from 42% in the first round to 78% in the second round; but a decrease to 34% was observed in the fourth round. malaria pt performance was 2% at first, but average scores increased between the second and fourth rounds, culminating in a fourth-round score of 39%. many participants requested training and mentoring. conclusions: there were gross deficiencies in the quality of laboratory services rendered across nigeria. in-country pt programmes, implemented in conjunction with mentoring, will improve coverage and diagnosis of hiv, tuberculosis and malaria. introduction top ↑ the importance of quality services in healthcare laboratories in developing countries has been recognised universally. 1, 2, 3 at present, the laboratory infrastructure and test quality for all types of clinical laboratories remains weak in most countries in africa. 4,5 laboratories applying the principles of a quality management system (qms) generate reliable and cost-effective results; moreover, quality management is one of the major building blocks of the accreditation process in the african region. 6,7 it has become necessary to strengthen the capacity of clinical laboratories in order to ensure the generation of quality results that are suitable for use by clinicians and which benefit patients.proficiency testing (pt) is an external quality assessment (eqa) programme involving sending a panel of samples to a group of participating laboratories.8 although the organisers issuing the panels know the result, the participating laboratory personnel do not. pt verifies that laboratories are proficient in their testing process and can obtain accurate and reliable results. 6 comparison of results between groups of laboratories may also be used to validate a particular measurement process. as beneficial as the pt programmes may be, they are not readily available to many laboratories in developing countries. some of the limitations to local laboratories' enrolment in foreign programmes are the high cost, challenges of transportation with respect to country regulations, suitable means of transport of specimens to sites, difficulty in interpretation of pt results and the absence of technical support with regard to identifying and correcting causes of poor performance. 6 since laboratory-confirmed diagnosis of hiv, tuberculosis (tb) and malaria is essential to public health prevention and support services, accurate and reliable laboratory results are critical.9 in addition, the world health organization's regional office for africa (who afro) has recommended that national public health reference laboratories develop and implement a qms,2 including participation in an eqa scheme.7 these reference laboratories, according to who afro, should, in turn, provide national eqa programmes to other laboratories within the country.7 as a result of the challenges encountered by laboratories, a national pt feasibility study for hiv, tb and malaria was undertaken at both public and private health laboratories in nigeria. this study was conducted to evaluate the level of implementation of qms in nigeria and to assess the proficiency of laboratories in the diagnosis of hiv, tb and malaria. research method and design top ↑ this was a prospective study carried out in two phases between 2009 and 2011. the first phase was a questionnaire survey to provide baseline information on quality practices in laboratories, whilst the second phase was the provision of pt services. phase 1: questionnaire survey in the first phase, six states were selected at random from each of the six geopolitical zones of nigeria. six focal persons who were laboratory state coordinators were then identified and recruited to serve as zonal coordinators. zonal coordinators identified 20 laboratories, each from different local government areas within the six states. structured questionnaires, adapted from the who template, 10 were developed and field tested prior to distribution of the survey (box 1). the questionnaire had five sections, including general questions about the laboratory, specific questions about hiv, tb and malaria units and questions on general quality issues. the laboratory head and respective heads of each unit completed the questionnaires. the questionnaires were sent to the zonal coordinators to administer to the 120 laboratories, 106 of which consented to participate and returned completed questionnaires. data entry and analysis were performed using the filemaker pro v10 (2009) database and microsoft® excel, respectively. phase 2: provision of proficiency testing service of the 106 laboratories that completed the questionnaires, 44 indicated their interest regarding participation in the joint pt programme for the three major diseases of public health importance, namely, hiv, tb and malaria. these laboratories were enrolled in the second phase of the study. by september 2010, pre-characterised panels were prepared for hiv, tb and malaria in the nigerian institute of medical research (nimr)’s reference laboratories and were distributed to the participating laboratories by courier. four rounds of panels each of hiv, tb and malaria were sent to each laboratory on a quarterly basis for a year. characterisation of panels hiv blood samples obtained from blood banks were characterised at the national reference laboratory by testing on the determine™ hiv-1/2 rapid test (abbott, usa), genscreen™ ultra hiv ag-ab enzyme immunoassay (bio-rad, france) and new lav-blot i and new lav-blot ii western blotting (bio-rad, france). the tests were all carried out according to their manufacturers’ instructions. 11,12,13 five panels, each comprising three positive and two negative samples, were sent to the participating laboratories for each round. the pt panels were scored based on the hiv-positive or -negative status assigned by the reference laboratory. the correct use of the national testing algorithm was also assessed. box 1: needs assessment questionnaire for the national external quality assessment programme for hiv, tuberculosis and malaria. box 1 (continues): needs assessment questionnaire for the national external quality assessment programme for hiv, tuberculosis and malaria. box 1 (continues): needs assessment questionnaire for the national external quality assessment programme for hiv, tuberculosis and malaria. box 1 (continues): needs assessment questionnaire for the national external quality assessment programme for hiv, tuberculosis and malaria. tuberculosis following informed consent, fresh sputum specimens were collected from patients who attended the directly observed treatment short-course (dots) clinic at nimr. panel slides were prepared as described by martinez-guarneros et al. 14 and each slide reading was carried out by two independent microscopists who arrived at a consensus. a total of five unstained slides per panel was sent to each laboratory (20 slides in total), with instructions to stain panels using the laboratory’s routine procedure to identify and quantify the acid-fast bacilli (afb) using the who or international union against tuberculosis and lung disease (iuatld) grading system. the ratio of positive to negative was varied randomly in each round of panel distribution. the participating laboratories were assessed on correct identification of the slide status and quantification of the afb, as compared with the assigned characteristics from the reference laboratory. malaria following informed consent, blood was collected in edta tubes from malaria parasite-positive patients with varying degrees of parasitaemia, as well as from malaria parasite-negative subjects. before samples were sent out, thick and thin films were made on the same slide and stained with giemsa stain at the reference laboratory, according to the standard method.15 the films were screened to ensure a good staining reaction. the slides were examined by two independent microscopists for cell distribution, parasite density count, species and stage identification. a limit of 30% was set to reach a consensus; however, where consensus was not reached, a third microscopist read the slide as a tiebreaker. the consensus information was recorded for each slide. for each round, the slides were packaged in the slide boxes in sets of three negatives and two positives. laboratories were expected to determine the parasite status and density as well as analyse each slide for species identification. results were assessed based on these parameters and the errors identified. reports were returned showing error types and suggestions for improvement. panel distribution for each shipment, the panels were parcelled in a triple packaging format, including instructions and reporting forms. the panels were sent in cold boxes through a courier agent to each zonal coordinator in order to save cost. each report form contained sections for results and comments, enabling participants to provide feedback. the zonal coordinators were responsible for distributing the panels to the participating laboratories within their zone. participants were instructed to return results within two weeks of receipt of the panels and the same channel was then used for returning the results and feedback forms to nimr. returned results were assessed and scored based on performance as compared with the expected results and individual performance scores were then returned to the participants through the same zonal coordinators. mentoring component after the first round of panel distribution, job aids (i.e., brief procedural instructions) for diagnosis of each disease type were prepared and sent to all the laboratories to help improve performance. at the end of the second round of panel distribution, because of cost constraints, 13 laboratories were recommended for personnel retraining as a result of poor performance. these laboratories were invited for a fully-sponsored training course at nimr. personnel from 11 of the 13 laboratories attended the week-long training, during which participants spent two days each on practical sessions on the laboratory diagnosis of tb and malaria and one day on hiv diagnosis. the training also consisted of didactic sessions on qms. panels were sent out to the laboratories for the third round immediately after the training. there was no mentoring session before the fourth round. data analysis results were scored based on the assigned pt provider ratings and characteristics. discordant results were assigned zero points, whilst concordant results were assigned 20 points per sample, for a total of 100 points per panel. scores of 80% and above were reported as satisfactory, which is the generally-accepted standard. 16 an unassigned score for a particular distribution indicated that a laboratory did not return the result. all scored results were entered into a filemaker pro v10 database where individual reports were generated for each laboratory. the data were then exported and analysed in a microsoft® excel spreadsheet. the feedback from the laboratories was analysed and suggested improvements for the pt services were implemented where possible. ethical considerations ethical approval was obtained from the institutional review board of the nimr (11 may 2009). only those sites that indicated an interest in participating were enrolled in the study, at no cost. laboratories were free to decline participation in the study at any point in time. results top ↑ questionnaire survey table 1 shows the results of the surveyed laboratories from the first phase of the study. this study used an abridged grading system of the who stepwise laboratory quality improvement process towards accreditation (slipta) to assess laboratories' adherence to the international organization for standardization (iso) 15189 standard, measuring laboratory quality on a scale of zero to five stars. 17 the laboratories attained, based on self-reporting, an average score of 65%, which is equivalent to two stars out of five. it was also found that of the 106 laboratories that completed the questionnaire, 68 (64%) reported having a system of result validation and only 34% (n = 36/106) provided scheduled maintenance of equipment. most of the laboratories did not have preventive maintenance policies, thereby possibly undermining the quality of results generated by the equipment. internet access was found to be uncommon (n = 36/106; 34%) amongst the respondents. the survey also showed that very few laboratories were registered for pt for hiv (n = 35/106; 33%), tb (n = 33/106; 31%) and malaria (n = 19/106; 18%). forty-four (42%) of the 106 laboratories indicated interest in participating in the pt for the three diseases offered in this study as they had not been registered for pt previously. table 1: survey results of the 106 surveyed laboratories. participation and response rate of laboratories of the 44 laboratories that indicated interest in participating in the joint pt programme, 10 (23%) were publicly-owned laboratories within hospital settings whilst 34 (77%) were private, stand-alone laboratories. in the second phase of the study, two laboratories opted out, one at the first round of panel distribution and the other after the second round of distribution. a few laboratories did not return results and were thus not assigned scores. laboratories that did not receive scores included 3/44 (7%) in the first round, 5/43 (12%) in the second, 2/42 (5%) in the third and 7/42 (17%) in the fourth round. on average, 10% failed to return results on one or more pt samples. proficiency testing panel testing results most laboratories returned the pt panel results within the two weeks stipulated, but the time had to be extended for some laboratories because of political insecurity. all the laboratories tested the hiv panel using rapid test kits, either serially or in parallel algorithms. some did not confirm a positive result with a second test kit. the pt results showed improvement in hiv pt scores from an average of 74% in the first round to 95% in the third round, but this was not sustained in the fourth round (figure 1). the major issue observed with hiv pt was the incorrect use of the national testing algorithm, which requires consistent results from two rapid test kits before confirming a positive hiv status; some laboratories used only a single reactive result (figure 2). figure 1: average performance of laboratories at different rounds of panel testing. figure 2: use of national testing algorithm in hiv diagnosis. for tb pt, all laboratories stained the slides using the ziehl-neelson staining technique. there was an improvement after the first round from an average score of 42% to 78% in the second round; however, the average dropped consistently from that point to 34% in the fourth round (figure 1). the issues observed with tb pt included quantification errors and a high level of false negative results (figure 3). an unusually high level of false positives was observed in the third round of the pt. figure 3: comparison of errors in tuberculosis diagnosis. although performance in malaria pt appeared poor, there was a steady increase in average scores from 2% in the first round to 39% in the fourth round (figure 1). participants appeared to continue to have difficulties with parasite detection and count throughout the testing period (figure 4). one laboratory confirmed the use of rapid test kits for malaria; its results were not included in the analysis. figure 4: frequency and types of errors identified in malaria diagnosis. feedback from participating laboratories a total of 81 persons provided feedback throughout the duration of the pt. the respondents were at liberty to express their concerns to nimr on any issues whatsoever. figure 5 shows the categorised comments from the participating laboratories. figure 5: categorised comments by participating laboratories. there was a great demand for training expressed by 25 (31%) of those who gave feedback. some requested on-site mentoring visits, whilst others wanted practical training sessions organised by the pt provider. some stated that training would enhance their competence and serve as a motivating factor for participation. twenty (25%) of the feedback responses provided useful suggestions to the provider for improvement. some requested that the time to return results be extended, whilst others requested that the quarterly exercise be replaced by samples being distributed every 2 months. fifteen (19%) respondents also requested the continuation of the programme and expansion of the scope to include more analytes for laboratory investigations of other diseases, aside from the hiv, tb and malaria, as well as inclusion of more laboratories. whilst 12 (15%) of the respondents requested the provision of reagents for pt, four (5%) complained about bad microscopes and needed some assistance, either financially or through direct provision of better microscopes and supervisory visits. a number of comments were actionable immediately and helped the provider to improve the quality of pt samples. the most prominent example was a series of complaints of leakage of plasma samples, which the provider responded to by changing the sample tubes. equally important complaints after the first round were broken slides and the quality of some of the malaria parasite slides. the provider improved on the packaging by ensuring proper sealing and positioning of the slide boxes so as to prevent damage during transportation. to address the slide quality, three or four readers reviewed the stained slides for subsequent panels for the second, third and fourth rounds and the best slides with the lowest inter-reader variability were selected and sent. an expert on malaria panel preparation from the national research centre, burkina faso, where the staff had previously acquired malaria panel preparation skills, visited the provider to ensure quality practices. discussion top ↑ the initial phase of the study indicates the prevalence of a poor culture of qms in nigerian laboratories. from the questionnaire survey, the laboratories earned an average of two stars, despite the fact that the grade was attained by self-reporting and not by an external audit. the lack of qms culture creates concern regarding the accuracy, reliability and timeliness of clinical results generated in laboratories. this finding supports previous observations that, in sub-saharan africa, laboratory infrastructure and personnel are affected adversely by the lack of resources and prioritisation, hampering laboratory system efforts in the fight against infectious and chronic diseases. 4,5 as a result, the accessibility of laboratory testing and the quality of available services remain a serious challenge.7 there is a dire need to create a culture of quality management in nigerian laboratories, which will help practitioners appreciate the necessity of results validation and participation in pt and other programmes. nimr plans to address this need with its newly-awarded training grant to build the culture of qms in both private and government-owned laboratories. this grant utilises the strengthening laboratory management toward accreditation (slmta) programme to develop capacity for laboratories not supported by the us president’s emergency plan for aids relief (pepfar) fundsa lack of resources as well as a poor understanding of the benefits of participation in an external pt programme may have been responsible for the low enrolment in this study. of the laboratories that did enrol, two private laboratories opted out of the pt programme after commencement. without any formal communication, the first laboratory was closed down at the point of delivery of a set of panels. the closure may have been related to the political crises in that region. the second laboratory communicated to the provider that the staff would no longer be able to participate in the study because the laboratory owner had gone back to school for full-time study. investing in qms is expensive and time consuming. as such, care should be taken in the selection of the laboratories enrolled in such pt projects in order to ensure their ability and willingness to provide the needed services. the rate of failure to return results varied despite the fact that the due dates for the results were extended at the request of some of the participating laboratories. one reason for this variation could possibly be a poor understanding of the importance of pt programmes. some of the laboratories’ staff members reported that they had to wait for the most senior laboratory scientist, often the owner of the laboratory, to be present during sample analysis. training will help improve understanding and increase participation. the incorrect use of the national hiv testing algorithm was common at the outset of this study. the nationally recommended algorithm includes serial testing, which requires a second test for an initial reactive sample. some laboratories used test kits outside the nationally-approved kits; some used a single test to confirm hiv infection; and others used parallel testing algorithms. correct use of the standardised hiv testing algorithm would reduce the risk of issuing false reactive results. when so much time and so many resources are spent on developing hiv testing algorithms, it is essential that countries ensure their proper dissemination to all levels where hiv testing is carried out. in this study, provision of a job aid with step-by-step instructions for hiv testing after the first round of panel distribution resulted in marked improvements. however, some laboratory scientists complained that their job aids were kept in the office of the head of the laboratory and not at the point of use in the laboratory. lack of available job aids in the laboratory may have affected the laboratories' performance in this study. provision of job aids may also have contributed to the improvements observed in the diagnosis of tb in the second round of panel distributions; however, this improvement was not sustained and quantification errors were common. this finding underscores the challenges of managing tb patients on therapy, as the efficacy of therapy would be difficult to monitor. other error types that could have a serious impact on the community were the high rate of false positive results observed in the third round and the persistent false-negative results observed throughout the study. these errors have serious implications: individuals diagnosed incorrectly as positive will most likely be placed on unnecessary therapy, whilst individuals who are diagnosed incorrectly as negative will be released into the community and will spread the infection. all measures must be put in place to halt this trend, especially as nigeria has been ranked 13th on a list of 22 countries with the highest burden of tb. 16 unlike the positive performances reported in an eight-year eqa of public reference laboratories, which recorded 82% acceptable results for malaria species identification,18 this study observed a comparatively low rate of acceptable malaria results. the poor performance observed for both tb and malaria diagnoses may be connected to the report in phase 1 of the study, in which only 34% of the laboratories reported having preventive maintenance for their equipment. it is important that preventive maintenance programmes be established in laboratories in order to ensure proper functioning of equipment so as to guarantee the accuracy and reliability of test results. confirming the need for better equipment maintenance, 5% of respondents complained about the quality of their microscopes. inadequate equipment maintenance, as observed in this study, is not limited to nigeria; poor maintenance culture is one of the major challenges in strengthening health systems in sub-saharan africa. 19,20 in spite of equipment limitations, there was continual improvement in performance on malaria panels, particularly with regard to parasite count and staging. this progression gives hope for improvement in the diagnostic proficiency of malaria microscopists if more efforts can be devoted to their training. to bridge this gap, nimr now provides annual training for malaria microscopists from all over the country. study participants demonstrated that they recognise that there are gaps in their knowledge and are willing to be trained for improvement. thirty-one per cent of the participants who gave feedback from phase 2 of the study requested further professional training. moreover, some of the participants who attended the resulting training commended the organisers, as they had not undertaken any prior in-service training. efforts directed at in-service training should be increased and extended to private laboratories that contribute a great deal to the health system, particularly in nigeria. those who had not participated in a pt programme previously also requested that the programme be sustained and expanded with regard to the scope of analytes and the number of participating laboratories. this request came despite the varying acceptable result rates obtained from the laboratories. such feedback is indeed a clarion call for more donor investment in eqa. it is essential that individual african countries be strengthened in order to take up the challenge of providing eqa in their respective countries, in order to extend pt programmes to these laboratories. currently available programmes are accessible to national public health or reference laboratories in africa, but do not benefit peripheral or private laboratories. access to pt programmes will also motivate the drive toward accreditation, as observed in south africa.7 the quest for accreditation can help laboratories address most other concerns. preparing for accreditation helps to strengthen laboratory management in and application of best practices throughout the laboratory system. awareness of laboratory accreditation is gathering momentum at present in nigeria, as is evidenced by the enrolment of 30 laboratories for accreditation preparedness training, the preparation for enrolment by others and the high demand for training by still more laboratories. there are currently 30 personnel who have been trained to roll-out the slmta programme in nigeria, three of whom qualified as master trainers;21 several other in-country training courses to prepare laboratories for accreditation are on-going. in summary, this study identified gross deficiency in the quality of laboratory services rendered across the country, indicating a poor state of qms. the pt study was well received, with demands to extend its scope. although most participants requested training and on-site mentoring, the training provided by this exercise was too short and did not have the desired impact for hiv and tb diagnoses. nevertheless, just as it has been reported from regional eqas that public health and reference laboratories in the african region are capable of the accurate determination of disease status,18,22 so it is believed that the laboratories that participated in this study also can perform satisfactorily if given the necessary support. for example, similar laboratories in uganda23 and other resource-constrained countries 24 have been supported in their endeavours to improve the quality of their services and have yielded remarkable improvements. there is, therefore, a need to strengthen laboratory systems in individual countries by providing pt programmes to clinical laboratories, including those that are privately owned with high volumes of work. the implementation of pt programmes will enhance the drive toward laboratory accreditation in the region. limitations of the study differences may have arisen from the self-administered questionnaires that could have influenced the findings reported in this study. also, there may have been varied readings by microscopists for the blood and sputum films, affecting study findings. conclusion there was gross deficiency in the implementation of qms which inadvertently affected the proficiency of the laboratories in the diagnosis of hiv, tb and malaria. concerted efforts are therefore required to train nigerian laboratories on qms, which would yield the desired outcome as observed from this study. acknowledgements top ↑ this project was supported by funds from the international association of national public health institutes (ianphi) under the sub-award no. 6-38223-g1. we appreciate ianphi for this support. we are also grateful to the zonal coordinators who assisted us in identifying the laboratories and who served as a bridge between us and the participating laboratories. we thank all the laboratories that made time to be part of this project. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. authors’ contributions r.a.a. (human virology laboratory) developed the proposal, coordinated the project and wrote the manuscript. c.c.o. (national tuberculosis reference laboratory) led the tb team in preparing the tb panel. r.n.o. (clinical diagnostic laboratory) led the malaria team in preparing the malaria panels and evaluating the reports. n.n.n. (national tuberculosis reference laboratory) prepared the tb panel and evaluated the reports. c.k.o. (human virology laboratory) was responsible for all paperwork and prepared reports for the participating laboratories. a.z.m (monitoring and evaluation unit) provided bio-statistical expertise for the team. t.y.r. (national tuberculosis reference laboratory) prepared the tb panel and maintained the link with the zonal coordinators. m.n.a. (human virology laboratory) prepared the hiv panel and evaluated the reports. s.j.n. (clinical diagnostic laboratory) prepared the malaria panel and also helped to evaluate the reports. i.-o.e. (clinical diagnostic laboratory) prepared the malaria panel and e.o.i. (national tuberculosis reference laboratory) was involved in designing the project. references top ↑ 1. maher d, harries ad. quality care: a link between clinical and public health approaches to hiv infection in developing countries. trop med int health. 2010;15(4):391–395. 2. maher d, raviglione m. the history of the dots strategy: achievements and perspectives. in: schaaf hs, zumla a, editors. tuberculosis – a comprehensive clinical reference, 2nd ed. london: elsevier, 2009; pp. 270–273. 3. colebunders r, decock r, mbeba mj. improving the quality of care for persons with hiv infection in sub-saharan africa. trop geogr med. 1995;47(2):78–81. 4. derua ya, ishengoma dr, rwegoshora rt, et al. users' and health service providers' perception on quality of laboratory malaria diagnosis in tanzania. malar j. 2011;10:78. http://dx.doi.org/10.1186/1475-2875-10-78 5. hailegiorgis b, girma s, melaku z, et al. laboratory malaria diagnostic capacity in health facilities in five administrative zones 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in serum/plasma by immunoblotting [document on the internet]. c2007 [cited 2014 mar 06]. available from: http://www.bio-rad.com/webroot/web/pdf/inserts/cdg/en/883521_en.pdf 14. martinez-guarneros a, balandrano-campos s, solano-ceh ma, et al. implementation of proficiency testing in conjunction with a rechecking system for external quality assurance in tuberculosis laboratories in mexico. int j tuberc lung dis. 2003;7(6):516–521. 15. world health organization. tuberculosis profiles by country [homepage on the internet]. c2014 [cited 2014 mar 07]. available from: http://www.stoptb.org/countries/tbdata.asp 16. forbes ba, sahm df, weissfeld as. bailey & scott's diagnostic microbiology. 12th ed. st. louis, mo: mosby, 2007; p 625–626. 17. world health organization regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation in the african region (with checklist) [document on the internet]. c2011 [cited 2014 mar 06]. available from: http://www.afro.who.int/index.php?option=com_docman&task=doc_download&gid=8642&itemid=2593 18. frean j, perovic o, fensham v, et al. external quality assessment of national public health laboratories in africa, 2002–2009. bull world health organ. 2012;90:191–199a. http://dx.doi.org/10.2471/blt.11.091876 19. penfold s, shamba d, hanson c, et al. staff experiences of providing maternity services in rural southern tanzania – a focus on equipment, drug and supply issues. bmc health serv res. 2013;13:61. http://dx.doi.org/10.1186/1472-6963-13-61 20. fonjungo pn, kebede y, messele t, et al. laboratory equipment maintenance: a critical bottleneck for strengthening health systems in sub-saharan africa? j public health policy. 2012; 33(1):34–45. http://dx.doi.org/10.1057/jphp.2011.57 21. ianphi public health institutes of the world. slmta training of trainers workshop: creating a culture of quality for nigerian laboratories [homepage on the internet]. c2013 [cited 2014 mar 06]. available from: www.ianphi.org/whatwedo/projects/list/nigeria5.html 22. cham f, maleka m, masango m, et al. the world health organization african region external quality assessment scheme for anti-hiv serology. afr j lab med. 2012;1(1), art. #39, 6 pages. 23. yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin pathol. 2010;134(3):401−409. http://dx.doi.org/10.1309/ajcpnbbl53fwuiqj 24. gaydos ca, rizzo-price p, balakrishnan p, et al. impact of international laboratory partnerships on the performance of hiv/sexually transmitted infection testing in five resource-constrained countries. int j std aids. 2011;22(11):645–652. http://dx.doi.org/10.1258/ijsa.2011.010527 abstract introduction methods results discussion acknowledgements references about the author(s) salisu b. muazu department of internal medicine, faculty of medicine, rasheed shekoni federal university teaching hospital, dutse, nigeria hauwa bako department of medical laboratory science, faculty of allied health sciences, ahmadu bello university zaria, zaria, nigeria ahmad m. bello department of medical laboratory science, faculty of allied health sciences, bayero university kano, kano, nigeria john n. onuche kogi state hospital management board, lokoja, nigeria faruk salami laboratory department, okene zonal hospital, okene, nigeria abimbola o. abioye department of clinical pathology, faculty of medicine, lagos university teaching hospital, lagos, nigeria zainab i. nadabo laboratory department, women and children hospital, gombe, nigeria richard j. banya faculty of medical laboratory science, taraba state college of health technology, takum, nigeria eni-yimini s. agoro department of biochemistry, faculty of basic medical sciences, federal university otuoke, bayelsa, nigeria saheed a. adekola department of medical laboratory science, faculty of clinical pathology, federal medical centre, lagos, nigeria citation muazu sb, bako h, bello am, et al. knowledge, competence, experience of healthcare practitioners using glucometers for patient care in nigeria. afr j lab med. 2025;14(1), a2770. https://doi.org/10.4102/ajlm.v14i1.2770 note: additional supporting information is available for download alongside the online version of this article as online supplementary document 1. original research knowledge, competence, experience of healthcare practitioners using glucometers for patient care in nigeria salisu b. muazu, hauwa bako, ahmad m. bello, john n. onuche, faruk salami, abimbola o. abioye, zainab i. nadabo, richard j. banya, eni-yimini s. agoro, saheed a. adekola received: 16 jan. 2025; accepted: 24 june 2025; published: 07 oct. 2025 copyright: © 2025. the author licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: despite the widespread acceptability of glucometers as a blood glucose self-monitoring and point-of-care device, their usage is confronted with operational, technical, regulatory, and quality control concerns. objective: this study assessed knowledge, estimated competence, and measured experience of healthcare practitioners using glucometers for patient care in two states of northern nigeria. methods: this cross-sectional, descriptive study used a total population sampling strategy and self-completed questionnaires. a total of 768 questionnaires were distributed to hospitals in jigawa and kogi states, nigeria, from december 2019 to april 2022. the questionnaire had three sections: collecting details about type of healthcare facility, sociodemographic characteristics and educational qualifications of participants, and assessment of knowledge, competence and practice. data were analysed and results expressed as frequencies and percentages. results: overall, 570 questionnaires were filled and retrieved, giving a response rate of 74.2%. most of the participants were male (312; 54.7%); female participants totalled 258 (45.3%). most participants were aged < 50 years (25–40 years, 215 [37.7%]; 41–50 years, 246 [43.2%]). the majority of participants were medical laboratory scientists (124, 21.8%]), technicians (151, 26.5%), or nurses (132, 23.2%). most participants (284, 49.8%) reported having no formal training prior to first use of glucometers in patient care. many participants (379, 66.5%) knew about glucometer calibrators; 235 (41.2%) did not know what specific purpose calibrators served. conclusion: this study found a lack of knowledge, competence and experience among healthcare practitioners, especially for hands-on use of glucometer calibrators and standard operating procedures for blood glucose testing using glucometers. what this study adds: the study brings to fore the need for training and retraining of healthcare practitioners on the theoretical and practical skills required for operating glucometers. periodic calibration of glucometers and provision of quality control materials should be incorporated into standard operating procedures at point-of-care testing workstations in health facilities. keywords: knowledge; competence; experience; glucometers; healthcare practitioners; nigeria. introduction the practice of using glucometers for self-monitoring of blood glucose (smbg) is an essential component for care and management of diabetes,1,2 which is supported by educational and clinical guidelines.3,4 information generated from smbg is shared with healthcare teams for modification of multiple self-care behaviours and simplifying problem-solving discussions.5,6 numerous sources of error have been reported to affect blood glucose testing using glucometers, including clinical, operators, reagent, environmental, software, and hardware.7 despite the myriad of challenges, glucometers have undergone evident technological and methodological improvements in recent times.8 although conflicting reports exist pertaining to the accuracy and reproducibility of glucometers, evaluation have showed decreased reliability and non-compliance with some international standards of certain brands,8,9 while increased sensitivity and specificity have been reported for other brands.10,11 compared with central laboratory testing for blood glucose using auto-analysers, enhanced performance has been observed for most glucometers, despite quality control concerns.9,12,13 outpatient glucometers differ from those found in hospital settings, resulting in heterogeneity of operators and multi-directional studies. because of the diversity of glucometer operators, some studies14,15,16,17,18,19 have assessed the knowledge, attitude and practice of individuals living with diabetes type 1 and 2 using glucometers for routine smbg, while other studies20,21,22,23,24,25 evaluated knowledge, competency and experience of healthcare practitioners (hcps) operating glucometers. researchers’ efforts to develop an operator’s assessment checklist26 and a self-management model27 for hcps and patients are yet to be fully recognised and accepted by international regulatory organisations. ensuring patient safety and reducing medical errors are key components of global best practices.28 integrating point-of-care tests (pocts) into laboratory management information systems is necessary in order to allow bidirectional information exchange and traceability.29 the lack of or inadequate hcp formal training, as well as the absence of or inadequate quality control materials and practices, have been reported as major concerns for using glucometers for blood glucose monitoring and patient care.30,31,32,33 studies conducted in northern nigeria have shown poor quality control practices for glucometers, and inadequate or lack of training for hcps, to be major challenges facing the use of glucometers as a point-of-care device in the region. although studies have accessed either knowledge, competence or experience among hcps, limited studies have investigated the trio in jigawa and kogi states of nigeria, where the study was conducted. the objectives of the study were to assess knowledge, estimate competence and measure experience of hcps using glucometers for patient care in two states of northern nigeria. methods ethical considerations an application for full ethical approval was made to the jigawa state health research ethics committee and kogi state health research ethics committee. ethical approval reference number moh/sec/3/784/1/12 received on 23 november 2019 and reference number moh/prs/465/v.1/011 received on 17 may 2021, were granted before the study commenced in kogi and jigawa states. we obtained written informed consent from all participants. information which revealed the identity of participants was masked for confidentiality purposes. study materials were stored in locked steel cabinets accessible only to the research team. study design and setting we performed a cross-sectional descriptive assessment conducted using a self-completed questionnaire (online supplementary questionnaire). the questionnaire was designed to cover important aspects of knowledge, competence and experience required by health professionals to appropriately use glucometers for patient care. the study was performed in 36 health facilities of kogi and jigawa states of nigeria. healthcare practitioners comprising physicians, medical laboratory scientists, pharmacists, physiotherapists, nutritionists and/or dietitians, medical laboratory technicians, community health workers, and nurses, spanning 48 local government areas, participated in the study between 02 december 2019 and 16 october 2020 in jigawa state, and from 18 may 2021 to 29 april 2022 in kogi state. sampling size and strategy using the total population sampling approach, all eligible hcps working in the study hospitals and involved in patient care, who served as the target population, were invited to take part in the study. an equal number of questionnaires was distributed to the respective departments in the study hospitals. healthcare practitioners who were on intern service or the mandatory national youth service corps were excluded. health professionals in administrative positions were also excluded, because they were not actively involved in direct patient care. data collection a questionnaire for self-completion was developed by the investigators through an iterative process involving several stages of drafting and revision of the questionnaire items. the drafting and redrafting process involved experts in different fields with expertise in survey research, laboratory quality management systems, and poct to ensure the questionnaire’s content and face validity. the questionnaire was pre-tested with a small group of health professionals (n = 30) from 25 november 2019 to 29 november 2019 at the accident and emergency ward of rasheed shekoni federal university teaching hospital, jigawa state, and the instrument was refined using the outcome of aggregate score for sections, as well as clarity and interpretation of the questions. participants of the pilot study were asked to make inputs to improve the questionnaire, and minimal adjustments which did not show need for significant changes were suggested. as a result, the questionnaire was adopted for the study. the questionnaire was designed to evaluate the knowledge, competence, and experience of hcps on the use of glucometers in patient-care settings. questions were numbered serially, did not follow a particular design, and were attached to options and sub-questions. the final questionnaire was structured into three sections; section ‘a’ comprised facility details, section ‘b’ contained personnel details (sociodemographic characteristics of respondents), and section ‘c’ comprised assessment questions for knowledge, competence and experience. questions in section ‘c’ included general knowledge about glucometers and calibrators, technical competence, and experiences of hcps actively operating glucometers in patient care. a total of 768 copies of the final paper-based questionnaires were distributed to participants at their various workstations. reminder/follow-up was done via phone calls at weekly intervals for three consecutive weeks, after which manual collection of paper-based completed questionnaires was performed. data analysis information from completed questionnaires was entered into a microsoft® excel database, (microsoft corporation, redmond, washington, united states). data entry was performed by two research assistants. entry by the first research assistant was cross-checked and validated by the second. missing data were not encountered during data entry by the research assistants and review by the research team. data entered were cleaned then exported to statistical package for the social sciences v25.0 (ibm corp., armonk, new york, united states) for subsequent management and analysis. gender, age range and educational qualifications were represented by a specific code prior to data entry for categorisation purposes. respondents’ characteristics are expressed in percentages and frequencies analysed using simple descriptive statistical tools. each knowledge question was scored as 0 for incorrect responses and 1 for correct responses. composite knowledge was calculated as an aggregate of the six knowledge questions, with a minimum of 0 and a maximum of 6 achievable by each respondent. respondents who achieved scores between 0 and 4 (< 70%) were considered to have low knowledge, while those who scored 5 and 6 (≥ 70%) were considered to have high knowledge. similarly, competence was operationalised by a mix of 6 questions which were scored and analysed in the same way as described for the knowledge domain. categorical variables were analysed using a 2 × 2 cross-tabulation analysis (table 1) to determine the level of knowledge and competence of respondents, hypothesised as a function effect of formal training and availability of standard operating procedures (sops) for glucometers. experience was measured by the number of positive responses related to skills and work experience pertaining to glucometer usage. table 1: cross-tabulation (2 × 2) of responses collected from 02 december 2019 to 16 october 2020 in jigawa state, nigeria, and from 18 may 2021 to 29 april 2022 in jigawa and kogi states, nigeria. results participant characteristics of the 768 questionnaires distributed, 570 were filled and retrieved, with a response rate of 74.2% recorded. the majority of the respondents were medical laboratory technicians (n = 151, 26.5%), nurses (n = 132, 23.2%), or medical laboratory scientists (n = 124, 21.8%) (table 2). the highest educational qualification of respondents was almost evenly split between the diploma level (n = 260, 45.6%) and graduate degree level (n = 245, 43.0%). table 2: participants’ sociodemographic characteristics, collected from 02 december 2019 to 16 october 2020 in jigawa state, nigeria, and from 18 may 2021 to 29 april 2022 in jigawa and kogi states, nigeria. knowledge and competence of respondents most respondents (379/570, 66.5%) were familiar with glucometers as a point-of-care device (table 3). despite availability of training for hcps in some hospitals (248/570, 43.5%), most hcps have no theoretical (332/570, 58.2%) and practical skills (368/570, 64.6%) on glucometer calibrators. table 3: general responses of healthcare professionals, collected from 02 december 2019 to 16 october 2020 in jigawa state, nigeria, and from 18 may 2021 to 29 april 2022 in jigawa and kogi states, nigeria. many (368/570, 64.6%) had never calibrated a glucometer used for patient care. standard operating procedures for blood glucose testing using glucometers were unavailable in most clinical settings (322/570, 56.5%). discussion our study found a paucity of theory and skills for glucometer calibrators and calibration in about half of the participants, which in turn impacts the frequency of calibration of the device by hcps, reliability and reproducibility of test results obtained, and the quality of life of the patients. unavailability of sops for blood glucose testing using glucometers was reported by many respondents, despite the accessibility of training in some healthcare facilities. about two-thirds of participants knew glucometers existed as a point-of-care device utilised for patient care. this is because over the years, glucometers have gained wide acceptability as an smbg tool and point-of-care device. a study23 at tygerberg hospital located in the northern suburbs of cape town, western cape, south africa, in 2016, which assessed knowledge and awareness of pocts, reported that 97% of participants had performed blood glucose testing using glucometers. however, by restricting participation to only doctors and nurses, the study excluded other hcps who were likely to operate the device. another study22 in nigeria, at the university of benin teaching hospital and central hospital, edo state, southern nigeria, in 2015, reported good knowledge of poct (50.6%) among doctors, but the finding is not comparable to our study because of non-specification of poct devices and restricted enrolment of study participants to only doctors. we observed that work experience, or years of service, for most respondents ranged between 1 and 10 years (41.2%) and 11 and 20 years (38.4%), showing that years of hcps’ service or work experience in patient care was not directly linked with knowledge about existence of the device and its use. this implies that hcps may not have to practise for many years before becoming acquainted with glucometers. similarly, an assessment of nurses’ competencies in diabetes care,20 in anambra state, southeast nigeria, in 2023, showed a positive association between duration of work experience and skills. however, the skills were diverse and not limited to blood glucose testing using glucometers. as a result, deductions from the study aren’t comparable to our study. nearly half of the respondents (49.2%) reported not securing formal training or certification prior to first use of glucometers in patient care. this is because most health facilities in the private or public sector in nigeria rely solely on device manual inserts provided by manufacturers to guide hcps as a substitute for formal training or certification. similarly, a multi-centre study by nnakenyi et al.,30 conducted in enugu southeast nigeria in 2017 across five health facilities, found documentation for personnel training in only 26% of poct sites, for which glucometers accounted for about 65% of the point-of-care devices in use. inadequate formal training22,23 for hcps remains a major setback for achieving accuracy and reliability in blood glucose testing using glucometers. lack of or inadequate training translates to limited theoretical knowledge20 and skills that may negatively impact quality assurance, quality control, and quality management systems, as a whole. limited knowledge of operators, particularly hcps, can lead to misdiagnosis, inaccurate test results, flawed research reports and wrong therapeutic interventions. when knowledge is poor, practice is hampered, hence the need to educate hcps on the importance of seeking knowledge and skills required for operating point-of-care devices such as glucometers. some studies have suggested in-service training on pocts for doctors, nurses and midwives20,21,22,23 in nigeria and south africa. the need to train other cadres of hcps is less advocated in studies, despite the presence of such professionals in a standard poct organogram as stipulated in international guidelines. three-quarters of participants (75.1%) reported in the affirmative regarding availability of a training forum for hcps on glucometers in their health facilities. despite the availability of the training forums, hcps do not deem it necessary to secure formal training or certification preceding first use in patient care. hospital in-service training provides an avenue for hcp continuous professional education and development. other sources, such as conferences, journal articles, and pharmaceutical companies, have also served as sources of information for hcps on poct.31 although more than half of respondents (66.5%) were aware of the existence of glucometer calibrators, many (41.2%) didn’t know what purpose calibrators specifically served in smbg. this may be attributed to the fact that most new glucometers do not come with calibrators in their packs, and those with calibrators lacked an operation manual.13 thus, the necessity to harmonise theorical knowledge and skills for most hcps. more than half of respondents (58.2%) did not possess theoretical knowledge about glucometer calibration, and a similar number of respondents (64.6%) had no practical experience of glucometer calibration, which is supported by reports of some studies11,23 conducted in south africa, 2020, and australia, 2025. theoretical knowledge is crucial for operators of glucometers in order to identify factors which may either affect the testing device or strips, leading to underestimation or overestimation of blood glucose when using the device. the essence of calibration is for early detection of device non-conformances and ensuring results obtained are accurate and reproducible. studies in douala, southwest cameroon,8 in 2014–2015 and calabar, southsouth nigeria,9,13 in 2021, have evaluated the functional capacity of glucometers using calibrators provided by some brands. the frequency of glucometer calibration varied among respondents and about half (48.2%) reported to have never calibrated a glucometer, despite years of service in patient care. responses showed that the ideal way of calibrating glucometers which is before first use, weekly, and after purchasing a new vial of test strips is not practised by most hcps in nigeria. likewise, inaku et al.,32 in a survey of poct use among doctors and nurses in calabar, southsouth nigeria, 2019, reported that 24.3% of participants performed quality control practices on pocts in use of which glucometers accounted for 91.4% of the pocts. the study reported poor quality control among doctors and nurses operating the devices. regulation of poct use was also suggested by the majority of respondents in the study. limited knowledge of glucometer calibration among hcps,9 as reported in a study in calabar, southsouth nigeria, 2019, translates to erroneous results and unsatisfactory patient outcomes,13 as supported by a study in enugu southeast nigeria, 2022. periodic calibration of glucometers is recommended in order to prevent misdiagnosis, improve test result quality, minimise mismanagement of patients and achieve beneficial outcomes that will impact quality of life of end-users. certain brands of glucometers have exhibited superiority in terms of precision and accuracy, even without necessarily meeting certain international standards for pocts.8,10,11 this has led to product bias among hcps and patients, with increased competition for manufacturing companies. healthcare practitioners are expected to know when results tend towards incorrectness, especially when managing high-risk patients such as those on admission in intensive care units, to prevent misdiagnosis.23 half of the participants (50.0%) reported observing inconsistency of test results obtained from the same glucometer operated at different times. the finding may be attributable to the myriad of technical challenges and sources of errors associated with blood glucose testing using glucometers. about half of respondents (43.6%) rated the accuracy of glucometers as 51.0% to 99.0%, and 17.6% reported its accuracy as 100.0% when compared to central laboratory testing for blood glucose. in recent times, increased accuracy and precision for some glucometers have been reported,2,10 although there are contrary findings.8,9 the comparison of our finding with other reports may not be accurate because of differences in study designs: descriptive cross-sectional versus comparative analytical prospective. glucometer operators should be knowledgeable about operational limitations of certain brands of the device used for blood glucose testing. suggestions to keep point-of-care devices, such as glucometers, under the laboratory department has been faced with a lot of opposition, because such devices are dedicated for near patient testing. healthcare practitioners should be equipped with knowledge of limitations of the device and when to refer testing for critical patients to the central laboratory. standard operating procedures are sets of step-wise instructions containing elements of quality assurance and quality control which conform with regulatory guidelines for end users. robust training, supervision and intensive quality assurance mechanisms are necessary to optimise blood glucose testing using glucometers.34 unavailability of sops for glucometers in most health facilities accounted for 56.5% of responses. provision of teaching aids and sops in the form of flow charts or simple checklists for glucometers is essential in achieving quality blood glucose testing. absence of sops for blood glucose testing using glucometers in health facilities may impact negatively on operators’ proficiency and quality of results generated using the device.23 most studies in sub-saharan africa22,23,30 have broadly assessed pocts as an entity and considered glucometers only as a subset of poct, with study participation limited to specific healthcare cadres: doctors, nurses, midwives, or laboratory professionals. for instance, an multi-centre study30 in enugu southeast nigeria, 2017, glucometers accounted for 65% of the pocts, and participation was restricted to doctors, nurses, and laboratory personnel. the notable findings were lack of traceable patient test records, troubleshooting faulty devices by non-laboratorians, and poor method validation observed at the study sites. absence of a poct committee in all study hospitals was observed in all study sites. another study22 in edo state, southern nigeria, in 2015, conducted at two tertiary health facilities assessed knowledge and utilisation of poct among doctors. the study did not specify the types of pocts assessed, what was assessed, the reason for the assessment, and participation was restricted to only doctors. a hospital audit23 at cape town, western cape, south africa, in 2016, which evaluated the knowledge of poct best practices among clinical staff, precisely doctors and nurses, suggested the need for formal training and competency assessment for operators. the reasons for limiting participation to doctors and nurses only was not clearly defined. a survey of poct devices among doctors and nurses32 at calabar, southsouth nigeria, in 2019, also showed that most operators used poct devices for disease monitoring. the pocts evaluated were glucometers, urine testing strips, haemoglobin meters, bilirubin meters, and cholesterol meters. the non-restriction of the pocts to glucometers, differences in study designs, and limitation of participation to specific cadres of hcps in other studies, may not allow for appropriate comparison with the present study. in nigeria, operating glucometers in hospital settings is not restricted to a specific cadre of hcps.22 all hcps involved in patient care, irrespective of training, years of experience and skills, automatically become operators of the device32 because of the lack of organograms for pocts in nigerian hospitals. the unavailability of hospital-based poct committees may also be responsible for some irregularities observed in some health institutions. our study was carefully designed to identify gaps and proffer solutions to the spate of blood glucose testing using glucometers in nigeria. currently, there is no regulatory agency or national guideline in nigeria dedicated to pocts or glucometers, and there are no poct committees in hospitals. organising centralised local, state or national poct training or certification in nigeria may encourage stakeholders in government, health, science, and technology, as well as the private sector, towards engagement of hcps in developing policies, creating hospital committees on pocts, and implementation of policies at the grassroots. recommendations we recommend compulsory formal training, on-the-job competency assessment, and certification on glucometers and other pocts for hcps. updating and assessing hcps’ knowledge, competency and skills is a professional gap that needs to be addressed by the relevant stakeholders. formation of hospital-based, state and national poct committees, provision of standard protocols, and quality control materials and engagement of central laboratories in establishing standard poct guidelines and implementation of its content should also be considered. limitations our study limitations include the restriction of participation to hcps employed only by the study hospitals which are directly under the state ministries of health, thereby excluding hcps employed in the private sector although actively involved in patient care. although the response rate was quite high, we observed in some hospitals that the available hcps were busy with official duties when the questionnaires were distributed and didn’t have the time to fill the questionnaires at follow-up and until completion of the study. the questionnaire was not designed to get feedback from hcps on how poct committees will be formed at hospitals, the likely members of the proposed committees, and the number of pocts to be included in local and national guidelines. the information would have provided insights into the opinions of hcps on pocts, and provided suggestions on how the state and federal ministries of health can establish a formidable poct programme. our findings are solely applicable to the study hospitals and may not be generalised for all health facilities in nigeria, especially in resource-constrained settings where testing of blood glucose is completely absent or only performed in central laboratories, as seen in some hospitals. conclusion the study found that although most participants possessed theoretical knowledge, the skills required for calibration of glucometers were absent. standard operating procedures, calibrators, formal training, and skills acquisition forums for glucometers should be made available in health facilities in order to improve test quality and minimise avoidable errors in blood glucose testing using glucometers in nigeria. our study is the first in nigeria conducted among a diverse cadre of hcps, numerous inter-state health facilities, and different tiers of health centres: cottage, primary, secondary, and tertiary hospitals. acknowledgements the authors sincerely appreciate the support of the jigawa and kogi state ministries of health. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions all authors, s.b.m., h.b., a.m.b., j.n.o., f.s., a.o.a., z.i.n., r.j.b., e-y.s.a. and s.a.a., were responsible for development of study protocol until submission to the respective state ethics committees and writing the first draft of the manuscript. s.b.m., h.b., a.m.b., j.n.o. and f.s. distributed and retrieved the questionnaires, and collected the data for entry onto the microsoft excel spreadsheet for data analysis. a.o.a., z.i.n., e.-y.s.a., r.j.b. and s.a.a. critically revised the manuscript in preparation for submission. all authors, s.b.m., h.b., a.m.b., j.n.o., f.s., a.o.a., z.i.n., r.j.b., e.-y.s.a. and s.a.a., approved the final version of the article prior to 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ene ab, isong ik, ita oi, inyama ma. survey of the use of point of care testing devices in a tertiary hospital in nigeria. saudi j pathol microbiol. 2019;4(10):712–718. https://doi.org/10.36348/sjpm.2019.v04i10.003 de block c, cheng ayy, christensen tb, patted urh, ginovker a. healthcare professionals’ knowledge of and attitudes towards the use of time in range in diabetes management: online survey across seven countries. diabetes ther. 2023;14:1399–1413. https://doi.org/10.1007/s13300-023-01429-x higioka as, rudolf-oliveira rcm, martinello f, bazzo ml. analytical performance of glucometers in a tertiary care hospital. accred qual assur. 2016;21:143–149. https://doi.org/10.1007/s00769-015-1188-2 about the author(s) noutin f. michodigni department of molecular biology and biotechnology, pan african university institute for basic sciences technology and innovation (pausti), nairobi, kenya department of reproductive health and biology, institute of primate research (ipr), nairobi, kenya atunga nyachieo department of reproductive health and biology, institute of primate research (ipr), nairobi, kenya juliah k. akhwale department of zoology, school of biological sciences, jomo kenyatta university of agriculture and technology (jkuat), nairobi, kenya gabriel magoma department of molecular biology and biotechnology, pan african university institute for basic sciences technology and innovation (pausti), nairobi, kenya department of biochemistry, college of health sciences, jomo kenyatta university of agriculture and technology (jkuat), nairobi, kenya abdoul-salam ouédraogo department of medical microbiology laboratories, souro-sanou teaching hospital, bobo-dioulasso, burkina faso andrew n. kimang’a department of medical microbiology, college of health sciences, jomo kenyatta university of agriculture and technology (jkuat), nairobi, kenya citation michodigni nf, nyachieo a, akhwale jk, magoma g, ouédraogo a-s, kimang’a an. corrigendum: formulation of phage cocktails and evaluation of their interaction with antibiotics in inhibiting carbapenemase-producing klebsiella pneumoniae in vitro in kenya. afr j lab med. 2023;12(1), a2028. https://doi.org/10.4102/ajlm.v12i1.2028 note: doi of original article published: https://doi.org/10.4102/ajlm.v11i1.1803 correction corrigendum: formulation of phage cocktails and evaluation of their interaction with antibiotics in inhibiting carbapenemase-producing klebsiella pneumoniae in vitro in kenya noutin f. michodigni, atunga nyachieo, juliah k. akhwale, gabriel magoma, abdoul-salam ouédraogo, andrew n. kimang’a published: 09 may 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. in the published article, michodigni nf, nyachieo a, akhwale jk, magoma g, ouédraogo a-s, kimang’a an. formulation of phage cocktails and evaluation of their interaction with antibiotics in inhibiting carbapenemase-producing klebsiella pneumoniae in vitro in kenya. afr j lab med. 2022;11(1), a1803. https://doi.org/10.4102/ajlm.v11i1.1803, on page 1 the following paragraph is updated as it was incorrectly formulated: the original incorrect wording: the precipitated bacteriophages were members of myoviridae, siphoviridae and podoviridae. the revised and updated wording: the precipitated bacteriophages were members of myoviridae and podoviridae. in addition, on page 7 the following paragraph is updated as it was incorrectly formulated: the original incorrect wording: this current study revealed the presence of lytic tailed klebsiella phages belonging to the family myoviridae, siphoviridae and podoviridae in nairobi sewage systems with relatively short latent periods and optimal burst sizes, indicating their therapeutic potential in composing phage cocktails and synergistic interaction in combination with non-sensitive antibiotic (imipenem) against carbapenem-resistant k. pneumoniae clinical isolate in vitro. the revised and updated wording: this current study revealed the presence of lytic tailed klebsiella phages belonging to the family myoviridae and podoviridae in nairobi sewage systems with relatively short latent periods and optimal burst sizes, indicating their therapeutic potential in composing phage cocktails and synergistic interaction in combination with non-sensitive antibiotic (imipenem) against carbapenem-resistant k. pneumoniae clinical isolate in vitro. the authors apologise for these errors. the corrections do not change the study’s findings of significance or overall interpretation of the study’s results or the scientific conclusions of the article in any way. about the author(s) trevor peter clinton health access initiative, gaborone, botswana mah-sere keita african society for laboratory medicine, addis ababa, ethiopia john nkengasong centers for disease control and prevention, atlanta, georgia, united states citation peter t, keita m-s, nkengasong j. building laboratory capacity to combat disease outbreaks in africa. afr j lab med. 2016;5(3), a579. http://dx.doi.org/10.4102/ajlm.v5i3.579 editorial building laboratory capacity to combat disease outbreaks in africa trevor peter, mah-sere keita, john nkengasong copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the major ebola outbreak in west africa in 2014 has brought the world’s attention to the critical demands of health systems strengthening in africa. global health responses to outbreaks have never before been tested to the extent driven by ebola; however, many lessons were learned and countries are actively working on bolstering capacity to detect and respond effectively to ebola and other outbreaks of global health security importance. laboratory testing played a key role in the response, and a number of key improvements in laboratory capacity have been made since 2014, with support from african and international partners, including the african society for laboratory medicine, the world health organization’s regional office for africa (who afro) and the united states centers for disease control and prevention, among others. moving forward, the continent needs to set a new trajectory in healthcare development that will prevent future ebola outbreaks from reaching the 2014 scale, as well as tackle the other disease challenges that africa disproportionately carries. cognisant of this and the critical role of laboratory testing in multiple aspects of disease control – surveillance and rapid detection, clinical management and patient care, and the programme management of outbreak responses – this issue of the african journal for laboratory medicine publishes here a set of seminal articles that provide a blueprint for countries and partners on strengthening laboratories in africa to address the urgent needs of the global health security agenda and the international health regulations. kennedy, wurie and colleagues provide excellent insights into the prior condition of laboratory services in two countries at the focus of the ebola epidemic, liberia and sierra leone. they highlight conditions that hampered more effective responses, including gaps in human resource capacity, laboratory systems and infrastructure, and argue for national policies, planning, operations and investment that will establish appropriate laboratory institutions and networks supportive of the health systems necessary for laboratory testing in outbreak situations to be impactful. their observations remind us of familiar challenges; however, in the post-ebola context their recommendations highlight how common laboratory capacity problems now have new urgency and require innovative solutions. philip onyebujoh from the world health organization’s regional office for africa presents a vision for integrating national laboratory networks, surveillance systems and health research institutions to strengthen public health systems. while these are important components of the armoury of national disease programmes, in many african countries these areas operate independently and fail to achieve much-needed synergy. this is a major limitation to countries achieving the global health security agenda and international health regulations requirements and steps need to be taken to address them. strong leadership, as well as political and financial support, will be required to move swiftly. close coordination among the key stakeholders will be essential. the articles by best, ondoa and cowan provide critical new frameworks to address many of the above needs and the tools needed to guide the laboratory strengthening process. best provides a framework for developing and strengthening functional, tiered laboratory networks, covering each of the systems necessary for optimal network operation and management. this paper also provides a much-needed update on the 2008 maputo declaration on strengthening laboratory services by defining a new list of essential laboratory tests required at different levels of the health system to address global health security agenda priorities. the maputo declaration originally established standards for test service delivery that have been adopted across many countries. the update presented here is timely and should become the new standard. ondoa presents a quantitative scorecard tool to assess this capacity and, importantly, assess the functionality and readiness of laboratory networks for outbreak situations. this tool is based on the functional, tiered laboratory framework presented by best and assesses core functions of laboratory networks essential for rapid and effective outbreak responses. the scorecard also provides an innovative outbreak laboratory-readiness scoring system that will be useful for governments, implementing partners, policy makers and donors. similarly, cowan provides a practical framework for regulating the use of diagnostics in outbreak situations. new tests for diseases like ebola and zika are often rapidly developed in times of outbreak. given the urgency of outbreak situations and the need for governments to make rapid decisions on the use of such diagnostic technologies, this guidance is invaluable. the articles by perovic, schultsz and okeke tackle the looming crisis of antimicrobial resistance. they highlight the critical role of laboratories in antimicrobial resistance detection and containment and note serious deficiencies in current capacity in africa. in light of this, they provide a practical stepwise approach for implementation of laboratory-based antimicrobial resistance surveillance in african countries. antimicrobial resistance in africa is a major but neglected public health priority; the guidance provided in these unique articles is a call to action and provides a foundation for building an effective response. the articles published in this issue of ajlm are timely and highly relevant as africa takes important steps toward strengthening its defences against the next outbreak. the tools, guidance and vision they provide should become the blueprint for laboratory capacity necessary to combat disease outbreaks in africa and for strengthening health security programmes internationally. hiv situation in uganda uganda’s laboratory infrastructure and hiv-related testing services quality assurance in uganda laboratory and point-of-care quality assurance policy and framework in uganda acknowledgements references about the author(s) steven aisu central public health laboratory, ministry of health, kampala, uganda wilson nyegenye central public health laboratory, ministry of health, kampala, uganda victor bigira clinton health access initiative, kampala, uganda charles kiyaga central public health laboratory, ministry of health, kampala, uganda michael dfendu central public health laboratory, ministry of health, kampala, uganda sam acellam clinton health access initiative, kampala, uganda richard walwema infectious disease institute, kampala, uganda lydia nakiyingi infectious disease institute, kampala, uganda isaac sewanyana central public health laboratory, ministry of health, kampala, uganda aida namakula central public health laboratory, ministry of health, kampala, uganda richard batamwita central public health laboratory, ministry of health, kampala, uganda william lali world health organization, kampala, uganda citation aisu s, nyegenye w, bigira v, et al. quality assurance as an integral component of diagnostic testing in clinical laboratories and point-of-care testing: the uganda experience. afr j lab med. 2016;5(2), a447. http://dx.doi.org/10.4102/ajlm.v5i2.447 country profile quality assurance as an integral component of diagnostic testing in clinical laboratories and point-of-care testing: the uganda experience steven aisu, wilson nyegenye, victor bigira, charles kiyaga, michael dfendu, sam acellam, richard walwema, lydia nakiyingi, isaac sewanyana, aida namakula, richard batamwita, william lali received: 24 mar. 2016; accepted: 11 aug. 2016; published: 17 oct. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. hiv situation in uganda uganda, with an estimated population of 35 million people, is a landlocked country that forms part of the east african union member states. the country is divided into nine political regions (figure 1), in which there are 112 administrative districts. in order to make the administration and coordination of health services more efficient, the country is further divided into 14 health regions. figure 1: key hiv statistics in uganda, 2014, showing hiv prevalence by region. uganda is classified as a high burden hiv country with an estimated 1.6 million people living with hiv/aids and 140 000 new cases reported annually (table 1; figure 1).1 being one of only two countries where incidence was on the rise in 2013, hiv remains a major public health problem in uganda.2 hiv prevalence varies across the country, being highest in the central region and near urban centres and lowest in the mid-eastern and west nile regions. table 1: key hiv statistics in uganda, 2014. in 2013, the world health organization changed the recommendation for initiation of antiretroviral therapy from a cd4 count of 350 cells/mm3 to 500 cells/mm3.3 uganda was one of the first countries to adopt these recommendations and began implementing the revised treatment guidelines in april 2014.4 under the revised antiretroviral therapy guidelines, cd4 enumeration remains critical to staging the need for antiretroviral therapy among hiv-positive patients aged 15 years and above.5 in addition, the world health organization recommended the use of viral load testing as the preferred antiretroviral therapy monitoring approach. the ministry of health in uganda subsequently adopted viral load monitoring for antiretroviral therapy patients into the national guidelines. uganda’s viral load programme has since scaled up rapidly, but universal access has not yet been achieved. for patients who do not have access to viral load services, cd4 testing remains essential for monitoring antiretroviral therapy response. as uganda scales up the new world health organization/unaids 90-90-90 strategy,6 there has been a steep rise in the number of people in need of hiv testing and viral load monitoring across the country. it is estimated that over 850 000 people will be on antiretroviral therapy by the end of 2016. meeting these targets will require innovative ways of developing laboratory systems, including scaling up of point-of-care (poc) tests for hiv testing, diagnosis of opportunistic infections and monitoring response to treatment or disease progression. uganda’s laboratory infrastructure and hiv-related testing services the laboratory network in uganda consists of a five-tiered system covering the following levels: national, regional, general hospital (district), health centre iv and health centre iii. laboratories at national, regional and district levels are relatively well developed, whereas laboratories at lower health centre iv and iii level generally have inadequate space, no running water, frequent power interruptions and inadequate working space. in order to overcome some of these challenges, the country has established a national sample and results referral network based on hubs. a hub is a high volume laboratory that functions as a referral laboratory for 30–40 lower level laboratories. complex tests that cannot be handled at the hub, such as early infant diagnosis (eid), viral load testing, culture for tuberculosis and outbreak investigation samples, among others, are centralised at the central public health laboratory (cphl) and other reference laboratories.7,8,9 although testing capacity has been increased through deployment of modern high-throughput equipment, national testing targets for eid and viral load have not yet been met. it can be argued that lack of, or limited access to, laboratory services may be overcome through centralisation of testing services and building of new laboratories. however, this course of action requires substantial investment and could result in a significant delay. moreover, there is a nagging fear that if donor funding is significantly reduced or entirely stopped, such projects may be rendered ‘white elephants’. cognisant of such possibilities, over the last few years, uganda has adopted a combination of conventional testing platforms at higher level health facilities and poc testing at lower level health facilities. thus, in uganda, eid and viral load testing use roche molecular diagnostics and abbott molecular laboratory methods, while cd4 and hiv testing use a mix of conventional analysers and poc devices, including bd facscalibur®, bd facscount®, partec cyflow® counter and alere pima™ for cd4 counts, and elisa and rapid diagnostics tests for hiv. in addition, bd mgit is used for tuberculosis cultures and cepheid genexpert® for pcr. poc tests are deployed in remote rural areas or where test volumes are low, while conventional analysers are used mainly at hubs and in urban areas. quality assurance in uganda current quality assurance situation the quality assurance programme in uganda is still in its infancy. like many african countries, uganda has adopted the strengthening laboratory management towards accreditation and stepwise laboratory quality improvement process towards accreditation approaches to improving quality systems in laboratories. however, the supporting components to this programme, such as internal quality control procedures and external quality assessment (eqa), are quite weak. although cphl has put in place some measures in collaboration with the uganda virus research institute, the national tuberculosis and leprosy control program, the infectious diseases institute and the national drug authority to carry out some eqa functions, major gaps still exist. there is poor eqa coverage, particularly for poc testing; low response rates from participating facilities and lack of a comprehensive corrective action plans to support poorly performing laboratories. only panels for hiv testing and tuberculosis smear microscopy are produced locally, whereas panels for clinical chemistry, haematology, bacteriology, cd4 counts and viral load are supplied by external agencies. the eqa schemes and suppliers are not centrally coordinated. because uganda anticipates a rapid scale up of poc testing, cphl has developed plans to review the poc quality assurance policies and framework. laboratory and point-of-care quality assurance policy and framework in uganda current status of point-of-care quality assurance policy and framework uganda has a national health laboratory policy and strategic plan that guides the implementation of laboratory quality management systems across the health laboratory services. however, the strategies do not explicitly address poc testing deployment or poc testing quality issues. therefore, there is a need to review the national laboratory policy and strategic plan so as to address quality systems for poc testing. table 2 shows the results of a rapid assessment of the poc testing quality assurance policy and framework. while there is top-level leadership engagement (estimated score 80%), major gaps still exist for coordination at the national level (60%), definition of roles and responsibilities (60%), policies and strategic plans (40%), and availability of national laboratory standards (30%). table 2: status of point-of-care quality assurance policy and framework in uganda external quality assurance reforms for point-of-care testing a functional eqa scheme consists of six key components (figure 2), specifically: plan, define, implement, monitor, improve and evaluate. these components are arranged in a cyclical manner to demonstrate the concept of continual improvement. figure 2: components of a functional eqa scheme. in uganda, implementation of the eqa scheme will be done in three phases, as outlined below. phase 1: plan an hiv-related point-of-care testing quality system planning and developing an hiv-related poc quality system requires the review of the current guidelines and standards, and therefore requires the engagement of relevant leaders to execute the activities. with leadership engagement, establishment of the national quality assurance coordination team is easier and the roles and responsibilities of the selected teams can be defined (figure 3). before defining the expected quality of poc tests, there is a related need to conduct a situational analysis that may include mapping disease prevalence, as well as selecting and assessing sites. with the abovementioned background activity, the implementation plan will be developed with the further incorporation of quality assurance and policies. finally, a financial plan will be drafted with an emphasis on human resources and cost–benefit analysis. figure 3: proposed national external quality assessment coordination structure. phase 2: implement quality assurance for point-of-care testing the implementation strategy for quality assurance of poc testing requires quality assurance training and certification. this will further involve activities such as the process control, audits, site support supervision and mentorship, all of which constitute the fundamental components of capacity building. establishment of eqa (active, intermediate, passive) production, packaging/shipping through hubs, programme management and outsourcing will be implemented when the need arises (figure 4). the quality assurance supply chain and logistics programme can continuously be strengthened for improvement. figure 4: proposed courier system for the flow of national external quality assessment materials, forms, results and feedback. phase 3: sustain the quality assurance programme for the sustainability of the quality assurance programme planning, allocation of resources and continuous encouragement of socio-economic entrepreneurship are required. sustainability implementation will require focused monitoring and evaluation initiatives, country ownership and involvement, a high level of advocacy, and mobilisation of the community. feedback, corrective action and inter-lab comparisons are vital for quality assurance programmes, as these are the basis for improvement. cost of activities and resources for quality assurance improvement estimating the cost of eqa activities will include understanding the country’s needs based on identified interventions to inform key policy decisions and reducing high costs. external support will be sought to price the final model of service provision and to establish a cost-effective poc testing eqa scheme that is integrated in a comprehensive national eqa program. in cases where it will be necessary to produce eqa panels locally, the activity will be undertaken by the uganda national health laboratory and respective reference laboratories. eqa materials will be distributed using the hub system. technical assistance and other resources will be mobilised both internally and externally to support the programme. conclusion and way forward uganda is actively working to implement a robust quality assurance programme that will address poc testing in the country. although the quality assurance programme is still in its infancy, uganda has already adopted strengthening laboratory management towards accreditation and stepwise laboratory quality improvement process towards accreditation to improve overall laboratory quality systems and is focusing on how to improve internal quality control procedures and eqa. uganda has always taken great strides to meet challenges with innovative approaches. the country established a national sample and results referral network based on hubs to improve eid services, which also resulted in cost savings for the programme and in turn improved other health services. more recently, uganda performed a rapid assessment of the poc testing quality assurance policy and framework to identify major gaps that should be addressed. uganda understands the strength of working in a collaborative manner with all institutions to implement a sustainable country-owned eqa programme. cphl is working with the uganda virus research institute, the national tuberculosis and leprosy control program, the infectious diseases institute and the national drug authority to plan and carry out eqa functions necessary to support quality cd4, eid, and viral load services. acknowledgements the authors thank the ministry of health, the development partners and all the implementing partners within the health system of the country. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. sources of support none. references joint united nations programme on hiv/aids (unaids). the gap report. geneva, switzerland: unaids; 2014. ministry of health, macro i. uganda aids indicator survey (ais) 2011. kampala, uganda; ministry of health; 2011. world health organization. who issues new hiv recommendations calling for earlier treatment [page on the internet]. c2013 [cited 2016 sep 22]. available from: http://www.who.int/mediacentre/news/releases/2013/new_hiv_recommendations_20130630/en/ ministry of health, uganda. addendum to the national antiretroviral treatment guidelines [document on the internet]. c2013 [cited 2016 sep 27]. available from: http://preventcrypto.org/wp-content/uploads/2012/07/uganda-national-art-guidelines_2014.pdf ministry of health, uganda. uganda national policy guidelines for hiv counselling and testing [document on the internet]. c2012 [cited 2016 sep 22]. available from: http://www.who.int/hiv/pub/guidelines/uganda_art.pdf joint united nations programme on hiv/aids (unaids). 90-90-90: an ambitious treatment target to help end the aids epidemic. geneva, switzerland: unaids; 2014. pepfar, usaid. increasing viral load monitoring of people living with hiv on art in northern uganda in line with the 90-90-90 global targets [document on the internet]. c2016 [cited 2016 sep 27]. available from: https://www.usaidassist.org/sites/assist/files/improving_vl_testing_in_northern_uganda_june2016_a4_ada.pdf kiyaga c, sendagire h, joseph e, et al. uganda’s new national laboratory sample transport system: a successful model for improving access to diagnostic services for early infant hiv diagnosis and other programs. plos one. 2013;8(11):e78609. http://dx.doi.org/10.1371/journal.pone.0078609. ecollection 2013. stevens ws, gelman r, glencross dk, et al. evaluating new cd4 enumeration technologies for resource-constrained countries. evaluating diagnostics: the cd4 guide. nature rev microbiol. 2008;6(11 suppl):s29–s38. http://dx.doi.org/10.1038/nrmicro2000 introduction challenges and future directions mriglobal training history mriglobal training programme designing a locally sustainable programme discussion summary acknowledgements references about the author(s) lance d. presser global engagement program, mriglobal, gaithersburg, maryland, united states citation presser ld. establishing diagnostic training programs in resource-poor settings: the case of sierra leone. afr j lab med. 2020;9(1), a889 https://doi.org/10.4102/ajlm.v9i1.889 opinion paper establishing diagnostic training programs in resource-poor settings: the case of sierra leone lance d. presser received: 08 aug. 2018; accepted: 21 feb. 2020; published: 15 june 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction outbreak and initial response the west africa ebola virus disease (evd) epidemic in 2014–2016 resulted in at least 28 652 total cases (15 261 laboratory confirmed), of which at least 11 325 were fatal (case fatality rate ~40%).1 during the epidemic, most of the cases were concentrated in liberia, guinea, and sierra leone, with some cases exported to the united states, nigeria, mali, and other countries around the world.2 cases of evd began appearing in sierra leone in may 2014. mriglobal first deployed to sierra leone in january 2015 and has maintained a presence in the country ever since, resulting in numerous deployments for diagnostics, engineering and, now, training teams (figure 1). mriglobal provided assistance to the government of sierra leone and international partners to implement diagnostic testing, training courses, and other outbreak-related activities (table 1). mriglobal supported the national and district evd surveillance databases and provided data for evd surveillance, contact tracing, case investigation, et cetera. it trained staff and offered support to members of the national rapid response team at the sierra leone central public health reference laboratory (cphrl). it established, managed, and staffed evd testing laboratories in both sierra leone and guinea (figure 1). initially, the mobile laboratory was in moyamba, sierra leone, but in april 2015 it moved to lakka in freetown, sierra leone, on the same grounds as the cphrl. figure 1: mriglobal mobile diagnostic laboratory in sierra leone. (a) setup of the mobile diagnostic laboratory in moyamba, sierra leone, january 2015 (aerial view). (b) setup of the mobile diagnostic laboratory at the sierra leone central public health reference laboratory in lakka, freetown (moved in april 2015, photograph is from november 2016). (c) interior of extraction laboratory unit. (d) interior of molecular diagnostics laboratory unit. table 1: number of confirmed, probable, and suspected ebola virus disease cases, number of deaths and number of mriglobal staff deployments during the ebola virus disease epidemic – guinea, liberia, and sierra leone, march 2014 – september 2016. international partner training programmes in sierra leone numerous international partners developed programmes in sierra leone during and after the west africa evd outbreak. the united states centers for disease control and prevention (cdc), china cdc (ccdc), association of public health laboratories (aphl), world health organization, public health england (phe) and a number of other organisations developed and conducted a variety of training events. the following is a brief summary of the training activities hosted by international partners in sierra leone. the aphl works to build laboratory systems in the united states and globally. its international work focuses on building national laboratory systems and expanding access to quality diagnostic testing systems. during the outbreak, aphl, in partnership with mriglobal, trained 26 national rapid response team laboratory scientists and provided consultation regarding the national strategic plan of sierra leone’s ministry of health and sanitation (mohs). the aphl training ranged broadly and included basic bacteriology courses, influenza diagnostics, etc. each of these trainings had its own challenges. influenza diagnostics, for example, relied upon using abi 7500 quantitative reverse-transcriptase polymerase chain reaction machines that were not well maintained, and it was extremely challenging to get reagents shipped in a timely fashion that maintained a cold chain. the aphl closed their offices in sierra leone on 26 february 2019. the united states cdc began working in sierra leone during the 1970s, establishing a long-running research programme on lassa fever.3 as part of the 2014–2016 evd outbreak response, more than 700 cdc staff served on over 1000 deployments and, in 2015, a permanent cdc country office was established to focus on the global health security agenda.4 the cdc has established and supported training programmes ranging from field epidemiology training programme to an ecology and molecular diagnostics training programme with a university in sierra leone whose goal is to identify the animal reservoir of the ebola virus.5 the cdc office in sierra leone has not published much information on their projects in sierra leone; however, their office remains open and runs surveillance, capacity building and epidemiology programmes. programmes like the ‘creation of a national infection control programme in sierra leone’ and the continuing field epidemiology training programme are indicative of the type of successful, ongoing engagements between cdc and sierra leone.6 china has a presence in sierra leone and the ccdc was a major international partner during the outbreak. the ccdc built a biological safety level 3-capable laboratory space in combination with a hospital in jui, a suburban neighbourhood to the east of freetown and has been operating both since the early stages of the outbreak. multiple teams of chinese researchers and clinicians have rotated through the facilities and have maintained a consistent presence following the end of the evd outbreak. in a recent press release, the director of the ccdc noted that more than 60 chinese experts have been sent to sierra leone, and 30 sierra leoneans have studied and trained in china. ccdc supports ongoing national surveillance for ebola, dengue fever, yellow fever, zika and lassa fever.7 public health england set out to renovate multiple sierra leone government laboratories in sierra leone, including the connaught hospital laboratory, the largest in freetown. the phe training programme focused on training national laboratory staff to international safety and quality standards, while teaching principles of molecular testing for ebola virus and other high-consequence pathogens. the training was broken down into theory training and practical training. theory training consisted of three sections: general information, a molecular theory course lasting two and half weeks and a molecular virology short course. theory training occurred on multiple occasions, and the usual number of trainees at each session was approximately 15. practical training lasted six weeks and was performed at three different government laboratories across sierra leone. at each site,8,9 trainees were trained and had supervised work experience and competence assessments performed by the phe trainers. additional support and training were given on alternative assays and platforms as well as maintenance support. unfortunately, phe has not published any reports on their training programmes, but it is the author’s opinion that the phe trainers were of good quality and had developed a quality training programme. public health england is still supporting the renovated hospital laboratories. it is the author’s opinion that renovating hospital laboratories provides better return on investment than the construction or renovation of central or national public health laboratories in many circumstances, including in sierra leone. challenges and future directions overall, there was little standardisation of programmes, materials or contact time with trainees between partner training programmes. training materials and schedules occurred with very minimal input from the mohs. also, although two trainees may have similar certificates, the lack of standardisation of training programmes makes it difficult to compare skills between trainees. to this end, the author thinks it would be valuable for both the host country and partners to work together to standardise all training programmes and materials for training purposes as much as possible. the adage ‘practice makes perfect’ rings true in all molecular diagnostics training events and continued refreshers are extremely valuable if possible. when possible, the mohs should require partners to use standardised procedures and assays. during the evd outbreak, numerous organisations brought in their own proprietary assays, many of which were not commercially available. trainees were trained on numerous assay platforms, and while this was necessary during the outbreak, it has been problematic during the post-outbreak capacity building phase. staffing, purchasing, logistics and refresher training would all be easier to achieve with standard assays in place, used by all partners, as dictated by the mohs. ideally, the mohs should be in charge of: developing and providing training materials and standard operating procedures that are easily adaptable to all laboratories; providing individualised training assessments to guide personalised future training as well as laboratory operations refresher training on a regular basis. mohs should also verify that implemented procedures are routinely performed. a comprehensive external quality assessment programme for all government laboratories would be an incredible accomplishment. this will more than likely happen very slowly, and there is always a risk that it may not happen at all. therefore, it is recommended that partners organise with the mohs to standardise and make the post-outbreak capacity building phase more efficient. mriglobal training history as the evd outbreak resolved and evd cases decreased, the rapid diagnostic response also evolved. the mriglobal mobile diagnostic laboratory was one of the laboratories that was moved (from moyamba to lakka). toward the end of the outbreak, there were far fewer blood samples being tested, and as the need for urgent response diminished, the focus turned to permanent transitioning of laboratories to the mohs and training of the mohs national rapid response team. mriglobal is an independent, not-for-profit organisation that performs aspects of laboratory design, operations, biosafety and security, research and diagnostics for government, academia and industry in the united states and internationally. mriglobal conducted training at the mobile diagnostic laboratory at cphrl in lakka during the outbreak. the duration of the training was six weeks and training components included didactic and kinesthetic training, laboratory simulations and continual refresher training based on molecular diagnostics testing for evd. a total of eight graduates were trained using a wide variety of materials. trainees also received quality assurance, quality control and biosafety training, which were rarely included in other partner training. mriglobal training programme disease surveillance systems in west africa grapple with the problem of how to function, train and persist in resource-poor settings. it is vital for surveillance systems, especially surveillance systems in resource-poor settings, to increase capacity efficiently by building or repurposing infrastructure. however, often funding for infrastructure is limited and can be difficult to sustain; therefore, comprehensive training of professional staff is more likely to give a better return on investment. with support from the united states defense threat reduction agency, and mriglobal, the sierra leone mohs has developed a training programme to assist in disease surveillance in west africa. the mriglobal molecular diagnostics training curriculum includes: powerpoint lectures, hardcopy handouts and notes, textbooks, quizzes and exams, as well as all the physical training materials (pipettes, appropriate personal protective equipment, molecular laboratory equipment, biosafety cabinets, etc.) to fulfil an immersive molecular diagnostics (specifically evd) training experience. the training programme utilises team mentoring (usually a team of two or three trainers) and supervision of trainees by subject matter experts, in which sierra leone mohs staff are trained by mriglobal staff. participants were given exit surveys throughout the training in 2015 and 2016 which showed a high degree of satisfaction with most aspects of the programme, including the length of the programme and the content (unpublished results). a key strength of the training programme is a true partnership approach, which utilises the use of onsite laboratory equipment to offer assorted training to sierra leone mohs staff, and a team model for mentorship and supervision. the author believes the molecular diagnostics and disease surveillance training partnership established at the sierra leone cphrl can be used as a model for sustainable capacity building and training in low-income and middle-income countries. molecular diagnostics training included, but was not limited to, the following topics: equipment overview, use, and maintenance laboratory workflow process pipetting decontamination personal protective equipment biological waste disposal introduction to rna/dna introduction to virology introduction to immunology introduction to epidemiology laboratory-acquired infections quality management systems specimen management designing a locally sustainable programme the mriglobal mobile diagnostic laboratory that served as an evd diagnostic testing laboratory during the epidemic includes a sample extraction laboratory with multiple biosafety cabinets for sample inactivation and nucleic acid extraction, a reagent preparation space, and a quantitative real-time reverse-transcriptase polymerase chain reaction space. the purpose of the diagnostic training being held at the mobile diagnostic laboratory is to support the development of laboratory personnel and regional staff associated with the mobile diagnostic laboratory and to help integrate it into the existing cphrl workflow. molecular diagnosis and surveillance require partnerships between laboratorians, public health experts and government officials. in order to adequately train personnel, numerous partnerships were established. developing these partnerships served as the base for the programme at the sierra leone cphrl. mriglobal subject matter experts were very mindful to consider feasibility, sustainability and local relevance during the design of the training programme. this required aligning with national priorities and resources. the major topics of the diagnostics training programme developed are: safety protocols; laboratory orientation; reagent preparation; sample receipt and inactivation; nucleic acid extraction; quantitative real-time reverse-transcriptase polymerase chain reaction; data review, analysis and reporting; proficiency testing and targeting mentoring. ethical considerations this study followed all ethical guidelines for research involving no human participants. discussion effective, operational laboratories are the pillar of effective clinical and public health systems, and are critical to the detection and diagnosis of infectious disease. in a recent publication, another international partner stated: the absence of staff, stuff, space, and systems needed to detect outbreaks of infectious disease such as the recent ebola epidemic in west africa, and diagnose other medical conditions has underscored the need to not only set up diagnostic equipment in places where it is scarce, but also invest resources into training laboratory personnel. (p. 102)10 laboratories worldwide suffer from scarcities of skilled or qualified staff. payment for laboratory technicians and other categories of laboratory workers is lower than other specialties, and periodically delayed. numerous times from 2014 to 2017 in sierra leone, government laboratory staff went unpaid for months due to the inability of the government to pay its workers. college-level and formal training opportunities are very limited or non-existent. a large proportion of laboratory staff are chosen without having the proper certificates, degrees or technical expertise necessary to carry out their responsibilities, resulting in systemic failures. training students in diagnostic techniques is not an easy task. expecting trainees to learn molecular diagnostic skills in short courses of two to six weeks is unreasonable and not sustainable. even the best trainees require more than six weeks of training to become truly proficient, which is why refresher training or continued oversight is necessary for success. in order to truly make a sustainable difference regarding staff training and performance, organisations interested in training should be very conscious of whom they select for training, be prepared to provide as much refresher training as necessary and be able to provide some financial incentive or balance the training with the daily work tasks of laboratory staff. additionally, laboratory staff often lack access to adequate tools and supplies. resource-poor laboratories often use obsolete technologies, expired reagents and improperly or uncalibrated equipment. the lack of equipment maintenance further erodes laboratory capabilities. electricity instability in many low-income countries results in power surges or outages that damage equipment. proper personal protective equipment is often lacking or compromised, resulting in hazardous work conditions for the staff. funding organisations need to have equipment maintenance and replacement plans, as well as personal protective gear and consumable requirements, in place before a training programme begins. in many low-income countries, adequate space is difficult to find. many laboratories are located in small, cluttered spaces in hospitals. often, laboratories consist of a single room, and operations meant to be done in separate spaces are done near one another. many laboratories do not have a good water or electrical supply. fuel for generators is expensive and, while useful to keep vital equipment running, is not sustainable. laboratories lacking trained staff, stuff and appropriate space often find it very difficult to develop robust systems. quality, biosafety, accurate recording and reporting and a culture of maintenance are all critical laboratory functions; however, they are often not clearly understood or are under-prioritised. national guidelines and policies are often inadequate by international standards. communication between ministries of health and international partners is often lacking. with sierra leone, as discussed above, numerous international organisations were training laboratory staff using a variety of different techniques and materials. communication was very important to limit training overlap, trainee poaching and a variety of other potential misunderstandings. maintenance is difficult to instil, and without service technicians, eventually equipment reaches obsolescence. rust and dirt are constant enemies of laboratory equipment, especially in non-climate-controlled environments. performance skills of laboratory staff can also decline without consistent use or refresher training. without active training, mentorship and quality management systems in the laboratory, performance can diminish. both equipment and staff performance decline, due to lack of maintenance or skills usage, and are important considerations when establishing a training programme. summary following the west africa evd outbreak, a high priority was placed on the training of staff and building or repurposing of infrastructure. mriglobal worked closely with the sierra leone mohs to develop a sustainable, replicable training programme for diagnostics. with the proper prioritisation by the sierra leone mohs and international partners, sustainable gains can be made in the area of clinical diagnostics, which will help mitigate future outbreaks. as stated previously, the author believes the molecular diagnostics and disease surveillance training partnership established at the sierra leone cphrl can be used as a model for sustainable capacity building and training in low-income and middle-income countries. it is also the author’s opinion that long-term (10–20 year) sustainable engagement plans will be ultimately the most successful in sierra leone. acknowledgements the author expresses his deep gratitude to many partners, including the sierra leone health authorities, various donors, and local and international organisations whose contributions have helped support efforts to build quality clinical laboratory systems. the author wants to thank the support staff at mriglobal, as well as staff at the united states embassy in sierra leone. the author also thanks scott poynter for his photographs used in this manuscript. competing interests the author declares that he has no financial or personal relationships that may have inappropriately influenced him in writing this article. authors’ contributions i declare that i am the sole author of this research article. sources of support this work was funded under defense threat reduction agency cooperative biological engagement program contracts hdtra1-08-d-0008 and hdtra1-15-c-0007. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views expressed in the submitted article are the author’s own and not an official position of mriglobal or the funding agency responsible and no official endorsement should be 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in sierra leone, 2015. bmj glob health. 2019;4(3):e001504. https://doi.org/10.1136/bmjgh-2019-001504 public health england. phe’s legacy public health work in sierra leone [homepage on the internet]. 2017 [cited 2017 nov 02]. available from http://sierraexpressmedia.com/?p=84573 baez s, pannetier d, oestereich l, et al. emergence of zaire ebola virus in guinea. n engl j med. 2014;371(15):1418–1425. https://doi.org/10.1056/nejmoa1404505 saez am, weiss s, nowak k, et al. investigating the zoonotic origin of the west african ebola epidemic. embo mol med. 2015;7(1):17–23. https://doi.org/10.15252/emmm.201404792 orozco jd, greenberg la, desai ik, et al. building laboratory capacity to strengthen health systems. clin lab med. 2018; 38(1):101–117. https://doi.org/10.1016/j.cll.2017.10.008 introduction xpert® mtb/rif: the unfulfilled promise? an african solution acknowledgements references about the author(s) christiaan mulder tuberculosis department, anti-persoonsmijnen ontmijnende product ontwikkeling (apopo), sokoine university of agriculture, morogoro, tanzania amsterdam institute for global health and development, amsterdam, the netherlands georgies mgode tuberculosis department, anti-persoonsmijnen ontmijnende product ontwikkeling (apopo), sokoine university of agriculture, morogoro, tanzania stewart e. reid centre for infectious disease research in zambia, lusaka, zambia division of infectious diseases, university of alabama at birmingham, birmingham, alabama, united states citation mulder c, mgode g, reid se. tuberculosis diagnostic technology: an african solution … think rats. afr j lab med. 2017;6(2), a420. https://doi.org/10.4102/ajlm.v6i2.420 opinion paper tuberculosis diagnostic technology: an african solution … think rats christiaan mulder, georgies mgode, stewart e. reid received: 30 jan. 2016; accepted: 20 sept. 2016; published: 31 mar. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction tuberculosis has now gained ranking alongside hiv as one of the two leading causes of death from infectious diseases worldwide.1 in 2014, it was estimated that 1.5 million people died as a result of and 9.6 million fell ill with tuberculosis.1 despite these alarming figures, efforts to reduce the annual tuberculosis incidence rate over the last decade have resulted in only a meagre 1.5% decline.1 in order to reach the ambitious targets of the sustainable development goals by 2030 of reducing tuberculosis deaths by 90%, reducing the tuberculosis incidence rate by 90%, and ensuring that no tuberculosis-affected family is facing catastrophic costs due to tuberculosis, a paradigm shift is urgently needed.1 recently, a series of papers was published in the lancet on how to eliminate tuberculosis, suggesting repacking current interventions into a comprehensive control strategy.2 the world health organization end tb strategy supports this and also emphasises the need for better adoption and rapid uptake of new tools to diagnose tuberculosis earlier, the systematic screening of high-risk populations, and the effective and rapid roll-out of these strategies in highly-affected countries.3 however, the practicality of achieving these components remains challenging because of the lack of a rapid, simple, accurate and affordable point-of-care diagnostic and screening algorithm that can be scaled-up to screen large numbers of individuals. nevertheless, achievement of these goals is necessary and must catalyse the development of new interventions in africa, for africa, the continent with the highest tuberculosis mortality and morbidity rates.1 xpert® mtb/rif: the unfulfilled promise? the most recent and widely-implemented new laboratory technology for the diagnosis of tuberculosis is the xpert mtb/rif® (cepheid, sunnyvale, california, united states). xpert mtb/rif has been rolled-out in many resource-limited, high tuberculosis-incidence countries through the support of international donors. although the xpert mtb/rif assay has proven to increase tuberculosis case detection,4 two recent randomised trials in southern africa suggest that introducing xpert mtb/rif alone may not significantly reduce tuberculosis-related morbidity and mortality,5,6 underscoring the fact that tuberculosis diagnostics do not function in isolation but are part of a cascade, which includes clinician assessment, treatment initiation decisions, and follow-up and adherence efforts. in addition, an xpert mtb/rif instrument costs $17 000 and relies on testing cartridges that, even with concession pricing for developing countries, cost just under $10.00 per unit. the significant ongoing electricity needs, the variable costs and technological upkeep (maintenance and calibration) pose major barriers for the sustainability of this technology at the point of care in resource-limited settings.7 a full scale-up of xpert mtb/rif in tanzania, for example, would require a 25% increase in the national tuberculosis programme’s budget and financial resources, which is unaffordable in the current fiscal climate.8 although there is clearly a role for xpert mtb/rif in diagnosing tuberculosis and providing drug-susceptibility results in specific populations and in specific settings, without higher sample throughput and reduced costs, as well as robust linkages to care, the burden of tuberculosis will not drop. we agree with the stop tb partnership’s global plan to end tb 2016–2020 that a paradigm shift is needed, one that includes new and innovative programmes that can rapidly and efficiently roll out diagnostics, drugs and vaccines to impact the tuberculosis epidemic, especially in the developing world.9 however, to date most of the new laboratory tests under development in the diagnostic pipeline are molecular methods, which can be expensive and are unable to efficiently handle high sample throughput in decentralised settings. the ideal tuberculosis diagnostic test would be low cost, patientand user-friendly, accurate for all forms of tuberculosis in both hiv-negative and hiv-positive populations, produce immediate results at the point of care, have high throughput, and be well embedded and sustainable in developing countries within existing health care delivery structures.10 an example of this would be a urine-based, lateral-flow assay similar to the standard home pregnancy test. however, to our knowledge, there is nothing in the five-year tuberculosis diagnostic pipeline for review by the world health organization that meets all of these requirements.11 an african solution in the last decade, tuberculosis scent detection studies have been performed with animal, insect and electronic noses and may be a unique ‘outside-the-box’ solution to fill the tuberculosis diagnostic need in developing countries.12 recently, research using specially-trained african giant pouched rats as detectors of pulmonary tuberculosis, has advanced to the stage where this technology is being used daily for tuberculosis screening for people living in tanzania and mozambique.13 sputum samples are collected from presumptive tuberculosis patients who test ziehl neelsen smear-negative at public and private tuberculosis clinics in dar es salaam and maputo. a team of detection rats subsequently re-screens these samples to identify tuberculosis through scent and diagnose tuberculosis cases that were missed by smear microscopy. using a threshold of at least one detection rat indication to determine a positive result provides a sensitivity of 72%–80% and a specificity of 59%–74%, at an inexpensive cost per screening (table 1).14,15 all the samples indicated positive by the rats currently undergo confirmation by concentrated light-emitting diode fluorescence microscopy (led-fm). confirmation with xpert mtb/rif is under consideration, given the higher sensitivity of xpert mtb/rif compared with led-fm. table 1: test characteristics of detection rats and tuberculosis diagnostics common in sub-saharan africa. the detection rat technology is part of a larger community response where results are reported on the same day to a community-based organisation which is then required to track the tuberculosis-positive patients and ensure that they initiate antituberculosis treatment. the overall advantage of rats is they can screen up to 100 samples in 20 minutes and can reduce workload by rapidly funnelling-down the number of smear ‘negative’ samples that require confirmatory testing to about 30% of the original number.16 samples indicated positive by the rats but which are led-fm negative are considered bacteriologically tuberculosis-negative but, at the discretion of the clinician, can: (1) undergo repeat tuberculosis testing; (2) be treated empirically for tuberculosis; (3) undergo further investigations/treatment for other conditions mimicking tuberculosis; or (4) simply be followed and asked to return to the clinic if symptoms do not resolve or worsen. using the tuberculosis detection rats in combination with led-fm has significantly increased tuberculosis case detection in tanzania since 2007 and in mozambique since 2013. to date, there have been an additional 10 000 patients (a 40% increase) missed by the public health system that were diagnosed by the rats and confirmed by led-fm.17 by providing same-day results, and with the support of community-based healthcare workers, a large number of patients can access tuberculosis treatment quickly, maximising individual clinical benefits and minimising community spread. specific studies comparing the morbidity and mortality of rat detection compared to the standard of care have not yet been done, but need to be undertaken. of relevance to africa, the sensitivity of rats does not seem to be affected by the hiv status of the patient15 (unlike smears, which have been found to have sensitivities as low as 26% in new hiv clinic enrolees18). detection rat technology has been rigorously tested and researched for over 10 years. the trained rats target a blend of specific volatile organic compounds produced by mycobacterium tuberculosis.24 the rat detects tuberculosis by walking through a rectilinear cage with holes in the floor where small pots with different patient’s sputum specimens are placed, each covered with a sliding lid. as the rat approaches a pot, the technician slides the lid open for the rat to sniff the sputum. in the proof-of-principle study, weetjens et al. showed that rats could be trained by operant conditioning (positive reinforcement) to pause for at least five seconds at holes where a sputum sample was positive for tuberculosis (as confirmed by solid culture), but not to pause at holes where the sputum sample was tuberculosis-negative.25 these research results were sufficiently promising that in 2007 the tanzanian ministry of health allowed the rats to be used for second-line screening of sputum samples from presumptive tuberculosis patients who presented at tuberculosis clinics.25 the mozambique ministry of health followed suit in 2013. other studies have shown that teams of rats, which are fast and affordable, improve case detection well above that of directly-observed treatment, short-course clinic microscopy.13,14,26 in tanzania, there is a functioning breeding programme run by a belgian non-governmental organisation – apopo (‘anti-persoonsmijnen ontmijnende product ontwikkeling’; www.apopo.org) – in collaboration with the sokoine university of agriculture which supplies rats ‘as needed’ for the training, research and detection programmes currently underway. training tuberculosis detection rats takes an average of nine months, but they have a working lifespan of up to seven years. training one rat costs about $1000 and the cost of having one sample evaluated by a team of four rats is around $0.92, assuming a throughput of 1000 samples a week, which is the current throughput for 24 clinics in dar es salaam (manuscript in preparation). the cost per sample screened could potentially drop to around $0.70 through scale-up as more samples are screened. while there are some variable costs associated with screening more samples by rats, they are far less than the costs associated with screening extra samples with, for example, xpert mtb/rif. this is because each xpert mtb/rif examination requires a standard cost for each cartridge used.23 the unlimited access to the rats from the region, combined with an intensive rat trainer accreditation programme, also run by apopo, that includes mentoring new handlers, helps to ensure that apopo’s work is sustainable and the impact is scalable. rat performance and accuracy is monitored daily by an intensive quality assurance programme. known positive and known negative samples are placed randomly for the rats to examine and individual rat performance is assessed. because of the rapid diagnostic speed and low variable costs of detection rats, this technology is particularly well suited to screening the large numbers of patients found in african cities where tuberculosis is concentrated in heavily-populated, informal settlements characterised by substandard housing and low income. in these settings, public health laboratories are typically unable to maintain the quality of smear microscopy due to the high throughput of tuberculosis samples, resulting in many false-negative results. using an efficient motorcycle sputum transportation system, it is possible to have all samples shipped to one quality-controlled central laboratory to undergo screening by detection rats and confirmation by led-fm, with a turn-around time of one day. large, congested metropolitan centres not only act as a focus of local tuberculosis infection, but may also facilitate the spread of tuberculosis infection countrywide due to the extensive migration in and out of the cities. innovative and affordable approaches, such as tuberculosis detection rats that offer high sample throughput, could be a solution to successfully tackle tuberculosis burden in these areas and would be in line with the zero tb cities project which focuses on finding the ‘how’ for tackling infectious diseases with local tools in local contexts, while not lowering standards (http://www.advanceaccessanddelivery.org/). detection rat algorithms may also be used as a cost-effective, active case-finding tool in key high-risk populations, such as correctional facility inmates, miners and refugees. whether increases in case detection through the use of detection rats has an impact on reducing tuberculosis morbidity and mortality and whether this is cost-effective needs further study. conclusion in order to accelerate the elimination of tuberculosis in sub-saharan africa, a multi-pronged approach is required. until new, sustainable and inexpensive laboratory tuberculosis diagnostic tests are discovered, commercialised, validated and scaled up, other innovative diagnostic solutions, such as tuberculosis detection-trained african pouched rats, must be used in combination and in collaboration with tuberculosis laboratories in africa. this will require countries and international donors to adopt and roll-out complementary technologies for the various settings where they work best. rat scent detection technology, working in concert with laboratories, provides an opportunity to play a major complementary role in improving tuberculosis detection rates in low resource, high tuberculosis-burden urban settings for the foreseeable future. acknowledgements competing interests c.m. and g.m. are full-time employees of apopo. s.e.r. is on the apopo scientific advisory committee. sources of support none. authors’ contributions c.m. and s.e.r. drafted the manuscript and g.m. reviewed it thoroughly. all authors approved the final version of the manuscript. references world health organization. global tuberculosis report 2015. geneva, switzerland: who; 2015. das p, horton r. tuberculosis—getting to zero. lancet. 2015 dec 5;386(10010):2231–2232. https://doi.org/10.1016/s0140-6736(15)00401-8 pubmed pmid: 26515677. world health organization. end tb strategy. geneva, switzerland: who; 2014. steingart kr, sohn h, schiller i, et al. xpert® mtb/rif assay for pulmonary tuberculosis and rifampicin resistance in adults. cochrane database syst rev. 2014;1:cd009593. https://doi.org/10.1002/14651858.cd009593.pub2 pubmed pmid: 24448973. theron g, zijenah l, chanda d, et al. feasibility, accuracy, and clinical effect of point-of-care xpert mtb/rif testing for tuberculosis in primary-care settings in africa: a multicentre, randomised, controlled trial. lancet. 2014:383(9915):424–435. https://doi.org/10.1016/s0140-6736(13)62073-5 pubmed pmid: 24176144. churchyard gj, stevens ws, mametja ld, et al. xpert mtb/rif versus sputum microscopy as the initial diagnostic test for tuberculosis: a cluster-randomised trial embedded in south african roll-out of xpert mtb/rif. lancet glob health. 2015 aug;3(8):e450–457. https://doi.org/10.1016/s2214-109x(15)00100-x pubmed pmid: 26187490. pantoja a, fitzpatrick c, vassall a, et al. xpert mtb/rif for diagnosis of tuberculosis and drug-resistant tuberculosis: a cost and affordability analysis. eur respir j. 2013 sep;42(3):708–720. https://doi.org/10.1183/09031936.00147912 pubmed pmid: 23258774. langley i, lin hh, egwaga s, et al. assessment of the patient, health system, and population effects of xpert mtb/rif and alternative diagnostics for tuberculosis in tanzania: an integrated modelling approach. lancet glob health. 2014 oct;2(10):e581–591. https://doi.org/10.1016/s2214-109x(14)70291-8 pubmed pmid: 25304634. stop tb partnership. the global plan to end tb 2016–2020. geneva, switzerland; 2015. world health organization. high-priority target product profiles for new tuberculosis diagnostics: report of a consensus meeting. geneva, switzerland: who; 2014. unitaid. tuberculosis diagnostics technology and market landscape. 4th ed. geneva, switzerland; 2015. suckling dm, sagar rl. honeybees apis mellifera can detect the scent of mycobacterium tuberculosis. tuberculosis (edinb). 2011 jul;91(4):327–328. https://doi.org/10.1016/j.tube.2011.04.008 pubmed pmid: 21546308. beyene n, mahoney a, cox c, et al. apopo’s tuberculosis research agenda: achievements, challenges, and prospects. tanzan j health res. 2012;14(2):121–130. mahoney a, weetjens b, cox c, et al. pouched rats’ detection of tuberculosis in human sputum: comparison to culturing and polymerase chain reaction. tuberc res treat. 2012; article id 716989, 5 pages. https://doi.org/10.1155/2012/716989 reither k, jugheli l, glass tr, et al. evaluation of giant african pouched rats for detection of pulmonary tuberculosis in patients from a high-endemic setting. plos one. 2015;10(10):e0135877. https://doi.org/10.1371/journal.pone.0135877 pubmed pmid: 26445086. pubmed central pmcid: 4596709. edwards tl, valverde e, mulder c, et al. pouched rats as detectors of tuberculosis: comparison to concentrated smear microscopy. eur respir j. 2016 aug;48(2):579–582. https://doi.org/10.1183/13993003.00264-2016 pubmed pmid: 27288036. poling a, mahoney a, beyene n, et al. using giant african pouched rats to detect human tuberculosis: a review. pan afr med j. 2015;21:333. https://doi.org/10.11604/pamj.2015.21.333.2977 pubmed pmid: 26587178. pubmed central pmcid: 4634033. henostroza g, topp sm, hatwiinda s, et al. the high burden of tuberculosis (tb) and human immunodeficiency virus (hiv) in a large zambian prison: a public health alert. plos one. 2013;8(8):e67338. https://doi.org/10.1371/journal.pone.0067338 pubmed pmid: 23967048. pubmed central pmcid: 3743881. world health organization. systematic screening for tuberculosis: principles and recommendations. geneva, switzerland; 2013. chihota vn, grant ad, fielding k, et al. liquid vs. solid culture for tuberculosis: performance and cost in a resource-constrained setting. int j tuberc lung dis. 2010 aug;14(8):1024-31. pubmed pmid: 20626948. steingart kr, ng v, henry m, et al. sputum processing methods to improve the sensitivity of smear microscopy for tuberculosis: a systematic review. lancet infect dis. 2006 oct;6(10):664–674. https://doi.org/10.1016/s1473-3099(06)70602-8 pubmed pmid: 17008175. unitaid. tuberculosis diagnostics technology and market landscape. 3rd ed. geneva, switzerland; 2014. vassall a, van kampen s, sohn h, et al. rapid diagnosis of tuberculosis with the xpert mtb/rif assay in high burden countries: a cost-effectiveness analysis. plos med. 2011 nov;8(11):e1001120. https://doi.org/10.1371/journal.pmed.1001120 pubmed pmid: 22087078. pubmed central pmcid: 3210757. mgode gf, weetjens bj, nawrath t, et al. mycobacterium tuberculosis volatiles for diagnosis of tuberculosis by cricetomys rats. tuberculosis (edinb). 2012 nov;92(6):535–542. https://doi.org/10.1016/j.tube.2012.07.006 pubmed pmid: 22883935. weetjens b, mgode gf, machang’u rs, et al. african pouched rats for the detection of pulmonary tuberculosis in sputum samples. int j tuberc lung dis. 2009 jun;13(6):737–743. pubmed pmid: 19460250. poling a, weetjens bj, cox c, et al. using giant african pouched rats to detect tuberculosis in human sputum samples: 2009 findings. am j trop med hyg. 2010 dec;83(6):1308–1310. https://doi.org/10.4269/ajtmh.2010.10-0180 pubmed pmid: 21118940. pubmed central pmcid: 2990050. article information authors: stephen balinandi1,2 barnabas bakamutumaho2 john t. kayiwa2 juliette ongus3 joseph oundo1 anna c. awor2 julius j. lutwama2 affiliations: 1field epidemiology and laboratory training program, kenya2uganda virus research institute, uganda 3department of medical laboratory sciences, jomo kenyatta university of agriculture and technology, kenya correspondence to: stephen balinandi postal address: uganda virus research institute, po box 49, entebbe, uganda dates: received: 07 may 2012 accepted: 09 apr. 2013 published: 24 june 2013 how to cite this article: balinandi s, bakamutumaho b, kayiwa jt, ongus j, oundo j, awor ac, lutwama jj. the viral aetiology of influenza-like illnesses in kampala and entebbe, uganda, 2008. afr j lab med. 2013;2(1), art. #65, 5 pages. http://dx.doi.org/10.4102/ ajlm.v2i1.65 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the viral aetiology of influenza-like illnesses in kampala and entebbe, uganda, 2008 in this original research... open access • abstract • introduction • research methods and design    • study locations and target population    • laboratory testing • ethical considerations • trustworthiness • results    • demographic and clinical characteristics of study participants    • frequencies of detected viral aetiologies    • mixed viral infections • discussion • limitations of the study • conclusion • acknowledgements    • competing interests    • authors' contributions • references abstract top ↑ background: as the threat of zoonoses and the emergence of pandemic-prone respiratory viruses increases, there is a need to establish baseline information on the incidence of endemic pathogens in countries worldwide. objectives: to investigate the presence of viruses associated with influenza-like illnesses (ili) in uganda. methods: a cross-sectional study was conducted in which nasopharyngeal swab specimens were collected from patients diagnosed with ili in kampala and entebbe between 14 august 2008 – 15 december 2008. a multiplex polymerase chain reaction assay for detecting 12 respiratory viruses was used. results: a total of 369 patients (52.3% females) was enrolled; the median age was 6 years (range 1–70). one or more respiratory viruses were detected in 172 (46.6%) cases and their prevalence were influenza a virus (19.2%), adenovirus (8.7%), human rhinovirus a (7.9%), coronavirus oc43 (4.3%), parainfluenza virus 1 (2.7%), parainfluenza virus 3 (2.7%), influenza b virus (2.2%), respiratory syncytial virus b (2.2%), human metapneumovirus (1.4%), respiratory syncytial virus a (1.1%), parainfluenza virus 2 (0.5%) and coronavirus 229e (0.5%). there were 24 (14.0%) mixed infections. conclusions: this study identified some of the respiratory viruses associated with ili in uganda. the circulation of some of the viruses was previously unknown in the study population. these results are useful in order to guide future surveillance and case management strategies involving respiratory illnesses in uganda. introduction top ↑ globally, influenza-like illnesses (ili), also known as acute respiratory illnesses, are common causes of morbidity and mortality in both developed and developing countries.1,2 in temperate climates, ili is reported throughout the year amongst hospital patients with a marked increase in cases recorded during winter periods. there is also evidence of sporadic background activity of ili transmission throughout the year amongst communities in tropical climates, with a slight increase in cases during the rainy season.3,4,5 however, little is known about the aetiologic agents of ili in some developing countries, making it a challenge to plan and implement effective patient management and disease prevention and control efforts.6,7as surveillance and monitoring programmes for ili scale up in many countries, primarily triggered by the increased threat of zoonosis and the emergence of pandemic-prone respiratory viruses, there is a need to identify and document the incidence of endemic and circulating pathogens. this information is important for differential diagnosis, outbreak investigations, trend analysis, early recognition of emerging and re-emerging viruses and implementation of specific public health interventions such as mass vaccination campaigns. in uganda, a tropical country lying along the equator, surveillance for influenza was started in july 2007. by june 2008, influenza viruses were confirmed in only 12% of patients presenting with ili at health facilities, implying that the aetiologies in the remaining 88% were unknown.8 there are a few studies in uganda that have identified influenza viruses, respiratory syncytial viruses, parainfluenza viruses, coxsackieviruses and echoviruses as being causative agents of ili – but these studies were conducted during the 1970s.9,10,11 more recent results from other countries including senegal, cote d’ivoire, kenya and madagascar have shown that these viruses are in circulation together with newly-discovered viruses such as coronaviruses, bocaviruses and polyomaviruses.12,13,14,15,16 in the current study, we identify the respiratory viruses that are associated with ili patients seeking healthcare in kampala city and entebbe town, both located in the central region of uganda. research methods and design top ↑ study locations and target population this study was carried out under the national protocol for the routine surveillance of human influenza in uganda. patients of all ages presenting with ili at the sentinel surveillance sites for influenza at kiswa health centre in the ugandan capital kampala and entebbe hospital in entebbe town were enrolled into this cross-sectional study between august and december 2008. these two study sites are separated from each other by approximately 35 km.using a world health organization (who)-modified criterion as used by others,17,18,19 an ili case was defined as any individual presenting with fever (≥ 38 °c) and any two of the following clinical signs: cough, sore throat, myalgia and headache. a maximum of five eligible cases was enrolled on each day at each of the study sites. patient demographic characteristics and clinical history were recorded on a standardised clinical form. the presentation of other related symptoms, such as shortness of breath, conjunctivitis, diarrhoea and vomiting, was also recorded. this information was obtained from the caregiver if the patient was unable to speak or was a child. laboratory testing all the laboratory testing was carried out at the national influenza centre (nic) located at uganda virus research institute, entebbe, uganda. this laboratory is the national reference centre for human influenza surveillance in uganda and participates routinely in the who external quality assessment panel programme for influenza viruses. a nasopharyngeal swab (pur-wraps®, puritan medical products company llc, maine, usa) was collected from each study participant. specimens collected at health centres were placed immediately in 500 µl of viral transport media (dulbecco’s modified eagle’s medium®, highveld biological ltd, lyndhurst, south africa; supplemented with 0.5% serum albumin, 100 u/ml of penicillin and 100 u/ml of streptomycin), stored in liquid nitrogen and transported to the nic for further analysis.a commercially-available multiplex pcr kit (seeplex® rv detection; seegene inc, rockville, md, usa) for the detection of adenovirus, influenza a and b viruses, respiratory syncytial viruses a and b, human metapneumovirus, parainfluenza viruses 1, 2 and 3, rhinovirus a, and coronaviruses 229e and oc43 was used. this kit has been used elsewhere to identify respiratory viruses in similar specimens.20,21 the assay was performed according to the manufacturer’s instructions. briefly, total nucleic acids (both ribonucleic acid [rna] and deoxyribonucleic acid [dna]) were extracted from the collected specimens (viral gene-spintm, intron biotechnology, gyeonggi-do, korea) and target sequences present in the extracts were then reverse transcribed (for the rna form) and amplified in two separate assays of multiplex polymerase chain reaction (pcr; mpcr) using virus-specific kit primers. one assay contained primers for adenovirus (amplicon size 534 bp), human metapneumovirus (469 bp), coronavirus 229e (375 bp), parainfluenza virus 1 (324 bp), parainfluenza virus 2 (264 bp), and parainfluenza virus 3 (219 bp), whilst the other mpcr assay had primer sets for influenza a virus (513 bp), influenza b virus (455 bp), respiratory syncytial virus b (391 bp), rhinovirus a (337 bp), respiratory syncytial virus a (273 bp) and coronavirus oc43 (231 bp). the amplified products were observed using agarose gel electrophoresis. specimens that had matching bands corresponding to the expected amplicon sizes for each type of virus relative to the molecular weight marker (seeplex®) were scored as positive for that virus. kit positive and negative controls were also included in every assay run. an internal positive control (seeplex®) was also included to evaluate the amplification efficiency of each tested specimen. ethical considerations top ↑ as these data were collected from patients as part of routine healthcare delivery and were anonymous, the uganda ministry of health guidelines did not require ethical review. informed (verbal) consent was obtained from each study participant or caregiver. trustworthiness top ↑ this study was conducted following the national guidelines for influenza surveillance as established by the ugandan ministry of health. thus, all the study participants were identified and recruited by trained healthcare workers using a modified who case definition for ili. in addition, all laboratory procedures were performed in a who collaborating reference laboratory following the kit manufacturer’s instructions. both the specimen collection and laboratory testing methods have been used elsewhere in similar studies.17,18,19,20,21 results top ↑ demographic and clinical characteristics of study participants between august and december 2008, a total of 369 study participants presenting with ili at the two study centres were recruited. of these, 286 (77.5%) participants were from entebbe hospital and 83 (22.5%) participants were from kiswa health centre (table 1). both genders were represented in almost equal proportions (52.3% females and 47.7% males) and the median age was 6 years (range: 1–70). over half of the study participants (61.5%) were aged 10 years or less, and had low or no form of education (74.5%). table 1: demographic characteristics of patients with influenza-like illness at kiswa health center and entebbe hospital, uganda, between august 2008 and december 2008. all patients were seen at the outpatient departments of the two study sites and none required hospital admission at the time of enrolment. apart from fever, the most common clinical symptoms were cough (98.4%), shortness of breath (43.1%) and headache (29.0%). only 3.8% (n = 14) of the study participants reported the presence of a chronic condition or illness such as active tuberculosis, chronic cough and chest pain. almost all of the participants (94.5%) reported to the clinic within three days (range: 1–31) of the onset of symptoms. frequencies of detected viral aetiologies out of 369 swab specimens collected and analysed, 172 (46.6%) were positive for one or more respiratory agents. the most frequently-detected respiratory virus amongst all study participants was influenza a (n = 71, 19.2%). others were adenovirus (n = 32, 8.7%), rhinovirus a (n = 29, 7.9%) and coronavirus oc43 (n = 16, 4.3%). the least-detected respiratory viruses were parainfluenza virus 2 (n = 2, 0.5%) and coronavirus 229e (n = 2, 0.5%) (table 2). approximately 63.0% of all viral detections, including all the parainfluenza viruses (1, 2 and 3) and 78.1% of adenovirus and 75.0% of coronavirus oc43, were detected from study participants who were aged 10 years or less. only rhinovirus a and all influenza viruses were detected across all ages. table 2: frequency and prevalence of respiratory viruses amongst patients presenting with influenza-like illness at kiswa health center and entebbe hospital, uganda, between august 2008 and december 2008; n = 369. mixed viral infections of the 172 that tested positive for respiratory agents, there were 24 (14.0%) cases with mixed infections of two or three viruses. no patient was infected with more than three viruses. twenty-one cases were infected with two viruses, with the most frequent mixture being adenovirus and influenza a virus (n = 5). other frequent viral mixtures were adenovirus and rhinovirus a (n =3) and influenza a virus and coronavirus oc43 (n =3) (table 3). adenovirus was present in 62.5% (n = 15) of mixed infections which was almost a half (46.9%) of all detections for this virus. respiratory syncytial virus b was the only aetiology that was not detected in a mixed infection. table 3: mixed infections amongst patients presenting with influenza-like illness attending kiswa health center and entebbe hospital, uganda, between august 2008 and december 2008; n = 24. discussion top ↑ this study identified viral aetiologies in 46.6% of all ili cases at two health facilities in kampala and entebbe, uganda, a prevalence level similar to that reported in other studies.22,23,24 the identified aetiologies include influenza a and b virus, adenovirus, rhinovirus a, coronavirus oc43 and 229e, parainfluenza virus 1, 2 and 3, respiratory syncytial virus a and b and human metapneumovirus. a number of these aetiologies have only recently been identified and their circulation in uganda was unknown previously. these include human metapneumovirus, whose discovery as a causative agent for ili has been recognised only in the last decade.25most of the detected viruses, including parainfluenza virus 1, 2 and 3, influenza b virus, adenovirus, human metapneumovirus and coronaviruses oc43 and 229e, were circulating at prevalence levels that were, in general, similar to those found elsewhere.12,14,26,27 influenza a virus was detected in 19.2% of ili cases, which was higher than the 12% prevalence level that was known to exist from previous observations in the same population.8 the higher prevalence observed for this virus could be attributed to the timing of this study which was conducted when rainfall is highest in uganda. it is probable that an outbreak associated with this virus was ongoing during the study period as observed previously.4,5,6 conversely, rhinovirus a was detected at 7.9% which is lower than the 10 % – 25% prevalence levels found in other ili surveillance studies within sub-saharan tropics.13,14,16,28 in the same studies, the prevalence of respiratory syncytial viruses a and b ranged between 5% – 21% which is higher than our 3.3% total prevalence for the same viruses. the low prevalence levels observed for these viruses could also be associated with their seasonality in the study population – a variable that could not be established with our current cross-sectional data. also, our ili case definition was focused more on influenza surveillance and could have been restrictive with regard to the signs and symptoms of other ili aetiologies.29,30 mixed infections amongst all cases that tested positive for respiratory agents accounted for 14.0% of the findings, with the majority being double infections. the prevalence of mixed infections ranging from 4.5% – 70% are reported from other studies, depending on the geographical location of the study area, the diagnostic method used or the general degree of illness in the study population.24,31,32,33 the high prevalence of mixed influenza a virus and adenovirus infections during a low circulation cycle of respiratory syncytial virus infection has previously been suggested.34 in our study, the number of mixed infections (n = 24) was not adequate to allow statistical analysis; a more comprehensive study is necessary in order to determine the associations and interactions between these viruses as well as the related clinical outcomes. limitations of the study top ↑ this study had a number of limitations. firstly, the number of viruses detectable by the multiplex pcr kit was limited to 12; it is possible that other respiratory viruses were circulating in the study population but were not identified. secondly, only nasopharyngeal swabs were collected, possibly missing viruses in the lower respiratory tract. thirdly, the study period was limited to four months and epidemiological aspects associated with these viruses such as seasonality could not be established. conclusion top ↑ we have shown the viral aetiologies of some of the ili in the study population that were not caused by influenza viruses. this information is vital for use in the future to guide policymaking on respiratory disease surveillance and case management in uganda. acknowledgements top ↑ we would like to thank all the staff at entebbe hospital and kiswa health centre, especially roselyn mutonyi, janet aliru, lucy wanyenze, grace kaija and molly busingye, who in addition to their routine hospital work, accepted the task of taking on the extra responsibility of collecting data for this study. we also thank robert downing, njenga m. kariuki and benjamin d. moser for their technical support. the study participants’ enrolment in this study is highly appreciated. we are also indebted to the centers for disease control and prevention, atlanta, usa who provided funding for this study through the african field epidemiology network and the kenya field epidemiology and laboratory training programme. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. authors’ contributions s.b. (feltp and uganda virus research institute), b.b. (uganda virus research institute), j.o. (jomo kenyatta university), j.o. (feltp) and j.j.l. (uganda virus research institute) conceived and provided technical design of the study; s.b., b.b. and j.t.k. (uganda virus research institute) performed both clinical and laboratory data collection; s.b. and a.c.a. (uganda virus research institute) performed computer data entry and analysis; and s.b., j.o., j.o. and j.j.l. wrote the article. references top ↑ 1. world health organization. the global burden of disease: 2004 update. [page on internet]. [c2009] [cited 2011 may 2]. available from: http://whqlibdoc.who.int/publications/2008/9789241563710_eng.pdf 2. williams bg, gouws e, boschi-pinto c, et al. estimates of world-wide distribution of child deaths from acute respiratory infections. lancet infect dis. 2002;2(1):25–32. http://dx.doi.org/10.1016/s1473-3099(01)00170-0 3. chew ft, doraisingham s, ling ae, et al. seasonal trends of viral respiratory tract infections in the tropics. epidemiol infect. 1998;121(1):121–128. http://dx.doi.org/10.1017/s0950268898008905, pmid:9747763, pmcid:2809482 4. moura fe, perdigão ac, and siqueira mm. seasonality of influenza in the tropics: a distinct pattern in northeastern brazil. am j trop med hyg. 2009;81(1):180–183. pmid:19556586 5. dosseh a, ndiaye k, spiegel a, et al. epidemiological and virological influenza survey in dakar, senegal: 1996–1998. am j trop med hyg. 2000;62(5):639–643. pmid:11289677 6. rudan i, lawn j, cousens s, et al. gaps in policy-relevant information on burden of 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kadjo ha, ekaza e, coulibaly d, et al. sentinel surveillance for influenza and other respiratory viruses in cote d’ivoire, 2003–2010. influenza other respi viruses. 2012;7(3):296–303. http://dx.doi.org/10.1111/j.1750-2659.2012.00389.x, pmid:22863403 17. boivin g, hardy i, tellier g, et al. predicting influenza infections during epidemics with use of a clinical case definition. clin.infect.dis. 2000;31(5):1166–1169. http://dx.doi.org/10.1086/317425, pmid:11073747 18. monto as, gravenstein s, elliott m, et al. clinical signs and symptoms predicting influenza infection. arch intern med. 2000;160(21):3243–3247. http://dx.doi.org/10.1001/archinte.160.21.3243, pmid:11088084 19. zambon m, hays j, webster a, et al. diagnosis of influenza in the community: relationship of clinical diagnosis to confirmed virological, serologic, or molecular detection of influenza. arch intern med. 2001;161(17):2116–2122. http://dx.doi.org/10.1001/archinte.161.17.2116, pmid:11570941 20. drews sj, blair j, lombos e, et al. use of the seeplex rv detection kit for surveillance of respiratory viral outbreaks in toronto, ontario, canada. ann clin lab sci. 2008; 38(4):376–379. pmid:18988931 21. yoo sj, kuak ey, shin bm. detection of 12 respiratory viruses with two-set multiplex reverse transcriptase-pcr assay using a dual priming oligonucleotide system. korean j lab med. 2007;27(6):420–427. http://dx.doi.org/10.3343/kjlm.2007.27.6.420, pmid:18160832 22. ahmed ja, katz ma, auko e, et al. epidemiology of respiratory viral infections in two long-term refugee camps in kenya, 2007–2010. bmc infect dis. 2012;12:7. http://dx.doi.org/10.1186/1471-2334-12-7, pmid:22251705, pmcid:3398263 23. sung ry, chan pk, tsen t, et al. identification of viral and atypical bacterial pathogens in children hospitalized with acute respiratory infections in hong kong by multiplex pcr assays. j med virol. 2009;81(1):153–159. http://dx.doi.org/10.1002/jmv.21364, pmid:19031443 24. thomazelli lm, vieira s, leal al, et al. 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in rural mozambique: a malaria endemic area with high prevalence of human immunodeficiency virus. pediatr infect dis j. 2011;30(1):39–44. http://dx.doi.org/10.1097/inf.0b013e3181f232fe, pmid:20805786 29. casalegno js, frobert e, escuret v, et al. beyond the influenza-like illness surveillance: the need for real-time virological data. euro surveill. 2011;16(1):1–2. 30. walsh ee, peterson dr, falsey ar. is clinical recognition of respiratory syncytial virus infection in hospitalized elderly and high-risk adults possible? j infect dis. 2007;195(7):1046–1051. http://dx.doi.org/10.1086/511986, pmid:17330796 31. lina b, valette m, foray s, et al. surveillance of community-acquired viral infections due to respiratory viruses in rhone-alpes (france) during winter 1994 to 1995. j clin microbiol. 1996;34(12):3007–3011. pmid:8940439, pmcid:229450 32. brunstein jd, cline cl, mckinney s, et al. evidence from multiplex molecular assays for complex multipathogen interactions in acute respiratory infections. j clin microbiol. 2008;46(1):97–102. http://dx.doi.org/10.1128/jcm.01117-07, pmid:17977985, pmcid:2224244 33. greensill j, mcnamara ps, dove w, et al. human metapneumovirus in severe respiratory syncytial virus bronchiolitis. emerg infect dis. 2003;9(3):372–375. http://dx.doi.org/10.3201/eid0903.020289, pmid:12643835, pmcid:2958536 34. forgie im, campbell h, lloyd-evans n, et al. etiology of acute lower respiratory tract infections in children in a rural community in the gambia. pediatr infect dis j. 1992;11(6):466–473. http://dx.doi.org/10.1097/00006454-199206000-00009, pmid:1608684 article information authors: john n. nkengasong1 deborah birx1 affiliations: 1division of global hiv/aids, center for global health, us centers for disease control and prevention, atlanta, united states correspondence to: john nkengasong postal address: 1600 clifton road, atlanta, ga 30333, united sates dates: received: 03 sept. 2014 accepted: 11 sept. 2014 published: 03 nov. 2014 how to cite this article: nkengasong jn, birx d. quality matters in strengthening global laboratory medicine. afr j lab med. 2014;3(2), art. #239, 4 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.239 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. quality matters in strengthening global laboratory medicine in this commentary... open access • introduction    • squaring the circle    • time to stop doing more of the same    • slipta and lqms-sip, the companions of slmta • evident evidence and the up-shot    • slmta, nearing the tipping point?    • conclusion • acknowledgements    • competing interests    • authors’ contributions    • disclaimer • references introduction top ↑ around 300 bc, during the time of the ancient greek physician, hippocrates, the first documented examination of human bodily fluids was conducted. this gave birth to laboratory medicine, which is the use of laboratory tests to guide clinical investigations.1 ever since, as a result of its multi-faceted nature, ensuring the quality of testing in laboratory medicine has remained a challenge, but is an evolving practice in many countries. in the developed world, laboratory medicine has been transformative and, in most cases, is the science behind clinical care and disease surveillance. central to the practice of laboratory medicine in the developed world is the recognition of the importance of quality assurance (qa). as such, there is regular review of existing policies in order to ensure the continuous improvement of quality systems. for instance, in the united states, reports by the institute of medicine, to err is human: building a safer health system (1999), 2 and the committee on quality of health care in america, crossing the quality chasm: a new health system for the 21st century (2001),3 helped refocus attention on the need to minimise medical errors and improve quality. in developing countries, qa in laboratory medicine has been severely neglected and has become a serious impediment to effective healthcare delivery and disease surveillance.4,5,6 in fact, a vicious cycle has been established whereby most physicians in developing countries rely on history-taking and physical examination for patient management, since they have little confidence in laboratory test results, even if laboratory facilities exist.5 as such, inadequate resources are allocated to laboratory services, which in turn results in less-than-optimal quality-assured results, further leading to the neglect of laboratory systems (figure 1). nonetheless, many countries are currently making great strides in implementing quality management systems (qms), which is leading to laboratory accreditation to international standards.7,8 the importance of quality in laboratory medicine cannot be overstated: it adds significant value to patient outcomes and management;9 reduces wastage, minimises sample rejection and enhances client satisfaction;10 prevents unneeded diagnostic testing; improves turnaround times for accurate diagnosis; and reduces the use of inappropriate treatment. because laboratory errors occur at a rate of 32% – 75% in the pre-analytic phase, 13% – 32% in the analytic phase and 9% – 31% in the post-analytic phase,11,12 it is vital that assuring the quality of laboratory medicine be considered a continuum of a total testing process of all analytical phases. errors that occur in the pre-analytical phase in the spectrum of laboratory testing can have a direct effect on patient outcomes in the post-analytical stage. figure 1: quality matters: a catalyst to a tipping point for strengthening laboratory medicine? squaring the circle because of the acute challenges of implementing qa in laboratory medicine, healthcare providers in developing countries have unwisely neglected the important role of laboratory diagnosis in patient care. as a result, achieving the international organization for standardization (iso) 15189 requirements for clinical laboratory has become a lofty aspiration. understandably, but unfortunately, these countries seem to find themselves at an impasse of practising laboratory medicine in the 21st century by squaring the circle: relying on inadequate quality-assured test results or empiricism for patient management, resulting in disproportionate administration of antibiotics and high cost to patients.5 in the last decade, there has been a massive focus on global health, with funding reaching an unprecedented us $28.2 billion in 2010.13 the surge in funding has resulted in the recognition that strengthening health systems, including laboratory services, is critical to healthcare delivery. thus, an unparalleled opportunity has presented itself to strengthen quality-assured laboratory medicine, using innovative approaches to addressing old, neglected challenges. time to stop doing more of the same increased funding for global health has placed a sense of urgency on stakeholders to act now and collectively, but in a different manner.4 previous approaches that attempted to strengthen qms did not use a holistic approach, focusing rather on individual activities: qa, proficiency testing and theoretical concepts. this approach has shown severe limitations with regard to advancing quality-assured laboratory medicine in developing countries. to continue with such strategies would be analogous to pounding square pegs into round holes. rather, novel holistic approaches that place emphasis on task-based and results-driven quality improvement projects are needed urgently. to achieve these goals, laboratory medicine in developing countries must innovate and create performance-enablers that would both incentivise and energise the field, thereby facilitating adoption. successful performance-enablers must focus on four chief aspects: implementation, measurement, reward and improvement. in order to ensure a sustainable culture of the practice of quality-assured laboratory medicine, countries need to embrace innovative qms programmes, which require commitment to the process of continuous quality improvement of laboratory medicine. some countries have made remarkable progress by using innovative approaches to implementing laboratory accreditation. for example, between 1961 and 1998, south korea endorsed the laboratory accreditation program (lap) of the college of american pathologists (cap); however, during that time only 11 laboratories were accredited.15 because of the challenges in implementation of lap, in 1999, south korea modified the cap-lap into a step-wise laboratory accreditation process known as the korea laboratory accreditation process (klap). in klap, laboratories with a score of > 90% received a two-year certificate; laboratories with scores between 60% and 89% received a one-year certificate; and those with a score of < 60% failed the certification. as a result of klap, 227 laboratories were certified between 1999 and 2006 and many laboratories were enrolled in the programme across the country. in 2001, thailand established a step-wise national accreditation programme as a local alternative for improvement of laboratory quality. the accreditation programme was established with standards based on iso 15189 and, from 2003 to 2009, 724 (50.6%) of 1432 laboratories in thailand were assessed. of these, 197 (27.2%) were accredited, primarily in the government sector.16 the programme has thus far been affordable, feasible, scalable, sustainable and effective.16 over the past five years, through global partnership, innovative performance-enablers have been developed to guide the implementation of a sustainable qms leading to accreditation in developing countries: (1) the stepwise laboratory quality improvement process towards accreditation (slipta);17 (2) the caribbean laboratory quality management system – stepwise improvement process (lqms-sip) towards accreditation;18 and (3) the task-based strengthening laboratory management toward accreditation (slmta) programme.19 slipta and lqms-sip, the companions of slmta both slipta and lqms-sip are innovative performance-enabling tools and companions of slmta. these tools were designed to motivate the implementation of qms with measurable delivery through slmta. slipta is a framework endorsed by the world health organization regional office for africa (who afro) and jointly implemented by the african society for laboratory medicine (aslm) for improving the quality of public health laboratories in african countries to achieve accreditation to the iso 15189 standard. the who afro slipta checklist is based on iso 15189/17025, with 111 items and a possible 258 points, which are further divided into five star levels: one to five.17 lqms-sip has been endorsed in the caribbean region and consists of a three-tiered system: the first tier represents the minimum requirements that correspond to mandatory criteria required for the granting of a licence based on legislation enacted by the ministries of health. the next two tiers are quality-improvement levels representing achievements in meeting specific requirements of a qms. the caribbean regional organization for standards and quality hosts the lqms-sip secretariat and works directly with countries and other laboratory stakeholders to coordinate the rollout and implementation of the regulatory activities and the recognition process. the caribbean public health agency (carpha) helps with the coordination of the caribbean public health laboratory network and participates in the development of quality standards. evident evidence and the up-shot top ↑ slmta is grounded in its ability to identify deficiencies in a laboratory, improve them and measure the outcomes. after completing a full slmta round, which typically lasts for up to 18 months, changes are evident and outcomes are visible. laboratories experience a net progression from one to five stars on the slipta scoring checklist upon completion of the slmta programme. more significant is the enduring impact on personnel who have undergone slmta training, achieving positive changes in attitude toward the culture of quality, as well as recognition of quality-assured laboratory medicine. in this special issue of the african journal of laboratory medicine, several countries have shared remarkable evidence regarding how slmta has been transformative in their laboratories7,8 and is beginning to stimulate changes in hospital management.20 since 2009, when slmta was launched in kigali, rwanda, it has expanded exponentially. as of the end of 2013 it has been implemented in 47 countries in africa, the caribbean, latin america and southeast asia. with the introduction of slmta, the prospects of implementing sustainable quality-assured laboratory medicine seem to be a reality in developing countries. in total, the 302 laboratories that have completed the slmta programme conduct approximately 43.5 million diagnostic tests annually. based on baseline audit scores, laboratories that had at least one quality star prior to slmta participation conducted only one out of every six tests. this number quadrupled to two out of three after slmta training. these gains have also proven to be sustainable; of the 92 laboratories that have conducted surveillance audits at five to 28 months after slmta completion, 62% showed a further increase in their score from the exit audit, with more than half increasing their score by more than 10 additional percentage points.8 at present, countries that have implemented slmta are caught between a state of cautious optimism and open-minded concern about the rollout and sustainability of the programme.d open-minded concern about the rollout and sustainability of the programme. slmta, nearing the tipping point? in order to ensure sustainability, it is urgent to identify system drivers that will enable the country to reach a tipping point; these include expanded coverage and demonstration of the impact on patient care. such a tipping point, defined in this context as the number of laboratories that will constitute a critical mass for the demand of the programme to become a nation-wide requirement, once attained, will begin to increase confidence in quality-assured laboratory medicine for evidence-based patient management. this may lead to an increased uptake and use of laboratory test results, encouraging greater investment of resources in laboratory services and, ultimately, breaking the vicious cycle of the neglected laboratory systems in developing countries (figure 1). potential drivers that could facilitate a tipping point for slmta include incorporating slmta into a pre-service curriculum for schools of medical laboratory sciences; strengthening the clinical-laboratory interface; developing country-specific national strategic plans for rolling out slmta and other qms tools; accelerating the process by engaging aslm or similar organisations to audit and reward laboratories that have undergone the slmta process; incorporating basic laboratory information systems as part of qms; designing slmta-like training tools for hospital and clinic certification and accreditations; and, lastly, encouraging donors and funders to prioritise qa and continuous quality improvement as a core component of laboratory health system strengthening. conclusion slmta eliminates redundant procedures by reorganising the laboratory set-up so that personnel spend less time handling and processing specimens. slmta is not a destination; rather, it is a journey that relies on ongoing cooperation at all levels, including senior management, laboratory staff and end users. in the 21st century, as the economies of developing countries continue to grow, many individuals will begin to seek affordable quality-assured healthcare; as such, there is bound to be increasingly consumer-oriented healthcare that holds physicians and laboratory workers more accountable for errors. because of improved quality-assured laboratory medicine, healthcare providers will broaden their practice from using laboratory tests to confirm clinical diagnoses to using tests to detect clinically unapparent diseases, as well as to support outbreak surveillance responses, such as the recent outbreak of ebola virus in west africa. in order to meet this demand, both slmta and slipta will need to be rolled out in developing countries so as to stimulate healthcare service providers to focus on a systematic work flow for quality services rendered to patients, resulting in increased efficiency and quality whilst lowering waste and cost and improving safety. critically, a patient-centered continuous quality improvement approach will become indispensable. hospital accreditation, as in thailand, will most likely drive the need for laboratory quality, as active involvement of managers will create collective organisational commitment of quality improvement and a focus on patient-oriented thinking. acknowledgements top ↑ competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions j.n.n. (cdc, atlanta) contributed to the conception and development of the manuscript. d.b. (cdc, atlanta) contributed to the writing and review of the manuscript. disclaimer the comments and conclusions in this report are those of the authors and do not necessarily represent the official position of the united states department of health and human services, public health service, or centers for disease control and prevention. references top ↑ 1.berger d. a brief history of medical diagnosis and the birth of the clinical laboratory. mlo med lab obs. 1999;31(7):28–30, 32, 34–40. 2.institute of medicine. to err is human: building a safer health system [document on the internet]. c1999 [cited 2014 sep 22]. available from: http://www.iom.edu/~/media/files/report%20files/1999/to-err-is-human/to%20err%20is%20human%201999%20%20report%20brief.pdf 3.committee on quality of health care in america. crossing the quality chasm: a new health system for the 21st century [document on the internet]. c2001 [cited 2014 sep 20]. available as ebook from: http://www.nap.edu/openbook.php?record_id=10027 4.petti ca, polage cr, quinn tc, et al. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006;42(3):377–382. http://dx.doi.org/10.1086/499363 5.polage cr, bedu-addo g, owusu-ofori a, et al. laboratory use in ghana: physician perception and practice. am j trop med hyg. 2006;75(3):526–531. 6.nkengasong jn, nsubuga p, nwanyanwu o, et al. laboratory systems and services are critical in global health: time to end the neglect? am j clin pathol. 2010;134(3):368–373. http://dx.doi.org/10.1309/ajcpmpsinq9brmu6 7.luman et, yao k, nkengasong jn. a comprehensive review of the slmta literature part 2: measuring success. afr j lab med. 2014;3(2), art. #276, 8 pages. http://dx.doi.org/10.4102/ajlm.v3i2.276 8.yao k, luman et, slmta collaborating authors. evidence from 617 laboratories in 47 countries for slmta-driven improvement in quality management systems. afr j lab med. 2014;3(2), art. #262, 11 pages. http://dx.doi.org/10.4102/ajlm.v3i2.262 9.carter jy, lema oe, wangai mw, et al. laboratory testing improves diagnosis and treatment outcomes in primary health care facilities. afr j lab med. 2012;1(1), art. #8, 6 pages. 10.zeh ce, inzaule sc, magero vo, et al. field experience in implementing iso 15189 in kisumu, kenya. am j clin pathol. 2010;134(3):410–418. http://dx.doi.org/10.1309/ajcpzirkdus5lk2d 11.leatherman s, ferris tg, berwick d, et al. the role of quality improvement in strengthening health systems in developing countries. int j qual health care. 2010;22(4):237–243. http://dx.doi.org/10.1093/intqhc/mzq028 12.bonini p, plebani m, ceriotti f, et al. errors in laboratory medicine. clin chem. 2002;48(5):691–698. 13.institute for health metrics and evaluation. financing global health 2012: the end of the golden age? [report on the internet]. c2012 [cited 2013 feb 05]. available from: http://www.healthmetricsandevaluation.org/publications/policy-report/financing-global-health-2012-end-golden-age 14.nkengasong jn. a shifting paradigm in strengthening laboratory health systems for global health: acting now, acting collectively, but acting differently. am j clin pathol. 2010;134(3):359–360. http://dx.doi.org/10.1309/ajcpy5asuejyq5rk 15.shin bm, chae sl, min wk, et al. the implementation and effects of a clinical laboratory accreditation program in korea from 1999 to 2006. korean j lab med. 2009;29(2):163–170. http://dx.doi.org/10.3343/kjlm.2009.29.2.163 16.wattanasri n, manoroma w, viriyayudhagorn s. laboratory accreditation in thailand: a systemic approach. am j clin pathol. 2010;134(4):534–540. http://dx.doi.org/10.1309/ajcpzyy19wmkmazt 17.world health organization regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation in the african region (with 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introduction methods results discussion acknowledgements references about the author(s) caroline a. fernando department of medical laboratory sciences, faculty of allied health sciences, general sir john kotelawala defence university, werahera, sri lanka deklanji t. dissanayake department of medical laboratory sciences, faculty of allied health sciences, general sir john kotelawala defence university, werahera, sri lanka uththara i. hewamana department of medical laboratory sciences, faculty of allied health sciences, general sir john kotelawala defence university, werahera, sri lanka shyamini rathnaweera department of medical laboratory sciences, faculty of allied health sciences, general sir john kotelawala defence university, werahera, sri lanka wickrama a. samanthilake department of quality management, national blood center, narahenpita, sri lanka ranga tudugala department of radiography and radiotherapy, faculty of allied health sciences, general sir john kotelawala defence university, werahera, sri lanka kithsiri b. jayasekara department of medical laboratory sciences, faculty of allied health sciences, general sir john kotelawala defence university, werahera, sri lanka kumudu kuruppu department of quality management, national blood center, narahenpita, sri lanka citation fernando ca, dissanayake dt, hewamana ui, et al. alternative methods for calculating percentage haemolysis of red cell concentrates in peripheral blood banks in sri lanka. afr j lab med. 2023;12(1), a1987. https://doi.org/10.4102/ajlm.v12i1.1987 original research alternative methods for calculating percentage haemolysis of red cell concentrates in peripheral blood banks in sri lanka caroline a. fernando, deklanji t. dissanayake, uththara i. hewamana, shyamini rathnaweera, wickrama a. samanthilake, ranga tudugala, kithsiri b. jayasekara, kumudu kuruppu received: 16 june 2022; accepted: 17 nov. 2022; published: 23 feb. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: haemolysis – one of the major limiting factors of red cell concentrate quality – must be measured as a quality-monitoring requirement. according to international quality standards, percentage haemolysis must be monitored in 1.0% of red cell concentrates produced monthly and maintained under 0.8%. objective: this study assessed three alternative methods for determining plasma haemoglobin concentration in peripheral blood banks that lack a plasma or low haemoglobin photometer – the gold-standard method – in sri lanka. methods: a standard haemolysate was prepared using an unexpired whole blood pack of normal haemoglobin concentration. a concentration series from 0.1 g/dl to 1.0 g/dl was prepared by diluting portions of standard haemolysate with saline. the alternative methods, namely visual haemoglobin colour scale, spectrophotometric calibration graph, and standard haemolysate capillary tube comparison, were designed using this concentration series and were used to test red cell concentrates received at the quality control department of the national blood center, sri lanka, from february 2021 to may 2021. results: a strong correlation was observed between the haemoglobin photometer method and the alternative methods (r = ~0.9). based on the linear regression model, the standard haemolysate capillary tube comparison method was the best of the three alternative methods (r2 = 0.974). conclusion: all three alternative methods are recommended for use in peripheral blood banks. the standard haemolysate capillary tube comparison method was the best model. keywords: blood banks; capillary tube comparison; haemoglobin colour scale; percentage haemolysis; red cell concentrate. introduction red cell concentrate (rcc) transfusion is essential to treat patients with anaemia and bleeding disorders, as well as in emergencies where there is severe blood loss during surgery or an accident.1 several clinical manifestations can occur in patients due to inappropriate blood transfusion. therefore, to ensure patient safety, rcc for transfusion must always be of good quality.1,2 quality of rcc depreciates with storage time, and haemolysis, the destruction of red blood cells, is one of its quality indicators.3,4 the morphology of red blood cells changes with an increase in osmotic fragility, resulting in deformity and rupture.5 haemolysis can occur due to the use of inappropriate methods during blood collection, processing,6 transportation, handling, and storage.7 upon rupture of the red blood cells, haemoglobin is released into the plasma, leading to a colour change8 that is indicative of haemolysis and can be detected in the supernatant plasma of centrifuged rcc.9 however, this does not totally define whether or not the blood pack is suitable for transfusion. rather, a quality parameter referred to as percentage haemolysis is used. according to international quality standards and those governed by the sri lankan national blood transfusion service quality control (qc) unit, rccs with percentage haemolysis greater than 0.8% towards the end of the storage period are unsuitable for transfusion.10,11 to calculate the percentage haemolysis, plasma haemoglobin concentration is required. the plasma or low haemoglobin photometer (lhbp) provides accurate results on the plasma haemoglobin concentration.11 the lhbp method relies on the oxidation of haemoglobin to haemoglobin by sodium nitrite, and the subsequent conversion of haemoglobin to hemiglobinazide by sodium azide, leading to a colour change that is measured by the photometric principle.12,13 presently, in the government sector of sri lanka, an lhbp is only available at the national blood center (nbc) because it is difficult to afford them at all other peripheral blood banks (pbbs). all pbbs in sri lanka face challenges in identifying haemolysed blood packs. when component storage refrigerators at these pbbs are subjected to temperature fluctuations or any malfunction, rccs are sent to the nbc for quality checks. when it is difficult to send the entire batch of rccs, they are discarded without quality assessment, leading to huge losses because the cost of a blood component includes the high costs of collection, preparation, testing, etc. in some pbbs, at times when blood collection rates are high and usage is low, excess blood packs are transported to other pbbs or to the nbc to be used before expiry. in such cases, the receiving pbbs have no means of assessing the quality of the rccs received. currently, pbbs only carry out visual detection of haemolysis in rccs before transfusion, and any rccs suspected of haemolysis are sent to the nbc to determine the percentage haemolysis. also, as a quality-monitoring procedure, pbbs send 1% of the monthly production of rcc to the nbc to detect haemolysis. however, possible mechanical damage to rccs due to poor transport facilities, difficulty in maintaining cold chain, high costs, and the time and labour requirements make this impractical, especially in emergencies. these problems are faced by many developing and low-income countries in the world, thus necessitating the establishment of alternative methods to detect haemolysis. this was an experimental study to design easy, reliable, and cost-effective methods for determining plasma haemoglobin concentration at pbbs where no lhbp is available. we introduce three alternative methods – visual haemoglobin colour scale (cs), spectrophotometric calibration graph (scg), and standard haemolysate capillary tube comparison (sctc) – for the determination of plasma haemoglobin concentration, which is required when calculating the percentage haemolysis of rccs. we also compare the performance, accuracy, and reliability of these methods to the gold-standard method, lhbp. methods ethical considerations ethical clearance was obtained from the research review committee of national blood transfusion services, narahenpita, sri lanka (ethical clearance number: nbts/moic/etru/co/2021/011). the ethical approval was obtained in a written format before beginning the study upon submission of a study proposal. all rccs used in this study were those obtained from voluntary blood donors and randomly selected and sent to the qc department at the national blood center, sri lanka, for haemolysis testing. therefore, no individual donor consent was required. permission to use the qc data of rccs received at the qc department was given by the senior medical laboratory technologist of the department. a barcode system was maintained on all blood components and no donor details were disclosed to the investigators of the study. raw data were stored by principal investigators on password-protected computers with restricted access. study setting this was a pilot experimental study conducted at the qc department of nbc, sri lanka, from february to may 2021. the nbc is the central hub and largest blood bank of the national blood transfusion service of sri lanka under the government sector. all other pbbs belonging to the national blood transfusion service in the country maintain a close link with the nbc for its services and their monthly quality checks. sampling the three developed alternative methods were used to determine the percentage haemolysis of rccs received at the qc department of the nbc between february and may 2021. red cell concentrates that were clotted, had leaky or physically damaged packs, or whose labels lacked necessary information were excluded from this study. the plasma haemoglobin concentrations of 68 rccs were measured using the lhbp method (standard method) and the three developed alternative methods (cs, scg and sctc methods), and their results were compared statistically. preparation of haemolysate the haemolysate was prepared using an unexpired, healthy whole blood pack. the whole blood was added into a sterile 50 ml falcon™ conical centrifuge tube (thermo fisher scientific, waltham, massachusetts, united states). to remove the plasma and buffy coat, the whole blood was centrifuged at 3500 rotations per minute (rpm) for 10 min using a thermo scientific™ labofuge™ 400 centrifuge (thermo fisher scientific, waltham, massachusetts, united states). afterwards, the supernatant containing the plasma and buffy coat was removed and discarded using a disposable samco™ fine tip transfer pipette (thermo fisher scientific, waltham, massachusetts, united states). the remaining blood was washed three times with equal volumes of 9 g/l nacl to ensure the complete removal of plasma, leukocytes, and platelets. the blood samples were mixed thoroughly between washes and the saline supernatant was carefully removed after centrifugation. fifty millilitres of distilled water and 50 ml of toluene (for red blood cells lysis) were added to the washed blood and homogenised using a mechanical shaker (~180 rpm) for 1 h (table orbital laboratory shaker flat platform-tos-4030p, mrc ltd; laboratory instruments, holon, israel). the mixture was stored at 4 ºc for 24 h – 48 h to allow the lipid and cell debris to form a semisolid surface between the toluene and the lysate. then after 48 h, the mixture was centrifuged at 3500 rpm for 10 min to remove the lysate layers. the lysate was pooled in a clean plain tube and centrifuged at 3500 rpm for 20 min. a sterile syringe was used to aspirate the required volume of lysate from the bottom into a clean sterile container, leaving the top 90% to be discarded. 5.35 ml of glycerol was added as a preservative to 12.5 ml of the lysate obtained, maintaining a 3:7 ratio. the broad-spectrum antibiotics amikacin sulphate (500 mg/2 ml; two drops) and gentamicin (80 mg/2 ml; two drops) were also added to prevent bacterial contamination. the lysate was then dispensed into clean sterile bottles, tightly capped, and stored at 4 ºc until the preparation of the concentration series. the preparation of the standard haemolysate was carried out according to the protocol mentioned in dacie and lewis’s practical hematology,14 but with modifications tailored to the laboratory setting and available resources. preparation of standard haemolysate concentration series the haemolysate stock was diluted using normal saline to prepare a standard haemolysate concentration series with haemoglobin concentrations of 0.1 g/dl, 0.2 g/dl, 0.3 g/dl, 0.4 g/dl, 0.5 g/dl, 0.6 g/dl, 0.7 g/dl, 0.8 g/dl, 0.9 g/dl and 1.0 g/dl. the haemolysate concentrations were prepared in clean 10 ml test tubes. a sysmex automated haematology analyser kx-21 (sysmex corporation, kobe, japan) was used to measure the haemoglobin concentration of the haemolysate stock, while the hemocue® lhbp (standard method) (hemocue, inc., brea, california, united states) was used to measure the lower haemoglobin concentrations of the series since low haemoglobin values (plasma haemoglobin) cannot be measured using a haematology analyser. visual haemoglobin colour scale method each concentrate (0.1 g/dl – 1.0 g/dl) was aspirated into five haematocrit capillary tubes (globe scientific inc., mahwah, new jersey, united states), which were then arranged in ascending order. high-quality photographs of each concentrate in the haematocrit capillary tubes were taken using the nikon d7500 camera (nikon inc., melville, new york, united states) under the same lighting conditions, from the same angle, and at the same place. colours from the photographs were used to design the visual haemoglobin cs using adobe® photoshop cc2017 software (2016, adobe inc., san jose, california, united states). the cs was then printed on 230 gsm (grams per square metre) glossy photo paper (printery company ltd., hong kong, china) using a three-colour laser printer (hewlett-packard colour laserjet pro mfp m281fdw, hewlett-packard company, palo alto, california, united states). the gloss lamination of the prepared cs protects it from mechanical damage and colour changes. this paper was also chosen because it was readily available in sri lanka at a low cost and can easily be printed. spectrophotometric calibration graph method the absorbance values of the standard haemolysate concentration series (0.1 g/dl to 1.0 g/dl) were measured for eight consecutive days using an enzyme-linked immunosorbent assay reader (bio-rad model 680, hercules, california, united states) at 450 nm. using microsoft excel software (2018, microsoft corporation, redmond, washington, united states), the average absorbance was plotted against the supernatant haemoglobin concentration values to prepare the scg. standard haemolysate capillary tube comparison method a portion of each concentrate from the standard haemolysate concentration series (0.1 g/dl – 1.0 g/dl) was aspirated into clean haematocrit capillary tubes, which were then anchored onto the concentration series holder made of hardboard with a white surface (figure 1). figure 1: standard haemolysate capillary tube comparison method developed at the quality control department of the national blood center, sri lanka, september 2020 – may 2021. sample preparation plasma obtained from rcc packs (suspected of haemolysis and received at the nbc qc laboratory, sri lanka) was used to determine plasma haemoglobin concentration by the standard method (lhbp) and the alternative methods (cs, scg and sctc). five millilitres of blood from the rcc was transferred into a clean test tube and centrifuged at 3500 rpm for a few seconds to separate the plasma. application of alternative methods visual haemoglobin colour scale method to estimate plasma haemoglobin for percentage haemolysis calculation, plasma from an rcc tubing is aspirated into a haematocrit capillary tube and visually compared to the scale (figure 2). the corresponding plasma haemoglobin value of the closest matching colour on the cs is selected and used in the percentage haemolysis calculation with the following equation: figure 2: visual haemoglobin colour scale printed on 230 gsm (grams per square metre) glossy laminated photo paper using a three-colour laser printer. the scale was developed at the quality control department of the national blood center, sri lanka, september 2020 – may 2021. spectrophotometric calibration graph method the absorbance values of the standard concentrations obtained on eight consecutive days were plotted against the plasma haemoglobin values of the standard concentration series (figure 3). also, a linear relationship between the mean plasma haemoglobin values and the mean absorbance values of the standard concentration series obtained on eight consecutive days was plotted to prepare the scg (figure 4). the resulting linear equation was used to determine the plasma haemoglobin values of rcc packs with unknown plasma haemoglobin concentrations: figure 3: plasma haemoglobin values of the standard concentration series versus the absorbance values obtained on eight consecutive days at the quality control department of the national blood center, sri lanka, september 2020 – may 2021. figure 4: spectrophotometric calibration graph developed at the quality control department of the national blood center, sri lanka, september 2020 – may 2021. the scg method requires access to an enzyme-linked immunosorbent assay reader, which is available in most pbbs in sri lanka. in sri lanka, pbbs are required to compute an scg and derive a linear equation using the standard concentration series issued to them by the nbc. the prepared scg can be used in subsequent measurements. also, in the absence of the graph, the linear equation obtained from the graph could be used to get a rough estimate of the plasma haemoglobin concentration. anyone who does not have sufficient knowledge of graphs can use the linear equation. data analysis statistical analyses were performed using ibm® spss® (version 20.0) statistical software (2011, ibm corp., armonk, new york, united states). the dataset was assumed to be normally distributed, and descriptive statistics such as mean, standard deviation, range, median, variance, and 95% confidence intervals were computed. we also determined the pearson product-moment correlations between the plasma haemoglobin values obtained using the different methods (lhbp vs cs, lhbp vs scg, lhbp vs sctc, cs vs scg, cs vs sctc, and scg vs sctc). thereafter, simple linear regression models were built based on the plasma haemoglobin values obtained when measured by the lhbp method (gold standard) (response variable – y-axis) and the alternative methods (explanatory variable – x-axis). results were considered statistically significant if p was 0.001 or less, with a 95% confidence interval. data obtained from the 68 rcc packs were filtered such that plasma haemoglobin values less than 0.1 g/dl or greater than 1.0 g/dl were not considered for statistical analysis. therefore, only results obtained from 46 rccs were considered (n = 46). since the measurable range of the developed alternative methods (cs, scg and sctc) was from 0.1 g/dl to 1.0 g/dl (that includes only the clinically significant range), any plasma haemoglobin value outside this range cannot be considered for statistical analysis purposes. results the mean plasma haemoglobin concentration of the rccs as determined by the lhbp method was 0.535 (standard deviation ± 0.277) (table 1). the mean plasma haemoglobin concentrations obtained using the cs, scg and sctc methods were 0.513 (standard deviation ± 0.274), 0.645 (standard deviation ± 0.286), and 0.530 (standard deviation ± 0.280). table 1: plasma haemoglobin concentrations (g/dl) of red cell concentrates received at the quality control department of the national blood center, sri lanka, february 2020 – may 2021. the correlation coefficient (r) between the lhbp and cs methods was 0.986 (p < 0.001), suggesting a statistically significant strong correlation between both methods (table 2). there were also statistically significant strong correlations between the lhbp and scg methods (r = 0.962; p < 0.001), the lhbp and sctc methods (r = 0.987; p < 0.001), the cs and scg methods (r = 0.937; p < 0.001), the cs and sctc methods (r = 0.982; p < 0.001), and between the scg and sctc methods (r = 0.939; p < 0.001). table 2: correlation between the plasma haemoglobin values determined using different methods at the quality control department of the national blood center, sri lanka, september 2020 – may 2021. all three alternative methods followed a simple linear regression. the sctc method had the highest coefficient of determination (r2) value (sctc = 0.974; cs = 0.972; scg = 0.926) (table 3). the sctc method also had the highest beta value (sctc = 0.987; cs = 0.986; scg = 0.962). all models based on the three methods were statistically significant (p < 0.001). table 3: linear regression for plasma haemoglobin values determined by the plasma or low haemoglobin photometer and three alternative methods developed at the quality control department of the national blood center, sri lanka, september 2020 – may 2021. discussion in this study, we developed three alternative methods for the determination of plasma haemoglobin concentration needed to calculate the percentage haemolysis of rccs in pbbs with limited resources. all three alternative methods correlated strongly with the gold-standard method, and they required only a very small volume of supernatant to determine the plasma haemoglobin concentrations of rcc samples. however, statistically, the sctc method gave the best results, having the highest coefficient of determination (r2 = 0.974; when the value of r2 is closer to 1, the model is considered more reliable) and the highest beta value (0.987; the degree of change in haemoglobin concentration measured by lhbp for every unit increase in haemoglobin concentration measured by the sctc method). nevertheless, the cs method was much simpler and practically easier to use than the sctc and scg methods. one limitation of the scg method is the excessive time consumption compared to the lhbp method. this is because each pbb must prepare its own scg graph and obtain the linear equation as absorbance values depend on the enzyme-linked immunosorbent assay reader used. one limitation of the sctc method is the difficulty in handling the 10 capillary tubes. to overcome that, a standard haemolysate capillary tube holder was developed. also, the sctc method consumed more time than the lhbp method. in 1995, a similar colour chart called the ‘haemoglobin colour scale’ was prepared as a simple alternative to assess anaemia and was intended for use when a haemoglobinometer was unavailable or impractical to use in the field.15 another colour chart was also previously developed for the quantitative estimation of methaemoglobinaemia in patients with propanil poisoning in rural areas and in hospitals with limited resources.11,16 as the cs is a visual method, results may vary with the eyesight of individuals, lighting, level of training, etc.17 another study introduced a world health organization haemoglobin cs17 for the diagnosis of anaemia in primary healthcare settings in low-income countries, and a meta-regression analysis was done to assess the impact of variables such as light source, level of training, population type, type of reference test, use of same or different samples for reference and colour scale testing, and the anaemia prevalence on the results.15,17 daylight coming over the shoulder of the observer was found to be the most appropriate light source for colour matching (r2 = 0.9386). inter-observer variation was measured by comparing the means of each group (r2 = 0.9500 for nurses, r2 = 0.9570 for laypeople, r2 = 0.9592 for students). the need for training was also statistically demonstrated (without training r2 = 0.8867; after training r2 = 0.9734). there was, however, no statistically significant difference between the reference method and the world health organization cs (f ratio 1.0198 at v = 99), and anaemia (haemoglobin < 12 g/dl) was efficiently diagnosed in 87% of cases.15 such a meta-analysis must be performed to further validate the methods developed in this study using larger rcc sample sizes. future statistical studies must be performed to prove that the three alternative methods introduced agree with the gold-standard method (lhbp) for detecting low plasma haemoglobin concentrations. apart from a simple linear regression analysis that estimates the linear relationship between plasma haemoglobin values determined by different methods, a level-of-agreement statistical analysis needs to be performed on the collected dataset to determine the degree of concordance. the stability of the standard haemolysate concentration series (used in the sctc method) must also be investigated to evaluate the stability of the colour references, and a repeated measures analysis of variance must be performed. limitations there are limited studies on methods to detect haemolysis in rcc packs. therefore, related articles were not found to compare our findings. furthermore, the preparation of a standard cs requires quality equipment like high-quality cameras, printers, and scanners. access to such high-quality technology and equipment was limited, and the technology and equipment used in this study may have affected the quality of the prepared cs. with better technological equipment, more accurate colours could be printed. also, a decrease in the number of blood donors due to the coronavirus disease 2019 pandemic resulted in the limited availability of rcc for haemolysis testing and method validation. another limitation of the study was that we evaluated the stability of the standard haemolysate series for only 8 days, which was a short time. a significant change in colour or absorbance after 8 days could have affected the results obtained using the scg and sctc methods. conclusion in this study, there was a strong and statistically significant correlation between the lhbp method (gold-standard method) and the three alternative methods (cs, scg and sctc) for the estimation of plasma haemoglobin concentrations. all three alternative methods were found to be suitable for the estimation of plasma haemoglobin concentrations, which is required for the calculation of percentage haemolysis in rccs. the three alternative methods can be introduced as cost-effective, and easy-to-use methods in pbbs with limited facilities. further validation of all methods is required. acknowledgements the authors are extremely grateful to dr lakshman edirisinghe, director, national blood transfusion service, mrs y.v.a.l. mangalika, acting superintendent medical laboratory technologist, national blood transfusion service, mr manuranga jayalath, medical laboratory technologist, quality control laboratory, national blood center, and nursing officer ms m.d. udula for the support given to carry out our research activities. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions c.a.f., d.t.d., u.i.h., and s.r. performed the experimental procedures and contributed to data analysis and writing and editing the manuscript. w.a.s. and s.r. developed the concept and performed the experimental analysis. r.t. contributed to statistical analysis of the data. k.b.j. and k.k. supervised the project and engaged in reviewing and editing the manuscript. all authors have read and agreed to the published version of the manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the authors confirm that the data supporting the findings of this study are available within the article. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references balentine jr. why would you need a blood transfusion? 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lanka. bull world health organ. 2016;94(8):622–625. https://doi.org/10.2471/blt.15.158147 cardigan r, smith k. evaluation of the hemocue plasma hemoglobin analyser for assessing haemolysis in red cell concentrates during storage. vox sanguinis. 2002;82(2):76–79. https://doi.org/10.1046/j.0042-9007.2001.00149.x hudson-thomas m, bingham kc, simmons wk. an evaluation of the hemocue for measuring haemoglobin in field studies in jamaica. bulletin of the world health organization. 1994;72(3):423–426. bain b, dacie j, lewis s. dacie and lewis practical haematology. edinburgh: churchill livingstone; 2012. lewis s, stott g, wynn k. an inexpensive and reliable new hemoglobin colour scale for assessing anaemia. j clin pathol. 1998;51(1):21–24. https://doi.org/10.1136/jcp.51.1.21 shihana f, dissanayake d, buckley n, dawson a. a simple quantitative bedside test to determine methemoglobin. ann emerg med. 2010;55(2):184–189. https://doi.org/10.1016/j.annemergmed.2009.07.022 marn h, critchley j. accuracy of the who hemoglobin colour scale for the diagnosis of anaemia in primary health care settings in low-income countries: a systematic review and meta-analysis. lancet global health. 2016;4(4):e251–e265. https://doi.org/10.1016/s2214-109x(16)00005-x article information authors: linda m. parsons1 akos somoskovi2 evan lee2 chinnambedu n. paramasivan2 miriam schneidman3 deborah birx1 giorgio roscigno2 john nkengasong1 affiliations: 1global aids program, us centers for disease control and prevention, atlanta, usa2foundation for innovative new diagnostics, geneva, switzerland 3the world bank, washington, dc, usa correspondence to: john nkengasong postal address: 1600 clifton road, atlanta ga 30333, usa dates: received: 15 apr. 2011 accepted: 19 jan. 2012 published: 11 june 2012 how to cite this article: parsons lm, somoskovi a, lee e, et al. global health: integrating national laboratory health systems and services in resource-limited settings. afr j lab med. 2012;1(1), art. #11, 5 pages. http://dx.doi.org/10.4102/ ajlm.v1i1.11 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. global health: integrating national laboratory health systems and services in resource-limited settings in this original research... open access • abstract • introduction • what does integration of laboratory systems and services mean? • advantages of integrated laboratory systems • disease-specific programmes impede laboratory services integration • strategies for integrating laboratory systems and services    • policy and strategic planning    • standardising testing and equipment and coordinating with partners    • joint coordination with clinicians    • implementation • practical examples of integrated laboratory capacity in the field    • integration of tb laboratory capacity in hiv laboratories    • integration of hiv testing in a tb laboratory    • avian influenza laboratory support in hiv laboratories • human resources training • recommendations and conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ laboratory systems worldwide are challenged not only by the need to compete for scarce resources with other sections of national health care programmes, but also with the lack of understanding of the critical role that laboratories play in the accurate diagnosis and monitoring of patients suffering from high-burdens of disease. an effective approach to establishing cost-effective laboratory systems that provide rapid and accurate test results for optimal impact on patient care is to move away from disease-specific programmes and establish integrated laboratory services. an integrated laboratory network provides all primary diagnostic services needed for care and treatment without requiring patients to go to different laboratory facilities for specific tests. such a network focuses on providing quality-assured basic laboratory testing through the use of common specimen collection, reporting and diagnostic platforms that can be used across diseases. an integrated laboratory system also provides specimen transport to specialised laboratories and an environment conducive to the introduction and use of new and more complex technologies that would benefit the patient population and public health systems as a whole. as such, this article described various strategies for, and practical examples of, the successful integration of laboratory services. introduction top ↑ human immunodeficiency virus (hiv) and/or aids, tuberculosis (tb) and malaria remain major threats that undermine the health of global populations and account for approximately five million deaths every year.1 a lack of adequate laboratory capacity in resource-poor, high-burden countries presents a significant barrier in providing appropriate diagnosis, care and treatment to patients infected with these and other emerging pathogens.1, 2, 3 modernisation and quality improvement of laboratory services would greatly improve the control of these diseases and support other initiatives.4 decades of underinvestment in national laboratory programmes have resulted in deficiencies in the infrastructure, equipment and human resources that are necessary for effective, quality-assured laboratory services.5 increases in international funding have been unprecedented in recent years,6,7 but programmes for major diseases are still organised vertically as silos, including the establishment of similar testing technologies for different diseases. this has led to wasted resources because of duplications of equipment and training and inefficient use of human capacity. during this time of increased awareness and programmatic scale-up, it is imperative to establish integrated national laboratory systems to optimise quality, efficient and cost-effective testing. what does integration of laboratory systems and services mean? top ↑ the demand for laboratory services to meet the diagnostic and treatment needs for hiv has helped drive investment in new and renovated laboratories to provide comprehensive laboratory diagnostic services for monitoring patients on anti-retroviral therapy with cd4, chemistry, haematology, molecular tests such as measuring plasma ribonucleic acid (rna) levels, testing of hiv-1 drug resistance mutations and testing for opportunistic infections. however, this expansion and investment in laboratory capacity should be optimised to serve the needs of other diseases of public health importance, provide general diagnostic services to support clinical care and support other public health priorities such as re-emerging infectious diseases and chronic diseases. an integrated laboratory network can be defined as one that has the ability to provide all primary diagnostic services needed for the care and treatment of patients without requiring them to go to different laboratories for specific tests. to meet these requirements the network should, (1) be focused on providing quality-assured basic laboratory testing, (2) use common specimen collection, timely reporting and diagnostic platforms that can be used across diseases within the same facility and (3) increase capacity for introducing and using new and more complex technologies. advantages of integrated laboratory systems top ↑ integrated systems have several advantages. firstly, and most importantly, they can support timely and comprehensive care and treatment without the costs and delays associated with referral to specialised facilities for particular diagnostic exams. an integrated system can benefit patients and clinical staff at hospitals located in urban centres, as well as patients served at the community level in remote areas. secondly, new diagnostic platforms, such as microscopes with dual light and fluorescent capacity and molecular diagnostic equipment, have the potential to perform assays for more than one disease. an integrated laboratory approach will ensure that these tools can be used optimally by cross-trained technical staff. thirdly, integrating diagnostic services for different diseases within the same facility helps avoid duplication of investments in infrastructure and laboratory supporting systems, such as specimen transport, supply chain management and information systems. lastly, an integrated approach to training can help ensure standardisation of core laboratory issues, such as quality assurance and standard operating procedures, as well as ensure the more efficient delivery of training. the examples below show how these approaches are both practical and successful. disease-specific programmes impede laboratory services integration top ↑ laboratory systems in resource-poor countries are challenged by, (1) dilapidated infrastructures, (2) lack of funding for developing and implementing national policies, strategic planning, and quality management systems, (3) unlinked referral and reporting services and (4) inadequate human resources, including lack of organised in-service training and long-term career pathways. the difficulty of dealing with all these challenges separately by each programme should provide sufficient reason to encourage integration of services. however, overcoming the traditional vertical approach in which higher level public health laboratories have been established to provide support for disease-specific ministerial directorates and programmes has proven difficult. furthermore, an unintentional negative effect of the disease-specific approach to laboratory strengthening has been the neglect of the core public health laboratory functions. most often, integrated laboratory services are usually found only at the district, sub-district and primary health centre levels. however, establishment of parallel laboratories within a national health care system is neither cost-effective nor conducive to efficient and coordinated patient care. strategies for integrating laboratory systems and services top ↑ integration of laboratory systems and services at different levels of the health system will ensure optimisation of the investments in laboratory-system strengthening by ministries of health, as well as those of international donors, such as the global fund to fight aids, tb, and malaria, the world bank, the us president’s emergency plan for aids relief (pepfar), and others. in the past, donor funding mechanisms have contributed to the fragmentation of laboratory capacities because they usually have been earmarked for disease specific efforts; although, recently, there is a trend towards greater flexibility. various strategies for integration are outlined in the subsections below. policy and strategic planning national laboratory systems (ideally composed of a tiered network of diagnostic laboratories and a national public health reference laboratory) must be capable of providing accurate, timely and cost-effective testing that is in line with each country’s programmatic goals and available clinical interventions. it is necessary to define at which level of a national laboratory network certain services or diagnostic platforms should be performed. these decisions should be based on testing complexity, throughput, specimen referral requirements and the needs of the public health programme and patient population being served. laboratories are complex systems that include components such as personnel, equipment, supplies and infrastructure, as well as support systems for information management, purchasing and inventory, and systems for evaluation and continuous improvement, such as internal and external quality assessment and occurrence management. developing and implementing a national laboratory policy and strategic plan that ensures integrated capacity at each level of the network can enable countries to work with partners and donors in defining specific objectives, setting standards and allocating appropriate funding and personnel for sustainable laboratory services.8 standardising testing and equipment and coordinating with partners laboratory infrastructure, test menus, technology, platforms and commodities should be standardised in each country to avoid duplication, as agreed upon by programme and laboratory representatives from 13 resource-poor countries in the maputo declaration of 2008.9 this approach requires strong leadership and coordination by the local ministries, along with partner and donor compliance, and includes many benefits, such as reduced procurement costs for commodities, easier implementation of quality assurance programmes and integration of multi-focused testing that uses shared equipment. for example, partners could work together in developing specialised facilities that would accommodate instruments for molecular diagnostics of tb, hiv and other diseases, rather than building separate labs dedicated to only one disease. training, equipment maintenance and quality management systems could be standardised to allow technicians to work using similar techniques across diseases. joint coordination amongst partners at the onset of a laboratory-related project could enable each partner to play a preferred role in areas where they have a relative advantage or expertise. joint coordination with clinicians strengthening of national laboratory systems depends on close partnerships – beyond the laboratory facility itself – with technical and clinical professionals, healthcare managers at the community, regional and national levels, and public health programmes. clinicians should support, facilitate and demand high-quality and responsive laboratory support for appropriate patient care. clinicians should also be involved in ensuring that testing algorithms are cost-effective, are based on sound evidence and have a relevant impact on clinical decision-making and patient-important outcomes.10 implementation there are two basic aspects to implementing integrated services within laboratories. the first is focused on provision of adequate services for patients presenting with particular clinical symptoms indicative of major infectious diseases. for example, an integrated laboratory should have the capacity to adequately monitor hiv-positive individuals for tb, malaria, or other opportunistic infections, or to provide rapid molecular testing for multi-drugresistant tb (mdr tb) in hiv and tb co-infected patients in order to improve infection control and treatment outcomes (figure 1). conversely, this laboratory should also be able to screen specimens from tb and malaria patients for hiv and provide other routine monitoring (clinical chemistry and haematology before and during treatment for hiv, tb, and malaria) when required. the second aspect focuses on the establishment of integrated diagnostic platforms or instruments that can be used for a variety of tests. the simplest example can be an integrated system for collecting and transporting specimens for tb and hiv testing or monitoring. a further step can be the integration of microscopy testing for tb, malaria and other parasitic pathogens. the most promising approach for cross-cutting disease testing is offered by the molecular platform. polymerase chain reaction (pcr) molecular testing can be used to detect rapidly and identify a wide range of viral and bacterial pathogens, including those that cause endemic diseases such as hiv, tb and malaria, and emerging infectious diseases such as new strains of influenza. practical examples of integrated laboratory capacity in the field top ↑ integration of tb laboratory capacity in hiv laboratories in 2006, a state of the art bio-safety level (bsl)-2 central laboratory was established at the ethiopian health and nutrition research institute (ehnri) in addis ababa through pepfar funding to support the laboratory diagnosis of hiv patients. since then, technical support has been provided by international partners to help establish testing and training capacity for hiv serology and external quality assessment (eqa), hiv incidence, cd4 counts, biochemistry, haematology and molecular testing for viral load and early infant diagnosis (eid). beginning in 2008, support was provided through the foundation for innovative new diagnostics (find) to create two bsl-3 tb testing laboratories in addis ababa: the first at the hiv laboratory at ehnri, to host the national reference laboratory for tuberculosis (nrlt), and the second at st. peter’s hospital, a central hospital responsible for the care of tb and hiv co-infected patients experiencing tb treatment failure or relapse. the testing capacity of the two laboratories now includes liquid and solid growth detection and drug susceptibility testing for tb, lateral-flow immuno-assay for identification of tb and rapid detection of mdr tb by the molecular line probe assay (lpa). the newly renovated molecular unit at st. peter’s will also be used to restart hiv viral load testing at the hospital, a function that had been stopped in the past because of problems related to inadequate infrastructure for molecular testing. in addition, molecular lpa testing for mdr tb will soon be introduced in four ethiopian regional laboratories that are currently performing hiv dna pcr for eid. in nigeria, a multifaceted, integrated, tiered laboratory programme has been established by the institute of human virology at the university of maryland to support a pepfar-funded scale-up of aids care and treatment in 26 states.11 services provided by the laboratory network include hiv rapid tests, adult cd4 counts, paediatric cd4 percentages, haematology, blood chemistries, syphilis serology, cryptococcal antigen test, tb smear microscopy, culture, drug susceptibility testing (dst), and molecular lpa. use of appropriate technology at all service levels and a robust quality assurance programme provide high quality integrated laboratory services. integration of hiv testing in a tb laboratory the national tb reference laboratory (ntrl) located at the queen elizabeth ii hospital in maseru, lesotho, was renovated by find and other partners to provide capacity for tb culture and molecular diagnostics. following strengthening of tb smear microscopy testing through training and the establishment of a quality assurance programme, the ntrl was first renovated to create a bsl-3 facility to implement tb culture, dst and rapid immunoassay-based identification of tb, with eqa provided from south africa.12 then tb lpa and hiv dna pcr were implemented in an adjacent, newly constructed clean-room facility, resulting in the establishment of integrated tb and hiv testing in a single facility. by using an integrated approach, implementation of novel molecular tb diagnostics paved the way to introduce hiv molecular testing capacity in the country. avian influenza laboratory support in hiv laboratories molecular hiv laboratories in africa have been called on to respond rapidly to laboratory surveillance for outbreaks of avian influenza. during the 2005 outbreak of avian influenza, the pepfar-supported global aids program laboratory in entebbe, uganda, provided training for about 60 laboratory experts on the use of pcr diagnosis for avian influenza. also in 2006, the pepfar-supported laboratory at asokoro hospital in abuja, nigeria, was instrumental in providing diagnostic support for an investigation of an avian influenza outbreak in nigeria.13 planning is ongoing for the provision of support for localised outbreaks of h1n1 (swine) influenza. figure 1: the framework of integrated laboratory services that addresses levels of a tiered laboratory network in developing countries. human resources training top ↑ the african centre for integrated laboratory training (acilt) was established in 2008 to develop and offer hands-on training courses for front-line laboratory staff. acilt’s vision is to provide for a healthier africa through quality laboratory practices to combat major infectious diseases. hosted by the south african national institute for communicable diseases and the national health laboratory service, acilt has a governance board consisting of experts from collaborating international institutions.before establishing acilt, needs assessments were performed in 10 resource-poor african countries to determine which topics would be most important for building capacity amongst laboratory personnel. courses were then developed collaboratively by acilt and international partners including the us centers for disease control and prevention (cdc), find and the world health organization to ensure standardisation. as of early august 2011, 720 participants from 29 countries had participated in more than 20 hands-on training courses covering: hiv dna pcr for eid, hiv incidence assay, tb culture and identification, eqa for hiv rapid testing, national laboratory strategic planning, biosafety, laboratory management, and laboratory accreditation. acilt satellite courses are offered at sites outside of south africa. a two-week course on tb culture and drug susceptibility testing was recently offered to south-east asian participants in bangkok and one-week courses on the molecular lpa testing for mdr tb have been hosted by the new laboratory facility at ehnri, at the national tb reference laboratory of lesotho, and at the institut pasteur in abidjan, côte d’ivoire. acilt tb culture and molecular lpa course materials have also been translated into french and portuguese through a partnership with the american society for microbiology. recommendations and conclusion top ↑ an integrated network of laboratories is indispensable for providing national support for global health initiatives for hiv, tb, malaria and other public health priorities. however, an integrated national laboratory network should be able to provide all needed primary diagnostic services for patients at each level of service without requiring patients to go to different facilities for specific tests. such laboratory services might be delivered either directly at defined levels of the network or indirectly by using an integrated specimen collection, referral and reporting system. the extent of integration at different levels of the laboratory system should always be based on the complexity, throughput and specimen referral requirements of the particular diagnostic platforms. last but not least, an integrated system allows increased capacity and preparedness for implementation of new technologies to improve the quality of current diagnostics and to address newly emerging public health concerns. this article has presented examples to demonstrate how some resource-limited countries have benefited from an integrated approach for clinical laboratory strengthening. these strategies could be replicated more broadly. acknowledgements top ↑ use of trade names is for identification only and does not constitute endorsement by the us department of health and human services, the public health service, or the cdc. financial support has been received from the global aids program of the national center for hiv, sexually transmitted diseases (std), and tb prevention at the cdc. the findings and conclusions in this article are those of the authors and do not necessarily represent the views of the funding agency. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. authors’ contributions j.n. (us centers for disease control and prevention) was the project leader. j.n. l.m.p. (us centers for disease control and prevention) a.s., (foundation for innovative new diagnostics) e.l. (foundation for innovative new diagnostics) c.n.p., (foundation for innovative new diagnostics) m.s., (the world bank) d.b.(us centers for disease control and prevention) and g.r. foundation for innovative new diagnostics) were responsible for experimental design and wrote the manuscript. references top ↑ 1. vittoria m. granich r. gilks cf, et al. the global fight against hiv/aids, tuberculosis and malaria. am j clin pathol. 2009;131:844–848. http://dx.doi.org/10.1309/ajcp5xhdb1pnaeyt, pmid:19461091 2. birx d, desouza m, nkengasong jn. laboratory challenges in the scaling up of hiv, tb and malaria programs. am j clin pathol. 2009;131:849–851. http://dx.doi.org/10.1309/ajcpgh89qdswfons, pmid:19461092 3. petti ca, polage cr, quinn tc, ronald ar, sande ma. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006;42:377–382. http://dx.doi.org/10.1086/499363, pmid:16392084 4. frieden tm, teklehaimanot a, chideya s, farmer p, kim jy, raviglione mc. a roadmap to control malaria, tuberculosis and human immunodeficiency virus/aids. arch intern med. 2009;169:1650–1652. http://dx.doi.org/10.1001/archinternmed.2009.309, pmid:19822819 5. marchal b, cavalli a, kegels g. global health actors claim to support health system strengthening – is this reality or rhetoric? plos med. 2009;6:1–5. 6. ravishankar n, gubbins p, cooley rj, et al. financing of global health: tracking development assistance for health from 1990 to 2007. lancet. 2009;373:2113–2124. http://dx.doi.org/10.1016/s0140-6736(09)60881-3 7. yu d, souteyrand y, banda ma, kaufman j, perriëns jh. investment in hiv/aids programs: does it help strengthen health systems in developing countries? global health. 2008;4:8. http://dx.doi.org/10.1186/1744-8603-4-8, pmid:18796148, pmcid:2556650 8. nkengasong jn, mesele t, orloff s, et al. critical role of developing national strategic plans as a guide to strengthen laboratory health systems in resource-poor settings. am j clin pathol. 2009;131:852–857. http://dx.doi.org/10.1309/ajcpc51blobbpakc, pmid:19461093 9. world health organization. the maputo declaration on strengthening of laboratory systems [document on the internet]. c.2008 [cited 2009 nov 16]. maputo: world health organization; 2008. available from: http://www.who.int/diagnostics_laboratory/maputo-declaration_2008.pdf 10. pai m, ramsay a, o’brian r. comprehensive new resource for evidence-based tb diagnosis. expert rev mol diagn. 2009;9:637–639. http://dx.doi.org/10.1586/erm.09.48, pmid:19817547 11. abimiku ag. building laboratory infrastructure to support scale-up of hiv/aids treatment, care and prevention. am j clin pathol. 2009;131:875–886. http://dx.doi.org/10.1309/ajcpelmg6gx6rqsm, pmid:19461097 12. paramasivan cn, lee e, kao k, et al. experience establishing tuberculosis laboratory capacity in a developing country setting. int j tuberc lung dis. 2009;14:59–54. 13. ortiz jr, katz ma, mahmoud mn, et al. lack of evidence of avian-to-human transmission of avian influenza a (h5n1) virus among poultry workers, kano, nigeria, 2006. j infect dis. 2007;196:1685–1691. http://dx.doi.org/10.1086/522158, pmid:18008254 abstract introduction methods results discussion acknowledgements references about the author(s) kome otokunefor department of microbiology, faculty of science, university of port harcourt, port harcourt, rivers state, nigeria tosanwumi v. otokunefor department of microbiology, faculty of science, university of port harcourt, port harcourt, rivers state, nigeria godwin omakwele department of microbiology, faculty of science, university of port harcourt, port harcourt, rivers state, nigeria citation otokunefor k, otokunefor tv, omakwele g. multi-drug resistant mycobacterium tuberculosis in port harcourt, nigeria. afr j lab med. 2018;7(2), a805. https://doi.org/10.4102/ajlm.v7i2.805 original research multi-drug resistant mycobacterium tuberculosis in port harcourt, nigeria kome otokunefor, tosanwumi v. otokunefor, godwin omakwele received: 21 mar. 2018; accepted: 14 sept 2018; published: 06 dec. 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: in past years, much focus has been on tackling the scourge and spread of tuberculosis worldwide. the recent emergence of multi-drug resistant (mdr) tuberculosis has, however, negatively threatened progress made so far. nigeria ranks fourth out of the 22 high tuberculosis burden countries in the world and has the highest burden of tuberculosis in africa. it is therefore necessary to monitor the mdr tuberculosis situation in the country. objectives: this study set out to assess the proportions of mdr tuberculosis in patients attending six directly observed treatment short-course centres in port harcourt, nigeria, from october 2015 to october 2016. methods: six hundred and nine participants between the ages of 18 and 75 years were enrolled in this study and comprised suspected and newly diagnosed tuberculosis cases. sputum samples obtained from the participants were screened for the presence of mycobacterium tuberculosis using standard culture and phenotypic biochemical techniques, and drug susceptibility testing was carried out using the 1% proportion conventional method. results: of the 609 participants enrolled, 30 (4.9%) were confirmed as m. tuberculosis-positive cases. a high prevalence of drug resistant tuberculosis was noted in this study (14/30, 46.7%), with 26.7% of isolates resistant to streptomycin. mdr tuberculosis, defined as being resistant to isoniazid and rifampicin, was detected in only one case (3.3%). conclusion: this study reports a low rate of mdr tuberculosis and contributes to the sparse data on drug resistant tuberculosis in nigeria. introduction tuberculosis, caused by mycobacterium tuberculosis, is still a major public health issue worldwide. this disease is particularly problematic in low and middle-income countries, where it is a major contributor to morbidity and mortality.1 of the 1 million people killed by tuberculosis each year, most are from these low and middle-income areas, with sub-saharan africa known to have the highest tuberculosis incidence.2 the problem of tuberculosis has been compounded in recent years by the emergence of multi-drug resistant (mdr) strains. this phenomenon has been increasingly observed round the globe. in 2015, the world health organization (who) noted about 480 000 cases of mdr tuberculosis globally,3 which accounts for about 5% of all global tuberculosis cases.4 the issue of mdr tuberculosis is particularly worrisome, because of its possible negative effect on treatment outcomes (such as treatment failures) and treatment options, with fewer drugs effective against the disease. the current who guidelines for the treatment of mdr tuberculosis include the use of at least five different drugs and a treatment time of up to 24 months.5 despite this, a high mortality rate is associated with mdr tuberculosis. one key step in the prevention of drug resistance is continuous monitoring of the situation through systematic surveillance and drug resistance testing. in a who 2016 publication, nigeria was shown to rank fourth out of the 22 high tuberculosis burden countries worldwide and has the highest burden of tuberculosis in africa. it is also one of 10 countries that account for 77% of the difference between who estimation and actual notifications due to underreporting and underdiagnosis.3 therefore, it is essential to monitor the mdr tuberculosis situation in the country. in the absence of the roll-out of nationwide tuberculosis data, current information is usually obtained from regional studies. two 2017 reviews analysing drug resistance and multi-drug resistance in nigeria and sub-saharan africa highlighted a gap in knowledge.6,7 of the papers included in these reviews, none reported on mdr tuberculosis in rivers state, nigeria. therefore, this study set out to assess the proportion of mdr tuberculosis in patients attending some directly observed treatment short-course centres in port harcourt, rivers state, nigeria. methods ethical considerations this study was approved by the rivers state ministry of health, port harcourt, rivers state, nigeria (study approval number: mh/prs/391/vol.2/385). informed verbal consent was obtained from all participants and names were omitted to protect participants’ privacy. study population participants between the ages of 18 and 75 years attending six randomly selected directly observed treatment short-course clinics and centres in port harcourt, rivers state, nigeria, were enrolled in this study from october 2015 to october 2016. these participants comprised people with suspected or presumptive tuberculosis and those recently diagnosed with less than one month duration of tuberculosis therapy. known tuberculosis patients on therapy for more than one month were excluded from this study. sample collection and processing three sputum samples of 5 ml volume were obtained from each participant. these samples were screened for the presence of m. tuberculosis using standard microscopic, culture and phenotypic biochemical techniques. preliminary detection for acid fast bacilli was carried out by direct microscopic examination following ziehl-neelsen staining using a standard protocol. next, sputum samples were processed for culture using the petroff’s method8 and culture was carried out on lowenstein-jensen agar slants. the set-up was incubated at 37 °c and inspected for characteristic growth weekly, for up to six weeks. phenotypic biochemical identification was then carried out based on catalase enzyme production, nitrate reductase and niacin production. drug susceptibility testing drug susceptibility testing was carried out using the lowenstein-jensen proportion method.9,10 this involved the use of critical drug concentrations of 0.2 µg/ml for isoniazid, 40 µg/ml for rifampicin, 2 µg/ml for ethambutol and 4 µg/ml for streptomycin. following a 28-day incubation, isolates were documented as resistant if a growth rate exceeding 1% of the control was observed. multi-drug resistance was defined as resistance to isoniazid and rifampicin. results a total of 609 participants were enrolled in this study, the majority of which were male (399, 65.5%). results of the direct microscopy showed that 26 of the participants were smear-positive for acid fast bacilli. however, based on culture and biochemical testing, 30 (4.9%) participants were confirmed as m. tuberculosis-positive cases. the rate of tuberculosis was slightly higher among women than among men (table 1). table 1: gender-based distribution of culture positive mycobacterium tuberculosis. in this study, 14 isolates (46.7%) were found to be resistant to at least one of the first-line drugs (isoniazid, rifampicin, ethambutol and streptomycin). the highest level of resistance (26.7%) was noted against streptomycin, while the lowest level (10%) was noted against rifampicin (figure 1). figure 1: rates of resistance to first-line tuberculosis drugs. in total, 11 isolates (36.7%) were mono-resistant and two isolates were resistant to three of the tuberculosis drugs tested (table 2). mdr tuberculosis was only detected in one case (3.3%). in total, seven different susceptibility profiles were identified in this study. table 2: drug resistance patterns of mycobacterium tuberculosis isolates. discussion with the high tuberculosis burden associated with nigeria, it could be assumed that a higher burden of mdr tuberculosis would also be reported. however, at present, data on mdr tuberculosis in nigeria are sparse. monitoring of this situation is essential for developing and analysing control strategies. varying rates of mdr tuberculosis have been reported worldwide. these have ranged from very low rates of 0.2% reported from japan,11 to mdr tuberculosis rates as high as 69% reported from pakistan.12 in china, which at 2017 was noted as having the second highest mdr tuberculosis burden,13 a study that same year reported a 10.1% total mdr tuberculosis prevalence.14 in addition to country to country variations, these rates could vary even within regions. the national indian mdr tuberculosis rate at 2010 was 2.1%, but a 2013 study analysing east delhi reported a 1.3% prevalence rate,15 while a 15.1% rate was reported among a tribe with a known high tuberculosis prevalence.16 other reported mdr tuberculosis rates from south east asia include 15.6% from nepal.17 african countries are not among the top three countries associated with over half the global burden of mdr tuberculosis.18 the mdr tuberculosis rates from this region have varied, with a range of rates reported: 4.5% from chad,19 12.0% from benin,20 5.1% from mozambique,21 18% from cameroon,22 11.5% from djibouti,23 and 1.2% and 3.37% from ethiopia,24,25 which are generally higher than the rate observed in this study. in addition to country and regional variations, the rate of mdr tuberculosis is also related to exposure of patients to tuberculosis drugs. higher rates of mdr tuberculosis are generally reported in retreatment cases as opposed to newly diagnosed cases.14,17,25,26 findings of this current study revealed a 3.3% mdr tuberculosis rate. this figure is lower but similar to the current reported nigerian mdr tuberculosis who data, which estimates a 4.3% rate among new cases.7,27 this low rate is encouraging, as a high level of prevalence would constitute a major public health issue. some previous studies assessing rates of mdr tuberculosis in nigeria had reported higher rates of 6.9%, 8%, 10.4% and 10.6% from awka, three different cities, cross rivers and kano respectively and extremely higher rates of 53.6%.28,29,30,31,32 others reported fairly similar rates of 4% from calabar and abuja, and 5.2% from cross rivers.33,34,35 with respect specifically to mdr tuberculosis rates in new cases, results of this study were quite similar to previous reported rates of 3%,32 4%33 and 5.2%,35 but lower than the pooled rate of 6.0% reported in a recent review on drug resistant tuberculosis in nigeria.7 despite the relatively low rate of mdr tuberculosis noted in this study, a 46.7% resistance rate against any of the first-line drugs was noted. this value was higher than a 2018 ethiopian report of a 23.3% resistance rate against any of the first-line drugs.24 it was, however, similar to reports from nearby benin of a 40% rate,20 as well as rates of 32.3% and 42% reported from nigeria.30,33 such a level of drug resistant tuberculosis in this region could complicate patient management by reducing treatment options. generally, the lowest rates of resistance have been reported against rifampicin,19,25,33 and this was also the finding in this study, with 10% of isolates resistant to rifampicin. the rates of resistance noted against the various drugs in this study (ethambutol, 20%; isoniazid, 13.3%; rifampicin, 10%; streptomycin, 26.7%) are similar to previous reports from nigeria. the highest level of resistance (26.7%) was noted against streptomycin. this trend has been reported by several other studies assessing drug resistance in tuberculosis cases.17,22,25,35 in the study by otu and colleagues, 47.6% resistance was noted against streptomycin,33 while pokam and colleagues35 noted a 27.6% resistance rate against streptomycin. this is thought to be linked with the fact that streptomycin is the oldest tuberculosis drug in use.36 while the use of streptomycin in clinical practice is supposed to be limited, this antibiotic is used both as a growth promoter and in therapy in both plant and animal farming.37 the development of resistance to this drug in bacteria of clinical importance in non-clinical settings following such use has been described,38 and such resistance has further been reported in bacteria of human origin.39,40 these may then have the potential to serve as a reservoir for further spread in the clinical setting. despite the high resistance to streptomycin, some studies from cameroon, ethiopia, and nepal have reported lower resistance rates of 6.0%, 7.87%, 24.4%, respectively, to streptomycin.17,22,25 one of these studies postulated that the lower rate of resistance against streptomycin could be linked with the fact that this drug is no longer a first-line drug for the treatment of tuberculosis in the test region.25 conclusion results of this study show a high level of drug resistance (46.7%) and low level of mdr (3.3%) tuberculosis. the high level of drug resistance is particularly worrisome considering that the test population comprised new cases. this study additionally contributes to the sparse data on drug-resistant tuberculosis in nigeria, which is necessary for the development of control measures to curb the spread of drug resistant tuberculosis. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. sources of support none authors’ contributions t.v.o. was the project leader. g.o. and k.o. were responsible for project and experimental design. g.o. carried out most of the laboratory investigations. k.o. managed the analyses of the data, managed the literature searches, and wrote the first draft of the manuscript. all authors read and approved the final manuscript. references gbd tuberculosis collaborators. the global burden of tuberculosis: results from the global burden of disease study 2015. lancet infect dis. 2017;18(3):261–284. https://doi.org/10.1016/s1473-3099(17)30703 glaziou p, sismanidis c, floyd k, raviglione m. global epidemiology of tuberculosis. cold spring harb perspect med. 2015;5(2):a017798. 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tuberculosis patients in and around ambo town, central ethiopia. plos one. 2018;13(2):e0193083. https://doi.org/10.1371/journal.pone.0193083 adane k, ameni g, bekele s, abebe m, aseffa a. prevalence and drug resistance profile of mycobacterium tuberculosis isolated from pulmonary tuberculosis patients attending two public hospitals in east gojjam zone, northwest ethiopia. bmc public health. 2015;15(1):572. https://doi.org/10.1186/s12889-015-1933-9 musa bm, adamu al, galadanci na, zubayr b, odoh cn, aliyu mh. trends in prevalence of multi drug resistant tuberculosis in sub-saharan africa: a systematic review and meta-analysis. plos one. 2017;12(9):e0185105. https://doi.org/10.1371/journal.pone.0185105 world health organization. multi-drug resistance tuberculosis (mdr-tb) 2013 update 2013. geneva: world health organization. kehinde ao, obaseki fa, ishola oc, ibrahim kd. multidrug resistance to mycobacterium tuberculosis in a tertiary hospital. j national med assoc. 2007;99(10):1185. okonkwo rc, onwunzo mc, chukwuka cp, et al. the use of the gene xpert mycobacterium tuberculosis/rifampicin (mtb/rif) assay in detection of multi-drug resistant tuberculosis (mdrtb) in nnamdi azikiwe university teaching hospital, nnewi, nigeria. j hiv retro virus. 2017;3(1). lawson l, yassin ma, abdurrahman st, et al. resistance to first-line tuberculosis drugs in three cities of nigeria. trop med int health. 2011;16(8):974–980. https://doi.org/10.1111/j.1365-3156.2011.02792.x thumamo bp, asuquo ae, abia-bassey ln, et al. molecular epidemiology and genetic diversity of mycobacterium tuberculosis complex in the cross river state, nigeria. inf genet evol. 2012;12(4):671–677. https://doi.org/10.1016/j.meegid.2011.08.011 adamu au, hafiz tr. multi-drug resistant tuberculosis pattern in kano metropolis, nigeria. j am sci. 2015;11:293–296. otu a, umoh v, habib a, ameh s, lawson l, ansa v. drug resistance among pulmonary tuberculosis patients in calabar, nigeria. pulm med. 2013;2013:235190. https://doi.org/10.1155/2013/235190 dosumu ea, osagie k, shuaib a, lawson l. multidrug resistant tuberculosis at the national hospital, abuja nigeria. afr j respir med. 2008;4(1):22–23. pokam bt, asuquo ae, abia-bassey ln, et al. multidrug resistance and demography of newly diagnosed tuberculosis patients in cross river state, nigeria. int j mycobact. 2013;2(2):89–93. https://doi.org/10.1016/j.ijmyco.2013.03.002 keshavjee s, farmer pe. tuberculosis, drug resistance, and the history of modern medicine. nejm. 2012;367(10):931–936. https://doi.org/10.1056/nejmra1205429 pezzella c, ricci a, digiannatale e, luzzi i, carattoli a. tetracycline and streptomycin resistance genes, transposons, and plasmids in salmonella enterica isolates from animals in italy. antimicrob agents chemother. 2004;48(3):903–908. https://doi.org/10.1128/aac.48.3.903-908.2004 scherer a, vogt hr, vilei em, frey j, perreten v. enhanced antibiotic multi-resistance in nasal and faecal bacteria after agricultural use of streptomycin. environ microbiol. 2013;15(1):297–304. https://doi.org/10.1111/1462-2920.12028 sundin gw, bender cl. dissemination of the stra-strb streptomycin-resistance genes among commensal and pathogenic bacteria from humans, animals, and plants. mol ecol. 1996;5(1):133–143. https://doi.org/10.1111/j.1365-294x.1996.tb00299.x barreto â, guimarães b, radhouani h, et al. detection of antibiotic resistant e. coli and enterococcus spp. in stool of healthy growing children in portugal. j basic microb. 2009;49(6):503–512. https://doi.org/10.1002/jobm.200900124 abstract introduction methods results discussion acknowledgements references about the author(s) wanda s. tshabalala department of internal medicine, grey’s hospital, university of kwazulu-natal, pietermaritzburg, south africa somasundram pillay department of internal medicine, edendale hospital, pietermaritzburg complex, university of kwazulu-natal, pietermaritzburg, south africa douglas p.k. wilson department of internal medicine, edendale hospital, pietermaritzburg complex, university of kwazulu-natal, pietermaritzburg, south africa citation tshabalala ws, pillay s, wilson dpk. diagnostic outcomes of bone marrow aspirate and trephine biopsies performed at a hospital in kwazulu-natal, south africa. afr j lab med. 2020;9(1), a1028. https://doi.org/10.4102/ajlm.v9i1.1028 original research diagnostic outcomes of bone marrow aspirate and trephine biopsies performed at a hospital in kwazulu-natal, south africa wanda s. tshabalala, somasundram pillay, douglas p.k. wilson received: 12 apr. 2019; accepted: 26 nov. 2019; published: 25 feb. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: bone marrow aspiration and trephine biopsy (bmat) are widely performed in adults to evaluate haematological and malignant conditions. however, the diagnostic yield from the procedure in unselected patients in the south african public sector has not previously been described. objectives: we identified the main indications and most common diagnoses encountered for bmat and described the demographic and blood profiles of patients, including hiv-positive patients, who had undergone the procedure at a tertiary hospital in kwazulu-natal. methods: we retrospectively reviewed laboratory data from january 2016 to december 2016 for all patients aged ≥ 13 years who underwent the procedure and stratified findings by demographic data. results: among 120 bmat biopsies studied, 80 (67%) cases were performed to evaluate suspected malignancy and a further 40 (33%) cases for non-malignant indications. the main indications for bone marrow examination were: cytopenias 38 (32%), lymphoma 35 (29%), leukaemia 21 (18%), and multiple myeloma 17 (14%). bmat results revealed that 60 cases (50%) were malignant in origin, 30 cases (25%) were non-malignant and 30 cases (25%) were classified as normal. the common diagnoses were: leukaemia, 24 (20%); multiple myeloma, 16 (13%) and lymphoma, 13 (11%). cases aged ≥ 50 years were more likely to have a malignant diagnosis (odds ratio: 5.8 (95% confidence interval: 2.2–17.1) p-value < 0.001). conclusion: the diagnostic yield of bmat was high, with significant abnormalities detected in three quarters of cases. haematological malignancy was the more common diagnosis. increasing age was associated with an increase in reporting of haematology malignancy. keywords: age; human immune virus; indications; outcomes; haematological malignant diseases; diffuse large b cell lymphoma; bone marrow aspirate and trephine biopsy. introduction bone marrow examination may provide vital information in the diagnosis and monitoring of haematological disease.1 bone marrow examination can be very effective when it is utilised in conjunction with a good clinical assessment. in some cases, diagnosis cannot be reached solely on an assessment of peripheral blood samples alone, and in these situations examination of the bone marrow is crucial.2 several south african studies have evaluated outcomes from the procedure in selected cohorts with hiv, tuberculosis or lymphoma. karsteadt et al., in their study examining the benefits of bone marrow examination in hiv-positive adults, showed that a low cd4 count (< 100 cells/μl) had a higher diagnostic yield in granulomatous disease of the bone marrow.3 in 2014, naidoo et al. studied the outcome of histological comparison to cytology in antiretroviral-therapy-naïve vs. antiretroviral-therapy-experienced hiv-positive patients presenting with peripheral blood cytopenias and found higher rates of granulomatous disease and pure red cell aplasia in antiretroviral-therapy-naïve patients.4 more recently in 2018, in a study conducted by philips and opie to assess the use of bone marrow examination in lymphoma sufferers in south africa, it was shown that out of the 1215 bone marrow aspiration and trephine (bmat) records, bone marrow involvement was most commonly present in non-hodgkin’s lymphoma (43.7%), followed by high-grade b subtypes (28.9%) and hodgkin’s lymphoma (35.7%).5 more than 200 bone marrow examinations are performed annually by our institution. a bone marrow examination is a crucial test that comes at a cost to the health institution. in the interest of healthcare cost effectiveness measures, we avoid performing bmat for treatable nutritional disorders with peripheral blood cytopenias. serum tests to confirm and assess for nutritional deficiencies are freely available at our institution. patients with cytopenias are therefore routinely screened for nutritional deficiencies such as vitamin b12 and folate deficiency, which can mimic leukaemia and had been reported in literature case studies.6 studies have demonstrated that the most common reasons for bone marrow examination were for diagnoses of haematological malignancies.7,8 the spectrum of haematological disease at our institution has not been previously studied and reported. we therefore undertook a retrospective analysis of sequential bmat biopsies performed over a 12-month period to evaluate the indications for and diagnostic yield from the procedure. methods ethical considerations ethical clearance was obtained from the biomedical research ethics committee, university of kwazulu-natal (ethical clearance number: be465/17). study site and design the haematology unit at grey’s hospital, pietermaritzburg, south africa, offers tertiary inpatient and outpatient services to the western half of kwazulu-natal and performs all the adult bmat procedures for the institution. we performed a retrospective cross-sectional study on results from bmat performed at grey’s hospital on individuals older than age 13 years from 01 january 2016 to 31 december 2016. data collection the following clinical, demographic, and biochemical parameters were extracted from the bmat records: age; sex; hiv status; cluster of differentiation 4 (cd4) lymphocyte count cells/µl; indication for bmat; final diagnostic outcome of bmat; bmat quality and trephine size; white cell count cells × 109/l; haemoglobin grams/dl; platelet count cells × 109. bone marrow and trephine sampling and definition we retrospectively analysed results from bmat performed by physicians at our institution’s clinical haematology unit during routine patient care. the national health laboratory service processed these biopsy specimens in accordance with the recommendations of the world health organization.9 the world health organization recommends a minimum of 1.5 cm length for a size of trephine core biopsy as adequate assessment.9 bmat results were included if: (1) patient was older than age 13 years at the time of the biopsy; and (2) bmat was reported to have adequate aspirate or trephine quality to reach final diagnoses. statistical analysis analyses were performed using analyse-it ultimate edition (analyse-it ltd, leeds, united kingdom). distributions of continuous data were determined using the shapiro wilk test and odds ratios with 95% confidence intervals for categorical data calculated using the fisher exact test. results a total of 204 cases were received from the national health laboratory service. one hundred and twenty cases met the inclusion criteria. eighty-four cases that did not meet the inclusion criteria included 68 cases excluded for being below the cut-off age of 13 years; 15 cases were excluded because they were out of the specified time range; and one case that had a poor trephine biopsy quality. seven of the 120 bmat cases had a poor bone marrow aspirate but had a good quality bone marrow trephine biopsy. these cases were treated as eligible, as a diagnosis could still be made on the information provided by the trephine biopsy. demographics malignant diagnoses were predominantly reported in all age groups older than age 30 years (figure 1). the study consisted of 63 (52.5%) male cases and 57 (47.5%) female cases with a median age of 40 years (interquartile range [iqr]: 30–60 years). age ≥ 50 years was associated with an increase in reporting of malignancy (odds ratio: 5.8; 95% confidence interval: 2.2–17.1; p < 0.001). figure 1: distribution of malignant and non-malignant conditions according to age groups of 120 bone marrow aspirates and trephine biopsies performed at a hospital in kwazulu-natal, south africa, january 2016 to december 2016. indications for performing a bone marrow aspirate and trephine biopsy the indications for bmat performed at our institution included 38 cases with cytopenias (32%), 35 with lymphoma (29%), 21 with leukaemia (18%), and 17 with multiple myeloma (14%) (table 1). table 1: indications for performing bone marrow aspirate and trephine biopsies on an unselected group of 120 patients at a hospital in kwazulu-natal, south africa, january 2016 to december 2016. indication of bone marrow aspirate and trephine biopsy among hiv-positive cases hiv-positive cases accounted for 46 (38%) of the 120 bmats reported with a median cd4 lymphocyte count of 241 cells/µl (iqr: 178.7–303.7). peripheral blood cytopenias and lymphomas were the common indications for bone marrow examination in the hiv-positive cohort (table 2). a diagnosis of diffuse large b cell lymphoma (dlbcl) made on a peripheral lymph node biopsy was an indication for bmat in 22 (47.8%) of 46 cases known to be hiv-positive. table 2: indications for bone marrow examination of 46† cases reported to be hiv-positive at a hospital in kwazulu-natal, south africa, january 2016 to december 2016. eighty-one percent (22/27) of the overall dlbcl cases were hiv-positive. less than 1% of the cases with dlbcl had bone marrow infiltration. sixty percent of hiv-positive cases with a diagnosis of dlbcl had a cd4 count ≥ 200 cells/µl. the median full blood count indices among hiv-positive cases were: white blood cell count, 6.43 × 109/l (iqr: 5.030–8.10); haemoglobin, 7.9 (iqr: 6.8–9.00) g/dl; and platelet count, 243.0 (iqr: 191.0–365.0) × 109/l. diagnostic outcomes of the bone marrow examination reported diagnoses for this study revealed 60 cases (50%) with malignancy, 30 cases (25%) were reported as non-malignant and a further 30 cases (25%) revealed a normal bone marrow (table 3). the most common diagnoses were (in descending order of frequency): 24 cases of leukaemia (20%); 16 of multiple myeloma (13%) and 13 of lymphoma (11%). acute leukaemia accounted for 46% (11/24 cases) of all diagnosed leukaemia cases when compared to chronic leukaemia. the median full blood count indices for leukaemia cases were: white cell count 93.00 × 109/l (iqr: 36.03–159.45); haemoglobin 7.3 g/dl (iqr: 7.00–11.30); and platelet count 130 × 109/l (iqr: 82.0–241.0). there were five cases reported to have granulomas, one case with a non-specific granuloma and a further four cases with tuberculosis granulomas. three of the cases diagnosed with tuberculosis granulomas were hiv-positive; all had cd4 counts less than 200 cells/µl. table 3: reported final diagnoses from bone marrow aspirate and trephine results of an unselected group of 120 patients at a hospital in kwazulu-natal, south africa, january 2016 to december 2016. discussion bone marrow aspirate and trephine biopsies in our institution were performed for a wide array of haematological conditions ranging from highly aggressive to non-benign malignancies. our findings were similar to some international studies conducted in nigeria, nepal, saudi arabia, iran, india, and ghana, all of which had reported haematological malignant disease as a common indication and outcome for a bone marrow examination.7,8,10,11,12,13 in our study there were 30 normal bone marrow results with the majority of these cases being bmat performed as part of the staging process for treatment of malignancies. since south africa is a country known to have a high prevalence of hiv infection, the use of bmat in the diagnosis of tuberculosis is not standard practise at our institution. we have access to diagnostic tools such as gene xpert and tuberculosis culture, which can be used in testing various body fluids of individuals with suspected tuberculosis infection, as well as urine lipoarabinomannan, which can assist with early diagnosis of disseminated tuberculosis. therefore, many patients with tuberculosis would have been diagnosed before bmat was considered by the attending clinicians. for this reason our study reported low rates (4/120) of tuberculosis diagnosis on bmat when compared to other south african studies that demonstrated higher tuberculosis diagnoses on bmat samples.4,14 these studies included: a study that looked at 410 case samples of adult and paediatric bmat submitted for excluding tuberculosis, which showed a 32.5% tuberculosis prevalence, and a study that assessed bone marrow morphology in 74 patients, in which comparing morphology between patients on antiretroviral therapy and patients naïve to antiretroviral therapy showed a 31% tuberculosis prevalance.4,14 both of these studies had higher tuberculosis prevalence due to their study populations being cases with high risk for tuberculosis. workup for cases presenting with cytopenias formed a great majority of bmat cases in our study. these results are similar to studies done to assess the use of bmat in hospitals in pakistan and india.15,16 cytopenias are well documented, multifactorial complications of hiv infection.4,5 our study demonstrated that 46% of hiv-positive participants had one or more forms of cytopenia: anaemia, pancytopenia, bicytopenia and thrombocytopenia. a study conducted in uganda demonstrated that 65% of antiretroviral-therapy-naïve hiv-positive participants had a form of cytopenia.17 we could not draw any conclusion with this result, as we had not assessed the status of antiretroviral therapy use by participants. age is known to be one of the important risk factors for developing haematological malignancy. in our study increasing age was associated with an increased prevalence of malignancy. the age group older than age 70 years had the highest frequency of malignancy when compared to the younger age study population groups for this study. this outcome is similar to other global studies that have assessed increasing age as a risk factor for developing malignancy.18,19,20 a study of lymphoid neoplasms in the united states demonstrated that the incidence of haematological malignancy in cases older than age 75 years increased by approximately 1.4% for dlbcl and 1.8% for follicular lymphoma per year.19 dlbcl is associated with high rates of hiv infection among cases.21 our hiv and dlbcl burdens are in keeping with the current rates in most countries throughout the world.4,21 hiv infection was highly prevalent at 81% among the cases that had undergone bone marrow examination for the staging of the dlbcl. this finding about prevalence of hiv infection in cases with dlbcl was similar to results of a study done at a south african hospital in 2016 which reported the prevalence of hiv as 81% in 139 patients with dlbcl.22 sixty percent of cases of dlbcl in our study had a cd4 lymphocyte count ≥ 200 cell/µl. the median cd4 count for our dlbcl group was 259 (iqr: 131.2–351.6) cells/µl. this outcome demonstrates that hiv infection placed patients at a higher risk for developing this malignancy and occurrence of the malignancy did not depend on low cd4 count. in the united states, a report on 41 patients diagnosed with dlbcl concluded that the blood cd4 level was a lesser factor in predicting the occurrence of dlbcl.23 the median cd4 count of their study was 400 cells/µl. it has been reported that a haemoglobin value of less than 10 g/dl and white cell count less than 4 × 109 significantly increases the likelihood of bone marrow involvement in non-hodgkin’s lymphoma.24 about 99% of cases reported for dlbcl in our study had no bone marrow infiltration. a large percentage (72%) of our cases without an infiltration had anaemia with a mean haemoglobin of 9.92 g/dl, a mean normal white cell count 9.91 × 109/l and mean platelet counts of 340 × 109/l. other reasons for the development of anaemia, such as: myelosupression secondary to chemotherapy, anaemia associated with chronic disease, autoimmune haemolytic anaemia, iron deficiency and tumour infiltration of bone marrow, need to be explored in patients with dlbcl to identify a potential aetiology.25 the majority of the bmats of cases with dlbcl were done as part of treatment planning. our hospital uses computerized tomography (ct) scanning and bone marrow examination for staging malignancy. our access to flourine-18-flourodeoxyglucose position emission tomography (pet) scanning is limited in our setting. pet scanning has been recommended in the literature as a superior staging modality for bone marrow involvement in lymphoma sufferers.26 medical literature has documented the limitations of bone marrow examination in patients with aggressive dlbcl and early lymphoma.26 other studies suggest that pet/ct scanning is more useful in assessing the involvement of the bone marrow.26,27 improved access to pet/ct scanning would reduce the frequencies of bone marrow examinations in lymphoma sufferers, as most studies suggest that it should be considered as a first-line examination of bone marrow in lymphoma patients.26,27 bone marrow infiltration in dlbcl can easily be missed as most dlbcl infiltration are focal rather than diffuse.26 pet/ct has a much more superior sensitivity (94% vs. 24%) and a higher negative predictive value (98% vs. 80%) when compared to a bone marrow examination.27 in our study there was only one case reported as nutritional deficiency and that was megaloblastic anaemia due to folate deficiency. our findings are in line with the current practice in south africa, which states that bone marrow examination is done to prove another diagnosis other than confirming nutritional deficiencies. in our current limited resource situation, the practice is to correct reversible factors such as nutritional disorders having a haematological impact first before subjecting patients to more expensive and invasive investigations. in the south african health system, both in the private and public sector, there is easy access to testing for nutritional deficiencies without having first to do bmat. this is possibly the reason why there was a paucity of bmat done in our study that showed nutritional deficiencies. we expect this trend to continue in our setting because of the required screening of nutritional deficiencies in our south african institutions. limitations as this was a retrospective study, we were not able to follow up on the end-point of the cases that had dlbcl or other malignancies nor were we able to assess the number of patients that were on antiretroviral medications. since the study site is a referral centre, the patients that are referred across come mostly with initial exclusion of a reversible cause for bone marrow failure. our results might also be influenced by the study population as it only represents patients who were treated by the haematologists with experience in such cases. recommendations based on the odds ratio of cases older than age 50 years in this study, we recommend treating physicians to have a low threshold for performing bmat in patients aged 50 years and older who present with any cytopenias, as this has shown an increased likelihood of a malignant diagnosis. we also recommend an early peripheral tissue biopsy in conjunction with bmat in hiv-positive individuals presenting with constitutional symptoms of malignancy, lymphadenopathy, or cytopenias for early identification of life-threatening malignancies. conclusion haematological malignant disease was the most common indication and outcome reported for bone marrow examination. the majority of bmat done for the staging of dlbcl did not reveal evidence of bone marrow infiltration. increasing age was found to be associated with reporting of haematological malignancies. nutritional disorders were rarely reported as an indication for bmat. acknowledgements the author would like to thank dr m.k. bizaare for initiating the concept of the study as well as the national health laboratory services staff, tinyiko ngobeni and dr y.l. naidoo for assistance in retrieving data competing interests the authors have declared that no competing interests exist. authors’ contributions w.s.t. contributed to the literature review, study protocol, ethics clearance and writing of the manuscript. s.p., assisted in the writing and editing of the manuscript and d.p.k.w. assisted with the writing of the manuscript and statistical analysis. sources of support no funding for this research project was received. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer views expressed in the article are by w.s.t. and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references burkhardt r, frisch b, bartl r. bone marrow biopsy in haematological disorders. j clin pathol. 1982;35(3):257–284. thelm h, diem h, haferlach t. colour atlas of hematology practical microscopic and clinical diagnosis. 2nd ed. stuttgart: thieme verlag, 2004; p. 20–27. karstaedt as, pantanowitz l, omar t, sonnendecker he, patel m. the utility of bone-marrow examination in hiv-infected adults in south africa. qjm. 2001;94(2):101–105. https://doi.org/10.1093/qjmed/94.2.101 naidoo s, naicker vl. retrospective comparison of cytological and histological bone marrow morphology in adult antiretroviral-naïve and antiretroviral experienced human immunodeficiency virus-infected patients with peripheral blood cytopaenias. s afr j infect dis. 2016;31(2):50–56. https://doi.org/10.1080/23120053.2016.1128147 phillips l, opie j. the utility of bone marrow sampling in the diagnosis and staging of lymphoma in south africa. int j lab hematol. 2018;40(3):276–283. https://doi.org/10.1111/ijlh.12782 cacoub p, gatfosse m, derbel a, chapelon c, verny c, godeau p. vitamin deficiency-induced pancytopenia mimicking leukemia. three cases. presse med. 1991;20(33):1603–1606. dapus do, damen jg. diagnostic outcome of bone marrow aspiration in a new center in nigeria. glob adv res j med med sci. 2012;1(7):166–177. jha a, suyami g, adhikari rc, panta ad, jha r. bone marrow examination in cases of pancytopenia. j nepal med assoc. 2008;47(169):12–17. swerdlow sh, campo e, harris nl, et al. who classification of tumours of haematopoietic and lymphoid tissues. lyon: iarc; 2008. bashawri la. bone marrow examination indications and diagnostic values. saudi med j. 2002;23(2):191–196. mirzai az, hosseini n, sadeghipour a. indications and diagnostic utility of bone marrow examination in different bone marrow disorders in iran. lab hematol. 2009;15(4):38–44. https://doi.org/10.1532/lh96.09009 shastry sm, kolte ss. spectrum of hematological disorders observed in one-hundred and ten consecutive bone marrow aspirations and biopsies. med j dr. d.y. patil uni. 2012;5(2):118–121. https://doi.org/10.4103/0975-2870.103334 bedu-addo g, ampem amoako y, bates i. the role of bone marrow aspirate and trephine samples in haematological diagnoses in patients referred to a teaching hospital in ghana. ghana med j. 2013;47(2):74–78. sedick q, vaughan j, pheeha t, alli na. bone marrow aspirates microscopy v. bone marrow trephine biopsy microscopy for detection of mycobacterium tuberculosis infection. s afr med j. 2015;105(9):773–775. https://doi.org/10.7196/samjnew.8171 ahmad sq, khan ou, zafar n. utility of bone marrow examination in a secondary care hospital. jrmc. 2011;15(1):40–41. bashir n, musharaf b, reshi r, jeelani t, rafiq d, angmo d. bone marrow profile in 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https://doi.org/10.1046/j.1440-1827.2002.01308.x hicks l, cheung m, boro j, ezzat h, leitch h. hiv-associated diffuse large b cell lymphoma: determinants of survival in the era of rituximab and haart. blood. 2010;116(21):2835. https://doi.org/10.1182/blood.v116.21.2835.2835 machialo jt. diffuse large b cell lymphoma in adults at chris baragwanath hospital [homepage on the internet]. johannesburg: witwatersrand university; 2016 [cited 2018 aug 3]. available from: http://hdl.handle.net/10539/23175 al-saleem ti, dulaimi e, millenson mm, smith mr, judd j, li t, borghaei h. low blood absolute cd4 counts is associated with inferior progression free survival in diffuse large b-cell lymphoma independent of age and ipi. blood. 2014;124(21):5420. https://doi.org/10.1182/blood.v124.21.5420.5420 lim st, tao m, cheung yb, rajan s, mann b. can patients with early-stage diffuse large b-cell lymphoma be treated without bone marrow biopsy? ann oncol. 2005;16(2):215–218. https://doi.org/10.1093/annonc/mdi050 sung hj, kim sj, lee jh, et al. persistent anemia in a patient with diffuse large b cell lymphoma: pure red cell aplasia associated with latent epstein-barr virus infection in bone marrow. j korean med sci. 2007;22:167–170. https://doi.org/10.3346/jkms.2007.22.s.s167 khan ab, barrington sf, mikhaeel ng, et al. pet-ct staging of dlbcl accurately identifies and provides new insight into the clinical significance of bone marrow involvement. blood. 2013;122(1):61–67. https://doi.org/10.1182/blood-2012-12-473389 berthet l, cochet a, kanoun s, et al. in newly diagnosed diffuse large b-cell lymphoma, determination of bone marrow involvement with 18f-fdg pet/ct provides better diagnostic performance and prognostic stratification than does biopsy. j nucl med. 2013;122(1):61–67. https://doi.org/10.2967/jnumed.112.114710 abstract background description of the intervention lessons learnt recommendations acknowledgements references about the author(s) timothy amukele icon clinical laboratories, icon clinical research plc, farmingdale, new york, united states ryland n. spence department of pathology, johns hopkins school of medicine, johns hopkins bayview medical center, baltimore, maryland, united states citation amukele t, spence rn. african countries established covid-19 testing in one month: here’s how they did it. afr j lab med. 2021;10(1), a1457. https://doi.org/10.4102/ajlm.v10i1.1457 lessons from the field african countries established covid-19 testing in one month: here’s how they did it timothy amukele, ryland n. spence received: 17 nov. 2020; accepted: 18 aug. 2021; published: 15 dec. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: as a novel and deadly acute respiratory syndrome, which later became known as coronavirus disease 2019 (covid-19), spread beyond china in late january 2020, there were no laboratories in africa that could test for the disease. however, in early march, just over a month later, 42 african countries had developed the expertise and resources to perform covid-19 testing. our goal was to document this public health success story, learn from it, and use it to inform future public health action. intervention: three groups were primarily responsible for establishing covid-19 testing capacity in africa. the first group comprised early test manufacturers who reacted with incredible speed and ingenuity early in the pandemic, such as the german company tib molbiol that developed a molecular test for covid-19 before the sars-cov-2 genome sequence was available. the second group included private and public donors such as the jack ma foundation, and the last were the coordinators of the rollout, such as the world health organization and the africa centres for disease control and prevention (cdc). lessons learnt: the first lesson was that speed is critical, especially during a crisis. it was also demonstrated that being a predictable and transparent trusted institution opens doors and improves effectiveness. africa cdc, which was only three years old, was able to secure significant resources from external partners and rapidly build substantial testing capacity within africa because it is a trusted institution. recommendations: lowand middle-income countries must build local trusted institutions to better prepare for public health challenges. keywords: testing capacity; covid-19; pandemic; coronavirus; africa; laboratory. background when côte d’ivoire had its first suspected coronavirus disease 2019 (covid-19) case in late january 2020, the nearest laboratory capable of performing covid-19 testing was in paris, france.1 yet just over a month later, by early march, 42 sub-saharan african countries had acquired the instruments, reagents and know-how to perform laboratory diagnostic testing for covid-19.2 broadly speaking, ongoing covid-19 infection can be diagnosed using two types of tests: antigen-based and nucleic acid-based tests. antigen-based tests detect the virus’s coronal spike proteins from which the virus derives its name. nucleic acid-based tests (also called molecular or polymerase chain reaction [pcr] tests) work by directly detecting severe acute respiratory syndrome coronavirus 2 (sars-cov-2) rna. these molecular tests are more specific and sensitive than antigen-based tests but are 3–10 times more expensive and require more complex instrumentation.3 nevertheless, in early 2020, the overwhelming majority of covid-19 tests available globally were molecular. in addition, although sub-saharan africa had less than 1% of the molecular testing capacity of that in the united states, thanks to hiv control efforts, every country had at least one location that could perform molecular testing once the sars-cov-2 molecular reagents were available.4 this article tells the story of how 42 african countries were able to acquire covid-19 pcr testing capacity in two months in early 2020. description of the intervention ethical considerations this study followed all ethical standards for research without direct contact with human or animal subjects. data collection we started by independently corroborating the ‘42 countries’ figure announced by the director of the africa centres for disease control and prevention (cdc) in his interview on 11 march 2020.2 specifically, we searched the internet and social media platforms such as twitter for announcements by individual countries that they could now test for covid-19. next, we used google searches to identify relevant sources (including those in the grey literature and popular press) that detailed how covid-19 testing capacity was established in africa in the first two months of 2020. data analysis raw data were collected as described above, entered into excel (microsoft corporation, redmond, washington, united states), and presented in this study without any transformation. no additional statistical analysis was performed. the figure displaying these data (figure 1) was also created using excel (microsoft corporation, redmond, washington, united states). figure 1: the total number of sub-saharan african countries reporting coronavirus testing results or laboratory testing capacity, january 2020 – may 2020. additional country-level data is available on the covid-19 testing dashboard of pathologists overseas (https://www.pathologistsoverseas.com/). lessons learnt as of march 2020, the total number of countries reporting coronavirus testing results or laboratory testing capacity in sub-saharan africa was 42 (figure 1). this public health success story involved three principal actors: early manufacturers of covid-19 tests, private and government donors, and coordinating bodies, including the world health organization (who) and africa cdc, that coordinated efforts within and across the african countries. the first lesson learnt from this experience was that speed is critical during a crisis, as with the much-criticised motto of facebook’s founder: ‘move fast and break things’. while this may not be good advice for every situation, it was the approach successfully adopted by all the actors in this story. the activities of these major players are discussed in greater detail below. covid-19 test manufacturers test manufacturers reacted with incredible speed and ingenuity in the early days of the pandemic. to illustrate, the announcement of the isolation of the sars-cov-2 virus on 07 january 2020 and the determination of its genome sequence on 10 january 2020, occurred less than a week after china first shared information about a new and deadly flu-like respiratory illness with the who and other countries on 03 january 2020.5,6 while the speed of virus isolation and genome sequencing was remarkable in itself, something even more prodigious was accomplished by an early covid-19 test developer. the german company tib molbiol (tib molbiol syntheselabor gmbh, berlin, germany), in collaboration with the charite hospital in berlin, announced a molecular test for covid-19 on 10 january, the same day the sars-cov-2 genome sequence became available.7,8 they accomplished this feat by creating their molecular sars-cov-2 test based on the sequences of other coronaviruses.9 this is akin to sewing custom clothing for a stranger based on pictures of their parents and siblings. tib molbiol made several versions of this blind test and vetted them using actual covid-19 patient samples before selecting the one that worked best. it was a gamble, but it paid off. the protocol for the best version was published by the who on 17 january 2020, and tib molbiol had sold four million tests by early march 2020.10,11 coordinating bodies many of the aforementioned tib molbiol tests were purchased by the who with funding from the bill and melinda gates foundation and sent to countries around the world to support the scale-up of the novel coronavirus diagnostic efforts.12 in particular, reagent kits were shipped to more than 20 countries in the african region by the who before 31 january 2020.13 this was to expand diagnostic capacity beyond the two referral laboratories in senegal and south africa that had it at the time. of note, the who had a much more expansive role in responding to the covid-19 pandemic.13 for example, on 31 january 2020, the who identified 13 top-priority countries that had either direct links with or a high volume of travel to china (algeria, angola, côte d’ivoire, the democratic republic of the congo, ethiopia, ghana, kenya, mauritius, nigeria, south africa, tanzania, uganda and zambia), and subsequent active screening was established in a majority of the airports in these countries.13 however, these initiatives that are not directly about the establishment of covid-19 diagnostic capacity in africa in the first month of 2020 will not be covered in this focused report. in africa, the who-sourced kits were primarily distributed by africa cdc, a technical institution of the african union that was established in 2016 but officially launched in january 2017.14 like other actors in this story, africa cdc acted swiftly. on 22 january 2020, five days after the publication of the tib molbiol protocol on the who website, africa cdc announced that it was working with member states to identify laboratories that were capable of receiving and testing specimens.10,15 on 05 february 2020, they created the africa task force for coronavirus preparedness and response,16 a multi-country multi-agency group designed to collaborate, communicate and coordinate efforts in response to the coronavirus pandemic. a day later and exactly a week after announcing that they were identifying laboratories in member countries, africa cdc, in collaboration with the pasteur institute in dakar, organised a workshop and training on covid-19 diagnosis for medical teams from 16 african countries, where each trainee received a kit that could run 100 tests (figure 2).17 the countries were côte d’ivoire, cameroon, the democratic republic of the congo, egypt, ethiopia, the gambia, gabon, ghana, kenya, nigeria, morocco, senegal, south africa, tunisia, uganda, and zambia. figure 2: first training on laboratory diagnosis of covid-19 in dakar, senegal, 06–08 february 2020. in partnership with the national institute for communicable disease, a second training on laboratory diagnosis of covid-19 was organised by africa cdc in south africa on 20–22 february 2020 (figure 3). each of the 12 countries that took part in the training received kits for the testing of 192 specimens, while egypt was provided with 700 additional kits, and nigeria and rwanda each received 1000 additional kits.18 figure 3: second training on laboratory diagnosis of covid-19, south africa, 21 february 2020. as many of these groups that were trained lacked the equipment to perform the testing in their home countries, the procurement and placement of equipment and biosafety protective gear were also addressed by the who and africa cdc. in addition, as stated in a media briefing on 04 february 2020, africa cdc planned to set up a referral system where the 16 laboratories that would be set up to perform covid-19 testing could receive samples and support other countries who were unable to test.19 it is not clear if this referral system was ever established but it likely was not needed for long because by 11 march 2020, john nkengasong, the director of africa cdc, had announced in an interview that there were 42 african countries with covid-19 testing capacity at the central level.2 like the who, the efforts of africa cdc transcended laboratory testing and included significant efforts in five other high-priority areas for coronavirus control, namely surveillance, infection prevention and control, clinical care, risk communication, and the distribution of donations by other groups and individuals.20 pre-existing disease surveillance networks also helped in the response to covid-19 in africa. for example, the who has 27 who collaborating centers (defined as ‘institutions such as research institutes, parts of universities or academies, which are designated by the director-general to carry out activities in support of the organization’s programmes’) in 14 african countries.21 other laboratory networks in africa include the regional integrated surveillance laboratory network, which is run by africa cdc and based in zambia, kenya, gabon, nigeria, and senegal; the west african network of biomedical analysis laboratories, which is a network of laboratories in francophone west africa supported by the mérieux foundation22,23; and many other disease-based laboratory networks. these laboratories were already in close contact and sharing surveillance and other information with the who or cdc before covid-19. thus, they were logical sites for situating covid-19 testing once the kits became available. private and government donors one of the most significant donations to help counter the impact of the coronavirus in africa was that made by the chinese billionaire and founder of alibaba, jack ma.24 jack ma’s massive operation had, by september 2020, shipped over 200 million units of personal protective equipment, testing kits and ventilators to over 150 countries and regions, including the united states and over 30 countries in africa. although this was not the largest covid-mitigating financial donation by a wealthy donor, it was arguably the most impactful as alibaba’s logistic muscle allowed the rapid delivery of life-saving face masks and ventilators to many countries that were outcompeted during the global jostle for life-saving equipment.25 on 16 march 2020, jack ma announced a donation of 100 000 masks, 20 000 test kits and 1000 sets of protective clothing and face shields to each of the 54 nations on the african continent.26 on 06 april 2020, an additional donation of medical supplies to all 54 countries of africa was announced.27 this second donation included 500 ventilators, 200 000 sets of protective clothing and face shields, 2000 thermometers, one million swabs and extraction kits, and 500 000 gloves. this brief article does not capture all the activities of all the partners that helped establish laboratory capacity for covid-19 diagnosis in the early days of the pandemic in africa. for example, many government and private partners, including the ethiopian government, the africa cdc, the united nations world food programme, the who, and ethiopian airlines, helped distribute jack ma’s large donation. there were many other groups involved in the early days of the coronavirus laboratory response by countries in africa, including wellcome, the department for international development, the mérieux foundation, oxford nanopore technologies, the united states cdc, and others.20,28,29 the denominating factor across the activities by all the players in this story was the speed with which they responded to build capacity. having a pandemic nipping at one’s heels is a strong incentive to move fast and stay focused. although the lightning-fast response by all partners to the covid-19 pandemic described in this article may not be possible under normal operating conditions, it does demonstrate what is feasible and gives us something to aspire to in terms of speed and efficiency. trusted institutions are crucial for moving africa forward another lesson from this experience is that trusted institutions are the key to effective public health interventions. much of the credit for the rapid establishment of testing capacity should go to the africa cdc as the main coordinating body for external partners (the who, donors, etc.) and countries within africa. but how was a three-year-old institution so effective at such a scale? the africa cdc had obvious advantages such as the resources to hire and retain world-class staff, deep institutional connections to the who and the united states cdc, as well as the pressure of a pandemic. however, our opinion is that the africa cdc was so effective because it is a trusted institution. being a trusted institution does not mean that an institution is perfect or even efficient. rather, a trusted institution has ‘policies and mechanisms that showcase their commitment to transparency, high standards, fiscal management, measurable results, and zero tolerance for corruption and mismanagement of funds’ (brough r, april 2018, personal communication). the establishment of covid-19 testing capacity in africa, while remarkable, was not an unmitigated success. african countries are still behind high-income countries in terms of the number of tests done per capita.30 nevertheless, it was a remarkable achievement and provides a clear example of the positive impact of trusted institutions in public health. recommendations we need to build more local trusted institutions. trusted institutions are characterised by predictability and transparency, which makes them more effective than other institutions because partners can engage without concerns that their donations may be diverted. more so, the influence of trusted institutions goes beyond donors and philanthropists as it also makes it easier for the recipients to engage. when invited by the africa cdc for training or capacity building, countries are much more likely to respond positively because they know that such offers are usually without any hidden motives or expectations. naturally, this begs the question, the answers to which are beyond the scope of this article but have been comprehensively addressed elsewhere31,32: how do we build trusted institutions? acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions t.a. conceived of the presented idea. t.a. and r.n.s. performed the research and found the original sources. both authors discussed the results and contributed to the final manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the data that support the findings of this study are available on request from the corresponding author, t.a. the data are not publicly available due to it containing information that could compromise the privacy of research participants. disclaimer the views expressed in this article are those of the authors and do not necessarily reflect the views of the institutions the authors represent. references washington post. africa has 1.2 billion people and only six labs that can test for coronavirus. how quickly can they ramp up? 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[https://twitter.com] jack ma [homepage on the internet]. c2020 [updated 2020 mar 16; cited 2020 may 12]. available from: https://twitter.com/jackma/status/1239581509125726210 merieux foundation. covid-19: the merieux foundation deploys diagnostic and research expertise on the ground [homepage on the internet]. https://www.fondation-merieux.org/. c2020 [cited 2020 jun 11]. available from: https://www.fondation-merieux.org/en/the-merieux-foundation-deploys-diagnostic-and-research-expertise-on-the-ground/ africa center for disease control. outbreak brief number 11: covid-19 pandemic – 31 march 2020 [homepage on the internet]. https://africacdc.org. c2020 [updated 2020 mar 31; cited 2020 jun 19]. available from: https://africacdc.org/download/outbreak-brief-11-covid-19-pandemic-31-march-2020/ our world in data. research and data to make progress towards the world’s largest problems [homepage on the internet]. [https://ourworldindata.org]. 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[homepage on the internet]. https://www.blogs.worldbank.org. c2014 [updated 2014 nov 13; cited 10 oct 2020]. available from: https://blogs.worldbank.org/governance/who-responsible-building-trust-institutions abstract introduction methods results discussion acknowledgements references about the author(s) habtamu asrat ethiopian public health institute, addis ababa, ethiopia department of medical laboratory sciences, college of health sciences, addis ababa university, addis ababa, ethiopia abebaw kebede ethiopian public health institute, addis ababa, ethiopia abnet abebe ethiopian public health institute, addis ababa, ethiopia abyot meaza ethiopian public health institute, addis ababa, ethiopia getinet hailu ethiopian public health institute, addis ababa, ethiopia adinew desale ethiopian public health institute, addis ababa, ethiopia andargachew gashu ethiopian public health institute, addis ababa, ethiopia wondwossen kassa ethiopian public health institute, addis ababa, ethiopia tesfaye mekonnen ethiopian public health institute, addis ababa, ethiopia ebisea abose ethiopian public health institute, addis ababa, ethiopia feven girmachew ethiopian public health institute, addis ababa, ethiopia dereje yenealem ethiopian public health institute, addis ababa, ethiopia achamyeleh mulugeta ethiopian public health institute, addis ababa, ethiopia gonfa ayana ethiopian public health institute, addis ababa, ethiopia kassu desta department of medical laboratory sciences, college of health sciences, addis ababa university, addis ababa, ethiopia citation asrat h, kebede a, abebe a, et al. performance evaluation of tuberculosis smear microscopists working at rechecking laboratories in ethiopia. afr j lab med. 2017;6(1), a590. https://doi.org/10.4102/ajlm.v6i1.590 original research performance evaluation of tuberculosis smear microscopists working at rechecking laboratories in ethiopia habtamu asrat, abebaw kebede, abnet abebe, abyot meaza, getinet hailu, adinew desale, andargachew gashu, wondwossen kassa, tesfaye mekonnen, ebisea abose, feven girmachew, dereje yenealem, achamyeleh mulugeta, gonfa ayana, kassu desta received: 02 nov. 2016; accepted: 31 jan. 2017; published: 21 apr. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: tuberculosis is an infectious disease caused by the bacillus mycobacterium tuberculosis. according to the ethiopian federal ministry of health’s 2013–2014 report, the tuberculosis case detection rate was 53.7%, which was below the target of 81% set for that year. objective: this study assessed the performance of tuberculosis smear microscopists at external quality assessment rechecking laboratories in ethiopia. methods: a cross-sectional study was conducted at 81 laboratories from april to july 2015. panel slides were prepared and validated at the national tuberculosis reference laboratory. the validated panel slides were used to evaluate the performance of microscopists at these laboratories compared with readers from the reference laboratory. results: a total of 389 external quality assessment rechecking laboratory microscopists participated in the study, of which 268 (68.9%) worked at hospitals, 241 (62%) had more than five years of work experience, 201 (51.7%) held bachelors degrees, and 319 (82%) reported tuberculosis smear microscopy training. overall, 324 (83.3%) participants scored ≥ 80%. sensitivity for detecting tuberculosis bacilli was 84.5% and specificity was 93.1%. the overall percent agreement between participants and reference readers was 87.1 (kappa=0.72). all 10 slides were correctly read (i.e., scored 100%) by 80 (20.6%) participants, 156 (40.1%) scored 90% – 95%, 88 (22.6%) scored 80% – 85% and 65 (16.7%) scored below 80%. there were 806 (20.7%) total errors, with 143 (3.7%) major and 663 (17%) minor errors. conclusion: the overall performance of participants in reading the slides showed good agreement with the reference readers. most errors were minor, and the ability to detect tuberculosis bacilli can be improved through building the capacity of professionals. introduction tuberculosis is an infectious disease caused by the bacillus mycobacterium tuberculosis. it remains a major global health problem, responsible for ill health among millions of people each year.1,2,3 it is the second leading cause of death among all infectious diseases worldwide after hiv.1,3 according to the 2014 world health organization global tuberculosis report, there were nine million new tuberculosis cases and 1.5 million tuberculosis deaths (1.1 million among hiv-negative people and 0.4 million among hiv-positive people) in 2013.1 one quarter of global cases and deaths occurred in the african region,1 and ethiopia ranked 10th in tuberculosis incidence among 22 high-burden countries.1,4 according to ethiopian ministry of health reports for 2012–2013 and 2013–2014, the targeted tuberculosis case detection rates were 82.7% for the 2012–2013 period and 81% for 2013–2014. however, the case detection rates achieved were 58.9% in 2012–2013 and 53.7% in 2013–2014, which were well below these targets.5,6,7 a low case detection rate is often associated with a lack of effective programme awareness, lack of active cough identification and lack of quality-assured routine diagnosis (such as sputum quality, reagent quality, knowledge, and capacity of professionals). in ethiopia, factors that are associated with low case detection rates have not been well studied. however, they are likely to be associated with these factors. therefore, the present study dealt with the performance of tuberculosis smear microscopists as one factor affecting quality-assured routine diagnosis. in most lowand middle-income countries, smear microscopy remains the foundation of tuberculosis diagnosis, despite its relatively low sensitivity. microscopy has also remained essential to monitoring of tuberculosis treatment. a microscopy network with adequate population coverage and high quality performance is therefore critical. bright-field sputum smear microscopy (i.e., conventional ziehl-neelsen staining) is widely available, simple to perform, inexpensive, and requires simple laboratory facilities.1,8 thus, one national tuberculosis control strategy recommended by the world health organization is to pursue expansion and enhancement of high-quality directly-observed treatment, short-course chemotherapy through early case detection and diagnosis at quality-assured laboratories.9 quality assurance of microscopy remains a critical activity of all laboratory networks, and a comprehensive external quality assessment (eqa) programme that includes on-site evaluation, random blinded rechecking, and panel testing should be implemented.1,8,10 eqa programmes are needed to ensure that smears are performed and stained properly, results are interpreted correctly and all microscopy centres achieve an acceptable level of performance. effective eqa programmes require dedicated and qualified staff for rechecking of smears. the implementation of eqa for microscopy has the advantage, not only of strengthening laboratory networks, but of improving diagnostic quality.11 the ethiopian federal ministry of health and the ethiopian public health institute decentralised eqa programmes to regional reference laboratories and have guided the regions to decentralise further into sub-regional laboratories and eqa rechecking laboratories. this decision was made with the assumption that all microscopy centres in the various regions would have a chance to participate in eqa programmes and that improved eqa coverage could be achieved. the mandate for conducting a rechecking programme was given to the eqa rechecking laboratories by regional health bureaus. following the endorsement of the regional health bureaus, eqa rechecking laboratories have the right to perform tuberculosis eqa blind rechecking by collecting slides from the microscopy centres in their catchment areas. the aim of this study was to produce baseline data about the performance of the tuberculosis rechecking laboratories and the microscopists who work there. methods ethical considerations leftover samples were collected anonymously from federal hospitals for panel preparation. all information about each tuberculosis eqa rechecking laboratory was kept confidential and used only for the purposes of this study and for the improvement of acid-fast bacilli (afb) microscopy. the research proposal was evaluated and approved by the departmental research and ethics review committee of the department of medical laboratory sciences, college of health sciences at addis ababa university with ref. no. mls/326/15 and protocol number: drerc 119/15/mls before the start of fieldwork. confidentiality was maintained during data collection, and written informed consent was obtained from each study participant. study design a cross-sectional study was conducted from april to july 2015 at tuberculosis eqa rechecking laboratories in ethiopia. validated panel slides were used to assess the performance of microscopists working in the laboratories. the study was conducted at 12 (100%) regional laboratories, three (75%) sub-regional laboratories, 46 (61%) hospital laboratories and 20 (59%) health centre laboratories among the 125 eqa rechecking laboratories in ethiopia. sputum samples for panel preparation were collected from federal hospitals anonymously, and panel slides were prepared in the national tuberculosis reference laboratory. each dilution panel was validated by six different readers following world health organization guidelines (reference readers).10 a set of 10 validated slides was distributed to participating laboratories to assess the reading and interpretation proficiency of smear microscopists; 50–70 minutes were allowed to complete the reading.10,12 the panel composition and bacilli load were: one grade 3+ slide, one grade 2+ slide, two grade 1+ slides, three 1–9 afb/100 field slides and three negative slides.10 the results were expressed as correct, minor error or major error. major errors were classified as high false positive, if a negative smear was misread as grade 1+ to 3+ positive, or as high false negative, if a grade 1+ to 3+ positive smear was misread as negative (table 1). minor errors were classified as a quantification error, when there was a difference of more than one grade in the reading of positive smear between the examinee and the reference readers, a low false positive, when a negative smear was misread as scanty (1–9 afb/100x field), or as low false negative, when a scanty slide (1–9 afb/100x field) was misread as negative.10,12,13 table 1: evaluation and interpretation of errors between rechecking laboratory microscopists and reference readers, ethiopia, april–july 2015†. each slide was worth 10 points. the total possible score was 100 points (for 10 slides), and based on national and world health organization guidelines, a passing score was considered to be ≥ 80%.10,12 committing major errors, like a high false positive or high false negative, was worth zero points, whereas minor errors, like low false positive, low false negative and quantification errors, were worth five points.8,10,12 data analysis all data were entered into a microsoft excel (microsoft, inc., redmond, washington, united states) spreadsheet and transported to spss (version 20.0; spss, inc., chicago, illinois, united states) for analysis. the percentages of agreements, differences and the different types of errors were calculated. the sensitivity, specificity, positive predictive value, and negative predictive value of smear reading for each tuberculosis eqa smear microscopist was calculated. the chi square test was used to assess associations between different variables. the strength of an agreement between participant readers and the reference readers were assessed using kappa statistics.14 results study participants a total of 389 microscopists (2 to 13 microscopists per rechecking laboratory) from 81 tuberculosis eqa rechecking laboratories participated in the study, of whom 263 (67.6%) were men and 126 (32.4%) were women (table 2). most of the study participants worked in hospital laboratories (n = 268, 68.9%); 241 (62%) participants had more than five years of work experience in tuberculosis smear microscopy services, 201 (51.7%) held a bachelors degree, and 319 (82%) had gone through tuberculosis smear microscopy in-service training. table 2: demographic characteristics of microscopists at tuberculosis external quality assessment rechecking laboratories in ethiopia (n = 389), april–july, 2015. panel testing among all 389 participants, 324 (83.3%) scored ≥ 80% (passing) (table 3). when stratified by place of work, more participants working in hospitals (n = 231, 86.2%) achieved a passing score (≥ 80 %) than participants who worked in other types of facilities. on the other hand, 21/23 (91.3%) microscopists with less than two years of work experience scored ≥ 80%, which was higher than microscopists with more than two years of work experience. the proportion of participants who scored ≥ 80% was higher among holders of a masters degree compared with participants with other educational backgrounds, and the proportion of participants who scored ≥ 80% was slightly higher among participants who had not had tuberculosis smear microscopy in-service training. in general, there were no statistically-significant associations between the performance of participants in tuberculosis bacilli detection and their sex, work experience, educational background, place of work or tuberculosis smear microscopy in-service training. table 3: relationship between demographic characteristics and scores of microscopists at tuberculosis external quality assessment rechecking laboratories in ethiopia (n = 389), april–july, 2015. a total of 3890 validated slides were read by study participants (table 4). the overall sensitivity for detecting tuberculosis bacilli was 84.5% and overall specificity was 93.1%. the overall percent agreement of participants with the reference readers was 87.1 (kappa=0.72). the percent agreement of participants working in health centres with the reference readers was 83.1% (kappa=0.64), which was slightly lower than participants working in hospitals or regional laboratories. the negative predictive values were quite low for participants working in all health facilities. table 4: overall sensitivity, specificity, predictive values and agreements of participants with reference readers in detecting tuberculosis bacilli, ethiopia, april–july, 2015. of the 389 participants, 80 (20.6%) participants correctly read all 10 slides and scored 100% (figure 1). a total of 156 (40.1%) scored 90% – 95%, which means they committed one major error or two minor errors. a total of 88 (22.6%) participants scored 80% – 85%, which means they committed three to four minor errors or two major errors or one major and one minor error or one major and two minor errors. finally, 65 (16.7%) participants scored below 80%, which means they had more than four minor errors or two major errors or one major and two minor errors. figure 1: concordance of tuberculosis external quality assessment rechecking laboratory microscopists with reference readers for detecting tuberculosis bacilli, ethiopia (n=389), april–july, 2015†. of the 3890 examined slides, there were a total of 806 (20.7%) errors, which included 143 (3.7%) major errors and 663 (17%) minor errors (table 5). of these, 89 (2.3%) errors were high false negatives, 54 (1.4%) were high false positives, 334 (8.6%) were low false negatives, 26 (0.7%) were low false positives and 303 (7.8%) were quantification errors. table 5: type of errors committed by external quality assessment rechecking laboratory microscopists in detecting tuberculosis bacilli by type of institution, ethiopia (n=3890 slides), april–july, 2015. discussion this cross-sectional study evaluated the performance of tuberculosis smear microscopists working at eqa rechecking laboratories and the status of the respective laboratories. in this study, the overall agreement of participants with reference readers for reading the validated slides was 87.1% (kappa=0.72), which was good agreement based on kappa statistics.14 however, lower agreement was observed when compared with a different study conducted in southern ethiopia, which found 96.8% agreement (kappa=0.936),15 and a study done in the town of hawassa, ethiopia, which found 95.2% agreement (kappa=0.73).16 when compared with a study done in the east and west amhara regions of ethiopia, higher agreement was also observed (98.4% in east amhara and 96.5% in west amhara [kappa=0.92]).17,18 thus, performance in our study was slightly lower than in similar studies conducted in other parts of ethiopia. this may have been due to the large number of laboratories and/or laboratory professionals included in our study, which was more of a nationwide study with wider representation. this may have made our study more prone to lower performance. in general, agreement in reading between participants and reference readers was slightly lower than in similar studies conducted in other countries. our finding was lower than studies done in india (98% agreement)19 and tanzania20 (89.2% agreement). however, agreement in our study was higher than studies done in ghana (73%)21 and the democratic republic of congo (74%).22 these differences may be attributable to differences in the composition of the panel slides, as we prepared a second degree of difficulty in our slides (three scanty and three negative slides).10 in our study, the overall sensitivity was 84.5% and specificity was 93.1%. the study in hawassa, ethiopia showed higher sensitivity (91.97%), but lower specificity (80.0%).16 on the other hand, both sensitivity (96.5%) and specificity (96.4%) were higher in the west amhara, ethiopia report.18 similarly, the study conducted in east amhara, ethiopia showed higher sensitivity (88.4%) and specificity (99.3%).17 both sensitivity and specificity were 96.8% in a southern ethiopia finding, which was higher than our study.15 sensitivity (88.5%) and specificity (100%) were also higher in the study conducted in tanzania.20 in our study, the lower sensitivity indicates that there were high false-negative rates (patients with tuberculosis bacilli misdiagnosed as negative). the consequence of this low sensitivity is that tuberculosis patients are not treated, which results in ongoing disease, disease transmission or death. the study in hawassa, ethiopia reported that 13.6% of microscopists correctly read all panel slides, which was slightly lower than our finding, and 86.4% committed at least one error in reading 10 slides, which was significantly higher than our finding.16 in the study done in india, 95% of readers reported no errors,19 demonstrating far greater proficiency than the present study. although the majority of our participants (83.3%) had an acceptable performance score (≥ 80%), we consider this a weak achievement, since study participants were from facilities with a responsibility to recheck other health institutions’ slides and provide support to them. considering this responsibility, microscopists at these facilities should have scored better than the current findings. in the present study, there were more low false negatives than quantification errors. false readings in southern ethiopia (3.2%), east amhara (1.6%) and west amhara (3.5%), ethiopia were lower than our finding.15,17,18 on the other hand, the percentage of errors in hawassa, ethiopia (29.75%) was higher than ours.16 in addition, the hawassa study had fewer major errors (2.22%), but more minor errors (27.5%) than our study.16 quantification errors were the biggest contributor to overall errors in the hawassa study, whereas in the present study, low false negatives were the most frequent errors.16 fewer errors were observed in india, where quantification errors were the most frequent and no high false positives were reported.19 in a similar study conducted in mexico, quantification errors were frequent (12.3%), followed by low false negatives (5.7%).23 a study conducted in the democratic republic of congo also reported frequent low false-negative errors.22 in another study conducted in taiwan, low false-positive errors were much higher (28.6%) than in the present study.24 false-negative errors could lead to failure to detect persons with infectious tuberculosis, who could continue to spread the disease in their communities; false positives could lead to unnecessary anxiety, exposure of patients to unwanted side effects of medications, and unnecessary expenditure.25 while lower rates of minor errors are acceptable due to the inherent problems of afb smear microscopy services, major errors are unacceptable. among minor errors, low false-positive and low false-negative errors both have a significant impact on patient management and tuberculosis control programmes, whereas quantification errors have no impact on patient management. hence, improving the competency of professionals through training, implementation of strong eqa programmes, supportive supervision and mentoring are critically important to reduce or avoid these types of errors and to maximise case detection rates.10 limitations this study has a few limitations, which should be considered when interpreting our results. first, unstained slides were not sent to participating laboratories. thus, the quality of the reagents used for routine afb microscopy was not assessed. additionally, information on the age of the participants was not collected in the demographic information. therefore, we could not evaluate the effects of age variability on the performance of the study participants. conclusion the overall performance of the tuberculosis eqa rechecking laboratories in reading afb slides showed good agreement with the reference readers (87.1%). overall, 20.7% of slides were misread, and most errors were minor. nevertheless, these errors are alarming, and our findings are a clarion call to tuberculosis control programmes to give needed support to eqa programmes. a large number of minor errors were noted; thus, continuous mentoring and supportive supervision for afb microscopy centres should be given priority to minimise errors and improve eqa activities in ethiopia. tuberculosis is a re-emerging global threat, and all steps to improve the accuracy of its diagnosis should be sought and implemented. acknowledgements the authors gratefully acknowledge the addis ababa university department of medical laboratory sciences, which provided the opportunity to perform this work. the authors would like to deeply acknowledge the ethiopian public health institute, regional laboratories capacity building directorate for financial support and the national tuberculosis reference laboratory for allowing the laboratory for panel preparation and validation. we also thank all study participant facilities and individuals. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support none. authors’ contributions h.a. conceived the initial idea, prepared the proposal, collected the data, analysed the data and wrote the manuscript. k.d., a.k. and g.a. gave advice and edited the proposal and the manuscript. a.a., a. meaza, g.h., a.d., a.g., w.k., t.m., e.a., f.g., d.y. and a. mulugeta participated in data collection and edited the manuscript. all authors read and approved the final manuscript. references 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addis ababa, ethiopia: fmoh; 2014. federal democratic republic of ethiopia, ministry of health. health sector development programme iv: annual performance report 2012/13. addis ababa, ethiopia: fmoh; 2013. parsons lm, somoskövi a, gutierrez c, et al. laboratory diagnosis of tuberculosis in resource-poor countries: challenges and opportunities. clin microbiol rev. 2011;24(2):314–350. https://doi.org/10.1128/cmr.00059-10 federal ministry of health of ethiopia. guideline for clinical and programmatic management of tb, leprosy and tb/hiv in ethiopia. 5th ed. addis ababa, ethiopia: fmoh; 2015. world health organization, association of public health laboratory, us centers for disease control and prevention, international union against tuberculosis and lung disease. external quality assessment for afb smear microscopy. washington dc, usa: aphl; 2002. ridderhof jc, deun av, kam km, et al. roles of laboratories and laboratory systems in effective tuberculosis programmes. bull world health organ. 2007;85(5):354–359. https://doi.org/10.2471/06.039081 ethiopian health and nutrition research institute, federal ministry of health. guidelines for quality assurance of smear microscopy for tuberculosis diagnosis. addis ababa, ethiopia: ehnri; 2009. international union against tuberculosis and lung disease. priorities for tuberculosis bacteriology services in low-income countries. 2nd ed. paris, france: iuatld, 2007; p. 56–57. viera aj, garrett jm. understanding interobserver agreement: the kappa statistic. fam med. 2005;37(5):360–363. shargie eb, yassin ma, lindtjørn b. quality control of sputum microscopic examinations for acid fast bacilli in southern ethiopia. ethiop j health dev. 2005;19(2):104–108. https://doi.org/10.4314/ejhd.v19i2.9978 hailemariam m, minuta a, bewoket g, et al. performance evaluation of laboratory professionals on tuberculosis microscopy at hawassa town, southern ethiopia. afr j microbiol res. 2015;9(16):1132–1138. https://doi.org/10.5897/ajmr2015.7402 mulat m. quality performance evaluation of laboratories on afb smears microscopy in eastern amhara region, ethiopia, 2011 [msc thesis] [document on the internet]. c2012 [cited 2014 dec 25]. available from: http://hdl.handle.net/123456789/2607 manalebh a, demissie m, mekonnen d, et al. the quality of sputum smear microscopy in public-private mix directly observed treatment laboratories in west amhara region, ethiopia. plos one. 2015;10(4):e0123749. https://doi.org/10.1371/journal.pone.0123749 dave pv, patel nd, rade k, et al. proficiency panel testing—a reliable tool in external quality assessment of sputum smear microscopy services in gujarat, india. indian j tuberc. 2011;58(3):113–119. basra d, matee min, mcnerney r. quality assessment of sputum smear microscopy for detection of acid fast bacilli in peripheral health care facilities in dar es salaam, tanzania. east afr med j. 2006;83(6):306–310. addo kk, owusu-darko k, dan-dzide m, et al. situation analysis of tb microscopy centres in ghana. int j tuberc lung dis. 2006;10(8):870–875. van rie a, fitzgerald d, kabuya g, et al. sputum smear microscopy: evaluation of impact of training, microscope distribution, and use of external quality assessment guidelines for resource-poor settings. j clin microbiol. 2008;46(3):897–901. https://doi.org/10.1128/jcm.01553-07 martinez-guarneros a, balandrano-campos s, solano-ceh ma, et al. implementation of proficiency testing in conjunction with a rechecking system for external quality assurance in tuberculosis laboratories in mexico. int j tuberc lung dis. 2003;7(6):516–521. wu m-h, chiang c-y, jou r, et al. external quality assessment of sputum smear microscopy in taiwan. int j tuberc lung dis. 2009;13(5):606–612. nnaji ga, chukwu jn. comparative analysis of errors in reading sputum smear microscopy by supervisors and peripheral laboratory technicians in southeastern nigeria. trop j med res. 2015;18(2):80–84. https://doi.org/10.4103/1119-0388.158399 article information authors: waqo g. boru1 gideon kikuvi2 jared omollo1 ahmed abade1 samuel amwayi1 william ampofo3 elizabeth t. luman4 joseph oundo1,5 affiliations: 1field epidemiology and laboratory training programme, kenya2institute of tropical medicine, jomo kenyatta university of agriculture and technology, kenya 3noguchi memorial institute for medical research, university of ghana, legon 4center for global health, u.s. centers for disease control and prevention, united states 5centre for disease control and prevention, kenya correspondence to: waqo boru postal address: ministry of health, field epidemiology and laboratory training program, box 20750–00200, nairobi, kenya dates: received: 07 may 2012 accepted: 11 oct. 2012 published: 03 sept. 2013 how to cite this article: boru wg, kikuvi g, omollo j. aetiology and factors associated with bacterial diarrhoeal diseases amongst urban refugee children in eastleigh, kenya: a case control study. afr j lab med. 2013;2(1), art. #63, 6 pages. http://dx.doi.org/10.4102/ ajlm.v2i1.63 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. aetiology and factors associated with bacterial diarrhoeal diseases amongst urban refugee children in eastleigh, kenya: a case control study in this original research... open access • abstract • introduction • research method and design    • study site    • study population    • definition of cases and controls    • sampling    • data and stool sample collection    • statistical analysis • ethical considerations • results    • demographic characteristics of study participants    • bacterial isolates    • antimicrobial susceptibility pattern    • risk factors associated with diarrhoea • trustworthiness    • reliability and validity of the research • discussion • limitations of the study    • recommendations • conclusions • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ introduction: kenya is home to over 400 000 refugees from neighbouring countries. there is scanty information about diarrhoea amongst urban refugees in kenya. objectives: we investigated the enteric bacteria causing diarrhoea amongst urban refugee children and described the associated factors. method: during the period of august–december 2010, urban refugee children between the ages of two and five who attended eastleigh county council health centre were enrolled into the study. diarrhoeal cases were compared with age-matched children with no diarrhoea (controls). stool specimens were collected and enteric bacteria isolated. a questionnaire was administered to identify risk factors. results: a total of 41 cases and 41 controls were enrolled in the study. the age and country of origin were similar for cases and controls. the bacterial isolation rates amongst the cases were: non-pathogenic escherichia coli 71%, shigella dysenteriae 2.4%, shigella flexneri 2.4%, salmonella paratyphi 5%. for the controls, non-pathogenic e. coli 90% and enterotoxigenic e. coli (etec) 2.4% were amongst the organisms isolated. all isolates were resistant to amoxicillin; resistance to other antibiotics varied by isolate type. factors associated independently with diarrhoea included children not washing their hands with soap (aor 5.9, p < 0.05), neighbour(s) having diarrhoea (aor 39.8, p < 0.05), children not exclusively breastfed for their first 6 months (aor 7.6, p < 0.05) and children eating food cooked the previous day (aor 23.8, p = 0.002). conclusions: shigella species, salmonella species and etec were found to be responsible for diarrhoea amongst the urban refugee children. measures to control and guide the use of antibiotics are critical for the prevention of antibiotic resistance. efforts to improve personal and domestic hygiene, including educational campaigns to promote appropriate handwashing, should be encouraged. introduction top ↑ diarrhoea is the second most frequent cause of death globally amongst children aged five and below,1 accounting for more than one-third of the deaths in this age group – more than hiv, malaria and measles combined.2 more than 80% of child deaths due to diarrhoea occur in africa and south asia, with only fifteen countries, including kenya, contributing to almost three-quarters of the total.2 in kenya, diarrhoea is a leading cause of morbidity, accounting for 17% of childhood illnesses.3 thirty per cent of the 9.2 million refugees in the world are found in sub-saharan africa, and of these, 80% are women and children.4 kenya hosts over 400 000 refugees from its neighbouring countries.5 eastleigh suburb in nairobi is a cosmopolitan commercial centre hosting a mixed population of native kenyans and a large proportion of urban refugees from somalia, eritrea and ethiopia. there is also a constant bidirectional movement of people between this suburb and the two refugee camps, kakuma and dadaab, located in the northern part of kenya. previous studies on diarrhoea amongst refugee populations in kenya were focused solely on the situation in the camps. there is, however, an increasingly large number of refugees dispersed in the urban areas. there is also limited information on the causes and associated risk factors of diarrhoea in the urban refugee children. for this study, a health facility-based, matched case–control study was conducted to determine bacterial aetiology and associated risk factors for diarrhoea amongst young urban refugee children in eastleigh, nairobi. research method and design top ↑ study site eastleigh is part of the pumwani division of nairobi with an approximate area of 7.4 km2 and a population of approximately 200 000 people.3 this study was conducted at eastleigh city council health centre, where refugees are able to access free medical services through the support of the german technical cooperation agency. study population children who were registered with the united nations high commission for refugees, were between two and five years of age and resided in eastleigh were enrolled in the study. definition of cases and controls children seen in eastleigh city council health centre with diarrhoea from august–december 2010 who had not started any antibiotic treatment were recruited as cases. controls were hospital-based and were defined as children who had sought medical attention for conditions other than diarrhoea, had no history of diarrhoea in the previous four weeks (based on the report of their caregiver) and were not on antibiotics. these children were recruited as controls upon informed and voluntary consent by their caregiver. the cases were then categorised as acute (defined as an episode lasting less than two weeks), persistent (diarrhoea lasting two weeks or longer) and bloody (defined as gross appearance of blood in the stool,6 as reported by the parent or guardian or noted by the study personnel, regardless of duration). sampling the sample size was determined using an assumed prevalence of 32% for bacterial aetiologies in children below the age of five years as described in previous studies done in sub-saharan africa.7 fleiss’s formula8 was used to generate the sample size, at a confidence interval of 95%. this yielded a sample size of 82 participants (41 cases and 41 controls). systematically, every second child meeting the case definition was enrolled in the study. controls were selected as the next age-matched child (±5 months from the age of a case) meeting the inclusion criteria who attended the health facility during the study period. data and stool sample collection we interviewed parents and guardians of the eligible participants using a standard questionnaire that addressed demographic and epidemiological data, duration of exclusive breastfeeding, hygiene practices, source and storage of drinking water and disposal of faeces. however, clinical information relevant to diarrhoea was obtained exclusively from the cases. stool specimens were collected using sterile plastic containers and transported in cary-blair transport media to the laboratory at the centre for microbiology research (cmr) at the kenya medical research institute (kemri) for bacterial culture and antimicrobial susceptibility testing. specimens were investigated for the isolation of enteric bacterial pathogens using conventional methods.9 all isolates identified as escherichia coli were characterised further by means of multiplex polymerase chain reaction (mpcr) targeting specific genes with the primers outlined in table 1. the pcr allowed for characterisation of the e. coli isolates as enterotoxigenic (etec), enteropathogenic (epec), enterohaemorrhagic (ehec), enteroinvasive (eiec) or enteroaggregative (eaec).10 the inocula for susceptibility testing were compared against the mcfarland 0.5 turbidity standard with the e. coli atcc 25922 strain being used as the test standard. antimicrobial susceptibility testing was done on mueller-hinton agar using the etest® minimum inhibitory concentration (mic) method. the interpretation of results was according to clinical laboratory standard institute (clsi) guidelines.11 the following antimicrobials were tested: amoxicillin, ampicillin, gentamycin, tetracycline, chloramphenicol, kanamycin, ciprofloxacin, ofloxacin, nalidixic acid, erythromycin, ceftriaxone and trimethroprim-sulphamethaxazole. table 1: primers sequence and target genes in the multiplex polymerase chain reaction (pcr) in the detection of diarrhoeagenic escherichia coli. statistical analysis data analysis was performed using epi info version 3.4.3 (cdc, atlanta, usa) software. bacterial isolation rates were calculated for both cases and controls and proportions were compared using the chi-square test corrected for matched case control. all variables with p≤0.1 in bivariate analysis were included in the initial multivariable conditional logistic regression model. standard backward elimination was conducted in order to obtain a final model of factors that were associated independently with diarrhoea at a p value of < 0.05. ethical considerations top ↑ the study was approved by the jomo kenyatta university of agriculture and technology and the national ethical review board at kemri. written informed consent was obtained from the parents or guardians of the children enrolled in the study. results top ↑ demographic characteristics of study participants the 41 cases and 41 controls were similar with respect to their mean age, 38 versus 37 months respectively. the age range was 24–59 and 24–55 months for cases and controls respectively. most of the study participants were from somalia (73% of cases and 51% of controls), with the remainder from ethiopia (27% of cases and 34% of controls) and eritrea (0 cases and 15% of controls). seven cases (17%) had received some form of treatment prior to arrival at eastleigh city council health centre. most of the cases (78%) were diagnosed with gastroenteritis, whilst the remaining were diagnosed with dysentery (2%), malaria (5%) or other conditions (6%) (table 2). ninety-three per cent of the cases had acute diarrhoea. the majority of the cases were prescribed antibiotics (76%) and/or flagyl (78%) (table 2). table 2: clinical information of urban refugee children with diarrhoea between the ages of two and five seen at eastleigh city council health centre, nairobi, august–december, 2010. bacterial isolates most of the isolates obtained (29 [71%] cases and 37 [90%] controls) were non-pathogenic e. coli. the pathogenic isolates were isolated from seven cases and one control. enterotoxigenic e. coli was the highest-ranking isolate, with three from the cases and one from a control. only two shigella spp. and salmonella spp. isolates were obtained from the cases. antimicrobial susceptibility pattern the heat-labile enterotoxin producing enterotoxigenic e. coli labile toxin (etec-lt) isolates were fully resistant to amoxicillin and many were resistant to ampicillin, erythromycin and tetracycline, but all were sensitive to gentamicin, nalidixic acid and ofloxacin (figure 1). the shigella spp. and salmonella spp. isolates were both resistant to ceftriaxone, trimethroprim-sulfamethaxazole, amoxicillin and erythromycin. the shigella spp. isolate was also found to be resistant to tetracycline, whilst the salmonella spp. isolate was also resistant to chloramphenicol. only four antimicrobials showed no resistance pattern, namely kanamycin, ciprofloxacin, ofloxacin and nalidixic acid. figure 1: antimicrobial resistance patterns of pathogenic isolates among urban refugee children in eastleigh in nairobi, august–december, 2010. risk factors associated with diarrhoea in the bivariate analysis, most of the exposure variables examined relating to lack of sanitary practices were associated significantly with diarrhoea. several factors associated with handwashing were statistically significant, with odds ratios ranging from 4–6. additional exposure within the home relating to breastfeeding, food preparation and water storage and treatment were statistically significant, with odds ratios ranging from 7–51. exposure outside the home, such as exposure to other cases of diarrhoea, were also significant, with odds ratios of 5–9 (table 3). table 3: bacterial enteric agents isolated in the urban refugee children between the ages of two and five as seen at eastleigh city council health centre, nairobi, august–december, 2010. eight variables were found to be associated independently with diarrhoeal illness in the final multivariate model. two were related to the handwashing category (children not washing hands with soap and/or not having a handwashing facility in the home: adjusted odds ratio (aor) 6); four were related to other exposure within the home (children not exclusively breastfed for their first six months; using a wide-mouthed container to store water; children having eaten food cooked the previous day; and not having a rack for drying dishes in the home: aor 7–40); and three were related to exposure outside the home (a neighbour having had diarrhoea during the previous two weeks; children sharing a toilet with a diarrhoea patient; and children drinking water from outside the home during the five days before enrolment: aor 10–40) (table 4). table 4: final multivariate model of factors independently associated with diarrhoeal diseases amongst urban refugee children between the ages of two and five seen at eastleigh city council health centre, nairobi, august– december, 2010. trustworthiness top ↑ the findings of this study are based on true findings as spelt out in the research protocol. the observations here outlined are as experienced during the study and are consistent with other research and related studies. reliability and validity of the research the experimental design of this study is reliable and valid. the procedures used in this research have been tested in other studies as cited in this article. it can be said with the strongest conviction that the results of this study are reproducible in any setting as long as these procedures are employed. discussion top ↑ the pathogenic organisms found among these urban refugee children with diarrhoea were etec, shigella flexneri, shigella dysenteriae and salmonella paratyphi. together, these known pathogens were more common among cases than controls, supporting their aetiological role. these findings agree with a previous study amongst children in kenya and senegal,12,13 as well as studies done in bangladesh and argentina,14 which determined that etec, salmonella spp. and shigella spp. were responsible for most cases of bacterial diarrhoea in children. proteus spp. and providencia spp., which are generally thought to be non-pathogenic, were isolated from both cases and controls. of note, however, is that aeromonas spp. were isolated from two diarrhoeal cases and none from the controls. although the sample size was small, this finding could be significant and can be recommended for further research to determine pathogenicity of this organism, as its presence has also been revealed in other studies.15 approximately 5% of the diarrhoeal cases amongst the urban refugee children were persistent. this finding is consistent with observations from a study conducted in india, which showed that persistent diarrhoea accounted for 5% of cases,16 and with a similar study in zaire.17 in bangladesh, persistent diarrhoea accounted for nearly half of the child diarrhoeal deaths.18 this finding agrees with a study done in rural western kenya.19all pathogenic bacterial isolates were found to be resistant to amoxicillin. etec isolates, the most common cause of diarrhoea, were resistant to both amoxicillin and tetracycline, and partially resistant to ampicillin and erythromycin. these antimicrobials are some of the first-line drugs that are prescribed commonly in kenya; resistance is therefore a cause of concern. this finding is consistent with a related study done in rural western kenya, which found a high level of isolates to be resistant to the antibiotics prescribed most commonly in kenya, such as amoxicillin, ampicillin and tetracycline20 and was also consistent with a study on antimicrobial resistance conducted in nigeria.21 the mushrooming of clinics and pharmacies in eastleigh, with no control over the prescription and use of antibiotics, may explain the antibiotic resistance observed. similarly, previous studies conducted in developing countries have established that, in some locations, antibiotics can be purchased from private hospitals, pharmacies and patent medicine stalls without a prescription, even when the practice is not legal.22 although oral rehydration salt therapy (ors) is recommended as the primary management for cases of uncomplicated diarrhoea,23 only 30% of the diarrhoea cases were managed with ors, whilst 76% were treated immediately with antibiotics. we found poor handwashing practices to be a strong risk factor for diarrhoea. this is in agreement with a study done on sanitation which established that washing one’s hands with soap is another important barrier to transmission24 and has been cited as being one of the most cost-effective public health interventions.25 a number of studies have also shown that handwashing with soap can reduce the incidence of diarrhoeal disease by over 40%.24 we also found that most of the children with diarrhoeal illness had eaten food cooked the previous day. although we were not able to find other studies related to this finding, the observation raises questions on food-storage practices in this community. use of wide-mouthed containers for water storage was also a strong risk factor for diarrhoea, creating a risk of contamination from the environment. additionally, non-exclusive breastfeeding was associated significantly with diarrhoeal illness (p = 0.015). despite the fact that our study participants were all above two years old and past the typical age for breastfeeding, this study suggests a long-term benefit of breastfeeding, as has been reported elsewhere.26 limitations of the study top ↑ this study was limited to determining bacterial aetiology with respect to diarrhoea, therefore parasites and viruses which are known to account for most cases of childhood diarrhoea were not investigated. stool sample processing was not real time since the laboratory is located quite a distance from the study site. however, measures were taken to ensure that sample integrity was maintained, as outlined in the research method and design. recommendations we identified the need to evaluate the use of antibiotics in diarrhoeal treatment in order to institute measures to control and guide their use and to minimise the risk of antibiotic resistance. there is a need to scale up the use of ors in the management of diarrhoea, both at home and in the hospital, as well as efforts to improve personal and domestic hygiene. this should include conducting educational campaigns to promote appropriate handwashing using soap, especially at all critical times. conclusions top ↑ infectious diarrhoea remains a major public health problem amongst refugee children under the age of five in kenya. the pathogenic bacterial agents responsible for diarrhoea amongst these children were etec, shigella dysenteriae, shigella flexineri and salmonella paratyphi. the commonly-prescribed antibiotics prescribed for diarrhoea, such as amoxicillin and ampicillin, are facing threat of resistance from the enteric pathogens. acknowledgements top ↑ i would like to extend my heartfelt and sincere gratitude to my supervisors, dr joseph oundo and dr gideon kikuvi, for their tireless support in developing my protocol and thesis to this final product. in addition, let me thank my mentors in the aslm manuscript-writing workshop, elizabeth luman and william ampofo, who worked tirelessly to refine this manuscript. many thanks to the field epidemiology and laboratory training programme (feltp) members, dr jared omollo, dr samuel amwayi and mr ahmed abade, for the relentless and invaluable support they accorded me throughout my project. i feel greatly indebted to the entire staff of nairobi city council eastleigh clinic, including the nurse in charge of the facility, sister esther sakella, ms isabell oluoch, ms dorothy mutanu and the laboratory manager, m. samuel senzo. special thanks to the members of the scientific steering committee and the national ethical review board at kemri, for their assistance in protocol review and final study approval respectively. many thanks to ibrahim sora, victor otieno and martin bundi, of the centre for microbiology research (cmr) kemri, for their great assistance in laboratory analysis. i also thank my fellow classmates for their support and encouragement throughout this study. my gratitude also goes to the institute of tropical medicine and infectious diseases (itromid) staff members for their support and to the centers for disease control and prevention (cdc) for funding this study. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions w.g.b. (feltp, kenya), was the principal author and j.o. (feltp and cdc, kenya) was the corresponding author. g.k. (jomo kenyatta university, kenya), as well as j.o., a.a. and s.a. (feltp, kenya) were responsible for protocol development, whilst e.l. (cdc, u.s.) and w.a. 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from nigeria. epidemiol infect. 1990;105(1):59–64. http://dx.doi.org/10.1017/s0950268800047646 21. bojalil r, calva jj. antibiotic misuse in diarrhea. a household survey in a mexican community. j clin epidemiol. 1994;47(2):147–156. http://dx.doi.org/10.1016/0895-4356(94)90019-1 22. victora cg, bryce j, fontaine o, et al. reducing deaths from diarrhoea through oral rehydration therapy. bull world health organ. 2000;78(10):1246–1255. pmid:11100619, pmcid:pmc2560623 23. fewtrell l, kaufmann rb, kay d, et al. water, sanitation, and hygiene interventions to reduce diarrhoea in less developed countries: a systematic review and meta-analysis. lancet infect dis. 2005;5(1):42–52. http://dx.doi.org/10.1016/s1473-3099(04)01253-8 24. jamison dt., breman jg, measham ar, et al., editors. disease control priorities in developing countries. 2nd edition. washington, d.c.: world bank and oxford university press, 1993. pmcid:pmc309549 25. vanderslice j, popkin b, briscoe j. drinking-water quality, sanitation, and breast-feeding: their interactive effects on infant health. bull world health organ. 1994;72(4):589–601. pmid:7923538, pmcid:pmc2486614 26. nguyen tv, phung lv, chinh lh, et al. detection and characterization of diarrheagenic escherichia coli from young children in hanoi, vietnam. j clin microbiol. 2005;43(2):755–760. http://dx.doi.org/10.1128/jcm.43.2.755-760.2005, pmid:15695676, pmcid:pmc548099 abstract background description of the intervention lessons learnt recommendations acknowledgements references about the author(s) mary kirk i-tech, department of global health, schools of public health and medicine, university of washington, seattle, washington, united states paul h. assoa international training and education center for health (i-tech), abidjan, côte d’ivoire casey iiams-hauser i-tech, department of global health, schools of public health and medicine, university of washington, seattle, washington, united states yves-rolland kouabenan international training and education center for health (i-tech), abidjan, côte d’ivoire jennifer antilla department of global health, university of washington, seattle, washington, united states caleb steele-lane i-tech, department of global health, schools of public health and medicine, university of washington, seattle, washington, united states greg rossum i-tech, department of global health, schools of public health and medicine, university of washington, seattle, washington, united states pascal komena international training and education center for health (i-tech), abidjan, côte d’ivoire patricia sadate ngatchou i-tech, department of global health, schools of public health and medicine, university of washington, seattle, washington, united states nadine abiola international training and education center for health (i-tech), abidjan, côte d’ivoire alain kouakou direction de l’informatique et de l’information sanitaire (diis), ministry of health, abidjan, côte d’ivoire adama pongathie direction de l’informatique et de l’information sanitaire (diis), ministry of health, abidjan, côte d’ivoire jean b. koffi division of global hiv and tuberculosis, centers for disease control and prevention, abidjan, côte d’ivoire christiane adje division of global hiv and tuberculosis, centers for disease control and prevention, abidjan, côte d’ivoire lucy a. perrone i-tech, department of global health, schools of public health and medicine, university of washington, seattle, washington, united states citation kirk m, assoa ph, iiams-hauser c, et al. adaptation of an electronic dashboard to monitor hiv viral load testing in côte d’ivoire. afr j lab med. 2021;10(1), a1284. https://doi.org/10.4102/ajlm.v10i1.1284 lessons from the field adaptation of an electronic dashboard to monitor hiv viral load testing in côte d’ivoire mary kirk, paul h. assoa, casey iiams-hauser, yves-rolland kouabenan, jennifer antilla, caleb steele-lane, greg rossum, pascal komena, patricia sadate ngatchou, nadine abiola, alain kouakou, adama pongathie, jean b. koffi, christiane adje, lucy a. perrone received: 03 june 2020; accepted: 06 jan. 2021; published: 17 may 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the ministère de le santé et de l’hygiène publique in côte d’ivoire and the international community have invested in health information systems in côte d’ivoire since 2009, including electronic laboratory information systems. these systems have been implemented in more than 80 laboratories to date and capture all test results produced from these laboratories, including hiv viral load (vl) testing. in 2018 the national hiv programme in côte d’ivoire requested international support to develop real-time tools such as dashboards to aggregate and display test-specific data such as hiv vl testing to support the country’s programmatic response to hiv. intervention: the vl dashboard was adapted in 2018 using source software code obtained from the kenyan ministry of health and modified for the ivorian context. the dashboard enables users to assess relevant clinical data from all ivoirians living with hiv who undergo vl testing through dashboard data visualisations, including the number of vl tests, kinds of samples tested, and vl levels stratified by demographics and geographic location. lessons learnt: the vl dashboard enables rapid analysis of vl testing data from across the country and enables the national hiv programme, donors and partners to respond rapidly to issues pertaining to access, turn-around times and others. recommendations: adapting existing open-source software is an effective and efficient way to implement transformative tools such as dashboards. the vl dashboard will likely be an essential tool for côte d’ivoire to meet the united nations programme on hiv/aids 90-90-90 targets. keywords: hiv viral load; electronic dashboard; patient monitoring; laboratory information system; côte d’ivoire. background as of 2018, côte d’ivoire had approximately 460 000 people living with hiv1 and an estimated hiv prevalence rate of 2.6%,1 the highest in west africa.2 to address unmet goals for the joint united nations programme on aids/hiv 90-90-90 targets, côte d’ivoire adopted the test and start approach in 2015, a proven cost-effective and lifesaving intervention.3 côte d’ivoire relies on viral load (vl) testing as the gold standard for clinical monitoring of hiv, yet vl testing remains limited in côte d’ivoire despite 17 laboratories having molecular testing capability. côte d’ivoire is working to scale up access to vl testing and optimise how test results are utilised for effective anti-retroviral therapy monitoring in pateints.4 the national hiv programme and the healthcare workforce in côte d’ivoire have been advocating with the international donor community for effective, user-friendly health information systems and monitoring tools.5 in response to this request, the united states centers for disease control and prevention engaged the international training and education center for health (i-tech) at the university of washington in 2009 to support the ministère de le santé et de l’hygiene publique and the national hiv programme (programme nationale de lutte contre le sida [pnls]) to improve the quality of care of people living with hiv by strengthening electronic laboratory information systems as part of the national health information system architecture. openelis (electronic laboratory information systems) (oe) is an open-source electronic laboratory information system that tracks laboratory processes and outputs and was created in the united states for public health laboratories as a software and business process framework.6 starting in 2009, oe was adapted for côte d’ivoire with i-tech’s support and 82 laboratories to date are implementing the system across the nation. although implementation of oe continues to expand and efforts are underway to interlink individual operating sites and their data through a centrally located consolidated server, obtaining aggregated hiv-related testing data from all sites remains challenging. in 2018 the ministère de le santé et de l’hygiene publique requested support to develop and deploy a dashboard solution for hiv vl testing. dashboards are increasingly used in clinic-based interventions7 as well as in population-based disease assessments.8 these dashboards can empower stakeholders to make decisions with greater efficiency over time.9 one primary function of dashboards is displaying data so that stakeholders can analyse key metrics to monitor progress to targets, identify challenges and mobilise to improve service quality.10 dashboards have been used for reporting purposes in hiv programme monitoring efforts. in uganda, option b+ usage has been documented since 2013 via a nationwide weekly reporting system using short message service technology.11 kenya also uses a dashboard to support the national review of vl and infant virologic test programme data through the integration of open-source laboratory information systems with cloud-based servers.12 namibia developed a pharmaceutical management information dashboard that interlinks four pharmaceutical information tools to serve as a platform for analysis and dissemination to improve anti-retroviral therapy delivery.13 here we describe the development of the vl dashboard for côte d’ivoire which is connected to the oe system and aggregates site-level oe data that can be viewed on a publicly available website. these visualisations permit the rapid examination of hiv-related testing data in côte d’ivoire, including spatial and temporal attributes of vl testing coverage, and support timely programme policymaking. description of the intervention ethical considerations ethical clearance for this project was given by the university of washington, united states centers for disease control and prevention and ivorian institutional review board. all de-identified data are publicly available on the dashboard website. dashboard development in october 2017, i-tech coordinated with the united states agency for international development, clinton health access initiative, and the kenyan ministry of health to access the software source code for the kenyan vl dashboard (https://viralload.nascop.org/).14 using this software source code as a starting point, the ivorian vl dashboard was developed to interface with the oe system in côte d’ivoire and designed to extract and aggregate vl test-specific data. populating the dashboard with data initially occurred through the manual extraction of a comma-separated value file from each oe system at each laboratory. trained staff then exported this file to data quality analysts through remote connection access or email. a data validation check was then conducted to ensure that data were not missing or inaccurate, otherwise it was sent back to the laboratories for correction in the oe system. once individual laboratory data were validated and complete, these cleaned data were then manually uploaded to the vl dashboard (note that a direct link from the oe systems to the vl dashboard through a national consolidated server is in its pilot phase as of september 2020, and this automation will remove the need for manual data management processes). the visualisations that populate the vl dashboard were adapted from the kenyan dashboard to the ivorian context, including geography, language, and data sources. dashboard features the vl dashboard was designed to display a range of data visualisations that can be sorted by month, year and region (figure 1). the primary source data for ‘trends by testing sample’ (test sample type by month) can be exported to microsoft excel (microsoft corp. redmond, washington, united states); the remaining data visualisations are available as graphic downloads. test sample type (dried blood spot or ethylenediaminetetraacetic acid plasma), demographic data (age, sex, and region), test results (≥/< 1000 copies, invalid, or undetectable), and clinical justification for the vl test can be visualised on the vl dashboard. the turn-around times of the clinical vl samples can also be visualised, with the graphic displaying times from collection of the sample to reception at a laboratory, reception to processing, and processing to sample validation. data submitted by each laboratory are available by regionally filtering data, with test numbers by results shown on the ‘region sites outcomes’ graph on the dashboard. a ‘visitor counter’ tracking feature was added to the dashboard in january 2020, showing the total number of visits, number of visits to a specific page of the dashboard, and the number of people online. figure 1: number of viral load tests in côte d’ivoire by region, october – december 2019. the 20 regions of the country are covered by 17 public laboratories that are capable of molecular testing for hiv viral load. the capital abidjan is divided into two jurisdictions based on geospatial parameters with the most people living with hiv located in abidjan 2. this graph was produced directly from the viral load dashboard. lessons learnt the vl dashboard was completed in june 2018 (figure 2). in july 2018, initial pilot data from the dashboard were presented to ministère de le santé et de l’hygiene publique and donor stakeholders at a technical working group meeting for final dashboard approval. the website became publicly available in july 2018 (https://chargevirale.openelisci.org/vl_dashboard/), with an initial 10 vl testing laboratories successfully transmitting data to the dashboard via oe. the remaining seven laboratories started data sharing from january 2019, and retrospective data from october 2016–2018 were obtained, cleaned, and uploaded into the dashboard. the vl dashboard successfully reported data for all people living with hiv who received vl testing services in côte d’ivoire, with each unique test event recorded in the dashboard. data from october 2016 to december 2019 indicated that a total of 622 500 samples were tested for vl, of which 452 896 (72.8%) samples were from women and 169 604 (27.2%) from men. regional disaggregation of the data accounts for 616 812 vl tests, with the remaining 5688 tests having lost location traceability and are currently under investigation. of the 616 812 traceable vl tests, the two abidjan catchment areas accounted for 40.6% (n = 252 510) of the tests in the dashboard. of the vl tests reported in the dashboard, 89.6% were from patients aged 25 years or older. since the dashboard became active, the sample processing rate has increased, reflecting increased vl testing access and scale-up and increased test demand after national prioritisation in 2016 (figure 3). data from october 2016 to december 2019 indicate that 56.9% (354 184 tests) of people living with hiv receiving anti-retroviral therapy have viral suppression. people living with hiv aged 25 years or older had the highest suppression rate (79%), and paediatric and adolescent patients (aged 0–19 years) had the lowest rates. paediatric (age < 10 years) viral suppression rates ranged from 47.6% to 56.9%. adolescent (age 10–19 years) suppression rates ranged from 53.9% to 55.2%. although the dashboard indicated that the national average turn-around time for ethylenediaminetetraacetic acid plasma samples decreased from more than 50 days to 16 days (figure 4), this does not yet meet the national target of under 14 days. viral load results obtained from tests performed on dried blood spots were introduced in 2018. figure 2: timeline of viral load dashboard development. the development of the viral load dashboard began in october 2017 and a pilot version was completed in july 2018. following adjustments to the software code the dashboard was finalised in january 2019 and is now populating data from all viral load testing laboratories. figure 3: number of viral load tests reported in côte d’ivoire, october 2016 – december 2019. the viral load dashboard captured all viral load testing data since its beginning in côte d’ivoire, october 2016. as the country committed to scaling up viral load testing access to people living with hiv, the number of total tests increased. the data represented above show this trend, which is expected to continue. figure 4: viral load testing turn-around time by sample type and by year that each type of viral load test was used. (a) ethylenediaminetetraacetic acid plasma 2016–2019; (b) dried blood spots 2018–2019, in côte d’ivoire. turn-around time is further disaggregated by collection to reception (c-r), reception to processing (r-p), and processing to validation (p-v). the adaptation, development and launch of the vl dashboard in côte d’ivoire is a collaborative success story and a milestone achievement for the national hiv programme and for international donors such as the united states president’s emergency plan for aids relief. the information available through the dashboard will help accelerate decision-making and programmatic response time,11 especially the ability to monitor key subpopulations. for example, paediatric and adolescent viral suppression rates are observably lower (53.4%) than adults aged 25 years or older (79.8%), and the dashboard is being used to monitor this population subset and track their clinical outcomes. it is noted that these vl suppression rates by age groups mirror trends among paediatric, adolescent, and adult patients in other resource-limited countries.15 this kind of information is critical to inform programmes and initiatives like the determined, resilient, empowered, aids-free, mentored, and safe, which serves adolescent girls and young women. in another example of the utility of the dashboard, although only 23.9% of all people living with hiv in côte d’ivoire1 live in the capital region of abidjan, dashboard data from october 2016 through december 2019 show that a large proportion of vl samples come from this region (n = 252 510 tests, 40.6%; figure 2). this finding suggests that more people from abidjan are receiving vl tests compared with other provinces and these data are now informing programmatic prioritisation. organisations in côte d’ivoire are now rapidly utilising vl dashboard information in programmatic and technical working group meetings. the pnls uses the data and visualisations from the dashboard in monthly reviews with implementing partners and other ministerial departments. the data from the vl dashboard is trusted in the pnls’s decision-making processes. one of the key success factors in the development of the dashboard was the collaborative nature of the work in each step of the process: from planning to design and implementation. the willingness of the developers of the kenyan dashboard to share the source code immensely benefited côte d’ivoire, as it shortened product launch time, reduced potential costs and prevented duplication of efforts to implement a dashboard for ivorian vl testing services. in the adaptation phase, i-tech worked closely with the direction de l’informatique et l’information sanitaire, the pnls, and the côte d’ivoire national hiv reference laboratory in monthly technical working group meetings. the technical working group first reviewed the data available in oe and mapped the data to the indicator calculations. after the vl dashboard was launched, these high-functioning technical working groups provided a forum to address challenges and improve the dashboard and included representatives from the direction de l’informatique et l’information sanitaire, pnls, laboratoire national de la santé public, and implementing partners. this ensured that all stakeholders were involved in the process. the vl dashboard has been a helpful tool in monitoring hiv trends towards meeting the ‘third 90’ united nations programme on hiv/aids goal and additional developments are underway to enhance features and improve clinical utilisation. the vl dashboard is also a model for the new early infant diagnosis dashboard launched in may 2020.16 completion of the consolidated national server, which is in its pilot phase since september 2020, will allow for rapid data importation from oe to the vl dashboard. plans are also underway to transfer ownership of the dashboard’s internet protocol address, currently owned by the university of washington, to the ministère de le santé et de l’hygiene publique-direction de l’informatique et l’information sanitaire. recommendations dashboards are helpful tools for public health programmes. the vl dashboard in côte d’ivoire is poised to be a transformational tool that improves the national response to hiv by displaying key data including the number of vl tests, kinds of samples tested, and vl levels stratified by demographics and geographic location. this dashboard enables rapid analysis of vl testing data from testing points across the country and will enable the pnls and other implementing partners to respond rapidly to vl testing access, delayed turn-around time, and data quality issues. the vl dashboard will help côte d’ivoire scale up vl monitoring to help meet the united nations programme on hiv/aids 90-90-90 targets. acknowledgements we thank colleagues at the united states centers for disease control and prevention for their support in developing the dashboard, colleagues in the ministère de le santé et de l’hygiene publique, clinical partner implementers, and all users of oe and the vl dashboard for providing valuable feedback for improving these tools. we thank clinton health access initiative/united states agency for international development kenya for sharing the vl dashboard software code. we thank eric zakpa, armande kouame, marisa van osdale, rama ouattara, and cat koehn for their programmatic and operational support. this project was determined to be non-research by the university of washington institutional review board, and the project was approved by the national institutional review board committee in côte d’ivoire, le comite’ national d’ethique des sciences de la vie et de la sante’. this project was also reviewed in accordance with the centers for disease control and prevention human research protection procedures and was determined to be a non-research programme evaluation. competing interests the authors have declared that no competing interests exist. authors’ contributions p.h.a., c.i.-h., c.s.-l., g.r., j.a., p.k., p.s.n., a.k., and a.p. conceived and designed the work. y.-r.k., m.k., n.a., and l.a.p. acquired, analysed, and interpreted data. p.h.a., c.i.-h., m.k., and l.a.p. wrote the original draft. j.b.k. and c.a. provided project funding and technical direction. y.r.k., m.k., n.a., g.r., and l.a.p. reviewed, revised, and critically edited the draft. all authors provided final approval of the version to be published and agree to be accountable for all aspects of the work. sources of support this work has been supported by the president’s emergency plan for aids relief through the united states centers for disease control and prevention under the terms of the cooperative agreement awarded to the university of washington-i-tech (6 nu2ggh001968-03-02). data availability the data that support the findings of this study are publicly available at: https://chargevirale.openelisci.org/vl_dashboard/. disclaimer the findings and conclusion in this report are those of the authors and do not necessarily represent the official position of the funding agencies. references unaids. aidsinfo: côte d’ivoire data sheet [homepage on the internet]. 2018 [cited 2020 jan 20]. available from: https://aidsinfo.unaids.org/ gbd 2015 collaborators. estimates of global, regional and national incidence, prevalence, and mortality of hiv, 1980–2015: the global burden of disease study 2015. lancet. 2016;3:e361–e387. estill j, egger m, blaser n, et al. cost-effectiveness of point-of-care viral load monitoring of antiretroviral therapy in resource-limited settings: mathematical modelling study. aids. 2013;27(9):1483–1492. https://doi.org/10.1097/qad.0b013e328360a4e5 maheu-giroux m, vesga j, diabaté s, et al. population-level impact of an accelerated hiv response plan to reach the unaids 90-90-90 target in côte d’ivoire: insights from mathematical modeling. plos med. 2017;14(6):e1002321. https://doi.org/10.1371/journal.pmed.1002321 rowan bh, robinson j, granato a, et al. workforce patterns in the prevention of mother to child transmission of hiv in côte d’ivoire: a qualitative model. hum resour health. 2018:16(4). https://doi.org/10.1186/s12960-018-0268-x university of washington. openelis global: a global lis. viewed 29 january 2021, from https://sites.google.com/site/openelisglobal/ enane la, vreeman rc, foster c. retention and adherence: global challenges for the long-term care of adolescents and young adults living with hiv. curr opin hiv aids. 2018;13(3):212–219. https://doi.org/10.1097/coh.0000000000000459 crofts j, moyo j, ndebele w, et al. adaption and implementation of local maternity dashboards in a zimbabwean hospital to drive clinical improvement. bull world health organ. 2014;92(2):146–152. https://doi.org/10.2471/blt.13.124347 cheng ck, ip dk, cowling bj, ho lm, leung gm, lau eh. digital dashboard design using multiple data streams for disease surveillance with influenza surveillance as an example. j med internet res. 2011;13(4):e85. https://doi.org/10.2196/jmir.1658 world health organization. considerations for developing a monitoring and evaluation framework for viral load testing. 2019; who/cds/hiv/19.5. radin ak, abutu aa, okwero ma, et al. confronting challenges in monitoring and evaluation: innovation in the context of the global plan towards the elimination of new hiv infections among children by 2015 and keeping their mothers alive. j acquir immune defic syndr. 2017;75(suppl 1):s66–s75. https://doi.org/10.1097/qai.0000000000001313 vrazo ac, sullivan d, ryan phelps b. eliminating mother-to-child transmission of hiv by 2030: 5 strategies to ensure continued progress. glob health sci pract. 2018;6(2):249–256. https://doi.org/10.9745/ghsp-d-17-00097 mabirizi d, phulu b, churfo w. implementing an integrated pharmaceutical management information system for antiretrovirals and other medicines: lessons from namibia. glob health sci pract. 2018;6(4):723–735. https://doi.org/10.9745/ghsp-d-18-00157 national aids/std control program (nascop), ministry of health government of kenya. viral load dashboard. viewed 29 january 2021, from https://viralload.nascop.org/ gous nm, onyebujoh pc, abimiku a, et al. the role of connected diagnostics in strengthening regional, national and continental african disease surveillance. afr j lab med. 2018;7(2):a775. https://doi.org/10.4102/ajlm.v7i2 ministère de le santé et de l’hygiène publique (mshp), government of côte d’ivoire. eid dashboard. 2020. viewed 29 january 2021, from https://chargevirale.openelisci.org/eid_dashboard/en/ abstract introduction methods results discussion acknowledgements references about the author(s) hyerim kim department of laboratory medicine, pusan national university hospital, busan, republic of korea jongmin kim department of laboratory medicine, pusan national university hospital, busan, republic of korea citation kim h, kim j. effect of delayed sample draw after blood collection for haemoglobin test in south korea. afr j lab med. 2023;12(1), a2008. https://doi.org/10.4102/ajlm.v12i1.2008 note: additional supporting information may be found in the online version of this article as online supplementary document 1 brief report effect of delayed sample draw after blood collection for haemoglobin test in south korea hyerim kim, jongmin kim received: 08 july 2022; accepted: 06 jan. 2023; published: 28 mar. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract between april and may 2022, 10 healthy adult non-patients were recruited from pusan national university hospital. venous blood drawn into a syringe was transferred into test tubes with a zero-to-45-minute delay. the transfer was done sequentially in two positions with the syringe and the needle adaptor end (1) heading downwards and (2) heading upwards. haemoglobin levels gradually increased over time in position 1 transfer while they gradually decreased in position 2. therefore, blood must be transferred quickly from a syringe to a tube for reliable test results. what this study adds: our findings confirm that delays between blood collection and transfer can affect haemoglobin levels. keywords: haemoglobin; blood specimen collection; erythrocyte count; specimen handling; phlebotomy. introduction blood collection is necessary since numerous tests are performed using blood specimens. inadequate blood sampling may lead to inaccurate results and mislead the clinician; therefore, collecting blood according to best practice guidelines is essential. current guidelines presented by the world health organization, clinical and laboratory standards institute, and european federation of clinical chemistry and laboratory medicine-latin america confederation of clinical biochemistry provides evidence-based recommendations for the stepwise process of adequate blood sampling.1,2,3 however, there is no detailed guidance on the time interval between collecting blood in the syringe and transferring blood to the test tube when using the needle and syringe system. since these guidelines recommend using vacuum extraction instead of the needle and syringe system, there seems to be no need for guidance on transferring blood to the test tube. nevertheless, the needle and syringe system is most commonly used in blood sampling,2 and delay in transferring blood to the test tube following sampling can occur, which may alter the results of blood tests, for instance haemoglobin levels. however, to the best of our knowledge, studies on the effect of delayed blood transfer to the test tube after phlebotomy on haemoglobin level results are lacking. we were asked to evaluate the effect of delayed blood transfer from the syringe to the test tube after receiving complaints of erroneous haemoglobin level changes in several patients (table 1). the physicians observed unanticipated changes in haemoglobin, such as a significant decline in haemoglobin in the absence of relevant events such as bleeding, surgery, or trauma. an incorrect result of haemoglobin level may mislead the clinician in monitoring the patient’s overall health and diagnosing haematologic diseases, including anaemia. the errors can be divided into pre-preanalytical, preanalytical, analytical, postanalytical, and post-postanalytical phases.4 up to 68% of errors are pre-preanalytical,4 including the well-known sample quality factors: haemolysis, cryoglobulin, lipaemia, and clotting issues.5,6 therefore, possible errors in all test phases were carefully examined. after excluding all potential causes of error, we found that all reported cases were sampled using the needle and syringe system and assumed that a delay between blood collection and blood transfer to the test tube may have resulted in a change in haemoglobin levels. this assumption is based on the hypothesis that the sedimentation of red blood cells occurred during the prolonged storage of sampled blood in syringes. red blood cell sedimentation is well known, as in the erythrocyte sedimentation rate (esr) test.7 however, red blood cell sedimentation in a phlebotomy setting and its effect on haemoglobin test results is understudied. thus, we conducted this study to evaluate the effect of delayed blood sample transfer on the haemoglobin level by analysing samples with different time delays under two conditions: drawing blood from the sedimented bottom portion or the non-sedimented upper portion of the syringe. table 1: reported cases of erroneous haemoglobin levels, south korea, april 2022 may 2022. methods ethical considerations an application for full ethical approval was made to the institutional review board of pusan national university hospital. the ethics approval number is 2204-004-113. all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional or national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards. written informed consent was obtained from all individual participants involved in the study. the collected personal data was saved on a password-protected computer that only the authors had access to. participant recruitment and data collection between april 2022 and may 2022, 10 healthy adult non-patients were recruited at pusan national university hospital, busan, south korea, using a recruiting poster. the volunteers were eligible for inclusion if they were aged between 20 and 60 years and weighed over 50 kg. each participant was asked a series of prepared questions to determine their eligibility and collect information about them, such as their gender, age, and medical history. volunteers were excluded if they were pregnant, had a history of a blood clotting disorder, or had inadequate venous blood collected. blood collection a trained laboratory technician collected blood from the veins in the antecubital fossa, once in each arm. first, blood was collected using a vacuum extraction system (multiple sample blood collection 21g needle and quick release needle holder, becton, dickinson and company, new jersey, united states). next, 3 ml of blood for the k2 ethylenediaminetetraacetic acid (edta) tube and 2.7 ml for the sodium citrate tube (becton, dickinson and company, new jersey, united states) were collected, which we defined as control samples. on the opposite arm, 3 ml of blood was collected using a needle and syringe system with a 24g winged butterfly needle (jms(k) medical supply, seoul, republic of korea) into ten 10 ml syringes (jung rim medical industrial, chungcheongbuk-do, republic of korea). the winged butterfly needle was used instead of the original needle connected to the syringe to minimise the number of venepunctures performed per participant. the syringes were placed horizontally on the ground without assistive devices. one ml of blood was transferred into two 3 ml k2 edta tubes at 0, 5, 10, 15, 20, 25, 30, 35, 40, and 45 min in two positions in the following order: (1) syringe vertically positioned with needle adaptor end heading downwards and (2) syringe vertically positioned with needle adaptor end heading upwards (figure 1). these edta tubes were defined as the test samples. figure 1: illustration of needle and syringe positioning during blood transfer, south korea, april 2022 – may 2022. (a) position 1, syringe vertically positioned with needle adaptor end heading downwards. (b) position 2, syringe vertically positioned with needle adaptor end heading upwards. outcome assessment the activated partial thromboplastin and prothrombin times were determined using sodium citrate samples in cs-5100 (sysmex corporation, kobe, japan), esr in edta tubes using test1 (alifax, padova, italy), and complete blood count in edta tubes using xn-9000 (sysmex corporation, kobe, japan). for test samples, complete blood count was examined, and the changes in haemoglobin levels were analysed to evaluate the effect of delayed blood transfer. statistical analysis the kruskal-wallis test was used to analyse haemoglobin levels over time. the kolmogorov-smirnov test and the shapiro-wilk test were adopted to test normality. all statistical analyses were performed by spss® 22 for windows (spss inc., chicago, illinois, united states). a p-value of less than 0.05 was considered statistically significant. the line graphs were generated using microsoft excel, and illustrations using microsoft powerpoint (microsoft corporation, redmond, washington, united states). results the baseline characteristics of 10 participants, three male and seven female (online supplementary table 1), show that none had comorbidities. the haemoglobin levels of the control samples were consistent with the 0 min sample of the test sample. in addition, the control samples’ prothrombin times, activated partial thromboplastin, and esr results were within the normal range except for those of two participants, which presented mildly elevated esr. among the control sample results, including esr, activated partial thromboplastin, prothrombin times, and international normalised ratio, none seemed to be correlated with the amount of haemoglobin change over time. the results of samples transferred while the needle adaptor end faced downwards had an increase in haemoglobin levels with time (figure 2, p < 0.001). the average haemoglobin level increased from 13.9 g/dl at 0 min to 20.2 g/dl at 45 min. compared to the 0 min sample, the changes appeared to be statistically significant at 35 min (p = 0.006), 40 min (p = 0.001), and 45 min (p < 0.001). figure 2: changes of haemoglobin level (g/dl) over delayed blood transfer in position, south korea, april 2022 – may 2022. (a) represents the 10 cases of position 1, and (b) shows the increase of average haemoglobin levels of 10 cases (p < 0.001, kruskal-wallis test). statistically significant changes between 0 min and 35 min (p = 0.006), 0 min and 40 min (p = 0.001), 0 min and 45 min (p < 0.001), 5 min and 35 min (p = 0.005), 5 min and 40 min (p = 0.001), 5 min and 45 min (p < 0.001), 10 min and 35 min (p = 0.011), 10 min and 40 min (p = 0.01), 10 min and 45 min (p < 0.001), 15 min and 35 min (p = 0.05), 15 min and 40 min (p = 0.008), and 15 min and 45 min (p = 0.002) were observed in the post hoc test. gradual decreases in haemoglobin levels were observed in samples transferred with the needle adaptor end facing up (figure 3, p < 0.001). the average haemoglobin level dropped from 14.2 g/dl at 0 min to 6.2 g/dl at 45 min. when compared with 0 min, the changes appeared to be statistically significant in 35 min (p = 0.033), 40 min (p = 0.006), and 45 min (p = 0.001). figure 3: change in haemoglobin level (g/dl) over delayed blood transfer in position 2, south korea, april 2022 – may 2022. (a) represents the 10 cases of position 2, and (b) shows the decrease of average haemoglobin levels of 10 cases (p < 0.001, kruskal-wallis test). statistically significant changes between 0 min and 35 min (p = 0.033), 0 min and 40 min (p = 0.006), 0 min and 45 min (p = 0.001), 5 min and 40 min (p = 0.013), 5 min and 45 min (p = 0.001), 10 min and 40 min (p = 0.02), 10 min and 45 min (p = 0.002), and 15 min and 45 min (p = 0.008) were observed in the post hoc test. discussion the findings of this study confirm that the interval between blood collection and blood transfer can affect haemoglobin levels. regardless of needle position, the delay between the blood draw and the blood transfer led to incorrect haemoglobin levels. when the syringe was positioned vertically with the needle adaptor end heading upwards when transferring, the haemoglobin levels dropped with time. on the other hand, the haemoglobin levels increased with time when transferring blood with the needle adaptor end heading downwards. we believe that the transfer delay may have induced sedimentation of the sample transferring the supernatant, that is, needle adaptor end heading upwards, resulting in low haemoglobin levels and transferring the lower part of sedimentation, that is, needle adaptor end heading downwards, results in increased haemoglobin levels. limitations there are several limitations. firstly, as all participants were between 31 and 41 years, the study samples may not represent the general population. the previously reported erroneous cases (table 1) were relatively older patients, aged from 38 to 81. secondly, blood transfer with the needle pointing downwards was performed after transferring blood with the needle pointing downwards; thus, the results of the former position may have been affected by the inversion. thirdly, we used a 24g butterfly needle instead of the original 21g needle connected to the 10 ml syringe, which is one of our institution’s general settings. therefore, the difference in needle gauges can be associated with erroneous factors, such as haemolysis.8,9 however, all 10 samples were obtained in the same position with the same needle. the needle change divulges the effects of delayed blood transfer. lastly, in this study, only 1 ml of blood was transferred into 3 ml edta tubes to minimise the amount of blood sampling per participant. however, a study reported that acceptable complete blood count values could be obtained with as little as 1 ml of blood in 4 ml k2 edta tubes.10 therefore, we believe that the method of this study using 1 ml of blood in 3.0 ml k2 edta tubes can be considered relevant. conclusion in conclusion, the findings of this study show that delayed blood transfer after venepuncture may lead to changes in haemoglobin levels; thus, blood should preferably be transferred directly into test tubes after collection. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions h.k. conceived of the presented idea and designed the experiments. j.k. performed the experiments, analysed data, and wrote the manuscript with support from h.k. sources of support this work was supported by a clinical research funding from pusan national university hospital in 2021. data availability the authors confirm that the data supporting the findings of this study are available within the article. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references institute cls. gp41: procedures for collection of diagnostic blood specimens by venipuncture; approved guideline. 7th ed. wayne, pa: clinical and laboratory standards institute; 2007. world health organization. who guidelines on drawing blood: best practices in phlebotomy. who; 2010 [cited 2022 june 13]. available from: http://whqlibdoc.who.int/publications/2010/9789241599221_eng.pdf simundic am, bölenius k, cadamuro j, et al. joint eflm-colabiocli recommendation for venous blood sampling. clin chem lab med. 2018;56(12):2015–2038. https://doi.org/10.1515/cclm-2018-0602 zandecki m, genevieve f, gerard j, godon a. spurious counts and spurious results on haematology analysers: a review. part ii: white blood cells, red blood cells, haemoglobin, red cell indices and reticulocytes. clin lab haematol. 2007;29(1):21–41. https://doi.org/10.1111/j.1365-2257.2006.00871.x de la salle b. preand postanalytical errors in haematology. int j lab hematol. 2019;41(s1):170–176. https://doi.org/10.1111/ijlh.13007 richard a, mcpherson mrp. henry’s clinical diagnosis and management by laboratory methods. 24th ed. st. louis, mo: elsevier; 2021. daniel b, yuan cs. effects of dietary supplements on coagulation and platelet function. thromb res. 2005;117(1):49–53. https://doi.org/10.1016/j.thromres.2005.04.017 lippi g, salvagno gl, montagnana m, brocco g, cesare guidi g. influence of the needle bore size used for collecting venous blood samples on routine clinical chemistry testing. clin chem lab med. 2006;44(8):1009–1014. https://doi.org/10.1515/cclm.2006.172 sharp mk, mohammad sf. scaling of hemolysis in needles and catheters. ann biomed eng. 1998;26(5):788–797. https://doi.org/10.1114/1.65 xu m, robbe va, jack rm, rutledge jc. under-filled blood collection tubes containing k2edta as anticoagulant are acceptable for automated complete blood counts, white blood cell differential, and reticulocyte count. int j lab hematol. 2010;32(5):491–497. https://doi.org/10.1111/j.1751-553x.2009.01211.x abstract introduction methods results discussion acknowledgements references about the author(s) promise c. nwaejigh department of chemical pathology and immunology, faculty of basic medical sciences, olabisi onabanjo university, sagamu, nigeria maria o. ebesunun department of chemical pathology and immunology, faculty of basic medical sciences, olabisi onabanjo university, sagamu, nigeria oluwaseye m. oladimeji department of medicine, lagos state university teaching hospital, ikeja, nigeria citation nwaejigh pc, ebesunun mo, oladimeji om. lecithin-cholesterol acyltransferase and paraoxonase-1 levels in atherosclerotic cardiovascular disease patients in nigeria. afr j lab med. 2024;13(1), a2286. https://doi.org/10.4102/ajlm.v13i1.2286 note: additional supporting information may be found in the online version of this article as online supplementary document 1. original research lecithin-cholesterol acyltransferase and paraoxonase-1 levels in atherosclerotic cardiovascular disease patients in nigeria promise c. nwaejigh, maria o. ebesunun, oluwaseye m. oladimeji received: 08 aug. 2023; accepted: 02 may 2024; published: 17 july 2024 copyright: © 2024. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: recent evidence has linked changes in plasma lecithin-cholesterol acyltransferase (lcat) and paraoxonase-1 (pon-1) levels with increased risk for development of premature atherosclerotic cardiovascular disease (ascvd) in different populations. however, studies on this in nigeria and sub-saharan africa are scarce. objective: this study assessed the association between reduced plasma lcat and pon-1 levels and an increased risk of ascvd, and their potential as biomarkers for ascvd. methods: atherosclerotic cardiovascular disease patients and healthy controls were randomly selected for this cross-sectional case-control study from the lagos state university teaching hospital, ikeja, lagos, nigeria, between march 2022 and march 2023. plasma lcat and pon-1 were determined by sandwich enzyme-linked immunosorbent assay, while the lipid profile was measured by spectrophotometry. results: a total of 153 ascvd patients (mean age: 52.92 ± 10.24 years) and 50 healthy controls (mean age: 46.96 ± 11.05 years) were included in the analyses. stastistically significant increases were observed in the mean body weight, hip circumference, waist circumference, waist-to-hip ratio, body mass index, diastolic and systolic blood pressure (all p ≤ 0.001), and pulse rate (p = 0.003) compared to the control values. statistically significant increases were also observed in the mean plasma total cholesterol, triglycerides, low-density lipoprotein cholesterol, and non-high-density lipoprotein cholesterol (all p ≤ 0.001). in contrast, the mean plasma high-density lipoprotein cholesterol, lcat, and pon-1 (p ≤ 0.001) were notably reduced compared to the control values. conclusion: the present study provides supportive evidence that changes in plasma lcat and pon-1 could predispose individuals to risk of premature ascvd. what this study adds: plasma lcat and pon-1 may serve as independent markers or complement other established cardiovascular disease markers to discriminate the risk of ascvd when it is unclear. keywords: atherosclerotic cardiovascular disease; lecithin-cholesterol acyltransferase; paraoxonase-1; lipid profile; anthropometric variables. introduction atherosclerotic cardiovascular disease (ascvd) is characterised by plaque buildup in the arteries, leading to reduced blood flow and an increased risk of heart attacks and strokes.1 it poses a major health concern worldwide due to its high morbidity and mortality rates.2 despite advances in treating ascvd, identifying novel risk factors for ascvd remains crucial for improving patient outcomes. in recent years, studies in europe3,4 and asia5,6 have focused on the role of plasma lecithin-cholesterol acyltransferase (lcat) and paraoxonase-1 (pon-1) levels as potential biomarkers for predicting cardiovascular risk. however, there is a paucity of information on the relationship between these biomarkers (lcat and pon-1) and ascvd in africa, especially in sub-saharan africa. paraoxonase-1 is an enzyme primarily synthesised in the liver and has been shown to have a protective effect against atherosclerosis,7 which is the underlying cause of several cardiovascular diseases, including ascvd. some studies have shown that individuals with low levels of pon-1 activity and concentrations have an increased risk of developing ascvd, while others have found no association between decreased pon-1 activity and ascvd.6,8 reduced pon-1 concentrations in ascvd patients suggest that pon-1 may play a protective role against the development of ascvd. low pon-1 concentrations have also been shown to be a predictor of ascvd risk.7 in one study, individuals with low pon-1 activity were found to have a 2.4-fold increased risk of developing ascvd.9 it is also important to note that the relationship between pon-1 activity and ascvd is complex and multifactorial. genetic variation, environmental factors, and lifestyle habits can also influence the regulation of pon-1 concentrations and, subsequently, ascvd risks.10,11 for example, smoking has been shown to reduce pon-1 activity,12 while diets high in antioxidants can increase pon-1 activity.11 on the other hand, lcat has long been recognised as a key regulator of high-density lipoprotein metabolism; however, the role of this enzyme in human atherogenesis and ascvd has remained controversial.13 earlier studies reported increases14,15 and decreases3,5 in plasma lcat activity and concentrations in patients with ascvd. studies have also reported that plasma lcat concentrations can be regulated by a complex interplay of several factors, such as genetics, hormonal, dietary, inflammatory, medication, and lifestyle factors.15,16 furthermore, research has suggested that there may be a correlation between pon-1 and lcat concentrations, as both enzymes are involved in lipid metabolism and may be regulated by similar factors. earlier studies have also linked lcat and pon-1 to several chronic diseases beyond ascvd, including diabetes, chronic kidney disease, and alzheimer’s disease.17,18,19 this study assessed the association between reduced plasma lcat and pon-1 levels and an increased risk of ascvd, and their significance as potential biomarkers for ascvd in nigeria and beyond. this study examined plasma lcat, pon-1, lipid profile, and anthropometric indices in ascvd patients. methods ethical considerations ethical approval for this study was obtained from the university’s health research and ethics committee of the lagos state university teaching hospital, ikeja, lagos, nigeria (reference number: lrec/06/10/1706). written informed consent was obtained from each enrollee to participate in the study; all participants were residents of lagos state, nigeria. participants’ anonymity was maintained using a barcode system, and the generated data were treated with the utmost confidentiality. the generated data were used only for this research. study design the study was a cross-sectional case-control study. the sample size of 138 was determined using the prevalence of a previous study in the region,20 based on a 10% prevalence rate of cardiovascular disease in nigeria.21 the study enrolled confirmed ascvd patients attending the cardio clinic and those admitted into the state university teaching hospital emergency medical wards (both located at lagos state university teaching hospital, ikeja, lagos, nigeria) between march 2022 and march 2023. a simple random sampling technique was used to select patients diagnosed with ischaemic heart disease (ihd), stroke, hypertensive heart disease, myocardial infarction and congestive heart failure as diagnosed by the attending consultant cardiologist based on the patient’s medical history, clinical presentation, electrocardiographic changes, echocardiographic changes, and biochemical characteristics. for the control group, 50 healthy volunteers with no history of chronic or degenerative diseases were selected from among patients’ relatives and staff at the state university teaching hospital. a sample frame was drawn from the controls recruited. a table of random numbers was then used to select the controls that participated in the study. a standard, structured questionnaire was administered via paper to all participants by the researchers (online supplementary figure 1). the questionnaire was used alongside patients’ medical records to address the general characteristics of all participants and to exclude participants on hormonal therapy, lipid-lowering medications, and those with chronic and degenerative diseases, such as diabetes, cancer, and chronic kidney and liver diseases, as these diseases and medications may alter the outcome of the results in the participants. figure 1: graded variations in the mean plasma lecithin-cholesterol acyltransferase concentrations in atherosclerotic cardiovascular disease subclasses and controls at lagos state university teaching hospital, lagos, nigeria, between march 2022 and march 2023. anthropometry anthropometric measurements (weight, height, waist, and hip circumference) were measured using standard procedures. heights were measured to the nearest centimetre with participants in standing positions, without footwear, using a non-stretchable measuring tape. each participant’s body weight was measured with light clothing and without shoes on a digital scale (omron h bf-514c, kyoto, japan). body mass index (bmi) was then deduced from the participants’ weight (kg) and their height (m); bmi = kg/m2. waist circumference (cm) was measured at the midpoint between the rib cage’s lower border and the hip bones’ iliac crest, measured in the horizontal plane with the participant standing during inspired expiration using a non-stretchable tape. the hip circumference (cm) was taken at the level of the greater trochanter, that is, around the point with the maximum circumference over the buttocks. both waist and hip circumferences were then used to determine the waist-hip ratio of participants. all participants’ systolic and diastolic blood pressures in millimetres of mercury were measured twice using an omron electronic blood pressure monitor (omron bp 7250, kyoto, japan) and the average was recorded and used in the analyses. blood sample collection about 7 ml of venous blood were collected by the researchers from patients and controls after an overnight fast of 8 h – 12 h and dispensed into a potassium-ethylene-diamine tetra-acetic acid sample container (1 mg/ml of blood). each sample was immediately placed in a cold chain until centrifuged at 3000 rpm for 5 min. the plasma was carefully separated from the cells into labelled screw-capped bottles and stored at –20 °c until assayed. biochemical analysis lipid profile parameters were assayed using commercially available kits (randox cholesterol, high-density lipoprotein cholesterol [hdlc], and triglycerides [tg]) from randox laboratories ltd., crumlin, united kingdom. the method of allain et al.22 was used for the estimation of total cholesterol (tc) and hdlc, while bucolo and david’s23 method was employed for the estimation of triglycerides. low-density lipoprotein cholesterol (ldlc) was calculated using friedewald et al.’s24 formulae. non-hdlc (nhdlc) is the sum of all the cholesterol present in each lipoprotein except hdlc, and it is derived by deducting hdlc from the tc of the conventional lipid panel.25 plasma human pon-1 and lcat concentrations were determined using the sandwich enzyme-linked immunosorbent assay method26 kit supplied by elabscience biotechnology limited company, wuhan, china. quality control sera were assayed along with the research samples during each batch of tests. data analysis all results were analysed using statistical package for social science software version 21 for windows 10 by ibm corporation (chicago, illinois, united states). the results were expressed as the mean and standard deviation (mean ± s.d.). a student’s t-test was used to compare the statistical difference between the two means, and the differences were regarded as significant at p < 0.05. an analysis of variance was used to determine differences within groups, and the differences were regarded as significant at p < 0.01. the relationships between lcat, pon-1, and conventional lipid parameters were assessed using pearson’s bivariate correlation and were regarded as significant at p < 0.01 and p < 0.05 (two-tailed). results general characteristics of study population a total of 153 confirmed ascvd patients (65 men and 88 women) aged between 22 years and 64 years were enrolled in the study. this comprised patients suffering from ihd (n = 35), stroke (n = 20), hypertensive heart disease (n = 43), myocardial infarction (n = 15), and congestive heart failure (n = 40). a total of 112 patients attended the cardio clinic and 41 were patients admitted into the state university teaching hospital emergency medical wards. controls comprised 50 healthy volunteers (22 men and 28 women) aged between 22 years and 64 years. there were statistically significant differences between ascvd patients compared to controls, with ascvd patients generally having higher values for mean age, weight, waist circumference, waist-hip ratio, bmi, systolic blood pressure, diastolic blood pressure (all p < 0.001), as well as hip circumference (p = 0.030) and pulse (p = 0.003) (table 1). when ascvd male patients were compared to female patients, statistically significant variations were only observed for mean height, waist circumference, waist-hip ratio (all p < 0.001), as well as weight (p = 0.006) and hip circumference (p = 0.013). no significant difference was observed in the mean height of ascvd patients compared to the controls. table 1: general characteristics, anthropometry, blood pressure, and pulse readings of atherosclerotic cardiovascular disease patients and controls at lagos state university teaching hospital, lagos, nigeria, between march 2022 and march 2023. lipid profile, lecithin-cholesterol acyltransferase, and paraoxonase-1 in all participants atherosclerotic cardiovascular disease patients had statistically significant differences compared to controls with higher values for mean plasma tc, triglycerides, ldlc, and nhdlc (all p < 0.001) (table 2). conversely, statistically significant differences were observed for mean plasma hdlc, pon-1, and lcat (all p < 0.001) with lower values for ascvd patients compared to control values. table 2: lipid profile, lecithin-cholesterol acyltransferase, and paraoxonase-1 of atherosclerotic cardiovascular disease patients and controls at lagos state university teaching hospital, lagos, nigeria, between march 2022 and march 2023. lipid profile, lecithin-cholesterol acyltransferase, and paraoxonase-1 in atherosclerotic cardiovascular disease subclasses statistically significant differences were observed in mean values of plasma tc, triglycerides, ldlc, and nhdlc (all p ≤ 0.001) with higher values for subclasses of ascvd compared with controls (table 3). on the other hand, statistically significant differences were noted in mean values for plasma hdlc, pon-1, and lcat (all p ≤ 0.001) with lower values for subclasses of ascvd compared with controls. the largest differences in atherogenic lipids (tc and triglycerides) and lipoproteins (ldlc and nhdlc) were observed among patients with myocardial infarction and ihd. conversely, these same two ascvd subclasses also recorded the lowest concentrations of plasma hdlc (anti-atherogenic lipoprotein). a similar trend was observed in the non-conventional lipid-related ascvd biomarkers (pon-1 and lcat), where myocardial infarction and ihd patients were noted to have the lowest concentrations of plasma pon-1 and lcat. table 3: lipid profile, lecithin-cholesterol acyltransferase, and paraoxonase-1 of different atherosclerotic cardiovascular disease subclasses and controls at lagos state university teaching hospital, lagos, nigeria, between march 2022 and march 2023. association between lecithin-cholesterol acyltransferase, paraoxonase-1, and conventional lipid profiles a statistically significant, negative correlation was observed between mean body weight and both pon-1 (r = -0.246, p = 0.002) and lcat (r = -0.236, p = 0.001) (table 4). a strong negative correlation was noted between lcat and atherogenic ldlc (r = -0.213, p = 0.004), tc (r = -0.197, p = 0.007), and nhdlc (r = -0.190, p = 0.009) among all ascvd patients. on the other hand, lcat correlated positively with pon-1 (r = 0.151, p = 0.031) among ascvd patients. table 4: pearson bivariate correlation between lecithin-cholesterol acyltransferase, paraoxonase-1 and conventional lipid profile of atherosclerotic cardiovascular diseases patients at lagos state university teaching hospital, lagos, nigeria, between march 2022 and march 2023. variations in lecithin-cholesterol acyltransferase and paraoxonase-1 concentrations in atherosclerotic cardiovascular disease subclasses graded variations were observed in the mean plasma lcat concentrations in the various subclasses of ascvd studied (figure 1). myocardial infarction patients had the lowest mean plasma lcat level. similarly, graded reductions were also observed in the mean plasma pon-1 concentration in ascvd subclasses (figure 2). the lowest mean plasma pon-1 concentration was noted in ihd patients, followed by patients suffering from myocardial infarction. figure 2: graded variations in the mean plasma paraoxonase-1 concentrations in atherosclerotic cardiovascular disease subclasses and controls at lagos state university teaching hospital, lagos, nigeria, between march 2022 and march 2023. gender and lecithin-cholesterol acyltransferase concentrations plasma lcat concentrations were significantly elevated in female participants compared to their male counterparts (online supplementary table 1). a marked gender discrepancy in lcat concentrations was also observed among ascvd patients and controls (figure 3). figure 3: stratification of lecithin-cholesterol acyltransferase concentrations based on gender in atherosclerotic cardiovascular disease patients and controls at lagos state university teaching hospital, lagos, nigeria, between march 2022 and march 2023. discussion the present study showed that the mean weight, waist circumference, hip circumference, waist-to-hip ratio, bmi, and systolic blood pressure and diastolic blood pressure were significantly higher in ascvd patients compared to the mean control values. these findings were consistent with previous studies conducted in europe that reported significant increases in the bmi, waist circumference, waist-to-hip ratio, and blood pressure in ascvd patients.27 although obesity (bmi > 30 kg/m2) was not a distinctive attribute in any of the ascvd subclasses, earlier studies have shown that the listed anthropometric variables are obesity-related parameters and well-established risk factors for ascvd.28 the present study also reported severe alterations in atherogenic lipids and lipoproteins in ascvd patients. there were significant increases in plasma tc, triglycerides, ldlc, and nhdlc, while hdlc (anti-atherogenic lipoprotein) was remarkably reduced compared to the control values. in addition, it is worth noting that the lipid profiles of the different subclasses of ascvd showed significant variations among the different groups assayed. notably, the highest increase in atherogenic lipids and lipoproteins (tc, triglycerides, ldlc, and nhdlc) concentrations was observed in myocardial infarction and ihd patients. conversely, these classes of ascvd also demonstrated the lowest reduction in the levels of the anti-atherogenic hdlc compared to other groups. these findings were similar to an earlier report by ebesunun et al.20 in southwest nigeria in 2013, and hedayatnia et al.29 in northeastern iran in 2020. both studies reported severe alteration in the atherogenic lipids and lipoproteins in patients suffering from myocardial infarction and ihd compared to other classes of ascvd. the findings in the present study further confirmed the severity of dyslipidaemia in these classes of patients. in addition, this study showed a remarkably significant reduction in the mean plasma pon-1 concentration in all the established cases of ascvd patients compared to the mean control values. this result is consistent with previous studies in other populations worldwide that associated low pon-1 concentration and activity with an increased risk of major cardiovascular events.6,8 the reduction in plasma pon-1 concentrations observed in the present study probably suggests impaired antioxidant and anti-inflammatory functions, which may contribute to the progression of atherosclerosis. oxidised ldlc can accumulate in arterial walls,30 leading to the formation of atherosclerotic plaques, and reduced pon-1 activity could further exacerbate this process. it is also important to note that the lowest reduction in plasma pon-1 concentrations occurred in patients suffering from ihd as well as myocardial infarction. the mean plasma pon-1 concentration in both cases was approximately 50% less than the mean control values. thus, it suggests the crucial role pon-1 plays in the presentation of ihd and myocardial infarction. furthermore, worthy of note is the significant reduction in the mean plasma lcat concentrations observed in the ascvd patients compared to the control values. this finding was at variance with some earlier studies that showed no association between lcat and cardiovascular disease,14,31 but on the other hand, it was in agreement with others,32,33 which reported reduced lcat activity and concentration in patients with cardiovascular disease and individuals at high cardiovascular risk. the reduction in the mean plasma lcat concentrations observed in ascvd patients may reflect impaired cholesterol esterification and hdl metabolism. this can lead to reduced hdl functionality and impaired reverse cholesterol transport, a process in which excess cholesterol is transported from peripheral tissues back to the liver for excretion.13 dysfunctional hdl and impaired reverse cholesterol transport are associated with an increased risk of atherosclerosis and ascvd.13 it is noteworthy that the mean plasma lcat concentrations of the different subclasses of ascvd showed significant variations among different groups, with the lowest value observed in myocardial infarction patients. this observation also agreed with earlier studies32,33 which reported a marked reduction in plasma lcat activity in patients suffering from myocardial infarction. the marked decrease in lcat concentrations observed in myocardial infarction patients may have a causal relationship with its aetiology in these patients. another important and striking finding in this present study is the marked gender discrepancy in the mean plasma lcat concentration of the participants studied. plasma lcat concentrations were remarkably elevated in the female participants compared to their male counterparts, both in the ascvd patients and the controls. this finding is consistent with that of earlier studies14,30 in other communities around the world that reported higher values of plasma lcat concentrations in females compared to their male counterparts. in addition, a significant negative correlation was observed between plasma pon-1, lcat, and the mean body weight (an established risk factor for ascvd). a strong inverse correlation observed between lcat and the atherogenic lipid and lipoproteins (tc, ldlc, and nhdlc) probably emphasised the important role played by lcat in part in countering the effect of atherogenic lipids and lipoproteins in ascvd patients. furthermore, the present study demonstrated a significant positive correlation between plasma lcat and pon-1, suggesting that the two lipoprotein-associated enzymes may play synergistic roles on hdl particles to counter the effects of atherosclerosis and ascvd progression. limitations one notable limitation of this study is the absence of recent prevalence data on ascvd in nigeria that could inform sample size calculations. this limitation was addressed using the available data, and the sample size was adjusted upward. we suggest that future studies may benefit from updated prevalence data on ascvd. conclusion the present study demonstrated significant decreases in plasma pon-1 and lcat levels with severe alterations in atherogenic lipids and lipoproteins in ascvd patients, especially the subtypes of myocardial infarction and ihd. these observations provide valuable supportive evidence for the interplay of pon-1, lcat, and lipid metabolism in the pathogenesis of this disease. these findings could potentially lead patients, in part, to develop therapeutic strategies for ascvd prevention and treatment in future research. acknowledgements the authors would like to acknowledge dr olanrewaju abdulazeez and his team at the lagos state university teaching hospital, nigeria, for their assistance in recruiting atherosclerotic cardiovascular disease patients for this study. we are also extremely grateful to mrs okereke doreen, deputy director of the medical laboratory unit at the national orthopaedic hospital, igbobi, nigeria, for her assistance in granting us permission to make use of their facility for some of our analysis. this article is partially based on the author’s thesis entitled ‘assessment of lipid profile, phospholipids, apolipoproteins, lecithin-cholesterol-acyltransferase and paraoxonase-1 among lagos residents with cardiovascular diseases’ towards the degree of phd in the department of chemical pathology & immunology, olabisi onabanjo university, nigeria, march, 2024, with supervisors, dr m.o. ebesunun and dr a.a. amballi. it is available at olabisi onabanjo university central library, ogun state, nigeria. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions p.c.n. developed the concept and performed the laboratory procedures, statistical analysis and interpretation of the data. p.c.n. also drafted and edited the manuscript. m.o.e. was involved in the conception, supervised the project, provided intellectual input, and engaged in reviewing and editing the manuscript. o.m.o. contributed to the selection of patients, the acquisition and interpretation of data, the reviewing and editing of the manuscript, and the approval of the final version to be published. sources of support the authors received no financial support for the research, authorship, and/or publication of this article. data availability the authors confirm that the data supporting the findings of this study is available within the article. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. it does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the authors are responsible for this article’s results, findings, and content. references makover me, shapiro md, toth pp. there is urgent need to treat atherosclerotic cardiovascular disease risk earlier, more intensively, and with greater precision: a review of current practice and recommendations for improved effectiveness. am j prevent cardiol. 2022;12:100371. https://doi.org/10.1016/j.ajpc.2022.100371 mahmood t, shapiro md. the questions on everyone’s mind: what is and why do we need preventive cardiology? methodist debakey cardiovasc j. 2021 sep 24;17(4):8–14. https://doi.org/10.14797/mdcvj.698 ossoli a, simonelli s, vitali c, franceschini g, calabresi l. role of lcat in atherosclerosis. 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2010;56:1128–1137. https://doi.org/10.1373/clinchem.2009.139931 abstract introduction methods results discussion acknowledgements references about the author(s) celeste swart faculty of health sciences, university of cape town, cape town, south africa national health laboratory service (nhls), groote schuur hospital, cape town, south africa surita meldau national health laboratory service (nhls), groote schuur hospital, cape town, south africa division of chemical pathology, university of cape town, cape town, south africa chad m. centner division of medical microbiology, university of cape town, cape town, south africa national health laboratory service (nhls), medical microbiology, groote schuur hospital, cape town, south africa adrian d. marais division of chemical pathology, university of cape town, cape town, south africa fierdoz omar division of chemical pathology, university of cape town, cape town, south africa citation swart c, meldau s, centner cm, marais ad, omar f. validation of phase for deriving n-acetyltransferase 2 haplotypes in the western cape mixed ancestry population. afr j lab med. 2020;9(1), a988. https://doi.org/10.4102/ajlm.v9i1.988 original research validation of phase for deriving n-acetyltransferase 2 haplotypes in the western cape mixed ancestry population celeste swart, surita meldau, chad m. centner, adrian d. marais, fierdoz omar received: 28 jan. 2019; accepted: 14 sept. 2020; published: 17 dec. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: there is a shortage of data on the accuracy of statistical methods for the prediction of n-acetyltransferase 2 (nat2) haplotypes in the mixed ancestry population of the western cape. objective: this study aimed to identify the nat2 haplotypes and assess the accuracy of phase version 2.1.1 in assigning nat2 haplotypes to a mixed ancestry population from the western cape. methods: this study was conducted between 2013 and 2016. the nat2 gene was amplified and sequenced from the dna of 100 self-identified mixed ancestry participants. haplotyping was performed by molecular and computational techniques. agreement was assessed between the two techniques. results: haplotypes were assigned to 93 samples, of which 67 (72%) were ambiguous. haplotype prediction by phase demonstrated 94.6% agreement (kappa 0.94, p < 0.001) with those assigned using molecular techniques. five haplotype combinations (from 10 chromosomes) were incorrectly predicted, four of which were flagged as uncertain by the phase software. only one resulted in the assignment of an incorrect acetylation phenotype (intermediate to slow), although the software flagged this for further analysis. the most common haplotypes were nat2*4 (28%) followed by nat2*5b (27.4%), nat2*6a (21.5%) and nat2*12a (7.5%). four rare single nucleotide variants (c.589c>t, c.622t>c, c.809t>c and c.387c>t) were detected. conclusion: phase accurately predicted the phenotype in 92 of 93 samples (99%) from genotypic data in our mixed ancestry sample population, and is therefore a suitable alternative to molecular methods to individualise isoniazid therapy in this high burden tuberculosis setting. keywords: nat2; haplotyping; isoniazid; pathology; molecular. introduction tuberculosis remains a global life-threatening infectious disease.1 patients in developing countries are particularly vulnerable, as informal settlements and overcrowded environments provide optimal conditions for the spread of tuberculosis. according to the national strategic plan, south africa has the third highest tuberculosis burden worldwide. with an annual incidence of approximately 1%.2 the western cape demonstrates nearly the highest global incidence of tuberculosis, with the mixed ancestry population being particularly affected.3 isoniazid is a key first-line antimicrobial drug in the treatment of tuberculosis. the dosage of isoniazid is usually standard and, in most settings, related to body mass in accordance with world health organization treatment guidelines.4 however, elimination of, and thus response to, the drug are affected by the patient-specific expression of metabolising enzymes, in particular arylamine n-acetyltransferase 2 (nat2), which catalyses n-acetylation of aryl amines and aryl hydrazines.5 these drugs include clinically important antimicrobial agents such as isoniazid and sulphonamides, caffeine, dapsone as well as some toxigenic substances and carcinogenic chemicals derived from the environment and human diet. expression of nat2 is limited to the intestine and liver.5 the nat2 gene is located on chromosome 8p22 and has two exons,6,7,8 one of which is transcribed.9 since the discovery of nat2 in 1990,10 extensive studies in various population groups have linked nat2 genetic variants to enzyme function, classifying individuals as either slow, intermediate or fast acetylators. the different acetylator phenotypes determine individual responses to toxins and prescribed medications, and also influence cancer susceptibility.11 the acetylation phenotype can affect clinical outcome on standard drug doses and may result in an increased risk of therapeutic failure or adverse drug reactions.12 both lack and excess of nat2 (slow and fast acetylation) can be associated with isoniazid hepatotoxicity. the major metabolites associated with isoniazid metabolism are thought to be responsible for isoniazid-induced liver injury, while isoniazid itself can also cause immune-mediated hepatotoxicity by binding to liver proteins and serving as a hapten.13 it is thus postulated that fast acetylators may have a poor response to treatment as a result of suboptimal exposure to isoniazid due to reduced isoniazid plasma levels, as well as liver injury due to an increased rate of isoniazid metabolite formation.13 conversely, slow acetylators achieve higher isoniazid plasma levels, since they cannot efficiently metabolise the drug, leading to hepatic injury.13 distinct nat2 haplotypes have been described, consisting of up to four single nucleotide variants (snvs) in various combinations. over 100 haplotypes have been classified into 20 groups according to defining mutations, for example, the nat2*5 group includes 29 different haplotypes and is defined by the c.341t>c snvs. each haplotype is associated with an acetylation phenotype. in human populations globally, the seven most prevalent nat2 haplotypes are: nat2*4, nat2*5b, nat2*6a, nat2*7b, nat2*12a, nat2*13a and nat2*14.14 these haplotypes include various combinations of nat2 snvs.15 nat2*4 is classified as the wild type and is associated with fast (normal) acetylator status, as are the nat2*12a (c.803a>g) and nat2*13a (c.282c>t) haplotypes. the other four common haplotypes are associated with either significantly decreased expression or decreased stability of the nat2 enzyme.16 heterozygotes for fast and slow alleles demonstrate intermediate acetylating activity. genotyping is typically performed using conventional restriction fragment length polymorphism (rflp) analysis to detect the most common variants only, or sanger sequencing to detect all snvs in the nat2 region. ambiguous nat2 genotyping results (the presence of more than one heterozygous snv in the same patient) commonly arise from sequencing data due to simultaneous analysis of both maternal and paternal alleles. this can cause uncertainty predicting the nat2 phenotype as it may be unclear if one or both alleles carry variants with functional effects. in these cases, more complex molecular techniques are required in addition to sanger sequencing for complete and accurate haplotyping.17 these include rflp or amplification-refractory mutation system (arms) amplification.18 these methods are tedious for implementation in a routine diagnostic laboratory, and therefore computational haplotype reconstruction programs have been developed that statistically predict the most likely haplotype present using algorithms that take into account established allele frequencies within the population. the computational algorithms offer a quicker, cheaper and more practical alternative. several approaches are available, including clark’s algorithm, the expectation maximum algorithm and the bayesian implementation, which estimate the maximum likelihood of haplotype frequencies.19 the software program phase (used in this study) is considered to be the reference standard for computational haplotype inference.20,21 it utilises the bayesian approach for reconstructing haplotypes based on population genotype data. using prior information (based on beliefs about patterns of haplotypes expected in certain population samples), as well as the information obtained from the observed data, the most likely haplotype is estimated and reconstructed for each allele in each individual.22 the proportions of slow and fast acetylators differ among various populations. in addition, the effect of different acetylation phenotypes on isoniazid-induced hepatic toxicity has been shown to be inconsistent across ethnic groups.23 there is limited nat2 genotypic data in african populations; the few known demonstrate nucleotide variants that differ from those commonly described in european populations. african populations are genetically diverse,24 and data can therefore not be extrapolated among the various population groups. the south african mixed ancestry population (8.9% of the south african population) is described as a distinct ethnic group with a complex genetic admixture,25 with over 60% living in the western cape and particularly in the cape town area. the major ancestral populations contributing to the genetic diversity of this group include khoisan and non-khoisan africans, with smaller contributions from both european and indian asian descent populations.26 the genotypic complexity and origin of this population not only influence disease susceptibility,27 but may also have implications when computational haplotype prediction algorithms are employed. it would be ideal to determine the nat2 acetylation status of each patient in order to individualise isoniazid therapy. however, the complex manual molecular haplotyping techniques are expensive, time-consuming and tedious,17 and are thus not realistically feasible in a high tuberculosis burden setting such as the western cape where this study was performed. while the phase computational algorithm is used routinely at many centres, its performance has not been validated in the south african mixed ancestry population. this study aims to describe the nat2 haplotypes identified in this population and to assess the accuracy of phase version 2.1.1 in assigning nat2 haplotypes in the mixed ancestry population of the western cape. methods ethical considerations the human research ethics committee (hrec ref: 327/2014), appointed by the faculty of health sciences at the university of cape town, approved this study. all the work conducted was performed on pre-extracted, stored dna from participants who gave informed consent for research conducted in a previous study, as well as for any future molecular testing. the dna samples were selected at random from participants of self-identified mixed ancestry by allocation of numbers only. study site and population this study was conducted between 2013 and 2016. the blood samples (n = 100) used in this study were sourced with informed consent from randomly selected, self-identified mixed ancestry adult patients attending the lipid outpatients’ clinic at groote schuur hospital in cape town, south africa. dna was extracted and stored. molecular techniques the nat2 gene was amplified by polymerase chain reaction (pcr), sequenced (using sanger sequencing) and haplotyped using molecular and computational techniques as described below. polymerase chain reaction the pcr was performed using primers designed to flank the entire coding region of the nat2 gene (pcr amplicon size 1003 bp). the reaction consisted of the appropriate forward (5′gtcacacgaggaaatcaaatgc3′) and reverse (5′agttgataattagtgagttgggtga3′) nat2 primers (0.5 µm), deoxynucleotide triphosphates (0.2 mm), supertherm gold taq dna polymerase (0.75 u) (jmr holdings, kent, united kingdom), 1 x buffer containing mgcl2 at 1.5 mm final concentration (supplied) and approximately 50 ng to 150 ng dna. polymerase chain reaction cycling conditions were as follows: 94 °c for 3 minutes; 35 cycles of: 94 °c for 30 s, 56 °c for 30 s and 72 °c for 40 s, followed by 72 °c for 8 min. reaction efficacy was assessed by agarose gel electrophoresis (using 2% agarose gel with the addition of ethidium bromide for visualisation) prior to further analysis. sequencing of polymerase chain reaction products sequencing of the resulting pcr products were outsourced to the central analytical facility (stellenbosch university, south africa). forward and reverse sequencing were performed. the sequencing data were compared to the nat2 genomic reference sequence obtained from genbank (ng_012246.1) (https://www.ncbi.nlm.nih.gov/genbank/) to identify all nat2 gene variants present (figure 1 and figure 2). figure 1: electropherogram demonstrating heterozygosity for the c.341t>c (p.i114t) single nucleotide variation, western cape, south africa, 2013–2016. single nucleotide variants depicted by the red ‘y’ highlighted in yellow. figure 2: electropherogram demonstrating heterozygosity for the c.282c>t (p.y94y) single nucleotide variation, western cape, south africa, 2013–2016. single nucleotide variants depicted by the red ‘y’ highlighted in yellow. restriction fragment length polymorphism restriction fragment length polymorphism was used to separate individual alleles in 25 samples with ambiguous haplotypes. the c.481c>t and c.191g>a variations destroy cleavage sites for kpn1 and msp1. kpn1 was therefore used in samples heterozygous for c.481c>t (n = 22), while msp1 was used in those with heterozygous c.191g>a (n = 3). while the normal allele is recognised and digested into two smaller fragments, the affected allele is not (figure 3). reaction products were separated by agarose gel electrophoresis (2%) followed by excision, agarose gel extraction and sequencing of the undigested allele. figure 3: kpn1 restriction fraction length polymorphism for c.481c>t wildtype and heterozygous variant, western cape, south africa, 2013–2016. agarose gel electrophoresis of nat2 pcr products digested with kpn1 restriction enzyme. the c.481c>t variant destroys a kpn1 recognition site. lanes 1 and 6: dna ladder (thermo scientific generuler dna ladder mix) with relevant corresponding bp sizes indicated. lane 2: patient sample (no c.481c>t variation). lane 3: normal control (no c.481c>t variation). lane 4: heterozygous control (c.481c>t variation on one allele). lane 5: blank. the arrow demonstrates the uncut allele (1003 bp) containing the c.481c>t single nucleotide variant. the two bands (553 base pairs and 450 base pairs) below this band demonstrate the normal (and therefore cut) allele. the restriction site on the normal allele is recognised and therefore cut into smaller fragments, while the affected allele is not digested, lending itself to excision for single allele sequencing. amplification-refractory mutation system polymerase chain reaction amplification-refractory mutation system pcr was performed for the remaining cases with ambiguous haplotypes containing heterozygous c.282c>t snvs (n = 42). an arms forward primer (5′ccacaatgttaggagggtatttttat3′) was designed to recognise and amplify only alleles containing the c.282t variant base. polymerase chain reactions were performed as before, using the same reverse primer, with the exception of an annealing temperature of 62 °c. after checking the reaction efficacy with agarose gel electrophoresis, the individual alleles were sequenced as before. data analysis sequence data were aligned to published reference sequences using the clustalw alignment function in bioedit version 7.2.528 and analysed. the court lab online calculator was used to assess whether the variants were in hardy-weinberg equilibrium,29 and haplotypes were assigned by molecular methods. phase computational algorithm results obtained from genotyping of both ambiguous (n = 67) and non-ambiguous samples (n = 26) were used as input data for the phase computational algorithm.21,22 the following data were inputted: number of variant positions, variant nucleotide positions and the genotypes for each sample at those positions (wild type, heterozygous, homozygous). the following parameters were specified: number of iterations (= 1000), thinning interval (= 1), burn-in (= 100). statistical analysis the information obtained by molecular and computational haplotyping was compared by kappa statistics using stata 13 (statacorp llc, college station, texas, united states). the p-values less than 0.05 were considered statistically significant. results genotypes from all 100 samples were found to be in hardy-weinberg equilibrium for each snv detected in the study population (table 1). four samples were heterozygous for both c.590g>a and c.857g>a, while another one was heterozygous for both c.341t>c and c.803a>g. no further work was done on these, although future studies should aim to analyse these with similar arms or rflp techniques and those employed here. an additional two ambiguous samples could not be assigned haplotypes due to degraded dna and repeatedly failed amplification – a total of seven samples were thus excluded from further analysis. table 1: frequencies of heterozygotes and homozygotes for each observed single nucleotide variant, with associated hardy-weinberg equilibrium probabilities, western cape, south africa, 2013–2016. of the 93 remaining samples, 26 were non-ambiguous genotypes and therefore did not require haplotyping. these 26 genotypes comprised either only wild type variants (n = 6), or were homozygous for any particular haplotype (n = 20). separation of the non-ambiguous alleles by rflp or arms pcr was thus not necessary and haplotypes could be assigned directly to these samples. allele separation and haplotype determination for the remaining 67 samples were obtained using the rflp and arms methods described earlier. the non-ambiguous samples demonstrated a predominance of haplotype nat2*5b (28%), followed by nat2*4 (25%), nat2*6a (17%), nat2*12a (12%), nat2*13a and nat2*5c (both 6%), nat2*5a (4%) and nat2*27 (2%). apart from the commonly described nat2 snvs, four additional variants were detected in our study population. a single sample was heterozygous for the c.622t>c (p.tyr208his) variant (rs56387565). in this instance, it co-occurred with c.803a>g, resulting in the less common haplotype nat2*12f. another was heterozygous for c.809t>c (p.ile270thr) (rs868725509). it co-occurred with c.341t>c and c.803a>g; molecular haplotyping could not be performed on this ambiguous sample due to the inability to delineate with available methodology. a single nonsense variant, c.589c>t (p.arg197*), resulting in a stop codon, was detected in isolation in one patient.30 two samples were heterozygous for a very rare variant, c.387c>t (p.ser129=) (rs144828000). in both of our patients, it co-occurred with the c.282c>t and c.590g>a variants (nat2*6a). the most common haplotypes in the 67 ambiguous samples, derived by molecular methods, were nat2*4 (29.1%) followed by nat2*5b (26.9%), nat2*6a (23.1%) and nat2*12a (6%). these frequencies were similarly predicted by phase (table 2). of 67 ambiguous samples, 15 (22%) required further molecular testing (i.e. rflp or arms pcr) to differentiate between slow and intermediate acetylation phenotypes. in 14 (93%) cases, the ambiguous genotype was resolved to predict a slow acetylation phenotype. table 2: haplotype frequencies as derived by molecular methods, as well as predicted by the phase computational algorithm, western cape, south africa, 2013–2016. there was a 94.6% agreement between the haplotypes obtained by molecular methods and those predicted by phase, with a strong correlation (kappa 0.94, p < 0.001). five haplotype combinations (from 10 chromosomes) were incorrectly predicted. of these, only one resulted in a change of acetylation phenotype (intermediate to slow). furthermore, the program flagged four of these five incorrect haplotype combinations (including the one incorrectly assigned to slow acetylator status) to indicate that uncertainty was present around one of the predicted variant positions. discussion nat2 genotypes were obtained by molecular methods for 100 samples. ninety-three genotypes were then used to validate the accuracy of phase in predicting haplotypes. twenty-six of these samples had non-ambiguous genotypes; of the remaining 67 samples, only 15 (22.5%) technically needed further allelic haplotyping, where the ambiguous genotype could lead to ambiguous acetylation phenotype prediction. this is in keeping with possible clinical scenarios, where non-ambiguous samples would not in fact need to be subjected to phase analysis to obtain haplotypes. four rare snvs were detected in our study population. the variant at position c.622t>c (rs56387565) results in a non-synonymous change (p.tyr208his); the functional effect of this variant is not well studied, although it has been linked to slow acetylation according to dbsnp.31 another variant at c.809t>c (rs868725509) is also non-synonymous (p.ile270thr) and has not been characterised. the nonsense variant c.589c>t (p.arg197*) results in a stop codon with a subsequent presumed lack of functional protein expression.30 these three snvs each occurred at a frequency of 0.5% in the study population. lastly, the very rare synonymous variant detected at position c.387c>t (p.ser129=) (rs144828000) has to date not been described in the context of the nat2 acetylation status, and occurred in 1% of the study cohort. further assessment of the protein and enzymatic activity of these snvs was not within the scope of this study. future studies, however, may investigate these less commonly detected snvs, whether ‘in silico’ or otherwise. there are limited data available on the most commonly occurring nat2 haplotypes in the mixed ancestry population of the western cape, although numerous studies have been performed in other population groups. fuselli et al. demonstrated a preponderance of nat2*4 (37%), nat2*7b (24%) and nat2*5b (23%) in 13 american and two siberian populations.32 our study showed a similar predominance of nat2*4 (28%) and nat2*5b (27.4%), while the frequency of nat2*7b was much lower (3.8%). while the wild type (nat2*4) was also the most commonly occurring haplotype in east asians,33 loktionov et al. described nat2*5b to be the most common haplotype in black south africans,34 followed by nat2*6a and nat2*12a (both 16.8%, vs 21.5% and 7.5% in our study population). in contrast to our findings, the wild type nat2*4 was shown to occur less frequently in black south africans (13.4%).34 in a cohort of 62 black and 54 mixed ancestry south africans, where 17% were found to be fast acetylators35, haplotype frequencies did not differ significantly between the two population groups (unpublished). the frequency of nat2 haplotypes obtained in this study serve as a priori information to strengthen future haplotype predictions by computational algorithms such as phase, specifically in this mixed ancestry population. the good correlation between phase and molecular haplotyping methods demonstrated in our mixed ancestry population is similar to previous studies in other populations. a study by golka et al. in a german population, where the statistical program phase version 2.1.1 was used to predict haplotypes from genotypes by analysing seven nat2 snvs commonly observed in the caucasian population, concluded that this program unambiguously derived haplotypes in over 99% of cases.36 similarly, other studies have shown that these computational algorithms (including phase) compare well with molecular haplotyping, and could thus be used as an alternative method for genetic phasing. sabbagh and darlu (2005) investigated the performance of several computational approaches in estimating individual haplotype phases in five different population groups (spanish, united kindom caucasians, black south africans, koreans and nicaraguans).17 they compared the results obtained from the computational programs with those obtained through direct molecular haplotyping and found that even the incorrectly predicted haplotypes did not result in phenotypic changes, resulting in a complete correlation of the acetylation phenotypes in all five population groups. the population group in which computational haplotype inference displayed the highest error rate was black south africans, although error rates were always low (less than 4%). the postulated reasons include the high number of ambiguous genotypes as well as the low rate of linkage disequilibrium between the various snvs in this sample, compared with the other four population groups.17 the authors suggest studying these data before computational haplotyping is employed, to attempt to predict the accuracy and reliability with which haplotypes will subsequently be inferred in such cases. the single incorrectly predicted phenotype (intermediate to slow) may be clinically significant. however, the computational program flagged the predicted haplotypes as being uncertain; in the clinical scenario, such a flag could serve as a prompt to confirm the haplotype by molecular methods instead of relying on phase. in our current setting, nat2 acetylation status is chiefly investigated by special request once the patient has hepatic complications of isoniazid therapy. apart from the adverse drug reactions that could be avoided by individualising isoniazid dosage according to nat2 phenotypes, incorrect dosage may contribute to the growing prevalence of multi-drug resistant tuberculosis.37 the unavailability of routine isoniazid genotyping could thus be considered a major limitation of current tuberculosis treatment protocols. n-acetyltransferase 2 genotyping should ideally be performed on all patients before commencing isoniazid therapy. the cost of genetic investigations as well as the limited infrastructure in the south african healthcare setting make it difficult to apply haplotyping without the phase computational approach. this approach could be supported by specific investigation of incompletely explored snvs and the development of a high throughput multiplex assay targeting common snvs relevant to the population being investigated. further studies are also recommended to investigate and monitor the isoniazid safety and treatment outcome in patients undergoing treatment for tuberculosis with different nat2 acetylation phenotypes. limitations due to budgetary and time constraints, only a small sample size of 100 was used. the arms pcr could not be set up for the following variants which may have improved the correlation: c.590g>a, c.857g>a, c.341t>c and c.803a>g. conclusion phase accurately predicted the nat2 acetylation phenotype in 92 of 93 samples (99%) from genotypic data in our mixed ancestry study population, and could be used as an alternative to molecular methods to individualise isoniazid therapy in our high tuberculosis burden setting. data obtained from this study may also be potentially used to predict haplotypes in future patients from this population group. acknowledgements competing interests the authors have declared that no competing interest exists. authors’ contributions c.s. was the primary author and performed the majority of the molecular work and subsequent analyses. all co-authors assisted with the reviewing of the manuscript. f.o. was the supervisor and assisted with the project design, and s.m. assisted with molecular bench work and interpretation of results. a.d.m. was responsible for the funding, and provided the source of samples utilised in this study. c.m.c. was responsible for running the phase computational haplotype predictions as well as the statistical analysis. sources of support research conducted in this study was supported by existing internal research funds from the university of cape town. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references mandal a. history of tuberculosis [homepage on the internet]. 2014 [accessed 2020 sep 7]. available from: 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https://doi.org/10.1093/cid/cis353 about the author(s) maurine m. murtagh international diagnostics centre, london school of hygiene & tropical medicine, london, united kingdom citation murtagh mm. quality assurance for point-of-care diagnostic testing: it is not negotiable. afr j lab med. 2016;5(2), a554. http://dx.doi.org/10.4102/ajlm.v5i2.554 editorial quality assurance for point-of-care diagnostic testing: it is not negotiable maurine m. murtagh copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. this special issue of the african journal of laboratory medicine (ajlm) is dedicated to quality assurance for in vitro diagnostics (ivds), which encompasses all aspects of the quality management of diagnostic testing, including quality control of the testing process as well as monitoring and control of the outcomes of that process, which are designed to ensure that test results are as accurate as possible. external quality assessment is an important element of this system and is the focus of most of the articles in this supplement. inaccurate ivd testing can have significant adverse impacts for patients. for an individual patient, it means not only a delay in receiving the correct diagnosis, but can also result in improper or unnecessary treatment, which may lead to long-term complications, additional clinic visits and additional cost. for public health programmes, failure to identify pathogens correctly in the event of an outbreak can lead, among other things, to a delay in determining the true cause and extent of an outbreak, and inadequate or incorrect control measures being put into place. in general, poor diagnostic quality leads to poor patient outcomes, increasing morbidity and mortality, and poses a barrier to effective public health interventions. although it should be self-evident that ivd testing is only valuable if it is accurate, quality assurance has often lagged behind scale-up of testing in resource-limited settings. with the high prevalence of disease in resource-limited settings, including hiv, malaria and tuberculosis, as well as unexpected outbreaks of infections such as ebola, dengue and zika, testing is critical and countries are often exhorted to scale-up testing quickly by the world health organization and other national and international stakeholders. however, the health systems in which testing must occur are often weak, fragmented and ill-prepared for any additional testing. to date, most testing in these countries, especially device-based testing, has taken place in centralised laboratories, while the majority of patients live, and present for treatment, in rural or peri-urban areas far from those facilities. therefore, effective scale-up of ivds requires not only increasing capacity for testing centrally, but also decentralising testing to take diagnostics closer to the point of care (poc), with measures to ensure the quality of testing, wherever it is performed. this has proven to be a difficult task. using hiv as an example, in addition to well-established laboratory-based platforms for cd4 testing, countries have had access to poc testing platforms for cd4 for more than five years, and many such platforms have been implemented. however, managing a network of poc sites has proven to be challenging, with significant operator and instrument errors, instrument breakdowns, reagent stock-outs and other difficulties plaguing the system. as countries now try to scale-up early infant diagnosis and viral load testing for hiv to reach the unaids 90-90-90 goals by 2020, it is expected that poc testing will play a significant role, and quality data will provide measurements for impact and success. as illustrated by the articles in this special issue of ajlm, countries are using lessons learned from their experiences with cd4 poc introduction to develop policies and strategic plans for early infant diagnosis and viral load scale-up in which quality assurance, including external quality assessment schemes, are embedded for both laboratory and poc testing. a pilot study in zimbabwe, for example, demonstrated the importance of using connectivity solutions to facilitate automated and timely reporting of external quality assessment results to monitor instrument and operator performance and to enable corrective actions to be taken as an integral part of a quality assurance system. quality assurance is often viewed as an added cost of an ivd system. this is particularly evident in resource-limited settings, where countries must make difficult decisions each year with respect to how to apportion budgets for diagnostics. the london school of hygiene & tropical medicine has developed a simple costing model that can quantify the significant value of diagnostic quality assurance and the serious consequences of not incorporating quality assurance into diagnostic programmes, both in terms of the cost of lives lost and the cost of unnecessary or incorrect treatment. the consequences of introduction of new diagnostics without quality assurance are dire in terms of lives and disability life years lost. the time has come for all stakeholders in global health to recognise that quality assurance for diagnostic tests and testing are not negotiable. investments in diagnostic quality assurance systems will pay dividends for years to come, improving both patient outcomes and healthcare economics in-country. abstract introduction methods results discussion conclusion acknowledgements references about the author(s) sofia o. viegas instituto nacional de saúde, ministério da saúde, maputo, mozambique khalide azam instituto nacional de saúde, ministério da saúde, maputo, mozambique carla madeira instituto nacional de saúde, ministério da saúde, maputo, mozambique carmen aguiar instituto nacional de saúde, ministério da saúde, maputo, mozambique carolina dolores instituto nacional de saúde, ministério da saúde, maputo, mozambique ana p. mandlaze instituto nacional de saúde, ministério da saúde, maputo, mozambique patrina chongo instituto nacional de saúde, ministério da saúde, maputo, mozambique jessina masamha centers for disease control and prevention, maputo, mozambique daniela m. cirillo irccs san raffaele scientific institute, who supranational tb reference laboratory, tuberculosis & mycobacteria unit, milan, italy ilesh v. jani instituto nacional de saúde, ministério da saúde, maputo, mozambique eduardo s. gudo instituto nacional de saúde, ministério da saúde, maputo, mozambique citation viegas so, azam k, madeira c, et al. mozambique’s journey toward accreditation of the national tuberculosis reference laboratory. afr j lab med. 2017;6(2), a491. https://doi.org/10.4102/ajlm.v6i2.491 note: sofia o. viegas and khalide azam listed under the author section are co-first authors for this article. lessons from the field mozambique’s journey toward accreditation of the national tuberculosis reference laboratory sofia o. viegas, khalide azam, carla madeira, carmen aguiar, carolina dolores, ana p. mandlaze, patrina chongo, jessina masamha, daniela m. cirillo, ilesh v. jani, eduardo s. gudo received: 15 may 2016; accepted: 15 dec. 2016; published: 31 mar. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: internationally-accredited laboratories are recognised for their superior test reliability, operational performance, quality management and competence. in a bid to meet international quality standards, the mozambique national institute of health enrolled the national tuberculosis reference laboratory (ntrl) in a continuous quality improvement process towards iso 15189 accreditation. here, we describe the road map taken by the ntrl to achieve international accreditation. methods: the ntrl adopted the strengthening laboratory management toward accreditation (slmta) programme as a strategy to implement a quality management system. after slmta, the mozambique national institute of health committed to accelerate the ntrl’s process toward accreditation. an action plan was designed to streamline the process. quality indicators were defined to benchmark progress. staff were trained to improve performance. mentorship from an experienced assessor was provided. fulfilment of accreditation standards was assessed by the portuguese accreditation board. results: of the eight laboratories participating in slmta, the ntrl was the best-performing laboratory, achieving a 53.6% improvement over the slmta baseline conducted in february 2011 to the stepwise laboratory quality improvement process towards accreditation (slipta) assessment in june 2013. during the accreditation assessment in september 2014, 25 minor nonconformities were identified and addressed. in march 2015, the ntrl received portuguese accreditation board recognition of technical competency for fluorescence smear microscopy, and solid and liquid culture. the ntrl is the first laboratory in mozambique to achieve iso 15189 accreditation. conclusions: from our experience, accreditation was made possible by institutional commitment, strong laboratory leadership, staff motivation, adequate infrastructure and a comprehensive action plan. introduction implementation of quality management systems (qms) ensures that laboratory services meet international standards and that results are accurate, reliable and timely, representing a vital role in diagnosis, monitoring of disease treatment, training, surveillance and disease prevention.1 however, in resource-limited settings, laboratories are poorly funded and suffer from chronic underinvestment, resulting in inadequate infrastructure, lack of equipment maintenance and calibration services, irregular training for laboratory workers and lack of qms.2,3 to improve the quality of laboratory services in africa, the world health organization’s regional office for africa and its international partners established a framework that allows for incremental implementation of qms. this process, called the stepwise laboratory quality improvement process towards accreditation (slipta) recognises laboratories’ efforts at each level toward full implementation of the iso 15189 standard requirements using a zeroto five-star grading system.4 in 2011, the mozambique ministry of health adopted this approach and implemented the first round of strengthening laboratory management toward accreditation (slmta), an innovative training and mentorship programme for continuous quality improvement, with the aim of improving patient care.5,6 the national tuberculosis reference laboratory (ntrl) was one of the eight participating laboratories enrolled in slmta. established in 1987, the ntrl falls under the mozambique national institute of health (mnih), within the ministry of health. the mnih’s mission is to contribute to the improvement of the well-being of the people of mozambique by generating and promoting scientific and technological solutions to the country’s principal health issues.7 beside the ntrl, the mnih hosts nine other reference laboratories, including two regional tuberculosis reference laboratories that are overseen by the ntrl, and reference laboratories for cellular immunology, molecular virology, virus isolation, parasitology, entomology, microbiology and serology. the ntrl provides laboratory reference services for the entire national health system in mozambique. these include diagnosis of tuberculosis, multi-drug resistant tuberculosis and extensively drug resistant tuberculosis and treatment monitoring and support of the laboratory network through training activities, technical assistance through on-site supervision, and provision of proficiency panels for external quality assessment. the ntrl also conducts surveillance for tuberculosis drug resistance and research activities. these services must be of high quality and meet international standards. in this article, we describe the ntrl’s road map, challenges and successes during the journey taken from february 2011 to march 2015 to attain international accreditation. methods setting the ntrl is in the city of maputo.7 at the time of accreditation, the ntrl had 18 staff members, including 10 biologists, four of whom had masters degrees, four laboratory technicians, two administrative staff and two cleaning staff (figure 1). the laboratory has an average workload of 50 samples per day, and performs tuberculosis culture in solid and liquid media, fluorescence smear microscopy, firstand second-line drug susceptibility testing (dst), acid-fast bacilli identification, identification of mycobacterium tuberculosis complex by immunochromatography, and molecular (cepheid genexpert® mtb/rif) and line-probe assays (hain genotype® mtbdrplus). figure 1: national tuberculosis reference laboratory organizational structure, mozambique. infrastructure improvement in 2009, the ntrl underwent renovation to improve biosafety and workflow. the renovation process was completed in 2010 and acted as a starting point in the process toward international accreditation. the renovation period was stressful for the laboratory staff as they twice had to move from one facility to another. to make matters worse, the ntrl was closed for four months to pave the way for renovations, which left the country without the reference services provided by the ntrl. pre-slmta period prior to slmta training, none of the staff from ntrl had previous experience in qms. no qms had been implemented in the laboratory, documentation and records were poorly controlled, equipment maintenance and calibration were poorly monitored, corrective actions were not undertaken and staff were not conscious about the importance of implementing qms. external proficiency panels were only available for smear microscopy and dst. only four quality indicators were monitored on a yearly basis: (1) quality of sputum samples; (2) smear microscopy and culture positivity rate; (3) contamination rate; and (4) workload. mnih strategy for achieving accreditation the mnih used a four-pronged approach to achieve accreditation of the ntrl. these included the creation of the national program for laboratory accreditation, the adoption of the slmta training pack, the slipta assessment, and the mentorship from expert assessors. some of the approaches were implemented concurrently (slmta, mentorship) and the slipta assessment was used at the end, to measure progress and determine readiness for accreditation. figure 2 outlines the timeline of key events in the pathway to accreditation. figure 2: timeline of events in the pathway to iso 15189 accreditation, mozambique, 2011–2015. national program for laboratory accreditation to support qms implementation, the ministry of health adopted slmta as its training strategy in 2011. to ensure institutionalisation and ownership of slmta in the health system, the ministry of health decided to translate the acronym of slmta into portuguese and adopted the acronym fogela, a direct translation of slmta to portuguese: fortalecimento da gestão de laboratórios para acreditação. the ministry of health also decided to establish the national program for laboratory accreditation, which represented the backbone for implementation of slmta. the main mandate of the national program for laboratory accreditation was to provide institutional leadership for slmta implementation at the ministry of health, as well as the framework for laboratory accreditation in mozambique. ntrl participation in slmta training together with seven other laboratories from mozambique, the ntrl participated in the first round of slmta implementation in mozambique between february 2011 and june 2012. in total, five staff from ntrl participated in the slmta training as trainees. none of the ntrl staff members became slmta master trainers. a baseline audit by slmta trainers was conducted in february 2011, using version 1 of the slipta checklist. that checklist comprised 12 sections that are aligned with the 12 quality system essentials. a total score of 250 points was possible.8 based on the recommendations and list of nonconformities from the baseline audit, an action plan was immediately drafted and approved. furthermore, the ntrl completed the three slmta workshops, intercalated by a total of six site visits and six quality improvement projects that were a mandatory part of slmta. table 1 presents the actions taken and outcomes for each improvement project. internal meetings were conducted after each workshop to engage all laboratory staff in implementing the improvement projects, and an exit audit was conducted in march 2012. table 1: national tuberculosis reference laboratory improvement projects during first round of strengthening laboratory management toward accreditation, 2011–2013, mozambique. mentorship mentorship was provided at all stages of the preparation for the ntrl accreditation and included technical mentorship to strengthen capacity for diagnostic procedures. during the slmta training, a mentorship programme was designed to support the implementation of the quality improvement projects. the programme included sixto eight-week periods when the mentor was in the laboratory, followed by an eight-week absence. supervisory visits were conducted to provide technical assistance, to monitor the implementation process and progress and to review laboratory action plans. after the slipta assessment, a mentor was assigned to support and guide the laboratory, working closely with the quality manager and head of the ntrl, in preparation for accreditation. the mentor had more than 15 years of experience in implementing qms in private and public clinical laboratories in portugal and, in line with the iso 9001 and iso 15189 standards, had a post-graduate degree in quality management and was also a senior certified auditor. slipta audit the ntrl was one of four laboratories selected by the national program for laboratory accreditation to pursue a slipta audit. the slipta audit took place in june 2013 and was a two-day process conducted by the african society for laboratory medicine’s certified external auditors (two expert international auditors and four locally-trained auditors). the updated version 2 of the slipta checklist based on 258 points was used.9 on day 1, the auditors reviewed the laboratory’s policies, procedures and records for its competency, completeness and compliance with the iso 15189:2012 standard. in addition to interviewing staff members, they also observed laboratory processes in the technical areas. on day 2, the assessor team and the laboratory staff discussed all audit findings, including recommendations from the assessors on how to close identified gaps. post-slipta assessment and preparation for the portuguese accreditation board audit following the slipta audit, an action plan was drawn up to address the audit findings, a management review with the mnih directorate was scheduled and several training activities were conducted. the training included iso 15189:2012 standard requirements, biosafety, several standard operating procedures and sample transportation. additional quality indicators were developed and/or monitored regularly for pre-analytical, analytical and post analytical phases. crucial laboratory indicators that were regularly monitored (monthly or quarterly) included: (1) sample rejection rate; (2) nonconformities in request forms; (3) culture contamination rate; (4) concordance of results between smear microscopy and culture versus type of patients (new cases or previously treated); (5) laboratory turn-around time; (6) client satisfaction; and (7) workload. both internal (performed by the mnih quality unit) and external audits (performed by an independent, external certified auditor) flagged the most challenging issues to be resolved as: (1) unavailability of certified external services and suppliers; (2) adequately maintaining laboratory equipment, reagents and consumables; (3) ensuring quality of examination results; and (4) post-examination processes (table 2). table 2: main challenges experienced and actions taken to accomplish iso 15189:2012 requirements for accreditation of the national tuberculosis reference laboratory, mozambique, 2011–2013. iso 15189 accreditation in march 2014, a year after the slipta audit, the ntrl decided to submit its application for an iso 15189 accreditation assessment for fluorescence smear microscopy and culture in solid and liquid media to the portuguese accreditation board (instituto português de acreditação; ipac). to apply, the ntrl followed all procedures, rules, criteria and methodology as described in ipac’s general regulations.10 for development of their accreditation activities, ipac has technical committees that comprise evaluators and external experts, as well as a consultative committee that supervises the impartiality of all processes. an appointed evaluation team examined the application to ensure that all required documentation was included. the application was then approved and the audit dates were scheduled. the assessment, conducted by two ipac auditors (one for qms and one technical), took place on 22–23 september 2014. following the assessment, a report, including all non-conformities that had to be corrected for demonstration of compliance with the accreditation standards, was discussed and delivered to the laboratory team. within six months all non-conformities had been resolved and evidence thereof was sent to ipac. costs associated with accreditation from the beginning of the process, the mnih directorate was committed to the accreditation, and efforts were made to ensure that sufficient resources and timely support were available to the process. discussions were also conducted with several partners to provide additional support. the main costs were related to laboratory renovation, followed by technical assistance/mentorship and equipment maintenance (table 3). table 3: approximate costs of national tuberculosis reference laboratory accreditation, mozambique, 2011–2013. results slmta training programme six quality improvement projects were conducted in the ntrl during the slmta training (table 1). from baseline to exit audit, the ntrl improved its score from 57 (22.8%) to 201 (77.9%) points (figure 3). improvements were observed across all 12 checklist sections, and the highest scores were achieved in the facilities and safety and client management and customer service sections, which made the ntrl the best-performing laboratory among the eight laboratories in the first slmta round. the high performance of the ntrl in the exit audit led to its selection for a slipta audit. mentorship and supervision were professional and effective at equipping staff with the skills required for high quality performance. figure 3: percentage scores for national tuberculosis reference laboratory performance in each section of the stepwise laboratory quality improvement process towards accreditation checklist at baseline, exit and final strengthening laboratory management toward accreditation audits. slipta programme during the slipta audit, the laboratory scored 197 points (76.36%), corresponding to three stars, which was similar to the slmta exit audit scores (figure 3). iso 15189 accreditation the ipac assessment revealed 25 minor nonconformities (table 2), most of which were related to document control. all nonconformities were addressed in a timely manner. on 27 march 2015, the ntrl was recognised by ipac as having implemented a qms and having the technical competency for fluorescence smear microscopy and solid and liquid culture. the ntrl thus became the first medical laboratory in mozambique to achieve iso 15189 accreditation. discussion internationally-accredited laboratories are recognised for their superior test reliability, operational performance, quality management and competence.11 in pursuit of this recognition, the ntrl began its journey by first renovating the laboratory as a foundation for establishing qms. the good infrastructure contributed to the high scores achieved for facilities and safety, an area that pulls down the scores of many laboratories in resource-limited settings.2,12 moving to a newly-renovated facility was also motivating for all ntrl staff. the starting point for implementing a qms at the ntrl was the slmta training. the ntrl improvement during the slmta training was outstanding. indeed, an evaluation of six of the eight laboratories participating in the first round of slmta showed that all had improved their scores.13 at baseline, all laboratories began with zero stars and at the slipta audit, only one laboratory remained at zero stars (though its score still increased from 14% to 44%), three laboratories were at one star, one laboratory was at two stars and the ntrl reached three stars.13 ntrl participation in the slmta programme was important for the introduction of basic qms concepts, and for demonstrating the importance of implementing qms and its impact on patient care. since several laboratories participated in the first slmta round, healthy competition between the laboratories was also a motivating factor, pulling each laboratory to work harder and better at implementing qms towards achieving the highest number of stars. although ntrl management were aware of the existence of other resources for attaining accreditation (e.g., the global laboratory initiative’s stepwise process towards tb laboratory accreditation),14 it was concluded that using different tools or checklists could disturb the team’s focus. thus, the ntrl decided to concentrate on using the slipta tool and the iso 15189 standard as a foundation to meet international standards and achieve iso 15189 accreditation. the scores achieved at the slipta assessment were slightly lower than the slmta exit assessment scores. one reason for that might be the use of a different checklist (version 2 versus version 1). another reason could be that the evaluation performed by highly-experienced auditors during the slipta audit was more rigorous. the slipta assessment increased staff motivation and confidence that iso accreditation was an achievable goal. at that point, the entire team believed that the ntrl could become the first internationally-accredited medical laboratory in mozambique. staff members were motivated to work together as a team, to learn more, and to develop a comprehensive calendar and action plan for gradual implementation of quality improvement plans. training further enhanced technical capacity and staff motivation to implement a qms. however, maintaining constant staff motivation was a challenge and required continuous investment and innovation. for example, small rewards were given in appreciation of staff members who made outstanding contributions to the accreditation process. building on this motivation, the mnih leadership, with financial support from partners, identified an external mentor to support the laboratory in its pursuit, and from that moment on, a continuous mentorship programme replaced the intermittent mentorship of the slmta training. the mentor’s presence and expertise were essential in mentoring the process towards accreditation. with regard to the iso 15189 application to ipac, ipac was selected as the accrediting body, primarily because of language, since mozambique is a portuguese-speaking country. not only did that facilitate communication between the audit team and the laboratory staff during and after the assessment, but it also negated the need for prior translation of qms documents into english. since the ntrl was just beginning implementation of a qms, the focus was on three basic assays to guarantee that the iso requirements were well established for smear microscopy and culture in solid and liquid media. additionally, during the slmta training, quality indicators for those assays were created and have been monitored regularly since then. good results on external quality assurance (eqa) were also considered as a selection criterion. recently, the ntrl submitted an application to expand its accreditation to include acid-fast bacilli identification, mycobacterium tuberculosis complex identification by immunochromatography and genexpert® mtb/rif. the overall goal is to have all assays performed by the ntrl accredited by 2018. during the accreditation process, internal and external audits were equally important for identifying areas that required attention and for informing the action plans. a clearly defined action plan mapped the pathway towards the goal, assigned responsibilities and built the platform for accountability toward achieving the common objectives, while keeping team members focused on activities and timelines. it was the experience of the ntrl that a very well-designed and comprehensive action plan was the key to successful implementation of a qms and achieving accreditation. although african countries have relied much on external support to strengthen public health laboratories and other health systems,15 country leadership represents a critical factor for achieving accreditation.3,16,17 in the ntrl’s experience, commitment at all levels of the organisation played the most important role in improving and sustaining continuous laboratory quality. mnih leadership not only provided financial support for the accreditation process but also participated actively in audit close-out meetings and the management review, as well as in engaging external partner support. to commemorate this outstanding achievement, a public ceremony led by the minister of health was held on 13 april 2015. the minister presented the accreditation certificate to the ntrl in the presence of civil society, implementing partners, several international agencies and stakeholders from the ministry of health and other public institutions. accreditation is a long journey that presents several challenges, especially in resource-poor countries. a major challenge experienced during the ntrl accreditation process included lack of local companies with proper expertise and certification to provide services for equipment maintenance and calibrations. other challenges encountered, as well as solutions, are outlined in table 2. the main objective of having a qms in place is to deliver quality laboratory services ensuring that patients receive timely and reliable results, thereby guaranteeing accurate diagnosis and treatment. with a qms implemented, the laboratory continuously monitors daily operations and easily identifies areas that require more attention for continuous and systematic improvement. the regular monitoring of quality indicators allows for rapid identification of system weaknesses and rapid resolution of problems. the experience of iso 15189 accreditation at the kenya medical research institute/centre for disease control hiv-research laboratory in kisumu, kenya showed a reduction in reagent wastage, leading to increased cost savings to the laboratory, as a major benefit of implementing a qms.16 although such an analysis was not performed at the ntrl, stock management system was improved and became more effective, enabling accurate forecasting of needs and avoidance of stock outs and reagents expiring, thus minimising waste. the accreditation of the ntrl also increased research partner’s confidence in the laboratory’s results and has increased the number of research collaborations and projects undertaken. implementation of a qms enhanced staff competency, provided by enhanced performance on external quality proficiency and internal competency assessments (data not shown). on a daily basis, the accreditation demonstrates that the laboratory is more competent in its mission to improve the health system in mozambique. in the journey toward accreditation, some aspects such as understanding and routine implementation of document control procedures could have been approached differently. for instance, increased training of ntrl staff on key quality concepts such as ‘the meaning of quality’, ‘the cost of not implementing quality’, ‘the importance of procedures to detect errors’ and ‘client-focused service provision’; proper understanding of quality concepts and the benefits of implementing quality which influences maintenance of daily documentation procedures in the laboratory; and multiple and routinely-scheduled internal audits looking at a few requirements at a time might have improved timely identification and resolution of weaknesses in laboratory processes. we believe these approaches may have helped establish a continuous quality improvement culture in the laboratory and would guarantee that accreditation is not only achieved but maintained. conclusion implementing a qms is a continuous process that needs to be improved constantly in order to maintain accreditation. the benefits of implementing a qms outweigh the challenges encountered and impact client and staff satisfaction, better service delivery and overall healthcare improvement. many factors contributed to achieving international accreditation in mozambique, including availability of financial resources, well-trained and motivated staff to implement the qms and robust action plans. most importantly, the commitment at all levels, especially from high-level leadership and stakeholders turned the dream of accreditation into a reality. box 1: lessons learned. acknowledgements the authors acknowledge the ntrl staff for their commitment in turning this dream into a reality. we also thank the ministry of health, the national institute of health of mozambique, the quality unit of the national institute of health and the fogela team for their support and encouragement during the pursuit of accreditation. we extend our thanks to all partners for their support, in particular the united states centers for disease control and prevention, united states agency for international development, family health international, association of public health laboratories, foundation for innovative new diagnostics, the american society for microbiology, and the american society for clinical pathology. we acknowledge wilber sabiiti for improving the use of english in the manuscript. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. sources of support none. authors’ contributions s.o.v. and k.a. conceived the study design and wrote the first draft of the article. j.m., i.v.j. and e.s.g. reviewed the drafts and provided writing support. information and data were gathered and analysed by c.m. and c.a. technical assistance through the whole accreditation process was provided by c.a., d.m.c., c.d., a.p.m. and p.c. all authors contributed to writing the article. all authors gave final approval of the version to be published and agree to be accountable for the accuracy and integrity of the work. references world health organization, clinical and laboratory standards institute, us centers for disease control and prevention. laboratory quality management system: handbook. geneva: who; 2011. petti ca, polage cr, quinn tc, et al. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006;42(3):377–382. https://doi.org/10.1086/499363 woodcock s, fine g, mcclure k, et al. the role of standards and training in preparing for accreditation. am j clin pathol. 2010;134(3):388–392. https://doi.org/10.1309/ajcp03tfpbkeyynt gershy-damet g-m, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. https://doi.org/10.1309/ajcptuuc2v1wjqbm world health organization regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation in the african region [with checklist] [document on the internet]. c2013 [cited 2017 feb 09]. available from: http://www.finddx.org/wp-content/uploads/2016/03/who-2013_guide_for_the_slipta.pdf yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin pathol. 2010;134(3):401–409. https://doi.org/10.1309/ajcpnbbl53fwuiqj instituto nacional de saude. instituto nacional de saude [homepage on the internet]. n.d. [cited 2016 oct 22]. available from: http://www.ins.gov.mz/ world health organization regional office for africa. [orientações da oms para o processo gradual de melhoria laboratorial com vista à acreditação na região africana (com lista de verificação)] [portuguese]. brazzaville, republic of congo: who afro; 2013. world health organization regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation in the african region (with checklist) [document on the internet]. c2015 [cited 2017 feb 09]. available from: http://www.who.int/tb/laboratory/afro-slipta-checklist-guidance.pdf instituto português de acreditação. instituto português de acreditação [homepage on the internet]. n.d. [cited 2016 oct 30]. available from: http://www.ipac.pt/ peter tf, rotz pd, blair dh, et al. impact of laboratory accreditation on patient care and the health system. am j clin pathol. 2010;134(4):550–555. https://doi.org/10.1309/ajcph1skq1hnwghf olmsted ss, moore m, meili rc, et al. strengthening laboratory systems in resource-limited settings. am j clin pathol. 2010;134(3):374–380. https://doi.org/10.1309/ajcpdqosb7qr5glr masamha j, skaggs b, pinto i, et al. working toward a sustainable laboratory quality improvement programme through country ownership: mozambique’s slmta story. afr j lab med. 2014;3(2), art. #253, 6 pages. https://doi.org/10.4102/ajlm.v3i2.253 global laboratory initiative. gli stepwise process towards tb laboratory accreditation [page on the internet]. n.d. [cited 2016 oct 30]. available from: http://www.gliquality.org/ alemnji ga, zeh c, yao k, et al. strengthening national health laboratories in sub-saharan africa: a decade of remarkable progress. trop med int health. 2014;19(4):450–458. https://doi.org/10.1111/tmi.12269 zeh ce, inzaule sc, magero vo, et al. field experience in implementing iso 15189 in kisumu, kenya. am j clin pathol. 2010;134(3):410–418. https://doi.org/10.1309/ajcpzirkdus5lk2d opio a, wafula w, amone j, et al. country leadership and policy are critical factors for implementing laboratory accreditation in developing countries: a study on uganda. am j clin pathol. 2010;134(3):381–387. https://doi.org/10.1309/ajcp6kmotclisgj3 abstract introduction case presentation management and outcome discussion acknowledgements references about the author(s) khadijat o. isezuo department of paediatrics, usmanu danfodiyo university teaching hospital, sokoto, nigeria usman m. sani department of paediatrics, usmanu danfodiyo university teaching hospital, sokoto, nigeria usman m. waziri department of paediatrics, usmanu danfodiyo university teaching hospital, sokoto, nigeria bilkisu i. garba department of paediatrics, usmanu danfodiyo university teaching hospital, sokoto, nigeria yahaya mohammed department of medical microbiology and parasitology, usmanu danfodiyo university teaching hospital, sokoto, nigeria joy f. legbo department of paediatrics, usmanu danfodiyo university teaching hospital, sokoto, nigeria nazish p. aquil department of surgery, usmanu danfodiyo university teaching hospital, sokoto, nigeria fatima i. abubakar department of paediatrics, usmanu danfodiyo university teaching hospital, sokoto, nigeria memuna omar department of paediatrics, usmanu danfodiyo university teaching hospital, sokoto, nigeria citation isezuo ko, sani um, waziri um, et al. ecthyma gangrenosum on the face of a malnourished child with pseudomonas sepsis: simulating cancrum oris. afr j lab med. 2018;7(1), a756. https://doi.org/10.4102/ajlm.v7i1.756 case study ecthyma gangrenosum on the face of a malnourished child with pseudomonas sepsis: simulating cancrum oris khadijat o. isezuo, usman m. sani, usman m. waziri, bilkisu i. garba, yahaya mohammed, joy f. legbo, nazish p. aquil, fatima i. abubakar, memuna omar received: 12 jan. 2018; accepted: 19 sept. 2018; published: 05 dec. 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract introduction: ecthyma gangrenosum (eg) is a cutaneous lesion commonly caused by pseudomonas aeruginosa that involves mainly the lower limbs and gluteal region, seen more in immunosuppressed patients with neutropenia. cancrum oris (noma) is a gangrenous necrosis of the face that begins as a gingival ulcer and progresses rapidly to destroy contiguous tissues in malnourished children. case presentation: this article reports a case of facial eg which was similar to noma in a malnourished child: a 16-month old girl with fever, cough, weight loss, watery stool and swelling on right cheek. she was febrile, pale, wasted with bilateral pitting pedal oedema. she had a solitary circumscribed round necrotic lesion, with surrounding hyperaemia on the right malar area which extended to destroy the right ala nasi. no intra-oral rashes but she had left ear discharge. she received blood transfusion, antibiotics, antiseptic wound care and nutritional rehabilitation. management and outcome: swabs of the lesion and ear discharge both revealed gram-negative bacilli and culture yielded p. aeruginosa. retroviral, mantoux and gene xpert tests were negative. she had moderate anaemia, normal white blood cell count, and neutropaenia. parenteral ceftriazone was changed to ciprofloxacin based on sensitivity results and lack of clinical response. the wound healed with residual scarring and partial destruction of right ala nasi. discussion: although this patient had facial necrosis to suggest noma, she did not have initial oral involvement, and clinical features such as pseudomonas sepsis and neutropaenia suggested eg. facial necrosis in malnourished children may be due to eg. introduction ecthyma gangrenosum (eg) is a cutaneous lesion commonly caused by pseudomonas aeruginosa,1,2 though it has been associated with other bacterial, viral and fungal aetiological agents.3,4,5 it occurs commonly in immunosuppression of various causes and is associated with neutropaenia.6 it may occur as a primary skin lesion which is the localised or non-septicaemic form or it may be the classical form which is a septicaemic illness associated with haematogenous spread of the causative bacterial organism.5,7 the typical appearance is that of a necrotic skin lesion with a central eschar surrounded by a hyperaemic halo.8 it commonly involves the lower limbs or genital area but may also involve other areas of the body occurring as solitary or multiple lesions. the sites commonly affected by eg lesions are the gluteal or perineal region (57%), extremities (30%), trunk (6%), and face (6%).9 some have reported facial involvement in 12% of cases.4 facial involvement has been reported to be common in neonates who are immunocompromised compared to older infants and children.10,11,12 cancrum oris (noma) is a severe oro-facial gangrene which occurs more in debilitated and malnourished patients.13 it usually starts as a gingival infected ulcer which rapidly becomes necrotic and spreads to produce extensive destruction of the tissues of the face in and around the oral cavity. aetiology is usually polymicrobial by opportunistic organisms related to malnutrition and immune dysfunction.14 some writers have described noma-like necro-ulcerative lesions involving the face for which, if diagnosed early, intensive therapy may limit progression and confer better prognosis.15 in this report, we highlight a case of a malnourished child who presented with a necrotic ulcer involving the face which was similar to noma but, on further review of the evolution of the ulcer and investigation of test results, was found more likely to be eg. we report the case to highlight that a necrotic facial ulcer presenting in a malnourished child may be eg and may mimic orofacial gangrene of noma if not aggressively managed. case presentation ethical considerations ethical approval for the study was obtained from the research and ethics committee of usmanu danfodiyo university teaching hospital (number: uduth/hrec/2018/699). written consent was obtained from the parents of the child. confidentiality was maintained as well as privacy, as the identity of the child was known only to the authors who partook in her management. the clinical pictures taken were selectively occluded except for the aspect showing the lesion so as to prevent recognition of the patient. method of data collection data was collected by history-taking, clinical examination, clinical photographs and anthropometric measurements (weight, height and occipitofrontal circumference); venous blood samples were collected via aseptic technique for electrolytes, complete blood count, retroviral screening and blood culture. a nasogastric tube was used to collect samples of gastric washings. a sterile swab stick was used to collect samples of the lesion and ear discharge. case report a 16-month old girl presented to usmanu danfodiyo university teaching hospital, sokoto, in october 2016 with complaints of fever, cough and weight loss of 2 months, watery stool of 6 weeks, swelling on right cheek of a week’s duration. the swelling initially started as a reddish rash which increased in size and darkened in colour. it was painless and not associated with discharge. she had poor nutritional and immunisation history. there was no preceding history of measles, though there was contact with other children who had pertussis. on examination, she was febrile (38.9 °c), pale, wasted with bilateral pitting pedal oedema and fluffy hair. her weight was 5 kilograms (kg), length was 68 cm (weight for length z-score of < −4 standard deviation, expected value of −2 standard deviation to +2 standard deviation), occipitofrontal circumference was 40 cm (expected range of 47 – 48 cm) and mid-arm circumference was 9.5 cm (expected normal value of >14 cm). she had a solitary circumscribed round necrotic lesion (2 cm × 2 cm) with a central black eschar and surrounding hyperaemia on the right malar area which had minimal purulent discharge but was not foul-smelling (figure 1). the central eschar sloughed off on the third day after admission to reveal a deep ulcer which had extended to destroy the right ala nasi (figure 2). there was no intra-oral communication of the ulcer from the external surface. she did not have any intra-oral rashes, gingival ulcers or oral thrush. figure 1: necrotic eschar on second day after admission. figure 2: deep ulcer extending to destroy the right ala nasi after sloughing of necrotic eschar. she developed purulent left ear discharge four days after admission. the external ear was normal; however, examination of the middle ear was not done as the child was not cooperative. her pulse was 124 beats per minute and respiratory rate was 46 cycles per minute. she had vesicular breath sounds with reduced intensity and normal heart sounds. abdominal examination revealed a non-tender hepatomegaly of 4 cm. neurological examination was normal. management and outcome swabs of the lesion and ear discharge showed pus cells and gram-negative bacilli, while culture of both yielded p. aeruginosa. it was sensitive to ciprofloxacin+++, ceftazidime++, ofloxacin++, and gentamicin++. blood culture was however negative, although anaerobic culture was not done. retroviral screening, mantoux and gene xpert tests were all negative. her packed cell volume was 22%, which dropped to 18% on the seventh day, on account of which she was transfused packed red cells, resulting in post-transfusion volume of 32%. her total white cell count was 7 × 109/l (reference range 4–11 × 109/l), with 16% constituting neutrophils (1120 cells, range: 2000 – 8000 cells), and 84% lymphocytes (5880 cells, range: 1000 – 6000 cells); platelets were 145 × 109/l. the erythrocyte sediment rate was 28 mm fall/hr. the diagnosis was severe acute malnutrition (oedematous type) with complications of sepsis, severe anaemia and possibly evolving cancrum oris. however, after further review of the patient’s history, evolution of the lesion, in conjunction with the investigation results suggesting neutropaenia and isolation of p. aeruginosa from the lesion, eg involving the face was considered to be more likely. she received blood transfusion, antibiotics, antiseptic wound care with normal saline soaks and dressing with honey. she also received nutritional rehabilitation in form of enriched pap and ready-to-use therapeutic food which is a nutritional rehabilitation diet manufactured mainly from peanuts. electrolyte derangements (hypokalaemia and hyponatremia) were corrected with oral potassium supplements added to feeds and oral rehydration solution. initial intravenous ceftriazone was changed to intravenous ciprofloxacin and intravenous gentamicin based on sensitivity results. the ulcer healed remarkably but with residual scarring, including destruction of right ala nasi (figure 3). oedema subsided four days after admission and her weight rose gradually during the subsequent 2 weeks to 6.8 kg. she spent about 3 weeks in the hospital. on a subsequent follow-up visit, she was doing well; however, the residual scar was still present, though much smaller. caregivers defaulted on an appointment to see the co-managing plastic surgeon for possible repair of the residual scar. figure 3: residual scarring after healing of the ulcer. discussion ecthyma gangrenosum lesions were first described in 1897 in association with pseudomonas septicaemia.16 eg usually begins as a haemorrhagic vesicle or pustule that gradually evolves into a necrotic ulcer with eschar. this progression is because the bacteria invades the walls of the dermal and sub-dermal vessels leading to vascular damage and interruption of blood supply to the skin causing necrosis of the skin with eschar and ulcer formation.17 although it has been recognised to be caused by a variety of other organisms, eg is most typically associated with severe systemic infection due to p. aeruginosa.17,18 it is most often seen in immunocompromised or neutropenic patients, though it can also occur in previously healthy individuals. our patient was immunocompromised as she was severely malnourished and she also had neutropaenia.19,20 the presence of acute otitis media caused by pseudomonas in the child in addition to the skin manifestation of eg supported pseudomonas sepsis even though the blood culture was negative. in a case report from brazil of eight cases of eg in children, facial involvement was less common in the series. one of the cases was also a female infant with facial involvement and malnutrition similar to our case.21 authors from india reported a case of a severely malnourished child with both eg involving the limbs caused by pseudomonas and noma; they suggested that both conditions can coexist with malnutrition as an underlying predisposing factor for both.22 a case was reported by biddeci23 from italy in a female infant who was previously healthy. she had a facial lesion similar to our patient which healed with a residual scar after treatment with anti-pseudomonal antibiotics based on a positive skin swab. blood culture was also negative, similar to findings in our patient. noma neonatorum (a distinct entity from noma in older children) which occurs in neonates, especially those with low birth weight and those born pre-term, is a rapidly progressive gangrenous lesion involving facial structures especially the mouth, nose and eyelids. it also involves the anal region and scrotum.24 it is also commonly associated with p. aeruginosa septicaemia. although some authors have recently questioned distinguishing noma in neonates from eg caused by pseudomonas,11 noma in older children is quite a distinct entity as it starts as a gingival ulcer and the ensuing gangrene rapidly spreads through muscles exposing the bone and teeth.13 also, in noma, the aetiological agents usually isolated include anaerobes like prevotella intermedia, fusobacterium nucleatum, peptostreptococcus micros, campylobacter and enteric gram-negative organisms, while eg is mainly caused by pseudomonas.13 however, these anaerobes cannot be completely ruled out in our case since anaerobic culture was not done. the findings in the index case suggest that the child likely had eg rather than noma given the evolution of the ulcerative process. however, without aggressive therapy or relatively early presentation of the patient, one may hypothesise that it could have spread, further simulating noma. in conclusion, this patient had facial necrosis to suggest noma; however, she did not have initial oral involvement, and clinical features suggested eg, which was supported by suspected pseudomonas sepsis and neutropaenia. facial necrosis in malnourished children may be due to eg. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. sources of support none. authors’ contributions k.o.i. conceptualised the report and wrote the initial draft. k.o.i., u.m.s., u.m.w. and b.i.g. were responsible for project design and reviewed the initial draft. y.m. performed most of the experiments. j.f.l., n.p.a., f.i.a. and m.o. made conceptual contributions and performed some of the experiments. references chan yh, chong cy, puthucheary j, loh tf. ecthyma gangrenosum: a manifestation of pseudomonas sepsis in three paediatric patients. singapore med j. 2006;47:1080–1083. fang lc, peng cc, chi h, lee ks, chiu nc. pseudomonas aeruginosa sepsis with ecthyma gangrenosum and pseudomembranous pharyngolaryngitis in a 5-month-old boy. j microbiol immunol infect. 2014;47:158–161. https://doi.org/10.1016/j.jmii.2012.05.010 reich h, williams fd, naik n, honig p, yan a. nonpseudomonal ecthyma gangrenosum. j am acad dermatol. 2004;50:s114–117. https://doi.org/10.1016/j.jaad.2003.09.019 zomorrodi a, wald e. ecthyma gangrenosum: considerations in a previously healthy child. pediatr infect dis j. 2002;21:1161–1164. https://doi.org/10.1097/00006454-200212000-00016 pathak a, singh p, yadav y, dhaneria m. ecthyma gangrenosum in a neonate: not always pseudomonas. bmj case rep [serial online]. 2013 [cited 2017 dec 07]. available from: http://casereports.bmj.com/content/2013/bcr-2013-009287.full.pdf bozkurt i, yuksel e, sunbul m. ecthyma gangrenosum in a previously healthy patient. indian dermatol online j. 2015;6:336–338. https://doi.org/10.4103/2229-5178.164479 el baze p, thyss a, vinti h, deville a, dellamonica p, ortonne jp. a study of nineteen immunocompromised patients with extensive skin lesions caused by pseudomonas aeruginosa with and without bacteremia. acta derm venereol. 1991;71:411–415. dorff gj, geimer nf, rosenthal dr, rytel mw. pseudomonas septicemia. illustrated evolution of its skin lesion. arch intern med. 1971;128:591–595. https://doi.org/10.1001/archinte.1971.00310220099014 elmariah s, ubriani r, james w, kageyama n, fish f. ecthyma gangrenosum [homepage on the internet]. [cited 2017 dec 10]. available from: http://www.emedicine.com/derm/topic539.htm foca md. pseudomonas aeruginosa infections in the neonatal intensive care unit. semin perinatol. 2002;26:332–339. https://doi.org/10.1053/sper.2002.36266 freeman af, mancini aj, yogev r. is noma neonatorum a presentation of ecthyma gangrenosum in the newborn? pediatr infect dis j. 2002;21:83–85. https://doi.org/10.1097/00006454-200201000-00025 parikh tb, nanavati rn, udani rh. noma neonatorum. indian j pediatr. 2006;73:439–440. https://doi.org/10.1007/bf02758572 enwonwu co, falkler wa, jr., phillips rs. noma (cancrum oris). lancet. 2006;368:147–156. https://doi.org/10.1016/s0140-6736(06)69004-1 raimondi f, veropalumbo c, coppola c, et al. noma neonatorum from multidrug-resistant pseudomonas aeruginosa: an underestimated threat? j pediatric infect dis soc. 2015;4:e25–e27. https://doi.org/10.1093/jpids/piu072 nash es, cheng lhh, smart k. cancrum oris-like lesions. br j oral maxillofac surg. 1991;29:51–53. https://doi.org/10.1016/0266-4356(91)90176-6 ecthyma gangrenosum [homepage on the internet]. [cited 2017 dec 10]. available from: https://emedicine.medscape.com/article/1053997-overview. duffill m. ecthyma gangrenosum [homepage on the internet]. new zealand: dermnet. [cited 2017 dec 10]. available from: https://www.dermnetnz.org/topics/ecthyma-gangrenosum/ bodey gp, bolivar r, fainstein v, jadeja l. infections caused by pseudomonas aeruginosa. rev infect dis. 1983;5:279–313. https://doi.org/10.1093/clinids/5.2.279 schaible ue, kaufmann she. malnutrition and infection: complex mechanisms and global impacts. plos med. 2007;4:e115. https://doi.org/10.1371/journal.pmed.0040115 segel gb, halterman js. neutropenia in pediatric practice. pediatr rev. 2008;29:12–24. https://doi.org/10.1542/pir.29-1-12 martínez-longoria ca, rosales-solis gm, ocampo-garza j, guerrero-gonzález ga, ocampo-candiani j. ecthyma gangrenosum: a report of eight cases. anais brasileiros de dermatologia. 2017;92:698–700. https://doi.org/10.1590/abd1806-4841.20175580 vaidyanathan s, tullu ms, lahiri kr, deshmukh ct. pseudomonas sepsis with noma: an association? indian j med sci. 2005;59:357–360. https://doi.org/10.4103/0019-5359.16653 biddeci g, cutrone m, mattei i, valerio e, favot f. ecthyma gangrenosum of the cheek in a 6-month-old infant. arch dis child. 2015;100:55–56. https://doi.org/10.1136/archdischild-2014-306852 ghosal sp, sen gupta pc, mukherjee ak, choudhury m, dutta n, sarkar ak. noma neonatorum: its aetiopathogenesis. lancet. 1978;2:289–291. https://doi.org/10.1016/s0140-6736(78)91691-4 abstract early infant diagnosis of hiv and virological monitoring of patients on antiretroviral therapy – ongoing challenges for sub-saharan africa the concept of pooled testing (‘pooling’) – some important considerations perspectives and outlook acknowledgements references about the author(s) wolfgang preiser division of medical virology, department of pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa national health laboratory service (nhls) tygerberg, cape town, south africa gert u. van zyl division of medical virology, department of pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa national health laboratory service (nhls) tygerberg, cape town, south africa citation preiser w, van zyl gu. pooled testing: a tool to increase efficiency of infant hiv diagnosis and virological monitoring. afr j lab med. 2020;9(2), a1035. https://doi.org/10.4102/ajlm.v9i2.1035 review article pooled testing: a tool to increase efficiency of infant hiv diagnosis and virological monitoring wolfgang preiser, gert u. van zyl received: 24 apr. 2019; accepted: 15 apr. 2020; published: 11 aug. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: pooled testing, or pooling, has been used for decades to efficiently diagnose relatively rare conditions, such as infection in blood donors. programmes for the prevention of mother-to-child transmission of hiv and for antiretroviral therapy (art) are being rolled out in much of africa and are largely successful. this increases the need for early infant diagnosis (eid) of hiv using qualitative nucleic acid testing and for virological monitoring of patients on art using viral load testing. while numbers of patients needing testing are increasing, infant hiv infections and art failures are becoming rarer, opening an opportunity for pooled testing approaches. aim: this review highlights the need for universal eid and viral load coverage as well as the challenges faced. we introduce the concept of pooled testing and highlight some important considerations before giving an overview of studies exploring pooled testing for eid and virological monitoring. results: for art monitoring, pooling has been shown to be accurate and efficient; for eid it has not been tried although modelling shows it to be promising. the final part attempts to place pooling into the context of current mother-to-child transmission of hiv and art programmes and their expected trajectories over the next years. conclusion: several points warrant consideration: pre-selection to exclude samples with an elevated pre-test probability of positivity from pooled testing, the use of dried blood or plasma spots, and choosing a pooling strategy that is both practically feasible and economical. finally, novel ideas are suggested to make pooling even more attractive. keywords: hiv; antiretroviral treatment; early infant diagnosis; pooling; pooled testing. early infant diagnosis of hiv and virological monitoring of patients on antiretroviral therapy – ongoing challenges for sub-saharan africa the prevention of mother-to-child transmission (pmtct) of hiv has made enormous progress over the past 15 years, with much-increased coverage and greatly improved programme components. according to estimates for 2017, 80% of hiv-positive pregnant women globally – and as many as 93% in eastern and southern africa – received antiretroviral drugs for pmtct. while 180 000 children aged up to 14 years were newly infected with hiv globally, 210 000 infant infections were averted due to pmtct.1 globally, 14.8 million hiv-exposed children remained uninfected.1 in south africa, the national pmtct programme has followed the world health organization option b+ policy since 2015: provision of lifetime combination antiretroviral therapy (art) for all hiv-positive pregnant women, regardless of their immunological or clinical stage.2 this has resulted in the reduction of the mother-to-child transmission rate to an estimated 4.6% overall in 2016.3 despite these successes, all hiv-exposed infants continue to need early infant diagnosis (eid). maternal antibodies persist up to age 18 months. therefore, eid relies on virological assays that detect either viral nucleic acid or viral proteins at different ages to diagnose cases of prenatal, perinatal and postnatal transmission.4 the prompt diagnosis of hiv infection is a prerequisite for starting infected babies on art early on, thus improving their prognosis.5 while infant hiv infection has now become or will soon become a relatively rare event,6 the sheer number of exposed infants in need of testing at different time points will continue to pose a major challenge to laboratory systems. another area that has seen enormous progress over the past two decades is art programmes, which have been implemented in much of africa and have been generally successful. of the 20 million people estimated to be living with hiv infection in eastern and southern africa in 2017, about 13 million or 66% were on art, of whom more than 10 million were estimated to have suppressed viral load (vl).1 for south africa, according to 2017 estimates, 90% of hiv-positive individuals know their status; 68% of these are on art and 78% of those on art achieve viral suppression.1 previously regarded as optional,7 routine virological monitoring of patients on art by hiv vl testing to detect art failure allows optimal patient management. addressing either adherence or presumed drug resistance (when there is no response to adherence interventions) secures long-term viability of art programmes by limiting emergence and transmission of antiretroviral drug resistance.8,9 optimal virological suppression is essential to prevent onward transmission; therefore, achieving an undetectable vl in 90% of those on art has been included as the last component of the joint united nations programme on hiv/aids (unaids) 90-90-90 targets to help end the hiv/aids epidemic.10 the need for virological monitoring is increasingly reflected in art guidelines.4 yet significant barriers remain: currently available vl tests are expensive, technically demanding and thus not widely available in many resource-limited settings (rls).11 while point-of-care vl tests may be an ideal solution and several systems are approaching clinical usability, their relatively high cost and other challenges remain unresolved.12 a recent forecast study underlines the need for a substantial rollout of both eid and vl testing.13 the concept of pooled testing (‘pooling’) – some important considerations pooled testing or ‘pooling’, also known as ‘group testing’, refers to mixing several samples together prior to performing a laboratory test. individual samples contained in a negative pool can be classified as negative without further testing, whereas samples contained in pools testing positive need to be re-tested individually. while pooling may seem rather crude and does not apply to measurements of analytes that are present in both physiological and pathological states, albeit, at different concentrations, pooling has a long tradition for infectious disease markers, for example for the screening of blood donations.14 here the physiological state is ‘negative’ (which means the laboratory marker, for example specific antibodies or pathogen-specific nucleic acid sequences, are undetectable), thus, the prevalence of positive samples among donors is low, thanks to the application of stringent donor selection criteria. used for antibody-based screening (e.g. syphilis) for decades, pooled testing was later adopted for nucleic acid testing (nat), too.15 the advantage of pooling lies in its ability to save on the number of tests needed and, thus, costs. as long as the condition to be detected occurs at low prevalence, most pools will test as negative. then, all samples contained in a negative pool can have a negative result reported while having used just one test. if a pool tests positive, it needs to be determined which of the constituent samples is responsible. this is achieved by re-testing the constituent samples individually (a process termed ‘deconvolution’ or ‘resolution’) which thus negates any saving for the samples concerned, in that the constituent samples together will, in the end, have used a number of tests equal to their number plus one (the test used for the pool). alternative strategies have been proposed that are more efficient than using straightforward pooling and deconvolution by re-testing constituent samples simultaneously. in two-dimensional or multidimensional matrix approaches, each sample is contained in a unique combination of different pools; the combination of pools that test positive resolves the sample responsible without re-testing.16 multistage or pyramid-type pooling strategies may likewise be more efficient than simple pooling.17 when a pooling approach is used for quantitative tests, a strategy of individually re-testing the constituent samples one after the other may be designed; when an individual sample’s quantitative test result can explain the quantitative result for the pool, the remaining samples may be regarded as negative.18 such an approach is, however, not without challenges, especially given the test-to-test variation of quantitative results. the main factors determining whether pooling may be advantageous are the prevalence of the condition to be tested for, and the pool size. pooling is predicated on a relatively low prevalence condition being sought. if a condition is highly prevalent, pooling will not save tests, due to frequent deconvolution and individual re-testing. the larger the pool size (i.e. the number of samples mixed together to form one pooled specimen for testing), the greater the potential saving. however, pool size is not only limited by practical considerations – test volumes and dilution factors, the latter linked to test sensitivity; but by the tested condition’s prevalence – a low prevalence ensures that a substantial proportion of test pools are negative. a simple example illustrates this: assuming a prevalence of 20%, a pool size of five specimens will yield almost no savings and a pool size of four, only marginal savings.19 by excluding patient samples with a high probability of positivity from pooling, the prevalence of the condition tested in the pooled samples is reduced invariably improving pooling efficiency.20 examples of such patients are children whose mothers did not receive pmtct, those who failed art previously or are clinically unwell, and those sent for confirmation of a previous positive result. several aspects need to be taken into account when considering pooled testing. the first aspect is the test sensitivity. pooled test sensitivity is often somewhat lower than the sensitivity of individual tests because of the required dilution of pooled specimens. however, this lowered may not be clinically relevant and counteracted to some degree by modifying the testing process (e.g. substituting some of the diluent or sample buffer with specimen). the sensitivity of pooled testing and any measures to improve it has to be considered and validated carefully before using pooling for diagnostic purposes. currently, available commercial vl assays have lower limits of detection below 50 copies/ml. this redundant sensitivity can be exploited by testing pools consisting of several mixed samples, if the world health organization’s recommended threshold is used for clinical decision-making. sample throughput is another important consideration. there needs to be a balance between volumes of samples (requests) received per day and the pool sizes (to allow for efficient pooling) without increasing test turn-around time while waiting for enough specimens to be received to allow for pooling. finally, the processes of pooling and deconvolution (of positive pools) add to the operator workload. on the other hand, operator workload is reduced through efficient pooling, as fewer tests need to be performed. however, performing pooling and deconvolution may also introduce clerical errors; the approach must therefore be designed well and conducted carefully. these factors need to be factored into calculations of pooling efficiency and assessing practicability. the interaction between the above-listed factors is rather complex. it is highly unlikely that anyone or a limited choice of pooling solutions, would be suitable for different scenarios. pooled testing for early infant diagnosis of hiv the earliest possible diagnosis of hiv infection in hiv-exposed infants is a prerequisite for early initiation of art, which markedly improves the prognosis of infants with hiv infection.5 this requires testing uncoagulated whole blood or dried blood spot (dbs) samples for viral genome (proviral dna, viral rna, or both) by means of polymerase chain reaction (pcr) or similar genome amplification technologies.4 pcr is technically demanding and costly, limiting access to it in many rls. dbs are widely used in rls, as they are easier to obtain, to store and to transport while performing generally well.21 recommendations about the optimal time points for eid testing are evolving, due to programmatic improvements and new scientific findings. the optimum would be three eid tests for each exposed infant: the first at or soon after birth (to detect infection acquired in utero), the second at around 6–10 weeks of age (to detect perinatal transmission; the preferred timing depends on the duration of postnatal post-exposure prophylaxis), and a third one 6 weeks after cessation of breastfeeding (to detect postnatal transmission, which would usually be via breast milk).2 not only is eid coverage across most of sub-saharan africa poor and the results commonly delayed,1 performing three tests for each exposed infant also places an enormous burden on health and laboratory services. measures to increase eid capacity and reduce costs are therefore sorely needed. point-of-care assays are increasingly being made available and have been piloted in several studies, but several drawbacks remain unresolved: their relatively high complexity requires trained staff; their relatively low throughput requires multiple instruments at busy clinics; and their relatively high cost which is and will likely remain higher than testing at centralised laboratories.22 interestingly, there does not seem to be much interest in pooled testing for eid. the first and so far only known study on this topic, published in february 2019, determined the clinical accuracy of pooled testing of infant dbs and developed a model to simulate the saving of reagent and consumable costs based on real data from a south african public health laboratory.23 the study found a high sensitivity (98.8%) for pooled testing. the one observed false-positive pool test result was confirmed through re-testing of the constituent patient samples. the savings of laboratory costs – 64% based on 2015 prices – were substantial. the article concludes that pooling eid pcr tests retains accuracy while substantially reducing costs, provided a laboratory receives sufficient numbers of samples and has a low to moderate infant hiv prevalence. the authors have developed and made available publicly an r-based web tool to assess the cost-efficiency of pooled pcr testing for infant hiv diagnosis for varying parameters (https://carivs.shinyapps.io/calculator). users will enter the average number of samples tested per day, the estimated positivity rate (maximum allowed is 10% as above that pooling would not be viable), the sample type (dbs or whole blood), the cost of reagents (if unknown, the model provides an estimated percentage saving) and the minimum number of samples required to run a batch for pooling and for individual testing. the model will then do a calculation, based on the above, and report the optimal pool size, the cost saving, and the reduction in daily batch runs both as a sentence (e.g. based on your input values, at the optimal pool size of 5, you will save 62% of costs and daily batched runs will decrease from 2 to 1) and graphically as a three-dimensional plot with axes for positivity (%), pool size and cost as a percentage of cost for testing individual samples. pooled testing for virological monitoring the sustained suppression of hiv replication in patients on art is the goal of hiv treatment. halting viral replication provides individual benefit as it allows immune reconstitution and reduces inflammation, thereby lowering the risk for hiv-associated infectious and non-communicable complications. moreover, on a population level, sustained virological suppression renders treated patients virtually non-infectious and thus prevents onward transmission.24 conversely, failing to achieve virological suppression puts the patient and the population at risk; this is not only through hiv disease and the transmission of hiv infection, but also through the emergence of antiretroviral drug-resistant hiv strains which may jeopardise future art options for the individual and, through the transmission of resistant virus strains could put the whole art programme at risk of failure.25 monitoring of patients on art by means of virological assays is therefore of paramount importance and after years of advocacy, this is now widely acknowledged and reflected in the third ‘90’ of the unaids’s 90-90-90 goals.26,10 in 2017, the proportion of people of all age groups on art who achieved virological suppression was estimated at 81% globally, at 79% for eastern and southern africa and at 73% for west and central africa. for botswana, the estimate is > 95% and for south africa, 78%. virological non-suppression appears to be increasingly rare in many settings and may have become a low prevalence condition in some places already.1 virological monitoring is usually done through hiv vl testing, that is, using assays that quantify the concentration of hiv rna in patient plasma. a vl result of less than 50 copies of hiv rna per millilitre of plasma is usually regarded as full suppression, which is the aim of art in industrialised countries. in rls, vl of 1000 copies/ml in patients on art for at least 6 months or a vl above 1000 copies/ml in patients after two consecutive measurements 3 months apart with adherence support defines virological failure (vf) and should trigger clinical action.4 the rationale for using 1000 copies/ml as the widely accepted public health threshold is twofold: below this level, there is a limited risk of both disease progression and hiv transmission.4 additionally, it avoids the irrelevant detection of viral ‘blips’, short episodes of low-level viraemia that may occur during effective art and are not associated with an increased risk of art failure.27 smith et al.28 was the first study to report the use of pooled vl testing to identify art failure. it was conducted on a small number of art patient samples in the united states and compared two different pooling strategies, namely mini pools of five samples each and 10 × 10 matrix pools, with individual vl testing as the gold standard. in a matrix pooling approach, each sample is contained in more than one pool and the combination of pools testing positive allows direct identification of the sample responsible in most instances when the prevalence of failure is low, making deconvolution unnecessary. even though 23% of samples had a vl of > 50 copies/ml, both pooling strategies combined with a search and retest algorithm, led to savings of > 50% compared with individual testing, with only a minimal decrease in accuracy. a subsequent modelling study assessed various pooling strategies for monitoring art patients with vf prevalences between 1% and 25% in terms of efficiency and accuracy.19 it also compared three different pooling approaches – mini pools, mini pools with algorithm-based deconvolution, and matrix pooling to individual testing. using the quantitative information, that is, vl measurements available, this study showed pooling efficiency at higher prevalences than for which a purely binary approach (vl either detectable or undetectable) would have been efficient. however, such relatively complicated approaches may not prove feasible in many rls. a third study, conducted in south africa, investigated three aspects. firstly, whether the use of basic data recorded on routine laboratory request forms would allow selection of art patients with a low probability of art failure for pooled testing. secondly, whether mini pool or matrix pooling strategies were more suitable, comparing them against individual vl testing in a laboratory study. thirdly, the use of dbs and dried plasma spots instead of liquid plasma samples for preparing mini pools.20 in the retrospective cohort studied, the vf rate was 14.2%.20 after applying age > 15 years and being on a first-line art regimen as selection criteria (information easily obtained from test request forms), this dropped to 8.7% making pooling of such pre-selected samples more feasible. four hundred plasma specimens were tested in 80 mini pools of five each, and 300 of these samples also with a 10 × 10 matrix strategy. pooled testing reduced the number of tests needed by 30.5% – 60.0%. even though the matrix pooling strategy was more efficient than mini pooling, the latter was deemed overall more suitable as it proved much easier to perform, taking less operator time and requiring less expertise for constitution and deconvolution of pools. in addition, waiting for 100 samples to be available for testing before a 10 × 10 matrix can be constructed may increase turn-around times in many laboratories; this is generally not a problem with mini pools of five samples. the study found a poorer specificity and efficiency of pooled dbs testing. factors such as the contribution of cell-associated hiv rna and the variable plasma fraction (depending on patients’ haematocrit values) could cause falsely high vl results, while the presence of haemoglobin and other inhibitory substances in dbs could lead to falsely low results. pooled dried plasma spot testing, however, showed less variability and excellent accuracy and an efficiency of 60% for a pool threshold of 200 hiv rna copies/ml. a large field study in malawi subsequently addressed two issues relevant to rls: a higher vf rate and the problems inherent in testing plasma samples.29 testing 350 patient samples, the study found that pooled testing of plasma samples had excellent sensitivity (96.4%) and reduced the number of tests required by 44.3%. using dbs, prepared either directly by finger prick (which would be the preferred option in real-life use of pooled testing in rls) or from venous blood, sensitivity was 78.6% and 89.3%, saving 28.6% – 32.9% of tests. it concluded that when using the nuclisens assay – which does not amplify the cell-associated hiv dna contained in dbs, in contrast to most other hiv vl assays – testing dbs is a feasible option. pooling for virological monitoring of art patients has been assessed in other settings: those with a low prevalence of vf such as south korea30 and in rls with typically much higher failure rates such as mexico,31 kenya32 and mozambique.33 even under rls conditions pooled testing was generally efficient with acceptable accuracy, although not as efficient as in settings with low failure rates. however, el bouzidi et al.34 reported that testing in mini pools of four samples could not reliably identify vf in a london, united kingdom, art population, due to insufficient diagnostic sensitivity. while almost one in three failing patients was missed, the vl values of failing patients were low and mostly irrelevant for rls. perspectives and outlook pooled testing has a long history of successful use in the field of infectious disease testing, the most notable application being for screening blood donors both by serology and since the 1990s by nat.15 more recently, a multitude of studies have investigated pooled nat as a tool to detect early hiv infection that would be missed by routine hiv screening tests. in africa, karim et al.35 were the first to propose such a strategy; yet even in a high risk setting such as described by gous et al.,36 pooled nat would still be relatively expensive per additional case detected, as very few patients will be in the nat-positive antibody-negative stage of recent infection.37 more recently, a study by dowling et al.38 demonstrated that dbs can be used for this purpose, which would facilitate its wide implementation in rls. with ongoing major advances in both eid and art programmes, it is surprising that the use of pooled testing has hardly been explored for the purposes of eid, while there is ample literature on pooled testing for monitoring patients on art in a variety of settings. the need for eid testing will remain unchanged even as mother-to-child transmission rates are in decline. point-of-care testing might appear as an ideal solution for a variety of reasons,39 yet it is questionable whether it will be the most affordable solution against the background of an enormous, only partially met need. pooled eid testing has been modelled to be efficient,23 and infant hiv infection is becoming less prevalent in most places. however, ongoing vigilance is required as the evolving nature of the pmtct programme makes establishing a definite diagnosis increasingly challenging.40,41 the need for art monitoring will increase in the foreseeable future. in principle, when the prevalence of vf is low, pooled testing is very efficient, saves costs and may even reduce the average turn-around time (time from obtaining sample to receipt of test result). it is expected that with the rollout of newer therapy regimens the prevalence of vf may drop further, to below 7%.42 this might cause pressure to decrease the frequency of vl testing further, when in actual fact failure needs to be identified more efficiently. the performance characteristics of modern laboratory-based vl assays are redundant for widely accepted vl thresholds and thus provide comfortable room for pooling; highly sensitive assays offer leeway when a threshold of 1000 copies/ml is used to define art failure. pooling would potentially increase throughput and reduce turn-around time. pooled hiv vl testing has the potential to reduce the reagent cost of vl testing. early initiation of art and the use of more tolerable high genetic barrier regimens for first-line art have the potential to further drive down the prevalence of vf.42 pooled vl testing is therefore likely to become even more efficient in the near future. nevertheless, large scale implementation of pooled testing requires overcoming several hurdles. the first is susceptibility to human error, when pooling and deconvolution are performed manually. the second is that pooling and deconvolution add to hands-on time and therefore increase labour costs. the third is that the efficiency of pooled testing is highly susceptible to test failure. important considerations as regards pooling for eid and monitoring art, a number of pivotal points are emerging from the published studies, and these issues need to be taken into account when considering the introduction of pooled testing. firstly, to ensure the highest possible efficiency of pooling, a pre-selection step should be considered to identify samples from patients with a high pre-test probability of vf (i.e. hiv-positive patients). this could be achieved by interrogating the information contained on most test request forms or the information contained in the laboratory information system, where possible. the purpose of this would be to exclude ‘high risk’ specimens from being pooled lest they ‘poison’ pools. secondly, to maximise the usefulness of pooled testing in rls, the use of dbs or dried plasma spot would be ideal. once dried onto filter paper, such samples are non-infectious and stable for prolonged periods, as long as they are not subjected to extreme temperatures and excessive humidity; they can therefore be stored and transported even over long distances easily and cheaply.43 dried blood spots are relatively easy to obtain using finger (or toe or heel, in infants) pricks and are already widely used for eid in rls. for eid, their sensitivity may need consideration as the proportion of infected infants with low vl values increases.44 regarding vl testing, dbs have certain intrinsic disadvantages that are unlikely to be overcame, such as the detection of intracellular viral genomes. on the other hand, dried plasma spots negate part of the attractiveness of dried samples as they require expert sample handling and manipulation at the clinical site. this expertise is often lacking in many peripheral clinics and also increases operator time, which in turn increases the risk of sample mix-ups, mislabelling, and the like. how much solution simple-to-use plasma separation devices will offer for this issue remains to be ascertained.45 thirdly, regarding the preferable pooling strategy, it appears that in rls straightforward mini pools and parallel deconvolution probably offer the best efficiency with ease of handling. in many rls it will be necessary to avoid placing too much extra strain on scarce laboratory staff in terms of work time and effort. a misconceived, over-ambitious approach may lead to an increase in clerical errors and other problems that could easily negate the efficiency gained through pooling. limitations apart from the factors outlined above, a clear limitation of pooling is that it needs to be done in a centralised laboratory in order to have access to preferably automated equipment and skilled staff to operate it and to assemble large enough numbers of specimens for testing. this necessitates sample transport, which is often challenging and increases turn-around times. point-of-care testing obviously has a number of advantages over any centralised testing, including pooled testing. however, it also poses a number of challenges such as price, operator training and supervision, quality control, etc., which still have to be resolved. ultimately, both will have a role to play: point-of-care testing for remote rural clinics (to avoid expensive shipping and lengthy delays) and pooling for urban areas and clinics with good transport links to major laboratories. suggestions for future research two opportunities exist that could make pooling even more attractive for rls. the first can be summarised by the question whether the ‘load’ in ‘viral load’ is actually needed. using a qualitative hiv pcr assay calibrated to detect down to approximately 1000 copies/ml per constituent sample and which generates a product covering most of the hiv pol gene for sanger sequencing, pooled testing could inform about vf (albeit without assigning a vl value) and the presence of the most relevant drug resistance mutations. this unconventional approach seems to work well in both developed countries and in rls,46,47,48 while addressing the urgent and largely unmet need for resistance testing in africa.49 the second opportunity addresses a major challenge for most african laboratories: the scarcity of well-qualified and trained staff. pooled testing has one principal disadvantage: it requires additional handling and, thus, increases hands-on time for qualified staff. this is necessary to perform the additional steps involved, namely the preparation of the pools and then the deconvolution of positive pools, involving retrieval of the samples that comprised a positive pool and re-testing them. unfortunately, both of these tasks are prone to human errors, which can cause sample mix-ups and other mistakes. automated pooling may help to overcome the important implementation hurdle posed by the shortage of trained laboratory staff. a pre-analytic sample robot could conduct the actual pooling process, then store samples (preferably cooled) until the pooled test result is available and lastly retrieve samples from positive pools for deconvolution. additionally, such a system could be integrated with the laboratory information system and also pre-screen patient samples with a low probability for failure, before preparing the pools educing the need for deconvolution. to conclude, pooling offers attractive prospects for african countries, not just for monitoring art but also for conducting eid testing. the time has come to conduct large-scale pilot studies, first on a limited scale, in parallel to individual testing to demonstrate non-inferiority of pooled testing in everyday use and subsequently in settings with suboptimal eid availability or which do not currently offer routine regular virological monitoring. acknowledgements competing interests the authors declare that no competing interests exist. authors’ contributions both authors shared all aspects equally, from developing the concept to writing and revising the review paper. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data 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zyl g, stich a, preiser w. a qualitative pcr minipool strategy to screen for virologic failure and antiretroviral drug resistance in south african patients on first-line antiretroviral therapy. j clin virol. 2014 aug;60(4):387–391. https://doi.org/10.1016/j.jcv.2014.05.011 boobalan j, dinesha tr, gomathi s, et al. pooled nucleic acid testing strategy for monitoring hiv-1 treatment in resource limited settings. j clin virol. 2019 aug;117:56–60. https://doi.org/10.1016/j.jcv.2019.05.012 van zyl gu, frenkel lm, chung mh, preiser w, mellors jw, nachega jb. emerging antiretroviral drug resistance in sub-saharan africa: novel affordable technologies are needed to provide resistance testing for individual and public health benefits. aids. 2014 nov;28(18):2643–2648. https://doi.org/10.1097/qad.0000000000000502 abstract introduction methods results discussion acknowledgements references about the author(s) iseimokumo c. peletiri department of medical microbiology, faculty of clinical sciences, college of health sciences, university of jos, jos, plateau state, nigeria medical microbiology & parasitology laboratories, national hospital, abuja, federal capital territory, nigeria rosemary c. nwachukwu medical microbiology laboratory, department of medical laboratory services, federal medical centre, yenagoa, bayelsa state, nigeria diweni c. peletiri medical biotechnology department, national biotechnology development agency, abuja, federal capital territory, nigeria esther q. onoja hematology and blood transfusion laboratory department, national hospital, abuja, federal capital territory, nigeria charity t. tulagha nursing services department, diete-koki memorial hospital, opolo, yenagoa, bayelsa state, nigeria ikaprite i. igbalibo medical microbiology & parasitology laboratories, national hospital, abuja, federal capital territory, nigeria grace m. ayanbimpe department of medical microbiology, faculty of clinical sciences, college of health sciences, university of jos, jos, plateau state, nigeria eugene i. ikeh department of medical microbiology, faculty of clinical sciences, college of health sciences, university of jos, jos, plateau state, nigeria citation peletiri ic, nwachukwu rc, peletiri dc, et al. epidemic forecast and preparedness for explosive-cerebrospinal meningitis outbreak in nigeria using the preventive vaccination strategy. afr j lab med. 2023;12(1), a2086. https://doi.org/10.4102/ajlm.v12i1.2086 review article epidemic forecast and preparedness for explosive-cerebrospinal meningitis outbreak in nigeria using the preventive vaccination strategy iseimokumo c. peletiri, rosemary c. nwachukwu, diweni c. peletiri, esther q. onoja, charity t. tulagha, ikaprite i. igbalibo, grace m. ayanbimpe, eugene i. ikeh received: 21 sept. 2022; accepted: 10 aug. 2023; published: 17 nov. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: within the african meningitis belt, yearly outbreaks of cerebrospinal meningitis (csm), with incidence rates of 10–100 cases per 100 000 population, are typically punctuated by explosive epidemics occurring every 8–12 years, with incidence rates that can exceed 1000 cases per 100 000 population. from 1928 to 2018, nigeria recorded the highest number (21%) of cases in the region. the reactive vaccination strategy, a protocol with major drawbacks, has been the vaccination method utilised in nigeria. aim: this review highlights the need for governments within the african meningitis belt to start preparations against the next explosive csm epidemic expected to occur between 2024 and 2028 using the preventive vaccination strategy. methods: we performed a literature search on the google scholar search engine using relevant search strings and included studies and reports between 1905 and 2022 that met set criteria. results: neisseria meningitidis serogroups a, b, c, w135, x, and y; haemophilus influenzae serotypes a, b, c, e, and f; and streptococcus pneumoniae serotypes 1, 4, 5, 6, 9, 19, 19f, and 20 were implicated as aetiologies. however, the reactive vaccination strategy was only used against n. meningitidis a or c, h. influenzae b, and pneumococcal conjugate vaccine. between 2011 and 2017, a polysaccharide vaccine (acw or acyw) active against serogroups a, c, w and y was used within the african meningitis belt for the first time. varying genotypes of n. meningitidis, h. influenzae and s. pneumoniae were identified. conclusion: our results revealed a very high success rate for the preventive vaccination strategy. what this study adds: in order to ensure reductions in the morbidity and mortality associated with invasive csm, the federal ministry of health, nigeria, should leverage existing knowledge of the circulating serogroups, serotypes, and genotypes of the primary bacterial aetiologies and commence the implementation of the preventive vaccination strategy. keywords: meningitis epidemic forecast; explosive-cerebrospinal meningitis outbreak; reactive vaccination strategy; preventive vaccination strategy; african meningitis belt; nigeria. introduction the occurrence of large epidemics of meningococcal meningitis in africa for a century led to the delineation of the african meningitis belt,1 comprising 26 countries: benin, burkina faso, burundi, cameroon, central african republic, chad, côte d’ivoire, democratic republic of the congo, eritrea, ethiopia, the gambia, ghana, guinea, guinea-bissau, kenya, mali, mauritania, niger republic, nigeria, rwanda, senegal, south sudan, sudan, tanzania, togo and uganda (figure 1).2 since 1905, when the first meningitis outbreak was reported to have occurred in zungeru, northern nigeria, meningitis outbreaks have become an annual occurrence and a household menace in nigeria.3 of the 36 states and the federal capital territory in nigeria, 25 states, including the federal capital territory, are now within the meningitis belt (figure 2).4 figure 1: the african meningitis belt, 1998. figure 2: states within the meningitis belt in nigeria, 2017. every year, a meningitis outbreak occurs in the dry season within the african meningitis belt. the outbreak typically occurs between december and june,5 with incidence rates of 10–100 cases per 100 000 population. these annual outbreaks are usually punctuated by explosive epidemics occurring every 8–12 years, with incidence rates that can exceed 1000 cases per 100 000 population.6,7 the 1996 cerebrospinal meningitis (csm) outbreak was the most serious epidemic ever recorded in nigeria, with 109 580 cases and 11 717 deaths, giving a case fatality rate (cfr) of 10.7%.8 from 1928 to 2018, nigeria recorded the highest number of csm cases (552 821 cases; 21% of the total) within the african meningitis belt.9 historically, a few bacterial aetiologies have been implicated in the major epidemics recorded in nigeria. before the introduction of the meningococcal a conjugate vaccine, nigeria encountered three major epidemics, all caused by neisseria meningitidis serogroup a. these epidemics occurred in 1977 (1257 cases; cfr = 8.3%),10 1996 (109 580 cases; cfr = 10.7%),8 and 2009 (55 626 cases; cfr = 4.1%).11 however, after the introduction of the meningococcal a conjugate vaccine, the aetiologic serogroup changed. two epidemics in 2015 (6394 cases; cfr = 5.0%)12 and 2017 (5595 cases; cfr = 10.9%)13 were caused by n. meningitidis serogroup c. vaccination efforts against these yearly meningitis outbreaks have been based on the ‘reactive vaccination strategy’, a methodology with identified major drawbacks such as delays in confirming outbreaks and deploying vaccines, difficulty in assessing some remote communities, limited capacity of health workers in the collection of cerebrospinal fluid (csf) specimens, certainty of deaths during epidemics, as well as confirmed cases of survivors of csm with sequelae, among others. with the looming spectre of a global explosive csm epidemic, improving infectious disease (e.g., meningitis) forecasting continues to be a major priority of global health preparedness.14,15,16 serotyping is vital in the development of vaccination strategies,17 as well as for the identification, containment and subsequent prevention of outbreaks. it also guides the determination of appropriate public health responses such as the administration of chemoprophylaxis for contacts of haemophilus influenzae type b (hib) cases or the administration of meningococcal conjugate versus serogroup b meningococcal (menb) vaccine during outbreaks of n. meningitidis.18,19,20 knowledge of the circulating bacterial genotypes are meant to establish if a disease outbreak is short term (local epidemiology) or long term (global epidemiology).21 this review aimed to highlight the major drawbacks of the reactive vaccination strategy, the benefits of the preventive vaccination strategy and the importance of serotyping as nigerian as well as other governments and health authorities within the african meningitis belt prepare for the next explosive csm epidemic expected to occur between 2024 and 2028. methods we performed a literature search on the google scholar search engine (https://scholar.google.com/) using the following search terms: ‘laboratory examination of cerebrospinal fluid (csf)’; ‘laboratory diagnosis of meningitis’; ‘control of meningitis outbreak in the african meningitis belt’; ‘epidemic meningitis in sub-saharan africa’; ‘clinical features in adults with bacterial meningitis’; ‘n. meningitidis serogroups found in nigeria’; ‘meningitis caused by h. influenzae serotypes in nigeria’; ‘meningitis caused by streptococcus pneumoniae serotypes in nigeria’; ‘n. meningitidis conjugate vaccines’; ‘h. influenzae type b routine childhood vaccination’; ‘s. pneumoniae vaccination plan’; ‘immunisation programmes against n. meningitidis’; ‘immunisation programmes against meningococcal meningitis’; ‘immunisation programmes against meningitis caused by h. influenzae’; ‘immunisation programmes against pneumococcal meningitis’; ‘vaccination against meningitis outbreak’; ‘impact of vaccination during meningitis outbreak’; ‘n. meningitidis genotypes in nigeria’; ‘h. influenzae genotypes in nigeria’; s. pneumoniae genotypes in nigeria’. the search was done from 2015–2022 with three active reviewers and four passive reviewers. the epidemic forecast was performed prospectively. inclusion criteria we included articles that reported results of laboratory examination of csf and laboratory diagnosis of csm, as well as articles that used a definite vaccination protocol either during or after a meningitis outbreak between 1905 and 2022. all articles that discussed the laboratory diagnosis of meningitis with results of the serogroups or serotypes of bacterial aetiologies of csm within nigeria were included. we also included articles that reported the circulating genotypes of the three major bacterial aetiologies (n. meningitidis, h. influenzae, and s. pneumoniae) in nigeria. finally, we included all the articles that reported the use of any of the vaccination strategies (reactive or preventive) globally. exclusion criteria we excluded articles that did not report on the serogroups, serotypes, or circulating genotypes of the bacterial aetiologies of meningitis within the meningitis belt of nigeria. we also excluded articles that did not report on vaccination strategies. forecast methodology the epidemic forecast was performed prospectively based on credible information that explosive meningitis epidemics occur every 8–12 years with incidence rates that can exceed 1000 cases per 100 000 population.6,7 since the last explosive epidemic was in 2017, by simple extrapolation, the next epidemic is expected to occur between 2024 and 2028. forecast accuracy evaluation the forecast accuracy evaluation method used relied on the documented evidence from moore in 19926 and the centre for disease control and prevention7 that explosive csm epidemics occur every 8–12 years,6,7 with the last occurrence being in 2017 as recorded by nigeria centre for disease control.13 the centre for disease control and nigeria centre for disease control are authorities responsible for the management of csm globally and locally, hence the method we used for this forecast is justified. results results generated from the google scholar search engine returned 68 articles that met the set criteria and were included in the final analysis. however, the results were grouped into four categories. the first category comprised studies that reported the laboratory diagnostic protocols used in the identification of bacterial aetiologies of csm in the african meningitis belt. in low-resource countries like nigeria, the laboratory diagnostic methods used are presumptive identification of aetiologies made on the basis of cytological examination of the csf, specific colony morphology on blood and or chocolate agar, staining properties on gram stain or by detection of specific antigens in the csf by latex agglutination test or a rapid diagnostic test,22 and the metagenomic protocol for use with molecular methods.23 there were 10 articles that fell within this category with prevalence rates ranging from 1.7% to 20.4%.24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44 two studies also utilised the molecular approach in the identification of bacterial aetiologies.25,33 the second category of studies included those that reported the varying serogroups of n. meningitidis, as well as the serotypes of h. influenzae and s. pneumoniae associated with meningitis in nigeria. between 2008 and 2021, 13 studies reported n. meningitidis serogroups a, b, c, w135, x, and y as responsible for csm infections in northern nigeria. seven of these studies12,25,34,35,36,37,38 reported n. meningitidis serogroup c as the only pathogen, while one study39 implicated n. meningitidis serogroup a. two studies40,41 implicated both n. meningitidis serogroups a and c. one study conducted in 2016 reported n. meningitidis serogroups a, c, and w135,32 while another study42 reported four serogroups (n. meningitidis serogroups a, b, c, and w135) as the circulating serogroups in jigawa state, northern nigeria. the last study33 reported all six serogroups of n. meningitidis, including serogroups a (sacb), b (synd), c (syne), w135 (syng), x (xcbb), and y (synf), as well as the not-typeable (non-groupable or serogroup-negative) strains during the 2017 and 2018 meningitis seasons. for h. influenzae, nnadi and colleagues41 reported h. influenzae type b as the only serotype implicated in their study, while another study33 reported encountering five serotypes of h. influenzae, including serotypes a (acsb), b (bcsb) c (ccsd), e (ecsh), and f (bexd). for s. pneumoniae, one study25 implicated s. pneumoniae serotypes 1, 5, and 19f, while another study43 reported s. pneumoniae serotypes 6, 19, and 20. the last study33 reported s. pneumoniae serotypes 1 (wzy1), 4 (wzy4), 5 (wzy5), and 9 (wzy9). from these three studies,25,33,43 the s. pneumoniae serotypes circulating in northern nigeria are serotypes 1 (wzy1), 4 (wzy4), 5 (wzy5), 6 (wcip), 9 (wzy9), 19 (wzy19), 19f (wzy19f), and 20 (wzy20). in nigeria, therefore, the circulating n. meningitidis serogroups are n. meningitidis a (sacb), b (synd), c (syne), w135 (syng), x (xcbb), and y (synf);12,25,32,33,34,35,36,37,38,39 h. influenzae serotypes a (acsb), b (bcsb), c (ccsd), e (ecsh), and f (bexd);33,38 and s. pneumoniae serotypes 1 (wzy1), 4 (wzy4), 5 (wzy5), 6 (wcip), 9 (wzy9), 19 (wzy19), 19f (wzy19f), and 20 (wzy20).25,33,43 the third category of articles were those that reported the circulating invasive genotypes of n. meningitidis, h. influenzae, and s. pneumoniae implicated in meningitis outbreaks in northern nigeria. there were three studies in this category. n. meningitidis genotypes abcz, adk, aroe, fumc, pdhc, and pgm;44 h. influenzae genotypes adk, fuck, and mdh;45 and s. pneumoniae genotypes aroe and gki46 have been reported to be circulating among csm patients in parts of northern nigeria. the final category comprises articles that reported vaccination during meningitis outbreaks (reactive vaccination strategy) and those that reported vaccination before any meningitis outbreak (preventive vaccination strategy). thirteen articles discussed the reactive vaccination strategy, whereas 21 studies discussed the preventive vaccination strategy. the reactive vaccination strategy has been faulted by some authorities owing to several identified major drawbacks. (1) there are response delays due to challenges with confirming the outbreak, rapidly deploying the vaccines, and organising the vaccination campaigns.47 (2) there are also gaps associated with laboratory confirmation during outbreaks.47 (3) limited and late laboratory confirmation is a bottleneck in the submission of international coordinating group (icg) vaccine requests and decision-making on vaccine release.48 (4) another identified drawback of the reactive vaccination strategy is that delays in vaccine deployment and campaign planning hamper response.47 (5) in 201249 and 2015,50 limited emergency stockpiles undermined the timeliness and effectiveness of outbreak response. (6) there was also the difficulty in accessing some of the more rural and remote communities experiencing the outbreak, which hampered early outbreak response activities.41 (7) healthcare workers’ limited capacity for csf specimen collection is another drawback.41 (8) there is also the certainty of between 8% and 15% deaths during epidemics even when the disease is diagnosed early and adequate treatment is started.51 (9) at the end of every meningitis season, 21% – 40% deaths are being recorded.52,53 (10) another major drawback is the unlikely reduction of the number of epidemic cases by more than half with the commencement of mass vaccination after an outbreak.54,55 (11) about 10% – 30% of survivors of csm have sequelae, such as hearing loss, neurological disability, impaired cognitive function, or loss of a limb.51,53,56,57 the preventive vaccination strategy is exemplified in the routine hib childhood vaccination programmes implemented in england and wales, following which hib is no longer a major cause of acute bacterial meningitis in children.58,59,60 following the introduction of the different hib immunisation strategies over the past decades globally, cases in toddlers, older children, and adults have continued to decline rapidly and have now become extremely rare.61 generally, the hib conjugate vaccine remains highly effective in preventing invasive disease in young children.62,63 the experiences gained over the years from the hib conjugate vaccination programme – the first conjugate vaccine to be introduced in the united kingdom – and the success of the vaccine in controlling what was once a devastating infection in young children have already contributed to the successful implementation of other conjugate vaccination programmes such as those against invasive meningococcal capsular group c and pneumococcal disease.64 effective vaccines are available for n. meningitidis serogroups a, c, w, and y65 and vaccines for serogroup b have been approved for usage since 2015.66 in the united states, the quadrivalent acwy vaccine (meningococcal conjugate effective against serogroups a, c, w, and y) was recommended for use in persons within the 11–12 years age bracket.67 a 2011 update from the advisory committee on immunization practices (acip) recommended administration of a booster dose at age 16.68 the joint committee on vaccination and immunization of public health england recommended that individuals aged 14–18 years should be vaccinated with the quadrivalent conjugate acwy vaccine with the goal of generating herd immunity in the overall population against serogroup w disease.69 following an outbreak of invasive meningococcal disease at a korean military training centre in 2011, the korean health authorities recommended that beginning in 2012, new military recruits should receive the quadrivalent acwy conjugate vaccine before reporting at the training centre.70 this is a clear demonstration of using the preventive vaccination strategy against outbreak of meningococcal disease. in the united states, the incidence of invasive meningococcal disease associated with serogroups c, w, and y among the 11–19-year-old population has been declining since the introduction of the quadrivalent vaccine.71 in the united kingdom, administration of the serogroup c conjugate vaccine was reported to have led to the reduction in serogroup c carriage in university students for up to two years after vaccine administration.72 in burkina faso, carriage studies two years after widespread use of the serogroup a conjugate vaccine showed that carriage of serogroup a had been nearly eliminated.73 the introduction and widespread use of meningococcal vaccines for serogroup c in europe and for serogroups a, c, w, and y led to reductions in invasive meningococcal disease associated with these serogroups.74 discussion immunisation programmes against the major csm bacterial aetiologies (n. meningitidis, h. influenzae, and s. pneumoniae) have been successfully implemented in africa, the americas, asia, australia, and europe.75,76,77 two types of vaccination programmes are being implemented globally: the reactive vaccination strategy or programme and the preventive vaccination strategy or programme. in nigeria, it is the reactive vaccination strategy that is been practised against n. meningitidis outbreak.78 the reactive vaccination strategies currently used globally, especially in the african meningitis belt, rely on the detection and timely reporting of suspected meningitis cases beyond a set threshold.79 the process involved in identifying and confirming the outbreak, a requirement for requesting vaccine from the icg, inevitably leads to delays in the overall public health response.80,81 even when the disease is diagnosed early and adequate treatment is started, about 8% – 15% of patients die, often within 24 h – 48 h after the onset of symptoms, or development of sequelae such as brain damage, hearing loss, neurological disability, or impaired cognitive function may occur in 10% – 30% of survivors.51,53,56,57 between 2011 and 2017, during all outbreaks that occurred within the african meningitis belt (burkina faso, cameroon, ethiopia, ghana, niger, nigeria, and togo) where reactive vaccination was conducted for the first time with the use of a polysaccharide vaccine that was effective against serogroups a, c, w or a, c, y, w for 4.7 million people. to ensure maximum potential impact (e.g., herd immunity or protection) and for monitoring purposes, conjugate vaccines were used under specific defined conditions: vaccination of whole districts, distribution of vaccination cards, coverage survey, and strengthened monitoring.47 as the term ‘preventive vaccination’ implies, this strategy intends to prevent a meningitis outbreak using vaccination. preventive medicine and public health share common goals, such as promoting general health, preventing specific diseases, and applying epidemiologic concepts and biostatistical techniques toward these goals. however, preventive medicine seeks to enhance the lives of individuals by helping them improve their own health, whereas public health attempts to promote health in populations through the application of organised community efforts.82 the major goal of primary prevention by specific protection involves prevention of specific diseases by using vaccines.83 vaccination against a limited range of serogroups or serotypes of a pathogen can lead to the selection of ‘escape variants’, thus leading to epidemics. for example, the mass vaccination against n. meningitidis using a polyvalent polysaccharide vaccine contributed to the selection of non-serogroup c meningococci, leading to outbreaks. thus, knowledge of genetic diversity is an important prerequisite for the development of successful vaccines.84 epidemiological surveillance is important as it provides the data on which national and regional health authorities can base their vaccination policies. therefore, to enhance further reductions in the morbidity and mortality associated with invasive csm, the introduction of serogroupor serotype-appropriate vaccines should be informed by epidemiologic data.74 it should be noted that the commencement of mass vaccination after an outbreak is unlikely to reduce the number of epidemic cases by more than half.54,55 this information underscores the need for a paradigm shift from a reactive vaccination strategy to a preventive vaccination strategy on mass vaccination to prevent further csm epidemics in africa. therefore, to ensure successful control of epidemics in the continent, a proper coordination among all the countries within the african meningitis belt is necessary. it is expected that african countries may incur a high financial burden in rolling out a preventive vaccination programme against meningitis. however, mass vaccination need not be undertaken yearly since several trials have shown that these vaccines may induce adequate antibody responses57,85 that may remain high for up to four years in persons aged > 5 years.86 booster vaccination has been recommended for persons who remain at increased risk of meningococcal disease. for individuals whose most recent dose was received at younger than 7 years, a booster dose should be given after three years. however, if the most recent dose was received at age 7 years or older, a booster dose should be administrated after 5 years and every 5 years thereafter as long as the person remains at increased risk for meningococcal disease.87 it should be noted that a surveillance and outbreak response system is more effective when the capacity to prevent, detect, and appropriately respond to outbreaks is readily available.88 the last explosive csm epidemic occurred during the 2017 meningitis season. relying on the documented evidence from moore in 19926 and the centers for disease control and prevention in 2011,7 and the fact that the yearly meningitis outbreak is punctuated by explosive epidemics occurring every 8–12 years with incidence rates that can exceed 1000 cases per 100 000 population, we forecast the next explosive epidemic in the african meningitis belt to occur between 2024 and 2028. in order to prevent experiencing the devastations usually associated with csm outbreaks, and with the identified major drawbacks of the reactive vaccination strategy, we recommend the following: countries within the african meningitis belt should as a matter of responsibility secure appropriate budgetary provisions in their 2023 budget cycle for the procurement of vaccines, medical devices and provision of logistics support to kick-start the preventive vaccination strategy. donor agencies engaged in the fight against meningitis should key into this preventive vaccination strategy by making funds available for the procurement and the actual administration of relevant and appropriate vaccines before outbreaks. adequate planning and preparedness against the next explosive csm epidemic expected to occur, likely from 2024, should commence early in 2023. there should be updated training of medical officers in the process of collection of csf samples through lumbar puncture from suspected meningitis patients before commencement of antibiotic therapy. the establishment and use of molecular laboratories in the molecular diagnosis (polymerase chain reaction assays) of bacterial and other aetiologies of meningitis is also recommended. there should be regular training and re-training of medical laboratory scientists (biomedical scientists) in the techniques of analysing csf samples by way of both the phenotypic and molecular methods. individual countries within the african meningitis belt should endeavour to utilise the available research results of the circulating serogroups of n. meningitidis and serotypes of h. influenzae and s. pneumoniae within their countries for commencement of the preventive vaccination strategy before the start of the 2024 meningitis season, which normally will commence in december 2023. effective vaccination is based on the knowledge of the circulating serogroups or serotypes as the case may be. there should be public health education (enlightenment) by relevant authorities to the general local populace on the importance of the preventive vaccination strategy against meningitis. this will enhance general acceptability of the meningitis vaccination programmes. finally, our recommendations in this review article are relevant for tackling both annual and explosive meningitis outbreaks. acknowledgements we acknowledge the federal ministry of health, nigeria, for their support by way of introducing the corresponding author to the various states’ health authorities. also acknowledged are the ethics committee members of national hospital, abuja, nigeria; kebbi state ministry of health, nigeria; plateau state ministry of health, nigeria; sokoto state ministry of health, nigeria; and zamfara state ministry of health for their support and approval to carry out the original research in their various states. all staff of the various laboratories where the original research samples were received and phenotypically processed are appreciated (peletiri et al. 2021a; 2021b).23,33 also, we wish to appreciate the managing director and chief executive officer of safety molecular pathology laboratory, enugu, enugu state, nigeria, dr emmanuel nna (doctor of philosophy), for training the principal investigator in the practical application of metagenomic protocol and general orientation in molecular diagnostic techniques in the polymerase chain reaction laboratory of his institute. also appreciated are all staff of safety molecular pathology laboratory, enugu, enugu state, nigeria, for the various roles they played in the course of running of all samples for preparation of metagenomic deoxyribonucleic acid template and actual molecular diagnostic procedures. the outcome of the main original research already published (peletiri et al. 2021a; 2021b; 2022a; 2022b; 2022c)23,33,44,45,46 led to this review article. finally, we appreciate pollett and colleagues89 for use of their guidelines and their guidance in reporting items for epidemic forecasting. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions i.c.p. conceptualised the research project with inputs from e.i.i., and g.m.a. i.c.p., r.c.n., g.m.a., and e.i.i. were responsible for the methodologies cited. i.c.p., r.c.n., d.c.p., e.q.o., c.t.t., i.i.i., g.m.a., and e.i.i. were responsible for formal analysis of all data. i.c.p., d.c.p., e.q.o., and e.i.i. co-wrote the article. i.c.p., r.c.n., d.c.p., e.q.o., c.t.t., i.i.i., g.m.a., and e.i.i. participated in the validation and interpretation of data. i.c.p., r.c.n., d.c.p., e.q.o., c.t.t., i.i.i., g.m.a., and e.i.i. critically revised specific aspects of the articles and approved the final article. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability accession codes, unique identifiers, or web links are provided for publicly available data sets and can be requested from the corresponding author, i.c.p. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references lapeyssonie l. cerebrospinal meningitis in africa. bull world health organ. 1963;28:1–114. centers for disease control and prevention. the african meningitis belt: control of epidemic meningococcal disease, who practical guidelines, world health organization, 1998. who/emc/bac/98.3. in centers for 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2011;29(49):9137–9140. https://doi.org/10.1016/j.vaccine.2011.09.107 heath pt, booy r, griffiths h, et al. clinical and immunological risk factors associated with haemophilus influenzae type b conjugate vaccine failure in childhood. clin infect dis. 2000;31(4):973–980. https://doi.org/10.1086/318132 collins s, ramsay m, campbell h, slack mp, ladhani sn. invasive haemophilus influenzae type b disease in england and wales: who is at risk after 2 decades of routine childhood vaccination? clin infect dis. 2013;57(12):1715–1721. https://doi.org/10.1093/cid/cit579 american academy of pediatrics committee on infectious diseases. updated recommendations on the use of meningococcal vaccines. pediatrics. 2014;134(2):400–403. https://doi.org/10.1542/peds.2014-1383 macneil jr, rubin l, folaranmi t, ortega-sanchez ir, patel m, martin sw. use of serogroup b meningococcal vaccines in persons aged ≥10 years at increased risk for serogroup b meningococcal disease: recommendations of the advisory committee on immunization practices, 2015. morb mortal wkly rep. 2015;64(22):608–612. https://doi.org/10.15585/mmwr.mm6441a3 bilukha oo, rosenstein n, national center for infectious diseases, centers for disease control and prevention. prevention and control of meningococcal disease. recommendations of the advisory committee on immunization practices (acip). mmwr recomm rep. 2005;54(rr-7):1–21. centers for disease control and prevention (cdc). updated recommendations for use of meningococcal conjugate vaccines – advisory committee on immunization practices (acip), 2010. mmwr. morb mortal wkly rep. 2011;60(3):72–76. public health england. meningococcal group w (menw) immunization advised for 14 to 18 year-olds [homepage on the internet]. 2015 [cited 2022 aug 22]. available from: https://www.gov.uk/government/news/meningococcal-group-w-menw-immunization-advised-for-14-to-18-year-olds lee so. commencement of the meningococcal vaccination for the republic of korea army. infect chemother. 2013;45(1):113–115. https://doi.org/10.3947/ic.2013.45.1.113 macneil j. considerations for use of serogroup b meningococcal (menb) vaccines in adolescents. presented to the advisory committee on immunization practices. june 24, 2015 [homepage on the internet]. 2015 [cited 2022 aug 22]. available from: http://www.cdc.gov/vaccines/acip/meetings/downloads/slides-2015-06/mening-03-macneil.pdf. maiden mcj, ibarz-pavón ab, urwin r, et al. impact of meningococcal serogroup c conjugate vaccines on carriage and herd immunity. j infect dis. 2008;197(5):737–743. https://doi.org/10.1086/527401 ala’aldeen da, oldfield nj, bidmos fa, et al. carriage of meningococci by university students, united kingdom. emerg infect dis. 2011;17(9):1762–1763. https://doi.org/10.3201/eid1709.101762 pelton si. the global evolution of meningococcal epidemiology following the introduction of meningococcal vaccines. j adolesc health. 2016;59(suppl 2):s3–s11. https://doi.org/10.1016/j.jadohealth.2016.04.012 conklin l, loo jd, kirk j, et al. systematic review of the effect of pneumococcal conjugate vaccine dosing schedules on vaccine-type invasive pneumococcal disease among young children. pediatr infect dis j. 2014;33(suppl 2):s109–s118. https://doi.org/10.1097/inf.0000000000000078 hajjeh r, mulholland k, schuchat a, santosham m. progress towards demonstrating the impact of haemophilus influenzae type b conjugate vaccines globally. j pediatr. 2013;163(1):s1–s3. https://doi.org/10.1016/j.jpeds.2013.03.022 trotter cl, lingani c, fernandez k, et al. impact of menafrivac in nine countries of the african meningitis belt, 2010-15: an analysis of surveillance data. lancet infect dis. 2017;17(8):867–872. https://doi.org/10.1016/s1473-3099(17)30301-8 nigeria centre for disease control (ncdc). vaccination: preventing meningitis outbreaks. in fmoh, ncdc, editors. preparedness and response to cerebrospinal meningitis outbreaks. a guide for health workers and authorities in nigeria, fmoh, ncdc, 2017; p. 19–20. trotter cl, cibrelus l, fernandez k, lingani c, ronveaux o, stuart jm. response thresholds for epidemic meningitis in sub-saharan africa following the introduction of menafrivac®. vaccine. 2015;33(46):6212–6217. https://doi.org/10.1016/j.vaccine.2015.09.107 maurice j. vaccine shortage threatens spread of meningitis in niger. lancet. 2015;385:2241. https://doi.org/10.1016/s0140-6736(15)61050-9 obaro sk, habib ag. control of meningitis outbreaks in the african meningitis belt. lancet infect dis. 2016;16:400–402. https://doi.org/10.1016/s1473-3099(16)00121-3 katz dl, elmore jg, wild dmg, lucan sc. introduction to preventive medicine. in: katz dl, elmore jg, wild dmg, lucan sc, editors. jekel’s epidemiology, biostatistics, preventive medicine, and public health, chapter 14. 4th ed., elsevier saunders, 2014; p. 173–180. katz dl, elmore jg, wild dmg, lucan sc. methods of primary prevention: health promotion. in: katz dl, elmore jg, wild dmg, lucan sc, editors. jekel’s epidemiology, biostatistics, preventive medicine, and public health, chapter 15. 4th ed., elsevier saunders, 2014; p. 181–195. virdi js, sachdeva p. genetic diversity of pathogenic microorganisms: basic insights, public health implications and the indian initiatives. curr sci. 2005;89(1):113–123. greenwood bm, whittle hc, bradley ak, fayet mt, gilles hm. the duration of the antibody response to meningococcal vaccination in an african village. trans r soc trop med hyg. 1980;74(6):756–760. https://doi.org/10.1016/0035-9203(80)90195-9 mohammed i, onyemelukwe gc, obineche en, gupta n, oyeyinka go. control of epidemic meningococcal meningitis by mass vaccination. ii. persistence of antibody four years after vaccination. j infect. 1984;9(2):197–202. https://doi.org/10.1016/s0163-4453(84)91468-3 mbaeyi s, duffy j, mcnamara la. meningococcal disease. in hall e., wodi ap, hamborsky j, et al., editors. epidemiology and prevention of vaccine-preventable diseases. centers for disease control and prevention. 14th ed. washington, d.c. public health foundation, 2021. p. 207–224. lo tq, marston bj, dahl ba, de cock km. ebola: anatomy of an epidemic. annu rev med. 2017;68:359–370. https://doi.org/10.1146/annurev-med-052915-015604 pollett s, johansson ma, reich ng, et al. recommended reporting items for epidemic forecasting and prediction research: the epiforge 2020 guidelines. plos med. 2021;18(10):e1003793. https://doi.org/10.1371/journal.pmed.1003793 abstract introduction methodology results discussion acknowledgements references about the author(s) winny koster amsterdam institute for global health and development, amsterdam, the netherlands department of public health, faculty of social and behavioural sciences, amsterdam institute for social science research, university of amsterdam, amsterdam, the netherlands albert g. ndione institut de recherche pour le développement (ird), transvihmi, crcf-hôpital fann, dakar, senegal mourfou adama centre national de lutte anti tuberculeuse (cnlat), laboratoire national de référence des mycobactéries, ouagadougou, burkina faso ibrehima guindo institut national de recherche en santé publique, bamako, mali iyane sow directorate of laboratory services, ministry of public health and social welfare, dakar, senegal souleymane diallo centre d’infectiologie charles mérieux, bamako, mali jean sakandé fondation mérieux, ouagadougou, burkina faso pascale ondoa amsterdam institute for global health and development, amsterdam, the netherlands department of global health, amsterdam university medical centers, amsterdam, the netherlands african society for laboratory medicine, addis ababa, ethiopia citation koster w, ndione ag, adama m, et al. an oral history of medical laboratory development in francophone west african countries. afr j lab med. 2021;10(1), a1157. https://doi.org/10.4102/ajlm.v10i1.1157 note: additional supporting information may be found in the online version of this article as supplementary document 1. original research an oral history of medical laboratory development in francophone west african countries winny koster, albert g. ndione, mourfou adama, ibrehima guindo, iyane sow, souleymane diallo, jean sakandé, pascale ondoa received: 20 dec. 2019; accepted: 20 oct. 2020; published: 16 mar. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: underdeveloped and underused medical laboratories in sub-saharan africa negatively affect the diagnosis and appropriate treatment of ailments. objective: we identified political, disease-related and socio-economic factors that have shaped the laboratory sector in senegal, mali and burkina faso to inform laboratory-strengthening programmes. methods: we searched peer-reviewed and grey literature from february 2015 to december 2018 on laboratory and health systems development from colonial times to the present and conducted in-depth interviews with 73 key informants involved in (inter)national health or laboratory policy, organisation, practice or training. this article depended on the key informants’ accounts due to the paucity of literature on laboratory development in francophone west african countries. literature and interview findings were triangulated and are presented chronologically. results: until around 1990 there were a few disease-specific research laboratories; only the larger hospitals and district health facilities housed a rudimentary laboratory. the 1990s brought the advent of donor-dictated, vertical, endemic and epidemic disease programmes and laboratories. despite decentralising from the national level to the regional and district levels, these vertical laboratory programmes biased national health resource allocation deleteriously neglecting the development of the horizontal, general-health laboratory. after the year 2000, the general-health laboratory system received more attention when, influenced by the world health organization, national networks and (sub-)directorates of laboratories were installed. conclusion: to advance national general healthcare, as opposed to disease-specific healthcare, national laboratory directors and experts in general laboratory development should be consulted when national policies are made with potential laboratory donors. keywords: oral history; medical laboratory; senegal; burkina faso; mali. introduction underdeveloped and underused medical laboratories in sub-saharan africa hamper the diagnosis and management of potentially epidemic infectious diseases as well as general public health conditions.1,2,3,4 recent national surveys of laboratory capacity conducted in senegal, mali and burkina faso – francophone west africa for short, and the countries this study focuses on – found that many laboratories still have insufficient personnel, a lack of or dysfunctional basic equipment, and operate in inappropriate rooms.5,6,7,8 studies in senegal on barriers to the uptake of the seven recommended routine maternal diagnostic tests found that laboratory problems were part of the reason why only around one-third of pregnant women received the complete set of tests.9,10 identifying the historical developments that have shaped the laboratory sector could help to understand the gaps and provide lessons for laboratory-strengthening programmes. as far as we know, this is the first study on this topic. the guiding question of our study was: what political, socio-economic, and disease-related factors have influenced the current status of medical laboratories in senegal, mali and burkina faso? the development of medical laboratories in africa cannot be dissociated from the development of the healthcare sector at large.11,12,13,14 the healthcare sector in colonial times was mainly directed controlling infectious disease control and keeping the colonial workforce healthy to ensure economic productivity.15 the years after independence saw optimism towards national economic growth that focused on improved healthcare for the general population.15 the global economic recession of the early 1980s and the structural adjustment programmes of the late 1980s–1990s had a significant negative impact on national economies and forced governments to cut spending and lower their development goals of early post-independence.15,16,17 the ensuing cuts in the health sector thus created institutional, technological and personnel capacity problems.13,17,18 the late 1990s saw the emergence of global health initiatives in response to worldwide health-related problems, mainly epidemic diseases, that could pose global security threats. our article progresses chronologically: it begins in late colonial times, goes on to the post-independence period, followed by the time of structural adjustment, and ends with the emergence of global health. in each period, we describe the developments in the laboratory sector and the influencing factors. methodology ethical considerations the objectives of the study were explained to all key informants; all gave verbal consent to be interviewed and recorded, as can be proven by the audio recordings. study design the data for this qualitative descriptive study are derived primarily from in-depth key informant interviews and a literature review. the study proposal identified themes in the interrelated factors influencing laboratory development. themes explored were: organisation of services, regulations, health policies and plans; economic conditions and by extension personnel, equipment, training and infrastructure development; endemic and epidemic disease contexts; and lastly, funding by national governments and (inter)national donors. data collection methods and study participants literature review between february 2015 and february 2016, three country teams of three to six social science researchers and laboratory professionals searched for peer-reviewed and grey literature on the study’s themes and identified factors in local libraries, organisations and government departments (online supplementary document – table 1), and freely accessible websites (online supplementary document – list 1). key informant interviews key informants, selected by the country team members, were in 2015 interviewed for 30 min to 3 h, mainly at their workplace. selection criteria were having an active role in the (inter)national health or laboratory policy, organisation, practice or training (past and present). for every key informant, a personalised question guide was prepared, considering their professional background, the period of their professional work and their involvement with laboratories. a summary of the 73 key informants’ background characteristics is presented in table 1, details are provided in the online supplementary document – table 2. key informants were trained in clinical or laboratory medicine (or both) and included retired (n = 8) and current (n = 65) staff members of various departments in the ministry of health (moh), international organisations, private and public health facilities, professional associations, training institutions, and universities; many had worked in several functions. one key informant’s professional career commenced during the colonial period, nine commenced in the two decades after the three countries’ independence in 1960, while the remainder commenced from the 1980s. table 1: background of the 73 key informants, francophone africa, february 2015 – february 2016. data analysis and presentation the audio-recorded key informant interviews (in french) were transcribed verbatim. nvivo qualitative data analysis software, version 10 (qsr international, melbourne, australia), was used for the thematic analysis of the interviews and a timeline was constructed for each country. each country team summarised their literature findings thematically and chronologically. in this article, the findings derived from the two data collection methods have been triangulated and are presented for the three countries combined. occasionally, differences and specificities across countries are pointed out. it should be noted that when the article refers to literature, the same information was usually corroborated by key informants. all quotations in the text are english translations of the french citations. results the study’s primary finding and challenge was the scarcity of written sources on laboratories; consequently, this history of laboratory development mainly hinges on the accounts of key informants, all of whom willingly recounted their experiences. notably, those involved in laboratory practice or policy welcomed the attention; as a senegalese biologist involved in laboratories since 1982 expressed: ‘the laboratory has habitually been the forgotten part of medicine, meaning one thinks of everything, and of the laboratory only after that’ (key informant 62, male, interview date 11 march 2015, dakar). descriptions of developments for colonial times and during the first decades after independence are briefer than for the periods afterwards because most of the key informants commenced their laboratory experience after these two periods. colonial times in colonial times, all three countries housed renowned research laboratories, including centre muraz in burkina faso, institut pasteur in senegal and laboratoire central de biologie in mali. these laboratories were involved in the research and the control of endemic and epidemic diseases – including trypanosomiasis, cholera, onchocerciasis, meningitis, malaria and syphilis – to preserve the labour and military force,15 and were funded by fides (fonds d’investissements pour le développement economique et social des territoires d’outre-mer).19 the staff were french doctors and local assistants – mainly nurses – trained on-the-job.19,20 a retired burkinabé pharmacist stated that everything related to laboratories in french west africa started with colonial doctors interested in tropical diseases; for instance, gaston muraz, a french military doctor, created centre muraz in bobo dioulasso in 1939. the few existing national and regional hospitals had small laboratories attached.19 in dakar, the capital of french west africa – which included present-day burkina faso and mali – principal, dantec and fann hospitals were among the first modern healthcare facilities in sub-saharan africa.21 mali had kayes, mopti, markala and point g hospitals. in burkina faso, the first hospital was a military facility at bobo dioulasso (now the university hospital sanou sourô). a retired director of institut national de recherche en santé publique (inrsp) in mali, who replaced the french director of the central laboratory in bamako in 1956, remembered that some pharmacies also had a small laboratory attached. post-independence (≈1960–1979) after independence, the colonial research laboratories remained – although some under another name (the malian laboratoire central de biologie became institut national de biologie humaine) – as did those attached to the few public national and regional hospitals.22 a few medical laboratories were attached to private pharmacies in burkina faso, while mali had 16 public stand-alone medical laboratories, mainly for technical support of disease control programmes.19 laboratory diagnostic tests were limited and aimed mainly at detecting parasites and measuring glycosuria. even in larger public hospitals, laboratory services were embryonic and only a few laboratory training programmes existed. in burkina faso, nurses could specialise in laboratory technology at ecole jamot and centre muraz. in mali, schools were opened for medical doctors and pharmacists, two of which offered biology: l’école des assistants médicaux (founded 1969) and l’école nationale de médecine et de pharmacie (founded 1974). in mali, a microbiology research laboratory was set up in 1973, where many of the malian key informants received training. the head of this laboratory had a wide network of international partners who helped provide the necessary resources to make the laboratory internationally renowned. laboratory assistants were trained in the secondary school of health (founded 1963) and the institute national de formation en sciences de la santé. in senegal, the faculty of medicine at cheikh anta diop university (founded 1962) trained pharmacists who worked all over west africa.21 in the three countries, assistants trained on-the-job represented a large part of the laboratory workforce. the national moh in senegal and burkina faso did not prioritise laboratories, resulting in a lack of equipment and supplies for the few existing public facilities. in mali, however, the minister of health acknowledged the importance of laboratories relatively early; he created the national research laboratory inrsp in 197323 and aimed to strengthen public hospital laboratories and establish a national coordinating body and policy. the former inrsp director, recounted that in 1974 this health minister created the division of laboratories and appointed him head, telling him to assess the status of laboratories in the country. based on his situation analysis, he recommended that laboratory services be decentralised to the regional level. disease outbreaks, in particular the 1974–1975 meningitis and cholera outbreaks, added to the health minister’s motivation to decentralise laboratory services. the former inrsp director remembered how cumbersome the control of these outbreaks had been, notably the transportation of suspected cases’ stool samples from the regional laboratories to the central laboratories for analysis. primary healthcare and structural adjustment (≈1980–the late 1990s) this period saw the implementation of two global strategies for health systems development: the 1978 alma ata primary health care declaration and the 1988 bamako initiative. these called for the decentralisation of basic health services, a focus on clinical diagnosis and the supply of essential drugs and equipment. however, structural adjustment programmes, with their efficiency-driven economic reforms, implied less state involvement, public spending cuts and privatisation of healthcare services.15,17 decentralisation of laboratories in the three countries, health centres at (sub-)district level were built, usually with a supporting laboratory to provide the stipulated minimum of primary healthcare diagnostic tests.24,25 the revolutionary sankara regime (1983–1987) aimed to bring healthcare, including rudimentary laboratories, to all corners of burkina faso, as the then minister of health and sports (1985–1987) explained. a pharmacist and current health inspector recounted that the reorganisation of the burkinabé national health system into districts became a fact in 1992–1993, with service norms set by level; laboratories were designated to the district level. medical doctors and laboratory personnel working at district level narrated that at this time if there was a laboratory at all, it was rudimentary and very few types of tests were done, usually in a room not designed to house a laboratory. a burkinabé laboratory technician depicted how, in the 1980s, a typical health centre laboratory was just a small room with a microscope, a manual centrifuge and a fridge with some reagents. ‘community people called us “stool doctors” because they only saw us examining stools’ (key informant 35, male, interview date 06 may 2015, ouagadougou), recounted the lecturer for laboratory technicians at ecole nationale de santé publique in ouagadougou. a challenge for peripheral laboratories was the absence or unreliability of electricity. a medical doctor, currently at conseil national de lute contre le sida, shared his experiences in rural burkina faso: ‘during the years 1984–1990, it was challenging. it was apparent that when you were in a place without electricity you could use very little equipment. only those working in private laboratories had solar microscopes. thus, we had to seek a window and work with sunlight; in that way, we could only do parasitology tests’. (key informant 46, male, interview date 04 may 2015, ouagadougou) he concluded: ‘sophisticated laboratory development followed electrification’. his midwife colleague at conseil national de lute contre le sida added that most health problems were diagnosed at this time through physical patient examination. two senegalese medical doctors, both working in district health centres, remembered that in the 1980s and 1990s, they only sporadically referred patients to regional hospital laboratories for diagnostic tests if the patients could afford the travel costs. key informants in mali and burkina faso reported that structural adjustment programmes had stimulated private practice; this was supported by government laws authorising private medical practice.26 consequently, some private laboratories were created, either stand-alone, like rive droite in bamako, or attached to private hospitals (six in burkina faso). this legal opening prompted some pharmacists to leave the public sector and open private pharmacies with a laboratory attached. the malian and burkinabé key informants generally applauded the development of private laboratories because it increased access to laboratory services, though they acknowledged quality control challenges. training opportunities and insufficiencies training opportunities at this time increased. the university of mali (founded in 1993) offered pharmacists and medical doctors additional training options in medical biology. cheikh anta diop university in dakar offered training for senior laboratory technicians. in burkina faso, a three-year training programme for laboratory technicians started in 1985 at the ecole nationale de santé publique, while during the sankara regime some nurses, including one key informant, were sent for laboratory medicine training in cuba. before 1985, most burkinabé laboratory technicians were trained in dakar, and some in canada and france. the year 1999 saw the start of the licence professionnelle option analyses at the university of ouagadougou. insufficient local training opportunities persisted for assistant and specialisation levels. also, no formal training existed for laboratory assistants and nurses trained on-the-job, who still formed a large portion of laboratory personnel.27 the saying went: ‘you are a laboratory worker and you die a laboratory worker’. due to the lack of laboratory training and career opportunities, many nurses left laboratory work to pursue further nursing training, although some nurses among our key informants stayed because they liked the work. a clinical biology university professor explained that because there was a lack of advanced specialisation training in medical biology or biochemistry in senegal, medical doctors such as himself had to go for further studies abroad, mainly to france. (in)visibility of laboratories in national policies and programmes the first national demographic and health surveys were conducted in the mid-1980s. national health plans focused on equity in service access through primary healthcare, the reduction of infant, child and maternal mortality, and family planning.28 these demographic and health survey reports and national plans made little reference to laboratories. only in 1999 did the malian public health sector set up a referral system for laboratory tests: from first-level centre de santé de référence to second-level regional hospitals to third-level university hospitals.24 no national funds were dedicated to laboratories, and therefore laboratory operations relied on budgetary allocations from health facility management. in those periods of economic austerity, the management of health facilities struggled to maintain all services, including laboratory services. limited budgets and the consequently erratic reagent availability made it difficult for laboratories to function well.28 biological pharmacists among the key informants explained that it was demotivating and boring to work in laboratories with so little support and rudimentary equipment when from their training they knew that the technology was more advanced in europe. however, some doctors in charge of health facilities took the initiative to strengthen their laboratories. a director at the reproductive health directorate senegal recounted how when he became the medical director of a district health centre (1995–1998), the laboratory gained reference in the area because he asked a french friend to help him equip the laboratory with full blood count and glycaemia machines. laboratories became more visible in senegal in 1990 as part of the direction de la pharmacie et des medicaments avec volet laboratoires,29 and in burkina faso in 1993, when they gained a place in the direction générale de la pharmacies, des medicaments traditionels et des laboratoires. a current health inspector reasoned that the burkinabé moh realised the need to coordinate and supervise laboratory activities because the number of laboratories had increased. disease and donors many informants pointed out that the rapid development of laboratories from the 1990s onwards was mainly linked to aids control programmes which, compared to tuberculosis and malaria control programmes, required more than reagents and microscopes. once hiv cases were discovered in the mid-1980s, national aids programmes were set up with large international donor support to gather epidemiological data. until the mid-1990s, hiv testing was centralised – either the suspected hiv-positive individuals had to go to central laboratories or the laboratory staff went to the regions. a director of the senegalese direction des laboratoires remembered that in cases of suspected hiv, laboratory staff had to travel to the regions to collect the blood samples and bring them to dakar for analysis. hiv serology and immunology tests were gradually decentralised to the regional hospital level. donors supported regional hospitals and selected district health centre laboratories with equipment and supplies and by training laboratory staff – often assistants – to execute specific tests. the president of the burkinabé association of technicians explained that in 1999 the entrance qualifications for laboratory technician training at the ecole nationale de santé publique were raised from a middle-school exam to baccalauréat-level (final exam of secondary school) because the sophisticated equipment and more complicated techniques required more highly trained laboratory personnel. two important drivers for laboratory development were the 1996 world health organization (who) meeting in ouagadougou and the 1998 meeting in bamako on epidemic preparedness and the vital role of laboratories. ministry of health representatives from 16 west african countries participated. key informants who attended these meetings remembered that the who stressed laboratory development, training of laboratory staff for early diagnosis of epidemic diseases, and setting up of national laboratory networks. some few years later, all mohs agreed to support medical laboratories to prevent and fight epidemic diseases. emergence of global health (late 1990s–present) increasing but suboptimal laboratory services in all three countries, more public regional hospitals and health centres were built from the late 1990s onwards, which included buildings or rooms dedicated to laboratory services.30 referral laboratories were connected to university hospitals and research laboratories were established for specific diseases. compared to mali and burkina faso, senegal had fewer private stand-alone laboratories. in 2012, while senegal had only 6, burkina faso had 80.31,32,33 with the arrival of more equipment and machines, the array of tests that laboratories could process increased.34,35 the head of the senegalese aids control programme and the head of the who hiv programme in ouagadougou explained that technological developments enabled further roll-out of the prevention of mother to child (hiv) transmission (pmtct) programme. around 2006, all health centre laboratories could perform hiv confirmation diagnostic tests. a burkinabé pharmacist noted doctors began to increasingly use laboratory services when they observed a significant increase in the number of laboratories, tests and more qualified laboratory personnel. other key informants observed that some younger doctors even overused the laboratory, with one expressing: if the person [clinician] knows he can have a haemoglobin count, he does not bother to check the eyes or tongue of the patient, because that would take him more time than to write a test request. (key informant 47, female, interview date 04 may 2015, ouagadougou) medical laboratories exist from the health centre level. however, many (public) laboratories are often substandard. they have inadequate qualified human resources, often face stock-outs of rapid tests, encounter recurrent machine maintenance problems and non-availability of supplies and reagents, and have inadequate laboratory space. the latter was particularly true in mali. a malian biological pharmacist described how a health centre would find a laboratory space: ‘here there is a spare storeroom that only has to be transformed to be a laboratory’ (key informant 28, male, interview date 14 august 2015, bamako). laboratory staff worried that working in a non-dedicated room compromises safety and biosecurity. for instance, a laboratory technician in a malian health centre complained that laboratory training was not practicable in these settings. for instance little space means that equipment is sometimes placed too close to the wall, or on top of each other. some health centre laboratories are still headed by inadequately trained staff. mali has a shortage of clinical biologists due to the lack of training opportunities. in burkina faso, biological pharmacists are available, but many opt to work in private pharmacies, so they are ‘lost for the [public] laboratory’. in senegal, the bottleneck is the result of an insufficient budget to recruit the many required trained laboratory technicians. improved organisation the three countries’ mohs realised the need to prepare and enable health facility laboratories to quickly respond to epidemic threats and to be less dependent on specialised research laboratories. national plans began to increasingly include laboratory services: national laboratory networks, directorates and sub-directorates. interestingly, an outbreak of meningitis in burkina faso in 2002 led directly to the installation of the direction des laboratoires as one of the four technical sub-directorates of the direction générale de la pharmacie, du médicament et des laboratoires (décret n°2011/pres/pm/ms). two key informants who were involved in this recounted what had made the moh realise the importance of laboratory service coordination. during the outbreak, the country’s meningitis research laboratory had identified the outbreak virus to be a different strain (w135) previously unknown in the country, brought by pilgrims from mecca. however, this discovery was not communicated to the moh, leading to a wasteful high-cost countrywide meningitis a vaccination campaign. in mali, the national network of laboratories was created in 2004, consisting of specialised laboratories, hospital and centre de santé de référence (district) laboratories, a few cscom (sub-district) laboratories and private laboratories. in 2011, the division des laboratoires was added to the directorate of pharmacy and medicines. in senegal, the laboratory was in 2002 included in the direction de la pharmacie et des laboratoires and in february 2005 the national network of laboratories was officialised. this network extended to laboratories at the health centre level.36 senegal created a direction des laboratories in 2012.31 its director narrated the 10-year process of its birth: he advocated for its creation in 2002, and the then minister of health had been in favour; however, she was replaced and the new minister was less interested. when the former health minister was reinstated in 2012, the directorate was created. a public health medical doctor at who dakar explained that the advantage of being a stand-alone directorate is visibility at the ministry level, which goes with resource allocations. the problem of not having a budget for the sub-directorate in burkina faso was explicated by its former director: ‘i was appointed director without materials, without anything. it is with my car that i visited all the laboratories in the country [for quality assessment]’ (key informant 30, male, interview date 04 may 2015, ouagadougou). the director of the division of laboratories in mali noted that the division still struggles to function well because of a lack of resources for personnel. the national laboratory networks, the (sub-)directorate and division depend heavily on donor funds for their outreach activities such as general supervision and quality control. however, these donors mainly support disease-focused programmes and outreach stops when the programme ends. for example, laboratory supervision activities in mali no longer take place since money from the global fund to fight aids, tuberculosis and malaria stopped. to improve organisation and monitoring, the réseau d’afrique de l’ouest des laboratoire was created in 2009. resaolab is a regional laboratory network that includes senegal, burkina faso and mali, domiciled in bamako funded by agence française de développement and fondation mérieux. national laboratory policies and plans the who guided the three mohs in developing national laboratory policies and strategic plans. the first step was to conduct a countrywide inventory and evaluation of the status of laboratories. the united states centres for disease control and prevention (cdc) funded this exercise in mali in 2012, and the malian national laboratory policy was accepted in the same year. in burkina faso, the national laboratory policy was endorsed by the government in 2007, including the first five-year strategic plan.37 senegal has no approved plan yet. the laboratory became more integrated into the health system through the specific mention of laboratory testing in management guidelines for certain conditions. for example, in senegal and mali, the antenatal care guidelines identify laboratory tests that all pregnant women should receive.38,39,40 increased training opportunities in-country training opportunities for all levels of laboratory personnel have increased since the late 1990s, and medical staff could specialise in laboratory sciences. public and some private schools (the latter more prevalent in senegal) were opened and existing schools or universities offered new curricula. since 2007, laboratory technicians can upgrade their qualification through the bachelor in applied medical biology (biologie médicale appliquée – bams) in bamako, organised in collaboration with the centre d’infectiologie charles mérieux, fondation mérieux, and université catholique de lyon. in 2010, a nine-month training programme started at ecole nationale de santé publique, burkina faso, for lower-trained technicians to upgrade to the same level as the later-trained baccalauréat-level technicians. the association of laboratory technicians had pushed for this training, and as of 2015 nearly all had been trained. specialised training in laboratory sciences for medical doctors and pharmacists (diplôme d’études spécialisées de biologie clinique [des bc]) is offered in ouagadougou (since 2004), dakar (since 2008) and bamako (since 2013). a diplôme d’études spécialisées lecturer in dakar explained that these regional training opportunities are cost-efficient because students no longer need to go overseas for diplôme d’études spécialisées training, as his generation had to do. since 2010, réseau d’afrique de l’ouest des laboratoires organises refresher training in dedicated institutes in all three countries for biologists and senior technicians working in laboratories, including modules on quality control, biosecurity and biosafety, and health information systems. a general problem reported for many laboratory training programmes is the scarcity of equipment and supplies for hands-on practice. a lecturer in dakar explained how lecturers have to buy equipment and reagents for the students’ practical work because since 2008 or 2009 the university does not receive government funding for research: ‘we do not even have a bottle of reagent for the practical work. the state has no money’ (key informant 60, male, interview date 10 march 2015, dakar). to tackle this problem, senegalese university and training school laboratories have been allowed to generate money by offering paid services to the public. donor support focusing on specific diseases donors have continued to support specific laboratories with training and equipment for the disease they focus on. hiv programmes have greatly increased the availability and level of laboratory services for diagnosis and treatment follow-up. international donors, including the united states cdc, the bill and melinda gates foundation, the clinton health access initiative, the world bank and the global fund, have financially supported or provided equipment, supplies and staff training. the global fund and world bank financially supported infrastructure, surveillance, and supervision of hiv programmes. the united states cdc gave technical and financial support for sentinel site surveillance of hiv and syphilis among pregnant women, while providing quality controls for the testing services. the who provided financial and technical support for laboratory services to fight (other) epidemic-threat diseases, focusing on bacterial meningitis, bacterial diarrhoeal diseases, yellow fever, measles and rubella. in 2004, the who conducted training on the stratégie de surveillance integrée de la maladie et la riposte in ouagadougou, which was attended by several key informants. the training covered, among other things, epidemiology, security, quality assurance and laboratory systems development. in burkina faso, this training led to the national external quality assessment programme in 2006, supervision of laboratory systems and several training programmes in biosecurity.41 the who published a document on laboratory development42 that all countries ratified – the key informant at the who burkina faso office was one of the authors. the west african ebola epidemic of 2013–2016 was a wake-up call for donors to support laboratories and for governments to establish national research laboratories. the inrsp already existed in mali, and the institute’s ex-director and a current lecturer both recounted how donors had supported the inrsp with equipment that enabled it to perform ebola diagnostic tests on regional samples. these key informants believed that this timely and efficient action contributed to the containment of ebola in mali. many key informants criticised donor support for its focus on specific diseases. the former head of several senegalese moh directorates (2004–2012) complained that donors for aids and malaria programmes only support selected laboratories with equipment and training. two biotechnologists active in the malian national association of laboratory technicians highlighted the lack of long-term vision and soundness of donor support during the ebola outbreak when just a few technicians were trained and general biosecurity was only maintained as long as ebola was a threat. a burkinabé pharmacist recounted how a donor had given 10 microscopes – even though the (vertical) programme only needed three – but had not allowed the extra seven microscopes to be used for other services. informants also criticised the fact that some donors imported their machines, not considering what was already available. for example, the head of the directorate for sexual and reproductive health in burkina faso recounted how, as a medical doctor in the region, she experienced that the laboratory equipment given by donors differed from what is normally used in the country. for instance, they received cd4 count machines that used different reagents and could be maintained or repaired by only one person in west africa when the machines were out of commission for three months. réseau d’afrique de l’ouest des laboratoires is one of the few donor-funded programmes focusing on the laboratory system. it supports the training of different levels of laboratory staff, the renovation and equipment of laboratories, and the equipment of training centres.43 involved key informants were very positive about the contributions of réseau d’afrique de l’ouest des laboratoires. some other programmes and funds also address laboratory systems, including the global health security agenda (launched 2014), the regional disease surveillance systems enhancement ii project and the west african health organisation which conducts large laboratory system strengthening through the world bank funding. discussion until the late 1990s, there were few investments in the expansion of laboratory capacity, partly as a result of economic austerity that affected the overall health sector, but also because laboratories were not considered a priority. from the 1990s onwards, the development of laboratories was mainly influenced by the emergence of potential pandemic diseases that require laboratory confirmation for treatment and control. new technology has led to the expansion of laboratory services. however, operating a laboratory (including machines and equipment) depends on the availability of amenities such as electricity and running water. at the time of this report, unavailable and erratic public amenities challenge proper laboratory functioning. not only do power cuts hinder testing, power fluctuations also cause equipment breakdown. the mohs in these economically constrained countries have had and still do have to operate with limited public finances, relying heavily on external financial aid for the running of public health services. donors have therefore played the most important role in how laboratories have been developmentally geared towards specific diseases; national policymakers did not take a leading role. dependence on donors puts the mohs in a weak position in terms of setting priorities for laboratory development and research. donor support for specific diseases did and might not strengthen the laboratory system for the diagnosis and follow-up of all health conditions. an unexpected finding was the big influence of ‘laboratory champions’: persons committed to laboratories, who have a ‘vision’ and ‘fight’ for the development of the national laboratory system. they are individuals who got ‘the laboratory virus’, as the director of the senegalese directorate of laboratories aptly described them. these champions included biologists and pharmacists who lobbied national ministers of health and donors to set up research laboratories, national health ministers and laboratory technicians active in their associations. these champions were often constrained in putting their vision into practice by the larger national context of unsupportive political leadership, general poverty and the lack of basic infrastructure and amenities. recommendations drawing lessons from the study findings, the following recommendations for laboratory-strengthening programmes are directed at political leaders, mohs, health facilities and donors: have a stand-alone directorate of laboratories with a dedicated national budget, as per the recommendation of the 2008 maputo declaration.44 the who regional office for africa, the african society for laboratory medicine and the africa cdc should continue to remind and support national policymakers who signed the declaration. national policymakers should dedicate more national budget to laboratory development and give the national directorate the mandate to coordinate and guide donor involvement in public and private laboratories at all levels. prioritise continuous professional development opportunities for laboratory personnel at all levels, including laboratory assistants trained on-the-job. professional councils and associations, and the african society for laboratory medicine, should play a role in regulating the profession while the national leadership supports with sufficient funding. national public health laboratories should be established – this is one of the goals of the africa cdc, supported by the african society for laboratory medicine – and should have political influence at the moh to set national research priorities. health facility management committees should establish a dedicated budget for the laboratory, reasoning that the laboratory is a source of direct income and needs to function optimally. as the director of a senegalese regional hospital expressed: ‘that will make the facility function. the laboratory and the radiology are the “lungs” of the health facility’ (key informant 65, male, interview date 16 march 2015, kaolack). limitations the authors acknowledge that personal accounts do not constitute hard, objective data, as is the norm in the medical sector, including laboratory medicine. the authors are also aware that respondents may have constructed this history of, and recommendations for, laboratory development to suit their own or their organisations’ interests. nevertheless, by triangulating literature and accounts of key informants from three countries and different health professions and levels, the authors opine that this oral history presents a true reflection of the development of medical laboratories in francophone west africa. conclusion this unique article on the long-term historical developments of the laboratory sector in francophone west africa demonstrates that by collecting and recording the experiences of people who lived through and have been actors in laboratory developments, a history that would have been otherwise forgotten has been constructed. many of the historical laboratory sector challenges still exist and will not be surmounted without national leaders prioritising the alleviation of national poverty and the development of basic infrastructure. as exemplified by the champions described in this article, national policymakers must be abreast of their population’s disease burdens and needs and play the important leadership role to donors ensuring donor programmes match the populace’s healthcare or laboratory needs, consequently advancing their population’s healthcare. ministries of health should see laboratories as an integral part of the health system, and not simply as part of vertical disease programmes. the retired director of inrsp in mali aptly reiterated: ‘the laboratory is the brain of the health system’ (key informant 24, male, interview date 13 august 2015, bamako). acknowledgements we thank the francophone west africa country team members – other than the authors of this article – who participated in the literature studies. in senegal, they are mouhamed ahmed badji and papa ngore sarr sadio; in burkina faso, dr nikiéma abdoulaye, dr paul somda, naby alphonse and zélé issa; in mali, prof. bourèma kouriba, dr seydou diarra and mamadou fadiala sissoko. we also thank the west african and dutch socialab members who contributed to the overall socialab study that this study is part of: dr aicha marceline sarr, prof. robert pool, prof. constance schultz. special thanks go to prof. robert pool for critically reading the final draft manuscript and oumou badji and sylvain yaméogo for transcribing the key informant interviews. we are most grateful to the key informants who gave their time and enthusiasm to share their experiences with us; without them this history could not have been written. we apologise for having had to sacrifice their often very rich contributions for the general history. competing interests the authors have declared that no competing interests exist. authors’ contributions w.k. was responsible for the study proposal and design, interviews (senegal and burkina faso), coordination, analysis of key informant interviews and drafting of manuscripts. a.g.n. drafted the literature study and report. m.a. and i.g. conducted interviews (mali) and drafted the literature study and report. i.s., s.d. and j.s. were involved in the study proposal and design, selecting key informants and the literature study. p.o. worked on the study proposal and design and was the principal investigator at socialab. all authors critically commented on the draft manuscripts and gave their approval of the final article. sources of support the study was funded by the netherlands organization for scientific research, science for global development (nwo/wotro) under w07.4.203.00. data availability statement upon request, transcripts of interviews can be provided. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references okeke in. divining without seeds. the case for strengthening laboratory medicine in africa. ithaca and london: cornell university press; 2011. petti ca, polage cr, quinn tc, ronald ar, sande a. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006;42(3):377–382. https://doi.org/10.1086/499363 bates i, maitland k. are laboratory services coming of age in sub-saharan africa? clin infect 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[reproductive health services protocol, senegal]. dakar: ministère de la santé; 2000. présidence de la république. loi n°02–044 /du 24 juin 2002 relative à la santé de la reproduction [law n°02–044 /of 24 june 2002 relating to reproductive health]. 2002. sakandé j, niekiema a, kabré e, et al. national external quality assessment for medical biology laboratories in burkina faso: an overview of three years of activity. ann biol clin. 2010;68(6):637–642. oms. capacités requises des laboratoires en vertu du règlement sanitaire international et leur mise en place dans la région africaine de l’oms [laboratory capacity requirements under the international health regulations and their implementation in the who african region]. brazzaville: bureau régional de l’oms pour l’afrique; 2013. fondation mérieux. rapport annuel 2011. lyon: fondation mérieux; 2011. world health organisation, regional office for africa. the maputo declaration on strengthening of laboratory systems. 2008 [homepage on the internet] [cited 2015 dec 18]. available from: https://www.who.int/diagnostics_laboratory/maputo-declaration_2008.pdf article information authors: eleanor a. ochodo1 mariska m.g. leeflang1 affiliations: 1department of clinical epidemiology, biostatistics and bioinformatics, academic medical center, university of amsterdam, amsterdam, the netherlands correspondence to: eleanor ochodo postal address: meibergdreef 9, 1105 az, academic medical center, university of amsterdam, the netherlands dates: received: 04 aug. 2011 accepted: 17 nov. 2011 published: 30 jan. 2012 how to cite this article: ochodo ea, leeflang mmg. systematic reviews of diagnostic test accuracy for evidence-based diagnostic practice in africa. afr j lab med. 2012;1(1), art. #7, 3 pages. http://dx.doi.org/10.4102/ ajlm.v1i1.7 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. systematic reviews of diagnostic test accuracy for evidence-based diagnostic practice in africa in this scientific letter... open access • acknowledgements    • competing interests    • authors’ contributions • references in 2009, a debate started about whether there was enough evidence to change current guidelines, from presumptive malarial treatment of children under 5 years who present with fever, to testing these children before treating them.1,2 a major argument against this policy change was the lack of confidence in the performance of the available rapid diagnostic tests to diagnose malaria in children accurately. this debate neatly illustrates one of the many diagnostic dilemmas with which resource-poor countries struggle. it also illustrates the need for more, and better, evidence about diagnostic questions in these countries. the ultimate question here may be whether we should test before treatment. the optimum study design would be a randomised controlled trial in which one arm is tested before treatment, and the other arm is treated presumptively. before such a trial will be granted by ethical committees, health-care workers, the public or the patients, however, more knowledge is needed on the accuracy and reliability of the available malaria tests. only when the accuracy of these tests is high enough will policy makers, ethical committees, health-care workers and patients put their trust in them. furthermore, it will be possible to make a sensible decision about which test to use in the trials only when the accuracy of the available tests is known. the accuracy of a diagnostic test depends on its ability to distinguish people with a target condition from people without the specified condition. a target condition can be either a disease or a specific stage of a disease. accuracy is expressed often as sensitivity (percentage of people carrying the disease, with a positive test result), and specificity (percentage of people who do not carry the disease, with a negative test result). other measures to express accuracy include the predictive values, likelihood ratios, area under receiver operating characteristic curve, and diagnostic odds ratios. in a study evaluating the accuracy of a test, the results of the test under evaluation are compared to the results of a reference standard. a reference standard is the best available test or method to diagnose a target condition accurately.3,4,5 a valid scientific evaluation of the accuracy of tests is required in order to assist health-care providers, researchers, and policy makers in making rational and evidence-based decisions about the use and interpretation of diagnostic tests. a valid scientific evaluation can be carried out by summarising the results of previously published studies systematically. such a systematic review is a scientific tool that objectively and methodologically identifies, appraises and summarises the existing data from quality primary studies.6 it constitutes the highest level of scientific evidence.7,8 a systematic review may, or may not, include a meta-analysis, which is a statistical method of summarising the results of primary studies into a single and precise estimate.6 the benefits of systematic reviews are substantial. firstly, they help to summarise a large volume of information into a form that can be read easily and can be used to make decisions. secondly, systematic reviews establish whether scientific findings are consistent and can be generalised across populations and settings, or whether findings vary significantly according to subgroups. in addition, systematic reviews are of use in identifying the risk of bias in primary studies. the rigorous methods used in systematic reviews limit bias and improve the reliability and accuracy of conclusions. as a result they can assist effectively in guideline development and in translating research into practice.9,10 systematic reviews of diagnostic test accuracy in particular are useful to establish why the accuracy of tests vary. test accuracy has been shown to vary with factors such as the patient population, spectrum of disease, disease stage, the type of test used, the method of test administration and the expertise of the people who administer the test.11 diagnostic test accuracy reviews are also useful in comparing tests, or combinations of tests, or different diagnostic strategies.12 effective diagnosis in africa is impeded by limited financial and human capacity, and poor laboratory infrastructure. poor laboratory infrastructure includes poor assurance mechanisms or a lack of quality assurance mechanisms, a lack of appropriate laboratory reagents and equipment, and logistical challenges in specimen collection, storage and transportation. these factors may lead to numerous variations or inconsistencies in diagnostic results; therefore, even when laboratory services are available, health workers perceive them as unhelpful and unreliable.13 these variations and inconsistencies constitute a hindrance to decision making and guideline development. in this setting, therefore, systematic reviews of available studies on the accuracy of diagnostic tests will be useful in evaluating inconsistencies objectively, and in identifying sources of bias and variation. more so, in a situation where resources are limited, both an accurate diagnosis and the choices made towards that diagnosis are crucial for the optimal use of those resources. the adoption of quality systematic reviews of diagnostic test accuracy will facilitate rational decisions related to which diagnostic tests resources should be allocated. unfortunately, the capacity to prepare systematic reviews in developing countries is limited by a shortage of skills and a lack of access to scientific literature because of insufficient finances to pay for subscriptions to medical journals.14 access to scientific literature is hampered further by slow internet connections that make it difficult to download articles.15 concerted efforts are therefore required to promote the use of scientific literature and to build internet capacity. in its bid to enhance the quality of laboratory practice in africa, the newly established african society for laboratory medicine can help to advocate for the use of high standard diagnostic test accuracy reviews to guide evidence-based diagnostic practice.16 currently, the most prominent organisation that is actively promoting the preparation and use of systematic reviews is the cochrane collaboration.17 although it is committed to global participation in the use of systematic reviews, the cochrane collaboration is still dominated by authors and articles from developed nations. the evidence from cochrane reviews, therefore, may not be applicable to developing countries, which have the largest burden of disease.18 in order to address this challenge, the cochrane collaboration has embarked on initiatives to promote participation from developing countries actively. these include building capacity of local authors through the provision of training opportunities, fellowships and by forming partnerships with local institutions. for instance, the reviews for africa programme (rap) trains and mentors african health researchers to prepare cochrane reviews. rap is a partnership between the south african cochrane centre and the liverpool school of tropical medicine. this programme focuses on systematic reviews on diseases that are applicable to africa, such as hiv and aids, malaria and tuberculosis.19 the collaboration for evidence based health care in africa (cebha) was formed recently to further promote evidence-based practice in africa. this organisation is the result of collaboration between international and local african researchers, with its main aim being the facilitation of knowledge and the implementation of evidence-based health care in africa. one of its pillars is the support of african scientists to develop systematic reviews through training and mentorship. it is currently focusing on eastern african countries and plans to expand to other african countries in the future.20 additionally, in order to enable scientists from developing countries to gain access to scientific literature, the world health organization (who), in collaboration with major publishers, set up the health internetwork access to research initiative (hinari). this programme offers free or low cost access to medical journals for eligible developing countries. this enhanced access to literature can enable african researchers to prepare systematic reviews.21 even though internet connectivity in africa is still limited, it has witnessed a marked improvement in penetration and speed. latest estimates report an increase of2633% in internet users between 2000 and 2011. broadband initiatives have also been put in place to increase bandwidth, with the latest being the availability of fibre-optic cables in certain african countries. as internet access and speed continue to improve, access to scientific literature will improve as well.22 in a nutshell, systematic reviews, when prepared rigorously, objectively summarise the findings from available studies and provide a strong source of evidence. in order to promote the use of diagnostic test accuracy reviews in africa, the african society for laboratory medicine can help to promote the use of high standard diagnostic test accuracy reviews to guide evidence-based diagnostic practice. secondly, the importance of these reviews ought to be disseminated to african researchers through workshops, and their capacity needs to be built through training and mentorship. these can be performed in partnership with the cochrane collaboration, cebha, or other researchers well versed in developing these reviews. finally, the use of the hinari programme that was set up to enable researchers from developing countries to access scientific literature should be encouraged. acknowledgements top ↑ we thank prof. dr bossuyt for reviewing an earlier draft of this manuscript. competing interests dr mariska leeflang is a co-convener of the cochrane screening and diagnostic test methods group and a contributor to the collaboration for evidence based health care in africa (cebha). views expressed in this article are not necessarily shared by either the cochrane collaboration or cebha. no funding was received for this project. the authors declare no financial competing interests. authors’ contributions e.a.o. (university of amsterdam) conceived this project, and e.a.o. and m.m.g.l. (university of amsterdam) drafted the manuscript. references top ↑ 1.english m, reyburn h, goodman c, snow rw. abandoning presumptive antimalarial treatment for febrile children aged less than five years – a case of running before we can walk? plos med. 2009;6(1):e1000015. http://dx.doi.org/10.1371/journal.pmed.1000015, pmid:19127977, pmcid:2613424 2.d’acremont v, lengeler c, mshinda h, mtasiwa d, tanner m, genton b. time to move from presumptive malaria treatment to laboratory-confirmed diagnosis and treatment in african children with fever. plos med. 2009;6(1):e252. http://dx.doi.org/10.1371/journal.pmed.0050252, pmid:19127974, pmcid:2613421 3.bossuyt pm, reitsma jb, bruns de, et al. towards complete and accurate reporting of studies of diagnostic accuracy: the stard initiative. ann intern med. 2003;138(1):40–44. pmid:12513043 4.van stralen kj, stel vs, reitsma jb, dekker fw, zoccali c, jager kj. diagnostic methods i: sensitivity, specificity, and other measures of accuracy. kidney int. 2009;75(12):1257–1263. http://dx.doi.org/10.1038/ki.2009.92, pmid:1934009 5.habbema jdf, eijkemans r, krijnen p, knottnerus ja. analysis of data on the accuracy of diagnostic tests. in: knottnerus ja, buntinx f, editors. the evidence base of clinical diagnosis: theory and methods of diagnostic research. 2nd ed. oxford: wiley-blackwell, 2002; p. 117–144. 6.green s. systematic reviews and meta-analysis. singapore med j. 2005;46(6):270–273. pmid:15902354 7.oxford center for evidence-based medicine-levels of medicine [homepage on the internet]. c2009 [cited 2011 jun 29]. available from: http://www.cebm.net/index.aspx?o=1025 8.the cochrane collaboration. evidence-based health care and systematic reviews [homepage on the internet]. no date [cited 2011 jul 06] available from: http://www.cochrane.org/about-us/evidence-based-health-care 9.mulrow cd. rationale for systematic reviews. bmj. 1994;309(6954):597–599. http://dx.doi.org/10.1136/bmj.309.6954.597, pmid:8086953, pmcid:2541393 10.cook dj, greengold nl, ellrodt ag, weingarten sr. the relation between systematic reviews and practice guidelines. ann intern med. 1997;127(3):210–216. pmid:9245227 11.irwig lm, bossuyt pm, glasziou pp, gatsonis c, lijmer jg. designing studies to ensure that estimates of test accuracy will travel. london: bmj publishing group, 2002; p. 95–116. 12.leeflang mm, deeks jj, gatsonis c, bossuyt pm. systematic reviews of diagnostic test accuracy. ann intern med. 2008;149(12):889–897. pmid:19075208, pmid:2956514 13.petti ca, polage cr, quinn tc, ronald ar, sande ma. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006;42(3):377–382. http://dx.doi.org/10.1086/499363, pmid:16392084 14.english m, opiyo n. getting to grips with grade-perspective from a low-income setting. j clin epidemiol. 2011;64(7):708–710. http://dx.doi.org/10.1016/j.jclinepi.2010.07.016, pmid:21316192 15.bukachi f, pakenham-walsh n. information technology for health in developing countries. chest. 2007;132(5):1624–1630. http://dx.doi.org/10.1378/chest.07-1760, pmid:17998362 16.the african society for laboratory medicine [homepage on the internet]. no date [cited 2011 may 17]. available from: http://www.afslm.org/ 17.the cochrane collaboration. cochrane reviews [homepage on the internet]. no date [cited 2011 jul 06]. available from: http://www.cochrane.org/cochrane-reviews 18.mcdonald s, turner t, green s. cochrane strategic session on ‘ensuring the cochrane collaboration enables better global participation.’ the cochrane collaboration 2011 [ homepage on the internet]. c2011 [cited 2011 jul 06]. available from: http://www.cochrane.org/news/tags/authors/cochrane-strategic-session-ensuring-cochrane-collaborationenables-better-global-p 19.reviews for africa programme (rap): training in the science of research synthesis for cochrane authors of systematic reviews [homepage on the internet]. no date [cited 2011 jul 06] available from: http://www.mrc.ac.za/cochrane/rap.htm 20.collaboration for evidence based health care in africa [homepage on the internet]. no date [cited 2011 nov 13]. available from: http://www.cebha.org/ 21.hinari access to research in health programme [homepage on the internet]. no date [cited 2011 jul 10]. available from: http://www.who.int/hinari/en/ 22.internet world stats – internet usage statistics for africa as at june 30, 2011 [homepage on the internet]. c2011 [cited 2011 nov 14]. available from: http://www.internetworldstats.com/stats1.htm next-generation sequencing where short reads come short long-read sequencing ‘nano-promises, giga-prospects’ situation report lessons from the past the illumina-pacbio merger outlook acknowledgements references about the author(s) boluwatife a. adewale medicine and surgery, faculty of clinical sciences, college of medicine, university of ibadan, ibadan, nigeria college research and innovation hub (crih), university of ibadan, ibadan, nigeria university college hospital, ibadan, nigeria citation adewale b.a. will long-read sequencing technologies replace short-read sequencing technologies in the next 10 years? afr j lab med. 2020;9(1), a1340 https://doi.org/10.4102/ajlm.v9i1.1340 opinion paper will long-read sequencing technologies replace short-read sequencing technologies in the next 10 years? boluwatife a. adewale received: 18 july 2020; accepted: 07 sept. 2020; published: 26 nov. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the year 1977 is quite remarkable in the history of genomics. it was the first time that the complete genome of an organism (phage φx174) would be sequenced – the advent of the first generation of sequencing technologies. of the two sequencing methods published that year, fred sanger’s ‘chain-termination’ method would become the mainstay of sequencing technology for the next three decades. because of its better usability compared to the maxam-gilbert method, it was widely preferred and became commercialised1 by applied biosystems inc. thanks to the collaborative efforts of scientists across the world,2 sanger sequencing eventually produced a reference human genome, courtesy of the us$2.7-billion (united states dollar) human genome project completed in 2003.3 next-generation sequencing the launch of pyrosequencing by 454 life sciences in 2005 marked the beginning of the second generation of sequencing. massively parallel or next-generation sequencing (ngs) technologies eliminated the need for multiple personnel working on a genome by automating dna cleavage, amplification and parallel short-read sequencing on a single instrument, thereby lowering costs and increasing throughput.1,4 pyrosequencing relies on the release of pyrophosphates when nucleotides are washed over fixed dna clones in a dna polymerase-mediated reaction (figure 1). pyrophosphate takes part in a cascade of reactions that eventually give off light which is detected and interpreted by a computer program. ion torrent employs a similar technique except that it uses ph instead of luminescence to detect nucleotide addition, whereas the applied biosystems inc. solid™ system is a sequencing--by-ligation technique which detects fluorescence that results from the action of dna ligase on oligonucleotides (figure 1).5,6 figure 1: overview of shortand long-read sequencing technologies. short-read sequencing methods are displayed on the left, long-read sequencing methods are shown on the right. short-read sequencing methods are classified into sequencing by synthesis and sequencing by ligation. they all require clonal amplification; emulsion polymerase chain reaction for 454 pyrosequencing, solid™ and ion torrent, ssba is peculiar to illumina, solid-phase template walking is used for certain solid™ technologies. illumina, fluorescent-labelled dntp added to bridge amplified dna template; 454 pyrosequencing, empolymerase chain reaction-generated microbead-bound dna clone in picotitre well, dna polymerase is added to the well, nucleotides are washed over in turn, deoxynucleoside triphosphate incorporation monitored via pyrophosphate release; ion torrent, similar to 454 pyrosequencing, deoxynucleoside triphosphate incorporation monitored via h+ ions release detected by a ph sensor that uses complementary metal-oxide-semiconductor technology. solid™, microbead-bound dna template flanked by adapters is hybridized to a growing complementary strand, under the action of dna ligase. cpal, another sequencing by ligation technique not described in this paper, employed by complete genomics, anchor sequence and probes hybridize to dna template in a series of ligation reactions taking place on a nanoball. pacbio single-molecule real-time, sequencing takes place on zero-mode waveguide chip. dna polymerase at the bottom of the well, fluorescent nucleotides being added to the strand. oxford nanopore technology relies on changes in ion flow as nucleotides pass through the nanopore. solid™ and ion torrent have been in the ngs market since 2007 and 2010, whereas roche announced the phaseout of 454 pyrosequencing in 2013 after facing stiff competition in the market. the reversible terminator sequencing currently sold by illumina is the predominant ngs platform: a sequencing-by-synthesis approach that uses bridge amplification and fluorescent signals from the addition of deoxynucleoside triphosphate (dntp) to a growing complementary strand (figure 1). the signal is detected in real time by a coupled charge device camera and interpreted by computer software.1 next-generation sequencing technologies have made sequencing much easier, faster and cheaper than sanger sequencing. the august 2019 report from the national human genome research institute put the cost of sequencing a complete human genome at $942.00 united states dollars (usd). thanks to ngs, the reduction in cost of genome sequencing has beaten moore’s law prediction (figure 2), more evidently since 2008, about the same time that ngs became popular in sequencing centres.7 figure 2: the cost of sequencing a single human genome from 2001 to 2019. the y-axis represents the cost of sequencing a human genome, the x-axis is labelled from year 2001 to 2019. moore’s law predicts a biennial doubling of transistors on a microchip. the line drawn in the graph above illustrates moore’s law prediction of the cost of sequencing per human genome. it is shown clearly that the cost has been consistently lower than predicted by moore’s law since 2008. illumina, introduced in 2006, is the most robust of all ngs technologies. it quickly gained traction among scientists, because of its high throughput and affordability compared to 454 pyrosequencing, solid™ and ion torrent. in fact, illumina was the first company to fulfil the ‘$1000 genome’ promise with its hiseq x ten sequencer in 2014. sequencing a genome now costs below $1000.00 usd with ongoing efforts to reach the $100.00 usd target.3 where short reads come short all second-generation sequencing technologies have a common limitation – the inability to sequence long stretches of dna. to sequence a large genome like human dna using ngs, the dna has to be fragmented and amplified in clones of between 75 base pairs and 400 base pairs, hence the term ‘short-read sequencing’ (srs). computer programs are then used to assemble the random clones into a contiguous sequence.2 aside from the fact that polymerase chain reaction – a necessary step in srs – causes preferential amplification of repetitive dna, srs fails to generate sufficient overlap sequence from the dna fragments. this constitutes a major challenge for de novo sequencing of a highly complex and repetitive genome like the human genome.2 the detection of large sequence changes is another area of difficulty that is encountered using srs – a major barrier to studying structural variations.8 long-read sequencing third-generation sequencing technologies which are otherwise called long-read sequencing (lrs) technologies address the shortcomings of ngs. whereas the sanger and srs approaches cannot exceed read lengths of 1 kilobase pair, third-generation sequencing technologies read lengths are between 5 kilobase pairs and 30 kilobase pairs. the longest read length ever generated by a third-generation sequencing technology is 2 gigabase pairs.9 long-read sequencing methods sequence a single molecule – thus abolishing amplification bias – and generate a reasonable length of overlap sequence for better sequence assembly.1 there are two prominent lrs technologies namely, nanopore sequencing and the single-molecule real-time (smrt) sequencing.10 single-molecule real-time sequencing was commercially released in 2011 by pacific biosciences (pacbio). a smrt genome sequencer consists of millions of zero-mode waveguides – microscopic wells with dna polymerase fixed at the bottom. within each zero-mode waveguide, two hairpin adaptors are ligated to both ends of the molecule to form a circular single-stranded dna. with the aid of a primer complementary to the adaptors, dna polymerase is used to sequence a complementary strand. when a fluorescent nucleotide is added to the strand, a fluorescence of wavelength corresponding to the added nucleotide is given off. this signal is captured in real time by a coupled charge device camera and interpreted by a computer program.1 nanopore sequencing, released by oxford nanopore technologies in 2014, works by a different principle: threading the dna molecule through a 1.5 nm wide bioengineered channel embedded in a biological membrane. electrical current across the channel depends on which nucleotide is traversing the channel at the time. this variation is used to determine the base sequence of the nucleic acid.1 single-molecule real-time and nanopore sequencing check all of the boxes in terms of suitability for de novo sequencing, resolution of repeat sequences (e.g. human leukocyte antigen gene, centromere), detection of structural variations, epigenetics and transcriptome sequencing, among others. however, the accuracy per read and the throughput of lrs is poor compared to srs.1,10 moreover, there are challenges in the adaptability of lrs for sequencing different genome lengths, as lrs data processing takes longer for organisms with large genomes than for viral genomes.10 the high error rates of nanopore sequencing are attributed to the poor sensitivity of the nanopore and the inability to control the speed of dna translocation through the pore. whereas the errors in nanopore sequencing are systematic, smrt base-calling errors are random and can be reduced using circular consensus sequencing (ccs), a method that allows the dna to have multiple passes through the zero-mode waveguides. increasing the accuracy of smrt via ccs comes at a higher cost than using ngs.3 nanopore sequencing has improved over the years, although it is still not as accurate as ngs. ‘nano-promises, giga-prospects’ minion, a small-sized nanopore sequencing device similar to a universal serial bus flash drive, was used to sequence the ebola virus during the 2014-2016 outbreak in west africa in 60 min.3 the minion sequencer is much more affordable when compared with illumina and smrt considering the cost of instruments and consumables.9 considering its portability, affordability and its ability to generate ultra-long reads, the possibility of using minion to accurately determine larger genome sequences is of immense value to researchers and clinicians. it holds the answer to affordable genome sequencing in low-resource settings and at the point of care. with improving nanopore technology aimed at decreasing minion’s high error rates, these promises may not be far-fetched. the sequel ii 8m smrt cell, which was launched in 2019 by pacbio, has a ccs read accuracy of 99% and above. promethion, also released in 2019 by oxford nanopore technologies, generates ultra-long reads, although with less accuracy than smrt. in their efforts to beef up quality, both companies update their products regularly. the latest -double-sensored nanopore cell r10 and linear consensus sequencing (a similar method to smrt ccs) are expected to improve the accuracy of oxford nanopore technologies products. interestingly, lrs technologies are approaching srs in terms of cost and accuracy. using the sequel ii 8m smrt cell that was released, ccs reads can now be obtained for about $1500.00 – an eight-fold reduction in costs.3 less accurate long reads from the promethion flow cell can be obtained at about the same price.3,5 error rates for lrs used to be 12% – 15%5 but have now fallen to less than 1% for smrt and less than 5% for nanopore sequencers10 compared to an error rate of approximately 0.1% in ngs.3,5 situation report notwithstanding srs market dominance, lrs has broadened its range of applications in recent years as a result of significant improvements in accuracy and throughput. for instance, there has been an increase in the use of minion in metagenomics studies. lrs technologies are increasingly being used in epigenetics and transcriptomics as well.10 the technical difficulties associated with de novo assembly of short reads for large complex genomes will likely persist for the foreseeable future. the question then becomes whether lrs technologies will improve on their own shortcomings to take over the market. a paradigm shift in the prevailing technology is likely to occur if there is a major scientific breakthrough, such as the discovery of a dna polymerase with a longer lifespan for higher throughput and accuracy in smrt base calling, or a drastic improvement in the sensitivity and dna translocation speed in nanopore sequencing that matches the accuracy of srs technologies. however, such improvements are more likely to take place gradually rather than suddenly; i do not see that occurring in the next 10 years. lessons from the past history suggests a difficult transition from short-read to long-read technologies. when 454 pyrosequencing was introduced in 2005, it had a clear advantage over sanger sequencing in terms of throughput and cost. however, funding agencies and scientists were not immediately receptive to the new technology. many of the researchers had got used to sanger sequencing, whereas investors were mindful of their stakes. thus, it took 3 years before ngs gained wide acceptance in the scientific community.4,7 even a major scientific breakthrough in sequencing technologies could still take about 2–3 years before finding its footing in the market for wide acceptability. as of 2014, illumina controlled about 70% of the market for dna sequencers and accounted for 90% of global dna data.3 it is the biggest company on the genomic sequencing market with a worth of $43.6 billion usd. the company’s large and increasing customer base makes it difficult to change sides by motivating customers to buy their stocks.11 currently, no technology compares to illumina in terms of cost, throughput and accuracy. even if one were to arise today, it would still have a hard time replacing illumina, considering the niche that illumina has carved for itself in the market. the illumina-pacbio merger the potential of lrs, particularly smrt, is not unnoticed by market giant illumina, who entered talks in 2018 on a $1.2 billion usd merger with pacbio. both parties, however, terminated the deal owing to concerns from the united kingdom competition and markets authority and the united states federal trade commission about an emerging monopoly.12 one is left to imagine why illumina wanted a merger. sheer desire for monopoly or the prospect of a robust hybrid technology? anyway, the synergy of both generations of sequencing technology is worth considering. hybrid sequencing combines the throughput and accuracy of srs with the long read length of lrs. it is highly effective for genome polishing10 and can produce reference genomes for most organisms.3 outlook while it may be difficult to predict with certainty major breakthroughs capable of altering the narrative in genomic sequencing, it is apparent that lrs holds the key to many unanswered questions in genomics. long-read sequencing technologies, although constantly improving and gradually closing the gap on ngs in terms of accuracy and costs,3,5 have yet to attain their full potential. in this decade, i expect that lrs technologies will gain more recognition and acceptance as they are adapted for sequencing a variety of organisms. in the absence of an innovation in lrs technology that can significantly increase accuracy and throughput and reduce costs far beyond what srs technologies offer, i would argue that it is unlikely that lrs technologies completely replace srs technologies in the next 10 years. meanwhile, in the middle of these improvements in srs and lrs technologies, i expect to see increased efforts towards making hybrid sequencing more scalable. acknowledgements i am immensely grateful to my mentors and teachers dr j.o. olugbami and dr o.o. bello for their invaluable guidance and support. this article was adapted from the winning essay for the sanger institute prize 2020 organised by the wellcome sanger institute, hinxton, cambridge, united kingdom. competing interests the author declares that no competing interests exist. author’s contributions the author b.a.a. wrote and edited the manuscript. ethical considerations i confirm that ethical clearance was not needed for this paper. sources of support the author received no financial support for the research, authorship or publication of this article. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the author and do not necessarily reflect the official policy or position of any affiliated agency of the author. references heather jm, chain b. the sequence of sequencers: the history of sequencing dna. genomics. 2016;107(1):1–8. https://doi.org/10.1016/j.ygeno.2015.11.003 mardis er. dna sequencing technologies: 2006–2016. nat protoc. 2017;12(2):213–218. https://doi.org/10.1038/nprot.2016.182 giani am, gallo gr, gianfranceschi l, formenti g. long walk to genomics: history and current approaches to genome sequencing and assembly. comput struct biotechnol j. 2020;18:9–19. https://doi.org/10.1016/j.csbj.2019.11.002 schuster sc. next-generation sequencing transforms today’s biology. nat methods. 2008;5(1):16–18. https://doi.org/10.1038/nmeth1156 kchouk m, gibrat j, elloumi m. generations of sequencing technologies: from first to next generation. biol med. 2017;9(3):1–8. https://doi.org/10.4172/0974-8369.1000395 suresh ps, venkatesh t, tsutsumi r, shetty a. 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https://www.forbes.com/sites/kenberman/2019/02/21/genome-sequencing-stocks-on-the-rise/#76a851951f51 illumina, pacbio scrap $1.2b merger, citing ‘continued uncertainty’ [homepage on the internet]. genetic engineering and biotechnology news; 2020 [cited 2020 mar 06]. available from: https://www.genengnews.com/news/illumina-pacbio-scrap-1-2b-merger-citing-continued-uncertainty/ article information authors: christopher kariuki1 john m. kagira2 victor mwadime1 maina ngotho1 affiliations: 1institute of primate research, kenya 2jomo kenyatta university of agriculture and technology, kenya correspondence to: christopher kariuki email: chriskinya@gmail.com postal address: po box 24481, karen 00502, nairobi, kenya dates: received: 27 aug. 2013 accepted: 18 aug. 2015 published: 05 oct. 2015 how to cite this article: kariuki c, kagira jm, mwadime v, ngotho m. virulence and pathogenicity of three trypanosoma brucei rhodesiense stabilates in a swiss white mouse model. afr j lab med. 2015;4(1), art. #137, 8 pages. http://dx.doi.org/10.4102/ajlm.v4i1.137 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. virulence and pathogenicity of three trypanosoma brucei rhodesiense stabilates in a swiss white mouse model in this original research... open access • abstract • introduction • methods    • animals    • trypanosomes    • experimental design    • statistical analyses    • ethical considerations • results    • parasitaemia and gross clinical observations    • parasite virulence and mouse survival    • parasite morphology    • histopathological findings • discussion    • limitations of the study    • recommendations • conclusion • trustworthiness    • reliability and validity of the research • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: a key objective in basic research on human african trypanosomiasis (hat) is developing a cheap and reliable experimental model of the disease for use in pathogenesis and drug studies. objective: with a view to improving current models, a study was undertaken to characterise the virulence and pathogenicity of three trypanosoma brucei rhodesiense stabilates, labelled as international livestock research institute (ilri)-2918, ilri-3953, and institute of primate research (ipr)-001, infected into swiss white mice. methods: swiss white mice were infected intraperitoneally with trypanosomes and observed for parasitaemia using wet blood smears obtained by tail snipping. induction of late-stage disease was undertaken using diminazene aceturate (40 mg/kg, berenil) with curative treatment done using melarsoprol (3.6 mg/kg, arsobal). results: the prepatent period for the stabilates ranged from three to four days with mean peak parasitaemia ranging from log10 6.40 to 8.36. first peak parasitaemia for all stabilates varied between six and seven days post infection (dpi) followed by secondary latency in ilri-2918 (15–17 dpi) and ipr-001 (17–19 dpi). survival times ranged from six dpi (ilri-3953) to 86 dpi (ipr-001). hindleg paresis was observed in both ilri-3953 (at peak parasitaemia) and ilri-2918 (after relapse parasitaemia). mice infected with ipr-001 survived until 54 dpi when curative treatment was undertaken. conclusions: this study demonstrated that the stabilates ilri-2918 and ilri-3953 were unsuitable for modelling late-stage hat in mice. the stabilate ipr-001 demonstrated the potential to induce chronic trypanosomiasis in swiss white mice for use in development of a late-stage model of hat. introduction top ↑ human african trypanosomiasis (hat), or sleeping sickness, is a parasitic infectious disease caused by infection with the haemoprotozoans trypanosoma brucei gambiense (chronic form/gambian hat) or t. b. rhodesiense (acute form/rhodesian hat), two morphologically-identical but epidemiologically-distinct subspecies of t. brucei. both parasites are transmitted cyclically by haematophagous tsetse flies of the genus glossina and are restricted to discrete foci within sub-saharan africa.1,2,3,4,5,6 hat is the archetype of a neglected disease, affecting the poorest people in africa.7 the disease runs an intricate course which can result in death if not promptly managed.8 the disease pathology consists of two distinct stages. during the early (haemolymphatic) stage, trypanosomes are present in the lymphatic fluid and blood, as well as the extravascular spaces of most organs. in the late (meningoencephalitic) stage, parasites are detected within the central nervous system (cns), where they cannot be treated by drugs that are incapable of crossing the blood-brain barrier.3 one of the major objectives in current basic research on hat is the development of a cheap and reliable experimental model of the disease that can be utilised to both increase our understanding of the disease and allow for the development of drug regimens for the management of late-stage hat.9 however, the development of such a model has proven to be difficult as most t. brucei subspecies tend to cause an acute rather than chronic infection in experimental animals.10,11 presently, drug efficacy trials against late-stage hat are performed in both murine models (mus musculus) and in non-human primates such as vervet monkeys (chlorocebus aethiops).9,12,13,14,15,16,17 whilst the vervet monkey model of rhodesian hat has been widely used in a number of drug evaluation and pathogenesis studies,9 it possesses unique disadvantages for the purposes of basic research. primates represent an expensive and ethically-challenging model, whose use is only justifiable under the auspices of avoiding unnecessary human involvement in most studies. on the other hand, most mouse models of rhodesian hat have been extensively developed using the non-human infective t. b. brucei, which may possess dissimilar pathogenicity and drug susceptibility profiles when compared to the human-infective t. b. rhodesiense or t. b. gambiens e.11 in particular, these mouse models, being non-human infective and parasite-based, may not depict accurately the cns involvement by human-infective parasites.10,11,12,15 thus, mouse models for rhodesian hat, particularly those based on t. b. rhodesiense, are essential in order to provide a more correct depiction of the disease's pathogenicity and drug susceptibility profiles.11 as different parasite strains from a particular subspecies may have dissimilar disease progression, it is of interest to perform preliminary studies that compare a number of strains in order to have a proper baseline for future studies involving the selected parasite strains. this study aimed to characterise the virulence and pathology of three t. b. rhodesiense stabilates, international livestock research institute (ilri)-2918, ilri-3953, and institute of primate research (ipr)-001, the target parasites for establishment of the rodent and primate models of hat, in outbred swiss white mice. to the best of the authors’ knowledge, these parasites have not yet been evaluated comparatively. methods top ↑ animals as the pathogenicity and virulence of two of the trypanosome subspecies used were known (ilri-2918 and ilri-3953), available data from previous studies were used to estimate the sample size per group.11,18,19,20 swiss white mice of either sex (n = 108), weighing 25–30 grams, were obtained from the rodent facility at the ipr and provided with mouse feed (mice cubes®, unga feeds, nairobi, kenya) and water ad libitum. the mice were kept in 14 cm x 30 cm x 15 cm macrolone cages. they were maintained at an ambient temperature of between 20 and 25°c and wood shavings (various timber mills, embulbul, kajiado, kenya) were used as bedding. trypanosomes stabilate ilri-2918 was clonally derived from kenya trypanosomiasis research institute (ketri)-2772, which was isolated from a natural infection in a human in alupe, busia, kenya and designated 2772.18 stabilate ilri-3953 was originally referred to as uganda trypanosomiasis organisation (utro)-310185, which was isolated from a naturally infected human in bugiri, uganda in 1982. the parasite has been passaged in mice and designated ilri-3953.19,20 stabilate ipr-001 was obtained from a cerebrospinal fluid sample from an infected human in bugiri, uganda. after three passages in outbred swiss white mice at the ipr, the stabilate was designated ipr-001. for the current study, all stabilates were obtained from storage in liquid nitrogen (–196 °c), thawed and diluted with phosphate saline glucose (psg) buffer (ph 8.0), then injected intraperitoneally (0.2 ml each) into three swiss white female mice that were immunosuppressed via irradiation with caesium chloride at 600 rads for five minutes. upon the onset and rise of parasitaemia, the imunosuppressed mice were euthanised and their blood collected via cardiac puncture. the collected blood was then diluted to a parasitaemia of approximately 104 trypanosomes/0.2 ml using psg, after which the diluted blood was injected intraperitoneally (ip; 0.2 ml each) into the 108 mice included in the study, as described below. six mice were used as positive controls and injected with psg buffer only (0.2 ml ip). experimental design the experiment was designed and executed as indicated in table 1. after infection, experimental mice were observed daily for parasitaemia using wet blood smears obtained by tail snipping. at the same time, thin blood smears were also prepared and giemsa stained for observation of pleomorphism via microscopy, as described by kagira et al.11 the parasitaemia was then estimated using the method described by herbert and lumsden.21 table 1: experimental design. for both ilri-2918 and ipr-001-infected mice, all surviving mice (both experimentally infected and controls) were treated with diminazene aceturate (40 mg/kg ip, berenil®, intervet, south africa) at 21 days post infection (dpi) as described previously by kagira et al.11 at this stage, the parasites were assumed to have invaded the cns. the diminazene aceturate therefore only clears the haemolymphatic trypanosome complement, because it cannot cross the blood-brain barrier in sufficient quantities to clear the cns. mice were then observed daily for relapse parasitaemia using wet smears obtained via tail snipping. relapse detection sensitivity was enhanced by examining the collected blood using the woo method.22 upon relapse of parasitaemia, all surviving mice (both experimentally infected and controls) were treated with melarsoprol (3.6 mg/kg ip, arsobal®, sanofi-aventis, paris, france) for four days consecutively. six mice per stabilate were euthanised at set time points (dpi 7, 14 and 21). all their major organs (heart, liver, spleen, brain, lungs and kidneys) were harvested and perfused with citrate saline buffer via the left ventricle through the hepatic portal vein until all the blood was completely drained. they were then preserved in 10% neutral buffered formalin for histopathology. statistical analyses data were managed and analysed using ms excel (microsoft excel® 2007, microsoft corporation, redmond, washington, united states). the means for parasitaemia and parasite morphology were calculated using the average function in ms excel and variability was calculated using the standard error of the mean function in microsoft excel, ‘= (stdev(a1:a2))/(sqrt(count(a1:a2))’, where a1 represents the first value in the series and a2 represents the last value in the series. ethical considerations all experiments were carried out at the ipr in karen, nairobi, kenya. the experiments were conducted in accordance with protocols approved and authorised by the institutional review committee of the institute. results top ↑ parasitaemia and gross clinical observations in ilri-2918-infected mice, patency was demonstrated at five dpi (figure 1). this was estimated at log10 3.07. all of the patent mice were active and had smooth coats at the onset of patency. first peak parasitaemia was observed at seven dpi (figure 1) and the mean peak parasitaemia was estimated at log10 6.4. secondary latency was observed at 17 dpi (figure 1), with mean peak parasitaemia estimated at log10 4.87. following treatment at 21 dpi with diminazene aceturate, parasitaemia dropped below microscopically-detectable levels between 22 and 28 dpi (figure 1). relapse parasitaemia was observed on 50 dpi. the parasitaemia at the onset of relapse was log10 0.6. figure 1: mean parasitaemia pattern for t. b. rhodesiense ilri-2918 infection in swiss white mice. the first peak occurred at seven dpi; the second peak occurred at 17 dpi. mean parasitaemia at the first peak was estimated at log10 6.4. mean parasitaemia at the second peak was estimated at log10 4.87. following treatment at 21 dpi with diminazene aceturate (40 mg/kg ip, berenil®, intervet, south africa), parasitaemia dropped below microscopically-detectable levels between 22 and 28 dpi. relapse parasitaemia was observed on 50 dpi. the parasitaemia at the onset of relapse was log10 0.6. from 10 dpi, the mice began to develop ruffled coats, lower appetite and lower activity levels. these symptoms occurred at varying levels of parasitaemia, with some groups experiencing the symptoms during the rising phase of parasitaemia and others during the dropping phase of the first parasitaemia peak. peri-orbital oedema also occurred during the rising and dropping phases of parasitaemia, with three mice having one or both eyes shut at all times. these symptoms persisted until the second rising phase of parasitaemia, at which time they disappeared and the mice appeared active and once again had smooth coats. at 26 and 27 dpi, hindleg paresis was observed in two mice. these mice were euthanised and their major organs collected for histopathology. at 27 dpi, after treatment with diminazene aceturate at 21 dpi, the surviving mice again had lowered activity and ruffled coats. the prognosis of these mice, however, improved steadily and the clinical symptoms slowly dissipated, disappearing by 31 dpi. at 50 dpi, all surviving mice relapsed into parasitaemia at log10 0.6 and at 51 dpi, the mice succumbed to hindleg paresis and were subsequently euthanised. in ilri-3953-infected mice, patency was demonstrated from three dpi, with peak parasitaemia observed at seven dpi (figure 2). this was estimated at log10 8.36. all of the patent mice were active and had smooth coats at the onset of patency. mice began to develop ruffled coats, lower activity level and paresis from seven dpi. one mouse also exhibited peri-orbital oedema at peak parasitaemia (log10 8.4; seven dpi). as the parasitaemia rose, mice developed hindleg paresis and were found dead in their cages, with some deaths occurring at six dpi. by 10 dpi, all experimental mice had been euthanised or were found dead in their cages, necessitating a termination of the experiment on this isolate. figure 2: mean parasitaemia pattern for t. b. rhodesiense ilri-3953 infection in swiss white mice. the first peak occurred at seven dpi. mean parasitaemia at the first peak was estimated at log10 8.36. all mice were found dead in their cages or were euthanised by 10 dpi. in ipr-001-infected mice, patency was demonstrated from three to five dpi (figure 3). all of the patent mice were active at the onset of patency. the first peak of parasitaemia was observed at six dpi (log10 8.36) and the second peak parasitaemia occurred at 18 dpi (log10 8.81). ruffled coats, lower appetite and lower activity level were observed from as early as the first parasitaemia peak. after treatment with diminazene aceturate, parasitaemia dropped below microscopically-detectable levels and clinical symptoms abated. relapse parasitaemia occurred at 39 dpi (log10 1.62) and peaked at 54 dpi (log10 6.8). following treatment with melarsoprol, parasitaemia was again observed to drop below microscopically-detectable levels until the termination of the experiment at 86 dpi. figure 3: mean parasitaemia pattern for t. b. rhodesiense ipr-001 infection in swiss white mice. the first peak occurred at six dpi; the second peak occurred at 18 dpi. mean parasitaemia at the first peak was estimated at log10 8.36. mean parasitaemia at the second peak was estimated at log10 8.81. following treatment at 21 dpi with diminazene aceturate (40 mg/kg i.p., berenil®, intervet, south africa), parasitaemia dropped below microscopically-detectable levels between 22 and 25 dpi. relapse parasitaemia was observed to occur at 39 dpi (log10 1.62) and peaked at 54 dpi (log10 6.8). following treatment with melarsoprol (3.6 mg/kg ip, arsobal®, sanofi-aventis, paris, france), parasitaemia was again observed to drop below microscopically-detectable levels until the termination of the experiment at 86 dpi. parasite virulence and mouse survival in ilri-2918-infected mice, survival was constant (100%) up to and after treatment with diminazene aceturate at 21 dpi (figure 4). upon treatment, mice developed hindleg paresis (50% of the experimental population at the time), at which time they were euthanised. upon relapse of parasitaemia, the remaining mice succumbed to the parasite, thereby causing termination of the experiment. figure 4: mean survival for ilri-2918, ilri-3953 and ipr-001 infected mice. in ilri-2918 infected mice, survival was 100% until after treatment with diminazene aceturate at 21 dpi, whereupon 50% developed hindleg paresis and were euthanised with the rest of the population succumbing to the parasite after parasitaemia relapse. in ilri-3953 infected mice, mortality was 100% by 10 dpi. in ipr-001 infected mice, survival was 97% before treatment with diminazene aceturate at 21 dpi, dropping and stabilising at 38% (24–31 dpi), followed by 35% (32–44 dpi), then 31% (45–56 dpi) and finally stabilising after treatment with melarsoprol to 27% (57–79 dpi). all mice surviving after the treatment were euthanised. in ilri-3953-infected mice, mortality increased with the rise in parasitaemia, with mice rendered moribund as the experiment progressed. by 10 dpi, all mice were dead in their cages or had been euthanised after hindleg paresis was observed. in ipr-001-infected mice, mortality occurred even before treatment with diminazene aceturate at 21 dpi. approximately 3% of the experimental population succumbed to the stabilate's parasitaemia, with 97% surviving before treatment with diminazene aceturate at 21 dpi. the survival dropped further through 38% (24–31 dpi), to 35% (32–44 dpi), then 31% (45–56 dpi), before finally stabilising after treatment with melarsoprol to 27% (57–79 dpi), whereupon it remained stable until the end of the experiment. at the end of the experiment, the surviving mice were euthanised and their organs harvested for histopathology. parasite morphology examination of the giemsa-stained thin blood smears indicated that pleomorphism occurred in two of the parasite strains used, namely, ilri-2918 and ipr-001. in these stabilates, two trypomastigote forms – long slender and short stumpy – were observed during the course of infection. from five dpi in ilri-2918 and three dpi in ipr-001, there was a predominance of long slender trypomastigotes (97.4% in ilri-2918 and 83.7% in ipr-001), with few short stumpy trypomastigotes observed (2.6% in ilri-2918 and 16.3% in ipr-001). for both strains, observation of the different morphological forms ceased at 23 dpi after treatment with diminazene aceturate at 21 dpi. in ilri-2918-infected mice, there was an increase in short stumpy forms (18.6%) with a decline in long slender forms (83.7%) (figure 5) during the first parasitaemia peak at seven dpi, compared to the populations at the beginning of the observation period. at eight dpi, there was a great increase in the number of short stumpy trypomastigotes (64.6%), with a decline in the long slender forms (35.4%). at the second peak (at 17–19 dpi), there was a predominance of short stumpy forms (up to 92.4%) and a decline in long slender forms (7.6%). figure 5: parasite morphology pattern for ilri-2918 infection in swiss white mice. during the first parasitaemia peak at seven dpi, there was an increase in short stumpy forms (18.6%) with a decline in long slender forms (83.7%). at the second peak at 17–19 dpi, there was a predominance of short stumpy forms (up to 92.4%) and a decline of long slender forms (7.6%). in ipr-001-infected mice, during the first peak at six dpi, there was a notable increase in the number of short stumpy trypomastigotes (33.6%) and a decline in the long slender forms (66.4%) compared to the populations at the beginning of the observation period (figure 6). in this isolate as well, at seven dpi, there was a great increase in the number of short stumpy trypomastigotes (54.4%) and a decline in the long slender forms (45.6%). at the second peak, at 18 dpi, there was a predominance of short stumpy forms (74.9%) and a decline of long slender forms (25.1%). however, the greatest increase in short stumpy forms during the ipr-001 infection occurred between 20 and 23 dpi (from 94.6% – 95.2%). figure 6: parasite morphology pattern for ipr-001 infection in swiss white mice. during the first peak at six dpi, there was a notable increase in the number of short stumpy trypomastigotes (33.6%) and a decline in the long slender forms (66.4%) compared to the populations at the beginning of the observation period. at the second peak at 18 dpi, there was a predominance of short stumpy forms (74.9%) and a decline of long slender forms (25.1%). histopathological findings gross anatomy observation revealed enlargement of the spleen and hyperaemia in the major organs. histopathological observations of the brain included discrete inflammation, with the foci of meningitis characterised by progressive mononuclear cell infiltration, perivascular cuffing (figure 7) and hyperaemia of meningeal blood vessels. these observations were seen to progress over time in all observed samples from experimental mice. infiltration of the brain parenchyma with mononuclear cells was rare and only began to be prominent in the late stages of infection. the choroid plexus was prominent in infected mice and contained inflammatory exudates, mainly of mononuclear cells. figure 7: perivascular cuffing (indicated by the shaded arrow) in a parasite-relapse mouse brain sample. in all groups of experimental mice, most organs were characterised by interstitial mononuclear cell infiltration into the parenchyma and perivascular cuffing. the most affected organs were the liver, spleen, heart and lungs. there were no marked differences between ilri-2918 and ipr-001-infected mice. the germinal centres of the spleen were active and contained many immature lymphocytes and plasma cells, especially in samples obtained at 14 and 21 dpi, although these cells were reduced in number by 28 dpi. the lung alveoli, as well as the interseptal spaces, were characterised by infiltration with inflammatory exudates and cells, including lymphocytes, macrophages and a few neutrophils. this was more prominent in samples from mice euthanised after 21 dpi. the liver indicated extensive lymphocytic infiltration coupled with necrosis of hepatocytes, especially in mice euthanised after 28 dpi. discussion top ↑ of the three stabilates investigated, we found that two possessed the necessary parasitaemic characteristics of a typical hat infection. both ilri-2918 and ipr-001 had a mean pre-patent period and parasitaemia pattern similar to that reported by other researchers.11 the survival times for these two stabilates and the pathological changes they induced in experimental animals demonstrated that these parasite strains are capable of causing a chronic infection. this is a feature particularly useful in models for drug efficacy testing. some of the symptoms observed in mice infected with ilri-2918 and ipr-001, particularly peri-orbital oedema, have also been described in higher primate models of hat.13,23 the deaths occurring at 31 dpi for ilri-2918 and at 21 dpi for ipr-001 could have resulted from an immunological crisis arising after massive trypanolysis occasioned by treatment with diminazene aceturate. the mortality and/or morbidity observed in trypanosome-infected mice could be attributed primarily to self-inflicted damage by a disproportionate immune and/or innate response.24 mortality and morbidity could also be caused by the immuno-depressant capabilities of a trypanosome infection on a mouse strain. this may have led to opportunistic infections, leading to the death of mice infected with a trypanosome strain.25 in this study, the parasitaemia pattern showed a predominance of two different morphological types of trypomastigotes, namely, short stumpy and long slender forms. in both ilri-2918 and ipr-001 infections, there was a predominance of short stumpy forms during or shortly after peak parasitaemia. this phenomenon was observed at both parasitaemia peaks and appeared to be alleviated by treatment with diminazene aceturate treatment. however, for both strains, the situation was reversed during the rising phase of the parasitaemia. in this case, the short stumpy form declined and the long slender form predominated. this observation is in line with what has been reported by others.11 pleomorphism is considered a pre-adaptation to transmission to the tsetse fly, with long slender, multiplicative forms being transformed into short stumpy, non-multiplicative forms capable of survival in the tsetse fly midgut.26 this process, which is a host antibody-independent response, is probably mediated by a density-sensing mechanism.27 the pathological changes described in most organs appeared to be characteristic of t. brucei infections. unlike most other trypanozoon genus members, t. brucei is particularly tissue invasive, causing cellular infiltration into the parenchyma of most organs.11,13 the cellular reactions we observed in most organs showed that a lymphoid immune reaction is critical in the pathogenesis of t. b. rhodesiense infections, as described previously by others.11,13,28 in the brain, it has been shown that the inflammatory reaction begins as meningitis and progresses into the virchow-robin spaces before spreading to the parenchyma.11 whilst the successful induction of the meningoencaphalitic stage was assumed here (because of the relapsing parasitaemia after treatment with diminazene aceturate, which clears only the haemolymphatic component of the parasite), further proof in terms of demonstration of trypanosomes within the cns, coupled with appearance of anti-trypanosomal antibodies as described by previous studies,29,30 would greatly improve the value of the reported model. in addition, the role played by the particular mouse strain can influence the disease progression and pathology encountered. this has been shown by others, with certain strains such as balb/c mice showing the greatest susceptibility, whereas c57bl/6 mice show the greatest tolerance.25,31 given that the mice used in this study were classified as outbred swiss white, there could be a significant deviation, particularly in the disease pathology, when the model is established with inbred strains. most murine models of trypanosomiasis for drug efficacy trials rely on the assumption that trypanosomes have invaded the brain parenchyma by 21 dpi.10,21 in this study, the same assumption was made with subcurative treatment with diminazene aceturate. relapses occurred in both ilri-2918and ipr-001-infected mice, coupled with severe meningoencephalitis as has been reported previously.10,11,32 limitations of the study as described in the preceding section, there were a number of limitations observed which can be summarised as follows. firstly, the strain of mouse greatly influences the disease progression and pathology encountered. there could thus be deviations if the model is established using a different mouse strain, particularly an inbred one. secondly, the study assumed the occurrence of cns involvement by relapse of parasitaemia after haemolymphatic clearance with diminazene aceturate, as has been done in previous studies. however, assessment of plasma concentrations of acute-phase protein, c-reactive protein and haptoglobin could also be used as a marker for experimental infections.16,33 thirdly, it is difficult to interpret brain pathology in terms of relapse following treatment with trypanocidals, as they, too, have been shown to induce brain pathology. another point of contention when using trypanocidals to stage the disease is the danger that subcurative treatment of a parasitic infection may lead to emergence of drug resistance, a position which would complicate the interpretation of efficacy data if the drug under investigation has a similar mode of action.11 finally, the parasites used in this study have not yet been characterised genetically. recommendations in order to circumvent the preceding limitations, an initial baseline study is required for any novel mouse model utilising the described parasite isolates, incorporating more extensive measures of parasitaemia as well as parasite staging. full genetic characterisation of the parasites used here is needed in order to ascertain the identity of the isolates during future experiments and in order to account for variations observed in subsequent studies. because of the heterogenous nature of outbred swiss mice, there may be significant deviations in results from mouse studies using the same isolates in the future. for this reason, studies using the more homogenous inbred mouse strains, which may not be susceptible to the isolates used here, are recommended in order to establish the late-stage hat model. conclusion top ↑ in conclusion, this study investigated infection with three different t. b. rhodesiense strains, ilri-2918, ilri-3953 and ipr-001, which led to the occurrence of acute, hyper-acute and chronic infections, respectively. ilri-2918 showed indications of causing a chronic infection in mice; however, its relapse parasitaemia induced hindleg paresis and death. this strain of the parasite was therefore classified as causing an acute infection in mice and was regarded by the investigators as being unsuitable for the development of a late-stage model of hat. ilri-3953 was observed to be highly virulent, with all the experimental mice found dead in their cages or humanely euthanised by 10 dpi. upon further passaging in immunocompetent swiss white mice (unpublished data), the parasite showed no indications of reducing its virulence. therefore, the parasite was classified as hyper-acute and unsuitable for the development of a late-stage model of hat. ipr-001, a field-isolated parasite, showed indications of causing a chronic infection in mice. the first peak of parasitaemia rose to log10 8.36 at six dpi. after subcurative treatment, mice relapsed and survived in good physical condition/health until curative treatment. this parasite was therefore classified as causing chronic infection and is proposed as the parasite of choice for further work in developing a late-stage model of hat. trustworthiness top ↑ in as far as the authors are concerned, and in view of the outcome of the t. b. rhodesiense infections in the mouse studies, the results obtained in this study appear to compare well with those arising from t. b. brucei and other related studies. reliability and validity of the research the authors consider the experimental design of this study to be reliable and valid for the purpose of establishing a mouse model in outbred swiss white mice. the procedures utilised in this study have been tested and validated in other studies as cited in this article. acknowledgements top ↑ this project was funded by the director of the ipr. the authors are grateful to the technical assistance provided by alex gaithuma, thomas adino and claire n. kimani of the department of animal sciences, ipr. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions m.n. (ipr) was the project leader and, along with j.m.k. (jomo kenyatta university of agriculture and technology), was in charge of animal welfare. m.n., c.k. (ipr) and j.m.k. were responsible for the experimental and project design. c.k., v.m. (ipr) and j.m.k. performed the experiments, whilst c.k. and j.m.k. performed the data analysis. all authors participated in the writing of the manuscript. references top ↑ kennedy pge. human african trypanosomiasis of the cns: current issues and challenges. j clin invest. 2004;113(4):496–504. http://dx.doi.org/10.1172/jci200421052 kennedy pge. diagnostic and neuropathogenesis issues in human african trypanosomiasis. int j parasitol. 2006;36(5):505–512. http://dx.doi.org/10.1016/j.ijpara.2006.01.012 kennedy pge. the continuing problem of human african trypanosomiasis (sleeping sickness). ann neurol. 2008;64(2):116–126. http://dx.doi.org/10.1002/ana.21429 fèvre em, odiit m, coleman pg, et al. estimating the burden of rhodesiense sleeping sickness during an outbreak in serere, eastern uganda. bmc public health. 2008:8:96. http://dx.doi.org/10.1186/1471-2458-8-96 simarro pp, jannin j, cattand p. eliminating human african trypanosomiasis: where do we stand and what comes next? plos med. 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eckersall pd, jennings fw. elevation of the concentration of acute phase proteins in dogs infected with trypanosoma brucei. acta trop. 1991;49(2);77–86. abstract introduction methods results discussion acknowledgements references about the author(s) lindi-marie coetzee national priority programme, national health laboratory service, charlotte maxeke johannesburg academic hospital, johannesburg, south africa department of molecular medicine and haematology, university of the witwatersrand, johannesburg, south africa naseem cassim national priority programme, national health laboratory service, charlotte maxeke johannesburg academic hospital, johannesburg, south africa department of molecular medicine and haematology, university of the witwatersrand, johannesburg, south africa deborah k. glencross national priority programme, national health laboratory service, charlotte maxeke johannesburg academic hospital, johannesburg, south africa department of molecular medicine and haematology, university of the witwatersrand, johannesburg, south africa citation coetzee l, cassim n, glencross dk. newly implemented community cd4 service in tshwaragano, northern cape province, south africa, positively impacts result turn-around time. afr j lab med. 2022;11(1), a1376. https://doi.org/10.4102/ajlm.v11i1.1376 original research newly implemented community cd4 service in tshwaragano, northern cape province, south africa, positively impacts result turn-around time lindi-marie coetzee, naseem cassim, deborah k. glencross received: 04 sept. 2020; accepted: 25 feb. 2022; published: 03 june 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the northern cape is south africa’s largest province with an hiv prevalence of 7.1% versus a 13.5% national prevalence. cd4 testing is provided at three of five national health laboratory service district laboratories, each covering a 250 km precinct radius. districts without a local service report prolonged cd4 turn-around times (tat). objective: this study documented the impact of a new cd4 laboratory in tshwaragano in the remote john taolo gaetsewe district of the northern cape, south africa. methods: cd4 test volumes and tat (total, pre-analytical, analytical, and post-analytical) data for the northern cape province were extracted for june 2018 to october 2019. the percentage of cd4 results within the stipulated 40-h tat cut-off and the median and 75th percentiles of all tat parameters were calculated. pre-implementation, samples collected at tshwaragano were referred to kimberley or upington, northern cape, south africa. results: pre-implementation, 95.4% of samples at tshwaragano were referred to kimberley for cd4 testing (36.3% of kimberley’s test volumes). only 7.5% of tshwaragano’s total samples were referred post-implementation. the tshwaragano laboratory’s cd4 median total tat decreased from 24.7 h pre-implementation to 12 h post-implementation (p = 0.003), with > 95.0% of results reported within 40 h. the kimberley laboratory workload decreased by 29.0%, and testing time significantly decreased from 10 h to 4.3 h. conclusion: the new tshwaragano cd4 service significantly decreased local tat. upgrading existing community laboratories to include cd4 testing can alleviate provincial service load and improve local access, tat and efficiency in the centralised reference laboratory. keywords: cd4 testing; district laboratory; turn-around-time; immunology; hiv; operational science. introduction the northern cape province occupies 30.0% of the land area of south africa (372 889 km2) yet is the most sparsely populated due to the arid climate, with a population of 1.26 million people (3.2 people/km2), making up only 2.2% of the national population. the province consists of five districts (the respective main towns where the district seats are situated are indicated in brackets): namakwa (springbok), pixley ka seme (de aar), zwelentlanga fatman mgcawu (upington), frances baard (kimberley), and john taolo gaetsewe (tshwaragano, previously known as kuruman) (figure 11,2). distances between major towns range from 236 km (drive time: 2 h and 24 min) to 775 km (drive time: 7 h and 10 min). figure 1: districts and sub-districts serviced by the community cd4 testing facilities in the northern cape province of south africa (september 2020). (a) major towns (b) major roads between district seats (towns). in 2019, the northern cape reported an hiv prevalence of 7.1% (i.e. 81 778 people living with hiv), with an estimated 90.0% of patients diagnosed and 83% on anti-retroviral treatment (art).3 this hiv prevalence is significantly lower than the national prevalence of 13.5% and at the lower end of provincial estimates, ranging from 18.2% (2 029 470 people living with hiv) in kwazulu-natal to 6.8% (452 210 people living with hiv) in the western cape province. the northern cape province has the lowest reported density of people living with hiv (0 per 5 km2) compared to gauteng which has over 13 000 people living with hiv per 5 km2.3 although it has the most healthcare facilities per 1000 people living with hiv (4.5), more than 10.0% of the population live more than a 2-h drive away from their nearest facility.1 in south africa, a universal ‘test-and-treat’ approach was adopted for the diagnosis and treatment of hiv/aids patients.4,5,6,7,8,9 late hiv presentation and a high disease burden8,9,10,11 remain challenges in the south african hiv programme. cd4 testing, although no longer required for eligibility for antiretroviral therapy initiation, still plays a valuable role in the identification of hiv-positive patients with advanced disease (cd4 counts < 200 cells/µl) or very advanced disease (cd4 counts < 100 cells/µl).4,5,6,7,8,10,12,13,14,15,16,17,18,19,20,21 the local standard of care in south africa recommends patients returning for care within seven days of hiv diagnosis.12 cd4 counts from this visit are used to fast-track patients onto antiretroviral therapy and for the treatment of other hiv-related opportunistic diseases such as tuberculosis and cryptococcal disease.15,18,22 in south africa, cd4 services are offered through the national health laboratory service (nhls) at 46 testing laboratories placed strategically to service public healthcare facilities consisting of hospitals and primary healthcare clinics in both urban and rural settings. in 2019, 2.8 million cd4 tests were performed across south africa, with 2.2% done in the northern cape province, compared to 34.0% in kwazulu-natal and 21.0% in the gauteng province. the 2020 mid-year population statistics and annual nhls cd4 testing data (2019) reported that an average of 4.4% of all south africans received a cd4 test, with 4.7% in the northern cape compared to 8.0% per capita in the kwazulu-natal province of south africa.23 as only 46 of the 268 nhls laboratories provide a cd4 testing service, samples are not always tested locally. all samples get registered onto the nhls laboratory information system (lis) upon arrival at one of these laboratories, even if cd4 testing is not provided. for this paper, two types of sample referrals are described: one where samples from primary health care facilities and hospitals within respective service precincts are received and registered at smaller, non-testing source laboratories and referred to a cd4 testing laboratory (inter-laboratory referral), and another where samples from hospitals and primary healthcare facilities within the immediate service precinct or catchment area are received, registered and tested at the same laboratory. before 2019, three cd4 testing laboratories serviced the entire northern cape province, with kimberley as the local referral testing site for the region (frances baard district) receiving samples via inter-laboratory referral or directly from its sister hospitals and other clinics within the direct service precinct. the de aar cd4 laboratory (pixley ka seme district) was implemented in 2012,24 and the upington cd4 laboratory (zwelentlanga fatman mgcawu district) in the following year, after identifying gaps in service by the integrated service delivery model.25 cd4 tests from the john taolo gaetsewe district (with tshwaragano as the seat town) were primarily referred to the kimberley laboratory. the tshwaragano laboratory is 236 km from kimberley, 266 km from upington, and 480 km from the de aar laboratory (figure 1) and provides general pathology services to 38 public health facilities within a 200 km service precinct radius. laboratory service efficiency and overall performance are typically measured by turn-around time (tat).26,27,28,29,30 in the nhls, total tat is reported and includes pre-analytical or laboratory-to-laboratory tat, that is from first registration onto lis to sample receipt at the testing laboratory; the analytical registered-to-test tat component, reporting the time between sample receipt/registration at the testing laboratory to test result entry into the lis,28,31 and the post-analytical tat, that is the time from result entry at the testing laboratory to authorisation/review by a senior technologist (test-to-review). tat components that are currently not captured include the time from venepuncture to sample dispatch, travel time to the nearest nhls laboratory, potential delays at the receiving laboratory before sample registration, and delays of report delivery to the requesting site after the review/release of results. these factors are incorporated in the nhls cd4 tat guidelines, requiring that 90% of cd4 results be reported within 40 h of first registration onto the lis to adhere to the local standard of care (return within seven days). in april 2018, a routine internal review of annualised provincial tat revealed that the burden of cd4 testing on the kimberley laboratory could be alleviated and service delivery improved by further decentralising cd4 testing to tshwaragano, john taolo gaetsewe district (data not published). this study aimed to document the impact of implementing a cd4 testing service in the existing tshwaragano laboratory on tat as a measure of laboratory performance. the preand post-implementation total and component cd4 testing tat and the percentage of samples reported within the nhls-stipulated tat threshold of 40 h are reported for tshwaragano and the centralised testing laboratory in kimberley, northern cape, south africa. methods ethical considerations ethics clearance was obtained from the university of the witwatersrand (m1706108, approved for five years from 13 july 2017). this was a retrospective study using cd4 sample data from the corporate data warehouse, with no patient consent required and no patient identifiers captured. study design a cross-sectional study design was used to analyse tat data from cd4 testing laboratories in kimberley, upington, de aar and tshwaragano, northern cape, south africa. only data with a reviewed cd4 result between june 2018 to october 2019 were analysed. cd4 testing at all of the northern cape laboratories was done using the beckman coulter aquios cl (continuous loader) cytometer system (beckman coulter, miami, florida, united states).32 this system is ideal for low to medium-high test volumes (10–120 samples per day) and is user-independent, with sample preparation and analysis performed in a closed system requiring minimal operator hands-on time, an especially useful feature for sites with limited dedicated/skilled staff. the aquios cl platform performance correlated well with previously used systems such as the coulter epics xl-mcl™33 and fc500 multi-plate loader (mpl)34 as per validations done before the national roll-out35,36 and instrument verifications done per testing site as part of the implementation process.37,38,39 data preparation data were extracted from the central data warehouse of the nhls as password-protected files. the extracted data included the following variables: episode number, referral laboratory, testing laboratory, facility name, province, health district, sub-district, date first registered on the lis, date tested, date reviewed (result released), total tat, laboratory-to-laboratory tat, registered-to-test tat, and test-to-review tat. data were categorised as ‘not referred’ where the referral and testing laboratory was the same and as ‘referred’ where the testing and referral laboratories differed. data with invalid dates and a total tat > 120 h or < 1 h were excluded. a total tat of 1 h is set as the minimum analytical acquisition and reporting time on the aquios platform, while a total tat > 120 represented the upper limit of the cd4 testing window (five days), beyond which a specimen is no longer suitable for analysis.40,41 data were categorised to distinguish between the pre-implementation (june 2018 to october 2018) and post-implementation (november 2018 to october 2019) periods; the month of implementation (october 2018) was categorised as pre-implementation as samples were still being referred to kimberley. implementation procedures at the tshwaragano laboratory all of the procedures undertaken to ensure that the tshwaragano laboratory was ready to offer quality cd4 testing were documented. typically, a site inspection was done by the service provider and an on-site operational checklist was completed by a cd4 trainer to assess requirements for implementation. several readiness checks were confirmed before the commencement of instrument implementation, training, verification, and reportable patient testing (figure 2). figure 2: summary of the standardised process for establishing new cd4 testing facilities in the national health laboratory service, south africa, september 2020. satisfaction questionnaire a likert scale satisfaction survey was circulated to the 15 health facilities with the highest test volumes referred to tshwaragano laboratory for cd4 testing. on a scale from ‘1’ (poor) to ‘5’ (very good), the facility manager/senior nurse at these clinics rated the overall service rating, average tat and cd4 test result delivery of the cd4 service pre-and post-implementation of local cd4 testing at the tshwaragano laboratory. data analysis and statistics microsoft excel (redmond, washington, united states) and sas® statistical software 9.4 (cary, north carolina, united states) were used to analyse the data. graphs were created with graphpad prism software, version 7.0 for windows (graphpad software, san diego, california, united states). pre-implementation of the cd4 testing laboratory at tshwaragano, the overall and monthly number of referred samples tested by the three then active cd4 laboratories in the region (kimberley, de aar and upington) were assessed. tat parameters to determine laboratory performance/efficiency included the total tat, the interquartile range (iqr) of the total tat, the percentage of all tested samples within the nhls-stipulated 40-h tat cut-off, and the median tat for the three tat components. due to the non-gaussian distribution of tat data,28 the median and 75th percentile of total tat was calculated and reported for this study; this was to allow the analysis of the majority of samples without the influence of outliers or, in gaussian terms, the long right tail, that is cd4 samples with a total tat exceeding 120 h (typically outside the 99th percentile). the monthly median and 75th percentile total tat for the tshwaragano and kimberley laboratories were reported. preand post-implementation test volumes and tat parameters (total tat and tat components) were compared at the provincial level (all northern cape results) and individual laboratory level using either the unpaired, non-parametric mann-whitney analysis for two groups or the unpaired, non-parametric kruskal-wallis test for three or more groups. p-values of < 0.01 were considered statistically significant and p < 0.001 highly significant. results exclusion criteria identified 10 131 samples (1.5%) with a total tat exceeding 120 h and two samples with a total tat of less than 1 h. after applying the exclusion criteria, cd4 data for 83 232 samples collected during the study period were analysed. of all cd4 samples tested in the northern cape province, the median cd4 count was 437 cells/µl (iqr: 237–658 cells/µl), the median total tat was 19.8 h (iqr: 11.9–31.9 h), and 96% of all tested samples were within the nhls-stipulated 40-h tat cut-off. preand post-implementation cd4 test volumes during the pre-implementation and implementation months (5 months), 23 754 cd4 tests were performed across the province: 75.9% (n = 18 030) at the kimberley laboratory, 12.6% (n = 2988) at de aar, and 11.5% (n = 2736) at the upington laboratory (figure 3). there were 13 714 samples registered at the tshwaragano laboratory during the study period, of which 95.4% were referred to and tested at the kimberley laboratory, and 4.6% were referred to and tested at the upington laboratory. the 12-month follow-up post-implementation period reported 59 478 samples tested across the four laboratories. of these, 42 879 (72.1%) were tested at the existing laboratories: kimberley (n = 30 575; 51.4%), de aar (n = 6356; 10.7%) and upington (n = 5948; 10.0%). the newly implemented tshwaragano cd4 laboratory tested 27.9% (16 599) of the total northern cape cd4 samples over the 12-month post-implementation period, becoming the second-largest testing site in the northern cape. overall, a slight increase in total provincial cd4 test numbers of 4% per month was noted post-implementation (figure 3). figure 3: total cd4 test volumes of laboratories in the northern cape, south africa, june 2018 to october 2019. the red rectangle indicates the implementation month of october 2018. the red dotted line shows total test volumes per month, with the linear trend line (black dotted line) and equation indicated. referral patterns during the preand post-implementation periods during the pre-implementation phase, 32.9% (7829/23 754) of all cd4 tests done in the northern cape province were referred from source laboratories (where samples were first registered on the nhls lis), while the remaining tests (n = 15 925; 67.0%) were registered and tested at the same laboratory. at the kimberley laboratory, 62.7% (n = 11 296) of samples tested were from local hospitals and health facilities. referred source laboratory samples tested at this laboratory came from the tshwaragano (36.3%; 6541), upington (0.7%; 131) and de aar (0.3%; 57) laboratories (table 1). five samples (0.02%) were referred from outside of the northern cape provincial borders. the de aar and upington laboratories tested most of the samples from their local precincts, contributing 75.8% (n = 2266 of 2988; de aar) and 86.4% (n = 2363 of 2736; upington) of their workloads. samples were only referred to kimberley by these laboratories due to local instrument or network downtime. table 1: cd4 testing workload at the four precinct laboratories in the northern cape province, south africa, during the pre-implementation and post-implementation of the tshwaragano cd4 laboratory between june 2018 and october 2019. post-implementation, 91.97% of all cd4 test samples received at the tshwaragano laboratory (n = 16 599) were tested locally (n = 15 266), with 1250 samples referred to the kimberley laboratory (7.5%) and 83 samples referred to the upington laboratory for testing (0.5%). for the same period, the kimberley cd4 workload was made up almost entirely of locally received samples (92.2%) (table 1); only 7.8% of their total cd4 test volumes (n = 2385 samples of 30 575) were received from referring laboratories, that is tshwaragano (4.1%), upington (2.1%) and de aar (1.6%). the proportion of local samples tested at the kimberley laboratory thus increased by 29.5% between the preand post-implementation periods, while the average number of tests performed monthly decreased from 3606 pre-installation to 2547 per month post-installation (29.4%). the overall median cd4 count pre-implementation was 442 cells/µl: 397 cells/µl in upington, 419 cells/µl in kimberley and 451 cells/µl at the de aar laboratory. the differences in median cd4 count between the laboratories were not clinically significant. the post-implementation overall median cd4 count was 436 cells/µl: 406 cells/µl in upington, 440 cells/µl in kimberley and tshwaragano, and 427 cells/µl at the de aar laboratory. differences in median cd4 count preversus post-implementation for the northern cape province were not statistically significant (kruskal-wallis, p = 0.139). turn-around time: preversus post-implementation the median total tat across the four northern cape province testing laboratories during the whole testing period was 19.8 h (iqr: 11.9–31.9 h). the pre-implementation phase median total tat for the three laboratories was 21.65 h (iqr: 16.7–34.1 h) compared to 18.5 h (iqr: 10.9–31.4 h) in the post-implementation phase across four laboratories (mann-whitney p = 0.0001, highly significant). for the tshwaragano laboratory, there was a significant decrease in median total tat from 24.7 to 12 h preversus post-implementation (mann-whitney p = 0.0009), mainly due to a sharp decline in the median testing time (registered-to-test) tat from 14 h to 3 h (figure 4). comparatively, the median total tat at the kimberley laboratory only decreased from 23.0 h to 22.2 h (not significant), although their analytical phase (registered-to-test) tat significantly decreased from 10 h to 4.3 h. for the new tshwaragano laboratory, 75th percentile total tat values were monitored monthly (figure 5) and observed to decrease significantly to below the stipulated cut-off of 40 h, stabilising around 32 h after july 2019. the proportion of samples reported within the stipulated 40-h threshold at tshwaragano changed significantly from 84.0% (pre-implementation) to 92.5% post-implementation. since november 2019, the efficiency of the tshwaragano laboratory has improved, with > 95.0% of tested samples within the nhls-stipulated 40-h tat threshold month-on-month (as per ongoing monthly tat dashboard reports). the kimberley, de aar and upington cd4 laboratories were able to sustain a 75th percentile total tat of < 40 h, with > 90.0% of samples meeting the < 40-h cut-off monthly after the reviewed period. figure 4: cd4 testing turn-around time components at the tshwaragano and kimberley laboratories in the northern cape, south africa during the preand post-implementation months (june 2018 to october 2019). lab-to-lab: time from first registration on the laboratory information system to sample receipt in the testing laboratory; reg-to-tst: time from registration/receipt at the testing laboratory to test result entry on the laboratory information system; tst-to-rvw: time from test result entry to verification by a senior staff member and release to requesting clinic/physician. figure 5: cd4 testing median total turn-around times at the tshwaragano laboratory in the northern cape province of south africa from june 2018 to october 2019. the orange rectangle indicates the month of cd4 implementation at the tshwaragano laboratory and the solid red line shows the organisational cd4 cut-off of 40 h. the test-to-review tat component did not impact the total tat as all laboratories reported tat times < 1 h (median: 10 min). questionnaire results the pre-implementation assessment score was 3 (performance rated as average), with delayed result delivery being the most common reason for the rating provided. the post-implementation score was 5 (very good), with faster result delivery (most within 24 h) being the most cited improvement. respondents observed that patients returned earlier than the stipulated seven days to collect their results (within 2–4 days). nonetheless, feedback received highlighted ongoing challenges of poor connectivity that affected access to results on the internet-based lis and delivery of sms printer results in remote outlying facilities. discussion this paper describes the positive impact of the implementation of a new cd4 testing facility in the tshwaragano laboratory of the northern cape, south africa, and the subsequent improved cd4 testing tat in this region. the implementation of the tshwaragano laboratory positively impacted the region’s referral laboratory at kimberley, where average monthly workload volumes decreased by 29.4 %, enabling a better workflow and more efficient service delivery. post-implementation of cd4 testing services at the tshwaragano laboratory, all the testing sites, that is the three community cd4 laboratories at de aar, upington and tshwaragano, and the referral laboratory at kimberley, could focus solely on managing their local precinct workload, with > 85.0% of samples registered and tested on-site per laboratory. expectedly, no significant changes were noted in the median cd4 count overall and per laboratory as the patient base accessing care remained unchanged. the most dramatic impact was seen in the testing phase (registered-to-test) tat, which decreased significantly at both the tshwaragano (14 h to 3 h) and kimberley (10 h to 4 h) laboratories, contributing to the overall decrease in median total tat at these laboratories. this could be attributed to a better-organised workflow, where all samples are collected, registered and available in the laboratory by the start of the testing shift (night). this is especially relevant as referred samples often get set aside and tested only after local sample testing is done. the average daily test volumes for the tshwaragano and kimberley laboratories (< 160 samples per day) are well within the capacity of the testing platform (aquios) and the number of instruments per laboratory, resulting in better instrument utilisation. the test-to-review tat component at all testing laboratories was < 1 h and did not impact total tats, that is, there were no delays associated with waiting for qualified staff members to review results. further streamlining of this process was made possible by the introduction of auto-review in march 2020.42 there was a slight increase (~4%) in the monthly average cd4 tests conducted in the northern cape province during the pre-and post-implementation periods. the latter could be attributed to the availability of local testing at tshwaragano and the addition of six additional referring clinics (personal communication with laboratory management) since the commencement of cd4 services. the outcomes of the satisfaction rating questionnaire were a testament to the success of this implementation, with an overall post-implementation rating of 5 (vs 3 pre-implementation). improved local cd4 report delivery (due to shortened local tat) could see patients return for their results earlier than the stipulated seven days. this is especially relevant in patients with severe hiv disease (cd4 < 100 cells/µl) to enable the fast-tracking of serum cryptococcal antigen testing and rapid initiation of treatment in cryptococcal antigen-positive cases. as monitored through the nhls weekly/monthly tat dashboard reports, the tshwaragano laboratory maintained a median total tat and 75th percentile total tat of < 20 h for the last financial year (april 2020 to march 2021), with > 98% of samples tested within the 40-h cut-off. this study supports the findings of previous studies24,28,31,43 and demonstrates how decentralising cd4 testing services to district or community facilities according to a tiered service delivery model25 can improve the tat at the decentralised site. smaller community laboratories with existing laboratory infrastructure offering more generalised pathology services can reliably implement cd4 services, effectively eliminating delays due to the transport of samples historically sent to centralised testing facilities. nhls community laboratories are usually located in the seat of the respective districts and generally serve large service precincts, typically supporting up to 40 or more health facilities. with minimal training effort and cost25,43 (benchtop installation, installation of lis interface with instruments, staff training, and testing platforms on national service level agreement), laboratories can be upgraded to include cd4 testing in their repertoire of services. decentralised services also improve coordinated technical efforts across the parent province (the northern cape in this instance), where cd4 laboratories can, in line with the integrated service delivery model service plan,25 operate as a consolidated network with two-way support between the main reference laboratory (in kimberley) and sister community laboratories, thereby ensuring uninterrupted self-sustaining service. limitations the reported tat only reflects laboratory tat and not the full sample journey from a patient to a result-in-hand, which may impact patient management (i.e., leading to patients returning for results, loss to follow-up, and/or delays in the initiation of therapy). however, this paper aimed to show the impact on tat at the laboratory level and did not assess the impact on patient management. conclusion using existing infrastructure, upgrading an existing district nhls laboratory to include cd4 testing is possible with minimal intervention. district cd4 testing laboratories can service their local health clinics and hospitals without delays due to sample referral, reducing the total tat and allowing earlier patient support/intervention. the implementation of a fourth district cd4 testing facility in the northern cape province resulted in the local testing of > 90% of samples from their catchment areas while maintaining excellent performance. acknowledgements the authors thank neo mokone and sithembile mojalefa for their assistance in the implementation and validation of the instrument, training of staff and ongoing support of this laboratory. they also re-visited clinics with the questionnaires for feedback after implementation. thank you to the nhls management and national department of health hiv coordinators and clinic staff for their ongoing support. competing interests the authors declare no conflict of interest nor any financial or personal relationships that may have inappropriately affected the content/publication of this article. authors’ contributions l.c. made substantial contributions to the conception and design of the work, acquisition of laboratory data, drafting the work and revising it critically for important intellectual content. n.c. made substantial contributions to the data analysis and revising the work critically for important intellectual content. d.k.g. revised the work critically for important intellectual content as well as overall supervision. all authors read and approved the final manuscript. sources of support the authors received no financial support for the research, authorship, and/or publication of this article. data availability the authors do not have permission to share the raw data, but summaries of data used for figures in this publication are available from the corresponding author, l.-m.c. disclaimer the views expressed here are those of the authors and not necessarily those of their sponsor and employer, the south african national 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https://doi.org/10.4102/ajlm.v7i1.665 valenstein pn, emancipator k. sensitivity, specificity, and reproducibility of four measures of laboratory turnaround time. am j clin pathol. 1989;91(4):452457. https://doi.org/10.1093/ajcp/91.4.452 hawkins rc. laboratory turnaround time. clin biochem rev. 2007;28(4):179–194. cassim n, tepper me, coetzee lm, glencross dk. timely delivery of laboratory efficiency information, part i: developing an interactive turnaround time dashboard at a high-volume laboratory. afr j lab med. 2020;9(2):947. https://doi.org/10.4102/ajlm.v9i2.947 beckman coulter. aquios cl flow cytometry system [homepage on the internet]. indianapolis, in: beckman coulter life sciences; 2020 [cited 2020 nov 20]. available from: https://www.beckman.co.za/flow-cytometry/instruments/aquios-cl beckman coulter. coulter® epics® xl™ flow cytometer; coulter epics xl-mcl™ flow cytometer; system ii™ software [homepage on the internet]. reference. ca. 2010 [cited 2021 oct 28]. available from: 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technology congress (smltsa); 25–27 oct; bloemfontein; 2019. cassim n, coetzee lm, schnippel k, glencross dk. estimating implementation and operational costs of an integrated tiered cd4 service including laboratory and point of care testing in a remote health district in south africa. plos one. 2014;9(12):e115420. https://doi.org/10.1371/journal.pone.0115420 ajlm 5_1.indb reviewer acknowledgement open accesshttp://www.ajlmonline.org page 1 of 1 we would like to take this opportunity to thank all of those who provided scientific and logistical support for this issue of the african journal of laboratory medicine: ajlm african journal of laboratory medicine in an effort to facilitate the selection of appropriate peer reviewers for the african journal of laboratory medicine, we ask that you take a moment to update your electronic portfolio on www. ajlmonline.org for our files, allowing us better access to your areas of interest and expertise, in order to match reviewers with submitted 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lecher segundo r. leon christophe longuet robert n. maina elisabet manasanch zirra m. mangoro sharon martin teferi mekonen abera william ampofo stephen balinandi allen bateman debrah i. boeras eileen burd abhijit chaudhury kalyani daita bakary drammeh dennis ellenberger glen fine peter fonjungo george gachara reba kanungo yenew kebede andrea kim charles kiyaga stephania koblavi deme kekoura kourouma elizabeth m. kutter shirley lecher segundo r. leon grace london charles g. massambu sikhulile moyo dennis mok chris murrill nicaise ndembi john n. nkengasong jack nyamongo brenda okech pascale ondoa bharat parekh linda parsons amy piatek lila rahalison pat riley gajendran sivakumar belete tegbaru adeola tomi-olugbodi raquel villegas lara vojnov musau wakabongo michael z. wang mick mulders jane mwangi nicaise ndembi clement b. ndongmo john n. nkengasong michael a. noble bryan nyary beldinah r. ochola igho ofotokun iruka n. okeke pascale ondoa ketan patel helen perry pedro c. pires zilma rey pierre rollin ahmed saleh michel segondy timothy su ralph timperi farouk a. umaru raquel villegas larry westerman katy yao ernest yufenyuy reviewers 2015 reviewers 2016 should names have inadvertently been excluded from this list, the publisher apologises and undertakes to amend the exclusion in the next issue. abstract introduction methods classification and structure of the mpox virus pathogenesis and pathophysiology of mpox virus epidemiology and historical outbreaks management, potential therapeutics, and vaccines laboratory diagnosis of mpox prevention and control challenges and future perspectives recommendations conclusion acknowledgements references about the author(s) chikwelu l. obi dean’s office, school of science and technology, sefako makgatho health sciences university, pretoria, south africa nqobile m. mkolo department of biology and environmental sciences, school of science and technology, sefako makgatho health sciences university, pretoria, south africa liziwe l. mugivhisa department of biology and environmental sciences, school of science and technology, sefako makgatho health sciences university, pretoria, south africa modupe o. ogunrombi department of clinical pharmacology and therapeutics, school of medicine, sefako makgatho health sciences university, pretoria, south africa mukhethwa m. mphephu department of biology and environmental sciences, school of science and technology, sefako makgatho health sciences university, pretoria, south africa clarissa m. naidoo department of biology and environmental sciences, school of science and technology, sefako makgatho health sciences university, pretoria, south africa citation obi cl, mkolo nm, mugivhisa ll, ogunrombi mo, mphephu mm, naidoo cm. mpox: an emerging or re-emerging infection with a potential colossal burden on healthcare globally. afr j lab med. 2025;14(1), a2644. https://doi.org/10.4102/ajlm.v14i1.2644 note: additional supporting information is available for download alongside the online version of this article as online supplementary document 1. review article mpox: an emerging or re-emerging infection with a potential colossal burden on healthcare globally chikwelu l. obi, nqobile m. mkolo, liziwe l. mugivhisa, modupe o. ogunrombi, mukhethwa m. mphephu, clarissa m. naidoo received: 11 oct. 2024; accepted: 28 mar. 2025; published: 31 may 2025 copyright: © 2025. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract the world health organization identified mpox (formerly known as monkeypox), as a resurgent zoonotic epidemic caused by the mpox virus. it is an emerging and re-emerging pathogen with a range of hosts and geographical distribution worldwide. peer-reviewed scientific articles from 1958 to 29 august 2024 related to global mpox research were extracted from web of sciencetm core collection and google scholar databases to gauge the extent of the infection. mpox is marked by a recent resurgence of infections across continents, with africa being the hardest-hit region. the mpox re-emergence has shown a new mechanism of transmission, with several causes such as a rise in the number of unvaccinated individuals, behaviour risk factors, waning immunity, genetic evolution, and environmental circumstances. preventive and control measures of mpox include vaccination and patient isolation, while treatment involves antivirals and antibiotics for secondary bacterial infections. laboratory diagnosis entailing polymerase chain reaction can be effective for routine purposes, but results of serological tests must be interpreted with caution, because of cross-reacting determinants among orthopoxviruses. the structure and classification of the mpox virus, clinical manifestations, pathophysiology, epidemiology, historical antecedent, therapeutics, vaccines, and laboratory diagnosis of the disease are explicated, showcasing mpox as an emerging or re-emerging infection with a potential colossal burden on healthcare, and its classification as an international public health emergency by the world health organization. what this study adds: this review provides the global situation of mpox as an emerging or re-emerging infection, warranting its designation as an international public health emergency. keywords: mpox; clades; antivirals; vaccines; laboratory diagnosis. introduction mpox, formerly known as monkeypox by the world health organization (who), is a zoonotic viral infection with a cycle of transmission involving the active participation of wild animals.1 the mpox virus is regarded as both an emerging and a re-emerging pathogen with a varied range of hosts.2 the re-emergence of the mpox virus version with enhanced virulence since its first identification and reporting in monkeys in 1958 is favoured by the alterations in the virus genome resulting from mutations.2 although mpox was identified 70 years ago, it has been ignored in medical literature because of its rarity.3 however, it has recently been accorded much attention because of a trail of morbidity and mortality in different age groups worldwide, although its foci were mainly in africa.2,4 the mpox name is likely to have originated from the fact that mpox was initially identified in monkeys (cynomolgus) from singapore used for research purposes in 1958 and then transported to copenhagen in denmark.3,5,6 mpox is caused by a diverse and large group of orthopoxviruses7 and is characterised by symptoms that resonate with those of smallpox and chickenpox, even though mpox usually presents milder symptoms compared to smallpox and has a 10% fatality rate in people who are not vaccinated against smallpox.3 mpox is a viral disease that has been added to the who list of communicable diseases by viruses with the possibility for widespread and epidemic repercussions and fears after the coronavirus disease 2019 outbreak, hence its classification as an international public health emergency.8,9,10 by 29 august 2024, a total of 89 596 established cases of mpox had been recorded across 114 countries, with 157 deaths,11 resulting in apprehension regarding the geographic spread.12 even though the exact causes and reasons for the re-emergence of mpox are unknown, it is postulated that it could be a result of several factors, such as the increase in the number of people not vaccinated against smallpox, risk factors of behaviours of men having sex with other men, decreasing immunity, genetic evolution, and ecological conditions.11 the global spread of the disease is a health hazard.13 additionally, the recent disease modelling proposes that pandemics which are usually caused by emerging diseases are expected to surge in severity and frequency in the upcoming decades.14 hence, the review aims to report on mpox as an emerging or re-emerging infection with a potential colossal burden on healthcare. methods on 29 august 2024, peer-reviewed scientific articles related to global mpox research were extracted from the web of sciencetm core collection and google scholar databases. the search included papers from 1958 to 29 august 2024, and included phrases associated with mpox, monkeypox virus, mpox classification and structure, mpox epidemiology, mpox vaccines, and mpox laboratory diagnosis. in every study that was retrieved, only english-language literature was included. classification and structure of the mpox virus classification of mpox virus the mpox virus belongs to the poxviridae family, which comprises 22 genera and 83 species.15 the family is subdivided into two subfamilies, namely chordopoxvirinae, with 52 species and 18 genera, and entomopoxvarinae, with 31 species and 4 genera. twelve members of the genus orthopoxvirus have been recognised to affect both humans and animals.15,16 the variola virus is the commonly known member that causes smallpox, while others are known as the cowpox virus, camelpox virus, skunkpox virus, volepox virus, taterapox virus, akhmeta virus, abatino macacapox virus, raccoonpox virus, vaccinia virus (vacv), ectromelia virus, and mpox virus.15,16 structure of mpox virus and its genome the lipoprotein outer membrane of the mpox enveloped virus is oval or brick-shaped.17 the normal size of the mpox virus varies from 200 nm to 250 nm, with a double-stranded dna genome of almost 197.2 kb, and encodes 181 proteins.18 the hairpin end of the linear genome is covalently closed and there are no free 3’ or 5’ ends.18 the genome ends consist of the 10 kb inverted terminal repeats and nucleotide homopolymers (figure 1). however, short tandem repeats have been observed in the genome.18 orthologous poxvirus genes are closely packed, and intergenic areas that are 100 bp in length are uncommon.18,19 the ‘housekeeping’ proteins that are encoded by orthologous poxvirus genes in the conserved central area are responsible for transcription, replication, and virion processes. in the terminal domains, the proteins encoded by orthologous poxvirus genes are linked to the pathogenesis and host range.19,20 figure 1: the structure of the mpox virus and its genome. the mpox virus consists of two main clades: clade i, formally identified as the congo basin clade, and clade ii, formally recognised as the west africa clade.21,22 these clades are split into two subclades, namely clade ia and clade ib in clade i, and clade iia and clade iib in clade ii (figure 2).21,22 lately, clade iii, consisting of hmpox-1 a, b.1, a.1.1, a.1, and a.2, has been reported in non-african countries, including mexico.22,23,24 clade iii varies primarily in regions of coding and is linked to the host’s recognition of antigenic determining factors and immune modulation.25 a total of 46 single-nucleotide polymorphisms are reported from clade iii.23,26 the mutation of mpox is because of the action of the apolipoprotein b mrna editing enzyme, catalytic polypeptide 3 family of cytosine deaminases.27 figure 2: phylogenetic visualisations of mpox virus clades generated with ggtree package: (a) all mpxv clades, (b) clade ia and (c) clade iib. pathogenesis and pathophysiology of mpox virus viral endocytosis, cell membrane fusion, and micropinocytosis accelerate viral transmission through oropharyngeal, subcutaneous, nasopharyngeal intramuscular, and intradermal pathways (figure 3).28 the virus replicates at the location of inoculation, which leads to the virus spreading to different organs of the lymph nodes, blood, bone marrow, tonsils, and spleen, triggering inflammatory immune-mediated phagocytosis.29,30 this signifies the period of incubation, which usually lasts for 7 days to 21 days. through the guidance of the enveloped and mature virions, the genome and proteins of the mpox virus are discharged into host cells. intracellular mature virions encompassing the dna encoding the virus get generated when the virus mrna transcription and translation occurs.31,32 intracellular mature virions enfolded in the golgi apparatus fuse with the host inner cell membrane to form cell-related virions before being released into extracellular areas to produce extracellular enveloped virions.29 figure 3: cytosolic mpox virus pathways depicting the enveloped virion entry to the host cell through fusion and the mature virion. the mpox virus can be spread in different modes from animals to humans, or humans to humans. however, the general mode is through human contact with the bodily fluids of the infected animals.33,34,35 the distinction between mpox and smallpox is that mpox virus infection triggers lymphadenopathy in humans while smallpox does not. the early symptoms of mpox virus infection are muscle aches, headache, diarrhoea, fever, chills, vomiting, fatigue, and backache, which advance to fatigue, developing into secondary bacterial infections of the skin or blood and lung infections.21 the body of the infected person can be affected by lesions that are initiated in the oropharynx, and inflammation of the heart, brain or other organs can also occur.36,37 moreover, patients can also have neurological concerns in the form of encephalitis.36 the onset symptoms occur mostly in all age groups, although infants or the elderly might have a less frequent or different presentation of symptoms.21 epidemiology and historical outbreaks mpox virus was identified first in monkeys in 1958 during the occurrence of outbreaks in colonies of captive monkeys that were kept for purposes of research in a danish research institute, and has since evolved into a zoonotic infection affecting the human population.37,38,39,40 the first detection of mpox in a human host was discovered on 01 september 1970 in a baby (9 months old) in the democratic republic of the congo.38,39,40,41,42 since the campaign for worldwide smallpox vaccination gradually ended between the 1970s and 1980s, and smallpox was eventually eradicated by 1977, the number of human mpox cases in africa has been on the increase.43,44,45,46 the who verified a case of mpox later in 1980, and since then the virus has spread globally with a ten-fold increase in the past decades.38,39 in 1982, active mpox surveillance programmes were started in the democratic republic of the congo. this increased the number of detected cases. a high number of approximately 386 cases was observed in the democratic republic of the congo alone, whereas only 18 cases were observed in other endemic countries, with children being most affected.47,48 in 1986, mpox surveillance programmes in the democratic republic of the congo were terminated because of the onset of another epidemic, termed aids, in africa. this prompted the who to redirect its resources on public health.49,50 there was a decline in the number of confirmed human mpox cases in the decade that followed. no case was reported to the who beyond 1992. it was only in 1996 and 1997 that a cluster of 344 cases were reported in the democratic republic of the congo among a cohort predominantly unvaccinated against smallpox.51 since then, outbreaks have been common in the democratic republic of the congo. annually, over 1000 cases have been reported since 2005.44 in nigeria, there was a complete hiatus of cases for 39 years until 2017, when mpox re-emerged. a sizable country-wide outbreak of over 120 laboratory-confirmed or -suspected infections involving clade ii was then recorded.51 several factors, including population density, shifting patterns of land use, and declining herd immunity provided by smallpox vaccination were reported to be providing opportunities for more zoonotic transmission and outbreaks.51 in contrast, between 1970 and 2018, case reports of human mpox infection from other countries were infrequent.43,44 mpox re-emerged as a global outbreak in may 2022, with almost 86 000 cases and 53 deaths confirmed from 110 countries around the world, especially in areas where the disease had not been common.13 in 2023, there was an occurrence of mpox in the united states3 with 47 human cases that might be a result of close interaction with prairie dogs which were alleged to have been infected during a shipment imported from ghana.1 a recent outbreak was also reported in europe and africa, with suspected cases and total deaths recorded across different countries between 2022 and 2024. the african region experienced a surge in cases in july 2024. these data outline the outbreak trends across various geographical locations from 2022 to 2024.21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42 the geographical location and demographics revealed mpox occurrences mainly in central, east, and west africa, as well as multi-country outbreaks for clade iib, lineage b.1. demographics indicated morbidities of 70% and above, except for adults in burundi, across all clades and sub-clades, with a preponderance in men (online supplementary table 1). sexual intercourse mode of transmission is reported to contribute more to the spread of the virus, especially by men who have sexual intercourse with other men.21 previously, the age of people infected with mpox ranged from 7 months to 40 years,47 where more than 80% of the mpox cases were seen in children under 10 years of age.47 the attributable deaths (17%) also occurred in children.47 the median age of the affected people moved from younger children of 4 years old in the 1970s to the median age of young adults of 21 years between 2010 and 2019.3 this could be because of the suspension of smallpox vaccinations which offered some form of herd immunity against mpox.3 however, recently in 2024, the age of people infected with mpox ranged from 0 years to 65 years, and men between 18 years and 44 years old continued to be excessively affected by the outbreak, accounting for 79.2% of reported cases, while women account for 3.6%. moreover, a rash (systemic, oral, genital, or unknown location) is reported in 88% of cases with at least one reported symptom, and is thus the most common symptom (figure 4).21 it was reported that the denotation of a symptom may differ throughout reporting systems in different countries as a result of a general lack of negative reporting and symptom classifications.21 figure 4: symptomatology of people infected with mpox, reported from january 2022. management, potential therapeutics, and vaccines the treatment of mpox infections involves community-based and clinical measures to limit its spread.52,53 in community and clinical settings, patients should stay at home, and avoid contact with infected people, and healthcare professionals should follow guidelines to reduce the risk of infection.8,54 the guidelines include avoiding direct contact with skin lacerations, treating skin rashes with personal protective equipment, and wearing gloves.55 infected individuals should be isolated for approximately 2–4 weeks to decrease the spread of the virus and mitigate the impact of possible outbreaks.56 in addition, supportive care is essential for patients with limited antiviral therapies, including hydration, nutrition, symptomatic management, and antibiotic treatment.57,58,59 in immunocompromised patients, superinfection and eye infections can be managed with antibiotics.60 intensive care management and mechanical ventilation are often used for respiratory and neurological issues.61 potential antiviral agents new antiviral agents and vaccines such as cidofovir (gilead sciences, foster city, california, united states), brincidofovir (chimerix, durham, north carolina, united states), tecovirimat (siga technologies, new york, united states), immunoglobulin (vaccinia immune globulin; siga technologies, new york, united states) acam2000® (emergent biosolutions, gaithersburg, maryland, united states), and jynneostm (bavarian nordic, kvistgård, denmark) offer new therapeutic prospects for orthopoxviruses (online supplementary table 2).62,63 smallpox (variola virus), mpox, and cowpox share collective genetic similarities, which include shared surface proteins such as antibodies, and t-cell receptors.62 in addition, some smallpox vaccines create antibodies against a range of epitopes that bind to the shared epitopes of mpox proteins.63 the antiviral resistance of cidofovir is slow, affecting poxviruses through serial passage.63,64,65 it is effective in treating compound orthopoxvirus infections and is used in humans for poxvirus infections, molluscum contagiosum, and aids-associated cytomegalovirus retinitis.66,67 brincidofovir, a lipid-conjugated cidofovir analogue, was united states food and drug administration (fda)-approved in 2021 for smallpox therapy because of its comprehensive activity against double-stranded dna viruses and lower ec50 compared to cidofovir.68,69 its lipophilicity allows efficient entry into host cells and a prolonged intracellular half-life.69,70 oral brincidofovir has shown potential in treating poxvirus infections and pre-proactive therapy of adenovirus viraemia.71,72,73,74,75 tecovirimat, fda-approved in 2018, targets the v061 gene in cowpox and the membrane protein p37, which is responsible for extracellular enveloped virus formation.76,77 it is available in intravenous and oral forms and has no documented cross-resistance.78,79 it is effective in treating orthopoxviruses in animal models, preventing death, and reducing viral distribution to distant tissues.80 tecovirimat is used to treat orthopoxvirus infections in human patients because of its tolerance levels. four clinical trials are ongoing to evaluate its safety, tolerability, and pharmacokinetics.81 vaccinia immune globulin intravenous (emergent biosolutions, gaithersburg, maryland, united states) is an fda-approved immunoglobulin used to manage smallpox vaccination and adverse effects in skin disorders.82 it provides passive immunity but is not recommended for immunocompromised patients, diabetic renal complications, or sepsis history.83 clinicians may consider vaccinia immune globulin intravenous for complex mpox infections or t-cell immunodeficiency patients; however, live attenuated vaccines should be avoided 3 months after vaccination.84 vaccines and immunisations first-generation smallpox vaccines were effective, but adverse effects led to subsequent vaccine development.85 currently, three licensed mpox vaccines are available: modified vaccinia ankara-bn (bavarian nordic, kvistgård, denmark), lc16-kmb (konica minolta, tokyo, japan), and orthopoxvac (university of florida, gainesville, florida, united states).86 the fda has authorised acam2000 and jynneos for smallpox prevention and cross-protective immunity.87 the vacv can be treated with vaccinations, either before or after exposure.88 post-exposure prophylaxis should be administered 4 days or less after exposure, to prevent symptomatic infection.89 if mpox symptoms are absent, post-exposure prophylaxis can be administered up to 14 days after exposure. the fda authorised jynneos intradermal injection as a route of administration for emergency use in august 2022, in addition to the previously approved subcutaneous injection.90,91 the jynneos vaccine, approved by the fda for alternative use in mpox virus-infected persons, works by eliciting humoral and cellular immune responses.92 preclinical research indicates that mpox vaccination is safe during pregnancy and breastfeeding, although there is no preclinical research available for paediatric patients, despite being safe in immunocompromised people such as hiv patients.93 the acam2000 is a licensed vaccine for smallpox and mumps, given in single doses and lyophilised for long-term storage.93,94 it aims to balance the risk of pathogenic mpox with potential side effects from replicative vaccinations. furthermore, acam2000 is not recommended for hiv-positive patients since they have immunosuppressed systems, as it is a live vaccine that can cause severe complications. attenuated vacv, also known as modified vaccinia ankara, requires two injection treatments. live vacv immunisation has been linked to skin infections, premature delivery, congenital abnormalities, stillbirth, and perinatal mortality.94 however, vacv immunisation has a safer profile and fewer side effects compared to acam2000.95,96 healthcare professionals and laboratory people who are at a high risk of exposure are also evaluated for pre-exposure prophylaxis.84 world health organization guidelines on therapeutic interventions and vaccines for mpox pre-exposure immunisation for high-risk persons and post-exposure vaccination within 4–14 days is suggested by the who’s normative recommendations for mpox, to avoid or minimise the seriousness of the disease.21,90 vaccination should be used in combination with public health interventions such as contact tracing, isolation, and surveillance.10,21 target product profiles, which outline required vaccination qualities, have also been produced by the who.21,94,95 clinical administration places significance on supportive care related to medicine, and antivirals according to who guidelines.21,54,62,63 to ensure the reasonable allocation of vaccinations, treatments, and diagnostics, the who has also set up access and distribution processes, and has modified its recommendations in response to new information.21,50 laboratory diagnosis of mpox a critical factor in the diagnosis of infections, including the mpox virus, rests on laboratory diagnosis.96,97,98,99 two of the definitive methods for the laboratory diagnosis of mpox infection are the direct and indirect methods. for the indirect method, specimens collected are screened for the virus, the nucleic acid, or the viral antigens. to identify the dna, nucleic acid amplification testing is employed for the direct method. on the other hand, immune responses to the viral antigens form the basis for the detection using the indirect method.98 specimen collection, transport, and storage the main sample for laboratory diagnosis of mpox infection is the skin lesions, such as swabs from lesion exudate or lesion crusts. ideally, lesions, crusts, and vesicular fluids should not be placed in a similar tube, to obtain good dna or prevent inhibitors.97,98,99 oropharyngeal swabs, rectal or genital swabs, semen, or urine may be collected based on clinical manifestations.98,99 blood samples are also used, especially when treated with ethylenediaminetetraacetic acid. this process may increase the concentration of the virus; however, it may not align with samples collected from lesions, as viraemia typically occurs during the early stages of infection or the prodromal phase. sample collection should be done by trained healthcare personnel in an appropriate clinical or field setting, using personal protective equipment, and then transported to laboratories with the appropriate level of biosafety for analysis.35,100 after collection of samples for laboratory diagnosis of mpox, samples should be placed in the fridge at 2 °c – 8 °c or kept chilled at –2 °c within an hour after collection, before transportation to the base laboratory. emphasis is usually placed on the storage and handling of specimens while being transported. if it is anticipated that it may exceed 7 days before processing of specimens following transportation, such samples must be stored at –20 °c or a lower temperature (± 2 °c), or at –70 °c if storage will exceed 60 days after collection, to obviate false negative results caused by, for example, inability to extract dna.98,99 virus isolation the first isolate of the mpox virus was obtained in 1958 from cynomolgus monkeys using monkey kidney (vero) and human amnion cells.101 in humans, the virus was first isolated from a patient with a skin infection resembling smallpox. this was achieved by infecting pig embryo kidney cells, homo sapiens epithelial carcinoma cells, and african green monkey kidney cells, where cytopathic effects were observed.102 although research laboratories have employed culture-based techniques for the identification of the mpox virus, the routine identification of the virus is not recommended by the who as it is not only laborious, but has low sensitivity and takes some days, and must be performed using a biosafety level-3 safety chamber.97,98 electron microscopy the use of electron microscopy is a known method for identifying structures, including the fine details of the virus after isolation. this method reveals that the mpox virus has a brick-shaped (200 nm – 250 nm) or ovoid format, displaying intricate internal structures such as a double-stranded dna genome (∼197 kilobases) and enzymes. although electron microscopy is useful in revealing progeny virions in different phases of assembly, immature and mature ones, in infected cells, it is not recommended for routine laboratory diagnosis of the mpox virus.102,103,104 serological methods the use of serology to ascertain immune responses, immunoglobulin m and immunoglobulin g antibodies to mpox infection by use of enzyme-linked immunosorbent assay, plaque reduction neutralisation test, complement fixation test, haemagglutination inhibition, and immunofluorescence are all of diagnostic value. however, results obtained should be interpreted with caution because of cross reactions or shared antigenic determinants among the orthopoxviruses or, in recent cases of vaccination, against smallpox.105,106 clinical laboratory findings and biomarkers to complement laboratory methods, variations of biochemical and haematological indices, such as thrombocytopenia, rated as the most common, hypoalbuminaemia, leucocytosis, and increased transaminase level are critical.1 other findings indicate that higher levels of aspartate aminotransferase, and alanine aminotransferase may be related to poor prognosis. in addition, cytokine modulation has been reported to correlate with the severity of mpox in humans.107,108 the cytokines comprise interleukins 1b, 2r, 4-8, and 13, among others.98,108 the polymerase chain reaction (pcr) test on lesions was, however, reported to give the highest clinical sensitivity, 91% – 100%,109,110,111,112 whereas the sensitivity of saliva, nasopharyngeal swab, and oral swabs was between 68% and 100%, seminal fluid was 78% – 100%,109,111 and rectal swabs were 78% – 97%.112 genome sequencing for epidemiological purposes or monitoring of transmission patterns as an adjunct to conventional laboratory analysis of mpox infection, whole genome dna sequencing is critical for observing differences in the viral genome over time. genome sequencing is not recommended for routine diagnosis due to its expensive outlay, high cost of reagents, and the advanced training required for the process. few samples of patients may require genome sequencing from samples as genome monitoring of circulating types may assist policymakers and healthcare personnel in terms of decision-making as well as the introduction of measures to reduce or abrogate the chain of transmission.99,113,114 real-time polymerase chain reaction it has been reported that real-time pcr is the gold standard molecular method for lab-based diagnosis of mpox.35,97,98 the real-time pcr workflow for mpox detection involves several key steps: denaturation, annealing, extension, and fluorescence readout. initially, the double-stranded dna is denatured to separate the strands into single strands, which facilitates the binding of primers to their respective target regions. the forward and reverse primers are designed to bind to specific sites on the single-stranded dna. the reverse primer plays a critical role in this process by binding to the complementary strand, enabling the synthesis of the complementary strand during the extension phase. during annealing, both the forward and reverse primers bind to their target sequences on the dna. following this, dna polymerase synthesises the complementary strand in the extension phase. the fluorescence signal is then released, allowing for real-time detection of the target sequence. regarding other tests involved, this real-time pcr assay specifically measures the amplification of target dna sequences, such as those within the g2r, b7r, b6r, n3r, and tnf receptor genes in the mpox virus genome, as previously reported.113,115 additionally, multiplex real-time pcr assays can be used to detect and differentiate various infectious agents and different subclades (iia, iib) of the mpox virus.113,114,115,116,117,118,119 loop-mediated isothermal amplification real-time pcr is a standard technique method for diagnosing mpox infection; however, its shortcomings comprise sample processing time, constant availability of electricity supply, and technical skills.113,120 consequently, in terms of point of care, a user-friendly, simple, inexpensive, and rapid method for the laboratory diagnosis of mpox in lowto middle-income countries or areas with simple laboratory infrastructure are warranted. loop-mediated isothermal amplification (lamp) is a recommended technique for the amplification of nucleic acid at a single and isothermal temperature of about 60 °c – 65 °c. while lamp requires less equipment than real-time pcr, it still relies on electricity, which may limit its utility in areas with inconsistent power supply. nevertheless, its simplicity and low-cost nature make it a valuable option for resource-limited settings.121,122,123,124,125,126,127,128 recombinase-based isothermal amplification assays recombinase-based isothermal amplification assays are also isothermal amplification methods involving enzyme-based dna amplification at a constant temperature of around 37 °c to 42 °c within 10 min,127 and, because of its simplicity, high sensitivity, and rapidity, this method is suitable for point-of-care or field applicability for mpox detection. sensors sensors are also used in laboratory diagnosis based on their ability to respond to stimuli, biological, physical, and chemical agents that elicit observable effects that are measurable through electrochemistry, colourimetry, and fluorescence. their applications are simple, rapid, inexpensive, and adaptable for use in rural or less-resourced settings.129 in terms of the method, the rna sensors or toehold switches can stimulate the translation of a reporter gene, such as beta-galactosidase or a green fluorescent protein, against a receptor rna trigger sequence present in the sample. this results in the activation of a reporter protein, followed by a light reflection that generates an observable colour change. alternatively, fluorescence-based methods may use fluorescent dyes such as 6-carboxyfluorescein or sybr green to detect the presence of the target rna, generating a measurable fluorescence signal.129,130,131 prevention and control for effective control of mpox outbreaks, rapid recognition and investigation of new cases, along with a comprehensive understanding of all possible routes and modes of transmission, are essential strategies for vaccine distribution.7 prevention of transmission of the mpox virus principally depends on hygienic practices and restrictions on contact with wild animals as the primary transmission mode of the virus to humans.1,84 direct contact with an infected animal should be prevented to avoid the animal-to-human route. additionally, physical contact with other people who are infected should be avoided, personal protective equipment should be used to reduce transmission, and there should be isolation of patients with mild cases of the disease.9 in cases where a person presents with lesions caused by mpox around the genitals, sexual contact must be avoided, or condoms should be used to avoid transmission.13 although the majority of mpox cases during the 2022 outbreak occurred in men who have sex with men, the infection can affect individuals of all genders and age groups. during pregnancy, special precautions are necessary to prevent vertical transmission from mother to child through the placenta. additionally, perinatal transmission can be avoided by preventing neonatal contact with mpox-related genital lesions during childbirth.131,132,133,134 other avenues of transmission include tourism and travelling.132 hence, prevention and control of the mpox virus can also be affected by controlling international air travel volume and entrenching policies regarding entry into countries. deng et al.40 showed that a reduction in the volume of air travel and strict policies on border entry contributed to a reduction of mpox introductions of infected persons into china.13 smallpox vaccine has been shown to provide almost 85% protection against mpox because of herd immunity.11 preand post-prophylaxis vaccination has also been recommended by the healthcare authorities for healthcare workers, laboratory workers and technicians, response teams against outbreaks, as well as scientists who research clinical samples, and vaccinators.84 post-exposure prophylaxis is suggested when there has been unprotected contact with infected people or there has been sharing of close spaces with infected people for extended periods (prolonged or repeated close contact) where there might be secretions of aerosols and the presence of the virus in the air particles.84 vaccines, antiviral agents, and drugs such as tecovirimat are used as the first line of treatment. challenges and future perspectives mpox prevention, treatment, and control face several challenges, including healthcare gaps and the limited availability of treatments, vaccinations, and resource environments.135,136 diagnostic challenges, such as access to advanced technologies, can hinder timely and accurate diagnosis.137 additionally, fever-like illnesses accompanied by a rash may be misdiagnosed as mpox.49 to effectively control mpox, strong public health systems are required to monitor and manage outbreaks, and trace contacts. however, underdeveloped systems in endemic areas make containment difficult.138 public mistrust of vaccines, especially in poorly literate areas, can impede mass vaccination campaigns. immunisation resistance, motivated by misleading information, remains a significant barrier.139 recommendations the following recommendations are of significance in the narratives concerning mpox: global emergency preparedness to tackle the potential pandemic of the disease is critical and should be actioned. training and re-training of healthcare professionals on the differential diagnosis and other intricate aspects of the disease. enhanced research activities to uncover emerging clades and their genomes, more insights into their epidemiology, and genomic surveillance to identify changes and adjust vaccines should be prioritised. increased provision of vaccines, especially to endemic areas to curb centrifugal spread, should be central to the strategies to tackle the disease. increased allocation of resources by countries in a bid to contain the menace of the disease and its anticipated health burden. development of simple screening and diagnostic test kits or methods especially for use in less-resourced countries. conclusion mpox is not a new disease, but an emerging and re-emerging disease caused, probably, by increased rates of zoonoses, deforestation, food insecurity, global mobility, and increased socialisation pressures. others include risky behaviours and decreased immunity to the smallpox virus, which shares cross-reactive antigenic determinants with the mpox virus, resulting in waning protection offered by herd immunity against mpox. the key drivers of infections across various age groups and countries over the past years were the various clades. the historical foci of the disease have been mainly in africa, in addition to reported cases in other continents, indicating varying degrees of morbidity and mortality in all affected countries. management of the disease may depend on the range of clinical manifestations, including the use of antivirals, and antibiotics for secondary bacterial infections. preventive and control measures are predicated on the isolation of patients, protective sexual intercourse, avoiding contact with fluids of infected persons, and vaccination, among others. laboratory diagnosis involves the use of pcr for routine purposes, but the use of serological tests should be interpreted with caution because of shared antigenicity among the orthopoxvirues. electron microscopy and genome sequencing are mainly for research-related endeavours. acknowledgements we sincerely thank the south african medical research council through its division of research capacity development under the research capacity development initiative. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. the author, c.l.o., serves as an editorial board member of this journal. the peer review process for this submission was handled independently, and the author had no involvement in the editorial decision-making process for this manuscript. the author has no other competing interests to declare. authors’ contributions c.l.o. conceived the idea of the study. c.l.o., n.m.m., l.l.m., m.o.o., m.m.m., and c.m.n. collected and analysed the data. c.l.o., n.m.m., l.l.m., m.o.o., m.m.m., and c.m.n. drafted the manuscript. c.l.o., n.m.m., l.l.m., m.o.o., m.m.m., and c.m.n. reviewed and revised the final article, and c.l.o., n.m.m., l.l.m., m.o.o., m.m.m., and c.m.n. agreed to the published version of the article. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support we acknowledge the assistance of the south african medical research council (grant number mkolo24/25). data availability in this study, the data sources 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vaccination hesitancy in non-endemic countries: concise literature review. vaccines. 2023;11(2):229. https://doi.org/10.3390/vaccines11020229 article information authors: fatim cham1 mahlatse maleka2 martin masango2 emma goetsch2 el h. belabbes1 beverley singh2 guy m. gershy-damet1 adrian puren2,3 affiliations: 1world health organization regional office for africa, brazzaville, republic of congo 2centre for hiv and sti, national institute for communicable diseases, south africa 3division of virology and communicable disease, university of the witwatersrand, south africa correspondence to: fatim cham postal address: inter-country support team for eastern and southern africa (ist/esa), world health organization regional office for africa, highlands, harare, zimbabwe dates: received: 26 apr. 2012 accepted: 21 sept. 2012 published: 07 nov. 2012 how to cite this article: cham f, maleka m, masango m, et al. the world health organization african region external quality assessment scheme for anti-hiv serology. afr j lab med. 2012;1(1), art. #39, 6 pages. http://dx.doi.org/10.4102/ ajlm.v1i1.39 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the world health organization african region external quality assessment scheme for anti-hiv serology in this original research... open access • abstract • introduction • ethical considerations • methods    • characterisation of bulk volume specimens    • utilisation of internal quality control    • analysis of data • results    • pt panel testing methodologies    • participation and response rate of laboratories    • pt panel testing results    • internal quality control • discussion • acknowledgments    • competing interests    • authors’ contributions • references abstract top ↑ a regional external quality assessment scheme (reqas) for anti-hiv serology aimed to objectively assess reliability and quality of hiv testing processes in the african region. this involved the distribution of proficiency testing (pt) panels to participating laboratories from 2002 to 2010. during the survey period, this included 16 distributions of pt panels to 49 laboratories in 30 countries, and the overall average score during the nine-year survey period was 98.9%, with a frequency of accurate detection, of anti-hiv-1 and/or anti-hiv-2 antibodies in the pt panels, ranging from 93% to 100%. problems highlighted included lack of human resources and frequent stock outs of test kits, reagents and consumables for routine hiv testing. the design of the reqas allowed appraisal of the reliability of anti-hiv serological testing methods utilised by laboratories for clinical assessment of patients and/or surveillance programmes. the reqas was able to demonstrate that laboratories participating in the reqas performed well and sustained their participation in the scheme. this bodes well for clinical diagnosis, surveillance and training activities at these reference laboratories. introduction top ↑ since the 2001 united nations general assembly 26th special session (ungass) declaration of commitment for access to treatment care and support services for people living with hiv and/or aids, there has been an unprecedented scaling up of integrated and comprehensive services for diseases of public health importance in the african region (www.unaids.org)1. as laboratory and non-laboratory testing for hiv, tb and malaria is one of the main entry points of access to prevention and support services, accurate and reliable laboratory results are essential for diagnosis and monitoring of diseases of public health importance.2 in accordance with world health organization regional office for africa (who afro) resolutions endorsed by member states to strengthen laboratory capacity in the african region, it was recommended that national public health reference laboratories (nphrls) develop and implement integrated quality management systems (qms) including participation in external quality assessment schemes (eqas) for all diagnostic and monitoring tests. additional recommendations included the implementation of integrated, cost-effective and sustainable national eqas. moreover, nphrls strengthening of their qms is a gateway to identifying training needs and technical support in addition to attaining accreditation based on international standards.3,4,5,6 external quality assessment (eqa) is an objective means of assessing the integrity of the entire laboratory testing process, and aims to educate and improve performance in quality assurance (qa) issues.7 eqa includes, among others, on-site assessments, and blinded rechecking of previously tested specimens and/or proficiency testing.8 proficiency testing (pt), an essential component of eqa, is an independent means of assessing the quality of the testing process whereby multiple well-characterised specimens are periodically sent to a group of laboratories for analysis and/or testing using routine laboratory procedures. thus, pt programmes can assist laboratory services to identify factors contributing to errors, in addition to determining whether a laboratory can reliably perform a given test when compared to its peers. in the african region, few public health laboratories are participating in eqas for diagnostic and monitoring tests. these laboratories are for the most part limited to central level laboratories, mainly because schemes coordinated by international or regional providers are not able to provide sufficient advice on remedial actions when necessary. moreover, these schemes are relatively expensive for national governments to extend participation to laboratories in the tiered laboratory network. hence, in response to requests from member states, who afro established the regional external quality assessment schemes (reqas) for anti-hiv serology in collaboration with the national institute of communicable disease (nicd) in south africa and the national reference laboratory (nrl) in senegal to support the anglophone and francophone countries, respectively. the reqas, established in march 2002, aim to assess the quality of anti-hiv-1 and hiv-2 serological testing for interested laboratories. additionally, the reqas allow comparison of testing facilities, in addition to evaluating the quality of serological testing using enzyme immunoassays (eia) and hiv rapid tests. participation in the reqas is voluntary and at no cost to laboratories, thus allowing participation of laboratories in at least one of the reqas components. the scope of this paper is limited to the reqas for anti-hiv serology coordinated by nicd and aims to present results of surveys conducted from 2002 to 2010 and challenges encountered. the present article describes results of a reqas for anti-hiv serology established for public health laboratories in the african region and discusses the implications for efforts aimed at assuring the quality of hiv testing and strengthening public health laboratories towards accreditations in the african region. ethical considerations top ↑ these laboratories applied to participate in the surveys. methods top ↑ characterisation of bulk volume specimens bulk volume blood obtained as plasma from the south african national blood services was converted to serum by recalcification and heat-inactivated at 56 °c for 60 minutes.9 serum samples were characterised by testing on at least three different 3rd generation anti-hiv-1/2 enzyme immunoassays (eias), three different anti-hiv-1/2 rapid tests and by western blotting. from 2002 to 2010, a total of 17 pt panel batches (001– 016), each comprised of ten serum specimens, were dispatched to laboratories. the pt panel batches 001–013, 015–016’ were comprised mainly of hiv-1 sero-positive and sero-negative samples. for pt panel batch 014, two batches (014–1 and 014–2) were dispatched: pt panel batch 014–1 was comprised of four hiv-1 and six sera-negative samples and pt panel batch 014–2 included two hiv-2, four hiv-1 and four hiv sero-negative samples. pt panel batch 014–2 was dispatched to 14 laboratories that responded to a survey confirming capacity to sero-type hiv-2. hiv-2 sero-positive samples were characterised by elisa and confirmed using the multispot rapid test kit (bio-rad laboratories, usa) and western blot using new lav blot ii (bio-rad laboratories, usa). all assays used in the characterisation of the pt panels were performed according to manufacturer’s instructions. furthermore, the env subtype of 38 hiv sero-positive samples included for pt panel batches 001 to 008 was determined by heteroduplex mobility assay (hma). of these, 36 were env subtype c whilst 2 were env subtype b. due to logistic and cost reasons, env sub-typing of panels was discontinued for pt panel batches 009 to 016. however, it is likely that panels sourced from the south african blood bank were either subtype b or c based on results of surveys of hiv-1 subtype conducted in south africa.10 characterised serum panels were aliquoted in 100µl volumes, labelled and stored at −80 °c until panels were ready for shipment to laboratories. the pt panels containing ten serum samples of known anti-hiv status together with instructions and report forms were distributed to participating laboratories twice a year, in march and october. in 2002 and 2004, the pt panels were dispatched once to 17 and 34 laboratories, respectively. however, the panels were sent out twice yearly in 2003 and from 2005 to 2010. for each shipment, ten serum specimens in united nations (un) approved packages including instruction notes and reporting forms were transported to laboratories by an iata-certified company.11 to reduce shipping costs to countries with more than one participating laboratory, pt panels were shipped to the respective central level laboratories for onward distribution to other regional or peripheral laboratories. furthermore, the central level laboratories were also responsible for collating results of the pt testing and reporting to the reqas coordinators. laboratories were instructed to return results by either fax or e-mail to the reqas coordinators within 30 days of receipt of the panels. additionally, upon receipt of data from laboratories, the reqas coordinator forwarded the expected results and reports of individual laboratory performance scores on the pt panels. in addition, a distribution report comprising comparative data from all laboratories, complete results of the panel characterisation, test readings as well as methodologies used for testing each panel preparation was generated and forwarded to laboratories. utilisation of internal quality control laboratories were requested to complete a questionnaire on their use of internal quality control (iqc) materials. the purpose of the questionnaire was to determine whether iqc was routinely performed and if it was, whether laboratories were using test kit controls supplied in the test kit or ’in-house’ produced controls. analysis of data pt panels were scored based on assigned hiv serology positive or negative status of each pt sample as characterised by the eqas provider. results that were discordant from the expected result were assigned 0 points whilst concordant results were assigned 2 points with a total possible score of 20 points for 10 samples in each pt batch. in addition, a combined score was calculated according to participant and a combined score was obtained for the group as well as the coefficient of variation of the score over time. results top ↑ pt panel testing methodologies responses relating to testing methodologies received from laboratories indicated a wide range of platforms used for screening and confirmation of anti-hiv antibodies. the who recommends three testing strategies (i, ii and iii) which aim to increase accuracy viz., prevalence or diagnostic testing whilst reducing costs for determining hiv sero-status12. laboratories select the most appropriate strategy, based on prevalence and purposes of testing. only eight laboratories out of 49 participants confirmed that they are currently using who strategy ii and iii, for hiv prevalence testing with serial or parallel testing algorithms for diagnostic purposes. three laboratories out of 49 participants reported using who strategy ii with serial testing algorithm, whereas 21 laboratories reported using the serial testing algorithm only. five laboratories reported using who testing strategy iii, which differs from strategy ii as it employs a third assay platform based on different antigen preparations and/or different test principles from assay platforms used in screening tests12. of the 49 laboratories, 15 performed rapid hiv testing only whilst seven performed elisa testing only. fifteen laboratories performed both elisa and rapid hiv testing. ten of 49 laboratories reportedly used line probe assays. of these, four laboratories used western blot assays, four used inno-lia assay and two used the hiv blot 2.2. twelve laboratories did not report on what type of testing was used (figure 1). figure 1: platforms used by participating laboratories to test the proficiency testing panels 2002-2010. participation and response rate of laboratories the reqas has to date registered 49 laboratories in 30 african countries (figure 2). thirteen countries, including botswana, comoros, eritrea, ivory coast, kenya, malawi, mozambique, rwanda, senegal, the united republic of tanzania, uganda, zambia and zimbabwe, have more than one laboratory participating in the reqas. from 2005 to 2010, 14 laboratories were newly enrolled in the scheme, including laboratories in angola, burundi, ivory coast, kenya, liberia, malawi, rwanda, sierra leone, uganda, the united republic of tanzania and zambia. in 2010, five regional laboratories in tanzania, currently preparing for accreditation, enrolled in the reqas. laboratories in cameroon, the democratic republic of congo (drc), the republic of congo and south africa were subscribing to the reqas coordinated by both nicd and the nrl in senegal. figure 2: countries currently participating in the who/reqas for anti-hiv serology. at the start of the surveys, 17 laboratories enrolled in the reqas; however the number of participating laboratories increased more than two-fold with 49 laboratories participating at the end of 2010. the response rate by participating laboratories was also observed to increase from 65% for pt panel batch 001 in 2002 to 95% for pt panel batch 016 in 2010. however, the response rate declined to 60% for pt panel batch 008 (figure 3). although, the need for routine testing of pt panels was emphasised by the reqas coordinators, in several facilities pt panels were not processed according to the routine testing algorithm or results of the pt panels were not submitted within the 30-day deadline stipulated by the reqas coordinators. for most laboratories, failure to use the national testing algorithm and/or to return results on time was mainly due to lack of sufficient financial resources, reagents and/or non-functional equipment to complete testing at the time of the external assessment. hence, in some cases laboratories tested pt samples using only one test kit, as opposed to the routine testing algorithm of the laboratory, whilst others reported using expired test kits. additional problems encountered related to improper handling and processing of pt panels as well as inadequate human resources, which invariably affected the post-analytical stages of data reporting and analysis. figure 3: summary of eqa results for the survey period during 2002–2010. pt panel testing results within the 30-day deadline, participating laboratories returned results of the pt panels to the coordinators of the reqas via email, fax or regular postage. a zero and two score was assigned for discordant and concordant results, respectively. hence a maximum score of 20 was attainable if participating laboratories results were 100% concordant with the expected results per distribution. a score ≥ 85% (≥ 17/20) was set as the cut-off for acceptable performance and a root cause analysis (rca) for unacceptable results was conducted for laboratories scoring ≤ 16/20. an unassigned score for a particular distribution indicates non-participation, late responses or non-return of results. during the survey period, 42 laboratories attained the passing score (≥ 85%) with only seven laboratories receiving unacceptable scores (figure 3). the overall average score during the survey period was 99% (19.8/20) with a coefficient of variation (cv%) of < 10% for all distributions, with the exception of pt panel batch 005. the cv% for pt panel batch 005 was 16.2% (figure 4). figure 4: coefficient of variation (%) of eqa results during the survey period (2002–2010). inter-laboratory variations were mainly due to reporting of discordant results that caused an overall decrease in the average score for pt panel batches 005, 010, 013, and 014, with pt panel batch 013 having the lowest average score of 19 out of 20. moreover, the high cv% for pt panel batch 005 was as a result of 20 discordant results from nine laboratories. of these discordant results, 30% were due to discordant results reported for a panel sample characterised as hiv sero-negative. for pt panel batch 013, a panel sample characterised as hiv-1 sero-positive was reported as sero-negative by 12 out of 29 laboratories. to establish the root cause, the sample was included in pt panel batch 014–1 and 014–2. it was noted that 13 out of 36 laboratories still reported the sample as sero-negative. results of the rca indicated that laboratories failing to correctly report the sero-status of this sample for pt panel batch 013 and 014 used a fourth-generation elisa kit as part of their testing algorithm. additionally, some laboratories also reported faint bands with rapid tests and falsely interpreted the sample as hiv-1 sero-negative. thirteen out of 14 laboratories that received pt panel batch 014-2 correctly identified the two hiv-2 specimens. one laboratory failed to return results to the reqas coordinators. for pt panel batch 010, some laboratories reported receiving leaked or empty samples upon arrival of the pt panel shipment. however, these samples were excluded from the overall scoring. hereafter, the reqas coordinators discontinued the use of these tubes for subsequent distributions. internal quality control results based on responses to the questionnaires relating to iqc indicated that 73% of laboratories routinely used iqc materials. of these, 92% used ‘in-house’ prepared controls, whereas 8% reported using iqc materials supplied with the test kits. furthermore, of the laboratories that responded using ‘in-house’ iqc material, 58% indicated that a single serum or plasma specimen was utilised as iqc material whilst 39% of the responding laboratories used multiple sera and/or plasma and 1 lab used ‘other’ (positive control is diluted with the negative control to make a weakly positive). furthermore, 45% of laboratories indicated that iqc materials were included in each eia plate run, 34% of the laboratories reported using iqc with each new batch of test kits, 16% of laboratories reported that iqc materials were included daily, and 5% of laboratories reported including iqc on weekly basis. discussion top ↑ the comparative performance data generated by the reqas illustrates the added value of quality assured laboratory services to strengthening health systems. in general, the reqas demonstrated that public health laboratories are able to accurately determine the hiv sero-status of clinically derived specimens, within the defined parameters of the scheme. the format of hiv serology testing methods varied over the survey period; microplate-based eias were mostly used at the start of the survey but were increasingly replaced with simple and rapid testing methods. the switch may have been driven by the numbers of specimens tested daily and/or budgets of participating laboratories. retrospective evaluation over the nine-year survey period indicated a 98.8% overall concordance between reported and expected results from participating laboratories, with the highest percent of overall errors found in eias. noticeably, during the survey period, fourth-generation (i.e. combined detection of hiv antigen and antibody) eia test kits were observed as giving the most discordant results. interestingly, most laboratories using the hiv vironostika uni-form ag/ab (biomérieux, france) switched to using either the murex hiv 1.2.0 (diasorin, italy) or the vironostika hiv uniform ii plus o (biomérieux, france). with the exception of challenges with rapid tests kits used by a few laboratories reporting discordant results for pt panel batches 013 and 014, minimal errors appeared with other testing platforms. the reason for discordant and/or equivocal results with the fourth-generation eias is not clear and is beyond the scope of this paper. however, pt panels were characterised using third-generation assays, genscreen hiv 1/2 (bio-rad, germany), vironostika uniform ii plus o (biomérieux, france) and murex hiv 1.2.o (diasorin, italy), hence it is important to determine the process of achieving cut-offs for testing using fourth-generation assays to understand the difference in performance to third-generation assays. other sources of false reporting may be due to inappropriate pt panel storage conditions upon receipt, test kit storage and handling, use of expired test kits as well as failure to follow the routine testing algorithm. testing algorithms are defined as the combination and sequence of specific test kits used in a given testing strategy; it also describes the sequence of tests to be performed. the key to achieving the final result is to always follow the sequence of the tests in the algorithm. from results of the nine-year survey, it was apparent that most laboratories failed to adhere to their national algorithms. some laboratories relied on one test for determining hiv-1 sero-status, whilst others were performing additional testing on pt specimens found to be non-reactive on the first screening test, which often led to inconclusive overall results. furthermore, laboratories indicated that confirmatory tests were routinely performed on site for all reactive specimens. hence, it was noted that 95% of laboratories performed confirmatory testing on reactive samples that were part of the pt panel whilst 5% reported performing screening tests on the pt panels. additionally, some laboratories failed to realise the importance of ongoing participation in an eqas, proper maintenance of records and application of corrective actions to improve testing services. to address the issues of non-response by participating laboratories which were classified as either panel received but not processed, panel received but problems with testing (either routine or panel-specific) or panel processed but results not submitted or submitted late, the coordinators of the reqas continued to encourage laboratories to report problems encountered with testing of the pt panels, in case assistance is required in resolving a specific laboratory problem. furthermore, the reqas coordinators established the rca concept for laboratories. rca aims to determine the reasons(s) for an inadequate or poor performance as well as non-responses to allow the reqas coordinators to provide the required technical support to solve problems that exist in the laboratories. on the other hand, the rca allowed laboratories to assess whether the pt panels were received in a satisfactory condition or whether the correct samples were tested using the appropriate method as stipulated in their site-specific standard operating procedures. the reqas is at no cost to participating laboratories, as the scheme is funded mainly by who afro through donor support. with increasing participation of laboratories, the reqas using liquid serum panels may not be sustainable, especially with high cost of courier transport particularly with the requirements for cold chain transport. to alleviate these challenges, the reqas coordinators plan to pilot dried tube specimen (dts) technology in 2011. dts technology is a practical and cost effective methodology for assuring the quality of serological testing in resource limited settings13. another limitation of the reqas was the distribution of mostly hiv-1 env subtype b and c panels to laboratories that routinely test non-subtype b or c samples. henceforth, the coordinators of the scheme have considered inclusion of non-subtype c hiv-1 and hiv-2 sera as part of the pt panel batches. in summary, analysis of results of the reqas presented in this report provides evidence that public health laboratories in the african region are capable of accurately determining hiv sero-status albeit with challenges related to human and financial resources. moreover, the high quality of results is evidence that the majority of african laboratories have the capacity to provide quality-assured data for clinical assessment of patients and/or surveillance programmes. acknowledgments top ↑ the who/nicd reqas surveys were supported by funds from the us centres for disease control and prevention (cdc) to who afro. the authors are grateful to laboratory personnel at all participating laboratories, colleagues at the who afro, who geneva and inter-country support teams for critical review of this manuscript. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this paper. views expressed in this paper are entirely those of the authors and not of who afro. authors’ contributions f.c. (world health organisation) g.m. (world health organisation), e.h.b. (world health organisation) and a.p. (national institute for communicable diseases) were responsible for project design. f.c. (world health organisation) a.p. (national institute for communicable diseases) and m.m. (national institute for communicable diseases) wrote the paper. m.m. (national institute for communicable diseases) and m.m. (national institute for communicable diseases) were responsible for coordinating the surveys. e.g. (national institute for communicable diseases) and b.s. (national institute for communicable diseases) made conceptual contributions to the surveys. references top ↑ 1. warner-smith m, rugg d, frescura l, moussavi s. monitoring the 2001 declaration of commitment on hiv/aids. j acquir immune defic syndr. dec 2009;52 suppl 2:s77-86.2. birx d, de souza m, nkengasong jn. laboratory challenges in the scaling up of hiv, tb, and malaria programs: the interaction of health and laboratory systems, clinical research, and service delivery. am j clin pathol. 2009;131(6):849-851. 3. burnett d. iso 15189:2003: quality management, evaluation and continual improvement. clin chem lab med. 2006;44(6):733-739. 4. gershy-damet gm, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393-400. 5. guzel o, guner ei. iso 15189 accreditation: requirements for quality and competence of medical laboratories, experience of a laboratory i. clin biochem. mar 2009;42(4-5):274-278. 6. zeh ce, inzaule sc, magero vo, et al. field experience in implementing iso 15189 in kisumu, kenya. am j clin pathol. 2010 ;134(3):410-418. 7. world health organization (who) tcfdcapcataophla. guidelines for appropriate evaluations of hiv testing technologies in africa 2001. 8. hertzberg ms, mammen j, mccraw a, nair sc, srivastava a. achieving and maintaining quality in the laboratory. haemophilia. 2006;12 suppl 3:61-67. 9. world health organization. guidelines for assuring the accuracy and reliability of hiv rapid testing: applying a quality system approach; 2005. 10. wilkinson e, engelbrecht s. molecular characterization of non-subtype c and recombinant hiv-1 viruses from cape town, south africa. infect genet evol. 2009;9(5):840-846. 11. pearson je. regulatory constraints for the transport of samples and compliance with the world organisation for animal health (oie) standards for biosecurity and biocontainment. dev biol (basel). 2007;128:59-68. 12. world heath organization. hiv assays: operational characteristics (phase i) report 14 simple/rapid: world health organization; 2004. 13. parekh bs, anyanwu j, patel h, et al. dried tube specimens: a simple and cost-effective method for preparation of hiv proficiency testing panels and quality control materials for use in resource-limited settings. j virol methods. 2010;163(2):295-300. abstract introduction methods results discussion acknowledgements references about the author(s) modupe a. kuti department of chemical pathology, faculty of basic clinical sciences, college of medicine, university of ibadan, ibadan, nigeria synlab nigeria laboratories, lagos, nigeria jokotade o. adeleye department of medicine, faculty of clinical sciences, college of medicine, university of ibadan, ibadan, nigeria joshua o. akinyemi department of epidemiology and medical statistics, faculty of public health, college of medicine, university of ibadan, ibadan, nigeria olajumoke a. ogundeji department of chemical pathology, university college hospital, ibadan, nigeria olusola o. omoyele synlab nigeria laboratories, lagos, nigeria department of chemical pathology, faculty of basic clinical sciences, lagos state university college of medicine, lagos, nigeria oluwadamilare a. obe synlab nigeria laboratories, lagos, nigeria department of medical microbiology, faculty of basic clinical sciences, lagos state university college of medicine, ikeja, nigeria ademola s. adewoyin synlab nigeria laboratories, lagos, nigeria department of haematology, faculty of basic clinical sciences, college of medicine, university of lagos, idi araba, nigeria oyetunji o. soriyan synlab nigeria laboratories, lagos, nigeria department of chemical pathology, faculty of basic clinical sciences, college of medicine, university of lagos, idi araba, nigeria department of chemical pathology, lagos university teaching hospital, idi araba, nigeria citation kuti ma, adeleye jo, akinyemi jo, et al. assessing the clinical implications of low-density lipoprotein cholesterol equations using nigerian data. afr j lab med. 2025;14(1), a2729. https://doi.org/10.4102/ajlm.v14i1.2729 original research assessing the clinical implications of low-density lipoprotein cholesterol equations using nigerian data modupe a. kuti, jokotade o. adeleye, joshua o. akinyemi, olajumoke a. ogundeji, olusola o. omoyele, oluwadamilare a. obe, ademola s. adewoyin, oyetunji o. soriyan received: 18 dec. 2024; accepted: 25 june 2025; published: 19 sept. 2025 copyright: © 2025. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: newer equations, which are more accurate than the friedewald formula (ff), have been published for the calculation of low-density lipoprotein (ldl) cholesterol. the impact of their adoption on decision-making has not been examined in nigerian laboratories. objective: this study examined the clinical implications of differences in estimating ldl cholesterol by the ff, martin-hopkins (mh), and sampson-national institutes of health (nih) equations. methods: between 01 january 2019 and 31 december 2023, lipid profile data, and the associated gender, were retrieved from the laboratory information system of synlab nigeria for persons aged 18–75 years. differences in ldl cholesterol estimates from the three equations, and agreement with category assignments that determine clinical decisions, were examined. results: lipid profile data from 19 126 records were retrieved. this included data from 8234 (43.1%) women. the difference between ff estimates of ldl cholesterol and the other two equations was less than 10% for over 96% of the data. this difference increased with triglyceride levels. there was at least substantial agreement in the clinical category assignment of the equations, (ĸ > 0.715, p < 0.001). however, when triglycerides were > 1.69 mmol/l, the ff classification of < 1.81 mmol/l was classified as > 1.81 mmol/l in 43.3% and 25.1% of cases by mh and sampson-nih, respectively. for triglycerides > 4.51 mmol/l, there was constant bias, with mh higher than sampson-nih. conclusion: using the ff formula may significantly impact primary prevention of atherosclerotic cardiovascular disease. switching to the mh or sampson-nih equation is advisable. what this study adds: this study provides a basis for nigerian laboratories to switch from the friedewald formula to one of the newer equations for the calculation of ldl cholesterol. keywords: calculated low density lipoprotein cholesterol; friedewald formula; martin-hopkins equation; sampson-nih equation; lipid profile; ldl cholesterol clinical categories. introduction low-density lipoprotein (ldl) cholesterol is a measure of the cholesterol mass transported within ldl and plays a prominent role in the pathogenesis of atherosclerotic cardiovascular disease. although it can be measured directly in the laboratory, laboratories usually calculate ldl cholesterol using the friedewald formula (ff) in persons whose plasma triglyceride concentrations are below 4.52 mmol/l.1 the ff obviates the need for the time-consuming, laborious and expensive preparative ultracentrifugation previously required for the determination of ldl cholesterol.2 furthermore, the ease of using the formula, its cost effectiveness, convenience and relatively good accuracy in most clinical scenarios led to its global adoption and continued wide usage in most routine laboratories over the last 50 years.3 the availability of direct assays for ldl cholesterol in the late 1990s had minimal impact on this practice, as their introduction to laboratory operations meant an additional testing step and, consequently, increased costs. these direct assays also demonstrated susceptibility to significant interferences from hypertriglyceridaemia.4,5 there are, however, challenges with the use of the ff. firstly, its inability to accurately estimate ldl cholesterol when the triglycerides are ≥ 4.52 mmol/l means that the laboratory needs to have an alternative means of determining and reporting ldl cholesterol in such scenarios. it has also been shown that the assumption in the formula of a fixed ratio of triglyceride levels to very low-density lipoprotein (vldl) cholesterol of 5:1, when triglycerides are < 4.52 mmol/l, is physiologically inaccurate. according to the lipid research clinics prevalence study, this ratio may actually vary between 5 and 9.6 a further implication of the above is that the ff may not be suitable when chylomicrons are present in the circulation. this may be particularly problematic with the increasing adoption of non-fasting specimens for initial lipid testing.7,8 the ff is also unsuitable for use in persons with dysbetalipoproteinaemia (type iii hyperlipoproteinaemia).2 in response to these limitations, several alternative equations have been developed. of these, the martin-hopkins (mh) equation9 and the sampson-national institutes of health (nih) equation10 are the most recent and pre-eminent. unlike the ff, these two equations do not use a fixed factor for the triglycerides:vldl cholesterol ratio. the original mh equation, published in 2013,9 determines the factor based on the ratio of triglyceride to non-high-density lipoprotein (hdl) cholesterol, and could be used with triglycerides levels up to 4.51 mmol/l. an updated version of the mh equation, which is able to report ldl cholesterol up to a triglycerides of 9.04 mmol/l, was subsequently released in 2021.11 the sampson-nih equation, released in 2020, also determines vldl cholesterol from the triglycerides and non-hdl cholesterol, and has been reported to be accurate even with triglycerides up to 9.04 mmol/l.10 the reliability and accuracy of these equations is also contributed to by the fact that their derivation involved the use of much larger sample sizes and study populations that were more diverse with regard to health status and ldl cholesterol concentrations compared to those used for the ff.1,9,10 the majority of the laboratories in nigeria continue to use the ff for the calculation of ldl cholesterol. for samples with a triglycerides value exceeding 4.51 mmol/l, either direct ldl cholesterol assays are employed, or no value is reported for ldl cholesterol and a comment is entered into the report. adoption of either of these two formulae (mh or sampson-nih) would mean that the laboratory would be able to report ldl cholesterol for a larger percentage of its clients without additional cost. this study compared ldl cholesterol results obtained with the mh and sampson-nih equations against that of the ff. it examined any clinically significant differences in the results obtained with the three equations from lipid profile values from a nigerian population. furthermore, for samples with a triglycerides value exceeding 4.51 mmol/l, an assessment of the comparability of the results obtained with the mh and sampson-nih equations in this population was also performed. methods ethical considerations this study was approved by the lagos university teaching hospital, health research ethics committee on 29 november 2024, and was performed in accordance with the declaration of helsinki. this was a review of historic data which did not involve contact with any human participants. patient consent was therefore not deemed required by the ethical board. all data were de-identified to maintain confidentiality and were stored on password-protected devices. only de-identified data were used for analysis. study design and source of data this was an observational cross-sectional study. data were retrieved from the laboratory information management system of synlab nigeria. all samples submitted to synlab nigeria for lipid studies between 01 january 2019 and 31 december 2023 were included in the analysis. only data for persons aged between 18 and 75 were included in the final analysis. the data sets were filtered so that only the first (i.e. the oldest) lipid profile data per patient was included in the analysis. synlab nigeria was selected for several reasons. it is the largest private medical laboratory in nigeria with the widest national footprint. it maintained its iso 15189 accreditation during the period of interest, and it receives samples collected from a diverse range of persons accessing care at primary, secondary and tertiary healthcare institutions. the easymedical® laboratory information system, developed by easy medical srl (cluj-napoca, romania), is health-level 7 compatible and provides comprehensive information processing services for synlab nigeria laboratories. the laboratory information system supports the electronic processing of all reportable patient testing performed by its laboratory including clinical, anatomic, and cytology test results. all completed test results are transmitted electronically from the performing machine to the laboratory information system for review and sharing with patients and doctors. data on the laboratory information management system are stored both in a cloud-based server and terrestrially. sample collection samples for lipid profile studies are collected within synlab nigeria into serum separator tubes. samples from referring institutions may be either serum or ethylenediaminetetraacetic acid plasma. individuals are normally advised to fast for at least 12 hours before presenting for sampling. laboratory measurements and ldl cholesterol calculation total cholesterol, hdl cholesterol, and triglycerides were measured on the beckmann au680 clinical chemistry analyser (beckman coulter, inc., brea, california, united states). low-density lipoprotein cholesterol is routinely calculated by ff for triglycerides < 4.52 mmol/l. no value is provided when triglycerides are ≥ 4.52 mmol/l. the ldl cholesterol is calculated by the ff thus: ldl cholesterol = total cholesterol – hdl-cholesterol – (triglycerides/2.2) [mmol/l]. an excel template for the calculation of ldl cholesterol by mh was graciously provided by professor seth martin, while that for sampson-nih equation was calculated from an excel template downloaded from https://nih.figshare.com/articles/code/equation_calculator_for_low-density_lipoprotein_cholesterol/11903274. the rendering of the sampson-nih equation is ldl cholesterol = total cholesterol/0.948 – hdl cholesterol/0.971 − (triglycerides/3.74 + [triglycerides*non-hdl cholesterol]/24.16 − triglycerides2/79.36) − 0.244 [mmol/l]. all samples with a triglycerides value > 9.04 mmol/l were excluded from the analysis. statistical analysis data are presented as mean (standard deviation) or median (interquartile range), depending on normality of the data. normality of data was assessed using the kolmogorov-smirnov test. differences in means were determined using the independent student t test, and for medians using the mann whitney u test. differences in the ldl cholesterol estimates of the equations was assessed using the nonparametric wilcoxon signed rank test. to assess the practical difference in interpreting cholesterol results, the kappa coefficient was used to assess the degree of agreement in classifying individuals into ldl cholesterol categories. for lipid profile data beyond the 4.51 mmol/l limit of the ff, the agreements between the ldl cholesterol estimates by the mh and the sampson-nih equations were assessed by spearman’s (rho) correlation coefficients and the bland-altman plot. all analyses were performed using the spss statistics for windows, version 28.0 (ibm corp., armonk, new york, united states). results were considered significant if p was 0.05 or less. results data characteristics a total of 56 669 lipid profiles were received and analysed during the study period. after removing data for repeated visits, 19 126 remained for persons between the ages of 18 and 75 years (table 1). the mean age of the women was 51.43 (s.d.: 12.99) years, and the mean age of men was 51.32 (s.d.: 12.56) years. persons aged between 35 and 64 years provided 72.42% of the data. for total cholesterol, 10 205 (53.4%) of the values were less than 5.17 mmol/l, and for triglycerides, 17 924 (93.7%) of triglycerides values were less than 2.26 mmol/l. regarding hdl cholesterol, 2561 (30.62%) women had values below 1.29 mmol/l, while 2328 (21.37%) men had values below 1.03 mmol/l. for lipid profiles with triglycerides < 4.52 mmol/l, the median (interquartile range) of all the ldl cholesterol estimates for the ff, mh, and sampson-nih equations were 3.10 (1.42) mmol/l for ff, 3.08 (1.40) mmol/l for mh, and 3.13 (1.45) mmol/l for sampson-nih, p < 0.001. table 1: characteristics of lipid profile data obtained from synlab nigeria laboratories between 01 january 2019 and 31 december 2023. differences in ldl cholesterol estimates the percentage difference between ldl cholesterol estimates was less than 10% for the ff versus mh equations in 18 388 profiles (96.1%) (table 2), and for the ff versus sampson-nih equations in 18 801 profiles (98.3%). the percentage difference exceeded 20% for ff versus mh in 258 profiles (1.3%), and for ff versus sampson-nih in 123 profiles (0.6%). the latter observations occurred when triglycerides were > 1.68 mmol/l. the median percentage difference between ldl cholesterol estimates of ff and mh, as well as between estimates of ff and sampson-nih, increased as triglycerides levels increased (figure 1). the median percentage difference between the ldl cholesterol estimates of the equations exceeded 10% at triglycerides > 3.39 mmol/l for ff/mh, and at > 3.95 mmol/l for ff/sampson-nih. table 2: percentage differences in ldl cholesterol estimates between the equations by triglyceride levels of lipid profile data obtained from synlab nigeria laboratories between 01 january 2019 and 31 december 2023. figure 1: median differences in ldl cholesterol estimates by triglyceride levels of lipid profile data obtained from synlab nigeria laboratories between 01 january 2019 and 31 december 2023. for triglycerides values between 1.69 mmol/l and 4.51 mmol/l, the kappa coefficient was 0.715 (substantial agreement) for agreement between the ldl cholesterol estimates of ff and mh, and 0.81 (almost perfect agreement) for agreement between ff versus sampson-nih. both coefficients were significant at p < 0.001. for ldl cholesterol classified as < 1.81 mmol/l by ff, 43.3% were classified as > 1.81 mmol/l by mh, and 25.1% were classified as > 1.81 mmol/l by sampson-nih (table 3). agreement with ff categorisation > 4.91 mmol/l was 100% for both mh and sampson-nih. table 3: agreement of categorisations of the ldl cholesterol estimates at triglycerides concentration 1.69 – 4.51 mmol/l lipid profile data obtained from synlab nigeria laboratories between 01 january 2019 and 31 december 2023. for the lipid profile data with triglycerides > 4.51 mmol/l, the correlation between the estimates for mean versus the difference of the mh and sampson-nih were strongly correlated; spearman’s rho = 0.992 and p < 0.01. for the same data, the median (interquartile range) estimate of ldl cholesterol was 2.86 (1.62) mmol/l for mh and 2.41 (1.69) mmol/l for sampson-nih (data not shown). figure 2 is a plot of the differences between mh and sampson-nih estimates against their means. it showed a constant bias with mh estimates higher than sampson-nih; however, almost all values were within the limits of agreement. the agreement of mh and sampson-nih ldl cholesterol categorisations had a kappa score of 0.336, p < 0.001 (data not shown). figure 2: bland-altman difference plot of ldl cholesterol estimates by mh and sampson-nih at triglycerides > 4.51 mmol/l of lipid profile data obtained from synlab nigeria laboratories between 01 january 2019 and 31 december 2023. discussion the findings in this study indicate that about 95.4% of the ldl cholesterol estimates by ff were within 0.26 mmol/l of those by the mh equation, and 99.4% for the sampson-nih equation. this represents a less than 15% difference for an ldl cholesterol concentration of 1.81 mmol/l. furthermore, the recently updated set of quality requirements for acceptable performance in proficiency testing programmes released by the clinical laboratory improvements act has a target value + 20% for ldl cholesterol.12 finally, estimates of the reference change value for ldl cholesterol, derived from biological variation data have been reported to be as high as 19%.13 the above suggests that, for the vast majority of ldl cholesterol estimates by the ff, the difference from the estimates by the newer equations may not have been clinically significant. this agrees with the observation of wang et al. among a large us cohort.14 these would suggest that the friedewald-calculated ldl cholesterol provides a reasonable estimation of ldl cholesterol and could guide treatment decisions for most patients, in addition to providing support for the recommendations of current guidelines on the continued use of this formula. our data revealed that, as triglycerides levels increased, the percentage difference between ldl cholesterol values calculated by ff and that calculated by mh increased. a similar pattern was observed between values calculated by ff and by sampson nih. this is probably because of the metabolism of vldl, the main contributor to fasting plasma triglycerides, and the structure of the different formulae. as plasma triglycerides increase, there is a change in the relative concentrations of the two vldl particle types. the proportion of large vldl1 particles, which contain about 70% triglycerides, significantly outstrip that of the smaller vldl2 particles, which consist of about 30% triglycerides. this difference in their relative concentrations widens with increasing triglycerides concentrations.15 consequent to this, the ff, which has a fixed triglycerides:cholesterol ratio, will increasingly underestimate the concentration ldl cholesterol compared to the mh and sampson-nih, which adjust their estimate of vldl cholesterol based on triglycerides concentrations. this triglycerides-influenced gradient in the difference between these equations has also been reported by others.16,17 hypertriglyceridaemia of at least 1.68 mmol/l was present in over 1 in 10 of the profiles retrieved for this study. in the context of primary prevention of atherosclerotic cardiovascular disease, hypertriglyceridaemia-related underestimation of ldl cholesterol could impact clinical decision-making. ldl cholesterol levels define the clinical category that is used to identify persons for whom statin therapy should be initiated, as well as those who are achieving the goal of therapy.18 from our data, about 50% of ldl cholesterol categorisation by ff as < 1.81 mmol/l, with triglycerides > 1.69 mmol/l, were classified in the > 1.81 mmol/l category by the mh equation. the implication of this is that persons either requiring the initiation or an intensification of therapy may not be offered the same. these findings are very similar to several other reports and indicate a major limitation to the continued and unqualified use of the ff for primary prevention of atherosclerotic cardiovascular disease.19,20,21 recognising this limitation of the ff, guidelines on the management of blood cholesterol for the primary prevention of atherosclerotic cardiovascular disease recommend the measurement of direct ldl cholesterol or the use of another equation, such as the mh or nih, to improve accuracy of risk assessment when ldl cholesterol is less than 1.81 mmol/l.1,22 although present to a lesser degree, significant and clinically consequential underestimation of ldl cholesterol was also observed at higher ldl cholesterol concentrations and with the nih equation. for the clinical laboratory seeking to improve its support for atherosclerotic cardiovascular disease primary prevention efforts and to replace the ff formula, there are items to consider. firstly, the laboratory must decide which of the several available formulae is to be adopted. a key consideration for this decision should be scientific evidence of superior analytical accuracy in determining the concentration of ldl cholesterol as compared to the gold standard method of beta quantification. with regard to the two pre-eminent equations used in this study, there are claims and counterclaims of superior accuracy. concerns have been raised over accuracy of the variant of the gold standard technique used, the population used for the development of the formulae, as well as the basis and reliability of the factor used in estimating the cholesterol content of vldl.23,24 for primary prevention efforts, what may be of more importance is the classification of individuals into the appropriate ldl cholesterol treatment category, especially in the above discussed context when there is hypertriglyceridaemia. from our data, although the level of agreement between the two equations with each other in this scenario was better than with the friedewald equation, there were still significant differences, especially when ldl cholesterol was < 1.81 mmol/l. very similar to our findings, sajja et al.,25 following their review of the electronic health record data from patients with atherosclerotic cardiovascular disease, reported clinically significant discordance in a pairwise comparison of the sampson-nih equation and the mh equation of about 23% at ldl cholesterol values of ≥ 1.81 mmol/l. at triglycerides > 4.52 mmol/l, our data indicate only fair agreement between the two equations. these suggest that the ldl cholesterol estimates from these two equations are not interchangeable. based on these, it has been suggested that laboratories should indicate on their report which equation they are using to calculate their ldl cholesterol estimates or, if feasible, report the estimates of multiple equations.26 another major consideration for the laboratory seeking to replace the ff is the ease of implementing the new formula. the ff is a simple arithmetic formula that requires little expertise to incorporate into a laboratory information system software. the sampson-nih equation is a more mathematical bivariate quadratic equation developed by multiple least squares regression. the actual equation may require marginally higher expertise than the ff, but should still be easily implementable.10 the mh equation is more complex than the above two. it is similar to the ff, but uses an adjustable factor to divide the triglycerides instead of the fixed 5. it is rendered as ldl cholesterol = total cholesterol – hdl cholesterol – (triglycerides/adjustable factor). the adjustable factor is sourced from a 240-cell stratification of triglycerides and non-hdl cholesterol. implementation of this formula would require some coding.9 in essence, implementation of the sampson-nih equation might be easier for the majority of laboratories. limitations the effect use of historic data which may have been sourced from a population that is diverse in clinical circumstances may be unpredictable. the size of the data employed for this study may have dampened the effect of this limitation. conclusion the ff is inadequate to support current efforts at primary prevention of atherosclerotic cardiovascular disease and should no longer be recommended by guidelines. laboratories must switch to more modern equations to provide more reliable ldl cholesterol estimates. depending on the capacity of the laboratory, the implementation of the sampson-nih equation might be easiest. acknowledgements the authors gratefully acknowledge the support of prof. seth martin, prof. alan remaley, and dr maureen sampson, who provided the microsoft excel templates for the two comparative equations used in this study. we also acknowledge the management of synlab nigeria laboratories, who granted access to their data. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions m.a.k. and o.a. ogundeji conceptualised the study. m.a.k., j.o. adeleye, j.o. akinyemi, and o.a. ogundeji developed the methodology, statistics and the interpretation of the same. m.a.k., j.o. adeleye, o.o.o., and o.a. ogundeji wrote the original draft. m.a.k., j.o. adeleye, j.o. akinyemi, o.o.o., o.a. obe, a.s.a., and o.o.s. reviewed and approved the final draft. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the data that support the findings of this study are not publicly available. however, the data can be made available upon reasonable request from the 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risk continuum. j am heart assoc. 2023;12(11):e028892. https://doi.org/10.1161/jaha.122.028892 samuel c, park j, sajja a, michos ed, blumenthal rs, jones sr, et al. accuracy of 23 equations for estimating ldl cholesterol in a clinical laboratory database of 5,051,467 patients. glob heart. 2023;18(1):36. https://doi.org/10.5334/gh.1214 whelton sp, meeusen jw, donato lj, jaffe as, saenger a, sokoll lj, et al. evaluating the atherogenic burden of individuals with a friedewald-estimated low-density lipoprotein cholesterol <70 mg/dl compared with a novel low-density lipoprotein estimation method. j clin lipidol. 2017;11(4):1065–1072. https://doi.org/10.1016/j.jacl.2017.05.005 martin s, ditmarsch m, simmons m, alp n, turner t, davidson m, et al. comparison of low-density lipoprotein cholesterol equations in patients with dyslipidaemia receiving cholesterol ester transfer protein inhibition eur heart j. 2022;43(supplement_2):ehac544.2359. https://doi.org/10.1093/eurheartj/ehac544.2359 grundy sm, stone nj, bailey al, beam c, birtcher kk, blumenthal rs, et al. 2018 aha/acc/aacvpr/aapa/abc/acpm/ada/ags/apha/aspc/nla/pcna guideline on the management of blood cholesterol: executive summary. j am coll cardiol. 2019;73(24):3168–3209. https://doi.org/10.1016/j.jacc.2018.11.002 sampson m, wolska a, meeusen jw, otvos j, remaley at. the sampson-nih equation is the preferred calculation method for ldl-c. clin chem. november 2023:hvad190. https://doi.org/10.1093/clinchem/hvad190 grant jk, kaufman hw, martin ss. extensive evidence supports the martin–hopkins equation as the ldl-c calculation of choice. clin chem. december 2023:hvad199. https://doi.org/10.1093/clinchem/hvad199 sajja a, li h-f, spinelli kj, blumenthal rs, virani ss, martin ss, et al. discordance between standard equations for determination of ldl cholesterol in ptients with atherosclerosis. j am coll cardiol. 2022;79(6): 530–541. https://doi.org/10.1016/j.jacc.2021.11.042 lam r, manemann sm, seehusen ke, remaley at, st sauver jl, jiang r, et al. the clinical impact of estimating low-density lipoprotein cholesterol (ldl-c) using different equations in the general population. lipids health dis. 2024;23(1):210. https://doi.org/10.1186/s12944-024-02188-9 abstract introduction pre-ebola virus disease laboratory system post-ebola virus disease laboratory strengthening initiatives capacity-strengthening initiatives coordination of transport and referral services lessons learned acknowledgements references about the author(s) stephen b. kennedy incident management system, emergency operations center, ministry of health, monrovia, liberia partnership for research on ebola virus in liberia, liberia-us clinical research partnership program, first floor, john f. kennedy medical center, monrovia, liberia christine l. wasunna partnership for research on ebola virus in liberia, liberia-us clinical research partnership program, first floor, john f. kennedy medical center, monrovia, liberia john b. dogba national reference laboratory, ministry of health, charlesville, margibi county, liberia philip sahr partnership for research on ebola virus in liberia, liberia-us clinical research partnership program, first floor, john f. kennedy medical center, monrovia, liberia national reference laboratory, ministry of health, charlesville, margibi county, liberia candace b. eastman africabio enterprises, inc., payne avenue, sinkor, monrovia, liberia fatorma k. bolay partnership for research on ebola virus in liberia, liberia-us clinical research partnership program, first floor, john f. kennedy medical center, monrovia, liberia liberia institute for biomedical research, ministry of health, charlesville, margibi county, liberia national research ethics board, partnership for research on ebola virus in liberia, first floor, john f. kennedy medical center, monrovia, liberia gloria t. mason national research ethics board, partnership for research on ebola virus in liberia, first floor, john f. kennedy medical center, monrovia, liberia mark w.s. kieh partnership for research on ebola virus in liberia, liberia-us clinical research partnership program, first floor, john f. kennedy medical center, monrovia, liberia citation kennedy sb, wasunna cl, dogba jb, et al. the laboratory health system and its response to the ebola virus disease outbreak in liberia. afr j lab med. 2016;5(3), a509. http://dx.doi.org/10.4102/ajlm.v5i3.509 lessons from the field the laboratory health system and its response to the ebola virus disease outbreak in liberia stephen b. kennedy, christine l. wasunna, john b. dogba, philip sahr, candace b. eastman, fatorma k. bolay, gloria t. mason, mark w.s. kieh received: 20 june 2016; accepted: 15 aug. 2016; published: 31 oct. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract the laboratory system in liberia has generally been fragmented and uncoordinated. accordingly, the country’s ministry of health established the national reference laboratory to strengthen and sustain laboratory services. however, diagnostic testing services were often limited to clinical tests performed in health facilities, with the functionality of the national reference laboratory restricted to performing testing services for a limited number of epidemic-prone diseases. the lack of testing capacity in-country for lassa fever and other haemorrhagic fevers affected the response of the country’s health system during the onset of the ebola virus disease (evd) outbreak. based on the experiences of the evd outbreak, efforts were initiated to strengthen the laboratory system and infrastructure, enhance human resource capacity, and invest in diagnostic services and public health surveillance to inform admittance, treatment, and discharge decisions. in this article, we briefly describe the pre-evd laboratory capability in liberia, and extensively explore the post-evd strengthening initiatives to enhance capacity, mobilise resources and coordinate disaster response with international partners to rebuild the laboratory infrastructure in the country. now that the evd outbreak has ended, additional initiatives are needed to revise the laboratory strategic and operational plan for post-evd relevance, promote continual human resource capacity, institute accreditation and validation programmes, and coordinate the investment strategy to strengthen and sustain the preparedness of the laboratory sector to mitigate future emerging and re-emerging infectious diseases. introduction the first laboratory policy in liberia was completed in 2010 through the efforts of the national diagnostic unit–laboratory technical working group. this led to the development of a comprehensive three-year laboratory strategic and operational plan (2011–2013) for the effective operation, management and sustainability of the laboratory system in liberia.1 however, this plan was not fully instituted due to the lack of adequate funding for its implementation and operationalisation as a key pillar for the rebuilding of the resilient health delivery system of the country. the lack of adequate funding and limited human resources to support diagnostic and laboratory services remain as major challenges. with the desired goal of disease surveillance and epidemic control of the ministry of health, the major emphasis of the laboratory strategic and operational plan was to develop a sustainable plan to build and strengthen the national laboratory system and its governance structure. the need for alignment of resources and coordination to develop an integrated laboratory system with clear definition of roles and responsibilities became a priority of the ministry of health. initiatives were set to review the current situation to avoid duplication, reduce the turnaround time of test results and strengthen an effective and efficient reporting mechanism. importantly, the critical need for laboratory human resource development to adequately respond to future outbreaks was highlighted. pre-ebola virus disease laboratory system prior to the ebola virus disease (evd) outbreak in liberia, the structure of the system regulating the laboratory environment was fragmented and not well coordinated. laboratory subunits within the ministry of health, including the national reference laboratory, the national diagnostics unit and the national blood safety program, were poorly coordinated. this led to duplication and overlapping of functions as there was a poorly-defined demarcation of duties and responsibilities among these subunits. a variety of equipment platforms were frequently out of service, with limited capacity for repairs and preventive maintenance. moreover, the stock-out of needed reagents and consumables often led to an interruption of diagnostic services, such as cd4 testing, clinical chemistry and haematology, throughout the country. diagnostic testing services were often limited to clinical tests performed in the hospitals and health facilities with the national reference laboratory functionality basically restricted to performing limited testing services for epidemic-prone diseases such as measles, rubella and yellow fever. the lack of testing capacity in-country for lassa fever, dengue, marburg and other haemorrhagic fevers affected the country’s health system during the onset of the evd outbreak. this therefore added to the environment that enabled the rapid spread of evd in a system with a weak diagnostics infrastructure and limited trained human resource capacity to combat such a devastating outbreak. an extensive description of the pre-evd laboratory structures and services, laboratory infrastructure and training institutions, including the laboratory-related gaps and key challenges in liberia are further detailed in a corresponding publication entitled ‘pre-ebola virus disease (evd) laboratory system and related challenges in liberia’.2 post-ebola virus disease laboratory strengthening initiatives between april 2015 and april 2016, there has been enhanced capacity to detect evd and new approaches3,4,5 have been implemented (figure 1). several evd-dedicated laboratories were set up across the country, in close proximity to an ebola screening and/or ebola treatment unit to inform admittance, treatment, and discharge decisions as well as to enhance routine surveillance of evd. figure 1: post-ebola virus disease technology advancement. biosafety and biosecurity considerations, as well as bio-risk management, were major strategic considerations employed during the determination for the establishment of an evd testing laboratory in liberia. the current evd-dedicated laboratories in liberia are biosafety level 2, with enhanced safety features for conversion to biosafety level 3 capabilities, including fluid-resistant positively-pressurised protective suits (tyvek suits) with self-contained breathing apparatus, full face-shield or goggles, stringent decontamination procedures, and considerable oversight by laboratory managers, the ministry of health and the incident management system (ims), the coordinating body for evd response in the country. to date, these laboratories have shifted from the use of biosafety level 2 cabinets with a collapsible negative-pressure viral isolation chamber to a glove box, thereby reducing the level of personal protective equipment required and resulting in significant cost-savings. all the ‘hot suites’ in these laboratories have stringent work flow and security features (figure 2). figure 2: research laboratory strengthening. the diagnostic options to enhance evd surveillance had evolved since the outbreak in 2014. traditionally, rna detection using quantitative realtime pcr (qrt-pcr) within 3–10 days after the onset of symptoms had been used; however, the liberian ministry of health recognised the need for a sensitive and selective rapid test to detect evd at the point of care. several point-of-care immunoassays (e.g., corgenix’s reebov™ antigen rapid test; orasure’s oraquick) were granted the united states food and drug administration’s emergency-use authorisation, and the world health organization listing for the presumptive detection of ebola zaire virus. these assays are able to detect ebola virus (ebov) antigen within 30 minutes. one of the regional laboratories in liberia participated in the validation of the oraquick® rapid diagnostic test (rdt), using routine surveillance whole blood and oral fluid samples. leveraging its simplicity and high containment, the cepheid genexpert® system is now the testing platform of choice by the ministry of health, having being validated elsewhere.6 this is an automated, single-cartridge system targeting the glycoprotein and nucleoprotein genes of ebov, with test results available within 90 minutes. this system was selected over traditional qrt-pcr as being more user-friendly and sustainable in a good public–private partnership programme. the use of the oraquick rapid antigen test was initiated in liberia by the us centers for disease control and prevention, the world health organization and implementing partners in 2015, and was primarily used for surveillance of dead bodies during and after one of the sporadic clustered outbreaks in liberia as the primary test, with qrt-pcr being the confirmatory test. the lessons learned in the implementation of the rdts resulted in continued training on the use of the rdt by potential operators, such as laboratory technicians, nurses, clinicians, environmental health technicians, and mortuary staff. it was also determined that there was a need to periodically review the implementation process and devise innovative ways for verifying results, such as taking photographs of the rdt results for secondary independent analysis. the aim of this pilot was to build confidence in the use of the ebov rdt in field situations as an ebov infection screening test for dead bodies during an outbreak, then to extend its use to suspected cases and their contacts following further validation. during the outbreak, capabilities for serology (breast milk, whole blood) and genomics were developed to help determine the source of an evd cluster and evd status. at present, the 2016–2021 laboratory strategic plan is being finalised. this strategy provides a road map for a robust and sustainable laboratory network throughout the country. an electronic laboratory information system is also under development to ensure that laboratory information is synthesised and utilised for timely decision making. capacity-strengthening initiatives in terms of workforce development, there has been significant investment in training and supervision, not only on the use of the equipment but also on preventive maintenance and servicing. several liberian laboratory technicians and technologists were initially trained on ebov diagnostics by different agencies who were operating these laboratories. now, there are at least 21 competent trained laboratory personnel in ebola diagnostics. each laboratory is staffed by five technicians, on average, and a data entry officer. the team is responsible for all aspects of infection, prevention and control, biosafety, biosecurity, sample reception, processing and testing, as well as interpretation and reporting of results. the laboratory staff have received extensive training on running a pcr laboratory and also in performing traditional qrt-pcr (whole blood, oral swab, vaginal swabs and semen) and troubleshooting. accordingly, there are now several evd-dedicated diagnostic testing laboratories in liberia: (1) the national reference laboratory in margibi county; (2) the tappita evd laboratory at jackson f. doe hospital in nimba county; (3) the elwa mobile laboratory – genexpert only for whole blood samples; (4) bong evd laboratory at phebe hospital complex in bong county; and (5) the redemption hospital’s central laboratory that has been transitioned from international expert-directed to liberian-led and expert-supported based on technical assistance and support, procurement of laboratory equipment and supplies, and inventory management (figure 3). approximately 18 technicians are currently undertaking training in biomedical equipment engineering such as servicing, maintenance and repair. a number of technicians have also been trained in genexpert system maintenance and calibration. figure 3: post-ebola virus disease technology enhancement at redemption laboratory. coordination of transport and referral services an intricate and well-coordinated specimen referral (i.e., collection, transport and delivery) system supported by riders for health has also been established. the structure of the transportation and referral network conforms to universally-acceptable practices7 to ensure that specimens are delivered to the laboratory within a few hours to a maximum of 3–4 days post-collection. this network is also responsible for the distribution of specimens to laboratories within their daily testing capacities to ensure that samples are tested in a timely manner and the integrity is not compromised as a result of the transport and environmental conditions. this referral system not only supports evd surveillance but also other diseases, as the liberian health system shifts from the evd emergency response to that of routine disease surveillance and preparedness. at present, specimen acceptance and rejection criteria are more stringent, based on sample eligibility criteria, unlike during the outbreak and the 90-day enhanced surveillance periods where efforts were directed at testing every specimen received by a laboratory, even if the patient identifiers were missing, as long as the presumed evd status of the samples was determined. lessons learned some of the laboratories in the country participated in the validation of oral swab, semen and vaginal fluid testing on the genexpert system in support of the evd survivor programme. there have been lessons learned from each of the laboratories in terms of the gene targets and sensitivity of the various testing platforms when performing semen testing; therefore, care must be taken in the interpretation of the results when using traditional qrt-pcr instruments and approved ebola assays. given its sensitivity, the genexpert ebola assay has largely been used to resolve the observed discrepancies. redemption hospital, a government-owned, free-service health facility, has adopted an integrated testing model since installation of the genexpert system in july 2015 for ebov detection, with full implementation four months later. cepheid’s xpert ebola assay had been extensively validated for use with whole blood. this has benefited redemption hospital, which was among the hardest-hit hospitals, where triage and other important clinical decisions were made within two hours of receiving an evd suspect’s specimen. additionally, collaborations with other vertical programmes, such as the national aids control program and the national tuberculosis & leprosy control program, has been fostered, thereby translating to better use of limited resources such as shared equipment and laboratory staff. plans are underway to roll-out the genexpert systems in laboratories in some of the political sub-divisions of the country based on disease burden (e.g., hiv, tuberculosis); geographic location; patient density; and number of ebov tests requested on a weekly basis. conclusion and recommendations the following recommendations are proffered to help mitigate challenges faced by the laboratory system: establishment of an integrated laboratory system with a well-coordinated mechanism for communication and interaction to ensure that duties and responsibilities are not duplicated. such a system will likely reduce the confusion and safeguard needed resources in order to meet the national goals. instead of multiple programmes, all laboratory or diagnostics functions should be coordinated under one umbrella. developing a comprehensive laboratory strategic plan and operational plan to meet current challenges is critical. the previous national laboratory strategic and operational plan was for the three years spanning 2011 to 2013. if done, this will guide the integrated laboratory system in aligning its stipulated goals with the country’s national health agenda and plans for rebuilding a resilient health system. resources can then be sourced to support this comprehensive laboratory strategic plan. formulate a comprehensive training plan for upgrading the skills of laboratory personnel incorporated within the strategic plan. this should clearly indicate the training of laboratory personnel to become specialists in various areas ranging, for example, from immunology, microbiology, molecular biology, zoonosis, parasitology, biochemistry, biomedical engineering, virology, pathology, field epidemiology, laboratory management and disaster management, to laboratory information systems. in addition, capacity is needed at national universities and laboratory training institutions to provide advanced degrees (e.g., msc, phd) in laboratory programmes, support accreditation and continual education, and create a platform for career advancement in the field of laboratory medicine. develop evd guidelines and standard operation procedures to regulate the donation of medical equipment, laboratory platforms and standardised laboratory equipment in-country, and solicit service contracts for the preventive maintenance and repair of broken equipment to ensure the continuity of diagnostic services. after standardisation, prioritise the issuance of contractual agreements to suppliers and manufacturers that have sales representatives and service centres in-country or within the sub-region, with restrictions stipulated regarding the time taken to respond to technical breakdown. ensure that biosafety and biosecurity, including quality control and assurance, become an integral part of the revised post-evd national laboratory strategic and operational plan. acknowledgements the authors acknowledge the contributions of the incident management system of liberia’s ministry of health and international organisations in strengthening the laboratory system in liberia. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support none. authors’ contributions the first draft of the manuscript was written by s.b.k., c.l.w., j.b.d., p.s. and f.k.b., and reviewed for scholarly content by c.b.e., g.t.m. and m.w.s.k. laboratory resources and materials were provided by s.b.k., p.s., c.b.e., f.k.b., g.t.m. and m.w.s.k. suggestions for modifications were made by j.b.d., c.l.w., p.s., c.b.e., f.k.b., g.t.m. and m.w.s.k., and subsequently incorporated by s.b.k. the final version of the manuscript was approved by all authors. references ministry of health and social welfare, republic of liberia. national diagnostics unit: strategic plan, 2011–2013. monrovia, liberia; 2011. kennedy sb, dogba jb, wasunna cl, et al. pre-ebola virus disease laboratory system and related challenges in liberia. afr j lab med. 2016;5(3), a508. http://dx.doi.org/10.4102/ajlm.v5i3.508 nouvellet p, garske t, mills hl, et al. the role of rapid diagnostics in managing ebola epidemics. nature. 2015;528(7580):s109–116. http://dx.doi.org/10.1038/nature16041 yen c-w, de puig h, tam jo, et al. multicolored silver nanoparticles for multiplexed disease diagnostics: distinguishing dengue, yellow fever, and ebola viruses. lab chip. 2015;15(7):1638–1641. http://dx.doi.org/10.1039/c5lc00055f kaushik a, tiwari s, dev jayant r, et al. towards detection and diagnosis of ebola virus disease at point-of-care. biosens bioelectron. 2015;75:254–272. http://dx.doi.org/10.1016/j.bios.2015.08.040 schnippel k, meyer-rath g, long l, et al. scaling up xpert mtb/rif technology: the costs of laboratoryvs. clinic-based roll-out in south africa. trop med int health. 2012;17(9):1142–1151. http://dx.doi.org/10.1111/j.1365-3156.2012.03028.x us centers for disease control and prevention. guidance for collection, transport and submission of specimens for ebola virus testing. updated january 2015. atlanta, ga: cdc; 2015. abstract history of laboratory networks in africa the global health security agenda and national laboratory networks major requirements for functional national laboratory networks acknowledgements references about the author(s) michele best corporate director of laboratories, dimensions healthcare system, cheverly, maryland, united states jean sakande laboratory of biochemistry, university of ouagadougou, ouagadougou, burkina faso citation best m, sakande j. practical recommendations for strengthening national and regional laboratory networks in africa in the global health security era. afr j lab med. 2016;5(3), a471. http://dx.doi.org/10.4102/ajlm.v5i3.471 lessons from the field practical recommendations for strengthening national and regional laboratory networks in africa in the global health security era michele best, jean sakande received: 18 apr. 2016; accepted: 15 aug. 2016; published: 31 oct. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract the role of national health laboratories in support of public health response has expanded beyond laboratory testing to include a number of other core functions such as emergency response, training and outreach, communications, laboratory-based surveillance and data management. these functions can only be accomplished by an efficient and resilient national laboratory network that includes public health, reference, clinical and other laboratories. it is a primary responsibility of the national health laboratory in the ministry of health to develop and maintain the national laboratory network in the country. in this article, we present practical recommendations based on 17 years of network development experience for the development of effective national laboratory networks. these recommendations and examples of current laboratory networks, are provided to facilitate laboratory network development in other states. the development of resilient, integrated laboratory networks will enhance each state’s public health system and is critical to the development of a robust national laboratory response network to meet global health security threats. history of laboratory networks in africa efforts to combat hiv, tuberculosis and malaria in sub-saharan africa in the past decade have underscored the inadequacy of laboratory systems in most african countries, as they were not equipped to perform vital testing for hiv care and prevention programmes. laboratory testing is required for the diagnosis and management of these diseases and many other communicable and non-communicable diseases endemic to the african region. for many years, there had been steady neglect of national health laboratories in africa, leading to a lack of trust in laboratory results and empiric treatment of patients. early improvement efforts employed a vertical approach, creating national hivand aids-specific laboratories. this approach has proved inadequate, as it created competition for resources and did not improve the entire laboratory system in the countries.1 it became clear that all laboratories in a country should be part of a fully-integrated laboratory network that can address both individual patient care needs and public health needs from the peripheral levels to the national level. in the past decade, there has been a focus on the development of a national laboratory network that is sustainable and will be more efficient in the use of limited resources. in 2008, a number of international meetings helped to focus efforts on an integrated network approach to laboratory system strengthening efforts. these included the maputo declaration in mozambique2 and the world health organization’s regional office for africa (who afro) resolution afr/rc/58/r2, which was issued during the 58th session of the regional committee held in yaoundé, cameroon, in september 2008.3 the creation of the stepwise laboratory (quality) improvement process towards accreditation system by who afro4 and the strengthening laboratory management toward accreditation programme in 2009 were major steps toward quality improvement of laboratories in the region.5 these actions led to the development of national laboratory strategic plans and a national laboratory policy in numerous countries, including botswana, burkina faso, eritrea, ethiopia, cameroon, kenya, liberia, mali, mauritius, senegal and tanzania. one focus of these countries has been the development of an integrated, tiered national public health laboratory network that can provide high-quality, accessible and efficient laboratory testing services for the entire population. there are many examples of functional laboratory networks in the african region. the polio laboratory network in african region has played a critical role in diagnosing poliovirus disease (poliomyelitis) and the detection of poliovirus transmission, resulting in many countries in africa having been declared free of polio.6 who afro supports a public health laboratory network, including national reference centres, sub-regional and regional reference laboratories for specific diseases and who collaborating centres for plague. this laboratory network-based surveillance of meningitis epidemics has played a significant role in the timely outbreak response in meningitis-belt countries.7 in response to the maputo declaration, a number of sub-regional networks dedicated to disease surveillance, prevention and response have been launched in africa with the support of several international development partners. one partner, the west african laboratory network, funded by the french development agency and foundation mérieux in 2009, focuses on three main areas of activity: training laboratory personnel, setting quality assurance, and strengthening epidemiological surveillance in seven west african francophone countries, including guinea, since 2013. the west african laboratory network allowed for the first diagnosis of ebola virus disease by the jean merieux p4 laboratory in lyon.8 another partner, the east africa public health laboratory network project, includes three mutually-reinforcing components which are supporting the five east africa community member states to diagnose communicable diseases of public health importance and to share information about those diseases to mount an effective regional response.9 in addition, effective national laboratory networks have been developed in a number of countries, including ethiopia and tanzania, as a result of the maputo declaration2 and resolution afr/rc58/r2.3 despite the above exemplary achievements in developing laboratory networks, the 2014–2015 ebola virus disease outbreak in west africa highlighted gaps and unmet needs in these countries and underscored the importance of redoubling disease prevention and control efforts in africa. while many countries have made progress in strengthening diagnostic and surveillance capacity, much more needs to be done, particularly to comply with the 2005 who international health regulations.10 consequently, there is a great need for a gap analysis assessment of current laboratory systems in africa to promote innovative approaches to achieving sustainable national laboratory networks throughout africa. to this end, a laboratory network assessment tool, the labnet scorecard, was developed by the african society for laboratory medicine to assist countries in assessing the development of their networks.11 the global health security agenda and national laboratory networks the global health security agenda was launched in february 2014 by a group of nations working collaboratively to promote global health security as an international priority. it is led by a global health security steering group of 10 member nations. the goal of the agenda is to accelerate progress toward a world safe and secure from global health threats posed by infectious diseases. diseases of recent global threat concern include ebola virus disease, middle east respiratory syndrome, avian influenza, yellow fever, and multi-drug resistant tuberculosis. in may 2014, a global health security commitment development meeting was held in helsinki and identified 11 global health security action packages, five of which have a laboratory component,12 including antimicrobial resistance, zoonotic disease, biosafety and biosecurity, immunization, and national laboratory system. the goal of the national laboratory system package is the development of safe and secure laboratory capacity in each country. it contains an action item to evaluate and improve capacity at national reference, provincial and district laboratories for 10 core laboratory tests. the core tests include bacterial culture for salmonella enteritidis serotype typhi, viral culture for poliovirus, pcr for influenza virus, serology for hiv, microscopy for mycobacterium tuberculosis, and rapid diagnostic testing for plasmodium spp., as well as at least four tests selected by the country on the basis of its major public health concerns. the five-year target is real-time biosurveillance with a national laboratory system and effective modern point-of-care and laboratory-based diagnostics. it is expected that the laboratory system will be able to reliably conduct testing on at least five of the 10 core tests on specimens transported to accredited laboratories from at least 80% of the districts in the country.12 in addition, laboratories must develop plans for detection and surveillance of antimicrobial resistance and surveillance of zoonotic diseases. in summary, there is a need to establish functional national laboratory networks for various reasons, including to provide laboratory confirmation for priority diseases, support national integrated disease surveillance, provide individual patient care, and implement various aspects of the global health security agenda. accurate laboratory data are essential for clinicians to accurately assess the status of patients’ health, make accurate diagnoses, formulate treatment plans, and subsequently monitor the effects of treatment. this applies to both communicable and non-communicable diseases. a tiered, integrated laboratory network with strong supporting core capabilities will provide the best model for efficient service delivery across various levels of the public health system.13 traditionally, the public health laboratory has been the sole response laboratory for public health emergencies; however, a new model has emerged,14 exemplified by the us centers for disease control and prevention’s laboratory response network, a national network of laboratories charged with identifying and characterising agents of bioterrorism and other threats to public health. in this new model, the national public health laboratory leads a network of clinical and other agency laboratories, such as food testing, veterinary, and local public health laboratories, to support public health response. major requirements for functional national laboratory networks effective implementation of national strategic plans the development of an integrated, functional, high-quality public health laboratory network requires the development of a national laboratory strategic plan to provide the vision and roadmap for its implementation. a guidance document to assist countries in development of national laboratory strategic plans was published by the who and us centers for disease control and prevention in 2008.15 this document, and samples of strategic plans from other countries, may be helpful in the development of a tiered laboratory network. each country must develop its own plan as it will be unique to its existing medical, laboratory and regulatory structure. the initial step in the development of the strategic plan is the appointment of a leadership team or working group by the country’s ministry of health that possesses deep knowledge of the current national laboratory situation.16 as part of strategic planning, the group will assess the current state of laboratories in the country with an assessment of strengths and weaknesses using the african society for laboratory medicine’s labnet scorecard for assessing the national laboratory network functionality.11 as part of the strategic planning process, leadership will determine priorities for strengthening the national laboratory network system and develop a vision, goals, objectives, expected results and strategies for the next three to five years to achieve the desired results. using the available resources and partners’ support, the working group will finalise the strategic/implementation plan with an assigned budget and time-frames. the working group should present the finalised strategic plan to ministry of health policy makers and planning authorities for approval. if approved, this plan should be incorporated into the health sector plan. following the approval of the strategic plan, obtaining essential resources, and preparation of the operational guide or a well-formulated constitution, a formational meeting should be organised. the next step is to launch the national laboratory network and begin to implement the plan. it is important to note that a strategic plan is not an implementation plan – it merely provides the strategic initiatives on which the implementation plan for the development of the laboratory network is based. based on the strategic plan initiatives, a variety of planned activities will be needed to address gaps in current systems. activities must be prioritised as certain activities are required in order for others to take place. for example, a commodities list cannot be developed until the equipment plan for the network is finalised. an implementation plan must be developed with action plans for activities that are required to accomplish key strategies. estimated costing information is required for all activities in the implementation plan for the first three years, at a minimum. adequate funding of the plan is essential for its success. ideally, an implementation team would be led by assigned laboratory leadership and a technical working group in consultation with implementation consultants. implementation of the plan is the most critical and challenging aspect of the strategic planning process. top laboratory leadership will need to own the implementation plan and monitor progress regularly. plan implementation requires a separate monitoring and evaluation plan to determine whether planned targets are being met; strategic plans must be reviewed at least annually for progress made toward objectives. the plan may need to be updated as circumstances change or at regular intervals. adequate financial support as part of the strategic and implementation plan, cost estimates must be developed for all activities in the plan. this will provide ministry of health officials with anticipated costs of the laboratory network development for the next three to five years. costs should include laboratory renovation, equipment, human resources, reagents and supplies, quality assurance, external quality assessment and accreditation, specimen referral, training, and other costs. an annual budget for the national laboratory network should be developed by the director of the division of laboratories and approved by the ministry of health as part of the health sector budget process. the budget should be adequate to cover the annual capital and operating expenses of the laboratory network. a national laboratory policy and regulatory framework as part of the health sector policy for the country, a national laboratory policy provides the regulatory framework for the laboratory network within the country. national laboratory standards are developed that apply to all laboratories operating in the country, including public, private, and mission-based laboratories. these standards cover a variety of areas, including human resources, infrastructure, equipment, in vitro diagnostic device (ivd) regulation, and analysis/activities by health system level. it should be aligned with the international health regulation and global health security agenda requirements. integrated, tiered national laboratory network development an integrated, tiered national laboratory network is an integrated system of laboratories organised in three or four tiers and aligned with the public health delivery system of the country (figure 1).17 levels of laboratories are determined by their test menus and functions, and a referral network is established in order to perform tests at the most appropriate level of the tiered system. the test menu available at each tier or level of the laboratory network would depend on infrastructure available and testing needs for patients. an example of test menus for a tiered network is shown in table 1, and an example of a grid mapping different details of key requirements by each tier of the network is shown in table 2. point-of-care and rapid test kits should be chosen carefully and used wherever possible to allow for ease of use and reliability. testing would be referred up the pyramid of laboratories with communications going both up and down. the number of tiers in the laboratory system and the test menu performed at each level may vary depending on service level needs, priority diseases, geographic coverage, referral capabilities, and resources available. it may be desirable to first prioritise the full development of the national public health laboratory (tier 4) and a good referral system, as the national reference laboratory could initially serve the testing needs for the country while the rest of the network is being developed. a plan for the geographic location and development of all laboratories in the network would be the next priority in the plan. figure 1: the integrated tiered laboratory network (level 4 in some countries includes the reference laboratories and the national public health laboratory is on top of the system). table 1: example of laboratory tests and services needed at each level of a tiered national laboratory network. table 2: example of mapping grid of laboratory requirements for different tiers. medical and public health laboratory services are a critical component of national health systems and are central to disease diagnosis, treatment, prevention, surveillance and outbreak investigations. when used optimally, laboratory testing generates knowledge that can facilitate patient safety, improve patient outcomes and lead to more cost-effective healthcare. in african countries, laboratory testing influences medical decision making in nearly half of the cases presenting to primary healthcare facilities. the vast majority of the population (except for the few who can afford private healthcare) depend on public health services for all their healthcare needs. laboratory services need to be fully integrated as a core component of health systems, yet very few countries in the african region have clearly defined the role of laboratory services at each level of the healthcare pyramid. most countries are not aware of what laboratory services are being offered to the population in terms of types of tests and their quality. as a consequence, national planning for laboratory personnel and support services is almost non-existent. in most countries, laboratory services are charged at subsidised prices and are a major income-generating unit in health facilities. however, much of the laboratory income is used to support other services, leaving the laboratory deficient in essential resources. there is an urgent need to re-examine the approach to formulating essential primary healthcare (district) laboratory packages to address the health problems in different local contexts, and to re-define issues of access, quality and how public laboratory services should be funded and sustained. for a national laboratory network to be fully functional, it must be part of the country’s universal health coverage and embody three related objectives. the first objective is to ensure equity in access to health services; everyone who needs services should get them, not only those who can pay for them. the second objective is that the quality of health services should be good enough to improve the health of those receiving services. the third objective is that people should be protected against financial risk, ensuring that the cost of using services does not put people at risk of financial harm. for a national laboratory service to meet universal health coverage goals, countries must define how a primary healthcare laboratory package should be formulated to contribute to universal health coverage in african country settings, including formulating quality laboratory packages at all levels of the national health system, ensuring equity in access to the laboratory services, and financing affordable laboratory services. both technical and administrative laboratory leadership are required to provide direction in the development of the laboratory network implementation plan. effective leadership must be in place to direct the development of the laboratory network and assure implementation of the strategic plan. the director of laboratory services in the ministry of health must have adequate authority and resources to implement the plans. an organisational chart for laboratory services should clearly define roles and responsibilities. initially, there must be a thorough assessment of the existing national laboratory network, and a technical working group should develop a thorough description of the current structure, practices, and functions of the laboratory system. the assessment should address laboratory infrastructure, equipment, staffing profiles, type of tests performed at each level, quality assurance systems, reporting, supervision and sources of funding. efforts spent in documenting these practices, including what works or does not work in each of the assessment domains, will ensure a systematic approach to improving deficient areas and building a solid foundation for a national laboratory network. in collaboration with stakeholders and public health programmes, the laboratory leadership group needs to assess the priority diseases, conditions and events that require laboratory confirmation and that are relevant to the country. consideration should be given to the 10 core tests defined in the global health security agenda national laboratory system action package.12 diseases that are top causes of mortality/morbidity in the country and have epidemic potential should be included. in addition, the leadership group would determine the individual patient care needs that require the support of clinical laboratory testing. these are the tests typically performed in association with hospital inpatient or outpatient visits. consideration should also be given to laboratory testing required for the clinical management of patients during potential epidemics, such as an outbreak of cholera or ebola virus disease. the working group would suggest which testing can be performed in the tiered network within the country and which testing must be sent to sub-regional, regional or global reference laboratories. a plan addressing the geographic coverage needs for laboratory testing, as well as the test menu, should be developed that is specific to each country. the group would also ensure the contribution of laboratory services to universal health coverage at all levels of the national health system. core capacities and infrastructure required to support effective laboratory networks in africa national laboratory networks must have core capabilities and adequate infrastructure in a number of important areas to function efficiently. the areas discussed below must be addressed in order to have a functional resilient laboratory network. universal health coverage it is crucial for each country to ensure that its national laboratory system is part of its universal health coverage. in fact, there is an urgent need to re-examine the approach to formulating essential primary healthcare (district) laboratory packages to address health problems in different local contexts, and to re-define issues of access, quality and how public laboratory services should be funded and sustained. governance and organisation to assure national ownership and leadership, governments should establish a department of laboratories within the ministry of health (maputo declaration)2 and appoint a national director of laboratories who oversees all the laboratories in the country, including public, clinical and private laboratories, as recommended by the 2008 who afro guide for national public health laboratory networking to strengthen integrated disease surveillance and response.18 the director must have the necessary authority and be able to communicate with the appropriate levels of the national health system as seen in a variety of countries, including senegal and burkina faso. the director assures that the private and public laboratories in the network are providing adequate service levels and meeting all standards for laboratory quality and safety. the national director for laboratory service is directly linked to and coordinates activities with other health sector programmes. the organogram for national laboratory services must be defined with identification of all management roles and responsibilities for the network and their relationships with other programmes. tiered laboratory network structure the structure of the tiered laboratory network must be established, taking into consideration the geographical distribution of the population in order to place laboratories in appropriate areas to assure patient care coverage. this network should be able to provide adequate laboratory testing coverage for > 95% of the population in order to meet individual patient care and public health needs. the distribution of laboratories in the country should be defined to match the distribution of the population. privateand public-sector laboratories should be included in the network. the network or national public health laboratory, in collaboration with other sectors, such as food and agriculture, should lead all laboratory functions covered by the one health initiative.19 the network must be integrated with the surveillance and public health activities of the country. channels of regular communication and specimen referral must be defined within and outside the network to assure maximum capacity to perform efficient testing. working relationships must exist between laboratories at the local, regional and international level. in addition, the network should be fully integrated into public health outbreak response protocols and surge capacity must be available. well-designed, safe laboratory facilities laboratories must have a safe and suitable physical environment with appropriate space, power, climate control, water, internet/communication system, and transport access. there should be an uninterruptible power supply supporting laboratory equipment in case of power surges. sufficient lighting, bench space, and clean water are also required. biosafety level 2, 3 or 4 requirements must be met depending on the types of testing that are to be performed. current laboratories must be assessed for the adequacy of their physical infrastructure, including alignment with existing building construction codes and biosafety levels required for laboratory testing. country standards should be defined for laboratory facility renovations and construction of new laboratories. an action plan to address laboratory facility upgrade needs should be developed and should include a prioritisation of facilities to be upgraded or built as part of the strategic plan. adequate funding must be allocated for this upgrade plan by the ministry of health and/or other partners. supply chain management system laboratory testing requires the consistent availability of hundreds of types of consumable supplies and reagents. supply chain management systems must exist at the central procurement and laboratory facility levels. the director in the division of laboratories must actively collaborate with and inform procurement and central medical stores, so that laboratory needs are met. there must be adequate and efficient procurement and distribution systems to provide adequate supplies of reagents, consumables and quality control materials. harmonisation of test equipment and assays will help limit the number of items that must be procured and kept in stock at all times. supply consumption and forecasting systems must be developed and laboratory staff trained on supply inventory management to avoid stock-outs and excessive waste. an effective procurement system must be in place that will be able to maintain supplies in central stores for distribution to all sites. surge capacity for reagents and supplies should be maintained. efficient distribution systems to the various regions and facilities must be developed, and the laboratory environment should have adequate space to store cold chain and non-cold chain supplies. in vitro diagnostic device regulation the quality of reagents is essential for quality results; however, low-quality reagents are circulating in africa. while the director of laboratories may not be directly responsible for this regulation, laboratory services is a major stakeholder. unfortunately few countries have the capacity to review each in vitro diagnostic device (ivd) for quality, safety and performance in a pre-market setting. baseline surveys of the ivd regulatory landscape in africa in 2012 revealed that regulation of ivd is a neglected area.20 the global harmonization taskforce model proposes a set of rules to place each ivd into a risk class dependent on the impact of the ivd on public and personal health.21 it is important to strengthen the capacity of countries for regulation of both preand post-market ivds. actions required include national ivd and medical device regulation policy, pre-marketing ivd registration, post-marketing surveillance and publication of a national authorised reagent list every year. in 2015, south africa released draft regulations to address ivd issues. equipment management plan laboratory equipment must be procured, maintained, and regularly replaced to assure that laboratory testing is consistently available at all sites within the network. an equipment management plan should be developed that lists the equipment required for each tier of the laboratory network. equipment should be harmonised and appropriate for the complexity of the laboratory at each tier in order to reduce the training, maintenance, and supply chain burdens for the country. harmonisation also allows for easy referral of specimens to other network laboratories, and makes the job of developing standard operating procedures, training, competency assessment, and reference intervals easier. this plan must be updated annually. all decisions on equipment for the network should be made in consultation with the director of the division of laboratories. the network should have an equipment manager to support management of all equipment placed in laboratories. a plan for equipment maintenance must be developed that assures responsive and efficient preventative maintenance (e.g., training of lab personnel) and corrective maintenance (e.g., recruitment of competent maintenance technicians, maintenance contracts with outside vendors). back-up analysers or a good specimen referral system should be in place to assure critical testing needs are met on a continuous basis. quality systems management the elements of an effective laboratory quality system include organisation, standard operating policies and procedures, a quality control system, planned quality improvement activities, external quality assessments, document control, and a plan for accreditation. these are described in detail below: organised quality unit: an organised quality unit reporting directly to the director of laboratory services must exist to develop and manage all aspects of the quality system in the country, including the external quality assessment system. laboratory quality officers should be identified at each laboratory to assist with quality system implementation. regular supportive supervision by quality unit staff or quality officers from a higher tier laboratory is required to assure that all elements of the quality system are in place in each laboratory. these visits provide mentoring and training of quality officers and laboratory staff on quality systems and test performance. standard operating procedures: standard operating procedures must be understood by staff and implemented to ensure overall test reliability, which includes accuracy and precision. after equipment and testing methods are determined and harmonised, standard operating procedures should be developed by the quality unit at the central level for all laboratories in the network. these standard documents can serve as templates for all laboratories and be modified to meet the needs of individual laboratories. this reduces the amount of time spent on developing standard operating procedures at each laboratory in the network. experts in the equipment system or assay are usually the best persons to develop the standard operating procedures for dissemination to the other network laboratories. a document control system must be in place for all laboratory documents. quality control: laboratory professionals must routinely perform and evaluate quality control samples to guarantee that test methods and equipment perform according to the established standards. quality control procedures should be standardised based on equipment and methods in use. quality control materials must be of high quality and available continuously. since these materials may have a short shelf life, special procurement methods must exist to assure continuous availability. patient testing should not be released by staff without running quality control according to standard operating procedures. quality control results must be reviewed regularly by laboratory supervisors to assure quality control procedures are run and corrective actions are taken when results do not meet standards. external quality assessment plan: an external quality assessment plan should be developed by the quality unit to specify the scope and participation of laboratories in the network. the plan should address the national external quality assessment requirements, including types of programmes required for each laboratory in the network. laboratories must participate in external quality assessment programs for critical assays in microbiology, haematology, immunology, microscopy and chemistry, in order to demonstrate that the laboratory has an acceptable level of quality. definitions of acceptable performance in external quality assessment programmes should be defined and reports available to all laboratories. laboratories must evaluate external quality assessment results and take appropriate corrective actions. the source of external quality assessment programmes (national or international) for each test should be defined in the plan. in order to reach all statewide laboratories, a national external quality assessment programme can be run by national health or reference laboratories (e.g., burkina faso).22 national health or reference laboratories should participate in an international external quality assessment programme. accreditation plan: a plan for accreditation of laboratories within the network by the national laboratory should exist using the who stepwise laboratory quality improvement process towards accreditation checklist23 and/or an international accreditation programme. the plan should define the type of accreditation planned for all laboratories in the network. national reference laboratories should try to achieve international accreditation. a timeline and roadmap for accreditation activities would be developed by the quality unit. workforce development the country should possess a human resource development strategy for the laboratory sector. job descriptions should be available that define roles, responsibilities and qualifications for the entire network. adequate numbers of well-trained, competent and motivated laboratory scientists and technologists/technicians must be available in-country to manage the laboratories in the network and to perform routine and specialised testing. strong pre-service curricula must be in place to train laboratory workers entering the field at diploma and/or degree levels. competency-based certification to perform tests within their scope of duties must be completed for all staff. national licensing or registration systems for laboratory staff would be ideal. opportunities for continuing professional development must exist to develop management, leadership, and higher level technical skills required by the network. these skills must exist to provide the required technical and administrative oversight of the laboratory network. programmes such as strengthening laboratory management towards accreditation are available to strengthen core management skills so that staff can manage technical operations and achieve accreditation. laboratory staffing appropriate workload-based staffing models should be developed for each laboratory in the system to assure adequate numbers of staff and correct skill mix. skill levels of testing personnel must be tied to the complexity of instrumentation and methods in use at each tier. once the staffing model is developed, an assessment must be made of the adequacy of technical staff in the country to meet the projected needs. plans will need to be developed to address the recruitment of the required numbers of laboratory technicians, technologists, scientists, and maintenance technicians. strategies must be developed to retain competent staff in laboratories such as career paths and adequate remuneration and incentives. information management and communication systems a system of immediate and regular reporting of district laboratory information to the provincial and to the national levels, as well as to patient care providers, would be expected. laboratories require adequate computer systems, internet access, and other electronic devices in order to transmit reports and data within the country. laboratories generate information that must be captured and transmitted either on written reports or electronically in a laboratory information system. within a national health system, a hospital information system with a laboratory component may be desirable to transmit laboratory orders and results electronically. the ability to meet patient care needs, integrated disease surveillance needs and laboratory testing/reporting with one health sector computer system will be most efficient and cost-effective for the country. an alternative is a laboratory information management system that interfaces with a variety of other systems/databases in the country. the laboratory information management system must be capable of generating a variety of management reports that provide data for public health surveillance and laboratory monitoring and evaluation purposes. these reports should be able to easily pull antimicrobial resistance and reportable disease data and reports. standardised laboratory request forms and public health report forms can be used if information systems are not available. the presence of paper forms makes email and fax capability extremely important in order for referral systems to return laboratory results back to the site of patient care and to public health authorities. free access laboratory information management system could be developed and installed in laboratories to ease the work of technicians and facilitate the transfer of epidemiological data (e.g., labbook used by west african laboratory network). it is also necessary to develop an information technology network infrastructure to support any laboratory computer system. specimen collection, referral and transport laboratories at peripheral sites in the network will usually have a more limited test menu. health facilities must have the ability for trained staff to collect and properly package specimens at one location and transport them to the next level of a tiered network for testing, without compromising the quality of the specimens or the safety of the packaging and transport staff. these specimen referral and transport systems should be integrated to include all specimens, where possible – at minimum, systems should at least be coordinated. adequate tracking and chain of custody systems must exist and standard operating procedures should exist for all specimens being referred. the ability to safely collect, package and transport specimens to another laboratory, as well as to get laboratory reports back to the originating facility or to public health authorities in a timely manner, is critical. special packaging guidelines published by the international air transport association are required for highly pathogenic materials. adequate transportation systems covering all necessary routes need to be defined for these functions. the utilisation of other existing logistics systems within the laboratory network, broader health system or private sector should be explored. biologic risk management, including waste management biologic risk management includes laboratory biosafety and biosecurity, which share the common goal of safe handling of biologic materials and keeping valuable biologic materials safe and secure during use, storage, and transport.23 biosafety. biosafety guidelines should be developed to establish safe work practices and containment levels for all laboratories. biosafety risk assessment must be done at each facility to determine the biologic risks that will be encountered in the laboratory. a determination of the biosafety level for each facility based on type of testing should guide the type of protective equipment and containment equipment required. biosafety guidelines should address all aspects of safety in the laboratory, including personal protective equipment, engineering controls, phlebotomy safety, sharps precautions, post-exposure prophylaxis, and waste management. in addition any special precautions unique to the type of pathogens being tested must be implemented. laboratories may reference the who laboratory biosafety manual, 3rd edition, for biosafety recommendations for laboratories.24 all laboratory staff must be trained on biosafety precautions specific to their work. laboratory safety audits should be performed by quality or biosafety officers to assure compliance with biosafety guidelines. biosecurity. countries will need to adopt national legislation for management of biosecurity and biosafety risks.25 these regulations would address the use and control of dangerous pathogens domestically, as well as measures to control the export of these pathogens. a biosecurity plan should be developed by the division of laboratories to establish accountability for the secure handling, storage and disposal of valuable biologic materials, such as patient specimens, reference strains, microbiologic cultures or dangerous pathogens. an initial risk assessment would be performed by laboratory management and scientific staff to determine the level of biosecurity measures needed based on the country’s needs. a distinction must be made between specimens and materials that require additional biosecurity measures and those that will be tested on a one-time basis, stored briefly then destroyed by the laboratory. standard operating procedures must be developed for handling and storage of routine specimens as well as valuable biologic materials, including dangerous pathogens. biosecurity plans will need to address all items listed in box 1. box 1: biosecurity plan features. box 2: lessons learned. priority disease surveillance a priority list of communicable diseases and dangerous pathogens should be used to determine laboratory service requirements for the country. national laboratory network laboratories should be able to conduct testing to support integrated disease surveillance for at least 10 priority infectious diseases, as described earlier. this requires an integrated, tiered laboratory network with modern diagnostic capabilities and real-time surveillance capabilities. data from laboratory test systems should be available electronically in real-time to all stakeholders in the public health sector through interconnected information systems or databases. laboratory-based surveillance must be fully implemented with a national reporting system for antimicrobial resistance. one reference laboratory in the country must be able to identify three of the seven who priority antimicrobial resistance pathogens. ideally, laboratories at regional and national levels should be able to identify pathogens and perform antibiotic susceptibility testing. a surveillance system should also be in place for the detection of three to five priority zoonotic diseases. in order to accomplish this, multi-sector collaboration must exist between the laboratory network and the agriculture sector. conclusion successful laboratory networks are the direct result of effective laboratory leadership, accurate assessment, strategic and operational planning, and significant financial investment, particularly in human resource capacity. the development of successful laboratory networks begins with a baseline assessment followed by a good strategic planning process. laboratory leadership must be empowered and provided with the resources to plan, develop and monitor the laboratory network to meet the public health needs of the country. each of the core capabilities described here must be systematically planned and implemented to assure a functional national laboratory network. the integration of the laboratory network with the public health system of the country is required to meet current and emerging disease threats in the era of global health security. acknowledgements competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support none. authors’ contributions m.b. and j.s. were both equally responsible for the writing of this article. references alemnji ga, zeh c, yao k, et al. strengthening national health laboratories in sub-saharan africa: a decade of remarkable progress. trop med int health. 2014;19(4): 450–458. http://dx.doi.org/10.1111/tmi.12269 world health organization regional office for africa. the maputo declaration on strengthening of laboratory systems. maputo, mozambique: who afro; 2008. world health organization regional office for africa. resolution afr/rc58/r2: strengthening public health laboratories in the who african region: a critical 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http://www.afro.who.int/en/clusters-a-programmes/hss/blood-safety-laboratories-a-health-technology/blt-highlights/3859-who-guide-for-the-stepwise-laboratory-improvement-process-towards-accreditation-in-the-african-region-with-checklist.html world health organization. laboratory biosafety manual, 3rd ed. geneva, switzerland: who; 2004. world health organization. biorisk management: laboratory biosecurity guidance. geneva, switzerland: who; 2006. abstract background description of the intervention lessons learnt recommendations acknowledgements references about the author(s) ado g. abubakar department of laboratory, apin public health initiatives, abuja, nigeria institute of human virology, nigeria, abuja, nigeria mary okoli department of laboratory, nigeria centre for disease control and prevention, abuja, nigeria oluwaseun o. akinmulero department of laboratory, apin public health initiatives, abuja, nigeria institute of human virology, nigeria, abuja, nigeria andrew thomas department of laboratory, institute of human virology, nigeria, abuja, nigeria temitope a. olaleye department of laboratory, apin public health initiatives, abuja, nigeria institute of human virology, nigeria, abuja, nigeria simon gabo department of laboratory, nigeria centre for disease control and prevention, abuja, nigeria samuel s. awala department of laboratory, institute of human virology, nigeria, abuja, nigeria felicia nwatu department of laboratory, institute of human virology, nigeria, abuja, nigeria blessing ugboaja department of laboratory, institute of human virology, nigeria, abuja, nigeria ifeanyi odoh department of laboratory, institute of human virology, nigeria, abuja, nigeria joseph dattijo department of laboratory, institute of human virology, nigeria, abuja, nigeria loveth akayi department of laboratory, institute of human virology, nigeria, abuja, nigeria mudiaga esiekpe department of laboratory, institute of human virology, nigeria, abuja, nigeria ayuba dawurung department of laboratory, institute of human virology, nigeria, abuja, nigeria olusola a. akanbi department of laboratory, nigeria centre for disease control and prevention, abuja, nigeria felix villeng department of laboratory, nigeria centre for disease control and prevention, abuja, nigeria item i. item department of laboratory, nigeria centre for disease control and prevention, abuja, nigeria olumide okunoye division of global hiv and tb, united states centers for disease control and prevention, abuja, nigeria william nwachukwu department of laboratory, nigeria centre for disease control and prevention, abuja, nigeria james avong department of laboratory, nigeria centre for disease control and prevention, abuja, nigeria olajumoke babatunde department of laboratory, nigeria centre for disease control and prevention, abuja, nigeria nishanth parameswaran united states centers for disease control and prevention, atlanta, georgia, united states stacie greby united states centers for disease control and prevention, atlanta, georgia, united states matthias alagi division of global hiv and tb, united states centers for disease control and prevention, abuja, nigeria mahesh swaminathan united states centers for disease control and prevention, atlanta, georgia, united states nwando mba nigeria centre for disease control and prevention, abuja, nigeria ernest yufenyuy united states centers for disease control and prevention, atlanta, georgia, united states melissa coughlin united states centers for disease control and prevention, atlanta, georgia, united states diana martin division of parasitic diseases and malaria, united states centers for disease control and prevention, atlanta, georgia, united states mcpaul okoye division of global hiv and tb, united states centers for disease control and prevention, abuja, nigeria nnaemeka c. iriemenam division of global hiv and tb, united states centers for disease control and prevention, abuja, nigeria chikwe ihekweazu nigeria centre for disease control and prevention, abuja, nigeria citation abubakar ag, okoli m, akinmulero oo, et al. lessons learnt from laboratory capacity building in supporting integrated disease serosurveillance using a multiplex bead assay in nigeria. afr j lab med. 2025;14(1), a2861. https://doi.org/10.4102/ajlm.v14i1.2861 lessons from the field lessons learnt from laboratory capacity building in supporting integrated disease serosurveillance using a multiplex bead assay in nigeria ado g. abubakar, mary okoli, oluwaseun o. akinmulero, andrew thomas, temitope a. olaleye, simon gabo, samuel s. awala, felicia nwatu, blessing ugboaja, ifeanyi odoh, joseph dattijo, loveth akayi, mudiaga esiekpe, ayuba dawurung, olusola a. akanbi, felix villeng, item i. item, olumide okunoye, william nwachukwu, james avong, olajumoke babatunde, nishanth parameswaran, stacie greby, matthias alagi, mahesh swaminathan, nwando mba, ernest yufenyuy, melissa coughlin, diana martin, mcpaul okoye, nnaemeka c. iriemenam, chikwe ihekweazu received: 07 may 2025; accepted: 16 sept. 2025; published: 09 dec. 2025 copyright: © 2025. the authors. licensee: aosis. this work is licensed under the creative commons attribution 4.0 international (cc by 4.0) license (https://creativecommons.org/licenses/by/4.0/). abstract background: serologic assays can monitor population exposure to pathogens and immunity to vaccine-preventable diseases; the multiplex bead assay (mba) can test for multiple analytes simultaneously. intervention: specimens collected during the nigeria hiv/aids indicator and impact survey (naiis) were used to estimate the seroprevalence of diseases of public health importance, and specimens collected from special studies were used to estimate the seroprevalence of the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) to improve the surveillance programmes. in 2019, luminex magpix® instruments were installed at the mba laboratory in the national reference laboratory, nigeria centre for disease control and prevention, in abuja, nigeria. dried blood spot specimens from the naiis, stored with consent for future testing, were prioritised for testing. between 2019 and 2022, the national reference laboratory mba laboratory conducted antibody and antigen assays for an estimated 130 000 naiis participants, completed two rounds of coronavirus disease 2019 (covid-19) serosurveys, and validated sars-cov-2 serological assays for use in nigeria. lessons learnt: the data generated by the mba laboratory supported funding requests for childhood immunisation and the application of the national malaria elimination programme global fund. the mba results strengthened the national hiv serial rapid testing algorithm. during the covid-19 pandemic, the laboratory validated a sars-cov-2 mba, which measured antibodies against three antigens. this multi-target assay was used to complete two additional rounds of the covid-19 household serosurvey in nigeria. recommendations: this strategy of investing in mba laboratory capacity building could serve as a model for other nations seeking to fortify their public health infrastructure. what this study adds: nigeria’s integrated disease surveillance capability was boosted through mba technology. keywords: multi-disease; serosurveillance; multiplex bead assay; capacity building; nigeria. background serosurveillance can be used to monitor population exposure to hiv, malaria, enteric pathogens, many neglected tropical diseases, and emerging pathogens and disease risks. it can also identify immunisation coverage gaps or waning immunity for vaccine-preventable diseases and risk factors associated with exposure.1,2,3,4 serosurveillance measures antibody responses to or antigens from pathogens, providing information on population immunity, prior disease exposure, or recency of infection.4,5,6 specimens collected and archived during surveys and other studies, stored appropriately in a biorepository, with consent for future testing, are potential sources of current and past pathogen exposure and immunity levels.1,6 public health programmes can leverage these data to identify changes in the epidemiology of diseases over time, monitor antibody levels in the population, and assess recent infections.7,8,9 serosurveillance data may be used to measure disease endemicity, making serosurveillance extremely useful for monitoring transmission interruption or recrudescence for diseases such as malaria, vaccine-preventable diseases and neglected tropical diseases in elimination settings.1,10 the united states centers for disease control and prevention (us cdc) support and strengthen global health security preparedness.11 the multiplex bead assay (mba) tests simultaneously for multiple analytes using a low volume of serum or dried blood spot (dbs) specimen, and it offers cost and time advantages compared to single-analyte immunoassays.12 multiplex bead assay panels developed by the us cdc have been transferred successfully to laboratories in multiple countries, resulting in a substantial public health impact. in addition, results have been used to help inform global vaccination policy, identify immunity gaps for targeted interventions, and support validation of disease elimination.8,9 the benefits of the use of the mba as an integrated serosurveillance platform across public health programmes include assessment of intervention effectiveness, guidance for evidence-based policy and strategy, and verification of disease control and elimination goals, providing cross-cutting opportunities for partnership and cost savings across organisational structures, increased feasibility of use in developing countries, and addressing the needs of traditionally neglected disease programmes.13,14,15 the nigeria hiv/aids indicator and impact survey (naiis) was a nationwide population-based household survey conducted in july–december 2018 with a sample size of over 200 000 participants between 0 and 64 years of age. the primary objectives were to estimate national and state-level hiv prevalence and viral load suppression in each of the 37 states in nigeria, as well as national hiv incidence.16,17 the nigeria multi-disease serologic surveillance using stored specimens, leveraged remnant naiis specimens stored with consent for future testing to expand surveillance for other diseases of priority interest in nigeria.16,17 we describe the process of developing mba capacity in nigeria to test stored naiis samples and leveraging that capacity to estimate a severe acute respiratory syndrome coronavirus-2 (sars-cov-2) seroprevalence during the pandemic. description of the intervention ethical considerations an application for full ethical approval was made to the national health research ethics committee and ethics consent was received on 27 august 2019. the ethics approval number is nhrec/01/01/2007-27/08/2019. the nigeria hiv/aids indicator and impact survey and nigeria multi-disease serologic surveillance using stored specimens were conducted under two different protocols, reviewed and approved by the national health research ethics committee, nigeria, the university of maryland, baltimore institutional review board, and the us cdc. the us cdc employees did not have access to personal identifying information or contact with the clients. the sars-cov-2 projects also received national health research ethics committee and us cdc approvals. these activities were reviewed by the us cdc, deemed not research, and were conducted consistent with applicable federal law and cdc policy§ (see, for example, 45 c.f.r. part 46.102(l)(2), 21 c.f.r. part 56; 42 usc; §241(d); 5 usc. §552a; 44 usc. §3501 et seq.) intervention the main objective of the nigeria multi-disease serologic surveillance using stored specimens project was to support the government of nigeria in conducting serosurveillance to understand disease epidemiology better and to provide information for public health action, specifically to control malaria, vaccine-preventable diseases, and other diseases of public health importance.18 discussions about using remnant samples to support this objective started during naiis planning stages in 2017. subsequently, the naiis protocol included consent for storage with future testing for diseases of public health importance; naiis funding included equipping and staffing the national biorepository at the national reference laboratory (nrl), nigeria centre for disease control and prevention. in january 2019, the mba laboratory of the nigeria centre for disease control and prevention started equipment procurement with support from the us cdc, the global fund to fight aids, tuberculosis and malaria, the united states president’s malaria initiative, and the bill and melinda gates foundation, through a grant from the cdc foundation. capacity building the international laboratory branch of the division of global hiv and tb and the division of parasitic diseases and malaria of the us cdc provided on-site training and technical assistance to mba staff, including sample processing (figure 1), data management, and the use and maintenance of the luminex magpix® (luminex corporation, austin, texas, united states) instruments. three mba management staff members were trained in project management at the us cdc, and luminex provided advanced equipment maintenance training. quarterly on-site mentorship was provided by a luminex-certified engineer, as well as routine and as-needed maintenance for the magpix® equipment and monthly technical assistance by subject matter experts from the us cdc atlanta to build the capacity of mba staff. the mba staff developed daily, weekly, monthly, and semi-annual equipment maintenance schedules. during the coronavirus disease 2019 (covid-19) pandemic travel restrictions, the team received virtual technical assistance from the us cdc. additional training included virtual training on scientific writing skills and a weekly journal club, where staff reviewed and presented selected articles on serosurveillance topics. figure 1: multiplex bead assay laboratory specimen flow. additional support included virtual training by the us cdc in atlanta on inter-laboratory comparison, assessing the precision and validation of a sars-cov-2 antibody test. public health england (now united kingdom health security agency) provided biosafety and risk assessment training. in addition, four mba staff members were trained on bead coupling for the mba through a collaboration between the african centre for excellence in genomics of infectious diseases and the nigeria centre for disease control and prevention. supplies and logistics the us cdc, the global fund, president’s malaria initiative, and the cdc foundation supported the commodities used in the mba laboratory. sample sets were prioritised into three tranches based on priority data needs (table 1). testing was completed between 2019 and 2022. supplies were received and maintained in the mba storeroom at the nrl and in an offsite warehouse. the weekly tracking of commodities was reported to all stakeholders to avoid stock-outs and to prevent the expiration of commodities before usage. table 1: nigeria hiv/aids indicator and impact survey (naiis) samples† tested in the multiplex bead assay laboratory, nigeria, 2019–2022. equipment maintenance initially, the mba laboratory started with two luminex magpix® machines. additional machines were added as resources became available. the laboratory now houses seven luminex magpix® machines, one of the largest in africa (figure 2). the luminex magpix® multiplexing system was chosen because of its affordability, compactness and durability. however, the equipment faced challenges such as clogging, probe jamming, xz axis assembly malfunctioning, and communication cable malfunctioning. these were addressed through capacity-building, training the mba staff to manage equipment challenges, including completing routine maintenance such as removing, sonicating, flushing, and replacing probes, syringes, and in-house tubing when maintenance staff could not travel to nigeria owing to covid-19 mitigation restrictions. in addition, the inadequate power supply to the nrl interrupted testing. however, upgrades to the nrl electrical services, including generator and rewiring upgrades, and installing appropriate uninterruptible power supplies, addressed most of the inadequate power supply problems. figure 2: a cross-section of the multiplex bead assay laboratory with six of the seven magpix® instruments at the national reference laboratory, abuja, nigeria, 2022. biosafety and biosecurity working with guidance provided by the us cdc, the nigeria centre for disease control and prevention adopted and implemented a safety plan for the mba laboratory at the nrl. multiplex bead assay laboratory staff members were trained on the appropriate handling and biosafety considerations of specimens (dbs and plasma samples). a safety team was established and a safety plan was created. safety operational practices were adopted, including bi-annual risk assessments, standard waste management protocols, and refresher training. during the covid-19 pandemic, a shift in laboratory staff operations, with longer hours, allowed activities to continue during lock-downs, with the implementation of social distancing and administrative controls. data management each mba run included a standard set of controls for quality control assessment. the assay was validated for detecting a range of antibodies and antigens. laboratory data were downloaded from the magpix® instrument, and results were exported using secure file transfer portals into a data file. each record was identified by the unique sample identification, which did not include personally identifiable information. a comma-separated values data backup system was instituted using an external storage device, managed and protected to avoid data loss and unauthorised access. quality assurance and quality control a quality assurance programme was implemented to provide credibility and to increase confidence in the mba laboratory results. external testing at the us cdc in atlanta was used to ensure accurate laboratory results and early problem identification. generally, there was good inter-laboratory reproducibility.18 implementation of iso 17025 standards it was recommended that the mba laboratory adopt the iso 17025 standard for calibration and testing laboratories. here, we outline the experience of implementing the two measures and the lessons learnt. the nrl management had enrolled the mba laboratory earlier in a mentorship programme towards achieving accreditation, the strengthening laboratory management toward accreditation, based on the iso 15189:2012 standard for medical laboratories to develop high-quality management systems. iso 15189:2022 is the international standard of choice for exhibiting competence and quality in clinical diagnostic laboratories.19,20,21 this necessitated the migration to the iso 17025 standard, similar to iso 15189. iso 17025 is suited for surveillance laboratories performing testing and calibration.22,23 impact the mba laboratory met critical malaria, measles and tetanus data call deadlines. the information from the generated data helped to strengthen the capacity to monitor and evaluate public health interventions, provided opportunities to measure changes and trends over time and, most importantly, provided opportunities to address cross-cutting issues that are difficult with vertical programmes. among these were the global fund to fight aids, tuberculosis and malaria concept note planning for the malaria grant in february 2020, the nigeria measles and rubella strategy workshop in march 2020, and the maternal and neonatal tetanus elimination planning discussions in july 2020 (women of reproductive age samples). seroprevalence estimates and modelling were used to assess disease exposure, the impact of public health interventions, supplementary immunisation activities, and routine vaccination coverage among children and women of reproductive age. the routine monitoring of this population allows one to ascertain the cumulative population immunity. using serologic testing to understand trends in the proportion of women who were exposed to covid-19 over time was helpful in determining the proportion of infected participants and in providing a valuable surveillance platform for women attending their antenatal care clinic. since the inception of the mba in june 2019, about 130 000 samples have been tested for antibodies across multiple diseases and malaria antigens using stored naiis specimens. the naiis dbs specimens allow estimation of the prevalence of malaria, vaccine-preventable diseases, and neglected tropical diseases across the age ranges 0–64 years. as a result of the testing performed by the mba team in nigeria, 13 articles have been published in peer-reviewed journals. the team has also presented abstracts at the american society of tropical medicine and hygiene annual conference. additional publications are also expected from this work. severe acute respiratory syndrome coronavirus 2 multiplex bead assay projects verification and evaluation was conducted of cross-reactivity of immunoglobulin g assay for sars-cov-2 in nigeria. the study verified the immunoglobulin g assay for sars-cov-2 with specimens collected from the nigerian population. previous independent verifications showed lower sensitivity and specificity of sars-cov-2 immunoglobulin g assays than reports and initial validations by manufacturers. further testing revealed a significant level of cross-reactivity for two commercial enzyme-linked immunosorbent assays for sars-cov-2, both of which targeted only the nucleocapsid protein. in contrast, the cdc in-house beads and the luminex xmap sars-cov-2 multi-antigen assay, targeting the spike, receptor-binding domain and nucleocapsid, performed better with less cross-reactivity, demonstrating the need to evaluate assays before implementation. the sample size included 200 nigerian institute of medical research specimens (100 sars-cov-2 positive and 100 sars-cov-2 pre-covid-19 pandemic).24,25,26 a cross-sectional survey was also conducted among healthcare workers across three states (rivers, plateau, and oyo) in nigeria on knowledge, attitude and practice. the seroprevalence of sars-cov-2 infection among medical laboratory scientists, doctors, pharmacists and nurses was assessed using a cdc-developed multi-antigen sars-cov-2 immunoglobulin g assay in a total sample size of 1271 individuals. serologic surveillance results of sars-cov-2 and other diseases of public health importance using dbs specimens from women attending antenatal clinics for their first visit in u.s. president’s emergency plan for aids relief-supported states in nigeria were also analysed for covid-19 seroprevalence. the sample size in this study was 14 400. a cross-sectional surveillance project was conducted using remnant viral load specimens from people living with hiv to assess the seroprevalence of sars-cov-2 among people living with hiv. a total of 37 400 viral load remnant plasma or dbs specimens from people living with hiv were tested from u.s. president’s emergency plan for aids relief-supported laboratories in nigeria. lessons learnt frequent quarterly supervisory visits by the cdc atlanta team ensured adherence to the protocol. which, in turn, led to quality results. international and regional partnership fostered supply chain logistics and assay standardisation significantly. periodic competency assessments for laboratory personnel reduced errors and improved the reliability of assay results. investments in training, mentorship and funding built a sustainable laboratory surveillance capacity for nigeria. this success has attracted two additional multi-pathogen surveillance grants, and an uninterrupted supply of laboratory commodities through local vendors during emergencies, such as the covid-19 pandemic, has sustained optimal laboratory operations. recommendations based on the success of the nigeria multi-disease serologic surveillance using stored specimens project, we recommend the implementation of a robust laboratory-based integrated disease surveillance in other african countries using mba technology. to create a serosurveillance hub in these countries, centralised expertise is highly recommended. training of bioengineers in magpix® maintenance is recommended for the sustainability of in-country mba services. advanced capacity building on local bead coupling, data management and the establishment of multi-pathogen mba proficiency testing programmes are recommended for country ownership and sustainability of the mba technology. acknowledgements the authors thank the government of nigeria for creating an environment that will enable the establishment of the mba. they also thank the nigeria centre for disease control and prevention, the us cdc atlanta, the us cdc nigeria, the cdc foundation, bill and melinda gates foundation, the global fund, united states president’s malaria initiative, national aids and stds control programme, national health policy for the development of primary health care, national agency for the control of aids, national biorepository centre at the nrl, institute of human virology, apin public health initiatives, and other stakeholders for their numerous supports. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions a.g.a. and m.o. contributed equally to this work. a.g.a., o.o., s.g., m.o., n.c.i., and c.i. conceived and designed the concept. a.g.a., o.o.a., t.a.o., o.a.a., and n.c.i. wrote the initial draft of the manuscript. e.y., m.c., n.p., d.m., and n.c.i. provided mentorship and technical support. a.g.a, m.o., s.g., o.o., m.o., n.c.i, and c.i. participated in administrative support of the project. a.g.a., m.o., o.o.a., a.t., t.a.o., s.g., s.a.a., f.n., b.u., i.o., j.d., l.a., m.e., a.d., o.a.a., f.v., i.i., o.o., w.n., j.a., o.b., n.p., s.g., m.a., m.s., n.m., e.y., m.c., d.m., m.o., n.c.i., and c.i. participated in the critical revision of the manuscript for intellectual content. n.c.i. supervised this work. all authors approved the final version of the manuscript for publication. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the authors confirm that the data supporting the findings of this study are available within the article and its references. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. they do not necessarily reflect the official policy or position of any affiliated institution, 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immunoassays in a nigerian population. microbiol spect. 2021;9:e00680-21. https://doi.org/10.1128/spectrum.00680-21 abstract introduction methods results discussion acknowledgements references about the author(s) naseem cassim department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa national priority programme, national health laboratory service, johannesburg, south africa lindi-marie coetzee department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa national priority programme, national health laboratory service, johannesburg, south africa deborah k. glencross department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa citation cassim n, coetzee l-m, glencross dk. modelling cd4 reagent usage across a national hierarchal network of laboratories in south africa. afr j lab med. 2023;12(1), a2085. https://doi.org/10.4102/ajlm.v12i1.2085 original research modelling cd4 reagent usage across a national hierarchal network of laboratories in south africa naseem cassim, lindi-marie coetzee, deborah k. glencross received: 21 sept. 2022; accepted: 15 feb. 2023; published: 15 may 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the national health laboratory service is mandated to deliver cost-effective and efficient diagnostic services across south africa. their mandate is achieved by a network of laboratories ranging from centralised national laboratories to distant rural facilities. objective: this study aimed to establish a model of cd4 reagent utilisation as an independent measure of laboratory efficiency. methods: the efficiency percentage was defined as finished goods (number of reportable results) over raw materials (number of reagents supplied) for 47 laboratories in nine provinces (both anonymised) for 2019. the efficiency percentage at national and provincial levels was calculated and compared to the optimal efficiency percentage derived using pre-set assumptions. comparative laboratory analysis was conducted for the provinces with the best and worst efficiency percentages. the possible linear relationship between the efficiency percentage and call-outs, days lost, referrals, and turn-around time was assessed. results: data are reported for 2 806 799 cd4 tests, with an overall efficiency percentage of 84.5% (optimal of 84.98%). the efficiency percentage varied between 75.7% and 87.7% between provinces, while within the laboratory it ranged from 66.1% to 111.5%. four laboratories reported an efficiency percentage ranging from 67.8% to 85.7%. no linear correlation was noted between the efficiency percentage, call-outs, days lost, and turn-around time performance. conclusion: reagent efficiency percentage distinguished laboratories into different utilisation levels irrespective of their cd4 service level. this parameter is an additional independent indicator of laboratory performance, with no relationship with any contributing factors tested, that can be implemented across pathology disciplines for monitoring reagent utilisation. what this study adds: this study provides an objective methodology to assess reagent utilisation as an independent measure of laboratory efficiency. this model could be applied to all routine pathology services. keywords: hiv; cd4; efficiency; reagents; laboratory. introduction the national health laboratory service (nhls) provides essential laboratory testing to the public health sector in south africa.1 the organisation is mandated to provide cost-effective, reliable diagnostics to all public health facilities, irrespective of size, location, or level of complexity.1 a network of well-placed laboratories, ranging from highly sophisticated and centralised academic facilities to small rural, hospital-based laboratories, provide essential services1 for both communicable (like hiv and tuberculosis) and non-communicable diseases (cancer, diabetes, and cardiovascular diseases).1 laboratory services support a vast testing repertoire, but this study focused on hiv-related diagnostics prescribed by the world health organization and the national treatment guidelines.2,3 people living with hiv are eligible for antiretroviral therapy irrespective of age, cd4 cell count, and clinical stage,2 and it is recommended that antiretroviral therapy is initiated within seven days if there are no clinical contraindications.2 laboratory services play a key role in the baseline clinical evaluation that includes confirming hiv status, screening for tuberculosis, cryptococcal disease, renal insufficiency and determining cd4 count (to assess immune status), among others.2 once patients are on antiretroviral therapy, routine hiv viral load monitoring is required, with cd4 monitoring only recommended for patients on cotrimoxazole preventive therapy.2 cd4 services in south africa are offered through an integrated tiered service delivery model that aims to extend coverage, improve turn-around time (tat), and contain programmatic costs.4,5 since the implementation of the integrated tiered service delivery model, multiple remote areas with existing community laboratories that previously referred samples have been upgraded to include cd4 services, such as in de aar, upington, and tshwaragano. the local cd4 testing in the newly established remote laboratories consequently reduced cd4 testing referrals, decentralising cd4 services, and caused a dramatic reduction in the tat of cd4 test results.6 one way of monitoring instrument performance is through the assessment of tat. turn-around time assessment measures and monitors laboratory testing efficiency and result delivery to support patient care. thus, substantial emphasis is placed on laboratory tat, with each test having a predetermined cut-off target to ensure accurate testing and timely results. various local studies reported a substantial improvement in tat performance across the network of cd4 testing facilities, with continuous monitoring through a weekly tat dashboard ensuring timely intervention where the tat target is unmet.7,8,9,10 for laboratories to meet their tat targets, they must streamline all aspects of their workflow; this includes optimising sample preparation and reagent use. effective reagent utilisation notably saves time and money and can be used as an additional measure of laboratory performance and efficiency. less efficient laboratories can be identified by assessing laboratory cd4 reagent utilisation, corrective practices implemented, and streamlined workflow processes introduced. furthermore, as all cd4 testing procedures are standardised across the nhls, including the adoption of good laboratory practice principles, practices of effective reagent utilisation can offer additional value in the quest for better service efficiencies. ‘six sigma’ is a quality management strategy that aims to improve the quality of processes and focuses on identifying and removing defects.11,12 this strategy includes ‘lean’, a methodology used to improve the efficiency of clinical laboratory procedures.11,12 one of the key factors is to identify the value stream and remove wastage.13 in the delivery of any efficient service, lean manufacturing concepts include the flow of raw material, work in progress, and finished goods, while in the case of this study, laboratory staff, analysers, and information elucidate efficiency.13,14 in a pathology setting, the flow of raw materials relates to the supply of test reagents and associated consumables required to perform a specific test. finished goods in a laboratory environment refer to the verified result delivered to the healthcare worker or originator of the test request. to produce a result, the laboratory requires trained staff to conduct testing on appropriate analysers before test results are verified on the laboratory information system (lis) for reporting. the study assessed laboratories’ optimal use of cd4 test reagents to identify laboratories that need to minimise wastage by improving and refining their workflow. the laboratories utilised national, standardised analyser platforms and protocols (standard operating procedures) and a national lis. this study aimed to model cd4 reagent use across a national network of laboratories, using data for the 2019 calendar year in south africa. a secondary objective was to review national and provincial efficiency of reagent use and reagent usage at the individual laboratory level. in laboratories where efficiency was suboptimal, a further objective was to assess whether there was any correlation between their reagent efficiency percentage and other service efficiency indicators. these service indicators included the number of service call-outs during instrument failure, days lost (as a consequence of instrument downtime), delays due to referrals (where samples are sent to sister laboratories for testing during instrument downtime), and tat performance of the affected laboratory. methods ethical considerations ethical clearance for this study was obtained from the university of the witwatersrand (m220163). only aggregate secondary laboratory data were used; our study did not require the use of any patient identifiers. all specific province and individual laboratory identifiers were removed, and sites were anonymised for this study. the human research ethics committee did not require patient consent. study design the cross-sectional study design assessed the cd4 reagent utilisation of 47 nhls laboratories in south africa for the 2019 calendar year. cd4 testing was performed on the beckman coulter fc500 mpl/cellmek and aquios cl cytometers (beckman coulter, miami, florida, united states) during the studied period. beckman coulter supplied all the cd4 reagents for both cytometers per the national tender agreement.15,16 irrespective of the instrument used, all laboratories utilised the same cd4 panleucogating reagents and national standard operating procedures.15 optimal efficiency percentage calculation the optimal efficiency percentage per 100 tests was calculated using over 18 years of cd4 testing data (from 2004) of the national priority programme.17,18 to determine the optimal efficiency percentage, we assumed an 8-h working day and a 5-day testing week and included error rates, the number of controls used (which count as one test per control tested), days lost (due to instrument downtime), and other indicators (such as electricity outages). data extraction beckman coulter (miami, florida, united states) provided aggregate data on raw materials (test reagent kits) delivered to each laboratory for 2019, and the number of tests provided from each delivery was estimated by multiplying the number of kits delivered by 300 (the number of tests per kit). the variables for this extract included: (1) reagent item number (the company’s reagent kit product identifier), (2) ‘ship-to location’ used for supply chain management (which indicates the laboratory that received the kits), and (3) number of kits delivered. in addition, beckman coulter also provided the number of service call-outs for each laboratory during 2019. in this study, finished goods were defined as the number of cd4 results authorised by qualified technical personnel on the lis (for this study, this process is termed ‘review’). the reported test volumes, identified per laboratory, were extracted by the corporate data warehouse. the specimen-level cd4 variables reported were: (1) episode number, (2) testing laboratory, (3) source laboratory, (4) province, (5) review date, (6) tat (in hours), and (7) referral status (referred or not referred). the source laboratory may not routinely offer cd4 services and would have to refer samples to a testing facility. data preparation the beckman coulter ‘ship-to location’ was matched to the respective equivalent-identified corporate data warehouse testing laboratory, for example ‘bongani hospital’ with ‘welkom’. the raw and finished materials were used to calculate the efficiency percentage (finished goods/raw materials). the analysis was conducted using microsoft excel (microsoft corp., redmond, washington, united states). all provinces and laboratories have been anonymised, for example laboratory 1 (p3), to ensure confidentiality. data analysis the following indicators were reported for each laboratory: (1) efficiency percentage, (2) number of call-outs (calls logged with the supplier helpdesk), (3) days lost, (4) sample referral percentage, (5) samples meeting tat cut-off percentage, and (6) capacity utilisation. microsoft excel was used for the capacity utilisation by calculating the throughput, for example 120 samples per 8-h shift for the aquois flow cytometer, and the test volumes performed for a defined period.15 for this analysis, we divided the annual test volumes by annual capacity, assuming hours worked and the number and type of flow cytometer platform used per laboratory. for each laboratory, the total number of call-outs logged with the beckman coulter call centre was reported. call-outs were categorised as field mentoring, field service visits, or modification type 2 (software or hardware update) reports. in consultation with beckman coulter, call-outs related to non-essential support from the supplier, including activities such as courtesy visits or initial instrument installation, were not deemed to impact service delivery and efficiency and were disregarded. daily laboratory test volumes were analysed to determine the total number of days lost. a lost day was defined as a weekday (tuesday to friday) where fewer than 15 tests were performed. test volumes over weekends, public holidays and mondays were excluded from this analysis.19 monday test volumes were excluded because, historically, there are limited hiv services on this day, resulting in low test volumes.19 the daily test volumes were coded using this threshold as 1 or 0 to determine the number of lost days. the laboratory data were used to determine the percentage of samples that were referred (where the source and testing laboratory were different in the lis). a referral was defined as a sample originating from a different laboratory from where it was tested. referrals involve the transportation of samples with delays that could affect sample stability. all references to referrals in the manuscript relate to the percentage of referred samples. a key laboratory efficiency report is the tat performance for various tests.20 turn-around time assesses laboratory service delivery speed, reliability, and efficiency.20 the nhls agrees to an annual performance plan with the national department of health, which includes key outcomes and outputs.20 the optimal national cd4 reporting tat target for 2019 was 90% of samples tested within 40 h. this tat allows timely baseline laboratory investigations to ensure antiretroviral therapy initiation within seven days.2 all laboratories within the national cd4 network are required to meet the organisation’s cd4 tat targets, which are monitored by measuring the percentage of samples that any given laboratory reports within the 40-h window. for this study, this measurement was also used and is shown as the percentage of samples that met the tat cut-off of 40 h, referred to as tat performance, for comparison to other efficiency parameters reported. a microsoft excel bubble chart was used to report the reagent efficiency percentage, the percentage of samples that met the organisational tat target, and annual raw material cost (in united states dollars). laboratory performance was then categorised as a scatter plot reporting the reagent use efficiency by tat performance. to assess any linear relationship to the percentage reagent efficiency and call-outs, days lost, referrals, and tat performance scatter plots were generated for all laboratories using stata® se (statacorp llc, college station, texas, united states). pairwise correlation coefficients between these variables were also determined. for the top and bottom performing provinces (with the lowest and highest percentage reagent efficiency), the following parameters were tabled for each testing laboratory within these provinces: (1) efficiency percentage, (2) number of call-outs, (3) days lost, (4) percentage of referrals, and (5) percentage of samples that met the tat cut-off. an analysis of the instrument capacity utilisation for these laboratories was also conducted. results we report the data for 2 806 799 cd4 tests performed in 2019 across 47 testing laboratories. there are nine provinces with 3–10 testing laboratories per province. optimal efficiency percentage we assumed an error rate of 8%, four controls per day, 4 days lost per year and 3% for other interruptions for an optimal efficiency percentage. the days lost were calculated using 50 weeks and five working days per week. two weeks were excluded to account for annual public holidays.21 thus, the calculated optimal efficiency percentage was 84.98% (table 1), and indicates that for a typical kit of 300 tests, each laboratory, in an optimal setting, should be able to produce 254 reportable cd4 results. the remaining 15% (46 tests per kit) accounts for other aspects such as daily controls, error rates, and other testing interruptions (i.e. power outages or instrument downtime). table 1: assumptions and calculation of the optimal cd4 reagent efficiency percentage, south africa, 2019. national and provincial analysis a national efficiency percentage of 84.5% was reported across all cd4 laboratories during 2019 (data not shown). the provincial analysis revealed that 53.7% of all finished materials (cd4 results) were generated by two provinces (data not shown). the provincial efficiency percentage ranged from 75.7% (p8) to 87.7% (p2) (figure 1). only two provinces reported an efficiency percentage of ≤ 80% (p8: 75.7% and p7: 79.9%). the percentage of samples that met the tat target ranged from 87.2% (p6) to 98.0% (p7). figure 1: bubble chart assessing provincial cd4 reagent utilisation of nine provinces in south africa for 2019. the efficiency percentage, percentage of samples that met the turn-around time target, and raw materials cost (united states dollars) are reported on the x-axis, y-axis, and as bubble size. laboratory reagent efficiency the laboratory efficiency percentages ranged from 66.1% (laboratory 13 [p3]) to 104.5% (laboratory 22 [p2]) (figure 2). other indicators of laboratory efficiency also varied; the percentage of samples that met the stipulated organisational cd4 tat cut-off varied from 79.5% (laboratory 19 [p4]) to 99.0% (laboratory 3 [p7]). the days lost to testing ranged from 1 day (laboratory 1 [p3]) to 52 days (laboratory 22 [p2]) (figure 2). overall, 21/47 laboratories met the optimal efficiency percentage target (44.7%) compared to 40/47 for tat performance (85.1%). quadrants a and c met the national stipulated tat target (> 90% of reported cd4 results with 40 h). quadrant a (comprising 36.2% or 17/47 laboratories) showed efficient reagent use (≥ 84.98%), while quadrant c (comprising the majority of laboratories, i.e. 53.2% or 25/47 laboratories) showed less efficient reagent usage (< 84.98%). quadrants b and d did not meet the stipulated tat; however, quadrant b (8.5% or 4/47 laboratories) showed efficient use of reagents ≥ 84.98%, while quadrant d (2.1% or 1/47 laboratories) reported < 84.98% reagent efficiency. figure 2: cd4 reagent utilisation of 47 laboratories across nine provinces in south africa for 2019. correlation of reagent efficiency percentage to other factors the scatter plot analysis for all laboratories did not reveal a linear relationship between efficient reagent use and call-outs, days lost, referrals, and tat performance (figure 3). a pairwise correlation coefficient of −1 or +1 would indicate a perfect linear relationship15; however, values of −0.0770, −0.0151, 0.0853 and −0.2596 were reported for these test parameters, confirming very weak correlation (values very close to 0). figure 3: correlation of cd4 reagent use efficiency of 47 laboratories in south africa for 2019. efficiency is plotted with (a) the number of call-outs, (b) the number of days lost, (c) the percentage of referrals, and (d) turn-around time performance. detailed analysis of individual laboratory efficiency for the top and bottom provinces the laboratories in the bottom province (p8) had an efficiency percentage ranging from 67.8% to 85.7% (table 2). only one laboratory in this province met the optimal efficiency percentage (laboratory 20). the number of call-outs for these laboratories varied from 42 to 64; however, days lost to service delivery in these same four sites ranged between 2 and 4 days. the percentage of referred samples tested in these sites also varied from 23.4% to 41.0%. conversely, all four laboratories had tat values ranging from 95.8% to 97.2%. the instrument capacity utilisation was below 85% for all testing sites (table 2). table 2: comparison of the laboratories with highest and lowest cd4 reagent use efficiency, south africa, 2019. in the best-performing province (p2), the reagent use efficiency percentage ranged from 80.7% to 104.9% across three laboratories. a range of 25–34 call-outs and 4–52 days lost was reported. only laboratory 22 reported a ≥ 90% tat performance. instrument capacity utilisation was under 62% for this province. discussion the work investigated how effectively cd4 laboratories used reagents to deliver cd4 services in south africa. the outcomes revealed that the national cd4 network reported a cd4 efficiency percentage of 84.5%, ranging from 75.7% to 87.7% at the provincial level. individual laboratory efficiency percentage went as low as 66.1%. the averaged national performance masked less efficient reagent use at provincial and laboratory levels, evident in the variability of efficiency percentages reported for provinces and laboratories. several laboratories had efficiency percentages below 18.9% of the calculated optimal reagent use percentage. thus, it is essential to assess efficiency at the decentralised level to identify the less efficient performers for appropriate corrective action and intervention. similar masking of laboratory performance was reported for tat in the cd4 testing network.10 one interesting observation was a laboratory with an efficiency reagent use of over 100%. this aberration indicates that some reagents from the previous year may have been used in 2019 (for example, careful provision for contingency and reagents for the 2018 holiday period, which was then used in 2019). diligent stock control will assist in standardising contingency reagent stocks held in laboratories. only 21 sites met the calculated optimal reagent efficiency percentage, indicating that perhaps the optimal efficiency percentage was too optimistic and failed to factor in laboratory testing volume differences. for example, a site with much higher cd4 processing volumes must perform relatively more control tests each day than a site with lower test volumes or that work only an 8-h shift versus two or three 8-h shifts. the detailed analysis for the top and bottom provinces also suggests that in addition to the standardised test platform, the workflow and stock management procedures need to be standardised across laboratories to ensure best practices. other contributing variables that could have affected the laboratory efficiency percentage include varying staff capacity (staff numbers) and expertise. these aspects are especially difficult to assess in smaller, community-type laboratories that typically employ multi-tasking rotations across pathology disciplines to deliver essential pathology services.4,6,7 instrument log data files were unavailable at the time of this study. it was, therefore, impossible to assess the number of repeated tests run, the frequency of controls performed (especially as a proportion of the total volume of tests performed), and the number of invalid runs, all of which could contribute to the lower percentage efficiency, especially in smaller cd4 volume laboratories. this study, however, highlights the value of instrument log data files and the need to collate this information on an organisation’s central data server to facilitate near real-time analysis, including reagent utilisation. other information, including days lost, duplicated testing, and the number of controls used, could also be logged and centrally monitored to identify more systemic problems in laboratories. here, instrument-collected data would be invaluable to identify practices employed in the better-performing laboratories that could be standardised across all testing sites. such an approach would make it easier to introduce corrective interventions and pair provinces with a poor efficiency percentage to their better-performing sister sites. ultimately all efficiency parameters mentioned in this article should ideally be monitored at a centralised level by a national overseeing, harmonising body that uses instrument logs and other relevant data to report on national service efficiency and performance.4,6,7 a comprehensive monitoring and evaluation plan should be developed that stipulates the indicators to be reported. the development of appropriate dashboards reporting reagent utilisation efficiency is needed to monitor performance for continual improvement of the national laboratory system. furthermore, this reagent efficiency model could be extended to other tests across a national laboratory network to improve reagent efficiency across all pathology disciplines.8,9 to accomplish this, a national lis is key to generating the data for real-time monitoring. well-narrated data provide far more granularity than aggregate data systems22 and ensure that data are continually visible from the national to the laboratory level for all tests. in our context, all laboratory data is stored in a data warehouse environment with massive server capacity for all tests in the national network.22 further investigation is required to understand how reagents are used in cd4 laboratories with higher and lower volumes to improve overall reagent usage. interestingly, the lack of linear correlation between reagent usage and other efficiency parameters, including call-outs, days lost, referrals, and tat performance, could indicate more systemic laboratory problems. although beyond the scope of this study, detailed on-site inspection at each laboratory, with full audits of procedures and practices, could address some of these questions. limitations a limitation of this study was the absence of instrument log file data. unfortunately, these data are stored on a local instrument-linked computer. in addition, the optimal efficiency percentage calculated was based on averages of laboratories studied with the assumptions stated in the methods section; outlines suggest this calculated percentage may have been too optimistic and requires further workflow studies. conclusion we reported a model of reagent efficiency across a pathology network with various service tiers. the work emphasises the importance of review at all hierarchical tiers of service, in this instance at the provincial or individual laboratory level, to detect masked poorer levels of efficiency and areas for corrective action. there was no correlation between the reagent use efficiency with days lost, call-outs, referrals, and tat performance, suggesting that although reagent usage and instrument function are both aspects that contribute to overall laboratory efficiency, these are separate issues that need to be monitored individually. underlying factors contributing to reagent wastage should be investigated to inform waste awareness strategies and standardised procedures to minimise missed diagnostic opportunities and cost implications. real-time monitoring of reagent use through instrument log data files can save costs, which is especially relevant in an expenditure-constrained setting. acknowledgements the authors would like to thank beckman coulter for providing expenditure data, and silence ndlovu of the national priority programme of the nhls for extracting the laboratory test volumes. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions n.c. and l.-m.c. developed and executed the research, conducted the data analysis, prepared the first draft and provided editorial input. d.k.g. gave editorial input and was the project leader. sources of support the authors received no financial support for the research, authorship, or publication of this article. data availability the authors do not have permission to share the laboratory data. disclaimer the views expressed in this manuscript are those of the authors and not those of the university of the witwatersrand or the nhls. references national health laboratory service (nhls). annual report 2018/19 johannesburg, south africa [homepage on the internet]: national health laboratory service (nhls). 2019 [cited 2020 feb 19]. available from: https://www.nhls.ac.za/key-documents/annual-reports/ national department of health (ndoh). art clinical guidelines for the management of hiv in adults, pregnancy, adolescents, children, infants and neonates pretoria, south africa [homepage on the internet]: national department of health (ndoh). 2019 [cited 2020 jan 18]. 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cd4 laboratory workload and related turn-around-time (tat) cape town, south africa [homepage on the internet]. 2012 [cited 2022 aug 20]. available from: https://www.researchgate.net/publication/267756209_using_central_data_warehouse_cdw_reports_for_monitoring_cd4_laboratory_workload_and_related_turn-_around-time_tat?channel=doi&linkid=5459d3fb0cf2cf516483e354&showfulltext=true national health laboratory service (nhls). annual performance plan 2020–2021 johannesburg, south africa [homepage on the internet]: national health laboratory service (nhls). 2020 [cited 2020 feb 19]. available from: https://www.parliament.gov.za/storage/app/media/docs/tpap/642703b7-8ff7-48c8-a758-d4a4292b6b28.pdf the republic of south africa. the public holidays act 36 of 1994 cape town, south africa [homepage on the internet]: republic of south africa; 1994 [cited 2022 aug 19]. available from: https://www.gov.za/sites/default/files/gcis_document/201409/act36of1994.pdf stevens ws, cunningham b, cassim n, gous n, scott le. cloud-based surveillance, connectivity, and distribution of the genexpert analyzers for diagnosis of tuberculosis (tb) and multiple-drug-resistant tb in south africa. mol microbiol 2016:707–718. abstract introduction research method and design results discussion conclusion acknowledgements references about the author(s) anneta naidoo department of virology, nelson r mandela school of medicine, school of laboratory medicine and medical sciences, university of kwazulu-natal, durban, kwazulu-natal, south africa national health laboratory services, inkosi albert luthuli central hospital, durban, kwazulu-natal, south africa raveen parboosing department of virology, nelson r mandela school of medicine, school of laboratory medicine and medical sciences, university of kwazulu-natal, durban, kwazulu-natal, south africa national health laboratory services, inkosi albert luthuli central hospital, durban, kwazulu-natal, south africa pravi moodley department of virology, nelson r mandela school of medicine, school of laboratory medicine and medical sciences, university of kwazulu-natal, durban, kwazulu-natal, south africa national health laboratory services, inkosi albert luthuli central hospital, durban, kwazulu-natal, south africa citation naidoo a, parboosing r, moodley p. real-time polymerase chain reaction optimised for hepatitis c virus detection in dried blood spots from hiv-exposed infants, kwazulu-natal, south africa. afr j lab med. 2016;5(1), art. #269, 6 pages. http://dx.doi.org/10.4102/ajlm.v5i1.269 original research real-time polymerase chain reaction optimised for hepatitis c virus detection in dried blood spots from hiv-exposed infants, kwazulu-natal, south africa anneta naidoo, raveen parboosing, pravi moodley received: 30 sept. 2014; accepted: 08 jan. 2016; published: 18 mar. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: there is a paucity of data on the prevalence of hepatitis c virus (hcv) in children, particularly in sub-saharan africa. a major obstacle in resource-limited settings for polymerase chain reaction (pcr) testing is the necessity for specimen transportation and storage at low temperatures. there are numerous recent studies of using real-time hcv pcr for diagnosis and screening of plasma and serum, but few have looked at using dried blood spot (dbs) specimens. objectives: the aim of this study was to optimise a real-time hcv pcr method to detect hcv rna from infant dbs specimens for use as a tool for hcv surveillance in kwazulu-natal, south africa. method: the lightcycler® 2.0 instrument was used for the hcv pcr using the lightcycler® rna master sybr green i kit. template volume, primer concentration and primer annealing temperatures were optimised and the method was used on 179 dbs specimens from hiv-exposed infants in kwazulu-natal. results: primer concentrations adjusted to 0.25 µm and a template volume of 10 µl improved the pcr amplification. primer annealing temperatures lowered from 65 °c to 58 °c resulted in higher quantities of amplified pcr product. the limit of detection of the optimised hcv pcr assay was between 1200 iu/ml and 3580 iu/ml of hcv rna. hcv was not detected in any of the 179 dbs specimens. conclusion: the optimised real-time hcv pcr on infant dbs specimens performed well, but hcv was not found in this surveillance study. hiv infection may have little impact on the vertical transmission of hcv in this region. introduction hepatitis c virus (hcv) infects 3% of the global population,1 but there is a paucity of data on hcv prevalence in children, particularly children in sub-saharan africa.2 hcv is usually transmitted to infants vertically, with a rate that varies from 3% to 7%, although it may increase twoto five-fold with hiv co-infection.3 the prevalence of hcv is higher amongst hiv-positive patients because of shared routes of transmission and common risk factors.4 the estimated prevalence of hcv in sub-saharan africa was 3% amongst hiv-negative individuals in 2002, but prevalence increased to 7% amongst individuals with hiv co-infection by 2014.5 the prevalence of hcv/hiv co-infection in south africa was 0.1% in 20026 compared to 3.5% amongst individuals infected only with hcv.7 there are reasons to suspect that the prevalence of hcv may be high in kwazulu-natal, south africa. firstly, kwazulu-natal has a high antenatal hiv prevalence of 40.1%.8 secondly, kwazulu-natal has a high overall adult hcv prevalence of 6.4% and a much higher prevalence of hcv/hiv co-infection (13.4%),9 which may imply a high prevalence of hcv amongst pregnant woman. the world health organization suggests that screening for hcv in regions with a high prevalence of hiv should be conducted, but depends on hcv prevalence and resources.10 in a region with a high prevalence of both hiv and hcv, especially amongst pregnant women,9 it is important to determine the prevalence of hcv amongst infants. although most children with chronic hcv infection remain asymptomatic, some may have mild abnormalities in liver function and chronic hcv infection may progress to severe liver disease, end-stage liver disease and death.2 liver disease resulting from hcv infection is a major cause of mortality and morbidity amongst adults co-infected with hiv and hcv, which further compounds the hepatotoxic effects of antiretroviral therapy.4 infant diagnosis of hcv by molecular-based technologies is superior to serological techniques for detecting hcv infection by means of maternal antibodies circulating in infants.2 a major obstacle in resource-limited settings for polymerase chain reaction (pcr) testing is the necessity of transporting and storing specimens at low temperatures. however, using dried blood spot (dbs) specimens, which do not require refrigeration, significantly reduces pre-analytical problems and is widely used in molecular virology for the detection of viral nucleic acids.11 numerous studies have reported on the use of real-time pcr for hcv diagnosis using plasma and serum,12,13,14,15,16,17,18,19,20 although few have looked at dbs specimens.21,22,23,24,25 however, a few recent studies have found good correlations for adult serum and plasma compared with dbs specimens analysed using commercial hcv pcr assays.26,27,28,29 the prevalence of hcv amongst hiv-exposed infants in kwazulu-natal, south africa is unknown. therefore, the aim of this study was to optimise a real-time pcr assay to detect hcv rna from hiv-exposed infant dbs specimens for use as a tool for hcv surveillance in kwazulu-natal, south africa. research method and design ethical considerations ethical clearance was obtained from the university of kwazulu-natal biomedical research ethics committee (be273/090). informed consent was not required since this was a retrospective study in which we used discarded specimens from routine testing. all data were recorded and then anonymised for the purposes of the study. specifically, hiv pcr results, hcv pcr results, age and clinical diagnosis were recorded anonymously in a spreadsheet. no patient identifiers were recorded. study design this study was conducted retrospectively in the laboratory at the department of virology, national health laboratory service/university of kwazulu-natal, at the inkosi albert luthuli central hospital in durban, south africa. this is a reference laboratory which receives all dbs specimens from public sector facilities in kwazulu-natal for pcr testing of dna from hiv-exposed infants for early diagnosis of hiv infection. the number of dbs specimens tested by the laboratory has steadily increased each year from 13 699 in 2005 to 73 033 in 2012. the uptake of hiv pcr testing amongst hiv-exposed infants in kwazulu-natal has also increased from 18% in 2005 to 97.4% in 2011.30 a sample size of 179 was calculated using epi-info™ 6 (centre for disease control, atlanta, georgia, united states, 1998) using the number of live births in kwazulu-natal,30 the prevalence of hiv amongst antenatal attendees (which approximates the percentage of hiv-exposed infants)8 and the approximate hcv seropositivity in infants, based on data from the laboratory information system. the sample size was representative of hiv-exposed infants who attended public health facilities in kwazulu-natal and who required hiv pcr testing because of suspected hiv exposure or clinical suspicion of hiv infection. specimen collection as part of routine early infant diagnosis of hiv, infants’ blood is collected by heel-prick and spotted onto whatman filter paper in four circles. in the laboratory, two spots are cut out and used for routine hiv pcr testing within four to five days of dbs specimen collection. after releasing the results, the card (with the two unused spots) is routinely stored at room temperature for at least three months and then discarded. these discarded dbs specimens were tested for hcv after specimens had been stored for at least three months after initial collection. specimens with inadequate volume (where the entire circle was not filled with blood) were excluded from the study. acceptable dbs specimens were sequentially selected from august to december 2010 until 179 specimens were obtained. external quality control specimens were purchased from quality control for molecular diagnostics (qcmd; glasgow, scotland) and internal quality control specimens were prepared in-house. laboratory methods before automated extraction, using the nuclisens® easymag® extraction (biomérieux, marcy i’etoile, france), two dbs (approximately 50 µl blood per spot) were cut with sterilised scissors into a 1.5 ml eppendorf tube and 2 ml nuclisens® easymag® lysis buffer containing 5 m guanidinium thiocyanate was added to the tube. the lysis buffer was also added to the quality control specimens. the specimens were agitated for 30 minutes on an orbital shaker and centrifuged at 1500 x g for two minutes. supernatant was pipetted into transfer wells, which were then loaded onto the automated extraction system. briefly, cell-bound nucleic acids were released with the chaotropic agent − guanidinium thiocyanate − and then bound to silica particles, which were immobilised on a filter. impurities were removed by several washes in buffers containing guanidinium thiocyanate, 70% ethanol and acetone, after which the purified nucleic acids were eluted in molecular grade water. the lightcycler® 2.0 instrument (roche diagnostics, mannheim, germany) was used to perform reverse transcription and amplification in a one-step assay using the lightcycler® rna master sybr green i kit (roche diagnostics, mannheim, germany), which contained the enzymes thermus thermophilus dna polymerase, dntps, reaction buffer and sybr green i fluorescent dye. the primers used in the reverse transcription pcr were ky80 (sense) 5’-gca gaa agc gtc tag cca tgg cgt-3’ and ky78 (antisense) 5’-ctc gca agc acc cta tca ggc agt-3’. these primers target the highly-conserved 5’-non-coding region of the hcv genome to produce a pcr product of 244 bp and are known to detect all hcv genotypes.31 the reverse transcription pcr master mix consisted of pcr-grade water, mn(oac)2, both primers and the lightcycler® rna master sybr green i mix. the reverse transcription and pcr parameters were set according to the lightcycler® rna master sybr green i package insert. reverse transcription of the template rna was performed at 61 °c for 20 minutes. complementary dna (cdna) was amplified over 40 cycles of denaturation, annealing and extension. denaturation and extension were performed at the temperatures on the package insert. the annealing temperature was calculated using the melting temperatures of the primers, namely, 65 °c (4 °c subtracted from 69 °c), using standard pcr optimisation guidelines, whereby a range of annealing temperatures (55 °c to 65 °c) was used to determine the optimal annealing temperature, where efficiency of pcr amplification was maximal.32 melting curve analysis to identify pcr products was performed as described on the package insert. the pcr products were detected with a sybr green i dye, which intercalates with double-stranded dna and is detected at a wavelength of 530 nm. primer concentrations and template volumes were optimised using the methods described on the lightcycler® rna master sybr green i package insert. primer concentrations ranging from 0.1 µm to 0.5 µm, using two-fold dilutions, were used to determine the optimal primer concentration capable of detecting the lowest concentration of hcv rna. template volumes of 1 µl, 5 µl and 10 µl were used to determine the optimal starting concentration of the template rna. the hcv quantitative pcr proficiency panel (qcmd, glasgow, scotland) was prepared in five-fold dilutions as an external quality control standard to calculate the lower limit of detection of the optimised hcv rna pcr assay. for the preparation of the in-house internal quality control specimens, hcv-negative whole blood was provided by the south african national blood services (durban, south africa). these dbs specimens were prepared by spiking the hcv-negative whole blood with hcv-positive plasma (3 580 000 iu/ml) obtained from the national institute of communicable diseases (sandringham, johannesburg, south africa). this plasma was tested for hcv using the cobas ampliprep/cobas taqman hepatitis c virus assay on the cobas ampliprep and cobas taqman 48 analyser (roche diagnostics, mannheim, germany). ten-fold serial dilutions were made, with a starting concentration of 3 580 000 iu/ml of hcv rna, resulting in values of 3 580 000 iu/ml, 358 000 iu/ml, 35 800 iu/ml and 3580 iu/ml. unspiked whole blood was used as a negative control. fifty microliters (50 µl) of the positive and negative internal control specimens were spotted onto whatman filter paper to mimic infant dbs specimens. results primer concentrations adjusted to 0.25 µm and a template volume of 10 µl improved pcr amplification. an annealing temperature of 58 °c resulted in higher levels of amplified pcr product compared with an annealing temperature of 65 °c. quality control specimens with hcv rna values of < 10 000 iu/ml were detectable at an annealing temperature of 58 °c. detection at a melting temperature of 86 °c was indicative of an hcv-positive result (figure 1) for quality control specimens with viral loads of 13 646 iu/ml (high), 7063 iu/ml (medium) and 3972 iu/ml (low). the external quality control specimens with known hcv viral loads of 30 000 iu/ml, 6000 iu/ml and 1200 iu/ml were detected by the pcr, whereas external quality control specimens with an hcv viral load of 240 iu/ml were undetectable (figure 2). the in-house hcv quality control specimens with viral loads of 3 580 000 iu/ml, 358 000 iu/ml, 35 800 iu/ml and 3580 iu/ml were all detected by the hcv pcr (figure 3). figure 1: melting curves at a melting temperature of 86 °c represent hcv-positive controls. sixteen specimens that tested negative for hcv by serology were used as hcv-negative controls. figure 2: external quality controls. the melting curves show results of five-fold dilutions of hcv-positive external quality control specimens. these specimens were used to determine the lowest hcv rna-positive concentrations that the optimised pcr assay could detect. hcv rna at concentrations of 30 000 iu/ml, 6000 iu/ml and 1200 iu/ml were detected by the assay, whereas concentrations of 240 iu/ml of hcv rna and lower were undetectable. figure 3: internal quality controls. internal quality control dbs specimens were prepared by spiking hcv-negative whole blood with hcv-positive plasma. ten-fold dilutions were prepared with a starting concentration of 3580 000 iu/ml of hcv rna and added onto whatman filter paper. hcv-negative whole blood was used as a negative control. an hcv rna concentration of 3580 iu/ml was the lowest concentration of rna detected for dbs using the optimised pcr assay. the lowest concentration detected by the optimised hcv pcr was 1200 iu/ml (figure 2) when using the qcmd quality control specimens. the in-house internal quality control dbs specimens were detected with viral loads as low as 3580 iu/ml of hcv rna (figure 3). of the 179 infant dbs specimens, 13 tested hiv-positive and 166 tested hiv-negative via pcr. all 179 dbs specimens tested negative for hcv using the optimised real-time pcr assay. discussion we optimised a real-time pcr test for hcv using dbs specimens from infants as a simple method for qualitative hcv detection. although our method was qualitative, it was able to detect at least 3580 iu/ml and 1200 iu/ml in various types of quality control specimens. this could be useful for detecting acute hcv infections, which generally have viral loads less than 105 iu/ml.33 the detection of amplified nucleic acid using sybr green i is inexpensive and is an easier technical approach in comparison to other real-time pcr formats such as taqman and molecular beacon detection methods.14 it is possible that hcv rna viral load in our infant dbs specimens was below the level of detection of the assay, since hcv rna viral loads amongst infants in the first year of infection may be too low for detection.3 dbs specimens have improved the cost-effectiveness, coverage and availability of molecular-based testing in resource-limited settings.11,24,34,35 this has resulted in the development of several optimised pcr techniques to detect hcv using dbs.21,22,24,25 as with our method, several of these techniques are based on real-time pcr and, like de crignis,22 we used lightcycler® technology with sybr green i as the fluorophore.21,24,25 probe-based detection methods are known to improve the sensitivity of target detection, but increase the cost of the testing.21 the limit of detection varies amongst assays. our assay had a limit of detection of 3580 iu/ml, which is higher than previously reported limits of detection of 2500 iu/ml22 and 250 iu/ml.21 this difference may be attributed to differences in storage conditions of the dbs specimens before testing and differences in methods of rna extraction, which may result in variations in the number of copies of starting rna.11,25,34 additionally, dbs specimen volumes are lower when compared with plasma and serum specimens, which reduces the amount of rna extracted.34 thus, the correlation between hcv viral load from dbs and hcv viral load from plasma or serum may be imprecise, suggesting that dbs specimens are better suited to diagnosis, whereas plasma or serum are better suited to monitoring of hcv antiviral treatment.34 these methods for hcv testing using real-time pcr have been applied to hcv treatment monitoring,24 screening for hiv/hcv co-infection22 and surveillance for hcv amongst intravenous drug users.36 our real-time hcv pcr was used on dbs specimens from an infant population, which may be useful for hcv surveillance amongst infants. the use of dbs specimens for hcv pcr has several advantages.11,34,35 firstly, they are easier to collect than whole blood and present a lower infectious hazard. secondly, specimen transportation and storage are cheaper, since refrigeration is not required. lastly, the long-term stability of nucleic acids in dbs has been proven for molecular analysis. therefore, real-time hcv pcr using dbs specimens for hcv surveillance has been used to understand the molecular epidemiology of hcv.21,22,23,24,25,36 limitations our hcv pcr optimisation has several limitations. firstly, the results are not representative of all infants, since only specimens from hiv-exposed infants who require an hiv pcr were tested. secondly, our optimised hcv pcr may not be optimal for routine clinical diagnostic purposes, although it may be convenient for surveillance work. thirdly, we did not compare our dbs specimen method to those using whole blood. however, we did use alternative methods to evaluate our pcr assay, which included testing external quality control specimens with known hcv viral loads ranging from 1081 iu/ml to 34 594 iu/ml and clinical hcv-positive and hcv-negative specimens from a reference laboratory. further, positive and negative controls were included in each run. lastly, we chose not to follow previously described methods but to optimise our own method, because it was more cost-effective than other real-time techniques reported. in addition, we used existing equipment and in-house pcr consumables that the technical staff in our laboratory were already proficient in using. given that our dbs specimens were from hiv-exposed infants in kwazulu-natal, we expected to find a moderate prevalence of hcv amongst these infants as reported by other african countries with populations co-infected with hiv and hcv.37,38,39 infection with hiv is a risk factor for hcv transmission in co-infected pregnant women and is known to increase vertical transmission.3,4 the fact that we did not detect hcv rna in our study specimens may be because of the prolonged storage (≥ 3 months) of our dbs specimens at room temperature, which may have resulted in degradation of hcv rna.25 furthermore, perinatal transmission of hcv requires a high maternal hcv viral load.3 detection of hcv rna at birth is usually indicative of intrauterine infection, whereas infection during delivery is associated with hcv rna being detected only after infants are aged six months; in addition, delivery by caesarean section is less frequently associated with hcv transmission than vaginal delivery.3 however, clinical information, such as maternal hcv viral load, mode of delivery of infants, infant liver function markers, risk factors (such as hbv co-infection) and laboratory results (such as cd4 cell counts and hiv viral loads) were not available for our study. conclusion we have demonstrated that using an optimised real-time hcv pcr assay on dbs specimens from hiv-exposed infants is a good technique for hcv detection and may be well suited for surveillance purposes in resource-limited settings. to our knowledge, this is the first surveillance in south africa using an optimised real-time hcv pcr to report the prevalence of hcv amongst hiv-exposed infants. considering that hcv was not detected in our dbs specimens, hcv prevalence in this population of infants in kwazulu-natal, south africa may be low and may have little impact on hiv-exposed infants. therefore, further surveillance for hcv amongst hiv-exposed infants in our setting may not be necessary. however, hcv surveillance may still be prudent for hiv-exposed infants in countries with moderate to high hiv and hcv prevalence and should include collection of clinical information and data about confounding factors. acknowledgements competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. sources of support we would like to thank the national institute of communicable diseases, gauteng for providing us with serum samples positive for hcv. we also appreciate the south african national blood services, durban providing us with hcv-negative blood. authors’ contributions r.p. (university of kwazulu-natal/national health laboratory services) designed the project and analysed the data. a.n. 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control trial. j viral hepat. 2008;15(4):250–254. http://dx.doi.org/10.1111/j.1365-2893.2007.00937.x njouom r, pasquier c, ayouba a, et al. low risk of mother-to-child-transmission of hepatitis c virus in yaounde, cameroon: the anrs 1262 study. am j trop med hyg. 2005;73(2):460–466. ogboghodo bc, aigbirior ma, bazuaye gn, et al. hepatitis c virus and human immunodeficiency virus-i (hiv) co-infection in children in benin city, nigeria. afr. j. biomed. res. 2009;12(1):1–6. sadoh ae, sadoh we, iduoriyekemwen nj. hiv co-infection with hepatitis b and c viruses among nigerian children in an antiretroviral treatment programme. s. afr. j. child health. 2011;5(1):7–10. abstract introduction methods results discussion acknowledgements references about the author(s) cailin nieuwenhuizen department of haematology, faculty of health sciences, university of pretoria, pretoria, south africa department of haematology, tshwane academic division, national health laboratory service, pretoria, south africa tshiphiri netshidzivhani department of haematology, faculty of health sciences, university of pretoria, pretoria, south africa department of haematology, tshwane academic division, national health laboratory service, pretoria, south africa johan potgieter department of haematology, faculty of health sciences, university of pretoria, pretoria, south africa department of haematology, tshwane academic division, national health laboratory service, pretoria, south africa citation nieuwenhuizen c, netshidzivhani t, potgieter j. establishment of haemoglobin a2 reference intervals in pretoria, south africa: a retrospective secondary data analysis. afr j lab med. 2022;11(1), a1841. https://doi.org/10.4102/ajlm.v11i1.1841 original research establishment of haemoglobin a2 reference intervals in pretoria, south africa: a retrospective secondary data analysis cailin nieuwenhuizen, tshiphiri netshidzivhani, johan potgieter received: 28 jan. 2022; accepted: 20 apr. 2022; published: 12 aug. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: haemoglobinopathies are one of the most common inherited diseases worldwide. quantification of haemoglobin a2 is necessary for the diagnosis of the beta thalassaemia trait. in this context, it is important to have a reliable reference interval for haemoglobin a2 and a local reference range for south africa has not been established. objective: this study aimed to establish reference intervals for haemoglobin a2 using stored patient laboratory data. methods: this descriptive study used retrospective data to evaluate haemoglobin a2 levels determined using high-performance liquid chromatography at the national health laboratory service haematology laboratory in pretoria, south africa. all tests performed from 01 october 2012 to 31 december 2020 were screened for inclusion; of these, 144 patients’ data met the selection criteria. the reference interval was calculated using descriptive statistics (mean and standard deviation) with a 95% confidence interval. results: analysed data from enrolled patients showed a normal distribution. the mean age of the patients was 40 years (range: 3–84 years). the reference interval for haemoglobin a2 calculated from this data was 2.3% – 3.6%. the minimum haemoglobin a2 was 2.3% and the maximum was 3.9% with a mean of 2.95% and a standard deviation of 0.357%. conclusion: a normal reference interval has been established for the population served by the laboratory that will assist with accurate diagnosis of the beta thalassaemia trait. this reference interval may also be useful to other laboratories that employ the same technology, especially smaller laboratories where obtaining a sufficiently large number of normal controls may be challenging. keywords: haemoglobin a2; reference range; reference interval; beta thalassemia; high-performance liquid chromatography. introduction reference intervals are an integral part of any functioning clinical laboratory. reference intervals are often the endpoint used by clinicians in the management of patients and in clinical decision-making. it has been estimated that 80% of medical decisions are made from laboratory results.1 the value of the results produced by a laboratory is largely influenced by the quality of the reference interval used in the interpretation of that result.2 an example of where reference intervals are of diagnostic utility is in the diagnosis of the beta thalassaemia trait. the increase in haemoglobin a2 (hba2) level is the most important laboratory parameter for the identification of carriers of beta thalassaemia, and is considered diagnostic in the appropriate clinical context.3 accurate quantification of hba2 in the haematology laboratory is therefore essential to allow for routine diagnosis of the beta thalassaemia trait.4 this highlights the value of reliable reference intervals. beta thalassaemia is inherited in an autosomal recessive pattern and the beta thalassaemia trait is estimated to have a prevalence of 1.5% worldwide, affecting approximately 80–90 million people.5 africa has a considerable disease burden in terms of haemoglobinopathies and in particular beta thalassaemia with 1520 conceptions affected annually, western africa accounting for the majority of cases.6 reference intervals are established through a validation process with a statistically adequate number, ideally 120, of healthy reference individuals.7 this needs to be done for all reagents and instrument combinations. however, even the clinical and laboratory standards institute guidelines recognise that this is not feasible for many laboratories and finding a cohort of 120 healthy individuals is not feasible for every test.8 alternatively, reference intervals may be verified, with only 20 samples needed, or transferred provided that the analytic system and the test population are comparable.8 however, verification and transfer of reference intervals is not ideal and these methods have their own disadvantages.8 every laboratory that performs hba2 testing is responsible for establishing its own reference interval. this is done by quantifying the hba2 percentage in a cohort of healthy adults who do not have iron deficiency or the thalassaemia trait.3 the reference interval is a range that should be calculated including individuals with characteristics that are comparable to the reference group so that the reference interval can be correctly applied to the population serviced by the laboratory.7 given the importance of hba2 reference ranges, considerable work has been done internationally on normal reference intervals for hba2. however, there is a paucity of literature from africa with no published reference intervals for hba2. in keeping with good laboratory practice, a need was identified to determine hba2 reference intervals in a local african population for the department of haematology of the national health laboratory service (nhls), tshwane academic division (tad), pretoria, south africa. using a sufficient number of results from medical records would provide a healthy cohort for establishing a reference interval without the need to recruit healthy individuals. this is particularly useful for a test that is not routinely requested and only offered by specialised laboratories. in order to establish a reference interval for hba2 without the limitations inherent to traditional methods of establishing reference intervals, we made use of previously reported normal high-performance liquid chromatography (hplc) results from the nhls department of haematology. methods ethical considerations approval was obtained from the academic affairs, research and quality assurance department of the nhls. the study protocol was approved by the faculty of health sciences research ethics committee of the university of pretoria (protocol number 518/2019). patient consent was waived by the ethics committee as the study was conducted using historical data. participants’ information was treated with confidentiality. each participant was allocated a unique study number to ensure anonymity. study design and setting this was a descriptive study using retrospective data from blood test results stored in the laboratory information system of the nhls at the department of haematology, tad, and vermaak and partners path care pathology group. study population and sampling strategy the study population comprised patients investigated by tad, nhls, and included patients from both high-income and low-income settings and represented a variety of ethnicities. all hplc results of tests performed at the nhls tad from 01 october 2012 to 31 december 2020 were screened for inclusion (figure 1). the following selection criteria were applied: hplc performed within the study period and normal hplc results of patients who also had a corresponding complete blood counts (cbc) were included in this study. results of individual patients were excluded if they were aged two years or younger, were anaemic (haemoglobin < 12.5 g/dl), had a mean cell volume < 75 fl or > 100 fl, had a mean corpuscular haemoglobin below 27 pg, had a red cell distribution width of > 15% or had variant haemoglobin, inclusive of haemoglobin s, detected in their haemoglobin electrophoretic result. participants that did not meet the selection criteria or met the exclusion criteria were excluded from the study and subsequent analysis. a minimum sample size of 120 patient is required in order to establish a reference interval.8 figure 1: flow diagram of the study design, pretoria, south africa, 01 october 2012 – 31 december 2020. data collection apart from hplc reports, corresponding results of cbc, thyroid stimulating hormone, serum folate, serum vitamin b12 and ferritin level, performed within a week of the taking of the hplc specimen, were evaluated. only normal hplc results were included in the analysis. the hba2 levels were determined in the haematology laboratory using the hplc d10 instrument (bio-rad® laboratories, hercules, california, united states). the d10 instrument uses ion-exchange hplc technology to analyse haemoglobin. all analyses were performed according to good laboratory practice and the manufacturer’s recommendations. the haematology laboratory at the nhls tad has been accredited by the south african national accreditation system. appropriate controls and calibrators were used throughout the study. the lyphochek® hemoglobin a2 control level 1 and 2 (bio-rad laboratories, irvine, california, united states) were used as controls and the d10tm dual program hba2/f/a1c calibrator/diluent set (bio-rad® laboratories, hercules, california, united states) was used for calibration. the blood test results listed above were captured in a microsoft excel (microsoft corporation, redmond, washington, united states) spreadsheet. exclusion criteria were applied after which 144 samples with normal hba2 levels were identified, and these were used for calculation of reference intervals. data analysis the descriptive statistics mean, median, standard deviation and inter-quartile range, with 95% confidence interval, and the 2.5th and 97.5th percentiles were used to describe the continuous variables such as hba2 levels. the two-sample t-test, or non-parametric alternative were used to compare group means. pearson’s correlation was used to measure correlations between hba2 levels and age, as well as other continuous variables such as cbc parameters. tests were evaluated at 5% level of significance. all analyses were done using stata 15 (statacorp, college station, texas, united states) software. results the mean age of the 144 patients included in this data analysis was 40 years (range 3–84 years). the study population comprised 67 female and 77 male patients. after a single outlier of 4.3% was excluded from the analysis, the mean hba2 value was 2.95% with the range between 2.2% and 3.9%. the standard deviation was 0.357% (figure 2). figure 2: box-plot of haemoglobin a2 distribution, pretoria, south africa, 01 october 2012 – 31 december 2020. data were normally distributed as indicated in the kernel density estimation (figure 3). the hba2 reference interval established from this data set was 2.3% – 3.6%. figure 3: kernel density estimate demonstrating the normal distribution of data, pretoria, south africa, 01 october 2012 – 31 december 2020. a sex comparison was performed by t-test to compare the mean hba2 of male patients to that of female patients in an attempt to identify possible bias. no significant difference was found (p = 0.1328). the correlation between age and hba2 was also assessed. a trend towards lower hba2 values with increased age was appreciated (figure 4), with a pearson correlation coefficient of –0.2826. figure 4: scatterplot of haemoglobin a2 and age distribution, pretoria, south africa, 01 october 2012 – 31 december 2020. discussion the hba2 reference interval established from this data set was 2.3% – 3.6%. normal reference intervals for hba2 have been published for other patient populations. a study performed at the leiden university in the netherlands, using the variant classic hplc (bio-rad®) platform, reported a hba2 reference interval of 2.3% – 3.5%.3 despite the difference in study population, the reference interval determined in this current study is comparable to that of the leiden group which used a similar method, that is, hplc (bio-rad®) technology. han et al. reported a reference interval of 2.3% – 3.1% for hba2 in a chinese population of reproductive age.9 however, these investigators used a capillarys2 instrument (sebia, france) to generate their data.9 most evidence suggests that hba2 of > 4% is indicative of beta thalassaemia trait with almost 100% sensitivity and 90% specificity.10 a grey zone between 3.1 and 3.9 is generally accepted as reported in a comprehensive review.11 this often poses a diagnostic challenge. studies have been conducted to identify the presence of mutations in these individuals.10,11,12,13,14 giambona et al. found that 80% of patients in this group in an italian population were negative for molecular defects, and the most significant finding was the presence of beta thalassaemia gene mutations found mostly in patients with hba2 in the region of 3.5–3.9 and mean cell volume < 80 fl.11 the upper limit of the normal reference interval established in this current study does fall within the previously described ‘grey zone’. however, in the presence of a normal cbc, the possibility of an underlying carrier state in these patients remains small. this highlights the importance of interpreting hba2 within the clinical context, taking into consideration the cbc parameters and iron studies. there appears to be a weak association between a decreasing hba2 value and increasing age; this was true even when looking at subsets of age and when excluding patients aged 70 years or older. although this trend was seen, when calculating the reference interval by age, the reference interval remained 2.3–3.6 when rounded to one decimal place. therefore, this was not a significant finding. in this current study we used data available on the laboratory information system in order to establish a reference interval. this represents a novel approach in our setting. data mining is emerging as an alternative to the traditional direct a priori method. data mining makes use of electronic data records and statistical techniques to determine the healthy population within a data set in order to establish a reference interval.15 the electronic data records may be obtained from insurance claims, electronic health records as well as a variety of other sources.16 all sources require data capturing platforms which allow for database management in order to deal with the enormous volume of information currently being generated as well as the complexity of analysing and interpreting the data. the analytical methods that have been employed to establish reference intervals include the hoffmann method, bhatacharya method and more recently the truncated maximum likelihood method.15 the truncated maximum likelihood method employs complex statistical algorithms that make use of maximum likelihood estimation and require 4000 data points in order to establish a robust reference interval.15 currently there is still hesitancy regarding the use of indirect methods but it is likely to be used to establish many reference intervals in the future. indirect methods may be particularly useful for tests that are not routinely performed as screening tests in the healthy population.17 data mining has many advantages: it is less costly as the blood results of large cohorts of individuals are readily available, it is faster, and it can even be considered more ethical.15 a study conducted by katayev et al. showed that reference intervals could be reliably and reproducibly established using data mining. reference intervals were calculated for eight analytes and were found to be comparable to already accepted published reference intervals.17 although our study was small and the data were captured manually, it does highlight the potential of using laboratory information records to glean valuable data with relatively low cost and fewer limitations than are inherent to establishing reference intervals in the conventional manner by direct population sampling. limitations one of the limitations of this study was the inability to exclude all confounding factors. although our study could be improved on by only including patients who have been tested for all confounding factors for hba2, this would only be feasible if a large number of data sets were included. another option would be to use a larger cohort and an algorithm that does not require the exclusion of all confounders. a normal cbc was used as a surrogate marker of a nutritional deficiency, and this remains a limitation. it should be noted that all published confounders are not routinely considered when establishing reference intervals for hba2 or when interpreting hplc results. conclusion a normal hba2 reference interval of 2.3% – 3.6% has been established for the population served by the laboratory. this will assist with the interpretation of results. the reference interval could also be useful to other laboratories, especially smaller laboratories where obtaining a sufficiently large number of normal controls may be challenging. the inability to exclude all of the confounding factors that influence hba2 levels needs further research. acknowledgements we thank prof. r. pool for his insight and guidance and dr d. toi for his assistance in proofreading the article. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions j.p. and t.n. conceived of the presented idea. c.n. developed the theory and performed the computations. j.p., c.n. and t.n. verified the analytical methods. j.p. supervised the findings of this work. all authors discussed the results and contributed to the final manuscript. sources of support funding has been provided by the university of pretoria development fund. data availability the data that support the findings of this study are available on request from the corresponding author, c.n. disclaimer the views expressed in the submitted article are the authors’ own and not an official position of the institution. references castellone dd. establishing reference intervals in the coagulation laboratory. int j lab hematol. 2017;39(s1):121–127. https://doi.org/10.1111/ijlh.12661 jones g, barker a. reference intervals. clin biochem rev. 2008;29(suppl 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methods results discussion acknowledgements references about the author(s) seth a. attoh j.m. wadhwani department of anatomical pathology, 37 military hospital, accra, ghana frederick hobenu j.m. wadhwani department of anatomical pathology, 37 military hospital, accra, ghana lawrence edusei department of pathology, korle-bu teaching hospital, accra, ghana kwasi agyeman-bediako j.m. wadhwani department of anatomical pathology, 37 military hospital, accra, ghana clement t. laryea department of medicine, 37 military hospital, accra, ghana edward o. nyarko public health division, 37 military hospital, accra, ghana michael k. amedi department of radiology, 37 military hospital, accra, ghana richard h. asmah department of molecular biology, university of health and allied sciences, ho, ghana edward asumanu department of surgery, 37 military hospital, accra, ghana mary mcaddy j.m. wadhwani department of anatomical pathology, 37 military hospital, accra, ghana anthony maison j.m. wadhwani department of anatomical pathology, 37 military hospital, accra, ghana godwin nyarko j.m. wadhwani department of anatomical pathology, 37 military hospital, accra, ghana raymond d. fatchu department of pathology, 37 military hospital, accra, ghana kafui akakpo department of pathology, university of cape coast, cape coast, ghana citation attoh sa, hobenu f, edusei l, et al. postmortem diagnosis of covid-19: antemortem challenges of three cases at the 37 military hospital, accra, ghana. afr j lab med. 2020;9(1), a1290. https://doi.org/10.4102/ajlm.v9i1.1290 original research postmortem diagnosis of covid-19: antemortem challenges of three cases at the 37 military hospital, accra, ghana seth a. attoh, frederick hobenu, lawrence edusei, kwasi agyeman-bediako, clement t. laryea, edward o. nyarko, michael k. amedi, richard h. asmah, edward asumanu, mary mcaddy, anthony maison, godwin nyarko, raymond d. fatchu, kafui akakpo received: 03 june 2020; accepted: 21 aug. 2020; published: 03 nov. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: consistency among clinical symptoms, laboratory results and autopsy findings can be a quality measure in the diagnosis of coronavirus disease 2019 (covid-19). there have been classic clinical cases that have met the case definition of covid-19 but real-time reverse-transcription polymerase chain reaction (rrt-pcr) tests of nasopharyngeal swabs were negative. objectives: this study aimed to share pathological observations of autopsies performed at the 37 military hospital’s department of anatomical pathology on three presumed covid-19 cases in accra, ghana. method: complete autopsies with detailed gross and histopathological analysis were conducted between april 2020 and may 2020 on three suspected covid-19 cases, of which two had initial negative (rrt-pcr) nasopharyngeal tests. postmortem bronchopulmonary samples of two cases were collected and tested by rrt-pcr for severe acute respiratory syndrome coronavirus 2 (sars-cov-2). results: the two postmortem bronchopulmonary samples tested for sars-cov-2 by rrt-pcr were positive. though no postmortem bronchopulmonary sample was taken from the third case, a close contact tested positive for sars-cov-2 in later contact tracing. for all three cases, lung histopathological findings were consistent with acute respiratory distress syndrome. conclusion: the outcome of covid-19 testing is dependent on the sample type and accuracy of sampling amongst other factors. histopathological findings vary and may be dependent on a patient’s modifying factors, as well as the duration of infection. more autopsies are required to fully understand the pathogenesis of this disease in ghanaians. keywords: covid-19; autopsy; postmortem diagnosis; false-negative; ghana. introduction the global estimate of confirmed cases of coronavirus disease 2019 (covid-19) as of 27 may 2020 stood at over 5.5 million in approximately 213 countries and territories with over 349 190 deaths, giving a mortality rate of 15.7%.1 in ghana, the first confirmed covid-19 case was reported on 12 march 2020. as at the end of may 2020, over 7303 cases, 34 deaths and 2412 recoveries had been recorded.2 covid-19 emerged from wuhan, hubei province, china in december 2019, and is clinically associated with viral pneumonia.3,4 clinical, laboratory and radiological features for covid-19 are non-specific; features are similar to other respiratory tract infections.5 thus, mild symptoms of covid-19 such as fever, cough, dyspnoea, myalgia and fatigue were initially treated and managed as pneumonia symptoms by healthcare workers. the world health orginization (who) situation report 94 defines a suspected covid-19 case as: [a] person presenting with acute respiratory illness (fever and at least one sign/symptom of respiratory disease, e.g. cough, shortness of breath) and no aetiology that fully explains the clinical presentation; or a patient with an acute respiratory illness and has been in contact with a confirmed or probable case of covid-19 in the last 14 days before the onset of symptoms; or a patient with severe acute respiratory illness (fever and at least one sign/symptom of respiratory disease, e.g. cough shortness of breath; and requiring hospitalization) and in the absence of an alternative diagnosis that fully explains the clinical presentation. (pp. 11, 12) the clinical presentation of covid-19 infection varies from mild to moderate to severe. the severe presentation is reportedly characterized by acute respiratory distress syndrome. in line with this, diffuse alveolar damage (dad) is a reported postmortem pathological feature of covid-19; dad is the histological correlate of acute respiratory distress syndrome. in the acute stage, dad is characterized by hyaline membrane formation in the alveoli and the organizing stage by interstitial widening due to oedema and fibroblast proliferation.7 however, there is an instance where dad was not reported.8 other pathological features that have been reported in the lung include: oedema, fibrinous exudates, reactive hyperplasia limited to some type ii pneumocytes and patchy inflammatory changes with scattered multinucleated giant cells.7 others report pathological similarities between these coronavirus infections: covid-19, severe acute respiratory syndrome (sars) and middle eastern respiratory syndrome.3 though other pathological features have been reported in other organs, including the liver and heart, the changes in these organs have been less significant than those reported in the lung. the prominent role of endotheliitis in covid-19 infection was highlighted in one such publication that reported the presence of viral inclusion in endothelial cells.9 the authors suggested that infection with sars coronavirus 2 (sars-cov-2) results in endotheliitis in several organs from the involvement of the virus and as part of the host inflammatory response. the authors hypothesized that a strategy that targets endotheliitis in susceptible patients (people with diabetes, hypertension, obesity) is likely to improve survival in such patients.9 among nucleic acid tests, the polymerase chain reaction (pcr) method is considered the ‘gold standard’ for the detection of covid-19, and it is characterized by rapid detection, high sensitivity and high specificity.10 the specificity of most real-time reverse-transcription (rrt) pcr tests is estimated at 100%, because the design of the primers is specific for the genome sequence of sars-cov-2. however, false-negative reports may occur and have been associated with intended and non-intended activities during case detection, patient preparation, sample collection, packaging, storage, transport and reporting.11 the nasopharyngeal swabs or other upper respiratory tract specimens, including throat swabs or, more recently, saliva are the commonly used specimens for rrt-pcr diagnosis of covid-19.11 howbeit, the positivity rates of these samples vary; bronchoalveolar lavage has the highest positivity rate. in one study of 205 confirmed covid-19 infections, the rrt-pcr positivity rate of bronchoalveolar lavage, sputum, nasal and oropharyngeal specimens were 93%, 72%, 63% and 32%, respectively.3 although confirmed deaths due to covid-19 are being recorded, autopsy findings of covid-19 reported deaths in africa, including ghana, are largely unavailable. this report shares pathological observations of autopsies performed at the 37 military hospital’s j.m. wadhwani department of anatomical pathology on three presumed covid-19 cases. it also highlights the challenges associated with managing presumed cases. methods ethical considerations due to the emergency nature of the pandemic, ethical review and approval was not conducted. however, all identifiers were removed from the report and verbal consent was also sought from the relations of the deceased, where required. study site the 37 military hospital, a 600-bed tertiary facility, is one of the largest hospitals in ghana. annually, the j.m. wadhwani department of anatomical pathology of the hospital conducts approximately 1500 hospital, medico-legal or coroner’s autopsies in its standard morgue facility. the notices of death were received by the department of anatomical pathology for two presumed covid-19 cases who were being managed within the 37 military hospital. also received at the department was a case referred from a peripheral hospital for a medico-legal or coroner’s autopsy to determine the cause of death. all cases were received between april 2020 and may 2020 and met the clinical case definition of covid-19; antemortem sars-cov-2 tests of the two tested cases were negative. all cases were transported to the morgue under covid-19 recommended protocols as published by the who.12 a complete autopsy was conducted on all cases with detailed gross and histopathological analysis. examinations were performed in the department’s state-of-the-art morgue following guidelines for performing autopsies on presumed covid-19 cases. except in the coroner’s autopsy case, postmortem bronchopulmonary samples were collected, immediately placed in viral transport media and sent to the noguchi memorial institute for medical research where rrt-pcr was performed. selected organs (lungs, heart, brain, kidneys, liver, spleen) were sampled and fixed in 10% buffered formalin for histopathological studies. organ slides were made, stained with haematoxylin and eosin and examined by certified histopathologists. tissue sections were retained in formalin and blocks appropriately stored. findings from the autopsy were corroborated by both clinical presentations and laboratory outcomes. results case 1 the first case was a man with hypertension and diabetes who was in his late thirties. he had a non-productive cough, fever (39.5 °c) and had experienced breathlessness for six days before hospital admission. he alleged that he had no contact with a confirmed or probable covid-19 case. a provisional diagnosis of covid-19 was made, the patient was isolated, and a nasopharyngeal swab was taken for sars-cov-2 testing. the sars-cov-2 test result came in after his demise and was negative. therefore, a coroner’s autopsy was requested and was performed to find out the cause of death (table 1). no postmortem bronchopulmonary specimen was taken. however, following contact tracing, it was discovered that he had prior contact with a confirmed covid-19 patient and that one of his caregivers tested positive for sars-cov-2. table 1: autopsy findings of three presumed covid-19 cases at the 37 military hospital, accra, ghana, april 2020 to may 2020. microscopic sections of the lungs show autolytic changes with bacterial colonization of the bronchioles (figure 1). the prominent pathological findings in fairly preserved areas of the lungs were severe oedema and dad with prominent hyaline membrane formation, infiltration of the interstitium by macrophages and scattered multinucleated giant cells. also, there was evidence of pneumonic changes in the lungs: moderate dense inflammatory cell exudate. in the final autopsy report, the cause of death was listed as bronchopneumonia most likely due to or as a consequence of covid-19; diabetes and hypertension were contributory causes. figure 1: haematoxylin and eosin staining of lung tissue samples from a 38 year old male patient presumed to have covid-19 (37 military hospital, accra, ghana, april 2020). case 1: (a) autolytic changes with bacterial colonization in the smaller airways (blue arrows) ×100. diffuse alveolar damage is noted with hyaline membrane formation (green arrows) ×100. (b) higher magnification showing hyaline membrane (black arrows) and alveolar macrophages (blue arrow) ×400. (c) diffuse alveolar damage with prominent hyaline membrane formation (black arrow) ×100. (d) hyaline membrane formation (black arrows) ×400. (e) pneumonic changes with mixed inflammatory cells exudate (blue arrow). hyaline membrane (black arrow) ×100. (f) area of microthrombi in smaller pulmonary capillaries ×400. case 2 the second case was a 60-year-old woman with hypertension who was morbidly obese and had in the past week been diagnosed with diabetes mellitus. she had a three-week history of worsening breathing difficulty, one day of unproductive cough and no history of asthma. she allegedly had no contact with a confirmed or probable case of covid-19. she was being managed for bronchitis or asthma with no improvement. an initial diagnosis of pulmonary thromboembolism was made. differential diagnoses were congestive cardiac failure due to hypertension or covid-19 infection. she was therefore transferred from the emergency centre to the isolation unit. she was managed with intravenous fluids, anticoagulants, antibiotics, insulin and anti-hypertensive medications. a nasopharyngeal swab was taken for a covid-19 test three days after admission. she experienced palpitation and dyspnoea but no orthopnoea, paroxysmal nocturnal dyspnoea, pedal oedema, calf tenderness or fever. she had an oxygen saturation of 85% at room air and 95% at non-rebreather oxygen, a normal pulse and a blood pressure of less than or equal to 140/90 mmhg throughout admission. she had a marginally increased neutrophil-white blood cell count and a high d-dimer of 1.26 ug/ml (fibrinogen equivalent unit; normal limit 0.0–0.5). her liver function test showed deranged liver enzymes. however, renal function tests, c-reactive protein (3.6) and troponin were normal. a computerised tomogram scan was done, the pulmonary vessels were reported as normal (figure 2). figure 2: high resolution computerised tomography scan of a 60 year old female patient with covid-19 (37 military hospital, accra, ghana, april 2020). case 2: axial image showing traction bronchiectasis in an area of ground-glass opacities (red arrows) and bilateral ‘crazy paving’ opacities (blue arrows). the pcr results for covid-19 were returned as negative three days after sample collection. she was therefore transferred from the isolation unit to a general medical ward. she later suddenly developed severe respiratory distress, started desaturating and later died. an academic autopsy was ordered by the attending clinical team who queried the pcr covid-19 result (table 1). a postmortem bilateral lung parenchymal swab was taken for sars-cov-2 testing. microscopic examination of the lungs showed severe congestion with foci of haemorrhage. there was a proliferation of fibroblasts and infiltration of macrophages within the interstitium and in the alveolar space; dad with characteristic hyaline membranes in the alveoli; and interstitial fibrosis and oedema. fibrin thrombi, mostly located in the subpleural region were noted. the liver and spleen were poorly preserved. microthrombi were also noted in some of the glomeruli (figure 3). in the final autopsy report, the cause of death was listed as acute pulmonary embolism due to or as a consequence of covid-19 pneumonia; diabetes mellitus and hypertensive heart disease were contributing causes. figure 3: haematoxylin and eosin staining of lung tissue samples from a 60 year old female patient with covid-19 (37 military hospital, accra, ghana, april 2020). case 2: (a) alveolar sacs filled with alveolar macrophages (green arrow) and multinucleated giant cells (blue arrow) ×400. (b) fibrous tissue proliferation in alveolar sacs (black arrow). also noted are alveolar macrophages (blue arrow) ×400. (b) fibroblast proliferation (green arrow) ×400. (d) diffuse alveolar damage with prominent hyaline membrane formation (black arrow) x400. (e) thrombus at the glomerulus (blue arrow) ×400. (f) thrombus in a small pulmonary artery (blue arrow) ×100. case 3 the third case was a 55 year old man with hypertension and diabetes who had a seven-day history of non-productive cough and dyspnoea. he had a fever, general weakness and headache but no chest pains, sore throat, leg swelling, paroxysmal nocturnal dyspnoea or orthopnoea. he had a recent travel history that suggested a covid-19 exposure. the man was in respiratory distress; oxygen saturation56% on room air, 82% on non-rebreather oxygen and his temperature was 37.6 °c. his blood pressure was 175/94 mmhg, and his pulse rate was 112 beats per minute regular. he had no pallor of mucous membranes, not jaundiced and hydration was fair. examinations of the chest revealed reduced air entry in lung bases with crepitations. there were bronchial breath sounds over lower zones. heart sounds i and ii were present, normal and no murmurs were heard. no bi-pedal oedema was also noticed. the abdomen was soft, non-tender and there was no organomegaly. a diagnosis of bilateral pneumonia to rule out covid-19 in a patient with diabetes and hypertension was made. pulmonary embolism was a differential diagnosis. he was managed on intravenous fluids, anticoagulants, antibiotics, insulin and anti-hypertensive medications. he had a low haemoglobin (11.5g/dl) and a high white blood cell count (15.58 × 1010/l) with platelets at 185 × 1010/l. liver and renal function tests were normal. a plain chest x-ray and a computerised tomography scan were requested (figure 4). later that day, his breathing became laboured and uneven. his temperature was 36.6 °c, pulse rate 100 beats per minute; respiratory rate was 29 per minute; oxygen saturation was 96% on intranasal oxygen. his blood pressure shot up to 220/140 mmhg but was controlled by intravenous labetalol. figure 4: radiological images of a 55 year old male patient with covid-19 (37 military hospital, accra, ghana, may 2020). case 3: (a) postero-anterior chest x-ray showing bilateral ground-glass opacification and right upper and middle zone peripheral patchy opacities. (b) high resolution computerised tomography scan, coronal view, showing bilateral, ground-glass opacities with thickened interlobular and intralobular lines with ‘crazy paving’ appearance. bilateral peripheral and subpleural opacities (red arrows). a nasopharyngeal swab for sars-cov-2 testing was to be collected the following day but the patient passed before the sample could be taken. an academic autopsy was therefore ordered by the attending clinical team (table 1). postmortem bilateral lung parenchymal swabs were positive for sars-cov-2. microscopic examination of the lungs showed severe congestion with haemorrhages. there were severe pulmonary oedema and moderately dense macrophage exudate. diffuse alveolar damage with hyaline membrane formation was striking, and large pneumocytes showing enlarged nuclei and granular amphophilic cytoplasm were present. microthrombi were also noted in some smaller pulmonary capillaries (figure 5). in the final autopsy report, the cause of death was listed as covid-19 pneumonia with diabetes mellitus and hypertension as contributory. figure 5: haematoxylin and eosin staining of lung tissue samples from a 55 year old male patient with covid-19 (37 military hospital, accra, ghana, may 2020). case 3: (a) severe pulmonary oedema, diffuse alveolar damage with hyaline membrane formation (blue arrow) ×100. (b) severe pulmonary oedema, diffuse alveolar damage with hyaline membrane formation (blue arrow) ×100. (c) large pneumocytes (blue arrow) ×400. (d) microthrombi in small pulmonary arteries (black arrows) ×100. (e) higher magnification (x400) of image (d) showing microthrombi (blue arrow) ×100. (f) interstitial widening, pulmonary oedema and prominent hyaline membranes (blue arrow). discussion though thousands of covid-19 cases have been reported in ghana with more than 30 deaths, no autopsies performed on such cases have been published in the medical literature. postmortem samples were taken for covid–19 testing in two of the cases that were autopsied. both lung parenchymal samples were positive for sars-cov-2. in two cases, an earlier test using a nasopharyngeal swab sample was negative. this suggests that there is the possibility of false-negative results and that there is a proportion of covid-19 patients who, because of their false-negative test results, will be managed in health facilities without protective protocols. similarly, for those that die without being tested or have false-negative test results, their remains will be handled without the necessary safety precautions. to ensure the safety of workers and prevent unconscious exposure, health facilities and funeral homes should ensure that adequate safety measures are put in place during this pandemic. in one case, the antemortem nasopharyngeal sample was negative for sars-cov-2, whereas the postmortem bronchopulmonary sample was positive for sars-cov-2. such false-negative errors for covid-19 may be the result of pre-analytical activities, such as a poorly collected sample, wrong sample labelling, mislabeling, interference from medications and improper storage or transport.11,13 it is therefore essential that great emphasis is made in ensuring adherence to proper sample collection and processing protocols across the entire cascade of activities in the diagnosis and reporting of covid-19. considering the inevitable occurrence of deaths within the global covid-19 pandemic, it is crucial for anatomical pathologists, while following due safety protocols, to look out for evidence of covid-19 in all autopsy cases. the most significant pathological findings in our patients were in the lungs. this finding is similar to previous findings in other covid-19 autopsies. in our patients, significant pathological findings that were present in all three patients included severe oedema, congestion, haemorrhages, the proliferation of pneumocytes, scattered multinucleated giant cells and dad with hyaline membrane formation.3,8,14 other pathological features including the proliferation of fibroblasts with interstitial fibrosis and microthrombi in the kidneys were seen in one patient. again, in a previous report, one patient had patchy inflammatory infiltrates that suggested a pneumonic process.4 though all our patients had diabetes, we did not find the reported endotheliitis that has been reported to be present in some diabetic individuals. although molecular testing for case 1 was not done, dad was observed. this combined with the anecdotal evidence – contact with a covid-19 patient and a later positive covid-19 test of his caretaker – is suggestive of covid-19. the death was thus ascribed to covid-19 infection, in line with who6 situation report 94 for certifying deaths due to covid-19 which states that: [w]here a definite diagnosis of covid-19 cannot be made but it is suspected or likely (e.g. the circumstances are compelling within a reasonable degree of certainty), it is acceptable to report covid-19 on a death certificate as ‘probable’ or ‘presumed’. (p. 12) the pathological presentation observed in the lungs were consistent with similar cases reported in beijing and oklahoma by barton et al.,7 and xu et al.14 limitations bronchopulmonary sampling was not done for sars-cov-2 testing for case 1 as a result of the psychological unpreparedness of the autopsy team for covid-19 autopsies. additionally, no immunohistochemical staining was done for histopathological samples collected to show the presence and distribution of immune cells in the lungs. also, the quality of photomicrographs could have been better if the bodies were better preserved after death. conclusion findings from this study indicate that autopsies are capable of reporting evidence of covid-19 and provides insights for proper sample management for purposes of diagnosis. more so, the diagnosis of covid-19 at autopsy is relevant in situations where molecular testing such as rrt-pcr is not available to inform relevant public health action. bronchopulmonary samples for sars-cov-2 testing during postmortem should be taken for presumed covid-19 cases. more autopsies are required to fully understand the pathogenesis of this disease in ghanaians. = acknowledgements competing interests the authors have declared that no competing interest exist. authors’ contributions s.a.a. conceived the idea and performed autopsies; k.a. and r.d.f. developed the initial manuscript; f.h., l.e. and k.a.-b. reviewed slides; k.a.-b. assisted in autopsies; c.t.l. managed clinical cases; m.k.a. reported x-rays and computerised tomography scans; a.m. and g.n. processed tissues and prepared slides. e.o.n., r.h.a., e.a. and m.m. did the literature review and discussed the findings of the study. all authors reviewed the manuscript and provided feedback. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references who. who covid-19 dashboard who covid-19 dashboard [homepage on the internet]. who; 2020 [updated 27 may 2020; cited 2020 may 27]. available from: https://covid19.who.int/?gclid=cj0kcqjwn7j2brdrarisahjkxmychap3bruknnxltl7keoklhmv1v91uttjfrby-hruu5_zpj4fopcuaaidaealw_wcb ghs. ghana covid-19 updates [homepage on the internet]. ghana health service (ghs); 2020 [updated 26 may 2020; cited 2020 may 27]. available from: https://www.ghanahealthservice.org/covid19/latest.php huang c, wang y, li xl, ren l, zhao j, hu y. clinical features of patients infected with 2019 coronavirus in wuhan, china. lancet. 2020;395(10223):497–506. https://doi.org/10.1016/s0140-6736(20)30183-5 singhal t. a review of coronavirus disease-2019 (covid-19). indian j pediatr. 2020;87(4):286. https://doi.org/10.1007/s12098-020-03263-6 samsami m, bagherpour z, nematihonar b, tahmasbi h. covid-19 pneumonia in asymptomatic trauma patients; report of 8 cases. arch acad emerg med. 2020;8(1):e46. who coronavirus disease. 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march 24, 2020 p. 6. https://www.who.int/publications/i/item/infection-prevention-and-control-for-the-safe-management-of-a-dead-body-in-the-context-of-covid-19-interim-guidance lippi g, simundic am, plebani m. potential preanalytical and analytical vulnerabilities in the laboratory diagnosis of coronavirus disease 2019 (covid-19). clin chem lab med. 2020;58(7):1070–1076. https://doi.org/10.1515/cclm-2020-0285 xu z, shi l, wang y, et al. pathological findings of covid-19 associated with acute respiratory distress syndrome. lancet respir med. 2020;8(4):422. https://doi.org/10.1016/s2213-2600(20)30076-x article information author: nicolas bouchet1 affiliation: 1quality assurance department, cnrfp (centre national de recherche et de formation sur le paludisme), burkina faso correspondence to: nicolas bouchet email: nikolas.bouchet@gmail.com postal address: 01 bp 2208, ouagadougou, burkina faso dates: received: 08 apr. 2014 accepted: 24 sep. 2014 published: 13 may 2015 how to cite this article: bouchet b. iso 15189: 2012: quels changements pour les laboratoires africains? afr j lab med. 2015;4(1), art. #181, 4 pages. http://dx.doi.org/10.4102/ajlm.v4i1.181 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. iso 15189: 2012 : quels changements pour les laboratoires africains? in this opinion paper... open access • introduction    • la norme iso 15189: 2012    • l'approche processus    • manuel qualité    • gestion des risques    • gestion des informations de laboratoire    • changements mineurs • conclusion • remerciements    • intérêts concurrents    • contributions des auteurs • références introduction top ↑ la norme iso 15189 est le standard international de référence en matière de biologie médicale,1 et l'accréditation est la reconnaissance d'un système qualité en pleine conformité avec cette norme. conscients des challenges actuels dans le domaine de la santé publique et de la recherche biomédicale, les laboratoires africains de biologie médicale pourraient être amenés à développer des systèmes de management de la qualité qui suivent les exigences de la norme iso 15189. sous l'impulsion de l'oms, les programmes slipta et slmta ont vu le jour ces dernières années en afrique, pour accompagner les laboratoires dans la mise en place de processus qualité. 2, 3, 4, 5 comme tout système qualité, les normes iso évoluent, et sont périodiquement révisées; c'est en décembre 2012 que l'iso, l'organisation internationale de normalisation, a publié la troisième édition de la norme iso 15189 6, qui remplace la version de 2007. 7 cet article a pour but de mettre en avant les changements majeurs entre les deux versions de cette norme, et les modifications que cela entraînera dans les systèmes qualité mis en place dans les laboratoires, et notamment les laboratoires africains. la norme iso 15189: 2012 la norme dans sa version 2012 est un document plus clair et mieux organisé que la version 2007. outre le titre qui s'est raccourci, la manière d'organiser les différentes rubriques a été changée, en suivant un ordre plus logique, ce qui permettra sans aucun doute aux utilisateurs de mieux appréhender les différentes exigences. lors de la présentation de la nouvelle version, l'iso a parlé de « révision technique »8, cela signifiant un document présentant chaque aspect avec plus de détails; mais la lecture de cette nouvelle version montre que les changements sont en réalité plus conséquents. l'approche processus un processus est « un ensemble d'activités corrélées ou interactives qui transforme des éléments d'entrée en éléments de sortie »9. l'approche processus décrit l'organisation du travail comme une suite d’étapes inter-reliées avec pour objectif la satisfaction optimale des clients et l'atteinte des objectifs fixés. le but est de rendre l'organisation plus fonctionnelle, et moins dépendante de la hiérarchie, avec l'introduction et le développement du management transversal, de la culture du résultat et du travail d’équipe au sein du laboratoire. la version 2007 abordait ce concept, mais laissait libre choix aux laboratoires de suivre ou non une approche processus dans leur système de management de la qualité (smq); cette approche existait déjà dans les smq basés sur la norme générale de la qualité, l'iso 9001, qui avait introduit ce concept dans sa version 20001, et l'avait maintenu dans sa version de 2008.1 la version 2012 de l'iso 15189 est beaucoup plus explicite, l'approche processus devenant une exigence (sous-chapitre 4.2.1); ceci pourrait s'avérer compliqué à mettre en œuvre pour les laboratoires africains. en effet, cette approche peut s'avérer difficile à mettre en place pour tous les laboratoires d'analyses de petite taille, avec un personnel peu nombreux, ces laboratoires représentant la majorité des laboratoires de biologie médicale en afrique sub-saharienne. l'approche processus implique en effet de cartographier les processus, c'est-à-dire de représenter l'ensemble des processus nécessaires pour le smq et leurs séquences en déterminant les interactions qui les lient, et en désignant des pilotes de processus avec une définition très claire des rôles de chacun. dans des structures de petite taille, une même personne peut avoir une vue d'ensemble et une vue détaillée des activités, et l'intérêt d'une cartographie précise des différents processus est souvent vu comme inutile. si par exemple, dans un laboratoire de 4 employés, chaque individu est impliqué dans tous les processus, la mise en place d'une cartographie peut paraître superflue. néanmoins, cette approche présente plusieurs avantages: tout d'abord, elle insiste sur le but final du smq du laboratoire, le résultat du patient, pour permettre par la suite de bien définir le rôle de chacun dans l'obtention du résultat du patient, et de le montrer clairement. elle permet enfin d'analyser et donc d'améliorer les performances du laboratoire. les laboratoires de biologie médicale ont un avantage pour mettre cette approche en œuvre, puisque leurs activités s'articulent autour de trois grands processus opérationnels, définis dans la norme: les processus pré-analytiques (chapitre 5.4), analytiques (5.5) et post-analytiques (5.7). nous pensons qu'en se concentrant sur ces trois processus majeurs, un laboratoire pourra être en mesure de rattacher les autres processus (comme les équipements et la gestion des stocks, les ressources humaines, la satisfaction des clients, l'amélioration continue, etc.) à une cartographie assez simple. manuel qualité alors que pour la rédaction du manuel qualité, la version 2007 (sous-chapitre 4.2.4) proposait un plan en 23 points, la version 2012 s'attache à décrire (sous-chapitre 4.2.2.2) les six grands points essentiels dans un manuel qualité de laboratoire: politique qualité, description du smq, organisation et structure du laboratoire, rôles et responsabilités de la direction du laboratoire, système documentaire et les politiques établies pour le smq avec référence aux activités sur lesquelles elles reposent. ce changement va donc laisser à chaque laboratoire le soin de définir le plan de son manuel en fonction de son propre système qualité, ce qui démontrera d'une part la maîtrise de la norme par le laboratoire, et d'autre part la capacité du laboratoire à adapter la norme aux réalités locales, ce qui s'avère être souvent le cas en afrique sub-saharienne. le tout en simplifiant l’écriture de ce document, pierre angulaire du smq. gestion des risques cet aspect est à nos yeux le changement majeur de la nouvelle version de la norme. il apparaît dans le sous-chapitre 4.1.1.4, relatif au directeur de laboratoire, où il est du rôle du directeur d’ « élaborer et appliquer un plan de fonctionnement dégradé » ; il est précisé que ces plans doivent être soumis à essai de manière périodique. le sous-chapitre 4.14.6 (gestion des risques) aborde également le sujet, en mettant l'accent sur les risques de défaillances éventuelles sur les résultats des analyses; tout doit être mis en œuvre pour réduire et/ou éliminer ces risques. le concept est également retrouvé dans le nouveau paragraphe consacré à la gestion des informations de laboratoire (5.10.3); il est précisé dans le point 5.3 que « le laboratoire doit disposer de plans de contingence […] en cas de défaillances ». ce concept de management des risques est une introduction importante de cette nouvelle norme; il implique un travail conséquent pour sa mise en œuvre, car toutes les situations d'urgence doivent être identifiées et évaluées (exemple: rupture en réactifs causée par un retard de livraison, panne d'un analyseur, coupure d’électricité, dysfonctionnement d'une imprimante, etc.). chaque situation doit être inscrite dans ce « plan de fonctionnement dégradé », avec pour chacune: la manière de résoudre ce problème (corrections = mesures palliatives); la manière de prévenir le patient et le médecin; la manière de communiquer le problème en interne; les actions correctives à mettre en œuvre pour éviter que le problème ne se reproduise; la manière de revenir à une situation normale. ces plans doivent ensuite être « testés périodiquement », et tout ce processus doit être documenté pour être justifié le cas échéant; ces tests doivent servir à améliorer le plan si nécessaire. ces plans doivent également être audités en cas d'audit interne du système qualité. si l'on prend des exemples de risques comme une coupure d’électricité ou le retard d'approvisionnement en réactifs, ce sont des évènements rares dans les pays industrialisés, mais ce sont des problèmes courants dans les pays d'afrique sub-saharienne. nombreuses sont les capitales africaines qui vivent encore au rythme des délestages durant certaines périodes de l'année, et les retards de livraison sont fréquents pour tout ce qui concerne les réactifs et consommables dans les laboratoires. ce qui sera un risque avec une probabilité d'apparition très faible dans un pays industrialisé sera un risque avec une fréquence d'apparition très élevée dans un pays de l'afrique sub-saharienne, ce qui va rendre la gestion des risques selon la norme iso plus difficile à mettre en œuvre pour les laboratoires africains. les plans de fonctionnement dégradé devront être mis en œuvre périodiquement pour certains aspects, cela faisant qu'une opération prévue pour être « exceptionnelle » deviendra une opération de routine. il est à noter que cette introduction de la notion de gestion des risques dans la nouvelle version de la norme iso 15189 s'inscrit dans une logique plus globale. ce concept, qui vient du monde industriel, a fait ces dernières années son entrée dans le monde réglementaire. dans le cadre du processus de révision de la norme iso 9001, qui est le référentiel de base en ce qui concerne les systèmes de management de la qualité (la nouvelle version est prévue pour 2015), le concept d'une approche de gestion des risques a été identifié par le groupe d'experts chargés de la révision de l'iso 9001 (26ème réunion de l'iso tc 176, tokyo, février 2010)8, et sera intégré à la nouvelle version. la maîtrise des risques est clairement l'un des objectifs de cette nouvelle version de la norme iso 9001, nécessitant de se projeter vers le futur pour anticiper tous les problèmes possibles qui pourraient empêcher la satisfaction client, objectif prioritaire de l'iso 9001. dans le domaine même d'application de l'iso 15189, dans les laboratoires de biologie médicale, ce concept a été introduit aux etats-unis par les nouvelles réglementations en matière de plan de contrôles de qualité internes, en suivant la ligne directrice du clsi (clinical and laboratory standards institute) ep23-a.12 cette ligne directrice a introduit le concept de gestion du risque dans le domaine des contrôles de qualité internes dans les laboratoires de biologie médicale13, 14, 15. gestion des informations de laboratoire la section 5.10 est une demi-nouveauté de cette version; il s'agit en effet de l'annexe b de la norme version 2007, qui passe donc du statut de ‘section informative’ à celui de ‘section normative’, avec obligation de mettre en œuvre ces exigences. toute cette section est centrée sur la protection des données et la gestion du système d'information; le laboratoire doit s'assurer que les outils informatiques impliqués dans la gestion des informations de laboratoire (collecte, traitement, enregistrement, compte-rendu, conservation des données) soient validés par les fournisseurs, soient régulièrement maintenus et soient sécurisés. les laboratoires devront tenir compte de ces nouvelles exigences et agir en conséquence, même si elles sont difficiles à mettre en œuvre, et tout aussi difficiles à documenter, encore plus dans notre contexte africain. changements mineurs le point 4.14, qui était dans l'ancienne version de la norme uniquement consacré aux audits internes, est dans cette version 2012 plus complet, puisqu'il y est question de tous les aspects des audits et évaluations: revue des prescriptions, procédures et exigences concernant les échantillons, les évaluations des retours d'informations de la part des clients, la prise en compte des suggestions du personnel, les audits internes et externes, la gestion des risques et le suivi des indicateurs qualité. tous ce processus d’évaluation / audit a pour objectif d'améliorer le système qualité du laboratoire, en s'assurant que tous les processus nécessaires à la qualité des résultats des analyses ont été réalisés pour répondre aux exigences des clients. un nouveau sous-paragraphe (4.1.1.3) présente des règles d’éthique au sujet des conflits d'intérêts potentiels, de l'intégrité du personnel, des considérations éthiques dans le cadre de la manipulation des échantillons d'origine humaine et de la confidentialité des patients. ce point est très important, car la culture de l’éthique des affaires / des entreprises est un concept peu développé en afrique sub-saharienne, contrairement au concept d’éthique médical, qui était traité dans la version 2007 de la norme en annexe c, ce qui a disparu dans la version 2012. conclusion top ↑ la norme iso 15189: 2012 reste le ‘gold standard’ en matière de qualité dans les laboratoires de biologie clinique. l'organisation plus claire de cette version permettra aux laboratoires de mieux s'imprégner du document pour mieux répondre aux exigences normatives. il est en effet impératif aux laboratoires d'appliquer ce modèle qualité pour obtenir l'accréditation pour s'assurer la confiance des patients et un respect national et international. le défi est de taille pour les laboratoires africains, qui doivent s'adapter à cette norme en prenant en compte les conditions particulières dans lesquelles ils évoluent. il est souhaitable que l'oms-afrique adapte sa liste slipta à cette nouvelle version de la norme, en adaptant de manière générale tous les programmes d'accompagnement pour inclure tous les changements de la version 2012. remerciements top ↑ j'adresse mes remerciements au dr issa nébié ouedraogo et au dr issiaka soulama (tous deux du cnrfp, burkina faso) pour leurs précieux commentaires et discussions autour du manuscrit, ainsi qu'au dr sodiomon b. sirima, administrateur délégué du cnrfp pour m'avoir permis de mener ce travail. intérêts concurrents aucun conflit d'intérêt: l'auteur déclare n'avoir aucun lien financier ou personnel l'ayant influencé de façon inappropriée pendant la rédaction de l'article. contributions des auteurs n.b. (centre de recherche et de formation sur le paludisme), rédaction de l'article. références top ↑ datema tam, oskam l, klatser pr. review and comparison of quality standards, guidelines and regulations for laboratories. afr j lab med. 2011;1(1), art.#3, 7 pages. gershy-damet g, rotz p, cross d. belabbes e, cham f, ndihokubwayo j, et al. the world health organization african region laboratory accreditation process. am j clin pathol. 2010; 134:393-400. http://dx.doi.org/10.1309/ajcptuuc2v1wjqbm maruta t, motebang d, wanyoike j, peter t, rotz pj. impact of mentorship on who-afro strengthening laboratory quality improvement process towards accreditation (slipta). afr j lab med. 2012;1(1), art. #6, 8 pages. yao k, mckinney b, murphy a, rotz p, wafula w, sendagire h, okui s, et al. improving quality management systems of laboratories in developing countries. am j clin pathol. 2010;134:401-409. http://dx.doi.org/10.1309/ajcpnbbl53fwuiqj mothabeng d, maruta t, lebina m, lewis k, wanyoike j, mengstu y. strengthening laboratory management towards accreditation: the lesotho experience. afr j lab med. 2012;1(1), art. #9, 7 pages. organisation internationale de normalisation (iso). iso 15189: 2012. laboratoires de biologie médicale – exigences concernant la qualité et la compétence. troisième édition 2012-11-01, version corrigée 2013-03-01. organisation internationale de normalisation (iso). iso 15189: 2007. laboratoires d'analyses de biologie médicale – exigences particulières concernant la qualité et la compétence. deuxième édition 2007-04-15, version corrigée 2007-09-15. site internet de l'organisation internationale de normalisation (iso). disponible à cette adresse: www.iso.org organisation internationale de normalisation (iso). iso 9000: 2005 systèmes de management de la qualité – principes essentiels et vocabulaire. troisième édition 2005-09-15. organisation internationale de normalisation (iso). iso 9001: 2000 systèmes de management de la qualité – exigences. troisième édition 2000-12-15. organisation internationale de normalisation (iso). iso 9001: 2008 systèmes de management de la qualité – exigences. quatrième édition 2008-11-15, version corrigée 2009-07-15. clinical and laboratory standards institute. ep23-a: laboratory quality control based on risk management; approved guideline. 2011. person n. developing risk-based quality control plans: an overview of clsi ep23-a. clin lab med, 2013, 33, 15-26. http://dx.doi.org/10.1016/j.cll.2012.11.003 westgard j. perspectives on quality control, risk management, and analytical quality management. clin lab med, 2013, 33, 1–14. http://dx.doi.org/10.1016/j.cll.2012.10.003 nichols j. laboratory quality control based on risk management. ann saudi med. 2011 may-jun; 31(3): 223–228. http://dx.doi.org/10.4103/0256-4947.81526 abstract introduction methods results discussion acknowledgements references about the author(s) kagiso m. masemola department of chemical pathology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa department of chemical pathology, national health laboratory service, johannesburg, south africa ngalulawa kone department of chemical pathology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa department of chemical pathology, national health laboratory service, johannesburg, south africa chemedzai chikomba department of chemical pathology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa department of chemical pathology, national health laboratory service, johannesburg, south africa siyabonga khoza department of chemical pathology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa department of chemical pathology, national health laboratory service, johannesburg, south africa citation masemola km, kone n, chikomba c, khoza s. validation of nova stat profile prime plus point-of-care testing for dialysis in south africa. afr j lab med. 2025;14(1), a2663. https://doi.org/10.4102/ajlm.v14i1.2663 note: additional supporting information is available for download alongside the online version of this article as online supplementary document 1. original research validation of nova stat profile prime plus point-of-care testing for dialysis in south africa kagiso m. masemola, ngalulawa kone, chemedzai chikomba, siyabonga khoza received: 24 oct. 2024; accepted: 17 apr. 2025; published: 08 july 2025 copyright: © 2025. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: chronic kidney disease is a global health crisis, and delays in laboratory testing worsen outcomes. point-of-care testing (poct) has shown utility in various settings, but its performance at high creatinine levels seen in advanced chronic kidney disease is variable. objective: we evaluated the analytical and clinical performance of the nova stat profile prime plus (spp+) point-of-care analyser among patients on maintenance haemodialysis. methods: a prospective study was conducted at chris hani baragwanath hospital, johannesburg, south africa. in phase one (01 june 2023 – 31 july 2023), precision, linearity, and accuracy of spp+ were assessed using remnant patient samples. blood gases, electrolytes, and metabolic parameters were compared with the gem premier 5000, while urea and creatinine were compared with the roche cobas c702. in phase two (01 november 2023 – 31 december 2023), spp+ was clinically validated among adults undergoing haemodialysis. whole blood was collected preand post-dialysis to assess dialysis adequacy and the creatinine index. results: in phase one, spp+ showed acceptable precision (coefficients of variation 0% – 2.7%), linearity, and correlation (r > 0.90). creatinine showed proportional bias (4.56% [0.33 – 8.80]) at higher concentrations. among 51 haemodialysis patients (22 women, 29 men; aged 32–51 years), spp+ showed 88.6% agreement for single pool kt/v and urea reduction ratio. however, creatinine index agreement was low (34.3%, cohen’s kappa = 0.24, p < 0.0001). conclusion: nova spp+ was comparable to central laboratory analysis, though caution is needed at high creatinine levels, where central laboratory analysis remains the preferred choice. what this study adds: this study provides the first south african data on poct in haemodialysis. the analyser has demonstrated potential for monitoring, but performance concerns remain at high creatinine levels. the findings offer practical guidance for integrating poct into advanced chronic kidney disease care in a resource-limited setting. keywords: creatinine index; critical care analysers; haemodialysis adequacy; nova stat profile prime plus; point-of-care testing. introduction point-of-care testing (poct) on blood gas analysers has evolved from solely measuring ph and blood gases to sophisticated analysers capable of assessing electrolytes and metabolic parameters.1 point-of-care testing enables testing closer to patients by decentralising certain laboratory diagnostic services.2 it provides rapid access to laboratory test results for clinical decision-making and potential for improved outcomes. however, despite these advantages, when managed by non-laboratory staff, poct is not error-proof, and can be susceptible to both preanalytical, analytical as well as post-analytical errors.2,3 the new iso 15189:2022, incorporating iso 15189:2012 and iso 22870:2016, mandates appointing trained individuals for poct quality.4 laboratory experts play a crucial role in overseeing quality, offering expertise in device selection, user training, quality assurance, risk reduction and management of the total testing process.5 comprehensive method verifications, such as health technology assessments, assist in establishing poct device performance and evaluating its clinical utility.6 while there is no consensus on the scope of method verification, clinical and laboratory standards institute (clsi) guidelines offer a structured framework for evaluating analytical performance.5 nova biomedical (nova biomedical, waltham, massachusetts, united states) has incorporated advancements in poct into the nova stat profile prime plus (spp+) analyser, utilising microsensor technology with capability to measure ph, partial pressure of carbon dioxide, partial pressure of oxygen, sodium, potassium, chloride, ionised calcium, ionised magnesium, glucose, lactate, creatinine, urea and total bilirubin. a south african study in 2020 evaluated that the spp+ demonstrated acceptable precision and correlation with other poct analysers.7 while literature supports spp+ as an analyser yielding satisfactory results across diverse clinical settings,8,9,10 a 2018 study in the united kingdom emphasises the need for caution in interpreting results, particularly in the context of screening for post-contrast acute kidney injury.8 this caution is warranted, because of the low level of agreement of the spp+ in comparison with other similar analysers.9 this finding is supported by a 2023 study in nicaragua and guatemala which showed that, as concentrations of measured creatinine increase in chronic kidney disease (ckd), point-of-care creatinine is less accurate.10 however, this has not been evaluated with the spp+. therefore, robust analytical and clinical verification is needed before considering broader implementation of this poct analyser. treatment for patients with advanced ckd through haemodialysis aims to improve quality of life, while also extending life expectancy as an additional goal. however, achieving haemodialysis adequacy does not equate to adequate patient care.11 haemodialysis-dependent patients require treatments beyond haemodialysis alone. this is reflected in the kidney disease outcomes quality initiative guidelines, which cover various aspects of care such as anaemia, nutrition, metabolic bone disease, diabetes, and cardiovascular disease.12 as a priority, the kidney disease outcomes quality initiative recommends assessing haemodialysis adequacy through formal urea kinetic modeling as a proxy for small solute clearance, specifically using single-pool kt/v (spkt/v).11,13 this calculation involves the dialyser’s urea clearance rate (k), haemodialysis hours (t), and total body water content represented by the urea distribution volume (v).11 for thrice-weekly schedules, adequate haemodialysis requires a minimum spkt/v of 1.2, with an optimal target of 1.4 or, in resource-limited settings, a urea reduction ratio (urr) of 65% per session.11,13 while small solute clearance is a priority, assessing dialysis adequacy should also consider other important outcomes in this population.11 protein-energy wasting (pew) is prevalent in advanced ckd and highest in patients with kidney failure.12,14,15 this is because pew results from factors other than reduced nutrient intake, such as catabolic illness, oxidative stress, or loss of biological substances in urine and dialysate.15,16 several studies emphasise pew’s prognostic significance as a strong predictor of morbidity and mortality, irrespective of haemodialysis adequacy. the kidney disease outcomes quality initiative guidelines recommend using creatinine kinetics to estimate muscle mass and, by extension, pew in adults with kidney failure.11 a simple tool is the creatinine index (cindex) reflecting dietary protein intake and skeletal muscle mass, with a reduced cindex (< 22 mg/kg/day) linked to poor patient survival.17 frequent assessments of haemodialysis adequacy are recommended,12 but the centralised laboratory system in south africa poses practical challenges.18 in this system, same-day test results are often unavailable because of pre-analytical challenges such as sample transportation and excessive laboratory workload. in response to these delays, healthcare professionals advise patients to schedule their appointments for a later time, thereby prolonging diagnostic turnaround times.3,18,19 measurements of urea and creatinine on blood gas analysers offer a potential solution, with demonstrated comparability to central laboratory analysers.20 however, no studies have evaluated the performance of spp+ for haemodialysis adequacy or pew. therefore, the aim of this study was to evaluate both the analytical performance of the spp+ blood gas analyser and its clinical performance in haemodialysis. methods ethical considerations the study received ethical clearance from the university of the witwatersrand human research ethics committee. approval for the use of remnant patient samples submitted for routine diagnostic testing was granted under clearance number m2111171, with the requirement for patient consent waived by the committee. for the prospective phase involving recruited participants, separate approval was obtained under clearance number m230207, and written informed consent was obtained prior to participation. demographic data were collected using de-identified questionnaires. all responses were recorded anonymously, and stored data were protected with password-secured access. study design a prospective, observational study was conducted from 01 june 2023 to 31 december 2023, at the national health laboratory services, which is affiliated with chris hani baragwanath academic hospital located in johannesburg, south africa. this facility is a tertiary healthcare institution. participants the study was conducted in two phases: the first phase evaluated the spp+ analyser’s precision,21 linearity22 and accuracy23 using clsi protocols with quality control material, external quality assessment materials, and remnant patient samples. this phase was conducted from 01 june 2023 to 31 july 2023. the second phase (clinical validation) involved consecutive recruitment of participants from the adult chronic haemodialysis unit at chris hani baragwanath academic hospital between 01 november 2023 and 31 december 2023. this unit caters for 72 patients weekly, all undergoing thrice-weekly haemodialysis. participants who did not complete a haemodialysis session for any reason, and those on acute dialysis, were excluded. individuals who agreed to participate provided two 5 ml whole blood samples before and after a haemodialysis session. phase two participants completed a questionnaire to collect information required for calculation of haemodialysis adequacy and cindex, including age, sex, and preand post-haemodialysis weight. the questionnaire was created by the authors and submitted to the ethics committee. it was administered by the primary researcher through an interview to ensure that patients understood the form. responses were recorded on paper forms. sample collection and analysis phase one: analytical verification precision study (clsi ep15): within-run and between-run imprecision were evaluated using stat profile prime plus® blood gas/co-oximeter controls 1, 2, and 3, as well as chemistry controls 4 and 5 from nova biomedical. five-replicate runs of each control level were run daily for five consecutive days.21 linearity study (clsi ep06): linearity across the measuring range was assessed utilising at least five concentration levels. for electrolytes and metabolic parameters, nova linearity levels 1–4 were analysed. because of the instability of blood gas analytes, external quality assessment materials from bio-rad external quality assurance services and the national health laboratory service proficiency schemes were used as additional concentration levels. these materials were lyophilised and reconstituted prior to analysis. linearity materials were analysed in triplicate.22 method comparison (clsi ep09): forty-five whole blood (sodium-heparin) remnant samples from in-hospital patients were used to compare the analyser to currently used routine analysers in our laboratory, namely gem premier 5000 blood gas analyser (werfen s.a., barcelona, spain) for blood gas and electrolyte tests, and the roche cobas 8000 c702 analyser (roche diagnostics, rotkreuz, switzerland) for urea and creatinine.23 potentiometric sensors were used for analysis of ph, partial pressure of carbon dioxide, sodium, potassium, chloride, and ionised calcium on spp+ and gem premier 5000 (online supplementary table 1). amperometry was used to evaluate partial pressure of oxygen, glucose and lactate. urea and creatinine were measured using coupled enzymatic reactions with electrochemical detection. on the roche cobas c702 module, urea and creatinine were measured using enzymatic principles coupled with spectrophotometric detection. phase two: clinical validation during the pre-haemodialysis step, 5 ml of peripheral blood was drawn into lithium-heparin syringes by the unit nursing staff. then, using the stop-dialysate-flow technique, nursing staff promptly collected an additional 5 ml of blood from the dialyser input port in the post-haemodialysis step. the whole blood samples were first analysed on the spp+ and thereafter aliquoted into 1 ml microcentrifuge tubes and centrifuged at 3500 rpm for 10 min. the plasma was then transferred into 3 ml hitachi cups for analysis on the roche cobas c702 module. statistical analysis results were captured on microsoft excel (version 2312, microsoft corporation, redmond, washington, united states). biochemical results were extracted from the analysers and questionnaire responses initially recorded on paper were entered manually into excel using single data entry which was later verified. analytical verification statistical tests were performed following clsi protocols.21,22,23 the national health laboratory services ep15 excel template adapted from the clsi, ep evaluator (version 12.3.0.2, clinical and laboratory standards institute, wayne, pennsylvania, united states) and medcalc® statistical software (version 22.009, medcalc software ltd., ostend, belgium) were used for statistical analysis. analytical performance specifications (aps) were chosen based on one of three models from the milan conference.2 biological variation data were obtained from the european federation of clinical chemistry and laboratory medicine and westgard databases. when the latest biological variation data for an analyte were unavailable, we used the total allowable error limits from the royal college of pathologists of australasia and the clinical laboratory improvement amendments. precision (clsi ep15): mean concentration, standard deviation (s.d.), coefficient of variation, and bias were calculated using the one-way analysis of variance test. the manufacturer’s performance claims (online supplementary table 2) and aps (online supplementary table 3) were used for acceptance criteria. linearity study (clsi ep06): using ep evaluator (12.3.0.2), linearity was acceptable if the deviation from the unweighted regression line was within the allowable deviation limits for each analyte. method comparison (clsi ep09): tukey’s rule was used to test for outliers. passing-bablok regression was used to show the correlation between methods. bland-altman plots were used to define method agreement intervals, and these were compared to total allowable error limits for each analyte. clinical validation haemodialysis adequacy was assessed using spkt/v and urr. spkt/v was calculated via the second-generation daugirdas equation11 (equation 1): urea reduction ratio was calculated as a ratio13 (equation 2): two cut-off points (a minimum spkt/v of 1.2 and an optimal spkt/v of 1.4) were used to assess the level of agreement between methods for haemodialysis adequacy. furthermore, participants were then classified into two groups based on haemodialysis adequacy targets determined by urr, with a minimum urr of 65% per session. protein-energy wasting was estimated utilising the creatinine index (cindex).17 calculation of cindex involved the modified cindex equation, incorporating the participant’s age, sex, pre-haemodialysis creatinine concentration, and calculated spkt/v.17 the shapiro-wilk test was used to assess normality of spkt/v, urr and cindex results from both analysers. statistical comparison was done using the wilcoxon signed rank test; p < 0.05 was considered statistically significant. inter-rater agreement was assessed with percentage agreement and the cohen’s kappa statistic. results analytical verification precision all analytes met manufacturer claims and aps criteria for within-run and between-run imprecision (table 1). table 1: assessment of within-run and between-run precision using stat profile prime plus ampouled controls (n = 25), johannesburg, south africa, june 2023 to july 2023. method comparison passing-bablok analysis: there was a good correlation between gem premier and spp+ for all analytes (r > 0.90), except for sodium (r = 0.88) (table 2). table 2: summary of passing-bablok regression parameters for method comparison and analytical precision assessment johannesburg, south africa, june 2023 to july 2023. a comparison of spp+ and gem premier 5000 revealed biases for sodium and chlorise. sodium showed a constant negative bias (intercept of −25.3 [−56.79–0.60]). with a regression slope of 1.2. chloride had a spearman rank correlation coefficient of 0.90, but showed a positive constant bias (intercept of 10.52) with a confidence interval that did not include the ideal of zero (0.84–19.82). comparison of urea and creatinine on spp+ and roche cobas c702 demonstrated an acceptable correlation (r > 0.95) for both analytes, but revealed systematic errors (figure 1). urea had a slope of 0.85 and a y-intercept of −0.56 when compared to plasma urea. creatinine on spp+ showed a positive constant error (y-intercept = 15.04) and a proportional error with a slope of 0.78. although the method comparison study covered the reference interval for all analytes, it did not assess extremes of the measuring range due to the limited availability of lithium-heparin remnant samples. figure 1: correlation of spp+ and roche cobas c702 results for urea and creatinine, johannesburg, south africa, june 2023 to july 2023. (a) passing–bablok regression analysis (y = 0.559 + 0.853x; n = 45) and (b) bland–altman plots for urea. regression analysis displays the regression line (solid), confidence intervals (dashed), and identity line (x = y, dotted). (c) passing–bablok regression analysis (y = 15.041 + 0.796x; n = 45) and (d) bland–altman plots for creatinine. bland-altman plots depict differences versus the mean with mean (solid blue) and limits of agreement (dotted red) lines. 95% confidence intervals for the limits of agreement are indicated by blue error bars. bland-altman plots: on evaluation of method agreement, all analytes had percentage bias within the stipulated aps except chloride (table 3). the average percentage difference for chloride between for spp+ and gem premier 5000 was –2.2% which is more than the 0.6% minimum bias analytical performance specification by the european federation of clinical chemistry and laboratory medicine and more than 1.5% minimum bias by the royal college of pathologists of australasia (rcpa). however, the bias was within clinical laboratory improvement amendments limits of 2.5%. table 3: bland–altman plots for method comparison and agreement analysis using the nova stat profile prime plus, johannesburg, south africa, june 2023 to july 2023. the spp+ urea and creatinine showed acceptable analytical agreement with the roche cobas c702. even though the bias noted with urea was within aps, we noted a concentration-dependent bias (figure 1). the spp+ had a negative bias at lower concentrations (< 5 mmol/l) and a positive bias to the roche cobas c702 at higher urea concentrations (> 10 mmol/l). linearity all analytes showed acceptable linearity except urea. urea did not meet aps at the lowest concentration level (2.3 mmol/l) in external quality assessment material. it exceeded the allowable non-linearity percentage of 13.3% and had a deviation of −12.20%. despite not meeting aps, the urea concentration at the lower level was within external quality assessment aps and was therefore deemed acceptable. clinical validation fifty-one (51) patients were recruited for estimation of dialysis adequacy and cindex by measuring urea and creatinine in preand post-haemodialysis samples (online supplementary figure 1). collection was completed for 35 haemodialysed patients: 16 women, 19 men; median age was 42.5 years (interquartile range 32–51 years). all data were not normally distributed. post-dialysis weight had a median of 66.0 kg (interquartile range 56.5 – 72.5 kg) for women and 64.0 kg (interquartile range 53.5 – 70.8 kg) for men. haemodialysis adequacy by spkt/v and urea reduction ratio the median of spkt/v for both the spp+ and roche cobas c702 analysers was 1.5 (p = 0.626) (table 4). the median urr for spp+ was 71.3 and for roche cobas c702, 71.5 (p = 0.844). when classifying the minimum spkt/v, there was 88.6% agreement between the two analysers, with a cohen’s kappa statistic of 0.44 (table 5). for classification of optimal spkt/v, the agreement was 77.1%, with a cohen’s kappa statistic of 0.47. table 4: comparison of roche cobas c702 and stat profile prime plus systems for haemodialysis adequacy assessment and creatinine index measurement, johannesburg, south africa, june 2023 to december 2023. table 5: clinical agreement between roche cobas c702 and stat profile prime plus for assessing minimum and optimal single pool kt/v and creatinine index in haemodialysis, johannesburg, south africa, june 2023 to december 2023. protein-energy wasting by creatinine index in evaluating cindex, the roche cobas c702 analyser showed a median cindex of 22.78 (table 4), whereas the spp+ analyser had a median cindex of 19.63 (p < 0.0001). for the minimum cindex, agreement between the two analysers was 34.3%, with a cohen’s kappa of 0.24 (table 5). discussion the spp+ demonstrated acceptable precision, with performance comparable to central laboratory analysers for blood gases, electrolytes, glucose and lactate. it showed good agreement in haemodialysis adequacy assessment, supporting the potential of poct in decentralising haemodialysis monitoring. however, a proportional bias was observed in creatinine measurements (4.56%), with poor agreement for cindex (34.30%) which may impact the assessment of pew. this study shows that spp+ is fit for purpose with acceptable performance on analytical verification.7 the slightly lower correlation in sodium measurements aligns with findings from a 2020 south african study (r = 0.898), which also reported a weaker correlation between the radiometer abl800 flex® (radiometer, copenhagen, denmark) and spp+.7 however, since correlation does not assess agreement between methods, further assessment of bias (−0.41%) confirmed that performance of the spp+ is within allowable performance limits and therefore the lower correlation was not clinically significant. the spp+ device shows good agreement in assessing haemodialysis adequacy, suggesting potential to expand poct applications and improve clinical decision-making. the study found no statistically significant median differences in the measurement of spkt/v and urr between the spp+ and roche cobas c702 analysers (p = 0.626 and p = 0.844, respectively), supporting the utility of the spp+ for haemodialysis adequacy. a good agreement was seen in haemodialysis adequacy assessment for minimum (agreement = 88.6% and cohen’s kappa = 0.44) and optimal spkt/v (agreement = 77.1% and cohen’s kappa = 0.47) and by urr (agreement = 88.6% and cohen’s kappa = 0.44). despite the low cohen’s kappa values, which typically indicate weak agreement, the high percentage agreement suggests that the spp+ and central laboratory analyser still show acceptable concordance in assessing haemodialysis adequacy. the discrepancy in kappa values is likely because of the non-parametric distribution of data, which can introduce bias and inflate the likelihood of chance agreement. our findings align closely with those of a 2021 study in france that demonstrated that poct haemodialysis assessment show acceptable agreement with the roche cobas c701 analyser and an ultra-performance liquid chromatography tandem mass spectrometry method.22 however, in this french study, acceptable agreement was also found for creatinine measurement and cindex using the abl90 flex plus® blood gas analyzer (radiometer medical aps, copenhagen, denmark). analyser. in our study, we observed a proportional bias of serum creatinine (bias 4.56% [0.33–8.80]), especially evident at higher concentrations. in comparison with roche cobas c702, spp+ had a constant systematic error, with a y-intercept of 15.04. although, we only report a comparison with one analyser, our findings were consistent with studies in guatemala and the united kingdom, where poct creatinine inaccuracies at high concentrations skewed clinical interpretations and required a correction factor.24 in our study, creatinine index (cindex) was used as a surrogate for pew. low cindex has been found to be associated with poor cardiovascular outcomes. our findings on the cindex showed statistically significant differences (p < 0.0001) and poor agreement between the spp+ and roche cobas c702 analysers (agreement = 34.3 %, cohen’s kappa of 0.24). the suboptimal agreement between the analysers could be attributed to reduced linearity at higher creatinine concentrations. our study findings parallel those of a 2023 study in nicaragua and guatemala that demonstrated a rise in the inaccuracy with increasing concentrations of creatinine.10,24 a second study conducted in guatemala in 2017 also showed that poct creatinine consistently overestimated the creatinine by an average of 22% and the disagreement appeared greater at higher concentrations of serum creatinine.24 the spp+ analyser shows promise for decentralising poct in haemodialysis, its performance in measuring creatinine and the cindex raises concerns, particularly in patients with high serum creatinine concentrations. the observed proportional bias in creatinine measurements and poor agreement in cindex could significantly impact clinical decisions related to pew and cardiovascular risk. given the increasing prevalence of end-stage kidney disease in regions with limited resources such as south africa, further research is necessary to refine poct technologies for biomarkers such as creatinine, to ensure their accuracy at high concentrations. future studies should explore broader analyser comparisons and clinical validation in more populations with advanced ckd to improve the reliability of these devices in clinical practice. strengths the strengths of our study include incorporation of established clsi guidelines adding to the quality and validity of the study. furthermore, the study evaluated the clinical utility of the spp+ analyser in haemodialysis. this clinical validation adds practical relevance to this evaluation and highlights the potential impact on patient care. limitations the method comparison was restricted to remnant patient samples, which did not cover the entire analytical range. despite being adequately powered (> 80%), the sample size of patients attending the haemodialysis unit remains relatively small. furthermore, the performance of the spp+ was only compared to two other analysers in our study. this could affect the extent to which our findings can be generalised beyond this specific patient population and set of analysers. finally, pre-analytical factors and implementation costs were not assessed in our study. conclusion as ckd and treated kidney failure become more common, our study supports the use of poct to assess acid-base status, electrolyte balance, metabolic parameters, and dialysis adequacy. however, it should not replace central laboratory testing for creatinine measurement in patients on chronic haemodialysis. we recommend using central laboratory analysis for this purpose. acknowledgements this article is a modified version of a preprint article, available at ssrn: https://doi.org/10.2139/ssrn.4815665. this article is partially based on the k.g.m.’s thesis entitled ‘validation of nova stat profile prime plus point-of-care testing for dialysis in south africa’ towards the degree of master of medicine in the department of chemical pathology, university of the witwatersrand, south africa, with supervisors siyabonga khoza, chemedzai chikomba and ngalulawa kone. the thesis has not yet been submitted at the time of publication. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions k.m.m., n.k., c.c., and s.k. contributed to the conceptualisation, methodology, formal analysis, writing, visualisation, and investigation of this article. c.c. and s.k. are joint senior authors and contributed equally to the supervision of the study. sources of support the authors received no financial support for the research, authorship, and/or publication of this article. data availability the data that support the findings of this study are available on request from the corresponding author, k.m.m. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. the article does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the authors are responsible for this article’s results, findings, and content. references vashist sk. point-of-care diagnostics: recent advances and trends. biosensors. 2017;7(4):62. https://doi.org/10.3390/bios7040062 khan ai, pratumvinit b, jacobs e, et al. point-of-care testing performed by healthcare professionals outside the hospital setting: consensus-based recommendations from the ifcc committee on point-of-care testing (ifcc c-poct). clin chem lab med. 2023;61(1):5–12. https://doi.org/10.1515/cclm-2023-0502 khan ah, shakeel s, hooda k, siddiqui k, jafri l. best practices in the implementation of a point of care testing program: experience from a tertiary care hospital in a developing country. j clin lab anal. 2019;33(5):e22853. 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wayne (pa): clsi; 2020. clinical and laboratory standards institute (clsi). measurement procedure comparison and bias estimation using patient samples; approved guideline. 3rd ed. wayne (pa): clsi; 2018. griffin br, butler-dawson j, dally m, et al. unadjusted point-of-care creatinine results overestimate acute kidney injury incidence during field testing in guatemala. plos one. 2018;13(9):e0204614. https://doi.org/10.1371/journal.pone.0204614 abstract introduction methods results discussion acknowledgements references about the author(s) nisha vadivelu department of microbiology, m.p. shah government medical college, jamnagar, india rashmika d. parmar department of microbiology, m.p. shah government medical college, jamnagar, india hitesh shingala department of microbiology, m.p. shah government medical college, jamnagar, india krunal d. mehta department of microbiology, m.p. shah government medical college, jamnagar, india citation vadivelu n, parmar rd, shingala h, mehta kd. comparison of chromogenic and cysteine lactose electrolyte deficient agar for identification of uropathogens in gujarat, india. afr j lab med. 2025;14(1), a2551. https://doi.org/10.4102/ajlm.v14i1.2551 original research comparison of chromogenic and cysteine lactose electrolyte deficient agar for identification of uropathogens in gujarat, india nisha vadivelu, rashmika d. parmar, hitesh shingala, krunal d. mehta received: 05 july 2024; accepted: 25 nov. 2024; published: 10 feb. 2025 copyright: © 2025. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: urinary tract infections (utis) are prevalent bacterial infections, necessitating rapid and accurate diagnosis for timely treatment. conventional culture techniques, such as cystine lactose electrolyte deficient (cled) agar, can delay treatment and contribute to inappropriate antibiotic use. not much is known about alternatives such as chromogenic uti agar. objective: the study aimed to assess the performance of chromogenic uti agar compared to conventional methods for identifying uropathogens, especially in polymicrobial infections, and to determine its sensitivity, specificity, time efficiency, and cost-effectiveness for uti diagnosis. methods: an observational cross-sectional study was conducted from march 2024 to june 2024 in the microbiology department of m.p. shah government medical college in jamnagar, gujarat, india. urine samples from patients with suspected utis (n = 250) were processed using both chromogenic uti agar and cled agar. the performance of chromogenic uti agar was assessed for pathogen identification, detection of polymicrobial infections, time to results, and cost-effectiveness. results: chromogenic uti agar detected single bacterial growth in 63/250 (25.2%) samples, and mixed bacterial growth in 24/250 (9.6%) samples, whereas cled agar showed single bacterial growth in 67/250 (26.8%) samples and mixed bacterial growth in 10/250 (4%). the chromogenic medium provided preliminary results 5.5 h earlier (p < 0.001) and final results 24 h earlier (p < 0.001) than conventional methods. cost analysis revealed a 33% reduction per-test cost using chromogenic uti agar (p < 0.001). conclusion: chromogenic uti agar demonstrated excellent performance in the rapid and accurate diagnosis of utis, including improved detection of polymicrobial infections. what this study adds: when it comes to diagnosing utis, chromogenic uti agar has several benefits over traditional techniques, such as high accuracy, enhanced detection of polymicrobial infections, and cost-effectiveness. the research backs up the inclusion of chromogenic medium in standard uti diagnosis procedures. keywords: urinary tract infections; chromogenic media; rapid diagnosis; cost-effectiveness; polymicrobial infections; clinical microbiology. introduction urine is the most frequently cultured specimen in laboratories globally, as urinary tract infections (utis) rank among the most prevalent health issues encountered in both hospital and community environments.1 they are a major cause of morbidity in both community and hospital settings and are responsible for a large percentage of infections linked to healthcare.2 quick and precise uti diagnosis is essential for prompt and effective treatment, which can avoid complications and lower medical expenses.3 in gujarat, india, where this study was conducted, utis represent an important health burden. a recent study at a tertiary care hospital in ahmedabad found that 45.6% of urine cultures were positive for organisms, with escherichia coli being the most common isolate.4 however, the diagnostic methods used in many regional laboratories still rely primarily on conventional culture techniques, which can delay treatment and contribute to inappropriate antibiotic use. there is a need to evaluate newer diagnostic methods, such as chromogenic media, in our local setting to potentially improve uti management. traditionally, the gold standard for uti diagnosis has been urine culture using conventional media such as cystine lactose electrolyte deficient (cled) agar, followed by biochemical tests for pathogen identification. however, because cled agar and macconkey agar lack differential genus-specific indicator features, mixed cultures are not detectable.5 while effective, this method is time-consuming, typically requiring 48 h – 72 h for definitive results. in an era of increasing antimicrobial resistance, there is a pressing need for faster and more efficient diagnostic methods.6 chromogenic media have emerged as a promising alternative for the rapid detection and identification of uropathogens. these media contain chromogenic substrates that are cleaved by specific bacterial enzymes, resulting in coloured colonies that allow for the presumptive identification of pathogens directly on the primary isolation plate.7 this approach has the potential to reduce turnaround time, decrease workload, and improve the accuracy of uti diagnosis.8 furthermore, in resource-limited settings such as many parts of india, the cost-effectiveness of diagnostic methods is a crucial consideration. while chromogenic media may offer diagnostic advantages, their higher upfront cost compared to conventional media has been a barrier to widespread adoption in some regions.9 therefore, a comprehensive evaluation of both performance and cost-effectiveness is essential to inform evidence-based decision-making in our local healthcare system. several studies have evaluated the performance of various chromogenic media for uti diagnosis, with many showing promising results.10,11 however, the performance of these media can vary depending on the specific formulation and the prevalence of different uropathogens in different geographic regions.12 therefore, it is important to evaluate the performance of chromogenic media in specific clinical settings before their widespread adoption. chromogenic uti agar is a commercially available chromogenic medium that has shown potential for rapid identification of uropathogens.13 however, its performance in comparison to conventional methods has not been studied extensively in our regional setting. furthermore, its ability to detect polymicrobial infections, which can be challenging to identify using conventional methods, warrants further investigation.14 the study evaluates the performance of chromogenic uti agar in comparison to conventional culture methods (cled agar plus biochemical tests) for the diagnosis of utis, and for the detection and identification of individual uropathogens and polymicrobial infections. this study determines the sensitivity, specificity, and positive and negative predictive values of chromogenic uti agar for uti diagnosis. furthermore, it compares the time to identification and cost-effectiveness of chromogenic uti agar versus conventional methods. the present study findings could inform decisions on incorporating this medium into routine diagnostic protocols for utis. methods ethical considerations the institutional ethics committee of m.p. shah government medical college and guru gobind singh hospital, jamnagar, india gave its approval to the project (reference number: 266/03/2023). written informed consent was obtained from all participants or their legal guardians before enrolment in the study. for participants under 18 years of age, assent was obtained in addition to parental/guardian consent. during the consent process, participants were informed about the purpose of urine sample collection, and the nature of the research study, and were assured of data confidentiality. they were also informed that participation was entirely voluntary, and they could withdraw from the study at any time without any impact on their medical care. patient data were anonymised to maintain confidentiality and were stored on password-protected devices. only the investigators of the study had access to the data. study design and settings this observational, cross-sectional study was conducted from march 2024 to june 2024 in the department of microbiology at m.p. shah government medical college, jamnagar, gujarat, india. sample size a power analysis was performed using g power 3.1 software (heinrich-heine-universität düsseldorf, düsseldorf, germany) to determine the sample size.15 assuming a medium effect size (w = 0.3), an alpha of 0.05, and a power of 0.80, the required sample size was calculated to be 238. we rounded up to 250 to account for potential exclusions. patient selection and sample collection the study included patients, of all ages and genders, with clinically suspected utis, who were seen in our tertiary care hospital’s outpatient and inpatient departments. the presence of at least one of the following symptoms – dysuria, frequency, urgency, suprapubic discomfort, or fever with no other apparent cause in catheterised patients – was included for clinical suspicion of a uti. after giving ambulatory patients thorough instructions on the correct collection method, clean-catch midstream urine samples were taken from them. for catheterised patients, urine samples were collected aseptically from the catheter port using a sterile syringe. all samples were collected in wide-mouthed, screw-capped, sterile containers. the samples were then taken to the microbiology laboratory within 2h of being collected from the corresponding wards and forwarded to the microbiology outpatient department. before culture, samples were kept in a refrigerator at 4 °c for no more than 4 h if processing could not be done right away.16 sample processing and media preparation upon receipt in the laboratory, samples were logged into a secure electronic database with a unique identifier. samples were then processed immediately or stored at 4 °c if there was a delay of more than 30 min before processing. hichrome uti agar (himedia laboratories, maharashtra, india; lot no: 0000510836, expiry: 2024-11) was made in compliance with the guidelines provided by the manufacturer. in short, 1000 ml of distilled water was used to dissolve 32.45 g of dehydrated powder, which was then heated to boiling to dissolve it fully, and autoclaved for 15 min at 121 °c. after cooling to 45 °c – 50 °c, the medium was transferred into sterile petri plates.17 cystine lactose electrolyte deficient agar (himedia laboratories, maharashtra, india; lot no. 0000403507, expiry: 2024-08) was prepared similarly according to the manufacturer’s instructions. cystine lactose electrolyte deficient agar powder and distilled water were autoclaved at 121 °c for 15 min, then the mixture was cooled to 45 °c – 50 °c, mixed and dispensed into sterile petri plates.18 inoculation and incubation a calibrated loop technique was used for semi-quantitative culture. a calibrated loop or measured piece of filter paper can be used to estimate the approximate number of bacteria per millilitre of urine. accepting that a single colony represents a single organism is the foundation of both approaches. for instance, if a 1 ml inoculum yields 20 colonies, then there are 20 organisms in 1/500 ml of urine or 10 000 organisms in 1 ml (500 × 20). if 20 bacterial colonies are discovered in the current study, the number of colonies per millilitre is 20 × 1000 = 20 000. so, a 1 µl sterile loop was used to inoculate urine onto chromogenic uti agar and cled agar plates. the inoculum was spread in a line down the centre of the plate and then streaked perpendicularly to yield isolated colonies. for 18 h to 24 h, all plates were incubated aerobically at 37 °c. extended incubation for up to 48 h was performed if no growth was observed after 24 h. colony counting and identification after incubation, plates were examined for growth. colony counting was performed manually using a colony counter. notable bacteriuria was defined as the growth of two distinct uropathogens at > 104 colony-forming units (cfu)/ml, or ≥ 105 cfu/ml of non-coliforms, or > 102 cfu/ml of coliforms in symptomatic women, or > 103 cfu/ml in symptomatic men. on chromogenic uti agar, presumptive identification was made based on colony colour and morphology according to the manufacturer’s colour chart. on cled agar, colonies were identified based on their characteristic appearance and lactose fermentation. definitive identification requires confirmatory molecular or biochemical testing. future studies should incorporate these validation methods to establish the accuracy of chromogenic-based identification.19 confirmatory tests all presumptive identifications were confirmed using a combination of gram staining and biochemical tests. the following tests were performed, as appropriate: catalase, coagulase, oxidase, indole production, urease production, citrate utilisation, and triple sugar iron agar. quality control measures the investigation employed quality control strains, specifically e. coli atcc 35218, enterococcus faecalis atcc 29212, staphylococcus aureus attc 29213, and pseudomonas aeruginosa atcc 27853. data collection and management all patient-related data were collected from the microbiology laboratory’s medical records, and sample collection and laboratory assays were performed by trained research staff following standard clinical protocols. double data entry was performed to ensure accuracy, and any discrepancies were resolved by referring to the original laboratory worksheets. cost analysis data collection cost data were collected from the hospital’s financial records and the microbiology laboratory’s purchase orders. direct costs included the prices of media, reagents, and disposable supplies. labour costs were calculated based on the average technician salary and the time required for each test, as measured by time-motion studies conducted over a 2-week period. indirect costs, such as equipment depreciation and utilities, were allocated based on the proportion of laboratory workload dedicated to urine cultures. all costs were converted to united states dollars (usd), using the average exchange rate for the study period. to account for potential price fluctuations, we collected cost data for the past 3 years and used the average values in our analysis. a cost comparison between chromogenic uti agar and conventional methods was performed, taking into account the costs of media, reagents, and labour. time to preliminary results (based on chromogenic agar or colony appearance on cled) and final results (after confirmatory tests) were recorded for each sample.20 statistical analysis statistical package for the social sciences version 26.0 was used for the statistical analysis (ibm corp., armonk, new york, united states). the two media’s uropathogen detection rates were compared using the chi-square test. to assess the two media’s capacity to identify mixed cultures, mcnemar’s test was applied to paired nominal data. statistical significance was defined as p < 0.05.21,22 using the findings from traditional techniques as the gold standard, sensitivity, specificity, positive predictive value, and negative predictive value were computed for chromogenic uti agar. results in total, 250 patients with suspected utis were involved in this investigation (table 1). the participants’ average age was 45.3 years (standard deviation [s.d.] ± 18.7), with a predominance of female patients (63.2%, n = 158) compared to male patients (36.8%, n = 92). the majority of patients (70.0%, n = 175) were from outpatient settings, while 30.0% (n = 75) were inpatients. of the total participants, 15.2% (n = 38) had urinary catheters at the time of sample collection. regarding comorbidities, the most common condition was hypertension (29.2%, n = 73), which was followed by diabetes mellitus (20.8%, n = 52) and chronic kidney disease (7.2%, n = 18). notably, 26.8% (n = 67) of patients reported having a uti within the past year, indicating that over a quarter of our study population experienced recurrent infections. at the time of sample collection, 18.0% (n = 45) of patients were currently using antibiotics, which could potentially influence the culture results. table 1: demographic and clinical characteristics of study participants, jamnagar, gujarat, india, march 2024 to june 2024. out of the 250 samples, chromogenic uti agar detected single bacterial growth in 63 (25.2%) samples, while cled agar showed single growth in 67 (26.8%) samples (p = 0.683, not significant) (table 2). notably, chromogenic uti agar identified mixed bacterial growth in 24 (9.6%) samples, more than double the number detected by cled agar (10 samples, 4.0%), and this difference was statistically significant (p = 0.011). the number of samples showing no growth was slightly higher in cled agar (173, 69.2%) compared to chromogenic uti agar (163, 65.2%); however, this difference (p = 0.339) was not statistically significant. table 2: comparison of growth of chromogenic urinary tract infections agar and cysteine lactose electrolyte deficient agar, jamnagar, gujarat, india, march 2024 to june 2024. escherichia coli was the most commonly isolated organism on both media, with slightly higher detection on cled agar (64.2% vs 58.7%) (table 3). chromogenic uti agar showed improved detection of enterococcus spp. (14.3% vs 7.4%, p = 0.212) and pseudomonas spp. (9.5% vs 7.4%, p = 0.668) and cled agar. interestingly, chromogenic uti agar did not detect any staphylococcus spp., while cled agar identified one isolate (p = 0.332). table 3: comparison of single uropathogen isolation, jamnagar, gujarat, india, march 2024 – june 2024. chromogenic uti agar detected mixed growth in all 24 samples where polymicrobial infections were present, achieving a 100% detection rate (table 4). in contrast, blood agar detected mixed growth in only 11 samples (45.8%), macconkey agar in nine samples (37.5%), and cled agar in 10 samples (41.7%). the differences in detection rates between chromogenic uti agar and each of the other media were highly statistically significant (p < 0.001 for all comparisons). table 4: rate of detection of mixed bacterial growth in different media, jamnagar, gujarat, india, march 2024 to june 2024. chromogenic uti agar detected a total of 24 polymicrobial infections, while cled agar only identified 10, a statistically significant difference (p = 0.011) (table 5). the most common polymicrobial combination on both media was e. coli + klebsiella spp., detected in 13 samples (54.1%) by chromogenic uti agar and eight samples (80%) by cled agar (p = 0.159). notably, chromogenic uti agar was able to detect combinations involving enterococcus spp. that were missed by cled agar, including five cases (20.8%) of pseudomonas spp. + enterococcus spp. (p = 0.124) and four cases (16.7%) of enterococcus spp. + e. coli (p = 0.612). table 5: comparison of polymicrobial uropathogen isolation, jamnagar, gujarat, india, march 2024 to june 2024. strong reliability in both positive and negative test results was indicated by the high positive and negative predictive values. the overall accuracy of 97.2% suggested excellent agreement between chromogenic uti agar and conventional methods. chromogenic uti agar demonstrated high sensitivity and specificity in detecting urinary tract pathogens when compared to conventional methods (table 6). table 6: diagnostic performance of chromogenic urinary tract infections agar compared to conventional methods, jamnagar, gujarat, india, march 2024 to june 2024 (n = 250). in terms of cost, chromogenic uti agar was found to be more economical. the cost per test was usd 2.50 ± 0.20 for chromogenic uti agar compared to usd 3.75 ± 0.30 for conventional methods, representing a 33% reduction (p < 0.001). when projected to an annual workload of 5000 tests, this translates to a potential saving of usd 6250 ± 1000 per year (p < 0.001). time efficiency was also markedly improved with chromogenic uti agar. chromogenic uti agar demonstrated statistically significant advantages in both cost and time metrics (table 7). preliminary results were available 5.5 h earlier on average (18.5 ± 2.5 h vs 24.0 ± 3.0 h, p < 0.001). more importantly, final results were obtained 24 h earlier (24.0 ± 3.0 h vs 48.0 ± 6.0 h, p < 0.001). additionally, the technician time required per test was reduced by 10 min when using chromogenic uti agar (15.0 ± 2.0 min vs 25.0 ± 3.0 min, p < 0.001). table 7: cost and time comparison between chromogenic and conventional media for urinary tract infections diagnosis, jamnagar, gujarat, india, march 2024 to june 2024. discussion the present study demonstrates several important advantages of chromogenic uti agar. it exhibited high sensitivity (95.4%) and specificity (98.2%) in detecting uropathogens, comparable to conventional methods. notably, chromogenic uti agar showed superior ability in detecting polymicrobial infections (9.6% vs 4.0%, p = 0.011), which is crucial for accurate diagnosis and appropriate treatment. a statistically significant improvement in time efficiency was observed, with chromogenic uti agar reducing time to preliminary results by 5.5 h and final results by 24 h compared to conventional methods. furthermore, it proved to be more cost-effective, with a 33% reduction in per-test cost. these findings collectively suggest that chromogenic uti agar offers substantial benefits in terms of accuracy, detection of polymicrobial infections, time efficiency, and cost-effectiveness in the diagnosis of utis, potentially improving patient care and laboratory workflow in our setting. chromogenic uti agar showed high sensitivity (95.4%) and specificity (98.2%) in detecting uropathogens, comparable to or exceeding the performance reported in previous studies conducted in the united kingdom by perry and freydière, and flores-mireles et al. on chromogenic media.7,23 this high accuracy is crucial for utis, which affect millions worldwide, and greatly contribute to illnesses linked to healthcare.24,25 rapidly and accurately identifying pathogens can lead to more timely and appropriate treatment, potentially reducing complications and the spread of antimicrobial resistance.26 the current study’s isolation rate and the majority of organisms identified were staphylococcus spp., e. faecalis, klebsiella spp., and e. coli. the findings are similar to those of a study conducted in sudan by fatema et al.27 improved identification of polymicrobial infections chromogenic uti agar found 24 (70.58%) of the 34 cases of mixed growth of two organisms, while cled agar detected 10 (29.41%). these results are comparable to those reported by biji et al. in thiruvananthapuram, india,28 and kaskar et al. in mumbai, maharashtra, india.29 one of our study’s most significant findings was chromogenic uti agar’s superior ability to detect polymicrobial infections compared to conventional methods (9.6% vs 4.0%, p = 0.011). this aligns with previous research in canada and the united states, suggesting that chromogenic media can enhance the detection of mixed cultures in urine samples.13,19 the improved detection of polymicrobial infections is particularly important given the challenges these infections pose in terms of diagnosis and treatment.30 time and cost efficiency our study demonstrated that chromogenic uti agar substantially reduced the time to both preliminary and final results compared to conventional methods. this rapid turnaround time is crucial in clinical settings, potentially allowing for early start of the right antimicrobial treatment.31 furthermore, the cost analysis revealed a 33% reduction in per-test cost using chromogenic uti agar. in an era of increasing healthcare costs, this cost-effectiveness could be an important factor in the adoption of chromogenic media in routine clinical practice.32 implications for clinical practice the combined benefits of improved accuracy, faster results, and cost-effectiveness suggest that chromogenic uti agar could be a valuable tool in the diagnosis of utis. its implementation in clinical laboratories could potentially streamline workflows, reduce turnaround times, and improve patient care. but it is crucial to remember that even while chromogenic medium can offer presumptive identification, confirmatory tests are still necessary for definitive identification and antimicrobial susceptibility testing.7,24 recommendations while our study demonstrates the advantages of chromogenic uti agar in a tertiary care setting in gujarat, future research could explore its performance across different healthcare settings and geographic regions in india. multi-centre studies would be valuable to confirm these findings across diverse patient populations. additionally, future studies could evaluate the performance of chromogenic uti agar in detecting less common uropathogens or anaerobic bacteria, potentially expanding its utility in uti diagnosis. furthermore, research investigating the impact of chromogenic media on clinical outcomes, such as more appropriate antibiotic prescribing, reduced length of hospital stays, or improved patient outcomes, would provide valuable evidence for its clinical utility beyond laboratory performance metrics. limitations our study contains several drawbacks despite its advantages. firstly, it was only carried out in one location, which might have limited how far it can be applied. multi-centre studies would be valuable in confirming these findings across different patient populations and geographic regions. secondly, we did not evaluate the performance of chromogenic uti agar in detecting less common uropathogens or anaerobic bacteria. while this study evaluated chromogenic media as a primary culture medium, standard practice often employs it as a secondary identification tool. future studies should assess its optimal placement in the diagnostic workflow, considering both standalone and sequential use with conventional media. conclusion our study demonstrates that chromogenic uti agar offers important advantages over conventional methods in the diagnosis of utis, including high accuracy, improved detection of polymicrobial infections, faster turnaround times, and cost-effectiveness. these findings support the integration of chromogenic media into routine diagnostic protocols for utis. however, as with any new diagnostic tool, its implementation should be accompanied by appropriate training and quality control measures to ensure optimal performance in clinical settings. acknowledgements we would like to thank prof. hitesh shingala, head of the department of microbiology, for providing resources to conduct the study. thanks to dr rashmika d. parmar for supervision and final approval of the article. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions n.v. contributed to the conceptualisation, visualisation, methodology, formal analysis, investigation, and writing of the original draft. r.d.p. contributed to project administration, supervision, and validation of the study. h.s. contributed to the project administration, validation, and resources of this study. k.d.m. contributed to the project administration, software, data curation, writing, reviewing, editing, supervision and final approval of the article. n.v., r.d.p., h.s., and k.d.m. contributed to the writing, editing and final approval of this article. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the data that support the findings of this study are available on request from the corresponding author, k.d.m. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. the article does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the authors are responsible for this article’s results, findings, and content. references khalid m. comparison of chromogenic (hicrome urinary tract infection agar) medium with cysteine lactose electrolyte deficient agar in a resource-limited setting. int j appl basic med res. 2021;11(1):9–13. https://doi.org/10.4103/ijabmr.ijabmr_306_19 magill ss, o’leary e, janelle sj, et al. changes in prevalence of health care-associated infections in u.s. hospitals. n engl j med. 2018;379(18):1732–1744. https://doi.org/10.1056/nejmoa1801550 bonkat g, pickard r, bartoletti r, et al. eau guidelines on urological infections 2018. arnhem, european association of urology; 2018. patel hb, soni st, bhagyalaxmi a, patel nm. causative 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culture media after one or two days of incubation compared with reference media. j clin microbiol. 2002;40(4):1500–1503. https://doi.org/10.1128/jcm.40.4.1500-1503.2002 leber al, editor. clinical microbiology procedures handbook. asm press; 2016. yousefi m, pourmand mr, fallah f, hashemi a, mashhadi r, nazari-alam a. characterization of staphylococcus aureus biofilm formation in urinary tract infection. iran j public health. 2016;45(4):485–493. fatema n, alam a, ahmed co. comparative study between chromogenic medium and conventional media for detection of uropathogens in urine. jalalabad med j. 2009;6:5–10. biji sm, sasikumari o, bai jr. a study on evaluation of different culture media for the isolation of routine urinary pathogens. int j curr microbiol app sci. 2017;6:195–201. https://doi.org/10.20546/ijcmas.2017.602.026 kaskar sn, tendolkar mr, vaidya sp, et al. comparative evaluation of performance and cost effectiveness of hicrome uti agar in detection of urinary tract pathogens. ijar. 2018;4:358–366. carricajo a, boiste s, thore j, aubert g, gille y, freydière a. comparative evaluation of five chromogenic media for detection, enumeration and identification of urinary tract pathogens. eur j clin microbiol infect dis. 1999;18(11):0796–0803. https://doi.org/10.1007/s100960050403 world health organization. global antimicrobial resistance surveillance system (glass) report: early implementation 2017–2018. geneva: world health organization; 2018. stefaniuk e, baraniak a, gniadkowski m, hryniewicz w. evaluation of the bd phoenix automated identification and susceptibility testing system in clinical microbiology laboratory practice. eur j clin microbiol infect dis. 2016;35(9):1543–1551. abstract introduction methods results discussion acknowledgements references about the author(s) iryna tanasiichuk department of modern technologies of medical diagnostics and treatment, institute of postgraduate education, bogomolets national medical university, kyiv, ukraine olha karaman laboratory of oncoimmunology and design of tumor vaccines, r.e. kavetsky institute of experimental pathology, oncology and radiobiology, national academy of sciences of ukraine, kyiv, ukraine larysa natrus department of modern technologies of medical diagnostics and treatment, institute of postgraduate education, bogomolets national medical university, kyiv, ukraine citation tanasiichuk i, karaman o, natrus l. key success factors for the implementation of quality management systems in developing countries. afr j lab med. 2023;12(1), a2058. https://doi.org/10.4102/ajlm.v12i1.2058 note: additional supporting information may be found in the online version of this article as online supplementary document 1. review article key success factors for the implementation of quality management systems in developing countries iryna tanasiichuk, olha karaman, larysa natrus received: 13 aug. 2022; accepted: 17 nov. 2022; published: 31 jan. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: despite the tremendous progress made in advancing laboratory medicine in lowand middle-income countries (lmics), inadequate quality management systems (qmss) remain a problem and barrier to provision of reliable laboratory services in resource-limited settings. therefore, it is useful to study the experience of medical laboratories in lmics that have successfully implemented qms. aim: this review identified key success factors (ksfs) for medical laboratories in lmics implementing qms in accordance with the international organization for standardization standard 15189 as a pathway to improving laboratory quality. methods: applying preferred reporting items for systematic reviews procedures, we conducted a targeted search of studies from lmics published between 2012 and 2022 to identify ksfs. thirty-two out of 952 references retrieved were considered relevant and included in this review. grounded theory was used to extract key features of the included studies to derive ksfs. results: ten ksfs for medical laboratories striving to implement qms were identified and described. these ksfs were integrated to create a model of success for laboratory qms implementation. the model consists of three underlying factors, namely preparing for change, resource availability, and effective project management, each comprising three separate ksfs. institutional commitment was identified as the core of the model and is integral to ensuring the quality of laboratory services. conclusion: laboratories planning to implement a qms can benefit from understanding the ksfs demonstrated in this study as this would help them to identify the necessary changes to implement and set realistic expectations about the outcomes of qms implementation. keywords: laboratory quality; accreditation of medical laboratories; iso 15189; strengthening laboratory quality management systems; quality improvement. introduction healthcare systems around the world are tasked with improving the quality of medical care and patient safety. high-quality laboratory services are critical to achieving this goal, considering the significance of laboratory testing results in physicians’ decision-making processes.1,2,3 currently, the international organization for standardization (iso) 15189:2012 ‘medical laboratories – requirements for quality and competence’ standard4 (hereinafter referred to as ‘the standard’) is the most demanding regulatory document related to medical laboratories. following the standard requires meeting both the principles of iso 9001:20085 for implementing a quality management system (qms) and the requirements of iso/international electrotechnical commission 17025:20056 for technical competency. numerous medical laboratories that have met the standard’s requirements and received accreditation have shown improvements in the quality of medical services and patient safety.1,7,8,9,10,11,12,13 at the same time, it is noteworthy that implementation of the standard is not an easy task, and preparation for accreditation is typically considered a multi-year, expensive, and labour-intensive project, even in developed countries.7,8,9,12,14 establishing and maintaining the standard’s requirements presents significant and often insurmountable challenges in resource-poor settings.3,7,15,16,17 starting with the maputo declaration in 2008, global efforts to strengthen laboratory medicine in lowand middle-income countries (lmics) and the unprecedented increase in international funding for these initiatives has made it possible to significantly improve the quality of laboratory services in lmics.18,19 however, despite these tremendous advances in laboratory medicine, inadequate qmss remain a problem and one of the barriers to the provision of reliable laboratory services in many lmics.3,15,16 therefore, it is useful to study the experience of medical laboratories in lmics that have successfully implemented qms and improved laboratory quality. awareness of the factors that affect the success of a qms implementation would help identify the main steps that medical laboratories or their parent organisations (hereinafter referred to as ‘the implementers’) need to take in implementing necessary changes. these factors are actively discussed in the literature. however, the respective studies are predominantly focused on individual laboratories, or a small number of laboratories located in specific countries. a systematic review would provide a balanced and unbiased summary of the accumulated studies. consequently, this research aimed to identify the key success factors (ksfs) for medical laboratories in lmics striving to implement qms in accordance with iso 15189 as a pathway to improving laboratory quality. methods reporting guidelines the preferred reporting items for systematic reviews (prisma) was used to guide the reporting of this review.20 this systematic review is registered in prospero with the registration number: crd42022338151. literature search for this systematic review, we searched medline (i.e., pubmed), web of science, and google scholar in june 2022. the terms ‘iso 15189 accreditation’, ‘laboratory quality management system’, and ‘medical laboratories’ were used in various combinations with the following words: ‘success’, ‘strengthening’, ‘improvement’, and ‘implementation’. additional search terms, namely ‘africa’, ‘asia’, ‘low-income countries’, and ‘countries with limited resources’ were used to narrow down the search results. the results were cross-referenced with the world bank’s 2022 list of low-, lower-middle-, and upper-middle-income countries.21 the reference lists of all the selected studies were thoroughly inspected to identify additional studies of interest. study selection and data extraction two reviewers screened the titles and abstracts against the eligibility criteria. all the potentially relevant articles were accessed in full-text format. both reviewers independently made the final decision on whether to include each of the articles in this review, and conflicting decisions were discussed and resolved. the articles were subjected to content analysis by two reviewers, each of whom extracted and documented the key findings of the included studies. inclusion сriteria articles that were published in english between 2012 and 2022 and contained primary data that demonstrated the results of qms implementation in medical laboratories in lmics according to the standard’s requirements were included in the review. no restrictions were placed on the research design. exclusion сriteria we excluded studies without a direct focus on the implementation of the standard’s requirements, as well as studies whose reports of such implementation did not include information about the immediate evidence of improvement in the quality of laboratory services. we also excluded studies conducted in non-medical laboratories (forensic, research, etc.). data synthesis we used the grounded theory to identify the ksfs, with the data analysed using a multistage procedure of open, axial and selective coding.22 conceptualisation and data categorisation were done by one of the reviewers, after which another reviewer assessed the theoretical relevance of the selected categories. any discrepancies in the reviewers’ definition, formulation or integration of the categories were resolved through discussions until an agreement was reached. assessment of the study quality one reviewer critically evaluated the methodological quality of the papers using the critical appraisal tool described by hawker et al.23 according to the tool, studies can be assessed based on nine criteria: abstract and title; introduction and aims; method and data; sampling; data analysis; ethics and bias; results; transferability or generalisability; implications and usefulness. each criterion was evaluated on a scale of 1 (very poor) to 4 (good) in accordance with the developed protocol.23 thus, each article could receive from 9 to 36 points, indicating the methodological rigour of the study. another reviewer cross-checked 30% of the assessed studies and expressed full agreement with the initial assessment of their quality. results literature search results a total of 952 records were identified through databases (n = 880) and citation (n = 72) searching. after removing the duplicates, 753 abstracts were retrieved and screened. after applying the inclusion and exclusion criteria, two independent reviewers narrowed down the references to 59 full-text versions of articles. the analysis of those articles led to 32 of them being included in the final analysis (figure 1). figure 1: flow diagram illustrating the literature review process to identify the key success factors for medical laboratories in lowand middle-income countries striving to implement a quality management system in accordance with international organization for standardization standard 15189, 2012–2022. characteristics of the identified studies most of the studies included in this review were conducted in africa: ethiopia (n = 6), kenya (n = 4), tanzania (n = 3), lesotho (n = 3), botswana (n = 2), nigeria (n = 2), cameroon (n = 2), mozambique (n = 2), rwanda (n = 1), zambia (n = 1), ghana (n = 1), zimbabwe (n = 1), and benin (n = 1).24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49 studies reporting data from the southeastern asian countries (cambodia [n = 2], vietnam [n = 2]),50,51,52,53 the caribbean region (n = 1),54 and armenia (n = 1)55 were also included in the analysis (supplementary table 1). we found no relevant studies conducted in eastern europe (ukraine, belarus, moldova, romania, etc.) or central asia (kazakhstan, uzbekistan, kyrgyzstan, tajikistan, turkmenistan). the 32 studies included in this review demonstrated the results of successful qms implementation in 280 medical laboratories; 266 showed quality improvement and 14 met the standard’s requirements and achieved accreditation (table 1). two hundred and seventy-eight out of the 280 laboratories were in the public sector. most (n = 237) of the studies were conducted in laboratories enrolled in the strengthening laboratory management toward accreditation programme. table 1: characteristics of laboratories that showed quality improvements in 32 identified studies conducted in lowand middle-income countries between 2012 and 2022. study quality the included studies varied in their methodological quality: two studies received the highest attainable score – 36 points, 13 scored between 33 and 35 points, 16 scored between 29 and 32 points, and just one scored 25 points. studies lost points mostly due to insufficient presentation of the methods of research (n = 5; 16%) and result bias (n = 22; 69%). model of success for laboratory quality management system implementation the use of open and axial coding techniques22 allowed the identification of 10 different categories, which represent the necessary activities, conditions, and strategies needed to improve the quality of laboratory services by implementing qms in accordance with the standard. those categories were named ‘key success factors’ as they are crucial to the implementation of the standard’s requirements (table 2). across the 32 studies, the most frequently identified ksfs were ‘mentorship’ (n = 27; 84%) and ‘trained laboratory staff’ (n = 26; 81%), while the least common factors were ‘personnel management’ (n = 8; 25%) and ‘qms implementation strategy’ (n = 8; 25%). table 2: key success factors for medical laboratories striving to implement a quality management system as identified by a review of 32 studies conducted in lowand middle-income countries between 2012 and 2022. using the selective coding technique,22 the 10 ksfs were integrated based on their properties and paradigmatic connections into a ‘model of success for laboratory qms implementation’ (figure 2). this model consists of three underlying factors, namely preparing for change, resource availability, and effective project management, each comprising three separate ksfs. these three underlying factors are united around their own central factor, which serves as a stabilising factor and the core of the model of success, without which other factors are ineffective. figure 2: model of success for laboratory quality management system implementation developed based on a review of 32 studies conducted in lowand middle-income countries between 2012 and 2022. preparing for change as the first underlying factor in the model of success for laboratory quality management system implementation hospital management dedication to laboratory qms implementation, laboratory personnel commitment to quality improvement, and trained laboratory staff are the three ksfs that ensure readiness for change among the contributing parties. a high level of readiness for change is crucial to the implementation of any organisational change,56 including in the medical sphere,57,58 and medical laboratories are no exception. the starting point of qms implementation and laboratory quality improvement should be sufficient preparation for the implementation initiative. sufficient preparation means that all stakeholders involved in the process of qms implementation must agree that there is a need for change and possess the required theoretical and practical knowledge. firstly, the need for change must be recognised at the top management level to ensure the provision of essential financial support25,28,40,45,52 and organisational conditions.29,37,40,44,51,52 nevertheless, top hospital management can only provide the required assistance when there is a clear understanding of the benefits of effective qms to the patients and the facility in general.28 the lack of such understanding creates a strong administrative barrier to the laboratory’s quest for accreditation.31 at the same time, the rejection of new standards and resistance to change29,31,39,43 by laboratory personnel could nullify the efforts of senior management aimed at implementing laboratory qms. laboratory personnel commitment to quality improvement is thus one of the ksfs for the improvement of laboratory service quality.24,26,29,30,34,37,38,39,40,41,43,45,52 however, one must realise that the implementation of the standard’s requirements in medical laboratories often calls for considerable changes in personnel’s daily routines. reorganising the existing processes and introducing new ones during routine laboratory practice often leads to increases in workload.25,29,31,41,42,45,53,55 this could significantly impact personnel’s attitude to change, more so when the personnel do not see the need for organisational changes. involving personnel in the decision-making process and teaching them the basics of qms could help overcome the reluctance to change and develop personnel commitment.29,30,37,38,40,49 staff awareness of qms is another component of the organisational readiness to implement changes and is also one of the two most common ksfs in the selected studies. implementing laboratory qms requires ensuring proper theoretical and practical preparation in advance. managerial and technical laboratory personnel must be conversant with the standard’s requirements and practical applications.24,27,28,29,30,31,34,37,39,40,42,43,44,46,47,48,49,50,51,52,54,55 technical personnel should also be aware of the standard’s requirements for technical procedures,22,24 and laboratory management personnel must be familiar with organisational management, leadership, and improvement activities.26,32,50,51 resource availability as the second underlying factor in the model of success for laboratory quality management system implementation physical facilities, staffing and qms implementation strategy are the three main resources that could either facilitate or inhibit the overall laboratory improvement process. physical facilities represented by adequate infrastructure26,29,30,31,39,48 and independent financial28,29,30,34,35,43,45,48,51,54 and material29,31,40,47,48 resources are basic requirements for good laboratory practice. the implementers must understand that substantial investments are necessary to comply with the technical requirements of the standard regarding laboratory and office facilities, environmental conditions, and laboratory equipment, reagents, and consumables. adequate staffing is the second valuable resource that greatly affects the qms implementation process. the lack of human resources25,28,29,37,42,55 and employee turnover30,31,32,40,41,49,54 can hinder the progress of qms implementation. laboratory management should keep this in mind and make appropriate management decisions in hiring the laboratory staff needed to efficiently handle the workload and prevent staff turnover. the qms implementation strategy is another essential resource that affects the efficiency of the system’s implementation. it is very important to choose the type of implementation that best fits the laboratory. our study shows that the use of a gradual, step-by-step qms implementation approach, such as the world health organization’s ‘laboratory quality stepwise implementation’ tool51 or the us centers for disease control and prevention and the world health organization – regional office for africa’s ‘stepwise laboratory quality improvement process towards accreditation’ framework,29,44,45,48,54 facilitates a laboratory’s success towards accreditation. for laboratory personnel, the unassisted procedure of creating an implementation plan is often problematic and could lead to demotivation and abandonment of the quality improvement project. the aforementioned tools provide a well-structured roadmap for qms implementation, thus mitigating the lack of personnel experience in planning. the use of the gradual step-by-step approach of qms implementation is also one way of dealing with the problem of financial and human resource shortage.29,44,45,48,51,54 effective project management as the third underlying factor in the model of success for laboratory quality management system implementation effective project management integrates the ksfs that are directly related to within-laboratory processes of qms implementation, sustenance, and improvement, including personnel management, personnel motivation, and mentorship. the implementation of laboratory qms, as well as any other quality systems, requires effective human resource management and leadership, which requires laboratory leaders to have the appropriate knowledge, skills, and abilities.26,28,29,30,34,37,51,52 the establishment of a managerial infrastructure and delineation of management responsibilities increase the efficiency of personnel management and contribute to the success of change implementation in medical laboratories.28,29,30,34,37 forty-one percent of the analysed studies revealed personnel motivation as key to successful qms implementation.26,28,30,31,37,41,42,44,45,47,48,49,53 our results correspond to the findings of other studies that have recognised the psychological aspect of organisational changes and individual change acceptance as key components of success in healthcare innovation.57,58 motivating and inspiring the personnel to act towards achieving a common goal is the laboratory managers’ responsibility, and this requires strong leadership skills. the problem is that most of the laboratory leaders have not received specific training in this area.59 at the same time, our study shows that it is important for laboratory managers to develop their leadership skills32,52 and demonstrate their commitment to quality by establishing a shared vision and encouraging employees to do their best to improve the quality of laboratory services.24,26,30 the lack of leadership skills among laboratory managers requires appropriate measures to be taken to solve this problem. one of the possible mechanisms to overcome this shortcoming can be mentorship. in 27 of the 32 studies included in this review, mentorship was identified as one of the ksfs for executing laboratory service quality improvement projects and receiving accreditation.25,26,27,28,30,31,32,33,34,35,36,37,39,40,42,43,44,45,46,47,48,49,50,51,52,53,54 the mentor’s duty is not only to ensure the qms implementation by planning and monitoring quality improvement activities27,32,40,50 and increasing the staff awareness of qms implementation mechanisms,27,35,36,40,45,46,50,51,54 but also to influence the attitude of laboratory managers to advocate and support all quality improvement efforts.28,32,33,35,46,51,52,54 thus, the implementers may consider mentorship as one of the additional tools of laboratory qms implementation, especially when dealing with personnel unpreparedness for change and weak leadership. institutional commitment as the central factor in the model of success for laboratory quality management system implementation fifty-three percent of the studies included in this review convincingly show that awareness of the essential role of laboratory medicine in a functioning healthcare system at the policy and governmental levels and the appropriate facilitation of the accreditation process of the medical laboratory are crucial factors in improving the quality of laboratory services.24,25,26,27,29,31,33,36,38,39,40,41,42,48,51,53,54 our study shows that commitment to laboratory quality improvement in lmics must come from the top down. if the healthcare regulators are not truly dedicated to improving laboratory service quality, any efforts by individual laboratories to achieve iso 15189 accreditation will fail. nevertheless, some accreditation requirements could be achieved by the laboratories on their own. for example, all procedures required by the standard can be documented, internal audit processes can be implemented, quality indicators can be established, and the inventory control system for reagents and consumables can be introduced. all of this will undoubtedly lead to some improvements in laboratory quality. however, for full compliance with the standard’s requirements, laboratories are dependent on the government. the medical laboratories in lmics are faced with common problems such as shortage of robust supply chains for reagents and consumables,26,31 deficit of equipment maintenance providers,29,31,40,47,48 lack of external quality assurance,24,40,48 insufficient workforce capacity,25,29,31,32,37,40,41,42,49,55 limited number of trained laboratory personnel,25,26,29,30,54 etc. these difficulties make it nearly impossible to meet the standard’s requirements. it is important to realise that resolving these issues is not under the control or purview of any single laboratory management team and requires governmental intervention. if these problems are far from being resolved, expecting a laboratory to achieve an internationally recognised accreditation will often be an unreasonable initial goal. thus, an understanding of the policy environment would allow the implementers to have realistic expectations about the outcomes of qms implementation. this understanding is also important to guide the choice of an implementation strategy, the development of effective timetables, and the decisions about the involvement of international or local partners. discussion this study shows that tangible laboratory quality improvement is achievable when the implementers can ensure а high level of staff readiness for change, resource availability, and effective project management. these three underlying factors in the model of success for laboratory qms implementation are attainable even in resource-limited settings. however, for full compliance with the standard’s requirements, certain conditions must be guaranteed at the policy and governmental levels. a top-down approach, which implies an initial dedication of governmental institutions, is critical for improving laboratory service quality and achieving iso 15189 accreditation in lmics. thus, institutional commitment is the central factor stabilising the model of success for laboratory qms implementation and is integral to ensuring the quality of laboratory services. this study had several limitations, the first of which is the small number of geographical regions included. most of the studies included were conducted in african countries. no studies from eastern europe or central asia could be identified. another limitation was that most of the studies included were on laboratories that had participated in the strengthening laboratory management toward accreditation programme, thus making it difficult to assess the success factors for improving laboratory service quality independently of the strengthening laboratory management toward accreditation programme. another limitation was the varying level of methodological quality of the included studies. studies that scored ‘poor’ or ‘very poor’ on any of the nine assessed criteria were not excluded from this review. due to the large heterogeneity of the design and research outcome of the included studies, we could not conduct a meta-analysis or comparisons across studies. despite these limitations, this study has demonstrated that laboratories planning to implement a qms can benefit from understanding the ksfs identified in this study as this would help to identify the main steps they need to take in implementing the necessary changes and set realistic expectations about the outcomes of qms implementation. acknowledgements the authors would like to thank dana bortnik for the graphic design of figure 2. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions l.n. conceived the presented idea. i.t. designed the study and led the data collection and writing of the results and the manuscript. i.t. and o.k. participated in data collection, analysis and interpretation. i.t. wrote the manuscript with input from all authors. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability data sharing is not applicable to this article, as no new data were created or analysed in this study. disclaimer the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the relevant institutions. references plebani m, sciacovelli l. iso 15189 accreditation: navigation between quality management and patient 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care. 2014 jun;26(3):321–329. https://doi.org/10.1093/intqhc/mzu047 world health organization. laboratory leadership competency framework. geneva: world health organization; 2019. abstract introduction ethical considerations case presentation management and outcomes discussion acknowledgements references about the author(s) katherine e. hodkinson department of molecular medicine and haematology, university of the witwatersrand, johannesburg, south africa national health laboratory services, johannesburg, south africa yvonne perner national health laboratory services, johannesburg, south africa department of anatomical pathology, university of the witwatersrand, johannesburg, south africa deborah k. glencross national health laboratory services, johannesburg, south africa department of haematology and molecular medicine, university of the witwatersrand, johannesburg, south africa tracey wiggill national health laboratory services, johannesburg, south africa department of haematology and molecular medicine, university of the witwatersrand, johannesburg, south africa adam botha national health laboratory services, johannesburg, south africa department of anatomical pathology, university of the witwatersrand, johannesburg, south africa janet poole department of paediatric oncology, university of the witwatersrand, johannesburg, south africa department of paediatric oncology, charlotte maxeke johannesburg academic hospital, johannesburg, south africa citation hodkinson ke, perner y, glencross dk, wiggill t, botha a, poole j. extranodal presentation of a lymphoma with precursor b-cell phenotype and translocation t(8;14) in south africa. afr j lab med. 2022;11(1), a1355. https://doi.org/10.4102/ajlm.v11i1.1355 case study extranodal presentation of a lymphoma with precursor b-cell phenotype and translocation t(8;14) in south africa katherine e. hodkinson, yvonne perner, deborah k. glencross, tracey wiggill, adam botha, janet poole received: 12 aug. 2020; accepted: 14 sept. 2021; published: 31 jan. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract introduction: a rare entity of a b-cell malignancy with precursor b-cell phenotype and concomitant translocation t(8;14) or variant myc translocation exists. these cases show clinical, pathological and molecular overlap between precursor b-lymphoblastic leukaemia or lymphoma and burkitt leukaemia or lymphoma (bll). case presentation: we report a case from february 2019 at the charlotte maxeke johannesburg academic hospital, south africa, of a 9-month-old infant with a predominantly extracranial soft tissue mass showing extradural extension. there was no involvement of the peripheral blood or bone marrow. fine needle aspiration and tru-cut biopsy of the soft tissue scalp mass showed the tumour to be of precursor b-cell phenotype. contrastingly, an immunophenotypic assessment revealed a high s-phase fraction and raised concern for bll. this prompted testing for the translocation t(8;14) by fluorescence in-situ hybridisation analysis, which confirmed this aberration. management and outcome: based on the published experience of other centres, the patient was initiated on a bll protocol. despite an excellent clinical response, the patient succumbed to neutropenic sepsis six months after diagnosis. conclusion: leukaemia or lymphoma with translocation t(8;14) or variant myc translocation and precursor b-cell phenotype is a rare entity with a varied clinical presentation. this poses a challenge for diagnosis and classification and a clinical dilemma for the choice of treatment. keywords: burkitt leukaemia/lymphoma; b-lymphoblastic leukaemia/lymphoma; translocation t(8;14); variant myc translocations; s-phase fraction; terminal deoxynucleotidyl transferase (tdt); extranodal. introduction the presence of the translocation t(8;14) or variant myc translocation in leukaemia with a precursor b-cell phenotype has been described in approximately 2% of paediatric cases.1,2 rarer still is a pure lymphomatous version (extranodal or nodal), which, to the best of our knowledge, has only been reported twice in the literature, one of which was in a paediatric patient.3,4 this entity has overlapping clinical and pathological features with burkitt leukaemia or lymphoma (bll) and precursor b-lymphoblastic leukaemia or lymphoma (b-all). it has been described across a spectrum of ages ranging from 32 months to 64 years, in both male and female patients. peripheral blood and bone marrow involvement have been reported in the majority of cases, with isolated extranodal disease reported in only a few.1,3,4,5 the immunophenotype is that of a precursor b-cell, with the expression of cd19, cd10 and terminal deoxynucleotidyl transferase (tdt), varied expression of cd34, and no expression of both surface light chain and cd20. molecular studies have confirmed the involvement of the myc gene in a translocation t(8;14) or variant translocation, which is the molecular hallmark of bll.2 this genetic aberration has largely directed the choice of therapy towards a mature b-cell or burkitt-like protocol in almost all reported cases. it is uncertain whether this represents the best therapeutic approach as there is very limited data on both the duration of remission in these patients and the exact underlying molecular behaviour of this tumour. this case study highlights this rare entity, as well as its associated diagnostic and therapeutic challenges. ethical considerations parental informed consent was provided for this case report. the ethical clearance was obtained from the university of the witwatersrand human research ethics committee (medical), johannesburg, south africa, under the approval number m190356. patient results were de-identified and stored in a secure database to ensure patient confidentiality. case presentation a 9-month-old male patient from angola presented to the charlotte maxeke johannesburg academic hospital, south africa, in february 2019, with a 3–4-week history of progressive bilateral proptosis and scalp masses. the computed tomography scan identified bilateral occipital and temporal scalp masses with extracranial extension into the orbital cavities and sinuses, and extradural extension into the anterior cranial fossa. there was no hepatosplenomegaly or lymphadenopathy. a fine needle aspiration sample of the soft tissue scalp mass was submitted for flow cytometry, and a tru-cut biopsy (manufacturer unknown) for histological assessment at the national health laboratory service. as part of the staging work up, bone marrow aspiration and a trephine biopsy were performed to exclude infiltration of the marrow. laboratory methods flow cytometry was performed on the fine needle aspiration sample from the scalp mass on a facs calibur instrument (bd biosciences, san jose, california, united states), using the paint-a-gate (becton dickinson, bd biosciences, san jose, california, united states) and modfit (verity software house, topsham, maine, united states) software. the following antibodies were used: cd19 fitc (becton dickinson, bd biosciences, san jose, california, united states), cd10 pe (beckman coulter inc., brea, california, united states), cd45 percp (becton dickinson, bd biosciences, san jose, california, united states), cd34 apc (becton dickinson, bd biosciences, san jose, california, united states), cd117 pe (beckman coulter inc., brea, california, united states), hla-dr fitc (becton dickinson, bd biosciences, san jose, california, united states), cd13 pe (beckman coulter inc., brea, california, united states), kappa fitc (dako, glostrup, denmark), lambda pe (dako, glostrup, denmark) and cytoplasmic tdt fitc (dako, glostrup, denmark). immunohistochemical work up was performed on the tru-cut biopsy from the scalp mass using the following stains: cd20 (dako, glostrup, denmark), pax5 (dako, glostrup, denmark), cd10 (leica biosystems, newcastle upon tyne, united kingdom), tdt (cell marque, rocklin, california, united states), cd34 (dako, glostrup, denmark), eber ish (roche, mannheim, germany), bcl2 (dako, glostrup, denmark), and ki67 (dako, glostrup, denmark). fluorescence in-situ hybridisation for the detection of translocation t(8;14) was performed using a vysis lsi myc/igh/cep8 tri-colour dual fusion probe (abbott, chicago, illinois, united states), with orange, green and aqua signals reflecting the myc, igh and centromere 8 regions. laboratory results histological examination of the tru-cut biopsy of the scalp mass revealed intermediate-sized tumour cells with irregular nuclear contours, a high nuclear-cytoplasmic ratio, dispersed nuclear chromatin, and one to four inconspicuous nucleoli present within a prominent background of tingible body macrophages (figure 1). the tumour immunophenotype based on flow cytometry and immunohistochemistry was that of a precursor b-cell, with the expression of tdt, absence of surface light chains and a high ki67 index (table 1, table 2 and figure 2). contrastingly, the high s-phase fraction detected was in a range that is typically seen in burkitt leukaemia.6 given the latter, fluorescence in-situ hybridisation analysis was requested and found to be positive for the translocation t(8;14)(q24;q32) (figure 3). figure 1: tru-cut biopsy of scalp mass of a 9-month-old male patient who presented to the charlotte maxeke johannesburg academic hospital in february 2019. image shows diffuse infiltration by intermediate-sized tumour cells with dispersed nuclear chromatin, irregular nuclear contours and one to four inconspicuous nucleoli. numerous tingible body macrophages impart a starry sky pattern. 7 mitotic figures per 40 hpf. haematoxylin & eosin (h&e) stain at ×40 magnification. figure 2: terminal deoxynucleotidyl transferase (tdt) immunohistochemical stain of tru-cut biopsy of scalp mass from a 9-month-old male patient who presented to the charlotte maxeke johannesburg academic hospital in february 2019. image shows ~40% positivity in the tumour. ×40 magnification. figure 3: fluorescence in-situ hybridisation analysis of fine needle aspirate from a 9-month-old male patient who presented to the charlotte maxeke johannesburg academic hospital in february 2019. image shows translocation t(8;14)(q24;q32) using the vysis lsi myc/igh/cep8 tri-colour dual fusion probe, as well as a 2f1o1g2a pattern: 2 fusion, 1 orange, 1 green, 2 aqua signals. the orange, green and aqua signals represent the myc, igh and centromere 8 regions. table 1: laboratory results at presentation of a 9-month-old male patient who presented to the charlotte maxeke johannesburg academic hospital in february 2019. table 2: laboratory results at presentation of a 9-month-old male patient who presented to the charlotte maxeke johannesburg academic hospital in february 2019. management and outcomes the patient was initiated on a bll regimen as per the fab lmb 95 protocol with excellent clinical response.7 there was complete resolution of the proptosis and the scalp masses were no longer clinically evident. sadly, the patient succumbed to neutropenic sepsis six months after diagnosis. discussion the co-existence of the translocation t(8;14) or variant myc translocation with a precursor b-cell phenotype in an extranodal malignancy is extremely uncommon and has been reported in only one paediatric and one adult patient in the literature.3,4 these individuals were both female and had no laboratory evidence of either peripheral blood or bone marrow involvement at presentation. the youngest, a 5-year-old, presented with abdominal, orbital and mandibular masses.4 the second, a 64-year-old, had multiple extranodal lesions and a chronic disease course.3 similarly, the clinical presentation of our patient was with isolated extranodal disease. this pattern of involvement is often seen in burkitt lymphoma and thus formed part of the differential diagnosis in our patient. laboratory work up demonstrated the co-expression of cd19 and cd10 by the tumour, with no expression of cd34 or light chains. because of this, the expression of tdt was assessed. terminal deoxynucleotidyl transferase is a dna polymerase that functions to provide junctional diversity in both b-cell and t-cell receptor genes at the precursor cell stage.8 it is a marker of immaturity and its expression in mature neoplasms such as bll would be aberrant. terminal deoxynucleotidyl transferase was found to be positive in ~30% – 40% of the tumour on both the fine needle aspiration and tru-cut biopsy specimens. while these results pointed towards a tumour at a precursor cell stage of development, the s-phase fraction analysis found the tumour proliferative activity to be very high. this measurement is performed by flow cytometry and uses dna content to determine the proportion of cells in each phase of the cell cycle. studies performed at our centre have shown the s-phase fraction of b-all to be characteristically around 10, whereas a fraction of more than 30, as seen in our patient, would be more typical of bll.6 comparisons could not be made with the other cases in the literature as s-phase fraction analysis was not reported. the ki67 index detects a protein associated with cellular proliferation, which is present in all active stages of the cell cycle. given that the ki67 index and s-phase analysis measure different aspects of proliferation, these parameters are not always comparable. notably, the ki67 index is unhelpful in distinguishing b-all and bll as values of over 95% can be anticipated in both.6 despite the conflicting laboratory findings in our patient, the high s-phase fraction in the context of extranodal disease prompted the testing for, and confirmation of, the translocation t(8;14). b-cell lymphoma 2 (bcl2) expression is typically seen in acute lymphoblastic leukaemia and the possibility of a double-hit lymphoma was excluded by the presence of tdt.2 s-phase fraction analysis may therefore serve as an early indicator of this disease entity, albeit further investigation of its clinical utility is required. in summary, a combination of diagnostic modalities with evaluation by a multidisciplinary team is required to confirm this rare entity. we propose a laboratory approach to the diagnosis of lymphomas with features of b-all and bll (figure 4). figure 4: laboratory approach to the diagnosis of lymphoma with features of precursor b-lymphoblastic leukaemia or lymphoma and burkitt leukaemia or lymphoma. the therapeutic management of bll differs significantly from b-all. the former requires high-intensity pulsed chemotherapy that is tailored to the high proliferative rate of the tumour. furthermore, the duration of therapy is shorter as compared to the extended maintenance used in b-all.7 our patient was instituted on a bll treatment protocol, in line with the therapeutic approach described in the literature.1,4,5 there are however limited cases and minimal long-term follow-up data from which meaningful conclusions can be drawn. on a molecular level, there is a lack of consensus as to whether this entity represents a precursor b-cell at an intermediate stage of maturation and with aberrant myc expression, or a mature b-cell with a less differentiated immunophenotype. furthermore, questions have been raised as to whether or not the presence of the translocation t(8;14) translates into an actual proliferation advantage. in an attempt to address these questions, one study analysed the molecular characteristics of neoplasms positive for ig-myc rearrangement and with precursor b-cell phenotype in 12 patients. aberrant variable diversity joining recombination was shown in five patients and activating nand k-ras mutations were detected in seven; all neoplasms had a dna methylation profile that clustered with that of precursor b-cells. in light of these findings, the authors of the study posed the question as to whether or not cases with myc rearrangement better fit a b-all profile.9 what is apparent from the literature is that further studies are required into the molecular behaviour of this entity to determine the prognostic implications and best therapy. conclusion leukaemia or lymphoma with translocation t(8;14) or variant myc translocation and precursor b-cell phenotype is a rare entity with varied clinical presentation. awareness of this entity and a high index of suspicion are required by both clinicians (haematologists, oncologists) and pathologists to prevent a misdiagnosis. further studies into the molecular and clinical behaviour of this tumour are required to optimise the therapeutic approach. acknowledgements thanking both ashleigh forsman and tshilidzi dzivhani from the somatic cell genetics unit (department of molecular medicine and haematology, national health laboratory service, johannesburg) for the analysis of the fluorescence in-situ hybridisation. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions k.e.h. drafted the manuscript. all authors were involved in the conceptualisation, structuring and editing of the final manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the author. references navid f, mosijczuk ad, head dr, et al. acute lymphoblastic leukemia with the (8;14)(q24;q32) translocation and fab l3 morphology associated with a b-precursor immunophenotype: the pediatric oncology group experience. leukemia. 1999;13(1):135–141. https://doi.org/10.1038/sj.leu.2401244 swerdlow sh, campo e, pileri sa, et al. the 2016 revision of the world health organization classification of lymphoid neoplasms. blood. 2016;127(20):2375–2390. https://doi.org/10.1182/blood-2016-01-643569 shiratori s, kondo t, fujisawa s, et al. c-myc rearrangement in b-cell lymphoblastic lymphoma with the involvement of multiple extranodal lesions. leuk lymphoma. 2011;52(4):716–718. https://doi.org/10.3109/10428194.2010.551158 meznarich j, miles r, paxton cn, afify z. pediatric b-cell lymphoma with lymphoblastic morphology, tdt expression, myc rearrangement, and features overlapping with burkitt lymphoma. pediatr blood cancer. 2016;63(5):938–940. https://doi.org/10.1002/pbc.25907 sakaguchi k, imamura t, ishimaru s, et al. nationwide study of pediatric b-cell precursor acute lymphoblastic leukemia with chromosome 8q24/myc rearrangement in japan. pediatric blood cancer. 2020;67(7):e28341. https://doi.org/10.1002/pbc.28341 glencross dk. flow cytometry in diagnostic haematopathology. johannesburg: university of the witwatersrand; 1992. patte c, auperin a, gerrard m, et al. results of the randomized international fab/lmb96 trial for intermediate risk b-cell non-hodgkin lymphoma in children and adolescents: it is possible to reduce treatment for the early responding patients. blood. 2007;109(7):2773–2780. https://doi.org/10.1182/blood-2006-07-036673 mccaffrey r, harrison ta, parkman r, baltimore d. terminal deoxynucleotidyl transferase activity in human leukemic cells and in normal human thymocytes. n engl j med. 1975;292(15):775–780. https://doi.org/10.1056/nejm197504102921504 wagener r, lópez c, kleinheinz k, et al. ig-myc (+) neoplasms with precursor b-cell phenotype are molecularly distinct from burkitt lymphomas. blood. 2018;132(21):2280–2285. https://doi.org/10.1182/blood-2018-03-842088 abstract introduction opportunities for system strengthening along the tuberculosis diagnosis and treatment cascade conclusion acknowledgements references about the author(s) ishani pathmanathan division of global hiv and tb, us centers for disease control & prevention, atlanta, georgia, united states epidemic intelligence service, us centers for disease control & prevention, atlanta, georgia, united states anand date division of global hiv and tb, us centers for disease control & prevention, atlanta, georgia, united states william l. coggin division of global hiv and tb, us centers for disease control & prevention, atlanta, georgia, united states john nkengasong division of global hiv and tb, us centers for disease control & prevention, atlanta, georgia, united states amy s. piatek global health bureau, united states agency for international development, washington dc, united states heather alexander division of global hiv and tb, us centers for disease control & prevention, atlanta, georgia, united states citation pathmanathan i, date a, coggin wl, et al. rolling out xpert mtb/rif® for tuberculosis detection in hiv-positive populations: an opportunity for systems strengthening. afr j lab med. 2017;6(2), a460. https://doi.org/10.4102/ajlm.v6i2.460 review article rolling out xpert mtb/rif® for tuberculosis detection in hiv-positive populations: an opportunity for systems strengthening ishani pathmanathan, anand date, william l. coggin, john nkengasong, amy s. piatek, heather alexander received: 06 apr. 2016; accepted: 03 aug. 2016; published: 31 mar. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: to eliminate preventable deaths, disease and suffering due to tuberculosis, improved diagnostic capacity is critical. the cepheid xpert mtb/rif® assay is recommended by the world health organization as the initial diagnostic test for people with suspected hiv-associated tuberculosis. however, despite high expectations, its scale-up in real-world settings has faced challenges, often due to the systems that support it. opportunities for system strengthening: in this commentary, we discuss needs and opportunities for systems strengthening to support widespread scale-up of xpert mtb/rif as they relate to each step within the tuberculosis diagnostic cascade, from finding presumptive patients, to collecting, transporting and testing sputum specimens, to reporting and receiving results, to initiating and monitoring treatment and, ultimately, to ensuring successful and timely treatment and cure. investments in evidence-based interventions at each step along the cascade and within the system as a whole will augment not only the utility of xpert mtb/rif, but also the successful implementation of future diagnostic tests. conclusion: xpert mtb/rif will only improve patient outcomes if optimally implemented within the context of strong tuberculosis programmes and systems. roll-out of this technology to people living with hiv and others in resource-limited settings offers the opportunity to leverage current tuberculosis and hiv laboratory, diagnostic and programmatic investments, while also addressing challenges and strengthening coordination between laboratory systems, laboratory-programme interfaces, and tuberculosis-hiv programme interfaces. if successful, the benefits of this tool could extend beyond progress toward global end tb strategy goals, to improve system-wide capacity for global disease detection and control. introduction the 2015 united nations sustainable development goals include an ambitious plan to end the tuberculosis and hiv epidemics by 2030.1 yet presently, fewer than two thirds of estimated people with tuberculosis are notified, and tuberculosis remains the greatest cause of morbidity and mortality among people living with hiv.2 to achieve a world free of tuberculosis deaths, disease and suffering by 2035, improved tuberculosis diagnostic capacity is critical.3 sputum smear microscopy, although widely used, has unacceptably poor sensitivity for detecting mycobacterium tuberculosis in people living with hiv.4,5 bacterial culture – the gold standard for tuberculosis diagnosis – is more sensitive, but is costly, technically challenging, and reliant on a sophisticated centralised laboratory infrastructure. moreover, it takes weeks to obtain results, which increases delays in treatment initiation and the period of potential disease transmission or loss to follow-up.6,7 efforts have therefore increasingly focused on developing rapid, near point-of-care (poc) tuberculosis diagnostic tools that can be easily utilised in resource-limited settings.7,8,9 in 2010, the world health organization (who) acknowledged the cepheid xpert mtb/rif® assay (sunnyvale, california, united states) as a major milestone in bringing rapid, simple, bacteriologically-confirmed diagnosis of tuberculosis disease and rifampicin resistance potentially closer to the patient poc, by strongly recommending it as the initial diagnostic test for people with suspected multi-drug-resistant (mdr) or hiv-associated tuberculosis.10,11,12,13 a 2014 cochrane review later determined it to be cost-effective for this indication, and verified that it significantly increases tuberculosis case detection compared with smear microscopy in people living with hiv. pooled sensitivity was 79% and pooled specificity was 98%, and sensitivity was higher among smear-positive (97%) than among smear-negative but culture-positive people living with hiv (61%).14 in 2010, the united states (us) president’s emergency plan for aids relief, the us agency for international development and the us centers for disease control and prevention issued a joint commitment to support the rapid and appropriate scale-up of this technology. in 2012, an agreement with cepheid was negotiated by the us president’s emergency plan for aids relief, the us agency for international development, unitaid and the bill and melinda gates foundation to reduce the test price by 40% in 145 eligible countries. by december 2014, the public sector in these countries had procured 3763 xpert® instruments (17 883 modules) and more than 10 million mtb/rif cartridges.4,9,15,16,17 despite initially high expectations, however, rapid scale-up of xpert mtb/rif has uncovered limitations, many due not to the test itself, but to the systems that support it. although initial modelling predicted that accurate same-day diagnosis by xpert mtb/rif could reduce tuberculosis mortality by 20%–35% by facilitating earlier treatment initiation,18,19 subsequent studies have failed to show a mortality benefit.9,20,21,22,23,24 in two multi-center, randomised-controlled trials from sub-saharan africa, while xpert mtb/rif significantly increased the proportion of tuberculosis patients starting treatment who had laboratory-confirmed diagnoses, the absolute number of patients initiating treatment remained unchanged. this was likely due to empiric treatment by clinicians with insufficient trust in the negative predictive value of available tuberculosis diagnostic algorithms20,23,24,25,26,27,28,29 (although notably, when xpert mtb/rif was located at the poc instead of a centralised laboratory, the proportion of bacteriologically-confirmed disease was higher, empiric treatment less frequent, and time to treatment shorter).29,30 while some studies have found that xpert mtb/rif availability reduces time to tuberculosis treatment initiation,21,23,31,32,33 others highlight persistent delays due to backlogs in machine module availability and inefficiencies in result processing and transfer.34,35,36 in one notable success story, decentralised xpert mtb/rif in a multi-country study reduced median time from sputum collection to tuberculosis treatment from 56 to five days; however, this was attributed to efficient specimen transport and result reporting systems.37 finally, although predicted to be cost-effective,13,19,38,39,40,41,42 routinely using xpert mtb/rif requires ongoing investments in trained staff, supplies and infrastructure.41,42 results from early programmatic implementation in nine tb reach (http://www.stoptb.org/global/awards/tbreach/) countries revealed increased tuberculosis case detection, but also multiple challenges, including a 10.6% test failure rate (partly due to difficulties maintaining a continuous power supply), heterogeneous result reporting, and difficulties with supply chain management and sputum transport.42 these experiences highlight that use of xpert mtb/rif technology itself is merely one component within a cascade of activities that must be successful to ensure that all tuberculosis patients are diagnosed and achieve successful and timely treatment and cure (figure 1). each process is critical in the programmatic management of tuberculosis (and hiv), and weaknesses in any may minimise the realised utility of any new diagnostic. in addition, they must all be supported by adequate funding, coordination and stakeholders engagement, as exemplified by the varied success of xpert mtb/rif implementation thus far. while this technology and newer assays certainly present opportunities for rapid, accurate diagnosis closer to the poc, to maximise their impact in programmatic settings it is crucial that we concurrently optimise other systemand programme-related factors necessary for tuberculosis diagnosis and treatment. scale-up of xpert mtb/rif for diagnosis of tuberculosis in people living with hiv presents a unique opportunity to leverage current tuberculosis and hiv laboratory, diagnostic and programmatic investments, and to coordinate with multiple stakeholders to strengthen laboratory systems, laboratory-programme interfaces, and tuberculosis-hiv programme interfaces overall. investments in evidence-based interventions at each step along the cascade and within the system as a whole will augment not only the utility of xpert mtb/rif, but also the successful implementation of future diagnostic tests (figure 2). figure 1: key steps in the cascade from tuberculosis diagnosis to successful treatment. figure 2: summary of challenges and opportunities along the tuberculosis care cascade. opportunities for system strengthening along the tuberculosis diagnosis and treatment cascade finding presumptive tuberculosis patients the who recommends xpert mtb/rif as the initial diagnostic test in individuals with suspected hiv-associated or mdr tuberculosis, which eliminates the additional clinic visit needed to perform sputum microscopy followed by xpert mtb/rif if negative.11,43 who guidelines also recommend that people living with hiv be evaluated at every clinical encounter for cough, fever, weight loss or night sweats, with positive symptom screens prompting further diagnostic evaluation; this screening algorithm has a 79% overall sensitivity (90% in clinical settings) and 50% specificity.44,45 subsequent xpert mtb/rif diagnostic testing further increases case detection sensitivity and specificity; however, to test the maximum number of people living with hiv for presumptive tuberculosis, correctly-performed symptom screening is often required first. universal uptake of the recommended screening algorithm will require incorporation into national guidelines and clinician training in most settings, and provides an opportunity for concurrent sensitisation to xpert mtb/rif. importantly, however, reliance on symptom screening before performing a tuberculosis diagnostic test can miss asymptomatic patients. the potential role for initial tuberculosis screening of people living with hiv using xpert mtb/rif (regardless of symptoms) may become cost-effective in high tuberculosis-burden settings, especially with the anticipated roll-out of xpert mtb/rif ultra, which is much more sensitive for smear-negative tuberculosis (94% sensitivity reported preliminarily among smear-negative, culture-positive patients).46 this highly-sensitive technology may also empower clinicians to reduce widespread empiric tuberculosis treatment among people living with hiv in the future. tuberculosis case detection among people living with hiv can be maximised by strategically placing genexpert® machines in facilities and areas with the highest tuberculosis and hiv prevalence. however, even under the best programmatic circumstances, clinical screening algorithms alone may be insufficient to identify tuberculosis among those who do not seek care, are contacts of known tuberculosis patients or are from remote or marginalised populations. randomised interventions, such as tuberculosis contact tracing, mobile vans, household tuberculosis and hiv counseling, as well as screening reduced tuberculosis prevalence in communities in zambia, south africa and zimbabwe,47,48 and active community-based tuberculosis case-finding endeavours, have improved tuberculosis case detection in other low-income settings.49,50,51,52,53,54 such community-based efforts have great potential to identify additional persons at risk for tuberculosis, who can subsequently benefit from diagnostic technologies such as xpert mtb/rif. collecting specimens the sensitivity of smear microscopy for one sputum specimen was 29% in a study of people living with hiv with presumptive tuberculosis in thailand and vietnam, and the incremental yield was 7% for two sputum specimens and 2% for three.55 most tuberculosis programmes routinely collect and examine at least two specimens per patient; however, the collective sensitivity of even three sputum smears remains low, and access to mycobacterial culture is limited in most low-resource settings. xpert mtb/rif offers a useful alternative, with particularly high sensitivity in sputum smear-positive people living with hiv (95% – 99%).4 in one study of patients with smear-negative tuberculosis, the sensitivity of xpert mtb/rif for one sample was only 72.5%, but this increased incrementally to 85.1% with two specimens and to 90.2% with three.56 in contrast to smear microscopy, however, cost considerations often limit xpert mtb/rif to one specimen per individual. under such resource constraints (and until the validation and widespread availability of xpert® mtb/rif ultra), the collection of a single sputum specimen must at least be optimised. although data regarding the impact of sputum quality on tuberculosis diagnosis is sparse and heterogenous,57 in theory any method that increases the quality and bacillary load of a specimen should improve diagnostic yield. mycobacterial load is the most significant predictor of xpert mtb/rif-positivity in pulmonary specimens and, in lieu of invasive specimen collection methods, patient instruction can increase microscopic detection of tuberculosis.58,59,60 additional yield can be achieved by supervised, physiotherapy-assisted collection.61 such simple and low-cost approaches are certainly warranted for all sputum specimen collection; even as more sensitive near-poc diagnostics become available, the quality of sputum samples will remain an important predictor of their diagnostic value.46 the healthcare worker training necessitated by xpert mtb/rif introduction provides an opportunity to re-evaluate and re-direct specimen collection techniques to the benefit of any sputum-dependent diagnostic assay. recent advances in transport media can further improve specimen quality. the primestore molecular transport medium® (longhorn vaccines and diagnostics, san antonio, texas, united states) inactivates organisms, preserves nucleic acids for molecular detection, and has been shown to enhance tuberculosis detection by xpert mtb/rif significantly in samples with low volume and/or bacterial load. staff familiarity with this medium may facilitate use of other decontaminating transport reagents that preserve organisms for culture.62,63 transporting specimens the ability of xpert mtb/rif to detect m. tuberculosis within two hours is a breakthrough; however, reduced turn-around time is not necessarily sufficient to adequately affect time to diagnosis.64,65 in particular, inefficient specimen collection and transport systems have been associated with increased patient attrition, time to appropriate treatment, and culture contamination rates.7,66 improving specimen referral and transport systems is a critical cross-cutting area to target in public health laboratory and tuberculosis systems strengthening efforts worldwide.67,68 although xpert mtb/rif may not be a classic poc test, several programmes are choosing to introduce genexpert machines in high-volume clinics where presumptive tuberculosis patients are screened and treated, thereby reducing specimen transport needs. however peripheral, low-volume sites may conversely place machines centrally, which increases demand for efficient specimen transport systems. in addition, the who recommends that individuals diagnosed with rifampicin-resistant tuberculosis have a specimen referred for laboratory culture and conventional drug susceptibility testing, and are monitored by sputum smear and culture.13 thus, despite the relative success of moving tuberculosis diagnostic capabilities closer to the patient (and even in the context of genexpert omni, which will likely offer true poc tuberculosis diagnosis in the foreseeable future),69,70 maintaining and strengthening specimen referral and transport systems remains critical. recently, us global health security agenda investments in specimen referral systems, such as safe, standardised sample packaging and shipping using the existing ugandan early infant diagnosis specimen transportation network, improved the speed and quality of sample transport to national reference laboratories.71 the introduction of the xpert mtb/rif diagnostic technology to hiv facilities provides a similar opportunity to evaluate specimen transport and referral systems for tuberculosis diagnosis and treatment monitoring – as well as for hiv viral load and early infant diagnosis that are also offered by cepheid as part of the multi-disease genexpert platform72 – and to potentially leverage or integrate these systems to ensure timely, safe delivery of biologic specimens to the point of testing. testing specimens while much excitement surrounding xpert mtb/rif has stemmed from its relative user-friendliness, it does have key and sometimes challenging operational requirements. these requirements include an uninterrupted power supply, stable ambient temperatures, waste disposal mechanisms, and equipment security against theft. supply chains must be reliable, and must control for backlogs in order processing and customs clearance, and limited cartridge shelf life. modules require annual calibration, machines need routine maintenance, and technical assistance must be readily accessible for trouble-shooting unanticipated challenges.6,13 costs for service and maintenance can be prohibitive, and planning and resource mobilisation must be assured. early results from xpert mtb/rif implementation in nine tb reach countries indicated a 42% module failure rate (10.6% xpert mtb/rif test failure rate), likely due to problems with irregular power, dust build up, overheating and staff quality control.42,73 finally, while each module can process a sample within two hours, backlogs can occur if samples exceed available modules, specimens are batched instead of processed as they are received, or throughput at sites remains below instrument capacity due to staffing or time constraints.10 the who and others offer recommendations for how to address these issues,6,11,12,13,42,74 but implementation of these recommendations requires advanced planning, ongoing coordination, and significant investments in infrastructure. in addition, xpert mtb/rif scale-up requires investments in labour. in one south african primary healthcare setting, xpert mtb/rif use increased tuberculosis screening and rapid detection, but poc placement increased logistical responsibilities for the clinic, requiring two to five staff members to provide same-day diagnostic evaluations for 16 patients per day.40 in the setting of minimal biosafety concerns, non-laboratory staff are being trained to run the assay in some cases.23,40,56 this approach may ease some pressures on limited laboratory human resource capacity; however, emphasis must be placed on testing quality. continuous quality improvement for any diagnostic test is critical for ensuring accuracy and reliability, detecting and reducing errors, and ensuring customer satisfaction.75 although the us centers for disease control and prevention currently provides dried-tube specimen-based proficiency panels to over 400 xpert mtb/rif testing sites, dried culture spots have been used by the national health laboratory service in south africa, and other proficiency testing panels have been developed and assessed, comprehensive external quality assessment programmes for xpert mtb/rif remain limited.76,77,78 however, tuberculosis laboratories are well-versed in external quality assessment schemes for sputum smear microscopy which, ideally, are run nationally and include blinded re-checking of slides, on-site supervisory visits, panel testing, feedback and corrective action.79 these systems are deemed so important that several key who laboratory policies are dependent on the presence of a quality-assured smear microscopy network; however, these can be costly and logistically difficult to implement and maintain.80,81 decentralised xpert mtb/rif testing shares many parallels with smear microscopy networks and thus, as programmes build quality assurance systems to support its implementation, they should capitalise on the opportunity to work within and improve existing smear microscopy external quality assessment programmes, and to create quality management systems, laboratory and testing site accreditation and certification initiatives. similarly, as genexpert instruments are placed within hiv facilities, plans for ensuring the accuracy and reliability of xpert mtb/rif testing should be aligned and/or integrated with systems for hiv-related poc test quality improvement.75 finally, external quality assessment programmes should include supplementation, when possible, with continuous performance monitoring via information systems. this has been accomplished with hiv viral load testing in south africa, and remote monitoring is a rapidly growing area of interest, supported by genexpert and other instrument-based tests.74,77,82 hiv programmes offer a well-established model and tools for a stepwise and continuous cycle to plan, implement and sustain quality assurance for poc testing, with emphasis on staff and site certification standards, supervision, and rigorous monitoring and evaluation.83 these can be emulated, and strengthened in conjunction with improvements in tuberculosis diagnostic testing continuous quality improvement. reporting and receiving results the laboratory-clinic interface is often challenged by lack of effective communication. in many settings, courier systems relied upon to transport specimens to the point of testing are also responsible for delivering test results to clinicians. however, the potential impact of rapid diagnostic tests such as xpert mtb/rif to improve clinical care cannot be realised if results are not received and interpreted rapidly.64 in a 2005 study of smear-positive tuberculosis patients who did not initiate treatment, respondents indicated delays in result receipt as a factor contributing to morbidity and mortality.7 the who recommends establishment of rapid reporting mechanisms for xpert mtb/rif results, including electronic systems, especially in the setting of incompletely decentralised xpert mtb/rif availability.13 this was highlighted in a notable cambodian study, where transmitting tuberculosis case-finding results directly to clinicians by text message the day they became available greatly shortened tuberculosis diagnostic delays.51 in the previously cited uganda global health security agenda project, enhancement of an existing online, open-source communication system to integrate data sources from laboratory, transportation and communication networks allowed real-time tracking of specimens and results.71 prevention of mother-to-child transmission of hiv programmes provide another example of how currently available technologies, such as mobile phones, web-based information systems, and text messaging, can decrease early infant diagnosis result reporting times.84 a diverse suite of potential mobile health solutions to expedite xpert mtb/rif test results for clinical and programme monitoring are emerging from device manufacturers and third-party innovators, and are increasingly maximising the use of laboratory data transmission via mobile telephony, data storage ‘in the cloud’, interoperability and encryption.85 however, unique challenges must be anticipated in terms of data ownership agreements, privacy standards, and the need for technological expertise and infrastructure. coordination with such existing and planned projects may benefit tuberculosis, hiv and other programmes through cost-sharing and expansion of rapid reporting networks. initiating and monitoring treatment assuming presumptive patient identification and specimen collection, transport, testing and result reporting all occur in a rapid and high-quality manner, clinicians receiving tuberculosis diagnostic results are then tasked with making treatment decisions. when xpert mtb/rif (or other genotypic) results are discrepant from phenotypic results, clinicians must be trained to interpret them and act based on available information.12,13 this training offers the added opportunity to refresh them on tuberculosis diagnosis and management. after interpreting xpert mtb/rif results, clinicians must then see patients through to treatment completion and cure. due to improved sensitivity of xpert mtb/rif over smear microscopy, and its capacity to detect rifampicin resistance, increased drug sensitive and mdr tuberculosis case detection among people living with hiv is anticipated with xpert mtb/rif scale-up. in one south african study, although xpert mtb/rif introduction reduced the time to mdr tuberculosis treatment initiation, higher case detection paradoxically increased the waiting list for treatment initiation and admission to a tuberculosis specialty hospital.86 appropriate planning and resource mobilisation is thus critical to accommodate this imminent increase in tuberculosis patients, especially for the management of mdr tuberculosis, which requires dedicated facilities or established community-based models of care, specialised staff and stable drug supplies.6 many lowand middle-income countries currently have limited capacity to provide quality mdr tuberculosis management, and scale-up of its treatment without quality control could fuel development of extensively drug-resistant tuberculosis. since xpert mtb/rif does not distinguish between live and dead bacteria, it cannot be used to monitor disease relapse or treatment failure.14,87 conventional tuberculosis microscopy, culture and drug susceptibility testing are still needed to assess for drug resistance and treatment failure; thus, these systems must concurrently be strengthened even as xpert mtb/rif use is scaled up.6,13 conversely, future developments in molecular and phenotypic drug susceptibility testing capabilities in response to newly-available pharmacotherapy options may benefit from improvements made to the tuberculosis diagnostic cascade to accommodate xpert mtb/rif.88 finally, improved tuberculosis case detection will increase the number of people living with hiv prioritised for antiretroviral therapy – even in the context of new who guidelines recommending antiretroviral therapy initiation regardless of immune status – and is also the gateway for other important tuberculosis/hiv interventions including tuberculosis infection control and preventive therapy.43,89,90 in many settings, tuberculosis and hiv care and treatment are provided at different locations within parallel systems. linking co-infected patients to both tuberculosis treatment and antiretroviral therapy therefore requires strengthened coordination and communication between national tuberculosis and hiv programmes, as well as consideration of integrated service delivery. supporting the cascade although each element in the cascade between tuberculosis symptom identification and successful treatment must be optimised individually to maximise the impact of xpert mtb/rif technology, there are also several overarching requirements. first, it is important that evidence-based recommendations are incorporated into clinical and laboratory guidelines and policies within national tuberculosis and hiv programmes. a recent survey of 22 high tuberculosis burden countries noted that, while 86% had a policy or algorithm to use xpert mtb/rif, most implementation was donor-supported, and not considered sustainable.74 adequate funding is clearly crucial to support initial investments in technology, as well as ongoing costs of cartridges, calibration, staff training and supervision. at current concessional prices, a four-module genexpert machine with a computer costs $17 000, cartridges $9.98 each, a calibration kit $450, and shipping an average of $1000 ($1 per cartridge). programmes also need to budget for service, maintenance and extended-warranty costs.4,12,13 all things considered, the total costs of investing in xpert mtb/rif technology for the first year are an estimated $61 000 per machine, with subsequent annual running costs of around $32 000.6 although shown to be cost-effective in many settings, cost-effectiveness does not necessarily imply affordability, especially in countries with yearly health expenditures often less than $20 per capita.86 despite increases in international donor funding for tuberculosis programmes since 2002, the yearly funding gap was predicted in 2013 to exceed $2 billion by this year.91 given the limited resources but known benefit, xpert mtb/rif implementation must be prioritised for maximum ease and impact and, most importantly, integrated whenever possible into other systems strengthening efforts currently underway.6,20,89 the second critical factor required to support the tuberculosis diagnostic cascade is increased local and international stakeholder commitment to tuberculosis programmes in general, and coordination of efforts between healthcare sectors, facilities and central governments, and healthcare settings and laboratories. xpert mtb/rif has generated significant interest and investment among ministries of health, research institutions, and donors. to ensure the success of initial phased implementation projects and national scale-up plans, it is important that tools, innovations and best practices are shared, and that all efforts within countries are coordinated and championed at the ministry level according to national priorities.92 finally, roll-out and national scale-up of projects must be planned in the context of overall tuberculosis and hiv programmes. in particular, as xpert mtb/rif use is scaled up, underlying systems must be able to accommodate additional patients expected to be diagnosed using the assay. after stakeholders are coordinated and implementation plans finalised, supplies must be procured, inventories organised, staff trained, and sites prepared for roll-out. supervision and quality assurance are needed at every step, as is robust monitoring and evaluation – currently not well established – to routinely collect and respond to data on programmatic performance, outcomes and impact, and to assess where instruments are used and where rateand quality-limiting steps within the cascade are occurring.92 like each individual part of the cascade, these efforts should capitalise on monitoring and evaluation systems already in place, or aim to strengthen those that may benefit from additional investments. conclusion in 2012, loveday et al. assessed a typical patient’s journey from diagnosis to treatment in kwa-zulu natal, south africa, to determine the effectiveness of decentralised care for mdr tuberculosis patients. although both patientand health system-related factors resulted in ultimately sub-optimal outcomes, most challenges encountered were due to health systems factors, including poor communication of laboratory results, incorrect provider implementation of clinical guidelines, and inadequate integration of tuberculosis and hiv services. this ‘typical journey’ highlights the fact that weaknesses at any step in the clinical cascade can compound deficits in others.93 conversely, improvements at any step can and should benefit the system as a whole. xpert mtb/rif is a major diagnostic breakthrough, but will only improve patient outcomes if optimally placed and implemented within the context of strong tuberculosis programmes and systems. the roll-out and rapid scale-up of this technology to people living with hiv and others in resource-limited settings offers a unique opportunity to address current challenges to maximise impact on the quality of tuberculosis programmes in general. ministries of health, funding agencies and implementing partners should capitalise upon this opportunity by investing in strong, patient-centred health systems, staff and programmes, not only to optimise the success of xpert mtb/rif (and other genexpert-supported platforms such as hiv viral load testing and early infant diagnosis), but also to allow any future technologies to be seamlessly incorporated and implemented.73,94 in particular, xpert mtb/rif implementation should be leveraged to strengthen collaborative tuberculosis hiv activities, laboratory networks, and the laboratory-clinic interface. if this is accomplished, the benefits of this diagnostic tool could extend beyond increased tuberculosis case detection and treatment, toward achieving global end tb strategy goals, improving system-wide capacity for disease detection and control, and promoting global health security. acknowledgements competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support none. authors’ contributions i.p. and h.a. were responsible for concept development, literature review, writing and revisions. a.d., w.l.c., j.n. and a.s.p. were responsible for writing and revisions. references united nations. sustainable development goals [homepage on the internet]. 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testing: a needs assessment and roadmap for action. lancet infect dis. 2013;13(5):449–458. https://doi.org/10.1016/s1473-3099(13)70025-2 date a, modi s. tb screening among people living with hiv/aids in resource-limited settings. j acquir immune defic syndr. 2015;68(suppl 3):s270–273. https://doi.org/10.1097/qai.0000000000000485 world health organization. guideline on when to start antiretroviral therapy and on pre-exposure prophylaxis for hiv [document on the internet]. c2015 [cited 2015 sep 30]. available from: http://apps.who.int/iris/bitstream/10665/186275/1/9789241509565_eng.pdf floyd k, fitzpatrick c, pantoja a, et al. domestic and donor financing for tuberculosis care and control in low-income and middle-income countries: an analysis of trends, 2002-11, and requirements to meet 2015 targets. lancet glob health. 2013;1(2):e105–115. https://doi.org/10.1016/s2214-109x(13)70032-9 alexander h. scaling up xpert mtb/rif as part of hiv care: progress, challenges, and the way forward? talk presented at the 19th core group meeting of the global tb/hiv working group. washington, dc; 11–12 february, 2014. loveday m, padayatchi n, voce a, et al. the treatment journey of a patient with multidrug-resistant tuberculosis in south africa: is it patient-centred? int j tuberc lung dis. 2013;17(10 suppl 1):56–59. https://doi.org/10.5588/ijtld.13.0101 kik sk, denkinger cm, jefferson c, et al. potential market for novel tuberculosis diagnostics: worth the investment? j infect dis. 2015;211(suppl 2):s58–66. https://doi.org/10.1093/infdis/jiu817 abstract introduction methods results discussion acknowledgements references about the author(s) thapelo mbhele molecular sciences institute, school of chemistry, faculty of science, university of the witwatersrand, johannesburg, south africa donald m. tanyanyiwa school of pathology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa department of chemical pathology, faculty of health sciences, sefako makgatho health sciences university, pretoria, south africa refilwe j. moepya molecular sciences institute, school of chemistry, faculty of science, university of the witwatersrand, johannesburg, south africa sindeep bhana school of medicine, faculty of health sciences, university of the witwatersrand, johannesburg, south africa maya m. makatini molecular sciences institute, school of chemistry, faculty of science, university of the witwatersrand, johannesburg, south africa citation mbhele t, tanyanyiwa dm, moepya rm, bhana s, makatini mm. relationship between amino acid ratios and decline in estimated glomerular filtration rate in diabetic and non-diabetic patients in south africa. afr j lab med. 2021;10(1), a1398 https://doi.org/10.4102/ajlm.v10i1.1398 original research relationship between amino acid ratios and decline in estimated glomerular filtration rate in diabetic and non-diabetic patients in south africa thapelo mbhele, donald m. tanyanyiwa, refilwe j. moepya, sindeep bhana, maya m. makatini received: 26 sept. 2020; accepted: 16 july 2021; published: 10 dec. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: diabetic kidney disease is a major complication resulting from type 1 and type 2 diabetes. currently, the microalbuminuria test is used to monitor renal function; however, it does not detect albumin until progressive loss of renal function has occurred. objective: this study analysed the relationship between changes in amino acid ratios and estimated glomerular filtration rate (egfr) decline in diabetic and non-diabetic patients. methods: urine samples were collected from participants between february 2019 to april 2019 and analysed from november 2020 to january 2021. diabetic (glycated haemoglobin > 6.4%) and non-diabetic patients (glycated haemoglobin ≤ 6.4%) from chris hani baragwanath hospital, south africa, were further categorised based on the degree of renal function predicted by the egfrs. amino acids were quantified using tandem mass spectrometry to determine the concentrations and ratios of tyrosine/phenylalanine, ornithine/arginine, arginine/citrulline and citrulline/ornithine at different stages of the chronic kidney disease. results: among diabetic patients, the tyrosine/phenylalanine ratio showed a statistically significant increase (p = 0.04) as the egfr declined from stage 1 to stage 4; the ornithine/arginine ratio showed a strong negative correlation with egfr. the citrulline/ornithine ratio differed between the diabetic and non-diabetic patients in stage 1 of chronic kidney disease. conclusion: amino acid ratios (ornithine/arginine and tyrosine/phenylalanine) are affected by the progression of diabetes and can be correlated to renal function. the citrulline/ornithine ratios differ between the studied groups in stage 1 of the disease and may be utilised to predict the onset of chronic kidney disease. keywords: diabetic nephropathy; albuminuria; amino acids; lc-ms/ms; chronic kidney disease; glomerular filtration rate. introduction diabetic nephropathy or chronic kidney disease (ckd), resulting from both type 1 and type 2 diabetes, is one of the leading causes of death in the world among non-communicable diseases.1,2 diabetic nephropathy usually develops about 10 years after the onset of diabetes.3 chronic kidney disease affects about half a million people globally, and people with ckd are at a high risk of developing stroke and cardiovascular disease. chronic kidney disease patients are most likely to die from cardiovascular disease than progress to end-stage renal failure.4 in south africa, diabetic nephropathy is diagnosed clinically using a digital community analyser vantage instrument that measures the amount of albumin and creatinine, as well as the ratio of albumin to creatinine (a/c) in urine. the diagnostic criteria based on the a/c ratio are as follows: normal range ≤ 3.5 mg/mmol (female) and ≤ 2.5 mg/mmol (male); microalbumin range 2.6 mg/mmol – 25.0 mg/mmol (female) and 3.6 mg/mmol – 35.0 mg/mmol (male); macroalbumin range 26.0 mg/mmol – 100.0 mg/mmol (female) and 36.0 mg/mmol – 100.0 mg/mmol (male).5 although the a/c ratio is a good indicator of ckd, the detected value needs to be sustained in the patient over 3 months to accurately diagnose ckd.6 it also lacks specificity and sensitivity during a progressive decline in renal function and can give false results, where patients with ckd could appear normal.7 the glomerular filtration rate (gfr) is an important marker in the assessment of kidney function. it can be calculated using the ckd epidemiology collaboration formula and the modification of diet in renal disease equation. the modification of diet in renal disease equation estimates the gfr (egfr) based on age, sex and serum creatinine (scr).7,8 renal decline in ckd is classified into five stages, which indicate the progression of the disease. these stages are based on the gfr and include stage 1 (≥ 90 ml/min per 1.73 m2), stage 2 (60 ml/min – 89 ml/min per 1.73 m2), stage 3 (30 ml/min – 59 ml/min per 1.73 m2), stage 4 (15 ml/min – 29 ml/min per 1.73 m2) and stage 5 (< 15 ml/min per 1.73 m2).9 the assessment of gfr using scr concentrations gives inconclusive results in the early stages of renal decline and lacks sensitivity because scr only becomes detectable after renal decline has progressed significantly. the ckd epidemiology collaboration formula provides a good indicator of the egfr in the early stages of ckd (> 90 ml/min per 1.73 m2); however, it also underestimates the gfr in diabetic patients.10 similarly, although the modification of diet in renal disease equation provides reliable results for patients with ckd, it may give false positives for healthy patients.8 measuring the concentration of amino acids in the body is important in the diagnosis and management of several metabolic abnormalities. amino acid concentrations can be used to determine the status of patients’ nutrition, renal function and tissue injury.11 the metabolism and excretion of amino acids such as phenylalanine and tyrosine have been reported to undergo considerable changes in patients with ckd.12 urine is the most preferred biofluid in metabolomics due to its ease of collection in large volumes and its lesser complexity compared to other body fluids such as blood.13,14 liquid chromatography tandem mass spectrometry eliminates lengthy sample preparations as amino acids can be analysed without the need for derivatisation.11 the advantages of using this technique include high sensitivity, specificity, and throughput.15 this technique is based on the structural characteristics of analytes obtained by fragmentation through collision, and it is a powerful technique for quantitation.16 this study aimed to assess the relationship between urinary amino acid concentration ratios and renal function decline as predicted by the egfr in diabetic and non-diabetic patients using liquid chromatography tandem mass spectrometry. methods ethical considerations ethical clearance was obtained from the human and animal rights chris hani baragwanath academic hospital medical advisory committee and the university of the witwatersrand human research ethics committee (reference number: m180798). a brief explanation of the study was communicated to the participants, who then provided written informed consent to allow the collection and analysis of their urine samples and medical records. medical practice and ethical guidelines were followed to ensure the confidentiality of the results obtained in this study. samples were coded to preserve anonymity. study design and sampling a total of 187 spot urine specimens were collected from study participants between february 2019 and april 2019 at the chris hani baragwanath academic hospital located in soweto, johannesburg, south africa. this is the biggest hospital in africa and serves a large community from urban to rural areas; it sees hundreds to thousands of patients annually. data on ethnicity, age, egfr, glycated haemoglobin (hba1c), scr, urinary creatinine, albumin, a/c and gender were obtained from participants’ medical records. samples from 36 participants were excluded from the analysis because of missing data (33 medical records without egfr data and 3 without hba1c data). all participants with complete medical data (n = 151) were of african descent, including women (n = 104) and men (n = 47) between the ages of 18 years and 90 years. urine samples were divided into two groups based on hba1c: people with diabetes (n = 129; hba1c > 6.4%) and people without diabetes (n = 22; hba1c ≤ 6.4%). these groups were further divided into sub-groups based on the stage of ckd as follows: 63 people with diabetes and 9 without diabetes were categorised as being in stage 1 (≥ 90 ml/min per 1.73 m2), 43 people with diabetes and 10 without diabetes were classified as being in stage 2 (60 ml/min – 89 ml/min per 1.73 m2), 15 people with diabetes and 3 without diabetes were classified as being in stage 3 (30 ml/min – 59 ml/min per 1.73 m2), 7 people with diabetes were classified as being in stage 4 (15 ml/min – 29 ml/min per 1.73 m2), and 1 participant was categorised as being in stage 5 (< 15 ml/min per 1.73 m2). the amino acids arginine, citrulline, isoleucine, ornithine, phenylalanine, and tyrosine were quantitatively analysed in each sample. using the modification of diet in renal disease equation, the egfr was calculated to assess the degree of kidney function. urine samples were collected into 20 ml sterile polypropylene screw-top containers and placed on wet ice in polystyrene containers before transportation to the national institute of occupational health biobank for long-term storage at −20 °c. before analysis, the samples were transported to the university of the witwatersrand school of chemistry mass spectrometry laboratory, where they were briefly stored at −20 °c for a few days before analysis. chemicals and materials all amino acids (purity ≥ 98.0%), as well as the formic acid (purity ≥ 98.0%), sigmatrix urine diluent (purity ≥ 98.0%) and hplc-grade acetonitrile (≥ 99.9%), were obtained from sigma-aldrich (johannesburg, south africa). deionised water, which was used as a solvent for dilution of analytes, was purified using a direct-q® 3 uv millipore system (molsheim, france). a stock solution of each amino acid (100 mg/l) was prepared by weighing 10 mg of the amino acid which was then dissolved to a final volume with millipore h2o in a 100 ml volumetric flask. from the stock solution, 10 calibration points (0.01 mg/l, 0.05 mg/l, 0.10 mg/l, 0.30 mg/l, 0.50 mg/l, 1.00 mg/l, 2.00 mg/l, 3.00 mg/l, 4.00 mg/l, and 5.00 mg/l) were prepared by diluting appropriate volumes of stock solution in a urine diluent–h2o (1:5 ratio) mixture. the urine diluent was used as a blank matrix to mimic human urine. quantitative analysis frozen urine samples were thawed for 30 min, then 1 ml of the urine was transferred into a 2 ml centrifuge tube and diluted with 1 ml of millipore h2o, and centrifuged at 3000 g for 10 min. afterwards, 0.2 ml of the supernatant was transferred into a 2 ml ultra-high performance liquid chromatography (uhplc) vial and 0.8 ml of millipore h2o was added to make up the final volume to 1 ml. standards and samples were analysed on a ymc-triart c18 uhplc column (1.9 µm beads, pore size 120 å, 2 mm id × 150 mm length) (analytical science technology pty ltd, cape town, south africa). all analyses were conducted in triplicate with quality control samples (lowest limit of quantitation [lloq]1 [0.1 mg/l], lloq3 [0.5 mg/l] and upper limit of quantitation [uloq] [4 mg/l]), which were analysed in the same way as the urine samples above and were used to assess the performance (linearity, accuracy, precision, and percentage recovery) of the method. the thermo scientifictm ultimatetm 3000 uhplc system coupled to a bruker compact qq-time-of-flight high-resolution mass spectrometer (bruker daltonics, bremen, germany) was used for the analysis. the samples and standards were placed in an autosampler set at 5 °c. a two-buffer system was employed, utilising formic acid as the ion-pairing agent; solvent a consisted of 0.1% formic acid in h2o (volume per volume), and solvent b comprised 0.1% formic acid in acetonitrile (volume per volume). twenty µl of the sample was injected into the c18 uhplc column and eluted using a gradient elution technique, starting from an h2o:acetonitrile ratio of 95:5% to 5:95% in 10 min; the standards and samples were injected three times. mass spectrometry parameters the electrospray ionisation source was operated in the positive ionisation mode at a temperature of 250 °c. the dry gas (n2) flow rate was set at 9.0 l/min, the nebuliser gas (n2) pressure was set to 1.8 bars, the capillary voltage was 4500 v, the end plate offset was -500 v, and the charging voltage was 2000 v. in the quadrupole, a multiple reaction monitoring method was applied with a mass range of 50 mass-to-charge ratio (m/z) to 1300 m/z to fragment arginine (175 m/z), citrulline (176 m/z), isoleucine (132 m/z), ornithine (133 m/z), phenylalanine (166 m/z), and tyrosine (182 m/z) at 26.00, 14.00, 22.00, 12.00, 15.00 and 12.00 ev collision energies. the time-of-flight detector voltage was set at 2242.9 v. data analysis all data processing was done on the bruker quantanalysis software (bruker daltonics, bremen, germany) to determine the concentration of amino acids. the dilution factor of 10 was used to determine the actual concentration of amino acids in the urine samples. the lloq was determined for each amino acid and found to be within the range of 0.52 mg/l to 0.58 mg/l for arginine, citrulline, and ornithine and 0.12 mg/l to 0.19 mg/l for isoleucine, phenylalanine, and tyrosine (table 1). statistical data analysis was carried out in microsoft excel version 2101 (microsoft corporation, redmond, washington, united states). mean and standard deviation values were obtained using descriptive statistics, significant differences in variables between diabetic and non-diabetic patients were assessed using analysis of variance, and linear correlations between biochemical and analytical data were determined using pearson’s correlation coefficient (r). a p-value 0.05 or less was considered significant. table 1: analytical method performance characteristics of three spiked blank matrices with amino acid standards at different concentrations, johannesburg, south africa, november 2019 to january 2021. results all amino acids were well separated on the c18 column and gave different fragment ions and retention times (arginine, 0.81 min; citrulline, 0.90 min; isoleucine, 2.10 min; ornithine, 0.74 min; phenylalanine, 4.44 min and tyrosine, 2.96 min), indicating good selectivity (figure 1). figure 1: chromatogram showing (a) the uhplc baseline separation of arginine, citrulline, isoleucine, ornithine, phenylalanine and tyrosine; and (b) the mass spectrum with retention times and collision energy required to fragment each amino acid to give the fragment ions (quant masses). data analysed between november 2019 and january 2021, johannesburg, south africa. in diabetic patients, the ornithine/arginine and tyrosine/phenylalanine ratios increased as the egfr decreased (table 2). this relationship was confirmed by strong negative correlations (r = –0.81; p = 0.19 and r = –0.96; p = 0.04) between the amino acid concentration ratios and the egfr. conversely, the arginine/citrulline ratio decreased with decreasing egfr, and this was supported by a statistically significant positive correlation (r = 0.71; p = 0.29). the concentrations of ornithine, isoleucine, tyrosine and phenylalanine decreased with decreasing renal function and this was confirmed by statistically significant correlations ([r = 0.83; p = 0.17], [r = 0.94; p = 0.06], [r = 0.94; p = 0.06] and [r = 1.00; p = 0.004]). the amino acid concentrations demonstrated statistically significant positive correlations to hba1c coefficient (r) and correlated negatively with urinary creatinine and age. there was no significant correlation between the amino acid concentrations and a/c ratio, albumin, or scr. haemoglobin a1c correlated strongly with concentrations of citrulline (r = 0.76), isoleucine (r = –0.83) and ornithine (r = –0.86), while age correlated negatively with the concentrations of isoleucine (r = –0.98), ornithine (r = –0.95), phenylalanine (r = –0.83) and tyrosine (r = –0.80). table 2: correlation between the amino acid concentrations and ratios and clinical data of patients with diabetes, johannesburg, south africa, november 2019 to january 2021. the trends observed between amino acid concentrations and ratios with clinical parameters for non-diabetic patients are within the normal range for hba1c and egfr, and hence do not show disease progression (table 3). serum creatinine correlated significantly (p = 0.45) with isoleucine concentration (r = 0.76) and citrulline/ornithine ratios (r = 0.81, p = 0.40) in non-diabetic patients. the tyrosine/phenylalanine ratios (r = 0.85), as well as the concentrations of tyrosine (r = 0.76) and ornithine (r = 0.83), decreased with decreasing egfr, while the concentrations of citrulline (r = –0.94) and isoleucine (r = –0.91) increased with decreasing egfr (table 3). table 3: correlation between the amino acid concentrations and ratios and clinical data of non-diabetic patients, johannesburg, south africa, november 2019 to january 2021. the citrulline/ornithine ratios (p = 0.01), as well as the concentrations of citrulline (p < 0.001) and isoleucine (p = 0.04), were significantly higher in the diabetic group compared to the non-diabetic group in stage 1 of ckd (table 4). similarly, the ornithine/arginine ratios (p = 0.02) and citrulline concentrations (p = 0.03) were significantly higher in the diabetic group compared to the non-diabetic group in stage 3. in stage 4, the tyrosine/phenylalanine ratios were significantly (p = 0.03) higher, while phenylalanine concentrations were lower in the diabetic group compared to the non-diabetic group. table 4: differences in amino acid concentrations and clinical parameters between diabetic and non-diabetic patients with declining estimated gfr, johannesburg, south africa, november 2019 to january 2021. discussion the quantitative method developed for the analysis of amino acids in urine was validated and found to be reliable, selective, sensitive, and accurate. we could relate the alterations in urinary amino acid ratios at different stages of ckd to the egfr and some clinical characteristics associated with the disease. the analysis showed that the amino acid ratios of ornithine/arginine, tyrosine/phenylalanine and arginine/citrulline are affected by the progression of diabetes and can be correlated to renal function. there were significant changes in amino acid ratios between the diabetic and non-diabetic patients in stage 1 of ckd, and this could be utilised to predict the onset of ckd. the accuracy of the analytical method used to obtain the lloq was determined to be within the acceptable range for all the amino acids analysed. the linearity (r2) ranged from 0.983 to 0.999, the percentage recovery ranged from 90% to 130%, and the precision (%rsd) was less than 30%, except for ornithine. the method was also selective and sensitive as shown by the lowest limit of detection and lloq, and all the analytes of interest were well separated in the liquid chromatography and accurately identified by the mass spectrometer. hence, the method is reliable and can be used to quantify the amino acids of interest within the analytical range of 0.01 mg/l to 5 mg/l. in diabetics patients, the concentrations of citrulline, isoleucine, phenylalanine and ornithine correlated positively with hba1c ([r = 0.76; p = 0.24)], [r = 0.83; p = 0.17], [r = 0.54; p = 0.46], [r = 0.86; p = 0.41]) and egfr ([r = 0.33; p = 0.67], [r = 0.94, p = 0.06], [r = 1.00; p = 0.004], [r = 0.83; p = 0.17]). the concentrations of these amino acids decreased with egfr decline, meaning that the disease and renal function may alter these amino acids. in non-diabetic patients, arginine, citrulline and isoleucine correlated positively with hba1c, meaning that these amino acid concentrations fluctuate with glucose levels. even though citrulline showed a positive correlation with hba1c in both the diabetic and non-diabetic groups, its concentrations levels were significantly (p < 0.001) higher in the diabetic group. a study conducted in france in 2014 reported that citrulline concentration and citrulline/ornithine ratio increased while the tyrosine/phenylalanine ratio decreased (in plasma samples) in the late stages of ckd.17 similarly, in this study, we observed (in urine samples) an increase in citrulline concentrations and citrulline/ornithine ratios in stage 1; however, tyrosine/phenylalanine ratios increased from stage 1 to stage 4 of ckd with decreasing concentrations of phenylalanine. amino acid concentrations have been reported to decrease in diabetic groups, and this decrease is attributed to the conversion of these amino acids to glucose.18 our findings show that phenylalanine follows this trend, as its concentrations decreased from stage 1 to stage 4 of ckd. the observed trend can be explained by looking at the metabolic pathways of the studied amino acids that occur in the liver and kidneys. arginine is converted to ornithine, ornithine to citrulline and citrulline to arginine, while phenylalanine is converted to tyrosine.17 thus, the declining kidney function may affect the proper conversion of citrulline to arginine, as shown also by the significant (p = 0.05) decrease in the ratio of arginine/citrulline in stage 3 of ckd. citrulline is a non-essential amino acid that is mainly synthesised in the gut and then taken up by the kidneys, wherein it is converted to arginine.19 the increased concentration of citrulline in diabetic patients could be attributed to the inability of the kidneys to carry out this function. the kidneys convert 50% of the total body phenylalanine to tyrosine through hydroxylation by the action of the phenylalanine-4-hydroxylase enzyme.19 at stage 4 of ckd, the tyrosine/phenylalanine ratios were significantly different between the diabetic and non-diabetic groups, and the phenylalanine concentrations were lower in the diabetic group. the metabolism of phenylalanine has two outcomes – the production of tyrosine, and the complete oxidation of phenylalanine to carbon dioxide and water to drive the production of glucose.20 we observed an increase in tyrosine/phenylalanine ratios with egfr decline, which suggests an increased production of tyrosine in the kidneys as renal function decreases and could explain the low phenylalanine concentrations in stage 4 of ckd. limitations the exact cut-off values for amino acid concentrations and ratios, which indicate the onset of diabetic nephropathy or ckd, could not be determined. also, the sample size for non-diabetic patients was below 30 (n = 22), which is the acceptable size for accurate statistical evaluation, and this may have influenced the results of comparisons between the two groups of patients. a more detailed statistical evaluation would have to be conducted to indicate early ckd using amino acid ratios. conclusion the amino acid concentrations and ratios in human urine samples could provide valuable information about the onset and progression of diabetic nephropathy as there appeared to be significant correlations between the amino acid concentrations and declining egfr. amino acid ratios (ornithine/arginine and tyrosine/phenylalanine) are affected by decreased renal function, and citrulline/ornithine ratios may predict early ckd. acknowledgements the authors would like to thank the chris hani baragwanath academic hospital chemical pathology staff for the collection and transportation of samples, and the national institute of occupational health staff for long-term storage, sorting, and transportation of samples. we also thank the witwatersrand mass spectrometry team for granting access to the analytical instruments. this article is partially based on the second author’s thesis21 of the degree of doctor of philosophy in division of human genetics, department of pathology, faculty of health sciences at the university of cape town, south africa, with supervisor prof. collet dandara, received september 2018, available here: http://hdl.handle.net/11427/30132. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions d.m.t. generated the hypothesis and planned the patient recruitment and sample collection logistics. t.m. ran the project, analysed the samples, carried out the statistical analysis and wrote the manuscript. m.m.m. supervised the project and provided expert analytical interpretation. r.j.m. assisted with the analytical processes. s.b. assisted with patient recruitment. sources of support the work was supported by the national research foundation, under the thuthuka award (gun 117975). data availability data sharing is not applicable to this article. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references brenneman j, hill j, pullen s. emerging therapeutics for the treatment of diabetic nephropathy. bioorg med chem lett. 2016;26(18):4394–4402. https://doi.org/10.1016/j.bmcl.2016.07.079 kanwar ys, wada j, sun l, et al. diabetic nephropathy: mechanisms of renal disease progression. exp bio med. 2008;233(1):4–11. https://doi.org/10.3181/0705-mr-134 moosa mr, van der walt i, naicker s, meyers am. important causes of chronic kidney disease in south africa. south african med j. 2015;105(4):1–8. https://doi.org/10.7196/samj.9535 adeniyi ab, laurence ce, volmink ja, davids mr. prevalence of chronic kidney disease and association with cardiovascular risk factors among teachers in cape town, south africa. clin kidney j. 2017;10(3):1–7. https://doi.org/10.1093/ckj/sfw138 motta la, shephard mds, brink j, lawson s, rheeder p. point-of-care testing improves diabetes management in a primary care clinic in south africa. prim care diabetes. 2017;11(3):248–253. https://doi.org/10.1016/j.pcd.2016.09.008 alhajim sa, sherhan aa. albumin to creatinine ratio is fairly correlated with albumin excretion rate in chronic kidney disease. am j lab med. 2018;3(6):48–52. colhoun hm, marcovecchio ml. biomarkers of diabetic kidney disease. diabetologia. 2018;61(5):996–1011. https://doi.org/10.1007/s00125-018-4567-5 lopez-giacoman s. biomarkers in chronic kidney disease, from kidney function to kidney damage. world j nephrol. 2015;4(1):57–73. https://doi.org/10.5527/wjn.v4.i1.57 canal c, pellicer r, rocha ci, et al. tables for estimating glomerular filtration rate from plasma creatinine. nefrologia. 2008;28(3):317–324. currie g. biomarkers in diabetic nephropathy: present and future. world j diabetes. 2014;5(6):763–776. https://doi.org/10.4239/wjd.v5.i6.763 le a, ng a, kwan t, cusmano-ozog k, cowan tm. a rapid, sensitive method for quantitative analysis of underivatized amino acids by liquid chromatography-tandem mass spectrometry (lc-ms/ms). j chromatogr b anal technol biomed life sci. 2014;944:166–174. https://doi.org/10.1016/j.jchromb.2013.11.017 kopple jd. phenylalanine and tyrosine metabolism in chronic kidney failure. j nutr. 2007;137(6 suppl 1):1586s–1590s. https://doi.org/10.1093/jn/137.6.1586s kemperman rfj, horvatovich pl, hoekman b, reijmers th, muskiet faj, bischoff r. comparative urine analysis by liquid chromatography – mass spectrometry and multivariate statistics: method development, evaluation, and application to proteinuria. j proteome. 2007;6(1):194–206. https://doi.org/10.1021/pr060362r kalantari s, jafari a, moradpoor r, ghasemi e, khalkhal e. human urine proteomics: analytical techniques and clinical applications in renal diseases. int j proteomics. 2015;2015:782798. https://doi.org/10.1155/2015/782798 tang x, gu y, nie j, fan s, wang c. quantification of amino acids in rat urine by solid-phase extraction and liquid chromatography/electrospray tandem mass spectrometry: application to radiation injury rat model. j liq chromatogr relat technol. 2014;37(7):951–973. https://doi.org/10.1080/10826076.2013.765451 waterval wah, scheijen jljm, ortmans-ploemen mmjc, habets-van der poel cd, bierau j. quantitative uplc-ms/ms analysis of underivatised amino acids in body fluids is a reliable tool for the diagnosis and follow-up of patients with inborn errors of metabolism. clin chim acta. 2009;407(1–2):36–42. https://doi.org/10.1016/j.cca.2009.06.023 duranton f, lundin u, gayrard n, et al. plasma and urinary amino acid metabolomic profiling in patients with different levels of kidney function. clin j am soc nephrol. 2014;9(1):37–45. https://doi.org/10.2215/cjn.06000613 durá-travé t, gallinas-victoriano f, cortes-castel e, moya-benavent m. amino acid plasma concentrations and urinary excretion in young diabetics. in: ahmed ag, editor. diabetes and its complications. london: intechopen, 2018; p. 109–118. https://doi.org/10.5772/intechopen.72080 van de poll mgc, soeters pb, deutz nep, fearon kch, dejong chc. renal metabolism of amino acids: its role in interorgan amino acid exchange. am j clin nutr. 2004;79(1):185–197. https://doi.org/10.1093/ajcn/79.2.185 williams ra, mamotte cds, burnett jr. phenylketonuria: an inborn error of phenylalanine metabolism. clin biochem rev. 2008;29(1):31–41. tanyanyiwa dm. type 2 diabetes mellitus and dyslipidaemia: effects of genetic variation in african populations [homepage on the internet]. cape town: university of cape town; 2018 [cited 2021 feb 24]. available from: http://hdl.handle.net/11427/30132 abstract introduction methods results discussion acknowledgements references about the author(s) martine e. oloume haematology laboratory, centre pasteur, yaoundé, cameroon abas mouliom haematology laboratory, centre pasteur, yaoundé, cameroon bernard f. melingui haematology laboratory, centre pasteur, yaoundé, cameroon suzanne belinga medical analysis department, centre pasteur, yaoundé, cameroon julie s. nana haematology and immunology department, catholic university of central africa, yaoundé, cameroon mathurin tejiokem epidemiology department, centre pasteur, yaoundé, cameroon francoise n. sack haematology department and blood bank, central hospital, yaoundé, cameroon jeanne manga quality management, centre pasteur, yaoundé, cameroon annie r. epote haematology laboratory, centre pasteur, yaoundé, cameroon citation oloume me, mouliom a, melingui bf, et al. haematological values in a healthy adult population in yaoundé, cameroon. afr j lab med. 2019;8(1), a852. https://doi.org/10.4102/ajlm.v8i1.852 original research haematological values in a healthy adult population in yaoundé, cameroon martine e. oloume, abas mouliom, bernard f. melingui, suzanne belinga, julie s. nana, mathurin tejiokem, francoise n. sack, jeanne manga, annie r. epote received: 22 june 2018; accepted: 21 jan. 2019; published: 31 oct. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: haematological values derived from local populations are useful in laboratories to improve diagnoses for local patients. in cameroon, these data are not yet available. moreover, there is great variation in baseline parameters pertaining to full blood cell count among medical laboratories. objectives: this study aimed to determine values for the complete blood cell count of a healthy adult cameroonian population for use in locally derived ranges in our medical laboratories. methods: a cross-sectional study was conducted among blood donors attending three blood banks in yaoundé from november 2015 to september 2016. we expected to obtain at least 120 venous blood samples from both men and women. tests were performed for (1) hiv, (2) complete blood cell count, (3) hepatitis b virus, (4) malaria, (5) syphilis, (6) c-reactive protein and (7) hepatitis c virus. results: we enrolled 294 healthy participants (161 men, 133 women) aged 18 to 55 years. the median haemoglobin concentration was 135 g/l in men and 114 g/l in women (p < 0.001). the median reticulocyte count was 60 × 109/l in men and 40 × 109/l in women (p < 0.001). significant variation by sex was observed for the platelet count. the median white blood cell count was 4.1 × 109/l in men and 4.6 × 109/l in women (p = 0.008). conclusion: this study provides locally derived ranges for complete blood cell and reticulocyte counts for a healthy adult population in yaoundé, cameroon. these results can be used pending larger studies. keywords: complete blood cell count; reticulocyte count; locally derived ranges. introduction a complete blood cell count is the most current and simplest method for assessing medullary function and haematopoiesis. it is a routine analysis that is very useful in medical practice; this test can reveal very diverse pathologies. a proper interpretation is therefore essential to guide the diagnosis and request an additional medical analysis. until now, haematological reference values have not been established for the cameroonian population. normal values used in our laboratories are provided from the literature or instrument manuals. these values usually come from industrialised countries and are not always suitable for our context. cameroon has a heterogeneous population consisting of 250 ethnic groups that can be classified into five main groups: (1) southern tropical forest peoples, (2) western highlanders, (3) coastal tropical forest peoples, and the (4) kirdi and (5) fulas ethnic groups. yaoundé is a multicultural city in which these communities can be found. factors such as age, sex and race in addition to environment can significantly influence blood count parameters.1,2,3 the clinical and laboratory standards institute recommends that laboratories develop their own local values.4 furthermore, there is a great variability in reference values between medical analysis laboratories in cameroon. this study was intended to determine the values of a complete blood cell count in a healthy adult population for better quality diagnosis, which could in some cases prevent unnecessary testing. methods ethical considerations ethical approval was obtained from the cameroon national ethical committee of research for human health (permit number: 2016/01/701/ce/cnersh/sp). every participant gave written informed consent before the sample collection. study population and sampling strategy this was a cross-sectional study conducted from november 2015 to september 2016. the target population constituted blood donors, because they were submitted to a rigorous medical screening that took into account several criteria for detecting pathology, thus ensuring a healthy population for the study. the recruitment sites were the blood banks of the most frequented hospitals, including the university teaching hospital of yaoundé, the yaoundé central hospital, and the hospital centre of essos. a stratified and clustered sampling strategy enabled us to divide the population into two groups (men and women) and collect around 50 samples per site and per sex. according to the clinical and laboratory standards institute,4 a study requires a minimum of 120 samples in each group in order to obtain valid reference values. a survey was administered before the blood donation that enabled the selection of participants and the collection of socio-demographic data. all of the eligible blood donors who gave their informed consent were included in the investigation. people who met one or more of the following criteria were excluded: (1) currently sick or being followed up for a known pathology, (2) pregnant women, (3) undergoing treatment for any disease, (4) history of blood donation or transfusion within the immediate past 4 months, (5) regular smokers and (6) hospitalisation within the immediate past month. in addition, we excluded people with a positive serology result for any one of the serological tests carried out at the blood bank, including hiv, syphilis, hepatitis b virus, hepatitis c virus, positivity to blood parasites through a thick or thin blood film, platelet aggregates, c-reactive protein > 6 mg/l, or inflammatory syndrome, as well as people with hyperthermia (˃ 37.8 °c) or hypothermia (< 35 °c) as determined by thermometer as a result of temperature control-related conditions. laboratory analysis for each participant, blood was drawn from a superficial vein in the upper limb using venepuncture sampling. this allowed the collection of 5 ml of blood in an ethylene diamine tetraacetic acid tube, which was used for full blood count, reticulocyte count and blood parasite analysis by a thick and thin blood film, and 4 ml of blood in a tube without anticoagulant for c-reactive protein analysis. those tests were performed at the laboratory of centre pasteur cameroon. samples were stored at room temperature in an air-conditioned room (20 °c – 23 °c). samples were transported in a cooler containing a tube rack to avoid thermal or mechanical shocks that could damage cells and cause haemolysis. analyses were done within 6 hours of blood collection. a peripheral blood smear and a thick film were prepared for every sample. serological tests for hiv, syphilis, hepatitis b virus, and hepatitis c virus were carried out by every blood bank. human immunodeficiency virus (hiv) antibodies were detected by two rapid tests that detect both hiv-1 and hiv-2 infections: (1) the immunocomb hiv 1&2 bispot (orgenics, courbevoie, france) and (2) the determine hiv-1/2 (abbott laboratories, chicago, illinois, united states), an immunochromatographic assay. samples reactive to only one of the two rapid testing procedures were considered indeterminate and further tested using an elisa method. hepatitis b virus was detected using a one-step, immunoassay-based diaspot hbsag test kit (diaspot diagnostics, jawa barat, indonesia) for qualitative detection of serum hepatitis b surface antigen. igg antibodies to hepatitis c virus were detected using diaspot hcv-ab test strips (diaspot diagnostics, jawa barat, indonesia), an immunochromatographic assay. syphilis was diagnosed using the venereal disease research laboratory test and the treponema pallidum hemagglutination assay test (omega diagnostic, alva, scotland, united kingdom). the full blood and reticulocyte counts were analysed using pentra dx nexus (horiba-abx, montpellier, france). this analyser allows for the differentiation and quantification of 28 parameters of a haematopoietic population with five reference methods of measurement: (1) flow cytometry, (2) cytochemistry, (3) impedance, (4) absorbance and (5) optical cytometry. the reticulocyte count was performed using fluoro-flow cytometry with orange thiazol. quality control levels of internal quality controls (horiba-abx difftrol, abx minotrol reti) were analysed daily on three samples: (1) low, (2) normal and (3) high. the performance of the laboratory was checked based on the external proficiency testing program with biology prospective (villers-lès-nancy, france). the laboratory has been audited as per the world health organization regional office for africa’s laboratory improvement process towards accreditation (slipta) checklist, and has achieved a four-star ranking on the slipta tier of recognition of laboratory quality management (valid: september 2017 to september 2019). statistical analysis data were registered in the excel 2013 software (microsoft corp., redmond, washington, united states) and analysed with epi info 7 (centers for disease control and prevention, atlanta, georgia, united states) and spss 19 software (ibm corp., chicago, illinois, united states). the data sets were not tested for a gaussian distribution, which was taken into account in the data analysis. for each parameter, the standard deviation and the median with the 2.5th percentile and 97.5th percentile were calculated. the 2.5th percentile was used to define the lower reference limit, while the 97.5th percentile defined the upper one. a kruskal–wallis test for the two groups was used to correlate parameters according to sex. the threshold for significance was set at p < 0.05 for all statistical analyses. results demographic characteristics the total number of people approached and asked to participate in the study was 375. after applying the exclusion criteria, 294 participants (161 men and 133 women) were maintained in order to determine normal values for the local population. a positive serology result for any one of the serological tests carried out at the blood bank, and positivity to blood parasites were major reasons participants were excluded (table 1). the sex ratio was 1:12. their ages ranged from 18 to 55 years for men and 18 to 53 years for women (figure 1). most participants identified as southern forest peoples (table 2). the majority of the population (59%) was between age 18 and 28 years and 31% was between age 29 and 39 years. in terms of blood donation, 61% of the population had never donated blood before. the distribution was approximately equal between the three sites. once the exclusions had been applied, around 45 donors of each sex were included per site. figure 1: distribution of study participants by age group, yaoundé, cameroon, november 2015 – september 2016. table 1: exclusion criteria applied to the study population in yaoundé, cameroon, november 2015 – september 2016. table 2: study population by main ethnic groups, yaoundé, cameroon, november 2015 – september 2016. red blood cell parameters the median haemoglobin concentration was 135 g/l in men and 114 g/l in women and the difference was statistically significant (p < 0.001) (table 3). a statistically significant difference between men and women was also noted for red blood cell and haematocrit values, with men having higher values than women. normal local values were found for erythrocyte parameters; no differences between men and women were observed for mean corpuscular volume, the mean corpuscular haemoglobin concentration, the mean haemoglobin concentration, and the red blood cell distribution index. the reticulocyte count was higher in men compared with women and the difference was statistically significant. table 3: erythrocyte parameters for men and women, yaoundé, cameroon, november 2015 – september 2016. platelet parameters the number of platelets was higher in women (median: 243 × 109/l) than in men (median: 211 × 109/l) (table 4). neither the mean platelet volume nor the platelet distribution index differed by sex. table 4: platelet parameters for men and women, yaoundé, cameroon, november 2015 – september 2016. white blood cell parameters the median white blood cell count was 4.1 × 109/l in men and 4.6 × 109/l in women (table 5). statistically significant differences between men and women were found in the levels of neutrophils, eosinophils and monocytes. no differences between men and women were observed for lymphocytes or basophils. table 5: white blood cell parameters for men and women, yaoundé, cameroon, november 2015 – september 2016. discussion this study took place in yaoundé, a cosmopolitan town that is located in the central region of the country. it consists of a number of hills that have an altitude of 600 m to 700 m above sea level, and a plateau. the highest mountain is 1221 m in altitude, where few people live. at an altitude above 2000 m, the effect that this has on haematological values is mainly an increase in the haemoglobin level.2 medical laboratories more often than not use data provided by an analysis or selected from literature data according to whether they consider these sources to be a relevant reference. several reviews have confirmed that reference values obtained in western countries differ significantly from african populations.2,3 during studies to obtain reference values, ensuring a healthy study population is a painstaking and expensive exercise because medical tests must be carried out to confirm this. blood donors could be considered an accessible population that was submitted to a rigorous medical selection. in addition to the blood donation serology, we carried out blood parasite analysis and eliminated all subjects presenting an inflammatory syndrome through c-reactive protein analysis. in most cases, the results correlate with studies carried out in other african countries.7,8,9,10 table 6 provides haematological locally derived ranges from our study compared with sources from other studies. the difference obtained between men and women during the red blood cell count and haemoglobin concentration determination is a confirmed fact. men have higher values than women. in our study, the haemoglobin concentration ranges from 110 g/l to 160 g/l in men and 98.9 g/l to 137 g/l in women. in a study carried out by dossoh et al. in ghana, the same results were observed in men with values ranging from 80 g/l to 140 g/l in women.11 these variations have been attributed to factors such as the effect of the androgen hormone on erythropoiesis and menstrual blood loss in women.2 in similar african studies,7,9 the haemoglobin concentration values were lower compared to data from a population in france.12 the prevalence of sickle cell disease in cameroon is around 2%.13 one of the limitations of the study is that we did not check for haemoglobin abnormalities among participants. table 6: haematological locally derived ranges from our study compared with sources from other studies. in an american study14 consisting of 166 black and 296 white patients, the average haemoglobin concentration was lower among black patients. as stated elsewhere, our neutrophil results progress in the same direction, although the values are more elevated in the populations of western countries12 compared to the african population.7,15 the reason that populations in western countries have higher values of neutrophils has not been well elucidated. the theory of an excess of marginated neutrophil pools, that these blood cells would be found more in myeloid organs and less in peripheral blood, is often suggested. however, a previous study seemed to disprove this hypothesis.16 our study demonstrates higher eosinophil values in men compared to women. the same result was observed in a study done in france.17 many reviews define eosinophilia as exceeding an upper limit of about 0.6 × 109/l for men and women.18 there is not supposed to be any ethnic variation in the normal eosinophil count and no physiological cause of an increased count has been offered. the most common causes of eosinophilia depend on socioeconomic and geographical factors. in the developing world, the most common cause is parasitic infection, whereas in the developed world, it is allergy.18 the overall prevalence of soil-transmitted helminthiasis was 25.12% and 10.71% for schistosomiasis in the central region of cameroon in 2012.19 those results concerned school-aged children, and the prevalence of these infections is probably lower in adults and in urban areas.20 the national programme for the control of schistosomiasis and soil-transmitted helminthiasis has made great strides in reducing the burden caused by these diseases on the cameroonian people. as far as platelets are concerned, we obtained higher values in women compared to men (p < 0.001). the same results were obtained in togo and uganda and in a united kingdom study7,9,21 and in france.12 however, our values were lower compared to the united kingdom and french studies. the platelet count seems to be lower among black people than in white people. the reticulocyte count is used to determine if the bone marrow is responding adequately to the body’s need for red blood cells. it helps to find the cause of anaemia and to classify hypo and hyper proliferative anaemias.22,23 a few studies in africa have determined the normal ranges of reticulocytes. in our study, the reticulocyte count was significantly higher in men than in women. in a study done by tarallo et al.,24 the observed ranges were also found to be different between men and women. nevertheless, our limit values were higher than those found in a population in france.24 this finding is interesting, because it could mean that the interpretation of reticulocyte counts should be based on sex, similar to those for haemoglobin parameters. conclusion haematological values of the local population are necessary in order to improve daily diagnosis. we conducted a small-scale pilot study due to limited financial support before launching a project. although the size of our sample was smaller than in other studies, the median values of many parameters, especially haemoglobin and platelet counts, were comparable to the ones found in other studies conducted in africa but different to those in the northern hemisphere countries. this study took place in the capital city, which is a cosmopolitan town. it would be more appropriate to use these results rather than the standards from foreign countries and to eliminate the variability in reference ranges between local laboratories. acknowledgements the authors would like to thank both the general and scientific staff of centre pasteur cameroon. special thanks go to dr sarah eyangoh, dr claude tayou, dr jules tatchuang and dr lawrence ayong. they also acknowledge the entire staff of the haematology laboratory of centre pasteur cameroon: ms tapita ndjongue, ms tecla nnomo, mr herve abene nnoma and mr jean-pierre ambobia. the authors acknowledge the blood donor volunteers, and all the staff of the three blood banks of the three different hospitals in yaoundé – the university teaching hospital, yaoundé central hospital and the hospital centre of essos – where participants were received and serological tests were performed. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions m.e.o., a.r.e., f.n.s., j.m. and s.b. designed the study. m.e.o., 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https://doi.org/10.1515/cclm.2010.292 tarallo p, humbert jc, fournier b, mahassen p, henny j. reticulocytes: reference limits. clin lab haematol. 1996;18 suppl 1:13–14. article information authors: bruce h. noden1,2 vincent nowaseb1,3 cornelia de waal-miller1 berta e. van der colf1 affiliations: 1department of health sciences, school of health and applied science, polytechnic of namibia, namibia 2department of entomology and plant pathology, oklahoma state university, united states 3national commission on research, science and technology, namibia correspondence to: bruce noden email: bruce.noden@okstate.edu postal address: 127 noble research center, oklahoma state university, ok 74078, united states dates: received: 10 sept. 2014 accepted: 30 june 2015 published: 20 aug. 2015 how to cite this article: noden bh, nowaseb v, de waal-miller c, van der colf be. profile, perceptions and future expectations of medical laboratory scientists in namibia. afr j lab med. 2015;4(1), art. #246, 7 pages. http://dx.doi.org/10.4102/ajlm.v4i1.246 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. profile, perceptions and future expectations of medical laboratory scientists in namibia in this original research... open access • abstract • introduction • research method and design    • materials and setting    • design and procedure    • analyses       • ethical considerations • results    • study population profile    • perceptions and experiences of the study population • future expectations and perceptions • discussion    • limitations • conclusions • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: public healthcare systems in sub-saharan africa are challenged by healthcare worker shortages, loss of trained staff and attrition to the private sector. studies have historically focused on medical doctors, nurses and pharmacists, with limited focus on medical laboratory scientists. objectives: this study addresses the professional perspectives and expectations of the first two classes of biomedical science students, who graduated from the polytechnic of namibia in 2012 and 2013. methods: a questionnaire was developed to capture qualitative and quantitative data from fourth-year students completing their final semester. data collected included: demographic information; students’ experience; professional expectations; and perceptions about the future of biomedical science education in namibia. results: amongst the 42 of 45 enrolled students who completed the questionnaire, nearly two-thirds anticipated working in government hospitals (29%) or industry (35%), with fewer planning careers in private hospitals (12%) or academia (14%). most expressed an interest in working abroad (64%) and/or in the capital (64%), with fewer interested in small urban areas (48%). only 7% expressed interest in working in a rural area. regarding their view of the future of biomedical science in namibia, 38% responded that it was encouraging, whereas the rest responded that it was uncertain (52%), negative (2%) or unknown (7%). conclusion: members of the first graduating classes of namibia’s nascent biomedical science degree programme reported a perceived lack of opportunity for professional advancement in the field if they remained in namibia. continued thought needs to be given to develop sustainable strategies and opportunities to retain namibian biomedical laboratory scientists in namibia. introduction top ↑ countries in sub-saharan africa are challenged by a lack of healthcare workers.1 on average, healthcare systems in african countries field less than 22.8 skilled healthcare workers per 10 000 population; far below the 59.4 per 10 000 population average documented in more developed countries.2 the already inadequate numbers of healthcare workers being trained are often further reduced by ’out‘-migration to other countries; it has been reported that 8000 nurses leave sub-saharan africa every year.3 this migration, commonly referred to as the “brain drain”, presents a difficult challenge for developing countries already facing substantial barriers to provide a minimal standard of healthcare services.4,5,6 for many healthcare workers, migration to affluent countries is driven by inadequate salaries, lack of training opportunities, limited chances for promotion, safety concerns and poor living conditions at home.4,5,7 it is also driven by healthcare worker shortages in developed countries.1 most evaluations of the “brain drain” phenomenon have focused on medical doctors,8,9,10 nurses5 and pharmacists.11 despite the important role of medical laboratory scientists in the medical diagnostic field, there is almost nothing in the current literature about the impact of this cadre’s out-migration on the health systems in developing countries.12 medical laboratory scientists perform the tests, generate the preliminary reports and, together with other qualified laboratory professionals, monitor the quality of clinical laboratories. altogether, this teamwork culminates in generating reliable results so that evidence-based diagnoses and effective patient care are delivered. the persistent shortage of medical professionals, including medical laboratory scientists, is well reported in countries such as the united kingdom and united states.13,14,15 there is, therefore, an increasing possibility that the “brain drain” phenomenon documented amongst doctors and nurses could also be extended to young african laboratory technicians and technologists and medical laboratory scientists. the resulting loss of these professionals would not only undercut the quality of healthcare delivery in africa, but will also drain the few resources available to the nascent laboratory science training programmes on the continent and may, eventually, cause them to be eliminated.16 namibia has historically relied on south african universities to train its medical personnel. the country also has a long history of reliance on foreign healthcare workers in all major health-related cadres (doctors, nurses, pharmacists and medical laboratory scientists). starting in 2008, the university of namibia (unam) has offered degree programmes in medicine and pharmacy. in the same year, the polytechnic of namibia (pon) introduced degree programmes in biomedical science and environmental health. the biomedical science programme at the pon was hosted by the school of health and applied sciences. prior to 2008, the technical staff of public and private laboratories comprised either foreigners or namibians who left the country to complete a three-year diploma in south african institutions. in 2005, the pon was approached by local stakeholders, as well as the ministry of health and social services, to create a training programme in-country. an advisory committee developed a curriculum for a four-year professional degree programme, which would allow graduates to move into a master’s programme and, ultimately, a doctorate in laboratory sciences. the four-year professional bachelor’s degree incorporated clinical chemistry, haematology, microbiology and molecular diagnostics with other important subjects, as well as a research project. after four years at the pon, the students complete a one-year internship in industry prior to registering as medical laboratory scientists. this programme, which partnered with cape peninsula university of technology (cput) in cape town, south africa, was the first such programme in southern africa. the curriculum and course were approved in 2007 and the first intake of students occurred in january 2008. as the programme developed, external assistance was provided by a president’s emergency plan for aids relief (pepfar) development grant through the local centers for disease control and prevention office and a twinning arrangement with university of arkansas for medical sciences. the new programme began with three faculty members teaching the first cohort of students. the programme grew each year, adding new faculty members, most recruited from industry. through time, the training laboratories were equipped for practical modules. the ministry of education offered study loans to students and the ministry of health and social services offered several bursaries. whilst starting off with excitement, there were considerable challenges in the first years of the programme. finding teaching venues in a dynamically-changing educational institution, in addition to challenges in finding faculty members, meant that practicals were not presented concurrently with theory sessions and international instructors were brought in to provide intense short practical modules. whilst all of these have now been addressed since the programme moved into its own building in 2014, the first two cohorts of students worked through the challenges of the early years. the pon graduated its first class of medical laboratory scientists in 2012. medical education is an important national investment, but the returns obtained are not always what are hoped for, or even expected.5,7 in namibia, where a trained, dedicated cadre of non-expatriate healthcare workers is desperately needed, it is important for nascent healthcare professional training degree programmes to understand what students expect from the education programme in which they have enrolled; and, more importantly, what they expect from their home county’s healthcare system in terms of career opportunities, remuneration and professional advancement after graduation. studies focused on the students, particularly in the early years of these valuable training programmes, provide namibian educators and policy makers with valuable information about how national investments in professional healthcare education may (or may not) benefit the national healthcare system.5,8,9,10,11 as an initial component of this research, the objective of this study was to describe and analyse the profile of the first two classes, which graduated in 2012 and 2013, in regard to personal and educational backgrounds, experiences whilst at the pon and future career expectations. research method and design top ↑ materials and setting consecutive fourth-year, final-semester students (study size: n = 22, 2011; n = 23, 2012) were enlisted from the first two classes graduating from the pon biomedical science programme in 2012 and 2013. only fourth-year students in the programme attending a tutorial during the preparation of their honours thesis research projects were provided an opportunity to complete the questionnaire. those who did not attend the tutorial (n = 3) were not given another opportunity. design and procedure a questionnaire was adapted from modipa and kambisya11 and nguyen et al.5 prior to filling out the questionnaire, the research team went through the questions with the students to ensure understanding. all students were provided as much time as necessary for full analysis of each question and a moderator was on hand to answer any questions. the questionnaire contained closedand open-ended questions concerning: the demographics and backgrounds of the students; their experiences and perceptions during the four years of training; and their expectations and final perceptions of the programme. analyses all data were entered into a microsoft excel™ spreadsheet and analysed using ibm spss statistics for windows, version 21.0 (ibm corp., armonk, ny 2012). for categorical data, pearson’s χ2 tests or fisher’s exact tests were used. binary logistic regression analysis was used to assess associations between population characteristics and desire to work abroad. p-values of less than 0.05 were considered to be statistically significant. ethical considerations the study and questionnaire were approved by the institutional research and publications committee of the pon, the ethical committee of the university, as per policy [irpc-poly/2011/7377/539]. the respondents voluntarily completed the questionnaire and all answers were recorded anonymously. completed questionnaires were securely organised and stored. results top ↑ study population profile of the 45 students enrolled in the programme, all 42 students who were present in the tutorial completed the questionnaire. three international students (angola, n = 1; botswana, n = 2) were not present the day the questionnaire was given, because they were still completing their in-service training commitments. amongst those who responded, the majority were either women (n = 32; 76%) or were between the ages of 21 and 25 years (n = 41; 98%) (table 1). two respondents (5%) were married. almost half of the namibian students were from northern regions (n = 20; 47%) and a third (n = 14; 33%) from central regions. the majority of the students had lived in an urban area before the age of 17 (n = 30; 71%), but most had rural experience (n = 27; 64%). in addition, almost two-thirds had attended government schools in urban areas (n = 27; 64%) and six (14%) had private education. the proportions of students with fathers (n = 14; 40%) or mothers (n = 12; 31%) with a tertiary educational experience were similar. table 1: characteristics of fourth-year students in the biomedical science programme at the polytechnic of namibia in 2011 and 2012. perceptions and experiences of the study population slightly more than half of the students learned of the programme through the media (newspaper or internet) or personal contacts (table 2). the choice to apply for the programme was predominantly self(n = 19; 42%) or family-influenced (n = 18; 40%). just over half (n = 24; 59%) chose biomedical science as their first choice. other students were interested in other medically-related programmes (24%), including medicine, forensics, pharmacy, dentistry and psychology, which were not yet offered in the country when the programme was initiated, or engineering (12%) and environmental health (5%), both of which were already being offered at the pon (data not included in table 2). table 2: perceptions and experiences of fourth-year students in the biomedical science programme at the polytechnic of namibia in 2011 and 2012. when asked why they chose biomedical science, the majority (n = 31; 78%) responded that it was because it was medically-oriented: ‘always wanted to do something medically related and stay in namibia and didn’t want pre-med at unam.’ (female, 22) ‘interested in medical field but didn’t want contact with ­patients.’ (female, 22) another six (15%) chose the programme because it sounded interesting: ‘i got amazed by the name. i never knew what it really was. went to try my luck at the pre-selection and just got in.’ ­(female, 23) ‘[g]ood in biology so pursued related courses. being a scientist sounded good.’ (male, 21) three of the students (8%) chose the programme because they wanted to help others: ‘[h]elp patients in order to save lives.’ (female, 25) ‘thought i would be more helpful and productive to work in a background career instead of an upfront one.’ (female, 22) with regard to living situations and financing of education, most students (n = 22; 49%) lived with family during their four years of study, whilst others rented accommodations off-campus (n = 18; 38%) or used the university housing (n = 7; 15%). forty per cent (n = 21) financed their education through student loans, whilst others were covered through bursaries (n = 15; 29%) or family support (n = 13; 25%). the studies of one student were financed through an agreement between unam and the pon, whilst another was financed through a trust fund. the main difficulties experienced were financial, with related mentions of transport (taking taxis to and from place of residence), supplies (textbooks, computers) and housing. future expectations and perceptions top ↑ most students indicated that they anticipated working in a government hospital setting (n = 15; 29%) or industry (n = 18; 35%) after completion of their training (table 3). a smaller group anticipated working in private hospitals n = 6; 12%) or in academia (n = 7; 14%). one student was focused on working in a forensic laboratory. when asked why they chose their particular settings for anticipated employment, responses could be summarised into four main areas. the first area centred on exposure to new things or new diseases, as can be seen from the following responses: table 3: future expectations and perceptions of fourth-year students in the biomedical science programme at the polytechnic of namibia in 2011 and 2012. ‘[e]xposure to all types of diseases as government [clinics] deal with patients with all backgrounds.’ (male, 24) ‘[e]xposure to wider range of more severe illnesses more common in low-income populations.’ (female, 23) the second area was focused on financial reasons or bursary repayment. the third area centred on the desire to improve one’s qualifications or personal growth and development, as shown by the following responses: ‘[i] believe these are the areas where my skills and qualifications will be utilised best and will also allow me to grow in my profession.’ (female, 22) ‘i have already worked in several med labs so for me to go into a different environment would be interesting and build my knowledge.’ (female, 23) finally, the fourth area was focused on personal professional choice (n = 2), such as a desire to improve the system: ‘so i can provide the best service possible which is not always done at government hospitals.’ (female, 22) ‘[t]o improve industry.’ (male, 22); personal choices (n = 3): ‘i don’t like hospitals and academia is not my field either.’ (female, 22) ‘i love to test water, to make sure it is sterile as this is the ­biggest need in all living beings.’ (male, 22) ‘i want to be a lecturer or training officer at pon one day so that i can train students very well and hopefully to [sic] make biomed the best course at pon.’ (female, 23); or altruism (n = 1): ‘[i] would like to help underprivileged people in society.’ (male, 25) with respect to location of practice, 55% of respondents provided more than one answer. sixty-four per cent (n = 27) wanted to work in the capital (windhoek), 48% (n = 20) in a small urban area 64% (n = 27); wanted to work abroad and 7% (n = 3) wanted to work in a rural area (table 3). of those considering working abroad, 48% (n = 13) preferred europe, 48% (n = 13) north america, 30% (n = 8) were considering working in another african country and 7% (n = 2) wanted to work in australia or new zealand. reasons given for wanting to work abroad included: (1) experiencing new ways of doing things; (2) exposure to new techniques; and (3) attraction to research possibilities not currently available in namibia. equal proportions of male (60%) and female (66%) students desired to work abroad. although not statistically significant, fewer students who started the programme in 2008 (52%) desired to work abroad compared with 2009 (76%). there were no regional differences amongst those desiring to work abroad. once more, though not statistically significant, more students with fathers (70%) or mothers (69%) with secondaryor tertiary-level education desired to work abroad compared with fathers (44%; p < 0.242) or mothers (43%; p < 0.225) with primary or unknown education (data not included in table 3). when asked whether they would have chosen to study biomedical science again, 24% (n = 10) answered in the affirmative; only half said they would recommend the programme to someone else (table 3). concerning their perception of the future of biomedical science in namibia, 38% (n = 16) responded that that it was bright and encouraging, whereas the rest were uncertain (n = 22; 52%), negative (n = 1; 2%) or unknown (n = 3; 7%). reasons given for being positive included: ‘[b]ecause new learning methods and training techniques are continuously developing.’ (male, 27) ‘we are needed, nip [namibia institute of pathology] is accredited. things [are] looking better. quality management is improving in labs.’ (female, 22) ‘[w]e are pioneers, so we can make changes as they need to be made.’ (female, 22) ‘i have faith in [the] country’s potential to develop.’ (female, 23) ‘[m]ore students in this field will make a difference.’ ­(female, 23) ‘[m]ore technologists = more development.’ (female, 24) most of those who responded with uncertainty focused on the lack of development in the industry and lack of opportunities arising from saturation of the market. of the 22 uncertain respondents, nine mentioned the limited job market and three mentioned a lack of alternatives for personal growth in the industry: ‘no means of profession ladder, small market, foreigners saturate the market, not many alternatives.’ (female, 23) ‘slow pace in namibia and there is very little interest by current professionals to develop the profession.’ (female, 22) ‘[the] current situation in the lab does not provide a very bright future for a young developing biomed scientists, but change is possible.’ (female, 22) one even mentioned a fear that positions would be replaced by automation in the near future. those who were ’unknown‘ in their responses cited lack of experience: ‘i can’t predict the future.’ (female, 23) ‘[n]o comment, as [i] am not in the industry yet.’ (female, 22) when asked what they anticipated doing in the future, the majority (n = 29; 58%) wished to further their education, whilst 28% (n = 14) wanted to explore other career fields and 14% (n = 7) planned to continue working in the laboratory. discussion top ↑ this study is the first of its kind to assess the characteristics, experiences and perceptions of medical laboratory science students in the biomedical science training programme in namibia. the implementation of a new health-related training programme in biomedical science in 2008 was unique for namibia as well the region. as such, a number of foreign students, albeit a small percentage, as well as students from all over namibia, were attracted to the programme. this novelty provided a diverse environment in which students from different cultures and socio-economic levels could interact professionally and support one another in their development. the characteristics of the first cohort of students were similar to other studies of medically-oriented students in other african countries.8,9,10,17,18,19 in a country where less than 1% of the adult population has a tertiary degree, one interesting component was the relatively high proportion of students whose parents had a tertiary degree (between 31% and 40%). whilst nguyen et al.5 found an association between desire to emigrate and mothers having a tertiary education, our study did not replicate this finding. this, possibly, speaks not only to their desire to work in a medically-focused profession but it could also be related to the pioneer spirit that these first two cohorts had as they worked through the challenges of a developing programme. having parents with tertiary degrees may have provided a stabilising factor which kept them pursuing their degrees. the results from the survey also highlighted some of the important challenges faced by students in those initial years. in fact, it was quite striking that 76% of the respondents indicated that they would not have chosen to study biomedical science again. there are a number of possible reasons for this result, some of which could be gleaned from their responses. first of all, biomedical science was not the first choice for over 40% of the respondents. respondents seemed to be attracted to medically-oriented careers but, for almost 25%, only after they were not able to get into other health-related programmes. they may have settled, instead, for a course that they did not know much about and were now looking at a career in something they were not sure about after having invested four years. secondly, financial difficulties, which were cited by almost one-third of the respondents, could have affected responses whilst reflecting on the questions. whilst some explicitly cited ’financial difficulties‘, the influence of finances was observed in the other challenges mentioned, such as transport, supplies and housing. for example, transport was critical, because 38% of the respondents lived off-campus and needed to take a taxi (us $2–3/day) to get to and from campus. the cost incurred by that alone meant that some did not have funds for a midday meal but had to wait to eat until they returned home in the evening. the housing conditions in which many students lived were also difficult. those living with relatives often lived in less-than-ideal situations, because the family members were distantly related or the students were an added burden to an already challenging family situation. this meant the students had to move often between supporting family members or put up with basic conditions that lacked running water or electricity. the challenges away from the campus often meant that classes were missed or stressful conditions were endured in order to achieve this degree. finally, the two-year period (2012–2013) during which the respondents were finishing the programme was filled with uncertainty as the namibian professional stakeholders, which first supported the programme, began to realise the implications of 23–24 graduates per year entering the workforce. this produced public questions concerning whether there would be enough positions for graduates. it is notable that shortly after these two groups of students graduated, the programme’s intake size was reduced from 30 to 15 to accommodate these industry concerns. taken together, it is still intriguing that 50% of the respondents would encourage someone else to study biomedical science at the pon. this may indicate that, whilst the programme may not have been what they had hoped for, they saw qualities that would benefit others. this aspect needs to be developed further in future studies focused on the alumni of the programme. in addition to the challenges faced by students, this study provides insights into the motivations and future expectations of this valuable cadre of health professionals and provides data suggesting the necessity of keeping them actively engaged in their home country. of the 42 students surveyed, 64% expressed interest in going abroad, even for a while, if given the chance. the main theme that resonated throughout their responses was that of perceived opportunity for advancement, either in experience or training. with respect to the preferred work location, respondents had a strong desire to work in urban areas or abroad. the reasons given were motivated primarily by a strong desire for opportunity and access to self-development. at that point in their training, the majority of respondents felt that the future of their profession was uncertain, mainly because they could only see job saturation in the near future as the industry is not growing fast enough to meet their needs. this desire for self-development opportunities resounds across sub-saharan africa as countries grapple with the out-movement of trained medical professionals to more affluent, opportunity-providing countries. according to our results, the responses of these namibian students to emigrate to more innovative environments in which to learn new technologies is similar to those of other african medical students,8,9,10,17,18,19 nurses,5,20 pharmacy students11 and medical laboratory professionals.6,16,21 this study provides a glimpse into yet another cadre of medical professionals in africa who are looking for ways to better themselves and move into positions where they can earn and achieve more from their training.5 the fact that a majority chose the programme themselves meant they saw potential to be satisfied in this profession. to keep them in it, however, careful consideration and innovative thinking is needed, if namibia wants to achieve its stated health development goals in the next 10 years.20,22 it was not surprising to find that only 7% of respondents were willing to work in rural areas. this is most likely because the majority grew up and attended schools in urban areas. less than 30% lived in rural areas and attended rural high schools before the age of 17. similar studies report that experience in urban areas creates a natural desire to stay in those areas.5 the same applies for rural experience and/or rural placement.11,23 often, this motivation is fuelled by more than just the higher earning potential.11 in the case of these students, the majority had lived and attended school in urban areas and, as a result, desired to stay connected, bringing in the theme of opportunity and future plans once again. the fact that most covered their education via loans means they have obligations to repay those debts. bursary arrangements often mean there is an expectation to pay back the sponsor by working for the sponsoring company for a certain number of years. those whose parents supplemented their tuition and living expenses may also have some kind of payback expectations, which keeps the student closer to the urban context where such opportunities are available. the equal desire of these namibian students to work either in europe or north america was notable. studies have reported that students often emigrate to their colonising countries because of shared national language and education systems.4 germany and south africa both have important roles in the historical development of namibia with both german and afrikaans being spoken by large portions of the population. now that namibia has developed medical training facilities in a number of disciplines, it will be important to monitor student emigration patterns.24 limitations one of the limitations of the study was the sample size. students in other years could have been included in the study group but the designers of the study felt it was important to capture the responses of the first two years of pioneering students in order to identify ways to improve the program. this served as a form of assessment, not only for the programme but also for the industry as a whole, as these students represented a cohort of highly-qualified namibians who did not leave the country to study abroad but rather persevered through challenges and difficulties. another limitation is the use of a cross-sectional approach to capture the perceptions and intentions of these students. as a questionnaire only monitors a student’s opinion on a particular day, it can only be interpreted as an idea of how they perceive their future at that point in time. conclusions top ↑ as the first study to assess the characteristics, experiences and perceptions of students in the nascent medical training programmes in namibia, these results provide a glimpse into yet another cadre of medical professionals in africa who are looking for ways to better themselves and move into positions where they can earn and achieve more from their training. the diversity of their backgrounds, challenges faced as they pursued training in a developing programme and concerns for the future have common threads with other health-related cadres in developing countries. the desire for meaningful engagement and opportunity and the uncertain view of how their training will integrate with the national programme means that merely having new medical training programmes in namibia is not enough. continued thought needs to be given by the stakeholders and government entities that originally envisioned the programmes in order to develop strategies and opportunities which will keep these professionals meaningfully engaged in the country. acknowledgements top ↑ the researchers would like to thank mr john pitman for editorial assistance. b.h.n. was partially supported by the oklahoma agricultural experiment station (okl-02909). competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions b.h.n. (polytechnic of namibia; oklahoma state university) conceptualised the study; developed the original proposal; obtained the ethical permissions for the study; developed the questionnaire and administered it; input the data; completed the analysis; and wrote the initial draft of the manuscript. c.dw.-m. (polytechnic of namibia), b.e.vdc. (polytechnic of namibia) and v.n. 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http://dx.doi.org/10.1186/1478-4491-8-21 ferrinho p, sidat m, fresta mj, et al. the training and professional expectations of medical students in angola, guinea-bissau and mozambique. hum resourc health. 2011;9:9. http://dx.doi.org/10.1186/1478-4491-9-9 modipa si, dambisya ym. profile and career preferences of pharmacy students at the university of limpopo, turfloop campus, south africa. educ health. 2008;21(3):164. reuter ml. the future of the medical technologist. lab med. 2000;31(11):598–599. http://dx.doi.org/10.1309/3cgx-c4qy-nln1-6a17 davis k. responding to the medical laboratory staffing shortage: the canadian perspective. clin leadersh manag rev. 2002;16(6):399–407. ward-cook k. medical laboratory workforce trends and projections: what is past is prologue. clin leadersh manag rev. 2002;16(6):364–369. beck s, doig, k. laboratory managers’ views on attrition and retention of laboratory personnel. clin lab sci. 2005;18(4):238–247. marinucci f, majigo m, wattleworth m, et al. factors affecting job satisfaction and retention of medical laboratory professionals in seven countries of sub-saharan africa. hum resour health. 2013;11:38. http://dx.doi.org/10.1186/1478-4491-11-38 fronteira i, rodrigues a, pereira c, et al. realities and professional expectations of medical students attending guinea bissau’s medical school in 2007 school year. acta med port. 2011;24(2):265–270. bailey n, mandeville kl, rhodes t, et al. postgraduate career intentions of medical students and recent graduates in malawi: a qualitative interview study. bmc med educ. 2012;12:87. http://dx.doi.org/10.1186/1472-6920-12-87 mandeville kl, bartley t, mipando m. future career plans of malawian medical students: a cross-sectional survey. hum resour health. 2012;10:29. http://dx.doi.org/10.1186/1478-4491-10-29 mutale w, ayles h, bond v, et al. measuring health workers’ motivation in rural health facilities: baseline results from three study districts in zambia. hum resour health. 2013;11:8. http://dx.doi.org/10.1186/1478-4491-11-8 mcclure k. student perceptions of the clinical laboratory science profession. clin lab sci. 2009;22(1):16–21. witt j. addressing the migration of health professionals: the role of working conditions and educational placements. bmc public health. 2009;9(suppl 1):s7.http://dx.doi.org/10.1186/1471-2458-9-s1-s7 kaye dk, mwanika a, sekimpi p, et al. perceptions of newly admitted undergraduate medical students on experiential training on community placements and working in rural areas of uganda. bmc med educ. 2010;10:47. http://dx.doi.org/10.1186/1472-6920-10-47 dambisya ym. the fate and career destinations of doctors who qualified at uganda’s makerere medical school in 1984: retrospective cohort study. bmj. 2004;329(7466):600–601. http://dx.doi.org/10.1136/bmj.38134.524387.ae introduction tuberculosis laboratory services in africa priorities for strengthening tuberculosis services in africa acknowledgements references about the author(s) philip c. onyebujoh world health organization, regional office for africa, harare, zimbabwe ajay k. thirumala tb laboratories, bangalore, india amy piatek global health bureau, usaid, washington, dc, united states citation onyebujoh pc, thirumala ak, piatek a. stronger tuberculosis laboratory networks and services in africa essential to ending tuberculosis. afr j lab med. 2017;6(2), a519. https://doi.org/10.4102/ajlm.v6i2.519 opinion papers stronger tuberculosis laboratory networks and services in africa essential to ending tuberculosis philip c. onyebujoh, ajay k. thirumala, amy piatek received: 30 june 2016; accepted: 15 nov. 2016; published: 31 mar. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction the recent transition from the millennium development goals to the sustainable development goals highlights a major paradigm shift in the global fight against tuberculosis.1 the world health organization (who) end tb strategy, approved by the world health assembly in 2014, calls for a 90% reduction in tuberculosis deaths and an 80% reduction in tuberculosis incidence rates by 2030, compared with 2015.2 globally, tuberculosis death rates have dropped by 22%, and an estimated 49 million lives were saved between 2000 and 2015.3 in africa, since 2000, the downward trends in tuberculosis prevalence, incidence and death rates are notable. between 1990 and 2012, the central african republic, egypt, eritrea, ghana, malawi, niger, rwanda and uganda were some of the best-performing countries, recording reductions of more than 50% in all three indicators. by contrast, cameroon, equatorial guinea, lesotho, liberia, mauritania, sierra leone, south africa and swaziland all more than doubled their 1990 rates for at least two of the above tuberculosis indicators.4 africa is home to more than a quarter of the estimated incidence of all tuberculosis cases (2.7 million; 26%) and has twice the global estimated incidence rates (239/100 000 people) of new tuberculosis cases.3 tuberculosis death rates in africa are three times greater than the global average.3 although estimated tuberculosis incidence rates are coming down from incidence rates in 2000 in sub-saharan africa, the large number of people living with hiv and the dual epidemic of tuberculosis/hiv challenge efforts to accurately diagnose tuberculosis and treat people suffering from both diseases, reflecting the relatively stable numbers of new tuberculosis case notifications since 2010 (figure 1). improving equitable access to tuberculosis diagnosis and treatment remains the most pressing need for africa where laboratory services play a pivotal role. tuberculosis laboratory services in africa public health laboratory organisational structures and services for the diagnosis of infectious diseases, including tuberculosis, are heterogeneous and suffer from several challenges in africa.5,6 assessment of the strengths, weaknesses, opportunities and threats regarding tuberculosis laboratory services provide possibilities for objective interventions at both the country and the regional level in africa (table 1). efforts to diagnose tuberculosis in africa are lagging behind estimated incidence of the disease, as reflected in the stable case detection rates for the last five years (table 2). in 2015, 48% of estimated incident tuberculosis cases (all forms) were detected and national tuberculosis control programmes were notified, which is significantly lower than the 61% global average.3 new tuberculosis case notifications have decreased since 2009 in line with decreases in estimated tuberculosis incidence rate and tuberculosis/hiv incidence rates. of the cases notified, 64% were bacteriologically confirmed, reflecting the challenge in the capacities of national tuberculosis control programmes to enable universal access to accurate molecular tuberculosis diagnostics, ensuring notification and prompt treatment to all those people who are diagnosed with tuberculosis. new strategies and policies to guide diagnostic interventions have been introduced in africa, in line with the global tuberculosis control policies of the who. increasing investments are being made by national governments and donor agencies to accelerate efforts for preventing, diagnosing, and treating tuberculosis, although large gaps remain in meeting need.3 since 2011, investments in molecular tuberculosis diagnostics have multiplied many fold, as reflected by deployment of an increasing number of cepheid genexpert® systems and modules across africa.7 however, these efforts were not being translated into increased diagnosis of tuberculosis cases or increased laboratory confirmation of cases that were notified. data reported to the who by the ministries of health in the african region, indicate that the numbers of laboratories providing tuberculosis diagnostic services using smear microscopy and genexpert have gradually increased from 2009. between 2009 and 2014, the number of microscopy laboratories increased from 10 501 to 15 233 (45%). furthermore, the number of laboratories able to diagnose tuberculosis with genexpert increased from zero to 817 genexpert systems (18 735 gx modules) since it was introduced for the first time in 2011 (table 3). however, the overall number of new laboratory-confirmed tuberculosis cases has not increased. as a more sensitive and accurate molecular diagnostic tool, the potential of the genexpert technology to improve diagnosis of tuberculosis/hiv cases has yet to be fully realised with regard to the overall increase in the number of hiv-positive tuberculosis cases detected. figure 1: tuberculosis incidence and notification rates. estimated tuberculosis incidence rates per 100 000 population and tuberculosis notifications reported to who (1) are presented, along with new hiv infections reported to the unaids database (2) between 1990 and 2013 in 18 sub-saharan african countries (southern and eastern africa, excluding south africa). table 1: key strengths, weaknesses, opportunities and threats for tuberculosis laboratory services in africa.† table 2: new tuberculosis and relapse cases notified in africa, 2010–2015.† table 3: tuberculosis laboratory services in africa, 2009–2014. table 4: regional distribution of firstand second-line tuberculosis drug susceptibility tests performed for notified cases reported in 2015 in africa. eight african countries, including angola, the democratic republic of congo, ethiopia, kenya, mozambique, nigeria, south africa and zimbabwe, are included on the who’s list of 30 multi-drug resistant (mdr), high tuberculosis-burden countries.3 it is necessary that africa develop tuberculosis culture and drug susceptibility testing (dst) laboratories (for firstand second-line anti-tuberculosis medicines) capable of treatment monitoring and diagnosis. the rapid implementation of genexpert technology in africa has resulted in an increase of laboratory-confirmed cases of rifampicin-resistant tuberculosis, a surrogate for mdr tuberculosis, of 128% between 2009 and 2014 (table 4). during the same period, the total number of laboratories providing dst also increased by 20%, and the number of laboratories offering line-probe assays increased by 221%. providing universal access to tuberculosis dst remains a newly-defined target of the end tb strategy. universal access to dst is currently defined as dst for at least rifampicin among all patients with bacteriologically-confirmed tuberculosis, and further dst for at least fluoroquinolones and second-line injectable agents among all tuberculosis patients with rifampicin resistance. preliminary assessment of existing gaps for achieving universal access to dst (figure 2), based on 2015 case notifications in africa, indicated that only 349 435 (54%) laboratory-confirmed tuberculosis patients had access to a first-line dst. in 2015, of the 16 434 patients who were diagnosed and treated for mdr tuberculosis, only 3898 (23.7%) could get a second-line dst test. thus, the challenge that lies ahead for achieving universal access to firstand second-line dst suggests a need to close the 46% gap in access to first-line dst, and the 76.3% gap for second-line dst among tuberculosis and mdr-tuberculosis patients on treatment. regional disparities exist in the gaps in access to universal dst, with the southern africa development community sub-region having better access than the rest of africa, mainly because of progress made in south africa proportionate to the number of tuberculosis patients (table 4). however, these gaps are expected to be bridged through the recent endorsement of rapid molecular line probe assay for second-line drugs and the introduction of shorter-term regimens for programmatic management of drug-resistant tuberculosis.8 the pace of establishing tuberculosis diagnostic services needs to be viewed in the context of quality assessment measures and proficiencies for testing. quality management systems were introduced in laboratory networks in africa as an integrated effort starting in 2008, and commendable efforts were made through support from the african society for laboratory medicine for strengthening laboratory management towards accreditation to the iso 15189 standard. tuberculosis-specific quality management roadmaps for accreditation of quality standards were advocated by the stop tb partnership’s global laboratory initiative. most peripheral laboratories in africa have at least one component of an external quality assessment programme for smear microscopy (usually panel testing). who data indicate that less than half of the smear microscopy laboratories performed adequately on external quality assessments (table 5). nearly two-thirds of dst laboratories reported meeting who-specified quality standards and about half of the laboratories providing xpert® mtb/rif test or line probe assay were routinely monitored for quality under programmatic conditions. figure 2: tuberculosis drug susceptibility testing. firstand second-line tuberculosis drug susceptibility tests performed for notified cases reported in 2015 in africa. table 5: quality of tuberculosis laboratory services in africa, 2009–2014. all of this taken together strongly suggests that available tuberculosis laboratory services need to be optimally utilised in africa as new technologies are introduced during the next few years. there is an urgent need to redefine the purpose, structure and use of diagnostic networks to diagnose tuberculosis and drug-resistant tuberculosis in an epidemiological context and within the operational capacities of the existing, resource-limited settings of africa without compromising quality standards. priorities for strengthening tuberculosis services in africa make use of new ambitious global strategies and goals, and diagnostic testing advances to accelerate global tuberculosis efforts, the world health assembly approved the end tb strategy9 in 2014 as a 20-year strategy with the vision of a ‘world free of tb’ and the goals of ‘zero deaths, disease and suffering due to tb’.2 the strategy’s milestones and targets are ambitious: by 2025, there should be a 75% reduction in tuberculosis deaths and 50% reduction in new tuberculosis cases, and by 2035, there should be nearly no deaths from tuberculosis and a 90% reduction in new tuberculosis cases. the global plan to end tb 2016–2020 was developed concurrently by the stop tb partnership as a costed plan to implement the end tb strategy and provide a path for policy makers to achieve the strategy’s milestones, including a roadmap for new diagnostics.10 both the end tb strategy and global plan clearly articulate that countries must design and implement diagnostic services that promote early and rapid diagnosis of tuberculosis and mdr tuberculosis by using new technologies and expanding the pool of people to be tested. it is fully expected that countries will provide universal dst to all people with tuberculosis. africa must use the opportunities within both the strategy and global plan and translate them into interventions to improve diagnostic services. compared to a few years ago, laboratory services in africa now have better access to advanced tuberculosis diagnostics, including the rapid molecular assays that can accurately diagnose tuberculosis and drug-resistant tuberculosis in less than a day, improved sensitivity for microscopy through fluorescent light-emitting diode technology and faster automated liquid tuberculosis culture systems. recent advancements in diagnostic tests to detect hiv, including advanced point-of-care rapid diagnostic tests, early infant diagnostic tests and viral load tests, are all critically important in africa as part of the continuum of care for people co-infected with tuberculosis and hiv. laboratory quality management and accreditation systems are now available, including the strengthening laboratory management towards accreditation and stepwise laboratory improvement process towards accreditation tools developed especially for africa.11 other enabling e-tools for tuberculosis diagnostics are increasingly made available, including: software platforms that transfer diagnostic outcomes and testing information in real-time to tuberculosis programme officers, clinicians and health workers; commercial specimen transport solutions that can reduce the potential for culture contamination; and mapping technology to allow the visualisation and overlay of all diagnostic networks in a country and the ability to optimise integrated networks in response to existing and future diagnostic algorithms. strengthen strategic policy, planning and health system components to improve tuberculosis diagnostic networks the data above suggest that increasing the number of laboratories may improve access to tuberculosis diagnostic services to an extent. however, it may not necessarily increase overall tuberculosis case notifications under programmatic conditions. enabling interventions such as appropriate changes in national diagnostic policies and algorithms, adequately resourced ambitious strategic plans, increased training for human resources, efficient sputum transport systems to avoid unnecessary diagnosticand patient-delayed testing, and increased participation of community level organisations are all needed. the need for early access to point-of-care diagnostic services and treatment monitoring is greatest at the primary healthcare level and at the community level, whereas more sophisticated extensive diagnostic capacity is needed at regionalor central-level facilities.12 suboptimal linkages with clinical services in many countries often result in either no diagnosis or diagnoses without treatment initiation or delayed treatment, which in turn lead to poor treatment outcomes, including death. it is expected that with increased electronic connectivity between new and emerging molecular diagnostic tools and treatment centres, the diagnostic-treatment gap will diminish significantly and clinical outcomes will improve. integrate public health laboratory and diagnostic networks to address the deficiencies in laboratories’ diagnostic capacity, the integration of public health laboratory networks and surveillance systems13 for infectious diseases remains an urgent requirement. this need became clear during the recent ebola outbreak in west africa, which had a devastating socioeconomic impact on the countries affected,14 as well as on their public health systems.15 well-designed, integrated systems would also meet the need for the implementation of the international health regulations16 and be in line with the global health security agenda with its current efforts to promote global health security as an international priority.17 integrating laboratory services under one roof would be enhanced by networking with adjunct public health institutions, disease-specific referral laboratories and centres of excellence, facilitating systematic engagement of all governmental and non-governmental health structures. it is envisaged that linking the capacity to detect and manage mdr and extensively-drug-resistant tuberculosis to both the global health security agenda and the recently forged declaration on combating antimicrobial resistance will strengthen control of mdr and extensively-drug-resistant tuberculosis globally. strengthening collaborative tuberculosis/hiv activities (i.e., an integrated approach for prevention, diagnosis, and treatment among co-infected patients) will remain critical in addressing the tuberculosis/hiv epidemic in sub-saharan africa. integrated laboratory services under one roof have the potential to reduce repeated visits of patients to health facilities by comprehensively screening for multiple infectious diseases, thus improving early case detection and management, in addition to optimising the use of laboratory resources. the fast-growing transition toward more non-communicable conditions in most settings can be tackled through integrated service provision. by default, several laboratory facilities at the district level in africa provide ‘integrated services’, in one form or other, due to resource constraints and sharing of facilities and personnel. the challenges are mainly related to inadequate infrastructure, equipment, skilled laboratory personnel and their mentoring and supervision, resulting in inadequate integration. the integrated approach demands systematic training and recruitment of high-quality and skilled laboratory scientists, technologists and other laboratory personnel. to achieve universal health coverage, as set out in the end tb strategy, interdependent, fully-integrated laboratory networks at the country and regional levels are needed, with a minimum set of diagnostic technologies for key infectious diseases prevalent at district and subdistrict levels. establish more regional collaboration a persuasive way to enhance diagnostic capacity is through the establishment of regional laboratory networks. the capacity of national reference laboratories for culture and drug resistance testing in many african countries has been developed through several multi-country collaborative efforts, such as the expandx-tb project and the east african laboratory network, established by the world bank and aimed at setting up cross-cutting laboratory services with a clear surveillance focus. these networks have been playing an increasingly critical supportive role in the diagnosis and management of drug-resistant tuberculosis. the strengthening of laboratory systems should be further facilitated through new african regional collaborative initiatives across intergovernmental health bodies and communities. regional health networks have an important role in facilitating adoption and adaptation of global policies and allowing better care and surveillance through increased domestic funding commitments within the region. the east central southern african health community regional tuberculosis laboratory networking supported by the global fund is one example of the right direction in this regard. prioritise research to inform african-specific solutions key components of the end tb strategy are the early and rapid diagnosis of tuberculosis, universal dst, and systematic screening of contacts and people who practise high-risk activities.16 research across the continuum from basic to new tool development and operational research is an essential requirement of the end tb strategy and should be strengthened, especially in africa. the existing laboratory and diagnostic systems at various levels in africa would greatly benefit from increased resource mobilisation for, and participation in, the development of new tuberculosis point-of-care diagnostics, shorter treatment regimens and, ultimately, an effective vaccine by 2025. while new rapid diagnostic technology has become increasingly available, further scale up must be informed through well-designed operational assessments, including the integration of testing platforms across diseases and with diagnostic algorithms employed in other diseases. african-specific operational research is needed to improve many components of the diagnostic network, including linkages from diagnostic to treatment centres, ideal testing algorithms for country-specific settings, expanding skills and capacities of laboratory technologists, operational requirements for technologies at the facility level, such as quality electricity supply, adequate logistics for diagnostic commodity procurement and distribution within the country, effective integrated sample transport networks and timely feedback of results. garner commitment and leadership from within african governments while concerted action from all stakeholders is needed, national governments must demonstrate stronger political will and provide stewardship. efforts must be enhanced to ensure that diagnosis and treatment systems for tuberculosis and drug-resistant tuberculosis are available and well aligned across the different levels of their public health systems. lower-level laboratories must be linked to higher-level laboratories for access to follow-up testing for efficient patient management, ensure optimal use of different technologies at different levels of the tiered network, and maintain staff competence in these techniques.12 ministries of health should ensure that national medical laboratory strategic plans – either as stand-alone documents or as part of national tuberculosis strategic plans – are put in place and implemented. strategic oversight at the country level should continuously ensure the effective management of laboratories, the quality of testing, and the efficient use of the network’s tuberculosis diagnostic services. conclusions tuberculosis laboratory services and networks suffer multiple deficiencies in africa. implementation of rapid molecular diagnostic laboratory tools for tuberculosis in the past five years, has resulted in an increased number of people diagnosed with and treated for drug-resistant tuberculosis in africa, although drug-sensitive tuberculosis cases and tuberculosis/hiv cases have not seen increased case notifications, primarily due to restricted diagnostic algorithms, and capacity and access limitations. there is an urgent need to redefine the purpose, structure and optimal use of diagnostic networks to diagnose tuberculosis within the resource-constrained setting of africa and tuberculosis epidemiology. well-planned and -resourced national strategies are needed to achieve the laboratory targets set forth by the global plan to strengthen services toward ending tuberculosis. appreciable efforts made in the introduction of new laboratory technologies in the last five years in several countries need to be multiplied at all service delivery levels to maximise technology utilisation to close the diagnostic gap in providing universal access to tuberculosis dst by 2020 in africa. integration of laboratory services within public health institutions and public–private collaborations would improve tuberculosis case detection. tuberculosis is preventable, treatable and curable, and governments need to swiftly demonstrate strong leadership in ending tuberculosis as a leading infectious disease in africa. acknowledgements the data used for generating the tables was extracted from the who global tuberculosis database. the authors wish to acknowledge the field experiences shared by the national tuberculosis control programmes and tuberculosis laboratories across africa. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. sources of support none. authors’ contributions p.c.o., a.p. and a.k.t. contributed to qualitative and quantitative analysis of the information. all three contributed to the conceptualisation, design, development of this opinion article. references united nations (2016). sustainable development knowledge platform: health and population [page on the internet]. c2016 [cited 2016 oct 28]. available from: https://sustainabledevelopment.un.org/topics/healthandpopulation world health organization. who end tb strategy: global strategy and targets for tuberculosis prevention, care and control after 2015 [page on the internet]. c2016 [cited 2016 oct 28]. available from: http://www.who.int/tb/post2015_strategy/en/ world health organization. global tuberculosis report 2016 [page on the internet]. c2016 [cited 2016 oct 28]. available from: http://www.who.int/tb/publications/global_report/en/ united nations development programme. mdg report 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international health regulations (2005). 2nd ed [document on the internet]. c2005 [cited 2016 oct 28]. available from: http://www.who.int/ihr/publications/9789241596664/en/ us centres for disease control and prevention. global health security agenda: action packages [page on the internet]. c2016 [cited 2016 oct 28]. available from: http://www.cdc.gov/globalhealth/security/actionpackages/ world health organization. the end tb strategy [document on the internet]. c2015 [cited 2016 oct 28]. available from: www.who.int/tb/end_tb_brochure.pdf article information author: corena de beer1 monika esser2 wolfgang preiser1 affiliation: 1department of pathology (division of medical virology), university of stellenbosch, south africa2department of pathology (immunology unit), university of stellenbosch, south africa correspondence to: corena de beer postal address: department of pathology, university of stellenbosch, po box 19063, tygerberg 7505, south africa dates: received: 09 dec. 2012 accepted: 15 aug. 2012 published: 15 oct. 2012 how to cite this article: de beer c, esser m, preiser w. optimising automation of a manual enzyme-linked immunosorbent assay. afr j lab med. 2011;1(1), art. #15, 3 pages. http://dx.doi.org/10.4102/ ajlm.v1i1.15 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. optimising automation of a manual enzyme-linked immunosorbent assay in this original research... open access • abstract • introduction • materials and methods    • automation of the mk016 enzyme-linked immunosorbent assay • results • discussion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ objective: enzyme-linked immunosorbent assays (elisas) are widely used to quantify immunoglobulin levels induced by infection or vaccination. compared to conventional manual assays, automated elisa systems offer more accurate and reproducible results, faster turnaround times and cost effectiveness due to the use of multianalyte reagents.design: the vacczyme™ human anti-haemophilus influenzae type b (hib) kit (mk016) from the binding site company was optimised to be used on an automated biorad phd™ system in the immunology laboratory (national health laboratory service) in tygerberg, south africa. methods: an automated elisa system that uses individual well incubation was compared to a manual method that uses whole-plate incubation. results: results were calculated from calibration curves constructed with each assay. marked differences in calibration curves were observed for the two methods. the automated method produced lower-than-recommended optical density values and resulted in invalid calibration curves and diagnostic results. a comparison of the individual steps of the two methods showed a difference of 10 minutes per incubation cycle. all incubation steps of the automated method were subsequently increased from 30 minutes to 40 minutes. several comparative assays were performed according to the amended protocol and all calibration curves obtained were valid. calibrators and controls were also included as samples in different positions and orders on the plate and all results were valid. conclusion: proper validation is vital before converting manual elisa assays to automated or semi-automated methods. introduction top ↑ quantitative analytical methods have advanced significantly since the development of the enzyme immunoassay (eia) and enzyme-linked immunosorbent assay (elisa) in 1971 by the groups of perlmann and engvall, and schuurs and van weemen, respectively.1,2,3 before this, the only method for performing immunoassays was the radioimmunoassay (ria), which was first described by yalow and berson in 1960.4however, the ria had several shortcomings, for example the potential health threat of radioactivity, short half-lives of radioisotopes, cumbersome radioactive waste disposal, expensive counting equipment, etc.3,5,6 an important shift from radioisotope-labelled liquid-phase assays to solid-phase assays occurred in 1968. miles and hales7 developed an immuno-radiometric technique, which used radioactively labelled antibodies instead of labelled antigens for measuring insulin in human plasma. plastic tubes were subsequently coated with the antigen or antibody to create a solid-phase or immunosorbent platform. 8 modern commercial elisa/eia kits use 96-well microtitre plates, where either an antigen or an antibody is noncovalently bound to a solid-phase support. these methods are widely employed by laboratories and manufacturing companies for microbiological, virological and other serological diagnostic tests, validation of assays and general quality control. although automated pipetting devices have been used for more than two decades, the high cost associated with the technique remains a major limiting factor in developing countries and smaller laboratories.3 materials and methods top ↑ the immunology unit of the national health laboratory service (nhls) in tygerberg, south africa, uses elisas for serological quantification of antibody levels. before installation of the biorad phd™ system, all assays had been performed manually. the system performs sample dilution, dispenses patient samples and reagents into the microplate, and performs temperature-specific incubation and washing according to pre-defined protocols. automation of the mk016 enzyme-linked immunosorbent assay the 96-well microtitre plate included in the vacczyme™ human anti-haemophilus influenzae type b (hib) kit (mk016) from the binding site company (birmingham, united kingdom) is precoated with the hib capsular polysaccharide antigen conjugated to human serum albumin. in addition to controls and other reagents, the kit also contains five calibrators (0.1 mg/l – 9.0 mg/l) to construct a five-point calibration curve. concentration (logarithmic scale) is plotted against optical density (linear scale) to produce the calibration curve. the quantification range for anti-hib antibody concentration is 0.11 mg/l – 9.0 mg/l.9prediluted samples and controls were pipetted into the plate and incubated for 30 minutes. unbound proteins were removed by a wash step before addition of conjugate (purified peroxidase-labelled rabbit anti-human g-chain-specific immunoglobulin g). after a further 30 minutes of incubation, another wash step was applied to remove all excess conjugate. substrate (3.3’,5.5’ tetramethylbenzidine) was then added, which induced a colour change (from the characteristic serum colour to blue) over 30 minutes. phosphoric acid was then added to stop colour development. the optical density (od) was measured spectrophotometrically at 450 nm and the intensity of the final colour is proportional to the concentration of antibody present in the sample. a list of assays that are validated on the phd™ system is available from biorad (www.bio-rad.com; phd™ validated assay list). results top ↑ analyses with elisa kits from the biorad list produced valid results in our laboratory and these methods were automated without any problems. however, when performing the non-validated mk016 assay on the phd™ system, the calibrators did not produce the required od values recommended by the quality control sheet included in the kit (figure 1). figure 1: vacczyme™ anti-hib enzyme-linked immunosorbent assays calibration curve as provided on the quality control sheet. the highest calibrator reached an od of only 1.417 ± 0.245 (range 1.252–1.813) instead of 2.500 as specified on the quality control certificate. although the lower calibrators were associated with smaller margins of error, they showed a similar trend. all results calculated from this calibration curve were therefore too low and hence invalid.comparison of the individual steps of the automated and manual methods identified a difference of 10 minutes in all incubation periods. the phd™ system times incubation for each well individually and proceeds to the next step only once the exact incubation time has been reached for that specific well. however, timing of manual assays usually starts only after reagents have been added to the last well of the plate; i.e. well incubation is timed rather than plate incubation. in an effort to address this difference, all incubation steps were increased from 30 minutes to 40 minutes on the phd™ system. the duration of the washing steps of the two methods was very similar and therefore not regarded as contributing to the discrepant results. the duration of the washing steps was therefore left unchanged. a total of 16 calibration curves were subsequently generated from the phd™ system to validate the adjusted protocol. all the od values obtained produced acceptable calibration curves (figure 2) and the mean od value for the highest calibrator reached 2.295 ± 0.171 (median = 2.405; range = 1.934–2.553). the recommended values for the high and low controls are 2.4 mg/l – 3.6 mg/l and < 0.35 mg/l, respectively. the high and low controls as used on the phd™ system produced results of 2.538 ± 0.094 mg/l and < 0.35 mg/l, respectively. figure 2: vacczyme™ anti-hib enzyme-linked immunosorbent assay calibration curves before and after optimisation. to confirm our findings, calibrators and controls were included in different positions and orders on the plate. values obtained for these tests were within 5% of the required ranges, which suggested that the amended protocol had a uniform effect on all individual wells of the plate (data not shown). discussion top ↑ automated systems, such as the biorad phd™ instrument, are extremely useful and accurate in assessing immune responses to specific antigens following disease or vaccination. the major advantages of automation or semi-automation include the use of volumes as low as 1 ml, increased accuracy and reproducibility of results, better use of expensive skilled labour, faster overall laboratory turnaround time, ability to perform multiple assays simultaneously and cost effectiveness due to use of multianalyte reagents. use of an automated system also eliminates pipette volume variation and handling errors.accurate, reproducible and reliable laboratory results are crucial for patient management and care, such as initiating treatment in patients with possible immunodeficiency and revaccination of children with insufficient protection following routine childhood vaccination. it is furthermore important for evaluation of study cohort results or establishing reference ranges for specific populations or age groups. this study emphasises the importance of optimisation and validation whenever changing protocols or reagents in order to produce valid and accurate results. in the case of the mk016 assay it was not possible to transfer the protocol established for the manual method to the automated system without modification. after troubleshooting all the individual steps of both methods, a methodological difference was identified and addressed. the modified protocol was scrutinised by repeated measurements of samples of known concentration in different assays and plate positions before amending the existing protocol. acknowledgements top ↑ we would like to thank allere healthcare (pty) ltd (bedfordview, south africa) for donating test kits for optimisation. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this paper. authors’ contributions c.d.b. was the project leader and responsible for experimental and project design, as well as performing the experiments and writing the manuscript. m.e. was the head of the laboratory where the experiments were conducted and w.p. provided valuable input into the final manuscript. references top ↑ 1. engvall e, perlman p. enzyme-linked immunosorbent assay (elisa). quantitative assay of immunoglobulin g. immunochemistry. 1971;8(9):871–874. http://dx.doi.org/10.1016/0019-2791(71)90454-x2. van weemen bk, schuurs ah. immunoassay using antigen-enzyme conjugates. febs lett. 1971;15(3):232–236. http://dx.doi.org/10.1016/0014-5793(71)80319-8 3. lequin r. enzyme immunoassay (eia)/enzyme-linked immunosorbent assay (elisa). clin chem. 2005;51(12):2415–2418. http://dx.doi.org/10.1373/clinchem.2005.051532 4. yalow r, berson s. immunoassay of endogenous plasma insulin in man. j clin invest 1960;39(7):1157–1175. http://dx.doi.org/10.1172/jci104130 5. gosling jp. a decade of development in immunoassay methodology. clin chem. 1990;36(8):1408–1427. 6. engvall e. the elisa, enzyme-linked immunosorbent assay. clin chem. 2010;56(2):319–320. http://dx.doi.org/10.1373/clinchem.2009.127803 7. miles lem, hales cn. labelled antibodies and immunological assay systems. nature 1968;219:186–189. http://dx.doi.org/10.1038/219186a0 8. engvall e, jonsson k, perlmann p. enzyme-linked immunosorbent assay, elisa. ii. quantitative assay of protein antigen, immunoglobulin g, by means of enzyme-labeled antigen and antibody-coated tubes. biochem biophys acta 1971;251:427–434. http://dx.doi.org/10.1016/0005-2795(71)90132-2 9. package insert, 24 may 2006. vacczyme™ human anti haemophilus influenzae type b (hib) enzyme immunoassay kit mk016. the binding site, birmingham, uk. abstract introduction methods results discussion acknowledgements references about the author(s) naseem cassim national health laboratory service(nhls), national priority programme, johannesburg, south africa department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa ahsan ahmad department of urology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa reubina wadee department of anatomical pathology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa jaya a. george department of chemical pathology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa deborah k. glencross national health laboratory service(nhls), national priority programme, johannesburg, south africa department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa citation cassim n, ahmad a, wadee r, george ja, glencross dk. using systematized nomenclature of medicine clinical term codes to assign histological findings for prostate biopsies in the gauteng province, south africa: lessons learnt. afr j lab med. 2020;9(1), a909. https://doi.org/10.4102/ajlm.v9i1.909 original research using systematized nomenclature of medicine clinical term codes to assign histological findings for prostate biopsies in the gauteng province, south africa: lessons learnt naseem cassim, ahsan ahmad, reubina wadee, jaya a. george, deborah k. glencross received: 11 sept. 2018; accepted: 24 june 2020; published: 28 sept. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: prostate cancer (pca) is a leading male neoplasm in south africa. objective: the aim of our study was to describe pca using systemized nomenclature of medicine (snomed) clinical terms codes, which have the potential to generate more timely data. methods: the retrospective study design was used to analyse prostate biopsy data from our laboratories using snomed morphology (m) and topography (t) codes where the term ’prostate’ was captured in the narrative report. using m code descriptions, the diagnosis, sub-diagnosis, sub-result and international classification of diseases for oncology (icd-o-3) codes were assigned using a lookup table. topography code descriptions identified biopsies of prostatic origin. lookup tables were prepared using microsoft excel and combined with the data extracts using access. contingency tables reported m and t codes, diagnosis and sub-diagnosis frequencies. results: an m and t code was reported for 88% (n = 22 009) of biopsies. of these, 20 551 (93.37%) were of prostatic origin. a benign diagnosis (icd-o-3:8000/0) was reported for 10 441 biopsies (50.81%) and 45.26% had a malignant diagnosis (n = 9302). an adenocarcinoma (8140/3) sub-diagnosis was reported for 88.16% of malignant biopsies (n = 8201). an atypia diagnosis was reported for 760 biopsies (3.7%). inflammation (39.03%) and hyperplasia (20.82%) were the predominant benign sub-diagnoses. conclusion: our study demonstrated the feasibility of generating pca data using snomed codes from national laboratory data. this highlights the need for extending the results of our study to a national level to deliver timeous monitoring of pca trends. keywords: prostate biopsy; systemized nomenclature of medicine; snomed; morphology; topography; prostate cancer and adenocarcinoma. introduction prostate cancer (pca) was a leading male cancer in south africa in 2012, while globally it is the second most frequently diagnosed neoplasm.1 a 2012 global cancer study reported an estimated age-specific incidence rate of 67.9 per 100 000 for south africa, with an associated mortality rate of 26.4 per 100 000.1,2 presentation with highly aggressive pca in african men in south africa has been described.3,4 a national cancer control programme was recommended by the world health organization, with the aim of identifying priorities and assigning resources to sustain progress towards the reduction of cancer incidence and mortality as well as to improve the quality of life for patients with cancer.5 this would be attained through the equitable implementation of evidence-based strategies for prevention, early detection, treatment and palliative care, in the context of optimal use of limited healthcare resources.6 the 2013–2020 global action plan for the prevention and control of non-communicable diseases aims to achieve a 25% reduction in the relative risk of premature mortality from cancers and other non-communicable diseases.7 this initiative emphasises the importance of both surveillance and disease registries that should be integrated into existing health information systems to improve the availability of high-quality data.7 current cancer registry reporting is not integrated into any hospital information system. the purpose of a cancer registry is to establish and maintain a cancer incidence reporting system that informs planning of cancer control programs.8 cancer registries should typically publish annual data within 28 months after the close of the year in which the incident case was diagnosed.9 a delay in cancer registry reporting is a major limitation for understanding pca trends.9 the national cancer registry of south africa is a passively reported registry that used the international classification of diseases for oncology (icd-o-3) — recommended international methodology — to manually code pathology reports.10,11,12 the icd-o-3 describes both the anatomical site and cell type and behaviour (malignant or benign biopsy).13 the national cancer registry (ncr) had only reported data for 2014 in 2018.14 cases are manually coded, and this results in a reporting delay. based on the surveillance, epidemiology, and end results programme standard, the 2015 report should have been published by 2018.9 the programme itself, on which the south african reporting standard is based, is comprised of 11 registries in five states and six metropolitan areas in the united states which generate annual cancer data approximately 28 months after diagnosis.8 both internationally and nationally, cancer surveillance is defined as an ongoing, timely, and systematic collection and analysis of cancer data to assess risk factors, screening, diagnosis, and cancer incidences and deaths.9 the aim of surveillance is to analyse and disseminate cancer data to identify challenges and opportunities in the delivery of timeous cancer control programmes.9 the cancer registry reporting is a time-intensive process requiring trained coders to review each narrative biopsy report individually and to manually add the applicable icd-o-3 codes to the reporting system.15 this can take hundreds of hours of manual database building to report pca data. the coders would have to add both topography and morphology icd-o-3 codes for each narrative biopsy report reviewed.15 this manually intensive process prolongs time between diagnosis and reporting of identified pca cases for surveillance. pca reporting is required at least 28 months after diagnosis (~2 years) to understand changes in incidence.8 without timely data this would not be possible. therefore, new approaches are required to reduce or automate the coding process to provide more timely cancer data. some studies have used approaches such as natural language processing and text mining.16,17,18 for our study, we decided to use the systematized nomenclature of medicine (snomed) clinical terms.19 the systematized nomenclature of medicine is a comprehensive and precise health terminology used globally that incorporates a structured list of health terms or concepts.19,20 one of the benefits of using snomed is that codes can be mapped to other coding systems to facilitate interoperability.19 all references to snomed relate to snomed ct.21 in an anatomical pathology setting, snomed ct is used to capture the histological finding in the form of morphology (m) and topography (t) codes. the m and t codes are captured directly in the laboratory information system (lis) by the pathologist after examining prostate cores. the same histological findings are also reported as a narrative pathology report.19 the m and t code(s) are captured separately in defined test items in the lis, which has a dictionary of all the snomed codes that may be reported and the anatomical pathologist selects the appropriate codes to add based on the histological findings. for each biopsy, more than one snomed m/t code may be captured. for example (personal communication, vreede h, august 12, 2017, sharing the laboratory information system snomed ct code table extract), for a biopsy of prostatic origin with an adenocarcinoma finding, the following codes would be reported (description in brackets); (1) t-28 000 or t-92 000 (prostatic structure), (2) m-80 003 (malignant neoplasm, primary) and (3) m-81 403 (adenocarcinoma, no subtype). studies outside south africa have used snomed codes to transform laboratory and other reported data for cancer registry reporting.22 a good example is the danish pca registry that analysed snomed data for biopsies with pca, that is histologically verified.22 this study confirmed that the snomed codes generated clinically useful data.22 our study described here is the first attempt in south africa to investigate reporting pca using snomed codes by collating this information contained in the national laboratory data repository. it is anticipated that this could, in future strengthen surveillance activities. additionally, using the snomed codes to report on patients without pca could provide important presentation information that is currently poorly understood. the majority of local studies have manually coded biopsy reports to extract pca information. the development of snomed ct lookup tables have the potential to automate this process and improve timely pca reporting. the objective of this study was to describe the methodology used to report pca data using snomed ct lookup tables. methods ethical considerations ethics clearance for this study was obtained from the university of the witwatersrand (m170419). this study used national laboratory data that does not contain any patient identifiers. no patient recruitment was required. study design this was a retrospective descriptive study that analysed prostate biopsy data between 2006 and 2016 for men ≥ 30 years in the gauteng province. data extraction and preparation retrospective prostate biopsy data for the period 2006–2016 were extracted from the national health laboratory repository of patient-related data where the term ‘prostate’ was captured in the narrative pathology report. simple text mining approaches were used in the netezza aginity (marlborough, massachusetts, united states) query tool which employed pattern matching by fuzzy string search function.23 two data extracts were received: (1) prostate biopsy, and (2) chained m and t code(s) captured for each biopsy. the prostate biopsy extract included the following variables: (1) episode number, (2) unique patient identifier (generated using a probabilistic matching algorithm24), (3) age, (4) gender, (5) race (where populated), (6) facility code, (7) facility name, (8) reviewed date, and (9) biopsy results text (unstructured narrative report detailing histological findings). the snomed data extract included the following variables: (1) episode number, (2) chained (comma separated) m code(s) (comma delimited), e.g. m-00 100, m-43 000, m-72 450, and (3) chained t code/s, e.g. t-92 000, t-74 000). from the prostate biopsy data extract, the unique snomed ct code combinations were extracted, and a lookup table was developed (figure 1). the lookup tables were developed in a two-step process: (1) code manipulation to combine descriptions, and (2) coding the lookup tables. the blue circles in the figure indicate which figures provide additional details for each step, that is: data manipulation (figure 2) and query to combine data extracts and lookup tables in a single database query (figure 3). figure 1: high-level overview of the steps taken to code the unique chained systemized nomenclature of medicine (snomed) code combinations extracted from the biopsy narrative data extract. there were two separate snomed data extracts from the laboratory information system: morphology (m) and topography (t). the colour coding indicates the various processes; (1) green: data extracts, (2) yellow snomed code manipulation in preparation for lookup table development, and (3) orange: lookup tables with coded variables. the extracted unique chained snomed code combinations were used to prepare the two lookup tables to generate the following new coded variables; (1) organ, (2) organ icd-o-3, (3) diagnosis, (4) diagnosis icd-o-3, (5) sub-diagnosis, (6) sub-diagnosis icd-o-3, and (7) sub-result (for an inflammation sub-diagnosis). the number of biopsies is indicated for each data extract. the blue circles indicate which figures provide additional details on each step. figure 2: six-step procedure used to transform the chained comma separated systemized nomenclature of medicine m codes into individual columns (leaving the original value intact) to add the laboratory information system code table descriptions (in preparation for lookup table development). the same procedure was conducted for t codes. the manipulation was achieved using standard microsoft excel functions (screenshots included next to each step). the steps are as follows: (1) copy unique chained codes to a new worksheet and then copy and paste to a new column for processing (leaving the original values intact), (2) use the microsoft excel text to column function to separate the chained codes and name new columns, e.g. m code 1-n, (3) insert a new column next to each code column and label as a description column, e.g. m code descr 1-n, (4) add the alphabetically sorted laboratory information system systemized nomenclature of medicine code description in a new worksheet, (5) use the microsoft excel vlookup function to add the code description (range lookup set at 1 for an exact match), e.g. m-00 100 code description is ‘normal tissue (finding)’, and (6) microsoft excel concatenate function used to combine code descriptions combined in a new column. figure 3: relational database diagram describes how the various tables were joined (left outer). for each table, the primary and foreign keys are provided. the lookup tables contain only the unique systemized nomenclature of medicine code combinations. the lines indicate a relationship join between tables. once the table joins are implemented, variables from any table can be reported. structured query language could be used to combine the required data for analysis. combining systemized nomenclature of medicine descriptions for the lookup table the chained snomed codes were provided in a format that could not readily be analysed and had to be separated into individual columns with the applicable descriptions added from the lis code table. data were prepared using microsoft excel (microsoft corporation, redmond, washington, united states)25 (figure 2). the unique code combinations and descriptions were extracted for the development of the lookup table. coding the systemized nomenclature of medicine m and t lookup tables we used the prepared m and t code descriptions to start populating the lookup tables. for the m lookup table, we used each unique code description combination to populate the following new variables: (1) diagnosis, (2) diagnosis icd-o-3 code, (3) sub-diagnosis, (4) sub-diagnosis icd-o-3 code, and (5) sub-result with guidance from an anatomical pathologist and a urologist. the team reviewed each code combination and assigned values to be captured in the lookup table. an example of the coding is provided for four biopsies in table 1. we captured the matching icd-o-3 codes for predominantly malignant findings. the diagnosis reports the overall biopsy finding, whereas the sub-diagnosis was used to differentiate the diagnosis, e.g. benign, negative for malignancy (icd-o-3: 8000/0) and ‘hyperplasia’, respectively (episode a). assigning the malignant code descriptions was fairly easy, but we struggled with benign findings given the number of findings reported and the order thereof. to clarify coding, we defined the reporting order to assign a benign sub-diagnosis as follows: (1) inflammation, (2) hypertrophy, (3) hyperplasia, (4) edema, (5) atrophy, and (6) adenosis. this made it easier to assign these finding in logical order. the sub-result was used to identify the type of inflammation reported, e.g. acute, chronic, granulomatous, etc. similarly, the t code lookup table reported the organ (prostate/other) and associated icd-o-3 code (c61.9 for prostate). table 1: example of four prostate biopsies where the systemized nomenclature of medicine code descriptions were assigned a diagnosis and sub-diagnosis including the allocation of icd-o-3 codes. combining the lookup tables with the prostate biopsy data microsoft access (microsoft corporation, redmond, washington, united states) was used to combine the datasets in a single query: (1) prostate data extract table, (2) snomed m lookup table, and (3) snomed t lookup table. tables were combined using a left outer join in a relational database (figure 2).26 this join type ensures that all rows from the prostate data extract table were populated with only the matching values from the other tables reported using referential integrity (table 1).26,27 this query contained all the variables for the data analysis. systemized nomenclature of medicine m code descriptive analysis the number of prostate biopsies with an m and t code populated was assessed as a contingency table using sas enterprise guide 7.1 (sas institute incorporated, cary, north carolina, united states).28 descriptive analysis was conducted for prostatic biopsies with the m code captured. test volumes for the top 10 m code combinations with their chained descriptions were reported (in descending order). the number of biopsies of prostatic origin was also reported. descriptive analysis of diagnosis and sub-diagnosis the diagnosis and sub-diagnosis volumes were reported for biopsies with m code populated of prostatic origin. for each diagnosis, the sub-diagnoses were then reported. where more than 10 sub-diagnoses were reported, the first 10 were reported and the remaining grouped as ‘other’. results using our methodology, 25 010 biopsy results were extracted and analysed. for the lookup tables, there were unique 1520 m and 702 t code combinations. systemized nomenclature of medicine m and t code descriptive analysis m codes were provided for 22 195/25 010 biopsies (89%; table 2). the t code was provided for 24 546/25 010 (98%) biopsies. there were 22 009/25 010 biopsies with both an m and t code populated (88%). there were 2815/25 010 (11%) biopsy reports that could not be analysed using lookup tables; they did not have both m and t codes (table 2). descriptive analyses were conducted for 20 551/25 010 (82%) biopsies of prostatic origin. of the 22 195 biopsies with m codes, m-40 000 (‘inflammation’) was the most commonly reported sub-diagnosis (n = 3400; 15.3%). this was followed by two adenocarcinoma combinations: m-81 403 (n = 2677; 12.1%) and m-81 403, m-80 003 (n = 2166; 9.8%) (table 3). table 2: contingency table to assess the percentage of systemized nomenclature of medicine m and t codes populated using the mapping table for prostate biopsies between 2006 and 2016 in the gauteng province, south africa. table 3: top 10 most commonly requested systemized nomenclature of medicine m code combinations from the prostate biopsy data between 2006 and 2016 in the gauteng province, south africa. descriptive analysis of diagnosis and sub-diagnosis using this approach, we noted 10 441 (50.81%) biopsies with a benign diagnosis (icd-o-3:8000/0) and 9302 (45.26%) biopsies with a malignant diagnosis (8000/3). atypia was reported for 760 biopsies (3.7%). an uncertain (whether benign or malignant) diagnosis (8000/1) was reported for 48 (0.23%) biopsies (table 4). table 4: descriptive analysis of diagnosis and sub-diagnosis where both a systemized nomenclature of medicine m and t code are populated of prostatic origin between 2006 and 2016 in the gauteng province, south africa. inflammation was reported as a sub-diagnosis for 4075 benign biopsies (39.03%). this was followed by no pathologic diagnosis and hyperplasia at 26.90% (n = 2809) and 20.82% (n = 2174) respectively. inflammation and hyperplasia were reported in two combinations contributing 7.85% of the top 10 benign sub-diagnoses. cumulatively, the top 10 sub-diagnoses reported represented 98.8% (n = 10 320) (table 4) of all benign diagnoses. the majority of samples with a malignant diagnosis reported an adenocarcinoma (8201, 88.16%) sub-diagnosis. there were 408 biopsies with a carcinoma (4.39%) sub-diagnosis. the malignant neoplasm sub-diagnosis was reported for 693 (7.45%) biopsies where it was not possible to differentiate the tissue type, reporting predominantly the m-80 003 code (malignant neoplasm, primary) (personal communication, vreede h, august 12, 2017). the majority of biopsies with an atypia/dysplasia diagnosis reported an atypia sub-diagnosis (n = 616; 81.05%) followed by dysplasia (n = 87; 11.45%). only 1.71% of biopsies with an atypia/dysplasia diagnosis reported a high grade intraepithelial lesion (n = 13). only 48 (0.23%) biopsies reported an uncertain sub-diagnosis (8000/1). discussion we showed that it was possible to automate prostate biopsy reporting using a commonly available relational database (microsoft access) and snomed lookup tables in the gauteng province. the use of icd-o-3 codes for malignant findings facilitate pca reporting similar to cancer registries.13 to routinely automate the registration and surveillance of pca in south africa, the lookup tables developed for this study would need to be introduced to the corporate data warehouse. lookup tables are routinely used by the corporate data warehouse (cdw) to transform laboratory data for reporting, for example the hiv serology results reported as ‘neg’, ‘n’ or ‘negative’ are transformed to a single value (‘neg’) for uniform reporting.28 the benefit of this mechanism for pca reporting is that as biopsies are reported in the lis, the data replicated to the cdw will be conformed to report the biopsy diagnosis and sub-diagnosis within three months of diagnosis. lookup tables would facilitate a constant feed of analysed pca data to the south african ncr to ensure timeous reporting. over time, any new snomed code combinations identified would have to be added to the lookup table. by providing this data at shorter intervals, it would be possible to triangulate against other local data sources (ncr and other). triangulation is the process used in public health to review and interpret data from multiple sources that answer the same question for decision making.30 unpublished data from this study revealed that between 2012 and 2016, pca incidence has increased from 44.92 to 57.31 per 100 000 compared to 46.53 reported by the ncr in 2012.31 another advantage of this approach is that pca data from other african countries using a lis could also be analysed using the developed lookup tables to dramatically improve pca reporting across africa. antoni et al. assessed the methods used for reporting the 2018 global cancer statistics estimates.32 for 14/51 african countries, pca incidence estimates were based on simple average rates from neighbouring countries (27%).32 for south africa, projections of national incidence were sourced from the ncr.32 the snomed lookup tables have the potential to improve both national and regional pca incidence reporting across the african continent providing more accurate data. with better data, cancer control initiatives could be better mobilised. the principles applied in our study could also be implemented for other cancers. the snomed codes are captured for all cancers of public health importance routinely. similar lookup tables could be developed to report on lung, breast and cervical cancers with incidence rates of 17.3, 49.0 and 13.5 per 100, 1000 respectively in 2018 in south africa.33 the approach described in our study is not unique. similar approaches have been employed in the danish cancer registry, where data reported for 161 525 biopsies for the period 1995–2011 were undertaken using snomed codes.22 the danish cancer registry predominantly reported data for pca, whereas our study reported data for negative biopsies as well. the combination of the lookup tables reported in our study with text mining to extract the gleason score reported in an unpublished study could be used to report data similar to the danish cancer registry, for example diagnosis of ‘neoplasm, malignant’, sub-diagnosis of ‘adenocarcinoma’ and a gleason score of 3 + 3 = 6 would be coded as ‘bgs3+3’.22 it is important to provide up to date pca data at both the national, provincial, district and health facilities levels to identify hotspots where programmatic interventions are required. snomed lookup tables could be used to focus programmatic interventions for geographic areas with a higher pca burden. similar initiatives using laboratory data have been undertaken locally for hiv and tuberculosis services.24,34,35 an example is the world bank report that described spatial clustering analysis of hiv viral load suppression at the national, provincial, district, sub-district and health facility levels.24 this study indicated that national laboratory data stored in the cdw has the potential to provide important strategic information on the quality and reach of the antiretroviral therapy programme by highlighting the geographical variation in the proportion of patients virally suppressed.24 this information can be accessed by healthcare workers using the epidemiological dashboard developed to identify health facilities that are performing poorly.24,34 similar work has also been published using cluster of differentiation 4 data to highlight areas where hiv-positive patients presenting for care have a higher burden of advanced disease.34 the assimilation of health data into workable and user-friendly dashboards has had a big impact on how health data is used locally.36,37,38 in the medium term, it would be possible to develop a pca epidemiological dashboard similar to the hiv example mentioned to report pca trends by age, race group and geographical boundaries routinely. the pca dashboard could facilitate the reporting of the number of incident cases. the dashboard could also provide insights into how and where health care services are being accessed to inform both guideline changes and programmatic improvements to facilitate equitable access to care across the country. it could also provide loss to follow up and waiting time data for patients who had presented with an elevated prostate specific antigen and who were confirmed with pca histologically using the cdw probabilistic matching algorithm.29 data reported in our study additionally demonstrated functionality by providing data for benign histological findings, potentially useful to identify trends for patients diagnosed with chronic inflammation who eventually progress to pca. while the icd-o-3 codes are particularly important for pca cases, the importance of additional benign findings is especially important for inflammation and hypertrophy that have been shown to be linked with many other cancers.39,40 several studies have indicated that chronic inflammation has a potential role in prostatic carcinogenesis and tumour progression.39,41,42 nelson et al. reported that chronic or recurrent inflammation may play a role in the development of pca.43 using the data generated using the lookup tables, patients with chronic inflammation could be followed up to identify whether they progress to pca in an african setting. finally, to improve snomed reporting, it is recommended that the m and t codes be defined as mandatory fields. this will ensure that 100% of biopsies include these codes. this will address the 12% of biopsies without this information. future research includes extending the lessons learnt with pca in the use of lookup tables to other common cancers, as patient-level data is already available in the cdw and could be easily unlocked for national cancer reporting. limitations one of the limitations is that the data for our study was limited to biopsies sent to the nhls and did not include data from private sector laboratories and thus limits the generalisability of our study. as private sector laboratories also use the snomed code, discussions will be initiated with them to share pca data for national reporting. an additional limitation of our data is the 10% of biopsies excluded a snomed m or t code. unfortunately, these fields are not mandatory and can be uncaptured. by amending rules and making these fields mandatory, all biopsies would prospectively be reviewed with at least one t and m code captured. the excluded data would also affect the generalisability of our study. we are not able to determine the findings for these biopsies. to address this gap, text mining and machine learning approaches are being investigated. a sample of the biopsy data will be used to train the machine learning models i.e. malignancy (1) and benign (0). the big data tools will be validated against well populated snomed data entailing grid search, k-fold cross validation, precision, recall and f-score. the combination of snomed, text mining and machine learning will hopefully address the missing data. with minor changes to the laboratory information system, this has the potential to report pca histological findings for all biopsies. key messages existing national laboratory data has been used for the first time in south africa to report pca diagnosis across a province using snomed lookup tables. this could be implemented across south africa to provide timely pca trends. conclusion our study has demonstrated that it is possible to automate pca reporting using snomed codes for 88% of biopsies. the value of national laboratory data as shown in our study can easily be extended to deliver the timely monitoring of pca trends across south africa and other african countries. this could also be applied to report data for other cancers of public health interest. acknowledgements the authors thank professors martin hale (anatomical pathology) and mohammed haffejee (urology) for their insights into pca diagnosis. we would also like to thank drs elvira singh and mazvita sengayi from the national cancer registry. for providing the data for the study, we would like to thank the academic affairs, research and quality assurance department, as well as tinyiko ngobeni, a data analyst at the corporate data warehouse. competing interests the authors declare that they have no conflicting interests. authors’ contributions j.a.g. and d.k.g. designed and supervised the study by providing leadership and oversight. r.w. and a.a. assisted with the snomed lookup table development. n.c. developed the methodology and conducted the research. all authors contributed to writing the initial draft. j.a.g. and d.k.g. were responsible for reviewing the article and overall project administration. sources of support the authors declare that they have not received any funding for this study. data availability statement the authors do not have permission to share the data generated for this study. disclaimer the authors declare that the views expressed in the submitted article are our own and not the official position of any institution or funder. references ferlay j, soerjomataram i, ervik m, et al. globocan 2012 v1.0, cancer incidence and mortality worldwide: iarc cancerbase no. 1 [homepage on the internet]. lyon: international agency for research on cancer (iarc); c2013 [cited 2018 feb 28]. available 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prognostic value of advanced lung cancer inflammation index in head and neck squamous cell carcinoma. eur arch otorhinolaryngol. 2019;276(5):1487–1492. https://doi.org/10.1007/s00405-019-05381-0 kobayashi s, karube y, inoue t, et al. advanced lung cancer inflammation index predicts outcomes of patients with pathological stage ia lung adenocarcinoma following surgical resection. ann thorac cardiovasc surg. 2019;25(2):87–94. https://doi.org/10.5761/atcs.oa.18-00158 sfanos ks, de marzo am. prostate cancer and inflammation: the evidence. histopathology. 2012;60(1):199–215. https://doi.org/10.1111/j.1365-2559.2011.04033.x leitzmann mf, rohrmann s. risk factors for the onset of prostatic cancer: age, location, and behavioral correlates. clin epidemiol. 2012;4(1):1–11. https://doi.org/10.2147/clep.s16747 nelson wg, de marzo am, isaacs wb. prostate cancer. n engl j med. 2003;349(4):366–381. https://doi.org/10.1056/nejmra021562 article information authors: susan githii1 gunturu revathi2 anne muigai3 samuel kariuki4 affiliations: 1jomo kenyatta university of agriculture and technology, itromid kemri, kenya2the aga khan university hospital, nairobi, kenya 3jomo kenyatta university of agriculture and technology, juja, kenya 4centre for microbiology research, kemri, kenya correspondence to: susan githii postal address: 010024092, thika dates: received: 26 apr. 2012 accepted: 20 sept. 2012 published: 20 may 2013 how to cite this article: githii s, revathi g, muigai a, kariuki s. carriage rate and serotype distribution of streptococcus pneumoniae amongst children in thika hospital, kenya. afr j lab med. 2013;2(1), art. #45, 5 pages. http://dx.doi.org/10.4102/ ajlm.v2i1.45 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. carriage rate and serotypes of streptococcus pneumoniae amongst children in thika hospital, kenya in this original research... open access • abstract • introduction • ethical considerations • methods    • study site    • study population    • study design    • study sample size    • study information and consent from parents or guardians    • obtaining nasopharyngeal swabs • laboratory procedures    • processing of nasopharyngeal swabs    • preservation of streptococcus pneumoniae strains    • antimicrobial susceptibility testing    • serotyping of pneumococci by latex agglutination method    • quellung reaction for identification of pneumococcal factors    • statistical analysis • results    • streptococcus pneumoniae carriage rate    • serotype distribution • discussion    • pneumococcal carriage rate    • antimicrobial susceptibility to penicillin    • chloramphenicol    • cotrimoxazole    • erythromycin    • cefotaxime    • tetracycline    • prevalent serotypes • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. rates of carriage are highest in infants and the elderly. the objectives of this study were to determine the rate of nasopharyngeal colonization by s. pneumoniae, and to describe the antibiotic resistant patterns and the serotypes of the carried isolates. a cross-sectional study design was used. nasopharyngeal swabs were collected from 315 children in the months of october and november 2010 and processed to isolate s. pneumoniae. the isolates were serotyped by the quellung reaction and their antibiotic susceptibilities assessed by the disc diffusion method. the overall nasopharyngeal carriage rate for s. pneumoniae was 17%. seventeen serotypes were detected amongst 55 strains analysed: 6a, 23f, 19f, 13, 6b, 14a, 20, 7c, 1, 15b, 35b, 19a, 11a, 34, 5, 3 and 23a. susceptibility testing revealed that nearly all (98%) were resistant to cotrimoxazole, 9% were resistant to penicillin and 7% to cefotaxime. resistance to chloramphenicol and erythromycin was 2% and 4%, respectively. all isolates were fully sensitive to tetracycline.high levels of cotrimoxazole resistance and some resistance to other antimicrobial agents commonly used in thika district hospital shows that there is need to revise antimicrobial policy in this region in the treatment of invasive pneumococcal infections. the frequent serotypes found in this study have previously been associated with pneumococcal infections in children. several of these serotypes are included in the ten-valent vaccine and therefore use of this vaccine will help reduce pneumococcal infections in thika. introduction top ↑ streptococcus pneumoniae is an inhabitant of the human upper respiratory tract.1 the main reservoir of s. pneumoniae is the human nasopharynx and this is the main source of person to person transmission.1 pneumococci are usually spread through respiratory secretions and aerosols and give rise to an invasive infection only after spreading to other areas of the respiratory tract, or, if they penetrate the nasopharyngeal mucosa, leading to systemic circulation via the cervical lymphatic.2 carriage rates differ depending on environment and age, with the highest carriage rates occurring in children between two and four years old.3 the risk of disease is highest amongst children below five years old, older adults, smokers, and persons with certain chronic illnesses.4 children under five years old are frequently colonised for prolonged periods with the same serotypes.5 only a small percentage of colonised children develop an invasive infection, but pneumococcal nasopharyngeal isolates reflect the strains currently circulating in the community.1 treatment of pneumococcal infections is dependent on the site of infection and currently involves administration of antimicrobial agents and immunisations with the pneumococcal polysaccharides vaccines.6 the most common agents currently being used by physicians to treat patients are the beta-lactam antibiotics which include penicillins, cephalosporins, monobactams and carbapenems.7 other antibiotics include the macrolides and fluoroquinolones.8 s. pneumoniae has demonstrated increasing resistance to the commonly used antibiotics, especially penicillin.9 these antibiotic-resistant strains are more frequently found in child carriers than in adult carriers.10 antibiotic-resistant s. pneumoniae strains are usually associated with a limited number of serotypes which are frequent causes of invasive paediatric infections.11 thus, an understanding of nasopharyngeal carriage prevalence, serotype distribution and antibiotic susceptibility patterns to commonly used antimicrobial agents in various communities is of great importance. ethical considerations top ↑ ethical clearance for the study was obtained from kenya medical research institute (kemri) scientific steering committee and ethical review committee. approval was also obtained from medical superintendent, thika district hospital. methods top ↑ study site the study was conducted in thika district hospital in thika, kenya, during the months of october and november 2010. study population the study population included children of five years and below who had been admitted to thika district hospital, pediatric ward and whose parent or guardian had given consent for participation in the study. study design a cross-sectional study design was used. study sample size the sample size calculation was based on the precision around the estimate of the expected (28%) nasopharyngeal carriage rate of s. pneumoniae, using the formula n = z2 p (1-p)/0.05.12 this resulted in a minimum sample size of 310 children. study information and consent from parents or guardians selection of the patients was done from the hospital’s database and it involved those children who had been admitted to the hospital on the previous day. the parents or guardians of the selected children were called to the procedure room and information sheets explaining the purpose of the study and the procedures involved were distributed. those who allowed their children to participate in the study were given a consent form to sign. obtaining nasopharyngeal swabs nasopharyngeal samples were collected by gentle insertion of a flexible swab stick with dacron-tipped flexible-wire swabs. this was done by tipping the child’s head slightly backwards, and passing the swab directly backward, parallel to the floor of the nasopharynx. the swab was passed till it reached the posterior pharynx. once in place, the swab was rotated through 180 degrees and left in place for two seconds to saturate the tip before removing it slowly. once the nasopharyngeal specimen was collected, it was placed in a bottle containing 3.0 ml of trypticase soy broth medium and labelled with a study specimen number. excess wire handle was cut off from the swab leaving the swab itself in the transport media and the cap was tightened. laboratory procedures top ↑ processing of nasopharyngeal swabs the specimen was vortexed for 20 seconds to disperse organisms from the swab stick. ten microlitres of the specimen was inoculated on columbia blood agar plates (ba) and incubated at 35 °c in 10% carbon dioxide overnight. the plates were checked for growth at 24 and 48 hours. the identity of the isolates was confirmed by standard laboratory methods, which included colony morphology, gram staining, catalase tests, susceptibility to ethylhydrocupreine hydrochloride (optochin) and bile solubility tests. preservation of streptococcus pneumoniae strains pneumococcal isolates were stored by picking from the optochin plate one or more colonies and streaking them as a lawn onto a ba plate. after overnight incubation, the growth was examined for purity. pure growth was harvested with a sterile swab and dispensed into tubes of trypticase soy broth medium. the suspension was then stored at −20 °c and −70 °c for later serotyping. antimicrobial susceptibility testing antimicrobial susceptibility testing was determined using kirby-bauer disc diffusion technique for the confirmed s. pneumoniae isolates. a small inoculum of each bacterial isolate was emulsified in 3 ml sterile normal saline in bijou bottles and the density was compared with a barium chloride standard (0.5 mcfarland). a sterile cotton swab was dipped into the standardised solution and used for evenly inoculating mueller–hinton plates. the plates were then allowed to dry. antibiotics with the following concentrations were placed on the plates: chloramphenical 30 µg, cotrimoxazole (sulfamethoxazole 23.7 µg, trimethoprime 1.25 µg), tetracycline 30 µg, cefotaxime 30 µg, erythromycin 15 µg and penicillin 10 iu. the antibiotics were well spaced in order to prevent the overlapping of inhibition zones. these antibiotics were selected based on the prescription practices in thika district hospital. the plates were incubated at 35 °c for 24 hours. quality control for susceptibility patterns was performed on a daily basis using s. pneumoniae atcc 49619. susceptibility results were interpreted according to the clinical laboratory standard institute guidelines, 2008. serotyping of pneumococci by latex agglutination method pneumococci were serotyped by the latex agglutination method using commercially available antisera from staten’s serum institute, copenhagen, denmark. the serotypes were defined by the quellung reaction. a loopful of overnight growth of pure s. pneumoniae was taken from a ba plate and suspended in 300 ml of phosphate buffered saline, ph 7.2. on a glass slide, 5 ml of latex reagents and 10 ml of cell suspension were mixed with a pipette tip and the slide rocked for one minute. the slide was observed for any sign of visible clumping of the suspension. clumping of the cells indicated a positive result implying presence of the pneumococci; smooth suspension indicated negative results, implying absence of the pneumococci. this procedure was repeated until the specific pool was known. once the pool was known, each type or group within the pool was tested individually. the types that historically occurred more often were tested first followed by the next ones in the order of their occurrence. all the pneumococcal strains were then identified finally by the quellung test. quellung reaction for identification of pneumococcal factors the cell suspension prepared above was mixed with the respective factor sera to determine its factors. four microlitres of bacterial cell suspension was dispensed onto a labelled slide. three microlitres of the antiserum were added and mixed thoroughly with the cell suspension. a cover slip was placed on the mixture whilst avoiding drying. using an oil immersion lens, the mixture was examined under a phase contrast microscope for the presence of apparent capsular swelling to identify the pneumococcal factors. statistical analysis data entry and analysis were performed using microsoft excel. results top ↑ streptococcus pneumoniae carriage rate nasopharyngeal swabs were collected from 315 children: 185 males and 130 females. s. pneumoniae was isolated from the nasopharynx in 55 of the 315 children, representing a carriage rate of 17%. there was no significant difference in carriage rates between males and females. the highest rate of carriage was found in children between one and two years old.antibiotic susceptibility testing was performed on all of the 55 isolates. the proportion of isolates classified as susceptible, intermediate or resistant to the antibiotics that were tested was determined (table 1). table 1: anti-microbial susceptibility of streptococcus pneumoniae isolates. serotype distribution serotyping was performed on all of the 55 isolates. a total of 17 serotypes were identified in this study: nine (16%) serotype 6a, eight (15%) serotype 23f, seven (13%) serotype 19f, five (9%) serotype 13, four (7%) serotypes 6b and 14a, two (4%) each of serotypes 20, 7c, 1, 15b, 35b, 19a and 11a, and one (2%) each of serotypes 34, 5, 3 and 23a (table 2). table 2: distribution of the identified streptococcus pneumoniae serotypes. discussion top ↑ pneumococcal carriage rate the 17% prevalence of nasopharyngeal colonisation by s. pneumoniae observed in this study was substantially lower than previous studies. the study was conducted in the months of october and november which was the rainy season. in a study conducted in kilifi, kenya, a carriage rate of 53% and 62% during the dry and rainy season was observed respectively,13 which was much higher than the findings in this study. a 36% carriage rate was observed in children attending the maternal child health clinic (mch) in eldoret, kenya, which was also much higher than the findings in this study.14 colonisation prevalence varies across different regions, depending upon climatic and geographic location.3 this fact may partially explain the differences in the carriage rates. antimicrobial susceptibility to penicillin in the present study, 9% of s. pneumoniae cases were resistant to penicillin. this is much lower than 25% resistance observed in a study amongst adult patients attending clinics in nairobi, kenya,15 but similar to the 10% resistance reported in canada.16 resistance to penicillin occurs when the target penicillin binding proteins become physically altered so that penicillin binds with reduced affinity.17 penicillin has been the drug of choice for pneumococcal infections because of its efficacy, safety and low cost; thus significant levels of resistance would have substantial implications for pneumonia treatment. chloramphenicol resistance to chloramphenicol was 2%. this correlates with findings in rural western kenya in which resistance to chloramphenicol was also found to be 2%.18 low resistance to chloramphenicol (4%) had also been found in the eldoret, kenya, study.14 chloramphenicol therefore still finds a role in controlling pneumococcal respiratory infection. resistance to chloramphenicol in the pneumococcus is mediated by the acquisition of a gene encoding chloramphenical acetyl transferase. this gene is located on a plasmid, probably derived from the staphylococcus, which has entered the pneumococcus by linearisation and inclusion on a transposable element.19 cotrimoxazole a high rate of cotrimoxazole resistance (98%) was observed amongst the pneumococcal isolates. a study on pneumococcal bacteremia conducted in rural western kenya also had observed similary high resistance of 95%. cotrimoxazole is amongst the most commonly available antibiotic in most public hospitals for treatment of acute respiratory tract infections and other bacterial infections, and the high level of resistance may reflect overuse and sometimes its misuse. also, in many developing countries, antimicrobials are purchased in single doses and taken only until the patient feels better, which may occur before the pathogen has been eliminated. erythromycin the study revealed 2 cases resistant to erythromycin. a study carried out on adult patients attending clinics in nairobi, kenya, did not find any erythromycin resistance.15 resistance to erythromycin varies worldwide with low resistant rates being observed. resistance to erythromycin can result from mutation in chromosomal genes or plasmids transferred from resistance bacteria. the low resistant rate observed in this study suggests that erythromycin can continue to be used as empiric therapy for pneumococcal infections. cefotaxime the 7% resistance to cefotaxime found in this study is higher than in a study conducted in italy.21 cefotaxime is used as an optional alternative to penicillin-resistant strains and also to those individuals who are allergic to penicillin. the resistance noted to cefotaxime in this study was low, perhaps because the drug is expensive and therefore less abused in this region. tetracycline a study carried out in kenya amongst adults with acute pneumonia had observed resistance of 32%.22 as the isolates in this study showed no resistance to tetracycline, this antimicrobial would be useful for treatment of pneumococcal infections, but as there have been reported cases of resistance elsewhere, regular monitoring of resistance to tetracycline will be necessary in thika. prevalent serotypes serotypes 1, 5, 6a, 6b, 14a, 19f and 23f identified in this study account for the majority of invasive infections in most regions and are commonly involved in invasive pneumococcal diseases.1 in a study conducted in kilifi, kenya, serotypes 19f, 6b, 23f and 6a were found to be the most prevalent, which is comparable to the findings in this study.13 serotype 19f was found in 13% of the samples. this serotype is more frequently found amongst children.24 the serotypes 19f, 23f, 6b, 1 and 5 identified in this study are included in the ten-valent vaccine and therefore they can be prevented by vaccination. serotype 6a was the most prevalent in this study but is not included in the ten-valent vaccine currently used in kenya. this warrants evaluation of alternative vaccines that may protect against a wider range of serotypes. conclusion top ↑ antimicrobial resistance observed in this study shows that there is need to revise antimicrobial policy in this region in the treatment of invasive pneumococcal infections. surveillance programmes should be carried out continuously to monitor the susceptibility levels of s. pneumoniae strains in this region in order to curb the problem of developing resistance at an early stage. some of the serotypes identified in this study are included in the ten-valent vaccine which is currently being used in kenya. therefore use of this vaccine will help in the prevention and reduction of pneumococcal infections in thika. vaccines that protect against other common serotypes would also be beneficial. acknowledgements top ↑ the authors wish to acknowledge dr. moses nderitu, dr. jonah mwangi, dr. muiruri and irene mutuku for their support and encouragement, and all participating children, parents and paediatric ward staffs for their voluntary contribution to the study. competing interests the authors have no competing interests. authors’ contributions s.g. (jomo kenyatta university) was the principal investigator. g.r. (the aga khan university), a.m. (jomo kenyatta university) and s.k. (centre for microbiology research) were the university supervisors. references top ↑ 1. faden h, stniacvich j, brossky l, bernstein j, ogra pl. changes in nasopharyngeal flora during otitis media of childhood. pediatr infect dis.j 1990;9(9):623–626.2. sanders la, rijkers gt, kuis w et al. defective antipneumococcal polysaccharide antibody response in children with recurrent respiratory tract infections. j allergy clin immunol. 1993;91(1 pt 1):110–119. 3. bogaert d, groot r, hermans pw. streptococcus pneumoniae colonisation, the key to pneumococcal disease. lancet infect dis. 2004;4:144–154. http://dx.doi.org/10.1016/0091-6749(93)90303-w 4. nuorti p, butler j, farley m. cigarette smoking and invasive pneumococcal disease. active bacterial core surveillance team. new engl j med. 2000;342(10):681–689. http://dx.doi.org/10.1056/nejm200003093421002 5. meats e, brueggemann ab, enright mc. stability of serotypes during nasopharyngeal carriage of streptococcus pneumoniae. j clin microbiol 2003;41(1):386–392. http://dx.doi.org/10.1128/jcm.41.1.386-392.2003 6. simell, korkeila b, pursiainen h, kilpi tm, kayhty h. pneumococcal carriage and otitis media induce salivary antibodies to pneumococcal surface adhesin a, pneumolysin, and pneumococcal surface protein a in children. j infect dis. 2001;183(6):887–896. http://dx.doi.org/10.1086/319246 7. john cc. treatment failure with use of a third-generation cephalosporin for penicillin-resistant pneumococcal meningitis: case report and review. clin infect dis. 1994;18(2):188–193. 8. barry a. fuchs p, brown s. in vitro activities of five fluoroquinolone compounds against strains of streptococcus pneumoniae with resistance to other antimicrobial agents. antimicrob agents chemother. 1996;40(10):2431–2433. 9. schrag s, beall j, dowell b. limiting the spread of resistant pneumococci: biological and epidemiologic evidence for the effectiveness of alternative interventions. clin microbiol rev. 2004;13(4):588–601. http://dx.doi.org/10.1128/cmr.13.4.588-601.2000 10. appelbaum pc. antimicrobial resistance in streptococcus pneumoniae: an overview. clin infect dis. 1992;15(1):77–83. 11. hausdorff wp, bryant j, paradiso pr, siber gr. which pneumococcal serogroups cause the most invasive disease: implications for conjugate vaccine formulation and use, part i. clin infect dis. 2000;30(1):100–121. 12. fisher a, laing j, stoeckel j, townsend j. handbook of family planning operations research design. new york: population council; 1998. 13. abdullahi o, lewa p, nyiro j, scott jag. the descriptive epidemiology of streptococcus pneumoniae and haemophilus influenzae nasopharyngeal carriage in children and adults in kilifi district, kenya. pediatr infect dis j. 2008;27(1):59–64. http://dx.doi.org/10.1097/inf.0b013e31814da70c 14. nyandiko w, greenberg d, shany e, yiannoutsos ct, musick b, mwangi aw. nasopharyngeal streptococcus pneumoniae among under-five year old children at the moi teaching and referral hospital, eldoret, kenya. east afr. med. j. 2007;84(4):156–162. 15. paul, bates j, kimari j, gilks c. serotypes and antibiotic susceptibilities of in nairobi, kenya. jinfect. 1996;32(2):139–142. http://dx.doi.org/10.1016/s0163-4453(96)91374-2 16. zhanel g, karlowsky j, palatnick l. prevalence of antimicrobial resistance in respiratory tract isolates of streptococcus pneumoniae: results of a canadian national surveillance study. the canadian respiratory infection study group. antimicrob agents chemother. 1999;43(10):2504–2509. 17. klugman kp. pneumococcal resistance to antibiotics. clinmicrobiolrev. 1990;3(2):171–196. 18. daniel r, geoffrey j, barrack a, et al. high rate of pneumococcal bacteremia in a prospective cohort of older children and adults in an area of high hiv prevalence in rural western kenya. jinfect dis. 2010;10:1471–2334. 19. widdowson, c, adrian p, klugman p. acquisition of chloramphenicol resistance by the linearization and integration of the entire staphylococcal plasmid pc194 into the chromosome of streptococcus pneumoniae. antimicrob agents chemother. 2000:44(2):393–395. http://dx.doi.org/10.1128/aac.44.2.393-395.2000 20. hamel mj, greene c, chiller t, ouma p, polyak c, otieno k, et al. does cotrimoxazole prophylaxis for the prevention of hiv-associated opportunistic infections select for resistant pathogens in kenyan adults? am j trop med. hyg. 2008;79(3):320–330. 21. marchese a, tonoli e, balistreri g, debbia e, schito g. antibiotic susceptibility patterns and serotypes of antibiotic resistant and/or invasive streptococcus pneumoniae strains circulating in italy. microb drug resist. 2000;6(2)163–170. 22. scott ja, hall aj, hannington a et al. aetiology, outcome, and risk factors for mortality among adults with acute pneumonia in kenya. lancet. 2000;355(9211):1225–1230. http://dx.doi.org/10.1016/s0140-6736(00)02089-4 23. bakir m, yagci a, akbenlioglu c, ilki a, ulger n, soyletir g. epidemiology of streptococcus pneumoniae pharyngeal carriage among healthy turkish infants and children. eur j pediatr. 2002;161(3):165–166. 24. brandileone mc, vieira vs, casagrande st, zanella rc, guerra ml, bokermann s. prevalence of serotypes and antimicrobial resistance of streptococcus pneumoniae strains isolated from brazilian children with invasive infections. pneumococcal study group in brazil for the sireva project. regional system for vaccines in latin america. microbdrugresist. 1997;3(2):141–146. article information authors: thomas gachuki1 risper sewe1 jane mwangi2 david turgeon3 mary garcia4 elizabeth t. luman3 mamo umuro1 affiliations: 1kenya ministry of health, national hiv reference laboratory, kenya2division of global hiv/aids, us centers for disease control and prevention, nairobi, kenya 3division of global hiv/aids, us centers for disease control and prevention, atlanta, georgia, united states 4clinical pathology laboratories, austin, texas, united states correspondence to: thomas gachuki postal address: kenyatta national hospital grounds off ngong road, po box 20750 00202, nairobi, kenya dates: received: 24 july 2014 accepted: 10 sept. 2014 published: 03 nov. 2014 how to cite this article: gachuki t, sewe r, mwangi j, et al. attaining iso 15189 accreditation through slmta: a journey by kenya’s national hiv reference laboratory. afr j lab med. 2014;3(2), art. #216, 9 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.216 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. attaining iso 15189 accreditation through slmta: a journey by kenya’s national hiv reference laboratory in this lessons from the field... open access • abstract • introduction • research method and design    • study site    • slmta process and evaluation    • team formation    • improvement projects    • training    • mentorship • results    • audit scores and accreditation    • improvement projects    • quality indicators and costs • discussion    • conclusion • acknowledgements    • competing interests    • authors’ contributions    • attribution of support    • disclaimer • references abstract top ↑ background: the national hiv reference laboratory (nhrl) serves as kenya’s referral hiv laboratory, offering specialised testing and external quality assessment, as well as operating the national hiv serology proficiency scheme. in 2010, the kenya ministry of health established a goal for nhrl to achieve international accreditation.objectives: this study chronicles the journey that nhrl took in pursuit of accreditation, along with the challenges and lessons learned. methods: nhrl participated in the strengthening laboratory management toward accreditation (slmta) programme from 2010–2011. improvement projects were undertaken to address gaps in the 12 quality system essentials through development of work plans, team formation, training and mentorship of personnel. audits were conducted and the scores used to track progress along a five-star grading scale. standard quality indicators (turn-around time, specimen rejection rates and service interruptions) were measured. costs of improvement projects and accreditation were estimated based on expenditures. results: nhrl scored 45% (zero stars) at baseline in march 2010 and 95% (five stars) after programme completion in october 2011; in 2013 it became the first public health laboratory in kenya to attain iso 15189 accreditation. from 2010–2013, turn-around times decreased by 50% – 95%, specimen rejections decreased by 93% and service interruptions dropped from 15 to zero days. laboratory expenditures associated with achieving accreditation were approximately us $36 500. conclusion: international accreditation is achievable through slmta, even for a laboratory with limited initial quality management systems. key success factors were dedication to a shared goal, leadership commitment, team formation and effective mentorship. countries wishing to achieve accreditation must ensure adequate funding and support. introduction top ↑ the burden of hiv in kenya is high, with 1.6 million people living with the infection as of december 2011, including 621 813 patients who had been placed on antiretroviral therapy (art) by 2010. in order to support diagnostic testing and laboratory monitoring of hiv patients, there is a high demand for quality laboratory services, as 5.7 million hiv tests were performed in 2012 alone.1gershy-damet et al. pointed out that high-quality laboratory testing is critical for patient care, disease prevention and surveillance.2 laboratory test results play a crucial role in medical decision making; and accurate and reliable diagnostic testing and monitoring are critical to the successful management of hiv. in order to ensure the reliability and accuracy of testing, a quality system that addresses all aspects of testing is essential. however, establishing and maintaining high-quality testing standards presents major challenges in resource-poor settings.3 key amongst these challenges is lack of adherence to international standards as a result of inadequate quality management systems (qms)4 that focus on achieving quality testing services. in addition, because most hiv diagnostic testing is done by non-laboratory staff, reference laboratories play a critical role in monitoring field testing.2,5 to ensure quality results at every level, the world health organization (who) recommends that national reference laboratories seek accreditation to international standards.6 in 2003, the kenya ministry of health established the national hiv reference laboratory (nhrl), a public health facility designed to monitor the quality of hiv testing by providing a serology proficiency scheme, conducting external quality assessment (eqa) testing and acting as the centre of excellence in the laboratory monitoring of hiv patients. initially, the nhrl did not have qms in place and was not benchmarking itself against international standards. the quality of analytical testing and services was not validated, limiting its ability and authority to act as a centre of excellence. in 2010, the nhrl adopted the ministry of health’s goal to accredit all national and regional level public laboratories in kenya to the international organization for standardization (iso) 15189 standard, which is specifically designed to encourage medical laboratories to develop a highly disciplined approach to improving the quality of services.7 iso 15189 assesses the competence of the qms within the laboratory,8 provides a framework for increased analytical quality9 and verifies that laboratories are not deviating from quality and competency standards.10 the accreditation journey at the nhrl began in 2009 when laboratory management invited a consultant from a global healthcare public foundation (aghpf) to review the current laboratory qms and provide advice on needed improvements. the findings of this review stirred the management to seek assistance in the development and implementation of a more robust qms. in 2010, nhrl adopted the strengthening laboratory management toward accreditation (slmta) programme and enrolled in kenya’s first cohort along with 12 other laboratories, with the goal of attaining iso 15189 accreditation. this paper chronicles the journey that the nhrl took in the pursuit of international accreditation, along with the challenges and lessons learnt. we show how management commitment, team formation, culture change and mentorship were instrumental in the successful completion of this journey. research method and design top ↑ study site the nhrl is located in the capital city of nairobi and consists of three main sections: serology, molecular, and art monitoring. in addition, there are two cross-cutting sections: logistics, and monitoring and evaluation. each section is managed by a team lead.in its role as an hiv referral laboratory and centre of excellence, the nhrl is responsible for strengthening laboratory systems for hiv diagnosis, care, treatment and surveillance. it provides leadership and support to the national hiv laboratory programme by formulating policy and guidelines on hiv laboratory-related issues and coordinating activities and partners. the nhrl offers reference services in hiv testing and laboratory art monitoring, including hiv viral load testing, early infant diagnosis, cd4 lymphocyte enumeration and the evaluation and monitoring of the quality of hiv testing reagents and equipment. it also provides and coordinates eqa services in hiv testing by running the national hiv serology quality assurance program for over 7000 laboratory and non-laboratory testing personnel. additionally, the nhrl is responsible for eqa programmes in cd4 lymphocyte enumeration, haematology and chemistry. the nhrl also provides support and mentoring to hiv testing and art monitoring personnel, as well as building in-country capacity to design, implement and evaluate hiv-related surveillance systems and surveys. slmta process and evaluation the slmta programme uses the stepwise laboratory quality improvement process towards accreditation (slipta) checklist in order to identify strengths and weaknesses and to measure progress. the slipta checklist provides an evaluation score based on laboratory quality in the 12 quality system essentials (qses). laboratories are assigned a ‘star’ level based on their scores: zero stars (0% – 54%), one star (55% – 64%), two stars (65% – 74%), three stars (75% – 84%), four stars (85% – 94%), and five stars (≥ 95%). laboratories that score five stars are encouraged to pursue iso 15189 accreditation.11a baseline audit was conducted in march 2010 by slmta in-country trainers using the slipta checklist. this was followed by the first slmta workshop in april 2010, then the second workshop in september of the same year and the third workshop in january 2011. an exit audit was conducted by auditors from the kenya accreditation service (kenas) in october 2011. in february 2012, a consultant from the south africa national accreditation service (sanas) performed a pre-accreditation assessment utilising the sanas 15189 checklist in order to determine readiness for accreditation. several quality indicators were monitored weekly, monthly or annually so as to assess the impact of the slmta programme on laboratory service quality and patient care. specimen turnaround times for viral load, enzyme-linked immunosorbent assay (elisa) and cd4 tests were calculated using data from the laboratory information management system (lis). information on service interruptions because of equipment downtime and stockouts was obtained from the lis monthly and averaged over a calendar year. customer satisfaction was estimated from patient feedback forms that were availed either in laboratory reception areas or by mailing to customers. specimen rejections were tallied from the lis. corrective actions and occurrence management were evaluated based on completed corrective action forms and quarterly reports. these were divided into three phases: pre-analytic, analytic and post-analytic. routine results from eqa panel tests for all analytes were collated and performance evaluated using microsoft® excel 2007, by aggregating the score achieved in every eqa challenge and obtaining a percentage score. a score of 100% was desirable, whilst any score below this would call for corrective action. costs of programme implementation were estimated in us dollars based on expenditures made by the laboratory on quality improvement. these costs include fees paid to the accrediting body, kenas, and the cost of various improvements such as access control, safety equipment, equipment service contracts, iso training, eqa enrolment, storage area renovation and electronic temperature-monitoring system. for this analysis, in-kind contributions such as mentorship provided by the us centers for disease control and prevention (cdc) and costs borne by the ministry of health, such as slmta training, were not included. the opportunity cost of staff time to participate in training, complete the improvement projects and prepare for accreditation was also not included. team formation to implement the qms, a strategic, tiered, accreditation team structure with a clear reporting mechanism was formed. the structure included a management team, a quality assurance (qa) team and section teams.the management team was composed of the laboratory manager, deputy laboratory manager/qa manager, section team leads, the safety officer and the logistician. this core group guided the accreditation process and held regular review meetings in order to track progress and monitor the quality indicators adopted by the laboratory. they also reviewed gaps identified in both internal and external audits and formulated plans for continuous quality improvement. the qa team, reporting to the management team, was chaired by the qa manager/deputy laboratory manager and two qa officers (one also serving as the safety officer). this team was responsible for monitoring the accreditation process, offering leadership and coordinating the implementation of various improvement projects. each member of this team was assigned to a section and mentored by the qa manager. section team leads were given authority to make decisions and were ultimately responsible for improvement projects within their section. the section teams held weekly meetings to discuss problems and possible solutions and to track the progress of improvement projects within their section. section team leads reported to the qa team on all critical issues pertaining to qms implementation. annual staff retreats were held at the beginning of each year, during which work plans were developed with clear timelines and action points that incorporated all 12 qses and were based on iso 15189 requirements. team building also took place during the annual staff retreats. these plans were posted on bulletin boards where they were visible to all staff. regular monthly staff meetings were held in order to review work plans and monitor progress of the quality improvement initiative. after every internal and external audit, work plans were modified so as to reflect progress made and to redirect efforts where needed. individual staff members set annual accreditation goals and targets against which they were appraised for their annual staff performance contracts. an employee recognition scheme was put in place and incentives were provided. laboratory management led the way by prioritising accreditation and making sure that all personnel were keenly aware of the accreditation goal; accreditation was the main agenda item in all meetings and took priority in budget considerations, ensuring that resources required for the process were secured. improvement projects improvement projects were undertaken for all 12 qses in order to address the gaps identified in the audits. each member of the nhrl staff was responsible for at least one project with clear timelines. the findings of routine audits were used to make continual improvements within the qms. the laboratory undertook more improvement projects (table 1) than required by kenya’s slmta team, including changes to the design of the laboratory and development of workflow diagrams. the plan–do–check–act cycle was adopted in implementation of the quality improvement projects.12 most importantly, method validation was performed in order to assess the methods and equipment utilised in the laboratory.13 table 1: gaps identified and corresponding improvement projects conducted for slmta implementation at kenya’s national hiv reference laboratory, 2010–2013. all staff members were actively involved in the quality improvement projects. work plans were developed at the beginning of each year and after every audit. the work plans involved establishing a strategic goal and objective, with responsibility and project timeline assigned. work plans were reviewed regularly in staff meetings and were located centrally in the laboratory for easy reference. the work plans served as valuable tools for setting realistic targets, measuring progress and enforcing individual responsibility, leading to a focused implementation of improvement projects. flow diagrams were developed to assist in identifying weak areas and making necessary improvements. training all laboratory personnel were trained on the iso 15189 standard by management sciences for health and on good laboratory practice by the kenya aids vaccine initiative. staff also underwent 14 days of mentorship training in three of kenya’s internationally-accredited research laboratories: the kenya medical research institute (kemri)/cdc hiv research laboratory in kisumu, the us army walter reed laboratory in kericho and the academic model for treatment laboratory in eldoret. the qa manager also attended internal audit training conducted by sanas. mentorship two mentors from cdc’s international laboratory branch, division of global hiv/aids in atlanta spent a total of eight weeks in the nhrl during the slmta process. an initial three-week visit was made in january 2011 following the second slmta workshop. to make effective use of the mentors’ time on site, a brief report was prepared by the nhrl in advance of the first visit and shared with the mentors, including information on test methods and equipment used in the nhrl. at the beginning of the visit an internal audit was performed and a work plan developed based on the findings, in collaboration with the qa team and individual members of the various laboratory sections. at the end of the visit another audit was performed and the entire team participated in development of another work plan for outstanding issues.long-distance support then followed via email for a six-month period. an additional two-week visit was made by one of the mentors, who is also a member, inspector and team lead for the college of american pathologists. a final three-week visit was made by both mentors in january 2012, three months after the exit audit, in order to prepare the laboratory for the iso accreditation pre-assessment. results top ↑ audit scores and accreditation at the baseline audit in march 2010 before slmta implementation, nhrl scored 45%, corresponding to zero stars. at the october 2011 exit audit, the laboratory more than doubled their score to 95%, earning five stars. in march 2013, three years after initiation of slmta, the nhrl achieved accreditation to iso 15189. improvement projects gaps were identified in all 12 qses after the baseline audit. improvement projects were undertaken to address these problems (table 1). some projects were one-time activities, such as development of policies and procurement; for example a policy on environmental control was developed and room thermometers were procured. other projects implemented more comprehensive on-going changes to laboratory procedures, such as quarterly analysis of occurrence management and keeping minutes at staff meetings. all the improvement projects that were undertaken were completed by the time the laboratory attained accreditation. quality indicators and costs average turn-around time for viral load testing decreased from 20 days in 2010 to six days in 2013 (70%). similarly, elisa turn-around time decreased from 191 days to 10 days (95%). cd4 turn-around time decreased from 24 hours to 12 hours (50%). the number of rejected specimens decreased from 133 in 2010 to nine in 2013 (93%) and the number of service interruption days decreased from 15 to zero (100%) (table 2). table 2: trends in quality indicators, kenya’s national hiv reference laboratory, 2010–2013. the cost to the laboratory to conduct slmta improvement projects and to continue through to iso 15189 accreditation was us$36 500 (table 3). table 3: expenditures by kenya’s national hiv reference laboratory to achieve iso 15189 accreditation discussion top ↑ the nhrl was successful in achieving accreditation to iso 15189 in march 2013, three years after beginning the quality improvement process. high-quality laboratory testing is critical for patient care, disease prevention and disease surveillance.5 although the majority of laboratory testing is done by public laboratories, no laboratory in the public sector had been accredited previously in kenya, as all eight accredited laboratories were private or research laboratories. in fact, in all of sub-saharan africa except south africa, only two public laboratories had been accredited previously to international standards: one in namibia and one in botswana.14the success of nhrl was a result of several factors. firstly, the team was built with a shared vision, all striving to meet iso 15189 requirements. collective involvement has been shown elsewhere to be important in implementing change.15,16 the slmta trainees shared their projects with all staff, who then took up responsibility; this helped to prevent the mentality that quality improvement was ‘someone else’s job’ and ensured shared ownership of the process. in the weekly section meetings, brainstorming led to development of local solutions and sharing of best practices, ensuring there was no slackening of momentum. these meetings also enhanced the cohesiveness of the entire nhrl staff team. secondly, the old adage is true: what gets measured, gets done. slipta scores and star levels provided a framework for identifying strengths and weaknesses and quantifying progress. the baseline audit offered an objective analysis of processes in the laboratory, revealed critical gaps in the system and guided the team in initiating a gradual process of preparedness for accreditation. the exit audit documented how far the laboratory had come, giving leadership and staff the motivation to continue improving and the confidence to seek international accreditation. thirdly, the slmta programme provided nhrl staff the training needed to make qms improvements quickly and to prepare for accreditation. the laboratory used slmta improvement projects as a springboard to implement additional projects with a wider scope in order to cover all aspects of the qms. changes to the design of the laboratory and workflow diagrams allowed efficient and logical flow of work processes. improvement in testing turn-around time was achieved by preventing service disruptions, ensuring uninterrupted reagent supply, establishing equipment service contracts and creating a back-up programme. fourthly, mentorship was key in helping the laboratory customise solutions. effective mentorship has been shown to be a success factor in the implementation of slmta in various settings.16,17 the two cdc mentors not only spent periods of time in the facility but also offered guidance and assistance remotely. the intense preparation conducted by laboratory staff before the visits enhanced focus and sustainability when the mentors left. the mentors did not perform tasks, but instead guided laboratory staff to do them, fostering ownership and building capacity. contact was maintained with mentors after they left, ensuring continuity. the mentees identified high-priority areas in which they required assistance, saving time onsite. a positive staff attitude facilitated the productive relationships with mentors; no time was wasted in finding common ground because all shared the same goal of nhrl accreditation. finally, continued focus on accreditation after slmta allowed the laboratory to reach even higher levels. the pre-accreditation assessment conducted by the sanas assessor offered an objective in-depth analysis using a different checklist and gave laboratory staff an idea of what to expect in the accreditation visit. findings from this assessment were used to address remaining gaps prior to the official inspection. nhrl faced many critical challenges in implementing qms, as summarised in table 4. one serious problem that remains unsolved is staff attrition. because the government handles staff deployment, trained staff members are often transferred to other laboratories. nhrl is working with the ministry of health to prioritise continuity of staff and training for new staff members in order to sustain quality levels. the nhrl spent approximately us$36 500 in pursuit of iso 15189 accreditation, in addition to that spent by the ministry of health on slmta training and by partners for mentorship and additional training. one of the largest expenses was the placement of equipment on service contracts. to reduce costs, the laboratory adopted the equipment placement model, whereby an equipment manufacturer places equipment in a laboratory at no cost, recovering their expenses by selling reagents to the laboratory. other substantial expenses included the renovation of a storage room to overcome space shortages and installation of a temperature-monitoring system in order to improve the archiving of specimens. the largest single expense was the purchase of a back-up generator; this purchase also benefited other users within the national public health laboratories complex. many key components of the programme were paid for by various partners and were thus not included in the cost estimate. for example, iso training was sponsored by management sciences for health and included staff from other laboratories. personnel were immunised by the division of vaccination in the ministry of health. finally, the aghpf consultant and cdc mentors, critical for readying the laboratory for accreditation, were sponsored by their respective organisations. cost considerations must be weighed against the benefits of quality improvement. some improvements will result in large cost savings over time. human resource management has been made easier as staff competencies are assessed annually; personnel are now more efficiently assigned to specific responsibilities based on their core competencies. the process of hiv results confirmation, which used to take more than one month, now takes less than 10 days, ensuring rapid resolution nationwide for clients with discrepant hiv results. hiv viral load results are now received in less than 10 days; this information is critical with regard to alerting clinicians to the need to change treatment regimens for patients with treatment failure, thus reducing their likelihood of developing drug resistance. services are no longer interrupted because of reagent shortages or equipment downtime and adherence to sample handling guidelines has greatly reduced rejected samples, decreasing both costs and wastage.18 accreditation also provides immeasurable benefits in enabling the nhrl to fulfil its mission as the country’s reference laboratory for hiv testing. it has accorded the nhrl international recognition and elevated customer confidence with respect to the reliability of services as they fulfil their mandate. pursuit of accreditation has led to significant improvement in the quality of both analytical test results and customer service. because of the central role the laboratory plays in kenya, these benefits have a direct impact on the quality of hiv testing and monitoring throughout the country. conclusion the experience of kenya’s nhrl shows that it is feasible to attain international accreditation through the implementation of the slmta programme, even in settings with poor resources and laboratories without initial systems. acknowledgements top ↑ the authors wish to thank the staff and management of the nhrl, kenya for their efforts leading to the first accreditation of a public laboratory in kenya. we would also like to thank the cdc-kenya and cdc-atlanta offices for their support and technical assistance. we would also like acknowledge the assistance provided by the division of vaccine (ministry of health, kenya) and the management sciences for health. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions t.g. (nhrl) led the slmta programme and accreditation process at nhrl and wrote the manuscript. r.s. (nhrl) provided assistance with the accreditation process and reviewed the manuscript. j.m. (international laboratory branch, cdc, kenya) supported slmta implementation and reviewed the manuscript. d.t. (international laboratory branch, cdc, atlanta) supported implementation of the qms and reviewed the manuscript. m.g. (clinical pathology laboratories) supported implementation of the qms and reviewed the manuscript. e.t.l. (international laboratory branch, cdc, atlanta) assisted with analysis and interpretation of the data and substantially revised the manuscript. m.u. (nhrl) led implementation of the qms and reviewed the manuscript. attribution of support this publication was made possible by support from the u.s. president's emergency plan for aids relief (pepfar) through cooperative agreement 5u19 gh000069-02 from cdc, division of global hiv/aids. disclaimer the findings and conclusions in this article are those of the author(s) and do not necessarily represent the official position of the cdc or the government of kenya. references top ↑ 1.national aids control council. the kenya aids epidemic: update 2012 [document on the internet]. c2012 [cited 2014 sep 15]. available from: http://nascop.or.ke/library/3d/final%20kenya%20update%202012,%2030%20may.pdf2.gershy-damet gm, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. http://dx.doi.org/10.1309/ajcptuuc2v1wjqbm 3.petti ca, polage cr, quinn tc, et al. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006;42(3):377–382. http://dx.doi.org/10.1086/499363 4.zeh ce, inzaule sc, magero vo, et al. field experience in implementing iso 15189 in kisumu, kenya. am j clin pathol. 2010;134(3):410–418. http://dx.doi.org/10.1309/ajcpzirkdus5lk2d 5.bates i, maitland k. are laboratory services coming of age in sub-saharan africa? [editorial] clin infect dis. 2006;42(3):383–384. http://dx.doi.org/10.1086/499368 6.world health organization. joint who – cdc conference on health laboratory quality systems, lyon, france, 9 – 11 april 2008. who/hse/ihr/lyo/2008.3 [document on the internet]. c2008 [cited 2014 sep 15]. available from: http://www.who.int/csr/ihr/lyon/report20080409.pdf?ua=1 7.international organization for standardization. iso 15189:2007: medical laboratories – particular requirements for quality and competence. 2nd ed. geneva, switzerland: international organization for standardization, 2007. 8.international laboratory accrediation cooperation. guidance for the implementation of a medical laboratory accreditation system. ilac-g26:07/2012 [document on the internet]. c2012 [cited 2014 sep 15]. available from: http://www.ats.rs/sites/default/files/download/ilac_g26_07_2012.pdf 9.peter tf, rotz pd, blair dh, et al. impact of laboratory accreditation on patient care and the health system. am j clin pathol. 2010;134(4):550–555. http://dx.doi.org/10.1309/ajcph1skq1hnwghf 10.rabinovitch a. the college of american pathologists laboratory accreditation program. accred qual assur. 2002;7:473–476. http://dx.doi.org/10.1007/s00769-002-0537-0 11.world health organization regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation in the african region (with checklist) [document on the internet]. c2011 [cited 2013 may 31]. available from: http://www.afro.who.int/index.php?option=com_docman&task=doc_download&gid=8642&itemid=2593 12.woodcock s, fine g, mcclure k, et al. the role of standards and training in preparing for accreditation. am j clin pathol. 2010;134(3):388–392. http://dx.doi.org/10.1309/ajcp03tfpbkeyynt 13.chan cc. principles and practices of analytical method validation: validation of analytical methods is time‐consuming but essential. qual assur j. 2011;14(3–4):61–64. http://dx.doi.org/10.1002/qaj.477 14.schroeder lf, amukele t. medical laboratories in sub-saharan africa that meet international quality standards. am j clin pathol. 2014;141(6):791–795. http://dx.doi.org/10.1309/ajcpq5ktkagsscfn 15.mcalearney as, terris d, hardacre j, et al. organizational coherence in health care organizations: conceptual guidance to facilitate quality improvement and organizational change. qual manag health care. 2013;22(2):86–99. http://dx.doi.org/10.1097/qmh.0b013e31828bc37d 16.audu ra, onubogu cc, nwokoye nn, et. al. improving quality in national reference laboratories: the role of slmta and mentorship. afr j lab med 2014. in press. afr j lab med. 2014;3(2), art. #200, 7 pages. http://dx.doi.org/10.4102/ajlm.v3i2.200 17.makokha ep, mwalili s, basiye f, et al. using institutional mentorship to roll out slmta in kenya. afr j lab med. 2014. in press. 2014;3(2), art, #220, 8 pages. http://dx.doi.org/10.4102/ajlm.v3i2.220 18.carlson ro, amirahmadi f, hernandez js. a primer on the cost of quality for improvement of laboratory and pathology specimen processes. am j clin pathol. 2012;138(3):347–354. http://dx.doi.org/10.1309/ajcpsmqyaf6x1hut abstract introduction methods results discussion acknowledgements references about the author(s) reena d. mohanlal department of anatomical pathology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa national health laboratory services, chris hani baragwanath laboratory, johannesburg, south africa citation mohanlal rd. endometrial sampling at an academic hospital in south africa: histological findings, lessons learnt and interesting surprises. afr j lab med. 2020;9(1), a1038. https://doi.org/10.4102/ajlm.v9i1.1038 original research endometrial sampling at an academic hospital in south africa: histological findings, lessons learnt and interesting surprises reena d. mohanlal received: 26 apr. 2019; accepted: 15 may 2020; published: 29 sept. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: outpatient sampling is used to investigate endometrial pathology. little is known about practice habits and local failure rates at chris hani baragwanath academic hospital in johannesburg, south africa. objective: this study assessed the frequency of samples that showed no or limited histological representation of endometrium, and described demographic and pathological features. methods: all endometrial sample histology reports from the national health laboratory services at the hospital from 01 july 2013 to 31 may 2017 were retrieved by searching the laboratory’s information system. clinical variables (age, menopausal state, indication for biopsy, endometrial thickness on ultrasound) and pathological findings (macroscopic amount of tissue, histological diagnosis, microscopic presence of endometrial tissue) were extracted and statistically analysed. results: a total of 1926 samples were included, 91% of which were submitted for abnormal or postmenopausal bleeding. no endometrium was observed in 25% of samples and 13% showed limited endometrium. benign diagnoses (86%) were most common, with proliferative or secretory changes, endometrial polyps and endometritis accounting for most of these. associations between the amount of sample received and the presence of endometrial tissue (p ≤ 0.001) and benign versus malignant diagnoses (p ≤ 0.001) were noted. the greater the endometrial thickness, the greater the likelihood of obtaining more sample (bulky vs scant p < 0.001) and making a malignant versus benign diagnosis (p = 0.005). conclusion: these findings are in keeping with literature outside africa. histology reports should be explicit when terms such as ‘inadequate’ or ‘insufficient’ are used, in order to facilitate clinical decision-making. keywords: endometrial sampling; inadequate endometrial biopsy; postmenopausal bleeding; pathology; histopathology; gynaecology. introduction outpatient blind endometrial sampling using a disposable, flexible aspiration device such as a z-sampler or pipelle is well tolerated by patients and is a quick and easy procedure to perform.1 in resource-limited settings, this modality for investigating suspected endometrial disease becomes especially relevant, as it eliminates the cost of a hospital stay, anaesthetic administration and post-operative complications inherent with in-theatre diagnostic dilation and curettage. most of the literature pertaining to endometrial sampling is from non-african countries. outpatient sampling compares well to other modalities and studies have shown a concordance rate of 95% between pipelle and diagnostic dilation and curettage for atypia and hyperplasia,1 agreement between office sampling and final diagnosis of 0.73, and no significant difference between hysteroscopic and office sampling.2 outpatient sampling has also been shown to avoid hysteroscopy, particularly in patients with postmenopausal bleeding and an endometrial thickness of more than 4 mm.3 there were no significant differences in the agreement rates for tumour type or grading among endometrial biopsy, curettage or hysteroscopy methods.4 however, outpatient sampling performs less well when there are focal endometrial lesions.1 the biopsy procedure may fail when insufficient tissue is obtained or there is an inability to access the endometrial cavity.5 failure rates of up to 53% have been cited in a meta-analysis.5 furthermore, the reporting of these biopsies is not standardised. there is poor agreement on what constitutes an ‘insufficient’ or ‘inadequate’ biopsy6,7 among both general and gynaecologic pathologists, as there are no standardised criteria for determining adequacy as shown in a study from the united kingdom.7 such ambiguous sign-outs or reporting may result in misinterpretation by the clinician and subsequent overtreatment of the patient.6 this study was undertaken to determine the frequency of biopsies of outpatient endometrial aspiration that showed no or limited amounts of endometrium and to describe demographic and pathological findings in a retrospective cohort of cases at a tertiary hospital in johannesburg, south africa. methods ethical considerations ethics approval for this study was obtained from the medical ethics committee of the university of the witwatersrand (m170697). study design this retrospective, descriptive cross-sectional study was conducted at the department of anatomical pathology, chris hani baragwanath hospital national health laboratory services, located on the hospital premises in soweto, south africa. endometrial biopsies are submitted to the laboratory from the gynaecology department at the hospital. a retrospective search of all endometrial biopsies reported from 01 july 2013 to 31 may 2017 was conducted using the search word ‘endometrium’ on the laboratory’s information system. a review of all histology reports identified using this search was then undertaken. all endometrial biopsy samples obtained with an aspiration device such as a z-sampler were included; while those biopsy samples obtained during hysteroscopy, dilatation and curettage and suction curettage were excluded. clinical and pathological variables were obtained from the histology reports only. no clinical records or patients’ files were accessed. age, menopausal status, endometrial thickness (in millimetres), clinical examination findings, the presence of risk factors for carcinoma (body mass index ≥ 25 kg/m2, hypertension, diabetes, tamoxifen use, nulliparity) and indications for biopsy were obtained from the clinical history section of the histopathology reports. the clinical history section on the histology report comprises information provided by the submitting clinician on the requisition slip that accompanies a specimen. pertinent laboratory findings including diagnosis, diagnostic category (benign, atypical, malignant), quantity of tissue at macroscopic assessment (scanty, moderate or bulky) and the presence or absence of endometrial tissue on microscopic examination were also recorded from the histology report. cases with superficial strips of endometrium or stroma recorded on histological examination were classified as limited (figure 1). diagnoses recorded as malignant were carcinomas and lymphomas; those recorded as atypical were hyperplasia with atypia and squamous intraepithelial lesions. all remaining cases, for example polyps, endometritis, and proliferative or secretory phase endometrium, were categorised as benign. as per laboratory standard operating procedures, the categories of tissue assigned at grossing, or macroscopic examination, were: bulky tissue (fills more than one tissue cassette), moderate (fills up to one tissue cassette) and scanty (minimal or small fragments of tissue submitted). slides of selected cases were retrieved for photography. figure 1: endometrial sample showing limited endometrial tissue comprising less than 10 endometrial strips (original magnification ×100). data analysis all data were recorded on a datasheet and entered into an excel (microsoft, redmond, washington, united states) spreadsheet for statistical analysis. where data were missing, no value was entered. there was no patient follow-up or clinical record review. statisticatm version 13.0 (dell, round rock, texas, united states) and stata version 15 (statacorp, college station, texas, united states) were used in the statistical analysis. categorical data (macroscopic tissue quantity, diagnostic category, diagnosis) were reported as frequencies. numerical data (age and endometrial thickness) were reported as medians with interquartile ranges, as these data were non-normally distributed. only endometrial thicknesses reported in postmenopausal patients were used. the chi-square and fisher’s exact tests were used to assess associations between the amount of tissue at grossing and diagnostic category, and the presence of endometrial tissue on histological examination. the kruskall–wallis test was used to assess differences in endometrial thicknesses recorded for macroscopic quantity of tissue and diagnostic category. pairwise comparison (dunn’s test) was used to assess associations between median endometrial thickness and diagnostic category, and macroscopic tissue quantity in the postmenopausal group. statistical significance was set at p < 0.05. results entire cohort a total of 1926 outpatient endometrial aspiration biopsies were received and reported in the laboratory from 01 july 2013 to 31 may 2017. the median age of the patients was 53 (interquartile range 48–62) years (table 1). most biopsies (n = 1625, 90.58%) were submitted for abnormal or postmenopausal bleeding. in 196 (10.18%) cases, the presence of risk factors for endometrial carcinoma was indicated (data not shown in table). scanty and moderate samples comprised the majority of samples received, with only 135 (6.82%) bulky specimens noted. for the entire group, 485 (25.18%) cases had no endometrium and 245 (12.72%) cases had limited amounts in the form of superficial strips of mucosa or stroma (figure 1). the majority of diagnoses were benign (n = 1725, 86.42%). of the 110 malignancies diagnosed, there were 32 cases of serous carcinoma of the endometrium, 24 endometrioid carcinomas and 13 malignant mixed müllerian tumours. cervical pathology was noted in 107 (5.56%) of the total biopsies, 70 cases of which were diagnosed with cervical squamous intraepithelial lesions, eight with squamous cell carcinoma and three with cervical adenocarcinoma. table 1: clinical characteristics and pathological findings for all endometrial samples received at chris hani baragwanath academic histopathology laboratory, south africa, 01 july 2013 – 31 may 2017. the distribution of samples with histologically determined endometrium present, absent or limited in amount and the diagnostic categories across macroscopic quantities was assessed. associations between the macroscopic quantity of tissue received and the presence of endometrial tissue (p ≤ 0.001) and diagnostic category (p ≤ 0.001) are shown in table 2. table 2: macroscopic quantities of endometrial tissue samples received at chris hani baragwanath academic hospital histopathology laboratory from 01 july 2013 to 31 may 2017 in relation to microscopically assessed endometrial tissue and diagnostic categories. cases of interest two cases of interest were noted. an endometrial sample from a 39-year-old woman with abnormal uterine bleeding revealed cytomegalovirus endometritis (figure 2). no clinical follow-up was available for this patient. a b-cell non-hodgkin lymphoma (figure 3) was noted in a 22-year-old woman with bilateral ovarian masses, hepatosplenomegaly and lymphadenopathy. in addition to endometrial involvement, previous histology also showed nodal and marrow infiltration by lymphoma. figure 2: endometrial breakdown and endometritis in an endometrial sample from a 39-year-old patient with abnormal bleeding (original magnification ×200). cytomegalovirus inclusions in endometrial epithelial cells are indicated by arrows in the inset (original magnification ×400). figure 3: diffuse large b-cell non-hodgkin lymphoma infiltrating endometrial stroma in a sample from a 22-year-old patient with lymphadenopathy and organomegaly (original magnification ×400). cd20 immunohistochemical stain showing cd20-positive b-cells stained brown (inset, original magnification ×200). actinomyces six biopsies from five patients, ranging in age from 54 to 78 years, showed infection with actinomyces bacteria (figure 4). all patients with actinomyces infection presented with abnormal bleeding, with one patient additionally complaining of per vaginal discharge. an intrauterine contraceptive device in situ was noted in only one patient and none of patients with actinomyces infection had microbiological confirmation. figure 4: actinomyces bacterial infection in an endometrial sample (original magnification ×400) from a 57-year-old female who presented with postmenopausal bleeding. the bacteria are seen as the large clumped mass of basophilic filamentous structures. tamoxifen use of the total biopsies studied, 11 were from patients on tamoxifen. three presented with postmenopausal bleeding, and the endometrial thickness reported in three women ranged from 4 mm to 18 mm (data not shown). two biopsies from patients on tamoxifen showed endometrial polyps (both patients presented with bleeding), two showed endometritis and one, atypical squamous cells (data not shown). postmenopausal subgroup a total of 994 patients (see table 1) of the entire cohort were postmenopausal, as indicated in the clinical history sections of the laboratory records. the median age in the postmenopausal group was 60 years (interquartile range 54–68). the majority of biopsies (n = 964, 98.27%), where an indication for biopsy was provided, were for postmenopausal bleeding. risk factors for malignancy were noted in 126 (12.7%) patients (data not shown). in the postmenopausal group, 267 (26.86%) had no endometrium and 152 (15.29%) had limited amounts (data not shown). malignant diagnoses accounted for 82 (8.27%) of postmenopausal cases (data not shown). other significant pathological findings included hyperplasia (n = 16), hyperplasia with atypia (n = 15), endometrial polyps (n = 115), inactive endometrium (n = 125) and endometritis (n = 71) (data not shown). endometrial thicknesses were specified in only 94 of postmenopausal patients, with an overall median of thickness of 11.0 mm (interquartile range 7–17) (data not shown). significant differences were noted in the median endometrial thicknesses recorded among the diagnostic categories and macroscopic quantities of tissue for postmenopausal women (table 3). on subsequent analysis, it was shown that the greater the endometrial thickness, the greater the likelihood of obtaining more tissue (bulky vs moderate p = 0.021 and bulky vs scanty p < 0.001) (data not shown). in addition, the thicker the endometrium measured on ultrasound, the more likely a diagnosis of malignant versus atypical (p = 0.048) and malignant versus benign (p = 0.005) (data not shown) was made. table 3: median endometrial thicknesses per diagnostic category and macroscopic quantity of tissue in postmenopausal women obtained by endometrial sample at chris hani baragwanath academic hospital, south africa, 01 july 2013 – 31 may 2017. discussion outpatient endometrial samples are regularly encountered in general pathology practice. one-quarter of samples in this study contained no endometrial tissue on histological examination. furthermore, in the postmenopausal group, endometrial thickness on ultrasound correlated with the amount of tissue obtained at sampling and the histologically confirmed diagnostic category. as per other studies from canada and turkey, abnormal bleeding was the most common presenting feature in our study.8,9 the importance of adhering to guidelines or indications for biopsy has been stressed, as endometrial sampling is not without risk. a tendency to over-investigate patients younger than 40 with abnormal bleeding has been shown.10 furthermore, sampling of the endometrium without a clear indication may contribute to higher failure rates. although larger samples show better agreement rates with final excisional diagnoses,4,11 bulky specimens formed the minority of samples submitted in our cohort. failure rates reported in the literature include cases where insufficient tissue was noted on histological examination or where the endometrium could not be accessed due to technical or patient-related factors.5 higher failure rates have been noted in postmenopausal, obese patients, those with advanced age or previous failed pipelle and when sampling was performed by a non-physician.9 the failure rate for this cohort is higher than some reported failure rates (6.33% – 18.4%)8,9,12 and lower than the overall failure rate of 42% reported in a meta-analysis where 11% of cases failed due to technical reasons and 31% had insufficient tissue.5 these figures need to be interpreted with caution, as the criteria for labelling biopsies as ‘insufficient’ may vary across institutions. in one study, complete agreement on classification as insufficient or diagnostic was shown in only 57% of cases.7 the terms ‘insufficient’ or ‘inadequate’ were proposed for cases with no endometrial tissue present and ‘unassessable’ if there was too little tissue.6 recently published canadian guidelines may facilitate standardising reporting. categories of ‘non diagnostic sample, no endometrial tissue present’ and ‘scant fragments of inactive endometrial surface epithelium and/or stroma (suboptimal for histopathological assessment)’ have been suggested.13 an endometrial surface area of at least 35 mm,2,11 or a minimum of 10 endometrial strips14 have also been proposed as cut-offs for a conclusive sample. limited endometrial tissue was noted in 245 (12.72%) of our biopsies. the histological finding of limited endometrial tissue should be correlated clinically with the endometrial thickness as this microscopic finding could be compatible with atrophy, requiring no further clinical intervention. adequacy is related to endometrial thickness. one study showed that 27% of samples were adequate among postmenopausal patients with an endometrial thickness of less than 5 mm, leading to the suggestion that sampling should not be done in this group.3 this study corroborates the findings of others in that the thicker the endometrium in postmenopausal women, the greater the likelihood of obtaining more sample and a malignant diagnosis.3,8 specificity of endometrial sampling is high, but its sensitivity is low.5 one study has shown that, following an inadequate or scant biopsy, 10% of patients had hyperplasia, 5% had malignancy on repeat sampling and 15% had malignancy on hysterectomy.7 worrisome histological findings such as atypia or necrosis on initial biopsy should be communicated as 43% of such patients in one study had malignancy on follow-up biopsy.15 the frequency of histological diagnoses noted in the entire cohort is similar to findings by inal et al.,8 with the exception that hyperplasia was diagnosed in fewer patients (2.02% vs their 9.7%). cervical pathology was noted in some of this cohort’s endometrial biopsies. histopathologists should be alert to the presence of, and pathological changes in, cervical tissue present in an endometrial sample, as this may direct clinicians to re-examine and rebiopsy the cervix. in the current study, hyperplasia or malignancy were diagnosed in 113 (11.37%) of postmenopausal women, which is in keeping with other studies.10,16 cytomegalovirus endometritis and endometrial involvement by non-hodgkin lymphoma were two interesting cases encountered. isolated case reports of cytomegalovirus endometritis from the 1990s have been noted, but the most recent report was in a postpartum uterus specimen.17 polymerase chain reaction for cytomegalovirus was negative and the diagnosis was based on immunohistochemistry.17 a caveat to bear in mind is that biotin present in postpartum endometrial epithelium interacts with the avidin-biotin-complex, resulting in false-positive staining on immunohistochemistry.18 in women, non-hodgkin lymphoma most commonly affects the ovaries, as it is postulated that the ovarian microenvironment is conducive to the growth of tumour cells. it is important to categorise lymphomas as either primary genital tract disease or secondary involvement, as this may have treatment implications.19 the prevalence of actinomycotic endometritis was 0.31% in this study, which is higher than the 0.0002% previously recorded.20 the association with prolonged use of an intrauterine contraceptive device is well known, but this history is not always forthcoming. although forming only a very small subset in this study, patients on tamoxifen with postmenopausal bleeding and with adequate endometrial tissue most commonly had endometrial polyps on biopsy, which is similar to another study.21 limitations this study included a review of laboratory-record histology reports from a large number of endometrial biopsies. clinical information submitted in some instances was minimal, with no information about the menopausal status or endometrial thickness. the postmenopausal group was determined using clinical history only and all cases with no comment on menopausal state were excluded from this subgroup. a large number of cases could therefore have been left out, but assumptions about the menopausal state based purely on age could not be justified. furthermore, there was no clinical record review to assess outcomes and final diagnosis for cases where there was no endometrium or limited endometrium. as this hospital is a tertiary public hospital in a metropolitan area, the cases are biased and therefore may not be a true reflection of the south african community. further areas of study could include a follow-up of cases with no or limited endometrium and to assess local reasons for the failure rates in this study. conclusion this study provides information about the indications, diagnoses and representation of endometrium in endometrial samples seen at a large tertiary hospital in soweto. there should be an awareness among pathologists and clinicians regarding terms such as ‘insufficient’, ‘inadequate’, ‘scant’ and ‘unassessable’ in an endometrial sample pathology report. if these terms are used, they should be accompanied by a clear description of the type and amount of tissue that is present as there is no consensus among pathologists regarding adequacy criteria.6,7 in the appropriate setting, repeat biopsy can be suggested by the pathologist when no or little endometrial tissue is present as there is a possibility of finding hyperplasia or malignancy on subsequent biopsy.7 ultimately, management following an ‘insufficient’ tissue biopsy will depend on clinical factors such as the persistence of bleeding and ultrasound findings.7,12,13 endometrial sampling is a useful investigation if done under the appropriate clinical circumstances, submitted with adequate history and reported mindfully by the histopathologist. acknowledgements the author wishes to thank dr innocent maposa (faculty of health sciences research office) for biostatistical support, mr eric liebenberg for assistance with photography, mr deon mahlathi for slide retrieval, and colleagues and staff at chris hani baragwanath academic hospital histopathology laboratory. competing interests the author declares that she has no financial or personal relationships that may have inappropriately influenced her in writing this article. author’s contributions i declare that i am the sole author of this research article. sources of support this research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. data availability statement data is available upon request from the author. disclaimer the views expressed in the submitted article are the author’s own and not an official position of the institution or funder. references demirkiran f, yavuz e, erenel h, bese t, arvas m, sanioglu c. which is the best technique for endometrial sampling? aspiration (pipelle) versus dilatation and curettage (d&c). arch gynecol obstet. 2012;286(5):1277–1282. https://doi.org/10.1007/s00404-012-2438-8 visser n, reijnen c, massuger l, nagtegaal i, bulten j, pijnenborg j. accuracy of endometrial sampling in endometrial carcinoma: a systemic review and meta-analysis. obstet gynecol. 2017;130(4):803–813. https://doi.org/10.1097/aog.0000000000002261 elsandabesee d, greenwood p. the performance of 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taking tamoxifen. j menopausal med. 2018;24(2):81–86. https://doi.org/10.6118/jmm.2018.24.2.81 article information authors: rosemary a. audu1 catherine c. onubogu2 nkiru n. nwokoye2 eke ofuche3 shirematee baboolal4 odafen oke5 elizabeth t. luman6 emmanuel o. idigbe2 affiliations: 1human virology laboratory, nigerian institute of medical research, nigeria 2national tuberculosis reference laboratory, nigerian institute of medical research, nigeria 3 aids prevention initiative in nigeria, nigeria 4american society for microbiology, united states 5us centers for disease control and prevention, nigeria 6us centers for disease control and prevention, united states correspondence to: rosemary audu postal address: nigerian institute of medical research, 6 edmond crescent, yaba, nigeria dates: received: 21 may 2014 accepted: 08 july 2014 published: 08 sept. 2014 republished: 07 nov. 2014 how to cite this article: audu ra, onubogu cc, nwokoye nn, et al. improving quality in national reference laboratories: the role of slmta and mentorship. afr j lab med. 2014;3(2), art. #200, 7 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.200 note: article republished with updated references relating the special issue. copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. improving quality in national reference laboratories: the role of slmta and mentorship in this original research... open access • abstarct • introduction • research method and design    • implementation sites    • slmta implementation    • evaluation of laboratory performance    • quality improvement projects    • mentorship and additional support • results    • overall performance    • performance of quality system essentials    • quality improvement projects    • effect of changes to the audit checklist • discussion    • limitations    • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstarct top ↑ background: the nigerian institute of medical research houses two reference laboratories: the virology and tuberculosis laboratories. both were enrolled in the strengthening laboratory management toward accreditation (slmta) programme. objective: to describe the impact of slmta and discuss factors affecting the results, with an emphasis on mentorship. methods: the slmta programme was implemented from april 2010 through november 2012. participants attended three workshops and executed quality improvement projects; laboratory auditors evaluated performance using a standard checklist. the virology laboratory did not receive mentorship; however, the tuberculosis laboratory had an international mentor who visited the laboratory four times during the programme, spending two to four weeks embedded within the laboratory during each visit. results: there was an overall improvement in the performance of both laboratories, with the virology laboratory increasing 13% (from 80% at baseline to 93% at exit audit) and the tuberculosis laboratory increasing 29% (from 66% to 95%). these scores were maintained nine months later at the surveillance audit. conclusion: the slmta programme resulted in improved and sustained quality management performance for both laboratories. mentoring was a possible factor in the substantial improvement made by the tuberculosis laboratory and should be considered in order to augment the training received from the slmta workshops. introduction top ↑ the level of implementation of laboratory standards in the african region, verified through the process of accreditation, has historically been very low.1 until recently, most laboratories in africa have not emphasised quality management systems (qms) in the provision of healthcare services. in addition, lack of staff training and education, poor physical infrastructure, climate extremes and financial constraints2 have limited implementation of laboratory quality systems. the absence of national laboratory strategic plans to provide roadmaps for the implementation of quality laboratory services, as well as the lack of national laboratory quality standards to guide the provision of quality clinical laboratory services and accreditation in nigeria, are also obstacles for the implementation of quality laboratory systems. an earlier study has also reported that the culture of qms is uncommon in nigerian laboratories;3 as such, there is a need to build this culture. to strengthen the laboratory systems of african countries in a systematic approach, the us centers for disease control and prevention (cdc), in collaboration with the american society for clinical pathology, the clinton health access initiative and the world health organization’s regional office for africa (who afro), launched the strengthening laboratory management toward accreditation (slmta) training programme in 2009.4 this programme, which focuses on strengthening laboratory management to achieve immediate laboratory improvement and accelerate accreditation preparedness, has been implemented in 47 countries worldwide and is expanding rapidly.5 the programme includes workshops, improvement projects, site visits and, in some cases, mentoring.6 nigeria embraced the slmta programme in 2009 and, by 2010, seven laboratory experts were trained to roll out the slmta programme in 24 of the 344 nigerian laboratories supported by the us president’s emergency plan for aids relief (pepfar). the nigerian institute of medical research (nimr) has two reference laboratories, namely, the human virology laboratory (virology laboratory) and the national tuberculosis reference laboratory (tb laboratory), under its mandate ‘to conduct basic, applied and operational research for the prevention and control of communicable and non-communicable diseases of public health importance in the country in collaboration with the federal and state ministries of health and other stakeholders’.27 both laboratories were amongst the 24 enrolled in the slmta programme. the two laboratories are similar in their institutional management and were supported by the same pepfar implementation partner. however, the tb laboratory was assigned a mentor to assist with slmta implementation, whilst the virology laboratory was not. we describe the impact of the slmta programme and discuss potential factors affecting the results, with an emphasis on mentorship. research method and design top ↑ implementation sites the virology laboratory has provided laboratory services to the nigerian hiv treatment programme since 2002 and similar services for the pepfar hiv treatment project which commenced in 2004. the laboratory has the following sections: serology, immunology, chemistry, haematology and molecular diagnostics, which includes resistance testing for hiv. a total of 81 758 tests were performed by the virology laboratory in 2010. the laboratory staff comprised 14 degree-holding professionals, one diploma-holding professional, one certificate-holding professional, two data clerks, two phlebotomists, two cleaners, one driver and eight other administrative staff. this laboratory had some previous experience implementing qms and, in 2008, had received international organization for standardization (iso) 9001 accreditation,3 a general organisational certification of management processes. in preparing medical laboratories for international accreditation, the slmta programme employs iso 15189, which specifies standards for qms particular to medical laboratories. to help it meet these standards, which are more relevant to clinical laboratories, the virology laboratory enrolled in the slmta programme. the tb laboratory was established to meet the institute’s research mandate. from 2005, the scope of services increased with inclusion of a ‘directly observed treatment short-course’ (dots) diagnostic centre, which brought about the expansion and renovation of the laboratory in order to meet the tb diagnostic service needs of both the private and public sectors. with the dots centre, many more tb suspects were referred to the laboratory for diagnosis, treatment and follow-up, and the diagnostic workload increased dramatically. the laboratory was commissioned as a national tb reference laboratory in 2007 and offers the following services: smear microscopy for acid-fast bacilli; solid and liquid culture; identification of mycobacterium tuberculosis complex and mycobacteria other than tuberculosis (motts); and drug susceptibility testing using solid, liquid and molecular-based techniques. the laboratory is also involved in national tb drug resistance surveillance. a total of 80 799 tests were conducted in 2010. during the course of the slmta programme, the tb laboratory included 14 degree-holding staff, four diploma-holding staff, three microscopists, one administrative staff, one data clerk and one cleaner. each laboratory also had a director, laboratory manager, quality assurance officer and dedicated personnel who had consistent job responsibilities throughout the duration of the programme. slmta implementation the slmta programme was implemented in the nimr virology and tb laboratories over two years and seven months (figure 1). three workshops were conducted within this period, with an average eight-month interval between them. the laboratory managers and quality assurance officers from both laboratories attended the workshops, after which they trained the other laboratory staff. figure 1: slmta implementation timeline in nigerian reference laboratories. evaluation of laboratory performance a baseline audit was conducted in each laboratory in april 2010, six months before the first slmta workshop (figure 1). intermediate audits were conducted after each workshop in order to monitor progress and to help identify any remaining gaps. an exit audit was conducted in november 2012, four months after the third workshop and a surveillance audit was conducted in august 2013, nine months after the exit audit, in order to determine the ability of the laboratories to sustain the quality advances made. the baseline and first intermediate audits were conducted using the laboratory accreditation preparedness checklist developed in 2009 by who afro.8 the checklist had a total score of 250 points distributed into 12 sections corresponding to the 12 quality system essentials. in 2012, who afro revised the checklist by adding more details in the requirements for documents and records and management review, as well as modifying the sectional scores, with a new total of 258 points.9 this revised checklist was used in parallel with the older version for the second intermediate and surveillance audits to allow us to evaluate the effect of the revision; results presented are from the revised checklist. for the exit audit, only the revised checklist was used. based on the audit scores, laboratories were assigned a zeroto five-star rating, whereby < 55% = zero stars, 55% − 64% = one star, 65% − 74% = two stars, 75% − 84% = three stars, 85% − 94% = four stars and 95% − 100% = five stars. independent laboratory experts who had taken the slmta training-of-trainers course, which included one day of training on auditing, were engaged as auditors; the same team conducted the audits in both laboratories, although different auditors were scheduled for each round of audit. quality improvement projects quality improvement projects were selected by each laboratory after the first and second workshops based on laboratories’ specific needs within topics addressed at the workshops. the projects were implemented by all staff members and were monitored for effectiveness by their supervisors using the who afro checklist; reports were presented at the next workshop by the quality managers. the virology laboratory embarked on three quality improvement projects after the first workshop. the first was that staff were trained on the importance of monitoring the autoclave with emphasis being placed on effective sterilisation and proper waste segregation. they were then assessed daily by means of an in-house-developed checklist in order to improve competency regarding sterilisation and waste disposal. in addition, provision was made for stock level on inventory cards, expired reagents were disposed of and general improvements were made to the organisation of the store; and the storage media for documents were monitored monthly to ensure ease of retrieval of records, documents and policies. after the second workshop, another set of improvement projects was conducted: complaint types, root causes, corrective actions and effectiveness were monitored in order to improve customer satisfaction; specimens in and out of the laboratory were clocked for three months to measure and improve turnaround time; and the number and duration of items out of stock were monitored in order to reduce stock-outs of materials, kits and reagents. the officer responsible for storage monitored the stock-outs from the weekly requisition and issue records. for the tb laboratory, two quality improvement projects were implemented after the first workshop: monitoring inventory of reagents in order to improve turnaround time for acid-fast bacilli smear microscopy; and training staff on how to conduct internal audits. the laboratory conducted three improvement projects after the second workshop: monitoring media preparation and reviewing sputum-collection records in order to reduce the contamination rate of cultured samples; administering and analysing questionnaires from clients and effecting corrective actions in order to improve customer satisfaction; and improving documentation of inventory and establishing a requisition system for the stores. mentorship and additional support contrary to slmta’s implementation roadmap,5 time constraints on the laboratory experts who rolled out the slmta programme in nigeria prevented follow-up site visits at the virology laboratory between the workshops, which would have assisted in linking the training curriculum with on-site activities. for the tb laboratory, an experienced international mentor from the american society for microbiology was assigned to work with the laboratory throughout the slmta process. only tb laboratories were assigned mentors in this round of the slmta programme in nigeria. the mentor had a postgraduate degree in quality management systems and a doctoral degree in microbiology with a tb specialty, had previously managed a laboratory that successfully attained international accreditation and had attended a slmta training-of-trainers workshop. a facility-based approach was adopted as the mentor visited the laboratory on four occasions for an average duration of three weeks at a time, allowing an in-depth understanding of the laboratory. the mentor provided daily assistance to the staff in the implementation of qms, which included training in practical skills, assisting in improving quality of testing and giving assignments to be completed between visits. nonconformities reported from each audit were addressed by the mentor at each visit and management review meetings were established to identify opportunities for improvement and to formulate action plans. the mentor had administrative support from institutional management and the federal ministry of health, as well as technical and logistical support provided by cdc’s office in nigeria. within the time frame of the slmta programme, the aids prevention initiative in nigeria organised a five-day training on accreditation preparedness, with emphasis on quality management systems. staff from both of the laboratories participated alongside staff from other laboratories that the organisation supports. results top ↑ overall performance there was an overall improvement in the performance of both laboratories during the slmta programme (figure 2). the virology laboratory moved from an overall score of 80% at baseline, representing three stars, to 93% at the exit audit, representing four stars. the tb laboratory improved steadily from 66% at baseline audit, representing two stars, to 95% at exit audit, representing five stars. both laboratories maintained these gains at the nine-month surveillance audit. figure 2: comparison of performance of virology and tuberculosis laboratories over time using the who afro checklist. performance of quality system essentials examining the 12 quality system essentials closely revealed specific areas of strength, weakness and improvement (figure 3). the greatest improvements for the virology laboratory were in purchasing and inventory (from 67% to 90%) and in process control and internal and external quality assessment (from 74% to 94%) (figure 3a). the virology laboratory achieved 100% scores in five quality system essentials (documents and records; client management and customer service; internal audit; corrective action; occurrence and/or incident management and process improvement); however no progress was made in organisation and personnel, which remained at 80% for the exit audit. the tb laboratory generally started with lower scores than the virology laboratory, leaving more room for improvement. it made substantial improvements in management review (from 42% to 100%); internal audit (from 50% to 100%) and occurrence/incident management and process improvement (from 50% to 100%) (figure 3b). the tb laboratory also had very good performance at the exit audit in all the quality system essentials, obtaining 100% scores in six sections (documents and records; management review; client management and customer service; internal audit; corrective action; and occurrence/incident management and process improvement). figure 3: comparison of performance of the virology and tuberculosis laboratories in the 12 quality system essentials using the who afro checklist. quality improvement projects the impact of the quality improvement projects on the quality system essentials is shown in table 1. for the virology laboratory, after the first workshop the projects on documents and records and on purchasing and inventory impacted positively at the first intermediate audit and progress was sustained through the exit audit. the project on organisation and personnel did not have a positive impact on the score for the corresponding quality essential, as there was a drop in the next audit score, which did not improve beyond baseline at the exit audit. of the projects conducted after the second workshop, only the client management and customer service project corresponded to sustained performance, with audit scores remaining at 100%, as they were at the baseline audit. performance in information management was maintained at 93% after the second intermediate audit, but dropped to 83% at the exit audit, whilst purchasing and inventory improved from 93% before implementation to 97% at the second audit, but dropped to 90% at the exit audit. at the surveillance audit, improvements were generally sustained, except for purchasing and inventory, which reverted to the baseline score of 67%. for the tb laboratory, after the first workshop there was an improvement in the performance of the information management section, which continued through to the exit audit, where it reached 94% (table 1). the internal audit score dropped initially, but improved subsequently, attaining 100% at the exit audit. after the second workshop, there was a gradual improvement for process control and internal and external quality assessment to 91% at the exit audit; and maintenance of client management and customer service at 100%. purchasing and inventory scores decreased initially following project implementation (from 77% to 70%), then increased to 87% at the exit audit. the surveillance audit showed sustained or improved performance in all areas. table 1: impact of quality improvement projects on quality system essentials in the virology and tuberculosis laboratories of the nigerian institute of medical research. effect of changes to the audit checklist parallel audit scores using the revised (2012) checklist were slightly lower than those using the original (2009) checklist (table 2). differences ranged from 0% to 7% and were smaller at the surveillance audit than at the second intermediate audit, where they resulted in a change of star category for both the virology and tb laboratories. table 2: comparison of audit scores based on the original 2009 who afro checklist and the revised 2012 checklist. discussion top ↑ both the virology and tb laboratories successfully improved their quality scores, increasing by 13% and 29%, respectively. the virology laboratory started with more experience and higher scores at the baseline audit. however, improvement in the tb laboratory was steady and the exit score exceeded that of the virology laboratory. the two laboratories were from same institution and had the same management commitment and partner support, with similar test menu diversity, test volume and staff strength. the major difference between slmta implementation in the two laboratories was the presence of a facility-based laboratory mentor in the tb laboratory. other countries, such as kenya and botswana, have found similar results when implementing an accreditation-readiness programme, with mentored laboratories showing greater improvement than their non-mentored counterparts.10,11,12 whilst conclusive evidence is lacking, as none of these programmes were designed as case-control studies (i.e., with mentors randomly assigned to laboratories), the combined anecdotal evidence strongly supports the benefit of such mentorship. mentors who spend extended, well-structured periods in the laboratory working alongside the staff and helping participants to put quality improvement into practice through direct, daily coaching, can provide the needed support to fast-track laboratories toward quality improvement. the laboratories faced several challenges with regard to slmta implementation. firstly, whilst the laboratory checklist was used to help identify and correct problems, an understanding of some of the requirements was often a challenge, especially in the virology laboratory where an experienced mentor was not available to assist. similarly, the virology laboratory reported challenges in interpreting iso 15189 standard requirements and auditor recommendations. secondly, though many of the quality improvement projects were implemented successfully and increased performance of the quality system essentials, some of these advances were not sustained, especially in the virology laboratory. the purchasing and inventory section was affected worst as some records were not maintained. sustainability is a common concern for any improvement programme; once the intense focus of implementation ceases, special efforts and continued supervision are required so as to ensure that old habits do not return. it is possible that the root causes of the deficiencies were not properly identified and addressed. nevertheless, improvements in total scores were sustained, suggesting that quality improvement overall was maintained. successful achievement of the four to five star levels reached by the two nimr laboratories indicates a high level of laboratory functioning and gives credibility to the quality of the laboratory test results produced for improved healthcare services. the 22 other laboratories in nigeria’s first slmta round had similarly impressive results, moving from an average baseline of 60% to 87% at exit audit; 16 of the laboratories achieved four to five stars.13 these successes inspired nigeria to implement a second round of slmta in 2013 and to begin discussions regarding further national expansion of the programme. because of the potential benefit of on-site mentorship, national experts are being trained in nigeria to play this critical role. limitations our observations are subject to several limitations. the first of these is that mentorship was not assigned randomly. whilst factors that we examined, such as management support, laboratory size and testing volume, were similar for the two laboratories, there may have been other unobserved factors that could account for some of the differences. for example, the laboratories chose different quality improvement projects to implement between workshops. a report by maina et al. suggests that internal audits (which were implemented by the tb laboratory after the first workshop) may be a catalyst for improvements in other areas, as conducting self-review can identify areas that need improvement.14 whilst the virology laboratory was already conducting internal audits before slmta, the tb laboratory started with a 50% score in this area and increased to 100%, potentially helping to explain their improvement in other areas. the second limitation is that the checklists used for the baseline and exit audits were not exactly the same, potentially introducing bias in the results. comparison of the scores obtained by the two checklists used in parallel at the second intermediate and surveillance audits revealed that the revised checklist produced slightly lower results than the original checklist, suggesting that our overall improvement results are possibly conservative. the final limitation is that the auditors engaged in this study had only undertaken one day of training on auditing, which is not adequate to fully qualify them as auditors. whilst the use of a checklist helps to standardise the auditing process, some variability may have been introduced because of inexperience. conclusion the slmta programm was successful in improving the quality of the laboratory systems in these two laboratories, as evidenced by improved and sustained audit scores. the laboratory with expert on-site mentorship improved farther and steadier, achieving a score of five stars. our results suggest that laboratories should consider using on-site mentorship in order to augment the impact of slmta in implementing quality improvement. acknowledgements top ↑ the authors would like to thank the federal ministry of health and the management of nimr for their political commitment; and cdc’s nigeria office and the aids prevention initiative in nigeria for their support in conducting the slmta programme. we also appreciate the work of the auditors and facilitators who executed the project. the findings and conclusions in this article are those of the authors and do not necessarily represent the views of the cdc. this research has been supported by pepfar through the cdc. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. authors’ contributions r.a.a. (human virology laboratory) analysed the data and initiated and wrote the manuscript. c.c.o. (national tuberculosis reference laboratory) provided the required information for the tb laboratory. n.n.n. (national tuberculosis reference laboratory) contributed to the write-up review. e.o. (aids prevention initiative in nigeria) coordinated the implementing partner support. s.b. (american society for microbiology) implemented the mentorship model. o.o. (cdc, nigeria) provided technical support. e.t.l. (cdc, united states) assisted with data analysis and interpretation and revised the manuscript extensively. e.o.i. (national tuberculosis reference laboratory) was responsible for the overall oversight with regard to the project implementation programme. references top ↑ 1.gershy-damet gm, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in african region. am j clin pathol. 2010;134(3):393–400. http://dx.doi.org/10.1309/ajcptuuc2v1wjqbm 2.petti ca, polage cr, quinn tc, et al. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006;42(3):377–382. http://dx.doi.org/10.1086/499363 3.audu ra, sylvester-ikondu u, onwuamah ck, et al. experience of quality management system in a clinical laboratory in nigeria. afr j lab med. 2012;1(1), art. #18, 5 pages. http://dx.doi. org/10.4102/ajlm.v1i1.18 4.world health organization [who representative’s office for rwanda]. press release: kigali host the launch of a program to accelerate national laboratory service capacity building towards accreditation in the african region [document on the internet]. c2008 [cited 2014 jul 30]. available from: www.who.int/hiv/amds/diagnostics/amds_kigali_pr_lab.pdf 5.yao k, maruta t, luman et, nkengasong jn. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. 2014;3(1). in press. 6.yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin pathol. 2010;134(3):401–409. http://dx.doi.org/10.1309/ajcpnbbl53fwuiqj 7.nigerian institute of medical research. development of a strategic plan (2011–2015) [document on the internet]. c2011 [cited 2014 jul 27]. available from: https://nimr.gov.ng/_data/nimr_strategic_plan.pdf 8.world health organization’s regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation in the african region (with checklist) [document on the internet]. c2011 [cited 2014 aug 10]. available from: http://www.afro.who.int/en/clusters-a-programmes/hss/bloodsafety-laboratories-a-health-technology/blt-highlights/3859-who-guide-for-thestepwise-laboratory-improvement-process-towards-accreditation-in-the-africanregion-with-checklist.html 9.world health organization’s regional office for africa. world health organization releases guide for the stepwise laboratory improvement process towards accreditation (slipta) in africa [page on the internet]. c2013 [cited 2014 jul 30]. available from: http://www.aslm.org/stay-informed/press-room/news-articles/world-healthorganization-releasesguide-for-the-stepwise-laboratory-improvementprocess-towards-accreditation-slipta-in-africa/ 10.gachuki t, sewe r, mwangi j, et al. attaining iso 15189 accreditation through slmta: a journey by kenya’s national hiv reference laboratory. afr j lab med. in press. 11.makokha e, mwalili s, basiye f, et al. using institutional mentorship to roll out slmta in kenya. afr j lab med. in press. 12.mokobela k, moatshe m, modukanele m. using slmta and mentorship to accelerate laboratory improvement in botswana. afr j lab med. in press. 13.yao k, luman e, slmta collaborating authors. evidence from 617 laboratories in 47 countries for slmta-driven improvement in quality management systems. afr j lab med. in press. 14.maina, rn, mengo dm, mohamud ad, et al. progressing beyond slmta: are internal audits the key drivers of accreditation? afr j lab med. in press. article information authors: elizabeth t. luman1 katy yao1 john n. nkengasong1 affiliations: 1international laboratory branch, division of global hiv/aids, center for global health, us centers for disease control and prevention, atlanta, georgia, united states correspondence to: elizabeth luman postal address: 1600 clifton road, atlanta, ga 30333, united states dates: received: 18 sept. 2014 accepted: 21 sept. 2014 published: 03 nov. 2014 how to cite this article: luman et, yao k, nkengasong jn. a comprehensive review of the slmta literature part 1: content analysis and future priorities. afr j lab med. 2014;3(2), art. #265, 11 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.265 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. a comprehensive review of the slmta literature part 1: content analysis and future priorities in this review article... open access • abstract • introduction • research methods and design • results and discussion    • literature search results    • previous experience with quality management systems    • the drive for action    • implementation    • capacity building    • partnership    • on-site support and mentorship    • success factors    • challenges    • limitations to the study    • feedback on the slmta programme • future directions    • progression (continued improvement in slmta laboratories)    • saturation (additional laboratories within countries that have implemented slmta)    • expansion (implementation in additional countries)    • extension (adapting slmta for implementation beyond the laboratory)    • transformation (impact on health systems and patient care) • acknowledgements    • competing interests    • authors’ contributions    • cdc disclaimer • references abstract top ↑ background: since its introduction in 2009, the strengthening laboratory management toward accreditation (slmta) programme has been implemented widely throughout africa, as well as in the caribbean, central and south america, and southeast asia.objective: we compiled results from local, national and global studies to provide a broad view of the programme and identify directions for the future. the review consists of two companion papers; this paper focuses on content analysis, examining various thematic components of the slmta programme and future priorities. methods: a systematic literature search identified 28 published articles about implementing the slmta programme. results for various components of the slmta programme were reviewed and summarised. results: local and national studies provide substantial information on previous experiences with quality management systems; variations on slmta implementation; building human resource capacity for trainers, mentors and auditors; the benefits and effectiveness of various types of mentorship; the importance of management buy-in to ensure country ownership; the need to instill a culture of quality in the laboratory; success factors and challenges; and future directions for the programme. conclusions: local, national and global results suggest that the slmta programme has been overwhelmingly successful in transforming laboratory quality management. there is an urgent need to move forward in four strategic directions: progression (continued improvement in slmta laboratories), saturation (additional laboratories within countries that have implemented slmta), expansion (implementation in additional countries), and extension (adapting slmta for implementation beyond the laboratory), to lead to transformation of overall health systems and patient care. introduction top ↑ the strengthening laboratory management toward accreditation (slmta) programme is a structured quality improvement curriculum designed to achieve immediate and tangible advances in health service delivery.1 the central feature of this intervention is its emphasis on the ‘how-to’ of implementing quality management systems (qms) by translating the ‘what’ (concepts, principles, theories, guidelines and standards) into practical behaviours, laboratory practices and daily routines, through hands-on practice during training and improvement projects in trainees’ home laboratories. the importance of practical training dedicated to building management capacity has been well deliberated in a series of working papers published by the world health organization (who) on strengthening leadership and management in low-income countries, titled ‘making health systems work’.2,3,4,5in the five years since its introduction in 2009, the slmta programme has been implemented widely throughout the developing world.1 in december 2012, slmta country coordinators and implementers gathered at a two-day symposium in cape town, south africa to discuss their experiences and lessons learned. it was evident that a substantial amount of programmatic expertise had been gained. a supplemental issue of the african journal of laboratory medicine (ajlm) was commissioned in order to document and share successes and challenges, summarize laboratoryand country-specific analyses, and publish global programme data. this systematic literature review aims to compile existing fragmented results into a comprehensive report, to provide a broad view of the programme and to identify directions for the future. because of the large volume of information collected, the review has been published in two parts. part 1 focuses on content analysis, examining various thematic components of the slmta programme and future priorities. part 2, published separately, compiles the quantitative data reported in the publications, examines scores and indicators, and uses meta-analysis to augment the results.6 research methods and design top ↑ a comprehensive search of electronic bibliographic databases was performed, including medline and the directory of open access journals, using the keyword ‘slmta’. slmta country programme leaders and partner agencies were contacted so as to identify additional sources. we included all published and in-press studies that discussed the slmta programme. the majority of the search results were in-press manuscripts being prepared for the supplemental issue of ajlm focusing on the slmta programme, called ‘transforming the quality of laboratory medicine through the strengthening laboratory management toward accreditation program’; authors of this review coordinated the issue. results and discussion top ↑ literature search results we identified 28 published manuscripts focusing on the slmta programme,1,7– 33 including 26 published concurrently with this article,1,7-20,22-31,33one previously-published article summarising the development and methodology of the slmta programme32 and one previously-published study regarding slmta implementation in lesotho21 (table 1). six papers presented experiences from a single laboratory7,10,17,26,28 or a single hospital,9 14 presented data from two to 45 laboratories within a single african country,8,12,– 15,18,– 22,25,29– 31 one discussed activities in two southeast asian countries,23 one covered data from four countries in the caribbean region11 and six had a general or global focus.1,16,23,24,32,33 in total, these studies included detailed information on slmta implementation in 211 laboratories in 18 countries. table 1: characteristics of published slmta studies. previous experience with quality management systems a recent study summarised the scarcity of accreditation in public laboratories throughout sub-saharan africa.34 however, the results from the slmta laboratories paint an even more problematic picture – not only are laboratories in developing countries not meeting the international quality standards needed for accreditation, but the level of quality management is extremely low. several authors reported that their laboratories had no prior experience with qms,7,10,17,22 with one study reporting that prior to slmta, ‘the idea of qms was entirely new to most laboratory staff in the selected facilities, where a culture of quality has been lacking’.22 the low level of baseline audit scores across the programme – 84% of laboratories did not reach even one star on the who regional office for africa’s (afro) stepwise laboratory quality improvement process towards accreditation (slipta) five-star scale and the mean baseline audit score was 39% – seems to confirm this assessment.33 these findings are supported by a study in kampala, uganda, which found that only 5% of audited laboratories in the city had reached one star on the slipta quality scale.35previous experience in establishing qms was limited in these countries and what had been done was largely reported to be ineffective. one study of slmta implementation in the caribbean region reported that ‘past laboratory assessments and training did not provide them with a structured roadmap to assist in implementation; as a result, the majority of these laboratories did not initiate the process of qms development and implementation’.11 another reported that ‘prior to the introduction of slmta, several trainings and quality improvement initiatives had been implemented in hospitals and laboratories in ethiopia, but little improvement was noted’.12 in botswana, there had been ‘slow progress in implementing qms’, and authors noted that ‘previous training of healthcare workers has focused on general management topics rather than identifying tangible tasks to bring about change, making the training difficult to apply in the laboratory’.28 the drive for action several authors report that implementation of slmta came after years of neglect of laboratories. in many cases, these circumstances improved with the advent of the us president’s emergency plan for aids relief (pepfar) in 2003,7,11,14,15,22,27 which emphasised the importance of quality laboratory tests and infused much-needed capital into the laboratory systems.36 other international programmes also played a key role, such as the global fund to fight aids, tuberculosis and malaria37 and the global health initiative.3838 concurrently, several regional and global policy statements called for strengthening public medical laboratories: the maputo declaration, which called on ‘national governments to support laboratory systems as a priority’;39 the lyon statement on the need for laboratory qms and accreditation of national reference laboratories;40 and the yaoundé resolution, in which who afro recognised the need to further strengthen public health laboratories and to encourage member states ‘to develop or strengthen comprehensive national laboratory policies’.41authors noted that also during this timeframe, countries began to develop five-year laboratory strategic plans – kenya14,15 and ethiopia12,13 in 2005; botswana20,28 and lesotho21 in 2008; zimbabwe30 in 2010; and ghana25 and namibia19 in 2012. these plans called for laboratory strengthening and development of qms, some specifying accreditation goals. for example, the laboratory strategic plan in botswana called for implementation of qms in all laboratories by 2014 and accreditation of four district-level laboratories by 2013 and two national-level laboratories by 2014.20 in kenya, the ministry of health set a goal to accredit all national and regional level public laboratories10,14 and established a national accreditation steering committee to coordinate accreditation activities. the rwanda ministry of health aimed to enroll all 48 central and district hospital laboratories in the accreditation preparation process,29 and the ministry of health in mozambique established a national laboratory policy, which outlined their commitment to implement qms and to pursue accreditation for reference and provincial hospital laboratories.18 the focus on laboratory quality improvement led these countries to implement the slmta programme in order to help them improve their quality management. implementation the standard slmta implementation model includes three workshops followed by periods of several months for laboratories to implement improvement projects, usually with onsite support and mentorship.1 the slmta programme is evaluated through audits based on the slipta checklist.1several countries have customised slmta implementation to meet their needs. in cameroon, slmta workshops were decentralised and conducted on site, allowing many more staff to participate in training.1,22,26 the slmta team in vietnam developed an electronic tool for slipta audits so as to improve timeliness and accuracy of audit results and reduce language barriers.23 in rwanda, performance-based financing was used in one cohort, in which payment was provided to laboratories based on slipta audit scores to incentivise continuous quality improvement.29 in several programmes, non-laboratory personnel participated in the slmta training, including hospital managers and administrators9,22,26 as well as clinicians.13,22,29 several countries have established departments or workgroups dedicated to the implementation of quality management. for example, zimbabwe’s national quality assurance program established a training and mentorship department;30 kenya’s ministry of health created a national accreditation steering committee to coordinate laboratory accreditation activities;14 and mozambique established a national laboratory technical working group to build a framework for a national laboratory quality improvement programme, to lead and coordinate its implementation and to monitor and maintain results.18 individual facilities also established quality improvement programmes. for example, a hospital in cameroon developed a quality improvement task force to coordinate quality improvement efforts.9 one laboratory in kenya formed a tiered accreditation team structure, including a management team, quality assurance team and section teams, to run the programme and lead the laboratory to international accreditation.10 capacity building with the rapid expansion of the slmta programme, the need to build capacity for more slmta trainers, mentors and auditors has been identified.1,16 maruta et al. summarise global efforts to develop trainers and master trainers using a training-of-trainers (tot) strategy with teach-back methodology.16 the tot course is an intensive two-week training course taught by master trainers, in which candidate trainers learn to teach the 44 activities in the slmta curriculum (through a combination of skills learning, practice and feedback) and to follow set guidelines for programme implementation. as of the end of 2013, 433 trainers and 38 master trainers have been produced, and the vast majority (97% and 87%, respectively) are based in developing countries. tot courses have been held in botswana, dominican republic, ethiopia, ghana, kenya, mozambique, nigeria, rwanda, south africa, tanzania, vietnam and zimbabwe and have been conducted in english, portuguese, spanish and vietnamese.critical considerations include ensuring that tot graduates are utilised effectively and that fidelity of implementation is maintained as the programme expands. the maruta et al. study found a 92% utilization rate of tot graduates, with 97% of participants reporting that the tot trained them either well or extremely well for implementing slmta.16 furthermore, global data suggest that training quality has been maintained, as the 132 laboratories that implemented slmta during the first year (2010) had the same mean improvement (24 percentage points) as the 170 laboratories that implemented slmta in subsequent years (2011–2013).33 the development of mentors and auditors has not been summarised globally, although several studies report country-specific efforts. in cameroon, 11 mentors and seven auditors have been trained to support programme scale-up.33 in rwanda, 17 local mentors were trained to roll out the slmta programme.29 in mozambique, 15 auditors were trained18 and in ghana, 15 mentors and 11 auditors were trained.25 several papers discussed national plans to train additional mentors and auditors.8,18,19,25,26,29 one critical consideration for auditor training is to ensure high qualifications and consistency between them. several studies discussed the variability of auditor expertise and reliability as a serious limitation of both the programme and their reported results.8,29,33 partnership the rapid and widespread expansion of the slmta programme could not have occurred without the active participation of an extensive network of partners. these various partners have been instrumental in all aspects of programme development and implementation (table 2). primarily funded through pepfar1 and developed under the leadership of the us centers for disease control and prevention (cdc), ministries of health have implemented the programme with support from international organisations, institutions and private companies, parastatal organisations and local non-governmental organisations. one study focused on the use of partnership to implement slmta in 15 laboratories in ghana, concluding that ‘building in-country capacity through local partners is a sustainable model for improving service quality in resource-constrained countries’ and that ‘local partners, when supported and managed adequately, can achieve great results at a reasonable cost’.25 table 2: partners contributing to the slmta programme as reported by published studies. table 2 continues: partners contributing to the slmta programme as reported by published studies. on-site support and mentorship on-site support and mentorship are key components of the slmta programme, as mentors are expected to provide in-depth support after workshops to assist laboratory personnel in implementing changes.32 support and mentorship models have ranged from no site visits or mentorship,8,20 occasional visits,14 periodic visits of several days to several weeks,8,10, 16, 28, 29, 30 to embedded mentors working full time in their assigned laboratories for extended periods11, 14, 18, 22, 25, 26, 29, 30 (table 3). kenya has piloted a novel mentorship model, ‘twinning’ public laboratories with local state-of-the-art research laboratories. this institutional mentorship approach holds promise not only for laboratory improvement, but also for fostering long-standing sustainable partnerships between public health and research laboratories.15 table 3: mentorship models reported by slmta studies. well-planned scientific studies are lacking with regard to the effectiveness of mentorship overall or the relative effectiveness of various models. all of the slmta-related studies conducted to date have serious methodological flaws, primarily the lack of random assignment to mentorship models, lack of control groups and small sample sizes. despite these limitations, several post-hoc analyses comparing results in mentored laboratories to those in non-mentored laboratories,8,20 as well as intensive mentorship to less intensive mentorship,15,29 concur that mentorship appears to be beneficial (table 3). these findings agree with an earlier study that found that slipta scores increased in four lesotho laboratories after mentorship, although no control laboratories were used on which to base a comparison.42 despite the lack of solid scientific evidence, there is a general belief that mentorship is an important component of the slmta programme and a critical factor for success. success factors numerous factors have been identified by authors as being critical to the success of slmta implementation. at the facility level, many authors reported the importance of engaging hospital and senior management from the beginning so as to ensure their buy-in and ownership of the programme7,12, 17, 20, 25, 26, 28, 29, 30 and to promote institutionalisation and thus sustainability.18 many also noted the importance of a strong commitment and team spirit amongst the laboratory staff7,9, 10, 18, 20, 28, 29 and a willingness to build a culture of quality17,26 and problem solving.20 the various components of the slmta programme – including the how-to guidance provided by the workshops;7,10, 17, 28, 32 mentorship7, 10, 11, 25, 30 and supervisory visits7, 12, 18, 21, 32 to keep laboratories on track; improvement projects;20, 26, 32 and ensuring that both staff11, 17, 32 and leadership17 are accountable and motivated – were viewed as critical. in addition, authors noted that it is essential to measure what has been accomplished through audits using the slipta checklist10, 11, 17, 18, 21, 32 and analysis of other key indicators.28 these data are powerful means to recognise and motivate staff, determine further actions needed17 and provide information as an advocacy tool.16 finally, ongoing communication with hospital management21 and clinical staff9, 17, 26, 28 is critical so as to ensure continued focus on patient care and support for future activities.at the country level, it is critical to ensure clear commitment and ownership within the ministry of health in order to improve laboratory quality at all levels, including development of a national laboratory policy and strategic plan, establishment of a laboratory technical working group and provision of financial support.1, 21, 25 early engagement of key stakeholders and partners,11,21, 25 followed by effective communication and continuous advocacy for laboratory quality,9, 12, 18 were identified as important components of success, as were the development of a programme implementation plan9, 10, 11 that includes human resource needs for trainers, auditors and mentors;1 a plan to ensure sufficient geographic coverage through careful site selection;1, 18 and establishment of specific programme goals.18 challenges challenges at both the facility and programme levels were identified, as well as those beyond the scope of the programme that affect the programme. at the facility level, common concerns surrounded the difficulty of engaging non-slmta-trained laboratory staff,1, 10, 12, 13 as well as hospital management and regional health bureaus1, 12, 13 and ensuring harmonisation with other hospital improvement programmes.12 it was noted that behavioural change requires time, commitment and consistent support.32 also documented were difficulties in: providing sufficient site support;1, 12 ensuring that staff understand the requirements of qms8 and the international organization for standardization (iso) 15189 standard;8, 10, 12 how to conduct internal audits;25 and the importance of establishing quality in laboratory testing.22 implementing a qms is a difficult process; particularly noted were the challenges of: balancing the requirements of multiple functions within a laboratory;20 establishing root causes of nonconformities;8, 14 equipment maintenance and outages;13, 22 development of method validation;10 space shortages;10 document review and maintenance;7, 20, 26 insufficient time to implement improvement projects;21 and the general concern that the entire process required more time and resources than anticipated.13at the programme level, the shortage of well-trained mentors11, 13, 15, 25 was a common concern, as were the lack of trainers22 and auditors.23 in addition, it was noted that mentors and other implementers have competing duties, since they are generally not dedicated solely to slmta activities,18, 30 which may lead to suboptimal utilisation rates of trainees.16 furthermore, language and communication barriers amongst mentors, trainers and auditors can be a challenge,23,25 exacerbating the shortages. also reported were higher-level challenges that had an impact on programme implementation. the most-commonly reported challenge was that of staff attrition.10, 11, 13, 17, 20, 21, 29, 31, 32 one study of programme implementation in the caribbean region reported the following: [e]nsuring a sufficient number of well-qualified laboratory workers is an ongoing challenge, exacerbated by high levels of attrition as staff that have benefitted from government-supported training leave the public sector for more lucrative jobs in the private sector, either locally or overseas. thus the remaining staff are overworked, reducing the amount of time available for training and quality improvement activities.11 in a study from rwanda, the only laboratory whose scores decreased from baseline to exit audit lost both their quality officer and laboratory manager during the programme;29 and a laboratory in kenya found that patient complaints increased as a result of high staff turnover.17 shumba et al. suggest several strategies to reduce staff attrition, including encouraging ministries of health and supervisors to agree not to reassign trained staff for a period of time, providing financial incentives to participants at the completion of slmta and using binding contracts in which participants agree to remain on the job for a specified period.31 decentralised training may also help reduce the effect of attrition; in cameroon, the authors concluded that, ‘[i]n the decentralised model, the majority of laboratory staff are trained to implement qms, reducing the impact of attrition of a few trained staff members’.22 in addition, studies reported a general shortage of qualified laboratory staff, especially staff with qms expertise.11,12,22, 25, 29 also noted was a lack of quality manuals, guidelines and procedures12 to provide clear direction, as well as a lack of national strategic plans22 to define stakeholders and facilitate coordination with partners.13 these issues, when coupled with institutional bottlenecks,22,25 slow procurement processes10 and lack of or limited accreditation preparation budgets,12,25 further hamper improvement efforts. finally, the existing low level of quality management in developing countries33 suggests that much work is needed to ensure sufficient quality of laboratory services to provide for public and personal health needs. limitations to the study this review is subject to several limitations. primarily, the results may not be representative of the programme as a whole, or a comprehensive account of all laboratories’ experiences. for example, whilst these studies mentioned some 30 partners who helped to develop or implement the slmta programme, others were undoubtedly missed. feedback on the slmta programme overall, the published literature was strongly in favour of the slmta programme, with investigators and public health officials reporting satisfaction with the results. nkengasong and birx suggest that ‘with the introduction of slmta, the prospects of implementing sustainable quality-assured laboratory medicine seem to be a reality in developing countries’.24 other investigators agreed, saying that slmta ‘was found to be a practical option that yielded positive results for strengthening laboratories’20 and also that ‘[t]he tremendous improvement… shows that slmta coupled with mentorship is an effective, user-friendly, flexible and customisable approach to implementation of laboratory qms’.11a study of attitudes of health professionals in ethiopia found that laboratory professionals had a supportive perception of slmta, whilst some hospital chief executive officers ‘were more sceptical of slmta and raised concerns regarding programme costs and the prolonged process associated with implementation’.13 a hospital director in cameroon disagreed, saying that: slmta is an invaluable tool for every lab director, every hospital manager and health policy maker because of its value in ensuring quality improvement within the laboratory and its potential in contributing to strengthening the entire health system at little or no cost.9 future directions top ↑ the slmta programme is expanding rapidly and authors have identified an urgent need to sustain the gains and move forward in four strategic directions to lead to transformation of overall health systems and patient care (figure 1). figure 1: future directions of the slmta programme. progression (continued improvement in slmta laboratories) several authors have discussed the need to ensure that laboratories sustain gains made and continue to move forward.8,11, 17, 20, 26, 27, 29 yao et al. point out that quality improvement should be seen as an ongoing journey and that slmta provides the tools needed to ensure better patient care;1 ntshambiwa et al. concur that slmta has ‘helped lay a firm foundation for further advancements in patient care’.28 rwanda’s first two slmta cohorts not only sustained their results at a surveillance audit a year after slmta completion, but increased their scores by a median of 10 percentage points.29 globally, 92 laboratories have completed surveillance audits; 62% further increased their score.33 nkrumah contends that indigenous capacity building is critical in order to ensure sustainability,25 whilst maruti et al. focus on the need to change the laboratory culture by establishing universal rules, teaching staff the principles and techniques of quality improvement, continually reinforcing the behaviours by integrating them into daily routines and engaging hospital stakeholders.17 audu et al. argue that ‘sustainability is a common concern for any improvement programme; once the intense focus of implementation ceases, special efforts and continued supervision are required so as to ensure that old habits do not return’.8 maina et al. also focus on post-slmta sustainability, identifying internal audits and corrective action as catalysts for continued improvement.14 noble et al. commend the achievements made by laboratories to date, cautioning:but to them we put forward this challenge: whilst it is a great achievement to reach a level of success where the requirements of accreditation are met, the true accomplishment is reaching the point where the level of quality is an everyday practice and expectation, and the laboratory is ‘accreditation-ready’ over and over. when the inevitable slips and mistakes occur in laboratories that are accreditation-ready, the processes of error detection, correction and improvement, and progress back to quality, must occur quickly, smoothly and sustainably.27 as the slmta programme matures, studies measuring the long-term sustainability of results and examining factors associated with continued progress will be critical for ensuring the enduring impact of the programme. saturation (additional laboratories within countries that have implemented slmta) several countries have established formal plans for country-wide qms implementation, using the slmta programme as the central improvement tool.18,25, 29 many have already implemented second (13 countries), third (six countries) and further cohorts (three countries) as they expand slmta nationally; of the 21 countries that began slmta in 2010–2011, 16 (76%) have thus far implemented additional cohorts.33 kenya has conducted six rounds of slmta, whilst lesotho has reached near saturation, with slmta implemented in 18 of its 19 laboratories.33several authors discuss the next steps toward achieving greater saturation within countries. most common amongst these is the need to develop additional local trainers, mentors and auditors,1,11, 13, 16, 22, 23, 25, 30 as well as educating the general workforce through pre-service training.24 guevara et al. suggest that ‘there is now a need to internalise the programme and transition it to local governments and other donors in order to facilitate expansion and ensure sustainability’,11 whilst lulie et al. argue that further efforts are needed to ‘decentralise responsibility from the government to the management at their facilities’.13 it is evident that not all laboratories in a country’s health system need to be accredited; however, all laboratories must maintain a culture of continuous quality improvement. nkengasong and birx discuss the need for countries to identify a ‘tipping point’ or threshold of laboratories that must be accredited in order to establish this culture and ‘increase confidence in quality-assured laboratory medicine for evidence-based patient management’.24 once accreditation goals are defined in national laboratory strategic plans, a slmta implementation plan can be developed with clear priorities to help guide laboratories in the tiered health system to achieve their goals. masamha et al. suggest that, in addition to accreditation, countries could track the progress of quality systems with indicators such as: [the] number of provinces with dedicated quality management officers; percentage of laboratories audited in the previous 12 months; percentage of audited laboratories demonstrating improvement as measured by the slipta checklist; and percentage of laboratories implementing external proficiency testing for select services.18 expansion (implementation in additional countries) slmta implementation started with 11 countries in 2010 and spread to an additional 10 in 2011, 15 in 2012 and 11 in 2013.33 because pepfar is the primary funding source, slmta to date has been rolled out largely in pepfar-supported countries43 (43 of the 47 countries implementing slmta are pepfar-funded, 91%). as of the end of 2013, 75% of the 57 pepfar-supported countries have implemented slmta, with most of the remaining countries located in asia.to date, slmta has been implemented in 38% of low-income countries and 26% of lower-middle-income countries, based on world bank classifications.44 further expansion beyond pepfar-supported countries to other lowand lower-middle-income countries will require the identification of additional global partners to provide funding and implementation support. extension (adapting slmta for implementation beyond the laboratory) to take full advantage of the benefits of improved laboratory quality, improvements will need to be made outside the scope of the laboratory as well. in her commentary, the laboratory director for namibia’s ministry of health and social services (mohss) explains:as quality improvements become institutionalised in hospital laboratories, it is becoming evident that entire hospital systems are in dire need of similar quality improvement programmes. the namibia mohss calls on international agencies to develop and adapt programmes such as slipta and slmta for use throughout hospital systems so as to ensure continuous quality patient care.19 along the same lines, eno et al. report on the experience of one hospital that adapted the slmta tool for wider implementation, inspired by successful implementation of the programme in their laboratory.9 results were encouraging, with ‘steady improvement in service delivery’; reduced patient wait times, infection rates and stillbirths; and increased hospital revenue. the authors concluded that ‘[s]uch a programme has the potential to impact positively on hospital quality systems; consideration should be made for development of a formal slmta-like programme for hospital quality improvement … expanding the strengthening laboratory management toward accreditation programme into one for strengthening hospital management toward accreditation’.9 transformation (impact on health systems and patient care) as slmta continues to grow, it has the potential to have a profound and lasting impact on health systems and patient care. in a report on the impact of pepfar, the institute of medicine concluded that:pepfar’s laboratory efforts have had a fundamental and substantial impact on laboratory capacity in countries. this laboratory infrastructure and capacity has been, and can continue to be, leveraged to improve the functioning of countries’ entire health systems.36 there is a growing movement toward establishing a culture of quality at all levels of service in order to care for patients,24 including not only the laboratory but the pharmacy, clinics, maternity, surgical rooms and others. as quality improves, there is a need to measure the impact on patient outcomes through well-defined and rigorous programme evaluation. such data will provide local, national and global decision makers with the evidence needed to justify expenditures and implement the most appropriate solutions for their given situations. acknowledgements top ↑ we would like to thank the lead authors of the in-press papers for allowing us to examine their results prior to publication, making it possible to publish this review simultaneously with their work: linda andiric, rosemary audu, laura eno, thomas gachuki, giselle guevara, tilahun hiwotu, adino lulie, robert maina, ernest makokha, talkmore maruta, phidelis maruti, jessina masamha, mary mataranyika, kelebeletse mokobela, juliana ndasi, thuong nguyen, bernard nkrumah, siyem nkwawir, michael noble, keoratile ntshambiwa, innocent nzabahimana, phoebe nzombe and edwin shumba. special thanks go to philip rotz, lee schroeder, bethanie rammer and penny smorenburg for their valuable feedback in manuscript revision. this research has been supported by pepfar through the cdc. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions e.t.l (cdc, atlanta) analysed the data and wrote the manuscript. k.y. (cdc, atlanta) and j.n.n (cdc, atlanta) provided substantial input to the revision of the manuscript. cdc disclaimer the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the cdc. references top ↑ 1. yao k, maruta t, luman et, nkengasong jn. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. 2014;3(2), art. #194, 8 pages. http://dx.doi.org/10.4102/ajlm.v3i2.1942. egger d, travis p, dovlo d, et al. strengthening management in low-income countries. making health systems work: working paper no. 1 who/hss/healthsystems/2005.1 [document on the internet]. c2005 [cited 2014 oct 04]. available from: http://www.who.int/management/working_paper_1_en_opt.pdf 3. egger d, ollier e. managing the health millennium development goals – the challenge of management strengthening: lessons from three countries: report on an international consultation on strengthening health leadership 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laboratory. afr j lab med. 2014;3(2), art. #216, 9 pages. http://dx.doi.org/10.4102/ajlm.v3i2.216 11. guevara g, gordon f, irving y, et al. the impact of slmta in improving laboratory quality systems in the caribbean region. afr j lab med. 2014;3(2), art. #199, 9 pages. http://dx.doi.org/10.4102/ajlm.v3i2.199 12. hiwotu tm, ayana g, mulugeta a, et al. laboratory system strengthening and quality improvement in ethiopia. afr j lab med. 2014;3(2), art. #228, 6 pages. http://dx.doi.org/10.4102/ajlm.v3i2.228 13. lulie ad, hiwotu tm, mulugeta a, et al. perceptions and attitudes toward slmta amongst laboratory and hospital professionals in ethiopia. afr j lab med. 2014;3(2), art. #233, 6 pages. http://dx.doi.org/10.4102/ajlm.v3i2.233 14. maina rn, mengo dm, mohamud ad, et al. progressing beyond slmta: are internal audits and corrective action the key drivers of quality improvement? afr j lab med. 2014;3(2), art. #222, 7 pages. http://dx.doi.org/10.4102/ajlm.v3i2.222 15. makokha ep, mwalili s, basiye fl, et al. using standard and institutional mentorship models to implement slmta in kenya. afr j lab med. 16. maruta t, yao k, ndlovu n, moyo s. training-of-trainers: a strategy to build country capacity for slmta expansion and sustainability. afr j lab med. 2014;3(2), art. #196, 7 pages. http://dx.doi.org/10.4102/ajlm.v3i2.196 17. maruti pm, mulianga ea, wambani ln, et al. creating a sustainable culture of quality through the slmta programme in a district hospital laboratory in kenya. afr j lab med. 2014;3(2), art. #201, 5 pages. http://dx.doi.org/10.4102/ajlm.v3i2.201 18. masamha j, skaggs b, pinto i, et al. working toward a sustainable laboratory quality improvement programme through country ownership: mozambique’s slmta story. afr j lab med. 2014;3(2), art. #253, 6 pages. http://dx.doi.org/10.4102/ajlm.v3i2.253 19. mataranyika mn, beukes lk. view from the top: involvement of namibia’s health ministry in laboratory quality improvement. afr j lab med. 2014;3(2), art. #195, 2 pages. http://dx.doi.org/10.4102/ajlm.v3i2.195 20. mokobela ko, moatshe mt, modukanele m. accelerating the spread of laboratory quality improvement efforts in botswana. afr j lab med. 2014;3(2), art. #207, 6 pages. http://dx.doi.org/10.4102/ajlm.v3i2.207 21. mothabeng d, maruta t, lebina m, et al. strengthening laboratory management towards accreditation: the lesotho experience. afr j lab med. 2012;1(1), art. #9, 7 pages. http://dx.doi.doi: 10.4102/ajlm.v1i1.9 22. ndasi j, dimite l, mbome v, et al. decentralised facility-based training as an alternative model for slmta implementation: the cameroon experience. afr j lab med. 2014;3(2), art. #231, 6 pages. http://dx.doi.org/10.4102/ajlm.v3i2.231 23. nguyen tt, mckinney b, pierson a, et al. slipta e-tool improves laboratory audit process in vietnam and cambodia. afr j lab med. 2014;3(2), art. #219, 5 pages. http://dx.doi.org/10.4102/ajlm.v3i2.219 24. nkengasong jn, birx d. quality matters in strengthening global laboratory medicine. afr j lab med. 2014;3(2), art. #239, 4 pages. http://dx.doi.org/10.4102/ajlm.v3i2.239 25. nkrumah b, van der puije b, bekoe v, et al. building local human resources to implement slmta with limited donor funding: the ghana experience. afr j lab med. 2014;3(2), art. #214, 7 pages. http://dx.doi.org/10.4102/ajlm.v3i2.214 26. nkwawir sc, batumani nn, maruta t, awasom cn. from grass to grace: how slmta revolutionised the bamenda regional hospital laboratory in cameroon. afr j lab med. 2014;3(2), art. #203, 6 pages. http://dx.doi.org/10.4102/ajlm.v3i2.203 27. noble ma, martin r, ndihokubwayo j-b. making great strides in medical laboratory quality. afr j lab med. 2014;3(2), art. #256, 2 pages. http://dx.doi.org/10.4102/ajlm.v3i2.256 28. ntshambiwa k, ntabe-jagwer w, kefilwe c, samuel f, moyo s. translating a national laboratory strategic plan into action through slmta in a district hospital laboratory in botswana. afr j lab med. 2014;3(2), art. #209, 5 pages. http://dx.doi.org/10.4102/ajlm.v3i2.209 29. nzabahimana i, sebasirimu s, gatabazi jb, et al. innovative strategies for a successful slmta country programme: the rwanda story. afr j lab med. 2014;3(2), art. #217, 6 pages. http://dx.doi.org/10.4102/ajlm.v3i2.217 30. nzombe p, luman et, shumba e, et al. maximising mentorship: variations in laboratory mentorship models implemented in zimbabwe. afr j lab med. 2014;3(2), art. #241, 8 pages. http://dx.doi.org/10.4102/ajlm.v3i2.241 31. shumba e, nzombe p, mbinda a, et al. weighing the costs: implementing the slmta programme in zimbabwe using internal versus external facilitators. afr j lab med. 2014;3(2), art. #248, 6 pages. http://dx.doi.org/10.4102/ajlm.v3i2.248 32. yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin pathol. 2010;134(3):401–409. 33. yao k, luman et, slmta collaborating authors. evidence from 617 laboratories in 47 countries for slmta-driven improvement in quality management systems. afr j lab med. 2014;3(2), art. #262, 11 pages. http://dx.doi.org/10.4102/ajlm.v3i2.262 34. schroeder lf, amukele t. medical laboratories in sub-saharan africa that meet international quality standards. am j clin pathol. 2014;141(6):791–795. 35. elbireer am, jackson jb, sendagire h, et al. the good, the bad, and the unknown: quality of clinical laboratories in kampala, uganda. plos one. 2013:8(5);e64661. 36. committee on the outcome and impact evaluation of global hiv/aids programs implemented under the lantos-hyde act of 2008; board on global health; board on children, youth, and families. evaluation of pepfar. washington, dc: the national academies press; 2013 [available from: http://www.nap.edu/catalog.php?record_id=18256] 37. the global fund to fight aids, tuberculosis and malaria [homepage on the internet]. n.d. [cited 2014 aug 28]. available from: http://www.theglobalfund.org/en/ 38. us department of health and human services. global health initiative [page on the internet]. n.d. [cited 2014 aug 28]. available from: http://www.globalhealth.gov/global-programs-and-initiatives/global-health-initiative/ 39. world health organization regional office for africa. the maputo declaration on strengthening of laboratory systems [document on the internet]. c2008 [cited 2013 jan 02]. available from: http://www.who.int/diagnostics_laboratory/maputo-declaration_2008.pdf 40. world health organization. joint who–cdc conference on health laboratory quality systems. who/hse/ihr/lyo/2008.3 [document on the internet]. c2008 [cited 2013 jan 03]. available from: http://www.who.int/ihr/lyon/report20080409.pdf 41. world health organization regional office for africa. who report calls for strengthening public health laboratories [page on the internet]. c2008 [cited 2014 jul 21]. available from: http://www.afro.who.int/en/media-centre/pressreleases/item/679-who-report-calls 42. maruta t, motebang d, mathabo l, et al. impact of mentorship on who-afro strengthening laboratory quality improvement process towards accreditation (slipta). afr j lab med 2012;1(1), art. #6, 8 pages. 43. the united states president’s emergency plan for aids relief. countries [page on the internet]. n.d. [cited 2014 aug 28]. available from: http://www.pepfar.gov/countries/ 44. the world bank. country and lending groups [page on the internet]. c2014 [cited 2014 aug 28]. available from: http://data.worldbank.org/about/country-and-lending-groups abstract introduction surveillance of antimicrobial resistance in healthcare-associated pathogens in africa surveillance of antimicrobial resistance in healthcare-associated pathogens in south africa acknowledgements references about the author(s) ashika singh-moodley centre for healthcare-associated infections, antimicrobial resistance and mycoses, national institute for communicable diseases, johannesburg, south africa husna ismail centre for healthcare-associated infections, antimicrobial resistance and mycoses, national institute for communicable diseases, johannesburg, south africa olga perovic centre for healthcare-associated infections, antimicrobial resistance and mycoses, national institute for communicable diseases, johannesburg, south africa citation singh-moodley a, ismail h, perovic o. an overview of antimicrobial resistance surveillance among healthcare-associated pathogens in south africa. afr j lab med. 2018;7(2), a741. https://doi.org/10.4102/ajlm.v7i2.741 review article an overview of antimicrobial resistance surveillance among healthcare-associated pathogens in south africa ashika singh-moodley, husna ismail, olga perovic received: 20 dec. 2017; accepted: 30 may 2018; published: 06 dec. 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract healthcare-associated infections are a serious public health concern resulting in morbidity and mortality particularly in developing countries. the lack of information from africa, the increasing rates of antimicrobial resistance and the emergence of new resistance mechanisms intensifies this concern warranting the need for vigorous standardised surveillance platforms that produce reliable and accurate data which can be used for addressing these concerns. the implementation of national treatment guidelines, policies, antimicrobial stewardship programmes and infection prevention and control practices within healthcare institutions require a platform from which it can draw information and direct its approach. in this review, the importance of standardised surveillance systems, the challenges faced in the application of a surveillance system and the condition (existence and nonexistence) of such systems in african countries is discussed. this review also reports on some south african data. introduction infections caused by healthcare-associated pathogens are a public health concern that results in morbidity and mortality particularly in developing countries.1 patients reporting to south african hospitals (both the public and private healthcare sectors) are at high risk of acquiring healthcare-associated infections and the costs of managing these infections cause an additional burden.2 the inappropriate use and overuse of antimicrobial agents leads to the selection of antimicrobial-resistant organisms and since these agents play a critical role in healthcare, increasing rates of resistance creates a serious threat in healthcare settings.3 the world health organization (who) released its first global antimicrobial resistance surveillance report in 2014 which stated that resistance to a wide range of antimicrobial agents is increasing in all six who regions. this report also highlighted that there was a significant gap in surveillance data and a lack of standards for methodology, data sharing and coordination in numerous countries worldwide, stressing the importance of and need for clearly defined, standardised surveillance systems.4 the centre for disease control and prevention (cdc) defines surveillance as the: ongoing systematic collection, analysis and interpretation of health data essential to planning, implementation and evaluation of public health practice, closely integrated with the timely dissemination of these data to those who need to know.5 in a hospital environment, surveillance is an important tool that requires the implementation of an effective and integrated programme encompassing antimicrobial resistance, an antimicrobial stewardship programme and infection prevention and control.2,6 the extent of antimicrobial resistance in healthcare-associated pathogens needs to be established. however, it is challenging for healthcare facilities to perform surveillance due to the lack of standardised definitions for healthcare-associated infections, data analysts, information technology (it) structure and staff trained on infection prevention, particularly in resource-limited settings like sub-saharan africa.7,8 a systematic review of 190 antimicrobial resistance surveillance studies conducted in sub-saharan africa from january 1990 to january 2013 by leopold and colleagues in 2014, further highlighted the flaws in currently available data and the challenges experienced when implementing antimicrobial resistance surveillance.9 although south africa has greater resources as compared to the rest of africa, the quality of these programmes varies across institutions and standardised surveillance systems are not in place in most south african healthcare facilities.2 nevertheless, the south african society for clinical microbiology (sascm) has published yearly surveillance reports from both the public and private healthcare sectors.10 although reports are not accessible in real time, the data are made available. the latest accessible data from the public healthcare sector reported on bloodstream infections over the period january to december 2015 for gram-negative organisms (acinetobacter baumannii complex, enterobacter cloacae complex, escherichia coli, klebsiella pneumoniae and pseudomonas aeruginosa) and gram-positive organisms (staphylococcus aureus, enterococcus faecalis and enterococcus faecium) from five provinces (gauteng, kwazulu-natal, free state, western cape and eastern cape). sascm extracted routine data from sentinel sites predominantly from large academic hospitals from an electronic database.11 sascm also reported information from the private healthcare sector for the period january to december 2013 from urine and bloodstream infections from the same five provinces.12 antimicrobial susceptibility for each of the pathogens was reported from both the public and private healthcare sectors in the country during these periods. the south african national department of health attributed the weaknesses of surveillance and reporting activities to a number of factors. these included the low number of trained microbiologists outside of major urban centres, limited funding, the lack of a national electronic prescribing system and lack of linkage to pharmacy, clinical and laboratory data systems in institutions resulting in incomplete and variably reported information on antimicrobial resistance and consumption.13 in the latest activities from the south african department of health, a national resistance map was released, and combined data for both public and private healthcare sectors on antimicrobial susceptibility levels are available for the first time.14 an example of a successful surveillance programme is the european antimicrobial resistance surveillance scheme (earss), which was developed in 1999 and monitored two bacterial pathogen types (staphylococcus aureus and streptococcus pneumoniae) in 12 laboratories. by 2015, the number of bacterial pathogens monitored increased to eight and included over 900 laboratories.15,16 from this, it is clear that it is important to start off with a manageable number of sites and provide good quality data that can then be expounded. in an effort to improve worldwide antimicrobial resistance surveillance, the global antimicrobial resistance surveillance system (glass) was developed to support the global action plan (gap) on antimicrobial resistance. it is coordinated with the national action plans of countries and aims at enabling countries to generate antimicrobial resistance data that is standardised, comparable and validated. glass combines patient, laboratory and epidemiological surveillance information which when together can aid in understanding the extent of antimicrobial resistance in individual and susceptible populations worldwide.17 currently, the glass early implementation manual focuses on selected bacterial pathogens together with their selected antimicrobial combinations from four clinical specimens.17 while surveillance systems have been established in europe, latin america, central asia and eastern europe, they are lacking in developing countries. the united kingdom department of health therefore launched the fleming fund, aligned with who and glass, to support these countries in developing antimicrobial resistance surveillance systems.18 the african continent and asia are included in this initiative. in addition, the african society for laboratory medicine (aslm), together with other international partners, is in the process of developing a holistic stepwise framework to coordinate laboratory-based antimicrobial resistance surveillance in africa, including south africa.19,20 surveillance of antimicrobial resistance in healthcare-associated pathogens in africa according to the who global report on surveillance,4 antimicrobial resistance is increasing in the african region, and there have been reports of a significant number of antimicrobial-resistant bacteria that are transmissible in the hospital and in the community. since surveillance of antimicrobial-resistant bacteria is performed only in a few african countries, there is a paucity of accurate and reliable information and consequently limited data concerning the true extent of the problem. an additional challenge noted during an external quality assessment of public health laboratories in africa21 showed that many countries experience problems with performing antimicrobial susceptibility testing. from this external quality assessment, a question on the consistency and accuracy of antimicrobial susceptibility testing data arises. furthermore, although some countries have established surveillance programmes, there is a lack of a formal framework across the region.4,21 as part of the who’s gap on antimicrobial resistance, the who has sent an open call for countries to enrol in the glass to participate in a structured surveillance programme and provide reliable and complete demographic data.22 for a national laboratory-based surveillance programme, government commitment and support is non-negotiable. this requires the drafting of policies and strategies and the securing of resources both, financial and human. glass participation also requires the establishment of a coordinating centre which will systematically collect, analyse and share data nationally and internationally. participation also requires a regional reference laboratory which will provide technical support including training, capacity building and strategy advice.20 a review of 12 published articles from 2005 to may 2015 on antimicrobial resistance in east africa23 showed that resistance to commonly used antimicrobial agents was prevalent and that multidrug resistance was increasing in the region. there was scarcity of data on the prevalence of antimicrobial resistance and hospital-acquired infections in developing countries in east africa and the authors suggest that intensive investigation and surveillance is warranted.23 this was again indirectly reiterated in a systematic review published in 2017 of antibiotic-resistant escherichia coli and salmonella data from 2004 to early 2015 in tanzanian healthcare settings.24from this review, a significant increase in resistance was observed. the authors began by stating in their abstract that reliable data are limited and recommend that proactive strategies in antimicrobial stewardship and infection control measures are crucial and must be implemented24, all of which can be solved by establishing a structured surveillance system. surveillance of antimicrobial resistance in healthcare-associated pathogens in south africa in south africa there is a high burden of infectious diseases consisting of a significant population that are of bacterial origin25. a prominent increase in extended-spectrum beta-lactamase (esbl) production and emergence of carbapenemase production in klebsiella pneumoniae and enterobacter spp., multidrug resistance and an increase of carbapenemase production in acinetobacter baumannii and pseudomonas aeruginosa, an increase in multidrug-resistant escherichia coli and resistance in gram-positive isolates with a decline in methicillin-resistant staphylococcus aureus (mrsa) and a steady increase in vancomycin-resistant enterococci (vre) have been noted in various reports26,27,28,29,30,31,32 making use of various methodologies. the need for a systematic and consistent approach in selecting antimicrobials for the treatment of these infections is essential. an established standardised surveillance system will provide information on causative pathogens and antimicrobial susceptibility patterns of these pathogens. in addition, the antimicrobial spectrum of a selected agent, the use of appropriate dosing schedules based on the selected agent’s pharmacokinetic and pharmacodynamic properties and pharmacological considerations in the patient should be included. an intervention strategy such as directing therapy to narrow-spectrum agents once microbiology results become available should also be established as well as the routes of transmission when a resistant strain is recovered from more than one individual. once this information is known, an intervention can be introduced and subsequently proved effective.2,7 a report by crowther-gibson and colleagues in 201125 aimed at summarising surveillance efforts in south africa. this report reviewed the national burden of disease and levels of antimicrobial resistance in common bacterial infections and showed that antimicrobial resistance in healthcare-associated infections varied among antimicrobial agents in the public and private healthcare sectors. this report indicated the need for continuous monitoring as an effort to observe and control the spread of resistance. limitations noted were that the causes of illness and deaths were not well documented, a common occurrence in low-resource countries. distinguishing viral from bacterial diseases requires a level of detail that did not exist in the majority of the cases.25 other efforts to publish south african data is the sentry antimicrobial surveillance programme which is an international programme documenting antimicrobial resistance in predominantly healthcare-associated pathogens.33,34,35,36,37,38,39,40,41,42,43 surveillance data, however, do not report findings from south africa exclusively hence findings specific to the country cannot be extracted. antimicrobial resistance surveillance in healthcare-associated infections is particularly important because it is crucial to understand the dynamics in a specific healthcare setting. however, as patients are often transferred between wards and hospitals it becomes problematic to establish a system that would work in all healthcare settings due to differences in staff complement, antimicrobial stewardship programmes and infection prevention and control practices. these differences highlight this challenge and is further reiterated in a 6-month surveillance study conducted in tygerberg children’s hospital, cape town, in 2015 whose aim was to evaluate three surveillance methods. point prevalence surveys, laboratory surveillance and tracking of antimicrobial prescriptions were evaluated against the reference method which was prospective clinical healthcare-associated infection surveillance. while a combination of antimicrobial prescription tracking and laboratory surveillance produced the best results in their setting, the authors concluded that healthcare facilities should select a suitable method subject to available resources and practice context.44 however, as indicated repeatedly in the current review, the problem that exists with such a recommendation is that methodologies are not standardised and in order to obtain a holistic picture of antimicrobial resistance in healthcare-associated infections in south africa, standardised methods need to be implemented. our group at the antimicrobial resistance laboratory, which is a national reference laboratory at the national institute for communicable diseases (nicd), established and commenced (through the germs-sa platform) a standardised laboratory-based antimicrobial resistance surveillance (lars) programme of pathogens causing healthcare-associated infections such as the eskape pathogens (enterococcus faecium, staphylococcus aureus, klebsiella pnemoniae, acinetobacter baumanii, pseudomonas aeruginosa and enterobacter species) at sentinel sites in 2010 and electronic surveillance from routine laboratories in 2013. the primary objectives of the lars programme were to determine the number of cases reported from selected hospitals for selected pathogens, to describe antimicrobial susceptibility of the significant treatment regimens for the pathogens45 and a secondary objective was molecular characterisation of resistance genes.26,27,30 in 2014, antimicrobial resistance surveillance data on klebsiella pneumoniae isolates from patients with bacteraemia were published30; isolates were submitted by sentinel laboratories in five regions of south africa from mid-2010 to mid-2012. this included 13 academic centres serving the public healthcare sector in the gauteng, kwazulu-natal, free state, limpopo and western cape provinces. findings from this study showed that of the 2774 isolates, 1895 (68%) expressed esbls and displayed resistance to cefotaxime, ceftazidime and cefepime. more so, 46% of all isolates were resistant to ciprofloxacin and 33% to piperacillin-tazobactam. susceptibility to aminoglycosides was variable: an average of 95% were susceptible to amikacin but only 31% were susceptible to tobramycin and gentamicin. susceptibility to ciprofloxacin (53%) was lower than to levofloxacin (75%) while susceptibility to cefoxitin was higher (87%). the minimal inhibitory concentration (mic) measures, the lowest concentration required to inhibit the growth of 50% (mic50) and 90% (mic90) of the isolates, were stable over the 3-year period and there was a small but statistically significant trend (p < 0.001–0.41) towards a decrease in susceptibility to many antimicrobial agents. molecular characterisation was performed on 270 phenotypically esbl-producing isolates. the presence of esbls (blactx-m, blashv and blatem genes) were confirmed in all 270 isolates with 93% of the isolates tested expressing more than one resistance gene. the majority of the isolates (95%) were phenotypically susceptible to the carbapenems tested, and no isolate contained blakpc or blandm-1, which were the only carbapenemases screened for. the authors stated that the high proportion of esbl-producing k. pneumoniae isolates and the prevalence of esbl genes is of great concern.30 more recently data from a cross-sectional study on carbapenem-resistant enterobacteriaceae (cre) isolates showed a 1.9% prevalence of carbapenemases in blood culture isolates.26 another publication from lars-generated surveillance data investigated staphylococcus aureus bacteraemia in academic hospitals from gauteng, south africa.46 in this study, the epidemiology of s. aureus bacteraemia was described and factors associated with mrsa infection were determined. cases of s. aureus bacteraemia over 1 year (september 2012 to september 2013) were reported from three sentinel sites and detailed clinical information was collected. statistical analysis included multivariable logistic regression to determine factors associated with mrsa infection and mortality. this study showed that 442 cases of s. aureus bacteraemia were reported and antimicrobial susceptibility testing was performed on 54% of the isolates (n = 240). methicillin-resistant staphylococcus aureus infection was noted in 36% (n = 86) of cases. independent predictors of mrsa included a longer hospital stay before positive specimen collection, hospitalisation in the last year, human immunodeficiency virus (hiv) infection and antimicrobial use in the previous 2 months. the only independent predictor of mortality among cases with s. aureus bacteraemia was being elderly. antimicrobial susceptibility testing results showed that mrsa isolates were non-susceptible to more antimicrobial agents compared to methicillin-susceptible s. aureus (mssa) isolates. all isolates were susceptible to daptomycin and linezolid. four isolates were non-susceptible to vancomycin and teicoplanin (mic 16 mg/ml) while 13 cases (6%) (five mrsa and eight mssa isolates) showed a susceptible breakpoint value (mic 2 mg/ml). for molecular characterisation, sccmec typing was performed on 82 mrsa isolates. nine isolates were non-typeable; the most common sccmec type was type iii (56%), followed by type iv (29%) typically associated with hospitaland community-acquired infections, respectively. type ii accounted for only four isolates and no type i isolates were found. clinical information was available for 140 cases. according to the authors, majority of patients (86%, 121/140) received one or more antimicrobial agents, 95 cases received empirical treatment while 42 received directed treatment. of those that received empirical treatment (n = 95), a greater proportion of mssa cases (95%, 55/58) received appropriate empirical therapy compared to mrsa cases (57%, 21/37) and this was statistically significant (p < 0.001). of those that received directed treatment (n = 42), all mssa cases (25/25) received appropriate directed therapy while this was true for only 59% (10/17) of mrsa cases; again, this was statistically significant (p < 0.001). this study demonstrated that antimicrobial prescription practices should be monitored and that knowledge of local epidemiology as well as factors predictive of mrsa infection is important as it will assist in guiding the use of appropriate empirical treatment. the importance of antimicrobial resistance surveillance as a public health measure was also highlighted.46 a more recent lars study published in 201527 investigating the prevalence and trends of s. aureus bacteraemia in 2709 hospitalised patients from 13 academic centres in south africa in 2010 to 2012 examined antimicrobial resistance and the molecular epidemiology for laboratory-based surveillance. forty-six per cent of cases were mrsa and there was a significant decline of mrsa from 53% to 40% over the 3-year period. geographical distribution showed that mrsa was significantly higher in gauteng compared to the other provinces and children younger than 5 years of age were associated with higher mrsa rates compared to all other age groups. methicillin-resistant staphylococcus aureus isolates were resistant to more classes of antimicrobial agents compared to mssa isolates. isolates were fully susceptible to glycopeptides, daptomycin, linezolid and quinupristin-dalfopristin. while ciprofloxacin and trimethoprim-sufamethoxazole resistance declined significantly over the surveillance period, resistance to macrolides, aminoglycosides, tetracycline, rifampin and mupirocin remained comparable. the mic50 and mic90 for all agents remained stable from 2010 to 2012. the most prevalent sccmec type distribution was consistent with the previous study,41 with type iii predominating (41%) followed by type iv (31%). further molecular studies found 47 different spa-types with the five most common spa-types accounting for 87% of the isolates and were t037, t1257, t045, t064 and t012. the most common multilocus sequence type (mlst) was st612 clonal complex 8 (cc8) (n = 7), followed by st5 (cc5) (n = 4), st36 (cc30) (n = 4) and st239 (cc8) (n = 3). overall, this study revealed the presence of a variety of hospital-acquired mrsa clones in south africa and a dominance of a few clones, a result that was similar to previous findings in south africa,47,48,49 indicating a slow evolution of circulating clonal types. this study demonstrated the importance of monitoring trends in resistance and molecular typing to detect changing epidemiological trends in antimicrobial resistance patterns.27 in south africa, the antimicrobial resistance laboratory at the nicd has been assigned as the who collaborating centre for antimicrobial resistance surveillance and in april 2017, responded to the first data call from the glass.22 germs-sa data for s. aureus isolated from blood culture specimens submitted from 2015 to 2016 showed a decrease in cefoxitin resistance, from 31.4% (n = 225/882) to 24.9% (n = 182/867) (unpublished data). secondary electronic surveillance for a select panel of bacteria (eskape) modelled on the glass pathogen-antimicrobial combination is utilised for the purpose of complementing the well-established germs-sa surveillance system. antimicrobial resistance data are channelled and formatted as antimicrobial resistance maps for both public and private healthcare sector laboratory results, and are made available on the nicd website.14 these antimicrobial resistance maps are summarised and published as annual reports on the federation of infectious diseases societies of southern africa (fidssa) website10. in 2016, klebsiella pneumoniae isolates demonstrated 65% susceptibility to cefepime, 44% to piperacillin-tazobactam and 41% to gentamicin, while less than 30% of escherichia coli isolates were non-susceptible to cephalosporins. for the non-fermentative gram-negative bacteria, more than 80% of acinetobacter baumannii isolates were non-susceptible to carbapenems, while 80% and 75% of pseudomonas aeruginosa isolates were susceptible to cephalosporins and carbepenems respectively. for the gram-positive bacteria, more than 99% of enterococcus faecalis and enterococcus faecium isolates demonstrated susceptibility to oxazolidinones and 1% and 5% of e. faecalis and e. faecium isolates demonstrated susceptibility to glycopeptides respectively. staphylococcus aureus isolates demonstrated higher susceptibility to cloxacillin, from 65% in 2015 to 69% in 2016 (unpublished data). the findings from these studies and other ongoing studies which have not as yet been published show that south african data is being generated from the expanding surveillance programme in the hope of elucidating the situation of antimicrobial resistance in healthcare-associated pathogens in south africa. conclusion antimicrobial resistance surveillance in healthcare-associated settings, e.g. hospitals, long-term care facilities, dialysis clinics, etc., is essential in order to gain an understanding of the prevalent healthcare-associated pathogens such as the eskape organisms, the antimicrobial resistance patterns of critical organisms and their mechanisms of resistance. national laboratory systems should support the surveillance for antimicrobial resistance and implement standardised practices, manuals and quality systems. in addition, this information will assist in the development of national treatment guidelines, inform policies and strategies for antimicrobial stewardship programmes and infection prevention and control. due to the lack of reliable and accurate information in african countries, there is an urgent need for the implementation of standardised surveillance programmes. acknowledgements competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support none. authors’ contributions a.s.-m. conceptualised and wrote the review manuscript. h.i. wrote and edited the manuscript. o.p. was instrumental in coordination and editing of the manuscript. references sonmezer mc, ertem g, erdinc fs, et al. evaluation of risk factors for antibiotic resistance in patients with nosocomial infections caused by pseudomonas aeruginosa. can j infect dis med microbiol. 2016;2016:1321487. brink a, feldman c, duse a, et al. guideline for the management of nosocomial infections in south africa. s afr med j. 2006;96(7 pt 2):642–652. https://doi.org/10.1080/10158782.2006.11441269 cui d, liu x, hawkey p, et al. use of and microbial resistance to antibiotics in china: a path to reducing antimcobial resistance. j int med res. 2017;0(0):1–11. https://doi.org/10.1177/0300060516686230 world health organization (who). 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2014. fortuin-de smidt mc, singh-moodley a, badat r, et al. staphylococcus aureus bacteraemia in gauteng academic hospitals, south africa. int j infect dis. 2015;30:41–48. https://doi.org/10.1016/j.ijid.2014.10.011 jansen van rensburg mj, madikane ve, whitelaw a, chachage m, haffejee s, elisha bg. the dominant methicillin-resistant staphylococcus aureus clone from hospitals in cape town has an unusual genotype: st612. clin microbiol and infect dis. 2011;17:785–792. https://doi.org/10.1111/j.1469-0691.2010.03373.x moodley a, oosthuysen wf, duse ag, marais e. molecular characterization of clinical methicillin-resistant staphylococcus aureus isolates in south africa. j clin microbiol. 2010;48(12):4608–4611. https://doi.org/10.1128/jcm.01704-10 orth h, dreyer zs, makgotlho e, oosthuysen wf, sinha a, wasserman e. characterisation of staphylococcus aureus bacteraemia at tygerberg hospital. south afr j epidemiol infect. 2013;28(1):22–27. https://doi.org/10.1080/10158782.2013.11441515 abstract introduction methods results discussion acknowledgements references about the author(s) walaa samy department of medical biochemistry, faculty of medicine, zagazig university, zagazig, egypt osama a. gaber department of medical microbiology and immunology, faculty of medicine, zagazig university, zagazig, egypt rania m. amer department of medical microbiology and immunology, faculty of medicine, zagazig university, zagazig, egypt nahawand a. el-deeb department of internal medicine, faculty of medicine, zagazig university, zagazig, egypt ahmed a. abdelmoaty department of tropical medicine, faculty of medicine, zagazig university, zagazig, egypt ahmed l. sharaf department of tropical medicine, faculty of medicine, zagazig university, zagazig, egypt ahmed m. el-gebaly department of tropical medicine, faculty of medicine, zagazig university, zagazig, egypt rasha mosbah department of infection prevention and control, zagazig university hospitals, zagazig university, zagazig, egypt maha e. alsadik chest department, faculty of medicine, zagazig university, zagazig, egypt amal fawzy department of medical biochemistry, faculty of medicine, zagazig university, zagazig, egypt alshymaa a. ahmed department of clinical pathology, faculty of medicine, zagazig university, zagazig, egypt citation samy w, gaber oa, amer rm, et al. role of ace2 and tmprss2 polymorphisms in clinical severity and outcomes of covid-19 in egypt. afr j lab med. 2024;13(1), a2375. https://doi.org/10.4102/ajlm.v13i1.2375 note: additional supporting information may be found in the online version of this article as online supplementary document 1. original research role of ace2 and tmprss2 polymorphisms in clinical severity and outcomes of covid-19 in egypt walaa samy, osama a. gaber, rania m. amer, nahawand a. el-deeb, ahmed a. abdelmoaty, ahmed l. sharaf, ahmed m. el-gebaly, rasha mosbah, maha e. alsadik, amal fawzy, alshymaa a. ahmed received: 21 nov. 2023; accepted: 23 may 2024; published: 27 aug. 2024 copyright: © 2024. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the clinical presentations of coronavirus disease 2019 (covid-19) exhibit significant variation, ranging from asymptomatic cases to mortality resulting from severe pneumonia. host genetics can partially explain this variation. objective: this study evaluated possible associations between severity and outcome of covid-19 and single nucleotide polymorphism (snp) rs2285666 in the ace2 gene and snp rs2070788 in the tmprss2 gene. methods: the study included a sample of 100 consecutive adult patients admitted to the covid-19 isolation and intensive care units of the zagazig university hospitals, zagazig, egypt from july 2021 to november 2021. for rs2285666, polymerase chain reaction-restriction fragment length polymorphism was carried out. for rs2070788, real-time polymerase chain reaction was performed. results: for rs2285666, the ga genotype was the most frequent among female patients (39% [16/41]) and the a genotype was more prevalent among male patients (54.2% [32/59]). for rs2070788, the aa genotype was the most frequent among all patients (46% [46/100]). no rs2285666 or rs2070788 genotypes or allele frequencies had significant associations with either severity or outcomes of patients. conclusion: this study found no significant associations of covid-19 severity or outcomes of patients with genotypes or allele frequencies of the rs2285666 snp in the ace2 gene or the rs2070788 snp of the tmprss2 gene. the search for other genetic associations with covid-19 infection is still required. what this study adds: the study reveals that host genetics explain the variation observed in the disease. specific genetic variants can confer either increased susceptibility or resistance to the disease. keywords: ace2 gene; tmprss2 gene; single nucleotide polymorphisms; covid-19; severity; outcome. introduction coronavirus disease 2019 (covid-19), which is caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2), has resulted in more than 6 million fatalities and 603 million confirmed cases since its identification in 2019 until 07 september 2022.1 the clinical presentations of the covid-19 infection showed significant variation, ranging from asymptomatic cases to mortality resulting from severe pneumonia. host genetics can account for part of this variation, as some genetic variants can confer either increased susceptibility or resistance to the disease.2 similar to all viruses, sars-cov-2 undergoes genetic mutations over time, which can potentially affect various aspects of the virus, including its ability to spread and infect, the severity of symptoms and disease, the efficacy of vaccines, diagnostic methods, and other public health interventions. since the outbreak of the pandemic, sars-cov-2 has been undergoing continuous evolution. omicron, the most divergent variant of concern, is still evolving, both genetically and antigenically. there is a possibility that it may be able to evade the existing population immunity compared with the pre-omicron strains. also, omicron prefers to involve the upper rather than the lower respiratory tract; therefore, ongoing research on the sars-cov-2 virus is still required.3 the sars-cov-2 virus binds to host cell surface receptors by utilising spike (s) protein to mediate the fusion of the viral envelope with the cell membrane. the s protein is composed of s1 and s2 subunits. the subunit s1 contains the receptor-binding domain, that interacts with the peptidase domain of the angiotensin-converting enzyme 2 (ace2) protein. this step is critical for the viral entry into the cell. the ace2 protein is a well-characterised negative regulator of the renin-angiotensin system, which catalyses the conversion of angiotensin ii to the heptapeptide angiotensin (1–7), which produces the opposite effect to that of angiotensin ii.4 the ace2 protein plays two opposing roles in covid-19 infection. firstly, it acts as a potent receptor for the virus to enter the cell. secondly, it has a protective function in maintaining the balance of angiotensin ii, a hormone involved in regulating blood pressure. however, when there is an excessive buildup of angiotensin ii, it can lead to the release of large amounts of cytokines, causing a cytokine storm and subsequent tissue damage.5 the interaction between sars-cov-2 and ace2 protein receptors results in a decrease in ace2 protein expression and a reduction in its protective function on the angiotensin ii pathway. therefore, the activation of ace2 has been proposed as a potential therapy for covid-19. this emphasises the significance of ongoing research on ace2 gene polymorphisms and their functional impacts. this is important not only because the ace2 protein receptor is involved in the development of infections, but also because it is suggested to be a potential target for therapy.6 the ace2 gene is located on chromosome xp22; a functional single nucleotide polymorphism (snp) in the ace2 gene ‘g8790a (rs2285666)’ in intron 3 could alter mrna splicing and so affect ace2 gene expression.7 in addition, rs2285666 is suggested to alter the binding affinity of the ace2 protein receptor for sars-cov-2.8 receptor binding is facilitated by the human enzyme transmembrane protease serine 2 (tmprss2),9 a family of membrane-anchored proteases expressed on the airway epithelial cell surface. it acts as a viral entry cofactor by cleavage of the viral spike s protein to s1 and s2 subunits to facilitate the interaction with the ace2 protein receptor’s extracellular domain.10 therefore, genetic variations in ace2 and tmprss2 nucleotide sequences may change the interaction of receptor-binding domain and peptidase domains, altering the host’s susceptibility to the virus and influencing the disease’s severity and outcome. the tmprss2 gene is located on chromosome 21q22. rs2070788 is a g to a transition snp located in intron 11–12 of the tmprss2 gene and was found to be associated with reduced tmprss2 gene expression.11 based on that, it was proposed that the aforementioned snps can influence the course of covid-19 infection. this work was performed to evaluate the possible association between the ace2 (rs2285666) and tmprss2 (rs2070788) snps and clinical severity and outcomes among covid-19 patients. methods ethical considerations the institutional review board of the faculty of medicine, zagazig university, approved this work (zu-irb#6775). participants voluntarily signed a written informed consent. procedures were carried out in agreement with the declaration of helsinki. patients’ data files and sample tubes were coded, so laboratory workers were blinded to patients’ information. study design and patient criteria this prospective cohort study was performed in the biochemistry and clinical pathology departments in collaboration with the scientific and medical research center, faculty of medicine, zagazig university, zagazig, egypt, from 01 july 2021 to 30 november 2021. the study included 100 consecutive patients, ≥ 18 years old, from both genders and the same ethnicity, admitted to the covid-19 clinic, isolation and intensive care units of the zagazig university hospitals, zagazig, egypt. patients who refused to participate were excluded from the study. diagnosis and routine investigations patients were selected based on the covid-19 case definition provided by the world health organization.12 nasopharyngeal and oropharyngeal swabs were collected and processed according to the centres for disease control guidelines.13 the diagnosis of covid-19 patients was made by real-time polymerase chain reaction (pcr) of the collected swabs, at the scientific and medical research centre according to the guidelines of the egyptian ministry of health and as described previously.14 cycle threshold values were used as an indicator of viral load.15 routine laboratory workup data including full blood counts, liver, kidney function tests, c reactive protein (crp), lactate dehydrogenase, plasma ferritin, troponin t, and d dimer were collected from patients’ files. additionally, all patients underwent routine chest computed tomography scans; results were collected from patients’ files after obtaining the required permissions. the egyptian ministry of health and population management roles were applied to stratify confirmed patients based on severity. mild cases include all symptomatic patients with no radiological signs of pneumonia. moderate cases are those with pneumonia manifestation with positive radiological signs and do not meet the criteria of severe cases. severe cases are those with a respiratory rate > 30, oxygen saturation at room air < 92, and chest imaging showing > 50% lesions or progressive lesions within 1 day or 2 days, while critically ill patients are severe patients who still have oxygen saturation at room air < 92 or respiratory rate > 30, despite receiving oxygen therapy.14 asymptomatic, mild, and moderate cases without risk were referred to home isolation with close follow-up. conversely, moderate cases were hospitalized in covid-19 areas, severe cases were hospitalized in intermediate care units, while critically ill patients were hospitalized in intensive care units. therapy was carried out according to the aforementioned guidelines. routine laboratory workup was performed in the clinical pathology department laboratories. based on the severity, patients were classified as mild, moderate, or severe cases, and based on the outcome patients were classified as survived and died. demographic criteria, history of vaccination, comorbidities, occurrence of complications, viral load, and laboratory results were compared in between each two groups. according to the guidelines of the egyptian ministry of health, parenteral prophylactic anticoagulants were prescribed for hospitalised patients to guard against cardiovascular complications. in case of suspected pulmonary embolism, as most of these patients are haemodynamically unstable, and because of the risk of medical staff exposure to infection, diagnosis was based on echocardiography and d-dimer level rather than ct-angiography.16 genotyping according to manufacturers’ guidelines, a volume of 2 ml of ethylenediaminetetraacetic acid anti-coagulated blood was obtained for dna extraction using the qiaamp blood kit (qiagen gmbh, hilden, germany). for the ace2 snp, rs2285666, pcr and restriction fragment length polymorphism were used with the primers: forward 5′-cat gtg gtc aaa agg ata tct-3′ and reverse 5′-aaa gta agg ttg gca gac at-3′. polymerase chain reaction mixture included 12.5 µl of dreamtaq green pcr master mix (thermo scientific™, vilnius, lithuania), 10 pmol of forward and reverse primers, 100 ng of extracted genomic dna, and nucleases-free water up to 25 µl. the thermal cycling conditions commenced with an initial denaturation at 95 °c for 5 min, then 25 cycles (95 °c for 30 s, 60 °c for 40 s, and 72 °c for 30 s), ended by a final extension for 3 min at 72°c. the pcr products were digested using alui (thermo fisher scientific inc., toronto, ontario, canada) at 37 °c for 3 h.17 the amplified and digested products were examined using 2% agarose gel electrophoresis for 30 min and visualized utilising an ultraviolet transilluminator. the amplicon size was 817 bp, after restriction; the g allele appears as one band at 817 bp, and the a allele appears as two bands at 589 bp and 228 bp. in women, the ag genotype appears as three bands at 817 bp, 589 bp, and 228 bp.18 for the tmprss2 snp, rs2070788, a real-time polymerase chain reaction assay was performed, snp genotyping assay that contains the specific primer-probe mixture and taqman universal pcr master mix were purchased from thermo fisher scientific inc. (vilnius, lithuania), amplification and detection were carried out on quantstdiotm5 real-time polymerase chain reaction system (applied biosystems, siemens healthcare diagnostics inc, singapore); all procedures were carried out according to manufacturers’ guidelines. the reaction mixture was as follows: 10 µl of (2x) taqman universal pcr master mix, 20 units (0.5 µl) of the primer-probe assay, 1 µl of the extracted genomic dna, and 8.5 µl nuclease-free water making the final volume of the mixture 20 µl. the real-time polymerase chain reaction cycling conditions were 60 °c for 2 min, initial denaturation at 95 °c for 10 min then 40 cycles of denaturation at 95 °c for 15 s, and annealing -extension at 60 °c for 1 min.19 data analysis and results interpretation were carried out automatically by the instrument’s software based on the default cycle threshold cutoff. data analysis the spss® statistical software version 22 (ibm corp., armonk, new york, united states) was used for data analysis. numeric values are presented as mean ± standard deviation if it was normally distributed (age, haemoglobin concentration, and platelet counts), while for not-normally distributed data (total and differential leukocyte counts, prothrombin time, kidney function tests, liver enzymes, lactate dehydrogenase, ferritin, crp, and cycle threshold value) the median (range) was used. student’s t-test and one-way analysis of variance with least significant differences post hoc test were used to compare means; mann-whitney u test and krusskal-wallis with bonferroni post hoc tests were used for medians comparison. frequencies were expressed as numbers (percentages). chi-square test and the two-tailed fisher’s exact test for results ≤ 5 were used to compare frequencies. p-values < 0.05 were considered statistically significant differences. after comparing all variables either qualitatively or quantitatively between different groups, all variables that have p < 0.1 were tested by the multivariate logistic regression, and significant results are presented as p-value, odds ratio and 95% confidence interval. because ace2 is an x-linked gene, ace2 g8790a genotypes were analysed separately in female and male patients. results this study included 100 patients with covid-19 admitted to zagazig university hospitals; their ages ranged from 35 years to 80 years, 59% (59/100) of them were male patients, and 41% (41/100) were female patients. apart from smoking (36%), no other substance abuse, including alcohol, has been reported; 56% of patients had history of other medical diseases including hypertension (31%), diabetes mellitus (19%), cardiovascular diseases (14%), chronic obstructive lung diseases (8%) and malignancies (6%) (table 1). prior to the globalisation of the vaccination programme in egypt, only 14% of patients had been administered the sinopharm vaccine, while 2% had received the oxford astrazeneca vaccine. among the patients, fever was observed in 95% of cases, pharyngitis in 94% of cases, and chest pain in 61% of cases; 4% were diagnosed by pcr before the appearance of symptoms. concerning severity, 51%, 34% and 15% were severe, moderate and mild cases, while 44% died by the end of the study. nineteen patients (19%) were complicated with secondary bacterial pneumonia; 12 (12%) were infected with methicillin-resistant staphylococcus aureus, 5% with klebsiella pneumoniae, and 2% with acinetobacter spp. seventeen (17%) patients had cardiovascular complications in the form of lower limb ischaemia (2%), deep vein thrombosis (7%), pulmonary embolism (5%), and cerebrovascular stroke (3%). ten (19.6%) of the severe patients group and 7 (14.2%) of the mild or moderate group had cardiovascular complications, 10 (58.8%) of whom had comorbidities in the form of hypertension (6/10; 60%), diabetes mellitus (4/10; 40%), cardiovascular diseases (3/10; 30%) and malignancy (1/10; 10%), and 7 of whom (41.2%) did not survive. table 1: patient characteristics (n = 100), faculty of medicine, zagazig university, zagazig, egypt, 01 july 2021 – 30 november 2021. there were statistically significant associations between age and severity (p = 0.03) (online supplementary table 1). complications occurred more frequently among severe patients (p < 0.001). statistically significant increases among severe cases were observed for both kidney function markers (p = 0.03) and crp (p < 0.001). there were no significant associations between either demographic features or laboratory data and patient outcome (online supplementary table 2). a statistically significant increase in mortality rate was observed in association with severity (p < 0.001) and the occurrence of complications (p = 0.02). for the ace2 snp rs2285666, the ga genotype was the most frequent among female patients (39%), while among male patients the a genotype was more prevalent (54.2%). for the tmprss2 snp rs2070788, the aa genotype was the most frequent among all patients (46%). ace2 rs2285666 and tmprss2 rs2070788 genotypes and allele frequencies had no significant associations with either disease severity or patient outcomes, as shown in table 2 and table 3. multivariate logistic regression revealed that crp level was the only independent predictor of covid-19 severity in this cohort: p = 0.001, odds ratio (95% confidence interval) were 1.1 (1.0–1.2) (data not presented). furthermore, it was found that severity was an independent predictor of outcome: p < 0.001, odds ratio (95% confidence interval) were 46.5 (10.2–211.2). table 2: associations between covid-19 severity and single nucleotide polymorphisms of the ace2 and tmprss2 genes, faculty of medicine, zagazig university, zagazig, egypt, 01 july 2021 – 30 november 2021. table 3: associations between covid-19 outcomes and single nucleotide polymorphisms of the ace2 and tmprss2 genes, faculty of medicine, zagazig university, zagazig, egypt, 01 july 2021 – 30 november 2021. discussion the mean age of patients who developed severe disease was significantly higher than mild or moderate cases. this aligns with many previous findings that age is a major risk factor for covid-19, and the most unfavourable outcomes are attributed to age-related comorbidities and immunocompromising status.20 patients with severe disease showed a slight but significant increase in serum urea and creatinine. hachim et al.21 studied the kidney function in 250 covid-19 patients in the middle east region. the study reported an association between covid-19 severity and renal impairment. additionally, these results agree with a study conducted by gabarre et al. in 2020 in paris, france,22 in which 42.9% of critically ill patients developed acute kidney injury. this finding highlights the value of evaluating kidney function tests in patient monitoring. although it was elevated in 94% of patients, crp showed a sharp increase in severe cases, and it was the only independent predictor for patients’ severity in this work. these results go with those of wang et al. in 2020 in china,23 who reported that all patients who developed severe diseases displayed elevated crp results and that crp was the only independent predictor of severity. potempa et al.24 revealed that crp was a reproducible, rapid, and cost-effective marker to evaluate the extent of tissue damage and inflammatory process in covid-19 patients. the viral load did not significantly influence disease severity. prior research has demonstrated conflicting findings on this topic, cho et al. in 2020 in hong kong, china,25 reported that viral load is not associated with either severity or outcome. abdulrahman et al.26 explain that the correlation between disease severity and crp level, rather than viral load, can be attributed to the influence of host factors on disease progression and the contribution of pro-inflammatory cytokines to the severity of the disease. the mortality rate was higher in severe cases than in mild or moderate cases, particularly in those who encountered complications during the disease course. this result agrees with previous studies in egypt and worldwide. gaber et al., in 2022 in egypt,27 reported that oxygen saturation and chest computed tomography severity score are predictors of mortality. furthermore, assal et al., in 2022 in egypt,28 found that the need for mechanical ventilation and the occurrence of secondary bacterial infections are associated with higher mortality. additionally, mahendra et al., in 2021 in india,29 indicated that severe pneumonia is associated with a higher fatality rate. angiotensin-converting enzyme 2 and tmprss2 gene polymorphisms have been implicated previously in altering the susceptibility to covid-19 infection and influencing severity and mortality rates.30 wang et al., in 2020 in china,31 demonstrated that ace2 snps-rs4646116, rs267606406, and rs143936283 were associated with higher affinity to the receptor-binding domain. in contrast, ace2 snps-rs1244687367, rs146676783, and rs961360700 were associated with lower binding affinity and hence decreased susceptibility to covid-19 infection. also, tmprss2 gene polymorphisms have been associated with susceptibility to covid-19 infection and outcome.32 in a study on italian patients in 2020, asselta et al. revealed that tmprss2 snps-rs2070788, rs9974589, and rs7364083 were associated with increased severity of covid-19.32 moreover, fuentes et al. reported that rs61735794 and rs61735792 were significantly associated with covid-19 infection outcome.33 in this work, no significant associations were detected between either the ace2 rs2285666 snp or the tmprss2 rs2070788 snp and covid-19 severity and outcome. the aa genotype of rs2285666 (g8790a) was found to increase ace2 gene expression up to 50%.34 however, controversies have been observed regarding the effect of this point mutation; srivastava et al., in 2020 in india,35 demonstrated a negative correlation between the alternate allele (a) and case fatality rate of sars-cov-2 infection, and möhlendick et al., in 2021 in germany,36 reported a threefold increase in severity in cases carrying the gg genotype. hence, they suggested that rs2285666 harbours a protective role against covid-19. a possible explanation for this finding is that the increased serum and tissue-bound ace2 protein in patients carrying the aa genotype counteracts the pro-inflammatory and fibrotic effects of angiotensin ii.37 however, martínez-gómez et al., in 2022 in mexico,38 and sabater et al., in 2022 in spain,39 found that the aa genotype was associated with critical outcome. the current work’s results agree with those of alimoradi et al., in 2022 in iran,18 karakaş et al., in 2021 in turkey,40 and elnagdy et al., in 2022 in egypt,41 that there is no significant association between rs2285666 and covid-19 severity and outcome. variations in findings between studies can be attributed to factors such as ethnicity variations, sample size, patients’ habits, and, notably, viral load. in addition, along with genetic variations, many other factors can influence the balance between the susceptibility and protective roles of ace2 protein, such as smoking, diet, comorbidities, age, sex, and cleavage of the ace2 protein by products of the tmprss2 and adam17 genes.5 pandey et al., in 2022 in india,42 and asselta et al., in 2020 in italy,32 observed that the tmprss2 snp rs2070788 had a statistically significant association with disease severity and fatality rate in american and italian populations, respectively. another multicentre study by irham et al. in 202011 showed that rs2070788 and another three snps, which affect the expression of the tmprss2 gene in lung tissues, were more prevalent in american and european populations than in asian populations; this finding can explain the higher susceptibility and fatality rates in american and european populations. schönfelder et al., in 2021 in germany,43 observed no significant association between rs2070788 and covid-19 severity. in addition, they attributed differences among several studies to variations in sample size, ethnicity, and the possibility of the presence of linkage disequilibrium with rs383510. limitations this study has some limitations that should be considered when interpreting the findings. firstly, this was a single-centre study with a relatively low sample size. thus, the results may be insufficiently powered and require further confirmation by future multicentre studies with larger cohorts. secondly, host genetics is not the only factor that can influence covid-19 disease: the host immunological status as well as sars-cov-2 strain variations have impacts on disease severity and outcome. in addition, an interplay between all of these factors exists; no data were available about patients’ antibody response or viral genotypes so these factors could not be included in the analyses. this may have resulted in insufficient data about the impact of these factors on disease behaviour. conclusion the genotypes and allele frequencies of the ace2 gene snp rs2285666 and the tmprss2 gene snp rs2070788 showed no significant associations with the severity and outcome of patients. further investigation into additional genetic correlations with covid-19 infection is still necessary. acknowledgements the authors would like to thank the staff members of the zagazig scientific and medical research center, dr manal abd alhafeez, dr mariem a. soliman, mrs samah alsaeed and mr peter maher, for their assistance during the laboratory work. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions n.a.e.-d., a.a. abdelmoati, a.l.s., w.s., a.m.e.-g., o.a.g., r.m., a.f., r.m.a., m.e.a. and a.a. ahmed contributed to the study’s conceptualisation and design. n.a.e.-d., a.a. abdelmoati, a.l.s., w.s., and a.m.e.-g. contributed to patient selection and data collection. w.s., o.a.g., r.m. and a.f. contributed to sample collection and processing and manuscript revision. r.m.a., a.f. and m.e.a. contributed to data collection and analysis, and manuscript revision. a.a. ahmed contributed to data analysis and manuscript writing. finally, the manuscript has been read and approved by all the authors. all authors are responsible for the reported research. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the data that support the findings of this study are available on request from the corresponding author, a.a. ahmed. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. it does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the authors are responsible for this article’s results, 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the novel coronavirus, and the renin-angiotensin system; the need for clinical trials. front med. 2020;7:248. https://doi.org/10.3389/fmed.2020.00248 martínez-gómez le, herrera-lópez b, martinez-armenta c, et al. ace and ace2 gene variants are associated with severe outcomes of covid-19 in men. front immunol. 2022;13:812940. sabater molina m, nicolás rocamora e, bendicho ai, et al. polymorphisms in ace, ace2, agtr1 genes and severity of covid-19 disease. plos one. 2022;17(2):e0263140. https://doi.org/10.1371/journal.pone.0263140 karakaş çelik s, çakmak genç g, pişkin n, et al. polymorphisms of ace (i/d) and ace2 receptor gene (rs2106809, rs2285666) are not related to the clinical course of covid-19: a case study. j med virol. 2021;93(10):5947–5952. https://doi.org/10.1002/jmv.27160 elnagdy mh, magdy a, eldars w, et al. genetic association of ace2 and tmprss2 polymorphisms with covid-19 severity; a single centre study from egypt. virol j. 2024;21(1):27. https://doi.org/10.1186/s12985-024-02298-x pandey rk, srivastava a, singh pp, chaubey g. genetic association of tmprss2 rs2070788 polymorphism with covid-19 case fatality rate among indian populations. infect genet evol. 2022;98:105206. https://doi.org/10.1016/j.meegid.2022.105206 schönfelder k, breuckmann k, elsner c, et al. transmembrane serine protease 2 polymorphisms and susceptibility to severe acute respiratory syndrome coronavirus type 2 infection: a german case-control study. front genet. 2021;12:667231. https://doi.org/10.3389/fgene.2021.667231 abstract introduction methods results discussion acknowledgements references about the author(s) victor n. fondoh administration/quality management, bamenda regional hospital laboratory, regional hospital bamenda, bamenda, cameroon department of medical laboratory sciences, school of health and medical sciences, catholic university of cameroon, bamenda, cameroon department of health economics policy and management, faculty of business management, university of cameroon, bamenda, cameroon richard m. fondoh administration/pharmaceutical management, north-west regional fund for health promotion, bamenda, cameroon charles n. awasom department of anatomy, school of health and medical sciences, catholic university of cameroon, bamenda, cameroon pefoule l. edith bamenda regional hospital laboratory, regional hospital bamenda, cameroon winlove a. ntungwen patient first laboratory, columbia, maryland, united states bong roland product safety/quality control mangement, geochim sarl, cameroon rebeca enow-tanjong department of medical laboratory science, school of health and medical sciences , catholic university of cameroon, bamenda, cameroon patrick njukeng global health systems solutions, limbe, cameroon judith shang laboratory service, center for disease control and prevention, yaoundé, cameroon egbe p. egbengu department of medicine and surgery, school of health and medical sciences, catholic university of cameroon, bamenda, cameroon talkmore maruta east central and southern africa health community, arusha, united republic of tanzania akazong etheline department of biochemistry, university of dschang, dschang, cameroon robert leke department of medicine and surgery, school of health and medical sciences, catholic university of cameroon, bamenda, cameroon ayuk leo tb-department, regional hospital bamenda, bamenda, cameroon denis nsame administration/management, regional hospital bemenda, bamenda, cameroon citation fondoh vn, fondoh rm, awasom cn, et al. haematological reference intervals for healthy adults in bamenda, cameroon. afr j lab med. 2020;9(1), a1193. https://doi.org/10.4102/ajlm.v9i1.1193 original research haematological reference intervals for healthy adults in bamenda, cameroon victor n. fondoh, richard m. fondoh, charles n. awasom, pefoule l. edith, winlove a. ntungwen, bong roland, rebeca enow-tanjong, patrick njukeng, judith shang, egbe p. egbengu, talkmore maruta, akazong etheline, robert leke, ayuk leo, denis nsame received: 07 apr. 2020; accepted: 01 july 2020; published: 21 dec. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: in the era of evidence-based medicine, haematological reference intervals are essential for the interpretation of data for clinical decision-making, monitoring of treatment and research. it is not uncommon that reference intervals used in most african countries have been obtained from published scientific literature, textbooks, reagent/instrument manuals. objective: the aim of this study was to determine haematological reference intervals of healthy adults in bamenda, cameroon. methods: this was a cross-sectional study conducted between june and november 2015. participants were voluntary blood donors at the blood bank service of the regional hospital bamenda aged between 18 and 65 years. the mean, median and standard deviation of the mean were calculated for each haematological parameter. the 95th percentile reference intervals were determined using the 2.5th and 97.5th percentile. the differences between gender for all the parameters were evaluated using the kruskal-wallis test. significance was determined at the 95% confidence level. results: out of a total of 340 participants, 202 (59.4%) were men and 138 (40.6%) were women. the median red blood cell, haemoglobin, haematocrit and mean cell haemoglobin concentration were significantly higher in men than women (p < 0.001). the median white blood cell, absolute lymphocytes count, absolute granulocytes and platelet counts for men were significantly lower than those for women (p < 0.011). conclusion: we propose that the present established haematological reference intervals in this study should be used for clinical management of patients and interpretation of laboratory data for research in bamenda. keywords: haematological reference intervals; african population; pathogenic infections; haematological abnormalities; cameroon; clinical and laboratory standard institute; local reference values; bamenda. introduction haematological reference intervals are essential for the interpretation of data for diagnosis, clinical decision-making and research in this era of evidence-based medicine. it is not uncommon that reference intervals used in most african countries have been obtained from published scientific literature, textbooks,1,2 the world wide web, reagent package inserts or instrument manuals.3 more often than not, these values have been established from ‘caucasian’ populations in europe or the united states and may not apply to local settings.4,5 there is published literature to confirm that haematological reference intervals established in african populations6,7,8,9,10,11,12,13 differ significantly from those obtained from caucasian populations.5,14,15 several factors, including interand intra-population variation among populations of the same race, age, sex, geographical origin, altitude, genetics, dietary patterns and ethnicity,7,16,17,18,19,20 account for the differences in these reference intervals. moreover, pathogenic infections such as hiv, hepatitis b virus (hbv), hepatitis c virus (hcv), syphilis and some haematological abnormalities generally influence the haematological intervals.9,21,22 besides, the clinical and laboratory standard institute recommends that clinical laboratories establish and/or verify their local reference values.21,23 cameroon is one of the countries that has been burdened by the malaria and hiv epidemics and that has received multilevel interventions, including access to drugs, and capacity building to manage prevention, treatment and clinical trials. there is little published literature on haematological reference intervals established for the population of yaoundé in cameroon.6 these intervals cannot be used nationwide since yaoundé is not representative of the average topography or ecological niche of cameroon, in general, and bamenda, in particular. besides the fact that bamenda is at a lower altitude than yaoundé city and differs from other settlements and ethnic groups, there is a need for clinical laboratories to establish and harmonise standard intervals in all localities23 for effective clinical decision-making, monitoring of treatment and management of interventions.24,25,26,27 the objective of this study was to determine the haematological reference intervals of healthy adults between april and september 2015 in bamenda, cameroon. methods ethical considerations ethical clearance to carry out this research was obtained from the institutional review board of regional hospital bamenda, cameroon (number: 029/app/rdph/rhb/irb). participants consented to participate in the study by signing the consent form. participants could withdraw from the study even after signing the consent form. study area the study was conducted at the regional hospital bamenda situated in bamenda, capital of the north-west region of cameroon, which lies at an altitude between 1100 m and 1430 m above sea level.28,29 because of its high socio-economic activity, bamenda is a cosmopolitan city with settlements of people from diverse ethic backgrounds,30 including mankon, nkwen, bamendakwe, nsongwa, mbatu, chomba and bandza.31 as it is situated in the grass fields, most of their diet includes varieties of vegetables.32 regional hospital bamenda has standard clinical laboratory and blood bank services. the laboratory has been implementing laboratory quality management systems since 2010 and obtained iso 15189 accreditation by the south african national accreditation services in 2017 for biochemistry, serology and haematology services.33 currently, the blood bank service is in the process of certification with the safe blood for africa foundation. research design this was a cross-sectional descriptive study conducted between april 2015 and september 2015. the participants were voluntary blood donors who presented during the regional hospital bamenda’s voluntary blood donation programme. blood samples were collected from the mankon, nkwen and bamendakwe settlements. a stratified and clustered sampling method was used. the population was divided into two groups (men and women) and at least 50 samples were collected from participants at each site and from each sex. the blood donors were subjected to several physical and medical screening protocols, as required by the national blood transfusion programme of the ministry of health, cameroon,34 in addition to the clinical and laboratory standards institute guidelines for the establishment of reference intervals21 using a questionnaire. the questionnaire was used to profile eligible donor. criteria include: the donor should be free from any non-communicable disease, should not have donated blood or had any sexually-transmitted diseases in the previous three months, should not have been sick or been vaccinated during the previous four months and should not have been on any medication for at least a week before sample collection. also, the donor should not have smoked on the day of donation or should not have drunk alcohol for at least 24 hours before donation. female donors should not be pregnant, breastfeeding, or on or expecting their menses within one week of the donation. furthermore, the donor should be between the ages of 18 and 60 years (women) and 18 and 65 years (men), with blood pressure of 100 mmhg – 140 mmhg/60 mmhg – 100 mmhg, weight greater than 50 kg, and temperature between 36.0 °c – 37.5 °c. blood specimens were collected from donors who were physically fit and who consented to be part of the study. we anticipated enrolling at least 150 participants from each sex to meet the minimum target of at least 120 or more participants after exclusions, as recommended by the clinical and laboratory standards institute.21 inclusion and exclusion criteria participants that met the inclusion criteria for voluntary blood donation were excluded if they were positive for hiv, hbv, hcv or syphilis. participants who were sickle cell disease carriers (had the as genotype) or who had sickle cell disease (had the ss genotype) were also excluded. participants who did not meet the inclusion criteria, who did not consent, or who withdrew their consent after consenting, were excluded. sample collection blood was collected by trained and competent personnel into two 5 ml vacutainer tubes containing dipotassium ethylene diamine tetraacetic acid (k2edta). samples were stored and transported to the blood bank service of the regional hospital bamenda in a cold chain between 2 °c and 8 °c within 2 h of collection. one tube was used for screening hiv, hbv, hcv, syphilis and haemoglobin electrophoresis, and the other for complete blood count analysis. the plasma was separated from the red blood cells in separate tubes within 1 h of the samples’ arrival at the blood bank. both tubes were stored at 4 °c – 8 °c for testing the following day. hiv, hepatitis b virus, hepatitis c virus, syphilis and haemoglobin electrophoresis testing plasma samples were screened at the blood bank department of the regional hospital bamenda. the national algorithm of a rapid test for hiv screening in cameroon was used.35 samples were screened for hiv using the hiv-1/2 ag/ab combo determine (alere medical co., ltd, matsuhidai, matsudo-shi, chiba-ken, japan) as the first-line test and oraquick (orasure technologies, inc., bethlehem, pennsylvania, united states) as the second-line test. all participants who were hiv-negative with the first-line test were confirmed as negative with the second-line test. participants who were positive for hiv with the first-line test only were declared positive and excluded. syphilis was screened using the rapid plasma reagin carbon slide agglutination assay (cypress diagnostics, langdorp, belgium) and the treponema pallidum haemagglutination test for the serodiagnosis of syphilis – immutrep® tpha (omega diagnostics ltd, alva, scotland, united kingdom). hepatitis b virus was screened for using the hbsag diaspot rapid diagnostic test (diaspot diagnostics, jawa barat, indonesia) while hepatitis c virus antigen was detected using the hcv ag diaspot rapid diagnostic test (diaspot diagnostics, jawa barat, indonesia). haemoglobin electrophoresis was done using the hospitex diagnostics (hospitex diagnostics srl, sesto fiorentino, italy) electrophoresis machine. the haematological analysis was done within 6 h of sample collection, using the urit 3300 auto-analyser (urit medical electronic [group] co., ltd, guilin, china). the instrument was calibrated using eurocell diagnostics internal controls (eurocell diagnostics, rennes cedex, france), following the protocol provided by the manufacturer. the analyser automatically counted and gave a print-out of results for: red blood cells (rbc); haemoglobin (g/dl); haematocrit (%); mean cell volume; mean cell haemoglobin; mean cell haemoglobin concentration); coefficient of variation for the standard deviation of red cell distribution (%); standard deviation of red cell distribution; white blood cells (wbc); proportion of lymphocytes (%), monocytes (%) and granulocytes (%); absolute count of lymphocytes (×109/l), monocytes (×109/l) and granulocytes (×109/l); platelets; mean platelet volume; platelet distribution width and plateletcrit. quality control the urit 3300 auto-analyser used for the analysis of the specimens went through a vigorous formal verification process following the clinical laboratory standards institute guidelines21 and the policies of the quality management system of the bamenda regional hospital laboratory. precision was monitored daily using commercial internal controls (eurocell diagnostics, rennes cedex, france) and reviewed using a levey-jennings control chart. randox international quality assurance scheme riqas (randox laboratories limited, crumlin, county antrim, united kingdom) external quality controls were done bi-monthly to monitor accuracy. the analysis was suspended if the daily commercial internal control failed. the analysis was done by a trained and competent technician. data collection data were collected by three trained personnel using a structured data collection format. data for age, sex and haematological parameters for the participants who were negative for hiv, hbv, hcv and syphilis, with no haemoglobin abnormalities, were collected from the print-out of the urit 3300 auto-analyser. data were entered into excel 2007 software (microsoft corp., redmond, washington, united states) and double-checked for data entry errors by a second person. statistical analysis the analysis was done using microsoft excel 2007 spreadsheet (microsoft corporation, redmond, washington, united states) and spss version 16 software (ibm corp., chicago, illinois, united states). outliers were eliminated using the box plot function. the median, mean and standard deviation were calculated for each haematological parameter. the 95th percentile reference intervals were determined using the 2.5th and 97.5th percentile. the differences between sexes for all the parameters were evaluated using the kruskal-wallis test. significance was determined at the 95% confidence level. results of the 487 individuals who presented for the blood donation campaigns, 147 were excluded as per the exclusion criteria (table 1). of the 340 participants included in the study, 202 were male (59.4%) and 138 were female (40.6%) within the age range of 18–60 years (95% confidence interval: 31.5 ± 10.9; median age = 29 years). one hundred and thirty-nine participants (40.9%) were aged 18–25 years, 97 (28.5%) were aged 26–35 years, 62 (18.2%) were aged 36–45 years, 35 (10.3%) were aged 46–55 years, and 7 (2.1%) were aged 56–65 years (figure 1). figure 1: percentage distribution of age group (years) and sex of participants in the blood donor population of bamenda, cameroon, april-september 2015. table 1: exclusion criteria applied to the blood donor population, bamenda, cameroon, april-september 2015. the median rbc, haemoglobin, haematocrit and mean cell haemoglobin concentration were significantly higher in men than in women (rbc: 5.31 × 1012/l vs. 4.60 × 1012/l, p < 0.001; haemoglobin: 14.6 g/dl vs. 12.6 g/dl, p < 0.001; haematocrit: 43.9% vs. 38.3%, p < 0.001; and mean cell haemoglobin concentration: 33.1 g/dl vs. 32.8 g/dl, p = 0.005). although the median mean cell volume and mean cell haemoglobin were higher in men than women (mean cell volume 27.6 fl vs. 27.3 fl, p = 1.000; and mean cell haemoglobin 27.6 pg vs. 27.3 pg, p = 0.147), the differences were not statistically significant (table 2). table 2: erythrocyte parameter reference intervals of healthy adults stratified by sex, bamenda, cameroon, april-september 2015. the median total wbc, absolute lymphocyte count and absolute granulocyte count were significantly lower in men than in women (wbc: 5.0 × 109/l vs. 5.5 × 109/l, p = 0.002; absolute lymphocyte count: 2.1 × 109/l vs. 2.2 × 109/l, p = 0.011; and absolute granulocyte count: 2.4 × 109/l vs. 2.8 × 109/l, p = 0.002). although the absolute median monocyte count for men was relatively higher than that of women, the difference was not statistically significant (table 3). table 3: leucocyte parameter reference intervals of healthy adults stratified by sex, bamenda, cameroon, april-september 2015. also, the median platelet count was significantly lower in men (231 × 109/l) than in women (253 × 109/l; p = 0.009). there was no statistically significant difference between the median mean platelet volume, platelet distribution width and plateletcrit for men as compared to women (table 4). table 4: platelet parameter reference intervals of healthy adults stratified by sex, bamenda, cameroon, april-september 2015. discussion the reference interval for haematological parameters, which may serve as a standard for decision-making on clinical laboratory results, treatments and research, were established from 340 participants from bamenda city, cameroon. the participants included 202 (59.4%) men and 138 (40.6%) women aged between 18 and 60 years. according to our findings, the median rbc, haemoglobin and haematocrit for men were significantly higher than for women. these variations may be attributed to the influence of the hormone androgen on erythropoiesis as well as menstrual blood loss in women.36 our findings are consistent with previous reports in africa, including oloume et al. in cameroon,6 awad et al. in sudan,38 addai-mensah et al. in ghana,7 bakrim et al. in morocco,8 mulu et al. in ethiopia13 and yalew et al. in ethiopia,12 miri-dashe et al. in nigeria,36 dosoo et al. in ghana10 and kueviakoe et al. in togo,9 karita et al. in eastern and southern south africa21 and menard et al. in central africa.20 similar findings have also been reported in the united states.5 according to oloume et al. in the yaounde study in cameroon, rbc, haemoglobin, haematocrit and mean cell haemoglobin concentration were lower compared to those obtained in this study.6 in their study, however, haemoglobin abnormalities were not excluded from their sample collection, considering the 2% prevalence of sickle cell disease in cameroon37 and may thus account for the low values reported. besides, bamenda is at a higher altitude than yaoundé29,39,40 and is situated in the grassland; also, its inhabitants are used to the consumption of vegetables32 which have a high iron content that may increase the erythrocyte parameters. at higher altitude, there are physiological changes to humans that compensate for the lower partial pressure of oxygen at higher altitudes.41,42 the same reason may account for the relatively lower intervals of rbc, haemoglobin and haematocrit in other countries at lower altitudes compared to those in this study,7,10,12,17,36 in contrast to higher intervals observed in a study conducted in ethiopia at higher altitudes.13,43 on the other hand, we observed relatively lower intervals of haemoglobin and haematocrit in this study compared to those reported in the united states (table 5).5 this may be attributed to lower ferritin and transferrin saturation among black participants.44 besides, our study showed a significantly higher mean cell haemoglobin concentration in men than in women, which had also been reported in previous work in ethiopia.12 table 5: comparison of reference intervals in bamenda, cameroon, with other african countries and the united states, 2002 and 2015. the median total wbc for men was lower than that for women, and the difference was statistically significant. this may be attributed to the significant difference in the immune system of men and women, associated with the presence of sex hormone receptors on the immune cells. these make women generally more prone to autoimmunity, resulting in lower rates of infection and chronic inflammatory disease.45,46 our findings are in concordance with those reported by oloune et al. in yaoundé, cameroon,6 bakrim et al. in morocco,8 tekkeşin et al. in turkey14 and mine et al. in botswana.47 the significantly higher median platelet count in women compared to men is suggestive of variations in hormone type and concentrations in the different genders as well as the effect of erythropoietin released in response to menstrual blood loss and cross-stimulation of megakaryopoiesis.10,36 our findings are consistent with other studies in africa: addai-mensah et al. in ghana,7 bakrim et al. in morocco,8 mulu et al. in ethiopia,13 miri-dashe et al. in nigeria,36 dosoo et al. in ghana10 and kibaya et al. in kenya.17 however, platelet counts in this study were relatively higher than those of other african countries in contrast to higher counts reported in the united states (table 5).5 this could be attributed to genetic factors, compounded by the increased consumption of platelets by plasmodium spp. in malaria-endemic areas.48,49 limitations a limitation for this study was that we could not screen for malaria, helminthes or all types of abnormal haemoglobin (except for the as and ss sickle cell genotypes), and our complete blood analyser could not differentiate the granulocytes into neutrophils, basophils and eosinophils. also, subclinical conditions which could affect blood parameters were not discernable during sample collection. furthermore, ethnic and cultural differences that may influence diet and nutritional practices could have affected the outcome of our haematological intervals. we could not control for potential selection bias for some people who visited bamenda and donated blood. recommendations we recommend that locally generated haematological values should be used as reference intervals in our locality and that each region in cameroon should determine their haematological reference intervals as recommended by the clinical laboratory standards institute.21 conclusion the haematological reference intervals established in this study are comparable to those obtained in yaoundé, cameroon and other studies within and outside of africa. any differences in values may be due to differences in latitudes of the localities, race and diet. we propose that the present established haematological reference intervals in this study should be used for clinical management of patients and interpretation of laboratory data for research in bamenda. acknowledgements we acknowledge all the participants of this study for voluntarily giving their blood for the establishment of haematological reference intervals for the bamenda community. thanks to the centre for disease control and prevention (cdc), cameroon, and the global health system solutions, limbe, for their technical support, the management of the department of health economics, policy and management, and the faculty of health and medical science of the catholic university of cameroon-bamenda for academic support during this study. lastly, we appreciate the staff and management of the bamenda regional hospital laboratory and the blood bank for their wonderful support during the study. competing interests the authors have declared that no competing interests exist. authors’ contributions v.n.f. (study leader) was responsible for the conceptualisation of the study, study design, supervision (of the experiments, specimen collection, testing, data collection), statistical analysis and final writing. c.n.a. and r.m.f. were responsible for the conceptualisation of the study and statistical analysis. v.n.f., p.l.e. and w.a.n. were responsible for performing the experiments, specimen collection, specimen testing and data collection. b.r. was responsible for providing technical equipment maintenance services. t.m., r.m.f., c.n.a., r.e-t., p.n., b.r., j.s., e.p.e., a.e., r.l, a.l. and d.n. were responsible to help shape the research, adding literature search and critical review of the manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability statement data sharing does not apply to this article as no new data were created or analysed in this study. disclaimer the views and 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kenya cecilia wanjala innovation technology transfer division, kenya medical research institute, nairobi, kenya jeremiah zablon kenyatta national hospital, nairobi, kenya moses orina innovation technology transfer division, kenya medical research institute, nairobi, kenya lucy muita innovation technology transfer division, kenya medical research institute, nairobi, kenya jacqueline thiga innovation technology transfer division, kenya medical research institute, nairobi, kenya lameck nyabuti innovation technology transfer division, kenya medical research institute, nairobi, kenya eunice wainaina innovation technology transfer division, kenya medical research institute, nairobi, kenya joseph mwangi centre for virus research, kenya medical research institute, nairobi, kenya alice mumbi innovation technology transfer division, kenya medical research institute, nairobi, kenya samuel omari innovation technology transfer division, kenya medical research institute, nairobi, kenya ann wanjiru innovation technology transfer division, kenya medical research institute, nairobi, kenya samson m. nzou innovation technology transfer division, kenya medical research institute, nairobi, kenya missiani ochwoto innovation technology transfer division, kenya medical research institute, nairobi, kenya citation kimotho jh, gosar aa, inyangala r, et al. pre-evaluation assessment of serological-based covid-19 point-of-care lateral flow assays in kenya. afr j lab med. 2021;10(1), a1317. https://doi.org/10.4102/ajlm.v10i1.1317 original research pre-evaluation assessment of serological-based covid-19 point-of-care lateral flow assays in kenya james h. kimotho, abdiaziz a. gosar, ronald inyangala, paulyne wairimu, fred siyoi, damaris matoke-muhia, cecilia wanjala, jeremiah zablon, moses orina, lucy muita, jacqueline thiga, lameck nyabuti, eunice wainaina, joseph mwangi, alice mumbi, samuel omari, ann wanjiru, samson m. nzou, missiani ochwoto received: 25 june 2020; accepted: 23 june 2021; published: 17 sept. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: timely testing is a key determinant of management outcomes of coronavirus disease 2019 (covid-19). real-time reverse transcription polymerase chain reaction tests are currently the mainstay for covid-19 testing. however, serological point-of-care tests (pocts) can be useful in identifying asymptomatic and recovered cases, as well as herd immunity. objective: the aim of this study was to assess covid-19 pocts in kenya to support the emergency use authorisation of these tests. methods: between march 2020 and may 2020, 18 firms, of which 13 were from china, submitted their pocts to the national regulatory authority, the pharmacy and poison board, who in turn forwarded them to the kenya medical research institute for pre-evaluation assessment. the tests were run with real-time reverse transcription polymerase chain reaction covid-19-positive samples. pre-covid-19 plasma samples that were collected in june 2019 were used as negative samples. the shelf lives of the pocts ranged from 6 to 24 months. results: only nine (50%) tests had sensitivities ≥ 40% (range: 40% – 60%) and the ability of these tests to detect igm ranged from 0% to 50%. many (7/18; 38.9%) of the kits had very weak igm and igg band intensities (range: 2–3). conclusion: serological-based pocts available in kenya can only detect covid-19 in up to 60% of the infected population. keywords: covid-19; point-of-care; igm; igg; sensitivity; specificity; sars-cov-2. introduction the world is grappling with one of the worst disease pandemics ever experienced in over 100 years, the coronavirus disease 2019 (covid-19). according to a world health organization situation report, as of 07 june 2020, the number of covid-19 cases had reached 6 931 000 people globally, with more than 400 121 deaths. in kenya, the number of reported cases as of 06 june 2020 was 2600 with 83 deaths.1 real-time reverse transcription polymerase chain reaction (rrt-pcr) is currently the gold standard for severe acute respiratory syndrome coronavirus 2 (sars-cov-2) diagnosis as it is highly sensitive and relatively easy to develop.2 however, rrt-pcr test protocols are complex, expensive, mainly suited to advanced laboratories, and typically take 4–6 h to complete. moreover, these tests are manufactured by predominantly european and american companies that have adopted a ‘me first’ policy due to the high numbers of covid-19-related deaths in their own countries, thereby successfully eliminating the chances of these test kits flowing to africa and kenya in quantities sufficient for use.3,4 on the other hand, serological-based point-of-care tests (pocts) that take 5–15 min to complete can pick up asymptomatic or recovered cases of covid-19, making them suitable to support disease surveillance and the determination of herd immunity.5 however, these pocts have low overall sensitivities (34% – 80%) and specificities (70% – 100%) compared to covid-19 rrt-pcr as the gold standard.6 serological-based pocts detect plasma levels of immunoglobin g (igg), immunoglobin m (igm), and, sometimes, iga against sars-cov-2. the levels of igm are elevated during the first week after sars-cov-2 infection, peak at 2 weeks, and then reduce to baseline levels in most patients. on the other hand, igg is detectable after 1 week and is sustained at a high level for a long period.7,8,9 currently, the world health organization does not recommend the use of covid-19 serological tests in the clinical setting. however, the potential of the use of antigen-based pocts at triage to rapidly detect cases is acknowledged.10 to evaluate in vitro serological-based pocts, the world health organization recommends the use of a minimum of 200 prospective covid-19 specimens (100 confirmed rrt-pcr positive samples and 100 prospective specimens from patients with signs and symptoms suggestive of covid-19). at least 30 specimens should be rrt-pcr positive at the time of specimen collection and within a week of symptom onset. the remaining data may be supplemented during the review process. for diagnostic specificity, 200 individual specimens from symptomatic patients that tested negative to covid-19 by rrt-pcr and at least 1000 specimens from the general population collected before november 2019 are used. at least 50% of data are requested for submission.11 manufacturers of commercial covid-19 pocts submit their pocts to the pharmacy and poisons board (ppb), nairobi, kenya, for evaluation to facilitate and enhance the issuance of emergency use authorisation (eua). upon receiving the eua, the supplier of a poct may opt to submit it for the full evaluation. in the face of the pandemic, the regulatory pathway of eua bypasses the often longer, data scrutinising pre-evaluation process conducted by the regulator (the ppb) before allowing market authorisation to a manufacturer. owing to the novelty of the coronavirus and the devastating risk of death of infected persons, the need to avail the pocts and rrt-pcr to the general population was paramount. the need for laboratory performance pre-evaluation was identified as a key component required in ascertaining the quality of the pocts, thus helping to ensure that in the interim period of use of the pocts, performance, efficacy and safety standards are met. this study aimed to conduct a preliminary evaluation of commercial pocts that had been presented to the kenya medical research institute (kemri), nairobi, kenya, by the ppb. methods ethical considerations this study was initially approved by the scientific ethical review unit at kemri under protocol kemri/seru/cbrd/209/4008. it was also reviewed and approved by the kenyatta national hospital ethical review committee under protocol number p274/05/2020. covid-19 patients or subjects had to provide written informed consent before blood sample collection. all samples were anonymised. sample collection a total of 50 anonymised blood samples were collected through purposive sampling from patients (male and female) of all ages with active infection as determined by covid-19 rrt-pcr. the study subjects were from the isolation and quarantine centre at the kenyatta national hospital infectious diseases unit. after consenting, 5 ml blood samples were drawn from the subjects and transported to the kemri innovation and technology division. the serum was separated and stored at –20 °c until the day of the pre-evaluation assessment. eighteen covid-19 pocts were received from the ppb, the kenyan drug regulatory authority, who had received the tests from various clients for pre-evaluation assessment and subsequent registration for eua. the kits included in the study were rapid-format antibody-based pocts that were meant to detect igm or igm/igg antibodies to sars-cov-2 nucleocapsid or spike 1 or 2 proteins. pre-covid human de-identified archived blood samples were collected from national blood transfusion centres and were randomly selected for the pre-evaluation of kits. they were stored at –80 °c at the kemri innovation and technology division to serve as covid-negative samples. test evaluation the tests were run according to the manufacturers’ instructions under a biosafety cabinet. briefly, for lateral flow assays, the procedure was as follows: blood samples were spun in a centrifuge and the serum and plasma were separated. the test cassette was removed from foil and allowed to equilibrate to room temperature. one drop (about 10 μl) of plasma sample was loaded using a micropipette into the sample well in the cassette. thereafter, about 60 μl (2–3 drops) of the assay solution (chase buffer) was pipetted into the sample well in the device. after about 10 min, the results were interpreted. only tests in which the colour of the control line changed were considered valid, and if a coloured line was observed for igm or igg, the test was considered positive. the intensity of the colour was compared with that of the colour reference card and semi-quantified. the immunofluorescent protocol was as follows: 150 μl of detector diluent was transferred into a vial containing detector crystal. 10 μl of plasma sample was then added, mixed, and, using a micropipette, 75 μl of the mixture was pipetted into the sample well in the cartridge. after 10 min, the cartridge was placed into the immunofluorescent reader and the results were read and interpreted. determination of sensitivity and specificity of the assays data generated were recorded and analysed using microsoft excel 2010 (microsoft corporation, redmond, washington, united states) and stata version 14.1 (statacorp llc, brownsville, texas, unites states). the specificity of the assays was determined using a panel of 50 pre-covid-19 pandemic serum samples from june 2019. sensitivity was determined for each assay using 50 rrt-pcr sars-cov-2-positive samples. the rna for confirmatory tests were extracted using a qiaamp viral rna mini kit (qiagen, germantown, maryland, united states). the pcr was done on an applied biosystems quantstudio™ 5 (thermofisher scientific, waltham, massachusetts, united states) pcr machine using a sacace biotechnologies sars-cov-2 real-tm detection kit (scalabrini, como, italy) with their recommended cycling conditions set at 35 °c for 20 min for reverse transcription, initial pcr activation at 94 °c for 10 s, and five cycles at 64 °c for 25 s. the cycling step was done at 94 °c for 10 s followed by 64 °c for 25 s for 45 cycles. analysis of the results was done using quantstudio design and analysis software (thermofisher scientific, waltham, massachusetts, united states) and interpreted using the sacace sars-cov-2 real-tm result analysis guide. the antibody tests were considered positive when the control band and igm, igg or both bands were visible. diagnostic sensitivity (%) was calculated as a/(a + c) × 100 while diagnostic specificity (%) was calculated as d/(b + d) × 100, where a represents true positive results, c represents false negatives, b represents false positives and d represents true negatives. the pocts were considered to have a high sensitivity when able to correctly identify all positive samples in a panel. tests that only detected 40% or lower of the positive samples in the panel were deemed to have lower sensitivity as they would miss positives and give higher false negative rates. results the study involved a total of 18 pocts from different manufacturers. the number of kits submitted by each manufacturer differed as the national policy on the number of kits required for eua pre-evaluation assessment had not been established at the time. the shelf lives allocated to the kits were highly varied: 6 months (2/18; 11.1%), 12 months (8/18; 44.4%), 18 months (1/18; 5.6%), and 24 months (7/18; 38.9%) (table 1). many (7; 38.9%) of the kits were manufactured in march 2020 (and the study commenced in may 2020). thirteen (72.2%) of the kits analysed were manufactured in china, 2 (11.1%) in korea, 1 (5.6%) in canada, 1 (5.6%) in the united states, and 1 (5.6%) in malaysia. table 1: characteristics of covid-19 point-of-care test kits submitted to the kenya medical research institute by the pharmacy and poisons board, nairobi, kenya, for pre-evaluation assessment, march–may 2020. most of the tests (15/18; 83%) were based on covid-19 igg or igm detection on separate bands; one was based on covid-19 igg or igm detection on a combined band, while two could only detect igm. the signal from 17 tests could be detected by the naked eye, while the signal of one kit (id 11) could be detected only using a fluorometric machine which is a semi-automated in vitro diagnostic device that detects analytes through fluorescent scanning. diagnostic sensitivity of the point-of-care test kits kit 2 (igm) and kit 14 showed low sensitivities of 26.7% and 11.1%; kit 5 showed no activity; kit 11, which was the only kit with igg and igm combined in a single band test, showed high sensitivity (figure 1). of the total 18 kits, 6 (33.3%) kits had sensitivities of at least 50%: kits 7, 10, 12, 13, 16, and 18. five kits had diagnostic sensitivities between 40% and 50% (kits 3, 4, 6, 8 and 17), while kits 1, 9 and 15 had sensitivities between 30% and 40%. only kits manufactured in china and the united states managed to score more than 40% diagnostic sensitivity. figure 1: diagnostic sensitivity of the pre-assessed covid-19 point-of-care tests submitted to the kenya medical research institute by the pharmacy and poisons board, nairobi, kenya, for pre-evaluation assessment, march–may 2020. combined igg and igm sensitivity was assessed using rrt-pcr as standard. diagnostic sensitivity of the igg and igm separate bands the diagnostic sensitivity of the igg and igm bands were considered separately for all the pocts (figure 2). the igm band of kit 1 did not demonstrate any capacity to detect igm. kit 2, an igm-only kit, had a low sensitivity of 19% and a specificity of 95%. the sensitivity of kit 13 was 55% for igg and 40% for igm. kit 12 showed higher diagnostic sensitivity for igg (40%) than for igm (5%). kit 3 was the only kit that showed higher diagnostic sensitivity for igm (43%) than igg (19%). interestingly, five kits (kits 4, 6, 8, 10, and 18) showed equal diagnostic sensitivity for igg and igm. there was a weak positive correlation between the igm and igg diagnostic sensitivities of the kits (pearson’s correlation coefficient [r] = 0.2527; r2 = 0.0639; p = 0.45). figure 2: diagnostic sensitivity of the pre-assessed covid-19 point-of-care tests submitted to the kenya medical research institute by the pharmacy and poisons board, nairobi, kenya, for pre-evaluation assessment, march 2020 – may 2020. separate igg or igm sensitivities were assessed using rrt-pcr as standard. diagnostic specificity of the point-of-care test kits eight kits (kits 1, 3, 8, 9, 12, 14, 16, and 18) had a diagnostic specificity of 100%, while five kits had low diagnostic specificities of 81% (kit 7), 92% (kit 17), 94% (kit 13), and 95% (kits 2 and 15). the remaining five kits were not tested for diagnostic specificity as they had run out of stock. igm and igg band signal intensities of the point-of-care test kits the intensities of the igm and igg bands of the pocts were analysed with the assistance of a colour reference chart (figure 3). the average intensities of the observed bands of many (7/18; 38.8%) of the kits were at score 3 on the scale (figure 3). the intensities of the igg bands were generally stronger than those of igm. kits 3, 6, 8, 10, 12, 13, 16, 17, and 18 displayed the strongest intensities, which corresponded with their higher diagnostic sensitivities compared to the remaining kits. figure 3: colour intensity of the positive control signal for the igm and igg bands of point-of-care tests assessed at kenya medical research institute between march 2020 and may 2020. discussion this study carried out a pre-evaluation assessment of poct kits submitted by manufacturers to the ppb for pre-evaluation. out of the kits evaluated, 50% had sensitivities of ≥ 40%, and most of the kits had low band intensities. the kits with sensitivities of ≥ 40% were given emergency approval. currently, rrt-pcr tests are the mainstay of sars-cov-2 diagnosis; however, they have complex protocols, are expensive, and require advanced laboratory equipment.2 the main goal of introducing a poct is to avail quick test results to the healthcare workers or the patient to support fast clinical management decisions and, ultimately, improve patient outcomes and overall public health. it is hoped that pocts will be useful in identifying asymptomatic and recovered cases of covid-19 as well as herd immunity.12 the pre-evaluation assessment of pocts is crucial to mitigating the risks associated with the introduction of kits that can cause public anxiety and false alarm to the market. the regulatory pathway for the evaluation and assessment of pocts per the stipulated turnaround time for in vitro diagnostics takes 1–3 years; however, the covid-19 pandemic presented increased pressure and the need to grant marketing authorisation to manufacturers, necessitating the development of a pre-evaluation assessment policy. according to unpublished reports from the ppb, and as a pre-requisite measure for issuance of the eua, those kits without satisfactory sensitivity and specificity results must be subjected to further evaluation. the shelf lives of the pocts in this study ranged from 6 to 24 months. the manufacturers did not provide reports of their stability data or their plans for generating such data.13 the study showed that 50% of the pocts that were submitted for pre-evaluation assessment did not meet the criteria to proceed to full evaluation as they were unsatisfactory. of the 18 pocts, one had no activity at all, two igm-detecting tests had both low sensitivity and low specificity, and three tests displayed high false-positive values. only nine (50%) tests had sensitivities of ≥ 40% (range: 40% – 60%). the capacity of these nine tests to detect igm ranged from 0% to 50%. the majority (57.1%) of the tests displayed very weak visual igm band intensities (scale scores of 2–3). the sensitivities observed in this evaluation were lower than those reported elsewhere, and the ability of the tests to detect igm varied, with one test failing to capture any covid-19 igm. there was also a weak positive correlation between igm and igg sensitivity for the kits. the abbott id now covid-19 test (abbott laboratories, abbott park, illinois, united states) is said to be the most sensitive and specific poct on the global market, with a sensitivity of 80.4% and specificity of 95.9% with rrt-pcr as the standard.14,15 a study conducted to assess the quality of a poct based solely on pcr-positivity among anonymous blood donors in uppsala university, uppsala, sweden, reported a high performance, with sensitivities of 69% for igm and 93.1% for igg, and specificities of 100% for igm and 99.2% for igg.16 another study conducted in china among covid-19-positive patients established the sensitivity and specificity of the test they assessed to be 88.66% and 90.63%.17 a study done at guangzhou eighth people’s hospital, china, among patients with confirmed sars-cov-2 infection found that the combined detection of nucleocapsid-specific and spike-specific igm and igg could identify up to 75% of sars-cov-2-positive cases in the first week using an enzyme-linked immunosorbent assay test kit.18 in consonance with the findings of this study, the organization for economic co-operation and development has emphasised the need to recognise that pocts have not been fully developed for sars‑cov‑2 and their true clinical performance is mostly unknown.19 a satisfactory quality assessment of covid-19 diagnostics is very critical. recently, quality failure-related issues have been reported globally. these include instances such as india stopping the use of 500 000 pocts from two chinese firms for questionable efficacy,20 a report of initial pcr test kits that were contaminated with coronavirus synthetic materials in the united states,21 britain buying millions of pocts that did not work,22 first samples of pocts manufactured in india failing the quality tests,23 and spain withdrawing chinese-manufactured kits with a sensitivity of 30%.24 it is noteworthy that kit 11, which failed in this study, is from the same company that made the kits that failed in india. all kits need to be re-evaluated using standard covid-19 igg and igm to establish a gold standard poct. the kits assessed in this pre-evaluation should be limited to use in research and sero-surveillance only. furthermore, there is a need for continued evaluation of these kits using whole blood samples with larger sample sets of at least 400 covid-19-positive and negative samples. this would be a full evaluation of the kits and would increase the power of the study. there is also a need to formulate national and international policies on the determination of shelf lives of products that are developed during medical emergencies such as the covid-19 pandemic. limitations this study only used plasma matrix. there is a need to assess the performance of these poct kits using whole blood samples, especially whole blood from finger pricks and serum as per the manufacturers’ instructions. the sample size used in this study was less than the 400 samples recommended by the world health organization. the suppliers of the pocts could not provide the adequate number of kits required. conclusion there was very poor igm detection by most of the poct kits assessed and this could affect timely detection of covid-19 early infection and spread as compared to rrt-pcr. similarly, the serological-based test kits available in the country can only detect up to 60% of the infected population. acknowledgements this work was supported by prof. yeri kombe, director general, kenya medical research institute, to whom we are very grateful. we are also grateful to dr fred siyoi, chief executive officer, pharmacy and poisons board, kenya, for his unreserved support for this work. the authors would like to confirm that the people mentioned in this section have granted permission as required. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions j.h.k., a.a.g., r.i., p.w., f.s., d.m-m., c.w., j.z., m. orina, l.m., j.t., l.n., e.w., j.m., a.m., s.o., a.w., s.m.n. and m. ochwoto contributed in different capacities to the design and implementation of the research, to the analysis of the results and to the writing of the manuscript. sources of support this work was supported by the kenya medical research institute under the grant award kemri/cov/innov/001. data availability repository: figure share https://doi.org/10.608/m9figshare12727037. this project contains the following underlying data: data file 1 (data for true negatives). data file 2 (data for true positives). data file 3 (pre-evaluation raw data). data are available under the terms of creative commons by 4.0 and can be requested from the corresponding author. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency or the authors. references john hopkins university. epidemic map updated in real-time. baltimore, md: center for systems science and engineering; 2020. jeong sae-im. korea approves 2 more covid-19 detection kits for urgent use. korea biomed rev. 2020. liang m, du l, liu j, et al. sars patients-derived human recombinant antibodies to s and m proteins efficiently neutralize sars-coronavirus infectivity. biomed environ sci. 2005;186(6):363. sheridan c.coronavirus and the race to distribute reliable diagnostics. nat biotechnol. 2020;38:382–384. https://doi.org/10.1038/d41587-020-00002-2 hopkins j. serology-based tests for covid-19. baltimore, md: johns hopkins; 2020. zainol r, othman s, abdul s, et al. diagnostic performance of covid-19 serology assays. malaysian j pathol. 2020;42(1):13–21. vabret n, graham j, conor g, et al. immunology of covid-19 current state of the science. immunity. 2020;42(2):910–941. srikrishna d, dhillon rs, beier d. we need a cheap way to diagnose coronavirus. harv bus rev. 2020. hou h, wang t, zhang b, et al. detection of igm and igg antibodies in patients with coronavirus disease 2019. clin translational immunol. 2020;9(5):e1136. https://doi.org/10.1002/cti2.1136 world health organization. advice on the use of point-of-care immunodiagnostic tests for covid-19: scientific brief. geneva: world health organization; 2020. world health organization. instructions for submission requirements: in vitro diagnostics (ivds) detecting antibodies to sars-cov-2 virus. geneva: world health organization; 2020. drain p, hyle e, noubary f, et al. diagnostic point-of-care tests in resource-limited settings. lancet infect dis. 2014;14(3):239–249. https://doi.org/10.1016/s1473-3099(13)70250-0 world health organization (b). establishing stability of in vitro diagnostic medical devices. 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[cited 2020 june 14]. available from: https://www.alere.com/en/home/ hogan c, sahoo m, huang c, et al. five-minute point-of-care testing for sars-cov-2 not there yet. j clin virol. 2020;128:104410. https://doi.org/10.1016/j.jcv.2020.104410 hoffman t, nissen k, krambrich j, et al. evaluation of a covid-19 igm and igg rapid test; an efficient tool for assessment of past exposure to sars-cov-2. infect ecol epidemiol. 2020;10(1):1754538. https://doi.org/10.1080/20008686.2020.1754538 li z, yi y, luo x, et al. development and clinical application of a rapid igm-igg combined antibody test for sars-cov-2 infection diagnosis. j med virol. 2020;92(9):1518–1524. https://doi.org/10.1002/jmv.25727 sun b, feng y, zheng p, et al. kinetics of sars-cov-2 specific igm and igg responses in covid-19 patients. emerg microb infect. 2020;9(1):940–948. https://doi.org/10.1080/22221751.2020.1762515 oecd. home testing for covid-19: a way to lift confinement restrictions. oecd. paris; 2020. hindustan times new delhi china. concerned as india decides to stop use of chines covid-19 test kits. new delhi; 2020. new york times. cdc labs were contaminated delaying corona virus testing official say. new york; 2020. the sunday times. britain has millions of coronavirus antibody tests, but they don’t work. london; 2020. nidheesh la. first samples of hll’s india-made rapid testing fail quality test in kerala. new delhi: livemint; 2020. the guardian. coronavirus test kits withdrawn in spain over poor accuracy rate. madrid; 2020. article information authors: siyem c. nkwawir1 nakeli n. batumani1 talkmore maruta2 charles n. awasom1 affiliations: 1bamenda regional hospital laboratory, cameroon2african society for laboratory medicine, ethiopia correspondence to: siyem nkwawir postal address: po box 818, regional hospital bamenda, north west region, cameroon dates: received: 27 may 2014 accepted: 20 aug. 2014 published: 03 nov. 2014 how to cite this article: nkwawir sc, batumani nn, maruta t, awasom cn. from grass to grace: how slmta revolutionised the bamenda regional hospital laboratory in cameroon. afr j lab med. 2014;3(2), art. #203, 6 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.203 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. from grass to grace: how slmta revolutionised the bamenda regional hospital laboratory in cameroon in this original research... open access • abstract • introduction • research methods and design    • phase 1: preparation    • phase 2: slmta programme implementation    • phase 3: evaluation • results • discussion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: public health laboratories form the foundation on which today’s clinical laboratory practice in cameroon is built. the advent of the strengthening laboratory management toward accreditation (slmta) programme in 2009 empowered the bamenda regional hospital laboratory (brhl) to improve its working culture, practices and management.objectives: to evaluate the results of slmta implementation at brhl and discuss lessons learned. method: in 2010, the slmta programme was rolled out in cameroon to improve laboratory quality management systems in five laboratories, including brhl. three workshops were conducted (the first centralised, the remaining two on-site at each laboratory) and improvement projects were implemented after each workshop with the assistance of mentors. audits were used in order to evaluate performance and to identify areas for further improvement. results: brhl had the lowest score (18%) amongst the cohort at the baseline audit and the highest (81%) at the official stepwise laboratory quality improvement process towards accreditation (slipta) audit conducted in august 2013 by the african society for laboratory medicine. improvements were observed in each of the 12 quality system essentials; improvement was especially noteworthy in the areas of facilities and safety, and purchasing and inventory. staff investment and pride in the quality of laboratory services increased. conclusion: brhl’s remarkable improvement was achieved with a combination of slmta training activities, intensive on-site mentorship and the collective focus of all laboratory staff. the experience at bamenda hospital illustrates what can be achieved when a laboratory successfully harnesses the energy of its staff and implements changes to improve the quality of services in a transformation taking them from grass to grace. introduction top ↑ the need to improve health outcomes regionally has led health systems and governments in africa toward evidence-based medicine, which requires access to high-quality information such as laboratory tests for decision making.1 given the emphasis on reliable data, increased attention has been focused on medical laboratories and their quality of services. in 2008, at a meeting in yaoundé, cameroon, organised by the world health organization’s regional office for africa (who afro), leaders from several african countries recognised the critical role of public health laboratories in disease control and prevention and resolved to strengthen public health laboratories throughout the region.2 in the following year, who afro launched a stepwise laboratory accreditation preparation scheme, later called the stepwise laboratory quality improvement process towards accreditation (slipta).3 slipta provides a framework for benchmarking progress using an audit checklist based on international organization for standardization (iso) 15189:2007 requirements. at the same meeting, the strengthening laboratory management toward accreditation (slmta) programme was launched in order to help support laboratory quality improvement. slmta is a laboratory management training programme which follows a multi-workshop implementation model4 and supports the development of the 12 quality system essentials (qses) of the clinical and laboratory standards institute (clsi) laboratory quality management system guidelines.5cameroon is a central african country with a population of approximately 20 million people living in 10 regions.6 bamenda is the headquarters of the northwest region, which has a population of approximately 1.8 million.6 bamenda regional hospital, a 400-bed public health hospital inaugurated in 1956, serves as the referral hospital for the 19 district hospitals of the northwest region. the field of laboratory medicine in cameroon has developed in several phases, starting with the paramedic ‘medical field unit’ introduced before independence in 1961. these laboratory medicine professionals received minimal training; their primary role was field identification of contagious diseases such as varicella, measles, yaws, smallpox, leprosy and tuberculosis. in most cases, they used basic identification techniques in mobile units.7 in 1968, a decree by the ministry of health led to the establishment of an organisational structure within public hospitals and health units.8 the hospital’s chief pharmacist became the laboratory head, under the supervision of the hospital director or a medical doctor who had specialised qualifications in microbiology, biochemistry, haematology or serology. at the time, laboratory services were either free or offered at minimal cost to the patient.8 two decades later, in 1990, a subsequent decree established organisational structures within private clinical laboratories.9 these statutes were designed to ensure the widespread provision of affordable laboratory services. they did not, however, address the quality of those services. in cameroon, the public health sector, in particular, has been characterised by a laissez-faire approach, with little concern for client satisfaction, limited specialised skills and mismanagement of both human and financial resources.10,11,12 in an effort to strengthen laboratory management so as to achieve rapid laboratory quality improvement, the slmta programme was launched in cameroon in october 2010. the bamenda regional hospital laboratory (brhl) was selected as one of five laboratories in the initial slmta cohort. laboratory personnel and other hospital staff participated in a series of training activities and quality improvement projects in an effort to enhance laboratory management, provide reliable patient services and create a lasting culture of quality. this article discusses slmta implementation at brhl, as well as results achieved and lessons learned. research methods and design top ↑ phase 1: preparation the us centers for disease control and prevention (cdc) served as the sponsor for slmta implementation in cameroon, with global health systems solutions (ghss) as the implementing partner. five laboratories (yaoundé central hospital laboratory, brhl, buea regional hospital laboratory, douala laquintinie hospital laboratory and laboratoire d’analyse medicales du centre yaoundé) were selected based on commitment from hospital laboratory management, appointment of a quality officer and the availability of participants who would maintain the same job responsibilities through the duration of the programme.4prior to slmta implementation, local capacity for auditors, trainers, and mentors was developed. in 2009, seven cameroonian auditors were trained to evaluate laboratory quality management systems (qms). five cameroonian laboratory personnel completed the slmta training-of-trainers course. in 2010, an experienced mentor and instructor from the clinton health access initiative (chai), lesotho, trained 11 cameroonian laboratory mentors. along with training on international standards, mentors were taught to build a sustainable culture of quality by transferring their knowledge and expertise to bench technologists. during the planning stages, cdc and ghss organised an advocacy meeting with hospital directors and laboratory managers of the five laboratories to ensure their buy-in and commitment to laboratory improvement. brhl further engaged hospital management for support in implementing qms, through meetings and active involvement in the laboratory improvement process. several hospital managers were also invited to attend the on-site slmta trainings, which helped encourage their support and collaboration. additionally, all hospital staff members were briefed on the laboratory improvement efforts. phase 2: slmta programme implementation the 12 slmta modules were taught in a series of three workshops, each lasting four days, over a period of eight months (table 1). the five in-country slmta trainers facilitated the programme. table 1: slmta workshop topics, bamenda regional hospital laboratory. the first workshop was conducted in mutengene, cameroon (october 2010) for staff from the five targeted laboratories, including four staff members from brhl. this workshop focused on the cross-cutting, productivity management, quality assurance, and documents and records management modules. following the first workshop, the slmta coordinators and trainers decided that it would be more effective and practical to hold the remaining two slmta training workshops at each of the enrolled laboratories so as to allow for more personnel to be trained. therefore, brhl’s second and third workshops were conducted on-site at the bamenda regional hospital. to maximise impact, 17 staff members attended the training, including the heads of the blood bank, reception and customer service, haematology, biochemistry, microbiology, serology and parasitology, as well as the safety officer, equipment officer, quality officer, laboratory director and hospital director. the 13 participants who missed the first centralised workshop were given materials to review. after each workshop, participants implemented improvement projects based on areas of weakness in the laboratory. all laboratory staff were involved in the improvement projects, including those who did not attend the training. following the training, ghss mentors were deployed to the five targeted laboratories, spending extended periods of time on site. the structured peer-to-peer, side-by-side mentorship approach was used to impart knowledge and expertise from the mentor to the laboratory staff following set guidelines.13,14 two additional mentors were assigned to brhl, alternating supervisory visits and working with all staff members to implement a change in culture and address gaps. iso 15189, the slipta checklist and the slmta toolkit were the primary tools used for follow-up. the mentors helped staff members address areas requiring improvement by examining the slmta toolkit, developing improvement projects, repeating activities from the training as needed, and then measuring improvement achieved using the checklist. phase 3: evaluation a series of audits using the slipta checklist were conducted and scores were calculated for each of the 12 qses so as to pinpoint problem areas and guide the development of improvement projects. overall scores were calculated as a percentage of the maximum possible points. to benchmark progress, these scores were also broken into ‘star’ levels, with zero stars corresponding to < 55%, one star 55% – 64%, two stars 65% – 74%, three stars 75% – 84%, four stars 85% – 94% and five stars 95% – 100%.the baseline audit was conducted 11 months prior to slmta implementation (november 2009). interim audits were conducted at various points (october 2010 before workshop 1; june 2011 before workshop 2; december 2011 after workshop 3; and february 2012). the exit audit was conducted in september 2012. these audits were performed by locally-trained auditors who were neither slmta trainers nor mentors for the brhl staff. finally, in august 2013, the african society for laboratory medicine (aslm) conducted an official slipta audit. results top ↑ brhl’s performance progressed steadily from 18% (zero stars) at baseline, where it was ranked last amongst the laboratories in its cohort, to 85% (four stars) in an interim audit eight months after completion of the three workshops, but then dropped to 67% (two stars) at the exit audit seven months later (figure 1). at the official aslm audit, the laboratory scored 81% (three stars), and was ranked first amongst the four laboratories audited, with the others scoring 60% – 72%. figure 1: figure 1: progress of the bamenda regional hospital laboratory in quality management systems as measured by the stepwise laboratory quality improvement process towards accreditation (slipta) checklist from november 2009 to august 2013. improvement was observed in each of the 12 qses measured in the slipta checklist (figure 2). from baseline to the last interim audit in february 2012, the most substantial improvements were in facilities and safety (93 percentage points), and purchasing and inventory (80 percentage points), whilst the least improved area was internal audit (6 percentage points). from the last interim to the exit audit, there were noted decreases in three areas: facilities and safety (37 percentage points), corrective action (33 percentage points), and equipment (27 percentage points). at the official aslm audit, the strongest areas were client management and customer service (100%), process control and internal and external quality assessment (94%), purchasing and inventory (93%) and facilities and safety (93%); whilst the weakest areas were corrective action (42%), management reviews (53%) and documents and records (56%). figure 2: performance of the bamenda regional hospital laboratory (%) across the 12 quality system essentials at baseline, last interim, exit and official aslm audit. improvement projects were selected and implemented after each slmta workshop (table 2). after each audit, the summary of non-conformities and recommendations was reviewed by the quality team and additional improvement projects were assigned, depending on what was feasible at the time, available resources and the seriousness of the non-conformity. table 2: a summary of improvement projects, bamenda regional hospital laboratory, cameroon. discussion top ↑ brhl’s substantial improvement was achieved by means of a combination of training activities, intensive on-site mentorship and collective focus of all laboratory staff. the experience at brhl illustrates what can be achieved when a laboratory successfully harnesses the energy of its staff and implements changes in order to improve the quality of services.engaging the management of bamenda hospital was key to ensuring financial support and sufficient human resources for the quality improvement process. the hospital director attended several of the slmta trainings, engaged directly with the mentor on quality management issues and increased the frequency of his visits to the laboratory. with management buy-in, the laboratory implemented infrastructure renovations and gained financial support for the purchase of reagents. since slmta completion, meetings between clinicians and laboratory staff have continued on a regular basis so as to enable laboratory staff to inform and consult with those they now see as critical clients and partners. because of these meetings, corrective actions have been easier to implement, customer satisfaction surveys have been facilitated and trust has been built between these users and providers of laboratory services. for brhl patients, the improvements have resulted in better service delivery because of procedure standardisation, fewer recalls of specimens, a safer environment, more reliability of testing and the ability to provide feedback on services offered. cameroon was the first country to decentralise slmta training,15 conducting the second and third workshops on-site at each enrolled laboratory. moving the slmta trainings to the facility allowed more staff to be trained and helped encourage universal participation and enthusiasm amongst all staff. whilst there was resistance to change in the beginning, by the third training there was a strong zeal to increase the laboratory’s star rating, as can be seen in the exit audit results. staff members became optimistic as the scores started to rise; their determination to improve the laboratory’s ratings was evidenced by their willingness to work extra hours, including weekends and public holidays. one young female laboratorian in the microbiology department said, ‘slmta has made us more ambitious. i now look forward to progressing in this profession and being able to help people of my community’. initially, the laboratory implemented what was possible at the time, rather than waiting for the ideal situation to arise. the quick successes of initial improvement projects bolstered staff confidence and inspired the desire for success, helping the laboratory sustain motivation for the more difficult projects. these successes also helped garner additional technical and financial assistance for further changes. whilst some improvement projects dealt with day-to-day activities, others sought to address larger challenges and attain more long-term goals. one noteworthy improvement project was to reorganise the reception area, which resulted in the creation of multiple reception sites, increased confidentiality and reduced turnaround time for test results. better organisation is a common theme in slmta training; one elderly female participant noted, ‘it has even extended to my home. now all my cupboards in the kitchen are labelled. all junk i removed’. laboratory staff members have reported a change in the culture of the laboratory. one commented that ‘our patients have ceased to be patients; they are now our clients’. the hospital director agreed, saying, ‘if i travel, i am now confident that work still continues, even in my absence’. he even took a leading role in directing improvement projects, personally supervising the closing of gaps in preparation for the official aslm audit. he met twice daily with quality team members for a period of three weeks; daily tasks were identified every morning and achievements and bottlenecks were reported every evening. a full-time mentorship approach allowed the on-site mentor to participate in daily activities in the laboratory and provide targeted training and guidance regarding the implementation of various aspects of the qms. the slmta-trained mentors helped build a working culture that emphasised quality and a sustainable qms. their continued presence over time helped them understand the local culture and practices, enabling them to design a tailored approach to assisting the laboratory in quality improvements. by working side-by-side with staff as peer coaches and role models, the mentors gained acceptance by laboratory personnel and accelerated implementation of quality requirements. the laboratory's performance increased steadily through slmta implementation, but the dramatic drop in the exit audit score 15 months after the last workshop highlights the importance of focusing on sustainability. staff had viewed the attainment of improved scores as the end of the struggle, rather than as part of an ongoing process of continuous quality improvement, requiring the maintenance of a new culture of quality. for example, client management and customer satisfaction, purchasing and inventory, process control and occurrence management all improved substantially from baseline to the last interim audit, but then declined once the slmta programme had ended and focus was lost. after the drop in score at the exit audit, the laboratory staff redoubled their efforts and results for these areas increased substantially once again by the official aslm audit. one area that did not improve after the exit audit was documents and records. whilst documentation improved from 6% at baseline to 72% at the last interim audit, it then dropped to 56% at exit where it remained at the official audit. the noted improvement resulted from the development and authorisation of many new policies and procedures, including a quality manual. however, by the time of the exit audit, the laboratory had begun to experience new challenges associated with the expanded documentation structure; the laboratory was overwhelmed by the number of documents that needed review and the ongoing maintenance required to retrieve documents, archive obsolete documents and communicate new changes. this experience highlights the need to plan ahead and to consider not only the development but also the maintenance of new systems. a new quality culture is growing at brhl as staff have embraced the need for continuous improvement and excellence in patient care. however, there is now a critical need to develop components outside the reach of the laboratory’s control: updated national guidelines for policies and procedures, professional boards for licensure, an up-to-date electronic database for proper document control and management, a hospital improvement programme to keep pace with improvements in the laboratory, regular audits of the qms and qualified service engineers. though the ultimate goals of reaching five stars and international accreditation have not yet been realised at brhl, the measurable improvement to date is a catalyst for greater achievements as the laboratory continues its journey from grass to grace. acknowledgements top ↑ the authors wish to acknowledge the commitment of the bamenda regional hospital management. we would also like to acknowledge the implementing partner (ghss), the sponsors (cdc) and all persons who supported slmta implementation at bamenda hospital. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions s.c.n. (brhl) wrote the manuscript. n.n.b. (brhl) was quality officer at the time and provided valuable data. t.m. (aslm) performed critical reviews and analyses. the article was conceived of and supervised by c.n.a. (brhl). references top ↑ 1. world health organization’s regional office for africa. resolution afr/rc59/r4: policy orientations on the establishment of centres of excellence for disease surveillance, public health laboratories, food and medicines regulation. in: final report: 59th session of the who regional committee for africa. brazzaville, republic of the congo: world health organization’s regional office for africa, 2009; p. 12–13.2.world health organization’s regional office for africa. resolution afr/rc58/r2: strengthening public health laboratories in the who african region: a critical need for disease control. in: final report: 58th session of the who regional committee for africa. yaounde, republic of cameroon: world health organization’s regional office for africa, 2008; p. 11–13. 3. world health organization regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation in the african region (with checklist) [document on the internet]. c2011 [cited 2013 may 31]. available from: http://www.afro.who.int/index.php?option=com_docman&task=doc_download&gid=8642&itemid=2593 4.yao k, mckinney b, murphy a, rotz p, wafula w, sendagire h, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin pathol. 2010 sep;134(3):401-9. pubmed pmid: 20716796. http://dx.doi.org/10.1309/ajcpnbbl53fwuiqj 5.clinical and laboratory standards institute. application of a quality management system model for laboratory services; approved guidelines – third edition. clsi document gp26-a3 [isbn 1-56238-553-4]. clinical and laboratory standards institute; 2004. 6.republic of cameroon. 3e rgph. la population du cameroun en 2010 [cameroon’s population in 2010] [document on the internet]. c2010 [cited 2014 sep 09]. available from: http://www.statistics-cameroon.org/downloads/la_population_du_cameroun_2010.pdf 7.awasum ss. evaluation of medical laboratory practice and training in anglophone cameroon; 2002 (unpublished presentation: 1st national scientific conference of medical laboratory technologists, ‘creating professional awareness in the new millennium’, 3–4 may 2002). 8.decree no. 68-df-419 of 15th october 1968: establishing the structural organization and the organic functioning of the hospital and health facilities in cameroon. government printers; 1968. 9.federal republic, cameroon. decree no 90-1465 of 9 november 1990: to lay down the organization and functioning of private clinical laboratories. government printers; 1990. 10.agendia a. cameroon’s free fall health system: two videos shock the world [page on the internet]. c2010 [cited 2014 sep 07]. available from: http://agendia.jigsy.com/entries/health/cameroon%e2%80%99s-free-fall-health-system-two-videos-shock-the-world 11.achanyi-fontem j. cameroon government x-rays health sector plan for 2012 [page on the internet]. c2008 [cited 2014 sep 07]. available from: http://www.leffortcamerounais.com/2008/03/cameroon-govern.html 12.the world justice project. cameroon healthcare access program [page on the internet]. c2008 [cited 2014 sep 09]. available from: http://worldjusticeproject.org/opportunity-fund/cameroon-healthcare-access-program-1 13.maruta t, motebang d, wanyoike j, et al. impact of mentorship on who-afro strengthening laboratory quality improvement process towards accreditation (slipta). afr j lab med. 2012;1(1), art. #6, 8 pages. http://dx.doi.org/10.4102/ajlm.v1i1.6 14.maruta t, rotz p, trevor p. setting up a structured laboratory mentoring programme. afr j lab med. 2013;2(1), art. #77, 7 pages. 15.ndasi j, dimite l, mbome v, et. al. decentralised facility-based training as an alternative model for slmta implementation: cameroon experience. afr j lab med. 2014;3(2), art. #231, 6 pages. http://dx.doi.org/10.4102/ajlm.v3i2.231 abstract introduction methods results discussion acknowledgements references about the author(s) shilpi gupta department of microbiology, military hospital, bhopal, india mahadevan kumar department of microbiology, bharati vidyapeeth university medical college, pune, india shelinder p.s. shergill department of microbiology, armed forces medical college, pune, india kundan tandel department of microbiology, command hospital (central command), lucknow, india citation gupta s, kumar m, shergill sps, tandel k. evaluation of ceftriaxone-sulbactam-disodium edetate adjuvant combination against multi-drug resistant gram-negative organisms. afr j lab med. 2020;9(1), a991. https://doi.org/10.4102/ajlm.v9i1.991 original research evaluation of ceftriaxone-sulbactam-disodium edetate adjuvant combination against multi-drug resistant gram-negative organisms shilpi gupta, mahadevan kumar, shelinder p.s. shergill, kundan tandel received: 04 feb. 2019; accepted: 27 aug. 2020; published: 10 dec. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: multi-drug resistant (mdr) gram-negative bacteria are an emerging threat, both in hospital and community settings. as very few antibiotics are effective against such infections, the need of the hour is a new antibiotic or drug combination which can overcome the effect of extended-spectrum β-lactamases (esbl) and metallo β-lactamases (mbl). a new antibiotic combination of ceftriaxone, sulbactam and disodium edetate (cse) has recently been proposed to tackle the mdr organisms. objective: our study was carried out to assess the susceptibility of esbland mbl-producing gram-negative organisms to cse. methods: the study was conducted in a tertiary-care hospital in delhi, india, from february 2017 to june 2017. a total of 179 mdr (85 esbl + 94 mbl) gram-negative isolates from various clinical samples, identified by an automated system (vitek 2) were tested against cse using the kirby-bauer disc diffusion method. susceptibility to cse was recorded based on interpretative zone sizes of ceftriaxone as per 2017 clinical and laboratory standards institute guidelines. results: the most common isolate was escherichia coli (76/179; 42.4%) followed by klebsiella pneumoniae (53/179; 29.6%) and acinetobacter baumanii (27/179; 15.1%). the in vitro susceptibility of esbland mbl-producing gram-negative isolates to cse was found to be 58/85 (68.2%) for esbl and 37/94 (39.4%) for mbl. conclusion: the in vitro susceptibility results obtained for cse against esbl-producing organisms is promising. it has the potential to emerge as a carbapenem-sparing antibiotic, active against esbl-producing strains. further clinical studies are required to establish the clinical efficacy of cse against mdr pathogens. keywords: ceftriaxone sulbactam, disodium edetate; multi-drug resistance; carbapenem-sparing. introduction increased antimicrobial resistance of gram-negative bacteria (gnb), both hospitaland community-acquired, is of great concern worldwide.1 according to the report of global antibiotic resistance partnership – india working group, the irrational and increased use of antibiotics, especially cephalosporins, in india has resulted in the emergence of multi-drug resistant (mdr) bacteria which were earlier known to be susceptible.2 the menace of the emerging threat has been realised by the indian government, hence it has called for effective action to address the increasing antimicrobial resistance. the indian ministry of health and family welfare, in collaboration with the world health organization, formulated the national health policy 2017, which calls for an urgent need for standardisation of antibiotic usage guidelines to minimise the emergence of antimicrobial resistance. thus, both organisations decided to tackle the issue as a priority collaborative work in 2018–2019.3 despite increasing antibiotic resistance, the common therapy for treatment of gnb infections includes use of β-lactams with β-lactamase inhibitors, third and fourth generation cephalosporins and carbapenems.4 production of β-lactamase enzymes is the most widespread mechanism of resistance adopted by gnb to counteract the effect of β-lactam antibiotics.5,6 extended-spectrum β-lactamases (esbls) are usually plasmid-mediated β-lactamases and hydrolyse oxyimino group-containing β-lactam antibiotics.7 metallo β-lactamases (mbls) are a class of broad-substrate spectrum enzymes that also hydrolyse most β-lactam antibiotics, except monobactams.8 other mechanisms that contribute to resistance are drug efflux systems, outer membrane protein changes, antibiotic-modifying enzymes and antibiotic-target modification.9 carbapenems are used against the esbl-producing organisms because of their stability against hydrolysis by esbls and broad-spectrum activity.10 however, with the emergence of carbapenem-hydrolysing enzymes, overexpression of efflux pumps and changes in outer membrane proteins, increases in carbapenem resistance have been reported among the members of enterobacteriaceae and non-fermenter gnb, such as the acinetobacter and pseudomonas group of pathogens.11,12 in india, the prevalence of esbl and mbl producers among gram-negative organisms range between 28% – 84% and 7.5% – 71%, respectively.13,14 the increasing resistance towards available antibiotics, as well as the lack of development of new antibiotics against gnb, could soon lead to the world experiencing the tough situations of the pre-antibiotic era with an increase in cases with untreatable infections. a newer approach to improving the efficiency of the existing antimicrobials is the use of antibiotic adjuvants. a novel antibiotic adjuvant entity of ceftriaxone, sulbactam with adjuvant disodium edetate (cse) is being used in indian hospitals against mdr infections.15,16 the antibiotic adjuvant entity is a combination of ceftriaxone plus sulbactam with disodium edetate and has undergone phase iii clinical trials under the aegis of the central drugs standard control organisation, india.17 this study aimed to study the in vitro susceptibility to cse of mdr gram-negative organisms isolated in our centre. thus, the present study aimed to study the in vitro susceptibility of esbland mbl-producing gnb to cse and to explore whether it could be utilised as a carbapenem-sparing drug. methods ethical considerations ethical clearance was obtained from the institutional ethics committee, army hospital (research and referral), new delhi, india (92/2016). study design and setting this cross-sectional study was conducted in the department of microbiology of a 900-bed, tertiary-care, super-speciality army hospital (research and referral), new delhi, india from february 2017 to june 2017. isolates were obtained from various clinical samples from both outand inpatients received in the laboratory for bacterial culture from different clinical departments. sample types included: urine, pus, cerebrospinal fluid, sputum, tissue from burn wound sites, endotracheal aspirate, semen, high vaginal swab and drains fluid. microbiological processing samples were processed using conventional methods. blood culture bottles were incubated in a fully automated blood culture system, the bact/alert 3d (biomérieux sa, marcy-l’étoile, france). after obtaining a pure bacterial growth, isolate identification and antibiotic sensitivity testing were carried out on a vitek 2 compact (biomérieux sa, marcy-l’étoile, france). an mdr isolate was defined as a gnb strain that showed resistance to at least three different categories of antibiotics.18 a total of 85 esbland 94 mbl-producing gnb were identified by phenotypic tests and confirmed by the vitek 2 system for inclusion in the study. the confirmatory test for esbl production was carried out using discs containing ceftazidime (30 µg) alone, along with ceftazidime/clavulanic acid (30/10 µg) discs. similarly, cefotaxime (30 µg) and cefotaxime/clavulanic acid discs (30/10 µg) were also used. an increase in zone diameter of ≥ 5 mm around the clavulanate disk compared to the zone of inhibition for the ceftazidime and cefotaxime disk alone was used to confirm and isolate as positive for esbl production as per clinical and laboratory standards institute guidelines.19 the modified hodge test19 was used for isolates identified as carbapenemase-producing gnb strains. a 10 µg meropenem disc was placed on a mueller-hinton agar plate previously inoculated with escherichia coli american type culture collection 25922 (the indicator organism). afterwards, the test organisms were streaked out in a straight line, starting from the edge of the meropenem disc, for at least 20 mm – 25 mm length. the enhancement of growth of the indicator organism along the test organism’s streak line and zone of inhibition of the disk to form a cloverleaf appearance was considered as a positive indicator for carbapenemase production as per clinical and laboratory standards institute guidelines.19 for detection of class b carbapenemases (mbl), the double-disc synergy test using imipenem and imipenem plus ethylenediaminetetraacetic acid disc was done. an organism with a zone size difference of 7 mm between imipenem and imipenem plus ethylenediaminetetraacetic acid discs was considered to be an mbl-producing strain.20 all 179 isolates were then further tested for antimicrobial susceptibility against cse (venus medicine research centre, baddi, himachal pradesh, india) by the kirby-bauer disc diffusion method on a mueller-hinton agar kept at 37 °c for 16 h – 18 h (figure 1). quality control of cse antibiotic discs was done using e. coli american type culture collection 25922 and a laboratory-characterised sensitive isolate of acinetobacter baumanii (strain no. ahrr1205/2017). susceptibility of the tested organisms against this combination was reported as sensitive, intermediate or resistant based on the zone of inhibition mentioned for ceftriaxone as per clinical and laboratory standards institute guideline m100s27: performance standards for antimicrobial sensitivity testing, 2017.19 the zone of inhibition around the disc was measured, and the organism was labelled as sensitive if the zone measured > 23 mm for enterobacteriaceae or > 21 mm for acinetobacter, intermediate if 20 mm – 22 mm (enterobacteriaceae) or 14 mm – 20 mm (acinetobacter), or resistant if < 19 mm for enterobacteriaceae or < 13 mm for acinetobacter.19 non-fermenters such as pseudomonas aeruginosa and burkholderia cepacia were not tested against cse as these are known to be inherently resistant to ceftriaxone and there are no testing standards mentioned in clinical and laboratory standards institute guidelines.19 figure 1: zone of inhibition around the ceftriaxone-sulbactam-disodium edetate disc, delhi, india, february 2017 to june 2017. data analysis statistical analysis was done using graph pad, a free online software offering by founder dr harvey motulsky (graphpad software, san diego, california, united states). associations between two factors were drawn through univariate logistic regression using the fischer exact test. p-values of less than 0.05 were considered statistically significant. results a total of 179 clinical isolates from 168 clinical cases (117 male patients and 51 female patients) were included in the study, with a mean patient age of 43.22 years (range: 4–85 years). most isolates were from urine, followed by pus and blood specimens, and these accounted for 136 (75.9%) of the total isolates (table 1). of the included pathogens, 127 (70.9%) were isolated from inpatients, and 29 (16.2%) were isolated from patients in intensive care units (figure 2). figure 2: ward distribution of samples included in the study at a hospital in delhi, india, february 2017 to june 2017. table 1: prevalence of individual pathogens in various samples at a hospital in delhi, india, february 2017 to june 2017. among the isolated pathogens, e. coli (n = 76; 42.4%) was the most predominant followed by klebsiella pneumoniae (n = 53; 29.6%) and a. baumanii (n = 27; 15%) (table 1). eighty-five (47.5%) of the tested isolates were esbl producers and 94 (52.5%) were mbl producers. fifty-eight (68.23%) of the esbl producers (table 2) and 37 (39.36%) mbl producers (table 3) showed in vitro sensitivity towards cse. table 2: antibiogram for ceftriaxone-sulbactam-disodium edetate against extended-spectrum β-lactamases producing gram-negative isolates at a hospital in delhi, india, february 2017 to june 2017. table 3: antibiogram for ceftriaxone-sulbactam-disodium edetate against metallo β-lactamases producing gram-negative isolates at a hospital in delhi, india, february 2017 to june 2017. among the identified esbl-producing gnb, 44 (73.3%) e. coli and 7 (53.8%) k. pneumoniae showed sensitivity towards cse, while 51 (85%) e. coli and 11 (84.6%) k. pneumoniae showed sensitivity towards meropenem. the most common mbl-producing gnbs, k. pneumoniae, a. baumanii and e. coli, showed 27.5% (n = 11), 48% (n = 12) and 31.3% (n = 5) sensitivity, respectively, towards cse and 2.5% (n = 1), 0% and 6.3% (n = 1) sensitivity, respectively, towards meropenem (data not shown). a statistically significant association was found when susceptibility to meropenem and cse were compared (p < 0.001) in esbl-producing e. coli. however, no statistically significant associations were seen when the cse susceptibility pattern was compared to meropenem susceptibility patterns for other esbland mbl-producing organisms. twelve (48%) of the mbl-producing a. baumanii isolates which were resistant to meropenem showed susceptibility to cse (data not shown). multi-drug resistant e. coli, k. pneumoniae and a. baumanii showed 64.5% (n = 49), 33.9% (n = 18) and 48.1% (n = 13) susceptibility, respectively, towards cse and 71.1% (n = 54), 20.8% (n = 11) and 7.4% (n = 2), respectively, against meropenem (figure 3). figure 3: in vitro antibiotic susceptibility pattern for most common gram-negative isolates against ceftriaxone-sulbactam-disodium edetate and meropenem (n = 156) at a hospital in delhi, india, february 2017 to june 2017. discussion this study included only those mdr gnb isolates which were proven to be pathogenic, obtained from clinically established cases of infection. the majority of isolates were from urine samples (n = 84; 47%), followed by pus (n = 34; 19%) and blood (n = 18; 10%). the identified pathogens included e. coli, k. pneumoniae and a. baumanii, in decreasing order of prevalence. similar distributions of isolates with similar specimen distribution have been reported by other studies.21,22 in the present study, e. coli (n = 49; 58.3%) was highly prevalent in urine samples, followed by k. pneumoniae (n = 22; 26.2%), indicating their significant role in urinary tract infections. similar findings were also reported by janifer et al. in chennai, south india23 and ruchika et al. in gurgaon, haryana, north india.24 klebsiella spp. and e. coli are known to be the major causative agents for hospital-acquired infections. according to the national health service report of 2017 on gram-negative bloodstream infections, e. coli, p. aeruginosa and klebsiella spp. were responsible for 72% of all gram-negative bloodstream infections, with e.coli accounting for 59% of the total cases.25 however, in the present study, e. coli and k. pneumoniae were implicated in 16 out of 18 (88.8%) gram-negative bloodstream infection cases, with k. pneumoniae identified in the majority (10/18; 55.5%) of cases as compared to e. coli (6/18; 33.3%), which is in contrast to the national health service report.25 acinetobacter baumanii is known to be an important pathogen in causing respiratory infections such as hospital-acquired pneumonia and bacteraemia, especially in intensive care patients, followed by skin and soft tissue infections and urinary tract infections.26,27 the present study has dissimilar findings in terms of isolation of a. baumanii, with maximum isolation from pus, because of skin and soft tissue infection cases (50%); followed by respiratory samples, because of respiratory infections (32.1%); and blood samples, because of bacteraemia (7.1%). in the present study, 95 out of 179 (53.1%) mdr gnb isolates showed sensitivity to cse in vitro. in similar studies conducted in the northern and western parts of india, higher susceptibility rates against cse have been reported by bhatia et al. (84% – 94%), kumar et al. (81.9% – 94.74%), bagga et al. (87.5% – 94.6%) and sachdeva et al. (74.2% – 80.5%).12,24,28,29 the lower rate in our study compared to other studies could be because the isolates tested in the present study have been identified as esbl or mbl producers, which was not specifically mentioned by other studies. the susceptibility of esbland mbl-producing isolates to the cse combination in the present study was from 68% for esbl producers and 39% for mbl producers. the range is close to a similar study conducted in mumbai, maharashtra, india by sahu et al.30 because of increased clinical use of carbapenems against mdr gnb, our study also compared the efficacy of cse against meropenem on gram-negative isolates to use this new combination as a possible carbapenem-sparing drug. carbapenems are considered to be the drug of choice for esbl-producing gnb. the present study showed that 58 (68.2%) of the esbl isolates were susceptible to cse, of which e. coli susceptibility to cse was statistically significant when compared with its susceptibility to meropenem (p < 0.001). the susceptibility profile to cse of the three most predominant pathogens in our study, e. coli (49/76, 64.5%), k. pneumoniae (18/53, 34%) and a. baumanii (13/27, 48.1%) was comparable for e.coli (54/76, 71.1%) when compared to meropenem and high for k. pneumoniae (11/53, 20.8%) and a. baumanii (2/27, 7.4%). however, several authors from different parts of india (haryana, western uttar pradesh, gujarat) have reported significantly higher susceptibility to cse when compared with meropenem.24,28,31 our study showed comparable sensitivity among esbl organisms to cse (58/85, 68.2%) and meropenem (64/85, 75.3%), which implies that if cse is tested against all esbl isolates and they are found to be susceptible, cse could be used as a drug of choice in place of carbapenems. most of the mbl-producing organisms are resistant to carbapenems and the drug of choice for such isolates is polymixins. in the present study, 39.4% (34) of such isolates were susceptible to cse; thus, cse instead of polymixins could be considered as a therapeutic option in these cases. one major concern is finding effective treatments for infection with acinetobacter spp., which is now commonly isolated from critical areas in most of the hospitals worldwide.24 our study found cse to be effective in 48.1% of mbl-producing a. baumanii infections, which is a fair number, and use of cse could be beneficial in such infections. a similar study conducted in pune, india found that cse was a superior antibiotic compared to other commonly used β-lactam antibiotics, including carbapenems, when tested against mdr gnb.32 a study conducted in faridabad, haryana, india, which evaluated the clinical use of cse on patients, concluded that cse should be used as a carbapenem-sparing drug and its combination with polymyxins can help to reduce mortality rates by successfully treating complicated mdr cases of intraabdominal, lower respiratory tract and urinary tract infections.12 limitations the limitation of our study was the relatively small number of isolates tested. larger sample size and diverse health facility level (primary to tertiary) studies would be required to rule out any referral bias that is expected in a tertiary-care hospital. further, this study can be extended with application to clinical situations to have a clinico-microbiological correlation to guide clinicians for the judicious use of cse against mdr pathogens. conclusion the results of this study show that cse can potentially be effective among esbl-producing bacteria, especially e. coli. the susceptibility of multi-drug resistant gram-negative microorganisms to cse suggests that cse could be a good option as a carbapenem-sparing drug and also against some of the mbl-producing organisms. acknowledgements we wish to thank venus remedies (india) for providing antibiotic discs of ceftriaxone-sulbactam-disodium edetate for testing. competing interests there are no competing interests, financial interests, relationships and affiliations relevant to the subject matter or materials discussed in the manuscript. authors’ contributions all authors contributed intellectually to the work. s.g. was involved in bench work and manuscript writing; m.k. was involved in the study concept and supervision, manuscript writing and final approval; s.p.s.s. was involved in the study concept and supervision; and k.t. was responsible for collection of mdr strains, and contributed to the bench work. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references kumar d, singh ak, ali mr, chander y. antimicrobial susceptibility profile of extended spectrum ß-lactamase (esbl) producing escherichia coli from various clinical samples. infect dis (auckl). 2014;7:1–8. https://doi.org/10.4137/idrt.s13820 global antibiotic resistance partnership (garp) – india working group. rationalizing antibiotic use to limit antibiotic resistance in india. indian j med res. 2011;134(3):281–294. national action plan on antimicrobial resistance (nap-amr) 2017–2021. april 2017 | ministry of health & family welfare-government of india. bush k. bench-to-bedside review: the role of β-lactamases in antibiotic-resistant gram-negative infections. bush crit care. 2010;14:224. https://doi.org/10.1186/cc8892 bush k, jacoby ga, medeiros aa. a functional classification scheme for βlactamases and its correlation with molecular structure. antimicrob agents chemother. 1995;39(6):1211–1233. https://doi.org/10.1128/aac.39.6.1211 bush k, jacoby ga. updated functional classification of beta-lactamases. antimicrobial agents chemother. 2010;54(3):969–976. https://doi.org/10.1128/aac.01009-09 bradford pa. extended-spectrum β-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. clin microbiol rev. 2001;14(4):933–951. https://doi.org/10.1128/cmr.14.4.933-951.2001 drawz sm, bonomo ra. three decades of β-lactamase inhibitors. clin microbiol rev. 2010;23(4):160–201. https://doi.org/10.1128/cmr.00037-09 chopra i, schofield c, everett m, et al. treatment of health-care associated infections caused by gram-negative bacteria: a consensus statement. lancet infect dis. 2008;8(2):133–139. https://doi.org/10.1016/s1473-3099(08)70018-5 pravin kn and michelle sv. prevalence of carbapenem resistant enterobacteriaceae from a tertiary care hospital in mumbai, india. j microbiol infect dis. 2013;3(04):207–210. https://doi.org/10.5799/ahinjs.02.2013.04.0110 rodrigues c. carbapenem-resistant enterobacteriaceae: a reality check. reg health forum. 2011;15(1):83–86. bhatia p. alternative empiric therapy to carbapenems in management of drug resistant gram negative pathogens: a new way to spare carbapenems. res j infect dis. 2015;3:2. http://doi.org/10.7243/2052-5958-3-2 basavaraj cm, jyothi p, peerapur basavaraj v. the prevalence of esbl among enterobacteriaceae in a tertiary care hospital of north karnataka, india. j clin diagn res. 2011;5(3):470–475. chaudhuri bn, rodrigues c, balaji v, et al. incidence of esbl producers amongst gram-negative bacilli isolated from intra-abdominal infections across india (based on smart study, 2007 data). j assoc physicians ind. 2011;59(5):287–292. de as, kumar sh, baveja sm. prevalence of metallo-β-lactamase producing pseudomonas aeruginosa and acinetobacter species in intensive care areas in a tertiary care hospital. indian j crit care med. 2010;4(1):217–219. https://doi.org/10.4103/0972-5229.76089 chaudhary m, payasi a. clinical, microbial efficacy and tolerability of elores, a novel antibiotic adjuvant entity in esbl producing pathogens: prospective randomized controlled clinical trial. j pharmacy res. 2013;7(4):275–280. https://doi.org/10.1016/j.jopr.2013.04.017 chaudhary m, payasi a. a randomized, open label prospective, multicenter phase-iii clinical trial of elores in lower respiratory tract and urinary tract infections. j pharmacy res. 2013;6(4):409–414. https://doi.org/10.1016/j.jopr.2013.04.011 magiorakos ap, srinivasan a, carey rb, et al. multidrug-resistant, extensively drug resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. clin microbiol infect. 2012;18(3):268–281. https://doi.org/10.1111/j.1469–0691.2011.03570.x clinical laboratory standard institute (clsi). performance standards for antimicrobial susceptibility testing (2017) clsi approved standards clsi m100-s23. wayne, pa: clsi; 2017. clinical laboratory standard institute. performance standards for antimicrobial susceptibility testing; twenty-second informational supplement. wayne, pa: clinical laboratory standard institute; 2013, p. 32. pop-vicas ae, d’agata em. the rising influx of multidrug-resistant gram-negative bacilli into a tertiary care hospital. clin infect dis. 2005;40(12):1792–1798. https://doi.org/10.1086/430314 subhedar v, jain sk. gram negative super bugs: a new generation of icu infections, an emerging challenge for health care settings. am j microbiol res. 2016;4(1):47–50. janifer j, geethalaksmi s, satyavani k, et al. prevalence of lower urinary tract infection in south indian type 2 diabetic subjects. indian j nephrol. 2009;19(3):107–111. https://doi.org/10.4103/0971-4065.57107 bagga r. retrospective analysis of antibiotic susceptibility and resistance patterns against nosocomial gram negative pathogens in fortis memorial research institute gurgaon. int j curr adv res, 2015;4(9):347–351. nhs_improvement. guidance on the definition of healthcare associated gram-negative bloodstream infections. in: nhs improvement, editor. public health england; 2017. gaynes r, edwards jr. overview of nosocomial infections caused by gram-negative bacilli. clin infect dis. 2005;41(6):848–854. https://doi.org/10.1086/432803 paul m, weinberger m, siegman-igra y, et al. acinetobacter baumannii: emergence and spread in israeli hospitals 1997–2002. j hosp infect. 2005;60(3):256–260. https://doi.org/10.1016/j.jhin.2005.01.007 kumar m, chaudhary s, makkar dk, garg n, chugh sk. comparative antimicrobial efficacy evaluation of a new product elores against meropenem on gram-negative isolates. asian j pharm clin res. 2015;8:251–254. neelam sachdeva. antibiotic sensitivity pattern of bacterial pathogens in rajeev gandhi cancer hospital, delhi. int j recent sci res. 2016;7(1):8480–8485. sahu m, sanjith s, bhalekar p, keny d. waging war against extended spectrum beta lactamase and metallobetalactamase producing pathogens – novel adjuvant antimicrobial agent cse1034an extended hope. j clin diagn res. 2014;8(6):dc20–dc23. https://doi.org/10.7860/jcdr/2014/8802.4504 makkar dk, kumar m, chaudhary s, goyal s, aggarwal p, garg n. comparative antimicrobial efficacy evaluation of a new product elores against meropenem on gramnegative isolates. asian j pharm clin res. 2015;8:1–4. arora s, munshi n. comparative assessment of antibiotic susceptibility pattern of gram negative pathogens isolated from intensive care unit patients in pune. bmrj. 2015;10(2):1–9. https://doi.org/10.9734/bmrj/2015/18199 abstract introduction methods results discussion acknowledgements references about the author(s) jason van rensburg department of biomedical sciences, faculty of health and wellness, cape peninsula university of technology, cape town, south africa saarah davids south african medical research council/cape peninsula university of technology cardiometabolic health research unit and biomedical sciences, faculty of health and wellness, cape peninsula university of technology, cape town, south africa carine smuts department of biomedical sciences, faculty of health and wellness, cape peninsula university of technology, cape town, south africa glenda m. davison south african medical research council/cape peninsula university of technology cardiometabolic health research unit and biomedical sciences, faculty of health and wellness, cape peninsula university of technology, cape town, south africa division of haematology, faculty of health sciences, university of cape town, cape town, south africa citation van rensburg j, davids s, smuts c, davison gm. use of full blood count parameters and haematology cell ratios in screening for sepsis in south africa. afr j lab med. 2023;12(1), a2104. https://doi.org/10.4102/ajlm.v12i1.2104 original research use of full blood count parameters and haematology cell ratios in screening for sepsis in south africa jason van rensburg, saarah davids, carine smuts, glenda m. davison received: 21 oct. 2022; accepted: 31 jan. 2023; published: 11 apr. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: sepsis is characterised by multi-organ failure due to an uncontrolled immune response to infection. sepsis prevalence is increased in developing countries and requires prompt diagnosis and treatment. reports, although controversial, suggest that full blood count parameters and cell ratios could assist in the early screening for sepsis. objective: the study evaluated the use of haematological cell ratios in screening for sepsis in a south african population. methods: the study retrospectively analysed the complete blood counts, blood cultures (bc) and biochemical test results of 125 adult patients who presented between january 2021 and july 2021 at a hospital in cape town. an iso15189-accredited laboratory performed all of the tests. we compared and correlated the automated differential counts, neutrophil, monocyte and platelet-to-lymphocyte ratios with procalcitonin levels. a p-value of < 0.05 was considered significant. results: sixty-two sepsis patients (procalcitonin > 2 ng/l and positive bc) were identified and compared to 63 non-sepsis controls. all cell ratios were significantly elevated in sepsis patients (p < 0.001). however, the two groups had no significant difference in absolute monocyte counts (p = 0.377). in addition, no correlation was detected between any cell ratios and procalcitonin. conclusion: in combination with complete blood count parameters, haematology cell ratios can be used for early sepsis detection. the full blood count is widely available, inexpensive, and routinely requested by emergency care clinicians. although procalcitonin and bc remain the gold standard, the calculation of cell ratios could provide a simple screening tool for the early detection of sepsis. what this study adds: this study adds evidence to the proposal that calculating haematological cell ratios assists in the early screening of sepsis in a south african setting. keywords: sepsis; full blood count; neutrophil-to-lymphocyte ratio; monocyte to lymphocyte ratio; platelet-to-lymphocyte ratio. introduction sepsis is a life-threatening condition characterised by multiple organ system failures due to a dysregulated immune response manifesting secondary to a bacterial or fungal infection.1 due to differing definitions of sepsis, global prevalence figures remain unclear; however, a minimum of 49 million sepsis cases are estimated to occur annually, with up to 11 million mortality.2 sepsis prevalence is high in lowand middle-income countries due to high poverty levels and lack of healthcare resources. immunocompromised individuals, the elderly and neonates are most at risk.3 sepsis is a medical emergency that requires prompt treatment to achieve a favourable clinical outcome. clinical symptoms are often non-specific making diagnosis difficult, variable and unclear. the diagnosis is predominantly based on the sequential organ failure assessment score, which measures the functional status of the respiratory, haemostatic, hepatic, renal, cardiovascular and nervous systems.4,5 the sequential organ failure assessment score is not specific to sepsis; thus, biological markers such as blood cultures (bc), procalcitonin, and c-reactive protein have been adopted in clinical practice.6 these biomarkers, although important, have limitations. procalcitonin is a precursor molecule to the thyroid hormone calcitonin and is undetectable in the serum of healthy individuals. procalcitonin levels correlate with sepsis severity, with serum concentrations between 0.5 ng/l and 2.0 ng/l indicative of bacterial infection and those > 2.0 ng/l indicative of sepsis.7,8 although highly specific, procalcitonin assays may not be widely available, have a longer turnaround time than a complete blood count and require specialised instrumentation and costly reagents. additionally, procalcitonin may be falsely elevated in conditions such as renal impairment, congestive heart disease, burn wounds, malignancies and acute pancreatitis, and all common sepsis complications.9 c-reactive protein is an acute-phase protein synthesised by the liver in response to inflammation, making it an early and sensitive marker of sepsis.10 c-reactive protein is elevated in infection, injury or chronic disease inflammations. thus, it is not specific, limiting its use as a marker of sepsis.11 on the other hand, bc is a specialised investigation that can detect microorganism growth in a patient’s blood. it uses an enriched growth medium to support microbial growth and a colourimetric detection system to detect carbon dioxide produced by the growing blood pathogens.12 any growth within the media indicates sepsis; thus, bcs are considered the gold standard of sepsis diagnosis. however, this test has limitations, including sample contamination due to non-sterile collection. also, antibiotics use before sampling may cause false results. in addition, although clinicians get results within 24 h, slow-growing organisms can take 5 days or longer to grow, which is unsuitable for early diagnosis.13 therefore, although these biomarkers and culture techniques are helpful, the need for a quick, efficient and cost-effective method to screen for early sepsis is still relevant. recent studies have reported using full blood count parameters and cell ratios to screen for sepsis.14,15 it is generally accepted that an abnormal white cell count with an increase in immature granulocytes indicates sepsis and has been included as one of the four systemic inflammatory response syndrome criteria.16 automated full blood and differential counts are essential in diagnosing sepsis and are often the first laboratory test to be requested. it has a fast turnaround time, is cost-effective and can potentially provide crucial information on peripheral blood cell populations if interpreted correctly. bacterial sepsis is complex and results in the initiation of an acute inflammatory response with the release of cytokines, chemokines and alterations in the proportions and numbers of innate and adaptive immune cells. these changes can be detected by analysing the automated full blood count and differential results.17 neutrophils, the most common peripheral leucocytes, form part of the innate immune response and are released by the bone marrow in response to several stimuli, including stress, smoking and inflammation.18 during sepsis or infection, they increase rapidly and can eliminate pathogens by phagocytosis and oxidative bursts. in severe cases, the neutrophil function can become dysregulated, leading to chronic inflammation.18 it is believed that after exposure to lipopolysaccharides, anti-apoptotic signals of the b-cell lymphoma 2 (bcl-2) family are upregulated, leading to neutrophils resisting apoptosis and increasing their lifespan.18 although a disruption in neutrophil numbers can indicate infection, on their own they are not a reliable indicator of sepsis. in africa, benign ethnic neutropenia, defined as a neutrophil count of < 1.5 × 109/l in normal healthy individuals, is common, and researchers have proposed that the reference interval for neutrophils in african populations is lower than in other population groups.19,20 this african neutrophil reference interval is essential for benchmarking limits at which the white cell count and neutrophil count can screen sepsis in african populations. on the other hand, lymphocytes make up 20% – 40% of circulating blood cells and form part of the adaptive immune system. during sepsis, this cell population, unlike neutrophils, undergoes apoptosis leading to severe lymphopenia. therefore, calculating the neutrophil-to-lymphocyte ratio (nlr) can be a far more valuable and reliable sepsis biomarker than the neutrophil count alone.21,22,23,24,25 monocytes, also measured by an automated cell counter, are innate immune cells that function as phagocytes and initiate the adaptive immune response by presenting bacterial antigens to t-cells and secreting proinflammatory cytokines. monocyte counts are known to climb rapidly during the early stages of infection; however, much like neutrophils they can become defective and undergo rapid apoptosis.26,27 it has been suggested that the monocyte-to-lymphocyte ratio (mlr) could add valuable information to early sepsis screening.28 however, studies examining this parameter are scarce and conflicting.29,30 platelets, the smallest circulating blood cells, lack a nucleus and are usually released by the bone marrow in response to bleeding and endothelial injury. platelets participate in haemostasis and the inflammatory response by secreting cytokines, recognising pathogen-associated molecular patterns and activating innate immune cells.31 thus, their involvement in both processes makes them critical in sepsis. thrombocytopaenia, defined by a platelet count of < 150 × 109/l, is common in sepsis and forms part of the sequential organ failure assessment score,4 which measures organ failure and forms part of the diagnostic criteria for sepsis. furthermore, the prognostic value of a severely decreased platelet count (< 50 × 109/l) in septic patients has been demonstrated,32 while a study of 5537 patients with sepsis reported that patients with an elevated platelet-to-lymphocyte ratio (plr) at diagnosis (> 200) were associated with increased mortality.33 the calculation of the nlr, mlr and plr are potential rapid screening tools to detect sepsis. although studies have demonstrated the value of these ratios, reports have been variable and often unclear. therefore, this study aimed to evaluate the utility of haematological cell ratios to detect gram-negative bacterial sepsis in patients presenting to a hospital in cape town, south africa, and to correlate the values with serum procalcitonin levels. methods ethical considerations this study was retrospective and thus no additional testing was conducted on participants. participant consent was waived and ethical clearance was granted by the cape peninsula university of technology’s ethics committee (clearance number: cput/bms-ec 2021/e32). permission to use the laboratory’s patient records was obtained from the laboratory. all patient records were handled according to the declaration of helsinki and confidentiality was maintained by removing all patient identifiers. data were stored on a password-protected computer which was only accessible to the primary investigator. study design this retrospective study analysed 125 adult patients (all > 18 years of age) with complete blood count parameters. the tests were conducted between january 2021 and july 2021 at an iso15189-accredited laboratory in cape town, south africa. based on procalcitonin levels (> 2 ng/l) and bc results (positive with confirmed gram-negative bacillary growth), participants were divided into two groups comprising those with confirmed sepsis (n = 62) and a control group (n = 63) which comprised hospitalised patients who had no clinical or laboratory evidence of sepsis. the haematological cell ratios and full blood count parameters were then compared and correlated using statistical tests. full blood count and haematological cell ratios peripheral blood was collected into vacutainer tubes containing ethylenediaminetetraacetic acid. automated full blood and white cell differential counts were performed by flow cytometric analysis on the sysmex xn series automated analyser (sysmex corporation, kobe, hyogo, japan). the nlr and mlr were calculated by dividing the relevant absolute cell line count by the absolute lymphocyte counts, whereas the plr was calculated by dividing the total platelet count by the absolute lymphocyte count. biochemical tests peripheral blood was collected into a serum separating tube and centrifuged at 4000 revolutions per minute for 5 to 10 min to remove the serum. procalcitonin values were then generated using the chemiluminescent microparticle immunoassay principle and processed on the abbott alinity ci and architect ci analysers (abbott laboratories, chicago, illinois, united states). c-reactive protein results were generated by immunoturbidimetry chemistry assay (abbott laboratories, chicago, illinois, united states). a procalcitonin cut-off value of > 2 ng/l was used to determine if a patient had sepsis.8 blood cultures blood culture samples were collected in duplicate for anaerobic and aerobic cultures. cultures were incubated and monitored for growth for 7 days on the biomérieux bactalert 3d system (biomérieux, marcy-l’etoile, france). cultures flagged for growth by the analyser were examined microscopically and subcultured on appropriate media based on organism morphology and gram stain results. culture plates were examined after 18 h to 24 h of incubation, and isolates were speciated by mass spectrometry using a biomérieux vitek-ms’s (biomérieux, marcy-l’etoile, france) principle of matrix-assisted laser desorption ionization time-of-flight. in cases where inadequate microbial material was available for mass spectroscopy, biochemical analysis and speciation were performed using the biomérieux vitek 2 (biomérieux, marcy-l’etoile, france). only cultures with gram-negative bacilli growth were considered for the sepsis group, as gram-positive cocci are most likely to be pre-analytical contaminants. data collection and statistical methods all archived full blood counts, differential counts and blood chemistry data were collected from the laboratory’s information system. consecutive patients, > 18 years of age, with a positive bc and a negative gram stain, were included in the sepsis group, while the controls comprised hospitalised patients with no evidence of sepsis and negative bc. results were collated on microsoft excel 2019 (microsoft corporation, redmond, washington, united states) and imported into jamovi version 2.3 (jamovi project, sydney, australia) for statistical analysis. subjects were assigned unique identifiers, and only haematology, chemistry, culture results, age and gender data were collected to prevent bias and protect confidentiality. the normality of the data set was established by jamovi’s frequency statistics, with the range of −1 to 1 considered to be normally distributed. skewed data were presented in quartiles, and significance was assessed using the kruskal-wallis’s test.34 normally distributed data were presented as standard deviations, and significance was determined using the one-way analysis of variance test with p < 0.05 considered significant. correlations between analytes were assessed by bivariate regression, and a p-value of < 0.05 was considered significant. receiver operating characteristic curves were constructed to evaluate cell ratios as possible diagnostic screening markers for sepsis. results general characteristics the study comprised 125 patients’ data. patients were over 18 years; 62 had sepsis, while 63 had no sepsis (control group). the 62 sepsis patients had gram-negative bacteria (pseudomonas aeruginosa, klebsiella pneumoniae, escherichia coli and serratia marcescens) isolated from their bc and had a procalcitonin value > 2 ng/l. of the 62 patients, 32 were male, and the group’s median age was 65 (range: 30–87). the results were compared to 63 controls over 18 years (19–77) with no evidence of sepsis (table 1). table 1: demographic data of sepsis and non-sepsis patients analysed between january 2021 and july 2021 in cape town, south africa. haematology patients with sepsis had a significantly elevated white cell and neutrophil count compared to the control group (p < 0.001). the absolute lymphocyte count was significantly reduced in the sepsis group (p < 0.001) and, similarly, the median platelet count was lower (p < 0.001). in contrast, although the median absolute monocyte counts varied, there was no statistical significance in monocyte counts between the sepsis and control groups (p = 0.377) (table 1). all three cell ratios varied significantly between the two study groups, most notably the median nlr being 8.62 times higher in the sepsis group, whereas the mlr and plr were 3.06 and 0.71 times higher. the differences in cell ratios were statistically significant, with p < 0.001 (table 1). neutrophil-to-lymphocyte ratio although the nlr varied in those with sepsis, values were statistically significantly elevated (p < 0.001) compared to the control group (figure 1a), which all fell within the reported nlr reference interval of 0.78 to 3.53 with a mean of 1.65.21 despite the high levels of both nlr and procalcitonin, no correlation was detected between the two tests (r = 0.218; p = 0.088) (figure 1b). figure 1: neutrophil-lyphocyte ratio characteristics among 62 patients with sepsis compared to 63 non-sepsis controls presenting between january 2021 and july 2021 in cape town, south africa. (a) the median and interquartile range of the neutrophil-lymphocyte ratio. (b) the correlation between the neutrophil-lymphocyte ratio and procalcitonin. monocyte-to-lymphocyte ratio similarly, although the mlr showed a large variation, it was significantly elevated when compared to the non-septic control group (p < 0.001) (figure 2a). no significant correlation could be detected between the mlr and procalcitonin levels (r = 0.202; p = 0.07) (figure 2b). figure 2: monocyte-lymphocyte ratio characteristics among 62 patients with sepsis compared to 63 controls diagnosed between january 2021 and july 2021 in cape town, south africa. (a) the median and interquartile range of the monocyte-lymphocyte ratio. (b) the correlation between the monocyte-lymphocyte ratio and procalcitonin. platelet-to-lymphocyte ratio the plr in those with diagnosed sepsis was significantly higher than in the control group (p < 0.001) (figure 3a); however, no significant correlation could be detected between the plr and procalcitonin levels (r = 0.069; p = 0.602) (figure 3b). figure 3: platelet-lymphocyte ratio characteristics among 62 patients with sepsis compared to 63 controls presenting between january 2021 and july 2021. (a) the median and interquartile range of the platelet-lymphocyte ratio. (b) the correlation between the platelet lymphocyte ratio and procalcitonin. receiver operating characteristic curve analysis among the three cell ratios assessed, the nlr was established as the best diagnostic screening marker (area under curve: 0.977; youden index: 0.855), while the mlr was considered to be good (area under curve: 0.877; youden index: 0.710). the plr only performed moderately (area under curve: 0.699; youden index: 0.455) (figure 4). figure 4: receiver operating characteristic curves for the neutrophil-lymphocyte ratio, monocyte-lymphocyte ratio and the platelet-lymphocyte ratio of patients analysed between january and july 2021 in cape town, south africa. discussion in this retrospective study of 62 sepsis and 63 control patients, the nlr, mlr, plr, white blood cell count and neutrophil count were significantly elevated in those with sepsis (p < 0.001). conversely, there was no significant difference in the absolute monocyte count between the two groups (p = 0.377). furthermore, despite the significantly increased full blood count parameters in the sepsis group, no correlation was detected between the cell ratios and procalcitonin. the receiver operating characteristic curve analysis established that the nlr was the best screening marker for sepsis amongst the three cell ratios. researchers have demonstrated that an elevated nlr indicates sepsis and predicts morbidity and mortality. for example, a study conducted on 60 000 intensive care patients from the beth israel deaconess medical center in the united states between june 2001 and october 2012 concluded that an nlr of > 20.25 in adults was associated with an elevated mortality risk after 28 days.22 although other studies support this finding,23,24 controversy over cut-off values remains. in a similar study to ours, a retrospective laboratory analysis of 1468 turkish patients recommended an nlr cut-off of > 5 as being highly suggestive of sepsis.35 in this current study, we defined sepsis based on a positive bc; eight of the 62 (13%) confirmed sepsis patients had an nlr < 5 suggesting that a cut-off value of > 5 is significant. however, an nlr of > 5 cannot be used in isolation to indicate sepsis in africa due to the high frequency of benign ethnic neutropenia which may result in a lower nlr in healthy individuals. although the nlr, in this current study, was significantly increased in patients with sepsis, there was no significant correlation with procalcitonin levels. one reason for this finding could be that the nlr and procalcitonin can be elevated in several conditions, with neither test being specific for sepsis.17,36 neutrophil counts and the nlr can be influenced by numerous variables, including age, obesity, hiv, cardiovascular disease, type 2 diabetes and malignancy.24 both procalcitonin and the nlr measure different processes, and therefore neither should be used in isolation to diagnose or predict the outcome in patients with sepsis. although our study revealed no significant difference in the monocyte count between those with and without sepsis, the mlr was significantly elevated in the sepsis group (p < 0.001), which was mainly due to a decreased lymphocyte count. this finding implies that the mlr could be an additional valuable indicator of sepsis. reports examining the use of the mlr are scant and unclear, with one study of 152 patients diagnosed with klebsiella pheumonia in china, between january 2014 and december 2017, demonstrating an association with negative bc28 and another prospective analysis of 392 patients diagnosed over a 4-year period in serbia reporting only a moderate association with sepsis.29 due to these conflicting observations, further studies in different population groups are recommended. in this current study, as expected, platelet counts in those with sepsis were significantly reduced (p < 0.001), while the plr, although showing a wide variation, was significantly different to the control group (p < 0.001). however, receiver operating characteristic curve analysis demonstrated that the plr only had moderate diagnostic value. these results support two previous research reports, one conducted between january 2011 and july 2013 in the netherlands and another undertaken between 2001 and 2008 in china, which demonstrated that low platelet count and elevated plr are associated with sepsis and a worse prognosis in cases of severe thrombocytopenia.32,33 calculating the plr could provide valuable information if analysed with other cell ratios and full blood count parameters. limitations in this study, we hypothesised that simple full blood count parameters, utilised together with calculating haematological cell ratios, could provide an early screening tool for sepsis. however, as this was a laboratory-based study, a significant limitation of this work was the lack of clinical information, and therefore we could not link these parameters with disease severity, the sequential organ failure assessment score or outcome. furthermore, emerging parameters such as the monocyte distribution width were not investigated. conclusion an automated full blood count and differential are widely available. it is a simple test that is easy to perform, inexpensive and is the first investigation requested by clinicians in the emergency ward. although procalcitonin and bc remain the gold standard for sepsis diagnosis, they are often only available in central laboratories. our results suggest that although each full blood count parameter should not be used alone, used together they could provide a simple screening tool for the detection of early sepsis in limited-resource settings. acknowledgements we would like to acknowledge dr niehaus for assistance in the collection of data. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions j.v.r. conceptualised the study, wrote the first draft of the manuscript, collected and analysed the data and approved the final manuscript for publication. s.d. performed statistical analysis, provided intellectual input and approved the final manuscript for publication. c.s. supervised the collection of data, provided intellectual input and approved the final manuscript for publication. g.m.d. supervised the project, performed analysis of the data, provided intellectual input and authorised the final draft for publication. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability all data generated from this study are available on request from the corresponding author, g.m.d. disclaimer the views and opinions expressed in this manuscript are those of the authors and do not reflect any official policy or postion of any affliated agency or authors. references gül f, arslantaş mk, cinel i̇, kumar a. changing definitions of sepsis. 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disease control and prevention, caribbean regional office, barbados 2african field epidemiology network, caribbean office 3princess margaret hospital, bahamas 4ladymeade reference unit, barbados 5african field epidemiology network, uganda 6clinton health access initiative, botswana correspondence to: giselle guevara postal address: us centers for disease control and prevention, caribbean regional office, us embassy, bridgetown, wildey business park, wildey, st. michael bb14006, barbados dates: received: 19 may 2014 accepted: 18 june 2014 published: 03 nov. 2014 how to cite this article: guevara g, gordon f, irving y, et al. the impact of slmta in improving laboratory quality systems in the caribbean region. afr j lab med. 2014;3(2), art. #199, 9 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.199 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the impact of slmta in improving laboratory quality systems in the caribbean region in this original research... open access • abstract • introduction • research method and design    • advocacy strategy with governments    • laboratory audits       • slmta workshops       • mentorship for the laboratories • results    • case studies       • case study 1 – inventory management       • case study 2 – improving documents and records management in the microbiology laboratory • discussion    • early engagement of key stakeholders    • an implementation roadmap    • structured improvement approach    • mentorship    • key challenges and recommendations    • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: past efforts to improve laboratory quality systems and to achieve accreditation for better patient care in the caribbean region have been slow. objective: to describe the impact of the strengthening of laboratory management toward accreditation (slmta) training programme and mentorship amongst five clinical laboratories in the caribbean after 18 months. method: five national reference laboratories from four countries participated in the slmta programme that incorporated classroom teaching and implementation of improvement projects. mentors were assigned to the laboratories to guide trainees on their improvement projects and to assist in the development of quality management systems (qms). audits were conducted at baseline, six months, exit (at 12 months) and post-slmta (at 18 months) using the stepwise laboratory quality improvement process towards accreditation (slipta) checklist to measure changes in implementation of the qms during the period. at the end of each audit, a comprehensive implementation plan was developed in order to address gaps. results: baseline audit scores ranged from 19% to 52%, corresponding to 0 stars on the slipta five-star scale. after 18 months, one laboratory reached four stars, two reached three stars and two reached two stars. there was a corresponding decrease in nonconformities and development of over 100 management and technical standard operating procedures in each of the five laboratories. conclusion: the tremendous improvement in these five caribbean laboratories shows that slmta coupled with mentorship is an effective, user-friendly, flexible and customisable approach to the implementation of laboratory qms. it is recommended that other laboratories in the region consider using the slmta training programme as they engage in quality systems improvement and preparation for accreditation. introduction top ↑ improving laboratory quality systems and attaining accreditation are important benchmarks in national health laboratory practice, as accreditation is a process that gives formal recognition of the technical competence of a laboratory to perform specific tests.1 in many cases, the added value of accreditation far outweighs the necessary investment in human resources, finances and time, since it is an independent method of determining and monitoring laboratory performance, whilst assuring the validity of the results to the users.2,3implementation of laboratory quality management systems (qms) and achievement of accreditation amongst laboratories in the caribbean region has been limited. available data report only three accredited government-owned or public clinical laboratories in the caribbean as of 2011.4 over the years, many caribbean laboratory staff have been provided with information on qms and accreditation in various forms, including training, conferences, meetings and printed material. however, using this knowledge collectively and developing a comprehensive plan in order to address quality gaps and begin the journey toward accreditation have been challenging. during a preliminary laboratory needs assessment survey conducted in 2009, laboratory managers and other stakeholders discussed the problems of an undertrained laboratory workforce, the lack of motivation and, most importantly, the perception that the quality improvement process was cumbersome.4 the need to put strategies in place to eliminate these hindrances as soon as possible was emphasised. the recommendation was that a more user-friendly, stepwise approach to quality systems implementation, in combination with task-based training tools to improve staff knowledge, could lead to more substantial improvement in quality systems. the strengthening laboratory management toward accreditation (slmta) programme was launched in 2009 and has been implemented in 47 countries worldwide.5 it is a management training programme that utilises a series of workshops interspersed with on-site projects designed to improve laboratory quality. evidence from other settings has shown that the slmta training programme yields observable and measurable laboratory improvements.6 furthermore, the training empowers laboratory staff and enhances management’s ability to improve their own laboratories by making use of existing resources.7 a laboratory quality improvement mentorship intervention programme in lesotho that incorporated the slmta training and a stepwise approach to accreditation preparedness has resulted in significant measurable improvements in the quality of enrolled laboratories over a period of 12 months.8 the reauthorisation of the us president’s emergency plan for aids relief (pepfar ii) in 2008 resulted in the establishment of the pepfar caribbean regional program and the development of the pepfar partnership framework with 12 caribbean countries (barbados; trinidad and tobago; belize; suriname; jamaica; the bahamas; st. lucia; st. vincent and the grenadines; grenada; antigua and barbuda; st. kitts and nevis; and dominica). since then, the pepfar laboratory-strengthening working group has worked closely with the ministries of health (mohs) in these countries to improve the quality and reliability of laboratory results and to offer basic testing services for persons living with hiv. the need to engage laboratories in these countries in quality improvement and accreditation was identified very early during this collaboration when it became apparent that laboratory services, systems and infrastructure in the region were weak, with various populations lacking access to timely, low-cost and high-quality laboratory services.4 with the aim of improving laboratory quality in the region, the us centers for disease control and prevention (cdc) caribbean regional office laboratory team, the international laboratory branch of the division of global hiv/aids at cdc atlanta and the african field epidemiology network (the laboratory implementing partner) collaborated to research options for effective laboratory quality improvement. the decision was made to use the slmta training programme, coupled with the world health organization regional office for africa’s (who afro) stepwise laboratory quality improvement process towards accreditation (slipta) checklist, along with mentorship, in order to improve the quality systems of five laboratories in four of the caribbean partnership framework countries. this article discusses improvements in the laboratory quality systems during the 18-month implementation of the slmta training programme and mentorship in these laboratories. research method and design top ↑ advocacy strategy with governments at the initiation of the regional laboratory strengthening activities, following the signing of the pepfar caribbean regional partnership framework in 2010, key sensitisation meetings were held with policymakers and other stakeholders in each of the four countries to highlight the need, importance and advantages of improved laboratory quality systems and accreditation. these meetings included chief medical officers, permanent secretaries, laboratory directors and other regional partners. in addition to discussing an overall strategy for collaboration and strengthening of the entire laboratory health system, a presentation was made highlighting the stepwise approach toward accreditation, the slmta training programme and the use of mentors as innovative approaches to implementing quality systems and eventually achieving accreditation. the proposed strategy for laboratory strengthening began by engaging the national reference laboratories in each of the four selected countries. although each laboratory was unique in its operation, size and workload, it was agreed that the challenges faced were similar and they would, therefore, all benefit from the proposed interventions. to ensure buy-in and to highlight the need for providing additional resources to address the deficiencies previously identified during the laboratory needs assessment survey in 2009 and the subsequent baseline audits in 2011, key senior officials from the human resources, procurement and maintenance departments of the mohs and hospitals were invited to attend the audit debrief meetings in their respective countries. laboratory audits periodic audits spanning three to four days were conducted in each of the five national reference laboratories by experienced auditors using the slipta checklist. the slipta programme uses a stepwise accreditation preparedness scheme that recognises laboratories according to their level of compliance with the the international standard iso 15189 – medical laboratories – particular requirements for quality and competence. the results of the laboratory audits were reported for each of the 12 sections of the checklist covering the 12 quality systems essentials (clsi gp 26-a3 [2004]), including 111 main items for a total of 258 possible points (table 1). the score obtained by each laboratory indicates the level of performance, which determines the star rating from 0 to five stars. table 1: slipta scoring system for laboratories. the audits were conducted in each of the five participating laboratories at baseline, after six months (mid-term audit), after 12 months (exit audit) and after 18 months (follow-up audit) to ensure continuous monitoring of the laboratories and their performance (figure 1). each laboratory audit began with an introductory meeting convening the laboratory director and departmental heads in order to summarise the proposed audit plan which would be used to identify areas for improvement. at the end of the audit, a formal debrief meeting was held with laboratory management, technical staff and key persons from the moh and hospital whose responsibilities affect the smooth functioning of the laboratories. after the baseline audit, a customised quality system implementation plan was developed in order to outline the nonconformities found, recommendations for follow-up actions, responsible persons, timeline for completion and status (table 2). table 2: example of quality systems implementation plan. throughout the programme the laboratories were audited at approximately six-month intervals, which allowed them to monitor their continued progress and update the quality system improvement plans originally developed at the baseline audit. the list of nonconformities found at the previous audit was also comprehensively reviewed and updated to determine the number of completed corrective actions over the period. open nonconformities were assigned for further follow-up by the laboratory and its management. exit audits were conducted using the slipta checklist three months after the last slmta workshop concluded (12 months after baseline). a follow-up audit was then conducted six months later to evaluate the longer-term effectiveness and sustainability of the programme. these audits allowed laboratories to determine their level of progress from the baseline to exit of the slmta training and mentorship programme. slmta workshops the slmta training programme was implemented as a series of three workshops which began in may 2011 and were conducted approximately three months apart (figure 1). a total of 24 participants (three to five per laboratory) from across the five focus laboratories were chosen based on the size of their laboratory and the testing needs of each country. these included staff from the various departments, (i.e., chemistry, blood bank, serology, etc.), as well as the quality manager or designee. participants were required to develop improvement projects and complete them during the hiatus between workshops. the improvement projects were generally chosen based on the areas of nonconformity indicated in the laboratory’s individualised quality systems implementation plan along with the needs of the laboratory at the time. each participant presented a summary of their completed improvement projects at the subsequent workshop, including the baseline data collected, the measure of progress within the study period and the challenges experienced during project execution. final improvement projects were presented orally by each participant and graduation certificates were awarded to them in the presence of officials from the moh and the hospitals in order to highlight the importance of this event to the process of accreditation preparedness. figure 1: laboratory strengthening implementation model for the caribbean region. mentorship for the laboratories each of the engaged laboratories was assigned a mentor to assist in developing and establishing their qms by providing technical assistance and coaching on implementing the improvement projects from the slmta training. three fulltime mentors were used for this activity across the five laboratories. each mentor had at least 10 years of experience in laboratory technology and development of qms. during the first few months of the programme, the mentors spent approximately one week each month embedded in the assigned laboratory. after approximately six months, the length of each mentor’s assignment was increased to two or three weeks, depending on the needs of the laboratories at that time. six-week mentorship action plans were developed to give direction to both the laboratory and the mentor, allowing for measurement of progress over the specific six-week period (table 3). since the mentor was physically on-site for only part of the six-week period, the laboratory had a period of self-management during which time they communicated with the mentor via email, internet conferencing and telephone. all management and technical procedures produced during the assignments were forwarded to the laboratory directors or department directors for final approval. table 3: example of six-week mentorship action plan for a laboratory. results top ↑ at the baseline audits the laboratory scores ranged from 19% to 52%, corresponding to 0 stars (figure 2). scores increased steadily throughout the programme and by 18 months each laboratory had improved, with three of the laboratories more than doubling their baseline scores. one laboratory reached four stars on the five-star scale, two attained three stars and the remaining two laboratories each attained two stars. of this group, one laboratory achieved accreditation through the college of american pathologists (cap) in september 2013; meanwhile three others have applied for accreditation and are preparing for the assessment within the next few months. figure 2: improvement in implementation of the laboratory quality systems and stars attained over 18 months. figure 3 shows the average percentage improvement across the five laboratories for each of the 12 sections of the checklist (i.e., the 12 quality system essentials), measured as the difference between the baseline and follow-up score after 18 months. the greatest improvements were in corrective action (66%), organisation and personnel (55%) and purchasing and inventory (54%). the sections showing the least improvement were process control (18%), occurrence management (25%), internal audits (30%) and equipment (36%). average final absolute scores were > 60% for all areas except occurrence management and internal audits. overall, between 141 and 735 standard operating procedures (sops) were completed and approved for each laboratory over the 18-month period (table 4), leading to an average increase on the checklist of 40% from the baseline score in the area of documents and records (figure 3). table 4: number of standard operating procedures (sops) completed for the 5 laboratories. figure 3: average performance improvement of all laboratories across the 12 sections of the who afro slipta checklist. improvement in each laboratory can also be measured by the change in the number of identified nonconformities (figure 4). nonconformities decreased by more than half during the intervention period. for each laboratory this translated into at least a 50% decrease in outstanding nonconformities over the entire implementation period. figure 4: change in number of nonconformities per laboratory over time. case studies each participant enrolled in the slmta training programme was required to choose, plan and execute at least three improvement projects over the duration of the programme. slmta trainers provided tools, techniques and examples in order to guide participants to design effective projects within their laboratory, whilst mentors provided implementation support. as a result of these projects, tangible improvements were observed in the qms and overall operations of the laboratories. two high-impact projects are presented here as case studies: case study 1 – inventory management laboratory 4 has three store rooms containing hundreds of supplies from various vendors. the baseline audit showed that management of stock was a challenge within this facility, with frequent stock-outs, lack of proper tracking forms in the storage areas and increased borrowing from other laboratories. upon investigation, factors such as unpredictable patient-testing workload, delivery delays and back-order issues consistently affected the supply levels. these issues were exacerbated by the poor record keeping and lack of an organised inventory management system, preventing effective forecasting. a key recommendation to the slmta trainee was to put a system in place to ensure sufficient stock levels of all supplies. hence, an improvement project was designed to enhance inventory management in all areas of the system, with the overall objective to reduce stock-outs to less than 5% within a four-month period. to achieve this objective, all staff were briefed on the project, including their specific roles in the success of the intervention. the following data collection and monitoring tools were developed: expired reagent record log; cardex for storage areas; order form; laboratory inventory card; inventory control colour chart; laboratory loan form; requisition order code form; quotation request form; receiving and inspection investigative checklist; regular receiving and inspection log; refrigerator cardex; section grading card; inventory and usage pattern data collection log; and a laboratory stores task assignment checklist. during the improvement project, 15 quality indicators were monitored (figure 5). the results showed that seven of the 15 areas either maintained or achieved 100% compliance, whilst two other areas achieved 90% and 80% compliance over the baseline results. other areas achieved appreciable improvements (figure 5). overall stock-outs were reduced to 5% as a result of the general improvements in the system. figure 5: improvement in laboratory stock management in laboratory 4. case study 2 – improving documents and records management in the microbiology laboratory laboratory 2 has had problems managing quality system documents and associated manuals in their microbiology section. this has resulted in limited progress toward achieving accreditation and difficulty in training new staff in the department. an improvement project was designed to address document and records management. a team of key organisational individuals was convened to work together on the development of the qms. this critical step helped to gain support for the project throughout the various sections in the department. section leaders had the ultimate responsibility of designating and distributing the assignments within their sections. the documents and records were grouped into four categories: technical sops; management sops; logs and checklists; and equipment (including the equipment list, preventative maintenance logs and sops for each item of equipment). figure 6 depicts the level of improvement in documentation after three months of this intervention. technical sops showed the highest level of improvement, from 0% to 67%, closely followed by equipment documentation, from 0% to 63%; the least improvement was in the management sops. figure 6: improvement in documentation for laboratory 2. discussion top ↑ although diverse in its geography, people, size and economy, the caribbean region shares a common challenge in achieving accreditation of its medical laboratories. previous didactic training programmes introduced laboratory staff to the basic quality management principles and the existence of the iso 15189 standard. despite this knowledge, limited progress was seen. an approach that encompassed slmta training, a stepwise evaluation process and mentorship has resulted in tremendous improvement in the quality systems of five national laboratories in four countries of the region within an 18-month period, one of them having attained accreditation. several factors may have contributed to the successes: early engagement of key stakeholders key to the success of global health interventions is full engagement of decision makers in the process from the beginning. in particular, facilitating meetings of policy makers – permanent secretaries, chief medical officers and top management officials of the hospital – along with technical staff, in order to identify challenges and opportunities to resolve nonconformities was important for this project, since these individuals subsequently provided the laboratories with the support and resources needed to ensure timely improvement of the quality systems. endorsement by top management for laboratory systems strengthening activities has proven to be important for the success of this stepwise approach. an implementation roadmap the process of accreditation can appear to be daunting, as extremely high levels of compliance with the quality requirements are essential for a successful assessment and a passing score. for a laboratory without an effective qms, identifying challenges and developing a quality improvement plan can seem like an insurmountable goal, which can lead to demotivation and subsequent inaction. the use of a stepwise improvement process, along with specialised guidance documents, has been shown to provide laboratory stakeholders with a clearer path toward quality systems improvement and accreditation.1 caribbean laboratory directors and managers emphasised that past laboratory assessments and training did not provide them with a structured roadmap to assist in implementation; as a result, the majority of these laboratories did not initiate the process of qms development and implementation.4 the slipta checklist was used to conduct an initial gap analysis in the participating laboratories, leading to the development of an implementation plan, which provided direction for improving the laboratory qms. this plan outlined the process to be taken and the indicators that would be used to measure tangible progress and outcomes over time. everyone involved, including hospital management, was assigned specific tasks relating to their functions and roles, with key deliverables and solid deadlines. use of the stepwise evaluation method enabled recognition of incremental improvements at each audit throughout the process, providing added motivation to all the staff. the scores achieved at each audit highlighted the status attained and the progress that the laboratories had made in building an effective qms, in eliminating nonconformities and in their readiness for accreditation. structured improvement approach prior approaches to laboratory strengthening in the region focused mainly on mass sensitisation to and training on the iso standards and quality management basics, but not on implementation. in some cases the persons trained had not previously been exposed to the principles of continuous quality improvement, total quality management, or development of a quality system specifically for the laboratory. the slmta programme taught the enrolled laboratories how to change the way they approached quality management and their daily operations. the programme also provided user-friendly tools that allowed staff to work more efficiently, as evidenced by their improved star ratings after 18 months. an important component of the slmta training is the improvement projects developed and implemented by the trainees. this promoted a culture of systematic problem solving and a strategic approach to the application of quality system requirements. these projects and their measureable results served as a tool for the laboratory to advocate with management and policymakers for continued support. with the changing economic priorities and limited resources in these developing countries, it was critical to document the impact of any quality improvement and accreditation preparations, so as to demonstrate for stakeholders that the benefits outweigh the costs.2 in the case of these caribbean laboratories, nonconformities were drastically reduced, with corresponding improvement in each of the quality management systems. for example, a 66% improvement was observed in the laboratories’ ability to perform corrective actions. a similar slmta intervention in lesotho6 reported a 34% improvement in corrective action application over an 11-month period. mentorship according to maruta, rotz and peter, ‘a laboratory mentoring program can be an important way to establish and solidify quality management systems and to help laboratories achieve accreditation goals’.9 the presence of the mentors in this programme served two main purposes. firstly, mentors provided needed technical assistance in order to aid the laboratory in the development and finalisation of the qms documentation. it has been documented that a strong foundation for quality assurance begins with development of a quality manual, sops and test methods, since they serve as a guide for both implementing and enhancing the quality system.10 the mentors played a critical role in bridging the gap between what was learnt in the workshops and what was implemented within the laboratories, drawing the team together to develop a strategy and guiding them to address the existing issues. for example, the majority of laboratory staff initially reported that their quality documents were delayed in the process of development for six or more months. the reduction in nonconformities recorded in these laboratories can be directly linked to the increase in the number of documents developed, completed and implemented as a result of the technical assistance provided by the mentors. secondly, mentors alleviated fears associated with preparation for accreditation and acted as a catalyst for enhanced communication. with the mentors present, communication improved greatly amongst the laboratory staff, laboratory management and medical staff. management was more open to presentations and discussions with the laboratory staff, since these consultations were centered on actual data, nonconformance reports and demonstrated improvement. staff often planned management review meetings whilst the mentor was on site, allowing the mentor to help facilitate communication with upper management and showcase the improvement in the laboratory qms as a result of the interventions. key challenges and recommendations the caribbean region is made up of small island nations with most country populations in the range of hundreds of thousands. ensuring a sufficient number of well-qualified laboratory workers is an ongoing challenge, exacerbated by high levels of attrition as staff that have benefitted from government-supported training leave the public sector for more lucrative jobs in the private sector, either locally or overseas. thus the remaining staff are overworked, reducing the amount of time available for training and quality improvement activities. there is also a shortage of qualified mentors who can provide the needed support to laboratories engaged in quality improvement efforts and accreditation preparation. these personnel challenges limit the laboratories’ opportunities for development of qms and achievement of laboratory accreditation. encouraging governments in the region to prioritise health system–strengthening strategies that lead to staff development and retention would benefit not only laboratories, but the health system overall. one of the main logistical challenges faced in this programme stemmed from the use of mentors based in different countries, who were required to travel by air to provide on-site support. thus, considerable funds needed to be invested and intervention was sometimes delayed because of travel issues. establishment of a cadre of in-country or regional slmta trainers and mentors would build local capacity and help reduce programme costs, especially as the programme expands. the momentum achieved through success of the slmta programme in these five laboratories must now be directed to further improvements in these laboratories, as well as expansion of the programme throughout the region. one of the participating laboratories recently achieved accreditation from cap and three more have subsequently applied for accreditation, as a direct result of the training and technical assistance received in the slmta programme. the remaining laboratory will continue to be monitored by means of slipta audits, whilst preparing actively for accreditation in the near future. introduction and implementation of the slmta programme in the caribbean region has been made possible by funding from the pepfar programme; however, there is now a need to internalise the programme and transition it to local governments and other donors in order to facilitate expansion and ensure sustainability. conclusion quality management interventions in the caribbean over the past 10 years had resulted in few improvements in the overall laboratory quality infrastructure, as evidenced by the low performance scores achieved at baseline audits and the limited number of previously-accredited laboratories in the region. a change of approach was thus needed in order to increase these numbers and put more laboratories on the path to accreditation. implementation of the slmta and mentorship approach in several laboratories in the region has achieved tangible improvements in qms development and overall quality within a very short period. continued improvement in these laboratories and expansion of this programme to other laboratories in the region are recommended. sustained improvement will require government funds to be invested in training resources, including development and establishment of local mentorship programmes. our results strongly support the growing body of evidence indicating that the slmta training programme is an important tool to empower laboratory staff, enhance management competence and achieve observable and measurable results for improved laboratory quality. acknowledgements top ↑ we would like to thank all of the laboratory staff who participated in the slmta programme and who worked diligently on the improvement projects. we thank the mentors for their hard work and persistence with each laboratory. in addition, we would like to acknowledge the technical assistance provided by the african field epidemiology network, the financial support from pepfar and the guidance and technical expertise provided by the cdc. cdc disclaimer: the findings and conclusions in this report are those of the author(s) and do not necessarily represent the views of the us centers for disease control and prevention. this research has been supported by the us president’s emergency plan for aids relief (pepfar) through the us centers for disease control and prevention. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. authors’ contributions g.g. (us centers for disease control and prevention, caribbean regional office) was the project leader and wrote the manuscript. f.g. and y.i. (both african field epidemiology network, caribbean office), as well as i.w. (princess margaret hospital, bahamas) and s.b. (ladymeade reference unit, barbados), provided data. k.p. and r.a. (both us centers for disease control and prevention, caribbean regional office) provided comments. t.m. (clinton health access initiative botswana) and n.n. (african field epidemiology network, uganda) provided technical input; and g.a. (us centers for disease control and prevention, caribbean regional office) provided comments and co-wrote the manuscript. references top ↑ 1.gershy-damet gm, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. http://dx.doi.org/10.1309/ajcptuuc2v1wjqbm2.zeh ce, inzaule sc, magero vo, et al. field experience in implementing iso 15189 in kisumu, kenya. am j clin pathol. 2010;134(3);410–418. http://dx.doi.org/10.1309/ajcpzirkdus5lk2d 3. peter tf, rotz pd, blair dh, et al. impact of laboratory accreditation on patient care and the health system. am j clin pathol. 2010;134(4):550–555. http://dx.doi.org/10.1309/ajcph1skq1hnwghf 4.alemnji ga, branch s, best a, et al. strengthening national laboratory health systems in the caribbean region. glob public health. 2012;7(6):648–660. http://dx.doi.org/10.1080/17441692.2012.670861 5.yao k, maruta t, luman et, nkengasong jn. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. 2014;3(2), art. #194, 8 pages. http://dx.doi.org/10.4102/ajlm.v3i2.194 6.mothabeng d, maruta t, lebina m, et al. strengthening laboratory management towards accreditation: the lesotho experience. afr j lab med. 2012;1(1), art. #9, 7 pages. http://dx.doi.org/10.4102/ajlm.v1i 1.9 7. yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin pathol. 2010;134(3):401–409. http://dx.doi.org/10.1309/ajcpnbbl53fwuiqj 8.maruta t, motebang d, wanyoike j, et al. impact of mentorship on who-afro strengthening laboratory quality improvement process towards accreditation (slipta). afr j lab med. 2012:1(1), art. #6, 8 pages. http://dx.doi.org/10.4102/ajlm.v1i1.6 9.maruta t, rotz p, peter t. setting up a structured laboratory mentoring programme. afr j lab med. 2013;2(1), art. #77, 7 pages. http://dx.doi.org/10.4102/ajlm.v2i1.77 10.abimiku ag. building laboratory infrastructure to support scale-up of hiv/aids treatment, care, and prevention: in-country experience. am j clin path. 2009;131(6):875–886. http://dx.doi.org/10.1309/ajcpelmg6gx6rqsm abstract introduction methods results discussion acknowledgements references about the author(s) phidelis m. marabi department of medical laboratory sciences, school of health sciences, kisii university, kisii, kenya department of biomedical sciences, faculty of public health and community development, maseno university, maseno, kenya paul m. kosiyo department of medical laboratory sciences, faculty of medicine, maseno university, maseno, kenya stanslaus k. musyoki department of medical laboratory sciences, school of health sciences, south eastern kenya university, kitui, kenya collins ouma department of biomedical sciences, faculty of public health and community development, maseno university, maseno, kenya citation marabi pm, kosiyo pm, musyoki sk, ouma c. thyroid and reproductive hormonal factors associated with menorrhagia among women in kenya. afr j lab med. 2025;14(1), a2653. https://doi.org/10.4102/ajlm.v14i1.2653 original research thyroid and reproductive hormonal factors associated with menorrhagia among women in kenya phidelis m. marabi, paul m. kosiyo, stanslaus k. musyoki, collins ouma received: 17 oct. 2024; accepted: 07 feb. 2025; published: 10 apr. 2025 copyright: © 2025. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: menorrhagia, characterised by menstrual blood loss exceeding 80 ml per cycle, is a common issue in western kenya. however, there are insufficient data on how hormonal disorders contribute to its occurrence. objective: this study aimed to examine the differences and associations between thyroid and reproductive hormone levels in women with menorrhagia versus those without, in bungoma county, kenya. methods: a comparative cross-sectional study was conducted among 428 women (214 with menorrhagia and 214 controls) aged 18–45 years, between 01 december 2022 and 31 september 2023 at bungoma county referral hospital. the analysis included thyroid stimulating hormone, total and free triiodothyronine, thyroxine, follicle stimulating hormone (fsh), luteinising hormone, prolactin, oestrogen, progesterone, and testosterone. results: women experiencing menorrhagia had statistically significant increases in levels of fsh (p < 0.0001), oestrogen (p < 0.001), and total testosterone (p < 0.001), while prolactin levels had a statistically significant decrease (p < 0.001) compared to those without menorrhagia. there were no statistically significant differences in total triiodothyronine (p = 0.384), free triiodothyronine (p = 0.610), total thyroxine (p = 0.127), free thyroxine (p = 0.360), or thyroid stimulating (p = 0.118). no associations were found between menorrhagia and either thyroid or reproductive hormones. conclusion: elevated levels of fsh, oestrogen, and testosterone, along with reduced prolactin, may serve as potential biomarkers for diagnosing menorrhagia in premenopausal or reproductively aged women. a screening tool that integrates these hormonal markers could improve the accuracy of diagnosis and optimise treatment strategies in primary healthcare settings. what this study adds: the study suggests that levels of fsh, oestrogen, total testosterone, and prolactin differ significantly between women with and without menorrhagia, indicating their potential use in predicting the condition. keywords: menorrhagia; thyroid; reproductive; hormone; women; kenya. introduction menorrhagia, characterised by excessive or extended menstrual bleeding, may result from a variety of underlying conditions.1 it is estimated to affect approximately 11% – 13% of women during their reproductive years, with prevalence increasing with age.2 in several surveys, heavy menstrual bleeding remains one of the primary reasons women seek primary care.3 it is the fourth-most prevalent complaint that necessitates referral to a gynaecologist for evaluation and treatment.4 menorrhagia has a profound impact on women’s physical, emotional, and social well-being, significantly reducing workplace productivity and overall quality of life.5 additionally, it contributes to increased healthcare costs because of the need for frequent and sometimes expensive medical interventions.2 hormonal imbalances are responsible for 80% of cases of abnormal uterine bleeding.6 deficiency in thyroid hormones and irregularities in thyroid stimulating hormone (tsh) can impact ovarian function via multiple mechanisms, potentially altering follicle stimulating hormone (fsh) and luteinising hormone (lh), and affecting the metabolism of peripheral steroid hormones, prolactin, and sex hormone-binding globulin.7 despite the known interaction between thyroid function and menstrual disorders, data specifically linking thyroid hormones with menorrhagia remain limited. oestrogen and progesterone, the hormones that regulate the growth and shedding of the uterine lining during menstruation, must remain balanced to prevent excessive bleeding. conditions such as polycystic ovarian syndrome, obesity, insulin resistance, or thyroid dysfunction can disrupt this balance, leading to heavy menstrual bleeding.8 globally, the prevalence of thyroid disorders varies,9 with women being more frequently affected than men.10 thyroid dysfunction, such as hyperthyroidism and hypothyroidism, can adversely affect women’s reproductive health.11 hypothyroidism affects between 0.2% and 5.3% of the european population and between 0.3% and 3.7% of the united states population, depending on demographic factors.12 the prevalence of hyperthyroidism is reported to be 0.7% in europe, 0.5% in the united states, and 0.3% in australia.9 in south africa, the incidence of hyperthyroidism among men is 0.00007 per 100 000, compared to a significantly higher rate of 0.009 among women.13 however, there are limited data on the prevalence of thyroid dysfunction in kenya. despite the global burden and recognised association between menstrual disorders and thyroid function, there is a lack of specific data on the link between thyroid hormones and menorrhagia in kenya. while global statistics suggest that hormonal imbalances affect 80% of women,14 kenyan local data are scarce. in kenya, while menorrhagia is estimated to affect around 48.9% of women,15 there remains a paucity of data on hormonal disorders among affected women. studies in western kenya report a significant burden of menorrhagia, affecting 35.3% of women.16 additionally, the bungoma county referral hospital has reported a substantial number of gynaecological patients seeking care for menorrhagia.17 however, data on hormonal abnormalities in menorrhagic women remain scarce. thus, this study explored the differences and associations in thyroid and reproductive hormone levels among women with and without menorrhagia at bungoma county referral hospital, kenya. methods ethical considerations the study was approved by the maseno university scientific and ethical review committee under reference number muserc/01166/22. research authorisation was also granted by the national commission for science, technology, and innovation with approval number nacosti/p/22/22573. additionally, data collection permission was obtained from bungoma county referral hospital (reference number bdh/tp/b/vol2). written informed consent was obtained from all participants following a concise explanation of the study’s purpose. participation in the study was entirely voluntary. data security protocols included storing data in a secured physical location and using password protection for the computer. participants’ personal information was kept confidential, with protocol numbers assigned for reference, and this information was used solely for research purposes. study area the study was conducted at bungoma county referral hospital, located in bungoma county, western kenya, between 01 december 2022 and 31 september 2023. bungoma county referral hospital, a level 5 facility, serves as the primary referral health facility for the county and its surrounding areas, catering to a population of approximately 1 670 570 over a total area of 2206.9 km2.18 the gynaecological clinic at bungoma county referral hospital serves approximately 6000 patients each year.17 study design and population this research utilised a comparative cross-sectional design. the sample size was calculated using a formula for quantitative comparative studies, yielding a total of 428 participants (214 from each group). a systematic random sampling technique was employed, where every second individual who met the inclusion criteria was selected until the target sample size was reached. inclusion criteria the study aimed to recruit adult women aged 18 to 45 years who visited the clinic seeking treatment for menorrhagia at bungoma county referral hospital. a comparison group was created by randomly selecting healthy women. a qualified gynaecologist assessed each patient’s menorrhagia, making a clinical diagnosis based on the definition of menorrhagia, which includes regular menstrual cycles with excessive blood loss (more than 80 ml per cycle or requiring changes of hygiene products more frequently than every 2 h) and/or a duration longer than 7 days.19 exclusion criteria participants were excluded if they had taken sylate, tranexamic acid, or any other medications for bleeding disorders in the last 3 months. women receiving hormone replacement therapy, oral contraceptives, or other hormonal treatments that could affect menstrual bleeding or hormonal levels, were also disqualified. those with diagnosed thyroid disorders (such as hyperthyroidism or hypothyroidism) or chronic illnesses (including diabetes, hypertension, or significant cardiovascular diseases) were not eligible. additionally, women with pelvic conditions (such as uterine fibroids, polyps, or cancers) that might influence bleeding patterns, as well as those who had recent gynaecological surgery affecting menstrual bleeding, were excluded. participants with blood disorders, such as haemophilia or thrombocytopenia, were also disqualified from the study. sample and data collection participants were divided into menorrhagic and non-menorrhagic groups based on a screening method as previously employed,20 which included self-reported menstrual blood loss and clinical evaluation. this ensured a standardised and reproducible approach to participant selection. a structured questionnaire was used to collect sociodemographic and clinical information, such as age, residence, parity, occupation, and literacy level. blood samples were collected in a plain vacuum tube (becton, dickinson and company, franklin lakes, new jersey, united states). the samples were promptly inverted gently and assigned a unique identification number for each participant. they were then quickly transported to the laboratory. in the laboratory, the samples were centrifuged at 3000 revolutions per minute for 5 min to isolate the serum within 4 h of collection. if immediate testing was not feasible, the separated samples were stored at 4 °c for a maximum of 24 h. laboratory analyses the thyroid and reproductive hormone profiles were evaluated using the maglumi 800 clia instrument (shenzhen new industries biomedical engineering co. [snibe] diagnostics, shenzhen, china). the analysis included tsh, total and free triiodothyronine, thyroxine, fsh, lh, prolactin, oestrogen, progesterone, and testosterone. the analyser uses the luminescence principle for clinical chemistry. the reference ranges for thyroid and reproductive hormones in adult women were obtained from the laboratory at bungoma county referral hospital: total triiodothyronine levels: 60 ng/dl – 180 ng/dl; free triiodothyronine levels: 130 pg/dl – 450 pg/dl; total thyroxine levels: 5.0 µg/dl – 12.0 µg/dl; free thyroxine levels: 0.7 ng/dl – 1.9 ng/dl; tsh levels: 0.5 milli-international units (miu)/l – 2.87 miu/l; fsh levels: 1.5 iu/l – 12.4 iu/l; lh levels: 5 iu/l – 25 iu/l; prolactin levels: 2 ng/ml – 29 ng/ml; oestrogen levels: 30 pg/ml – 400 pg/ml; progesterone levels: 5 ng/ml – 20 ng/ml; and testosterone levels: 0.5 nmol/l – 2.4 nmol/l. reagents, control materials and standards were stored at 2 °c – 4 °c and allowed to reach room temperature prior to testing, ensuring thorough mixing according to the manufacturer’s guidelines. control materials with both normal and pathological levels were analysed before the sample testing commenced. data analysis the analysis was conducted using the statistical package for social science (v.26) software (ibm corporation, armonk, new york, us). descriptive statistics, such as frequency, median, and dispersion, were presented in tables. the shapiro-wilk test was employed to assess normality, while histograms and box plots were used for visual confirmation of the data distribution. the chi-square test was applied for categorical descriptive data, including residence, parity, and occupation. the mann-whitney u test was used to compare continuous descriptive data, such as age. since the data did not adhere to a normal distribution, the mann-whitney u test was also utilised to compare thyroid and reproductive hormone levels between menorrhagic and non-menorrhagic women. to assess the relationship between menorrhagia and hormonal parameters, binary logistic regression was conducted, as the dependent variable was categorical (menorrhagic vs non-menorrhagic). participants with potential confounding factors, such as body mass index, dietary factors, and other variables that could influence hormonal levels or menstrual patterns, were excluded from the analysis. a p-value of ≤ 0.05 was considered statistically significant. results demographic characteristics of the study population a total of 428 adult women aged 18 years to 45 years (median [interquartile range]: 33.5 [11.0] years) were included in the study (table 1). among them, there were 214 participants with menorrhagia and an equal number of healthy non-menorrhagic women. no significant differences were observed between the groups regarding urban or rural residence (p = 0.139), parity (p = 0.390), and occupation (p = 0.846) (table 1). table 1: demographic characteristics of the study participants at bungoma county referral hospital, kenya, december 2022 – september 2023. comparison of thyroid hormones between menorrhagic and non-menorrhagic women there were no significant differences between menorrhagic and non-menorrhagic women regarding total triiodothyronine (p = 0.384), free triiodothyronine (p = 0.610), total thyroxine (p = 0.127), free thyroxine (p = 0.360), and tsh levels (p = 0.118) (table 2). table 2: comparison of thyroid hormones between menorrhagic and non-menorrhagic women, bungoma county referral hospital, kenya, december 2022 – september 2023. comparison of reproductive hormones between menorrhagic and non-menorrhagic women menorrhagic women were more likely to have increased fsh (59/214; 27.6%), oestrogen (214/214; 100%) and testosterone (214/214; 100%) levels than non-menorrhagic women (fsh: 10/214, 4.7%; oestrogen: 186/214, 86.5%; testosterone: 210/214, 98.1%) (table 3). table 3: comparison of reproductive hormones between menorrhagic and non-menorrhagic women, bungoma county referral hospital, kenya, december 2022 – september 2023. menorrhagic women (16/214; 7.5%) were more likely than non-menorrhagic women (7/214; 3.3%); to have reduced prolactin levels (table 3). association between thyroid hormones and menorrhagia menorrhagia was not associated with total triiodothyronine (odds ratio [or] = 1.001, 95% confidence interval [ci] = 0.996 – 1.006, p = 0.541), free triiodothyronine (or = 1.001, 95% ci =0.999 – 1.002, p = 0.371), total thyroxine (or = 1.080, 95% ci = 0.993 – 1.175, p = 0.092), free thyroxine (or = 1.467, 95% ci = 0.845–2.561, p = 0.233), and tsh (or = 0.891, 95% ci = 0.705 – 1.25, p = 0.390) (table 4). table 4: association between menorrhagia and thyroid hormones, bungoma county referral hospital, kenya, december 2022 – september 2023. association between reproductive hormones and menorrhagia menorrhagia was not associated with fsh (or = 0.849, 95% ci = 0.712 – 1.013, p = 0.069), lh (or = 1.021, 95% ci = 0.808 – 1.291, p = 0.860), prolactin (or = 1.202, 95% ci = 0.984 – 1.469, p = 0.071), oestrogen (or = 1.015, 95% ci = 0.988– 1.043, p = 0.279), or progesterone (or = 0.951, 95% ci = 0.749 – 1.207, p = 0.678). however, the model did not determine the association between total testosterone and menorrhagia (table 5). table 5: association between menorrhagia and reproductive hormones, bungoma county referral hospital, kenya, december 2022 – september 2023. discussion in this study, we found that menorrhagic women exhibited statistically significant, increased levels of fsh, oestrogen, and testosterone, while prolactin levels were statistically significantly lower compared to non-menorrhagic women. however, no statistically significant differences were observed in thyroid hormone levels, and none of the evaluated hormonal parameters had statistically significant associations with menorrhagia. in the current study, menorrhagic women had an identical median age to non-menorrhagic women. the median age of all participants in the study was 33.5 years, which was closely similar to the study population of a 2018 study done in pakistan, whose mean age was 35.56 years.21 however, the age range in the current study differed from one conducted in tanzania, published in 2022,22 which focused on the age range of 15 to 59 years. in this study, the living environments (rural or urban) of women with menorrhagia did not significantly differ from those of women without menorrhagia, a finding that aligns closely with research conducted in tanzania and published in 2022.22 the present study revealed that a greater percentage of women with menorrhagia reported having had at least one child, although no significant difference in parity was observed. these results contrast with those from a study conducted in the united states, published in 2016,23 where only one person was parous. the difference may be because of the sample size, with the latter study involving only 38 participants. the present study revealed that a significant proportion of women with menorrhagia were engaged in informal employment, showing no notable difference compared to women without menorrhagia. these results align with those of a study conducted in tanzania and published in 2022,22 which reported that 52.5% of women with menorrhagia are employed as farmers. this study indicated that levels of total triiodothyronine, free triiodothyronine, total thyroxine, free thyroxine, and tsh were similar between menorrhagic and non-menorrhagic women. additionally, it found no significant association between total triiodothyronine, free triiodothyronine, total thyroxine, and free thyroxine hormones and menorrhagia. the current findings contrast with those of an egyptian study, published in 2006,24 which identified a notable difference in the levels of total triiodothyroninee, free triiodothyronine, total thyroxine, and tsh between women with menorrhagia and the control group. the differences in the results may be a result of variations in the study populations concerning geography and ethnicity. the findings also contradict those of an iraqi study, published in 2019,25 which found that as thyroxine reduces and tsh increases, the likelihood of menorrhagia is increased. however, the current study found that 1.9% of menorrhagic women had total triiodothyronine levels that were greater than the expected reference range. about 5.6% of menorrhagic women had higher levels of free triiodothyronine, whereas 4.2% had lower levels below the anticipated reference range. some 3.7% of menorrhagic women had total thyroxine levels that exceeded the expected reference range. about 6.1% of menorrhagic women had higher levels of free thyroxine than the anticipated reference range, whereas 2.3% had lower tsh. the findings were consistent with those of an indian study, published in 2015,26 which showed that 9% of patients had hypothyroidism and 3% had hyperthyroidism. the present findings can be explained by the fact that the onset of hypothyroidism or hyperthyroidism is so gradual that characteristic clinical manifestations may not present for months or years. furthermore, in hypothyroid women, menorrhagia may be the only presenting complaint.27 the abnormal thyroid hormone levels (both total and free triiodothyronine and thyroxine) observed in a small percentage of menorrhagic women in this study suggest that thyroid dysfunction may be an underlying factor contributing to this condition. clinicians should consider routine thyroid function tests for women presenting with menorrhagia to identify potential endocrine contributors. understanding the thyroid status of menorrhagic patients may guide treatment decisions.28 for instance, if hypothyroidism or hyperthyroidism is diagnosed, appropriate management of thyroid disease could potentially alleviate menorrhagic symptoms and improve overall menstrual health.29 the findings align with previous studies, indicating a possible commonality in the prevalence of thyroid dysfunction among women with menorrhagia.28 this consistency suggests that similar screening practices may be beneficial across different populations and healthcare settings. the present findings highlight the need for further research to explore the causal relationship between thyroid disorders and menorrhagia. understanding whether thyroid dysfunction directly contributes to menstrual irregularities could enhance clinical approaches and treatment strategies. the present study was, however, limited in the sense that being a cross-sectional study, the present study did not assess how thyroid hormonal parameters change over time in relation to menorrhagia. future longitudinal studies on how thyroid hormones change over time in relation to menorrhagia are recommended so as to help understand how these hormones evolve over time and their impact on treatment outcomes. in light of the present findings, public health initiatives that focus on increasing awareness of thyroid health in relation to menstrual disorders, potentially leading to earlier diagnosis and intervention, are recommended. the present findings further suggest that women experiencing menorrhagia may benefit from a comprehensive evaluation that includes hormonal profiling to tailor treatment options based on their specific endocrine status. in the current study, fsh levels were considerably greater in menorrhagic patients compared to non-menorrhagic cases, with around 27.6% of menorrhagic cases having raised fsh levels and 0.9% having lower fsh levels than the normal range. it was additionally found that fsh levels were not significantly associated with menorrhagia. this lack of association may be attributed to the multifactorial nature of menorrhagia, which is influenced by a combination of hormonal, haematological, and structural factors. while fsh plays a crucial role in ovarian function, excessive menstrual bleeding is more commonly driven by imbalances in oestrogen and progesterone, abnormalities in endometrial haemostasis, or underlying uterine conditions such as fibroids and adenomyosis.30 the interplay of these factors may overshadow the role of fsh in determining bleeding severity. the current results are consistent with those of a study conducted in egypt, published in 2006,24 that found a substantial difference in fsh levels between menorrhagic women and the control group. the current findings are also in agreement with those reported in a study by eldred and thomas, published in 1994,31 that demonstrated no significant differences in the plasma concentrations of fsh between those with objectively heavy loss and those with normal loss. these present findings may be explained by the fact that shorter menstrual cycles could be an early sign of a woman entering the menopausal transition, while longer cycles tend to appear later, often associated with elevated fsh levels, which suggest a decrease in active ovarian follicles.32 clinically, elevated fsh levels in menorrhagic women in the present study could serve as a potential marker for identifying women at risk for heavy menstrual bleeding. however, since fsh is not significantly associated with the severity of menorrhagia, it may not be reliable for diagnosing the condition itself. from a clinical perspective, the elevated fsh levels in menorrhagic women might suggest a potential endocrine disruption, prompting clinicians to explore hormonal treatments or further investigate ovarian function in these patients.33 the corroboration of the current findings with studies from different populations (like the egyptian study)24 reinforces the idea that menorrhagia might be associated with hormonal imbalances across diverse groups, which could have implications for global health approaches to managing menstrual disorders. the lack of significant association with the condition in the current study emphasises the need for a broader understanding of the aetiology of menorrhagia. the present data revealed significantly lower prolactin levels in the menorrhagic group compared to the non-menorrhagic group, with about 7.5% of menorrhagic patients exhibiting reduced prolactin levels. it further demonstrated no significant association between prolactin and the likelihood of experiencing menorrhagia. this lack of association may be because of variations in individual physiological responses to hormonal fluctuations. some women with altered prolactin levels may exhibit compensatory endocrine mechanisms that help maintain normal menstrual function, thereby masking a direct relationship with menorrhagia. additionally, factors such as stress, medication use (e.g. dopamine agonists or antagonists), and body mass index can influence prolactin levels, contributing to variability in findings.34 however, these factors were not evaluated in the present study. these results are consistent with those of a study conducted in egypt, published in 2006,24 which showed a significant difference in prolactin levels between menorrhagic and non-menorrhagic women. the present findings, however, contradict an indian study, published in 2017,35 that demonstrated higher prolactin levels in cases. the reduced prolactin levels observed in this study may be due to the lower tsh levels in the menorrhagic group, as previous research has established a connection between tsh and prolactin levels.35 the lack of significant association between prolactin levels and the likelihood of experiencing menorrhagia implies that clinicians should not rely on prolactin measurements alone when assessing patients with heavy menstrual bleeding. this could shift the diagnostic focus towards other hormonal or systemic factors, such as thyroid function (given the noted link between tsh and prolactin levels). the association of lower tsh and prolactin levels in the menorrhagic group could suggest a broader endocrine imbalance, and the interrelationship between tsh and prolactin could be an avenue for further exploration. clinicians might consider evaluating thyroid function more closely in patients presenting with menorrhagia. the discrepancies between the current findings and other studies (e.g. the egyptian study24 showing higher prolactin levels, and the indian study35) highlight the need for further research to understand the diverse physiological mechanisms behind menorrhagia in different populations. this variability may be influenced by genetic, environmental, or lifestyle factors. the present findings underscore the complexity of hormonal interactions in menorrhagia and suggests a need for a more nuanced approach to diagnosis and treatment that considers individual patient profiles rather than relying solely on prolactin levels. further research is warranted to clarify these relationships and guide clinical practice. in the present study, oestrogen levels demonstrated significantly higher levels in menorrhagic cases compared to non-menorrhagic cases. the findings contradicted the findings of eldred and thomas’s study, published in 1994,31 which demonstrated no significant difference between those with heavy blood loss and those with normal bleeding. the lack of association observed in our study may be due to individual variations in endometrial receptor sensitivity to oestrogen. some women may exhibit heightened endometrial responsiveness to circulating oestrogen, leading to excessive proliferation and bleeding, while others may experience resistance, reducing oestrogen’s impact on menstrual flow.36 additionally, other factors such as coagulation disorders, vascular abnormalities, and inflammatory mediators could contribute to menorrhagia independently of oestrogen levels, potentially masking a direct hormonal effect.37 the findings differ from those of a study conducted in egypt, published in 2006,24 that showed reduced oestrogen in menorrhagic cases compared to the control group. this discrepancy highlights the need for further research to clarify the role of oestrogen in menorrhagia, as well as potential variations in population characteristics, methodologies, or underlying conditions that could influence these outcomes. this finding may be explained by the fact that unopposed oestrogen makes the endometrium fragile, highly vascular, and lacking adequate stromal support, leading to prolonged and excessive uterine bleeding.19 this insight can inform both preventive and therapeutic strategies, suggesting that managing oestrogen levels might mitigate bleeding in affected individuals. the higher oestrogen levels in menorrhagic cases in the present study suggest that hormonal imbalance, specifically elevated oestrogen, may play a crucial role in the pathophysiology of heavy menstrual bleeding. this could help identify hormonal factors that contribute to menorrhagia, enhancing diagnostic accuracy and treatment options. understanding the hormonal dynamics involved in menorrhagia could guide clinicians in choosing appropriate treatments, such as hormonal therapies that balance oestrogen and progesterone, potentially reducing bleeding episodes and improving patient outcomes. these findings not only contribute to the understanding of the hormonal underpinnings of menorrhagia but also underscore the importance of further investigation into the mechanisms and potential treatments for this condition. in this study, testosterone levels were notably elevated in menorrhagic women compared to those without menorrhagia. however, no significant association was found between testosterone and menorrhagia. this lack of association may be attributed to the complex, multifactorial nature of menorrhagia, where various hormonal and non-hormonal factors interact to influence menstrual bleeding. while elevated testosterone levels could indicate a hormonal imbalance contributing to menstrual disorders, other underlying factors such as ovarian dysfunction, endometrial pathology, or co-existing conditions may obscure the relationship between testosterone and menorrhagia.6 these findings are consistent with an egyptian study, published in 2006,24 which also reported higher testosterone levels in the menorrhagic group compared to the non-menorrhagic group. the findings also concur with redmond’s38 study, published in 2004, that showed elevated testosterone levels in the menorrhagic group relative to the non-menorrhagic group. in the current study, elevated testosterone levels in the menorrhagic group could be linked to slightly elevated lh levels in the same group, as prior research has shown that lh influences androgen synthesis by theca cells.39 in the 1970s, it was found that theca cells in developing follicles synthesised androgens (including androstenedione, testosterone, and dihydrotestosterone) in response to lh. additionally, it was noted that androgens produced by theca cells were transformed into oestradiol by the aromatase (cyp19a1) enzyme within granulosa cells.39 the present findings highlight the need for additional studies to explore the mechanisms by which testosterone and lh interact in the context of menorrhagia. future research could examine the role of theca cell function and aromatase activity, potentially identifying new biomarkers for diagnosis or targets for therapy. the higher testosterone levels observed in menorrhagic women in this study may suggest a hormonal imbalance that could be linked to menstrual disorders. understanding this relationship could help clinicians identify potential hormonal causes of menorrhagia in affected patients. although the study found no significant association between testosterone and menorrhagia, the elevated testosterone levels observed in menorrhagic patients might still serve as a potential biomarker for further investigation. clinicians might consider evaluating testosterone levels in patients presenting with heavy menstrual bleeding, particularly if other symptoms of hormonal imbalance are present. the elevated testosterone levels in menorrhagic individuals, alongside slightly increased lh levels, may suggest a dysregulation in the hypothalamic-pituitary-gonadal axis. understanding this relationship is crucial for clinicians as it points to potential underlying endocrine disorders that could contribute to menorrhagia. recognising that elevated testosterone levels may be part of the pathophysiology of menorrhagia could open avenues for targeted therapies. for example, interventions that modulate androgen levels or lh action may provide new options for managing symptoms in affected individuals. recommendations this study recommends developing a risk assessment tool for menorrhagia in primary healthcare settings, integrating hormonal markers such as fsh (> 10 iu/l), oestradiol outside normal cycle ranges, total testosterone (> 70 ng/dl), and prolactin (> 25 ng/ml). these thresholds, alongside clinical symptoms, would help identify at-risk women for early intervention. limitations being a cross-sectional study, the present study did not assess how hormonal parameters change over time in relation to menorrhagia. conclusion follicle stimulating hormone, prolactin, oestradiol, and testosterone are potential markers for menorrhagia. although no direct associations were found, the significant hormonal differences suggest that monitoring these markers could help identify hormonal imbalances and guide targeted treatment, improving early detection and management of menorrhagia. acknowledgements leonard lusenaka contributed to the recruitment of the participants, thomas wekesa contributed by performing the sample collection exercise, and antony sangolo and emmanuel khisa assisted in the analysis of the samples and the data collection. competing interests the research received funding from south eastern kenya university that may be affected by the research reported in the enclosed publication. the authors have disclosed those interests fully and have put in place an approved plan for managing any potential conflicts arising from that involvement. the terms of these funding arrangements have been reviewed and approved by the affiliated university in accordance with its policy on objectivity in research. authors’ contributions p.m.m., s.k.m., p.m.k., and c.o. planned the study and participated in all areas. s.k.m., p.m.k., and c.o. oversaw the study. p.m.m. and p.m.k. conducted the data analysis and developed the article. s.k.m., p.m.k., and c.o. reviewed the article for philosophical insights. p.m.m., s.k.m., p.m.k., and c.o. reviewed the final article and approved it for publication. sources of support this research received partial funding support from south eastern kenya university. data availability the data sets collected and analysed during the current study are not publicly available due to ethical reviewing guidelines, but they are available from the corresponding author, p.m.m., upon reasonable request. disclaimer the views and opinions expressed in this article are those of the authors and are the product 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follicular theca cell recruitment, differentiation, and impact on fertility: 2017 update. endocr rev. 2017;39(1):1–20. https://doi.org/10.1210/er.2017-00164 abstract introduction methods results discussion acknowledgements references about the author(s) jonathan gwasupika department of clinical sciences, tropical diseases research centre, ndola, zambia victor daka department of public health, school of medicine, copperbelt university, ndola, zambia justin chileshe department of biomedical sciences, tropical diseases research centre, ndola, zambia moses mukosha department of pharmacy, school of health sciences, university of zambia, lusaka, zambia steward mudenda department of pharmacy, school of health sciences, university of zambia, lusaka, zambia bright mukanga department of public health, school of medicine, copperbelt university, ndola, zambia ruth l. mfune department of public health, school of medicine, copperbelt university, ndola, zambia gershom chongwe tropical diseases research centre, ndola, zambia citation gwasupika j, daka v, chileshe j, et al. covid-19 positive cases among asymptomatic individuals during the second wave in ndola, zambia. afr j lab med. 2023;12(1), a2119. https://doi.org/10.4102/ajlm.v12i1.2119 original research covid-19 positive cases among asymptomatic individuals during the second wave in ndola, zambia jonathan gwasupika, victor daka, justin chileshe, moses mukosha, steward mudenda, bright mukanga, ruth l. mfune, gershom chongwe received: 11 nov. 2022; accepted: 18 apr. 2023; published: 31 may 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: coronavirus disease 2019 (covid-19) is a worldwide public health concern for healthcare workers. about 80% of cases appear to be asymptomatic, and about 3% may experience hospitalisation and later die. less than 20% of studies have looked at the positivity rate of asymptomatic individuals. objective: this study investigated the covid-19 positivity rates among asymptomatic individuals during the second covid-19 wave at one of zambia’s largest testing centre. methods: this was a retrospective cross-sectional study conducted on routine surveillance and laboratory data at the tropical diseases research centre covid-19 laboratory in ndola, zambia, from 01 december 2020 to 31 march 2021. the study population was made up of persons that had tested for severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection as a requirement for travel. microsoft excel was used to come up with an epidemiological curve of daily covid-19 positive cases; proportions for gender were described using frequencies and percentages. results: a total of 11 144 asymptomatic individuals tested for sars-cov-2 were sampled for the study and 1781 (16.0%) returned positive results. the median age among those tested was 36 years (interquartile range: 29–46). testing for covid-19 peaked in the month of january 2021 (37.4%) and declined in march 2021 (21.0%). the epidemiological curve showed a combination of continuous and propagated point-source transmission. conclusion: the positivity rate of 16.0% among asymptomatic individuals was high and could imply continued community transmission, especially during january 2021 and february 2021. we recommend heightened testing for sars-cov-2 among asymptomatic individuals. what this study adds: this study adds critical knowledge to the transmission of covid-19 among asymptomatic travellers who are usually a key population in driving community infection. this knowledge is critical in instituting evidence-based interventions in the screening and management of travellers, and its control. keywords: asymptomatic individuals; covid-19 disease; positivity rate; sars-cov-2; zambia. introduction coronavirus disease 2019 (covid-19), caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2), is a worldwide public health concern for healthcare workers, including physicians, public health specialists and researchers.1 the world health organization declared the outbreak of covid-19, which was first reported in wuhan, china, as a public health emergency of international concern on 30 january 2020,2 as it was posing a high risk to countries with vulnerable health systems.3 almost all 55 countries in africa have been affected by the coronavirus pandemic, with sub-saharan africa being the most affected.4 zambia recorded its first case of covid-19 on 18 march 20205 and has since recorded a total of 333 555 covid-19 cases and 4017 deaths as of 04 october 2022.6 severe acute respiratory syndrome coronavirus causes a number of human respiratory disease conditions, ranging from mild cold to severe respiratory distress syndrome, and it largely spreads between persons by respiratory droplets and contact routes.7 on the other hand, sars-cov-2 has been seen to spread faster than sars-cov, which was first reported in 2003 and caused previous outbreaks. accumulating evidence showed that sars-cov-2, unlike sars-cov, is transmitted by persons without symptoms.8 about 80% of cases appear to be asymptomatic,4,9,10 and about 3.3% may experience hospitalisation and later die. based on global biological, epidemiological and modelling evidence, asymptomatic covid-19 may play a substantial role in the pandemic trajectory.11 despite public health preventive measures such as hand hygiene, social distancing, quarantine and travel restrictions which were instituted, transmission of covid-19 seemed to be ongoing.12 vaccination against covid-19 has been shown to be effective against contracting severe forms of the disease.13 however, vaccination does not protect an individual from transmitting or becoming infected with sars-cov-2.14 zambia has experienced four waves of covid-19.1 during the first wave to about the fourth wave of covid-19, it was a requirement to have a negative polymerase chain reaction covid-19 certificate by everyone intending to travel.15,16 this study aimed to assess the positivity rate among asymptomatic travellers during the second wave of the covid-19 pandemic, and its determinants. methods ethical considerations ethical approval to carry out the study was obtained from the tdrc research ethics committee (irb registration number: 00002911). permission to carry out the study and access to patient information was obtained from the tropical diseases research centre management. informed consent was not obtained from any individual as there was no active participation in the study. confidentiality of patient information was adhered to and data were de-identified prior to analysis. study design and site this was a retrospective cross-sectional study conducted on surveillance and laboratory data collected at the tropical diseases research centre (tdrc) covid-19 laboratory in ndola, zambia, from 01 december 2020 to 31 march 2021. the tdrc is a national health research institution specialising in both infectious and non-infectious diseases. the tdrc covid-19 laboratory is accredited for certification of travellers by the african society for laboratory medicine and conducts approximately 400 covid-19 tests per day. study population and eligibility criteria the study population was made up of persons who were tested for sars-cov-2 infection during the second wave of covid-19. complete enumeration of the data set comprising individuals tested for covid-19 was obtained for analysis. eligibility for testing was based on getting tested for covid-19 as a mandatory requirement for international travel, regardless of age. tests of individuals that were collected from outside the tdrc and those that were done outside the stipulated period of the second wave were not included in the analysis. additionally, tests that were done after vaccination had begun were excluded. data collection data were collected from an already-prepared microsoft excel spreadsheet (microsoft corporation, redmond, washington, united states) and a case investigation form comprising the following information: date the test was done, age, gender, and results. an extraction data tool was used for data collection. variables with no clear labels and missing data were removed from the data set. data analysis microsoft excel was used to come up with an epidemiological curve of daily covid-19-positive cases, whereas proportions for gender were described using frequencies and percentages. age was described as a continuous variable and the mean, median, mode and range were used. to test for differences on the covid-19 test result, the chi-square test was used once assumptions were met to analyse binary variables; otherwise fisher’s exact test was used. for continuous variables; the mann-whitney ranksum test was used for skewed data. after stratifying covid-19 positivity by months, the one-way analysis of variance test was used to analyse for differences in age among groups. to predict factors associated with a positive test for covid-19, logistic regression methods were used. stata® software, version 14 se (stata corp., college station, texas, united states) was used for analysis. a p-value less than 0.05 was considered statistically significant at a confidence interval of 95%. results a total of 11 144 asymptomatic travellers tested for covid-19 were sampled for the study and 1781 tested positive, resulting in a positivity rate of 16.0% (table 1). the study participants had a median age of 36 years (interquartile range: 29 to 36 years). the test for covid-19 was noted to be done mostly by travellers in the age group 19 to 50 years. the youngest participant was 1 year old while the oldest was 92 years old. a majority of those tested were male travellers (73.2%; 8152/11 144); female travellers accounted for the remaining 26.8% (2992/11 144). the highest number of tests were completed in january 2021 (4170/11 144). table 1: basic characteristics of participants, ndola, zambia, december 2020 – march 2021. the proportion of female travellers that tested positive for covid-19 (18.4%) was greater than the proportion of male travellers (15.1%) with a p-value of 0.027 (table 2). among individuals who tested for covid-19 prior to travelling, about 7303 (83.2%) tested negative and were ndola residents, whereas among individuals that tested positive, 304 (12.9%) were not ndola residents. there were no positive results among individuals whose samples were collected orally. of the monthly tests done, 30/1557 (1.9%) were positive in december 2020, 1060/4170 (25.4%) were positive in january 2021, 532/3079 (17.3%) positive in february 2021 and 159/2338 (6.8%) positive in march 2021. table 2: covid-19 positivity rate and socio-demographics of participants in ndola, zambia, december 2020 – march 2021. when stratified by month of visit to the testing centre, there were more ndola residents seeking testing services at the tdrc laboratory in january 2021 (n = 948) than any other month included in the study (table 3). an equal peak number of non-ndola residents (n = 111) was seen in january 2021 and february 2021. the lowest number of travellers was seen in december 2020. table 3: covid-19 positivity rate stratified by months in ndola, zambia december 2020 – march 2021. coronavirus disease 2019 cases began to rise on 05 january 2021 and reached a peak on 26 january 2021 (figure 1). the cases remained high until 23 february 2021, when there was a reduction of 150 in the number of positive cases reported. figure 1: daily covid-19-positive cases in asymptomatic travellers in ndola, zambia, december 2020 – march 2021. female travellers had a 16.0% (adjusted odds ratio: 1.16; 95% confidence interval: 1.03 – 1.30; p = 0.012) increased chance of testing positive for sars-cov-2 compared to male travellers, after adjusting for age, residence, and month in which the test was done (table 4). table 4: predictors of positive covid-19 during the second wave in ndola, zambia, december 2020 – march 2021. discussion this study found a positivity rate of 16.0% (1781/11 144), with 69.1% (1231/1781) of male travellers being affected. the months of january 2021 and february 2021 recorded the highest rate of positivity. the epidemiological curve showed that the second wave of covid-19 lasted from december 2020 to the end of march 2021. further, the chances of testing positive for sars-cov-2 if an individual was female increased by about 16% (95% confidence interval: 1.03–1.30) compared to being male, after controlling for other variables. the positivity rate found in this study was similar to the positivity rate at the national level in zambia during the same period.6 despite the similarity, the national level positivity rate comprised both symptomatic and asymptomatic cases. the positivity rate found could have been higher if control measures of isolation and quarantine of cases and testing of people before travel were not put in place and followed.11 on the other hand, the positivity rate in this study was higher than the national rate of 10.6% during the first wave.15 this could have been due to differences in the attack rate and rate of transmission of the strain of coronavirus.16 conversely, a study in nigeria reported a higher positivity rate of 20.8% in the second wave which lasted from 25 october 2020 to 03 april 2021, with asymptomatic cases being the majority.17 a study done by avadhanula et al. showed a positivity rate of 11.4% among asymptomatic patients during the second wave between 18 march 2020 and 15 august 2020 in houston, texas, united states, which was much lower than the rate found in this study.18 this could have been due to differences in region, rate of transmission, adherence to recommended guidelines and utilisation of covid-19 vaccine as it was introduced in some countries earlier than others.17 our study and a study by ghosh, sarkar and chouhan, done in india between march 2021 and may 2021, thus confirmed the presence of covid-19 among asymptomatic individuals.19 our study found that a rise in covid-19 cases during the second wave of the pandemic was observed from december 2020 and ended in march 2021. the epidemiological curve for the daily cases of covid-19 showed a peak on 26 january 2021. in italy, different findings were reported in which the second wave began in august 2020 and continued to february 2021.20 a study in spain demonstrated that the second wave started on 01 july 2020 and ended on 15 october 2020, indicating that this period was different to what was obtained in our study.21 in india, the peak of cases was observed around 01 march 2021.19 these differences could be a result of differences in geographical locations and climatic conditions across the globe.22,23,24 this study found a statistically significant difference in positivity rate between female travellers compared with male travellers, with female travellers more likely to test positive. these findings are consistent with those in nigeria, where more asymptomatic female individuals than male tested positive.17 other studies have also reported similar findings in which female individuals had higher odds of positivity than male individuals.25,26 this could be due to female patients having a higher health-seeking behaviour than male patients.25 in a study done in netherlands, on data collected from march 2020 to august 2020, there was no significant difference in positivity rates between female patients and male patients.27 the study also found the age between 19 to 50 years to have a higher positivity rate compared to those who were 18 years or younger and those older than 50 years. this finding was not different from the study done in wuhan, china, and bahrain, ireland, that reported a higher prevalence of covid-19 in individuals who were less than 45 years old in wuhan, and 20 to 49 years in bahrain.28,29 this could be because those aged 18 years and younger were less susceptible to covid-19 during the second wave.30 in addition, control measures such as closure of school, colleges and universities may have contributed to the age group 18 years and younger having a low positivity rate.31 on the other hand, most of the individuals older than 50 years were symptomatic and prone to hospitalisation as compared to younger individuals.32 limitations this study had some limitations, one of which was that there was no control on the variables as this was a retrospective analysis of previously collected data. findings in this study may not be generalisable, as the data were obtained from one testing site. however, the study had good power and the results are a true reflection of the country’s positivity rate. a prospective study with more variables is recommended. conclusion the positivity rate was found to be 16.0%, implying that there was continued community transmission despite the instituted public health guidelines. age was not a predictor of a testing positive for covid-19, whereas the month in which a test was done, the sex of the individual and their place of residence were good predictors. the positivity rate reported in this study suggests the need to heighten testing of sars-cov-2 among asymptomatic individuals. acknowledgements we would like to acknowledge the staff in the molecular laboratory at the tropical diseases research centre for availing the data that was used in this study. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions j.g. conceptualised the study, conducted the formal analysis and wrote the first draft. v.d. conducted the formal analysis and review and editing of the manuscript. j.c., s.m., b.m. and r.l.m. performed data curation and reviewed the manuscript. m.m. performed the formal analysis and reviewed the manuscript. g.c. performed the editing, review of the manuscript and supervised the conduct of the study. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the data supporting the findings of this study are available from the corresponding author, v.d., upon request. disclaimer the views and opinions expressed in this article are solely of the authors and do not reflect the official policy or position of any affiliated organisation of the authors. references sokolovska l, sultanova a, cistjakovs m, murovska m. covid-19: the third wave of coronavirus infection outbreak. j transl sci. 2021;7(1):e1–e5. https://doi.org/10.15761/jts.1000389 bwire gm, paulo ls. coronavirus disease-2019: is fever an adequate screening for the returning travelers? trop med health. 2020;48(1):1–3. 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https://doi.org/10.1136/bmjgh-2021-007076 avadhanula v, nicholson eg, ferlic-stark l, et al. viral load of sars-cov-2 in adults during the first and second wave of covid-19 pandemic in houston, tx: the potential of the super-spreader. j infect dis. 2021;223(9):1528–1537. https://doi.org/10.1093/infdis/jiab097 ghosh dd, sarkar a, chouhan dp. covid-19 second wave: district level study of concentration of confirmed cases and fatality in india. environ challenges. 2021;5:100221. https://doi.org/10.1016/j.envc.2021.100221 coccia m. the impact of first and second wave of the covid-19 pandemic in society: comparative analysis to support control measures to cope with negative effects of future infectious diseases. environ res. 2021;197:111099. https://doi.org/10.1016/j.envres.2021.111099 iftimie s, lopez-azcona af, vallverdu i, et al. first and second waves of coronavirus disease-19: a comparative study in hospitalized patients in reus, spain. plos one. 2021;16(3):e0248029. https://doi.org/10.1101/2020.12.10.20246959 mudenda s. the second wave of covid-19 and risk of the third wave: factors affecting the continuous transmission, spread of, and increased mortality associated with coronavirus disease 2019 (covid-19). eur j environ public health. 2021;5(2):em0081. https://doi.org/10.21601/ejeph/11056 de cos o, castillo v, cantarero d. facing a second wave from a regional view: spatial patterns of covid-19 as a key determinant for public health and geoprevention plans. int j environ res public health. 2020;17(22):8468. https://doi.org/10.3390/ijerph17228468 chen s, prettner k, kuhn m, et al. climate and the spread of covid-19. sci rep. 2021;11(1):9042. https://doi.org/10.1038/s41598-021-87692-z stall nm, wu w, lapointe-shaw l, et al. sexand age-specific differences in covid-19 testing, cases, and outcomes: a population-wide study in ontario, canada. j am geriatr soc. 2020;68:2188–2191. https://doi.org/10.1111/jgs.16761 lapointe-shaw l, rader b, astley cm, et al. web and phone-based covid-19 syndromic surveillance in canada: a cross-sectional study. plos one. 2020;15(10):e0239886. https://doi.org/10.1371/journal.pone.0239886 ballering av, oertelt-prigione s, olde hartman tc, et al. sex and gender-related differences in covid-19 diagnoses and sars-cov-2 testing practices during the first wave of the pandemic: the dutch lifelines covid-19 cohort study. j womens health. 2021;30(12):1686–1692. https://doi.org/10.1089/jwh.2021.0226 yu c, zhou m, liu y, et al. characteristics of asymptomatic covid-19 infection and progression: a multicenter, retrospective study. virulence. 2020;11(1):1006. https://doi.org/10.1080/21505594.2020.1802194 almadhi ma, abdulrahman a, sharaf sa, et al. the high prevalence of asymptomatic sars-cov-2 infection reveals the silent spread of covid-19. int j infect dis. 2021;105:656–661. https://doi.org/10.1016/j.ijid.2021.02.100 starke kr, reissig d, petereit-haack g, schmauder s, nienhaus a, seidler a. the isolated effect of age on the risk of covid-19 severe outcomes: a systematic review with meta-analysis. bmj glob health. 2021;6(12):e006434. https://doi.org/10.1136/bmjgh-2021-006434 rotevatn ta, nygård k, espenhain l, et al. when schools were open for in-person teaching during the covid-19 pandemic – the nordic experience on control measures and transmission in schools during the delta wave. bmc public health. 2023;23(1):1–12. https://doi.org/10.1186/s12889-022-14906-y crimmins em. age-related vulnerability to coronavirus disease 2019 (covid-19): biological, contextual, and policy-related factors. public policy aging rep. 2020;30(4):142–146. https://doi.org/10.1093/ppar/praa023 abstract introduction ethical considerations case presentation management and outcome discussion acknowledgements references about the author(s) bakani a. siwele department of orthopaedics, sefako makgatho health science university, pretoria, south africa ndivhuho a. makhado national health laboratory service – dr george mukhari tertiary laboratory, department of medical microbiology, pretoria, south africa department of microbiological pathology, sefako makgatho health sciences university, pretoria, south africa global health institute, university of antwerp, wilrijk, belgium department of biomedical sciences, mycobacteriology unit, institute of tropical medicine, antwerp, belgium matodzi t. mariba department of orthopaedics, sefako makgatho health science university, pretoria, south africa citation siwele ba, makhado na, mariba mt. late diagnosis of multidrug-resistant tuberculosis in a child at dr george mukhari academic hospital, ga-rankuwa, south africa: a case report. afr j lab med. 2019;8(1), a783. https://doi.org/10.4102/ajlm.v8i1.783 case study late diagnosis of multidrug-resistant tuberculosis in a child at dr george mukhari academic hospital, ga-rankuwa, south africa: a case report bakani a. siwele, ndivhuho a. makhado, matodzi t. mariba received: 02 feb. 2018; accepted: 09 jan. 2019; published: 29 july 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract introduction: south africa has one of the top ten tuberculosis burdens in the world, only lagging behind countries with significantly larger populations. increased awareness of extrapulmonary tuberculosis, specifically spinal tuberculosis, is necessary, because of the hiv epidemic. case presentation: this report describes the case of a 9-year-old male patient who was suspected of having multidrug-resistant (mdr) tuberculosis, based on failure to recover clinically and radiologically after 6 months on first-line anti-tuberculosis treatment. pus samples were sent to an accredited academic laboratory for histopathology, microscopy, culture, line-probe assay (mtbdrplus assay) and phenotypic mgit 960 drug susceptibility tests. second-line mdr tuberculosis treatment was introduced. clinical, radiological, physical processes and more laboratory tests were conducted to document whether or not there was improvement in the patient. management and outcome: after laboratory diagnosis of mdr tuberculosis, the patient was started on mdr tuberculosis treatment. the patient started improving remarkably after the introduction of anti-tuberculosis treatment and rehabilitation, although he also required surgery to stabilise the spine. neurological improvement was observed in the patient and he fully recovered. discussion: although the diagnosis of spinal mdr tuberculosis may not be achieved easily by culture, the well-known gold standard method of tuberculosis diagnosis, it is of great importance to rapidly initiate an effective anti-tuberculosis treatment of drug-resistant strains to reduce the deformity of the spine. keywords: spinal tuberculosis; extrapulmonary tuberculosis; multidrug-resistant tuberculosis; laboratory diagnosis; radiological improvement. introduction globally, tuberculosis is the most common infectious disease and is responsible for a high rate of morbidity and mortality when not properly managed.1 co-infection with hiv and tuberculosis, and the increasing emergence of drug-resistant (dr) strains of tuberculosis, especially multidrug-resistant (mdr) tuberculosis and extensively drug-resistant (xdr) tuberculosis, present a major threat to effective tuberculosis control.2,3 not only is south africa one of the top 10 countries in the world for prevalence of tuberculosis, but it also has the highest number of patients with dr tuberculosis in the world. however, it does lag behind countries with significantly larger populations, such as india and china.4 tuberculosis is a highly infectious disease of the lungs. pulmonary tuberculosis, caused by the mycobacterium tuberculosis complex, can also affect other body sites as extrapulmonary tuberculosis; spinal tuberculosis is a frequently encountered form of extrapulmonary tuberculosis.5,6 despite the availability of anti-tuberculosis therapy, the vertebrae and spinal cord often have a delayed diagnosis leading to devastating, irreversible complications, for example paraplegia.7,8,9 furthermore, it has been proven that half of extrapulmonary tuberculosis cases have been previously reported, and delayed diagnosis may further enhance the risk of transmission of tuberculosis via contacts with more people, as well as compromising healthcare workers.10 in this case study, factors associated with clinical aspects, radiology and late diagnosis of mdr tuberculosis of the spine and its clinical outcome are reported. ethical considerations oral consent was obtained from the patient’s parents along with assent from the patient and ethical clearance from the sefako makgatho health sciences university’s research and ethics committee (smurec/m/147/2017:j) for the publication of this case study, including any images in any abstract or publication. all personal identifiers were anonymised for confidentiality before data processing was performed. there were no patient identity links to the radiological images used. case presentation a 9-year-old male patient with a history of backache was referred from a district hospital to the dr george mukhari academic hospital, gauteng province, south africa, due to severe upper back pain and gibbus (kyphosis) deformity that emerged over 8 months prior to admission. he had no history of tuberculosis contact or constitutional symptoms (cough or fever, loss of weight or loss of appetite). upon physical examination at admission, the patient was ambulatory but limping, had tenderness at the gibbus area, with pain in the waist. he was cooperative and appeared to have a normal skin condition. there was no bacillus calmette-guerin scar observed. the neurological examination was normal and intact. x-rays revealed kyphosis at the bottom of the thoracic spine (t12 vertebra) and the first vertebra of the lumbar spine (l1 vertebra). his t12 had vertebral destruction with preserved posterior elements. the l1 upper end plate and t12 lower end plate destruction with a peri-vertebral shadow were indicative of an abscess (figure 1). figure 1: x-rays showed (a) l1 upper end plate and (b) t12 lower end plate destruction with peri-vertebral shadow. plain radiography of the chest was unremarkable (figure 2). furthermore, the haemoglobin was low (11.5 g/dl), with a normal-range white blood cell count and an elevated c-reactive protein level of 18 mg/l. the erythrocyte sedimentation rate was a bit high at 20 mm/hour; alkaline phosphatase was 211 u/l. the alanine aminotransferase (19 u/l) and aspartate aminotransferase (31 u/l) were within the normal ranges. a serology test for hiv enzyme linked immunosorbent assay was negative. a superficial pus swab microscope analysis was acid-fast bacilli-negative when stained with auramine-o. an mgit 960 culture was also negative after 42 days. figure 2: plain x-ray demonstrating unremarkable features of the chest. magnetic resonance imaging of the entire spine revealed features suggestive of tuberculosis infection (figure 3). there was kyphotic deformity and pre-vertebral fluid collection at t12/l1 extending to the epidural space. the fluid collection was low on t1, high on t2 and became enhanced post-contrast. there was also an abnormal signal of l1 and t12, as well as associated involvement of the superior end plate of l1. the t12/l1 intervertebral disc was not clearly delineated with the distortion of normal anatomy. spinal canal stenosis was observed at the level of kyphotic deformity with cord compression, although there was spinal cord edema. posterior elements were preserved and facet joints were normal (figure 3). figure 3: the magnetic resonance imaging of the spine revealed (a) kyphotic deformity and (b) pre-vertebral fluid collection at t12/l1 extending to the epidural space. a second set of the magnetic resonance images of the entire spine, which was done eight months later, revealed progression of the disease (infective process), increased kyphosis, and a paravertebral abscess, as well as complete destruction of lower and upper end plates of t12 and l1 with cord compression (figure 4). figure 4: the magnetic resonance imaging of entire spine revealed (a = green arrow) an increased kyphosis, and (b = dark blue arrow) a paravertebral abscess. furthermore, it demonstrated a complete vertebral destruction of lower and upper end plates of t12 (c = orange arrow) and l1 (d = light blue arrow). management and outcome the patient was admitted and subsequently started on empirical first-line anti-tuberculosis treatment as recommended by the world health organization, as well as other supportive treatment, including thoracic lumber spinal orthosis. the patient did not improve clinically after 1 month of administering the empirical treatment. non-operative treatment included a thoracic lumber spinal orthosis brace, analgesia and first-line tuberculosis treatment to prevent further collapse and back care. the patient’s mother consented to operative management, which included spinal decompression and posterior instrumentation, with rods and screws, as well as a biopsy. the patient was neurologically intact post-surgery (figure 5). the operation was successful. figure 5: x-rays revealed intact spine post-surgery and decompression (a and b). the pathological diagnosis of the spine (t12/l1) on the vertebral lesion biopsy revealed the presence of chronic necrotising granulomas with multinucleated langhans-type giant cells. ziehl-neelsen staining, used to confirm acid-fast bacilli, was negative. the patient’s first-line anti-tuberculosis treatment, included pyrazinamide, rifampin, isoniazid and ethambutol. the patient’s condition deteriorated despite a total of 6 months of first-line anti-tuberculosis treatment, and the surgical wound started to open leaving space between the sides of the incision (gaping). x-rays showed failed implants; therefore, the pedicular screws were backed off from the distal part of the vertebra (t12/l1) with increased kyphotic deformity of the spine (figure 6). figure 6: x-rays showing failed implants backing off from the (a) distal part of vertebra (t12/l1) and (b) increased kyphotic deformity of the spine. when the patient was on rehabilitation, he developed an abscess on the forehead, which was later drained in the ward, and sent to the national health laboratory services – dr george mukhari academic hospital tertiary laboratory for further mycobacteriological tests. fluorescent microscopy of the smear stained with auramine-o was negative for fluorescing acid-fast bacilli. the mgit 960 (becton dickinson diagnostics, sparks, maryland, united states) culture was also negative after 42 days. the patient was sent to surgery for incision and drainage of the abscess on the gibbus area, where from thick pus was drained and sent for tuberculosis microscopy, culture, line-probe assay (mtbdrplus assay, hain life sciences, nehren, germany) and phenotypic mgit 960 drug susceptibility testing. the auramine-o smear results were negative, whereas the mgit culture was positive after 35 days. the line-probe assay result showed rifampin monoresistance, which was later confirmed by the phenotypic mgit 960 drug susceptibility testing as rifampin monoresistant, ethambutol resistant and isoniazid sensitive after 9 months of first-line anti-tuberculosis treatment. the second-line mgit 960 drug susceptibility testing showed that the isolate was susceptible to moxifloxacin and kanamycin. this led to an mdr tuberculosis diagnosis, since tuberculosis caused by rifampin-resistant strains is presumed to be mdr tuberculosis and treated as such (95% of rifampin-resistant strains are mdr3). the patient was started on the mdr tuberculosis treatment, including moxifloxacin, ethionamide, amikacin, pyrazinamide, terizidone and pyridoxine; he started improving remarkably on the treatment and rehabilitation. at 15 months, the patient fully recovered and the implants were later removed. discussion the diagnosis of extrapulmonary tuberculosis is not clinically easy due to the non-specific manifestations of the condition; thus, a high index of suspicion is required to accurately diagnose the condition.7 patients who present with focal or spinal abnormalities, with or without a chest radiograph showing previous or active tuberculosis, should be investigated for spinal tuberculosis. spinal tuberculosis seems to be due to an arterial or venous route of infection. in the early stages, the infection affects the anterior portion of the vertebra.5 osseous tuberculosis may spread to an anterior type involving the vertebral bodies.7,11,12 commonly, patients with spinal tuberculosis present in the first three decades of life with subtle symptoms, such as backache, malaise, loss of appetite and weight, night sweats and fever, as observed in this study. in this case, the patient had progressive back pain over several months with localised tenderness of the spine and weight loss. although signs and symptoms of systemic infection are often missing, severe back pain is the typical presenting symptom of early spinal tuberculosis disease and should encourage further investigation of a definite diagnosis. symptoms such as fever and weight loss in spinal tuberculosis patients are present in less than 40% of cases and are often not specific.7 common symptoms of spinal tuberculosis include leg weakness (69%), gibbus deformity (46%), pain (21%), and palpable masses (10%).9 in this study, the patient had no spinal cord involvement despite the delayed diagnosis. in the advanced stages of spinal tuberculosis disease, a significant proportion of patients present with neurological impairment.9,13 kyphotic deformity is seen in 95% of cases, although the deformity may not develop in the early stages or in less severe cases.2,14 less than 40% of patients present with constitutional symptoms of generalised body malaise, loss of appetite, weight loss, night sweats and fever.5,7 the majority of spinal tuberculosis cases can be managed non-surgically. radical surgery at a younger age, combined with anti-tuberculosis treatment, has been associated with favourable patient outcomes.15 the surgical procedures can be done anteriorly or posteriorly with instrumentation. other studies have reported that about 0.7% of patients can have implant failure following a spinal tuberculosis operation.12 in this study, surgery was performed and anti-tuberculosis treatment was given. the patient fully recovered after 15 months. conclusion in conclusion, drug-resistant spinal tuberculosis cannot be easily diagnosed by gold standard methods. a high index of suspicion followed by clinical examination, radiological examination and laboratory investigations will help to promptly diagnose the condition and allow for effective patient management. with emerging mdr tuberculosis, the osseous abnormality is also at risk of involvement. a diagnosis of mdr tuberculosis of the spine should be borne in mind, if the patient does not respond to the first-line of treatment. drug-resistant anti-tuberculosis treatment and radical surgery can lead to a reduction of spinal tuberculosis complications such as paraplegia. acknowledgements the authors would like to thank the national health laboratory services – dr george mukhari tertiary laboratory and fellow medical doctors in the department of orthopaedics for their support during the study period. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions all authors contributed to the writing of the article. b.a.s. and m.t.m. initiated the case report and designed the study. 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https://doi.org/10.1016/j.tcmj.2012.07.005 park dw, sohn jw, kim eh, et al. outcome and management of spinal tuberculosis according to the severity of disease: a retrospective study of 137 adult patients at korean teaching hospitals. spine. 2007;32(4):e130–e135. https://doi.org/10.1097/01.brs.0000255216.54085.21 abstract introduction methods results discussion acknowledgements references about the author(s) naome mugabiirwe department of medical laboratory, kyazanga health centre iv, lwengo, uganda rogers kalyetsi department of medical laboratory science, faculty of medicine, mbarara university of science and technology, mbarara, uganda richard ayella department of medical laboratory, kyazanga health centre iv, lwengo, uganda james obote department of medical laboratory, kyazanga health centre iv, lwengo, uganda frank ssedyabane department of medical laboratory science, faculty of medicine, mbarara university of science and technology, mbarara, uganda citation mugabiirwe n, kalyetsi r, ayella r, obote j, ssedyabane f. hepatitis b virus infection and hbeag positivity among pregnant women in south west uganda. afr j lab med. 2022;11(1), a1784. https://doi.org/10.4102/ajlm.v11i1.1784 original research hepatitis b virus infection and hbeag positivity among pregnant women in south west uganda naome mugabiirwe, rogers kalyetsi, richard ayella, james obote, frank ssedyabane received: 09 nov. 2021; accepted: 10 may 2022; published: 15 aug. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: hepatitis b virus is a public health burden in uganda, yet little is known about its epidemiology in pregnancy. objective: this study aimed at determining the prevalence and associated risk factors of hepatitis b virus infection among pregnant women attending antenatal care at the kyazanga health centre iv in lwengo district, uganda. methods: this cross-sectional study was conducted from april 2021 to june 2021 and analysed qualitative data that were collected using a structured in-person questionnaire. aseptically collected blood specimens were screened for hepatitis b virus infection using an immunochromatographic rapid diagnostic test kit. participants who were positive for the hepatitis b surface antigen (hbsag) were further screened for hepatitis b envelope antigen (hbeag) using commercial rapid diagnostic test kits. results: out of 384 pregnant women studied, eight tested positive for hbsag. this gave a prevalence of 2.1% (95% confidence interval: 1.0% – 4.1%); 5/8 (62.5%) were positive for hbeag. none of the variables studied were significantly associated with hbsag positivity among pregnant women. conclusion: hepatitis b viral infection is still a public health challenge in pregnant women with possible risk for vertical transmission to their babies in the study area. we recommend routine screening for hepatitis b virus in pregnancy in addition to strengthening current strategies aimed at controlling and preventing hepatitis b infection spread and transmission. keywords: risk factors; hepatitis b; pregnant women; prevalence; uganda. introduction hepatitis b is caused by hepatitis b virus (hbv) infection. hepatitis b virus belongs to the family hepadnaviridae and is highly hepatotropic, causing acute and chronic liver diseases including cirrhosis and hepatocellular carcinoma.1 hepatitis b virus is estimated to have caused 820 000 deaths worldwide2 by contributing to chronic hepatitis and hepatocellular carcinoma.3 the prevalence of hbv infection in sub-saharan africa is reported to range from 5% to 20%,4 with perinatal transmission estimate rates ranging from 1% to 5%.4 uganda is known to be a highly endemic area of the infection with an estimated national prevalence of 10.0%.5 the regional distribution of disease varies, with the highest prevalence in the northern region; prevalence is 19% in the northwest and 25.0% in the northeast.5 the national prevalence of hbv infection among women attending antenatal care was 4.1% in the year 2018.6 studies done in uganda around 2018 estimated the prevalence of hepatitis b virus infection among pregnant women attending antenatal care to be 2.9% (95% confidence interval: 1.58% – 5.40%) at mulago national referral hospital in central uganda,5 11.8% at two hospitals in northern uganda7 and 3.1% at mbarara regional referral hospital in western uganda.6 some risk factors for hepatitis b infection include tattooing or use of contaminated sharp instruments, reuse of needles, intravenous drug use, percutaneous as well as mucosal exposure to infected blood or any other body fluids such as saliva, vaginal or seminal fluids.5 mothers who are positive for hepatitis b surface antigen (hbsag) have a 10% to 40% risk of passing the infection onto their newborn babies and the risk increases to 90% among mothers who are positive for the hepatitis b envelope antigen (hbeag).8 babies who are infected perinatally have a 90% risk of developing chronic infection.9 antiviral therapy during late pregnancy and immunisation of babies exposed to hbv within the first 12 h of life may reduce mother-to-child transmission by 75% to 90% and reduce risk of developing chronic infection.10,11 the perinatal transmission risk is 70% to 90% for hbeag-positive mothers, 25% for hbeag-negative/anti-hbe-negative mothers and 12% for mothers who are positive for anti-hbe while negative for hbeag.5 a number of studies have been conducted in south western uganda on the epidemiology of hbv infection, though little data exists on prevalence and associated risk factors among pregnant women attending antenatal care at the kyazanga health centre iv (hciv). therefore, this study aimed to determine the prevalence and risk factors associated with hbv infection among pregnant women attending antenatal care at kyazanga hciv, lwengo district, south western uganda. methods ethical considerations ethical approval was sought from the department of medical laboratory science, faculty research committee faculty of medicine mbarara university of science and technology (must/mls/030). permission was also obtained from the district health officer, lwengo district and the office of medical officer health sub-district, kyazanga health centre iv. informed consent was obtained from all study participants. participants were identified with numbers, not names, for prevention of breach of confidentiality. all completed questionnaires were archived in the department of medical laboratory science under lock and key. the generated computer database was kept inaccessible for non-study persons; for study personnel, access was restricted with a password. study design and setting this cross-sectional study was conducted between april 2021 and june 2021 at the kyazanga hciv antenatal clinic located in kyazanga town council, lwengo district in south western uganda. the kyazanga hciv is a public health facility which serves buktoto west, including referrals from five other low-level health facilities. sample size, study population and sampling strategy we calculated a sample size of 384 participants based on a presumed 50% prevalence rate of hbv6 using an allowable standard error of ±0.05 at 95% level of confidence. the study population included pregnant women who attended the antenatal clinic during the study period. we used a systematic random sampling method to recruit all participants. individuals were selected at regular intervals, where every second pregnant woman who arrived at the clinic was selected from the sampling frame. data collection with the help of clinic nurses, we administered an in-person structured questionnaire to collect socio-demographic data (age, place of residence, level of education, primary [completed primary school year seven], secondary [completed senior school year six], or tertiary [reached any tertiary institution]), participant’s place of birth and gravidity (primigravida [first pregnancy], multigravida [2–4 pregnancies], grand multigravida [≥ 5 pregnancies]) and risk factors for hbv (history of blood transfusion, history of abortion or miscarriage, history of hospital admission, history of tooth extraction, history of body piercing, history of unprotected sexual intercourse, history of sharing sharp materials and history of injection drug abuse) from study participants. laboratory testing immediately after consent and completion of the questionnaire, three millilitres (3 ml) of venous blood was drawn aseptically by venipuncture from the mid-cubital vein, into ethylenediaminetetraacetic acid-vacutainers. specimens were labelled using identification numbers (codes) and immediately taken to the laboratory for centrifugation (3000 rpm for 5 min) to separate plasma from blood cells. sd bioline hbsag immunochromatographic rapid diagnostic test kits (abbott diagnostics korea inc, gihueng-gu, yongin-si, republic of korea) were used for qualitative detection of hbsag in serum from each study participant. plasma was added to the sample pad on the hbsag test strip and timed for 15 min for the reaction to occur. results were then read, interpreted and recorded. positive samples were then tested for the hbeag, using the commercial sd bioline hbeag testing kit (abbott diagnostics korea inc, gihueng-gu, yongin-si, republic of korea) to show the level of infectivity and risk of infection. all laboratory testing was carried out within the health unit laboratory, following standard operating procedures as well as manufacturer’s instructions. quality assurance and control the questionnaire was pre-tested and validated on five pregnant women attending antenatal care at the kyazanga hciv. each batch of the rapid dipstick tests was pre-tested with positive and negative controls (known positive and known negative plasma samples) for quality assurance. positive and negative controls were run along with each batch of samples. the sensitivity and specificity of the rapid test strip were 98.84% and 98.94%, respectively. data management and analysis data were entered into a microsoft excel spread sheet (microsoft office professional plus 2013, version 15.0.4675.1003, microsoft inc, redmond, washington, united states) and then imported into stata 13 (statacorp llc, college station, texas, united states) software for analysis. demographic data were presented in the form of frequencies and percentages. prevalence was presented as percentages and using pie charts. associations between risk factors and hepatitis b were determined using regression analysis and a p-value of < 0.05 was considered to be statistically significant. results socio-demographic characteristics of pregnant women attending antenatal clinic at kyazanga health centre iv we recruited 384 pregnant women attending the antenatal clinic at kyazanga hciv from april 2021 to june 2021 (table 1). the participants’ mean age was 27 years, with a standard deviation of 7.34 years. participants’ ages ranged from 17 to 48 years, with the majority aged between 26 and 28 years. most study participants were from urban areas (51.0%), had completed a primary seven level of education (65.0%), had been born in a hospital setting (61.2%) and were multigravidas (54.2%). table 1: demographic characteristics of participants attending the antenatal clinic at kyazanga health centre iv, south western uganda, between april 2021 and june 2021. prevalence of hepatitis b virus infection of the 384 participants, eight participants tested positive for hbsag for an overall prevalence of viral hepatitis of 2.1% (table 2). of the eight hbsag-positive participants, five tested positive for hbeag (62.5% of hbsag-positive participants). of the eight participants who tested positive for hbsag, 75.0% (6/8) were aged 24–35 years, 25.0% (2/8) were aged younger than 24 years (table 3). additionally, 62.5% (5/8) lived in an urban setting, and 37.5% (3/8) lived in a rural setting. a majority (62.5%; 5/8) had completed a primary seven level of education, 25.0% (2/8) had attained secondary education, 12.5% (1/8) had tertiary education. most participants had been born at home (62.5%; 5/8), while 37.5% (3/8) had been born at the hospital. most participants 75.0% were multigravida and 25.0% were primigravida. among women who were hbsag-positive, 50.0% (4/8) had a history of blood transfusion, 62.5% (5/8) had a history of sharing sharp instruments, all had a history of unprotected sexual intercourse, none had any history of drug abuse by injection, 87.5% (7/8) had a history of hospital admission, 37.5% (3/8) had a history of abortion or miscarriage, 62.5% (5/8) had a history of tooth extraction, 50.0% (4/8) had multiple sexual partners and none had any history of body piercing. table 2: overall prevalence of hbsag and hbeag positivity among participants attending antenatal at kyazanga health centre iv, south western uganda, between april 2021 and june 2021. table 3: prevalence of hbv infection by risk factor among pregnant women attending kyazanga health centre iv, south western uganda, between april 2021 and june 2021. risk factors for hepatitis b virus infection on univariate analysis, place of birth (odds ratio: 1.06; p = 0.018), a history of blood transfusion (odds ratio: 1.037; p = 0.043) and a history of hospital admission (odds ratio: 1.03; p = 0.04) were associated with hbv infection (table 4). on multivariate analysis, no variable was significantly associated with hbv infection. table 4: risk factors predisposing pregnant women to hbv at kyazanga health centre iv, south western uganda, between april and june 2021. discussion prevalence of hbv infection we report the prevalence of hbsag at 2.1% among pregnant women attending antenatal care at kyazanga hciv, lwengo district, uganda. the epidemiology of hbv infection can be described in terms of the prevalence of hbsag positivity in a population. this can be broadly classified as high (> 8.0% hbsag prevalence), intermediate (2.0% – 7.0% hbsag prevalence) and low (< 2.0% hbsag prevalence).12 this study reports intermediate endemicity of hbsag and high prevalence of hbeag of 62.5% (5/8) among the hbsag-positive women, which indicates a high risk of perinatal transmission. in comparison to other studies done within uganda, the prevalence reported in this study is comparable to infection levels reported at mbarara regional referral hospital of 2.5% in 201813 and 3.12% in 2019.6 the finding of our study is also comparable to 2.9% prevalence which was reported at mulago regional referral hospital.5 this similarity may be because the same risk group was studied and the tests used to ascertain hbv infection were similar (i.e., rapid tests). however, this study’s result does not correlate with the reported prevalence of 11.8% in two hospitals located in northern uganda where similar diagnostic methods were used.7 this high prevalence of hepatitis b in northern uganda could be due to a difference in socio-demographic factors like level of education. a majority of people in northern uganda are said to have not gone beyond primary seven.14 within the east african countries, this study closely correlates with the finding of 3.8% in mbagathi district in kenya in 2014,15 3.8% at nyamagana district hospital in mwanza, tanzania in 2014,16 3.9% in dar es salaam in 201417 and 3.9% in rwanda in 2018.18 it is lower than the reported prevalence of hbsag at khartoum teaching hospital, sudan, 7.5% in 2010,19 and juba teaching hospital, south sudan, 11.0% in 2012.20 this intra-regional variation could be due to geographical variation, sample size and laboratory methods used for diagnosis. the prevalence of hbeag positivity of 62.5% among hbsag-positive pregnant women reported in this study is higher than the prevalence reported by a study in northern uganda at 14.9%.7 hbsag-positive mothers who test positive for hbeag are known to be highly infectious as the virus is actively replicating.21 therefore, they have a 40.0% to 90.0% risk of transmitting the hbv infection to their babies at birth.22 however, our study did not test for anti-hbe or anti-hbc antibody levels, which would further help predict the risk of perinatal transmission in these women. risk factors for hbv infection none of the risk factors studied had statistically significant associations with hbsag positivity. this concurs with studies done in buea in cameroon in 2012,23 and benin city in nigeria in 2009.24 however, not all of the risk factors considered by those studies were assessed in the current study. we also report here that there was a high prevalence of hbsag among pregnant women aged 24–35 years, although the association with hbsag positivity was not stastically significant; this agrees with a study done in egypt between 2010 and 2011.25 this observation of increasing age with hbsag infection can be attributed to increased likelihood of contracting hbv during each cycle of pregnancy. however, this contrasts with findings from a study done by bayo and his colleagues in 2012, who reported a high prevalence of hbv infection among pregnant women aged 20 years old or less in northern uganda.7 the current study also found no association between participants’ history of blood transfusion and hbsag positivity. this agrees with several studies conducted in various places, including mbarara regional referral hospital in uganda in 201813 and 2019,6 in egypt between 2010 and 2011,25 in mulago, uganda between 2018 and 2019,5 and juba teaching hospital, south sudan in 2012.20 this finding contrasts with studies done in khartoum, sudan, in 2010 which found a positive association between participant’s history of blood transfusion and hbsag positivity.19 this observation may be due to implementation of good blood screening strategies for transfusion-transmissible infections in blood bank donations, reducing incidence of infection by blood transfusion. this study showed a high prevalence of hbsag positivity among the multigravida pregnant women, although the association was not statistically significant. this contrasts with a study done in mwanza, tanzania, in 2014, which showed that multigravidity is associated with hbsag.16 it is thought that chances of exposure to hbv increase as one progresses through the cycle of pregnancy from conception to child birth, and the same happens as the gravidity rises from primagravidity to multigravidity. history of hospital admission was not found to be significantly associated with hbv infection. this contrasts with a study from egypt between 2010 and 2011, which found a significant association between hospital admission and hbv infection, since hospital admission may expose pregnant women to surgical procedures which can lead to hbv infection.25 neither a history of abortion nor history of multiple sexual partners were significantly associated with hbsag positivity in this study. however other studies have reported significant association of hepatitis b infection with history of abortion and multiple sexual partners.26,27 this may be due to increases in sexual activity and sexual contact creating more opportunities for acquiring hbv infection this needs further study to test the level of this significance in this area. no significant association with hbv infection was found with a history of injection drug abuse, body piercing or body tattooing among these women. this is in agreement with a study conducted in southwestern nigeria in 2013.28 however, these factors are considered to increase the chances of becoming infected with hbv.29 the lack of a significant association may be attributed to the fact that practices of drug abuse by injection and tattooing are uncommon in this study setting. recommendations in view of high hbeag positivity among hbsag-positive pregnant women, there is need for routine screening of pregnant women for hbsag and hbeag to predict the risk of perinatal transmission and strengthen immunisation strategies for babies at risk. there is also a need to adapt treatment and prevention strategies to achieve the world target of free communities without hbv infection. limitations this study did not explore all the risk factors that may predispose pregnant women to hbv. for example, information about a history of jaundice, vulvular ulcerations, history of sexually transmitted infection, and a family history of hbv were not collected. the self-reported risk factor data used in this study could be subject to recall bias. conclusion hepatitis b virus was found to have intermediate endemicity among the pregnant women attending antenatal care at kyazanga hciv. however, this is not too low to cause morbidity and mortality among the population. none of the proposed risk factors were significantly associated with hbv infection; explanations for this observation need to be explored in future. acknowledgements we acknowledge the staff and administrators of kyazanga health centre iv for accepting our study and giving us the opportunity to explore the world of science today, thank you so much. we also acknowledge the department of medical laboratory science under the leadership of the head of department, mr rugera simon peter, and the faculty of medicine under the leadership of faculty dean, associate professor getrude n. kiwanuka. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions n.m., r.k., r.a., j.o. and f.s. have contributed substantially to this work and met the criteria for authorship. n.m., r.a. and j.o. conceived and developed the idea. they also performed data collection and prepared the first draft of the manuscript. r.k. and f.s. supervised the entire project and approved the final version. sources of support this research project was funded exclusively by the authors. this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the datasets used during the current study are available from the corresponding author, n.m., on reasonable request. disclaimer the views and opinions expressed in this artice are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references kao j-h, chen d-s. global control of hepatitis b virus infection. lancet infect dis. 2002;2(7):395–403. https://doi.org/10.1016/s1473-3099(02)00315-8 who. fact sheet on hepatitis b. geneva; world health organisation; 2019. kamenya t, damian dj, ngocho js, philemon rn, mahande mj, msuya se. the prevalence of hepatitis b virus among hiv-positive patients at kilimanjaro christian medical centre referral hospital, northern 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infection among pregnant women in bahir dar city, northwest ethiopia: a cross sectional study. bmc infect dis. 2014;14(1):1–7. https://doi.org/10.1186/1471-2334-14-118 abstract introduction results and discussion acknowledgements references about the author(s) passoret vounba economic community of central african states (eccas) commission/fourth phase of the regional disease surveillance systems enhancement project (redisse iv), libreville, gabon severin loul economic community of central african states (eccas) commission/fourth phase of the regional disease surveillance systems enhancement project (redisse iv), libreville, gabon ludovic f. tamadea economic community of central african states (eccas) commission/fourth phase of the regional disease surveillance systems enhancement project (redisse iv), libreville, gabon joël f.d. siawaya department of laboratory services, chu mère-enfant fondation jeanne ebori, libreville, gabon regional integrated surveillance and laboratory network (rislnet) for central africa, libreville, gabon citation vounba p, loul s, tamadea lf, siawaya jfd. microbiology laboratories involved in disease and antimicrobial resistance surveillance: strengths and challenges of the central african states. afr j lab med. 2022;11(1), a1570. https://doi.org/10.4102/ajlm.v11i1.1570 note: additional supporting information may be found in the online version of this article as online supplementary document 1. review article microbiology laboratories involved in disease and antimicrobial resistance surveillance: strengths and challenges of the central african states passoret vounba, severin loul, ludovic f. tamadea, joël f.d. siawaya received: 27 feb. 2021; accepted: 10 jan. 2022; published: 31 mar. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract laboratory systems have been largely neglected on the margins of health systems in africa. however, since the 2000s, many african countries have benefited from massive investments to strengthen laboratory capacities through projects fighting priority diseases (hiv/aids, tuberculosis, malaria). this review examined the laboratory capacities of the economic community of central african states (eccas). online research using specific terms was carried out. studies published between 2000 and 2021 on the role of the laboratory in disease and antimicrobial resistance surveillance in the 11 eccas countries were considered. the number of human and animal health laboratories meeting international standards was very low in the sub-region. there were only seven international organization for standardization (iso) 15189-accredited human health laboratories, with five in cameroon and two in rwanda. there were five high biosafety level (bsl) laboratories (one bsl3 laboratory each in cameroon, the central african republic, democratic republic of congo and the republic of congo, and one bsl4 laboratory in gabon) and three iso 17025-accredited laboratories in the eccas sub-region. only six countries currently have whole-genome sequencing devices, which is insufficient for a sub-region as large and populous as eccas. yet, a plethora of pathogens, particularly haemorrhagic viruses, are endemic in these countries. the need for laboratory capacity strengthening following a one health approach is imperative. since emerging and re-emerging zoonotic infectious diseases are projected to triple in frequency over the next 50 years and given the inextricable link between human and animal health, actors in the two health sectors must collaborate to preserve world health. keywords: laboratory capacity; economic community of central african states (eccas); laboratory strengthening; one health; epidemics; antimicrobial resistance. introduction laboratory results guide evidence-based clinical decisions. in 1923, louis pasteur declared that ‘without laboratories, men of science are soldiers without arms’.1 a standard medical laboratory is essential for patient care and communicable disease surveillance. the laboratory is indispensable, both for routine diagnosis of infections and for the rapid identification of epidemic outbreaks. in addition, antimicrobial resistance (amr) surveillance, food safety and water quality assessment, analysis of environmental samples, etc. are also laboratory dependent. in sub-saharan africa, unfortunately, laboratory service has been a sidelined health service that receives very little government budgetary allocation. in 2008, representatives of african governments recognised that: in resource-limited settings, several challenges have resulted in inadequate laboratory systems to support the scale-up of programs. these include a lack of leadership and advocacy, human resources, career path and retention of staff, national laboratory policy, strategic planning (budgetary concerns), sufficient physical infrastructure, supply chain management, and quality management systems (quality assurance). (p. 1)2 the limited investment in laboratory systems impairs the quality of laboratory services. for instance, sub-saharan african laboratories are deficient in terms of qualified staff, modern equipment, a regular supply of quality reagents, water, or electricity, standard operating procedures, and quality assurance systems.3,4 this review aimed to highlight the roles, strengths, and challenges of the human and animal laboratories in the economic community of central african states (eccas). the eccas is an integrated african space created in 1983 and includes 11 countries: angola, burundi, cameroon, central african republic, chad, democratic republic of congo, equatorial guinea, gabon, republic of congo, rwanda, and são tomé and príncipe. according to the 2014 estimates, the population of the eccas is about 161 million inhabitants, spread over an area of 6 640 490 km2.5 a systematic search was carried out using terms ‘role of the laboratory in africa’, ‘role of laboratory in disease surveillance in africa’, ‘role of veterinary laboratories in africa’, ‘role of the laboratory in amr surveillance in africa’, ‘iso [international standardization organization] 15189-accredited laboratory in africa’, ‘iso 17025-accredited laboratory in africa’, ‘health reference laboratories in africa’, ‘veterinary reference laboratories in africa’, and each term accompanied by the name of each of the 11 eccas countries. these terms were searched on google, google scholar, pubmed, african journals online and international organisations’ websites: (world health organization [who], world organization for animal health [oie], food and agriculture organisation of the united nations, africa centres for disease control and prevention, african society for laboratory medicine [aslm]). all studies published between 2000 and 2021 in the four official languages used by the eccas (english, french, spanish and portuguese) on the role of the laboratory in the surveillance of diseases and amr in the 11 countries were included. results and discussion role of human health laboratory in patient management medical laboratories provide a precise diagnosis for patient management. early diagnosis and treatment reduce the risk of long-term complications in patients and prevent further transmission.6 thus, proper patient management requires close tripartite collaboration between the patient, clinicians and laboratory staff.7 in sub-saharan africa, access to reliable diagnostic tests is very limited and misdiagnosis occurs frequently, leading to physicians’ distrust of laboratory results.6 limited access to reliable diagnosis results in no or inadequate treatment, increased mortality, and an inability to determine the true prevalence of diseases.8 in the democratic republic of congo, the laboratories were unable to diagnose the diseases frequently encountered in this country.4 the lack of laboratory infrastructure may delay patients’ recovery. delay may occur due to referrals to other laboratories that are further away and often more expensive, or due to inappropriate treatment that lacks laboratory diagnosis. arsuaga et al.9 reported an example of the latter: the case of a missionary symptomatically diagnosed and unsuccessfully treated for malaria in cameroon and equatorial guinea, but was laboratory diagnosed and successfully treated for babesiosis, a malaria-like illness, eight months later in spain. in rwanda, a strong laboratory network was built in the early 2000s. internal and external laboratory control and assurance activities are regularly conducted at all levels of the network by the national reference laboratory (nrl).10 the external quality assessment focuses on enteric and meningitis pathogens, tuberculosis, malaria, and hiv/aids. in 2003, a concordance of 100% was reported for the unlinked, anonymous hiv/aids testing of all 288 samples sent by the rwandan nrl to the united states centre for disease control and prevention.10 nevertheless, rwandan laboratories are not exempt from cross-cutting problems, such as service interruptions due to reagent stock-out and equipment breakdown.11 the positive impact of laboratory capacity building on reducing infections and improving patient management has been reported in cameroon. eleven months after implementing capacity strengthening activities, the regional hospital of buea reported a reduced patient wait time at the reception from 3 h to less than 30 min.12 similarly, laboratory improvement capacities in the bamenda regional hospital laboratory resulted in fewer specimen recalls, improved test reliability, and the provision of feedback channels on services offered.13 from the eccas sub-region, gabon, cameroon, the democratic republic of congo, and the central african republic are part of the who’s emerging dangerous pathogens laboratory network.14 all four countries have national viral haemorrhagic fever (vhf) testing capacity, while gabon hosts the who afro eccas regional vhf reference laboratory.15 all eccas countries have influenza laboratories except chad, equatorial guinea, and são tomé and príncipe. however, the influenza laboratory network in the republic of congo, angola, and rwanda can easily be upgraded to include vhf testing capacities.15 in cameroon, the centre pasteur du cameroun is a reference centre for the network of quantitative polymerase chain reaction diagnostic laboratories for buruli ulcer. this network brings together 11 laboratories located in nine west and central african countries where buruli ulcer is endemic.16 ultimately, other neglected tropical diseases such as leprosy and cutaneous leishmaniasis will be integrated into the buruli ulcer platform. the network also plans to implement activities such as clinical trials evaluating new treatments, assays validating new molecular diagnostic tools, and surveillance of amr. role of laboratories in disease surveillance in the eccas sub-region the information provided by the laboratory is critically important for disease surveillance and response programmes. for efficient management of an epidemic and its containment, a strong laboratory system should be operational before, during, and after the epidemic.17,18 before an epidemic, the laboratory collects early warning signals and identifies the aetiological agent. during the outbreak, the laboratory is involved in the response and management measures for the containment of the epidemic, and after the outbreak, the laboratory monitors disease trends, evaluates interventions, and monitors progress towards control objectives. apart from disease outbreaks, laboratories monitor microbial genetic changes of public health concerns, such as changes that confer amr in bacteria or changes in rna viruses (such as ebola or coronavirus) that lead to the emergence of genetically diverse strains (variants) with high pathogenicity or transmissibility.19 thus, during certain outbreaks, such as the ebola or coronavirus outbreaks, the aetiological agent must be laboratory-characterised to detect the emergence of variants and guide response decisions.18 detection of extremely dangerous pathogens, such as the ebola virus, requires higher biosafety level (bsl3 or 4) laboratories. bsl4 laboratories are built to ensure biosafety and biosecurity when studying class 4 pathogens; pathogens transmitted via aerosols or unknown mechanisms and are often lethal, without known treatment or vaccine to fight them. however, in the absence of a bsl4 laboratory such as in the democratic republic of congo, bsl3 laboratories, for studying class 3 pathogens, usually, viruses or bacteria that infect humans or animals through inhalation and could be lethal, with reinforced biosafety and biosecurity, have been used to diagnose ebola cases. africa experiences approximately 100 public health events every year, of which 80% are caused by infectious agents.14 many of these events involve extremely dangerous pathogens. for example, since 1994, the eccas region has regularly recorded ebola epidemics, mainly in the democratic republic of congo, the republic of congo, and gabon,20 which due to their ecosystems are at high risk of vhf.21 unfortunately, despite the high health risks evident in african countries, resources for epidemic surveillance and response such as laboratory capacity are mostly lacking. as of 2015, across the entire african continent, only three countries (nigeria, kenya and south africa) had fixed bsl3 laboratories, while only two countries had bsl4 laboratories22: one in gabon, centre interdisciplinaire de recherches médicales de franceville (cirmf), and the other in south africa. as at march 2021, the number of operational bsl4 laboratories on the continent has not much changed: two are under construction in south africa and côte d’ivoire23; the number of bsl3 laboratories has increased, particularly in the eccas sub-region. bsl3 laboratories were recently built, including the institut national de recherches biomédicales in the democratic republic of congo, the centre pasteur du cameroun in cameroon, the institut pasteur de bangui in the central african republic,14 and the bsl3 laboratory dedicated to the management of multidrug-resistant tuberculosis in the republic of congo.24 the presence of the bsl3 and bsl4 laboratories in some eccas countries is a great asset for the sub-region to effectively monitor and respond to epidemics. for instance, the bsl4 laboratory in the cirmf, gabon, actively surveils emerging and re-emerging diseases not only in gabon but also in the other eccas countries.25 the objectives assigned to the cirmf includes diagnosing suspected vhf cases, developing new diagnostic methods, monitoring deaths in animal reservoir hosts, and conducting laboratory techniques training at national, regional and international levels. the cirmf has established a research partnership with the national public health laboratory in brazzaville, republic of congo, and the institut national de recherches biomédicales in kinshasa, democratic republic of congo, to study infectious diseases transmitted by animals in the tropical rainforest regions of equatorial africa.25 capacities of the human health laboratories in eccas sub-region to comply with international health regulations in 2015, the who recommended that member states annually report their progress in implementing the revised international health regulations (2005 ihr)26 and conduct a self-assessment of their capacity, followed by a joint external evaluation (jee).26 the jee tool is developed using the who instruments as well as different strategies and initiatives including the global health security action programme and the oie tool for the evaluation of the performance of veterinary services (pvs).26 the jee assesses ihr capacities in 19 technical areas, grouped into four main themes: ‘prevention’, ‘detection’, ‘response’, and ‘entry points and other ihr risks’ (chemicals and radiation). a national laboratory system is one of the four technical areas of the domain ‘detection’. the 2005 ihr capacity scores are classified from level 1 (no capacity) to level 5 (sustainable capacity). in the eccas region, except angola and equatorial guinea, all countries have completed the jee of their public health capacity to meet the requirements of the 2005 ihr. as per figure 1, in the laboratory area, most eccas countries scored low in several indicators both in terms of disease and amr surveillance capacities.24,27,28,29,30,31,32,33,34 figure 1: country scores for laboratory capacity assessment in the eccas countries. capacities of the animal health and food laboratories in the eccas sub-region to comply with the international standards in animal health and food safety, the performance evaluation of the veterinary services has shown that laboratory reliability and quality assurance are major issues in most african countries.35 for eccas countries, evaluation or gap reports reveal that laboratory capacities are very low for disease and amr surveillance. these weaknesses are both qualitative (very low performance scores, around 1–2 for the majority of countries) and quantitative (often only one regional or national veterinary laboratory per country, rarely two) (supplementary table 1). table 1: role of the laboratory system in achieving the goals of the who’s global plan against amr. to achieve the goal of eliminating dog-mediated human rabies deaths by 2030, many african veterinary laboratories, including the laboratoire national vétérinaire (lanavet) in cameroon and the veterinary laboratory of kinshasa, democratic republic of congo, recently benefited from increased capabilities for rabies diagnosis. the staff were trained to diagnose rabies using the direct fluorescence antibody test and conventional real-time polymerase chain reaction at the food and agriculture organisation of the united nations rabies reference centre in italy.36 mobile laboratories as a solution to the lack of fixed laboratory infrastructures in sub-saharan africa, particularly eccas countries, the few bsl3 and bsl4 laboratories are located in large urban centres. thus, they could be located far from epidemic outbreaks or areas at risk of emergence or re-emergence of epidemics. in the case of an epidemic such as ebola, the safe delivery of samples to the laboratory, reliable diagnosis, and prompt communication of results are crucial for a successful response.37 a mobile laboratory can alleviate this problem by shortening the time required to obtain results. mobile laboratories circumvent fixed laboratory construction delays, particularly in times of emergencies, as they can be deployed almost immediately. this has been demonstrated in some countries in the sub-region. during the 2005 ebola epidemics in the democratic republic of congo and the 2007 marburg fever epidemic in angola, mobile laboratories confirmed suspected cases within 4 h, consequently facilitating the work of the contact-tracing team.38 more recently, a portable sequencer in one of the mobile laboratories was used to investigate the date of introduction and geographical origin of the zika virus in angola.39 however, field mobile laboratories are capitaland logistics-intensive, and are thus best suited for providing limited services for brief periods.18 therefore, it is necessary to develop additional fixed and sustainable bsl3 and bsl4 laboratories in the eccas. role of laboratories in amr surveillance overview of the challenges of amr surveillance the advent of antibiotic therapy, which began with the discovery by 1928 of penicillin, completely revolutionised medicine and significantly reduced infectious disease mortality and disability. the use of antimicrobials has also increased animal production by improving animal welfare. unfortunately, amr seriously undermines the hopes raised by the discovery of antimicrobials. antimicrobial resistance is considered one of the most significant threats to human, animal and ecosystems health. antimicrobial resistance is exacerbated by the overuse and misuse of antimicrobials; 30% – 50% of antimicrobial prescriptions in human medicine are unnecessary.40,41,42 in animal health, the irrational use of antimicrobials is compounded by the use of growth promoters in animals or the use of antimicrobials for metaphylaxis. some growth promoters contain antimicrobials of critical importance to human health.43 in addition, in veterinary medicine metaphylaxis is rampant; metaphylaxis is the administration of antimicrobials to a herd of animals to treat sick individuals and prevent the disease in healthy individuals. antimicrobial resistant microorganisms in animals can subsequently be transmitted to humans through direct or indirect contact.44 indeed, there is a much higher risk of human colonisation through cattle, pigs and poultry infected with methicillin-resistant staphylococcus aureus.45 furthermore, genetic determinants of amr can be transferred from commensal or pathogenic animal bacteria to pathogenic human bacteria. last resort antimicrobials used to fight multidrug-resistant infections such as fluoroquinolones, third generation cephalosporins or colistin are becoming ineffective globally. in parallel to the extensive misuse of antimicrobials, the discovery of new antimicrobials has become increasingly seldom. as a result, the feared therapeutic impasse is becoming increasingly real.46 according to projections, by the year 2050, amr will be the world’s leading cause of annual death, with 10 million deaths per year, ahead of cancers (8.2 million) or diabetes (1.5 million).47 also, africa and asia will likely be the most affected continents. this is why, on the margins of the 71st session of the united nations general assembly in 2016, the alarm bell was sounded on amr.48 on this occasion and for the first time, heads of states and governments came together to adopt a common approach to fight the causes of amr in human and animal health, as well as in the environment. in this noble fight, the laboratory has a prominent part to play. role of the laboratory in fighting amr the early symptoms of an infectious disease may be non-specific and may combine clinical signs of several infectious diseases. for example, the first manifestations of an ebola virus infection include fever, headache, myalgia, and gastrointestinal disorders.49 to increase the chance of effective antibiotic therapy, a common approach is to use broad-spectrum antimicrobials while waiting for antimicrobial susceptibility test results, which are not usually available before 72 h.50 this practice runs contrary to the goal of the who global plan to optimise antimicrobial use.51 this empirical usage selects for antimicrobial-resistant microorganisms. therefore, medication before laboratory diagnosis should only be used when the disease is life-threatening and, even so, microbiological sampling should be performed before treatment is initiated.52 the focus should be on the development of innovative rapid diagnostic techniques that allow clinicians to identify the pathogen in minutes rather than days.53 antibiotic susceptibility testing is a key indicator for the design of effective interventions and rational use of antibiotics.54,55 reporting of antibiotic susceptibility results by medical laboratories is necessary to monitor emerging resistances and develop appropriate antimicrobial stewardship guidelines.54,56 therefore, antimicrobial susceptibility testing capacity is essential.57 in the 2018–2023 amr framework,58 the africa centre for disease control and prevention, in collaboration with existing partners, aimed to increase laboratory capacity for the detection of resistant microorganisms in humans and animals. the who’s global plan of action defines five strategic objectives for amr containment.45 as per table 1, the laboratory system has a key role to play in achieving these goals, both in terms of clinical and public health activities.45,51,59,60,61,62 in the eccas countries, probably due to weak laboratory capabilities, there is a huge amr data gap from the human,63 animal and environmental health sectors.64 however, the literature suggests that substantial effort is being made to achieve some of the objectives of the who’s global plan against amr. scientific articles contribute to public awareness, understanding, and knowledge building on amr. in belgium, for example, as a result of national awareness campaigns, streptococcus pneumoniae penicillin resistance decreased from 18% in 2000 to 7% in 2009.65 thus, in the eccas zone, research and public awareness should be at the heart of global amr control strategies. in animal health, the lanavet in cameroon and the institut de recherche en elevage pour le développement in chad are major vaccine production laboratories in africa66 aimed at preventing infections, thereby reducing antimicrobial use in animals. next-generation sequencing capacity in the eccas region next-generation sequencing (ngs) offers the potential to provide more accurate and timely information, thereby increasing the likelihood of meeting the 2005 ihr recommendations. the ihr recommends that urgent events are reported within 48 h to determine whether an event is ‘notifiable’. this information will rapidly inform the necessary control measures to prevent national and international transmission. diagnosis and surveillance of pathogens are the core capacity of public health systems.67 the whole-genome sequencing is a leading technique in the response, not only to the ongoing coronavirus disease 2019 pandemic but to future emerging and re-emerging infections. in the eccas region, countries with ngs devices are gabon (four devices), the democratic republic of congo and rwanda (two devices each), angola and cameroon (one each),68 and the republic of congo and equatorial guinea (unknown number each).69 the illumina platform is by far the most used in these countries, followed by ion torrent and nanopore. iso 15189or iso 17025-accredited laboratories in the eccas region the who afro through the african society of laboratory medicine (aslm), implemented the stepwise laboratory management towards accreditation (slmta), to improve medical laboratories in africa.70 the slmta was launched in 2009 to improve the quality of public and private health laboratories in african countries to achieve iso 15189 standards accreditation. the framework to audit the implementation of the slmta in laboratories is the stepwise laboratory improvement process towards accreditation (slipta).70 until october 2021, there were only two eccas countries with iso 15189-accredited laboratories: cameroon had five accredited laboratories while rwanda had two.71 although cameroon scored very low on most capacity attributes in the jee (figure 1), it has the largest number of accredited laboratories in the eccas region. the likely explanation could be that these currently accredited laboratories were not included in the jee cohort or that they rigorously accelerated their certification process after the jee. the last decade has seen the emergence of several projects supporting laboratory systems in low-income countries, notably as part of the fight against priority diseases (malaria, hiv/aids and tuberculosis). these projects have positively impacted laboratory services. the accreditation of veterinary laboratories is subject to iso 17025 standards. by the end of the european union-funded central african quality infrastructure project (piqac) in 2019, two food safety laboratories had been audited and were in the process of iso 17025 accreditation.72 six others were in the process of capacity building for accreditation, including the microbiology laboratory in the centre de contrôle de la qualité des denrées alimentaires (cecoqda) in n’djamena, chad.72 as of march 2021, the cecoqda’s microbiology laboratory with the congolese office control laboratory in the democratic republic of congo and the africa improved food laboratory in rwanda were the few iso 17025-accredited laboratories in the eccas region. in addition, the lanavet in cameroon is considered a centre of laboratory excellence by the food and agriculture organisation of the united nations.73 this laboratory organises training sessions on animal disease diagnosis for technicians in the sub-region and provides african swine fever diagnostic services for chad. capacity building needs of laboratories in the eccas zone the eccas countries need to incite both medical and veterinary laboratories to register in iso 15189 or iso 17025 accreditation processes. accreditation assessments are snapshot measurements of laboratory compliance, creating the risk that the efforts may weaken after an assessment.74 therefore, for already accredited laboratories, the most important challenge is maintaining their status. according to 2017 forecasts, the world’s population is expected to increase by 2.2 billion by 2050, with 1.3 billion of this growth occurring in africa.75 predictions also suggest that health risks will increase dramatically in africa, with the endemic rate of zoonotic viruses more than tripling by 2070.76 consequently, health services, particularly laboratory services, will have to support this demographic growth and high risk of emerging infectious diseases by providing services at low cost while maintaining quality. given the low scores recorded and the few numbers of accredited and bsl3 and bsl4 laboratories, it appears that the eccas countries have weak laboratory capacities in both human and veterinary medicine and must be strengthened. building the laboratory workforce is another challenge that eccas countries face since laboratory work has long been down on the lists of priorities of most health ministries in the eccas region. the central africa’s regional integrated surveillance and laboratory network (rislnet) meeting, which took place in malabo in march 2019,77 revealed that only a few countries including burundi, the democratic republic of congo and cameroon have their laboratory policies and strategic plans drawn up and validated. the republic of congo and são tomé and príncipe laboratory strategic plans remain to be validated. the central african republic, chad, gabon and equatorial guinea have no laboratory policies and no laboratory strategic plans. for those with laboratory policies and strategic plans, implementing them remain a challenge due to budgetary constraints. capacity strengthening through laboratory quality enhancement the information provided by the laboratory must be accurate, timely and subjected to quality assurance procedures. in other words, laboratory results must be accurate. to this end, all aspects of laboratory activities must be reliable and the reporting of results must be correct to be used for clinical or public health purposes. the iso divides the various laboratory analysis activities into three processes: pre-analytical, analytical, and post-analytical. laboratory errors that may negatively impact patient management or public health policies occur at 32% – 75% in the pre-analytical phase, 13% – 32% in the analytical phase and 9% – 31% in the post-analytical phase.78 therefore, as shown in figure 2, capacity building strategies need to be designed to address all aspects of the laboratory analysis and organisation.79 figure 2: fishbone diagram for medical laboratory analysis showing the focal elements in the capacity strengthening strategy. the best way to strengthen capacity is to enrol laboratories within the who’s slmta process. this process allows a substantial improvement in the quality of laboratories even if they do not reach the end of the accreditation process.11,12,13,80 unfortunately, few countries in the eccas zone have engaged their laboratories in the slmta process. and even for those countries that have signed up, the number of both public and private laboratories involved in the process is very low. for example, between 2012 and 2019, only 16 public and private laboratories in cameroon were enrolled in slmta, but this country has 3279 public laboratories.81 also, only 1.34% of the 1113 public and private laboratories in burundi were engaged in the slmta process.81 to avoid a decline in performance, slmta-enrolled laboratories must continue follow-up performance and apply the lessons learned during the process and, most importantly, attract national political commitment.82 moving towards integrated laboratory systems and networks the maputo declaration called for the development of national laboratory policies and national strategic laboratory plans. the call prioritises laboratory systems in the national health development plan.2 an integrated laboratory network can provide all primary diagnostic services needed for the care and treatment of patients without requiring them to go to different laboratories for specific tests.83 in resource-limited settings, such as in some african countries, the who recommends four operational levels of laboratories to better provide services in a national laboratory network84: level i or primary (health post and health centre laboratories that primarily serve outpatients), level ii or district level (laboratories in intermediate referral facilities), level iii or regional or provincial level (laboratories in a regional or provincial referral hospital that may be part of a regional or provincial health bureau), and level iv or national or multi-country reference laboratory (reference laboratory for one or more countries). thus, laboratory levels are determined by their diagnostic platforms as well as their functions. the national reference level carries out the most complex tests. a tiered, integrated laboratory network should meet the following criteria83: (1) provide quality-assured basic laboratory testing, (2) collect the common specimens, report results timeously, and use diagnostic platforms to detect different diseases within the same facility, and (3) increase capacity for introducing and using new and more complex technologies. an integrated laboratory should have the capacity to adequately monitor people with hiv/aids for tuberculosis, malaria or other opportunistic infections. it should also provide rapid molecular tests for multidrug-resistant tuberculosis in patients co-infected with hiv and tuberculosis to improve infection control and treatment outcomes. therefore, the integrated network avoids wastage of already limited resources and the referral of patients outside the network for certain laboratory tests.83 in 1993, the who afro established an integrated laboratory network in 15 african countries, including cameroon, the central african republic and the democratic republic of congo, to support the global polio eradication initiative.15 the polio laboratories of these three eccas countries have also integrated measles, yellow fever, and rotavirus programmes. more recently, the africa centre for disease control and prevention established rislnet within defined geographic regions of africa including central africa.77 the rislnet aims to effectively support prevention, rapid detection, and response to current and emerging public health threats. the rislnet operates under the one health concept, integrating human and animal health laboratories and surveillance assets. the materialisation of the one health concept is critical to efficiently prevent and respond to public health threats. as 65% of the recent major epidemics in the world have a zoonotic origin,85 there should be no division between disciplines in the human and animal health sectors. the one health approach will enable the early identification of emergent zoonosis. this can be achieved through the simultaneous surveillance of both human and animal disease in integrated surveillance and laboratory systems or networks. the one health approach, thus, mutualises resources and cuts costs. this was illustrated in uganda and nigeria, where during avian influenza outbreaks, hiv/aids diagnostic laboratories provided diagnostic support for avian influenza cases.83 in the fight against emerging amr threats, a stronger laboratory system will allow the detection of resistance and provide data for better trend tracking and infection control. also, standardised isolates banks, which would result from such a system, would support the research for better diagnostics and treatment. limitations in response to the coronavirus disease 2019 pandemic, several countries must have increased their response capacity by improving their laboratory capacity and biosafety levels. the laboratory capacities reported here may not have considered all the newly acquired capacities. this study was based on data available online; thus, a country’s laboratory capacity may not necessarily have been the subject of a study published on the internet. within the framework of the regional disease surveillance systems enhancement project (redisse iv) currently underway in the eccas countries, an inventory of laboratory capacities is being conducted and will provide exhaustive data on laboratory capacities. conclusion laboratory services are an essential component of a health system. in africa, particularly in the eccas sub-region, the need for reliable laboratory systems is greatest due to the higher risk of vhf. unfortunately, from this review, it is evident that laboratory capacity for disease and amr surveillance and response is weak. indeed, the capacities of laboratories in eccas countries are weak given the who’s jee scores and the very limited number of high biosafety levels (bsl3 and bsl4) and accredited laboratories. there is, therefore, a pressing need to strengthen the laboratory capacities in the sub-region to cope with the risk of disease emergence, which is predicted to triple in the coming decades. acknowledgements we sincerely thank dr skander hathroubi (from humboldt university of berlin, germany) for his contribution by reading and correcting the first draft of the manuscript. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions p.v. was responsible for conceptualisation, methodology, formal analysis, investigation, writing of the original draft, visualisation, project administration, validation, resources, writing of the review, and editing and supervision. s.l. was involved in the investigation, visualisation, validation, writing, review and editing. l.f.t. contributed to the methodology, investigation, validation, writing, review and editing. j.f.d.s. was involved in the validation, data curation, writing, review and editing. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references leng ll, luke lsg. ‘without laboratories, men of science are soldiers without arms’. singapore fam physician. 2017;43(4):3–4. world health organization-regional office for africa. the maputo declaration on strengthening of laboratory systems. 2008; [homepage on the internet]. 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[cited 2021 feb 26]. available from: https://pdf4pro.com/amp/download?data_id=4b9409&slug=consultation-on-technical-and-operational-recommendations wendt a, kreienbrock l, campe a. zoonotic disease surveillance – inventory of systems integrating human and animal disease information. zoonoses pub health. 2015;62(1):61–74. https://doi.org/10.1111/zph.12120 abstract introduction methods results discussion acknowledgements references about the author(s) temitope o. obadare department of medical microbiology and parasitology, obafemi awolowo university teaching hospitals complex, ile-ife, nigeria department of medical microbiology and parasitology, faculty of basic clinical sciences, obafemi awolowo university, ile-ife, nigeria adeyemi t. adeyemo department of medical microbiology and parasitology, obafemi awolowo university teaching hospitals complex, ile-ife, nigeria department of medical microbiology and parasitology, faculty of basic clinical sciences, obafemi awolowo university, ile-ife, nigeria oluwaseun a. ibrahim department of medical microbiology and parasitology, obafemi awolowo university teaching hospitals complex, ile-ife, nigeria naheemot o. sule department of epidemiology, faculty of biostatistics and occupational statistics, mcgill university, montreal, quebec, canada center for clinical epidemiology, lady davis institute, jewish general hospital, montreal, quebec, canada mayowa m. adeyemo department of hematology, pinderfields general hospital, mid yorkshire nhs trusts, wakefield, united kingdom olusegun i. alatise department of surgery, faculty of clinical sciences, obafemi awolowo university, ile-ife, nigeria department of general surgery, obafemi awolowo university, teaching hospitals complex, ile-ife, nigeria citation obadare to, adeyemo at, ibrahim oa, sule no, adeyemo mm, alatise oi. bacterial agents and antibiotic resistance in febrile neutropaenia in africa: a systematic review and meta-analysis. afr j lab med. 2025;14(1), a2816. https://doi.org/10.4102/ajlm.v14i1.2816 note: additional supporting information is available for download alongside the online version of this article as online supplementary document 1. review article bacterial agents and antibiotic resistance in febrile neutropaenia in africa: a systematic review and meta-analysis temitope o. obadare, adeyemi t. adeyemo, oluwaseun a. ibrahim, naheemot o. sule, mayowa m. adeyemo, olusegun i. alatise received: 25 mar. 2025; accepted: 21 may 2025; published: 26 aug. 2025 copyright: © 2025. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: febrile neutropaenia (fn) is an oncology emergency, but there is a paucity of data on it in africa. aim: this study aimed to review and aggregate data on fn in the context of antibiotic resistance. methods: published original articles between 1991 and 2024 were systematically searched in google scholar, pubmed, and african journals online databases (grey literature excluded). ‘febrile neutropenia’ was combined by boolean terms ‘or’ and ‘and’ with individual countries for the searched terms. data aggregation on bacteria isolates and antibiotics was done using microsoft excel. results: of 16 637 articles retrieved, 15 (from nine countries) with 1216 non-duplicate isolates were included in the analyses after exclusion of irrelevant and duplicate articles. there were 57.0% (698/1225) gram-positive and 43.3% (527/1225) gram-negative bacteria. aggregated resistance to antibiotics for gram-positive bacteria was 71.8% (163/227), for ampicillin, 74.3% (226/304), for cefoxitin, 64.1% (25/39), and 54.0% (47/87) for oxacillin, while that of gram-negative bacteria was 35.5% (184/519) for ciprofloxacin, 60.6% (168/277) for ceftriaxone, 65.9% (89/135) for cefuroxime, and 38.2% (153/401) for imipenem. staphylococcus aureus had 68.8% (22/32) resistance to oxacillin/methicillin and 10% (1/10) resistance to vancomycin. klebsiella spp. was 50% (9/18) resistant to quinolones, 75.9% (22/29) resistant to third-generation cephalosporins, and 25.0% (4/16) resistant to carbapenems, while acinetobacter spp. was 85.7% (6/7) resistant to gentamycin. conclusion: this review highlighted the paucity of data and the emergence of multidrug resistance in fn in africa. there is a need for antibiotic-resistance surveillance and antibiotic stewardship to optimise therapy in fn in africa. what this study adds: to the best of our knowledge, this is the first systematic review of fn in africa in the context of available laboratory resources across the african regions. keywords: febrile-neutropaenia; bloodstream-infection; bacterial agents; multi-drug resistance; africa. introduction cancer is one of the leading causes of morbidity and mortality globally and the fifth leading cause of mortality in africa.1 according to rajesh sharma and colleagues using the globocan 2020 estimate, about 1.1 million new cancer cases occurred in 2020, with over 700 000 mortalities in africa.2 this figure might increase by 102% to 2.12 million annually by 2040, with an accompanying doubled mortality rate if nothing is done to reverse the trend.3 the lowand middle-income countries (lmics) bear the brunt of cancer death, with overall 70% of mortality occurring in these regions because of delayed diagnosis, poor health infrastructure for cancer support, scarcity of oncology specialists, exorbitant cost, or lack of access to anti-cancer chemotherapy.4 in 2011, the brazzaville declaration on non-communicable diseases prevention and control in the world health organization african region was adopted, where african countries acknowledged non-communicable diseases, including cancer, as public health issues of concern, but national cancer control plans have been poorly funded and coordinated.5 neutropaenia is an absolute neutrophil count of less than 1.0 × 109 cells/l in individuals of central european descent, or less than 1.2 × 109 cells/l in african people.6 this is a common phenomenon in patients with cancer because of complications of cytotoxic chemotherapy, bone marrow metastatic replacement, and disruption of haematopoiesis by malignant cells. febrile neutropaenia (fn) is defined as a single oral temperature greater than or equal to 101 °f (38.3 °c), or a temperature greater than or equal to 100.4 °f (38 °c) for at least an hour, with an absolute neutrophil count of less than 1500 cells/l.7 febrile neutropaenia is an oncological emergency associated with sepsis, high treatment cost, treatment delay, and dose reduction, with potential compromise of cure in the patient.8 the severity and incidence of fn are dependent on various factors such as the content of the therapeutic chemotherapy, the cycle of therapy, age of the patient, type of malignancy, and other comorbidities of the patients.9 the fn annual incidence in the united kingdom is 9.4 per 1000 oncology admissions.10 in the united states, the incidence of fn ranges from 7.83 cases per 1000 (solid tumours) to 43.3 cases per 1000 (haematological malignancies).11 the incidence of mortality is high in patients with fn, ranging from 50% to 75%.12 early appropriate prophylaxis and empiric antibiotic use have significantly contributed to reduced morbidity and mortality in patients with fn.8 however, the emergence of multidrug-resistant (mdr) bacteria from misuse and overuse of antibiotics has reduced the effectiveness of broad-spectrum antibiotic use in patients with fn.13 it is documented that many antibiotics used in cancer patients are inappropriate and do not follow evidence-based standard guidelines.14 moreover, the increased frequency and duration of contact with healthcare settings, breached anatomical barriers, and invasive procedures predispose patients with fn to healthcare-associated infections, which are mostly mdr pathogens, lower the survival rate in patients with fn.13 the world health organization predicts that the incidence of malignancy would increase by 48% by 2030 from the previous survey in 2018 in lmics. this estimate could be less than the actual value, as there is an underestimation of cancer in africa because of diagnostic capacity and cancer reporting.15 this would invariably increase the number of patients with fn in africa. unlike the developed countries, there is a dearth of data on the epidemiology of bacterial pathogens implicated in fn and their antibiotic susceptibility in africa, which is responsible for the lag in the development of treatment protocols in many healthcare facilities in many african countries. in addition, the role of mdr, as well as its implication in fn, has not been well investigated, despite the high burden of infections by mdr pathogens in africa, with challenges of poor infection prevention and control practices, indiscriminate antibiotic use, as well as inadequate diagnostic infrastructure. this study aimed to review the studies on fn conducted in africa, and aggregate the data to document the patients’ clinical parameters, implicating bloodstream bacteria pathogens and their pattern of antibiotic resistance. methods this systematic review was performed according to the guidelines provided by the cochrane handbook for systematic reviews of interventions and reported by the preferred reporting items for systematic reviews and meta-analyses guidelines.16 data sources and search strategy google scholar, pubmed, and african journals online library databases were systematically searched using relevant keywords (and indexed words) combined by boolean terms ‘or’ and ‘and’ to form a final search strategy. the search term for google scholar [‘febrile neutropenia or neutropenia sepsis and antibiotic or antibacterial or antimicrobial and sensitivity or resistance or susceptibility and the name of the individual african countries’], pubmed [‘febrile neutropenia or neutropenia sepsis and the name of the individual african countries’], african journals online library [‘febrile neutropenia or neutropenia sepsis and the name of the individual african countries’] were tailored to each database, with year of publication restrictions from january 1991 to december 2024 for optimised performance at pulling out original articles published in africa. moreover, the references of the identified studies were hand-searched for other relevant studies not pulled by the searched terms from the databases. selection criteria studies included in the synthesis of this systematic review were original observational studies published in peer-reviewed journals conducted in africa on fn (or infection in oncology patients with fn components), which included a minimum of blood culture results with or without susceptibility pattern, with availability of the full texts. review articles, letters, editorials, case reports, case series, conference summaries/abstracts, clinical trials and articles not published in the english language were excluded from the synthesis of this review. these selection criteria were predetermined by all the authors before the search. the study’s validity was ensured by strict compliance with the determined selection criteria. study identification and article selection a total of 16 637 articles were pulled out by the search engines from the searched databases. upon screening by titles, 16 570 were found to be irrelevant based on the predetermined parameters for this study. the remaining 67 articles were screened using mendeley citation manager software (elsevier, amsterdam, netherlands), and 16 were excluded for being duplicates. twenty-eight articles were further excluded after screening of the abstracts and subsequent sorting based on inclusion criteria by two independent reviewers (t.o.o. and a.t.a.). where there was disagreement, another reviewer (o.a.i) was brought in to break the tie. at the end of the process, 15 eligible articles were selected for the final synthesis. the details of the preferred reporting items for systematic reviews and meta-analyses16 process for the selection are documented in figure 1. figure 1: preferred reporting items for systematic reviews and meta-analyses flowchart describing literature search and identification of articles on febrile neutropenia in africa, january 1991 – december 2024 for synthesis. data extraction the relevant qualitative and quantitative data were extracted from the selected articles using a piloted microsoft excel worksheet software (microsoft corporation, redmond, washington, united states) individually by two investigators (t.o.o. and a.t.a.). qualitative parameters extracted were the study design, location, region of africa (based on regional grouping by the african union; https://au.int/en/member_states/countryprofiles2), duration of study (in months), study time frame, sampled population, blood culture system, and the technique for bacteria identification and antibiotic susceptibility pattern of the bacteria associated with fn. the quantitative data extracted were the number of patients, the number of isolates obtained from the blood culture, the proportion of blood culture positivity, volume of blood sampled for microbiology investigation, antibiotic susceptibility, and patient outcome. discrepancies in the data extracted were corrected by referring to the original studies. data analysis the data on isolates and antibiotic susceptibility patterns were aggregated from the selected articles and presented in proportions. risk factors for fn were not included in this study, because of the varied study designs of the eligible articles with non-comparable study outcomes. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. results description of eligible articles the article search done in february 2024 and yielded a total of 16 637 articles from the three databases (google scholar [n = 16 500; pubmed [n = 103]; african journals online [n = 34]), of which 13 eligible original articles were selected for analysis. an additional two articles17,18 were included after a manual search of the references of the 13 eligible articles, resulting in a total of 15 articles for the synthesis after the selection criteria were applied. the 15 eligible studies were from nine countries, including egypt (5), south africa (3), kenya (1), sudan (1), tunisia (1), ghana (1), morocco (1), ethiopia (1), and uganda (1) (figure 2). the studies were conducted between 1991 and 2013 (figure 2). figure 2: distribution and number of eligible articles about febrile neutropenia in africa (january 1991 to december 2024) across african union member states. regions shown are as defined in the organisation of african union in 1976 (cm/res. 464qcxvi). the majority of the studies were prospective (10 studies; 66.7%) (supplementary table 1), conducted at a single centre (14 studies; 93.3%) (data not shown in table). many of the eligible articles had university teaching hospitals as study locations (seven studies; 46.7%). four studies (26.7%) were from cancer institutes and specialist hospitals. six studies (40%) were conducted among paediatric patients only, and among both paediatric and adult patients, while two studies (13.3%) were conducted among adult patients alone. nine studies (60.0%) were conducted among a patient population with both haematological and solid tumours, and two studies (13.3%) were from a population of patients with stem cell transplantation. medical microbiology laboratory methods the medical microbiology laboratory methods used were stated in many of the studies; 10 studies stated the blood culture system used (automated blood culture system bactec/bact alert, n = 7, and manual/conventional, n = 3). ten studies stated bacterial identification methods used (conventional biochemical method only, n = 1; vitek, n = 2; phoenix, n = 1; analytical profile index [api], n = 2; sensititre, n = 1; microscan, n = 2; bbl [becton dickinson bioscience] crystal id, n = 1). antibiotic susceptibility testing in 40% (6/11) of the studies was by disc diffusion methods (supplementary table 1). bacteria agents the review included 2062 patients, 2106 febrile episodes and a positive blood culture rate of 22.3% (1270/5675) (table 1). a total of 1216 non-duplicate isolates were recovered from the selected studies, of which 57.0% (698/1225) were gram-positive and 43.0% (527/1225) were gram-negative isolates. the most common isolates were coagulase-negative staphylococcus spp. (cons; n = 281; 2.9%); staphylococcus aureus (n = 153; 12.5%); klebsiella spp. (n = 126; 10.3%); escherichia coli (n = 99; 8.1%); streptococcus spp. (n = 87; 7.1%); acinetobacter spp. (n = 74; 6.0%) and pseudomonas spp. (n = 69; 5.6%). table 1: blood culture isolates from 15 studies on febrile neutropaenia in africa, 1991–2024. antibiotic susceptibility patterns antibiotic susceptibility patterns were reported in 11 studies (supplementary table 2). of these studies, four reported the antibiotic susceptibility patterns based on the isolated organisms tested,18,20,25,27 four articles reported the antibiotic susceptibility based on gram reactions of the isolated organisms,17,19,24,28 and three studies reported the proportion of resistance to tested antibiotics only without disaggregation-based isolated organisms.22,23,29 the overall rates of antibiotic resistance to antibiotics used for gram-negative bacteria included ertapenem (1.9%; 1/52), meropenem (2.1%; 3/142), cefepime (32.8%; 141/430), piperacillin/tazobactam (40.9%; 133/325), tobramycin (4.8%; 3/63), ciprofloxacin (35.5%; 184/519), and colistin (12.5%; 7/56). the pulled antibiotic resistance rate for antibiotics used for gram-positive bacteria included amoxicillin/clavulanic acid (71.8%; 163/227), vancomycin (2.8%; 7/321), clindamycin (50.7%; 37/73), and linezolid (2.8%; 1/30). antibiotic resistance profile for the most prevalent bacteria to the tested antibiotics, 66.7% (8/12) of cons isolates were resistant to oxacillin/methicillin, mostly susceptible to vancomycin (resistance rate 5.3%; 2/38), and susceptible to linezolid (table 2). in the same manner, s. aureus isolates were highly resistant to oxacillin/methicillin (68.8%; 22/32), and were mostly susceptible to vancomycin (10.0%; 1/10). klebsiella spp. isolates were mostly resistant to ampicillin (93.8%; 15/16), piperacillin/tazobactam (85.7%; 6/7), and third-generation cephalosporins (75.9%; 22/29), and a quarter (4/16) showed resistance to the carbapenems. escherichia coli showed similar high resistance to ceftazidime (72.7%; 8/11), and other third-generation cephalosporins (66.7%; 24/36). non-fermenters such as acinetobacter spp. showed resistance rates of between 50% and 100% to tested antibiotics, including gentamicin and ciprofloxacin. however, pseudomonas spp. showed low resistance to ciprofloxacin (7.7%; 1/13). table 2: antibiotic resistance to commonest isolates in febrile neutropaenia in africa, january 1991 to december 2024. discussion febrile neutropaenia is a medical emergency with attending consequences of morbidity, mortality and economic burden on both the patients and healthcare systems globally. the consequences of fn are further made difficult to deal with as the evolution of antimicrobial resistance escalates in lmics amid a dilapidating healthcare system, complicated with layered drivers of antibiotic overuse and limited/absolute access to recommended newer antibiotics appropriate for treating mdr infections.31 while various international guides on the management of fn have been published, many lmics are yet to develop their local guidelines. this may hamper the management of fn in these resource-constrained settings, where management of fn infections depends largely on empiric antibiotic therapy without recourse to the local epidemiological data and laboratory diagnostic capacity. the burden of fn in africa might not be fully described because of the paucity of data from this region. this review highlighted the low data output on fn compared to other regions, such as high-income countries, where oncology practice is more advanced, and is prioritised to be included in their health-benefit package for the public health sector, as there was no eligible study for this review in the last decade from africa.32 in addition to this, there is skewness to the data on fn in africa, as the north africa region and east and southern africa were equally represented with seven eligible articles, and west and central africa region had one eligible article, which reflects on the quality of health investment and sophistication in healthcare systems available from these regions.1 this review also corroborates the already documented health disparity divide across the human development index that exists between high-income countries and lmics in healthcare systems and deliveries.32 there is a need for more composite data on fn in africa through research, funding, and improved transparent health systems, in addition to data infrastructures to bridge the cancer care inequalities.1 blood cultures are a highly dependent test for the investigation of bloodstream infections, including fn in clinical microbiology laboratories.33,34 however, the procedure is limited by frequent contamination, as up to a third to half of all blood cultures are judged to be contaminants by infectious diseases physicians. the principal source of the contaminants is from the dermis commensals such as cons, corynebacterium spp., bacillus spp, micrococcus spp., and cutibacterium spp. because of poor disinfection of the skin.35 in this review, cons (9 of 15 studies) accounted for over a fifth (23.1%) of the total isolates, which is similar to the contamination rate (23.0%) reported by rupp et al.36 this may underscore the issue of contamination, which is a major problem in blood culture tests. while blood culture is the ‘gold standard’ for the investigation of blood stream infections, its accuracy hinges on effective compliance with the aseptic techniques through the preand analytical stages of the procedure (i.e. the selection of body site for sampling, aseptic cleaning of the venipuncture site, use of trained phlebotomist and downing of personal protective equipment).35 these underpin the need for specialised training for blood culture sample collection to improve the quality of diagnostic stewardship in the diagnoses of fn, isolating the pathogen involved to ensure targeted therapy for the treatment and improve treatment outcome in fn. six out of the 10 eligible studies for this review made use of an automated blood culture system, while the others (four studies) used ‘manual’ blood culture systems. the ‘manual’ blood culture system reduces the culture yield of the investigation test and negatively impacts the promptness of the isolation, antibiotic susceptibility patterns, including timely and appropriate commencement of targeted therapy, as well as epidemiological surveillance of pathogens involved in fn.34 the uses of automated blood culture systems in africa are usually restricted to major referral centres or reference laboratories, which are mostly powered by external collaboration from high-income countries. while the availability of the ‘automated’ blood culture system had significantly helped in the investigation of fn in lmics of africa, its sustainability remains in doubt after the funders’ collaboration because of the costly required regular equipment maintenance, complicated consumable logistics, and supply chain,37 as well as compatibility of this equipment with the tropical environment and infrastructural architecture.1,34 clinical laboratory standards institute and european committee on antimicrobial susceptibility testing guidelines on antibiotic susceptibility have stipulated specific antibiotics that are relevant for testing against specific bacteria. however, this was not followed in all cases. this could be because of the common use of a generic multidisc – a single-paneled antibiotic disc for gram-negative and gram-positive organisms, which might not accommodate the unique approach offered by a single antibiotic disc as stipulated by antibiotic susceptibility guidelines for the testing of organisms such as acinetobacter spp, pseudomonas spp. and streptococcus spp.38 this review revealed that the clinical outcome of fn can be improved in africa by increasing the diagnostic capacity of the laboratories infrastructures to isolate bacterial in bloodstream infection in fn, continuous training of the laboratory staff to improve their competencies to optimise blood culture testing, accurate pathogen identification, and antibiotic susceptibility testing to inform local policy development as well as adherence to international guidelines for comparable practice and result.39 the epidemiological distribution of bacteria in fn varies with geographical location, medical procedures, and antibiotic consumption in the locality. in this review, the significant variability across decades was not obvious. gram-positive bacteria were the most isolated (56.7%) organism in this review, which is similar to the pattern documented over six years (1995–2000)40 in the united states, with a gram-positive bacteria rate of 61.0% and a significant shift from gram-negative to gram-positive bacteria over two decades in europe (1985–2000).41 this is in contrast to what was documented in central america (from 2012 to 2014) where the most isolated group of bacteria in fn were gram negative (78.0%),42 as well as in india (from 2012 to 2014; 57.8%) and iran (from 2019 to 2020; 66.7%). the reasons for the predominance of gram-positive bacteria in africa in this review are unknown. however, it could be because of quinolone prophylaxis43 use in this setting, choice of mucositis-producing chemotherapy agents, as well as use of invasive devices such as central-line catheters and significant use of antacids.44 the use of prophylactic antibiotics (especially fluoroquinolone) has reduced the overall mortality in patients with an intermediate-to-high multinational association for supportive care in cancer fn risk index. however, it is cautioned against by the european organisation for research and treatment of cancer and the american society of clinical oncology, especially in cases with a low multinational association for supportive care in cancer fn risk index (≥ 21), because of the risk of emergence of mdr.45 the aggregated resistance rate to ciprofloxacin and amoxycillin/clavulanic acid (which are the initial empiric antibiotics for low-risk patients with fn) from this review is more than a third (ciprofloxacin) and about two-quarters (amoxycillin/clavulanic acid) of the isolates, and implies that these antibiotics might not be effective empiric therapy for fn in many africa settings because of the prevailing high antibiotic resistance situation driven by increasing antibiotic consumption in the hospital and community, as support for a failing, fractured heath system and antibiotic stewardship intervention.31,46 the use of cefepime and piperacillin/tazobactam as a monotherapy or as part of combined antibiotics for initial empiric therapy in high-risk patients with fn needs caution when escalating for treatment of fn, as two-fifths of the isolates were resistant to piperacillin/tazobactam and a third to cefepime. this underscores the need for optimised medical microbiology support for appropriate blood culture, pathogen isolation and determination of antibiotic susceptibility patterns to guide the escalation and de-escalation of antibiotic use in fn.47,48 commonly isolated gram-negatives in fn in this review, such as klebsiella spp, and e. coli, had high resistance to piperazilin/tazobactam, third-generation cephalosporins, and even last-result drugs such as carbapenems. in addition, there is a high prevalence of methicillin resistance in gram-positive cocci; however, vancomycin is still effective, as only about 10% of s. aureus isolates, and about 20% of cons, are resistant to vancomycin. the pattern of antibiotic resistance distributed across the spectrum of bacterial pathogens isolated in the fn from this review revealed that antibiotic resistance, especially to last-resort antibiotics such as carbapenems in gram-negative bacilli organisms, is more widespread. this is a huge concern that the mdr organisms are increasingly being implicated in fn, and this poses a significant challenge to treatment in such patients. in the light of this, especially with increasing morbidity and mortality because of carbapenem resistance in enterobacterales and non-glucose-fermenting gram-negative bacilli, there is an increasing role in the use of beta-lactams (and non-beta-lactams) in combination with beta-lactam-inhibitors such as ceftazidime-avibactam, ceftolozane-tazobactam, imipenem/relebactam, and others. there is, however, a challenge regarding access to this newer combination of antibiotics in lmics because of their high cost, and limited data on their use for fn in such climes.46 therefore, it is imperative to prioritise prevention of infection in patients with neutropaenia by modification of intensity of cytotoxic medication, addition of myeloid-growth-factor, systematic selection of patients for chemoprophylaxis, colonisation surveillance for mdr, and proper infection prevention and control.46,47,49 limitations there is an inherent limitation with systematic review, as its outcome is based on an aggregation of the secondary data from synthesised original articles. it was observed that some eligible articles did not follow the antibiotic susceptibility guidelines to select the antibiotics tested on the bacterial agents. moreover, in some cases, the breakpoints, the panels of antibiotics for some bacteria, had changed over time. these made the articles heterogeneous and presented challenges to getting a common denominator for the antibiotic susceptibility results for some analyses. however, these limitations did not nullify the essence of this study to report the situation analysis on the state of fn in africa in the context of antibiotic resistance. conclusion this review highlighted the paucity of data on fn in africa, which corroborated the health disparity in cancer treatment across the economic divide of the nations. our findings underscore the need for good laboratory infrastructures as well as correct blood culture technique in investigating fn to ensure isolation of true pathogens and appropriate ‘drug-bug’ combinations based on standard antibiotic susceptibility guidelines to reflect the appropriate epidemiological picture for the development of local guidelines in managing fn in africa. there is a high level of mdr strains among the organisms isolated in fn in this review, which makes a case for continuous need for antimicrobial resistance surveillance, improved laboratory capacity and stewardship. there is also a need for the review of international guidelines and suggested antibiotic prophylaxis and empiric therapy for fn in the light of local epidemiological findings. there might be a need for improved access to newer agents, such as beta-lactam-beta-lactamase inhibitors, in africa to treat fn caused by mdr, causing resistance to last-resort antibiotics (such as carbapenems) for improved outcomes in fn patients. this review should encourage further studies on bacterial agents of fn in this clime to generate needed pieces of evidence to support improvement in medical microbiology laboratory capacity for the diagnosis of infections in fn to inform local empirical guidelines and definitive therapy for optimised management of cancer patients. acknowledgements we acknowledge taiwo ogunbiyi for his role in facilitating the logistics during the data collection and literature search for this manuscript. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions t.o.o. and o.i.a. conceived and conceptualised this review. t.o.o., a.t.a., n.o.s., and o.a.i. searched the literature, and collated the data. the article was drafted by t.o.o., a.t.a., n.o.s., o.a.i., and m.m.a. o.i.a. provided leadership and critically reviewed the article. t.o.o., a.t.a., o.a.i., n.o.s., m.m.a., and o.i.a. agreed on the final draft of the article. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability the authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials. disclaimer the views and 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environmental repositories compliant biorepository outcomes of implementation measures context, successes, challenges and future plans acknowledgements references about the author(s) alash’le g. abimiku institute of human virology nigeria, abuja, nigeria institute of human virology, university of maryland, baltimore, maryland, united states talishea croxton institute of human virology nigeria, abuja, nigeria institute of human virology, university of maryland, baltimore, maryland, united states petronilla j. ozumba institute of human virology nigeria, abuja, nigeria ndidi agala institute of human virology nigeria, abuja, nigeria olasinbo balogun institute of human virology nigeria, abuja, nigeria emmanuel jonathan institute of human virology nigeria, abuja, nigeria enzenwa onyemata institute of human virology nigeria, abuja, nigeria kachimi ndifon institute of human virology nigeria, abuja, nigeria sunji nadoma institute of human virology nigeria, abuja, nigeria thankgod anazodo institute of human virology nigeria, abuja, nigeria sam peters institute of human virology nigeria, abuja, nigeria christine m. beiswanger coriell institute for medical research, camden, new jersey, united states independent contractor, philadelphia, pennsylvania, united states citation abimiku ag, croxton t, ozumba pj, et al. blueprint for building a biorepository in a resource-limited setting that follows international best practices. afr j lab med. 2019;8(1), a722. https://doi.org/10.4102/ajlm.v8i1.722 lessons from the field blueprint for building a biorepository in a resource-limited setting that follows international best practices alash’le g. abimiku, talishea croxton, petronilla j. ozumba, ndidi agala, olasinbo balogun, emmanuel jonathan, enzenwa onyemata, kachimi ndifon, sunji nadoma, thankgod anazodo, sam peters, christine m. beiswanger received: 21 nov. 2017; accepted: 22 mar. 2019; published: 28 aug. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: genetic diversity is abundant on the african continent. however, genomic research has been hampered by a lack of high quality and extensively annotated biospecimens and the necessary infrastructure to support such a technology-intensive effort. objective: the institute of human virology nigeria (ihvn) partnered with the h3africa consortium and the coriell institute for medical research to build an internationally recognised biorepository for the receipt, processing, storage and distribution of biospecimens for biomedical research. here, the authors describe the procedures, challenges and results encountered. results: key requirements for a high-quality biorepository were identified: (1) institutional support of infrastructure and services, (2) on-site trained staff with primary commitment to the biorepository, (3) reliance on best practices from globally recognised biorepository groups, (4) early implementation of a quality management system, (5) adoption of a laboratory information management system with demonstrated versatility in functions, (6) collaboration with external experts and sharing of experience through abstracts, newsletters, published manuscripts, and attendance at meetings and workshops, (7) strict adherence to local and national ethical standards and (8) a sustainability plan that is reviewed and updated annually. conclusion: utilising published best practices of globally recognised experts in the biorepository field as a benchmark, ihvn expanded and reorganised its existing laboratory facility and staff to take on this new purpose. keywords: biorepository; biobank; international guidelines; best practices; developing country. introduction although the african continent is rich in genetic diversity that informs the search for determinants of disease, genomic research in africa has lagged due to shortfalls in trained scientists and the necessary infrastructure. the hallmark of genomic research in more developed regions has been high-capacity computing networks and the existence of large collections of well-annotated biospecimens of exceptional quality and integrity in biorepositories that strictly adhere to best practices prescribed by leaders in biorepository science such as the national cancer institute, united states,1 the international society for biological and environmental repositories (isber)2 and the public population project in genomics and society (p³g).3 in 2010, a partnership between the national institutes of health (nih; united states) and the wellcome trust (united kingdom) fostered the development of the human heredity and health in africa (h3africa) initiative for genomic research to be led by african scientists. it would involve the training of young african scientists and the establishment of resources to study the interplay of complex genetics and environment in disease susceptibility, progression, and treatment.4 one unique and important component of the h3africa consortium is the establishment of regional biorepositories for the collection, quality control (qc), and future distribution of samples and associated data to researchers, using best practices, as new insights into genomic medicine are gained. the three regional h3africa biorepositories are: the institute of human virology nigeria (ihvn) h3africa biorepository (i-hab) in abuja, nigeria, serving western africa; clinical laboratory services in johannesburg, south africa, serving southern africa; and the integrated biorepository of h3africa uganda (ibrh3au) in kampala, uganda, serving eastern africa.5,6,7,8,9,10,11 the ihvn, the largest non-governmental organisation in nigeria providing hiv and tuberculosis prevention, care and treatment programmes under the us government president’s emergency plan for aids relief programme and the global fund partnered with the coriell institute for medical research, camden, new jersey, united states, in 2012 to achieve international biorepository recognition by adopting the best practices, in particular those of the isber.1,2,3 this was to be accomplished through a combination of staff training, auditing by an independent expert consultant, renovation and acquisition of additional dedicated storage space, and upgrading of critical equipment and information systems. the principal goal of i-hab is to provide reliable sample processing support, secure storage and shipping of high quality biological samples. here we report on the processes, successes and challenges of establishing such a biorepository in the context of a resource-limited setting. the knowledge gained from this undertaking can inform the establishment of laboratory facilities serving a variety of clinical and research purposes in lowand middle-income countries, with a committed staff, creative problem-solving and careful planning as we have shown. strategy and planning for an international society for biological and environmental repositories compliant biorepository ethical considerations ethical clearance was obtained from the national health research ethics committee, approval number (nhrec)/biob/01/02/2018 (22 march 2018–22 march 2020). institutional support for staffing and infrastructure to establish the new biorepository, renamed i-hab, a professor-level laboratory director, management staff and three technical staff were reassigned from the ihvn to i-hab. in addition to funding the initial renovation and expansion of the existing laboratory and storage facilities based on the audit reports, ihvn committed resources and staffing for maintaining and securing the facility, and for information technology support. readiness assessment the i-hab conducted a number of baseline assessments of its readiness to operate according to international biorepository practices using the isber self-assessment tool.5 the isber checklist evaluated the biorepository’s records, management reviews, organisation and personnel, client management, equipment, internal audit, purchasing and inventory, information management, process control and external quality assurance, corrective actions, occurrence and incident management, and facilities and safety (figure 1). the assessment analysis received from isber was incorporated into a remediation plan and the categorised outcomes and overall score were used to monitor progress. figure 1: flowchart of the process to bring ihvn h3africa biorepository to international biorepository standards. activities undertaken by the institute of human virology nigeria-h3africa biorepository to achieve compliance with biorepository ‘best practices’ are shown in coloured boxes: dark aqua are initial activities and final outcomes; pink are activities undertaken by ihvn h3africa biorepository staff; medium blue are activities carried out by the independent consultant; yellow are major policy determinations. additionally, the world health organization stepwise laboratory quality improvement process towards accreditation (slipta) checklist6 was used to ensure compliance with iso 15189 standards suitable for i-hab’s added role in supporting clinical sites. the slipta checklist assessed management, security, accuracy, record keeping, and standard operating principles (sops) but from a slightly different perspective that augments the isber self-assessment tool. independent biorepository consultant in partnership with the consultant from coriell, i-hab proposed a strategy of cyclic assessments and remediation to identify areas of improvement and to bridge identified gaps. during several trips to i-hab, the consultant worked with i-hab to identify disparities between existing ihvn practices and the widely accepted practices of biorepositories serving international studies. once the gaps were identified, plans for improvement were drawn up and implemented. figure 1 captures the overall process to upgrade the practices at i-hab using an audit tool developed by the consultant as detailed in table 1. the audit tool addressed areas of operation important to the mandate of the h3africa biorepositories. for example, qc of dna was included, but not processing of formalin-fixed tissue samples. table 1: audits by the independent consultant show incremental improvement. operations requiring immediate remediation received a score of 3; necessary, but not critical, improvements received a score of 2; strongly suggested improvements scored 1 and acceptable ‘as is’ practices received a score of 0. scores were totalled in each operational area and subtracted from the possible number of points in that area and a percentage of the possible score calculated (figure 2). the full report was used as a reference to address non-conformities prior to the next audit site visit, and to track improved compliance with isber best practices over time. figure 2: audit topics and results provided by ihvn h3africa biorepository consultant. this graph presents a quantitative assessment of ihvn h3africa biorepository performance on each external audit by the independent consultant. horizontal axis: proportion of the maximum possible score. vertical axis: major operational areas examined during each audit. adoption of a quality management system the i-hab already followed the principles of a quality management system (qms) in line with its role as the biorepository for other ihvn research studies. however, the following critical quality components were enhanced. external quality assurance scheme enrolment in the integrated biobank of luxemburg (ibbl)7 external quality assurance scheme meant dna extraction, quantification, and purity assessment from whole blood was performed annually. after verification of results, ibbl issues certification of proficiency. so far, i-hab has received a certificate of satisfactory performance from ibbl for all four annual reports since 2015. continuous quality improvement regularly scheduled meetings for review of all operations and planning activities are fundamental to reach and sustain quality. i-hab has three categories of review meetings. first is the weekly meeting of all i-hab staff, second is a bi-weekly meeting with the director, technical advisors and ihvn top management, and third is an alternating bi-weekly meeting with other regional h3africa biorepositories when the sponsors participate. in addition, i-hab staff attended the bi-annual h3africa consortium meetings to ensure an appreciation of the broader consortium’s opportunities and challenges, to learn from each other’s experiences and to develop consortium-wide strategies. collaborations with other h3africa investigators9,10 facilitated i-hab’s internal review and mastery of unique biobanking needs. training/mentoring training in biorepository principles and practices was provided to different categories of staff as necessary: the i-hab management staff, technical staff, support staff from other ihvn departments, and research staff at h3africa-supported clinical sites. training consisted of didactic presentations and discussions of biorepository policies and practices, interactive development of cost of service schedules, and wet labs. all training materials (presentation slides, review versions of sops, electronic links to background materials) were made available to participants for future use. implementation of a biospecimen inventory and laboratory information management system the i-hab adopted freezerworks (©2015, dataworks development inc., mountlake terrace, washington, united states) as a biospecimen inventory system. freezerworks is a commercially available laboratory information management system (lims) with good technical support, user-defined fields and reports, easy modifiability and frequent upgrades by the company to remain current with new technologies and features of a biorepository lims. it is able to track qc and distribution of samples as well as in-house inventory. additionally, freezerworks is compatible with most data sharing protocols and automated data capture for laboratory equipment. ethics and governance of banking of biological samples the i-hab serves as the ‘custodian’ of biospecimens stored at its facilities. the i-hab can return biospecimens to the submitter, redistribute or share them, use them for research, or dispose of them only upon authorisation by the original submitter of the biospecimens or an authorised agent. the governance of the h3africa biospecimens deposited with i-hab is guided by approved informed consents for the various studies and the policies of the h3africa consortium. sustainability planning and costing of biorepository services the nih funding supporting the h3africa biorepository programme will eventually cease and so planning for continuation of i-hab is crucial. sustainability of i-hab will require a multipronged approach with excellent revenue tracking and careful management of partnerships with commercial enterprise. the i-hab developed a costing model to determine the actual costs of its services. this tool can be used for a fee-for-service scheme ensuring that pricing does not fall short of covering its operational costs. for each service, a direct cost based on labour plus reagents and supplies and pro-rated cost of instrumentation was calculated. labour included technical and management time at a ratio of 90:10. services were costed by individual sample or by a minimum batch size. an ‘overhead cost’ as a percentage of the direct cost and a mark-up cost that would cover contingencies and future service development were added. this mark-up was considered to be negotiable for i-hab to remain competitive and depending on the potential customer’s resources or opportunity for collaborative efforts. outcomes of implementation measures staffing and management organisation the i-hab is guided and directed by a director who benefits from input from an advisory committee and technical experts to set goals in accordance with the i-hab and ihvn missions, the h3africa objectives and client requirements, as well as to develop and strengthen policies and procedures, and provide mentorship. a biorepository manager has the primary responsibility of organisational management, overseeing the qms and safety, ensuring activities are conducted ethically, contributing to the development and maintenance of a sustainability model, and training. two scientists with expertise in molecular biology were employed in light of the anticipated increase in biospecimen processing and qc associated with supporting h3africa projects. these positions as well as others are shown in i-hab’s organisational chart (figure 3). all staff members are cross-trained and involved in the overall qms activities, in addition to attending internal and external meetings, presenting at workshops and trainings, contributing to document creation and review, preparing manuscripts, and ensuring security of the facility, biospecimens and data. figure 3: ihvn h3africa biorepository organisational chart and staff responsibilities. the ihvn staff positions shared with ihvn h3africa biorepository are indicated by the purple outline; solely ihvn h3africa biorepository staff by the aqua outline; university of maryland, baltimore by the red outline; dual ihvn/umb by the double red outline; technical advisor and consultant by white boxes. biorepository infrastructure and security infrastructure the 1309 ft2 i-hab facility shown in the black boxes in figure 4 was expanded to 2077 ft2 to improve workflow and separation of processes, in anticipation of 100 000 dna samples to be processed and stored each year for h3africa projects in west africa. the green and red boxes in figure 4 depict the completed two expansions. major re-organisation created the following laboratory spaces: (1) a general biospecimen processing laboratory, (2) archival rooms, (3) dna processing room, (4) rna processing room, and (5) amplification room. facility upgrades include: restructuring of the manager’s office, installing epoxy flooring to prevent spills from being trapped and to enable floors to be sanitised, installing additional power and internet outlets to support more equipment and prevent usage of extension cords, elevating electrical outlets from the base of the floor as a safety precaution, installing a generator with a second one as backup to supplement the public power supply, and installing two inverters and numerous uninterruptible power supply devices to ensure constant power and to minimise power fluctuations. the two expansion spaces accommodate nine ultra-low temperature freezers, two refrigerators, three liquid nitrogen tanks, and one auto-fill liquid nitrogen tank. overall, i-hab utilises about 7 983 360 watts of power each week. only 58.3% of the necessary power is provided by the public power grid; the rest (41.7%; 70 hours per week) is provided by backup generators with an automatic switch during local power grid outages. the backup to ensure 24/7 power has been utilised for a total of 20 993 hours since 2012, when i-hab began its journey to achieve international best practices, until early 2016, when this manuscript was put together. the power generators are tested at full capacity for 72 hours once a year to ensure uninterrupted service in case of prolonged power outage. furthermore, backup plans include supply of liquid nitrogen or off-site storage of critical biospecimens in case of prolonged power outage. figure 4: expansion of ihvn h3africa biorepository infrastructure to support an international biorepository. the initial 1309 ft2 biorepository space is shown in the black outline of the left of the figure. this space was reconfigured and a 768 ft2 cryogenic room was added (green outline). a second expansion (red outline) added 773 ft2 of secure biorepository storage space. security written policies, sops, and upgrades were implemented to ensure that there are processes and procedures established that minimise risk for security breaches and dictate response in case of emergency. biometric devices (zkteco, tangxia town, dongguan, china) and closed circuit television cameras (swann communications, santa fe springs, california, united states) and monitors were installed to prevent unauthorised access and improve staff, biospecimen and data safety and security (figure 5). automatic data backups were initiated both to off-site servers and on-site external drives to prevent data loss. the i-hab provided formal training in emergency response to all ihvn security staff charged with 24-hour security of i-hab. figure 5: security installations at institute of human virology nigeria h3africa biorepository. (a) biometric access control panel. (b) motion-activated closed circuit television camera. (c) monitor display from closed circuit television camera showing both interior and exterior areas of biorepository. self-assessment results the i-hab scored 66.9% at baseline in december 2012 using the isber self-assessment tool (table 2). comments returned from isber are described in column 2 of table 2 and issues identified were addressed with mentorship from the consultant for a second isber self-assessment conducted in january 2014, when i-hab scored 88.2%. following remedial action to again address the issues raised, a final isber self-assessment conducted in july 2015 had an improved score of 95.0% (table 2). this underscores the need for continuous self-assessment and improvement to ensure quality and adherence to international best practices especially in light of newly added activities or processes. table 2: international society for biological and environmental repositories self-assessments show incremental improvement from 2012 to 2015. an assessment was also performed by ihvn laboratory quality assurance officers in february 2013 using the world health organization slipta checklist.6 while the score of 76.3% (table 3) is not directly comparable to the isber self-assessment tool score, it does suggest improvement in the management and operational activities of i-hab over the previous year’s score of 66.9%. table 3: world health organization laboratory self-assessment. audits of policy and practices by independent consultant beginning in october 2012, the coriell consultant implemented a schedule of rigorous formal audits of biorepository activities during three on-site visits. each audit was followed by a detailed report, which the staff used to address gaps identified during the audit (table 4). the initial assessment by the independent consultant indicated that i-hab exhibited reasonably good compliance (70%) with the best practices recommended by isber and nih in the areas of management, specimen processing, information technology systems, client and collection site interactions, informed consent and ethical reviews, and policies for distribution of biospecimens (figure 2). each subsequent audit exhibited an improvement in scores (81.9% in may 2013 and 93.6% by may 2015), suggesting that the remedial activities and formal biorepository training were effective tools for improving biorepository operations (table 1). the ethics and distribution areas were in total compliance from the onset of the h3africa phase i, because i-hab had been operating in these areas in earlier contracts from united states government funded grants11,12 where i-hab followed all the ethical requirement of the contracts. table 4: major deficiencies during formal audits. in summary, over the course of two and a half years, i-hab had demonstrated consistently improving operation as a biorepository in achieving international best practices. a summary of the major deficiencies found during the independent consultant audits is captured in table 4. there were 14 significant non-conformances at the first audit in october 2012. half of these were resolved by the second audit in may of 2013 and only two critical gaps remained by the third audit in may of 2015. both issues, automatic notification of staff regarding temperature conditions in biospecimen storage units, and a plan to regularly document perfect restoration of the biospecimen database from a backup copy, relied on external suppliers. changes/enhancements to quality management system general biorepository operations shipping policies, practices, forms and sops were written as generic documents for i-hab and the h3africa consortium before being customised for specific projects. included in the documents prepared to assist submitters in the deposition of biospecimens are: (1) sops on sample preparation, labelling, shipping conditions, form preparation, and communication with the biorepository, (2) material transfer agreement templates that can be adjusted to meet the regulatory requirements of the submitter’s country, (3) pre-shipment checklists to ensure that all supplies and forms are available and that all arrangements have been established with the biorepository and with the chosen shipping courier, (4) a shipment manifest form indicating the sample type, sample identification number and accompanying minimum sample essential data, (5) a shipment notification form to inform the biorepository of the shipment date and other arrangements, and (6) a shipment receipt confirmation and query form used to address any discrepancies in the shipment. in addition, more than 150 policy documents, sops and forms were created or revised in compliance with best practices and in response to site assessments and client needs. all documents include information on unique identification, versioning, review and sign-off by staff, and are in a document inventory for tracking. to more effectively update and review newly created and revised documents, meetings were held where authors presented the document to staff in an interactive session. questions and other feedback were addressed before adoption of documents. staff training and continuing education formal training sessions were provided to all i-hab staff as detailed below and in table 5. preand post-training assessment quizzes were used to determine the success of the training. staff were also re-evaluated annually. training included: table 5: summary of i-hab and ihvn support staff training of ihvn h3africa biorepository staff training. basic laboratory operations: good laboratory practices and workflow, specimen management, quality management, quality assurance, quality control, quality assessments, continuous quality improvement, sop writing, and safety. introduction to biobanking: biospecimen receipt, processing, aliquoting, labelling, storage, retrieval, transport, dna extraction, quality control methods, international air transport association regulations, data management, documentation and record keeping. biorepository management workshop: expanded training on some of the above topics, including accreditation processes, biorepository best practices, biorepository services, cost modelling for services, disaster planning, critical samples, stakeholders, funding and establishing policies. modern research bioethics: history of research ethics and research regulation; legal, moral and philosophical foundations of modern bioethics; informed consent, exploitation; benefits, inducements and compensation for research injuries and vulnerable populations; ethics committees; types of ethical review; scientific integrity and research misconduct; evolution of and process for ethical review in nigeria. bioinformatics course: attended by the information technology officer, this course included linux operating systems, python programming, advanced dataset management with linux, population and statistical genetics and next generation sequence analysis. grants manager course: participation in a three-week residency training component of biomedical research administration development at nih. the biomedical research administration development programme is designed to: help participants to navigate and understand the nih structure and programme operations, introduce participants to nih grant policy and compliance requirements, and provide an overview of the knowledge base and tools for building a strong research administration support and management infrastructure at the participant’s institution. freezerworks: the i-hab’s bioinformatics specialist, laboratory manager, and technical advisor attended freezerworks update training sessions provided by the manufacturer. step-down training was provided to all i-hab technical staff who were invited by dataworks as beta tester for freezerworks 2015. this experience provided an excellent opportunity for the staff to suggest feedback to the manufacturer. information technology and laboratory information systems as mentioned in the methods section, the i-hab uses freezerworks to manage biospecimen and associated data. there have been several upgrades since freezerworks was initially installed at i-hab in 2012. the most recent version, freezerworks 2015 ascent (©2015, dataworks development inc., mountlake terrace, washington, united states), is a networkable system with web access that enables tracking of samples from biospecimen collection through storage and distribution. all processes carried out on a biospecimen, such as testing, shipping, freeze-thaw cycles and other frequently required biospecimen modifications, were documented. the security feature was used to assign authorities for specific activities like viewing, modifying, adding data, as well as administrative duties such as customisation of the program. all activities are tracked by an audit trail and can be traced to the individual user. other useful functions used were import, export, and batch update of data, and generation of reports in excel, word or pdf. the i-hab purchased a temperature monitoring system to permit remote monitoring of all ultra-low temperature freezers and for automated remote alarm notification in case of a temperature excursion beyond preset limits (figure 6). the smartvue (thermo fisher scientific, waltham, massachusetts, united states) remote temperature monitoring and alert system did not work in nigeria due to incompatibility with the existing network system operating in the country, but the tutela (tutela monitoring systems, hampshire, united kingdom) worked. the system provided mechanisms for: digital visual readings, web accessible readings and graphs for remote monitoring in real time, and email and text alerts that enable the staff to respond. i-hab established an on-call schedule and response communication tree to ensure that staff members monitored and responded to emergencies during nights, weekends and holidays as well. in addition, ihvn security guards were trained in appropriate response protocols and have 24-hour access to respond to audible temperature alarms in the i-hab facility and log their responses. figure 6: remote temperature monitoring. features of the smartvue temperature system are shown. (a) digital temperature display available on individual ultra-low temperature freezers. (b) continuous temperature readout for each freezer accessible on web. (c) an email alert is sent whenever the temperature of an individual freezer exceeds limits set by biorepository management. governance and ethical considerations the national health research ethics committee (nhrec)13 has guided i-hab in developing practices that are in line with the national code of health research ethics of nigeria. regular interactions with members of nhrec and the h3africa funded research projects on ethical, legal, and society issues and the guidance documents produced by these groups has enabled i-hab to carry out its operations ethically according to the regulatory and social norms of nigeria and the other african countries it serves. the nhrec visited i-hab to observe and inspect its practices before licensing it to function as the first nigerian biorepository meeting the special requirements of the functions of a biorepository. sustainability and costing the i-hab sustainability plan is a multipronged approach to reduce or contain current costs. contracts were established with couriers and vendors whenever possible. i-hab management identified consumables and equipment required for expansion and sustainability and investigated vendors that provided those products and support to nigeria, those with offices in african countries and others that were well-known international third-party distributors. contracts for discounted pricing and services were negotiated with thermo fisher scientific and dna genotek (dna genotek, ottawa, ontario, canada) and discussions are ongoing with others. the i-hab negotiated prices and services for transport of biological materials. couriers were compared for their ability to transport biospecimens between the h3africa collection and processing sites and the i-hab. the duration and cost of the shipments as well as the provision of supplies for ambient, refrigerated and frozen shipments were assessed. due to varied and high costs associated with dry ice supply and replenishment required for some biomaterials, credo reusable shippers (pelican biothermal, llc, plymouth, minnesota, united states), equipped with robust insulation to maintain temperatures of −20 oc to −45 oc for many days were preferred. the i-hab also developed a model for pricing all biorepository services that was flexible enough to adapt to both rising costs for labour and supplies and that could respond to individual circumstances of potential clients. the exhaustive list of services offered by i-hab would permit potential clients to customise their contract to include only the minimal requirements for safe and secure processing and storage, or the high-end quality control and customised reporting. the mark-up percentage was adjusted up or down to reflect potential collaborative efforts or other benefits that i-hab might realise as a result of the contract. context, successes, challenges and future plans the advent of genomic medicine has underscored the need for mechanisms to make large cohorts of biospecimens and their associated datasets available to researchers across a variety of biomedical fields.4,14,15,16 these ‘data-driven’ research efforts are supported by the thousands of biospecimens stored in biorepositories. however, despite its burden of disease, africa has lagged in the development of biorepositories to provide high-quality samples for collaborative research across biomedical disciplines. african biorepositories have tended to be focused on single diseases in narrowly defined populations and for short periods of time. some operate as partners in united statesor european-funded collaborative studies17,18,19,20 of specific diseases. others are part of hospitals or academic centres that have restrictions on the use of biospecimens collected and stored for specific studies or clinical use.21,22,23,24 like most institutions in africa, ihvn had created a biorepository network to support its funded clinical and research activities related to hiv, tuberculosis, and hiv-associated malignancies in 2009. training in clinical protocols and biorepository activities was provided through its collaboration mainly with the institute of human virology, university of maryland school of medicine, baltimore, united states.11,12 the biorepositories had the ability to extract peripheral blood mononuclear cells, and extract dna from whole blood, qc the samples, store, and ship some of samples for investigative analysis at institute of human virology, university of maryland school of medicine using policies and practices specifically mandated by the individual investigator’s research. with the advent of the h3africa and expectation to process, ensure the quality of, provide long-term storage for, and distribute biospecimens for international genomic studies, it became necessary for ihvn to scale up its in-house biorepository operations to achieve international best practices. this upgrade of policies and practices ensures that biospecimens passing through i-hab meet the stringent needs of international biomedical research. this is essential to fulfil i-hab’s mission to promote population and personal health, by facilitating cutting-edge research and collaborations among african communities and globally, through the provision of high-quality, affordable biobanking services, while maintaining the integrity and confidentiality of these samples as an honest custodian. achieving international expertise in biobanking required a substantial revision and the development of a more complex and broader appreciation of biorepository operations. in terms of both selfand external assessments, i-hab’s performance increased significantly by around 23% from 2012 to 2015 due to improvements including but not limited to staffing, infrastructure, process, and security. the process of achieving international best practices revealed a number of contributing strengths and some serious challenges. however, to obtain such success it was important that the senior management of ihvn and i-hab were fully committed and willing to obtain the necessary resources to support the development and growth of the biorepository that will outlive h3africa. this prompt engagement of management staff is a major contributing factor to the success of i-hab and was the hallmark for sustainability. for example, ihvn provided financial resources for the acquisition and remodelling of the laboratory and cryogenics facilities, information technology services, security systems and staffing, training for i-hab staff in clinical laboratory practice and other infrastructure operations. likewise, i-hab strengthened its own expertise in biorepository operations by offering training courses to other ihvn staff and staff at clinical sites supported by ihvn and other investigators, thus creating a cycle of capacity building and sustainability. it is important to note that many of the laboratory skills and practices necessary for the operation of a clinical laboratory are also essential biorepository activities. i-hab had the advantage of a thorough grounding in quality management systems, safety protocols, and equipment maintenance and validation through pepfar. hence the appreciation of strict adherence to sops and documentation of activities was an advantage, as well as well-developed bench skills and the application of universal precautions for the handling of human biospecimens, which all became very useful when training and mentoring clinical sites who subsequently shipped biological specimens to the biorepository for storage. the interaction with h3africa researchers and experts, through the h3africa consortium and two other regional h3africa biorepositories,25,26,27,28,29,30,31 has been invaluable in developing international policies and practices now in place at i-hab. a number of collaborative manuscripts from the h3africa biorepository working group have highlighted several relevant concepts, including the role of genomics in african health, how biorepositories in africa directly support african genomics research, the value of piloting biorepository operations across national boundaries and the complex governance of ‘sharing’ biospecimens for secondary research.28,29,30,31 although i-hab has successfully developed an isber-compliant biorepository, there were a number of challenges that it had to overcome to achieve this. firstly, resource limitations with regard to infrastructure, especially uninterrupted electrical power and internet access, have been especially challenging to the development of i-hab: power outages eventually shorten the lifespan of freezer compressors and other instrumentation, the cost of fuel to operate the backup generators fluctuates and must be factored into the expenses of running the repository, the interruption of internet access by power failure exerts a substantial toll on both communications and operations, and modern science is becoming increasingly reliant on international web-based conferences and electronic data transfers, which are all dependent on reliable power and internet. secondly, technical support from international suppliers can be responsive and timely or it can be expensive and slow or even non-existent. obtaining the necessary technical help takes assertiveness, persistence and consistent follow-up. thirdly, acquiring the necessary laboratory supplies and instrumentation can also be expensive and experience delays. materials that are a quick online catalogue order in a developed country often require additional permits or other paperwork for imports and are likely to have additional costs associated with their delivery and installation. fourthly, compliance with ethical, legal, and social understandings and regulations also presents challenges to biorepository activities. thus far, i-hab has followed ethical practices specified by the nigerian government,13 (http://www.nhrec.net/nhrec/nchre_aug%2007.pdf) and its current contractors, nih as published by the office for human research protections (available at http://www.hhs.gov/ohrp/humansubjects/index.html), and the h3africa consortium (http://www.h3africa.org/consortium/documents). a full discussion of these issues is beyond the scope of this report, but additional insight can be found in other published articles.32 fifthly, i-hab has experienced major delays (up to 6–9 months) in obtaining ethical and government approval of material transfer agreements and import permits from a minority of partnering countries. on occasion the wording of a document will need to be changed, but in other cases, there is no existing body of law. it is essential that all transfers of biospecimens to and from a biorepository begin with a careful consideration of ethical, legal, and society issue actions and engagement to be taken and ensure the earliest possible application for required permits. finally, reliance on external funding support is increasingly uncertain in a climate of increasing demands to cut government spending. many funders of research infrastructure in africa (such as h3africa funders made up of the united states nih and the wellcome trust) emphasise grants and contracts designed to support the initiation of a programme with a built-in sustainability plan for that reason. to address this, a fee-for-service model has been developed at i-hab to support future biorepository activities. after projecting costs for labour, supplies, equipment maintenance and replacement, and overhead, a biorepository needs to add a mark-up to generate revenue for future investments in infrastructure upgrades and cost-of-operating increases. while academic and institutional-industrial partnerships are often considered to be a stable source of revenue, they are subject to the constraints of changing business goals. in addition, there are concerns about maintaining a strict ethical and logistical separation of biospecimens collected for ‘commercial’ programmes and those collected for ‘open collections’ accessible to the research community with ethical review and informed consent ensuring respect for patient confidentiality and privacy and a few other restrictions on the type of research. the most feasible approach to sustainability of an independent biorepository of quality will most likely involve a combination of the approaches that circumvent the challenges described above and that incorporate a strong and flexible business model. conclusion we have described the procedures, challenges and outcomes encountered in upgrading a study-specific sample storage facility to an internationally recognised biorepository supporting researchers globally with a focus on west africa. during the process, key requirements for successful biobanking were identified: (1) institutional support for development of infrastructure and technical services, (2) scientific management staff on-site with primary commitment to the biorepository, (3) reliance on best practices from globally known biorepository groups, (4) early development and implementation of a qms, (5) adoption of a lims with demonstrated versatility in user-defined fields and reporting functions and interoperability with other lims systems, (6) formal collaboration with external experts and sharing of experience through abstracts, newsletters, and published manuscripts, (7) strict adherence to local and national ethical guidelines, and (8) a sustainability plan that is reviewed and updated annually. the i-hab biorepository at ihvn is now ideally positioned to utilise internationally recognised best practices to receive, process, store, and ethically redistribute high-quality biospecimens for research, innovation, discovery, and diagnostics for several years to come. acknowledgements the authors acknowledge the institutional support from the institute of human virology nigeria, the suggestions and advise from the h3africa consortium members, and cooperation from the clinical sites of h3africa projects supported by the ihvn h3africa biorepository (i-hab). competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions a.g.a. and c.m.b. applied for the funding for this project and drafted the manuscript. t.c. provided managerial oversight of the biorepository and substantial contributions and revisions to the manuscript. p.j.o. supervised all aspects of the biorepository operations, including acquisition and installation of equipment, and revised the manuscript. n.a., o.b., e.j., e.o., k.n., s.n. and t.a. carried out all biorepository operations, including sample receipt, processing, storage, data management, and quality control, and revised the manuscript. s.p. coordinated i-hab activities within ihvn and revised the manuscript. k.n. contributed to the initial upgrade of the biorepository according to best practices. all authors gave final approval of the version to be published and agree to be accountable for the accuracy and integrity of the work. sources of support the research reported in this publication was supported by the national human genome research institute of the national institutes of health under award number uh2hg007008 and uh3hg007008 (pi: alash’le abimiku). data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institutes of health. references national cancer institute national institutes of health u.s. department of health and human services, nci best practices for biospecimen resources biorepositories and biospecimen research branch, 2016. international society for biological and environmental repositories. best practices for repositories: collection, storage, retrieval and distribution of biological materials for research international society 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https://doi.org/10.4102/ajlm.v8i1.743 original research morphological patterns of anaemia among pregnant women from sudan abuobieda b. abusharib received: 21 dec. 2017; accepted: 11 jan. 2019; published: 14 oct. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: morphological patterns of anaemia in pregnancy are considered essential for classification, diagnosis and management of patients, especially in regions with high maternal mortality like sudan. objectives: this study evaluated morphological patterns of anaemia among pregnant women in sudan and morphological differences across characteristics of participants. methods: this cross-sectional study was conducted from september 2016 to february 2017. a total of 200 women were selected according to specific criteria. laboratory tests were performed for complete blood count, blood smears were performed for morphology and vitamin b12, folate and iron levels were measured. participants were classified as: normochromic normocytic, microcytic hypochromic, macrocytic or dimorphic. further classification based on haemoglobin levels was also performed. results: a total of 116 participants (58%) had a dimorphic pattern, followed by 50 participants (25%) with a microcytic hypochromic pattern, 20 participants (10%) with a macrocytic pattern and 14 participants (7%) with a normochromic normocytic pattern. participants with the dimorphic pattern also had low levels of iron and folate. the majority of dimorphic participants presented with mild anaemia, whereas the majority of participants with the microcytic hypochromic pattern presented with moderate or severe anaemia. a high percentage of participants in late pregnancy had the dimorphic pattern, and there were significant differences in the degree of anaemia by parity, gestational age and regular intake of haematinic supplements. conclusion: the most frequent morphological pattern of anaemia in this study was dimorphic, followed by microcytic hypochromic, macrocytic and normochromic patterns. morphological patterns appeared to predict types of vitamin and mineral deficiency and the degree of anaemia. keywords: anaemia; pregnancy; morphological; pattern; clinicopathological. introduction anaemia affects more than 55% of women in sudan; most of them are pregnant.1 according to world health organization (who) guidelines, anaemia in pregnancy is defined as a haemoglobin level under 11 gm/dl for the first and third trimester and under 10.5 gm/dl for the second trimester.2,3,4 both red blood cell (rbc) mass and plasma volume expand in pregnancy, reaching the maximum level in the second trimester. however, the expansion of 35% – 40% in plasma volume exceeds the 20% – 25% increase in rbc mass; as a result, there is a dilutional drop in haemoglobin concentration, haematocrit and rbc count. additionally, there is a 2to 3-fold increase in iron requirements and 10to 20-fold increase in folate requirements.5,6 in africa, nutritional deficiency is a common cause of anaemia. three factors contribute to the pathogenesis of vitamin and mineral deficiencies in pregnancy. firstly, both the growing foetus and maternal tissues use the entire maternal stores of minerals and vitamins (i.e. there is an increase in demand). secondly, in developing countries, there is a lack of vitamin and mineral supplement use or inadequate food intake during pregnancy. thirdly, folic acid and vitamin b12 absorption are usually impaired during pregnancy. infectious diseases, such as malaria, helminths and hiv, besides poor quality of health services, poverty and ignorance, indirectly participate in the aetiology of anaemia in pregnancy.5 anaemia during pregnancy is one of the leading causes of maternal and foetal morbidity and mortality in almost all developing countries.6 even moderate bleeding in an anaemic pregnant woman could be a risk for preterm delivery. moreover, foetal growth restriction and low birth weight are increased when haemoglobin drops. also, maternal anaemia adversely affects cognitive, behavioural and physical development in infants. anaemia also depresses immune status and increases morbidity from infections in neonates. in 2011, the who published a new classification for anaemia in pregnancy based on haemoglobin concentration and gestational age as follows: in mild anaemia, haemoglobin concentration is 10.00–10.99 g/dl for the first and third trimester, and 10.49–10.99 g/dl for the second trimester. in moderate anaemia, the haemoglobin concentration is 7.00–9.99 g/dl regardless of trimester, whereas in severe anaemia, the haemoglobin concentration is less than 7.00g/dl irrespective of trimester.6 based on the morphology of rbcs and blood cell indices, anaemia is classified into normocytic normochromic, microcytic hypochromic, macrocytic and dimorphic anaemia. each type suggests specific aetiological factors, so an evaluation of the morphology of rbcs and clinical features among pregnant women could help in the diagnosis and management of patients. however, there is little research on the characteristic morphology of rbcs in anaemic pregnant women in sudan.1,7 the objective of this study was to evaluate the morphology of rbcs among anaemic pregnant women and examine differences in morphological features across some clinical characteristics of study participants. methods ethical considerations khartoum north teaching hospital research committee issued an ethical approval for conducting this study (study approval number: 14/2016). written consent was obtained from the participants, and a high level of protection was taken to maintain confidentiality of the collected data and usages for the study only. study design this was a cross-sectional study conducted at khartoum north teaching hospital in sudan from september 2016 to february 2017. two hundred anaemic, pregnant women attending antenatal clinics in the hospital were included, after fulfilling specific selection and exclusion criteria. to be included in the study, participants had to be anaemic (according to who 2001 classification of anaemia in pregnancy) and have no history of recent transfusions, bleeding or chronic disease. data collection a questionnaire was designed to collect information on age, parity (primigravida: first pregnancy, multigravida: 2–4 pregnancies, or grand multipara: ≥ 5 pregnancies), gestational age (first trimester, second trimester, third trimester), level of education (illiterate, did not complete primary school, completed primary school, or above primary school) and economic status (annual income in us dollars, very poor: < 700, poor: 700–1400, enough: 1400–2800, good: > 2800), in addition to marital status (married, separated, or divorced), past medical history, haematinic supplement use (yes or no) and other obstetrical information. furthermore, laboratory data were collected, including complete blood count, rbc morphology, iron profile, rbc folate and serum vitamin b12 level. statistical analysis the collected data were transferred to the statistical package for social sciences (spss) computer program (ibm® spss® statistics 19; ibm company, philadelphia, pennsylvania, united states). the mean age ± standard deviation, distribution and frequencies of the characteristics were calculated, in addition to differences in the severity of anaemia among morphological patterns, using the chi-squared test. p-values less than 0.05 were considered significant for differences in the degree of anaemia among categories of parity, gestational age, haematinic levels and regular intake of haematinic supplements. laboratory analysis nine ml of venous blood was collected and divided into three containers: ethylene diamine tetraacetic acid (edta), lithium heparin and clear containers. roche/hitachi cobas systems (cobas 6000 analyzer series, 2012 june; roche diagnostics corporation, indianapolis, indiana, united states) were used for measuring serum iron, iron binding capacity, serum ferritin, serum vitamin b12 and rbc folate. a quality control protocol was implemented, using normal, low and high range control sera. additionally, complete blood count was measured using sysmex kx20 automated analyser, manufacturer by sysmex corporation, september 2015 (bellport, new york, united states), and direct peripheral smears were taken to be compared with ethylene diamine tetraacetic acid (edta) smears; both types of smears were stained with leishman’s stain and examined under the microscope for rbc morphological classification. classifications of the study based on complete blood count parameters, mean corpuscular volume (mcv), mean corpuscular haemoglobin (mch) and mean corpuscular haemoglobin concentration (mchc) and their correlation with the morphology of smears in which the size of rbcs was compared to the nucleus of lymphocytes, the participants were grouped into four patterns of anaemia: microcytic hypochromic, macrocytic, normocytic normochromic and dimorphic (mixed macrocytic and microcytic). also, both red cell distribution width and mcv were compared to rbc morphology. the normal reference range was 11% – 15% for red cell distribution width, 76–96 fl for mcv, 27–33 picograms for mch and 33–36 g/dl for mchc. consequently, mcv less than 76 fl suggested microcytosis and (low mcv, low mch and/or mchc) indicated a microcytic hypochromic pattern, confirmed by morphology, while mcv greater than 96 fl with normal or elevated mchc suggested macrocytic pattern, also confirmed by morphology. however, normal mcv, mch and mchc indicated a normocytic normochromic pattern, confirmed by morphology. the dimorphic pattern was suggested by the presence of mixed macrocytic and microcytic pattern in morphology and dual population in rbcs histogram, while mcv could be normal, low or high. participants were also categorised as having mild, moderate or severe anaemia, according to who guidelines for haemoglobin concentration in pregnancy. in mild anaemia, haemoglobin concentration was 10.00–10.99 g/dl for the first and third trimesters and 10.49–10.99 g/dl for the second trimester. in moderate anaemia, the haemoglobin concentration was 7.00–9.99 g/dl regardless of trimester. similarly, in severe anaemia, the haemoglobin concentration was less than 7.00 g/dl irrespective of trimester. to simplify the analysis across some variables, participants were further categorised by considering participants with moderate or severe anaemia in one group, participants who were multigravidas or grand multiparas in another group and second or third trimester participants in a third group. results participants’ mean age (± standard deviation) was 28.5 years (± 7.1 weeks), and the age range was 17–46 years, 44% of participants had not completed primary school and 39% were illiterate, 25% had very poor income and 60% had poor income (table 1). table 1: distribution of socio-demographic and clinical variables, sudan, september 2016 to february 2017. morphological patterns of anaemia based on morphology and blood cell indices, 116 (58%) participants had the dimorphic pattern, 50 (25%) had the microcytic hypochromic pattern, 20 (10%) had the macrocytic pattern and 14 (7%) had the normocytic normochromic pattern (figures 1 and 2). figure 1: distribution of morphological patterns of anaemia, sudan, september 2016 to february 2017. figure 2: photos of morphological patterns of anaemia, sudan, september 2016 to february 2017. (a) hypochromic microcytic, (b) macrocytic pattern, (c) normochromic normocytic and (d) dimorphic pattern. dimorphic (microcytic and macrocytic) pattern representing 116 participants (58%), dimorphic was the most common morphological pattern of anaemia in our study; 74 participants (63.8%) had mild and 36 (31%) had moderate anaemia, and only 6 (5.2%) had severe anaemia. moreover, 76 participants (65.6%) with the dimorphic pattern were multigravidas, and only 20 participants (17.2%) were primigravidas. the majority of participants (102; 88%) were in their third trimester. in this group, 96 participants (82.8%) had a low iron profile, 90 (77.6%) had low folate and 16 (13.8%) had low vitamin b12 levels (tables 2 and 3). table 2: morphological patterns of anaemia across participant characteristics, sudan, september 2016 to february 2017. table 3: difference in morphological patterns of anaemia with iron profile, folate, vitamin b12 levels, sudan, september 2016 to february 2017. microcytic hypochromic pattern the microcytic hypochromic morphological pattern of anaemia was the second most frequent with 50 participants (25%). of them, 26 (52%) had mild anaemia and 20 (40%) had moderate anaemia. the distribution of parity in this pattern revealed 28 (56%) were multigravidas, 10 (20%) were grand multiparas, and only 12 (24%) were primigravidas. additionally, 24 (48%) participants were in their last trimester. none of the participants with this pattern had taken routine haematinic supplements during pregnancy. all participants with this pattern had a low iron profile, normal rbc folate and normal serum vitamin b12 (tables 2 and 3). macrocytic pattern the macrocytic pattern was the third most common, representing 20 participants (10%). all 20 were in the third trimester and 16 of them (80%) had mild anaemia. none had taken routine mineral or vitamin supplements during their pregnancy (table 2). fourteen (70%) had low vitamin b12 and 16 (80%) had low folate levels (tables 2 and 3). normocytic normochromic pattern the normocytic pattern was the least common, with 14 participants (7%). all were regularly taking mineral and vitamin supplements. all participants had only mild anaemia. moreover, all had a normal iron profile, normal serum vitamin b12 and normal rbc folate levels (tables 2 and 3). degree of anaemia across variables based on the haemoglobin level, 130 participants (65%) had mild anaemia, 60 (30%) had moderate anaemia and 10 (5%) had severe anaemia (table 4). a total of 96 participants (73.9%) in the mild anaemia group presented in the second or third trimester, whereas 34 (26.1%) participants were in the first trimester. however, 60 participants (85.7%) in the moderate or severe anaemia group were in the second or third trimester, and 10 (14.3%) participants were in the first trimester; the difference between the two groups was significant (p = 0.025). analysis of the degree of anaemia and parity showed 100 participants (76.9%) in the mild anaemia group were multigravidas or grand multiparas, whereas 30 participants (23.1%) were primigravidas. however, 64 participants (91.4%) in the moderate or severe anaemia group were multigravidas or grand multiparas, and only 6 participants (8.6%) were primigravidas; the difference between the two groups was significant (p = 0.034). table 4: differences in the degree of anaemia with gestational age, parity and haematinic supplement intake, sudan, september 2016 to february 2017. the analysis of the degree of anaemia and regularity of intake of haematinic supplements showed that 104 participants (80%) in the mild anaemia group were not regularly taking haematinic supplements during pregnancy, whereas 26 participants (20%) were taking haematinic supplements daily. on the other hand, 66 (94.3%) participants in the moderate or severe anaemia group were not regularly taking haematinic supplements during pregnancy, and only 4 (5.7%) were taking them daily. the difference between the groups for severity of anaemia with regularity of haematinic supplement intake was significant (p = 0.026). analysis of the degree of anaemia with vitamin and iron levels showed that 102 participants (78.5%) in the mild anaemia group had low serum iron, 76 participants (58.5%) had low folate and 10 participants (7.9%) had low vitamin b12, while 53 participants (88.4%) in the moderate anaemia group had low serum iron, 23 (38.4%) participants had low red cell folate and 8 (13.3%) participants had low vitamin b12. however, 8 participants (80%) in the severe anaemia group had a low iron profile, 7 (70%) had low red cell folate, and all had normal levels of vitamin b12. the difference between the two groups was significant (p = 0.031). on the other hand, neither rbc folate nor vitamin b12 levels showed significant differences with the degree of anaemia (p = 0.222 for rbc folate level and p = 0.112 for b12 level) (table 5). table 5: differences in the degree of anaemia with iron and vitamin levels, sudan, september 2016 to february 2017. discussion generally, the study participants had low socio-economic status, which agrees with what was stated by nirmala et al.,8 and zaheer et al. in india.9 also, 39% of the entire group were illiterate; this was consistent with findings of melku et al. from ethiopia,5 but it was a little higher than reported in another ethiopian study by lelissa et al.10 twenty-five per cent of the participants were divorced or separated, which is consistent with what was reported in india.11 these findings suggest that socio-economic status could be an early predictor or an indication of anaemia among pregnant women in sudan. additionally, an increasing prevalence of high parity among participants was observed (82% of the participants were multigravidas or grand multiparas). similarly, 78% of the participants were in late pregnancy (third trimester). this observation is consistent with the findings of amardeep et al.12 it indicates that the higher the parity, or the more advanced the pregnancy, the higher the likelihood of having anaemia; however, it can be explained by the depletion of mineral and vitamin stores in repeated pregnancies and increasing demands of late pregnancy. morphological patterns of anaemia in pregnancy seem substantially diverse across the globe, because of the diversity in aetiological factors. this study showed 58% of participants with a dimorphic (microcytic and macrocytic) pattern, followed by 25% of participants with a microcytic hypochromic pattern, 10% of participants with a macrocytic pattern and 7% of participants with a normocytic normochromic pattern. low socio-economic status and multi-nutritional deficiency (based on low vitamin and iron levels) in the participants may explain the increased prevalence of the dimorphic pattern among them; comparable results were found in malawi13 and palestine.14 surprisingly, melku et al. in neighbouring ethiopia reported a lower prevalence of the dimorphic pattern (4%) among pregnant women.5 however, results from rajasthan state in india showed the microcytic hypochromic pattern to be the most frequent (51%), followed by normocytic normochromic (32%), dimorphic (13%) and macrocytic (4%).15 these results were also supported by a further study from maharashtra state in india, which demonstrated microcytic hypochromic as the most common pattern in pregnancy (55.4%);16 the latter findings can be attributed to the fact that most of the indian participants were vegetarian, and the tendency to develop iron anaemia rather than mixed deficiency anaemia is greater in this population. although amardeep et al., in a study conducted in a rural population of central india, postulated a causal relationship in which iron deficiency results from folate deficiency,12 we believe the association is linked to the coexistence of a lack of folate and iron in the participants’ diet. this study demonstrated that the dimorphic pattern is tightly associated with low levels of both iron and rbc folate; therefore, it is assumed that the presence of a dimorphic pattern could be used as an early predictor of deficiency or as a first clue for empirical haematinic therapy in countries where full investigation is unavailable or expensive. similarly, the microcytic hypochromic pattern was strongly related to a low iron profile and hence can be an indicator for empirical iron therapy in the management of anaemia. however, empirical administration of folic acid to anaemic patients with a macrocytic or dimorphic (microcytic or macrocytic) pattern could potentially result in undesirable consequences such as neuropathy due to excessive use of vitamin b12 to facilitate entering of serum folate to the cells.5 on the other hand, our results showed that the dimorphic pattern tended to present as mild anaemia (64%), whereas the microcytic hypochromic pattern presents as moderate or severe anaemia, which agrees with what was reported by nirmala et al.8 these findings are contradictory to the postulations that the more severe the anaemia, the more likely it is to be a result of multiple deficiencies and not related solely to iron deficiency.12 moreover, a relatively high prevalence of the dimorphic pattern (88%) among those who did not regularly take haematinic supplements was observed and can be attributed to the lack of a national programme for providing free haematinic supplements to pregnant sudanese women. also, our results showed an increase in the dimorphic pattern in late pregnancy: 88% of the dimorphic pattern group was in the third trimester, a finding consistent with what was reported by nirmala et al.8 our study demonstrated a low percentage of the microcytic hypochromic pattern (25%) compared with what was reported by babu et al. in india (64%) and melku et al. in ethiopia (60%).5,11 this finding could be attributed to diversity in nutritional habits across these countries, for example, excessive drinking of iron inhibitors in tea and coffee in ethiopia or being vegetarian in india, or because of insufficient intake of foods or drinks rich in vitamin c, such as citrus fruits, and low bioavailability of dietary iron. although thalassemia contributes to the prevalence of microcytic hypochromic anaemia in the area, the finding of a low iron profile in our participants suggests that iron deficiency rather than thalassemia was the cause of the microcytic hypochromic pattern. on the other hand, the microcytic hypochromic pattern was highly prevalent among multigravidas, which is consistent with what was reported by amardeep et al.,12 and could be explained by the depletion of iron stores over repeated pregnancies. ten per cent of participants had the macrocytic pattern, 80% of them had low rbc folate and 70% had low vitamin b12; the majority of participants with this pattern had both vitamin deficiencies. thus, it is difficult to interpret the coexistence of low levels of both vitamins without considering the malabsorption state in pregnancy. generally, folate deficiency is more common than vitamin b12 deficiency in pregnancy, because increasing demands in pregnancy causes folate deficiency more than vitamin b12, whereas the latter results more commonly from malabsorption. however, both can be worsened by poor diet and low intake of milk and vegetables.12 the normocytic normochromic pattern was the least frequent; only 7% of participants presented with this pattern, and all of them had average vitamin and iron levels. although the study was not designed to detect the exact aetiology, parasitic infestation or micro-mineral deficiency (e.g. zinc and copper) could be involved. this postulation was proposed by two different studies conducted in sudan17 and malawi.13 several studies from around the globe suggest that deficiencies of other haematinics and chronic disease can contribute to the normocytic normochromic pattern. indonesian research assumed that lack of vitamin a might help cause the normocytic normochromic pattern in pregnancy.18 our study revealed that all participants exhibiting this pattern had average levels of iron, vitamin b12, and folate, and were taking haematinic supplements daily during pregnancy. therefore, we assumed microelements might be the cause, as chronically ill patients were excluded from the study. on the other hand, our study demonstrated statistically significant differences in the degree of anaemia across parity, gestational age and haematinic supplement intake; that is, increased parity, later gestational age and lack of regular haematinic supplement intake were associated with increased prevalence of severe anaemia. these findings were consistent with those of shankar et al.2 and al-farsi et al.19 additionally, the degree of anaemia revealed statistically significant differences with iron levels; that is, severe anaemia substantially increased with a low iron profile, but not with rbc folate or with vitamin b12 levels. these findings may explain why the dimorphic pattern tended to present as mild anaemia, whereas the microcytic hypochromic pattern presented as moderate or severe anaemia. limitations the most crucial weakness in the study was that it was conducted in one hospital in khartoum city; stronger results would be obtained if it was a multicentre study. moreover, the setting and design of the study were not sufficient to detect the exact aetiology of the morphological pattern. recommendations we recommend using rbc morphology for accurate diagnosis, management and follow-up of anaemia during pregnancy in all primary healthcare centres in sudan (at least, doing a peripheral smear for morphology). also, we recommend doing further studies on the possibility of using the morphological pattern to predict folate and iron deficiency and hence how and what haematinic supplements should be used in areas like sudan where a full investigation is not available. further studies on the topic are recommended in developing countries. conclusion the most frequent morphological pattern of anaemia among the pregnant women in this study was the dimorphic (mixed) pattern, followed by microcytic hypochromic, macrocytic and normochromic normocytic, which is in line with evidence for global diversity in the frequency of morphological patterns of anaemia in pregnancy. morphological patterns may predict the type of vitamins and mineral deficiency and we found some evidence to support this as a possibility; that is, the dimorphic pattern may be a predictor of mixed folate and iron deficiency, whereas the microcytic hypochromic pattern may be a predictor of iron deficiency during pregnancy. moreover, morphological patterns may predict the degree of anaemia as the dimorphic pattern tended to present as mild anaemia, whereas the microcytic hypochromic presented as moderate or severe anaemia. the study also concluded that there are significant differences in the severity of anaemia with parity, gestational age and lack of regular haematinic supplement intake. acknowledgements the author would like to acknowledge the administration and medical staff at khartoum north teaching hospital for their help in conducting this work. competing interests the author declares that he has no financial or personal relationships that may have inappropriately influenced him in writing this article. author contributions a.b.a. performed this research as a single-author project and was responsible for the project design, the laboratory work and quality control procedures, writing of the first draft, revisions, editing and approval of the final version of the manuscript. sources of support none. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references 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https://squ.pure.elsevier.com/en/publications/anemia-in-pregnancy-consequences-and-challenges-a-review-of-liter. al-farsi ym, brooks dr, werler mm, cabral hj, al-shafei ma, wallenburg hc. effect of high parity on occurrence of anemia in pregnancy: a cohort study. bmc pregnancy childbirth [serial online]. 2011;11(1):7. available from: http://www.biomedcentral.com/1471-2393/11/7. abstract introduction surveillance objectives antimicrobial resistance surveillance approaches how to enable microbiology laboratories to introduce antimicrobial resistance surveillance implementation of laboratory-based antimicrobial resistance surveillance discussion and conclusion acknowledgements references about the author(s) olga perovic national institute for communicable diseases, national health laboratory service and department of clinical microbiology, university of witwatersrand, johannesburg, south africa constance schultsz department of global health, amsterdam institute for global health and development, academic medical center, university of amsterdam, amsterdam, the netherlands citation perovic o, schultsz c. stepwise approach for implementation of antimicrobial resistance surveillance in africa. afr j lab med. 2016;5(3), a482. http://dx.doi.org/10.4102/ajlm.v5i3.482 lessons from the field stepwise approach for implementation of antimicrobial resistance surveillance in africa olga perovic, constance schultsz received: 02 may 2016; accepted: 15 aug. 2016; published: 31 oct. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: antimicrobial resistance (amr) has reached an end point, prompting a worldwide scare as no new antibiotics are in the pipeline, particularly for treatment of gram-negative bacteria. to prevent further development and spread of amr and to inform empirical treatment guidelines, surveillance of amr is necessary. objective: we aim to provide a framework for a stepwise approach toward implementation of laboratory-based surveillance for amr in african countries. methods and results: building up a surveillance system is a robust process that begins with a gap analysis in each participating country. this framework provides practical guidance on how to set up surveillance, identify responsibilities and set timelines in sustainable manner for african countries. it addresses sampling strategies, human resources, procurement and maintenance issues for amr testing at routine clinical and national reference and public health laboratories involved in amr surveillance. key issues such as laboratory capacity building, training and continuous education, quality and diagnostic stewardship are discussed in detail. discussion: there are several priorities for amr surveillance that need to be addressed in a comprehensive manner at regional and national levels, whilst keeping in line with current and proposed initiatives for laboratory capacity building, in order for african countries to achieve goals for combatting the real and current threat of amr. introduction antimicrobial resistance (amr) to bacterial pathogens is a global threat that ignores borders between countries. the emergence of multidrug-resistant bacteria means that very few effective antibiotics are available for the empirical or targeted treatment of many infectious diseases,1,2 including severe diseases such as bacterial bloodstream infections, bacterial meningitis and gonorrhoea. the impact that amr and its current pace of expansion will have on economic and health outcomes cannot be underestimated.3 amr is now considered a threat to global health security and, as such, is a key element of the global health security agenda (ghsa).4 the control of amr requires a holistic approach involving human, animal and environmental domains. the strategies necessary to deal with the amr threat developed by the world health organization (who) include: (1) to create awareness of amr; (2) to introduce antimicrobial stewardship and appropriate use of antibiotics; (3) to restrict and regulate overuse of antibiotics in human and animals; (4) to improve infection prevention and control measures; and (5) to enhance surveillance of amr.5 for some countries, amr is already a high priority, whereas other countries are still in the initial stages of recognising it as a threat.6 however, we have now reached the point at which all countries, including lowand middle-income countries (lmic), are required to develop strategies for containment of amr as part of the who’s global action plan (gap) for amr.5 this plan contains a number of features aimed at avoiding the emergence of new resistance and preventing transmission of existing resistance (table 1).7 table 1: strategic planning for antimicrobial resistance interventions aligned with the world health organization global action plan.10 most information on amr prevalence comes from high-income countries, and very little is known about amr in african countries. a systematic review of amr in sub-saharan africa by leopold et al. revealed a high prevalence of amr to commonly-used antibiotics in clinical bacterial isolates.8 that study also underlined the flaws in currently-available data and the challenges faced in lmics when implementing amr surveillance, including a lack of allocated human and economic resources.8 as one of its multifaceted responses to combat amr, the first step proposed in the who gap is to perform a baseline assessment of the amr prevalence in all countries.9 the who has sent an open call to all countries to enroll in the global amr surveillance system and to participate in a structured surveillance programme.6 lmics are encouraged to use every opportunity to apply for transfer of resources and funds in order to comply with the gap. there are clear and sometimes large differences between countries in the african region as to what extent amr surveillance is currently taking place. these differences are dependent on many factors, including socio-economic factors; however, surveillance of amr is most likely to be successfully implemented when approached using a stepwise process and when tailored to the country’s level of preparedness.10 here we address the essential components of an amr surveillance implementation plan. in general, an amr strategy is a holistic approach that includes monitoring resistance trends, strengthening diagnostic laboratories, applying infection prevention and control measures to increase social awareness and international collaboration, strengthening drug regulation and supply chains and development of novel drugs. all of these factors are aimed at promoting antimicrobial stewardship and encouraging novel research in diagnostics and drug development. amr surveillance is an important process for estimating the extent of the resistance burden in each country. amr surveillance relies on diagnostic laboratories; in sub-saharan africa, the need for laboratory improvement is evident, with some countries in need of laboratory system built from the ground up. this prevalent lack of laboratory resources and the subsequent difficulty of obtaining accurate results on antimicrobial susceptibility testing is an important challenge to address when proposing a stepwise approach. in addition, an amr strategy should consider surveillance for resistance of antibiotics used in animal feed and structure the strategy the same way as for humans. the who gap emphasises the one health approach,5 which is a multi-sectorial and multi-disciplinary way to optimise use of antimicrobials among both humans and animals. monitoring and evaluation of all tasks should be implemented with clear indicators and targets within the amr strategic framework. finally, the benefits of amr surveillance for clinically-relevant reporting should be made apparent (table 2). table 2: benefits of antimicrobial resistance surveillance. surveillance objectives the key objectives of amr surveillance are to estimate trends in amr rates and to detect the emergence and potential spread of amr, in order to inform programmes that formulate guidelines for treatment and prevention of infections and prevention of amr transmission. in africa, the development and improvement of laboratory capacity, including standardised testing, external quality assessment programmes, procurement, and timely and cost effective reports, should eventually lead to the establishment of an integrated and coordinated surveillance system for amr in the region, which will strengthen knowledge about amr through surveillance and research. this surveillance should increase understanding of the implications of amr and its epidemiology and allow for monitoring the effectiveness of guidelines and policy implementation. in addition, it would permit research and development of new diagnostics and novel technologies, optimisation of treatment, and other interventions as well as build human resource capacity as a secondary objective. collaboration within a team of human and animal experts in amr surveillance would allow for a one health approach. this requires the establishment of coordinated surveillance in animals, the ability to control and regulate antibiotics use in animals, and the identification of alternative options for growth promoters in agriculture. antimicrobial resistance surveillance approaches there are several approaches toward surveillance of amr, including population-, sentineland laboratory-based surveillance. the general perception is that laboratory-based surveillance is currently the most efficient method of surveillance of amr, which is what the who and ghsa advocate.1,4,5 however, laboratory-based surveillance is often biased, because of the potential barriers to and selection processes for submission of clinical specimens to laboratories for culture and susceptibility testing, particularly in resource-constrained settings.2 this bias may result in laboratory-based surveillance data being skewed toward a higher prevalence of amr. therefore, countries may choose to perform targeted population-based surveillance for specific diseases or pathogens, where there is a clear need to follow amr trends over time. given the current recommendations from the who and ghsa, the focus of this paper will be on laboratory-based surveillance. for laboratory-based surveillance to yield comprehensive data, a functional infectious disease diagnostic cycle is required. this cycle includes clinicians submitting samples for culture and susceptibility testing, a bacteriology laboratory that can generate quality culture and susceptibility test results, and a reporting system that includes not only the clinician requesting the test, but also a laboratory information system (lis) that can inform the surveillance programme, which may be steered by a central body (figure 1). in addition, for laboratory surveillance programmes that are technically carried out by national public health laboratories, a crucial element of the cycle is the rapid and complete transfer of all required materials, isolates and/or data from peripheral laboratories to the national site, and back reporting of results to ensure continuous engagement of laboratories and clinicians. figure 1: laboratory processes in relationship to electronic collection of antimicrobial resistance data. for a national laboratory-based surveillance programme to be successful, government commitment to support the surveillance programme for amr at the country level is essential (figure 2). such commitment can best be demonstrated by the design of dedicated policies and plans for amr control at government level, which include implementation plans and budgets. governments are required to provide human and financial resources, which may come from a variety of sources. governments of some countries may need to address specific deficiencies in supplies of water, electricity and infrastructure to allow laboratories to function appropriately. the national amr surveillance implementation plan could be used as an advocacy tool for government funding to achieve this. departments of health and other governmental structures should work in conjunction with partners, such as the who, ghsa and the african society for laboratory medicine (aslm), on amr programmes that require an inter-sectorial approach. the roles of who and aslm should be in capacity building, financial support, promoting and communicating abroad on the amr framework. figure 2: surveillance programme summary. establishment of a national laboratory-based surveillance system at the country level, as established by each country’s national department or ministry of health, also includes the establishment of a coordinating centre with the responsibility to systematically collect, analyse and share data at the national and international level. governments may decide to set up their coordinating centres differently, but all will need expertise in epidemiology, clinical microbiology, infectious diseases, veterinary medicine, data management and governance. such expertise may be built and subsequently sustained through continuous education programmes that take place locally or within the region. for example, south africa, kenya, ghana, uganda, nigeria and burkino faso all provide a field epidemiology training programme. establishment of at least one reference laboratory that can undertake infectious disease diagnostics and antimicrobial susceptibility testing, and that has competence in phenotypic and genotypic confirmation of the presence of resistance genes, is required. initially, in the absence of capacity to perform advanced phenotypical and molecular testing in the national reference laboratory, a clinical hospital bacteriology laboratory with demonstrated capacity to perform these functions, could be selected as a reference laboratory. in addition, instead of performing genotyping at the national level, molecular typing and gene characterisation could take place in another reference laboratory in the african region. national reference laboratories perform their role in close collaboration with provincial and district clinical laboratories, which generally receive samples directly from patients. together, these laboratories form the national surveillance network. countries should also consider including private laboratories in their network. although specifics vary by country, such laboratories may receive large numbers of samples from a substantial proportion of the population. strategies on how to involve and include private laboratories in national amr surveillance networks must be developed to achieve this inclusion. setting up a regional network for amr surveillance in africa to provide locally relevant amr data to serve as a bench mark may be the eventual goal. whilst several regional networks exist, the central asia and eastern european surveillance of antimicrobial resistance network is one model for the african region. this network is a joint initiative of the who regional office for europe, the european society of clinical microbiology and infectious diseases, and the dutch national institute for public health and the environment. the central asia and eastern european surveillance of antimicrobial resistance network includes all countries in the who european region that are not part of the european antimicrobial resistance surveillance network, which is coordinated by the european centre for disease prevention and control in the european union.11 the organisation of regional amr surveillance programmes creates opportunities for concentrating specific expertise in dedicated national laboratories, thus potentially reducing costs and increasing efficiency. in addition, such regional programmes may provide support for solving logistical issues, such as those described below. how to enable microbiology laboratories to introduce antimicrobial resistance surveillance laboratories that are participating in amr surveillance as sentinel or reference sites should comply with baseline requirements as indicated under this heading. the quality of laboratory-based amr surveillance data depends to a large extent on the microbiology laboratory. firstly, the quality of a laboratory system should be assessed in order to identify gaps and areas for improvements. in order for participating laboratories to meet minimal quality standards requirement as defined by the network (e.g., accreditation, external quality assessment programme, and other standards), establishment of a laboratory quality system is essential. however, the logistical difficulties of joining external quality assessment programmes and receiving quality control materials present significant challenges to countries in sub-saharan africa, because of customs and other policies for shipment of samples and microorganisms between different countries. laboratory assessments should include pre-analytical, analytical and post analytical steps for reporting of antimicrobial susceptibility testing (ast) and its limitations. for the pre-analytical phase, diagnostic stewardship is critically important. diagnostic stewardship is the process that guides and improves the use of microbiological tests to ensure they are timely, appropriate and accurate. reports sent to clinicians should include a feedback form to help on-going improvements. to apply required standards for ast, laboratories must have the required infrastructure, equipment, supplies and staffing in place. laboratories should be able to identify gaps and recommend improvements. for example, some laboratories may have all the equipment needed but no supply of reagents or other consumables needed for basic microscopy, culture and ast implementation. this, in turn, affects diagnostic stewardship as clinicians may lose confidence in laboratory reporting when timely, appropriate and accurate results cannot be achieved. a country’s national amr surveillance plan and the regional amr surveillance network may provide for policies and procedures that make timely ordering and receipt of consumables and other supplies feasible and affordable. aslm could play an active role in such advocacy activities. laboratory experts should be involved in ordering supplies to avoid receiving supplies that are irrelevant, not useful or of poor quality. the use of a lis is limited in most lmics, which is unfortunate, as it improves amr surveillance by reducing workload and errors, thus improving the overall quality of microbiology laboratories. laboratories should use lis to retrieve and share ast data with national and regional bodies (figure 1). whilst databases preferably allow sharing of data, confidentiality must also be guaranteed. the whonet data management software provides these features and is freely available.12 other initiatives such as those from the resaolab network13 have created their own open source lis that includes an option to store and report amr data. as mentioned, national and regional networks will require (inter)national standardisation of methods. for standardisation of bacteriology test methods, guidelines are available and are often used, e.g. those from the clinical and laboratory standards institute14 or from eucast,15 the latter of which is freely available. for additional standardisation of data collection and reporting to a central data collection point, a regional network would require additional software that can be linked to local liss, including newly-designed or whonet, european antimicrobial resistance surveillance network, or central asia and eastern european surveillance of antimicrobial resistance network software. this software may be expensive to set up, and maintenance is an additional consideration. it is also important to consider that these software systems may not be sufficient for outbreak detection due to the lack of an alert system. in addition, such systems must be compatible with existing or future national lis systems, in order to prevent time-consuming transfers of data, as is currently needed for whonet. thus, the available data collection systems for each country and the costing structure for implementation of region-wide amr surveillance need further investigation. human resource capacity may be the most challenging issue, as shortages of qualified and trained professionals can have a serious negative impact on the ability of laboratories to perform testing. human resources are a weak spot in microbiology and have been ignored. quality microbiology requires sufficient number of qualified technicians, as well as supervision by md or msc level clinical microbiologists who are capable of reporting results to clinicians. finally, veterinarians should be involved in the process of developing surveillance systems to strengthen diagnostic stewardship in animal health, provide assistance about status of amr in animal health, and implement preventative measures in animal health, if needed at a later stage. for capacity building that includes education, training and refresher courses and other forms of networking, the who and aslm should be excellent sources of support. implementation of laboratory-based antimicrobial resistance surveillance the following components should be considered when designing an amr surveillance plan (table 3). table 3: design summary for development of laboratory-based surveillance matrix. gap analysis a gap analysis that includes all levels of the health system must be performed in order to implement or further expand existing amr surveillance activities. for example, it was recently reported that many countries still lack reference to laboratories in their health policies and plans and do not consider laboratories at all in their health budgets.16 an analysis of existing surveillance systems should be undertaken before development of a new system is considered. there are currently several tools available to assess laboratory capacity and quality that could be used as a part of a gap analysis. these include the stepwise laboratory improvement process towards accreditation tools developed by who,17 as well as by aslm as part of the ghsa.18 these tools are targeted at different levels of the health system. the who stepwise laboratory improvement process towards accreditation tool is a generic quality system assessment tool for laboratories and is not designed to look specifically at amr surveillance capacity.19 the audit checklist tools developed by aslm are established to assess laboratory capacity at national level and include a bacterial amr testing module. whichever tool is used, it should be applied for the purpose for which it was designed. priority pathogen-antibiotic combinations in order to ensure amr surveillance of relevant pathogens, it is important to establish case definitions. for example, standard definitions should be established for patients with symptoms of syndromes that have high clinical importance, such as central nervous system infections and septicaemia. the selection of specimens should be directed according to local, national, and regional relevance, after considering site of infection, such as meningitis or bacteraemia. isolates from cerebrospinal fluid and blood cultures, with particular pathogens of focus, including both gram-negative and gram-positive bacterial organisms, should be included. in addition, exclusion criteria, such as de-duplication based on one specimen per patient in 21 days or based on global amr surveillance system criteria,6 should be established. in addition, there are number of priority organisms and antibiotics recommended by who that should be considered in a stepwise process. these include enterococcus faecium, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa, enterobacter spp., streptococcus pneumoniae, salmonella spp., shigella spp. and neisseria gonorrhoea. each country should prioritise which drug/bacteria combination to test and report according to local relevance. surveillance methods there are several types of surveillance and current recommendations are to perform laboratory-based surveillance. for each drug/bacteria combination, the approach toward laboratory-based surveillance should be decided on (figure 3). whichever approach is chosen, the availability of basic microbiology and core patient data is imperative and methods to calculate aggregate data based on sampling methods (periodic or continuous) are required. each country should decide how to approach the surveillance methodology that is sustainable in the defined period of time available. figure 3: surveillance methods. african regional antimicrobial resistance reference laboratory for technical support and strategy advice, an amr reference laboratory for african region should be established. the role of an african region amr reference laboratory would be multi-faceted. firstly, the regional reference laboratory should provide training of laboratory personnel from network member countries to help build capacity for development of surveillance systems in the trainees’ home countries. secondly, the regional reference laboratory should take the lead in introducing and providing training on new techniques. thirdly, it should provide confirmation of difficult ast results and offer advice when necessary. finally, it should manage other laboratory networks in the region. a public/private partnership should be sought as the funding source. discussion and conclusion considering the growing impact of amr worldwide and the lack of quality surveillance systems in the african region, an enormous effort must be undertaken through international and multi-sectorial collaboration. a recent report analysing the amr surveillance capacity of the east africa public health laboratory network by the center for disease dynamics, economics & policy, commissioned by the world bank, summarised the following key issues for laboratories to carry out amr surveillance:20 lack of demand of bacteriology diagnostics from clinicians, related to length of time to get results (at least two days); lack of trust in results; lack of laboratory capacity for blood cultures, which are needed for many of the most serious, life-threatening infections. low priority given to bacteriology diagnostic supplies by hospitals and other decision makers who control purchasing. the multi-component nature of bacterial culture and antimicrobial susceptibility testing, which makes it especially vulnerable to weak supply chains and frequent stock outs; results cannot be obtained if essential components are unavailable when testing is needed. lack of recognition that microbiology requires dedicated, trained personnel, leading some facilities and/or ministries of health to rotate staff in and out of microbiology. few options for automated testing relative to haematology, chemical pathology or hiv testing, which may be more satisfying to staff, leading to low morale. requirement for patients to pay out of pocket for diagnostic tests in many countries. whilst some of these may be generic to lmics, others may be more specific to certain countries or settings.21,22 whichever the situation, solving these issues will require collaborative efforts of multiple stakeholders. aslm could play a key role in facilitating these efforts due to its central position between ministries of health, policy makers, laboratory professionals and, increasingly, clinicians and professional societies. in conclusion several key steps should be implemented for an amr surveillance programme such as: assessment of laboratory capacity to perform surveillance for amr; identification of gaps; adoption of the global amr surveillance system manual or other guidelines; designation of national reference laboratories for amr and organisation of advisory committees; implementation of laboratory-based amr surveillance; and reporting to the global amr surveillance system. acknowledgements we thank aslm and who afro for organising the meeting in sierra leone and initiating discussions about amr in west african countries. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support none. authors’ contributions o.p. was the project leader. o.p. and c.s. were both responsible for project design and writing of the manuscript. references world health organization. surveillance standards for antimicrobial resistance [document on the internet]. c2002 [cited 2016 oct 07]. available from: http://www.who.int/csr/resources/publications/drugresist/whocdscsrdrs20015.pdf?ua=1(07/10/2016) laupland kb, ross t, pitout jd, et al. investigation of sources of potential bias in laboratory surveillance for anti-microbial resistance. clin invest med. 2007;30(4):e159–166. review on antimicrobial resistance [homepage on the internet]. available from: www.amr-review.org global health security agenda [homepage on the internet]. available from: https://ghsagenda.org world health organization. global action plan on antimicrobial resistance [document on the internet]. c2015 [cited 2015 nov 13]. available from: http://www.who.int/antimicrobial-resistance/publications/global-action-plan/en/ world health organization. call for participation: global antimicrobial resistance surveillance system (glass) [page on the internet]. c2016 [cited 2015 oct 27. available from: http://www.who.int/drugresistance/surveillance/glass-enrolment/en/ magiorakos ap, srinivasan a, carey rb, et al. multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. clin microbiol infect. 2012;18(3):268–281. http://dx.doi.org/10.1111/j.1469-0691.2011.03570.x. epub 2011 jul 27. leopold sj, van leth f, tarekegn h, et al. antimicrobial drug resistance among clinically relevant bacterial isolates in sub-saharan africa: a systematic review. j antimicrob chemother. 2014;69(9):2337–2353. http://dx.doi.org/10.1093/jac/dku176. epub 2014 may 30. barlam tf, cosgrove se, abbo lm, et al. implementing an antibiotic stewardship program: guidelines by the infectious diseases society of america and the society for healthcare epidemiology of america. clin infect dis. 2016;62(10):e51–e77. http://dx.doi.org/10.1093/cid/ciw118. epub 2016 apr 13. smith rd, coast j. antimicrobial resistance: a global response. bull world health organ. 2002;80:126–133. world health organization regional office for europe. central asian and eastern european surveillance of antimicrobial resistance (caesar) [page on the internet] c2016 [cited 2016 oct 07]. available from: http://www.euro.who.int/en/health-topics/disease-prevention/antimicrobial-resistance/antimicrobial-resistance/central-asian-and-eastern-european-surveillance-of-antimicrobial-resistance-caesar whonet. who collaborating centre for surveillance of antimicrobial resistance [homepage on the internet]. available from: http://www.whonet.org fondation mérieux. resaolab: strengthening the quality and accessibility of diagnostics in west africa [homepage on the internet]. available from: http://www.fondation-merieux.org/reinforcing-access-and-quality-of-biological-diagnosis-in-west-africa-resaolab clinical and laboratory standards institute. performance standards for antimicrobial susceptibility testing. 26th ed. clsi supplement m100s. wayne, pa: clsi; 2016 eucast [homepage on the internet]. available from: www.eucast.org african society for laboratory medicine. medical laboratories: mapping the landscape of national health policies and plans in sub-saharan africa [document on the internet]. c2014 [cited 2016 oct 06]. available from: http://www.waspalm.org/uploads/content_files/files/aslm_newsletter_lab_culture_issue_12.pdf [pp. 30–33] world health organization regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation (slipta) in the african region (with checklist) [document on the internet]. c2015 [cited 2015 sep 30]. available from: http://www.afro.who.int/en/clusters-a-programmes/hss/blood-safety-laboratories-a-health-technology/blt-highlights/3859-who-guide-for-the-stepwise-laboratory-improvement-process-towards-accreditation-in-the-african-region-with-checklist.html african society of laboratory medicine. settling the score: how a laboratory network assessment tool can be used to score functionality [page on the internet]. c2015 [cited 2016 oct 07]. available from: http://aslm.org/stay-informed/press-room/news-articles/settling-the-score-how-a-laboratory-network-assessment-tool-can-be-used-to-score-functionality ndihokubwayo jb, yahaya aa, dester at, et al. antimicrobial resistance in the african region: issues, challenges and actions proposed. african health monitor. 2013;16:27–30. world health organization. antimicrobial resistance, a68/20 [document on the internet]. c2015 [cited 2016 oct 07]. available from: http://apps.who.int/gb/ebwha/pdf_files/wha68/a68_20-en.pdf gelband h, martinez e. east africa public health laboratory networking project [document on the internet]. c2016 [cited 2016 oct 07]. available from: http://www.cddep.org/publications/east_africa_public_health_laboratory_networking_project#sthash.rfdnssfy.dpbs world health organization regional office for africa. guide for establishing laboratory-based surveillance for antimicrobial resistance. brazzaville, democratic republic of congo; 2013. article information authors: kelebeletse o. mokobela1 mpho t. moatshe1 mosetsanagape modukanele2 affiliations: 1ministry of health, botswana2us centers for disease control and prevention, botswana correspondence to: mosetsanagape modukanele postal address: po box 90, gaborone, botswana dates: received: 26 june 2014 accepted: 15 sept. 2014 published: 03 nov. 2014 how to cite this article: mokobela ko, moatshe mt, modukanele m. accelerating the spread of laboratory quality improvement efforts in botswana. afr j lab med. 2014;3(2), art. #207, 6 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.207 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. accelerating the spread of laboratory quality improvement efforts in botswana in this original research... open access • abstract • introduction • research methods and design    • the slmta programme    • additional training and mentorship in selected laboratories    • evaluation    • results • discussion    • conclusion • acknowledgements    • competing interests    • authors’ contributions    • cdc disclaimer • references abstract top ↑ background: in 2002, the ministry of health (moh) of botswana began its journey toward laboratory accreditation in an effort to enhance the quality of laboratory services. after a difficult start, the moh recognised the need for a more practical and sustainable method for change that could be implemented nationally; they therefore adopted the strengthening laboratory management toward accreditation (slmta) programme.objective: this study describes the process and lessons learned in implementing slmta and the role of supplemental training and mentoring so as to achieve botswana’s national laboratory quality improvement goal. methods: eight laboratories were enrolled into the slmta programme in 2010, which included a series of workshops and improvement projects conducted over nine months. four of these laboratories received supplementary training and focused mentorship from the botswana bureau of standards (bobs). laboratory performance was measured at baseline and exit using the world health organization regional office for africa’s stepwise laboratory quality improvement process towards accreditation (slipta) checklist. one laboratory did not receive an exit audit and was thus excluded from the analysis. results: an 18 percentage-point improvement was observed when comparing the median baseline score (53%) to the median exit score (71%) for the seven laboratories. laboratories that received additional training and mentorship from bobs improved 21 percentage points, whilst non-bobs-mentored laboratories improved eight percentage points. hospital management buy-in and strong laboratory staff camaraderie were found to be essential for the positive changes observed. conclusion: slmta facilitated improvements in laboratory quality management systems, yielding immediate and measurable results. this study suggests that pairing the slmta programme with additional training and mentorship activities may lead to further increases in laboratory performance; and that slmta is a practical approach to extending quality improvement to moh laboratories. introduction top ↑ laboratory quality management systems (qms) provide a strong foundation for promoting excellence in laboratory services that support fundamental components of effective healthcare systems.1in many resource-limited countries, laboratories lack robust quality systems, as they have historically been afforded low priority and few resources. this situation has led to poor-quality patient care and health outcomes, as well as loss of revenue resulting from inefficient and redundant processes. however, in recent years, an increased focus has been placed on the delivery of quality services as governments have moved toward initiating improvements in laboratory services.1,2in 2002, the botswana ministry of health’s (moh’s) laboratory services developed a five-year work plan with the goal of accrediting 16 laboratories, which resulted in the initial introduction of qms in select laboratories. six years later, limited improvements in the quality of laboratory management and service were noted because of high workload, inadequate staffing and poor infrastructure, amongst other factors. by 2011, after nine years of implementation and extensive partner and consultant support, four laboratories had attained international accreditation. whilst this accomplishment was commendable, it had been clear for some time that this approach was too costly (as consultants from outside botswana were employed) and too slow to be a sustainable option for long-term quality improvement on a national level; botswana needed a more viable strategy. in 2008, the moh developed a five-year (2009–2014) national laboratory strategic plan,3which called for implementation of qms in all laboratories by 2014 and accreditation of four district-level and two national-level laboratories by 2013 and 2014, respectively. the strategic plan directed country laboratory qms activities by incorporating a mentoring approach for laboratory accreditation. shortly after initiating this plan, the moh adopted the strengthening laboratory management toward accreditation (slmta) programme so as to catalyse the operation of the strategic plan and provide a platform to promote quality management of laboratories. in accordance with the strategic plan, key laboratories throughout the country were identified to participate in the slmta programme, which consists of a comprehensive management framework, training and mentoring toolkit, and a multi-workshop implementation model.4 whilst the slmta programme formed the cornerstone of botswana's laboratory improvement strategy, the moh theorised that combining slmta with additional qms training and targeted mentoring might achieve superior results.5 therefore, additional training and mentorship were offered to four top-priority laboratories designated as future centres of excellence. this article describes the process and lessons learned in implementing slmta and the role of supplemental training and mentoring so as to achieve botswana's national qms and accreditation goals. research methods and design top ↑ the slmta programme the moh enrolled eight national, regional, district and primary level laboratories throughout botswana in the slmta programme, beginning in august 2010. profiles of each laboratory (a to h) are listed in table 1. implementation followed the standard slmta process with a series of three workshops delivered over a period of nine months.5a total of 24 laboratory staff, including laboratory managers, quality officers and section heads, participated in the training. table 1: profiles of laboratories enrolled in the botswana slmta programme, 2010. each workshop was followed by a period of three months to allow participants to implement improvement projects. laboratory staff members were allowed to choose improvement projects that were relevant to their local environment and priorities. each laboratory was encouraged to involve all laboratory staff members in improvement project implementation. after each workshop, two follow-up visits were conducted by moh/slmta trainers in order to provide further training and coaching on improvement projects. the trainers spent one day in each laboratory. the visits included meetings with hospital management so as to create awareness and solicit support for the laboratory improvement process. additional training and mentorship in selected laboratories four of the eight laboratories (e, f, g and h) had been recently relocated to new facilities designated as centres of excellence in medical specialties. these facilities received additional training by the botswana bureau of standards (bobs), a certified international organization for standardization (iso) training organisation. training focused on understanding the auditing and documentation requirements for iso 15189. bobs also provided extra mentoring to these four laboratories from april to june 2011; monthly visits lasted one week in each laboratory. bobs provided mentorship on system documentation, covering the development of quality manuals, standard operating procedures (sops) and other quality documents as required by the iso standard. bobs mentors, together with the laboratory staff, conducted a gap analysis and developed a work plan with deliverables for the mentee laboratories. once a task from the work plan was completed (e.g., writing a quality manual), the laboratory would share it with the mentor who would then make corrections. the mentor provided guidance on the document’s layout, as well as on interpretation of different clauses of the iso standard. after finalising the documents, the mentor assisted with implementation. the laboratory and the mentor established a relationship that allowed both parties to communicate via email or telephone, as needed, between visits. evaluation slmta in-country trainers conducted audits in order to measure laboratory improvements using the world health organization regional office for africa’s stepwise laboratory quality improvement process towards accreditation (slipta) checklist.6the slipta checklist is organised around the 12 quality system essentials (qses).7 for each qse, a score is obtained by calculating the points that a laboratory has received from each item on the checklist. an overall score is used to rate laboratories on a zeroto five-star rating scale, with a score of < 55% as zero stars, 55% – 64% as one star, 65% – 74% as two stars, 75% – 84% as three stars, 85% – 94% as four stars and ≥ 95% as five stars. slipta was used for both the baseline (july 2010) and exit (november 2011) audits.from july 2012 to february 2013, bobs mentors conducted trial assessments in their laboratories using the south african national accreditation system’s (sanas) checklist in order to determine the laboratories’ readiness for accreditation by sanas, a key regional accreditation body for southern africa.9this checklist yields qualitative results rather than a numerical score and highlights areas of focus for accreditation preparation. one of the eight laboratories (laboratory h) did not receive an exit audit as a result of a schedule conflict and therefore is not included in the slipta data analysis in this article; it did, however, participate in the trial assessment using the sanas checklist. results at baseline, four of the seven laboratories had a zero-star rating, two had one star and one had two stars. at exit, two laboratories remained at zero stars, four laboratories were rated at two stars and one laboratory was rated at three stars. the median slipta audit scores were 53% at baseline and 71% at exit, a median increase of 18 percentage points. the three bobs-mentored laboratories improved by 21 percentage points from a median score of 53% at baseline to 74% at exit, whilst the non-bobs-mentored laboratories increased eight percentage points from a median score of 49% at baseline to 57% at exit. the greatest improvements were in two of the bobs-mentored laboratories (e and g), which gained 27 percentage points each. among non-bobs-mentored laboratories, one (d) had a slight decrease in score, whilst the others each increased by three to 15 percentage points (figure 1). figure 1: slipta scores and star levels at the baseline and exit audits, botswana slmta programme 2010–2011. comparing the median performance scores at baseline with those at exit for each qse across the seven laboratories, the greatest improvement was in occurrence management and process improvement (40 percentage points), management reviews (25 percentage points), client management and customer service (25 percentage points), documents and records (20 percentage points) and internal audit (20 percentage points). corrective action, however, had a 13 percentage-point drop from baseline to exit (figure 2). figure 2: mean qse scores at the baseline and exit audits, botswana slmta programme 2010-2011 (n = 7). figure 3a shows the performance of the three bobs-mentored laboratories (e, f and g) in the 12 qse sections of the slitpa checklist. for these laboratories, the greatest gains were in the areas of occurrence management and process improvement (50 percentage points), internal audit (40 percentage points), client management and customer service (38 percentage points) and documents and records (36 percentage points). for the non-bobs-mentored laboratories (a, b, c and d), documents and records (26 percentage points) and client management and customer service (25 percentage points) had the greatest improvement (figure 3b). none of the qse scores dropped for bobs-mentored laboratories; for non-bobs laboratories, however, scores dropped in three areas: corrective action (25 percentage points); information management (4 percentage points); and process control and internal and external quality assessment (3 percentage points). figure 3: mean qse scores of (a) bobs-mentored laboratories (n = 3) and (b) non-bobs-mentored laboratories (n = 4) at the baseline and exit audits, botswana slmta programme 2010-2011. in the trial assessment using the sanas checklist after the exit audit in the bobs-mentored laboratories (e, f and g), the laboratories had made additional improvements in many areas (table 2). for example, quality manuals were now complete, internal audits were executed and safety procedures were in place in most of the audited laboratories. some notable deviations included: incomplete finalisation of critical documents; out-of-date and incomplete equipment services and calibrations; and incomplete external quality assessment (eqa) programmes. following this audit, laboratory (e) and laboratory (h) were encouraged to apply for international accreditation. table 2: qualitative results from trial assessment using the sanas checklist in bobs-mentored laboratories enrolled in the botswana slmta programme 2010-2011. discussion top ↑ the introduction of the slmta programme in hospitals in botswana was found to be a practical option that yielded positive results for strengthening laboratories. all but one laboratory demonstrated improvements and, as a whole, laboratories improved in all qses except corrective action.supplemental mentorship and training may have contributed to the success amongst bobs-mentored laboratories, which showed greater median improvements in the slipta audit results. however, it is important to note that the small number of laboratories and lack of random assignment to bobs mentorship limits the ability to draw definitive conclusions regarding this comparison. other studies have also noted that supplementing slmta with additional training and mentoring may be beneficial.9,10,11our findings, when joined with these others, suggest that combining slmta with other strategies in a qms programme may lead to synergistic improvement. one of the keys to success of the roll-out of slmta in the laboratories was strong staff commitment and involvement. during the training sessions, staff involvement was cultivated by the formation of teams that brainstormed improvement projects and outlined specific implementation tasks. this practice fostered a culture of problem solving and boosted confidence amongst laboratory staff, who felt empowered to implement improvement projects previously considered beyond their capability. these projects were developed by the trainees and responsibility was shared across the laboratory team. laboratories showed improvements in areas in which they had previously struggled, such as internal audit and management reviews. some laboratories also improved their inventory control systems and anecdotally reported decreased turnaround times. support and buy-in from hospital management for slmta was another critical component of the quality improvement process. as understanding and ownership of the process increased amongst hospital management, managers became more willing to assist laboratories in improvement projects that required additional funds or involvement from multiple hospital departments. for example, some projects required infrastructure modifications; hospital management provided resources for sink relocations, trunks to hold electrical cords and facility painting. one hospital hired extra temporary staff to assist with phlebotomy duties whilst laboratory staff concentrated on quality improvement activities. as a result, laboratory staff morale and commitment improved. three laboratories had minimal improvements or decreased scores. laboratory a, the national health laboratory, serves multiple functions: as a national reference laboratory, a procurement agency and a training agency. balancing the requirements of these diverse functions was a unique challenge that will require resolution before tangible results can be achieved. laboratory f had initially shown improvements, but had difficulties in getting documents reviewed and authorised by laboratory management prior to the exit audit. in laboratory d, both of the slmta-trained staff members transferred to other facilities during the implementation of the programme, which impacted negatively on performance. limited progress was observed across all the laboratories in the areas of equipment; facilities and safety; and organisation and personnel, indicating that further work is needed. these areas may require greater funding and management involvement at higher levels, which the moh is attempting to address. for example, throughout the country medical equipment maintenance and calibration programmes are weak and national eqa programmes and laboratory information systems do not meet the needs of laboratories. as the five-year national laboratory strategic plan comes to an end, the moh is in the process of launching a follow-up plan for 2015 to 2019. this plan seeks to address remaining challenges and to identify ways of maintaining and increasing the improvement that was achieved in all participating laboratories. one new approach will be to use the many documents (e.g., sops and safety manuals) developed during the slmta programme as the standard national documents for distribution to all moh laboratories. in addition, to ensure sustainability and continuous quality improvement amidst reduced donor funding, an additional 18 local mentors have been trained to help accelerate the spread of laboratory quality improvement efforts. conclusion the effort for widespread improvement of laboratory services in botswana has gained momentum in the past few years following the introduction of the slmta programme. the programme was well received by staff for its practicality and measurable impact, as improvement was demonstrated in most of the enrolled laboratories. whilst positive gains have been achieved, progress still needs to be made in struggling areas to minimise system disruptions in the laboratory and improve the national service programmes that support them. this study adds to the growing body of evidence that a combined strategy of slmta plus targeted training and mentorship can lead to further effectiveness of a qms programme and higher-quality laboratory service delivery. acknowledgements top ↑ we thank the moh for continued support in the laboratory endeavour to attain accreditation. we also acknowledge the us centers for disease control and prevention (cdc) for providing funding for the programme through the us president’s emergency plan for aids relief (pepfar). thanks also go to the bobs staff and management as implementing partners for this programme. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions k.o.m. (moh) provided input to the manuscript. m.t.m. (moh) drafted the manuscript. m.m. (cdc, botswana) provided input to the manuscript and is the corresponding author. cdc disclaimer the findings and conclusions in this report are those of the authors and do not necessarily reflect the official position of the cdc. references top ↑ 1. petti c, polage cr, quinn tc, et al. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006;42(3):377382.2. gershy-damet gm, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. http://dx.doi.org/10.1309/ajcptuuc2v1wjqbm 3. botswana ministry of health. health sector laboratory strategic plan (hslsp): 2009–2014 (unpublished); n.d. 4. yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin pathol.2010;134(3):401–409. http://dx.doi.org/10.1309/ajcpnbbl53fwuiqj 5. maruta t, motebang d, mathabo l, et al. impact of mentorship on who-afro strengthening laboratory quality improvement process towards accreditation (slipta). afr j lab med 2012;1(1), art. #6, 8 pages. 6. world health organization regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation in the african region (with checklist) [document on the internet]. c2011 [cited 2014 may 14]. available from: http://www.afro.who.int/en/clusters-a-programmes/hss/blood-safety-laboratories-a-health-technology/blt-highlights/3859-who-guide-for-the-stepwise-laboratory-improvement-process-towards-accreditation-in-the-african-region-with-checklist.html 7. clinical and laboratory standards institute. application of a quality management system model for laboratory services; approved guidelines – third edition. clsi document gp26-a3 [isbn 1-56238-553-4]. wayne, pa: clinical and laboratory standards institute; 2004. 8. south african national accreditation system [homepage on the internet]. n.d. [cited 2013 jun 12]. available from: http://www.home.sanas.co.za/ 9. audu ra, onubogu cc, nwokoye nn, et al. improving quality in national reference laboratories: the role of slmta and mentorship. afr j lab med. 2014;3(2), art. #200, 7 pages. http://dx.doi.org/10.4102/ajlm.v3i2.200 10. nzabahimana i, sebasirimu s, gatabazi jb, et al. innovative strategies for a successful slmta country programme: the rwanda story. afr j lab med. 2014;3(2), art. #217, 6 pages. http://dx.doi.org/10.4102/ajlm.v3i2.217 11. makokha ep, mwalili s, basiye fl, et al. using standard and institutional mentorship models to implement slmta in kenya. afr j lab med. 2014;3(2), art. #220, 8 pages. http://dx.doi.org/10.4102/ajlm.v3i2.220 summary background disease surveillance in africa connected diagnostics in disease surveillance a bottom-up approach to continent-wide antimicrobial resistance surveillance confidentiality and privacy of health information conclusion acknowledgements references about the author(s) natasha m. gous systemone, llc, johannesburg, south africa systemone, llc, springfield, massachusetts, united states philip c. onyebujoh africa centres for disease control and prevention, african union, addis ababa, ethiopia alash’le abimiku international research center of excellence, institute of human virology, abuja, nigeria chris macek systemone, llc, johannesburg, south africa jeff takle systemone, llc, johannesburg, south africa citation gous nm, onyebujoh pc, abimiku a, macek c, takle j. the role of connected diagnostics in strengthening regional, national and continental african disease surveillance. afr j lab med. 2018;7(2), a775. https://doi.org/10.4102/ajlm.v7i2.775 opinion paper the role of connected diagnostics in strengthening regional, national and continental african disease surveillance natasha m. gous, philip c. onyebujoh, alash’le abimiku, chris macek, jeff takle received: 30 jan. 2018; accepted: 22 aug. 2018; published: 06 dec. 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. summary the africa centres for disease control and prevention (africa cdc) and world health organization (who) regional office for africa (afro) are building a global health security programme that aims to strengthen both regional and national health through networked, collaborative efforts to improve infectious disease and antimicrobial resistance surveillance. to achieve this, the africa cdc is calling for a data-sharing platform that can be leveraged across member states and disease areas, strengthening the ability to collate, analyse and interpret data, and to respond with the appropriate action. although numerous disease intelligence and surveillance systems exist, they are plagued with inaccurate or untimely data. we contend, furthermore, that it was this lack of data quality – and not the lack of surveillance systems or networks – that prevented the global community from acting earlier in response to the ebola outbreak in 2014–2016. the new field of ‘connected diagnostics’ is one solution to this concern, as it automates data collection directly from the diagnostic instruments to multiple levels of stakeholders for real-time decision-making and policy response. this article details how the intervention of ‘connected diagnostics’ could solve the primary underlying failure in existing surveillance systems – the lack of accurate and timely data – to enable difficult political decisions earlier. the use of connectivity solutions can enable critical health and operational data to empower the africa cdc, regional hubs, and each country with a consistent and automated data feed while still maintaining country privacy and controls. background in an interconnected and interdependent world, the threat of any infectious disease is no longer simply a national health concern. globalisation, despite all of its advantages, has resulted in a world where infectious pathogens can effortlessly traverse borders and threaten global public health security. outbreaks of infectious diseases are increasing globally with a resurgence of epidemics such as cholera, ebola and dengue, particularly in africa. so too, the rise of antimicrobial resistance (amr) partly due to the increasing use of antibiotics, particularly in low-income countries, poses a worldwide biosecurity and economic threat. the number of deaths globally due to amr is projected to reach a staggering 10 million per year by 2050.1 the requirement for timely reporting of infectious diseases has been highlighted on several occasions. a poignant reminder of the impact of poor data quality and timeliness in the management of disease outbreaks is the ebola outbreak in 2014–2016 – an outbreak that resulted in the loss of $2.2 billion us dollars gross domestic product in guinea, sierra leone and liberia2 as well as 11 325 lives.3 in 2015, an outbreak of a ‘strange disease’ occurred in kampala, uganda. the outbreak was thought to have begun in january 2015 or earlier, but was not recognised and reported as a typhoid outbreak until february 2015 due to the lack of surveillance systems; this delay led to thousands being affected and several deaths.4 some countries are reporting an average delay of 22 days from collection of infectious disease specimens to return of results to facilities.5 similarly, in the context of hiv, the return of laboratory result to referring facilities can take up to 90 days6 with up to 50% loss of results,7 thus, severely delaying initiation of treatment, adherence counselling and switch to second-line treatment. monitoring of infectious diseases and amr through surveillance systems is the cornerstone of any outbreak and response network and the availability of a robust and timely system will ensure a country’s propensity to manage and promptly contain an outbreak.8 time, more than anything, determines the success of a response. however, surveillance systems are largely fragmented and lack input of timely, accurate data. disease surveillance systems disease surveillance systems serve two key functions: first, to provide an early warning system for potential threats and, second, to provide a system to monitor diseases, trends, and progress towards control and elimination strategies.9 worldwide, different disease surveillance strategies and systems have been adopted. traditional systems typically rely on a notification-based approach, whereby health care providers regularly submit data to relevant health authorities on a specified infectious disease within a specified territory.8 this requires passive reporting on disease information and, although inexpensive, can result in sequential errors, reflecting underand over-reporting by health care providers of diseases and cases. active surveillance systems, on the other hand, rely on proactive searches for cases with established criteria, risk factors or events, but still require manual data collection. by design, both of these strategies, although employing structured reporting mechanisms, suffer numerous challenges, including the lack of or inaccessibility of real-time, quality data, under-reporting or delayed reporting of cases and deaths,8 and complex management.10 advancements such as the internet have revolutionised how disease data is collected and now, web-based surveillance systems such as healthmap, allow automated and rapid collection of large amounts of unstructured data from electronic sources.10,11,12 however, even with advancements, web-based surveillance is still challenging, as the information collected from diverse sources is not structured,12 has uncertain and varying data quality, and may lead to inaccurate interpretation and predictions.10 disease surveillance in africa africa has been plagued by numerous and recurring epidemics over the past several decades. not only do these diseases share the ability to decimate entire towns and villages, they also place enormous economic strain on countries.13 global estimates by the world bank place the annual global cost of a moderately severe to severe pandemic at roughly $570 billion.14 responses to many of these diseases have been hampered by weak health care systems, lack of policies that encourage integration and coordination within countries and across borders, and the absence of accurate and timely diagnostic data for decision-making. when the who afro attempted to provide an inventory of all epidemics reported in africa between 1970 and 2016, the effort was impeded by limited data, inconsistencies in reporting of occurrences and magnitudes of outbreaks, and variability in description of outbreak locations.15 the increasing burden of amr threatens the effectiveness and success of infectious disease treatment programmes.16 africa is thought to contribute a large proportion of the global amr burden due to limited control and monitoring of use of antibiotics and the high rates of communicable diseases such as hiv and tuberculosis.17 however, the scarcity of data coming from the region on amr surveillance reflects the absence of tools to collect valuable information, reliance on passive reporting from centres and lack of training and expertise needed for continuous monitoring and reporting.16 one review on the status of amr in africa found that recent data on amr was not available for 42.6% of african countries and what was available were lacking in quality, even though resistance to commonly prescribed antibiotics was very high in the african continent.18 a review of 135 current antibiotic prescribing guidelines also found that, in general, most do not consider resistance patterns for highly prevalent infectious syndromes such as community-acquired pneumonia and urinary tract infections.19 this largely reflects the lack of collection of accurate resistance data and limits our true understanding of amr burden. the role of the africa centres for disease control and prevention in antimicrobial drug resistance surveillance to strengthen both regional and national health, the africa cdc and who afro are building a global health security programme, focusing on rapid response surge teams, starting at the africa cdc and stemming out to the national public health institutes (nphis). five regional collaborating centres (rccs) hosted by zambia, kenya, gabon, nigeria and senegal, in addition to key nphis in those sub-regions, make up the africa cdc regional integrated surveillance laboratory network or rislnet,20 with headquarters in ethiopia. the goal of this integrated system is to drive networked, collaborative efforts to strengthen and improve disease surveillance linked to laboratory confirmation, disease preparedness and response. a further expected outcome of the africa cdc is the establishment of the antimicrobial resistance and surveillance network or amrsnet that will standardise the approach to amr surveillance and strive to achieve quality data.20 in order for both of these efforts to be fruitful, a data-sharing platform is needed which can be leveraged across member states and disease areas to strengthen the ability to collate, analyse and interpret the generated data, and to respond appropriately. connected diagnostics in disease surveillance to ensure an effective response to a potential outbreak event, timeliness is of paramount importance. this requires surveillance, assessment and communication mechanisms to be in place to increase awareness of management strategies, and to facilitate their initiation in the early phases.21,22 when a passive, paper-based surveillance system was implemented in the public health sector in south africa for health care-associated infections, the main challenge was incomplete data collection.23 nurses felt that manual collection and recording of data added to their workload, thus highlighting the need for systems that are able to automatically collect data and communicate it to various levels of the health care system. interconnected diagnostic networks can address these needs by facilitating the interactions between laboratory confirmation, automated data collection, interpretation, delivery of results and real-time monitoring of disease as well as analysis. also, by providing accurate and reliable diagnostic information directly to the point of patient care, more timely patient management and appropriate use of antimicrobials can be facilitated. real-time reporting of laboratory-confirmed cases a critical component of disease-specific surveillance and early warning systems is their reliance on laboratory confirmation of the disease. high-quality, reliable laboratory detection has been emphasised by the africa cdc as the central component to rapid response.20 with the evolution of medical diagnostic instruments and availability of digital health platforms in many african countries, the tools for detection and response are already in place and ready to be used and connected for surveillance. as an example, the genexpert platform (cepheid, sunnyvale, california, united states) is a molecular diagnostic tool commonly used for tuberculosis and first-line drug resistance detection, but also allows detection of a range of other diseases including hiv, hepatitis c virus, ebola, methicillin-resistant staphylococcus aureus and flu through the use of pathogen-specific cartridges. the platform produces a wealth of electronic diagnostic and operational data that can be collected and transmitted in real-time, directly from the instrument, utilising connectivity solutions such as the gxalert/aspect platform (systemone, llc, springfield, massachusetts, united states) or c360 (cepheid, sunnyvale, california, united states).24 in the context of tuberculosis, the move towards connected diagnostic platforms has been strongly recommended by the who in the latest global laboratory initiative guideline on connectivity and as part of the end tb strategy. the guideline indicates that all sites that use who-recommended rapid tuberculosis diagnostics should be transmitting results electronically to clinicians and to information management systems, using data connectivity solutions by no later than 2020.24 currently, the existing genexpert footprint in africa is extensive; the who afro accounted for 42% of the global genexpert module procurement in 2016 and almost 65% of total cartridge procurement.25 by leveraging this existing footprint, connected genexpert systems could shorten the detection-response gap, reduce patient loss to follow-up and facilitate early antimicrobial therapy and thus reducing morbidity and mortality.26 automated alerts and triggers the paper-based systems still found in many nations today have notoriously slow reporting cycles. disease surveillance in a nphi may consist of manual tabulation of vital registries and death certificates. these sources are of questionable accuracy (e.g. cause of death listed as ‘fever’ instead of ‘ebola infection’) and typically tabulated on an annual or quarterly basis, which is far too slow to identify an outbreak in progress and prevent it from reaching critical mass. lack of knowledge regarding disease thresholds has also been found to greatly restrict early identification of disease outbreaks.27 connectivity solutions can assist by providing an in-built system to trigger automated electronic alerts when reported cases of a specific disease exceed a predefined threshold. this trigger could be sent to an epidemiologist for specialist analysis or to field staff to verify the occurrence of outbreaks and ensure that prompt control measures and case findings are instituted. setting these thresholds is a crucial component to early warning and outbreak systems and needs to be based on characteristics of the local disease.28 during the 2014–2016 ebola outbreak in west africa, genexpert devices located in mobile laboratories in guinea and sierra leone were interfaced to gxalert to enable real-time collection and management of diagnostic data. gxalert automatically reported ebola-positive cases directly to the laboratory directors in conakry and freetown via sms and email alerts to provide key decision-makers with accurate, reliable and timely information to decrease the time needed to coordinate a response. once the results from diagnostic instruments are digitised, several patient-centred interventions also become immediately possible: faster treatment enrolment: results can be transmitted in real-time via sms, email, online dashboard or connection to another database (e.g. electronic patient record) for faster treatment enrolment. in malawi, systemone’s aspect platform is used to transmit hiv viral load results from the central laboratory directly to the referring clinics in real-time. clinicians confirm receipt to acknowledge that they are acting on the results.29 in the context of amr, the timely feedback of patient results supports clinicians in providing prompt patient management and decision-making on antibiotic use.30 immediate contact tracing: when results are tagged at the laboratory with patient name, phone number and address, this information can be transmitted immediately (or after approval) to contact tracing teams to contain outbreaks earlier. training of staff to maintain confidentiality is critical for the optimal operation of such a system. establishment of ‘sentinel sites’ for outbreak containment and amr surveillance: diagnostic positives in neighbouring countries can alert their national emergency operations centres and the africa cdc’s rccs. by connecting sentinel sites along borders or key points of entry, neighbouring countries can better prevent outbreaks from crossing their borders. developing capacity for amr surveillance and data collection at sentinel sites can help inform antibiotic prescription guidelines and infection control policies, inform intervention needs and help develop an understanding of emergence, transmission and dissemination of pathogens.30 tagging and geo-locating sample transportation: connected, integrated sample transport will contribute to solving the problem of sample loss, delays and misplacement. geo-tagging sample containers with similar identifiers with patient source and point of origin will rapidly improve the speed of sample access and eliminate the administrative bottlenecks of tracing missing samples. operational dashboards and interoperability a further need of the africa cdc is to link various surveillance mechanisms to create a holistic, integrated system. connectivity solutions are not designed to replace existing systems, but rather to feed them faster and with more accuracy than manual collection with critical data. existing disease surveillance systems and laboratory information systems (e.g. global antimicrobial resistance surveillance system, global early warning system, disa, district health management information system-2) are robust tools with well-established processes. what they need most are better sources of timely and accurate data. connected diagnostics help these systems better fulfil their designed purpose. even without complex laboratory information systems, connectivity solutions provide an immediate subset of structured data that is sufficient for core activities. programme managers, the ministry of health, the national reference laboratory and other stakeholders can remotely monitor reliable and accurate patient diagnostic data, rates of positive cases, trends and geospatial information depending on their different levels of access to operational dashboards. these dashboards can provide some data analytic capabilities to support interpretation and exploration of trends and anomalies, and data can also be easily aggregated to multiple levels of stakeholders for real-time decision-making and response. a bottom-up approach to continent-wide antimicrobial resistance surveillance building disease surveillance on connected diagnostics will enable a coordinated response to potential public health threats. in a scenario where a country has diagnostic instruments connected to a system such as gxalert or aspect, for example, digital copies of geo-located, disease-positive cases are stored on a country-level database. each country as well as the africa cdc rccs and headquarters could have access to their own operational dashboard based on the permissions set by the ministry of health (figure 1). this type of system would serve the africa cdc’s need for real-time, geospatial reporting of quality results and enable them to monitor de-identified results of importance, while still preserving the country privacy and controls in place. it would also serve to strengthen the coordinating and supporting role for the rcc in specific sub-regions. figure 1: potential flow of information between national, regional and africa cdc. at each level, the server manages what information and triggering conditions determine further reporting. automated escalations from the national to the rcc level, based on certain thresholds, can initiate effective coordination and allow cross-border surveillance (table 1). significant value is obtained from the ability to receive real-time statistics on disease burden, outbreaks, at-risk populations and other epidemiological metrics for further public health threats. table 1: bottom-up approach to a continent-wide disease surveillance network showing how access can be restricted based on permissions. confidentiality and privacy of health information the protection of both patient privacy and national sovereignty are, naturally, strong concerns. the international health regulations31 provide the legal framework and political agreement needed to help the international community prevent and respond to any potential cross-border threats. however, in practice, creating an effective mechanism for sharing this critical data in a transparent and timely manner has been challenging. the advent of connected diagnostics introduces a new opportunity. the technology is capable of alerting all of the necessary organisations in real-time. the africa cdc has the mandate and the legal underpinnings within the international health regulations to work in concert with nations to negotiate what levels of data access are acceptable and under what conditions (figure 1). the rccs do not need to see details that identify an individual patient and might not need to even see specific results, but could be alerted if more than 10 positive lassa fever results (or plague or ebola, etc.) occurred within a single month. the alert may simply facilitate a conversation between the rcc and the national emergency operations centre about the cases to see if resources are needed. although a diagnostic result is not the same thing as a ‘positive patient’, the information is close enough that for pathogens of public health importance, the appearance of an unexpected number of positive results warrants a conversation between the public health bodies involved, at the minimum. the legal framework supporting this has existed for some years. the technology now exists to establish such a surveillance network very affordably; for example, based on the experience of installing and maintaining connectivity services for more than 2100 cepheid genexperts, abbott m2000s, becton dickinson mgits, alere pimas and qs, and others by systemone, savics and the foundation for innovative new diagnostics (find), the cost to connect the entire continent of africa’s hiv viral load instruments is only around $5 m per year (or $91 000 per african country). doing so would provide the systems needed to give every country full, digital access to their hiv viral load testing results at a national, regional, district or site level, the rcc reduced and de-identified access to hiv data in their region, and the africa cdc the real-time summary information necessary to guide effective policy. putting the entire continent’s hiv drug resistance testing into a multi-tiered amr surveillance network is extraordinarily inexpensive and yields extraordinary amr insights around one of the deadliest diseases in history. the costs for connecting diagnostics for pathogens of concern is, in our opinion, at or below the cost of doing so for hiv. this is attainable in the short term. conclusion as we prepare for future outbreaks and monitoring of amr, we should use the advanced technologies that are available to us in order to evolve and strengthen key factors in ensuring a well-functioning surveillance strategy: laboratory detection, reporting and response systems. the opportunity exists to take advantage of the ability to collect unprecedented amounts of electronic data from diagnostic platforms through connectivity solutions. by connecting the deployed devices at the beginning of an outbreak or, better yet, prior to new outbreaks during simulations of preparedness, a highly reliable and real-time system for detection and response can be activated at ‘patient zero’. immediate reports via any electronic means of positive cases can be sent directly to key decision-makers and response coordinators, reducing the time to response, improving data quality issues and stopping unnecessary deaths due to prolonged disease transmission. reliable, automated data, sent directly from diagnostic systems, enables national, regional, and continent-wide political and health decision-makers to act confidently, as all stakeholders will be utilising high-quality data generated in real-time. acknowledgements competing interests authors n.m.g., j.t. and c.m. are all either employees, shareholders or both of systemone llc, a company that provides a disease intelligence software currently operating in the industry. sources of support 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and response system (cidars). bmc public health. 2017;17:570. https://doi.org/10.1186/s12889-017-4488-0 gous n, chirwa f, takle j, schooley a. reducing time for hiv viral load result delivery to art facilities in malawi. conference on retroviruses and opportunistic infections (croi) 2018; 2018 march 4–7; boston, ma: croi foundation. seale ac, gordon nc, islam j, peacock sj, scott jag. amr surveillance in low and middle-income settings – a roadmap for participation in the global antimicrobial surveillance system (glass). wellcome open r. 2017;2:92. https://doi.org/10.12688/wellcomeopenres world health organization. international health regulations (2005). 3rd ed. geneva: who press, world health organization; 2016. response from attoh et al. acknowledgements references about the author(s) rujittika mungmungpuntipantip private practice, bangkok, thailand viroj wiwanitkit department of community medicine, dy patil university, pune, india citation mungmungpuntipantip r, wiwanitkit v. post-mortem diagnosis of covid-19. afr j lab med. 2021;10(1), a1471. https://doi.org/10.4102/ajlm.v10i1.1471 scientific letter post-mortem diagnosis of covid-19 rujittika mungmungpuntipantip, viroj wiwanitkit received: 26 nov. 2020; accepted: 23 dec. 2020; published: 24 mar. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. we would like to share our impression on the report ‘postmortem diagnosis of covid-19: antemortem challenges of three cases at the 37 military hospital, accra, ghana’.1 attoh et al. concluded that ‘the outcome of covid-19 testing is dependent on the sample type and accuracy of sampling amongst other factors’1 and suggested that ‘more autopsies are required to fully understand the pathogenesis of this disease in ghanaians’.1 indeed, post-mortem diagnosis of coronavirus disease 2019 (covid-19) is possible and there are many reports of the existence of pathogenic viruses in autopsy specimens.2,3 autopsy is also very useful for understanding the pathogenesis of this new disease. however, it must be performed with high caution. while there are no confirmed cases of the pathogen being spread from deceased patients, infection of forensic pathology workers has been reported.4 more autopsies might be recommended, but adequate biosafety and biosecurity, and other infection control precautions must be in place for these to occur. response from attoh et al. in our article we did not go into details on the methodology. although covid-19 is a category 3 infectious agent, negative pressure systems are unavailable in our country. therefore, a few modifications were made. post-mortems were performed using world health organization’s interim guidance for infection prevention and control for the safe management of a dead body in the context of covid-19.5 the structural design of the autopsy suites used lacked negative pressure systems with filters. as a result, extractors were fitted to provide unidirectional airflow away from the anatomical pathology team into the atmosphere as an appropriate technology. also, members of the anatomical pathology team were carefully selected to exclude those with chronic health conditions such as diabetes mellitus, and cardiovascular and respiratory diseases. it is interesting to note that none of the forensic pathologists nor the anatomical pathology technicians involved in the study have shown covid-19 symptoms up to today. acknowledgements competing interests the authors have declared that no competing interests exist. authors’ contributions r.m. and v.w. have equal contributions in giving ideas, drafting, analysing, writing and giving final approval for this submission. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references attoh sa, hobenu f, edusei l, et al. postmortem diagnosis of covid-19: antemortem challenges of three cases at the 37 military hospital, accra, ghana. afr j lab med. 2020 nov 3;9(1):a1290. https://doi.org/10.4102/ajlm.v9i1.1290 sawant ob, singh s, wright 3rd, re, et al. prevalence of sars-cov-2 in human post-mortem ocular tissues. ocul surf. 2020;nov 8;s1542-0124(20)30168–3. zijlstra jg, van meurs m, moser j. post-mortem diagnostics in covid-19 aki, more often but timely. j am soc nephrol. 2021;32(1):255. https://doi.org/10.1681/asn.2020091263 sriwijitalai w, wiwanitkit v. covid-19 in forensic medicine unit personnel: observation from thailand. j forensic leg med. 2020 may;72:101964. https://doi.org/10.1016/j.jflm.2020.101964 world health organization (who). infection prevention and control for the safe management of a dead body in the context of covid-19: interim guidance [homepage on the internet]. world health organization, 2020; p. 6. who reference number: who/2019-ncov/ipc_dbmgmt/2020.2. available from: https://www.who.int/publications/i/item/infection-prevention-and-control-for-the-safe-management-of-a-dead-body-in-the-context-of-covid-19-interim-guidance abstract introduction methods results discussion acknowledgements references about the author(s) xoliswa l. mpumlwana department of quality control, south african national blood service, roodepoort, south africa winnie kruger department of biomedical sciences, faculty of health sciences, university of johannesburg, johannesburg, south africa ute jentsch department of specialized lab services and quality control, south african national blood service, roodepoort, south africa citation mpumlwana xl, kruger w, jentsch u. establishment of a stable proficiency testing matrix in transfusion microbiology in south africa. afr j lab med. 2023;12(1), a2095. https://doi.org/10.4102/ajlm.v12i1.2095 original research establishment of a stable proficiency testing matrix in transfusion microbiology in south africa xoliswa l. mpumlwana, winnie kruger, ute jentsch received: 20 oct. 2022; accepted: 13 june 2023; published: 30 aug. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: all medical laboratories must participate in proficiency testing (pt) programmes to ensure high-quality results. proficiency testing samples mimic clinical samples; however, pt programmes for detection of bacteria in blood products are not routinely performed due to unavailability of matrix-equivalent samples. objective: the aim of this study was to develop and test a matrix-equivalent pt programme using blood products as the basis matrix. methods: a prospective cross-sectional study was conducted from april 2021 until june 2021, using 52 blood products comprising 36 pooled platelet and 16 red blood cell products at the south african national blood service pt laboratory in gauteng. products were manipulated into matrix-equivalent pt samples by spiking 42 products with known bacterial strains at specific concentrations and treating the remaining 10 products with preserving fluid containing antibiotics. the level of agreement between the researcher results and participating laboratories’ results was assessed. results: of the prepared matrices, 568 out of 572 (99%) were stable for 30 days. bacteria could correctly be identified in spiked samples for up to 23 days. samples treated with preserving fluid remained negative until day 30. for spiked samples, an average of 98% agreement (153/156) was achieved between the three participating laboratories when compared with the researcher’s results; 100% agreement was achieved for unspiked samples. the kappa scores obtained from all tested variables presented with scores between 0.856 and 1.000, and the p-value was < 0.001 throughout. conclusion: the developed pt matrix was therefore stable and suitable to be implemented in transfusion microbiology. what this study adds: this study demonstrated that a stable microbiology pt programme using platelets and red blood cells can be developed for use on bacterial detection analysers and could help to close the gap presented by unavailability of a blood pt matrix for transfusion microbiology. keywords: south african national blood service; bacterial contamination; transfusion-transmissible bacteria; proficiency testing matrix; bacterial stability testing. introduction blood products provide therapeutic and life-saving benefits to the recipients. international best practice guidelines provide evidence-based information on how to mitigate against transfusion-transmitted bacterial infections and assure blood safety from collection to issuing. despite these measures, which include screening of donors prior to blood donation, use of effective antiseptics, screening of donated blood for viral markers and aseptic technique during processing of blood and blood products, bacterial contamination of blood products, especially of platelets, remains a significant infectious hazard in blood transfusion.1,2,3 while viral causes of transfusion-transmissible infections are effectively screened for, bacterial contamination of blood products remains a significant risk. platelets, when compared to other blood products, are more prone to bacterial contamination because of their storage conditions at 20 °c – 25 °c, which are conducive to bacterial growth.3,4 quality control testing of platelets and other blood products in microbiology using bacterial culture and bacterial identification methodologies have been implemented in the blood transfusion field as a further mitigating practice.5 these screening methodologies have shown effectiveness and have decreased events of transfusion-transmitted bacterial infections due to early detection of bacteria in contaminated products.6,7 rapid classification and detection of microbes is essential for the prevention of treatment failures and rapid spread of antimicrobial drug resistance.8 the south african national blood service (sanbs) quality control testing for bacterial screening is performed on 1% of the donated apheresis and pooled platelet (pp) products.9 the sanbs and other blood transfusion services are experiencing a quality gap as there are no formal microbiology proficiency testing (pt) matrix programmes for blood product safety testing. lyophilised and frozen commercial pt programmes were validated for use in verification of the sensitivity of bacterial detection instruments.10 these commercial pt programmes do not fully cover the matrix effect as some human manipulation is still required during the testing process; they require either thawing or making up volume with sterile water. the pt matrix is the totality of components within the sample in addition to what is being measured and represents real-time samples in terms of viscosity, turbidity, composition and colour.11 this study developed and tested a blood product-based pt matrix as a foundation to develop a full programme in the future. the developed matrix did not require any manipulation by the participating laboratories; all matrix component requirements were met. the requirements of this matrix are that it must be stable and support bacterial growth. methods ethical considerations this study was approved by the faculty of health sciences, university of johannesburg (hdc-01-3-2019), and the sanbs human research ethics committee (hrec, sanbs: 2019/0476). donors provided consent prior to the blood donation process for using some of their blood for blood safety improvement purposes. the participating laboratories were invited and gave consent to take part in the study as independent evaluators of samples. anonymity and confidentiality were maintained by giving each donation a unique identifier and data were collated in a microsoft excel (microsoft corp., redmond, washington, united states) spreadsheet, decoded, and stored electronically in a password-controlled folder. sample population the study took place in the sanbs proficiency testing laboratory in johannesburg, south africa, between april 2021 and june 2021. whole blood units donated by eligible blood donors were collected by qualified phlebotomists using sanbs-approved protocols. donated whole blood units were couriered at temperatures between 1 °c and 10 °c by the sanbs transport department from the donor centres to a centralised sanbs processing site. each whole blood unit was processed and separated into red blood cells (rbc), platelets and plasma components. thereafter, five platelet units from five different whole blood donors with the same blood group were pooled together to make one pp unit. the statistical value for sample size was calculated based on a method used on two-rater studies to detect a statistically significant kappa on dichotomous variables. based on the availability at the time of sampling, blood group a+ units were mostly used in this study as there were excess units available and using these would not have a negative impact on blood supply.12 pooled platelet units (n = 36) and excess a+ rbc packed-cell units (n = 16) were produced for the purposes of this study and sent to the pt laboratory for the production of blood matrix pt samples. study design, sample testing, materials and instruments this was a prospective cross-sectional study that was approached in two phases, namely the preparation of the pt matrix and the testing of the prepared pt matrices. preparation of proficiency testing matrices blood product units consisting of pp and rbc concentrates were obtained and tested for baseline sterility prior to manipulation into pt matrices. no additional supplements were added to the concentrates while conducting the study. after sampling for baseline sterility testing, 42 of the 52 units, comprising 29 pp and 13 rbc concentrates, were each spiked with 1 ml of a known bacterial strain using coupled spikes. the selected bacterial strains known to cause transfusion-transmitted bacterial infections included staphylococcus epidermidis, staphylococcus aureus, streptococcus pyogenes, streptococus bovis, streptococcus dysgalacticae, bacillus cereus, bacillus thuringiensis, listeria monocytogenes, enterobacter cloacae, serratia marcescens, escherichia coli, morganella morganii, pseudomonas fluorescens, klebsiella pneumoniae and serratia liquefaciens.13 the bacterial concentrations ranged between 0.5 and 0.63 macfarland standard, utilising a variety of common gram-positive and gram-negative bacteria. only one bacterial strain was introduced into each product unit or concentrate during inoculation. the bacterial strains used were validated by the paul ehrlich institute and are available at the world health organization as reference strains for use in blood transfusion services.14 the remaining unspiked 10 units were treated with 1 ml preserving fluid containing bacteriostatic agents. the use of the preserving fluid was to ensure that the non-spiked units remain negative until 7 days after the closing date.13 the pp units were stored at 20 °c – 25 °c while rbc units were stored at 2 °c – 6 °c for 48 h, which are the standard storage conditions for these products to allow bacterial proliferation to take place. confirmation of intended results of manipulated units was performed after 48 h storage using gram staining, bacterial culture, bacterial identification, and antimicrobial susceptibility testing (ast) by the researcher. this was followed by splitting of units; each unit was split into 15 samples (n = 780 pt samples in total). samples were tested at three different stages, namely establishment of reference results (n = 52), stability testing (n = 572) and laboratory testing for evaluation purposes (n = 156). sample identification is shown in figure 1. figure 1: sample split summary conducted at the south african national blood service in johannesburg, south africa, between april 2021 and june 2021. safety precautions and prevention of cross-contamination of samples during preparation aseptic technique was followed throughout the preparation of the pt matrix samples to prevent cross-contamination. the samples were prepared inside a class ii biosafety cabinet (esco lifescience, singapore) with one unit or bacterial strain at a time. the environment in which the preparation took place was monitored by taking samples from the air, surfaces and researchers’ hands. testing of proficiency testing matrix samples testing of the prepared pt matrix was conducted in two stages, namely testing by the researcher and testing by the participating laboratories. all samples were subjected to bacterial culture and, if positive, gram staining, bacterial identification and ast were performed. two approved and commonly used bacterial culture testing platforms, where bacterial strains metabolise the substrates of the culture media, were used, namely bact/alert 3d (biomérieux, craponne, france) and bactec 9120 (becton dickinson, san jose, california, united states), and vitek ii compact (biomérieux, craponne, france) was used for ast. testing to establish reference results reference results of analysed spiked and non-spiked units had to confirm the absence and presence of bacterial growth and, if bacterial growth was observed, bacterial identification as well as ast pattern were determined. the reference results were expected to agree with the expected result per process. after confirmation of the reference results, the pt matrix samples were aliquoted into smaller 5 ml vials to prepare them for storage and independent testing by participating laboratories. stability testing stability testing involved storing of samples at different temperature ranges (2 °c – 6 °c, 20 °c – 25 °c, and 37 °c) and random selection of these samples for testing on different days over a period of 30 days. a time period of 30 days was selected to ensure maximum time for analysis and stability testing. this time frame also allowed for sample transportation to participating laboratories, storage at participating laboratories prior to testing, analysis and submission of results to the researcher. the results obtained from all samples tested for stability on scheduled testing days were expected to be similar to the reference results. testing by participating laboratories replicas of the same aliquoted samples were selected, three vials from each of the 52 units (n = 156). the 156 samples were further split into three sets of 52 identical samples which included spiked and unspiked samples. these samples were packed and transported at ambient temperature to three independent participating laboratories according to international air transport association regulations.15 these laboratories are south african national accreditation system accredited to international organization for standardization standard 15189:2012. to ensure maintenance of temperature chain and validity of results, the participating laboratories were instructed to store the pt samples at ambient temperature until testing time. the samples were to be tested within 14 days of receipt according to their respective laboratory protocols. after testing, the participants sent the results to the researcher for evaluation against preset limits of acceptability and the reference results. statistical analysis qualitative data obtained from the participating laboratories and from stability testing included two dependent variables, such as positive/negative, sensitive/resistant, correct/incorrect. post capturing, qualitative data were coded and converted into a quantitative form, for example 1 for positive and 2 for negative, and analysed using cohen’s kappa (k) statistical method. the statistical package for social sciences program (ibm, bengaluru, india) was used to measure cohen’s kappa. the inter-rater reliability agreement and the p-value cut-off points were calculated as: k > 0.75 and p ≤ 0.05, indicating statistical significance. the kappa score and p-value were evaluated between the researcher and the participating laboratories as well as for stability testing results. results reference and stability testing results reference testing results by the researcher were similar to results obtained during preparation of the matrices for spiked and the non-spiked units, thus 100% (52 out of 52) compliant results were obtained. table 1 indicates the number of products spiked with a specific bacterial strain; for example, s. epidermidis was used to spike three pp and one rbc concentrates. table 1: bacterial strains used to spike products at the south african national blood service in johannesburg south africa between april 2021 and june 2021. for stability testing, 52 out of 52 (100%) compliant results were obtained until day 14 for both spiked and unspiked units; thereafter, only pp and rbc units spiked with l. monocytogenes started showing signs of deterioration. from day 23, m. morganii failed to grow on pp and rbc units. a 99% (206 out of 208) level of agreement was achieved for stability testing from all variables tested. bacterial culture and gram stain level of agreement for stability testing a high level of agreement was obtained across all testing days for bacterial culture results stability detection with kappa scores of > 0.75 (figure 2). the p-value for all testing days was < 0.001. from day 1 until day 9, k = 1.000 was maintained. a decrease in the kappa score was observed from day 9 to day 14, where k = 0.942, and a further decline on day 30, with k = 0.856. figure 2: kappa scores for bacterial culture and gram stain stability testing for spiked and unspiked concentrates at the south african national blood service in johannesburg, south africa, between april 2021 and june 2021. bacterial identification level of agreement for stability testing the stability testing for bacterial identification indicated a cohen’s kappa score of 1.000 on day 1 versus day 3, day 1 versus day 5 and day 1 versus day 9, as illustrated in table 2. day 1 versus day 14, as well as day 1 versus day 23, had a kappa score of 0.955, while day 1 versus day 30 indicated a score of 0.842. the p-value obtained across all the testing days was < 0.001. table 2: bacterial identification stability testing kappa statistics for pairs of measures at the south african national blood service in johannesburg, south africa, between april 2021 and june 2021. antimicrobial susceptibility testing level of agreement for stability testing gram-positive and gram-negative results obtained from all ast cards utilised had a constant statistical value from day 1 until day 30. a high level of agreement for ast stability testing was obtained with k = 1.000, p < 0.001 across all testing days. comparative results between the researcher (reference results) and participating laboratories a high level of agreement (98% to 100%) between the reference standard and participating laboratories was obtained. two percent of discrepant results were obtained for gram stain and bacterial identification, while 98% of the results were comparable to the reference results. the results for all tested variables are summarised in table 3. table 3: antimicrobial susceptibility testing kappa statistics for pairs of measures between the researcher and the participating laboratories at the south african national blood service in johannesburg, south africa, between april 2021 and june 2021. bacterial culture level of agreement between researcher and participating laboratories eighty-one percent (42 out of 52) of the samples were expected to yield positive bacterial culture results while 19% (10 out of 52) were expected to give negative results during testing by both participating laboratories and the researcher. a high level of agreement was obtained between all participating laboratories and the reference results, with k = 1.000 and p < 0.001 across all participating laboratories (table 3). gram stain level of agreement between researcher and participating laboratories the samples for gram stain consisted of 16 out of 52 (30.8%) gram-positive cocci, 7 out of 52 (13.5%) gram-positive bacilli, 19 out of 52 (36.5%) gram-negative bacilli and 10 out of 52 (19.2%) with no bacteria observed. laboratory 1 obtained a kappa score of 0.947, and laboratory 2 and 3, 0.920, when compared to the researcher. laboratory 2 and laboratory 3 had kappa scores of 0.974 and 0.867 when compared to laboratory 1, while laboratory 3 had a 0.841 kappa score when compared to laboratory 2. a high level of agreement, shown in table 3, between the researcher and all participating laboratories was obtained as well as a p-value of < 0.001, across all participating laboratories. bacterial identification level of agreement between researcher and participating laboratories a high level of agreement, summarised in table 3, was obtained across all participating laboratories and the researcher for bacterial identification. laboratory 1 and laboratory 2 had a 0.958 kappa score when compared to the researcher, while laboratory 3 had a score of 0.894. laboratory 3 had a kappa score of 0.894 when compared to laboratory 1 and laboratory 2. a kappa score of 1.000 was obtained between laboratory 1 and 2. a statistically significant p-value of < 0.001 was obtained across all participating laboratories. antimicrobial susceptibility testing level of agreement between researcher and participating laboratories all participating laboratories obtained results similar to the researcher for ast, with k = 1.000, p < 0.001, indicating a high level of agreement and statistical significance across all participating laboratories, as illustrated in table 3. discussion in this study, a stable microbiology pt programme using platelets and red blood cells as the basis matrix has been developed to be used on bacterial detection analysers. unlike the commercially available lyophilised and frozen pt programmes, detection of active bacterial growth metabolism on blood products was observed in this study.10 there were no discrepancies observed between the reference results and the participating laboratories for bacterial culture and ast results. all positive and negative cases were identified correctly, showing a high level of agreement. the deviations from the reference results observed do not discredit the newly developed pt matrices; however, non-complying results must be investigated. participating laboratories must address non-complying results promptly according to appropriate quality management systems.16 the results obtained during stability testing demonstrated that storage time of up to 23 days did not influence the outcome of results for most bacterial strains, except for l. monocytogenes. the laboratory test results obtained during storage in different temperature ranges also confirmed that the newly developed pt matrices were stable. two out of 42 bacterially spiked samples started showing some degree of deterioration as they could not give a positive signal, from days 14 and 23. the two non-compliant results were from both pp and rbc, and these were both spiked with l. monocytogenes. listeria monocytogenes was unable to grow beyond day 14 as it requires specific growth substances in addition to what was provided by the pp and rbc concentrates. these specific growth substances include amino acids such as leucine, methionine, arginine as well as vitamins such as riboflavin, thiamine, biotin and thioctic acid.17 in a study conducted by prax et al., it is stated that l. monocytogenes was slow growing in cold stored red cells from days 7 to 28.18 both platelet and rbc bags were not supplemented with the required amino acids and vitamins to support the growth of l. monocytogenes. brain heart infusion broth which has been confirmed to be effective in the cultivation of a wide range of microbes, could be considered as the base medium to augment growth. this broth contains protease peptone and other infusions which serve as sources of amino acids, vitamins as well as vital growth factors.18 at day 30, acceptable agreements were obtained; however, 5 out of 42 bacterial spiked samples showed signs of deterioration for bacterial culture. blood products spiked with l. monocytogenes and m. morganii were mostly affected. morganella morganii could not be identified on day 30 by either the researcher or the participating laboratories. this bacterial strain was validated and accepted during enlargement of the world health organization international repository for platelet transfusion.19 during that validation, m. morganii was spiked into platelet concentrates and stored for up to seven days at 20 °c – 25 °c; in comparison, the storage in this matrix-equivalent pt establishment study was for up to 30 days at 20 °c – 25 °c. based on observation from this study, one can then conclude that m. morganii is stable for 23 days, as all pp and rbc concentrates spiked with this bacterial strain had growth until day 23. staphylococcus aureus and s. pyogenes showed signs of deterioration at day 30. one participating laboratory identified staphylococcus aureus as staphylococcus sciuri and streptococcus pyogenes as alicyclobacillus acidoterrestris, on day 30. in a real case scenario, misidentification of bacteria could lead to patient mistreatment or incorrect treatment or no treatment at all; for example, when a gram-positive coccus is identified instead of a gram-negative bacillus, incorrect antimicrobial agents effective for killing or inhibiting only the growth of gram-positive cocci may be used for treatment of patients. bacterial identification discrepancies observed on day 30 could have been caused by poor aseptic technique during testing on that day by the participating laboratory. the pp and rbc treated with preserving fluid remained negative throughout the study. it can therefore be concluded that no contamination occurred during storage of the pt matrices. limitations the study is currently limited to pt samples transported by road as stability testing was not performed on samples transported by air. the pt samples were only tested on bacterial culture methods detecting active bacterial metabolism and not on other platforms such as flow cytometry and rapid bacterial detection methods. further findings indicated a limitation on l. monocytogenes’ inability to grow on platelet and rbc concentrates; this could be due to method limitation. it is recommended that growth of l. monocytogenes be enhanced by the addition of emulsified microbe into brain heart infusion enrichment media prior to spiking of concentrates.17 conclusion it was concluded that the majority of the newly developed blood pt matrices were stable for up to 23 days at all storage conditions between 2 °c and 37 °c; therefore, pt matrices will be suitable for sending to areas where delivery can be achieved within 7 days from dispatch. listeria monocytogenes will be excluded from the pt matrices and further studies will be conducted. this matrix will cover gram staining, bacterial screening and identification as well as antimicrobial susceptibility testing. the blood product matrix will eliminate matrix-related problems such as biases and uncertainties caused by use of the inappropriate matrix (lyophilised and frozen) while performing pt. the outcome of this study, therefore, provided a possible solution for closing the gap of the unavailable blood pt matrix for transfusion microbiology. further validation and comparison for reproducibility before being offered as an official pt programme for transfusion microbiology are suggested. once implemented successfully locally, these pt samples will be validated for shipment to african countries and other countries beyond south african borders to support blood safety. acknowledgements the authors would like to thank the south african national blood service proficiency testing and quality control departments for availing their premises and resources, as well as the national health laboratory service for taking part in the study. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions x.l.m. devised the project, the main conceptual ideas and proof outline, performed the experiments, processed the experimental data, interpreted results and performed analysis, drafted the manuscript and designed all the figures and tables. w.k. supervised the entire project while u.j. co-supervised. both w.k. and u.j. analysed the data, reviewed and edited the manuscript. all authors discussed the results and commented on the manuscript. sources of support the funding of this study was provided by the south african national blood service as part of the operational budget. data availability the data that support the findings of this study are not openly available due to ethical reasons, but are available from the corresponding author, x.l.m., upon reasonable request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references agzie m, niguse s, 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red blood cells. vox sang. 2021;116(6):692–701. https://doi.org/10.1111/vox.13057 spindler-raffel e, benjamin rj, mcdonald cp, et al. enlargement of the who international repository for platelet transfusion-relevant bacteria reference strains. vox sang. 2017;112(8):713–722. https://doi.org/10.1111/vox.12548 abstract introduction signs and symptoms of inherited metabolic disorders basic methods for the laboratory investigation of inborn errors of metabolism purine metabolism disorder conclusion acknowledgements references about the author(s) john i. anetor department of chemical pathology, faculty of basic medical sciences, university of ibadan, ibadan, nigeria bose e. orimadegun department of chemical pathology, faculty of basic medical sciences, university of ibadan, ibadan, nigeria gloria o. anetor department of public health science, faculty of health sciences, national open university of nigeria, abuja, nigeria citation anetor ji, orimadegun be, anetor go. a pragmatic approach to the diagnosis of inborn errors of metabolism in developing countries. afr j lab med. 2023;12(1), a1946. https://doi.org/10.4102/ajlm.v12i1.1946 review article a pragmatic approach to the diagnosis of inborn errors of metabolism in developing countries john i. anetor, bose e. orimadegun, gloria o. anetor received: 30 apr. 2022; accepted: 05 apr. 2023; published: 30 may 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract inborn errors of metabolism (iem) are a group of genetically derived diseases that are individually rare but collectively common and can be very severe. while high-income countries usually employ modern scientific technologies like tandem mass spectrometry for iem investigation, these disorders are, in contrast, only rarely screened for in developing countries due to misconceptions that the required facilities are beyond the reach of these countries. this paper attempts to educate scientists and clinicians in developing countries on low-technology iem screening methods that only require moderate facilities. although a definitive diagnosis of iem may require specialised laboratory investigations and attendant interpretation, in most cases, the basic facilities available in the average clinical chemistry laboratory in developing countries can allow the early detection of iem. this early detection would facilitate critical early decision making, thus leading to better management, optimised treatment, and reduced morbidity and or mortality of iem in these resource-limited countries. with this approach, a few referral centres for confirmatory investigation, comparable to those existing in developed countries, could be established. this can be integrated into creative health education for healthcare professionals and families who have individuals with iem. what this study adds: iems are important enough that every country, developed or developing, should have screening plans and basic laboratory facilities that are adequate for initial iem diagnosis. no country should therefore give up on testing for iems on the excuse of a paucity of advanced facilities. keywords: basic laboratory investigations; developing countries; inborn errors of metabolism; health education; optimisation of treatment. introduction inborn errors of metabolism (iem), also referred to as inherited metabolic disorders, are mutational disorders that impair the function of proteins in every way (mostly enzymatic functions). archibald garrod, who was the first to explain the relationship between genotype and phenotype, helped bring attention to iem through his pioneering work on one of the first types of iem discovered – alkaptonuria.1,2 his work helped to elucidate iem, thus paving the way for the realisation that every aspect of human anatomy and physiology is determined by biochemical reactions catalysed by specific enzymes, and that enzymes are determined by genetic constitution. since then, several additional types of iem have been described, and many more are being investigated (table 1).3 although iems are quite common collectively, individual iem disorders are rare. the most frequently encountered iems are monogenic forms.4,5 in most cases, iems are autosomal recessive. table 1: types of inborn errors of metabolism commonly investigated using laboratory procedures. according to the 2003 institute of medicine report ‘reducing birth defects: meeting the challenge in the developing world’,6 birth defects are the leading cause of infant mortality in many regions of developing countries.7 in these countries, malnutrition, poverty, disease, and lack of access to healthcare elevate the prenatal risk of birth defects and impose enormous personal and societal consequences. experts in developed countries often have similar concerns because birth defects are also the highest single cause of infant death in many regions.8 there is a link between birth defects and genetic abnormalities such as iems,7 and environmental and genetic factors are inextricably linked. as these gene-environment interactions are important in iem, people in developing countries are at a greater risk of developing an iem.9 this highlights the need for scientists and doctors to prioritise iem detection, management, and prevention through research aimed at identifying environmental red flags. a holistic and integrated approach is also required for the effective investigation and treatment of iem. to achieve this, collaboration across the distinct disciplines of genetics, evolutionary and developmental biology, environmental science, and sociology is necessary.7 this article is, in part, an extended response one of the authors gave at a laboratory medicine congress in north africa in late 2019, where a questioner inferred that iems are not screened for or diagnosed in developing nations because the needed specialist laboratory facilities are not available. although a recent report from colombia (a developing country) by echeverri et al.10 outlines the activities in their reference centre that support the questioner’s position, their diagnostic approach is not pragmatic or applicable to most developing countries but may apply to referral centres in developing countries. nevertheless, they did make a notable point about the importance of health education for both health and non-health professionals, who are critical and underappreciated in the field of iem. a recent report by civallero, de kremer and giugliani11 proposed the use of a few simple and affordable instruments such as spectrophotometer and gas chromatography-mass spectrometry for iem investigations, as these can give an enormous amount of information that can be adapted to the needs of health facilities in developing countries. this approach contrasts that of echeverri et al.10 and may be more relevant to health facilities in developing countries. the mayo clinic has also outlined another diagnostic approach that can be modified for the qualitative and quantitative determination of diagnostic markers of iem (table 2).12 table 2: general methods for the qualitative and quantitative determination of diagnostic markers of inborn errors of metabolism. clinically, iem can be grouped into three categories, namely, disorders that cause intoxication, disorders that affect energy metabolism, and disorders that affect complex molecules.13,14 in disorders of intoxication, metabolites accumulate at toxic concentrations and the accumulating metabolites are usually proximal to the metabolic block. consequently, identifying these metabolites provides a basis for diagnosis using basic laboratory facilities, which are readily affordable in resource-limited settings. therefore, this article aims to raise awareness of basic investigations that can detect iem early and do not require expensive or sophisticated equipment commonly used in developed countries. a quasi-systematic review was conducted where the science citation index and pubmed databases were searched, independent of date of publication, for articles on iem. we prioritised articles that were published in developing countries and articles published by leading authorities and institutions. we also included the early lectures of the ‘father of inborn errors of metabolism’, archibald garrod.1,2 we conducted a limited synthesis of all retrieved articles and found evidence of the paucity of investigations of iem in developing countries due to the lack of facilities, which we have tried to address in this article. a major limitation of our approach is that it only provides a cursory overview of the topic. signs and symptoms of inherited metabolic disorders there are some common signs and symptoms that raise the index of suspicion of iem.15 these include acute life-threatening crisis, lethargy or coma with or without a symptom-free period, seizures, persistent vomiting, respiratory distress syndrome (apnoea), poor feeding/failure to thrive, hypotonicity/hypertonicity (ataxia, posturing), hepatosplenomegaly/jaundice, dysmorphic features (facial coarsening), macroglossia, ocular features (cataract, corneal clouding, abnormal eye movements), coarse hair with abnormal texture, abnormal odour of urine/body, loss of developmental milestones, unusual responses to fasting and intercurrent infection (von gierke’s disease, type 1 glycogenosis), and fluctuating neurological status. basic methods for the laboratory investigation of inborn errors of metabolism alkaptonuria alkaptonuria is one of the earliest iems described by garrod.2 this is a benign disorder that poses no danger to life except for ochronosis (darkening) of the cartilage, and patients with this condition are prone to arthritis from the fourth decade of life. alkaptonuria is characterised by the darkening of standing urine, and a simple benedict’s reaction on the urine sample will give a positive reaction. all that is required for this test is copper sulphate in an alkaline medium, which is inexpensive. phenylketonuria some basic investigations can also aid in the early detection of phenylketonuria, which is one of the most well-known and potentially fatal iems that is associated with profound intelligence quotient loss and severe mental retardation (mr). phenylketonuria can easily be diagnosed using the fecl3 test (on which the phenistix test is based) that requires only a 10% fecl3 solution, the guthrie test, amino acid analysis using high-performance liquid chromatography or tandem mass spectrometry. the latter two methods may be expensive but could be made available in a referral centre. the guthrie screening test for increased phenylalanine in serum is one alternative to specialised high-performance liquid chromatography for amino acid analysis that is within the reach of many laboratories in developing countries. it is a microbiological test that relies on phenylalanine’s ability to counteract the effects of a metabolic antagonist on the growth of a bacillus subtilis strain that requires phenylalanine as a growth factor. the test procedure involves the suspension of b. subtilis spores in an agar medium containing a minimum amount of growth nutrients plus a fixed amount of the metabolic antagonist, β-2-thienylalanine, which is a non-protein amino acid similar in structure to phenylalanine. when the infant is 6 days old (or as soon as possible if the baby is being treated with antibiotics that interfere with the test), blood is drawn by heel prick onto filter paper, dried, and sent to the laboratory. these blood spots are cut into uniform discs and then placed on agar plates inoculated with the b. subtilis isolate, with the only source of phenylalanine being the blood spot. the blood spot discs are compared to discs containing known amounts of phenylalanine. if the serum contains a high level of phenylalanine, the phenylalanine will diffuse from the sample disc into the bacterial medium to counteract the inhibitory effect of thienylalanine, resulting in a ring of bacterial growth around the disc. if the phenylalanine level is less than 2 mg/dl, the growth inhibition will not be overcome, and no bacterial growth will be observed. as false positives can occur, positive results should be confirmed using a chemical method or chromatography. to avoid false positive results due to liver immaturity, tests should not be performed on premature infants or full-term babies immediately after birth. the infant should also be on a phenylalanine-containing diet for at least 24 h before the sample is collected.3 maple syrup urine disease in maple syrup urine disease, there is a deficiency of the branched chain α-ketoacid dehydrogenase enzyme that decarboxylates the branched chain amino acids (leucine, isoleucine, and valine). this results in the accumulation of these amino acids and their metabolites (a-ketoacids) in the blood. maple syrup urine disease is a severe iem that begins in the first year of life and progresses to severe mr, acidosis, coma, and death within 5 years if left untreated. as an alternative to tandem mass spectrometry and high-performance liquid chromatography analysis of organic acids, maple syrup urine disease can easily be detected in the laboratory using the rothera’s test. this requires a simple sodium nitroprusside reagent (a common reagent in most clinical chemistry laboratories) and ammonium hydroxide. when mixed and pulverised, the test retains its positive reaction, unlike in cases of metabolic derangement such as type 1 diabetes mellitus (diabetes ketoacidosis), where the ketone bodies (acetoacetate, acetone, and 3-hydroxy butyrate) are responsible for the positive reaction. the clinical observation of the characteristic offensive odour of the urine (isovaleric aciduria), which may be the first suggestive sign of abnormal metabolite excretion, may be an indication for this investigation. alpha-1-antitrypsin deficiency another iem that can readily be investigated with the moderate facilities available in most developing countries, is alpha-1-antitrypsin deficiency. alpha-1-antitrypsin is an antiprotease synthesised in the liver, and its deficiency arises from point mutations in the serpina1 gene (single amino acid substitution), with over 70 alleles described.16 the normal genotype is pimm, and pimz is the heterozygous genotype for the z gene. alpha-1-antitrypsin deficiency frequently arises from pizz (the homozygous genotype for the z gene).17 this genetic defect causes the protein to form aggregates in the liver that cannot be excreted, thus causing liver damage. the absence of its antiprotease activity also causes emphysema and cirrhosis in children. although alpha-1-antitrypsin deficiency is commonly investigated using polymerase chain reaction in advanced laboratories, the combination of traditional liver function tests and cellulose acetate serum protein electrophoresis, when the α-band is either significantly reduced or completely absent, is highly suggestive. wilson’s disease wilson’s disease (hepatolenticular degeneration) is another iem that can easily be investigated in a basic clinical laboratory. the disease is due to a mutation in the atp7b gene, which encodes the copper-transporting protein, ceruloplasmin. wilson’s disease is easily identified by kayser fleischer’s rings (almond-coloured rings around the cornea). in the laboratory, wilson’s disease can be diagnosed by determining serum copper levels using simple flame atomic absorption spectrophotometry and ceruloplasmin by standard spectrophotometric methods. the result will show low ceruloplasmin concentrations and serum copper levels accompanied by massive copper excretion in the urine. if necessary, a liver biopsy can be done to detect excessive amounts of copper in the liver. cystic fibrosis cystic fibrosis is a common iem that affects one out of every 2500 live births in the united kingdom.18 cystic fibrosis is inherited in a recessive mendelian pattern, and individuals with this disease experience a generalised exocrine secretion disorder that is characterised by abnormally viscous secretions. the functional defect is caused by the cystic fibrosis transmembrane conductance regulator protein, which controls transmembrane chloride transport.19 surprisingly, cystic fibrosis patients who do not have functional copies of the protein have a slew of lung and digestive problems.20 symptoms include recurrent respiratory infections, irreversible lung disease, and pancreatic insufficiency leading to malabsorption. in neonates, intestinal obstruction can occur due to the increased viscosity of faecal material, which is useful in laboratories for disease screening. cystic fibrosis can be identified clinically through slit-lamp examination of affected patients’ eyes, which may reveal pathognomonic crystals of cystine in the cornea. this corneal defect can cause photophobia, beginning around the age of two. cystic fibrosis can be screened for using immunoreactive trypsin in blood. also, faecal pancreatic elastase-1 is the most widely used test to diagnose pancreatic insufficiency in people with cystic fibrosis. sweat electrolyte analysis is used to make the final diagnosis; when sweat chloride level is less than 30 mmol/l, cystic fibrosis is not likely, but when it is greater than or equal to 60 mmol/l, it is diagnostic.21 pilocarpine iontophoresis (using an electric device to stimulate sweating) is also a standard test used in the diagnosis of cystic fibrosis. galactosaemia galactosaemia is caused by a lack of galactose-1-phosphate uridyltransferase, a rate-limiting enzyme that causes hypoglycaemia. this leads to increased levels of galactose-1-phosphate due to blockage of the typical metabolic pathway, and this may lead to alternative metabolism and direct tissue damage. some key biochemical features of galactosaemia include impaired bilirubin uptake and conjugation, increased unconjugated hyperbilirubinemia, hepatomegaly, jaundice, severe mr, accumulation of free galactose, and deposition of galactose-1-phosphate in renal tubules with attendant renal damage and associated generalised aminoaciduria. simple and practical tests that can be used to investigate galactosaemia include benedict’s test to detect the presence of reducing substances, thin-layer chromatography to determine the presence of galactose in urine, and galactose-1-phosphate uridyltransferase enzyme assay to detect elevated blood galactose. amniocentesis may be used for prenatal diagnosis, and the galactose tolerance test may be performed if the expected rise in the blood glucose level after galactose administration is not observed. in advanced laboratories, molecular analysis is available.22 the diagnosis of galactosaemia is well within the capabilities of most basic medical facilities and the treatment is simple, only requiring the elimination of galactose from the diet. von gierke’s disease in von gierke’s disease, there is deficiency of glucose-6-phosphatase, an enzyme that splits glucose from glucose-6-phosphate (glucose-6-po4 + g-6-phosphatase → glucose + po4). owing to this metabolic block, glucose-6-phosphate accumulates in the liver. the presence of fasting hypoglycaemia and hepatomegaly are strongly suggestive of von gierke’s disease and clearly distinguishes it from other glycogenoses such as pompe’s, mcardle’s, and many others. urea cycle disorders type 1 hyperammonaemia type 1 hyperammonaemia is one of the common urea cycle disorders (ucds). it is an autosomal recessive disorder that is caused by a deficiency of the carbamoyl phosphate synthetase 1 enzyme. the main biochemical characteristics of ucds are extremely high blood ammonia levels, mr, and a low urea level. because of the mr that comes with this metabolic disorder, early detection is critical, and the diagnosis is within the capabilities of a typical laboratory. to screen for type 1 hyperammonaemia, advanced methods such as amino acid analysis using high-performance liquid chromatography or tandem mass spectrometry may be required. in a resource-limited medical facility, blood gas and electrolyte abnormalities are important for detecting ucds. the presence of reduced blood urea levels, increased ammonia levels, elevated transaminase (alanine transaminase and aspartate transaminase) activity, and coagulopathy (increased prothrombin time and partial thromboplastin time) should be strongly suggestive of a urea cycle defect and risk of encephalopathy and should be sufficient basis for referring patients for specialist investigation and initiating early patient management. hyperammonaemia type ii the biochemical lesion of hyperammonaemia type ii is a deficiency of the ornithine transcarbamoyl transferase enzyme, and the inheritance pattern is x-linked. the key features of this disorder include elevated ammonia levels in the blood, cerebrospinal fluid and urine, as well as orotic aciduria caused by carbamoyl phosphate channelling into pyrimidine synthesis. hyperammonaemia is caused by a lack of any of the urea cycle enzymes. if the metabolic block occurs in one of the earlier steps of the urea cycle, ammonia, which is neurotoxic, accumulates and causes a more severe condition. conversely, the absence of enzymes catalysing reactions in the later stages of the urea cycle results in the accumulation of less toxic intermediates and less severe symptoms. hence, the exact point of the metabolic block needs to be identified as it determines the prognosis. because the brain is sensitive to ammonia, hyperammonaemia, as well as elevated ammonia levels in other body fluids, causes toxic symptoms and neurological damage. understanding the situation is critical because it guides treatment, which primarily consists of a low-protein diet and frequent small feeds. hippuric acid formation (conjugation product between glycine and benzoyl is a detoxification step) or phenylacetyl glycine can also eliminate amino nitrogen. citrullinaemia citrullinaemia is another example of ucds. citrullinaemia is caused by arginosuccinate synthetase deficiency, which results in elevated ammonia and citrulline levels in the blood. breast milk should be avoided in this condition because it contains high amounts of citrulline. hyperornithinaemia another urea cycle enzyme deficiency is hyperornithinaemia, which is an ornithine transport defect. it is an autosomal recessive condition in which ammonia and ornithine levels in the blood are elevated. hyperargininaemia hyperargininaemia, which results from arginase deficiency and causes arginine accumulation in the blood and cerebrospinal fluid, is another metabolic abnormality seen in ucds. it is worth noting that in this condition, cysteine and lysine are excreted in the urine instead of arginine. mass spectrometry can be used to screen for hyperargininaemia. purine metabolism disorder purine metabolism disorder is best illustrated by lesch-nyhan’s (l-n) syndrome, an x-linked inherited purine metabolism disorder. the biochemical lesion is caused by a hypoxanthine-guanine phosphoribosyltransferase deficiency, which impairs the purine salvage pathway. this results in the accumulation of phosphoribosyl pyrophosphate, an intermediate in the purine synthetic pathway, and other purine synthetic cycle intermediates in the biosynthetic pathway. hypoxanthine-guanine phosphoribosyltransferase activity is normal in the brain and at low levels in the liver and spleen.23 some of the neurological manifestations of l-n syndrome are thought to be due to the toxicity of the purine degradation products to the developing central nervous system or the absence of hypoxanthine-guanine phosphoribosyltransferase, which results in an imbalance in adenine and guanine nucleotides at a critical developmental phase for the infant.24 self-mutilation, mr, raised uric acid levels (described as petit gout), nephrolithiasis (renal stone), and gout later in life are the main characteristics of l-n syndrome. the elevated uric acid level in relation to the patient’s age and other clinical manifestations, such as mr, are sufficient indications for l-n syndrome diagnosis in resource-limited countries. molecular investigation for confirmation may be requested from referral centres. tangier’s disease tangier’s disease is one of the common iems of lipid metabolism. tangier’s disease is an autosomal dominant metabolic disorder that derives from the deficiency of adenosine triphosphate-binding cassette transporter-1. the key biochemical and clinical characteristics of tangier’s disease include defective efflux of cholesterol from cells, reduction in high-density lipoprotein levels in the blood, absence of the α-band in serum protein electrophoresis, accumulation of cholesterol esters in tissues, presence of large orange-yellow tonsils, and muscle atrophy associated with recurrent neuropathies and atherosclerosis. the aforementioned features of the disease are sufficient indicators for the early detection of tangier’s disease before the onset of the later features, such as neuropathy and atherosclerosis. porphyrias the porphyrias are a group of iems associated with the absence of genes encoding enzymes that catalyse reactions in the haem biosynthetic pathway (porphyria means purple). the porphyrias are characterised by increased production and excretion of porphyrin precursors (delta aminolaevulinic acid and porphobilinogen). most are inherited as autosomal dominant traits and are classified into three broad groups: hepatic porphyrias, erythropoietic porphyrias, and combined erythropoietic and hepatic abnormalities. the classification is based on the major site where the enzyme deficiency is manifested. the clinical manifestations of porphyrias vary and are not usually associated with anaemia. one of the most common clinical manifestations is acute intermittent porphyria, which is an inherited genetic disorder.25 the biochemical lesion in acute intermittent porphyria is a lack of porphobilinogen deaminase (uroporphyrinogen-1-synthetase), which causes a secondary increase in aminolaevulinic acid synthase activity (negative feedback mechanism abolished). delta aminolaevulinic acid and porphobilinogen, two key intermediates in the haem biosynthetic pathway, are elevated in blood and urine of patients with porphyrias. the porphobilinogen assay should be conducted using fresh urine samples transported in dark bottles. the key reagent required for porphobilinogen is p-dimethylaminobenzaldehyde (ehrlich’s reaction), which is commonly employed in testing for urobilinogen, a basic test conducted in all laboratories. elevated urinary porphobilinogen excretion confirms the presence of hepatic porphyria. if porphobilinogen excretion exceeds aminolaevulinic acid excretion, lead poisoning can be ruled out.26,27 acute abdominal pain and neurological manifestations, such as sensory and motor disturbances, including confusion and agitation, are among the clinical symptoms that may be intermittent and vague (dubbed ‘little imitators’). a simple ultraviolet fluorescence test with a wood’s lamp to demonstrate porphyrin is also useful and easily accessible. congenital hypothyroidism all newborns in every hospital should be screened for congenital hypothyroidism before they are discharged. congenital hypothyroidism is a clinically significant disorder, especially due to its adverse neurological outcome. this adverse neurological outcome can however be easily reversed if detected early. most infants are asymptomatic at birth because thyroxin from the maternal circulation diffuses across the placenta. congenital hypothyroidism may be suggested by clinical features such as lethargy, umbilical hernia, slow movement, hoarse cry, macroglossia, hypothermia, and hypotonia.28 a simple panel of thyroid function tests, especially thyroid-stimulating hormone, are useful investigations to confirm these clinical findings where they manifest. the thyroid-stimulating hormone value is particularly important as it is very sensitive.29 congenital adrenal hyperplasia congenital adrenal hyperplasia is an inherited endocrinopathy caused by the genetic absence of certain enzymes involved in steroidogenesis, particularly the cortisol pathway. due to the lack of a negative feedback mechanism, this biochemical lesion results in the accumulation of intermediates, some of which are potent androgens such as 17-hydroxyprogesterone, which accounts for virilism and genital ambiguity in affected patients. the most common of these enzyme deficiencies is a 21-hydroxylase deficiency. the enzyme responsible for producing the mineralocorticoid aldosterone may also be altered, which could explain the salt-loss presentation (hyponatraemia and hyperkalaemia). some of the key clinical and biochemical features include precocious growth, increased testosterone, increased 17-hydroxyprogesterone, suboptimal cortisol level with basal and adrenocorticotropic hormone stimulation tests, small testes, and shock. though genetic tests like karyotyping may be used to detect congenital adrenal hyperplasia, basic tests such as sodium assay (hyponatraemia), potassium assay (hyperkalaemia), urinary sodium test (increased sodium level), and plasma renin activity (increased plasma renin activity level) may be within the reach of laboratories in resource-limited countries. owing to the severity of congenital adrenal hyperplasia, we think it should be a candidate for newborn screening programs in these countries. inherited hyperbilirubinaemias congenital hyperbilirubinaemias arise from the absence of enzymes involved in bilirubin metabolism and transportation, especially uridine diphosphate glycosyltransferase. some of these hyperbilirubinaemias include crigler najjar, gilbert, dubin-johnson, and rotors. these can be easily diagnosed by basic bilirubin determination (total and conjugated) and related to clinical presentation. a combination of blood and urine findings helps in the differential diagnosis. newborn screening programmes newborn screening programmes are useful for the early detection of conditions with a presumptive period during which treatment can dramatically improve the outcome. these programmes are especially useful in resource-poor countries where facilities for managing ensuing complications are unavailable in delayed cases. while screening programmes are available in many developed countries, they are not available in many developing countries, and those that were previously available have degenerated. the assumption that the observed degeneration is due to a lack of advanced scientific equipment required for investigation is false as many iems can be detected using basic clinical chemistry laboratory facilities; this has been demonstrated in this brief article and supported by brown and lo14 and, more recently, civallero, de kremer and giugliani.11 even in the absence of tandem mass spectrophotometry in these countries, basic laboratory investigations can accomplish a great deal. inherited metabolic disorders such as congenital hypothyroidism (cretinism), phenylketonuria, and cystic fibrosis are commonly screened for in high-income countries like the united kingdom. in developing countries, these disorders, as well as others such as maple syrup urine disease, galactosaemia, alpha-1-antitrypsin deficiency, ucds, and l-n syndrome, can also routinely be investigated using basic laboratory facilities. for early diagnosis and to avoid the serious and irreversible consequences of iem, close collaboration between clinics and metabolic laboratories, or just clinical laboratories, is critical. a structured approach should be in place and may include targeted laboratory investigation and a comprehensive patient examination to assess drug use, feeding history, transfusion history, and family medical history. at the minimum, an iem team consisting of clinicians (paediatricians), a clinical chemist or laboratory scientist, and a dietician must be available. interpretation of findings should take a multidisciplinary approach as this is one area where clinicians require a great deal of guidance from laboratory specialists. many physicians who request laboratory tests are unfamiliar with the metabolic derangements associated with various iems. brown and lo14 propose that metabolic investigation reports should include age-related reference intervals for quantitative results. with a differential diagnosis derived from relevant abnormal and/or normal data, recommendations for further specialist investigations, and sufficient contact information for such institutions, there should be enough collaboration for clinicians to feel free to contact the laboratory if questions arise. laboratory reports are best interpreted when clinical and relevant laboratory information are provided along with the test requisition, as is well-known in pathology and laboratory medicine. results pointing to multiple iems should be correlated with the patient’s clinical and laboratory data to narrow the differential diagnosis before further investigation.14,30 it is recommended to conduct basic investigations that are useful in the diagnosis of iems as these are beneficial to the community (table 3).31 table 3: basic investigations useful in the diagnosis of inborn errors of metabolism. conclusion although the individual types of iems are rare, this category of diseases is common and often severe. because severe morbidity and/or mortality can be avoided in some forms of iem, it is critical to make accurate diagnoses early, especially in developing countries with limited complication management facilities. though the definitive diagnosis of iems may require specialised laboratory investigations and interpretation, basic investigations that can be conducted in the average clinical chemistry laboratory in developing countries can provide valuable information for critical and early decision making in most cases. referrals to specialised centres for further investigation can then be done. appropriate interpretation of investigation findings requires interdisciplinary collaboration between clinical laboratory scientists and clinicians and is critical for the early diagnosis of patients with iem and improved management of complex cases. a good team of paediatricians, clinical chemists (or laboratory scientists), and a dietician can be extremely useful. it is critical to emphasise that education and training for both health and non-health professionals are both essential and beneficial. health facilities in developing countries need support from international organisations like the international federation of clinical chemistry to improve current diagnostic capacity. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions j.i.a., b.e.o. and g.o.a. contributed equally to the conceptualisation, review and final approval of the manuscript. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability data sharing does not apply to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this manuscript are those of the authors and do not reflect the official policy or position of any affiliated agency of the authors. references garrod ae. inborn errors of metabolism. the croonian lectures. london: oxford university press; 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and molecular basis of inherited disease. 8th ed. new york, ny: mcgraw-hill; 2001. peters tj, sarkany r. porphyria for the general physician. clin med. 2005;5(3):275–281. https://doi.org/10.7861/clinmedicine.5-3-275 anderson ka, sassa s, bishop d, desnick r. disorders of heme biosynthesis: x-linked sideroblastic anemia and the porphyrias. in: sciver c, beaudet a, sly w, valle d, childs b, editors. metabolic and molecular bases of inherited disease [homepage on the internet]. new york, ny: mcgraw-hill, 2001; pp. 2991–3062. shedlofsky si. young woman with recurrent abdominal pain. in: scott mg, grownowski am, eby cs, editors. tietz’s applied laboratory medicine. hoboken, nj: john wiley, 2007; pp. 513–522. ain. the fatigued attorney. in: scott mg, grownowski am, eby cs, editors. tietz’s applied laboratory medicine. hoboken, nj: john wiley, 2007; pp. 513–522. demers lm, spencer ca. laboratory medicine practice guidelines: laboratory support for the diagnosis and monitoring of thyroid disease. clin endocrinol (oxf). 2003;58(2):138–140. https://doi.org/10.1046/j.1365-2265.2003.01681.x anetor ji, durojaiye oo, iyanda a, adeniyi a, agbedana eo. a basic investigation for inherited metabolic disease in two institutions for the handicapped in ibadan: indication for genomics. j biomed invest. 2006;4(1):23–27. https://doi.org/10.4314/jbi.v4i1.30411 kruszka p, regier d. inborn errors of metabolism: from preconception to adulthood. am fam physician. 2019;99(1):25–32. abstract introduction methods results discussion acknowledgements references about the author(s) neema camara epidemiology and disease control section, ministry of health, dodoma, united republic of tanzania nyambura moremi department of bacteriology, national public health laboratory, dar es salaam, united republic of tanzania janneth mghamba epidemiology and disease control section, ministry of health, dodoma, united republic of tanzania eliudi eliakimu health quality assurance unit, ministry of health, dodoma, united republic of tanzania edwin shumba african society for laboratory medicine, addis ababa, ethiopia pascale ondoa african society for laboratory medicine, addis ababa, ethiopia beverly egyir department of bacteriology, noguchi memorial institute for medical research, college of health sciences, university of ghana, legon, ghana citation camara n, moremi n, mghamba j, et al. surveillance of antimicrobial resistance in human health in tanzania: 2016–2021. afr j lab med. 2023;12(1), a2053. https://doi.org/10.4102/ajlm.v12i1.2053 review article surveillance of antimicrobial resistance in human health in tanzania: 2016–2021 neema camara, nyambura moremi, janneth mghamba, eliudi eliakimu, edwin shumba, pascale ondoa, beverly egyir received: 08 aug. 2022; accepted: 09 mar. 2023; published: 22 may 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: antimicrobial resistance (amr) surveillance plays an important role in early detection of resistant strains of pathogens and informs treatments decisions at local, regional and national levels. in 2017, tanzania developed a one health amr surveillance framework to guide establishment of amr surveillance systems in the human and animal sectors. aim: we reviewed amr surveillance studies in tanzania to document progress towards establishing an amr surveillance system and determine effective strengthening strategies. methods: we conducted a literature review on amr studies conducted in tanzania by searching google scholar, pubmed, and the websites of the tanzania ministry of health and the world health organization for articles written in english and published from january 2012 to march 2021 using relevant search terms. additionally, we reviewed applicable guidelines, plans, and reports from the tanzanian ministry of health. results: we reviewed 10 articles on amr in tanzania, where studies were conducted at hospitals in seven of tanzania’s 26 regions between 2012 and 2019. nine amr sentinel sites had been established, and there was suitable and clear coordination under ‘one health’. however, sharing of surveillance data between sectors had yet to be strengthened. most studies documented high resistance rates of gram-negative bacteria to third-generation cephalosporins. there were few laboratory staff who were well trained on amr. conclusion: important progress has been made in establishing a useful, reliable amr surveillance system. challenges include a need to develop, implement and build investment case studies for the sustainability of amr surveillance in tanzania and ensure proper use of third-generation cephalosporins. what this study adds: this article adds to the knowledge base of amr trends in tanzania and progress made in the implementation of amr surveillance in human health sector as a contribution to the global amr initiatives to reduce amr burden worldwide. it has highlighted key gaps that need policy and implementation level attention. keywords: surveillance; antimicrobial resistance; covid-19; one health; tanzania; africa. introduction antimicrobial resistance (amr) is a global public health threat with extensive social, health and economic impacts.1,2 globally, it accounts for about 700 000 deaths annually.3 antimicrobial resistance threatens the lives of 10 million people and an economic loss of up to $100 trillion (united states dollar [usd]) per year by 2050, if there are no effective interventions.3 it is estimated that the magnitude of the amr burden falls on lowand middle-income countries.3 antimicrobials are the mainstay of modern medicine; without them, medical procedures, including surgeries, joint replacements, and treatments, such as cancer chemotherapy, could become too risky to be undertaken as healing would take a long time and become costly.3 in 2015, the world health assembly, through its 68th session, adopted the global action plan on amr to ensure the treatment and prevention of infectious diseases with quality-assured, safe, and effective medicines available.4 the global action plan outlines five strategic objectives, which are: (1) to improve awareness and understanding of amr; (2) to strengthen knowledge through surveillance and research; (3) to reduce the incidence of infection; (4) to optimise the use of antimicrobial agents; and (5) to ensure sustainable investment in countering amr.4 to support the implementation of the global action plan, during the same year, the world health organization (who) launched the global amr surveillance system (glass), the first global collaborative effort to standardise amr surveillance.5 the glass provides a standardised approach for collecting, analysing, and sharing amr data and documents the status of existing or newly developed national amr surveillance systems.5 in 2016, tanzania developed the national action plan for amr (2017–2022) following the who and global health security agenda joint external evaluation recommendation. subsequently, a holistic one health amr surveillance framework was developed to guide the establishment of amr surveillance systems in the human, animal and environmental health sectors. the country is bordered by more than eight countries, which poses a high risk of pathogen importation into the country. in addition, several socioeconomic, demographic and environmental factors also facilitate the emergence and spread of microorganisms; thus, robust health systems are paramount for detecting, responding, and mitigating the effects of the resistant microbes. in this current global coronavirus disease 2019 (covid-19) pandemic situation, where scientists are struggling to find an effective treatment for covid-19, antibiotics have been widely used to manage covid-19, either to treat covid-19 itself or co-infections.6 in fact, recent studies have shown the rampant use of antibiotics by most covid-19 patients without bacterial co-infections.7 this paper reviewed amr surveillance studies and documents the progress made in establishing the amr surveillance system in the human health sector in tanzania and provides recommendations for strengthening it. the literature review was essential to contextualise the amr situation in the past decade and the need to strengthen amr surveillance. methods data collection we searched google scholar, pubmed, and websites of the tanzania ministry of health and who written in english and published from january 2012 to march 2021. we used the search terms: ‘antimicrobial resistance’, ‘bacterial resistance’, ‘antibiotic resistance’; ‘amr surveillance’, or ‘surveillance’ or ‘cross-section’ or ‘review’; and ‘tanzania’. all words were searched together, and, in some instances, two of the three words were used. we reviewed guidelines, plans, and reports from the ministry of health to describe the tanzania amr surveillance system’s objective, surveillance sites, data collection, reporting, analysis, interpretation, and dissemination, coordination of amr surveillance activities, and funding of amr surveillance. setting and structure of healthcare system in tanzania the united republic of tanzania comprises tanzania’s mainland and the semiautonomous islands of zanzibar, and it lies on the east african coast. the tanzania 2012 population census was 44 928 923.8 tanzania mainland has 26 administrative regions, 139 districts and 184 councils. the council divides into divisions, then wards, and streets/villages. the local government authorities (or councils) are the most important administrative and implementation units for public services. health services in tanzania are decentralised into three categories: district (primary level), regional (secondary level), and zonal and national hospital (tertiary level). the district level provides primary health care services through dispensaries at the village level, health centres at the ward level, and level 1 hospital at the council level.9 dispensaries provide preventive and curative out-patient services. in contrast, health centres admit patients and sometimes provide surgical services. council hospitals provide healthcare to referred patients and provide medical and basic surgical services. regional referral hospitals provide specialist medical care. zonal and national hospitals offer advanced (super specialist) medical care and are teaching hospitals for medical, paramedical, and nursing training.9 public, private and faith-based organisations health facilities, private pharmacies, and accredited drug dispensing outlets provide pharmaceutical services.10,11 results antimicrobial resistance trends of priority pathogens in tanzania a total of 10 articles on amr in tanzania were retrieved and reviewed. these studies were conducted at either the zonal or regional referral hospitals between 2012 and 2019. four of the 10 studies were conducted at kilimanjaro christian medical centre in the kilimanjaro region, three at bugando medical centre in mwanza region, two at muhimbili national hospital in dar es salaam region, and one study each at maweni regional referral hospital in kigoma region, musoma regional referral hospital in mara region, sumbawanga regional referral hospital in rukwa region, st. benedict ndanda hospital in mtwara region, bagamoyo district hospital in pwani region, sekou toure regional referral hospital, nyamagana district hospital, and sengerema district designated hospital in mwanza region (figure 1). blood, pus and wound swabs, and urine were the most common laboratory samples analysed (table 1). all of the studies performed antimicrobial susceptibility testing (ast) using the disk-diffusion method per the clinical laboratory standards institute guidelines.12 the most frequently isolated microorganisms from blood were staphylococcus aureus, klebsiella pneumoniae and escherichia coli; and from pus, pseudomonas aeruginosa (table 1). s. aureus resistance to clindamycin ranged between 33.3% to 68.4% and erythromycin between 35.6% to 76.3%, while resistance to cotrimoxazole was 82.6% and ampicillin was 100%.13,14,15,16 the studies reported low rates of resistance to cefoxitin (27.3%), tetracycline (34.9%), cotrimoxazole (26.5%) and ceftriaxone (11.1%).14,17,18 prevalence of methicillin-resistant s. aureus decreased from 41.2% in 2013 to 9.5% in 2015, but rose to 66.7% in 2018.14,15 k. pneumoniae was resistant to ampicillin (100%), cotrimoxazole (96.3%), ceftriaxone (95.7%), amoxicillin/clavulanate (94.6%), ceftazidime (90.9%), gentamycin (86.4%) and cefepime (75.6%).13,16,19 compared to other gram-negatives, e. coli was more resistant to ampicillin, amoxicillin-clavulanic acid, gentamycin, tetracycline, ciprofloxacin, amikacin, third-generation cephalosporins (ceftazidime and ceftriaxone) and cefepime.,13,15,16,19,20 notably, p. aeruginosa was resistant to cefepime (93.8%).13 overall, most studies documented high resistance rates of gram-negative bacteria to third-generation cephalosporins.17,21,22 figure 1: antimicrobial resistance study locations and sentinel surveillance sites, august 2021, tanzania. table 1: antimicrobial resistance trends of glass priority pathogens in tanzania, 2012–2021. progress in implementation of antimicrobial resistance surveillance coordination in 2018, tanzania took the first step of developing a one health national amr surveillance framework to guide the establishment of amr surveillance programmes in the country. the framework provides a holistic approach to monitor trends of infections and resistance that will inform standard treatment guidelines that support best practices for patient care, and links amr information from the human, animal and environmental health sectors.23 the objectives of amr surveillance are to routinely collect, analyse, and interpret quality amr data to generate evidence on trends and the burden of the who priority pathogens, and monitor amr interventions’ effectiveness. the country also established a national multi-sectoral coordinating committee (mcc) to oversee and coordinate all amr-related activities in all sectors. the chief medical officer of the ministry of health and the director of veterinary services, ministry of livestock and fisheries alternate as co-chair of the committee. the committee is composed of representatives from the human, animal and environmental health sectors, as well as livestock and food production stakeholders, and those from medical and agricultural universities. the who, food and agriculture organization, united states centres for disease control and prevention, management science for health and world organization for animal health, are also represented in the mcc. there are designated national amr focal points from animal and human sectors that form part of the mcc secretariat, as well as four multisectoral technical working groups on (1) awareness, effective communication and education; (2) knowledge, surveillance, research and sustainable investments; (3) sanitation, hygiene and infection prevention and control; and (4) antimicrobial use stewardship. the mcc and surveillance twg meets at least once every quarter of the year. the whole coordination structure operates under the ‘one health’ and whole-of-government approach. antimicrobial resistance surveillance system tanzania started with laboratory-based amr surveillance in healthcare settings, as laboratory-based surveillance is the most efficient amr burden determination method.24,25 in the first phase of the national tanzania amr surveillance there were two laboratory levels: coordinating laboratory and site (sentinel/participating) laboratories. as of 2022, there are a total of nine amr sentinel sites which are active and functional. the sentinel sites include muhimbili national hospital, mbeya zonal referral hospital, bugando medical centre, kilimanjaro christian medical centre, mnazi mmoja hospital in zanzibar, temeke regional referral hospital in dar es salaam region, morogoro regional referral hospital in morogoro region, maweni regional referral hospital in kigoma region, and benjamin mkapa hospital in dodoma region (figure 1). the national health laboratory (nhl) is the national coordinating laboratory, and its primary roles include: developing amr national standard operating procedures; training, mentoring and supervising sentinel laboratories; conducting external quality assurance and monitoring internal quality assurances done by sentinel laboratories; and compiling, aggregating, analysing, visualising and disseminating national amr surveillance data to the national mcc and the glass. on the other hand, sentinel laboratories isolate and identify organisms; perform susceptibility tests; store isolates as per national standardised operating procedures; produce and share timely antibiograms with clinicians; and conduct internal quality assurances. antimicrobial resistance surveillance involves the routine collection of blood and urine specimens from in and out-patients with clinical signs and symptoms attending the hospitals. clinicians decide whether to take samples for microbiological culture based on the patient’s clinical assessment. presently, the participating laboratories employ phenotypic methods for pathogen identification and disk diffusion for ast. the ast is a laboratory procedure to identify effective antimicrobial agents that kill or prevent the growth of isolated pathogens recovered from samples of individual patients.26 antimicrobial susceptibility testing results guide clinicians and service providers to decide on target therapy, preserve drugs, and evaluate treatment services.26 notably, continuous surveillance for resistance patterns is crucial due to the mutations in bacterial dna.26 the ast is performed and interpreted according to international guidelines such as the clinical and laboratory standards institute guidelines. the ast results are categorised into either susceptible (s) or non-susceptible, which include intermediate (i) and resistant (r) according to clinical breakpoints defined by clinical and laboratory standards institute. patient clinical data, including infection origin (community or hospital), age, gender and admission types (out-patient, in-patient, general ward or intensive care unit) are collected regardless of culture positivity or negativity. infection origin are categorised as hospital-acquired (specimen from an in-patient admitted for > 2 days) or community-acquired (specimen from an out-patient or in-patient admitted for ≤ 2 days).23 clinical data and ast positive culture results are recorded in the reporting forms and entered into the laboratory information system and the world health organization network (whonet), a freely available system for capturing, analysing and sharing amr data in a standardised format. data import into whonet can be semi-automated using the add-on baclink software (who collaborating centre for surveillance of antimicrobial resistance, boston, massachusetts, united states), which allows for import from other data sources, for example, text files exported from a laboratory information management system (lims) or directly from a laboratory instrument.27 however, whonet intentionally provides only a solution for basic laboratory specimen management and result reporting and does not have comprehensive lims functionality.27 laboratory departments communicate ast results immediately to clinicians as well as the infection prevention and control and amr teams for appropriate treatment and control programs in the local setting. target pathogens for monitoring and reporting as per the national and who priorities include e. coli, k. pneumoniae, acinetobacter baumannii, s. aureus, neisseria meningitidis, streptococcus pneumoniae, salmonella spp., shigella spp., pseudomonas spp. and neisseria gonorrhoeae. data analysis, interpretation and dissemination the participating laboratories must clean, collate, analyse, and create site-specific bacterial antibiograms every month. in addition, annual amr surveillance reports are shared with the relevant clinical departments and hospital committees to increase hospital and community amr awareness, inform treatment policies at the health facility, and encourage continued participation in the surveillance system. antimicrobial resistance data from surveillance sites are centrally stored and managed at the nhl. the nhl conducts data quality checks, analysis, and visualisation, generates official amr reports, and provides long-term data storage. antimicrobial resistance surveillance reports, including trends and resistant pathogen prevalences, are generated at least twice a year and disseminated to stakeholders after approval by the amr surveillance technical working group and the national mcc. at the same time, the clean amr data set is transmitted to the glass (figure 2). the amr surveillance data guides strategies and policies for combating amr. it also provides opportunities for in-depth scientific research that generates additional knowledge on amr. however, sharing of surveillance data among sectors is yet to be strengthened, that is, environment, health and animal. tanzania started reporting amr data to glass in june 2021. figure 2: antimicrobial resistance data flow in tanzania (9 january 2022). quality assurance and standards all nine sentinel sites participate in the external quality assurance which is being supported by nhl and african society for laboratory medicine under the project called external quality assessment for africa. external quality assurance is done twice a year for all sites and involves most of the glass priority pathogens, including e. coli, k. pneumoniae, a. baumannii, s. aureus, n. meningitidis, s. pneumoniae and salmonella spp. as per the amr surveillance framework, all isolates are to be stored at −70 °c for future studies. isolates with unusual, unexpected, or indeterminate resistance patterns are sent to the nhl for confirmatory testing and ast. also, every 10th isolate from each site is sent to nhl for quality assessment. funding funds to run the nine amr surveillance sentinel laboratories are contributed by the government of the united republic of tanzania, the fleming fund, and the united states agency for international development fund under the infectious disease diagnostic and surveillance project. therefore, there is a need to establish a sustainable funding mechanism for amr activities in tanzania. discussion this review has shown that the most frequently isolated microorganisms from blood were s. aureus, k. pneumoniae and e. coli; from urine, e. coli; and from pus, p. aeruginosa. most studies documented high resistance rates of gram-negative bacteria to third-generation cephalosporins. importantly, significant progress has been made in establishing amr surveillance; nine sentinel sites across tanzania have been established and are generating data and there are suitable and clear coordination structures and platforms for multisectoral engagement and collaboration under one health. however, sharing of surveillance data between sectors is yet to be strengthened. there are also few laboratory staff well trained on amr practices. studies in tanzania reveal increasing bacterial resistance to third-generation cephalosporins. in tanzania, ceftriaxone is reportedly prescribed excessively and inappropriately in hospital settings,28 and this may explain the observed third-generation cephalosporins resistance trends. if this trend continues, clinicians will resort to broad-spectrum antibiotics, such as carbapenem, which are the last resort according to the tanzania treatment guideline.29,30 if such a situation occurs, effective, quality, and affordable healthcare provisions, the core fundamentals for universal health coverage, will be far from being realised. progress made in establishing the amr surveillance system in the country is commendable. a total of nine sentinel sites have been established. there are suitable coordination structures and platforms for multisectoral engagement and collaboration under the ‘one health’.31 the amr sentinel surveillance sites are representative and provide amr trends and burden data. amr surveillance system has helped to standardise routine microbiological cultures and ast in hospitals, particularly those participating in amr surveillance according to global standards (mcc meeting minutes of 11 may 2021, unpublished). however, the facilities still face challenges while implementing amr surveillance, including a lack of interoperability between the sentinel site laboratory information system and whonet to enable automatic data transfer between the two systems. the double entry of the same information in two different systems exhausts and overworks the laboratory staff. a lack of fit-for-purpose lims and open-source lims software with technical standards and functionality for amr surveillance is a particular concern in lowand middle-income countries.27 although whonet is a functional and useful repository for microbiological data with capabilities for standardised data sharing, it lacks full lims functionality.27 thus, there is an urgent need for investment in laboratory information technology infrastructure and data management systems that can capture high-quality laboratory and patient management data. there are few well-trained laboratory staff at the sentinel sites for data analysis and the production of antibiograms, which can be shared with the clinicians and amr teams to inform on the appropriate treatment and measures to tackle amr at the hospital or community. at the surveillance sites, frequent stock out of ast reagents and analysis, inadequate resources, and poor laboratory infrastructure for phenotypic and genotypic analysis is commonplace. although amr surveillance receives much support from the government as per human resources and infrastructure, there are also funds from donors. a sustainability plan is essential to prevent over-dependence on donors over time. these challenges are unique in tanzania and have also been reported elsewhere in africa.32,33 however, despite the challenges, amr surveillance is still ongoing in the country. sharing of surveillance data between sectors is yet to be strengthened. antimicrobial resistance is a broad and complex issue affecting the animal, human and environmental sectors; thus, a multisectoral and multidisciplinary combat approach is needed. antimicrobials are also widely used in animals for treatment and growth promoters. in addition, evidence suggests that antimicrobial use in animals contributes significantly to the development of amr in humans,34 necessitating a comprehensive and coordinated amr surveillance system that can continuously share amr data between sectors to inform public health interventions. this study has some limitations. the review was based on reports only. we did not seek additional inputs and insights from amr stakeholders through a standardised questionnaire or interview. as such some comprehensive views and perspectives may have been missed out. the amr trends in tanzania presented here should be interpreted with caution as the review was only based on 10 surveillance articles. conclusion tanzania is currently implementing amr surveillance in nine hospitals, and reporting of amr data to glass has commenced. there are well-established amr coordination mechanisms at health facilities and national levels to effectively implement and utilise the amr information. although there are challenges affecting implementation, the current amr surveillance system in place is useful, reliable and capable of better performance. there is a need to develop, implement and build an investment case study for the sustainability of amr surveillance in tanzania. we recommend that the government creates a fit-to-purpose laboratory information system with functionality able to link with other systems; develops a mechanism for sustainable financing for laboratory infrastructure development and continuous supply of reagents, commodities, and laboratory materials; and invests hugely in building human capacities for bacterial identification and ast and data analysis, interpretation and utilisation. acknowledgements the authors would like to acknowledge the government of tanzania and partners for the efforts to implement amr surveillance activities. also, we appreciate authors of the surveillance articles and contributors of the amr reports, minutes, guidelines, and frameworks which informed this writing. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions n.c., e.s., p.o. and b.e. were involved in the conceptualization of the project. n.c. and b.e, were involved in the writing of an initial draft, reviewing and editing the manuscript. n.c., n.m., j.m, e.e., e.s., p.o., b.e. reviewed and edited the manuscript. all authors read and approved the final version of the manuscript. ethical considerations this paper has no ethical concerns as it does not have human or animal subjects or data, and as such ethical approval from an ethics review board was not required. sources of support this work was part of the activities of a fellowship (surveillance in human health) workplan, which was supported by the fleming fund through the fellowship scheme in tanzania. data availability the authors confirm that the data supporting the findings of this study are available within the article. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the 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2021;10(3):273. https://doi.org/10.3390/antibiotics10030273 kariuki s, keddy kh, antonio m, okeke in. antimicrobial resistance surveillance in africa: successes, gaps and a roadmap for the future. afr j lab med. 2018;7(2):924. https://doi.org/10.4102/ajlm.v7i2.924 ndihokubwayo jb, yahaya aa, desta at, et al. antimicrobial resistance in the african region: issues, challenges and actions proposed. african health monitor. 2023;(16):4. carrique-mas jj, choisy m, van cuong n, thwaites g, baker s. an estimation of total antimicrobial usage in humans and animals in vietnam. antimicrob resist infect control. 2020;9(1):16. https://doi.org/10.1186/s13756-019-0671-7 reviewer acknowledgement http://www.ajlmonline.org open access page 1 of 1 the editors and guest editors would like to take this opportunity to thank all of those who provided scientific and logistical support for this issue of the african journal of laboratory medicine: should names have inadvertently been excluded from this list, 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for the african journal of laboratory medicine. please do not hesitate to contact us if you require assistance in performing this task. publisher: publishing@aosis.co.za tel: +27 21 975 2602 fax: +27 21 975 4635 teferi m. abera aaron o. aboderin avelin aghokeng gilbert akankwasa maka akhalaia george alemnji gilda alves timothy amukele aje j. anejo-okopi rosemary a. audu oluwatoyin a. babalola colleen bamford sonia boender valerie f. boltz jane carter jaya d. chidambaram nelson chimbiya alpha diallo khadim diongue romolo dorizzi jeroen eikenboom dennis ellenberger ana e. farfán jonathan fletcher lisa gerwing-adima b. gu jeannette guarner xu-xiao guo hiroshi ichimura seth inzaule gnimintakpa joseph phyllis kanki yenew kebede andrea kim charles kiyaga stéphania koblavi dème olayinka kotila adjane koura kekoura kourouma zirra mangoro margherita morpurgo jane mwangi keiki nagaharu anneta f. naidoo vusumuzi ncube john n. nkengasong asa’ah nkohkwo monica odhiambo igho ofotokun j. olufemi ogunbiyi iruka n. okeke j.a. onaolapo pascale ondoa judith owen bharat parekh ketan patel robert perry pedro pires b. pratumvinit bethanie rammer kristina i. rother david j. sambian lee schroeder ritu shrivastava svetoslav n. slavov william c. smith ali tiss willy urassa lara vojnov diane waku-kouomou brooke weckselblatt meseret workineh katy yao acknowledgement to reviewers http://www.ajlmonline.org www.ajlmonline.org www.ajlmonline.org http://www.ajlmonline.org http://www.ajlmonline.org http://www.ajlmonline.org/index.php/ajlm/user http://www.ajlmonline.org/index.php/ajlm/user http://www.ajlmonline.org/index.php/ajlm/user abstract introduction research method and design results discussion acknowledgements references about the author(s) margaret l. mcnairy icap, columbia university, new york, new york, united states weill cornell medical college, new york, new york, united states charon gwynn icap, columbia university, new york, new york, united states miriam rabkin icap, columbia university, new york, new york, united states gretchen antelman icap, columbia university, new york, new york, united states yingfeng wu icap, columbia university, new york, new york, united states bereket alemayehu icap, columbia university, new york, new york, united states travis lim centers for disease control and prevention, atlanta, georgia, united states rubina imtiaz centers for disease control and prevention, atlanta, georgia, united states fausta mosha ministry of health and social welfare, dar es salaam, republic of tanzania michael mwasekaga centers for disease control and prevention, dar es salaam, republic of tanzania asha a. othman ministry of health, zanzibar, republic of tanzania jessica justman icap, columbia university, new york, new york, united states citation mcnairy ml, gwynn c, rabkin m, et al. increased utilisation of pepfar-supported laboratory services by non-hiv patients in tanzania. afr j lab med. 2016;5(1), art. #318, 7 pages. http://dx.doi.org/10.4102/ajlm.v5i1.318 original research increased utilisation of pepfar-supported laboratory services by non-hiv patients in tanzania margaret l. mcnairy, charon gwynn, miriam rabkin, gretchen antelman, yingfeng wu, bereket alemayehu, travis lim, rubina imtiaz, fausta mosha, michael mwasekaga, asha a. othman, jessica justman received: 03 apr. 2015; accepted: 12 oct. 2015; published: 16 feb. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: it is unknown to what extent the non-hiv population utilises laboratories supported by the president’s emergency plan for aids relief (pepfar). objectives: we aimed to describe the number and proportion of laboratory tests performed in 2009 and 2011 for patients referred from hiv and non-hiv services (nhss) in a convenience sample collected from 127 laboratories supported by pepfar in tanzania. we then compared changes in the proportions of tests performed for patients referred from nhss in 2009 vs 2011. methods: haematology, chemistry, tuberculosis and syphilis test data were collected from available laboratory registers. referral sources, including hiv services, nhss, or lack of a documented referral source, were recorded. a generalised linear mixed model reported the odds that a test was from a nhs. results: a total of 94 132 tests from 94 laboratories in 2009 and 157 343 tests from 101 laboratories in 2011 were recorded. half of all tests lacked a documented referral source. tests from nhss constituted 42% (66 084) of all tests in 2011, compared with 31% (29 181) in 2009. a test in 2011 was twice as likely to have been referred from a nhs as in 2009 (adjusted odds ratio: 2.0 [95% confidence interval: 2.0–2.1]). conclusion: between 2009 and 2011, the number and proportion of tests from nhss increased across all types of test. this finding may reflect increased documentation of nhs referrals or that the laboratory scale-up originally intended to service the hiv-positive population in tanzania may be associated with a ‘spillover effect’ amongst the general population. introduction investment in strengthening laboratory systems in resource-poor countries is critical to meet health needs across major diseases such as hiv/aids and to meet the united nations millennium development goals.1 in the past decade, the us government has invested over $15 billion in hiv prevention, care and treatment in lowand middle-income countries via the president’s emergency plan for aids relief (pepfar).2 this support has included a wide range of activities aimed at strengthening health services, including laboratories, to provide services for persons living with hiv (plwh). although the positive impact of these targeted health services on plwh is undeniable, the effect of hiv service scale-up on broader health systems, including services for patients without hiv, has been debated.3,4,5,6,7 since 2006, pepfar has provided over $440 million to strengthen laboratory systems through improved infrastructure and equipment, human resources and training, quality improvement, and technical assistance.8 this investment has expanded laboratory services such as diagnostic and monitoring tests for plwh. because these laboratory investments support health facilities serving a broad population of patients, not just plwh, it is plausible that they may have affected, or in the future could affect, the coverage and quality of laboratory services used by the general population – that is, individuals with no known hiv infection.9 to our knowledge, no studies have explored this question yet. in an effort to describe pepfar’s investment in laboratory services for the general population, we analysed routinely collected programmatic data from selected public laboratories in tanzania. specifically, we selected a convenience sample of pepfar-supported laboratories in tanzania, which are supported through icap at columbia university.10 in these laboratories, the only information distinguishing the hiv status of the patient from whom the test was collected was the test’s referral source; that is, an hiv service or a non-hiv service (nhs) (e.g. general medical or outpatient services). although referral source is not a definitive diagnosis of hiv status, it was the only routinely recorded information available as a proxy for hiv status. our primary objective was to describe the number and proportion of selected core laboratory tests performed for patients referred from the respective services in 2011. a secondary objective was to compare changes in proportions of tests performed for patients referred from nhss in 2009 and 2011. research method and design ethical considerations this study was approved by the columbia university medical center institutional review board, the us centers for disease control and prevention, the tanzania national institute for medical research and the zanzibar medical research and ethics committee. study population we conducted a retrospective cross-sectional analysis of laboratory tests from 2009 and 2011 in a convenience sample of pepfar-supported public laboratories in tanzania. all laboratories received pepfar support from icap at columbia university. laboratories that were included were all categorised as public sector, offered integrated laboratory services for all laboratory samples (i.e. using the same staff and equipment for hiv and non-hiv patients), performed at least haematology testing over the study period, and had available laboratory register data on preliminary assessment. data abstracted from laboratory testing registers did not include patient-identifying information. definitions of laboratory tests and outcomes a laboratory test was defined as the presence of a documented haematology, chemistry, tuberculosis or syphilis test result in a laboratory register located at the laboratory facility. a haematology test result was defined as any automated or manual test for haemoglobin or a complete blood count (e.g. celldyne 1800, coulter). a chemistry test result was defined as creatinine or liver function tests (alanine aminotransferase, aspartate aminotransferase or alkaline phosphatase) or other blood chemistry panel results from an automated machine (e.g. humastar 80, hitachi, reflotron). a tuberculosis test included a microscopy smear or culture. a syphilis test result was defined as a test from a venereal disease research laboratory or a rapid plasma reagin or antibody test. on-site registers were used to classify samples as from hiv services, a nhs, or an unknown referral source (i.e. did not have a documented referral source). the primary outcome of this study was the proportion of laboratory tests with documented nhs referral sources amongst all tests with a referral source (either hiv or nhs referral). other outcomes included the proportion of laboratory tests performed with documented nhs referral sources amongst all tests, including tests with and without referral sources. site-level variables programmatic information was used to provide contextual information about included laboratories. routinely collected quarterly monitoring and evaluation data from the co-located hiv care and treatment facilities were used to quantify the number of years each facility had provided hiv care services and the number of hiv-positive patients enrolled in the hiv care service. information from facility-based surveys completed in 2009 and 2011 at laboratories included the location type (urban vs rural) and type of facility (primary, secondary or tertiary); the 2011 survey also described the number of trained laboratory personnel working in each laboratory. data collection between march and july of 2013, study staff extracted de-identified laboratory data from on-site hard-copy registers at included laboratories. study staff met briefly with laboratory personnel to assess the availability of laboratory registers for each of the aforementioned tests. study staff reviewed the available laboratory registers to tally the number of each type of test conducted per month. totals were aggregated by the type of referral source. if an hiv clinic was indicated as a referral source, the test was categorised as coming from an hiv service. if another clinic or unit within the facility was documented as the source in the register, the test was categorised as coming from a nhs. if no source was documented for the patient, the test was categorised as coming from an unknown referral source. statistical analysis proportions of tests conducted amongst all the laboratories were calculated for specimens referred from hiv services, nhss and those with an unknown referral source. proportions were calculated by year and by test type. a generalised linear mixed model was constructed to predict the odds that a laboratory test was referred from a nhs, taking into account intrafacility correlations. we used a generalised linear mixed model without confounders to account for intrafacility correlation, and an adjusted generalised linear mixed model that controlled for key facility-level variables including year, facility location and total volume of tests performed at each facility as fixed effects, with the laboratory treated as the random effect. key contextual variables hypothesised to affect the proportion of tests from an hiv service compared with from a nhs, such as location (rural vs. urban), region, facility type and service size (e.g. number of patients enrolled in hiv care) were assessed individually to determine an unadjusted odds ratio. candidate confounders (p < 0.25 when unadjusted) were entered and examined in generalised linear mixed models, but only the significant variables (p < 0.05) were kept in the final models for the purposes of calculating the adjusted odds ratios. all analyses were conducted using sas version 9.3 (sas institute, cary, north carolina, united states). results amongst the 127 pepfar-supported laboratories in tanzania during the study period, 94 laboratories had testing data available from registers in 2009 and 101 laboratories had testing data available from registers in 2011 (figure 1). a total of 93 laboratories had testing data for both 2009 and 2011. when the analysis was restricted to laboratories whose registers included tests with a referral source, a total of 51 in 2009 and and 61 laboratories in 2011 remained in the sample. figure 1: location of laboratories in tanzania receiving pepfar support from icap during 2009 and 2011 (n = 94 laboratories in 2009 and 101 in 2011). characteristics of laboratory facilities the majority of laboratories were located in an urban area (table 1). in 2009, there were 59 (63%) primary level laboratories, whereas in 2011 there were 66 (65%). in 2009, 70% of laboratories had been providing hiv care for up to one year, compared with 2% in 2011; in 2011, 67% had been providing hiv care for two to three years and 31% had been providing hiv care for at least four years. the median number of plwh enrolled in care at pepfar-supported hiv facilities increased from 139 in 2009 to 269 in 2011. data were not available on the median number of laboratory technicians in 2009, but 40% of laboratories had one technician in 2011, 32% of laboratories had two or three technicians in that year and 29% had at least four technicians. the median number of laboratory tests documented in available on-site registers was 415 tests (interquartile range [iqr]: 108–1211) in 2009 and 652 tests (iqr: 217–2034) in 2011. the number and proportion of laboratories conducting more than 1500 tests per year increased from 18 (19%) in 2009 to 28 (28%) in 2011. table 1: characteristics of pepfar-supported laboratories in tanzania for which data were provided for 2009 and 2011. the completeness of available data at laboratories varied according to the type of test and over time. the proportion of laboratories providing any data on haematology tests increased from 64% (60/94) in 2009 to 92% (93/101) in 2011 (p < 0.001) (table 1). the proportion of laboratories providing any data on other tests increased measurably but not significantly from 2009 to 2011: 13% (12/94) vs. 23% (23/101) for chemistry tests (p = 0.07), 85% (80/94) vs. 88% (89/101) for tuberculosis tests (p = 0.53), and 32% (30/94) vs. 46% (46/101) for syphilis tests (p = 0.05). of the 94 laboratories providing data in 2009, 61% (57/94) provided data for 12 months of the year compared with 75% (75/101) in 2011 (data not shown in table 1). characteristics of laboratory tests the total number of tests recorded increased from 94 132 in 94 laboratories in 2009 to 157 343 in 101 laboratories in 2011 (table 2). the proportion of all tests performed for patients referred from a nhs increased from 31% (29 181) in 2009 to 42% (66 084) in 2011 (figure 2). in both years, less than one-fifth of all tests were documented as being referred from hiv services: 14% (13 178) in 2009 and 11% (17 308) in 2011. approximately half of all tests lacked a documented referral source: 56% (52 714) in 2009 and 47% (73 951) in 2011. figure 2: referral sources for laboratory tests performed by pepfar-supported laboratories in tanzania, 2009 and 2011. the graph depicts the overall proportion of laboratory tests performed for patients from different types of referral source (94 laboratories in 2009; 101 laboratories in 2011). table 2: tests conducted by pepfar-supported laboratories in 2009 and 2011 by type of test haematology tests constituted the majority of all tests documented in the two study periods, accounting for 58% (54 499) of all tests in 2009 and 67% (104 693) of tests in 2011 (table 2; figure 2). the proportion of haematology tests performed for patients referred from a nhs increased from 34% in 2009 to 45% in 2011. less than 10% of haematology tests in either year were performed for patients with a documented referral from hiv services (8% in 2009 and 9% in 2011). in contrast, chemistry tests represented a much smaller proportion of the total number of tests: 13% (12 607) in 2009 and 11% (17 680) in 2011. however, the proportion of chemistry tests performed for patients referred from hiv services was much larger than any other test type (45% in 2009 and 40% in 2011). the vast majority of all tuberculosis tests in 2009 and 2011 had an unknown referral source: 78% (13 648) in 2009 and 64% (13 792) in 2011. syphilis testing increased from 9528 tests in 2009 to 13 420 tests in 2011. the proportion of syphilis tests recorded for patients referred from hiv services decreased from 24% (2287) in 2009 to 2% (268) in 2011 and was accompanied by a large increase in the proportion of tests performed for patients with an unknown referral source: from 44% (4192) in 2009 to 62% (8320) in 2011. when analyses were restricted only to tests with a documented referral source, the sample of laboratories decreased from 94 to 51 in 2009 and from 101 to 61 in 2011. amongst this sample, the proportion of all laboratory tests performed for patients referred from nhss increased from 69% (28 722) in 2009 to 76% (63 462) in 2011 (figure 3). the proportion of haematology tests performed for patients referred from nhss increased modestly from 82% (18 722) in 2009 to 84% (46 936) in 2011, yet dramatically in net number. the proportion of chemistry tests performed for patients referred from nhss increased from 40% (3795) in 2009 to 52% (7461) in 2011, and the proportion of tuberculosis diagnostic tests increased from 78% (3046) to 81% (6323) during this same period. the proportion of syphilis tests performed for patients referred from nhss increased the most: from 58% (3107) in 2009 to 95% (4810) in 2011. figure 3: proportion of tests referred from non-hiv services in 2009 and 2011 from amongst tests with a documented referral source. after excluding tests without a documented referral source, a total of 51 laboratories in 2009 and 61 laboratories in 2011 remained for the analysis. the total number of tests for which any referral status was available is shown for each year below the graph for each test category. for all laboratory tests, tests were approximately twice as likely to be referred from nhss in 2011 compared with 2009, based on the unadjusted odds ratio of 1.9 (95% confidence interval [ci]: 1.8–1.9) (table 3). a similar odds ratio was estimated after adjusting for year, location and number of tests conducted (adjusted odds ratio: 2.0 [95% ci: 2.0–2.1]). when stratified by test type, we found that amongst all types of test, chemistry and syphilis tests were the most likely to have been referred from nhss in 2011 compared with 2009 (adjusted odds ratios: 1.9 [95% ci: 1.7–2.0] for chemistry; 13.0 [95% ci: 11.0–17.0) for syphilis]). haematology tests performed in urban laboratories were more likely to be referred from nhss than haematology tests from rural laboratories (adjusted odds ratio: 6.8 [95% ci: 1.2–40.0]). samples that came from laboratories that conducted fewer tests (≤ 500 and 501–1500 per year) were more than twice as likely to be referred from nhss compared with those from laboratories that conducted a larger number of tests (> 1500) per year (adjusted odds ratios: 2.5 [95% ci: 1.9–3.3] for ≤ 500 tests and 2.6 [95% ci: 2.3–2.9] for 501–1500 tests). table 3: odds ratios of a laboratory test being performed for a patient referred from non hiv-services, 2009 and 2011.†,‡ discussion we investigated the potential impact of pepfar-supported laboratory scale-up on the general (non-hiv) patient population in a country in sub-saharan africa. the results describe the number of laboratory tests performed in 2009 and 2011 in a convenience sample of pepfar-supported laboratories in tanzania and the proportion of tests performed for patients referred from hiv services, nhss and unknown sources. a key finding in this analysis is the substantial increase in the proportion of all tests referred from nhss from 2009 to 2011 – both when including all laboratory tests and when including only tests with known referral sources. there was considerable variation in the number of tests performed by each facility (iqr: 108–1211 tests in 2009 and 217–2034 in 2011). also of note was the substantial variation in testing volume across different types of laboratory tests. haematologic tests were the most common type and are the laboratory cornerstone for antenatal care, malaria diagnosis and treatment, routine outpatient diagnostics for infectious diseases, and hiv care. syphilis tests were the least common test; however, the volume of syphilis tests increased substantially from 2009 to 2011, reflecting in part a tanzania ministry of health recommendation for rapid tests kits (sd bioline), which enabled routine point-of-care syphilis screening to become more feasible, as opposed to rapid plasma reagin, which require cold-chain analysis and trained laboratory staff. chemistry tests were performed most often for patients referred from hiv services, which may reflect the clinical practice of assessing renal function amongst hiv patients before and during antiretroviral therapy.11 it is difficult to interpret changes in tuberculosis testing given that the majority of laboratory registers did not record a referral source in this category. referral sources were not reported for any tests by 46% (43/94) of laboratories in 2009 and 40% (40/101) of laboratories in 2011. it was not possible to infer the hiv status of these patients. in laboratories with unknown referrals amongst some tests, the intermittent lack of referral documentation may be due to random missing data at the laboratory. alternatively, in some laboratories, technicians may prioritise documentation of samples from hiv clinics and leave all other referral sources blank, leading to samples referred from nhss and those without a referral source being grouped together. in this scenario, patients with unknown referral sources would be more likely to represent the general population. if unknown referral sources were actually non-hiv patients, our data suggest little meaningful change in the proportion of tests referred from nhss between 2009 and 2011, relative to tests referred by hiv services. limiting the analysis to laboratories that did record a referral source restricted the sample size to 51 in 2009 and 61 laboratories in 2011. within this subgroup, the proportion of tests referred from nhss increased across all test types, similar to analyses including the full sample of laboratories. a model was used to predict the odds that an individual test was referred from a nhs, given that individual tests are not independent of each other in a laboratory. using this model, the probability of a test being referred from a nhs was two times higher in 2011 than in 2009. notably, haematology and tuberculosis tests were less likely to be referred from a nhs in 2011 than in 2009. this finding may be due to the model taking into account the correlation of the laboratory tests within a specific site when estimating the odds ratios. for example, larger laboratories may have skewed the results reported in figures 2 and 3, but once intrafacility correlation is accounted for, the proportion of tests referred from nhss for haematology and tuberculosis appeared to decrease over time. these findings suggest a large amount of site-level variation in the odds of a test being referred from nhss. in addition, the observed odds ratio for all tests was likely driven by the increases in nhs testing in chemistry and syphilis between 2009 and 2011. limitations this study has several limitations. firstly, the data comprised a non-random convenience sample of laboratories. thus, the results may not be generalisable to other pepfar-supported laboratories in tanzania or in other pepfar-supported countries. it is also unknown, in the absence of a comparison group, whether the volume of laboratory tests referred from hiv services and nhss would have changed in the absence of pepfar or at comparable public laboratories not supported by pepfar. secondly, it would have been advantageous to describe the change in laboratory tests over a longer period. however, this was not feasible, as icap support for most study laboratories began in 2009. thirdly, because the sources of the laboratory data did not record identifiable patient information, the unit of analysis in this study was a laboratory test and not an individual patient, who could have had multiple tests. as stated previously, the hiv status of the patient for whom each test was performed was unknown. future analyses evaluating utilisation of laboratory services at the patient level would provide additional information as to whether there are differences in laboratory usage according to patients’ hiv status. fourthly, our data did not include information on the reason for a test being ordered for samples referred from nhss. thus, we could draw no conclusions as to whether or how guidelines for laboratory testing amongst non-hiv patients influenced utilisation of laboratory services. finally, we were limited by the availability of hard-copy laboratory registers. the absence of a register did not necessarily mean that tests were not performed in a given laboratory, but merely that we were unable to access documentation of the test being performed. even when registers were available, only 54% (51/94) of laboratories in 2009 and 61% (61/101) of laboratories in 2011 recorded referral sources; amongst those that did, we could not verify the referral source against other records. however, data availability and quality are unlikely to have changed notably over the study period. this study provides descriptive data as a departure point for answering the question of how pepfar’s investment in laboratory services may have influenced utilisation of laboratory services by the general population. a systematic impact evaluation would be beneficial and would require prospective data or comparison groups and should include data on other variables about serviced populations, including the hiv status of patients for whom laboratory tests are performed, and laboratory characteristics, including staffing, equipment, training, quality improvement and costs. conclusion this retrospective study found that in a convenience sample of pepfar-supported laboratories in tanzania, the number and proportion of tests performed for patients referred from nhss increased for all tests from 2009 to 2011 compared with referrals from hiv services. the increase was driven in part by chemistry and syphilis testing. although these findings are descriptive and may not be generalisable to other hiv-supported laboratories in tanzania and other resource-limited countries, this finding may reflect increased documentation of referrals from nhss in laboratory registers over time. another possibility is that laboratory scale-up originally intended to service the hiv-positive population in tanzania may be associated with a ‘spillover effect’ on laboratory use amongst the general population in the sampled facilities. these data may inform subsequent prospective studies to evaluate the impact of pepfar-supported laboratory scale-up on utilisation of laboratory services and the impact on health outcomes amongst the general population acknowledgements the findings and conclusions in this paper are those of the authors and do not necessarily represent the official position of the centers for disease control and prevention. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. sources of support this research was financially supported by pepfar through the us centers for disease control and prevention under the terms of grant number 5u48dp001916-05 sip10-038. support was provided by the national center for injury prevention and control, the national center for chronic disease prevention and health promotion, the coordinating office of global health, the national center for birth defects and developmental disabilities, the national center for immunization and respiratory diseases and the national center for hiv, viral hepatitis, stds and tb prevention. authors’ contributions m.l.m. (columbia university; weil cornell medical college) was the project leader. c.g. (columbia university) and y.w. (columbia university) performed data analysis. m.l.m., c.g., m.r. (columbia university), g.a. (columbia university), y.w., b.a. (columbia university), t.l. (centers for disease control and prevention, atlanta), r.i. (centers for disease control and prevention, atlanta), f.m. (ministry of health and social welfare, republic of tanzania), m.m. (centers for disease control and prevention, tanzania), a.a.o. (ministry of health, republic of tanzania) and j.j. (columbia university) assisted in design, implementation, data interpretation and manuscript writing. references birx d, de souza m, nkengasong jn. laboratory challenges in the scaling up of hiv, tb, and malaria programs: the interaction of health and laboratory systems, clinical research, and service delivery. am j clin pathol. 2009;131(6):849–851. http://dx.doi.org/10.1309/ajcpgh89qdswfons unaids. unaids report on the global aids epidemic 2012. geneva: unaids; 2012. justman je, koblavi-deme s, tanuri a, goldberg a, gonzalez lf, gwynn cr. developing laboratory systems and infrastructure for hiv scale-up: a tool for health systems strengthening in resource-limited settings. j acquir immune defic syndr. 2009;52 suppl 1:s30–33. http://dx.doi.org/10.1097/qai.0b013e3181bbc9f5 world health organization maximizing positive synergies collaborative group. an assessment of interactions between global health initiatives and country health systems. lancet. 2009;373(9681):2137–2169. http://dx.doi.org/10.1016/s0140-6736(09)60919-3 biesma rg, brugha r, harmer a, walsh a, spicer n, walt g. the effects of global health initiatives on country health systems: a review of the evidence from hiv/aids control. health policy plan. 2009;24(4):239–252. http://dx.doi.org/10.1093/heapol/czp025 rabkin m, el-sadr wm, de cock km, bellagio hiv/health systems working group. the impact of hiv scale-up on health systems: a priority research agenda. j acquir immune defic syndr. 2009;52 suppl 1:s6–11. http://dx.doi.org/10.1097/qai.0b013e3181bbcd69 yu d, souteyrand y, banda ma, kaufman j, perriëns jh. investment in hiv/aids programs: does it help strengthen health systems in developing countries? global health. 2008;4:8. http://dx.doi.org/10.1186/1744-8603-4-8 unaids. unaids report of the global aids epidemic 2010. geneva: unaids; 2010. nkengasong jn, mesele t, orloff s, kebede y, fonjungo pn, timperi r, et al. critical role of developing national strategic plans as a guide to strengthen laboratory health systems in resource-poor settings. am j clin pathol. 2009;131(6):852–857. http://dx.doi.org/10.1309/ajcpc51blobbpakc icap columbia university [homepage on the internet]. new york: columbia university; c2014 [cited 2014 march 20]. available from: http://icap.columbia.edu/ tanzania national aids control programme (tnacp). national guidelines for the mangement of hiv and aids. third edition (revised february 2009). dar es salaam: tanzania national aids control programme (tnacp); 2009. available from: http://www.who.int/hiv/pub/guidelines/tanzania_art.pdf abstract introduction nontyphoidal salmonella typhoidal salmonella vibrio cholerae shigella species escherichia coli acknowledgements references about the author(s) anthony m. smith centre for enteric diseases, national institute for communicable diseases, national health laboratory service, johannesburg, south africa faculty of health sciences, university of the witwatersrand, johannesburg, south africa citation smith am. review of molecular subtyping methodologies used to investigate outbreaks due to multidrug-resistant enteric bacterial pathogens in sub-saharan africa. afr j lab med. 2019;8(1), a760. https://doi.org/10.4102/ajlm.v8i1.760 review article review of molecular subtyping methodologies used to investigate outbreaks due to multidrug-resistant enteric bacterial pathogens in sub-saharan africa anthony m. smith received: 23 jan. 2018; accepted: 25 sept. 2018; published: 14 mar. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: in sub-saharan africa, molecular epidemiological investigation of outbreaks caused by antimicrobial-resistant enteric bacterial pathogens have mostly been described for salmonella species, vibrio cholerae, shigella species and escherichia coli. for these organisms, i reviewed all publications describing the use of molecular subtyping methodologies to investigate outbreaks caused by multidrug-resistant (mdr) enteric bacterial infections. objectives: to describe the use of molecular subtyping methodologies to investigate outbreaks caused by mdr enteric bacterial pathogens in sub-saharan africa and to describe the current status of molecular subtyping capabilities in the region. methods: a pubmed database literature search (english language only) was performed using the search strings: ‘africa outbreak mdr’, ‘africa outbreak multi’, ‘africa outbreak multidrug’, ‘africa outbreak multi drug’, ‘africa outbreak resistance’, ‘africa outbreak resistant’, ‘africa outbreak drug’, ‘africa outbreak antibiotic’, ‘africa outbreak antimicrobial’. these search strings were used in combination with genus and species names of the organisms listed above. all results were included in the review. results: the year 1991 saw one of the first reports describing the use of molecular subtyping methodologies in sub-saharan africa; this included the use of plasmid profiling to characterise salmonella enteritidis. to date, several methodologies have been used; pulsed-field gel electrophoresis analysis and multilocus sequence typing have been the most commonly used methodologies. investigations have particularly highlighted the emergence and spread of mdr clones; these include salmonella typhi h58 and salmonella typhimurium st313 clones. in recent times, whole-genome sequencing (wgs) analysis approaches have increasingly been used. conclusion: traditional molecular subtyping methodologies are still commonly used and still have their place in investigations; however, wgs approaches have increasingly been used and are slowly gaining a stronghold. african laboratories need to start adapting their molecular surveillance methodologies to include wgs, as it is foreseen that wgs analysis will eventually replace all traditional methodologies. introduction for molecular epidemiological investigation of outbreaks in sub-saharan africa caused by antimicrobial-resistant (including multidrug-resistant [mdr]) enteric bacterial pathogens, most published data describe pathogens belonging to the genus or species of salmonella, vibrio cholerae, shigella and escherichia coli. by ‘molecular epidemiological investigation’, i refer to the use of molecular subtyping techniques that analyse bacterial strains at the level of their nucleic acid and so gives an indication of genetic similarity of strains.1 molecular subtyping is vital for accurate epidemiological investigations of bacterial infections.2 molecular subtyping allows one to segregate unlike strains of the same species or serotype of bacteria and identify clones or clusters of bacteria (genetically related strains). molecular subtyping allows one to track the spread of strains or clones and determine how local strains compare to those circulating worldwide.3 molecular subtyping data is critical for successful epidemiological investigation of outbreaks of disease; in particular, outbreak-related cases of disease can be differentiated from sporadic cases of disease.4 knowledge of the molecular epidemiology of bacterial infections provides information of major circulating (infecting) clones, so that in times of antimicrobial treatment and vaccine interventions, well-informed and educated decisions can be made to combat the disease.5 the mid-1990s saw the start of noteworthy publications describing the use of molecular subtyping techniques to investigate the molecular epidemiology of antimicrobial-resistant enteric bacterial pathogens in sub-saharan africa.6 these molecular subtyping techniques included plasmid profiling, ribotyping (southern blotting of restricted genomic dna and probing with ribosomal genes), random amplified polymorphic dna analysis (also called arbitrarily primed polymerase chain reaction [pcr]), enterobacterial repetitive intergenic consensus elements pcr, pulsed-field gel electrophoresis (pfge) analysis, multiple-locus variable-number tandem-repeats analysis (mlva), multilocus sequence typing (mlst), multilocus sequence analysis and whole-genome sequencing (wgs) analysis. molecular subtyping data from sub-saharan africa is summarised in table 1 and the countries involved are highlighted in figure 1. to date, the most popular and well documented molecular subtyping techniques have included pfge, mlva, mlst and wgs analysis. traditional molecular subtyping methodologies, such as pfge, mlva and mlst, have their advantages and disadvantages; these have been well reviewed elsewhere on numerous occasions.1,2,7,8,9,10,11 some molecular subtyping techniques (such as ribotyping, random amplified polymorphic dna analysis and enterobacterial repetitive intergenic consensus elements pcr) may only allow a local comparison of bacterial strains within a single laboratory, because the workings of the methodology are difficult to standardise between laboratories, and so the data that is produced cannot be truly and accurately compared between laboratories. in contrast, a technique such as pfge analysis, has been standardised by pulsenet international (http://www.pulsenetinternational.org/) and is successfully used for inter-laboratory comparison of subtyping data and global comparison of bacterial strains.4 figure 1: map of africa highlighting (with shading) countries where antimicrobial-resistant enteric bacterial pathogens have been isolated and further investigated using molecular subtyping methodologies. table 1: summary of data from publications that have described the use of molecular subtyping methodologies to investigate antimicrobial-resistant enteric bacterial pathogens isolated in sub-saharan africa. newer approaches to molecular subtyping involve wgs analysis.12,13,14 analysis of wgs data for molecular epidemiological purposes can include multiple approaches; however, the more popular methods are whole-genome mlst and single nucleotide polymorphisms (snp) analysis.15 wgs data is electronically portable and can easily be shared between laboratories, allowing for a global inter-laboratory comparison of bacterial strains. current challenges surrounding wgs include how to standardise the quality of wgs data generated and how to standardise the analysis of wgs data, in order to allow for a successful inter-laboratory comparison of analysed wgs data. pulsenet international has taken on this challenge and published their vision for implementation of wgs for global foodborne disease surveillance and global analysis of enteric pathogens.15 the aim of the present manuscript is to review all publications that have described the use of molecular subtyping methodologies to investigate outbreaks due to multidrug-resistant enteric bacterial pathogens in sub-saharan africa. the review focuses on enteric pathogens belonging to the genus and species of salmonella, vibrio cholerae, shigella and escherichia coli, because most molecular epidemiological investigations have been described for these microorganisms. the aim of this review is to inform the readers about the current status of molecular subtyping capabilities and activities in sub-saharan africa and finally suggest that the way forward for molecular subtyping in african laboratories is the implementation of wgs analysis as soon as possible. a pubmed database literature search (english language only) was performed using the search strings: ‘africa outbreak mdr’, ‘africa outbreak multi’, ‘africa outbreak multidrug’, ‘africa outbreak multi drug’, ‘africa outbreak resistance’, ‘africa outbreak resistant’, ‘africa outbreak drug’, ‘africa outbreak antibiotic’, ‘africa outbreak antimicrobial’. these search strings were used in combination with genus and species names of the organisms listed above. all results were included in the review. nontyphoidal salmonella for nontyphoidal salmonella (nts), molecular epidemiological analysis of salmonella typhimurium and salmonella enteritidis are most commonly described in sub-saharan africa. one of the first reports using molecular subtyping techniques to investigate enteric bacterial pathogens isolated in sub-saharan africa was a 1991 study that used plasmid profiling (following restriction endonuclease digestion) to distinguish african isolates of salmonella enteritidis from united states (us) isolates.16 molecular subtyping of nts then progressed to pfge analysis of mdr salmonella typhimurium isolated in kenya, where 64 isolates were grouped into eight pfge clusters and were described as multiclonal.17 salmonella typhimurium accounts for a significant proportion of reported cases of nts-associated invasive disease in sub-saharan africa. nts-associated invasive disease has developed to become a leading public health challenge in sub-saharan africa.18,19 a predominant type of invasive salmonella typhimurium in sub-saharan africa is a mdr strain that has been designated st313 (based on its mlst profile). clonal spread of this mdr st313 strain was first reported from kenya and malawi in 2009, with the strain showing resistance to ampicillin, chloramphenicol, sulphamethoxazole, trimethoprim, streptomycin and kanamycin.20 in kenya, a more recent analysis of salmonella typhimurium outbreak isolates, using wgs-snp analysis, identified a single clade of mdr st313 strains showing resistance to ampicillin, ceftriaxone, chloramphenicol, sulphamethoxazole and trimethoprim; extended-spectrum beta lactamase (esbl) genes blactx-m-15, blatem-1 and blaoxa-1 were also harboured by the isolates.21 the mdr st313 has further been reported from malawi, nigeria, democratic republic of the congo and south africa.22,23,24 leekitcharoenphon and coworkers22 used mlst, pfge and wgs-snp analysis to describe a close relationship between mdr st313 strains from nigeria and democratic republic of the congo; strains were isolated from invasive (blood) and non-invasive (stool) specimens; all isolates harboured blatem1b, cata1, stra/b, sul1, and dfra1 genes coding for resistance to various classes of antimicrobial agents. keddy and coworkers23 used mlst to investigate salmonella typhimurium isolates from south africa and reported that nts meningitis in south africa was highly associated with the mdr st313 strain. besides salmonella typhimurium, salmonella enteritidis is also a major player with regard to nts-associated invasive disease in sub-saharan africa. feasey and coworkers25 used wgs-snp analysis to investigate 675 salmonella enteritidis isolates from 45 countries (including 28 african countries), to describe the existence of one global epidemic clade and two ‘african’ clades, of which the african clades are geographically confined to specific regions of sub-saharan africa. both african clades show mdr, with an enlarged mdr virulence plasmid. both african clades also show patterns of genomic degradation with a similarity to those shown by other host-restricted invasive salmonella serotypes described in africa: patterns of genomic degradation similar to the african salmonella typhimurium st313 clade. pulsed-field gel electrophoresis analysis is commonly used to investigate outbreaks of salmonella enteritidis.26,27,28 niehaus and coworkers26 reported on a salmonella enteritidis foodborne outbreak in south africa, where human isolates and a food isolate were shown to have an indistinguishable pfge pattern. salmonella enteritidis investigations have also included nosocomial outbreaks; vaagland and coworkers28 investigated a nosocomial outbreak of neonatal salmonella enteritidis in tanzania, with pfge analysis of mdr (ampicillin, chloramphenicol and cefuroxime) isolates suggesting a clonal outbreak. more recently, mlva has proven a suitable method to investigate the molecular epidemiology of salmonella enteritidis in south africa; mlva was used to investigate multiple foodborne outbreaks over the period 2013–2015; mlva was able to cluster outbreak isolates according to distinct mlva profiles.29 nosocomial outbreaks have also been investigated in south africa, where salmonella isangi and salmonella typhimurium have been involved.30,31 wadula and coworkers30 used pfge analysis to show clonality among esbl-producing salmonella isangi in paediatric hospital wards. smith and coworkers31 used pfge analysis, mlva and mlst to investigate an esbl-producing salmonella typhimurium outbreak in a paediatric hospital ward; outbreak isolates showed indistinguishable molecular subtyping profiles including an mlst subtype of st34, and showed resistance to ampicillin, ceftriaxone, trimethoprim, sulphamethoxazole, chloramphenicol and tetracycline. typhoidal salmonella in 2004, kariuki and coworkers32 documented one of the first reports using molecular subtyping techniques to characterise mdr salmonella typhi isolated in africa. they used pfge analysis to investigate 102 kenyan outbreak isolates of salmonella typhi and identified two distinct subtypes among the infecting isolates; 82% of the isolates were mdr with resistance to ampicillin, chloramphenicol, tetracycline, streptomycin and cotrimoxazole. when pfge analysis is standardised, as is the case with pulsenet methodology employed by participating pulsenet international laboratories, then inter-laboratory comparison of pfge patterns can occur, to allow for a global investigation of outbreak isolates and tracking of emerging strains. such was the case reported by smith and coworkers,4 who used pfge analysis to investigate an outbreak of salmonella typhi associated with a restaurant in south africa; the source of the infecting strain was a restaurant worker who was tracked to australia where a salmonella typhi isolate was recovered from the individual; the isolate shared an indistinguishable pfge pattern as compared to the pattern of the outbreak strain. using the same scenario as described above (comparison of pfge patterns within the pulsenet international laboratory network), keddy and coworkers33 investigated a mdr (including resistance to fluoroquinolones) salmonella typhi isolate recovered from a south african patient who had interacted with a person who had recently travelled to bangladesh; the pfge pattern of the isolate had never previously been seen within the south african pfge database, but the pfge pattern was typical of that seen of isolates from the indian subcontinent, evidence to support the view that the isolate originated in bangladesh. global tracking of salmonella typhi through inter-laboratory comparison of molecular subtyping data is particularly important in establishing the source (and spread) of mdr (and virulent) strains of salmonella typhi. an example of such a strain is salmonella typhi h58, a highly clonal mdr haplotype of salmonella introduced from asia into africa and now currently spreading through many sub-saharan african countries.5 further investigation of salmonella typhi outbreaks using pfge analysis have been reported from the malawi-mozambique border,34 in uganda35,36 and in south africa.37 in uganda, walters and coworkers36 investigated a prolonged waterborne outbreak in two neighbouring districts; they used pfge analysis to document the clonal spread of mdr salmonella typhi from the kasese district to the bundibugyo district. analysis of selected isolates showed mdr to ampicillin, chloramphenicol, cotrimoxazole, streptomycin and tetracycline. in south africa, keddy and coworkers37 investigated a waterborne outbreak in the delmas area of south africa in 2005; they used pfge analysis and mlva to show high relatedness among outbreak isolates. these outbreak isolates from 2005 were also shown to be highly related to isolates associated with a similar waterborne outbreak in the same area in 1993. besides pfge analysis, mlva is often described as a useful molecular subtyping method for discrimination of strains belonging to the salmonella species. tau and coworkers38 described the development and evaluation of a mlva assay for molecular subtyping of salmonella typhi in sub-saharan africa. they evaluated the mlva assay on a panel of african isolates and showed that it had higher discriminatory power as compared to pfge; the mlva assay was able to differentiate outbreak isolates from sporadic isolates. whole-genome sequencing – single nucleotide polymor-phisms analysis has been increasingly used to investigate the molecular epidemiology of salmonella typhi in sub-saharan africa; a common theme has been the description of the mdr salmonella typhi h58 strain.5,39 wong and coworkers5 used wgs-snp analysis to investigate 1832 salmonella typhi isolates from 63 global countries, to identify one major mdr lineage (named h58) that has arisen and spread across asia and africa over the past 30 years. multiple h58 transfers have occurred, moving from asia into africa. an mdr h58 epidemic has been described in sub-saharan africa where h58 lineages are displacing antimicrobial-susceptible strains. mdr h58 lineages are associated with resistance to ampicillin, trimethoprim, sulphonamides, chloramphenicol, streptomycin and tetracycline, including reduced susceptibility to fluoroquinolones.5,39 this mdr h58 lineage has now been described in the eastern and southern regions of sub-saharan africa including kenya, tanzania, malawi and south africa.5,38,39,40 hendriksen and coworkers41 also used wgs-snp analysis to investigate an outbreak of salmonella typhi in zambia; most isolates showed resistance to ampicillin, chloramphenicol, streptomycin, sulphamethoxazole and trimethoprim, while some isolates also had reduced susceptibility to fluoroquinolones. isolates belonged to a new variant of the h58 haplotype, namely haplotype h58b. interestingly, investigation of mdr salmonella typhi from nigeria -western africa, using wgs-snp analysis, has not detected the h58 lineage; instead the majority of isolates belonged to a different lineage -h56 lineage, which carries genes coding for resistance to ampicillin, tetracycline, chloramphenicol and sulphamethoxazole (blatem1, cata1, tetb, dfra1, sul1). this h56 lineage is relatively common across africa, predominantly in western and central regions of sub-saharan africa.42 vibrio cholerae ribotyping and enterobacterial repetitive intergenic consensus elements pcr were among the first molecular subtyping techniques used to characterise v. cholerae o1 in africa.43,44 in 1996, dalsgaard and coworkers43 documented the first molecular epidemiological investigations of mdr v. cholerae o1 in africa. they used ribotyping to investigate outbreak isolates in guinea-bissau; strains isolated in 1994–1995 showed a ribotype pattern which was different fro strains isolated in 1987, suggesting that the 1994–1995 outbreak was as a result of the introduction of a novel strain into the country. later, ribotyping analysis of v. cholerae o1 outbreak isolates from senegal showed the same ribotype pattern as the 1994–1995 guinea-bissau outbreak strain, suggesting that senegal acquired their outbreak strain from guinea-bissau.45 in 2001, dalsgaard and coworkers46 reported on the clonal relationship (using ribotyping) of cholera isolates from mozambican migrant workers associated with a 1998 outbreak that occurred in south african provinces bordering mozambique. mdr v. cholerae o1 isolates were associated with the outbreak among the migrant workers and the isolates showed resistance to furazolidone, streptomycin, sulphamethoxazole, trimethoprim and tetracycline; isolates also showed the presence of class 1 integrons and the sxt element. another analysis of mozambican mdr isolates associated with the 1998 mozambique/south africa cholera outbreak confirmed the presence of the sxt element among isolates, with enterobacterial repetitive intergenic consensus elements pcr dividing isolates into two different molecular subtypes.47 mobile genetic elements are mostly responsible for the molecular basis of mdr v. cholerae o1 in sub-saharan africa. mobile genetic elements include transposable elements (sxt elements), integrons and conjugative plasmids. the sxt element is a self-transmissible element that integrates into the chromosome and carries genes encoding resistance to several antimicrobial agents, including chloramphenicol, sulfamethoxazole, streptomycin, trimethoprim and furazolidone. the sxt element has been reported from numerous countries in sub-saharan africa.46,48,49,50,51 in 2006, scrascia and coworkers52 investigated v. cholerae o1 isolates from multiple outbreaks that occurred in kenya during 1998 and 1999. most isolates showed an identical ribotype profile and similar random amplified polymorphic dna analysis profile suggesting a clonal origin for the outbreaks; isolates were resistant to chloramphenicol, spectinomycin, streptomycin, sulphamethoxazole and trimethoprim. over the period 2008–2009, published data from ghana, cameroon, ethiopia and somalia reported similar trends (as described above) for mdr v. cholerae o1 isolates following analysis using ribotyping and random amplified polymorphic dna analysis that of each country reporting a clonal origin for outbreak isolates within their respective country.50,53,54,55 from 2007 onwards, trends in molecular subtyping of v. cholerae, started shifting to an increased use of pfge analysis. in 2007, keddy and coworkers56 used pfge analysis to compare the relatedness of south african v. cholerae o1 isolates from a 2001–2002 epidemic to that of a 1980–1987 epidemic; pfge analysis showed that isolates from the 1980–1987 epidemic were distinctly different to isolates from the 2001–2002 outbreak. in 2008, smith and coworkers57 reported on the first cholera outbreak in namibia over the period 2006–2007; mdr v. cholerae o1 isolates showed an indistinguishable pfge pattern and showed resistance to streptomycin, sulphamethoxazole and trimethoprim. in 2011, ismail and coworkers48 investigated an outbreak of esbl-producing mdr v. cholerae o1 in south africa which occurred over the period may to july 2008; pfge analysis showed a clonal relationship among isolates, with isolates showing resistance to ampicillin, cotrimoxazole, chloramphenicol, nalidixic acid, tetracycline, kanamycin and streptomycin. the molecular basis of antimicrobial resistance was explained by proving the presence of the sxt element and the blatem gene encoding tem-63 β-lactamase. later in 2008, a v. cholerae o1 outbreak in zimbabwe spilled over into south africa, which triggered a very large outbreak in south africa over the period november 2008 to april 2009. mdr outbreak isolates showed similar pfge patterns: isolates showed the presence of the sxt element encoding multidrug resistance; some isolates also showed esbl activity due to the presence of the blatem gene encoding tem-63 β-lactamase.58 in october 2010, the haiti cholera outbreak started, with involvement of mdr v. cholerae o1.59,60 pfge analysis of haiti outbreak strains isolated over the period october 2010 to february 2011 showed that a single pfge profile predominated. this predominant profile was also shown in mdr strains from cameroon, south africa, india, nepal, pakistan, afghanistan and oman61; this global comparison of v. cholerae o1 pfge patterns was facilitated via pulsenet international laboratories. further investigation with wgs-snp analysis showed that the haiti outbreak strain was a hybrid-type of el tor strain encoding a classical-type of cholera toxin and that the outbreak strain was most closely related to strains originating from india and cameroon.62 the haiti cholera outbreak identified the need for higher resolution molecular subtyping methodologies for comparison of v. cholerae o1 isolates; this sparked the increased use of nucleotide sequencing approaches including wgs, to generate nucleotide sequence data for comparison of isolates. in 2011, thompson and coworkers63 characterised v. cholerae o1 outbreak isolates from ghana using multilocus sequence analysis of housekeeping genes and identified two major clusters in ghana. in 2011, mutreja and coworkers64 provided evidence for multiple waves of global transmission (from the bay of bengal) within the seventh cholera pandemic, which included multiple waves of transmission into sub-saharan africa; these data were made possible by analysis of wgs data (wgs-snp analysis approach) from a global collection of v. cholerae o1 isolates, including african outbreak isolates. in 2013, kiiru and coworkers,65 reported a wgs-snp analysis on kenyan v. cholerae isolates which included environmental isolates (serogroups o1 and non-o1) and v. cholerae o1 clinical isolates associated with outbreaks; they found that a clade comprising clinical isolates and some environmental isolates mapped back onto wave three of the monophyletic seventh cholera pandemic phylogeny, while some other environmental isolates were phylogenetically very different from the monophyletic seventh pandemic lineage of v. cholerae o1. the continued presence of mdr associated stx elements were documented on the genomes of mdr clinical and mdr environmental isolates. recent isolates of v. cholerae o1 from tanzania have also been found to map back onto wave three of the monophyletic seventh cholera pandemic phylogeny, as determined by wgs-snp analysis.66 interestingly, wgs-snp analysis of mdr v. cholerae o1 outbreak isolates recently sourced from cameroon has found that these cameroonian isolates do not map onto the phylogeny of isolates from other african countries such as kenya, tanzania, zambia, zimbabwe and mozambique; instead, cameroonian isolates are of their own distinct clonal cluster (clade), forming part of an isolated reservoir of mdr v. cholerae o1 in the lake chad basin.67 although wgs approaches for comparison of v. cholerae isolates have gained a stronghold in sub-saharan africa, the traditional molecular subtyping methodologies of pfge, mlva and mlst are still commonly used to investigate the molecular epidemiology of v. cholerae in sub-saharan africa, as these technologies still remain more accessible and more affordable for most laboratories66,68,69,70,71,72,73. for these recent reports, the central themes remain the same as previous studies. the major conclusions for these studies are summarised as follows. the most commonly reported cholera-causing organism is characterised as v. cholerae o1 biotype el tor, the so-called ‘atypical’ el tor variant housing genetic determinants coding for a ‘classical-type’ of cholera toxin. the majority of v. cholerae o1 isolates show the presence of sxt elements encoding a large part of the mdr phenotype. the majority of isolates show mdr. a common profile includes resistance to ampicillin, chloramphenicol, sulphamethoxazole, trimethoprim, spectinomycin and streptomycin, including reduced susceptibility to ciprofloxacin. molecular subtyping of v. cholerae o1 isolates shows examples of geographical clustering, as already described above in the mention of cameroonian isolates characteristic of the lake chad basin region; moore and coworkers72 found that isolates from western africa (togo and guinea) form a closely related group which is separated from a distinctive cluster associated with the african great lakes region (democratic republic of the congo and zambia). smith and coworkers71 found that a mozambican cluster of isolates was distinctly different (including a different antimicrobial resistance profile) as compared to isolates from western africa (togo, guinea and côte d’ivoire) and central africa (democratic republic of the congo). shigella species shigella dysenteriae was the first shigella species in sub-saharan africa that was characterised by molecular subtyping techniques; these details were first documented in the years 1996 and 1997.6,74 in 1996, kariuki and coworkers6 documented an outbreak of dysentery due to mdr s. dysenteriae serotype 1 in kenya: all 22 outbreak isolates were resistant to ampicillin, chloramphenicol, sulphamethoxazole, trimethoprim, streptomycin and tetracycline; pfge and plasmid profiling showed clonality among the isolates. in 1997, pillay and coworkers74 documented a nosocomial outbreak of dysentery caused by mdr s. dysenteriae serotype 1 which transpired in a psychiatric institution in south africa. isolates were resistant to ampicillin, chloramphenicol, tetracycline and cotrimoxazole; ribotyping showed clonality among the isolates. later in 1996, a report documented how pfge analysis was used to investigate mdr s. dysenteriae serotype 1 isolates associated with two separate dysentery outbreaks in the central african republic; pfge analysis provided evidence to show that each outbreak was associated with a different clone.75 in 2009, smith and coworkers76 reported a cluster of 29 watery diarrhoea cases associated with mdr shigella boydii serotype 2 isolates in south africa. isolates were resistant to ampicillin, trimethoprim, sulphamethoxazole and streptomycin; pfge analysis showed clonality among the isolates. more recent molecular epidemiological investigations of shigella species in sub-saharan africa has involved wgs-snp analysis of isolates. connor and coworkers77 used wgs-snp analysis to investigate 351 global (including africa) isolates of shigella flexneri, to show that s. flexneri is composed of seven distinct phylogenetic groups. each phylogenetic group contains geographically restricted sub-lineages that seem to have constantly inhabited areas for several decades; there is limited evidence of intercontinental transmission. antimicrobial resistance determinants have been acquired and maintained locally on several episodes – to summarise, s. flexneri from sub-saharan africa cluster together and form a distinct lineage as compared to lineages from other parts of the world. njamkepo and coworkers78 used wgs-snp analysis to investigate 331 global (including africa) isolates of s. dysenteriae serotype 1, to show that s. dysenteriae serotype 1 is composed of four distinct lineages. lineage iv contains most of the recent (last few decades) isolates from africa and the indian subcontinent; lineage iv has been transmitted in several waves to africa; most recent (since 1990) outbreaks in africa have been caused by this lineage iv; compared to other lineages, lineage iv show antimicrobial resistance including mdr. escherichia coli surprisingly very little published data exist concerning molecular epidemiological investigation of outbreaks in sub-saharan africa caused by e. coli enteric pathogens (diarrhoeagenic e. coli). diarrhoeagenic e. coli includes the following six main categories: shiga-toxin producing e. coli (which includes the enterohaemorrhagic e. coli), enteropathogenic e. coli, enteroinvasive e. coli, enterotoxigenic e. coli, enteroaggregative e. coli and diffusely adherent e. coli. in 2003, okeke and coworkers79 reported on the etiology of acute diarrhoea in adults in nigeria: shiga-toxin producing e. coli and enteroaggregative e. coli were significantly associated with the diarrhoea. pfge analysis of shiga-toxin producing e. coli isolates provided evidence to suggest that an outbreak had occurred. in 2012, tau and coworkers80 reported on the characterisation of e. coli o104 isolates from south africa for the years 2004–2011. this study was undertaken in response to the 2011 outbreak of bloody diarrhoea and haemolytic uraemic syndrome which occurred in germany, caused by an enteroaggregative shiga-toxin producing e. coli o104:h481. the south african investigation found two antimicrobial-susceptible enteropathogenic e. coli o104:non-h4 isolates and five enteroaggregative e. coli o104:h4 isolates (showing resistance to ampicillin, sulphamethoxazole and trimethoprim). pulsed-field gel electrophoresis analysis showed that these south african isolates were unrelated to the germany outbreak strain. conclusion whole-genome sequencing is undoubtedly the way forward for investigation of enteric pathogens in diagnostic and public health laboratories. numerous publications have analysed data sets to compare results obtained using old-fashioned molecular subtyping methodologies versus results obtained using newer wgs analysis methodologies.14,82,83,84 conclusions from these studies are unanimous and convincing, in that wgs trumps all other methodologies. old-fashioned methodologies offer limited genetic resolution and limited discriminatory ability and there can sometimes be a lack of accuracy; all these hinder epidemiologic investigations. for example, data from old-fashioned methodologies can incorrectly conclude that unrelated isolates are indistinguishable, resulting in false alerts and unnecessary investigations. whole-genome sequencing provides a supreme unparalleled discriminatory ability, can reflect phylogeny and can provide an evolutionary context. in addition, wgs can provide valuable information related to genetic determinants conferring antimicrobial resistance. numerous publications have reported how analysis of wgs data can be used to infer antimicrobial resistance in bacterial pathogens;85,86,87,88,89 these studies have reported high concordance between wgs resistance predictions and conventional phenotypic antimicrobial susceptibility testing methodologies. even so, conventional phenotypic susceptibility testing methodologies still have their place in the laboratory and must continue to be used for the foreseeable future. for now, wgs antimicrobial resistance prediction is best used as an adjunct to conventional phenotypic susceptibility testing. in summary, public health laboratories in africa need to urgently reconsider and adapt their molecular surveillance methodologies to include wgs. it is foreseen that wgs analysis will eventually replace all traditional methods currently used in the microbiology laboratory, both traditional phenotypic methods and traditional genotypic methods including molecular subtyping methods such as pfge, mlva and mlst.90,91,92,93 within the next year, the pulsenet usa programme is planning to have wgs (and analysis of wgs data) fully implemented as its primary method for molecular epidemiological investigation of enteric pathogens.94 the next challenge is how to effectively implement wgs analysis globally in public health laboratories, how to standardise the quality of wgs data generated and how to standardise the analysis of wgs data, all in order to allow for a successful inter-laboratory comparison of analysed wgs data. pulsenet international has taken this challenge and published their vision for implementation of wgs for global foodborne disease surveillance and global analysis of enteric pathogens.15 pulsenet africa (a regional network of pulsenet international) believes and supports this vision for implementation of wgs for global analysis of 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france. emerg infect dis. 2017;23(9):1462–1470. https://doi.org/10.3201/eid2309.170336 zankari e, hasman h, kaas rs, et al. genotyping using whole-genome sequencing is a realistic alternative to surveillance based on phenotypic antimicrobial susceptibility testing. j antimicrob chemother. 2013;68(4):771–777. https://doi.org/10.1093/jac/dks496 mcdermott pf, tyson gh, kabera c, et al. whole-genome sequencing for detecting antimicrobial resistance in nontyphoidal salmonella. antimicrob agents chemother. 2016;60(9):5515–5520. https://doi.org/10.1128/aac.01030-16 zhao s, tyson gh, chen y, et al. whole-genome sequencing analysis accurately predicts antimicrobial resistance phenotypes in campylobacter spp. appl environ microbiol. 2016;82(2):459–466. https://doi.org/10.1128/aem.02873-15 stoesser n, batty em, eyre dw, et al. predicting antimicrobial susceptibilities for escherichia coli and klebsiella pneumoniae isolates using whole genomic sequence data. j antimicrob chemother. 2013;68(10):2234–2244. https://doi.org/10.1093/jac/dkt180 mason a, foster d, bradley p, et al. accuracy of different bioinformatics methods in detecting antibiotic resistance and virulence factors from staphylococcus aureus whole-genome sequences. j clin microbiol. 2018;56(9):e01815–17. https://doi.org/10.1128/jcm.01815-17 koser cu, ellington mj, cartwright ej, et al. routine use of microbial whole genome sequencing in diagnostic and public health microbiology. plos pathog. 2012;8(8):e1002824. https://doi.org/10.1371/journal.ppat.1002824 long sw, williams d, valson c, et al. a genomic day in the life of a clinical microbiology laboratory. j clin microbiol. 2013;51(4):1272–1277. https://doi.org/10.1128/jcm.03237-12 bertelli c, greub g. rapid bacterial genome sequencing: methods and applications in clinical microbiology. clin microbiol infect. 2013;19(9):803–813. https://doi.org/10.1111/1469-0691.12217 allard mw. the future of whole-genome sequencing for public health and the clinic. j clin microbiol. 2016;54(8):1946–1948. https://doi.org/10.1128/jcm.01082-16 oakeson kf, wagner jm, mendenhall m, rohrwasser a, atkinson-dunn r. bioinformatic analyses of whole-genome sequence data in a public health laboratory. emerg infect dis. 2017;23(9):1441–1445. https://doi.org/10.3201/eid2309.170416 abstract introduction methodology and data analysis review findings implications and recommendations acknowledgements references about the author(s) adesola olalekan department of medical laboratory science, university of lagos, idiaraba, lagos, nigeria centre for genomics of non-communicable diseases and personalized healthcare (cgnph), university of lagos, akoka, lagos, nigeria bamidele iwalokun centre for genomics of non-communicable diseases and personalized healthcare (cgnph), university of lagos, akoka, lagos, nigeria department of molecular biology and biotechnology, nigerian institute of medical research, yaba, lagos, nigeria oluwabukola m. akinloye department of medical laboratory science, oulton college, moncton, new brunswick, canada olayiwola popoola department of medical laboratory science, university of lagos, idiaraba, lagos, nigeria centre for genomics of non-communicable diseases and personalized healthcare (cgnph), university of lagos, akoka, lagos, nigeria titilola a. samuel centre for genomics of non-communicable diseases and personalized healthcare (cgnph), university of lagos, akoka, lagos, nigeria department of biochemistry, university of lagos, idiaraba, lagos, nigeria oluyemi akinloye department of medical laboratory science, university of lagos, idiaraba, lagos, nigeria centre for genomics of non-communicable diseases and personalized healthcare (cgnph), university of lagos, akoka, lagos, nigeria department of molecular biology and biotechnology, nigerian institute of medical research, yaba, lagos, nigeria citation olalekan a, iwalokun b, akinloye om, et al. covid-19 rapid diagnostic test could contain transmission in lowand middle-income countries. afr j lab med. 2020;9(1), a1255. https://doi.org/10.4102/ajlm.v9i1.1255 review article covid-19 rapid diagnostic test could contain transmission in lowand middle-income countries adesola olalekan, bamidele iwalokun, oluwabukola m. akinloye, olayiwola popoola, titilola a. samuel, oluyemi akinloye received: 26 apr. 2020; accepted: 07 aug. 2020; published: 30 sept. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: coronavirus disease 2019 (covid-19) caused by severe acute respiratory syndrome coronavirus-2 (sars-cov-2) has impacted heavily on global health. although real-time polymerase chain reaction (rt-pcr) is the current diagnostic method, challenges for lowand middle-income countries (lmics) necessitate cheaper, higher-throughput, reliable rapid diagnostic tests (rdts). objective: we reviewed the documented performance characteristics of available covid-19 rdts to understand their public health utility in the ongoing pandemic, especially in resource-scarce lmic settings. methods: using a scoping review methodology framework, common literature databases and documentary reports were searched up to 22 april 2020, irrespective of geographical location. the search terms included ‘sars-cov-2 and serological testing’ and ‘covid-19 and serological testing’. results: a total of 18 rdts produced in eight countries, namely china (6; 33.33%), the united states (4; 22.22%), germany (2; 11.11%), singapore (2; 11.11%), canada, kenya, korea and belgium (1 each; 5.56%), were evaluated. reported sensitivity ranged from 18.4% to 100% (average = 84.7%), whereas specificity ranged from 90.6% to 100% (average = 95.6%). the testing time ranged from 2 min to 30 min. of the 12 validated rdts, the igm/igg duo kit with non-colloidal gold labelling system was reported to elicit the highest sensitivity (98% – 100%) and specificity (98% – 99% for igg and 96% – 99% for igm). conclusion: we found reports of high sensitivity and specificity among the developed rdts that could complement rt-pcr for the detection of sars-cov-2 antibodies, especially for screening in lmics. however, it is necessary to validate these kits locally. keywords: coronavirus disease; covid-19; sars-cov-2; rapid diagnostic test; lowand middle-income countries. introduction coronavirus disease 2019 (covid-19) is an emerging respiratory disease that was first reported to the world health organization (who) as a cluster of pneumonia of unknown origin from wuhan, china, in december 2019.1 the unknown causative agent was found through deep sequencing to be severe acute respiratory syndrome coronavirus-2 (sars-cov-2) on 7 january 2020 and the disease covid-19 was named on 11 february 2020. in response, who declared covid-19 as a public health emergency of international concern on 30 january 2020 and a pandemic on 11 march 2020.1 as of 22 april 2020, an estimated 2 572 805 confirmed cases and 178 551 confirmed deaths from covid-19 had been reported.2 the first 10 cases in africa were reported in five countries (nigeria, algeria, morocco, egypt and senegal).3 although earlier cases of covid-19 in many lowand middle-income countries (lmics) were described as imported by travelers from china, italy, the united kingdom and germany, community transmission has now become the major cause of new covid-19 infections.3,4 early, rapid, large-scale diagnosis and accurate diagnosis of covid-19 is one of the key interventions for covid-19 containment in both high-income and lmic settings.3 the availability of the sars-cov-2 genome has led to the development and validation of various reverse transcriptase real-time polymerase chain reaction (rt-pcr) in vitro diagnostic test kits by different manufacturers for covid-19 diagnosis.5,6 this diagnostic test is based on the detection of genes encoding the envelope (e), spike (s), nucleoplasid (n), rna-dependent rna polymerase and open reading frame 1a/b (e.g. orf1ab, orf1a, orf1b) polyproteins within the genomic rna of sars-cov-2.5,6,7 due to lack of culture facilities, the rt-pcr method is currently the reference standard method of confirming covid-19 diagnosis in suspected cases globally. for epidemiological investigation, public health and clinical actions, rt-pcr has been shown to be very reliable at screening and confirming the diagnosis of covid-19 using upper respiratory (e.g. nasopharyngeal swab, oropharyngeal swab, throat swab and nasal swab) and lower respiratory (e.g. sputum and bronchioalveolar lavage) samples.7,8 real-time pcr has also been useful for monitoring viral rna shedding dynamics during the acute phase of the disease and viral rna decay and disappearance during the convalescence stage of the disease among survivors.9,10 however, in spite of its high analytical sensitivity its detection range is limited to 3.2 – < 10.0 rna copies per reaction.6,7,8 the rt-pcr method has been reported from studies done inside and outside china to also be prone to giving false negative results under certain conditions, thereby missing some covid-19 cases. these missed cases are therefore not isolated increasing community transmission.8,9,10 these conditions include insufficient or inappropriate sample for viral rna isolation, poor sample transportation to the laboratory, poor storage of the isolated rna samples, poor quality of the rt-pcr assay and poor timing for sample collection. the asymptomatic phase of sars-cov-2 infection – the first few days post infection onset and the convalescence phase ≥ 14 days post infection onset, especially in a missed infection, have been indicated as times when cases can be missed.7,8,9,10 poor quality rt-pcr assay is characterised by an inconsistent cycle threshold value and/or lack of amplification signal for one or two targeted genes. these missed cases are therefore not isolated increasing for sar-cov-2 detection.6,7,8 also, due to limited financial resources, the limited number of accredited molecular laboratories of biosafety level 2/3 and limited number of technical experts, the scaling up of rt-pcr for covid-19 diagnosis is limited in lmics.7,8,9,10 taken together, the above challenges of rt-pcr have necessitated the deployment of serological rapid diagnostic tests (rdts) for covid-19 diagnosis, which could identify asymptomatic and convalescent covid-19 cases undiagnosed by rt-pcr. covid-19 serological rdts are antigen-antibody based tests that detects sars-cov-2 igm and/or igg in human blood samples or sars-cov-2 viral antigen from respiratory samples within 15 min.8,11,12 unlike the rt-pcr protocols, serological tests require less expensive equipment, no technical expertise or electricity to run and very minimal biosafety requirements. also, unlike rdts that use small amounts of biological sample (10 ul – 20 ul) and have an average run time of 15 minutes, the rt-pcr protocols use large amounts of samples (150 ul – 200 ul) and have an average run time of about 90 minutes.6,7,8 these advantages of serological rdts have attracted serious attention for their use in large-scale covid-19 serological rdts are antigen-antibody based tests that detects sars-cov-2 igm and/or igg in human blood samples or sars-cov-2 viral antigen from respiratory samples within 15 min testing especially at the peripheral level of the health system and outside hospital settings in lmics. data from worldmeters show that african countries compared to other countries conducted fewer tests per population (figure 1). this lower testing power means relatively fewer cases can be detected. thus, the rollout of various rdt kits by different manufacturers could be a favourable development particularly for lmics as rdts can be easily scaled up for rapid covid-19 diagnosis.11,12 besides, rdts can provide additional sero-epidemiological data that will be used to determine the magnitude of covid-19 spread within a population. rdts achieve this by identifying active and previous symptomatic or asymptomatic cases; these data are then used to calculate case-fatality rate and determine the anti-sars-cov-2 immunity level of a community.11,12 however, to harness the various epidemiological and clinical usefulness of currently available covid-19 serological rdts, it is important to determine and/or validate their performance levels. in the present scoping review, the following research questions will be answered: (1) what are the currently available serological rdts for testing, (2) to what extent have these serological rdts been validated by their manufacturers and (3) what is the level of performance characteristics of these serological rdts? presently, the level of accuracy of many serological rdts available for use in lmics remains unclear, coupled with insufficient information about their strengths and limitations. this review will provide insight into the performance characteristics of these kits and enable evidenced-based decisions for their possible use in large-scale covid-19 testing and containment strategies in lmics. figure 1: distribution of severe acute respiratory syndrome coronavirus-2 burden and test per population in selected african countries. methodology and data analysis a scoping review was conducted using a methodology framework by arksey13 with modification as described by adhikari et al.14 this includes: (1) identifying a clear research objective and search strategies, (2) identifying relevant research articles, (3) selecting research articles, (4) extracting and charting of data, and (5) summarising, discussing, analysing and reporting the results. the online databases searched included google scholar, medrxiv, biorxiv and pubmed, as well as documentary reports and white paper publications from relevant online websites including who, the united states centers for disease control and prevention (cdc) and the nigeria centre for disease control (ncdc) for information on new rdts for covid-19 published up to 22 april 2020. the search terms used include ‘sars-cov-2 and testing’, ‘covid-19 and rapid test’ and ‘covid-19 and diagnostic kits’. diagnostic kits published for the confirmation of other coronaviruses, such as the coronavirus associated with the 2003 sars outbreak in asia and middle east respiratory syndrome-coronavirus, were excluded. all the members of the review teams were involved in paper search and selection and a consensus was reached through peer review. duplicated publications and those with insufficient information were removed. the extracted data included the name of the diagnostic kit, manufacturer, test performance based on sensitivity, specificity, predictive positive and negative values, test principles and special characteristics and testing time. the data were entered into excel (microsoft, redmond, washington, united states) and exported to statistical product and service solution version 23 (ibm spss inc., chicago, illinois, united states) for cleaning and analysis. review findings overall, 28 publications on coronavirus-based diagnostic kits that matched the goal of this publication were included in this study (figure 2). articles were excluded based on duplication and lack of information on detection principle, type of kit, performance characteristics and manufacturers’ details. all eligible publications on covid-19 diagnostic kits by country and performance as of 22 april 2020 were summarised in numbers and percentages using descriptive analysis. on the whole, a total of 18 serological rdt kits were included for analysis. of these, four were antigen rdts (22.2%), nine were total immunoglobulin rdts (50%) and five were igm/igg serological rdts (27.8%) (figure 3). these kits were produced in eight countries, namely china (6; 33.33%), the united states (4; 22.22%), germany (2; 11.11%), singapore (2; 11.11%) and kenya, canada, korea and belgium (1 each; 6.56%) (table 1). fourteen of the rdt kits are antibody-detection kits for use with blood, plasma or serum (77.8%), and four were antigen-detection kits for use with swab, sputum or blood (22.2%). the majority of these kits (13; 72.22%) use lateral flow membrane technology, whereas the remaining five (27.78%) use colloidal gold (figure 4). figure 2: prisma flow diagram showing the scoping review process. figure 3: distribution of the serological rapid diagnostic tests by testing principle. figure 4: distribution of the serological rapid diagnostic tests by testing platform. table 1: performance characteristics of newly developed severe acute respiratory syndrome coronavirus rapid diagnostic kits analysed in this review, 22 april 2020. in general, the sensitivity of the test kits irrespective of sample specification ranged from 18.4% to 100% and their specificity ranged from 90.6% to 100%. the pooled analysis revealed an average (range) sensitivity of 81.6% (72.9% – 88%) and specificity of 94.4% (88.2% – 97.5%). the sensitivity and specificity of lateral flow immunoassay membrane type rdt kits were in the range (average) of 84.4% – 100% (92.7%) and 90.6% – 100% (96%), respectively, and that of lateral flow immunoassay colloidal gold type were 18.4 – 99.1% (67.7%) and 91.7% – 100% (98.3%), respectively (figure 4). three of these kits, namely bodysphere rapid test (los angeles, california, united states), thermogenesis rapid covid-19 test kit (rancho cordova, california, united states) and nadal® covid-19 test kit (regensburg, germany), had a sensitivity of 99% – 100%. these three kits also had a specificity range of 91% – 100%. asides their better sensitivity and specificity compared to other rdts, these kits are for use with blood samples only, detect both igg and igm, and have shorter testing time of 2 – 10 min. the testing time for all the identified kits ranged from 2 to 30 min with an average testing time of 13.5 min (95% confidence interval = 10.8 min – 16.1 min). only two of the kits provided information on positive predicted value and negative predictive value (range = 87.5% – 100.0% to 26.2% – 96.2%). out of the 18 rdts identified, 6 (33%) were not subjected to performance validation by the manufacturers of the kits. two of four antigen detection kits, seven of nine total immunoglobulin and three of five igm + igg serological kits were validated for sensitivity and specificity using rt-pcr assay as the reference method (table 1).15 on the whole, eight of the 13 lateral flow immunoassay membrane type and four of the five lateral flow immunoassay colloidal gold type kits were validated. of the 12 serological rdts validated by rt-pcr, the igm/igg duo kit with non-colloidal gold labelling system was found to elicit the highest and acceptable sensitivity (98% – 100%) and specificity (98% – 99%) values for igg and specificity of 96% – 99% for igm compared to other rdt types and the counterpart colloidal gold system-based igm/igg duo kit (figure 5). figure 5: performance characteristics of the different serological rapid diagnostic tests by testing platform; lfia membrane (a) and colloidal gold devices (b). implications and recommendations the need to expand diagnostic testing in order to cope with the current spread of covid-19 infection in many settings in lmics where resources for rt-pcr are limited and difficult to sustain has made rdt kits for sars-cov-2 an important tool in the global fight against the covid-19 pandemic. for patients with suspected infection, rt-pcr is used to detect sars-cov-2 in sputum, throat and nasopharyngeal swab, and secretions of the lower respiratory tract samples such as bronchoalveolar lavage and bronchial washings.16,17,18 however, limited facilities and human resources for molecular testing using rt-pcr tends to slow down testing for covid-19 in resource-limited countries. it has been argued that rdts do not have sufficient evidence to support their use in the covid-19 pandemic and hence should be used only in a research setting.19 cassaniti et al. have earlier reported low sensitivity and specificity of serological assay which led to misdiagnosis of covid-19 in the vast majority of the patients in their study population.20 the who has emphasised that tests with inadequate quality may miss patients with active infection or falsely categorise patients as having the disease, further hampering disease control efforts, hence the need for questioning the performance of sars-cov-2 rdt kits.19,21 most manufacturers of the rdts have performance characteristics of the kits validated using the rt-pcr technique as the reference method. however, several publications have reported the possibility of false-negative results using rt-pcr.22 thus, the sensitivity and specificity data of reviewed kits should be understood in light of this bias. the declaration of covid-19 as a global pandemic and the huge concern of its transmission in lmics where hiv, tuberculosis and malaria are currently endemic have necessitated the need to scale up diagnostic testing to mitigate further spread and the rising number of covid-19 deaths outside china.23,24 in many settings in lmics, such as small communities, riverine areas, health posts and primary health centres, resources for rt-pcr are absent.23,24 this has made the development of serological rdts for the detection of specific sars-cov-2 antigens, anti-sars-cov-2 igm and anti-sars-cov-2 igg an attractive and very important tool in the global fight against the covid-19 pandemic in lmics. findings from the 18 serological rdt kits analysed in this review imply that three different types of serological rdts, antigen, total immunoglobulin, and combined igm and igg-based rdt with the ability to provide results between 2 min and 30 min are currently available for potential large-scale testing in lmics using five types of biological samples (nasopharyngeal swab, throat swab, whole blood, plasma and serum). due to challenges associated with more sensitive biological samples such as bronchoalveolar lavage and sputum, both nasopharyngeal and throat swabs are used for covid-19 testing by rt-pcr in many settings.7,8,9 also, whole blood, plasma or serum is often used as biological sample for rt-pcr for monitoring viremia to predict covid-19 severity during the acute stage of infection and viral clearance during the convalescent stage.7,8 the latter is currently used to inform hospital discharge decisions in many countries; use of different samples for diagnosis and viral clearance determination can negatively impact on discharge decision-making.8,9,10,12 a potential way of circumventing discharge decision errors is to employ a diagnostic tool that uses the same type of sample for both diagnosis and viraemia monitoring such as the sars-cov-2 antigen and specific anti-sars-cov-2 igm/igg duo detection kit identified in this review. this can be integrated into the local covid-19 management guidelines in lmics. this guideline is currently being used in malaysia and europe.25,26 the primary weakness of rt-pcr for covid-19 diagnosis lies in its inability to detect infection using nasopharyngeal samples collected outside the viral rna shedding period. the shedding period is characterised by presence of low viral rna, such as seen in asymptomatic, pre-symptom days (~2 days prior to symptom onset) and post-infection days (~14 post infection onset).26,27 also, the rt-pcr, may also miss infections due to poor sample collection and preparation as well as poor storage of isolated rna. these weaknesses can be addressed by serological rdts, which detect the more stable viral immunogenic proteins such as the s and n proteins, which persist more than rna or anti-sars-cov-2 igm and igg which have been reported to peak between 2 and 3 weeks and 17 days post infection onset.26,27 guo et al.28 reported an improvement of covid-19 identification by rt-pcr from 51.9% to 98.6% with the integration of an igm-based immunoassay. however, the results of sensitivity (18.4% – 100%) and specificity (90.6% – 100.0%) reported for 12 of the 18 reviewed serological rdt kits by their manufacturers imply that the currently available covid-19 rdts are not equally accurate and only a few of them pass the sensitivity and specificity benchmark of 95%. zainol et al.29 recently reported a sensitivity range of 72.7% – 100.0% and specificity range of 98.7% – 100.0% for igm/igg duo-based serological rdt kits for covid-19 in their review in which nine serological kits were analysed. the authors also reported a sensitivity range of 86.4% – 90.6% and a specificity of 99% for total immunoglobulin-based rdts. in brazil, castro et al.30 reported a mean (range) anti-sars-cov-2 igm sensitivity of 82% (76% – 87%) and specificity of 97% (96% – 98%) and anti-sars-cov-2 igg sensitivity of 97% (90% – 99%) and specificity of 98% (97% – 99%). although in this review, only 5 of the 18 serological rdt kits offered combined igm and igg detection, we also found a better performance characteristic for this type of rdt kit compared to the antigen and total immunoglobulin kits using non-colloidal gold labelling system with acceptable sensitivity (98% – 100%) and a specificity (98% – 99%) values for igg and specificity of 96% – 99% for igm, suggesting the ability of these kits to detect past infections, confirm true negative results and rule out false positive covid-19 testing results by rt-pcr. however, the performance of these kits to confirm recent infections seems to be below the benchmark of 95%, since they had a sensitivity range of 85% – 94%, which was even lower for colloidal gold labelling systems at 57.1%. meanwhile, the improvement offered by the antigen-based rdt kits in this review can be said to be none or marginal at 84.4% – 96%. another implication of these findings is that more than one serological rdt kit may be needed for a sars-cov-2 detection algorithm to improve confirmation and diagnosis of covid-19 by rt-pcr, if deployed in lmics. it is also important to note that 6 of the 18 reviewed serological rdt kits lacked reports on sensitivity and specificity, thus the accuracy in diagnosising covid-19 is unknown as at the time of this review. this finding further reiterates the difficulty associated with sars-cov-2 serological rdt kit validation by manufacturers, since rt-pcr the reference method targets viral rna instead of specific sars-cov-2 antibodies or antigens. a similar opinion has been shared by castrol et al.,30 given the well-documented differences in the kinetics of the viral rna (even between samples) and anti-sars-cov-2 antibodies in infected individuals. as of 01 april 2020, the death toll for covid-19 was over a million globally and the need for accurate intervention to stop transmission and re-infection of covid-19 is now extremely necessary. the who advises countries to improve the rate of testing to identify an infected individual for appropriate isolation and treatment. the availability of efficient and rapid diagnostics for covid-19 has been indicated as one of the mitigation strategies to control the pandemic. rapid diagnostic tests are cheaper and more readily available; thus, they might be more useful stopping transmission by rapidly identifying positive and previous cases particularly in lmics. these data will in turn be useful for both disease diagnosis and surveillance. the rdt will either detect the presence of viral proteins (antigens) expressed by the covid-19 virus or the presence of antibodies in the blood of covid-19-infected people.31,32 the performance of the kits has been shown to depend on several factors such as the onset of illness, the viral load in the specimen, the integrity of the specimen collected from suspected cases, processing, age, nutritional status, the severity of the disease, and certain medications or underlying disease condition, especially immune suppression diseases and the precise formulation of the reagents in the test kits.19 the lmics reported the lowest rate of testing per population with corresponding lower numbers of cases compared with developed countries. this may be an indication of limited testing resources and facilities due to the challenges associated with rt-pcr. therefore, there may be several cases in this population that are not detected with antecedent clinical implications. the use of rdts will not only help to detect currently infected or previously exposed individuals who have developed immunity as well as identify asymptomatic carriers. these will inform decisions for public health measures, for example, cases among a more igm-positive population may be an indication of a subclinical outbreak. the economic impact of movement restrictions and lockdowns in many of these countries is not well managed, adding unimaginable suffering in an already impoverished population. the use of rdts for the screening of covid-19 may help to determine individuals who are at lower risk and may be permitted to go back to work. when coupled with clinical symptoms and molecular testing, rdts may serve as a first-line tool for diagnosis and help to better understand the spread of diseases. although the covid-19 test kit market is in its infancy, the global covid-19 outbreak and up-surging cases are driving the demand for rdts, hence researchers throughout the world are striving to develop rdts to track infected people. to date, very few countries have succeeded in developing sars-cov-2 testing kits, while some are still working on improving the performance of their products. with the dedicated global efforts on preventing the spread of covid-19 and flattening the curve, significant improvement must have occurred in improving the performance of covid-19 test kits. increasing accessibility to testing among other interventions has improved the containment and transmission of the infection. while algorithms have been developed to limit testing to individuals that fulfil certain criteria, such as contact with the infected patients, clinical symptoms of covid-19, travelling history to epidemic countries, etc., testing the entire population has been recommended.33 resource limitations means most lmics can not cope with the up-surge of infection and transmission. while testing per population is high in developed countries with over 3 million tested in the united states, testing per population is still very low in developing countries with less than 10 000 tested in nigeria as of 22 april 2020. therefore, the addition of validated serological-based rdt even with lower performance characteristics compared to rt-pcr may serve as complementary tools to increase the rate of testing per population, especially in lmics where community transmission is now on the rise. therefore, the use of rdts operated as lateral flow immunochromatographic assays to detect both igm and igg on separate test lines using whole blood, plasma or serum samples is desirable for lmics. wang et al. reported that the combination of rt-pcr testing and clinical features for diagnosis of covid-19 facilitated the management of the sars-cov-2 outbreak in china,22 and covid-19 mass testing facilities have been strongly advocated to end the epidemic rapidly.34 the use of rdt will not only allow mass testing facilities in lmics but coupled with clinical features in symptomatic patients and molecular testing (rt-pcr) in asymptomatic populations may help to contain transmission in lmics. conclusion considering the peculiarity of lmics, especially their economic situation, the standard rt-pcr may not be able to cope with the testing needs of these countries because of limited infrastructure and human resources. generally, it is agreed that rapid testing techniques are useful for screening for early detection of symptomatic cases, which is crucial for averting community or hospital transmission and strengthening contact tracing and active surveillance. this review revealed considerable good performance of the rdt with manufacturer sensitivity and specificity using varieties of samples including blood samples. hence, the use of rdt kits in lmics may increase access to testing and better triaging of covid-19 patients. we, however, identified that most of the proposed rapid kits have not been optimised and validated. it is important that the kits undergo further validation with samples from countries of proposed use in reference to rt-pcr before use. acknowledgements competing interests the authors have declared that no competing interest exists. authors’ contributions o.a. conceived the idea for the study. o.a., b.i., and a.o. designed the study. a.o., t.a.s., o.m.a. and o.p. searched for published work. o.a. and b.i. reviewed and made the selection of eligible studies. a.o., t.a.s., o.m.a. and o.p. extracted and compiled the data. o.a., b.i. and a.o. analysed the data while b.i. and a.o. prepared the first draft of the article. o.a. did the final editing of the article. all authors contributed to the writing of the article and have seen and approved the final version. ethical considerations ethical clearance was not required for this study. funding information this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references zhu n, zhang d, wang w, et al. a novel coronavirus from patients with pneumonia in china, 2019. n engl j med. 2020;382(8):727–733. https://doi.org/10.1056/nejmoa2001017 dong e, du h, 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[cited 2020 apr 06]. available from: https://www.ncbiotechorg/news/biomedomics-teams-bd-launch-rapid-covid-19-test kit tbtc-. nadalr covid-19 igg/igm test instruction manure [homepage on the internet]. 2020 [cited 2020 apr 20]. available from: https://www.dailymailcouk/news/article-8128327/test-test-covid-kits-10-minute-finger-prick-tests-mask-diagnose-instantly.html abstract introduction methods results discussion acknowledgements references about the author(s) fales z. mwamba department of laboratory, levy mwanawasa university teaching hospital, lusaka, zambia mwansa mwape department of laboratory, levy mwanawasa university teaching hospital, lusaka, zambia global health supply chain procurement and supply management, chemonics international, lusaka, zambia brenda c. simfukwe department of laboratory, levy mwanawasa university teaching hospital, lusaka, zambia nowella m. musunga department of laboratory, levy mwanawasa university teaching hospital, lusaka, zambia evans m. mathebula abbott rapid diagnostics (pty) ltd, pretoria, south africa school of health systems and public health, faculty of health sciences, university of pretoria, pretoria, south africa geofrey mupeta department of laboratory, levy mwanawasa university teaching hospital, lusaka, zambia department of pathology and microbiology, school of medicine and health sciences, mulungushi university, livingstone, zambia citation mwamba fz, mwape m, simfukwe bc, musunga nm, mathebula em, mupeta g. usability of a blood-based hiv self-test kit in lusaka province, zambia: a cross-sectional analysis. afr j lab med. 2025;14(1), a2903. https://doi.org/10.4102/ajlm.v14i1.2903 original research usability of a blood-based hiv self-test kit in lusaka province, zambia: a cross-sectional analysis fales z. mwamba, mwansa mwape, brenda c. simfukwe, nowella m. musunga, evans m. mathebula, geofrey mupeta received: 16 june 2025; accepted: 30 sept. 2025; published: 20 dec. 2025 copyright: © 2025. the authors. licensee: aosis. this work is licensed under the creative commons attribution 4.0 international (cc by 4.0) license (https://creativecommons.org/licenses/by/4.0/). abstract background: despite progress toward the joint united nations programme on hiv/aids (unaids) 95-95-95 targets, zambia faces persistent gaps in hiv testing coverage. the ministry of health implemented blood-based hiv self-testing (hivst) to improve accessibility. this study evaluated the checknow™ hivst kit’s usability in lusaka province health facilities. objective: to determine usability, awareness and user-friendliness of the checknow™ hivst among zambian adults. methods: we conducted a cross-sectional study from 04 september 2023 – 22 september 2023 across four high-volume healthcare facilities. a total of 323 checknow™ hivst kits were distributed, with 316 consenting adults successfully enrolled in the study. data were collected through structured questionnaires administered via face-to-face interviews following test completion, capturing information on socio-demographics, hiv testing history and user perception of the self-testing process. descriptive statistics were employed for data analysis. results: among 316 participants, 56.3% (178/316) were female, and 41.5% (131/316) were aged 25–34 years. the majority (95.0%, 300/316; p < 0.001) found the checknow™ kit easy to use, while 65.0% (206/316) had prior awareness of hivst. additionally, 83.6% (264/316; p < 0.001) followed the test instructions correctly and independently. a high proportion (98.7%, 312/316; p < 0.001) expressed willingness to test again, and 99.7% (315/316; p < 0.001) would recommend it to others. conclusion: the checknow™ blood-based hivst kit demonstrated high usability and ease of use, supporting its potential to expand hiv testing coverage in zambia. however, increased awareness efforts are necessary to maximise uptake and ensure broader accessibility. what this study adds: this study provides the first evidence that blood-based hiv self-testing is feasible and acceptable within zambian clinical settings. it offers a critical new strategy to expand testing coverage and reach key populations by integrating self-testing into routine health services. keywords: checknow™; hiv self-test; usability; awareness; user-friendliness. introduction hiv self-testing (hivst) has become an essential component of global strategies to increase hiv testing accessibility, particularly in resource-limited settings where healthcare infrastructure is constrained.1 recognising its potential, the world health organization recommends hivst as a complementary approach to conventional facility-based testing, with recent data indicating that 85% of countries had incorporated hivst into their national hiv policies by 2023.2,3 among available options, blood-based hivst, such as the checknow™ hivst, demonstrate superior clinical performance. this rapid lateral flow immunoassay is designed for the qualitative detection of antibodies to hiv-1 and hiv-2 using a finger-prick blood sample, and has manufacturer-reported sensitivity and specificity of 99.5% and 98.5%.4 studies have shown that these tests achieve 99.1% sensitivity compared to 92.3% for oral fluid tests during the critical acute infection phase.5 this enhanced detection capability makes blood-based tests particularly valuable for early hiv diagnosis and transmission prevention. the effectiveness of hivst programmes depends heavily on product usability. recent systematic reviews highlight persistent challenges with blood-based hivst, including difficulties with finger-prick blood collection (reported by 15% – 30% of users) and result interpretation errors that reduce testing frequency by up to 40% in some populations.6,7,8 these barriers are most pronounced among individuals with limited health literacy and older adults, underscoring the need for optimised test designs and clearer instructional materials.9,10 zambia’s hiv epidemic reflects these global challenges while presenting unique local considerations. with an adult hiv prevalence of 11.3%,11 the country has made significant progress in testing coverage, yet persistent gender disparities remain: male participants represent only 35% of facility-based tests despite comparable seroprevalence rates to female participants.11,12 in response, zambia’s ministry of health launched its 2020–2025 hivst scale-up plan, prioritising workplace and community-based distribution to reach underserved populations.13 this strategy builds on formative research demonstrating > 80% acceptability of hivst across demographic groups in zambian urban and peri-urban settings.14,15 this study therefore aimed at evaluating the user-friendliness, awareness, and usability of the blood-based checknow™ hivst kit in lusaka province. by assessing real-world implementation challenges and user experiences, this study can inform the ongoing national scale-up of hivst services and contribute to global understanding of blood-based self-testing optimisation. methods ethical considerations the ethical clearance for this study was obtained from the tropical disease research centre ethics committee, located at the tropical disease research centre, zambia, under reference number tdrc irb # tdrec/095/07/23. permission to conduct the research was granted by the zambia national research authority with reference number nhra021/10/08/2023. the principal investigator maintained active researcher registration (nhrar-r-389/17/05/2023) throughout the study period (04 september 2023 – 22 september 2023). written informed consent was obtained from all participants after a detailed explanation of the study’s objectives, procedures, potential risks, and benefits. participation was entirely voluntary, with assurances that individuals could withdraw at any time without penalty or negative repercussions. the study collected socio-demographic data from participants but did not involve the collection or analysis of bio-samples. this approach ensured ethical compliance while focusing on self-reported experiences with the hivst kit. to maintain confidentiality, all participant data were de-identified and securely stored, with exclusive access granted to the study team; no third-party access was permitted. the selection of participants was unbiased, enrolling only those who met the predefined inclusion criteria. study design we conducted a cross-sectional study at four high-volume health facilities in lusaka province (04 september 2023 – 22 september 2023) to assess the user-friendliness, awareness, and usability of the checknow™ blood-based hivst kit (abbott rapid diagnostic jena [gmbh], germany). a cross-sectional approach was chosen because it allows for a snapshot analysis of participants’ experiences, perceptions, and usability challenges at a single point in time. participants were randomly allocated into assisted and unassisted groups, with the assisted group receiving guidance from a trained healthcare worker or peer educator, while the unassisted group performed the self-test independently following the instructions for use. the structured study design ensured that usability factors could be compared between the two groups to assess variations in ease of use, interpretation accuracy, and willingness to recommend the test. study site and population the study was conducted in four healthcare facilities selected for their high hiv prevalence and testing volume: kanyama general hospital (urban). chawama general hospital (urban). nangongwe clinic (rural). chongwe district hospital (rural). the study population included all adults aged ≥ 18 years who sought hiv testing at these facilities and were willing to use the checknow™ hivst kit. sample size and sampling a total of 323 checknow™ hivst kits were systematically distributed across four study facilities, with allocation proportional to each site’s average monthly testing volume. participant recruitment occurred through consecutive random sampling during routine hiv testing services over a 14-day implementation period (04 september 2023 – 22 september 2023). inclusion and exclusion criteria inclusion criteria adults aged ≥ 18 years, with no prior hivst experience, seeking hiv testing, and willing to use the checknow™ hivst kit. exclusion criteria minors, or those declining to use self-test. data collection and quality control the study implemented rigorous quality control measures to ensure standardised data collection. the research questionnaire underwent formal validation (21 august 2023 – 25 august 2023) through a two-stage process involving content review by hiv testing experts followed by cognitive testing with 30 pilot participants. this validation process confirmed the instrument’s clarity and appropriateness for the study population. trained data collectors administered all study procedures following a comprehensive five-day training programme (28 august 2023 – 01 september 2023). the training curriculum covered proper hivst administration, ethical consent procedures, and standardised data collection techniques. only staff demonstrating full competency through practical assessments were certified to participate in the study. following test completion, trained data collectors conducted face-to-face interviews to collect comprehensive data using a structured electronic questionnaire. this included detailed socio-demographic characteristics (age, gender, urban-rural), clinical testing histories (previous hiv tests, results, and frequency), and qualitative user feedback about their testing experience. all physical documents, including signed consent forms and original test results, were securely stored in locked filing cabinets with restricted access limited to authorised study personnel only. all testing occurred in private designated spaces to ensure confidentiality and minimise external influences. statistical analysis data were validated using microsoft excel (version 2016, microsoft, redmond, washington, united states) before being analysed in spss statistics v26.0 (ibm corp., armonk, new york, united states). descriptive statistics summarised participant socio-demographic characteristics, hiv testing history, usability, and awareness outcomes. the pearson’s chi-square or fisher’s exact tests was used to explore associations between two categorical variables, while mann-whitney u test was used to compare means between two groups of participants. statistical analysis was performed using spss statistics v26.0 with the level of significance set at p < 0.05. qualitative feedback from open-ended responses was categorised thematically, identifying trends related to ease of use, challenges, and willingness to recommend the kit. results participant characteristics the study enrolled 316 participants across four lusaka province facilities, demonstrating significant demographic variations. gender distribution showed a statistically significant female predominance (56.3%, 178/316 vs male: 42.1%, 133/316; p = 0.002), with minimal missing data (1.6%, 5/316), as shown in table 1. age groups differed significantly from national census distributions (p < 0.001), with 25–34-year-olds representing the largest cohort (131/316, 41.5%), followed by 18–24-year-olds (35.7%, 113/316) and ≥ 35-year-olds (22.8%, 72/316). urban residents were over-represented (60.1%, 190/316 vs rural: 39.9%, 126/316; p < 0.001). testing history revealed exceptionally high prior engagement (95.6%, 302/316; p < 0.001 versus the national rate of 89%), with most recent tests occurring ≤ 1 year prior (61.9%, 187/302; p < 0.001), demonstrating frequent engagement with testing services. all four facilities demonstrated high testing uptake (97.8%, 316/323). site-specific distribution patterns are shown in table 2. table 1: participant socio-demographic characteristics, lusaka province, zambia, 04 september 2023 – 22 september 2023. table 2: kit utilisation by study site, lusaka province, zambia, 04 september 2023 – 22 september 2023. user-friendliness and awareness the checknow™ hivst kit exhibited outstanding usability and acceptability in the study. as shown in table 3, 95.0% (300/316; p < 0.001) of participants found the test easy to use, with only 4.4% (14/316) reporting difficulties. prior awareness of hivst varied significantly (65.0%, 206/316; p < 0.001), and among those familiar with self-testing, 58.3% (120/206) had prior testing experience. notably, a pronounced gender disparity was observed among previous users (p < 0.001): 69.2% were female participants (83/120), 29.2% were male participants (35/120), and gender information was unavailable for 1.6% (2/120). the kit demonstrated near-universal acceptability, with 98.7% (312/316; p < 0.001) of participants willing to reuse it and 99.7% (315/316; p < 0.001) recommending it to others, underscoring its potential for widespread adoption. table 3: usability and awareness outcomes, lusaka province, zambia, 04 september 2023 – 22 september 2023. user experience with checknow™ hiv self-test kit figure 1 indicates participants’ experiences with the checknow™ hivst kit. positive feedback includes responses indicating ease of use, confidence in result interpretation, satisfaction with privacy, and willingness to use or recommend the kit. negative feedback includes reports of difficulty in blood collection, challenges with self-pricking, uncertainty in result interpretation, or preference for assisted testing. over three-quarters (78%, 246/316) of respondents described the test as simple, though 22% (70/316) noted challenges with blood collection. more than half (65%, 205/316) expressed high trust in the accuracy of their results, while 35% (111/316) desired clearer instructions for interpretation. the majority (82%, 259/316) valued the discretion of self-testing, with 18% (57/316) suggesting improvements in packaging for enhanced confidentiality; 89% (281/316) would endorse the kit to others, citing convenience and reliability. figure 1: user experience themes with checknow™ hiv self-testing kit, lusaka province, zambia, 04 september 2023 – 22 september 2023. usability of the checknow™ hiv self-test the checknow™ hivst kit demonstrated excellent performance in our evaluation, with statistically significant findings, as shown in table 4. participants showed significantly higher rates of unassisted administration (58.9%, 186/316) compared to assisted use (23.7%, 75/316; p < 0.001), indicating successful independent implementation. the accuracy of participants’ interpretation of the test results was exceptionally high at 97.5% (308/316; p < 0.001), with only 1.6% (5/316) errors. this was assessed by comparing each participant’s recorded result against a trained healthcare worker’s independent reading of the same used test device. while 83.6% (264/316) of users comprehended instructions independently, the 16.1% (51/316) requiring assistance remained statistically significant (p < 0.001), highlighting a need for enhanced instructional materials. test results followed expected distributions, with 90.8% negative (287/316), 7.9% positive (25/316), and no invalid results. these outcomes, combined with minimal missing data and perfect test validity, confirm the kit’s robust real-world performance while identifying key opportunities for optimising user support through refined instructional aids for the 16.1% needing assistance (p < 0.001), and targeted training for healthcare workers supporting the 23.7% assisted cases. table 4: hiv self-testing performance metrics, lusaka province, zambia, 04 september 2023 – 22 september 2023. discussion our evaluation of the checknow™ blood-based hivst kit in zambia revealed several key findings with important implications for hiv testing programmes. the exceptionally high acceptability rates (95.0% ease of use, 98.7% willingness to reuse, and 99.7% recommendation rate) demonstrate the potential of well-designed blood-based hivst kits to expand testing access. these results align with recent multi-country studies showing that optimised hivst kits can increase testing uptake by 40% – 60% among hard-to-reach populations, including men and adolescents.16,17,18 the near-universal recommendation rate is particularly noteworthy, as peer-driven distribution has been shown to increase first-time testing threefold in similar settings.19,20 the gender distribution of our participants (56.3% female) reflects a well-documented pattern across sub-saharan africa, where women are consistently 1.8 times more likely to utilise hivst services than men.21 this disparity stems from multiple factors, including men’s greater occupational mobility (particularly in mining and transportation sectors), and perceptions that clinical settings are ‘female spaces’.22,23 successful interventions from uganda’s male-friendly hivst programme, which increased testing rates among men by 35% through workplace distribution, could be adapted for zambia’s context.24,25 targeted outreach to male-dominated workplaces and social venues should be prioritised in scale-up plans. the 97.5% success rate in performing all testing steps correctly suggests that previous concerns about the complexity of blood-based hivst may be overcome through improved kit design. key features contributing to this success likely include: intuitive pictorial instructions (shown to improve accuracy by 22% in low-literacy populations),26 pre-filled reagents that minimise procedural steps,27 and clear visual indicators for result interpretation.28 however, our findings contrast with studies showing 15% lower success rates among older adults in tanzania,29 highlighting the need for age-specific usability assessments during implementation. the 65% pre-test awareness level we observed, while comparable to rates in south africa (69.9%),30 indicates substantial room for improvement. zambia could adapt successful awareness strategies from other settings, including the use of community-based ‘hivst champions’ (shown to increase awareness to 81% in pilot programmes31), integration with mobile health platforms for results interpretation support,32 and targeted messaging through male-oriented community networks.33 study limitations while this study provides valuable insights into hivst usability, three key limitations should be considered. first, the single-site urban design may limit generalisability to rural populations where national literacy rates are 37% lower than urban populations. second, reliance on self-reported data introduces potential social desirability bias, suggesting future research should incorporate direct observational methods for more objective assessment. finally, the cross-sectional design cannot evaluate critical longitudinal outcomes including sustained testing frequency over time, linkage-to-care rates following self-testing, or patterns of repeat test utilisation. these limitations highlight the need for multi-site longitudinal studies that combine usability assessments with clinical outcome tracking in diverse population groups. conclusion and recommendations the checknow™ hivst kit’s strong performance, evidenced by its 95.0% usability rate and 99.7% recommendation score, supports its inclusion in the national hiv testing strategy in zambia. to maximise impact, we recommend: gender-sensitive distribution through workplaces and male-oriented community venues, and targeted awareness campaigns addressing persistent knowledge gaps. these findings contribute to growing evidence that well-designed blood-based hivst can play a crucial role in achieving universal testing targets, particularly among populations that face barriers to facility-based testing. acknowledgements the authors would like to thank the invaluable contributions of abbott rapid diagnostics (pty) ltd, which were key in ensuring the effective coordination and management of the project. the authors also acknowledge the support and contribution by staff and management at kanyama first-level hospital, chawama first-level hospital, chongwe district hospital and nangongwe urban clinic. competing interests the authors declare that there was no competing interest that may have inappropriately influenced them into writing this article. credit authorship contribution fales z. mwamba: conceptualisation, methodology, writing – original draft, project administration, validation, data curation, writing – review & editing, supervision, funding acquisition. mwansa mwape: conceptualisation, methodology, project administration, validation, data curation. brenda c. simfukwe: conceptualisation, methodology, project administration, validation, data curation. nowella m. musunga: conceptualisation, methodology, project administration, validation, data curation. evans m. mathebula: writing – original draft, writing – review & editing. geofrey mupeta: conceptualisation, methodology, formal analysis, writing – original draft, visualisation, project administration, software, validation, data curation, writing – review & editing. sources of support this research was funded by abbot rapid diagnostics (pty) ltd through the ministry of health zambia. data availability the data that support the findings of this study are available on request from the corresponding author, geofrey mupeta the data are not publicly available due to restrictions as they contain information that could compromise the privacy of research participants. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. the article does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the authors are responsible for this article’s results, findings, and content. references world health organization. guidelines on hiv self-testing and partner notification [homepage on the 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dzinamarira t. approaches for improving linkage to hiv care among hiv self-testing individuals in sub-saharan africa. bmj glob health. 2023;8:e012664. https://doi.org/10.1136/bmjgh-2023-012664 sikombe k, beres lk, simbeza s. male testing barriers in zambia: a mixed-methods analysis. j int aids soc. 2022;25(7):e25982. https://doi.org/10.1002/jia2.25982 mbele w, dako-gyeke p, ndapewa frans a, ndayishimiye jc, ching j. empowerment and utilization of hiv testing among partnered women in zambia: evidence from the zambia demographic and health survey 2018. plos glob public health. 2024;4(12):e0002791. https://doi.org/10.1371/journal.pgph.0002791 world health organization. consolidated guidelines on hiv prevention, testing, treatment, service delivery and monitoring: recommendations for a public health approach [homepage on the internet]. geneva: who; 2021 [cited 2025 mar 01]. available from: https://www.who.int/publications/i/item/9789240031593 ngoc bv, majam m, green k, et al. acceptability, feasibility, and accuracy of blood-based hiv self-testing: a cross-sectional study in ho chi minh city, vietnam. plos glob public health. 2023;3(2):e0001438. https://doi.org/10.1371/journal.pgph.0001438 ndyabakira a, getahun m, byamukama a, et al. leveraging incentives to increase hiv testing uptake among men: qualitative insights from rural uganda. bmc public health. 2019;19:1763. https://doi.org/10.1186/s12889-019-8073-6 nangendo j, katahoire ar, karamagi ca, obeng-amoako go, muwema m, okiring j, et al. uptake and perceptions of oral hiv self-testing delivered by village health teams among men in central uganda: a concurrent parallel mixed methods analysis. plos glob public health. 2023;3(6):e0002019. https://doi.org/10.1371/journal.pgph.0002019 zanolini a, chipungu j, vinikoor mj, et al. hiv self-testing in lusaka province, zambia: acceptability, comprehension of testing instructions, and individual preferences for self-test kit distribution in a population-based sample of adolescents and adults. aids res hum retroviruses. 2018;34(3):254–260. https://doi.org/10.1089/aid.2017.0156 kelvin ea, george g, romo ml, et al. the impact on hiv testing over 6 months when free oral hiv self-test kits were available to truck drivers in kenya: a randomized controlled trial. front public health. 2021;9:635907. https://doi.org/10.3389/fpubh.2021.635907 zeleke ea, stephens j, gesesew ha, et al. acceptability and use of hiv self-testing among young people in sub-saharan africa: a mixed methods systematic review. bmc prim care. 2024;25:369. https://doi.org/10.1186/s12875-024-02612-0 terefe b, jembere mm, reda gb, et al. knowledge, and utilization of hiv self-testing, and its associated factors among women in sub-saharan africa: evidence from 21 countries demographic and health survey. bmc public health. 2024;24:1960. https://doi.org/10.1186/s12889-024-19529-z simwinga m, gwanu l, hensen b, et al. lessons learned from implementation of four hiv self-testing (hivst) distribution models in zambia: applying the consolidated framework for implementation research to understand impact of contextual factors on implementation. bmc infect dis. 2024;22:977. https://doi.org/10.1186/s12879-024-09168-5 kelvin ea, george g, mwai e, et al. mobile health support for hiv self-testing in low-literacy communities: a randomized controlled trial. j int aids soc. 2021;24(3):e25690. https://doi.org/10.1002/jia2.25690 hlongwa m, mashamba-thompson t, makhunga s, et al. effectiveness of male-targeted hivst promotion through social networks in south africa. front public health. 2023;11:1125325. https://doi.org/10.3389/fpubh.2023.1125325 abstract introduction methods results discussion acknowledgements references about the author(s) abass abdul-karim tamale zonal public laboratory, ghana health service, tamale, ghana david opare national public health laboratory, ghana health service, accra, ghana ulysses balis department of pathology, university of michigan school of medicine, ann arbor, michigan, united states lee f. schroeder department of pathology, university of michigan school of medicine, ann arbor, michigan, united states citation abdul-karim a, opare d, balis u, schroeder lf. providing specimen transport through an online marketplace in the northern region of ghana. afr j lab med. 2023;12(1), a2062. https://doi.org/10.4102/ajlm.v12i1.2062 note: additional supporting information may be found in the online version of this article as online supplementary document 1. original research providing specimen transport through an online marketplace in the northern region of ghana abass abdul-karim, david opare, ulysses balis, lee f. schroeder received: 12 nov. 2022; accepted: 22 may 2023; published: 20 july 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: integrated diagnostic networks, which are themselves dependent on robust specimen transport solutions, are fundamental to effective healthcare systems. objective: this study aimed to pilot an online marketplace for the transport of specimens throughout a laboratory network in ghana. methods: independent drivers were matched with health facilities that required specimen transport using a suite of mobile applications and web portals developed for this study. this marketplace was piloted with seven drivers, two laboratories, and five health facilities in ghana’s northern region from march 2019 to october 2019. results: during the pilot, 182 deliveries were completed for 691 patients, including 4118 laboratory tests for antenatal care, disease surveillance, and clinical testing. testing included 34 tests for communicable and non-communicable diseases. all but two specimens (laboratory cancellations) were successfully delivered and tested. the median time from request to encrypted emailing of results was 19.7 h, while that for a drop-off request was 0.9 h. in the midwife registry, the median time from patient visit to result recording was 1 day, compared to 4 days in the same months in 2018, and the number of mothers without documented testing decreased from 41 to 3. similarly, the proportion of tuberculosis specimen deliveries from buipe polyclinic to tamale zonal laboratory taking over 1 day fell from 62% at baseline to 3% during the pilot. conclusion: an online marketplace successfully orchestrated the delivery of laboratory specimens under a variety of clinical circumstances, reducing overall turn-around time without diminution of the overall specimen delivery process. what this study adds: this study established the efficacy of an online marketplace to orchestrate timely and high-quality delivery of specimens within a laboratory network. keywords: laboratory network; specimen transport; specimen referral; public health laboratory; essential diagnostics; diagnostic network. introduction fundamental to any effective national healthcare system is a functional laboratory network with hierarchical tiers.1,2 through a laboratory network, peripheral health centres without full testing capacity can access a larger portfolio of diagnostics by sending patient specimens to higher-tier laboratories. a robust courier solution that ensures specimens are shipped efficiently and reliably is essential to the laboratory network but is often non-existent in many lowand middle-income countries, except for some select diseases of public health importance.3,4 in ghana, specimen transport is limited but critical for clinical testing and disease surveillance and is a particular concern in the northernmost regions of the country.5,6 the maputo declaration of 2008 called for the strengthening of laboratory networks throughout sub-saharan africa.1 this was in response to the escalating efforts to curb endemic conditions like hiv, tuberculosis, and malaria. since then, other major global efforts for the surveillance of antimicrobial resistance and epidemic-prone diseases2 like coronavirus disease 2019 have identified the need for tiered laboratory networks.7,8,9 tiered laboratory networks permit knowledge sharing between tiers, strengthened reagent supply chains, shared resources for instrument servicing, and testing access for remote health facilities through specimen transport.10 recent efforts to strengthen specimen transportation revealed the feasibility of a wide variety of strategies, with perhaps the greatest barrier being a lack of engagement.3,11,12,13,14,15,16,17 in ghana, various ad-hoc approaches to specimen transport are utilised and the results are not always satisfactory. for instance, the time to deliver specimens for meningitis testing, a disease requiring rapid results, varied between 3 and 7 days in the yendi and talensi districts in ghana.5,18 the rate of laboratory-confirmed meningitis is thus low in ghana and throughout the entire meningitis belt, where fewer than 10% of cases are confirmed through testing.19 online marketplaces like uber™ and airbnb™ have had major impacts in multiple industries. uber technologies incorporated (san francisco, california, united states) revolutionised the transportation industry by tapping into the sharing economy. this study aimed to create and pilot an online marketplace in ghana to match drivers with health facilities that need specimens sent to higher-tier laboratories. our goal was to harness the existing transportation resources in ghana to efficiently move specimens throughout the laboratory network. this approach also allowed for the most effective use of diagnostic resources in a health system where instruments are rarely used to full capacity due to a lack of locally available specimens. methods ethical considerations this study was conducted according to the ethical approval provided by ghana health service ethical review committee (ghs-erc 002/05/18). written consent was obtained for participation from the prospective survey participants, as well as from patients submitting samples to be used in the study. all data were anonymised and maintained on health insurance portability and accountability act-compliant servers and password-protected laptops. development of the mobile and web-based application a mobile and web-based application suite formed the technological foundation for our online marketplace (figure 1). the custom suite was created for this study by app emporio (rajkot, india) on a technological stack including nodejs (https://nodejs.org/en), angular2 (https://angular.io/), and mongodb (https://www.mongodb.com/). the suite included different persona-based applications for health facilities, drivers, and laboratories. there was also a web portal for the participating laboratories, where test menus, turn-around times (tats), and prices could be added. similarly, an administrative portal provided real-time visualisation of operations, including global positioning system locations of drivers and active deliveries. figure 1: screenshots of the health facility mobile phone application developed for the specimen transport network piloted at health facilities in the northern region of ghana between march 2019 and october 2019. the application includes (a) the laboratory test menus from which healthcare staff select tests, (b) the tracking features of the application, and (c) visualisation of real-time driver location during the delivery. workflow of the specimen transport network patients were identified through routine clinical care and brought to the health facility’s laboratory for phlebotomy and possible participation in the pilot. there, specimens were collected from the patients and packaged in triple containers with ice packs and paper requisitions to facilitate proper identification of samples and confirmation of tests to be performed.20 the packages were then sealed to prevent exposure of drivers to potential biohazards. drivers were thus not required to use personal protective equipment. a trained health facility liaison then used the application to request a driver from a list of active drivers, sorted by distance. if the desired driver did not accept, the application automatically queried other drivers in order of distance from the pick-up point, based upon real-time geospatial telemetry from the driver-persona-based application. once a driver accepted the task, they travelled to the health facility, picked up the package, and delivered the same to the selected laboratory. during this time, the driver was trackable in real time through the application or web interface. upon delivery, the driver confirmed completion through the application, and the laboratory confirmed receipt of the adequate samples for testing, as well as the availability of the required testing reagents. laboratory test results were delivered to the health facilities electronically through proton ag (geneva, switzerland), a secure end-to-end encrypted email service. quality assurance staff from the project team monitored the requested deliveries and ensured that samples arrived safely and that the laboratories performed the tests and communicated the results. the application also included a rating system for health facilities and drivers. a pilot study of the online marketplace was conducted from march 2019 to october 2019 at five public health facilities in the northern region of ghana (online supplementary document – supplementary methods), and a medical records review collected data on test utilisation and tats (online supplementary document – supplementary methods). additionally, prospective surveys of patients (online supplementary document – supplementary survey 1) and providers (online supplementary document – supplementary survey 2) collected data on challenges and preferences for laboratory testing. data analysis all data on specimen delivery were stored on our servers and made available through our web administration dashboard. no private health information was stored on this server. data from the abstraction of medical records and laboratory registers were entered into microsoft excel spreadsheets (microsoft corporation, redmond, washington, united states) and checked for consistency. data were imported to the r statistical environment (v3.6.1)21 for statistical analysis. for dichotomous outcomes, statistical evaluations were conducted using the mcnemar test (mcnemar.test function within the r stats package) with continuity correction, and tats were compared using the two-sample kolmogorov-smirnov test (ks.test function within the r stats package), with approximated p-values where exact computation was not possible due to ties (p < 0.05 were considered significant). due to technical difficulties, deliveries were often cancelled and had to be re-requested by facilities after the driver had already started the collection or delivery. in these instances, timestamps would underestimate actual delivery times. therefore, in the tat calculations, any deliveries with collection or delivery durations of less than 10 min were excluded, as the shortest driving time between the health facilities and laboratories was about 10 min. results in the medical record review of 15 health facilities (pre-pilot), 23 laboratories were identified that performed external testing, with a median of four laboratories per health facility (online supplementary document – supplementary figure 1). outside tamale, test results for 99% of in-house tests were delivered by the laboratory within 1 day of request, compared to 82% of tests sent to external laboratories (p < 0.001). in tamale, the proportions of test results resulted by the laboratories within 1 day of test request were similar for in-house tests (100%) and tests conducted at other laboratories (98%). for health facilities outside tamale, test results for 90% of in-house tests were recorded by providers within the same day of the test request compared to 74% of tests performed outside the facility (p < 0.001). within tamale, test results for 51% of in-house tests were recorded on the same day of the test request compared to 40% of tests performed outside the facility (p = 0.50). within tamale, the median tat from test request to recording of results was 3.1 h for in-house testing compared to 1.7 h for send-out testing (p < 0.001). likewise, the median tat from request to recording by the provider was higher for in-house testing (12.7 h) compared to send-out testing (4.8 h; p = 0.08). the types of tests conducted in-house were similar to those conducted elsewhere, with a total of 23 different tests identified. the most common of these were malaria, complete blood count, haemoglobin, typhoid, urinalysis with microscopy, sickle solubility, urine pregnancy, and hepatitis b surface antigen tests, which collectively accounted for 95% of testing. other less-commonly ordered tests included stool microscopy, hepatitis c test, glucose-6-phosphate dehydrogenase test, tuberculosis sputum microscopy, liver function tests, renal function tests, glucose tests, and hiv tests. interviews were conducted with 73 patients, including representatives from each of our pilot sites. when asked about the frequency with which they experienced different barriers to testing, 58% of patients described test availability as ‘often’ or ‘always’ a barrier, 15% reported test expense as ‘often’ or ‘always’ a barrier, and 1% reported the physician not ordering tests as ‘often’ or ‘always’ a barrier (figure 2). there were no responses of ‘other’ reasons. nearly equal numbers of patients responded that they would either transport the samples to a laboratory themselves (44%) or that a specimen would be sent by some other means (46%). others (8%) reported that they had experienced both, while 1% reported that they had experienced neither. due to the structure of the question, it was difficult to interpret how patient samples were transported to outside laboratories (e.g., by a system organised by the health facility or by the patient’s family or friends). nearly all patients (96%) said they would be interested in the specimen transport service being piloted, mostly to avoid travel or reduce transport costs; two patients were, however, concerned about the possibility of mislabelled specimens or loss of confidentiality. figure 2: results of a patient survey conducted at health facilities in the northern region of ghana between march 2019 and october 2019. patients reported that test availability was the most common cause preventing laboratory testing. seven providers were interviewed, with representation from each health facility (figure 3). twenty-nine percent responded that tests are ‘often’ or ‘always’ returned promptly. when testing was not available in their health facility, 57% of providers responded that samples were ‘often’ or ‘always’ sent to outside laboratories, 43% stated that patients were transferred to higher-tier facilities, and 14% stated that patients travelled themselves to outside laboratories. the most commonly experienced barriers to accessing diagnostic testing were tat (80% responded ‘often’ or ‘always’) and cost (50% responded ‘often’ or ‘always’). all seven providers said that the specimen transport services would be useful if made available at their facilities. figure 3: results of a survey conducted among health providers at health facilities in the northern region of ghana between march 2019 and october 2019. related questions are grouped between dashed lines. over the 8 months of the pilot, 182 deliveries were conducted for 691 patients, including 4118 laboratory tests (table 1). other than laboratory cancellations for two specimens due to spillage (samples were re-collected), all deliveries were completed, and results were posted. of the 182 deliveries, 120 were excluded from tat calculations as their collection or delivery durations were less than 10 min, indicating a technical difficulty with the application that required the resubmission of delivery requests. at the individual testing level, the median time from health facility request to sending of results by encrypted email was 19.7 h, with an interquartile range (iqr) of 18.6 h – 20.7 h. the median time from request to collection of the specimen by the driver was 21 min (iqr: 11 min – 38 min), and the median time from specimen pick-up to drop-off at the laboratory was 28.8 min (iqr: 19 min – 65 min). the median time from drop-off to reporting of results was 18.5 h (iqr: 16.2 h – 19.4 h), accounting for 94% of the total tat (iqr: 89% – 96%). turn-around times observed during the pilot could not be compared with those observed during the retrospective analysis because most medical records only contained dates, not times (hours and minutes). driver fees varied from $1.75 united states dollars (usd) to $8.77 usd per delivery, depending on the requesting facility. multiple patients were pooled for each delivery. some surveillance testing for tuberculosis required variably higher fees for delivery due to longer travel times (> 2 h; up to $17.54 usd). the direct payments to drivers on average were $0.77 usd per patient and $0.11 usd per test, excluding the longer travel to the buipe district for surveillance testing (18 deliveries). table 1: characteristics and performance of the specimen transport network piloted at health facilities in the northern region of ghana between march 2019 and october 2019. one hundred and ten deliveries (60% of all deliveries) included samples for clinical testing (figure 4). deliveries were conducted mostly for haemoglobin (n = 146 tests), malaria (n = 142), and urinalysis (n = 103) testing. surveillance testing initiated by disease control officers was included in 18% of deliveries, and these included tests for tuberculosis (n = 63), meningitis (n = 5), measles (n = 7), and yellow fever (n = 4). figure 4: laboratory tests ordered during the pilot of a specimen transport network at health facilities in the northern region of ghana between march 2019 and october 2019. the figure shows tests performed for antenatal care, general clinical care, and public health surveillance. antenatal care guidelines recommend a specific set of tests, which explains the consistent ordering patterns for that patient population. however, much general clinical testing included the same tests as for antenatal care. surveillance testing was largely for tuberculosis but also included testing for bacterial meningitis and other priority public health targets. at the antenatal care (anc) facility reviewed, there were 248 mothers at baseline and 250 in the pilot phase. more mothers were without any documented testing in the anc facility during the baseline months in 2018 (41 not tested; 207 tested) compared to the corresponding months during the pilot (3 not tested; 247 tested; mcnemar test: p < 0.001). in the baseline months, 136 mothers had dates recorded in the midwife register for when their laboratory test results were entered. during the pilot, laboratory test result dates were entered in the register for 115 mothers. the median time from the initial visit to result recording in the anc register (which may be later than the actual return of results) was significantly shorter during the pilot compared to the baseline months (1 day vs 4 days; two-sample kolmogorov-smirnov test: p < 0.001) (figure 5). figure 5: antenatal care testing times and screening rates as extracted from a midwife registry in the northern region of ghana between may 2018 – august 2018 (baseline) and may 2019 – august 2019 (on-demand delivery). the histograms represent the number of patients (y-axis) with documented test results within a given time frame from the initial patient visit (x-axis). the bars are overlaid on top of each other. in the review of zonal laboratory records for tuberculosis testing, there were 77 specimens during the baseline months and 32 specimens during the pilot. during the pilot, the median delivery time for specimens was 0 days (same day) compared to a median of 2 days at baseline. this translated to 97% of specimens being delivered in less than 1 day (time from specimen collection to receipt in laboratory) during the pilot compared to 38% of specimens being delivered in less than 1 day at baseline (two-sample kolmogorov-smirnov test: p < 0.001) (figure 6). figure 6: specimen transport times for tuberculosis testing as documented in the public health laboratory register from the buipe polyclinic in the northern region of ghana between 2009 and 2014 (baseline) and march 2019 – october 2019 (on-demand delivery). the histogram shows the number of specimens (y-axis) and the time intervals within which they were delivered for testing after specimen pick-up (x-axis). the bars are overlaid on top of each other. discussion this pilot of an online marketplace for the transport of laboratory specimens revealed that specimens could be successfully delivered under a variety of clinical circumstances, including for general clinical testing at health facilities and hospitals, anc testing with daily deliveries of bundled patient specimens, and surveillance testing as requested by disease control officers. for tuberculosis testing, tats improved from a baseline period, and for anc testing, both tat and the number of mothers screened improved. a 2021 study found that most health facilities in the northern region of ghana did not offer the majority of tests recommended in the world health organization essential diagnostics list.6 our review of medical records revealed that the testing that does occur is performed by a combination of on-site and external laboratories. outside of testing for hiv early infant diagnosis and some limited diseases for public health surveillance, none of the facilities reviewed operated formalised specimen transport systems for clinical testing (a.a.-k., 2021, personal communication, march 31). presumably, therefore, samples tested at external laboratories were mostly transported through ad-hoc means (e.g., patient presentation to the laboratory, delivery of the specimen by family or friends, etc.). more on-site tests were completed within 1 day of request compared to external testing, and this difference was greater outside tamale. this suggests that specimen transport should be prioritised in rural areas, where there are longer distances to external laboratories. nevertheless, patients in both urban and rural settings would likely benefit from such services. the sustainable development goals demand quality essential healthcare services with universal health coverage, where timeliness is a key component of quality.22,23,24,25 in line with this, the benefits of integrated laboratory networks to healthcare delivery include increased availability, accuracy, and tat of laboratory testing.26,27 during the pilot, the time from the initial visit to recording of test results in the midwife register at the anc decreased from 4 days to 1 day, and tuberculosis testing times decreased from 2 to 0 days (same day). as specimen transport systems are a key component of laboratory networks, multiple models have been implemented in different settings, and although the published models are not directly comparable (different metrics and different geographic and disease characteristics), they provide some context to the findings in this study. in a 2013 study in uganda, a motorcycle hub-and-spoke delivery system for centralised hiv early infant diagnosis led to a reduction in tat from 1–2 months to 5–10 days.11 the same service led to a tat reduction for tuberculosis testing from 21 to 3 days.3 in ethiopia, a private–public partnership involving the ethiopian postal service enterprise led to hiv early infant diagnosis tats decreasing from 7 to 2 days in addis ababa and from 10 to 5 days in the amhara region.3,11,12 the current pilot performed comparably, if not better than these. in addition to tat, laboratory networks can also improve the rates at which patients receive testing and downstream interventions. for example, in a 2015 study in haiti, the number of patients enrolled on antiretroviral therapy for new sites joining the specimen referral network increased by 182% within 6 months.28 antenatal care screening rates for mothers increased in this study compared to the prior year (3 out of 250 mothers not tested compared to 41 out of 248 mothers not tested at baseline). antenatal care screening is important for the prevention of mother-to-child transmission of infectious diseases, monitoring for disorders such as pre-eclampsia, and ensuring that expectant mothers with anaemia deliver at facilities with transfusion capabilities. while this study did not follow such patient outcomes, it is reasonable to expect that there would be a commensurate positive impact with improved screening.29,30 regarding tuberculosis, at least six previously undiagnosed patients were diagnosed during the pilot. one of these patients reported prior symptoms for 1 year, and another described her son’s recent death from a condition with similar symptoms. the early detection of tuberculosis, enabled through laboratory networks, can have an important impact on transmission (and thus prevalence), patient outcomes, and the economic burden on affected families.31,32 this pilot was designed to assess the feasibility and effectiveness of the transport model, not the costs. nonetheless, when a health system is determining whether to provide testing on site or via specimen transport, specimen transport cost is a factor that must be considered. in some settings, overall testing costs may be lower with external testing. in the 2013 study in uganda, the implementation of the specimen transport system for centralised testing led to a 62% reduction in operational costs.3 in a 2023 study in uganda, researchers found similar per-test costs in the decentralised versus centralised model for tuberculosis testing.33 although we documented driver fees, which varied based on distance travelled, we did not perform a comprehensive cost analysis. these deliveries often included batches of patient specimens, thus reducing the per-specimen transportation cost considerably. in this study, excluding the longer deliveries for surveillance in buipe district, average driver fees were $0.73 usd per patient and $0.11 usd per test. in other settings, this amount would vary based on the use of taxis versus motorcycles (with motorcycles likely costing less), the number of patients per delivery, and the number of tests per delivery. another cost was for smartphones, as most of the drivers had phones, but not smartphones. the study required providing them with smartphones and data. however, this augmented provisioning to drivers may not be necessary if smartphone adoption increases over time.34 a full analysis would need to include the costs of application development and maintenance, platform operation, as well as the economies of scale that depend on the number of deliveries per period, among other costs. ghana was the first sub-saharan african country to introduce a tax-funded national health insurance scheme (nhis 2003). the nhis provides payments to accredited public and private laboratories for testing. however, nhis reimbursement to laboratories is often less than what laboratories would charge when patients pay ‘out of pocket’. a 2021 study in ghana found that the greater the difference between the nhis reimbursement and the market rate, the less available the test was in health facilities and laboratories.6 nonetheless, the nhis represents a foundation upon which payments for diagnostic testing can be provided. the nhis provides a single payment per test and does not pay for specimen transport, the cost of which would thus be borne by the health facility, the patient (who might otherwise have to pay for transport themselves), or the laboratories, as they desire more specimens. because such a transport system would increase testing demand, laboratories may be willing to pay for some, or possibly all, of the transport costs. this would be particularly true if specimens are transported in batches such that per-test delivery costs are low compared to testing costs. regarding sustainability, future work would be needed to further explore the scenarios (e.g., frequency, distance, and urgency of pick-ups) in which this service would be optimal for both the ‘buyer’ and ‘seller’. this includes understanding the ‘willingness to pay’ of the potential buyer. it is also important to understand the ‘willingness to sell’ of the potential seller of this service under each scenario (i.e., drivers). payment could be improved through telebanking, and different payment contracts could be explored (e.g., simple payment per delivery or flat rates plus per-delivery fee). limitations there were challenges experienced during this pilot. first, facilities often had to resend requests and that may have impacted tat estimates, although this was mitigated by excluding unreasonably short collection or delivery durations of less than 10 min. these difficulties could have been due to unstable cellular networks leading to the cancellation of active deliveries. in the next phase, cellular message event buffering with a closed-loop confirmation messaging protocol could mitigate this shortcoming. second, while the surveillance testing pilot was successful, disease control officers sometimes made requests from very distant locations (> 2-h drive), thus leading to unsustainably high driver fees. recruitment of bus drivers in a daisy-chained fashion to allow deliveries to and from bus stations and subsequent bus delivery between cities is a possible solution. third, requests from district hospitals were fewer than anticipated. this may have represented a reluctance to engage in a change of procedure. it is also possible that providers preferred to refer patients to nearby private laboratories where the providers had existing relationships. further investigation would be necessary to determine the root causes of low district hospital order rates. conclusion one path towards improving diagnostic testing is additional in-house testing, but a complementary approach using specimen transport could build on the laboratory network approach. instead of investing in fleets of drivers for this purpose, an online marketplace taps into existing transportation resources that are often under-utilised. through such a specimen transport service as piloted in this study, existing laboratory capacity could be better harnessed for patient care and disease surveillance. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions a.a.-k. contributed to study conceptualisation, methodology, investigation, project administration, writing, review and editing. d.o. contributed to the supervision of the project as well as writing, review, and editing of the article. u.b. contributed to conceptualisation, methodology, writing, review and editing. l.f.s. contributed to conceptualisation, methodology, formal analysis, writing of the original draft, review and editing, software, resources, and supervision of the project. sources of support funding for the study was received from the bill and melinda gates foundation (opp1182131). data availability the datasets generated and analysed during the current study are available from the corresponding author, l.f.s., on reasonable request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of 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[cited 2023 jan 23]. available from: https://www.who.int/publications-detail-redirect/who-htm-stb-psi-2011.21 zwerling a, shrestha s, dowdy dw. mathematical modelling and tuberculosis: advances in diagnostics and novel therapies. adv med. 2015;2015:907267. https://doi.org/10.1155/2015/907267 thompson rr, nalugwa t, oyuku d, et al. multicomponent strategy with decentralised molecular testing for tuberculosis in uganda: a cost and cost-effectiveness analysis. lancet global health. 2023;11(2):e278–e286. https://doi.org/10.1016/s2214-109x(22)00509-5 broadband commission for sustainable development. working group report on smartphone access: strategies towards universal smartphone access. geneva: itu; 2022. abstract introduction methods results discussion acknowledgements references about the author(s) talishiea croxton institute of human virology nigeria, abuja, nigeria institute of human virology, university of maryland, baltimore, maryland, united states ndidi agala institute of human virology nigeria, abuja, nigeria emmanuel jonathan institute of human virology nigeria, abuja, nigeria olasinbo balogun institute of human virology nigeria, abuja, nigeria petronilla j. ozumba institute of human virology nigeria, abuja, nigeria enzenwa onyemata institute of human virology nigeria, abuja, nigeria shefiya lawal department of internal medicine, university of abuja teaching hospital, abuja, nigeria manmak mamven department of internal medicine, university of abuja teaching hospital, abuja, nigeria samuel ajayi department of medicine, university of abuja teaching hospital, abuja, nigeria sylvia e. melikam department of medicine, university of ibadan, ibadan, nigeria mayowa owolabi center for genomic and precision medicine, college of medicine, university of ibadan, ibadan, nigeria bruce ovbiagele department of neurology, university of ghana medical school, accra, ghana dwomoa adu department of neurology, medical university of south carolina, charleston, south carolina, united states akinlolu ojo department of internal medicine, university of michigan, ann arbor, michigan, united states christine m. beiswanger coriell institute for medical research, camden, new jersey independent contractor, philadelphia, pennsylvania, united states alash’le abimiku institute of human virology nigeria, abuja, nigeria institute of human virology, university of maryland, baltimore, maryland, united states citation croxton t, agala n, jonathan e, et al. h3africa partnerships to empower clinical research sites to generate high-quality biological samples. afr j lab med. 2020;9(1), a935. https://doi.org/10.4102/ajlm.v9i1.935 lessons from the field h3africa partnerships to empower clinical research sites to generate high-quality biological samples talishiea croxton, ndidi agala, emmanuel jonathan, olasinbo balogun, petronilla j. ozumba, enzenwa onyemata, shefiya lawal, manmak mamven, samuel ajayi, sylvia e. melikam, mayowa owolabi, bruce ovbiagele, dwomoa adu, akinlolu ojo, christine m. beiswanger, alash’le abimiku received: 01 dec. 2018; accepted: 12 dec. 2019; published: 18 mar. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the institute of human virology nigeria (ihvn) – human heredity and health in africa (h3africa) biorepository (i-hab) seeks to provide high-quality biospecimens for research. this depends on the ability of clinical research sites (crs) – who provide biospecimens – to operate according to well-established industry standards. yet, standards are often neglected at crss located in africa. here, i-hab reports on its four-pronged approach to empower crss to prepare high-quality biospecimens for research. objectives: i-hab sought (1) to assess a four-pronged approach to improve biobanking practices and sample quality among crss, and (2) to build human capacity. methods: i-hab partnered with two h3africa principal investigators located in nigeria and ghana from august 2013 through to may 2017 to debut its four-pronged approach (needs assessment, training and mentorship, pilot, and continuous quality improvement) to empower crss to attain high-quality biospecimens. results: close collaborations were instrumental in establishing mutually beneficial and lasting relationships. improvements during the 12 months of engagement with crss involved personnel, procedural, and supply upgrades. in total, 51 staff were trained in over 20 topics. during the pilot, crss extracted 50 dna biospecimens from whole blood and performed quality control. the crss shipped extracted dna to i-hab and i-hab that comparatively analysed the dna. remediation was achieved via recommendations, training, and mentorship. preanalytical, analytical and post-analytical processes, standard operating procedures, and workflows were systematically developed. conclusion: partnerships between i-hab and h3africa crss enabled research sites to produce high-quality biospecimens through needs assessment, training and mentorship, pilot, and continuous monitoring and improvement. keywords: biobank; training; africa; developing country; biotechnology. introduction biobanking is underdeveloped in africa.1,2 obvious obstacles include sparse financial resources, challenging operating environments, underdeveloped infrastructure, inferior logistics, and an unreliable electrical power supply.2,3,4,5 also, preanalytical processes occurring at collection sites affect biospecimen quality.3 past reports documented destitute laboratory systems, and the hope for advancements through international declarations, funding, networking, training, and mentorship.6,7,8,9 the united states national institutes of health (nih) and the wellcome trust founded human heredity and health in africa (h3africa) (www.h3africa.org) to promote genomic research in africa through funding of african researchers, and provision of bioinformatics core and biorepositories located in nigeria, south africa, and uganda.6 the nih required biorepositories to complete a feasibility phase (phase i) to qualify for the implementation phase (phase ii). the nih also assigned h3africa research projects to a biorepository and required projects to deposit an aliquot of all dna. the bioinformatics core stored data. beiswanger et al.10 described the process for accessing biospecimens. the nih funded the institute of human virology nigeria h3africa biorepository (i-hab). i-hab’s primary pursuit in phase i was to achieve international standards according to the international society for biological and environmental repositories (isber) best practices.11 for example, isber provides requirements for biospecimen collection, processing, quality control (qc), storage and transport, infrastructure, quality management, safety, training, and a laboratory information management system (lims).11 the lims documents and tracks biospecimen attributes from collection through to use, depletion, and destruction. i-hab also referred to the international organization for standardization (iso) 15189 standards for clinical laboratories (https://www.iso.org/standard/56115.html) and strengthening laboratory management toward accreditation (slmta) (https://slmta.org) for requirements pertaining to quality management, as appropriate. regarding specific protocols, i-hab referred to manufacturer specifications and journals. considering the potential imbalance between standardised preanalytical processes according to isber, iso, and slmta and the lack of such standards in laboratories in africa, i-hab sought to evaluate assigned clinical research sites (crss) and implement improvement strategies. i-hab established a training and mentorship programme to bridge gaps and to ensure researchers obtain biospecimens of the highest quality. this article describes i-hab’s experience in engaging two h3africa projects from august 2013 through may 2017 in preparation for phase ii.10 methods the nih assigned i-hab six research projects including project a – a crs located in tanzania (not included in this study), nigeria, and ghana, with the project’s central laboratory hub (clinical research sites sends biospecimens to laboratory hubs for processing, testing or storage) in ghana – and project b, with a crs and laboratory hub in nigeria. i-hab employed a four-pronged approach of needs assessment, training and mentorship, piloting, and continuous quality improvement to help sites to collect, process, store, and transport high-quality biospecimens for future research. a working group of nih experts and representatives from each h3africa biorepository developed guidelines and protocols to guide the h3africa consortium on biorepository processes for harmonisation and consistency. the documents included biospecimens deposit and access requirements, and specific procedures for biospecimen collection, processing, transport, and shipping (www.h3africa.org). for example, the group established minimal criteria for dna purity and concentration to ensure quality and consistency. the guidelines required submitters to maintain: ethical standards: ethical approval, informed consent, and material transfer agreements (mta). legal requirements: shipping regulations and import and export permits. h3africa requirements: dna quality, minimal data set, shipping checklist, and manifest and query forms. the ‘minimal data set’ included a biospecimen identification code, de-identified participant identification code, study name, specimen type, date of collection, gender, age at collection, and storage box position. the working group standardised the format of each element to ensure consistency and decrease errors. the working group based the contents of the standard operating procedures (sops) on international air and transport association regulations, isber best practices, manufacturer’s specifications, and well-established practices as specified. iso 15189 standards were useful for creating sops and documents regarding quality management and for defining the structural elements required for all sops. i-hab recommended that sites customise sops to their laboratories. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. needs assessment the i-hab and research teams met to discuss project descriptions, processes, and procedures that potentially impact a biospecimen’s quality. i-hab reviewed study protocols and other documents in consideration of responsibilities and procedures impacting a biospecimen’s integrity. i-hab assessed facilities, equipment, personnel, documents and records, specimen management, organisation, purchasing, inventory, and quality assurance measures in line with underlying principles of isber best practices and iso 15189 to ensure the sites could meet protocol requirements according to industry standards. assessments included a combination of discussion, sop review, site observation, and competency assessments for specifically assigned tasks. i-hab offered document development, training and mentorship, biospecimen storage, distribution, and qc to resolve problems identified by assessments. training and mentorship i-hab customised training and mentorship for project a’s clinical site in nigeria and its hub, and project b’s hub site according to the outcome of the needs assessment. training and mentorship occurred at i-hab, crss and hubs for groups and individuals. training began with lectures to introduce theories, followed by exercises to provide practical experience, and concluded with participant assessments to test competency. mentorship provided additional time to practise procedures, to address problems revealed through training, and to review processes that were required but were not covered during training. mentorship areas included document development and revision, workflow design, supply chain management, biological transportation, and laboratory procedures. pilot study following training and mentorship, i-hab piloted exercises with project a and project b to determine preparedness for biospecimen deposition. sites tested processes preceding biospecimen deposit including participant recruitment, biospecimen collection, processing, qc, temporary storage, transport to i-hab, and documentation. i-hab tested biorepository processes succeeding biospecimens deposit including: biospecimen receipt, qc, aliquoting, storage, transport, and documentation. i-hab and the sites processed mtas, and i-hab acquired copies of ethical approvals and blank consent forms to ensure biospecimen sharing was according to ethical requirements. investigators transferred biospecimens collected from geographically dispersed crss to the study’s hub site and the hub site transferred dna biospecimens to i-hab for comparative qc and storage. project a’s crs in nigeria was the only one to transfer biospecimens directly to i-hab, because it required assistance to temporarily store and ship non-dna biospecimens to its hub in ghana. thus, i-hab piloted shipments from project a’s and project b’s hubs to i-hab, from project a’s site in nigeria to i-hab and from i-hab to project a’s hub. shipping sites shipped biospecimens according to international air and transport association regulations under class 6.2 biologicals, infectious substances, and class 9 for dry ice (unless otherwise stated). shipments contained temp tale4 quantitative, reusable temperature loggers, (sensitech, beverly, massachusetts, united states) and 3tm warmmark time temperature indicators (qualitative, disposable cards) (telatemp, anaheim, california, united states) unless otherwise indicated. biospecimens were documented upon receipt. quality control i-hab required sites to submit qc results for the dna deposited. i-hab also evaluated dna for concentration (absorbance at 260 nanomole [nm]) and purity (260/280 absorbance ratio) by spectrophotometry (nanodrop, thermoscientific, waltham, massachusetts, united states), and for quality by gel electrophoresis.12,13 dna purity of 1.7–2.0 was acceptable. dna with unacceptable purity was stored but flagged for suboptimal quality. where specified, i-hab and project a’s nigerian site investigated plasma and serum for haemolysis using i-hab’s protocol for visual grading. laboratorians classified biospecimens from 0 (normal) to 4 (extreme haemolysis) by comparing them to a picture gradient of five adjacent plasma samples (numbered 0 to 4), beginning with a normal, non-haemolysed sample and gradually increasing to an extremely haemolysed sample. i-hab documented and communicated outcomes and recommendations to the projects’ principal investigators and staff. continuous quality improvement i-hab engaged sites in continuous quality improvement. continuous improvement is a cyclical process to improve quality by identifying problems and resolving them. i-hab reviewed biospecimen qc, and shipment packaging, temperature, and documentation. i-hab provided additional training or pilot exercises where appropriate to address nonconformities. the teams implemented corrective and preventive measures before initiating phase ii shipments to i-hab. i-hab determined improvement by the elimination of previously identified problems, improvements in dna quality, and overall improvements in efficiency. following pilot deposition at i-hab, project a and project b continued to identify opportunities for improvement. project hubs monitored temperatures in shipments between i-hab and project hubs. i-hab monitored biospecimens and corresponding data for all subsequent deposits and communicated discrepancies to the sender within one week of discovery. similarly, hubs continued to qc 100% of the dna deposited. to ensure accuracy, i-hab re-tested 10% of the dna received. if more than 10% of the re-tested dna was beyond acceptable range or inconsistent with i-hab’s results, then i-hab tested the remaining 90% (total 100%). in such instances, i-hab tested 100% of subsequent batch shipments from the project, until the project completed two consecutive shipments for which the qc for at least 90% of the dna tested was within acceptable range and consistent with i-hab’s results. i-hab continued to meet with the projects’ staff to discuss future activities, challenges, and strategies of improvement. results engagement activities occurred over the three years of phase i (2013–2016), in preparation for phase ii. i-hab observed improvements in staff proficiency, procedural efficiency, and supply upgrades. i-hab trained 51 persons in over 20 topics. i-hab further determined improvements in supplies by the elimination of previously identified issues that resulted in poor sample quality. preanalytical, analytical, and post-analytical processes, documents, and workflows were systematically created and modified based on needs assessment, training, mentorship, pilot exercises, and continuous quality improvement. project a the study’s investigators established a system for phase ii. the crs in nigeria collected, processed, temporarily stored, and shipped biospecimens to the project’s hub in ghana. an independent, commercial contractor extracted and qc tested dna. the hub stored dna and shipped the aliquots required for deposition to i-hab. i-hab performed needs assessment, training, and the pilot of the entire study process with the site in nigeria, then piloted dna shipment from ghana to i-hab. the process of engagement with project a’s crs in nigeria is summarised in figure 1. figure 1: partnership to empower project a’s clinical sites in nigeria and ghana. i-hab engaged clinical sites through needs assessment, training and mentorship, pilot exercise, and continuous monitoring in preparation for sample deposition. needs assessment in march 2014, i-hab’s manager, supervisor and qc officer assessed the crs in nigeria to compare infrastructural, commodity, and staffing resources to the laboratory protocol, h3africa requirements, and best practices according to isber. the staff included an entry-level laboratory technician and data clerk. i-hab evaluated the laboratory, staff, equipment, supplies, procedures, and workflows. the i-hab manager communicated assessment outcomes and recommendations to the site principal investigator, study coordinator, laboratory technician, and data clerk as appropriate. i-hab and project a agreed that training, mentorship, and document development were required for phase ii preparation. training and mentorship i-hab provided training and mentorship for project a’s clinical site laboratory technician and data clerk: the laboratory technician attended a five-day workshop at i-hab, september 2013 (table 1). i-hab provided customised five-day training for the laboratory technician, incorporating her responsibilities per the study protocol and h3africa specifications, january 2014 (table 1). the i-hab manager, supervisor, and biospecimens qc officer mentored the laboratory technician and data clerk for one day at their facility during a dry run on february 2014. the dry run simulated activities from participant arrival through to mock biospecimen collection, processing, temporary storage, and transport, including documentation. the laboratory technician packed and transported the mock biospecimens and the manifest using i-hab’s daily shuttle from project a to i-hab. (the drivers were trained in biological safety and biospecimens transport.) i-hab verified all mock biospecimens against the manifest. i-hab assisted in the development or modification of worksheets as appropriate. the dry run revealed that the lab technician was not proficient in computer operations. the technician participated in a four-day introductory computer workshop, march 2014. table 1: training topics to build knowledge and skills – project a in nigeria. during the two-day dry run, improvements maximised client and staff safety, specimen integrity, and efficiency. i-hab rearranged the biospecimen collection and processing area to reflect the natural flow of activities. samples were inadequately separated during processing, and storage vials popped once they were frozen. thus, i-hab made recommendations that improved biospecimen integrity: (1) upgrade the centrifuge to enable staff to process biospecimens with adequate centrifugal force; (2) separate and freeze plasma, red cells, and buffy coat immediately, rather than freeze whole blood in ethylenediaminetetraacetic acid vacutainers without processing them, and (3) replace flip cap tubes with cryovials for biospecimen storage. i-hab also provided project a with order information for biological shipping materials. pilot i-hab performed two pilots with project a. the first pilot tested the crs’s processes of enrolment, samples processing, temporary storage, and shipment from i-hab to ghana. the second pilot contained one aliquot of all project a’s dna. the pilot tested the quality of the dna and shipment from ghana to abuja. dna was extracted at a commercial laboratory, stored at the hub and shipped to i-hab on dry ice by dhl nigeria (lagos, nigeria). i-hab provided the hub with temperature loggers and indicators to monitor the shipment. upon receipt, i-hab investigated dna quality using nanodrop (thermoscientific, waltham, massachusetts, united states) and agarose gel electrophoresis. outcomes were discussed with project personnel and recommendations made. project a, clinical site (nigeria) the clinical research site pilot occurred in february 2014. the mta took seven months to process, due to unfamiliarity with such agreements; subsequent mtas took one to three months. the site collected and processed biospecimens, performed visual grading for haemolysis for plasma and serum, and documented the results in the manifest. upon delivery, i-hab inspected all biospecimens and imported the data into freezerworks (dataworks development, mountlake terrace, washington, united states). i-hab’s qc officer blindly re-evaluated 40 of the plasma and serum using the same visual grading methods for comparison. 92.5% of the results were consistent. also, i-hab temporarily stored and subsequently shipped on dry ice 1075 biospecimens to the hub in ghana using dhl nigeria (lagos, nigeria): plasma, red cells, buffy coat, urine, and oral fluid. the shipment duration was two days and temperature monitors confirmed that targeted temperature ranges were maintained. i-hab received favourable feedback regarding the shipment. project a amended practices at its other crss to reflect improvements made during the pilot. in march 2015, i-hab trained 12 persons from five project a crss for one week at i-hab (table 1). trainees were five laboratorians, one coordinator, two nurses, and four data clerks. nine (90%) trainees who took pre-tests and post-tests increased in performance (figure 2). the average pre-test scores were 62% (range 30% to 80%) while average post-test scores were 84.4% (range 63% to 100%). as expected, laboratorians performed better than non-laboratorians on average. figure 2: project a’s training scores in nigeria march 2015. project a’s pre-test and post-test training scores according to staff designation. two persons were absent during the pre-test; thus, those scores are missing. project a, hub site (ghana) during project a’s hub’s pilot in september 2015, project a shipped 20 unique dna biospecimens (extraction and qc at a commercial laboratory) to i-hab by dhl on dry ice in four days. ghana transferred data via a template comma-separated values file. the dna was still frozen upon receipt, but temperature monitors were not included in the shipment. also, there were inconsistencies between site versus biorepository results. only 5% of the dna was within the acceptable purity range per ghana’s result versus 25% per i-hab (table 2). the average purity reported by both was below the acceptable standard. furthermore, dna concentration averaging 2.0 ng/µl (0.1–7.1) was lower than the recommended for deposition by h3africa consortium. nanodrop 2000 and 8000 series have lower detection limits of 2 ng/µl and 2.5 ng/µl; thus, measurements below these limits are unreliable. the dna was not evaluated by agarose gel electrophoresis due to poor dna integrity. these samples were tagged as low quality before storage and results were communicated and discussed with project a. the major benefits of the pilot were knowledge of project a’s contractor’s poor dna quality and non-compliance with the shipping procedures. table 2: dna quality control results for projects a and b. continuous quality improvement through remediation, problems in dna concentration and purity, and temperature monitoring improved as observed from project a’s hub’s subsequent shipment to i-hab. the first phase ii shipment occurred in april 2017. the shipment contained 402 dna samples, 100% met h3africa recommendations for concentration (table 2). purity improved to 38% acceptability per ghana’s results and 45% per i-hab. temperature monitors confirmed that cold chain was maintained. phase ii is ongoing. thus, i-hab continues to monitor compliance with shipping regulations and h3africa requirements, and i-hab will partner with project a to devise resolutions if challenges arise. through july 2019, project a has deposited 15 333 non-dna biospecimens at i-hab for temporary storage and shipment to ghana and deposited 7535 dna samples for long-term storage. project b (nigeria) needs assessment project b staff and i-hab teams met three times: may, july and august 2014. the meetings facilitated an understanding of each other’s goals, requirements, expectations, and services. consequently, project b requested that i-hab introduce biospecimens management during its staff orientation (figure 3). the i-hab manager and supervisor reviewed project b’s study protocol to determine appropriate training topics. figure 3: partnership to empower project b’s clinical site in nigeria. i-hab engaged clinical sites through needs assessment, training and mentorship, pilot exercise, and continuous monitoring in preparation for sample deposition. training and mentorship the i-hab supervisor attended project b’s five-day study-wide orientation in july 2014, which included 35 research staff. the supervisor facilitated a biospecimen management workshop for one day, which spanned 14 topics (table 3). she also reviewed supplies required for biospecimen collection, processing, and storage, and made recommendations, such as for laboratory supplies. for example, i-hab suggested supplies for shipment of biological samples, freezerworks basic lims software for sample management, and software for remote temperature monitoring and alerts, which remain in use by project b. the i-hab supervisor trained the project analyst in freezerworks customisation and operations over five days, in late july 2014. consequently, the analyst successfully customised the software, and adopted freezerworks as the lims for biospecimen management. table 3: training topics to build knowledge & skills – project b in nigeria†. pilot in january 2015 i-hab drafted a protocol outlining pilot procedures. the teams attained ethical, legal, and regulatory requirements for the pilot. the mta took one month to execute. in june 2015 project b extracted, qc tested, and shipped biospecimens to i-hab by road via tranex (lagos, nigeria) in one day. there were two shipments: one on dry ice (dna-5, plasma-4, and serum-5) and one at a controlled ambient temperature (dna-5). i-hab conducted dna qc as specified above. outcomes and recommendations were communicated. the pilot identified areas that required improvement before phase ii. the hub forgot to send the manifest and the query form prior to the shipment. both shipments contained insufficient refrigerant and were warm. the qualitative temperature indicators were not activated prior to shipment and the quantitative temperature loggers were misplaced. the logger for controlled ambient temperature was placed in the frozen shipment, while the logger for frozen shipments was placed in the controlled ambient temperature shipment. nevertheless, 100% of the dna had acceptable purity and concentration (table 2). the benefit of the pilot was to learn of issues with biospecimen shipping procedures. continuous quality improvement i-hab provided remedial training for the laboratory manager and study coordinator in july 2015 (table 3). the training occurred over five days and included biological packaging and shipping, operations of temperature indicators and monitors, dna extraction, and dna qc using nanodrop. i-hab demonstrated proper document completion over skype calls. following these preventative measures, the first phase ii shipment, containing 1000 dna specimens, commenced without errors in documentation or packaging (november 2016). temperature monitors demonstrated that the target temperature was maintained. also, the concentration of dna improved from 15.00 ng/ml – 69.66 ng/ml to 78.72 ng/m – 514 ng/ml and 100% of dna was within acceptable range (table 2). project b deposited 6000 dna specimens as of september 2019. for each shipment i-hab monitored the documentation, packaging, temperature monitors, and dna qc. in may 2017, i-hab noticed a decrease in dna quality. investigation revealed that a new employee had extracted the dna; thus, i-hab trained him in august 2018. dna quality improved in the next shipment received in september 2018 and has remained within the recommended concentration and purity. i-hab will continue to monitor related activities and liaise with project b to investigate and resolve identified problems. discussion researchers inexperienced with processes that influence biospecimen integrity may attain high-quality biospecimens for research by partnering with a biorepository and establishing a system of needs assessment, training and mentorship, pilot, and continuous quality improvement that is based on best practices. it is beneficial to start with general meetings and needs assessments to understand the intended goals and procedures and whether resources and capabilities are well aligned. even if a scientist or contractor claims to be to be proficient, they should be subjected to a standardised assessment to ensure competency. had project a subjected its contractor to assessment, it would have learned of the contractor’s poor performance prior to study activation. the contractor had not resolved issues with purity that were indicative of protein contamination (purity ratio below 1.7) as of the first phase i shipment. likewise, project b’s new hire experienced issues with dna quality that could be circumvented via an assessment. both examples demonstrate that assumptions of competency risk financial and biological resources. they also demonstrate the imperativeness of training and monitoring. the key to effective training is to establish a foundation of harmonised guidelines, procedures, and minimal requirements that align with project goals and best practices. the biorepository should utilise standard and customised training that encompasses theory, practical exercises, and post-tests. training achieved staff competency and eliminated issues revealed by needs assessments, pilot exercises, and continuous monitoring. both projects improved in performance following training; however, biological shipping may require refresher training or additional practical exercises. training and mentorship contribute to the future cadre of knowledgeable researchers and generate business towards sustainability. non-laboratorians demonstrated knowledge of biospecimen management after training. freezerworks training empowered project b’s analyst to customise and operate the lims. also, the knowledge gained by project a’s technologist empowered her to recommend changes to the study protocol, suggest areas of training for all sites, and train laboratory staff on project a’s other clinical research sites. project a and project b requested additional services such as additional training and storage of non-dna biospecimens. both projects also gave public testimonials at scientific meetings that prompted interest and trust of other researchers. the pilot process allows biorepositories to identify challenges in the cascade of processes leading to biospecimen deposit that may only be revealed by a real-time trial that involves all direct and indirect processes. for example, the pilots identified and eliminated errors in shipping that can only be unmasked by full trial. due to the peculiarities of packaging and international air transport association regulations associated with international biological shipments, on-site mentorship is useful to reinforce procedures learned during training, according to the trainees’ true environment and resources. consequently, i-hab incorporated site visits to assist sites to execute their first shipment to i-hab. our outcomes mirrored the successes and challenges observed in other laboratory development programmes in sub-saharan africa that incorporate assessments, training, and mentorship. generally, training and mentorship improved laboratory performance and empowered a new cadre of professionals.14,15 problems in biospecimen acceptability,3 documents and records, information management, and supplies were common; however, training and mentorship facilitated improvements.16,17 literature suggests that relapses may occur after training, particularly in documentation.14 thus, i-hab’s method of continuous improvement shall be critical to maintain improvements. conclusion biobanking in africa is expected to increase and improve with greater knowledge and resources.6 however, crss with abilities to meet best practices to collect, process, and store biospecimens of good integrity and quality are scarce. partnerships between competent regional biorepositories and research investigators may build human capacity and improve biobanking practices and biospecimen quality by adopting an approach of needs assessment, training and mentorship, pilot, and continuous quality improvement. the improvements in quality may improve performance, increase staff morale, and contribute to sustainability of the biobank, research entity, and biobanking industry. acknowledgements the authors acknowledge the institutional support from the institute of human virology nigeria, the suggestions and advice from the h3africa consortium members, and cooperation from the clinical sites of h3africa projects supported by the ihvn h3africa biorepository (i-hab). competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions as lead author, t.c. wrote the initial draft. c.m.b. and a.a. provided guidance for the second draft. a.a., t.c., p.j.o. and c.m.b. conceived the strategy for the site engagement and field study. m.m., s.a., m.o., b.o., d.a., and a.o. devised strategies of implementation specific to local, project, and facility peculiarities, and revised the manuscript. p.j.o., e.o., n.a., e.j., and o.b. participated in implementation, collected and analysed data, contributed to tables and figures, and revised the manuscript. s.e.m. and s.l. participated in implantation and data collection and revised the manuscript. sources of support support was provided by the national institutes of health (united states) through the h3africa initiative. the research reported in this publication was supported by the national human genome research institute of the national institutes of health under award numbers uh2hg007008 and uh3hg007008 (pi: alash’le abimiku). data availability statement data are available within this article. disclaimer the views expressed herein are our own and not an official position of the funder or affiliated institutions. references mayne es, croxton t, abimiku a, et al. genes for life: biobanking for genetic research in africa. biopreserv biobank. 2017;15(2), 93–94. http://doi.org/10.1089/bio.2017.0007 gasmelseed n, elsir aa, deblasio p, et al. sub-saharan centralized biorepository for genetic and genomic research. sci total environ. 2012;423,210–213. https://doi.org/10.1016/j.scitotenv.2010.07.054 croxton t, swanepoel c, musinguzi h, et al. lessons learned from biospecimens shipping among the human heredity and health in africa biorepositories. biopreserv biobank. 2017;15(2), 103–110. http://doi.org/10.1089/bio.2017.0009 matimba a, tybring g, chitereka j, et al. practical approach to biobanking in zimbabwe: establishment of an inclusive stakeholder framework. biopreserv biobank. 2016;14(5), 440–446. https://doi.org/10.1089/bio.2015.0043 vaught j, abayomi a, peakman t, et al. critical issues in international biobanking. clin chem [serial online]. 2014;60(11), 1368–1374. [cited 2020 jan 14] available from: http://clinchem.aaccjnls.org/content/60/11/1368.long vaught j. biobanking and biosecurity initiatives in africa. biopreserv biobank 2016;14(5), 355–356. https://doi.org/10.1089/bio.2016.29009.jjv bridging biobanking and biomedical research across europe and africa [homepage on the internet]. c2016 [cited 2018 sep 11]. available from: http://www.b3africa.org/?page_id=2 yao k, maruta t, luman et, et al. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. 2014;3(1), art. #194, 8 pages. pmid: 2675233. https://doi.org/10.4102/ajlm.v3i2.194 nkrumah b, van der puije b, bekoe v, et al. building local human resources to implement slmta with limited donor funding: the ghana experience. afr j lab med. 2014;3(2), art. #214, 7 pages. pmid: 26937417. https://doi.org/10.4102/ajlm.v3i2.214 beiswanger cm, abimiku a, carstens n, et al. accessing biospecimens from the h3africa consortium. biopreserv biobank [serial online]. 2017;15(2), 95–983. [cited 2020 jan 14] available from: https://www.liebertpub.com/doi/10.1089/bio.2017.0008 abimiku a, mayne es, joloba m, et al. h3africa biorepository program: supporting genomics research on african populations by sharing high-quality biospecimens. biopreserv biobank. 2017;15(2), 99–102. http://doi.org/10.1089/bio.2017.0005 lai e, birren bw, clark sm, et al. pulsed field gel electrophoresis. biotechniques. 1989;7, 34–42. https://doi.org/10.1016/b978-0-12-101290-8.50006-7 maniatis t, fritsch ef, sambrook j. molecular cloning – a laboratory manual. new york: cold spring harbor laboratory press, 1989; 545. audu ra, onubogu cc, nwokoye nn, et al. improving quality in national reference laboratories: the role of slmta and mentorship. afr j lab med [serial online]. 2014;3(2), art. #200, 7 pages. https://doi.org/10.4102/ajlm.v3i2.200 mbah h, ojo e, ameh j, et al. piloting laboratory quality system management in six health facilities in nigeria. plos one. 2014;9(12), e116185. https://doi.org/10.1371/journal.pone.0116185 ndihokubwayo jb, maruta t, ndlovu n, et al. implementation of the world health organization regional office for africa stepwise laboratory quality improvement process towards accreditation. afr j lab med. 2016;5(1), a280. https://doi.org/10.4102/ajlm.v5i1.280 mesfin ea, taye b, belay g, et al. factors affecting quality of laboratory services in public and private health facilities in addis ababa, ethiopia. ejifcc. 2017;28(3), 205–223. abstract introduction methods results discussion acknowledgements references about the author(s) lucius c. imoh department of chemical pathology, faculty of medical sciences, university of jos, jos, plateau state, nigeria abdulazis s. longwap department of chemical pathology, faculty of medical sciences, university of jos, jos, plateau state, nigeria favour e. haruna department of chemical pathology, faculty of medical sciences, university of jos, jos, plateau state, nigeria oghale j. asieba department of chemical pathology, faculty of medical sciences, university of jos, jos, plateau state, nigeria joy p. istifanus department of chemical pathology, faculty of medical sciences, university of jos, jos, plateau state, nigeria joy a. imoh department of geography and planning, faculty of environmental science, university of jos, jos, plateau state, nigeria mathilda e. banwat department of community medicine, faculty of medical sciences, university of jos, jos, plateau state, nigeria citation imoh lc, longwap as, haruna fe, et al. practices and barriers to screening for hyperglycaemia in pregnancy among providers of antenatal care in jos, nigeria. afr j lab med. 2022;11(1), a1845. https://doi.org/10.4102/ajlm.v11i1.1845 original research practices and barriers to screening for hyperglycaemia in pregnancy among providers of antenatal care in jos, nigeria lucius c. imoh, abdulazis s. longwap, favour e. haruna, oghale j. asieba, joy p. istifanus, joy a. imoh, mathilda e. banwat received: 04 feb. 2022; accepted: 09 june 2022; published: 31 oct. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: screening for hyperglycaemia in pregnancy (hip) is an important component of comprehensive antenatal care. screening practices for hip in nigeria and factors that influence these practices are not well understood. objective: we examined the screening practices for hip and their correlates among antenatal healthcare providers (ahps). methods: this descriptive cross-sectional study of ahps providing all levels of antenatal care was conducted between august 2019 and september 2019 in jos, nigeria. eligible ahps completed a semi-structured, self-administered questionnaire, and data were analysed for adherence to recommended screening practices such as world health organization, international association of diabetes and pregnancy study groups and national institute for health and care excellence guidelines. results: of the 128 respondents included in the analysis, 59 (46.1%) were male and 69 (53.9%) were female. the mean participant age was 35.7 years (standard deviation: ± 8.5 years). most (68.0%) screened all pregnant women (universal screening) for gestational diabetes mellitus. fasting blood glucose (77.0%) and random blood glucose (55.7%) were the most common tests used. only 27 respondents (22.1%) screened using the 75 g oral glucose tolerance test, and most were doctors, ahps in faith-based or government institutions, tertiary institutions and facilities with availability of automated glucose analysers (p < 0.05 for all). conclusion: screening practices for hip among the ahps do not generally conform to best practices. hence, there is an urgent need for implementation of universal guidelines and provision of regular updates and basic glucose measuring devices for ahps at all healthcare levels. keywords: hyperglycaemia in pregnancy; gestational diabetes mellitus; guidelines for gestational diabetes mellitus; screening practices; oral glucose tolerance test; low middle-income countries. introduction the incidence of gestational diabetes mellitus (gdm) and overt diabetes in pregnancy are rising globally in tandem with rising trends of obesity, diabetes mellitus (dm) and metabolic syndrome. the epidemiological transition, which is associated with rise in incidence of non-communicable diseases in lowand middle-income countries, suggests that diseases like dm will increasingly become more prevalent in nigeria.1 indeed, the greatest rise in noncommunicable diseases and deaths due to noncommunicable diseases is predicted to occur in developing and poor-resource countries like nigeria.2,3 recent studies have demonstrated an increase over time in the prevalence of gdm in nigeria.4 remarkably, the prevalence of gdm in jos, plateau state, in recent studies has been shown to be comparable with prevalence in regions of the world with a high burden of dm.5,6 hyperglycaemia in pregnancy (hip) has the potential to worsen maternal and child health indices, thus making it of public health concern. the international federation of gynecology and obstetrics recommends that hip and gdm be considered a global health priority.7,8 the international federation of gynecology and obstetrics advocates for prevention, screening, early diagnosis and management of hip as an important intervention towards achieving sustainable development goal 3, which partly focuses on reducing maternal and child mortalities.9,10 the modalities for screening and diagnosis of diabetes outside of pregnancy are universally accepted. however, the same cannot be said for the pregnant population. although stakeholders agree on the need for diagnosing and managing hip, contentious issues regarding screening for diabetes in pregnancy include: the type of test to use for screening, the criteria for diagnosis, whom to screen (universal vs risk-based or selective screening) and when to screen, among others.9,10 these can be influenced by several factors including age and experience of the healthcare worker, cadre of antenatal healthcare provider (ahp), specialty of ahp, socio-economic determinants like cost of screening or availability of laboratory facilities and administrative or organisational factors such as availability of policies, guidelines and standard operating procedures.11,12,13 there appears to be no universally acceptable guideline for screening in nigeria, as the modalities for screening are left to the discretion of the health provider. the disparity in knowledge and screening practices across health centres in nigeria has implications for early diagnosis, referral and management of women with hyperglycaemia. first-line healthcare providers, particularly nurses and midwives, are usually the first point of contact for pregnant women. the screening practices they adopt may make a significant difference in identifying pregnant women with potential adverse outcomes. the demand to streamline the screening diagnosis and management of hip in nigeria has to begin with examination of the current practices and their determinants. unfortunately, there is a paucity of data on the subject of screening practices for hip in nigeria. it is therefore important to assess the current screening practices for hip by first-line ahps in healthcare facilities. most studies on gdm and dm in pregnancy have focused on women accessing care in tertiary and academic healthcare centres. not much is known regarding practices in private or rural settings. this study hopes to evaluate the practices concerning screening, diagnosis and management of hip in different cadres of healthcare centres in jos, nigeria, with a view to understand the underlying determinants that may be associated with them and their implication for maternal health. methods ethical considerations this study was carried out after due approval from the human research ethical committee of the jos university teaching hospital (dcs/adm/127/xxix/1692). written permission was obtained from the plateau state ministry of health. appropriate permission was obtained from the relevant authorities of the health facilities. written informed consent was obtained from all participants after due explanation of the research work and procedures. anonymity and confidentiality of the information obtained from the participants in this study was assured and maintained. study design this was a cross-sectional descriptive study conducted between august 2019 and september 2019 to survey the screening practices and their correlates among ahps. study area this study was conducted in jos north local government area (lga) in plateau state. plateau state is one of the 36 states in nigeria located in the north central geographical zone. jos north is one of the lgas of jos, the capital city of plateau state with a cosmopolitan nature, inhabited by people from all tribes in nigeria. antenatal care is provided at primary, secondary and tertiary healthcare facilities and accessed by persons in the entire plateau state and also people living in the neighbouring states in the north central and north east regions of nigeria. study participants and selection criteria this study was conducted among ahps of different cadres at all levels of primary, secondary and tertiary healthcare centres in jos north lga. this included doctors, nurses and community health providers such as community health extension workers and community health officers, and front-line staff directly involved in providing antenatal care in primary, secondary and tertiary healthcare centres in jos north lga who gave consent to participate in the study. any ahps on leave or unavoidably absent during the period of the study or who had worked < 3 months at the current health facility were not included in the study. the sample size was determined by the formula for cross-sectional study and finite population correction:14 in equation 1, n is the desired sample size, z is the standard normal deviation corresponding to a 95% level of confidence. the value obtained from a standard normal distribution is 1.96. p is the prevalence of screening for hip. using an assumed prevalence of screening for hip of 50%: further correction of the sample size was done, because the population of ahps in jos north lga is less than 10 000. the formula for this correction is: in equation 3, nf is the desired sample size when the population is less than 10 000. n is the desired sample size when the population is more than 10 000 = 384 and n is the population size of ahps in jos north lga = 140. therefore: a value of 10% of the minimum sample size was added to the study to account for non-response and incomplete data, bringing the total sample size to 115 ahps. sampling technique a multi-stage sampling technique was employed. there are 69 health facilities (private and public; primary, secondary and tertiary) that provide antenatal care services in jos north lga according to data from the state ministry of health. all the health facilities and ahps who gave consent were included in the study. potential participants were provided with a semi-structured questionnaire to complete at their convenience. data collection instrument data were collected using a pretested self-administered structured questionnaire organised in four parts (part a: socio-demographics; b: screening practice and diagnosis of hip; c: diagnostic criteria for hip; d: constraints to screening for hip). the questionnaire was developed following extensive review of the available literature on recommended screening practices such as world health organization, international association of diabetes and pregnancy study groups and national institute for health and care excellence guidelines.10 it was written in english, given the high level of english fluency among the target participants. the participants were allowed to select all options that applied to a given question and the freedom to select ‘not sure’ if they were uncertain of their response. for questions related to challenges and constraints to adequate screening and diagnosis for gdm and dm in pregnancy, participants responded on a five-point likert scale: ‘strongly agree’, ‘agree’, ‘neutral’, ‘disagree’ and ‘strongly disagree’. procedure for data collection at each selected health facility, each eligible respondent was given a detailed explanation of the research by the researcher or trained research assistants. after obtaining informed consent the questionnaire was distributed to the participants who self‑administered them independently and returned them to the researcher on completion. the researcher and trained research assistants served as a supervisor to ensure data quality by checking the completeness of questionnaires. data analysis data were cleaned and entered into microscoft excel® version 15.0 (microsoft corp. 2013, redmond, washington, united states) and exported to statistical product and service solutions version 23.0 (ibm corp. 2015, armonk, new york, united states) software for statistical analysis. descriptive statistics were presented as mean values ± standard deviation or medians with interquartile ranges for non-normal continuous variables, and proportions (as percentages) for categorical variables. tables and graphical representations were used to summarise the data. statistical associations of dependent and independent variables were assessed using chi-square tests or yate’s correction test for continuity where the chi-square test would not be appropriate. all tests were two-tailed, a 95% confidence interval was used and p-values < 0.05 were considered statistically significant. results a total of 193 questionnaires were returned by ahps in 60 health facilities. of these, only 159 respondents had worked for at least three months in the current health facility and 128 (80.5%) responded that they screen for gdm and were selected for further analysis. socio-demographic characteristics of the study participants fifty-nine (46.1%) of the respondents were male and 69 (53.9%) were female healthcare workers (table 1). the ages of the participants ranged from 21 to 60 years with a mean of 35.6 ± 8.2 years. most (47.6%, 61/128) of the respondents worked in private health facilities and 44.5% (57/128) provided primary healthcare. forty-four (34.4%) of the participants were doctors, and obstetrics and gynaecology (76.5%, 13/17) was the most common specialty among doctors with clinical specialties. table 1: general characteristics of antenatal healthcare providers in healthcare facilities in jos north, plateau state, nigeria, august to september 2019. screening practices of respondents the most common period for screening was the second trimester (75.0%; 96/128) (table 2). most (68.0%; 87/128) of the respondents screened all pregnant women for gdm (universal screening). the majority (49.2%; 63/128) of the respondents practised a laboratory screening method only, while (46.1%; 59/128) used a combination of in-clinic and laboratory methods. table 2: summary of screening practices and challenges according to antenatal healthcare providers in healthcare facilities in jos north, plateau state, nigeria, august to september 2019. risk factors and prompting for screening among those who practised in-clinic screening (n = 62), the most common risk factors that prompted screening were: maternal obesity (43.5%; 27/62), history of macrosomic baby (40.3%; 25/62), family history of dm (40.3%; 25/62), past history of gdm or impaired glucose tolerance (35.5%; 22/62) and intrauterine foetal death (33.9%; 21/62) (figure 1). figure 1: risk factors prompting screening for hyperglycaemia in pregnancy among antenatal healthcare providers in healthcare facilities in jos north, plateau state, nigeria, august to september 2019. factors associated with universal and risk-based screening the bivariate analysis of factors associated with type of screening practice (universal vs risk-based screening) showed that provider gender, category and level of institution, job designation and cadre of doctors were each significantly associated with screening practice (p < 0.05) (table 3). however, multivariate analysis showed no independent predictors of this screening practice. table 3: factors associated with universal or risk-based screening using binary logistic regression analysis among antenatal healthcare providers in healthcare facilities in jos north, plateau state, nigeria, august to september 2019. method of testing among those who practised laboratory screening (n = 122), the most common glucose testing method was the glucose meter (glucometer) (88.5%; 108/122) (table 2). this was followed closely by dipstick (82.0%; 100/122). only 16/122 (13.1%) had automated chemistry analysers. laboratory test used among those who practised laboratory screening (n = 122), the most commonly used test for diagnosing gdm was fasting blood glucose (fbg) (77.0%; 94/122), followed by random blood glucose (rbg) (55.7%; 68/122) (figure 2). very few used the recommended oral glucose tolerance test (ogtt): 22.1% (27/122) used the 75 g ogtt and 10.7% (13/122) used the 100 g ogtt. postpartum glucose testing was mostly done using fbg (68.9%; 84/122). figure 2: laboratory testing for hyperglycaemia in pregnancy among antenatal healthcare providers in healthcare facilities in jos north, plateau state, nigeria, august to september 2019. among the 94 ahps who used fbg for diagnosing gdm, the most common threshold value cited was 7.0 mmol/l (37.2%; 35/94), 6.0 mmol/l (12.8%; 12/94) and 5.1 mmol/l (5.3%, 5/94) (figure 3). there were 29 different cut-off values in all ranging from 2.5 mmol/l to 11.0 mmol/l. among the 68 ahps who used rbg for diagnosing gdm, there were 13 different threshold values ranging from 2.0 mmol/l to 15.0 mmol/l. fourteen (20.6%; 14/68) ahps mentioned a cut-off value of 11.0 mmol/l. figure 3: threshold values of fasting blood glucose and random blood glucose for diagnosing gestational diabetes mellitus among antenatal healthcare providers in healthcare facilities in jos north, plateau state, nigeria, august 2019 to september 2019. (a) fasting blood glucose threshold (b) random blood glucose. further analysis (not illustrated) showed that only eight of the 27 ahp (29.6%) who used ogtt for diagnosing gdm mentioned the correct diagnostic thresholds for the three time points (0 h, 1 h and 2 h) for glucose measurement. of the eight who used the correct diagnostic thresholds, seven aligned with the international association of diabetes and pregnancy study groups criteria, while one aligned to the national institute for health and care excellence criteria. there were 20 different combinations of threshold values mentioned by the ahps. factors associated with diagnostic testing the private and public hospitals (p = 0.014), primary and secondary healthcare facilities (p < 0.001), community health extension workers (p = 0.001), community health officers and nurses (p = 0.001) and facilities with available urine dipstick testing (p < 0.001) were more likely to use urine glucose for diagnosis of gdm (table 4). private institutions (p = 0.001) and facilities with available glucose meters (p = 0.005) were more likely to use fbg or rbg for diagnosing gdm. table 4: factors associated with type of diagnostic test for gestational diabetes mellitus among antenatal healthcare providers in healthcare facilities in jos north, plateau state, nigeria, august 2019 to september 2019. antenatal healthcare providers in faith-based or government institutions (p = 0.001) and tertiary institutions (p < 0.001) were more likely to use ogtt for diagnosing gdm. doctors compared to other categories of ahps were more likely to use ogtt (p < 0.001). antenatal healthcare providers in facilities with availability of automated glucose analyser were more likely to use ogtt (p = 0.011); however, ahps in facilities where urine dipstick is used for glucose testing were less likely to use ogtt for diagnosing gdm (p = 0.005). challenges to adequate practice late booking for antenatal care was cited as the most common challenge to screening and diagnosis of hip identified by the respondents (85.2%; 104/122) (table 2). this was followed by lack of guidelines (75.4%; 92/122), lack of information on gdm screening (63.9%; 78/122), lack of qualified personnel for testing (46.7%; 57/122) and lack of testing equipment (43.4%; 53/122). discussion the screening practice observed in this study showed that the majority of the respondents screen for gdm. screening is carried out at all trimesters of pregnancy but most commonly during the second trimester. thus, most ahps follow the recommended practice of screening between 24 and 28 weeks. however, a large number of ahps screen during first trimester. if this is done routinely for all pregnant women, it may reflect lack of knowledge or familiarity of recommended practice in screening guidelines. on the other hand, recent guidelines such as the world health organization and international federation of gynecology and obstetrics, advise early screening for women with risk factors.7,8 targeting screening at only 24–28 weeks might cause logistical challenges when considering that a substantial number of women in low-income countries may drop out or miss scheduled visits for various reasons such as lack of finance and disruption of services and poor accessibility to healthcare facilities, which is common in these settings.15,16 the high frequency of late screening for gdm observed in this study may be due to such factors, as uncertainty about gestational age and late presentation for antenatal care among nigerian women is common, as reported by previous studies.17,18,19,20 in our study, almost 70% of the ahps practised universal screening for hip, whereas almost a quarter practised risk-based screening. this is similar to a survey of health workers in morocco where more than two-thirds of the participants were in favour of universal screening and about one-third of providers preferred risk-based screening.21 in a belgian survey, 83.9% of primary care physicians preferred universal screening.22 this finding suggests a trend toward universal screening. in a review article, utz et al. published that as many as 80% of guidelines recommended universal screening.12 universal screening for gdm provides broad coverage of screening for all pregnant women as part of comprehensive antenatal care. however, implementation of universal screening with the gold standard testing method, ogtt, may be problematic in lowand middle-income countries due to cost, equipment and staffing challenges, among others. this is amplified when considering that more than 95% of ahps screened in our study used a laboratory method with or without an in-clinic method. also, universal screening is the easier way out for this cadre of ahps, who may lack the requisite knowledge and experience to sort pregnant women for screening based on risk factors. selective screening based on risk factors was practised by a quarter of the ahps in our study, mostly doctors in clinical specialties or in tertiary settings. the majority of doctors in a survey in india preferred risk-based screening despite a national recommendation of universal screening.17 a previous study reported an established practice of risk-based screening in a teaching hospital in ibadan, western nigeria.23 this may reflect greater knowledge of selecting women at high risk for gdm using risk factors. the most common risk factors prompting screening for gdm in our study include maternal obesity, history of macrosomic babies, family history of dm, previous history of gdm or impaired glucose tolerance and intrauterine foetal death. this is in keeping with an earlier study in jos, nigeria, where the top four risk factors among women that were screened for gdm included history of macrosomic babies, maternal obesity, family history of dm and intrauterine foetal death.24 selective screening based on risk factors is anchored on the need for cost-effectiveness, considering the high cost of screening all pregnant women. the critics of this approach have cited the fact that the prevalence of undiagnosed diabetes is rising among women of childbearing age and as much as 40% – 60% of women without the traditional risk factors for gdm may end up having gdm or overt diabetes in pregnancy and may be missed if only risk-based screening is adopted.7,25 in addition, failure to identify risk factors may occur due to gaps in historical recall of risk factors by patients, as well as poor, incomplete or unavailable medical records, which are common challenges in health systems in lowand middle-income countries.12 furthermore, low compliance to risk-factor-based screening guidelines has been reported. as many as 70% of pregnant women with existing risk factors were not screened for gdm in a study in northern sweden.26 the use of a structured, risk-factor checklist has been suggested to increase the effectiveness of identifying women with gdm in ibadan, nigeria.23 glucometer and dipstick urine glucose testing were the most available testing methods in our study. this is not surprising considering the simplicity, affordability and availability of these testing methods. in our study, only a minority (13.1%) of the ahps had access to automated chemistry analysers and, as expected, almost 70% of these were in tertiary care centres and none in primary healthcare settings. this finding underscores the claim that inability to test for glucose is a challenge for screening and management of hip. the international federation of gynecology and obstetrics has advocated the use of glucose meters in settings where it will be practically difficult to test with automated analysers.7 bhavadharini et al. suggested that in settings where estimations by venous blood are not practicable, capillary blood glucose can be used as an initial screening test for gdm, using lower 2-h capillary blood glucose cut-off points to maximise the sensitivity.27 however, use of hand-held glucometers possesses some challenges, such as abuse by poorly trained personnel, need for calibration with a laboratory method, challenges with use of multiple glucometers from different vendors, quality control issues and the need for validation of current diagnostic criteria in different lowand middle-income country settings. therefore, it is important that the validity and diagnostic performance of this method is explored in the nigerian settings. the availability of glucose meter and urine dipstick testing may also explain why many of the ahps in our study adopted a universal screening strategy. while on the face of it, this is commendable, our results suggest that the majority of women were not screened according to the recommended screening method. the method for diagnosis of gdm preferred by ahps in our study included urine glucose, fbg and rbg. these testing methods, although simple and readily available, lack sensitivity and run the risk of missing women with gdm. a similar finding was reported by utz et al. in a moroccan study where fbg was the most common test used by the respondents and urine glucose was commonly used by nurses.21 these methods also trade off diagnostic sensitivity compared to the gold standard method, ogtt. a previous study in nigeria showed that as many as one in five women would be misclassified as normal glucose tolerant, if only fbg were used.5 only a quarter of the ahps in our study used the recommended diagnostic method of either 75 g or 100 g ogtt. the majority of these had good knowledge of hip and practised in a tertiary care setting. on the other hand, practising in a primary care setting and availability of dipstick for glucose testing were associated with diagnosing gdm with urine glucose. although there was good agreement regarding the test used by ahps in a given facility, there appeared to be some confusion with respect to the threshold used for making a diagnosis of gdm. antenatal healthcare providers who worked within specific health facilities used different discriminatory cut-off values for diagnosing gdm. this is in agreement with previous studies in india by utz et al. and agarwal et al.21,28 the disparities in threshold for making a diagnosis of gdm portends grave danger of misclassification in managing women with hip. also, there are implications for referral of further care, if women are misclassified at lower cadre centres. the post-partum follow-up for women with gdm is important for identifying sustained impaired glucose tolerance and those likely to progress to type 2 dm. it is recommended that pregnant women identified with gdm be tested 6–12 weeks post-partum.14,29 there are debates as to the most effective or suitable type of glucose testing during the post-partum period. although a 75 g ogtt is widely recommended by major international guidelines, the use of rbg, fbg and glycated haemoglobin has been advocated in some quarters.21 it is not surprising though that, given the simplicity of fbg and rbg, these tests were the most preferred by ahps in our study. it is important to address identified challenges to adequate screening and diagnosis of gdm, if management of hip is to be improved. the ahps in our study were most concerned about late booking for antenatal care, which implies late testing for hip. the consequence is that ahps are afforded little time to intervene in cases of gdm or hip in order to improve the pregnancy outcome. three-quarters of the ahps in our study agreed that issues concerning guidelines for screening and diagnosis of gdm, as well as general knowledge of hip, present an arduous challenge to screening and diagnosis of hip. it is commendable that in the last decade gains have been made towards harmonious international guidelines. it is therefore crucial that stakeholders in nigeria work at developing a consensus for addressing the peculiarities of screening in the nigerian context. lack of testing equipment and qualified personnel for testing were also identified as challenges to adequate screening and diagnosis of gdm in our study. it is important that laboratory personnel have the right testing methods and requisite knowledge to conduct the recommended ogtt for gdm. glucose meters should be made widely available, but even then, it would be more appropriate that a 75 g ogtt be conducted with three glucose measurements than a once-off glucose measurement. this would require training of the ahps at primary and secondary care levels. recommendations there is an urgent need for a harmonised screening and management guidelines for hip that address the peculiar challenges of low-income settings. lower cadre ahps, especially those in primary healthcare settings, should be trained on standardised screening procedures for hip like ogtt. the utility of hand-held glucose meters in identifying hip and the impact of this in rural communities should be explored by stakeholders including obstetricians, endocrinologists and laboratorians. this should include studies to determine thresholds for local populations that will maximise the diagnostic capacity for identifying women with hip using capillary blood glucose by available devices like glucose meters. limitations this study may have had some limitations. because this is a cross-sectional study, it is difficult to establish temporal relationships. also, the response of the ahps on the issues concerning screening practices may be influenced by social desirability bias, although an effort was made to affirm the anonymous nature of the study. some responses may have been influenced by the level of understanding of the operations in the facilities of employment. however, for such responses, only those who had worked for at least three months at the current facility were included in the analysis. also, this study did not seek to elicit from the ahps their reasons for their screening practices. exploring these items would have made the number of questions too many which could have discouraged overall response. however, inferences were made from known factors that generally influence screening practices. conclusion in conclusion, this study found that the recommended testing methods are not available at lower levels of the healthcare system in jos, north-central nigeria. primary and secondary government or public health facilities lack even basic glucose testing devices like a glucose meter. the screening methods used for hip generally involve simple testing methods like urine glucose tests and glucose meters, and the screening tests used do not generally conform to internationally recognised best practices. acknowledgements the authors appreciate the study participants for their participation in this study, mr yahaya khalid yusuf for assisting in collecting the data for this project and mrs elizabeth taiwo adeyemo for helping in compiling the data. the authors thank support of training and mentoring in nigeria for academics for providing mentoring for the research. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions l.c.i. served as the lead investigator. l.c.i., a.s.l., f.e.h., o.j.a., j.p.i., j.a.i. and m.e.b. contributed to study conception and design, and data acquisition. l.c.i., a.s.l., f.e.h., o.j.a., j.p.i., j.a.i. and m.e.b. contributed to data analysis and interpretation, and writing of the article. l.c.i., a.s.l., f.e.h., o.j.a., j.p.i., j.a.i. and m.e.b. contributed to editing, reviewing and final approval of article. all authors read and approved the final version of the manuscript. sources of support this research project was supported in a mentored research by the fogarty international centre of the united states national institutes of health and also the office of the director, national institutes of health, united states national institutes of nursing research and the united states national institutes of neurological disorders and stroke under award number d43tw010130. data availability the data sets used and analysed during the current study are available from the corresponding author, l.c.i., on reasonable request. disclaimer the content is solely the responsibility of the authors and does not necessarily represent the official views of the funding organisations. references ciccacci f, orlando s, majid n, marazzi c. epidemiological transition and double burden of diseases in low-income countries: the case of mozambique. pan afr med j. 2020;37:49. https://doi.org/10.11604/pamj.2020.37.49.23310 bigna jj, noubiap jj. the rising burden of non-communicable diseases in sub-saharan africa. lancet glob health. 2019;7(10):e1295–e1296. https://doi.org/10.1016/s2214-109x(19)30370-5 yuyun mf, sliwa k, kengne ap, mocumbi ao, bukhman g. cardiovascular diseases in sub-saharan africa compared to high-income countries: an epidemiological perspective. glob heart. 2020;15(1):15. 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classification and diagnosis of diabetes: standards of medical care in diabetes. diabetes care. 2019;42(suppl 1):s13–s28. https://doi.org/10.2337/dc19-s002 ajlm 9(2)_2020_contents.indd http://www.ajlmonline.org open access table of contents i lessons from the field driving the usage of tuberculosis diagnostic data through capacity building in lowand middle-income countries natasha gous, alaine u. nyaruhirira, bradford cunningham, chris macek african journal of laboratory medicine | vol 9, no 2 | a1092 | 18 november 2020 review article pooled testing: a tool to increase efficiency of infant hiv diagnosis and virological monitoring wolfgang preiser, gert u. van zyl african journal of laboratory medicine | vol 9, no 2 | a1035 | 11 august 2020 opinion paper building diagnostic systems in sierra leone: the role of point-of-care devices in laboratory strengthening rashid ansumana, fatmata bah, kan biao, doris harding, mohamed b. jalloh, ann h. kelly, francess koker, zikan koroma, mambu momoh, mohamed h. rogers, james rogers, alice street, eva vernooij, isatta wurie african journal of laboratory medicine | vol 9, no 2 | a1029 | 01 april 2020 opinion paper nigeria’s efforts to strengthen laboratory diagnostics – why access to reliable and affordable diagnostics is key to building resilient laboratory systems dhamari naidoo, chikwe ihekweazu african journal of laboratory medicine | vol 9, no 2 | a1019 | 26 august 2020 opinion paper preparing national tiered laboratory systems and networks to advance diagnostics in africa and meet the continent’s health agenda: insights into priority areas for improvement pascale ondoa, nqobile ndlovu, mah-sere keita, marguerite massingaloembe, yenew kebede, collins odhiambo, teferi mekonen, aytenew ashenafi, amha kebede, john nkengasong african journal of laboratory medicine | vol 9, no 2 | a1103 | 21 september 2020 35 41 48 53 58 page i of i table of contents i editorial connected diagnostics systems: the future of disease control in africa noah fongwen, debi boeras, rosanna w. peeling, timothy amukele african journal of laboratory medicine | vol 9, no 2 | a1365 | 21 december 2020 original research field evaluation of a mobile biosafety laboratory in senegal to strengthen rapid disease outbreak response and monitoring cheikh fall, aurélie cappuyns, oumar faye, steven pauwels, gamou fall, ndongo dia, moussa m. diagne, cheikh t. diagne, makhtar niang, alassane mbengue, martin faye, idrissa dieng, babacar gningue, abdoulaye bousso, ousmane faye, rudi pauwels, amadou a. sall african journal of laboratory medicine | vol 9, no 2 | a1041 | 20 august 2020 lessons from the field timely delivery of laboratory efficiency information, part i: developing an interactive turnaround time dashboard at a high-volume laboratory naseem cassim, manfred e. tepper, lindi m. coetzee, deborah k. glencross african journal of laboratory medicine | vol 9, no 2 | a947 | 29 april 2020 lessons from the field timely delivery of laboratory efficiency information, part ii: assessing the impact of a turn-around time dashboard at a high-volume laboratory naseem cassim, lindi m. coetzee, manfred e.e. tepper, louella perelson, deborah k. glencross african journal of laboratory medicine | vol 9, no 2 | a948 | 29 april 2020 lessons from the field an open-source molecular diagnostic platform approach for outbreak and epidemic preparedness devy m. emperador, laura t. mazzola, cassandra kelly-cirino african journal of laboratory medicine | vol 9, no 2 | a1017 | 28 september 2020 1 3 12 21 29 vol 9, no 2 (2020) special collection: the future of diagnostics issn: 2225-2002 (print) | issn: 2225-2010 (online)african journal of laboratory medicine background observations acknowledgements references about the author(s) pascale ondoa african society for laboratory medicine, addis ababa, ethiopia amsterdam institute for global health and development, academic medical centre, department of global health, university of amsterdam, amsterdam, netherlands nqobile ndlovu african society for laboratory medicine, addis ababa, ethiopia mah-sere keita african society for laboratory medicine, addis ababa, ethiopia marguerite massinga-loembe africa centres for disease, control and prevention, addis ababa, ethiopia yenew kebede africa centres for disease, control and prevention, addis ababa, ethiopia collins odhiambo african society for laboratory medicine, addis ababa, ethiopia teferi mekonen african society for laboratory medicine, addis ababa, ethiopia aytenew ashenafi africa centres for disease, control and prevention, addis ababa, ethiopia amha kebede african society for laboratory medicine, addis ababa, ethiopia john nkengasong africa centres for disease, control and prevention, addis ababa, ethiopia citation ondoa p, ndlovu n, keita m-s, et al. preparing national tiered laboratory systems and networks to advance diagnostics in africa and meet the continent’s health agenda: insights into priority areas for improvement. afr j lab med. 2020;9(2), a1103 https://doi.org/10.4102/ajlm.v9i2.1103 opinion paper preparing national tiered laboratory systems and networks to advance diagnostics in africa and meet the continent’s health agenda: insights into priority areas for improvement pascale ondoa, nqobile ndlovu, mah-sere keita, marguerite massinga-loembe, yenew kebede, collins odhiambo, teferi mekonen, aytenew ashenafi, amha kebede, john nkengasong received: 20 sept. 2019; accepted: 29 may 2020; published: 21 sept. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. background access to diagnostics remains sub-optimal in africa due to limited human, financial and technical resources that affect various components of the health system.1,2 additionally, the lack of standardised systems for evaluation and registration of diagnostics3 cripples the introduction of better technologies, representing missed opportunities to address healthcare challenges. examples of poor access to diagnostics and its dramatic consequences are numerous. despite strong vertical control programmes, 40% of hiv-infected patients on antiretroviral therapy do not receive the recommended yearly hiv viral load monitoring test.4 in 2016, a dramatic 21% of children born to hiv-positive mothers were reported as not receiving early infant diagnosis testing before age 8 weeks in west and central africa.5 data from the world health organization (who) global tuberculosis database of 20166 also indicate that drug-resistant tuberculosis is largely missed in africa. in addition to 70% of patients not being notified, a rifampicin susceptibility test was available to less than 10% of patients in 23 of 47 countries and second-line resistance testing was available only in 60% of the countries on the continent.7 the picture is equally worrisome for diseases that are poorly or not supported through dedicated programmes. nine in 10 individuals carrying the hepatitis b or c virus have never been tested, while these infections are estimated to cause 60% of liver cancers and an epidemic larger than that caused by hiv.8 in a study conducted in senegal in 2015–2016, less than 30% of pregnant women attending antenatal care at the primary healthcare level had access to the minimum panel of screening tests for the most common clinical conditions threatening maternal and child health.9 almost half of the mortality cases associated with cervical cancer are due to late detection of the disease.10 more recently, it appeared that when the first coronavirus disease 2019 (covid-19) case was reported in egypt in february 2020, only 2 of the 55 countries on the continent were capable of detecting the severe acute respiratory syndrome coronavirus 2 (sars-cov-2). insufficient access to in vitro diagnostics (ivd) at all levels of healthcare delivery in low-income and middle-income countries reduces access to life-saving treatments, impairs the delivery of quality healthcare and compromises progress towards universal health coverage in africa. implementation of simplified, more robust and more affordable diagnostic options has been proposed to increase access to diagnostics in low-resource settings. rapid diagnostic tests that can be performed at the community level or through self-testing have transformed the management and prevention of several diseases such as hiv infection, malaria and diabetes.11,12,13 point-of-care molecular technologies, such as the genexpert® or the m-pima®, have been demonstrated to increase specificity and simplicity of testing, while reducing the turnaround time for test results14,15 for improved patient retention and management. despite addressing critical testing processes or delivery gaps, innovative or conventional diagnostic technologies frequently fail to translate into tangible public health outcomes.16,17,18 the failure of diagnostics to reach a large proportion of the population in need of it can partly be linked to implementation approaches that are designed for the site level, with oversight of the tiered laboratory network requirements and insufficient attention given to the underlying laboratory systems (e.g. supply chain, workforce, finance, etc.). complementing the maputo declaration of 2008,19 the freetown declaration of 201520 underscores that delivering diagnostic services in the context of functional, integrated national laboratory networks is the recommended strategy for providing maximum population coverage and cost-effective utilisation and delivery of diagnostic services in resource-limited settings. apart from selected disease-dedicated investments (e.g. hiv, tuberculosis), national tiered laboratory networks generally remain grossly underfunded with mild to critical dysfunctions in the underlying laboratory systems. in addition to falling short in the provision of optimal access to essential clinical diagnostics, the current sub-optimal capacity of tiered laboratory networks translates into weaknesses in the detection component of the ‘prevent, detect, respond’ framework of health security21,22 missed opportunities to stop onward transmission of infectious diseases and to detect and prevent non-communicable diseases hinder efforts to reduce the burden of illnesses in africa. the who estimates that the resulting loss of annual productivity due to the heavy disease burden in africa equalled $2.4 trillion and 630 million years of healthy lives in 2015.23 despite the call for more attention in building effective public health laboratory systems,24 which was made in the aftermath of the ebola epidemic of 2015, few data exist that allow quantification of the overall performance of national laboratory networks and systems across diseases. this lack of information prevents the design of impactful interventions and hinders the uptake of the otherwise large and relevant range of diagnostic technologies available. in this report, the african society for laboratory medicine (aslm) and the africa centres for disease control and prevention (africa cdc) share a unique insight into some of the most critical areas for improvement to bridge the gap between the capacity of laboratory networks and the promises of diagnostic technologies. observations root causes of dysfunction in laboratory networks are not sufficiently measured or addressed actionable data on integrated laboratory network functionality are scarce. the who joint external evaluation25 tool provides a high-level overview of the performance of the national laboratory network across four domains: (d1.1) laboratory testing for the detection of priority diseases, (d1.2) specimen referral and transport system, (d1.3) effective national diagnostic network and (d1.4) laboratory quality system, as part of the evaluation of entire national health systems to support compliance with international health regulations requirements. data from 54 countries21 indicate that quality management and sample referral systems are the most neglected areas across the continent with 68.1% and 50.0% of countries having none to only a basic capacity in place (figure 1), further aggravating the gaps in diagnostic networks with issues related to coverage and reliability of testing. more than 48.0% of countries have gaps in defining or providing access to tier-specific laboratory testing strategies, while more than 84.0% of the countries assessed demonstrated sustainable capacity for laboratory testing to support the surveillance of 10 priority diseases. the global health security agenda laboratory network (labnet26) scorecard was developed to complement the joint external evaluation tool and to provide more granularity on nine core capabilities of the laboratory network and systems, enabling the identification of specific gaps and related root causes. in eight countries where this evaluation was conducted, the average performance of laboratory networks ranged from none to basic capacity across the nine core capabilities assessed (figure 2), highlighting the presence of many common disabling factors (table 1), including: weak laboratory governance. in several countries, no directorate of laboratories exists or it is not directly placed under the authority of the ministry of health, preventing adequate coordination of laboratory services and limiting the sphere of operation of the laboratory vis-à-vis the other health sectors. unclear assignment of administrative versus technical tasks and mandates between directorates and national public health institutes and national reference laboratories also prevents the overall development and enforcement of regulations related to various aspects of diagnostics (such as ivd evaluation and registration, definition and updates of tier-specific minimal testing packages, laboratory staffing norms or definition and implementation of quality standards). figure 1: proportion of african countries (n = 54) at various levels of capacity in the area ‘laboratory network’ of the joint external evaluation tool. figure 2: overall level of laboratory network functionality in eight countries assessed. table 1: most critical gaps in laboratory network core capabilities were identified through the laboratory network assessment of eight sub-saharan african countries between 2017 and 2019, and their implications for diagnostic services. two countries with strong laboratory governance include ethiopia, through the ethiopian public health institute, and uganda, through the directorate of laboratory services of uganda. they offer good examples of best practices in the management of the laboratory systems and networks. these countries report updated national policies and strategic plans, budget lines earmarked for laboratory functions and regulations defining and enforcing laboratory clinical and public health functions from the reference level to the community level: missed opportunities to prioritise diseases for both surveillance and care. countries do not have access to up-to-date and fit-for-purpose epidemiological data to prioritise diseases of public health importance using a risk-based approach. often, the who list of 43 pathogens for surveillance serves as a de facto national list of ‘priority’ pathogens. the who recommends prioritising 10 pathogens27 to ensure that communicable diseases with the most severe morbidity and mortality are effectively screened, confirmed and the treatment monitored at the various tiers of the diagnostic network. countries implementing the global health security agenda prioritise 10 core tests covering international health regulations immediately notifiable diseases among the who top 10 causes of death in low-income and middle-income countries. the integrated disease surveillance and response framework of the who regional office for africa recommends prioritising epidemic-prone diseases targeted for eradication and elimination, and endemic diseases, resulting in a list of 19 diseases. in practice, these overlapping recommendations are difficult to interpret. the plethora of ‘prioritised’ diseases complicates the implementation of robust tier-specific packages and surveillance systems that can support both epidemiology reporting and clinical care. insufficient attention to evidence-based management of laboratory networks. none of the countries assessed had well-defined processes to routinely monitor the performance of the laboratory network without the support of external partners. this situation precludes the establishment of a quality assurance loop of laboratory networks and systems. ultimately, the lack of monitoring and evaluation systems (i.e. lack of key performance indicators for the laboratory sector and of responsible units to collect, analyse and act upon the data) for laboratory networks undermines the return on investment of most capacity strengthening interventions aimed at advancing diagnostic services. recommendations strengthening of integrated quality management and specimen referral systems are the most urgent interventions needed, the outcomes of which should be evaluated against the advancement of joint external evaluation scores. various initiatives funded by global stakeholders are ongoing (e.g. united states agency for international development, united states president’s emergency plan for aids relief, global fund) with the potential to be transitioned to and sustained by stronger and empowered national laboratory leadership with the political support of africa cdc. the aslm and their partners can work together at formulating clearer guidance on the respective mandates of directorates of laboratories and national public health institutes (including the monitoring and evaluation framework for laboratory network functions) as well as advocating to empower the laboratory sector. these efforts align with the recommendations of the maputo and the freetown declarations. assisting countries to define tier-specific testing packages that address the needs of clinical diagnostics and disease surveillance is another important intervention with the potential to guide the introduction of diagnostics at the levels where they are most needed and cost-effective. we recommend that every country conduct an external or self-evaluation of their laboratory systems once a year or once every two years as part of a continuous quality improvement cycle for their national tiered laboratory network. while the joint external evaluation tool provides high-level dashboards for a set of four indicators, the labnet scorecard is designed to guide countries in selecting specific and feasible interventions most likely to tackle the root causes of the identified dysfunctions across a comprehensive set of indicators. laboratory networks are not configured to support diagnostic services that are integrated, cost-effective and with maximum population coverage mutualising scarce resources for most cost-effective health services is (or should be) a constant concern in low-resource settings. a data-driven configuration of a national laboratory network can support the design of faster and more affordable sample transportation routes towards testing hubs. from the specimen referral landscape, assessments that were performed during 2015 and 2016 in eight countries by aslm under the global health security agenda laboratory strengthening effort revealed that a certain level of integration of the specimen transport system (sts) can exist, often between hiv and tuberculosis programmes. the stss are generally fragmented, working in parallel, using different transport mechanisms depending on the disease programme, are funded by various donors and have challenges in terms of cost-effectiveness, turnaround time and coverage. a few countries, such as ethiopia, rwanda and uganda, have highly integrated specimen referral networks that cater to any disease programme and span across diagnosis to surveillance, detection and response. ideally, this disease-agnostic approach should be developed such that individual stss are effectively and efficiently networked, and that any type of specimen can be easily and seamlessly moved from where it originates to the appropriate diagnostic equipment. addressing the common problem of fragmented and disease-specific stss, aslm coordinated the development of a standardised sts assessment and development toolkit,28 addressing multiple diseases, surveillance and clinical diagnostic needs, and factoring in different modes of transportation. one country, burkina faso, was able to use the findings of the assessment to establish a new specimen referral system for surveillance that is now being built upon, scaled up and integrated to cover any disease programme.29 the availability of up-to-date geo-localised information about laboratory network capacity provides evidence on which to base the process of defining optimal service configurations, including the shortest routes for sample transportation, maximum population coverage for testing services, cost-effective supply chains, or opportunities for testing integration. initiatives aimed at collecting geographical information system data on laboratory capacity are gaining momentum to improve the performance of hiv and tuberculosis control programmes.30,31 the aslm and africa cdc have implemented a continent-wide laboratory capacity mapping programme (labmap32) across diseases, based on open-source tools (ona [ona systems inc., nairobi, kenya], planwise [instedd, sunnyvale, california, united states]33) and fostering country ownership. this system allows the easy collection, curation and analysis of geospatial data to make informed decisions on national laboratory networks and is interoperable with the district health information system (dhis2, university of oslo, norway) 234. thirteen of the 54 countries on the continent (24%) are currently using the system under the coordination of africa cdc’s regional coordinating centres.35 among the 101 level 4 and level 3 laboratories assessed, only 40% had the capacity to conduct culture or polymerase chain reaction-based tuberculosis diagnosis, 11% and 34% could perform hiv drug resistance genotyping and early infant diagnosis, and 36% could run confirmatory testing for meningitis through culture or polymerase chain reaction (figure 3). using labmap data from 2018, we determined that in niger,36 eight facilities at level 3 are conducting meningitis testing, including serology, bacterial culture and nucleic acid testing, covering a total of 7.9 million people (40% of the nigerien population) within a 2-hour drive radius. only one facility (covering 2.8 million people, 14% of the population) is equipped to conduct polymerase chain reaction for the differential diagnosis of pathogens causing meningitis, which is critical for the swift adjustment of antibiotic therapy. planwise calculated that three strategically located level 3 laboratories involved in meningitis testing could be upgraded with a maximum impact reaching out to an additional 4.1 million (20%) inhabitants. planwise and other similar software like the supply chain guru from llamasoft37 offer the opportunity to calculate the best options to improve the laboratory network testing capacity and coverage, including upgrading existing facilities, building new laboratories or configuring specimen referral and supply chain routes. figure 3: gaps between actual diagnostic capacity and world health organization recommendations for minimal testing package in 101 level 4 and level 3 laboratories in 12 african countries (data from 2018 to 2019). recommendations data-driven optimisation of laboratory networks is an important management activity that can support essential public health and clinical functions. key considerations of any optimisation exercise should include at least: the structure of the tiered laboratory networks, (essential) testing needs for both clinical diagnosis and disease surveillance, and opportunities for test integration. specimen referrals are important systems underlying national laboratory networks. taking a network approach for developing integrated stss may provide greater cost savings, efficiencies, increased coverage and increased access. this approach can begin by mapping and optimising the overall referral network based on the existing diagnostics network (or any upcoming changes), reducing redundancies where possible and leveraging existing or new systems to create economies of scale, clearly planning and budgeting for the optimised network and implementing the new approach. any potential benefits should be clearly estimated and used to convince governments of the approach and gain buy-in from other stakeholders as well. regular collection and updating of (geo-located) laboratory capacity information is necessary to inform network optimisation exercises. such activities could be enforced as part of laboratory registration or licensing and re-licensing processes under the coordination of the directorate of laboratories. most diagnostic services delivered through laboratory networks are not quality assured unreliable diagnostic tests can compromise the quality of healthcare delivery. a laboratory quality management system (lqms) is a formalised system that documents processes, procedures and responsibilities for achieving an international standard of quality. the implementation of lqms was identified as a priority to strengthen laboratory services in africa38,39 a decade ago. as part of this momentum effect, various tools and frameworks have been developed and disseminated to guide laboratories towards lqms implementation and international organization for standardization 15189 accreditation. some of these resources have a regional or a disease-specific focus; these include the strengthening laboratory management toward accreditation, the stepwise laboratory quality improvement process towards accreditation (slipta), which also includes a tuberculosis-specific checklist, the lqms training tool kit and the laboratory quality services international group. to date, around 520 laboratories have received international accreditation on the continent. more than 370 of these accredited facilities are in south africa and most of them belong to the public sector, highlighting geographic differences and the lagging behind of private laboratories. the slipta is a who regional office for africa programme assessing laboratory progress towards the international organization for standardization 15189 standards and with aslm serving as the secretariat. since its launch in 2012, around 430 laboratories implementing lqms have been assessed across 17 countries (table 2), illustrating the increasing awareness of national stakeholders with regard to quality requirements, as well as the commitment of the laboratory community to advance diagnostic services. however, this number is below the original target of 2500 enrolled laboratories set at the programme onset40 and represents a modest outcome at both the continental and country scale. a couple of countries have demonstrated impressive coverage of the slipta programme (e.g. botswana and namibia, table 2). however, assuming that only 50% of the total number of government laboratories are in hospitals (the target facilities to implement the international organization for standardization 15189 norm), the current number of laboratories engaged in slipta represent only 0.3% of the total number of government laboratories in nigeria and 3% in kenya. some bottlenecks with lqms implementation using slipta are inherent to the programme itself. the handling of certification requests and processes through countries’ ministry of health prioritises government over private laboratories. the lack of core funding supporting the programme translates into insufficient capacity for aslm to cover the needs of an entire continent, not only to conduct audits but also to mentor laboratories towards quality improvement and accreditation. benefitting from more stable united states president’s emergency plan for aids relief funding, and despite reaching an impressive 1333 laboratories across various continents,41 the mentorship-focused strengthening laboratory management toward accreditation programme is equally unable to comprehensively cover the needs of entire national laboratory networks. advocacy efforts towards policymakers are also impaired by the lack of simple indicators linking lqms implementation with improved health outcomes. country-specific roadblocks have also been identified such as low political commitment and the lack of regulatory and policy frameworks guiding the expansion of lqms as national programmes, adapted for each tier of laboratory networks. recently, continuous quality improvement initiatives targeting disease-specific testing (such as hiv) have been implemented. although continuous quality improvement directly addresses the quality of diagnostic testing through a problem-solving approach linked to patient outcomes, it still needs to be embedded into larger quality management programmes in order to contribute to sustainable outcomes supporting universal healthcare coverage and international health regulations. table 2: stepwise laboratory quality improvement process towards accreditation coverage of facilities in the public and private sector in 17 african countries (as of august 2019). in addition to the poor coverage of lqms implementation, most countries do not have systems in place to ensure that all testing sites comply with the basic components of quality assurance such as external quality assessments, proficiency testing schemes42 and various quality checks linked to post-market surveillance of ivd. collectively, these observations suggest that dramatically high proportions of diagnostic tests delivered in african countries are not adequately quality assured. this inevitably results in laboratory test results that are incorrect or fraudulent, causing a lack of trust among clinicians and communities. recommendations to overcome these challenges, the aslm and who regional office for africa launched slipta version 2.0,43 which proposes to institutionalise slipta at the country level, harness partner resources for slipta funding and redefine the role of aslm as a coordinating rather than an implementing body. this strategic approach is currently being implemented through regional collaborations where the west african health organization and east, central and southern africa health community are providing leadership to advance lqms using the slipta programme in west, central east and southern africa, with aslm serving as a high-level coordinator. according to this model, aslm and its partners work at generating a sufficient pool of local slipta auditors and laboratory mentors who can be mobilised to advance lqms and conduct slipta audits upon regional or national request. the efforts of africa cdc to establish the regional integrated laboratory network (rislnet) is also contributing to the extension of slipta version 2.0, through the implementation of lqms in national public health institutes, with subsequent deployment of the system in lower tiers of the national laboratory networks and across the private sector. a couple of african countries like ethiopia have stepped forward to ‘franchise’ the slipta model as national programmes aiming to cover all laboratory facilities from the public and the private sector. the foundation of a country-led slipta programme is the definition of national quality standards for diagnostic services at each level of service delivery, clearly describing which laboratory has the vocation to be accredited or certified and against which set of minimum quality standards. critical shortage of pathologists compromises present and future benefits of laboratory diagnostics clinical pathologists are physicians trained in various disciplines of laboratory medicine, such as haematology, medical microbiology, transfusion medicine, clinical biochemistry or cytopathology.44 they provide highly specialised knowledge and leadership, ensuring the quality of the pre-analytical, analytical and post-analytical phases of testing, as well as critical information on the severity and prognosis of diseases based on test results. clinical pathologists make sure that patients are started and maintained on the correct treatment regimen. in the united states and united kingdom, the pathology workforce varies between 3 and 5 per 100 000 inhabitants.45 a quick desk survey conducted by aslm reveals critical gaps in the clinical pathology profession in 10 countries of sub-saharan africa. firstly, the mere definition of this profession is often not well understood, with most survey respondents only aware of anatomic pathologists but not clinical pathologists. data from the college of pathologists in east, central and southern central africa show a bias towards anatomical pathology (60%) compared to clinical pathology (18%) of the 119 registered pathologists (dr shahin sayed, personal communication). some countries also use pharmacists as ‘pathologists’,46 although this profession requires a background in medical studies, suggesting insufficient clinical interpretation of laboratory test results that might compromise patient outcomes. secondly, an up-to-date inventory of pathologists by national professional councils seems to be lacking in many countries, suggesting that these highly specialised professionals are not adequately registered or certified in their respective countries. thirdly, the ratio of pathologists (regardless of their specialty) is 5–350 times lower than ratios observed in the united states (table 3), translating into gaps of more than 4000 pathologists for a country like ethiopia or more than 6000 for a country like nigeria. in most countries sampled, the number of pathologists is lower than the number of tier 2 and tier 3 hospitals (where pathology services are required), suggesting that clinical pathologists would have to serve in more than one facility to reduce the gaps. for example, 61 pathologists in ethiopia have to support a total of 400 hospitals. the number of medical microbiologists (a sub-specialty of clinical pathology), at 0.5, is even lower in all countries sampled. this worrisome situation raises concerns about the sustainability and impact of current global and national efforts to establish diagnostic capacity for the control of antibiotic resistance on the african continent. the introduction of novel diagnostic technologies such as point-of-care testing at the community level or next generation sequencing at the reference level will only increase the need for specialised laboratory medicine professionals who are able to ensure the correct use and interpretation of diagnostic tests for improved patient and public health outcomes. collectively, these data highlight severe gaps in general and clinical pathology, in particular in sub-saharan african countries. table 3: overview of numbers, coverage and needs of pathologists in nine surveyed countries of africa (data from august 2019). recommendations in-country capacity for pathology training is commonly reported, with efforts by the college of pathologists in east, central and southern central africa and other organisations to advocate for training of more pathologists and the laboratory workforce. however, the magnitude of the gaps highlighted here and by others demands that many more resources be deployed to produce higher numbers of pathologists at a faster pace in the disciplines associated with the most severe disease burdens, and to provide acceptable solutions for task shifting at each tier of the laboratory network. key interventions to reduce the shortage of clinical pathologists include: the formulation of staffing norms in national laboratory strategic plans and healthcare human resources development strategies by defining the number and profile of pathologists at each relevant tier of the laboratory network and increasing opportunities for education, training and mentorship at the regional level, including innovative digital, remote training options. this could be done as part of the objective of the workforce development institute of africa cdc,47 with the establishment of certification and qualification programmes that ensure standard levels of competency, at least in national public health institutes. regulatory bottlenecks slow down introduction of useful diagnostics the past decade has seen the introduction of game-changing technologies for major public health diseases such as hiv, tuberculosis and malaria, which promise easier access, use and impact of diagnostics at the community level where most patients seek care. the who’s ivd prequalification process is a standardised procedure to determine whether products meet requirements for safety, quality and performance. the findings of this prequalification are used to provide guidance to countries in selecting laboratory diagnostics to be implemented at the programme level. the who prequalification represents the ‘ticket’ for any ivd to penetrate national markets, and is a process that takes 2–3 years. additionally, national regulatory frameworks often foresee additional evaluations to verify that the prequalified diagnostic is adapted to specific contexts. however, the relevance of multiple, in-country evaluations is not always clear, represents unnecessary repetition, and has no additional value compared to who prequalification results or to a well-designed single evaluation conducted in one centre of excellence located in a region with similar disease epidemiology. delayed registration of ivds in-country prevents access to reliable existing diagnostics for many priority diseases including those associated with outbreaks. regulatory bottlenecks for ivds during country registration compromise the implementation of essential diagnostics, and prevent universal health coverage and african health security. recommendations an innovative approach to facilitate the swift evaluation and registration of useful and performant ivd and support the advancement of the diagnostic agenda in african regions is needed. leveraging existing networks of excellence that can quickly conduct the standardised evaluation of ivds and support collaborative registration procedures on behalf of entire regions of africa can lead to critical benefits in access to ivd, while waiting for or to complement who prequalification. the africa collaborative to advance diagnostics, led by africa cdc, was launched in abuja during the biannual aslm conference in 201848 with the overall aim of advocating for appropriate investment in diagnostics as well as accelerating regulations to facilitate timely and wide access to essential diagnostics. one of the goals of the africa collaborative to advance diagnostics is to work towards speedier registration of diagnostic technologies, through a pan-african approach that complements who prequalification, leverages existing continental expertise and provides opportunities for manufacturers and other relevant stakeholders to support the process. conclusion in addition to advancing the development of increasingly relevant, reliable, specific and affordable ivd products, the future of diagnostics in africa also depends on our collective ability to comprehensively and swiftly address the systemic weaknesses in national laboratory networks. under the leadership of the africa cdc and who regional office for africa, african technical agencies at the continental or regional level (e.g. aslm, the west african health organization and east central and southern africa health community) or national level (e.g. nigeria cdc) have a critical role to play in providing direct technical assistance and vision for advancing national laboratory network diagnostic capacity and on setting adequate priorities for international cooperation. implementing the african union recommendations on domestic investment in healthcare49 is critical to ensure that laboratory systems and networks are sustainably prepared for the future of diagnostics in africa. acknowledgements the authors thank the african society for laboratory medicine and the africa centres for disease control and prevention team members samba diallo, edwin shumba and anafi mataka for their input in data collection and analysis. we thank dr kameko nichols for her contribution to the section on sample transportation systems. we thank dr ali elbireer for his contribution to the initial design of the manuscript. we thank professor oni emmanuel idigbe for his support in collecting data on african pathologists. we thank the ministries of health of niger, cameroon, congo, ethiopia, democratic republic of congo, chad, zambia, zimbabwe, sao tome and principe, gabon, central african republic and malawi for sharing data on laboratory mapping. we thank the ministries of health of ethiopia, uganda, burkina faso, senegal, cameroon, tanzania, democratic republic of congo and nigeria for sharing data on the labnet scorecard assessment. competing interests the authors declare that they have no conflicts of interest. authors’ contributions all authors contributed equally to the work. ethical considerations ethical approval is not required for this opinion article. sources of support this work would not have been possible without funding from the bill and melinda gates foundation, the president emergency plan for aids relief, the global health security agenda, unitaid and vital strategies. data availability statement data sharing is not applicable to this article as no new 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[cited 2019 july 29]. available from: https://www.afro.who.int/news/cervical-cancer-common-amongst-african-women angotti n, bula a, gaydosh l, kimchi ez, thornton rl, yeatman se. increasing the acceptability of hiv counseling and testing with three c’s: convenience, confidentiality and credibility. soc sci med. 1982;68(2009):2263–2270. https://doi.org/10.1016/j.socscimed.2009.02.041 visser t, daily j, hotte n, et al. rapid diagnostic test for malaria. bull world health organ 2015;93:862–866. https://doi.org/10.2471/blt.14.151167 schell o, crocker b, weng j. impact of hba1c testing at point of care on diabetes management. j diabetes sci technol. 2017;11(3):611. https://doi.org/10.1177/1932296816678263 world health organization. automated real-time nucleic acid amplification technology for rapid and simultaneous detection of tuberculosis and rifampicin resistance: xpert mtb/rif assay for the diagnosis of pulmonary and extrapulmonary tb in adults and children. geneva: who; 2013. bianchi f, cohn j, sacks e, et al. evaluation of a routine point-of-care intervention for early infant diagnosis of hiv: an observational study in eight african countries. lancet hiv. 2019;6(6):e373–e381. https://doi.org/10.1016/s2352-3018(19)30033-5 di tanna gl, khaki ar, theron g. effect of xpert mtb/rif on clinical outcomes in routine care settings: individual patient data meta-analysis. lancet glob health. 2019;7(2):e191–e199. https://doi.org/10.1016/s2214-109x(18)30458-3 burchett hed, leurent b, baiden f, et al. improving prescribing practices with rapid diagnostic tests (rdts): synthesis of 10 studies to explore reasons for variation in malaria rdt uptake and adherence. bmj open. 2017;7(3):e012973. https://doi.org/10.1136/bmjopen-2016-012973 rao vb, schellenberg d, ghani ac, rao vb, schellenberg d, ghani ac. overcoming health systems barriers to successful malaria treatment. trends parasitol. 2013;29(4):164–180. https://doi.org/10.1016/j.pt.2013.01.005 world health organization. maputo declaration [homepage on the internet]. [cited 2019 july 29]. available from: https://www.who.int/diagnostics_laboratory/maputo-declaration_2008.pdf african society for laboratory medicine. freetown declaration [homepage on the internet]. [cited 2019 july 29]. available from: http://www.aslm.org/what-we-do/global-health-security/freetown-declaration/ who african region: jee mission reports [cited 2019 july 29]. available from: https://www.who.int/ihr/procedures/mission-reports-africa/en/ nuche-berenguer b, kupfer lej. readiness of sub-saharan africa healthcare systems for the new pandemic, diabetes: a systematic review. diabetes res. 2018;18:9262395. https://doi.org/10.1155/2018/9262395 world health organization regional office for africa. a heavy burden: the productivity cost of illnesses in africa [homepage on the internet]. 2019 [cited 2020 july 25]. available from: https://www.afro.who.int/publications/heavy-burden-productivity-cost-illness-africa nkengasong jn, skaggs ba. are post ebola reconstruction efforts neglecting public health laboratory systems? lancet glob health. 2015;3(11):e678. https://doi.org/10.1016/s2214-109x(15)00159-x bell e, tappero jw, ijaz k. joint external evaluation – development and scale-up of global multisectoral health capacity evaluation process. emerg infect dis. 2017;23 (suppl 1):s33–s39. https://doi.org/10.3201/eid2313.170949 ondoa p, datema t, keita-sow ms, et al. a new matrix for scoring the functionality of national laboratory networks in africa: introducing the labnet scorecard. afr j lab med. 2016;5(3), a498. https://doi.org/10.4102/ajlm.v5i3.498 mehand ms, millet p, al-shorbaji f, rith c, kieny mp, murgue b. world health organization methodology to prioritize emerging infectious diseases in need of research and development. emerg infect dis. 2019;24(9):e171427. https://doi.org/10.3201/eid2409.171427 stop tb partnership. gli specimen referral toolkit. [homepage on the internet]. [cited 2020 july 25it]. available from: http://www.stoptb.org/wg/gli/srt.asp dama e, nikiema a, nichols k, et al. designing and piloting the first sample referral system in burkina faso using the national postal service. health security. 2020; 18(s1):s98–s104 nichols be, girdwood sj, cropton t, et al. monitoring viral load for the last mile: what will it cost? j int aids soc. 2019;22(9):e25337. https://doi.org/10.1002/jia2.25337 girdwood sj, nichol be, moyo c, crompton t, chimhamhiwa d, rosen s. optimizing viral load testing access for the last mile: geospatial cost model for point of care instrument placement. plos one. 2019;4(8):e0221586. https://doi.org/10.1371/journal.pone.0221586 african society of laboratory medicine. laboratory mapping program (labmap) [homepage on the internet]. [cited 2019 sep 18]. available from: http://www.aslm.org/what-we-do/laboratory-mapping/ instedd. planwise: geographic optimization of health system coverage [homepage on the internet]. [cited 2019 sep 18]. available from: https://instedd.org/project/planwise/ dhis2. covid-19 surveillance package [homepage on the internet]. [cited 2020 july 25]. available from: https://www.dhis2.org/ africa centres for disease control and prevention. strategic plan 2017–2021 [homepage on the internet]. [2020 july 25]. available from: http://www.ianphi.org/_includes/documents/strategy%20africa%20english%20cdc.pdf worldpop. open spatial demographic data and research [homepage on the internet]. [2019 may 19]. available from: www.worldpop.org.uk llamasoft inc. llamasoft [homepage on the internet]. [cited 2020 may 19]. available from: https://llamasoft.com/ world health organization. joint who-cdc conference on health laboratory quality systems, lyon, 9–11 april 2008. who/hse/ihr/lyo/2008.3. accessed at https://www.who.int/ihr/publications/who_hse_ihr_lyo_2008.3/en/ [cited 2020 july 25] world health organization regional committee for africa. resolution afr/rc58/r2 to strengthen public health laboratories in who african region at all levels of the healthcare system: a critical need for disease control. in: final report 58th session of who regional committee for africa, yaounde, september 2008. african society of laboratory medicine. aslm2020: strategies and vision to strengthen public health laboratory medicine in africa [homepage on the internet]. [cited 2019 dec 29]. available from: https://www.eiseverywhere.com/file_uploads/e46acabb2952cbecd2d610ca4cbfd45c_aslm2020.pdf strengthening laboratory management toward accreditation. slmta [homepage on the internet]. [cited 2019 aug 16]. available from: www.slmta.org carter jy. external quality assessment in resource-limited countries. biochem med. 2017;27(1):97–109. https://doi.org/10.11613/bm.2017.013 nqobile n. slipta 2.0 – the next ‘country driven’ frontier for laboratory quality improvement [homepage on the internet]. lab culture. 2018;18:12–13. [cited 2019 july 29]. available from: http://www.aslm.org/stay-informed/press-room/lab-culture-newsletter/ fleming ka, naidoo m, wilson m, et al. an essential pathology package for low and middle income countries. am j clin pathol. 2017;147(1):15–32. robboy si, weintraub s, horvath ae, et al. pathologist workforce in the united states. arch pathol lab med. 2013;213:1723–1732. https://doi.org/10.5858/arpa.2013-0200-oa hance p. repenser la formation des biologistes. le laboratoire en zone tropicale. colloque organisé par le spe, 7 juin 2006. bull soc pathol exot. 2006;99:409–417. africa cdc. africa cdc institute for workforce development [homepage on the internet]. [cited 2019 sep 18]. available from: https://africacdc.org/africa-cdc-institutes/africa-cdc-institute-for-workforce-development/ african union. africa centres for disease control and prevention and partners launch the africa collaborative initiative to advance diagnostics – afcad to address existing barriers towards advancing the diagnostic agenda in africa [homepage on the internet]. [cited 2020 july 25]. available from: https://au.int/en/pressreleases/20181116/africa-centres-disease-control-and-prevention-and-partners-launch-africa africa health business. africa leadership meeting. investing in health [homepage on the internet]. 2019 executive report [cited 2020 may 19]. available from: https://ahb.co.ke/wp-content/uploads/2019/03/alm-report.pdf abstract introduction methods conclusion acknowledgements references about the author(s) nqobile ndlovu division of chemical pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa african society for laboratory medicine, johannesburg, south africa rajiv t. erasmus division of chemical pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa south african medical research council (samrc) research unit on cardiometabolic health, cape peninsula university of technology, cape town, south africa annalise e. zemlin division of chemical pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa national health laboratory service, tygerberg hospital, cape town, south africa citation ndlovu n, erasmus rt, zemlin ae. narrative review: continuous professional development training programmes in africa and their limitations. afr j lab med. 2025;14(1), a2602. https://doi.org/10.4102/ajlm.v14i1.2602 review article narrative review: continuous professional development training programmes in africa and their limitations nqobile ndlovu, rajiv t. erasmus, annalise e. zemlin received: 02 aug. 2024; accepted: 18 feb. 2025; published: 16 may 2025 copyright: © 2025. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract continuous professional development (cpd) represents a cornerstone in the advancement of professional skills and knowledge across various sectors. it is globally recognised as a transformative process that unlocks potential, increases capacity, and fosters personal growth. this narrative review article aimed to understand how the cpd training programmes for laboratory professionals are implemented and sustained in africa. a narrative review was conducted where a comprehensive search was conducted across pubmed, embase, and web searches for white and/or grey literature, facilitated by a custom python script. a combination of keywords, truncations, and subject headings targeted four key themes: continuing professional development (and related terms), laboratory professionals, african countries, and aspects of implementation and scoring. the search was restricted to articles in english published from 2009 to 2024. while the actual training needs and gaps for cpd programmes are widely known, the actual implementation of cpd has remained a challenge. in the past, cpd training programmes have been implemented to address the lack of skills and the insufficient and skewed distribution of these health workers. this approach is not sustainable and has led to some challenges with coordination, quality assurance, and regulation. each country has its unique context and training needs; therefore, cpd needs to be more coordinated and tailored so that professionals are given the right training for their needs. what this study adds: addressing training gaps for laboratory professionals in africa will require a well-structured, coordinated and tailored approach that will deliver a continent-wide cpd programme. keywords: continuing professional development; laboratory professionals; training; african countries; health professional boards. introduction medical laboratory professionals play a key role in the detection, diagnosis, and monitoring of diseases in today’s healthcare environment.1,2 they are the backbone of quality diagnostics, providing essential services that range from routine testing to specialised analyses critical for patient care.3,4 their work informs clinical decisions, guides treatment, and contributes to disease surveillance and control by allowing correct diagnosis, proper management of patients, and also by improving public health outcomes.1,5 despite this, the african continent continues to grapple with an inadequately skilled and competent laboratory workforce.3,6,7,8,9 with the growing need for a competent work force, health professional boards around the globe are increasingly demanding that practitioners demonstrate their participation and commitment to continuous professional development (cpd). it will, therefore, be important that opportunities for cpd be made available and relevant to the cadre of healthcare professionals.10,11,12 basic education and training alone are no longer enough, as knowledge is rapidly evolving and changing.13 it is common practice, in many countries, that laboratory professionals need to be registered and licensed to practise – although implementation has varied across the african continent. registration and licensure should be structured in a manner that includes new knowledge acquisition, skills and competence improvement given the ever-changing landscape of medical practice and technology. to promote and encourage the culture of learning, developed countries have adopted new strategies where remuneration increases and career growth for medical laboratory professionals are linked to participation in cpd programmes.14 understanding the role of continuous professional development programmes continuous professional development represents a cornerstone in the advancement of professional skills and knowledge across various sectors, and is globally recognised as a transformative process that unlocks potential, increases capacity and fosters personal growth.15,16,17 it provides an important opportunity for progressing and developing one’s career. health professionals will need to continuously advance their knowledge, learn new competencies, and build on existing ones.18,19,20 the main purpose of cpd is to promote better patient care and satisfaction by ensuring that health professionals are knowledgeable and empowered with latest skills.21,22,23 in some african countries, there is a lack of opportunities and structures that will ensure that learning for medical laboratory professionals is happening routinely and continuously.10,24,25,26 the implementation of cpd in africa varies, with some regions developing robust policies and frameworks, while others are still in the nascent stages of policy development.27,28,29 in some places where these programmes exist, they are fragmented, neither curated nor accredited, and eventually no recognition is provided to learners to contribute towards their cpd.30 for many laboratory practitioners, the only training events available for cpd are the ad hoc peer-to-peer trainings organised by employers.10 to compound the situation, there is little to no monitoring or evaluation of cpd implementation to understand what works and what does not. as such, both policymakers and those responsible for resource allocation are left with limited evidence to support scale-up of cpd programmes. in europe, for example, the cpd programme is well structured, and this has been demonstrated by a cpd programme established among the 13 members of european federation of clinical chemistry and laboratory medicine, with cpd credits being used for renewing the practising licence.31 one of the success factors for the implementation of cpd is that it should be driven by laboratory professionals themselves, employers, and training institutions, and should be well organised by national professional institutions, who are able to deliver well-structured cpd activities.24,31 countries have different levels of institutional support for promoting and encouraging human resource development through cpd programmes, and studies have shown that there is a low participation of the concerned laboratory professionals in the existing cpd programmes.19,32 in addition to the reduced access to courses, there is also a lack of confidence in the quality of the training being conducted by many stakeholders.19 continuous professional development programmes that are not well designed to address identified training needs and current programmes often result in low motivation for cpd.30 studies have highlighted the qualitative benefits of cpd, emphasising its role in improving service delivery and patient outcomes, particularly in the fields of hiv and tuberculosis, which are of high priority in sub-saharan africa.30,33 however, there is limited published information on the implementation of cpd programmes in africa. additionally, because of limited mechanisms and infrastructure to maintain professional registrations, certification, licensure, and re-licensure, most of the laboratory workforce is not obliged or motivated to acquire or maintain relevant skills and competencies. the need for cpd in africa is increasingly becoming critical, considering the threats that new microbes pose, as well as the rapidly evolving technologies being developed in response. few national cpd providers that provide training for medical laboratory professionals exist across the african continent.24,33 as countries in africa set up their own cpd programmes, harmonisation of these programmes across countries will be desirable and necessary to facilitate movement of laboratory professionals within the region as has been done in the european union.34 this should also extend to in-country situations, where standardisation of cpd requirements and credit systems is done across all implementing in-country partners and regulatory bodies. countries should make every effort to provide clear guidance to users and all providers of cpd and make sure that these guidelines for cpd development, implementation, monitoring and evaluation are clear.1 continuous professional development practice in africa continuous professional development implementation for laboratory professionals in africa presents a complex and varied picture. for instance, for the majority of african nations, the cpd activities predominantly fall under the umbrella of professional organisations. this includes a diverse array of entities such as medical associations, academies, professional bodies, government organisations, academic and training institutions, which are responsible for initiating, conducting, and promoting cpd programmes.35 the credit point system is integral to this structure, serving as a quantifiable measure of the time invested in cpd, thereby facilitating monitoring and record-keeping.36 however, a prevalent challenge is the potential reduction of cpd to a mere collection of attendance certificates, which may not necessarily reflect a genuine engagement with the content or an actual interest in the subject matter. such a practice risks engendering a misleading sense of security regarding the true impact and outcomes of cpd efforts.30 it is imperative that cpd initiatives not only provide educational opportunities but also inspire real interest and relevance to the professionals’ practice to ensure meaningful professional growth. research questions this article aims at understanding how cpd training programmes for laboratory professionals are implemented and sustained in africa. specifically, it describes which african countries have mandatory cpd programmes and how they are implemented. we further explore the requirements that are essential for the effective deployment of cpd programmes in africa. lastly, we review what facilitates and hinders the implementation of cpd in africa. methods this study employed a narrative review where a comprehensive search was conducted across pubmed, embase, and web-based grey literature repositories, utilising a custom python script to automate query execution across multiple databases.37,38 the review aimed to evaluate the effectiveness, challenges, and implementation of cpd training programmes in africa. the search strategy included boolean operators and medical subject headings terms were tailored for each database to maximise relevant results, and it included: (‘continuous professional development’ or ‘cpd’ or ‘medical education’ or ‘professional training’). it also included ‘africa’ or ‘sub-saharan africa’ or ‘lowand middle-income countries’ or ‘training effectiveness’ or ‘barriers’ or ‘evaluation’ or ‘capacity building’. the inclusion criteria involved all peer-reviewed or grey literature published from 2009 to 2024. studies evaluating cpd models, implementation, impact, or challenges within african healthcare systems were included. these were both quantitative and qualitative evaluations of cpd programmes. the following were excluded: commentary articles, non-evaluative literature, and articles not in english. the process involved title/abstract screening, followed by full-text review. data extracted included study characteristics, cpd intervention types, training duration, effectiveness outcomes, and barriers identified. experiences of continuous professional development programmes in africa mandatory continuous professional development programmes and their implementation in africa countries in africa are at different levels of development for institutional support of promoting and encouraging human resource development through a cpd programme. while the actual training needs and gaps for cpd programmes are widely known – based on a few country surveys, the actual implementation of cpd has remained a challenge. for example, the full implementation of the policy for cpd in nigeria that was established in 2012 is not yet well known. in nigeria, it is noted that its implementation has been affected by lack of awareness by institutions and individual laboratory professionals themselves.26 concerning training needs, an assessment of cpd needs in nigeria showed that training on quality management systems was a priority. a study on laboratory professions in botswana observed that the participants wanted cpd to focus on quality management systems, technical and practical cases, skills assessment and customer care.10 a study in ghana also identified quality management systems as a priority focus for cpd programmes.27 these studies found that holding workshops, conducting practicals, and face-to-face presentations were the preferred mode of conducting cpd. little to no reference in the studies was made about which institutions should be responsible for delivering the cpd programmes in botswana.10 to address this training need on quality management systems, a continent-wide cpd training programme called stepwise laboratory quality improvement process towards accreditation (slipta) was launched by the world health organization’s regional office for africa and other partners in 2011.2 the aim of the slipta programme was to build quality systems for medical laboratories to ensure laboratories produce timely, reliable and accurate laboratory results.39 as of december 2023, over 500 african laboratories had been audited and awarded star ratings for the stage of their laboratory quality (using a 0to 5-star award system) according to the slipta programme.40 this programme has tremendously increased laboratory professionals’ knowledge and expertise in implementing quality management on the continent. another programme that was launched at the same time by world health organization’s regional office for africa and partners to address quality issues was the strengthening laboratory management toward accreditation (slmta) programme. the slmta is a laboratory training programme with practical activities designed to equip laboratory professionals with skills and competencies for establishing and building quality management systems in laboratories that are working towards general quality improvement and/or international accreditation. the difference between the two programmes is that slipta is a tool for auditing laboratories while slmta is a training and mentorship toolkit.39,41,.42,43,44,45,46 regional trainings on slmta were conducted and, in the process, created a grassroots movement on quality, as laboratory staff felt competent and empowered to improve their laboratory processes. despite the implementation of the slipta and slmta programmes, challenges with achieving accreditation have persisted. a study in 2015 in ethiopia and another study in rwanda done in 2018 showed that inadequate skilled laboratory personnel in ethiopia and lack of funding in rwanda were found to be challenges in attaining accreditation and sustaining laboratory quality management systems.47,48 laboratory accreditation and quality management systems are not usually covered during pre-service training.49 laboratory professionals will therefore need ongoing cpd courses on laboratory management and quality management systems to overcome challenges with the accreditation journey. in one case in rwanda, out of a total complement of 45 staff, only four staff members had cpd training on quality management systems.48 availability and access to ongoing cpd training that supports laboratory professionals to improve quality systems towards accreditation will therefore be critical. experiences in malawi, tanzania and south africa noted that non-governmental organisations and other partners continue to coordinate, provide, track and monitor cpd activities.30 however, this has led to some challenges with overall coordination and harmonisation of cpd activities being conducted by many different stakeholders. most of the donor-funded cpd activities are usually fragmented and delivered to address disease-specific agendas that are designed around the goals and funding cycles of donors.30 in addition, these become a template across many countries and do not solve country-specific training needs.30 there is need for close involvement of countries, especially the recipients of cpd programmes, so that these trainings can feed into the overall capacity building targets and training goals. many african countries continue to struggle with the implementation of cpd training programmes. according to the 2015–2020 health sector transformation plan in ethiopia, the federal ministry of health reported challenges in the implementation and actual realisation of the cpd programme in the country.50 this shortcoming on human resource capacity development affected all the health workers.50 also, there have not been efforts to ensure that cpd activities are used for re-licensure of health professionals. in 2018, the federal ministry of health took some policy decisions to ensure that the cpd programme gained traction.50 unfortunately, the gap between the policy and cpd implementation remains. the same challenges observed in the other countries, such as lack of standardisation, and regulation of cpd programmes, continue to hinder progress in ethiopia.1,19 in ghana, a study was done to describe training needs of medical laboratory professionals; however, no institutions or programmes were identified to deliver these trainings.27 essential requirements for effective deployment of continuous professional development programmes in africa according to several studies, limitations on utilisation of cpd range from time constraints, travel distance and perception of its importance.10,27,28 in addition, the format of cpd delivery has recently come into question. lectures or seminars have been used for a long time as delivery methods for training and, thereafter, awarding of cpd points. however, cpd trainings are now being delivered in other ways, such as in-house lectures, short workshops, safety sessions, quality assurance programmes, and journal clubs.10 it has been shown that the delivery style of cpd is strongly linked to improvement of competence. for example, the educational formats that are engaging to participants (e.g. case discussion, role-play, hands-on practice sessions) can produce the desired learning outcomes as compared to didactic and formal instruction.10 the advent of online resources and learning opportunities have resulted in many professionals being able to conduct training in the comfort of their homes or in their offices. over the last decade, the pace at which technology is changing in terms of patient management and diagnosis has led to professionals leaning towards online resources to be able to stay abreast. the online cpd programmes are considered to be cost effective as compared to being physically present at seminars or workshops.51 this has gradually changed the learning behaviour, especially as laboratory practitioners get more comfortable with digital platforms.51 clinicians in sub-saharan africa have been shown to prefer online courses as compared to workplace or external locations – citing the convenience it brings for busy professionals.29 slow and limited internet access, however, will remain a huge challenge to creating online cpd opportunities for most african countries. as such, a blended approach that utilises both virtual and in-person cpd trainings will remain important so as to fulfil all learning needs and preferences. laboratory professionals are most likely to benefit from cpd programmes that allow them to return to their workplace and practise what they have learnt.30 most importantly, cpd should be accompanied by equipment and resources to enable professionals to put what they learnt into practice. this is not usually the case, as seen in a study among nurses in malawi, tanzania and south africa, where apparent lack of test kits, microscopes and other equipment severely limited the ability to put cpd into practice.30 additionally, this may result in discouraging laboratory professionals from participating in cpd programmes, as they will not see the value of such trainings. where such trainings are happening, they are becoming theoretical exercises and just a fulfilment of trainings arranged without input from beneficiaries. such scenarios demonstrate a lack of consideration for local priorities or individual needs. the picture is, however, different for cpd topics that are donor driven, as these will have facility-based resources that are programme specific. funding for such cpd is readily available and inadvertently has excluded most professionals who may not be involved in these programmes. currently, large public health programmes such as for tuberculosis and hiv are driving most of the cpd programmes on the continent.30 countries will have to adapt and learn to leverage these resources to advance other cpd training needs. factors that hinder implementation of continuous professional development programmes many professional regulatory institutions in developing countries are now pushing for proper regulation and enforcement of cpd requirements for re-licensing.10 however, the efforts to have these requirements actually implemented often varies from country to country. for example, in uganda, botswana and south africa, health professional boards will not register professionals without a certain number of cpd credits.10 in other african countries, these requirements for re-licensing are not yet mandatory and therefore many laboratory professionals do not need to attend cpd trainings nor are they required to document any cpd trainings they do attend.10 in 2021, the african society for laboratory medicine (aslm), through a cpd project called ‘sustaining antimicrobial resistance (amr) surveillance qualification framework’ conducted a landscape analysis on available cpd providers in 14 african countries.52 this allowed insights into the current status of workforce regulation, competency requirements and the associated cpd across the 14 african countries (table 1). it is important to note that although 12 of the countries have documented guidelines on cpd requirements for laboratory professionals, the implementation of these requirements is still a work in progress, as most countries are still developing platforms to deliver accredited cpd programmes. without funding and technical support, the countries will not be able to implement these cpd programmes. table 1: regulatory councils and professional associations involved in continuous professional development in 14 african countries, october 2021. this cpd project on the qualification framework aimed to equip the amr surveillance workforce (microbiologists and epidemiologists) with the knowledge and skills to be able to implement key functions of the national amr surveillance system and activities under the ‘one health’ approach across 14 priority african countries and three asian countries.52 to explore options for continuity and sustainability of the cpd qualifying the workforce for amr surveillance in africa and asia (qwars) qualification framework post-project implementation, aslm engaged the country cpd stakeholders, including those involved in training of laboratory and epidemiology professionals in the priority countries.52 all the countries (14/14) indicated that they will recognise the cpd qwars professional qualification. of these, 6/14 (43%) were likely to also recognise the university of witwatersrand cpd mechanism associated with the course, while 8/14 (57%) have more advanced implementation of national cpd programmes and are more likely to review, adopt and adapt the qwars curriculum and assign their own cpd points.52 by adopting the cpd qwars professional qualification and establishing roles for the qwars qualified trainees and master trainers, it is envisaged that the participating countries will access cpd of their amr surveillance workforce. with a pool of qualified personnel, the countries will be better equipped to implement their national action plans for amr surveillance to address the increasing threat from amr at national, regional and global levels.52 professional qualification and experience play a role in cpd attendance. in many developing countries, experiences show that differences in basic qualifications will also present unequal availability and opportunities to cpd programmes for laboratory professionals.31 it is usually the most qualified and those with many years of experience that get these training opportunities.30 the newly recruited professionals, even though in most need of cpd training, are usually left out of these opportunities; supervisors attend most cpd training as they happen to get the invitations and have the deciding power on who attends.30 this vicious cycle continues as in most institutions there are no training or capacity-building plans that have been developed and followed through.30 in other instances, the same individuals are repeatedly given the opportunity to attend cpd programmes, thereby demotivating others in the process.30 other barriers for laboratory professionals interested in participating in cpd include workplace locations that are remote and rural. unfortunately, these remote locations have the majority of the laboratory personnel who could benefit from the cpd programmes. health workers who are normally posted in rural and often neglected places face challenges related to travel distance, as well as a lack of communication and updates on upcoming cpd training opportunities.19 in some settings, laboratory professionals cannot take time to attend cpd training largely because there are only a few staff available to remain working in their absence.24 this situation therefore leads to inconsistent participation in cpd programmes.24,33 organising cpd training workshops where participants are required to come to a central site or a particular country has been the norm for a while in many settings. this approach of travelling individuals is, however, proving to be expensive and not sustainable. as such there is limited training that can be delivered and may not be sufficient in addressing the training gaps. the extensive travelling logistics, especially for participants attending from different countries, adds to the challenges of in-person cpd training programmes.24 it is therefore imperative that administrators of cpd programmes consider alternative and effective approaches to delivering cpd training at low costs. the current training packages designed and offered to increase the knowledge and skills of the recipients continue to face challenges with uptake. instead of learning and skills development being owned and driven by the professionals themselves, learning is currently happening through a prescribed type of approach to training (top-down approach) and this has limited effects.18 decentralising cpd programmes to increase their reach in africa is another approach that can be promoted. workplace cpd training opportunities could be adopted for rural and remote settings to improve opportunities – reducing barriers of distance and time.53 however, experiences from ethiopia indicate that there is hesitancy to support the decentralised provision of cpd training programmes. most laboratory professionals are uncomfortable with decentralising the cpd training programmes as they believe this will lead to poor quality and not achieve the intended goals of the programme. they also fear the possibility of enhancing illegal certification with a decentralised approach.19 hence, many cpd programmes remain centralised and coordinated by established institutions. to scale up training on the continent and create a critical pool of well-trained laboratory professionals with the aim of improving laboratory quality, several critical factors need to be addressed in any cpd approach. addressing these factors will require recognition that individuals prefer an environment of continuous education that has a flexible format, relevant content and is delivered at a convenient time, as participants have many other commitments. they prefer to drive their own learning, especially that which is applicable to their life and work environment. lastly, cpd programmes will work well when the credit system is familiar and understood by the professionals. this is because participants can calculate the cpd points they can earn and, as a result, are able to chart their learning journey to achieving the requirements on credit units.54 as cpd training programmes become more and more available, laboratory professionals will need to avoid attending trainings that are just a fulfilment of a requirement. first and foremost, a clear evaluation and understanding of the actual training needs will be important. secondly, defining what trainings could be done internally and what can be outsourced or conducted externally to the workplace will be critical in establishing sustainable cpd programmes that are accessible in all locations.55 there are few studies that have defined and documented specific cpd training needs in africa. study results in botswana and ethiopia indicate that the preferred training areas for attention include technical, laboratory management, and leadership skills.10,28 this is not surprising, given the ever-changing technology and testing needs that were experienced because of emerging and reemerging diseases such as coronavirus disease 2019. the testing needs for the 21st century have become more complex with the increasing disease burden on the continent. another topic identified is laboratory quality management systems.28 this interest in quality management systems has been created because of the scale-up of accreditation programmes on the african continent. the recognition and pride that comes with working in an accredited laboratory has spurred the demand for such trainings. national laboratory quality programmes have been initiated across the continent and many of these have been heavily funded by donors; hence, the increased interest and trainings. interestingly, in ghana, it was observed that laboratory professionals from the private sector preferred topics on quality management systems while those from public health facilities prioritised topics on technical areas.27 it is important to note that many cpd training opportunities have focused on the public sector professionals while ignoring private sector health workers.28 this can be attributed to the predominant nature of donor funding that is geared towards the public sector. scaling cpd training programmes in africa will require an all-inclusive approach, as the private sector also serves a huge population in africa. another important aspect has been whether cpd can improve the professional’s working environment. studies have observed that job satisfaction and retention are increased when well-designed cpd programmes are made available to professionals.56 without increased opportunities for cpd, laboratory professionals may become demotivated and experience restricted career growth.56 laboratory professionals who have been upskilled, and therefore have certain opportunities because of additional competencies, are more likely to be happy and remain in their jobs. currently on the continent, health workers struggle to find the right cpd programmes for their career growth. this is compounded by a lack of sufficient data, experience and guidance on how to design and implement relevant and strong cpd programmes.57 as already noted, one of the persistent barriers for many cpd programmes on the continent is funding. the scale, quality, effectiveness and reach of these cpd programmes will hinge on the availability of resources. on one hand, you have individual laboratory professionals without funding and resources to attend cpd opportunities. on the other hand, institutions are struggling to support, sponsor and meet some of these cpd training goals.30 mulvey adds a third component that is critical in the implementation and funding of the cpd programme – the employer.17 in what the author calls the cpd ‘triad’, namely the individual, the institution and the employer, the author argues that the employer is important, as the cpd programme costs money and time off work.17 the employer will have to understand that an up-to-date, well-trained and efficient employee will increase productivity and confidence in the product or service being provided. it is therefore worthwhile for employers to invest in cpd.17 unfortunately, the prevailing status is that most of the cpd programmes in africa have been funded by donors, local and international non-governmental organisations, with less support coming from governments.30 in ethiopia, this has been attributed to lack of commitment from upper-level management in supporting cpd trainings, citing cost issues.19 in a survey in nigeria, almost 90% of the interviewed participants indicated lack of funding to attend cpd training programmes and, as result, there was low attendance for cpd training.26 therefore, lack of sustainable financial resources is a huge barrier to cpd planning, implementation, evaluation and continuity.30 a new cpd programme launched under the aslm academy is envisioned to play a pivotal role in restructuring cpd in africa by addressing the implementation challenges.58 the aslm academy is expected to implement a continent-wide standardised scoring system that recognises diverse learning activities and accommodates the varied needs of professionals. the aslm academy will create and distribute comprehensive learning materials that are accessible even in low-resource settings, providing a mix of online and face-to-face training options to cater to different learning preferences and connectivity issues. conclusion in summary, implementation of cpd programmes in africa remains a challenge. linking cpd training to licensure and implementing mandatory cpd programmes will be critical in developing a well-skilled and up-to-date laboratory workforce. acknowledgements this article will partially contribute to the author’s thesis entitled ‘strategies for laboratory continuous professional development in africa region’ towards a phd in the department of pathology, division of chemical pathology, with supervisors annalise e. zemlin and rajiv t. erasmus. competing interests the primary author, n.n., is the chief executive officer of the african society for laboratory medicine, and this manuscript will contribute to a phd study. r.t.e. declares that he is an editor for the african journal of laboratory medicine. the peer review process for this submission was handled independently, and the authors had no involvement in the editorial decision-making process for this manuscript. the authors have no other competing interests to declare. authors’ contributions n.n. was the principal investigator and was responsible for the conceptualisation, methodology development, data collection and review of articles, and writing the original manuscript. r.t.e. contributed to the conceptualisation and methodology approach and was also involved in the critical review and approved the final version to be published. a.e.z. also contributed to the conceptualisation and methodology approach, and was involved in the critical review and approved the final version to be published. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the authors confirm that the data supporting the findings of this study are available within the article. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. the article does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. 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references about the author(s) tarsizio chikaonda department of molecular medicine and haematology, faculty of health sciences, school of pathology, university of the witwatersrand, johannesburg, south africa unc project, lilongwe, malawi irene ketseoglou department of molecular medicine and haematology, faculty of health sciences, school of pathology, university of the witwatersrand, johannesburg, south africa nelson nguluwe unc project, lilongwe, malawi robert krysiak unc project, lilongwe, malawi isaac thengolose unc project, lilongwe, malawi felix nyakwawa malawi national tuberculosis programme, lilongwe, malawi nora e. rosenberg unc project, lilongwe, malawi christopher stanley unc project, lilongwe, malawi james mpunga malawi national tuberculosis programme, lilongwe, malawi irving f. hoffman unc project, lilongwe, malawi university of north carolina at chapel hill, chapel hill, north carolina, united states maria a. papathanasopoulos department of molecular medicine and haematology, faculty of health sciences, school of pathology, university of the witwatersrand, johannesburg, south africa mina hosseinipour unc project, lilongwe, malawi university of north carolina at chapel hill, chapel hill, north carolina, united states lesley scott department of molecular medicine and haematology, faculty of health sciences, school of pathology, university of the witwatersrand, johannesburg, south africa wendy stevens department of molecular medicine and haematology, faculty of health sciences, school of pathology, university of the witwatersrand, johannesburg, south africa citation chikaonda t, ketseoglou i, nguluwe n, et al. molecular characterisation of rifampicin-resistant mycobacterium tuberculosis strains from malawi. afr j lab med. 2017;6(2), a463. https://doi.org/10.4102/ajlm.v6i2.463 original research molecular characterisation of rifampicin-resistant mycobacterium tuberculosis strains from malawi tarsizio chikaonda, irene ketseoglou, nelson nguluwe, robert krysiak, isaac thengolose, felix nyakwawa, nora e. rosenberg, christopher stanley, james mpunga, irving f. hoffman, maria a. papathanasopoulos, mina hosseinipour, lesley scott, wendy stevens received: 08 apr. 2016; accepted: 05 oct. 2016; published: 31 mar. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: availability and access to the detection of resistance to anti-tuberculosis drugs remains a significant challenge in malawi due to limited diagnostic services. the xpert® mtb/rif can detect mycobacterium tuberculosis and resistance to rifampicin in a single, rapid assay. rifampicin-resistant m. tuberculosis has not been well studied in malawi. objectives: we aimed to determine mutations in the rifampicin resistance determining region (rrdr) of the rpob gene of m. tuberculosis strains which were defined as resistant to rifampicin by the xpert mtb/rif assay. methods: rifampicin-resistant isolates from 43 adult patients (≥ 18 years) from various districts of malawi were characterised for mutations in the rrdr (codons 507–533) of the rpob gene by dna sequencing. results: mutations were found in 37/43 (86%) of the resistant isolates in codons 511, 512, 513, 516, 522, 526 and 531. the most common mutations were in codons 526 (38%), 531 (29.7%) and 516 (16.2%). mutations were not found in 6/43 (14%) of the resistant isolates. no novel rpob mutations other than those previously described were found among the rifampicin-resistant m. tuberculosis complex strains. conclusion: this study is the first to characterise rifampicin resistance in malawi. the chain-termination dna sequencing employed in this study is a standard method for the determination of nucleotide sequences and can be used to confirm rifampicin resistance obtained using other assays, including the xpert mtb/rif. further molecular cluster analysis, such as spoligotyping and dna finger printing, is still required to determine transmission dynamics and the epidemiological link of the mutated strains. introduction tuberculosis remains an important public health problem especially in the developing world. the global impact of tuberculosis is significant, with an annual estimate of 9.6 million tuberculosis cases and over 1.5 million deaths due to tuberculosis in 2014.1 the tuberculosis burden is worsened by the emergence and spread of multi-drug resistant (mdr) tuberculosis cases, defined as simultaneous resistance to at least rifampicin and isoniazid, with or without resistance to any other drug.1 in malawi, there were 5564 new smear-positive cases of tuberculosis registered in 2014.1 patients diagnosed with tuberculosis are treated with the standard quadruple antibiotic therapy recommended for drug-susceptible tuberculosis (rifampicin, isoniazid, ethambutol and pyrazinamide). rapid detection of drug resistance is crucial in choosing the most effective treatment to avert morbidity and mortality of infected individuals and reduce the risk of mdr tuberculosis transmission.1 rifampicin, if the isolate is susceptible, is a very important component of the current tuberculosis treatment regimen and has proved to be effective to both susceptible strains and strains resistant to streptomycin and isoniazid.1 however, there is growing resistance to rifampicin, largely due to particular genomic mutations in the rpob gene of mycobacterium tuberculosis.2 the rpob gene encodes the β subunit of rna polymerase, which is involved in chain initiation and elongation. a signature sequence for m. tuberculosis identification is contained in this region.3,4 mutations in the rifampicin resistance determining region (rrdr) of this gene (codons 507–533) are associated with rifampicin resistance.5 detection of such mutations indicates rifampicin-resistant tuberculosis strains and can be used as a predictor for mdr tuberculosis, although not a complete surrogate marker.6 globally, it is estimated that 3.3% of new cases and 20% of previously-treated cases have mdr tuberculosis and that 9.7% of these cases have pre-extensively drug resistant tuberculosis.1 a substantial percentage (37%) of new tuberculosis cases and a staggering percentage (74%) of the global estimate of mdr tuberculosis incident cases were not reported or remained undiagnosed in 2014. determination of the pattern of drug resistance is performed in less than 3% of people diagnosed with tuberculosis worldwide.1,7,8 phenotypic drug susceptibility testing (dst) relies on detection of growth and is performed using an antibiotic susceptibility testing set consisting of a growth control and one tube for each anti-tuberculosis test drug, with known concentration.9 phenotypic dst is widely used in limited-resource settings. this method is inexpensive and accurate, though time consuming due to its reliance on the growth of m. tuberculosis, which takes a long time to obtain results.10 mutation(s) in the genes relevant to responses to each drug are associated with resistance to tuberculosis drugs.10 genotypic dst methods target well-characterised resistance-associated mutations. genotypic dst determines such mutations in the tested gene region only and as such, unknown or less frequent mutations might be missed.11,12 molecular tools for rapid dst are developed upon a better understanding of mutations responsible for drug resistance in the bacterial genome.13,14 several molecular methods, including the xpert® mtb/rif (cepheid, sunnyvale, california, united states), have been developed for the detection of m. tuberculosis complex dna and rrdr mutations associated with rifampicin resistance.15,16,17 the world health organization strongly recommends the use of xpert mtb/rif as the initial diagnostic test for use on pulmonary specimens from adults and children suspected of having hiv-associated tuberculosis or mdr tuberculosis.1 the use of dna sequencing complements the above assay in detecting new mutations, as well as confirming the presence of the most frequent mutations that could be associated with drug resistance, and has conferred excellent benefits to patient care due to the larger dna fragment that is sequenced.18,19 dna sequencing determines mutations by comparing the differences between the gold standard (h37rv reference strain) and the test nucleotide sequence.20,21 we aimed to assess the rrdr of the rpob gene for mutations in m. tuberculosis strains that were defined as rifampicin resistant by the xpert mtb/rif assay and to establish the prevalence of such mutations in tuberculosis-infected malawians. methods ethical considerations approvals for this study were granted by university of the witwatersrand human research ethics committee (m120256), national health sciences research committee (nhsrc) in malawi (nhsrc # 999) and the university of north carolina (unc; chapel hill) institutional review board (cid 1211). obtaining consent was waived because the study used previously-stored and residual sputum pellets, and consent from patients was given during recruitment and/or this was part of routine testing for tuberculosis drug resistance. study population we conducted this study using processed sputum sediments (pellets) from new and previously-treated patients, ≥ 18 years of age. demographic and clinical information was collected from the respective laboratory tuberculosis registers and entered in excel (microsoft, inc., redmond, washington, united states) spreadsheets. both retrospective and prospective pellets (n = 995) were used in the current study. retrospective pellets retrospective sputum pellets (n = 351) were collected between april 2011 and july 2012 from a study conducted in outpatients initiating tuberculosis treatment at martin preuss centre at bwaila hospital (bwaila) in lilongwe, which was looking at the prevalence of drug-resistant tuberculosis at this hiv/tuberculosis clinic.22 sputum samples were processed for routine culture at the unc project laboratory following the n-acetyl-l-cysteine and sodium hydroxide (nalc-naoh) method. pellets were inoculated on both mycobacterium growth indicator tubes (mgit) and löwenstein jensen media. dst was performed using the hain mtbdrplus assay (hain lifescience gmbh, nehren, germany). residual pellets were stored at -80°c and selected at random for use in this study without any special criteria to eliminate bias. prospective pellets prospective sputum pellets came from patients suspected of drug-resistant tuberculosis (n = 644) who presented consecutively to district hospitals in malawi, and sputum samples were sent to the national tuberculosis reference laboratory (ntrl) in lilongwe for conventional dst between june 2012 and may 2014. sputum was processed using the nalc-naoh method and the pellet was split in two. the first sample was used for routine culture and conventional dst at the ntrl, while the other was stored for up to two weeks at 2°c – 8°c for xpert mtb/rif testing and mgit culture at the unc laboratory. mycobacterium cultures at the unc laboratory pellets were re-suspended in 1.5 ml of phosphate buffer and split into different volumes. one part was processed for routine culture using mgit and the other was processed on xpert mtb/rif (0.5 ml). mgit tubes were inoculated with 500 µl of the re-suspended sample as previously described23 and were monitored daily for growth using a hand-held bactec micromgit reader for up to 42 days. smears were prepared from positive cultures, stained using ziehl-neelsen stain (becton, dickinson & company, sparks, maryland, united states) and examined for acid-fast bacilli. determination of eligibility for dna sequencing was dependent on results from the xpert mtb/rif assay (software version g4). dna was extracted from corresponding mgit cultures if rifampicin resistance was detected by xpert mtb/rif. xpert mtb/rif assay samples (n = 995) were processed as per the manufacturer’s instructions. a sample reagent buffer was added to 500 µl of the re-suspended pellet in the ratio of 3:1 (sample reagent buffer:specimen) as described previously.24 the container was closed tightly and then vigorously shaken for 15 seconds. the mixed specimen was left to stand for 10 minutes followed by another vigorous shaking for 15 seconds and left to stand for a further five minutes. thereafter, 2 ml of the mixed specimen was loaded into a single-use xpert mtb/rif cartridge. the closed cartridge was loaded into the genexpert® instrument, where extraction, amplification and detection of m. tuberculosis and screening for resistance to rifampicin were performed automatically and simultaneously. results were available within two hours. the xpert mtb/rif probe and cycle threshold (ct) value was documented for each tuberculosis strain that was detected as resistant to rifampicin. dna extraction and amplification genomic bacterial dna was extracted from cultures at the unc laboratory using the hain genolyse kit (hain lifescience gmbh, nehren, germany). from mgit liquid media, 1.0 ml was transferred to a sarstedt micro-centrifuge tube. the tube was centrifuged for 15 minutes at 14 000 rpm. the supernatant was carefully discarded and the pellet was re-suspended in 100 µl lysis buffer from the kit, vortexed thoroughly and incubated at 95°c in a heating block for five minutes. tubes were removed and briefly centrifuged to remove condensation. to each tube, 100 µl of neutralization buffer was added, vortexed for five seconds and then centrifuged for five minutes at 14 000 rpm. supernatant was transferred to a new tube and the pellet was discarded. the extracted dna was stored at -80°c for use as dna template. mtbdrplus line probe assay the genotype®mtbdrplus line probe assay (version 2) was performed according to the manufacturer’s instructions. in brief, an appropriate number of pcr tubes, one for each sample and one each for a pcr positive (m. tuberculosis dna) and negative (dh2o) control were labelled. a master mix was prepared for one reaction and the final volume adjusted according to the total number of samples, with some excess to allow for pipetting errors. the solution was mixed by inversion and an aliquot of 45 µl of the prepared master mix was transferred to each pcr tube. 5 µl of each sample or control dna was added to the appropriate pcr tube. pcr was performed in a 9700 dna thermocycler (applied biosystems, foster city, california, united states), using the cycling protocol as described by the manufacturer. dna pcr pcr amplification of the rpob gene, which included the rrdr, was carried out using forward primer rpobf2 (5’-gag ggt cag acc acg atg ac-3’; nucleotide positions 1030 to 1049 according to h37rv numbering, genbank accession number cab09390.1) and reverse primer rpobr (5’-gag ccg atc aga ccg atg t-3’; nucleotide positions 1460 to 1478 according to h37rv numbering) in a geneamp pcr system 9700 thermocycler (applied biosystems, foster city, california, united states). the total volume of the pcr reaction used was 50 µl, containing: 5 µl of 10x high fidelity buffer, 1 µl of 10 mm dntp mix, 2 µl of 50 mm mgso4, 1 µl of each primer, 0.2 µl of taq hifi (invitrogen, carlsbad, california, united states), 36.8 µl of molecular grade water and 3 µl of extracted genomic dna. amplification conditions were set at: 94°c for two minutes; followed by 35 cycles of 94°c for 30 seconds, 55°c for 30 seconds, 68°c for 40 seconds; followed by a 10-minute final elongation at 68°c. pcr products were purified using the genejet pcr purification kit (thermo scientific, waltham, massachusetts, united states) following the manufacturer’s instructions. 5 µl of the amplified product was visualised on a 1% agarose gel. dna sequencing dna sequencing was performed in an automated dna sequencer abi 3700, (applied biosystems, foster city, california, united states) using the bigdye terminator v3.1 sequencing kit with forward primer rpobs (5’-gca gac gtt gat caa cat cc-3’) and reverse primer rpobr (5’-gag ccg atc aga ccg atg t-3’). the total volume of the sequencing reaction was 20 µl, containing: 4 µl of bigdye terminator, 2 µl of 10x sequencing buffer, 2 µl of water, 1 µl of primer and 11 µl of sample. amplification conditions were set for: one minute at 96°c; followed by 25 cycles of 96°c for 10 seconds, 50°c for five seconds, 60°c for four minutes; followed by a 10-minute final extension at 72°c. the completed sequencing reaction mixture was purified using 75% isopropanol as per the manufacturer’s instructions. the sample plate was loaded onto the abi 3700 automated dna sequencer and the resulting sequences were analysed and compared to the wild-type sequence of the well-characterised m. tuberculosis h37rv reference strain using sequencher software v4.8 (genecodes corporation, ann arbor, michigan, united states). the well-defined rrdr region (codons 507–533) corresponded to codons 426–452 (nucleotides 1276–1356) in the downloaded h37rv reference sequence. statistical analysis all study data were entered into an excel spreadsheet and then exported and analysed using stata version 12 (statacorp, college station, texas, united states). we calculated proportions and percentages based on district, patient category, xpert mtb/rif probes and codons in the rrdr of the rpob gene to determine resistance mutations and their prevalence. results the 644 specimens from ntrl were categorised clinically as previously treated cases (574/644; 89.1%), new (27/644; 4.2%), or not known (43/644; 6.7%). from bwaila, 24/351 (6.8%) suspected tuberculosis cases were categorised as retreatment, and 327/351 (93.2%) were from patients presenting with tuberculosis for the first time. the xpert mtb/rif assay detected rifampicin resistance in 64/995 (6.4%) specimens, which were selected for further dna sequencing of the larger region of the rpob gene. three specimens (3/64) were from bwaila, while the rest (61/64) were collected from ntrl. most of the rifampicin-resistant cases detected by xpert mtb/rif were associated with probe b (23/64) and probe e (23/64). fifteen (15/64) samples were detected by probe d, 2/64 by probe a, while probe c detected 1/64 samples (table 1). of these, 43/64 were successfully sequenced. the remainder (n= 21; 32.8%), all from ntrl, were not sequenced, either due to failure of pcr amplification (n = 19) or they failed sequencing (n = 2) (figure 1). the pcr amplification failure could not be explained, although in part this could be due to insufficient template dna, a an attempt was made to extract dna from pellets (n = 7) which had not grown on culture. figure 1: workflow and isolate selection for dna sequencing from tuberculosis cultures and processed sediments. table 1: rpob gene mutations associated with xpert mtb/rif probes used. seven different mutations were detected in 37/43 (86%) specimens in codons 511, 512, 513, 516, 522, 526 and 531. mutations were common in codons 526 (38%), 531 (29.7%) and 516 (16.2%) (table 2). no insertion, deletion or double point mutations were observed. however, mutations were not detected in 6/43 (14%) of the strains by dna sequencing, despite being detected by xpert mtb/rif probe b (4/6) and by probe e (2/6) as rifampicin resistant. repeat xpert mtb/rif testing was not done for these strains due to insufficient left-over pellets. delayed ct values between 17.0 and 32.7 were observed in 4/6 strains which had no mutations. the observed ct values were markedly higher than the δct max cutoff of > 4 for the automated detection of rifampicin resistance by the xpert mtb/rif assay.25 the remainder (n = 2) gave an undetectable result on probe b (n = 1) and probe e (n = 1). table 2: distribution of mutations in codons 491–574 of the rpob gene and amino acid changes in the rifampicin resistance determining region, malawi, 2011 and 2014†. when stratified by district, the prevalence of mutations was high in tuberculosis patients from ntchisi 2/5 (40%), nkhotakota 4/13 (30.8%), nsanje 5/20 (25%) and balaka 2/12 (16.7%). lilongwe, the capital city of malawi, had the lowest prevalence of mutations 9/466 (1.9%), followed by mulanje 2/57 (3.5%). as expected, the number of mutations was highest among patients registered as retreatment 29/598 (4.8%), compared with new cases 8/354 (2.3%). there were no real trends of mutations across classified retreatment cases (recurrent, default, etc.) in relation to new tuberculosis cases. mutations in codon 531 were found in almost all patient groups, except for those classified as recurrent; codon 526 in all groups, except in the ‘new patient’ category in which mutations at codon 516 were predominant. a single strain from a patient with no history of prior tuberculosis treatment was detected with a mutation at codon 522. all 43 specimens that were successfully sequenced were also tested on genotype mtbdrplus and two strains gave discordant results. both strains showed susceptibility to rifampicin on genotype mtbdrplus, but resistance to rifampicin on xpert mtb/rif. mutations were not detected in these strains by dna sequencing. discussion this study is the first to characterise rifampicin resistance in malawi. the majority of rpob gene mutations in this study analysed by direct sequencing correlated well with rifampicin resistance observed on xpert mtb/rif. our findings reveal that 86% of all rifampicin-resistant isolates harboured mutations in the rrdr of the rpob gene with codon 526 (cac → tac) as the most frequent, followed by codon 531 (tcg → ttg/tag). by contrast, mutations were not detected in 6/43 (14%) of the strains following nucleotide sequencing. false-positive rifampicin resistance was most likely considering the observed wild-type sequences in these strains. data on drug-resistance mutations involving the rrdr of the rpob gene, which relates highly to rifampicin resistance, is new for malawi as no previous studies have analysed the rpob gene among tuberculosis strains circulating in the country. previous reports demonstrated that strains requiring high rifampicin concentrations in phenotypic dst have been associated with mutations at codons 526, 516, and 531,26,27 corroborating that these are the most prevalent rpob mutations worldwide.28 several studies of rifampicin-resistant tuberculosis have reported the presence of novel and common rpob gene mutations, especially in the rrdr.29,30,31 in addition, other studies have documented the presence of common and novel rpob mutations outside the rrdr.29,32 based on our amplicon size, we only evaluated the most frequently-mutated codons between positions 491–574, which include the rrdr. the contribution of additional mutations to resistance at positions outside the region that was sequenced, for example codon 176, cannot be ruled out. future work should focus on sequencing the entire rpob gene. it is of particular interest that the xpert mtb/rif assay detected rifampicin resistance in six isolates, but partial sequencing of the rpob gene did not detect any changes in the nucleotide sequences, such that the mechanisms/pathways of resistance of these strains remain unknown. since unprocessed sputum was not sequenced, it was difficult to confidently exclude hetero-resistance in these strains. in low rifampicin-resistance prevalence areas, the xpert mtb/rif assay would be expected to falsely diagnose some cases,8 which could be the case with these strains. sequencing the entire rpob gene might help to understand the mechanism of resistance of the strains. in contrast, results obtained in swaziland show that the xpert mtb/rif assay did not detect the rpob i491f mutation (outbreak strain) in 38/125 (30%) of the isolates tested as compared to dna sequencing, raising fears about the assay’s unreliability due to under-diagnosis and potential treatment inadequacy, since it does not detect mutations outside the rrdr.33 furthermore, a study by theron et al. observed that five strains were rifampicin resistant on xpert mtb/rif, rpob sequencing and/or genotype mtbdrplus, but susceptible on phenotypic dst.34 it should be noted, however, that pcr amplicons were different in the xpert mtb/rif assay versus what was used for the sanger-based sequencing. the probes may have bound to minority variants in the xpert mtb/rif amplicons, resulting in a positive signal, whereas a combination of pcr bias and population-based sanger sequencing (where the limit of detection is approximately 20% of the quasispecies and only the predominant population is reported) might have resulted in only the wild-type sequence being detected.35 moreover, different fitness of drug-resistant tuberculosis under culture conditions can allow for the outgrowth of wild-type tuberculosis,36 accounting for the discordant results between the molecular and phenotypic assays. drug resistance was detected in some new tuberculosis cases and these results were confirmed by detecting mutations in the rrdr of the rpob gene. detecting resistance to rifampicin in new tuberculosis cases highlights the need for testing resistance to drugs in this category especially if the patient was in contact with a known mdr tuberculosis patient. our results show that the prevalence of mutations associated with rifampicin resistance among new tuberculosis cases was 2.3%. in malawi, the prevalence of mdr tuberculosis is low (0.4%) among new smear-positive cases and is 4.8% among retreatment cases. testing for tuberculosis drug resistance in malawi is not routinely done except for those highly suspected of drug resistance.37 results obtained by the xpert mtb/rif assay were in agreement with genotype mtbdrplus results on rifampicin resistance, with an exception of 2/43 (4.7%), which were sensitive on the genotype mtbdrplus but rifampicin resistant on xpert mtb/rif. ct values of 18.5 and 24.4 on xpert mtb/rif, both on probe b, were interpreted as rifampicin resistant. mixed infection with multiple tuberculosis strains was excluded in these strains given the wild-type rpob gene sequences and no observed underlying peaks on the dna sequence chromatograms. phenotypic dst is difficult to standardise and expensive and time-consuming to maintain routinely. in the absence of molecular techniques, several critical questions about tuberculosis remain unanswered, including recognition of acquisition of drug resistance resulting from gene mutations versus transmission of drug resistant strains.38 limitations the relatively small number of rifampicin-resistant samples observed in the current study limited our ability to establish the overall prevalence of rpob gene mutations in tuberculosis strains circulating in malawi. moreover, we sequenced only an approximately 450 bp amplicon; thus, the contribution of mutations in regions outside of the amplicon sequenced to overall resistance could not be determined. with the available data, an assessment of the epidemiological link between the rifampicin-resistant strains was not possible, as such further molecular cluster analysis is still required to determine transmission dynamics of the mutated strains. future studies should consider performing phenotypic dst to detect mutations outside the rrdr (full length rpob gene) and compare to dna sequencing, considering that sanger sequencing has a limit of detection where the minority population may not be detected. conclusion the chain termination dna sequencing employed in this study is a standard method for the determination of nucleotide sequences. it is conclusive and can be used to confirm rifampicin resistance obtained by other assays, including the xpert mtb/rif assay, despite having a limit of detection with minority resistant populations. although phenotypic dst is recommended to verify rifampicin resistance when using xpert mtb/rif, dna sequencing can be used to detect the frequency and exact site of mutations in the rrdr. all tuberculosis strains with mutations in the current study had one of the previously-described rpob gene mutations containing nucleotide changes. no new mutations were identified. findings from this study emphasise the need for a national representation of sputum specimens from tuberculosis-infected patients to assess the magnitude of tuberculosis rpob gene mutations that are responsible for rifampicin resistance in malawi. acknowledgements the authors thank the unc project laboratory staff, national tuberculosis reference laboratory (malawi) and laboratory staff at the hiv genotyping laboratory at university of the witwatersrand for assisting with the study. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support this project was funded by the national commission for science and technology (malawi) through the health research capacity strengthening initiative (hrcsi). additional funds were made available by the unc project (malawi), unc fogarty aitrp (d43 tw001039), unc centre for aids research (p30 ai50410) and malawi hiv implementation research scientist program (d43 tw010060). authors’ contributions t.c. conceptualised the study. t.c., i.k., n.n. and i.t. performed the experiments. m.h., l.s., w.s., f.n., r.k., m.a.p., j.m. and i.f.h. oversaw the operational aspects of the study. t.c., n.e.r. and c.s. conducted the data analysis and t.c. drafted the manuscript. all authors revised the manuscript and approved the final draft. references world health organization. global tuberculosis report 2015. geneva, switzerland: who; c2015 [cited 2016 jan 27]. available from: http://apps.who.int/iris/bitstream/10665/191102/1/9789241565059_eng.pdf heep m, brandstätter b, rieger u, et al. frequency of rpob mutations inside and outside the cluster 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identifies compensatory mutations in rna polymerase. nat genet. 2011;44(1):106–110. https://doi.org/10.1038/ng.1038 mariam dh, mengistu y, hoffner se, et al. effect of rpob mutations conferring rifampin resistance on fitness of mycobacterium tuberculosis. antimicrob agents chemother. 2004;48(4):1289–1294. malawi national tuberculosis programme. national tuberculosis control programme manual. 7th ed. lilongwe, malawi: ministry of health; 2012. mathema b, kurepina ne, fallows d, et al. lessons from molecular epidemiology and comparative genomics. respir crit care med. 2008;29(5):467–480. https://doi.org/10.1055/s-0028-1085699 article information authors: amase nyamngee1 luke d. edungbola1 olajide o. agbede1 alakija k. salami2 charles nwabuisi1 aliu a. akanbi ii1 olatunde o. k. ibrahim3 muchae tilahun4 douglas b. moser5 affiliations: 1department of medical microbiology and parasitology, university of ilorin, nigeria 2department of medicine, university of ilorin, nigeria 3department of pathology, university of ilorin, nigeria 4ethiopian health and nutrition research institute, ethiopia 5division of global hiv/aids, u.s centers for disease control and prevention, usa correspondence to: amase nyamngee postal address: university of ilorin, pmb 1515, ilorin, nigeria dates: received: 07 may 2012 accepted: 09 apr. 2013 published: 26 nov. 2013 how to cite this article: nyamngee a, edungbola ld, agbede oo, et al. prevalence, intensity and complications of microsporidium spores amongst hiv patients in ilorin, nigeria. afr j lab med. 2013;2(1), art. #66, 6 pages. http://dx.doi.org/10.4102/ ajlm.v2i1.66 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. prevalence, intensity and complications of microsporidium spores amongst hiv-positive hospital patients in ilorin, nigeria in this original research... open access • abstract • introduction • research method and design    • description of the study area    • study design    • sampling method    • sample collection and processing    • determination of t-cell count    • data analysis • results • ethical considerations    • ethical clearance    • informed consent    • data protection • trustworthiness • discussion • limitations of the study    • recommendations • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: microsporidiasis, which is of great concern for immunocompromised patients, is poorly studied in developing countries. objectives: a study was carried out amongst hiv-positive hospital patients and hiv-negative hospital controls in ilorin, nigeria, between january 2009 and july 2010 to determine the prevalence and intensity of microsporidium spores and the complications associated with their presence. method: stool samples from 750 hiv-positive patients and 375 hiv-negative patients were studied using the chromotrope-2r staining technique. determination of cd4+ count was performed on the partec cyflow sl-3 cd4/8 instrument. intensity of spores was determined by counting the total number of the spores in a 10 µl stained smear of stool. images were captured with phenix microimage analysis software and data obtained were analysed using the statistical package for the social sciences. results: the prevalence of microsporidium isolates amongst the hiv-positive hospital patients was significantly higher (42.4%) than amongst the hiv-negative controls (19.2%) (p < 0.05). the intensity of microsporidial spores amongst hiv-positive hospital patients was also significantly higher than amongst the controls (p < 0.05). however, the difference in the intensity of spores amongst hiv-positive patients who were on antiretroviral therapy (n = 411) and those who were not (n = 339) was not significant (p = 0.236). microsporidiasis in hiv infection infection was common amongst patients with with low cd4+ counts, diarrhoea, body rashes and cough. conclusion: both the prevalence and intensity of microsporidiasis are high amongst hiv-positive hospital patients; campaigns to promote awareness, prevention and control are required. laboratory testing for microsporidia in hiv patients should be performed routinely so as to identify the organism for prompt medical attention. introduction top ↑ microsporidiasis is recognised by the world health organization as being a globally-important medical, public health and socio-economic problem.1,2 microsporidia are obligate and opportunistic intracellular spore-forming parasites which belong to the phylum protozoa.3,4,5although there are more than 1000 known microsporidium species and as many as 100 genera of the group affecting both vertebrates and invertebrates,6 there are only 12 species, belonging to 8 genera, that are known to be pathogenic to humans.7,8the first well-documented human case of microsporidiasis, where encephalitozoon spp. was detected in a stool sample of a diarrhoea patient, was reported in 1959.9 since then, microsporidia have become increasingly better recognised as opportunistic human pathogens,10,11,12 particularly in patients with suppressed immunity, including organ transplant patients and hiv patients.5,13,14 microsporidia can also infect people with competent immune systems, resulting in latent infections.6,15,16 in hiv patients, microsporidia have been implicated in intestinal, ocular, pulmonary and renal diseases, and have been shown to cause diarrhea and other adverse health conditions.17,18,19 chronic diarrhoea is common in hiv-positive patients but the enteric pathogens are identified in fewer than 50% of cases.5,7,8,20,21,22 the medical and public health implications of chronic diarrhoea in hiv-positive patients are problematic, especially with regard to the management of hiv with antiretroviral therapies.23,24 with the detection of more cases of chronic diarrhoea in hiv-positive patients,4,12,25,26enterocytozoonbieneusi (a species of microsporidium) is increasingly suspected to be an aetiological agent, and has also implicated in the excessive morbidity and mortality.18,26,27,28,29 in portugal, ferreira et al.30 reported a 41.2% prevalence of microsporidial diarrhoea in hiv patients, calling for better management of diarrhoea in these patients. there is a paucity of information on microsporidia, particularly in developing countries where no concerted effort has been made to ascertain the prevalence, intensity, mode of transmission and tissue pathology caused by microsporidiasis, especially amongst hiv patients. in nigeria, there is no documented information on human microsporidia and microsporidiasis. the objective of this study was to determine the prevalence and intensity of microsporidium spores in hiv-positive hospital patients in ilorin, nigeria, and to determine the association of microsporidiasis with other medical conditions amongst these patients. research method and design top ↑ description of the study area this work was carried out at the university of ilorin teaching hospital, a tertiary and referral hospital located in ilorin, kwara state, nigeria. study design a cross-sectional, hospital-based study was designed and implemented so as to determine which pathogenic microsporidia could be detected in both hiv-positive and -negative patients attending the university of ilorin teaching hospital between january 2009 and july 2010. a sample size of 1125 was calculated using fisher’s formula and based on an estimated 47% prevalence of diarrhoea amongst hiv-positive patients.5,31 the sample comprised 750 hiv-positive and 375 hiv-negative patients, matched 2-to-1 for age, sex and other socio-economic variables. informed consent was obtained from participants before being enrolled for this study. a structured questionnaire was administered to each patient to collect demographic variables and arv status, as well as information about other concomitant infections. the patients completed the questionnaire before the samples were collected. sampling method simple random-sampling by number randomisation was used to select hiv-positive patients for this study. hiv status of patients was determined based on rapid hiv testing kits. hiv-negative controls were selected by number randomisation amongst all possible matches. sample collection and processing all participants were screened for hiv-status prior to stool collection. the screening and the collection of the stool samples were done by the researcher and two trained laboratory assistants. three screening methods were used concurrently to test and confirm hiv-sero-status: (1) antec hiv test (antec international ltd, u.k.), (2) determine hiv 1/2 test (inverness medical, beijing, china) and (3) uni-gold hiv test (trinity biotech, ireland). confirmation of hiv positivity occurred only when all three tests were reactive for hiv antibodies and confirmation for hiv negativity was when all three tests showed no reaction. for the purposes of this study, patients who showed reactions on one or two of the three test kits were retested, but were excluded if the results remained discordant.stool specimens were collected in sterile universal bottles from all patients. for the identification of microsporidium spores, unconcentrated formalin-fixed stools were stained using the chromotrope2r staining method.5,32 each stained smear was examined by light microscope under an oil immersion lens.5 the microsporidium photomicrographs and measurement of the spore size were taken according to the manufacturer’s instruction, using phenix microimage analysis software (pmias 3.3), microscope model me 100. a positive control slide from the division of parasitic diseases of the centers for disease control and prevention (cdc), atlanta, georgia, reference no. un3373, was compared against the positive samples in this study. in addition, three positive slides were sent to the cdc in atlanta for confirmation. the intensity of the spores in the stool samples (figure 1) was determined by counting the total number of spores in a 10 µl stained smear of stool on a glass slide.1 counting and authentication of the spores was carried out manually by the lead investigator and three independent co-researchers after blinding on hiv sero-status. the average of the 4 results was taken to be the mean intensity of the spores per 10 µl of stool. figure 1: images of microsporidium spores in stool samples from our study. determination of t-cell count the cd4+ count was determined using a cd4/8 cyflow machine (partec cyflow sl-3, germany) according to the manufacturer’s instructions. a 3 ml sample of blood in an ethylenediaminetetraacetic acid (edta) tube was inserted into the machine and the cd4/cd8 count of each individual sample was determined. data analysis the statistical package for the social sciences (spss version 16.00) was used for data anlysis. prevalence of infection was given in percentages in line with the variables. chi-square and 95% confidence interval analysis were the statistical tools used to determine significance at a cut-off value of 95% (p = 0.05). results top ↑ we enrolled 750 (66.7%) hiv-positive and 375 (33.3%) hiv-negative patients. the prevalence of microsporidium infection amongst the hiv-positive patients was 42.4%, which was significantly higher than the corresponding 19.2% prevalence in the hiv-negative patients (p < 0.05; table 1a). amongst hiv-positive patients, prevalence of microsporidiasis was significantly lower amongst men than amongst women (39.6% vs. 44.9%, p < 0.05). differences in prevalence between men and women amongst hiv-negative patients (19.9% vs. 18.6%) and amongst all patients (33.1% vs. 36.1%) did not reach statistical significance (p > 0.05) (table 1b). the prevalence amongst hiv-positive patients who had been on arvs for between 3–18 months was similar to that amongst patients not on arvs (67.6% vs. 68.2%, p >0.05) (table 2). all hiv-positive patients aged 42–61 who were not on arvs had microsporidiasis; their counterparts who were on arvs also had a high prevalence. the prevalence of microsporidiasis increased with age amongst both groups. the mean intensity of microsporidium spores per 10 µl of stool amongst the hiv-positive patients who were on arvs showed no significant difference when compared with those who were not on arvs (table 3). the 52–61 age group demonstrated the highest intensity of microsporidium spores (mean±standard deviation: 277±94.0) amongst hiv-positive patients who were on arvs. patients not on arvs had the highest intensity of spores (234 ± 88.0) in the 32–41 age group. the difference in intensity amongst those patients on arvs and those not on arvs in relation to age was not statistically significant (p > 0.05). in both groups, intensity of microsporidium spores was inversely associated with cd4+ count; age groups with low cd4+ had high intensity of microsporidium (table 3). amongst hiv-positive patients, those with diarrhoea had the highest prevalence of microsporidiasis amongst all those with a single condition (95.6%) (table 4). this was followed closely by those with body rashes (91.7%). amongst those with multiple conditions, those with diarrhoea and body rashes (with and without cough) had 100% prevalence of microsporadiasis. table 1a: prevalence of microsporidiasis by age amongst hiv-positive and -negative patients (n = 1125). table 1b: prevalence of microsporidiasis amongst hiv-positive and -negative patients by gender (n = 1125). table 2: prevalence of microsporidiasis amongst hiv-positive patients by age and arv status (n = 750). table 3: intensity of microsporidium spores in relation to age, cd4+ count and antiretroviral therapy status amongst hiv-positive patients (n = 750). table 4: association of microsporidiasis with other disease conditions in hiv-positive cases. ethical considerations top ↑ ethical clearance ethical clearance was sought and obtained from the ethical review committee of the university of ilorin teaching hospital with reference number, uith/cat/189/817. informed consent the purpose of this study was explained in detail to all subjects during the study. participation was voluntary and the confidentiality of information given for the purpose of this study was guaranteed.all subjects were also apprised of the benefits of knowing their hiv status, cd4+ counts, and medical complications. data protection all specimen collected were preserved according to international standards and kept under iced hydrogen refrigeration in the data storage chamber of the university of ilorin teaching hospital. trustworthiness top ↑ independent researchers confirmed and counted the spores to address the issue of credibility and the average of the four counts was recorded. we also obtained a control slide of the organism from the cdc in order to ensure that the organism was identified correctly. to address the issues of dependability and confirmability, we relied on an average independent count and the control slide.  discussion top ↑ our findings revealed a high prevalence (42.4%) and significant intensity of microsporidiasis amongst the hiv-positive patients, thus complicating morbidity and mortality in areas where hiv is common. the infection was detected across genders and age groups indicating that microsporidia are ubiquitous as suggested previously.6,16 whilst prevalence increased with age amongst the hiv-positive patients, there was was not a clear trend amongst the hiv-negative controls. we observed that the intensity of spores was highest amongst the hiv-positive patients, especially in the very old and in those with very low (< 10) cd4+ counts, suggesting a relationship with the level of cd4+ counts in hiv-positive patients19. previous studies have reported a high intensity of microsporidia in hiv-positive patients due to their low immune status, which created a condition for the rapid replication of microsporidium spores12.amongst the hiv-positive patients, it was observed that 95.6% of those reporting with diarrhoea were positive for microsporidium, which may implicate microsporidia as an aetiologic agent in the diarrhoea that is seen in these immunocompromised patients. this is in line with previous suggestions concerning the need for more investigations of the aetiologic agent of this diarrhoea amongst hiv-postive patients.5,18 a 100% prevalence of microsporidium spores was seen amongst hiv-positive patients who had both diarrhoea and body rashes (with or without cough). other conditions associated with microsporidium were body rashes only (91.7%), diarrhoea with cough (77%) and body rashes with cough (70.0%), stimulating interest about the involvement of microsporidiasis in other complications amongst hiv patients. this is contrast with the findings of nkinin et al.16who reported an unexpectedly high prevalence (67.5%) of microsporidiasis in 126 immunocompetent patients in cameroon in 2007, but found no clinical manifestations in these individuals. presently, there is no effective treatment for treating microsporidiasis which has made control difficult, even in areas where there is knowledge regarding microsporidium infections in humans.23,24 limitations of the study top ↑ this study is subject to several limitations. first, measuring the intensity of spores in stool samples is somewhat subjective and variable; to reduce these issues, we had 4 experts independently count spores and averaged the results. second, the study was hospital-based, limiting our inference to the general population. third, irregular power supply may have adversely affected some of our samples; this was reduced through the provision of an alternative power supply. recommendations we recommend that routine laboratory screening be performed for microsporidia in the hospital setting and that positive isolates be made notifiable. more studies on the aetiology of microsporidiasis (under lightand electron microscopy) should be undertaken, which should include histopathology study of different organs at autopsy. conclusion top ↑ we found a high prevalence and intensity of microsporidia amongst hiv-positive patients, especially amongst those with diarrhoea, body rashes and/or cough. these findings should provide base-line information for further studies and provoke interest in investigations of microsporidia that aim at determining the mode of infection, pathogenicity and treatment as well as prevention and control. health education campaigns to promote awareness, prevention and control of microsporidiasis should be conducted regularly, particularly amongst hiv-positive patients as well as medical, public health and laboratory personnel. positive results for microsporidia obtained through routine laboratory screening in hospitals should be notifiable. finally, based on the prevalence and implications of microsporidial infection as observed in this study, there is a need for the development of an ideal drug for the treatment of microsporidiasis. acknowledgements top ↑ i would like to acknowledge the help received from the division of parasitic diseases of the cdc, atlanta, usa, for providing the control slide of microsporidium species which was essential for the success of this study. i also appreciate the support of mr tunde odewale, the chief laboratory scientist of the department of medical microbiology and parasitology, university of ilorin, for his laboratory assistance in the preparation of permanent slides. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. authors’ contributions a.n. (university of ilorin) designed the study, conducted the research work, collected the samples, analysed the data, wrote the draft and submitted the final manuscript. l.d.e. (university of ilorin) supervised the entire work from the beginning and was involved fully in both the identification of the organisms and the write-up. o.o.a. (university of ilorin) supervised the study design and implementation. a.k.s. (university of ilorin) participated in the sample collection and identification of the complications, as well as contributing to the draft.c.n. (university of ilorin) participated in the study design, as well as in the sampling, examination and identification of the positive samples. he also edited the write-up. a.a.a. (university of ilorin) participated in the sample collection and analysis, as well as editing the final write-up. o.o.k.i. (university of ilorin) participated in both sample collection and analysis. m.t. (ethiopian health and nutrition research institute) read the draft and made corrections with regard to the laboratory procedures. d.b.m. 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2006;42(1):115–125. http://dx.doi.org/10.1086/498510, pmid:16323101, pmcid:2683841 30. ferreira fm, bezerra l, santos mb, et al. intestinal microsporidiosis: a current infection in hiv-seropositive patients in portugal. microbes infect. 2001;3(12):1015–1019. 31. araoye mo. research methodology with statistics for health and social sciences. nathadet, 2004. isbn 978-36450-8-0. http://dx.doi.org/10.1016/s1286-4579(01)01465-4 32. chioralia g, trammer t, kampen h, et al. relevant criteria for detecting microsporidia in stool specimens. j clin microbiol. 1998;36(8):2279–2283. pmid:9666005 pmcid:105031 abstract introduction methods results discussion acknowledgements references supplementary materials supplementary document 1: search strategy supplementary document 2: analysis template supplementary document 3: code list about the author(s) pascale ondoa department of global health, amsterdam institute for global health and development, academic medical center, university of amsterdam, amsterdam, the netherlands ankie van der broek royal tropical institute, amsterdam, the netherlands christel jansen royal tropical institute, amsterdam, the netherlands hilde de bruijn ministry of education, culture and science, international policy unit, the hague, the netherlands constance schultsz department of global health, amsterdam institute for global health and development, academic medical center, university of amsterdam, amsterdam, the netherlands citation ondoa p, van der broek a, jansen c, et al. national laboratory policies and plans in sub-saharan african countries: gaps and opportunities. afr j lab med. 2017;6(1), a578. https://doi.org/10.4102/ajlm.v6i1.578 original research national laboratory policies and plans in sub-saharan african countries: gaps and opportunities pascale ondoa, ankie van der broek, christel jansen, hilde de bruijn, constance schultsz received: 05 oct. 2016; accepted: 11 apr. 2017; published: 31 july 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the 2008 maputo declaration calls for the development of dedicated national laboratory policies and strategic plans supporting the enhancement of laboratory services in response to the long-lasting relegation of medical laboratory systems in sub-saharan africa. objectives: this study describes the extent to which laboratories are addressed in the national health policies and plans created directly following the 2008 momentum for laboratory strengthening. method: national health policies and plans from 39 sub-saharan african countries, valid throughout and beyond 31 december 2010 were collected in march 2012 and analysed during 2013. results: laboratories were addressed by all countries. human resources were the most addressed topic (38/39) and finances and budget were the least addressed (< 5/39). countries lagging behind in national laboratory strategic planning at the end of 2013 (17/39) were more likely to be francophone countries located in west-central africa (13/17) and have historically low hiv prevalence. the most common gaps anticipated to compromise the implementation of the policies and plans were the disconnect between policies and plans, under-developed finance sections and monitoring and evaluating frameworks, absence of points of reference to define gaps and shortages, and inappropriate governance structure. conclusion: the availability of laboratory policy and plan implementation can be improved by strictly applying a more standardised methodology for policy development, using harmonised norms to set targets for improvement and intensifying the establishment of directorates of laboratory services directly under the authority of ministries of health. horizontal programmes such as the global health security agenda could provide the necessary impulse to take the least advanced countries on board. introduction laboratory services are key to the quality of healthcare but have remained a historically-neglected component of health systems in lowand middle-income countries. the need for quality medical laboratory services to form an integrated part of the health system has been widely acknowledged by key national and international players during the past decade, resulting in the mobilisation of substantial funding earmarked for laboratory improvement in resource-limited settings.1,2 alongside these efforts, the development of a national laboratory policy and strategic plan (nlsp) relevant to each country’s needs, and aligned with its health policy(ies) and plan(s), has also been advocated.3,4,5 a national laboratory policy defines the vision and the mission of a country’s laboratory system, whereas a strategic plan provides the corresponding roadmap guiding the process of the practical implementation of the necessary laboratory system improvement. the coherence of nlsps with other national health guidance documents, such as national health policies, plans for development of human resources for the health sector, or disease-specific policies or plans, increases the likelihood that laboratory development strategies will be implemented. despite the recent prioritisation of laboratory services in global and national health agendas, various aspects of the laboratory system fail to meet standards in several countries of sub-saharan africa. clinical diagnostic capacity often remains insufficient for the control of hiv, malaria, tuberculosis and other infectious diseases and for responding to the rise in incidence of non-communicable diseases.6,7,8 in addition, the lack of laboratory-based surveillance precludes the timely identification of emerging infectious disease threats.9,10 the recent ebola virus disease epidemics11 and the paucity of systematic data on the antimicrobial resistance12,13 of key bacterial pathogens are two dramatic examples illustrating the lack of laboratory capacity to either respond to or anticipate public health events of national or international concern. the persistent shortage of laboratory workers in many countries of sub-saharan africa14 also highlights that crucial needs are still unmet. the primary aim of this study was to describe to what extent laboratory services were addressed in the round of national health policies and strategies created by sub-saharan african countries following the 2008 maputo declaration.5 the profile of countries lagging behind the process of laboratory strategic planning was explored. methods we conducted a desk review of national health policy documents. eligible documents had to be valid throughout and beyond 31 december 2010 and were collected in march 2012. no updated or new policies were added after document collection was closed. the analysis was completed at the end of 2013. selection of countries and documents sub-saharan african countries identified by the world health organization as experiencing a crisis in human resources for health15 or receiving support from the united states president’s emergency plan for aids relief (pepfar) were included in the study,16 which led to a review of 41 countries in total. a search was carried out, per country for the following documents: national health strategy (or policy in case a strategy was not found). national human resources for health policy and/or strategy. national laboratory policy and/or strategy and/or operational plan. national hiv policy and/or strategy. national tuberculosis policy and/or strategy. national malaria policy and/or strategy. searches were carried out on the websites of the ministries of health, thematic websites (such as www.aidstar-one.com and the roll back malaria website http://www.rbm.who.int/countryaction/index.html), google, and within the networks of the fondation mérieux, the association of public health laboratories, the royal tropical institute of the kingdom of the netherlands, and the amsterdam institute for global health and development. details on the search strategies and the web sites visited are provided in supplementary document 1. additional information data on the political, economic and health status of each country were retrieved from the world bank (www.worldbank.org), world health organization17 and unaids18 databases. information on pepfar collaboration, the existence of a department of laboratory services within the ministry of health and the degree of advancement of national laboratory strategic planning were retrieved from the pepfar (www.pepfar.gov) and world health organization databases (http://www.who.int), ministry of health websites and through direct questioning of in-country laboratory stakeholders. document review a data collection form was developed (supplementary document 2) and translated into a list of 88 codes pertaining to various aspects of national laboratory policy and strategic planning (supplementary document 3). the codes were grouped in broader families of codes, representing specific themes, e.g., ‘attention to the public and the private sector’, and according to a set of questions guiding the analysis. e.g., ‘to which aspects of the laboratory do the policy and plans pay attention?’. the coding framework was applied to all the documents available for review. a core team of two members searched the documents, entered the data in the form and discussed and reviewed each other’s work to reduce the risk of differences in interpretation of documents and coding of data. following the review, and whenever necessary, the documents were screened for specific key words to ensure that all relevant sections had been properly coded and to allow description of content insufficiently covered by the coding list. data with assigned meanings in atlas.ti (atlas.ti, version 6.2; atlas.ti scientific software development gmbh, berlin, germany) were transferred to spss (ibm spss statistics, version 20 for windows; ibm corp., armonk, new york, united states). analysis for each country, we evaluated the compliance of policies and plans with guidance documents for laboratory policy and strategic planning,19 and the availability of concurrent and aligned national laboratory policies, strategies and operational plans according to the planning cycle. we examined the degree of harmonisation between policies and plans addressing the laboratory, the overall health sector and the development of human resources for health for each country. countries were categorised into groups of ‘not started’ versus ‘started’ with respect to the development and implementation of their nlsp at the time of the analysis in 2013 (supplementary table 1). these groups of countries were compared for proportions of low-, middleand high-income countries or against their average hiv prevalence using the chi-square or student’s t-test. table 1: cross references between national laboratory, national human resources for health and national health policies or plans. the analysis of the coded data focused on the identification of gaps and opportunities in selected areas of the laboratory system: governance, private sectors, quality and accreditation, human resources, finances and monitoring and evaluation. attention to laboratory services was defined by the number of times that the laboratory was specifically addressed, and was quantified by calculating the frequency of each code or group of codes per document type, country or group of countries. chronological alignment between policies and plans was evaluated by plotting inception and expiry dates of the documents against each other for each country. results in total, 126 documents from 39 countries were found for review (figure 1). figure 1: countries and documents included in the study. availability of aligned national laboratory policies, strategies and operational plans of the 10 countries (25.6%) with a national laboratory policy, strategy, or operational plan available for review, one had developed a combined policy and strategic plan within a single document and nine had developed either a laboratory policy or a laboratory strategic plan. the start and end years of the national laboratory policies, strategies or operational plans; national health policies; and national human resources for health policies or strategies of the 10 countries with a laboratory document available for review are depicted in figure 2. lack of chronological overlap was identified between some policies and plans. in south africa, for instance, the foreseen implementation time frame of the human resources policy lay largely outside the foreseen implementation timeline of the national health policy. in sierra leone, the human resources policy had become obsolete shortly after national laboratory policy implementation started. in rwanda, the timeline of the national policies for laboratory, for human resources, and for health were not aligned. in contrast, the democratic republic of congo had perfectly coordinated the timelines of all three national policies. figure 2: overlap of validity time frames in 10 countries with a laboratory policy or plan available for review. the 10 national laboratory policies were screened for any reference to either the national health policy or the human resources for health plan (table 1). nine of 10 countries referred to their national health policy in their national laboratory plan. six of the 10 national laboratory policy documents did not refer to human resources for health plans. factors associated with advancement of national laboratory policy and strategic planning in sub-saharan africa information gathered through the association of public health laboratories, pepfar, fondation mérieux, the royal tropical institute and the amsterdam institute of global health and development networks, indicated that 17 countries were not yet engaged in initial discussions regarding national laboratory strategic planning and were categorized as ‘not started’. the 23 other countries (of which 10 had a national laboratory policy available for review) were found to be either engaged in discussions, have developed a first draft, or (for the most advanced) have implemented, reviewed and evaluated their nlsp. these countries were classified as ‘started’. an update of the situation in early 2017 indicated that six additional countries are now categorised as ‘started’, making for a total of 29 (supplementary table 1). countries that had not yet started with their nlsp were more likely to be french-speaking, be classified as lower income, or not be receiving pepfar support compared to countries that had already started with the process of strategic planning (supplementary figure 1a and 1b). countries categorised as ‘not started’ were also those with a lower hiv prevalence as compared to countries categorised as ‘started’ (average prevalence = 1.9% versus 9.5%, p = 0.001). there were no differences between the two groups of countries in terms of gross domestic product, percentage of gross domestic product spent on health, health expenses per capita, and percentage of external resources for health (supplementary figure 1c). main laboratory themes addressed in health policies and plans the most common component of laboratory services addressed across various health policies (38/39 countries) was human resources for laboratory, mostly in terms of shortages (32/39) (figure 3). all national laboratory and tuberculosis policies, strategies, and operational plans mentioned the issue of human resources for laboratory as compared with 25% of national health policies, 24% of the human resources for health policies, 19% of hiv policies, and 15% of malaria policies (data not shown). figure 3: attention to laboratory aspects in country policies and plans (n = 39). other main cross-cutting components of laboratory strengthening were mentioned as follows: governance (35/39); organizational structure and management capacity (36/39); laboratory infrastructure, equipment and supplies (36/39); and quality assurance (34/39), all issues were predominantly mentioned in the specific context of hiv/aids (34/39). biosafety was the least addressed cross-cutting component of laboratory services, being overlooked by 21 of the 39 countries (53.8%). coverage of the eight main cross-cutting components of laboratory strengthening19 was achieved in the 10 countries with a national laboratory policy. among the 29 countries without a dedicated laboratory policy or plan, four addressed all core aspects of laboratory strengthening through other type(s) of plans, while the majority overlooked one (n = 15) or several (n = 10) of the eight cross-cutting components concerning medical laboratories. budget allocation and donor support for laboratory services were scarcely covered (figure 3), regardless the type of document studied. the percentage of health budgets earmarked for laboratory services or human resources was mentioned by only two countries. among the 39 countries, donor support was hardly addressed in the context of the organisational structure of laboratories (n = 2), biosafety (n = 2), or management capacity (n = 4), and more frequently in the context of laboratory equipment (n = 10) and supply chain (n = 11). most significant weaknesses identified in policies and plans little attention to financing regardless of the type of plan, very limited attention was devoted to the budget available or needed for the provision of laboratory services or for the implementation of laboratory improvement strategies. evidence for a budget line dedicated to laboratory services was found in two out of the 39 countries. of the 37 national health plans, three reported a specific part of the health budget allocated to support services and one indicated budget allocation for laboratory staff in general. eleven countries provided a budget dedicated to specific strategies to improve laboratory services in their national health plans. all 10 national laboratory policies reported insufficient government funding for laboratory services. at the same time, seven of these did not provide the percentage of the overall government health budget available for laboratory services. an overview of donor contributions was also lacking in all of the documents reviewed. information on costing, budgeting and identification of funding sources for the implementation of the overall as well as specific components of the nlsp were largely missing. funding from external partners was often mentioned in the context of the provision of equipment and supplies, or in relation to specific diseases, but rarely in the area of human resources development. lack of practical framework to monitor and evaluate laboratory services and national laboratory policy implementation no indicators were proposed to measure the performance of laboratory services in any of the national laboratory policies. a monitoring and evaluation section to follow the implementation of the plan was included in four of the seven nlsp (democratic republic of congo, malawi, tanzania and zimbabwe), with only one (democratic republic of congo) providing sufficiently detailed indicators linked to a budget and a chronogram. insufficient attention to the creation of laboratory departments within ministries of health only five countries described streamlining the administration of their laboratory systems through a laboratory directorate falling directly under the authority of the ministry of health, as recommended by the maputo declaration (table 2). the other countries organised their laboratory services through a department responsible for, but not dedicated to, laboratories (n = 13), through a single service provider or research institution (n = 4), or through multiple entities within the ministry of health and provider institutions (n = 7). for 10 countries, no information on the laboratory coordinating body could be found. table 2: type of laboratory governance in the 39 sub-saharan african countries studied. of the seven national laboratory policies of countries with no directorate of laboratories, only two national laboratory policies explicitly aimed at raising the profile of laboratory services through one department within the ministry of health. nineteen of the 21 national health policies (90.5%) addressing laboratory governance were from countries that did not yet have a directorate of laboratory services under the ministry of health. of these, only two proposed the creation of a directorate of laboratories within the ministry of health as a way to improve the governance of laboratory services. poorly-informed situation analysis of the private laboratory sector private laboratories were addressed by 28 of 39 countries (71%) and by all national laboratory policies (data not shown). five national laboratory policies described private laboratories as being separate from the public laboratory network. six national laboratory policies, two human resources for health plans, one hiv policy and one malaria policy from eight of the 39 countries (84%) provided some information on the number of facilities operating in the private sector or the percentage of the population they serve or the laboratory staff they employ. the governance of the private sector and private sector links with the national laboratory network were left largely unclear in most of the policies and plans reviewed. only three national laboratory policies provided some indication that the private sector was functioning as part of the overall laboratory network. the list of participating stakeholders and summaries of meeting proceedings attached to the policies and plans suggested that the private laboratory sector and economic actors susceptible to support the implementation of the plan were only marginally involved in the development of the laboratory documents. insufficient points of reference for establishing workforce shortage reduction strategies twenty-six countries reported laboratory workforce shortages in their national health policies and/or human resources for health policies, four mentioned overstaffing and nine did not explicitly discuss shortages in the laboratory workforce. among the countries indicating shortages, six did not provide any information on the category of laboratory workers concerned, 10 did not specify a point of reference to define the shortage and 20 did not have any information on the attrition rate of the laboratory workforce (supplementary table 2). three countries defined shortages against positions actually available (vacancies), three against current or projected workload, and 11 against national norms based on tier-specific staffing requirements. national laboratory policies generally included more aspects of human resources for laboratory in their plans as compared to any other type of policy and plan (supplementary figure 2). however, eight of 10 nlp did not provide data on the categories of staff affected nor on points of reference to define the shortages. clear strategies, targets and funding sources to improve the availability, capacity and performance of the workforce were not specified. vision and plan for laboratory accreditation not referred to in national laboratory policies and plans nine of the 10 national laboratory policies planned to use laboratory accreditation as a way to promote quality laboratory services, in line with the recommendations of the maputo declaration. however, roadmaps explaining how countries intend to move toward accreditation across diseases, at different tiers of the laboratory system and given the resources available were rarely provided or referred to (box 1). ethiopia was an exception, detailing an initial achievement of disease-specific international accreditation of polio and hiv reference laboratories, followed by a progressive expansion to other diseases and down to the regional level. certification of laboratories based on compliance to national standards was never mentioned as a strategy to ensure the quality of laboratory services at lower tiers. box 1: strategies for laboratory accreditation described in the 10 national laboratory policies and plans. discussion the attention devoted to medical laboratories in general, and to human resources in particular, in the round of national health policies and strategic plans that appeared shortly after the international momentum for the strengthening of laboratory services underscores the robust uptake of the recommendations of the maputo declaration in the majority of sub-saharan african countries. however, 17 countries (43%) were still lagging behind the process at the time of the analysis in 2013. strikingly, countries with less advanced laboratory policies and plans were mainly located in west and central francophone africa with a history of low hiv prevalence and limited pepfar investment. combined with the observation that most laboratory issues were addressed in the context of hiv/aids, this finding illustrates the key role that international, vertical, disease-specific programmes, such as pepfar and the global fund, have played in prioritising and funding the development of laboratory systems in the region. at the same time, the report suggests that areas with smaller hiv epidemics benefited less from available opportunities to advance their national laboratory policy and strategic planning. more equitable access to technical and financial resources to advance laboratory systems through adequate policy and planning could be achieved by diagonalising vertical and horizontal programmes in such a way that disease-wide, multi-sectoral and whole-of-government programmes for health improvement include the improvement of laboratory systems for the control of specific diseases. the global health security agenda goal to accelerate the achievement of international health regulations targets could offer a momentum for this paradigm shift, by incorporating laboratory-specific targets in the effort to achieve global health security.20 this report identifies several inadequacies which should be addressed in the upcoming round of nlsp to increase the likelihood of implementation of laboratory policies and plans and support the achievement of national and global health targets. recommendations application of standardised methodologies consistently apply existing standardised methodologies for the development of national laboratory policies. this would ensure that no policy is formulated without a strategic plan or vice versa and that national laboratory policies are adequately aligned with other related health policies and integrate all key elements necessary for implementation. in addition, a (self-applied) national laboratory system assessment using available standardised tools, for example, the world health organization laboratory assessment tool (lat)-system assessment,21 laboratory network (labnet) scorecard,22 and a strengths, weaknesses, opportunities, threats (swot) analysis of laboratory cross-cutting areas by a large inter-sectoral group of laboratory stakeholders conducted prior to the policy formulation, could facilitate the adequate prioritisation of strategic areas for improvements. such a strictly standardised approach is ongoing for the development of national laboratory policies in eastern europe and central asia, under the ‘better lab for better health initiative’23 of the world health organization’s regional office for europe, with promising results. harmonised national norms and standards define and disseminate harmonised national norms and standards for various aspects of laboratories as a priority for international partners in laboratory development. regional bodies, such as the african society for laboratory medicine or the world health organization’s regional office for africa, could facilitate the development or the sharing and adjustment of national norms and standards available (e.g., staffing norms based on anticipated workload defined in botswana; norms for infrastructure and equipment) from countries in similar settings. international technical partners could coordinate the development of generic costing tools for key items such as the implementation of quality management systems or laboratory information management systems or sample referral systems. renewal of advocacy efforts renew advocacy efforts for the creation of a directorate of laboratory services directly under the authority of the ministry of health, as recommended by the first call of the maputo declaration. with the correct mandate, a directorate can leverage improvements in several cross-cutting areas of laboratory systems such as development and enforcement of regulations, legislation (including the private sector), and development and application of normative standards (e.g., staffing, quality, infrastructure). the directorate would delineate important roadmaps such as national laboratory accreditation plans with linkage to laboratory-dedicated budget lines. regional bodies and international development partners can significantly contribute to advocacy efforts, as illustrated by the resaolab (réseau d’afrique de l’ouest des laboratoires) programme from fondation mérieux, which promoted the creation of laboratory directorates in burkina faso and senegal.24 conclusions the conclusions and recommendations stated here are mainly based on the analysis of documents that were available for review. the authors acknowledge the possibility that the outcome of the review could have been different, if all national policies had been available for the analysis. additionally, the documents available from public sources may have been short versions that lacked budget sections. finally, the present situation of national laboratory policy and strategic planning is different from that in 2010, with west and central francophone african countries such as mali, burkina faso, burundi and the republic of congo currently implementing their first nlsp. in addition, six countries on the continent are implementing their second round of nlsp. moving forward, the lessons reported here can be taken on board when countries develop or revise their laboratory policies, or initiate the next round of nlsp. acknowledgements the authors thank ralph timperi, tsehaynesh messele and trevor peter for their input to the design of the research question and the interpretation of the results. we are indebted to julien schijver and merel lammer for their contribution to the data analysis. we thank jean-louis abena, esther gathinji and cleophas malaba for sharing documents. we thank john nkengasong for his critical review of the manuscript. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support this research was supported by the african society for laboratory medicine and the african field epidemiology network. authors’ contributions p.o. and a.v.d.b. conceived and designed the research question and the analysis framework. p.o., a.v.d.b., c.j. and h.d.b. collected the documents and analysed the data. p.o., a.v.d.b., c.j. and c.s. contributed to the interpretation of the results. p.o. drafted the manuscript. a.v.d.b. and c.s. critically reviewed the manuscript. references piot p, kazatchkine m, dybul m, et al. aids: lessons learnt and myths dispelled. lancet. 2009;374(9685):260–263 [erratum in 2009;374(9690):608]. https://doi.org/10.1016/s0140-6736(09)60321-4 the world bank. cross-country laboratory network to raise east africa’s defenses against disease. washington, dc: world bank; 2010. nkengasong jn, mesele t, orloff s, et al. critical role of developing national strategic plans as a guide to strengthen laboratory health systems in resource-poor settings. am j clin pathol. 2009;131(6):852–857. https://doi.org/10.1309/ajcpc51blobbpakc nkengasong jn, nsubuga p, nwanyanwu o, et al. laboratory systems and services are critical in global health: time to end the neglect? am j clin pathol. 2010;134(3):368–373. https://doi.org/10.1309/ajcpmpsinq9brmu6 world health organization. the maputo declaration on strengthening of laboratory systems. geneva, switzerland: who; 2008. world health organization. global tuberculosis report 2015. geneva, switzerland: who; 2015. joint united programme of hiv/aids. 90-90-90 – an ambitious treatment target to help end the aids epidemic. geneva, switzerland: unaids; 2014. mcnerney r. diagnostics in developing countries. diagnostics. 2015;5(2):200–209. https://doi.org/10.3390/diagnostics5020200 tambo e, ugwu ec, ngogang jy. need of surveillance response systems to combat ebola outbreaks and other emerging infectious diseases in african countries. infect 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[cited 2016 apr]. available from: www.pepfar.gov/countries/index.htm world health organization. health accounts [page on the internet]. c2010 [cited 2016 apr]. available from: http://www.who.int/health-accounts/en/ joint united programme of hiv/aids. unaids report on the global aids epidemic | 2012 [document on the internet]. c2012 [cited 2017 may 03]. available from: http://www.unaids.org/sites/default/files/media_asset/20121120_unaids_global_report_2012_with_annexes_en_1.pdf world health organization – regional office for africa; us centers for disease control and prevention. guidance for development of national laboratory strategic plans. atlanta, ga: cdc; 2010. us centers for disease control and prevention. global health security agenda: action packages (ghsa). atlanta, ga: cdc; 2014. world health organization. laboratory assessment tool. annex 1: laboratory assessment tool/system questionnaire [document on the internet]. c2012 [cited 2016 apr]. available from: http://www.who.int/ihr/publications/annex1_lat.pdf?ua=1 ondoa p, datema t, keita-sow m-s, et al. a new matrix for scoring the functionality of national laboratory networks in africa: introducing the labnet scorecard. afr j lab med. 2016;5(3), 9 pages. https://doi.org/10.4102/ajlm.v5i3.498 brown cs, zwetyenga j, berdieva m, et al. new policy formulation methodology paves the way for sustainable laboratory systems in europe. who public health panorama. 2015;1(1):41–47. delorme l, machuron jl, sow i, et al. [resaolab: west african network of laboratories to enhance the quality of clinical biology] [article in french]. bull soc pathol exot. 2015;108(1):36–40. https://doi.org/10.1007/s13149-014-0378-1 supplementary materials table of contents supplementary document 1: search strategy documents national plans, strategies, costing frames valid (strategies and frameworks) or published (evaluations and reports) after the expiry date of 31 december 2010 national health policy or plan national health strategy national human resources for health (hrh) strategy (or policy) national laboratory strategy (or policy) national hiv strategy (or policy) national tuberculosis strategy (or policy) national malaria strategy (or policy) costing frameworks for the above strategies websites (all accessed april 2013) http://www.who.int/countryfocus/cooperation_strategy/listofccs/en/index.html (for country cooperative strategies of world health organization) http://www.erepublic.org/egovincountriesmmo/mali.html (to assess websites of all governments of countries) http://www.globalhivmeinfo.org/systemsmanagement/lists/ministries%20of%20health/allitems.aspx (for all ministries of health) http://www.kit.nl/smartsite.shtml?ch=fab&id=29313&part=resources (for lots of general information on human resources) http://www.kit.nl/smartsite.shtml?ch=fab&id=32449 (eldis human resources for health dossier) http://www.kit.nl/smartsite.shtml?ch=fab&id=32467 (hrh global resource center: a global library of human resources for health) http://www.kit.nl/smartsite.shtml?ch=fab&id=32196 (human resources for health) http://www.kit.nl/smartsite.shtml?ch=fab&id=32432 (world health organization collaborating centres global database. collaborating centres dealing with human resources for health development) http://www.kit.nl/smartsite.shtml?ch=fab&id=32194 (world health organization human resources for health) www.theglobalfund.org/ (per country proposals can be read) also visit the websites of international donor organisations and initiatives: usaid, fhi, roll back malaria, unaids, kncv, msh, partners in health, pepfar, jica, usaid, msh, institut pasteur, fondation mérieux and ird (institut de recherche pour le développement). if documents are not yet found, do a google search for: “health national plan” – country “human resources for health” – country “strategic health plan” – country “health policy” – country “workload study health” –country “health workers” – country “health evaluation” – country “laboratory services” – country supplementary document 2: analysis template country name: documents reviewed: give proper documentation details: author, publisher, title, date of publishing national governmental documents: .. .. national non-governmental documents .. .. multilateral agencies .. .. bilateral agencies .. .. global health initiatives (gfatm, gavi) .. .. world bank .. .. other .. .. is there evidence of existing documents on this subject that are not reviewed? yes/no if yes: which documents websites reviewed: mention link and date accessed general hiv/aids prevalence/ incidence: according to unaids according to current national strategic/health plan tuberculosis prevalence/ incidence: according to stop tb according to current national strategic/health plan malaria prevalence/ incidence: according to roll back malaria according to current national strategic/health plan country identified with a human resources for health crisis? yes/no low income country: yes/no middle income country: yes/no national health budget: proportion of the health budget contributed by donors: a: participation in development of plans: 1: out of the documentation is there evidence that the following stakeholders have been participating in the development of national plans? representative of: government ministry of health ministry of education ministry of finance ministry of defence other public sector laboratory services private non-profit sector laboratory services (e.g., non-governmental organisations, faith-based organisations) private sector laboratory services professional health association for microbiologists/ medical doctors/ laboratory staff research groups others: unknown 2: within the ministry of health is there a special department for: a: human resources for health: yes/no b: diagnostic services including the lab services: yes/no c: a separate laboratory services: yes/no b: national health policies and plans: 3: which policies and plans exist in the country? national health policy: yes/ no/ don’t know national health strategic plan: yes/ no/ don’t know human resources for health plan: yes/ no/ don’t know national laboratory plan: yes/ no/ don’t know other: [please provide] 4: which national health policies and plans pay attention to laboratory services? national health policy: yes/ no/ don’t know national health strategic plan: yes/ no/ don’t know human resources for health plan: yes/ no/ don’t know national laboratory plan: yes/ no/ don’t know other: [please provide] 5: if yes to any of the above, what type of laboratories are mentioned? a: public laboratory services? a.1: primary care level a.2: secondary care level a.3: tertiary care level a.4: national reference laboratory b: private laboratory services? b.1: primary care level b.2: secondary care level b.3: tertiary care level b.4: other 6: is there attention for strengthening of the public private mix in laboratory services? yes/ no describe : 7: which aspects in respect to laboratory services are mentioned in the plan? (indicate in which plan information is found) organisational structure of laboratory services (organogram): yes/ no management capacity for laboratory services: yes/ no laboratory infrastructure: yes/ no laboratory equipment: yes/ no supply chain (e.g. consumables): yes/ no human resources for laboratory services: yes/ no safety measurements: yes/ no working environment: yes/ no quality assurance (e.g. accreditation/ standardisation of testing, supervision): yes/ no 8: are the following laboratory services mentioned separately?: laboratory services in the context of hiv/aids: yes/ no laboratory services in the context of tuberculosis: yes/ no laboratory services in the context of malaria: yes/ no blood transfusion services yes/ no other: [please provide] c: human resources for laboratory services: (for this section the documents of all stakeholders will be analysed) (indicate in which plan information is found) 9: is there evidence that there is a shortage on laboratory personnel? yes/ no/ not clear 10: if yes, on what kind of indicators/criteria is this evidence based? information on current staffing coverage (lab workers related to inhabitants) in the country?: yes/ no/ not clear information on the establishment for laboratory workers (the staff for which, in the public sector, a position and salary is reserved): yes/ no/ not clear information on targets for numbers of laboratory staff: yes/ no/ not clear based on workload studies: yes/ no/ not clear based on minimum packages of activities for health services: yes/ no/ not clear international comparison: yes/ no/ not clear other: [please provide] source: 11: what kind of laboratory staff is lacking? a: medical laboratory scientist/medical technologist with university degree (#available/ # needed) b: medical laboratory technicians (#available/ # needed) c: laboratory assistants (#available/ # needed) d: others (#available/ # needed) source: 12a: are there strategies to decrease the shortage on hr for health laboratories mentioned in the documents? yes/ no/ not clear 12b: are these strategies accompanied by indicators? yes/ no/ not clear 12c: are these strategies accompanied by a budget? yes/ no/ not clear 12d: are responsibilities regarding the implementation of these strategies indicated? yes/ no/ not clear 13: if strategies are mentioned, what kind of strategies are mentioned in the documents? hr development pre-service (formal) education: yes/ no/ not clear recruitment strategies: yes/ no/ not clear tackling of gender inequality: yes/ no/ not clear hr management career development in-service training retention strategies (salary and other financial incentives) allowing dual practice other incentives (e.g. housing, transport) staff distribution (urban/rural) task shifting other: [please provide] 14: does the plan provide investment in the training institutions for laboratory workers? yes/ no/ not clear 15: if yes: a: investment in the infrastructure (e.g. buildings, equipment)/ learning materials of these training institutions b: investment in lecturers/ tutors of these schools c: other d: not specified 16: does evidence exist about high attrition of lab personnel? yes/ no/ not clear 17 : if available, what is the attrition rate of laboratory staff? rate…/ unknown 18: if disaggregated, what is the attrition rate of: a: medical laboratory scientist/medical technologist with university degree: b: medical laboratory technicians: c; laboratory assistants: d: others: 19: is there information on where laboratory staff migrate to? yes/ no/ not clear 20: if yes, do they migrate a: from public health sector to private for profit health sector b: from public health sector to international ngo/ agencies (health) c: to the non-health laboratory sector d: out of the country e: from private non-profit sector to public sector f: from private for-profit sector to public sector g: other d: financing for laboratories (indicate in which plan information is found) 21: what is the total health budget for the country? a: which proportion of the total government budget is earmarked for the health sector? budget:… unknown (source from: http://www.who.int/nha/country/en/index.html#u) b: how much does the government spend on health per capita in us dollar terms?budget:… unknown (same source as a) c: which proportion of the total health budget is planned/used for medical support services (lab, pharmacy and radiology?) budget:… unknown e: which proportion of the total health budget is planned/used for laboratory services? budget:… unknown f: which proportion of the budget for laboratories is planned/used for hrh? budget:… unknown costing: 22: are laboratory services in the national health plans costed? national health strategic plan: yes/ no/ don’t know human resources for health plan: yes/ no/ don’t know national laboratory plan: yes/ no/ don’t know other namely 23: are there national or international donors that invest in laboratory services? yes/ no/ not clear source: 24: if yes, which donors? 25: which laboratory services are supported by these donors? organizational structure of laboratory services (organogram): yes/ no management capacity for laboratory services: yes/ no laboratory infrastructure: yes/ no laboratory equipment: yes/ no supply chain (e.g. consumables): yes/ no human resources for laboratory services: yes/ no safety measurements: yes/ no working environment: yes/ no quality assurance (e.g. accreditation/ standardisation of testing, supervision): yes/ no blood transfusion services yes/ no technical expertise other: [please provide] 26: do these donors invest in remuneration of lab staff (top up of salary/ incentives)? yes/ no/ not clear e: implementation of plans 27: are there monitoring and evaluation reports or other documents that give evidence that laboratory components of plans implemented? a: evidence of implementation of laboratory components of nlsp yes/ no/ not clear b: evidence of implementation of laboratory components of donor plans yes/ no/ not clear 28: if yes: which elements of laboratory services are implemented? a: describe the implemented laboratory components of nlsp: b: describe the implemented laboratory components of donor plans supplementary document 3: code list 0. this relevant document was not found for review 1.0 plan developed with multiple ministry of health departments 1.1 plan developed with multiple ministries 1.2 plan developed with public or private laboratory service providers 1.3 plan developed with professional associations 1.4 plan developed with researchers or universities 1.5 plan developed with others 1.6 unknown who participated in plan development 2.1 the ministry of health has a special hrh department 2.2 the ministry of health has a department that includes laboratory services 2.3 the ministry of health has a separate department for lab services 3.1.1 public sector lab services mentioned in the plan 3.1.2 private sector lab services mentioned in the plan 3.1.3 attention for strengthening the public-private mix 3.2.1 primary care level labs mentioned 3.2.2 secondary level labs mentioned 3.2.3 tertiary care level labs mentioned 3.2.4 national reference laboratory mentioned 4.1 organisational structure of lab services mentioned in the plan 4.2 governance and management capacity for lab services mentioned in the plan 4.3 lab infrastructure mentioned in the plan 4.3.6 hrh for lab services mentioned in the plan 4.4 laboratory equipment mentioned in the plan 4.5 supplies and supply chain mentioned in the plan 4.6 safety levels mentioned in the plan 4.8 quality assurance of laboratory services mentioned in the plan 4.9 other aspects of laboratory services mentioned in the plan 5.0.1 laboratory services mentioned in the context of hiv/aids 5.0.2 laboratory services mentioned in the context of tuberculosis 5.0.3 laboratory services mentioned in the context of malaria 5.0.4 laboratory services mentioned in the context of blood transfusion services 5.0.5 laboratory services mentioned in the context of non-communicable diseases 5.0.6 laboratory services mentioned in the context of food or product safety 5.0.7 laboratory services mentioned in the context of epidemiological surveillance 5.0.8 laboratory services in the context of quality control of medicines/ medical devices 5.0.9 laboratory services mentioned in the context of hygiene and environmental health 5.0.91 laboratory services mentioned in other context 5.1.1 different cadres mentioned, including laboratory personnel 5.1.2 different cadres mentioned, excluding laboratory personnel 5.1.3 different cadres mentioned, unclear whether laboratory personnel is included 6.0 there is a surplus of laboratory personnel 6.1 there is a shortage of (qualified) laboratory personnel 6.1.1 there is a shortage of medical laboratory scientists/ medical technologists with university degree 6.1.2 there is a shortage of laboratory technicians 6.1.3 there is a shortage of laboratory assistants 6.1.4 there is a shortage of other laboratory personnel 7.1 the evidence for the shortage of laboratory personnel is based on information on current staffing coverage (laboratory workers per no. inhabitants or per facility) in the country 7.2 the evidence for the shortage of laboratory personnel is based on available positions for laboratory personnel in the public sector 7.3 the evidence for a shortage of laboratory personnel is based on the mentioning of training needs 7.4 the evidence for the shortage of laboratory personnel is based on other targets/ sources 7.5 unknown where the qualification ‘shortage’ is based on 8 exact attrition rate of laboratory technicians 8.01 strategy accompanied by indicator 8.02 strategy accompanied by budget 8.03 strategy accompanied by responsibilities 8.1.1 gender issues related to hrh in laboratories 8.1.2 retention strategy 8.1.3 motivation strategy 8.1.4 performance strategy 9.1 pre-service (formal) education 9.2 recruitment strategies 9.3 in-service training 9.4 continuous professional development 9.5 investment in career development of laboratory staff 9.6 supportive supervision 9.7 management 9.8 increase salaries 9.9 other incentives (housing, transport, non-financial) 9.91 enhancing working conditions 9.92 allowing dual practice 9.93 urban-rural distribution of staff 9.94 task shifting 9.95 other 91.1.2 % of the health budget allocated to support services 91.1.3 % of the health budget allocated to laboratories 91.1.4 % of the health budget allocated to laboratory hrh 92.2.1 laboratory services in general are costed 92.2.2 this specific laboratory service is costed 93.3.1 this plan shows evidence of a donor committing to invest in organisational structure 93.3.2 this plan shows evidence of a donor committing to invest in management capacity for laboratories 93.3.3 this plan shows evidence of a donor committing to invest in lab infrastructure 93.3.4 this plan shows evidence of a donor committing to invest in lab equipment 93.3.5 this plan shows evidence of a donor committing to invest in supply chain 93.3.6 this plan shows evidence of a donor committing to invest in hrh 93.3.7 this plan shows evidence of a donor committing to invest in safety levels 93.3.8 this plan shows evidence of a donor committing to invest in environment 93.3.9 this plan shows evidence of a donor committing to invest in quality assurance 93.3.91 this plan shows evidence of a donor committing to invest in other aspects of laboratory services supplementary table 1: status of the national laboratory strategic plan of sub-saharan african countries in 2013 and 2017. supplementary table 2: evidence for laboratory workforce shortage and attrition provided in national policies and plans supplementary figure 1: comparison of groups of countries that have started versus not started the process of implementing a national laboratory policy and strategic plan. supplementary figure 2: aspects of human resources development addressed in 10 nlsp and 25 hrh. abstract introduction methods results discussion acknowledgements references about the author(s) naadira vanker graduate school of business, university of cape town, cape town, south africa norman h. b. faull graduate school of business, university of cape town, cape town, south africa citation vanker n, faull nhb. laboratory test result interpretation for primary care doctors in south africa. afr j lab med. 2017;6(1), a453. https://doi.org/10.4102/ajlm.v6i1.453 original research laboratory test result interpretation for primary care doctors in south africa naadira vanker, norman h. b. faull received: 30 mar. 2016; accepted: 26 aug. 2016; published: 24 mar. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: challenges and uncertainties with test result interpretation can lead to diagnostic errors. primary care doctors are at a higher risk than specialists of making these errors, due to the range in complexity and severity of conditions that they encounter. objectives: this study aimed to investigate the challenges that primary care doctors face with test result interpretation, and to identify potential countermeasures to address these. methods: a survey was sent out to 7800 primary care doctors in south africa. questionnaire themes included doctors’ uncertainty with interpreting test results, mechanisms used to overcome this uncertainty, challenges with appropriate result interpretation, and perceived solutions for interpreting results. results: of the 552 responses received, the prevalence of challenges with result interpretation was estimated in an average of 17% of diagnostic encounters. the most commonly-reported challenges were not receiving test results in a timely manner (51% of respondents) and previous results not being easily available (37%). when faced with diagnostic uncertainty, 84% of respondents would either follow-up and reassess the patient or discuss the case with a specialist, and 67% would contact a laboratory professional. the most useful test utilisation enablers were found to be: interpretive comments (78% of respondents), published guidelines (74%), and a dedicated laboratory phone line (72%). conclusion: primary care doctors acknowledge uncertainty with test result interpretation. potential countermeasures include the addition of patient-specific interpretive comments, the availability of guidelines or algorithms, and a dedicated laboratory phone line. the benefit of enhanced test result interpretation would reduce diagnostic error rates. introduction laboratory services play an integral role in the healthcare system from primarythrough to tertiary-level care – since diagnostic tests can either confirm or exclude a tentative diagnosis, or screen for potential diseases.1 the underlying purpose of laboratory testing lies in the association between laboratory test results and the potential to improve a patient’s health status. this requires ordering a test when appropriate and necessary, accurate interpretation, and acting upon the result.2 primary care doctors are usually the point of entry into a healthcare system, and are consequently exposed to a variety of medical conditions that range in both complexity and severity. it is postulated that these doctors are therefore at a higher risk of making medical errors than specialists.3 due to the variety and intricacies of laboratory tests available, there is the potential for test-related errors to occur in a range of clinical conditions, that may result in significant patient harm.4 studies have shown that between 15% and 54% of errors occurring at a primary healthcare level are related to the testing process.5 diagnostic errors can be due to three underlying causes, namely: no identifiable fault, system-related, and cognitive. cognitive errors are caused by incorrect interpretation of available information and may be caused by faulty knowledge, faulty data gathering, or faulty synthesis of data. a large-scale study found that up to 74% of diagnostic errors are either completely or in part due to cognitive failures.6 this suggests that many diagnostic errors are related to misunderstanding or misinterpreting the available information. studies have shown that primary care doctors face uncertainty when interpreting clinical laboratory reports.7,8,9,10 a nationwide study involving 1768 primary care physicians was conducted in the united states to determine the challenges that this group faces with laboratory test ordering and result interpretation. the findings were that clinicians experienced uncertainty due to inconsistencies in the receipt of results, problems with the report format, and difficulties with interpretation of results in 8.3% of diagnostic encounters.7 research conducted in south africa also demonstrated the uncertainty that doctors experience with laboratory test result interpretation. this study investigated how confident interns were with requesting biochemical tests and interpreting the results. the study found that although these junior doctors were fairly confident when dealing with common investigations, they experienced challenges with interpreting the results of more complex and less common tests. of the 61 respondents, 23% reported a lack of confidence in interpreting the results of complex tests.8 this explorative study investigated the problems and challenges that primary healthcare doctors in south africa face with the interpretation of clinical laboratory test results. a secondary aim of the study was to identify potential countermeasures to address these challenges. methods ethical considerations ethical approval was granted by the commerce faculty ethics in research committee, graduate school of business, university of cape town. anonymity was maintained throughout the process. informed consent was obtained through a cover letter containing the survey link. the surveys were completed online, with all responses anonymised through the system. the researchers did not have access to respondents’ identifying or personal information. study design this research was based on a study conducted by hickner et al., entitled ‘primary care physicians’ challenges in ordering clinical laboratory tests and interpreting results’.7 the original survey was developed through an inductive approach, using information obtained from three focus groups comprising 27 primary care doctors, as well as from a panel of experts working in primary healthcare and laboratory medicine. the questionnaire was authorised for use by the office of the associate director for science at the us centers for disease control and prevention. the original nineteen-part questionnaire was reduced to nine sections to focus on the challenges that primary healthcare doctors face with clinical laboratory result interpretation. the survey themes included doctors’ uncertainty with interpreting test results, mechanisms they use to overcome this uncertainty, challenges with appropriate result interpretation, and perceived solutions to interpreting test results. the questionnaire categories were as follows: (1) demographic information; (2) information about the doctor’s practice; (3) interpretation uncertainty; (4) the diagnostic evaluation process; (5) laboratory consultation; and (6) test utilisation enablers. questions related to the doctor’s practice included: whether the doctor was a general practitioner or specialist; the number of years in practice; the predominant categories of tests ordered (i.e. diagnostic tests, chronic disease monitoring, or routine screening); the number of patients seen per week; the number of tests ordered per week; and the number of tests per week that were associated with interpretation uncertainty. two questions were added to the demographic section of the questionnaire to determine whether the south african doctor worked in a rural, semi-urban or urban practice and whether he/she predominantly made use of private pathology laboratories or the parastatal (national health laboratory service) laboratory. however, it was not ascertained whether the majority of patients seen were hospitalised or out-patients. responses were predominantly chosen from a list of five-point graded options, but there was space given for open-ended responses. response options ranged from: ‘extremely useful’ to ‘not at all useful’, ‘extremely important’ to ‘not at all important’, ‘extremely well’ to ‘not at all well’, and ‘extremely problematic’ to ‘not at all problematic’. survey administration for this cross-sectional study, questionnaires were sent out electronically using a survey link to the approximately 7800 primary care doctors in the south african medical association database. the survey was sent on 13 october 2015 and remained open for responses until 13 november 2015. analysis response data from the surveys were exported to microsoft excel (microsoft corp., redmond, washington, united states) and analysed using the ibm spss statistics® package (ibm spss statistics for macintosh, version 22.0.; ibm corp., armonk, new york, united states). the qualitative responses were analysed quantitatively using descriptive statistics to determine relative frequencies. results presented were based on the number of respondents who selected the top two responses from the five-point scale – namely, ‘extremely and very useful’, ‘extremely and very important’, ‘extremely and very well’, or ‘extremely and very problematic’. open-ended responses received were reported as ‘other’ in the figures below. results overview and respondent characteristics of the approximately 7800 questionnaires sent out, 552 completed questionnaires were received, equating to a response rate of 7%. incomplete questionnaires were excluded from the analysis, so as not to skew the results. although the survey was sent to doctors registered in the south african medical association database as general and/or independent practitioners, this database included a few doctors who were either in training or were qualified specialists. table 1 describes the doctors’ practice characteristics and test utilisation information. of note, respondents saw an average of 115 patients per week, ordered an average of 24 tests per week, and experienced uncertainty in result interpretation for four of these tests. this equates to challenges in the interpretation of approximately 17% of test results. table 1: respondents’ practice characteristics and laboratory test utilisation information, south africa, 13 october 2015–13 november 2015. challenges with laboratory test results the challenges that doctors experienced with laboratory test results (figure 1) were selected from a list of possible options and rated using the five-point scale. the most prominent problems were related to accessing results with 51% of respondents reporting that not receiving results in a timely manner was very problematic and 37% reporting problems with availability of previous test results. (the timeliness of the receipt of results was as perceived by the respondents and was not quantified or defined in the question.) the next-highest reported type of challenge related to the result being incompatible with the patient’s clinical picture, which could either be seen as an inconsistent result (27%) or a laboratory error (28%). figure 1: challenges that doctors face when using laboratory test results, south africa, 13 october 2015–13 november 2015. although not strictly related to challenges with laboratory test results, a few respondents did report challenges with laboratory access and financing in the open-ended part of this section. these challenges included: difficulties for rural hospitals or practices to get samples to a laboratory, the unavailability of specialised tests (e.g., b-type natriuretic peptide and helicobacter pylori igg), and medical aids not authorising or paying for tests. diagnostic evaluation process the majority of doctors (66%) typically used a core set of 20 or fewer clinical laboratory diagnostic tests. when faced with diagnostic uncertainty, most respondents (67%) reported always double-checking with another doctor or electronic resources (e.g., uptodate, webmd, patient.co.uk, etc.) if they doubted their decision. even when confident in their pre-test diagnoses, 42% of doctors would still think ‘what else could it be?’. while 58% of clinicians were concerned about over-testing their patients, only 33% were concerned about under-testing patients. interpretation uncertainty when faced with diagnostic uncertainty in a difficult or unusual case (figure 2), most respondents would either follow-up and reassess the patient (84%) or review the patient’s history and physical findings (82%). eighty-four percent of primary care doctors also found it very useful to discuss the case with a specialist. figure 2: tactics employed by doctors to deal with test interpretation uncertainty, south africa, 13 october 2015–13 november 2015. laboratory consultation in a variety of contexts, most respondents found communication with the laboratory to be useful (figure 3). of note, 82% of doctors found it very useful for the laboratory to contact the clinician with critically abnormal results. figure 3: usefulness of laboratory communication/consultation, south africa, 13 october 2015–13 november 2015. in general, only about one-third of respondents noted very important reasons as to why they did not frequently contact laboratory professionals. these reasons included: difficulties in contacting the person who could answer their questions (36%); not knowing whom to contact (33%); difficulties in getting through to the laboratory (32%); and not having a working relationship with laboratory professionals (27%). only 11% reported that they did not contact the laboratory because they felt that they had received unreliable information during previous interactions. a small number (2%) of individuals reported specific problems with public sector laboratories wherein they felt that laboratory staff were unhelpful regarding lost or rejected specimens, and inaccurate, delayed or urgent results. a lack of access to pathologists at certain regional laboratories was also noted as a problem. test utilisation enablers test utilisation enablers (figure 4) are methods that have been developed to assist clinicians in using diagnostic laboratory testing more effectively. interpretive comments – comments provided with the test result to give additional information on the meaning of the results – were reported by 78% of respondents as very useful. seventy-four percent of doctors found guidelines – aids published by specialty organisations or societies for the interpretation of patient’s test results based on clinical presentation usually guided by decision trees – to be useful. similarly, clinical algorithms – guidelines used within local practices or institutions – were reported as very useful by 67% of doctors. seventy-two percent of the respondents reported that a dedicated laboratory phone was useful; however, only 39% of respondents had access to a dedicated laboratory phone line. information on test performance characteristics, such as sensitivity, specificity, and likelihood ratios, were also reported as very useful by 59% of respondents, but were only available to 32%. the test utilisation enablers considered useful were selected from the options presented in the five-point scale questions, and no additional enablers were suggested by respondents in the open-ended response section. figure 4: usefulness and availability of test utilisation enablers, south africa, 13 october 2015–13 november 2015. when compared to urban and peri-urban respondents, rural doctors experienced considerably lower availability of interpretive comments, information on test performance characteristics, trending of laboratory results (when previous results are compared with current results), and reflex testing (a test performed by the laboratory in response to results from a previous test) (table 2). in contrast , the rural doctors reported increased availability of a dedicated laboratory phone line. table 2: availability of test utilisation enablers for the urban, peri-urban and rural doctor cohorts,† south africa, 13 october 2015–13 november 2015. further comments open-ended feedback on result interpretation was also elicited from respondents. results included acknowledgement that the ‘interpretation of results are [sic] critical to reliably apply the blood results to our patients in terms of diagnosis, screening and monitoring their pathology’. a number of respondents felt that the interpretive comments currently received were not specific to patients’ age, sex, clinical picture or previous results, but were instead based on general information. furthermore, these comments did not include recommendations for further testing or treatment. specific challenges were noted in the interpretation of microbiology, serology (especially hepatitis b), and discordant hiv results. in contrast to the challenges noted, certain private laboratories were identified and commended for the inclusion (where necessary) of interpretive comments written by pathologists. discussion this study found that primary care doctors in south africa experience challenges with laboratory test result interpretation in approximately 17% of their diagnostic encounters. by comparison, a similar study conducted in the united states found that primary care physicians reported uncertainty in 8.3% of diagnostic encounters. this emphasises the need for improved mechanisms and countermeasures to aid south african doctors with result interpretation. the most common general challenges with laboratory test results reported by primary care doctors are related to receiving and accessing results – namely, not receiving results timeously and previous results not being easily available. literature shows that over 80% of laboratories receive complaints about turn-around times,11 yet there are no universal evidence-based goals for laboratory processing times, and clinicians’ expectations have often been found to be unreasonable. nevertheless, laboratories should acknowledge customer dissatisfaction, and aim to provide results within a timeframe that is achievable by the laboratory and optimal for patient care.11 trending of results is the displaying of a patient’s previous results alongside the current test result to identify patterns of change and to enhance result interpretation. over half of all respondents in our study found this to be an extremely or very useful test utilisation enabler, but less than a third reported its availability. result trending has been shown to decrease time spent by clinicians on a case.12 laboratories can play a role in addressing these challenges by determining appropriate turn-around times and communicating these times to the doctors, as well as by including previous test results on current reports to enable result trending. however, the availability of previous results requires integrated health information technology systems, which are not available in all healthcare environments.12 although this study focused on the post-analytical phase of laboratory testing, the survey raised two questions around the analytic testing process and whether the clinicians experienced challenges with ‘suspected errors in laboratory results’ and ‘results inconsistent with the patient’s symptoms and history’. inconsistent results were reported as a challenge by 27% of respondents and possible laboratory errors by 28%. however, this did not include inquiry into errors occurring in the pre-analytical phase of testing, such as the mislabeling of samples. it has been found that pre-analytical errors account for 55% of laboratory errors causing a missed or delayed diagnosis.13 therefore, clinicians should be aware that suspected errors or inconsistent results might be due to failures that occur outside the control of the laboratory. our study found that primary care doctors find consultation with other clinicians or laboratory professionals to be an important mechanism in aiding test result interpretation. the majority of respondents reported a dedicated laboratory phone line to be an important test utilisation enabler, and, although this was only available to less than 40% of the total study population, over half of the rural doctor cohort had access to this service. this suggests that laboratories based in rural areas are trying to leverage their limited resources. a review of literature found that failures in communication between clinicians and the laboratory could negatively impact patient safety.5 improving communication channels between the laboratory and clinical practitioners could lead to improved patient care and reduce unnecessary specialist referrals, which are at times requested purely for test result interpretation.7 the majority of survey respondents reported interpretive comments to be the most useful test utilisation enabler. interpretive comments are added to a laboratory report in order to provide further information on the result and to aid in the diagnostic process. these comments can be provided by a qualified pathologist through technology-based interpretive algorithms and expert systems or through the addition of a ‘canned’ comment. a ‘canned’ comment is pre-written text that is added onto all results for a specific test, regardless of the actual result or the patient’s clinical history, and is considered to be the least useful form of interpretive commenting.14 respondents in our study supported this view when noting that interpretive comments that were not specific to the patient’s age, sex, clinical picture or previous results, were not particularly useful. a study assessing the impact of narrative interpretations for complex laboratory tests found that the comments reduced the time taken and the number of tests required to reach a diagnosis and had an impact on the differential diagnosis. furthermore, most respondents in that study felt that the interpretive comments helped prevent a misdiagnosis.15 the provision of high quality, patient-specific interpretive comments should improve patient care, decrease diagnostic errors, reduce costs, and enhance appropriate specialist referrals.16 the majority of respondents also reported guidelines or algorithms to be useful test-utilisation enablers. clinical algorithms and practice guidelines are developed to provide a standardised, evidence-based approach to clinical processes in order to reduce error rates, improve clinical effectiveness, and enhance the quality of patient care.17 clinical algorithms are particularly helpful in the interpretation of results for conditions that require a complex panel of tests for diagnosis, management, and monitoring of disease progression (e.g., diabetes mellitus).18 however, the availability of guidelines does not always ensure their use or result in changes in medical practices and behaviours. it is recommended that guidelines be disseminated through systems or accompanied by tools to facilitate their use and effectiveness.19 a study that compared the use of a technology-based expert system with conventional (non-computer-based) guidelines, found that the computer-based guideline system shortened the time taken to reach a diagnosis from (on average) 3.2 days to one day.20 the availability of guidelines or algorithms would be a useful countermeasure to aid doctors in interpreting complex tests and recommending further investigations that would guide management decisions. these guidelines may be integrated into the existing health information technology system, which may not be developed in certain settings, or can be in the form of applications that are uploaded onto independent mobile devices.7 it has been found that clinicians are often unaware of whether their diagnoses at the time when they are making them are correct or erroneous.21 therefore, interventions to reduce errors, such as medical decision support systems, should be embedded in a system rather than being made available only when perceived to be needed.21 to standardise quality and improve efficiency, particularly in areas where human capital is limited, information technology can be leveraged. studies have shown that health information technology can enhance delivery of care, reduce errors, and decrease utilisation of potentially inappropriate care.12,19,20 twenty-two per cent of respondents work in rural areas and the availability of test utilisation enablers (including interpretive comments) in these areas is lower compared to urban and peri-urban areas. furthermore, it was reported that certain regional laboratories lack access to a pathologist. in these cases, embedded technology-based solutions (such as expert systems for interpretive comments or integrated guidelines) may be particularly useful in assisting primary care doctors with test result interpretation. limitations response rates to surveys are reported to be 10%–20%,22 but the response rate in our study was 7%. this could be because the electronic platform used for the survey administration dissuaded doctors uncomfortable with the technology from participating; additionally, the length of time required to complete the survey (15 minutes) may have been considered too long.23 however, the study by hickner et al.,7 on which our study is based, had a response rate of 5.6%, suggesting that this type of research may be associated with low response rates. a possible further limitation in this study is that the challenges faced by clinicians with result interpretation may have been under-reported. research has shown that medical doctors have a tendency to display overconfidence, which can impact self-reported findings.24 conclusion primary care doctors in south africa acknowledge that they experience uncertainty when interpreting certain clinical laboratory test results. the most useful countermeasures and mechanisms identified by the doctors to improve this included: the addition of patient-specific interpretive comments; the availability of national or international guidelines or local clinical algorithms; and enhanced communication with the laboratory through a dedicated phone line. the ultimate benefit of enhanced test result interpretation would be reduced diagnostic error rates and a more efficient and effective primary healthcare system, which would reduce the rates of referral for secondary and tertiary levels of care. acknowledgements we would like to thank all the respondents who took the time to complete this survey. we also wish to thank ms marilyn myburgh from the south african medical association for her assistance with distributing the survey and dr julie taylor of the us centers for disease control and prevention for allowing us use of the questionnaire. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support none. authors’ contributions n.v. was the project leader who co-designed the project, collected and analysed the data, and contributed to writing and preparing the manuscript. n.h.b.f. was the academic supervisor who co-designed the project and contributed to preparing the manuscript. references forsman rw. why is the laboratory an afterthought for managed care organizations? clin chem. 1996;42(5):813–816. jackson br. managing laboratory test use: principles and tools. clin lab med. 2007;27(4):733–748. https://doi.org/10.1016/j.cll.2007.07.009 singh h, giardina td, meyer an, et al. types and origins of diagnostic errors in primary care settings. jama intern med. 2013;173(6):418–425. https://doi.org/10.1001/jamainternmed.2013.2777 clayton pd, evans sr, pryor t, et al. bringing help to the clinical laboratory – use 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african journal of laboratory medicine – advancing laboratory medicine and science in africa. afr j lab med. 2012;1(1), art. #86, 1 pages. http://dx.doi.org/10.4102/ ajlm.v1i1.86 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the african journal of laboratory medicine – advancing laboratory medicine and science in africa in this editorial... open access laboratory medicine has evolved rapidly on the african continent. the growth of state-of-the art research institutions and public health agencies has led to ground-breaking research, cross-cutting management techniques, and both formal and informal policy statements. disease-specific and regional laboratory networks have been critical in ensuring that information and practices are shared between neighboring countries. networks of excellence have formed in each of the four regions of sub-saharan africa in order to support research infrastructure and strengthen south-south collaboration. in northern africa, well-established professional societies, such as the egyptian society of laboratory medicine and the moroccan society of clinical chemistry and laboratory medicine, play critical roles in the transformation of the laboratory profession. additionally, laboratory-strengthening activities have been supported by various established organisations, including the south african national accreditation system (sanas).from this fertile intellectual ground have come some of the brightest scientists in the world. africans on the continent and in the diaspora are leading the way to find preventive measures, tests, and cures for both infectious and non-communicable diseases. never before have partnerships for global health existed as they do in africa today, with funding, research, and implementing partners working hand-in-hand with national governments and laboratory systems. international agencies, research institutes, commercial companies, and governmental and regional bodies are joining forces with a common goal: strengthening laboratories and improving health on the continent. africa has been the catalyst for the development of revolutionary new molecular approaches to address the high burden of infectious diseases, and technologies that can be used closer to the site of patient management. innovative information tools are being used to support data management, instrument performance, and timely distribution of laboratory results. this is a momentous time in history for laboratory medicine and science in africa. the landscape of public health is changing throughout africa. new infectious diseases are emerging, and as people live longer, non-communicable diseases such as cancer and diabetes are becoming increasingly important, fuelled in this region by high rates of chronic inflammation from diseases such as tb and hiv. as the next tidal wave of challenges for global health rolls in, we must ensure that laboratories are prepared to meet the diagnostic challenges that they will be faced with in the future. the african society for laboratory medicine (aslm) was officially launched in march 2011 as an umbrella organisation for african laboratory personnel. aslm takes an aggressive, forward-looking approach to advancing laboratory services by fostering new research; providing opportunities for mentorship; and building capacity to develop and sustain laboratory practice, science, and policy for better programme implementation and outcomes. the society’s newly unveiled strategic vision, aslm2020, has set aggressive targets to be achieved by the year 2020: enhancing workforce development by supporting the training and certification of 30 000 laboratory staff; strengthening 25 national and regional regulatory bodies; and enrolling 2500 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laboratory medicine, aslm’s official scientific journal, is the first all-inclusive peer-reviewed journal specifically designed to cover all laboratory-related topics, with a focus on implications for the african continent. the journal balances cutting-edge basic laboratory science with vital discussions of policy debates, whilst highlighting success stories in which laboratory interventions have made a critical contribution to healthcare programs. the journal focuses on the role of the laboratory and its professionals in the clinical and public health sectors, and supports a data-driven approach to translating research in practice and policy. at this historical crossroad, the african journal of laboratory medicine is well-positioned to provide a critical forum for discussion that advances science and informs policy and programmatic recommendations. we hope that you find this inaugural edition of the african journal of laboratory medicine enlightening, inspirational, and 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hunç hiroshi ichimura lucius c. imoh emmanuel o. irek http://www.ajlmonline.org� https://ajlmonline.org� mailto:submissions@ajlmonline.org mailto:submissions@ajlmonline.org https://ajlmonline.org� https://ajlmonline.org� https://ajlmonline.org/index.php/ajlm/user https://ajlmonline.org/index.php/ajlm/user page 2 of 2 reviewer acknowledgement http://www.ajlmonline.org open access 3. it is good practice as a reviewer to update your personal details regularly to ensure contact with you throughout your professional term as reviewer to the african journal of laboratory medicine. please do not hesitate to contact us if you require assistance in performing this task. publisher: publishing@aosis.co.za tel: +27 21 975 2602 prenika jaglal thumeka p. jalavu feyisayo e. jegede zacceus jeremiah jaco joubert jean f. kloppers adjane k. koura modupe a. kuti lynnette t. kyokunda sunil k. maharana caroline maluleka talkmore maruta altaf a. mir reena d. mohanlal margaret mokomane modisa s. motswaledi wilson mtotela devon muir pierre mukadi-kaningu stephanie murugan graham mutandi benard m. mutua farai muzambi pieter nel apeksha niraula chinwe a. nnabugwu arnold m. nyaga lucy o. nyangau simon o. nyangena collins o. odhiambo collince o. ogolla chukwubike u. okeke onyekachukwu f.i. okeke mitchel o. okumu denis omali royronald o. ongong’a nyamongo onkoba chin-yih ou john r.p. padde iseimokumo c. peletiri joachim potgieter pasipanodya i. ruredzo jean sakande yahya j. salman salam sawadogo nathan shaviya aramide r. shehu anthony m. smith carmen swanepoel khine swe swe han fabio timeus farouk a. umaru rochelle van wijk reubina wadee musau wakabongo justyna wojno abdourahamane yacouba raj n. yadav rania a. zayed azza a. zulfu http://www.ajlmonline.org� mailto:publishing@aosis.co.za acknowledgement to reviewers abstract introduction methods results discussion acknowledgements references about the author(s) justine d. niandat department of medical laboratory science, faculty of health sciences and technology, university of jos, jos, nigeria department of chemical pathology, federal medical centre keffi, keffi, nigeria caroline a. okoli department of medical laboratory science, faculty of health sciences and technology, university of jos, jos, nigeria citation niandat jd, okoli ca. erythrocyte glucose-6-phosphate dehydrogenase deficiency-induced anaemia in children in jos, north-central nigeria. afr j lab med. 2025;14(1), a2733. https://doi.org/10.4102/ajlm.v14i1.2733 original research erythrocyte glucose-6-phosphate dehydrogenase deficiency-induced anaemia in children in jos, north-central nigeria justine d. niandat, caroline a. okoli received: 20 dec. 2024; accepted: 24 june 2025; published: 14 oct. 2025 copyright: © 2025. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: glucose-6-phosphate dehydrogenase deficiency (g6pdd), a common inherited enzyme defect, associated with severe neonatal anaemia and hyperbilirubinaemia, can result in permanent neurologic damage or death. prevalence of g6pdd-induced anaemia in vulnerable groups, like children, is not known in our setting. objective: this study was aimed at determining the prevalence of erythrocyte g6pdd-induced anaemia among children aged 0–5 years old seen at jos university teaching hospital, north-central nigeria. methods: this was a hospital-based cross-sectional study conducted from february to june 2023. glucose-6-phosphate dehydrogenase and haemoglobin levels were analysed colourimetrically. data were analysed; p < 0.05 was considered significant. results: out of 100 children aged 0–5 years (54 male, 46 female), 40 (40%) were g6pd deficient. nineteen (35.2%) of the g6pd-deficient children were male and 21 (45.7%) were female. fifty-one (51%) children were anaemic, 23 (57.5%) were g6pdd-induced; 85 (85%) of the parents had no knowledge of g6pd and its deficiency. conclusion: this study showed a high prevalence of g6pdd-induced anaemia among children in jos. this suggests that there may be a need for early routine g6pd screening in children for early detection and proper intervention in those with the deficiency. what this study adds: this study has objectively established high prevalence of anaemia, g6pdd and g6pdd-induced anaemia in children aged 0–5 years in jos, nigeria, highlighting the importance of g6pd screening in children. keywords: prevalence; glucose-6-phosphate dehydrogenase deficiency-induced-anaemia; children; awareness; jos, nigeria. introduction anaemia and glucose-6-phosphate dehydrogenase (g6pd) deficiency are causes of childhood morbidity and death, especially in malaria-endemic areas such as nigeria. deficiency of g6pd, an oxidoreductase that catalyses the oxidation of glucose-6-phosphate to 6-phosphogluconate or the corresponding lactose, is the most common and important enzyme deficiency in red blood cells. mediterranean, asian, and african g6pd deficiency variants have been reported.1 about 7.5% of the world’s population is estimated to have at least one g6pd deficiency variant, with 15% to 26% occurring in west africa, and 15.3% in nigerian children.2,3,4 glucose-6-phosphate dehydrogenase is an antioxidant enzyme. it is present in all body cells, including the erythrocytes, where it helps protect the cells from damage and premature destruction by reactive species.1 the first step in glucose metabolism via the pentose-phosphate shunt is catalysed by g6pd, with nicotinamide adenine dinucleotide phosphate (nadph) and ribose-5-phosphate ultimately being produced. ribose-5-phosphate is an important component of nucleotides.4,5 glucose-6-phosphate dehydrogenase deficiency is an x-linked disorder, where the enzyme deficiency occurs more in male than in female individuals, because the males only have one x chromosome when compared to the females, who have two x-chromosomes.6 glucose-6-phosphate dehydrogenase deficiency is usually asymptomatic. however, certain factors, such as malaria infestation, consumption of flavin-containing foods, administration of oxidative antibiotics, or antimalarial drugs can trigger the haemolytic crisis.1 if not promptly diagnosed and properly managed, g6pd deficiency can cause severe hyperbilirubinaemia and its associated complications, such as anaemia, kernicterus, and even death in neonates.7 the world health organization2 recommends routine g6pd screening of newborns in areas where the prevalence of g6pd deficiency is as high as 3% – 5%, or more. studies have shown that early detection reduces morbidity and mortality in newborns by establishing the aetiological diagnosis of jaundice and facilitating therapeutic choices.1 the prevalence of g6pd deficiency-induced anaemia among children seen in jos university teaching hospital, nigeria, has, to the best of our knowledge, not been established.8 anaemia is one of the major causes of paediatric death and can affect a child’s mental, physical, and social development. with the decrease in iron levels, it can lead to poor cognitive function, thus negatively affecting a child’s academic performance, and creating further complications in later life.9 thus, this study aims to determine the prevalence of g6pd deficiency-induced anaemia among children seen in jos university teaching hospital. this knowledge could guide the clinician to have the right index of suspicion in children presenting with some of the clinical symptoms of the disease. this can also establish the need to include a g6pd test among the battery of tests for neonates presenting with non-specific signs and symptoms of g6pd deficiency, such as prolonged jaundice (often the first week of life), anaemia, hyperbilirubinaemia, sudden jaundice, haemoglobinuria, poor feeding, lethargy, tachycardia, and others, thus helping in the early establishment of correct diagnosis and treatment, and thereby helping to reduce the morbidity and mortality resulting from g6pd deficiency. methods ethical considerations ethical approval was sought and obtained from the ethical review committee of jos university teaching hospital and jos university teaching hospital research ethics committee before the commencement of the study, with reference number juth/dcs/irec/127xxxi/437 and registration number nhrec/juth/05/10/22. data were anonymised and stored on a hard drive. written informed consent was obtained from the parents or legal guardians of the participants. the consent forms contained information on the study’s purpose, procedures, potential risks, benefits, confidentiality, and publishing of the study’s results. study design and population this was a hospital-based cross-sectional study. the study was carried out from 10 february to 28 june 2023 in the paediatrics clinic, units, and wards of jos university teaching hospital, located at lamingo, jos, in the north local government area of plateau state, north-central nigeria. the minimum sample size to obtain data that is scientifically acceptable was calculated using the thrustfield10 formula. the expected proportion in population based on previous studies10 was 8%. one hundred children aged 0 to 5 years, seen at the paediatrics clinic, units, and wards of jos university teaching hospital, and whose legal parent(s) or guardian(s) gave their assent, were recruited consecutively for this study until the required sample size was reached. sample and data collection questionnaire administration a structured questionnaire was administered to each child’s legal parent or guardian by a trained member of the research team. the questionnaires were filled by the educated parents/guardians or individually interpreted for the uneducated ones. the questionnaire contained sections that requested information on aspects of socio-demography and g6pd deficiency awareness. blood sample collection and preparation a 2 ml venous blood sample was aseptically collected from each child by an experienced paediatrician into ethylenediaminetetraacetic acid plastic sterile vacutainer containers (bd vacutainer®; becton dickinson, ltd., franklin lakes, new jersey, united states), mixed gently, and transported to the laboratory in a cold box for immediate laboratory analysis of g6pd and erythrocyte indices. laboratory analysis the haemoglobin concentration (g/dl), erythrocyte values (x1012/l) and packed cell volume (%) were obtained using an automated haematology analyser (accu cell dx360 3parts; shenzhen icubio biomedical technology co., ltd, shenzhen, china). the reference range for haemoglobin concentration for children aged ≤ 5 years is ≥11.0 g/dl.11 children with a haemoglobin concentration lower than 11 g/dl were considered anaemic12 in this study. glucose 6-phosphate dehydrogenase estimation was carried out quantitatively using the icubio ichaem-535 chemistry automated analyser (shenzhen icubio biomedical technology co., ltd, shenzhen, china), based on the principle as explained by pferffer et al.6 glucose-6-phosphate dehydrogenase catalyses the first step in the pentose phosphate shunt, oxidising glucose-6-phosphate (g-6-p) to 6-phosphogluconate (6-pg) and reducing nadp+ to nadph. the rate of formation of nadph is proportional to the g6pd activity, and is measured as an increase in absorbance at 340 nm. erythrocyte g6pd activity reference range was 6.97 iu/ghb – 20.5 iu/ghb (at 37°c) for children.13 children with erythrocyte g6pd activity lower than 6.97 iu/ghb were considered g6pd deficient. data analysis data were entered into excel spreadsheet software and analysed using the international business machine statistical package for social science version 26 (ibm corp., armonk, new york, united states). test for normality was done using kolmogorov-smironv test with lilliefors correction. chi-square was used to analyse for the categorical variables. the z-test was used to test for significance in the mean values of the numerical variables. the means for participants’ erythrocyte g6pd activity were compared using one-way analysis of variance, and p < 0.05 at a 95% confidence interval were considered a significant difference. results distribution of the study population one hundred children participated in this study (table 1). two-thirds (66/100) were aged less than 1 year. there were slightly more boys (n = 54/100) than girls (n = 46/100). table 1: distribution of the study population (100 children seen in paediatrics units of jos university teaching hospital, nigeria, from april 2023 to june 2023) by age and gender. mean erythrocyte glucose-6-phosphate dehydrogenase activity of the children forty patients (40%) were g6pd deficient, with a mean g6pd activity of 4.07 iu/ghb ± 1.08 iu/ghb, while 60 (60%) had normal g6pd activity, with a mean of 11.58 iu/ghb ± 4.28 iu/ghb (p < 0.001) (table 2). table 2: mean erythrocyte glucose-6-phosphate dehydrogenase activity of 100 children seen in paediatrics units of jos university teaching hospital, nigeria from april 2023 to june 2023. erythrocyte glucose-6-phosphate dehydrogenase status distribution by gender of the children fifty-four (54%) of the 100 children were male. of the 40 g6pd-deficient children, 19 (n = 19/40) were male (p = 0.29) (table 3). table 3: erythrocyte glucose-6-phosphate dehydrogenase status distribution by gender of 100 children seen in paediatrics units of jos university teaching hospital, nigeria from april 2023 to june 2023. mean activity of erythrocyte glucose-6-phosphate dehydrogenase by age and gender of the children mean g6pd activity was non-significantly (p = 0.83) higher among the children aged 1 to 3 years compared to the other age groups, and lower (p = 0.29) among the female patients (7.82 iu/ghb ± 4.91 iu/ghb) compared to the male patients (9.24 iu/ghb ± 5.08 iu/ghb). glucose-6-phosphate dehydrogenase activity was non-significantly associated with both age and gender (f = 0.34, p = 0.83 [age]; f = 1.13, p = 0.29 [gender]) (table 4). table 4: the mean activity of erythrocyte glucose-6-phosphate dehydrogenase by age and gender of 100 children seen in paediatrics units of jos university teaching hospital, nigeria from april 2023 to june 2023. prevalence of glucose-6-phosphate dehydrogenase deficiency-induced anaemia in the children mean packed cell volume was lower in anaemic children (30.92% ± 6.07%) compared to non-anaemic children (34.50% ± 7.11%), which was statistically significant (p = 0.008). glucose-6-phosphate dehydrogenase deficiency-induced anaemia was found in 23 (57.5%) of the children, while 28 (46.7%) of the anaemic children were non-g6pd deficiency induced. seventeen (42.5%) of g6pd-deficient children were not anaemic and 32 (53.3%) children with normal g6pd activity had normal packed cell volume values (p = 0.29) (table 5). table 5: prevalence of glucose-6-phosphate dehydrogenase deficiency-induced anaemia in 100 children seen in paediatrics units of jos university teaching hospital, nigeria, from april 2023 to june 2023. parental level of knowledge of glucose-6-phosphate dehydrogenase deficiency by glucose-6-phosphate dehydrogenase activity of children thirty-four (40%) children of 85 parents who had no knowledge of g6pd and its deficiency were g6pd deficient. six children of 14 parents who had slight knowledge of g6pd were g6pd deficient (table 6). table 6: parental level of knowledge of glucose-6-phosphate dehydrogenase deficiency by glucose-6-phosphate dehydrogenase activity of children seen in paediatrics units of jos university teaching hospital, nigeria, from april 2023 to june 2023. discussion we report the prevalence of erythrocyte g6pd deficiency-induced anaemia in children in a tertiary hospital setting in north-central nigeria. from this study, there was a high (40%) prevalence of g6pd deficiency, with a mean g6pd activity score of 4.07 iu/ghb ± 1.08 iu/ghb for g6pd-deficient children. glucose-6-phosphate dehydrogenase deficiency was not ageor gender-dependent (p = 0.83 [age]; p = 0.29 [gender]). forty (40%) of the children were anaemic and 23 (57.5%) had g6pd deficiency-induced anaemia. the high prevalence of g6pd deficiency observed in this study is similar to that (40.7%, and mean 3.79 iu/ghb ± 1.37 iu/ghb) reported by jatau et al. in 2019,11 in jos, north-central nigeria, among icteric neonates. however, it is higher than the 1.50 iu/ghb ± 0.02 iu/ghb reported by uko et al.14 in calabar, south-south nigeria. the discrepancy between the result of this study and the cited work may be because of differences in assay methods, promptness in assaying for g6pd after sample collection, and level of malaria endemicity in the geographical locations. the nigeria malaria indicator survey15 reported that malaria was more endemic in north-central nigeria compared to the south-south region of the country. the association between g6pd deficiency and malaria is particularly significant.16 glucose-6-phosphate dehydrogenase-deficient individuals have a selective advantage against malaria infection, as the malaria parasite (plasmodium spp.) is vulnerable to oxidative damage. the altered red blood cells in g6pd-deficient individuals create an inhospitable environment for the parasite, making it more difficult for the parasite to survive and reproduce. as a result, individuals with g6pd deficiency have a reduced risk of developing severe forms of malaria caused by p. falciparum. this selective advantage has contributed to a higher prevalence of g6pd deficiency in regions where malaria is endemic.17 thus, it may be necessary to carry out a further study on the occurrence of malaria-induced anaemia in g6pd-deficient children in our region. in addition, g6pd deficiency was neither age nor gender-dependent in this study. glucose-6-phosphate dehydrogenase deficiency is an x-linked disorder, where the enzyme deficiency occurs more in male than in female individuals, because the males only have one x-chromosome when compared to the females, who have two x-chromosomes.11 however, from this study, more of the female patients (45.7%) were g6pd deficient (x = 7.82 iu/ghb ± 4.91 iu/ghb) compared to the males (35.2%) (x = 9.24 iu/ghb ± 5.08 iu/ghb). this agrees with the finding by iheanacho et al. in 2017,18 on g6pd deficiency by gender in children at vwang village, vom, jos south, plateau state, nigeria, where g6pd deficiency was found to be higher in girls (23.3%) when compared to boys (19.8%). ours, however, is contrary to the finding by albagshi et al.,19 who reported a higher prevalence among male children in eastern saudi arabia. cappellini and fiorelli, in 2008,13 noted that in geographical regions where the frequency of the g6pd-deficient allele is high, homozygous females are not rare. thus, there is a need for molecular and genetic characterisation of the g6pd-deficient children within our region and beyond to provide clarity on the comprehensive aetiology of the deficiency. also, in this study it was observed that there was a non-significant (p = 0.83) increase in serum g6pd activity from 8.54 iu/ghb ± 5.41 iu/ghb in children less than 1 year to 9.12 iu/ghb ± 4.08 iu/ghb as age increased between 1 and 3 years; however, there was a decrease in the activity (8.58 iu/ghb ± 5.01 iu/ghb) with further rise in age. age was noted not to have a significant (f = 0.34, p = 0.83) effect on g6pd activity. this implies that g6pd deficiency does not depend on age, as was also noted by iheanacho et al.18 it is a genetic condition present from birth. therefore, the g6pd activity remains relatively stable throughout a person’s lifetime.12 there was a high (57.5%) prevalence of g6pd-induced anaemia among children attending jos university teaching hospital, nigeria. anaemia is one of the major causes of paediatric death and can affect a child’s mental, physical, and social development. with the decrease in iron levels, it can lead to poor cognitive function, thus negatively affecting a child’s academic performance and further complications in later life.9 this implies that it may be necessary to include the g6pd test as part of paediatrics routine screening tests. this will help in early detection of g6pd deficiency and in creating awareness early regarding crisis-predisposing factors such as malaria infestation, consumption of flavin-containing foods, administration of oxidative antibiotic or antimalarial drugs, and the required preventive measures. this will also aid in prompt proper treatment and case management in times of oxidative stress crisis in these subjects. anaemia could also be caused by other factors, such as poor nutrition,20 as 28 (46.7%) of the anaemic children were non-g6pd deficient. this study also showed a poor level of awareness of g6pd and its deficiency among the people, as almost 99% of them had either ‘no’ or ‘slight’ knowledge of g6pd and its deficiency before enlightenment talks from the research team. this tallies with the report of williams et al.,21 who stated that there is a low level of awareness of g6pd deficiency among lay people in nigeria. limitation of the study the limitations of this study include small sample size and recall bias, which is inherent in this type of study. also, because of financial restrictions, we were not able to conduct a molecular analysis of g6pd. this could have helped to differentiate the g6pd genotypes. conclusion there is a high prevalence of g6pd deficiency and g6pd deficiency-induced anaemia among the children in this study, and a very poor level of awareness of this defect among the parents and guardians. this could suggest the need to screen children, especially neonates, for g6pd deficiency, to allow for early detection of this abnormality. acknowledgements we acknowledge the tremendous assistance received from the staff of the paediatric clinic and units in the course of sample collection for this study. our special thanks go to mr philip lekshak, mr bitrus for helping in the sample collection, mrs gladys for helping to organise the patients, mrs hope zanko for running the laboratory analysis, mr ishaya zhakom and mr deffi favour for being involved in statistical analysis and interpretation and prof. james adisa for reviewing and editing the work. this article is partially based on the author’s undergraduate research project entitled ‘cross sectional study on the prevalence of erythrocyte glucose-6 phosphate dehydrogenase activity in children attending jos university teaching hospital’ towards the degree of bachelor of medical laboratory science in the department of medical laboratory science, university of jos, nigeria, on 13 august 2023, with supervisor dr caroline amuche okoli. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions j.d.n. was involved in data collection, sample collection, laboratory analysis, and write-up of the original draft. c.a.o. conceptualised the study, supervised it, validated the methods and results, was involved in statistical analysis and interpretation, and was involved in editing and reviewing the article. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the data that support the findings of this study are available on request from the corresponding author, c.a.o. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. the article does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the authors are responsible for this article’s results, findings, and content. references muzaffar ih, boundaqji kr, khatib ha, et al. an overview of the most common enzyme defect, glucose-6-phosphate-dehydrogenase deficiency. ejhm. 2018;70(1):102–108. https://doi.org/10.12816/0042970 world health organization. screening for g6pd deficiency in children and neonates in malaria-endemic regions. geneva: who; 2019. howes re, dewi m, piel fb, et al. spatial distribution of g6pd deficiency variants across malaria-endemic regions. malar j. 2013;12(1):1–15. https://doi.org/10.1186/1475-2875-12-418 eyo-ita eu, ogunsanya b, osazee em, eyo-ita ia, omoigberale a. glucose-6-phosphate dehydrogenase deficiency in a nigerian neonate with acute bilirubin encephalopathy triggered by ciprofloxacin. j med case rep case series. 2023;4(13). https://doi.org/10.38207/jmcrcs/2023/aug04130385 world health organization. haemoglobin concentration for the diagnosis of anaemia and assessment of severity [homepage on the internet]. vitamin and mineral nutrition information system. who/nmh/nhd/mnm/11.1. geneva: world health organization; 2011 [cited 2025 jan 20]. available from: https://www.who.int/publications/i/item/who-nmh-nhd-mnm-11.1 pferffer m, daniel a, ley b, et al. quantification of glucide-6 phosphste dehydrogensr ctivity by spectrophotometry: a systematic review and meta-analysis. plos med. 2020;17(5):1003084. https://doi.org/10.1371/journal.pmed.1003084 pengboon p, thamwarokun a, changsri k, kaset c, chomean s. evaluation of quantitative biosensor for glucose-6-phosphate dehydrogenase activity detection. plos one. 2019;14(12):226–927. https://doi.org/10.1371/journal.pone.0226927 eziokwu as, angelini d. new diagnosis of g6pd deficiency presenting as severe international journal of healthcare sciences issn 2348-5728 (online) rhabdomyolysis. cureus. 2018;10(3):1–4. https://doi.org/10.7759/cureus.2387 mann dm, swahn mh, mccool s. undernutrition and malaria among under-five children: findings from the 2018 nigeria demographic and health survey. pathog glob health. 2021;115:423–433. https://doi.org/10.1080/20477724.2021.1916729 thrustfield m. veterinary epidemiology. 2nd ed. oxford: blackwell science ltd.; 1995, p. 178–198. jatau e, toma o, egesie o, et al. glucose-6-phosphate dehydrogenase activity in newborn in jos: a necessary evaluation for icteric neonates. j biomed res clin pract. 2019;2(4):191–196. https://doi.org/10.46912/jbrcp.127 luzzatto l, ally m, notaro r. glucose-6-phosphate dehydrogenase deficiency. blood. 2020;136(11):1225–1240. https://doi.org/10.1182/blood.2019000944 cappellini md, fiorelli g. glucose-6-phosphate dehydrogenase deficiency. lancet. 2008;371(9606):64–74. https://doi.org/10.1016/s0140-6736(08)60073-2 uko ek, agwunobi sn, udoh jj. glucose-6-phosphate dehydrogenase levels in jaundiced neonates in calabar. niger j med. 2003;2:98–102. national malaria elimination programme (nmep), national population commission (npopc), national bureau of statistics (nbs), and icf international. nigeria malaria indicator survey 2015. abuja, nigeria, and rockville, maryland, usa: nmep, npopc, and icf international. 2016. [cited 2025 jun 02]. available from: https://www.nigerianstat.gov.ng/pdfuploads/mis20.pdf jeremiah z, jeremiah t, emelike f. frequencies of some human genetic markers and their association with plasmodium falciparum malaria in the niger delta, nigeria. j vector borne dis. 2010:47(1):11. taleb m, brahim at, yebouk c, et al. epidemiology of glucose-6-phosphate dehydrogenase deficiency in 10 malaria-endemic countries in sub-saharan africa: a systematic review. pan afr med j. 2025;50:7. https://doi.org/10.11604/pamj.2025.50.7.45066 iheanacho c, etuka c, ugwu bk, nnanna o. glucose-6-phosphate dehydrogenase deficiency in children at vwang village, vom, jos south, plateau state, nigeria. ijhs. 2017;5(1):588–593. albagshi mh., alomran s, sloma s, alsuweel a, alkhalaf h. prevalence of glucose-6-phosphate dehydrogenase deficiency among children in eastern saudi arabia. cureus. 2020;12(10):1–3. https://doi.org/10.7759/cureus.11235 morsy n, alhady s. nutritional status and socio-economic conditions influencing prevalence of anaemia in pregnant women. ijstr. 2014;3(7):54–60. williams o, gbadero d, eduwnorum g, brearkey a, shasher t, clundu t. glucose-6-phosphate dehydrogenase deficiency in nigeria children. plos one. 2013;8(7):e68800. https://doi.org/10.1371/journal.pone.0068800 abstract introduction methods results discussion acknowledgements references about the author(s) jean f. kloppers department of haematology and cell biology, school of pathology, faculty of health sciences, university of the free state, bloemfontein, south africa department of haematology and cell biology, universitas academic unit, national health laboratory services, bloemfontein, south africa andré de kock department of haematology and cell biology, school of pathology, faculty of health sciences, university of the free state, bloemfontein, south africa department of haematology and cell biology, universitas academic unit, national health laboratory services, bloemfontein, south africa johané cronjé department of haematology and cell biology, school of pathology, faculty of health sciences, university of the free state, bloemfontein, south africa department of haematology and cell biology, universitas academic unit, national health laboratory services, bloemfontein, south africa anne-cecilia van marle department of haematology and cell biology, school of pathology, faculty of health sciences, university of the free state, bloemfontein, south africa department of haematology and cell biology, universitas academic unit, national health laboratory services, bloemfontein, south africa citation kloppers jf, de kock a, cronjé j, van marle a-c. molecular characterisation of npm1 and flt3-itd mutations in a central south african adult de novo acute myeloid leukaemia cohort. afr j lab med. 2021;10(1), a1363. https://doi.org/10.4102/ajlm.v10i1.1363 original research molecular characterisation of npm1 and flt3-itd mutations in a central south african adult de novo acute myeloid leukaemia cohort jean f. kloppers, andré de kock, johané cronjé, anne-cecilia van marle received: 17 aug. 2020; accepted: 19 mar. 2021; published: 30 june 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: recognition of molecular abnormalities in acute myeloid leukaemia (aml) has improved our understanding of its biology. npm1 and flt3-itd mutations are recurrent in aml and clinically significant. npm1 mutations are associated with a favourable prognosis, while flt3-itd mutations are an independent poor prognostic factor in aml. objective: this study described the prevalence and molecular characteristics of the npm1 and flt3-itd mutations in a newly diagnosed aml patient cohort in central south africa. methods: the study included 40 de novo aml patients. an npm1 and flt3-itd multiplex polymerase chain reaction assay was optimised to screen patients for the respective mutations and were confirmed using sanger sequencing. the prevalence of the npm1 and flt3-itd mutations were determined, and mutation-specific characteristics were described in relation to patients’ demographic information and aml classifications. results: the patients’ median age was 38.5 years, with 77.5% (n = 31) of patients being self-proclaimed black africans. aml with recurrent genetic abnormalities was most prevalent (57.5%; n = 23), of which acute promyelocytic leukaemia (apl) was most common (40.0%; n = 16). none of the patients had the npm1 mutation. flt3-itd was present in 37.5% (6/16) of apl patients and in one (20.0%) of five aml patients with a t(8;21) translocation. most patients had an flt3-itd allele ratio of ≥ 50% and itd lengths of > 39 bp. conclusion: flt3-itd mutations were mainly found in apl cases at a similar prevalence as reported in the literature. high flt3-itd allele ratios and long itd lengths predominated. no npm1 mutations were detected. keywords: acute myeloid leukaemia; aml; npm1; flt3-itd; frequency; south africa. introduction over the past decade, the field of acute myeloid leukaemia (aml) diagnostics has shifted from a primarily clinicopathological assessment to an integrated approach, including morphology, immunophenotyping, cytogenetics, and molecular genetics.1,2 in an era of precision medicine, the use of molecular genetic data, in particular, has fundamentally shaped the diagnostic approach to patients with aml.3 genetic profiling has led to more accurate diagnostic classification and improved risk stratification and identification of potential therapeutic targets.1 acute myeloid leukaemia is a genetically heterogeneous disease.2 recurrent structural chromosomal aberrations detected by conventional cytogenetics were previously considered the most relevant prognostic variable in addition to the patient’s age and performance status.2,3 three main cytogenetic risk groups (low, intermediate, and high) were defined, with approximately 50% of patients having a normal karyotype and falling into the intermediate cytogenetic risk group.3,4 the recognition of additional molecular abnormalities, especially in cytogenetically normal aml (cn-aml), has helped to improve our understanding of the disease biology and better predict outcomes.3,4 mutational analysis performed on diagnostic samples of aml patients enrolled in the eastern cooperative oncology group e1900 trial demonstrated somatic mutations in as many as 97.3% of these patients.5 the cancer genome atlas research group6 has shown that, on average, patients with aml have 13 acquired mutations at the time of diagnosis across the entire genome. two genes in which recurrent mutations, which often co-occur, with known clinical significance have been identified in patients with aml include the gene encoding nucleophosmin protein-1 (npm1) and one of the signalling genes, fms (feline mcdonough sarcoma gene)-related tyrosine kinase 3 (flt3).3,6 npm1 mutations are associated with a favourable prognosis,3 while the flt3-internal tandem duplication (itd) mutation is considered an independent poor prognostic factor in patients with aml.7 however, the co-occurrence of these two gene mutations modulates their prognostic impacts.3,7 nucleophosmin is a nucleolar phosphoprotein that shuttles between the nucleus and the cytoplasm.3 nucleophosmin protein-1 is thought to be involved in many cellular functions, including protein synthesis, dna replication, and regulation of the cell cycle.3 the reported frequency of npm1 mutations in patients with aml varies between 27% and 30% and is considered the most frequently identified molecular abnormality in cn-aml, occurring in 45% – 60% of cases.4,6,8,9 four common mutations cause mutant npm1 (one 4 bp duplication and three separate 4 bp insertions). these mutations are all restricted to exon 12, the region that encodes the c-terminus of npm-1, and result in a frame shift.10 the flt3 gene encodes a class iii receptor tyrosine kinase. mutations in signalling genes such as flt3, which lead to constitutive activation of the receptor tyrosine kinase, confer survival and proliferative advantage to cells.3,9 fms-related tyrosine kinase 3 mutations occur in approximately one-third of patients with aml, and the itd mutation is considered the second most common molecular abnormality in cn-aml (28% – 34% of cases).4,9 fms-related tyrosine kinase 3-itd mutations usually occur between exons 14 and 15 and can range between 3 bp and more than 400 bp in size.11 the limited reports on the prevalence of the flt3-itd and npm1 mutations in african aml populations indicate lower frequencies than reported internationally. in an egyptian study that included 123 aml patients, flt3-itd and npm1 mutations were detected in 17.9% and 19.5% of patients.12 another study by sofan et al. (2014) detected npm1 mutations in 28% of cn-aml.13 contrary to these findings, no npm1 mutations were found in a study of 100 sudanese aml patients.14 the frequency of npm1 and flt3-itd mutations in a south african adult de novo aml cohort was 7.5% and 12%.15 this study investigated the presence and molecular characteristics of the npm1 and flt3-itd mutations in a newly diagnosed aml population in central south africa. the study further aimed to describe the npm1 and flt3-itd mutations in relation to patient demographics and specific aml classifications. methods ethical considerations approval for the study was obtained from the health sciences research ethics committee from the university of the free state (study approval number: ufs-hsd2018/1174/2711) and the free state province department of health (study approval number: fs_201810_016). written informed consent was obtained from all participants prior to sample collection. samples were allocated study numbers to ensure patient confidentiality, and patient data were stored on password-protected devices, which were only accessible to the researchers. study population all adult patients (n = 40; coded fn1–fn40) that were diagnosed with de novo aml at the universitas academic hospital in bloemfontein, south africa, during the study period (november 2018 – december 2019), and who were able to give written informed consent were included in this study. all aml patients were included regardless of cytogenetic findings. patients’ demographic variables (age, sex, and self-proclaimed ethnic background) and disease-specific characteristics (aml classification according to the world health organization’s classification of tumours of haemopoietic and lymphoid tissues, revised 4th edition16) were obtained from patients’ medical records. based on the laboratory information records that were reviewed, none of the patients had any previous clonal myeloid disorder. blood samples were obtained from each participant for this study during routine blood collection by the treating physician. dna extraction genomic dna was extracted from whole blood samples using the wizard® genomic dna purification kit (promega, madison, wisconsin, united states). dna samples were quantified using the biodrop™ µlite instrument (biodrop, cambridge, united kingdom). npm1 and flt3-itd multiplex pcr optimisation anonymised samples that were positive and negative for the npm1 and flt3-itd mutations and formed part of an external quality control programme (generously donated by the department of haematology at charlotte maxeke johannesburg academic hospital, national health laboratory service, johannesburg, south africa) were used to optimise the assay. published primers, fluorescently labelled with hexadecimal colour and fluorescein amidites fluorophores17 were used in the assay (npm1 forward: 5’gtt tct ttt ttt ttt ttt cca ggc tat tca ag3’; reverse: 5’hex cac ggt agg gaa agt tct cac tct gc3’; and flt3-itd forward: 5’famagca att tag gta tga aag cca gcta3’; reverse: 5’ctt tca gca ttt tga cgg caa cc3’). primers were evaluated for target-specificity using the basic local alignment search tool (available at https://blast.ncbi.nlm.nih.gov/blast.cgi) from the national center for biotechnology information (ncbi, bethesda, maryland, united states). a polymerase chain reaction (pcr) mixture was prepared with the gotaq® dna polymerase (promega madison, wisconsin, united states) reagents. components of the kit were manually mixed and the mixture consisted of 5 µl 5х gotaq buffer, 2 µl of mgcl2, 0.5 µl of dntps, and 0.2 µl of gotaq dna polymerase (5 u/µl). additionally, 1 µl of genomic dna (100 ng/µl), 14.3 µl of nuclease-free water, and 1 µl each of the forward and reverse primers (5 µm) (for flt3 and npm1) were added to the pcr mixture. a temperature gradient experiment was used to determine the optimal pcr annealing temperature for the primers. the gradient temperatures ranged between 65 °c and 70 °c. the subsequent pcr cycling conditions were one cycle at 95 °c for 10 min, 45 cycles of 95 °c for 20 s, 65 °c for 40 s, and 72 °c for 40 s, and a final step of one cycle at 72 °c for 32 min. polymerase chain reaction products were subjected to capillary electrophoresis (3500 genetic analyser, applied biosystems, foster city, california, united states). briefly, 1 µl of each sample was mixed with 9.5 µl of hi-di™ formamide (applied biosystems, foster city, california, united states) and 0.5 µl genescan™ 600 liz™ size standard (applied biosystems, foster city, california, united states). the optimal primer concentrations (5 µm) and dna limit of detection were determined as part of the optimisation procedure. the npm1 and flt3-itd multiplex pcr assay had a lower limit of detection of 0.8 ng/µl. capillary electrophoretic results were analysed using genemapper version 6 software (applied biosystems, foster city, california, united states). npm1 and flt3-itd detection in acute myeloid leukaemia patients the optimised npm1 and flt3-itd multiplex pcr was used to screen the 40 de novo aml patients for the respective mutations. positive and negative controls, as well as a no-template control, were included in each subsequent run for quality control purposes. the npm1 and flt3-itd wild-type amplicon sizes were expected to be 170 base pairs (bp) and 330 bp. the npm1 positive samples were expected to have an amplicon at 170 bp for the wild-type and an additional amplicon at 174 bp. fms-related tyrosine kinase 3-itd positive samples were expected to have an amplicon at 330 bp and an additional amplicon larger than 330 bp. the allele ratio for the respective mutations in each patient was calculated by determining the area under the curve for the mutant to wild-type alleles, expressed as a percentage. the respective positive and negative controls yielded the expected amplicon sizes upon evaluation of the capillary electrophoresis results (figure 1). figure 1: observed capillary electropherograms of the npm1 and flt3 control samples (february 2020, bloemfontein, free state, south africa). (a) npm1 positive control with an additional amplicon at 174 base pairs representing the 4 base pairs insertion, and wild-type flt3-itd at 330 base pairs. (b) flt3-itd positive control with an additional amplicon at 405 base pairs, representing the 75 base pairs internal tandem duplication and npm1 wild-type at 170 base pairs. (c) npm1 and flt3-itd negative control with expected amplicons at 170 base pairs and 330 base pairs. sanger sequencing of npm1 and flt3-itd amplicons npm1 and flt3-itd control samples, as well as the amplicons from the 40 patient samples, were sequenced to confirm and validate the pcr results. sequencing reactions for the npm1 and flt3-itd mutations were prepared separately using primers as listed above (primers for sequencing were not fluorescently labelled). the pcr products were purified using the exosap-it® express pcr product clean-up (affymetrix, santa clara, california, united states) as per the manufacturer’s instructions. sanger sequencing reactions were prepared using the bigdye™ terminator version 3.1 kit (applied biosystems, foster city, california, united states) as per the manufacturer’s instructions. briefly, the sequencing reaction consisted of 2 µl sequencing reaction mix (bigdye™ terminator v3.1), 1 µl sequencing buffer (bigdye™ terminator v3.1), 5 µl nuclease-free water, 1 µl forward primer (flt3 or npm1), 1 µl reverse primer (flt3-itd or npm1) and 1 µl pcr product. the sequencing reaction was subjected to the following cycling conditions in a 2720 thermal cycler (applied biosystems, foster city, california, united states): one cycle at 96 °c for 1 min, and 25 cycles at 96 °c for 10 s, 50 °c for 5 s, and 60 °c for 4 min. sequence reactions were cleaned up with the zymo research dna sequencing clean-up kit™ (promega madison, wisconsin, united states) as per the manufacturer’s instructions. sequence raw reads were loaded onto the 3500 genetic analyser (applied biosystems, foster city, california, united states). for comparison, sample sequences were aligned against npm1 (genbank accession: ng_016018.1) and flt3-itd (genbank accession: ng_007066.1) reference sequences retrieved from the national center for biotechnology information using the local pairwise sequence alignment algorithm software (available at https://embnet.vital-it.ch/software/lalign_form.html). sanger sequence analysis confirmed that the npm1 positive control had a 4 bp insertion (bases: catg), and the flt3-itd positive control had a 75 bp itd. the mutation-negative controls were compared to the respective reference sequences, and were found to be 100% similar. data analysis fragment analysis electropherograms (figure 1) were generated using the genemapper v6 software (applied biosystems, foster city, california, united states), and displayed the size of the alleles obtained for the target pcr fragments (applied biosystems, foster city, california, united states). flt3-itd allelic ratios were determined by calculating the ratio of the area under the curve of the flt3-itd mutant allele to the flt3 wild-type allele as displayed by genemapper. the sequence was analysed using the sequencing analysis program version 5.3.1 (applied biosystems, foster city, california, united states) and chromas version 2.6.6 (technelysium pty ltd, brisbane, australia). tables were generated using microsoft word 2016 (microsoft, redmond, washington, united states). patients’ demographic data and aml classification were summarised in table format. ethnicity, defined as either black, white or mixed race, was based on patients’ self-proclaimed identity and accordingly documented. results the aml patients (fn1 to fn40) were all successfully screened with the npm1 and flt3-itd multiplex pcr assay. the aml patient cohort had a median age of 38.5 years (range 18–85 years) at presentation and the ratio of women to men was 1:1.2. the cohort included 31 black african patients, four white patients, and five patients of mixed ethnicity. acute myeloid leukaemia subtypes in our patient cohort comprised of aml with recurrent genetic abnormalities, aml with myelodysplasia-related changes, and aml not otherwise specified (also referred to as cn-aml). acute myeloid leukaemia with recurrent genetic abnormalities was most prevalent (57.5%; n = 23), of which acute promyelocytic leukaemia (apl) was most common (40.0%; n = 16). none of the patients had the npm1 mutation. fms-related tyrosine kinase 3-itd was present in 37.5% (6/16) of apl patients and in one (20.0%) of five aml patients with a t(8; 21) translocation (table 1). patients with an flt3-itd mutation had a median age of 30.8 years at presentation, and the majority of patients were black africans (n = 5/7) (table 2). internal tandem duplication lengths of the flt3 mutations observed in this study ranged between 19 bp and 75 bp, and the flt3-itd allele ratios ranged between 17.5% and 94.9%. table 1: classification of acute myeloid leukaemia patients’ npm1 and flt3-itd genotypes (august 2020, bloemfontein, free state, south africa). table 2: acute myeloid leukaemia patients positive for the flt3-itd mutation (august 2020, bloemfontein, free state, south africa). discussion acute myeloid leukaemia is a genetically heterogeneous disease2 with numerous clinical and genetic factors influencing the final diagnosis. the recognition of additional molecular abnormalities, such as npm1 and flt3-itd in aml, has ensured that the disease biology is better understood and has allowed for improved risk stratification to be included in the diagnostic algorithms.3,4 this is the first study to investigate the presence of npm1 and flt3-itd in a central south african aml population. the npm1 was absent in this study population, while the flt3-itd mutation predominated in the apl subtype. furthermore, the majority of patients that were positive for the flt3-itd mutation had long itd lengths and allelic ratios above 50%. it was notable that the median age of our patient cohort was 38.5 years. the global median age at presentation for aml is 67 years18 and, in comparison, the current patient cohort presented with the disease at a considerably younger age. this finding was similar to a previous south african study, where adult aml patients presented with the disease at a median age of 41 years.15 most patients in this study were self-proclaimed black africans, and there was no predominant disease presentation based on sex. two other african studies found a similar age distribution and sex ratio.19,20 the flt3-itd mutation was only detected in patients with aml with recurrent genetic abnormalities, of which apl predominated. compared to a reported frequency of flt3-itd mutations in apl of up to 40%,16 our results were not surprising. however, flt3-itd mutations in aml with a t(8;21) translocation, detected in one of our patients, is thought to be uncommon, occurring in less than 10% of aml cases.21 the reported frequency of flt3-itd mutations in cn-aml in african populations varies significantly from 11% in south african patients to up to 34.6% in an egyptian cohort, which is more in line with data from high-income countries.9,15,22 the absence of flt3-itd in cn-aml patients in our study was unexpected, but could likely be attributed to the small study cohort. we recommend that the flt3-itd frequency should be investigated in a larger central south african cn-aml population. internal tandem duplication lengths of the flt3 mutations observed in this study ranged between 19 bp and 75 bp and were found in the juxtamembrane domain of the gene. according to liu et al.,23 longer itd lengths (more than 39 bp) have been associated with a worse prognosis, the possible reason being that longer insertions in the juxtamembrane domain may cause more significant disruption of the domain’s auto-inhibitory function.23 in our study, 71.4% (5/7) of the patients with flt3-itd mutations had an itd length of 39 bp or longer. further investigations are needed to determine whether the high prevalence of flt3-itd mutations in our apl cohort confers a worse prognosis. this investigation would be justified considering that the majority (4 of 6) of apl patients with flt3-itd mutations had allele ratios above 50% and in other aml subgroups, allelic ratios above 50% are stratified as an adverse prognostic risk.24 an interesting observation was the unusually high number of apl cases among our aml cohort, which, according to the revised 4th edition of the classification of tumours of haemopoietic and lymphoid tissues, only accounts for 5% – 8% of aml cases.16 notably, eight of our apl patients presented within a single month. several studies, including a south african study, have alluded to the association between apl clustering and seasonality.25,26,27 with such a small study cohort, one can only speculate that seasonality may have contributed and warrants further investigation. similar to a sudanese study involving 100 aml patients,14 none of the patients in this study had an npm1 mutation. however, npm1 mutations were detected in 28.3% of egyptian cn-aml patients and 7.5% of south african aml patients.13,15 the complete absence of the npm1 mutation in this study could be attributed to the low median age at disease presentation in the study cohort, especially considering that the frequency of npm1 mutations increases proportionally with age.28 in addition, npm1 mutations are more commonly associated with cn-aml,16 which accounted for only 35% (n = 14) of all cases in this study.4,9 the cancer genome atlas research network reported that nearly 50% of all aml patients are cn-aml cases, and the observed prevalence of cn-aml in the current study was lower.6 the lower prevalence of cn-aml relative to aml with recurrent genetic abnormalities in our study, and, by association, the lower prevalence of these two mutations commonly linked with cn-aml, might be attributed to race, as black patients were previously found to be more likely to have aml associated with cytogenetic abnormalities.18,29 limitations this study was limited by a small sample population. in addition, the predominance of apl cases in our cohort may have contributed to a biased prevalence of the flt3-itd mutation. an association between phenotype and genotype was also not determined. conclusion the flt3-itd mutation was mainly found in apl cases at a similar prevalence as reported in the literature. high flt3-itd allele ratios and long itd lengths predominated. no npm1 mutations were detected. the absence and lower frequency of npm1 and flt3-itd mutations could possibly be attributed to the low median age at presentation and the majority of patients presenting with aml with recurrent genetic abnormalities. acknowledgements the authors acknowledge the national health laboratory service for funding the project, ms leandi du plessis for technical assistance and dr daleen struwig for assistance with the technical and editorial preparation of the manuscript. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions j.f.k. was involved in the study design, assisted in performing the study, performed data analysis and contributed to writing the manuscript. a.-c.v.m. analysed clinical data and contributed to writing the manuscript. j.c. assisted in performing the study and critically evaluated the manuscript. a.d.k. was involved in the study design, supervised the study and critically evaluated the manuscript. all the authors approved the final version of the article prior to submission. sources of support funding was received from the national health laboratory service research development grant (grant004_94767). data availability data are available from the corresponding author upon reasonable request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references shanmugam v, kim as. molecular versus morphological classifications of myeloproliferative neoplasms: you don’t know jak! the hematologist: ash news and reports [serial online]. 2019;16(1):12–13. 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2005;106(12):3747–3754. https://doi.org/10.1182/blood-2005-05-2168 sekeres ma, peterson b, dodge rk, et al. differences in prognostic factors and outcomes in african americans and whites with acute myeloid leukemia. blood. 2004;103(11):4036–4042. https://doi.org/10.1182/blood-2003-09-3118 abstract introduction methods results discussion acknowledgements references about the author(s) carla m. madeira national tuberculosis reference laboratory, instituto nacional de saúde, marracuene, mozambique khalide i. azam national tuberculosis reference laboratory, instituto nacional de saúde, marracuene, mozambique daisy n. sato american society for microbiology, são paulo, brazil celso khosa centro de investigação e treino em saúde da polana caniço, instituto nacional de saúde, marracuene, mozambique nilesh bhatt centro de investigação e treino em saúde da polana caniço, instituto nacional de saúde, marracuene, mozambique sofia o. viegas department of the laboratory network and reference services, instituto nacional de saúde, marracuene, mozambique citation madeira cm, azam ki, sato dn, khosa c, bhatt n, viegas so. evaluation of the ogawa-kudoh method for tuberculosis isolation in two health units in mozambique. afr j lab med. 2020;9(1), a929 https://doi.org/10.4102/ajlm.v9i1.929 original research evaluation of the ogawa-kudoh method for tuberculosis isolation in two health units in mozambique carla m. madeira, khalide i. azam, daisy n. sato, celso khosa, nilesh bhatt, sofia o. viegas received: 30 oct. 2018; accepted: 07 apr. 2020; published: 20 july 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: mozambique is among the highest tuberculosis, tuberculosis–hiv and multidrug-resistant-tuberculosis burden countries. although molecular technologies are available in-country, mycobacterial isolation through culture remains an important tool for tuberculosis diagnostics and drug susceptibility testing. objective: we evaluated the use of the ogawa-kudoh (ok) mycobacterial culture, a simple technique, to isolate mycobacterium tuberculosis in two health units, in maputo city, mozambique. methods: from may to december 2014, 122 patient samples were collected in chamanculo general hospital and polana caniço general hospital. the specimens were first tested in the health units using the ok method and afterwards shipped to the national tuberculosis reference laboratory for mycobacterial culture using the nalc-naoh-citrate (nalc) decontamination method followed by inoculation in lowenstein jensen (lj) solid media as the reference standard. results: among 107 samples with valid results, 98 (91.6%) had concordant results in both methods; 9 (8.4%) had discordant results. the contamination rate was 4.1% (5/122) for the ok and 9.0% (11/122) for the nalc/lj methods. the sensitivity of ok was 80% (95% confident interval [ci]: 51.4–94.7) and the specificity was 94% (95% ci: 85.8–97.3). the degree of agreement between both methods was moderate (kappa: 0.68; 95% ci: 0.48–0.89). conclusion: the ok method showed satisfactory sensitivity and specificity. the method also had a lower contamination rate when compared to the nalc/lj. similar to other studies in resource-limited settings, our findings showed that the ok method can effectively be implemented in settings with limited laboratory capacity to isolate tuberculosis bacteria by culture for further testing. keywords: ogawa-kudoh; mycobacterial culture; tuberculosis diagnostics; mozambique; laboratory. introduction mozambique is amongst the highest tuberculosis, tuberculosis–hiv and multidrug-resistant-tuberculosis burden countries.1 at present, the country has a tuberculosis laboratory network comprised of three tuberculosis reference laboratories, 85 sites performing xpert® mtb/rif (cepheid, sunnyvale, california, united states) and 400 smear microscopy laboratories. mycobacterial culture is only performed in the three tuberculosis reference laboratories, using both solid lowenstein jensen (lj) media and liquid mycobacterial growth indicator tubes for simultaneous analysis. the national testing algorithm recommends mycobacterial culture for xpert® mtb/rif-resistant cases and for treatment monitoring of resistant cases in order to isolate mycobacterium tuberculosis for further drug susceptibility testing. mycobacterial culture is considered the reference standard for tuberculosis diagnosis. however, one of its major constraints is sample transportation from the collection site to the reference laboratory. in most cases, long transportation distances are associated with the need for a cold chain, prompting high contamination rates in the presence of fast-growing bacteria, and that can lead to under-diagnosis of tuberculosis. another constraint on the performance of mycobacterial cultures is the need for high-level containment laboratories, because of testing procedures, and the need for qualified laboratory staff.2,3,4 these requirements come with high costs, which contribute to challenges associated with implementing mycobacterial culture in rural areas.4 in order to detect and treat all tuberculosis cases, it is necessary to expand simple and accessible diagnostic laboratory services, especially in resource-limited countries such as mozambique. in 1974, kudoh and kudoh described a simple, rapid and inexpensive mycobacterial culture method, the ogawakudoh (ok) method,5 which does not require high-level laboratory containment or extensive technical training.5 evaluating the applicability of the ok mycobacterial method opens up the possibility for less specialised laboratories to carry out tuberculosis culture, increasing the diagnosis capacity in the country. the aim of this study was to evaluate the accuracy and applicability of the ok method, as compared to lj mycobacterial culture as the reference standard, for m. tuberculosis isolation in two peri-urban health units in maputo city, mozambique. methods ethical considerations ethical approval to conduct the study was obtained from the national bioethics committee (cnbs), ministry of health, mozambique, with the reference number 368/cnbs/13. the patients were included after understanding the study procedures and signing a written informed consent form. study setting the study was conducted in two health units in maputo city, chamanculo general hospital and polana caniço general hospital. both are level ii health units, located in peri-urban areas of maputo. the distance from the national tuberculosis reference laboratory in maputo to chamanculo general hospital is 4.5 km and to polana caniço general hospital is 2.3 km. at the time of the evaluation, both laboratories were only performing tuberculosis diagnosis by smear microscopy. requests for mycobacterial cultures from the two health units were sent to the national tuberculosis reference laboratory in maputo. patients and specimens this cross-sectional study was conducted between may 2014 and december 2014. a total of 122 samples, one per patient, categorised as new (i.e. patients with presumptive pulmonary tuberculosis who had never been treated for tuberculosis or had been treated for less than 30 days) or previously-treated (i.e. patients with presumptive pulmonary tuberculosis who had been treated for tuberculosis for more than 30 days), were consecutively included in the study. the specimens were first tested in the health units using the ok method and the remaining samples were shipped to the national tuberculosis reference laboratory in maputo for mycobacterial culture using the n-acetyl-l-cysteine (nalc)-sodium hydroxide (naoh)-citrate decontamination method, followed by inoculation in lj solid media, which was used as a reference standard. auramine-o staining was also performed at chamanculo general hospital, and a ziehl-nielsen smear microscopy was performed at polana caniço general hospital. a 5-day training schedule was performed for laboratory technicians from the two health units, which also included biosafety and ok technical training. staff technical competency was also evaluated. standard operational procedures and registration forms were implemented for the study and supervision visits were performed once a week at each participating site. basic patient demographics were collected from laboratory request forms. ogawa-kudoh method the ok method was performed as described by kudoh and kudoh.5 briefly, sputum samples were impregnated in a swab. the impregnated swab was placed in a sterile tube containing 3 ml of 4% naoh solution for 2 minutes and then inoculated with rotary movements in the ok media. mycobacterial cultures were incubated at 36 ± 1 °c for up to 60 days. nalc-naoh-citrate/lj method equal volumes of sputum sample and nalc-naoh-citrate reagent were added to a 50 ml conical tube, after which the mixture was stirred and allowed to stand for 15 min at room temperature. after 15 min, 35 ml 0.067m phosphate buffer (ph 6.8) was added to the mixture. the mixture was then mixed by inversion and centrifuged at 3000 g for 15 min. the supernatant was discarded and the pelleted material was resuspended in 1 ml of buffer. from the suspension, 0.5 ml was inoculated into solid lj medium and incubated at 37 °c for up to 8 weeks. tubes were read weekly to verify the presence of mycobacteria.6 the use of lj as the reference standard method, instead of the liquid mycobacterial growth indicator tube culture, which had higher sensitivity and higher contamination rates when compared to lj, was made in order to establish a direct comparison between the two solid media methods (ok and lj). a smear microscopy examination of the suspension was also performed. statistical analyses the culture contribution to diagnostics was calculated based on the recovery rate for both methods, ok and lj, as the ratio between positive cultures and negative smear microscopy. a database was created in microsoft office access (2007, version 12; microsoft corp, redmond, washington, united states), and was later exported to ibm spss statistics for windows (2015, version 23.0; ibm corp., armonk, new york, united states) for analysis of sensitivity, specificity, predictive values (positive and negative) and the degree of agreement between the methods. concordance between the tests was analysed using the friedman kappa test. to verify whether there were associations between the performance of the methods, and the demographic characteristics and categories of patients, the chi-square test was performed. the level of statistical significance was set to 0.05 (two-sided) for all analyses. formulas to calculate sensitivity, specificity, positive predictive value, and negative predictive value were used as previously described.7 levels of agreement were interpreted as follows: values greater than or equal to 0.75 were interpreted as having ‘excellent’ concordance between the two variables; values between 0.4 and 0.75, ‘sufficient to good’ agreement; and values smaller than 0.40, ‘weak’ agreement. results of the 122 samples analysed, 60 were from female patients and 62 were from male patients. the median age was 36 years (range:8–70 years). regarding the category of the patients, most cases had previously been treated (n = 84, 68.9%) and 38 cases were new (31.1%) (data not shown in tables). a total of 98 (80.3%) patients had concordant culture results for both methods, 9 (7.4%) had discordant results and 15 (12.3%) had contaminated results (10 samples were contaminated on lj only, 4 contaminated on ok only, and 1 was contaminated on both methods). the contamination rate for ok was 4.1% (5/122) and for nalc/lj, 9.0% (11/122). against the nalc/lj method as the reference standard, and excluding contaminated results, the sensitivity of the ok method was 80% (12/15; 95% ci: 51.4–94.7), the specificity was 93.5% (86/92; 95% ci: 85.8–97.3), the proportion of patients with true-negative results in both techniques was 96.6% (86/89; 95% ci: 89.9–99.1), and the proportion of patients with true-positive results using both techniques was 66.7% (12/18; 95% ci: 41.2–85.6). the agreement between the two methods was kappa = 0.68 (95% ci: 0.48–0.89) (table 1). table 1: comparison between ogawa-kudoh and nalc-naoh/lowenstein jensen methods, mozambique, 2014. for the comparison between the two culture methods, contaminated results were also excluded from the analysis. mycobacterial culture positivity was 17/111 (15.3%) for nalc/lj and 18/117 (15.4%) for the ok method. the recovery rate was similar for both methods, 17.1% (18/105) for ok and 17.9% (17/95) for lj (tables 2 and 3). table 2: results ratio of mycobacterial culture and direct microscopy, mozambique, 2014.† table 3: results ratio of mycobacterial culture and direct microscopy, mozambique, 2014.† discussion in this study, sensitivity, specificity, positive predictive value and negative predictive value were acceptable for the ok method. in addition, there was a high degree of agreement and the recovery rate was similar between the ok and nalc/lj methods. the contamination rate of the nalc/lj method was twice as high as the ok method. this finding reinforces the capacity of ok to kill other contaminants, since the ok media is slightly more acidic compared to lj and neutralises the high concentration of naoh used to decontaminate the sample. similar studies performed in brazil (in 1999, 2006, and 2018) and peru (in 2007) also found significant differences between the contamination rates obtained with ok and with standard mycobacterial culture methods, again showing that the ok method is efficient in recovering mycobacteria and efficiently killing contaminants.8,9,10,11 these results indicate the value of the ok mycobacterial method for diagnosis of pulmonary tuberculosis as a possible alternative for the nalc/lj method. the ok mycobacterial method has been used successfully in other countries, particularly in latin america, including brazil, as an alternative to traditional culture methods.4,12,13 the pan-american health organization recommends the use of ok for tuberculosis diagnosis in areas with limited laboratory capacity.14 a study conducted in uruguay, with the purpose of assessing the efficacy of the ok method for mycobacterial culture in sputum samples, found that after conserving and shipping specimens at room temperature without the addition of any substances to prevent the overgrowth of contaminating bacteria, the ok method was appropriate for culturing mycobacteria, even when processing was delayed for 2–4 days from collection.15 furthermore, the study showed that the duration of the decontamination time was not critical and satisfactory outcomes can still be obtained by increasing the decontamination time up to 4 min.15 in general, mycobacterial culture on lj, when performed on sputum samples, generates approximately 20% more positives than smear microscopy,4 because the sensitivity of smears is lower when compared to culture. access to mycobacterial culture is a challenge in mozambique, since there are only three tuberculosis reference laboratories, some health units are hard to access by road, the distances are long and the laboratory capacity is limited. additionally, mozambique is a tropical country, where the temperature can reach 40 °c during summer. the sample transport system is very mixed and relies on motorbikes, bicycles, ambulances or partners, and samples are not shipped daily. all of these factors contribute to delays in samples reaching the reference laboratories and to sample contamination, leading to inconclusive results. although advanced molecular methods, such as the xpert® mtb/rif, are available in mozambique, mycobacterial culture remains the reference standard for tuberculosis diagnosis. furthermore, culture is needed to isolate the bacteria for other tests, including drug susceptibility tests and sequencing in this environment of increasing resistance to tuberculosis drugs. the mycobacterial culture using the ok method is a simple-to-perform, low-cost method and its biosafety requirements present a lower risk to laboratory technicians, since it does not require agitation or centrifugation steps, and thus reduces the production of aerosols. however, establishment of this assay in peripheral laboratories requires biosafety training and awareness of the risks and precautions for manipulation of class iii pathogens. additionally, triple packing biosafety procedures must be implemented to ship positive samples from these laboratories to reference laboratories for further diagnostic assays, such as identification of the m. tuberculosis complex and drug susceptibility tests. limitations the present study was conducted at only two health facilities in maputo city, which might not provide the study with the power to generalise the findings. in addition, there may have been delays in shipping samples from the health centres to the national tuberculosis reference laboratory in maputo, as a result of transportation constraints. this can affect the quality of the specimen, allowing the growth of other bacteria and leading to contamination. liquid (mycobacterial growth indicator tube) culture results were not evaluated, and were not compared with the ok or lj mycobacterial culture methods. liquid culture is known to have higher sensitivity, and could have had implications for the ok or lj findings. re-decontamination and re-inoculation of the contaminated tubes, to increase valid results and to reduce contamination rates, was not considered for the present study. conclusion the ok method showed satisfactory sensitivity and specificity, with lower contamination rates and higher detection rates when compared to the nalc/lj method. the ok method can effectively be implemented in settings with limited laboratory capacity to isolate tuberculosis bacteria by culture for further testing. acknowledgements the authors acknowledge the national tuberculosis reference laboratory staff for their support during the study and their commitment to improve tuberculosis diagnosis in mozambique. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions c.m.m. conceived the study design, performed the laboratory tests, and wrote the first draft of the article. k.i.a. supervised the laboratory work and supported the data analysis. d.n.s. and s.o.v. provided general supervision during the study implementation and reviewed the manuscript draft. c.k. and n.b. participated in the data analysis, reviewed the draft and provided writing support. all authors gave final approval of the version to be published and agree to be accountable for the accuracy and integrity of the work. sources of support none. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views expressed in the submitted article are the authors’ own and not an official position of the institution or funder. references world health organization. 2018. global tuberculosis report 2018. world health organization. geneva, switzerland. european centre for disease prevention and control. handbook on tuberculosis laboratory diagnostic methods in the european union – updated 2018 [https://ecdc.europa.eu]. stockholm: ecdc, 2018;p. 115. 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[cited 2015 january 28]; available from: https://www.researchgate.net/publication/272817141_analise_de_dados_para_ciencias_sociais_a_complementaridade_do_spss_6_edicao_revista_atualizada_e_aumentada_maria_helena_pestana_joao_nunes_gageiro coelho ag, zamarioli la, vicente mp, reffo e silva r. avaliaçäo do método de ogawa: kudoh para o isolamento de micobactérias/evaluation of the ogawa: kudoh method for the isolation of mycobacteria. rev inst adolfo lutz. 1999;2(58):57–61. parimango rodríguez d, chávez castillo m, luján velásquez m, otiniano garcía m, robles castillo h, muñoz ganoza e. comparación de los medios ogawa y löwenstein jensen en el aislamiento de mycobacterium tuberculosis de pacientes con tuberculosis pulmonar. hospital regional docente de trujillo, perú. rev med vallejiana. 2007;4(1):24–31. https://doi.org/10.18050/revistamedicavallejiana.v4i1.2217 jaspe rc, rojas ym, flores la, sofia toro e, takiff h, de waard jh. evaluation of the kudoh swab method for the culturing of mycobacterium tuberculosis in rural areas. trop medint health. 2009;14(4):468–471. https://doi.org/10.1111/j.1365-3156.2009.02236.x costa rr da, silva sf da, fochat rc, et al. comparison between ogawa-kudoh and modified petroff techniques for mycobacteria cultivation in the diagnosis of pulmonary tuberculosis. einstein (são paulo) [www.scielo.br]. 2018 [cited 2019 may 23]; 16(2). available from: http://www.scielo.br/scielo.php?script=sci_arttext&pid=s1679-45082018000200209&lng=en&tlng=en aily dcg, shikama m de lm, oliveira mlf, silva rm. perfil bacteriológico da tuberculose pulmonar na região de campinas e das diretorias regionais de saúde xii, xv e xx – 1999/2000. rev inst adolfo lutz. 2003;62(1):5–10. borges m, cafrune pi, possuelo lg, valim ar de m, ribeiro mo, rossetti mlr. análise molecular de cepas de mycobacterium tuberculosis provenientes de um centro de saúde ambulatorial em porto alegre, (rs). j braspneumol. 2004;30(4):358–364. https://doi.org/10.1590/s1806-37132004000400010 organización panamericana de la salud. manual para el diagnóstico bacteriológico de la tuberculosis parte ii – cultivo [https://www.paho.org/en]. 2008, 107 p. [acessed on january 19, 2015]; available from: http://www1.paho.org/spanish/ad/dpc/cd/tb-labs-cultivo.pdf. rivas c, coitinho c, dafond v, corbo m, baldjian m. performance of the ogawa-kudoh method for isolation of mycobacteria in a laboratory with large-scale workload. rev argentmicrobiol. 2010;42(2):87–90. abstract introduction methods results discussion acknowledgements references about the author(s) allan n. njau department of pathology and laboratory medicine, aga khan university hospital, nairobi, kenya samuel m. gakinya department of pathology and laboratory medicine, aga khan university hospital, nairobi, kenya shahin sayed department of pathology and laboratory medicine, aga khan university hospital, nairobi, kenya zahir moloo department of pathology and laboratory medicine, aga khan university hospital, nairobi, kenya citation njau an, gakinya sm, sayed s, moloo z. xpert® mtb/rif assay on formalin-fixed paraffin-embedded tissues in the diagnosis of extrapulmonary tuberculosis. afr j lab med. 2019;8(1), a748. https://doi.org/10.4102/ajlm.v8i1.748 original research xpert® mtb/rif assay on formalin-fixed paraffin-embedded tissues in the diagnosis of extrapulmonary tuberculosis allan n. njau, samuel m. gakinya, shahin sayed, zahir moloo received: 31 dec. 2017; accepted: 08 feb. 2019; published: 18 sept. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: diagnosis of extrapulmonary tuberculosis continues to be a challenge due to the complexity of the causative organism and the wide array of pathologic features seen in this infection. xpert mtb/rif can be used on fresh or frozen tissue specimens for diagnosis of tuberculosis with good results. however, there is little data on its use with formalin-fixed paraffin-embedded (ffpe) tissues. objectives: the aim of this study was to demonstrate the potential utility of xpert mtb/rif and to compare its performance to ziehl-neelsen staining for the detection of mycobacterium tuberculosis from ffpe tissues using histological features from haematoxylin and eosin staining as the gold standard. methods: eighty randomly selected archival ffpe tissues exhibiting histological features of tuberculosis were included in the study. after deparaffinisation and lysis, all the tissue specimens were subjected to the xpert® mtb/rif assay. the outcome measures were proportions of positively identified cases by each test. results: using histology as the gold standard, the sensitivity of ziehl-neelsen staining was 20.3% (95% confidence interval: 12% – 30.8%), and the sensitivity of the xpert® mtb/rif assay was 53.2% (95% confidence interval: 41.6% – 64.9%); the difference was statistically significant (p = 0.002). none of the cases tested positive for rifampicin resistance. conclusion: with prior deparaffinisation and lysis, ffpe tissues are amenable to testing by xpert® mtb/rif assay. a validation study to determine the clinical utility, analytical optimisation and cost implications of this assay for ffpe tissues is recommended. keywords: xpert mtb/rif; extrapulmonary tuberculosis; formalin-fixed paraffin-embedded tissues; kenya. introduction globally, extrapulmonary tuberculosis (eptb) represented 15% of the 6.3 million cases and in africa 16% of the 1.2 million cases that were notified in 2016. children and the immunosuppressed population have been reported to be at a higher risk of eptb than the general population.1,2 diagnosis of eptb involves analysis of fresh or fixed tissue biopsies using culture, histological examination or molecular tests. culture is regarded as the gold standard; however, its main disadvantages are the long turnaround time – up to 6 weeks – and the fact that only fresh tissue can be used. in addition, the sensitivity of cultures has been found to be variable (0% – 80%).3 histological diagnosis of tuberculosis on haematoxylin and eosin (h&e) stained sections usually relies on the presence of classical necrotising granulomas with langhans giant cells. however, tuberculosis can exhibit other patterns of inflammation such as non-necrotising granulomatous inflammation, non-specific chronic inflammatory processes and suppurative inflammation.3,4,5 in hiv and tuberculosis co-infection for example, there may be both delay and poor or no formation of granulomas.6 in addition, other agents and pathologic conditions can mimic tuberculosis. examples include infections caused by nontuberculous mycobacteria, bacteria other than mycobacteria, fungi, some viruses and parasites as well as non-infective causes such as vasculitides and crohn’s disease.7,8 presence of acid-fast bacilli (afb) on ziehl-neelsen (zn) staining is widely utilised as a confirmatory test for eptb in a wide spectrum of formalin fixed paraffin embedded (ffpe) tissues because it is quick and relatively cheap; however, its drawback is its low sensitivity (40%).3,9,10 both commercial and in-house polymerase chain reaction (pcr) based assays for tuberculosis have been used to improve detection rates of eptb in ffpe tissues and to overcome some of the limitations encountered by the aforementioned tests.11,12 polymerase chain reaction based assays have been found to be more powerful in ruling in rather than ruling out a diagnosis of tuberculosis.10 a major reason for this is the fact that most eptb tissue specimens tend to be paucibacillary. moreover, the bacilli are usually not uniformly distributed throughout the specimens.12,13,14 the xpert® mtb/rif assay (cepheid, sunnyvale, california, united states) is an automated semi-nested real time pcr in which sample lysis, dna extraction, amplification and detection takes place within a single plastic cartridge. the assay detects the presence of the m. tuberculosis complex and rifampicin resistance within 2 hours.15,16 the world health organization initially endorsed the assay in 2010 as a rapid nucleic acid amplification assay that simultaneously detects the presence of the m. tuberculosis complex and rifampicin resistance in sputa. however, a wide spectrum of tissues are now being analysed using the xpert® mtb/rif assay.17,18 meta-analysis done on the accuracy of xpert mtb/rif in detecting tuberculosis shows that the median pooled sensitivity and specificity in lymph nodes was 84.9% and 92.5%, while in tissues other than lymph nodes, the median pooled sensitivity and specificity was 81.2% and 98.1%.19 in these studies, the nature of the specimens was reported to be either fresh or frozen. a zambian study published in 2017 by polepole et al. showed xpert® mtb/rif assay to be potentially useful for the diagnosis of tuberculosis in ffpe tissues. however, the sensitivity was found to be too low. using histology as the gold standard, the accuracy of zn, xpert mtb/rif, and in-house pcr were assessed in 100 ffpe tissues. the sensitivity of xpert mtb/rif within lymph nodes and non-lymph node tissues was 30% and 35%.20 when dealing with ffpe tissues as the starting specimen on pcr based assays, pretreatment procedures for deparaffinisation and mitigation of the effects of pcr inhibitors is necessary.21,22,23,24 on the other hand, only minimal bio-safety requirements are needed for ffpe tissues since they do not contain live bacilli. the xpert mtb/rif platform, when compared to other pcr platforms, offers additional benefits that include ease of conducting the test and interpreting the results. in addition, only minimal time is required to train laboratory staff who can operate the system.16,18 the xpert mtb/rif technology is also widely available and accessible as opposed to the other technologies that tend to be found mainly in reference laboratories. on many occasions, tissues are submitted to the laboratory while fixed in formalin and with no possibility of culture. the low sensitivity of zn often results in negative zn staining. not infrequently, tissues also lack the typical histologic features of tuberculosis. in these situations, xpert mtb/rif can be a useful adjunct or ‘add-on’ test for eptb. methods ethical considerations the health research ethics committee, aga khan university, provided approval for the study [2014/rec-56(v3)]. study design and setting this was a retrospective cross-sectional laboratory based study conducted at the aga khan university hospital, nairobi, in the department of pathology and laboratory medicine. the laboratory receives samples from the university hospital as well as from other county and mission hospitals within the country. only 17% of the samples analysed in this study were from the university hospital and these were fixed in 10% neutral buffered formalin for 6–12 h prior to processing. the remaining 83% were from the other hospitals and the dilution of formalin used and duration of fixation could not be determined. the patient’s biodata, site of biopsy and relevant clinical history, when provided, were obtained from the laboratory specimen inventory and information system. sampling the study included specimens received between january and december 2014 and with a diagnosis of eptb based on the original histology features from h&e staining (necrotising granulomatous inflammation, non-necrotising granulomatous inflammation, chronic inflammation, necrotising inflammation or suppurative inflammation). the specimens were included irrespective of their zn results; however, specimens in which a diagnosis of eptb had been ruled out, such as cases of fungal infection, were not considered. out of the 132 archived ffpe tissue blocks that fit the above description, 17 were excluded because the amount of tissue preserved was too small to produce an adequate sample for h&e staining, zn staining and the xpert® mtb/rif assay. out of the 115 remaining blocks, 80 ffpe blocks were randomly selected for analysis. laboratory procedures from each ffpe block, h&e and zn stained slides were prepared on 3 µm thick sections. the histological pattern of inflammation for each case was recorded. the zn stained slides were screened for afb under oil at ×1000 magnification and the results recorded as either positive or negative. the principal investigator first screened all the slides and then every fifth case had the h&e and zn stained slide screened by a second pathologist. for the xpert® mtb/rif assay, up to 10 sections of 10 µm thickness were cut from the ffpe blocks and the scrolls placed in a 1.5 ml micro-centrifuge tube. deparaffinisation was done using a modified xylene method, which involved two washes in 1 ml of xylene, two washes in 1 ml of absolute ethanol followed by two rinses in 1 ml of phosphate buffered saline. lysis of the tissue fragments followed with resuspension of the tissue fragments in 1 ml of phosphate buffered saline and the addition of 20 µl of qiagen proteinase k, then the mixture was incubated at 56 °c for 12 hours. if there were tissue fragments visible on inspection after the initial incubation, another 20 µl of proteinase k was added and re-incubated for 1 or 2 h. after complete lysis was achieved, the proteinase k was deactivated by increasing the temperature to 94 °c for 10 min.25 the xpert® mtb/rif assay was then performed following the manufacturer’s instructions using the lysate and without any further attempt to extract dna from the lysate. as a quality control measure, the microtome blade was changed and the microtome overlay cleaned with absolute alcohol after each case to prevent carryover contamination.6,20,21 xpert mtb/rif results were recorded as either detected, not detected or error. one case resulted in an xpert® mtb/rif assay error. the bacilli load in each specimen was also recorded following the assay’s semi-quantitative estimate of the bacilli load as either high, medium, low or very low. rifampicin resistance was recorded as either detected, not detected or indeterminate.15 data analysis the data were analysed using the statistical package for social sciences version 19, (ibm corp, armonk, new york, united states). sensitivities were calculated from proportions of cases positive on xpert mtb/rif and those positively detected by zn against histology as the gold standard. a chi-square test was applied to test for statistical differences and p values less than 0.05 were considered statistically significant. results only 79 cases were analysed after the single case without xpert mtb/rif results was excluded from further analysis. the mean age of patients was 27 years with an age range of 6 to 85 years. forty-four (55.7%) specimens were from female patients, and 35 (44.3%) were from male patients. twenty-six different types of tissues were analysed and classified into nine groups according to the topographic site of the biopsy (table 1). lymph nodes were the most common site, 30 specimens (38%), followed by the female genital tract, 15 specimens (19%), and the abdominal cavity, 11 specimens (13.9%). the least prevalent sites were the male genital tract and the central nervous system with only one specimen each. clinical information was available for 48.1% (38/79) of the specimens, of which 13.2% (5/38) were reported to have confirmed tuberculosis in a site other than the tissue submitted. xpert mtb/rif was positive for all five specimens, and zn was positive for three of the five specimens. in 36.8% (14/38) of the specimens with clinical information, tuberculosis was clinically suspected; 5 out of the 14 were positive by xpert mtb/rif and 1 by zn. hiv infection was reported in six (15.8%) of the specimens, of which four were positive by xpert mtb/rif and two by zn. table 1: demographic and topographic sites of tissues. necrotising granulomatous inflammation was the predominant histological pattern of inflammation contributing to 91.1% (72/79) of the specimens (table 2). in this group, 53% (38/72) were positive by xpert mtb/rif and 22% (16/72) by zn. the contribution of granulomatous inflammation, chronic inflammation, necrotising inflammation and suppurative inflammation was marginal. in this latter group of inflammation combined, 57% (4/7) were positive by xpert mtb/rif and none by zn. table 2: yield of ziehl-neelsen and xpert mtb/rif by patterns of inflammation. in lymph node tissues, 16 out of 30 were positive by xpert mtb/rif compared to 8 by zn, whereas in non-lymph node tissues, 26 of 49 were positive by xpert mtb/rif compared to 8 by zn (table 3). xpert mtb/rif was positive in 87.5% (14/16) of the specimens that were zn positive. two specimens that were zn-positive gave a negative xpert mtb/rif result. using h&e histology results as the gold standard, the overall sensitivity of zn was 20.3% (95% confidence interval [ci]: 12% – 30.8%) and that of xpert mtb/rif was 53.2% (95% ci: 41.6% – 64.9%), significantly better than zn (p = 0.002). table 3: yield of ziehl-neelsen and xpert mtb/rif test by tissue type. of the 42 xpert mtb/rif positive specimens, the bacilli load in 26 (61.9%) was quantified as ‘very low’, 11 (26.2%) as ‘low’ and only 5 (11.9%) as ‘medium’. none of the cases showed a ‘high’ bacilli load and neither was there any case of rifampicin resistance detected. however, 7 of the 42 xpert mtb/rif-positive cases showed an ‘indeterminate’ rifampicin resistance, all of which had a ‘very low’ bacilli load. discussion in this study, we compared the xpert mtb/rif assay to histology for the detection of m. tuberculosis from archived ffpe tissues. the results obtained show that xpert mtb/rif had a higher sensitivity (53.2%) than zn (20.3%). similar to other studies, we found that zn had a low sensitivity for tuberculosis in ffpe tissues.3 the sensitivity of xpert mtb/rif in our study was comparable to, though slightly higher (53.2% vs 50%) than, a previous study, in which the performance of the qiagen artus m. tuberculosis rg pcr assay was compared to xpert mtb/rif among 40 ffpe tissues with histopathological features consistent with tuberculosis.26 similarly, the sensitivity of xpert mtb/rif in our study for lymph node and non-lymph node specimens (53.1% and 53.1%) was higher than that found by polepole et al. (30% and 35%).20 in further support of the usefulness of xpert mtb/rif, its performance was found to be better than zn in specimens from patients reported to have tuberculosis in other sites and hiv infection. in this study, we could not determine the specificity of xpert mtb/rif, because we only included tissues with histological features of tuberculosis. the two cases that were zn positive and xpert mtb/rif negative may represent false negatives or may have been cases of nontuberculous mycobateria. we could however not confirm the true status, because culture was not performed. another possible explanation is that cases of eptb tend to be paucibacillary. moreover, the bacilli may not be evenly distributed within the tissue,3 raising the possibility that the sample came from a part of the tissue that may have not harboured any organisms. multiple (up to 10 sections, 10 µm thick) sections of tissue were processed for xpert mtb/rif testing with the aim of increasing the chances of obtaining sections with the bacilli; however, we do not know to what extent this mitigated the above factors. the quality of dna is also likely to have been reduced due to the untoward effects of formalin fixation.22 a study by jonathan et al. conducted to investigate the effects of fixatives and fixation times on pcr showed that formalin-fixed tissues gave reliable pcr results, if the tissue was fixed for less than 48 h.23 in this study, standardisation of fixation times and dilution of formalin used could not be determined for 83% of the specimens that had been referred from other hospitals in different parts of the country. a semi-quantitative estimate of bacilli load based on the cycle threshold value is another parameter that is obtained from the xpert® mtb/rif assay. in this study, we found that more than three-quarters of the cases (61.9% and 26.2%) had a quantification of either ‘very low’ or ‘low’. this finding is probably due to the above-mentioned pre-analytical factors: the paucibacillary nature of eptb samples and formalin fixation. the secondary objective of our study was to determine the prevalence of rifampicin resistance. in this study, no case of rifampicin resistance was detected. indeterminate rifampicin resistance results were however obtained for seven of the 42 cases positive on xpert mtb/rif. not surprisingly, all had a very low bacilli load. therefore, only in the 35 cases with rifampicin resistance results could we truly say that there was no case of rifampicin resistance detected. in the study by polepole et al., a similar problem of a large number of ‘indeterminate’ results for rifampicin resistance was also encountered. therefore, a very low bacilli load as quantified by xpert mtb/rif, which subsequently limits the assessment of rifampicin resistance, may have led to this finding. it is also likely that this finding was due to the small sample size of this study. limitations this study was limited by its small sample size and its retrospective nature. the duration of fixation and the strength of the formalin used were not known for the majority of the specimens. these are important factors, because they affect the quality of the dna extracted. lack of culture results and additional clinical information are additional limitations, because they would have aided in the determination of the clinical utility of this assay in the context of ffpe tissues. conclusion histological assessment of h&e and zn stained slides forms a readily accessible and affordable diagnostic tool for eptb. however, the inability to make a definitive diagnosis of eptb is common due to its low sensitivity. in our study, we found that culture for eptb was rarely done and all the samples we analysed were received in the laboratory already fixed in formalin, thereby precluding culture as a diagnostic test. since the xpert mtb/rif assay is widely available in many parts of kenya, it may potentially be an excellent ‘add-on’ or adjunct test for the detection of m. tuberculosis in fixed tissues. this is especially true in cases that are zn-negative or those that lack the typical histologic features of tuberculosis. we recommend a validation study to determine the clinical utility, analytical optimisation and cost implications of this assay. acknowledgements we would like to express our gratitude to geoffrey omuse, gunturu revathi and nelson kuria for advice and organisational support. we are also grateful for the technical support of christine ndaya and sarah mugo. we would also like to thank the aga khan university for funding this study. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions a.n.n. and s.m.g. conceived the idea. all the authors made conceptual contributions to the study. a.n.n. performed the tests, analysed the data and prepared the draft. s.m.g. reviewed the results. s.m.g., s.s. and z.m. critically revised the draft and gave final approval. sources of support funding for the study was provided by the aga khan university, nairobi, kenya. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the author(s) and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references world health organisation (who). global tuberculosis report. 2017. geneva: who; 2013. world health organisation (who). policy statement on xpert mtb-rif 2013. geneva: who; 2013. promod k, raj a, singh n, gopal k. diagnosis of extrapulmonary tuberculosis by pcr. fems microbiol immunol. 2012;66:20–36. https://doi.org/10.1111/j.1574-695x.2012.00987.x kitinya jn, richter c, perenboom r, chande h. influence of hiv status on pathological changes in tuberculous pleuritis. tuber lung dis. 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postmortem diagnosis of tuberculosis in paraffin-embedded tissues. mikrobiyol bul. 2014;48(4):577–584. https://doi.org/10.5578/mb.8299 abstract introduction methods results discussion acknowledgements references about the author(s) mohamed b. jalloh research unit, joint medical unit, republic of sierra leone armed forces, freetown, sierra leone department of community health, faculty of clinical sciences, college of medicine and allied health sciences, university of sierra leone, freetown, sierra leone eva vernooij school of social and political sciences, university of edinburgh, edinburgh, united kingdom department of interdisciplinary social sciences, faculty of social and behavioural sciences, utrecht university, utrecht, the netherlands alice street school of social and political sciences, university of edinburgh, edinburgh, united kingdom citation jalloh mb, vernooij e, street a. invisible and undervalued: a qualitative study of laboratory workers’ experiences and perceptions of laboratory strengthening in sierra leone. afr j lab med. 2024;13(1), a2292. https://doi.org/10.4102/ajlm.v13i1.2292 original research invisible and undervalued: a qualitative study of laboratory workers’ experiences and perceptions of laboratory strengthening in sierra leone mohamed b. jalloh, eva vernooij, alice street received: 13 aug. 2023; accepted: 22 feb. 2024; published: 31 may 2024 copyright: © 2024. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the 2013–2016 west africa ebola outbreak highlighted the importance of laboratory capacity to outbreak response while also revealing its long-standing neglect. the outbreak prompted massive international investment into strengthening laboratory services across multiple healthcare settings. objective: in this article, we explore hospital-based clinical laboratory workers’ experiences and perceptions of their everyday working environment in sierra leone, and how recent investments in laboratory strengthening have shaped these. methods: this qualitative study draws on in-depth interviews with eight laboratory workers and participant observation of laboratory practices at a tertiary referral hospital in freetown between april 2019 and december 2019. interview and observational data were coded and analysed using a reflexive thematic approach. results: the ebola outbreak prompted international investments in automated devices, biosafety training, and a new dedicated infectious diseases laboratory. however, little investment was made in the infrastructure and supply systems needed to sustain routine laboratory work or keep machines functioning. laboratory workers perceived their work to be under-recognised and undervalued by the government, hospital managers and clinical staff, a perception compounded by under-use of the hospital’s laboratory services by clinicians. conclusion: understanding laboratory technicians’ views, experiences, and priorities is essential to any sustainable laboratory-strengthening effort. investments in personnel should match investments in technologies and infrastructure for outbreak response. what this study adds: this study contributes to an understanding of how clinical laboratory personnel in sierra leone view and experience their work, and introduces the concept of social invisibility to explain these experiences. keywords: ebola outbreak; neglect; perceptions; investment; laboratory technicians; laboratory capacity; infrastructure. introduction the ebola outbreak exposed significant gaps in sierra leone’s laboratory system and prompted a surge in international assistance for laboratory strengthening and emergency preparedness.1,2,3 during and after the outbreak, the public laboratory was a key target for interventions aimed at improving laboratory services, including training of personnel, provision of equipment and infrastructural improvements.4,5,6 several publications have explored the experiences of international laboratory teams concerning their diagnostic achievements and laboratory support during and after the outbreak.7,8,9,10,11,12,13 other publications have focused on the experiences of frontline health workers, including nurses and doctors.14,15,16 however, far less attention has been given to the west african laboratory workers’ experiences during ebola and in its aftermath, or their priorities for improving routine and emergency clinical laboratory services. laboratory workers can provide unique insight into the changes to clinical laboratory work that have occurred since the ebola outbreak, especially the impact of international assistance on everyday laboratory work in the country. the few studies published in this area point to the need for more in-depth qualitative research to understand the sociocultural and institutional contexts within which laboratory work becomes valued or devalued and in which careers are developed.17,18,19 this article aims to understand the experiences of laboratory workers in a government-funded military hospital in freetown, sierra leone, their reflections on the ebola outbreak, and their views on how management, government, and international partners can best support and improve their work. it contributes to a growing literature around laboratory strengthening in african countries,1,2,19 by providing on-the-ground perspectives from a country that has been the locus of intensive laboratory-strengthening interventions in a post-epidemic context. we argue that such efforts must consider the experiences and priorities of laboratory workers on the ground, since this group can best advise on the everyday realities of laboratory work and which interventions are most likely to improve morale, workflows, and capacity. we aim to generate new insights into everyday laboratory work that will be valuable to a growing range of laboratory-strengthening efforts in africa, including those spearheaded by pan-african organisations such as the african society for laboratory medicine and the africa centres for disease control and prevention. methods ethical considerations ethical approval for the study was granted by the sierra leone ethics and scientific review committee (approval date 10 september 2018) and the university of edinburgh research ethics and integrity committee. we also obtained approval from the 34 military hospital (mh 34) (reference jmu/1000/1) where the data on which this article is based were collected. formal written consent was obtained from all interview participants. to maintain patient confidentiality in the study, all identifying information was anonymized and replaced with unique codes and access to the data was restricted to authorized personnel only. setting this study forms one component of a larger research project on post-ebola laboratory strengthening in sierra leone, funded by the european research council. the total research project included ethnographic research on laboratory work and clinical diagnostic pathways at a public referral hospital in sierra leone,19,20 a historical review of policy and planning relating to laboratory strengthening in sierra leone,3,20 ethnographic research on diagnostic processes and workflows at community health centres,20 survey-based research on community-based fever management, and a district level survey of diagnostic availability and useability.20,21 this sub-study involved qualitative research at the 34 military hospital (mh 34), freetown, sierra leone, which played a central role in the medical response to the ebola outbreak. it was the first hospital to manage ebola cases in freetown and sent its health workers, including the first author of this article, to support several ebola treatment centres and community care centres across sierra leone. its laboratory technicians were crucial in collecting and transporting samples from patients to designated laboratories to detect the ebola virus. the mh 34 was also the site of intensive international laboratory-strengthening efforts during and after the epidemic, including the establishment of a chinese government-funded infectious diseases laboratory.9,22 the mh 34 in freetown, sierra leone, was established during british colonial rule to provide medical services to serving military personnel and their dependents. currently, the hospital offers free care to military personnel and their dependents, while charging the public for services. the hospital’s core funding comes from the sierra leone government’s allocation to the ministry of defence. it, therefore, treats patients from across the spectrum of ethnic, religious, and economic groups in sierra leone. the mh 34 is the leading infectious disease facility in sierra leone for treating patients infected with high-consequence pathogens.23 it also hosts the country’s rapid deployable isolation and treatment facility that responds to outbreaks in any part of sierra leone within 72 h to 96 h. the mh 34 has two clinical laboratories: the main and infectious diseases laboratories. the main laboratory is as old as the hospital and includes phlebotomy, hiv/aids, serology, haematology, biochemistry, blood banking, and microbiology sections. it has 20 publicly employed personnel, and the government of sierra leone funds its equipment and consumables, with help from non-governmental organisations and international donors. study participants were drawn from formally employed and volunteer laboratory technicians. volunteers receive transport refund, are recruited by the hospital, and do not receive any salary. paid staff, however, receive salaries and other benefits of employment and are only recruited by the human resources management officer of the government of sierra leone. the infectious diseases laboratory is part of the infectious disease department built in 2018, with funding from the government of the people’s republic of china. the infectious disease laboratory provides advanced services in haematology, microbiology, and serology for patients admitted to the adjacent 30-bed infectious disease ward, performing routine clinical testing without a surveillance mandate. staff members work in both the infectious disease laboratory and the main laboratory, and are rotated between the two, with three sierra leonean staff working in the infectious disease laboratory at any time. the infectious disease laboratory also has two permanently posted chinese staff. its equipment and consumables are funded and supplied by the chinese military. in addition to these two clinical laboratories, a biosafety level 2 research laboratory, also built by the chinese government in 2018, supports the prompt diagnosis of emerging high-consequence pathogens in sierra leone and conducts studies on infectious disease burden in the country.23 sample purposive sampling was used to ensure that sierra leonean laboratory workers with a wide range of demographic characteristics and employment experiences were included in the study. for selection, we categorised laboratory workers based on which clinical laboratory they worked in, which laboratory section they worked in, whether they were employed as payroll staff or as volunteers, and whether they worked on the laboratory bench or as a technical lead. we purposefully selected participants within each of these employment groups to ensure overall representation from a wide range of demographic groups, including gender, ethnicity, religion, and age. sample size was determined by a combination of inclusion of participants from each of these groups and qualitative data saturation, defined as the point at which no new themes were emerging from the data. data collection the lead author, m.b.j., who is employed at the 34 military hospital as a physician, conducted data collection for this study between april 2019 and december 2019. his status as a hospital employee provided valuable insight into the institution’s historical and social context, and was essential to the ability to translate research findings rapidly into impactful institutional changes. during data collection, measures were implemented to minimise both participant and researcher bias. to minimise any potential for participant bias arising from m.b.j.’s employment in the hospital, participants were fully informed about the objectives of the study, namely to understand their experiences, efforts that would be made to anonymise their data, and limitations to these efforts given the small sample size, and their rights as research subjects. to mitigate researcher bias arising from m.b.j.’s prior knowledge of the institutions, co-authors were consulted regularly to obtain a perspective and ensure that m.b.j.’s biases and assumptions did not unduly influence the research process. semi-structured interviews were conducted using a topic guide developed for the broader study and adapted for mh 34 by e.v., a.s., and m.b.j. the interviews focused on topics such as career history, experiences during the ebola outbreak, daily workflows in the laboratory, changes in laboratory work over their careers, challenges faced in the laboratory, and priorities for improvement. the interviews took place in the interviewee’s workspace, ensuring privacy, and lasted an average of 40 min. trained translators translated the transcripts from krio to english. to verify the accuracy of the translation, m.b.j., who speaks krio fluently, reviewed the english translations against the audio recordings for any unclear areas before approving the data for thematic analysis. all interviewees provided consent for audio recording. ethnographic observation by m.b.j. was also conducted in the laboratory, by observing working practices, interactions, equipment usage, and the use of the workspace throughout the working day. these visits lasted between 2 h and 4 h, and occurred on different days and times to gather a comprehensive understanding of laboratory life. during these visits, m.b.j. also asked laboratory workers questions about their work, equipment, and infrastructure to uncover additional insights not covered in the formal interviews. data analysis a reflexive thematic analysis approach was employed to allow collaborative interpretation of the qualitative data and to identify patterns and themes across the data set.24,25 due to the small sample size, this analysis was undertaken using microsoft word (microsoft corporation, redmond, washington, united states). the authors a.s. and m.b.j. each read all the interview transcripts and coded them. after that, a meeting was held with a.s., m.b.j. and e.v. to review the coding for each transcript, ensure consistency across the transcripts, check for researcher bias, and resolve any differences between the coders. following a further review to check the coding schema’s consistency, coherence and logic, e.v., a.s., and m.b.j. collectively collated the codes into larger thematic groupings. the team then aligned individual codes, themes, and example quotations and cross-checked for overlaps between themes and the overall coherence of the thematic structure. results a total of eight laboratory staff participated in interviews for this study (table 1). we interviewed six laboratory technicians who worked across all laboratory sub-departments. the two laboratory leads we interviewed had responsibility for managing the two clinical laboratories. of the eight interview participants, only one was female, reflecting the broader demographics of laboratory staff, with 19 male and 2 female employees at the time of the study. all participants had experience working in an outbreak, and seven had direct experience working during the ebola outbreak. all participants had diplomas in medical laboratory sciences at the eastern technical university of sierra leone. table 1: characteristics of study participants at the 34 military hospital, freetown, sierra leone, april 2019 to december 2019. career experiences laboratory technicians were often encouraged by family members to pursue laboratory medicine: ‘my initial interest was in engineering science, but they [mum and friends in healthcare] unanimously encouraged me … to do laboratory medicine.’ (participant 2, laboratory technician, 20 june 2019) three staff experienced financial difficulties after writing the national high school qualifying exams and felt pressured to study laboratory medicine as a route to economic security. four opted to become laboratory technicians after volunteering or being employed as nurses or military officers for several years. at the time of data collection, one of the staff had volunteered for seven years and had not yet progressed to formal employment. towards the end of the civil war, several programmes were introduced into the military to convert the fighters into professionals as a part of efforts supported by the british government, and some participants reported receiving laboratory training through these schemes. for the few who had initially trained as nurses, their primary motivation to become laboratory technicians was that they would be able to support medical care for patients more effectively through laboratory work than through nursing: ‘[s]o i decided to enter the lab because … i saw that the laboratory is the basis of medicine because it really tells you what a person’s disease is, and when you know about a person’s disease, that’s when you’ll be able to fight it; if you don’t know what that person’s disease is and you give medication, that medicine will endanger that person’s health.’ (participant 4, laboratory technician, 23 june 2019) all but one of the technicians had experience working during the 2013–2016 west african ebola outbreak, and, in many cases, this was described as a significant career moment that influenced later decisions and perceptions about laboratory work. several technicians described their willingness to work during ebola in terms of a sense of public duty. many also described this as a period of self-sacrifice, which often involved significant personal costs regarding separation from relatives or the risks involved. health workers known to have contact with ebola patients and/or samples described being stigmatised by colleagues and the public. two study participants stopped working as laboratory technicians during the ebola outbreak. the first changed his role to a quarantine officer, while the second stopped working altogether because his boss died of ebola, and his family insisted that he could not continue to work. some interviewees also described this as a period of intensive training, which provided opportunities for learning new skills: ‘i had issues with my home because of my ebola work, but i was determined to serve the people.’ (participant 8, laboratory technician, 14 september 2019) ‘i was attached to the [ebola] treatment centre that was managed by msf [médecins sans frontières]. i attended training, for which they gave me a certificate before i started working. we were trained on infection prevention and control and the use of personal protective equipment [ppe] when collecting blood samples.’ (participant 5, laboratory technician, 09 september 2019) some were motivated to continue their service as laboratory technicians, having seen the central role that laboratory medicine played in detecting and responding to outbreaks: ‘if we have another outbreak, no one will be able to detect the causative organism unless the laboratory technician runs the sample and provides answers as to what the causative organism is.’ (participant 5, laboratory technician, 09 september 2019). impact of ebola outbreak on laboratory work participants described the 2013–2016 west african ebola outbreak as highlighting the central role that the laboratory plays in clinical care. participants described increased investment in high-end laboratory equipment in the main lab, especially automated machines, during the outbreak as one outcome of this increased visibility: ‘specifically, the genexpert was brought here due to the ebola.’ (participant 7, laboratory lead, 12 september 2019) staff also received much training on biosafety, which improved practices like hand washing, waste segregation and use of gloves during sample collection, transportation, and analysis: ‘before ebola, some of us were even handling samples with bare hands because we didn’t believe in difficult infectious disease, we just thought of diseases like syphilis, gonorrhoea because it’s curable we didn’t take care too much. because when we contract those types of diseases within a few days after taking treatment, it’s treated. in fact, i think it was ebola that made the majority of people become aware of even rvs [retroviral status], which is hiv and aids and hepatitis because they’re all viral diseases.’ (participant 4, laboratory technician, 23 june 2019) as ebola outbreak-related work increased, most other patients avoided coming to public hospitals, which led to a decline in the routine functions of the laboratory. unlike other components of laboratory medicine, little investment went into improving the existing hospital laboratory space and infrastructure where laboratory services are delivered. participants pointed out that the main hospital laboratory did not undergo any renovation or expansion during or after the ebola outbreak. a notable difference was the dedicated infectious diseases laboratory space built post-ebola as part of a 30-bed chinese government-sponsored infectious disease department. participants described the infectious disease laboratory as having a ‘modern’ space, new equipment, a reliable supply of reagents and other utilities and a more comprehensive range of tests, and notably, a separate repair and maintenance system, financed and operated by the chinese government, which was unavailable to the main laboratory, leading to regular machine breakdowns in the main laboratory, despite the investments in more high-end automated machines during ebola: ‘at the other laboratory department, they don’t do biochemistry tests because of the machine [breakdown]. but here at the infectious disease department, we conduct all necessary tests precisely for patients.’ (participant 6, laboratory technician, 11 september 2019) training training was a recurrent theme in the interviews, including discussing positive training experiences and the need or desire for training in particular areas. many participants positively described the additional training they had received during the ebola outbreak. despite this, they highlighted the need for additional and routine training in biosafety and equipment maintenance areas. some hoped for additional professional training abroad, citing that overseas training is often more structured and that staff sent on such training would be more focused than when training is delivered locally: ‘we actually are expecting both international and national training. specifically, we are out of someone who is trained in biomedical engineering. it’s really important, really important.’ (participant 7, laboratory lead, 12 september 2019) ‘they just trained us to do the basic maintenance that is clean[ing] … we are not trained on that [thorough check of equipment]. so, we wait these people who we call from guinea. they too call for american embassy before ever they can even come and see about these machines.’ (participant 1, laboratory technician, 07 june 2019) another participant suggested providing equipment and training to laboratory staff for fire prevention or management: ‘well, to improve also the safety, safety talking about the security risk, the case of fire outbreak. fire extinguishers, people are trained about fire safety. … well, i say this because we do have experience with electrical problems.’ (participant 7, laboratory lead, 12 september 2019) laboratory infrastructure the laboratory service at the time of the study was characterised by infrastructural challenges, such as erratic electricity supply, the absence of stabilisers, hot storage spaces for reagents, and a lack of maintenance for workspaces, leading to a short lifespan for laboratory equipment and supplies: ‘even in our store here, you will realise upon entering that it is hot for you; what do you think its effect would be for supplies in cartons?’ (participant 2, laboratory technician, 20 june 2019) failure to expand the laboratory infrastructure or renovate the existing spaces for over a decade despite the ebola outbreak meant that staff, equipment, and the public were exposed to rain due to the leaking roof: ‘they only do the repainting and nothing more.’ (participant 5, laboratory technician, 09 september 2019) ‘the building leaks, and in the rainy season, the machines get wet whenever it rains.’ (participant 8, laboratory technician, 14 september 2019) the absence of a biosafety cabinet and regular maintenance for laboratory equipment leaves laboratory staff vulnerable with a sense of neglect and marginalisation. at the time of the interview, there was no functioning biochemistry, haematology or hormone analyser in the main laboratory, and the staff interviewed pointed to infrastructural gaps as the primary cause for the breakdown of the analysers: ‘at the main laboratory, there are so many non-functional machines there. the building needs to be expanded.’ (participant 8, laboratory technician, 14 september 2019) ‘faulty equipment such as the haematology analyser or printer limit the quality of services that we provide … the building itself is not ideal; it is archaic now and too small for us.’ (participant 2, laboratory technician, 20 june 2019) several automated machines in the laboratory were procured in the years following the ebola outbreak, including a sysmex haematology analyser, electrolyte analyser and electrophoresis machine. however, the sysmex machine was used for less than a month before it broke down. the electrolyte analyser and electrophoresis machine were never fully installed. altogether, three machines were not working at the time of the interview: ‘yeah, we have, we have two, ok three, like even the hb [haemoglobin] electrophoresis had the same problem. the haematology analyser has the same problem, and the biochemistry analyser has the same problem. so, we have three machines that are down right now.’ (participant 1, laboratory technician, 07 june 2019) staff contrasted the issues with physical infrastructure and maintenance of the main laboratory with the modern equipment and space at the infectious disease laboratory. interviewees characterised the infectious disease laboratory in terms of regular maintenance routines and the exercise of quality controls that the laboratory staff were trained and supervised to perform by laboratory professionals deployed to sierra leone as part of the chinese military medical expert group: ‘in the morning [at the infectious disease laboratory], i will have to clean the machines, cross-check the waste and distil [sic] water from the haematology machine. i will also check the printer to see if it’s functional. if there is a need to add or change the distilled water, i will do so afterwards. if there is a need to empty the waste, i have to do so to avoid the machine breakdown. i will also do the same in the biochemistry department, checking the waste, the machine’s temperature, and the pins that help to operate the machine. i also need to check all the pins and needles of the biochemistry machine to avoid breaking them. and quantity. if we run low on some reagents, i will mix more using the manuals provided.’ (participant 6, laboratory technician, 11 september 2019) supply chain equipment procurement was a sensitive topic, and some laboratory staff hesitated to discuss it. the interviewees reported little or no consultation on the quantity and type of equipment and reagents required by the lab. some reported receiving reagents that did not match the equipment, leading to equipment breakdown: ‘we may receive a call, and at times our boss may not even be informed on the procurement or coming of the new machine and at times, they may even double a specific machine because they have failed to discuss the matter with us, and they know not exactly what the lab requires.’ (participant 2, laboratory technician, 20 june 2019) at the point of data collection, the laboratory had not received any reagents for several months. stockouts of reagents were common at the main laboratory, leading to redundant equipment. the laboratory relies on only one vendor to supply reagents to the laboratory, and interviewees described this ‘monopoly’ as the cause of erratic supplies: ‘the government provides these supplies through a private contractor. the problem we have is that machines that are in frequent use receive little or no reagents, while we continue receiving reagents for machines that are faulty or dysfunctional. we, therefore, always encounter stockouts of reagents before another batch of supplies is received.’ (participant 5, laboratory technician, 09 september 2019) some donor-supported tests, such as hiv tests, were also reported as unsustainable and afflicted by nationwide stockouts: ‘for instance, the cartridge for the genexpert machine ran out of stock for a long time and replenishing it may take up to 3 or 4 months. at times, there is stockout of such cartridges in the entire country.’ (participant 2, laboratory technician, 20 june 2019) the infectious diseases laboratory received equipment and reagents from the chinese military and was reported as largely having a sustainable supply. even so, interviewees reported that culture and sensitivity testing was not done at the infectious disease laboratory because the cassettes are expensive and unavailable in the local market. workplace relationships a major challenge highlighted by participants was staff shortages that resulted in a high daily workload and long turn-around times: ‘we actually don’t have enough staff, but those of us here, we are doing all we can to complete our task before the day ends. most of the time, i don’t even return home. i will have to sleep in our department. so, at times, i will wake up at night and run some of the tests before daybreak.’ (participant 6, laboratory technician, 11 september 2019) the poor allocation of human and material resources to public clinical laboratories and the perceived lack of career opportunities in laboratory medicine is evidence to the staff that their work is undervalued by other clinical staff and the health sector: ‘we have been ignored for a long time, that when opportunities are available, since we work together with you, we desire to have discussions with you, the clinicians. but since i came to this hospital, it’s been three years, and there has never been a meeting between clinicians and lab workers. and when we discuss, we would know the issues, and you would be informed on which machines are faulty to ensure that all the lab investigations you requested are performed.’ (participant 2, laboratory technician, 20 june 2019) there is a vibrant private laboratory sector in freetown, and doctors at mh 34 refer patients to private facilities for diagnostic tests that the mh 34 laboratory cannot perform. due to the absence of a memorandum of understanding between the mh 34 laboratory and the private laboratories, patients are referred to the private sector, where they usually pay for the services out of pocket and return to the hospital with a printed copy of their results for the attention of the referring clinician. even though external referrals are viewed by laboratory technicians as justified when the mh 34 laboratory cannot perform such tests, they also perceive clinicians to have poor levels of trust in the quality of their work. ethnographic observations and informal interactions outside of the formal interview context suggested a pervasive sense, reported among laboratory workers, that clinicians did not sufficiently value their expertise. it was also difficult to demonstrate the quality of their work when supply chain and infrastructural challenges prevented them from carrying out tests. regardless of the challenges, laboratory staff reported going the extra mile to process and report urgent results to doctors quickly: ‘like when we are done in the laboratory, especially if the doctor needs an urgent result like hb [haemoglobin] level of the patient, immediately we’re done we should make sure that the clinician receives the results. if it’s a ward patient, we make sure that we take the results urgently and tell the nurse on duty that, hmm, this patient is alarming; please let the doctor know urgently.’ (participant 3, laboratory lead, 22 june 2019) the laboratory staff interviewed, especially the volunteers, also requested improved pay and working conditions: ‘the doctors should be aware that the lab technicians are exposed to much risk in their work setting. we are exposed to several viruses and if we get sick, we expose our family members. we, therefore, need to be given incentives to work well. … i have advocated so they can increase my salary, but that has not come to effect even after repeatedly reminding my superiors. i have worked for over a year, and they have refused to add to my salary. however, other colleagues, such as the cleaners, are receiving more pay than myself, including the electrician. i am distressed to learn that the electrician gets more pay than me, who works as a laboratory technician. i work very hard but get a little incentive in return. now i am even thinking of getting a second job.’ (participant 6, laboratory technician, 11 september 2019) discussion our study findings show that laboratory staff hold positive valuations of their laboratory work. however, laboratory technicians report feeling undervalued by policymakers and government officials. despite an increased focus on laboratories during the ebola crisis, laboratory workers do not view this attention as translating into sustainable progress within the sector, and feel their insights are overlooked when making critical decisions. this perception of neglect is exacerbated by inadequate training opportunities and a lack of investment in laboratory infrastructure, contributing to low morale among laboratory technicians. positive perceptions of laboratory work by family members contributed to an understanding of their work as valuable and important, both for the family and for society. this positive sense of the value of their work had, in many cases, been enhanced by their experience of the ebola outbreak, in which laboratory workers perceived testing had played a crucial role in saving lives and bringing the outbreak to an end. our finding that laboratory workers had a strong sense of their work’s important contribution to the health sector confirms findings from elsewhere in west africa that laboratory workers refer to themselves as the ‘brains’ of the healthcare system,17 and as the ‘sorcerers’ of the clinicians, who investigate the underlying problem causing their patients’ illness.19 however, this sense of pride in their expertise and positive view of their work was contradicted by perceptions of how others viewed it. our thematic analysis showed that laboratory workers often feel undervalued and under-recognised within the sierra leone health system, results that mirror qualitative research on laboratory workers in other institutions in the country18,19 and align with research that has shown that laboratories have been historically neglected within health system strengthening efforts in africa.26,27 for the laboratory workers included in this study, the lack of investment in the laboratory infrastructure and equipment, limited career growth opportunities, and poor pay and working conditions evidenced how poorly the government valued their work. the fact that they had little input in the procurement of equipment, were not trained to maintain the equipment they received, and did not receive sufficient supplies to carry out basic tests led to a sense among laboratory staff that their views and experiences were not seen as essential and that little attention was being paid to the logistical and infrastructural systems that enable laboratories to function. the neglect of laboratory infrastructure in the main laboratory was contrasted with how the chinese government was viewed as valuing and resourcing the work undertaken in the dedicated infectious disease laboratory. beyond their relationship with the government, laboratory staff also perceived the laboratory as a neglected space within the hospital. the fact that many clinicians sent patients to private laboratories rather than the hospital’s main laboratory for testing demonstrated the poor regard their clinical colleagues had for them. these tensions echo findings from the laboratory-clinical interface elsewhere in africa.28,29 in tanzania, for example, a study found that in-person contact between clinicians and laboratory professionals is seldom institutionalised, and collaboration is rare.30 as other scholars have shown, relationships between clinicians and laboratory workers must be understood regarding the socio-political, economic, and cultural context in which staff operate.31 our findings suggest that in sierra leone, these relationships and a mutual understanding of one another’s professional viewpoints are undermined by the lack of investment in infrastructure, supply chains and human resources. one key issue raised was the appropriateness of donated laboratory equipment and the establishment of supply systems, training, and maintenance to ensure that those machines continued to be usable in the future. the hierarchical nature of public institutions like the military often leads to decisions being made by top-ranking officials from the ministry of defence rather than consulting the laboratory technicians who are familiar with the day-to-day operational needs. also, it is common for institutions like the military hospital to be approached by non-profits, corporate entities, or foreign government agencies with an offer to donate equipment. structures and systems to vet these donations and determine whether the items given meet the actual needs and conditions of the receiving institution are non-existent. selection of donated equipment, therefore, does not typically undergo a systematic evaluation to ensure compatibility with existing infrastructure, availability of necessary supplies, and the hospital staff’s ability to operate and maintain the equipment effectively. another issue was the perceived resource inequality between the ‘main laboratory’ supported by the sierra leone military and the infectious disease laboratory financed by the chinese military. as others have shown, post-ebola laboratory-strengthening efforts have often resulted in donations of high-end equipment, including more automated diagnostic devices, and infrastructural investments. but rather than integrating those investments within existing laboratory structures, separate units and parallel vertical systems have often been set up, which fall into disrepair after donors leave.3 our findings show that laboratory staff are acutely aware of these dynamics and that they impact their perception of how their work is valued by others. overall, across a wide range of areas, including career and training opportunities, procurement processes, infrastructure investment, clinician-laboratory interactions, laboratory staff described their work as ignored, underpaid, undervalued, and neglected. we suggest that these experiences are best understood as a form of social invisibility in which laboratory workers perceive that the value of their contribution to society is not validated and recognised by others, whether that be fellow hospital staff, managers or government.32 we found that social invisibility may be experienced through spatial isolation within the laboratory, lack of consultation on decisions, or physical neglect as when staff work amidst dilapidated infrastructure. this finding that social invisibility is a key aspect of the post-ebola laboratory worker experience in mh 34 confirms findings from other studies in the region about experiences of hidden labour and undervalued contributions to the response.14,15,16,17,18,19 it also mirrors the findings from a qualitative study of healthcare workers in sierra leone, which found that a lack of recognition for healthcare providers by superiors and the health system in general led to poor morale and contributed to poor care.33 perhaps the most striking aspect of our findings is that only three years after the end of the ebola outbreak, laboratory workers in sierra leone see their work as undervalued and unrecognised. laboratories are increasingly recognised as key to disease control programmes, elimination efforts, and epidemic preparedness and response.29 the west africa ebola outbreak was a watershed moment that highlighted the extent to which government and international agencies had overlooked the region’s laboratory systems and ushered in intensive laboratory-strengthening efforts.1 our research shows that the ebola experience in the career of laboratory workers reinforced their sense of public service and the importance of their work. nonetheless, laboratory workers expressed disappointment that the highly visible nature of their contribution during ebola, when laboratory testing was widely recognised as vital to the response, was not reflected in subsequent investments in infrastructure and training. since these findings were based on a small study with a limited sample size, further research is needed to establish how widely this affected the laboratory sector in the region. in terms of priorities for policy changes and investments, laboratory workers articulated the need for improvement in training and employment conditions, physical space, infrastructure, restructuring of supply chains and laboratory-driven equipment procurement and maintenance, and the need for improved relationships with clinical staff. the hospital management and the different ministries concerned should consider the alignment and integration of the two laboratories, such as integrating both units as a single ‘main’ laboratory. it is important that these measures are not only viewed as material investments but that they are acknowledged as a key part of the process of social visibility and recognition and as making a potentially key contribution to staff morale. at mh 34, some of these priorities have already begun to be addressed in response to the findings of this study. in november 2020, the mh 34 initiated a project to enhance laboratory services by identifying and implementing interventions focused on five target areas: staff, supplies, systems, space, and services.34,35,36 most of the interventions directly targeted the concerns raised by laboratory workers as part of this study. staffing to improve staffing, we recruited additional technicians, data clerks, and laboratory interns, identified gaps in technical competence through regular surveys, and facilitated rotation, in-house training, or collaboration with external partners for specialised training courses.34,35 supply chains and maintenance we brokered a public-private partnership with a local laboratory equipment vendor, which allowed us to acquire new equipment, consumables, and reagents with the possibility of deferring payment. we also secured free maintenance for the equipment at the hospital. by shifting from international to local vendors, we achieved a steady and sustainable source of reagents and equipment and broadened our laboratory’s repertoire of services. our testing capability grew to encompass various fields, such as biochemistry, haematology, hormones, and microscopy, which resulted in a substantial reduction in referrals of inpatients to private laboratories.35 physical infrastructure we undertook several renovations to create a more favourable working environment and boost the morale of laboratory technicians. in addition to repainting, we replaced all the air conditioning units, repaired the ceiling to eliminate leaks, increased the water storage capacity to guarantee a reliable water supply, and furnished the on-call room with new furniture, including air conditioning and beds. other measures we implemented to enhance safety in the workplace included fitting breakers, stabilisers, and inverters (for certain equipment) to safeguard against erratic voltages and ensure optimal performance.36 workplace relationships lastly, we arranged guided tours of the laboratory for hospital clinicians and frequently presented and arranged for laboratory leads to present on aspects of laboratory work at the continuous medical education seminars for hospital staff. exhibiting the laboratory’s operations and constraints has significantly increased the number of patients that clinicians refer to our laboratory.35,36 limitations this research design encompassed several limitations. we acknowledge the limitation imposed by the small sample size. while we sought to mitigate this through a measure of data saturation, the cohort of technician participants represented here may not fully capture the diversity of experiences and perspectives that exist among laboratory workers at large. to safeguard anonymity in a small sample size, we were unable to disclose full demographic and employment details for individual participants. expanding the sample size in future research could potentially yield more diverse insights and amplify the reliability of the thematic constructs identified. the study’s setting is limited to a single hospital. while this provides an intimate understanding of the technicians’ experiences within this particular environment, the unique operational and environmental factors specific to this hospital may not be representative of other institutions. comparison to similar studies, as detailed in the discussion, suggests that the findings have wider applicability. nonetheless, the findings are best viewed as a detailed case study and further research is needed to establish the extent to which they can be generalised. while qualitative research inherently involves interpretative elements by the researchers, m.b.j.’s employment at the hospital may introduce additional unconscious biases stemming from their personal experiences and internal knowledge of the hospital environment. to mitigate this, a.s. and e.v. reviewed m.b.j.’s input into the research process and made recommendations for alternative questions, interpretations, and analysis where data collection and data analysis processes were perceived to risk bias. as outlined in the methodology section, steps were also taken to limit participant bias stemming from m.b.j.’s position as a hospital employee. while some bias may nonetheless have persisted, the results indicate that participants felt safe to mention several concerns related to clinicians, the infrastructure, hospital administration and even the way the ministry of defence undertakes procurement of supplies. the highlighted limitations underscore the need for cautious interpretation of the study’s outcomes. to enhance the validity and extend the applicability of the findings from this research, future studies with larger sample sizes, involving multiple hospitals with varying characteristics, and including research multiple team members, including researchers who are not affiliated with the study settings, are recommended. conclusion the neglected state of laboratories in african healthcare systems has been improving since the ebola outbreak in west africa, but maintaining investments after the end of an emergency and the exit of many donors has been a challenge. laboratories are essential in outbreak response, but this study shows that laboratory workers feel undervalued due to a lack of investment, limited career opportunities, poor pay, pressured working conditions, and clinicians sending patients to private labs. it is important that laboratory workers’ perceptions and experiences are understood, listened to and acted upon when setting priorities for national and international health system strengthening efforts. laboratory staff at mh 34 identified investment in training, improved employment conditions, physical space, integration of donor and local infrastructures, equipment maintenance, supply chain restructuring, and clinical staff relationships as key priorities that should be addressed if they are to feel valued and contribute effectively to routine patient care and emergency response. integrating research findings from this study into laboratory restructuring and investment at mh 34 provides an example of how qualitative research, which focuses on people’s experiences, relationships, and practices, can contribute to strengthening laboratory systems in efficient ways. acknowledgements we would like to express our sincere gratitude to all those who have contributed to the completion of this article. without their support, this work would not have been possible. first and foremost, we would like to thank dr stephen sevalie, commanding officer and colonel hassan s. kamara, finance officer of the joint medical unit for facilitating the roll-out of this study at the joint medical unit and for providing feedback throughout the entire research process. we would also like to extend our appreciation to all the laboratory workers who generously dedicated their time to be a part of this study. their cooperation and willingness to share their experiences and perspectives have provided vital insights that have contributed significantly to the findings and conclusions presented in this article. additionally, our heartfelt thanks go to members of staff of the 34 military hospital research unit for their assistance with data collection, data analysis, or any other form of technical support. their expertise and unwavering commitment have played a pivotal role in ensuring the accuracy and reliability of the research results. furthermore, we are grateful to our colleagues and peers who provided valuable discussions, suggestions, and encouragement throughout the writing process. their contributions have shaped our understanding and further enriched the content of this article. lastly, we would like to acknowledge the funding support provided by the european research council under the european union’s horizon 2020 research and innovation programme. their financial assistance was instrumental in facilitating the research, data collection, and publication of this article. we are sincerely thankful to all those mentioned above, and anyone not explicitly mentioned but who has contributed to this work in any other form. your valuable contributions have played a vital role in making this article a reality, and we are deeply appreciative of your involvement. thank you all once again for your unwavering support and dedication. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions each author of this study made significant contributions to various aspects of the research process. m.b.j. played a crucial role in the adaptation of the design of the study for the joint medical unit. he was involved in framing the research questions and designing the qualitative methodology. additionally, he conducted data collection, which involved conducting interviews and structured observations of laboratory technicians in their workstations. he also participated in data analysis by coding, categorising and interpreting the qualitative data. furthermore, m.b.j. contributed extensively to the writing of the manuscript, including drafting sections of the introduction, methodology, and results, while also providing critical revisions and feedback throughout the writing process. e.v. provided valuable expertise in the literature review aspect of the study. she contributed to the conceptualisation and design of the study and in determining the theoretical framework for this study. she conducted comprehensive literature searches, critically reviewed existing scholarly works, and synthesised relevant literature. e.v. also participated in the thematic analysis and interpretation of qualitative data, contributing to the development of key themes and findings. additionally, she played an essential role in structuring the discussion section of the manuscript, incorporating relevant theoretical frameworks, and comparing the study’s findings with existing literature. she actively participated in the revision process, providing constructive feedback, and contributing to the final version of the article. a.s. conceptualised the study and mentored the lead author in all phases of the study and its publication. she made substantial contributions to the data analysis and interpretation phase of the study. she was involved in coding, categorising and analysing qualitative data to identify themes and patterns. a.s. actively contributed to the development of conceptual frameworks and the integration of qualitative findings into the larger study objectives. she also provided critical insights and suggestions for refining the data analysis process. additionally, a.s. contributed significantly to the writing of the introduction, methodology and results section and parts of the discussion section, ensuring accurate representation and interpretation of the qualitative data. all authors have thoroughly reviewed and approved the final version of the manuscript for submission, acknowledging their accountability for the accuracy and integrity of the research presented. in summary, the authors collectively contributed their unique expertise, knowledge, and efforts to successfully complete this qualitative study. each author’s individual contribution was valuable in shaping the research from its inception to the final manuscript, reflecting a collaborative endeavour. sources of support this research received support from the european research council under the european union’s horizon 2020 research and innovation programme under grant agreement no. 715450. data availability data for this study have not been made publicly available to protect the identity of the participants in accordance with ethical requirements. the requirements of the sierra leone ethics and scientific review committee were followed to ensure that all findings are based on de-identified aggregate data and all published results shared in group form. de-identified data supporting the findings of this study are available from the corresponding author, m.b.j., on request. disclaimer the opinions and views expressed in this article are solely those of the authors and do not necessarily represent the official position of any organisation, institution, or entity with which the authors may be affiliated. the authors acknowledge that their perspectives and conclusions are based on their interpretation of the available data and sources. while every effort has been made to ensure the accuracy and reliability of the information presented, the authors do not guarantee the completeness or comprehensiveness of the content. the article is intended for informational purposes only and should not be considered as professional advice or definitive guidance. readers are encouraged to consult additional sources and seek expert advice in relevant areas to make informed decisions or judgments. furthermore, the authors cannot be held responsible for any consequences or damages that may arise from the use, implementation, or interpretation of the information provided in this article. the content is offered without any warranties, explicit or implied, regarding its correctness, reliability, or suitability for any purpose. the authors observe ethical standards and have made every effort to appropriately reference and acknowledge all sources used in this research. however, if any unintentional oversight or error has occurred, the authors apologise and would appreciate being notified in order to rectify it. readers are advised to exercise critical thinking and independent judgment when interpreting the findings, recommendations, or opinions presented in this article. the authors welcome constructive dialogue, differing viewpoints, and further discussion to enhance ongoing scholarly discourse on the topic. in summary, the authors disclaim any liability, 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https://doi.org/10.1016/s0140-6736(16)00119-7 willott c, boyd n, wurie h, et al. staff recognition and its importance for surgical service delivery: a qualitative study in freetown, sierra leone. health policy plan. 2021;36(1):93–100. https://doi.org/10.1093/heapol/czaa131 jalloh mb. laboratory services progress report at the joint medical unit. unpublished report. 2020. jalloh mb. special report on laboratory priorities, functions, and resources at the joint medical unit. unpublished report. 2021. jalloh mb. annual report of interventions for the promotion of laboratory services at the joint medical unit. unpublished report. 2022. abstract introduction methods results discussion reliability acknowledgements references about the author(s) faieqa adams western province blood transfusion service, cape town, south africa gregory r.m. bellairs western province blood transfusion service, cape town, south africa arthur r. bird western province blood transfusion service, cape town, south africa oluwafemi o. oguntibeju department of biomedical sciences, cape peninsula university of technology, cape town, south africa citation adams f, bellairs grm, bird ar, oguntibeju oo. metabolic effects occurring in irradiated and non-irradiated red blood cellular components for clinical transfusion practice: an in vitro comparison. afr j lab med. 2018;7(1), a606. https://doi.org/10.4102/ajlm.v7i1.606 original research metabolic effects occurring in irradiated and non-irradiated red blood cellular components for clinical transfusion practice: an in vitro comparison faieqa adams, gregory r.m. bellairs, arthur r. bird, oluwafemi o. oguntibeju received: 24 jan. 2017; accepted: 20 july 2018; published: 06 dec. 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: storage lesions occur in red blood cell products when potassium ions, haemoglobin and lactate dehydrogenase are released into the extracellular plasma due to post-irradiation storage or cellular degeneration. the south african blood transfusion establishments do not comply with the universal leucocyte-reduction policy due to cost and the current hiv pandemic. various studies regarding storage lesions have been completed in well-developed countries but not in cape town, south africa. objective: this study aimed to determine cellular degeneration occurring in non-irradiated and irradiated red blood cells (rbc) by comparing the measured biochemical and haematological indices during storage of up to 42 days. method: eighty whole blood units were collected from voluntary, non-remunerated donors. blood components tested weekly until expiry were whole blood, rbc concentrate, leucocyte-reduced rbc concentrate (pre-storage) and paediatric rbc concentrate (n = 20). ten units per product were irradiated and 10 were not. evaluations included potassium, sodium, glucose, lactate dehydrogenase, phosphate, haemoglobin, haematocrit, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentrate, mean cell volume and plasma haemoglobin. plasma haemolysis levels were calculated using an approved formula. results: the haemolysis levels evaluated on day 35 and day 42 were higher than the recommended 0.8%, whereas results for the non-irradiated components up to expiry were all below 0.8%. conclusion: this study confirms that gamma irradiation aggravates the rbc storage lesions. the products tested yielded similar results to other studies in developed countries, however the south africa transfusion medicine policy should remain unchanged. introduction the administration of red blood cell (rbc) products enhances intravascular oxygen-carrying capacity and is considered a vital treatment for patients with anaemia triggered by haemorrhage due to surgery, trauma or various haemoglobinopathies or malignancy.1 most clinicians agree that rbc products deteriorate as soon as they leave the donor, despite an ongoing debate about increasing the storage period of blood products while still ensuring the safety and efficacy of stored blood.2 storage lesions include, but are not limited to, acidosis due to lowering of ph, decreased 2,3-diphosphoglyceric acid concentrations, low adenosine triphosphate levels, a decrease in intracellular potassium and increase in cytoplasmic sodium concentrations, lipid peroxidation and oxidative stress to band 3 structures and partial membrane loss via vesicle formation.3 damage to the cell membrane causes haemolysis and potassium leakage, although these increases are not regarded as being clinically significant except in the following situations: when patients present with renal failure, at the onset of hyperkalaemia or when aged whole blood is transfused to neonates. when dealing with ill neonates, the potassium levels on storage may become significant, especially when large volumes are transfused. haemoglobin, lactate dehydrogenase (ldh) and potassium cations are released into the supernatant due to oxidative damage caused by storage lesions.4 formation of haemoglobin microparticles due to the loss of rbc membrane structural integrity are caused by glucose consumption. the membrane becomes deformed due to an increase in osmotic fragility and spheroechinocytes are formed. while some storage lesions may occur within days or weeks, elevated potassium and ldh levels may be detected within hours of storage.3 all these changes affect blood rheology and may add to transfusion difficulties.5 whole blood and component therapy whole blood (wb) is collected from the donor, and is the primary material used for component processing with an expiry period of 35 days when stored at 1 °c to 6 °c. wb is normally administered for neonatal exchange transfusions to treat hyperbilirubinaemia as a result of haemolytic disease of the newborn. this treatment decreases the neonatal bilirubin level by removing the rh-d-positive rbcs and the circulating maternal allo-anti-d.6 in adults, it is also clinically indicated to rectify massive haemorrhage due to trauma, surgical or obstetrical causes.7 component therapy is based on the transfusion of a specific component as this allows more patients to benefit from a single wb donation, while reducing the need for entire wb transfusions.8 the rbc concentrate (rbcc) is the component indicated for patients suffering from anaemia, obstetric haemorrhage, surgery or patients with an acute blood loss of more than 30% total blood volume to improve oxygen delivery to body tissues.9 rbc products can be obtained by processing a wb donation by either using a centrifugation or apheresis method.10,11,12 in south africa, the standard procedure used is the centrifugation method. the plasma and buffy coat are extracted and distributed between the sterile, interconnected but separate satellite bags. saline-adenine-glucose-mannitol is then added to the packed red cells which allows the rbcc to be stored for 42 days. the buffy coat-poor method produces a better quality plasma yield due to the high centrifugal forces used, and leucocyte levels in rbcc are reduced without filtration.12 another advantage is the reduction of micro-aggregate formation during storage.10 this is the standard product prepared for the majority of rbc transfusions in south africa and all rbc products are stored at 1 °c to 6 °c.6 a normal rbcc unit may contain about 1 to 2 billion leucocytes, which may cause various transfusion-associated complications.13 leucocytes are known to increase cellular damage and cause adverse transfusion reactions in recipients, such as allo-immunisation to human leucocyte antigens, non-haemolytic febrile transfusion reactions, transfusion-associated graft versus host disease and transfusion-associated lung injury. leucocytes may also be a pathogenic haven for certain transfusion-associated leukotropic viruses such as epstein-barr virus and cytomegalovirus. b-lymphocytes may be regarded as vectors for the variant creutzfeldt-jakob disease prions.14 transfusion-related immunomodulatory effects, which include post-operative infections leading to mortality due to multi-organ failure, is another transfusion reaction.6,15 in order to minimise or prevent these adverse reactions, blood transfusion establishments prepare leucocyte-reduced and irradiated blood component products. blood is filtered soon after collection as fragmenting granulocytes may cause cellular damage, immune complications or transmit leukotropic viruses from donor to patient.12,15 the rbcc unit may be filtered by physically inserting the spike of the filter into the rbcc unit via a port on the bag, but this reduces the expiry time to 24 h post-filtration. another method is to add the filter to the rbcc using a sterile connecting device to maintain sterility. the latter procedure is used to produce a pre-storage leucocyte-reduced rbcc, with a 42-day expiry when stored at 1 °c to 6 °c. unlike many well-developed countries, south africa does not abide by the universal leucocyte-reduced policy, as the pre-storage leucocyte-reduced rbcc component is produced only when needed. this is due to the high cost involved and the hiv pandemic. there is also no evidence to suggest that patients transfused with filtered rbcc will evade the transmission of creutzfeldt-jakob disease.16 the filtered adult rbcc unit may be further modified by equally dividing the adult unit to produce two paediatric units of approximately 130 ml each or four infant rbcc units of about 55 ml each. blood transfusion services produce small volume units in order to reduce cost and wastage. clinicians who expect premature neonates with low birth weights of less than 1500 g to be multiply transfused will place these patients on the neonatal limited donor exposure programme where units processed from one donor are reserved for a particular infant. this reduces the risk of infection and the production of allo-antibodies as patients will then only be exposed to one set of donor antigens.6,17 it is known that blood stored for long periods contain storage lesions that are not well tolerated in neonates requiring multiple transfusions.18 hyperkalaemia and arrhythmia have been associated with ill infants receiving large volumes of blood. this may be due to high potassium levels present in stored rbc products, which may be caused by environmental heating or cellular degeneration caused by irradiation.19 guidelines stipulate that blood used for an exchange or intrauterine transfusion for hyperkalaemic neonates to be less than 5 days old and infused within 24 h post-irradiation.6,20,21 irradiation gamma irradiation prevents the cells from engrafting and initiating an immune response against the host, as it inhibits the proliferation of t lymphocytes when lymphocyte dna becomes damaged. irradiation mainly affects rbc components and not granulocytes or platelets.22 as irradiation is the preferred method to prevent transfusion-associated graft versus host disease, most blood transfusion establishments use the freestanding 137c irradiator, where the cellular components are exposed to irradiation dosages between 25 gy and 50 gy. blood may be irradiated 14 days post donation and stored for another two weeks without losing rbc viability.6 clinical indications include cellular donations from blood relatives, donors who are homozygous for shared human leukocyte antigen haplotypes, neonatal intrauterine or exchange transfusions and allogeneic bone marrow transplant recipients.23 rbc membrane impairment due to irradiation causes an increase in supernatant potassium ions, plasma haemoglobin and ldh levels with a decrease in ph concentration, indicating that gamma irradiation exacerbates storage lesions.24,25 percentage plasma haemolysis rbc haemolysis may be due to factors such as the storage period, the presence of leucocytes in unfiltered blood, mechanical injury during filtration, bacterial contamination during donation or component processing malpractices.26,27 haemolysis increases the oxygen affinity of haemoglobin, which reduces oxygen transfer to the tissues. the biochemical reaction of plasma haemoglobin and nitric oxide may cause endothelial dysfunction, leading to intravascular thrombosis, leucocyte adhesion and possible vasoconstriction.28 south african blood transfusion establishments concur with the european council ruling that stipulates that the haemolysis levels of stored rbc products should be less than 0.8% at the end of the storage period, whereas the united states food and drug administration specifies less than 1%. both councils insist on a 75% survival rate for transfused rbcs 24 h post-transfusion.3 despite various methods available to measure haemolysis, no standardised method exists as each criterion may be measured in various ways. one study reported a coefficient of variation of approximately 55% between 14 laboratories measuring percentage of plasma haemolysis.27 the common practice of visually checking rbc products before they are issued from the blood bank is often incorrect, subjective and may result in gross overestimation. this may lead to these units being discarded, as dark pink supernatant discoloration may demonstrate plasma haemoglobin levels as low as 0.09% haemolysis.29 monitoring the quality of blood a quality management system ensures that the quality of blood and blood components is continuously monitored to allow patients to receive the safest possible blood and to provide a service to stakeholders (donors, clinicians, patients, business partners and employees). management ensures that facilities are adequately equipped and have trained or qualified staff on site to collect blood donations and subsequently process, test and issue blood and blood products. in south africa, procedures are completed according to a departmental standard operating procedure document and staff must comply with the relevant acts and the standards of practice for blood transfusion. quality management systems also ensure that all equipment used for laboratory testing have been validated and routinely calibrated and that reagents utilised are quality controlled according to a prescribed method. deviations from documented work procedures are recorded, investigated and appropriate corrective action taken. the quality indicators are continuously monitored and documented, such as during annual internal and external audits, staff proficiency tests, competency assessments and the monthly blood components quality control assessments. the purpose of this study was to compare biochemical and haematological storage lesions in irradiated and non-irradiated rbc components that occur during the standard storage period to assess the effect of irradiation on wb and paediatric rbbc, both products are used in neonatal transfusion, and to determine whether current south african policies regarding the storage period of irradiated products need be modified. methods ethical considerations each donor completed a donor questionnaire prior to wb donation where they agreed that their donation may be used for research purposes. available levels of blood stock were not compromised and donor confidentiality was maintained by using barcoded serial numbers. the chief executive officer or medical director at the blood transfusion establishment provided a letter of consent for the research study to take place at the establishment’s headquarters in cape town, south africa. ethical approval from the health and wellness sciences research ethics committee from the cape peninsula university of technology (study approval number: cput/hw-rec 2014/h10) was obtained. all ethical, scientific standards and good clinical practice were maintained throughout the study. study population voluntary, non-remunerated donors residing in the western cape region of south africa were selected according to the criteria for the protection of the donor and recipient as indicated in the standards of practice for blood transfusion in south africa.30 donors unable to fulfil the criteria and first-time donors were excluded from the study. study design this was a random sample research design, and the products tested included wb, buffy coat-poor rbcc, pre-storage leucocyte-reduced rbcc and paediatric rbcc. eighty wb units were collected into collection bags containing citrate phosphate dextrose terumo (quadruple pack with diversion pouch) on the same day. wb units were randomly selected for processing into the different products. each test group consisted of 10 irradiated rbc units per product and the control group comprised 10 non-irradiated counterparts. a wb unit was separated into components via centrifugation using a sorvall rc 12 bp plus centrifuge (thermo fisher scientific, waltham, massachusetts, united states) and centrifuged at 3140 rpm for 12 min at 4 °c to produce rbcc, fresh frozen plasma and cryoprecipitate products. a wb unit was also centrifuged at 3140 rpm for 10 min at 22 °c to produce rbcc, fresh frozen plasma and platelets. this was done to not compromise the stock levels. plasma and buffy coat were extracted using a t-ace ii extractor (terumobct, lakewood, colorado, united states). the units placed in the test group were gamma irradiated the day after processing using a gammacell 3000 irradiator (nordion inc., ottawa, ontario, canada). the irradiator was loaded with two components at a time with a minimal gamma exposure of 2574 cgy and a central dose of 2932 cgy (range: 25 gy to 50 gy). a machine-cycle printout was attached to each irradiated product. sampling a diversion pouch was aseptically attached to the rbc unit by using a tscd® ii sterile tubing welder (terumobct, lakewood, colorado, united states). after the pouch was filled with the required amount of blood, the tubing was hermetically sealed using the tube sealer and the unit was returned to the fridge to be stored at 1 °c to 6 °c. supernatant for testing was obtained by centrifuging the test sample at 3000 rpm for three min. this procedure was repeated on days 7, 14, 21, 28, 35 and 42. in vitro measurements sample supernatant was used to test potassium, phosphate, sodium, glucose and ldh using the abx pentra 400 chemistry analyser (horiba abx sas, montpellier, cedex, france). the ph was measured using a desktop ph meter (amtech laboratory services, cape town, south africa). the haematological levels for the full blood count, haemoglobin, haematocrit, mean cell volume, mean corpuscular haemoglobin and the mean corpuscular haemoglobin concentrate were measured using the abx pentra xl 80 (horiba abx sas, montpellier, cedex, france). the hemocue® plasma/low hb system (se-262-71, hemocue ab, ängelholm, sweden) was used to measure the plasma haemoglobin levels. the formula for calculating the percentage of plasma haemolysis is detailed below. equation 1: calculation for percentage plasma haemolysis (adapted from leitner, 2001)22 testing for the haematological factors, biochemical indices and routine donor testing commenced within the accepted 72 h post-collection and is known as day 1. statistical analysis data were documented using an excel spreadsheet (excel 2013, microsoft corporation, redmond, washington, united states). statistical analyses of the test results were performed using excel 2013, as well as prism 5, 2007 (graphpad software, inc. san diego, california, united states). statistical comparison of the non-irradiated (control group) and irradiated (test group) was completed using paired t-test for single time points and analysis of variance for multiple time points during the storage period. multi-comparisons to define specific differences between the control and test groups at specific time points were performed using the post-hoc bonferroni statistical calculations. results are expressed as mean ± standard deviation; a p-value of less than 0.0 (p < 0.01) was considered significant. results the results for wb observed for the testing of biochemical analytes and haematology factors for the non-irradiated and irradiated units during the storage period are listed below, together with graphical illustrations. biochemistry statistically significant increases in serum potassium results were observed (figure 1), except for wb day 28 (p = 0.01) and paediatric rbcc on day 42 (p = 0.13). sodium levels decreased during storage, whereas the phosphate levels increased, except for the leucocyte-reduced non-irradiated rbcc and paediatric rbcc on day 28 due to outliers. most results were not statistically significant (p > 0.01), except for paediatric rbcc on day 42 (p = 0.002). a reduction in serum glucose levels indicating glycolytic metabolism was observed for all non-irradiated and irradiated products, albeit with no statistical significance. ldh concentrations increased with statistical significance for leucocyte-reduced rbcc on days 14, 28 and 42 (p = 0.002) and paediatric rbcc on day 42 (p = 0.002) (figure 2). ldh levels for non-irradiated wb increased by 164% and irradiated wb increased by 215%, whereas non-irradiated paediatric rbcc increased by 132% and irradiated units increased by 436%. a gradual decrease in ph levels was observed with no statistical significance (figure 3). figure 1: potassium levels. figure 2: lactate dehydrogenase (ldh) levels. figure 3: ph levels. haematology the haemoglobin levels for non-irradiated wb and rbcc decreased by 8% and 3% (p-values: 0.02–0.88) (figure 4). the percentage of plasma haemolysis results indicate that by expiry all irradiated products exceeded the 0.8% haemolysis level as stipulated by the european council, whereas non-irradiated levels were all below 0.8% (figure 5). statistically significant increases in plasma haemoglobin for rbcc were indicated on days 28 and 42 (p = 0.001) and on days 28 and 42 (p = 0.002) for leucocyte-reduced rbcc (figure 6). mean corpuscular volume results for irradiated wb decreased from 88.5 fl (day 1) to 87.6 fl (day 35). the haematocrit levels showed a gradual increase, whereas all products displayed p-values higher than 0.01 for both mean corpuscular volume and mean corpuscular haemoglobin levels during the storage period. mean corpuscular haemoglobin concentrate levels displayed similar trends except for paediatric rbcc on day 28 (p = 0.005), and p-values for erythrocyte levels ranged from 0.01 to 1.00. a reduction in leucocyte and thrombocyte levels for both non-irradiated and irradiated products was observed. statistically significant values were observed for leucocyte-reduced rbcc on day 1 (p = 0.006), day 14 (p = 0.002) and day 28 (p = 0.001). figure 4: haemoglobin (hb) levels. figure 5: percentage plasma haemolysis levels. figure 6: plasma haemoglobin levels. discussion this study aimed to determine whether there are any significant differences between non-irradiated and irradiated rbc products produced in cape town and to establish whether the south african policy regarding irradiated rbc products should be amended. while current literature indicates that gamma irradiation aggravates storage lesions in rbc products, less is known about irradiated leucocyte-reduced components.31 the blood transfusion establishments in south africa do not comply with the universal leuco-reduction policy due to the cost.6 although gamma irradiation exacerbates storage lesions, it is the accepted method to prevent transfusion-associated graft versus host disease.24 the irradiated rbc products in our study had higher percentage of haemolysis levels from days 28 to 42, which has also been observed by earlier studies.22,25,29,32,34,35,36 blood transfusion establishments should be aware that there are many factors contributing to rbc haemolysis. some of these factors include analyte differences between donors, diverse preand post-transfusion abilities of various donors, possible genetic inconsistencies, pre-analytical factors and component processing strategies.3,26,34 all used units were sent to the microbiology laboratory to be examined for bacterial contamination. the results were negative. the results for non-irradiated paediatric rbcc indicate statistically significant potassium results on day 28. while top-up neonatal transfusions are mostly small volume (10–20 ml/kg), it is suggested that blood be used before day 28 even though the risk to hyperkalaemic neonates is minimal due to the small plasma volume. however, for large volume transfusions such as neonatal exchange transfusions or in acute blood loss, wb less than 5 days is used to prevent hyperkalaemia and low 2, 3-diphosphoglycerate levels.6 our results confirm most previous outcomes regarding filtered rbcc that have been irradiated not later than day 14.32,33 it also proves that leucocyte-reduction of rbc products moderate haemolysis as a decrease in haemolysis was observed when non-irradiated day 42 rbcc was compared to non-irradiated, pre-storage, leucocyte-reduced day 42 rbcc. our results also indicate that cellular degeneration is lower when leucocyte-reduced non-irradiated rbc products are compared to their irradiated counterparts and confirms that rbc leucocyte-reduced products improve blood safety and efficacy.34 limitations donor blood was not tested prior to component separation and subsequent irradiation; thus, there were no baseline measurements. rbcc were produced by centrifuging wb at two different centrifugation temperatures, instead of a single temperature of 4 °c. an improved strategy would be to use one rbc product and divide it into two equal aliquots where one bag is irradiated and the second bag used as the control. additionally, it was an oversight not to include the irradiation of blood on day 14 and keep it stored until expiry (per united states guidelines). recommendations despite controversial debates regarding the transfusion of rbcc products, it still remains a popular treatment resource. the published results of meta-analysis regarding storage lesions should be carefully reviewed before policies relating to transfusion medicine are amended. the scientific community should be contemplating improvements to storage strategies, such as anaerobic storage, nutritive additives, proteomic-based biomarkers and an alternative to gamma irradiation for the prevention of graft versus host disease. oxidative stress may occur, and therefore the addition of antioxidants to units of blood could possibly decrease lipid peroxidation and prevent leakage of potassium, ldh and haemoglobin into the plasma. conclusion the outcome of this study confirms that gamma irradiation exacerbates rbc storage lesions during storage at 1 °c to 6 °c for up to 42 days. increases in potassium, ldh and haemolysis levels were observed, which may result in serious implications when transfused to immunocompromised patients. this study further observed significant differences between irradiated wb and paediatric rbcc due to differences in plasma volume. significant differences were also demonstrated between adult irradiated, pre-storage, leucocyte-reduced rbcc and paediatric rbcc for glucose, ph levels, plasma haemoglobin levels and plasma haemolysis. although these products are both leucocyte-reduced, the irradiated paediatric rbcc undergoes further product modification. the products tested yielded similar results to other studies in developed countries that leucocyte-reduction improves blood safety and efficacy. while south african blood transfusion services do not comply with the universal leuco-reduction policy, it is recommended that for now, the south african transfusion medicine policy remain unchanged, as it is not cost-effective to only produce leucocyte-reduced products due to the country’s current health issues. reliability the evaluations of the testing performed in this study were completed using equipment, methods and corresponding reagents according to the manufacturers’ instructions and relevant standard operating procedures. blood and samples were collected from the donor population in the western cape region of south africa. the equipment and analysers with readily available corresponding reagents are well known in the field of immunohaematology and therefore the methodology used in this study can be applied elsewhere in the world. acknowledgements competing interests the authors declare that they have no conflict of interest regarding the publication of this article. sources of support the financial assistance of the cape peninsula university of technology and western province blood transfusion service towards this research is acknowledged. opinions expressed in this article and the conclusions arrived at are those of the authors and are not to be attributed to cape peninsula university of technology or western province blood transfusion service. authors’ contributions o.o.o. was the project leader and made conceptual contributions. g.r.b. and a.r.b. were responsible for experimental and conceptual design. f.a. prepared samples, performed all the experiments and calculations. references sparrow rl. red blood cell storage and transfusion-related immunomodulation. blood transfus. 2010;8 (suppl 3):s26–s30. https://doi.org/10.2450/2010/005s isbister jp. is the clinical significance of blood storage lesions underestimated? tatm. 2003;5(3):356–362. https://doi.org/10.1111/j.1778-428x.2003.tb00175.x/pdf d’alessandro a, liumbruno g, grazzini g, zolla l. red blood cell storage: the story so far. blood transfus. 2010;8(2):82–88. https://doi.org/10.2450/2009.0122-09 chaudhary r, katharia r. oxidative injury as contributory factor for red cells storage lesion during twenty eight days of storage. blood transfus. 2012;10(1):59–62. https://doi.org/10.2450/2011.0107-10 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https://doi.org/10.1136/bmj.38103.735266.55 bird ar, crookes r. leucocyte depletion of blood components-guidelines of the blood transfusion services of south africa. s afr med j. 2006;96(5):288–289. josephson cd. neonatal and pediatric transfusion. in: roback jd, combs mr, grossman bj, hillyer cd editors. aabb technical manual. 16th ed. bethesda, maryland: aabb, 2008; p. 639–663. ratcliffe jm, elliott mj, wyse rkh, hunter s, alberti kgmm. the metabolic load of stored blood. implications for major transfusions in infants. arch dis child. 1986;61:1208–1214. https://doi.org/10.1136/adc.61.12.1208 parshuram cs. prospective study of potassium-associated acute transfusion events in pediatric intensive care. pediatr crit care. 2003;4(1):65–68. https://doi.org/10.1097/00130478-200301000-00013 treleaven j, gennery a, marsh j, et al. guidelines on the use of irradiated blood components prepared by the british committee for standards in haematology blood transfusion task force. br j haematol. 2010;152:35–51. https://doi.org/10.1111/j.1365-2141.2010.08444.x australian and new zealand society of blood transfusion ltd (anzsbt). guidelines for prevention of transfusion-associated graft-versus-host disease (ta-gvhd). 1st ed. sydney: anzsbt, 2011. leitner gc, neuhauser m, weigel g, kurze s, fischer mb, höcker p. altered intracellular purine nucleotides in gamma-irradiated red blood cell concentrates. vox sang. 2001;81:113–118. https://doi.org/10.1046/j.1423-0410.2001.00082.x shaz h, hillyer cd. transfusion-associated graft-versus-host disease and microchimerism. in: murphy mf, pamphilon dh, editors. practical transfusion medicine. 3rd ed. oxford, united kingdom: wiley-blackwell, 2009;107–116. mintz pd, anderson g. effects of gamma irradiation on the in vivo recovery of stored red blood cells. ann clin lab sci. 1993;23(3):216–220. agarwal p, ray vl, choudhury n, chaudhary rk. effects of pre-storage gamma irradiation on red blood cells. indian j med res. 2005;122(5):385–387. sowemimo-coker so. red cell haemolysis during processing. transfus med rev. 2002;16(1):46–60. https://doi.org/10.1053/tmrv.2002.29404 han v, serrano k, devine dv. a comparative study of common techniques used to measure haemolysis in stored red cell concentrates. in: vox sang journal compilation. amsterdam: international society of blood transfusion, 2009. aubron c, nichol a, cooper dj, bellomo r. age of red blood cells and transfusion in critically ill patients. ann intensive care. 2013;3:2. https://doi.org/10.1186/2110-5820-3-2 sawant rb, jathar sk, rajadhyaksha sb, kadam pt. red cell haemolysis during processing and storage. asian j transfus sci. 2007;1(2):47–51. https://doi.org/10.4103/0973-6247.33446 bellairs grm, ingram c. standards of practice for blood transfusion in south africa. 6th ed. johannesburg/cape town: sanbs/wpbts, 2013. zubair ac. clinical impact of blood storage lesions. am j haematol. 2010;85:117–122. winter km, johnson l, kwok m, et al. understanding the effects of gamma-irradiation on potassium levels in red cell concentrates stored in sag-m for neonatal red cell transfusion. vox sang. 2015;108:141–150. https://doi.org/10.1111/vox.12194 zimmermann r, wintzheimer s, weisbach v, strobel j, zingsem j, eckstein r. influence of prestorage leukoreduction and subsequent irradiation on in vitro red blood cell (rbc) storage variables of rbcs in additive solution saline-adenine-glucose –mannitol. transfus. 2009;49:75–80. https://doi.org/10.1111/j.1537-2995.2008.01920.x hess jr. an update on solutions for red cell storage. vox sang. 2006;91:13–19. https://doi.org/10.1111/j.1423-0410.2006.00778.x makroo rn, raina v, bhatia a, et al. evaluation of the red cell haemolysis in packed red cells during processing and storage. asian j transfus sci. 2011;5(1):15–17. https://doi.org/10.4103/0973-6247.75970 zimmermann r, schoetz am, frisch a, et al. influence of late irradiation on the in vitro rbc storage variables of leucoreduced rbcs in sagm additive solution. vox sang. 2011;100:279–284. https://doi.org/10.1111/j.1423-0410.2010.01410.x abstract introduction methods results discussion acknowledgements references about the author(s) ellen gobusamang department of medical laboratory sciences, faculty of health sciences, university of botswana, gaborone, botswana naledi g. nyepetsi department of medical laboratory sciences, faculty of health sciences, university of botswana, gaborone, botswana modisa s. motswaledi department of medical laboratory sciences, faculty of health sciences, university of botswana, gaborone, botswana ishmael kasvosve department of medical laboratory sciences, faculty of health sciences, university of botswana, gaborone, botswana citation gobusamang e, nyepetsi ng, motswaledi ms, kasvosve i. hyperuricaemia is associated with dyslipidemia but not hba1c among type 2 diabetes mellitus patients in botswana. afr j lab med. 2019;8(1), a786. https://doi.org/10.4102/ajlm.v8i1.786 brief report hyperuricaemia is associated with dyslipidemia but not hba1c among type 2 diabetes mellitus patients in botswana ellen gobusamang, naledi g. nyepetsi, modisa s. motswaledi, ishmael kasvosve received: 07 feb. 2018; accepted: 28 june 2019; published: 07 nov. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract medical records and residual samples from 334 type 2 diabetes mellitus patients attending a clinic in gaborone, botswana, during the period september–december 2016 were analysed for the effects of hyperuricaemia on biochemical markers of adverse outcomes. the patients were stratified as having hyperuricaemia (> 400 µmol/l) or normal serum uric acid (≤ 400 µmol/l). we compared glycated haemoglobin, triglycerides, low-density lipoprotein-cholesterol, high-density lipoprotein-cholesterol, total cholesterol and serum creatinine between the two serum uric acid categories. hyperuricaemia was detected in 28% of patients (95% confidence interval 23.1–32.9) and was associated with increased serum triglycerides, triglyceride to high-density lipoprotein-cholesterol ratio and creatinine concentration, but not with glycated haemoglobin. keywords: hyperuricaemia; diabetes mellitus; glycated haemoglobin; lipids; africans. introduction epidemiologic studies suggest that hyperuricaemia plays a role in the aetiology and pathogenesis of a number of diseases including diabetes mellitus (dm).1,2 serum uric acid (sua) has a putative role in the development of cardiovascular disease.3,4 in a recent study conducted in south africa, hyperuricaemia was associated with an increased risk of mortality among acute myocardial infarction patients.5 however, it remains unclear whether increased sua concentration contributes to the aetiology or if it is a consequence of these conditions. the role of sua in type 2 diabetes mellitus (t2dm) is inconclusive. however, hyperuricaemia is associated with obesity and insulin resistance,6 and cross-sectional data have shown that hyperuricaemia is prevalent among t2dm patients.7 other studies have also reported that hyperuricaemia increases the risk of t2dm.8,9,10 in a meta-analysis of 11 cohort studies, participants with hyperuricaemia had a 17% increased risk of developing diabetes per 1 mg/dl increase in sua concentration.9 in another meta-analysis of eight prospective cohort studies, a 1 mg/dl increase in sua resulted in a 6% increase in the risk of incident t2dm.8 other studies have reported that hyperuricaemia predisposes individuals to the development of dm complications.11 among dm patients, poor glycaemic control increases the risk of microvascular complications.12 the measurement of glycated haemoglobin (hba1c) and lipids are used to monitor dm patients, and adverse changes are associated with the risk of dm complications. hba1c is a biochemical indicator of chronic glycaemia, and high values indicate poor control.13 the determination of total cholesterol, low-density lipoprotein (ldl) and high-density lipoprotein (hdl) cholesterol, and triglycerides provide valuable information for the prediction of coronary heart disease. triglyceride concentration has an inverse relationship with hdl cholesterol concentration in dm patients.13 both hypertriglyceridemia and low serum hdl cholesterol concentration independently increase the risk of developing coronary heart disease.13 findings on the role of sua in the pathogenesis of dm are inconsistent, and studies on associations between hyperuricaemia and the development of microvascular diseases among dm patients in african populations are lacking. in this study we determined the prevalence of hyperuricaemia and its association with hba1c and lipids in t2dm patients in botswana. methods ethical considerations the study was approved by the university of botswana’s institutional review board, the ministry of health and wellness health research unit (permit number hpdme: 13/18/1 vol x [619]) and the research and ethics committee of princess marina hospital (permit number pmh 5/79 [268-2-2016]). no individual consent was obtained from the patients. study population we conducted a cross-sectional study of residual samples from 334 t2dm patients attending a dm clinic in gaborone city, during the period september–december 2016. the criteria for inclusion were age ≥ 18 years and a known history of t2dm. minors (< 18 years) with t2dm were excluded from the study. the samples enrolled in the study were collected as part of clinical care and were de-identified prior to inclusion in the study, as recommended in the international organization for standardization’s 15189 standard. the clients’ identities were not revealed to study staff and confidential information was only made accessible to authorised persons. laboratory investigations laboratory tests were performed on au480 chemistry analyzer (beckman coulter inc, brea, california, united states). in some cases, the sample volume was insufficient to perform all the tests or the data were missing from the patient’s records. the analyser was calibrated and samples processed according to the manufacturer’s instructions using reagents provided by the manufacturer. serum uric acid was measured using the uricase/peroxidase enzymatic method, and hyperuricaemia was defined as sua > 400 µmol/l. this cut-off is the upper reference limit used to define hyperuricaemia in botswana. hba1c was measured using a latex agglutination assay. briefly, a synthetic polymer containing multiple copies of the immunoreactive portion of hba1c agglutinates with latex coated with hba1c-specific mouse monoclonal antibodies. total cholesterol, hdl cholesterol, ldl cholesterol and triglycerides were determined by routine enzymatic methods. creatinine was measured using a kinetic modification of the jaffe procedure.14 statistical analysis the results were analysed using the statistical package for social sciences version 24 statistical software (ibm corporation, new york city, new york, united states). all quantitative variables were expressed as mean ± standard deviation or median with interquartile range (iqr). categorical variables were expressed as proportions with estimated 95% confidence intervals (ci). comparisons of hba1c concentration according to sex or sua categories were conducted using an independent samples t-test, or the mann-whitney test was used to compare medians for non-parametric data. results were considered statistically significant if p < 0.05. box-and-whisker plots were used to show differences in serum creatinine concentration and hba1c according to uric acid category. results samples from 334 patients (236 women) with t2dm of unknown duration were studied. ninety-two patients (28%, 95% ci 23.1–32.9) had hyperuricaemia and the prevalence was comparable between sexes (25% [95% ci 19.4–30.6] in women compared to 33% [95% ci 23.5–42.5] in men, p = 0.182). hba1c levels were comparable between sua categories; median 7.1% (iqr 6.4–8.9) in t2dm patients with hyperuricaemia compared to median 7.4% (iqr 6.3–9.3) in t2dm patients with normal sua, p = 0.379 (table 1). similarly, hyperuricaemia had no effect on total and ldl cholesterol levels, p ≥ 0.321. there was a tendency for lower hdl cholesterol among patients with hyperuricaemia, p = 0.090. median triglyceride levels were higher among patients with hyperuricaemia (1.99 mmol/l [iqr 1.48–2.57] vs 1.72 mmol/l [iqr 1.19–2.57], p = 0.032); similarly, t2dm patients with hyperuricaemia had higher ratios of triglycerides to hdl cholesterol (median: 1.89 [iqr 1.33–2.79]) compared to t2dm patients with normal sua concentration (median: 1.71 [iqr 1.00–2.44], p = 0.021). hyperuricaemia was associated with higher median creatinine concentration, 78 µmol/l (iqr 61–115) vs 62 µmol/l (iqr 52–73) in patients with normal uric acid, p < 0.001 (figure 1). figure 1: comparison of (a) serum creatinine concentration and (b) glycated haemoglobin (hba1c) according to uric acid category in type 2 diabetes mellitus patients receiving treatment in gaborone, botswana, september–december 2016. table 1: comparison of laboratory characteristics according to serum uric acid categories in type 2 diabetes mellitus patients receiving treatment in gaborone, botswana, september–december 2016. discussion the prevalence of hyperuricaemia among t2dm patients in botswana is high and is comparable to findings in north africa.15 in our study, hyperuricaemia was not associated with poor glycaemic control as measured by hba1c. this is in contrast to data from the national health and nutrition examination survey iii conducted nationwide in the united states.16 our data corroborates findings from another study conducted on a cohort of newly diagnosed t2dm patients in china that reported no relationship between sua and hba1c after adjusting for insulin concentration.17 lipids are a risk factor for cardiovascular disease.13 in our study, t2dm patients with hyperuricaemia had significantly higher triglyceride concentrations and triglyceride to hdl cholesterol ratios. it has been suggested that a high triglyceride to hdl cholesterol ratio is a risk factor for coronary heart disease.18 the triglyceride to hdl cholesterol ratio can be used to predict the presence and degree of coronary atherosclerosis.19 although high triglyceride to hdl cholesterol ratio has been reported to be a marker of insulin resistance,20 a study of african americans did not corroborate this finding.21 other lipids were not associated with hyperuricaemia in our study. hyperuricaemia was associated with an increased creatinine concentration. although hyperuricaemia may simply be a marker of renal disease, there are studies which suggest that elevated sua levels might contribute to the development and progression of renal dysfunction.3 recently (2014), toda et al. reported that hyperuricaemia was an independent risk factor for the development of chronic kidney disease,22 and in studies in japan, elevated baseline sua and increases in sua increased the risk of developing chronic kidney disease.23 in another study, hyperuricaemia was associated with incident diabetic retinopathy among male patients with t2dm.24 limitations we acknowledge that missing data on dm duration is a limitation to the interpretation of our findings, as the evolution of nephropathy and microvascular diseases may be influenced by the duration of dm. adjusting for the duration of t2dm and the severity of dm might have sharpened our findings. in a study by korpachev et al. in 2009, hyperuricaemia with decreased uric acid excretion was characteristic of severe dm, with a reduced kidney filtration rate.25 conditions that reduce erythrocyte survival time may affect hba1c values. although we did not screen for the effect of haemoglobinopathies or malaria on hba1c, it is unlikely that these conditions were confounders in our study. malaria transmission in southern botswana is low to absent, and there is no literature on haemoglobinopathy in botswana. conclusion in conclusion, hyperuricaemia was prevalent among t2dm patients in our study and was associated with elevated triglyceride concentrations, a high triglyceride to hdl cholesterol ratio and elevated serum creatinine concentration, but it was not associated with hba1c concentration. as recommended elsewhere, the monitoring of t2dm should include the measurement of sua and the institution of interventions that lower sua.3 acknowledgements we would like to thank the laboratory staff of the national health laboratory service and lancet laboratories for providing the serological and molecular testing. competing interests the authors have declared that no competing interests exist. authors’ contributions e.g. and i.k. were responsible for the conceptualisation, experimental design, data acquisition, analysis, interpretation of data and writing of the manuscript. n.g.n. and m.s.m. contributed to the experimental design and revising the manuscript for important intellectual content. all authors read and approved the final manuscript. sources of support this research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references kivity s, kopel e, steinlauf s, et al. the association between serum uric acid and diabetes mellitus is stronger in women. j women health. 2013; 22(9):782–789. https://doi.org/10.1089/jwh.2012.4043 freedman ds, williamson df, gunter ew, et al. relation of serum uric acid to mortality and heart disease. the nhanes i epidemiologic follow-up study. am j epidemiol. 1995;141:637–644. https://doi.org/10.1093/oxfordjournals.aje.a117479 martinez-quintana e, tugores a, rodriguez-gonzález f. serum uric acid levels and cardiovascular disease: the gordian knot. j thorac. 2016;8(11):e1462–e1466. https://doi.org/10.21037/jtd.2016.11.39 kivity s, kopel e, maor e, et al. association of serum uric acid and cardiovascular disease in healthy adults. am j cardiol. 2013;111:1146–1151. https://doi.org/10.1016/j.amjcard.2012.12.034 ranjith n, myeni nn, sartorius b, et al. association between hyperuricemia and major adverse cardiac events in patients with acute myocardial infarction. metab syndr relat disord. 2017; 15:18–25. https://doi.org/10.1089/met.2016.0032 baker jf, krishnan e, chen l, et al. serum uric acid and cardiovascular disease: recent developments, and where do they leave us? am j med. 2005;118:816–826. https://doi.org/10.1016/j.amjmed.2005.03.043 heinig m, johnson rj. role of uric acid in hypertension, renal disease, and metabolic syndrome. cleve clin j med. 2006;73:1059–1064. lv q, meng xf, he ff, et al. high serum uric acid and increased risk of type 2 diabetes: a systematic review and meta-analysis of prospective cohort studies. plos one. 2013;8(2):e56864. https://doi.org/10.1371/journal.pone.0056864 kodam s, saito k, yachi y, et al. association between serum uric acid and development of type 2 diabetes. diabetes care. 2009;32:1737–1742. https://doi.org/10.2337/dc09-0288 jia z, zhang x, kang s, et al. serum uric acid levels and incidence of impaired fasting glucose and type 2 diabetes mellitus: a meta-analysis of cohort studies. diabetes res clin pract. 2013;101:88–96. https://doi.org/10.1016/j.diabres.2013.03.026 lee jj, yang ih, kuo hk, et al. serum uric acid concebtration is associated with worsening in severity of diabetic nephropathy among type 2 diabetic patients in taiwan – a 3-year prospective study. diabetes res clin pract. 2014;106 (2):366–372. https://doi.org/10.1016/j.diabres.2014.07.027 klein r, klein be, moss se. relation of glycemic control to diabetic microvascular complications in diabetes mellitus. ann intern med. 1996;124:90–96. https://doi.org/10.7326/0003-4819-124-1_part_2-199601011-00003 nih consensus conference: triglyceride, high-density lipoprotein and coronary heart disease. jama. 1993;269:505–510. bowers ld, wong et. kinetic serum creatinine assays. ii. a critical evaluation and review. clin chem. 1980;26 (5):555–561. fouad m, fathy h, zidan a. serum uric acid and its association with hypertension, early nephropathy and chronic kidney disease in type 2 diabetic patients. j bras nefrol. 2016;38:403–410. https://doi.org/10.5935/0101-2800.20160065 bandaru p, shankar a. association between serum uric acid levels and diabetes mellitus. int j endocrinol. 2011;604–715. https://doi.org/10.1155/2011/604715 cui y, bu h, ma x, et al. the relation between serum uric acid and hba1c is dependent upon hyperinsulinemia in patients with newly diagnosed type 2 diabetes mellitus. j diabetes res. 2016:7184123. https://doi.org/10.1155/2016/7184123 da luz pl, favarato d, faria-neto jr jr, et al. high ratio of triglycerides to hdl-cholesterol ratio predicts extensive coronary disease. clinics. 2008;63:427–432. https://doi.org/10.1590/s1807-59322008000400003 gaziano jm, hennekens ch, o’donnell cj, et al. fasting triglycerides, high density lipoprotein, and risk of myocardial infarction. circulation. 1997; 96:2520–2525. https://doi.org/10.1161/01.cir.96.8.2520 hadaeghi f, khalili d, ghasemi a, et al. triglyceride/hdl-cholesterol ratio is an independent predictor for coronary heart disease in a population of iranian men. nutr metab cardiovsac dis. 2009;19(6):401–408. https://doi.org/10.1016/j.numecd.2008.09.003 sumner ae, finley kb, genovese dj, et al. fasting triglyceride and the triglyceride-hdl cholesterol ratio are not markers of insulin resistance in african americans. arch intern med. 2005;165(12):1395–1400. https://doi.org/10.1001/archinte.165.12.1395 toda a, ishizaka y, tani m, et al. hyperuricemia is a significant risk factor for the onset of chronic kidney disease. nephron clin pract. 2014;126(1):33–38. https://doi.org/10.1159/000355639 kuwabara m, bjornstad p, hisatome i, et al. elevated serum uric acid level predicts rapid decline in kidney function. am j nephrol. 2017;45:330–337. https://doi.org/10.1159/000464260 kuwata h, okamura s, hayashino y, et al. serum uric acid levels are associated with increased risk of newly developed diabetic retinopathy among japanese male patients with type 2 diabetes: a prospective cohort study (diabetes distress and care registry at tenri [ddcrt 13]). diabetes metab res rev. 2017;33(7) https://doi.org/10.1002/dmrr.2905 korpachev vv, hurina nm, korpacheva ti, et al. peculiarities of uric acid balance disorders in patients with type 2 diabetes and metabolic syndrome. fiziol zh. 2009;55(3):133–140. abstract introduction methods results discussion acknowledgements references about the author(s) dame yenyetou laboratoire national de référence des résistances aux antimicrobiens, centre hospitalier universitaire souro sanou, bobo-dioulasso, burkina faso laboratoire des pathogènes emergents et reémergents (lapather), école doctorale sciences de la santé, université nazi boni, bobo-dioulasso, burkina faso emmanuel zongo laboratoire national de référence des résistances aux antimicrobiens, centre hospitalier universitaire souro sanou, bobo-dioulasso, burkina faso laboratoire des pathogènes emergents et reémergents (lapather), école doctorale sciences de la santé, université nazi boni, bobo-dioulasso, burkina faso institut de recherche en sciences de la santé, ouagadougou, burkina faso emilie dama us centers for disease control and prevention, ouagadougou, burkina faso merci muhigwa laboratoire national de référence des résistances aux antimicrobiens, centre hospitalier universitaire souro sanou, bobo-dioulasso, burkina faso laboratoire des pathogènes emergents et reémergents (lapather), école doctorale sciences de la santé, université nazi boni, bobo-dioulasso, burkina faso issouf sanou laboratoire des pathogènes emergents et reémergents (lapather), école doctorale sciences de la santé, université nazi boni, bobo-dioulasso, burkina faso service des systèmes d’information, et de l’évaluation de la qualité, centre muraz, ouagadougou, burkina faso charles sawadogo direction des laboratoires de biologie médicale, ministère de la santé, ouagadougou, burkina faso soumaya ouangraoua jhpiego burkina faso office, ouagadougou, burkina faso ibrahim sangare laboratoire des pathogènes emergents et reémergents (lapather), école doctorale sciences de la santé, université nazi boni, bobo-dioulasso, burkina faso abdoulaye nikiema integrated quality laboratory services, ouagadougou, burkina faso anicet g. dahourou us centers for disease control and prevention, ouagadougou, burkina faso abdoul s. ouedraogo laboratoire national de référence des résistances aux antimicrobiens, centre hospitalier universitaire souro sanou, bobo-dioulasso, burkina faso laboratoire des pathogènes emergents et reémergents (lapather), école doctorale sciences de la santé, université nazi boni, bobo-dioulasso, burkina faso citation yenyetou d, zongo e, dama e, et al. sentinel laboratory compliance with best practices in burkina faso’s antimicrobial resistance surveillance network. afr j lab med. 2024;13(1), a2259. https://doi.org/10.4102/ajlm.v13i1.2259 original research sentinel laboratory compliance with best practices in burkina faso’s antimicrobial resistance surveillance network dame yenyetou, emmanuel zongo, emilie dama, merci muhigwa, issouf sanou, charles sawadogo, soumaya ouangraoua, ibrahim sangare, abdoulaye nikiema, anicet g. dahourou, abdoul s. ouedraogo received: 14 july 2023; accepted: 20 nov. 2023; published: 30 jan. 2024 copyright: © 2024. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: standardising procedures is the best way to harmonise and strengthen the quality of laboratory-based antimicrobial resistance surveillance. since 2018, burkina faso has developed and disseminated the national manual of procedures for performing antibiotic susceptibility tests in sentinel laboratories within its national antimicrobial resistance surveillance network. objective: our study aimed to assess these sentinel laboratories’ compliance with good practices for antibiotics susceptibility tests. methods: four teams evaluated the antimicrobial resistance sentinel sites laboratories throughout burkina faso from 19 to 28 september 2022. eighteen out of 19 sentinel laboratories were evaluated. a four-member technical committee designed and validated the evaluation tool composed of three microsoft excel sheets. the evaluation emphasised quality controls for culture media, antibiotic discs and compliance with antimicrobial susceptibility testing procedures by the laboratories. excel software was used for data recording and graphs and table design. the free r software version 4.2.0 was used for descriptive statistics. an overall score below 80% was considered noncompliance. results: most (83.33%) of the sentinel laboratories conducted at least one quality control activity for culture media, and 66.67% conducted at least one quality control activity for antibiotic discs. over three-quarters (76.47%) of the laboratories were more than 80% compliant with the modified kirby bauer antimicrobial susceptibility testing method. conclusion: the evaluation revealed the noncompliance of sentinel laboratories with the national procedure manual, particularly in the quality control component. what this study adds: this study has provided baseline data on the sentinel laboratories’ compliance with the national antimicrobial susceptibility testing procedures manual, particularly in areas performing quality control checks or meeting quality indicators for culture media and antibiotic discs. keywords: culture media; antibiotic susceptibility tests; quality control; sentinel laboratories; burkina faso. introduction antimicrobial susceptibility testing (ast) allows for appropriate antibiotic therapy and monitoring of bacterial antibiotic resistance that threatens our ability to treat potentially fatal infections.1,2 for monitoring to happen, susceptibility testing results must be of good quality. however, in africa, the quality of microbiological results on antimicrobial resistance (amr) is a significant challenge.3 indeed, very unusual results combinations have been found in several african studies. for instance, a study conducted in egypt revealed that over 80% of proteus isolates were resistant to imipenem, 80% were sensitive to ceftriaxone, and 50% were sensitive to vancomycin.4 other studies in ethiopia, namibia, and rwanda have shown resistance to penicillin in streptococcus pyogenes and vancomycin resistance in staphylococcus aureus isolates from cerebrospinal fluid.5,6,7 the susceptibility test result quality depends on the equipment (antibiotic discs and culture media), compliance with standard operating procedures, and good laboratory practices.8 to improve data quality, the world health organization encourages countries to develop a national action plan to tackle amr.1 one of the priorities of this national action plan is the implementation of laboratory-based surveillance for amr. establishing a laboratory network under the coordination of a reference laboratory is essential to provide factual data to estimate the impact and evolution of amr in a country. the quality of the data collected from laboratories is one of the significant challenges of this surveillance. the quality of the data collected depends on the availability of qualified human resources, appropriate equipment, and suitable methods. harmonisation of practices and use of standardised procedures improve the quality of the data collection and correct interpretation. they also allow inter-laboratory data comparison and sharing at several levels (national, regional, and international) through the data entry and processing software provided by world health organization.9 in 2017, burkina faso’s ministry of health developed an amr national action plan, which included the implementation of laboratory-based surveillance of amr.10 as part of the global health security agenda programme, the african society for laboratory medicine, with financial and technical support from the united states centers for disease control and prevention, supported the ministry of health in establishing the laboratory-based surveillance system of amr in 2018. the overarching goal of the system is to provide comparable and validated data on the prevalence and trends of amr in a group of bacteria of interest for both the world health organization global antimicrobial resistance surveillance system and the country.11 a national guideline for laboratory-based surveillance of amr and a standardised national ast procedure was developed and endorsed by the ministry of health12 to support the implementation of this surveillance system. to ensure the accuracy and reliability of susceptibility testing results, the quality of the reagents used, and the competence of the staff performing and interpreting the tests, the guideline required laboratories to carry out weekly quality control of antibiotic discs and the culture media after preparation.13,14 however, no study data on quality control of sentinel laboratories for amr have been found since implementing laboratory surveillance in burkina faso. to ensure compliance with the national ast procedure and the quality of data produced by sentinel laboratories, the national antimicrobial resistance surveillance laboratory, with support from the united states centers for disease control and prevention through the african society for laboratory medicine and johns hopkins program for international education in genecology and obstetrics (jhpiego), initiated this first quality control study of sentinel amr laboratories. this study aimed to assess the compliance of sentinel laboratory practices with the national ast procedure and propose an action plan to correct non-conformities. methods ethical considerations ethical approval was obtained from the burkina faso ministry of health (no. 004725ms/sg/dgap/dlbm/dmp – 24 november 2021). this study did not involve human subjects or animal research. all the methods and procedures carried out in this study were in accordance with the declaration of helsinki and adhered to burkina faso national health research and ethics guidelines. all participating laboratories received detailed information about the study, including the purpose of the assessment and the audit plan. laboratories included eighteen amr surveillance sentinel site laboratories across burkina faso were evaluated (figure 1). sentinel site laboratories were selected based on their human (biomedical technologists and biologists) and material resources to effectively detect resistance using standardised methods and their geographical distribution to cover the whole country. there were three private laboratories and 15 public laboratories: eight from the district level and seven from the intermediate level. figure 1: location of the evaluated antimicrobial resistance sentinel sites in burkina faso, 2022. development and validation of assessment tool a technical committee developed and validated the assessment tool. the committee is composed of representatives from the department of medical biology laboratories of the ministry of health, an international expert in the evaluation of biology laboratories, specialists in quality management and microbiologists from the national antimicrobial resistance surveillance laboratory. the committee reviewed the existing literature on ast and quality control to select the indicators for the assessment tool.11,12,15 the assessment tool was composed of three excel sheets. the first sheet contained four indicators on the performance of quality control of culture media (control of the ph, measuring the thickness of the culture medium, checking culture media fertility, and sterility). the second sheet had seven indicators on the presence of reference strains and the performance of quality control of the antibiotic discs (having escherichia coli atcc 25922 (cip 7624), staphylococcus aureus atcc 25923 (cip 7625), and pseudomonas aeruginosa atcc 27853 (cip 76110), monitoring the condition of desiccants, defining the periodicity of quality control of the discs and the number of quality controls carried out over a given period, and verifying if the diameters of the inhibition zones are checked). the last sheet included the stages of carrying out ast according to the modified kirby bauer method with 4 stages and 27 sub-stages (table 1). table 1: key indicators measured to check compliance of antimicrobial resistance sentinel site laboratories’ practices with burkina faso’s standardised national antimicrobial susceptibility testing procedure from 19 september 2022 to 28 september 2022 in burkina faso. evaluation team four teams evaluated the amr sentinel sites laboratories from 19 september 2022 to 28 september 2022. each team comprised two evaluators (a microbiologist and a quality manager with proven experience in medical biology) and a supervisor from the directorate of biomedical biology laboratories at the ministry of health. all the evaluation team members were trained to use the assessment tool appropriately to evaluate each activity. evaluation method and period the chosen approach was a two-day on-site observation of each sentinel laboratory and review of documents. the evaluation started with an introduction meeting followed by the questionnaire administration. as part of the process, the evaluation verified the existence of critical technical and administrative documents and essential points or steps of the pre-analytical, analytical and post-analytical phases. the evaluation of ast practice focused on the following steps: preparation of the 0.5 mcfarland bacterial inoculum, time and techniques for inoculating the bottles after preparing the bacterial suspension, choice and arrangement of antimicrobial discs, incubation times, temperature, and incubation condition after inoculation. for the interpretative reading, the following aspects were evaluated: reading times after incubation, measurement of inhibition diameters, interpretation of tracer discs, and detection and reporting of phenotypes of interest for amr monitoring. in the end, a debriefing meeting was held with the hospital management and laboratory staff to present the evaluation findings before the final report. study variables the records showing that activities to implement key quality control indicators for culture media and antibiotic discs were completed were checked to verify their compliance. then, evaluators observed laboratory practices and assigned each quality indicator or stage a score. a stage completed per the requirements was worth 1 point. if the requirement was partially met, it was worth 0.5. a requirement not met was worth 0 points. all points obtained were used to calculate each laboratory’s compliance rate with the steps for carrying out the ast. data analysis microsoft excel software (microsoft corporation, redmond, washington, united states) was used for data entry and to produce graphs and tables. the free software r version 4.2.016 was used to perform descriptive statistics. score levels were defined to evaluate the overall performance of the laboratories. an overall score below 80% was considered noncompliance.17 a report resulting from the data analysis with corrective measures had been sent to the participating sentinel sites to improve their practices. results number and checking rate of culture media quality indicators regarding culture media quality indicators, 15 of 18 laboratories performed at least one quality control indicator (table 2). all 15 laboratories tested the sterility and fertility of culture media, two laboratories checked media ph after preparation, and one measured the thickness of the agar. table 2: number and rate of culture media quality indicators checked in antimicrobial resistance sentinel laboratories in burkina faso obtained through questioning from 19 september 2022 to 28 september 2022. checking rate of antibiotic disc quality indicators a total of 12 of 18 laboratories performed at least one antibiotics disc quality control check (table 3). only 16.67% (n = 2 of 12) of the laboratories monitored the desiccant activity; 58.33% (n = 7 of 12) of the laboratories had defined periodic quality control check times for antibiotic discs, while 71.43% (n = 5 of 7) checked these quality indicators at the indicated frequency. in addition, 83.33% (n = 10 of 12) of the laboratories documented the inhibition diameters according to the critical diameters defined for the reference strain or antibiotic association. table 3: number and rate of laboratories verifying the antibiotic discs’ quality indicators checked in antimicrobial resistance sentinel laboratories in burkina faso obtained through questioning from 19 september 2022 to 28 september 2022. availability of reference strains all (n = 12 of 12) laboratories performing quality control of antibiotic discs had the reference strain e. coli atcc 25922 (cip 7624) (figure 2). two laboratories had the reference strains of e. coli atcc 25922 (cip 7624) and s. aureus atcc 25923 (cip 7625), and two laboratories had the reference strains of e. coli atcc 25922 (cip 7624) and p. aeruginosa atcc 27853 (cip 76110). seven laboratories had all three reference strains. figure 2: availability of reference strains in the antimrobial resistance sentinel laboratories in burkina faso obtained through questioning from 19 september 2022 to 28 september 2022 in burkina faso. compliance rate with modified kirby bauer method most (n = 17 of 18) laboratories performed the routine asts according to the modified kirby bauer method described in the national procedure manual (figure 3). the average compliance rate for all laboratories was 85.24%. the majority (n = 13 of 17) of laboratories had compliance rates above 80%. the highest and lowest rates obtained were 97% and 75%, respectively. figure 3: compliance rate of antimicrobial resistance sentinel site laboratories in burkina faso with the modified kirby bauer method obtained through observation from 19 september 2022 to 28 september 2022 in burkina faso. discussion this study is the first to assess the implementation of quality indicators in the amr sentinel site laboratories since burkina faso implemented the amr surveillance in 2018. this activity has provided baseline data on the sentinel laboratories’ compliance with the national ast procedures manual, particularly in areas performing quality control checks or meeting quality indicators for culture media and antibiotic discs. the ministry of health meets the requirement to promote good practices in sentinel laboratories by conducting this assessment. an impartial review of documents and records and direct observation of practices allowed an objective assessment of compliance with the national procedure. we analysed the non-conformities identified during the evaluation to determine their impact and severity on operations, processes, patient outcomes, and safety considerations. thus, the assessors proposed urgent actions depending on the criticality of each detected non-conformity. each laboratory received reports with proposed improvement plans and recommendations. a too-acidic agar ph increases ß-lactam activity, while an alkaline ph favours aminoglycoside and macrolide activity. agar thickness has an impact on the diffusion gradient of the antibiotic.18 nevertheless, a few laboratories controlled these parameters in our context. maintaining the required agar thickness and ph depends on the medium dispensing technique, which requires a calibrated dispenser and a ph meter; however, only a few laboratories had these. fertility and sterility are the most important indicators in medium quality control. the medium must be carefully checked to avoid contamination before culture.19 all sentinel laboratories checked the culture medium quality indicators to ensure each medium met the microbiological characteristics before use. åhman et al. found that many discs exhibited significant quality issues, with 33% (n = 26 of 78) out-of-range readings in the first study and 17% (n = 20 of 120) in the second. furthermore, discs from certain manufacturers showed unexpected variations in inhibition diameter zones (4 mm – 9 mm) for discs from the same vial.20 in our study, only 67% of our sentinel laboratories monitored the quality indicators of antibiotic discs; this situation could increase the chances of obtaining inaccurate results and, consequently, ineffective antibiotic therapy.21 this lack of disc quality control is concerning given the already alarming global resistance situation for some anitbiotics. indeed, the resistance of e. coli to colistin, currently used as a last-resort antibiotic, has been observed in asia, europe, america, africa, and oceania (specific percentages for each continent: 66.72%, 25.49%, 5.19%, 2.27%, and 0.32%, respectively).22,23 the low rate of laboratories complying with these indicators can be attributed to staff mobility due to various assignments, lack of staff involvement, and the fact that, in most laboratories, personnel selection for training is not based on expressed training needs or competence assessments. in fact, in many laboratories, the staff trained in microbiological testing by the ministry of health and its partners did not necessarily come from the microbiology department due to an established internal rotation for various continuous training programmes. staff were selected without necessarily considering the specific field covered by the training. we believe that the ministry of health should keep trained staff as long as possible. furthermore, although participation in training is a source of motivation for staff, it would be good to take into account the specific field covered by the training in the selection of participants. the reference strains are used to assess the overall performance of the test. they are used for quality control of all steps of culture and the study of bacterial sensitivity to antibiotics.18 quality control of antibiotic discs using reference strains should be performed at least every two weeks.12 performing checks at short intervals allows for a retrospective analysis of previous results to identify issues in the event of discrepant results, allowing for corrective actions to be taken in case of non-conformity results. despite this, only 58.83% of the laboratories carrying out the quality controls of the antibiotic discs had defined periodicity of passage, and only 71.43% respected this predefined periodicity. this situation demonstrates that implementing the procedures in the national ast manual is still weak. the ministry of health should strengthen awareness, communication, and especially laboratory staff training. limitations the methodology used in our study did not verify the corrective measures taken in the event of non-conclusive control. also, one laboratory was not evaluated for security reasons. despite these methodological limitations, we obtained sufficient results that could be evaluated. conclusion the evaluation revealed non-conformities with the national ast procedure for carrying out ast at the sentinel laboratories level, particularly in the quality control component. we formulated proposals for improvement plans to address these non-conformities to provide reliable, accurate, and timely results, and to contribute effectively to the fight against amr. regular monitoring of quality indicators will ensure the reliability and accuracy of results from the sentinel sites laboratories. acknowledgements we would like to thank the ministry of health, through the direction of medical biology laboratory, for the information letters sent to the sentinel site laboratories and the mission orders for the evaluators. thanks also go to the national reference laboratory for antimicrobial resistance for the practical organisation of the evaluations and to all the participating sentinel site laboratories. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions d.y., e.z., e.d., m.m., i. sanou, c.s., s.o., i. sangare, a.n., a.g.d. and a.s.o. participated in the writing and final approval of the article. sources of support this work was financially and technically supported by united states centers for disease control and prevention through the african society for laboratory medicine and jhpiego. data availability data from the conformity assessment of sentinel laboratory practices for antimicrobial resistance in burkina faso are available from https://docs.google.com/spreadsheets/d/16dxq2ntpmjgwkpgti9pmuabzgiaiwfta disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any agency affiliated with the authors. references seale ac, gordon nc, islam j, peacock sj, scott jag. amr surveillance in low and middle-income settings – a roadmap for participation in the global antimicrobial surveillance system (glass). wellcome open res. 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abstract introduction situation analysis of drug resistant tuberculosis in africa towards ending drug resistant tuberculosis in africa acknowledgements references appendix 1 about the author(s) nazir ismail center for tuberculosis, national institute for communicable diseases, national health laboratory services, johannesburg, south africa department of medical microbiology, university of pretoria, pretoria, south africa department of internal medicine, university of witwatersrand, johannesburg, south africa farzana ismail center for tuberculosis, national institute for communicable diseases, national health laboratory services, johannesburg, south africa department of medical microbiology, university of pretoria, pretoria, south africa shaheed v. omar center for tuberculosis, national institute for communicable diseases, national health laboratory services, johannesburg, south africa linsay blows center for tuberculosis, national institute for communicable diseases, national health laboratory services, johannesburg, south africa yasmin gardee center for tuberculosis, national institute for communicable diseases, national health laboratory services, johannesburg, south africa hendrik koornhof center for tuberculosis, national institute for communicable diseases, national health laboratory services, johannesburg, south africa philip c. onyebujoh africa centres for disease control and prevention, addis ababa, ethiopia citation ismail n, ismail f, omar sv, et al. drug resistant tuberculosis in africa: current status, gaps and opportunities. afr j lab med. 2018;7(2), a781. https://doi.org/10.4102/ajlm.v7i2.781 review article drug resistant tuberculosis in africa: current status, gaps and opportunities nazir ismail, farzana ismail, shaheed v. omar, linsay blows, yasmin gardee, hendrik koornhof, philip c. onyebujoh received: 01 feb. 2018; accepted: 12 sept. 2018; published: 06 dec. 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the world health organization end tb strategy targets for 2035 are ambitious and drug resistant tuberculosis is an important barrier, particularly in africa, home to over a billion people. objective: we sought to review the current status of drug resistant tuberculosis in africa and highlight key areas requiring improvement. methods: available data from 2016 world health organization global tuberculosis database were extracted and analysed using descriptive statistics. results: the true burden of drug resistant tuberculosis on the continent is poorly described with only 51% of countries having a formal survey completed. in the absence of this data, modelled estimates were used and reported 92 629 drug resistant tuberculosis cases with 42% of these occurring in just two countries: nigeria and south africa. of the cases estimated, the majority of patients (70%) were not notified, representing ‘missed cases’. mortality among patients with multi-drug resistant tuberculosis was 21%, and was 43% among those with extensively drug resistant tuberculosis. policies on the adoption of new diagnostic tools was poor and implementation was lacking. a rifampicin result was available for less than 10% of tuberculosis cases in 23 of 47 countries. second-line drug resistance testing was available in only 60% of countries. the introduction of the short multi-drug resistant tuberculosis regimen was a welcome development, with 40% of countries having implemented it in 2016. bedaquiline has also been introduced in several countries. conclusion: drug resistant tuberculosis is largely missed in africa and this threatens prospects to achieve the 2035 targets. urgent efforts are required to confirm the true burden of drug resistant tuberculosis in africa. adoption of new tools and drugs is essential if the 2035 targets are to be met. introduction global declines in tuberculosis incidence1 provide evidence that political commitment together with aggressive plans to curb the disease can make a difference. these efforts not only stopped the upward spiral of tuberculosis incidence but reversed the trend. the world health organization (who) has now set targets to end tuberculosis by 2035 and is directing efforts at accelerating the rate of decline, with the expectation of reducing the incidence rate by 90% and mortality by 95% compared to levels in 2015.2 africa is home to over 1 billion people and is disproportionately affected by tuberculosis with 2.6 million of the 10.4 million global tuberculosis cases,3 making the continent a key geographical area for health interventions. sub-saharan africa, in particular, saw rates rapidly escalate in the early 1990s due to a delayed response to the emergent hiv epidemic at the time.4,5 these failures resulted in incidence rates that are the highest in the world and have made the task to end tuberculosis even more challenging. nonetheless, the tide has changed with the rapid expansion of anti-retroviral therapy resulting in sharp declines in hiv-associated tuberculosis incidence in countries in sub-saharan africa, thus offering a window of hope.6,7,8 several factors threaten the potential to realise these targets, and key among these is drug resistant tuberculosis.9 unfortunately, much like the global situation, drug resistant tuberculosis in africa is largely missed with 93 000 cases estimated in 2016, while only 27 828 (30%) were diagnosed.3 even when the diagnosis is made, only 59% achieve a successful treatment outcome. we present a review of the current status of drug resistant tuberculosis in africa using data from 2016 who global tuberculosis database,10 focusing on epidemiology, diagnostic tools and therapeutic options. we also highlight and discuss potential priority areas that require strengthening leading up to attaining the 2035 targets. situation analysis of drug resistant tuberculosis in africa the best estimate of the burden of drug resistant tuberculosis in africa requires population-based survey findings, as testing for all tuberculosis cases for rifampicin drug resistance is not done routinely in most countries. only three countries – mauritius, south africa and swaziland – had over 80% of cases tested for rifampicin (table 1). drug resistance surveys are thus the main source of data and are applied based on robust methodologies.11 such surveys have however been completed for only 24 of 47 (51%) countries (table 2). when restricted to the last five years, the figure is even lower at 23% (11/47). for 21 countries, no data are available, making planning a response much more challenging and potentially less effective. no country in africa had a repeat survey in the five-year window recommended, and assessing trends was thus largely not possible based on survey data alone. table 1: rifampicin resistance detection in africa from policy to application. table 2: comparison of rifampicin-resistant and multi-drug-resistant tuberculosis burden estimated with notified, ordered from highest to lowest estimated burden: 2016. in light of the above limitations, who applies modelling using available data sources, including surveys.12 this serves as the best estimate of the burden of drug resistant tuberculosis. as modelling is based on selected assumptions, its use is not meant to replace the ideal of robust routine data. the estimated drug resistant tuberculosis burden is shown in table 2, and countries are ranked from highest to lowest. nigeria (20 000) together with south africa (19 000) account for 42% (39 000/92 629) of the total estimated burden, and thus their achievements or failures regarding drug resistant tuberculosis control will have a great impact on the overall picture for africa. when the next three highest burden countries – democratic republic of congo (7600), mozambique (7600) and ethiopia (5800) – are added, the cumulative figure accounts for 65% (60 000/92 629) of the estimated burden of cases. this highlights the obvious heterogeneity of disease burden among african countries and the need for a targeted response rather than a generalised one. an interesting modelling study was undertaken by musa et al.13 using available data sources, including surveys and published studies in sub-saharan africa, to estimate the trends and regional prevalence of drug resistant tuberculosis. the region with the highest prevalence was in the south of africa with 3.1% (2.1% – 4.2%) of tuberculosis cases being drug resistant. this was followed by central (2.1%; 1.1% – 3.0%), western (1.9%; 1.2% – 2.6%) and eastern (1.7%; 1.1% – 2.2%) regions of sub-saharan africa. these prevalence estimates correlate well with recent surveys in the region. another modelling study by sharma et al.,14 which included four geographically diverse countries, predicted that there will be an increase in multi-drug resistant (mdr) tuberculosis across all four countries analysed and a decline in the relative contribution of acquired drug resistance. the mdr tuberculosis prevalence for south africa come 2040 is predicted to be 5.7% (95% prediction interval 3.0–7.6) compared with 12.4% (9.4–16.2) for india, 8.9% (4.5–11.7) for the philippines and 32.5% (27.0–35.8) for russia. although the estimated prevalence is higher in other parts of the world, this needs to be seen in the context of the high incidence of tuberculosis in sub-saharan africa, thus, single digit changes in prevalence constitute substantial changes in absolute numbers. furthermore, the prediction of a global increase has implications for africa, and the authors indicated that existing activities through the green light committee will not be sufficient to change this trend. the most recent survey in south africa (2012–2014), compared to the previous one conducted just over 10 years earlier, showed an almost doubling of the rate in rifampicin resistance (1.8% to 3.4%)15 also a similar increasing rate of resistance was observed in botswana from 2.0% to 3.6% between 2002 and 2007–2008.16 the increase observed was primarily in new cases for both countries and driven by rifampicin mono-resistant tuberculosis, particularly in the south african survey. the increase in rifampicin resistant (rr) tuberculosis among new cases highlights the role of primary drug resistant tuberculosis transmission, which is likely to occur due to missed diagnostic opportunities when patients are not tested and treated for drug-resistant tuberculosis, or never reach health services. this has been the major obstacle in the era preceding the introduction of molecular diagnostics (xpert® mtb/rif [cepheid, sunnyvale, california, united states] and line probe assays), which still continues to persist. of importance as well is the higher rate of isoniazid mono-resistance (6.1%) compared to any rifampicin resistance (4.6%) in the 2012–2014 south africa survey, with the former also associated with poorer treatment outcomes. comparing the estimated burden of drug resistant tuberculosis to cases notified, there is an alarming notification gap, as only 27 828 of the 92 629 estimated cases were notified. this suggests that 70% of cases in africa are being missed. in addition, the proportion of missed rr and mdr tuberculosis cases are estimated to be above 50% for 43 of the 47 african countries (table 1). it should be noted that the confidence intervals are relatively wide, yet using the lower estimate the gap is 45%. the extremely drug resistant tuberculosis estimates are not available for comparison; however, a total of 1092 extremely drug resistant tuberculosis cases were notified, of which south africa reported 89% (967) of all extremely drug resistant tuberculosis cases on the continent.17 this disproportionality is most likely due to lack of capacity to diagnose extremely drug resistant tuberculosis in africa. only 53% of rr and mdr tuberculosis cases in africa had second-line drug susceptibility testing performed, and 47% (22/47) of member states do not have a laboratory that can perform second-line resistance testing (table 3). table 3: second-line resistance detection in africa from policy to application. mortality reduction is another important target of who’s 2035 end tb strategy. missed cases contribute to mortality directly when cases are not detected through current programmes, or indirectly due to late presentation or testing for resistance being unavailable. the overall mdr tuberculosis mortality for the 2014 africa cohort (n = 16 231) was 20%, while treatment success was only 58% (figure 1). the situation was far worse for the extremely drug resistant tuberculosis cohort in 2014 (n = 623) with mortality at 42% and treatment success at only 27%. outcomes for the 2014 cohort of mdr tuberculosis by country are shown in figure 1. figure 1: multi-drug-resistant tuberculosis outcomes by country grouped by size of cohort: 2014. countries with no data or no reported cases are not shown. reviewing the diagnostic landscape for drug resistant tuberculosis in africa, encouraging signs are noted with 45% (21/47) of countries having progressive policies, which include the use of the xpert mtb/rif assay as the initial who-endorsed rapid diagnostic test (table 1). comparing policy to practice, clear gaps emerge. across the continent only 35% of newly diagnosed tuberculosis cases had a rifampicin drug susceptibility test performed (table 1), implying that primary drug resistant tuberculosis cases are largely being missed. rifampicin drug susceptibility testing was available for less than 10% of new cases in 23 countries. in contrast, mauritius (94%), south africa (91%), swaziland (82%), senegal (69%) and rwanda (66%) were ranked as the top five countries with high coverage for rifampicin drug susceptibility testing among notified cases. on the continent, the xpert mtb/rif is available in 43/47 (91%) countries, comprising 1740 testing sites (table 1). adjusted for population size, there is large variability ranging from less than 1 to 24 sites per million people. albeit a crude measurement, it does highlight important gaps in coverage for many countries. however, coverage of drug susceptibility testing does not directly imply utilisation, which is likely to be even poorer. policies on universal drug susceptibility testing are also lacking with 19 (40%) of the 47 countries having a policy in place (table 3). the genotype mtbdrsl (hain life sciences, nehren, germany) for second-line drug susceptibility testing is available in 20 of 47 (43%) countries at 29 sites (table 3). this proportion is unsurprisingly low as the genotype mtbdrsl has only been recently endorsed to address gaps in second-line drug susceptibility testing. overall, 28 (60%) countries have second-line drug susceptibility testing available, using either line probe assay or phenotypic drug susceptibility testing (table 3). availability of second-line drug susceptibility testing has, however, not translated into practice with only 43% (12/28) of these countries testing more than half their notified rifampicin-resistant tuberculosis cases. however, these percentages are not truly reflective, as they are based on the number of detected rather than estimated cases. another important issue with respect to laboratories is quality assurance. among all african countries, 26 (55%) national reference laboratories (nrls) reported having an iso 15189 accreditation status. although this does seem encouraging, this reporting may not be a true reflection of the situation, as the reference laboratories that are formally recognised by slmta18 as being accredited based on external evaluation are the nrls of ethiopia, mozambique, south africa and uganda. iso 15189 accreditation should be a basic requirement for this level of laboratory. in the absence of accreditation, participation in external quality assurance programmes provides a basic measure of quality evaluation and competence while countries progress towards accreditation and has been introduced for high burden countries through the who. apart from diagnostics, treatment is an essential component of tuberculosis control as well and affects a country’s ability to reach the who end tb strategy targets. drug resistant tuberculosis treatment is often prolonged and uses less effective regimens with more adverse events compared to first-line treatment. the number of cases reported on treatment per country varies (figure 1), with all but one treating less than 600 cases in a year (south africa: > 10 000). the treatment success overall in africa was 59%, 20% died and 16% were lost to follow-up (appendix 1). the introduction of the short-course regimen is a major improvement and, in 2016, 36% (17/47) of countries used this patient friendly regimen (figure 1). countries leading the implementation of this regimen were democratic republic of congo (555), cote d’ivoire (349) and cameroon (135) with 31%, 68% and 100% of rr and mdr tuberculosis patients in these countries, respectively also having second-line drug susceptibility testing performed. thus, adoption of the short-course regimen is not strictly linked to second-line drug susceptibility testing; however, in light of the recent rapid who guidance, the need for early identification of fluoroquinolone resistance is important. the top three countries with at least 50 cases or more and having the highest treatment success were countries implementing the short-course regimen: burundi (90%), rwanda (88%) and cote d’ivoire (85%). bedaquiline is a new and welcome addition to drug resistant tuberculosis management and is reported to be highly effective.19,20 it is currently being used in 11 african countries as well as in south africa where its use is on a large scale (figure 1). this new agent also offers a potential for scale-up in addressing poor treatment outcomes for drug resistant tuberculosis. towards ending drug resistant tuberculosis in africa drug resistant tuberculosis poses a major hurdle to achieving the who end tb strategy targets. acting early and decisively will be a determining factor in either future success or failure. encouragingly, the past 5 years have seen new diagnostic technologies and treatment options become available, as well as strong global political commitment to end tuberculosis. despite the obstacles threatening the realisation of the who end tb strategy targets in africa, there are equally effective tools available for achieving success. primary among the urgent needs is a clear understanding of the burden of tuberculosis and drug resistant tuberculosis, which will greatly impact planning and efficient resource allocation, a key issue in resource-constrained environments. progress has been made with just over half of the countries ever having completed a survey. however, there are still large gaps in the data available and these need to be urgently addressed. furthermore, in order to assess progress and respond appropriately, trend data is essential. the use of routine data reported to the who has added benefits, but concerns about accuracy and completeness result in this information being treated with circumspection. modelled estimates using this routine data would also be impacted as a consequence. an alternative approach to address gaps is the use of sentinel surveillance for tracking annual trends in strategically selected sites. this is a potential hybrid solution, which is being considered by the hiv programme.21 such a system has been used in south africa for tuberculosis, and unpublished data do support the value of such an approach. this approach can be achieved with far fewer resources and has the potential to strengthen existing routine systems and serve as a starting point for replication of developed sites in other areas. ensuring routine standardised algorithms detecting rifampicin and second-line resistance is the ideal and should be facilitated in the era of the xpert mtb/rif and genotype mtbdrsl. a few countries have shown high uptake of rapid diagnostic tools for early rifampicin and second-line resistance detection. these efforts should be standardised to ensure data comparisons between countries and regions of africa. another key concern is missing cases, which are estimated to be equal to or greater than the notified burden. although the numbers are staggering, modifying current diagnostic algorithms to adopt new technologies could dramatically reduce this gap in a short period of time and can be seen in some countries already. the utilisation of the xpert mtb/rif has been lacklustre on the continent, with many countries limiting its use to selected cases and thus diminishing the impact of this critical molecular diagnostic platform. this not only reduces the detection of tuberculosis by approximately 10% – 20%,22 but also means that by design, rifampicin resistance is missed almost completely in the vast majority of tuberculosis cases. it should be noted that although the prevalence of drug resistant tuberculosis is higher in previously treated cases compared to new cases, the absolute number of drug resistant tuberculosis patients among previously treated cases is far lower than the numbers in new tuberculosis cases. for the 2015 cohort, 1 200 078 new and relapsed drug resistant tuberculosis cases were notified, of which only 38 059 were previously treated cases.3 the net effect is a similar burden in absolute numbers of drug resistant tuberculosis between new and previously treated cases. this is because previously treated cases, despite having a much higher prevalence of drug resistant tuberculosis cases, contribute only to a small part of the total tb burden. aggressive scale-up of the use of rapid molecular diagnostic tools (e.g. xpert) as the primary test will be key to finding missing drug resistant tuberculosis cases and is all the more urgent as primary transmission is a major contributor to the propagation of the disease.15,22 missing resistance may result in poorer outcomes, and further compromises the health and well-being of the population at large. in addition, stigma and lack of access to health services are known obstacles to care and likely contribute to missing cases. some of these cases will most likely end up at a health facility and the unavailability of quality diagnostic technologies would lead to a catastrophic treatment failure. several countries other than south africa have taken the approach of using the xpert mtb/rif as the primary test for the detection of tuberculosis and drug resistant tuberculosis, and thus models for implementation are available. the south african approach uses a central model with important emphasis on logistics, which has worked well. in regions where transport infrastructure is limited, the use of the genexpert omni (cepheid, sunnyvale, california, united states) could be an effective solution. the greater focus now is on multiplex diagnostic platforms that will also facilitate integration at different levels of the health system, thereby providing improved diagnostic yield. irrespective of the approach, careful planning and budgeting will be needed in order to achieve the necessary returns. a second issue relates to second-line testing. while this is being achieved in several african countries with good overall coverage among notified rr tuberculosis cases (table 3), it is often a challenge in most settings across africa with 43% (20/47) of member states having no coverage. as the overall reported burden of drug resistant tuberculosis is not particularly high in many countries, the need and operational feasibility to set up such systems may be better served through regional collaboration. there are three who-certified supranational reference laboratories in africa24 and several nrls that have adequate capacity to provide such services. while this would lead to increase in volumes of second-line testing, it will also ensure operational efficiency and allow skills to be developed and sustained. linked to such services is also the need for supporting surveys on the continent. the three supranational reference laboratories are limited by capacity to provide for the large needs of the continent. expanding the number of supranational reference laboratories is required and nrls with potential should be supported to achieve this status. the number of nrls complying with iso 15189 is still unacceptably low, considering that nrls are expected to be the standard against which sub-national laboratories are compared. the introduction of the strengthening laboratory management toward accreditation programme has seen 54 laboratories achieve accreditation by the end of 2017 over a 5-year period;25 however, only two were national tuberculosis reference laboratories. prioritisation of nrls to achieve accreditation should be a short-term goal using strengthening laboratory management toward accreditation. the emergence of the new africa centres for disease control and prevention portends hope through the introduction and implementation of the regional integrated laboratory and surveillance network hosted through the five regional collaborating centres in the five geographical sub-regions of the african union. it is anticipated that these centres, and the associated network, can be used to improve and strengthen diagnostic capabilities within the africa region. the continuum of care requires not only good diagnostics but also effective early treatment. successful outcomes among mdr tuberculosis patients are exceptionally low with death and loss to follow-up being common endpoints. these are complex issues underpinned by late presentation, poor access to services and overburdened health services. treatment options are another important determinant of patient outcomes. therapy is often prolonged; however, a short-course regimen has shown promise in reducing the loss of patients to follow-up. the highest success rates observed in africa were among those applying this approach and, importantly, a large evidence base for this policy came from africa and confirms its value.25 the introduction of the short regimen requires greater impetus to ensure it is accessible and widely used, as currently only 36% of countries have introduced it. more positive news out of africa is the use of bedaquiline, with significant reductions observed in mortality for both mdr and xdr tuberculosis cases using this agent.26 this too is a timely improvement in addressing urgent issues in the management of drug resistant tuberculosis. south africa has scaled up its use, but it has only recently been introduced in 11 countries. time lost due to slow scale-up will result in preventable deaths and further transmission. additionally, skills in dealing with complex drug interactions and adverse events are available, particularly in south africa, and south-south collaboration can facilitate the safe and effective introduction of the new agents. the availability of a second agent – delamanid – is another promising advancement and opens the way for potentially effective combination therapy to be standardised for xdr tuberculosis treatment. this would simplify management of these complex cases, especially for areas where skills may be lacking. the global drug facility is an important mechanism for access to new drugs for the treatment of drug resistant tuberculosis in africa and can be utilised to improve the management of drug resistant tuberculosis. as highlighted, south-south collaborations are important, and findings reported by cain et al.27 demonstrate the impact of migration and management of drug resistant tuberculosis. these authors showed that a large and increasing case load of mdr tuberculosis patients from specific areas of somalia crossed borders to kenya, seeking care due to lack of services. migration is an important contributor to missing cases and may lead to poor outcomes. similar migratory behaviour has been documented in other regions on the continent related to employment seeking in the mining sector.28 dealing with migration and the continuum of care requires common standards of care to be available, good communication and referral mechanisms, as well as a unique identifier to link patients across countries and regions. although this issue has received some attention from the regions, the impetus has been slow and coordination weak. the launch of the africa centres for disease control as well as prevention and its regional collaborating centres and target national public health institutes does offer a potential coordination mechanism and should be prioritised. the innovation in laboratory engineered accelerated diagnostics project is a new initiative and will use biometrics29 as the unique patient identifier across regions. if successful, it will provide a model to allow patients to be managed through care irrespective of where they are diagnosed. ending tuberculosis and specifically drug resistant tuberculosis cannot be fully realised without dealing with hiv infection and disease. hiv/aids is a major contributor to the burden of both tuberculosis and drug resistant tuberculosis, and current efforts at also ending hiv are encouraging. achieving viral suppression at a population level is an important tuberculosis prevention strategy and will likely lead to continued declines in hiv-associated tuberculosis and drug resistant tuberculosis. this would, however, result in a relatively higher proportion of hiv-negative tuberculosis and drug resistant tuberculosis. this group is less likely to die and, consequently, can transmit for longer periods. thus, missing such cases will have long-term consequences, and messages to ensure this group is also investigated will be increasingly important. another issue that should be appreciated is that the burden of tuberculosis and drug resistant tuberculosis has for many years exceeded the existing medical care capacity, leading to failures in health delivery. in spite of this, the declines seen offer a window of hope where the developed capacity and burden may align once again. thus, the need to maintain funding and capacity during this period is needed if we are to end tuberculosis by 2035. limitations it is important to contextualise the limitations of this analysis. the data used were taken from the who global tuberculosis database, which derived data from countries through unverified self-reporting. countries are usually given time to verify their data before submission and, in addition, anomalies identified by who result in queries sent back to countries for checking before the data are accepted. additionally, the estimates included are based on established mathematical models adopted for use by who and are inherently influenced by the assumptions applied to the model. the wide confidence intervals account for the uncertainty in deriving these estimates. conclusion drug resistant tuberculosis is difficult to manage even in the best of environments and is likely to pose a major challenge for africa as it works toward achieving the who end tb strategy targets. any delays in addressing drug resistant tuberculosis will mean lost ground, which will make the challenge even greater. our ability to end tuberculosis and, specifically, drug resistant tuberculosis, by 2035 will require a major uphill effort, but it is achievable given the right strategic focus complemented by strong leadership and adequate resources. the adage ‘know your epidemic, and know your response’ serves as a guiding principle leading up to 2035 and we have provided detailed data clearly highlighting areas of success and failure. it is clear that the burden is highly heterogeneous, and focusing on key countries will be greatly rewarding, if available resources are used wisely. gaps in data are also large and certainty needs to be achieved on the true burden of tuberculosis and drug resistant tuberculosis throughout the continent. experience gained in addressing the deficiencies identified here could influence prioritisation within the tuberculosis control programme in the future. the advent of new and improved diagnostics constitutes a major advancement, although adoption has not been aggressive enough in many parts of africa, and this needs greater impetus. despite shortcomings as a continent, african countries have played a leading role for both the evaluation of drug resistant tuberculosis diagnostic tools and treatment options, which include the short regimen and bedaquiline. these findings need to move to scale rapidly for us to accelerate progress in dealing with drug resistant tuberculosis and ultimately end the disease. acknowledgements we would like to thank all the national tuberculosis control programmes that contributed important public health data to the who global tuberculosis database. we also thank the who headquarters and regional offices for ensuring the data collected were standardised and reviewed before making the information publicly available. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. sources of support none. authors’ contributions n.i., f.i. and p.c.o. were involved in the conception and design. n.i. and l.b. did the data analysis. n.i., f.i., s.v.o., l.b., h.k., y.g. and p.c.o. wrote the first draft. n.i., f.i., s.v.o., l.b., h.k., y.g. and p.c.o. provided important intellectual input. references who. global tb report. 2016 [cited 2018 january 22]. available from: http://www.who.int/tb/publications/global_report/gtbr2016_executive_summary.pdf who. who end tb strategy. 2015 [cited 2018 january 22]. available from: http://www.who.int/tb/end_tb_brochure.pdf?ua=1 who. global tb report. 2017 [cited 2018 august 27]. available from: http://www.who.int/tb/publications/global_report/gtbr2016_executive_summary.pdf abdool karim ss, churchyard gj, karim qa, lawn sd. hiv infection and tuberculosis in south africa: an urgent need to escalate the public health response. lancet. 2009;374(9693):921–933. https://doi.org/10.1016/s0140-6736(09)60916-8 harries ad, hargreaves nj, kemp j, et al. deaths from tuberculosis in sub-saharan african countries with a high prevalence of hiv-1. lancet. 2001;357(9267):1519–1523. https://doi.org/10.1016/s0140-6736(00)04639-0 takarinda kc, harries ad, sandy c, mutasa-apollo t, zishiri c. declining tuberculosis case notification rates with the scale-up of antiretroviral therapy in zimbabwe. public health action. 2016;6(3):164–168. https://doi.org/10.5588/pha.16.0029 haumba s, dlamini t, calnan m, et al. declining tuberculosis notification trend associated with strengthened tb and expanded hiv care in swaziland. public health action. 2015;5(2):103–105. https://doi.org/10.5588/pha.15.0008 nanoo a, izu a, ismail na, et al. nationwide and regional incidence of microbiologically confirmed pulmonary tuberculosis in south africa, 2004–2012: a time series analysis. lancet infect dis. 2015;15(9):1066–1076. https://doi.org/10.1016/s1473-3099(15)00147-4 mariandyshev a, eliseev p. drug-resistant tuberculosis threatens who’s end-tb strategy. lancet infect dis. 17(7):674–675. https://doi.org/10.1016/s1473-3099(17)30246-3 who. who’s global tb database. geneva, switzerland: who; 2016. who. guidelines for surveillance of drug resistant tuberculosis. 5th ed. geneva, switzerland: who; 2015. who. methods used by who to estimate the global burden of tb disease. 2016 [cited 2017 december 16]. available from: http://www.who.int/tb/publications/global_report/gtbr2016_online_technical_appendix_global_disease_burden_estimation.pdf musa bm, adamu al, galadanci na, zubayr b, odoh cn, aliyu mh. trends in prevalence of multi drug resistant tuberculosis in sub-saharan africa: a systematic review and meta-analysis. plos one 2017;12(9):e0185105. https://doi.org/10.1371/journal.pone.0185105 sharma a, hill a, kurbatova e, et al. estimating the future burden of multidrug-resistant and extensively drug-resistant tuberculosis in india, the philippines, russia, and south africa: a mathematical modelling study. lancet infect dis. 2017;17(7):707–715. https://doi.org/10.1016/s1473-3099(17)30247-5 nicd. south african tb drug resistance survey. 2015 [cited 2017 december 01]. available from: http://www.nicd.ac.za/assets/files/k-12750%20nicd%20national%20survey%20report_dev_v11-lr.pdf menzies hj, moalosi g, anisimova v, et al. increase in anti-tuberculosis drug resistance in botswana: results from the fourth national drug resistance survey. int j tuberc lung dis. 2014;18(9):1026–1033. https://doi.org/10.5588/ijtld.13.0749 who. global tb report. 2017 [cited 2018 august 25]. available from: http://www.who.int/tb/publications/global_report/gtbr2016_executive_summary.pdf slmta. slmta laboratories that have achieved accreditation. 2017 [cited 2018 august 30]; available from: https://slmta.org/accredited-labs/ diacon ah, pym a, grobusch mp, et al. multidrug-resistant tuberculosis and culture conversion with bedaquiline. n engl j med. 2014;371(8):723–732. https://doi.org/10.1056/nejmoa1313865 who. the use of bedaquiline in the treatment of multidrug-resistant tuberculosis. 2013. [cited 2016 october 04]. available from: http://apps.who.int/iris/bitstream/10665/84879/1/9789241505482_eng.pdf raizes e, hader s, birx d. the us president’s emergency plan for aids relief (pepfar) and hiv drug resistance: mitigating risk, monitoring impact. j infect dis. 2017;216(suppl_9):s805–s807. steingart kr, schiller i, horne dj, pai m, boehme cc, dendukuri n. xpert® mtb/rif assay for pulmonary tuberculosis and rifampicin resistance in adults. cochrane database syst rev. 2014;(1):1–166. shah ns, auld sc, brust jc, et al. transmission of extensively drug-resistant tuberculosis in south africa. n engl j med. 2017;376(3):243–253. https://doi.org/10.1056/nejmoa1604544 who. who announces new tb supranational reference laboratory in the african region. 2017 [cited 2018 january 22]. available from: http://www.who.int/tb/features_archive/tb_supranational_reference_laboratory_afro/en/ trebucq a, schwoebel v, kashongwe z, et al. treatment outcome with a short multidrug-resistant tuberculosis regimen in nine african countries. int j tuberc lung dis. 2018;22(1):17–25. https://doi.org/10.5588/ijtld.17.0498 schnippel k, ndjeka n, maartens g, et al. effect of bedaquiline on mortality in south african patients with drug-resistant tuberculosis: a retrospective cohort study. lancet respir med. 2018;6:699–706. https://doi.org/10.1016/s2213-2600(18)30235-2 cain kp, marano n, kamene m, et al. the movement of multidrug-resistant tuberculosis across borders in east africa needs a regional and global solution. plos med. 2015;12(2):e1001791. https://doi.org/10.1371/journal.pmed.1001791 rees d, murray j, nelson g, sonnenberg p. oscillating migration and the epidemics of silicosis, tuberculosis, and hiv infection in south african gold miners. am j ind med. 2010;53(4):398–404. https://doi.org/10.1002/ajim.20716 ilead. innovation: laboratory engineered accelerated diagnostics. 2017. [cited 2018 january 22]. available from: http://ilead.org.za/ appendix 1 table 1-a1: mdr-tb outcomes in africa: 2014. global targets are not enough cooperation hinges on wealthy nations doing more acknowledgements references about the author(s) lukoye atwoli east african medical journal, nairobi, kenya abdullah h. baqui journal of health population and nutrition, baltimore, maryland, united states of america thomas benfield danish medical journal, copenhagen, denmark raffaella bosurgi plos medicine, cambridge, united kingdom fiona godlee the bmj, london, united kingdom stephen hancocks british dental journal, london, united kingdom richard horton the lancet, london, united kingdom laurie laybourn-langton uk health alliance on climate change, london, united kingdom carlos augusto monteiro revista de saúde pública, são paulo, brazil ian norman international journal of nursing studies, london, united kingdom kirsten patrick canadian medical association journal, ottawa, ontario, canada nigel praities pharmaceutical journal, london, united kingdom marcel g.m. olde rikkert the dutch journal of medicine, amsterdam, netherlands eric j. rubin the new england journal of medicine, boston, massachusetts, united states of america peush sahni national medical journal of india, new delhi, delhi, india richard smith uk health alliance on climate change, london, united kingdom nicholas j. talley medical journal of australia, sydney, new south wales, australia sue turale international nursing review, geneva, switzerland damián vázquez pan american journal of public health, washington, dc, united states of america citation atwoli l, baqui ah, benfield t, et al. call for emergency action to limit global temperature increases, restore biodiversity, and protect health. afr j lab med. 2021;10(1), a1707. https://doi.org/10.4102/ajlm.v10i1.1707 editorial call for emergency action to limit global temperature increases, restore biodiversity, and protect health lukoye atwoli, abdullah h. baqui, thomas benfield, raffaella bosurgi, fiona godlee, stephen hancocks, richard horton, laurie laybourn-langton, carlos augusto monteiro, ian norman, kirsten patrick, nigel praities, marcel g.m. olde rikkert, eric j. rubin, peush sahni, richard smith, nicholas j. talley, sue turale, damián vázquez copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. wealthy nations must do much more, much faster the un general assembly in september 2021 will bring countries together at a critical time for marshalling collective action to tackle the global environmental crisis. they will meet again at the biodiversity summit in kunming, china, and the climate conference (cop26) in glasgow, uk. ahead of these pivotal meetings, we—the editors of health journals worldwide—call for urgent action to keep average global temperature increases below 1.5°c, halt the destruction of nature, and protect health. health is already being harmed by global temperature increases and the destruction of the natural world, a state of affairs health professionals have been bringing attention to for decades.1 the science is unequivocal; a global increase of 1.5°c above the pre-industrial average and the continued loss of biodiversity risk catastrophic harm to health that will be impossible to reverse.2,3 despite the world’s necessary preoccupation with covid-19, we cannot wait for the pandemic to pass to rapidly reduce emissions. reflecting the severity of the moment, this editorial appears in health journals across the world. we are united in recognising that only fundamental and equitable changes to societies will reverse our current trajectory. the risks to health of increases above 1.5°c are now well established.2 indeed, no temperature rise is “safe.” in the past 20 years, heat related mortality among people aged over 65 has increased by more than 50%.4 higher temperatures have brought increased dehydration and renal function loss, dermatological malignancies, tropical infections, adverse mental health outcomes, pregnancy complications, allergies, and cardiovascular and pulmonary morbidity and mortality.5,6 harms disproportionately affect the most vulnerable, including among children, older populations, ethnic minorities, poorer communities, and those with underlying health problems.2,4 global heating is also contributing to the decline in global yield potential for major crops, falling by 1.8–5.6% since 1981; this, together with the effects of extreme weather and soil depletion, is hampering efforts to reduce undernutrition.4 thriving ecosystems are essential to human health, and the widespread destruction of nature, including habitats and species, is eroding water and food security and increasing the chance of pandemics.3,7,8 the consequences of the environmental crisis fall disproportionately on those countries and communities that have contributed least to the problem and are least able to mitigate the harms. yet no country, no matter how wealthy, can shield itself from these impacts. allowing the consequences to fall disproportionately on the most vulnerable will breed more conflict, food insecurity, forced displacement, and zoonotic disease—with severe implications for all countries and communities. as with the covid-19 pandemic, we are globally as strong as our weakest member. rises above 1.5°c increase the chance of reaching tipping points in natural systems that could lock the world into an acutely unstable state. this would critically impair our ability to mitigate harms and to prevent catastrophic, runaway environmental change.9,10 global targets are not enough encouragingly, many governments, financial institutions, and businesses are setting targets to reach net-zero emissions, including targets for 2030. the cost of renewable energy is dropping rapidly. many countries are aiming to protect at least 30% of the world’s land and oceans by 2030.11 these promises are not enough. targets are easy to set and hard to achieve. they are yet to be matched with credible short and longer term plans to accelerate cleaner technologies and transform societies. emissions reduction plans do not adequately incorporate health considerations.12 concern is growing that temperature rises above 1.5°c are beginning to be seen as inevitable, or even acceptable, to powerful members of the global community.13 relatedly, current strategies for reducing emissions to net zero by the middle of the century implausibly assume that the world will acquire great capabilities to remove greenhouse gases from the atmosphere.14,15 this insufficient action means that temperature increases are likely to be well in excess of 2°c,16 a catastrophic outcome for health and environmental stability. critically, the destruction of nature does not have parity of esteem with the climate element of the crisis, and every single global target to restore biodiversity loss by 2020 was missed.17 this is an overall environmental crisis.18 health professionals are united with environmental scientists, businesses, and many others in rejecting that this outcome is inevitable. more can and must be done now—in glasgow and kunming—and in the immediate years that follow. we join health professionals worldwide who have already supported calls for rapid action.1,19 equity must be at the centre of the global response. contributing a fair share to the global effort means that reduction commitments must account for the cumulative, historical contribution each country has made to emissions, as well as its current emissions and capacity to respond. wealthier countries will have to cut emissions more quickly, making reductions by 2030 beyond those currently proposed20,21 and reaching net-zero emissions before 2050. similar targets and emergency action are needed for biodiversity loss and the wider destruction of the natural world. to achieve these targets, governments must make fundamental changes to how our societies and economies are organised and how we live. the current strategy of encouraging markets to swap dirty for cleaner technologies is not enough. governments must intervene to support the redesign of transport systems, cities, production and distribution of food, markets for financial investments, health systems, and much more. global coordination is needed to ensure that the rush for cleaner technologies does not come at the cost of more environmental destruction and human exploitation. many governments met the threat of the covid-19 pandemic with unprecedented funding. the environmental crisis demands a similar emergency response. huge investment will be needed, beyond what is being considered or delivered anywhere in the world. but such investments will produce huge positive health and economic outcomes. these include high quality jobs, reduced air pollution, increased physical activity, and improved housing and diet. better air quality alone would realise health benefits that easily offset the global costs of emissions reductions.22 these measures will also improve the social and economic determinants of health, the poor state of which may have made populations more vulnerable to the covid-19 pandemic.23 but the changes cannot be achieved through a return to damaging austerity policies or the continuation of the large inequalities of wealth and power within and between countries. cooperation hinges on wealthy nations doing more in particular, countries that have disproportionately created the environmental crisis must do more to support low and middle income countries to build cleaner, healthier, and more resilient societies. high income countries must meet and go beyond their outstanding commitment to provide $100bn a year, making up for any shortfall in 2020 and increasing contributions to and beyond 2025. funding must be equally split mitigation and adaptation, including improving the resilience of health systems. financing should be through grants rather than loans, building local capabilities and truly empowering communities, and should come alongside forgiving large debts, which constrain the agency of so many low income countries. additional funding must be marshalled to compensate for inevitable loss and damage caused by the consequences of the environmental crisis. as health professionals, we must do all we can to aid the transition to a sustainable, fairer, resilient, and healthier world. alongside acting to reduce the harm from the environmental crisis, we should proactively contribute to global prevention of further damage and action on the root causes of the crisis. we must hold global leaders to account and continue to educate others about the health risks of the crisis. we must join in the work to achieve environmentally sustainable health systems before 2040, recognising that this will mean changing clinical practice. health institutions have already divested more than $42bn of assets from fossil fuels; others should join them.4 the greatest threat to global public health is the continued failure of world leaders to keep the global temperature rise below 1.5°c and to restore nature. urgent, society-wide changes must be made and will lead to a fairer and healthier world. we, as editors of health journals, call for governments and other leaders to act, marking 2021 as the year that the world finally changes course. competing interests: we have read and understood bmj policy on declaration of interests and fg serves on the executive committee for the uk health alliance on climate change and is a trustee of the eden project. rs is the chair of patients know best, has stock in unitedhealth group, has done consultancy work for oxford pharmagenesis, and is chair of the lancet commission of the value of death. none further declared. provenance and peer review: commissioned; not externally peer reviewed. this editorial is being published simultaneously in many international journals. please see the full list here: https://www.bmj.com/content/full-list-authors-and-signatories-climate-emergency-editorial-september-2021 this is an open access article distributed in accordance with the terms of the creative commons attribution (cc by 4.0) license, which permits others to distribute, remix, adapt and build upon this work, for commercial use, provided the original work is properly cited. see: http://creativecommons.org/licenses/by/4.0/ acknowledgements competing interests we have read and understood bmj policy on declaration of interests and fg serves on the executive committee for the uk health alliance on climate change and is a trustee of the eden project. rs is the chair of patients know best, has stock in unitedhealth group, has done consultancy work for oxford pharmagenesis, and is chair of the lancet commission of the value of death. none further declared. authors’ contributions laurie laybourn-langton conceived of the idea, coordinated its development and delivery, and led drafting along with richard smith. all authors contributed significantly to the editorial content. funding information laurie laybourn-langton’s time was funded by the climate and health council. respective authors were paid by their employers. data availability the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. disclaimer this editorial is being published simultaneously in many international journals. please see the full list here: https://www.bmj.com/content/full-list-authors-and-signatories-climate-emergency-editorial-september-2021. references in support of a health recovery [homepage on the internet]. available from: https://healthyrecovery.net intergovernmental panel on climate change. summary for 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covid-19 pandemic and the social determinants of health. bmj. 2021;372:n129. https://doi.org/10.1136/bmj.n129 abstract introduction methods results discussion acknowledgements references about the author(s) barbara tornimbene amr division, surveillance, prevention and control department, world health organization, geneva, switzerland sergey eremin amr division, surveillance, prevention and control department, world health organization, geneva, switzerland reuben abednego national health laboratory quality assurance and training centre (nhlqatc), tanzania, dar es salaam, united republic of tanzania elamin o. abualas national public health laboratory, federal ministry of health, khartoum, sudan ilhem boutiba faculty of medicine, university of tunis el manar, tunis, tunisia abiodun egwuenu nigeria center for disease control, abuja, nigeria walter fuller antimicrobial resistance (amr) world health organization, regional office for africa, brazzaville, congo laetitia gahimbare antimicrobial resistance (amr) world health organization, regional office for africa, brazzaville, congo susan githii national microbiology reference lab, national public health laboratories, nairobi, kenya watipaso kasambara ministry of health, lilongwe, malawi chileshe lukwesa-musyani lusaka district laboratory, university of zambia, lusaka, zambia fidy a. miamina department of health watch, epidemiological surveillance and response (dvsser), antananarivo, madagascar sekesai mtapuri-zinyowera national microbiology reference laboratory, zimbabwe, harare, zimbabwe grace najjuka department of microbiology, joint clinical research centre (jcrc), kampala, uganda olga perovic centre for healthcare-associated infections, antimicrobial resistance and mycoses (charm), johannesburg, south africa bassem zayed world health organization, regional office for east mediterranean, cairo, egypt yahaya a. ahmed world health organization, regional office for africa, brazzaville, congo maha t. ismail world health organization, regional office for east mediterranean, cairo, egypt carmem l. pessoa da silva amr division, surveillance, prevention and control department, world health organization, geneva, switzerland citation tornimbene b, eremin s, abednego r, et al. global antimicrobial resistance and use surveillance system on the african continent: early implementation 2017–2019. afr j lab med. 2022;11(1), a1594. https://doi.org/10.4102/ajlm.v11i1.1594 original research global antimicrobial resistance and use surveillance system on the african continent: early implementation 2017–2019 barbara tornimbene, sergey eremin, reuben abednego, elamin o. abualas, ilhem boutiba, abiodun egwuenu, walter fuller, laetitia gahimbare, susan githii, watipaso kasambara, chileshe lukwesa-musyani, fidy a. miamina, sekesai mtapuri-zinyowera, grace najjuka, olga perovic, bassem zayed, yahaya a. ahmed, maha t. ismail, carmem l. pessoa da silva received: 28 mar. 2021; accepted: 20 apr. 2022; published: 31 aug. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: antimicrobial resistance (amr) is becoming a critical public health issue globally. the world health organization launched the global antimicrobial resistance and use surveillance system (glass) to support the strengthening of the amr evidence base. objective: the article describes the evolution of national amr surveillance systems and amr data reporting of countries in the african continent between 2017 and 2019, and the constraints, perceived impact and value of the participation in glass. methods: data on implementation of national surveillance systems and amr rates were submitted to glass between 2017 and 2019 and summarised though descriptive statistics. the information on constraints and perceived impact and value in glass participation was collected though a set of questionnaires. results: between 2017 and 2019, egypt, ethiopia, madagascar, malawi, mali, mozambique, nigeria, south africa, sudan, tunisia, uganda and zambia submitted data to glass. the main constraints listed are linked to scarce laboratory capacity and capability, limited staffing, budget issues, and data management. moreover, while the data are not yet nationally representative, high resistance rates were reported to commonly-used antibiotics, as the emerging resistance to last treatment options. conclusion: despite the limitations, more and more countries in the african continent are working towards reaching a status that will enable them to report amr data in a complete and systematic manner. future improvements involve the expansion of routine surveillance capacity for several countries and the implementation of surveys that allow to effectively define the magnitude of amr in the continent. keywords: amr; surveillance; africa; implementation; who. introduction antimicrobial resistance (amr) is defined as ‘the presence of resistance to antimicrobial medicines in infectious agents such as bacteria, viruses, fungi and parasites’1. this resistance can be inherent or acquired by the inappropriate use of medicines. recent studies position amr as a leading cause of death around the world, with the highest burdens in low-resource settings.2 africa remains the continent most afflicted by infectious diseases and amr can dramatically hamper treatment effectiveness and greatly amplify disease burden and its complications.3 a 2017 systematic review from tadesse et al.,4 documenting the status of amr in africa, found that ‘recent amr data was not available for more than 40% of countries’. in countries for which data were available, ‘the level of resistance to commonly prescribed antibiotics was significant’, and that the ‘quality of microbiological data is of serious concern’.3 a second systematic review, also published in 2017 and targeting west africa, found amr to be common in this subregion. it particularly occurred in hospitalised patients with bloodstream infections (bsi), and both outpatient and hospitalised patients with urinary tract infection (uti).5 two reviews of amr in sub-saharan africa and one review in east africa also revealed ‘a high prevalence of amr to commonly-used antibiotics in clinical bacterial isolates’.6,7 the studies also flagged the flaws in available data and the challenges faced in lowand lower middle-income countries when implementing amr surveillance. in 2015, the world health organization (who) launched the global antimicrobial resistance and use surveillance system (glass) to support strengthening of the amr evidence base. as stated in the glass report 2020, glass encourages countries to move to surveillance approaches based on systems that includes epidemiological, clinical, and population-level data, rather than only on laboratory data. in addition to data collection, glass promotes strengthening of national amr surveillance systems to ensure that reliable and representative information is produced. it is supported by who collaborating centres network, involving strong commitment from participating countries and close collaborations with who headquarter, regional and country offices.8 during the early implementation phase (2015–2019), glass focused on collecting information on the status of existing or newly developed national amr surveillance systems and to provide a standardised approach to the collection of amr rates for selected bacteria causing generic infections in humans. the first data call was opened in may 2017, and it recurs every year in the same period, between 1 may and 31 august. this article aims to describe the evolution of national amr surveillance systems and amr data reporting capacity of african countries participating in glass for the first three annual glass data calls (2017–2019), together with a summary of reported amr rates for selected indicators. the article also describes, for a subset of african countries, the constraints, perceived impact, and value linked to reporting data to glass. methods ethical considerations ethics approval was not required for this study. each country has its own ethical approval for amr surveillance and in many cases routine surveillance does not require ethical clearance. data sources the glass database is the source of information of countries participation and reporting, implementation of the national surveillance system, and amr rates. the data were submitted by counties during three data calls, between 2017 and 2019. the information on constraints and perceived impact and value in glass participation was collected though a set of questions sent via email to countries’ amr national focal points (nfps) and who regional offices’ staff in charge of amr activities. information gathered thorough global antimicrobial resistance and use surveillance system data calls the glass amr data call is open yearly between may and august, and countries submit the information on the implementation of the national amr surveillance systems for the data call year, and amr rates for the previous year. information on the implementation of the national surveillance system in this article, key indicators on the implementation of the national surveillance system are summarised and presented to reflect changes during three data calls (2017–2019). as stated in the glass report 2020,7 glass collects information on the implementation of national amr surveillance systems through a standardised questionnaire filled in every year by the nfp. a set of indicators is then used to measure the development and strengthening of national amr surveillance:7 the establishment of a national coordinating centre and the national reference laboratory. these two bodies are in charge of data management and capacity building, and are key to the coordination of the national systems. number of surveillance sites and local laboratories performing antimicrobial susceptibility testing (ast) that report amr data to glass. this information allows a better understanding of the structure and capacity of the national surveillance system structure. surveillance sites can be hospitals, clinics, or in-and outpatient community healthcare facilities with access to relevant epidemiological and laboratory support and information. provision of external quality assessment. this allows a better understanding of the diagnostic capacity of the surveillance system, by checking the provision of external quality assessment to the national reference laboratory and clinical local laboratories, and the use of international standards for diagnostics and ast. antimicrobial resistance data as stated in the glass reports, glass requires amr data to be collected through a surveillance system that gathers results from ast for common human bacterial pathogens in four infection sites, specifically bsis caused by acinetobacter spp., e. coli, k. pneumoniae, salmonella spp., s. aureus and s. pneumoniae, utis caused by e. coli and k. pneumonia, gastrointestinal infections caused by salmonella spp. and shigella spp., and genital infections caused by n. gonorrhoeae. data are generated by the collation of results from specimens that are routinely sent to laboratories for clinical testing and includes blood, urine, stool and cervical and urethral samples. the rationale for selection of these particular specimens is that the growth of a pathogen is a proxy of infection in the associated anatomical sites. the target population under surveillance is the national population of patients seeking care in hcfs. data to be collected includes: ‘numbers of patients with susceptible, non-susceptible, intermediate, and resistant isolates, as well as numbers of isolates with unknown susceptibility.8,9,10 two types of unknown results are recorded. the first, ‘unknown_no_ast’, is the number of isolates with ast results that were not reported (or not performed) for a specific antibiotic. the second, ‘unknown_no_breakpoints’, is the number of isolates for which ast was performed but which had no interpretation of results available for a specific antibiotic. additionally, countries are invited to report patients’ microbiological results (bacterial isolation and identification, ast), as well as demographic and epidemiological variables such as age, gender, and origin of infection in tested patients, in aggregated format.8 the distribution of infections and bacteria analysed and submitted to glass by countries during the three data calls (2017–2019) is summarised by year and shown in table format. antimicrobial resistance rates: proportions of patients with resistant infections reported by countries during three data calls are presented for: bloodstream infections caused by escherichia coli and klebsiella pneumoniae resistant to carbapenems and third-generation cephalosporins (3gc), and bsis caused by methicillin-resistant staphylococcus aureus (mrsa). urinary tract infection caused by e. coli and k. pneumoniae resistant to carbapenems and ciprofloxacin. as aligned to the method outlines in the glass report 2016–2017, ‘rates are shown only if [countries reported] results for > 10 patients, and for pathogen–antibiotic combinations with > 10 ast results and < 30% unknown ast results’11. box-and-whisker plots are used to summarise the reported median rate of resistance for specific specimen–pathogen–antibiotic combinations. the plots portray the distribution of the submitted data, outliers, and the median. the box within the chart displays where around 50% of the data points fall and it contains the lower quartile, the upper quartile, and the median in the centre. the median is the value separating the higher half from the lower half of the results. information gathered through countries’ and who regional offices’ feedback after the end of the first glass data call in august 2017 and the last data call in august 2019, enrolled african countries, the who regional office for africa and the who regional office for the eastern mediterranean were asked to provide feedback to a set of questions covering three themes: constraints: the difficulties encountered to participate in glass data calls. impact: the positive impact that the three data calls might have had to foster data generation. value: the added value of participating in glass. the qualitative data obtained from countries’ and regional offices’ feedback is summarised in the article using a set of codes identified for each theme and shown in pie chart format. coding was done by identifying a passage in the text, searching, and identifying concepts, and finding relationships between them. data analysis data on information on the implementation of the national surveillance system were exported from the glass information technology platform into microsoft excel (microsoft corporation, california, united states), and bar charts were used to visualise the proportion of each variable outcome for all reporting countries in order to interpret the data. antimicrobial resistance data were exported from the glass information technology platform and validated and analysed using stata (statacorp, college station, texas, united states) software. for each country, the number of patients with confirmed bacterial infection was calculated by collapsing all the laboratory results (susceptible, non-susceptible, intermediate, and resistant isolates, as well as numbers of isolates with unknown susceptibility) for a specific pathogen and choosing the antibiotic with the highest number of results reported. the number of patients with ast results by pathogen is calculated in the same way, but unknown susceptibility results are not included. the proportion of patients with resistant infection for a specific indicator (see ‘antimicrobial resistance rates’ mentioned earlier) is then calculated for each country using the following formula: all countries’ results were pulled together, and the median rate of resistance was calculated. tableau software (tableau, mountain view, california, united states) was used to visualise the data though box-and-whisker plots. the questionnaires’ contents, with countries’ and who regional offices’ feedback, were pulled together in microsoft excel and screened for key words to define codes for the feedback themes. the proportion of respondents for each code was then calculated, based on the total number of questionnaires received. results global antimicrobial resistance and use surveillance system data calls countries’ participation and reporting enrolment and reporting varied during the three data calls, both on submission on the information of the status of the national amr surveillance systems and amr data. by the end of the last data call in august 2019, 23 out of 54 (43%) african countries were formally enrolled in glass: cote d’ivoire, egypt, ethiopia, gabon, gambia, ghana, kenya, liberia, libya, madagascar, malawi, mali, mauritania, mauritius, mozambique, nigeria, south africa, sudan, tanzania, tunisia, uganda, zambia and zimbabwe. table 1 shows progress in country participation and reporting from 2017 to 2019, with almost a 100% increase in number of countries over the three years. table 1: number of countries on the african continent enrolled in glass and which reported information on their national surveillance systems and amr data during the three glass data calls (2017–2019). most countries have been able to report amr data with age and gender stratification (table 2). the reporting of the number of tested patients, the denominator used to calculate frequency of amr infection in patients with suspected bacterial infection, has increased from one (17%) reporting country in 2017, to 10 (100%) reporting countries in 2019. infection origin has proved to be the least reported variable throughout the three data calls. table 2: number of countries on the african continent enrolled in glass and reporting ast results during the three data glass calls (2017–2019), as well as availability of data stratification by age, gender and infection origin and data on the number of patients from which a diagnostic sample was taken (tested). information of the status of national antimicrobial resistance surveillance systems data show that the national coordinating centre is established, or is in the process of being established, and the national reference laboratories are nominated in around 80% of countries participating in the three data calls. the number of surveillance sites reporting to glass over the three years went from 52 hospital and five outpatient facilities in 2017, to 63 hospital and 62 outpatient facilities in 2019. in certain cases, the number of surveillance sites could not be retrieved so the number of laboratories supporting the surveillance systems was reported instead; this was done for five laboratories in 2018, and 12 laboratories in 2019. overall, the total number of surveillance sites increased from 57 in 2017 to 137 in 2019. almost 80% of countries reported in three data calls having the national reference laboratories participating in an external quality assessment scheme, while external quality assessment for clinical laboratories that contribute to the national amr surveillance programmes went from being performed by 27% of countries in 2017, to 61% of countries in 2018, and 48% of countries in 2019. in around 70% of the countries, clinical laboratories performed ast according to recognised standards, from either the clinical & laboratory standards institute or the european committee on antimicrobial susceptibility testing. antimicrobial resistance data distribution of infections and bacteria analysed: compared to the first data call, with six countries reporting ast results for 11 060 patients with confirmed bacterial infections, in 2019 glass received ast results from 10 countries for 32 117 patients, three times the number from 2017 (table 3). bloodstream infection is the most frequent infection reported for the three years, followed by uti, gastroenteric infection and gonorrhoea (table 3). bloodstream infections caused by s. aureus and k. pneumoniae appeared to be the most recurrent, while e. coli is the most frequent etiological agent of reported utis. rate of gastroenteric infections caused by shigella species and salmonella species were reported equally through the years. table 3: summary of confirmed bacterial infections and patient with ast results, by infection site and pathogen, reported by egypt, ethiopia, madagascar, malawi, mali, mozambique, nigeria, south africa, sudan, tunisia, uganda, and zambia during three in glass data calls (2017–2019). antimicrobial resistance rates: the patterns of resistance of e. coli and k. pneumoniae to different antibiotics by infections sites and specific organisms between 2016 and 2018, as reported in 2017–2019 data calls, are summarised in table 4. results show that resistance to 3gc in bsis is above 50% for e. coli and 81% for k. pneumoniae, while carbapenem resistance reaches a maximum of 8% for e. coli and 24% for k. pneumoniae; mrsa is found to be the cause of about 20% of bsis. similarly, resistance of e. coli and k. pneumoniae to carbapenems is generally found to be below 10% in utis, while resistance to ciprofloxacin is between 36% and 60%. table 4: proportion of resistance (median) for selected infection site, by antibiotics and pathogen, in egypt, ethiopia, madagascar, malawi, mali, mozambique, nigeria, south africa, sudan, tunisia, uganda and zambia between 2016 and 2018. boxplots for each pathogen–antibiotic combination in different infections sites (bsi and uti) are presented in figure 1. although the list and number of countries reporting on specific pathogen–antibiotic combination varied throughout the three data calls, data show a certain consistency in the reported rates. figure 1: boxplots showing proportion (median) of infection syndrome due to bacteria resistant to selected antibiotics in egypt, ethiopia, madagascar, malawi, mali, mozambique, nigeria, south africa, sudan, tunisia, uganda, and zambia between 2016 and 2018. the number of countries reporting for each year and the number of patients with antimicrobial susceptibility testing (ast) results are shown in the x-axis. the list and the number of countries reporting each year may vary. each red dot corresponds to a single country result. (a) boxplots showing proportion (median) of bloodstream infections due to escherichia coli resistance to third-generation cephalosporins in egypt, ethiopia, madagascar, malawi, mali, mozambique, nigeria, south africa, sudan, tunisia, uganda and zambia between 2016 and 2018. the number of countries reporting for each year and the number of patients with ast results are shown below the x-axis. the list and the number of countries reporting each year may vary. each red dot corresponds to a single country result. (b) boxplots showing proportion (median) of bloodstream infections due to escherichia coli resistance to carbapenems in egypt, ethiopia, madagascar, malawi, mali, mozambique, nigeria, south africa, sudan, tunisia, uganda and zambia between 2016 and 2018. the number of countries reporting for each year and the number of patients with ast results are shown below the x-axis. the list and the number of countries reporting each year may vary. each red dot corresponds to a single country result. (c) boxplots showing proportion (median) of bloodstream infections due to klebsiella pneumoniae resistance to third-generation cephalosporins, in egypt, ethiopia, madagascar, malawi, mali, mozambique, nigeria, south africa, sudan, tunisia, uganda and zambia, between 2016 and 2019. the number of countries reporting for each year and the number of patients with ast results are shown below the x-axis. the list and the number of countries reporting each year may vary. each red dot corresponds to a single country result. (d) boxplots showing proportion (median) of bloodstream infections due to klebsiella pneumoniae resistance to carbapenes in egypt, ethiopia, madagascar, malawi, mali, mozambique, nigeria, south africa, sudan, tunisia, uganda and zambia, between 2016 and 2019. the number of countries reporting for each year and the number of patients with ast results are shown below the x-axis. the list and the number of countries reporting each year may vary. each red dot corresponds to a single country result. figure 1 (continues...): boxplots showing proportion (median) of infection syndrome due to bacteria resistant to selected antibiotics in egypt, ethiopia, madagascar, malawi, mali, mozambique, nigeria, south africa, sudan, tunisia, uganda, and zambia between 2016 and 2018. the number of countries reporting for each year and the number of patients with antimicrobial susceptibility testing (ast) results are shown in the x-axis. the list and the number of countries reporting each year may vary. each red dot correspond to a single country result. (e) boxplots showing proportion (median) of bloodstream infections due to methicillin-resistant staphylococcus aureus (mrsa) in egypt, ethiopia, madagascar, malawi, mali, mozambique, nigeria, south africa, sudan, tunisia, uganda and zambia, between 2016 and 2019. the number of countries reporting for each year and the number of patients with ast results are shown below the x-axis. the list and the number of countries reporting each year may vary. each red dot corresponds to a single country result. (f) boxplots showing proportion (median) of urinary tract infections due to escherichia coli resistance to ciprofoloxacin in egypt, ethiopia, madagascar, malawi, mali, mozambique, nigeria, south africa, sudan, tunisia, uganda and zambia between 2016 and 2019. the number of countries reporting for each year and the number of patients with ast results are shown below the x-axis. the list and the number of countries reporting each year may vary. each red dot corresponds to a single country result. (g) boxplots showing proportion (median) of urinary tract infections due to escherichia coli resistance to carbapenems in egypt, ethiopia, madagascar, malawi, mali, mozambique, nigeria, south africa, sudan, tunisia, uganda and zambia between 2016 and 2019. the number of countries reporting for each year and the number of patients with ast results are shown below the x-axis. the list and the number of countries reporting each year may vary. each red dot corresponds to a single country result. (h) boxplots showing proportion (median) of urinary tract infections due to klebsiella pneumoniae resistance to ciprofloxacin in egypt, ethiopia, madagascar, malawi, mali, mozambique, nigeria, south africa, sudan, tunisia, uganda and zambia between 2016 and 2019. the number of countries reporting for each year and the number of patients with ast results are shown below the x-axis. the list and the number of countries reporting each year may vary. each red dot corresponds to a single country result. figure 1 (continues...): boxplots showing proportion (median) of infection syndrome due to bacteria resistant to selected antibiotics in egypt, ethiopia, madagascar, malawi, mali, mozambique, nigeria, south africa, sudan, tunisia, uganda, and zambia between 2016 and 2018. the number of countries reporting for each year and the number of patients with antimicrobial susceptibility testing (ast) results are shown in the x-axis. the list and the number of countries reporting each year may vary. each red dot correspond to a single country result. (i) boxplots showing proportion (median) of urinary tract infections due to klebsiella pneumoniae resistance to carbapenems in egypt, ethiopia, madagascar, malawi, mali, mozambique, nigeria, south africa, sudan, tunisia, uganda and zambia between 2016 and 2019. the number of countries reporting for each year and the number of patients with ast results are shown below the x-axis. the list and the number of countries reporting each year may vary. each red dot corresponds to a single country result. countries’ and who regional offices’ feedback eleven countries, as well as the who regional office for africa and the who regional office for the eastern mediterranean, provided feedback on the first three years of glass implementation. the feedback from nfps was provided either at the end of the first data call, the third data call, or both (table 5). responses by nfps and regional offices are presented in figure 2 by the three themes (constraint, perceived impact, and value) and identified codes. limited resources, economic issues, bureaucratic bottlenecks, and political instability were reported as having a major impact on the roll out of national amr surveillance systems. for example, the partial absence of quality assurance provision and tools to extract, clean and aggregate data were listed as important limitations to countries’ participation. scarce availability of trained professionals and resources to hire new staff was also indicated as major issue. both who regional offices noted that glass data generation, validation, and processing are centralised and headed by a single nfp. however, due to the shortage of manpower, nfps frequently oversee other activities, which results in competing priorities that might delay data reporting. lack of it tools, technical training for data management and data preparation, and complex it system interoperability, were also mentioned as important constraint for data reporting. in some countries, where a national laboratory-based surveillance programme has been in place for a long period of time, an enhanced surveillance system had to be adapted to meet glass’s capacity to include population data. table 5: list of countries and years for which countries national focal points provided feedback to glass. figure 2: pie charts summarising the proportion of countries’ national focal points and who regional offices’ responses related to constraints, perceived impact, and value associated to reporting data to global antimicrobial resistance and use surveillance system (glass). feedback was received by kenya, madagascar, malawi, nigeria, south africa, sudan, tanzania, tunisia, uganda, zambia, zimbabwe and the who regional office for africa and regional office for the east mediterranean, in relation to 2017 and 2019 glass data calls. (a) pie chart summarising the proportion of countries’ national focal points and who regional offices’ responses related to constraints associated to reporting data to glass. feedback was received by kenya, madagascar, malawi, nigeria, south africa, sudan, tanzania, tunisia, uganda, zambia, zimbabwe and the who regional office for africa and regional office for the east mediterranean, in relation to 2017 and 2019 glass data calls. (b) pie chart summarising the proportion of countries’ national focal points and who regional offices’ responses related to perceived impact associated to reporting data to glass. feedback was received by kenya, madagascar, malawi, nigeria, south africa, sudan, tanzania, tunisia, uganda, zambia, zimbabwe and the who regional office for africa and regional office for the east mediterranean, in relation to 2017 and 2019 glass data calls. figure 2 (continues...): pie charts summarising the proportion of countries’ national focal points and who regional offices’ responses related to constraints, perceived impact, and value associated to reporting data to global antimicrobial resistance and use surveillance system (glass). feedback was received by kenya, madagascar, malawi, nigeria, south africa, sudan, tanzania, tunisia, uganda, zambia, zimbabwe and the who regional office for africa and regional office for the east mediterranean, in relation to 2017 and 2019 glass data calls. (c) pie chart summarising the proportion of countries’ national focal points and who regional offices’ responses related to perceived value associated to reporting data to glass. feedback was received by kenya, madagascar, malawi, nigeria, south africa, sudan, tanzania, tunisia, uganda, zambia, zimbabwe and the who regional office for africa and regional office for the east mediterranean, in relation to 2017 and 2019 glass data calls. nevertheless, the glass surveillance methods proposed, and the it tools offered, were found to be well-defined and easy to use. countries were able to reform data sharing systems to suit the glass data reporting model. mostly, this was done by establishing local laboratories data systems, which were electronically linked to clinical data and to national network. according to one nfp, feedback was also sent to hospitals and outpatient clinics, resulting in treatment changes. the application of whonet, a laboratory information system software, to glass data preparation was found to be very useful and succeeded to strengthen data management capability.12 the national reports produced using data collected for glass were used by countries to develop and/or implement amr national plans and policies, to develop proposals, to orient partners, and to direct the necessary technical assistance. participation in glass has helped to launch strategies on data use and development of policies on antimicrobial use and amr. the experience gained during the data calls has been used to establish the mechanism of monitoring and evaluation of the amr surveillance system. the evidence has also collectively brought together one health partners and ministries to implement multisectoral projects and integrated surveillance. discussion african countries have responded to the first glass three data calls with a high level of interest and dedication, and the glass framework has proven to be a vital tool to the establishment and/or development of national amr surveillance systems, as reflected by the overall positive feedback of nfps to glass participation. the main constraints encountered by countries during the data calls were linked to lack of specific national activities to tackle amr, scarce laboratory capability, staffing and budget issues, and data management. however, through support from glass, countries were also able to revitalise their laboratory components and microbiological output, both for infectious diseases and amr diagnostics. in order to respond to the glass data call, countries improved the collection, analysis and presentation of standardised data generated from healthcare facilities, which in some cases also resulted in improved patient treatment. countries without an amr surveillance system in place used the glass manual for early implementation to model the structure of the new national system.13 the initial steps of the participation and the data call also pushed countries to assess the capacity of their national amr reporting system(s). this allowed the identification of gaps to address in the future, and it acted in participating countries as platform for the establishment of the national surveillance core components. this is key, considering that the data reported to all three glass data calls (2017–2019) might suggest the presence of high rates of amr in the continent. as expected, ast results for bsi were most frequently reported, followed by uti, gastroenteric infection and gonorrhoea. this is in line with the available evidence which shows that, in africa, bsi is a major cause of morbidity and mortality, and utis are some of the most frequent bacterial infections affecting people, both in the community and in hospitals.7 reported high resistance to 3gc is particularly worrying in some parts of africa, where diagnostic facilities are scarce and antibiotics such as carbapenems and semi-synthetic aminoglycosides (e.g. amikacin) are either unavailable or prohibitively expensive.7 in many sub-saharan africa hospitals, limited nursing capacity favours the use of broad-spectrum antimicrobials with a once-daily dosing regimen and this has led to the widespread adoption of 3gc for the empirical management of hospitalised patients with suspected sepsis.14 moreover, extended spectrum beta-lactamase-producing enterobacterales, for which resistance to 3gc is a marker, are also resistant to penicillins and therefore represent an important threat to the treatment of bsis in these settings.7 the reported rate of bsi caused by mrsa was also high (between 21% and 24%). methicillin-resistant s. aureus has been linked to significant morbidity and mortality and it carries an evident threat to african countries, since there might be limited access to antibiotics effective against hospital-associated mrsa, such as linezolid and daptomycin.15 furthermore, the scarce implementation of infection prevention and control measures and widespread hiv infection and tuberculosis can amplify the difficulty of dealing with the mrsa epidemic in africa. escherichia coliand k. pneumoniae-reported resistance to ciprofloxacin in utis was found to be consistently high (between 36% and 60%). this could potentially be linked to samples obtained from complicated and hospitalised patients, as in almost all countries reporting to the glass community, utis are not tested for and treated empirically.3,16 this is important, as fluoroquinolones have an significant role in treating of severe infections, such as septicaemia, and therefore increasing resistance can have severe health consequences.17 finally, reported resistance of e. coli and k. pneumoniae to carbapenems was high in both bsis and utis. this is worrying as, until recently, carbapenems were the last-resort antibiotics used for managing multidrug-resistant bacterial infections.18 moreover, the organisms that are resistant to carbapenems are frequently resistant to many other classes of commonly-used antimicrobial agents; thus, managing infections caused by them poses a substantial challenge in clinical practice and their public health impacts cannot be over-emphasised.19,20 both ciprofloxacin and carbapenems are on the ‘watch’ list of the who 2019 aware classification, that comprises antibiotics with higher potential to induce resistance; ciprofloxacin is also on the who model list of essential medicines, where it is listed as a firstor second-choice empirical treatment option for definite infectious syndromes.21 considering the reported amr data, the spread of all listed resistant patterns needs to be carefully monitored, and every country should apply measures for continuous data collection, by strengthening surveillance activities or implementing population-based studies (e.g. prevalence survey). limitations due to the quality of the data reported to glass, and associated potential bias, no trends analysis was performed with presented amr data, nor comparisons among infection types, or the identification of risk factors linked to age, gender or the source of infection. as stated in the glass report 20208: [d]ata aggregation is a major limitation, as it considerably limits options for epidemiological characterization, obviating the detection and validation of data from countries … with unusual antimicrobial patterns. furthermore, … [l]ack of a sampling strategy results in selection bias, which may affect the representativeness and precision of results. cases are found and tested only in the population that seeks medical care, … and most data are still generated in laboratories, with no epidemiological insight. antimicrobial susceptibility testing varied widely among countries for the specimen–pathogen–antibiotic combinations chosen. the numbers of patients screened for resistance were still very low, suggesting that most data come from complicated and hospitalised patients. unfortunately, it was also not possible to show the frequency of amr for these syndrome-pathogen–antibiotic combinations in the tested population, as the needed denominator – the population of the patient for which a diagnostic sample is taken – was not always available.8 finally, it was not possible to obtain feedback from all of the african countries participating in the glass data calls. however, responses showed a homogeneous consensus to the global system participation and the benefits associated to it. conclusion although some african countries listed in this article still face important constraints while building their national amr surveillance systems, and even if not all of them have provided amr data, countries have shown a willingness to share information with glass, particularly the status and the development of their surveillance systems. countries on the continent are working towards reaching a status that will enable them to report data in a complete and systematic manner, through the establishment of surveillance core components and by assuring the quality of amr diagnostics. although reported data are still not representative at a national level, and ast varied considerably among countries, the participation in glass is clearly linked to improved national surveillance systems, which will also result in better clinical care in prescribing the appropriate antibiotics, one of the most challenging objectives of the global action plan-amr. future improvements involve the expansion of routine surveillance capacity for several countries and the implementation of surveys that allow for effective definition of the magnitude of amr on the continent. meanwhile, the evidence generated is supporting the identification of areas for further research – amr burden in healthcare settings, improvement of diagnostic stewardship, and amr in the human animal environment interface – and it is advocating for the continuous support of actions directed to amr monitoring and control. together with both national and regional partners, african countries’ participation in glass is leading the way towards the further development of an efficient and reliable global surveillance system, which will be able to function in various economic and socio-political contexts, and provide vital and actionable data. addressing antimicrobial resistance through glass is part of the ongoing efforts of member states to strengthen health security, improve health systems and ensure universal health coverage.22 acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions b.t., s.e., r.a., e.o.a., i.b., a.e., w.f., l.g., s.g., w.k., c.l-m., f.a.m., s.m-z., g.n., o.p., b.z., y.a.a., m.t.i. and c.l.p.d.s. contributed to the generation of content and the development of the manuscript. sources of support this research received no specific grant from any funding agency in the public , commercial, or not-for-profit sectors. data availability the data that support the findings of this study are openly available at https://docs.google.com/spreadsheets/d/1lqcx6wno4pul4kre6tqehtrn0ultumdu/edit#gid=1028268226 (glass 2016 amr data). disclaimer the authors alone are responsible for the views expressed in this publication and they do not necessarily represent the views, decisions or policies of the institutions with which they are affiliated. references who. antimicrobial resistance. 2017 [cited 2022 july 25]. available from: https://www.who.int/news-room/questions-and-answers/item/antimicrobial-resistance murray cj, ikuta ks, sharara f, et al. global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. lancet. 2022;399(10325):629–655. https://doi.org/10.1016/s0140-6736(21)02724-0 horton r. gbd 2010: understanding disease, injury, and risk. lancet. 2012;380(9859):2053–2054. https://doi.org/10.1016/s0140-6736(12)62133-3 tadesse bt, ashley ea, ongarello s, et al. antimicrobial resistance in africa: a systematic review. bmc infect dis. 2017;17:616. https://doi.org/10.1186/s12879-017-2713-1 bernabé kj, langendorf c, ford n, ronat jb, murphy ra. antimicrobial resistance in west africa: a systematic review and meta-analysis. int j antimicrob agents. 2017;50(5):629–639. https://doi.org/10.1016/j.ijantimicag.2017.07.002 leopold sj, van leth f, tarekegn h, schultsz c. antimicrobial drug resistance among clinically relevant bacterial isolates in sub-saharan africa: a systematic review. j antimicrob chemother. 2014;69(9):2337–2353. https://doi.org/10.1093/jac/dku176 lester r, musicha p, van ginneken n, et al. prevalence and outcome of bloodstream infections due to third-generation cephalosporin-resistant enterobacteriaceae in sub-saharan africa: a systematic review. j antimicrob chemother. 2020;75(3):492–507. https://doi.org/10.1093/jac/dkz464 world health organization. global antimicrobial resistance and use surveillance system (glass) report early implementation 2020 [homepage on the internet]. 2020 world health organization. global antimicrobial resistance surveillance system (glass) report: early implementation 2017-2018. 2019. available from: https://www.who.int/glass/resources/publications/early-implementation-report-2017-2018/en/ world health organization. global antimicrobial resistance and use surveillance system (glass) report: 2021. 2021 [cited 2020 jan 01]. available from: https://www.who.int/publications/i/item/9789240027336 world health organization. global antimicrobial resistance surveillance system (glass) report: early implementation 2016-2017. 2018 [cited 2020 jan 01]. available from: http://www.who.int/glass/resources/publications/early-implementation-report/en/ whonet. who collaborating centre for surveillance of antimicrobial resistance [homepage on the internet]. no date [cited 2021 jun 01]. available from: http://www.whonet.org/ world health organization. glass manual for early implementation. 2015 [cited 2020 jan 01]. available from: http://apps.who.int/iris/bitstream/10665/188783/1/9789241549400_eng.pdf musicha p, cornick je, bar-zeev n, et al. trends in antimicrobial resistance in bloodstream infection isolates at a large urban hospital in malawi (1998–2016): a surveillance study. lancet infect dis. 2017;17(10):1042–1052. https://doi.org/10.1016/s1473-3099(17)30394-8 falagas me, karageorgopoulos de, leptidis j, korbila ip. mrsa in africa: filling the global map of antimicrobial resistance. plos one. 2013;8:e68024. https://doi.org/10.1371/journal.pone.0068024 seifu wd, gebissa ad. prevalence and antibiotic susceptibility of uropathogens from cases of urinary tract infections (uti) in shashemene referral hospital, ethiopia. bmc infect dis. 2018;18:30. https://doi.org/10.1186/s12879-017-2911-x fasugba o, gardner a, mitchell bg, mnatzaganian g. ciprofloxacin resistance in communityand hospital-acquired escherichia coli urinary tract infections: a systematic review and meta-analysis of observational studies. bmc infect dis. 2015;15:545. https://doi.org/10.1186/s12879-015-1282-4 elshamy aa, aboshanab km. a review on bacterial resistance to carbapenems: epidemiology, detection and treatment options. future sci oa. 2020;6:fso438. https://doi.org/10.2144/fsoa-2019-0098 doi y, paterson dl. carbapenemase-producing enterobacteriaceae. semin respir crit care med. 2015;36(1):74–84. https://doi.org/10.1055/s-0035-1544208 nordmann p, naas t, poirel l. global spread of carbapenemase-producing enterobacteriaceae. emerg infect dis. 2011;17(10):1791–1798. https://doi.org/10.3201/eid1710.110655 world health organization. who model list of essential medicines [homepage on the internet]. 2021 [cited 2020 jan 01]. available from: https://www.who.int/publications/i/item/who-mhp-hps-eml-2021.02 who, the world bank. tracking universal health coverage: 2017 global monitoring report [homepage on the internet]. 2017 [cited 2020 aug 20]. available from: http://documents1.worldbank.org/curated/en/640121513095868125/pdf/122029-wp-revised-public.pdf abstract introduction ethical considerations case presentation management and outcome discussion acknowledgements references about the author(s) barend mitton department of medical microbiology, university of pretoria, pretoria, south africa tshwane academic division, department of medical microbiology, national health laboratory service, pretoria, south africa roxanne rule department of medical microbiology, university of pretoria, pretoria, south africa tshwane academic division, department of medical microbiology, national health laboratory service, pretoria, south africa nontombi mbelle department of medical microbiology, university of pretoria, pretoria, south africa tshwane academic division, department of medical microbiology, national health laboratory service, pretoria, south africa wesley van hougenhouck-tulleken division of nephrology, department of internal medicine, university of pretoria, pretoria, south africa department of internal medicine, steve biko academic hospital, pretoria, south africa mohamed said department of medical microbiology, university of pretoria, pretoria, south africa tshwane academic division, department of medical microbiology, national health laboratory service, pretoria, south africa citation mitton b, rule r, mbelle n, van hougenhouck-tulleken w, said m. post-procedural bacillus cereus septic arthritis in a patient with systemic lupus erythematosus. afr j lab med. 2020;9(1), a1119 https://doi.org/10.4102/ajlm.v9i1.1119 case study post-procedural bacillus cereus septic arthritis in a patient with systemic lupus erythematosus barend mitton, roxanne rule, nontombi mbelle, wesley van hougenhouck-tulleken, mohamed said received: 06 nov. 2019; accepted: 27 may 2020; published: 20 aug. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract introduction: bacillus species are often considered as contaminants when cultured from clinical samples. bacillus cereus may be a pathogen in certain circumstances and is known to cause musculoskeletal infections. this report aims to educate clinicians and clinical microbiology laboratories on b. cereus musculoskeletal infections and to heighten awareness that bacillus species should not always be dismissed as contaminants. case presentation: we report the case of a patient who presented to a tertiary hospital in pretoria, south africa, in november 2018 with b. cereus septic arthritis and underlying systemic lupus erythematosus (sle). the isolate would otherwise have been dismissed as a contaminant had it not been for the crucial interaction between the laboratory and the treating clinicians. to our knowledge, this is the first case report of septic arthritis caused by b. cereus in an sle patient where the organism was cultured from the joint specimen. identification of the organism was performed using matrix-assisted laser desorption/ionisation mass spectrometry. management and outcome: definitive treatment was with intravenous vancomycin, continued for four weeks, in addition to arthroscopy and management of the underlying sle. the patient had a good clinical outcome and regained full mobility. conclusion: musculoskeletal infections, specifically septic arthritis caused by b. cereus, are exceedingly rare infections. immune suppression, trauma, prosthetic implants and invasive procedures are important risk factors for b. cereus musculoskeletal infections. close collaboration with a multi-disciplinary team approach will effect the best outcome for complicated patients with b. cereus infections. keywords: bacillus cereus; septic arthritis; systemic lupus erythematosus; matrix-assisted laser desorption/ionisation mass spectrometry; maldi-tof ms; musculoskeletal infection; arthroscopy. introduction as the vast majority of bacillus species are non-pathogenic and ubiquitous in the environment, many clinical microbiologists and clinicians dismiss bacillus species cultured from clinical specimens as contaminants. occasionally, this results in a missed diagnosis and inappropriate clinical decision-making. this case is important, because it illustrates the importance of communication between clinicians and the clinical laboratory staff in determining the significance of culture results. this report aims to educate healthcare workers on bacillus cereus joint infections and further endeavours to assist healthcare practitioners in distinguishing when this organism should be dismissed as a contaminant and when it should be considered as a pathogen. ethical considerations written, informed consent was obtained from the patient. this research was approved by the university of pretoria, faculty of health sciences, research ethics committee (ethics reference number 133/2019). case presentation in november 2018, a 32-year-old male was referred to a tertiary academic hospital in pretoria, south africa, with a non-resolving septic arthritis of his right knee. the patient presented to a secondary hospital 10 days prior with a tender, swollen right knee, with no history of trauma. he underwent an arthroscopy at that centre and received intravenous amoxicillin-clavulanic acid, with a suboptimal response. in addition, he developed symptoms suggestive of systemic lupus erythematosus (sle), which included polyarthritis, xerostomia, raynaud’s phenomenon, proteinuria and confusion. on examination he was haemodynamically stable with a pulse rate of 100 beats per minute, a blood pressure of 117/81 mmhg and a temperature of 36.5 °c. he had asymmetric polyarthritis, involving the right elbow, left wrist, right knee and both ankles. the right knee was the worst affected, with swelling, erythema and tenderness on examination. an emergency arthroscopy of the right knee was performed, revealing a purulent effusion. intravenous ceftriaxone (1 g twice daily) was started empirically. x-rays of all affected joints revealed no accompanying osteomyelitis. no other imaging of the joints was done. the diagnosis of sle was confirmed based on a systemic lupus international collaborating clinics score1 of five (anti-nuclear antibody positive, lupus nephritis class 3, arthritis, low c3 and neurologic sle). laboratory investigations admission blood test revealed a white cell count of 8.04 × 109 cells/l with neutrophilia (73%), a c-reactive protein of 107 mg/l, a positive anti-nuclear antibody (titre 160) and a low c3 (0.50 g/l). in addition, an epstein–barr virus viraemia of 530 copies/ml was found. admission blood cultures had no growth. a pus sample taken during arthroscopy showed numerous gram-positive bacilli on the direct gram stain. culture revealed large, flat, grey, beta-haemolytic colonies on 5% horse blood agar (figure 1), which also grew on chocolate agar and macconkey agar. this isolate was identified as bacillus species and reported as a possible contaminant. the patient had a poor clinical response to the empiric ceftriaxone after 6 days of treatment, and a request was made by the attending clinicians for antimicrobial susceptibility testing on the isolate. the isolate was therefore referred for further identification to species level using matrix-assisted laser desorption/ionisation mass spectrometry by means of vitek® ms (biomérieux, marcy l’etoile, france), instrument software version 1.5.0.4, myla® version 4.5.1 (biomérieux), knowledge base (database) version 3.2. the isolate was identified as bacillus cereus. antibiotic susceptibilities were performed by etest® (biomérieux, marcy l’etoile, france) and interpreted using the clinical & laboratory standards institute m45 (2015) breakpoints.2 the isolate was resistant to penicillin (minimum inhibitory concentration [mic] 32 µg/ml), and cefotaxime (mic 32 µg/ml), but susceptible to vancomycin (mic 4 µg/ml) and imipenem (mic 4 µg/ml). figure 1: typical morphology of bacillus cereus on 5% horse blood agar (pretoria, south africa, 19 march 2019). management and outcome based on the report, the patient was started on intravenous vancomycin 1 g twice daily. the dose was adjusted to achieve a target vancomycin blood trough level of 15 mg/ml – 20 mg/ml. trough levels were monitored roughly every 3 days over the treatment period, and the dosage adjusted accordingly. no vancomycin-related adverse events occurred during this time. vancomycin was continued for 4 weeks; over this period, the pain and swelling improved dramatically, inflammatory markers normalised and the patient regained mobility. management of the sle included prednisone, mycophenolate mofetil and chloroquine, with good response. discussion bacillus species are gram-positive, aerobic or facultative anaerobic sporulating bacilli, which are ubiquitous in the environment.3 over 100 species are known to belong to the genus.3 bacillus cereus is a common cause of food poisoning and occasionally causes opportunistic infections, usually in vulnerable hosts. these infections include ophthalmic infections, wound infections, septicaemia, endocarditis, meningitis, necrotising pneumonia and orthopaedic infections.3,4 important virulence factors of b. cereus include production of toxins and the formation of biofilms and spores.4 except for b. cereus and b. anthracis, the genus bacillus is rarely associated with disease.3,5,6,7 therefore, bacillus species are often reported as contaminants, even when cultured from sterile specimens. this may result in a delay in correct diagnosis and inappropriate treatment. bacillus cereus grows well on most routine culture media such as blood agar (figure 1), chocolate agar and macconkey agar. routine laboratory tests will reveal large boxcar-shaped, gram-positive bacilli (figure 2) that are catalase positive. however, identification to species level requires specialised techniques such as matrix-assisted laser desorption/ionisation mass spectrometry or molecular techniques.7 figure 2: microscopic image at 1000 × magnification showing boxcar-shaped, gram-positive bacilli, typical of bacillus cereus (pretoria, south africa, 19 march 2019). musculoskeletal infections caused by b. cereus are rare but have been previously reported in literature.4,5,6,7 åkesson, hedströum and ripa.4 reported 12 cases of b. cereus orthopaedic infections, all occurring in post-operative or post-traumatic wounds, whilst dubouix et al.5 reported 41 cases with b. cereus wound infections associated with open fractures. gallo et al.6 reported two cases of b. cereus prosthesis-related septic arthritis, which were culture negative but identified using pcr-mass-spectrometric-technology and fluorescence in situ hybridisation of tissue. ha et al.7 reported a single case of late prosthetic joint infection with b. cereus that occurred 13 years after total hip replacement surgery, confirmed by 16s ribosomal ribonucleic acid (rrna) sequencing. in total, we found 56 cases reported over 25 years, highlighting the rarity of b. cereus musculoskeletal infections and emphasising the difficulty in making a definitive diagnosis. the scarcity of these infections may actually be the result of under-reporting, as bacillus species are often reported as contaminants. bacillus cereus produces β-lactamases and is resistant to most β-lactam antibiotics, except carbapenems.3 classically, bacillus species are susceptible to vancomycin, aminoglycosides and fluoroquinolones, whilst resistance to erythromycin, tetracycline and even carbapenems have been reported.3 the empiric antibiotic of choice for invasive b. cereus infections is vancomycin.3,7 in addition to administering antibiotics, it is imperative to obtain source control at the infected site, as b. cereus is known to form biofilm. the removal of implanted medical devices may be necessary.7 the concomitant sle may be considered a notable risk factor for invasive b. cereus infection in this patient. it is uncertain if the epstein–barr virus viraemia played a significant role in predisposing the patient further to this infection. however, both sle and epstein–barr virus infection are known to be immune–modulatory, down-regulating the innate and humoral systems and placing the patient at increased risk of opportunistic infections.8,9 an additional risk factor was the preceding arthroscopy, which may have introduced spores into the joint space. to our knowledge, this is the first case of septic arthritis in an sle patient where the organism was cultured from the joint specimen. the communication between the clinical and microbiology teams ensured that the organism was identified to species level and that antibiotic susceptibility testing was performed, resulting in a favourable outcome for the patient. conclusion bacillus species are often regarded as contaminants and receive little attention from the medical community. in certain high-risk patient groups, however, b. cereus may be a formidable pathogen. clinical microbiology laboratorians and clinicians should have a high index of suspicion in these patients and identify bacillus species in cultures from sterile sites to species level as well as perform antibiotic susceptibility when b. cereus is identified. immune suppression, trauma, prosthetic implants and invasive procedures are important risk factors for b. cereus musculoskeletal infections. bacillus cereus is universally resistant to most β-lactam antibiotics, with the exception of carbapenems. treatment with vancomycin was successful in the case described. close collaboration with a multi-disciplinary team approach will effect the best outcome for complicated patients with b. cereus infections. acknowledgements competing interests the authors have declared that no competing interests exist. authors’ contributions b.m. designed the data collection tools, collected case data, analysed the data, and drafted and revised the paper. b.m. was also the guarantor. r.r. collected case data, and drafted and revised the paper. n.m. drafted and revised the paper. w.v.h-t. collected case data, analysed the data, and drafted and revised the paper. m.s. designed the data collection tools, analysed the data, and drafted and revised the paper. sources of support this research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references petri m, orbai a–m, alarcón gs, et al. derivation and validation of the systemic lupus international collaborating clinics classification criteria for systemic lupus erythematosus. arthritis rheum. 2012;64(8):2677–2686. https://doi.org/10.1002/art.34473 clinical and laboratory standards institute (clsi). methods for antimicrobial dilution and disk susceptibility testing of infrequently isolated or fastidious bacteria. 3rd ed. clsi guideline m45. wayne, pa: clinical and laboratory standards institute; 2015. bottone ej. bacillus cereus, a volatile human pathogen. clin mircobiol rev. 2010;23(2):382–398. https://doi.org/10.1128/cmr.00073-09 åkesson a, hedströum så, ripa t. bacillus cereus: a significant pathogen in postoperative and post-traumatic wounds on orthopaedic wards. scand j infect dis. 1991;23(1):71–77. https://doi.org/10.3109/00365549109023377 dubouix a, bonnet e, alvarez m, et al. bacillus cereus infections in traumatology – orthopaedics department: retrospective investigation and improvement of healthcare practices. j infect. 2005;50(1):22–30. https://doi.org/10.1016/j.jinf.2004.05.012 gallo ph, melton-kreft r, nistico l, et al. demonstration of bacillus cereus in orthopaedic-implant-related infection with use of a multi-primer polymerase chain reaction-mass spectrometric assay: report of two cases. j bone joint surg. 2011;93(15):e85. https://doi.org/10.2106/jbjs.j.01181 ha j, park yj, kim yj, oh hc. late prosthetic joint infection and bacteremia by bacillus cereus confirmed by 16s rrna sequencing and hip joint tissue pathology. ann clin microbiol. 2016;19(2):54–57. https://doi.org/10.5145/acm.2016.19.2.54 ning s. innate immune modulation in ebv infection. herpesviridae. 2011;2(1):1. https://doi.org/10.1186/2042-4280-2-1 tikly m, navarra sv. lupus in the developing world – is it any different? best pract res clin rheumatol. 2008;22(4):643–655. https://doi.org/10.1016/j.berh.2008.05.003 reviewer acknowledgement open accesshttp://www.ajlmonline.org page 1 of 1 we would like to take this opportunity to thank all reviewers for the african journal of laboratory medicine, volume 2: abiy b. ambaye ahmed mohamed andrew thaiyah atunga nyachieo barbara marston beldinah r. ochola burton w. wilcke charles g. massambu chin-yih ou christophe longuet david turgeon debola olayinka dennis ellenberger diane waku edward kamau elizabeth luman eric opiyo fengxiang gao fredrick n. nindo gajendran sivakumar georges dahourou heather alexander helen perry henry s. limula jack nyamongo jane mwangi jean l. sankale jemal ali jesse kwiek john n. nkengasong john sorkin kapila bhowan keith p. klugman larry westerman lawrence barker linda de gouveia linda oskam luc kestens michael aidoo miguel e. quiñones-mateu milijaona radrianarivelojosia musau wakabongo naomi maina olumide ogundahuns pascale ondoa patty wilkins paul klatser paula fernandes polyxeni potter sam kariuki samoel khamadi sanon souleymane seema meloni segundo r. leon sharon martin sheba gitta stephania k. deme tjeerd datema tom shinnick walter r. campos william lali zilma rey should names have inadvertently been excluded from this list the publisher apologises and undertakes to amend the exclusion in the next issue. ajlm african journal of laboratory medicine in an effort to facilitate the selection of appropriate peer reviewers for the african journal of laboratory medicine, we ask that you take a moment to update your electronic portfolio on www. ajlmonline.org for our files, allowing us better access to your areas of interest and expertise, in order to match reviewers with submitted manuscripts. if you would like to become a reviewer, please visit the journal website and register as a reviewer. to access your details on the website, you will need to follow these steps: 1. log into the online journal at http://www. ajlmonline.org 2. in your ‘user home’ [http://www.ajlmonline. org/index.php/ajlm/ user] select ‘edit my profile’ under the heading ‘my account’ and insert all relevant details, bio statement and reviewing interest. 3. it is good practice as reviewer to update your personal details regularly to ensure contact with you throughout your professional term as reviewer to the african journal of laboratory medicine. please do not hesitate to contact me if you require assistance in performing this task. chantal parkins submissions@ajlmonline. org tel: +27 (0)21 975 2602 fax: +27 (0)21 975 4635 91 abstract introduction methods results discussion acknowledgements references about the author(s) waganeh sinshaw tuberculosis research unit/national tuberculosis reference laboratory, ethiopian public health institute, addis ababa, ethiopia department of microbiology, immunology and parasitology, school of medicine, addis ababa university, addis ababa, ethiopia department of medical laboratory science, college of health sciences, addis ababa university, addis ababa, ethiopia abebaw kebede tuberculosis research unit/national tuberculosis reference laboratory, ethiopian public health institute, addis ababa, ethiopia department of microbial, cellular, and molecular biology, college of natural and computational sciences, addis ababa university, addis ababa, ethiopia adane bitew department of medical laboratory science, college of health sciences, addis ababa university, addis ababa, ethiopia mengistu tadesse tuberculosis research unit/national tuberculosis reference laboratory, ethiopian public health institute, addis ababa, ethiopia zemedu mehamed tuberculosis research unit/national tuberculosis reference laboratory, ethiopian public health institute, addis ababa, ethiopia ayinalem alemu tuberculosis research unit/national tuberculosis reference laboratory, ethiopian public health institute, addis ababa, ethiopia bazezew yenew tuberculosis research unit/national tuberculosis reference laboratory, ethiopian public health institute, addis ababa, ethiopia misikir amare tuberculosis research unit/national tuberculosis reference laboratory, ethiopian public health institute, addis ababa, ethiopia biniyam dagne tuberculosis research unit/national tuberculosis reference laboratory, ethiopian public health institute, addis ababa, ethiopia getu diriba tuberculosis research unit/national tuberculosis reference laboratory, ethiopian public health institute, addis ababa, ethiopia ephrem tesfaye tuberculosis research unit/national tuberculosis reference laboratory, ethiopian public health institute, addis ababa, ethiopia dinka f. gamtesa tuberculosis research unit/national tuberculosis reference laboratory, ethiopian public health institute, addis ababa, ethiopia yeshiwork abebaw tuberculosis research unit/national tuberculosis reference laboratory, ethiopian public health institute, addis ababa, ethiopia helina mollalign tuberculosis research unit/national tuberculosis reference laboratory, ethiopian public health institute, addis ababa, ethiopia getachew seid tuberculosis research unit/national tuberculosis reference laboratory, ethiopian public health institute, addis ababa, ethiopia muluwork getahun tuberculosis research unit/national tuberculosis reference laboratory, ethiopian public health institute, addis ababa, ethiopia citation sinshaw w, kebede a, bitew a, et al. effect of sputum quality and role of xpert® mtb/rif assay for detection of smear-negative pulmonary tuberculosis in same-day diagnosis strategy in addis ababa, ethiopia. afr j lab med. 2022;11(1), a1671. https://doi.org/10.4102/ajlm.v11i1.1671 note: additional supporting information may be found in the online version of this article as online supplementary 1. original research effect of sputum quality and role of xpert® mtb/rif assay for detection of smear-negative pulmonary tuberculosis in same-day diagnosis strategy in addis ababa, ethiopia waganeh sinshaw, abebaw kebede, adane bitew, mengistu tadesse, zemedu mehamed, ayinalem alemu, bazezew yenew, misikir amare, biniyam dagne, getu diriba, ephrem tesfaye, dinka f. gamtesa, yeshiwork abebaw, helina mollalign, getachew seid, muluwork getahun received: 08 july 2021; accepted: 24 may 2022; published: 31 aug. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: there is limited information on the performance of the xpert® mtb/rif test for diagnosis of smear-negative pulmonary tuberculosis (snpt) and rifampicin resistance (rr) in the same-day diagnosis approach. the effects of sputum quality and other factors affecting the xpert performance are also under-investigated. objective: this study aimed to determine the performance of the xpert® mtb/rif test for detection of snpt and rr in the same-day diagnosis strategy and the effect of sputum quality and other factors on its performance. methods: a cross-sectional study was conducted from august 2017 to january 2018 across 16 health facilities in addis ababa, ethiopia. two spot sputum samples were collected from 418 presumptive snpt patients, tested with xpert® mtb/rif, then compared to tuberculosis culture. additionally, culture isolates were tested for rr by bactec mgit™ 960 drug susceptibility testing (dst) and mtbdrplus version 2. results: the xpert® mtb/rif test detected 24 (5.7%) snpt cases, with a sensitivity of 92.3% (75.9% – 97.9%) and specificity of 99.2% (97.8% – 99.7%) compared with tuberculosis culture. xpert® mtb/rif also detected three (11.58%) rr strains with 100.0% concordance with bactec mgit™ 960 dst and mtbdrplus results. three blood-stained snpt samples were positive by xpert (30.0%), which was 6.9 times higher compared to salivary sputum (odds ratio: 6.9, 95% confidence interval: 1.36–34.96, p = 0.020). conclusion: the performance of the xpert® mtb/rif to detect snpt and rr in same-day diagnosis is high. however, snpt positivity varies among sputum qualities, and good sample collection is necessary for better test performance. keywords: smear-negative pulmonary tuberculosis; xpert® mtb/rif test; sputum quality; tuberculosis culture; diagnostic performance. introduction smear-negative pulmonary tuberculosis (snpt) occurs when a presumptive pulmonary tuberculosis (ptb) patient tests negative by acid-fast bacilli microscopy but tests positive by more accurate diagnostic techniques.1 it is one of the most problematic issues in tuberculosis diagnosis.2 patients with snpt contributed 17.3% – 41.0% of community tuberculosis transmission in vancouver, canada, from january 1995 to march 19993 and 13.0% in the netherlands from 1996 to 2004.4 also, 55.4% of belarus tuberculosis cases were smear-negative but culture-positive in 2012.5 in ethiopia, bacteriologically confirmed snpt prevalence was reported as high as 23.9% in 2007.6 although snpt is assumed to be less contagious and have lower mortality compared to smear-positive tuberculosis, 50.0% – 71.0% of snpt patients develop active tuberculosis disease.3,4 these snpt patients also harbour a high proportion of drug-resistant tuberculosis strains. the prevalence of smear-negative multidrug-resistant (mdr) tuberculosis among presumptive ptb patients enrolled with other patients was 47.0% in belarus in 20125 and 11.5% in ethiopia august 2017 to january 2018.7 mortality due to snpt is also substantial, especially among the immunocompromised. in mozambique, the mortality of hiv co-infected patients reached 55.8%.8 thus, early detection with sensitive diagnostic tools that simultaneously detect drug-resistant tuberculosis is critical. however, diagnosis of snpt and smear-negative drug-resistant tuberculosis is challenging in lowand middle-income countries (lmics) like ethiopia,9 mainly due to a lack of sensitive diagnostic tools. in most lmics, smear microscopy, which has low sensitivity, is still the first line of diagnosis. the xpert® mtb/rif test is a cartridge-based, fully automated dna testing platform that, in less than 2 h, simultaneously detects tuberculosis and mutations conferring rifampicin resistance (rr).10 the technology detects rr using five probes targeting mutations in the rpob region of the mycobacterium tuberculosis genome11,12: rpob mutations are responsible for rr in over 99.5% of rr strains.10 the xpert® mtb/rif test currently resolves the challenges of initial smear microscopy in tuberculosis case detection, which improves the clinical management of tuberculosis cases.13,14 in addition, the xpert® mtb/rif is advantageous over smear microscopy and m. tuberculosis culture by its higher sensitivity, simultaneous detection of rr, shorter turnaround time (tat) (2 h), and a minimal safety requirement.15 by comparison, culture-based rr confirmation takes more than two weeks to get results.16 nevertheless, various factors impact the xpert® mtb/rif’s performance, such as the type of specimen,17 sputum quality,18,19,20 sample collection and diagnosis strategy,21,22,23 and clinical characteristics of patients.17,24,25 since 2012, more than 314 genexpert® instruments have been installed in ethiopia. also, the diagnostic strategy changed from spot-morning-spot, which required three samples collected over two days, to same-day diagnosis (spot-spot diagnosis), which requires two samples collected on the same day.26 however, information is lacking on the performance of the xpert® mtb/rif test to diagnose snpt, smear-negative rr tuberculosis, the effects of sputum quality and other factors in the same-day tuberculosis diagnosis approach in addis ababa, ethiopia. therefore, this study aimed to determine the performance of the xpert® mtb/rif test to diagnose snpt and rr against conventional tuberculosis culture and drug susceptibility testing (dst) in the spot-spot diagnostic strategy. it also intended to determine the snpt positivity in sputum samples with varying quality and the effect of sputum quality and other factors on xpert® mtb/rif test performance. methods ethical considerations the study was reviewed and approved by the institutional review board of the ethiopian public health institute (ref. no. ephi/613/535) and the departmental research and ethics review committee of the department of medical laboratory science, addis ababa university (ref. no. mls/364/17). written informed consent from the participants and assent from the guardians of participants less than 18 years of age were obtained. permission was requested with a legal letter and received from participanting health facilities. unique patient identifiers were used instead of patient names to conceal patient identity. the laboratory testing results and other patient information were locked. laboratory results were reported to the clinicians who ordered the test for further patient management. study setting and design this cross-sectional study was conducted from august 2017 to january 2018 in addis ababa, ethiopia. addis ababa is the federal capital of ethiopia and has a population of more than three million.27 the study was conducted in 16 systematically selected governmental and private directly observed treatment short-course tuberculosis sites. these sites participate in blind rechecking, and most participate in the on-site supervision programme by the addis ababa city administration health research and laboratory service. data collection socio-demographic information, clinical presentations, comorbidities, and other factors were collected using a structured questionnaire by trained clinicians through interviews. the questionnaire was piloted following the on-site training of the data collectors. patient enrollment a total of 418 presumptive snpt patients who were negative for two consecutive spot sputum smears were enrolled. adults and children with presumptive ptb who visited the sites during the study period were eligible. however, those taking anti-tuberculosis drugs for more than one week or unable to submit two spot sputum samples were excluded. trained laboratorians gave patients detailed instructions on sputum collection. each presumptive snpt patient collected two sputum spot ≥ 3 ml samples into sterile 50 ml falcon tubes. sputum samples were transported with a triple packaging system to the national tuberculosis reference laboratory (ntrl) of the ethiopian public health institute (ephi) for laboratory investigation. sputum samples were stored at 2 °c – 8 °c until transported by triple packaging system for safety, with a thermometer inside for temperature monitoring. the transportation from study sites to ntrl takes less than one hour since it is within the city. samples were submitted to ntrl within a maximum of two days. macroscopic sputum quality was evaluated and categorised as blood-stained, purulent, mucoid, and salivary based on the global laboratory initiative (gli) guidance.28 laboratory investigations the two consecutive spot sputum samples collected from each patient were pooled into one and homogenised. afterwards, each sputum pool was split into two; one for xpert® mtb/rif test (cepheid, sunnyvale, california, united states) and the other for culture testing on bactec™ mgit™ 960 system (becton-dickinson and company, sparks, maryland, united states) and in laboratory-made lowenstein jensen (lj). drug susceptibility testing (phenotypic and genotypic) was performed in the ntrl of ephi, as explained in sinshaw et al., 2019.7 quality assurance and quality control the sterility and performance of lj media-manufactured in-house were verified before use for testing using controls by randomly selecting some prepared lj medium, putting it in the lj incubator and monitoring for any contaminant growth. if no growth was observed within 56 days, it was considered as sterile or safe for use. likewise, a new lot or batch of bactec™ mgit™ 960 media was also verified. each of the culture and identification procedures was performed in a certified class ii biosafety cabine. preventive maintenance of the equipment, temperature monitoring, and instrument operation checks were performed. in each batch of sample cultured, reagent sterility and process contamination check control were incorporated based on the ntrl standard procedures. a proficiency testing scheme continuously monitored all study test methods. also, the ntrl is international organization for standardization 15189 accredited by the ethiopian national accreditation office for xpert® mtb/rif. data entry and analysis double data entry was perfomed on epidata statistical software version 3.0 (epidata association, odense, denmark). the clean data were transferred to and analysed using ibm statistical package for social sciences software version 20.0 (chicago, illinois, united states). data entry and cleaning were performed by the ntrl data manager. the characteristics of the study participants were analysed using descriptive statistics. the sensitivity and specificity of the xpert® mtb/rif test were calculated at 95% confidence intervals (ci). pearson chi-square, fisher’s exact test, or binary logistic regression was used to determine the associations between dependent and independent variables; variables with a p ≤ 0.2 were selected for multivariable analysis. the strength of associations was measured by odds ratios, and a p < 0.05 was taken as statistically significant. results socio-demographic and clinical characteristics of study participants most of the participants (231; 55.3%) were female. the average participant age was 36 years (standard deviation [s.d.] ± 18). most of the participants (257, 61.8%) had completed some schooling (grades 1–12), while 112 (26.9%) were uneducated, and more than half of the participants (218; 52.3%) were married. cough was the leading symptom (413; 98.8%), followed by fever (255; 61.2%). of the 225 participants interviewed or laboratory tested, 57 (25.3%) were positive for hiv (table 1 and table 2). table 1: socio-demographic characteristics of the participants and their association with snpt detection by xpert mtb/rif test in addis ababa, ethiopia, from august 2017 to january 2018. table 2: clinical manufestations and behavioural characteristics of the presumptive snpt patients in addis ababa, ethiopia, from august 2017 to january 2018. sputum quality and performance characteristics of xpert® mtb/rif test salivary sputum was the leading sample quality (147, 35.2%), while blood-stained sputum was the least (10; 2.4%) sputum quality submitted (table 3). the majority of the sputum submitted (310; 74.3%) had 3 ml – 5 ml volume, while 16.8% had 6 ml – 7 ml and 8.9% had 8 ml – 9 ml volume. table 3: positivity of xpert mtb/rif assay in different sputum sample qualities in addis ababa, ethiopia, from august 2017 to january 2018. the majority of the positive xpert® mtb/rif tests, 14 (51.9%), were low and very low in bacillary load. there were 20 (4.8%) unsuccessful xpert mtb/rif results (sum of errors, invalids and no results). the most unsuccessful results were errors (15; 3.6%) (table 4). table 4: semi-quantitative bacilli dna quantification and unsuccessful xpert mtb/rif test results in addis ababa, ethiopia, from august 2017 to january 2018. the error code 5007 was the most common (table 5). mucoid sputum accounted for seven (46.7%) errors, while purulent sputum accounted for five (33.3%). the other three (20.0%) errors were from salivary sputum. table 5: xpert mtb/rif test post-run analysis error results and possible causes in addis ababa, ethiopia, from august 2017 to january 2018. the xpert® mtb/rif test detected three rr cases. two of the rr cases were caused by probe e (codons 529–533) missing mutations and one by probe b (codons 511–518) missing mutation. sputum m. tuberculosis positivity rate mycobacterium tuberculosis positivity rate was 6.1% for salivary sputum and 4.2% for mucoid sputum. however, the m. tuberculosis positivity rate was higher (30.0%) in blood-stained sputum. blood-stained sputum was 6.9 times more m. tuberculosis positive than salivary sputum (95% ci: 1.36–34.96; odds ratio [or]: 6.9; p = 0.02), while purulent sputum was the second most positive (7.7%) (table 3). detection of snpt and rr by xpert® mtb/rif test of the 418 smear-negative presumptive ptb patients enrolled in this study, 27 (6.5%; 95% ci: 4.10–8.81) were m. tuberculosis positive by xpert® mtb/rif. on the other hand, 26 (6.4%; 95% ci: 4.00–8.74) were culture-positive. twenty-four (5.7%; 95% ci: 3.51% – 7.97%) sputum samples were positive by both xpert® mtb/rif and tuberculosis culture (lj and mgit) (figure 1). figure 1: xpert® mtb/rif assay performance reference to the mgit and lj mycobacterium tuberculosis culture in addis ababa, ethiopia, from august 2017 to january 2018. five samples were discordantly positive by both methods; one sample was lj culture-positive but mgit culture-negative. on the other hand, four samples were mgit culture-positive, two of which were lj culture-negative, while the other two were lj culture contaminated. besides, of the two xpert® mtb/rif negative culture-positive samples, one was mgit negative and the other mgit positive, but both were positive by lj solid culture. moreover, three sputum samples were xpert® mtb/rif positive but culture-negative. thus, the diagnostic sensitivity and specificity of the xpert® mtb/rif test relative to tuberculosis culture was 92.3% (75.9% – 97.9%) and 99.2% (97.8% – 99.7%). the overall diagnostic accuracy was 98.8% (97.2% – 99.5%) (figure 1). three (11.54%) rr snpt cases were detected by the xpert® mtb/rif test; two of the rr detected were from a new case and one from previously treated cases. these strains were later confirmed as mdr by genotype mtbdrplus version 2 and bactec™ mgit™ 960 dst. most of the snpt cases were detected from men (95% ci: 1.29–11.44; or: 3.85; p = 0.015]) (table 1). patients with weight loss had 4.05 times more risk of being diagnosed with snpt, in 95% ci (p = 0.007) compared to those without weight loss (supplementary table 1). discussion smear microscopy is still a front-line tuberculosis diagnostic tool in developing countries like ethiopia.1,21,29 however, because of its low sensitivity, smear microscopy misses many tuberculosis cases, resulting in delayed diagnosis and treatment initiation.15,30,31 therefore, this study evaluated the effect of sputum quality and xpert® mtb/rif performance for snpt detection in same-day diagnosis. sputum quality has effects on the detection of ptb.19,20,32,33 however, many studies do not consider attributes of sputum quality in mtb testing by the xpert® mtb/rif test.34,35 in the present study, the m. tuberculosis positivity by xpert® mtb/rif significantly varied across sputum qualities. the majority of sputum, 147 (35.2%), submitted was salivary with a positivity rate of 6.1%. although the blood-stained sample was the least submitted, the positivity was very high at 30.0%. in 95% ci, blood-stained samples showed 6.9 (p = 0.020) times more positive than salivary. many laboratories reject blood-stained sputum, assuming that it brings unreliable xpert results due to polymerase chain reaction inhibition; however, this study revealed the highest snpt positivity in blood-stained sputum. contrary to the present study, a study showed that the xpert result is only valid at less than 2.0% blood contamination of the sputa. if sputum contamination with blood is beyond 5.0%, the result will be unreliable and absolute inhibition occurs at 20.0% blood contamination.18 however, studies found that patients who are mtb positive in a blood sample have a higher risk of death.36 therefore, we are missing the most important sputum quality. the current finding is different from a study in uganda, where snpt was more in salivary sputum and lowest in blood-stained sputum.20 this might be as a result of the fact that the majority of the ugandan study participants were hiv positive and most of them had a very low cd4 count (≤ 200 cells/µl). the second highest snpt positivity, 7.7%, was diagnosed from purulent sputum. although the difference is not statistically significant, blood-stained sputum showed greater positivity than purulent sputum, whereas purulent sputum is considered the best sputum for mtb detection. the difference in macroscopic sputum appearance significantly varied for mtb positivity by xpert® mtb/rif test, implying a need for proper sputum collection, similar to other reports.20,33,34 in ethiopia, the sputum sample collection strategy for mtb diagnosis was changed from spot-morning-spot to spot-spot (same-day diagnosis) in 2017.26 same-day diagnosis stops multiple visits to the health facilities by the patient to submit sputum and receive a result. however, it is 2.8% less sensitive with a lower dropout rate than the conventional -spot-morning-spot strategy.21 in the spot-morning-spot strategy, three smear slides are made. the first slide made from the first spot sputum, the second slide made from the morning sputum and the third slide from the second spot sputum.22 this change increases the possibilities of being smear-negative. in the spot-spot approach, in the current study, xpert® mtb/rif assay detected an extra 24 (5.7%) snpt and three rr strains in comparison with smear microscopy. the ability of the xpert® mtb/rif test to detect snpt and rr is high in this study which might be because the missed morning sputum increased the snpt cases. the xpert® mtb/rif testing was performed on the direct sputum before culture to evaluate the performance of the xpert® mtb/rif in the peripheral health facilities where direct sputum only is used for xpert® mtb/rif testing. sputum processing, including decontamination, neutralisation, and pellet concentration before culture, is only possible in the tuberculosis culture reference laboratories. the diagnostic sensitivity of the xpert® mtb/rif test in this study was 92.3%, while specificity was 99.2% reference to tuberculosis culture. the present study revealed higher sensitivity and specificity than the uganda report20 because most of uganda’s study participants were hiv-positive and used a spot-morning-spot diagnostic approach. another justification might be tuberculosis prevalence is higher in ethiopia. similarly, the present study revealed a very high sensitivity compared to a study in jigjiga, ethiopia: 48.5% for smear-negative ptb.37 likewise, the current study showed higher sensitivity and specificity relative to a review conducted in liverpool, united kingdom, in 2013, which was 67.0% – 74.0% pooled sensitivity and 99.0% pooled specificity.38 however, the sensitivity of the xpert® to diagnose snpt showed considerable variability in different studies.39,40,41 the possible reasons for the variabilities in sensitivity and specificity might be differences in study design, study population, sample collection strategy, study period, study area or location, tuberculosis prevalence, comorbidity and laboratory performance. the three (11.54%) rr strains detected by the xpert® mtb/rif test in this study concords 100.0% with genotype mtbdr plus version 2 and phenotypic bactec™ mgit™ 960 dst results. in addition, studies reported high xpert® mtb/rif detection performance: 95.0% – 97.0% sensitivity and 98.0% – 99.0% specificity, for culture positives.38 the findings in this study are commendable since they facilitate early tuberculosis diagnosis and universal access to dst,42 which are pivotal to the endtb strategy. furthermore, the world health organization approved that tuberculosis diagnostic technologies such as xpert® mtb/rif need to be scaled up to become the first line of diagnosis. with the scale-up, patients will benefit from early diagnosis and initiation of treatment. more than half of the positive xpert® mtb/rif results (14; 51.9%) had a low or very low bacillary load, implying that snpt patients have a paucibacillary load. twenty (4.8%) xpert® mtb/rif results were unsuccessful (the sum of errors, invalids and no results), which is lower than a report from nigeria.43 the nigeria study did not include only presumptive smear-negative snpt cases. most of our unsuccessful results were due to error results (15; 3.6%), but in the nigerian study, these were considered invalid results.43 the error rate in the present study was higher than the gli recommendation (< 3.0%),44 but lower than a report from addis ababa, which reported 8.9%.45 the discrepancy in the study reports might result from the difference in the testers’ expertise and experience, study population, sample type, and study period. the error code 5007 was the most common (12; 2.9%), often caused by viscous sputum or wrong sample volume, improper filling of cartridge reaction tube, bubbles, or probe integrity issues. errors are a loss of valuable time and money for the patients and the laboratory. thus, patient training on quality sputum collection and laboratory staff refresher training may minimise these errors. of the three rr cases detected by the xpert® mtb/rif test, two were due to probe e (codons 529–533) missing, which is the most frequent type of mutation in the rpob region of the mycobacteria.46,47 the other was probe b missing (codons 511–518; mutation). limitations the current study did not explain the performance of the xpert® mtb/rif test in the same-day diagnosis to diagnose smear-negative rr as it did not include enough rr cases, which is a limitation of the study. conclusion the performance of the xpert® mtb/rif test to detect snpt in spot-spot samples and rapid detection of rr were high. however, the diagnostic performance of the test significantly varied across different sputum qualities. thus, good patient instruction or training and close follow-up on sputum sample collection are essential for getting quality sputum for better xpert® mtb/rif yield. we recommend testing sputum samples by xpert® mtb/rif irrespective of sputum quality. acknowledgements we acknowledge the ethiopian public health institute for their material and technical support of the study. we are also thankful for the study site staff and study participants. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions w.s. designed and conceptualised the study, performed the data analysis and interpretation of the results, and drafted the manuscript. a.k., a.a. and a.b. reviewed and edited the manuscript. a.k. was also involved in the study’s conceptualisation. z.m. performed the research methods, data analysis, and interpretation of results. e.t., m.t., b.y., m.a., b.d., g.d., a.a., h.m., y.a., g.s., m.g. and w.s. performed the laboratory analysis and reporting, site supervision, data collection, and data entry. d.f.g was responsible for data entry, data curation and analysis. sources of support this research received no specific grant from any funding agency in the 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assay in ethiopia. plos one. 2019;14(11):e0225205. https://doi.org/10.1371/journal.pone.0225205 alemu a, tadesse m, seid g, et al. does xpert® mtb/rif assay give rifampicin resistance results without identified mutation? review of cases from addis ababa, ethiopia. bmc infect dis. 2020;20(1):1–6. https://doi.org/10.1186/s12879-020-4817-2 ochang ea, udoh ua, emanghe ue, et al. evaluation of rifampicin resistance and 81-bp rifampicin resistant determinant region of rpob gene mutations of mycobacterium tuberculosis detected with xpert mtb/rif in cross river state, nigeria. int j mycobacteriol. 2016;5(2016):s145–s146. https://doi.org/10.1016/j.ijmyco.2016.09.007 abstract introduction methods results discussion acknowledgements references about the author(s) chido w. bvumbi department of laboratory diagnostic and investigative sciences, faculty of medicine and health sciences, university of zimbabwe, harare, zimbabwe vinie kouamou department of primary care and health sciences, internal medicine unit, faculty of medicine and health sciences, university of zimbabwe, harare, zimbabwe biomedical research and training institute, harare, zimbabwe ngalulawa kone department of chemical pathology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa department of chemical pathology, national health laboratory service, johannesburg, south africa trust zaranyika department of primary care and health sciences, internal medicine unit, faculty of medicine and health sciences, university of zimbabwe, harare, zimbabwe lloyd bowora department of laboratory diagnostic and investigative sciences, faculty of medicine and health sciences, university of zimbabwe, harare, zimbabwe hilda t. matarira department of laboratory diagnostic and investigative sciences, faculty of medicine and health sciences, university of zimbabwe, harare, zimbabwe raylton p. chikwati department of chemical pathology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa sydney brenner institute for molecular bioscience, faculty of health sciences, university of the witwatersrand, johannesburg, south africa citation bvumbi cw, kouamou v, kone n, et al. diagnostic cut-off value of haemoglobin a1c for diabetes mellitus in harare, zimbabwe. afr j lab med. 2024;13(1), a2373. https://doi.org/10.4102/ajlm.v13i1.2373 note: additional supporting information may be found in the online version of this article as online supplementary document 1. brief report diagnostic cut-off value of haemoglobin a1c for diabetes mellitus in harare, zimbabwe chido w. bvumbi, vinie kouamou, ngalulawa kone, trust zaranyika, lloyd bowora, hilda t. matarira, raylton p. chikwati received: 15 nov. 2023; accepted: 20 feb. 2024; published: 23 apr. 2024 copyright: © 2024. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract very little is known about the diagnostic performance of the american diabetes association glycated haemoglobin (hba1c) cut-off of 6.5% in resource-limited settings. this study, conducted between february 2023 and may 2023, aimed to determine the optimal hba1c cut-off for the diagnosis of diabetes mellitus by measuring hba1c and fasting plasma glucose levels in 120 adults attending care at a tertiary hospital in harare, zimbabwe. the optimal hba1c cut-off was 6.1% and glucose levels were strongly correlated with hba1c values. the prevalence of diabetes mellitus was higher (28.3%) at our derived hba1c cut-off than with the american diabetes association criterion (21.6%). what this study adds: this study highlights the need for population-specific cut-off hba1c values in the diagnosis of diabetes mellitus. keywords: glycated haemoglobin; diabetes mellitus; fasting plasma glucose; receiver operating characteristics curves; zimbabwe. introduction the prevalence of diabetes mellitus (dm) in zimbabwe has increased from 0.4% (95% confidence interval [ci]: 0.0–1.9) before 1980, to 5.7% (95% ci: 3.3–8.6) after 1980.1 while the reasons behind these changes are multifactoral, a key factor could be the transition in the diagnostic criteria of dm from urine to blood testing. from the eight studies evaluated, three used urine for glucose testing and were all conducted before 1980.1 when comparing the prevalence between these studies, it is apparent that the transition from urine to blood testing had an impact on the observed increase. it is therefore important to evaluate the diagnostic criteria in this population to improve diabetic care. currently, a glycated haemoglobin (hba1c) of 6.5% is used for diagnosing dm, as guided by the american diabetes association (ada).2 however, the diagnostic cut-off value of hba1c has been shown to vary across different races and ethnicities.3 studies have shown that hba1c levels are higher in african americans, african brazilians, british african caribbeans, asians and latinos than in populations of european ancestry.4,5 given this evidence, a universal hba1c cut-off may underdiagnose dm in some populations. we therefore aimed to determine an optimal hba1c cut-off value for dm in a black zimbabwean population of african ancestry. methods ethical considerations this study was approved by the joint research ethics committee of the university of zimbabwe (jrec 48/2023) and the parirenyatwa group of hospital’s clinical director. informed written consent was obtained from all participants. to protect privacy and confidentiality, all collected blood specimens were de-identified to ensure that no individual could be linked to the study information. all data were captured on a password-protected excel spreadsheet (microsoft corp., redmond, washington, united states) and printed results were stored in a cabinet only accessible to the study investigators. study design in this cross-sectional study, we recruited adults (≥ 18 years) from harare, zimbabwe, with a body mass index ≥ 25 kg/m2. recruitment of study participants was done from 15 february 2023 to 14 may 2023 at the diabetic clinic, parirenyatwa group of hospitals, a tertiary hospital. a minimum sample size of 119 participants was calculated using the dobson formula. participants who self-reported pregnancy or had anaemia, renal disorders or haemoglobinopathies based on a previous diagnosis by a healthcare practitioner were excluded. specimen processing blood specimens were collected by trained nurses and only used for this study. from each participant, approximately 8 ml of blood were collected after an 8 h overnight fast. participants were advised to have their dinner before midnight and not ingest any food or drink, except for water, until the next day when blood was drawn. blood specimens were collected in sodium fluorideand ethylenediamine tetraacetic acid-containing vacutainer tubes (becton, dickinson and company, plymouth, united kingdom). all blood specimens were transported from the clinic to the interpath medical laboratories, where they were processed, analysed and stored. at the laboratory, blood specimens in sodium fluoride tubes were immediately centrifuged at 3500 revolutions per minute for 5 min (yingtai instrument, changsha city, china). testing of fasting plasma glucose (fpg) was done within 2 h from blood collection. ethylenediamine tetraacetic acid blood specimens were stored at 2 °c – 8 °c before transportation on dry ice to the national health laboratory service in johannesburg, south africa, for hba1c measurement. anthropometric variables body weight was measured using a 120 kg scale (mcp camry, medicare products inc., new delhi, india). participants were asked to wear light clothing before standing on the scale. standing height was measured using a wooden board which had a sliding headpiece (united nations children’s fund, new york, new york, united states). participants were asked to maintain a straight posture against the wall before a reading was taken. body mass index was calculated as a ratio of the measured weight in kg and height in m2. fasting plasma glucose fasting plasma glucose levels were measured using the hexokinase methodology on a mindray analyser (mindray bs200, shenzhen, china). mindray reagents, calibrators, and quality control materials were used. diabetes mellitus was defined as a fpg ≥ 7.0 mmol/l.2 glycated haemoglobin hba1c levels were measured in ethylenediamine tetraacetic acid whole blood using a bio-rad high-performance liquid chromatography analyser, reagents, calibrators, and quality control materials (bio-rad variant ii, montreal, quebec, canada). statistical analyses all statistical analyses were performed using stata 16.1 (statacorp lp, college station, texas, united states). continuous variables were expressed as mean ± standard deviation if data followed a gaussian distribution, or median and interquartile range (iqr) if data were skewed. continuous variables were compared using the student’s t-test and mann-whitney u test, as appropriate. pearson correlation was used to assess the relationship between fpg and hba1c levels. statistical significance was set at p < 0.05. receiver operator characteristic curves were plotted to identify the diagnostic cut-off for hba1c using the highest youden’s j index.6 sensitivity and specificity were calculated for both the derived and ada hba1c cut-off values. results characteristics of study population we recruited a total of 120 participants, comprising 62 women and 58 men, from a pool of 138 eligible participants. non-participation was primarily due to the inability to commute to the study clinic and failure to provide a fasting blood specimen. their mean (± standard deviation) age was 43.2 (±14.0) years. median (iqr) body mass index was 29.0 (26.5–31.3) kg/m2. body mass index was higher in women than men. no other differences were observed (table 1). table 1: characteristics of study participants, harare, zimbabwe, february 2023 to may 2023. fasting plasma glucose and hba1c the median (iqr) fpg levels were 5.21 (4.35 – 7.00) mmol/l and hba1c, 5.55% (5.05% – 6.30%) (table 1). when the ada criterion (fpg ≥ 7.0 mmol/l) was applied, 31 (26.0%) participants had dm. their median (iqr) fpg levels were 7.50 (7.23 – 8.12) mmol/l and hba1c, 6.80% (6.35% – 8.40%) (data not shown). furthermore, there was a linear relationship between fpg and hba1c levels (r = 0.84, p < 0.001) (figure 1). figure 1: correlation between hba1c and fpg, harare, zimbabwe, february 2023 to may 2023. for all participants, the optimal hba1c cut-off for dm was 6.10% (figure 2). when stratified according to sex, the optimal hba1c cut-off was unchanged in men, at 6.10%, but lower in women, at 5.95% (figure 3). figure 2: receiver operating characteristic curve for the determination of the optimal cut-off value of hba1c for the diagnosis of diabetes mellitus in all study participants, harare, zimbabwe, february 2023 to may 2023. figure 3: receiver operating characteristic curves for optimal hba1c cut-off values for the diagnosis of diabetes mellitus in men and women, harare, zimbabwe, february 2023 – may 2023. (a) the optimal cut-off value for men was 6.1%. sensitivity = 84.6%, specificity = 89.5%. the area under the curve was 0.89, 95% confidence interval (ci): 0.783–0.994. (b) the optimal cut-off value for women was 5.95%. sensitivity = 94.4%, specificity = 94%. the area under the curve was 0.96, 95% ci: 0.810–1.000. diagnostic performance the derived hba1c cut-off of 6.1% had a higher sensitivity (87.1%) than the ada criterion (71.0%) (online supplementary table 1). however, specificity was slightly higher in the ada criterion (95.5% vs 93.2%). the prevalence of dm was higher (28.3%) when the 6.1% cut-off was applied, but lower with the ada criterion (21.6%) (online supplementary table 1). discussion our results showed that a 6.1% hba1c cut-off had high sensitivity and specificity in black zimbabwean participants with body mass index ≥ 25 kg/m2. when we applied the ada criterion, the sensitivity decreased, and we identified 6.7% fewer diabetics. in support of our findings, a meta-analysis from 2022 including seven african studies showed that the sensitivity for hba1c in the diagnosis of dm was highest (74.9%) at 6.0%.7 two global meta-analyses showed that the 6.5% cut-off was associated with lower sensitivity (59.4% and 52.7%).8,9 other studies from south africa (6.1%),10 india (6.1%),11 and haiti (6.3%)12 have also shown lower hba1c cut-offs than the ada criterion. our findings and others’ indicate that the optimal hb1ac cut-off differs from the ada criterion. some factors, such as hb1ac analytical methods and haemoglobin structure, may contribute to these differences.13 furthermore, different inclusion criteria may contribute to heterogeneity between studies.14,15 we also observed a positive linear relationship between fpg and hba1c levels. these findings are consistent with a meta-analysis conducted in 2015 which reported a similar trend (r = 0.74, p < 0.001).16 the slightly lower hba1c cut-off found in our study, of 5.95% in women compared to 6.10% in men, has not been reported previously. however, studies have shown that black women of african ancestry have lower hepatic clearance of insulin, differential ß-cell responsiveness and therefore higher mean blood glucose levels than white women of european ancestry.17,18 however, there is scarcity of data comparing black women and black men. strengths of our study include using the high-performance liquid chromatography method and recruiting from one urban population, where lifestyle and environmental factors were likely to be homogenous. in addition, all specimens were collected within the same weather season (autumn), thus the seasonal influence on hba1c was minimal. limitations limitations of our study include its small sample size, and sampling from a tertiary clinic; therefore, findings may not be generalisable. additionally, due to limited resources, we did not have specific symptomology, such as diabetic retinopathy, to confirm dm. we only collected data on body mass index, but not other risk factors that include family history, dietary intake, alcohol consumption, and hypertension. these could have been valuable in assessing the impact of the diagnostic performance of hba1c against fpg. another limitation was that we relied on ordered fasting which could not be standardised since participants would present at the study clinic after an overnight fast. it is possible that not all participants were compliant to the instructions given, as some could have misinterpreted that fasting only applies to meals, not beverages and sweets before the test. furthermore, we did not perform the oral glucose tolerance test, which is known for its higher sensitivity and specificity in the diagnosis of dm. conclusion this study demonstrates a higher diagnostic performance of an hba1c cut-off value of 6.1% in a black population from harare, zimbabwe, and that the ada cut-off was not optimal for this population. more studies are warranted to confirm these findings in a larger population with better representation of geographical location and more standardised methods of collecting fasting specimens. acknowledgements we would like to acknowledge the study participants and nurses at parirenyatwa group of hospitals. we also thank the staff and management at the interpath medical laboratories and national health laboratory service where the biochemical testing was performed. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions c.w.b., t.z., l.b. and h.t.m. conceptualised the study. c.w.b. and r.p.c. performed the experimental procedures. n.k. sourced hba1c reagents. c.w.b., l.b. and r.p.c. performed the data analysis. c.w.b., r.p.c. and v.k. drafted the first version of the manuscript. r.p.c., v.k., l.b., h.t.m., n.k. and t.z. reviewed intellectual content. all authors approved the final version of the manuscript for submission. sources of support this study did not receive a grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the authors confirm that de-identified raw data supporting the findings of this study are available from the corresponding author, r.p.c., on request. disclaimer the views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references mutowo m, gowda u, chamunorwa j, lorgelly p, owen a, renzaho a. prevalence of diabetes in zimbabwe: a systematic review with meta-analysis. int j public health. 2015;60(1):1–11. https://doi.org/10.1007/s00038-014-0626-y american diabetes association. diagnosis and classification of diabetes mellitus. diabet care. 2014;37(suppl.1):81–90. https://doi.org/10.2337/dc14-s081 ford cn, leet rw, kipling lm, et al. racial differences in performance of hba1c for the classification of diabetes and prediabetes among us adults of non-hispanic black and white race. diabet med. 2020;36(10):1234–1242. https://doi.org/10.1111/dme.13979 khosla l, bhat s, fullington la hrm. hba1c performance in 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diagnosis: a pooled analysis of 96 population-based studies with 331,288 participants. lancet diabet endocrinol. 2015;3(8):624–637. https://doi.org/10.1016/s2213-8587(15)00129-1 kaur g, lakshmi pv, rastogi a, et al. diagnostic accuracy of tests for type 2 diabetes and prediabetes: a systematic review and meta-analysis. plos one. 2020;15(11):e0242415. https://doi.org/10.1371/journal.pone.0242415 zemlin ae, matsha te, hassan ms, erasmus rt. hba1c of 6.5% to diagnose diabetes mellitus – does it work for us? – the bellville south africa study. plos one. 2011;6(8):e22558. https://doi.org/10.1371/journal.pone.0022558 kumar pr, bhansali a, ravikiran m, et al. utility of glycated hemoglobin in diagnosing type 2 diabetes mellitus: a community-based study. j clin endocrinol metab. 2010;95(6):2832–2835. https://doi.org/10.1210/jc.2009-2433 exebio jc, zarini gg, vaccaro j, exebio c. use of hemoglobin a1c to detect haitianamericans with undiagnosed type 2 diabetes. arq bras endocrinol metabol. 2012;56(7):449–455. https://doi.org/10.1590/s0004-27302012000700007 chen z, shao l, jiang m, ba x, ma b, zhou t. interpretation of hba1c lies at the intersection of analytical methodology, clinical biochemistry and hematology (review). exp ther med. 2022;24(6):707. https://doi.org/10.3892/etm.2022.11643 holman r. a brief history of the uk prospective diabetes study: rury holman, for the ukpds group. bjd. 2022;22(suppl 1):s32–35. https://doi.org/10.15277/bjd.2022.359 the dcct research group. diabetes control and complications trial (dcct): results of feasibility study. diabet care. 1987;10(1):1–19. https://doi.org/10.2337/diacare.10.1.1 ketema eb, kibret kt. correlation of fasting and postprandial plasma glucose with hba1c in assessing glycemic control; systematic review and meta-analysis. archiv public health. 2015;73:43. https://doi.org/10.1186/s13690-015-0088-6 chung st, de la cruz mg, aldana pc, et al. postprandial insulin response and clearance among black and white women: the federal women’s study. j clin endocrinol metab. 2019;104(1):181–192. https://doi.org/10.1210/jc.2018-01032 cree-green m. postglucose hyperinsulinemia in black women is not what we thought. j clin endocrinol metab. 2019;104(2):266–268. https://doi.org/10.1210/jc.2018-02213 abstract introduction methods results discussion acknowledgements references about the author(s) mary e. schmitz united states centers for disease control and prevention, dar es salaam, united republic of tanzania asph/cdc allan rosenfield global health, dar es salaam, united republic of tanzania karen chang united states centers for disease control and prevention, dar es salaam, united republic of tanzania asph/cdc allan rosenfield global health, dar es salaam, united republic of tanzania nichole arnett division of global hiv and tb, united states centers for disease control and prevention, atlanta, georgia, united states luciana kohatsu division of global hiv and tb, united states centers for disease control and prevention, atlanta, georgia, united states ruth lemwayi african field epidemiology network (afenet), dar es salaam, united republic of tanzania michael mwasekaga united states centers for disease control and prevention, dar es salaam, united republic of tanzania john nkengasong division of global hiv and tb, united states centers for disease control and prevention, atlanta, georgia, united states omotayo bolu division of global hiv and tb, united states centers for disease control and prevention, atlanta, georgia, united states fausta mosha african field epidemiology network (afenet), dar es salaam, united republic of tanzania tanzania ministry of health, community development, gender, elderly and children, dar es salaam, united republic of tanzania larry westerman division of global hiv and tb, united states centers for disease control and prevention, atlanta, georgia, united states citation schmitz me, chang k, arnett n, et al. onsite healthcare worker acceptability and performance of the point-of-care pima cd4 assay in dar es salaam, tanzania. afr j lab med. 2019;8(1), a740. https://doi.org/10.4102/ajlm.v8i1.740 original research onsite healthcare worker acceptability and performance of the point-of-care pima cd4 assay in dar es salaam, tanzania mary e. schmitz, karen chang, nichole arnett, luciana kohatsu, ruth lemwayi, michael mwasekaga, john nkengasong, omotayo bolu, fausta mosha, larry westerman received: 21 dec. 2017; accepted: 21 mar. 2019; published: 21 nov. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: healthcare workers’ acceptance of and ability to perform point-of-care testing is important for reliable and accurate results. the alere pima™ cd4 assay (pima cd4) is the cd4 point-of-care test for hiv management in tanzania. objectives: to evaluate healthcare workers’ acceptance and performance of pima cd4 testing. methods: the study was implemented in five high volume sites in dar es salaam, tanzania, in 2011. trained healthcare workers performed pima testing using three whole-blood specimens collected from each patient: venous blood, fingerstick blood directly applied to a pima cartridge (capillary-direct), and fingerstick blood collected in a microtube (capillary-microtube). using a semi-structured interview guide, we interviewed 11 healthcare workers about specimen collection methods and pima cd4 acceptability. quantitative responses were analysed using descriptive statistics. open-ended responses were summarised by thematic areas. pima cd4 results were analysed to determine variation between cadres. results: healthcare workers found pima cd4 user-friendly and recommended its use in low volume, peripheral facilities. both venous and capillary-direct blood were considered easy to collect, with venous preferred. advantages noted with venous and capillary-microtube methods were the ability to retest, perform multiple tests, or delay testing. pima cd4 results were trusted by the healthcare workers and were in agreement with laboratory pima testing. conclusion: in this point-of-care testing setting, the pima cd4 assay was accepted by healthcare workers. both venous and fingerstick capillary blood specimens can be used with pima cd4, but fingerstick methods may require more intensive training on technique to minimise variation in results and increase acceptability. keywords: point-of-care; cd4; hiv; user acceptability; microtube. introduction testing for cd4 count was previously a widely used method for determining the timing of antiretroviral therapy (art) initiation among hiv-positive individuals.1,2,3 in 2016, the world health organization recommended art initiation for all hiv-positive adults and children, regardless of cd4 count. however, cd4 testing for baseline and ongoing monitoring of clients on art where resources allow or if viral load testing is not available is recommended.4 in many resource-limited settings, the availability of cd4 testing is challenging due to poor laboratory infrastructure, human resource limitations, instrument maintenance issues, supply chain failures, poor result reporting systems, and insufficient means to ensure quality of testing.5,6,7,8 these challenges, coupled with inefficient specimen referral systems, previously resulted in delayed treatment decisions at clinics.9,10,11 high attrition rates between hiv diagnosis and art initiation have been observed with laboratory-based cd4 testing.9,10,11,12 consequently, patients may have progressed to severe or advanced clinical stages of hiv before seeking treatment, dramatically decreasing survival outcomes.9,10 while the current tanzanian guidelines have removed the need for cd4 testing to initiate art, they still recommend baseline cd4 testing to assess the need for cotrimoxazole preventive therapy and monitoring for any patient with a cd4 level below 350 cells/μl or where viral load testing is not available.13 thus, the testing barriers described will still limit the use of cd4 as a monitoring test, as well as influence wider laboratory monitoring, most importantly hiv viral load. in addition, the shortage of trained medical personnel remains a barrier in many resource-limited settings attempting to scale up art programmes.9,12,14 task-shifting from trained laboratorians to other cadres of healthcare workers is a potential solution to the limited access to cd4 testing and other laboratory monitoring for hiv-positive individuals living in remote and isolated areas.12,14 the 2016 world health organization guidelines also include a recommendation for task-shifting for trained and supervised non-laboratory staff to conduct blood fingersticks for sample collection.15 these factors and recommendations for task-shifting underscore the importance of timely laboratory monitoring and user-friendly, point-of-care (poc) testing technologies to allow for decentralised care. one such technology is the alere pima™ cd4 assay (pima cd4) (alere inc., waltham, maryland, united states). point-of-care cd4 assays potentially decrease the turnaround time for the reporting of cd4 results, improving linkage to care and, when cd4 is a criteria, increasing timeliness in art initiation and treatment switches.10,11,12,14,16,17,18,19 though findings have shown that healthcare workers are able to perform poc testing for patient care,20 there is little documentation about the acceptability of the technology among healthcare workers. this study assessed healthcare workers’ acceptance, ease-of-use, and specimen collection preferences for the pima cd4 assay in the clinical setting in tanzania. we compared the pima testers, healthcare workers to laboratorians, to determine agreement with or differences between pima cd4 results from these cadres. this evaluation was part of a clinical validation of specimen types for the pima cd4 assay compared to the facscalibur platform; the primary study findings have been reported separately.21 methods ethical considerations informed consent was obtained from all participants (patients and healthcare workers) in accordance with the united states department of health and human services’ 45 cfr 46 protection of human subjects regulation. ethics approval was obtained from the tanzania national institute for medical research, the tanzanian ministry of health and social welfare and the united states centers for disease control and prevention, associate director of science (protocol number u26gps002728). study design five healthcare sites in dar es salaam, tanzania, providing hiv care and treatment and prevention of mother-to-child transmission services, were purposefully selected for this study. site selection criteria were high patient volume and proximity to the reference laboratory, national health laboratory quality assurance training center (nhlqatc), in dar es salaam, tanzania. male and female hiv-positive patients aged between 8 and 65 years attending the selected clinics and requiring a routine cd4 test were eligible for the primary clinical validation study.21 the study was implemented over a five-week period in 2011. healthcare workers at the study sites collected specimens via three different methods: fingerstick capillary blood directly applied to a pima cd4 cartridge (capillary-direct), fingerstick capillary blood collected into an ethylenediaminetetraacetic acid (edta) microtube (capillary-microtube), and venipuncture blood collected into an edta tube (venous). a study trainer certified in phlebotomy trained healthcare workers on fingerstick collection followed by competency testing for all healthcare workers. peripheral venous whole blood was collected via venipuncture into edta vacuum tubes (becton, dickinson and company [bd] vacutainer, bd diagnostics, san jose, california, united states). pima cd4 testing was performed at all study sites by healthcare workers using all three specimen collection methods on the day of specimen collection. after pima cd4 testing at the healthcare site, the edta venous whole-blood specimens were transported to the nhlqatc, and pima cd4 and standard-of-care testing was performed on facscalibur (becton dickinson, san jose, california, united states) equipment on either the day of specimen collection or the following day by laboratory technicians. the healthcare workers and nhlqatc laboratory technicians conducting pima cd4 testing were trained on the pima cd4 assay and the use and interpretation of quality control materials. patient participants in the study only received the facscalibur cd4 results. patient participants did not receive the pima cd4 results because the assay was not approved for use in tanzania at the time of the study. healthcare worker survey all healthcare workers involved in blood collection and performing the pima assay during the study were invited to participate in a voluntary, individual interview at the end of the study implementation. the study coordinator individually interviewed healthcare workers using a semi-structured interview guide. interviews were conducted in kiswahili and translated into english for analysis. questions about each specimen collection method for pima cd4 testing included: (1) an ease-of-use score on a scale of 1 to 5, with 1 being the easiest and 5 being the most difficult, (2) a ranking of the three sample collection methods in order of preference, and (3) likes and dislikes for each method. the healthcare workers were also surveyed on their experience with the pima cd4 assay including: (1) the use of pima cd4 cartridge, (2) performing the pima cd4 testing, (3) quality control testing, and (4) errors and malfunctions experienced with pima cd4 testing. the healthcare workers were asked whether they trusted the pima cd4 results acquired from each of the three specimen collection methods. finally, healthcare workers were asked open-ended questions about what they liked and disliked about pima, experiences using the pima cd4 assay, and recommendations for rollout in tanzania. data analysis quantitative healthcare workers’ survey responses were analysed using microsoft excel (microsoft corporation, redmond, washington, united states) using frequencies and tallies of rankings for mean ease-of-use ranking. open-ended responses were summarised by thematic areas. for each theme, comparisons between common or divergent views were made. all pima cd4 results were analysed in ibm statistical package for social sciences 21 software (spss) (ibm corporation, armonk, new york, united states) and microsoft excel. healthcare worker and nhlqatc pima cd4 results were compared by using scatter plot and best-line analysis with linear regression to estimate the errors by determining coefficient of determination (r2), slopes, and y-intercepts. in a bland-altman analysis, systematic bias and errors between healthcare workers and laboratory technician pima cd4 testing were estimated.22 for bland-altman plots, the nhlqatc reference laboratory’s pima cd4 counts were plotted on the x-axis, and the difference between the healthcare workers’ and nhlqatc laboratorian pima cd4 counts for each pair of venous blood specimens were plotted on the y-axis. the average differences (bias) and limits of agreement (bias ± 2 standard deviations) were calculated and shown on the bland-altman plots. results the validation study enrolled 1060 patients. characteristics of study participants; number of invalid tests by specimen type, error type, and site type; number of results per participant by specimen type by site; and performance data compared to the gold-standard facscalibur for each specimen type have been reported separately.21 three sample types were collected from each patient: venous blood, capillary-direct, and capillary-microtube. of the 1060 samples collected by capillary-microtube fingerstick, 25 (2.8%) were of insufficient volume and six (0.6%) were clotted, while for the capillary-direct fingerstick collections, five (0.5%) were of insufficient volume. thus, during this study, the quality of the specimens collected was acceptable for pima cd4 testing, with only a few compromised specimens occurring with fingerstick collection. venous specimens were sufficiently collected and tested for cd4 counts for all patients.21 healthcare worker survey participants of the 20 onsite healthcare workers who supported specimen collection and pima cd4 testing across the five sites, 11 healthcare workers were interviewed after study enrolment ended to assess user acceptability. the other nine hcws were unavailable at the time of the interview. the cadre of interviewees included nurse counsellors (5), clinical officers (3), phlebotomists (2), and a laboratory technician (1). results from healthcare worker interviews were summarised for three topic areas: specimen collection methods, pima cd4 testing, and recommendations for use of pima in tanzania. healthcare worker feedback: specimen collection methods healthcare workers rated capillary-microtube specimens as more difficult to collect when compared to the venous and capillary-direct methods (table 1). on the ease-of-use ranking, the capillary-microtube method scored an average of 3.0 (95% confidence interval [ci] 2.54–3.46) versus 1.73 (95% ci 1.26–2.19) for the venous method and 1.82 (95% ci 1.18–2.46) for the capillary-direct method. eight of the 11 healthcare workers interviewed rated the capillary-microtube as the most difficult specimen collection method, with three noting the longer collection time as a disadvantage. table 1: results of the healthcare worker survey on ease-of-use for collection and testing ranking of specimen collection methods, and trust of pima cd4 results (n = 11), tanzania, 2011. the majority (8/11, 73%) of healthcare workers selected the venous specimen as their most preferred method and three (3/11, 27%) selected capillary-direct as their most preferred method (table 1). the capillary-microtube method was selected as the ‘least preferred method’ by seven (7/11, 64%) of the healthcare workers and the capillary-direct methods was selected by four (4/11, 36%). interviewees thought the venous method was the easiest and noted that it is the most common method they have been using for other tests performed at the clinic. healthcare worker feedback: pima cd4 testing the pima cd4 assay was viewed positively by all healthcare workers interviewed, with nine specifically noting that it was user-friendly (table 2). the healthcare workers liked that it was portable, did not require extra reagents, and retained battery life for a long time; however, electricity was still required for charging and could limit the length of time that the pima cd4 device was operational. they also liked that it required minimal training and could be used by lower cadres. they felt the user guide was helpful; however, several noted that the user guide should be translated into kiswahili. two interviewees commented that training should be practical-focused to assess competency. while prompt results were advantageous, several noted the challenge of low machine throughput, particularly for high volume facilities. table 2: selected qualitative responses from healthcare worker survey, tanzania, 2011. while most healthcare workers reported that the capillary-direct method was easy to collect and apply to the cartridge, three interviewees cited difficulty applying capillary blood directly to the pima cd4 cartridge. with both the venous and capillary-microtube specimens, blood was transferred from a tube via a transfer pipette and applied to the pima cd4 cartridge. only two (2/11, 18%) of the healthcare workers felt it was difficult to transfer venous blood to the pima cartridge. similar to the venous specimen, only one (1/11, 9%) healthcare worker felt that the capillary-microtube was difficult to transfer to the pima cartridge. nearly all respondents mentioned that an advantage of the venous and capillary-microtube methods was that they both allowed for retesting or delayed specimen testing if the pima analyzer was in use at the time of sample collection. conversely, healthcare workers noted that the capillary-direct method required the pima analyzer to be free immediately after fingersticking, and retesting was only possible by pricking the client a second time. this aspect of the capillary-direct method could increase a client’s waiting time and burden. nine (9/11, 82%) of the 11 healthcare workers trusted all pima cd4 results for all three specimen collection methods (table 1). all healthcare workers trusted pima cd4 results with venous specimens. of the two (2/11, 18%) healthcare workers who expressed some distrust with the pima cd4 results using fingerstick specimens, one trusted capillary-microtube but not capillary-direct, while the other was indecisive. the former healthcare worker also expressed difficulty obtaining fingerstick samples and a general preference for venous blood as a sample type. among the nine healthcare workers trusting all three methods, one noted they only trusted capillary-microtube if the client is a ‘good bleeder and has a good flow’. healthcare worker feedback: recommendations for use of pima in tanzania all interviewees recommended the use of the pima cd4 assay in tanzania; however, use should be tailored to specific sites and situations (table 2). the majority (8/11, 73%) of the healthcare workers recommended targeting use of the pima cd4 assay to peripheral sites such as health centres and dispensaries or low patient volume sites. the major reason for targeting peripheral sites was due to a general lack of onsite cd4 testing at those levels and delays in acquiring cd4 results from a referral laboratory. many healthcare workers expressed concerns related to increased patient wait time if the pima cd4 assay were placed in high volume facilities and used capillary-direct specimens, as the pima analyzer can process only one sample at a time. other healthcare workers mentioned that the pima cd4 assay could be used at higher-level facilities for targeted populations (such as prevention of mother-to-child transmission of hiv) or used for seriously ill patients in need of urgent cd4 counts. one healthcare worker mentioned that clients would like to receive cd4 results on the same day and this would improve hiv client care. healthcare workers’ performance with pima cd4 compared to laboratory the healthcare workers performed 3317 pima cd4 tests on whole-blood specimens during this study at the five healthcare sites. the capillary-direct method accounted for 1055 of these tests and included 111 (10.5%) invalid tests. the healthcare workers performed 1118 pima cd4 tests with capillary-microtube specimens with 110 (9.8%) invalid tests and 1144 pima cd4 tests with venous specimens including 95 (8.3%) invalid tests, while the laboratorian performed 1219 pima cd4 tests using venous specimens only. the overall pima cd4 invalid test rate for healthcare workers was 9.5% (316/3317), while the laboratorians invalid test rate was 12.6% (153/1219). for invalid tests where residual samples were sufficient, tests were repeated and the valid repeat result was included in the analysis. also, six of the valid tests carried out by laboratorians were repeated and the results were also included. a valid test was repeated for a number of reasons such as the wrong specimen id number being entered during testing and then repeated with the correct id or the operator not realising a specimen was already tested. pima cd4 testing by healthcare workers with fingerstick and venipuncture whole-blood specimens yielded absolute cd4 counts that were in close agreement with paired venous specimens tested by laboratorians with the pima cd4 assay (figure 1). the scatter plot analysis of venous blood tested by both healthcare workers and laboratorians, with the pima cd4 estimated the best-fit line to have a slope of 0.97 and y-intercept of +0.2 with a r2 value of 0.90. bland-altman analysis with the same specimens estimated a bias of -14 cells/ul with acceptable limits of agreement of -145 to +115, indicating good agreement between both cadres, healthcare workers and laboratorians, with venous blood tested on the pima cd4 assay. figure 1: healthcare worker pima cd4 results at point-of-care site compared to laboratory pima cd4 results. scatter and bland-altman plots of healthcare worker pima cd4 testing with venous blood (a and b), capillary blood collected in ethylenediaminetetraacetic acid microtube (c and d), and capillary blood applied directly to pima cd4 cartridge (e and f) compared with laboratory pima cd4 testing with matched venous blood, tanzania, 2011. pima cd4 results by healthcare workers using fingerstick blood, for both the capillary-direct and capillary-microtube methods, when compared to pima cd4 results by laboratorians using venous blood, also indicated good agreement between the cadres and different specimen types (figure 1). when comparing capillary-direct specimen testing results by the healthcare workers to venous blood testing by laboratorians, there was greater variation (more random error) in the pima cd4 results as indicated by a scatter plot analysis, with a lower r2 value of 0.77 and bland-altman analysis with wider limits of agreement range of 396, (-225 to 171). the r2 values for the capillary-microtube method (by healthcare workers) was 0.86 and for the venous method (by the laboratorians) was 0.90, while the bland-altman limits of agreement ranges were 294 for the capillary-microtube method and 260 for the venous method. discussion the lack of human resources is a recognised barrier to the continuum of hiv care.23 in resource-limited settings, this challenge is evident in all cadres of healthcare workers, including phlebotomists and laboratorians. task-shifting has been shown to be effective in resource-limited settings and thus may partially address shortages of healthcare workers.24,25,26 the innovation of poc testing lies in maximising available resources, so that existing cadres of healthcare workers not only collect specimens but also perform testing for clinical laboratory results. in studies focusing on the accuracy and reliability of poc testing, the test quality achieved for pima cd4 assay is reported to be as good as that performed by laboratory-based cd4 assays.12,17,21,27,28,29,30 nevertheless, similar to laboratory-based testing, the use of poc testing can be problematic due to procedural errors during specimen collection, sampling, and testing, lack of clear guidelines for implementation and lack of quality control and quality monitoring.31,32,33 our study assessed the pima cd4 assay feasibility, acceptability, and validity at the poc or clinical setting with the premise that acceptance and trust of the poc testing is necessary for reliability in testing and accurate results. all of the healthcare workers interviewed recommended the introduction of the pima cd4 assay in tanzania. they expressed that introduction of this poc cd4 assay would improve patient care, which was not a focus of this evaluation; however, other studies have shown improved linkages and earlier art initiations following the introduction of poc cd4 testing.10,11,18,19,34 the healthcare workers in this study appreciated the fact that it required minimal training and could be used across all cadres. relevant documents should be translated into the dominant local language, in this case kiswahili, particularly when task-shifting occurs to lower cadres that may not have the technical background and language skills to navigate non-local-language user manuals and training guides. the pima cd4 assay has been shown to produce comparable results to standard reference methods of cd4 measurement when using fingerstick capillary and venous blood sample collection methods.12,14,16,17,20,21,27,30 while our study had a cadre mix of nurses, phlebotomists, clinical officers, and laboratorians at the site level, a similar study in kenya compared only lay counsellors to laboratory technicians and found commensurate performance, suggesting that pima cd4 assays are suitable for use across cadres.20 our study also indicated that various cadres could perform the pima cd4 assay and achieve similar results as would laboratorians. poc testing requires that specimen collection and testing be performed within a short period of time. for our study, training for the pima cd4 assay specimen collection and pima testing was done simitaneously. as with conventinal laboratory cd4 testing with flow cytometry, the quality poc cd4 test results depends on the quality of the specimen. with training from a certificied phelobotomist, the healthcare workers were able to collect specimens by all three methods in this study. these specimens were of good quality and pima cd4 results were obtained for almost all study participants.21 the healthcare workers were able to correctly perform the pima cd4 testing on all three specimen types. the pima invalid test rates from the healthcare workers were similar to those of the trained laboratorians. the invalid test rate for the healthcare workers was 9.5%. as the healthcare workers become more familiar to performing the assay, we believe this invalid test rate will be lower. several respondents expressed greater confidence in venous and capillary-microtube results as compared to capillary-direct; however, only one said they did not trust capillary-direct results. this greater confidence with the venous and capillary-microtube methods corroborates healthcare workers’ expression of difficulty in applying the sample directly to the pima cartridge. relative to the venous and capillary-microtube methods, capillary-direct had a slightly higher frequency of invalid pima tests and more results that were variable as evidenced by the lower r2 and wider limit of agreement range as compared to the venous and capillary-microtube methods. a similar increase in invalid test rates and variability for capillary samples compared to venous samples has been reported in other pima evaluations.16,17,21,35 the invalid pima tests were primarily due to problems with specimen integrity, improper filling of blood in the cartridge, incorrect handling of the cartridge, or abortion of the assay process by the operator. in this study, client perspectives on the pima cd4 assay and the preferred sample collection methods were not obtained. in other studies, clients shared similar preferences for venous blood collection to avoid multiple fingersticks or stated that fingersticks were more uncomfortable than venipuncture.27,30 however, a south african study found multiple fingersticks were acceptable by the clients.36 limitations this study has several limitations. participating healthcare workers were few (only 20) and among those, only 11 were available for an interview, which was only about half of an already small pool of healthcare workers. however, to ensure participation in interviews was voluntary, healthcare workers had the opportunity to decline participation. as a result, our sample of healthcare workers included only those who had strong opinions about the pima assay and were willing to participate. in addition, the study sites were not randomly selected, introducing potential selection bias. all sites were high volume urban facilities where phlebotomy is performed on upwards of 100 clients per day. although the venous and capillary-direct methods were both seen as easier to use than capillary-microtube, the overall preference for venipuncture may have been influenced by the high volume of phlebotomy at all the sites. therefore, the user group was not representative of potential pima users in rural, peripheral facilities. all healthcare workers were interviewed separately; however, it is possible that they built consensus among themselves through shared experiences and training, which might have influenced their individual preferences. another limitation is that the questionnaire used had not been validated for this type of study. in addition, the questionnaire did not specifically seek views on the length of time to collect pima samples, run the test, or the overall work burden. furthermore, due to the small sample size and the qualitative nature of the study interviews, we were not able to conduct multivariate quantitative analysis to adjust for confounders and identify factors associated with outcomes, such as healthcare worker cadre. lastly, there was a significant delay between study implementation and publication, approximately 8 years; thus, conclusions should be interpreted with awareness that contextual factors may have changed since the time of study implementation. conclusion overall, healthcare workers found the pima™ cd4 assay to be user-friendly and recommended its use for hiv care. the healthcare workers were able to successfully collect venous and fingerstick specimens and perform poc pima cd4 testing, with results comparable to reference laboratory pima results. many healthcare workers noted that collecting whole blood in an edta tube, either by venipuncture or by fingerstick, was advantageous as it allowed for specimens to be tested at a later time or to be retested without going back to the patient for another specimen. despite the strong caveat of this study, its findings suggest that venous or capillary-microtube specimen collection may be more suitable for poc testing workflow with pima cd4 testing, rather than direct application of fingerstick capillary blood to a pima cd4 cartridge. although healthcare workers recognised that the capillary-microtube specimens provided similar benefit to the venous method for retesting, more training and familiarisation with this method of capillary blood collection may be required to increase healthcare worker acceptability. future studies could consider collecting a larger, more representative sample of healthcare worker feedback, as well as client preferences for sample collection methods for pima and other devices with options for capillary and venous blood use. in addition, our findings regarding preference for venous or capillary-microtube specimen collection may be applicable to other types of poc testing. acknowledgements we wish to thank the study participants and the site staff of mnazi moja, sinza, temeke, mbagala, and mwanayamala tanzanian health facilities which supported the study implementation. in memory of gilly arthur. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions m.e.s., l.k., m.m., j.n., o.b. and l.w. contributed to the study’s conceptualisation; m.e.s., k.c., f.m. and l.w. conducted the formal analyses; l.k., o.b. and l.w. supported funding acquisition. m.e.s., k.c., n.a., l.k., r.l., m.m., o.b., f.m. and l.w. supported investigation, methodology implementation and supervision. m.e.s., k.c. and l.w. prepared the original manuscript draft and all authors reviewed and approved the final manuscript. sources of support this research was supported by a grant to the african field epidemiology network (grant #u2gps002728) under the president’s emergency plan for aids relief through the united states centers for disease control and prevention. data availability statement data is available on request from the corresponding author. disclaimer the findings and conclusions in this article are those of the authors and do not necessarily represent the views of the united states centers for disease control and prevention. references world health organization. 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testing for hiv anti-retroviral treatment initiation and monitoring from multiple or single fingersticks. plos one. 2013;8(12):e85265. https://doi.org/10.1371/journal.pone.0085265 drain pk, rousseau c. point-of-care diagnostics: extending the laboratory network to reach the last mile. curr opin hiv aids. 2017;12(2):175–181. https://doi.org/10.1097/coh.0000000000000351 abstract introduction methods results discussion acknowledgements references about the author(s) emmanuel o. babafemi department of pharmacy and biomolecular sciences, faculty of science, liverpool john moores university, liverpool, united kingdom benny p. cherian department of microbiology, royal london hospital, barts health nhs trust, london, united kingdom khalid rahman department of pharmacy and biomolecular sciences, faculty of science, liverpool john moores university, liverpool, united kingdom gilbert m. mogoko department of microbiology, ipp pathology first, dobson house, bentalls, basildon, united kingdom oluwatoyin o. abiola department of computer science, faculty of science, afe babalola university, ado ekiti, nigeria citation babafemi eo, cherian bp, rahman k, mogoko gm, abiola oo. diagnostic accuracy of real-time polymerase chain reaction assay for the detection of trichomonas vaginalis in clinical samples: a systematic review and meta-analysis. afr j lab med. 2025;14(1), a2522. https://doi.org/10.4102/ajlm.v14i1.2522 note: additional supporting information is available for download alongside the online version of this article as online supplementary document 1. review article diagnostic accuracy of real-time polymerase chain reaction assay for the detection of trichomonas vaginalis in clinical samples: a systematic review and meta-analysis emmanuel o. babafemi, benny p. cherian, khalid rahman, gilbert m. mogoko, oluwatoyin o. abiola received: 05 june 2024; accepted: 24 jan. 2025; published: 16 apr. 2025 copyright: © 2025. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: vaginal trichomoniasis is a highly prevalent parasitic infection associated with hiv acquisition and preterm birth. the ‘gold standard’ for its diagnosis requires 3–7 days to detect by culture. rapid and accurate diagnosis, such as by nucleic acid amplification testing, is key to manage the disease, and control and prevent its transmission. aim: this review aimed to assess the overall accuracy of real-time polymerase chain reaction (rt-pcr)-based assays, for routine diagnosis of trichomonas vaginalis in clinical vaginal samples from women with symptomatic/asymptomatic trichomoniasis, using trichomonads culture as the gold standard. methods: medline, pubmed, embase, and other sources were used to search for included studies published between 01 january 1995 and 31 july 2023. the search terms ‘real-time polymerase chain reaction’, ‘real-time’, ‘polymerase chain reaction’, ‘trichomonas vaginalis’, ‘trichomonas’, ‘vaginalis’, ‘humans’, ‘rt pcr’, ‘nucleic acid amplification test’, ‘naat’, ‘trichomonad culture’, ‘women’ were included. summary estimates were calculated for the overall accuracy of the assay compared to trichomonads culture as the reference standard. meta-analysis was conducted using a bivariate meta-regression model. results: twenty-seven eligible studies met our inclusion criteria: sensitivity 99% (95% confidence interval [ci] 99–100), specificity 100% (95% ci 100–100), positive likelihood ratio 350.67 (167.42–734.49), negative likelihood ratio 0.02 (0.01–0.03), diagnostic odds ratio 23 064.05 (95% ci 8532.13–62 346.77), and area under receiver operating characteristics curve 0.99. there was significant heterogeneity in sensitivity and specificity (p < 0.001). conclusion: our results suggested that rt-pcr assays could be useful for the diagnosis of vaginal trichomoniasis with high sensitivity and specificity. what this study adds: this article provides a comprehensive review of the effectiveness of rt-pcr assays for the diagnosis of trichomoniasis with high sensitivity and specificity in comparison to other methods in clinical laboratory practice. the goal is to present awareness/evidence that this assay is more accurate and rapid than other techniques. keywords: trichomoniasis; trichomonas vaginalis; real-time polymerase chain reaction assay; vaginal swabs; systematic review; meta-analysis. introduction trichomonas vaginalis, a protozoan, causes trichomoniasis, which is a common sexually transmitted disease (std) that affects approximately 156 million persons globally per year, with the majority in low-income settings.1 it has been estimated that 7.4 million new cases occur annually in the united states.2 trichomonas is the most common non-viral std agent in the world, with an overall prevalence of 3.1%.3 trichomoniasis occurs in both men and women, causing infection; however, symptoms are widespread in women. symptomatic women present a malodorous, diffuse, vulvar irritation, with yellow-green vaginal discharge that may be mistaken for bacterial vaginosis. trichomonas vaginalis is known to cause vaginitis, cervicitis, and many infections that may go symptomless, with likely consequences such as premature birth, underweight at birth, tubal infertility, and pelvic inflammatory disease when left untreated.4 trichomoniasis may lead to adverse birth outcomes, such as increased risk and transmission of hiv infection, and premature rupture of the membranes, in women.5 hormonal changes predispose to a higher incidence of lower genital tract infections caused by trichomoniasis during pregnancy, which can lead to perinatal and maternal complications.6 tests with improved sensitivity and specificity are of great significance and essential for diagnosing trichomoniasis. the gold standard for the diagnosis of t. vaginalis infection is culture of the organism using vaginal specimens and is reported to have 75% to 89% sensitivity; however, it requires between 2 days and 7 days of incubation, resulting in substantial delays before obtaining the results.7 direct microscopic examination of the vaginal fluid using wet preparations remains the most widely utilised diagnostic test for t. vaginalis infection, despite its limited sensitivity in asymptomatic patients.8 microscopic examination is inexpensive and quick, but depends on the microscopist’s skill, and on the prompt transport and processing of the sample, which relies on the viable organisms, to avoid the loss of parasite motility.9 in addition, several authors have reported the use of nucleic acid amplification tests, including real-time polymerase chain reaction (rt-pcr). these have shown an improved sensitivity and specificity method for detecting t. vaginalis compared to microscopic examinations and culture.10,11 real-time pcr assays provide an improvement in medical screening for the parasite.11 therefore, a simple, rapid, and accurate diagnostic test with acceptable sensitivity and specificity is important in diagnosing t. vaginalis infection. this cannot be accurately diagnosed based on the clinical picture, because clinical symptoms of trichomoniasis may be similar to those of other stds.12 all the available published primary research studies were used in this review to provide summary estimates of the diagnostic accuracy of rt-pcr assay for detecting t. vaginalis from clinical samples. the study summarises current evidence-based clinical practice that can help diagnose t. vaginalis during pregnancy to prevent perinatal and maternal complications. the findings will help in choosing the most appropriate tool for rapid and accurate detection of t. vaginalis in pathological samples on a routine basis, medical screening, future guidelines and healthcare policy. methods study protocols the preferred reporting items for systematic reviews and meta-analyses statement guidelines13 were followed to conduct this systematic review and meta-analysis (online supplementary text 1). the preferred reporting items for systematic reviews and meta-analyses checklist was used to ensure that all the relevant information from studies (published 01 january 1995 to 31 july 2023, in any language) and unpublished articles was eligible to identify trichomoniasis among women in the analysis. we registered our systematic review protocol in prospero (international prospective register of systematic reviews): prospero crd42023435253. the quality assessment of diagnostic accuracy studies-2 (quadas-2)14 was used to assess the quality of the included studies. there was no need for institutional ethical review approval for this study. searching strategies the search was conducted with the aid of carefully selected terms. the search strategy included ‘real-time polymerase chain reaction’, ‘real-time’, ‘polymerase chain reaction’, ‘trichomonas vaginalis’, ‘trichomonas’, ‘vaginalis’, ‘humans’, ‘rt pcr’, ‘nuclei acid amplification test’, ‘naat’, ‘trichomonad culture’, and ‘women’. they were used distinctly and in combination, using boolean operators such as ‘or’ or ‘and’ to generate a list of primary studies. there was no language limitation to the search. a librarian information specialist familiar with the topic validated the search strategy for each database. two of the investigators independently and systematically searched the electronic bibliographic databases (medline, pubmed, embase, and other relevant databases) to identify additional records15,16 (online supplementary text 2). ‘google translate’ was used to screen abstracts and articles in languages other than english. eligibility criteria observational studies (cross-sectional and cohort) and case-control designs for detecting t. vaginalis from women clinical samples of any age were included. the studies were eligible for inclusion if they reported the total number of patients tested, described original research, contained positive/negative results that allowed the calculation of true positives, true negatives, false positives, and false negatives, and compared rt-pcr assay to a reference/gold standard method – culture-based assay. exclusion criteria included studies where rt-pcr assay was not used, t. vaginalis was detected in men, involvement of animals, and duplicate publication. study selection process two of the investigators screened full-text articles independently through careful reading of the title and abstract, for eligibility for use in the study to minimise bias in selection. the remaining three authors independently evaluated the quality of the studies against the checklist. any discrepancies in the inclusion of initially screened studies were resolved through discussion and, where needed, by a third reviewer. any rejected studies were documented. the overall study selection process is presented using the preferred reporting items for systematic reviews and meta-analyses statement flow chart13 (figure 1). figure 1: the preferred reporting items for systematic reviews and meta-analyses (prisma) 2020 flow diagram. data extraction and quality assessment after the appropriate articles were selected, data were extracted independently by two of the investigators using a data extraction template and presented using microsoft word 2016 containing author and year, participants, country, index test, reference test, and target sequence for detection of t. vaginalis dna (table 1). table 1: characteristics of included studies. the methodological quality for the included studies was assessed independently according to the four domains (patient selection, index test, reference standard, and flow and timing) of the quadas-2 tool (figure 2).14 the study quadas-2 quality criteria are presented in online supplementary text 3. figure 2: risk of bias and applicability concerns graph: review of authors’ judgements about each domain presented as percentages across included studies. data synthesis and meta-analysis measures of test accuracy were computed using standard methods recommended for meta-analysis of diagnostic studies. these are sensitivity, specificity, negative likelihood ratio (nlr), positive likelihood ratio (plr), diagnostic odds ratio (dor), and 95% confidence intervals (ci).17,18,19 the 2 × 2 data (true positives, false positives, true negatives, and false negatives) were extracted directly from source papers. the dersimonian-laird random-effect model was used to assess the overall accuracy and dor, which accounts for both random error (within-study variability) and heterogeneity (between-study variability). a bivariate model was used to estimate the area under the summary receiver operating characteristic (sroc) curve.18,19 the model uses the correlation between binary tests (sensitivity and specificity) and potential threshold effects. these measures were pooled using the random-effects model.18,19 individual articles in the meta-analysis contributed a pair of numbers, sensitivity and specificity, and summarised their joint distribution using an sroc curve. a global measure of the overall performance of the test employs the area under the sroc curve, the value of 1 indicates the perfect discriminatory ability of the test, while the curve value of 0.5 means that the test does not have a discriminating ability.18,19 we analysed data using meta-disc (version 1.4; http://www.hrc.es/investigacion/metadisc_en.htm) and reviewing manager version 5.4 (cochrane collaboration, oxford, united kingdom).19,20,21 the data were displayed graphically on forest plots and sroc plots.22 since publication bias is not recommended in the meta-analysis for diagnostic test accuracy study, we did not evaluate it.23 no p-value for authors and publishers of diagnostic accuracy study was used, since they do not test the hypothesis that may influence decisions about publication based on the statistical significance of the results.24 heterogeneity the heterogeneity tests for the included studies were explored with chi-squared (χ2) and i-squared (i2) statistics. stratified or subgroup analyses were used to investigate the source of heterogeneity. the studies of rt-pcr assays across continents (america, africa, asia-middle east, and europe [table 1, table 2, online supplementary table 1]) were specified a priori as potential sources of heterogeneity. the interpretations of heterogeneities among the studies are: i2 = 0, no heterogeneity; i2 < 25, low heterogeneity; i2 < 50, moderate heterogeneity; i2 < 75, high heterogeneity; and i2 < 90 considerable heterogeneity.25 they were also assessed visually, with forest plots and sroc curves with 95% prediction regions. table 2: summary of statistical results for trichomoniasis clinical samples. results study selection a total of 1022 articles were identified through the major electronic databases and other potentially relevant sources. from all identified studies, 50 articles were selected based on their relevance to the study topic. an additional seven studies were identified from grey literature and references of full-text articles. after screening all the titles and abstracts, removing the duplicates, and excluding the ineligible studies, 27 articles (22 742 samples/patients)26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50 were selected for full-text review and meta-analysis (figure 1). characteristics of the included studies in this systematic review and meta-analysis, 22 742 clinical samples obtained from 15 countries were included. the summary of the main characteristics of the included studies and the types of rt-pcr-based assays used is shown in table 1. there were 13 studies from america, 5 studies from africa, 5 studies from asia/middle east, and 4 studies from europe. the overall study quality assessment and methodological quality of studies by the quadas-2 tool are presented in online supplementary figure 1 and online supplementary figure 2. it showed a low risk of bias, except for studies using a case-control design. the methodological quality of studies (assessed by the quadas-2 tool) was generally high, with 27 of the studies meeting all four domains of the criteria (figure 2). the majority of the included studies used the principle of rt-pcr assay as the index test, demonstrating culture-based assay as the reference test. meta-analysis meta-analysis results were presented as 95% ci values for samples as follows: overall sensitivity 99% (95% ci 99–100) and specificity 99% (95% ci 99–100). area under the curve (auc) of the receiver operating characteristics was 0.99 for samples. the summary estimates of trichomoniasis for heterogeneity with chi-squared (χ2) using 95% ci were 46.19 (sensitivity), 197.18 (specificity), 159.14 (plr), 50.71 (nlr), and 68.07 (dor), with p < 0, indicating significant heterogeneity across studies. i2 was between 43.7% and 86.8%, showing a significant heterogeneity. there were considerable heterogeneities from the reviewed studies (table 2, online supplementary figure 3, online supplementary figure 4, online supplementary figure 5, and online supplementary figure 6). subgroup analyses of real-time polymerase chain reaction-based assay of trichomoniasis across the continents subgroup analyses were assessed by sources of data for these graders as seen below. an important note for all groups is that a test with perfect discrimination has a receiver operating characteristic curve that passes through the upper left corner (100% sensitivity, 100% specificity). the closer the receiver operating characteristic curve to the upper left corner, the higher the overall accuracy of the test. with the america subgroup (table 1) as the rt-pcr-based assay (13 studies, 10 796 specimens), the results were as follows: sensitivity 99% (95% ci 98–100), specificity 100% (95% ci, 99–100), and auc 0.99. the summary estimates of the performance of rt-pcr-based assay in america heterogeneity, with chi-squared (χ2) using 95% ci, were 32.20 (sensitivity), 82.52 (specificity), 46.44 (plr), 37.20 (nlr), and 43.06 (dor), with p ≤ 0.001, indicating significant heterogeneity across studies. i2 was between 62.7% and 85.50%, showing significant heterogeneity. there was considerable heterogeneity for the subgroup analysis by rt-pcr-based assay in america (online supplementary table 1; online supplementary figure 7, panels a–f). with the africa subgroup (table 1) as the rt-pcr-based assay (5 studies, 1178 specimens), the results were as follows: sensitivity 99% (95% ci 99–100), specificity 99% (95% ci, 97–99), and auc 0.99. the summary estimates of performance of rt-pcr-based assay in africa heterogeneity, with chi-squared (χ2) using 95% ci, were 7.10 (sensitivity), 34.88 (specificity), 27.77 (plr), 6.09 (nlr), and 5.42 (dor), with p ≤ 0.001–0.247, indicating significant heterogeneity across studies. i2 was between 26.2% and 88.50%, showing significant heterogeneity. there was considerable heterogeneity for the subgroup analysis by rt-pcr-based assay in africa (online supplementary table 1; online supplementary figure 8, panels a–f). with asia/middle east subgroup (table 1) as the rt-pcr-based assay (5 studies, 3967 specimens), the results were as follows: sensitivity 100% (95% ci 98–100), specificity 100% (95% ci, 100–100), and auc 0.99. the summary estimates of the performance of rt-pcr-based assay in asia/middle east heterogeneity, with chi-squared (χ2) using 95% ci, were 0.00 (sensitivity), 0.00 (specificity), 1.95 (plr), 4.58 (nlr), and 3.99 (dor), with p = 1, indicating significant heterogeneity across studies. i2 was between 0.00% and 12.70%, showing mild heterogeneity. there was considerable heterogeneity for the subgroup analysis by rt-pcr-based assay in asia/middle east (online supplementary table 1; online supplementary figure 9, panels a–f). with the europe subgroup (table 1) as the rt-pcr-based assay (4 studies, 6830 specimens), the results were as follows: sensitivity 100 (95% ci 97–100), specificity 100 (95% ci, 99–100), and auc 0.99. the summary estimates of the performance of rt-pcr-based assay in europe subgroup heterogeneity, with chi-squared (χ2) using 95% ci, were 0.00 (sensitivity), 0.00 (specificity), 2.04 (plr), 2.91 (nlr), and 3.22 (dor), with p = 1, indicating significant heterogeneity across studies. i2 was between 0.00% and 6.8% showing mild heterogeneity. there was considerable heterogeneity for the subgroup analysis by rt-pcr-based assay in europe (online supplementary table 1; online supplementary figure 10, panels a–f). discussion the primary aim of this study was to conduct a systematic review and meta-analysis of the relevant literature to synthesise evidence for the accuracy of rt-pcr-based assays for the diagnosis of trichomoniasis from clinical samples among women. trichomonas vaginalis infection is the most common non-viral std worldwide; only genital human papillomavirus is more prevalent.51 diagnosing the aetiology of most stds using culture methods is infamously difficult and can take several days to complete. the most widely used diagnostic test for vaginal trichomoniasis remains the wet-mount microscopy. culture-based assay remains the ‘gold standard’ to detect t. vaginalis. for the laboratory diagnosis of t. vaginalis, the use of pcr-based assays is more sensitive than culture and wet-mount microscopy. the uptake of the rt-pcr-based assays for routine diagnosis of t. vaginalis infections remains a challenge in resource-limited settings.39 it has been reported that ‘the sensitivity of culture compared with rt-pcr assay ranges from 34.9% to 78%, while the specificity is usually 100%’.52 likewise, the wet-mount microscopy specificity is usually high; however, sensitivity of 34.2% to 58.5% was reported when compared to rt-pcr assays.53 it was reported that, ‘rt-pcr assay is sensitive, specific, shortened turn-around time, and is reproducible, and automation of the procedure reduces hands-on time and decreases the risk of cross-contamination’.54 edwards et al. reported: the development of sensitive nucleic acid amplification tests for t. vaginalis has opened the possibility of testing asymptomatic patients, who often have low organism loads, undetectable with less sensitive diagnostic methods. in the uk, the cost of offering this service in a sexual health screen is thought to outweigh the benefit of detecting these asymptomatic infections, due to the relatively low prevalence of this organism in the general population. (p. 414)55 in this study, we have seen the evidence that using rt-pcr-based assays for the detection of t. vaginalis from pathological specimens among women is rapid and accurate. the review is extensive in scope and involves different rt-pcr-based assays. our study findings showed high specificity of 99% (95% ci 99–100), plr of 350.67 (167.42–734.49) and nlr of 0.018 (0.009–0.033) for trichomoniasis using rt-pcr-based assays. a plr of 350 specifies that a t. vaginalis infection is 350-fold more likely to be positive for an rt-pcr-based assay in comparison to patients free from the infection. table 2 confirms that rt-pcr-based assays were excellent for the laboratory diagnosis of trichomoniasis, as shown by auc and dor values. our study showed a considerable level of heterogeneity which led us to perform subgroup analyses to investigate the likely sources of heterogeneity. factors such as the t. vaginalis target gene sequence, sample size, study design, clinical settings of the primary studies, and the different rt-pcr-based assays could be responsible for the variations observed across the included studies. strengths and limitations this study’s diligent methodology, adopting the preferred reporting items for systematic reviews and meta-analyses guidelines, using comprehensive search strategy and assessment in this review across different search engines for identifying published and unpublished articles is an important strength. another strength of the review is non-restriction of studies to any language, thereby reducing bias. potential publication bias and heterogeneity were explored using the study guidelines.56,57 the review had some limitations that may affect its applicability, such as the inclusion of only a few studies in our subgroup analysis, which could also impact the precision of our estimation, cost-effectiveness assessment, and the likelihood of not publishing non-significant or unfavourable results.58 other limitations from this review include non-generalisation of the rt-pcr-based assay performance because of the many target genes and protocols used in the included studies. it was reported that, ‘diagnostic studies in general seem to be beset by these problems’59; therefore, the outcomes of this study should be interpreted with care considering the conditions, reporting, and discrepancy in study quality. ‘the use of guidelines such as the standards for reporting of diagnostic accuracy might improve the quality of reporting of primary studies’.60 further work should be considered to establish a simple, efficient and cost-effective rt-pcr assay that can be adapted for t. vaginalis detection from clinical samples in resource-limited countries. conclusion in conclusion, we can summarise that the present study identified rt-pcr as a highly sensitive and specific diagnostic assay compared to the reference culture-based methods for the detection of t. vaginalis. furthermore, the sensitivity and specificity were 99% (95% ci 99–100) confirming the rt-pcr-based assays’ accuracy for detecting trichomoniasis in clinical samples among women. therefore, as a result of our findings, we recommend that healthcare practitioners and policymakers in all countries adopt the use of this type of assay on a routine basis and in std clinics, particularly in lowand middle-income countries with a high disease burden, because of its rapid results with robust and good diagnostic accuracy when used to detect t. vaginalis in the clinical samples of both symptomatic and asymptomatic women. acknowledgements we would like to thank katherine stephan of the library services at liverpool john moores university for her support with the search strategy for this systematic review and meta-analysis. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions e.o.b. formulated the research question and designed/conceptualised the study. e.o.b. developed the search strategy. e.o.b. and g.m.m. drafted the article. e.o.b., b.p.c., k.r. and o.o.a. critically reviewed the article for content. e.o.b. reviewed the statistical analysis. e.o.b., b.p.c., k.r., g.m.m. and o.o.a. have read and approved the final version of the article. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support this review received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the authors confirm that the data supporting the findings of this study are available within the article. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. the article does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the authors are responsible for this article’s results, findings, and content. references world health organization. global progress report on hiv, viral hepatitis and sexually transmitted infections [homepage on the internet]. 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authors: verena gounden1,2 yashna rampursat1,2 affiliation: 1department of chemical pathology, national health laboratory services, inkosi albert luthuli central hospital, south africa2nelson r mandela school of medicine, university of kwazulu-natal, south africa correspondence to: verena gounden postal address: national health laboratory services, inkosi albert luthuli central hospital, 800 bellair road, cato manor, durban 4058, south africa dates: received: 24 june 2013 accepted: 09 july 2014 published: 08 oct. 2014 how to cite this article: gounden v, rampursat y. an audit of immunofixation requesting practices at a south african referral laboratory. afr j lab med. 2014;3(1), art. #91, 5 pages. http://dx.doi.org/10.4102/ ajlm.v3i1.91 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. an audit of immunofixation requesting practices at a south african referral laboratory in this original research... open access • abstract • introduction • research method and design • results • discussion    • limitations of the study    • recommendations    • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: it is common practice in most chemical pathology laboratories for reflective immunofixation electrophoresis (ife) to occur following the detection or suspicion of a paraprotein on serum protein electrophoresis (spep). the chemical pathology laboratory at inkosi albert luthuli central hospital (ialch) in durban, south africa, is currently the only non-private laboratory in the kwazulu natal province that performs spep analysis, with current practice requiring that the clinician request ife following suggestion by the laboratory after a suspicious spep result.objectives: to review the current process for ife at ialch in the context of reflective testing and to examine the use of the alpha-2-globulin/alpha-1-globulin ratio as a predictor of a positive ife result. methods: data for 1260 consecutive spep tests performed at the ialch national health laboratory service were collected between february and july 2011. spep and ife were performed with a sebia hydrasys automated electrophoresis system. the alpha-2-globulin/alpha-1-globulin ratio was calculated using density of corresponding fractions on spep. results: analysis revealed that of the 1260 speps performed during the analysis period, 304 ifes were suggested by the reviewing pathologist. a total of 45 (15%) of the suggested ifes were subsequently requested by the attending clinicians. almost half (46.5%) (n = 20) of the suggested ifes that were performed revealed the presence of a paraprotein. there was no statistically-significant difference between the alpha-2-globulin/alpha-1-globulin ratio for patients with positive or negative ifes (p-value = 0.2). conclusions: this study reveals the need for reflective addition of ife testing by the laboratory following suspicious findings on spep. introduction top ↑ overproduction of a single abnormal clone of a plasma cell or b lymphocyte results in the presence of a monoclonal gammopathy.1 disorders associated with the presence of a monoclonal protein (m protein) include b-cell lymphomas and leukaemias; amyloidosis and waldenstrom’s macroglobulinaemia; and plasma cell dyscrasias, which include multiple myeloma, monoclonal gammopathy of undetermined significance (mgus) and plasmacytoma.2 mgus is a premalignant plasma cell disorder with an associated risk of progression to multiple myeloma (mm). mm is currently recognised as being largely incurable; the ability to identify the premalignant condition (mgus) is important for strategies that attempt to delay or prevent progression to mm. it is also important to distinguish between those patients with mm and mgus as the management of these two groups of patients differs, with those with mgus being treated conservatively.1the m protein is identified by either serum protein electrophoresis (spep) or urine protein electrophoresis (upep) as a band of restricted migration.1 spep is utilised to identify and monitor patients with plasma cell dyscrasias, in particular mm. immunofixation electrophoresis (ife) is performed to confirm the presence of the monoclonal protein and to characterise its immunoglobulin heavy chain class and light chain type.1in some patients with a monoclonal gammopathy, spep may show a normal pattern or only hypogammaglobulinaemia. the monoclonal protein may also be masked on spep if it migrates in the beta or alpha-2 regions. in these patients, an ife can reveal or exclude the presence of the monoclonal protein.3 ife is a relatively expensive laboratory test requiring greater technologist time and input compared with other chemistry tests that utilise automated platforms. nevertheless, the practice of routine reflex or reflective ife testing following the detection of a suspected monoclonal band or other suspicious findings on spep or upep is commonplace in many laboratories.4 reflex testing refers to the practice of automatic addition of laboratory tests to an existing test request on the basis of laboratory-defined algorithms. reflective testing refers to tests added on by pathologists or clinical biochemists after consideration of a wider range of information (e.g. demographic data, clinical information, previous results and results of other accompanying tests requested).5 for the successful practice of reflective testing, laboratory knowledge of this wider range of information, particularly clinical information, is needed. reflex testing is based almost exclusively on laboratory results and utilises algorithms that may include some limited clinical information such as demographic data. reflective testing, on the other hand, depends on the expertise and knowledge of the reviewing pathologist, providing the advantage of incorporating other clinical information that reflex testing does not take into account. the soaring costs of laboratory testing, accompanied by increased demand, result in laboratory management having to perform intense scrutiny of test request practices, including reflex and reflective testing. the chemical pathology laboratory at inkosi albert luthuli central hospital (ialch) in durban, south africa, is currently the only laboratory that provides spep or upep services to all state-run healthcare facilities in the province of kwazulu natal (estimated population of over 10 million people).6 current practice at the laboratory for all spep samples requires that the clinician directly request the ife in addition to the spep (figure 1). neither reflex nor reflective ife is performed routinely by the laboratory; however, following pathologist review, the result report may suggest that the clinician order an ife for further workup of the patient. although this above-described practice may reduce costs by preventing unnecessary ife from being requested by the laboratory, it could result in the delayed diagnosis of a plasma cell dyscrasia, contributing to increased healthcare costs associated with hospitalisations and complications. spep and upep samples for patients from the hospital‘s clinical haematology department are exempt from the current laboratory ife policy and reflective addition of ife occurs where indicated. figure 1: flow diagram illustrating ialch laboratory ife requesting process. serum-free light chain (sflc) measurement is a widely-used screening test for plasma cell dyscrasias.7 however, this test is still relatively expensive and not offered in many laboratories serving the greater south african population. in addition to abnormal monoclonal proteins, other normal proteins are visualised on spep, including proteins that migrate in the alpha-2 and beta regions of the electrophoresis gel. lakshminarayanan et al. describe 'a two-fold increase in the odds ratio for a positive ife result when the alpha-2-globulin/alpha-1-globulin ratio was elevated'.8 the authors further recommend reflex ife testing in patients with a normal spep pattern and hypogammaglobulinaemia when an elevated alpha-2globulin/alpha-1-globulin ratio is detected.8 the aims of this study were to determine the number of ifes requested by clinicians following suggestion by the laboratory and to assess whether the determination of the alpha-2-globulin/alpha-1-globulin ratio, regardless of the presence of hypogammaglobulinaemia, could assist in predicting which of the patients in this population, with no paraprotein detected on spep, were more likely to have positive ife results. a secondary objective was to determine how many spep requests were accompanied by clinical histories provided by the clinician or test requestor. research method and design top ↑ data were collected from february 2011 to july 2011 for 1260 consecutive spep samples analysed at the ialch, national health laboratory services chemical pathology laboratory (durban, south africa). samples analysed consisted of routine samples with spep orders received by the laboratory from the laboratory hospital and referring hospitals within kwazulu natal province.the sebia hydrasys (sebia, norcross, ga, usa) automated electrophoresis system was used to perform spep and ife with polyclonal anti-human serum to identify immunoglobulin heavy and light chains.5 quantitation of spep fractions was performed using the sebia hydrasys densitometer system and phoresis software (sebia, norcross, ga, usa). the same pathologist interpreted all spep runs. requisition by the laboratory of ife testing was suggested on the result report if a monoclonal band or any other abnormal bands, such as restriction bands (abnormal areas of restriction on protein electrophoresis which may indicate the presence of a paraprotein), were identified on spep. ife testing was also suggested if the spep revealed hypogammaglobulinaemia in the absence of a monoclonal band. the total number of specimens analysed, spep and ife results, patient demographics, clinical histories and requesting clinician details were retrieved from laboratory or hospital information systems. relevant data from the information systems were analysed and statistical analysis was performed using microsoft® excel (microsoft® office 2007, microsoft, usa). a p-value of < 0.05 was considered to be of statistical significance. results top ↑ a total of 1260 speps were performed from 1 february 2011 to 31 july 2011. analysis revealed that the reviewing pathologist suggested 304 ifes; these comprised 87 internal (ialch wards and clinics) samples and 217 referred samples (see figure 2). fifteen per cent (n = 45/304) of the suggested ifes were requested subsequently by attending clinicians and all ifes were performed by the laboratory, except for two patients with insufficient samples. of the 87 internal samples for which ifes were suggested, 14% (n = 12/87) were requested subsequently by the clinicians. of the 217 referred samples for which ifes were suggested, 15% (n = 33/217) were requested subsequently by the clinician. almost half (46.5%; n = 20/43) of all the suggested ifes that were performed revealed the presence of a paraprotein, indicating the possible presence of a plasma cell dyscrasia. figure 2a: figure 2a: number of ife tests (n = 304) suggested by the laboratory from among total spep requests (n = 1260). 2b: percentage of ife tests requested following laboratory suggestion for referred (n = 33 of 217) and internal (n = 12 of 87) samples. clinical histories were available on the laboratory information system (lis) for only 53% (n = 162/304) of the patients for which ifes were suggested. table 1 summarises the clinical histories (n = 13) that were available for all ifes performed (n = 43). of the six positive ifes for which clinical histories were supplied, four had the clinical diagnosis of 'query myeloma', one was peripheral neuropathy and one was spinal tuberculosis. table 1: clinical histories for the 43 laboratory-suggested ife tests requested by clinicians and performed. all the speps resulting in an ife suggestion by the laboratory were new tests for each of the 304 patients. for 11% (n = 33/304) of the patients, a repeat spep rather than an ife was submitted by the attending clinician. the time period between the initial spep request and the repeat spep ranged from one to 182 days (median 28.5 days). analysis revealed that of the repeat speps ordered on patients, protein patterns for only three patients changed such that the restriction bands were no longer visualised. the alpha-2-globulin/alpha-1-globulin ratios were calculated for performed ifes for patients with and without hypogammaglobulinaemia, as only four of the 43 patients had hypogammaglobulinaemia. the alpha-2-globulin/alpha-1-globulin ratio mean and standard deviation (sd) for all patients with positive ifes was 3.2 and 0.9, respectively; for those with negative ifes, the mean was 3.58 (sd 1.0). student’s t-test revealed no statistically-significant difference between the two groups (p-value = 0.2). discussion top ↑ this study revealed that only a minority of clinicians request immunofixations following laboratory suggestion for follow-up testing as a result of suspicious spep findings. the reasons for this need to be investigated further, but there are several plausible explanations. one explanation may be that not all clinicians, particularly junior clinicians, are familiar with the terms 'paraprotein' and 'immunofixation', or with the implications thereof. furthermore, clinicians may find it inconvenient to phone the laboratory to request the addition of an ife test. it must also be noted that requisition of ife testing was uncommon amongst all clinicians apart from clinical haematologists and haematologist-oncologists. during the study, only two ifes were requested by general practitioners/clinicians from other specialities.with regard to the above clinical practices, it is difficult to ascertain the baseline prevalence of monoclonal bands for samples received from the general population. no prevalence data exist for the presence of monoclonal gammopathies in the south african population. van vuuren et al. reported a prevalence of 3.2% for monoclonal gammopathies in a group of hiv-infected patients in south africa.9 spep, ife and sflc have been recommended as screening tests for plasma cell disorders (except primary amyloidosis) by the international myeloma working group. in his study, katzmann reported evidence to substantiate the use of a simplified screening panel for mm detection, consisting of spep and sflc alone.10 however, sflc studies are not currently available in this setting; furthermore, a large number of patients with mgus were missed in the study by katzmann because ifes were not performed for these individuals.10 worldwide, different studies have shown the prevalence of mgus to range from 1% to 10% and the prevalence of mm to be around 1% of all cancers diagnosed.11,12,13,14 in view of these data, this study’s finding of the presence of monoclonal bands for almost half of those ifes requested following laboratory suggestion, indicates that the prevalence of monoclonal bands in patients with suspicious spep findings is greater than what would be found in the general population. this highlights the need for reflective testing and the addition of reflective immunofixation by the performing laboratory for suspicious findings on spep. the results of this study indicate that reflective testing is of particular importance for spep requests from clinicians who are not haematologists. this finding is of particular importance because the majority of patients with monoclonal gammopathies present initially to general practitioners and non-specialist clinicians.15 reflective testing requires input such as clinical history in order for a test to be added. the task is made more difficult if clinical histories are not readily available at the time of test review. the findings indicated that clinical histories accompanied only 53% of all speps that had suspicious findings and required possible reflective testing. these findings correlate with another study, which showed that 60% of laboratory request forms did not provide clinical information regarding diagnosis or patient clinical history; however, the availability of clinical information in this study was higher than reported previously in another south african referral laboratory.16,17 the lack of clinical information is a significant additional issue affecting interpretation and reflective testing for all laboratory testing. further education of healthcare workers with regard to inclusion of clinical histories when requesting tests may be needed. this education should ideally start with undergraduate healthcare worker training but should continue as part of continued education and communiqués provided by the laboratory services, particularly to medical interns, phlebotomists and nurses. another possible method to promote provision of clinical histories could be through utilisation of the laboratory and hospital information system to prevent the complete placement of a test order request if a clinical history is not provided. in order to assist with the process of reflective testing for spep/ife, the possible use of another parameter, the alpha-2-globulin/alpha-1-globulin ratio, was examined in patients with and without hypogammaglobulinaemia. this parameter is calculated from spep densitometry results and thus requires no added cost. the findings in this population did not show any statistically-significant correlation between the ratio and the presence of positive ife findings. the elevated ratio of alpha-2-globulin/alpha-1-globulin ratio may still have a role in patients with hypogammaglobulinaemia, as described by lakshminarayanan et al.,8 however, the findings could indicate that the ratio is unlikely to assist with detection of a monoclonal protein amongst the population examined in this study. limitations of the study the use of a single pathologist to report on speps allows for greater consistency. a potential shortfall of this approach is the possibility of overcalling or undercalling of suspicious bands on spep by the single pathologist. however, it should be noted that almost half of the suggested ifes requested were found to be positive. this finding, together with the poor rate of ife requisition following suggestion, substantiates the need for reflective testing for spep. recommendations further studies involving immunofixation should be performed for all patients with suspicious spep. conclusion in conclusion, both the laboratory and the user need to ensure that sufficient clinical information is provided to optimise reflective testing in a resource-limited environment. acknowledgements top ↑ we would like to acknowledge the laboratory technologists at ialch who performed the electrophoresis testing that made this study feasible. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions v.g. (inkosi albert luthuli central hospital; nelson r mandela school of medicine) conceived of the study and y.r. 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incomplete laboratory request forms: the extent and impact on critical results at a tertiary hospital in south africa. ann clin biochem. 2008;45(pt 5):463–466. abstract background systematic search methods literature search and characteristics of the studies included in the systematic review results discussion acknowledgements references about the author(s) emmanuel o. irek department of medical microbiology and parasitology, obafemi awolowo university teaching hospitals complex, ile-ife, osun, nigeria adewale a. amupitan department of medical microbiology and parasitology, obafemi awolowo university teaching hospitals complex, ile-ife, osun, nigeria temitope o. obadare department of medical microbiology and parasitology, obafemi awolowo university teaching hospitals complex, ile-ife, osun, nigeria aaron o. aboderin department of medical microbiology and parasitology, obafemi awolowo university teaching hospitals complex, ile-ife, osun, nigeria department of medical microbiology and parasitology, obafemi awolowo university, ile-ife, osun, nigeria citation irek eo, amupitan aa, obadare to & aboderin ao. a systematic review of healthcare-associated infections in africa: an antimicrobial resistance perspective afr j lab med. 2018;7(2), a796. https://doi.org/10.4102/ajlm.v7i2.796 review article a systematic review of healthcare-associated infections in africa: an antimicrobial resistance perspective emmanuel o. irek, adewale a. amupitan, temitope o. obadare, aaron o. aboderin received: 01 mar. 2018; accepted: 20 sept. 2018; published: 06 dec. 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: healthcare-associated infection (hcai) is a global health challenge, not only as an issue of patient safety but also as a major driver of antimicrobial resistance (amr). it is a major cause of morbidity and mortality with economic consequences. objective: this review provides an update on the occurrence of hcai, as well as the contribution of emerging amr on healthcare delivery in africa. methods: we searched pubmed, cochrane database, african journals online and google scholar for relevant articles on hcai in africa between 2010 and 2017. preferred reporting items of systematic reviews and meta-analyses guidelines were followed for selection. thirty-five eligible articles were considered for the qualitative synthesis. results: of the 35 eligible articles, more than half (n = 21, 60%) were from east africa. klebsiella spp., staphylococcus aureus, escherichia coli and pseudomonas spp. were the common pathogens reported in bloodstream infection, (catheter-associated) urinary tract infection, surgical site infection and healthcare-associated pneumonia. among these various subtypes of hcai, methicillin-resistant s. aureus (3.9% – 56.8%) and extended-spectrum beta-lactamase producing gram-negative bacilli (1.9% – 53.0%) were the most reported antimicrobial resistant pathogens. conclusion: this review shows a paucity of hcai surveillance in africa and an emergence of amr priority pathogens. hence, there is a need for a coordinated national and regional surveillance of both hcai and amr in africa. background healthcare-associated infection (hcai) is a global health challenge, not only as an issue of patient safety but also as a major driver of antimicrobial resistance (amr). the emergence and spread of amr threatens effective control and treatment of various infections worldwide.1,2 these infections, often caused by multidrug-resistant organisms, take a heavy toll on patients and their families by causing illness, prolonged hospital stay, potential disability, excess costs and sometimes death.3,4,5 thus, hcais are major causes of preventable morbidity and mortality in lowand middle-income countries where infection rates are relatively higher due to poor infection control practices, inappropriate use of limited resources, under-staffing of healthcare facilities and overcrowding of hospitals.6 healthcare-associated infections rank among the 10 leading causes of death in the united states, accounting for 1.7 million affected individuals and about 99 000 deaths in 2002 and resulting in up to usd33 billion of excess medical costs every year.7 in england, more than 100 000 cases of hcai are estimated to cost £1 billion and directly cause over 5000 deaths annually.5 although data are sparse, evidence suggests hcai exerts greater burden in developing countries. pooled prevalence of hcai in developing countries is 15.5 per 100 patients (95% confidence interval; 12.6–18.9), with surgical site infections being the leading hcai, caused mainly by gram-negative organisms and multidrug-resistant organisms. multidrug-resistant organisms account for 25% of hcai globally.2,5 there are different types of hcais as highlighted by the national healthcare safety network patient safety component manual of the united states centers for disease control and prevention.8,9 these include urinary tract infection (uti), which is usually catheter-related, surgical site infection (ssi), bloodstream infection (bsi) – laboratory-confirmed bloodstream infection or central-line associated bloodstream infection – and pneumonia (clinically-defined pneumonia or ventilator-associated). other hcais occur in the bones, joints, central nervous system, cardiovascular system (e.g. endocarditis) and in the skin and soft tissue. generally, hcai pervades all health systems across the divide of developed and developing economies globally. for every 100 hospitalised patients at any given time, 7 in developed and 10 or more in developing countries will acquire at least one hcai.7 moreover, about 5% – 10% of patients admitted to hospitals in developed countries acquire one or more hcais, with 15% – 40% of those admitted into the intensive care unit being most affected.2,5 antimicrobial resistant pathogens involved in hcai include methicillin-resistant staphylococcus aureus (mrsa), penicillin-resistant pneumococci, vancomycin-resistant enterococci, extended-spectrum beta-lactamase (esbl)-producing enterobacteriaceae and carbapenem-resistant enterobacteriaceae. the epidemiological gaps leading to the absence of reliable estimates of the global burden are mainly because surveillance of hcai consumes time and resources and requires expertise in data collection, analysis and interpretation.5,10 previous systematic reviews of hcai in developing countries11 covered the period between 1995 and 2008, while another, which focused on the world health organisation (who) african region, covered between 1995 and 2009.12 these reviews highlighted the need for boosting microbiological diagnostic capacity for hcai, increased infection prevention and control (ipc) practices, as well as frequent surveillance of hcai. only a few african countries have established national surveillance systems for hcai as emphasised by the who patient safety module.10 furthermore, pockets of data on hcai from different healthcare facilities across africa differ in methodological approach. this review provides an update (2010–2017) on the occurrence of hcai, as well as the contribution of emerging antimicrobial resistance in healthcare delivery in africa. systematic search methods this systematic review was conducted in line with the preferred reporting items for systematic reviews and meta-analyses (prisma), guidelines.13 pubmed, cochrane database, and african journals online databases were primarily searched for relevant articles using specific search terms (figure 1). other articles were obtained from google scholar. the literature search included articles from january 2010 to january 2017. the review included articles written only in the english language, as well as articles on all types of patient populations. we excluded duplicate articles, publications reporting the same data, outbreaks of hcai and data of surveillance beyond africa. we obtained the full text of potentially relevant studies and scrutinised them independently. then we screened the potentially relevant studies for further eligibility figure 2. figure 1: search terms used in the systematic review. figure 2: summary of article selection. criteria for selecting the articles included definitions used for hcai diagnosis, reported hcai prevalence or incidence, identified microbiological isolates and patterns of antimicrobial resistance (when documented). we only judged microbiological data suitable for assessment when the number of bacterial isolates was reported in relation to in-patients having suspected hcai. healthcare-associated infections included in this review were as defined by the united states centers for disease control and prevention,9 that is, infections that develop in in-patients on or after the third (> 48 h) day of admission. hence, we identified catheter-associated bsi, ssi, uti (catheter-associated or not), pneumonia (ventilator-associated or not) as the major subtypes of hcai, and we categorised other infections associated with healthcare service delivery, such as gastroenteritis, skin and soft tissue infection, as ‘others’. literature search and characteristics of the studies included in the systematic review we identified 8410 records from the search of the electronic databases. the number of full text articles screened was 7003 after the removal of duplicate studies, of which 193 studies were potentially eligible. however, only 35 articles were finally selected for qualitative synthesis for this review according to the aforementioned inclusion criteria.9,11 data were pooled from both prevalence and incidence studies and afterwards summarised in table 1. the prevalence of infection refers to infected patients per patients present in the hospital or ward at a given point in time. table 1: summary of eligible reviewed articles on healthcare-associated infection in africa published between 2010 and 2017. table 1: (continues...): summary of eligible reviewed articles on healthcare-associated infection in africa published between 2010 and 2017. results two-thirds of the reviewed articles were from pubmed, one-third were from african journals online and google scholar, and none were retrieved from the cochrane library. further, more than half (n = 21, 60%) of the synthesised articles were from east africa, whereas the rest were shared across northern, western, southern and central africa (figure 3). only one article reported an incidence study,14 whereas the rest were prevalence studies (retrospective or prospective). five (14.3%) of the reviewed articles based their categorisation on specific microorganisms isolated,15,16,17,18,19 whereas some others were based on specific hcai.14,16,17,20,21,22,23,24,25,26,35,37,38,39,44,45,46,47,48 only eight studies (22.9%)28,29,30,31,32,33,34,35 covered hcai in entirety and conducted full surveillance of the different types enumerated by previous published protocols.8,36 eight articles (22.9%), however, did perform hcai surveillance without the mention of the microorganisms implicated.24,34,35,37,38,39,40,41 the identification of antimicrobial resistance in the panel of laboratory investigation was included in less than half (n = 16, 46%) of all the reviewed articles14,15,17,18,20,23,25,26,27,29,31,32,33,42,43,44 with only four articles broadly identifying amr as multidrug-resistant organisms without further characterisation.17,20,42,43 in addition, only three of the articles specified the prevailing microorganisms of the various subtypes of hcai in surveillance.31,32,33 the phenotypic method was mostly utilised for the identification of the microorganisms in the laboratory according to clinical and laboratory standards institute guidelines.15,16,19,22,23,25,27,31,41,42,43,44,45,46,47,48 figure 3: distribution and number of eligible published articles on healthcare-associated infections in different african countries. furthermore, surveillance on (central line-associated) bsi was recorded in 14 (40%) of the reviewed articles14,15,18,23,28,29,30,31,32,33,34,44,49 and these were confirmed with blood culture. some of these articles, however, evaluated only bsi,14,23,34,44 whereas others included bsi with other hcai surveillance subtypes (table 1). bsi in some individuals was episodic and some others were central line-associated. diverse microorganisms implicated in bsi in order of decreasing frequency included klebsiella spp., s. aureus, e. coli, pseudomonas spp. and acinetobacter spp. (table 1). klebsiella spp. and staphyloccocus spp. were the most frequently identified causes of bsi. esbl producers and methicillin-resistant staphylococcus spp. were the most identified antibiotic-resistant microorganisms in the bsi articles (that mentioned amr within their panel of laboratory investigation).23,29,31,32,33,44 only one article reported vancomycin-resistant enterococci in bsi,18 and another recorded an escalating antibiotic resistance of acinetobacter baumannii to the carbapenems.32 surveillance for ssi was common among the reviewed articles. over half of the reviewed articles had ssi within the context of their surveillance, of which only 13 focused solely on ssi.16,19,21,22,24,25,27,37,38,39,43,46,48 wound swabs and wound biopsies were specimens taken for microbiological investigation. some occurrence of ssi were associated with caesarean sections or orthopaedic manoeuvres. the microorganisms most commonly isolated were s. aureus, e. coli, klebsiella spp., pseudomonas spp., in the order of decreasing frequency. common antimicrobial resistant organisms identified in ssi articles reviewed were mrsa and esbl-producing gram-negatives. in this systematic review, catheter-associated uti was seen mainly in urologic conditions such as prostatic enlargement and post-gynaecological procedures. some reviewed articles (n = 10; 29%) categorised healthcare-associated uti (catheter-associated inclusive) as a subset of other hcai surveillance types.15,18,29,30,31,32,33,34,40,41 common microorganisms isolated from healthcare-associated uti included klebsiella spp., e. coli, enterococcus spp., and pseudomonas spp. in addition, mrsa, vancomycin-resistant enterococci and esbl-producing gram-negative bacilli were the most common antimicrobial resistant pathogens noticed in some identified bacteria for healthcare-associated uti among the reviewed articles. only one study included healthcare-associated pneumonia as a lone subtype of hcai,20 whereas many others included it as a subset of hcai surveillance types.15,28,29,30,32,35,40 common microorganisms reported among these articles included klebsiella spp., pseudomonas spp., s. aureus and e. coli. as with other hcai subtypes in this review, mrsa and esbl-producing gram-negative bacilli were the most common antimicrobial resistant pathogens seen. other hcai studied in this systematic review were gastroenteritis,18,30,31 and skin and soft tissue infection.17,30,31,33,35,49 overall, for the reviewed articles that identified amr, the prevalence of methicillin-resistant staphylococcus spp. ranged between 3.9% and 80% among the staphylococcus spp. (s. aureus and coagulase negative staphylococci) reported.16,23,25,27,29,33 the prevalence of gram-negative bacteria producing esbl23,25,26,27,31,32,44 ranged between 1.9% and 53%, whereas vancomycin-resistant enterococci15,18,33 was between 2.54% and 100%. discussion until recently in africa, evidence on the enormity and debilitating effects of hcai on patients (and relatives of patients) has been low. the resultant effect of many studies conducted in developed countries was to propose a singular surveillance platform for hcai across their sub-regions.8,50 this was intended to identify gaps and target control of hcai. however, the gravity of hcai is yet to be fully understood in africa due to the enormous resource requirements for surveillance and diagnoses.51 this was evident by the paucity of studies identified in this review (figure 3). although a well-documented protocol for hcai has been proffered by the united states centres for disease control and prevention,8,9 only a few studies we reviewed adhered to it or any other protocol of interest. also, the robustness, reproducibility and inferences from methodology used in the reviewed articles differed considerably from study to study, thus limiting comparability and robust analysis. this systematic review was also limited with search only done in english. additionally, a follow-up for trends on the hcai surveillance was rarely conducted in the different healthcare facilities where these studies were conducted. this would have given a clue to either the reduction or the increment of hcai in such centres, as seen in the archival documentation of the united states cdc. the prevalent bacteria identified in bsi in this review were klebsiella spp., s. aureus, e. coli, acinetobacter spp., in order of decreasing frequency, which slightly contradicts the order of occurrence in a previous review conducted in south east asia,52 where acinetobacter spp. was found to be the most prevalent organism causing bsi. a similar frequency of identification was seen in ssi surveillance, with s. aureus being the most common across reviews with different ecologies but similar healthcare issues of poor funding.11,52 again, as with bsi, klebsiella spp. was the most commonly identified pathogen in this review, which concurs with a similar review by ling et al.52 although few studies were identified in this review for ventilator-associated pneumonia, klebsiella spp. still remained highly prevalent among other bacteria identified, as noted in a similar study.52 these similarities in the bacteria isolated may be due to the likened levels of ipc practices and antibiotic usage, which can influence bacterial fitness.53 in addition, the range of occurrence of the amr patterns in the review articles was quite alarming, considering there have been few or no previous reviews on amr patterns in hcai pathogens in africa. the range of mrsa in this review was higher than that reported in the joint european surveillance of mrsa in hcai.54 this can be adduced to the relatively low ipc practices in africa,55 especially during surgeries or invasive procedures. also, the occurrence of esbl-producing gram-negative bacilli was higher than that obtained by flokas et al.56 that reported 14% in a systematic review of esbl in paediatric utis. moreover, an increased trend of esbl has been observed in the united states with recent incidence of about 16.64 infections in 10 000 discharges.57 the inadequate ipc strategies instituted in these healthcare facilities to prevent hcai compromise the quality of healthcare service delivery, hence the prevalence.11 previous reviews on hcai in developing countries and in the who african sub-region11,12 have emphasised the need for improved ipc in healthcare facilities to drastically reduce hcai prevalence. many of the selected studies14,20 mentioned the need for the establishment of ipc, whereas others identified bundle implementation (of the different subtypes of hcai)14,20,38,26 in curbing hcai in their centres. only one article studied the aftermath reduction of hcai using ipc measures.20 thus, studies on interventional ipc measures in the reduction of hcai are still quite juvenile in africa. this has been advocated by who as a means of measuring and sustaining progress on patient safety.51 in this review, most of the reviewed articles highlighted the corresponding amr patterns of the microorganisms implicated in hcai,15,16,18,23,25,26,27,29,31,32,33,44 whereas others simply mentioned them as multidrug-resistant organisms.17,20,42,43 this may be due to inadequate laboratory capacity to identify the specific amr patterns. this also gives a foreknowledge of the existing prevalence of amr microorganisms in the healthcare facilities in africa and a possible spread to the communities if not curtailed. mrsa was identified in a previous review by allengrazi et al.11 as the most prevalent amr pattern implicated in hcai. this concurs with the amr pattern in this review. the presence of esbl was also noticed to be prevalent alongside mrsa in this review. carbepenem-resistant organisms have been known globally to cause much mortality and morbidity,1,58 and are widely implicated in hcai,50,58 but were rarely mentioned in the synthesised articles. however, one study highlighted carbapenem resistance in acinetobacter baumannii as hcai in an intensive care unit in libya.32 furthermore, antimicrobial stewardship has also been identified as a major solution to the rising rates of amr worldwide.59,60 the recognition of this was, however, of little priority in the articles reviewed, with only a limited number of studies15,18,23,25,27,30,37,31,33 highlighting the importance of antimicrobial stewardship in the reduction of amr in hcai. countries in africa have a wide variation in the capacity to combat amr in hcai, but have been greatly hampered by the availability of funds for research, innovation and capacity building.61,62 this is worsened by a lower percentage of total health spending in african countries.63,64 thus, the true burden of hcai in this systematic review is likely to be under-reported and is perhaps greater in countries with weaker health infrastructures. however, this narrative is changing with increasing commitment in africa to respond to the global threat of amr. in-country technical capacity with support from partners is now evolving not only to develop national action plans to combat amr, but also to institute national surveillance for amr. the who global antimicrobial surveillance system provides a tool to standardise data gathering, sharing and analysis through participating institutions and countries at the global level to monitor trends and implement controls.61,62,65,66 moreover, with the current global attention and high–level political commitment to control amr, funding support for amr control in africa is coming from various organisations, which include the who, react africa, the center for disease dynamics, economics and policy and the fleming fund.61,67,68 for sustainability, countries also should have budget lines for amr control activities either as stand-alone or, more realistically, as part of existing systems such as ipc, maternal and child health and health systems strengthening. monitoring and evaluation has to be incorporated as the systems develop. current platforms to do this include the global antimicrobial surveillance system,66 which accepts annual surveillance data that have been aggregated in-country, and the global point prevalence survey,69 which monitors antibiotic prescription patterns to enhance stewardship. hence, report on surveillance and trends of hcai and amr occurrence should inform regular updates on guidelines (treatment and ipc) and antibiotic stewardship protocols at the national level, while at the institutional level, evidence will inform accreditation for services or training. conclusion although prevention and evolution of hcai and the reduction of the occurrence of amr globally have been a primary focus of who,51 little has been done to combat it in africa. in addition, surveillance has been known to reduce the burden of hcai in developed healthcare facilities,7 where conscious means of prevention have been instituted accordingly. the inadequate coordination of regional and intra-continental surveillance in africa led to inconsistencies and non-uniformity in many reported studies of hcai in this review. this made it difficult to interpret data to display true representativeness. hopefully, there will be a coordinated national and sub-regional hcai surveillance as an agenda of the newly created africa centres for disease control and prevention. finally, this systematic review has compiled all relevant, accessible and eligible studies on hcai in africa as a baseline for further insight into developing a concrete surveillance system and strengthening local data collection at healthcare facilities. there seems to be a higher number of studies on hcai compared to previous reviews.12 klebsiella spp. was prevalent across all the hcai subtypes. mrsa and esbl-producing gram-negative bacilli were the notable resistant pathogens identified with worrisome occurrences. these make a strong case for increased laboratory capabilities in the identification of microorganisms and determination of resistance profiles (especially in who priority pathogens)70 implicated in hcai. henceforth, it is desirable that a periodical review of amr and hcai in africa be conducted in view of current interest. acknowledgements we wish to acknowledge the contribution of nigeria centres for disease control to this manuscript. we also thank professor iruka n. okeke and professor a.t. olayinka for the materials provided from the situation analysis for the nigeria action plan on antimicrobial resistance (http://www.ncdc.gov.ng/themes/common/docs/protocols/77_1511368219.pdf). competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support none. authors’ contributions a.o.a was the project leader and also made the conceptual contribution. e.o.i., t.o.o. and a.a.a. performed the search of literature. e.o.i and t.o.o compiled the figures and the table. a.a.a contributed to the editorial changes and a.o.a critically reviewed the manuscript. references world health organization. antimicrobial resistance. 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[serial online]. 2017 oct 31 [cited 2018 feb 25];38(10):1209–15. available from: https://www.cambridge.org/core/product/identifier/s0899823x17001568/type/journal_article centers for disease control and prevention (cdc). facility guidance for control of carbapenem-resistant enterobacteriaceae (cre). atlanta, ga: national center for emerging and zoonotic infectious diseases; 2015, p. 24. ecdc. point prevalence survey of healthcare-associated infections and antimicrobial use in european acute care hospitals. version 5. eur centre dis prevent control; 2016. 90 p. cdc. antibiotic use in the united states, 2017: progress and opportunities. atlanta, ga: department of health and human services, cdc;2017. mendelson m, dar oa, hoffman sj, laxminarayan r. global antimicrobial conservation fund for lowand middle-income countries. int j infect dis. 2016;51:70–2. world health organisation. source>global antimicrobial resistance surveillance system [homepage on the internet]. world health organisation. 2015. available from: http://apps.who.int/iris/bitstream/10665/188783/1/9789241549400_eng.pdf?ua=1 clarke g, desai r, hallward-driemeier m, et al. world development report 2005: a better investment climate for everyone [homepage on the internet]. 2005 [cited 2018 august 20]. available from: http://documents.worldbank.org/curated/en/876951468158729105/relatorio-sobre-o-desenvolvimento-mundial-2005-um-melhor-clima-de-investimento-para-todos university of washington center for health trends and forecasts. financing global health 2017 | institute for health metrics and evaluation [homepage on the internet]. 2017 [cited 2018 aug 20]. available from: http://www.healthdata.org/infographic/financing-global-health-2017 world health organisation. global action plan on antimicrobial resistance [homepage on the internet]. geneva: world health organization; p. 28. 2015 [cited 2018 august 20] available from: http://apps.who.int/iris/handle/10665/193736 world health organization. global antimicrobial resistance surveillance system [homepage on the internet], 36 p. 2015 [cited 2018 aug 20] available from: http://apps.who.int/iris/bitstream/10665/188783/1/9789241549400_eng.pdf?ua=1 fundingabout us – react africa [homepage on the internet]. [cited 2018 sep 18]. available from: https://www.reactgroup.org/about-us/funding/ hellen g, okeke in, aboderin ao, martinez e, matu m. east africa public health laboratory networking project strengthening the role of laboratories in tracking antimicrobial drug resistance in east africa [homepage on the internet], p. 40. 2016 [cited 2018 aug 20] available from: http://cddep.org/sites/default/files global pps [internet]. [cited 2018 oct 25]. available from: http://www.global-pps.com/ knols bg, smallegange rc, tacconelli e, magrini n, kahlmeter g, singh n. global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics. lancet infect dis. 2016;9(9):535–6. acknowledgements references about the author(s) collins o. odhiambo african society for laboratory medicine, addis ababa, ethiopia anafi mataka african society for laboratory medicine, addis ababa, ethiopia marguerite massinga loembe african society for laboratory medicine, addis ababa, ethiopialaboratory division, africa centres for disease control and prevention, addis ababa, ethiopia pascale ondoa african society for laboratory medicine, addis ababa, ethiopiaamsterdam institute for global health and development, department of global health, university of amsterdam, amsterdam, the netherlands citation odhiambo co, mataka a, massinga loembe m, ondoa p. maintaining routine hiv and tuberculosis testing services in sub-saharan african countries in the context of covid-19: lessons learnt and opportunities for improvement. afr j lab med. 2021;10(1), a1413. https://doi.org/10.4102/ajlm.v10i1.1413 opinion paper maintaining routine hiv and tuberculosis testing services in sub-saharan african countries in the context of covid-19: lessons learnt and opportunities for improvement collins o. odhiambo, anafi mataka, marguerite massinga loembe, pascale ondoa received: 29 sept. 2020; accepted: 24 mar. 2021; published: 17 june 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. since being declared a public health emergency of international concern on 30 january 2020, the coronavirus disease 2019 (covid-19) has spread internationally, reaching the stage of a global pandemic.1 african countries quickly put in place social and public health measures to limit the spread of the disease, with some of the most ‘visible’ measures being lockdowns, physical distancing and the overall surge of healthcare services to support the covid-19 response. the director-general of the world health organization, calling for increased testing, recommended ‘test, test, test’ as a critical step to contain the spread of the disease.2 when the first african case was reported in egypt in february 2020, only two centres of excellence laboratories on the continent were capable of conducting severe acute respiratory syndrome coronavirus 2 polymerase chain reaction testing, the gold standard assay recommended by the world health organization. one key strategy to swiftly scale-up the testing capacity for severe acute respiratory syndrome coronavirus 2 has been the re-tooling of existing nucleic acid amplification testing platforms toward the control of covid-19. nucleic acid amplification testing platforms available from hiv and tuberculosis control programmes offered a unique opportunity given their large footprint across several countries and the availability of in-service training programmes for the operators. at least three covid-19 molecular diagnostic assays listed under the emergency use authorisations of the united states food and drug administration or the world health organization can be used on platforms commonly available within the hiv and tuberculosis programmes – the abbott m2000 realtime system, cepheid’s genexpert® system, and the roche cobas 6800/8800 system.3 although covid-19 testing volumes in most african countries remain below or at the lower threshold required for relaxing the containment measures (10–30 tests per confirmed case),4 have we gone a step too far in refocusing most of the laboratory capacity to covid-19 testing? mounting evidence suggests that the ‘covidisation’ of the healthcare system and, more specifically, diagnostics poses a risk to maintaining other routine testing services for the control and prevention of endemic diseases like hiv, malaria, syphilis and tuberculosis.5 additionally, lockdowns imposed in most countries prevent patients from accessing healthcare and this may result in increased treatment failure among those on medication, and increased maternal and child mortality in the context of poorly attended childbirth and reduced antenatal care. without intervention, there is a risk of reversing many of the gains achieved towards meeting the targets of the joint united nations programme on hiv/aids 95:95:95, the end tb strategy, and the united nations sustainable development goals, including ending the ‘big three’ diseases – hiv, tuberculosis and malaria. hiv, tuberculosis and malaria programmes have made substantial progress towards achieving global targets due to massive donor funding. however, the joint united nations programme on hiv/aids estimates that there could be hundreds of thousands of extra deaths from hiv if routine services, including hiv screening, viral load and early infant diagnosis, are disrupted.6 eighty-five percent of national-level respondents from 61 countries participating in a world health organization, united nations children’s fund and global aids vaccine initiative poll reported lower vaccination proportions in may 2020 compared to the level in january 2020 – february 2020.7 additionally, disruption of maternal and child health services is estimated to contribute to at least 8% more deaths per month.8 a survey of the global fund’s supported programmes across 106 countries revealed that at least 85%, 80% and 76% of hiv, tuberculosis and malaria control services, respectively have been disrupted by the covid-19 pandemic.9 a recent modelling analysis from the stop tb partnership further illustrated how an additional lockdown period of three months could lead to 6.3 million more cases of tuberculosis globally by 2025, causing a setback of at least five years on the progress achieved thus far.10 more specifically, the survey revealed that at least 20% of hiv and tuberculosis laboratory services are facing high disruption with much of the advanced diagnostic equipment repurposed and the workforce reassigned for covid-19 testing. a world health organization survey of essential services revealed that hiv testing (n = 38) and viral load monitoring (n = 23) were the two most frequently interrupted testing services in the 61 countries that responded.11 it is important to note that laboratory testing is key to identifying disease cases, breaking transmission chains and monitoring patients on treatment. in outbreak situations, healthcare stakeholders at the national level must be able to quickly address testing needs and, at the same time, identify the testing threshold needed to ensure that routine testing services are maintained and health targets for the control of essential diseases remain on track. however, data about the overall function of health services are usually fragmented and rarely realtime on the continent. furthermore, movement restrictions have further hampered the collection of information on the continuation of essential healthcare services, particularly diagnostics. the african society for laboratory medicine through the laboratory systems strengthening community of practice (labcop)12 conducted an online survey (june 2020) among labcop member countries to seek a deeper understanding of the disruptions in hiv and tuberculosis testing services during covid-19 diagnosis scale-up. the labcop is funded by the bill and melinda gates foundation and is composed of multidisciplinary teams (clinicians, laboratorians and civil society) with strong collaboration with the ministries of health from 14 sub-saharan african countries.12 consensus responses to the survey questionnaire were received from the multidisciplinary teams of 10 of the 14 countries. among those, nine countries reported a decline in viral load testing volumes, eight reported a decline in tuberculosis genexpert testing volumes, while seven reported a decline in early infant diagnosis volumes during the covid-19 response, thereby confirming the global fund survey findings.9 in uganda, one of the few labcop participant countries with a publicly available viral load dashboard for testing services, there was a 28% reduction in viral load test volume (from 104 474 to 74 841 tests) between march 2020, when the first covid-19 case was reported in uganda, and may 2020.13 the main reason provided by most countries (9 of 10 countries) for the decline in testing volumes was either the inability of patients to visit the health facilities to access hiv and tuberculosis services due to movement restrictions aimed at limiting covid-19 transmission (at least 42 african countries had imposed partial or complete lockdowns as of may 2020) or patient fear of contracting the disease while visiting health facilities. five countries reported stock-outs of testing kits, reagents and laboratory consumables as a result of border closures. four countries indicated that the surge in covid-19 testing volumes caused or exacerbated the shortage of healthcare workers, as they were reassigned to support the covid-19 response (especially personnel skilled in molecular testing), thereby affecting the provision of other essential diagnostics based on polymerase chain reaction technology. whereas 8 of 10 countries reported re-purposing between 25% to 83% of the total hiv and tuberculosis testing equipment capacity in-country for covid-19 testing, only two countries identified insufficient molecular testing capacity as a barrier to maintaining hiv and tuberculosis diagnostic services. two separate analyses indicated that most hiv and tuberculosis instruments are often operated below their full capacity,14,15 indicating that available instruments in most countries may be sufficient to support both covid-19 and hiv and/or tuberculosis testing. moreover, under the impulse of strong hiv and tuberculosis disease control programmes funded by the united states president’s emergency plan for aids relief and the global fund, the equipment is used almost exclusively for one disease area due to vertical programming, despite the instruments’ multiplexing capability and the recommendation to ‘integrate’ testing.16 additionally, many of the countries who either repurposed hiv and/or tuberculosis equipment for covid-19 testing or refocused testing still experience challenges in long turn-around times for results, quality assurance and procurement issues, among others, indicating systemic weaknesses that need attention. the difficulty in scaling the covid-19 response while maintaining routine testing services for other essential diseases indicates that many countries are still struggling to achieve a multipurpose, resilient and effective laboratory network to address the needs of clinical and surveillance testing services and meet the prevention and control targets for all essential diseases.17 with more than a decade of large initiatives aimed at laboratory system capacity building in africa, what are we still missing to ensure that laboratory networks can more effectively forecast and organise laboratory services in both routine and emergency situations? through the labcop country annual self-assessment reports, we have been able to identify some of the inherent weaknesses in the viral load testing cascade, including laboratory network optimisation and monitoring and evaluation (m&e). laboratory stakeholders at the central level often do not have a comprehensive overview of available resources and capacities across the network, or how these can be leveraged to strengthen the various functions of the laboratory system; for example, how higher-tier laboratories can organise external quality assessment for those in lower tiers. further, at the laboratory level, managers and directors often lack the managerial skills and leadership needed to organise and implement comprehensive laboratory management systems that enable laboratories to conduct the tiered functions of the network for multiple diseases; these include knowledge in determining staffing levels, testing capacity, and roles and responsibilities, as well as skills in the use of geographic information system tools like labmap18 and labready to map, analyse and optimise the laboratory network capacity, functions and services. an inclusive approach that fosters strong collaboration across sectors (i.e., public and private, academic, civilian and military, human and animal health, vertical disease programmes, etc.), as well as an evidence-based decision-making process based on available laboratory data, should be used to inform scale-up of testing for covid-19 and any emerging disease. our survey has highlighted that evidence was seldom leveraged to inform scale-up of covid-19 diagnostic services; only one of three countries that reported the procurement of additional molecular testing equipment to support the surge in covid-19 testing identified a shortage of testing platforms as the reason for the reduction in hiv/tuberculosis testing, whereas only two of the four countries that reported hiring additional laboratory staff identified insufficient staff capacity as a gap. to address some of these gaps, the african society for laboratory medicine is working with various stakeholders, including the association of public health laboratories, the clinton health access initiative, the foundation for innovative new diagnostics, the world health organization, and the united states centers for disease control and prevention, among others, to develop a leadership and mentorship programme focused on leading and managing tiered laboratory networks to offer clinical care and public health services during routine and emergency situations. a guidance document for scaling up covid-19 diagnosis within the laboratory network while maintaining essential testing has been developed.19 additionally, the african society for laboratory medicine is convening an m&e sub-community of practice of the labcop articulated around the delivery of a fit-for-purpose m&e training and mentoring curriculum with the opportunity for direct technical assistance to labcop country teams. the key outcome of this initiative is the development or improvement of national dashboards to monitor and evaluate the implementation and performance of the hiv viral load testing cascade and diagnosis of other diseases, including covid-19. additionally, it is anticipated that the multi-country discussion will highlight best practices and identify actual needs that will inform the update of the current guidance document for establishing an m&e framework.20 the difficulty of scaling up covid-19 diagnostics while maintaining routine testing of hiv, tuberculosis and other essential diseases relates to pre-existing laboratory systemic weaknesses. solutions to durably tackle the gaps crippling diagnostic services include better knowledge, management and optimisation of national tiered networks to become more resilient in the face of health emergencies. attaining skills in the management of laboratory networks and strengthening of m&e systems in-country will contribute to better and faster identification of strengths and bottlenecks and mutualisation of existing resources across the laboratory network for routine and emergency testing needs. additionally, robust political commitment and sufficient provision of domestic funding are needed to ensure that all local health needs are adequately covered and are not compromised when addressing global priorities. acknowledgements the authors thank members of the labcop country teams for their responses to the survey questionnaire. we also thank dr george alemnji, dr clement zeh and jason williams for the insightful discussion on barriers to maintaining routine hiv and tuberculosis testing in the context of covid-19. we are grateful to mr michael waweru and mr getachew kassa for supporting the design of the questionnaire and for reviewing this manuscript. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions c.o.o. and p.o. conceptualised the idea, c.o.o. drafted the original manuscript, and p.o., a.m. and m.m.l. made substantial revisions to the manuscript for intellectual content. all authors reviewed and approved the manuscript. ethical considerations ethical clearance was not required for the study. this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support this work would not have been possible without funding from the bill and melinda gates foundation (investment number inv-003603) and resolve to save lives through the project ‘strengthening national capacity for covid 19 surge testing in selected countries across africa’ (surge cov19 testing, grant code: 4406-20-1-vs-covid-19-response). data availability statement data sharing is not applicable to this article. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references cucinotta d, vanelli m. who declares covid-19 a pandemic. acta biomed. 2020;91(1):157–160. who calls for more coronavirus testing: ‘test, test, test’ [homepage on the internet]. 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[cited 2020 jul 24]. available from: https://www.theglobalfund.org/media/9819/covid19_mitigatingimpact_report_en.pdf?u=637293077390000000 stop tb partnership. the potential impact of the covid-19 response on tuberculosis in high-burden countries: a modelling analysis. 2020 [cited 2020 sept 28]. available from: http://www.stoptb.org/assets/documents/news/modeling%20report_1%20may%202020_final.pdf who. disruption in hiv, hepatitis and sti services due to covid-19 [homepage on the internet]. 2020 [cited 2020 sept 28]. available from: https://www.who.int/docs/default-source/hiv-hq/disruption-hiv-hepatitis-sti-services-due-to-covid19.pdf?sfvrsn=5f78b742_6 aslm. labcop [homepage on the internet]. [cited 28 sept 2020]. available from: https://aslm.org/what-we-do/labcop/ uganda viral load dashboard [homepage on the internet]. [cited 22 jun 2020]. available from: https://vldash.cphluganda.org lecher s, williams j, fonjungo pn, et al. progress with scale-up of hiv viral load monitoring – seven sub-saharan african countries, january 2015–june 2016. mmwr morb mortal wkly rep. 2016;65(47):1332–1335. https://doi.org/10.15585/mmwr.mm6547a2 cazabon d, pande t, kik s, et al. market penetration of xpert mtb/rif in high tuberculosis burden countries: a trend analysis from 2014–2016. gates open res. 2018;2:35. https://doi.org/10.12688/gatesopenres.12842.1 who. considerations for adoption and use of multi-disease testing devices in integrated laboratory networks [homepage on the internet]. 2017 [cited 2020 sept 28]. available from: https://apps.who.int/iris/bitstream/handle/10665/255693/who-htm-tb-2017.06-eng.pdf?sequence=1 who regional office for africa. the maputo declaration on strengthening of laboratory systems [homepage on the internet]. 2008 [cited 2011 aug 18]. available from: http://www.who.int/diagnostics_laboratory/maputo-declaration_2008.pdf african society of laboratory medicine. laboratory mapping program (labmap) [homepage on the internet]. [cited 2021 feb 22]. available from: http://www.aslm.org/what-we-do/laboratory-mapping/ labcop cookbook of best practices. decentralizing covid-19 pcr diagnostic capacity to sub-national level [homepage on the internet]. [cited 28 sept 2020]. available from: https://aslm.org/wp-content/uploads/2020/09/bookletlabcopcookbook4-2020-09-02-webquality.pdf?x64533 considerations for developing a monitoring and evaluation framework for viral load testing [homepage on the internet]. geneva: world health organization; 2019 [cited 2020 sept 28] (who/cds/hiv/19.5). license: cc by-nc-sa 3.0 igo. available from: https://apps.who.int/iris/bitstream/handle/10665/324745/who-cds-hiv-19.5-eng.pdf ajlm 5_2 introduction.indd reviewer acknowledgement open accesshttp://www.ajlmonline.org page 1 of 1 we would like to take this opportunity to thank all of those who provided scientific and logistical support for this issue of the african journal of laboratory medicine: should names have inadvertently been excluded from this list, the publisher apologises and undertakes to amend the exclusion in the next issue. ajlm african journal of laboratory medicine in an effort to facilitate the selection of appropriate peer reviewers for the african journal of laboratory medicine, we ask that you take a moment to update your electronic portfolio on www. ajlmonline.org for our files, allowing us better access to your areas of interest and expertise, in order to match reviewers with submitted manuscripts. if you would like to become a reviewer, please visit the journal website to register. to access your details on the website, you will need to follow these steps: 1. log into the online journal at http://www. ajlmonline.org 2. in your ‘user home’ [http://www.ajlmonline. org/index.php/ajlm/ user] select ‘edit my profile’ under the heading ‘my account’ and insert all relevant details, bio statement and reviewing interest. 3. it is good practice as reviewer to update your personal details regularly to ensure contact with you throughout your professional term as reviewer for the african journal of laboratory medicine. please do not hesitate to contact us if you require assistance in performing this task. publisher: publishing@aosis.co.za tel: +27 21 975 2602 fax: +27 21 975 4635 avelin aghokeng jemal ali abiy b. ambaye walter r. campos jeremiah chakaya karidia diallo elisabeth gerwing-adima sheba gitta edward kamau christopher k. kariuki sam kariuki stephania koblavi deme william lali elizabeth luman john n. nkengasong jean louis sankale tom shinnick ajlm 5_3 introduction_grey.pdf ajlm_5(3).pdf abstract introduction methods results discussion acknowledgements references about the author(s) andré trollip foundation for innovative new diagnostics (find) south africa, cape town, south africa renuka gadde becton, dickinson & company, franklin lakes, new jersey, united states tjeerd datema datos, leiden, the netherlands kamau gatwechi becton, dickinson & company, nairobi, kenya linda oskam datos, leiden, the netherlands zachary katz foundation for innovative new diagnostics (find), geneva, switzerland andrew whitelaw department of pathology, faculty of medicine and health sciences, stellenbosch university, south africa national health laboratory service, tygerberg hospital, cape town, south africa peter kinyanjui national public health laboratory, kenyatta national hospital, nairobi, kenya patrick njukeng global health systems solutions, isokolo, cameroon dawit a. wendifraw national clinical bacteriology and mycology reference laboratory, ethiopian public health institute, addis ababa, ethiopia ibrahimm mugerwa ministry of health, national health laboratories and diagnostic services-amr-national coordination centre, kampala, uganda grace najjuka national health laboratories and diagnostic services, kampala, uganda nicholas dayie department of medical microbiology, university of ghana medical school, accra, ghana japheth a. opintan department of medical microbiology, university of ghana medical school, accra, ghana heidi albert foundation for innovative new diagnostics (find) south africa, cape town, south africa citation trollip a, gadde r, datema t, et al. implementation of a customised antimicrobial resistance laboratory scorecard in cameroon, ethiopia and kenya. afr j lab med. 2022;11(1), a1476. https://doi.org/10.4102/ajlm.v11i1.1476 original research implementation of a customised antimicrobial resistance laboratory scorecard in cameroon, ethiopia and kenya andré trollip, renuka gadde, tjeerd datema, kamau gatwechi, linda oskam, zachary katz, andrew whitelaw, peter kinyanjui, patrick njukeng, dawit a. wendifraw, ibrahimm mugerwa, grace najjuka, nicholas dayie, japheth a. opintan, heidi albert received: 30 nov. 2020; accepted: 11 mar. 2022; published: 20 june 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: in low-resource settings, antimicrobial resistance (amr) is detected by traditional culture-based methods and ensuring the quality of such services is a challenge. the amr scorecard provides laboratories with a technical assessment tool for strengthening the quality of bacterial culture, identification, and antimicrobial testing procedures. objective: to evaluate the performance of the amr scorecard in 11 pilot laboratory evaluations in three countries also assessed with the stepwise laboratory quality improvement process towards accreditation (slipta) checklist. methods: pilot laboratory evaluations were conducted in cameroon, ethiopia and kenya between february 2019 and march 2019. assessors with previous slipta and microbiology experience were trained. assessors performed the laboratory assessments using the slipta and amr scorecard tools. results: weaknesses in technical procedures and the quality management systems were identified in all areas and all laboratories. safety had the highest mean performance score (slipta: 68%; amr scorecard: 73%) while management review had the lowest (slipta: 32%; amr scorecard: 8%) across all laboratories. the amr scorecard scores were generally consistent with slipta scores. the amr scorecard identified technical weaknesses in amr testing, and slipta identified weaknesses in the quality management systems in the laboratories. conclusion: since the amr scorecard identified important gaps in amr testing not detected by slipta, it is recommended that microbiology laboratories use slipta and the amr scorecard in parallel when preparing for accreditation. expanding the use of the amr scorecard is a priority to address the need for quality clinical microbiology laboratory services in support of optimal patient care and amr surveillance. keywords: antimicrobial resistance; laboratory; clinical; blood; urine; faeces. introduction antimicrobial resistance (amr) is a global problem, with resistant infections currently claiming at least 50 000 lives each year across europe and the united states alone and hundreds of thousands more in other areas of the world.1 knowledge of amr patterns is essential for optimal individual patient care, antimicrobial stewardship and amr surveillance.2 reviews of available data from africa have found a high level of resistance to commonly used antibiotics in the region.3,4 despite nine new african countries joining the world health organization’s global antimicrobial resistance surveillance system in 2020/2021, amr data are generally lacking in many lowand middle-income countries (lmics).5 in addition to this limited availability, there are also concerns over the quality of existing amr data.6 during the current coronavirus disease 2019 pandemic, antimicrobial stewardship activities have been impacted globally, requiring coordinated strategies to inform actions to reduce the potential longer-term impact on amr.7 while recent advances in molecular methods to detect amr are being increasingly implemented in high-income settings,8,9 these are not available in many lmics, and traditional culture-based diagnostic methods, performed by clinical microbiology services, remain the gold standard. ensuring the quality of such services is a challenge because, in addition to the pre-analytical, analytical and post-analytical phases that take place within the laboratory, there are numerous other key drivers of overall diagnostic quality,10 including clinical question formulation and test selection, test ordering, sample collection and transportation to the laboratory, testing and results reporting, test results interpretation, and patient follow-up for clinical management or referral for further testing. laboratories with weak systems have higher levels of errors, which can affect patient care and undermine the confidence that clinicians have in laboratory services.11,12 to address the coronavirus disease 2019 pandemic, laboratories are receiving new molecular and point-of-care technologies, thus increasing the number of samples processed and reinforcing the need for high-performing and high-quality laboratories and systems, particularly in lmics. significant advances have been made towards improving laboratory capacity and quality in disease areas such as hiv and tuberculosis. one example is the stepwise laboratory quality improvement process towards accreditation (slipta) initiative developed by the united states centers for disease control and prevention in collaboration with the american society for clinical pathology, the clinton health access initiative, and the world health organization regional office for africa to promote the uptake of quality improvement initiatives in lmic laboratories.13,14,15 however, only a few clinical microbiology laboratories in lmics have achieved any form of accreditation, that is, a formal recognition that their quality management system (qms) complies with international standards.16,17,18 although the slipta initiative has, to some extent, facilitated laboratory improvement in africa, it may not specifically address the quality of processes in amr laboratories, including sample culture, species identification, and susceptibility testing. while implementing qms elements is critical, improving compliance with a technical standard of testing is equally important. in recognition of the potential gap in the quality of amr-related testing, we developed the amr laboratory scorecard (amr scorecard),19 which aims to improve the appropriate use of diagnostics to identify pathogens and guide patient treatment and management, and to optimise the surveillance and early detection of amr. the amr scorecard focuses on priority specimen types such as blood, urine, and faecal samples, and includes the culture, pathogen detection, species identification, and antimicrobial susceptibility testing (ast) processes. the amr scorecard is designed to assess these technical processes for the priority pathogens reported to the global antimicrobial resistance surveillance system. this includes pathogens associated with hospital and community-acquired infections in which amr is reportedly increasing, threatening the use of key drugs.5 in this article, we describe the performance of the developed amr scorecard during pilot laboratory evaluations in three countries. methods ethical considerations this article describes the performance of the amr scorecard during pilot evaluations and does not require ethical clearance. this article followed all ethical standards for research without direct contact with human or animal subjects. study design pilot evaluations of the amr scorecard were conducted in cameroon, ethiopia, and kenya between february and march 2019. the countries were selected based on their enrolment in the global antimicrobial resistance surveillance system and engagement with becton dickinson or foundation for innovative new diagnostics (find), the global alliance for diagnostics, in ongoing laboratory strengthening activities. the laboratories in these five countries are representative of microbiology services across the diagnostic network from central level laboratories to district-level laboratories. assessors were selected and trained on the use of the amr scorecard before performing the pilot laboratory assessments. antimicrobial resistance scorecard the amr scorecard was developed based on the latest guidance and requirements for amr testing obtained from a review of existing tools, checklists, and guidelines, including those of the healthcare-associated infection surveillance india20 and the united states centers for disease control and prevention.21 the amr scorecard is based on the world health organization regional office for africa slipta checklist version 2:2015 and incorporates clinical microbiology laboratory-specific requirements linked to sub-clauses in the slipta checklist. it consists of three scorecard modules that are used to assess the technical procedures for processing blood, urine, and faecal samples. assessment of technical procedures includes questions to determine if isolation procedures (e.g. ‘are media used for primary culture of faeces incubated at 35 °c – 37 °c for at least 18 hours?’), identification procedures (e.g. ‘are gram stains performed for all blood cultures showing any sign of positive growth [e.g. turbidity, haemolysis, or gas production]?’) and ast procedures (e.g. ‘does the laboratory use combination disk test or another equivalent method for carbapenemase screening?’) are being performed according to microbiology best practices. the amr scorecard is designed to be used as a stand-alone internal assessment tool or as part of a comprehensive slipta assessment to ensure the application of slipta requirements to these test methods. the amr scorecard uses the same scoring convention and the same 12-section structure as the slipta checklist. individual amr testing modules are scored according to the percentage of requirements met in each modular checklist. however, unlike slipta, whose laboratory assessment is based on the international standards organization (iso) 15 18922 standard, the amr scorecard assessment compares technical laboratory practices against best practices for microbiology and amr. an amr scorecard etool was developed to supplement the hard-copy technical modules and slipta checklist and to allow automated analyses and reporting of the laboratory assessments.22 the etool consists of a general amr testing spreadsheet for recording responses to questions common to all the technical modules, spreadsheets for information specific to faeces, urine and blood, a spreadsheet for previous audit information, and a summary spreadsheet with automated analysis and visualisation of the technical assessment scores. a spreadsheet corresponding to the 12 sections of slipta is also provided to allow simultaneous slipta evaluations. the summary spreadsheet with automated analysis provides a detailed overview and visualisation of the slipta assessment scores. assessor training trainee assessors who had microbiology experience and had participated in slipta assessments in their laboratories (but were not necessarily african society for laboratory medicine slipta certified) were chosen to attend the training workshops. training on the amr scorecard was conducted in ethiopia from 11 to 13 february 2019, in kenya from 25 to 26 february 2019, in uganda on 18 february 2020, and in ghana on 26 february 2020. facilitators trained two assessors from cameroon, eight assessors from ethiopia, and six assessors from kenya on the interpretation of amr scorecard questions, the use of the etool, and the procedures for conducting the assessments. as all the trainee assessors were already familiar with the use of the slipta checklist, training on slipta was not provided. theoretical training was supplemented by practical assessments of three facilities (two in ethiopia and one in kenya). the national or reference laboratories were chosen for the practical assessment to provide trainee assessors exposure to all the procedures evaluated using the scorecard (table 1). all three laboratories performed basic urine and faeces culture. automated blood cultures were performed by two laboratories, one in ethiopia and one in kenya. the reference laboratories in ethiopia and kenya are iso 15189:2012 certified and perform automated identification and ast. the practical assessments were overseen by the facilitators. due to time constraints, slipta assessments were not performed during the training assessments. table 1: antimicrobial resistance laboratory scorecard assessment activities in 14 laboratories in cameroon, ethiopia, and kenya between february 2019 and march 2019. pilot laboratory assessments before commencing the pilot assessments at the respective laboratories, assessors introduced the amr scorecard to the laboratory head and quality officer. all the assessments were conducted using the slipta checklist and the amr scorecard and transferred to the etool for further analysis. assessors evaluated the laboratory operations based on the slipta checklist and amr scorecard items, recording scores for each item and documenting findings in detail. during the assessment, assessors reviewed laboratory documentation to verify that policies, manuals, and standard operating procedures were complete, current, and accurate. they also reviewed records and observed laboratory procedures to verify that amr policies were being followed and that laboratory procedures used were appropriate for the testing performed. in addition, the assessors determined the availability of functional and well-maintained equipment, reviewed data on the number of processed samples, isolates, contaminated cultures, and negative cultures for each sample type, and reviewed the internal quality control and external quality assessment results. these data provided assessors with an overview of the laboratories’ operations and allowed the identification of systemic technical issues not easily determined using slipta alone. following the assessments, the assessors provided feedback to the laboratory head, quality officer, and laboratory technologists. non-conformities with the iso 15189:2012 standard identified by slipta23 and non-conformities with microbiology best practices identified by the amr scorecard were tabulated and presented to the laboratory along with copies of the completed checklists. data management the results of the pilot laboratory assessments were transferred to the etool, which then automatically calculated the scores and totals for each section and generated a bar graph of laboratory performance by section. the amr scorecard results for blood, faeces, and urine were analysed with the slipta scores. results antimicrobial resistance laboratory scorecard pilot assessments in the pilot assessments conducted in this study, weaknesses in technical procedures and the qms were identified in all areas and all laboratories (figure 1). the mean amr scorecard assessment scores ranged between 8% (section 2: management reviews) and 73% (section 12: facilities and safety), and the mean slipta scores ranged between 32% (section 2: management reviews) and 68% (section 12: facilities and safety). figure 1: mean performance scores of 11 microbiology laboratories assessed with the antimicrobial resistance laboratory scorecard and slipta in cameroon, ethiopia, and kenya between february 2019 and march 2019. based on the amr scorecard assessments, all 11 laboratories performed best with all sample types in section 12: facilities and safety (range: 25% – 100%; mean: 73%). the weakest performance for all sample types in all laboratories was in section 2: management reviews (range: 0% – 13%; mean: 8%), followed by section 11: occurrence management (range: 0% – 71%; mean: 12%), and section 6: evaluations and audits (range: 0% – 100%; mean: 22%). technical issues with the processing of all sample types (isolation, identification, and ast) were identified in section 8: process control and internal and external quality assessment of the amr scorecard (range: 3% – 72%; mean: 46%). in 10 of 11 laboratories, data on the number of isolated pathogens and cumulative ast patterns were not collected and reported to the relevant oversight committees such as the antimicrobial stewardship committee, or hospital surveillance or outbreak team. technical issues with the processing of urine samples included failure to perform cell counts or wet preparations (6 of 11 laboratories), lack of rejection criteria (8 of 11 laboratories), lack of quality controls for media (4 of 11 laboratories), lack of antibiotic discs for ast (9 of 11 laboratories), and failure to use purity plates or standardised inocula for ast (7 of 11 laboratories). technical issues with the processing of faeces samples included failure to perform wet preparations for parasites (5 of 11 laboratories), shortage of selenite f broth and lack of sub-culture testing (8 of 11 laboratories), and failure to perform serological identification of either salmonella or shigella species (5 of 11 laboratories). technical issues with the processing of blood samples included failure to perform extended-spectrum beta-lactamase and carbapenemase detection tests (11 of 11 laboratories). as with the results of the amr scorecard assessments, the slipta assessment also identified section 12: facilities and safety to be the strongest area (range: 40% – 95%; mean: 68%) in all the laboratories, followed by section 3: organization and personnel (range: 9% – 91%; mean: 57%) and section 7: purchasing and inventory (range: 0% – 88%; mean: 56%). the weakest area was section 2: management reviews (range: 0% – 43%; mean: 28%). some reasons for low slipta scores included failure to conduct regular management reviews or audits (6 of 11 laboratories), and failure to collect and analyse quality indicators (6 of 11 laboratories). weaknesses specific to the qms were identified using slipta as these are not assessed by the amr scorecard. for example, the content of the quality manual (documents and records) was identified as a weakness in 7 of 11 laboratories. comparisons between the amr scorecard and slipta scores the assessment scores obtained using the amr scorecard and slipta were disaggregated by laboratory area and laboratory level (tables 2–4). two laboratories were designated as central, five as regional and four as district laboratories. the central level laboratories had a mean amr scorecard assessment score of 37% and a mean slipta score of 45%. the regional-level laboratories had a mean amr scorecard assessment score of 40% and a mean slipta score of 55%. the district-level laboratories had a mean amr scorecard assessment score of 29% and a mean slipta score of 39%. table 2: stepwise laboratory quality improvement process towards accreditation and amr laboratory scorecard mean assessment scores in two central microbiology laboratories (a and b) in cameroon and ethiopia between february 2019 and march 2019. table 3: stepwise laboratory quality improvement process towards accreditation and amr laboratory scorecard mean assessment scores in five regional microbiology laboratories (a–e) in cameroon, ethiopia, and kenya between february 2019 and march 2019. table 4: stepwise laboratory quality improvement process towards accreditation and amr laboratory scorecard mean assessment scores in four district microbiology laboratories (a–d) in cameroon, ethiopia, and kenya between february 2019 and march 2019. at least one laboratory performed poorly at each level. at the central level, laboratory a had the lowest performance, with a mean amr scorecard assessment score of 30% and a mean slipta score of 35%. at the regional-level, laboratory a had a mean amr scorecard assessment score of 6% and a mean slipta score of 8%. at the district-level, laboratory a had a mean amr scorecard assessment score of 15% and a mean slipta score of 17%. as these results suggest, the overall amr scorecard scores were similar to the overall slipta scores, irrespective of the laboratory performance. however, differences between the slipta and amr scorecard assessment scores in the different areas of the laboratory were noted at all laboratory levels. at the central laboratories, the largest differences between the amr scorecard and slipta scores occurred in section 2: management reviews (0% vs 25%), section 9: information management (42% vs 63%) and section 10: corrective action (10% vs 39%). at the regional laboratories, the largest differences between the amr scorecard and slipta scores occurred in section 1: documents and records (33% vs 59%), section 2: management reviews (10% vs 36%), section 9: information management (22% vs 60%), and section 11: occurrence management (7% vs 50%). finally, at the district laboratories, the largest differences between the amr scorecard and slipta scores occurred in section 1: documents and records (26% vs 47%), section 4: client management (17% vs 40%), and section 7: purchasing and inventory (18% vs 48%). where there were large differences (> 20%) in the scores, the slipta score was always higher. discussion quality clinical microbiology services are an essential element of the amr response that enable the appropriate use of antibiotics, improve amr surveillance, and reduce the development of resistance.24 while implementing qms elements in the microbiology laboratory is critical, improving compliance to a technical standard of testing is equally important. the amr scorecard provides laboratories with a specific technical assessment tool for strengthening the quality of culture, identification, and ast laboratory procedures. in the pilot assessments conducted in this study, the amr scorecard scores generally correlated well with the slipta scores. safety in the laboratory has been identified as an increasingly important subject because of the emergence of highly infectious diseases, including coronavirus disease 2019. although safety has traditionally been regarded as a low-priority issue in developing countries,25 in both the amr scorecard and slipta assessments, section 12: facilities and safety was found to be the strongest area in the laboratories. this focus on safety, even in laboratories with weak systems (e.g. regional laboratory a), is encouraging. the slipta assessments identified weaknesses in the qms of the microbiology laboratories assessed. in 6 of the 11 laboratories, weaknesses identified included failure to conduct regular management reviews or audits. these findings are consistent with previous reports that some of the weakest areas in the laboratory are internal auditing and the collection of quality indicator data.11,15,16,26 overall, six laboratories (55%) received zero stars (< 55%) using the official slipta system, with only two laboratories (18%) scoring two stars (between 65% and 74%; regional laboratory b) and three stars (between 75% and 84%; regional laboratory d). the slipta scores in this study are consistent with the slipta scores from 47 countries worldwide, including 23 countries in africa, assessed using the strengthening laboratory management toward accreditation methodology prior to the initiation of laboratory strengthening activities (i.e. at baseline). yao et al.16 found that the mean score in these laboratories at baseline was 39% (median 37%), with 84% of the laboratories scoring zero stars (i.e. score < 55%). it has been suggested that microbiology laboratories are trailing other clinical laboratories in achieving accreditation.27 the overall poor performance recorded at each laboratory level suggests that providing quality microbiology services is a challenge across the tiered network. the exclusion of national or reference microbiology laboratories in the assessments likely resulted in the lower overall scores observed in the pilot. while national or reference microbiology laboratories were assessed using the amr scorecard during the training, slipta assessments were not conducted due to time constraints and thus the amr scorecard results were not included in this report. in settings with limited resources, strengthening technical testing and qms (including accreditation) is often initiated at the national level, suggesting that overall scores may have been higher had they been included. by providing a specific technical focus, the amr scorecard identified important gaps in amr technical testing not detected by slipta alone. the amr scorecard assesses the step-by-step procedures for sample processing, bacterial isolation and identification, and ast. approximately 44% of the amr scorecard focuses on these procedures in contrast to slipta which has a limited focus on technical procedures. topics covered by slipta are also covered by the amr scorecard but the latter focuses on the specific details related to amr. for example, slipta assesses whether standard operating procedures for laboratory functions and technical and managerial procedures are available, while the amr scorecard assesses whether the laboratory has, for example, documented procedures for microscopic examination and urine cell count. as the amr scorecard identified important gaps in amr testing not detected by slipta alone, and slipta identified specific weaknesses in the qms that were beyond the scope of the amr scorecard, it is recommended that microbiology laboratories that require a comprehensive assessment or are developing their qms through continuous improvement toward accreditation be assessed using both the slipta and the amr scorecard in parallel. thus, the amr scorecard can be used as an entry point into the qms journey, with laboratories choosing to apply for slipta certification (or iso certification) after reaching a satisfactory level (equivalent to 3–4 stars on slipta). it should be noted that the official star recognition system provided by african society for laboratory medicine can only be obtained through the slipta certification provided by the african society for laboratory medicine secretariat.28 the importance of data collection and analysis is also highlighted in several questions in the amr scorecard (e.g. ‘are the following performance indicators collected – number and percentage of urine cultures with cell counts > 105 cells/ml?’) and assessors are encouraged to assist laboratories to collect and analyse their data. the clinical laboratory is a major source of healthcare data that can be used to inform health system-wide actions meant to improve diagnostic test utilisation, service efficiency, and patient outcomes.29 in these evaluations, cumulative quality indicator data on isolated pathogens and ast were not collected by 10 of the 11 laboratories assessed. this was due to the lack of automated instruments or an electronic laboratory information system, meaning that staff were required to manually record and calculate isolation rates and ast patterns. in one laboratory, the compilation of data by the assessors exposed a very low isolation rate of enteric pathogens that required further investigation. quality indicator data, if available, could have been used to identify the cause of the low isolation rate, thereby improving the quality of enteric bacterial culture. in addition to improving the quality of laboratory testing, laboratory data can also be used to influence clinical decisions. for example, cumulative data on pathogens and ast results can be used to inform treatment guidelines. however, for laboratory data to impact health systems in such a way, the laboratory needs to carefully consider how the data are collated, communicated, and disseminated. data from the assessments in this pilot revealed that 10 of the 11 laboratories failed to collect data on cumulative ast patterns and report these data to oversight committees, thereby missing the opportunity to inform antimicrobial stewardship decisions with laboratory data. in the pilot assessments, where there were differences between the amr scorecard assessment and slipta scores, the slipta scores were consistently higher. in addition to the differences in the content of the two assessment tools, other factors may contribute to this finding. first, the laboratories included in the pilot were not part of any active and ongoing programmes to improve laboratory quality such as the strengthening laboratory management toward accreditation programme.15 only 2 of the 11 laboratories reported a previous slipta assessment. it is expected that laboratories participating in quality improvement programmes are more likely to score higher on both assessment tools. second, the amr scorecard is based on microbiology best practices and not on an iso standard such as slipta. without a standard to guide preparations, it may not be clear to laboratories what requirements need to be in place to assure quality amr testing. in this respect, the amr scorecard has a role to play in educating laboratories regarding the technical requirements for amr testing. there were several challenges to implementing the amr scorecard in the initial cohort of laboratories, including procurement of funding support for quality improvement and provision of cover for the trainers and mentors during programme-related absences. mentoring of laboratories has been reported to be an important component of successful and sustainable quality improvement initiatives;30 thus, it is important to ensure that enough resources in terms of funding and personnel are put in place to allow mentoring to take place. ideally, laboratories should be mentored by reference laboratories within the amr surveillance network. based on feedback from facilitators and assessors, the amr scorecard needed to be revised to strengthen identified weaknesses. suggested changes included revising the language of some questions and adding ‘not applicable’ options to others. two additional questions to determine whether laboratories were performing extended-spectrum beta-lactamase and carbapenemase screening on faecal samples were added to the faeces module. this increased the score of the faeces module by four points. the total score of the blood and urine modules remained unaltered in the revised amr scorecard. in 2020, the structure of the amr scorecard was changed, and additional scorecards were added to allow for assessments of other sample types including pulmonary, genital, and wound samples. evaluations of the revised amr scorecard were performed in three laboratories in ghana and uganda. limitations the microbiology laboratories selected for the pilot of the amr scorecard do not represent the scope of microbiology technical abilities across africa. while care was taken during the selection of laboratories for the study, the amr scorecard may be less useful in identifying and addressing gaps that impact the quality of testing in certain settings. in addition, all assessments, except those performed in cameroon, included find and becton dickinson facilitators. as these facilitators were involved in the development of the amr scorecard, they may have influenced the outcomes of the assessments in favour of the amr scorecard. conclusion this study showed that a customised scorecard to guide the establishment and strengthening of amr testing quality in resource-limited settings can assist in identifying and addressing quality gaps. the amr scorecard, used in conjunction with slipta, found important gaps in the procedures for identification and ast of priority pathogens that were not identified by slipta alone. expanding the use of this scorecard will help address the need for quality clinical microbiology laboratory services to support optimal patient care and amr surveillance. acknowledgements the authors are grateful to the ethiopia public health institute, kenya national public health laboratory service, and the ministry of health, cameroon. we wish to thank john nkengasong from the africa centres for disease control and prevention (africa) for his contribution to the conceptualisation of the amr scorecard and his guidance and support during its development. we are also grateful to cecilia ferreyra and cassandra kelly-cirino from find, and courtney maus, namita singh, kartik sharma, christine claire cruz, nermin hamurcu, and nuphar rozen-adler from becton dickinson for their support on this project. we wish to acknowledge the participating laboratories and facilities, the fleming fund, and uk united kingdom aid direct for support of the uganda and ghana sites, as well as the assessment teams in cameroon, ethiopia, and kenya. editorial assistance for later drafts was provided by rachel wright, phd, funded by find. competing interests the authors have the following competing interests: becton dickinson and find provided support for this study in the form of salaries for employees and financial support to the partner organisations and assessors. foundation for innovative new diagnostics and becton dickinson jointly developed the amr scorecard. foundation for innovative new diagnostics has partnership agreements with various diagnostic manufacturers for the development and implementation of amr diagnostic tools. foundation for innovative new diagnostics receives funding from multiple public and private donors for tuberculosis projects in technology development and clinical research. limited funding is occasionally provided by industry partners; acceptance of these funds is subject to review by an independent scientific advisory committee or another independent review body. becton dickinson is a global medical technology company that manufactures and supplies products and solutions pertaining to medical discovery, diagnostics, and the delivery of care. becton dickinson global health has longstanding collaborations with health agencies and is currently implementing projects in africa to strengthen laboratory quality including the use of the amr scorecard and improve laboratory capacity, including installation of becton dickinson equipment and providing broader systems. authors’ contributions a.t., t.d., l.o., a.w., k.g., z.k., r.g., and h.a. contributed to the development of the amr scorecard. a.t., d.a.w., g.n., j.a.o., k.g., i.m., p.k., p.n., and n.d. were responsible for the assessments. a.t., h.a. k.g., and r.g. were responsible for data analysis and preparation of the manuscript. all authors reviewed the manuscript and agreed with its content. sources of support this study was funded by becton dickinson and find through a grant from the department for international development, united kingdom. data availability the recorded assessment results and data sets generated or analysed during the current study are available from the corresponding author, a.t., on request. disclaimer the findings and conclusions in this publication are those of the authors and do not necessarily represent the official position of becton dickinson or find. references o’neill j. review on antimicrobial resistance: tackling a crisis for the health and wealth of nations [homepage on the internet]. 2014 [cited 2020 nov]. available from: https://amr-review.org/sites/default/files/amrreviewpaper-tacklingacrisisforthehealthandwealthofnations_1.pdf tangcharoensathien v, chanvatika s, sommanustweechaia a. complex determinants of inappropriate use of antibiotics. bull world health organ. 2018;96:141–144. https://doi.org/10.2471/blt.17.199687 ampaire l, muhindo a, orikiriza p, mwanga-amumpaire j, bebell l, boum y. a review of antimicrobial resistance in east africa. afr j lab med. 2016;5(1):432. https://doi.org/10.4102/ajlm.v5i1.432 leopold sj, van leth f, tarekegn h, schultsz c. antimicrobial resistance among clinically relevant bacterial isolates in sub-saharan africa: a systematic review. j antimicrob chemother. 2014;69(9):2337–2353. https://doi.org/10.1093/jac/dku176 who. global antimicrobial resistance and use surveillance system (glass) report 2021. geneva: world health organization; 2021. tadesse b, ashley e, ongarello s, et al. antimicrobial resistance in africa: a systematic review. bmc infect dis. 2017;17(1):616. https://doi.org/10.1186/s12879-017-2713-1 rawson tm, ming d, ahmad r, et al. antimicrobial use, drug-resistant infections and covid-19. nat rev microbiol. 2020;18:409–410. https://doi.org/10.1038/s41579-020-0395-y anjum m, zankari e, hasman h. molecular methods for detection of antimicrobial resistance. microbiol spectr. 2017;5(6):1–17. https://doi.org/10.1128/microbiolspec.arba-0011-2017 boolchandani m, d’souza a, dantas g. sequencing-based methods and resources to study antimicrobial resistance. nat rev genet. 2018;20(6):356–370. https://doi.org/10.1038/s41576-019-0108-4 mesfin e, taye b, belay g, ashenafi a, girma v. factors affecting quality of laboratory services in public and private health facilities in addis ababa, ethiopia. ejifcc. 2017;28(3):205–223. albert h, trollip a, erni d, et al. developing a customised approach for strengthening tuberculosis laboratory quality management systems toward accreditation. afr j lab med. 2017;6(2):576. https://doi.org/10.4102/ajlm.v6i2.576 peter tf, rotz pd, blair dh, khine a-a, freeman rr, murtagh mm. impact of laboratory accreditation on patient care and the health system. am j clin pathol. 2010; 134(4):550–555. https://doi.org/10.1309/ajcph1skq1hnwghf alemnji ga, zeh c, yao k, fonjungo pn. strengthening national health laboratories in sub-saharan africa: a decade of remarkable progress. trop med int health. 2014;19(4):450–458. https://doi.org/10.1111/tmi.12269 gershy-damet gm, rotz p, cross d et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. https://doi.org/10.1309/ajcptuuc2v1wjqbm yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin pathol. 2010;134(3):401–409. https://doi.org/10.1309/ajcpnbbl53fwuiqj yao k, luman et, slmta collaborating authors. evidence from 617 laboratories in 47 countries for slmta-driven improvement in quality management systems. afr j lab med. 2014;3(2):262. https://doi.org/10.4102/ajlm.v3i2.262 kibet e, moloo z, ojwang p, sayed s, mbuthia a, adam r. measurement of improvement achieved by participation in international laboratory accreditation in sub-saharan africa: the aga khan university hospital nairobi experience. am j clin pathol. 2014;141(2):188–195. https://doi.org/10.1309/ajcpv8a9mrwhgxef petti ca, polage cr, quinn tc, et al. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006;42(3):377–382. https://doi.org/10.1086/499363 find. amr laboratory scorecard package [homepage on the internet]. [cited 2020 nov]. available from: https://www.finddx.org/amr-lab-scorecard-package/ healthcare associated infection surveillance india. all india institute of medical sciences (aiims), centers for disease control and prevention (cdc), indian council of medical research (icmr). laboratory antibacterial resistance surveillance readiness tool [homepage on the internet]. 2017 [cited 2020 nov]. available from: https://www.haisindia.com/upload/fileuploads/1527836102_lab.%20assessment.pdf centers for disease control and prevention. lab assessment of antibiotic resistance testing capacity (laarc) [homepage on the internet]. 2021 [cited 2020 nov]. available from: https://www.cdc.gov/drugresistance/intl-activities/laarc.html iso 15189. medical laboratories – requirements for quality and competence. geneva: international organization for standardization; 2012. african society for laboratory medicine. who afro slipta checklist [homepage on the internet]. 2007 [cited 2020 nov]. available from: http://www.afro.who.int/en/clusters-a-programmes/hss/blood-safety-laboratories-a-health-technology/blt-highlights/3859-who-guide-for-the-stepwise-laboratory-improvement-process-towards-accreditation-in-the-african-region-with-checklist.html wilson ml. assuring the quality of clinical microbiology test results. clin infect dis. 2008;47(8):1077–1082. https://doi.org/10.1086/592071 ejilemele aa, ojule ac. health and safety in clinical laboratories in developing countries: safety considerations. niger j med. 2004;13(2):182–188. maina rn, mengo dm, mohamud ad, et al. progressing beyond slmta: are internal audits and corrective action the key drivers of quality improvement? afr j lab med. 2014;3(2):222. https://doi.org/10.4102/ajlm.v3i2.222 jacobs j, hardy l, semret m, et al. diagnostic bacteriology in district hospitals in sub-saharan africa: at the forefront of the containment of antimicrobial resistance. front med (lausanne). 2019;6:205. https://doi.org/10.1086/592071 african society for laboratory medicine. slipta [homepage on the internet]. 2021. available from: https://aslm.org/what-we-do/#slipta shirts b, jackson b, baird g, et al. clinical laboratory analytics: challenges and promise for an emerging discipline. j pathol inform. 2015;6:9. https://doi.org/10.4103/2153-3539.151919 maruta t, motebang d, mathabo l, rotz pj, wanyoike j, peter t. impact of mentorship on who-afro strengthening laboratory quality improvement process towards accreditation (slipta). afr j lab med. 2012;1(1):6. https://doi.org/10.4102/ajlm.v1i1.6 references about the author(s) iruka n. okeke department of pharmaceutical microbiology, university of ibadan, ibadan, nigeria citation okeke in. honing in on disease etiology. afr j lab med. 2017;6(1), a679. https://doi.org/10.4102/ajlm.v6i1.679 editorial honing in on disease etiology iruka n. okeke copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. what are the causes of disease? how best can laboratories detect them? and what can we do with the knowledge that laboratories generate to improve disease prevention, containment and treatment? these are questions that fall within the african journal of laboratory medicine’s scope and for which some poignant answers were supplied by articles in this volume. our cover features a photomicrograph of cylindrocarpon lichenicola from irek and colleagues’ report of a rare but debilitating case of keratomycosis1. a skilled diagnostician can only confirm this infection with access to mycology laboratory resources, which are uncommon on the african continent. this case report of a soil-acquired infection in a farmer points to the need for diagnostic laboratory vigilance, as well as a need to advocate for broader fungal diagnostics and treatments for african patients. for more common conditions, access to laboratory testing is critical for public health, as well as patient care. inadequate access to testing stands in the way of hiv care for roughly half of africans infected with the virus. inadequate access was also a significant factor in the challenge to contain the recent ebola outbreak in west africa. articles in this issue report on how the introduction of testing interventions positively impacted disease care on the continent for both life-threatening infections. in one example of how diagnostic test development and deployment can overcome health worker shortages, kaindjee-tjituka et al.2 found that introduction of point-of-care cd4 testing in namibia allows testing to be performed by nurses and lay community counsellors. thus, patients received test results and counselling on the same day and in some cases antiretroviral therapy was speedily initiated. louis and co-workers3 detail the parallel rapid diagnostic testing for malaria and ebola for febrile patients and corpses in forécariah, west guinea, in 2015. healthcare workers and red cross volunteers performed the tests, and their ability to care for patients and deliver healthcare safely was positively impacted. however, they faced some challenges such as: patients equating ebola testing with being diseased and the limited sensitivity of the ebola rapid diagnostic test, which meant that corpses that tested negative were still buried according to safe haemorrhagic fever burial practices. one of the criteria for promoting tests that are evaluated at the point of care is ease of interpretation, which allows trained lay people as well as health professionals to administer testing and respond appropriately to results. however, not all test encounters are this straightforward. primary healthcare physicians use a wide variety of tests to inform their diagnoses, and 17% of those surveyed by vanker and faull4 in south africa reported that they were unsure of how to interpret or use test results. vanker and faull state that this percentage is roughly double that reported from the united states, and their research points to the need for better physician education and support and clear test reporting, particularly in those areas that pinpoint disease etiology: microbiology, serology and discordant hiv tests. good testing is essential for optimal patient management, particularly in resource-limited settings, and also informs surveillance. as uwimana et al.5 report, under 30% of all leprosy cases in rwanda within the 17-year period between 1995 and 2011 were verified by laboratory results. the good news from that article is that leprosy detection rates are dropping – a testament to improved surveillance – and that laboratory verification of cases is becoming more frequent. in 2010, roughly 70% of cases were laboratory-verified, whereas 10 years prior, under 5% of reported cases were supported by laboratory results. almost all of the articles in this issue emphasise the need for continued improvement in access to laboratory testing and in surveillance for infectious diseases across the continent. thus, it is refreshing to learn from the guest editorial in this issue by amukele6 that the africa centres for disease control has made a strong start. this is certainly an exciting time for me to have commenced my tenure as african journal of laboratory medicine’s editor-in-chief. references irek eo, obadare to, udonwa pa, laoye o, abiri ov, adeoye ao, et al. cylindrocarpon lichenicola keratomycosis in nigeria: the challenge of limited access to effective antimicrobials. afr j lab med. 2017;6(1):612. kaindjee-tjituka f, sawadogo s, mutandi g, et al. task-shifting point-of-care cd4+ testing to lay health workers in hiv care and treatment services in namibia. afr j lab med. 2017;6(1), a643. louis fj, huang jy, nebie yk, koivogui l, jayaraman g, abiola n, et al. implementation of broad screening with ebola rapid diagnostic tests in forécariah, guinea. afr j lab med. 2017;6(1):1–6. vanker n, faull nhb. laboratory test result interpretation for primary care doctors in south africa. afr j lab med. 2017;6(1):453. uwimana i, bizimungu n, ingabire f, mukamukwiye e, sharangabo o, ngabonziza sc, et al. trends in leprosy case detection in rwanda, 1995–2011: analysis of 17 years of laboratory data. afr j lab med. 2017;6(1):426. amukele t. africa cdc: establishing integrated surveillance and laboratory networks for rapid disease detection and response, control, prevention, and clinical care in africa. afr j lab med. 2017;6(1):638. abstract introduction methods results discussion acknowledgements references about the author(s) adedayo o. faneye department of virology, faculty of basic medical sciences, university of ibadan, ibadan, nigeria oyeteju s. babalola department of virology, faculty of basic medical sciences, university of ibadan, ibadan, nigeria georgina n. odaibo department of virology, faculty of basic medical sciences, university of ibadan, ibadan, nigeria juwon arotiba department of oral and maxilofacial surgery, faculty of dentistry, university of ibadan, ibadan, nigeria olufemi d. olaleye department of virology, faculty of basic medical sciences, university of ibadan, ibadan, nigeria citation faneye ao, babalola os, odaibo gn, arotiba j, olaleye od. oral human papilloma virus infection among dental clinic attendees in ibadan, nigeria. afr j lab med. 2022;11(1), a1555. https://doi.org/10.4102/ajlm.v11i1.1555 original research oral human papilloma virus infection among dental clinic attendees in ibadan, nigeria adedayo o. faneye, oyeteju s. babalola, georgina n. odaibo, juwon arotiba, olufemi d. olaleye received: 17 feb. 2021; accepted: 26 may 2022; published: 25 nov. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: human papilloma virus (hpv) is associated with a subset of oropharyngeal squamous cell carcinoma and mouth or throat warts. however, there is currently limited information about oral hpv infections in nigeria. objective: this study aimed to provide information on the occurrence and circulating genotypes of hpv among patients attending three (one government and two private) dental clinics in ibadan, nigeria. methods: an oral swab was collected from 231 dental clinic attendees in ibadan between january 2016 and march 2017 and tested for hpv dna by polymerase chain reaction targeting the e6/7 genes of the virus. results: twenty-three of the 231 swab samples were hpv dna positive comprising 16 mono-infections and seven co-infections in 13 males and ten females. genotype 16 was present in ten patients, genotype 6/11 in five, genotype 18 and genotype 33 in four each, genotype 31 in three and genotype 39 in one. twenty-one cases were high-risk hpv genotypes, while two were low-risk. samples had co-infection and five had low risk type 6/11 either as single or as co-infection. persons who had engaged in oral sex as well as those aged 21-30 years has significantly higher prevalence. conclusion: this study showed that although hpv genotype 16 is the most common type among dental clinic attendees in ibadan, other genotypes are also circulating and that oral sex is a risk factor for the infection. therefore, introducing a multivalent hpv vaccine will reduce the risk of hpv-associated oropharyngeal carcinoma and other cancers in nigeria. keywords: oral hpv infection; dental clinic attendees; molecular detection; hpv vaccine; nigeria. introduction human papilloma viruses (hpv) are associated with anogenital cancer, including vulval, penile, vaginal, anal, and cervical cancer. these viruses have also been associated with a subset of head and neck cancers causing oropharyngeal squamous cell carcinoma.1 apart from hpv genotypes associated with oropharyngeal carcinoma, infections with a few other genotypes of hpv can cause warts in the mouth or throat.2 oral hpv infection is a common infection worldwide. a 4.9% oral hpv infection prevalence among healthy individuals worldwide has been reported. southern europe has the highest oral hpv prevalence (9.5%), while central america has 6.6% and east asia 0.6%. from the few studies in africa, the prevalence of oral hpv ranges from 2.3% to 6.5%.3 the situation of hiv infection has caused an increase in the prevalence of oral hpv infection, especially among hiv-infected individuals in developed countries.4 however, information on the effect of hiv on the prevalence of oral hpv in africa, and nigeria in particular, is limited. human papilloma viruses are small, non-enveloped, double-stranded dna viruses belonging to the papovaviridae family. the family has five major genera: alpha, beta, gamma, mu, and nu, with those in the alpha genus having the most medical importance. human papilloma viruses are further categorised into high risk or low risk based on their epidemiological associated malignancies.5,6,7,8 there are 14 hpv genotypes classified as high risk and they include hpv types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68, while genotypes 6, 11, 42, 43 and 44 are classified as low-risk types. human papilloma virus infection of the mouth and the oropharynx, like hpv infection of the uterine cervix, is associated with high-risk sexual behaviours (orogenital sex). high-risk hpv genotypes, especially hpv-16, are present in many oral and oropharyngeal squamous cell carcinoma where, in some cases, they play an essential aetiological role.7 presently, the prevalence of anogenital hpv infection in africa ranges from 9.8% in central africa to 19.5% in western africa9; in nigeria, it ranges from 3.5% to 28.8%. the common anogenital hpv genotypes circulating in nigeria are hpv types 16, 18, 31, 35, 33, 39, 45, 51, 52, 56, 59, 70 and 82,10 while those associated with cancer in nigeria include genotypes 16, 18, 33, 35, 45, 52 and 56.10 although various hpv strains are circulating in nigeria, the easily available vaccine is only against genotypes 16 and 18. yet, the available hpv vaccine is not included in the national vaccination programme, and only a few informed individuals make private arrangements for the vaccination. the population of people making this arrangement is also low due to cost and awareness11,12; thus, the hpv vaccine coverage in nigeria is meagre. there is limited information on the prevalence and circulating genotypes of oral hpv in africa and especially nigeria. this information is essential to know if available vaccines will be effective in preventing oral hpv infection and associated oral cancers in nigeria. this study was carried out to provide information on oral hpv and the circulating genotypes among patients presenting in dental clinics in ibadan, nigeria. methods ethical considerations this study was approved by the ethics review committee of the university of ibadan and university college hospital ibadan with approval number 160057, and participants provided written informed consent. confidentiality of the study participants was maintained; participants’ identities were codified, and only the authorised party could link the identity of the participants to the given code when the result is given to the dentist to notify the participants of their result. study design this study was a descriptive cross-sectional study carried out among 231 patients attending three dental clinics comprising one government (university college hospital ibadan dental clinic) and two private dental clinics in ibadan, oyo state, nigeria, from august 2016 to february 2017. government dental clinics in ibadan provide dental services for the population of ibadan and its environment. the minimum sample size calculated was 196 but a total of 231 individuals was recruited for the study. the sample size was determined using the formula: a prevalence of 15%13 at 95% confidence interval and 0.02 level of variability of the target population (n = the sample size, a = 1.96, p = prevalence, q = 1 – p, and d = 0.05). study population a total of 231 individuals who consented and were aged 18 years or older were recruited for the study. the participants were first-time attendees of dental clinics (one public and two private) in ibadan, oyo state, nigeria, presenting with various dental complaints. a convenient sampling method was used.14 individuals were excluded if they were unable or unwilling to provide consent or were in severe pain or with symptoms of oropharyngeal cancer. sample collection and processing socio-demographic and risk behaviour information (presence of oral warts, smoking, number of sexual partners, previous oral infection, and engaging in oral sex) was collected from each participant using a semi-structured questionnaire. a dental technician trained in collecting oral swabs for hpv testing collected oral swab samples from all participants. briefly, the tip of the swab stick was rubbed several times in the mouth and base of the tongue. the swab sticks were then placed in vials containing 500 µl of transport medium containing minimum essential medium and 2% bovine serum albumin with antibiotics (gentamicin) in a secured capped vial. samples were stored and transported at 4 °c to the laboratory. initial processing in the laboratory was done within 24 h of sample collection, and included vortexing each vial containing transport medium, removing the swab; the medium was aliquoted and stored at –80 °c until analysed for hpv dna by polymerase chain reaction (pcr). laboratory analysis total dna was extracted from each sample using a commercially available dna purification kit (jena bioscience, jena, germany) per the manufacturer’s instruction. the extracted dna was tested for the presence of the e6/e7 hpv viral gene by pcr using previously described primers (gp e6-3f, gp-e6-5b and gp-e6-6b) and protocol.15 amplification was achieved using a abi 9700 geneamp thermal cycler (applied biosystems®, waltham, massachusetts, united states) using the following cycling condition: 5 min at 95 °c for dna denaturation followed by 65 cycles of 30 s of denaturation at 95 °c, 30 s of annealing at 45 °c, 30 s of elongation at 68 °c and a final elongation of 5 min at 72 °c. the amplified products (602 base pairs – 666 base pairs) were detected using agarose gel electrophoresis. human papilloma viruses isolates were typed using genotype specific primers targeting the e6/e7 hpv virus gene as previously described15 (table 1). the primers used could not differentiate between genotypes 6 and 11, thus, it is referred to as genotype 6/11. table 1: list of primres used and the amplicon size. data analysis the data anlalysis was done using statistical package for social sciences (spss) version 18.2 (ibm corp. armonk, new york, united states). all the data generated were analysed using descriptive statistics such as mean and standard deviation and results were analysed using chi-square at α = 0.05. results characteristics of the study population a total of 231 study participants were recruited for this study, out of which 129 (55.9%) were female. their mean age was 42.59 (range: 18–62 years) (table 2). all the participants reported having sexual experience, while 46 (20.0%) had engaged in oral sex. thirty-six (15.6%) of the participants had warts, either oral or genital, while 24 (10.4%) were smokers. fifty-four (23.4%) of the participants had more than one sexual partner, and none had received any hpv vaccination. table 2: age distribution of dental clinic attendees in ibadan, nigeria, january 2016 to march 2017. human papilloma virus dna prevalence and genotypes of the 231 oral swabs tested, 23 (9.9%) had hpv dna, of which 21 were high-risk and two were low-risk hpv. of the 23 hpv dna-positive participants, 13 (56.5%) were male and 10 (43.5%) were female. also, 18 (78.3%) of the 23 participants with hpv had previously engaged in oral sex. participants in the age group 21–40 years had the highest rate of positivity (14.5%), whereas those aged 60 years or older had the lowest rate (2.1%) (p = 0.241) (table 3). table 3: distribution of human papilloma virus infection among dental clinic attendees in ibadan, nigeria, january 2016 to march 2017. of the 23 participants with hpv infection, 16 had mono infections while seven had co-infections with either other high-risk or low-risk genotypes. the most common genotypes detected in this study were genotypes 16 and 18. hpv genotype 16 was detected in ten participants, eight of which were single infection and one co-infection with genotype 33, the other one were the low risk group (6/11). genotype 18 was also detected in four participants, of which three were single infection and one co-infection with genotype 31. genotypes 31 and 33 were each detected in three participants, two as single infection and one each as co-infection with other genotype. genotype 39 was the least prevalent genotype detected in only one participants. a total of 21 participants had high-risk hpv dna. discussion the prevalence of oral hpv infection and the circulating genotypes among dental clinic attendees in ibadan, nigeria, was determined in this study. an oral hpv prevalence of 9.9% was observed. previous reports on the rates of oral hpv infection among asymptomatic individuals varied between different geographic areas. it ranged from 12.0% in south africa to 5.0% in the united states. the prevalence obtained among dental clinic attendees in this study is lower than the 12% reported from south africa among individuals attending hiv testing centres13 and higher than the 7.3% and 6.9% reported from the general population in the united states.16,17 the differences obtained could be due to the population tested. the higher prevalence in the south african population could be due to the underlying hiv infection. it was also noted that the prevalence of oral hpv among hiv-positive individuals in the united states is also high. the primers used for the detection of hpv dna in this study target the e6/7 region of the virus. this region is part of the viral oncogenes and is usually integrated into the host genome. thus, primers targeting this region are more sensitive than the l1 region most commonly used. other studies have shown that pcr protocols targeting the l1 region of the hpv genome are likely to miss some infections if the viral dna has been integrated, as the l1 region of the viral genome is usually deleted during viral integration.18,19 in addition, the detection of the e6/7 genes of the hpv can suggest a persistent hpv infection, which could be used to predict people at risk of oropharyngeal carcinoma. in this study, people aged 60 years and older had the lowest prevalence (2.1%) of hpv infection, while those in the age group of 21–40 years had the highest rate (14.5%) (table 3). the 21–40 age group is the most sexually active age group; thus, the pattern may result from sexual activities and engagement in oral sex among the persons within this age range. studies have shown that this is the age group most likely to engage in oral sex. in addition, oral sex was not as common in the past as it is now. this may be why the prevalence of oral hpv infection is higher among younger participants. the age distribution found in this study is similar to what was reported by antonsson et al.20 in australia but different from the pattern reported in the united states by sanders et al.,16 where the highest prevalence (11.3%) was observed among the age group 55–64 years. gender differences have been observed in the distribution of oral hpv.8,9 in this study, there are no significant differences in the gender distribution of hpv dna.21,20 furthermore, findings from this study showed that participants with more than one sexual partner had a higher prevalence of oral hpv infection. positive oral hpv testing is significantly positively correlated with an increase in vaginal sexual partners.21 there was also no significant difference in prevalence of hpv dna as per marital status of the participants in this study, although kero et al.22 reported that stable marital relationships protect men from oral and genital hpv infection. this study also noted that participants who engaged in oral sex had a significantly higher prevalence of oral hpv infection. oral sex is a major route for hpv transmission.21,23,24 although oral sex and having multiple sexual partners are shown to be significantly associated with oral hpv positivity, other behaviours such as kissing can transmit hpv.25 this study detected low risk hpv genotype 6/11 and high-risk genotypes 16, 18, 31, 33 and 39 among dental clinic attendees in oyo state, nigeria. among the previously reported circulating genital hpv types among women in ibadan, nigeria, according to nejo et al. and thomas et al.26,27 the most common hpv genotype (type 16) in this study is similar to the findings of thomas et al. but different from the report of nejo et al., which reported hpv genotype 31 as the most prevalent. human papilloma viruses genotype 16 has also been reported from other parts of the world as the most common oral hpv type. finally, the inclusion of the hpv vaccine into the national vaccination programme to cover both boys and girls in nigeria will reduce the prevalence of oral hpv infection and, ultimately, hpv-associated oropharyngeal cancer in nigeria. presently, the most common vaccine in nigeria (cervarix marketed by glaxosmithkline) targets hpv 16 and 18 and gardasil (marketed by merk & co.) targets only hpv 16, 18, 6 and 11 whereas this study and previous nigerian studies have identified other genotypes. thus, introducing hpv vaccine that will cover other circulating strains in addition to the ones they cover presently will make the vaccination programme more effective. the study by herrero et al.28 has demonstrated the efficacy of hpv vaccination in reducing prevalence of oral hpv after four years of vaccinating women in costa rica. limitations patients with symptoms of oropharyngeal cancer were not included in this study to identify the virus genotypes associated with oropharyngeal cancer in this region. information about the number of kissing partners of the participants was also not accessed in the study. the size of the study population is also a limitation of the study. conclusion this study describes a high prevalence of oral hpv infection among dental clinic attendees in ibadan, oyo state, nigeria, by detecting hpv dna from the oral swabs of the study participants using pcr. a prevalence of 9.9% of hpv infection was identified and also showed that hpv type 16 and 18 are the most common types detected among the study participants. oral sex was also significantly associated with hpv infection. the hpv vaccine for use in nigeria should cover at least the commonest circulating genotypes to reduce the risk of hpv-associated oropharyngeal and other cancers in nigeria. acknowledgements we are grateful to the members of staff of oyo state dental clinic, ibadan, for their assistance in collecting samples analysed, as well as the participants in this study. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them, as well as the participants in writing this article. authors’ contributions a.o.f. was responsible for conceptualisation of the idea, sample collection, sample analysis, writing of the draft manuscript and manuscript review. o.s.b. contributed to conceptualisation of the idea, sample collection, sample analysis and manuscript review. g.n.o. and o.d.o. were involved in conceptualisation of the idea, sample analysis and manuscript review, while j.a. contributed to conceptualisation of the idea, sample collection and manuscript review. sources of support this study was partly funded by the college of medicine seed award 2014. the manuscript writing of the project described was supported by the medical education partnership initiative in nigeria project funded by fogarty international, the office of aids research, and the national human genome research institute of the united states national institutes of health, the health resources and services administration and the office of the united states global aids coordinator under award number r24tw008878. data availability the authors confirm that the data supporting the findings of this study are available within the article. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. the content is solely the responsibility of the authors and does not necessarily represent the official views of the funding organisations. references gillison ml, koch wm, capone rb, et al. evidence for a causal association between human papillomavirus and a subset of head and neck cancers. j natl cancer inst. 2000;92(9):709–720. https://doi.org/10.1093/jnci/92.9.709 greenspan d, de villiers em, greenspan js, de souza yg, zur hausen h. unusual hpv types in oral warts in association with hiv infection. j oral pathol. 1988;17(9–10):482–488. https://doi.org/10.1111/j.1600-0714.1988.tb01321.x wood nh, makua ks, lebelo rl, et al. human papillomavirus prevalence in oral and oropharyngeal rinse and gargle specimens of dental patients and of an hiv-positive cohort from pretoria, south africa. adv virol. 2020;2020:2395219. https://doi.org/10.1155/2020/2395219 beachler dc, weber km, margolick jb, et al. risk factors for oral hpv infection among a high prevalence population of hiv-positive and at-risk hiv-negative adults. cancer epidemiol biomarkers prev. 2012;21(1):122–133. https://doi.org/10.1158/1055-9965.epi-11-0734 cogliano v, baan r, straif k, grosse y, secretan b, el ghissassi f. carcinogenicity of human papillomaviruses. lancet oncol. 2005;6(4):204. https://doi.org/10.1016/s1470-2045(05)70086-3 kurose k, terai m, soedarsono n, et al. low prevalence of hpv infection and its natural history in normal oral mucosa among volunteers on miyako island, japan. oral surg oral med oral pathol oral radiol endod. 2004;98(1):91–96. https://doi.org/10.1016/j.tripleo.2003.12.029 d’souza g, kreimer ar, viscidi r, et al. case-control study of human papillomavirus and oropharyngeal cancer. n engl j med. 2007;356(19):1944–1956. https://doi.org/10.1056/nejmoa065497 de villiers e-m, gunst k, stein h, scherübl h. esophageal squamous cell cancer in patients with head and neck cancer: prevalence of human papillomavirus dna sequences. int j cancer. 2004;109(2):253–258. https://doi.org/10.1002/ijc.11685 bruni l, albero g, serrano b, et al. human papillomavirus and related diseases in africa. summary report [homepage on the internet]. 2021 [cited 2017 mar 29]. available from: http://www.hpvcentre.net/statistics/reports/xfx.pdf bruni l, barrionuevo-rosas l, albero g, et al. executive summary [document on the internet]. ico/iarc information centre hpv cancer, hpv information centre; 2017 [cited 2018 july 15]. available from: http://www.hpvcentre.net/statistics/reports/nga.pdf world health organization. human papillomavirus (hpv) and cervical cancer: fact sheets [homepage on the internet]. 2019 [cited 2019 sep 11]. available from: https://www.who.int/news-room/fact-sheets/detail/human-papillomavirus-(hpv)-and-cervical-cancer world health organization regional office for africa. nigeria’s call to action – time to eliminate cervical cancer in nigeria [homepage on the internet]. 2019 [cited 2021 june 8]. available from: https://www.afro.who.int/news/nigerias-call-action-time-eliminate-cervical-cancer-nigeria vogt sl, gravitt pe, martinson na, hoffmann j, d’souza g. concordant oral-genital hpv infection in south africa couples: evidence for transmission. front oncol. 2013;3:303. https://doi.org/10.3389/fonc.2013.00303 taherdoost h. sampling methods in research methodology: how to choose a sampling tech-nique for research [homepage on the internet]. 2016 [cited 2021 may 20]. available from: https://hal.archives-ouvertes.fr/hal-02546796 sotlar k, stubner a, diemer d, et al. detection of high-risk human papillomavirus e6 and e7 oncogene transcripts in cervica scrapes by nested rt-polymerase chain reaction. j med virol. 2004;74(1):107–116. https://doi.org/10.1002/jmv.20153 sanders ae, slade gd, patton ll. national prevalence of oral hpv infection and related risk factors in the u.s. adult population. oral dis. 2012;18(5):430–441. https://doi.org/10.1111/j.1601-0825.2011.01892.x gillison ml, broutian t, pickard rkl, et al. prevalence of oral hpv infection in the united states, 2009–2010. jama. 2012;307(7):693–703. https://doi.org/10.1001/jama.2012.101 schneider-maunoury s, croissant o, orth g. integration of human papillomavirus type 16 dna sequences: a possible early event in the progression of genital tumors. j virol. 1987;61(10):3295–3298. https://doi.org/10.1128/jvi.61.10.3295-3298.1987 de andrea m, kiyono t, pal a, kundu r. human papillomavirus e6 and e7: the cervical cancer hallmarks and targets for therapy. front microbiol. 2020;10:3116. https://doi.org/10.3389/fmicb.2019.03116 antonsson a, cornford m, perry s, davis m, dunne mp, whiteman dc. prevalence and risk factors for oral hpv infection in young australians. plos one. 2014;9(3):e91761. https://doi.org/10.1371/journal.pone.0091761 dalla torre d, burtscher d, sölder e, widschwendter a, rasse m, puelacher w. the impact of sexual behavior on oral hpv infections in young unvaccinated adults. clin oral investig. 2016;20:1551–1557. https://doi.org/10.1007/s00784-015-1633-y kero km, rautava j, syrjänen k, kortekangas-savolainen o, grenman s, syrjänen s. stable marital relationship protects men from oral and genital hpv infections. eur j clin microbiol infect dis. 2014;33(7):1211–1221. https://doi.org/10.1007/s10096-014-2061-7 chung ch, bagheri a, d’souza g. epidemiology of oral human papillomavirus infection. oral oncol. 2014;50(5):364–369. https://doi.org/10.1016/j.oraloncology.2013.09.003 d’souza g, cullen k, bowie j, thorpe r, fakhry c. differences in oral sexual behaviors by gender, age, and race explain observed differences in prevalence of oral human papillomavirus infection. plos one. 2014;9(1):e86023. https://doi.org/10.1371/journal.pone.0086023 touyz lzg. kissing and hpv: honest popular visions, the human papilloma virus, and cancers. curr oncol. 2014;21(3):e515. https://doi.org/10.3747/co.21.1970 nejo yt, olaleye do, odaibo gn. molecular characterisation of genital human papillomavirus among women in southwestern, nigeria. plos one. 2019;14(11). https://doi.org/10.1371/journal.pone.0224748 thomas jo, herrero r, omigbodun aa, et al. prevalence of papillomavirus infection in women in ibadan, nigeria : a population-based study. 2004:638–645. https://doi.org/10.1038/sj.bjc.6601515 herrero r, quint w, hildesheim a, et al. reduced prevalence of oral human papillomavirus (hpv) 4 years after bivalent hpv vaccination in a randomized clinical trial in costa rica. plos one. 2013;8(7). https://doi.org/10.1371/journal.pone.0068329 article information authors: dianna edgil1 jason williams2 peter smith2 joel kuritsky1 affiliations: 1united states agency for international development (usaid), washington, united states 2the partnership for supply chain management (pfscm), arlington, united states correspondence to: dianna edgil postal address: 1300 pennsylvania ave. nw, washington dc, united states dates: received: 14 mar. 2013 accepted: 03 apr. 2014 published: 05 sept. 2014 how to cite this article: edgil d, williams j, smith p, kuritsky j. optimising the laboratory supply chain: the key to effective laboratory services. afr j lab med. 2014;3(1), art. #101, 7 pages. http://dx.doi.org/10.4102/ ajlm.v3i1.101 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. optimising the laboratory supply chain: the key to effective laboratory services in this original research... open access • abstract • introduction • research method and design    • pepfar procurement    • cd4 cost-per-test analysis    • country-level cost analysis • results    • pepfar procurement    • calculated cd4 cost-per-test analysis    • country-level calculated cost analysis       • countries a and b       • country c • discussion • limitations of the study    • recommendations • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: the supply chain management system (scms) is a contract managed under the partnership for supply chain management (pfscm) consortium by the united states agency for international development (usaid). scms procures commodities for programmes supported by the us president’s emergency plan for aids relief (pepfar). from 2005 to mid-2012, pepfar, through scms, spent approximately $384 million on non-pharmaceutical commodities. of this, an estimated $90m was used to purchase flow cytometry technology, largely for flow cytometry platforms and reagents.objectives: the purpose of this paper is to highlight the cost differences between low, medium and high utilisation rates of common cd4 testing instruments that have been procured though pepfar funding. method: a scale of costs per test as a function of test volume through the machine was calculated for the two most common cd4 testing machines used in hiv programmes: becton dickinson (bd) facscount™ and bd facscalibur™. instrument utilisation data collected at the facility level in three selected countries were then used to calculate the onsite cost-per-test experienced in each country. results: cost analyses indicated that a target of at least 40% utilisation for facscount™ and 15% utilisation for facscalibur™, respectively, closely approach maximal per-test cost efficiency. the average utilisation rate for cd4 testing instruments varies widely by country, level of laboratory and partner (0% − 68%). conclusion: our analysis indicates that, because cost-per-test is related inversely to sample throughput, the underutilisation of flow cytometry machines is resulting in an increase in average cost-per-test for many instruments. introduction top ↑ the us president’s emergency plan for aids relief (pepfar) has directed significant resources toward diagnosing and treating hiv in selected developing countries. in support of this programme, the united states government established a supply chain contract mechanism that is implemented by the partnership for supply chain management (pfscm) and managed by the united states agency for international development (usaid). the purpose of this contract is to provide technical assistance to host countries with procurement of pharmaceuticals, laboratory products and other items for country programmes.the global hiv community has recognised that increasing the efficiency of current programmes through better management, data-driven decision making and appropriate resource allocation are key to the continued scale-up of hiv programmes necessary for universal access to care and treatment.1,2 laboratory diagnostic services are a critical component of hiv programmes and are key to programmes’ ability to scale up treatment for hiv.3 building laboratory capacity requires resources and is accompanied by supply chain challenges.4,5 optimising laboratory procurement by using an evidence-based strategy to inform the procurement of laboratory instruments that are appropriate to the throughput (i.e., tier) of the laboratory in which they are to be placed, offers an opportunity to increase value for money6 and ensures that programmes can maximise the number of patients with access to reliable laboratory diagnostic services. the purpose of this article is to analyse costs associated with flow cytometry platforms by studying utilisation rates in three countries and comparing them to the maximum throughput recommended by manufacturers. the data will help inform country purchases and direct optimal deployment strategies of this technology. research method and design top ↑ pepfar procurement procurement data were collected from the pfscm orion procurement system. the orion system is pfscm’s integrated procurement system that controls, monitors and records all procurement activity for scms/pepfar. all figures represent the value of delivered commodities in us dollars ($) from september 2005 to june 2012. data are categorised into pharmaceuticals (pharma; 66%), rapid test kits (9%), analysers and/or reagents (13%), laboratory consumables (lab; 8%) and ‘other’ (4%). cd4 cost-per-test analysis we established a unit cost-per-test for the benton dickinson (bd) facscount™ and bd facscalibur™ instruments based on current prices paid by pfscm and manufacturer-established consumption amounts.7,8 consumption ratios were calculated for each product defined by the instrument manufacturer and end-user experiences (commodity list, consumption ratios and pricing in table 1). third-party control samples were not included in the overall pricing because of inconsistency in use. total costs were then calculated based on the product requirements needed to conduct cd4 testing over a one-day period. per-test costs were calculated using the following formula: table 1: cd4 count platform reagents and usage rates. cost-per-test = [(σ (unit quantity commodity cost/usage rate per test)a-e * tests per day) + daily control costf]/tests per day {eqn 1} estimated costs do not include product wastage, start-up and shutdown product consumption, or any additional equipment maintenance costs, which can vary considerably by end users and across countries. country-level cost analysis demographic, morbidity and service-statistics data were collected to inform multi-year (threeto five-year) laboratory instrumentation commodity requirements for three national quantification workshops in 2011 and 2012. instrument test numbers were gathered through data collection exercises at all laboratory sites in countries a and b and at a subset of sites in country c. data were provided by national laboratory leadership in each country, as well as pepfar implementing partners and united states government missions (usaid/us centers for disease control and prevention). where information on instrument service interruptions as a result of commodity stock-outs was available, diagnostic consumption was adjusted in order to account for a reduction in the number of days of operation. demographic and morbidity data, adjustments to test numbers associated with programme scale-up and general assumptions were documented. final forecast estimates were then validated through consensus at each quantification workshop. quantification outputs were used to develop first-year supply plans; to determine overall laboratory network commodity resource requirements, hiv diagnostics, care and treatment monitoring tests; and to prioritise laboratory spending when funding gaps were identified.to determine the overall efficiency of cd4 testing instruments within the laboratory system, cd4 facscount™ and facscalibur™ testing data were extracted from the quantification and forecast data gathered during the country’s first-year forecast period. for each country, the number of individual instruments and where they were located within each national laboratory network were determined. we compared the number of tests by instrument with the manufacturer-recommended average instrument throughput capacity (50 sample tests per day for facscount™ and 350 per day for facscalibur™) to determine instrument utilisation rates.7,8 the rates were not adjusted for instrument breakdowns because of a consistent lack of data in each represented country at the time of data review. diagnostic contribution was calculated by comparing individual machine throughput with total service provision estimates (service statistics), disaggregated by instrument type and level within the laboratory networks. for country c, we calculated product use to diagnostic contribution by the seven implementing partners using bd instruments (five partners using other brands were removed from the analysis). the total cd4 commodity cost was established for 2011 based on actual testing services provided during that year. the 2012 unit prices were calculated by projecting programmatic growth, planned instrument procurements and testing demand increases based on pepfar care and treatment targets. results top ↑ pepfar procurement as of june 30, 2012, pfscm assisted in procuring pharmaceuticals (antiretrovirals, treatment for opportunistic infections) and other products with a value of about $1.1 billion (figure 1). thirty-four per cent of pfscm’s overall commodity procurement, $384m, was spent on non-pharmaceutical products to supply pepfar-supported laboratories and testing sites in 53 countries. $150m of the non-pharmaceutical commodity amount was spent on reagents for analytical testing, with the majority ($90m) going to cd4 testing. procurement of analytical testing products, specifically for cd4 testing, has increased by almost 20% annually for the past five years and in 2011 accounted for 8% of all procurement (figure 2). figure 1: partnership for supply chain management (pfscm) total spending as of june 30, 2012. pfscm historical procurement data were used to determine the total expenditures for the supply chain management systems life of project (lop). all figures represent the value of delivered commodities in us dollars from september 2005 to june 2012. data are categorised into pharmaceuticals (pharma [66%], hiv rdts [9%], analysers and/or reagents [13%], lab/clinical supplies [8%] and other [4%]). figure 2: partnership for supply chain management (pfscm) spending on laboratory commodities delivered through june 2012. pfscm historical procurement data are displayed as expenditures per year from 2007 through june 2012. pepfar flow-cytometry expenditures show continual increases on a per-year basis. pepfar’s largest procurement of laboratory reagents during the period of september 2005 to june 2012 was for flow cytometry (64% of total reagent costs). bd products ($81m) represented 54% of the overall reagent costs and 90% of flow cytometry costs. for this reason, the two most commonly used bd flow cytometry platforms, facscount™ and facscalibur™, were chosen for further analysis in order to identify areas of cost savings or cost efficiency. calculated cd4 cost-per-test analysis we hypothesised that the cost for a test performed using these platforms would depend on volume, as was seen in a previous analysis.9 the cd4 cost-per-test analysis was limited to pfscm prices paid for the reagents required for the bd facscount™ and facscalibur™ platforms (table 1).using pfscm pricing for necessary testing commodities, we found that a higher throughput did result in a lower cost-per-test, with the rate of cost savings decreasing as the volume approached the manufacturer-recommended maximum throughput of the instrument (figure 3). the costs per test in this analysis were found to range from $14.64 to $7.29 for the facscount™ systems and $14.06 to $8.67 for the facscalibur™ system. we chose a rate of return of less than $0.01 per additional test per day as the point at which further investment in scale-up does not gain significant returns. whilst in both cases the rate of return diminishes to less than $0.01 per additional test added per day after a critical volume is achieved, for facscount™ the volume must exceed 40% (n > 20 tests) of the maximum daily throughput (50 tests), whereas for facscalibur™, the critical volume is approximately 15% (n > 45 tests) of maximum throughput (350 tests). figure 3: facscount™ versus faccalibur™ utilisation price per test in us dollars. the reagent unit cost-per-test for the bd facscount™ and bd facscalibur™ instruments was based on historical partnership for supply chain management (pfscm) pricing and manufacturer-established consumption amounts,7,8 using the reagents and consumption ratios in table 1. total costs were then calculated based on the product requirements needed to conduct cd4 testing over a one-day period. the rate of return diminishes to less than $0.01 per additional test added per day after a critical volume is achieved: facscount™, n > 20 tests or 40% throughput; facscalibur™, n > 45 tests or 15% throughput. several variable costs drive the cost-per-test of the bd systems, especially at low throughput volumes. in this analysis, the bd control and/or calibration kit is an important cost driver for both instruments in that unit pricing reductions are based on maximising use of the control kit by increasing the volume of tests processed per day. in comparison with the facscount™, the facscalibur™ has an initially lower baseline cost-per-test cd4 reagent (table 2). in fact, although the cost-per-test for both machines is similar at very low throughput, because of the lower baseline cost-per-test for facscalibur™ cd4 reagents, at extremely low volumes (n < 2) the facscalibur™ is slightly less expensive than the facscount™. however, as shown in figure 3, for volumes of more than two tests per day, the cost-per-test of the facscount™ system drops below that of the facscalibur™ system. the facscalibur™ system maintains a unit pricing per test at about $1.40 higher than that of the facscount™ as efficiency is gained. these results indicate that for all bd platforms, cost savings can be achieved by maximising daily testing volumes per machine with optimal targets of 40% throughput for facscount™ and 15% throughput for facscalibur™ systems. additionally and significantly, for volumes of tests of n > 2, the facscount™ system is the more cost efficient testing platform. table 2: facscount™ and facscalibur™ reagent unit price per test. country-level calculated cost analysis countries a and b to estimate potential cost savings that might be achieved by maximising throughput volumes, data from two countries were used to compare existing and recommended targeted throughput in each country. the cost-per-test as a function of throughput, as described above, was compared with actual utilisation rates collected from testing facilities in two pepfar countries in order to calculate the average cost-per-test of a cd4 test performed on facscount™ machines. for facscount™, country a (table 3) is shown to maintain an aggregated average instrument utilisation rate of 47%, which translates to an average cost-per-test of $7.46. country b (table 4) is shown to have an average facscount™ instrument utilisation rate of 10%, resulting in a calculated average cost of $8.64 per test. between countries a and b, the average cost-per-test difference for tests performed using a facscount™ system amounts to an estimated $1.18 in unit pricing overall. table 3: country a cd4 testing equipment utilisation. to identify areas in which the greatest cost efficiencies could be gained through increased utilisation of cd4 testing equipment within the tiered laboratory system, we performed a more targeted analysis of country b. in our review of the data for country b, we observed that laboratories in regional and provincial laboratories had 51 facscount™ machines. these sites had an utilisation rate of 9% (n < 5 tests per day), but contributed 30% of the total cd4 tests conducted for the country (table 4). costs in these laboratories averaged $8.83 per unit test. according to our analysis of cost-per-test as a function of instrument utilisation, country b would achieve a cost-per-test of $7.89 were it to increase its utilisation of these machines to 20%; a per-test savings of $0.94. moreover, were country b to increase utilisation to 40%, the target for maximising efficiency, the cost-per-test would be $7.51, a per-test savings of $1.32. extrapolating the savings accrued by country b for increasing cd4 equipment utilisation to 20% or 40% in regional and/or provincial laboratories as a percentage of the total budget spent on cd4 testing would reduce expenditures by 14% and 17%, respectively. overall, small increases in utilisation rates, when targeted to facilities with large diagnostic contribution to the total number of tests performed, can result in significant cost savings. table 4: country b cd4 testing equipment utilisation. country c in country c we calculated cd4 test costs by seven different implementing partners. targeting implementing partners with high cd4 testing contribution and seeking ways to increase utilisation rates may be one way to reduce testing costs and establish further commodity consumption efficiencies. the analysis of implementing partners in country c provided an opportunity to investigate implementing partner cd4 testing contributions for pepfar in 2011 and to examine projected growth into 2012. partners 2, 4 and 5 use facscount™ machines and contribute significantly to cd4 testing services within the pepfar country c programme for 2011 and 2012 (table 5). instrument utilisation rates for partners 2, 4 and 5 are, respectively, 30%, 4% and 43% in 2011 and 5%, 4% and 57% in 2012. the model for optimal instrument utilisation is partner 5, with a cost-per-test average of $7.49, which further gained efficiency in 2012 by increasing instrument utilisation to 57% with an average per-test cost of $7.40. in contrast, partner 4 had a throughput of 4% capacity in 2011 and 2012, with a calculated cost of $10.89 per test. table 5 indicates that the projected number of tests performed has increased substantially (by 13%), yet the percent utilisation remains unchanged. this indicates that, rather than increasing throughput on existing machines, additional machines have been procured by this partner such that an exceedingly high cost-per-test is maintained over time (10 new facscount™ instruments planned for 2012). given the extremely low throughput observed for partner 4, even a 2% increase in utilisation (an average of one more test per day) could reduce per-test costs by over $1 ($10.89 − $9.64). here, consolidating testing into existing machines to increase efficiency rather than procuring new equipment would result in significant cost savings over time. table 5: country c comparison of cd4 testing equipment utilisation in 2011 and 2012. for country c, product use and diagnostic contribution were disaggregated according to those implementing partners using bd instruments (five partners using other brands were removed from the analysis). total cd4 commodity costs for 2011 were based on actual testing services provided. 2012 unit prices were based on projected programmatic growth and planned instrument procurements. instrument utilisation rates for partners 2, 4 and 5 are, respectively, 30%, 4% and 43% in 2011 and 5%, 4% and 57% in 2012, indicating appropriate growth for partner 5, but a decrease or no increase in efficiency (reduced cost-per-test) for partners 2 and 4. similarly, in 2011 partner 2 operated with an average instrument utilisation of 30% and a cost-per-test of $7.64 (table 5). ideally, for 2012, partner 2 would increase instrument utilisation to 40% to gain efficiency and lower the cost-per-test for its cd4 testing programme. however, in this case, despite a significant projected increase in tests performed, instrument utilisation decreases to 5%, giving an average cost-per-test in 2012 of $10.27. this is an increase of $2.63 per test over 2011 costs. once again, the decrease in utilisation likely indicates the procurement of additional equipment (43 new facscount™ instruments planned for 2012) within a small testing pool that reduces the average volumes for all machines operated by partner 2. using the 2011 per-test cost ($7.64) to calculate the budget needed for partner 2 to perform the 55 798 tests projected for 2012 results in an estimated budget of $426 297 for reagent procurement. in this case, the decrease in utilisation and subsequent increase in per-test cost in 2012 (to $10.27) results in an actual budgetary requirement of $573 045. the underutilisation of cd4 testing equipment results in an additional $146 748 in reagent procurement needed to perform the same number of tests. these results indicate that efficient systems seeking to expand coverage must consider the cost implications of reducing volumes through existing equipment. discussion top ↑ our work in reviewing procurement and participation in laboratory commodity quantification exercises indicates that cost savings can be realised with better utilisation of cd4 testing instruments. in this analysis of national quantification exercises in three countries, we identify one area in which greater efficiency may be established by maximising cd4 instrument utilisation rates to reduce the cost-per-test of cd4 testing (represented by bd facscount™ and facscalibur™). these results indicate that for all bd platforms, cost savings can be achieved by maximising daily testing volumes per machine with optimal targets of 40% throughput for facscount™ and 15% throughput for facscalibur™ systems. this is particularly important for testing sites with large diagnostic contribution where small increases in utilisation rates can result in significant cost savings. for programmes seeking to expand coverage, acquisition of additional cd4 machines should consider the need to increase utilisation by consolidating testing into existing machines where existing referral networks are adequate.considering instrument placement before procurement, including accurate estimation of the appropriate demand at deployment locations, ultimately increases consumption efficiencies and reduces overall costs. to that end, it is particularly important to understand the cost implications of decentralising services to sites that underutilise instruments that contribute little to overall diagnostics. underutilising instruments that contribute less to the overall testing uptake has less of an impact on overall commodity costs, whereas underutilising instruments that have higher overall testing contributions has a higher impact on cost. for example, a facscount™ instrument operating at $9.00 per test that contributes to only 4% of the national testing target will have a lower impact on overall programme costs than if that same machine were contributing to 35% of the national testing targets. strategically relocating existing instruments could improve utilisation, as could replacing underutilised equipment with lower capacity point-of-care (poc) tests. it is important to either match site-level demand to the instrument (placing smaller instruments in low-volume clinics) or to place underutilised instruments into higher volume sites as back-up instruments in order to add redundancy in the event of equipment breakdown. planned expansion should first seek to efficiently utilise existing equipment and minimise the appropriation of tests from currently functioning sites. throughput of a selected cd4 testing machine should match testing consumption at the service delivery point. for certain facilities, diagnostics consumption falls below the optimal throughput volume for the more commonly-used cd4 platforms into the range of the low-throughput poc cd4 testing platforms, for which the cost-per-test is a flat rate from a commodity consumption perspective and does not vary with volume. in these cases, it may be more cost efficient to use a poc cd4 testing platform. this decision would require selecting a single supported poc instrument that is included in the national health laboratory strategic plan and strategically integrated into the tiered laboratory network to optimise existing instrumentation, balancing access to service. it should be noted that this analysis shows that for nearly all levels of throughput, the facscount™ platform is less expensive on a per-test basis than the facscalibur™ system. choosing to implement the facscalibur™, however, does have some benefit in very high-throughput facilities because it is fully automated and processes samples more quickly than the facscount™. thus, use of the facscalibur™ has the potential to reduce overall laboratory costs by allowing for higher testing volumes at service provision sites with less dependence upon laboratory staff time. whilst an acceptable level of instrument utilisation may appear to be achieved at the national level, for some countries disaggregating instrument utilisation for cd4 testing equipment by tiered level, region, or possibly by implementing partner, can assist in developing a more targeted intervention. for example, at the level of an individual implementing partner, diagnostic throughput may be very low, resulting in a high cost-per-test for testing sites managed by that implementing partner. for these partners, it may be useful to consolidate testing to existing equipment or to consider the suitability of new equipment for proposed expansion sites. further comparative analysis could potentially guide appropriate instrument deployment based on the site-specific demands, maximising partner cost efficiencies and further reducing the overall cd4 testing unit pricing scheme. limitations of the study top ↑ the study presented here has several limitations. the first is that the cost-per-test analysis considers only the cost of reagents. this likely results in an underestimation of the true cost-per-test, which would also include the cost of the equipment, human resources, service maintenance contracts and of expired and wasted product because of low equipment throughput and instrument breakdown. the second limitation to the analysis is a lack of information on efficiencies gained at very-low-volume sites by batching test samples collected over several days. very-low-volume sites using batch testing would at some level increase throughput and decrease the daily cost-per-test, perhaps significantly, at very low volume. finally, the analysis centres on bd cd4 testing platforms; whilst these are the most commonly-used cd4 testing platforms in pepfar-supported hiv testing programmes, other platforms with considerably lower reagent costs are used, albeit far less commonly, throughout africa. recommendations the 2008 maputo declaration on strengthening of laboratory systems6 called for harmonisation and standardisation of tests, reagents, consumables and instruments at each level within a tiered laboratory system. since that time, harmonisation has proven to be a challenge for many reasons, including evolving diagnostic coverage during scale-up, system maturation, existing procurement policies and changes in demographic and morbidity demands. nonetheless, in support of the maputo declaration, we must pursue a strategy of optimising laboratory procurement through informed decision making in order to advance harmonisation and maximise consumption efficiencies. such a strategy can advance harmonisation at all levels within the tiered system by using site-level data that informs the selection of equipment based on need or consumption at the point of use (consumption efficiencies); including the platform within the national testing algorithm, if one exists (hiv, tuberculosis and malaria); and understanding the sustainability of the platform within the regional setting (adequate infrastructure, reagent supply, maintenance and training curricula). the overall objective of optimising laboratory procurement is to develop instrument placement strategies that will increase appropriate coverage, increase overall commodity consumption efficiencies and ensure that instruments are operational, accounted for and, ultimately, maximise return on investment.as donor support and financial resources must stretch further to meet mounting global health needs, it is critical that potential savings be sought wherever possible. recognising the commitment and financial obligation required to support instrument life cycles amongst many competing priorities requires a broad perspective and regular re-evaluation of needs. in this way, translating historical laboratory procurement and quantification data into service performance indicators across all laboratory instrumentation and across different platforms can provide visibility into the critical operational aspects and create opportunities to establish further efficiencies of a laboratory network over time. product consumption and testing numbers can identify commodity wastage, supply chain management inefficiencies and the underutilisation of machines based on their potential testing capacity. armed with this information, those directing and managing laboratory networks are better equipped to develop responsive laboratory optimisation strategies that enable the best use of every donor dollar. conclusion top ↑ donors, implementing partners and host-country governments must make deliberate, transparent and coordinated efforts to advance evidence-based laboratory optimisation processes. countries must be positioned to take into account how best to balance costs, increase consumption efficiencies and ensure overall access to services. to realise further cost savings and continue to increase access to patient testing services, the development of laboratory optimisation strategies should serve as a critical step in further advancing overall healthcare delivery. fundamental to this strategy is ensuring a clear understanding of how effectively laboratory-related commodities are consumed in support of healthcare programmes. acknowledgements top ↑ we thank vincent wong for his thoughtful comments and sue carrington for her technical assistance.this research has been supported by the president’s emergency plan for aids relief (pepfar) through usaid under the terms of project number gpo-i-00-05-00032-00. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. authors’ contributions the opinions of the authors of this article do not represent the opinions of usaid. the authors’ contributions are as follows: j.k. (usaid) was the project leader and d.e. (usaid) wrote the manuscript and was responsible for project design. j.w. and p.s. (both pfscm) collected and analysed the data (at country level and global expenditures, respectively) and made conceptual contributions regarding the optimisation of cd4 diagnostics in order to maximise return on investment on equipment procurement. references top ↑ 1.el-sadr wm, holmes cb, mugyenyi p, et al. scale-up of hiv treatment through pepfar: a historic public health achievement. j acquir immune defic syndr. 2012;60 suppl 3:s96–104. http://dx.doi.org/10.1097/qai.0b013e31825eb27b2.larson hj, bertozzi s, piot p. redesigning the aids response for long-term impact. bull world health organ. 2011;89(11):846–852. http://dx.doi.org/10.2471/blt.11.087114 3.nkengasong jn, nsubuga p, nwanyanwu o, et al. laboratory systems and services are critical in global health: time to end the neglect? am j clin pathol. 2010;134(3):368–373. http://dx.doi.org/10.1309/ajcpmpsinq9brmu6 4.thairu l, katzenstein d, israelski d. operational challenges in delivering cd4 diagnostics in sub-saharan africa. aids care. 2011;23(7):814–821. http://dx.doi.org/10.1080/09540121.2010.541416 5.birx d, de souza m, nkengasong jn. laboratory challenges in the scaling up of hiv, tb, and malaria programs: the interaction of health and laboratory systems, clinical research, and service delivery. am j clin pathol. 2009;131(6):849–851. http://dx.doi.org/10.1309/ajcpgh89qdswfons 6.the world health organization. the maputo declaration on strengthening of laboratory systems [document on the internet]. c2008 [cited 2013 mar 13]. available from: http://www.who.int/diagnostics_laboratory/maputo-declaration_2008.pdf 7.bd biosciences. bd facscount system: technical specifications [document on the internet]. c2009 [cited 2013 mar 13]. available from: http://www.bdbiosciences.com/external_files/is/doc/tds/instruments/live/web_enabled/23-6575-02.pdf 8.bd biosciences. bd facscalibur flow cytometry system: technical specifications [document on the internet]. c2010 [cited 2013 mar 13]. available from: http://www.bdbiosciences.com/documents/facscalibur_flowcytometry_techspec.pdf 9.jani iv. cost comparison of point-of-care and laboratory cd4 testing in resource-limited settings. presented at the 6th ias conference on hiv pathogenesis and treatment: 2011 july 17–20; rome, italy. ajlm 7(2)_contents.indd http://www.ajlmonline.org open access table of contents i original research clostridium difficile in patients attending tuberculosis hospitals in cape town, south africa, 2014–2015 brian r. kullin, sharon reid, valerie abratt african journal of laboratory medicine | vol 7, no 2 | a846 | 06 december 2018 original research antibiotic resistance trends of eskape pathogens in kwazulu-natal, south africa: a five-year retrospective analysis yogandree ramsamy, sabiha y. essack, benn sartorius, miriam patel, koleka p. mlisana african journal of laboratory medicine | vol 7, no 2 | a887 | 06 december 2018 original research multi-drug resistant mycobacterium tuberculosis in port harcourt, nigeria kome otokunefor, tosanwumi v. otokunefor, godwin omakwele african journal of laboratory medicine | vol 7, no 2 | a805 | 06 december 2018 original research differential infectivity of gametocytes after artemisinin-based combination therapy of uncomplicated falciparum malaria dinkorma t. ouologuem, cheick o. kone, bakary fofana, bakary sidibe, amadou h. togo, demba dembele, sekou toure, sekou koumare, ousmane toure, issaka sagara, abdoulaye toure, adama dao, ogobara k. doumbo, abdoulaye a. djimde african journal of laboratory medicine | vol 7, no 2 | a784 | 06 december 2018 lessons from the field leveraging donor support to develop a national antimicrobial resistance policy and action plan: ghana’s success story japheth a. opintan african journal of laboratory medicine | vol 7, no 2 | a825 | 06 december 2018 lessons from the field antimicrobial resistance surveillance in ethiopia: implementation experiences and lessons learned rajiha a. ibrahim, amete m. teshal, surafel f. dinku, negga a. abera, abebe a. negeri, feven g. desta, eyasu t. seyum, adugna w. gemeda, wubshet m. keficho african journal of laboratory medicine | vol 7, no 2 | a770 | 06 december 2018 scientific letter need for better adherence to optimal incubation temperature for quality laboratory diagnostics and antibiotic resistance monitoring cristina gutierrez, akos somoskovi, kris natarajan, david bell african journal of laboratory medicine | vol 7, no 2 | a789 | 06 december 2018 brief report diagnosis of rifampicin-resistant tuberculosis: discordant results by diagnostic methods winnie mwanza, deborah milimo, maureen m. chilufya, nkatya kasese, maina c. lengwe, stembiso munkondya, petra de haas, helen ayles, monde muyoyeta african journal of laboratory medicine | vol 7, no 2 | a806 | 06 december 2018 49 58 66 70 76 80 84 86 page i of i table of contents i editorial antimicrobial resistance surveillance in africa: successes, gaps and a roadmap for the future samuel kariuki, karen h. keddy, martin antonio, iruka n. okeke african journal of laboratory medicine | vol 7, no 2 | a924 | 06 december 2018 review article an overview of antimicrobial resistance surveillance among healthcare-associated pathogens in south africa ashika singh-moodley, husna ismail, olga perovic african journal of laboratory medicine | vol 7, no 2 | a741 | 06 december 2018 review article drug resistant tuberculosis in africa: current status, gaps and opportunities nazir ismail, farzana ismail, shaheed v. omar, linsay blows, yasmin gardee, hendrik koornhof, philip c. onyebujoh african journal of laboratory medicine | vol 7, no 2 | a781 | 06 december 2018 review article a systematic review of healthcare-associated infections in africa: an antimicrobial resistance perspective emmanuel o. irek, adewale a. amupitan, temitope o. obadare, aaron o. aboderin african journal of laboratory medicine | vol 7, no 2 | a796 | 06 december 2018 review article antimicrobial resistance of vibrio cholerae from sub-saharan africa: a systematic review yahaya mohammed, aaron o. aboderin, iruka n. okeke, adebola t. olayinka african journal of laboratory medicine | vol 7, no 2 | a778 | 06 december 2018 opinion paper africa centres for disease control and prevention’s framework for antimicrobial resistance control in africa jay k. varma, john oppong-otoo, pascale ondoa, olga perovic, benjamin j. park, ramanan laxminarayan, rosanna w. peeling, constance schultsz, han li, chikwe ihekweazu, amadou a. sall, baboucarr jaw, john n. nkengasong african journal of laboratory medicine | vol 7, no 2 | a830 | 06 december 2018 opinion paper antimicrobial resistance surveillance with whole genome sequencing in africa: it’s (about) time hajo grundmann, hellen gelband african journal of laboratory medicine | vol 7, no 2 | a761 | 06 december 2018 opinion paper the role of connected diagnostics in strengthening regional, national and continental african disease surveillance natasha m. gous, philip c. onyebujoh, alash’le abimiku, chris macek, jeff takle african journal of laboratory medicine | vol 7, no 2 | a775 | 06 december 2018 1 3 9 20 29 36 40 43 vol 7, no 2 (2018): african laboratories in antimicrobial resistance surveillance issn: 2225-2002 (print) | issn: 2225-2010 (online)african journal of laboratory medicine abstract introduction methods results discussion acknowledgements references about the author(s) mercy n. okunorobo department of medical laboratory science, faculty of health sciences and technology, nnamdi azikiwe university, awka, nigeria nwakasi k. nnamah department of chemical pathology, faculty of clinical medicine, nnamdi azikiwe university, awka, nigeria ugomma a. ude department of medical laboratory science, faculty of health sciences and technology, ebonyi state university, abakaliki, nigeria enyioma a. ude department of medical laboratory science, faculty of health sciences and technology, nnamdi azikiwe university, awka, nigeria citation okunorobo mn, nnamah nk, ude ua, ude ea. lipids and apolipoproteins c-iii and e among treatment-naïve and treatment-experienced persons with hiv in nigeria. afr j lab med. 2023;12(1), a2018. https://doi.org/10.4102/ajlm.v12i1.2018 original research lipids and apolipoproteins c-iii and e among treatment-naïve and treatment-experienced persons with hiv in nigeria mercy n. okunorobo, nwakasi k. nnamah, ugomma a. ude, enyioma a. ude received: 18 july 2022; accepted: 12 apr. 2023; published: 17 july 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: dyslipidaemia is a known cause of cardiovascular mortality. persons living with hiv are at high risk of developing cardiovascular disease due to lipid metabolism disorders associated with hiv or its therapy. objective: this study evaluated concentrations of lipoproteins and apolipoprotein c-iii and e, as a way of assessing cardiometabolic risks among hiv patients. methods: we enrolled 50 hiv-negative persons and 100 hiv-positive patients, 50 on antiretroviral therapy (art) and 50 treatment-naïve persons, from the central hospital and the stella obasanjo hospital, benin city, edo state, nigeria, between may 2015 and november 2015. participants with a history of metabolic abnormalities were excluded. apolipoproteins were assessed by enzyme-linked immunosorbent assay, while lipids were measured by spectrophotometry. results: there were significant abnormalities in the lipid profile of patients with hiv. triglycerides levels of hiv patients (art-naïve: 1.44 ± 0.65 mmol/l; p < 0.001 and art-experienced: 1.49 ± 0.70 mmol/l; p = 0.001) were significantly higher than among controls (0.95 ± 0.54 mmol/l). hiv patients had higher concentrations of apolipoprotein c-iii than controls (p < 0.001) and higher low-density lipoprotein cholesterol levels (treatment-naïve: 2.83 mmol/l and art-experienced patients: 3.59 mmol/l) than controls (2.50 mmol/l; p = 0.003). conversely, hiv patients had significantly lowered high-density lipoprotein cholesterol levels compared to controls (p < 0.001). conclusion: dyslipidaemia was observed among hiv participants, irrespective of their art experience. therefore, it is crucial that the lipids of hiv patients be closely monitored to enable early intervention and decrease cardiovascular death. what this study adds: this study affirms that dyslipidemia is a complication of hiv or the prolonged use of art. keywords: dyslipidaemia; apolipoprotein c-iii; apolipoprotein e; hiv; antiretroviral therapy; cardiometabolic disorders; lipid profile. introduction hiv is a global pandemic associated with aberrations in the immune system and biochemical parameters, which increases the vulnerability of the infected individual to diseases.1,2 according to the nigeria hiv/aids indicator and impact survey, hiv is highly prevalent among adults in the south-south region of nigeria.3 the use of antiretroviral therapy (art) in the management of hiv has improved the life expectancy of persons living with hiv4 but has also been associated with an increased risk of metabolic abnormalities, including dyslipidaemia and cardiovascular death.5,6,7 a higher burden of cardiovascular disease has been reported among hiv patients on highly active antiretroviral therapy treatment compared to highly active antiretroviral therapy-naïve patients.8 dyslipidaemia refers to abnormal plasma lipid concentrations, including elevated plasma total cholesterol, low-density lipoprotein cholesterol (ldl-c), triglycerides, and reduced high-density lipoprotein cholesterol (hdl-c). these could occur as single entities or in combination.9,10 dyslipidaemia is a known predictor of endothelial dysfunction, cardiovascular disease, and atherosclerosis. it is a common non-communicable disease among persons living with hiv. thirty percent of persons living with hiv have been reported to have dyslipidaemia. the use of art may contribute to the development of dyslipidaemia in persons living with hiv.8 dyslipidaemia has been reported among both treatment-naïve and art-experienced hiv-positive patients,10 suggesting that lipid abnormalities among hiv patients may be caused by hiv itself and may be worsened by art.11 changes in lipid synthesis or transportation can cause dyslipidaemia.12 consequently, changes in apolipoproteins, protein components of lipoproteins that bind and transport lipids for metabolism or utilisation, could modulate serum lipid concentrations causing dyslipidaemia. apolipoprotein e (ae) is essential for the catabolism of remnant (triglyceride-rich) lipoproteins as it is a major transporter of cholesterol and triglyceride-rich lipoproteins.13 apolipoprotein c-iii (aciii) plays a crucial role in the synthesis and catabolism of very low-density lipoproteins.13 these proteins have been associated with hypertriglyceridaemia,14,15 a known predictor of cardiovascular disease.16 aside from its role in lipid and lipoprotein metabolism, ae is also involved in the immune response by stimulating or inhibiting antigen-t-lymphocyte proliferation.17 studies assessing serum lipid levels of hiv-positive subjects abound. however, there is a paucity of research evaluating apolipoprotein concentrations, despite their importance in lipid concentrations and metabolism. little is known about the concentration of apolipoproteins and their role in the course of hiv infection. the study aimed to assess the risk of cardiometabolic disorder and determine the correlation between lipids and the studied apolipoproteins of hiv-positive patients. methods ethical considerations ethical study approval was sought and obtained from the hospital management board of edo state, nigeria (study approval number: ch/a406 voliii/177a). the study participants were educated on the study’s details and assured of the confidentiality and anonymity of the information and data obtained during the study. we enrolled only participants who consented in writing. data that could be traced back to the participants were not collected. all information obtained from the participants was treated with the utmost confidentiality and used strictly for research. study location the study was carried out at the heart to heart clinic of central hospital and stella obasanjo hospital, both in benin city, edo state of nigeria, between may 2015 and november 2015. both hospitals have major hiv and aids centres serving patients from all over the state. study design this cross-sectional study compared the lipid profile and aciii and ae levels of hiv-positive and hiv-negative participants. for the case group, 100 participants were hiv-positive and grouped based on their treatment experience. group one included 50 hiv-positive art-naïve individuals. in contrast, group two included 50 hiv-positive individuals receiving first-line art made up of a combination of two nucleotide reverse transcriptase inhibitors (zidovudine or tenofovir and lamivudine) with one non-nucleotide reverse transcriptase inhibitor (efavirenz). all case participants were adults and enrolled for care in the study’s hospitals. for the control group, 50 hiv-negative adults with no history of organ or metabolic disease were recruited from among staff and patient relatives in the hospitals. any individual with a known history of diabetes mellitus or on treatment for dyslipidaemia was excluded from the study. also excluded were current smokers and excessive alcohol drinkers. excessive alcohol drinking was defined as a pattern of alcohol use, including binge consumption (four drinks or 70 g [for men] and 56 g [for women] in two hours), frequent or heavy consumption, hazardous use and dependency18. sample processing and analytical methods five millilitres of blood was obtained after an overnight fast from each of the participants. the blood sample was withdrawn from the antecubital vein using sterile hypodermic syringes into plain containers. all samples were transported from the collection site in the cold chain to the chemical pathology laboratory of a teaching hospital in south east nigeria, where the analysis was done. the blood was allowed to clot, and the serum separated from the cells by centrifuging at 1500 revolutions per minute for 5 min. the serum was stored at −20 ° c for one week before the samples were analysed. hiv screening all enrolled participants were tested to confirm their hiv status per the national hiv testing algorithm19 using hiv determine™ (alere medical co. limited, tokyo, japan), uni-gold recombigen hiv 1/2 test (trinity biotech, bray, ireland), and hiv 1/2 stat-pak (chembio diagnostic systems, medford, new york, united states). anthropometric variables anthropometric data of the participants, including height and weight, waist circumference, and systolic and diastolic blood pressure, were measured, and body mass index was computed using the ratio of weight (kg) to height (m2). lipid and apolipoprotein measurement lipid profile parameters were estimated using commercially available reagents (randox cholesterol, triglycerides, hdl-cholesterol) from randox laboratories, crumlin, ireland. to estimate of total cholesterol and hdl-c, the method of allan et al.20 was employed, while the method of bucolo and david21 was used to estimate triglyceride. low-density lipoprotein cholesterol was calculated using the friedewald equation:22 in equation 1, ldl-c is low-density lipoprotein, tchol is total cholesterol, tg is triglyceride and hdl-c is high-density lipoprotein. apolipoprotein c-iii and ae were measured by the enzyme-linked immunosorbent assay method using commercially available reagents from abcam (cambridge biomedical campus, cambridge, united kingdom). commercially sourced control sera (randox laboratories, crumlin, ireland) were included in every test, and the routine quality control procedures were maintained. dyslipidaemia was defined as elevated total cholesterol (≥ 5.0 mmol/l), elevated ldl-c (≥ 3.4 mmol/l), elevated triglyceride (≥ 1.6 mmol/l) or as low hdl-c (≤ 1.04 mmol/ l). hyperglycaemia was defined as fasting blood glucose ≥ 6.2 mmol/l. elevated blood pressure was defined as systolic and diastolic pressure greater than 130/85 millimetre of mercury. statistical analysis data analysis was conducted using statistical package for social sciences statistical software version 20 for windows 7 (ibm corp., chicago, illinois, united states). the results were expressed as mean ± standard deviation (s.d.). data obtained from this study were analysed using one-way analysis of variance, and post hoc tests were used to compare means. pearson bivariate correlation was used to assess associations between protein levels and lipid profiles. a p-value was considered significant at ≤ 0.05. results characteristics of study population the mean ages were 39.36 ± 7.02 years for art-naive, 38.46 ± 7.70 years for art-experienced and 31.20 ± 7.26 years for hiv-negative participants (table 1). the mean sbp of the art-naive participants was 135.52 ± 24.79 millimetres of mercury, 143.26 ± 27.98 millimetres of mercury for art-experienced participants and 130.72 ± 14.56 millimetres of mercury for the hiv-negative participants. patients on art had significantly higher sbp compared to the control group (p = 0.023). no significant difference was observed when the sbp of the art-naïve participants was compared with that of controls (p = 0.905) or with the art-experienced participants (p = 0.291). table 1: socio-clinical characteristics and lipid profile of controls, antiretroviral therapy-naïve and treatment-experienced hiv-positive patients, benin city, nigeria between may 2015 and november 2015. fasting lipid profile levels in patients and controls all hiv-positive patients irrespective of art experience had significantly higher mean values for total cholesterol (p = 0.003), triglycerides (p < 0.001), ldl-c (p = 0.003), aciii (p < 0.001), and ae (p < 0.001) levels compared to the hiv-negative participants (table 1). conversely, the hdl-c concentrations was significantly lower (p < 0.001) among the hiv-negative patients compared to the hiv-positive patients. when the art experience was considered, hiv-positive art-experienced patients had significantly higher total cholesterol (p = 0.054), ldl-c (p = 0.054) and ae (p = 0.049) compared to the art-naïve hiv-positive patients. no significant difference was noted when the triglycerides, hdl-c and aciii values were compared among the art-naïve and art-experienced hiv-positive patients. table 2: therapy consideration of the socio-clinical characteristics of the controls, antiretroviral therapy-naïve and treatment-experienced hiv-positive patients, benin city, nigeria, between may 2015 and november 2015. therapy duration characteristics there was no significant difference in the age, sbp, diastolic blood pressure, weight, height, and body mass index was of the various art-duration groups (table 2). lipids and apolipoprotein levels based on therapy duration there was a statistically significant difference when total cholesterol and ldl-c were compared among the various groups (table 3). when the values of triglycerides, hdl-c, aciii, and ae were compared for hiv-positive patients based on the duration of therapy, there were no statistically significant variations (table 3). table 3: therapy consideration of lipids and apolipoproteins for therapy duration of the controls, antiretroviral treatment-naïve and treatment-experienced hiv-positive patients, benin city, nigeria, between may 2015 and november 2015. lipids and body mass index for patient staging we observed no significant difference in the body mass index, total cholesterol, hdl-c, ldl-c, aciii, and ae among both art-experienced and art-naïve hiv-positive patients in different stages of the disease. stage 3 patients had significantly lower triglycerides levels (0.92 ± 0.27 mmol/l) compared with stage 4 patients (1.82 ± 0.78 mmol/l) but not significantly different compared to stages 1 and 2 patients (table 4). table 4: lipid and apolipoprotein levels among antiretroviral treatment-naïve and treatment-experienced hiv-positive participants per disease staging (who, 2005), benin city, nigeria, between may 2015 and november 2015. associations between apolipoproteins c-iii and e and lipid profiles the triglycerides of the healthy controls and art-naïve groups correlated positively with ae and aciii. among the art-experienced group, triglycerides correlated positively with aciii and negatively with ae (table 5). there was a negative correlation between the total cholesterol and ae in the healthy controls and the treatment-naïve patients, while a positive correlation was observed with the art-experienced group. with aciii, total cholesterol correlated positively among the healthy control and treatment-experienced group and negatively with the treatment-naïve group. among the healthy controls, a positive correlation was observed between hdl-c and the studied apolipoproteins. in the hiv-positive groups, hdl-c correlated negatively with the apolipoproteins. the ldl-c correlated negatively with ae among healthy controls and treatment-naïve group and positively in treatment-experienced groups. low-density lipoprotein cholesterol correlated positively with aciii in all studied groups. in all the parameters in the various groups, the associations between the apolipoprotein and lipids were not statistically significantly (p-value > 0.05). table 5: pearson bivariate correlation between apolipoproteins c-iii and e and the lipid profile of the controls, antiretroviral therapy-naïve and treatment-experienced hiv-positive patients, benin city, nigeria, between may and november 2015. discussion the present study assessed and compared the lipid profile, aciii, and ae among hiv-positive art-naïve patients and those on art. we found that the studied apolipoproteins and lipid profiles of hiv-negative participants were often lower than those of hiv-positive participants irrespective of the treatment experience. this suggests that hiv itself disrupts apolipoprotein concentrations, consequently disrupting lipid metabolism. we observed a significantly high sbp among the art-experienced compared to the healthy control participants. hypertension is known to increase the risk of cardiovascular disease and atherosclerosis. previous studies conducted in ethopia in 2018 and uganda between 2011 to 2014 have reported hypertension to be highly prevalent among hiv patients on art treatment.23,24,25,26 hiv patients on art are highly susceptible to developing hypertension compared to hiv-negative persons. the pathophysiology of hypertension among hiv patients may be directly or indirectly influenced by the virus or treatment-related factors. chronic inflammation and lipodystrophy associated with hiv infection have effects on the renin-angiotensin-aldosterone pathway, which may lead to kidney damage, vascular dysfunction, alterations in sympathetic nervous outflow, and hence hypertension.26 the typical risk factors of hypertension include genetic predisposition and lifestyle.23 the results from this study suggest that hiv increases the risk of isolated systolic hypertension, which increases a person’s chance of cardiovascular-related mortality.27 metabolic changes may contribute to the risk of developing isolated systolic hypertension.28 the serum concentrations of aciii and ae were significantly higher in both art-experienced and treatment-naïve patients compared to the controls, similar to the findings of bonnet et al.,29 who also reported a marked increase of aciii among patients on intensive art receiving care in france in 1999. apolipoprotein c-iii plays a major role in the metabolism of triglyceride-rich lipoproteins, such as chylomicrons and very low-density lipoproteins. apolipoprotein c-iii impairs lipolysis by inhibiting lipoprotein lipase and also prevents the binding of ae and apolipoprotein b to receptors on the liver, thereby impeding hepatic clearance of these triglyceride-rich lipoproteins. in addition, aciii also favours hepatic assembly and secretion of very low-density lipoproteins.30 hence, increased serum concentration of aciii is associated with decreased clearance of triglyceride-rich lipoproteins. triglyceride levels were unsurprisingly higher in both art-experienced and treatment-naïve hiv-positive participants compared to the control group due to increased aciii concentration. serum triglyceride concentration was not significantly different between art-naïve and art-experienced hiv-positive patients. previous studies conducted in nigeria in 2010 and in india in 2014 have reported hypertriglyceridaemia in hiv patients31,32 and increasing serum triglyceride concentration with art-duration.32,33,34 thus, hypertriglyceridaemia may be influenced by hiv itself or art use. hiv causes inflammation with the subsequent release of cytokines and decreased hepatic clearance, which may result in hypertriglyceridaemia.6 however, we did not observe a difference in the triglyceride concentration when therapy duration was considered; this may be attributed to our low sample size, which limits statistical strength. although an increase in age increases the risk of dyslipidaemia,35,36 prolonged use of art may be a stronger predictor of dyslipidaemia in hiv patients. our results did not record any difference in the lipid status of patients with different age groups. a high prevalence of dyslipidaemia has been associated with the prolonged use of first-line art.6 dyslipidaemia observed among patients on antiretroviral drugs in this study may be attributed to the effects of art on lipid metabolism, as there was significantly high total cholesterol (p < 0.033) and ldl-c (p < 0.025) among patients on art for more than two years. an increased ldl-c strongly predicts cardiovascular risk.30,31 apolipoprotein e is a multifunctional protein secreted primarily in the liver. it plays a significant role in the plasma clearance of lipoproteins.37 high plasma concentrations of ae are a reflection of high circulating ldl-c and triglycerides. it has been described as an early and specific indicator of cardiovascular disease.38 apolipoprotein e serum concentration between hiv-positive treatment-experienced and treatment-naïve groups was statistically significantly higher when compared to the hiv-negative population. this is similar to previous studies carried out in a south-eastern state of nigeria in 2021, which reported significantly higher ae in hiv-positive patients compared to hiv-negative persons.39 similar to our finding, ezeugwunne et al.40 in 2019 reported a reduction in serum concentration of ae as the length of art treatment progressed among hiv-positive patients in nnawi, nigeria. the reason for reduced ae with increased length of treatment is unclear but may be related to the immune system.41 limitations the small sample size used for the analysis limited the statistical strength of the study. conclusion from this study, dyslipidaemia is associated with hiv infection with or without art, although the pattern of dyslipidaemia may vary. prolonged use of art is a significant risk of dyslipidaemia among hiv-positive patients. patients with hiv, therefore, are at a high risk of developing cardiometabolic diseases; hence, routine monitoring of these patients’ lipid profiles will benefit their management. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions all listed authors contributed to the development of this manuscript. m.n.o. participated in designing the work as well as the data collection, investigations, data curation and writing of the manuscript. n.k.n. conceptualised and designed the work, and was involved in supervision of data curation and proofreading of the manuscript. u.a.u. was involved in the investigatory stages of this study, data curation and manuscript writing. e.a.u. participated in designing the methodology, data curation and manuscript writing. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability the data generated and analysed during this study are not publicly available but are available from the corresponding author on reasonable request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references kumar a, sathian b. assessment of lipid profile in patients with human immunodeficiency virus (hiv/aids) 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brazzaville, republic of congo ali a. yahaya world health organization regional office for africa, brazzaville, republic of congo hieronyma n. gumede-moeletsi world health organization regional office for africa, brazzaville, republic of congo jean b. ndihokubwayo world health organization regional office for africa, brazzaville, republic of congo citation boeras di, peeling rw, onyebujoh p, yahaya aa, gumede-moeletsi hn, ndihokubwayo jb. the who afro external quality assessment programme (eqap): linking laboratory networks through eqa programmes. afr j lab med. 2016;5(2), a560. http://dx.doi.org/10.4102/ajlm.v5i2.560 lessons from the field the who afro external quality assessment programme (eqap): linking laboratory networks through eqa programmes debrah i. boeras, rosanna w. peeling, philip onyebujoh, ali a. yahaya, hieronyma n. gumede-moeletsi, jean b. ndihokubwayo received: 31 aug. 2016; accepted: 31 aug. 2016; published: 17 oct. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract external quality assessment (eqa) surveys performed by the world health organization regional office for africa (who afro) revealed the need for the strengthening of public health microbiology laboratories, particularly for testing of epidemic-prone diseases in the african region. these surveys revealed common issues such as supply chain management, skilled personnel, logistical support and overall lack of quality standards. for sustainable improvements to health systems as well as global health security, deficiencies identified need to be actively corrected through robust quality assurance programmes and implementation of laboratory quality management systems. given all the pathogens of public health importance, an external quality assessment programme with a focus on vaccine-preventable diseases and emerging and re-emerging dangerous pathogens is important, and should not be stand-alone, but integrated within laboratory networks as seen in polio, measles, yellow fever and rubella. in 2015, who afro collaborated with the us centers for disease control and prevention, the london school of hygiene & tropical medicine and partners in a series of consultations with countries and national and regional eqa providers for the development of quality assurance models to support hiv point-of-care testing and monitoring. these consultations revealed similar challenges as seen in the who afro surveys. who afro brought forth its experience in implementing quality standards for health programmes, and also opened discussions on how lessons learned through such established programmes can be utilised to supporting and strengthening the introduction of early infant diagnosis of hiv and viral load point-of-care testing. an optimised external quality assessment programme will impact the ability of countries to meet core capacities, providing improved quality management systems, improving the confidence of diagnostic network services in africa, and including capacities to detect events of international public health importance. an external quality assurance programme (eqap) is critical for laboratories to provide high-quality test results. the eqap encompasses: (1) investments in building human capacity; (2) investments in building laboratory management systems, infrastructure and management of quality systems; (3) well-written policies and procedures; (4) a quality control system, quality improvement (qi) and external quality assessment (eqa); and (5) accreditation standards1. laboratory professionals should routinely perform quality control testing to guarantee that test methods and equipment perform according to established standards. laboratory professionals must participate in eqa/proficiency testing programmes in order to demonstrate that acceptable systems are in place and that specimens are collected and processed appropriately. the objectives of the world health organization regional office for africa (who afro) eqap are: to support countries in their efforts to improve their laboratory quality management systems, including accreditation. to collect, analyse, and report laboratory eqa feedback data and use this information for corrective action and laboratory capacity strengthening. to establish recognised, accurate, and sustainable diagnostic practices using appropriate algorithms. world health organization regional office for africa laboratory networks who afro has established and monitored several national/regional laboratory networks linked by eqa programmes, including polio, measles and rubella, yellow fever, rotavirus and influenza laboratories, the emerging and dangerous pathogens laboratory network, and the epidemic-prone bacterial diseases laboratory network, including tuberculosis. the polio laboratory surveillance network was established in africa in 1993 to support the global polio eradication initiative. this laboratory network is made up of 16 laboratories in 15 countries, with each laboratory supporting at least one other country. of these, 12 polio laboratories have integrated functions supporting measles, yellow fever and rotavirus programmes (figure 1). figure 1: vaccine-preventable disease networks in africa. the emerging and dangerous pathogens laboratory network has been established for the confirmation of viral haemorrhagic fevers in the who african region. the network consists of laboratories in 17 countries, categorised based on their capacity, as described in figure 2. figure 2: emerging and dangerous pathogens laboratory network. the laboratory capacity to diagnose influenza infections by real-time pcr in the african region has grown since the 2009 pandemic. presently, the regional influenza laboratory network comprises 30 national influenza reference laboratories in 30 countries. currently, 14 countries are registered as national influenza centres and are members of the who global influenza surveillance network in the african region (figure 3). since 2010, data on virological surveillance of the influenza virus have been received weekly from 22 countries. presently, 17 countries have a functional national influenza-like illness and severe acute respiratory infections surveillance system. in 2015, more than 35 000 samples were tested, around 15% of which were positive. as of this writing, more than 21 000 samples have been tested, and more than 10% were positive. this kind of information aids in identifying seasonal patterns of influenza in the region and informing influenza vaccination to prioritise high-risk groups. figure 3: the world health organization african region influenza laboratory network. external quality assessment programmes for the world health organization regional office for africa who afro collaborated with the national institute for communicable diseases (nicd) in south africa as the eqa provider.2 the who/nicd microbiology eqap was started in may 2002 and currently, over 90 laboratories in 45 african countries participate in this programme at a cost of usd 150 000/year (figure 4). figure 4: world health organization-national institute for communicable diseases microbiology external quality assessment programme for potential bacterial epidemics, tuberculosis and malaria in the african region (since 2002). its objective is to assess laboratory capacity to detect epidemic-prone diseases (enteric and meningitis pathogens and plague). in addition, this programme assesses laboratory capacity to diagnose malaria and tuberculosis.3 the nicd has the capacity to produce, perform quality control of and distribute samples for the eqap. the resulting data goes to both nicd and who afro, belonging to the who afro eqap programme. the eqap conducts three surveys (shipments) per calendar year, with 2 to 4 disease components per survey: bacteriology discipline: six simulated specimens containing organisms isolated from enteric, meningeal and routine clinical samples for microscopy, culture, identification and/or antimicrobial susceptibility testing. plague discipline: two simulated samples for diagnostic tests for yersinia pestis, including microscopy. tuberculosis microscopy: 10 slides per panel for reporting on the presence of acid-fast bacilli using the international union for tuberculosis and lung disease smear-grading system. malaria microscopy: 20 slides per panel to assess ability to diagnose malaria species and to count parasites. laboratories for enteric and meningitis pathogens were graded based on a score depending on their capacity to do the following: microscopy: clinically important, basic skill, relevant. culture and identification. serotyping/serogrouping: important to evaluate disease prevention and control strategies. antimicrobial agents selected for testing. antimicrobial susceptibility testing. in the region, it was observed that errors in antimicrobial resistance testing included inappropriate selection of antibiotics, guideline availability, no adherence to guidelines, no control/reference strains, and incorrect results and interpretation.4 these surveys reflect common issues, such as shortages of supplies and human resources, as well as the critical need for national standards and quality assessments.5 diagnostic challenges for tuberculosis control as shown in figure 5, there are a total of 15 847 laboratories in the region that have the capacity to perform tuberculosis smear microscopy, with less than half (6798) subscribing to an eqa programme. of the 7923 laboratories that can perform tuberculosis culture and drug-susceptibility testing, only 91 subscribed to an eqa programme for culture and 53 for drug-susceptibility testing services. it also highlights the enormous challenges inherent in meeting the who ‘end tb’ strategy which advocates for universal drug-susceptibility testing coverage. now, instead of performing cultures, many facilities are moving to molecular testing, the quality of which can be assured through a fee-based national health laboratory services tuberculosis programme for the cepheid genexpert®.6 figure 5: facilities available in the african region for tuberculosis diagnostic testing subscribing to an external quality assessment programme. external quality assessment programme corrective action as part of eqa, laboratories with low performance in the eqap receive feedback, training and technical support aimed at instituting corrective action. in a joint collaboration, the eqap and nicd provide feedback and corrective action to all participating laboratories in the three official working languages in the who african region (english, french and portuguese). on-site and regional training sessions are also provided. as part of technical support, standard operating procedures and guidelines are made available. specifically, guidelines and standard operating procedures for laboratories performing antimicrobial susceptibility testing are made available in addition to laboratory bench training workshops. way forward in 2015, who afro collaborated with the us centers for disease control and prevention, the london school of hygiene & tropical medicine and partners in a series of consultations with countries and international, national and regional eqa providers for the development of quality assurance models to support hiv point-of-care testing. participants consulted revealed similar challenges with supply chain management, skilled personnel, logistical support and overall lack of quality standards. who afro provided experiential oversight in implementing quality standards for health programmes, as well as facilitating discussions on how lessons learned through such established programmes should be utilised in supporting and strengthening the introduction of hiv early infant diagnosis and viral load point-of-care testing. conclusion the eqap has provided a clearer understanding of the capacities of participating laboratories to produce accurate results. this international eqap provides a unique opportunity for african national public health laboratories to continuously assess and improve their laboratory performance, identifying their strengths, limitations and on-going challenges. the eqap facilitates an improvement in the confidence of test results from diagnostic networks that impacts on treatment capacities by the requesting treatment centres. given all the pathogens of public health importance, the eqap should not be stand-alone, but rather integrated within laboratory networks, as seen with polio, measles, yellow fever and rubella. grouping them together provides economy of scale for cost-effectiveness and efficiencies for ease of training and reporting. the eqap needs to emphasise the utility of such programmes in improving services for both vaccine-preventable diseases and emerging and re-emerging dangerous pathogens. finally, an optimised eqap will impact the ability of countries to meet international health regulations (2005) core capacities, including the capacity to detect events of international public health importance. this programme has allowed who afro to identify the capacities and resources needed by countries for sustainable improvement and competency of diagnostic services throughout africa. improved quality of laboratory networks produces highly-accurate surveillance data which is required to estimate disease burden in countries, identify outbreaks and inform strategies for disease control and prevention. acknowledgements competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support none. authors’ contributions j.b.n. led the conception and design of the work, as well as the acquisition, analysis, and interpretation of data for the work. p.o., a.a.y. and h.n.g.-m. made substantial contributions to the acquisition, analysis, and interpretation of data for the work. d.i.b., r.w.p. and j.b.n. drafted the work and revised it critically for important intellectual content. references world health organization. who manual for organizing a national external quality assessment programme for health laboratories and other testing sites [document on the internet]. c2016 [cited 2016 sep 27]. available from: http://www.who.int/diagnostics_laboratory/160622_eqa_manual_2016_vl.pdf?ua=1 world health organization. who/nicd microbiology external quality assessment programme in africa years 1 to 4 2002-2006. geneva: world health organization; 2007. association of public health laboratories. external quality assessment for afb smear microscopy [document on the internet]. c2011 [cited 2011 dec 01]. available from: http://www.aphl.org/aboutaphl/publications/documents/external_quality_assessment_for_afb_smear_microscopy.pdf clinical and laboratory standards institute (clsi). performance standards for antimicrobial susceptibility testing. 19th informational supplement m100-s19. wayne, pa: clsi; 2009. frean j, perovic o, fensham v, et al. external quality assessment of national public health laboratories in africa, 2002–2009. bull world health organ. 2012;90:191–199a. http://dx.doi.org/10.2471/blt.11.091876 scott l, albert h, gilpin c, et al. multicenter feasibility study to assess external quality assessment panels for xpert mtb/rif assay in south africa. j clin microbiol. 2014;52(7):2493–2499. http://dx.doi.org/10.1128/jcm.03533-13 epub 2014 apr 30. abstract introduction methods results discussion acknowledgements references about the author(s) simon p. rugera department of medical laboratory science, faculty of medicine, mbarara university of science and technology, mbarara, uganda hope mudondo department of medical laboratory science, faculty of medicine, mbarara university of science and technology, mbarara, uganda jazira tumusiime department of medical laboratory science, faculty of medicine, mbarara university of science and technology, mbarara, uganda rahma udu department of pure and applied sciences, school of applied and health sciences, technical university of mombasa, mombasa, kenya ritah kiconco department of medical laboratory science, faculty of medicine, mbarara university of science and technology, mbarara, uganda department of biochemistry, school of health sciences, soroti university, soroti, uganda sylvia a. lumumba department of medical laboratory science, faculty of medicine, mbarara university of science and technology, mbarara, uganda department of medical science, school of applied and health sciences, technical university of mombasa, mombasa, kenya charles n. bagenda department of medical laboratory science, faculty of medicine, mbarara university of science and technology, mbarara, uganda citation rugera sp, mudondo h, tumusiime j, et al. association of obesity with hyperuricaemia among hiv-positive patients on antiretroviral therapy in south-western uganda. afr j lab med. 2025;14(1), a2565. https://doi.org/10.4102/ajlm.v14i1.2565 original research association of obesity with hyperuricaemia among hiv-positive patients on antiretroviral therapy in south-western uganda simon p. rugera, hope mudondo, jazira tumusiime, rahma udu, ritah kiconco, sylvia a. lumumba, charles n. bagenda received: 16 july 2024; accepted: 15 nov. 2024; published: 19 feb. 2025 copyright: © 2025. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: hyperuricaemia is a risk factor for gout and independently predicts hypertension, diabetes, and chronic kidney disease development. while elevated uric acid levels occur in hiv patients, and weight gain is linked to dolutegravir-based therapy, data on the obesity-hyperuricaemia relationship in this population remain limited. objective: the objective of our study was to evaluate the association between obesity and hyperuricaemia among hiv-positive patients on antiretroviral therapy in south-western uganda. methods: between april 2024 and june 2024, this study conducted a secondary analysis of data on uric acid level and factors associated with obesity from a 2023 cross-sectional study of hiv-positive participants. we used logistic regression to assess the factors associated with hyperuricaemia, and receiver operating characteristic curve analysis to assess the predictive performance of body mass index for hyperuricaemia. results: among 328 participants, hyperuricaemia prevalence was 23.48% (95% confidence interval [ci]: 19.19–28.39%) higher in male participants (31.6%) than female participants (20.0%, p = 0.023). overweight (adjusted odds ratio [aor]: 2.01; 95% ci: 1.01–4.00; p = 0.046), obesity (aor: 2.50; 95% ci: 1.09–5.73, p = 0.030), and male gender (aor: 2.31; 95% ci: 1.07–5.01, p = 0.033) were significantly associated with hyperuricaemia. conclusion: our findings indicate a relationship between hyperuricaemia and obesity in hiv patients on antiretroviral therapy in uganda. nationwide studies using primary data are needed to better understand this relationship’s epidemiological spread. what this study adds: this study is the first to link obesity with hyperuricaemia among hiv-positive ugandans on antiretroviral therapy, highlighting obesity as a key metabolic complication of hiv treatment. keywords: hyperuricemia; obesity; human immunodeficiency virus; antiretroviral therapy; dolutegravir. introduction serum uric acid (sua) is the end product of purine metabolism in humans.1 almost two-thirds of sua is produced endogenously, and the rest as a result of a diet abundant in purines.2,3 the kidneys excrete more than 70% of uric acid, with intestinal and biliary secretion accounting for a lesser amount.3 hyperuricaemia and gout development most commonly result from abnormalities in sua metabolism and its decreased excretion by the kidneys.4 the prevalence of hyperuricaemia has been rising rapidly, worldwide, in recent decades. reports indicate that the prevalence of hyperuricaemia ranges from 0.1% to 10% globally, with an incidence of 0.3 to 6 cases per 1000 person years.5 other data indicate that hyperuricaemia is prevalent not only in developed countries,6,7 but is also rising more frequently in lowand middle-income countries.5,8,9 lifestyle factors such as physical exercise, obesity, high purine diet, and alcohol consumption have been highlighted as independent indicators of the emergence of hyperuricaemia.10,11,12 in addition, increased endogenous uric acid synthesis and exogenous protein consumption are associated with hyperuricaemia in obese individuals.13 hyperuricaemia is a significant pathological condition that not only causes gout, but also acts as an independent risk factor for the onset of several other illnesses,14 such as diabetes mellitus,15 hypertension,16,17 chronic kidney disease,18 end-stage kidney disease,19 obesity,20 and metabolic syndrome,21 which are defined as a collection of risk factors for cardiovascular disease, including low levels of high-density lipoprotein cholesterol, hypertriglyceridaemia, central obesity, hypertension, and high glucose levels.22 among these disorders, obesity has been found to be spreading quickly worldwide.23 according to the world health organization, 16% of adults were living with obesity in 2022.24 in addition to having a negative impact on a person’s health, obesity places a heavy burden on the healthcare system.25 furthermore, it has been identified as a risk factor linked to several detrimental health outcomes, such as diabetes, hypertension, and high sua levels.6 a number of studies on metabolic syndrome indicate that sua is strongly associated with a number of indices, including waist circumference, body mass index (bmi), and dyslipidaemia.20,26 consequently, hyperuricaemia is thought to be a prevalent lifestyle condition associated with obesity.6,26 obesity in the last decade has been linked to various clinical conditions, including hyperuricaemia.27 because obesity has detrimental metabolic effects that lower life expectancy, increase morbidity and mortality, and negatively impact quality of life, it poses a severe danger to public health and the expense of healthcare globally.23 following world health organization recommendations in 2018, dolutegravir-based antiretroviral therapy (art) was adopted as the first-line treatment for all people living with hiv in uganda.28 despite its benefits, recent literature indicates a strong association between the initiation on dolutegravir-based art and subsequent weight gain and obesity.29,30 however, research about the relationship of obesity and sua in the hiv patients receiving antiretroviral medication is scarce in our setting. therefore, the aim of our study was to evaluate the prevalence of hyperuricaemia and its relationship with obesity among people with hiv on art. methods ethical considerations ethical clearance was obtained from the research ethics committee of mbarara university of science and technology (reference no.: must-2023-1039) on 13 september 2023 for the cross-sectional study whose data set was used in the secondary data analysis of the current study. further clearance to use the primary data for secondary data analysis was obtained from the research ethics committee of mbarara university of science and technology (reference no.: 36/05-17/must-2023-1039). in addition, the principal investigator and the research assistants of the primary study obtained informed written consent from each participant before enrollment into the study. standard clinical procedures and regulations were followed, and privacy, anonymity, and confidentiality were protected during data collection and handling in the primary study. the participants were selected using simple random sampling and allotted identification numbers to replace names. the questionnaires were locked in a cabin accessed by only the principal investigator. the data set obtained from the primary study was used purposely for secondary data analysis based on the ethical clearance. all data were handled according to national and international data protection regulations, to ensure that the participants’ rights and privacy were respected. study design, setting and population this study was a secondary data analysis of a data set from a cross-sectional study conducted among hiv-positive participants on art from 21 september 2023 to 21 october 2023 at mbarara municipal council health centre iv, south-western uganda. the title of the cross-sectional study whose data set has been used for secondary analysis was ‘correlation of serum uric acid/serum creatinine ratio with estimated glomerular filtration rate in predicting kidney dysfunction among people living with hiv at mbarara municipal council health centre iv, uganda’.31 the source population of the cross-sectional study was all adult hiv-positive patients on art, while those systematically selected hiv-positive patients attending mbarara municipal council health centre iv art clinic during the period of data collection were the study population. sample size and sampling technique the primary study used the kish and lesly formula to calculate the sample size. the sample size of 328 study participants was established in the cross-sectional study whose major variable was kidney dysfunction. a systematic sampling technique was used to select adult hiv-positive patients receiving art in the clinic to participate in the cross-sectional study. eligibility criteria the cross-sectional study included all adult hiv-positive patients who were aged 18 years and above, and who had been on art for at least 12 months, using a systematic selection procedure. however, the study excluded adult hiv-positive patients receiving art who had known kidney-related illnesses, as well as those who were unable to communicate. variables and equipment the major outcome variable in the cross-sectional study was kidney dysfunction, which was defined as an estimated glomerular filtration rate (egfr) < 90 ml/min, and the main exposure variable was sua/creatinine ratio. in this secondary data analysis study, the main outcome variable was hyperuricaemia. body mass index is categorised into < 25 kg/m2 (normal weight), 25 kg/m2 – 29.9 kg/m2 (overweight), and ≥ 30 kg/m2 (obesity), and was the major independent variable. the other independent variables that were explored in the data set for their relationship with hyperuricaemia included social demographics (gender, marital status, education level, religion, employment status, residence), smoking status, alcohol consumption status, blood pressure (bp), physical activity, family history of hypertension, family history of kidney disease, family history of cardiovascular disease, duration on art, viral load, egfr, and creatinine level. the information on social demographics, family background for chronic diseases (hypertension, kidney disease, cardiovascular diseases), and behavioural factors was collected by trained research assistants who directly interviewed study participants in the local language using a semi-structured questionnaire. the trained research assistants also reviewed the medical records of the consented study participants to obtain data regarding the duration of art, recent viral load results, and medication. the measurement of height and weight that were required for the calculation of bmi, waist/hip circumferences needed to calculate waist-to-hip ratio, and bp were also performed by the two research assistants. a digital sphygmomanometer was used to take bp measurements. two bp readings were recorded at a 5 min interval, and their mean value was taken as the bp. the weight and height of the participants were measured using a portable weight and height scale. approximately 4 ml of venous blood was collected from each study participant, using red top (serum) vacutainer vacuum tubes, by the principal investigator and a laboratory assistant attached to the art clinic. serum was separated by centrifugation at 3000 rpm for 4 min within a period not more than 1 h after blood collection. the serum concentrations of uric acid and creatinine were measured using the cobas© 111 analyser (roche diagnostics international, rotkreuz, switzerland) at the clinical chemistry department laboratory at mbarara city health centre iv. this instrument is designed to perform spectrophotometric measurements of the analyte level at a specific set of wavelengths using specific reagents. the analyser automatically performs all reagent and sample pipetting, incubation, photometric measurements, and calculations. calibration and quality control of this chemistry analyser were performed before the samples were run. the manufacturer’s instructions for the machine and the reagents were strictly followed. in order to contribute to the quality control process, 10% of the samples were run at mbarara regional referral hospital for comparison, using the humastar 200 (human diagnostics worldwide, wiesbaden, germany). there was no statistical difference between the results from both laboratories. statistical analysis data were analyzed using stata 17.0 (statacorp llc, college station, texas, united states). creatinine and sua:creatinine ratio were tested for normality using the shapiro-wilk normality test; neither was normally distributed. they were therefore summarised using the median with interquartile range, and their medians were compared between the participants with hyperuricaemia and those without hyperuricaemia using the wilcoxon-rank sum test. uric acid levels were categorised into hyperuricaemia present (uric acid > 7 mg/dl in men and uric acid > 6 mg/dl in women) and hyperuricaemia absent. the egfr rate was also categorised into kidney dysfunction present (egfr < 90 ml/min) and kidney dysfunction absent (egfr ≥ 90 ml/min). a categorical variable, hypertension (normal bp = systolic bp < 140 mmhg and diastolic bp < 90 mmhg; systolic hypertension only = systolic bp ≥ 140 mmhg and diastolic bp < 90 mmhg; diastolic hypertension only = systolic bp < 140 mmhg and diastolic bp ≥ 90 mmhg; and both systolic and diastolic hypertension = systolic bp ≥ 140 mmhg and diastolic bp ≥ 90 mmhg), was generated from both continuous systolic and diastolic bp variables. all categorical variables were summarised using frequencies and percentages. the prevalence of hyperuricaemia, together with its 95% confidence interval (ci), was obtained by dividing the number of participants with hyperuricaemia by the total number of participants that were recruited in the study (sample size), expressed as a percentage. to assess the association between categorised bmi and hyperuricaemia, we used logistic regression. hyperuricaemia was the binary dependent variable. all independent variables, including the main independent variable (categorised bmi) at the bivariate level, were compared with hyperuricaemia (uric acid levels that were dichotomised into high and normal levels). the associations were determined using odds ratios (or) together with their 95% ci, and the statistically significant ors were indicated by a p-value of < 0.2 at the bivariate level. the variables that were clinically and/or statistically significant at this level were also included in the multivariable model. the hosmer-lemeshow test was used to test the suitability of the final model in predicting the outcome variable: hyperuricaemia. in the final multivariable model, associations were considered significant at a p-value ≤ 0.05. to assess the predictive performance of bmi for hyperuricaemia, we used a receiver operating characteristic curve. predictive power was evaluated using the area under the curve (auc) and its 95% ci; a value near to 1 indicates better performance and a 95% ci that included 0.5 indicates a non-significant ability to discriminate between those with hyperuricaemia and those without hyperuricaemia. we used the youden index to estimate the optimal cutoff point of bmi for hyperuricaemia prediction at maximum sensitivity and specificity. results characteristics of the study participants a total of 328 participants were recruited in this study, of which 97.3% were on dolutegravir-based art. the median age of the study participants was 38 years, with an interquartile range of 31 years – 47 years. the majority of the study participants were women (230/328; 70.1%), married or cohabiting (185/328; 56.6%), had completed the primary level of education (179/328; 54.6%), were protestant by religion (155/328; 47.3%), and unemployed (173/328; 52.7%) (table 1). table 1: characteristics of study participants stratified by hyperuricaemia status among hiv-positive patients in south-western uganda, april 2024 to june 2024. prevalence of hyperuricaemia out of the 328 study participants, 77 had hyperuricaemia, giving an overall prevalence of 23.48% with a 95% ci of 19.19% to 28.39%, as indicated in figure 1. the prevalence of hyperuricaemia was significantly higher among the male participants (n = 31; 31.6%) than the female participants (n = 46; 20.0%), p-value 0.023. we also observed a significantly higher prevalence of hyperuricaemia among employed participants (n = 44; 28.4%) in comparison to the unemployed (n = 33; 19.1%), p-value 0.047 (table 1). figure 1: a bar graph showing prevalence of hyperuricaemia among hiv-positive patients in south-western uganda, april 2024 to june 2024. association between obesity and hyperuricaemia overweight (adjusted or: 2.01; 95% ci: 1.01–4.00; p = 0.046), obesity (adjusted or: 2.50; 95% ci: 1.09–5.73, p = 0.030), and male gender (adjusted or: 2.31; 95% ci: 1.07–5.01; p = 0.033) were significantly associated with the prevalence of hyperuricaemia among hiv-positive patients (table 2). table 2: factors associated with hyperuricaemia among hiv-positive patients in south-western uganda, april 2024 to june 2024. predictive performance of body mass index for hyperuricaemia body mass index did not show a significant predictive power for identifying hyperuricaemia, as the 95% ci includes a null value of 0.5, which randomly classifies participants in the two groups (table 3). however, a significant difference in the predictive performance of bmi for hyperuricaemia was noted between male participants (auc = 0.4128) and female participants (auc = 0.657), p-value 0.0029 (table 3 and figure 2). body mass index showed a significant predictive performance for hyperuricaemia among female participants (auc: 0.657; 95% ci: 0.568–0.746), while the predictive performance was not significant among male participants (auc = 0.4128; 95% ci: 0.279–0.546). at an optimal cutoff point of ≥ 26.806 kg/m2, bmi can significantly separate female participants with or without hyperuricaemia at both a sensitivity and specificity of 65% (table 3). figure 2: the receiver operating characteristic curves of the body mass index for separating participants with and without hyperuricaemia among (a) male and (b) female hiv-positive patients in south-western uganda, april 2024 to june 2024. table 3: predictive performance of body mass index for hyperuricaemia among hiv-positive patients in south-western uganda, april 2024 to june 2024. discussion our findings indicate for the first time the relationship between obesity and hyperuricaemia in uganda. in our study, the overall prevalence of hyperuricaemia was 23.48% (95% ci: 19.19–28.39), which falls within the worldwide prevalence, which is reported to range from 2.6% to 36.0% in different populations by a study carried out in bangkok in 2011.32 our overall prevalence is also in agreement with the hyperuricaemia prevalence of 25.2% reported among hiv-positive patients receiving stable highly active art in perugia in 2018.33 however, our study prevalence is significantly lower than a 2021 study on south indian adult patients with stable coronary artery disease (46.5%).34 limited studies done in africa have tried to establish the prevalence of hyperuricaemia among hiv patients. a study done in zimbabwe in 2017 found a prevalence of 13.0%, which was lower than our finding.35 since literature is scanty, we had to compare our results with dissimilar studies that did not deal with hiv patients. our results are in agreement with the 20.5% prevalence reported among adults in nigeria in 2016,36 and the 28.0% prevalence among outpatients at bafoussam regional hospital in gabon in 2024,37 but are in disagreement with the prevalence of 31.0% reported by a cross-sectional study conducted in ethiopia among adult staff members of a public health institute.34 these discrepancies may be because of differences in ethnic background and study populations. a significantly higher prevalence of hyperuricaemia was observed among male participants than female participants, as was also reflected in previous studies.38,39,40 our study notes a significant association between male gender and the presence of hyperuricaemia among hiv patients on art. this association is in agreement with the findings of a study conducted in kenya in 2018 among university workers that found that hyperuricaemia was threefold higher in male participants (adjusted or 2.938; 95% ci, 1.909–4.522, p < 0.01) than female participants.39 a related study in ethiopia in 2021, though in a different study population, also observed a significant association between male gender and hyperuricaemia.34 our findings in respect to the odds of hyperuricaemia (which were 2.01 times higher in overweight participants and 2.5 times higher in obese participants than in participants with a bmi < 25 kg/m2) were consistent with results from a zimbabwean study in 2017 that showed a significant association between hyperuricaemia and a bmi (an indicator of obesity) > 30 kg/m2,35 and a cross-sectional study among male adults in nigeria in 2015.41 these findings were also in agreement with a study conducted on bangladeshi adults in 2018 that found that even after adjusting for lipid profile, age and sex, the prevalence of obesity increased progressively across the sua quartiles. the sua levels were also closely and independently associated with obesity.4 another recent nationwide study in china done in 2024 also documented a significant association between obesity indices, including bmi and risk of hyperuricaemia.42 also, in line with our findings, numerous studies have demonstrated a positive association between sua and obesity in various populations. for instance, a 10-year follow-up research study in 2007 revealed that, across all race-sex-groups, rising sua levels were substantially correlated with higher bmi.43 studies from different ethnicities also found a positive relationship between hyperuricaemia and obesity. for example, tanaka et al. established that, after correcting for genetic and environmental variables in both genders, there was a significant correlation between bmi and sua levels in japanese adult twins in 2015.26 another study by wang et al. reported that, among healthy people in the chinese province of jiangsu in 2014, there was a positive correlation between bmi and sua levels.20 more recent research has also shown a strong positive correlation between sua levels and obesity in the chinese population in 2017 and 2018,44,45,46 japan in 2006,47 india in 2014,6,48 pakistan in 2009,49 iraq in 2015,50 and the united states in 2004.51 although earlier research has shown a positive association between sua levels and obesity, the exact mechanism by which obesity causes an increase in uric acid is not yet thoroughly understood. the relationship between obesity and sua may be explained using two factors: overproduction and poor renal excretion. an investigation carried out on a japanese population who had visceral fat obesity revealed that elevated uric acid levels are significantly impacted by its excessive synthesis, with decreased excretion and clearance of urates in the urine.52 furthermore, the buildup of visceral fat causes an increased flow of free fatty acids from plasma into the liver and hepatic portal vein, which in turn promotes triglyceride synthesis and subsequently increases the production of uric acid by activating the uric acid synthesis pathway.53,54 our study also indicates a significantly higher predictive performance of bmi for hyperuricaemia in female participants (auc = 0.657) than in male participants (auc = 0.513), p-value 0.0029. our finding is consistent with findings from a large taiwanese population study done in 2023 that also found bmi to have a higher predictive power for hyperuricaemia in female participants (auc = 0.728) than in male participants (auc = 0.655).55 limitations the study is a secondary data analysis; hence, we had limited control over the data collection process. as the study used a data set from a cross-sectional study, we could not establish the cause-effect relationship. the generalisability of our findings is limited to hiv-positive patients on dolutegravir-based art, and the results may not apply to those on different art regimens or other populations. conclusion our findings are the first of their kind to indicate a relationship between hyperuricaemia and obesity in hiv patients on art in uganda. the high prevalence of hyperuricaemia in these patients requires the attention of policymakers with regard to the institution of routine measurement of sua in obese and overweight people to prevent hyperuricaemia and its related complications. a study that does not rely on secondary data should be carried out country wide to further enhance our understanding of the epidemiological spread of this relationship. acknowledgements this article is a secondary analysis study of s.a.l’s thesis entitled ‘correlation of serum uric acid/serum creatinine ratio with estimated glomerular filtration rate in predicting kidney dysfunction among people living with hiv at mbarara municipal health centre iv’, towards the degree of master of medical laboratory science (clinical chemistry) in the department of medical laboratory science, mbarara university of science and technology, uganda, on 27 october 2023, with supervisors ritah kiconco, simon peter rugera, and rahma udu. competing interests the authors reported that they received funding from mbarara university of science and technology faculty of medicine research fund, which may be affected by the research reported in the enclosed publication. the authors have disclosed those interests fully and have implemented an approved plan for managing any potential conflicts arising from their involvement. the terms of these funding arrangements have been reviewed and approved by the affiliated university in accordance with its policy on objectivity in research. authors’ contributions s.p.r., c.n.b., and s.a.l. contributed to the conceptualisation of the study, data analysis, and result interpretation. c.n.b., s.a.l., h.m., j.t., r.k. and s.p.r., and r.u. contributed to the methodology. s.p.r., r.k., c.n.b., s.a.l., j.t., and h.m. contributed to the writing of the original draft article. c.n.b., s.a.l., h.m., j.t., r.k. and s.p.r., and r.u. contributed to the writing, review and editing. s.p.r., c.n.b., and r.k. contributed to supervision and project administration. s.p.r., h.m., j.t., r.u., r.k., s.a.l., and c.n.b. have read and agreed to the publication of the article. sources of support this research received funding from the mbarara university of science and technology faculty of medicine research fund to analyse primary data and 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et al. asociación entre la concentración de ácido úrico en suero y la obesidad entre los estudiantes universitarios (china). nutr hosp. 2015;31(6):2407–2411. ewenighi chinwe o, uchechukwu d, uchechukwu e, et al. prevalence of hyperuricemia and its risk factors in healthy male adults from abakaliki metropolis, nigeria. j mol pathophysiol. 2015;4(3):95. https://doi.org/10.5455/jmp.20150915011842 chen d, lu c, chen k, et al. association between anthropometric indices and hyperuricemia: a nationwide study in china. clin rheumatol. 2024;43(3):907–920. https://doi.org/10.1007/s10067-024-06884-w rathmann w, haastert b, icks a, giani g, roseman jm. ten-year change in serum uric acid and its relation to changes in other metabolic risk factors in young black and white adults: the cardia study. eur j epidemiol. 2007;22(7):439–445. https://doi.org/10.1007/s10654-007-9132-3 chen m-y, zhao c-c, li t-t, et al. serum uric acid levels are associated with obesity but not cardio-cerebrovascular events in chinese inpatients with type 2 diabetes. sci rep. 2017;7(1):40009. https://doi.org/10.1038/srep40009 yang c, yang s, feng c, et al. associations of hyperuricemia and obesity with remission of nonalcoholic fatty liver disease among chinese men: a retrospective cohort study. plos one. 2018;13(2):e0192396. https://doi.org/10.1371/journal.pone.0192396 zhu c, cui r, gao m, et al. the associations of serum uric acid with obesity-related acanthosis nigricans and related metabolic indices. int j endocrinol. 2017;2017:5438157. https://doi.org/10.1155/2017/5438157 oyama c, takahashi t, oyamada m, et al. serum uric acid as an obesity-related indicator in early adolescence. tohoku j exp med. 2006;209(3):257–262. https://doi.org/10.1620/tjem.209.257 sivakumar k, thamarai r, pragatha rj. screening of serum uric acid in obese individuals in rural population. int j sci study. 2014;2(2):1–4. haq au, mahmood r, safi aj, rehman ju, jillani g, haq iu. association of serum uric acid with obesity. j postgrad med instit. 2009;23(3):227–230. essa sa, mishari ak, kadhom qi. association between serum uric acid and obesity. j babylon univ. 2015;2(23):899–903. choi hk, atkinson k, karlson ew, willett w, curhan g. purine-rich foods, dairy and protein intake, and the risk of gout in men. n engl j med. 2004;350(11):1093–1103. https://doi.org/10.1056/nejmoa035700 matsuura f, yamashita s, nakamura t, et al. effect of visceral fat accumulation on uric acid metabolism in male obese subjects: visceral fat obesity is linked more closely to overproduction of uric acid than subcutaneous fat obesity. metabolism. 1998;47(8):929–933. https://doi.org/10.1016/s0026-0495(98)90346-8 fabregat i, revilla e, machado a. short-term control of the pentose phosphate cycle by insulin could be modulated by the nadphnadp ratio in rat adipocytes and hepatocytes. biochem biophys res comm. 1987;146(2):920–925. https://doi.org/10.1016/0006-291x(87)90618-8 fox ih. metabolic basis for disorders of purine nucleotide degradation. metabolism. 1981;30(6):616–634. https://doi.org/10.1016/0026-0495(81)90142-6 su s-y, lin t-h, liu y-h, et al. sex difference in the associations among obesity-related indices with hyperuricemia in a large taiwanese population study. nutrients. 2023;15(15):3419. https://doi.org/10.3390/nu15153419 abstract introduction slmta implementation pathway in the knbts slipta audit performance lessons learned acknowledgements references about the author(s) eric n. wakaria global communities, nairobi, kenya charles o. rombo kenya ministry of health, kenya national blood transfusion service, nairobi, kenya margaret oduor kenya ministry of health, kenya national blood transfusion service, nairobi, kenya serah m. kambale global communities, nairobi, kenya kimberly tilock global communities, nairobi, kenya daniel kimani division of global hiv and tb, united states centers for disease control and prevention, nairobi, kenya ernest makokha division of global hiv and tb, united states centers for disease control and prevention, nairobi, kenya peter m. mwamba global communities, nairobi, kenya jane mwangi division of global hiv and tb, united states centers for disease control and prevention, nairobi, kenya citation wakaria en, rombo co, oduor m, et al. implementing slmta in the kenya national blood transfusion service: lessons learned. afr j lab med. 2017;6(1), a585. https://doi.org/10.4102/ajlm.v6i1.585 lessons from the field implementing slmta in the kenya national blood transfusion service: lessons learned eric n. wakaria, charles o. rombo, margaret oduor, serah m. kambale, kimberly tilock, daniel kimani, ernest makokha, peter m. mwamba, jane mwangi received: 13 oct. 2016; accepted: 22 nov. 2016; published: 24 apr. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the kenya national blood transfusion service (knbts) is mandated to provide safe and sufficient blood and blood components for the country. in 2013, the knbts national testing laboratory and the six regional blood transfusion centres were enrolled in the strengthening laboratory management toward accreditation (slmta) programme. the process was supported by global communities with funding from the united states centers for disease control and prevention. methods: the slmta implementation at knbts followed the standard three-workshop series, on-site mentorships and audits. baseline, midterm and exit audits were conducted at the seven facilities, using a standard checklist to measure progress. given that slmta was designed for clinical and public health laboratories, key stakeholders, guided by global communities, tailored slmta materials to address blood transfusion services, and oriented trainers, auditors and mentors on the same. results: the seven facilities moved from an average of zero stars at baseline to an average of three stars at the exit audit. the average baseline audit score was 38% (97 points), midterm 71% (183 points) and exit audit 79% (205 points). the occurrence management and process improvement quality system essential had the largest improvement (at 67 percentage points), from baseline to exit, whereas facilities and safety had the smallest improvement (at 31 percentage points). conclusion: slmta can be an effective tool for preparing a blood transfusion service for accreditation. key success factors included customising slmta to blood transfusion activities; sensitising trainers, mentors and auditors on operations of blood transfusion service; creating slmta champions in key departments; and integrating other blood transfusion-specific accreditation standards into slmta. introduction a functional blood transfusion service (bts) is a critical component of a comprehensive healthcare system. blood transfusion is an essential and lifesaving intervention which is key to patient treatment and management. bts involves interrelated processes carried out by various cadres of medical and non-medical professionals. errors may occur at any point, resulting in dire consequences for the blood donors, patients and the public at large.1,2 to ensure the high quality and safety of blood and blood components, a national bts needs to have in place a comprehensive quality management system (qms) covering the entire process from donor selection through blood donation to utilisation. this aligns with the world health organization’s strategy on universal access to safe blood transfusion, which promotes the safety and accessibility of blood and blood components, and the reduction of transfusion-associated risks.3 the strategy recommends implementation of effective quality systems for bts, including quality management, development and implementation of quality standards, effective documentation systems, staff training and regular quality audits.3 the kenya national blood transfusion service (knbts), under the ministry of health, is mandated to provide safe and adequate blood and blood components in the country. knbts is managed through a national coordinating unit, six regional blood transfusion centres (rbtcs) and 11 satellites. the coordinating office also houses the knbts national testing laboratory (ntl), which mainly conducts confirmatory testing for transfusion transmissible infections for the rbtcs, quality assurance and reference checking. the ntl also serves as a backup testing facility for the rbtcs. the rbtc scope of service includes: (1) blood donor mobilisation, education, recruitment and retention; (2) blood collection and donor care, and laboratory testing of donated blood; (3) blood component preparation; (4) donor counselling and notification; (5) blood banking and distribution; and (6) haemovigilance. kenya’s blood need is approximately 420 000 units per year based on the world health organization’s formula of 1% of the country’s total population, currently estimated at 42 million.4 in 2009, the world health organization’s regional office for africa introduced the stepwise laboratory quality improvement process towards accreditation (slipta) and strengthening laboratory management toward accreditation (slmta) programmes. slipta provides a benchmark framework that measures a laboratory’s compliance with iso 15189 on a five-star scale using a comprehensive audit tool.5 slmta, on the other hand, provides training and mentoring to support laboratories’ implementation of the qms to achieve immediate, measurable improvement in laboratories in resource-limited settings. to evaluate progress, the slipta audit tool is used before and after the slmta process. slmta has been applied to clinical and public health laboratories with great success in moving the laboratories toward accreditation and, hence, improving the quality of laboratory services.6 kenya decided to apply this framework to improve quality within the bts. while slipta is based on iso 15189 requirements and designed for clinical and public health laboratories,7 it is applicable to bts given the significant laboratory aspects of blood transfusion. in addition, slipta has a significant management component derived from iso 9001, which is applicable to all organisations. knbts enrolled the ntl and the six rbtcs in slmta in january 2013 with support from the technical assistance for the implementation and expansion of blood safety program, implemented by global communities and funded by the us centers for disease control and prevention under the president’s emergency plan for aids relief. this paper describes the slmta process at knbts. it shares experiences and lessons learned, and details strategies necessary to use slmta to promote a safe and sufficient blood supply. slmta implementation pathway in the knbts planning at the start of the bts slmta process, global communities held sensitisation meetings with the nbts management and other stakeholders to promote buy-in and share the implementation plan. the roles and responsibilities of the various players, criteria for selection of workshop participants, and mentorship approach were agreed upon. four champions were selected from each of the seven facilities to participate in the slmta workshops. these included the rbtc head, quality manager, laboratory manager and blood donor services in-charge. the selection ensured that the management, quality, laboratory (testing, component preparation, sorting and distribution) and blood donor services (collection, notification and donor care) departments were represented. in addition, the national managers in charge of administration, procurement and commodities, training and biomedical engineering from the coordination office, participated in the workshops to ensure responsibility for development and roll-out of policies, budgeting and human resource allocation, among other critical functions. adaptation of slmta to accommodate blood safety activities since slmta was designed for clinical and public health laboratories, it had to be adapted to address most of the blood transfusion processes. while slmta focuses on the laboratory, bts has processes outside the laboratory, such as blood donor recruitment, donor care, donor notification, blood component processing, blood banking and distribution to transfusing facilities. training materials were customised to fit bts based on available blood-specific accreditation standards, such as those from the africa society of blood transfusion. all departments of the bts (including those outside the laboratory) were invited to the trainings. improvement projects were selected across all of these departments. slmta implementation the standard slmta implementation pathway of three workshops, on-site mentorships and audits was employed.7 the details of the workshops, mentorships, audits and other activities are summarised in figure 1. figure 1: kenya national blood transfusion service slmta implementation process. prior to the first training, the slmta trainers from the kenya medical research institute, a key slmta implementing partner in kenya, were oriented on bts, since most of them were drawn from clinical laboratories. the orientation resulted in customisation of the training, improvement projects and mentorship for bts activities, as well as in facilitating content inclusion to address the implementation needs of non-laboratory staff. improvement projects after each workshop, participants implemented improvement projects related to the training content as summarised in table 1. the improvement projects were structured to enable their implementation in various departments of knbts, thereby facilitating across-the-board application. the four participants trained per facility spearheaded the improvement projects implemented in their respective departments. table 1: list of improvement projects at the national testing laboratory and regional blood transfusion centres in kenya, 2013–2015. mentorship global communities used a hybrid mentorship model encompassing supervision and embedment8 of the mentors on site for the five days following the conclusion of each workshop and for the 10 days preceding the next workshop. mentorship methods included demonstration, side-by-side mentoring, and re-teaching of some of the aspects learnt during the workshops. briefing meetings between the mentors and knbts management were held after each mentorship cycle to review progress and identify solutions to challenges faced. furthermore, the slmta coordinator provided ongoing targeted mentorship that addressed emerging challenges. mentorship was conducted by six trained mentors and the global communities slmta coordinator with each mentor assigned a knbts facility. the slmta coordinator mentored the coordinating office. audits the knbts facilities were audited at baseline, midterm and exit to establish status, measure progress in quality improvement, identify areas of strengths and weakness, and measure the preparedness of the facilities for accreditation. the kenya accreditation service, the sole accreditation body in the country, conducted the baseline audit in january 2013 and the exit audit in february 2015. slmta-trained mentors conducted the midterm audit in july 2014. the audits were based on the slipta checklist, which addresses 12 quality system essentials9 and enables the quantification of quality improvement, allowing for determination of progress. slipta assigns stars to rate the level of compliance with the iso 15189:2007 standard, based on scores. the rating system is: zero stars (< 55%), one star (55% – 64%), two stars (65% – 74%), three stars (75% – 84%), four stars (85% – 94%), and five stars (≥ 95%).5,7 complementary activities global communities facilitated additional trainings to support the accreditation process. these included internal audit, iso 15189:2012, method validation and measurement uncertainty. the trainings were based on needs identified during audits, slmta workshops and mentorship sessions, and targeted staff positions that would ensure greater impact in addressing identified gaps. gaps in blood transfusion services, such as blood donor care, were addressed by training nurses in donor care, counselling and notification. knbts training curriculum was used during the training in blood transfusion activities. in addition, global communities supported knbts to develop a quality manual, safety manual, qms standard operating procedures and technical procedures. these tools were distributed to all knbts facilities. global communities also assisted knbts to institute a centralised document control model to standardise documentation. additional support global communities supported knbts in other activities, including computerisation of the bts through installation of a blood establishment computer system and strengthening its financial, administrative, procurement and human resources systems. these activities were in line with slmta principles of quality improvement, and complemented the slmta activities. programme ownership was a key consideration; hence, slmta activities were included in the performance contracts of the heads of rbtcs and the quality managers. this ensured their commitment to the process and the integration of slmta in their day-to-day activities. slipta audit performance the maximum score possible based on the slipta checklist is 258 points. overall, the seven facilities showed steady improvement in the audit scores from an average baseline score of 38% (97 points, zero stars), to 71% (183 points, two stars) at midterm, and 79% (205 points, three stars) at exit (figure 2). this represents an increase of 41 percentage points from baseline to exit. figure 2: average slipta checklist scores at the slmta baseline, midterm and exit audits for kenya national blood transfusion service facilities. scores were calculated from the slipta checklist based on points earned for each quality system essential out of a possible total of 258. slmta audit stars are awarded based on the percentage of total points earned according to the following rating system: zero stars (< 55%), one star (55% – 64%), two stars (65% – 74%), three stars (75% – 84%), four stars (85% – 94%), and five stars (≥ 95%). while all of the facilities improved from baseline to exit, the level of improvement varied (figure 3). baseline scores ranged from 61 (24%) to 120 (47%), whereas exit scores ranged from 180 (70%) to 227 (88%). at baseline, none of the seven facilities achieved a star rating. at the midterm, one facility achieved four stars, five facilities attained two stars and one facility got one star. at the exit audit, two facilities attained four stars, two facilities had three stars and three facilities garnered two stars. one facility experienced a 7% decrease and two facilities had a 1% decrease in performance from mid-term to exit. figure 3: progress of kenya national blood transfusion service facilities in the slmta process. for each slmta audit, scores were calculated from the slipta checklist based on points earned for each quality system essential out of a possible total of 258. overall, improvements were observed in each of the 12 quality system essentials in the slipta checklist (figure 4). at the baseline audit, the average score for all quality system essentials was 38%, and only the score for facilities and safety was above 50%. during midterm audit, the average score for all quality system essentials increased to 71%. for purchasing and inventory, facilities and safety and information management, the sites registered scores of 80% and above, and the highest performance was for purchasing and inventory at 86%. however, at the midterm audits performance for management reviews (47%) and internal audit (43%) remained below 50%. at the exit audit, all seven facilities registered the highest performance in information management (92%), facilities and safety (85%) and client management and customer service (84%). comparing the baseline and exit audits, marked percentage point improvements were observed for occurrence management and process improvement (67 percentage points), internal audit (59 percentage points) and corrective action (58 percentage points). areas with the least improvement across the bts facilities were facilities and safety (31 percentage points), purchasing and inventory (38 percentage points), and equipment (38 percentage points). figure 4: mean quality system essential scores for kenya national blood transfusion service facilities. the slmta process resulted in substantial quality improvements in the knbts. the seven facilities, which started with an average score of 38% at baseline, improved to 79% at the exit assessment, registering an increase of 41 percentage points within two years. by comparison, results from 302 laboratories participating in the slmta programme globally showed a 25 percentage point increase from baseline to exit audit over an average of 16 months.6 clinical laboratories enrolled in the slmta programme in kenya generally realise a three-star rating within nine months but stagnate at three stars; that is, the majority are unable to attain a higher star rating.10 several factors contributed to the successful application of slmta in the knbts. identifying participants from key departments in bts to champion slmta ensured across-the-board quality improvement. this approach also ensured that there was buy-in and that all staff were involved in the slmta process in line with the world health organization’s integrated strategy to promote the safety and accessibility of blood and reduce the risks associated with transfusion.3 cross-cutting staff involvement also ensured that the slmta process was embraced across the facilities and helped to overcome the notion that slmta was only for the laboratory department. in some departments, the champions created quality teams to oversee the improvements. both iso 15189 and iso 9001 recognise the involvement of people as one principle of quality management.11 our experience suggests that the impact of a qms is greatest when managers draw on the participation, experience, and knowledge of the entire staff. this created ownership of the qms in the knbts. improvement projects are a critical pillar of the slmta programme. for bts, the improvement projects were contextualised to address the interlinked processes involved in blood transfusion. as such, the improvement projects were segmented and customised per department. segmentation ensured that the improvement projects were relevant, focused on measurable changes and used department-derived data to stimulate departmental changes. this approach not only ensured institutionalisation of quality improvements but also increased the capacity and efficiency of service delivery. for example, the improvement projects for the blood donor services department focused on increasing blood collection, improving the turn-around time for delivery of donor cards, reducing the number of unsafe donors (and subsequently blood discard rates), and increasing donor notification to meet set standards. slmta learning activities were also adapted to blood transfusion. slmta has 44 training activities that need to be covered in approximately 60 hours spread over three workshops.7 as some of the slmta learning activities were not wholly applicable to bts, bts-specific learning activities were developed using the same principles used to derive slmta learning activities. for example, process mapping in the cross-cutting module was modified to include mapping of blood donor recruitment and mobilisation and blood issuance and distribution, as opposed to mapping the process from the ordering of a test as per processes in clinical laboratories. despite these improvements, challenges were observed, some of which were facility-specific, whereas others were cross-cutting. the six rbtcs share the same qms, and have similar management structures, test menus and staffing. therefore, it would be expected that performance would be similar among them; however, this was not the case. the facilities that scored the highest demonstrated teamwork and greater ownership of the slmta programme; involvement of all staff in activities led to institutionalisation of slmta and integration into day-to-day activities. unlike lower-scoring facilities, these facilities had regular staff meetings, frequent internal audits and structured management reviews, demonstrating greater understanding and application of a qms. ownership and teamwork also resulted in innovations to address identified nonconformities. one of the facilities with four stars at the exit audit had experienced a recurring non-conformity in equipment maintenance. they addressed it by negotiating a service agreement with the biomedical engineer of the neighbouring hospital to service the equipment. another facility that achieved four stars instituted an internal quality control programme for blood components by agreeing with a neighbouring county’s referral hospital to use their haematology analyser to perform quality checks for selected blood components. the ntl, which performs routine and confirmatory testing for transfusion-transmissible infections for the satellites and occasionally for the rbtcs, was among the lowest-star-rated facilities, despite its proximity to the head office and its limited scope of service. several factors may have contributed to this suboptimal performance, key among these being that the laboratory was assessed as part of the coordinating office and the non-conformities of the coordinating office were thus reflected in its audit. in addition, the laboratory occasionally experiences a heavy workload, so staff prioritise the workload instead of quality improvement activities. a similar pattern of performance was noted for the rwanda national reference laboratory.12 across the board, the knbts facilities experienced challenges in achieving improvement in some of the quality system essentials. it was observed that the least-improved quality system essentials – facilities and safety, purchasing and inventory, equipment, and documents and records – all require substantial involvement of the coordinating office and therefore any failures at that office affects all facilities. common non-conformities observed in these quality system essentials included lack of monitoring of supplier performance, frequent depletion or lack of supplies, lack of equipment calibration and service, weakness in document control, failure to perform method verification, and a deficient fire-safety programme. these issues require resources and commitment from the senior management of knbts. knbts, like many institutions implementing slmta in several countries, relies heavily on funding from the us president’s emergency plan for aids relief for its activities.13 lessons learned while the initial focus of slmta was on clinical and public health laboratories, our results indicate that it can be used effectively for bts with some adjustments to the implementation approach. the trainers, mentors and auditors need to be oriented on bts and activities to make them more effective in the application of slmta in bts. for slmta to be effective and sustainable within bts, all cadres and departments of the bts should be engaged. the programme built in-house capacity by training four quality managers as slmta trainers. referring to other blood transfusion-specific accreditation standards during customisation of the training materials ensured that areas such as donor management were appropriately addressed. the africa society for blood transfusion stepwise accreditation programme14 was a key reference standard during the customisation. coordination of activities was enhanced by joint planning among all stakeholders. this not only ensured that agreed-upon timelines were observed, it also resulted in the leveraging of resources from various partners to support knbts. limitations this is a descriptive study based on programmatic activities and as such is subject to limitations as described below. with only seven facilities under observation, the sample size was small. however, knbts has only seven facilities, and all seven implemented slmta and were included in the study. auditing the ntl as part of the national office resulted in the non-conformities of the national office being attributed to and reflected on the scores of the ntl, which may not have reflected the true performance of the ntl. the exit audit was conducted four months after workshop three; this period included the christmas season and as such, kntbs facilities did not have sufficient time to fully implement the lessons learned in the final workshop. the exit audit was conducted so soon after the workshop due to a change in the grant cycle by the donor, which resulted in reduction of the programme’s operating period by four months and subsequent close out of technical assistance support. conclusion slmta can be used to measurably improve qms in bts facilities and effectively prepare them for iso accreditation. the knbts successfully used slmta to advance toward accreditation, progressing from zero stars to an average of three stars at the exit audit within a period of 24 months. innovative approaches, including customising slmta to bts activities; sensitising trainers, mentors and auditors on bts and appointing slmta champions in key departments, contributed to the successful application of slmta within the knbts. with the realignment in the president’s emergency plan for aids relief to focus more exclusively on hiv care and treatment, funding for blood safety has decreased significantly. knbts needs to identify sustainable funding sources to address these issues, sustain the gains achieved and apply for accreditation. slmta remains a practical and effective approach for improving quality systems with bts and should be considered in resource-constrained settings. acknowledgements the findings and conclusions in this paper are those of the author(s) and do not necessarily represent the official position of the us centers for disease control and prevention or the government of kenya. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this paper. sources of support this publication was made possible by support from the us president’s emergency plan for aids relief (pepfar) through cooperative agreement #u2gps001992 from the us centers for disease control and prevention, division of global hiv and tb. authors’ contributions e.n.w. was the slmta coordinator and led the writing of the manuscript. p.m.m., s.m.k. and k.t. participated in implementation of slmta activities and contributed to conceptual design and writing of the manuscript. c.o.r. and m.o. coordinated and oversaw implementation of slmta in the knbts and contributed to writing of the manuscript. e.m. and j.m. contributed to writing and review of the manuscript. d.k. was the activity manager and contributed to writing the manuscript. references vuk t. quality management in blood establishments. isbt science series. 2009;4(1):45–51. strengers p. key elements of a blood transfusion quality management system, the tools and objectives. isbt science series. 2011;6(1):21–25. https://doi.org/10.1111/j.1751-2824.2011.01430.x world health organization. universal access to blood transfusion. geneva, switzerland: who; 2008. national council for population and development. kenya population situation analysis [document on the internet]. c2013 [cited 2017 jan 23]. available from: https://www.unfpa.org/sites/default/files/admin-resource/finalpsareport_0.pdf world health organization regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation in the african region (with checklist) [document on the internet]. c2011 [cited 2015 apr]. available from: http://www.afro.who.int/en/clusters-a-programmes/hss/bloodsafety-laboratories-a-health-technology/blt-highlights/3859-who-guide-for-thestepwise-laboratory-improvement-process-towards-accreditation-in-the-africanregion-with-checklist.html yao k, luman et, slmta collaborating authors. evidence from 617 laboratories in 47 countries for slmta-driven improvement in quality management systems. afr j lab med. 2014;3(2):262. https://doi.org/10.4102/ajlm.v3i2.262 yao k, maruta t, luman et, et al. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. 2014;3(2):194. https://doi.org/10.4102/ajlm.v3i2.194 maruta t, rotz p, trevor p. setting up a structured laboratory mentoring programme. afr j lab med. 2013;2(1):77. https://doi.org/10.4102/ajlm.v2i1.77 clinical and laboratory standards institute. application of a quality management system model for laboratory services; approved guidelines – third edition. clsi document gp26-a3 [isbn 1-56238-553-4]. wayne, pa: clinical and laboratory standards institute; 2004. makokha e, kimani d, njeru m, et al. the tipping point in slmta implementation: kenya’s experience. (abstract/oral session 2.5). presented at the 2nd african society for laboratory medicine international congress, december 2, 2014, cape town, south africa. international organization for standardization. iso 9001:2008 quality management systems-requirements. geneva: iso; 2008. nzabahimana i, sebasirimu s, gatabazi jb, et al. innovative strategies for a successful slmta country programme: the rwanda story. afr j lab med. 2014;3(2):217. https://doi.org/10.4102/ajlm.v3i2.217 luman et, yao k, nkengasong jn. a comprehensive review of the slmta literature part 1: content analysis and future priorities. afr j lab med. 2014;3(2):265. https://doi.org/10.4102/ajlm.v3i2.265 africa society for blood transfusion: afsbt step-wise accreditation standards. document number: omd-e-001-1. pinetown, south africa: afsbt; 2014. article information authors: talkmore maruta1 david motebang2 lebina mathabo2 philip j. rotz1 joseph wanyoike1 trevor peter1 affiliations: 1clinton health access initiative, maseru, lesotho2ministry of health and social welfare, lesotho correspondence to: talkmore maruta postal address: p.o. box 14671, maseru 0100, lesotho dates: received: 15 june 2011 accepted: 17 nov. 2011 published: 16 feb. 2012 how to cite this article: maruta t, motebang d, wanyoike j, peter t, rotz pj. impact of mentorship on who-afro strengthening laboratory quality improvement process towards accreditation (slipta). afr j lab med. 2012;1(1), art. #6, 8 pages. http://dx.doi.org/10.4102/ ajlm.v1i1.6 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. impact of mentorship on who-afro strengthening laboratory quality improvement process towards accreditation (slipta) in this original research... open access • abstract • introduction • methods    • mentorship sites    • mentor qualification and experience    • mentorship model       • facility-based approach       • time allocation per laboratory       • structured mentorship    • mentoring methods       • side-by-side mentoring       • targeted mentoring    • group discussions    • presentations    • staff meetings    • measuring laboratory progress    • data analysis • results • discussion    • outline of results    • practical implications    • limitations of the study    • recomendations • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: the improvment of the quality of testing services in public laboratories is a high priority in many countries. consequently, initiatives to train laboratory staff on quality management are being implemented, for example, the world health organization regional headquarters for africa (who-afro) strengthening laboratory management towards accreditation (slmta). mentorship may be an effective way to augment these efforts.methods: mentorship was implemented at four hospital laboratories in lesotho, three districts and one central laboratory, between june 2009 and december 2010. the mentorship model that was implemented had the mentor fully embedded within the operations of each of the laboratories. it was delivered in a series of two mentoring engagements of six and four week initial and follow-up visits respectively. in total, each laboratory received 10 weeks mentorship that was separated by 6–8 weeks. quality improvements were measured at baseline and at intervals during the mentorship using the who-afro strengthening laboratory quality improvement process towards accreditation (slipta) checklist and scoring system. results: at the beginning of the mentorship, all laboratories were at the slipta zero star rating. after the initial six weeks of mentorship, two of the three district laboratories had improved from zero to one (out of five) star although the difference between their baseline (107.7) and the end of the six weeks (136.3) average scores was not statistically significant (p = 0.25). after 10 weeks of mentorship there was a significant improvement in average scores (182.3; p = 0.034) with one laboratory achieving who-afro three out of a possible five star status and the two remaining laboratories achieving a two star status. at queen elizabeth ii (qe ii) central laboratory, the average baseline score was 44%, measured using a section-specific checklist. there was a significant improvement by five weeks (57.2%; p = 0.021). conclusion: the mentorship programme in this study resulted in significant measurable improvements towards preparation for the who-afro slipta process in less than six months. we recommend that mentorship be incorporated into laboratory quality improvement and management training programmes such as slmta, in order to accelerate the progress of laboratories towards achieving accreditation. introduction top ↑ clinical laboratories form the foundation of evidence-based patient treatment and care, and are a fundamental component of disease surveillance, diagnosis and monitoring at every level of the health care system.1 in many low-resource settings, including many african countries, laboratory services have suffered from inattention and chronic under-development. however, in recent years, ministries of health have increasingly prioritised the quality of testing services by implementing quality management systems and building quality improvement activities into laboratory service work plans.2,3,4 in the african region, the vital importance of laboratories in public health surveillance is part of the framework for the world health organization regional headquarters for africa’s (who-afro) integrated disease surveillance and response (idsr) strategies that are adopted in nearly all african countries.in response to these goals, in 2009 who afro established the who-afro laboratory accreditation process5 and laboratory management training programmes such as strengthening laboratory management towards accreditation (slmta1), and supported the launch of the african society for laboratory medicine in 2011.6 the who-afro laboratory accreditation process was subsequently replaced by the who-afro strengthening laboratory quality improvement process towards accreditation (slipta) in 2011. laboratory management training has been identified as one of the six building blocks in the implementation of a quality management system.3 however, pre-existing training programmes failed to result in measurable changes in laboratory practices.1 yao and others identified three key limitations to existing laboratory training programmes, (1) curriculum content, (2) lack of follow up of trainees to assist with application of knowledge into practice and (3) training focusing more on theory around generic management topics and less on practical aspects that can lead to direct implementation. establishing well-structured laboratory mentorship programmes has been suggested as a means of accelerating a laboratory’s path toward accreditation.3 various methods of laboratory mentorship have been described and implemented in both developing and developed countries.7,8,9 often mentorship is conducted over either short (less than one week) or long (six months to one year or longer) time periods. we believe that these mentorship models often do not achieve the desired impact. spending shorter periods of time in the laboratory does not enable the mentor to better understand the rhythms, patterns, practices and personalities of the laboratory in order to foster positive change in process and behaviour. long term continuous mentorship does not provide the opportunity to assess how well a laboratory is able to sustain or even extend quality improvement in the absence of the mentor. the purpose of this study was to pilot test a model of laboratory mentorship. a two step 10 week mentorship model was developed in lesotho in order to address the aforementioned limitations and deliver high-impact mentoring that supports the requirements of the iso 15189 laboratory quality standard and helps prepare laboratories for the who-afro slipta process. the purpose of this study was to measure the improvement in quality systems of laboratories receiving this model of mentorship. methods top ↑ mentorship sites mentorship was implemented at four laboratories in lesotho over an 18 month period between june 2009 and december 2010 (see table 1 for the profile of the laboratories). three of the laboratories were mafeteng, motebang and scott hospital laboratories. scott is a christian health association of lesotho (chal) hospital laboratory and the other two are owned by the government. the fourth facility was lesotho’s qe ii central laboratory, located at queen elizabeth ii hospital in the capital, maseru. the district laboratories typically had three modestly-sized rooms dedicated to testing and a small store room. these laboratories were comprised of the following sections: chemistry, haematology, cd4+ t-cell count and tb microscopy. these sections had automated analysers for chemistry, haematology, and cd4 + t-cell count and typically performed tests such as liver function tests, creatinine, cd4 count, full blood count, malaria smears, blood grouping and cross-match, tb microscopy, and a small range of serology rapid tests. one of the three district laboratories had microbiology with culture. the central laboratory had one large room compartmentalised into chemistry, cd4+ t-cell count, haematology, cytology, blood transfusion and histology. at the time of mentorship, all four laboratories used a paper based laboratory information system. table 1: profiles of the four laboratories where the mentorship model was implemented. during the mentorship period, mafeteng had three technologists and two microscopists, motebang seven technologists and two microscopists and scott had four technologists, one laboratory aide and one microscopist. the staff had three-year diploma qualifications from the local national health training college (nhtc). the central laboratory had 16 staff members, consisting of 13 diploma-holders and three bsc degree-holders, of whom three were supervisory staff. none of the laboratories had administration staff dedicated to the laboratory; therefore technologists performed their own data entries. during the period of mentorship, no other improvement initiatives were undertaken at the laboratories besides the routine six monthly supervisory check-in visits by the ministry of health laboratory representative. typically these were at most 2–3 hour supervisory visits by the national quality officers from the quality assurance unit of ministry of health laboratory directorate. mentor qualification and experience the laboratory mentor was an experienced laboratorian from the southern african region and had significant experience in quality systems building and training. the mentor was a trained medical laboratory scientist with a four year degree in bsc medical laboratory sciences. at the start of the mentorship, the mentor had eight years of laboratory working experience, with two of these as a trainer in laboratory quality management systems. the mentor had previously worked in a reference laboratory that was preparing for accreditation by the south african national association of standards (sanas). mentorship model we will now present the mentorship programme characteristics under subheadings. facility-based approach to foster a team approach to quality, the mentorship model employed a facility-based approach. the mentor did not focus on specific individuals (e.g. the laboratory supervisor) but rather worked with all laboratory staff, including supervisors, technologists, microscopists and sample transporters. the mentor was embedded within the operations of the laboratory in order to understand its processes, challenges, and the strengths and capabilities of the staff. the mentor worked alongside the laboratory staff as an experienced peer. time allocation per laboratory the mentorship was designed to ensure a significant amount of time with the mentor embedded within the laboratory in a series of mentoring engagements. each laboratory received a total of 10 weeks of full time, on-site mentorship. the time was divided between an initial six-week mentoring engagement, a break of between six and eight weeks, and a subsequent four week engagement (figure 1). the initial six week engagement commenced with a baseline assessment using the who-afro slipta checklist. the mentor then used the findings of this assessment to determine priority areas for quality improvement in the laboratory. during the initial six-week engagement the mentor worked alongside the laboratory staff to help them address the nonconformities revealed in the assessment. a second measurement with the same checklist was administered at the conclusion of the initial six-week engagement. the 6–8 week gap between the two mentoring engagements was purposely built into the model to provide an opportunity to observe how the laboratory functioned on its own after the establishment of the quality initiatives with the mentor. a third assessment with the who-afro slipta checklist was performed at the conclusion of this gap period to determine laboratory progress or regress and inform the work plan for the second mentoring engagement. during this second engagement, which lasted four weeks, the performance gains that were already realised were reinforced and areas of continuing concern were pointedly addressed. at the conclusion of the second mentoring engagement the laboratory was again evaluated with the who-afro slipta checklist. figure 1: schematic representation of the 10 weeks of mentorship split into two blocks of six and four weeks with six to eight weeks in between. structured mentorship to ensure an approach that could be standardised and scaled-up across laboratories, standard laboratory action plans and mentor schedules were formulated for all laboratories. the action plans were formulated following assessments using the who-afro slipta checklist. a summary of assessment findings was formulated by the mentor (see example in table 2) and discussed with the laboratory staff before a laboratory action plan was jointly formulated (see example in table 3). the laboratory action plan consisted of a list of activities to be done, the responsible person, timeline, the signature of the responsible person and the review dates. these became the working documents for the laboratory and defined its improvement path. table 2: example summary of assessment findings table formulated after each assessment to be used by the laboratory and mentor to generate action plans. table 3: example of laboratory action plan derived from the summary of assessment findings. to allow prioritisation of tasks, streamlining and focusing mentor activities, the mentor also formulated a ’mentor schedule‘ based on the summary of each assessment finding and the laboratory action plan. the mentor schedules listed by weeks the activities that the mentor would focus on to help the laboratory implement its action plan and hence resolve its nonconformities. if the laboratory did not meet requirements of the checklist this was considered as a nonconformity. for example, if the checklist required that the laboratory perform routine stock counts and this requirement was not met, this was considered a nonconformity. the mentor schedule prioritised the resolution of nonconformities that would build a foundation for resolving the other nonconformities during and beyond mentorship. for example, documentation is a priority for any of the subsequent quality improvements, hence this should appear early in the mentorship schedule. another example would be investigation and documenting corrective actions; the mentor should schedule this early on in order to allow the laboratory to begin implementation under the guidance of the mentor and then continue independently. mentoring methods whilst on site, the mentor employed a number of methods and techniques to implement the aforementioned action plans and scheduled activities. the methods will be described in the next section. side-by-side mentoring the mentor was part of the daily routines of the laboratory and provided coaching whilst working side by side with the laboratory staff, such as coaching on how to perform internal quality controls, calibration, plotting and reviewing of levy-jennings (l-j) charts for cd4+ t-cell count testing. during this time the mentor demonstrated a strong work ethic, efficient and professional job performance, and dedication to quality whilst conducting testing alongside the laboratory staff. this enabled the mentor to intimately understand the laboratory and teach by example, and targeted on-the-bench interventions. targeted mentoring special mentoring emphasis was also given to laboratory staff with greater levels of responsibility and specific duties within the laboratory, for example laboratory technical staff assigned to the roles of supervisor, quality officer, safety officer and inventory officer. these individuals received direct mentorship on their specific duties. group discussions discussions on specific topics were done with small groups either at a section level (for example haematology) or for a small team assigned to specific tasks. topics for discussion were drawn from nonconformity findings of assessments done at baseline and at different time points within the mentorship period such as inventory control, investigating and documenting corrective actions and the reviewing of l-j charts. presentations presentations on selected topics were made for the entire laboratory team once every week on a fixed day and time. topics for presentation were based on the nonconformities indentified during the baseline and/or exit assessments. examples include: corrective action investigation and reporting, external quality assurance (eqa), plotting and review of l-j charts, inventory control at facility level and competency assessments. staff meetings regularly scheduled laboratory meetings were held during the mentorship periods. during these meetings, the mentor provided coaching and provided advice on issues arising from the laboratory. these meetings were led by the laboratory supervisor and were an opportunity to reinforce the utility of staff meetings for communication and the need to document discussions and the resulting actions.the aforementioned mentoring methods were used together in all four laboratories and none were individually assessed for efficacy. measuring laboratory progress standardised measures of performance to gauge laboratory progress and mentoring effectiveness was conducted at specific time points within the 10 week mentorship period: at initial baseline, at the end of the first six week engagement, and at both the start and end of the second four week engagement (see figure 1).for the three district laboratories, the who-afro slipta checklist was used to collect and measure performance. the mentor, who was a who-afro trained assessor, made all measurements. the checklist, based on the iso 15189:2007(e) standard and the clsi guideline gp26-a3,10 quantitatively measures adherence to accreditation requirements for quality and competency. the scored checklist (total possible score is 250) is divided into 12 sections that cover the 12 quality system essentials (qse)11 (table 4). the scoring allows the checklist to assign the laboratory a zero to five star rating. the who-afro slipta checklist star rating was as follows: 0–137: 0 stars, 138–160: 1 star, 161–185: 2 stars; 186–211: 3 stars, 212–236: 4 stars and 237–250: 5 stars (table 4). table 4a: summary of who afro slipta checklist that covers the 12 quality system essentials and the weighted marks of each section out of the 250 total points. table 4b: summary of who afro slipta checklist that covers the 12 quality system essentials and the weighted marks of each section out of the 250 total points. for the central laboratory sections of chemistry, haematology, cd4 count and cytology, mentorship was conducted and progress monitored for each section individually. a section-specific checklist was developed, covering the 12 quality system essentials and used to monitor progress for five different sections. data analysis data on laboratory performance was measured using the who-afro slipta checklist for the three district laboratories and the section-specific checklists for qe ii central laboratory. checklist scores were analysed with the paired t-test to compare baseline performance with performance after six weeks of mentorship, at the start of the second mentorship period, and at the end of the 10 weeks of mentorship. results top ↑ for the three district laboratories, the average baseline score was 107.7 out of a possible 250 (43.6%) (range 109–138) (see table 5). at baseline, all three laboratories scored zero stars on the who-afro slipta star scale. after six weeks of mentorship, the average score was 136.3 (54.5%) (range 119–146). whilst the scores were numerically not significantly higher than the baseline (p = 0.25), two of the three laboratories had shifted to a one star status on who-afro slipta scale by six weeks. the scores at the start of the second mentorship engagement had remained stable (average 160.5; p = 0.096) and the one star status of the two laboratories was maintained. however, after an additional four weeks of mentorship, the average score was 182.3 (range 165–195), a significant increase of 74.7 points on average over the baseline scores (p = 0.034). two laboratories had achieved two stars and the third had achieved a three star status. table 5: assessment scores at specific time points during the mentorship periods at three district laboratories in maseru, lesotho. we illustrate the progress of the three district laboratories at the baseline, at the end of six weeks, the beginning of the four weeks of engagement and at 10 weeks (see figure 2). figure 2: performance of the three district laboratories at baseline, end of six weeks, beginning of four weeks follow-up visit and at 10 weeks using the slipta checklist. the who-afro slipta checklist has 12 sections, each with a different weight in marks, all adding up to 250. the progress shown by the 3 laboratories across the 12 sections of the slipta checklists is also illustrated (see figure 3a, b and c). figure 3: performance of (a) mafeteng district laboratory, (b) motebang district laboratory and (c) scott district laboratory on the 12 sections of the slipta checklist over 10 weeks of mentorship. after 10 weeks of mentorship, all three district laboratories improved their scores in client management from an average of 58% to 100% and achieved more than 80% scores in management reviews, facilities and safety and occurrence management from baseline scores of 33%, 57% and 56%, respectively. average scores for implementation of corrective actions improved from 25% to 67%. management reviews and internal audits showed the highest percentage change, 46% and 43% respectively (see figure 4). figure 4: average performance (based on the who-afro slipta checklist) of the three district laboratories over the 10 weeks mentorship measured at the four time points during the study: (1) initial baseline, (2) exit after first six weeks mentorship, (3) start of second mentorship period and (4) at the end of the 10 weeks of mentorship. average marks of the three laboratories are expressed as a percentage of the weighted total for each of the 12 sections of the checklist. improvements in the areas of management reviews, internal audits and corrective actions were important, as these areas are critical in the continual improvement process. through management reviews and internal audits the laboratory is able to continuously review and self-evaluate its quality management system. the identified opportunities for improvement need the laboratory to have the ability to implement and document corrective actions. for the five sections of the qe ii central laboratory, the section specific checklist was scored in percentages. at five weeks, the average score was 58.8%, which was significantly different from the baseline score of 44% (p = 0.017) (table 6). table 6: assessment scores at specific time points during the mentorship periods at queen elizabeth ii central laboratory. discussion top ↑ outline of results the mentorship programme in this study was associated with measurable improvement in the laboratory performance, as measured by the who afro slipta assessment tool. the mentored district laboratories moved from zero stars on the who-afro slipta scale to two or three out of a possible five stars (average increase of 74.7% from baseline score; p = 0.034). this represents a substantial improvement achieved over a relatively short time with a moderate investment of mentor time (10 weeks of mentorship over a six month period). based on the findings of this study, mentorship may be an effective mechanism to assist progress towards accreditation. we believe that mentorship is complementary to and can be implemented in conjunction with other management training programmes such as slmta for optimal impact (slmta is a task based training launched by who in 2009 that train laboratory managers in the implementation of the quality management system requirements of the who afro slipta process and eventual international accreditation11). client service and customer satisfaction improved faster than other sections by reaching, on average, 100% by 10 weeks. at the beginning of the mentorship, all four laboratories had laboratory hand-book, suggestion boxes and appropriately trained staff. mentorship built on these areas by assisting with conducting of customer surveys, reviewing findings and implementing improvements. having specific individuals tasked with safety, contributed to improvements to average mark of over 80% after 10 weeks as these individual received targeted mentorship. in the mentor work schedules, internal audits and management reviews appeared in the first six weeks engagement and were mentored on throughout the 10 weeks. this contributed to the observed improvements of an average of over 80%. the support from the laboratory directorate and the quality assurance unit (qau) of the ministry of health of lesotho contributed significantly to the improvements observed. the laboratory director introduced the mentor to the hospital managers and the laboratories. it was important for the mentor to be seen as an extension of the ministry of health. the mentor was allowed direct access to the laboratory director with regular meetings and reporting. the qau reviewed and authorised the quality documents that were introduced at these four laboratories. in addition, the mentor had access to strong logistical support for transport, accommodation and communication ensuring the smooth operation of the mentorship. this allowed the mentor to concentrate on mentoring. practical implications in this study, the district laboratories did not show significant improvement before completing 10 weeks of mentorship. this was likely because early improvement initiatives took time to implement. during the initial engagement, the mentor schedules prioritised areas that allowed the laboratory to set the basis for resolving nonconformities. for example, initial engagement prioritised identification of the areas that needed standard operating procedures (sops), training on how to write sops and then to have staff start to develop and implement these sops. in terms of assessment scores, improvement would only be noted when these sops were in place and being implemented. in a similar way, training on identification, investigation and documenting of corrective actions was done during the early phases of mentorship. corrective actions form the basis of resolving all nonconformities encountered in the laboratory such as internal quality control (iqc), eqa and customer complains to stock management. once the staff are trained and the system put in place, the improvements in terms of assessment scores will be evident when these improvements are constantly being implemented.the limited measurable improvement before 10 weeks was evidence that mentorship can only be effective if conducted over a sustained period of time. at the qe ii central laboratory where sections had smaller staff complements and smaller test menus than district laboratories, improvements were reflected faster within five weeks. limitations of the study one of the limitations of this study was the absence of control laboratories that were followed over the same period of time. accordingly it was not possible to compare the improvements with laboratories that did not receive mentorship. whilst it is possible that the improvements observed in this study were random or due to secular influences, we believe that this is unlikely given the magnitude of the observed change and the fact that no other training or laboratory management initiatives were implemented at these sites at the time of the study. it also remains to be determined how cost-effective this type of mentorship is when compared with other initiatives to assist laboratories in achieving accreditation. recomendations to ensure continued monitoring of laboratory progress beyond the mentorship period, it is recommended that assessments be conducted every six months with remote assistance where needed. mentorship programmes should also be aligned with the laboratory accreditation goals and the objectives of the ministry of health. findings from mentorship should also inform the national quality assurance system. as we have shown in this study (see figure 3), mentoring can yield many service level benefits, but a number of critical areas may require action on systems that are coordinated at national management level, often beyond the level of influence of individual laboratories. hence, parallel national strengthening initiatives for areas such as service and maintenance of instruments, supply chain, proficiency testing and routine assessment of laboratory performance are essential. conclusion top ↑ the use of standard tools for assessments allowed comparison across laboratories and aligned improvements to the who-afro slipta process. data collection at specific points allowed the mentor to track progress and gauge how much time should be spent on site in order to achieve significant improvements. embedding the mentor within the daily routines of the laboratory reduced the supervisory nature of the mentorship and encouraged peer-to-peer relationships to develop between the mentor and the laboratory mentees. this may have created an environment for the mentor to foster positive changes in the laboratory. based on these findings we believe that this laboratory mentorship model provides an opportunity for rapid laboratory quality improvement. the method is less dedicated to training in specific technical skills but more focused on training laboratory staff on the management of laboratories related to accreditation. by being iterative and workplace-based, it has an advantage over purely didactic based training in that it provides an extension of learning beyond the classroom to the workplace. this approach seeks to translate knowledge gained into daily practices and hence, re-enforces behaviour change. we recommend that mentorship become a key component of laboratory quality improvement programmes, especially those targeting accreditation. acknowledgements top ↑ competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. authors’ contributions t.m. (clinton health access initiative) is the main author of the manuscript, and designed and implemented the mentorship model. d.m. (ministry of health and social welfare) managed the project from the ministry of health side whilst j.w. (clinton health access initiative) managed the programme. t.p. (clinton health access initiative) and p.r. (clinton health access initiative) assisted in programme design and review of the manuscript. references top ↑ 1. yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries. an innovative training approach to accelerate laboratory accreditation. am j clin path. 2010;134:401–409. http://dx.doi.org/10.1309/ajcpnbbl53fwuiqj, pmid:20716796 2. lesotho ministry of health and social welfare. laboratory services national policy of 2008. 3. gershy-damet g-m, rotz p, cross d, et al. the world health organization african region laboratory accreditation process. improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. 4. guzel o, guner ei. iso 15189 accreditation: requirements for quality and competence of medical laboratories, experience of a laboratory i. clin biochem. 2009;42(4-5):274–278. http://dx.doi.org/10.1016/j.clinbiochem.2008.09.011, pmid:19863920 5. world health organization. kigali host the launch of a program to accelerate national laboratory service capacity building towards accreditation in the african region [press release]. kigali: who-afro; c2009 [cited 2011 jun 10]. available from: http://www.who.int/hiv/amds/diagnostics/amds_kigali_pr_lab.pdf 6. african society of laboratory medicine [homepage on the internet]. c2011 [cited 2011 jul 20] available from: http://www.afslm.org/ 7. buddeberg-fischer b, herta kd. formal mentoring programmes for medical students and doctors – a review of the medline literature. med teach. 2006;28(3):248–257. 8. frei e, stamm m, buddeberg-fischer b. mentoring programs for medical students – a review of the pubmed literature 2000–2008. bmc med educ. 2010;10:32. http://dx.doi.org/10.1186/1472-6920-10-32, pmid:20433727 9. guidance for structured laboratory mentoring. unpublished. included as additional information in the 2011 slmta training of trainers toolkit available from cdc atlanta. 10. kubono k. [quality management system in the medical laboratory – iso15189 and laboratory accreditation]. rinsho byori. 2004;52(3):274–278. japanese. pmid:15137330 11. world health organization: regional office for africa. laboratory accreditation checklist for clinical and public health laboratories. draft report. december 2009. available from: http://afslm.org/resources/checklist.pdf abstract introduction methods results discussion acknowledgements references about the author(s) joyce a. kubi department of clinical microbiology, school of medical science, kwame nkrumah university of science and technology, kumasi, ghana virology department, noguchi memorial institute for medical research, university of ghana, accra, ghana mohamed mutocheluh department of clinical microbiology, school of medical science, kwame nkrumah university of science and technology, kumasi, ghana joseph h.k. bonney virology department, noguchi memorial institute for medical research, university of ghana, accra, ghana william k. ampofo virology department, noguchi memorial institute for medical research, university of ghana, accra, ghana john k. odoom virology department, noguchi memorial institute for medical research, university of ghana, accra, ghana citation kubi ja, mutocheluh m, bonney jhk, ampofo wk, odoom jk. molecular detection of enterovirus d68 among children with acute respiratory tract infection in ghana. afr j lab med. 2019;8(1), a732. https://doi.org/10.4102/ajlm.v8i1.732 original research molecular detection of enterovirus d68 among children with acute respiratory tract infection in ghana joyce a. kubi, mohamed mutocheluh, joseph h.k. bonney, william k. ampofo, john k. odoom received: 30 nov. 2017; accepted: 02 nov. 2018; published: 26 june 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: acute respiratory tract infections of viral origin remain a leading cause of morbidity, mortality and economic loss regardless of age or gender. a small number of acute respiratory tract infection cases caused by enterovirus d68 (ev-d68) have been reported regularly to centers for disease control and prevention since 1987 by countries in north america, europe and asia. however, in 2014 and 2015, the number of reported confirmed ev-d68 infections was much greater than in previous years. the national influenza centre (nic), ghana carries out surveillance of respiratory infections, focusing on those caused by influenza virus; however, there is inadequate information on other viruses causing respiratory infections in ghana, including ev-d68. objectives: to investigate the association of ev-d68 with severe acute respiratory infections (sari) and influenza-like illness (ili) in ghana. methods: this was a retrospective cross-sectional study which involved archived human respiratory specimens stored at –80 °c at the nic from 2014 to 2015. using a random sampling method, oropharyngeal and nasopharyngeal swabs from patients with sari and ili that were negative by real-time pcr for human influenza viruses were screened for ev-d68 using real-time reverse transcription-polymerase chain reaction (rrt-pcr). results: enterovirus d68 was detected in 4 (2.2%) out of 182 sari samples tested. ev-d68 was detected in children younger than 5 years (4 – 100% of positives) and was not detected in children older than 5 years. enterovirus d68 was detected more frequently in sari cases (3%) than in ili cases (1.2%). conclusion: this study has shown for the first time the presence of ev-d68 in acute respiratory infections in ghana. the results confirmed minimal ev-d68 circulation in the ghanaian population. keywords: ev-d68; acute; respiratory tract infection; ghana. introduction enterovirus d68 (ev-d68) belongs to the family picornaviridae. it is part of the many types of enteroviruses, a group of single-stranded rna (ssrna), non-enveloped viruses. distinct from all other enteroviruses, ev-d68 exhibits acid lability and a lower optimum growth temperature which is similar to that of human rhinoviruses. the classified enterovirus 68 is a member of group d enteroviruses, hence the name ev-d68. the ev-d68 genome contains a single open reading frame which codes for a polyprotein that is cleaved into four viral capsid proteins vp1–vp4 and seven non-structural proteins 2a–2c, 3a–3d.1 during the past few years, ev-d68 has emerged as a major viral pathogen leading to heightened alertness with recent outbreaks occurring in the united states,2 canada,3 chile,4 as well as in several other countries in europe5 and asia.6 the virus is known to cause a spectrum of symptoms including sore throat, cough, breathing difficulties and central nervous system (cns) clinical signs that may be confused with influenza.7,8,9 the virus has also been shown to represent a considerable proportion of the pathogens associated with acute respiratory infections (ari), mostly upper respiratory tract infections (urti).10 ari is a very serious infection of the upper respiratory tract and presents mostly with difficulty in breathing. it is mainly common in children younger than 5 years and shares symptoms with influenza-like illness (ili) – which is commonly associated with patients younger than 2 years – such as fever or sore throat, cough and nasal congestion with pneumonia being a complication. ili causes a set of common symptoms that could be influenza or other illness; it represents the outpatient department cases specifically for this study. severe acute respiratory infection (sari) is an ari that shows a recent onset of fever (≥ 38 °c) within 7 days, cough and shortness of breath or difficulty in breathing which requires hospitalisation. children younger than 5 years and others with asthma are prone to the predisposing factors,11 as are adults with asthma and immunosuppression. enterovirus d68 caused outbreaks of respiratory illnesses in the united states in august 2014; in the middle of october 691 people in 46 states and the district of columbia were attacked with a respiratory tract infection (rti) brought about by ev-d68.11 a huge number of children in canada also had the disease, with a base loss of life of 14.12 for over two decades now, phylogenetic analyses of the sequences of all ev-d68 reported to cdc and who show that multiple clades of the virus have emerged and are currently circulating and contributing to respiratory disease globally.13 the ev-d68 strain responsible for the 2014 episode was new and had no or little reference, even though similar outbreaks had occurred over the years,14,15. consequently, phylogenetic analysis of ev-d68 strains from the 2014 episode could give cutting-edge data in regard to the development status of the infection. however, epidemiological data about this virus especially on the african continent is limited. an analysis of archived and novel ev-d68 strains from patients with respiratory disease in africa and the united states was performed and the results indicated that several recently emerged distinct clades are circulating globally.16 as there is currently no vaccine and definite treatment against ev-d68 infection, management of the infection is through symptomatic treatment. the national influenza centre (nic) in ghana conducts laboratory surveillance for rtis in ghana which mainly focuses on influenza viruses. data generated so far indicated a high burden of ari in children. between january 2014 and march 2015, the nic received 2801 presumptive ili and 2856 presumptive sari cases across the country for processing. real time transciption-polymerase chain reaction (rt-pcr) results indicated positivity rates of 10.5% among ili cases and 14.5% among sari cases, leaving a large proportion of samples with unknown aetiology. the recent ev-d68 outbreak has necessitated the need to re-examine the role that the virus may play as a potential cause of severe respiratory illness and the possible neurologic effects. as part of a retrospective study to identify the aetiology and clinical characteristics of viral rtis in influenza virus-negative samples, we probed these negative influenza cases in all children, both outpatients and inpatients, from influenza sentinel hospitals throughout ghana in 2014 for ev-d68 to determine and describe their association with aris. methods ethical considerations the study was approved by the ethical and protocol review committee of the noguchi memorial institute for medical research (nmimr), college of health sciences, university of ghana, accra, ghana; protocol identification number 6(3)2016–17. patients’ identities were de-linked from their names and given identification numbers for the purposes of confidentiality. all participants were anonymised for the study. study design and sample selection this was a cross sectional study involving archived respiratory specimens stored at -80 °c at the nic. as part of the routine influenza surveillance programme in the country, respiratory specimens (nasopharyngeal [np] and oropharyngeal [op] swabs) from patients presenting to health care facilities with ili or sari are sent to the nic. samples are tested for influenza and the negatives are selected for ev-d68 screening. the laboratory study was carried out to determine the potential introduction and circulation of ev-d68 in ghana. sample selection was carried out to ensure an even distribution. a total of 82 samples were selected from ili and 100 from sari cases. samples from ili cases were selected by a random sampling technique using microsoft excel version 2013 (microsoft corp., redmond, washington, united states). briefly, samples that had tested negative for influenza a and b viruses were stratified according to age groups (≤ 1, 1–4, 4–14), adapted from recommendations for global epidemiological surveillance standards for influenza.17 samples were then randomly selected from each group in representative proportion to the respective age group numbers in the total samples for the period of interest (figure 1). for sari, 100 samples that were selected from november 2014 to march 2015 were used in this study. all 182 samples were retrieved from a –80 °c freezer and screened for ev-d68. figure 1: monthly distribution of acute respiratory infection cases investigated. samples were selected from archived samples of patients with acute respiratory infection received from january 2014 to december 2015. ribonucleic acid extraction and real-time reverse transcription-polymerase chain reaction for detection of enterovirus d68 viral rna was extracted using the qiaamp® viral rna mini kit (qiagen, hilden, germany) according to the manufacturer’s recommendations (qiagen gmbh 1999). strain-specific real-time reverse-transcription-polymerase chain reaction (rrt-pcr) for ev-d68 2014 outbreak assay was used following the centers for disease control and prevention (cdc) protocol, version 10/14/2014.18 in brief, 5 µl of rna was added to a mixture of rt-pcr reaction with a full volume of 25 µl that was made up of 1x reaction buffer and ss iii rt/platinum taqmix (superscript iii platinum one-step quantitative rt-pcr system; life technologies, grand island, new york, united states). primers and probes were synthesised by a commercial supplier (eurofins mwg operon, huntsville, alabama, united states) based on sequences detailed in table 1. the cycling conditions for rrt-pcr were performed in an order of 50 °c for 30 min, 2 min at 95 °c for activation of polymerase, 45 cycles of 95 °c for 15 s, 55 °c for 1 min, and finally 72 °c for 5 s on an abi 7500 fast dx rt-pcr instrument by life technologies. as recommended in molecular laboratory settings, a unidirectional workflow technique was used to prevent contamination and ensure reliability of all laboratory testing. negative control constituted rna extracts known to be negative for pcr targets. four mm mgcl was added to the final reaction mixture. the instrument was used in a mode of a standard run with no passive reference dye and analysis with a manual threshold setting. rna derived from cdc (ev-d68 2014) rrt-pcr positive control was used as an external positive control. in each rrt-pcr run, this positive control and a non-template negative control were included. samples showing exponential amplification and with a cycle threshold (ct) value of 40 or less were considered positive for ev-d68. table 1: enterovirus d68 real-time reverse-transcription polymerase chain reaction panel primer and probe sequences [132]. data analysis data was entered in microsoft excel version 2013 and imported into statistical package for the social sciences which is known as spss (ibm corporation, armonk, new york, united states) for statistical analysis. results a total of 182 archived respiratory samples (op or np) were obtained from the nic between january 2014 and december 2015 (figure 1). the mean age was 8 years within a range of 1 month to 15 years, of which 102 (56%) were male with an average age of 1 year. females contributed 80 (44%) samples with an average age of 1 year (table 2). there were more specimens (45.5%) from patients in the 1 year or younger age group than any other age group. specimens were from 9 out of 10 regions in ghana. one hundred and sixty-three (163/182) patients showed fever with no less than one respiratory manifestation. the most common respiratory symptom was cough (89.6%) with each of other symptoms revealed in under half of samples (figure 2). myalgia was the slightest detailed indication (2.2%). two specimens were from patients with pre-existing medical conditions of asthma (1) and pneumonia (1). the majority (99%) of samples were collected within 7 days of ailment onset. figure 2: frequency of clinical symptoms reported from 182 patients tested. table 2: demographic characteristics of patients screened for enterovirus d68. detection of enterovirus d68 by real-time reverse transcription-polymerase chain reaction three (75%) of the 4 ev-d68 positive cases identified in this study were detected in sari cases while only 1 (25%) was found in ili cases (table 3). table 3: details of patients with enterovirus d68 infection. (acute respiratory infections cases received from 2014 to 2015). although all 4 ev-d68 cases were detected in the northern zone, there was no statistically significant difference in the detection rate between this zone and the southern zone. as shown in table 3, ev-d68 infections were all found in males. all ev-d68 positive samples were patients with urti; three were sari cases and one was an outpatient. cough and fever were the most common symptoms in these patients. there were no pre-existing medical conditions in positive cases (table 3). the median age of ev-d68-infected patients was 2 years (range 1–3 years). discussion the prevalence of ev-d68 infections in respiratory specimens reported in literature range from 1% to 36% mainly due to differences in study areas and studied populations.16 in this study, ev-d68 was detected in 2.2% of archived respiratory specimens from patients with ari in ghana which is consistent with the range in literature. our findings are consistent with another study conducted in spain where the laboratory confirmed that ev-d68 could be established in approximately 2.5% of tested episodes in both hospitalised children and outpatients.19 contrary to this, a study in france in 2009–2010, however, detected 63% ev-d68 among hospitalised children aged 6 months to 10 years.20 association of ev-d68 infections with seasonality have been published in several studies, although there are variations in regional and annual circulation of different ev-d68 types.18 although there is not enough evidence to describe seasonality of ev-d68 in relation to climate in the northern and southern parts of ghana, data from this study interestingly reveal that circulation of ev-d68 occurs all year round in ghana as shown in figure 1. detection frequency peaked in the third and fourth quarters with no detection in the first and second quarters. midgley et al (2014)11 reported a higher rate of ev-d68 detection among children in the 1–5 years range. similarly, this study detected ev-d68 more frequently among children younger than 5 years (table 3). the highest number of ev-d68 positive cases was recorded for the 1–5 years age group although this was statistically insignificant (p > 0.05) as shown in figure 3. as shown in table 2, ev-d68 was detected more frequently in sari cases (1.6%) than in ili case (0.5%). this correlates with other studies where detection of ev-d68 in ili cases was less but infection was high in hospitalised patients. the low ev-d68 detection rate in outpatients is similar to some studies published.20 cdc reports urti as the most common clinical presentation during ev-d68 infections. in this study, all ev-d68 positive samples were from patients with urti, which confirms the contribution of ev-d68 as a major viral agent in urti. clinical signs reported in ev-d68 associated ari included fever, cough, sore throat, rhinorrhoea, and headache, which is consistent with other studies.11,21 figure 3: occurrence of enterovirus d68 in the different age groups of aris investigated. limitations limitations to this study were that the prevalence of ev-d68 may have been underestimated due to the selection of ili samples that were negative for influenza infections and excluded the detection of co-infections with influenza viruses. the use of a highly specific ev-d68/2014 variant assay may have contributed to the low ev-d68 prevalence; a type-specific assay should be considered in the next phase of the study. also, the study did not allow the association of described clinical symptoms with only ev-d68 infections as infections by other respiratory pathogens were not ruled out. the seasonality of ev-d68 could not be fully described as this requires a systematic collection of samples over a long period of time. conclusion this study has shown the presence of ev-d68 in aris in ghana. the results from this study provide evidence of the circulation of ev-d68 in ghana in 2014. this study also provides minimal evidence of possible ev-d68 association with ari. we however acknowledge several limitations with our study including characterising the strains detected and the shorter period of the study. the nic’s platform for influenza virus surveillance could be used to monitor ev-d68 as well as other respiratory viruses. a more comprehensive study with systematic sample collection over a longer period should be established to determine the seasonal pattern of ev-d68. acknowledgements ev-d68 external positive control was provided by center for disease control and prevention (cdc). we would like to thank the picornavirus laboratory of cdc for providing us with ev-d68 positive control. we are also much indebted to the staff of the virology department, nmimr, university of ghana, especially prince kofi parbie, elijah p.a. quansah, gustavus m. hansen, christopher zab-yen abana and gifty mawuli for their support. competing interests we declare that we have no financial or personal relationships that may have inappropriately influenced us in writing this paper. authors’ contributions j.a.k. participated in the study design, sample processing, analysis of results and manuscript writing. m.m. was involved in analysis of results and writing of the manuscript. j.h.k.b. was involved in sample processing, data interpretation and editing of the manuscript. w.k.a. participated in data analysis and editing of the manuscript. j.k.o. supervised the study protocol, analysis, editing and interpretation of data and manuscript writing. all authors read and approved the manuscript. sources of support this study was partially funded by the polio laboratory of the virology department, noguchi memorial institute for medical research, university of ghana, accra, ghana. references liu y, sheng j, fokine a, et al. structure and inhibition of ev-d68, a virus that causes respiratory illness in children. science. 2015;347:71–74. https://doi.org/10.1126/science.1261962 midgley cm, jackson ma, selvarangan r, et al. severe respiratory illness associated with enterovirus d68 – missouri and illinois, 2014. mmwr morb mortality wkly rep. 2014;63(36):798–799. drews sj, simmonds k, usman hr, et al. characterization of enterovirus activity, including that of enterovirus d68, in pediatric patients in alberta, canada, in 2014. j clin microbiol. 2015;53(3):1042–1045. https://doi.org/10.1128/jcm.02982-14 torres jp, farfan mj, izquierdo g, piemonte p, henriquez j, o’ryan ml. enterovirus d68 infection, chile, spring 2014. emerg infect dis. 2015;21(4):728–729. https://doi.org/10.3201/eid2104.141766 bal a, schuffenecker i, casalegno js, et al. enterovirus d68 nosocomial outbreak in elderly people, france, 2014. clin microbiol infect. 2015;21(8):61–62. https://doi.org/10.1016/j.cmi.2015.05.008 zhang t, ren l, luo m, et al. enterovirus d68-associated severe pneumonia, china, 2014. emerg infect dis. 2015;21(5):916–918. https://doi.org/10.3201/eid2105.150036 farrell jj, ikladios o, wylie km, et al. enterovirus 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ichiyama t, shirabe k. enterovirus 68 infection in children with asthma attacks: virus-induced asthma in japanese children. allergy. 2011;66(12):1618–1620. https://doi.org/10.1111/j.1398-9995.2011.02725.x article information authors: tjeerd a.m. datema1 linda oskam1 paul r. klatser1,2,3 affiliations: 1kit biomedical research, royal tropical institute, amsterdam, the netherlands2athena institute, vu university amsterdam, amsterdam, the netherlands 3amsterdam institute for global health and development, academic medical centre of the university of amsterdam, amsterdam, the netherlands correspondence to: linda oskam postal address: meibergdreef 39, 1105 az amsterdam, the netherlands dates: received: 26 may 2011 accepted: 11 nov. 2011 published: 13 dec. 2011 how to cite this article: datema tam, oskam l, klatser pr. review and comparison of quality standards, guidelines and regulations for laboratories. afr j lab med. 2011;1(1), art. #3, 7 pages. http://dx.doi.org/10.4102/ ajlm.v1i1.3 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. review and comparison of quality standards, guidelines and regulations for laboratories in this original research... open access • abstract • introduction • methodology    • selection of laboratory quality documents    • analysis of quality documents • results    • clinical quality documents       • iso 15189       • clsi gp26       • the clinical laboratory improvement amendments       • jci clinical laboratory standard    • non-clinical quality documents • discussion    • clinical laboratory quality documents    • non-clinical laboratory quality documents    • national versus international laboratory quality documents    • study limitations    • recommendations • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: the variety and number of laboratory quality standards, guidelines and regulations (hereafter: quality documents) makes it difficult to choose the most suitable one for establishing and maintaining a laboratory quality management system. objectives: there is a need to compare the characteristics, suitability and applicability of quality documents in view of the increasing efforts to introduce quality management in laboratories, especially in clinical diagnostic laboratories in low income and middle income countries. this may provide valuable insights for policy makers developing national laboratory policies, and for laboratory managers and quality officers in choosing the most appropriate quality document for upgrading their laboratories. method: we reviewed the history of quality document development and then selected a subset based on their current use. we analysed these documents following a framework for comparison of quality documents that was adapted from the clinical laboratory standards institute guideline gp26 quality management system model for clinical laboratory services. results: differences were identified between national and international, and non-clinical and clinical quality documents. the most salient findings were the absence of provisions on occurrence management and customer service in almost all non-clinical quality documents, a low number of safety requirements aimed at protecting laboratory personnel in international quality documents and no requirements regarding ethical behaviour in almost all quality documents. conclusion: each laboratory needs to investigate whether national regulatory standards are present. these are preferred as they most closely suit the needs of laboratories in the country. a laboratory should always use both a standard and a guideline: a standard sums up the requirements to a quality management system, a guideline describes how quality management can be integrated in the laboratory processes. introduction top ↑ after the development of the plan-do-check-act cycle by deming (based on the work of shewart) in the 1920s,1,2 the principles of quality management (qm) spread and became especially important in private industry. qm has many advantages, including protecting the product quality, customer safety and the company’s reputation. requirements for regulation of processes are recorded in documents called ‘standards’. national and international quality standards were developed by several organisations. simultaneously, governments forced the introduction of qm by means of laws and regulatory standards.in laboratory practice the necessity of qm also became apparent, although later than in industry. laboratory quality standards first focused only on testing laboratories and calibration laboratories, with the first international standard (iso guide 25) launched in 1978 by the international organization for standardization (iso). nowadays, many national and international quality standards for laboratory practice exist and quality laboratory practice has become the norm. in the case of full compliance with standard requirements a laboratory may be accredited: a formal recognition of competence and compliance to a quality standard. documents other than standards are important as well. quality standards are lists of requirements that need to be met in order to ensure quality practice. these standards do not contain explanations on how to implement the requirements or comply with them. in addition, standards are mostly not measurable. therefore, a laboratory needs guidelines describing how to implement the standard requirements and a checklist to visualise the degree to which it has met the standard. since 2008 the initiatives to strengthen laboratories in low and middle income countries (lmic) have evolved rapidly, especially in the clinical diagnostic laboratory sector.3 whilst efforts to increase health in lmic initially focused on improving treatment and care, the focus has now broadened to also improving laboratory diagnosis. stakeholders encourage countries to develop national standards, guidelines and regulations (hereafter called ‘quality documents’) to improve the quality of laboratory testing, contributing to meeting the millennium development goals (mdg) to achieve better health.4,5,6 in 2009 the world health organization (who) regional office for africa (who-afro) launched an accreditation checklist based on international quality documents5,7,8,9,10,11 and in 2005 the who regional office for south east asia (who-searo) recommended the expansion of the national accreditation scheme of thailand to member countries.2,12 the number of international laboratory quality documents is large and their scopes are diverse. as a result, deciding which document to use for implementing qm may be difficult: the authors have observed clinical laboratories implementing quality standards meant for non-clinical laboratories. this points to a lack of knowledge of decision makers regarding the existence of different types of quality documents for different types of laboratories. no review of widely used laboratory quality documents has been published to describe their characteristics and differences. we believe that there is a need for such information in the context of the rapidly increasing efforts to introduce qm in clinical laboratories. here we review documents that are widely used in qm implementation in laboratories, followed by an analysis of selected documents. the target audience of this study is the clinical laboratory sector. however, non-clinical laboratory quality documents were also included to provide perspective by showing their differences when compared with clinical laboratory quality documents (explaining why they should not be used by clinical laboratories). this analysis is intended for policy makers developing national laboratory quality policies, and for laboratory managers and staff with the ambition to implement a quality management system (qms). methodology top ↑ selection of laboratory quality documents laboratory qm development was reviewed using an unstructured internet search to identify quality documents that are internationally important and widely used. of these, seven quality documents were selected for further study – five international standards or guidelines and two national us regulations.iso 17025 – general requirements for test and calibration laboratories was included because it was the first internationally published standard (in 1978) on laboratory qm, originally named iso guide 25.13 this standard is presently widely used for testing and calibration laboratories. in 1979 in the usa, the food and drug administration (fda) enacted a national regulation called good laboratory practice for nonclinical laboratory studies (21cfr58) (hereafter called fda-glp).14,15 in 1981 the organization for economic cooperation and development (oecd) translated the fda-glp into international requirements for testing and calibration laboratories in its member states, titled principles on good laboratory practice (oecd-glp), consisting of a series of documents focusing on the different aspects of accreditation.16 the oecd-glp could be regarded as the second quality document to ever be developed specifically for international use. in 1988 a national regulation made expressly for clinical laboratories was enacted in the usa. this regulation is known as clinical laboratory improvement amendments (clia), coded 42cfr493.17 the clia is a national regulation, but the college of american pathologists (cap) uses it as the basis for its accreditation18 that is provided to clinical laboratories worldwide. therefore, the clia is also of international significance. iso 15189 – medical laboratories – particular requirements for quality and competence is the most widely used clinical laboratory standard. it was published for the first time in 2003.19 the version used in our analysis is from 2007. in 1999 the clinical and laboratory standards institute (clsi) developed gp26-a1, a guideline for establishing a qms in clinical laboratories. the development of the guideline started in 1997 by combining all the requirements of six quality documents (current at that time) into one document.20 in 2006 the iso 15189 requirements were incorporated into the third edition of this guideline (gp26-a3) titled: application of a quality management system model for laboratory services; approved guideline.21 in the spring of 2011 the fourth edition was published (gp26-a4). this guideline is used internationally to implement the requirements of iso 15189 in clinical laboratories. the joint commission international (jci) accreditation standard for clinical laboratories (second edition published in 2010) has a rather different background from the other quality standards included in this study. the jci provides accreditation to hospitals. as part of this accreditation, the jci developed the standard to include qm practices in hospital laboratories.22 because jci hospital accreditation is widespread, the significance of jci laboratory accreditation may also increase (table 1 provides an overview of the selected quality documents for analysis, including background information). table 1: overview of analysed quality documents with their most important characteristics. analysis of quality documents to guide the analysis, a framework was constructed consisting of three steps to analyse the selected quality documents. in step 1 the nature of the quality document was determined. we used the following definitions: • international standard: a consensus document developed for international use, summing up all the requirements for a qms. • international guideline: a more descriptive document than a standard, developed for international use, defining the intent of standard requirements and how these should be integrated in the laboratory processes. • national regulation: regulatory standard written by a national government. in step 2 a framework of 12 quality system essentials (qses) was adapted from the clsi guideline for implementing qm in clinical laboratories: gp26-a3.21 these 12 qses together cover all aspects of a qms (figure 1). when analysing the selected quality documents, their articles or sub-parts were allocated to one of the 12 qses to yield an indication of the level of coverage of total quality by each quality document (table 2). finally, in step 3 additional characteristics of each quality document were recorded. figure 1: the framework with the 12 quality system essentials used to analyse to which extent the content of quality documents covers all aspects of total quality management. table 2a: analysis of contents of national quality documents using the quality system essentials framework. table 2b: analysis of contents of international quality documents using the quality system essentials framework. results top ↑ the results of the analysis of the nature of the quality documents (analysis step 1) are provided (table 1). the fda-glp, developed for non-clinical research laboratories, and the clia, developed for clinical diagnostic laboratories, are both national regulatory standards. the quality documents developed for international use included the oecd-glp, iso 17025:2005, iso 15189:2007, clsi gp26-a3 and the jci clinical laboratory standard, 2nd edition. the oecd-glp and iso 17025 were developed for use in non-clinical laboratories and the iso 15189, clsi gp26 and jci clinical laboratory standard were developed for use in clinical laboratories.the results of determining the level of coverage of total quality by each quality document (analysis step 2) are provided (table 2). clinical quality documents in general, customers play an important role in clinical laboratory quality documents. whereas the non-clinical quality documents are generally written from the perspective of protecting the process and its product, the clinical quality documents are written to protect the customer from flawed results.the attention given to continuous improvement is much higher in clinical quality documents than in non-clinical quality documents. also, occurrence management (correct handling of nonconformities/accidents, followed by improvement measures to prevent similar occurrences in the future) receives much more attention in clinical quality documents than in non-clinical quality documents. iso 15189 of all clinical laboratory quality documents studied, iso 15189 is probably the most widely used standard worldwide. regional and international accreditation organisations such as the international laboratory accreditation cooperation (ilac), the interamerican accreditation cooperation (iaac), the asia pacific laboratory accreditation cooperation (aplac) and the european cooperation for accreditation (ea) recommend accreditation of clinical laboratories to iso 15189.23a notable characteristic of the iso 15189 standard is, besides the requirements to a qms, the inclusion of two annexes with recommendations: one contains all recommendations for a laboratory information system and another is completely dedicated to different aspects of ethics.24 clsi gp26 as a standard, iso 15189 is a document that only contains requirements for a qms, but no further explanation on why and how these requirements should be complied with. the clsi gp26 guideline contains much more information for laboratories on what qm is, how it is integrated in the laboratory work, and why certain activities should be performed. the clsi gp26 approaches every laboratory activity from a process workflow perspective. it contains a detailed description of this workflow by discussing each phase of the process using flow charts and process tables. the remainder of the document describes the requirements per qse, which are highly focused on continuous improvement (when compared to other clinical laboratory quality documents). the clinical laboratory improvement amendments the clia is a us federal document that applies to all clinical laboratory testing performed on humans except for clinical trials and fundamental research. this regulation is comprehensive and contains many discipline specific requirements (i.e. special requirements for histopathology, genetic testing, molecular techniques, etc.). remarkably, the nature of the clia is more similar to non-clinical quality documents as it is primarily focused on protecting the analysis process rather than customers and personnel. however, the clia, in contrast to most non-clinical standards, covers all 12 qses, which indicates that it is specifically designed for clinical laboratory practice. jci clinical laboratory standard the jci accreditation standard is slightly different from the aforementioned standards as it is a highly elaborate document that combines a standard with a guideline, describing the intent of each standard requirement and, uniquely, the measurable elements of each requirement. it is most elaborate on safety requirements, but it is the only clinical laboratory document which does not cover all 12 qses: information management is not covered as a topic in itself, although some requirements related to information management are present as part of different sections in the document. this standard is, similarly to the clia, highly elaborate on sub disciplines within laboratory practice providing quality assurance and quality control standards for each specific discipline. non-clinical quality documents the most salient findings are that the non-clinical quality documents are less customer focused and, instead, written from the perspective of protecting the process and its product.non-clinical quality documents are also less focused on continuous improvement; both the fda-glp and oecd-glp lack requirement on this qse. in addition, the fda-glp also lacks requirements on the purchasing and inventory element of the qms. another salient characteristic of most non-clinical quality documents (fda-glp, oecd-glp, and iso 17025) is the requirement to have a study plan or validated protocol in place besides standard operating procedures (sops). discussion top ↑ there are a number of quality documents specific for clinical laboratories and for non-clinical laboratories. we compared the characteristics, suitability and applicability of these quality documents in view of the increasing efforts to introduce qm in laboratories. clinical quality documents focus on both protecting the process, and the safety of the customer and the laboratory staff, whereas non-clinical documents aim primarily at protecting the safety and integrity of the analyses performed. moreover, non-clinical quality documents are generally not focused on continuous improvement, an aspect that could be regarded as one of the major goals of a qms. clinical laboratory quality documents the fact that iso 15189 includes a complete annex on ethics is exceptional when compared to all other quality documents. in clinical laboratory practice patient confidentiality and proper behaviour towards the patient are obvious ethical requirements. however, regulations regarding financial arrangements of the laboratory staff with external organisations or persons, or protection of the environment through correct waste-management are also ethical requirements. therefore, we recommend including a paragraph containing specific ethical norms with regard to laboratory practice in every quality document.it was observed that the clia is highly comprehensive when compared to other clinical quality documents. this may be related to the fact that the clia is adapted to a national situation (with laboratories in the usa generally having abundant resources to facilitate good quality practice), whilst iso 15189, as an international standard, has to maintain a certain level of generality to make it applicable in multiple countries that may have different standards of practice that are often determined by resource availability. the clia is more focused on protecting the process rather than the customers and personnel, making it different compared to other clinical quality documents. this may be a typical characteristic of a regulation that can have a narrower focus because other national regulations cover, for example, occupational safety (e.g. the usa 29 cfr part 1910 25): clinical laboratories in the usa following the clia need to comply with multiple other national regulations, whereas international standards and guidelines have to provide complete sets of requirements covering all aspects of laboratory practice. non-clinical laboratory quality documents most non-clinical laboratory quality documents are primarily process-focused. this may be the reason for the total absence of personnel safety requirements: the paragraphs of non-clinical quality documents shown in table 2 in the qse facilities and safety are all related to proper facilities and actions enabling safety of the process, not directed at the safety of personnel. this is illustrated by the requirement of the fda-glp in sub-part b, 58.29 (d): ‘personnel shall take necessary personal sanitation and health precautions designed to avoid contamination of test and control articles and test systems’.a characteristic found to be specific for non-clinical quality documents is the requirement to have a study plan or validated protocol in place besides sops. often, non-clinical laboratories perform research in addition to routine procedures. this research cannot be standardised in sops. notable, and probably related to the nature of test and calibration laboratories, is the low number of requirements aiming at the qse process improvement. in clinical laboratories, processes consist mainly of routinely performed procedures. continuous improvement is necessary to keep the performance of these procedures as efficient and effective as possible. in calibration laboratories, techniques should be performed with as little variance as possible, whereas variation in activities is inherent to the work of testing laboratories. incorporating continuous improvement is therefore complicated, if not impossible. qses that are left uncovered in all but one of the non-clinical quality documents are occurrence management and customer service. establishing procedures on customer service and occurrence management may be advisable in any environment. only iso 17025 contains requirements related to both qses, probably due to the incorporation of the highly customer-focused iso 9001 standard during its development.13, 26 national versus international laboratory quality documents in many countries, international quality documents serve as the basis for national quality documents. such adaption leads to documents that vary by country with regard to comprehensiveness. for example, the national guideline for clinical laboratories in the netherlands is more extensive than the iso 15189 standard.27 the same applies to the usa clia and fda-glp regulations. in contrast, the chinese standard is less extensive than the iso 15189, which makes it more feasible for laboratories in china to attain the standard.28 the required level of iso 15189 was considered to be too high in relation to the resources available, with the consequence that few laboratories in china tried to become accredited. by making the national standards easier to achieve, chinese laboratories were encouraged to implement qm leading to a substantial increase in accredited laboratories.28 on one hand some efforts on qm are better than no efforts at all, the concern however is the question in how far such simplified national standards can maintain adequate quality. this should be a topic of further research.nevertheless, it is recommended to use national quality regulations, if available, as they are often more detailed and optimally adapted to the national situation, and take into account the available resources. the problem is that national standards are currently almost exclusively available in high-income countries. in the last decade, several middle-income countries, for example thailand, mexico and argentina, have developed simplified national accreditation schemes based on international standards.29,30,31,32 recently, who-afro together with, among others, the usa centers for disease control and prevention (cdc) has launched an accreditation checklist based on clsi gp26 and iso 15189, which is tailor-made for implementation in clinical laboratories in lmic, and has started the roll-out in sub-saharan africa.7 a strong point is that the absence of national regulations is taken into account in this initiative: the who-afro accreditation checklist contains lots of questions regarding laboratory safety that would otherwise need to be covered by national regulations on occupational safety.11 iso 15189 only refers to national or regional regulations for safety requirements in such instances; these are absent in many countries. study limitations a potential weakness of this study is that the framework for analysis was adapted from a clinical laboratory quality document. this means that this framework was tailor-made for clinical laboratory quality documents and thus biased towards a clinical laboratory qms. although we therefore were able to correctly identify gaps in non-clinical quality documents compared to clinical quality documents, we were not able to identify gaps in clinical laboratory quality documents as compared to non-clinical quality documents. recommendations the type of laboratory and the resources available determine which document suits the practice of the laboratory best. national regulations are generally more detailed and tailor-made to the national laboratory system. international guidelines may be less detailed in order to remain applicable in multiple countries.laboratories planning to establish a qms need both a standard and a guideline. a standard provides no information on the reasons for implementation of the requirements, and standards are generally not measurable. the jci document illustrates the need for more than a standard: in this document the requirements (standard) are supplemented with a description of the intent of the requirements to prevent misinterpretation (guideline). in addition, measurable elements help the laboratory to determine whether each requirement is complied with. it is important that documents are chosen which suit the practice of a laboratory best. hence, clinical laboratories should choose a clinical laboratory standard, not the fda-glp, oecd-glp, iso 17025 or any other non-clinical laboratory document. several suggestions and recommendations remain to developers of quality documents. it was observed that non-clinical quality documents generally lack provisions on occurrence management, customer service and process improvement; we recommend including requirements on these aspects. ethics is also an element that applies to all types of organisations. increasing attention to ethical behaviour is highly recommended. acknowledgements top ↑ we would like to thank mirjam engelberts for useful suggestions. competing interests the authors declare that they have no financial or personal relationship(s), which may have inappropriately influenced them in writing this article. authors’ contributions t.a.m.d. (royal tropical institute) datema was involved in designing the study, analysing the documents and writing of the article. l.o. (royal tropical institute) was involved in designing and supervising the study and writing of the article. p.r.k. 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[foundation to promote the quality of the laboratory and the accreditation of laboratories in health care (cckl). fourth practical guideline for a quality system for a laboratory in the health care system]. 4th ed. bilthoven, the netherlands: cckl; 2005. dutch. 28. yang zh. the laboratory accreditation and regulations of clinical laboratory in china. clin biochem. 2009;42:310. http://dx.doi.org/10.1016/j.clinbiochem.2008.09.027, pmid:19863937 29. mazziotta d. accreditation of clinical laboratories in the latin-american region. clin biochem. 2009;42:309. http://dx.doi.org/10.1016/j.clinbiochem.2008.09.026, pmid:19863936 30. sierra-amor ri, lópez-martinez m. medical laboratory accreditation according to iso 15189:2003. the mexican experience. biochem med. 2007;17(2):1 88–192. 31. sierra-amor ri. mexican experience on laboratory accreditation according to iso 15189:2003. clin biochem. 2009;42:318. http://dx.doi.org/10.1016/j.clinbiochem.2008.09.095, pmid:19863944 32. wattanasri n, manoroma w, viriyayudhagorn s. laboratory accreditation in thailand: a systemic approach. am j clin pathol. 2010;134(4):534–540. http://dx.doi.org/10.1309/ajcpzyy19wmkmazt, pmid:20855633 abstract introduction methods results discussion acknowledgements references about the author(s) candice l. hendricks department of paediatric haematology, faculty of health sciences, school of medicine, university of kwazulu-natal, durban, south africa department of paediatric haematology, inkosi albert luthuli central hospital, durban, south africa ashen naidoo department of haematology, faculty of health sciences, university of kwazulu-natal, durban, south africa national health laboratory service, inkosi albert luthuli central hospital, durban, south africa rajendra thejpal department of paediatric haematology, faculty of health sciences, school of medicine, university of kwazulu-natal, durban, south africa department of paediatric haematology, inkosi albert luthuli central hospital, durban, south africa nadine rapiti department of haematology, faculty of health sciences, university of kwazulu-natal, durban, south africa national health laboratory service, inkosi albert luthuli central hospital, durban, south africa beverley neethling department of paediatric haematology, faculty of health sciences, school of medicine, university of kwazulu-natal, durban, south africa department of paediatric haematology, inkosi albert luthuli central hospital, durban, south africa yasmin goga department of paediatric haematology, faculty of health sciences, school of medicine, university of kwazulu-natal, durban, south africa department of paediatric haematology, inkosi albert luthuli central hospital, durban, south africa suvarna buldeo department of haematology, faculty of health sciences, university of kwazulu-natal, durban, south africa national health laboratory service, inkosi albert luthuli central hospital, durban, south africa citation hendricks cl, naidoo a, thejpal r, et al. childhood aplastic anaemia with paroxysmal nocturnal haemoglobinuria clones: a retrospective single-centre study in south africa. afr j lab med. 2022;11(1), a1537. https://doi.org/10.4102/ajlm.v11i1.1537 original research childhood aplastic anaemia with paroxysmal nocturnal haemoglobinuria clones: a retrospective single-centre study in south africa candice l. hendricks, ashen naidoo, rajendra thejpal, nadine rapiti, beverley neethling, yasmin goga, suvarna buldeo received: 08 feb. 2021; accepted: 10 mar. 2022; published: 06 june 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: paroxysmal nocturnal haemoglobinuria (pnh) clones in children are rare but commonly associated with aplastic anaemia (aa) and myelodysplasia. objective: this study aimed to determine the prevalence of pnh clones in paediatric patients with idiopathic aa, identify differences in clinical and laboratory features and outcomes, and determine the impact of clone size on clinical presentation. methods: patients with confirmed idiopathic aa who were tested for pnh between september 2013 and january 2018 at the inkosi albert luthuli central hospital, durban, kwazulu-natal, south africa, were included. pnh clones were detected in neutrophils and monocytes by flow cytometry using fluorescent aerolysin, cd24, cd66b and cd14. results: twenty-nine children with aa were identified and 11 were excluded. ten patients (10/18, 55.6%) had pnh clones ranging from 0.11% to 24%. compared to the pnh-negative group, these children were older (median: 10 years vs 4 years, p = 0.02) and had significantly lower total white cell counts (median 1.7 × 109/l vs 3.2 × 109/l; p = 0.04). there was no difference in median absolute neutrophil count or haemoglobin concentration. four patients in each group received immunosuppressive therapy (ist). at six months, all four patients with pnh clones had responded, compared to one in the pnh-negative group. conclusion: more than half of children with aa had a pnh clone. the size of the clone did not impact clinical severity; however, ist use may positively impact prognosis. we recommend early initiation of ist in patients with aa to avoid delays associated with human leukocyte antigen typing. keywords: paroxysmal nocturnal haemoglobinuria clones; aplastic anaemia; paediatrics; flow cytometry; hla typing. introduction paroxysmal nocturnal haemoglobinuria (pnh) is an acquired haematopoietic stem cell disorder.1 it is rare in children and results from a somatic mutation in the glycophosphatidylinositol (gpi) glycan a gene on the short arm of the x chromosome.1,2,3 the gpi glycan a gene encodes gpi-anchored markers, including cd55 and cd59, which protect cells from complement-mediated attack.4,5 although the condition is classically described as presenting with nocturnal haemoglobinuria, this finding is not generally common and is particularly rare in children.4,6 patients may also present with bone marrow failure, and the presence of pnh clones has been shown in a large percentage of children with aplastic anaemia (aa).2,7 this association was described by ware et al. in 1991,8 where the largest initial paediatric pnh cohort was found to have features very different from their adult counterparts. since pnh clones are often found in aa, and patients with clinical pnh often progress to aa,9 what is the theory of association? the prevailing theory is that haematopoietic stem cells harbouring the pnh clone are resistant to the immune-mediated damage that occurs during aa and this promotes the expansion of these ‘unaffected’ cells,5,10 also termed the “escape mechanism”. according to luzatto,10 the progression of aa to pnh is thus “the rule rather than the exception”. very little knowledge exists on the difference in clinical presentation and response to treatment in children with and without pnh clones, particularly on the african continent. though aa is the most common presentation, there have been case series where thromboses and haemoglobinuria are documented.4 flow cytometry-based tests are the most accurate tests for the detection of pnh clones.7,11 classically, granulocytes and monocytes are analysed to determine the absence of gpi-linked proteins.3 red blood cells are not thought to represent the true size of the clone, as ongoing haemolysis and the presence of transfused cells usually confound results. paroxysmal nocturnal haemoglobinuria clone sizes < 1% require validation within each laboratory to determine accuracy. these smaller clone sizes are, however, becoming more important to identify, as their presence allows for future monitoring of clone size and the potential development of classic pnh.2 a previous study described the 10-year probability of developing pnh disease from a pnh clone to be 10.2%.2 historically, patients with aa were managed either supportively with transfusions or cured with a haematopoietic stem cell transplant (hsct).12,13 before immunosuppressive therapy (ist) became available, early reports from africa showed very high mortality rates in symptomatically treated patients.14,15 according to the north american pediatric aplastic anemia consortium, most centres would only opt for hsct as first-line therapy in patients presenting with acquired severe aa and who have a matched sibling donor.16 in the absence of a sibling donor, ist with anti-thymocyte globulin (atg) and cyclosporin becomes the best option.17,18 haematopoietic stem cell transplant from a matched unrelated-donor is only recommended for patients who do not respond to first-line ist. in our setting, the majority of patients are of recent african ancestry, where significant human leukocyte antigen (hla) diversity exists.19,20 all patients diagnosed with aa in our unit routinely have hla typing performed. if siblings from the same parents are present, they too are tested, and if they are a match, patients are referred for hsct. a donor search for local (south african) donors is also conducted via the south african bone marrow registry. uninsured patients treated within the state/public health system can be transplanted within the state/public sector if there is a compatible unrelated local donor identified from the registry. insured patients can in some instances access compatible international donors. human leukocyte antigen typing and the subsequent registry searches to find matches take time, and there is a low likelihood of finding an hla match for transplantation. this prolongs symptomatic treatment with the risk of multiple transfusion exposures. our study was conducted at a quaternary hospital in durban, south africa. the haematology laboratory provides in-house flow cytometry services and has been using the fluorescent aerolysin method to detect the presence of pnh clones in patients since september 2013. this study aimed to determine the prevalence of pnh clones in paediatric patients with aa, identify differences in clinical and laboratory features and treatment responses between patients with and without pnh clones, and determine the impact of clone size on clinical presentation. methods ethical considerations ethics approval was obtained from the biomedical research ethics committee of the university of kwazulu-natal (bca325/18). individual patient consent was not required due to the study being retrospective. data were password-protected, and patient confidentiality was maintained by only including patient hospital numbers on the final datasheets. study population the electronic database of the paediatric haematology-oncology unit of the inkosi albert luthuli central hospital, durban, south africa, was screened to identify patients with a diagnosis of aa between 01 september 2013 and 31 january 2018. the diagnosis was based on pancytopenia secondary to bone marrow hypoplasia in the absence of a clonal malignant disorder. patients who had an inherited bone marrow failure syndrome such as fanconi anaemia, or who were not tested for pnh, were excluded. the electronic medical files of these children were searched on the hospital information system (meditech, westwood, massachusetts, united states) as well as the laboratory information system (trak care, intersystems, cambridge, massachusetts, united states) to retrieve demographic and clinical information, as well as laboratory test results. patients with and without a pnh clone were compared with respect to demographics, laboratory parameters, presenting complaints, management and outcomes. the severity of aa was determined based on camitta’s criteria (cited by marsh et al.).21 haematuria was measured using a urine dipstick and therefore was not distinguished from haemoglobinuria. immunosuppressive therapy was administered in the form of rabbit or horse atg, where appropriate. all patients received premedication with antihistamines and antipyretics prior to the daily infusion, as well as intravenous methylprednisone daily during the administration of the drug course, followed by a course of oral prednisone and cyclosporin. response to ist was monitored at 6 months and 1-year post-administration. a response was deemed complete if full blood count parameters normalised (absolute neutrophil count [anc] ≥ 1.5 × 109/l, haemoglobin ≥ 11 g/dl, and platelet count ≥ 100 × 109/l), partial if there was transfusion independence in the presence of cytopenias, and none if there was continued transfusion dependence.22 laboratory analysis flow cytometry (facscanto ii, becton dickinson, san jose, california, united states) was performed to detect pnh clones using non-gpi-linked markers (cd15 and cd33) to identify the monocytes and neutrophils, and gpi-linked markers (fluorescent aerolysin, cd24, cd66b and cd14) to detect the loss of gpi anchors in a sequential gating strategy (figure 1). after performing doublet discrimination using forward scatter-area vs forward scatter-height and removing debris, all white blood cells were gated using cd45. neutrophils were then discriminated by gating on cd15-positive, cd33-dim and high-side scatter white blood cells. monocytes were discriminated by gating on cd33-bright, cd15-negative and low-side scatter white blood cells. neutrophil pnh clones were defined as clones negative for cd24, cd66b and fluorescent aerolysin, and monocyte pnh clones were clones negative for cd14 and fluorescent aerolysin. figure 1: gating strategy for paroxysmal nocturnal haemoglobinuria clones, south africa, september 2013 – january 2018. (a) all white blood cells gated using cd45; (b) gated neutrophils; (c) gated monocytes; (d, e, f) the dual-negative granulocytes; (g, h) dual-negative monocytes. a loss of two gpi-linked markers on both the monocyte and neutrophil lineage was required to detect a clone. all clone sizes were included as per the latest guidelines of the international clinical cytometry society/european society for clinical cell analysis,23 which classify clones as “pnh clone” for pnh populations > 1.0%, “minor population of pnh cells” or “minor pnh clone” for pnh populations between 0.1% and 1.0%, and “rare cells with gpi deficiency” or “rare cells with pnh phenotype” for pnh populations < 0.1%. for uniformity, we refer to all pnh populations, even if < 1.0%, as clones in this publication. data analysis statistical analysis was performed using graphpad prism version 8.4.3 (2020, san diego, california, united states). a multiple t-test which was used to determine the statistical difference between numerical variables using means (holm-sidak method) and a chi-square test or fisher’s exact test which was used for categorical variables. a p < 0.05 was considered statistically significant. results twenty-nine patients were diagnosed with aa during the study period, ten of whom had fanconi anaemia and were subsequently excluded (figure 2). of the remaining 19 patients, only one patient did not have pnh testing performed. eighteen patients were thus included. ten patients had a pnh clone (10/18; 55.6%); six of which were classified as having a “pnh clone” and four as having a “minor population of pnh cells”. figure 2: study inclusion and exclusion criteria for patients with aplastic anaemia at the inkosi albert luthuli central hospital, durban, south africa, september 2013 – january 2018. patients with a pnh clone were older compared to those without the clone (median age: 10 years vs 4 years; p = 0.02) (table 1). female patients made up the majority of patients in both the pnh-negative group (5/8; 62.5%) and the pnh-positive group (6/10; 60.0%). the full blood count results showed no difference in haemoglobin concentration and platelet count between the two groups. paroxysmal nocturnal haemoglobinuria-positive patients had a significantly lower median total white cell count of 1.7 × 109/l compared to 3.2 × 109/l in pnh-negative patients (p = 0.04). however, the median anc was similar in both groups: 0.26 × 109/l in the pnh-positive group and 0.32 × 109/l in the pnh-negative group. lactate dehydrogenase results were known in 80% (8/10) of pnh-positive and 50% (5/10) of pnh-negative patients and all results were within the normal range. all patients in the pnh-positive and pnh-negative groups had complete information relating to the reticulocyte production index, which was low in all patients. hypocellularity was also confirmed in all patients in both groups by bone marrow aspiration and trephine biopsy. table 1: clinical characteristics of paroxysmal nocturnal haemoglobinuria-positive and -negative patients at the inkosi albert luthuli central hospital, durban, south africa, september 2013 – january 2018. the majority of patients in both groups presented with severe or very severe aa. two patients without the pnh clone presented with haematuria, but this was not a presenting feature in any patients with the clone. bleeding from a peripheral site was noted in 70.0% of pnh-positive and 87.5% of pnh-negative patients, while no patients presented with evidence of thromboses. four pnh-positive (4/10; 40.0%) and four pnh-negative patients (4/8; 50.0%) were given ist. fifty percent of patients with the pnh clone died, compared to 37.5% among patients without the clone. seven of the ten (7/10; 70.00%) pnh-positive patients presented with bleeding (table 2). clone sizes in the granulocyte population and monocyte population in each patient ranged between 0.11% and 24.00%. five of the total pnh-positive patient cohort survived (5/10; 50.00%), four (40.00%) of whom received atg. the other survivor went on to receive an hsct and is still alive. the remaining five pnh-positive patients (5/10; 50.00%) died, two from sepsis and three from bleeding. one of these patients died while awaiting hla results. among those who died, the pnh clone sizes ranged from 0.30% to 15.00%. table 2: paroxysmal nocturnal haemoglobinuria clone sizes and clinical presentation and outcome of patients with aplastic anaemia at the inkosi albert luthuli central hospital, durban, south africa, september 2013 – january 2018. eight patients (four pnh-positive and four pnh-negative patients) in our cohort received atg. all patients received equine atg, and one patient subsequently received an additional dose of rabbit atg. one patient in the pnh-negative group died from sepsis a few days after infusion and was excluded from further analysis (figure 3). at both six and 12 months, one patient in the pnh-positive group had a complete response and the other three patients had a partial response. in the pnh-negative group, one patient had a partial response at six months while two did not respond. at 12 months, the patient with a partial response still had a partial response, one of the patients with no response had developed a partial response, while the other patient remained non-responsive despite receiving a second dose of atg. figure 3: response to immunosuppressive therapy among paroxysmal nocturnal haemoglobinuria population-positive and paroxysmal nocturnal haemoglobinuria population-negative patients at the inkosi albert luthuli central hospital, durban, south africa, september 2013 – january 2018. discussion paroxysmal nocturnal haemoglobinuria is rare in children and does not present classically. the presentation is normally bone marrow failure,12 which exists as a continuum with pnh.10,24 in this study we found a 55.6% prevalence of pnh clones in those children presenting with aa. the clone size had no impact on the severity of the clinical presentation, but patients appeared to respond well to ist. those with a pnh clone were also older than their pnh-negative counterparts and had lower total white cell counts. the differences in the presentation of pnh between adults and children have been described.8 a study reported in 1991 from the united states described that haemoglobinuria was detected in 50.0% of adult pnh patients compared to 15.0% among children, and only 25.0% of adult pnh patients had bone marrow failure compared to 58.0% among children.8 paroxysmal nocturnal haemoglobinuria clone sizes have been reported to be much lower in aa than in pnh disease,25 requiring high-sensitivity fluorescent aerolysin analysis to detect clones < 0.1%.26 no patients in our study presented with clinical pnh, in keeping with a report from japan also describing pnh clones in aa patients.2 the pnh clone prevalence in this study, at 55.6%, is much higher than the 12.9% prevalence observed in a paediatric cohort in india in 201527 that used a pnh clone cut-off of > 1.0%. when a pnh clone size of < 1.0% is included, as in our study, some studies show higher prevalence rates such as 41.0%28 and 46.0%.29 a study conducted in the united states30 found that 40.0% of patients were affected, even at a cut-off of 1.0%. in an adult cohort from tanzania, a 42.0% prevalence of pnh clones in aa patients was found.31 interestingly, this study also showed an overall higher aa prevalence of six cases per million per year, compared to two per million per year in europe and north america. a study among a paediatric cohort from egypt showed the presence of a pnh clone in 36.0% of patients using the cd59 immunohistochemical staining on bone marrow trephine biopsies.32 it must be noted that although we included very small clones in our study, the evidence for the impact of these minor clones on clinical presentation is not yet well established.27 it has also been shown that small populations of granulocytes bearing the pnh phenotype can even be found in healthy individuals33; any specific cut-off value may thus be arbitrary. the median age of children presenting with a pnh clone was significantly higher (p = 0.02). there is also evidence in the literature suggesting that pnh clones increase with age in children.27,34 in the current study, children without the pnh clone presented with a higher incidence of haematuria. as all patients presented with thrombocytopenia, the true reason for the haematuria could not be elucidated, particularly because a urine dipstick is unable to distinguish this from haemoglobinuria, the true mark of intravascular haemolysis. similarly, the median haemoglobin concentration of > 7 g/dl observed in all patients is likely due to blood transfusions received prior to referral. according to camitta’s criteria for the classification of aa severity (cited by marsh et al.),21 anc (< 0.5 × 109/l), reticulocyte count (< 20 × 109/l) and platelet count (< 20 × 109/l) are the predictors of severity. the presence of at least two of these three criteria indicates severe aa. a study in korea highlighted that the anc should be more heavily weighted, as it has the biggest impact on patient complications, is not influenced by transfusions, and can be readily used to classify severity in patients.35 in our cohort, 15 of the 18 patients had a neutrophil count of < 0.5 × 109/l, while the haemoglobin and platelet counts remained above the defined cut-off likely due to transfusions received, suggesting that anc may be a more important factor to consider in the diagnosis and severity grading of patients with aa. this study also found no association between pnh clone size and severity of clinical presentation. a study from india published in 2018 also showed no correlation between clone size and pnh symptoms in a large paediatric cohort.1 only three of the 100 patients in the study presented with haemolysis, all of whom had clone sizes > 10%, and one patient with a very small clone presented with thrombosis. this highlights the need for screening all patients for signs of clinical pnh, regardless of clone size. importantly, only one child in our cohort received an hsct. two patients had already developed platelet refractoriness and this may be due to the continuation of platelet transfusions while awaiting the hla results. our findings show an improved atg response in the pnh-positive cohort; however, the small size of the study population makes it impossible to reach a definitive conclusion. some studies have either shown, suggested or referenced an improved response to ist in patients with aa presenting with a pnh clone,18,27,36,37 or have found no difference in response.1,28,30 however, a recent meta-analysis that included 1236 participants from 11 studies showed a statistically significant improved response to ist in patients with pnh clones.38 in determining ist response in aa as a whole, the north american pediatric aplastic anemia consortium published results from 314 paediatric aa patients wherein complete response was observed in 59.8% of patients, and the 5-year event-free survival was 64.0%.39 relapsed or refractory disease is thus still an important factor to consider in the outcomes of these patients, with many either needing a second course of ist or an hsct. this group found that hsct in relapsed/refractory disease was the preferred treatment modality, rather than a second course of ist. while hsct has historically only been the preferred front-line therapy in patients having a matched sibling donor, a recent report by the united kingdom paediatric bmt working party, paediatric diseases working party and severe aplastic anaemia working party of the european society for blood and marrow transplantation showed that using a matched unrelated-donor upfront had similar results to matched sibling donors.40 importantly, patients with an upfront-matched unrelated-donor hsct had superior outcomes than patients who initially failed ist and then went on to receive a matched unrelated-donor hsct. regarding the timing of ist administration, it is important to note that in south africa, the genetic diversity of the population is such that less than 20.0% of patients (non-european) will have an hla match for hsct.41,42 although the paucity of african donors is certainly a problem, the diversity within the hla region is significant, even among typed donors.20,43 once abnormal cytogenetics, inherited bone marrow failure syndromes and malignancies have been excluded, early initiation of ist is recommended as a delay may lead to increased morbidity and risk of mortality. some studies suggest that the presence of a pnh clone in and of itself rules out an inherited bone marrow failure syndrome.1,29 this may further aid the timeous initiation of therapy. limitations the lower limit of quantitation or detection for pnh clones has not been defined by our laboratory. the quantitative values below 1.0% must thus be interpreted cautiously as the external quality assurance samples analysed in the laboratory have clone sizes greater than 1.0%. however, the external quality assurance programme used for minimal residual disease monitoring verifies the accuracy of the laboratory in defining clone sizes below 0.1% and the identification of very small clones in patients with bone marrow failure has been consistently reported in the literature. for this reason, we have opted to include all clones. the sample size is small; however, this is attributable to the rarity of pnh in the paediatric population and the lack of testing in all paediatric patients with aa. it is thus not possible to make any definitive conclusions regarding any observed differences between the two populations. additionally, the pnh-negative group was not monitored for the subsequent development of a pnh clone and the clone sizes in pnh-positive patients were not monitored and correlated to clinical outcomes. with clear evidence of the risk of developing clinical pnh in future,2 these are important factors to consider in the future management of these patients. conclusion our study found a very high prevalence of pnh clones in patients with aa. patients with pnh clones were older at presentation and had significantly lower total white cell counts. a higher clone size was not associated with a worse clinical presentation. patients with pnh clones who received ist survived. in similar settings where significant hla diversity exists and in the absence of a potential sibling donor, once malignancies and inherited bone marrow failure syndromes have been excluded, patients presenting with aa should be initiated on ist even while awaiting hla typing results. larger studies are needed to further determine the impact of pnh clones on the disease course in paediatric patients with aa. acknowledgements the authors acknowledge dr hamida van staaden who assisted with data collection for the project. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions c.l.h., a.n. and s.b. were responsible for conceptualisation of the study, protocol preparation, data analysis and write up of the manuscript; r.t., n.r., b.n. and y.g. were responsible for data analysis, and write up of the manuscript; c.l.h. collected the data. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability data supporting the findings of this study are available from the corresponding author, c.l.h., on request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references rahman k, mittal n, gupta r, et al. clinicopathological profile of paroxysmal nocturnal haemoglobinuria 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alessandrini m, pepper ms. factors influencing the umbilical cord blood stem cell industry: an evolving treatment landscape. stem cells transl med. 2018;7(9):643–650. https://doi.org/10.1002/sctm.17-0244 tshabalala m, ingram c, schlaphoff t, borrill v, christoffels a, pepper ms. human leukocyte antigen-a, b, c, drb1, and dqb1 allele and haplotype frequencies in a subset of 237 donors in the south african bone marrow registry. j immunol res. 2018;2018:1–8. https://doi.org/10.1155/2018/2031571 cao k, moormann am, lyke ke, et al. differentiation between african populations is evidenced by the diversity of alleles and haplotypes of hla class i loci. tissue antigens. 2004;63(4):293–325. https://doi.org/10.1111/j.0001-2815.2004.00192.x abstract introduction methods results discussion acknowledgements references about the author(s) kabiru abdullahi department of morbid anatomy & forensic medicine, faculty of basic clinical sciences, college of health sciences, usmanu danfodiyo university, sokoto, nigeria department of histopathology, usmanu danfodiyo university teaching hospital, sokoto, nigeria mohammed umar department of morbid anatomy & forensic medicine, faculty of basic clinical sciences, college of health sciences, usmanu danfodiyo university, sokoto, nigeria department of histopathology, usmanu danfodiyo university teaching hospital, sokoto, nigeria saddiku m. sahabi department of morbid anatomy & forensic medicine, faculty of basic clinical sciences, college of health sciences, usmanu danfodiyo university, sokoto, nigeria department of histopathology, usmanu danfodiyo university teaching hospital, sokoto, nigeria citation abdullahi k, umar m, sahabi sm. utilisation of fine needle aspiration cytology and biopsy in sokoto, nigeria: a five-year review. afr j lab med. 2019;8(1), a809. https://doi.org/10.4102/ajlm.v8i1.809 brief report utilisation of fine needle aspiration cytology and biopsy in sokoto, nigeria: a five-year review kabiru abdullahi, mohammed umar, saddiku m. sahabi received: 02 apr. 2018; accepted: 20 nov. 2018; published: 30 may 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract the histopathology department of usmanu danfodiyo university teaching hospital in sokoto, nigeria, conducted a total of 1435 fine needle aspiration biopsies from 1 january 2011 to 31 december 2015, constituting 30% of cytology specimens received. the most common site for the procedure was the breast (655 cases, 45.6%), and 893 cases (63.3%) were neoplastic lesions. even in resource-poor settings, this underutilised procedure is useful for patient management. keywords: fine needle; aspirate, cytology. introduction fine needle aspiration cytology (fnac) refers to a procedure whereby cells are obtained from a lump, mass or suspicious lesion using a ‘fine bore’ needle (22–27 g) with the aim of microscopic examination of the cells after appropriate staining techniques. the fnac procedure may be done with or without the use of a syringe. it may be performed guided by palpation or by an ultrasound scan.1,2,3,4,5 the first reports of the fnac procedure date back to the 19th century and since then, there have been tremendous developments in its acceptability and utility as part of the diagnostic armamentarium.3,4,5 the procedure is a simple, cheap, reliable, fast and fairly accurate tool, which, in most parts of the developed world, has become routine in medical practice.2 however, this may not be the case in resource-constrained settings; for example, it has been underutilised in nigeria, as reported by malami and others.4,6 there is a need to increase awareness of the procedure, especially with regard to its pivotal role as a cost-effective means of dramatically modifying the course of disease management in patients suspected of having neoplasms. also, information about its availability and usefulness will assist healthcare policymakers to channel resources towards developing this field of pathology practice in developing countries.6,7,8 virtually every organ or region of the body is accessible to the fine needle for aspiration biopsy. it is minimally invasive, gives the least discomfort (even in children) and does not require elaborate patient preparation, such as anaesthesia. results can be available within a few hours.5,6,8,9 methods ethical considerations ethical clearance for the study was sought and obtained from the hospital research and ethics committee of usman danfodiyo university teaching hospital, sokoto, nigeria. the ethical approval number is: uduth/hrec/2018/649. setting the study was conducted in the department of histopathology at usmanu danfodiyo university teaching hospital, sokoto, a tertiary health institution situated in the north-west region of nigeria. it provides tertiary healthcare services to (but not exclusively) the sokoto, kebbi, zamfara and niger states. it also receives referrals from the republic of niger, a neighbouring country. services rendered in the histopathology department of the hospital include histopathology of surgical biopsies and research specimens, cytopathology (including fnac), frozen section, immunohistochemistry and autopsy. population most of the patients presented at the histopathology department for the procedure were on referral after initially being seen by attending physicians in the outpatient units. a few were inpatients, which required the histopathologist to perform the fnac in the wards. in both cases, the requesting physicians filled out histopathology requisition forms indicating the site for the procedure and other relevant clinical details. the fine needle aspiration was done by the histopathologist, predominantly using a palpation guide. sampling universal sampling was employed, in which all cases registered as fine needle aspiration biopsy in the departmental records (reception registers, bench books, request forms, etc.) over the period 1 january 2011 to 31 december 2015 (five years) were consecutively selected specifically with respect to the patients’ age, sex, and site of biopsy. corresponding microscope slides prepared in quadruplicate (each replicate stained with papanicolaou stain, giemsa and hematoxylin or eosin) were retrieved from the departmental archive and re-viewed via light microscopy, using organ specific guidelines. faded slides were re-stained where possible; missing slides were excluded from the study. the data generated were entered into a microsoft excel 2007 edition (microsoft corporation, redmond washington, united states) spreadsheet, validated, exported to spss version 20 (ibm spss statics for windows, version 20.0; ibm corp, armonk, new york, united states) for analysis and results were presented as frequency distribution tables. results over the five-year period of the study, a total of 1685 fnac procedures were performed, of which 1435 were included in the study. this represented 30.0% of all cytology specimens seen during the same period. the age range of patients was two weeks to 100 years, with a mean age of 32.7 years. the procedure was most frequently performed in patients within the age range of 21–30 years and least performed in those less than one year old (table 1). a male-to-female sex ratio of approximately 1:3 was observed, depicting a female preponderance. this ratio changed to approximately 1:1 when breast fnac was excluded. table 1: sex and age distribution of fine needle aspiration biopsy in sokoto. the breasts were the most common site for fnac, with 655 (45.6%) cases, followed by 378 (26.3%) head and neck cases and 206 (14.4%) lymph node cases. abdominal viscera were the least accessed organs with 27 (1.9%) cases (table 2). non-neoplastic lesions (‘inflammation’ and ‘reactive’) accounted for a total of 250 (17.4%) cases, while neoplastic lesions (‘benign’ and ‘malignant’) accounted for 893 (62.3%) cases. specifically, there were 740 (51.6%) benign lesions and 153 (10.7%) malignant lesions. there were 218 (15.2%) lesions with suspicious features. table 2: distribution of fine needle aspiration biopsy by diagnosis, site and malignant cytologic diagnosis in sokoto. discussion a total of 1435 fnac procedures were performed over the five-year period studied, constituting approximately 30% of all cytology specimens seen in the department. this figure is much higher than that reported from north-central nigeria where a rate of 97 fnac per year (over three years) was observed.9 however, it is comparable to faduliye’s study from south-west nigeria, where a total of 1855 cases (representing fnac specimens and cytology specimens obtained by other means) were performed over a period of six years (although that study also included cytology specimens obtained by other means).1 these studies show that the procedure is still in the early stages of widespread use as compared to figures from centres in developed countries that report up to 1000 cases per year.5 we observed that the highest frequencies were seen in the 21–30 years and 31–40 years age ranges. fnac was also done in individuals at extreme ages (9 [0.6%] who were younger than 1 year and 17 [1.2%] who were older than 71 years) showing that the procedure is applicable irrespective of the patient’s age. these compare favourably with studies from nigeria and other countries.1,5,9,10 the frequency of fnac among women was higher with 1043 (72.1%) cases as compared to men, and this may reflect the higher frequency of breast fnac, which accounted for 655 (45.6%) of all fnac cases. this finding compares with the observations of mohammed et al. from north-east nigeria and vhriterhue from north central nigeria.7,8,9 only 153 (10.7%) of the cases were malignant, whereas 740 (51.6%) were benign. this is an important feature to note as this outcome underscores the usefulness of the procedure for the immediate triage of patients, which significantly alters the clinical management outline. other workers have also noticed a similar usefulness of the procedure.1,8,9,11 as vhriterhue and others have noted, a high frequency of ‘grey zone’ diagnosis (e.g. ‘indeterminate’) potentially undermines the usefulness of the fnac diagnosis. in our centre, we had 15.2% of such ‘grey zone’ (‘suspicious’) cases and this needs to be improved upon to reduce the dilemma and further distress placed on surgeons and patients.9,11 to achieve this, it is recommended that the fnac (especially for barely palpable lesions or lesions located in anatomically difficult sites to access) be performed under ultrasound guide, for example. this will further improve the yield of the aspiration. conclusion fine needle aspiration cytology is a useful tool in medical practice. even in nascent centres like ours, in north-west nigeria, it has found a place in pathology practice. it can be performed on all patients irrespective of age and sex. it is a cost-effective simple procedure, not usually requiring anaesthesia and can be performed on outpatients as well as inpatients, usually with the pathologist being guided by palpation of the lesion. its outcome immediately makes a difference in clinical judgment and patient management. it is recommended that there should be capacity building by encouraging the training and retraining of pathologists, and thus encouraging sub-specialisation in this underutilised area of pathology in centres practising in resource-poor regions. in the same vein, resources need to be channelled towards establishing fnac clinics even at the level of primary healthcare, thereby preventing the frequently reoccurring theme of patients presenting with advanced stage malignant neoplasms. there is a need to correlate cytological and histological findings to further encourage confidence in the procedure. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this paper. authors’ contributions k.a. developed the concept of the study and drafted the literature review. m.u. drafted the literature review and proofread the final manuscript. s.m.s. drafted the literature review and proofread the final manuscript. source of support none. disclaimer the views expressed in the article are those of the authors and not an official position of the institution. references faduyile fa, soyemi ss, oyewole oo. cytopathology practice in lagos, nigeria: our experience. ann trop pathol. 2016;7(2) july–december:117–122. bibbo m, wilbur dc, editors. comprehensive cytopathology. 3rd ed. philadelphia, pa: saunder elsevier; 2008. shehu sm, rafindadi ah. use of fine needle aspiration biopsy (fnab) in the management of breast diseases in ahmadu bello university teaching hospital, zaria. niger j surg. 1999;6;6–9. pindiga uh, abubakar h. fine needle aspiration cytology: appropriate technology, that is quick, safe and cheap. sahel med j. 2001;4:95–96. das dk. fine needle aspiration cytology: its origin, development and present status with special reference to a developing country, india. diagn cytopathol. 2003;28:345–351. https://doi.org/10.1002/dc.10289 malami sa, ochicha o. a review of the utilization of fine needle aspiration in clinical practice and research in nigeria. cytojournal. 2011;8:12. https://doi.org/10.4103/1742-6413.82277 bhursnamath b. fine needle aspiration biopsy cytology (fnab) in diagnosis of tumours. west afr j med. 1986;5:41–47. ngadda ha, ajayi na, ahidjo a, pindiga uh, mustapha sk, khalil mi. fine needle aspiration cytology diagnosis of liver diseases in the university of maiduguri teaching hospital, maiduguri. afr j med sci. 2004:33:255–257. vhriterhire ra, orkuma ja, ngbea ja, eke ba, jobo gta, adekwu a. cytology services utilisation pattern in north central nigeria. br j med med res. 2016:18(11):1–6. https://doi.org/10.9734/bjmmr/2016/29774 mcpherson k, steel cm, dixon jm. breast cancer – epidemiology, risk factors, and genetics. br med j. 2000;321(7261):624–628. https://doi.org/10.1136/bmj.321.7261.624 duduyemi bm, owusu-afiyie o, dnagquah ko, osakunor dn. cytopathology practice in kumasi: a 2 year retrospective audit. j cytol. 2017;34:22–26. https://doi.org/10.4103/0970-9371.197593 about the author(s) lebogang skosana national health laboratory services, tshwane academic division, pretoria, south africa department of medical microbiology, university of pretoria, pretoria, south africa farzana ismail department of medical microbiology, university of pretoria, pretoria, south africa centre for tuberculosis, national institute of communicable diseases, johannesburg, south africa nontombi mbelle † national health laboratory services, tshwane academic division, pretoria, south africa department of medical microbiology, university of pretoria, pretoria, south africa mohamed said national health laboratory services, tshwane academic division, pretoria, south africa department of medical microbiology, university of pretoria, pretoria, south africa citation skosana l, ismail f, mbelle n, said m. corrigendum: brucellosis – laboratory workers’ nightmare come true: a case study. afr j lab med. 2021;10(1), a1690. https://doi.org/10.4102/ajlm.v10i1.1690 note: doi of original article published: https://doi.org/10.4102/ajlm.v9i1.1114. †, 1956–2021. correction corrigendum: brucellosis – laboratory workers’ nightmare come true: a case study lebogang skosana, farzana ismail, nontombi mbelle, mohamed said published: 22 nov. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. in the version of this article initially published, skosana l, ismail f, mbelle n, said m. brucellosis – laboratory workers’ nightmare come true: a case study. afr j lab med. 2020;9(1), a1114. https://doi.org/10.4102/ajlm.v9i1.1114, the authors’ second affiliation was omitted in the ‘author’ and ‘affiliation’ sections. the authors’ affiliations are now corrected as: authors: lebogang skosana1,2 farzana ismail2,3 nontombi mbelle1,2† mohamed said1,2 affiliations: 1national health laboratory services, tshwane academic division, pretoria, south africa 2department of medical microbiology, university of pretoria, pretoria, south africa 3centre for tuberculosis, national institute of communicable diseases, johannesburg, south africa. this correction does not alter the study’s findings of significance or overall interpretation of the study’s results. the authors apologise for any inconvenience caused. abstract background description of the intervention lessons learnt recommendations acknowledgements references about the author(s) barbara s. van deventer department of forensic medicine, faculty of health sciences, university of pretoria, pretoria, south africa lorraine du toit-prinsloo department of forensic medicine, faculty of health sciences, university of pretoria, pretoria, south africa chantal van niekerk department of chemical pathology, faculty of health sciences, university of pretoria, pretoria, south africa citation van deventer bs, du toit-prinsloo l, van niekerk c. practical tips to using formalin-fixed paraffin-embedded tissue archives for molecular diagnostics in a south african setting. afr j lab med. 2022;11(1), a1587. https://doi.org/10.4102/ajlm.v11i1.1587 note: additional supporting information may be found in the online version of this article as online supplementary documents 1, 2 ,3 and 4. lessons from the field practical tips to using formalin-fixed paraffin-embedded tissue archives for molecular diagnostics in a south african setting barbara s. van deventer, lorraine du toit-prinsloo, chantal van niekerk received: 16 mar. 2021; accepted: 21 mar. 2022; published: 23 june 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: formalin-fixed paraffin-embedded (ffpe) tissue archives in hospitals, biobanks, and others offer a vast collection of extensive, readily available specimens for molecular testing. unfortunately, the use of tissue samples for molecular diagnostic applications is challenging; thus, the forensic pathology ffpe tissue archives in africa have been a largely unexploited genetic resource, with the usability of dna obtainable from these samples being unknown. intervention: the study, conducted from january 2015 to august 2016, determined the usefulness of ffpe tissue as a reliable source of genetic material for successful post-mortem molecular applications and diagnostics. formalin-fixed paraffin-embedded tissue samples were collected and archived from autopsies conducted over 13 years in the forensic medicine department of the university of pretoria (pretoria, south africa). deoxyribonucleic acid from ffpe tissue samples and control blood samples was amplified by high-resolution melt real-time polymerase chain reaction before sequencing. the procurement parameters and fixation times were compared with the quantity and quality of the extracted dna and the efficiency of its subsequent molecular applications. lessons learnt: this study has shown that ffpe samples are still usable in molecular forensics, despite inadequate sample preparation, and offer immense value to forensic molecular diagnostics. recommendations: ffpe samples fixed in formalin for more than 24 h should still be used in molecular diagnostics or research, as long as the primer design targets amplicons not exceeding 300 base pairs. keywords: autopsy; deoxyribonucleic acid; formalin-fixed paraffin-embedded tissue; formalin-fixed paraffin-embedded tissue archive; high-resolution melt analysis; molecular diagnostics; polymerase chain reaction; post-mortem genetic testing; sequencing. background advances in molecular biology have enabled the detection of preventable and treatable diseases at a genetic level and it is a field of study receiving growing attention over the years.1,2,3 globally, formalin-fixed paraffin-embedded (ffpe) tissue samples are the largest available and most used source of biological material for molecular applications – diagnostic purposes or research.3,4,5 formalin-fixed paraffin-embedded tissue archives in hospitals, private pathology institutions, biobanks, and tertiary academic pathology departments offer a vast collection of extensive, readily available specimens for molecular testing.6,7,8 the critical information these specimens contain and their undeniable usefulness to necessary health-related investigations are considered by many researchers to be invaluable for molecular diagnostics.4,8,9 unfortunately, the low quality and efficiency of dna extracted from ffpe samples in molecular diagnostic applications limits the use of archived ffpe tissue samples.10,11,12 potential factors influencing the limited molecular utility include different parameters used in the procurement, fixation and processing of these samples.1,6,11 particularly, formalin fixation causes dna fragmentation, formaldehyde exposure causes dna-protein cross-linking, and paraffin in dna samples inhibits polymerase chain reaction (pcr) amplifications. these factors impair the dna quality and efficiency for molecular applications, such as pcr and sequencing.9,11,12,13 although blood collected in ethylenediaminetetraacetic acid or fresh frozen tissue samples are the best source of dna and the preferred material used for genetic testing, numerous studies have reported on the successful use of ffpe tissue samples.2,14,15 the successful use of ffpe tissue samples is particularly encouraging because african forensic pathology still faces the dire reality of inadequate funding and resource allocation required to maintain freezer preservation systems for routine collection and storage of blood or tissue samples.15,16,17 forensic molecular pathology is an emerging field with significant clinical impact; its techniques are used to diagnose preventable and inherited causes of death.18,19,20 the role of the forensic pathologist in the medico-legal investigation of death includes determining the cause and manner of death. despite the implementation of forensic molecular investigations and research in many high-income countries, there is still a significant lack of it in lowand middle-income countries, mainly due to financial and resource constraints.9,15,21 several south african universities’ forensic medicine departments conduct valuable research, mainly focusing on possible causes of sudden deaths; however, there is still a remarkable paucity of publications on molecular forensic studies and their applications in africa.22,23,24 social and medico-legal issues challenge molecular forensic research, including personal and public concerns.25 fortunately, in most large forensic pathology centres, which are often linked to tertiary academic institutions, forensic pathologists have established extensive ffpe tissue archives through routine histology casework. these archives are excellent and sometimes the only archives for conducting large retrospective genetic epidemiological studies.9,15,20 until now, these forensic pathology ffpe tissue archives have mainly been unexploited; thus, the quality of dna obtainable from these samples is unknown.9,15,24 african countries, particularly south africa, have some of the highest numbers of unnatural deaths per year, compared to other countries in the world.26,27 unfortunately, this increases the burden on the already-scarce africa-practising forensic pathologists. consequently, the increased caseload delays case investigation, prolonging formalin fixation times by days, weeks, and even months.26,27,28 according to recommended guidelines for optimised molecular testing, tissue samples should be fixed in 10% buffered formalin for 14 h – 24 h.9,14 hence, most forensic pathology departments doubt the value and efficacy of their decades worth of ffpe tissue archives in the context of molecular diagnostic applications or research.9,15,20 this study aimed to determine the utility of ffpe tissue samples as a reliable source of genetic material for post-mortem molecular applications and diagnostics. firstly, the study evaluated the influence of the procurement parameters, fixation time, and storage periods of the archived ffpe tissue samples on the quantity and quality of the dna extracted and its efficiency for subsequent molecular applications. secondly, the study aimed to determine which dna extraction kits yield the best quality and quantity of ffpe dna. description of the intervention ethical considerations ethics approval was obtained from the faculty of health sciences research ethics committee university of pretoria (reference number 142/2014) to use the retrospective and prospective ffpe tissue samples and the control blood samples. the retention and use of tissues obtained at medico-legal post-mortem examinations were guided by the south african legislation (inquests act 58 of 1995, national health act 61 of 2003 and the regulations regarding the rendering of forensic pathology service r636). thus, patient and family consent was not required. healthy volunteers provided written informed consent before blood sample collection. all tissue and blood samples were assigned a case number, with no identifying features linked to any of these samples. study location and design this retrospective observational study was conducted in the department of forensic medicine, the university of pretoria, pretoria, south africa, from january 2015 to august 2016 using ffpe myocardial tissue samples obtained over 13 years from post-mortem examination of sudden unexplained infant deaths in the pretoria medico-legal laboratory. the samples were stored for further ancillary investigations to determine the cause of death, including genetic testing for genes linked to inherited cardiac arrhythmogenic disorders and sudden unexplained infant deaths, such as scn5a. specimen description the study included a total of 58 ffpe myocardial tissue samples. these ffpe tissue samples were obtained from sudden unexplained infant death complete post-mortem investigations between january 2002 and january 2015 and identified from the department of forensic medicine electronic repository. additionally, in january 2015, two prospective myocardial tissue samples (samples 59 and 60) were also obtained from autopsy and, starting in march 2015, venous blood from nine healthy volunteers was drawn into two 5 ml ethylenediaminetetraacetic acid tubes. specimen processing when retained, the archived ffpe myocardial tissue samples were immediately fixed in 10% neutral buffered formalin solution per routine and processed using the shandon pathcentre from thermo scientific (waltham, massachusetts, united states) and the tissue-tek® tec from sakura finetek (torrance, california, united states). the ffpe processing included dehydration in ethanol, clearing in xylene, and embedding in paraffin blocks. when obtained, the prospective myocardial tissue samples (samples 59 and 60) were immediately fixed in formalin for 14 h – 24 h, per qiagen protocol. subsequently, they were cleared in xylene, embedded in paraffin blocks (following the same procedure as the archived samples), and stored for a month. all ffpe tissue samples were cut into 20 μm thick sections using a microtome (leica biosystems, wetzlar, germany). the first three to four cuts of each ffpe specimen were discarded because the sample surface had been exposed to air. then, to ensure sufficiency, six to eight sections of each sample were put into a well-labelled sterile 1.5 ml microcentrifuge tube for dna extraction. during this specimen preparation process, the microtome’s blade and the cutting surface were cleaned with 100% ethanol between each specimen to prevent cross-contamination. deoxyribonucleic acid extraction deoxyribonucleic acid was extracted from all ffpe samples (archived and prospective) using two different extraction kits; the qiaamp dna ffpe tissue kit (qiagen, hilden, germany) and the isolate п ffpe dna kit (bioline, london, united kingdom). thus, two dna extraction procedures were performed on every ffpe tissue sample. the two kits differed in the type of deparaffinisation solution and incubation time at 90 °c for the reversal of dna cross-linking. the qiaamp dna ffpe tissue kit used standard qiagen-provided deparaffinisation solution and required an incubation time of 2 h at 90 °c, while the isolate п ffpe dna kit used xylene and incubation of 1 h at 90 °c.29 for the blood samples drawn into two 5 ml ethylenediaminetetraacetic acid tubes, the buffy coat was isolated (200 μl) and stored at – 80 °c until dna extraction using the qiaamp dna blood mini kit (qiagen, hilden, germany) per manufacturer’s instructions. further on, the concentration and purity of all dna samples (archived ffpe, prospective ffpe and blood) were determined spectrophotometrically (nanodrop, thermo scientific [waltham, massachusetts, united states]). for this study, a dna concentration range of 40 ng/μl – 75 ng/μl and a purity ratio above 1.75 were deemed sufficient. high-resolution melt real-time pcr and sequencing the dnas were diluted to concentrations between 40 ng/μl and 75 ng/μl. thirty-four amplicon primer pairs (online supplementary document table 1) for the scn5a gene30 generating 152 base pairs (bp) to 514 bp amplicon sizes amplified all extracted dna (60 ffpe tissue samples: two prospective and 58 archived; and nine blood samples). amplification was by high-resolution melt real-time pcr using sensifast high resolution melt master mix (bioline, london, united kingdom) on the rotorgene q (qiagen, hilden, germany). successful real-time pcr results had sigmoidal amplification and single melt curves. afterwards, the pcr amplicons were sanger sequenced by inqaba biotec, pretoria, south africa. sequencing chromatograms were analysed using clc main workbench 5 software (clc bio®, aarhus, denmark). a successful sanger sequencing had evenly spaced peaks and a minimal noise chromatogram. analysis the tissue retention date and the 10% formalin fixation duration for each ffpe tissue sample (online supplementary document table 2) were used for student’s t-test in microsoft excel 2013 (redmond, washington, united states) to determine a possible correlation with the quality of pcr amplifiability. lessons learnt deoxyribonucleic acid yield deoxyribonucleic acid extracted from all nine control blood samples yielded concentrations ranging from 15 ng/μl to 56 ng/μl, with sufficient 260/280 ratios between 1.75 and 2.0. comparison of dna yield obtained from two different extraction kits the maximum period of tissue sample storage (embedded in paraffin blocks) was 13 years, while the minimum was one month. the minimum period of tissue fixation in formalin wax was two days and the maximum, 271 days. the average value obtained for the fixation period of all 58 tissue samples was 26.1 days, with a median of 11.5 days. our findings showed an extensive delay in the department’s processing of ffpe tissue samples, particularly long tissue fixation in formalin (online supplementary document table 2). these findings are in keeping with similar ffpe archive conditions reported by other forensic pathology departments practising in resourceand fund-limited settings.9,15,16,24 the qiagen ffpe kit yielded much higher dna concentrations, 50 ng/μl – 900 ng/μl. after extraction, an elution volume of 50 μl of dna of each sample was obtained, with purity ratios between 1.7 and 2.1. the bioline ffpe kit yielded lower dna concentrations, 33 ng/μl – 137 ng/μl, with 260/280 ratios between 2.0 and 2.1 and an elution volume of 50 μl. for this study, a dna concentration range of 40 ng/μl – 75 ng/μl and a purity ratio above 1.75 were deemed sufficient. although the two different kits yielded dna concentrations of quite different ranges, no difference in pcr amplification results was observed. furthermore, both kits’ pcr amplification quality correlated; thus, successful or failed/unsuccessful amplification was recorded for each tissue sample. therefore, we concluded that the two kits yielded the same quality of extracted dna and pcr amplification and, by extension, the exact molecular analysis results. does size matter? amplification of all 34 amplicons, high resolution melt analysis, gel electrophoresis, and sequencing were successful for all dna extracted from blood. polymerase chain reaction amplified several amplicons from the ffpe dna; however, there was a significant reduction in the amplification of amplicons with a length greater than 300 bp (figure 1 and online supplementary document table 3). in contrast to vitosevic et al.,31 our study (as indicated in online supplementary document table 2) did not find a correlation between longer sample storage time and pcr amplification failure, as successful amplification was observed for samples stored between 1 month and 13 years. instead, we found that prolonged periods of formalin fixation, which causes dna fragmentation, correlated with failed pcr amplification (figure 2). figure 1: effect of amplicon size on pcr amplification success rate of dna extracted from ffpe tissue samples, pretoria, south africa, june 2015 – may 2016 (online supplementary document table 3). figure 2: laboratory results showing the association between a prolonged formalin fixation period and a decline in pcr amplification success rate, pretoria, south africa, march 2015 – july 2016 (online supplementary document table 2). formalin fixation time approximately half of the ffpe dna samples produced large amplicons; an association existed between successful amplification and a shorter (approximately four days) formalin fixation duration (figure 2, online supplementary document table 2 and online supplementary document table 4). converse to archived ffpe dna, which had been fixed in formalin for a period exceeding 24 h, the two prospective ffpe samples (fixed in formalin for only 24 h) yielded all 34 targeted amplicons, including a 514 bp amplicon (the largest amplicon). only four retrospective ffpe dna samples yielded the 514 bp amplicon. on analysis, these four ffpe dna samples, all stored for six years, were subjected to shorter periods of formalin fixation (figure 1 and online supplementary document table 2). this bar graph shows the significant decrease in successful pcr amplification by using dna extracted from ffpe tissue samples that have been fixated in formalin for periods longer than the prescribed 24 h. dna samples extracted from those ffpe tissue samples which adhered to the prescribed 24-h formalin fixation illustrates a 100% success rate in pcr amplification. samples were grouped according to days in formalin (see online supplementary document table 4). downstream applications sanger sequencing of amplicons was successful for both blood and ffpe tissue samples. formalin-fixed paraffin-embedded tissue sequences that showed variations upon alignment to the reference sequence were re-amplified and re-sequenced to validate these variations and exclude the possibility of dna cross-linking. results showed no aberrant variations in ffpe dna samples compared to amplicons resulting from blood dna, indicating that the fixation method does not seem to increase the chance of interpreting these as legitimate variations. recommendations cut your losses? this study showed that dna extracted from ffpe tissue samples could successfully be both amplified and sequenced. however, it is essential to note that dna measurements (concentration and purity ratio) obtained using a spectrophotometer do not assure successful pcr amplification and sequencing because it does not measure the extent of dna fragmentation. commercially available kits, such as the quantifiler® trio dna quantification kit, are designed to detect and help overcome external factors affecting pcr amplifiable quality. however, in a resource-poor setting, a spectrophotometer, which most laboratories have, will indicate the ‘usability’ of extracted dna.32 polymerase chain reaction success was limited to amplicons smaller than 300 bp in cases of prolonged formalin fixation. thus, ffpe samples could, and should, still be used in molecular diagnostics or research, as long as the primers are designed to generate pcr amplicons not exceeding 300 bp. our study confirmed that ffpe samples are still useful for molecular forensics and diagnostics despite inadequate sample preparation. thus far, ffpe tissue archives in most african forensic pathology departments have been an underexploited resource for conducting large retrospective and prospective genetic epidemiological studies.14,15,16 it is time to utilise these resources at our disposal to the advantage of the african population. the most significant benefit of post-mortem molecular testing is the high disease-specific diagnostic, therapeutic, and prognostic benefits derivable from subsequent genetic analysis.1,2,3 understanding the genetics of inherited diseases specific to african populations informs the development of more meaningful and relevant risk stratification techniques.3,15,33 this includes the development of more targeted molecular tests for various challenges in the forensic setting, including population genetics, human identification, and post-mortem interval estimation.9,15 acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions b.s.v.d., l.d.t.-p. and c.v.n. conceived and planned the experiments/study. b.s.v.d. carried out the experiments. b.s.v.d., l.d.t.-p. and c.v.n. contributed to the interpretation of the results. b.s.v.d. took the lead in writing the manuscript. all three authors provided critical feedback and helped shape the research, analysis, and manuscript. sources of support the authors would like to thank the genomics research institute (university of pretoria) for funding this project. we would also like to thank the south african national research foundation for funding part of this project. data availability the data that support the findings of this study are available from the corresponding author, b.s.v.d., upon request. disclaimer the views expressed in this article are those of the authors and do not necessarily reflect the official 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https://doi.org/10.1007/s12024-020-00327-z gouveia n, brito p, serra a, et al. validation of quantifiler® trio dna quantification kit in forensic samples. forensic sci int genet. 2015;5:24–25. https://doi.org/10.1016/j.fsigss.2015.09.010 laing m, kraus sm, shaboodien g, et al. an overview of the genetic basis of cardiovascular disease. samj. 2019;109(6):364–370. https://doi.org/10.7196/samj.2019.v109i6.14069 abstract introduction methods results discussion acknowledgements references about the author(s) lavendri govender department of biomedical and clinical technology, faculty of health sciences, durban university of technology, durban, south africa molecular research and development department, specialised laboratory services, south african national blood service, durban, south africa rosaley d. prakashchandra department of biomedical and clinical technology, faculty of health sciences, durban university of technology, durban, south africa pavitra pillay department of biomedical and clinical technology, faculty of health sciences, durban university of technology, durban, south africa ute jentsch medical department, specialised laboratory services, south african national blood service, durban, south africa citation govender l, prakashchandra rd, pillay p, jentsch u. molecular red cell genotyping of rare blood donors in south africa to enhance rare donor-patient blood matching. afr j lab med. 2021;10(1), a1400. https://doi.org/10.4102/ajlm.v10i1.1400 original research molecular red cell genotyping of rare blood donors in south africa to enhance rare donor-patient blood matching lavendri govender, rosaley d. prakashchandra, pavitra pillay, ute jentsch received: 21 sept. 2020; accepted: 14 may 2021; published: 27 sept. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: molecular red cell genotyping is devoid of serology limitations such as the scarcity of rare antisera and the possibility of inconclusive results due to biological interferences. blood incompatibility can result in immune transfusion reactions such as haemolytic transfusion reactions or haemolytic disease of the foetus and newborn. objective: the study aimed to use molecular red cell genotyping to identify rare blood group donors among south african blood donors. methods: red cell genotyping data were extracted retrospectively from the bids xt genotyping software in the immunohaematology reference laboratory from january 2015 to august 2016. the id core xt genotyping assay was used to identify the single nucleotide polymorphisms of 10 blood groups system alleles in 150 donors. associations between the resultant genotypes and predicted phenotypes, abo group, rhd type, race group and gender were studied. results: significant red cell genetic variability was noted among the numerous south african donor genotypes identified in this study. genotyping further confirmed the presence of at least one of the 16 rare genotypes in 50 donors. group o black donors were associated with two rare blood types, while several other rare blood types were found only in white donors, supporting an association between abo/rh subtype, race group and rare blood types. conclusion: targeted screening of donors for antigen-negative rare blood units for patients should be done to reduce the risk of haemolytic transfusion reactions and haemolytic disease of the foetus and newborn. keywords: red cell genotyping; genetic variations; rare blood types; donor-patient blood matching. introduction the antigens found on the surface of red blood cells determine the blood type of an individual. blood grouping is essential for donor-patient blood transfusion match.1 red cell antigen mismatch between donor and patient can result in alloimmunisation, which is the formation of red cell antibodies in the patient following transfusion with red cells that the patient lacks. clinically, alloimmunisation can result in mild to fatal haemolytic transfusion reactions.2 in neonates, alloimmunisation may cause mild to fatal haemolytic disease of the foetus or newborn.3,4 the current serological phenotyping method of red cell antibody detection used in south africa is simple and inexpensive. however, inconclusive serology results may result due to the interference from donor red blood cells in chronically transfused patients, a rare blood type causing false-positive results or autoantibodies in patients with autoimmune diseases.5 also, sourcing blood for patients with rare blood types is challenging due to the scarcity of commercial rare antisera for serological testing.6 the term rare blood type describes the absence of an antigen that is found normally in 99% of the country’s population and is described as: ‘negative for high-frequency-antigens (hfa)’ or the presence of antigens found in only 1% of the population termed ‘positive for low-frequency-antigens’ or the presence of an unusual or rare rh subtype in a very small percentage of the population (1:1000, 1:10 000).7 hence, rare commercial antisera are often not readily available and are expensive. therefore, they are not cost-effective for large-scale blood type screening.6 molecular genotyping overcomes the limitations of serology. the mapping of the human genome provided knowledge on the molecular backgrounds and polymorphisms of the blood groups that enabled the development of dna-based assays for red cell genotyping.8 genotyping utilises patients’ dna information to infer the red cell phenotype. genotyping assays have been widely used in transfusion medicine for over 20 years.8 this technology only became an affordable service in south africa in 2015 and has to date not translated into the genetic identification of rare and unique red cell genotypes in south africa or the wider african context. smaller studies in some african countries have been completed; however, these were limited to group-specific antigen coverage. moreover, the sample numbers were too low across the regions to be representative of the african diaspora and its multiple ethnic groups.9 therefore, this study aimed to provide the first comprehensive red cell genotyping of rare blood donors referred to the south african national blood service, immunohaematology reference laboratory (irl). methods ethical considerations full approval (ethical clearance number irec 011/17) was granted by the durban university of technology to complete the study as part of a master’s thesis. ethical approval was also granted by the south african national blood service human research ethics committee (certificate clearance number 2016/06). initial donor consent was provided by donors when completing the blood donor questionnaire at the time of blood donation. however, there were no human participants in this study as it was a retrospective review of red cell genotyping data only. all genotyping results were anonymised from donor details and blinded using designated study numbers only accessible to the researcher. study participants the south african national blood service provides a vein-to-vein blood service in eight of the nine south african provinces and the majority of the blood donations are successfully cross-matched for transfusion to patients. where routine cross-matching cannot be completed due to inconclusive serology results, the blood donation is referred to the national immunohaematology reference laboratory. this observational study retrospectively reviewed red cell genotyping data of 150 south african national blood service donors whose blood samples were referred to the immunohaematology reference laboratory between january 2015 and august 2016. the study population of 150 donors comprised 58 conveniently selected serology-determined rare blood type donors and 92 randomly selected group o, rhd+ donors (table 1). the selection bias for group o+ donors was because the south african national blood service donor recruitment strategy focuses on sourcing mainly group o+ blood since it is a common blood type in south africa and can be transfused to a+, b+, ab+ and o+ patients. table 1: percentage occurrence of the id core xt predicted phenotypes and genotypes for 150 south african blood donors genotyped at the south african national blood service immunohaematology reference laboratory from january 2015 to august 2016. the self-proclaimed race groups of the donors as stated on the blood donation donor questionnaire were collated to establish whether there is an association between red cell genotypes, rare blood types and race groups. donors indicated their self-proclaimed race groups as either black, mixed race, indian or white.10 these racial groups were introduced by apartheid and remain a description of the south african society with black south africans being the native black african population, mixed race south africans describing a person of mixed european (‘white’) and african (‘black’) or asian ancestry, as officially defined by the south african government from 1950 to 1991, indian south africans are south africans who descend from migrants who arrived from british-ruled india during the late 1800s and early 1900s and white south africans are south africans of european descent, as well as from certain parts of west asia. since the samples of donors were collected nationally within south africa, all race groups have representation in the study although not of equal distribution. dna extraction dna was extracted from blood collected in ethylene-diamine-tetraacetic-acid anticoagulated blood tubes using the maxwell as2000 (promega, madison, wisconsin, united states) instrument and the promega maxwell standard elution volume assay. dna was quantified using the nanodrop 2000 (thermofisher scientific, waltham, massachusetts, united states) then diluted to obtain a standard dna concentration of 20ng/µl with a purity falling between 1.7 and 1.9 as required for completion of the id core xt assay (progenika, derio, spain). id core xt assay to maximise resources, the immunohaematology reference laboratory utilised the id core xt assay kit (progenika/grifols, san antonio, texas, united states). the id core xt kit covers red cell antigens or alleles of 10 blood grouping systems: rhce, kell, kidd, duffy, mns, diego, dombrock, colton, cartwright and lutheran, totalling 37 red cell antigens in a single test. the id core xt kit was assayed in a luminex 200is analyser (luminex corporation, austin, texas, united states) to detect beads coated with specific single nucleotide polymorphisms. the bidsxt assay was used to exclude all invalid red cell genotypes that did not yield a bead count of greater than 30 or a median fluorescent intensity of 1000. data analysis the predicted phenotypes and genotypes were exported from the bids xt (progenika/grifols, san antonio, texas, united states) software to a powerbi software programme. simultaneously, demographic details of gender, race, abo group and rhd types were exported from the organisation’s information system to the business intelligence information technology database to study associations between these characteristics. database collation the occurrence of the genotypes and predicted phenotypes were analysed from the most to the least occurring among the white, black, indian and mixed south african race groups. rare blood types are either negative for hfa, positive for low-frequency-antigens or unusual, rare rh subtypes that occur in less than 1% in a population. however, due to the small sample size of the study and the 34 rare blood types that were conveniently added to the study, we could not use the less than 1% guideline. instead, the rare blood types currently listed on the south african rare donor file was used to separate the rare blood types – hrb–, hrs–, k–, jsb–, u–, kpb–, yta–, lub–, hy–, joa–, uvar+, cw+, r’r”, r”r, r’r’ and rzr1 – genetically tested in this study. results most of the study participants were men (63%, n = 95/150) with 80% of the race group distribution comprising 41% (n = 61) white donors and 39% (n = 59) black south african donors. the 2015–2016 red cell genotyping data was skewed by a selection bias for group o+ (71%, n = 107) donors which accounted for 33 rare blood types found in 32 donors. in comparison, group o– donors that made up 13% (n = 20) of the study population showed the presence of 15 rare blood types in 14 donors. the remaining 16% (n = 23) of the group a+/− and group b+/− had four rare blood types in four donors, cumulatively resulting in a total of 50 rare donors with 52 rare blood types at a molecular level. of the 58 serological known rare donors, 44 donors had the serologically defined rare blood types hrb–, hrs–, k–, u–, u-variant+, jsb, kpb–, r̎ r̍, rzr1, lub– and yta– that are covered by the id core xt. however, molecular genotype correlated with serotype in only 34 of the 44 donors (figure 1). ten of the 44 serology-defined rare blood types were absent upon molecular testing giving a 23% discrepancy rate between serotyping and genotyping. more so, rare blood types previously not detected by serology were found in two of these 10 donors (figure 1). fourteen of the 58 serologically known rare donors had one of the eight rare blood types: bombay oh, vel–, kna–, in(lu), lan–, dantu–, milton (mi ii) and henshaw that were not covered in the id core xt assay. therefore, these could not be confirmed genotypically (figure 1). figure 1: breakdown of the study population and the resultant total of 50 genetically identified donors based on retrospective red cell genotyping obtained from the south african national blood service, immunohaematology reference laboratory over the period january 2015 – august 2016. from among the 92 random donors, genetic and molecular screening identified an additional 14 donors with a total of 16 rare blood types. this brings the total number of rare donors confirmed using genotyping to 50, which comprises 33.3% of the study population (figure 1). rhd/rhce the most occurring rhd/rhce phenotype was the rh-positive ro (cde/cde) phenotype and was found in a majority (72%, n = 42/59) of the south african black donors. six black ro donors were associated with the rare blood genotype rhce*cear, rhce*cear and rhce*cear, rhce*ce[712g], representing the rare negative hfa, hrs–. in comparison, the white south african donors showed the most genetic variability, having all of the rh-negative and rh-positive subtypes. a rare rzr1 (cde/cde) phenotype occurred in one mixed race south african donor. the four black and three mixed race south african donors with the r’r (cde/cde) phenotype were associated with the rare blood genotype rhce*ce[733g,1006t], rhd*r’s-rhce*ce[733g,1006t] that is serologically referred to as the known negative for a hfa rh:-34 or hrb–. predominant among the indian south african donors was the rhce*ce, rhce*ce, r1r1 (cde/cde) genotype and predicted phenotype. kell the single nucleotide polymorphisms in id core xt cover the k/k, kpa/kpb and jsa/jsb antigens from the kell blood group system (table 1). the homozygous kel*k_kpb_jsb, kel*k_kpb_jsb was the most predominant kell blood group genotype found across the ethnicities in 84% (n = 126) of the study population. the remaining five kell genotypes were distributed among 16% (n = 24) of the race groups with the kel*k_kpa_jsb, kel*k_kpa_jsb (serologically referred to as the kpb-) phenotype being the least common kell genotype in this study. kidd the most occurring kidd genotype in the study population was the homozygous jk*a, jk*a kidd blood type. it was observed in 50% (n = 75) of the study population and the majority of the black and indian donors. the remaining 50% of the donors had the heterozygous jk*a, jk*b and the homozygous jk*b, jk*b. the three kidd genotypes were found in almost equal spread among the mixed race donors and the jk*a, jk*b was more common among the white donors. duffy the predominant occurrence of the gata mutation indicates the predicted fya–, fyb– phenotype as present in 31.3% (n = 47) of the study population (table 1). the fy*b_gata, fy*b_gata was predominant among the black donors while the fy*b, fy*b[265t]_fy*x was observed in one white donor. mns in the id core xt assay, the gypa and gypb genes identifying the mn and s-s-u alleles of the mns system are identified and reported individually. the relationship between the ss null alleles resulting in u-antigen variants (u-variant) and the gypb gene deletion resulting in a u-predicted phenotype is evident (table 1). the rare s-s-u– was found in only the black donors in this study. diego no di*a genotype was detected among study participants; however, the homozygous di*b, di*b was detected (table 1). dombrock, colton, cartwright, lutheran there was a slightly higher occurrence of the homozygous do*b, do*b than the do*a, do*b genotype, and there were four dombrock variants that occurred in less than 1% of the donors. the homozygous co*a, co*a, yt*a, yt*a and lu*b, lu*b genotypes occurred in more than 90% of the study population. a small percentage (2.7%, n = 4) of white donors had the co*a, co*b genotype while the yt*b, yt*b. lu*a, lu*a occurred in less than 1% of donors. the international society of blood transfusion’s categories of rare blood types include: negative for hfa, positive for low-frequency-antigens and rare unusual rh subtypes. there were 10 negative for hfas detected in 38 donors of which the hrb– and hrs– hfa were most common being found in 12 and 6 donors. the two positive for low-frequency-antigensme and four rare rh subtypes were found among seven donors. of the 52 rare blood types listed on table 2, 1% (n = 16) rare blood types were newly identified from 14 donors; two donors had two rare blood types each: hrs– with jsb– and u– with dob–. the 1% new rare blood types comprised of 3 hrb–, 3 jsb–, 2 u-variant (uvar+), 2 dob– (do*a, do*a_jo), 2 r’r”, 1 hrs–, 1 cecw, 1 r’r, and 1 u–. table 2: rare blood types by antigen type and frequency found among 150 donors genotyped between january 2012 and august 2016 at the south african national blood service, immunohaematology reference laboratory. discussion studying the red cell genetic variation among the four major south african race groups showed that the rh subtype ro is found in 42 out of 59 black donors, r1r1 in 10 out of 19 indian donors while six different rh subtypes are spread among the white donors. also, rare blood types such as the hrb– and hrs– were found more in black donors while other rare types such as k–, kpb– were found among white donors suggesting genetical association between rh subtype, race group and blood type. this knowledge allows for more effective rare donor screening strategies to be implemented to enhance rare donor-patient blood matching in south africa. in this study, screening of random group o+ donors identified 14 new rare donors that would have been missed by routine testing. hence, identifying markers suggesting a rare blood type in donors such as rh subtype, race and patterns of genetic variations will assist the development of algorithms for rare blood type testing and identification in donors. on comparing our results to those from historical serological prevalence studies,11 we observed that white and indian south africans share similar alleles for the rhce*ce/ce, *ce/ce, kel*k_kpb_jsb (k–), fy*a, fy*a and yt*a, yt*b genotypes.11 similarly, common red cell antigens among the black and mixed race south africans were the rhce*cde/cde, kel*k_kpb_jsb/jsa and fy*a/b_gata.11 according to the department of statistics south africa,10 the four major race groups are defined as black, white, indian and mixed race people while internationally and outside africa, the broadly defined ethnic groups are termed caucasian, african american, asian, other or mixed race. it has been proposed that global emigration patterns account for the red cell antigens of white south africans being related to caucasians, whereas black and mixed race south africans are similar to african americans and indian south africans similar to asians, and in some cases, caucasians.11,12,13,14 in this study, the presence of the rhce*cde/cde (r’r), *cde/cde (r’r”), *cde/cde(ro), kel*k (k–), kel*kpa (kpb–) and lu*a (lub–) in white south africans matched the findings in caucasian populations of italy, netherlands, america, spain, germany and austria.15,16,17,18,19 further, the rare e-hrband e-hrsfound among black south africans matched findings in the african american population.20,21,22 the js*a,js*a genotype (jsb–) was found in three black south africans and one indian donor which is not common in south africa. however, this phenotype is present in america and japan and is reportedly associated with asian populations.12,23 the detection of the rhce*cde/cde (ro) in all the race groups represented in the study contradicts the assumption of a race-specific blood type. interestingly, the rhce*cde/cde (ro) was most detected in the black south africans but only found in the netherlands.16 this finding suggests that this is a very rare blood type associated with african ancestry. the absence of the yta(yta–) cartwright antigen shown by the yt*b,yt*b genotype, was found in one indian south african but was not detected in a study by kahar and patel,24 which surveyed the prevalent red cell phenotypes in central india. the n of the yta(yta–) can be attributed to the small study size or area of india sampled. the ytaantigen was also identified in italy,15 america,17 spain,18 japan23 and canada25 prompting a decision to expand screening for this rare antigen in south africa to all race groups. similar to the european study by finning et al.,26 three of the negative for hfa js(b–), kp(b–) and jk(b–) were also found in the current study. although there have been suggested similarities between south african white donors and caucasians or europeans, it is evident that some uniqueness exists between the two geographic regions. the dib–, rhce*cern, dia–, mia+, cob+, lua+, dob+, which were unique to the european study by finning,26 were absent in the present study possibly due to the small sample size. more studies will be required to interrogate and investigate the underlying reasons for these potential differences at a genetic level or whether this pattern changes and becomes more similar when more donors are tested. the rhce*ce/ce genotype is a rare rh subtype (rzr1) that was found in one mixed race south african. a study completed by sharma et al.27 reported rzr1 to be found in 6% of native americans and 2.2% of indians in central india. the low prevalence antigen rhce*cw was found in two white south africans only. the id core xt assay does not differentiate the hrb– (rh:-34) from the hrb– (rh:-31). the formation of anti-hrb is due to the absence of the high-frequency antigen hrb (hrb–) and anti-hrb (rh:-31) is an ‘anti-e-like’ antibody. the absence of the ‘e’ antigen together with the hrb– (e-hrb)– indicates the true rare blood type. besides, the id core xt assay does not differentiate the hrs– (rh:-18) from hrs– (rh:-19) subtype. similar to the hrb– subtype, the absence of the ‘e’ antigen combined with the hrs– subtype indicates the true rare antigen type. this study had revealed a possible differentiation of the two hrb–/hrb– and hrs–/hrs– types due to the presence of specific single nucleotide polymorphisms in the genotype and in comparison to genotype patterns produced when historically known hrb– (rh:-34) samples are genotyped. this is reflected in table 2 where the r’s haplotype with the presence of the 733g and 1006t pointed to the rare hrb– type while the presence of homozygous cear or with the single nucleotide polymorphism 712g was an indication of the hrs– rare blood type. a total of 12 hrb– and six hrs– donors were reported in this study as these two rare types originated in south africa, hence found in more of our south african donor population.20,21 the rare negative high-frequency antigens k–, kp(b–), jk(b–), jo(a–), hy– and lu(b–) were found only in white donors and this is consistent with reports in europeans and caucasians from america and europe.28 the fy(a-b–) rare phenotype was not identified in the current study and may be attributed to the purposive sampling techniques used in the study. the gata mutation identified by the id core xt assay predicts fy(a–,b–); however, this is not the true rare phenotype, but rather an assay limitation. the genotype gypb*s, gypb*s or s– phenotype is currently not listed as a rare type in south africa as it is not difficult to locate; however, s– is listed on the rare donor files of spain18 and japan.23 although the s-s-u– phenotype predicted by the gypb*deletion is rare in caucasians and found mainly in african americans,17 it was found in one white and one black donor in the present study. this suggests the presence of this rare blood type among white south africans. interestingly, this study revealed many null alleles among the ss alleles of the mns blood group system, which was missed by serology. these are important as the null alleles were mainly associated with the presence of the u-variants. patients with u-variant phenotypes can only be given blood matched from u-variant donors or risk the formation of anti-u antibodies that will result in transfusion reactions. three red cell antigens, the jk(a-b–), co(a–) and ko, were not identified in the present study which could be explained by the small sample size. however, donors for these three antigens were previously active rare donors on the south african rare donor file but became lapsed donors having not donated blood in over 3 years.29 this highlights the need to increase stores of rare blood units in frozen storage so that patient-donor blood matching is not challenged by the lack of active blood donors. mass-scale cost-effective red cell genotyping can be used to screen donors routinely for this purpose in line with international practice as in the united states.30 the most important finding of this study was that among the mixed race group, 73% (8/11) of the donors showed the presence of rare antigens. thus, mass-scale rare donor screening among mixed race donors can increase the pool of donors with rare blood types. limitations a limiting factor of the id core xt assay is that several rare red cell antigens in south africa such as the rhnull, kn(a–), lan–, bombay oh, vel–, adult i–, ge–, inb–, rare abo subtypes and rhd/rhce hybrids are not covered by the assay. due to the small study sample size, genotypic findings must be confirmed in large-scale studies that include all south african ethnic groups that will be representative of the south african donor population. the study is the first comprehensive red cell genotyping study undertaken at the south african national blood service and for south africa. the extensive gene/antigen coverage of 10 blood groups systems in a single test translates to cost efficiencies, the possibility of high-throughput testing29 and easier detection of rare blood types present in more than one blood group. this study is also hypothesis-generating, and a few critical areas for future studies have been identified. conclusion this study has highlighted that random screening using molecular red cell genotyping increased the chances of finding rare donors by 15% (n = 14/92 random donors screened). it was further concluded that targeted screening using rh subtypes associated with race will increase the likelihood of identifying more rare blood donors. the competition between various commercial companies to provide faster, cost-effective genotyping kits with multiplex ability and high-throughput volumes has made it possible to introduce affordable mass-scale genotyping in south africa. this will also enable the creation of a substantial local south african red cell genotype database that can be expanded to include the rest of africa. acknowledgements we acknowledge the staff of the immunohaematology reference laboratory for the completion of red cell genotyping using the id core xt assay. we wish to express our gratitude to the supervisors of l.g.’s master’s thesis for their intellectual assistance. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions l.g. completed the study as part of her dissertation for her master’s in medical laboratory science completed in 2019 and is the main author of this manuscript. r.d.p., p.p. and u.j. supervised the findings of this work. all authors discussed the results and contributed to the final manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the authors confirm that the data supporting the findings of this study are available within the article. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references daniels g. human blood groups. john wiley & sons, hoboken, nj; 2008. suddock jt, crookston kp. transfusion reactions. instatpearls, treasure island, fl; 2019. daniels g, hadley a, soothill p. blood group antibodies in haemolytic disease of the fetus and newborn. alloimmune disorders of pregnancy. 2002;1:21–40. https://doi.org/10.1017/cbo9780511527043.004 urbaniak sj, greiss ma. rhd haemolytic disease of the fetus and the newborn. blood rev. 2000;14(1):44–61. https://doi.org/10.1054/blre.1999.0123 kutner j, mota m, conti f, et al. blood genotyping for improved outcomes in chronic transfusion patients: current and future perspectives. int j clin transfus med. 2014;2014(2):65–72. https://doi.org/10.2147/ijctm.s48394 wilkinson ds. clinical utility of genotyping human erythrocyte antigens. lab med. 2016;47(3):e28–e31. https://doi.org/10.1093/labmed/lmw014 nance st. global definitions of rare donors. isbt sci ser. 2013;8(1):23–27. https://doi.org/10.1111/voxs.12006 anstee dj. red cell genotyping and the future of pretransfusion testing. blood. 2009;114(2):248–256. https://doi.org/10.1182/blood-2008-11-146860 granier t, beley s, chiaroni j, et al. a comprehensive survey of both rhd and rhce allele frequencies in sub-saharan africa. transfusion. 2013:53(11pt2): 3009–3017. https://doi.org/10.1111/trf.12409 department of statistics south africa. community survey 2016, statistical release p0301, east london, eastern cape. (statssa.gov.za) [homepage on the internet]. statistical release report 2016. 2016 [cited 2020 feb 21]. available from: http://cs2016.statssa.gov.za/wp-content/uploads/2016/07/nt-30-06-2016-release-for-cs-2016-_statistical-releas_1-july-2016.pdf tishkoff sa, reed fa, friedlaender fr, et al. the genetic structure and history of africans and african americans. science [serial online]. 2009 [cited 2018 jul 19];324(5930):1035–1044. available from: http://science.sciencemag.org/ reid me, lomas-francis c, olsson ml. the blood group antigen factsbook. academic press, cambridge, ma; 2012. patin e, lopez m, grollemund r, et al. dispersals and genetic adaptation of bantu-speaking populations in africa and north america. science. 2017;356(6337):543–546. https://doi.org/10.1126/science.aal1988 ramerini m. dutch portuguese colonial history. marco ramerini, cape town; 1998. revelli n, villa ma, paccapelo c, et al. the lombardy rare donor programme. blood transfus. 2014;12(suppl 1):s249. luken js, danovic f, de haas m, et al. requests for red cells with rare blood types in the netherlands. immunohematology. 2016;32(2):51–52. https://doi.org/10.21307/immunohematology-2019-042 flickinger c. the american rare donor program. immunohematology. 2016;32(2) :71–73. https://doi.org/10.21307/immunohematology-2019-049 muñiz-diaz e, castro a, flores e, et al. the spanish program for rare blood donors. immunohematology. 2016;32(2):59–61. https://doi.org/10.21307/immunohematology-2019-046 hustinx h, lejon crottet s, scharberg ea. rare donor programs in switzerland, germany, and austria. immunohematology. 2016;32(2):63–66. https://doi.org/10.21307/immunohematology-2019-047 moores p. rh18 and hrs blood groups and antibodies. vox sanguinis. 1994;66(3):225–230. https://doi.org/10.1159/000462513 moores p, smart e. serology and genetics of the red blood cell factor rh34. vox sanguinis. 1991;61(2):122–129. https://doi.org/10.1159/000461336 poole j. the international rare donor panel. isbt sci ser. 2006;1(1):209. https://doi.org/10.1111/j.1751-2824.2006.00031.x tani y. rare donor program in japan. immunohematology. 2016;32(2):49–50. https://doi.org/10.21307/immunohematology-2019-041 kahar ma, patel rd. phenotype frequencies of blood group systems (rh, kell, kidd, duffy, mns, p, lewis, and lutheran) in blood donors of south gujarat, india. asian j transfus sci. 2014;8(1):51. https://doi.org/10.4103/0973-6247.126693 goldman m, st croix l. rare donor program: canadian blood services. immunohematology. 2016;32(1):15–16. https://doi.org/10.21307/immunohematology-2019-033 finning k, bhandari r, sellers f, et al. evaluation of red blood cell and platelet antigen genotyping platforms (id core xt/id hpa xt) in routine clinical practice. blood transfus. 2016;14(2):160. sharma dc, singhal s, rai s, et al. incidence of rh antigens, phenotype & probable genotype in the population of gwalior and chambal region, central india. int blood res rev. 2013;1(1):29–43. https://doi.org/10.9734/ibrr/2013/4616 avent nd, martinez a, flegel wa, et al. the bloodgen project of the european union, 2003–2009. transfus med hemother. 2009;36(3):162–167. https://doi.org/10.1159/000218192 smart e. south african rare donor panel. isbt sci ser. 2006;1(1):210–212. https://doi.org/10.1111/j.1751-2824.2006.00032.x noumsi gt, billingsley kl, mccaskill d, et al. effectiveness of high-throughput blood group genotyping on building a rare donor database. tranfusion. 2014;54:57a–57a. abstract introduction methods results discussion acknowledgements references about the author(s) samuel e. nnukwu department of medical laboratory science, faculty of allied medical sciences, university of calabar, calabar, cross river state, nigeria simon j. utsalo department of medical laboratory science, faculty of allied medical sciences, university of calabar, calabar, cross river state, nigeria olufunmilayo g. oyero institute for advanced medical research and training, college of medicine, university of ibadan, ibadan, oyo state, nigeria michel ntemgwa health products and food branch, health canada, ottawa, ontario, canada james a. ayukekbong section for clinical virology, redeem biomedical, buea, south west region, cameroon metabiota, nanaimo, british columbia, canada citation nnukwu se, utsalo sj, oyero og, ntemgwa m, ayukekbong ja. point-of-care diagnosis and risk factors of infantile, rotavirus-associated diarrhoea in calabar, nigeria. afr j lab med. 2017;6(1), a631. https://doi.org/10.4102/ajlm.v6i1.631 original research point-of-care diagnosis and risk factors of infantile, rotavirus-associated diarrhoea in calabar, nigeria samuel e. nnukwu, simon j. utsalo, olufunmilayo g. oyero, michel ntemgwa, james a. ayukekbong received: 08 apr. 2017; accepted: 08 sept. 2017; published: 08 dec. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: rotaviruses are the primary cause of acute gastroenteritis in young children worldwide and a significant proportion of these infections occur in africa. objectives: in the present study, we determined the prevalence and risk factors of rotavirus infection among children younger than age 5 years with or without diarrhoea in calabar, nigeria, using a rapid point-of-care test. methods: two hundred infants younger than age 5 years presenting with acute gastroenteritis and a control group of 200 infants without diarrhoea were tested for rotavirus. each stool sample was homogenised in an extraction buffer and the supernatant added into the sample well of the rida quick rotavirus test cassette and allowed to run for 5 minutes at room temperature. when both the control band and test band were visible on the test cassette a positive result was recorded, whereas when only the control band was visible a negative results was recorded. results: rotavirus was detected in 25 (12.5%) of children with diarrhoea and in no children without diarrhoea. our results demonstrated that children who were exclusively breast-fed by their mothers were not infected with rotavirus and that 92% of the infants infected with rotavirus experienced vomiting. conclusion: these data demonstrate that asymptomatic rotavirus infection is rare and that rotavirus is commonly detected in stool samples of children suffering from diarrhoea with concomitant vomiting. use of point-of-care rotavirus tests will enhance early diagnosis of rotavirus-associated diarrhoea and reduce irrational use of antibiotics. introduction diarrhoea is a major cause of infantile morbidity and mortality in developing countries and it is increasingly recognised as a disease of poverty.1 annually, more than 1 billion episodes of diarrhoea occur among children younger than age 5 years resulting in about 2.5 million deaths.1,2 more than 40% of global diarrhoea-associated deaths occur in africa;3,4 the risk of contracting diarrhoeal diseases has been suggested to be higher in developing countries compared to developed countries, due in part to unsafe water supplies, sub-optimal sanitation and unhygienic conditions.5 diarrhoea is a manifestation of intestinal dysfunction that causes frequent watery stool, resulting in loss of water, electrolytes and nutrients.6 the infectious cause of diarrhoea is multifactorial, with viruses, bacteria, protozoans or helminths often involved.7,8,9 in developing-country settings, microscopy (for the detection of protozoa and the ova of helminths) and bacteria culture are routinely performed to determine the cause of diarrhoea. virus investigation is often neglected. acute infantile diarrhoea has been shown to be commonly caused by viruses, notably group a rotaviruses, which are transmitted primarily via the faecal-oral route.10 the incubation period is usually between 2 and 6 days and symptoms may last for up to 5–7 days.11,12 rotavirus illness usually starts with an acute onset of fever and vomiting, followed by mild watery diarrhoea to frequently profuse diarrhoea that can result in severe dehydration, electrolyte imbalance and death.13,14 by age 5 years, nearly all children have been infected with rotavirus at least once,11 with severe infections occurring between age 6 months and 2 years, particularly in immunocompromised children.15,16 to date, there are two oral rotavirus vaccines: a pentavalent bovine human reassortment vaccine (rv5; rotateq, merck, new jersey, united states) and a monovalent (g1p8 attenuated human rotavirus vaccine rv1; rotarix, glaxosmithkline biologicals, belgium).15,17,18 however, there is limited access to routine vaccination, especially in rural communities, coupled with the fact that testing of rotavirus is often neglected in routine medical practice in nigeria. contrary to therapeutic guidelines, most diarrhoea cases are treated with antibiotics or anti-parasitic drugs and the contribution of rotavirus to diarrhoeal disease is widely neglected in routine clinical practice. globally, molecular diagnostic methods such as real-time polymerase chain reaction and enzyme-linked immunosorbent assay (elisa) have improved the detection of rotavirus over the years.19,20 however, the routine use of these assays in developing countries is limited due to the cost and skill required to perform the analysis.21 the rida quick rotavirus assay is a useful alternative to polymerase chain reaction and elisa.22 this assay is cheap (< $2 per test) and easy to perform, provides results within 5 min and does not require the use of sophisticated equipment or skilled training. compared to polymerase chain reaction, the specificity of this assay has also been evaluated to be 95% and the sensitivity to be 100%.22 the aim of this study was to determine the prevalence and risk factors of rotavirus infection among children younger than age 5 years with or without diarrhoea in calabar, nigeria, using a rapid point-of-care test. methods ethical considerations ethical clearance was obtained from the cross river state ministry of health ethics committee (rp/rec/2015/108) and participants (parents or guardians of children) provided written or oral inform consent. study design we conducted a cross-sectional study on the prevalence of rotavirus among children younger than age 5 years with diarrhoea and without diarrhoea in calabar, nigeria, from august to december 2015. sample size was calculated based on a retrospective estimate of the number of admissions due to diarrhoeal disease in children younger than age 5 years at the selected sites (calculated standard deviation was 25), with a 95% confidence interval and a 5% margin of error. two hundred infants younger than age 5 years presenting with acute gastroenteritis at four health establishments in calabar were tested. a control group of 200 infants without diarrhoea was also included. data on breastfeeding, hygiene practices, daycare/nursery school enrolment, drinking water sources were collected through questionnaires by trained personnel in a designated private area within the hospitals. sample collection, processing and test procedure two hundred stool samples were collected by trained health workers in sterile, leak-proof containers from infants younger than age 5 years with acute diarrhoea at the sentinel hospitals. in parallel, 200 stool specimens were collected from a control group of infants younger than age 5 years without diarrhoea. these controls were matched for age, gender and/or enrolment location. about 50 mg of stool sample was collected from the stool container using a sterile applicator and transferred into a sample tube containing 1 ml of extraction buffer and mixed gently to make a homogeneous mixture. about 2 to 3 drops of the supernatant from the homogeneous were was added into the sample well of the rida quick rotavirus combi test cassette (r-biopharm ag, darmstadt, germany) and allowed to run for 5 min at room temperature. when both the control band and test band were visible on the test cassette a positive result was recorded, whereas when only the control band was visible a negative result was recorded (figure 1). figure 1: rida quick rotavirus combi test cassette (r-biopharm ag, darmstadt, germany) showing negative (a) and positive (b) results for rotavirus. statistical comparison a two-sided fisher’s test was used to compare proportions with alpha set at 0.05. odd ratios and 95% confidence intervals were calculated using the spss software package v. 17.0 for mac (spss inc., chicago, illinois, united states). results demographic characteristics of participants amongst the 200 cases, 56.5% (113/200) were male and 43.5% (87/200) female (table 1). children younger than age 2 years constituted 61% of the study population, and the overall mean age was 22 months (range: 3–44 months). in the control group of 200 children younger than age 5 years, the mean age was 25 months (range: 3–47 months). male children constituted 55% (110/200) of the control group. table 1: demographic characteristics and rotavirus prevalence among children with and without diarrhoea in calabar, nigeria, august to december 2015. rotavirus detection by rida quick combi immunochromatographic test rotavirus was detected in 25/200 (12.5%) of the children with acute gastroenteritis, with no difference between male and female patients (15/113, 13.3% vs 10/87, 11.5% p = 0.1) (table 1). however, the prevalence was higher among children between ages 7 and 12 months (13/43; 30.2%) compared with other age groups (table 1). a total of 23 of the 25 children (92%) diagnosed with rotavirus infection reported vomiting (data not shown). in terms of study centre, both the university of calabar teaching hospital and the general hospital had a high prevalence of rotavirus infection compared with the abasi and ansa primary healthcare centres. meanwhile, rotavirus was not detected in any of the 200 control children without diarrhoea for the same age ranges. risk factors for rotavirus infection in order to investigate the relationship between feeding method of children and the occurrence of rotavirus, we compared the prevalence of rotavirus infection among children who were exclusively breastfed to those who were not exclusively breastfed (that is, children in this category received breast milk plus solid food or solid food only). the results showed that no rotavirus infection was detected among children who were exclusively breastfed, whereas a 12.9% rotavirus prevalence was observed in children who were not (table 2). there was also a statistically significant difference in the prevalence of rotavirus among children who attended nursery schools (21/99, 21.4%) compared with those who were not enrolled in any institution (4/101, 3.9%; p = 0.0001). we also investigated the relationship between hand washing before meals and the source of drinking water and the risk of rotavirus infection in children younger than age 5 years. there was no statistical difference in rotavirus prevalence among children who washed hands either independently or with the help of their parents or guardian before meals (20/161, 12.4%) compared with those who did not (5/39, 12.8%; p = 0.5). there was no statistically significant difference in rotavirus prevalence based on the source of drinking water. table 2: risk factors of rotavirus infection among children younger than age 5 years in calabar, nigeria, august to december 2015. discussion in this cross-sectional study, a rapid immunochromatographic point-of-care rotavirus detection assay was used to investigate the prevalence and risk factors of rotavirus infection among children younger than age 5 years in south-eastern nigeria. the prevalence of 12.5% for rotavirus among children with diarrhoea in this study is consistent with that of another study in nigeria where a prevalence of 13.8% was observed.23 this study also revealed that there was no statistically significant difference in the prevalence of rotavirus infection between male children and female children which is consistent with a previous report.23 a striking finding was the fact that no child who was exclusively breastfed tested positive for rotavirus. this corroborates evidence that breast milk may offer specific protection against rotavirus by the ‘decoy’ action of human milk glycans.24,25 this finding is compatible with the work of quigley et al.,26 who stated that breastfed babies are four times less likely to experience diarrhoea associated with rotavirus than bottle-fed babies. also, the highest prevalence of rotavirus infection was observed in children between age 7 and 12 months, a period that coincides with the weaning period from breastfeeding of most infants. this further corroborates the protective effect of breast milk on rotavirus infection. this observation is also consistent with studies that suggest that most severe rotavirus infections occur between ages 6 months and 2 years.27,28 we observed that the act of washing hands before meals did not confer any protection against rotavirus infection in the children studied. also, children who attended day care were more prone to rotavirus infection. this is consistent with previous findings where outbreaks of rotavirus infection have been more commonly reported from day care centres and nursery schools.27 overcrowding in most day care and nursery schools may facilitate transmission of rotavirus infection among children. there was no difference in rotavirus infection among children who consumed different sources of water, such as tap water, borehole or well water, and bottled water. this finding is in contrast with our previous report where the risk of rotavirus infection was higher among consumers of water from boreholes or wells compared with consumers of tap water.19 the fact that rotavirus was not detected in children without diarrhoea suggests that asymptomatic rotavirus infection is rare. also, 92% of children with rotavirus infection suffered from vomiting. this finding is consistent with previous scientific and clinical observations that diarrhoea and vomiting are the hallmarks of rotavirus infection.13,29 although rotavirus was more prevalent in hospital settings compared with primary health centres, the results were not statistically significant. a plausible explanation is that due to the lack of local capacity in most primary health centres, serious health issues are referred to hospitals for proper management. limitations this study, although carefully conducted, was subject to some limitations. perhaps the most compelling is that the study was limited to a small population in calabar and thus may not reflect the general population. secondly, because of lack of capacity, the investigators could not genotype the 25 rotavirus-positive samples, which could have conveyed further interesting findings on the epidemiology of the infection in the region. thirdly, other diarrhoea-causing pathogens were not investigated; therefore, the cause of diarrhoeal episodes cannot solely be linked to rotavirus infection alone. despite these limitations, the study is the first of its kind in south-eastern nigeria and suggests that asymptomatic rotavirus infection in children younger than age 5 years is rare. the study also provides additional knowledge on the risk factors of rotavirus infection and corroborates findings that exclusive breastfeeding may confer a protective advantage to infants under age 6 months against rotavirus infection. it also confirms the fact that day care and nursery school settings predispose children to rotavirus infection, as they are likely to be infected by other children. conclusion taken together, this study suggests that routine use of a rapid immunochromatographic test may enhance early detection of rotavirus and guide clinical management. this will ultimately reduce the burden of the disease in children and prevent the irrational prescription of antibiotics. acknowledgements the authors gratefully acknowledge all study participants enrolled in this study for their great cooperation and patience during the long examination days. we also express gratitude to the staff of university of calabar teaching hospital, ekpo abasi and ikot ansa primary healthcare centres for their continuous support, without whom this study could not have been completed. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect those of health canada or metabiota inc. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support none. authors’ contributions j.a.a. drafted the manuscript, which was reviewed and 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rotavirus and serotonin cross-talk in diarrhea. plos one. 2016;11:e0159660. https://doi.org/10.1371/journal.pone.0159660 bucardo f, mercado j, reyes y, gonzález f, balmaseda a, nordgren j. large increase of rotavirus diarrhoea in the hospital setting associated with emergence of g12 genotype in a highly vaccinated population in nicaragua. clin microbiol infect. 2015;21:603.e1–7. https://doi.org/10.1016/j.cmi.2015.01.022 gosselin v, généreux m, gagneur a, petit g. effectiveness of rotavirus vaccine in preventing severe gastroenteritis in young children according to socioeconomic status. hum vacc immunother. 2016;12:2572–2579. https://doi.org/10.1080/21645515.2016.1189038 thomas pd, pollok rc, gazzard bg. enteric viral infections as a cause of diarrhoea in the acquired immunodeficiency syndrome. hiv med. 1999;1:19–24. https://doi.org/10.1046/j.1468-1293.1999.00004.x patel m, shane al, parashar ud, jiang b, gentsch jr, glass ri. oral rotavirus vaccines: how well will they work where they are needed most? j infect dis. 2009;200 suppl 1:s39–s48. https://doi.org/10.1086/605035 patel mm, parashar ud. assessing the effectiveness and public health impact of rotavirus vaccines after introduction in immunization programs. j infect dis. 2009;200 suppl 1:s291–s299. https://doi.org/10.1086/605059 ayukekbong ja, andersson me, vansarla g, et al. monitoring of seasonality of norovirus and other enteric viruses in cameroon by real-time pcr: an exploratory study. epidemiol infect. 2014;142:1393–1402. https://doi.org/10.1017/s095026881300232x nordgren j, bucardo f, svensson l, lindgren p-e. novel light-upon-extension real-time pcr assay for simultaneous detection, quantification, and genogrouping of group a rotavirus. j clin microbiol. 2010;48:1859–1865. https://doi.org/10.1128/jcm.02288-09 parashar ud, nelson eas, kang g. diagnosis, management, and prevention of rotavirus gastroenteritis in children. br med j. 2013;347:f7204. https://doi.org/10.1136/bmj.f7204 weitzel t, reither k, mockenhaupt fp, et al. field evaluation of a rotaand adenovirus immunochromatographic assay using stool samples from children with acute diarrhea in ghana. j clin microbiol. 2007;45:2695–2697. https://doi.org/10.1128/jcm.00562-07 junaid sa, umeh c, olabode ao, banda jm. incidence of rotavirus infection in children with gastroenteritis attending jos university teaching hospital, nigeria. virol j. 2011;8:233. https://doi.org/10.1186/1743-422x-8-233 bode l. human milk oligosaccharides: every baby needs a sugar mama. glycobiology. 2012;22:1147–1162. https://doi.org/10.1093/glycob/cws074 yu y, lasanajak y, song x, et al. human milk contains novel glycans that are potential decoy receptors for neonatal rotaviruses. mol cell proteomics. 2014;13:2944–2960. https://doi.org/10.1074/mcp.m114.039875 quigley ma, cumberland p, cowden jm, rodrigues lc. how protective is breast feeding against diarrhoeal disease in infants in 1990s england? a case-control study. arch dis children. 2006;91:245–250. https://doi.org/10.1136/adc.2005.074260 ford-jones el, wang e, petric m, corey p, moineddin r, fearon m. rotavirus-associated diarrhea in outpatient settings and child care centers. the greater toronto area/peel region presi study group. pediatric rotavirus epidemiology study for immunization. arch pediatr adolesc med. 2000;154:586–593. https://doi.org/10.1001/archpedi.154.6.586 ramani s, kang g. burden of disease & molecular epidemiology of group a rotavirus infections in india. indian j med res. 2007;125:619–632. hagbom m, istrate c, engblom d, et al. rotavirus stimulates release of serotonin (5-ht) from human enterochromaffin cells and activates brain structures involved in nausea and vomiting. plos pathogen. 2011;7:e1002115. https://doi.org/10.1371/journal.ppat.1002115 abstract introduction methods results discussion acknowledgements references about the author(s) seth twum west african centre for cell biology of infectious pathogens, accra, ghana department of biochemistry, cell and molecular biology, university of ghana, accra, ghana kwadwo fosu west african centre for cell biology of infectious pathogens, accra, ghana department of biochemistry, cell and molecular biology, university of ghana, accra, ghana robin a. felder department of pathology, the university of virginia, charlottesville, virginia, united states kwabena a.n. sarpong west african centre for cell biology of infectious pathogens, accra, ghana department of biochemistry, cell and molecular biology, university of ghana, accra, ghana citation twum s, fosu k, felder ra, sarpong kan. bridging the gaps in newborn screening programmes: challenges and opportunities to detect haemoglobinopathies in africa. afr j lab med. 2023;12(1), a2225. https://doi.org/10.4102/ajlm.v12i1.2225 review article bridging the gaps in newborn screening programmes: challenges and opportunities to detect haemoglobinopathies in africa seth twum, kwadwo fosu, robin a. felder, kwabena a.n. sarpong received: 25 may 2023; accepted: 27 sept. 2023; published: 14 dec. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: haemoglobinopathies, including sickle cell disease and β-thalassaemia, are monogenic disorders with a relatively higher prevalence among malaria-endemic areas in africa. despite this prevalence, most african countries lack the necessary resources for diagnosing and managing these debilitating conditions. aim: this study provides a critical review of newborn screening for detecting haemoglobinopathies in africa, highlighting challenges and proposing strategies for improved diagnosis and management. methods: a literature search on haemoglobinopathies in africa was conducted in pubmed, google scholar and sciencedirect, using specific keywords and boolean operators, including articles published from january 1981 to december 2022. results: the data show that sickle cell disease is prevalent among populations in central and west africa; however, β-thalassaemia is prevalent among people in the northern parts of africa. newborn screening pilot initiatives for haemoglobinopathies were being implemented in angola, nigeria, ghana, the democratic republic of congo and the republic of benin. the cost of testing, lack of sufficient and accessible medical records, and inadequacy in healthcare infrastructure pose significant challenges in bridging the gaps in newborn screening. furthermore, the stigmatisation and lack of awareness of haemoglobinopathies and access to newborn screening programmes pose additional challenges. conclusion: this review highlights the challenges associated with haemoglobinopathy testing, effective strategies for mitigating these challenges, and future perspectives for expanding efforts toward detecting and managing these disorders across africa. providing affordable diagnostic tools, mobile clinics, government subsidies, education campaigns, and the implementation of electronic medical records systems could help bridge the gaps in newborn screening in africa. what this study adds: the study presents a comprehensive view of newborn screening of haemoglobinopathies in africa, provides a detailed outline of the challenges faced by newborn screening for haemoglobinopathies in africa, and offers strategies for better diagnosis and care. keywords: haemoglobinopathies; sickle cell disease; newborn screening; africa; thalassaemia. introduction haemoglobinopathies such as sickle cell disease (scd) and β-thalassaemia are global health monogenic disorders with a significantly higher prevalence among people in malaria-endemic areas owing to natural selection.1 about 70% of the recorded global haemoglobinopathy cases are in africa. additionally, africa has the highest under-five mortality for haemoglobin disorders among affected children.2 these haemoglobinopathies are inherited autosomal recessive haemoglobin abnormalities. clinically significant haemoglobinopathies include αand β-thalassemia, scd, haemoglobin e and haemoglobin c.3 in sub-saharan african populations, scd is prevalent (10.68%) and is caused by abnormal haemoglobin resulting from the substitution of valine for glutamic acid at position six of the β-globin chain.4 at low oxygen tension, sickle blood (hbs) polymerises easily, affecting red blood cell structure, function and life span.5 heterozygous hbs individuals are generally asymptomatic. in contrast, homozygous hbs individuals show clinical symptoms of scd.6 since the development of hbs newborn screening (nbs) programmes, it has become easier to identify babies with haemoglobin abnormalities at birth or in the first few days of life, before they exhibit clinical signs.7 according to the world health organization, haemoglobinopathy screening aims to identify carriers to estimate the risk of birthing children with haemoglobinopathies, followed by genetic counselling and offering options to avoid such births. in high-income countries, patients are engaged in programmes that provide strategies for complete care, resulting in better outcomes than in poorly resourced countries where comprehensive nbs programmes are not implemented. in 2019, africa recorded 38 403 scd deaths, accounting for, approximately, a 26% increase in scd mortality in children compared to 2000.8 without appropriate interventions, it is estimated that 90% of people suffering from scd in africa might not survive beyond 18 years of age.9,10,11 early nbs for scd allows for adopting a complete care strategy that includes preventative therapy, parental education, and the start of a tracking and follow-up programme for diagnosed individuals.12 sickle cell diseases are among the most important illnesses because of their prevalence and effects in sub-saharan africa, making this group of disorders the most vital to test for nbs in a significant portion of the continent,13 yet resources have not been made available by governments in sub-saharan africa and other high-prevalence countries.14 haemoglobinopathy diagnosis is achieved using the sickling test as a screening modality, followed by haemoglobin electrophoresis. isoelectric focusing and high-performance liquid chromatography are considered gold standards for sickle cell diagnosis as they differentiate between normal sickle cell trait and scd.15 other diagnostic tests, such as multiplex ligation probe amplification and gap polymerase chain reaction, are also offered at selected facilities. the development of point-of-care testing for scd15,16 is very beneficial, especially in locations with limited capabilities for central laboratory testing or where the transfer of blood samples to a centralised laboratory is not practical or convenient. the prevalence of haemoglobinopathy, genetic variability and cost efficiency are the primary variables influencing the choice of a specific haemoglobinopathy screening technique. mass screening programmes targeting people at various phases of life can be implemented where prevalence is high.17 however, testing for haemoglobin disorders in africa is associated with numerous challenges that require attention and practical solutions. this article examines interventions related to diagnosing haemoglobinopathies in africa, including various diagnostic methodologies. it highlights valuable lessons from past experiences, focusing on the challenges associated with haemoglobinopathy testing and effective strategies for mitigating them. additionally, the article provides future perspectives for expanding efforts toward detecting and managing these disorders across africa. methods literature search and data sources the literature search was carried out through pubmed® (https://pubmed.ncbi.nlm.nih.gov/), google scholar (https://scholar.google.com/), and sciencedirect (https://www.sciencedirect.com/), employing a combination of boolean operators (and, or) and predetermined keywords. our search terms were ‘hemoglobinopathies and africa or blood disorders and africa’, ‘sickle cell disease and africa’, and ‘thalassemia disease and africa’. to ensure the relevance of our findings, we restricted the search to publications between january 1981 and december 2022. the titles of the articles were selected based on the following eligibility criteria: (1) the study location must be within africa, and (2) the study must have reported the detection of sickle cell or thalassaemia disease or both. for an article to be considered, it must have reported the number of newborns screened, the number of haemoglobinopathies detected, and the testing method employed to identify the specific haemoglobinopathy. studies without a direct focus on haemoglobinopathies in africa and studies that did not provide information on the method used for screening were excluded (figure 1). research articles and systematic reviews with metadata that suited the inclusion criteria were retrieved. in addition, we conducted supplementary validation by thoroughly reviewing the references cited in the eligible articles. figure 1: flow chart of manuscripts reporting on haemoglobinopathies in africa. electronic literature search between 1981 and 2022 was performed using specific keywords, inclusion and exclusion criteria to identify manuscripts that have published on haemoglobinopathies in africa. data extraction, analysis, and visualisation data were extracted into a microsoft office excel 2016 (microsoft corporation, redmond, washington, united states) spreadsheet. for each eligible study, data extracted included study location or geographical zone, publication year, screening period, newborns screened, haemoglobinopathies tested, and the testing method used. the percentage prevalence of sickle cell and thalassaemia diseases reported for each study was calculated by dividing the number of haemoglobinopathies detected by the total number of newborns screened for haemoglobinopathies, multiplied by 100. packages found in r-studio (posit, public-benefit corporation, vienna, austria), such as ggplot2 and tidyverse, were used to generate the geographical zones with colour codes, using the calculated prevalence. results intervention: haemoglobinopathies tested in africa according to the world health organization, in 2010, defective haemoglobin genes affected around 5% of the world’s population; more than 300 000 infants are born with clinically severe haemoglobin abnormalities yearly. research shows these figures will increase from 305 800 in 2010 to 404 200 in 2050.11 although about 80% of affected children are born in underdeveloped countries, 83% are born with scd, with the remainder having thalassaemia syndromes.16 about 50% to 80% of children with scd and more than 50 000 children with major thalassaemia die in lowand middle-income countries.11 fortunately, nbs with comprehensive care for scd has been found to reduce early child mortality.17 as of 2023, no african country had succeeded in making scd a universal national health intervention.18,19,20 however, pilot screening programmes have been conducted by the member countries of the consortium on newborn screening in africa, such as kenya, ghana, liberia, nigeria, tanzania, zambia and uganda, and by other non-consortium members, such as benin, burkina faso, democratic republic of congo, gabon and mali. the data analysis from nbs programmes in africa shows that scd is prevalent among populations in central and west africa (figure 2a). more scd prevalence exists in individuals residing in the democratic republic of congo. conversely, β-thalassemia is prevalent among people in the northern parts of africa (figure 2b), with algeria and morocco having the highest prevalence.21 in algeria and morocco, ~75% are caucasians of arabic origin, while the congolese are multi-ethnic, with a significant percentage of black people. it is well documented that arab countries, such as morocco, have consanguinity of up to 30% and contribute significantly to the increased prevalence of inherited metabolic errors.22 conversely, while scd affects approximately 1 in 365 live births among africans, the sickle cell trait is found in about 1 in 13 africans.23 apart from tunisia, where the prevalence of scd is 9 in 10 000, the incidence of scd is high in the western and central parts of africa. figure 2: prevalence of haemoglobinopathies in africa (a) sickle cell disease (b) αand β-thalassaemia, 1981–2022. although the lack of testing and the limited literature have contributed to the scarcity of data on haemoglobinopathy testing in africa, some countries have been making massive efforts to pilot robust nbs programmes to identify a selected number of these conditions. our literature search made it increasingly apparent that obtaining data for haemoglobinopathy testing and disease prevalence for more than 50% of african countries became difficult. from 2014 to 2019, uganda screened more than 220 000 newborns in africa (table 1) and is one of the african countries involved in mass screening through pilot programmes. in addition, other countries such as ghana and angola participate in mass testing via pilot programmes (table 1). isoelectric focusing has been africa’s most widely used diagnostic haemoglobinopathy technique due to its higher resolution, low cost, and effortless performance.24 table 1: haemoglobinopathies tested in africa and the test method obtained from electronic literature search between 1981 and 2022. a study was conducted in the northern part of algeria and α-thalassemia was prevalent.25 in morocco, a study was conducted in three of the northern provinces, and α-thalassemia was also prevalent.38 for the republic of congo, a study was conducted in 12 departments in the entire national territory of congo, and scd was prevalent.26 in malawi, a study was conducted in the central region of malawi, and α-thalassemia was prevalent.37 in sierra leone, a study was conducted on selected participants from the rural and urban areas, and β-thalassaemia was prevalent.39 discussion challenges and mitigation strategies for haemoglobinopathy testing testing for haemoglobinopathies is essential for early diagnosis and appropriate treatment. however, several challenges and limitations are associated with haemoglobinopathy testing in africa. here, we discuss the critical challenges with haemoglobinopathy testing in africa and strategies to mitigate these challenges. point-of-care technologies many african countries lack the necessary resources, equipment and personnel with advanced training to perform testing and accurately interpret test results. most african countries may not have financial or technical support to establish and maintain a robust laboratory infrastructure for haemoglobinopathy testing.41 a potential solution to this challenge is using affordable diagnostic tools, such as rapid diagnostic tests, that are simple and easy to use.42 sickle cell analysis (biomedomics incorporated, morrisville, north carolina, united states),43 lateral flow immunoassays,44 and hemotype sc (silver lake research corporation, azusa, california, united states)45,46 are affordable, user-friendly, quick diagnostic tests that can successfully be used in resource-limited settings. investing in affordable point-of-care diagnostic tools can help improve the diagnosis and management of haemoglobinopathies in africa. mobile clinics in africa, a significant portion of the population resides in areas that are geographically distant from testing facilities,47 making it difficult to access testing centres or clinics that diagnose haemoglobinopathies. as a result, they may not have access to essential health services, including diagnostic testing, which can lead to increased health risks and delays in treatment. mobile clinics could solve this challenge by directly bringing healthcare services to underserved communities, including remote areas. using mobile clinics and community-trained laboratory personnel, healthcare providers can improve access to essential health services for people living in remote areas. furthermore, mobile clinics can meet the specific healthcare needs of different communities, and they can offer a wide range of services beyond diagnostic testing, including vaccination, health education, and counselling. additionally, africans’ rapid internal and external migration has made it difficult to estimate and control newborn haemoglobinopathies accurately. rapid migration changes epidemiology since people from high-prevalence areas move to low-prevalence areas.45 migrations without universal electronic health records management make it difficult to follow up with parents with these haemoglobinopathies and their newborns. specifically, there is a high-prevalence of hbs in africa and haemoglobin c in parts of west africa.46 without proper measures, heterozygous haemoglobin c may spread due to migration if scd is not controlled, increasing the likelihood of more hbs cases in africa. through an influx of migrants from west and central africa, internal migration in africa has caused scd, a previously rare disease, to be introduced in south africa.4 government subsidies the cost of testing and treating various medical conditions in africa can be prohibitively expensive for many low-income patients.48 this financial burden can prevent people from seeking the care they need, leading to delayed diagnoses and life-threatening consequences. governments in africa have a role to play in addressing this problem. by creating programmes subsidising the cost of testing and treatment for low-income individuals, they can ensure everyone has access to quality healthcare regardless of their ability to pay. these programmes could take various forms, including public health insurance schemes, direct subsidies for medical expenses, or partnerships with private healthcare providers to offer discounted rates for low-income patients. in addition to making care more affordable, these initiatives could also help to improve the overall quality and availability of healthcare services across the continent. by prioritising access to healthcare for all, african governments can help address scd, one of the most pressing challenges facing the continent today. awareness campaigns despite the prevalence of haemoglobinopathies, many africans are unaware of these disorders and their potential impact on health.48 one consequence of this lack of awareness is a low demand for testing, which can result in delayed diagnosis and treatment. also, access to newborn babies within days of birth or non-newborns is challenging in africa because women living in rural areas underutilise formal and evidence-based maternal health delivery services.49,50 awareness campaigns through various traditional and digital media platforms can help address these issues. traditional media platforms such as television, radio, and print media can reach a broad audience, especially in rural areas with limited access to the internet. these campaigns can educate people about the symptoms of haemoglobinopathies, the importance of testing, and the available treatment options. in addition, digital media platforms, such as social networks and mobile applications, can also be used to raise awareness.19 creating awareness of haemoglobinopathies is crucial for ensuring that people affected by these disorders receive adequate and timely medical care. targeted educational programmes some african communities stigmatise individuals with haemoglobinopathies due to cultural beliefs and misconceptions.51 this stigmatisation often arises from a lack of understanding of the genetic and biological basis of haemoglobinopathies, which can lead to harmful and discriminatory attitudes toward individuals with the condition.52 some communities view haemoglobinopathies as a result of witchcraft, curses, or other supernatural causes, leading to fear, ostracism and discrimination.53 education campaigns can address these beliefs and reduce stigmatisation. by raising awareness of the genetic and biological causes of haemoglobinopathies and the impact of stigmatisation on individuals and communities, education campaigns can help dispel myths and misconceptions surrounding the condition.54 education campaigns can also promote acceptance and support for individuals with haemoglobinopathies, encouraging communities to recognise and value the contributions of all members.55 by challenging stereotypes and promoting inclusion, education campaigns can help to create more inclusive and supportive communities that prioritise the health and well-being of all members, regardless of their health status. secure but accessible electronic medical record systems furthermore, in most african countries, medical records are not easily accessible, making it difficult to track the prevalence of haemoglobinopathies and monitor a patient health outcomes.50 implementing electronic medical records systems is a potential way to overcome this challenge. these electronic medical records are digital versions of patients’ medical records that healthcare providers can access and update in real time. electronic medical records can help healthcare providers collect and store patient health information, including diagnostic test results, medical histories, and treatment plans. electronic medical records can help to improve the accuracy and completeness of patients’ medical records and facilitate communication between healthcare providers, which is particularly important for patients with chronic conditions, such as haemoglobinopathies. they can also help to improve patient outcomes by enabling healthcare providers to monitor patient’s health over time, tracking medication adherence, and identifying potential complications or drug interactions. accessible electronic medical records can lead to earlier interventions and more personalised care, ultimately improving patients’ quality of life and reducing the burden of haemoglobinopathies on healthcare systems. standardised quality control material additionally, the quality of testing for haemoglobinopathies in africa is a major concern due to poor quality assurance practices. quality assurance is crucial in haemoglobinopathy testing to avoid misdiagnosis, which can have severe consequences for patients, including incorrect treatment and poor health outcomes. unfortunately, many testing facilities in africa lack quality assurance programmes or have inadequate ones, which can lead to errors in testing and misdiagnosis.56 quality assurance programmes not only need specimens with known values to measure along with patient specimens but also involve regular checks of equipment, reagents, and personnel performance, as well as ongoing training and education of staff on quality assurance practices. quality assurance includes standardisation and harmonisation in testing protocols between laboratories and between countries.19 perspectives for expanding the determination of haemoglobinopathies in africa in africa, interventions to control haemoglobinopathies focus on using primary healthcare and health promotion strategies to ensure policy formulation and implementation, legislation and regulation, and improving primary and secondary prevention measures. one of the main strategies is to partner with local health organisations.19 partnerships with local health organisations involve working with hospitals, clinics, community health centres, and other organisations to develop educational materials and outreach programmes to raise awareness about haemoglobinopathies and their impact on health. national programmes for haemoglobinopathies, especially scd and thalassaemia disorders, must be established and strengthened to prevent and control non-communicable diseases and should work together with national programmes for maternal and child health.57 key focus areas include advocacy, prevention and counselling, early detection and management, data collection and monitoring, research, community education, and collaboration. an integrated, multi-sectoral, and multi-disciplinary team of healthcare and social workers, teachers, parents, and concerned non-governmental organisations could address these areas. this team would focus on practical aspects of care delivery and programme implementation and monitoring. additionally, establishing genetic counselling and testing measures is essential.58 through genetic counselling, carriers of haemoglobinopathies can learn the risks of passing the disease on to their children, and receive guidance on family planning and reproduction options; this can help prevent the transmission of haemoglobinopathies through informed decision-making by families. widespread community participation and support are required to ensure individuals have access to these services and promote awareness and education about haemoglobinopathies. implementing community-based actions, such as surveillance and supervision, monitoring at all levels of operation, and periodic national review, will ensure that accurate and timely data is collected and used to guide decision-making.57 community-based surveillance and supervision can involve training community health workers to identify and report cases of haemoglobinopathies and providing education and support to affected individuals and families. monitoring at all operation levels, from the local to the national level, can help ensure that data are collected consistently and accurately and that appropriate data-backed interventions are implemented. periodic national reviews can identify haemoglobinopathy prevalence and incidence trends and surveillance system gaps. these data can then inform policy making and day-to-day programme management decision-making, such as allocating resources for prevention and treatment programmes, developing guidelines for screening and diagnosis, and providing training and support for healthcare providers. promoting pilot programmes is another strategy for expanding haemoglobinopathy detection in africa. newborn screening programme pilot initiatives for haemoglobinopathies are being implemented in angola,27 nigeria,59 ghana,60 the democratic republic of congo,26,61 and the republic of benin.30 however, many african countries do not have universal national nbs programmes for scd and other haemoglobinopathies. summary haemoglobinopathies, such as scd and β-thalassemia, are genetic disorders caused by abnormal haemoglobin affecting red blood cells. in africa, several challenges limit the testing for these disorders, including a lack of resources, limited access to testing facilities, migration, financial constraints, lack of awareness, and stigma. to improve access to quality healthcare services and raise awareness of african haemoglobinopathies, we propose using affordable diagnostic tools, mobile clinics, government subsidies, education campaigns, and establishing records or registries. the future may see these proposed strategies implemented to improve healthcare services for african haemoglobinopathies. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions this work was conceptualised by k.a.n.s., s.t., k.f. and r.a.f. analysis was done by k.a.n.s. and s.t. the original draft was written by k.f., k.a.n.s., s.t. and r.a.f. editing and reviews were done by r.a.f., k.f. and k.a.n.s. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support this work was supported by funds from a world bank african centres of excellence grant (waccbip+ncds: awandare) and a developing excellence in leadership, training, and science (deltas) africa grant (del-15-007: awandare). data availability data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy of any affiliated agency of the authors. references mulumba ll, wilson l. sickle cell disease among children in africa: an integrative literature review and global recommendations. int j afr nurs sci. 2015;3:56–64. https://doi.org/10.1016/j.ijans.2015.08.002 fattoum s. evolution of hemoglobinopathy prevention in africa: results, problems and prospect. mediterr j hematol infect dis. 2009;1(1):e2009005. https://doi.org/10.4084/mjhid.2009.005 murray cjl, vos t, lozano r, et al. disability-adjusted life years (dalys) for 291 diseases and injuries in 21 regions, 1990–2010: a systematic 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[cited 2023 aug 23]. available from: https://www.afro.who.int/news/african-health-ministers-launch-drive-curb-sickle-cell-disease-toll wonkam a, makani j. sickle cell disease in africa: an urgent need for longitudinal cohort studies. lancet glob health. 2019;7(10):e1310–e1311. https://doi.org/10.1016/s2214-109x(19)30364-x tshilolo l, kafando e, sawadogo m, et al. neonatal screening and clinical care programmes for sickle cell disorders in sub-saharan africa: lessons from pilot studies. public health. 2008;122(9):933–941. https://doi.org/10.1016/j.puhe.2007.12.005 ranque b, kitenge r, doucoure ndiaye d, et al. estimating the risk of infant and juvenile mortality attributable to sickle cell anemia in sub-saharan africa: a case control study. 2022;9(3):e208–e216. https://doi.org/10.1016/s2352-3026(22)00004-7 grosse sd, odame i, atrash hk, amendah dd, piel fb, williams tn. sickle cell disease in africa: a neglected cause of early childhood mortality. am j prev med. 2011;41(6 suppl.4):s398–s405. https://doi.org/10.1016/j.amepre.2011.09.013 world health organization. sickle-cell disease: a strategy for the who african region report of the regional director. geneva: who; 2010. weatherall dj. the challenge of haemoglobinopathies in resource-poor countries. br j haematol. 2011;154(6):736–744. https://doi.org/10.1111/j.1365-2141.2011.08742.x kanter j. point-of-care testing in sickle cell disease. in: inusa bpd, editor. sickle cell disease – pain and common chronic complications. london: intech; 2016. available from: http://www.intechopen.com/books/5318 bond m, hunt b, flynn b, huhtinen p, ware r, richards-kortum r. towards a point-of-care strip test to diagnose sickle cell anemia. plos one. 2017;12(5):e0177732. https://doi.org/10.1371/journal.pone.0177732 goonasekera hw, paththinige cs, dissanayake vhw. population screening for hemoglobinopathies. annu rev genom hum genet. 2018;19:355–380. https://doi.org/10.1146/annurev-genom-091416-035451 hsu l, nnodu oe, brown bj, et al. white paper: pathways to progress in newborn screening for sickle cell disease in sub-saharan africa. j trop dis public health [serial online]. 2018;6(2). available from: https://pubmed.ncbi.nlm.nih.gov/30505949/ paintsil v, amuzu ex, nyanor i, et al. establishing a sickle cell disease registry in africa: experience from the sickle pan-african research consortium, kumasi-ghana. front genet. 2022;13:802355. https://doi.org/10.3389/fgene.2022.802355 archer nm, inusa b, makani j, et al. enablers and barriers to newborn screening for sickle cell disease in africa: results from a qualitative study involving programmes in six countries. bmj open. 2022;12(3):e057623. https://doi.org/10.1136/bmjopen-2021-057623 kamal sm, fouad am. hepatitis c infection in patients with hemoglobinopathies. in: kamal s, editor., hepatitis c in developing countries. london: elsevier. 2018; p. 177–190. anwar wa, khyatti m, hemminki k. consanguinity and genetic diseases in north africa and immigrants to europe. eur j public health. 2014;24(suppl_1):57–63. https://doi.org/10.1093/eurpub/cku104 us centers for disease control and prevention. data & statistics on sickle cell disease [homepage on the internet]. cdc; [cited 2023 sep 01]. available from: https://www.cdc.gov/ncbddd/sicklecell/data.html arishi wa, al-hadrami ha, zourob m. techniques for the detection of sickle cell disease: a review. micromachines (basel). 2021;12(5):519. https://doi.org/10.3390/mi12050519 henni t, bachir d, tabone p, jurdic p, godet j, colonna p. hemoglobin bart’s in northern algeria. acta haematol. 1981;65(4):240–246. dokekias ae, ocko gokaba lt, louokdom js, et al. neonatal screening for sickle cell disease in congo. anemia. 2022;2022:9970315. https://doi.org/10.1155/2022/9970315 mcgann pt, ferris mg, ramamurthy u, et al. a prospective newborn screening and treatment program for sickle cell anemia in luanda, angola. am j hematol. 2013;88(12):984–989. https://doi.org/10.1002/ajh.23578 katawandja al, nzengu-lukusa f, nzongola-nkasu dk, muepu lt. neonatal screening for sickel cell disease in urban areas with limited resources: case of kindu city, east of the democratic republic of congo. open access libr. 2021;08(01):1–10. https://doi.org/10.4236/oalib.1107097 nkya s, mtei l, soka d, et al. newborn screening for sickle cell disease: an innovative pilot program to improve child survival in dares salaam, tanzania. int health. 2019;11(6):589–595. https://doi.org/10.1093/inthealth/ihz028 rahimy mc, gangbo a, ahouignan g, alihonou e. newborn screening for sickle cell disease in the republic of benin. j clin pathol. 2009;62(1):46–48. https://doi.org/10.1136/jcp.2008.059113 kafando e, sawadogo m, cotton f, vertongen f, gulbis b. neonatal screening for sickle cell disorders in ouagadougou, burkina faso: a pilot study. j med screen [serial online]. 2005;12(3):112–114. https://doi.org/10.1258/0969141054855300 sack ffn, njangtang dm, chemegni bc, djientcheu vdp. prevalence of sickle cell disease in newborns in the yaounde central hospital. j med res. 2017;3(6): 277–279. https://doi.org/10.31254/jmr.2017.3607 ragb s, elrahim m, el fotoh wm, ibrahim ra. screening of β-thalassemia carriers in high school students in shebin el-kom, menoufia governorate. menoufia med j. 2021;34(1):237. https://doi.org/10.4103/mmj.mmj_358_19 ropero p, molina l, gonzález fa, villegas a. incidencia de la anemia de células falciformes en guinea ecuatorial. med clin (barc). 2011;136(7):309–310. https://doi.org/10.1016/j.medcli.2009.04.021 martella m, campeggio m, pulè g, et al. distribution of hbs allele and haplotypes in a multi-ethnic population of guinea bissau, west africa: implications for public health screening. front pediatr. 2022;10:826262. https://doi.org/10.3389/fped.2022.826262 tegha g, topazian hm, kamthunzi p, et al. prospective newborn screening for sickle cell disease and other inherited blood disorders in central malawi. int j public health. 2021;66:629338. https://doi.org/10.3389/ijph.2021.629338 laghmich a, alaoui ismaili fz, barakat a, ghailani nourouti n, khattab m, bennani mechita m. alpha-thalassemia in north morocco: prevalence and molecular spectrum. biomed res int. 2019;2019:2080352. https://doi.org/10.1155/2019/2080352 wirth jp, ansumana r, woodruff ba, koroma as, hodges mh. association between sickle cell and β-thalassemia genes and hemoglobin concentration and anemia in children and non-pregnant women in sierra leone: ancillary analysis of data from sierra leone’s 2013 national micronutrient survey. bmc res notes. 2018;11(1):43. https://doi.org/10.1186/s13104-018-3143-x hajer s, neila t, sondess hf, et al. a lower-cost protocol for sickle cell disease neonatal screening in tunisia. ann saudi med. 2012;32(1):49–52. https://doi.org/10.5144/0256-4947.2012.49 hernandez ag, kiyaga c, howard ta, et al. operational analysis of the national sickle cell screening programme in the republic of uganda. afr j lab med. 2021;10(1):a1303. https://doi.org/10.4102/ajlm.v10i1.1303 segbefia ci, goka b, welbeck j, et al. implementing newborn screening for sickle cell disease in korle bu teaching hospital, accra: results and lessons learned. pediatr blood cancer. 2021;68(7):e29068. https://doi.org/10.1002/pbc.29068 nnodu oe, osei-akoto a, nembaware v, et al. skills capacity building for health care services and research through the sickle pan african research consortium. front genet. 2022;13:805806. https://doi.org/10.3389/fgene.2022.805806 mcgann pt, schaefer ba, paniagua m, howard ta, ware re. characteristics of a rapid, point-of-care lateral flow immunoassay for the diagnosis of sickle cell disease. am j hematol. 2016;91(2):205–210. https://doi.org/10.1002/ajh.24232 st john a, price cp. existing and emerging technologies for point-of-care testing. clin biochem rev. 2014;35(3):155. quinn ct, paniagua mc, dinello rk, panchal a, geisberg m. a rapid, inexpensive and disposable point-of-care blood test for sickle cell disease using novel, highly specific monoclonal antibodies. br j haematol. 2016;175(4):724–732. https://doi.org/10.1111/bjh.14298 adegoke sa, oladimeji oi, akinlosotu ma, akinwumi ai, matthew ka. hemotypesc point-of-care testing shows high sensitivity with alkaline cellulose acetate hemoglobin electrophoresis for screening hemoglobin ss and sc genotypes. hematol transfus cell ther. 2022;44(3):341–345. https://doi.org/10.1016/j.htct.2020.11.010 bukini d, nkya s, mccurdy s, et al. perspectives on building sustainable newborn screening programs for sickle cell disease: experience from tanzania. int j neonatal screen. 2021;7(1):12. https://doi.org/10.3390/ijns7010012 uyoga s, george ec, bates i, et al. point-of-care haemoglobin testing in african hospitals: a neglected essential diagnostic test. br j haematol. 2021;193(5):894–901. https://doi.org/10.1111/bjh.17431 adatara p, strumpher j, ricks e, mwini-nyaledzigbor pp. cultural beliefs and practices of women influencing home births in rural northern ghana. int j womens health. 2019;11:353–361. https://doi.org/10.2147/ijwh.s190402 fantaye aw, gunawardena n, yaya s. preferences for formal and traditional sources of childbirth and postnatal care among women in rural africa: a systematic review. plos one. 2019;14(9):e0222110. https://doi.org/10.1371/journal.pone.0222110 buser jm, bakari a, seidu aa, et al. stigma associated with sickle cell disease in kumasi, ghana. j transcult nurs. 2021;32(6):757–764. https://doi.org/10.1177/10436596211008216 leger rr, wagner ld, odesina v. stigma in adults with sickle cell disease and family members: scale development and pilot study in the usa and nigeria. int j afr nurs sci. 2018;9:23–29. https://doi.org/10.1016/j.ijans.2018.06.003 tusuubira sk, nakayinga r, mwambi b, odda j, kiconco s, komuhangi a. knowledge, perception and practices towards sickle cell disease: a community survey among adults in lubaga division, kampala uganda. bmc public health. 2018;18(1):561. https://doi.org/10.1186/s12889-018-5496-4 canatan d, aydinok y, kilinç y, et al. ulusal talasemi önleme kampanyasi: talotir projesi. turk j hematol. 2013;30(1):91–92. https://doi.org/10.4274/tjh.2012.0121 mccormick m, osei-anto ha, martinez rm. addressing sickle cell disease. washington, d.c.: national academies press; 2021. green ns, zapfel a, nnodu oe, et al. the consortium on newborn screening in africa for sickle cell disease: study rationale and methodology. blood adv. 2022;6(24):6187–6197. https://doi.org/10.1182/bloodadvances.2022007698 therrell bl, lloyd-puryear ma, ohene-frempong k, et al. empowering newborn screening programs in african countries through establishment of an international collaborative effort. j community genet. 2020;11(3):253–268. https://doi.org/10.1007/s12687-020-00463-7 ghosh k, colah r, manglani m, et al. guidelines for screening, diagnosis and management of hemoglobinopathies. indian j hum genet. 2014;20(2):101–119. https://doi.org/10.4103/0971-6866.142841 odunvbun me, okolo aa, rahimy cm. newborn screening for sickle cell disease in a nigerian hospital. public health. 2008;122(10):1111–1116. https://doi.org/10.1016/j.puhe.2008.01.008 ohene-frempong k, bonney a, nkrumah f. 270 newborn screening for sickle cell disease in ghana. pediatr res. 2005;58(2):401. https://doi.org/10.1203/00006450-200508000-00299 tshilolo l, aissi lm, lukusa d, et al. neonatal screening for sickle cell anaemia in the democratic republic of the congo: experience from a pioneer project on 31 204 newborns. j clin pathol. 2009;62(1):35–38. https://doi.org/10.1136/jcp.2008.058958 references about the author(s) john n. nkengasong international laboratory branch, division of hiv & tb, us centers for disease control and prevention, atlanta, georgia, united states citation nkengasong jn. the diagnostic–clinical chasm: work in progress? afr j lab med. 2016;5(1), a586. http://dx.doi.org/10.4102/ajlm.v5i1.586 editorial the diagnostic–clinical chasm: work in progress? john n. nkengasong copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the african journal of laboratory medicine (ajlm) has completed another exciting year by publishing a series of high impact articles that address key aspects of disease prevention and management. the driving forces that underpin the fundamental values of a journal are the felt impact the journal has on the practice of the discipline, in this case laboratory medicine in africa, and how it guides and helps shape the future of the profession. the series of papers published in this year’s issue satisfy these requirements. the articles published in ajlm in 2016 fall into four broad categories of diagnostics: availability, access, uptake, and impact on patient care or disease surveillance. with regard to access, several papers address various aspects of the quality of diagnostics and ways to improve the critical interface between laboratorians and clinicians. for example, jegede and colleagues describe the completeness of laboratory request forms submitted by clinicians in nigeria.1 this is an area that needs considerable work, as clinicians cannot be provided with accurate and timely diagnostic test results for effective management of patients, if laboratory test request forms are not completed accurately. related to this, mahomed and colleagues show that management of laboratory expenditures in primary care clinics in south africa resulted in significant cost savings.2 this is vital information, as cost poses a considerable barrier to access to quality diagnostic tests. more solutions are needed and the chasm between availability of diagnostics and clinicians’ use of the test results will continue to be a work in progress. will the availability of innovative approaches for diagnostics narrow the chasm? ajlm looks forward in 2017, albeit with anxiety, to how the acceleration of access to diagnostics in africa through the use of modern approaches could be a game changer in addressing this chasm and impacting public health practice in africa. for instance, as precision medicine – the ability to individualise patient care with a combination of information unique to an individual to identify prevention and treatment strategies based on genetic, environmental and lifestyle factors – continues to take centre stage in the 21st century in the developed world, laboratory diagnostic testing will play an even greater role. however, the availability of laboratory testing to meet the demands of precision medicine in africa in the 21st century will require disruptive innovation processes to bridge the diagnostic–clinical chasm. this is reminiscent of how mobile phones became game changers in africa, making communications affordable and easy. similarly, the new generation of non-sanger-based sequencing technologies, also called next generation sequencing, are becoming a game changer in dna sequencing due to their extraordinary speed, thus facilitating remarkable achievements and applications, and may also contribute to narrowing the diagnostic–clinical chasm. in developed countries, the increasing availability and affordability of next-generation sequencing technologies is rapidly changing the practice of microbiology. next-generation sequencing has the potential to revolutionise the practice of public health in africa by rapidly and accurately providing in-depth details on outbreak-causing pathogens, and decreasing dependence on more time consuming and expensive conventional diagnostic techniques. the question is how could such technologies be effectively established in public health laboratories in africa to better prepare them for outbreak investigations and other applications? for instance, how would next-generation sequencing technologies add value to babalola salu and colleagues’3 work on identifying imported zaire ebola virus disease from liberia into nigeria and subsequent contact tracing, or the rapid molecular confirmation of lassa fever imported into ghana as reported by bonney and colleagues?4 bridging the diagnostic-clinical chasm will continue to be a keen area of implementation science research to identify technological and cultural barriers that must be addressed in order to facilitate uptake and use of patient results and disease surveillance in africa. references jegede f, mbah ha, dakata a, et al. evaluating laboratory request forms submitted to haematology and blood transfusion departments at a hospital in northwest nigeria. afr j lab med. 2016;5(1), a381. http://dx.doi.org/10.4102/ajlm.v5i1.381 mahomed oh, lekalakala r, asmall s, et al. implications of the introduction of laboratory demand management at primary care clinics in south africa on laboratory expenditure. afr j lab med. 2016;5(1), art. #339, 6 pages. http://dx.doi.org/10.4102/ajlm.v5i1.339 salu ob, james ab, oke bo, et al. biosafety level-2 laboratory diagnosis of zaire ebola virus disease imported from liberia to nigeria. afr j lab med. 2016;5(1), a468. http://dx.doi.org/10.4102/ajlm.v5i1.468 bonney jhk, nyarko eo, ohene s-a, et al. molecular confirmation of lassa fever imported into ghana. afr j lab med. 2016;5(1), a288. http://dx.doi.org/10.4102/ajlm.v5i1.288 acknowledgements references about the author(s) zohreh jadali department of immunology, school of public health, tehran university of medical sciences, tehran, iran citation jadali z. pathobiology of hiv-related metabolic and cardiovascular comorbidities: towards a unifying mechanism. afr j lab med. 2024;13(1), a2582. https://doi.org/10.4102/ajlm.v13i1.2582 scientific letter pathobiology of hiv-related metabolic and cardiovascular comorbidities: towards a unifying mechanism zohreh jadali received: 24 july 2024; accepted: 04 sept. 2024; published: 25 oct. 2024 copyright: © 2024. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the recently published african journal of laboratory medicine article by okunorobo et al. brings to light a significant change in the lipid profile of hiv-positive patients versus healthy controls, emphasising the importance of dyslipidaemia in the development of cardiovascular diseases (cvds).1 these results are important, because dyslipidaemia is not only a common metabolic complication for hiv-positive individuals, but it also increases risk for cvds.2 therefore, understanding the causes of abnormal lipid metabolism is a critical step toward disease management. unfortunately, there is no definite explanation for why this happens. comorbidities like dyslipidaemia and cvds among hiv patients are multifactorial and have no single cause, but insights indicate that immune dysregulation plays a central role in executing pathological conditions.3 in essence, there seems to be a connection between dyslipidaemia, cvds, hiv, and immune-related pathological processes. among the possible mechanisms, the progressive loss of the cd4+ t-cell population is one of the most important.4 evidence indicates that hiv-positive individuals with low cd4 count have endothelial dysfunction and hypertension that is accompanied by increased cvd risk.4 these clinical conditions are strongly influenced by both elements of the immune system and a broad group of lipid components.4 hiv-positive individuals also have elevated proportions of t-helper 17 cells, which may promote inflammation, and senescent cells, which may increase risk of cardiovascular disease, in hyperlipidemic conditions.4 interleukin-2 production has also been shown to increase in these patients and can affect lipid metabolism and cvd risk.4 cells of the monocyte-macrophage lineage appear to also play a role in the complicated connection between the immune system and hiv disease, dyslipidaemia, and cvds.5 this is supported by elevation of intermediate/inflammatory monocytes with proatherogenic properties in hiv-positive patients.5 these cells play a detrimental role in cvd progression and can express abundant tissue factor, a risk marker for cvds.5 lipidic environment modification is another reason for production of tissue factor in plaque macrophages; thus, interplay of lipids and immune cells might in part be responsible for the cvd progression in infected patients.5 elevation of macrophage activation markers, soluble cd14, in hiv-positive patients provides additional evidence for the importance of the monocyte-macrophage lineage, because they can be associated with clinical cvd events and dyslipidaemia.6 monocyte and macrophage dysfunction in cholesterol handling as a cause of hiv infection may also affect the cvd risk.5 this may occur by down-regulation of adenosine triphosphate-binding cassette transporter a1, which is essential for cholesterol homeostasis and high-density lipoprotein metabolism.5 dysregulated inflammation is another risk factor for both cvd and dyslipidaemia in hiv-positive patients.7 this is supported by evidence, including (1) the increased level of high-sensitivity c-reactive protein as a nonspecific inflammatory marker that increases the cvd risk and dyslipidaemia, (2) the increased levels of d-dimer that can indicate a positive association with lipid indicators in patients with coronary artery disease, and (3) the increased production of interleukin-6 that occurs during hiv replication and is associated with dyslipidaemia and increased cvd risk.7 loss of gut barrier function and the ensuing microbial translocation that is usually associated with hiv disease progression can also elicit an inflammatory cascade.8 this inflammatory response may induce cvd through alteration in the balance of t-helper 17/regulatory t-cells and their associated cytokines.9 t-helper 17/regulatory t-cells appear to be a bridge linking the gut microbiota to host metabolic disorders.9 t-helper 17 and regulatory t-cells have antagonistic effects. t-helper 17 cells secrete pro-inflammatory cytokines and promote intestinal inflammation, whereas regulatory t-cells are anti-inflammatory.10 overproduction of pro-inflammatory cytokines could contribute to endothelial damage, and cardiovascular events.5 moreover, lipid profile abnormality due to disturbances in inflammatory and immune processes may exacerbate the process of cvds in infected individuals.5 microbial translocation is often associated with a reduction in microbial diversity, losing the beneficial microbes, and expansion of the pathogenic microbes.4 these changes can result in gut and peripheral t-cells activation and increases in plasma pro-inflammatory factors including tumour necrosis factor-α and soluble cd14.4 increased levels of these molecules confer an increased cvd risk in chronic hiv infection and have a strong link with dyslipidaemia.4 overall, the information above covers only small parts of the whole, but provides evidence for the crucial role of the immune system in dyslipidaemia and cvd risk among hiv patients. although definite disease mechanisms need to be established, the emergence of unifying mechanisms for hiv and related disorders is not far-fetched, because the immune system is a system of relations that interacts with other body systems. lipid metabolism and immune system responses regulate each other, and immune-metabolic dysregulation can contribute to hiv-associated pathogenesis and clinical observations related to hiv.3 alternatively, the heart and the immune system are highly integrated systems and immune dysregulation plays an essential role in the development of many cvds.3 therefore, the harmful effect of hiv infection on an individual’s immune system can disrupt proper functioning of different body systems. however, further investigations are needed to elucidate how the irregularities of the immune system contribute to altered lipid profile and increased risk for cvd among hiv patients. acknowledgements competing interests the author declares that they have no financial or personal relationships that may have inappropriately influenced them in writing this scientific letter. author’s contribution z.j. is the sole author of this scientific letter. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability data sharing is not applicable to this scientific letter as no new data were created or analysed. disclaimer the views and opinions expressed in this scientific letter are those of the author. it does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the author is responsible for this scientific letter’s content. references okunorobo mn, nnamah nk, ude ua, ude ea. lipids and apolipoproteins c-iii and e among treatment-naïve and treatment-experienced persons with hiv in nigeria. afr j lab med. 2023;12(1):2018. https://doi.org/10.4102/ajlm.v12i1.2018 wańczura p, aebisher d, iwański ma, myśliwiec a, dynarowicz k, bartusik-aebisher d. the essence of lipoproteins in cardiovascular health and diseases treated by photodynamic therapy. biomedicines. 2024;12(5):961. https://doi.org/10.3390/biomedicines12050961 ketelhuth dfj, lutgens e, bäck m, et al. immunometabolism and atherosclerosis: perspectives and clinical significance: a position paper from the working group on atherosclerosis and vascular biology of the european society of cardiology. cardiovasc res. 2019;115(9):1385–1392. https://doi.org/10.1093/cvr/cvz166 lembas a, załęski a, peller m, mikuła t, wiercińska-drapało a. human immunodeficiency virus as a risk factor for cardiovascular disease. cardiovasc toxicol. 2024;24(1):1–14. https://doi.org/10.1007/s12012-023-09815-4 nou e, lo j, grinspoon sk. inflammation, immune activation, and cardiovascular disease in hiv. aids. 2016;30(10):1495–1509. https://doi.org/10.1097/qad.0000000000001109 obare lm, temu t, mallal sa, wanjalla cn. inflammation in hiv and its impact on atherosclerotic cardiovascular disease. circ res. 2024;134(11):1515–1545. https://doi.org/10.1161/circresaha.124.323891 hmiel l, zhang s, obare lm, et al. inflammatory and immune mechanisms for atherosclerotic cardiovascular disease in hiv. int j mol sci. 2024;25(13):7266. https://doi.org/10.3390/ijms25137266 trøseid m, nielsen sd, vujkovic-cvijin i. gut microbiome and cardiometabolic comorbidities in people living with hiv. microbiome. 2024;12:106. https://doi.org/10.1186/s40168-024-01815-y liu j, liu f, liang t, et al. the roles of th cells in myocardial infarction. cell death discov. 2024;10:287. https://doi.org/10.1038/s41420-024-02064-6 tolomeo m, cascio a. the complex dysregulations of cd4 t cell subtypes in hiv infection. int j mol sci. 2024;25(14):7512. https://doi.org/10.3390/ijms25147512 ajlm 5_2_2014.indb reviewer acknowledgement open accesshttp://www.ajlmonline.org page 1 of 1 we would like to take this opportunity to thank all of those who provided scientific and logistical support for this issue of the african journal of laboratory medicine: should names have inadvertently been excluded from this list, the publisher apologises and undertakes to amend the exclusion in the next issue. ajlm african journal of laboratory medicine in an effort to facilitate the selection of appropriate peer reviewers for the african journal of laboratory medicine, we ask that you take a moment to update your electronic portfolio on www. ajlmonline.org for our files, allowing us better access to your areas of interest and expertise, in order to match reviewers with submitted manuscripts. 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we appreciate the time taken to perform your review(s) successfully. mahsa abassi kabir abdullahi teferi m. abera oyekola v. abiri olusola a. adejumo olukemi adekanmbi adeyemi adeyemo ezekiel rachel akinkunmi george alemnji r. joseph alexis william k. ampofo timothy amukele kingsley anukam mustafa arasa kevin arien george aryee rosemary a. audu george awungafac funmilola ayeni françois–xavier babin tony badrick fay betsou stephanie bispo debrah i. boeras andre st. aubyn bowers qi chen linda cherepow nelson chimbiya richard b. clark oksana debrah kassaye t. desta alpha a. diallo wayne dimech jienchi dorward robert eley mohammed a. elsheikh zahra eslamirad emmanuel fajardo samuel a. fayemiwo noah fongwen adeola fowotade tiago selbach garcia maria gazouli lisa gerwing-adima philip m. giffard mark a. giffen jr kjell grankvist alex greninger tomáš hanke lucia hans bernice harris william p. hausdorff marianne k. henderson pamela hepple susan horton ekua houphouet offiong f. ikpatt zeynep ozturk inal md. sadequl islam sam kariuki aderemi kehinde stephen b. kennedy luc kestens charles kiyaga jerzy klijanienko kekoura kourouma chika kusano george boateng kyei majda laboudi sylvia lacourse william lali kim lewis raphael lihana direk limmathurotsakul smijlika de lussigny megha maheshwari alexander martin-odoom talkmore maruta patrick mateta kamran mirza meade morgan anneta f. naidoo nicaise ndembi brooke nichols nicholas i. nii-trebi jean n. nikiema patrick njukeng michael a. noble jacinta nwogu iheanyi o. okonko ahmed olowo-okere sien ombelet pascale ondoa japheth opintan philip o. oshun collins o. otieno chin-yih ou pragyan s. panda ursula panzer ketan patel mario plebani nira pollock oluwafemi akinyele popoola philomena raftery michele ramsay sarah riley lucy j. robertson julia k. rohr debra de assis rosa ahmed saleh david jahbiuk sambian paul sandstrom christine e. schaner-tooley mary e. schmitz heidi schütt-gerowitt yan shen adebayo shittu cassandra soo derek r. stein timothy su anthony p. sunjaya catherine g. sutcliffe tomas szotkowski rosemary tambouret maria a. telles merih tesfazghi ajaykumar k. thirumala ralph timperi cristina touriño florette treurnicht raquel villegas lara vojnov hongping wei shannon whitmer peter r. williamson feng xu nicole young peter young anissa zaouak http://www.ajlmonline.org� https://ajlmonline.org� https://ajlmonline.org� https://ajlmonline.org� https://ajlmonline.org/index.php/ajlm/user https://ajlmonline.org/index.php/ajlm/user mailto:publishing@aosis.co.za abstract introduction methods results discussion acknowledgements references about the author(s) deep madkaiker department of transfusion medicine, advanced centre for treatment, research and education in cancer, tata memorial centre, homi bhabha national institute, mumbai, india department of immunohematology and blood transfusion, kasturba medical college, manipal, manipal academy of higher education, manipal, karnataka, india shashank ojha department of transfusion medicine, advanced centre for treatment, research and education in cancer, tata memorial centre, homi bhabha national institute, mumbai, india arunkumar n department of transfusion medicine, advanced centre for treatment, research and education in cancer, tata memorial centre, homi bhabha national institute, mumbai, india kalpesh chawan department of transfusion medicine, advanced centre for treatment, research and education in cancer, tata memorial centre, homi bhabha national institute, mumbai, india citation madkaiker d, ojha s, na, chawan k. effects of pre-storage leukoreduction on erythrocyte concentrates and performance of newer generation leuko-filters at a tertiary care oncology hospital in western india. afr j lab med. 2025;14(1), a2723. https://doi.org/10.4102/ajlm.v14i1.2723 original research effects of pre-storage leukoreduction on erythrocyte concentrates and performance of newer generation leuko-filters at a tertiary care oncology hospital in western india deep madkaiker, shashank ojha, arunkumar n, kalpesh chawan received: 10 dec. 2024; accepted: 28 mar. 2025; published: 25 june 2025 copyright: © 2025. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: leukoreduction is a post-processing technique that reduces residual leukocytes in cellular blood components. previous studies have evaluated these parameters mainly among older generation leuko-filters. objective: this study evaluated the immediate effects of pre-storage leukoreduction on red cell indices and the performance efficacy of two newer generation leuko-filters. methods: this retrospective analysis collected quality control data before and after leukoreduction for erythrocyte concentrates (ecs) from laboratory registers from the blood transfusion laboratory at the advanced centre for treatment, research and education in cancer in mumbai, india, for the period january 2015 to december 2019. data related to red cell indices and performance characteristics for fresenius and macopharma filters were included. results: a total of 500 records was included in the study. all ec units demonstrated a 99.99% leukocyte log reduction, with both filters showing equal efficacy. post-leukoreduction haemoglobin concentrations were lower than the pre-leukoreduction for all units (p < 0.001). of those prepared from 350 ml units, 11.6% (28/240) had haemoglobin levels under 40 g/bag as compared to 1.1% (3/260) among those prepared from 450 ml units. all indices exhibited statistically significant changes after leukoreduction (p < 0.001) except for mean corpuscular haemoglobin (p = 0.215). the fresenius filter required less time for leukoreduction compared to the macopharma filter (p < 0.001). conclusion: red cell indices show several changes following leukoreduction. further studies are needed to assess the microscopic and functional impact of leukoreduction. leuko-filters vary in their performance characteristics, which may influence vendor selection. what this study adds: this study found changes among several red cell indices after leukoreduction of ecs, which have not been extensively studied in previous literature. further, we found that the newer generation leuko-filters differ in specific performance characteristics, which may influence vendor selection. keywords: leukoreduction; red cell indices; quality control; whole blood; leuko-filter; red cell concentrate. introduction the field of transfusion medicine has experienced several challenging periods, which have culminated in the introduction of newer technological advancements to improve the safety of allogenic blood. leukoreduction was one such paradigm shift in transfusion practices which gained momentum in the 1990s in response to the variant creutzfeldt-jakob disease outbreak.1 this resulted in the implementation of universal leukoreduction in the united kingdom as a risk reduction measure. universal leukoreduction was also implemented by france to counter the potential spread of transmissible spongiform encephalopathies by donor leukocytes, and, during a general reorganisation of transfusion services, in canada.2 residual viable donor leukocytes are present in cellular blood components and have been implicated in several complications such as febrile, non-haemolytic transfusion reactions; human leukocyte antigen alloimmunisation and subsequent platelet transfusion refractoriness; transfusion related immuno-modulation; and transmission of infectious leukotropic agents such as cytomegalovirus and human t-lymphotropic virus i/ii, and transfusion-associated graft-versus-host disease.3 leukocyte depletion is known to reduce transfusion-related immunomodulation effect in patients, which depends on the degree of leukoreduction, with leuko-filtration being far more effective than buffy coat depletion.4 additionally, white blood cells (wbcs) are known to damage red cells, which further exacerbates red cell storage lesions during their storage.5 presently, leukoreduction is achieved via leuko-filters, comprising an external plastic housing which fits tightly around the filtration medium.2 the current generation of leuko-filters are highly efficient, as they have an excellent leukocyte removal efficacy and are able to achieve a 3–4 log reduction (99.9% – 99.99%) as compared to 1st and 2nd generation filters (90% – 96%).6 barrier filtration and cell adhesion are the major contributing mechanisms leading to a very high efficacy of leukocyte removal. most previous studies on leukoreduction filters have focused on evaluating erythrocyte concentrate (ec) quality parameters and filter performance characteristics, primarily among older generations of leukoreduction filters.7,8 fewer studies have reported changes in the red cell indices.5,9,10 blood transfusion services in india are decentralised, with varying practices across the country. leukoreduction is not a mandatory requirement in india. however, it may be implemented by the blood centre based on selective patient profiles. at our tertiary care oncology setup at the department of transfusion medicine at the advanced centre for treatment, research and education in cancer in mumbai, india, we cater to all types of oncology patients, and leukoreduced blood components are specifically indicated for haemato-oncology patients, since they are at risk of human leukocyte antigen alloimmunisation and future risk of poor transplant outcomes. in our current study, we assessed the immediate effects of pre-storage leukoreduction on red cell indices of ecs, and the performance efficacy of two newer-generation leuko-filters available at our centre. methods ethical considerations this study was approved by the institutional ethics committee-iii at the advanced centre for treatment, research and education in cancer, mumbai, india (project number 900713 and institutional ethics committee approval number 566/2020 dated 19 october 2020). waiver of consent was approved for this study, since it involved technical anonymised data (not involving human or animal research) delinked from original blood donor records, not available in the public domain. these quality control (qc) records were maintained as hard copies, which were recorded in laboratory registers, and the complete blood count (cbc) printouts from the haematology analyser, which were filed and stored in locked cabinet sections in the component laboratory. furthermore, access to these documents was restricted to the respective section staff, technical supervisors, and transfusion laboratory in-charge personnel. study design and setting this retrospective analysis of leukoreduction qc records for ecs recorded from 01 january 2015 to 31 december 2019 was conducted at the department of transfusion medicine (blood transfusion laboratory) at the advanced centre for treatment, research and education in cancer, mumbai, india. this hospital serves as a tertiary care oncology hospital catering to all types of cancer patients, including haemato-oncology. all ec units subjected to leukoreduction are prepared from whole blood which has been collected from voluntary blood donors in accordance with the national blood donor selection guidelines approved by the directorate general of health services, india, which is the regulatory authority for blood safety in india. quality control is mandated by the indian regulatory authorities for 1% of all leukoreduced ecs prepared from 350 ml or 450 ml whole blood. quality control is performed as a routine measure in the laboratory, to ensure the quality of blood components and that leukoreduction was performed by trained and competent staff as per standard operating procedures in the department of transfusion medicine, and documented in the respective qc registers. inclusion/exclusion criteria erythrocyte concentrates undergoing leukoreduction between 01 january 2015 and 31 december 2019 were included in the study, whereas ecs not subjected to leukoreduction, qc records with missing data, and units that were leukoreduced outside designated 5-year study period were excluded from the study. sample and data collection representative samples were collected from the tubing of the ec unit. the cbc samples were collected in ethylenediaminetetraacetic acid vacutainers (bd sciences, mumbai, maharashtra, india) and were analysed using the siemens advia® 2120i (siemens ag, erlangen, germany), a 5-part differential haematology analyser. these ethylenediaminetetraacetic acid samples were tested immediately and subsequently discarded as per national biomedical waste management guidelines. quality control data, including filter performance data for leukoreduced ecs, were recorded manually in physical laboratory registers maintained in department of transfusion medicine. red cell indices were recorded from the cbc report printouts from the siemens advia® analyser. laboratory analyses the data obtained from the qc records were cbc parameters of pre-leukoreduction and post-leukoreduction samples from red cell concentrates, namely wbc, red blood cell count (rbc), haemoglobin, haematocrit, mean corpuscular volume (mcv), mean corpuscular haemoglobin (mch), mean corpuscular haemoglobin concentration, cellular haemoglobin, and platelet count. post-leukoreduction wbc count was measured using nageotte’s haemocytometer (paul marienfeld gmbh & co. kg, lauda-königshofen, germany) as per validated departmental standard operating procedures, by trained technical staff. demographic data related to donors were not captured in the qc laboratory registers; only the date of collection and donor unit numbers were recorded, along with other qc data, while the actual donor records were stored separately. the two newer generation leuko-filters (macopharma leucolab lcg2b: macopharma, tourcoing, france; fresenius bior: fresenius kabi ag, homburg, germany) used at our centre were also assessed for their performance efficacy using the following parameters: wbc log reduction, hold-up volume of the filter, and time taken for filtration. these leuko-filter performance characteristics were also recorded in the qc laboratory registers. data analysis data were captured from the laboratory registers in a case record form, checked by the investigators during entry and cleaned using microsoft excel (redmond, washington, united states). data were analysed using ibm spss statistics for windows, version 24.0 (ibm corp., armonk, new york, united states). data were descriptively summarised using median and interquartile range (iqr) for continuous data, while categorical data were reported as frequency and percentage. normality assumptions were tested using shapiro-wilk’s test. the preand postoutcomes in the study were compared using the paired wilcoxon signed rank test for pre-leukoreduction and post-leukoreduction comparisons of red cell indices, and the mann u whitney test for inter-filter comparisons, depending on the normality of the data. the level of significance for all statistical analysis was established at p < 0.05. results a total of 500 leukoreduction qc records for ecs were reviewed during the study period, with 34 qc records with missing cbc data excluded from the final analysis. of these, 52% (260/500) were derived from 450 ml whole blood donations (r450), while the rest were prepared from 350 ml whole blood donations (r350). the majority, 69.2% (346/500), of ecs were filtered using macopharma leucolab lcg2b, with the remaining 30.8% (154/500) filtered using the fresenius bior leuko-filter. among the ecs filtered with the macopharma filter, 53.8% (186/346) were r450 units, while the rest were r350. the fresenius bior filter was used for almost an equal number of ecs units prepared from 350 ml (74/154; 48.1%) and 450 ml (80/154; 51.9%) whole blood donations. comparison of pre-leukocyte reduction and post-leukocyte reduction red cell indices there were no instances of leukoreduction failure, even with a wide range of pre-leukoreduction wbc loads (12.4 × 103/ul – 0.6 × 103/ul) in the ec units. additionally, the post-leukoreduction units showed a mean reduction in platelet count of approximately 97.2% ± 6.0%. in terms of haemoglobin concentration, a statistically significant reduction was seen in the post-leukoreduction units (p < 0.001). the difference in pre-leukoreduction haemoglobin content of the r350 units compared to the r450 units was statistically significant (r350 [median = 54.9 g/bag, iqr = 50.3–63.7] vs r450 [median = 62.5 g/bag, iqr = 58–70], p < 0.001). post-leukoreduction haemoglobin concentrations were statistically significant and lower than the pre-leukoreduction haemoglobin levels for all ec units (p < 0.001). post-leukoreduction haemoglobin concentrations were also noted to be lower and statistically significant in the r350 units compared to the r450 units (r350 [median = 47.5 g/bag, iqr = 42.8–56.8] vs r450 [median = 55.3 g/bag, iqr = 51.1–61.1], p < 0.001). despite all units meeting the qc criteria for leukoreduced red cell concentrates in india, we found that 11.6% of r350 units (28/240) had haemoglobin levels under 40 g/bag (as per european and united kingdom guidelines).11 meanwhile, only 1.1% (3/260) of r450 units had haemoglobin levels below this threshold. all red cell indices exhibited statistically significant changes after leukoreduction (p < 0.001), except for mch (p = 0.215) (table 1). table 1: red cell indices for preand post-leukoreduction samples, at the advanced centre for treatment, research and education in cancer, mumbai, india, 01 january 2015 to 31 december 2019. among the remaining parameters, all indices showed a statistically significant decrease (p < 0.001), except for mcv, which showed a statistically significant increase (p < 0.001). however, the absolute magnitude of difference between the pre-leukoreduction and post-leukoreduction for all red cell indices was found to be small. the most affected haematological parameters were haemoglobin, rbc, and haematocrit, with haematocrit showing a considerable narrowing of its range after leukoreduction (figure 1). figure 1: pre-leukoreduction and post-leukoreduction distribution of haemoglobin, haematocrit and red blood cell counts of erythrocyte concentrates, at the advanced centre for treatment, research and education in cancer, mumbai, india, 01 january 2015 to 31 december 2019. total red blood cell content in erythrocyte concentrate units (red blood cell count × volume of erythrocyte concentrate) the total rbc content was higher in all pre-leukoreduction units (r350 and r450) than the post-leukoreduction units (figure 2). another notable finding was the narrower range of red cell count in the post-leukoreduction ecs. furthermore, as expected, mean total rbc counts in r450 units were higher than those in r350 units, both before and after leukoreduction. figure 2: distribution of a) pre-leukoreduction and b) post-leukoreduction, total red blood cell content in bag for the two erythrocyte concentrate configurations (r450 and r350), at the advanced centre for treatment, research and education in cancer, mumbai, india, 01 january 2015 to 31 december 2019. both filters demonstrated an equal efficacy in obtaining a 3–4 log wbc reduction post-filtration, with all units in the study meeting the leukoreduction criteria of < 5 × 106 wbcs per bag. nevertheless, only 33.4% (167/500) of the units achieved a stricter leukoreduction target of < 1 × 106 wbcs per bag. erythrocyte concentrates filtered using the macopharma filter comprised a higher proportion of such units than those filtered using the fresenius filter. among those filtered using the macopharma filter, 76 out of 186 were derived from 450 ml whole blood and 56 out of 160 from 350 ml whole blood. whereas for those ecs filtered using the fresenius filter, 17 out of 80 were prepared from 450 ml whole blood and 18 out of 74 from 450 ml whole blood wb, which failed to meet this stricter criterion. leuko-filter data the lower red cell recovery observed in the post-filtration units is attributable to the hold-up volume retained in the filter housing medium. although no statistically significant difference was noted between the two manufacturers (p = 0.88 for r350 units and p = 0.193 for r450 units), there was a statistically significant lower hold-up volume in the r350 units compared to the r450 units, p < 0.001 (table 2, figure 3). table 2: summary of leuko-filter performance characteristics, at the advanced centre for treatment, research and education in cancer, mumbai, india, 01 january 2015 to 31 december 2019. figure 3: distribution of filter hold-up volumes for the two erythrocyte concentrate configurations (r450 and r350), at the advanced centre for treatment, research and education in cancer, mumbai, india, 01 january 2015 to 31 december 2019. the difference in time taken for filtration of r350 and r450 units was not found to be statistically significant (p = 0.343). in contrast, inter-filter comparisons revealed that ecs undergoing leukoreduction with the fresenius filter required statistically significantly less time compared to those processed with the macopharma filter, p < 0.001 (figure 4). figure 4: distribution of leuko-filtration time for the two different manufacturers of leuko-filters, at the advanced centre for treatment, research and education in cancer, mumbai, india, 01 january 2015 to 31 december 2019. discussion our study showed that both leuko-filter brands were equally efficacious in achieving leukoreduction to a residual leukocyte count of less than 5 × 106/bag. post-leukoreduction haemoglobin concentrations were statistically significant and lower in all units as a result of the hold-up volume in the leuko-filter. we noted that 11.6% of r350 units (28/240) had post-leukoreduction haemoglobin levels under 40 g/bag (as per european and united kingdom guidelines).11 meanwhile, only 1.1% (3/260) of r450 units had haemoglobin levels below this threshold. all red cell indices exhibited statistically significant changes after leukoreduction except for mch. these changes were noted to be statistically significant and lower, post-leukoreduction, except for mcv which showed a statistically significant increase. another notable finding was the narrower range of red cell count in the post-leukoreduction ecs. although no statistically significant difference in hold-up volume was noted between the two manufacturers, there was a statistically significant lower hold-up volume in the r350 units compared to the r450 units. lastly, the time taken for filtration of r350 and r450 units was not found to be statistically significant. however, in contrast, inter-filter comparisons showed that ecs undergoing leukoreduction with the fresenius filter required statistically significantly less time compared to those processed with the macopharma filter. our leukoreduction efficacy aligns with previous studies from india published in 202112 and 202213 which showed that the current generation of leuko-filters is highly effective at leukoreduction of red cell concentrates. two indian studies from 2014 to 2023 have also reported bag haemoglobin less than 40 g following leukoreduction and it would therefore be advisable that red cells from 450 ml units are allocated for leukoreduction to offset the losses occurring in the filter casing.14,15 among other red cell indices assessed, all indices showed a statistically significant change after leukoreduction, except for mch. however, the pre-leukoreduction and post-leukoreduction differences for all red cell indices were noted to be small in absolute magnitude. these findings are in accordance with two previous studies – an indian study by sonker, dubey and chaudhary in 2014,15 and an american study by aubuchon et al. in 1997,7 which have reported a decrease in the rbc content, haemoglobin and haematocrit. the increase in mcv found in our study was similar to a chinese study by ran et al. in 201116 and the egyptian study from 2016 by mahmoud and hassan.10 an italian study by pertinhez et al. from 2016 showed two intriguing findings, the first being that non-leukoreduced ecs showed a greater increase in mcv. the other finding was that the increase in mcv exhibits a linear increment when measured for leukoreduced ecs, as compared to non-leukoreduced units. these non-leukoreduced units showed a sigmoidal change in mcv, speculating that leukoreduction may select a homogeneous population of rbcs and support the hypothesis that leuko-depleted rbcs are characterised by a higher deformability that would favour their passage through the filter and are, therefore, more resistant to cell volume impairment during storage.17 the beneficial effects of leukoreduction on red cell storage was also reflected in a canadian study in 2005, wherein leukoreduction was proposed to improve the quality of red cell concentrates by demonstrating less haemolysis than conventional, unfiltered products.18 however, ghezelbash et al.9 from iran in 2017 did not find a statistically significant difference in the mch of leukoreduced units, while our study found a statistically significant decline in post-leukoreduction mch. this study further evaluated the changes in red cell indices over the storage duration, showing a decline in mch and mean corpuscular haemoglobin concentration. meanwhile, the study by antonelou et al. from greece in 2012 had shown an increase in the mch parameter in both young and senescent red cell units.5 the chinese study by ran et al. showed that irradiation, filtration, and combined irradiation and filtration in pre-storage units can cause damage in red cells and intensify the rbc storage lesions which could occur in a very short time frame (< 1 day).16 furthermore, our study was comparable to the french study in 1988 by andreu et al. which showed a similar 1-log platelet reduction in leukoreduced ecs, which might contribute to the beneficial effects of leukoreduction.8 however, an american study by heaton et al. in 1994 reported a much higher platelet reduction in their study, which might be attributable to use of a different leukoreduction filter.19 steneker et al., in their study from the netherlands in 1993, had shown that the presence of viable platelets may contribute to an improved performance of leukofiltration of the blood components.20 the two manufacturers evaluated in our study demonstrated equal efficacy, consistently achieving high levels of leukoreduction, as evidenced by the indian study by sonker et al. in 2014 and the american study by heaton et al. in 1994.15,19 while some units (33.4%) did not meet the more stringent council of europe guidelines,11 the necessity of reducing residual wbcs below 5 × 106/bag lacks substantial evidence, especially considering that complete elimination of residual wbcs is not possible. attempts to further reduce residual leukocytes to levels < 1 × 106/bag in the absence of confirmatory evidence (which exists for residual counts < 5 × 106/bag) is unlikely to be cost effective, especially in the setting of developing countries. an inherent drawback of the leuko-filtration process is the hold-up volume retained in the filter housing medium. our study found that the hold-up volume was statistically significantly higher in ecs filtered by macopharma filters (10.3% ± 3.5%) compared to fresenius filters (9.56% ± 2.72%). while most filtered units exhibited post-filtration losses of less than 15%, approximately 4.4% of the units had hold-up volumes exceeding 15% within the leuko-filters. despite this, all units complied with national qc guidelines, which require 75% of tested units to meet the qc standards. similar red cell losses in leuko-filters have been reported in previous studies by andreu et al. in 1988, and aubuchon et al. from the united states in 1997.7,8 we also noted that the hold-up volume was statistically significant and lower in the r350 units compared to r450 units. lastly, the inter-filter comparisons also showed a difference in the time taken for leukoreduction, with the fresenius filters demonstrating higher efficiency with faster filtration rates compared to the macopharma filter. leuko-filtration rates documented in published literature exhibit considerable variability, influenced by factors such as filter composition, ambient processing temperature, and the initial specifications of red cells prior to filtration, as shown in the study from the netherlands by pietersz, steneker and reesink in 1993.21 as anticipated, there was a noticeable difference in filtration times between r350 and r450 units, although this difference was not statistically significant. the time taken for filtration depends on the filter-housing medium, and may impact the workload distribution in a large-volume hospital. along with the cost, this may be a deciding factor when different leuko-filters with equivalent performance characteristics are evaluated for hospital supply. our study is the first indian study to evaluate two newer-generation filters for their performance efficacy and to compare the pre-leukoreduction and post-leukoreduction changes in red cell indices. our findings support previously published data and also present new data, especially with regard to changes in red cell indices where prior comparative research has been lacking. limitations the retrospective design of the research study precluded the evaluation of storage duration-related changes in red cell indices. further, we were only able to include those units which had been subject to a qc check. donor characteristics such as age, sex and other medical information which are known to influence ec characteristics were not evaluated as part of this study. additionally, parameters such as supernatant potassium levels, haemoglobin levels, and haemolysis percentage were not investigated. lastly, patient outcomes following the administration of leukoreduced red cells were not monitored as part of this study. conclusion red cell indices show several changes following leukoreduction, but further studies are needed to assess the microscopic and functional impact of leukoreduction. the newer-generation filters show a good performance efficacy for leukoreduction. erythrocyte concentrates prepared from 450 ml whole blood units should be preferred for preparation of leukoreduced units subject to local operational constraints, because of the higher red cell recovery. leuko-filters vary in their performance characteristics such as filtration time and hold-up volume, which may influence vendor selection for hospital purchase. acknowledgements we would like to thank all the staff in the department of transfusion medicine, advanced centre for treatment, research and education in cancer, who supported us in this research. this article is partially based on the author’s thesis entitled ‘retrospective assessment of leukoreduced red cell concentrates and leukocyte filter performance characteristics at a tertiary care haemato-oncology hospital’ towards the degree of md immuno-haematology and blood transfusion, in the department of transfusion medicine at the homi bhabha national institute, mumbai, india in 2021, with supervisor dr shashank ojha. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions d.m. and s.o. conceptualised the study, sought ethics committee approval, and supervised the conduct of the study. a.n. and k.c. performed sampling, lab analysis, and data compilation along with d.m. d.m., a.n. and k.c. compiled the article draft and the article was scrutinised by s.o. this final version of the article has been approved by d.m., s.o., a.n. and k.c. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the data that support the findings of this study are available from the corresponding author, d.m., upon reasonable request. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. the article does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the authors are responsible for this article’s results, findings, and content. references bassuni wy, blajchman ma, al-moshary ma. why implement universal leukoreduction? hematol oncol stem cell ther. 2008;1(2):106–123. https://doi.org/10.1016/s1658-3876(08)50042-2 harris sb, hillyer cd, editors. blood manufacturing: component 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https://doi.org/10.1111/j.1365-2141.1994.tb04923.x steneker i, prins hk, florie m, loos ja, biewenga j. mechanisms of white cell reduction in red cell concentrates by filtration: the effect of the cellular composition of the red cell concentrates. transfusion. 1993;33(1):42–50. https://doi.org/10.1046/j.1537-2995.1993.33193142309.x pietersz rn, steneker i, reesink hw. prestorage leukocyte depletion of blood products in a closed system. transfus med rev. 1993;7(1):17–24. https://doi.org/10.1016/s0887-7963(93)70029-2 abstract introduction methods results discussion acknowledgements references about the author(s) jude o. okoye department of medical laboratory science, school of public and allied health, babcock university, ilishan-remo, ogun state, nigeria department of medical laboratory science, faculty of health sciences and technology, college of medicine, nnamdi azikiwe university, nnewi campus, anambra, nigeria anthony a. ngokere department of medical laboratory science, faculty of health sciences and technology, college of medicine, nnamdi azikiwe university, nnewi campus, anambra, nigeria charles c. onyenekwe department of medical laboratory science, faculty of health sciences and technology, college of medicine, nnamdi azikiwe university, nnewi campus, anambra, nigeria olaposi omotuyi department of biochemistry, centre for biotechnology, adekunle ajasin university, akungba-akoko, ondo, nigeria deborah i. dada department of medical laboratory science, school of public and allied health, babcock university, ilishan-remo, ogun state, nigeria citation okoye jo, ngokere aa, onyenekwe cc, omotuyi o, dada di. epstein-barr virus, human papillomavirus and herpes simplex virus 2 co-presence severely dysregulates mirna expression. afr j lab med. 2021;10(1), a975. https://doi.org/10.4102/ajlm.v10i1.975 brief report epstein-barr virus, human papillomavirus and herpes simplex virus 2 co-presence severely dysregulates mirna expression jude o. okoye, anthony a. ngokere, charles c. onyenekwe, olaposi omotuyi, deborah i. dada received: 20 jan. 2019; accepted: 20 oct. 2020; published: 16 mar. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract this cross-sectional study evaluated the expression of mir-let-7b, mir-21, mir-125b, mir-143, mir-145, mir-155, mir-182, mir-200c, p53 gene, ki67, scca1 and cd4+ t-cell counts among 319 women, to epstein-barr virus, human papillomavirus and herpes simplex virus 2 mono-infections and co-infections, using enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction methods. this study suggests that malignancies associated with viral co-infection could be diagnosed early by monitoring cluster of differentiation 4+ t-cell counts and serum expression of mir-145 and mir-182. keywords: epstein-barr virus; human papilloma virus; herpes simplex virus 2; epithelial-mesenchymal transition; micrornas. introduction evidence shows that epstein-barr virus (ebv) infection favours human papillomavirus (hpv) fusion to host genome resulting in genetic instability, initiation and amplification of epithelial-mesenchymal transition (emt) to cancer in the host.1,2 the mechanisms by which they synergistically promote cancer invasiveness and metastasis are yet to be fully understood. although controversies still exist concerning the role of type 2 herpes simplex virus (hsv-2) in cervical carcinogenesis, mounting evidence suggests that the persistence and reactivation of hsv-2 not only facilitates ebv and hpv infection entry but also increases the risk of cervical squamous cell carcinoma by 60 times.3,4 this viral co-infection may elicit diverse, overwhelming dysregulation of the host’s oncomirs-cancer-associated micro-ribonucleic acids (mirnas) or the tumour suppressor mirnas. microrna is a short unit of non-coding rna that post-transcriptionally regulates gene expression.5 viruses are known to cause overexpression or underexpression of certain mirna, especially in individuals with cancer.5 in this study, we ascertained whether the extent of mirna overexpression or underexpression is higher or lower in viral co-infection than in mono-infection, and which type of co-infection is associated with the highest and lowest expression of mirna when compared with uninfected individuals. hence, this study evaluated the expression of normally downregulated biomarkers (mir-21, mir-155, mir-182, mir-200, p53 gene, ki67, and squamous cell carcinoma antigen 1 [scca1]) and normally upregulated biomarkers (mir-let-7b, mir-125b, mir-143, mir-145, and cd4+ t-cells) in individuals with single through ternary viral infections and those without the targeted viral infections (ebv, hpv and hsv-2). overexpressed and underexpressed mirnas could serve as biomarkers for identifying and or monitoring individuals who are at risk of developing cervical lesions.6 methods ethical considerations for this study, ethical approvals were obtained from the state hospital abeokuta ethics committee (sha/res/vol.2/177) and babcock university health research committee (buhrec549/18). written consent was obtained from all participants. the questionnaires filled by the participants and samples taken following selection were given specific numbers in place of names to protect the privacy of participants. personal information and results of the participants remained undisclosed to other personnel, except the investigators and respective participants. study area and participants this cross-sectional study was carried out between may 2017 and august 2018 during a free screening programme. using the convenience sampling technique, this study included active commercial sex workers (n = 319; mean age = 33.84 ± 8.38 years) living in selected brothels in abeokuta and ilishan-remo communities. interested participants visited the family planning clinic at their own expense. sex workers who voluntarily visited the family planning clinic were selected by researchers based on the description provided by the reports of the united states centers for disease control and prevention, the united nations for aids and world health organization.7,8 the demographics were collected using an interview-based questionnaire. inclusion criteria included being a self-proclaimed direct (commercial; receive money for sex) and sexually active female sex workers aged 18–54 years. individuals with any history of cervical, breast and oral cancers or those that are transgender or below 18 years or above 54 years were excluded. sample collection and handling the majority of samples were taken at the family planning unit, state hospital ijaiye, abeokuta and the phlebotomy unit, babcock university teaching hospital, nigeria, by nurses and researchers. some samples were collected at the brothels only on the insistence of concerned participants. all samples (blood and pap smears) were collected according to the standard operating procedure of the hospitals. participants were screened for cervical cancer using liquid base preparation and papanicolaou techniques. smears were taken by nurses and clinicians and evaluated by two cytopathologists. five millilitres (ml) of peripheral blood was drawn between the hours of 09:00 and 10:00, from each participant, by medical laboratory scientists; 3 ml was drawn into an ethylenediaminetetraacetic acid vacutainer tube for cd4+ t-cell counts and 2 ml was drawn into a plain tube for enzyme-linked immunosorbent assay (elisa). samples taken at the abeokuta collection centre were transported to the chemical pathology unit, babcock university teaching hospital, in a specimen transport cooler containing some ice packs. all samples were analysed at the chemical pathology unit, babcock university teaching hospital. after allowing the blood in the plain tube (2 ml) to stand for not more than 2 h, clear serum was separated into another plain tube and stored at −20 °c until analysed. cd4+ t-cell counts and pap smears were investigated daily while other test were carried out at intervals (≤ 2 months). sample assays the cd4 easy count kit and cyflow counter (sysmex partec gmbh, gorlitz, germany) were used for counting cd4+ t-cells in whole blood, not more than 2 h after collection. to maintain cd4+ t-cell day-to-day counts, two internal quality controls were employed: previously tested whole blood samples and stabilised whole blood samples. the separated sera were tested for antibodies against hiv-2, ebv, hpv, and hsv-2 by elisa (qingdao hightop biotech co. ltd, qingdao, shandong, china, and calbiotech inc., el cajon, california, united states) according to manufacturers’ instructions. according to the manufacturers instructions, a 96% to 99% agreement (performance) was observed between elisa kits and the reference elisa method. serum samples from participants whose cervical cells had hpv and hsv-2 viral cytopathic effects –koilocytes and viral cytoplasmic inclusions– were considered as internal positive controls. previously tested samples from laboratory and clinically confirmed hiv positive and negative cases were used as internal positive and negative controls for hiv testing. p16 regulates the cell cycle by acting as a cyclin-dependent kinase inhibitor and has been used as an alternative biomarker for hpv testing.10,11 thus, serum samples that were positive and negative for p16 (from previous a study) were also used as internal positive and negative controls for hpv testing. p16 kit-included positive and negative controls were considered as external controls (bioassay technology laboratory, yangpu, shangai, china). the cut-off values for positive and negative controls were ≥ 8100 ng/l and ≥ 8099 ng/l, respectively. both internal and external positive and negative controls were used in the calibration and calculation of cut-off values for hpv and hsv-2 antibodies. furthermore, serum samples from cervical cancer patients whose cervical tissues or biopsies tested positive for ebv (lmp-1), hpv and hsv-2 antigen by immunohistochemistry were used as internal positive controls while those whose cervical tissues or biopsies tested negative for the viral antigens by immunohistochemistry were used as internal negative controls (using a commercial kit from bio sb inc., santa barbara, california, united states). in all four positive and four negative controls (based on koilocyte presence, p16 positivity, hpv immunohistochemistry status and manufacturer’s hpv control sample) were used for hpv testing while three positive and three negative controls (based on viral inclusion in cervical cells, hsv-2 immunohistochemistry status, and manufacturer’s hsv-2 control sample) were used for hsv-2 testing. more so, two positive and two negative controls (based on ebv immunohistochemistry status and manufacturer’s ebv control sample) were used for ebv testing. the controls were included during assay (in order to eliminate false positives and forestall false negatives), prior to calculation of cut-offs. the sera were also tested by the elisa method for human proliferation-related ki-67 antigen (ki67; using kits from melsin medical co. ltd, changchun, jilin, china) and squamous cell carcinoma antigen 1 (scca1; using kits from elabscience biotechnology co. ltd, wuhan, hubei, china). using the internal and external control samples, tests for viral antibodies were considered valid when the mean negative control optical density was 0.1 or lower and the mean positive control optical density was 0.8 or higher. based on the formula provided by the manufacturers of the elisa kits, test samples were considered positive when their viral antibody estimates were greater than the cut-off values. the positive cut-off values for hiv, hpv igg, hpv igm, hsv-2 igg, hsv-2 igm, ebv igg, and ebv igm antibodies were 1.077, 1.071, 0.438, 1.520, 0.102, 1.570 and 1.030 or higher, respectively, while the negative cut-off values for the parameters were 1.076, 1.070, 0.438, 1.519, 0.101, 1.569 and 1.029 or lower, respectively. the positive cut-off values for ki67 and scca1 were 7849 ng/ml and 1670 pg/ml or higher while the negative cut-off values were 7848 ng/ml and 1669 pg/ml or lower. gene expression profiling rna isolation and complementary dna synthesis using the optimised phenol-chloroform rna extraction method,12 50 µl of serum was added to eppendorf tubes containing 50 µl trizol reagent and vortexed at 2500 revolutions per minute for 15 min. chloroform (100 µl) was added to the mixture which was subsequently vortexed and centrifuged for 30 min at 1500 revolutions per minute. the supernatants containing the rna were aspirated into new labelled tubes. iso-amyl alcohol (100 µl) was added to the supernatant with subsequent centrifugation for 30 min at 1500 revolutions per minute. the rna was recovered in pellet form following decantation of the supernatant. the rna pellet was washed thrice with 70% ethanol. fifty µl of 70% ethanol was added to the tube and the tube was centrifuged for 5 min at 1500 revolutions per minute. after which, the supernatant was decanted. after washing all tubes were allowed to air dry. fifty microlitre (µl) of nuclease-free water was added to the total rna to form the rna solution. the total rna concentration (48 µl of deionised distilled water + 2 µl of rna solution) was quantified spectrophotometrically at 260 nanometre. the rna limit of importance was set at 0.05–1.00. the complementary dna synthesis was carried out by adding mirna-universal stem-loop primer cocktail (containing nuclease-free water, the reverse transcriptase buffer, the reverse transcriptase, mirnauniversal stem-loop primer specific oligos, and oligo deoxyribonucleotide triphosphate) to 20 µl of total rna of each sample. the samples were then incubated at room temperature overnight. the concentration of complementary dna was determined spectrophotometrically at 260 nanometre and homogeneity was carried out on every sample. reverse transcriptase polymerase chain reaction the amplification was performed using optimisation and the universal stem-loop primer method.13 in order to validate our primers for mirna quantification listed in table 1, we test ran the primers against samples from known cervical cancer patients (as a positive control; n = 10) and patients with normal cervix (negative control; n = 10). the expression of oncomirs were at least fourfold higher in cancer patients than in healthy individuals while the expression of tumour suppressors were at least threefold higher in healthy individuals than in cancer patients. template (complementary dna, 2 µl), nuclease-free water (3 µl), forward primer (0.5 µl) and reverse primer (0.5 µl; inqaba biotechnical industries ltd, hatfield, pretoria, south africa) and master mix (4 µl; new england biolabs gmbh, frankfurt, germany) were added to each sample in no specific order. all reactions were done as singleplexes for each biomarker. amplification conditions were: 94 °c pre-denaturation for 5 min, 94 °c for 30 s, annealing 55 °c for 30 s and extension 72 °c for 30 s and then 5 min at 72 °c by 45 cycles. table 1: list of primer sequences. gel electrophoresis products from polymerase chain reaction (pcr) were electrophoresed in 0.5% of agarose gel using 0.5× tris-borate-ethylenediaminetetraacetic acid buffer of ph 8.3 (2.6 g of tris base, 5.0 g of tris boric acid and 2 ml of 0.5 m ethylenediaminetetraacetic acid; thermo fisher scientific johannesburg pty, germiston, south africa) with 0.2 µl ethidium as a fluorescent tag. the expression products were visualised as bands (amplicons) using an ultraviolet-transilluminator. to minimise image variations, the camera of an iphone (set at 10 cm above the surface of the transilluminator and without flash or zoom; 326 pixels per inch) was used in acquiring snapshots of the amplicons (figures 1e, 2e and 3e). densitometrical analysis of the snapshots was carried out using imagej software (version 1.49; national institute of mental health, bethesda, maryland, united states). using the imagej software adjustment tool, the snapshots were scanned and background impurities (including non-ethidium bromide luminescence and other reflections from gel particles) were eliminated. only the full area of the bands (specific ethidium bromide luminescence) was selected and quantified by the software. results were expressed in figures; three decimal places. relative expression of mirnas and the p53 gene were calculated following endogenous normalisation using mir-16 for mirnas and β-actin for the p53 gene.16 figure 1: relative expression of oncogenic mirnas among individuals with and without viral infection, ogun state, nigeria, 2018. (a) mir-21 expression was higher among participants with hpv mono-infection; (b) mir-182 expression was higher among participants with ebv and hsv-2 bi-infection; (c) mir-155 expression was higher among participants with ebv mono-infection; (d) mir-200c expression was higher among participants with hpv mono-infection; (e) snapshots of gel amplicons of oncomirs and normalising gene (mir-16) based on viral status (plotted as a–d). figure 2: relative expression of tumour suppressor mirnas in individuals with and without viral infection, ogun state, nigeria, 2018. (a) mir-let-7b expression was higher among participants with ebv and hpv bi-infection and lower among participants with ebv mono-infection; (b) mir-143 expression was higher among participants with ebv, hpv and hsv-2 tri-infection and lower among participants with hpv mono-infection; (c) mir-125b expression was higher among participants with ebv mono-infection and lower among participants with ebv, hpv and hsv-2 tri-infection; (d) mir-145 expression was higher among participants with hsv-2 mono-infection and lower among participants with ebv and hpv bi-infection (d); (e) snapshots of gel amplicons of tumour suppressors and normalising gene (mir-16) based on viral status (plotted as a–d). figure 3: biomarker levels and cd4+ t-cell counts among individuals with and without viral infection, ogun state, nigeria, 2018. (a) p53 gene expression was higher among participants with hpv mono-infection and lowest in control group, participants who had no viral infections (none); (b) cd4+ t-cell counts were higher among the control group (none) and lower among those with ebv and hsv-2 co-infection; (c) ki67 expression was higher among participants with hpv infection and lower in the control (none); (d) scca1 expression was higher among participants with hpv mono-infection and lower in those with ebv mono-infection; (e) snapshots of gel amplicons of p53 gene and normalising gene (β-actin) based on viral status (plotted as a). statistical analyses descriptive statistics were carried out to categorise participants into the eight groups: group 1 (none): ebv, hpv and hsv-2 seronegative participants; group 2: ebv mono-infection; group 3: hpv mono-infection; group 4: hsv-2 mono-infection; group 5: ebv/hpv bi-infection; group 6: ebv/hsv-2 bi-infection; group 7: hpv+hsv-2 bi-infection; and group 8: ebv/hpv/hsv-2 tri-infection. the relative expression of mirnas and the p53 gene in the healthy control group (uninfected commercial sex workers) and in the different viral status groups were compared using analysis of variance, partial correlation and pearson’s (bivariate) correlation in statistical package for social sciences (version 21; ibm corporation, armonk, new york, united states). in terms of viral status (coded as ‘0’ for no infection, ‘1’ for ebv mono-infection, ‘2’ for hpv mono-infection and ‘3’ for hsv-2 mono-infection, ‘4’ for ebv and hpv bi-infection, ‘5’ for ebv and hsv-2 bi-infection, ‘6’ for hpv and hsv-2 bi-infection and ‘7’ for ebv, hpv and hsv-2 tri-infection), partial correlation was carried out to determine the relationship between the expression of the biomarkers in all participants. results were presented as mean ± standard error of the mean. statistical significance was set at p-values less than 0.05. results the number (%) of participants (n = 319) in the eight groups were: group 1 (control group/none; virus negative group) = 81 (25.4%), group 2 (ebv mono-infection) = 15 (4.7%), group 3 (hpv mono-infection) = 30 (9.4%), group 4 (hsv-2 mono-infection) = 67 (21.0%), group 5 (ebv and hpv bi-infection) = 5 (1.6%), group 6 (ebv and hsv-2 bi-infection) = 46 (14.4%), group 7 (hpv and hsv-2 bi-infection) = 22 (6.9%) and group 8 (ebv, hpv and hsv-2 tri-infection) = 53 (16.6%). expression of oncomirs in viral infection mir-21 was significantly overexpressed among participants with ebv mono-infection (p = 0.047), hpv mono-infection (p < 0.001), and ebv-hpv bi-infection (p = 0.015) when compared with the control group (figure 1a). a statistically significant overexpression of mir-182 among participants with hsv-2 mono-infection (p = 0.043), ebv-hsv-2 bi-infection (p = 0.002), hpv-hsv-2 bi-infection (p = 0.011), and ebv-hpv-hsv-2 tri-infection (p = 0.018) was observed when compared with the control group (figure 1b). a significant mir-155 overexpression among participants with ebv mono-infection was observed when compared with the control group (p < 0.001; figure 1c). mir-200c overexpression in ebv mono-infection (p = 0.025) and hpv mono-infection (p = 0.003) when compared with the control group was statistically significant (figure 1d). expression of both mir-21 (figure 1a) and mir-200c (figure 1d) were higher among participants with hpv mono-infection than participants with other viral status (test groups) at p = 0.009 and p = 0.073, respectively. in terms of expression level, irrespective of viral status, results showed significant direct relationships between mir-21 and mir-200c (p < 0.001), between mir-182 and scca1 (p = 0.051) and between the p53 gene and some mirnas (mir-21, p < 0.001; mir-155, p < 0.001; mir-200c, p = 0.001). statistically significant inverse relationships were observed between mir-143 and mir-200c (p = 0.025) and between mir-200c and cd4+ t-cell counts (p = 0.048; figures 1d and 3b). in terms of virus status, (infected groups), significant direct relationships were observed between mir-21 and mir-200c (p = 0.053), mir-21 and ki67 (p = 0.045), mir-125b and mir-143 (p < 0.001), and p53 gene and mir-155 (p = 0.039). significant inverse relationships were also observed between mir-200c and mir-143 (p = 0.016), and mir-145 and ki67 (p = 0.001). insignificant direct relationships were also observed between mir-21 and p53 (p = 0.075) and mir-200c and p53 (p = 0.330), while an insignificant inverse relationship was observed between the mir-182 and p53 gene (p = 0.826). expression of tumour suppressors in viral infection higher expression was observed for mir-let-7b in ebv and hpv bi-infection (p = 0.315), mir-143 in ebv-hpv-hsv-2 tri-infection (p = 0.668), mir-125b in ebv mono-infection (p = 0.450) and mir-145 in hsv-2 mono-infection (p = 0.002). lower expression was observed for mir-let-7b in ebv mono-infection, mir-143 in hpv mono-infection and mir-125b in hsv-2 mono-infection (figure 2a–c), when compared with other viral mono-infections. in terms of expression level, significant direct correlations were observed between the following biomarkers: mir143 and mir-145 (p = 0.041), mir-143 and scca1 (p = 0.009), and mir-143 and mir-125b (p = 0.052), irrespective of viral status (figure 2). higher expression of p53 was observed among participants who were seropositive for ebv and hpv mono-infections (p = 0.014) but lower among those seropositive for ebv-hpv-hsv-2 tri-infection when compared with those who were seronegative for the three viruses (figure 3a). the result showed that hsv-2-related infections had lower cd4+ t-cell counts (p = 0.102; 3b). higher and lower serum levels of ki67 were observed in hpv and hsv-2 mono-infections (p = 0.208), when compared with those who were seronegative for the three viruses (figure 3c). lower serum level of scca1 was observed in ebv mono-infection compared with other groups (p = 0.005; figure 3d). expression of biomarkers in viral mono-infection and co-infection data analysis showed a higher expression of the mir-21, mir-155, mir-200c and p53 gene among those with viral mono-infection while a higher expression of mir-182 and lower cd4+ t-cell counts were observed among those with viral co-infection when compared with the control group (figure 4). the expression of mir-155 and mir-200c among women with a viral co-infection were insignificantly higher when compared with their expression in non-infected women. individuals with a viral mono-infection had a higher mir-145 expression, while those with viral co-infection had a lower expression of mir-145 when compared with participants who were without any viral infections (p = 0.015; figure 4). figure 4: expression of biomarkers among individuals with viral mono-infection or co-infection, in the absence of the target viruses, ogun state, nigeria, 2018. expression of mir-21, mir-155, mir-200c, p53 gene and ki67 were higher among participants with viral mono-infection than among those with viral co-infection; mir-182 and mir-143 were higher among participants with viral co-infection than among those with mono-infections; mir-let-7b was lower among participants with viral co-infection than among those with mono-infections; mir-125b expression was higher among participants with viral mono-infection than among those with viral co-infection; mir-145 expression was higher among participants with viral mono-infection and lower among those with viral co-infection, cd4+ t-cell counts was lower among participants with viral co-infection than among those with viral mono-infection; scca1 level was higher among participants with viral co-infection and lower among those with viral mono-infection; when compared with those who had no viral infections. discussion this study estimated the expression of normally downregulated biomarkers (mir-21, mir-155, mir-182, mir-200, p53 gene, ki67 and scca1) and normally upregulated biomarkers (mir-let-7b, mir-125b, mir-143, mir-145 and cd4+ t-cells) in viral mono-infection through tri-infection.6 since overexpression of oncomirs and underexpression of tumour suppressors are usually found in all cancers, especially cervical cancer,17,18,19,20 this study compared the level of biomarkers in groups 1 to 8 in a bid to identify the biomarkers that are severely affected by viral co-infection. although higher expression of mir-21 was observed among participants with hpv mono-infection, overexpression of mir-21 among participants with ebv mono-infection suggests that the virus could play an active role in emt as well. ebv-hpv bi-infection was not associated with higher expressed mir-21 beyond that observed in any of the individual mono-infections, however, it could be inferred that both viruses synergistically dysregulate mir-21. the findings of this study revealed that hsv-2 mono-infection was associated with minimal mir-21-related oncogenic activity when compared with ebv and hpv infection. in other words, this study suggests that hsv-2 mono-infection or its co-infection does not exert substantial oncogenic activity through mir-21 modulation. additionally, we posit that the oncogenic pathway of each virus varies and may slightly be counteractive during viral co-infection, hence the less expression of mir-21 and mir-155c in viral co-infection. this warrants more study. in this study, the overexpression of mir-200c in ebv mono-infection, agrees with the findings of motch et al. which show higher mir-200c expression in ebv-positive tumours than in ebv-negative tumours.17 the expression of mir-200c was insignificantly higher in women with viral co-infection than in those who were uninfected. additionally, the expression of mir200c was significantly higher in women with hpv mono-infection than in women with co-infection, including hsv-2 mono-infection. this also suggests that mir-200c does not play any active roles in oncogenicity associated with viral co-infection. the reason for this is still unknown, but it could be related to differences in the mirna regulatory pathway or the targeted protein of the investigated viruses. the high expression of mir-200c in hpv mono-infection could be due to its tumourigenic e6/e7 protein.18 again, hsv-2 infection was not associated with substantial dysregulation of mir-200c. thus, it could be inferred that mir-200c is not directly involved in hsv-2-associated cervical carcinogenesis. in this study, the significant overexpression of mir-155 only in ebv mono-infection suggests that the mirna expression could be virus dependent and also could be an early indication of ebv infection, immune activation, genetic instability or imminent tumourigenesis.19,20 an inverse relationship was observed between mir-155 and mir-let-7b expression in ebv mono-infection. this is in line with the findings of an earlier study which show that the expression of the let-7 family is suppressed in ebv-associated cervical cancer.21 since studies have shown that hpv induces genetic instability and emt by upregulating mir-182, it could be inferred that hsv-2 plays a similar role in cervical carcinogenesis by upregulating mir-182.18,22 the higher upregulation of mir-182 in viral co-infection when compared with viral mono-infection suggests that ebv, hpv and hsv-2 co-infection synergistically modulates the biomarker, possibly through a similar target protein. the triple impact of the viruses also suggests that the rate of emt could be faster in women with viral co-infection than in women with mono-infection. again, further studies are required. the findings of the current study also revealed that, although not statistically significant, the expression of ki67 and scca1 was relatively higher among participants with a viral infection and that a significant increase in these biomarkers could be indicative of cancer. viruses, individually or synergistically, have developed unique mechanisms for hijacking or inhibiting the tumour suppressive functions of wildtype p53.23 since there were direct relationships between the p53 gene, mir-21 and mir-200c in terms of viral infection, the higher expression of the p53 gene (tumour suppressor) in participants with ebv and hpv mono-infections could be an immunologic response to higher mir-21 and mir-200c (oncogenes) among participants with mono-infection. this could be an early indicator of cell cycle dysregulation. this higher expression of the p53 gene in hpv mono-infection was also observed by zatonski et al.24 continual reduction in its expression or its mutation during viral co-infection due to immune exhaustion, as seen in this study, or underexpression may trigger cervical carcinogenesis. microrna-125b is abundantly expressed in the healthy cervix. it increases shortly after hpv infection and is inactivated or relatively decreased as cancer develops.25,26 decreases in mir-125b expression and the development of cancer may occur due to viral co-infection, especially ebv-hpv-hsv-2 co-presence. underexpression of mir-143 and mir-145 has also been reported in cervical cancer, especially in the presence of hpv type 31 e7 oncoprotein.18,27,28,29 in this study, while mir-143 overexpression was associated with viral co-infection, mir-145 underexpression was associated with viral co-infection, irrespective of hsv-2 status. this suggests that mir-143 overexpression and mir-145 underexpression could be early indicators of viral co-infection and emt. it also suggests that ebv and hpv may synergistically and more aggressively promote emt than either of them could have done unilaterally. although mir143 and mir145 are tumour suppressors, the divergent expression of both mirnas in different viral status suggests that they may have dual functions. the latter may be an explanation for the discordant reports on the expression of both biomarkers in cervical cancer.18,22,30,31 furthermore, the higher expression of mir-143 and mir-145, including mir-let-7b and mir-125b, among women with hsv-2 mono-infection when compared with the control group suggests that the mirnas do not play any significant role in hsv-2-associated oncogenic activity. however, it could be argued that hsv-2 infection contributes to cervical carcinogenesis by reducing cd4+ t-cell counts. this is underscored by the finding that a decrease in cd4+ t-cell counts by 100 cells/mm3 increases the risk of cervical lesions by 13% to 18%.32 limitations the expression of mirnas in a few cases of ebv-hpv bi-infection (n = 5) and ebv mono-infection (n = 15) as seen in this study may not allow for conclusive generalisation in the investigated population. in other words, a clear and significant impact of ebv-hpv bi-infection and ebv mono-infection on mirnas may be observed when a larger population is investigated. however, this study could serve as a baseline for future studies. additionally, the use of conventional reverse transcriptase pcr in this study is a limitation, as expression levels had to be determined by densitometric analysis. we believe that the use of quantitative real-time pcr could have yielded superior results than reverse transcriptase pcr. conclusion the findings of this study show that while ebv and hpv infections may synergistically target mir-21 and mir-200c, hsv-2 and its related co-infections may target mir-182 and cd4+ t-cell activity to initiate emt. thus, this study suggests that viral co-presence increases the risk of cervical carcinogenesis by downregulating mir-145, upregulating mir-182 and suppressing cd4+ t-cell activity. acknowledgements special thanks are extended to staff of the centre for biocomputing and drug development, adekunle ajasin university and hiv counseling and testing and family planning clinics, state hospital abeokuta, for their technical assistance. competing interests the authors have declared that no competing interests exist. authors’ contributions j.o.o. and a.a.n. designed the study and wrote the manuscript. c.c.o. made conceptual contributions. j.o.o. and c.c.o. carried out elisa investigations and interpreted biochemical results. j.o.o. and d.i.d. carried out the cervical screening exercise and literature searches. j.o.o. and o.o. carried out the mirna laboratory investigations and interpreted results. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references khenchouche a, sadouki n, boudriche a, et al. human papillomavirus abd epstein-barr virus co-infection in cervical carcinoma in algerian women. virol j. 2013;10:340. https://doi.org/10.1186/1743-422x-10-340 kahla s, oueslati s, achour m, et 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control genome amplification. j virol. 2013;87(10): 6037–6043. https://doi.org/10.1128/jvi.00153-13 lee jw, choi ch, choi jj, et al. altered microrna expression in cervical carcinomas. clin cancer res. 2008;14(9):2535–2325. https://doi.org/10.1158/1078-0432.ccr-07-1231 dreher a, rossing m, kaczkowski b. differential expression of cellular micrornas in hpv 11, -16, and -45 transfected cells. biochem biophys res commun. 2011;412:20–25. https://doi.org/10.1016/j.bbrc.2011.07.011 omar t, schwartz s, hanrahan c, et al. progression and regression of premaligant cervical lesions in hiv-infected women from soweto: a prospective cohort. aids. 2011;25(1):87–94. https://doi.org/10.1097/qad.0b013e328340fd99 article information authors: edwin shumba1 phoebe nzombe1 absolom mbinda1 raiva simbi2 douglas mangwanya2 peter h. kilmarx3 elizabeth t. luman4 sibongile n. zimuto1 affiliations: 1zimbabwe national quality assurance programme (zinqap) trust, zimbabwe 2ministry of health and child welfare (mohcw), zimbabwe 3us centers for disease control and prevention (cdc), zimbabwe 4us centers for disease control and prevention (cdc), united states correspondence to: edwin shumba postal address: po box a1955, avondale, harare, zimbabwe dates: received: 09 sept. 2014 accepted: 13 sept. 2014 published: 03 oct. 2014 how to cite this article: shumba e, nzombe p, mbinda a, et al. weighing the costs: implementing the slmta programme in zimbabwe using internal versus external facilitators. afr j lab med. 2014;3(2), art. #248, 6 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.248 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. weighing the costs: implementing the slmta programme in zimbabwe using internal versus external facilitators in this original research... open access • abstract • introduction • research methods and design    • slmta programme    • expenditure analysis    • theoretical estimate of expenditures    • projected financial costs of national expansion • results • discussion    • limitations    • conclusion • acknowledgements    • competing interests    • authors’ contributions    • cdc disclaimer • references abstract top ↑ background: in 2010, the zimbabwe ministry of health and child welfare (mohcw) adopted the strengthening laboratory management toward accreditation (slmta) programme as a tool for laboratory quality systems strengthening. objectives: to evaluate the financial costs of slmta implementation using two models (external facilitators; and internal local or mohcw facilitators) from the perspective of the implementing partner and to estimate resources needed to scale up the programme nationally in all 10 provinces. methods: the average expenditure per laboratory was calculated based on accounting records; calculations included implementing partner expenses but excluded in-kind contributions and salaries of local facilitators and trainees. we also estimated theoretical financial costs, keeping all contextual variables constant across the two models. resource needs for future national expansion were estimated based on a two-phase implementation plan, in which 12 laboratories in each of five provinces would implement slmta per phase; for the internal facilitator model, 20 facilitators would be trained at the beginning of each phase. results: the average expenditure to implement slmta in 11 laboratories using external facilitators was approximately us$5800 per laboratory; expenditure in 19 laboratories using internal facilitators was approximately $6000 per laboratory. the theoretical financial cost of implementing a 12-laboratory slmta cohort keeping all contextual variables constant would be approximately $58 000 using external facilitators; or $15 000 using internal facilitators, plus $86 000 to train 20 facilitators. the financial cost for subsequent slmta cohorts using the previously-trained internal facilitators would be approximately $15 000, yielding a break-even point of 2 cohorts, at $116 000 for either model. estimated resources required for national implementation in 120 laboratories would therefore be $580 000 using external facilitators ($58 000 per province) and $322 000 using internal facilitators ($86 000 for facilitator training in each of two phases plus $15 000 for slmta implementation in each province). conclusion: investing in training of internal facilitators will result in substantial savings over the scale-up of the programme. our study provides information to assist policy makers to develop strategic plans for investing in laboratory strengthening. introduction top ↑ public health laboratories play a central role in disease detection, prevention and control. high-quality diagnostics and monitoring are critical to ensure better patient outcomes. in response, developing countries have started paying more attention to efforts to expand laboratory capacity, strengthen laboratory systems and improve laboratory quality.1 the strengthening laboratory management toward accreditation (slmta) programme was launched in 20092and has since been implemented in 47 countries in africa, southeast asia, the caribbean and latin america.3 the programme has demonstrated measurable improvement in laboratories as a result of an approach that incorporates improvement projects and structured supervisory visits, allowing extension of learning beyond the classroom.4 zimbabwe is a resource-limited country with an annual per capita health expenditure of us$16,5 well below the world health organization’s recommended minimum of $34.6 inadequate numbers of well-trained laboratory staff and lack of training on laboratory quality management systems are major challenges facing the laboratory system in zimbabwe.1,7 to address these gaps, the us president’s emergency plan for aids relief (pepfar), through the us centers for disease control and prevention (cdc), in 2010 made available a five-year grant to the zimbabwe ministry of health and child welfare (mohcw). this funding was earmarked for strengthening laboratory systems through a cooperative agreement with the zimbabwe national quality assurance program trust (zinqap), a local not-for-profit organisation which focuses on laboratory quality assurance issues in zimbabwe. in 2010, the mohcw’s department of laboratory services embarked on a five-year strategic plan, one objective of which was to implement quality management systems.8 the laboratory services directorate adopted slmta as a tool for laboratory quality systems strengthening and engaged zinqap as the programme provider. evaluation of financial costs, or expenditures, of implementing the slmta programme is essential in order to guide policy makers and prioritise scarce resources.9 cost analysis is a critical tool in understanding the value of programmes and projecting budgets for scale up.10 the aim of this study is to provide a partial financial expenditure analysis of the slmta programme in zimbabwe from the programme provider’s perspective, with a focus on expenditure comparison between using external (international) versus internal (country-based mohcw) facilitators. these two models have substantial upfront and ongoing cost implications and zimbabwe has used both in its slmta implementation and expansion. we evaluate the financial costs of slmta implementation using both external and internal facilitators and project the financial costs of scaling up the programme nationally. research methods and design top ↑ slmta programme details of slmta programme implementation have been described elsewhere.2 to date, the slmta programme in zimbabwe has been implemented in three cohorts; only cohorts i (implemented in 2010–2011) and iii (implemented in 2012–2013) are included in this analysis. cohort ii was conducted using a different model and detailed cost data were not available. the slmta programme in zimbabwe included four components: (1) baseline audits conducted by zinqap staff; (2) three slmta workshops of four days each; (3) supervision of the implementation of improvement projects following each workshop; and (4) exit audits. mentorship, which is often incorporated into slmta implementation, was not conducted for these two cohorts during the training because of financial constraints and availability of staff.two implementation models were used – one with external workshop facilitators and auditors; and the other with internal workshop facilitators and auditors following extensive training. for cohort i, 11 laboratories were trained using an external facilitator based in uganda, along with three local facilitators. the exit audit was conducted by three external auditors from uganda, lesotho and botswana. cohort iii used four internal facilitators who were trained in a 13-day training-of-trainers course followed by a six-day auditor training. the exit audit was also conducted by these internal facilitators. this cohort included 19 laboratories (13 in zimbabwe and six in namibia). expenditure analysis a partial expenditure analysis was conducted of the implementation of the slmta programme, using both external and internal facilitators. we evaluated the expenditures from a programme perspective, including only direct costs borne by zinqap, the programme provider, whilst excluding in-kind contributions and salaries of local facilitators and trainees. expenditures were collected using a detailed inventory (payment vouchers and general ledger) of all resources associated with the slmta programme and the training of local facilitators. expenditures were tallied for each of the four programme components and then categorised into training equipment, training (facilities and materials), trainers and supervisors (transport; accommodation and per diem; and fees) and participants (transport; accommodation and per diem). for cohort iii, expenditures for supervision and auditing of the six laboratories from namibia were not available. we therefore estimated costs as though they had been in-country, using an inflation factor based on average expenditures for these activities for the zimbabwean laboratories. we also calculated the cost of training local facilitators in training-of-trainers and auditing courses. these expenditures were entered into costit software version 4.5 (world health organization 2007) and analysed in the financial analysis mode. costit is software designed to record and analyse economic and financial data. all expenditures are expressed in us dollars, which has been the official national currency of zimbabwe since 2009. we used a ‘top-down’ cost accounting approach, which starts with total expenditures and then divides by the number of trained individuals and the number of laboratories to yield the average cost per trained individual and average cost per trained laboratory, respectively. theoretical estimate of expenditures expenditures of the two cohorts had considerable inherent variability resulting from contextual factors. for example, the rental cost of the training facility varied from workshop to workshop because of seasonality and fluctuations in the economy. also, for cohort i, zinqap paid per diems to participants, whilst for cohort iii, no participant per diem was paid because of budget constraints. to better compare the two models, we estimated slmta implementation costs based on a theoretical scenario, keeping all contextual variables constant across the two models. contextual variables included facility rental fees, training materials, number of laboratories and participants. expenditure assumptions for this model were based on the expert estimation of zinqap programme implementers, using lessons learned from conducting the two previous cohorts and a normative budgeting approach. projected financial costs of national expansion outputs for the two models based on theoretical financial costs when holding constant contextual variables were used to project the cost of scaling up the slmta programme nationally. we based these estimates on a two-phase implementation plan in which half of the 10 provinces in zimbabwe would implement slmta in each phase. slmta would be rolled out in a series of five cohorts (one per province) of 12 laboratories each, for a total of 60 laboratories per phase. for the internal facilitator model, one facilitator training would be held per phase, training four people per province to conduct the slmta programme locally. time required to implement a phase will depend on resources available; we therefore did not specify a timeframe. results top ↑ the total expenditures for implementation of the slmta programme in 11 laboratories using external facilitators were approximately $64 000 (table 1). the total expenditures in 19 laboratories using internal facilitators were approximately $28 000, plus $84 000 for facilitator training. this yields an average cost per laboratory of approximately $5800 using external facilitators and $5900 using internal facilitators ($3200 and $4700 per person trained, respectively). table 1: partial expenditure estimates for the slmta program in zimbabwe. when keeping all contextual variables constant across the two models, the theoretical financial cost of implementing slmta in 12 laboratories using external facilitators would be approximately $58 000, whilst the theoretical cost of implementing slmta in the same 12 laboratories using internal facilitators would be approximately $15 000, plus $86 000 to train 20 facilitators (table 2). the estimated financial cost to implement subsequent slmta cohorts would remain $58 000 per cohort using external facilitators and would be approximately $15 000 per cohort using the previously-trained internal facilitators, yielding a break-even point of 2 cohorts, at $116 000 for either model. table 2: theoretical cost estimates for the slmta program in zimbabwe. in the external facilitator model, the majority of the financial costs would be spent on slmta workshops (59%) and exit audits (37%) (figure 1a). for the internal facilitator model, the majority of the cost for the first slmta cohort would be spent on conducting the facilitator training (85%), with approximately 12% being spent on conducting workshops. for subsequent cohorts with internal facilitators, the majority of the financial cost would go toward the workshops (83%), with approximately 9% for supervision. figure 1: projected distribution of costs by (a) slmta component, and (b) expenditure category. examining expenditures by expenditure category, the majority of the cost of the external facilitator model would be fees for trainers and supervisors (78%), whilst the remainder would be spent on training (22%) (figure 1b). for the first cohort using the internal facilitator model, financial costs would be divided fairly evenly between participant expenses (44%), training (30%) and trainers and supervisors (22%). in subsequent cohorts using internal facilitators, training costs would account for the majority of expenditures (83%), with trainers and supervisor expenses accounting for the remaining 17%. the projected financial cost of scaling up the slmta programme nationally is shown in figure 2. implementation in 120 laboratories (12 in each of the 10 provinces) would cost approximately $580 000 using the external facilitator model and $322 000 using the internal facilitator model. the initial investment of training 20 internal facilitators ($86 000) will pay for itself by the second slmta cohort, after which the programme will benefit from a cost saving of $43 000 (74%) per cohort for the remainder of the cycle. figure 2: projected cost of scaling up strengthening laboratory management toward accreditation to 10 provinces using external and internal facilitators. discussion top ↑ this is the first detailed expenditure analysis of slmta implementation to be published. we found that programme expenditures to implement slmta were less than $6000 per laboratory. yao et al. have shown that slmta implementation results in substantial improvements in laboratory quality.3 collectively, zimbabwe’s cohort i laboratories conduct more than 1.5 million tests each year and their median audit scores more than doubled from preto post-slmta.3 given the dramatic improvements and relatively low financial cost of slmta implementation found in the current study, it can be argued that slmta is well worth the investment in time and resources.whilst the estimated financial cost to implement a single slmta cohort is lower using external facilitators, the upfront investment of training local facilitators pays for itself by the second cohort, resulting in a 44% cost savings over national scale up of the programme. in addition, training local laboratorians to be slmta facilitators builds long-term indigenous capacity of the laboratory programme, enabling these individuals to conduct in-country laboratory audits, assist laboratory staff in other activities and improve the overall quality of laboratory management throughout the country. in this case, it is critical to minimise staff turnover between the training and slmta implementation so as to ensure a return on investment. some strategies may include an agreement between the mohcw and supervisors not to reassign the trainers for a period of 2 years; financial incentives to the participants upon completion of the slmta programme; and binding contracts in which the participant agrees to remain on the job for a specified period or until the conclusion of slmta implementation in their province. bringing in external facilitators requires high transport costs as well as facilitators’ fees. because we assumed that the internal facilitators would be employed by mohcw and work within their own provinces, these costs would be minimised. we found that the financial costs of slmta implementation are sensitive to participant and trainer travel and per diems. in cohort i, when participants were provided accommodation and per diem, this cost was nearly 40% of the entire financial cost of slmta implementation. hence, decentralising the slmta programme to each province, minimising necessary travel for participants, will reduce costs substantially. a study in cameroon also found that decentralised slmta training enables more laboratory staff to be trained, increasing local capacity and sustainability.11 use of internal facilitators will make decentralisation more feasible, as facilitators can be selected strategically from a wide geographic distribution. slmta is often coupled with on-site mentorship in a variety of models so as to boost improvements even further.12 our analysis did not include mentorship costs, because mentorship was not incorporated in the slmta programme for these two cohorts. bringing in external mentors would increase the cost of the programme substantially; however, given that internal facilitators can receive mentorship training, they could also be used in this capacity to reduce costs and have a positive impact on programme implementation. limitations this study is subject to some limitations. firstly, several expenses associated with slmta implementation, such as overhead costs, vehicles and salaries, were provided in kind and thus not included in this analysis. our results may therefore underestimate the financial cost of implementing slmta in other settings. also not included was the cost of conducting improvement projects within the laboratories, as this was financed by the individual laboratories; however, slmta is designed to focus on management behaviours and participants are encouraged to identify solutions to problems within their existing resources. expenditures were reported as incurred at the time of implementation, not adjusted for inflation or annuitised over years of useful life. because inflation has varied widely over the past decade in zimbabwe,13 we did not attempt to adjust for it, but based estimates on approximate costs in 2013–2014. secondly, there is a level of uncertainty inherent in the assumptions of our theoretical models. however, the resulting financial cost estimates were fairly close to the actual financial costs experienced by the programme, suggesting that they may indeed be realistic. finally, this analysis is not meant to be an exhaustive assessment of all potential models, but rather a comparison of two models that have been used in zimbabwe. additional studies are needed to identify the most cost-effective models for various situations and to conduct an overall economic evaluation of the slmta programme. countries considering the implementation of slmta will need to examine the expected costs of each of the various components of programme implementation, taking into account local parameters and potential support from partners. conclusion our study suggests that national scale up of the slmta programme in zimbabwe would cost approximately $322 000. countries considering slmta implementation should weigh the pros and cons of investing in training of internal staff to act as programme facilitators. in zimbabwe, investing in training of local facilitators will result in a nearly 50% decrease in the costs of national expansion, as well as develop in-country capacity of laboratory managers and mentors, supporting programme sustainability. acknowledgements top ↑ we would like to acknowledge the zinqap finance office staff for their support and sharing data. this research has been supported by pepfar through the cdc. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions e.s. (zinqap) designed the study, collected and analysed data and wrote the manuscript. p.n. (zinqap), r.s.1 (mohcw), d.m. (mohcw), p.h.k. (cdc, zimbabwe) and s.n.z.1 (zinqap) implemented the programme and revised the manuscript. a.m. (zinqap) reviewed the manuscript and assisted in the data analysis. e.t.l. (cdc, united states) designed the study and substantially revised the manuscript. cdc disclaimer the findings and conclusions in this paper are those of the authors and do not necessarily represent the official position of the cdc. references top ↑ 1. nkengasong jn, nsubuga p, nwanyanwu o, et al. laboratory systems and services are critical in global health: time to end the neglect? am j clin pathol. 2010;134(3):368–373. http://dx.doi.org/10.1309/ajcpmpsinq9brmu62. yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative approach to accelerate laboratory accreditation. am j clin pathol. 2010;134(3):401–409. 3. yao k, luman et, slmta collaborating authors. evidence from 617 laboratories in 47 countries for slmta-driven improvement in quality management systems. afr j lab med. 2014;3(2), art. #262, 11 pages. http://dx.doi.org/10.4102/ajlm.v3i2.262 4. yao k, maruta t, luman et, nkengasong jn. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. 2014;3(2), art. #194, 8 pages. http://dx.doi.org/10.4102/ajlm.v3i2.194 5. community working group on health in zimbabwe. post health budget analysis 2013. harare; 2013 (unpublished). 6.commission on macroeconomics and health. macroeconomics and health: investing in health for economic development. report of the commission on macroeconomics and health (chaired by jeffrey d. sachs) [document on the internet]. c2001. [cited 2014 sep 26]. available from: http://whqlibdoc.who.int/publications/2001/924154550x.pdf 7. birx d, de souza m, nkengasong jn. laboratory challenges in the scaling up of hiv, tb, and malaria programs: the interaction of health and laboratory systems, clinical research, and service delivery. am j clin pathol. 2009;131(6):849–851. http://dx.doi.org/10.1309/ajcpgh89qdswfons 8. zimbabwe ministry of health and child welfare. laboratory service national strategic plan 2010–2014. harare; 2010 (unpublished). 9. de allegri m, marschall p, flessa s, et al. comparative cost analysis of insecticide-treated net delivery strategies: sales supported by social marketing and free distribution through antenatal care. health policy plan. 2010;25(1):28–38. http://dx.doi.org/10.1093/heapol/czp031 10. guinness l, levine r, weaver m. 10 best resources in … cost analysis for hiv/aids programmes in low and middle income countries. health policy plan. 2004;19(4):242–245. http://dx.doi.org/10.1093/heapol/czh029 11. ndasi j, dimite l, mbome v, et al. decentralised facility-based training as an alternative model for slmta implementation: the cameroon experience. afr j lab med. 2014;3(2), art. #231, 6 pages. http://dx.doi.org/10.4102/ajlm.v3i2.231 12. nzombe p, luman et, shumba e, et al. maximising mentorship: variations in laboratory mentorship models implemented in zimbabwe. afr j lab med. 2014;3(2), art. #241, 8 pages. http://dx.doi.org/10.4102/ajlm.v3i2.241 13. central intelligence agency. the world factbook [page on the internet]. c2013 [cited 2014 aug 31]. available from: https://www.cia.gov/library/publications/the-world-factbook/index.html abstract introduction methods results discussion acknowledgements references about the author(s) katherine klein centers for disease control and prevention, national center for hiv/aids, viral hepatitis, std and tb prevention, division of tb elimination, atlanta, georgia, united states kyle degruy centers for disease control and prevention, division of global hiv and tb, international laboratory branch, atlanta, georgia, united states zilma rey centers for disease control and prevention, division of global hiv and tb, international laboratory branch, atlanta, georgia, united states patricia hall centers for disease control and prevention, division of global hiv and tb, international laboratory branch, atlanta, georgia, united states andrea kim centers for disease control and prevention, division of global hiv and tb, epidemiology and surveillance branch, atlanta, georgia, united states steve gutreuter centers for disease control and prevention, division of global hiv and tb, health informatics, data management and statistics branch, atlanta, georgia, united states heather alexander centers for disease control and prevention, division of global hiv and tb, international laboratory branch, atlanta, georgia, united states citation klein k, degruy k, rey z, et al. a global proficiency testing programme for xpert® mtb/rif using dried tube specimens, 2013–2015. afr j lab med. 2020;9(1), a1167. https://doi.org/10.4102/ajlm.v9i1.1167 original research a global proficiency testing programme for xpert® mtb/rif using dried tube specimens, 2013–2015 katherine klein, kyle degruy, zilma rey, patricia hall, andrea kim, steve gutreuter, heather alexander received: 14 jan. 2020; accepted: 12 aug. 2020; published: 27 nov. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: proficiency testing (pt) is an important quality assurance measure toward ensuring accurate and reliable diagnostic test results from clinical and public health laboratories. despite the rapid expansion of the xpert® mtb/rif assay for the detection of tuberculosis in resource-limited settings (rls), low-cost pt materials for xpert mtb/rif external quality assessment (eqa) are not widely available. objective: we sought to determine whether a dried tube specimen (dts)-based pt programme would be a feasible option to support xpert mtb/rif eqa in rls. methods: between 2013 and 2015, the united states centers for disease control and prevention developed and conducted a voluntary eqa programme using dts-based pt material. eight rounds of pt, each comprising five dts samples, were provided to enrolled testing sites. after each round, participant results were compared to expected results, scored as satisfactory or unsatisfactory, and sites were provided with performance reports. results: programme enrolment increased from 102 testing sites in seven countries to 441 testing sites in 14 countries over the course of three years. in each pt round, approximately 90% of participating sites demonstrated satisfactory performance. in seven of the 14 enrolled countries, the proportion of sites with a satisfactory score increased between the first round of participation and the most recent round of participation. conclusion: this programme demonstrated that it is possible to implement an xpert mtb/rif pt programme for rls using dts, that substantial demand for xpert mtb/rif pt material exists in rls, and that country performance can improve in a dts-based pt programme. keywords: external quality assessment; xpert® mtb/rif; proficiency testing; dried tube specimens; resource-limited settings. introduction the world health organization recommends the xpert® mtb/rif (mycobacterium tuberculosis/rifampicin) assay (cepheid, sunnyvale, california, united states) as one of the initial diagnostic tests for all individuals evaluated for tuberculosis.1,2 the xpert mtb/rif assay is an integrated sputum processing and real-time polymerase chain reaction system that rapidly and simultaneously detects m. tuberculosis and the presence of mutations conferring resistance to rifampicin.3 the roll-out of the xpert mtb/rif assay to resource-limited settings (rls) with a high tuberculosis burden has been remarkable. by the end of 2016, 6659 genexpert instruments had been purchased at concessional prices for use at multiple levels of the tiered laboratory network.4 external quality assessment is a critical component of a laboratory quality management system. proficiency testing (pt) is one means of conducting external quality assessment for tests in clinical laboratories. proficiency testing provides objective evidence of accurate testing and may identify areas in the diagnostic process, including pre-analytic, analytic, and post-analytic phases, where quality improvement is needed.5,6 the rapid scale-up of xpert mtb/rif testing in rls has not coincided with the same investment in quality assurance activities for the assay. few pt providers for the xpert mtb/rif assay offer materials to sites in rls, leaving a substantial gap in external quality assessment implementation that could otherwise help to confirm site competency in assay administration and accuracy of test results for limited-resource countries.7,8,9 the united states centers for disease control and prevention (cdc) developed a pt panel for xpert mtb/rif, using a dried tube specimen (dts)-based method.10 the dts technique has successfully been used to develop pt panels for hiv, malaria and syphilis diagnostic assays in rls.11,12,13 our goal in this evaluation was to assess the performance and utility of cdc-prepared dts panels in an xpert mtb/rif pt programme. methods ethical considerations no patient specimen collection was required and no patient identifiers were recorded on quality assurance tools. participation in the xpert mtb/rif quality assurance and pt programme was voluntary and free of charge. no incentives were provided. this study was approved by the cdc center for global health division of human research protection (cgh hsr tracking number 2014-082). dried tube specimen production the method for producing dts was described previously by degruy et al.10 briefly, dts were produced by chemically inactivating liquid cultures of well-characterised mycobacterial strains using equipment and supplies commonly found in laboratories conducting tuberculosis liquid culture.14 mycobacteria were grown in bactec® mgit 7 ml tubes (becton, dickinson and company, sparks, maryland, united states) and inactivated using a 2:1 ratio of xpert mtb/rif sample reagent (cepheid, sunnyvale, california, united states) to mgit culture incubated for 2 hours with intermittent vortexing. inactivated cultures were washed, concentrated, resuspended, homogenised and diluted, before being dispensed into cryovials and air-dried in a class ii biological safety cabinet within a biosafety level iii tuberculosis containment laboratory. inactivation verification was performed by inoculating 0.5 ml of undiluted stock solution into mgit tubes supplemented with polymyxin b, amphotericin b, nalidixic acid, trimethoprim, and azlocillin (panta) (becton, dickinson and company, sparks, maryland, united states) and incubating for a total of 84 days over two incubation cycles in the bactec® mgit 960® instrument (becton, dickinson and company, sparks, maryland, united states) to confirm a lack of growth. dried tube specimens were validated prior to distribution by testing 10% of dts produced with the xpert mtb/rif assay (cepheid, sunnyvale, california, united states), as described previously.10 to test dts, the dts were rehydrated with 2.5 ml of sample reagent, shaken vigorously 20 times and incubated at room temperature for 15 minutes with additional shaking repeated after 10 min. the xpert mtb/rif cartridges were inoculated with approximately 2.0 ml of rehydrated samples using the transfer pipette provided with the kit, and tested immediately on a genexpert iv or genexpert viii using genexpert dx software version 4.0 (cepheid, sunnyvale, california, united states) according to manufacturer instructions.15 proficiency testing programme in 2013, select countries in sub-saharan africa, southeast asia, and the caribbean, supported by the united states president’s emergency plan for aids relief were invited to participate in cdc-atlanta’s pilot dts xpert mtb/rif pt programme at no cost. seven countries elected to participate in the pilot, designated a pt country coordinator, and enrolled testing sites that routinely conducted xpert mtb/rif testing on patient specimens. these countries continued to enrol additional sites throughout 2013, and four additional countries enrolled in subsequent 2013 pt rounds. all countries remained enrolled throughout 2014. in 2015, enrolment was extended to additional sites in all participating countries plus three new countries in africa. the pt kits included a panel of five dts samples, five transfer pipettes, testing instructions, and a paper form for recording results. four pt rounds were distributed quarterly in 2013 (2013-a, 2013-b, 2013-c and 2013-d). only one pt round was distributed at the end of 2014 (2014-a) to allow for evaluation of 2013 data. three pt rounds were distributed in 2015 (2015-a, 2015-b and 2015-c). country coordinators were responsible for distributing the panels to enrolled testing sites, collating results (including transferring data from paper forms to a template microsoft excel file (microsoft corp, redmond, washington, united states) and submitting data to cdc-atlanta via email within nine weeks of panel receipt. a sufficient number of pt kits were sent to enrolled countries to allow for distribution of one pt kit to each enrolled site; enrolled sites that did not submit results were categorised as enrolled but not participating in a given pt round. participant data were analysed by cdc-atlanta using microsoft excel. a report was generated for each participating site; this compared the site’s reported results to expected results (cdc-atlanta validation results) and included the consensus results from all the participating testing sites in all the ountries. consensus results detailed the number of testing sites detecting and not detecting m. tuberculosis complex, detecting and not detecting rifampicin resistance, reporting indeterminate rifampicin resistance, reporting uninterpretable tests (error, invalid, or no result), and not reporting m. tuberculosis complex and/or rifampicin results for each sample in the panel. to establish a consensus result for each pt sample in the round, we required at least 80% of participant results for that sample to match the expected result. in the 2013 pilot phase, participating sites were provided with qualitative reports indicating overall concordance of submitted results with the expected results. beginning in 2014, participating sites were assigned a quantitative score for each pt round. scores were calculated by assigning a value of 20 points to each of the five dts, for a total of 100 possible points. if the site’s qualitative m. tuberculosis complex and rifampicin resistance detection results matched the expected results for a given sample, 20 points were awarded. if either the m. tuberculosis complex detection result or the rifampicin resistance detection result did not match the expected results, zero points were awarded. if the m. tuberculosis complex detection result matched the expected result but the site’s rifampicin resistance detection result was indeterminate, 10 points were awarded. if a site reported an uninterpretable test, five points were awarded. if a site did not report a result, zero points were awarded. a total score of greater than, or equal to, 80 points was considered satisfactory for the pt round. for this evaluation, 2013 results were retrospectively scored to compare performance across all years of the programme. to further investigate concordance and discordance of site results with expected results, additional analyses were conducted in sas version 9.3 (sas institute, cary, north carolina, united states). results were categorised as concordant if both the qualitative m. tuberculosis complex and rifampicin results matched the expected results. if discordant, results were categorised according to the type of discordance (false-positive m. tuberculosis complex detection, unsuccessful run, etc.). tests were considered unsuccessful when the test was started, but a definitive result was not obtained due to an error, invalid result, power failure during testing, or similar problem. tests were also considered unsuccessful if the site chose not to test the sample, was unable to test the sample due to unavailability of xpert mtb/rif kits, or failed to report either the m. tuberculosis complex or rifampicin detection result. frequencies for results in each category were calculated by pt sample and in aggregate for all samples. the mycobacterial strain used to produce the pt sample and the mean cycle threshold (ct) of probe a during validation of the pt sample were included in the analysis to look for trends between these variables and the amount and type of discordance observed. in line with previous studies, probe a was selected for most analyses as it was the first probe to reach the detection threshold.7 we conducted additional statistical analyses to test for an association between the mean ct of probe a during validation and several types of discordance including false-negative m. tuberculosis complex detection, false-positive rifampicin resistance, and indeterminate rifampicin resistance. logistic regression of the type of discordance on mean ct of probe a was conducted using r glm (r foundation for statistical computing, vienna, austria).16 results enrolment in the dts-based xpert mtb/rif pt programme increased steadily from round 2013-a to round 2015-c, with 102 sites in seven countries enrolled in round 2013-a and 441 sites in 14 countries enrolled in round 2015-c (figure 1). participation by enrolled sites increased from 68 sites in six countries in round 2013-a to 342 sites in 13 countries in round 2015-c. the participation among sites ranged from a minimum of 66.7% in round 2013-a to a maximum of 83.8% in round 2015-b. figure 1: number of sites enrolled, participating, and achieving a passing score in dried tube specimen-based xpert® mtb/rif proficiency testing programme, 2013–2015. the proportion of participating sites with a satisfactory score ranged from a minimum of 88.1% in round 2015-b to a maximum of 93.1% in round 2014-a (table 1). individual countries had as few as 50% and as many as 100% of their participating sites achieve satisfactory scores. in seven of the 14 enrolled countries, the proportion of sites with a satisfactory score increased between the first round of participation and the most recent round of participation. of the remaining seven countries, the proportion of sites with a satisfactory score remained constant in three countries but decreased in four countries (table 1). table 1a: number and percentage of sites participating and achieving passing scores in dried tube specimen-based xpert® mtb/rif proficiency testing programme for participating countries, 2013–2015. table 1b: number and percentage of sites participating and achieving passing scores in dried tube specimen-based xpert® mtb/rif proficiency testing programme for participating countries, 2013–2015. all 40 pt samples met the criteria for establishing a consensus result except 2015-b-1. 2015-b-1 was below 80% concordance with expected results, primarily due to a high rate of false-negative m. tuberculosis complex detection results (19.9%) (table 2). this sample is further discussed in the limitations section. for 29 of the 39 pt samples with a consensus result, over 90% of dts results received were concordant with the expected result. five of the 10 pt samples with < 90% concordance had false-negative m. tuberculosis complex detection as the most frequently observed cause for discordance, four of the 10 samples had unsuccessful tests (errors, invalids, and other causes) as the most frequently observed cause for discordance, while one sample had an equal number of unsuccessful tests and indeterminate rifampicin results. error rates ranged from 0% to 5.9% across pt samples. table 2: comparison of dried tube specimen results with expected results for xpert® mtb/rif proficiency testing programme, 2013–2015. there were 765 (9.4%) discordant results out of 8150 dts results received throughout the eight pt rounds. of those, 350 (46%) discordant results were unsuccessful tests (figure 2). false-negative m. tuberculosis complex results were the second-largest contributor to discordant results 224 (29%), followed by indeterminate rifampicin results 79 (10%). the remaining discordant results consisted of: false-negative rifampicin results 45 (6%), false-positive rifampicin results 34 (4%), and false-positive m. tuberculosis complex results 33 (5%). figure 2: distribution of 765 total discordant results in xpert® mtb/rif proficiency testing programme among participating countries, 2013–2015. logistic regression of the proportion of false-negative m. tuberculosis complex detection results on the mean ct of probe a during validation yielded an odds ratio of 1.42 (95% confidence interval [ci] 1.32–1.52). however, sample 2015-b-1’s unusually high mean probe a ct, combined with its unusually high proportion of false-negative m. tuberculosis complex detection results, may have unduly influenced this estimate. when this sample was removed from the calculation, the odds ratio decreased to 1.14 (95% ci 1.04–1.26). logistic regression of the proportion of indeterminate rifampicin results and the proportion of false-positive rifampicin resistance results on the mean ct of probe a during validation yielded odds ratios of 1.14 (95% ci 1.02–1.25) for indeterminates and 0.96 (95% ci 0.79–1.16) for false positives. discussion we have shown that it is feasible to implement an xpert mtb/rif pt programme for rls using dts. over the eight rounds of pt panels provided across a period of three years, the number of countries participating in the programme doubled and the number of participating sites increased five-fold. in each pt round, at least two-thirds of enrolled sites participated in pt panel testing and reported test results. lower participation rates were seen in rounds during which certain countries or their enrolled sites were subsequently unable to participate due to logistical challenges, such as a lack of funding for in-country panel distribution, lack of staff, pt kits being lost in the mail between the country coordinator and the enrolled testing site, non-functioning xpert instruments or computers, and stockouts (inability to procure test cartridges). further work is needed to investigate and address the reasons for non-participation in proficiency testing as part of a comprehensive continuous quality improvement package. most participating sites performed well in the dts-based xpert mtb/rif pt programme. approximately 90% of participating sites in each pt round demonstrated satisfactory performance, and over 90% of returned results during the study period were concordant with expected results. the most common reason for a result that was not concordant with the expected result was an unsuccessful test. some of the reported causes of unsuccessful tests, such as power failures and stockouts of testing kits, are recurring challenges in rls.17 the use of a similar sample type, dried culture spots, for xpert mtb/rif external quality assessment in south africa has been described.7,8 we observed similar error rates to those reported by scott et al. and gous et al.; however, we observed a greater proportion of discordant results.7,8 the average ct of probe a obtained from dried culture spots was lower than that of many dts pt samples. lower cts correlate with greater amounts of m. tuberculosis complex dna present in the sample, indicating that a higher concentration of m. tuberculosis complex dna was present in the dried culture spots. this may account for the smaller proportion of false-negative m. tuberculosis complex detection results from dried culture spots. we observed that false-negative m. tuberculosis complex detection results were often more common among dts samples with higher probe a cts during validation. our analyses using logistic regression confirmed a statistically significant positive association between higher probe a cts and false-negative m. tuberculosis complex detection. for dts samples produced at cdc during the study period, an increase of 1 cycle in the mean probe a ct resulted in an estimated 42% increase in the proportion of false-negative m. tuberculosis complex detection results (the estimate decreased to 14% when sample 2015-b-1 was excluded). similarly, we found a statistically significant positive association between higher probe a cts and indeterminate rifampicin resistance results, with an estimated 14% increase in the proportion of indeterminate rifampicin resistance results for every 1 cycle increase in a sample’s mean probe a ct. no association was found between mean probe a ct and false-positive rifampicin resistance, and we did not observe any trends between the strain used to produce dts and the proportion and types of discordance during the study period. based on these results, we are investigating modifications to the dts preparation method that will increase the concentration of m. tuberculosis complex dna present in dts. however, it is not possible to rule out the presence of or determine the frequency of other site-specific factors, such as adherence to standard operating procedures, transcription errors, instrument maintenance and calibration, and dts and test cartridge storage and shipping conditions that may influence the accuracy and reliability of xpert mtb/rif test results. the large increase in enrolment we observed over the course of three years may have been influenced by several factors. as the world health organization endorsed xpert mtb/rif in 2010 and funds became available for the purchase of instruments, the number of laboratories in rls utilising xpert mtb/rif increased dramatically.4 the world health organization’s expanded recommendations for use of xpert mtb/rif in 2013 also encouraged rapid adoption of the technology.1 over the same time period, the number of laboratories in rls electing to pursue external accreditation also increased, facilitated by such programmes as strengthening laboratory management toward accreditation, and stepwise laboratory improvement program towards accreditation.18,19 thus, an increasing number of laboratories in rls are in need of xpert mtb/rif pt material to demonstrate proficiency in line with recommendations and requirements for continuous quality improvement and accreditation programmes. limitations several challenges arose during operation of the dts-based xpert mtb/rif pt programme. the participant consensus for sample 2015-b-1 was below 80% concordance with expected results, mainly due to false-negative m. tuberculosis complex results (failure to detect m. tuberculosis complex when the expected result was ‘mtb detected’). although this sample was produced using the same method as other dts samples, the probe a cts during validation were on average higher than those of other m. tuberculosis complex samples produced. this indicates 2015-b-1 contained fewer inactivated organisms and thus less dna than other samples, potentially contributing to the higher proportion of false-negative m. tuberculosis complex results received.20 to investigate the low concordance of 2015-b-1, three remaining aliquots were tested and confirmed the lower than average semi-quantitative result. due to the low participant consensus, the decision was made to award all sites that submitted results for 2015-b-1 full credit (20 points). based on this experience, we have implemented additional dts validation criteria to ensure dts contain enough inactivated organism to perform reliably. beginning in 2016, only dts samples with an average ct value of less than 23 for the first probe detected are included in the distributed pt panels. the mean ct value of 23 was selected based on the findings of previous investigators, who observed good concordance with mean ct values up to 23.7 in addition, recording and reporting of results was also a recurring challenge. at times, no explanation was provided for missing or incomplete results, and it was not possible to determine whether a site attempted to test the sample or if the test was unsuccessful and the result not reported. more clearly defined reporting language and procedures were added to report forms in future pt rounds to reduce the reliance of free text data entry and improve our ability to provide assistance on determining the root causes of discordant results and unsuccessful tests. recommendations and next steps the steady increase in enrolment and number of sites participating in this dts-based xpert mtb/rif pt programme demonstrates that there is a substantial demand for routine pt material for the xpert mtb/rif assay. the ease of large-scale batch preparation of dts using readily available supplies and equipment, and the successful establishment of country coordinator roles for in-country management of pt programme operations, indicate that a dts-based xpert mtb/rif pt programme could be a feasible option for countries wanting to manage their own xpert mtb/rif pt programme. future work will focus on piloting the transfer of the dts-based pt package to national and regional programmes as part of a comprehensive continuous quality improvement package, as well as developing a web-based pt data entry and reporting system. conclusion a continuous quality improvement package for xpert mtb/rif in rls that includes routine pt material is an important contributor to ensuring provision of accurate results. a dts-based pt programme for xpert mtb/rif is a useful tool for monitoring and improving xpert mtb/rif performance. the simplicity of producing dts and use of common mycobacteriology laboratory supplies make dts a feasible way for countries to provide xpert mtb/rif pt material to their testing sites. acknowledgements we thank bharat parekh for his inspiration and guidance, and all testing sites for their willingness to participate in the proficiency testing program. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions k.d. and h.a. conceived and designed the experiments, which were performed by k.d. and k.k. also, k.k., k.d. and s.g. analysed the data. k.k., k.d., z.r., p.h. and a.k. wrote the article and all authors reviewed and approved the manuscript. sources of support this research has been supported by the president’s emergency plan for aids relief through the united states centers for disease control and prevention, atlanta georgia. data availability statement data access level is public without identifying information of proficiency testers, testing sites and country. disclaimer the findings and conclusions in this article are those of the authors and do not necessarily represent the official position of the united states centers for disease control and prevention. references in this manuscript to any specific commercial products, process, service, manufacturer, or company do not constitute its endorsement or recommendation by the united states government. references world health organization. rapid communication: molecular assays as initial tests for the diagnosis of tuberculosis and rifampicin [homepage on the internet]. geneva: who press; c2020 [cited 2020 sep 1]. available from: https://www.who.int/publications-detail/rapid-communication-molecular-assays-as-initial-tests-for-the-diagnosis-of-tuberculosis-and-rifampicin-resistance world health organization. global tuberculosis report [homepage on the internet]. geneva: who press; c2019 [cited 1 september 2020]. available from: https://www.who.int/tb/publications/global_report/en/ helb d, jones m, story e, et al. rapid detection of mycobacterium tuberculosis and rifampin resistance by use of on-demand, near-patient technology. j clin microbiol. 2010;48(1):229–237. https://doi.org/10.1128/jcm.01463-09 world health organization. cumulative number of genexpert instrument modules and xpert mtb/rif cartridges procured under concessional pricing [homepage on the internet]. geneva: who press; c2016 [cited 2020 sep 1]. available from: https://www.who.int/tb/areas-of-work/laboratory/status_xpert_rollout_dec_2016.pdf?ua=1 world health organization. laboratory quality management system: handbook [homepage on the internet]. geneva: who press; c2011 [cited 2020 sep 1]. available from: http://apps.who.int/iris/bitstream/10665/44665/1/9789241548274_eng.pdf global laboratory initiative. guide for providing technical support to tb laboratories in lowand middle-income countries [homepage on the internet]. n.d. [cited 2020 may 20]. available from: http://stoptb.org/wg/gli/assets/documents/guideforprovidingtechnicalsupport_gb_web.pdf scott le, gous n, cunningham be, et al. dried culture spots for xpert mtb/rif external quality assessment: results of a phase 1 pilot study in south africa. j clin microbiol. 2011;49(12):4356–4360. https://doi.org/10.1128/jcm.05167-11 gous n, cunningham b, kana b, stevens w, scott le. performance monitoring of mycobacterium tuberculosis dried culture spots for use with the genexpert system within a national program in south africa. j clin microbiol. 2013;51(12):4018–4021. https://doi.org/10.1128/jcm.01715-13 scott l, albert h, gilpin c, alexander h, degruy k, stevens w. multicenter feasibility study to assess external quality assessment panels for xpert mtb/rif assay in south africa. j clin microbiol. 2014;52(7):2493–2499. https://doi.org/10.1128/jcm.03533-13 degruy k, rey z, alexander h. dried tube specimens (dts) for xpert mtb/rif proficiency panels [poster]. presented at: african society for laboratory medicine. 1st international conference; 2012 december 1–7; cape town. benzaken as, bazzo ml, galban e, et al. external quality assurance with dried tube specimens (dts) for point-of-care syphilis and hiv tests: experience in an indigenous populations screening programme in the brazilian amazon. sexually trans inf. 2014;90(1):14–18. http://doi.org/10.1136/sextrans-2013-051181 nguyen s, ramos a, chang j, et al. monitoring the quality of hiv-1 viral load testing through a proficiency testing program using dried tube specimens in resource-limited settings. j clin microbiol. 2015;53(4):1129–1136. https://doi.org/10.1128/jcm.02780-14 parekh b, anyanwu j, patel h, et al. dried tube specimens: a simple and cost-effective method for preparation of hiv proficiency testing panels and quality control materials for use in resource-limited settings. j virol methods. 2010;163(2):295–300. https://doi.org/10.1016/j.jviromet.2009.10.013 morlock gp, plikaytis bb, crawford jt. characterization of spontaneous, in vitro-selected, rifampin-resistant mutants of mycobacterium tuberculosis strain h37rv. antimicrob agents chemother. 2000;44(12):3298–3301. https://doi.org/10.1128/aac.44.12.3298-3301.2000 xpert mtb/rif assay product insert [homepage on the internet]. sunnyvale, ca: cepheid; version 301-1404, rev. f august 2019 [cited 2020 sep 1]. available from: https://www.cepheid.com/package%20insert%20files/xpert-mtb-rif-english-package-insert-301-1404-rev-f.pdf r core team. r: a language and environment for statistical computing [homepage on the internet]. vienna: r foundation for statistical computing; c2019 [cited 2020 sep 1]. available from: http://www.r-project.org/ ondoa p, datema t, keita-sow ms, et al. a new matrix for scoring the functionality of national laboratory networks in africa: introducing the labnet scorecard. afr j lab med. 2016;5(3):a498. https://doi.org/10.4102/ajlm.v5i3.498 yao k, maruta t, luman e, nkengasong j. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. 2014;3(2):a194. https://doi.org/10.4102/ajlm.v3i2.194 datema tam, oskam l, van beers sm, klatser pr. critical review of the stepwise laboratory improvement process towards accreditation (slipta): suggestions for harmonization, implementation and improvement. trop med int health. 2012;17(3):361–367. https://doi.org/10.1111/j.1365-3156.2011.02917.x blakemore r, story e, helb d, et al. evaluation of the analytical performance of the xpert mtb/rif assay. j clin microbiol. 2010;48(7):2495–2501. https://doi.org/10.1128/jcm.00128-10 abstract introduction ethical considerations case presentation management and outcome discussion acknowledgements references about the author(s) herbert makgopa department of chemical pathology, faculty of health sciences, university of pretoria, pretoria, south africa department of chemical pathology, national health laboratory service, pretoria, south africa tanja kemp division of endocrinology, department of internal medicine, faculty of health sciences, university of pretoria, pretoria, south africa surita meldau division of chemical pathology, department of pathology, university of cape town, cape town, south africa department of chemical pathology, national health laboratory service, cape town, south africa engela m. honey division of human genetics, department of biochemistry, genetics and microbiology, university of pretoria, pretoria, south africa bettina chale-matsau department of chemical pathology, faculty of health sciences, university of pretoria, pretoria, south africa department of chemical pathology, national health laboratory service, pretoria, south africa citation makgopa h, kemp t, meldau s, honey em, chale-matsau b. maternally inherited diabetes and deafness with a variable presentation across three generations within a pedigree, south africa. afr j lab med. 2024;13(1), a2384. https://doi.org/10.4102/ajlm.v13i1.2384 case study maternally inherited diabetes and deafness with a variable presentation across three generations within a pedigree, south africa herbert makgopa, tanja kemp, surita meldau, engela m. honey, bettina chale-matsau received: 05 dec. 2023; accepted: 20 mar. 2024; published: 29 may 2024 copyright: © 2024. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract introduction: maternally inherited diabetes and deafness (midd) is caused by the m.3243a>g pathogenic variant in maternally inherited mitochondrial dna. diabetes is prevalent in our setting; however, midd is rarely diagnosed. this study, undertaken in pretoria, south africa, highlights the variable presentation of midd in different patients within the same family. case presentation: a 45-year-old man (proband) with hearing impairment was referred to the endocrine unit in july 2015 due to poor glycaemic control (hba1c = 13%). his clinical and biochemical features were in keeping with midd. a genetic study of accessible maternal relatives was pursued. his mother had difficulty hearing and reportedly died from an unspecified cardiovascular cause. two sisters with diabetes and deafness died of cardiac-related conditions. one nephew had diabetes (hba1c = 7.7%), hearing loss and tested positive for m.3243a>g. a third sister tested positive for m3243a>g, but aside from bilateral mild hearing loss in higher frequencies, showed no other signs of target organ damage. her daughter developed end-stage kidney failure necessitating a transplant, while her son had no biochemical abnormalities and was negative for m.3243a>g. management and outcome: a multidisciplinary team managed and screened for complications of the patient and his maternal relatives. proband died prior to genetic testing. conclusion: most midd patients initially present with symptoms of diabetes only, and it is probable that many cases remain undiagnosed. a high index of suspicion is necessary when encountering a family history of both diabetes and impaired hearing, and screening should be offered to the patient’s maternal relatives. what the study adds: this study demonstrates the importance of proper assessment when evaluating a patient with diabetes and a family history of hearing loss. keywords: diabetes; mitochondrial dna; maternally inherited diabetes and deafness; heteroplasmy; hearing loss. introduction primary mitochondrial disease is a heterogeneous group of disorders resulting from inherited defects in mitochondrial energy production.1 maternally inherited diabetes and deafness (midd) is a form of primary mitochondrial disease, characterised by diabetes and progressive sensorineural hearing impairment.2 it is linked to various pathogenic variants in mitochondrial dna (mtdna), including the m.3243a>g point mutation in the mt-tl1 gene, which has emerged as the most common cause of midd, accounting for nearly 85% of all cases.3 other features associated with the m.3243a>g pathogenic variant are myopathy, gastrointestinal tract symptoms, nephropathy, cardiomyopathy, and neuropsychiatric features with heterogeneous manifestation even in individuals within the same family due to varying mtdna heteroplasmy levels between different tissues, thus developing different phenotypes. the estimated prevalence of midd is between 0.5% and 5.9% among patients with diabetes mellitus.4 diagnosis of midd is important to ensure appropriate treatment, continuous monitoring and that maternal relatives are identified and managed in a timeous manner. diabetes in midd, unlike in type 1, initially responds to secretagogues, but may later progress to require insulin for management. metformin is not suitable in these patients as it may cause or exacerbate lactic acidosis, which is a typical finding in many patients with primary mitochondrial disease.5 maternally inherited diabetes and deafness is rarely diagnosed in clinical practice, and it may be missed due to its complex genetic and phenotypic heterogeneity.6 while many studies have been conducted in europe, australia, asia, and north america, midd remains severely underdiagnosed on the african continent, with only limited studies available.7,8,9 in this context, we describe three generations of south african patients with midd within the same pedigree. ethical considerations written consent was obtained from the family and ethics approval obtained from the research and ethics committee at the university of pretoria (no. 496/2023). confidentiality was ensured by avoiding any use of names, initials, pictures or personal identification numbers in the preparation of this case study. case presentation the proband, a 45-year-old man, was referred to the endocrine unit of a tertiary hospital in pretoria, south africa, in july 2015 due to poor glycaemic control (glycated haemoglobin [hba1c] = 13%; abnormal > 6.5%). glycated haemoglobin was assessed using the national glycohaemoglobin standardization program-certified high performance liquid chromatography (bio-rad, hercules, california, united states) method. he was diagnosed with diabetes (presumed type 2), at age 37 years, and hearing impairment was noted a year later. he had a previous admission for congestive cardiac failure and an echocardiogram revealed an ejection fraction of 13% – 15%. at age 42 years, he developed proximal myopathy, leading to reliance on a wheelchair for mobility. he was grossly underweight, with a body mass index of 14.48 kg/m2. the patient had established renal impairment (urea = 18.8 mmol/l, reference interval = 2.1–7.1; creatinine = 139 µmol/l, reference interval = 64–104), and an estimated glomerular filtration rate of 47 ml/min per 1.73 m2 (chronic kidney disease, stage 3)10 with frank proteinuria. urea was analysed using the urease enzyme assay, creatinine the alkaline picrate while urine protein was tested with the turbidimetric method that utilises benzethonium chloride (abbott laboratories, chicago, illinois, united states) (table 1). type 1 diabetes mellitus was excluded (anti-glutamic acid decarboxylase and islets tyrosine phosphatase 2 antibodies were negative), and c-peptide levels were normal (2.6 µg/l; reference interval = 0.9–7.1), suggesting residual insulin secretion. as c-peptide levels may be decreased in patients with kidney failure, looking for features of monogenic diabetes can assist to reduce misclassification.11 he had not experienced any episodes of diabetic ketoacidosis. his clinical and biochemical features were in keeping with midd. table 1: laboratory results of the proband with maternally inherited diabetes and deafness for assessment of complications, obtained between july 2015 and june 2016, south africa. genetic investigations were pursued for some of his maternal relatives. genetic investigation of the proband was not done as the patient demised prior to testing. only one of his siblings and two of their children were available for testing. the presence of the m.3243a>g variant was investigated using polymerase chain reaction (pcr) followed by the restriction fragment length polymorphism method with apai (thermo scientific, waltham, massachusetts, united states) in dna extracted from urine samples. generated fragments were compared against a generuler dna ladder mix (thermo scientific, waltham, massachusetts, united states) (figure 1). figure 1: restriction enzyme digest results from the maternal nephew (iii 7) (lane 2, in gel image [a]), and sister (ii 4) (lane 2 in [b]) of proband with maternally inherited diabetes and deafness in south africa, 2016. the m.3243a>g variant creates an apai restriction site in the 407 bp pcr amplicon, resulting in 174 bp and 233 bp fragments in affected patients. in unaffected individuals there is 407 bp only (restriction enzyme did not cut) and affected (heteroplasmic) individuals have three bands (wild type [407 bp] and two minor bands [174 bp and 233 bp]). lanes 1 (both a and b), and 3 and 4 (in a) were other patients that tested negative for the variant. lane 5 (in a) and lane 3 (in b) are known positive controls. lane 6 (in a) and lane 4 (in b) are known negative controls. lane 7 is the no-template control in image a and lane 5 is the no-template control in image b. lanes l in both images contain generuler dna ladder mix (thermo scientific) used as a reference to estimate the size of the unknown fragment. the family pedigree (figure 2) and characteristics (table 2) indicated variable presentations across three generations. the proband’s mother had hearing difficulties and died from a suspected cardiovascular cause at age 43 years. among his three sisters, two were diagnosed with diabetes and deafness, and died prematurely, one at age 46 (ii 5) due to congestive cardiac failure, and the other at 54 years (ii 6) due to acute myocardial infarction. a son of one of these sisters (iii 7) was diagnosed with diabetes (hba1c = 7.7%), hearing loss and had the m.3243a>g variant. the third sister (ii 4) also tested positive for the m.3243a>g variant; however, apart from a recent decline in hearing, she had no features of target organ damage. in contrast, her daughter (iii 3) developed end-stage kidney failure requiring transplant in her 20s. her other child, a son (iii 4), tested negative for the m.3243a>g variant and has remained free of any related symptoms. heteroplasmy levels were not determined for any of these cases. figure 2: pedigree of proband’s family with maternally inherited diabetes and deafness showing variable presentation across three generations in south africa, 2016. while one of the family members (ii 4) only developed mild disease later in life, another (iii 7) presented with severe disease early in life. the proband’s mother (i 2) and two of his sisters (ii 5 and ii 6) had hearing loss and all died of cardiovascular related diseases. table 2: clinical and laboratory features of proband with maternally inherited diabetes and deafness and available maternal relatives obtained in 2015 and 2016, south africa. management and outcome a multidisciplinary team was involved in the management of the patients discussed in this study. insulin secretagogues were considered for the treatment of diabetes. the proband had severe myopathy requiring a wheelchair. the patient was mostly managed in primary care, and was referred very late to a specialist when he had already developed severe complications, including cardiovascular issues. he sustained a femur fracture with presumptive pulmonary embolism and died before genetic confirmation could be obtained. his maternal relatives were offered genetic counselling and screening for diagnosis and management of complications. discussion the pathogenic variant m.3243a>g of the mt-tl1 gene is one of the identified causes of mtdna-related primary mitochondrial disease worldwide. it was initially identified in patients with mitochondrial encephalopathy, lactic acidosis and stroke-like episodes in 1990 and a couple of years later in a large pedigree with maternally transmitted diabetes mellitus and deafness.12 the exact mechanism by which the m.3243a>g variant causes disease is still incompletely understood. however, decreases of pancreatic beta cell mass and declines in glucose-stimulated insulin secretion have been implicated in the development of diabetes in affected patients.13 maternally inherited diabetes and deafness is exclusively inherited through the maternal line, as the paternal contribution of sperm mitochondria to the fertilised egg is minimal, about 1000-fold less than that of the oocyte, and there are selective mechanisms targeting paternal mtdna for destruction, further reinforcing the maternal inheritance pattern.14 patients with midd range in age from 12 years to 67 years when they present with diabetes, and diabetes is often the presenting problem, although in some patients, onset of sensorineural deafness may precede presentation of diabetes.13 a definitive diagnosis is only possible through genetic confirmation. screening for the common m.3243a>g variant is best performed using dna from epithelial cells in urine or muscle, as heteroplasmy levels tend to decrease with age in actively dividing cells in blood.15 in the case of the proband, the midd diagnosis was inferred from a combination of clinical and laboratory features, a strong family history, and the positive identification of the m.3243a>g variant in some of his maternal relatives. the clinical spectrum of midd exhibits heterogeneity, encompassing variation in terms of age of onset, clinical manifestation and severity.5 as observed in this study, the proband’s sister tested positive for the m.3243a>g pathogenic variant but remained asymptomatic throughout most of her adult life. she only developed decreased hearing after reaching age 60 years. complications in patients with midd follow a distinct pathophysiology compared to complications in patients with type 1 and type 2 diabetes. proteinuria arises as a consequence of the mitochondrial dysfunction.2,5 the renal impairment is frequently misdiagnosed as diabetic kidney disease; however, in some patients, kidney failure may even develop before the onset of diabetes. as shown with the proband’s niece, end-stage kidney disease necessitating kidney transplantation can develop at a young age. pathogenic mtdna variants, such as the m.3243a>g variant, tend to be heteroplasmic in all affected individuals. one of the proband’s nephews tested negative for this variant, but had no features of end-organ damage. these diverse patterns of heteroplasmy underscore the complex nature of mitochondrial genetics and their impact on phenotypic expression. in this study, the pcr-restriction fragment length polymorphism method employed is still used in clinical laboratories for detection of the m.3243a>g variant. however, it is unable to detect heteroplasmy levels below approximately 5% and is not quantitative.15 in contrast, methods such as next generation sequencing, pyrosequencing, real-time pcr and ligation-mediated pcr have the advantage of accurately detecting much lower heteroplasmy levels,16 although due to their higher costs, these techniques are not routinely available in most public clinical laboratories. there is no cure for midd, and treatment mainly focuses on managing disease progression and minimising severity of complications in order to enhance the quality of life for affected individuals.13 as is evident from this study, cardiac abnormalities are common in patients with midd and can contribute to premature death. thus, echocardiography and regular electrocardiography should form part of the standard evaluation for midd. optimal patient management in midd requires a multidisciplinary approach, including supportive therapy and genetic counselling. these should be offered to all available maternal relatives to provide information on the mode of inheritance and enable informed choices and considerations for family planning.17 considering that symptoms of diabetes are often the reason for the initial health-seeking behaviour in most midd patients, it is probable that many cases remain undiagnosed in our local setting. a high index of suspicion should be maintained when encountering a family history of diabetes and impaired hearing. all maternal offspring and relatives of an individual with midd are potential carriers of a pathological variant and should be offered screening. acknowledgements competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions h.m. drafted the original article. t.k. performed clinical examinations and investigations of patients, and editing of the article. s.m. was responsible for the genetic studies, interpretation of genetic results, and editing of the article. e.m.h. performed genetic counselling and investigations of patients, and editing of the article. b.c.-m. conceptualised, edited the article and supervised the study. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the authors confirm that the data supporting the findings of this study are available within the article. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated institutions or organisations of the authors and the publisher. references schlieben ld, prokisch h. the dimensions of primary mitochondrial disorders. front cell dev biol. 2020;8:600079. https://doi.org/10.3389/fcell.2020.600079 ali as, ekinci ei, pyrlis f. maternally inherited diabetes and deafness (midd): an uncommon but important cause of diabetes. endocr metab sci. 2021;2:100074. https://doi.org/10.1016/j.endmts.2020.100074 wang m, liu h, zheng j, et al. a deafness-and diabetes-associated trna mutation causes deficient pseudouridinylation at position 55 in trnaglu and mitochondrial dysfunction. j biol chem. 2016;291(40):21029–21041. https://doi.org/10.1074/jbc.m116.739482 iwasaki n, babazono t, tsuchiya k, et al. prevalence of a-to-g mutation at nucleotide 3243 of the mitochondrial trna leu (uur) gene in japanese patients with diabetes mellitus and end stage renal disease. j hum genet. 2001;46: 330–334. https://doi.org/10.1007/s100380170068 yang m, xu l, xu c, et al. the mutations and clinical variability in maternally inherited diabetes and deafness: an analysis of 161 patients. front endocrinol. 2021;12:728043. https://doi.org/10.3389/fendo.2021.728043 ahmed st. understanding the molecular mechanisms of mitochondrial muscle disease using a quantitative, quadruple immunofluorescent assay. newcastle university, newcastle upon tyne; 2020. meldau s, owen ep, khan k, riordan gt. mitochondrial molecular genetic results in a south african cohort: divergent mitochondrial and nuclear dna findings. j clin pathol. 2022;75(1):34–38. https://doi.org/10.1136/jclinpath-2020-207026 ameh j, godwin i, obi i, puepet f, aminu b, suleiman t. the search for mitochondrial trnaleu (uur) a3243g mutation among type 2 diabetes mellitus patients in the nigerian population. afr j biotechnol. 2011;10(62):13383–13389. https://doi.org/10.5897/ajb11.1556 mkaouar-rebai e, tlili a, masmoudi s, et al. mutational analysis of the mitochondrial trnaleu (uur) gene in tunisian patients with mitochondrial diseases. biochem biophys res commun. 2007;355(4):1031–1037. https://doi.org/10.1016/j.bbrc.2007.02.083 meyers a. significance, definition, classification and risk factors of chronic kidney disease in south africa. s afr med j. 2015;105(3):233–236. https://doi.org/10.7196/samj.9412 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diabetes and deafness is associated with the degree of heteroplasmy in blood leukocytes. j clin endocrinol metab. 2009;94(8):3025–3030. https://doi.org/10.1210/jc.2008-2680 urata m, wakiyama m, iwase m, et al. new sensitive method for the detection of the a3243g mutation of human mitochondrial deoxyribonucleic acid in diabetes mellitus patients by ligation-mediated polymerase chain reaction. clin chem. 1998;44(10):2088–2093. https://doi.org/10.1093/clinchem/44.10.2088 müller pl, treis t, pfau m, et al. progression of retinopathy secondary to maternally inherited diabetes and deafness – evaluation of predicting parameters. am j ophthalmol. 2020;213:134–144. https://doi.org/10.1016/j.ajo.2020.01.013 abstract introduction methods results discussion acknowledgements references about the author(s) alicia naidoo department of medical microbiology, rk khan laboratory, national health laboratory service, durban, south africa department of medical microbiology, school of laboratory medicine and medical sciences, college of health sciences, university of kwazulu-natal, durban, south africa afsana kajee department of medical microbiology, school of laboratory medicine and medical sciences, college of health sciences, university of kwazulu-natal, durban, south africa department of medical microbiology, inkosi albert luthuli central hospital, national health laboratory services, durban, south africa nomonde r. mvelase department of medical microbiology, rk khan laboratory, national health laboratory service, durban, south africa department of medical microbiology, school of laboratory medicine and medical sciences, college of health sciences, university of kwazulu-natal, durban, south africa khine swe swe-han department of medical microbiology, school of laboratory medicine and medical sciences, college of health sciences, university of kwazulu-natal, durban, south africa department of medical microbiology, inkosi albert luthuli central hospital, national health laboratory services, durban, south africa citation naidoo a, kajee a, mvelase nr, swe-han ks. antimicrobial susceptibility of bacterial uropathogens in a south african regional hospital. afr j lab med. 2023;12(1), a1920. https://doi.org/10.4102/ajlm.v12i1.1920 original research antimicrobial susceptibility of bacterial uropathogens in a south african regional hospital alicia naidoo, afsana kajee, nomonde r. mvelase, khine swe swe-han received: 12 apr. 2022; accepted: 13 dec. 2022; published: 03 mar. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: urinary tract infections are common bacterial infections affecting millions worldwide. although treatment options for urinary tract infections are well established, with ciprofloxacin long considered one of the antibiotics of choice, increasing antibiotic resistance may delay the initiation of appropriate therapy. while this increase in antimicrobial resistance has been demonstrated in multiple studies around the world, there is a dearth of information from developing countries. objective: this study aimed to describe the antimicrobial susceptibility patterns of commonly isolated bacterial uropathogens in a south african hospital. methods: antimicrobial susceptibility data of isolates obtained from urine specimens at the rk khan hospital, a regional hospital in kwazulu-natal, south africa, between january 2018 and december 2020 were retrieved from the hospital’s laboratory information system and analysed to determine the differences in resistance rates between the most frequently isolated bacterial uropathogens. results: of the 3048 bacterial urinary pathogens isolated between 2018 and 2020, escherichia coli (1603; 53%) was the most common, followed by klebsiella spp. (437; 14%). both e. coli and klebsiella spp. showed high rates of resistance to amoxicillin/clavulanic acid (29.8% and 42.3%) and ciprofloxacin (37.7% and 30.4%). nitrofurantoin resistance was low among e. coli (6.2%) but high among klebsiella spp. (61.3%). conclusion: e. coli and klebsiella spp. in this study were highly resistant to amoxicillin/clavulanic acid and ciprofloxacin, two of the frequently prescribed oral treatment options. what this study adds: this study highlights the importance of regular local antimicrobial resistance surveillance to inform appropriate empiric therapy. keywords: antimicrobial susceptibility patterns; uropathogens; urinary pathogens; antibiotic resistance; urinary oral treatment. introduction although the urinary tract has multiple mechanisms in place to keep it sterile, urinary tract infections (utis) are one of the most common bacterial infections affecting nearly 150 million people worldwide and amounting to more than $6 billion united states dollars in healthcare costs, with approximately 95% of all utis occurring because of periurethral contamination by enteric uropathogens.1,2 international studies indicate that due to the high incidence of utis and the widespread practice of empirical treatment, the rate of antibiotic prescriptions is also increasing.3 as antibiotic use is known to be the main driver of the evolution of resistance, the increasing use of antibiotics in the treatment of utis has contributed to the alarming increase in antimicrobial resistance among frequently isolated urinary pathogens, thus further limiting treatment options, particularly with orally administered drugs.2,4,5,6 escherichia coli, klebsiella spp., and enterococcus spp., which are all members of the normal gut flora, are the most typically isolated organisms from urine, with e. coli being the most frequently isolated.7,8 empiric treatment options for utis are well established globally, and these include ciprofloxacin, amoxicillin/clavulanic acid, nitrofurantoin, fosfomycin, and cephalosporins.9,10 however, studies conducted locally and internationally demonstrate that an increase in the use of most of these drugs has caused a surge in antimicrobial resistance and a concomitant increase in treatment failures.2,11 this exerts greater pressure on an already overburdened healthcare system. antimicrobial resistance surveillance studies thus need to be conducted regularly to identify bacterial uropathogens and their antimicrobial susceptibility patterns to guide the empiric treatment of utis, which is a crucial part of antimicrobial stewardship. therefore, this study aimed to determine the prevalence of bacterial uropathogens and their antimicrobial susceptibility patterns to assist antimicrobial stewardship in a regional hospital in kwazulu-natal, south africa. methods ethical considerations ethics approval was granted by the university of kwazulu-natal biomedical research ethics committee, with permit number brec/00001578/2020. informed patient consent was not required by the university of kwazulu-natal ethics committee as this was a retrospective study and there was no patient interaction. patient privacy and confidentiality of data were protected in accordance with the declaration of helsinki. study design and setting a retrospective study was performed by retrieving three years of laboratory data from 01 january 2018 to 31 december 2020 at the national health laboratory services, rk khan hospital, durban, kwazulu-natal, south africa. the 543-bed hospital ranks as both a regional and district hospital and serves many internal and external clinics. data, which included patient location (inpatient and outpatient) and isolated uropathogens, were collected from the laboratory information system and captured onto a microsoft excel 365 spreadsheet, version 2205 (microsoft corporation, redmond, washington, united states). interpreted antimicrobial susceptibility data (‘resistant’, ‘intermediate’, ‘sensitive’) for the retrieved isolates were also extracted from the laboratory information system. inclusion criteria data on all positive urine bacterial cultures processed between january 2018 and december 2020 were retrieved from the laboratory information system. we then determined the distribution of bacterial uropathogens and analysed the antimicrobial susceptibility patterns of the two most common enterobacterales, namely e. coli and klebsiella spp. considering the study setting and the current literature, enterococcus faecalis, enterococcus faecium, group b streptococcus, and staphylococcus spp. were also chosen for analysis as they are the most common gram-positive bacterial causes of uti. antibiotics analysed in the study were based on the recommended empiric therapy as per the south african standard treatment guideline.9 exclusion criteria as this study was on bacterial uropathogens, all data on yeasts were excluded. to exclude duplicate entries, we used the following patient variables: patient name, surname, date of birth, and organism name. the organism name was included to remove duplicate entries from the same patient with the same organism. laboratory analysis urine specimens were processed according to the laboratory’s routine standard operating procedure. bacterial identification and antimicrobial susceptibility testing were carried out using the vitek® 2 automated system (biomerieux, marcy l’ètoile, rhône-alpes, france). as cephalothin was not part of the antibiotics tested on the vitek® 2 n255 card, cephalothin susceptibility was analysed using the kirby bauer disk diffusion method. for gram-negative bacteria, the antibiotics analysed were amikacin, amoxicillin/clavulanic acid, cefotaxime or ceftriaxone, ceftazidime, cephalothin, ciprofloxacin, gentamicin, meropenem, nitrofurantoin, piperacillin/tazobactam and trimethoprim/sulfamethoxazole. for the gram-positive bacteria, the antibiotics analysed include ampicillin, cloxacillin, penicillin, and vancomycin. results for cefotaxime and ceftriaxone were reported together as they have the same mechanism of action and resistance patterns and were therefore used interchangeably at rk khan hospital. all antimicrobial susceptibility results were interpreted using the clinical and laboratory standards institute guidelines.12 an extended-spectrum beta-lactamase producer was defined as an organism that was resistant to all cephalosporins, while carbapenem-resistant enterobacterales were defined as isolates that were resistant to any carbapenem. data analysis we determined the antimicrobial resistance rates of the most commonly isolated gram-negative (e. coli and klebsiella spp.) and gram-positive bacteria (e. faecalis, e. faecium, group b streptococcus, and staphylococcus spp.). additionally, for e. coli, we compared the antibiotic resistance rates between strains obtained from inpatients and outpatients and analysed the trends in antibiotic resistance rates over the three-year study period (2018–2020). we also determined the statistical significance of differences observed in the resistance rates between e. coli and klebsiella spp., as well as differences in the resistance rates of e. coli strains isolated from inpatients and outpatients. these statistical comparisons were done using the chi-square test, where a p-value less than 0.05 was considered statistically significant. the p-values for the pairwise comparisons were adjusted using the bonferroni correction method (r statistical computing software, version 3.6.3, r core team, auckland, new zealand). results from january 2018 to december 2020, the national health laboratory services rk khan laboratory analysed 3804 urine samples from patients with suspected utis. after removing duplicate data and data on yeasts, data on a total of 3048 bacterial isolates were included in this study. escherichia coli (n = 1603; 53%) was the most frequent urinary pathogen isolated, followed by klebsiella spp. (n = 437; 14%), including 398 (13%) klebsiella pneumoniae and 39 (1%) klebsiella oxytoca (figure 1). enterobacter spp. (n = 97; 3.2%) (including 82 [2.7%] enterobacter cloacae and 15 [0.5%] enterobacter aerogenes), pseudomonas aeruginosa (n = 61; 2%), acinetobacter baumannii (n = 84; 3%), and other gram-negative bacilli (n = 189; 6%) made up a further 14%. enterococcus faecalis (n = 372; 12%) was the most frequent gram-positive organism isolated, followed by e. faecium (n = 109; 4%), group b streptococcus (n = 55; 2%), and staphylococcus spp. (n = 41; 1%). figure 1: distribution of the 3048 bacterial urinary pathogens isolated at the rk khan hospital, durban, kwazulu-natal, south africa, january 2018 – december 2020. as e. coli and klebsiella spp. made up the majority of the gram-negative bacilli isolated, it was important to determine their antimicrobial resistance rates. the total number of cephalothin susceptibility results differed from the other antibiotics because cephalothin susceptibility was determined manually and the result was not always entered into the laboratory information system. similarly, the total number of results was different for each antimicrobial tested due to missing results linked to random terminations that can sometimes occur during automated antimicrobial susceptibility testing with the vitek® 2 instrument. e. coli demonstrated low levels of resistance to meropenem (0.56%; n = 9), amikacin (2.9%; n = 47), nitrofurantoin (6.2%; n = 99) and piperacillin/tazobactam (5.3%; n = 85) (figure 2). a very high level of resistance was noted in recommended oral treatment options, namely trimethoprim/sulfamethoxazole (61%; n = 988), ciprofloxacin (38%; n = 605) and amoxicillin/clavulanic acid (30%; n = 478). similarly, klebsiella spp. strains were highly resistant to nitrofurantoin (61%; n = 268), trimethoprim/sulfamethoxazole (48%; n = 208), amoxicillin/clavulanic acid (42%; n = 185), and ciprofloxacin (30%; n = 133). twenty-six percent of e. coli (n = 424) isolates were extended-spectrum beta-lactamase producers and 0.6% (n = 9) were carbapenem-resistant. among the klebsiella spp. isolates, 41.8% (n = 184) were extended-spectrum beta-lactamase producers and 5% (n = 22) were carbapenem-resistant. figure 2: antimicrobial susceptibility rates of e. coli and klebsiella spp. isolated from patients with suspected urinary tract infections at the rk khan hospital, durban, kwazulu-natal, south africa, january 2018 – december 2020. the total number of isolates is different for each antimicrobial due to missing results linked to random terminations that can sometimes occur during automated antimicrobial susceptibility testing with the vitek® 2 instrument. overall, klebsiella spp. was significantly more resistant than e. coli to all the tested antimicrobials except cephalothin (e. coli – 56% vs klebsiella spp. – 50%), ciprofloxacin (38% vs 30%), and trimethoprim/sulfamethoxazole (61% vs 48%). for all comparisons except cephalothin (p = 0.03) and ciprofloxacin (p = 0.005), the p-values were less than 0.001. among the gram-positive bacteria, e. faecium isolates showed high levels of resistance to both ampicillin (83.5%; n = 91) and penicillin (83.5%; n = 91) (table 1). conversely, e. faecalis isolates had low rates of resistance to ampicillin (0.8%; n = 3) and penicillin (2.4%; n = 9). in addition, the staphylococcus spp. isolates had high resistance rates to ampicillin (n = 38; 92.7%) and penicillin (n = 38; 92.7%). vancomycin was the most effective antibiotic against e. faecium (97.2% susceptibility) and staphylococcus spp. (100% susceptibility), while ampicillin and penicillin were most effective against group b streptococci (100% susceptibility). table 1: antimicrobial susceptibility patterns of commonly isolated urinary gram-positive cocci at the rk khan hospital, durban, kwazulu-natal, south africa, january 2018 – december 2020. for analysis of inpatient and outpatient data, only e. coli was studied as it constituted more than half of the bacterial pathogens. five hundred and eleven e. coli isolates (32%) were isolated from inpatients, while 1092 (68%) were from outpatients (table 2). low level of resistance was observed in both inpatient and outpatient wards for meropenem (0.8%; n = 4 and 0.1%; n = 1), amikacin (3.1%; n = 16 and 2.8%; n = 31), piperacillin/tazobactam (5.5%; n = 28 and 4.7%; n = 51) and nitrofurantoin (6.3%; n = 32 and 5.5%; n = 60). however, there were high rates of resistance to trimethoprim/sulfamethoxazole (inpatients: 69.7%; n = 355, outpatients: 57.6%; n = 625), ciprofloxacin (inpatients: 34.7%; n = 177, outpatients: 39%; n = 426), and amoxicillin/clavulanic acid (inpatients: 32.9%; n = 168, outpatients: 28.4%; n = 310). the prevalence of extended-spectrum beta-lactamases and carbapenem resistance in e. coli was higher among inpatients (31.0%; n = 158 and 0.8%; n = 4) compared to outpatients (24%; n = 261 and 0.1%; n = 1). table 2: antimicrobial susceptibility patterns of e. coli isolated from inpatient and outpatient wards at the rk khan hospital, durban, kwazulu-natal, south africa, january 2018 – december 2020 there were no statistically significant differences in resistance rates to recommended oral antibiotics, namely amoxicillin/clavulanic acid (p = 0.07), ciprofloxacin (p = 0.10) and nitrofurantoin (p = 0.54) between the e. coli isolates from inpatients and outpatients. however, for trimethoprim/sulfamethoxazole, e. coli isolates from inpatient wards were significantly more resistant than those from outpatient wards (p < 0.001). of the 1603 e. coli strains isolated between 2018 and 2020, 625 (39.1%) strains were isolated in 2018, 539 (33.6%) were isolated in 2019, and 439 (27.4%) were isolated in 2020, demonstrating slight decreases in prevalence over the study period (figure 3). amikacin and meropenem resistance rates, while low, increased slightly between 2018 (2.4% and 0.5%) and 2020 (3.6% and 0.9%). conversely, the e. coli isolates had higher resistance rates to the more commonly prescribed oral antibiotics, but these rates decreased steadily between 2018 and 2020 (cephalothin – 63.9% vs 49.8%; ciprofloxacin – 42.9% vs 30.4%; amoxicillin/clavulanic acid – 36.6% vs 22.4%). figure 3: antimicrobial susceptibility trends of e. coli isolated from patients with suspected urinary tract infections at the rk khan hospital, durban, kwazulu-natal, south africa, january 2018 – december 2020. the total number of isolates is different for each antimicrobial due to missing results linked to random terminations that can sometimes occur during automated antimicrobial susceptibility testing with the vitek® 2 instrument. over the three years (2018–2020), there were no statistically significant differences in resistance rates to amikacin (p = 0.50), meropenem (p = 0.75), nitrofurantoin (p = 0.16), piperacillin/tazobactam (p = 0.34), and trimethoprim/sulfamethoxazole (p = 0.2). in contrast, resistance rates to amoxicillin/clavulanic acid, ceftazidime, cephalothin and ciprofloxacin decreased significantly between 2018 and 2020 (p < 0.001). the e. coli isolates also demonstrated a significant decrease in resistance to cefotaxime or ceftriaxone (p = 0.04) and gentamicin (p = 0.02). discussion in this study, we found that e. coli was the most frequently isolated uropathogen at the rk khan hospital, durban, kwazulu-natal, south africa, between 2018 and 2020, followed by klebsiella spp. and e. faecalis. we also found that the commonly identified urinary pathogens had relatively high resistance rates to the widely administered antibiotics for uti treatment. our findings are consistent with those of previous local and international studies.13,14 a study conducted among patients with community-acquired utis in india showed that e. coli (55.1%), e. faecalis (15.8%), and k. pneumoniae (13.7%) were the most prevalent uropathogens isolated.13 similarly, a six-year (2011–2016) study conducted in the obstetric departments of six public-sector hospitals in durban, kwazulu-natal, south africa, showed that e. coli (54.2%) was the most common uropathogen, followed by k. pneumoniae (12.9%).14 in 2010, the infectious diseases society of america and the european society of microbiology and infectious disease published a clinical practice guideline outlining their recommendations for the treatment of uncomplicated utis, with nitrofurantoin, fosfomycin, trimethoprim/sulfamethoxazole, and ciprofloxacin recommended as the drugs of choice.6 similarly, in the south african standard treatment guideline, published in 2019, fosfomycin, ciprofloxacin, amoxicillin/clavulanic acid, and nitrofurantoin are also considered as empiric oral treatment options for utis.9 in their guidelines, both the infectious diseases society of america and the european society of microbiology and infectious disease recommend that ciprofloxacin should be used as an empiric antibiotic therapy provided that resistance rates remain below 10%.6 it was therefore disconcerting to discover that, in our study, both e. coli and klebsiella spp. showed high resistance rates against ciprofloxacin and amoxicillin/clavulanic acid. over 30% of the e. coli and klebsiella spp. isolates were resistant to ciprofloxacin and amoxicillin/clavulanic acid. in contrast, while only 6.2% of e. coli were resistant to nitrofurantoin, klebsiella spp. showed a high rate (61.3%) of nitrofurantoin resistance. although nitrofurantoin is still an option for treating utis caused by e. coli, our study suggests that it is ineffective against the majority of klebsiella spp. infections, leaving physicians with limited empiric oral therapy alternatives. similar to our findings, in a 9-year (2011–2019) study conducted in europe,15 resistance rates to ciprofloxacin and amoxicillin/clavulanic acid exceeded the 10% threshold for both e. coli (42.1% and 14.3%) and klebsiella spp. (68.7% and 38.8%). although the e. coli nitrofurantoin resistance rate (4.8%) in our study was below this 10% threshold, the resistance rate for klebsiella spp. (46.0%) far exceeded the threshold. these high rates of resistance to recommended oral empiric therapy are of major concern as this could lead to increased treatment failure rates. as our study was laboratory-based and the relevant information was not available on the laboratory information system, we were unable to distinguish between community-acquired and hospital-acquired utis. we, therefore, examined the antimicrobial patterns of e. coli isolates from the inpatient wards versus the outpatient wards. although resistance rates were generally higher (between 1% and 12% difference) among inpatients than outpatients, these differences were not statistically significant, except for trimethoprim/sulfamethoxazole. a japanese study done in 2020 showed that the differences in e. coli resistance rates between hospital-acquired and community-acquired utis were between 5% and 10%.16 according to the south african public healthcare system, new patients do not directly present to hospitals. instead, they are initially seen at local clinics and only referred to hospitals if required.9 as a result, patients who are seen at hospitals are either known hospital patients with chronic diseases or patients who need a higher level of care than the primary healthcare offered in the clinics. this may explain the high resistance rates in frequently isolated urinary bacteria in our study. it is however interesting to note that in a local study conducted in kwazulu-natal, south africa, between 2011 and 2016, lower levels of resistance to recommended empiric oral antimicrobials were reported among e. coli isolates from pregnant, but otherwise healthy, individuals who were generally not on antimicrobial therapy.14 despite these rates being lower than those of our study, they are still above the empirical antibiotic resistance threshold of 10% recommended by the infectious diseases society of america and the european society of microbiology and infectious disease. limitations because this was a retrospective study, the reliability of the data presented depended on the accuracy of the recording of the initial patient results. the resistance rates in this study may have been overrepresented as clinicians are unlikely to request susceptibility testing for patients that respond to empiric therapy. also, due to a lack of clinical information, we were unable to determine the difference between hospital-acquired and community-acquired infections. conclusion our study revealed that e. coli was the most predominant urinary pathogen isolated and that antimicrobial resistance levels in commonly isolated uropathogens remain elevated, especially against frequently prescribed oral antimicrobials. our findings indicate that the current empiric therapy used in the treatment of utis would fail in a large proportion of affected individuals, thus highlighting the need for more research to evaluate alternate oral drugs such as fosfomycin, which is not in use in the public sector despite being recommended in the south african standard treatment guideline.9 acknowledgements we would like to acknowledge the management and staff of rk khan laboratory (national health laboratory services) as well as the academic affairs and research office (national health laboratory services) for all their assistance while completing this project. competing interests the authors declare that they have no financial or personal relationship that may have inappropriately influenced them in writing this article. authors’ contributions a.n. was involved in conceptualisation and implementation of the research project, data collection, data analysis, literature review, writing of the first draft, writing, and editing of all subsequent drafts and the writing of the final draft. a.k. was responsible for editing the first and second drafts. n.r.m. contributed to conceptualisation of the research project and provided input on all drafts, editing and analysis. k.s.s.-h. provided input on implementation of the project and editing the first and final drafts. sources of support this research did not receive a grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the authors confirm that the data supporting the findings of this study are available within the article. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references van schoor j. urinary tract infection in women. s afr fam pract. 2016;58(1):6–10. https://doi.org/10.4102/safp.v58i1.4434 raja ns. oral treatment options of patients with urinary tract infections caused by extended spectrum beta-lactamase (esbl) producing enterobacteriaceae. j infect public health. 2019;12(6):843–846. https://doi.org/10.1016/j.jiph.2019.05.012 schmiemann g, kniehl e, gebhardt k, matejczyk mm, hummers-pradier e. the diagnosis of urinary infection. deutsches ärzteblatt int. 2010;107(21):361–367. https://doi.org/10.3238/arztebl.2010.0361 zur wiesch pa, kouyos r, engelstädter j, regoes rr, bonhoeffer s. population biological principles of drug-resistance evolution in infectious diseases. lancet infect dis. 2011;11(3):236–247. https://doi.org/10.1016/s1473-3099(10)70264-4 read af, woods rj. antibiotic resistance management. evol med public health. 2014;28(1):147. https://doi.org/10.1093/emph/eou024 gupta k, hooton tm, naber kg, et al. international clinical practice guidelines for the treatment of acute uncomplicated cystitis and pyelonephritis in women: a 2010 update by infectious diseases society of america and the european society for microbiology and infectious diseases. clin infect dis. 2011;52:e103–e102. https://doi.org/10.1093/cid/ciq257 flores-mireles al, walker jn, caparon m, hultgren sj. urinary tract infections: epidemiology, mechanisms of infection and treatment options. nat rev microbiol. 2015;13:269–284. https://doi.org/10.1038/nrmicro3432 elmanama aa, alreqeb aa, kalloub hk, et al. bacterial etiology of urinary tract infection and their antimicrobial resistance profiles. j al azhar university-gaza (nat sci). 2018;20:81–98. national department of health, south africa. essential drugs programme. hospital level (adults) standard treatment guidelines and essential medicines list. 5th ed. pretoria: ndoh; 2019. tan cw, chlebicki mp. urinary tract infections in adults. singapore med j. 2016;57(9):485–490. https://doi.org/10.11622/smedj.2016153 lewis da, gumede lye, van der hoven la, et al. antimicrobial susceptibility of organisms causing community acquired urinary tract infections in gauteng province, south africa. s afr med j. 2013;103:377–381. https://doi.org/10.7196/samj.6722 clinical & laboratory standards institute. performance standards for antimicrobial susceptibility testing 29th ed. wayne, pa: m100; 2019. gupta s, kapur s, padamavathi dv. comparative prevalence of antimicrobial resistance in community acquired urinary tract infection cases from representative states of northern and southern india. j clin diagn res. 2014;8:9–12. bhola p, mvelase nr, balakrishna y, milisana kp, swe swe-han k. antimicrobial susceptibility patterns of uropathogens isolated from pregnant woman in kwazulu natal province: 2011–2016. s afr med j. 2020;110(9):872–876. https://doi.org/10.7196/samj.2020.v110i9.14468 hrbacek j, cermak p, zachoval r. current antibiotic resistance trends of uropathogens in central europe: survey from a tertiary hospital urology department 2011–2019. antibiotics. 2020;9(9):630. https://doi.org/10.3390/antibiotics9090630 kanda n, hashimoto h, sonoo t, et al. gram-negative organisms from patients with community acquired urinary tract infections and associated risk factors for antimicrobial resistance: a single centre retrospective observational study in japan. antibiotics (basel). 2020;9(8):438. https://doi.org/10.3390/antibiotics9080438 abstract introduction methods results discussion acknowledgements references about the author(s) lindi-marie coetzee national health laboratory service (nhls), johannesburg, south africa department of molecular medicine and haematology, university of the witwatersrand, johannesburg, south africa naseem cassim national health laboratory service (nhls), johannesburg, south africa department of molecular medicine and haematology, university of the witwatersrand, johannesburg, south africa deborah k. glencross national health laboratory service (nhls), johannesburg, south africa department of molecular medicine and haematology, university of the witwatersrand, johannesburg, south africa citation coetzee l-m, cassim n, glencross dk. weekly laboratory turn-around time identifies poor performance masked by aggregated reporting. afr j lab med. 2020;9(1), a1102. https://doi.org/10.4102/ajlm.v9i1.1102 note: additional supporting information may be found in the online version of this article as supplementary document 1. original research weekly laboratory turn-around time identifies poor performance masked by aggregated reporting lindi-marie coetzee, naseem cassim, deborah k. glencross received: 18 sept. 2019; accepted: 14 sept. 2020; published: 21 dec. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: high-level monthly, quarterly and annual turn-around time (tat) reports are used to assess laboratory performance across the national health laboratory service in south africa. individual laboratory performances are masked by aggregate tat reporting across network of testing facilities. objective: this study investigated weekly tat reporting to identify laboratory inefficiencies for intervention. methods: cd4 tat data were extracted for 46 laboratories from the corporate data warehouse for the 2016/2017 financial period. the total tat median, 75th percentile and percentage of samples meeting organisational tat cut-off (90% within 40 hours) were calculated. total tat was reported at national, provincial and laboratory levels. provincial tat performance was classified as markedly or moderately poor, satisfactory and good based on the percentage of samples that met the cut-off. the pre-analytical, testing and result review tat component times were calculated. results: median annual tat was 18.8 h, 75th percentile was 25 h and percentage within cut-off was 92% (n = 3 332 599). corresponding 75th percentiles of component tat were 10 h (pre-analytical), 22 h testing and 1.6 h review. provincial 75th percentile tat varied from 17.6 h to 34.1 h, with three good (n = 13 laboratories), four satisfactory (n = 24 laboratories) and two poor performers (n = 9 laboratories) provinces. weekly tat analysis showed 12/46 laboratories (28.6%) without outlier weeks, 31/46 (73.8%) with 1–10 outlier weeks and 3/46 (6.5%) with more than 10 (highest of 20/52 weeks) outlier weeks. conclusion: masked tat under-performances were revealed by weekly tat analyses, identifying poorly performing laboratories needing immediate intervention; tat component analyses identified specific areas for improvement. keywords: cd4; turnaround time; laboratory performance; outliers; weekly reporting. introduction in south africa, public health facilities across 52 districts provide patient care through primary healthcare (phc) services, district, regional and tertiary hospitals. a wide spectrum of tests can be requested and submitted to the nearest pathology laboratory of the national health laboratory service (nhls). the nhls is the choice laboratory service provider of the south africa national department of health (ndoh). a network of more than 266 laboratories are strategically placed around the country to optimally accommodate the needs of local communities (urban and rural).1,2 routine laboratory tests have a predetermined total turn-around time (tat) cut-off that ensures that tests are processed within the required timeframe to effect the appropriate clinical intervention. total tat is defined as the time from first registration of a sample on the laboratory information system (lis) to the time a result is reviewed and released to the requesting physician. tat is in part determined by (1) the window of testing from venepuncture as prescribed by the test manufacturers, (2) time validity of sample integrity (e.g. how many hours or days before erythrocytes in blood samples die and cannot bind to antibodies effectively) and (3) the clinician timeline (emergency or quick-resulted laboratory testing vs routine laboratory testing).3,4,5 laboratory tat reflects the time taken for processing a sample and is a direct indicator of laboratory performance and an integral measure of efficiency where delays can impact patient management.3,4,5,6,7 hiv-associated tests like hiv viral load (vl) and cd4 counts, like all nhls laboratory tests, have strict predetermined organisational tat cut-offs, set to reflect treatment guidelines requirements and standards of care for hiv management by local authorities and the world health organization (who)8,9. these guidelines call for the availability of a cd4 result within 7–14 days.9 to meet this standard, the nhls has set a within-organisation standard of 40 h for 90% of all cd4 testing to be completed and results released. the accurate reporting of tat depends on the quality of data collected through the lis, that is, the inclusion of automated system date and timestamps at various time points in the journey from patient venesection to result review.10 for samples sent to nhls laboratories, four major date and timestamps are electronically collected and used to calculate tat components2,10: (1) pre-analytical time (lab-to-lab), that is, the time from first registration at any nhls source laboratory to referral receipt at the designated testing laboratory, (2) analytical time (reg-to-test), time from registration at the testing laboratory to result transmission to the lis, and (3) post-analytical time (test-to-review), time from test transmission onto the lis to result review and verification by a senior laboratory staff member (i.e. results become available for the requesting physician or nurse to access). total tat is the summation of all three tat components. the limitation of current tat reporting is that it commences when a sample is registered at a source laboratory (nearest to testing site), that is, the time lapse from patient venesection to sample arrival at the laboratory is not included in the pre-analytical tat.10 the majority of cd4 samples tested in nhls laboratories originate from public health clinics of the ndoh, with no electronic system linked to lis. for true clinical tat assessment and impact on patient care, the time from sample collection to result receipt should be tracked, but this remains a challenge.11 laboratory test tat in the nhls is monitored at national, provincial and laboratory level, with annual,12 quarterly and monthly reports generated routinely.10 traditionally, the mean tat is reported, but retrospective data analysis confirmed a non-gaussian tat distribution.4 taking this into account, cd4 tat reporting was upgraded to include the median, 75th percentile and percentage within cut-off to better reflect performance. the concept of classifying laboratory performance was also developed: laboratories are reported as good, satisfactory or poor, based on the 75th percentile and percentage within tat cut-off value quadrants as described in a recent publication.10 the current monthly reports are effective at giving management a snapshot of the cd4 programme and overall (global) laboratory performance10 but cannot be used for timely interventions. underlying problems with tat are not detected in real time, thus corrective actions are taken retrospectively, days or weeks after they occurred.10 more frequent reporting was thus recommended in addition to traditional tat reporting to enable more meaningful and timely interventions to improve laboratory performance. using these described tat parameters and classification, a weekly tat dashboard was developed and rolled out nationally in 2018 for monitoring the tat of some tests (the most requested hiv, tuberculosis and non-communicable diseases tests). turn-around time data will inform corrective action such as additional test operator training.13,14 although the cut-off values for cd4 testing changed from 85% within 48 h to 90% within 40 h in the 2016/2017 financial year, the concept and wording of laboratory performance classification were retained as managers were well acquainted with these terms. the aim of this article is to describe how weekly review of cd4 tat analysis can enable the identification of non-compliant laboratories to facilitate effective and timely corrective action and ensure continuous quality management for improved service delivery. data analysed represents performance prior to the national implementation of the weekly tat dashboard. methods ethical considerations ethics clearance was obtained from the university of the witwatersrand (m1706108). no patient identifiers were used for this study and laboratories and provinces were anonymised. cd4 turn-around time data cd4 tat data were extracted from the corporate data warehouse for the financial period april 2016 to march 2017 (2016/2017 financial year) for 46 cd4 testing laboratories. total tat was calculated for each sample tested and reported for 52 weeks, together with the tat component data. data analysis included the calculation of the median, 75th percentile and the percentage of samples with a tat within the stipulated organisation cut-off per week. this was reported per laboratory and per province (aggregated data of laboratories within each of the nine provinces). performance classification was introduced at provincial level and based on the percentage samples within tat cut-off as follows: (a) ≥ 95%: good performance, (b) 90.0% – 94.9%: satisfactory performance; (c) 85.0% – 89.9%: moderate to poor performance and (d) 80.0% – 84.99%: poor performance. performance thus refers to the degree of compliance with nhls tat cut-off. the number of weeks that provinces and laboratories did not achieve the 40 h cut-off was reported. outlier weeks were defined as weeks where the total tat of all samples tested did not achieve 90% with a tat under 40 h. additional data analysis was done on the weekly laboratory data to describe the tat component contribution to total tat per laboratory per week and included: (1) lab-to-lab tat, (2) reg-to-test tat and (3) test-to-review tat. the target times set for each tat component are (1) 14 h, (2) 24 h and (3) 2 h. although tat component analysis by laboratory is distributed weekly, for this study, only specific laboratories were selected to represent different levels of compliance and performance categories to demonstrate how individual tat components affects total tat. outlying laboratory tat components (> 24 h and < 2 h) were correlated with beckman coulter engineer logs to verify the impact of instrument downtime on prolonged tat (data not shown). laboratory site visits were conducted to assess root cause analysis for below standard tat (< 90% processed for > 40 h) performance identified. statistical analysis data were prepared and analysed using sas version 9.4 (cary, north carolina, united states) and graphpad software (san diego, california, united states). the nine provinces were numbered 1–9, with individual laboratories within a province assigned a number and labelled accordingly (i.e. 1.5 represents province 1 and laboratory 5). box and whisker plots were created for individual laboratory data over 52 weeks. national total test volumes and tat was plotted against the 50th and 75th percentiles in a bar graph. provincial total tat was plotted as 75th percentile per performance category per week. individual laboratory distribution of 75th percentiles per performance category was plotted, indicating high (> 350 samples per day), medium (150-350 samples per day) and low volume facilities (< 150 samples per day). component tat was plotted as stacked bar graphs, showing the 75th percentile for pre-analytical, testing, and review tat for selected laboratories representing the four performance categories. results global annual cd4 total turn-around time overview in this study 3 332 599 cd4 test tat were analysed. for the 2016/2017 financial year, the national median tat for all cd4 tests was 18 h with a 75th percentile of 23 h (table 1). overall, 91% of all samples met the stipulated cut-off of 40 h, indicating good overall laboratory performance for meeting organisational criteria for cd4 testing. the matched organisational component median (and tat 75th percentiles) for lab-to-lab tat was 6.3 (10 h), reg-to-test tat was 17.3 (22 h) and test-to-review tat was 1.3 (2.1 h). table 1: national annual national health laboratory service cd4 total turn-around time for all samples tested during the 2016/2017 financial year. national weekly total turn-around time the weekly distribution of the 50th percentile (median) and 75th percentile showed good consistency despite fluctuations in test volumes across the network of testing laboratories (n = 52) (figure 1). the median ranged from 14 h to 18 h, while the 75th percentile ranged from 21 h to 26 h (figure 1). test volumes fluctuated between 23 681 to 80 821 per week (mean of 64 088 tests weekly). figure 1: national total turn-around time of national health laboratory service cd4 tests per week for the 2016/2017 financial year. the 50th percentile (median, green circles), 75th percentile (red circles) and volume of samples (grey bars) are depicted. provincial total turn-around time distribution (75th percentile) per testing week annual global tat distribution did not identify any poor performance over 52 weeks. to identify poor performances, national tat were analysed per province. the number of cd4 tests ranged from 65 395 (lowest) to 1 066 137 (highest) (table 2). the percentage of samples tested within the 40-h tat cut-off ranged from 82% to 98%. provincial performance classification was made based on the latter percentage per province, as a to d (as described above). a minimum of three laboratories represented each province. table 2: annual provincial national health laboratory service cd4 data, indicating test volumes, the 75th percentile total turn-around time, the percentage of samples within turn-around time cut-off and the number of representative laboratories for 2016/2017 financial year. three provinces (3, 7 and 9) were classified as category a (good performance). these laboratories were able to maintain all cd4 reporting within organisation-stipulated tat at greater than 95% and 75th percentile tat of 21.7 h (province 3), 17.6 h (province 7) and 2.08 h (province 9). four provinces (1, 2, 4 and 8) were categorised b (satisfactory performance) having 90% – 94.9% of samples meeting the tat cut-off; 75th percentile values for these provinces were 23 h, 25.1 h, 23.4 h and 18.9 h, respectively. two provinces failed to meet the tat cut-off and were classified as categories c (moderate poor performance; province 6) and d (markedly poor performance province 5), indicating that less than 90% of samples met the cd4 tat. within the latter provincial performance clusters (c and d), the 75th percentile reported was 28.8 h and 34.2 h. no weekly outliers (weeks where total tat did not meet 90% < 40 h) were noted in the three good performance provinces (3, 7, and 9; comprising n = 13 individual laboratories; figure 2a). the 75th percentile total tat for these provinces never exceeded 30 h during the test period. figure 2b describes the four provinces with satisfactory performance (1, 2, 4 and 8, representing 24 individual laboratories; table 2). among this group, province 2 and 4 showed better consistency, easily meeting the tat cut-off throughout the test period. province 1 had a week with 75th percentile value of 37 h while province 8 had two weeks with 75th percentile values of 38 h and 40h. figure 2c represents province 6 comprising five laboratories, categorised as a moderate or poor performer, due to inconsistency, especially during weeks 24–34 of 2016, with two weeks having a 75th percentile tat of over 40 h. after week 35 of 2016, performance stabilised and 75th percentile values corrected to within cut-off values. one province (four individual laboratories; figure 2d) was classified as a markedly poor performer and characterised by inconsistency and repeated failure to meet the cut-off with less than 85% of reported tests meeting stipulated organisational tat cut-off (10 weeks exceeding 40 h). figure 2: weekly national 75th percentile cd4 turn-around time of nine provinces per performance category for the 2016/2017 financial year. (a) good performance (n = 3 provinces); (b) satisfactory performance (n = 4 provinces); (c) moderate to poor performance (n = 1 province) and (d) markedly poor performance (n = 1 province). individual laboratory total turn-around time by week and performance category the different performance levels identified at provincial level still masked the performance and contribution of individual laboratories to provincial performance. scatter plots were constructed to visualise the performance of individual laboratories over 52 weeks per provincial performance category. results showed that irrespective of the provincial performance classification (figures 3a–d), individual laboratory performance included good, satisfactory and poor performance laboratories. figure 3: scatter plots of the 75th percentile total turnaround time of individual laboratories in the national health laboratory service within the provincial performance classification groups a to d for the 2016/2017 financial year. the overall 75th percentile total turn-around time per laboratory is indicated above each plot. (a) good performance (n = 13 laboratories); (b) satisfactory performance (n = 24 laboratories), (c) moderate to poor performance (n = 5 laboratories), and (d) markedly poor performance (n = 4 laboratories). high-volume (blue circles), medium volume (light blue squares) and low-volume (pink triangles) laboratories are indicated. the red line in each graphs represents the target total tat. individual median total tat is indicated above each representative laboratory. the 75th percentile across 52 weeks for the good performance provinces (3 provinces and 13 laboratories) showed good overall compliance (tight clumping of weekly 75th percentile values) where the overall 75th percentile for the whole period ranged from 5.9 h (laboratory 7.1) to 24.8 h (laboratory 9.2) (figure 3a). similarly, the satisfactory performance provinces (n = 24 laboratories) (figure 3b) had a 75th percentile ranging from 10 h (laboratory 4.3) to 34.2 h (laboratory 4.4). the provinces having moderately poor performance had variable tat between 9 h (laboratory 6.4) to 35.7 h (laboratory 6.2) (figure 3c) while markedly poor performance provinces had tat 75th percentile values ranging from 17.2 h (laboratory 5.4) to 39.7 h (laboratory 5.5) (figure 3d). overall, four laboratories recorded a 52-week median tat of more than 30 h. the number of weeks that laboratories did not meet the cut-off criteria of 90% with tat under 40 h varied among categories and laboratories (0–22 weeks), where 12 of 46 laboratories (irrespective of performance category) had zero outlying weeks (26%), 25/46 (54%) more than 5 outlying weeks and 6 (13%) between 6 and 10 outlying weeks. only three laboratories showed outliers for more than 10 weeks where cut-off was not met (6.5%). case examples of individual laboratory component turn-around time analysis figure 4a shows a good performer high-volume laboratory, doing more than 350 samples per day with no outlying weeks (exceeding 40 h cut-off). over the 52 weeks, 86 559 tests were performed by this laboratory. a lab-to-lab 75th percentile of 9.5 h was reported (ranging from 3 h to 16 h), with a reg-to-test of 8.8 h (range from 6 h to 17 h) and a test-to-review of 1.3 h (range from 0 h to 4 h). more than 98% of all samples tested had a total tat of under 40 h. figure 4: stacked bar graphs showing examples of individual laboratory weekly performance for 2016/2017 financial year. 75th percentile turn-around time components color-coded: lab-to-lab (orange), reg-to-test (blue) and test-to-review (green), with cut-off of 40 h (red dotted lines). (a) good performance (laboratory a); (b) satisfactory performance (laboratory b); (c) moderate to poor performance (laboratory c) and (d) markedly poor performance (laboratory d). figure 4b represents a satisfactory performance laboratory, doing 63 998 samples for the period. it reported nine non-consecutive weeks of outliers (exceeding 40 h). of these outlier weeks, eight were due to prolonged reg-to-test (testing delay), where this component contributed between 27 h and 87 h to the total tat reported for these weeks. one week (week 51 of 2016) had an extended lab-to-lab value of 65 h, due to confirmed challenges with logistics. test-to-review ranged from 0 h to 6 h and, as such, had no contribution to the outlying weeks. extended reg-to-test (within laboratory tat) seen in weeks 6–11 of 2017 correlated with instrument downtime based on data provided by beckman coulter call centre log on engineers dispatched. the laboratory represented in figure 4c (moderate to poor performance) tested 77 640 samples during the 52 weeks and had an overall lab-to-lab 75th percentile of 17.7 h (ranging from 10 h to 78 h per week), with 11 weeks exceeding the target total tat (highest recorded total tat of 129 h). the lab-to-lab component (orange bars) contributed to total tat outlying weeks during weeks 21, 30–32, and 48 of 2016 and weeks 11 and 18 of 2017. reg-to-test (within laboratory tat) was the leading cause of outliers noticed during weeks 30–32, 47–48 of 2016 and weeks 11, 15 and 18 of 2016. the combined extended tat in two components (i.e. pre-analytical or lab-to-lab and analytical or reg-to-test) for weeks 30–32 of 2016 and weeks 15 and 18 of 2017 contributed to the total tat for this laboratory to fall into a category of 85% – 90% of samples within the tat target of 40 h. the laboratory contributing the most outlying weeks to group d (figure 3d) was analysed for component tat. this laboratory had 18 weeks of exceeding the target total tat. this was for the most part due to prolonged within laboratory tat (blue bars, figure 4d). from week 31 to 45 of 2016 the reg-to-test component contributed as much as 81 h (week 40 of 2016) to the weekly tat. during this period, some weeks also experienced prolonged test-to-review times of up to 25 h (week 1 of 2017). discussion tat remains a key performance indicator of laboratory service efficiency.4,14 the parameters reported (mean vs median) and time intervals of reporting impacts the utilisation of tat as a means to identify and address non-compliance to organisational cut-offs. definitions of tat may vary and depend on the test (routine or emergency), priority of reporting (immediate or delayed clinical intervention), the population served and activities or components measured.4 the clinical outcome, needs and responsibilities of management determine how tat information is used to ensure that there are no unnecessary delays in result reporting. across the nhls, tat information typically remains the jurisdiction of the testing laboratory where the laboratory manager uses this data to identify problems and initiate corrective action; the individual laboratory has sole and direct access to its own daily or weekly tat data.3,4,5 tat monitoring is however critical for priority programmes, such as hiv and tuberculosis,2,12 where individual laboratories monitor their respective test tat, while the organisation is responsible for reporting performance of the network of laboratories. relevant updated information on the efficiency of service delivery is vital in this context for risk assessment and timely intervention to ensure the continued excellence of service delivery10 and meeting dire local hiv and tuberculosis programme needs.9 ideally, sample-by-sample real-time reporting of tat would be the preferred way to monitor and assist laboratories in the identification of specific service delivery and related tat challenges. hierarchical global overview (usually annual) tat reporting is however the simplest and most widely used, but masks poor performance, as confirmed by data from this study. interrogating the weekly data by drilling down to laboratory level at weekly intervals, enables the identification of outliers and poor performers. this study showed that lower hierarchical levels, as well as shorter time periods, can unveil problematic and inefficient testing laboratories (figure 2 and figure 3). adding weekly tat component analysis at laboratory level further identifies problematic testing weeks and possible causes of prolonged tat (figure 4 and box 1). box 1: summary of study findings. the most common issues that impacted on prolonged lab-to-lab (pre-analytical) tat were delays in transport or sample collection from clinics to testing laboratories and changes in courier routes and pick up times. reg-to-test times (laboratory tat) were prolonged due to instrument downtime, lack of trained staff to operate testing instruments, delayed sample registration in the testing facility, challenges with reagent availability and timely delivery, and staff strikes.15 delays in the test-to-review phase, were mostly due to lack of result auto-authorisation in high-volume laboratories, nonavailability of result authorising staff when samples are analysed during night shifts or where problems with connectivity of the trackcare lis were reported. several references focus on the main causes of tat delays.5,16,17,18,19,20,21,22 the main objective of this study, however, was not to describe causes of delayed tat, but to emphasise the importance of tat monitoring with shorter time intervals as a means of more proactive interventions for sustained good performance across network of laboratories. data reported here demonstrate the need for more frequent tat reporting in effective performance management.10,13,15,23,24 an interactive weekly tat dashboard was introduced nationally in 2018 for the most requested tests across the nhls. frequent performance reporting can be effective in identifying challenges with meeting target tat cut-offs allowing for timely interventions.13 weekly assessment of tat and tat components not only identifies problematic testing laboratories or days with tat challenges, but also enables the identification of individual outlier samples that can be investigated (root cause analysis) to assess causes of tat delays. based on the data presented in this study, further refinement of the current reporting platforms is recommended to include daily reporting for rapid proactive intervention. a further recommendation is to integrate daily quality control tests, external quality assessment testing and equipment downtime supplier call-out data into the reporting to assist with focused troubleshooting and interventions. turn-around time monitoring and reporting are however guided by the requirements of the end user and will continue to be available at various time intervals for laboratory network management to assess overall trends, with weekly or daily reports to laboratory and programme managers enabling timely proactive intervention to ensure optimal laboratory performance and timely patient result reporting. limitations the monitoring of tat in the nhls is currently limited as end-to-end sample tracking system is absent. the tat reported in this article thus only represents the time from first registration on the lis to review of the result. conclusion national and provincial analysis of tat mask individual laboratory performance; therefore, tat analysis by week and by laboratory is recommended to highlight laboratory tat inefficiencies. root-cause analyses were able to identify pre-analytical, analytical or post-analytical factors contributing to performance. tat data was used to categorise performance at the provincial and laboratory level. this study used the concept of zooming in to lower levels and shorter times of tat reporting to identify possible non-compliant laboratories. in conclusion: (1) outlying weeks are not prescribed by provincial or laboratory classification of performance, (2) performance did not correlate to the size of the laboratory (i.e. test volumes of high, medium and low), (3) there were laboratories that had no outlier weeks during the analysed period that can serve as model laboratories for setting performance standards and good reproducibility of week-on-week performance across a network of testing laboratories. acknowledgements this article was in part presented as a poster at the african society for laboratory medicine meeting in 2018, abuja, nigeria (id:ps-2-3b-070), 10–13 december 2018. the authors thank area, business and laboratory managers in the nhls for their feedback on the use of the weekly tat dashboard. thank you to the central data warehouse for the availability of data. competing interests the authors have declared that no competing interests exist. authors’ contributions d.k.g. supervised the study by providing leadership and oversight as the project leader. l.-m.c. and n.c. designed the study, developed the methodology and conducted the research, data analysis, initial write-up and review. d.k.g. reviewed the data, provided editorial comments and technical input. all authors contributed to the manuscript development. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability statement data is available as online supplementary document 1. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references national health laboratory service (nhls). nhls annual report 2014/15 sandringham [homepage on the internet]. johannesburg: national health laboratory service; 2015 [cited 2017 jul 26]. available from: http://intranet.nhls.ac.za/assets/files/policy/nhls_annual_report_2015.pdf glencross dk, coetzee l, cassim n. an integrated tiered service delivery model (itsdm) based on local cd4 testing demands can improve turn-around times and save costs whilst ensuring accessible and scalable cd4 services across a national programme. plos one. 2014;9(12):e114727. https://doi.org/10.1371/journal.pone.0114727 goswami b, singh b, chawla r, gupta vk, mallika v. turn around time (tat) as a benchmark of laboratory performance. indian j clin biochem. 2010;25(4):376–379. https://doi.org/10.1007/s12291-010-0056-4 hawkins rc. laboratory turnaround time. clin biochem rev. 2007;28(4):179–194. pati hp, singh g. turnaround time (tat): difference in concept for laboratory and clinician. indian j hematol blood transfus. 2014;30(2):81–84. https://doi.org/10.1007/s12288-012-0214-3 valenstein p. laboratory turnaround time. am j clin pathol. 1996;105(6):676–688. https://doi.org/10.1093/ajcp/105.6.676 valenstein pn, emancipator k. sensitivity, specificity, and reproducibility of four measures of 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high-volume laboratory. afr j lab med. 2020;9(2):a947. https://doi.org/10.4102/ajlm.v9i2.947 coetzee l, cassim n, tepper m, glencross dk. standardizing individual laboratory turnaround time (tat) performance amongst laboratories in a cd4 testing network. pathcape 2018: 56th international fsasp congress; 2018 aug 16–18; stellenbosch. abstract introduction methods results discussion acknowledgements references about the author(s) victor n. fondoh bamenda regional hospital laboratory, regional hospital bamenda, cameroon faculty of health and medical sciences, catholic university of bamenda, bamenda, cameroon charles n. awasom faculty of health and medical sciences, catholic university of bamenda, bamenda, cameroon rebecca enow-tanjong faculty of health and medical sciences, catholic university of bamenda, bamenda, cameroon richard m. fondoh north-west regional fund for health promotion, bamenda, cameroon patrick njukeng global health systems solutions, limbe, cameroon judith shang center for disease control and prevention, yaounde, cameroon julianna ndasi global health systems solutions, limbe, cameroon moses samje faculty of health sciences, university of bamenda, bamenda, cameroon claris n. muluh administration, regional hospital bamenda, bamenda, cameroon thompson n. kinge bamenda regional hospital laboratory, regional hospital bamenda, cameroon citation fondoh vn, awasom cn, enow-tanjong r, et al. evaluation of corrective actions of feedback from clinicians on clinical laboratory services at bamenda regional hospital laboratory, cameroon. afr j lab med. 2020;9(1), a843. https://doi.org/10.4102/ajlm.v9i1.843 original research evaluation of corrective actions of feedback from clinicians on clinical laboratory services at bamenda regional hospital laboratory, cameroon victor n. fondoh, charles n. awasom, rebecca enow-tanjong, richard m. fondoh, patrick njukeng, judith shang, julianna ndasi, moses samje, claris n. muluh, thompson n. kinge received: 31 may 2018; accepted: 05 dec. 2019; published: 23 mar. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: customers’ satisfaction is imperative for success. clinical laboratories continuously strive to attain very high levels of customer satisfaction to serve their clients and maintain accreditation. the concept of customer satisfaction has not yet been asserted in most clinical laboratories in cameroon. objectives: our objectives were to assess the satisfaction of clinicians with the laboratory services at the bamenda regional hospital laboratory, identify important challenges, corrective actions implemented and changes in satisfaction. methods: this retrospective study reviewed secondary data from clinician satisfaction survey records from march 2017 and november 2017. challenges and implemented corrective actions were identified for assessed statements of dissatisfaction (dissatisfaction rates ≥ 20%) on the march 2017 survey. satisfaction rates in march 2017 and november 2017 were compared. results: high levels of dissatisfaction were observed for general satisfaction, waiting time, communication, duty consciousness, specimen collection and approach on the march 2017 survey. the main challenges identified were: lack of respect for the expected length of the waiting time, poor attitude, inadequate information, staff shortage and inadequate supervision. statistically significant reductions in rates of dissatisfaction were observed for general satisfaction, waiting time, communication, response to emergencies, issuing of results, specimen collection, approach and duty consciousness. conclusion: waiting time is a major cause of clinician dissatisfaction with laboratory services. the identification of clinicians’ challenges and the effective implementation of corrective actions contribute to improvements in clinician satisfaction. keywords: evaluation; feedback; corrective actions; clinician satisfaction; clinical laboratory services. introduction for every business to succeed, customer satisfaction is imperative. the satisfaction of customers at health institutions such as clinical laboratories, which includes clients (patients) and clinicians (doctors, nurses), is evaluated by using customer satisfaction surveys to continuously improve services.1,2,3,4 besides the continuous striving to attain very high levels of customer satisfaction, clinical laboratories must maintain their customers and competence in compliance with the international standards and their accreditation. this is done through the effective implementation of the international organization for standardization (iso) 15189 and iso 17025 standards.4,5,6 feedback from laboratory customers is a compulsory quality indicator in the preparation of the balanced scorecard for monitoring the effectiveness of the quality management system.4,6 one of the tools used to assess feedback is the clinician satisfaction survey. many previous studies have assessed clinicians’ satisfaction with clinical laboratories in europe, america7,8 and africa.9 findings from these studies reveal that most clinicians were satisfied with the cleanliness of the environment, quality of the results, presentation of the results, sample collection and handling of samples, whereas others were dissatisfied with the waiting times and attitude of the staff.8,9,10,11 most of these studies did not identify the corrective actions implemented or document changes in satisfaction. although clinician satisfaction surveys are conducted for clinical laboratories in cameroon that are enrolled in the stepwise improvement process towards accreditation,12,13,14 there is little published literature on the topic. the regional hospital bamenda is a public hospital that uses feedback from customer satisfaction surveys from patients to identify departments in the hospital that they are dissatisfied with. feedback from the surveys are presented to the clinicians and heads of the departments during the weekly hospital coordination meetings. however, corrective actions are not evaluated, and the questionnaires used are not standard. besides, apart from the laboratory services, the concept of a ‘clinician survey’ has not yet been established by other departments of the hospital. the bamenda regional hospital laboratory is a business centre in the regional hospital bamenda. it is a newly accredited clinical laboratory15 that routinely makes use of customer satisfaction surveys as a tool to assess customer feedback. surveys are done twice a year and corrective actions implemented in order to continuously improve on the quality of service delivery. our objectives were to assess clinicians’ satisfaction with the laboratory service, identify challenges, corrective actions implemented and document changes in satisfaction. methods ethical considerations ethical approval to carry out this study was obtained from the institutional review board (irb) of the bamenda regional hospital (irb reference number: 18/app/rdph/rhb/irb). consent to participate in the survey was obtained verbally (by agreeing to fill out the questionnaire). study area the study was conducted at bamenda regional hospital laboratory (brhl), which is a department at the regional hospital bamenda. the regional hospital bamenda is a level ii referral hospital in the north-west region of cameroon with a capacity of 400 beds and a bed occupancy rate of about 90%. the brhl was chosen for this study for the following reasons: it is the largest public laboratory in the north-west region with high quality and specialised services. it has a central laboratory made up of six departments and three satellite laboratories within the hospital premises. it has the capacity to run 118 different tests and a staff of 35. it serves about 170 patients (inpatients and outpatients) and about 90 clinicians (doctors, senior nurses, charge nurses and consulting nurses). it performs customer satisfaction surveys to continuously improve the services and the presence of records. besides, the haematology, biochemistry and serology services are newly accredited for competence and compliance with the iso 15819:2012 standard by the south african national accreditation system.15 research design this retrospective study reviewed secondary data collected from clinician satisfaction surveys that were carried out at the brhl in march 2017 and november 2017. information from the survey questionnaires and corrective action records were the primary data from which secondary data for the study were collected. the brhl has an advanced quality management system and conducts customer satisfaction surveys twice a year as a quality indicator for its balanced scorecard. the designed questionnaires were reviewed by the laboratory management and medical adviser16 and were administered to clinicians selected at random at the brhl by the head of the customer service in march 2017 and again in november 2017 to the same group of participants. both surveys were completed within a week during the hospital coordination meetings and the responses collected at the end of the meeting or at the offices of those who were not present in the meetings and those who were not on duty. the content of the questionnaires was explained to the participants who personally responded to the questions. the respondents were free to provide a phone number (optional) and signature (optional) so they could be contacted in case clarification was needed. each questionnaire was made up of rating statements and open-ended questions. the statements were assessed using a 5-point likert ranking as follows: poor (1), fair (2), good (3), very good (4), excellent (5).17 the statements covered the following areas: accuracy (correctness of laboratory results), reliability (trustworthiness of laboratory results), waiting time (time between sample collection and dispatch of results), communication (management of information by the laboratory), response to emergencies (attention to emergency tests), staff-client relationship (the bond between laboratory staff and clinicians), cleanliness (neatness of the laboratory and staff), duty consciousness (awareness of laboratory staff to perform their duty promptly), specimen collection (effectiveness of phlebotomists), issuing of results (effectiveness of delivery of results), approach (the way laboratory staff attended to patients and clinicians) and general satisfaction (the overall assessment of the service). open-ended questions included: what did the clinician like most about the laboratory? what did the clinician dislike about the laboratory, recommendations for improvements and suggestions of the tests that could be included on the laboratory test menu? at the end of the surveys, reports were presented to laboratory staff during the staff meetings and to clinicians in the laboratory-clinic interface (hospital coordination and scientific) meeting. action plans with corrective actions were implemented for the non-conformities (challenges) identified. evidence of the closed gaps were maintained in action plan reports, communication logs, complaint logs and meeting notes. inclusion criteria data from clinicians who were part of the surveys were included in the study. this included clinicians who were present at the hospital coordination meetings and clinicians who were on duty during the weeks that the surveys were conducted. participation was based on a random selection with verbal consent (by agreeing to respond to the questionnaire). exclusion criteria data from clinicians who were not part of the surveys were excluded from the study. this included clinicians who were not present at the hospital coordination meetings and clinicians who were not on duty during the weeks the surveys were conducted. data collection data were collected by three reviewers trained to collect secondary data for the study using a structured data collection format. all available questionnaires from the two surveys were used. the data were classified by level of satisfaction as follows: responses of excellent and very good were classified as ‘satisfied’, good was classified as ‘neutral’, and fair and poor were classified as ‘dissatisfied’. data for statements assessing: accuracy, reliability, waiting time, communication, response to emergencies, staff-client relationship, cleanliness, duty consciousness, specimen collection, issuing of results, approach and general satisfaction were decoded and entered into a microsoft excel 2010 spreadsheet (microsoft corporation, redmond, washington, united states). action plans (the plan of activities in the laboratory), communication logs (records of communication with clinicians), complaint logs (records of complaints from clinicians) and meeting notes (minutes from laboratory staff meetings and laboratory-clinic interface meetings) were reviewed to capture the corrective actions implemented. key informants (doctors, nurses, laboratory staff, laboratory management and hospital management) were contacted in case a clarification or further explanation or information was needed. data analysis percentage frequency distributions were calculated to rate the levels of satisfaction (satisfied, neutral and dissatisfied) for each of the assessed statements. dissatisfaction with an assessed statement was defined as a percentage score of 20% or more of dissatisfied responses. chi-square and fisher exact tests were used to compare the rates of satisfaction of the two surveys. a p-value of 0.05 or less was considered to be statistically significant.8 assessed statements with 20% or higher dissatisfaction score on march 2017 were selected for review. qualitative content analysis was conducted for the open-ended question responses by using calculated frequencies from two reviewers.8,16,18 responses that were related to assessed statements with a dissatisfied score were identified as challenges. clarification on the challenges and the corrective actions implemented were identified through a review of clinician survey reports, meeting notes, communication logs, complaint log action plans and interrogation with key informants (doctors, nurses, brhl staff and laboratory management and hospital management). results customer satisfaction scores out of 85 questionnaires administered to the clinicians in march 2017 and 89 administered in november 2017 during the clinician satisfaction surveys, 83 and 88 clinicians responded (response rates: 97.6% for march 2017 and 98.9% for november 2017) (table 1). following the assessed statements in march 2017, waiting time (34.9%) registered the highest dissatisfied rate. high dissatisfaction rates were also registered for general satisfaction (26.3%), communication (24.4%), responses to emergencies (24.1%), issuing of results (22.0%), specimen collection (21.5%), approach (21.3%) and duty consciousness (21.0%). the following areas fell below the dissatisfied rate: reliability (14.5%), staff-patient relationship (11.4%) and cleanliness (7.2%) (table 2). table 1: summary of content analysis of the clinician satisfaction survey of march 2017 at the regional hospital bamenda, cameroon. table 2: distribution of responses to the assessed statements exploring clinicians’ views on laboratory services from the same population in march 2017 and november 2017 at bamenda regional hospital laboratory, cameroon. in november 2017, no assessed statements had a dissatisfied score. between march 2017 and november 2017, statistically significant reductions in dissatisfied scores were registered for: waiting time (34.9% to 19.3%, p = 0.014), general satisfaction (26.3% to 9.6%, p < 0.001), communication (24.4% to 12.5%, p = 0.036), issuing of results (22.0% to 8.1%, p = 0.005), specimen collection (21.5% to 13.8%, p = 0.020), approach (21.3% to 8.4%, p = 0.007), and duty consciousness (21.0% to 4.7%, p < 0.001). although not statistically significant, there was a general decrease in dissatisfied scores between the two surveys for responses to emergencies, reliability and the staff-patient relationship. in addition, accuracy and cleanliness had reduced dissatisfied scores on the november 2017 survey, although they were not considered for evaluation, since they did not register dissatisfied rates below 20% on the march 2017 survey. open-ended responses waiting time: waiting time received the highest number of open-ended responses (table 2). respondents cited the lack of respect for waiting time for some emergencies, single or grouped tests, no explanation when the waiting time was delayed, inadequate information about the laboratory services, shortage of staff and lack of relaxation facilities such as a television set, video or audio player. some clinicians did not use the stat request forms (lab-rap-form) or indicate the emergency code ‘urgent!’ on request forms in cases of emergency. thus, their requests were treated as routine. for corrective actions, clinicians were oriented on the various processes and procedures in the laboratory, the waiting time for some tests was re-established, a test menu with waiting times was redistributed and posted in all wards, issuing of results at the top of each hour was intensified, laboratory staff were re-trained on good customer service practice, more staff were hired and a staff retention policy proposed to the hospital management. additionally, a proposal for the purchase of a television and a sound system was made to the hospital management. duty consciousness: concerning duty consciousness, clinicians commented on lateness to work and ineffective presence at the duty post. although the hospital had a biometric time clock system that was used to monitor staff attendance, the details were evaluated at the end of the month. thus, the effective presence of staff on duty was not promptly monitored using the biometric system. for corrective actions, the laboratory staff were cautioned about having a poor attitude and an attendance register was introduced in the laboratory by the laboratory management. approach: for approach, clinicians identified as challenges the lack of politeness of laboratory staff and inadequate explanations to patients or clinicians. for corrective actions, the laboratory staff were cautioned about having a poor attitude and re-trained on the importance of good customer service. issuing of results: clinicians identified inadequate follow-up on pending results and delay in the dispatch of some results, especially for patients admitted in the wards, as the main challenges. for corrective actions, the laboratory staff were cautioned about having a poor attitude, the laboratory management regularly made supervision tours around the wards to monitor the activities of the laboratory, and the results dispatch registers were monitored daily. specimen collection: for specimen collection, clinicians reported poor patient consent, poor venepuncture, many specimen rejections and task shifting (laboratory phlebotomist to nurses for patients admitted in the wards) as challenges. for corrective actions, the laboratory staff and clinicians were educated and trained on the specimen collection procedures and the need for proper specimen collection. communication: for communication, clinicians identified the following challenges: an inadequate explanation of procedures or difficulties and unavailability of laboratory information (phone numbers of some of the laboratory managers and test menu) to some clinicians. for corrective actions, adequate information on the laboratory services was provided and the hospital intercommunication system was maintained. response to emergencies: clinicians identified the following challenges related to response to emergencies: lack of respect for the procedures and waiting time for emergency requests. for corrective actions, laboratory staff were cautioned about having a poor attitude towards work, clinicians were oriented on the procedure for handling emergency requests, emergency requests were tracked to monitor waiting times and laboratory staff were cautioned about the need for adequate communication. discussion the purpose of this study was to assess clinicians’ satisfaction, identify what challenges clinicians faced when accessing laboratory services, identify corrective actions implemented in response to those challenges and document changes in satisfaction. a good response rate16 was registered for both surveys, which was attributed to the fact that most of the questionnaires were issued to the respondents (clinicians) during hospital coordination meetings, where almost all of the respondents were present and the responses were collected at the end of the meeting. for both surveys, some of the questionnaires administered to clinicians at their offices were not returned. respondents knew the surveys were routine and that they were an avenue for them to give their feedback, express their challenges and make proposals for the improvement of the laboratory services.8 the dissatisfaction rate in relation to waiting time in the march 2017 survey was the highest. although the laboratory had an advanced quality management system in place for the management of patients, the system was not respected by the laboratory staff. in addition, the absence of the hospital intercommunication system affected the flow of information from the laboratory to the wards and clinicians. due to the implementation of some of the corrective actions, there was a significant reduction in the dissatisfaction rate concerning waiting time on the november 2017 survey. the clinicians (especially those newly hired) created greater awareness of the activities and relationships between clinicians and the laboratory. they understood why the waiting time of some tests were longer than others and could, in turn, inform their patients. furthermore, the maintenance of the hospital intercommunication system strengthened communication between the laboratory and clinicians. similar dissatisfaction rates have also been identified in many other studies. oja et al., reported a dissatisfactory score for waiting time by clinicians in a study in finland.8 they attributed this to inadequate information about the waiting time of emergency requests, lack of respect for procedures for emergency requests and high prices for emergency tests.8 for corrective action, the laboratory informed the clinicians about the waiting time and advised them to order emergency tests for emergency cases.8 steindel and howanitz in a survey of clinicians in the united states also reported low satisfaction rates for waiting time.7 they observed that the waiting time for laboratory services was too long and the clinicians did not consider the laboratory sensitive enough to handle their emergency requests.7 allen and harris19 and boyde et al.,20 in the united kingdom also reported dissatisfactory rates for waiting time by clinicians. teklemariam et al. reported a satisfactory score for the waiting time for notification of critical values and timely results for hiv testing, but this was for a single test,9 whereas the report from our study was for the entire laboratory. zarbo et al. and zarbo, in another study in the united states, reported low satisfaction scores by clinicians that were related to timeliness of reporting,11, 21 and although corrective actions were implemented in the study in finland, there was no significant change in the dissatisfaction rate. duty consciousness received a high dissatisfaction rate score on the march 2017 survey. the corrective actions and the introduction of the laboratory attendance register by the laboratory management significantly reduced the dissatisfaction rate. this improved supervision of staff by the laboratory management, because details of staff attendance and movements, including when they went on break, were monitored promptly and daily. oja et al. in finland, observed a low dissatisfaction rate for the attitude that was not appropriate for comparison during their statistical analysis.8 the approach by the laboratory staff also had a high dissatisfaction rate score in march 2017, this was attributed to a poor attitude on the part of some laboratory staff, possibly due to burnout from high workload. the corrective actions implemented significantly reduced the dissatisfaction rate in november 2017. the re-training and re-orientation of laboratory staff and the hiring of more workers also reduced burnout. other studies did not assess the approach.7,8,11 clinicians were also dissatisfied with the issuing of results on the march 2017 survey. the laboratory staff were cautioned on attitude, and the corrective actions implemented significantly reduced the dissatisfaction rate. oja et al., in finland, reported a non-significant rate of dissatisfaction for missing results8 and zarbo21 reported a dissatisfaction rate for notification of abnormal results. specimen collection registered a high dissatisfaction rate on the march 2017 survey. according to key informants, this was attributed to inadequate training and orientation and failure to respect laboratory policies and procedures. many laboratory staff and clinicians were newly hired and were not trained on specimen collection. the corrective actions implemented significantly reduced the dissatisfaction rate. the clinicians became aware that they have to assist in specimen collection in emergency cases, including difficult cases, during routine working hours (7:30 to 15:30 pm). oja et al., in finland, reported a significant reduction in dissatisfaction rate for phlebotomist rounds, after the rounds were rescheduled in cooperation with the in-patient unit and other clinical units adjusted their work pattern to integrate better with the laboratory service.8 communication registered a high rate of dissatisfaction among the assessed statements in the march 2017 survey. according to key informants, this was attributed to the fact that some of the wards were recently renovated and information posted on the walls had been removed. some of the clinicians were newly hired or recruited and had not received an adequate orientation on the laboratory procedures and policies. although the main communication tools of the laboratory were in place, the laboratory did not adequately use them and verbal communication was still a problem. the laboratory did not register some of the verbal complaints presented by the clinicians for follow-up, and the complaints were persistent. information from key informants identified poor verbal communication of laboratory staff as the main cause of most of the challenges, which included long waiting time, many specimen rejections and poor follow-up of pending results. normally, after specimen collection in the wards, the phlebotomist was supposed to inform the patient of the exact hour of the day to collect results, but some phlebotomists informed them of the duration (for example, after 2 hours). this caused some of the patients to speculate the exact hour of the day to collect their results and resulted in numerous complains about waiting time even when the time had not been exceeded. besides, the laboratory results of patients admitted to the wards were dispatched directly to the clinicians, but some patients and caregivers presented to the laboratory separately to collect results that had already been issued to the wards. this was due to the failure to provide adequate information to the patient or caregiver during specimen collection by the laboratory. the corrective actions implemented significantly reduced the dissatisfaction rate of communication in the november 2017 survey. clinicians could better orient their patients on the functioning of the laboratory. oja et al.,8, in finland reported a low dissatisfaction rate in their study that was not appropriate for comparison during statistical analysis. response to emergencies registered a high dissatisfaction rate in the march 2017 survey. key informants attributed this to the poor attitude of the staff and lack of adequate information on laboratory services provided to clinicians. the corrective actions implemented significantly reduced the dissatisfaction rate. oja et al., in finland, observed a non-significant reduction in dissatisfaction rate for scheduling of phlebotomists during emergency hours. general satisfaction was the general impression of the clinicians about the laboratory services. general satisfaction registered a high rate of dissatisfaction among the assessed statements on the march 2017 survey. this was due to the accumulation of challenges from all the other assessed statements. the corrective actions implemented following the identification of all the challenges accounted for the significant reduction in the dissatisfaction rate in the november 2017 survey. several other studies did not consider the general satisfaction with laboratory services.7,8,11 recommendations a significant increase in customer (clinicians) satisfaction level of the brhl can be achieved through cooperation among hospital management, laboratory management, clinicians and other hospital staff. it is recommended that: the government should improve laboratory infrastructure to boost patient flow, employ more staff to replace those who transfer or retire and provide more automated machines to increase efficiency and reduce waiting times. the hospital management should: provide an interface system to link laboratory equipment to the laboratory information system to reduce clerical errors, maintain the internal communication system in the hospital, provide a web-based communication system that links the laboratory service to patients and clinicians, notify them when their results are ready, remind them of their appointments with the laboratory and allow logging of issues with laboratory services, provide equipment such as a television set, video or audio player to entertain customers, especially when they are waiting to be served, hire more staff to handle the workload, sign engagements with hired workers to guarantee their job security and motivation, and retain existing staff to maintain continuity, train other hospital staff on the importance of customer satisfaction, support the laboratory in the effective implementation of corrective actions, and plant close-circuit television cameras in the departments of the laboratory and hospitals to ease supervision and monitoring of staff activities and customers. clinicians should respect the policies and procedures of the laboratory and hospital. they should follow their appointments and report any incidents to the laboratory management, educate and counsel their patients before sending them to the laboratory, communicate well with their patients and the laboratory management in case of any incident, and receive and direct patients properly to avoid cross-aggregation with other services. the laboratory management and staff should positively change their attitude, respect all laboratory policies and procedures, intensify supervision, regularly train new and old staff, effectively implement corrective and preventive actions in real-time, intensify meetings (laboratory-clinic interface) with the users of the laboratory, educate the public on the use of laboratory services by making more frequent public talks over the radio and television, and revise the questionnaire to include more open-ended questions. limitations since this is a retrospective study, we could not control the consistency of the data collected. our study was based on the data collected, so we were limited to the assessed statements or questions. the fact that some participants provided their phone numbers might not be a bias since they knew that they could be called up as key informants and this was optional. conclusion according to our study, waiting time was a major cause of clinician dissatisfaction with laboratory services. to maintain reduced dissatisfaction rates in clinical laboratories, it is important to continuously identify challenges and effectively implement corrective actions. acknowledgements we acknowledge the staff and management of the regional hospital bamenda laboratory, present and former directors and management of the regional hospital bamenda and the ministry of public health for their support towards the satisfaction of customers and their wonderful input in terms of financial and management commitment towards the improvement of the laboratory services. thanks to the global health system solution, limbe and center for disease control and prevention (cdc) cameroon for technical support towards the improvement of the laboratory. lastly, we appreciate the staff of the bamenda regional hospital laboratory, users of the laboratory services (patients or caregivers, doctors and nurses) for their wonderful cooperation towards the satisfaction of patients. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions v.n.f. (study leader) was responsible for conceptualisation of the study, study design, statistical analysis and final writing of the manuscript, c.n.a. and r.e.-t. were responsible for conceptualisation of the study and correction of the manuscript, r.m.f. was responsible for correction of the manuscript and data collection, j.s., p.n., j.n., m.s. and t.n.k. assisted in the correction of the manuscript and c.n.m. did the data collection. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references kaye ad, okanlawon oj, urman rd. clinical performance feedback and quality improvement opportunities for perioperative physicians. adv med educ pract. 2014;5:115. https://doi.org/10.2147/amep.s62165 veloski j, boex jr, grasberger mj, et al. systematic review of the literature on assessment, feedback and physicians’ clinical performance: beme guide no. 7. med teach. 2006;28(2):117–128. https://doi.org/10.1080/01421590600622665 mugford m, banfield p, o’hanlon m. effects of feedback of information on clinical practice: a review. bmj. 1991;303(6799):398–402. https://doi.org/10.1136/bmj.303.6799.398 iso, e., 15189. medical laboratories – particular requirements for quality and competence. geneva: international organization for standardization. college of american pathologists. laboratory general checklist: laboratory accreditation program. northfeld, il: college of american pathologists, 2005. iso, i. iso/iec17025: 2005 general requirements for the competence of testing and calibration laboratories. iso, geneva; 2005. steindel sj, howanitz pj. physician satisfaction and emergency department laboratory test turnaround time: observations based on college of american pathologists q-probes studies. arch pathol lab med. 2001;125(7):863–871. oja pi, kouri tt, pakarinen aj. from customer satisfaction survey to corrective actions in laboratory services in a university hospital. int j qual health care. 2006;18(6):422–428. https://doi.org/10.1093/intqhc/mzl050 teklemariam z, mekonnen a, kedir h, et al. clients and clinician satisfaction with laboratory services at selected government hospitals in eastern ethiopia. bmc res notes. 2013;6(1):15. https://doi.org/10.1186/1756-0500-6-15 jones bj, bekeris lg, nakhleh rf, et al. physician satisfaction with clinical laboratory services: a college of american pathologists q-probes study of 138 institutions. arch pathol lab med. 2009;133(1):38–43. zarbo rj, nakhleh re, walsh m, et al. customer satisfaction in anatomic pathology: a college of american pathologists q-probes study of 3065 physician surveys from 94 laboratories. arch pathol lab med. 2003;127(1):23–29. yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin pathol. 2010;134(3): 401–409. https://doi.org/10.1309/ajcpnbbl53fwuiqj yao k, maruta t, luman et, et al. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. 2014;3(2), art. #194, 8 pages. https://doi.org/10.4102/ajlm.v3i2.194 ndasi j, dimite l, mbome v., et al. decentralised facility-based training as an alternative model for slmta implementation: the cameroon experience. afr j lab med. 2014.3(2), art. #231, 6 pages. https://doi.org/10.4102/ajlm.v3i2.231 sanas. 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african union member states, through the efforts of africa cdc and partners, have received technical support to use existing real-time polymerase chain reaction (rt-pcr) instruments to conduct testing, mostly at national reference or equivalent laboratories. although this technology may be inadequate to entirely meet the scale of testing required for covid-19 (because of limited numbers of instruments), these instruments are within the existing tiered laboratory network. leveraging existing rt-pcr instruments for covid-19 diagnosis is an important step in strengthening health systems on the continent for future emergency pandemics. responding to the current pandemic in ways that strengthen health systems and that go beyond emergency solutions to consider long-term solutions will benefit the continent as a whole. the ebola outbreak in west africa provides useful lessons on how emergency responses can impact health systems.2 during the ebola outbreak, novel technologies were provided to countries without consideration to the existing tiered laboratory network. as a consequence, some countries have been unable to incorporate those novel technologies into their laboratory networks, which impacts the overall sustainability of their health systems. it is time to remind both national and regional communities on the continent to think beyond the current covid-19 pandemic so that when africa emerges on the other side, its health systems will be stronger and more prepared to respond to the next one. central questions to keep in mind during the covid-19 response include: how will countries absorb multiple novel technologies within their health systems post-covid-19? how will emergency-use-authorised in vitro diagnostics be part of national tiered laboratory systems post-pandemic? what role will manufacturers play in initiating long-term evaluation procedures for covid-19 technologies? will these technologies be left to countries to manage without adequate support, guidance or capacity? answers to these questions are critical now. it is therefore imperative that national regulatory agencies, diagnostics manufacturers and national diagnostics technical working groups not ‘rush’ into issuing or adopting euas for new and untested devices outside their networks, but to consider the long-term impact of those technologies on their health systems. some of these approaches may include: update the current rt-pcr instruments to incorporate covid-19 testing. as the gold standard for viral testing, countries must work with their existing rt-pcr technology manufacturers to upgrade reagents, kits and software to accommodate covid-19.3 the latest eua from the united states food and drug administration for the cepheid xpress cartridge on genexpert instruments (cepheid, sunnyvale, california, united states) and the abbott r-sars-cov-2 reagents on abbott m2000 instrument (abbott laboratories, chicago, illinois, united states) are typical examples.4 national regulatory agencies should develop guidelines that outline clear and unambiguous procedures for issuing eua for new technologies. these guidelines should incorporate manufacturers’ plans to work with national agencies to incorporate new devices into existing tiered networks as euas expire. national regulatory agencies should limit eua approvals to devices that employ the gold standard of rt-pcr in their technologies over antigen-antibody-based, lateral-flow rapid diagnostic test kits, which may not demonstrate comparable sensitivity and specificity to sars-cov-2 as with rt-pcr instruments. in cases where rapid diagnostic tests are considered (because of urgency to scale up testing), scientifically prudent testing algorithms must be developed by national stakeholders and enforced. in this algorithm, any positive covid-19 sample from a rapid diagnostic test should be accompanied an rt-pcr-based confirmatory test. in addition, a percentage of negative test samples should also be confirmed with rt-pcr, in order to continuously monitor and confirm the specificity and sensitivity of rapid diagnostic tests. national regulatory agencies should seek the support of international technical partners, including the world health association, africa cdc, the african society for laboratory medicine and other non-governmental organisations such as the united states pharmacopeia and foundation for innovative new diagnostics, to help support and build capacity to rapidly scale up testing for enhanced case management and long-term emergency preparedness. these strategies and others, supported by national stakeholders, will support african countries in strengthening systems and improve preparedness for emerging pandemics, while building sustainable laboratory systems to help support better healthcare across the continent. acknowledgements the manuscript went through internal united states pharmacopeia technical and editorial process workflow. no need to mention individuals. competing interests the author has declared that no competing interest exists. authors’ contributions f.a.u. was the sole author of this article. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sector. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the author and do not necessarily reflect the official policy or position of any affiliated agency of the author. references world health organization. in vitro diagnostics and laboratory technology: prequalification of ivds and medical devices. [homepage on the internet]. geneva: world health organization; c2020 [cited 2020 apr 8]. available from: https://www.who.int/diagnostics_laboratory/200408_imdrf_collated_table_8_april_2020.pdf?ua=1 kieny m-p, evans db, schumets g, et al. health-system resilience: reflection on the ebola crisis in western africa. geneva: world health organization 2014;92:850. https://doi.org/10.2471/blt.14.149278 world health organization. emergency use listing (eul), weekly update. [homepage on the internet]. date [cited 2020 apr 4]. available from: https://www.who.int/diagnostics_laboratory/200403_eul_covid19_ivd_update.pdf?ua=1 united states food & drug administration (fda). xpert xpress sars-cov-2 test. [email discussion on the internet] 2020 march 20 [cited 2020 apr 8]. available from: https://www.fda.gov/media/136316/download abstract background ethical considerations case presentation management and outcomes discussion acknowledgements references about the author(s) rachel s. kamgaing medical analysis laboratory, chantal biya international reference centre for research on hiv/aids prevention and management, yaoundé, cameroon yagai bouba section appui au secteur sante, national aids control committee, yaoundé, cameroon chantal biya international reference centre, yaoundé, cameroon department of experimental medicine, faculty of medicine, university of rome tor verga, rome, italy samuel m. sosso medical laboratory analysis, chantal biya international reference centre for research on hiv/aids prevention and management, yaoundé, cameroon jeremiah e. gabisa medical laboratory analysis, chantal biya international reference centre for research on hiv/aids prevention and management, yaoundé, cameroon aubin nanfack laboratory of immunology and microbiology, chantal biya international reference centre for research on hiv/aids prevention and management, yaoundé, cameroon joseph fokam laboratory of virology, chantal biya international reference centre for research on hiv/aids prevention and management, yaoundé, cameroon department of medical laboratory sciences, faculty of health sciences, university of buea, buea, cameroon laure ngono laboratory of virology, centre pasteur du cameroun, yaoundé, cameroon nadine fainguem medical laboratory analysis, chantal biya international reference centre for research on hiv/aids prevention and management, yaoundé, cameroon michel c.t. tommo medical laboratory analysis, chantal biya international reference centre for research on hiv/aids prevention and management, yaoundé, cameroon catholic university of central africa, yaoundé, cameroon krystel n. zam medical analysis laboratory, chantal biya international reference centre for research on hiv/aids prevention and management, yaoundé, cameroon centre de biotechnologie de l’université de yaoundé 1, yaoundé, cameroon junie f. yimga chantal biya international reference centre for research on hiv/aids prevention and management, yaoundé, cameroon désiré k. takou chantal biya international reference centre for research on hiv/aids prevention and management, yaoundé, cameroon alexis ndjolo chantal biya international reference centre for research on hiv/aids prevention and management, yaoundé, cameroon citation kamgaing rs, bouba y, sosso sm, et al. challenges faced by the hiv testing system in lowand middle-income countries. afr j lab med. 2023;12(1), a1974. https://doi.org/10.4102/ajlm.v12i1.1974 case study challenges faced by the hiv testing system in lowand middle-income countries rachel s. kamgaing, yagai bouba, samuel m. sosso, jeremiah e. gabisa, aubin nanfack, joseph fokam, laure ngono, nadine fainguem, michel c.t. tommo, krystel n. zam, junie f. yimga, désiré k. takou, alexis ndjolo received: 06 june 2022; accepted: 08 nov. 2022; published: 31 jan. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract introduction: determining the hiv status of some individuals remains challenging due to multidimensional factors such as flaws in diagnostic systems, technological challenges, and viral diversity. this report pinpoints challenges faced by the hiv testing system in cameroon. case presentation: a 53-year-old male received a positive hiv result by a rapid testing algorithm in july 2016. not convinced of his hiv status, he requested additional tests. in february 2017, he received a positive result using immunocomb® ii hiv 1 & 2 bispot and roche cobas electrochemiluminescence assays. a sample sent to france in april 2017 was positive on the bio-rad genscreen™ hiv 1/2, but serotyping was indeterminate, and viral load was < 20 copies/ml. the roche electrochemiluminescence immunoassay and inno-lia hiv i/ii score were negative for samples collected in 2018. a sample collected in july 2019 and tested with vidas® hiv duo ultra enzyme-linked fluorescent assay and geenius™ hiv 1/2 confirmatory assay was positive, but negative with western blot; cd4 count was 1380 cells/mm3 and hiv proviral dna tested in france was ‘target-not-detected’. some rapid tests were still positive in 2020 and 2021. serotyping remained indeterminate, and viral load was ‘target-not-detected’. there were no self-reported exposure to hiv risk factors, and his wife was hiv-seronegative. management and outcome: given that the patient remained asymptomatic with no evidence of viral replication, no antiretroviral therapy was initiated. conclusion: this case highlights the struggles faced by some individuals in confirming their hiv status and the need to update existing technologies and develop an algorithm for managing exceptional cases. keywords: hiv diagnosis; counselling; hiv testing system; testing technologies; algorithm; cameroon. background hiv-1 infection is still a significant public health concern in sub-saharan africa, where over two-thirds of the world’s burden is found. nevertheless, the number of new infections has dropped from about 1 950 000 in 2000 to approximately 870 000 in 2020.1 following this remarkable achievement, the joint united nations programme on hiv/aids 2025 goals of 95-95-95 and eradicating hiv/aids by 2030 are now feasible targets.2 efforts in early diagnosis and the scale-up of antiretroviral therapy use have played a prominent role in this achievement. however, several issues remain to be addressed,3 including the high diversity of circulating hiv strains and the limited availability of appropriate and advanced technology to determine hiv status of exceptional cases.4 for example, in cameroon, the hiv testing algorithm comprises determine hiv 1/2 (abbott molecular inc, des plaines, illinois, united sates), hexagon hiv 1/2 (human biochemica, wiesbaden, germany), and oraquick hiv 1/2 (orasure technologies inc, petchabun, thailand). however, the weakness of the sample referral system, and the limited local availability of advanced technologies, among other reasons, continue to be a challenge in confirming the hiv status of some individuals in several african countries, including cameroon.5 furthermore, poor adherence to the national guidelines of an hiv testing programme also significantly affects the consistency and reliability of hiv results.6 this poor adherence might play a role in some individuals’ denial of hiv results.7,8 thus, we present this case report to outline one of the challenges some individuals face in confirming their hiv status in cameroon. ethical considerations this diagnostic report was conducted per the declaration of helsinki principles and national regulations. the patient provided a signed informed consent, declaring his will to freely participate and authorising the usage of his medical laboratory findings in this report. no patient-identifying information was used throughout the paper. case presentation we present the case of a 53-year-old married and asymptomatic man who received a confirmatory positive hiv diagnosis in 2016. the different tests performed, the methods used, and the corresponding time point are summarised in figure 1. figure 1: summary of the testing performed in the patient’s history from 2016 to 2021, cameroon. after a consultation, he was determined to be hiv-positive following an hiv test request using rapid diagnostic tests: determine hiv 1/2 (abbott molecular inc, des plaines, illinois, united sates), hexagon hiv 1/2 (human biochemica, wiesbaden, germany), and oraquick (orasure technologies, inc, petchabun, thailand); included in the cameroon screening algorithm as per the world health organization hiv testing guidelines. however, his wife tested hiv-seronegative. not convinced of his status, he sought another test in a laboratory with a more advanced technique. the laboratory tested his sample collected in july 2016; the results were positive using the immunocomb® ii hiv 1 & 2 bispot (orgenics ltd, yavne, israel) and vidas® hiv duo ultra (biomérieux, marcy-l’etoile, france) tests. a few months later, in another laboratory, an hiv-positive status was obtained on a sample collected in february 2017 using the immunocomb® ii hiv 1 & 2 bispot (orgenics ltd, yavne, israel) and the elecsys® hiv combi pt electrochemiluminescence immunoassay techniques (roche diagnostics gmbh, mannheim, germany) (figure 1). about two weeks later, he presented at one of cameroon’s national reference laboratories in yaoundé for serotyping and viral load testing. unfortunately, the serotyping test was indeterminate, and the viral load test was < 390 copies/ml by reverse transcription polymerase chain reaction (biocentric inc, collingswood, new jersey, united states), while the enzyme-linked immunosorbent assay, genscreen hiv1/2 (bio-rad diagnostics ltd, hercules, california, united states), was positive on this same sample. about six weeks later, the viral load test was repeated in france with a more sensitive reverse transcription polymerase chain reaction cobas® 8800 assay (roche diagnostics ltd, new south wales, australia), and the result was < 20 copies/ml (which corresponds to the lower limit of the detection of this test). following these results, new samples were ordered by the consultant and sent to france again; the elecsys® hiv combi pt electrochemiluminescence immunoassay and inno-lia hiv 1/2 score assay (fujirebio diagnostics inc, malvem, pennsylvania, united states) were performed on two samples collected over a three-month interval; the first gave an indeterminate result and the second was concluded as a negative result. this result comforted the patient in his doubt about his hiv status, but on a new sample collected in july 2019, the vidas® hiv duo ultra enzyme-linked fluorescent assay (biomérieux, france), and geenius™ hiv1/2 assay (geenius™ diagnostic, inc, redmond, washington, united states) were still positive. his viral load was < 40 copies/ml, the western blot was negative, and serotyping was indeterminate. the patient remained asymptomatic with a normal immunological status (cd4 t-cells: 1380 cells/mm3, cd8 t-cells: 661 cells/mm3). hiv proviral dna was not detectable in a sample collected on 02 october 2019. about 10 months later, serological tests were still hiv-positive, with a cd4 count of 945 cells/mm3. in january 2021, he was sent to the chantal biya international research centre for the prevention and management of hiv/aids, yaoundé, for viral load testing using the abbott m2000rt realtime hiv-1 assay (abbott molecular inc, des plaines, illinois, united sates), and the result was ‘target-not-detected’. of note, on this same sample, determine and oraquick were positive, while serotyping remained indeterminate. as of october 2021, his viral load remained ‘target-not-detected’, and he is still asymptomatic. noteworthy, he stated that he did not have any chronic diseases, nor had he engaged in any hiv-related risk behaviour. furthermore, his serological tests were negative for hepatitis b surface antigen (hbsag) and hepatitis c virus antibodies using rapid tests. management and outcomes so far, given that the patient remained asymptomatic with no evidence of viral replication, no antiretroviral therapy was initiated. we collected a new sample in our laboratory on 08 october 2021, and performed rapid testing according to the national algorithm, serotyping and hiv-1 viral load, but the outcome was serology-positive, viral load ‘target-not-detected’. however, he was advised to avoid any hiv risk behaviour, and we continue to monitor his serological and virological status. discussion efforts have been made towards improving laboratory medicine in africa, especially after the 2008 maputo declarations. some authors think a remarkable transformation has occurred in some areas of laboratory medicine, especially in those targeting the fight against hiv/aids in sub-saharan africa.3,9 nonetheless, numerous challenges, including weaknesses in the quality management systems, laboratory networks, human resources, and technological issues, must be appropriately addressed.9,10,11,12 despite testing progress, many factors influence the acceptance or refusal of provider-initiated testing and including self-trust, not being at risk for hiv, not being ready, and insufficient privacy and counselling.8 the aforementioned factors could have also influenced the man’s denial of his hiv test results of 08 july 2016 and 15 february 2017. moreover, a study conducted in cameroon between 2013 and 2014 reported that using lay counsellors in hiv testing units is a setback to delivering quality hiv testing services, where counselling and acceptance are cornerstones.7 confirming hiv status is still challenging in some cases, especially in western african countries, characterised by many hiv-1 subtypes, as highlighted by aghokeng et al., who found an inaccurate diagnosis of specific hiv-1 subtypes, notably in group m and o.5 therefore, there is a need for more sophisticated confirmatory tests.5 in the case described here, most of these tests, such as the sensitive viral load threshold test and the detection of hiv-1 proviral dna, were performed in europe. complex cases are sometimes encountered in diagnostic laboratories, but unfortunately, due to the absence or limited access to these tests, patients are without any conclusive hiv status. therefore, more sensitive diagnostics are essential, especially in this ‘test and treat’ era, where all patients testing seropositive are encouraged to start antiretroviral treatment as soon as possible to limit false positive cases. false positive tests have been reported in some developing countries, including cameroon.13,14 a well-functioning system to detect complex cases that might require further confirmation should be implemented to avoid unnecessary treatment. we advocate that national public health laboratories should be equipped and strengthened to handle complex situations. furthermore, besides the financial cost, there is the psycho-social burden posed on the patient due to an uncertain hiv status. the psycho-social burden goes beyond the individual level and affects the family and socio-economic dimensions. there are still unresolved issues about the hiv status of this man. the antibodies are persistently reactive, serotyping is continuously indeterminate, and the viral load measurement is ‘target-not-detected’. at this stage, the best way to demonstrate the presence of the virus would be to isolate it, but this will be challenging because of the undetectable viral load. it is still to be demonstrated, but we might also hypothesise that this man might be a rare case of an ‘elite controller’ or a ‘long-term non-progressor’. conclusion overall, this case highlights the struggles faced by some individuals in confirming their hiv status. in addition, it calls for the need to adapt existing technologies and develop an algorithm for managing special cases in lowand middle-income countries. acknowledgements the patient contributed in providing the research team with required data. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions r.s.k., y.b., and s.m.s. conceived the idea for the writing of the case report. j.e.g., l.n., a. nanfack, n.f., m.c.t.t., k.n.z., and j.f.y. collected the data and performed part of the sample processing and analysis. y.b., r.s.k., s.m.s., j.e.g. and d.k.t. drafted the paper. y.b., j.f., and a. ndjolo. critically revised the manuscript. all of the authors accepted the final version of this manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability all of the available data are presented within the text and in the figure included in this article. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references unaids_fact sheet [homepage on the internet]. [cited 2021 oct 16]. available from: https://www.unaids.org/sites/default/files/media_asset/unaids_factsheet_en.pdf unaids issues new fast-track strategy to end aids by 2030 – egpaf [homepage on the internet]. elizabeth glaser pediatric aids foundation; 2014 [cited 2021 mar 26]. available from: https://www.pedaids.org/2014/11/20/unaids-issues-new-fast-track-strategy-to-end-aids-by-2030/ nkengasong jn, yao k, onyebujoh p. laboratory medicine in low-income and middle-income countries: progress and challenges. lancet. 2018;391(10133):1873–1875. https://doi.org/10.1016/s0140-6736(18)30308-8 ragupathy v, zhao j, wood o, et al. identification of new, emerging hiv-1 unique recombinant forms and drug-resistant viruses circulating in cameroon. virol j. 2011;8:185. https://doi.org/10.1186/1743-422x-8-185 aghokeng af, mpoudi-ngole e, dimodi h, et al. inaccurate diagnosis of hiv-1 group m and o is a key challenge for ongoing universal access to antiretroviral treatment and hiv prevention in cameroon. plos one. 2009;4(11):e7702. https://doi.org/10.1371/journal.pone.0007702 consolidated guidelines on hiv testing services: 5cs: consent, confidentiality, counselling, correct results and connection 2015 [homepage on the internet]. world health organization; 2015 [cited 2021 mar 26]. available from: https://pubmed.ncbi.nlm.nih.gov/26378328/ ngangue p, gagnon m-p, bedard e. challenges in the delivery of public hiv testing and counselling (htc) in douala, cameroon: providers perspectives and implications on quality of htc services. bmc int health hum rights. 2017;17(1):9. https://doi.org/10.1186/s12914-017-0118-2 abdurahman s, seyoum b, oljira l, weldegebreal f. factors affecting acceptance of provider-initiated hiv testing and counselling services among outpatient clients in selected health facilities in harar town, eastern ethiopia. hiv aids (auckl). 2015;7:157–165. https://doi.org/10.2147/hiv.s81649 nkengasong jn, mbopi-keou f-x, peeling rw, et al. laboratory medicine in africa since 2008: then, now, and the future. lancet infect dis. 2018;18(11):e362–e367. https://doi.org/10.1016/s1473-3099(18)30120-8 world health organization. the maputo declaration on strengthening of laboratory systems [homepage on the internet]. 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https://doi.org/10.1080/15284336.2018.1429846 abstract introduction methods results discussion acknowledgements references about the author(s) jenipher g. mwakyabala department of microbiology and immunology, weill bugando school of medicine, catholic university of health and allied sciences, mwanza, united republic of tanzania conjester i. mtemisika molecular biology laboratory, central pathology laboratory, bugando medical centre, mwanza, united republic of tanzania stacy mshana department of microbiology and immunology, weill bugando school of medicine, catholic university of health and allied sciences, mwanza, united republic of tanzania adam a. mwakyoma department of clinical microbiology, kilimanjaro christian medical centre, moshi, united republic of tanzania vitus silago department of microbiology and immunology, weill bugando school of medicine, catholic university of health and allied sciences, mwanza, united republic of tanzania citation mwakyabala jg, mtemisika ci, mshana s, mwakyoma aa, silago v. characterisation of genes encoding for extended spectrum β-lactamase in gram-negative bacteria causing healthcare-associated infections in mwanza, tanzania. afr j lab med. 2023;12(1), a2107. https://doi.org/10.4102/ajlm.v12i1.2107 brief report characterisation of genes encoding for extended spectrum β-lactamase in gram-negative bacteria causing healthcare-associated infections in mwanza, tanzania jenipher g. mwakyabala, conjester i. mtemisika, stacy mshana, adam a. mwakyoma, vitus silago received: 30 oct. 2022; accepted: 27 jan. 2023; published: 12 apr. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract healthcare-associated infections (hcais) caused by extended spectrum β-lactamase-producing gram-negative bacteria (esbl-gnb) increase morbidity and mortality. this cross-sectional study characterised esbl genes (blactx-m, blatem and blashv) among 30 ceftriaxone-resistant gnb causing hcais between january 2022 and july 2022 by multiplex polymerase chain reaction assay at the zonal referral hospital in mwanza, tanzania. twenty-five (83.3%) had at least one esbl gene, of which 23/25 (92.0%) carried the blactx-m gene. seventy-two percent (18/25) of the gnb-esbl isolates carried more than one esbl gene, of which the majority (88.8%; n = 16/25) carried the blactx-m and blatem genes. extended spectrum β-lactamase genes, particularly blactx-m, are common among ceftriaxone-resistant gnb causing hcais. what this study adds: this study revealed the distribution of genes (blactx-m, blatem and blashv) coding for esbl production among ceftriaxone resistant gnb causing hcais however, all esbl producing gnb were susceptible towards ceftriaxone-sulbactam indicating that ceftriaxone-sulbactam may be empirically prescribed for treating patients with hcais. keywords: beta lactamases; extended spectrum beta-lactamase; gram-negative bacteria; healthcare-associated infections; multiplex pcr assay. introduction healthcare-associated infections (hcais), also referred to as nosocomial infections, are infections acquired by patients while receiving healthcare services from ≥ 48 h after admission to a healthcare facility.1,2,3 admission into intensive care units increases the risk of acquiring hcais due to (1) chronic diseases which lower body immunity; (2) surgical procedures which interfere with natural body defenses; and (3) medical invasive devices such as urinary catheters, central lines and intubators, which provide bacteria with direct entry into bodily tissues.4 escherichia coli, klebsiella aerogenes, enterobacter spp., acinetobacter baumannii and pseudomonas aeruginosa are the most common gram-negative bacteria (gnb) known to cause hcais.5,6,7,8 healthcare-associated infections are associated with significant increased cost of healthcare services, days of hospitalisation and mortality.9 healthcare-associated infections caused by multidrug-resistant bacteria phenotypes, such as extended spectrum β-lactamase-producing gnb (esbl-gnb), further exaggerate morbidity and mortality. at the study site in mwanza, tanzania, the prevalence of hcais in surgical site infections ranges from 10% to 26%.5,9,10 staphylococcus aureus accounts for nearly one-third of these, of which about 16% to 19% are methicillin resistant.5,9 on the other hand, only one study reported 13% of implicating gnb showed esbl phenotypes.9 to date, the distribution of esbl genes among esbl-gnb phenotypes causing hcais is not clearly known. this study unravels the distributions of esbl genes (blactx-m, blatem and blashv) among ceftriaxone-resistant gnb causing hcais at a zonal referral hospital in mwanza, tanzania. methods ethical considerations this study received ethical approval from the joint catholic university of health and allied sciences and bugando medical centre research ethics and review committee. the study approval number is crec: 2368/2022. all participants voluntarily provided written informed consent before being enrolled in the study. unique identification numbers were used to ensure confidentiality. laboratory results were communicated in a timely manner to attending doctors in order to guide rational therapy. study design, population, setting and duration this was a cross-sectional laboratory-based study of ceftriaxone-resistant gnb isolated from different hcais between january 2022 and july 2022 (unpublished data) at bugando medical centre – a zonal referral hospital located in mwanza, tanzania. the bacterial isolates, which had been archived in 20% glycerol stocks stored in a –40 °c freezer in the microbiology laboratory as part of a biorepository, were recovered for this study in july 2022. the duration of archive ranged from 1 to 6 months before recovery for molecular characterisation of esbl genes. clinical information related to each isolate, namely ward or clinic of origin, sample type, bacterial species name, and susceptibility towards third-generation cephalosporins, notably ceftriaxone, was extracted from the laboratory database. laboratory procedures were conducted in microbiology research laboratory and molecular biology research laboratory at the catholic university of health and allied sciences located at bugando medical centre in mwanza, tanzania. definition of healthcare-associated infection in the current study, hcai was defined as an infection that a patient develops after 48 h of hospital admission, while receiving healthcare for another disease or condition.11 laboratory procedure recovery of cro-r-gnb causing healthcare-associated infections and phenotypic detection of esbl production ceftriaxone-resistant gnb causing hcais were recovered by sub-culturing on plates of macconkey agar with salt (mca; himedia, mumbai, india). plates were incubated aerobically at 35 °c ± 2 °c for 20 h – 24 h followed by phenotypic detection of esbl production and dna extraction for multiplex polymerase chain reaction (pcr) assay. the disk combination method (dcm) from the clinical and laboratory standards institute12 was used for phenotypic detection of esbl production among recovered ceftriaxone-resistant gnb. dna extraction from 5 to 10 fresh colonies (≤ 24 h) of ceftriaxone-resistant gnb on plates of mca were used for dna extraction. a protocol for dna extraction from gnb by qiamp min dna extraction kit (qiagen, wuerzburg, germany) was used according to manufacturer’s instructions. dna samples were stored at −20 °c. multiplex pcr assay a multiplex pcr assay described by monstein et al.13 was used for amplification and detection of esbl genes (blactx-m, blashv, and blatem). briefly, 2 µl of each dna sample was added into a pcr reaction tube containing hotstartaq dna polymerase master mix (new england biolabs; hitchin, hertfordshire, united kingdom) and a set of primers (table 1), resulting in a final pcr reaction volume of 25 µl. the thermal cycler (t100™, bio-rad, kaki-bukit, singapore) was run with the following conditions: initial denaturation at 95 °c for 5 min; 30 cycles of denaturation at 94 °c for 30 s, annealing at 56 °c for 30 s, and extension at 72 °c for 1 min; and a final extension at 72 °c for 10 min. products were detected by using a 1% agarose gel with tris-acetate-edta buffer stained with safeviewtm dna stain (abm; richmond, british colombia, canada) and visualised under ultraviolet light. table 1: sequences of primers used for multiplex polymerase chain reaction assays for extended spectrum β-lactamase genes, bugando medical centre, mwanza, tanzania, january 2022 – july 2022. data management and analysis quantitative data were descriptively analysed by using microsoft excel (microsoft office; redmond, washington, united states) and stata version 15.0 (statacorp llp; college station, texas, united states; https://www.stata.com/stata15/). results a total of 30 ceftriaxone-resistant gnb causing hcais were recovered during this study period. most of the recovered bacteria were e. coli 43.3% (n = 13). the majority of ceftriaxone-resistant gnb were isolated from the burn unit (40%; n = 12), and from pus/pus swab samples (56.6%; n = 17). by dcm, all (100%; n = 30) ceftriaxone-resistant gnb had positive esbl phenotypes. multiplex pcr assay revealed that about 83.3% (n = 25) had at least one esbl gene, of which the majority (92.0%; n = 23) harboured the blactx-m gene. out of 25 gnb carrying esbl genes, 18 (72.0%) carried multiple genes; of these, 88.8% (n = 16) carried blactx-m and blatem genes (table 2 and figure 1). five isolates with negative pcr were e. coli (n = 3), isolated from pus/pus swab samples in the burn unit, and acinetobacter spp. (n = 2), one isolated from a urine sample in the medical ward and the other isolated from a pus/pus swab sample from the neonatal intensive care unit (table 3). figure 1: molecular characterisation of extended spectrum β-lactamase genes by multiplex polymerase chain reaction assay, bugando medical centre, mwanza, tanzania, january 2022 – july 2022. table 2: description of ceftriaxone-resistant gnb recovered for multiplex polymerase chain reaction amplification and detection of extended spectrum β-lactamase genes, bugando medical centre, mwanza, tanzania, january 2022 – july 2022. table 3: results of disk combination method and multiplex pcr assay, and distributions of esbl genes, bugando medical centre, mwanza, tanzania, january 2022 – july 2022. discussion the current study characterised the proportions and distributions of esbl genes (blactx-m, blatem, and blashv) among ceftriaxone-resistant gnb which were isolated from different hcais between january 2022 and july 2022 at a tertiary zonal referral hospital in mwanza, tanzania. the majority of ceftriaxone-resistant gnb were recovered from the burn unit, from patients with burn injuries who were prone to infections because of the breached skin barrier.14 moreover, e. coli accounted for the majority of recovered bacterial species, suggesting the patients’ own gut flora as an endogenous source of infection.3 however, e. coli can also be acquired from exogenous sources, such as contaminated inanimate surfaces, whenever hospital environmental cleaning and decontamination are poor.15 this study observed that all ceftriaxone-resistant gnb had positive esbl phenotypes by dcm, even though four out of five (83.3%) esbl phenotypes had at least one esbl gene on multiplex pcr assay. our findings are similar to a study by silago et al., conducted in mwanza, tanzania, in 2020, which reported a proportion of 93.3% esbl among gnb isolated from the hospital environment and hospitalised patients at the same setting.16 our findings are, however, higher than a study by said et al., which was conducted in 2021 in mwanza, tanzania, which reported that about 65.9% of gnb colonising children, of whom the majority were not hospitalised, harboured esbl genes at the same setting.17 therefore, the difference in study populations between the studies may explain the difference observed. similar to previous studies published in 2020 and 2021 in mwanza and in 2021 in morogoro, tanzania,16,17,18 the majority of ceftriaxone-resistant gnb were harbouring the blactx-m gene. the predominance of blactx-m may be a result of successful dissemination by conjugative epidemic plasmids, which facilitates its horizontal and vertical transmission.16,19,20,21 five confirmed esbl phenotypes by dcm did not harbour any of the three esbl genes (blactx-m, blatem, and blashv) by multiplex pcr assay. this observation is in line with previous studies conducted from the same setting, mwanza, tanzania, in 2020 and 2021.16,17 the isolates may be harbouring other esbl families which are non-cefotaximase-munich beta-lactamase (non-ctx-m), non-temoniera beta-lactamase (non-tem), and non-sulfhydryl reagent variable beta-lactamase (non-shv), such as oxacillinase beta-lactamase, pseudomonas extended resistant, vietnam extended-spectrum β-lactamase, tlahuica indianand guiana-extended spectrum families.22 limitations the small sample size of ceftriaxone-resistant gnb isolates obtained for this study is a weakness but did not affect the interpretation of the results. conclusion extended spectrum β-lactamase genes, to be specific blactx-m, are common among ceftriaxone-resistant gnb causing hcais. therefore, rational management of patients with hcais, guided by culture and sensitivity, is warranted. acknowledgements authors appreciate the support from microbiology research laboratory and molecular biology laboratory of the catholic university of health and allied sciences. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions v.s. and a.a.m. conceptualised the idea of the manuscript; j.g.m. and s.m. retrieved laboratory data, recovered bacteria isolates, and performed laboratory procedures; j.g.m. and c.i.m. interpreted and analysed data; c.i.m. wrote the first draft of the manuscript, which was critically reviewed by all co-authors who also approved the final manuscript. v.s. supervised protocols and every step of this research. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the data that support the findings made in this study can be made available from the corresponding author, c.i.m., on request. disclaimer the views expressed in this study are those of the authors and are not an official position of the affiliation institutes of the authors. references danasekaran r, mani g, annadurai k. prevention of healthcare-associated infections: protecting patients, saving lives. int j community med public health. 2014;1(1):67–68. peleg ay, hooper dc. hospital-acquired infections due to gram-negative bacteria. n engl j med. 2010;362(19):1804–1813. https://doi.org/10.1056/nejmra0904124 nagarjuna d, mittal g, dhanda rs, verma pk, gaind r, yadav m. faecal escherichia coli isolates show potential to cause endogenous infection in patients admitted to the icu in a tertiary care hospital. new microbes new infect. 2015;7:57–66. https://doi.org/10.1016/j.nmni.2015.05.006 world health organization. report on the burden of endemic health care-associated infection worldwide. world health organization: geneva, switzerland. 2011. mawalla b, mshana se, chalya pl, imirzalioglu c, mahalu w. predictors of surgical site infections among patients undergoing major surgery at bugando medical centre in northwestern tanzania. bmc surg. 2011;11(1):1–7. https://doi.org/10.1186/1471-2482-11-21 agarwal s. study of postoperative wound infection. indian j surg. 1972;34:314–320. nichols rl. surgical wound infection. am j med. 1991;91(3):s54–s64. https://doi.org/10.1016/0002-9343(91)90344-w kotisso b, aseffa a. surgical wound infection in a teaching hospital in ethiopia. east afr med j. 1998;75(7):402–405. mpogoro fj, mshana se, mirambo mm, kidenya br, gumodoka b, imirzalioglu c. incidence and predictors of surgical site infections following caesarean sections at bugando medical centre, mwanza, tanzania. antimicrob resist infect control. 2014;3(1):1–10. https://doi.org/10.1186/2047-2994-3-25 moremi n, claus h, vogel u, mshana se. surveillance of surgical site infections by pseudomonas aeruginosa and strain characterization in tanzanian hospitals does not provide proof for a role of hospital water plumbing systems in transmission. antimicrob resist infect control. 2017;6(1):1–8. https://doi.org/10.1186/s13756-017-0216-x haque m, sartelli m, mckimm j, abu bakar m. health care-associated infections – an overview. infect drug resist. 2018 nov 15;11:2321–2333. https://doi.org/10.2147/idr.s177247 clsi. performance standards for antimicrobial susceptibility testing. 32nd ed. clsi supplement m100. clinical and laboratory standards institute: berwyn, pennsylvania. 2022. monstein hj, ostholm-balkhed a, nilsson mv, nilsson m, dornbusch k, nilsson le. multiplex pcr amplification assay for the detection of blashv, blatem and blactx-m genes in enterobacteriaceae. apmis. 2007;115(12):1400–1408. https://doi.org/10.1111/j.1600-0463.2007.00722.x church d, elsayed s, reid o, winston b, lindsay r. burn wound infections. clin microbiol rev. 2006;19(2):403–434. https://doi.org/10.1128/cmr.19.2.403-434.2006 odoyo e, matano d, georges m, et al. ten thousand-fold higher than acceptable bacterial loads detected in kenyan hospital environments: targeted approaches to reduce contamination levels. int j environ res public health. 2021;18(13):6810. https://www.mdpi.com/1660-4601/18/13/6810 silago v, kovacs d, samson h, et al. existence of multiple esbl genes among phenotypically confirmed esbl producing klebsiella pneumoniae and escherichia coli concurrently isolated from clinical, colonization and contamination samples from neonatal units at bugando medical center, mwanza, tanzania. antibiotics. 2021;10(5):476. https://doi.org/10.3390/antibiotics10050476 said mm, msanga dr, mtemisika ci, et al. extended spectrum β-lactamase producing lactose fermenting bacteria colonizing children with human immunodeficiency virus, sickle cell disease and diabetes mellitus in mwanza city, tanzania: a cross-sectional study. trop med infect dis. 2022;7(8):144. https://doi.org/10.3390/tropicalmed7080144 moremi n, silago v, mselewa eg, et al. extended-spectrum β-lactamase blactx-m-1 group in gram-negative bacteria colonizing patients admitted at mazimbu hospital and morogoro regional hospital in morogoro, tanzania. bmc res notes. 2021;14(1):1–7. https://doi.org/10.1186/s13104-021-05495-x xia s, fan x, huang z, et al. dominance of ctx-m-type extended-spectrum β-lactamase (esbl)-producing escherichia coli isolated from patients with community-onset and hospital-onset infection in china. plos one. 2014;9(7):e100707. https://doi.org/10.1371/journal.pone.0100707 rossolini g, d’andrea m, mugnaioli c. the spread of ctx-m-type extended-spectrum β-lactamases. clin microbiol infect. 2008;14:33–41. https://doi.org/10.1111/j.1469-0691.2007.01867.x zhuo c, li xq, zong zy, zhong ns. epidemic plasmid carrying bla ctx-m-15 in klebsiella penumoniae in china. plos one. 2013;8(1):e52222. https://doi.org/10.1371/journal.pone.0052222 paterson dl, bonomo ra. extended-spectrum β-lactamases: a clinical update. clin microbiol rev. 2005;18(4):657–686. https://doi.org/10.1128/cmr.18.4.657-686.2005 abstract background excess laboratory capacity challenges with the scale-up of viral load testing in sub-saharan africa solutions: procurement and supply chain management acknowledgements references about the author(s) jason williams supply chain division, united states agency for international development (usaid), crystal city, virginia, united states dianna edgil supply chain division, united states agency for international development (usaid), crystal city, virginia, united states matthew wattleworth global health supply chain program, procurement and supply management (ghsc-psm), arlington, virginia, united states clement ndongmo global health supply chain program, procurement and supply management (ghsc-psm), arlington, virginia, united states joel kuritsky supply chain division, united states agency for international development (usaid), crystal city, virginia, united states citation williams j, edgil d, wattleworth m, ndongmo c, kuritsky j. the network approach to laboratory procurement and supply chain management: addressing the system issues to enhance hiv viral load scale-up. afr j lab med. 2020;9(1), a1022. https://doi.org/10.4102/ajlm.v9i1.1022 review article the network approach to laboratory procurement and supply chain management: addressing the system issues to enhance hiv viral load scale-up jason williams, dianna edgil, matthew wattleworth, clement ndongmo, joel kuritsky received: 01 apr. 2019; accepted: 15 may 2020; published: 13 aug. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract investment in viral load scale-up in order to control the hiv epidemic and meet the joint united nations programme on hiv and aids (unaids) ‘90-90-90’ goals has prompted the president’s emergency plan for aids relief and countries to increase their investment in viral load and infant virological testing. this has resulted in the increased procurement of molecular-based instruments, with many countries having challenges to effectively procure and place these products. in response to these challenges, the global laboratory stakeholder community has developed an informed ‘network approach’ to guide placement strategies. this article defines and describes the ‘network approach’ for laboratory procurement and supply chain management to assist countries in developing a strategic instrument procurement and placement strategy. the four key pillars of the approach should be performed in a stepwise fashion, with regular reviews. the approach is comprised of (1) laboratory network optimisation, (2) forecasting and supply planning, (3) the development of effective procurement and strategic sourcing to develop ‘all-inclusive’ contracts that provide transparent pricing, and the establishment of clear service and maintenance expectations and key performance indicators and (4) performance management to increase communication and planning, and promote issue resolution. investments in the network approach will enable countries to strengthen laboratory systems and ready them for future laboratory needs. these disease-agnostic networks will be poised to improve overall national disease surveillance and assist countries in responding to disease outbreaks and other chronic diseases. keywords: laboratory networks; molecular scale-up; optimisation; supply chain; laboratory. background of the 36.9 million people living with hiv, approximately half (21.7 million) are on antiretroviral therapy, and of those, four out of five are virally suppressed.1 ensuring patients are on the most effective treatments relies on the availability and use of viral load (vl) testing. for this to be successful, clinicians must order the test, samples must be transported to the laboratory and results must be returned. the achievement of the third ‘90’ of the ‘90-90-90’ strategy of the joint united nations programme on hiv and aids (unaids), to ensure that 90% of patients that are on hiv treatment are virally suppressed, depends on the scale-up of laboratory capacity with an effective sample transport network and an efficient laboratory–clinic interface that facilitates responses to patient management issues related to adherence and treatment failure. in 2013, the world health organization included vl monitoring in its treatment guidelines, with subsequent guidance and a recommendation for its use in 2014 and 2015.2,3,4 the addition of vl testing as the cornerstone of the unaids, ‘90-90-90’ strategy has resulted in an investment in vl testing globally.5 investments have been made to assist ministries of health in revising treatment policies, building laboratory capacity, and training and sensitising clinicians and patients on testing. to facilitate scale-up, there has been an effort to increase efficiencies by promoting procurement coordination between donors, to improve transparent pricing for reagents, and to implement procurement principles to address service and maintenance. the goal for coordination is to create a network of diagnostic capability that is nested within a broader public health response towards laboratory development. as countries have attempted to take vl testing to scale, reoccurring challenges continue to surface, which include difficulty with procurement and sample transport, delays in the return of results and the need to increase clinical demand.6,7,8 these challenges have an impact on the ability to increase testing and ensure quality services. in order to address this, we promote a ‘network approach’. by this we mean the use of a systematic strategy that aligns capacity with utilisation, promotes efficiency in the procurement and placement of machines, enables collaboration between donors and countries, and focuses on the development of efficient sample transport and result return. the purpose of this article is to identify the key aspects of this approach and provide critical considerations for countries to improve performance and create network efficiencies in order to reach their diagnostic goals. excess laboratory capacity in most countries, instrument capacity is higher than needed, requiring significant growth in testing in order for these products to be optimally used. even with a phased approach to the scale-up of vl testing, as recommended by the world health organization,3 only a portion of coverage goals have been achieved. in zimbabwe, for example, the national vl testing coverage target was established at 21% in 2015, but only 5.6% coverage was achieved by year’s end, largely due to challenges with resource mobilisation and coordination, equipment procurement and specimen transport.9 by june 2016, of seven countries surveyed, four (tanzania, côte d’ivoire, malawi and uganda) were performing less than 25% of the necessary vl tests needed for patients on antiretroviral therapy.10 in 2016, médecins sans frontières (msf) estimated the coverage of vl monitoring across seven sub-saharan african countries to be variable, ranging between 32% and 91%.6 data on infant virological testing (ivt) show less than 50% coverage within the first six weeks of life in many sub-saharan african countries.7 the world health organization’s survey of data on diagnostic instruments from 2013, which assessed the scale-up of vl testing in 127 countries, showed that vl testing capacity was available to conduct 1.2 tests per person on antiretroviral therapy, but only 0.5 tests per person were conducted. this results in a capacity utilisation rate of only 36.5%.11 more recently, reports from major molecular instrument manufacturers demonstrate that countries continue to increase the number of instruments. between may 2016 and may 2018, testing capacity in 21 african countries increased from over 15 million to 20.5 million tests, with an increase of 164 large molecular instruments (manufacturer reported, see figure 1 and figure 2). in most countries, existing instrument numbers and capacity are not limiting factors associated with the scale-up of vl testing. figure 1: reported manufacturer instrument counts in sub-saharan africa (may 2016 to may 2018, an increase from 405 to 569 instruments). figure 2: diagnostic capacity estimates in sub-saharan africa (may 2018 – 20 580 136 tests). past scale-up efforts for cd4 testing resulted in uncoordinated procurement and underutilisation of instruments, suboptimal network expansion and a lack of service maintenance coverage across laboratory networks.11,12 as vl testing replaces cd4 in most sub-saharan countries to monitor antiretroviral therapy, many of these issues are again resurfacing, including uncoordinated instrument management strategies.6,13 lessons learned from the implementation of cd4 testing indicate the need for a more efficient model of procurement and service provision for vl and ivt programmes. challenges with the scale-up of viral load testing in sub-saharan africa we have identified four challenges that programmes must address in order to take vl testing to scale: (1) donor and stakeholder coordination and transparent pricing, (2) inconsistency of reagent availability (forecasting and supply planning), (3) ensuring functional instrumentation and (4) suboptimal laboratory network planning and sample transport strategies. challenge 1 – donor and stakeholder coordination and transparent pricing coordination between partners and governments to ensure the distribution of resources according to programme needs has been challenging, frequently resulting in the over-procurement or under-procurement of instruments and reagents that do not meet the testing needs of programmes.14,15 one core aspect of the alignment of effective global procurement is to create transparency in pricing, leveraging volumes and donor investments as part of negotiating influence. pricing variability across countries has been described as a limiting factor to scale-up,16 creating challenges with budgeting. many countries with budget limitations have historically paid more per test due to lower testing volumes, with more difficult infrastructural challenges to overcome as part of service delivery. without coordination, donors can inadvertently undermine the ability to negotiate cost-effective testing strategies, with an end result of diminishing testing pools across instrument types, limiting negotiating influence and undermining volume pricing for tests performed nationally. to clearly understand pricing, it is important to unpack costs for fair comparisons. for example, per test costs could be calculated based on the primary reagent only, or may include reagents, consumables, shipping and distribution. pricing depends on volumes, instrument type, sample type, local versus international procurement, mode of import, inclusive service, maintenance costs, logistics costs, vendor management of reagent inventories and reagent rental or leasing arrangements. all of these components influence pricing for comparative purposes. the global fund (gf) has negotiated global access pricing for low and middle-income countries. the two most commonly used molecular brands for vl testing and ivt are roche molecular diagnostics and abbott molecular inc. commodity-related prices are set at a rate of $9.40 per test for roche, which includes reagents and consumables, with ex-works terms (goods are available at the seller’s or manufacturer’s site and must be transported by the buyer), whereas abbott’s pricing is based on volumes and duration commitments.17 the abbott’s approach has resulted in pricing variability across countries of between $10.50 and $22.50 per test for core reagents, with an additional $2.50 for the necessary calibrators, controls and added consumables. this brings abbott’s pricing to between $13.50 and $23.60 per test. yet, based on volumes and multi-year commitments as well as national negotiating influence, some countries have further reduced these prices. it should be noted that pricing schemes offered by roche can also have country-specific variability due to ‘free carrier’ pricing (where the seller arranges and pays for shipping to the country of export) included in the reagent pricing, with shipping details not separately itemised. this creates challenges during budgetary planning sessions, since it becomes difficult to predict shipping costs and ensure that the global ex-works $9.40 reagent and consumable pricing is adhered to. transparency in total cost breakdown is needed, as there is a perception that the pricing offered is different from the gf-published $9.40 per test pricing. challenge 2 – inconsistent reagent availability (forecasting and supply planning) reagent availability has been highlighted as one core obstacle to the scale-up of vl testing.6,7,16 although reagent availability is a critical aspect of ensuring vl testing, stock-outs are a symptom of broader supply chain system issues and data flow challenges that have a negative impact on scale-up. challenge 3 – instrument functionality due to inadequate or absent service and maintenance ensuring adequate service and maintenance, warranty and preventive maintenance coverage for equipment is a significant challenge. to date, instrument and vendor oversight has been managed on an instrument-by-instrument basis, with limited coordination of management strategies, sometimes independently by stakeholders in the same country. this has resulted in multiple, separate contracts for individual instruments, often negotiated on different timelines, using non-standardised terms and with limited consistency in contract oversight and management. key performance indicators and reporting requirements that can be used to monitor vendor performance have not historically been included in contracts. this makes adhering to existing service contracts and the monitoring of vendor performance difficult, limiting both vendor accountability and the development of appropriate maintenance strategies. challenge 4 – weak laboratory and sample referral networks given the ever-changing laboratory network environment, sample transport and referral networks have grown organically, and do not necessarily reflect an efficient network approach. these networks quickly become outdated and require adjustments to not only reflect national needs (e.g. the addition of other diagnostics demands, point-of-care, the integration of new specimens, backup sample transport in the event of equipment breakdown), but also to look for efficiencies, and possible integration. ultimately, the effects of a fragmented sample referral network can be far-reaching, ranging from increased operational costs across the entire network to improperly placed instruments and limited instrument utilisation. solutions: procurement and supply chain management to effectively address these existing challenges, a holistic approach or network approach is needed, which spans four major building blocks or elements that are described below and summarised in figure 3. figure 3: the network approach to laboratory procurement and supply management. diagnostic network optimisation the concept behind a network approach is to shift to an all-inclusive reagent rental scheme (rrs) or reagent service scheme (rss) that serves all existing and new instruments. this approach would be national, and not specific to a stakeholder, donor or disease. a vendor-specific instrument contract would contain terms and conditions that are informed collectively by all stakeholders. this approach would require all stakeholders to take stock of existing instruments, service contracts and procurement pricing schemes, and establish jointly renegotiated terms that take all stakeholder investments and contributions to the overall network into consideration. revised pricing schemes could potentially include: a national cost and contract structure that allows for volume growth and instrument expansion within a complete network, irrespective of the disease type or programme area, and that can be accessed by all stakeholders. a cost structure translated into an ‘all-inclusive per test cost’ spread across all instruments of the same brands within the network to include: cost options as part of network expansion that would account for existing legacy instruments and the development of new contract models (e.g. leasing and rentals) that facilitate the introduction of new instruments under standardised pricing schemes inclusive service and maintenance data solutions that would include patient result transmission, as well as instrument and user performance network staff training and consistency additional technology support that could assist in site-level efficiencies (barcode use, sample processing and workflow evaluations) enhanced commodity management strategies to ensure reagent availability (to include vendor-managed inventory) the goal of a network approach is to improve instrument utilisation by aligning capacity with demand: introducing standardised national pricing schemes, irrespective of the procurement mechanism, thereby enabling continuous service contract coverage providing opportunities to amortise instrument costs into reagent costs, in order to lower startup costs associated with scale-up sharing and assigning the longer-term management and mitigation of risks associated with instrumentation onto manufacturers and local vendors providing a no-cost option for instrument replacements due to high failure rates, capacity issues (upgrading) or even outdated technology. a network approach focuses on developing a baseline understanding of the current national vl testing network, including capacity and equipment utilisation, then exploring more efficient network options. once a refined network is adopted, planning and procurement must be coordinated among all stakeholders to avert the addition of more instrumentation that may not be included in the planned diagnostic network, and ensuring the constant supply of reagents and consumables. in support of coordinated planning, it is important to develop criteria for the placement of additional machines, including point-of-care or near to point-of-care platforms and higher throughput platforms which all stakeholders would be required to adhere to. negotiated agreements should look to the bundling of services (including connectivity). contractual requirements for data sharing (downtime, testing protocols, specimen types, etc.) will facilitate management of the network in real time and improve vendor accountability. to advance a network approach, it is important to look beyond a lowest price per test and focus on the total cost of ownership; initial per test costs will likely be higher, but the longer-term strategy will benefit the network. evidence-based optimisation of laboratory network factors determining the success of vl and ivt testing programmes include laboratory infrastructure and instrumentation, logistics, specimen transport, clinical implementation, and monitoring and evaluation. while it is helpful to coordinate procurement and service maintenance under a network approach, a limited understanding of reagent consumption, testing demand, laboratory performance and human resource capacity can undermine the functionality of a network. in cases of network expansion or revision, it may be necessary to carry out an analysis toward the goal of optimising the network. an approach to laboratory network optimisation would focus on the use of geographical information systems mapping tools (e.g. laboratory efficiency and quality improvement planning [labeqip] software and supply chain guru™ – llamasoft18,19), to map laboratory network parameters, including instrument locations and utilisation, testing demands, quality assurance, human resources, sample transport lanes, specimen types, demographic needs, costing components and partner performance data. labeqip is a software tool developed by united states agency for international development (usaid) and llamasoft, which is managed by the global health supply chain – procurement and supply management (ghsc-psm). it is a geographical information, systems-based solution that can improve laboratory network efficiency and advance quality service delivery through data-driven optimisation and modelling. virtual modelling, prior to instrument placement, or as part of formalising an overall shift in testing strategies, is a critical component in informing the approaches to laboratory network optimisation. labeqip has most often been used to strategically plan the design of laboratory networks, the placement of equipment, the planning of sample referrals and the improvement of instrument utilisation. labeqip and supply chain guru™ have been used in nigeria, cameroon, rwanda, eswatini, zimbabwe and zambia with support from the president’s emergency plan for aids relief (pepfar), gf, the clinton health access initiative (chai) and ghsc-psm, to develop virtual strategies to integrate hiv-tuberculosis sample transport, reduce instrument footprints to improve operational costs, and virtually place instruments to determine the impact on laboratory testing demands and instrument capacity requirements. labeqip can also be used to inform the integration of point-of-care technologies, and to assist in prioritisation and instrument rebalancing due to overburdened or underburdened testing demands. demand forecasting and supply planning in the initial phase of scale-up, programmes often use demographic or target-based forecasts. a demographic forecast takes the number of patients who are on antiretroviral treatment and multiplies that number by the number of vl tests per patient; a target-based forecast does the same, but uses the national or programme annual treatment numbers. both types of forecast invariably overestimate commodity demand, as they do not account for unreliable laboratory or logistics information systems and poor reporting, poor site-level stock management practices, uncoordinated instruments and instrument failure.6 further, during a period of rapid scale-up, historical consumption and procurement are not reliable indicators of future consumption. usaid and pepfar, through its supply chain implementing partner, ghsc-psm, has increased procurement of vl testing reagents from just over $7 million in 2014 to nearly $90 million in 2018. a linear projection of vl testing demand based on historical procurement in 2016 would have forecast approximately $37 million of vl testing-related procurement by 2017, increasing to $47 million in 2018. actual 2017 vl testing procurement data reflected almost $6 million in ghsc-psm expenditures, with over $90 million in procurement moving into 2018, an underestimation of about 44% and 48% if linear projections were used (figure 4). figure 4: linear projection of all the president’s emergency plan for aids relief viral load testing demand based on 2013 to 2016 historical procurement (projected 2016), compared to actual 2017/2018 procurement data. to address forecasting challenges, usaid promotes forlab (forlabplus.com), which was developed by usaid and chai and is managed by ghsc-psm, for national laboratory forecasting. forlab has been used in more than 21 countries since its launch in 2013. forlab includes forecasting commodity needs using a mixed methodology approach to improve accuracy and to provide consistent and greater transparency in national forecasting exercises. forlab includes demographic and morbidity data, service statistics and logistics data on commodity consumption in an effort to triangulate multiple forecasting methods to derive a best-fit procurement plan that can be used by stakeholders to establish realistic budgets and supply planning activities.19,20,21,22,23,24 forlab is a data-driven tool and works well when data are available and, when data are of a high quality, it can precisely predict need. however, poor site-level reporting can reduce its forecasting accuracy. when highlighting stock-outs as a limiting factor associated with the scale-up of vl testing, it is important to acknowledge that there are many factors outside the supply chain that can impede improvements in reagent availability, which must be addressed concurrently. as programmes scale up, site-level storage space can become a challenge, causing the dispersal of reagents across various locations within a particular laboratory. product dispersion can make routine stock management tasks more laborious and reduce reporting frequency and accuracy. as programmes scale up, it may be necessary to increase reagent distribution frequency to sites, if commodity storage is limited, for example from quarterly to monthly. for this to be successful, there is a need to ensure consistent and reliable stock status visibility. an additional factor not related to supply chains that has an impact on reagent availability includes early visibility into new instrument introductions, as without coordination additional reagents may not be available to support extra instrumentation. in order to prevent stock-outs, programmes need effective data flow from testing sites to the central warehouse to guide inventory management practices and product distribution mechanisms. effective supply chains are data-driven and require constant input on service delivery performance. accurate and consistent commodity stock levels and consumption reporting improves supply chain systems, allowing for accurate forecasting, timely procurements and improved visibility for manufacturers to assist with manufacturing lead times for large order quantities. without these consistent and reliable inputs through logistic management information systems or laboratory information management systems, it becomes increasingly difficult to prescribe effective procurement and supply chain interventions to reduce stock-out situations. strategic procurement and sourcing to address pricing variability within countries, it is critical to negotiate national pricing schemes. national testing volumes should be aggregated to negotiate a consistent price that all stakeholders can achieve. donors, host-country counterparts and manufacturers can work collaboratively with aggregated volumes to derive transparent pricing schemes and mutually agreed upon prices that include additional service offerings outside of just reagents and consumables. recent coordination with the gf has resulted in price transparency in haiti, the democratic republic of congo and cameroon, with initial price reductions of approximately $21.00 to $16.50, and then further to $13.50 for reagent costs. efforts are still in process to promote further reductions as scale-up continues in these countries, as well as to include more comprehensive service packages. this includes service, maintenance and data management, as well as standardised reporting requirements informed by agreed upon key performance indicators as part of a price-per-test scheme. the pepfar has currently renegotiated all of its existing vl/eid procurement contracts to significantly lower all-inclusive pricing schemes. a formal press-release will be announced shortly after the publication of this paper. the pepfar has engaged gf to push further transparent pricing reductions, with additional itemised system costing options, including all-inclusive reagent rental, service and maintenance, data management systems, as well as possible vendor-managed inventory. all future instrument investments and reagent procurement strategies should use rrs for new instruments, as well as inclusive rss for existing instrumentation. the pepfar’s current country operational planning technical guidance emphasises the use of rrs for instrument expansion, driving countries towards a more systems-focused approach. currently, south africa, kenya, uganda21,23 and, more recently, mozambique, haiti and nigeria are taking advantage of rrs or a combination of rrs and rss. currently, usaid is working with ghsc-psm to introduce more dynamic rrs agreements in nigeria, mozambique, haiti and zambia. by moving to a rrs or a rss approach, countries can amortise their initial capital investment for the scale-up and servicing of vl-testing instruments within their reagent pricing scheme, offsetting initial scale-up costs and expanding instrument coverage, as well as ensuring complete service contract availability. in order to assist countries in this approach, usaid developed a ‘12 question’ approach designed to help countries think through the use, placement and servicing of laboratory instruments prior to initiating procurement or rrs or rss arrangements (box 1, figure 5).18 figure 5: approach for negotiating reagent rental agreements (linked to 12-question approach). box 1: the ‘12 question’ approach to instrument procurement and placement. monitoring instrument and vendor performance when considering rrs or rss contracts, it is critical to establish defined expectations. contracts should be negotiated collaboratively with all stakeholders and donors. a harmonised set of key performance indicators (table 1) should be developed and should include: minimum response times for instrument repairs, training, logistics, and instrument and end-user performance. clear thresholds should be established for instrument failure frequencies, and service providers should be held accountable for responding to site-level failures that go beyond these established thresholds. contracts should dictate a standardised monthly and quarterly reporting format to assist in addressing site or instrument-specific challenges, as well as vendor service delivery issues. the contract should also define at least quarterly meetings with the supplier to review performance and work collaboratively to solve problems and address any performance issues. contracts should also clearly delineate lists of parts to be made available in-country for high-failure parts, minimum service technician requirements, possible data solutions for patient result transmission, and monitoring instrument and end-user performance. table 1: illustrative key performance indicators used to monitor vendor service and instrument performance through service contracts. conclusion the current effort to scale up vl testing and ivt has been significant. gains have been achieved within national laboratory networks to scale up vl testing and ivt, but there is still a need to ensure sound investments in laboratory infrastructure and instrumentation, without overlooking the supportive structures of logistics, clinical components, and monitoring and evaluation protocols. there are lessons learned from past scale-up efforts for cd4 testing, with the current global strategy to ensure procurement coordination across donors, standardising and ensuring transparent pricing for reagents, and implementing general procurement principles that aim to address some of the main supply chain and service challenges. however, these global strategies must be translated into operational plans at a country level. to be successful, all stakeholders will need to embrace the full cycle of the network approach for laboratory procurement and supply chain management; take stock of existing instruments, service contracts and procurement pricing schemes; and establish jointly renegotiated terms that leverage all stakeholder investments. countries that have successfully scaled up vl testing and ivt have focused on making these commitments and have thereby reduced the risk of equipment failure and commodity stock-outs two critical challenges to the success of vl testing and ivt programmes. while each of the four pillars of the network approach for procurement and supply management can support elements of the supply chain, true transformation of the laboratory network is only possible through embracing all four of the strategic pillars in a stepwise approach, with each phase in the cycle continuing to inform the next step. in the longer term, these investments and the broader network approach will not only address some of the more immediate challenges, but will also enable countries to strengthen laboratory systems and ready themselves for implementing future laboratory needs. these disease-agnostic molecular networks will be poised to improve overall national disease surveillance and assist countries in responding to disease outbreak responses and other chronic diseases. in addition, such networks will position countries to address sustainable strategies for laboratories in future health agendas. acknowledgements competing interests we declare that we have no financial or personal relationships that may have inappropriately influenced us in writing this article. authors’ contributions j.w. was the nigeria and zimbabwe laboratory network optimisation lead, d.e. was the eswatini laboratory network optimisation lead, m.w. was the zimbabwe procurement and supply management technical lead in network optimisation, and c.n. was the eswatini procurement and supply management technical lead in network optimisation. all leads contributed to the development and implementation of the laboratory network approach strategy. all authors, including j.k., were involved in technical content review and narrative development. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the content in this manuscript is that of the authors and does not necessarily reflect the view of the united states president’s emergency plan for aids relief, the united states agency for international development or the united states government. references joint united nations programme on hiv and aids (unaids). global hiv & aids 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[cited 2019 jul 30]. available from: http://www.stoptb.org/wg/gli/assets/documents/gli_guide_specimens_web_ready.pdf roberts t, cohn j, bonner k, hargreaves s. scale-up of routine viral load testing in resource-poor settings: current and future implementation challenges. clin infect dis. 2016;62(8):1043–1048. https://doi.org/10.1093/cid/ciw001 marinucci f, medina-moreno s, paterniti ad, wattleworth m, redfield r. decentralization of cd4 testing in resource-limited settings: 7 years of experience in six african countries. cytometry. part a: j int soc anal cytol. 2011;79(5):368–374. https://doi.org/10.1002/cyto.a.21064 abstract introduction methods results discussion acknowledgements references about the author(s) modupe a. kuti department of chemical pathology, faculty of basic medical sciences, college of medicine, university of ibadan, ibadan, nigeriadepartment of chemical pathology, university college hospital, ibadan, nigeria olabisi t. bamidele department of chemical pathology, university college hospital, ibadan, nigeria chioma t. udeh department of chemical pathology, university college hospital, ibadan, nigeria bola j. eseile department of chemical pathology, university college hospital, ibadan, nigeria olajumoke a. ogundeji department of chemical pathology, university college hospital, ibadan, nigeria citation kuti ma, bamidele ot, udeh ct, eseile bj, ogundeji oa. appropriate use of plasma glucose tests for diagnosis of diabetes mellitus in ibadan, nigeria. afr j lab med. 2022;11(1), a1433. https://doi.org/10.4102/ajlm.v11i1.1433 original research appropriate use of plasma glucose tests for diagnosis of diabetes mellitus in ibadan, nigeria modupe a. kuti, olabisi t. bamidele, chioma t. udeh, bola j. eseile, olajumoke a. ogundeji received: 23 oct. 2020; accepted: 07 feb. 2022; published: 29 apr. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: diabetes mellitus is a growing epidemic in africa. its diagnosis relies exclusively on laboratory evidence, which differs based on clinical circumstances. objective: the study described the appropriateness of plasma glucose test requests per the american diabetes association criteria. methods: we reviewed the plasma glucose test requests received by the chemical pathology laboratory of the university college hospital, ibadan, nigeria between june 2018 and november 2018. the american diabetes association diabetes diagnostic criteria were used to define the appropriateness of test requests and determine the potential for ill-informed clinical decisions. results: four hundred and twenty-three requisition forms were included, with the majority from the medical wards/clinics (72.3%); the most frequent reason for a plasma glucose test was systemic hypertension (28.6%). fasting plasma glucose was most requested (254; 60.0%). one hundred and sixteen (27.4%) requests were potentially inappropriate, with the 2-h postprandial plasma glucose (2hppg) test requests (83; 71.6%) being the most inappropriate. the difference in the proportion of inappropriate requests was not statistically significantly between medical or surgical wards/clinics (odds ratio 1.131, 95% confidence interval 0.709–1.803, p = 0.605). inappropriate requests in six cases may have triggered inappropriate action. conclusion: a third of the glucose tests requested for querying diabetes mellitus may have been inappropriate. results of such testing may trigger inappropriate clinical action. to improve the quality of care and for economic reasons, laboratories should have programmes to improve the appropriate use of their services. keywords: diabetes mellitus; glucose; utilisation management; laboratory; guidelines; fasting; postprandial; random. introduction the socio-economic burden of a diabetes mellitus (dm) diagnosis is substantial for persons in low and middle-income countries relative to persons in higher-income countries.1 the absence of robust national health insurance schemes in low and middle-income countries means that this burden is borne mainly by patients and their families. the total healthcare cost of persons with dm has been estimated to be about four times higher than for persons with normal glucose tolerance.2 this cost includes those incurred from admissions, outpatient visits, laboratory tests, medication, and treatment trips, as well as productivity losses by patients and caregivers. the rapidly rising prevalence of dm imposes an increasing significant strain on fragile national health systems,which are already buckling under a huge infectious disease burden. estimates of dm prevalence from urban populations in kenya, cameroon, and south africa range between 10% and 12%.3,4,5 a recent lancet commission on the burden of dm in sub-saharan africa reported that dm and its complications are costly to patients and that national health systems are largely unable to cope with the current burden of the disease.6 one of the fundamental requirements for appropriate public health response to this epidemic is accurate diagnosis of dm. the diagnosis of dm relies exclusively on biochemical evidence of a specific degree of glucose intolerance. the current accepted diagnostic criteria for dm in the non-pregnant adult were initially published by the american diabetes association in 1998 and were adopted by the world health organization in 1999.7,8 the laboratory tests referenced in the criteria are fasting plasma glucose (fpg), 2-h post-load glucose (2hplg) during an oral glucose tolerance test (ogtt), and random plasma glucose (rpg). in 2010, glycated haemoglobin was listed as a further diagnostic criterion.9 other than glycated haemoglobin, all the glucose criteria require that specific patient preparation steps or clinical symptoms be present. the fpg sample is collected after an overnight fast of at least 8 h, while the 2hplg sample is obtained 2 h after the patient consumes a standard glucose load of 75 g of anhydrous glucose. for rpg to be used as a dm diagnostic criterion, the presence of either classic symptoms of dm or hyperglycaemic crisis is required. this suggests that rpg is not recommended for screening of the asymptomatic person and 2hplg should not be used interchangeably with 2-h postprandial plasma glucose (2hppg). the latter test is performed on a sample taken after the consumption of the individual’s regular diet, which will vary significantly from person to person. two-hour postprandial plasma glucose has utility in assessing glycaemic control in persons with known dm.10 the study aimed to determine the appropriateness of the glucose test requests per the american diabetes association dm diagnosis criteria.11 furthermore, for tests that were ordered contrary to recommended guidelines (inappropriate tests), we examined the potential for consequent inappropriate clinical action based on the result of such tests. methods ethical considerations ethical approval was obtained from the university of ibadan/university college hospital ethics committee with a national health research ethics committee nhrec/05/01/2008a. the ethical committee assigned number for the study was ui/ec/19/0630. this was an analysis of secondary data and did not retrieve any patient-identifiable information or involve contact with any human participants. patient consent was therefore not required. all glucose results were anonymised from patient details and study numbers accessible only to the researchers were used during analysis. study setting the university college hospital, ibadan is a public tertiary hospital located in an urban setting in the south western region of nigeria. the hospital is served by a chemical pathology laboratory that receives about 33 000 samples annually, including blood, urine, and cerebrospinal, ascitic and pleural fluids. tests provided include those assessing for renal, liver, metabolic, endocrine, and neoplastic diseases. for a non-pregnant adult, the laboratory offers two stand-alone glucose tests – fpg and rpg – as well as two glucose profiles – fpg/2hppg and fpg/2hplg. study design this cross-sectional study reviewed all request forms from the wards/clinics of the hospital from june and november 2018 for plasma glucose tests. to be included in this study, the section for clinical information was required to contain information about the patient such as presenting symptoms, signs, working diagnosis, or treatment. any form with no clinical information or with clear information that the patient was previously diagnosed as having dm was excluded. definition of test request inappropriateness the appropriateness of the tests was defined using the american diabetes association dm diagnostic criteria (box 1).12 the following test requests were deemed as probably inappropriate requests for dm diagnosis: all 2hppgtest requests. all rpg test requests in which the clinical details on the requisition form included none of the classic symptoms of dm (polyuria, polydipsia, and weight loss). box 1: criteria for the diagnosis of diabetes mellitus by the american diabetes association (2018). the potential for inappropriate clinical action was deemed to be present if: the result of an inappropriately requested rpg exceeded 11.1 mmol/l, and/or the fpg was < 7.1 mmol/l and the 2hppg was > 11.1 mmol/l. data analysis statistical analysis was performed using ibm® statistical package for social sciences (spss®) version 25 (2017, ibm corp, armonk, new york, united states). proportions are presented as numbers (percent) and were compared using the chi-square test. results were considered significant if p was 0.05 or less, with a 95% confidence interval. results four hundred and twenty-three request forms satisfied the inclusion criteria. medical outpatient clinics and wards accounted for 306 (72.3%) of these requests. overall, the most common reason for a glucose test request was to evaluate systemic hypertension (table 1). the surgical clinics and wards most commonly requested a glucose test for evaluating neoplastic disease (benign or malignant). a one-off fpg was the most requested glucose test in both specialities, accounting for 254 (60.0%) of all requests (table 2). the usage pattern for the different tests/profiles was significantly different between both specialities (x2 = 138.911 [p < 0.001]); the surgical specialities requested more ogtt, mostly for postnatal assessment of persons with previous gestational dm. table 1: clinical information on requisition forms received in the chemical pathology laboratory, university college hospital, ibadan nigeria (june 2018 – november 2018). table 2: distribution of glucose requests by speciality of source ward/clinics of the university college hospital, ibadan, nigeria (june 2018 – november 2018). the fpg level was normal (< 5.5 mmol/l) in 77.7% (303) of cases, impaired (5.5 mmol/l – 6.9 mmol/l) in 15.1% (n = 59), and diabetic (≥ 7.0 mmol/l) in 7.2% (n = 28). the glucose level was < 11.1 mmol/l for 88% (n = 73) 2hppg, 88.7% (n = 47) 2-h ogtt and 100% (n = 33) rpg requests. one hundred and sixteen (27.4%) of the test requests were potentially inappropriate, comprising 83 (71.6%) requests for fpg/2hppg and 33 (28.4%) requests for rpg (table 3). all of the ogtt requests were appropriate. inappropriate test requests from the medical wards and clinics (n = 82; 70.7%) were higher than those from the surgical wards and clinics (48 tests; p = –0.629) (table 2). of the 83 inappropriately requested fpg/2hppg, the fpg was < 7 mmol/l while the 2hppg was > 11.1 mmol/l in 6 (7.2%) cases. the fpg and accompanying 2hppg were < 7 mmol/l and < 11.1 mmol/l in 71 (85.5%) cases. fasting plasma glucose > 7 mmol/l/2hppg < 11.1 mmol/l was seen in one case and fpg > 7 mmol/l/2hppg > 11.1 mmol/l was seen in another five cases. table 3: clinical information against inappropriately requested glucose tests in the chemical pathology laboratory, university college hospital, ibadan nigeria (june 2018 – november 2018). discussion despite the annual publication of the american diabetes association standards for the laboratory diagnosis of dm for over 20 years, this study provides evidence that there exists a need to increase the uptake of its recommendations in routine practice in our setting. nearly one out of every three requests for glucose tests, in our setting, was found to be potentially inappropriate. inappropriate requests were mostly due to requests for 2hppg as a dm screening test. reports from both nigeria and cameroon indicate that primary care physicians, who represent the bulk of physicians providing dm screening and care, have poor knowledge of the diabetes clinical guideline criteria.13,14 over 70% of the participants in both studies were not reliably diagnosed with dm using the requested glucose tests.13,14 the primary consequence of an inappropriate glucose test is the waste of the patient’s money for an unnecessary test. the wastage is particularly costly in a region where most patients are uninsured and thus pay out of pocket.15 money spent on unnecessary testing may reduce money for necessary treatment. there is also a cumulative cost to the health systems. glucose tests are relatively inexpensive, however, increased requests over time will drain laboratory resources.16, 17 a further consequence of inappropriate testing is a false negative test result. as observed in this study, the majority of the requests for rpg returned values below a ‘diagnostic’ threshold. there is no guarantee that the screened patients were all glucose tolerant. reports suggest that the sensitivity of an rpg value greater than or equal to 7 mmol/l for identifying asymptomatic persons with dm may be less than 70%.18,19 these reports concluded that it would be inappropriate to use the rpg test alone as a dm screening test. thus, an additional test is required for definitive diagnosis of dm, thereby increasing costs incurred by patients. we examined whether the requests from surgical wards and clinics were more likely to be inappropriate compared to those from medical wards and clinics. reports suggest that certain practices by surgeons may increase the likelihood of inappropriate test requests. charani et al.20 reported that surgeons frequently left care decisions perceived as non-surgical to junior team members. this practice is reportedly associated with an increased likelihood of inappropriate laboratory test requests.21,22 our study, however, did not observe a significant difference in rates of inappropriate test requests from the surgical wards and clinics compared to the medical ones. a possible explanation for this observed lack of significant difference in inappropriate test requests between both wards and clinics in this study might be due to, the much higher number of test requests from the medical areas consequently increasing the number of inappropriate requests. this may thus cancel out the effect of the delegation of duties within the surgical areas. we also note that, although a 2hppg > 11.1 mmol/l may be suggestive of the presence of dm, it may not be used as conclusive evidence of the disease, as current criteria for dm diagnosis do not include it. glycaemia > 11.1 mmol/l may be observed as part of a physiologic stress response.23 the appropriate response, therefore, to such a result should be an evaluation for dm using tests listed in the currently accepted criteria. any other clinical course, such as commencing treatment or re-ordering the same test, would be inappropriate. the present study has demonstrated that a significant percentage of requests for plasma glucose, a routine and frequently test, may be inappropriate. similarly, estimates from a teaching hospital in austria suggest that as many as 60% – 70% of high throughput tests such as potassium and lactate dehydrogenase may have been ordered inappropriately or in clinical situations where they had only minor relevance.24 depending on the criteria used, estimates of request inappropriateness from the united states range between 5% and 95% of tests.25 we have also shown here the potential for inappropriate clinical action following an inappropriate laboraory test requests. a 20-year review of malpractice claims in insurance companies that provide cover for patients in over 40 academic and non-academic hospitals in the united states found 181 claims that involved diagnostic errors and resulted in harm to the patient. over 50% of these claims were attributable to a failure to order an appropriate test.26 inappropriate test usage wastes resources and may result in actual harm to the patient. predictably, a system to manage physician test utilisation has value for patients, the physicians, and the health care system, with significant positive yields in quality of care and economic savings.27,28 such a system should be characterised by an understanding of the multitude of factors that influence test requisition. these range from diagnostic, therapeutic/prognostic, patient-related, physician-related and policy/organisation factors.29 depending on the specific intention of such a management programme, a wide range of approaches is available.30,31 these approaches include the use of utilisation audits, as in the current study, analytical algorithms, test guidelines, formularies and, where applicable, clinical decision support systems and changes to computerised provider order entry. implementing utilisation management initiatives requires the collaboration of key stakeholders, including pathologists, laboratorians and other professionals who contribute to the health care process.28 pathologists, whose background training enables them to observe testing behaviour patterns, suggest alternatives, manage testing algorithms and provide interpretative services, could show leadership in this regard.28,32,33 limitations the study was limited by its reliance on the information included in the submitted requisition forms. this information may not have correctly reflected the entire clinical status of the patient being evaluated. conclusion in conclusion, we have demonstrated the suboptimal usage of glucose tests in dm screening, with the speciality of the requesting physician as a potential factor. this provides evidence that laboratories should engage in programmes, such as standard diagnosis awareness campaigns, directed at improving the appropriate use of their services. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions m.a.k. conceived the article and analysed the data; o.t.b. and c.t.u. conceived the article and 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perceived hindrances and recommendations conclusion acknowledgements references about the author(s) beverly egyir department of bacteriology, noguchi memorial institute for medical research, accra, ghana alfred bortey department of bacteriology, noguchi memorial institute for medical research, accra, ghana kwabena o. duedu college of life sciences, birmingham city university, birmingham, united kingdom gifty boateng national public health and reference lab, korle-bu teaching hospital, korle-bu, ghana franklin a. bekoe disease surveillance unit, ghana health service, accra, ghana george hedidor world health organization country office, ghana, accra, ghana michael adjabeng world health organization country office, ghana, accra, ghana nicholas t.k.d. dayie department of medical microbiology, university of ghana medical school, accra, ghana noah obeng-nkrumah department of medical laboratory sciences, school of biomedical and allied health sciences, accra, ghana japheth a. opintan department of medical microbiology, university of ghana medical school, accra, ghana citation egyir b, bortey a, duedu ko, et al. implementation of antimicrobial resistance surveillance in ghana using the integrated disease surveillance and response strategy. afr j lab med. 2024;13(1), a2404. https://doi.org/10.4102/ajlm.v13i1.2404 opinion paper implementation of antimicrobial resistance surveillance in ghana using the integrated disease surveillance and response strategy beverly egyir, alfred bortey, kwabena o. duedu, gifty boateng, franklin a. bekoe, george hedidor, michael adjabeng, nicholas t.k.d. dayie, noah obeng-nkrumah, japheth a. opintan received: 05 jan. 2024; accepted: 10 june 2024; published: 13 aug. 2024 copyright: © 2024. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction the integrated disease surveillance and response (idsr) strategy is aimed at enhancing public health surveillance and response in the african region by establishing connections across community, health facility, sub-district, district, regional, and national levels.1 the technical guidelines for idsr were originally developed by the world health organization (who) regional office for africa, and the united states centers for disease control and prevention for the broader african context.2,3 the idsr strategy optimises resource utilisation by consolidating and streamlining common surveillance activities, including detection, reporting, analysis, interpretation, feedback, and action. this approach leverages shared structures, processes, and personnel to enhance efficiency in public health surveillance and response. the strategy also integrates the one health perspective, addressing events that intersect human, domestic animal, wildlife, and the environment.4 the idsr approach was adapted by ghana to provide public health personnel, managers and decision-makers with relevant, adequate and timely data that helps in determining disease burdens, ensuring proper allocation of resources for early detection of outbreaks and to provide rapid public health response. ideally, the strategy should also include monitoring of antimicrobial susceptibilities to promote efficient use of information for decision-making to combat antimicrobial resistance (amr). it’s worth noting that systematic monitoring of antimicrobial susceptibilities of bacterial species is not integrated into the existing public health structures with the idsr strategy in ghana. this is due to challenges related to logistics, infrastructure and human resource support to ensure the comprehensive implementation of amr surveillance by laboratories within the network of a sentinel surveillance system using the idsr approach. antimicrobial resistance is an important global health threat and considered a public health emergency that requires attention. in response to this threat, countries have developed action plans and instituted measures to reduce its impact. considering the huge clinical and economic impact caused by amr pathogens, it is imperative to purposively implement amr surveillance for a more holistic integrated surveillance system to combat amr and infectious diseases. in this article, we advocate for and offer recommendations regarding the implementation of amr surveillance within the idsr framework to facilitate real-time monitoring of amr pathogens as well as the timely identification of threats posed by amr pathogens to critical antimicrobials. the need: purposeful implementation of antimicrobial resistance surveillance using the integrated disease surveillance and response approach over the past 20 years, 94% (44/47) of the countries in the who african region have been using the idsr approach to intensify surveillance for specified priority diseases and conditions which have been met with great improvements.5,6,7 as at 2020, the data available show how the idsr approach aided in the identification, investigation and response to outbreaks of measles, yellow fever, hepatitis e virus, circulating vaccine-derived poliovirus type 2, and coronavirus disease 2019 in many countries.8 over time, there has been an increase in the resistance of bacteria to various antimicrobials.9 according to murray et al., about 4.95 million deaths globally were associated with amr, where 1.27m deaths were specifically attributed to amr in 2019.10 western sub-saharan africa recorded the highest mortality rates globally (27.3 deaths per 100 000) attributable to amr, which shows that it is a matter of great concern in the sub-region.10 mortality rates related to amr in the sub-saharan african region result from various factors, including ineffective infection prevention and control practices, inadequate management of healthcare systems, and insufficient data on the amr burden in the region. therefore, it is prudent and crucial to deliberately implement amr surveillance through the idsr to address the amr challenge in sub-saharan african countries. to a large extent, several healthcare facilities in ghana, tanzania and zambia do not adequately use the idsr technical guidelines for identification of cases in laboratories, and are not properly equipped for identification and confirmation of pathogens for priority diseases/conditions, including antimicrobial susceptibility testing of bacteria species.11,12,13 turner et al. noted that high-quality surveillance is essential to cover the major themes of the specific objectives of the who global antimicrobial resistance surveillance system, which include the estimation of amr burden, trends over time, and the impact of interventions.8,14 ideally, an amr surveillance system must have both clinical and laboratory data, and fortunately, the idsr case-based surveillance system of ghana collects clinical and laboratory data. however, the existing laboratory tests conducted are just to confirm the suspected diseases and not necessarily to keep track of pathogens and amr profiles of bacteria species in the communities or regions.15 exceptional steps are taken in this direction during meningitis outbreaks, where there is constant monitoring of the susceptibility of meningococcal bacteria causative agent to available antibiotics to inform case management. in clinical settings, data from amr surveillance are essential to inform empiric treatments.14,16 the selection of appropriate antimicrobial therapy has the potential to reduce the further rise and spread of amr as well as the rate of healthcare-associated infections and multi drug-resistant organisms. the integrated disease surveillance and response framework and antimicrobial resistance surveillance the idsr strategy involves the collection of health-associated data using standardised tools via two major methods: the indicator based surveillance and event based surveillance.4 the indicator based surveillance channel focuses on identification, collection, monitoring, analysis, and interpretation of structured data from health-based formal sources. on the other hand, the event based surveillance is designed for rapid capture of information about events that are of potential risk to public health. it starts as an alert which is triggered based on an early warning and response system. the existing idsr matrix comprises eight vital core functions and activities that are to be performed by health system levels (figure 1).2 step 1 includes identifying and recording cases, conditions, and events using standard and simplified case definitions at health facility and community levels. step 2 entails reporting of suspected cases and any potential public health emergency of international concern, conducting investigations, and submitting detailed reports. step 3 involves analysing and interpreting surveillance data based on person, place, and time for public health actions. step 4 entails investigating and confirming suspected cases or outbreaks through epidemiological investigation and laboratory testing. hereafter, organisms detected from suspected samples should be recorded and reported on the priority diseases. the distributions and prevalence should be analysed and communicated with experts and officials (this serves as interlink between step 4 and step 7). step 5 involves preparing by having emergency response plans, stockpiling essential supplies, and training personnel in advance. step 6 involves responding to outbreaks by determining the cause, mobilising resources, and establishing coordination mechanisms. step 7 entails engaging in risk communication with experts, officials, and at-risk communities to exchange information and advice. step 8 includes monitoring, evaluating, and providing feedback to improve the surveillance system’s effectiveness, involving community representatives, the private sector, and non-governmental organisations in evaluation activities. figure 1: diagram showing a suggested workflow incorporating antimicrobial resistance surveillance into the integrated disease surveillance and response matrix, using the one health approach. considering the gravity of the amr threat, we advocate for the implementation of amr surveillance in the idsr framework via the indicator based surveillance and event based surveillance channels on a sentinel basis. the indicator based surveillance component can include defining country-specific priority pathogens and antimicrobials, among others. ghana, as a signatory to the united nations and the who, will naturally defer to the who-defined priority pathogens and antimicrobials. further to this, we recommend that the country uses its existing surveillance structures and research data to conduct an assessment, and to identify any other pathogen or antimicrobial agent that is of importance to be included into ghana’s list of priority pathogens and antimicrobials. ghana already has an electronic digital disease surveillance platform in use; the existing case-based reporting platform is already integrated into the surveillance outbreak response management and analysis system. the case-based forms for amr surveillance can be integrated into the surveillance outbreak response management and analysis system platform which has already received significant investment for its deployment and training of health personnel to use it. during the coronavirus disease 2019 pandemic, the use of the surveillance outbreak response management and analysis system reduced the reporting time lapse from result generation in the lab to the consulting room. it also provided real-time updates for public health responses. we predict that the data on antimicrobials and pathogens will be huge; hence, an artificial intelligence-driven algorithm will be needed to analyse data in real time to identify patterns of concern which will be sent to designated public health officials to review and report for response action. the type of feedback from the artificial intelligence algorithm should allow classification of threats. in response to the indicator data, response plans can be developed and deployed. the event based surveillance, on the other hand, will encompass swift collection of data related to events that may pose a potential risk to public health. it commences as an alert, triggered by an informal and not well-defined early warning and response system. integrated disease surveillance and response and microbiology facilities in ghana according to the idsr framework, health facilities are supposed to identify suspected cases of diseases, notify, confirm them through laboratory testing and report on them as well. however, there are districts in ghana with neither hospitals nor microbiology laboratories. the absence of such needful capacity is a great limitation to the nationwide success of amr surveillance. out of 32 health facilities sampled in the ga west municipality, only six facilities had idsr guidelines in their facilities, while nine out of 32 submit weekly reports, and 19 submit monthly reports.17 such information highlights the need for improvement and capacity building of laboratories at health facilities in ghana to promote accurate, efficient, and effective disease and amr surveillance in ghana. figure 2 shows microbiology laboratories involved in testing of priority conditions in ghana. figure 2: map of ghana showing microbiology laboratories involved in integrated disease surveillance and response testing of priority conditions in ghana, may 2024. perceived hindrances and recommendations in april 2018, ghana introduced the antimicrobial use and resistance policy and national action plan on amr, aligning with the objectives of the global action plan on amr.18 ghana has established national platforms, including the national amr platform and the national one health platform, which comprise representatives from the human, animal, and environmental sectors. these platforms continuously deliberate on amr, one health, and related issues to address public health impacts. to enhance understanding of amr through surveillance and research, ghana secured the initial fleming country grant. this grant supported a government-led initiative to collect, analyse, and report amr and antimicrobial use data from humans and animals across 11 sentinel sites. the goal was to generate a comprehensive national amr overview to guide treatment decisions at both local and national levels. extensive engagement, training of laboratory staff, and preparation for a pilot surveillance were carried out at 11 sentinel sites. the sentinel sites or clinical microbiology laboratories received infrastructure upgrades with assistance from the fleming fund. this successful approach can be expanded and sustained within the framework of the idsr for amr surveillance in the country. it is worth noting that implementing effective or sustainable amr surveillance programmes in africa, including ghana, comes with a plethora of challenges. these could range from the lack of resources (material and human), improper or inadequate infrastructure, poor supply chain for laboratory consumables and inadequate data generation, collation, analysis and sharing. also, in places where there are laboratory facilities that can help in amr surveillance, health workers are not motivated enough to go beyond their normal duties due to the constant work overload in their facilities. some facilities in ghana can produce antibiograms from their laboratories, but the lack of sufficient biodata hinders the comprehensive assessment and analysis of the amr data. this calls for continuous training of health personnel and laboratory staff on data management, and the importance of surveillance. training programmes should cover all categories of personnel within the health delivery system, extending from the local to the regional and national levels. facilities lacking sufficient capacity to confirm the identity of pathogens can collaborate with national reference laboratories. in all the 16 regions of ghana, there are 11 regional or designated hospitals with microbiological labs. facilities without the relevant infrastructure can send isolates, along with appropriate epidemiological and clinical data to reference labs, for the identification and susceptibility testing of these pathogens. the reference laboratories must provide feedback to the submitting facilities regarding amr patterns of the pathogens, and where sequencing instruments are available in reference laboratories, whole genome sequencing can also be performed on the pathogens to generate granular data to inform surveillance activities as well as monitor the rise and spread of epidemic and pandemic clones. with support from the fleming fund’s regional grant, the seqafrica consortium is offering whole genome sequencing and data analysis services in ghana (and other african countries). this initiative has led to the sequencing of several amr bacterial species from the environment, animals, and humans to strengthen amr surveillance in the country. antibiogram information should be generated at the local, regional, and national levels to guide empirical antibiotic prescribing in hospitals. for example, a 6-month antibiogram data plot generated from clinical microbiology laboratories in hospitals can be pasted in consulting rooms to inform antimicrobial choices for patients. information on the overall microbiological cultures performed, the number of positive cultures, prevalence of amr pathogens, and whether the infections are community or hospital acquired, are additional information (currently not collected), and can be obtained as part of the core indicators of the existing idsr framework. antimicrobial susceptibility testing data from private hospitals and laboratories can also be harnessed within the idsr framework to gain a deeper understanding of the amr profiles of bacterial pathogens in the country. conclusion implementing amr surveillance within idsr strategy is a pivotal move towards identifying amr pathogens, particularly those implicated in outbreaks. furthermore, this implementation aligns with global health initiatives, reinforcing ghana’s commitment to combating amr and infectious diseases. by providing timely and accurate data on amr trends, the idsr framework will facilitate informed decision-making in healthcare policies, resource allocation, and public health interventions. ultimately, this endeavour will significantly contribute to safeguarding public health, improving patient outcomes, and curbing the spread of amr pathogens, marking a major stride in ghana’s ongoing battle against amr and infectious diseases. acknowledgements the authors express gratitude to all individuals whose works or articles have been referenced in this manuscript. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions b.e. conceived of and initiated the work. b.e. and a.b. prepared the original draft. k.o.d., g.b., f.a.b., g.h., m.a., n.t.k.d.d., n.o.-n. and j.a.o. participated in the critical review and editing of the manuscript and have given their approval for the content. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support no specific grant was received for this research from any funding agency in the public, commercial, or not-for-profit sectors. data availability data sharing is not applicable to this article as no new data were generated or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. it does not necessarily reflect the official 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[cited 2024 jun 03]. available from: https://www.reactgroup.org/news-and-views/news-and-opinions/year-2018/ghana-national-action-plan-on-amr-react-africa-supports-the-process/ introduction an important epidemiological reminder in this current context what we know what we should do acknowledgements references about the author(s) khadim diongue parasitology-mycology service, faculty of medicine, pharmacy and odontology, cheikh anta diop university, dakar, senegal laboratory of parasitology and mycology, aristide le dantec university hospital, dakar, senegal mamadou a. diallo laboratory of parasitology and mycology, aristide le dantec university hospital, dakar, senegal citation diongue k, diallo ma. covid-19 during malaria transmission season in africa and why we should be prepared: an example from senegal. afr j lab med. 2020;9(1), a1332. https://doi.org/10.4102/ajlm.v9i1.1332 opinion paper covid-19 during malaria transmission season in africa and why we should be prepared: an example from senegal khadim diongue, mamadou a. diallo received: 08 july 2020; accepted: 14 sept. 2020; published: 18 nov. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction coronavirus disease 2019 (covid-19) is not just a global health crisis; it is also a socio-economic crisis1 with a huge number of associated deaths.2 on 30 june 2020, the world health organization reported 10 185 374 cases and 503 862 deaths related to covid-19 globally, of which 297 290 cases and 6010 deaths were recorded in africa.1 in senegal, malaria transmission is seasonal with high vector populations observed during the rainy season – from mid-june to october. the heaviest rainfalls are observed in the months of july, august and september. on 30 march 2020, as effort to combat the spread of covid-19 in senegal, a state of emergency was declared banning religious gatherings, closing schools and universities and imposing a curfew between 22:00 and 06:00. as of 30 june 2020, the day the state of emergency was lifted, the authorities had reported 6698 confirmed covid-19 cases and 108 covid-19 related deaths1,3,4 thus the country was confronted with managing the expected rainy-season malaria epidemic and the on-going covid-19 pandemic. hence, this article aims to draw the attention of authorities to this current situation. an important epidemiological reminder in this current context before and after the ease of lockdown measures on 04 june 2020, senegal had been experiencing constantly increasing covid-19 cases, as evidenced by a 22% increase in cases during the month of june 2020. there were 1838 covid-19 cases on 01 june 2020, which had reached 2249 cases by 30 june 2020. however, the most detrimental event during the covid-19 pandemic is that of the general population deserting the health facilities that are hosting infected covid-19 individuals for fear of being tested or infected. since the decentralisation and proliferation of covid-19 care centres around the country, covid-19 hosting centres are being avoided by the populace; worse still, patients have now deserted the isolation centres. senegalese people avoid going to places where any test that could suggest covid-19 is done, even a body temperature measurement. however, experience has shown that in an outbreak such as the covid-19 pandemic, it is essential to not ignore other diseases such as malaria. the recent west africa ebola epidemic revealed the deleterious impact of increased health service demand on an already fragile health system, as well as on the control of other diseases. consequently, the ebola outbreak led to a substantial increase in morbidity and mortality of other diseases, including malaria.5 the world health organization has warned of a twofold devastation by the covid-19 pandemic: the first is its direct effect on health – covid-19 morbidity and mortality – and the second is the related increase in morbidity and mortality of other diseases due to lack of or diversion of adequate health response for other diseases.5 for example, during the ebola epidemic year in guinea, health facilities recorded a lower malaria patients’ visitation than was expected – an estimated deficit of 74 000 visitations. conversely and sadly, malaria caused 1067 deaths in 2014 – an ebola year – compared to 108 deaths reported in 2013 – a non-ebola year.6 more worryingly, in liberia, sierra leone and guinea, about 7000 other deaths related to malaria were recorded among children under age 5 years, which were attributed to the 2014–2016 ebola outbreak.6 what we know malaria illness presents some similar symptoms to the covid-19 illness: fever, respiratory distress, fatigue and an acute onset headache.5,7 the major sign between both illnesses is fever. thus, a malaria case or a covid-19 case may each be confused for the other, if symptoms alone are used to define a case during this current pandemic. also, since the beginning of the pandemic in senegal, fever is the major symptom of infected individuals8; febrile individuals are isolated before samples are taken. the sampling team is deployed after contact with the senegalese medical emergency assistance service; however, the emergency assistance centre has been overwhelmed (more than 726 000 emergency calls between 02 march 2020 and 29 june 20209), which could be the reason why, unfortunately, sampling teams can sometimes take more than 24 h to intervene. this delays the differential diagnosis of malaria and similar illnesses, as well as increases the risk of development of severe malaria, if malaria diagnosis is correct but delayed. therefore, clinicians should go back to the good old method of ‘any case of fever must be considered as malaria until proven otherwise’. confirming either malaria or severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection with a diagnostic test does not rule out the possibility of the other in a patient. indeed, both false-positive and false-negative results have been reported worldwide with rapid diagnostic tests.10 likewise, approximately 50% of infected cases are likely missed by current screening approaches for covid-19, even in countries with good healthcare systems and available diagnostic capacities. as covid-19 can be transmitted even by asymptomatic people, and personal social distancing cannot be effective when performing a malaria test, such as a rapid diagnostic test,11 other infection prevention and control measures must be carefully put in place.7 these measures include but are not limited to: proper donning and doffing of personnel protection equipment, especially face masks; provision and use of soap and water for handwashing; provision and use of alcohol-based hand sanitiser and the practice of good respiratory hygiene (i.e. covering of the mouth and nose with a disposable tissue when sneezing or coughing or sneezing into the crease of the elbow).7 thus, health providers should also be more careful. what we should do in senegal, according to the last national malaria control programme report published in 2018, malaria caused 395 706 cases with 284 deaths including 95 deaths (33.45%) among children younger than age 5 years.12 however, the malaria burden was more than 50% lower between 2009 and 2015, permitting the country to achieve the objectives of roll back malaria in 2015.3 therefore, to not lose this positive momentum in the fight against malaria, the senegalese national malaria control programme should scrupulously follow the recommendations of the world health organization, which include tailoring malaria interventions in the covid-19 response5 with particular regard to the following points: case management, chemoprevention (intermittent preventive treatment in pregnancy and seasonal malaria chemoprevention); extraordinary interventions, including presumptive treatment of fever and mass drug administration, as well as the assurance of continued access to and use of recommended insecticide-treated mosquito nets; and implementation of core vector-control activities to the greatest extent possible (current and planned insecticide-treated nets campaigns should go ahead, if at all possible). malaria prevention and treatment is even more important during the covid-19 pandemic than under normal circumstances. rapid diagnosis plays an important role in the outcome of both malaria and covid-19; thus, in addition to these recommendations, quick, easy, reliable and cheap diagnostics requiring minimal invasion must be performed, especially in cases of likely overlap with malaria and covid-19. this is the time for malaria-endemic countries challenged with the covid-19 pandemic to apply modern techniques including point-of-care tests such as the loop-mediated isothermal amplification assay for rapid detection of both plasmodium and sars-cov-2.13,14 meanwhile, health authorities should also centralise the management of covid-19 infected individuals so that populations suffering from other diseases (diabetes, high blood pressure, cardiovascular disorders, arthritis, etc.) could return to health facilities without fear of being infected. however, number of tests as well as the test centers for covid-19 detection should be multiplied in order to attend benefits of early case identification. acknowledgements we are thankful to dr mamane nassirou garba for his english review and all the staff of the parasitology and mycology laboratory at le dantec university of dakar, especially to prof. daouda ndiaye. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions k.d. was responsible for the conception and design of the study and drafting the article. m.a.d. reviewed the article. all authors approved the final version to be submitted. ethical considerations the work did not involve the use of human subjects or animal experiments. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references world health organization (who). coronavirus disease (covid-19): situation report – 162. who; 2020 [cited 2020 jun 30]. available from: www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports gangneux j-p, bougnoux m-e, dannaoui e, cornet m, zahar jr. invasive fungal diseases during covid-19: we should be prepared. j mycol méd. 2020;30(2):100971. https://doi.org/10.1016/j.mycmed.2020.100971 programme national de lutte contre le paludisme (pnlp). sénégal – plan stratégique de lutte contre le paludisme 2016–2020 [homepage on the internet]. pnlp; 2015 [cited 2020 jun 30]. http://www.pnlp.sn/wp-content/uploads/2016/08/pnlp_psn_vff_03-02-2016.pdf agence nationale de la statistique et de la démographie/service régional de la statistique et de la démographie de dakar (ansd/srsd). situation économique et sociale régionale – 2012 [homepage on the internet]. ansd/srsd; 2015 [cited 2020 jun 30]. available from: http://www.ansd.sn/ressources/ses/ses_dakar-2012.pdf world health organization (who). tailoring malaria interventions in the covid-19 response [homepage on the internet]. who; 2020 [cited 2020 jun 30]. available from: http://www.who.int/malaria wang j, xu c, wong yk, et al. preparedness is essential for malaria-endemic regions during the covid-19 pandemic. lancet. 2020;395(10230):1094–1096. https://doi.org/10.1016/s0140-6736(20)30561-4 chanda-kapata p, kapata n. covid-19 and malaria: a symptom screening challenge for malaria endemic countries. int j infect dis. 2020;94:151–153. https://doi.org/10.1016/j.ijid.2020.04.007 seydi m, lakhe na. profil épidémiologique, clinique et évolution des cas de covid-19 au sénégal. ministère de la santé et de l’action sociale [homepage on the internet]. 2020 [cited 2020 jun 30]. available from: http://familyplanning2020.org/sites/default/files/resources/covid/covid-19%20afrehealth%20webinar%20talk_cmit_dakar_senegal_pr.%20seydi_dr.%20lakhe_march19_2020_french%20version.pdf ministère de la santé et de l’action sociale (msas). message à la nation de sem le président sall levée de l’état d’urgence instauré dans le cadre de la lutte contre la maladie à coronavirus covid-19 [homepage on the internet]. msas; 2020 [cited 2020 jun 30]. available from: http://www.sante.gouv.sn/sites/default/files/discours%20du%20pr.pdf kafai nm, odom john ar. malaria in children. infect dis clin n am. 2018;32(1):189–200. https://doi.org/10.1016/j.idc.2017.10.008 diallo ma, diongue k, ndiaye m, et al. evaluation of carestart™ malaria hrp2/pldh (pf/pan) combo test in a malaria low transmission region of senegal. malar j. 2017;16:328. https://doi.org/10.1186/s12936-017-1980-z programme national de lutte contre le paludisme (pnlp). bulletin épidémiologique annuel 2017 du paludisme au sénégal [homepage on the internet]. pnlp; 2018 [cited 2020 jun 30]. available from: https://fr.africacheck.org/wp-content/uploads/2018/04/senegal-paludisme-bulletin-annuel-2017-pnlp.pdf lucchi nw, gaye m, diallo ma, et al. evaluation of the illumigene malaria lamp: a robust molecular diagnostic tool for malaria parasites. sci rep. 2016;6:36808. https://doi.org/10.1038/srep36808 l’helgouach n, champigneux p, schneider fs, et al. easycov: lamp based rapid detection of sars-cov-2 in saliva [homepage on the internet]. 2020 [cited 2020 jun 30]. https://www.skillcell-alcen.com/sites/skillcell-alcen/files/pdf/press/clinical-assessment-of-easycov-v10_1.pdf abstract background methods results discussion acknowledgements references about the author(s) dinkorma t. ouologuem malaria research and training center, department of epidemiology of parasitic diseases, university of science, techniques and technologies of bamako, bamako, mali cheick o. kone malaria research and training center, department of epidemiology of parasitic diseases, university of science, techniques and technologies of bamako, bamako, mali bakary fofana malaria research and training center, department of epidemiology of parasitic diseases, university of science, techniques and technologies of bamako, bamako, mali bakary sidibe malaria research and training center, department of epidemiology of parasitic diseases, university of science, techniques and technologies of bamako, bamako, mali amadou h. togo malaria research and training center, department of epidemiology of parasitic diseases, university of science, techniques and technologies of bamako, bamako, mali demba dembele malaria research and training center, department of epidemiology of parasitic diseases, university of science, techniques and technologies of bamako, bamako, mali sekou toure malaria research and training center, department of epidemiology of parasitic diseases, university of science, techniques and technologies of bamako, bamako, mali sekou koumare malaria research and training center, department of epidemiology of parasitic diseases, university of science, techniques and technologies of bamako, bamako, mali ousmane toure malaria research and training center, department of epidemiology of parasitic diseases, university of science, techniques and technologies of bamako, bamako, mali issaka sagara malaria research and training center, department of epidemiology of parasitic diseases, university of science, techniques and technologies of bamako, bamako, mali abdoulaye toure malaria research and training center, department of epidemiology of parasitic diseases, university of science, techniques and technologies of bamako, bamako, mali adama dao malaria research and training center, department of epidemiology of parasitic diseases, university of science, techniques and technologies of bamako, bamako, mali ogobara k. doumbo malaria research and training center, department of epidemiology of parasitic diseases, university of science, techniques and technologies of bamako, bamako, mali note: †, 1956-2018 abdoulaye a. djimde malaria research and training center, department of epidemiology of parasitic diseases, university of science, techniques and technologies of bamako, bamako, mali citation ouologuem dt, kone co, fofana b, et al. differential infectivity of gametocytes after artemisinin-based combination therapy of uncomplicated falciparum malaria. afr j lab med. 2018;7(2), a784. https://doi.org/10.4102/ajlm.v7i2.784 original research differential infectivity of gametocytes after artemisinin-based combination therapy of uncomplicated falciparum malaria dinkorma t. ouologuem, cheick o. kone, bakary fofana, bakary sidibe, amadou h. togo, demba dembele, sekou toure, sekou koumare, ousmane toure, issaka sagara, abdoulaye toure, adama dao, ogobara k. doumbo, abdoulaye a. djimde received: 02 feb. 2018; accepted: 30 sept. 2018; published: 06 dec. 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: most malaria-endemic countries use artemisinin-based combination therapy (act) as their first-line treatment. acts are known to be highly effective on asexual stages of the malaria parasite. malaria transmission and the spread of resistant parasites depend on the infectivity of gametocytes. the effect of the current act regimens on gametocyte infectivity is unclear. objectives: this study aimed to determine the infectivity of gametocytes to anopheles gambiae following act treatment in the field. methods: during a randomised controlled trial in bougoula-hameau, mali, conducted from july 2005 to july 2007, volunteers with uncomplicated malaria were randomised to receive artemether-lumefantrine, artesunate-amodiaquine, or artesunate-sulfadoxine/pyrimethamine. volunteers were followed for 28 days, and gametocyte carriage was assessed. direct skin feeding assays were performed on gametocyte carriers before and after act administration. results: following artemether-lumefantrine treatment, gametocyte carriage decreased steadily from day 0 to day 21 post-treatment initiation. in contrast, for the artesunate-amodiaquine and artesunate-sulfadoxine/pyrimethamine arms, gametocyte carriage increased on day 3 and remained constant until day 7 before decreasing afterward. mosquito feeding assays showed that artemether-lumefantrine and artesunate-amodiaquine significantly increased gametocyte infectivity to anopheles gambiae sensu lato (s.l.) (p < 10−4), whereas artesunate-sulfadoxine/pyrimethamine decreased gametocyte infectivity in this setting (p = 0.03). conclusion: different act regimens could lead to gametocyte populations with different capacity to infect the anopheles vector. frequent assessment of the effect of antimalarials on gametocytogenesis and gametocyte infectivity may be required for the full assessment of treatment efficacy, the potential for spread of drug resistance and malaria transmission in the field. background malaria is still a major public health problem in numerous parts of the world. malaria still affects 216 000 million individuals each year with 445 000 deaths worldwide.1 the global agenda for malaria elimination and eradication may never succeed without a thorough understanding of gametocyte biology and the true effect of the various interventions on malaria transmission. gametocyte development and viability are essential for the perpetuation of plasmodium life cycle by enabling both transmission from the human host to the mosquito vector2,3 and the spread of resistant parasites. plasmodium gametocyte development within the human host is a tedious process involving the differentiation from asexual to sexual forms to accommodate metabolic requirements, environmental changes and sexual reproduction.4,5 plasmodium gametocytes are conventionally classified into five distinct stages (stages i–v) but only the immature stage i gametocytes and the mature stage v gametocytes are detectable in the peripheral blood of a malaria-infected patient.6,7 the other stages (stage ii, iii, iv) are sequestered in the bone marrow and possibly other internal organs.6,8,9 gametocytes do not cause any symptoms in the infected human host, but the presence of competent circulating gametocytes and their duration in the bloodstream, which varies from 3 to 4 weeks,10 are directly responsible for malaria parasite transmission to the anopheles vector.11 however, gametocytogenesis and gametocyte transmission to the mosquito vector constitute a population bottleneck in the plasmodium life cycle as only a minute number of parasites enter the mosquito bloodmeal and gut.12 monitoring the density and infectiousness of circulating gametocytes is necessary for a better assessment of malaria transmission in endemic areas. gametocyte development within the human host is influenced by various factors, including host and parasite genetic factors, immune response, mosquitoes’ gut microbiota and the exposure to antimalarial drugs.13,14,15 several clinical and in vitro studies reveal that most antimalarial drugs currently in use can promote or impair gametocytogenesis and to some extent affect sexual reproduction within the mosquito vector.16 the 8-aminoquinoline primaquine is presently the only clinically used antimalarial drug displaying potent activity against all plasmodium species and gametocyte stages,16 but its side effects on glucose-6-phosphate dehydrogenase-deficient individuals hinder its use in large-scale elimination strategies.17,18 the 4-aminoquinoline chloroquine was shown to increase the production of fully competent gametocytes both in vitro and in vivo.19,20 in contrast atovaquone, artemisinin and the antifolates (sulfadoxine and pyrimethamine) have been shown to impair gametocyte development and infectivity.20,21,22,23,24 the gametocyte developmental stages affected by antimalarial drugs is poorly understood. the antimalarial treatment represents a stress factor that triggers differentiation of the asexual form into the gametocytes.25,26 this process may be more prevalent with drug-resistant parasites compared to sensitive ones.27 hence, the selective pressure exerted by the antimalarial drugs on the parasite may contribute to the spread of resistant parasites through the development and transmission of drug-resistant gametocytes.28 therefore it becomes essential to assess the emergence of resistant strains and the impact of treatment on gametocytogenesis and gametocyte infectivity. artemisinin-based combination therapies (act) are recommended in most malaria-endemic countries29 with the expected benefit to reduce gametocyte carriage.30 indeed, the fast killing action of artemisinin and derivatives on asexual parasites results in the decrease of circulating stage v gametocytes.30 artesunate has been reported to reduce post-treatment transmission of gametocyte to anopheles mosquitoes but does not abolish gametocyte infectivity entirely.31 with the increased interest in malaria elimination, understanding the impact of act regimens and other antimalarial drugs with different pharmacodynamic properties on gametocyte development and transmission becomes a key issue. here, we report an in vivo assessment of the impact of artemether-lumefantrine, artesunate-amodiaquine and artesunate-sulfadoxine-pyrimethamine on circulating gametocytes density and their infectivity. methods ethical considerations the protocol (nct00452907 on clinicaltrials.gov) was reviewed and approved by the ethical committee of the faculty of medicine, pharmacy, and dentistry, university of bamako (no 05-20 dated 22 june 2005). each participant (or legal guardian for minors) gave fully informed written consent before enrolment. study sites this study was conducted in bougoula-hameau, a peri-urban village of approximately 7000 people located near the city of sikasso in southern mali. plasmodium falciparum is hyperendemic with seasonal peaks in this village. parasitemia prevalence rates range from 40% to 50% during the dry season (january–april) and 70% to 85% during the rainy season (may–december).32 approximately 10% – 20% of the local population are gametocyte positive with an average gametocyte density of 23 gametocytes/µl.33 the main malaria vectors in the sikasso region are anopheles gambiae and anopheles funestus with a sporozoite rate of 6.4% at the end of the rainy season and an entomological inoculation rate (eir) of 0.032 infected bites per person per night.34 study design, volunteer follow-up and sample collection this is a sub-study of a previously published randomised controlled clinical trial, which was conducted from july 2005 to july 2007 to compare the efficacy of three act regimens: artemether + lumefantrine (coartem®, novartis, basel, switzerland), artesunate + amodiaquine (arsucam®, sanofi-aventis, paris, france), and artesunate (sanofi-aventis, paris, france) + sulfadoxine-pyrimethamine (fansidar®, roche, burlington, north carolina, united states).35 briefly, patients aged 6 months and above were enrolled in the clinical study if they satisfied the following inclusion criteria: weighed ≥ 35 kg, resided in the study village, were able to receive oral treatment, had an axillary temperature ≥ 37.5°c, and had plasmodium sp. infection with a parasite density between 2,000 and 200,000 asexual forms per microlitre of blood. as described previously by sagara et al.,35 780 volunteers were enrolled and 260 were randomly assigned to one of the three treatment arms and drug efficacy was assessed according to the world health organization 2003 protocols.36 briefly, blood samples were collected on days 1, 2, 3, 7, 14, 21, 28 and any day of recurrent illness. smears were made, giemsa-stained and read on site for asexual parasites and gametocytes quantification. parasite count was performed against 300 leucocytes and gametocyte count was performed against 1000 leucocytes. to evaluate gametocyte carriage following act administration, all participants who were gametocyte carriers at day 0, 1, 2, 3, 7, 14, 21 and 28 were selected. direct skin feeding procedures gametocyte infectivity was defined as the presence of oocyst in the midgut of the anopheles mosquito 8 days after a gametocyte containing blood meal. the infectivity of circulating gametocytes following treatment was evaluated by direct mosquito feeding experiments performed on gametocyte carriers before and after oral act administration on day 0 and days 3, 7, 14, 21 or 28. the test group was recruited among patients randomised in the main study,35 while the control group was recruited among patients presenting with malaria symptoms, who were gametocyte positive by microscopy but were not included in the main study. gametocytes carriers from the control group did not receive any antimalarial drugs at the time of infectivity assessment. to avoid repeated exposure to direct skin feeding, each volunteer underwent direct skin feeds only once during the entire study. in addition, the infectivity experiments were only performed on those days when adequate numbers of 3–5 days old mosquitoes were available to the team. wild female anopheles gambiae s.l. collected from the same study site were allowed to lay eggs. mosquitoes were kept in semi-natural conditions in field insectaries set up in the village. for each feeding experiment, 3–5 days old offspring (f1) reared in the insectaries were used. up to 60 f1 female mosquitoes were starved for about 12 h and held in 2 small screened-cups containing 30 mosquitoes each. they were then allowed to blood-feed for 5–10 min on the leg of a 6–18 years old consenting volunteer. after feeding, unfed mosquitoes were removed the same day, and only fed mosquitoes were kept in the insectaries as described above. 8 days post feeding, a group of at least 15 mosquitoes per carrier were dissected, their midguts pulled out in 0.5% mercurochrome for oocyst detection and quantification. oocyst numbers, abdomen stages, date and other observations were recorded on data sheets. oocysts were counted under light microscopes 10 times for each mosquito. statistical analysis for the analysis of the direct skin feeding experiment, feeding experiments for days 3–28 were lumped together in each treatment arm. to calculate the oocyst prevalence, for each treatment arm, the number of positive mosquitoes was divided by the total number of mosquitoes dissected for that treatment arm). the oocyst positivity for each act regimen was compared to that of the control group using the chi-square test. all data were analysed and reported using stata software version 14.0 (statacorp. 2015, college station, texas, united states). results evolution of gametocyte carriage and gametocyte density following artemisinin-based combination therapy treatment a total of 129 volunteers were gametocyte carriers in the artemether-lumefantrine arm, 123 in the artesunate-amodiaquine arm and 124 in the artesunate-sulfadoxine-pyrimethamine arm. following treatment initiation, gametocyte carriage significantly increased in the artesunate-amodiaquine and artesunate-sulfadoxine-pyrimethamine arms (figure 1). from day 0 to day 3, gametocyte carriage rose from 6.1% to 10.2% in the artesunate-amodiaquine arm (p = 0.005) and from 7.0% to 9.8% in the artesunate-sulfadoxine-pyrimethamine arm (p = 0.02). overall, the prevalence of gametocyte carriage in the artemether-lumefantrine arm steadily decreased from day 0 to day 28, while for the artesunate-amodiaquine and artesunate-sulfadoxine-pyrimethamine arms the prevalence of gametocyte carriage increased from day 0 to day 7 before declining (figure 1). figure 1: gametocytemia carriage evolution by treatment arm. infectivity of gametocytes before and after treatment with artemisinin-based combination therapy regimens overall 21 consenting volunteers in the control arm, 8 in the artemether-lumefantrine arm, 6 in the artesunate-amodiaquine arm, and 15 in the artesunate-sulfadoxine-pyrimethamine arm underwent direct skin feeding. the mean age of volunteers was comparable between groups (table 1). table 1: characteristics of the volunteers subjected to direct skin feeding assays. overall we dissected 698, 253, 174, and 602 mosquitoes to measure oocyst positivity at baseline, post-artemether-lumefantrine, post-artesunate-amodiaquine, and post-artesunate-sulfadoxine-pyrimethamine treatment, respectively (table 2). table 2: mosquitoes dissected per treatment arm and days of feeding. the oocyst positivity rate in the control group was 11.7% (82/698) (figure 2). for volunteers treated with artemether-lumefantrine, the overall positivity rate was significantly higher than that of the control group (11.7%, n = 698 vs 30.0%, n = 253; p < 10−4) (figure 2). likewise the oocyst positivity rate following artesunate-amodiaquine treatment was significantly higher than that of the control group (11.7%, n = 698 vs 40.2% n = 174; p < 10−4) (figure 2). in contrast, the oocyst positivity rate following artesunate-sulfadoxine-pyrimethamine treatment was significantly lower than that of the control group (11.7%, n = 698 vs 7.9%, n = 602; p = 0.03) (figure 2). figure 2: oocyst positivity rate by treatment arm. discussion the goal of this study was to determine the impact of different act regimens on plasmodium falciparum gametocyte carriage, density and infectivity to anopheles gambiae s.l. following artemether-lumefantrine, artesunate-amodiaquine and artesunate-sulfadoxine-pyrimethamine treatment. we measured p. falciparum gametocyte density of consenting volunteers suffering from acute uncomplicated malaria and gametocyte infectivity to anopheles mosquitoes before and after act administration. from the direct skin feeding experiments of this study, we show that post-artemether-lumefantrine and post-artesunate-amodiaquine treatment gametocytes are more infectious to anopheles gambiae s.l. than post-artesunate-sulfadoxine-pyrimethamine treatment gametocytes or non-drug treated controls. since artemisinin derivatives, including artesunate and artemether, are rapidly metabolised in vivo into dihydroartemisinin, the observed differences in infectivity could be dependent on the partner drugs (i.e. lumefantrine, amodiaquine and sulfadoxine-pyrimethamine). however, one cannot rule out the effect of differences in artemisinin derivative pharmacokinetics and pharmacodynamics between regimens. co-exposure to artemisinin derivative and the partner drugs albeit for a short period could impact gametocyte biology differently from any of the individual drugs. in addition, drug administration timing differs between artemether-lumefantrine and artesunate-amodiaquine or artesunate-sulfadoxine-pyrimethamine. while artemether-lumefantrine was given twice daily for 3 days, artesunate-amodiaquine and artesunate-sulfadoxine-pyrimethamine were administered once daily for 3 days. a number of studies have investigated the effect of antimalarial drugs on gametocyte density and infectivity.19,31 beavogui et al. revealed that sulfadoxine-pyrimethamine considerably increased gametocyte carriage in the population but the infectivity of these circulating gametocytes was very low.22 further investigation by kone et al. showed that the low infectivity following sulfadoxine-pyrimethamine treatment was due to pyrimethamine, which is known to prevent male gametocyte exflagellation.23 in addition, 4-aminoquinoline chloroquine, which is chemically related to amodiaquine, has been shown to induce the production of fully infective gametocytes both in vitro and in vivo.16 these studies support the observed decrease of infectivity with artesunate-sulfadoxine-pyrimethamine treatment and the increase of infectivity with artesunate-amodiaquine seen in this study. although gametocyte infectivity was different between the tested act regimens, the small number of carriers tested may be a limitation of this study. additional infectivity studies with higher numbers of gametocyte carriers need to be conducted to further investigate these findings. in addition, the mechanisms involved in antimalarial drugs and gametocyte biology ought to be thoroughly studied. to investigate gametocyte dynamics in the peripheral blood, gametocytes carriers were included in this study.35 baseline gametocyte carriage was significantly higher in the artemether-lumefantrine arm compared to both artesunate-amodiaquine and artesunate-sulfadoxine-pyrimethamine. that observation may have occurred by chance as the study was a randomised controlled trial; therefore this could not be attributed to a selection bias.35 there was no difference in gametocyte density between the treatment arms at baseline. gametocyte carriage significantly decreased in all treatment arms between day 0 and day 28. this result confirms numerous previous findings.30 however, while gametocyte prevalence in the artemether-lumefantrine arm decreased steadily from day 0 to day 28, there was a significant increase in the artesunate-amodiaquine and artesunate-sulfadoxine-pyrimethamine arms from baseline to day 3 with a plateau until day 7. this rise after act administration corroborates previous work38,39 and could be a consequence of the stress induced by the treatment.26,40,41 these data suggest that artemether-lumefantrine may affect gametocytogenesis differently from artesunate-amodiaquine or artesunate-sulfadoxine-pyrimethamine.24,42,43 conversely, the dosing schedule of artemether-lumefantrine, which is taken every 8 h instead of once daily as for artesunate-amodiaquine and artesunate-sulfadoxine-pyrimethamine, could also play a role in the above observations. we show that artesunate-amodiaquine and artemether-lumefantrine decreased gametocyte carriage in the treated population while both acts increased gametocyte infectivity to the mosquitoes. the effect of these acts in reducing gametocyte carriage can be a direct result of their active and fast killing action on asexual forms, leaving them no or little chance to differentiate into gametocytes.30 however, the few that manage to differentiate into gametocytes appear to be well fit for infecting the mosquitoes, hence their increased infectivity. conversely, sulfadoxine-pyrimethamine sharply increases gametocytes but those gametocytes appear to be less fit for infecting the mosquitoes.22, 23 pyrimethamine was also shown to hamper sporogonic development of p. falciparum in the anopheles mosquitoes.44 these could explain the decrease in infectivity of post artesunate-sulfadoxine-pyrimethamine gametocytes. gametocytes were detected and quantified by light microscopy, which is an important limitation of this study. thus, gametocyte carriage and gametocyte density are likely to be underestimated with our approach. the use of molecular tools7,45,46,47 and gametocyte quantification after purification38 could have yielded higher proportions of gametocyte carriers and gametocyte density. we also cannot exclude the possible modulation of the mosquitoes’ gut microbiota by the acts used in this study. the anopheles gut microbiota is known to influence the sporogonic cycle15 and some drugs, such as antibiotics, have been shown to change the composition of mosquitoes’ gut resident microbes.48 conclusion antimalarial drugs influence gametocytogenesis and their impact on gametocyte density and viability are likely to differ from one combination therapy regimen to another. evaluation of transmission potential in malaria-endemic areas requires more studies assessing the influence of current antimalarial treatment on gametocyte development and clearance in vivo, and the infectivity of post-treatment gametocytes. understanding the potential impact of antimalarial drugs on the spread of resistant strains and malaria transmission will require a fine assessment of their effects on gametocyte biology and the mechanisms involved. acknowledgements we thank all the volunteers who participated in this study, the local guides of bougoula-hameau, the data management staff of the malaria research and training center, university of science, techniques and technologies of bamako, mali, for managing the clinical data and the staff of the regional hospital of sikasso, mali, for their great collaboration during the study. we are indebted to m. siaka traore who allowed us to use one his huts as field insectaries. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support this work was supported by the european and developing countries clinical trial partnership (edctp) fellowship grant (2004.2.c.f1 to a.d.) and by sanofi (arten-l-00848). authors’ contributions d.t.o. conducted the data analysis and data interpretation, and drafted the manuscript. s.t., s.k., b.f., b.s., a.h.t., and d.d. contributed to the clinical studies. c.o.k., a.d., and a.t. contributed to the entomological studies. o.t. and i.s. contributed to the study design and the statistical analysis. o.k.d. 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discussion acknowledgements references about the author(s) cathy robinson international consulting services, louisiana state university alexandria, alexandria, louisiana, united states james johnson school of health sciences, central michigan university, mount pleasant, michigan, united states katy yao division of global hiv and tb program, centers for disease control and prevention, atlanta, georgia, united states hien bui centers for disease control and prevention, hanoi, vietnam citation robinson c, johnson j, yao k, bui h. critical success factors for vietnamese laboratories striving to implement quality management systems. afr j lab med. 2020;9(1), a937. https://doi.org/10.4102/ajlm.v9i1.937 brief report critical success factors for vietnamese laboratories striving to implement quality management systems cathy robinson, james johnson, katy yao, hien bui received: 05 dec. 2020; accepted: 27 aug. 2020; published: 18 dec. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract accurate laboratory reporting is crucial to patient diagnosis and treatment. this study identified critical success factors (csf) for implementing a laboratory quality management system (qms). this descriptive research used qualitative and quantitative methods to collect and analyze data from laboratory managers and staff employed in vietnamese hospital laboratories implementing a qms. the top five csfs identified were: (1) staff qms knowledge, (2) manager leadership, (3) staff commitment, (4) mentorship, and (5) hospital administration support. identifying csfs is critical to successful planning and implementation of qms. keywords: critical success factors; medical laboratories implementing qmss; laboratories earning iso 15189 accreditation; quality management system helps laboratories improve quality; accuracy; reliability of patient results. introduction medical laboratories provide critical services which are used by physicians to accurately diagnose, treat and monitor patient health. grycotis, the infectious advisor, emphasises the value of laboratory test results to clinicians in making an accurate diagnosis and monitor treatment regimes.1 equally important is the detrimental effect inaccurate results have including wrong diagnosis, wrong treatment, and patient death. likewise, nkengasong et al., emphasise that quality laboratory systems are needed to achieve the millennial development goals for health and meet universal access for treatment of hiv/aids, tuberculosis and malaria.2 similarly, the international standardization organization (iso) 15189 accreditation is recognised as the gold standard for measuring laboratory quality globally.3 one pathway to attaining this accreditation is by implementing a quality management system (qms). in addition to a qms training programme, the world health organization developed a checklist entitled stepwise laboratory quality improvement process towards accreditation to measure quality improvement within laboratories and provide continued recognition and motivation of staff to continue and sustain improvements.4 an international effort was fielded in developing countries to educate and train laboratory management and staff on improving the accuracy and reliability of laboratory results. qms training is designed to teach participants how to identify deficiencies in their laboratories, design improvement projects to fill those gaps, and enact standard practices and processes into daily laboratory practices. following the training, managers and staff return to their respective laboratories to begin improvement. intermittent self-assessments serve to monitor the improvement process, show improvements and motivate to continue improvement. several previous studies5,6,7 have evaluated the outcome of these training programmes on laboratory quality by comparing laboratory assessments before and after qms implementation. while this is a good first step, quantitative statistics do not identify ‘why’ a particular standard was or was not met. adding a qualitative component allows for the identification of ‘why’ qms implementation assessment scores often showed wide variability. in 1979, rockart worked with executives in manufacturing organisations to develop a set of factors aimed to guide goal development which proved to significantly contribute to project success. rockart referred to these factors as critical success factors (csfs).8 the primary benefit csfs offer any organisation is the ability to focus organisation efforts for project success. however, there is a noticeable lack of published literature identifying csfs for success in implementing qms projects in medical laboratories. this study aimed to identify csfs for medical laboratories implementing a qms project and ‘why’ a laboratory’s score may or may not improve over time. methods ethical considerations this research received approval from the institutional review board at central michigan university. this activity was reviewed per the centers for disease control and prevention (cdc) human research protection procedures and determined to be a non-research, public health programme activity. the vietnam administration of medical services agreed to this study and wrote letter no. 1506/kcb-qlcl to each hospital granting data collection permission to the researcher. written informed consent was obtained from all participants. results were reported in aggregate form only. study participants the vietnam administration for medical services assisted in selecting four laboratories (three city level and one district level, coded as h1–h4) based on their managers’ and staffs’ willingness to share their experiences while implementing a qms project. each laboratory reported their various stages of implementation at the time of the study (table 1). shi’s9 convenience sampling process was utilised to randomly select participants from each of the laboratories. participants included laboratory managers and staff; participation was voluntary. eleven participants from each lab (n = 44) completed the demographic survey and interview questions. table 1: vietnamese hospitals participating in this study (2017). study design the study was descriptive and employed a mixed design utilising qualitative and quantitative methods. qualitative data were collected from staff employed in the study’s laboratories currently implementing a qms project. qualitative data collected from responses to the semi-structured interview provided participant insight to answer ‘why’ their laboratory scores did or did not improve over time. during the interview, participants were asked to list the top five factors they felt were most important in meeting qms project goals and improving scores. for added clarity, each was asked to define the factors they listed. a quantitative demographic survey was used to gather participant data such as age, gender and education levels. with no previous csf studies found related to qms projects and medical laboratories, qms experts from three countries outside of vietnam served as benchmark panellists. benchmark panel of laboratory quality management system experts to validate this study’s findings, a panel of three experts from kenya, tanzania and ukraine agreed to serve as benchmark experts and review the vietnam study findings. each was a practising medical laboratory scientist experienced in the subject matter, that is, laboratory quality in resource-limited countries, qms, training and stepwise laboratory quality improvement process towards accreditation checklist. the expert panellists endorsed the qms implementation methods used in the vietnamese study as those similarly used in their respective countries. statistical analysis the researchers used content analysis10 to review and sort all responses (220 listed factors) into exhaustive and mutually exclusive categories. ten categories were identified. after identifying the content categories, the researcher and assistants sorted each of the factors into one of the content categories. cohen’s kappa statistic was used to measure inter-coder reliability between the researchers. this statistic provides a quantitative measure of reliability for two or more raters coding or sorting the same thing, corrected for how often the raters may agree by chance. applying frequency percentages, the top five categories were identified (table 2). though not the aim of the study, barriers were also identified using the same frequency percentage calculations. to look for bias between participants’ interview responses and demographic survey results, the chi-square test was applied. the same statistical analyses were applied to the data collected from the three expert panellists. table 2: top five critical success factor categories identified during the data content analysis. results and discussion applying cohen’s kappa statistic, reliability values were greater than 0.85, indicating excellent reliability between the researchers in coding and sorting the csfs.11 likewise, the chi-square test found no relevant bias between the demographic responses and the factors listed by the participants. five top csfs were identified from the vietnamese participants’ responses via content analysis by the researchers.12 the strength of this study lies in the close alignment of the identified top five csfs by the researchers and the expert panel after the analysis of participants’ responses. the top five success factors identified from the vietnamese study and the expert panellists were identical although the individual rankings varied between the top five positions (table 3). table 3: comparative ranking of critical success factors between the vietnam study and expert panel. the number one csf identified was staff knowledge of qms. specifically, all groups felt strongly that continuing education was crucial to ensuring current and new staff received qms training and skills. staff participants reported they wanted to improve the quality of their laboratories, but felt they lacked qms knowledge to engage in the improvement processes. qms knowledge included specific steps to follow when implementing a new task, why the specific task was important to test result accuracy and the importance of quality control monitoring. although qms was defined in the training, staff felt frequent reminders explaining the qms concept, how and why qms would improve their laboratory’s quality, and the benefit to patients would be beneficial and motivational. without continuing education, participants reported staff turnover often left the laboratory without knowledgeable staff to continue the implementation process. laboratory manager leadership was ranked as csf number two by the study participants, whereas the expert panel ranked laboratory manager leadership as csf number four. staff commitment to the change process was the third csf by the study participants; however, with the experts its ranking varied with expert #3 ranking it as third, and expert #1 and expert #2 ranking it as second. mentorship, as a csf, was in varying positions between all groups. the variation in ranking may be due to many types of mentorship options. expert #1 specifically listed embedded mentorship, whereas the others simply listed ‘mentorship’. often, due to time constraints with both the laboratory staff and mentor, email communication emerged as a valuable part of the mentorship package. discussing the mentorship experience at their laboratories, study participants agreed on the value of the mentorship without regard to whether the mentor visited the laboratory weekly or monthly. several participants commented that their mentors provided motivation and quick responses to their questions on qms implementation, often keeping them moving forward in implementation. without a mentor, resource staff indicated improvement projects stalled and were often discontinued. previous articles suggest that embedded and longer mentorships result in better outcomes for the laboratories, although this was not the finding in this study.5,6,7 hospital administration support received a wide range of scores. hospital support was defined as either financial support for qms resources or hospital-wide recognition for staff efforts. even without financial support, staff reported recognition from administrators and other hospital staff to be motivational. based on comments from the study participants, all agreed hospital support was critical but often lacking. this may account for the fifth-place ranking of hospital administration support. the panel of experts endorsed the implementation methods employed by the vietnamese laboratories. methods and processes included qms training, baseline assessments, development of a strategic plan and improvement projects to meet the strategic plan objectives (gaps). previous data reported quantitative outcome information based on individual laboratory assessments (stepwise laboratory quality improvement process towards accreditation scores in each of 12 sections and a total score). current and active involvement throughout the qms implementation process made these participants uniquely qualified to share their experiences as well as identify those factors considered critical to qms implementation success. combining qualitative data from this study with previous quantitative findings offers valuable information to laboratory managers explaining ‘why’ implementation or improvements often stalled and failed to move forward. one of the factors mentioned by both participants and experts is a perceived, or real, lack of knowledge by both laboratory managers and laboratory staff on qms. the expert panellists confirmed a similar knowledge deficit of qms principles and skills in staff attempting to implement unfamiliar processes in their laboratories. managers reported that they had received some management training but felt they would benefit from additional training related to staff orientation, conflict management, and quality control. study participants at all laboratories said the formation of teams greatly improved both morale and motivation. one interview question asked participants to list gaps identified during their project implementation as well as training they felt would strengthen their ability to successfully implement qms and continue moving towards iso 15189 accreditation (table 4). with significant changes in daily laboratory functions due to qms implementation, repetitive short training sessions for new and current staff seem reasonable to provide support and motivation until the changes become a normal daily routine. table 4: examples of staff reported knowledge gaps and requests for training (2017). limitations this study focused on a small number of laboratories located in vietnam. a larger study would offer more insight into csfs that affect medical laboratory qms success. asking study participants to make a forced ranking by only listing one csf for each position is another limitation. participants were unable to give equal rank to two csfs. conclusion this study is the first to identify csfs for the global medical laboratory sector. equipped with qms training, and now csfs, managers can more precisely focus their time, resources and implementation strategy to address staff needs and move their implementation plan forward. utilising csfs, implementation success should exhibit less variability between laboratories, and qms projects should begin to move forward. data from this study may strengthen qms success throughout vietnam and serve as a guide for managers in over 1000 laboratories in low-income and middle-income countries implementing qms.4,13 laboratory managers and staff considering implementation of a qms as a pathway to improving quality aspects of their laboratory would benefit from the results of this study. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions c.r. conducted research and wrote the article and manuscript for dissertation. j.j. was the dissertation chair and mentor and assisted with the manuscript. h.b. and k.y. were the subject matter experts and assisted with the manuscript. sources of support the strengthening laboratory management towards accreditation (slmta) programme was supported by the president’s emergency plan for aids relief through the centers for disease control and prevention (cdc). data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the findings and 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albany, ny: delmar publishers; 1997. neuendorf k. the content analysis guidebook online-cleveland state [homepage on the internet]. 2002. available from: http://academic.csuhio.edu/kneuendorf/content landis j, koch g. march. biometrics [serial online]. 1977;3:169. available from: https://www.jstor.org/stable/25293106 robinson cd. a multi case analysis of critical success factor in vietnam laboratories implementing quality management systems to earn international accreditation [dissertation]. mount pleasant, mi: central michigan university; 2018. iso 15189:2012. retrieved from: http://www.iso.org/obp/ui/#iso:15189:ed-3-v2:en abstract introduction methods results discussion acknowledgements references about the author(s) promise c. nwaejigh department of medical laboratory science, school of allied health sciences, babcock university, ilishan-remo, nigeria maria o. ebesunun department of medical laboratory science, faculty of basic medical sciences, olabisi onabanjo university, sagamu, nigeria stephen s. udofia department of medical laboratory science, school of allied health sciences, babcock university, ilishan-remo, nigeria adebusola a. shakunle department of obstetrics and gynaecology, lagos state university college of medicine, ikeja, nigeria citation nwaejigh pc, ebesunun mo, udofia ss, shakunle aa. apolipoproteins and high-density lipoprotein phospholipids as indicators of atherosclerotic cardiovascular disease in nigeria. afr j lab med. 2025;14(1), a2942. https://doi.org/10.4102/ajlm.v14i1.2942 note: additional supporting information is available for download alongside the online version of this article as online supplementary document 1. original research apolipoproteins and high-density lipoprotein phospholipids as indicators of atherosclerotic cardiovascular disease in nigeria promise c. nwaejigh, maria o. ebesunun, stephen s. udofia, adebusola a. shakunle received: 15 july 2025; accepted: 05 nov. 2025; published: 17 dec. 2025 copyright: © 2025. the author(s). licensee: aosis. this work is licensed under the creative commons attribution 4.0 international (cc by 4.0) license (https://creativecommons.org/licenses/by/4.0/). abstract background: altered apolipoproteins and high-density lipoprotein (hdl) phospholipids are linked to premature atherosclerotic cardiovascular disease (ascvd). objective: this study investigated associations between plasma apolipoprotein a1, apolipoprotein b, hdl phospholipids, and ascvd risk in nigeria, assessing their potential as early diagnostic markers. methods: this cross-sectional case-control study was conducted from november 2021 to november 2022 at lagos state university teaching hospital in nigeria. atherosclerotic cardiovascular disease patients and healthy controls were randomly selected. the plasma apolipoprotein a1 and b levels were determined via a sandwich enzyme-linked immunosorbent assay, and the lipid profile was measured via spectrophotometry. statistical analyses included t-tests, analysis of variance, analysis of covariance, and pearson’s correlation. results: in total, 172 confirmed ascvd patients (mean age: 54.01 ± 8.70 years) and 55 healthy controls (mean age: 44.55 ± 11.60 years) were included in the analyses. compared with the control values, ascvd patients showed significantly elevated apolipoprotein b, apolipoprotein b/a1 ratio, atherogenic lipid indices, total cholesterol, low-density lipoprotein cholesterol, non-hdl cholesterol, and triglycerides (p ≤ 0.001). in contrast, plasma hdl phospholipids, apolipoprotein a1, and hdl cholesterol were markedly lower (p ≤ 0.001). conclusion: these findings indicate that altered apolipoproteins and hdl phospholipids are associated with premature ascvd risk, with the apolipoprotein b/a1 ratio emerging as a superior marker for disease stratification. what this study adds: this study identifies the apolipoprotein b/a1 ratio as a strong early marker of ascvd risk in nigeria. keywords: atherosclerotic cardiovascular disease; apolipoprotein a1; apolipoprotein b; apolipoprotein b/a1 ratio; hdl phospholipids; lipid profile. introduction atherosclerotic cardiovascular disease (ascvd) represents a significant health challenge globally, with a rising incidence in nigeria and other developing regions.1,2 high-density lipoprotein (hdl) and its associated apolipoproteins, namely apolipoprotein a1 (apo a1) and apolipoprotein b (apo b), along with hdl phospholipids, have garnered considerable attention for their intricate involvement in ascvd progression. although research has explored lipid dysregulation and atherogenesis extensively, most of these studies have focused predominantly on populations from western countries,3,4 and asian populations.5,6 however, it is imperative to recognise the unique genetic, environmental, cultural dietary habits and lifestyle factors that contribute to ascvd susceptibility in diverse ethnic groups, particularly african populations. nigeria, as a key part of africa, is experiencing a growing ascvd epidemic as a result of urbanisation, westernised diets and changing lifestyles.7,8 despite the increase in ascvd in nigeria and africa, comprehensive studies are scarce on the specific lipid profiles associated with atherosclerosis. apolipoprotein a1, the major protein component of hdl, is integral to the process of reverse cholesterol transport,9,10 exerting anti-inflammatory and antioxidant effects that confer protection against ascvd.11,12 in contrast, elevated levels of apo b, which is found predominantly on low-density lipoprotein particles, are associated strongly with increased ascvd risk,13,14 reflecting the proatherogenic properties of low-density lipoprotein cholesterol (ldlc).15 the apo b/a1 ratio is used increasingly to assess cardiovascular risk. a lower ratio indicates a better balance between atherogenic and protective lipoproteins, which correlates with a lower risk of ascvd.16 recent studies have also emphasised this ratio as a more accurate predictor of cardiovascular events than traditional lipid measurements.17 advances in genomics have revealed that genetic variations influence apo a1 and apo b levels, affecting ascvd.18,19,20 furthermore, the composition and functionality of hdl, particularly its phospholipid content, have garnered attention for their potential impact on ascvd risk. hdl phospholipids, comprising a diverse array of molecular species, contribute significantly to hdl functionality, modulating its anti-inflammatory, antioxidative, and vasoprotective properties.21,22 alterations in the composition and functionality of hdl phospholipids have been implicated in ascvd.23,24 higher levels of specific hdl phospholipids are linked to a lower risk of major adverse cardiovascular events, suggesting that they could enhance traditional lipid markers for better risk stratification.23,25 emerging evidence suggests that hdl phospholipid composition may be a cardiovascular risk biomarker.26 this study assessed atherogenic lipid profiles in nigerian patients with ascvd, with a specific focus on apolipoproteins and hdl phospholipids. this study examined plasma apo a1, apo b, total and hdl phospholipids, and lipid profiles in ascvd patients. methods ethical considerations this study was approved by the health research and ethics committee of lagos state university teaching hospital in nigeria (reference number: lrec/06/10/1706). before participation, all individuals provided written informed consent. all procedures followed the ethical principles of the 2024 declaration of helsinki. to ensure anonymity, a barcode system was employed, and all data were handled with strict confidentiality. the collected data were used solely for this study. study design this cross-sectional case-control study determined a sample size of 138 based on prevalence data from a previous regional study,27 which reported an 8% – 12% prevalence of cardiovascular disease in sub-saharan african countries.28 in total, 172 confirmed ascvd patients, aged 20 years to 64 years, were enrolled. participants were selected using a simple random sampling technique at the lagos state university teaching hospital, lagos, nigeria. we enrolled participants diagnosed with hypertensive heart disease with significant atherosclerosis, ischaemic heart disease (ihd), myocardial infarction (mi), ischaemic stroke, or congestive heart failure linked to prior ihd or mi. recruitment took place during routine outpatient visits at the cardiology clinic, lagos state university teaching hospital, lagos, nigeria, from november 2021 to november 2022. eligible participants were identified through consecutive clinic record reviews and were enrolled at most six months post-diagnosis, with clinical stability confirmed by no acute cardiovascular events (e.g. mi or stroke) in the prior three months, as verified by a cardiologist and medical records. the ihd group comprised patients with stable conditions, such as stable angina, without a documented mi. the mi group included those with a prior mi, defined per the 2012 universal definition of myocardial infarction,29 based on clinical signs of ischaemia, elevated cardiac biomarkers (e.g. troponin) and echocardiographic findings. to minimise confounding effects on lipid-related biomarkers, we excluded patients on lipid-lowering therapy at recruitment or within the prior six months, as well as those on hormonal therapy, pregnant women, or individuals who declined participation. in our setting, some patients were not on lipid-lowering therapy owing to clinical contraindications (e.g. statin intolerance) or non-adherence, verified through medical records and patient interviews. we also excluded patients with acute mi, stroke, or other cardiovascular events within three months before sampling, to focus on early-stage ascvd and to support our aim of evaluating plasma biomarkers for early detection. the control group consisted of 55 healthy volunteers with no history of chronic or degenerative diseases, recruited from relatives of patients and staff at the lagos state university teaching hospital. all participants completed a standardised, structured paper questionnaire (online supplementary questionnaire) to capture general characteristics, inclusion/exclusion criteria, and potential confounding factors that could influence study outcomes. control participants were further evaluated for clinical examination, and basic laboratory screening, including fasting plasma glucose, lipid profile, and blood pressure measurements, to confirm their apparently healthy status and exclude subclinical chronic or metabolic conditions blood sample collection blood samples from ascvd patients were collected within 0–6 months of diagnosis to capture early-stage disease, consistent with the aim of the study to evaluate apolipoprotein a1, apolipoprotein b, and hdl phospholipids as potential early diagnostic markers. approximately 7 ml of venous blood was drawn from each participant after an overnight fast (8 h – 12 h). samples were collected in potassium-ethylenediaminetetraacetic acid (1 mg/ml) tubes, maintained at 4 °c, and processed within two hours. plasma was separated by centrifugation at 3000 rpm for 5 min, aliquoted into labelled vials, and stored at –20 °c until analysis, conducted within 1–12 months. the use of potassium-ethylenediaminetetraacetic acid minimised lipid peroxidation and was appropriate for apolipoprotein and phospholipid assays. although minor effects on total cholesterol and ldlc might occur, consistent use across all samples ensured data comparability and analyte stability. biochemical analysis lipid profile parameters (total cholesterol, high-density lipoprotein cholesterol [hdlc] and triglycerides), were measured via commercial kits (randox laboratories ltd., crumlin, northern ireland, united kingdom). total cholesterol and hdlc (following the removal of all non-hdl lipid fractions with phosphotungstic acid and magnesium ions) were estimated using the methods of allain et al.30 triglycerides were measured via the method developed by bucolo and david.31 the calculation of ldlc was performed using the formula provided by friedewald, levy and fredrickson,32 selected for its validation, cost-effectiveness, and suitability in resource-limited settings such as sub-saharan africa, where direct methods (e.g. ultracentrifugation or the martin-hopkins equation) are often impractical. all samples had triglyceride levels below 4.52 mmol/l, confirming the validity of the equation. based on these measurements, we further evaluated derived lipid indices, including ldlc, non-hdlc, castelli’s risk indices (cri-i, cri-ii), atherogenic coefficient (ac), and atherogenic index of plasma (aip), using standard lipid panel measurements (total cholesterol [tc], triglycerides, hdlc). formulas and clinical relevance are provided in online supplementary table 1.32,33,34 table 1: general characteristics and biochemical parameters of study population at lagos state university teaching hospital, lagos, nigeria, between november 2021 and november 2022. plasma total phospholipids were analysed via the wako phospholipid c assay kit (fujifilm wako pure chemical industries, ltd., osaka, japan), which employs the n-ethyl-n-(2-hydroxy-3-sulfopropyl) 3,5-dimethoxy aniline sodium salt method.35 high-density lipoprotein phospholipids were assayed via this method after treatment of participants’ plasma with phosphotungstic acid and magnesium ions (to remove all non-hdl phospholipids fractions).36 the plasma levels of human apo a1 and apo b in the participants were determined using the sandwich enzyme-linked immunosorbent assay technique,37 with assay kits sourced from elabscience biotechnology limited, wuhan, china. analyses were performed on a bio-rad imark™ microplate reader (bio-rad laboratories, hercules, california, united states) at 450 nm. the system performs optical calibration and data processing automatically, following the manufacturer’s instructions. quality control sera were included in each batch to ensure analytical precision and reliability. sandwich enzyme-linked immunosorbent assay was selected for apolipoprotein analysis because of its high specificity, sensitivity and practical advantages over western blotting and radioactive immunoassays. spectrophotometry was chosen for lipid profiling because of its efficiency and cost-effectiveness, providing direct measurement of lipid fractions. chromatographic methods, although precise, require more resources and technical skill, making spectrophotometry a more accessible option for routine analysis in sub-saharan africa. data analysis statistical analyses were conducted using ibm spss statistics (version 21, ibm corp., armonk, new york, united states). data completeness and normality were verified, with continuous variables reported as means ± standard deviation, and categorical variables as frequencies (percentages). normality of lipid and apolipoprotein outcomes (apo a1, apo b, hdl phospholipids, tc, ldlc, hdlc, triglycerides, non-hdlc, cri-i, cri-ii, ac, aip) was assessed using shapiro–wilk tests and q–q plots (n = 227). non-normal variables were log-transformed. levene’s test confirmed homogeneity of variances (p > 0.05). group differences (ascvd vs control) were analysed using independent samples t-tests and one-way analysis of variance with bonferroni correction (p < 0.001). effect sizes were reported as cohen’s d, r2, and partial η2. separate analysis of covariance models, with age as the covariate, were used for each lipid and apolipoprotein outcome owing to its significant correlation with outcomes (p < 0.05, pearson’s correlation; online supplementary table 2) other potential confounders (sex, bmi, smoking, hypertension) were non-significant (p > 0.05) and excluded, along with diabetes and lipid-lowering medication use. analysis of covariance assumptions were met: linearity (via scatterplots and pearson’s correlation), homogeneity of regression slopes (group × covariate interaction, p > 0.05), normality of residuals (shapiro–wilk and q–q plots), homoscedasticity (breusch-pagan test, p > 0.05), and independence of residuals (durbin–watson ≈ 2.0). age-adjusted means and partial eta-squared (η2) values for lipid parameters are presented in online supplementary table 3 to online supplementary table 9. results general characteristics of the study population a total of 172 confirmed ascvd patients (73 men, 99 women) aged 20–64 years were enrolled, including those with ihd (n = 40), ischaemic stroke (n = 25), hypertensive heart disease with significant atherosclerosis (n = 45), mi (n = 20), and congestive heart failure with a history of ihd or mi (n = 42). the control group comprised 55 healthy volunteers (24 men, 31 women) aged 20–64 years. atherosclerotic cardiovascular disease patients showed significantly higher mean plasma tc, ldlc, non-hdlc, and triglycerides levels than controls (all p < 0.001, table 1). similarly, all conventional lipid indices (cri-i, cri-ii, ac, and aip) (p < 0.001), apo b (p = 0.002), and the apo b/a1 ratio (p < 0.001) were elevated significantly. conversely, mean plasma hdlc, apo a1, and hdl phospholipid levels were significantly lower in ascvd patients (all p < 0.001). after adjusting for age using analysis of covariance, significant differences remained in all lipid and apolipoprotein parameters between ascvd patients and controls. full statistical details are provided in online supplementary table 3 to online supplementary table 10. conventional lipid profiles of atherosclerotic cardiovascular disease subclasses significant increases were observed in the mean plasma levels of tc, ldlc, non-hdlc, and triglycerides (p < 0.001) across ascvd subclasses and controls (table 2). similarly, all conventional lipid indices (cri-i, cri-ii, ac, and aip) showed marked elevations (p < 0.001). in contrast, plasma hdlc levels were significantly reduced (p < 0.001) across the ascvd subclasses and controls. patients with ihd and mi had the highest levels of atherogenic lipids (triglycerides and tc) and lipoproteins (ldlc and non-hdlc), with mean values significantly elevated compared to controls and other ascvd subgroups (p < 0.001). these groups also presented the lowest hdlc levels (p < 0.001), indicating a more atherogenic lipid profile. table 2: conventional lipid profile and indices in atherosclerotic cardiovascular disease subclasses and controls at lagos state university teaching hospital, lagos, nigeria, between november 2021 and november 2022. apolipoproteins and high-density lipoprotein phospholipid levels in atherosclerotic cardiovascular disease subclasses significant increases in mean plasma apo b, and the apo b/a1 ratio (p < 0.001) were observed across ascvd subclasses and controls (table 3). conversely, mean plasma apo a1 and hdl phospholipid levels were significantly reduced (p < 0.001). the highest apo b and apo b/a1 ratios were found in patients with mi and ihd, who also had the lowest levels of apo a1 and hdl phospholipids. the apo b/a1 ratio proved to be a superior marker for ascvd stratification compared to apo a1 or apo b alone.38 for instance, apo b levels did not differ significantly when patients suffering from stroke and heart failure were compared with the control group (online supplementary table 10), whereas the apo b/a1 ratio showed statistically significant differences across all ascvd subclasses when each subclass is compared with the control group (online supplementary table 11). table 3: apolipoproteins and high-density lipoprotein phospholipids in atherosclerotic cardiovascular disease subclasses and controls at lagos state university teaching hospital, lagos, nigeria, between november 2021 and november 2022. associations between apolipoproteins, high-density lipoprotein phospholipids and conventional lipid profiles a significant negative correlation was observed between mean plasma apo a1 levels and atherogenic lipids, including triglycerides (r = –0.386, p < 0.001), tc (r = –0.471, p < 0.001), ldlc (r = –0.483, p < 0.001), and non-hdlc (r = –0.500, p < 0.001) (table 4). in contrast, apo a1 showed a strong positive correlation with antiatherogenic hdlc (r = 0.386, p < 0.001). table 4: association between apolipoproteins, high-density lipoprotein phospholipids and conventional lipid profiles at lagos state university teaching hospital, lagos, nigeria, between november 2021 and november 2022. apo b was positively correlated with atherogenic lipids and lipoproteins (triglycerides, tc, ldlc, and non-hdlc). in addition, the apo b/a1 ratio exhibited strong positive correlations with conventional lipid indices, including cri-i (r = 0.626, p < 0.001), cri-ii (r = 0.601, p < 0.001), ac (r = 0.613, p < 0.001), and aip (r = 0.604, p < 0.001). high-density lipoprotein phospholipid levels correlated positively with apo a1 (r = 0.524, p < 0.001) but negatively with apo b (r = –0.174, p = 0.009) and the apo b/a1 ratio (r = –0.586, p < 0.001). variations in the concentrations of apo a1, apo b, and the apo b/a1 ratio across atherosclerotic cardiovascular disease subclasses graded variations were observed in mean plasma apo b concentrations across ascvd subclasses (figure 1), with the highest levels found in mi patients. similarly, the mean plasma apo b/a1 ratio showed a graded increase (figure 2), peaking in ihd patients. in contrast, mean plasma apo a1 levels exhibited a graded decline (figure 3), with the lowest levels recorded in mi patients. figure 1: graded variations in the mean plasma apolipoprotein b concentrations in atherosclerotic cardiovascular disease subclasses and controls at lagos state university teaching hospital, lagos, nigeria, between november 2021 and november 2022. figure 2: graded variations in apolipoprotein b/a1 ratio in atherosclerotic cardiovascular disease subclasses and controls at lagos state university teaching hospital, lagos, nigeria, between november 2021 and november 2022. figure 3: graded variations in the mean plasma apolipoprotein a1 levels in atherosclerotic cardiovascular disease subclasses and controls at lagos state university teaching hospital, lagos, nigeria, between november 2021 and november 2022. comparison of the total and high-density lipoprotein phospholipids levels across different classes of atherosclerotic cardiovascular disease the mean total plasma phospholipid levels remained within a narrow range across ascvd patients and controls (figure 4), with no significant difference between the groups (table 1). however, the mean plasma hdl phospholipid fraction was significantly higher in controls than in ascvd patients, with the lowest levels observed in those with mi and ihd. figure 4: plasma total and high-density lipoprotein phospholipids levels in atherosclerotic cardiovascular disease subclasses and controls at lagos state university teaching hospital, lagos, nigeria, between november 2021 and november 2022. discussion the present study demonstrated significant disruptions in atherogenic lipids and lipoproteins among ascvd patients. notably, there were substantial increases in plasma triglycerides, tc, ldlc, and non-hdlc levels, whereas hdlc (an antiatherogenic lipid) was markedly reduced compared with the control values. similarly, conventional lipid parameters (cri-i, cri-ii, ac, and aip) were markedly elevated in ascvd patients compared with controls. these findings are consistent with earlier studies,20,39 reporting elevated levels of atherogenic lipids and lipid indices in cardiovascular disease patients. in addition, this study revealed significant variations in lipid profiles and indices across different subclasses of ascvd. notably, the greatest elevations in atherogenic lipid and lipid indices were observed in patients with myocardial infarction and ihd. conversely, these groups also presented the lowest reduction in antiatherogenic hdl cholesterol levels compared with the other ascvd subclasses. these findings align with previous reports demonstrating severe dyslipidaemia in patients with mi and ihd compared with other ascvd subtypes.8,40 the results of this study further underscore the pronounced severity of dyslipidaemia in these patient groups. furthermore, this study revealed a substantial decrease in the mean plasma apo a1 concentration, and notable increases in apo b levels and the apo b/a1 ratio in ascvd patients, compared with those in the control group. these findings are consistent with prior research across diverse populations worldwide,13,14,16,17 which revealed that low apo a1 levels, elevated apo b levels, and a higher apo b/a1 ratio are associated with a heightened risk of major cardiovascular events. elevated apo b levels in ascvd patients probably indicate an increased burden of atherogenic particles, which is correlated with increased cardiovascular risk.38 concurrently, a higher apo b/a1 ratio suggests an imbalance favouring atherogenic particles over antiatherogenic particles, highlighting an adverse lipid profile in the ascvd group.16 notably, the lowest reduction in plasma apo a1 levels and the greatest increases in both apo b levels and the apo b/a1 ratio were observed in patients with mi and ihd. thus, these findings underscore the critical role of apo b in the manifestation of mi and ihd, as elevated levels of apo b-containing lipoproteins primarily drive atherosclerosis.38 moreover, reduced apo a1 levels in these ascvd subclasses (mi and ihd) suggest impaired reverse cholesterol transport, which may contribute to the lipid build-up and instability of plaques.13 plaque rupture is a common cause of mi,41 making apo a1 a crucial factor in these patients. furthermore, the present study revealed a marked reduction in the mean hdl phospholipid levels in ascvd patients compared with those in the controls. this finding is in agreement with previous studies that reported a reduction in hdl phospholipid levels in ascvd patients compared with those in the control group.23,26 these findings suggest a potential impairment in hdl functionality, which could contribute to the pathophysiology of ascvd. moreover, the reduction in hdl phospholipid levels was particularly pronounced in patients suffering from mi and ihd. these findings could imply that these ascvd subclasses are associated with more severe alterations in lipid metabolism and hdl function, possibly exacerbating the risk and severity of ascvd. in addition, the present study revealed significant inverse correlations between apo a1 levels and atherogenic lipids (tc, triglycerides, ldlc, and non-hdlc), and a positive association with hdlc, suggesting a protective effect against atherosclerosis. conversely, elevated levels of apo b correlate with increased levels of atherogenic particles, indicating increased cardiovascular risk. this study also identified the apo b/a1 ratio as a comprehensive marker for lipid-related ascvd risk, indicating that it is a superior marker for ascvd stratification and demonstrating a strong correlation with traditional lipid indices. higher ratios were associated with elevated levels of atherogenic lipids and decreased levels of hdlc, effectively integrating both proatherogenic and antiatherogenic lipid particles. this ratio provides a more complete view of an individual’s lipid profile and their associated cardiovascular risk.16,17 strengths, contributions, and implications for future research the findings of this study carry significant clinical and public health relevance for nigerian and sub-saharan african populations. the elevated apolipoprotein b/a1 ratio, coupled with changes in apolipoprotein a1, apolipoprotein b, and hdl phospholipids, indicates a higher risk of premature ascvd. the apolipoprotein b/a1 ratio emerges as a key marker for ascvd risk, reinforcing the need for earlier diagnosis and targeted interventions. the findings also underscore the importance of early screening and prevention programmes tailored to these regions. future research should focus on tracking changes in the apolipoprotein b/a1 ratio over time and assessing targeted interventions for ascvd prevention in sub-saharan africa. developing tailored screening programmes and investigating genetic and environmental factors influencing ascvd risk are crucial. comparative studies across sub-saharan africa can refine risk assessment, while public health initiatives and policy research may help to reduce ascvd mortality in these regions. limitations while the findings provide important insights, certain factors warrant consideration. unmeasured variables such as diet, physical activity and genetic predisposition may have influenced apolipoprotein and hdl phospholipid levels. the mean age difference of about 10 years between cases and controls, despite statistical adjustment, may also introduce residual confounding. in addition, the moderate areas under curve observed in preliminary analyses for apo a1, apo b, and hdl phospholipids probably reflect the sample size and exploratory design of the study. further research with larger, diverse cohorts is needed to validate these findings and to refine clinically relevant cut-off values through advanced modelling approaches. conclusion this study identified associations between lipid profile disruptions and ascvd in nigerian patients, characterised by elevated atherogenic lipids (triglycerides, tc, ldlc, and non-hdlc) and reduced hdlc, particularly in those with mi and ihd. apolipoprotein analysis showed decreased apo a1 and increased apo b, leading to a higher apo b/a1 ratio and indicating a greater atherogenic burden. reduced hdl phospholipid levels further suggest impaired hdl functionality. the apo b/a1 ratio emerged as a key marker for lipid-related ascvd risk, highlighting its potential for early diagnosis and risk stratification. these findings underscore the need for targeted screening and prevention strategies to improve cardiovascular outcomes in this population. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. credit authorship contribution promise c. nwaejigh: conceptualisation, writing – original draft, data curation, writing – review & editing. maria o. ebesunun: conceptualisation, writing – review & editing. stephen s. udofia: formal analysis, data curation, writing – review & editing. adebusola a. shakunle: formal analysis, investigation, writing – review & editing. all authors reviewed the article, contributed to the discussion of results, approved the final version for submission and publication, and take responsibility for the integrity of its findings. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the authors confirm that the data 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macronutrient, micronutrient, and nutrigenetic factors impacting cardiovascular risk markers apolipoprotein b and apolipoprotein a1: a narrative review. nutr rev. 2024;82(7):949–962. https://doi.org/10.1093/nutrit/nuad102 wan h, wu h, wei y, et al. novel lipid profiles and atherosclerotic cardiovascular disease risk: insights from a latent profile analysis. lipids health dis. 2025;24:71. https://doi.org/10.1186/s12944-025-02471-3 abera a, worede a, hirigo at, alemayehu r, ambachew s. dyslipidemia and associated factors among adult cardiac patients: a hospital-based comparative cross-sectional study. eur j med res. 2024;29(1):237. https://doi.org/10.1186/s40001-024-01802-x palasubramaniam j, wang x, peter k. myocardial infarction – from atherosclerosis to thrombosis. arterioscler thromb vasc biol. 2019;39(8):e176–e185. https://doi.org/10.1161/atvbaha.119.312578 abstract introduction objective 1: improve awareness and understanding objective 2: strengthen the knowledge and evidence base through surveillance and research objective 3: reduce the incidence of infection objective 4: optimise the use of antimicrobial medicines objective 5: develop the economic case for sustainable investment ways forward acknowledgements references about the author(s) iruka n. okeke department of pharmaceutical microbiology, university of ibadan, ibadan, oyo state, nigeria citation okeke in. laboratory systems as an antibacterial resistance containment tool in africa. afr j lab med. 2016;5(3), a497. http://dx.doi.org/10.4102/ajlm.v5i3.497 review article laboratory systems as an antibacterial resistance containment tool in africa iruka n. okeke received: 18 may 2016; accepted: 05 aug. 2016; published: 31 oct. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract introduction: as crucial as clinical laboratories are to preventing, identifying and managing resistance problems, laboratory scientists are among the most overlooked stakeholders. this review outlines the contributions that diagnostic laboratory systems should make toward all five of the world health organization’s 2015 strategic objectives for antimicrobial resistance containment. laboratory systems in resistance containment: antimicrobial susceptibility testing and surveillance are central to antibacterial resistance management and control and need to be implemented more commonly and closer to sick patients. however, the scope of tests that promote judicious antimicrobial use extend beyond susceptibility testing. laboratory tests for pathogens or their associated biomarkers confirm or rule out specific causes of signs and symptoms associated with infection. laboratory systems also provide critical support to infection control programmes. all of these functions promote rational antimicrobial use and contain the spread of resistance. routine laboratory data supports the development of vaccines and other technologies that could ease the pressure placed by antimicrobials. laboratories are also a rich source of information for health professionals, policymakers and the general public about the urgency of the resistance problem and progress in containing it. conclusion: laboratory systems are integral to antimicrobial resistance containment and contributions from african laboratories to addressing resistance need to be enhanced. introduction antimicrobial resistance is a grand global challenge that is reducing the success and increasing the cost of treating infections. as with many complex problems, there are many stakeholders in resistance – patients, prescribers and dispensers are often cited.1 however, much less visible but high on the list are those stakeholders that develop and deploy diagnostic tests. diagnostic testing is key to resistance containment and may in fact be the most important tool for increasing appropriate access to antimicrobials while simultaneously containing resistance.2,3 when appropriate tests are judiciously used within the context of an effective laboratory system, they lower selective pressure for resistance by promoting targeted and rational use of antimicrobials. they also lower drug and healthcare costs and can identify treatment failure caused by resistance, thereby unveiling the nature and scope of resistance and preventing its spread.4,5 laboratory systems and laboratory professionals are key to identifying, mapping, quantifying and communicating about resistance. patients with resistant infections can only be effectively and efficiently managed with laboratory support. the definitive evidence that resistance is a problem, as well as the fine description of the problem, comes from laboratories.6 most critically, attempts to treat infections or presumed infections without laboratory input drive resistance by increasing unwarranted antimicrobial use, which is a needless selective pressure for resistant strains. infectious patients, treated without laboratory support, are also more likely to remain infectious for longer and therefore spread their diseases. laboratory system strengthening for resistance containment is needed everywhere on the globe, but particularly in african countries, where infection is the leading cause of disease and death. it is now universally acknowledged, within and outside the scientific community, that deliberate and forceful steps across different sectors must be taken to contain resistance. the recent world health organization (who) global action plan on antimicrobial resistance7 outlines five strategic objectives for resistance containment, as follows: improve awareness and understanding of antimicrobial resistance through effective communication, education and training. strengthen the knowledge and evidence base through surveillance and research. reduce the incidence of infection through effective sanitation, hygiene and infection prevention measures. optimise the use of antimicrobial medicines in human and animal health. develop the economic case for sustainable investment that takes account of the needs of all countries, and increase investment in new medicines, diagnostic tools, vaccines and other interventions. most readers of the plan will appreciate that clinical laboratories and laboratory professionals will lead the second objective of surveillance and research. however, even though their role toward goals are less prominent, as summarised in table 1, laboratory systems are essential for meeting every one of these objectives, as discussed below. table 1: the role of the laboratory system in meeting the objectives of the world health organization global action plan on antimicrobial resistance. objective 1: improve awareness and understanding dissemination of information about antimicrobial resistance is key to effecting the behavioural change necessary to contain resistance.8 communications from laboratorians are easily evidence-based. diagnostic laboratory professionals in general, and microbiologists in particular, are in an excellent position to disseminate information on antimicrobial resistance to health professionals and the general public. resistance is a health problem but is also a microbiological phenomenon brought about by the genetic change and selective pressure from antibiotics. resistant organisms circulate among humans but also in other domesticated and wild animal species, as well as in the environment. microbiologists can and should highlight the mechanisms by which resistance evolves and spreads and how these connect to risk factors for resistant infections in both humans and animals. some microbiologists have played a pivotal role in public engagement projects and educational initiatives to drive home the message that antimicrobials must be conserved.8,9 for example, the uk microbiology society focused one issue of its comic, ‘marvellous microbes’, on antimicrobial resistance.10 microbiologists and other scientists need to do more to communicate the urgency of the problem as well as the pivotal contributions that laboratories make to contain it. this is particularly true in africa, where much of the general public and a significant proportion of health professionals have very little awareness of what diagnostic laboratories actually do, or of the biological basis for antimicrobial resistance. objective 2: strengthen the knowledge and evidence base through surveillance and research resistant strains are identified by antimicrobial susceptibility testing antimicrobial susceptibility testing by minimum inhibitory concentration (mic) measurement or disc diffusion is the gold standard for identifying resistance.11,12 for bacteria, once a laboratory is capable of isolating and identifying pathogens from clinical specimens, very few additional resources are needed to obtain a susceptibility profile. mics are measured in a broth dilution assay or on solid media by agar dilution.13 to obtain mics, standardised test and control cultures are inoculated into or onto media containing different dilutions of antimicrobial agent. for diagnostic testing, doubling dilutions are the most commonly-used format, although any geometric or arithmetic progression can be used to broaden the test range or increase precision. the mic is the minimum concentration preventing growth under test conditions. media composition, incubation temperature, inoculum size and quality, antibiotic format and incubation time can all affect the mic and must be standardised. disc tests are the most commonly-used methods of bacterial susceptibility testing worldwide. they are simple and reproducible tests that determine whether a bacterial isolate is sensitive or resistant to a given drug or drug class. standard discs containing specific concentrations of each antibacterial are placed on a calibrated lawn of bacteria. after incubation, a clear zone of no growth appears if the concentration of antibiotic around the disc is greater than the mic. as the concentration of antibiotic in the medium decreases with distance of diffusion through the agar from the disc, the level of resistance is inversely proportional to the diameter of the clear zone of inhibition. diffusion kinetics of the antibacterial in the test medium are also determinants of zone size, therefore test standardisation is key. earlier challenges with reproducibility have been overcome by standardising methodologies and interpretations and by using commercially-available media, discs and inoculum standardisation tools.14,15 thus today, disc diffusion tests are straightforward protocols that are easy to quality assure. if diffusion from a calibrated strip containing graded concentration of antibiotic, rather than a circular disc, an mic can be obtained by a diffusion protocol. mic strips are a somewhat costly but cost-effective and simple way to obtain an mic, particularly in laboratories that are set up for disc testing. the resistance crisis makes it impossible to continue to argue that antibiotic susceptibility testing is superfluous, a point that was made in many venues, even by experts, as recently as a decade ago.16,17 however, a common misconception in some parts of africa is that antibacterial susceptibility testing is a reference laboratory-level technique. susceptibility testing is best supported from central facilities and requires external quality assurance, but susceptibility testing of aerobic bacteria is designed to be performed at routine microbiology laboratories with minimal bacteriology facilities.18 the closer the site for culture and susceptibility testing is to the patient and the provider, the greater the chance that it will impact care for that specific patient and affect empiric prescribing at the relevant facility. disc diffusion tests in particular are robust, reproducible and capacity can easily be built.19 disc diffusion tests can even be rigorously performed in field laboratories equipped with a portable autoclave and a small incubator. compiling and disseminating facility-level data, in the process of providing prescribers with a valuable tool for resistance control, can also help to garner the support of clinicians for laboratory services. simple software, such as whonet,20,21 can be used at the facility level to aggregate and analyse susceptibility data and have the added advantage of allowing these data to be forwarded to and integrated with national, regional and global surveillance data. whonet will run on the most basic of computers, independent of platform, and capacity to enter, retrieve and analyse data can be built with ease. the software is available free, can be used onor off-line, and the who collaborating centre for antimicrobial resistance provides free technical support. a ‘can do’ narrative is essential for extending access to antibacterial susceptibility testing in africa. in decades past, the perception that actual ‘high-tech’ methods, such as flow cytometry for cd4+ counts and molecular tests for viral load, were highly specialised, caused reservations about appropriately deploying hiv-management plans, including antiretroviral therapy in africa. with carefully thought-out programmes that built capacity in parallel with infrastructure, many more patients than was originally envisaged are today able to access these services at the point of care. in western countries, laboratories below the level of those that offer diagnostic support for highly-active antiretroviral therapy provide routine susceptibility testing, and the same is true historically of many african facilities that no longer offer testing today. similar stories have unfolded around multidrug tuberculosis and malaria, for which the impracticability and long duration of testing has led to the development of rapid molecular xpert® tests and rapid diagnostic tests, respectively, providing many patients today with rapid diagnosis at the point of care.22,23 for hiv, tuberculosis and malaria, new technologies had to be developed and deployed to provide testing options in africa. antibacterial susceptibility testing, the much lesser challenge, based on much older methodology, has been less successfully rolled out to african patients and its neglect has important consequences for the delivery of care and resistance containment.4 antibacterial susceptibility testing uses basic techniques that can and should be offered by a secondary or even primary care level laboratory with minimal microbiology resources. they are methods that any medical laboratory scientist with microbiology training can perform. the required equipment is small, robust and inexpensive and the costs of consumables and quality assurance are not high. determining the aetiology of infections contains resistance bacterial infections can be life-threatening and while antibiotic use has a huge societal cost, most antibacterials are devoid of serious side effects. for these reasons, a significant number of people receive an antimicrobial prescription ‘just in case’. as soon as it can be shown that a patient’s signs and symptoms do not emanate from a bacterial infection, antibiotics can be withheld with confidence. spot tests for viral causes of childhood diarrhoea and acute upper respiratory tract infection reduce unnecessary empiric use in those conditions. simple point-of-care streptococcal (strep) throat swab tests have reduced antibiotic use in many parts of the world, because antibiotics are only ordered if the test is positive.24,25 for tests available in rapid point-of-care formats, results are available before initial therapy is begun and therefore unnecessary causes of empiric therapy can be prevented. in veterinary infections, point-of-care tests that delineate diarrhoea of bacterial aetiology from viral infections can be used on a farm to make an informed decision on the necessity for antibiotics.4 rapid point-of-care tests have not been as effective as they might be because patients and their providers often disregard the results and administer antibiotics when these tests suggest that they are not necessary.26 point-of-care testing may also be viewed as burdensome and even undermine care when it uncovers the fact that available therapies may not be effective choices.27 nonetheless, testing and surveillance are essential for revealing inadequacies to policymakers as well as informing available options. point-of-care tests, in particular, are exceptionally valuable in settings where the patient cannot easily return for follow-up, as is typical for many african health systems.4,28 laboratory professionals have a key role to play in educating stakeholders and building their confidence in tests that optimise patient treatment and conserve antimicrobials. going forward, development and deployment of multiplex tests that could help detect or rule out multiple causes of fever, diarrhoea or respiratory distress could further help to optimise antimicrobial use. biomarker tests can rapidly narrow differential diagnoses culture and susceptibility testing remain the only reliable and affordable option for many clinics. the challenge of slow turn-around time will soon become a problem of the past. rapid molecular and mass-spectrometry tests have now been developed and are being pre-tested and deployed in clinical laboratories that do not face severe resource limitations.29,30,31 while efforts are underway to increase the sensitivity and specificity of these tests and broaden their access, rapid aetiologic agent tests are only available for a few pathogens and in some cases are too expensive to be used routinely today in african settings. much-needed multiplex tests are non-existent or rare. this is a pressing need that many african researchers are engaged in addressing but to which much more research activity needs to be devoted. in severe infections of likely bacterial aetiology, antibacterial chemotherapy must be instituted swiftly to avoid unnecessary mortality or death before susceptibility testing results are obtained. for this reason, biomarker tests that provide rapid results and can reliably identify a bacterial infection, then antibacterial therapy is only prescribed when a bacterial infection is present. c-reactive protein and procalcitonin are among useful markers of systemic bacterial infection that are not used, or are underused in africa.32,33,34 in some cases, the sensitivities, specificities and positive and negative predictive values in african settings have yet to be evaluated making it difficult to advocate for their use.34 similarly, markers of invasive diarrhoea, such as lactoferrin,35,36 could potentially help restrict excessive use of antibiotics in viral and other self-resolving gastroenteritis and need to be assessed and deployed in african settings. surveillance of resistance is critical for qualifying and quantifying the problem, and for informing essential empiric prescribing of medication. to a large extent, the precise magnitude of resistance is not known. however, we know there is a problem and most of what we know about its scope and volume comes from laboratory testing and surveillance, both core functions of a laboratory system.37 in europe, for example, where surveillance is routine and has good coverage, there are good estimates of resistance and surveillance data have been used to prevent resistance rises or even bring resistance rates down.38,39 by contrast, where systematic surveillance is absent or rare, it is not clear what the major problems are, how big the problems are, or how best to tackle them. surveillance data are least available from africa40 and as such, the resistance problem is largely sketched from research data from small studies and clinical case reports. those data sources indicate a worrisomely large and growing problem, the cost of which is difficult to estimate. as of 2015, with the absence of surveillance, only a handful of african countries had demonstrated little policy activity or evidenced interest in addressing resistance.40,41 in countries where resistance is at the forefront of policy-makers’ priorities, some kind of surveillance information is available. one justification for swiftly and decisively applying appropriate alternate diagnostic tools is the long time that culture and susceptibility testing takes. severely-ill patients must be treated empirically, so is the cost of tests that will yield results justified? as resistance becomes increasingly common, the justification increases. surveillance data, if available, becomes less predictive when new resistances emerge. and when initial empiric therapy fails, the only fail-safe method to suggest an alternate chemotherapeutic course is to use laboratory testing information. the time to test results by conventional methods is long but, in most cases, it is well within the ideal time to make the necessary switch that could save a patient’s life. surveillance and laboratory testing are critical surveillance and laboratory testing are critical to implementing alternatives to antibiotics that are technological solutions to the resistance problem. surveillance determines the burden of a given infectious disease as well as the predominant pathogenic subtypes of its aetiological agent. only with this information can vaccines be developed and deployed. access to antimicrobial susceptibility testing has to increase, as do ways to integrate representative samples of infection testing into surveillance networks. only with these developments will susceptibility information be optimally exploited in patient care and antimicrobial conservation. as we work to expand the reach of good testing and build fine surveillance networks, complementary technologies could be brought to bear on this problem. difficulties with urban traffic and poor rural roads in warm moist tropical countries hamper the use of sentinel laboratories that could collect and process specimens from vast areas. improving specimen and data transport systems, for example through application of aerial drones to convey specimens (building on an idea from moses bangura http://falling-walls.com/lab/news/next+einstein+forum+sends+african+innovator+to+falling+walls+lab+finale+2016-7409) and mobile phones to return data could change the present situation in which susceptibility data are rarely available from poor countries and rural areas. objective 3: reduce the incidence of infection laboratory systems are central to infection control antimicrobial resistant infections are increasingly hospital acquired. laboratories can confirm outbreaks and are key to determining what happened and why. importantly, the data they provide help to prevent the spread of infections within health facilities so that fewer individuals require curative, typically antibiotic-based interventions. veterinary microbiology laboratories can help contain agricultural outbreaks and prevent infected meat products entering the human food chain. surveillance allows for better burden estimates laboratory-based surveillance makes important contributions to information that is essential for disease control policy decisions. it was malaria surveillance data that were used to model the impact of antimalarial interventions and which ultimately revealed that the most valuable antimalarial intervention has been insecticide treated bed nets. there is thus rigorous evidence supporting a non-drug preventive strategy with significant impact on one of africa’s most burdensome diseases. models based on resistance surveillance could provide more insight about bacterial infections and the trajectory for drug resistance and thereby inform disease and resistance control. laboratory systems as justification for development and deployment of disease control tools for streptococcus pneumoniae, decades of laboratory-based surveillance demonstrated that only a handful of serotypes accounted for most of the disease and most of the antibacterial resistance. these data aided the development of conjugate vaccine cocktails that are preventing resistant s. pneumoniae infections, an important cause of childhood illness and death in africa, and continue to drive vaccine policy worldwide.42,43 the application of susceptibility information from laboratories to vaccinology is by no means limited to pneumococci. until recently, oral antibacterials were the key tool used to control cholera outbreak size. when resistance became commonplace, not only were antibiotics less effective, but use of these drugs in outbreaks increased selective pressure for resistance. it is now acknowledged that while they cost more per dose than most antibacterial medicines, vaccines are the more cost-effective means for preventing pathogen spread in an outbreak and protecting the un-infected from the disease.44 vaccines are so effective in this regard that scientists were able to justify building a stockpile of vaccines for use during outbreaks.45 a vaccine-based approach to outbreak control is particularly valuable in africa, where the same geographic areas have seen repeated outbreaks in recent years.46 it is imperative that drawings are only made from the stockpile in the event of an outbreak that is verifiably cholera. therefore, countries can request vaccines from this life-saving and resistance-averting stockpile only when aetiologic confirmation of cholera is obtained after laboratory testing.47 what this means is that laboratory systems will improve vaccine access. availability of cd4+ counting and viral load testing to africans living with hiv has been crucial for the appropriate staging of infections and commencement of antiretroviral therapy. testing will also help to identify antiretroviral resistance promptly, limiting its spread. assuring effective antiretroviral therapy also prevents the dissemination of particularly problematic clones of opportunistic pathogens, epidemics of which are a matter of concern in africa.48,49 an additional, often unstated benefit of laboratory-supported highly-active antiretroviral therapy programmes is that they relieve pressure from trimethoprim-sulphamethoxazole and other antibacterials that were used to prevent opportunistic infections in aids. prophylactic antibacterials were the main tool used to prolong lives on the continent prior to the 2000s and were most probably selected for resistant bacterial clones and elements that have since narrowed antimicrobial options.50,51 objective 4: optimise the use of antimicrobial medicines informed prescribing for life-threatening invasive disease for some life-threatening human infections, antibacterials must be prescribed as soon as possible after patient presentation, before the results of antibacterial susceptibility testing are available. cases in point are bacteraemia and meningitis. in these infections, the patient is best served when recent local susceptibility information is available since this is the best way to guide empiric prescribing. when appropriate data are not available, more expensive reserve drugs are overused and/or treatment failures are more common. even when such data are available, blood or cerebrospinal fluid cultures should still be performed for two key reasons. firstly, patient-specific culture and susceptibility information allows caregivers to switch to a more appropriate antibacterial than the initial empiric choice. such changes are most likely to be implemented for in-patients and are most valuable in resource-limited settings. secondly, performing culture and susceptibility testing, even when empiric treatment is instituted, also accumulates vital information for treating other patients. blood and cerebrospinal fluid culture-derived susceptibility data are, for these reasons, among the most valuable types of susceptibility information for clinicians. indeed, where resource limitations preclude culture and susceptibility testing for all specimens, prioritising blood and cerebrospinal fluid is the most cost-effective alternative.51,52 this is true even though blood culture is more tedious and more expensive than urine or stool culture, and more prone to invalid results. high-quality, life-saving blood culture information can be obtained even if automated blood culture machines are not available.53 blood culture data can also be disseminated in geographic-specific ways to resistance-data networks (for example, resistancemap; http://resistancemap.cddep.org/). sadly, many more physicians practising in africa have access to culture and susceptibility testing for non-emergency samples than for blood. national and international drug policy the availability of surveillance data can influence drug-use policy on national and even international levels. in ghana, antibacterial resistance patterns of vibrio cholerae isolates were the basis for altering national treatment guidelines and resistance reports from other areas to support the idea that vaccines, rather than antibacterials, should be the mainstay for cholera outbreak control.54 similarly, archival resistance data from nigeria refutes the hypothesis that chloroquine may select for fluoroquinolone-resistant bacteria, making it unnecessary to factor in bacterial resistance in future policy decisions about this antiplasmodial drug.55 drug policies that are relevant to resistance include how different agents are used as well as which drugs are selected. more evidence is needed to determine whether antibacterial cycling or combinations might curtail resistance, and to guide drug use in veterinary settings to minimise the threat of resistance evolving in animal as well as human pathogens. novel antimicrobial strategies evolutionary theory and emerging data suggest that treatments that diminish or clear infections without actually killing bacteria or inhibiting growth may have a lower propensity to be overcome by resistance. thus, when preclinical candidates that inactivate bacterial toxins or prevent microbial adherence become available, they could have a significant impact on resistance containment.52,56,57 narrow-spectrum drugs, peptides or even bacteriophages also offer promise because the selective pressure they exert does not extend to a significant proportion of the normal flora. similar to vaccines and disease prevention tools, antivirulence, bacteriophage and other non-antibiotic therapies rely on precise identification of the aetiologic agent in every infection, before appropriate treatment can be selected and administered. objective 5: develop the economic case for sustainable investment antimicrobial resistance is a ‘tragedy of the commons’: a destructive paradigm created by a cheap, effective and widely-available resource (the antimicrobial drugs themselves), overuse of which incurs few costs for individuals but profound costs to society.58,59 a first step in preventing a tragedy of the commons is achieving a recognition of necessity. for antimicrobial resistance, the essential evidence for recognition comes from laboratories.37 with this evidence, it is possible to compute the losses from drug resistance as well as what might be saved if antimicrobials are conserved.3,60,61 less extensive economic information is available from african countries but the economic justification for investment in resistance containment, in general, and laboratory contributions, in particular, is illustrated by a case of prolonged febrile illness in a nigerian child with a history of empiric treatment with amoxicillin, fluoroquinolones and artesunate.62 when the child did not recover and was referred, blood culture revealed the aetiologic agent in the infection to be an extended-spectrum beta-lactamase-producing klebsiella pneumoniae strain that was susceptible to imipenem, which cleared the infection. during her six-week illness, chemotherapy and supportive care cost almost $600.00. only $387 was for the intravenous imipenem/cilastatin that eventually cleared the infection. diagnostic testing, including microbiological tests, blood chemistry, radiology that supported this patient and uncovered the extended-spectrum beta-lactamase -producing aetiological agent cost $90.00.62 challenges with antimicrobial access are as important to dealing with resistance as those produced by ‘excess’ in antimicrobial use. far too many african patients do not have access to antimicrobials at all and/or the best option for treating their infections and minimising the threat of resistance.63 for those that do have access to some antimicrobials, surveillance data will indicate whether the access is to effective drugs – an important means for containing resistance. among the justifications given for inadequate application of laboratory systems for infectious disease management and resistance control are the cost of testing. however, as the aboderin et al.62 case demonstrates, multiple empiric courses of antibacterial chemotherapy are costly, and supportive management of patients that are sickened by prolonged infections is even more expensive. most importantly, in cases such as that, the correct course of therapy, when it is itself expensive, cannot be initiated without diagnostic support. in addition to the high monetary cost of diagnostic insufficiency of these practices, significant disability-adjusted life years are lost when clinicians attempt to manage infections without the necessary information. ways forward without containing antimicrobial resistance, the sustainable development goals and the global health security agenda cannot be met.64 this paper illustrates how laboratories are central to resistance containment and especially focuses on the need for laboratory system strengthening on the african continent where, if nothing changes, 4.15 million people are predicted to be at risk of dying from resistant infections annually by 2050.65 healthcare providers need to make better use of existing laboratory services and demand necessary improvements. robust laboratory systems will actually reduce medicine and care expenses, therefore the perceived high cost of laboratory services should not be a balance to better building and integrating laboratories into the health system. african laboratories need to be better resourced so that they can deliver susceptibility information. this includes equipment, consumable resources and their supply chains, as well as development of the human resources to perform tests, store, curate and disseminate data, and apply these data to human and veterinary medicine and public health. recent audits indicate that these resources are lacking in many african health systems, as is access to both internal and external quality assurance.4,66 importantly, these are well-recognised needs, with well-known modalities for delivering them. therefore, these shortfalls can and should be addressed. the data, and their application, also need to be worked into functional surveillance systems. data from african countries are few and difficult to get, such that programmes that agglomerate data have described localities within the continent as being ‘data deserts’.67 oases must be built through national surveillance programmes, something that could potentially be done through the who’s global antimicrobial resistance surveillance system.68 expansion of access to currently-available susceptibility testing methods and techniques is needed, as well as better agglomeration and curation of existing data. for example, gandra, merchant and laxminarayan point out that in lowand middle-income countries, where public-sector laboratories may not be able to perform testing, private-sector laboratories often provide this service. however, data from those laboratories, while useable for patient care, is often inaccessible for public health purposes.67 where public sector data are available but largely serve lower-income patients than the private sector, careful laboratory-based surveillance could in fact be a useful pointer to differences in susceptibility rates among patients in different social strata.69 connecting susceptibility testing data to patient care and public health is a weak point in many laboratory systems that do provide susceptibility testing. moving away from notebooks that make data retrieval and agglomeration difficult may be one way to improve connections between susceptibility data generators and other healthcare providers. when electronic systems are introduced, resource-limited health facilities will be better served by free software systems with fewer capabilities than more complex systems requiring expensive subscriptions and difficult-to-access technical support. in the not-too-distant future, innovation around laboratory testing for resistance and surveillance promises faster diagnostic testing and more responsive surveillance next generation sequencing is increasingly affordable and can produce aetiologic information and extensive susceptibility profiles in a fraction of the time that is currently used to obtain more limited information by culture and susceptibility.70 cost, the major barrier to sequencing, is rapidly dropping, and improved tools that allow scientists without bioinformatics skills to use genome information are being developed. in a few years, genome sequencing should be the method of choice for life-threatening infections, and outbreaks in particular. similarly, it has been demonstrated that the major capital investments necessary to implement multiplex pcr tests or matrix-assisted laser desorption ionisation time-of-flight in clinical laboratory settings in north america can be offset by savings in medicines and hospital care.71 the scope and performance of point-of-care tests is improving and advances in nanoscience and microfluidics promise cheaper tests here as well.33 better links between african research and clinical laboratories may help move these newer technologies into patient care. conclusion the limited information available suggests that those resistance-containment interventions that draw heavily from laboratory systems – such as infection control and surveillance programmes – when properly implemented, comprise the greatest evidence base for effectiveness. african health systems have a greater proportion of patients with infectious diseases, a greater incentive than others to conserve antimicrobials and therefore the greatest need to prioritise laboratory resources for antimicrobial resistance containment. african laboratories offering diagnostic testing and participating in resistance surveillance will need to be early adopters of some of these technologies, which will ultimately further optimise antimicrobial use and contain costly resistance. acknowledgements i.n.o. is co-recipient of an african research leader’s award jointly funded by the uk medical research council (mrc) and the uk department for international development (dfid) under the mrc/dfid concordat agreement and also part of the edctp2 programme supported by the european union. i thank a. oladipo aboderin for helpful discussions. competing interests the author declares that she has no financial or personal relationships which may have inappropriately influenced her in writing this article. sources of support none. references laxminarayan r, chaudhury rr. antibiotic resistance in india: drivers and opportunities for action. plos med. 2016;13(3):e1001974. http://dx.doi.org/10.1371/journal.pmed.1001974 okeke in, peeling rw, goossens h, et al. diagnostics as essential tools for containing antibacterial resistance. drug resist updat. 2011;14(2):95–106. http://dx.doi.org/10.1016/j.drup.2011.02.002 mendelson m, røttingen j-a, gopinathan u, et al. maximising 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school of pharmacy, faculty of health sciences, university of namibia, windhoek, namibia helen morrison school of pharmacy, faculty of health sciences, university of namibia, windhoek, namibia lydia irengeya school of pharmacy, faculty of health sciences, university of namibia, windhoek, namibia roger k. verbeeck school of pharmacy, faculty of health sciences, university of namibia, windhoek, namibia citation singu bs, morrison h, irengeya l, verbeeck rk. therapeutic drug monitoring of phenytoin and valproic acid in critically ill patients at windhoek central hospital, namibia. afr j lab med. 2022;11(1), a1628. https://doi.org/10.4102/ajlm.v11i1.1628 original research therapeutic drug monitoring of phenytoin and valproic acid in critically ill patients at windhoek central hospital, namibia bonifasius s. singu, helen morrison, lydia irengeya, roger k. verbeeck received: 22 may 2021; accepted: 14 apr. 2022; published: 21 july 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: phenytoin and valproic acid, anticonvulsants, have a low therapeutic index and are highly plasma protein bound, mainly to albumin. hypoalbuminaemia is common in critically ill patients and increases the unbound drug concentration. thus, monitoring unbound rather than total plasma drug concentrations is recommended to optimise the dosing of these drugs. objective: this retrospective study determined unbound plasma concentrations of phenytoin and valproic as a more accurate value of drug levels than total plasma drug concentrations. methods: total plasma concentrations were retrieved for 56 intensive care unit patients for phenytoin and 93 for valproic acid. total drug concentrations were converted to unbound concentrations using a serum albumin-based normalising equation. results: total phenytoin plasma concentration was below (41.1% of patients), within (46.4%) or above (12.5%) the therapeutic range (10 μg/ml – 20 μg/ml). however, the predicted unbound plasma concentration of phenytoin was above the therapeutic range (1 μg/ml – 2 μg/ml) in the majority of patients (57.1%). for valproic acid, the total plasma concentration of most patients (87.1%) was below the therapeutic range (50 μg/ml – 100 μg/ml); among remaining patients (12.9%), it was within the therapeutic range. in the majority of patients (91.4%), the predicted unbound plasma concentration of valproic acid was between 2.5 μg/ml and 20 μg/ml. conclusion: the usefulness of monitoring the total phenytoin or valproic acid levels for dose optimisation is limited as it is an inaccurate indicator of a patient’s drug therapeutic state. thus, the unbound plasma drug concentrations should be quantified experimentally or predicted in resource-limited settings. keywords: phenytoin; valproic acid; critically ill patients; therapeutic drug monitoring; unbound concentration. introduction the incidence of seizures in north american general intensive care units (icus) was reported in 2013 to range from 3.3% to 34.0%.1 risk factors of seizures include head trauma, stroke, brain tumour, infections, hypoglycaemia, electrolyte abnormalities, and drug overdose. phenytoin and valproic acid are anticonvulsants frequently used to prophylax or treat seizures in critically ill patients.2,3 however, both drugs have a low therapeutic index; thus, drug monitoring is required to minimise the risk of toxicity and optimise therapeutic efficacy.4,5,6 following oral administration, both drugs are well-absorbed, highly bound to plasma albumin and eliminated by metabolism.2,4,5 the cytochrome p450 2c9 and 2c19 enzymes metabolise phenytoin to the inactive hydroxy-phenytoin, following first-order kinetics. however, at therapeutic concentrations, the rate of phenytoin metabolism approaches saturation and hence shows capacity-limited elimination (zero-order kinetics).5 metabolism of valproic acid proceeds mainly by glucuronidation (50.0%) and βand ω-oxidation (40.0%) and, to a much lesser extent, by cytochrome p450-catalysed oxidation (10.0%).6 the β-oxidation of valproic acid has been reported to be saturable and subject to autoinduction.6 the pharmacokinetic behaviour of phenytoin and valproic acid is complicated because their elimination kinetics are nonlinear. in addition, the pharmacokinetics of both drugs show a high degree of interindividual variability as a result of pharmacogenetic differences in enzyme and transporter activities, as well as high interpatient variability in plasma protein binding, in the case of valproic acid.7,8,9 as a result of this high interpatient variability, there is a poor correlation between the dose of these drugs and patient plasma concentrations. hence, monitoring patients’ phenytoin or valproic acid plasma concentrations is common, even in critically ill patients.2,7 phenytoin and valproic acid are highly bound to serum albumin (≥ 90%). the binding of these drugs depends on the drug plasma and albumin concentrations and the presence of other albumin-binding substances that may compete for binding sites with these drugs.10 thus, hypoalbuminaemia, a condition in which concentrations of the plasma protein albumin are lower than 35 g/l, increases the unbound proportion of these drugs because of the decreased albumin (binding sites) concentrations.11,12,13 hence, monitoring total plasma concentrations of phenytoin and valproic acid hypoalbuminaemia patients, such as critically ill patients, as the clinical indicator for therapeutic efficacy and safety, is not as reliable as utilising the unbound concentrations.14,15 since it is only the unbound drug that can cross biological membranes and cause pharmacological action through interaction with the drug target, the therapeutic range and dosage individualisation in hypoalbuminaemia patients should be informed by the unbound plasma drug concentrations to prevent toxicity or therapeutic failure.10,12,16,17 unbound plasma concentrations can be determined by equilibrium dialysis and ultrafiltration. however, the use of unbound plasma concentrations in clinical practice is limited because it is time consuming and costly. therefore, equations have been formulated to estimate the unbound concentrations of phenytoin and valproic acid using a patient’s measured total drug plasma concentration and serum albumin level.5,18,19,20,21,22 the therapeutic range for the total phenytoin plasma concentration is 10 μg/ml to 20 μg/ml, and 1 μg/ml to 2 μg/ml for the unbound phenytoin plasma concentration.4,5 for valproic acid, the therapeutic range for the total plasma drug concentration is 50 μg/ml to 100 μg/ml; for the unbound plasma concentration, a therapeutic range of 2.5 μg/ml to 20 μg/ml has been suggested.4,5 the windhoek central hospital in windhoek (namibia) conducts therapeutic phenytoin and valproic acid monitoring in critically ill patients in the icu facility by measuring only total plasma concentrations of the drugs. this study aimed to retrospectively estimate unbound plasma concentrations as a more accurate measure of the therapeutic plasma levels of two drugs. methods ethical considerations the research and ethics committees of the ministry of health and social services, the republic of namibia, approved this study; reference numbers hm2019 and lni2019. data were retrieved from patient record archives; no patients were required to participate in the data collection procedure of this study, and the ethics committee waived the requirement to obtain consent from patients before accessing their records. furthermore, names were excluded from the data collected for this study to protect patients’ privacy. patient population and data collection this retrospective analysis utilised data from the icu of windhoek central hospital, namibia. records of patients admitted between january 2013 and july 2019 were reviewed. patients who received phenytoin or valproic acid while at the icu and whose anticonvulsant plasma concentrations and serum albumin levels had been recorded were included in the study. the following information was extracted from the records: age, gender, diagnosis, total plasma concentrations of phenytoin or valproic acid, serum albumin concentration, and concomitant medication. serum albumin concentrations were measured at the namibia institute of pathology using the bromocresol purple method (cobas® 6000 analyser, roche diagnostics international, rotkreuz, switzerland). the total plasma concentrations of phenytoin and valproic acid were measured by fluorescence polarisation immunoassay (abbott architect i2000, abbott laboratories, chicago, illinois, united states). data analysis for each patient, the unbound plasma concentration of phenytoin (cu pht) was calculated based on the measured total plasma concentration of phenytoin (cp pht) and the serum albumin concentration (alb), in grams/decilitre, by using the original winter-tozer equation5 (equation 1). the therapeutic range for the total phenytoin plasma concentration is 10 μg/ml to 20 μg/ml, and 1 μg/ml to 2 μg/ml for the unbound phenytoin plasma concentration:4,5 the unbound plasma fraction of valproic acid (α vpa) was calculated based on the serum albumin concentration (alb) in μmol/l, by use of the hyperbolic equation proposed by parent et al.16,17 (equation 2): the unbound plasma concentration of valproic acid (cu vpa) was then estimated for each patient based on the measured total valproic acid plasma concentration (cp vpa) (equation 3). the therapeutic ranges were 50 μg/ml to 100 μg/ml for total valproic acid plasma concentration and 2.5 μg/ml to 20.0 μg/ml for unbound valproic acid plasma concentration:4,5 descriptive statistics were used to summarise the results. all statistical calculations were carried out using the excel software package of windows 10 (microsoft corporation, redmond, washington, united states). results in total, 1661 files of patients hospitalised between january 2013 and july 2019 at the icu of windhoek central hospital were reviewed. fifty-six patients, given phenytoin and 93 patients given valproic acid, who had data recorded for total plasma concentrations and serum albumin levels, were included in the study. patients were admitted to the icu for the following conditions: head trauma, hypoxic brain injury, infectious diseases such as meningitis and acute gastroenteritis, renal failure, and hepatic failure. the age of the patients ranged from two months to 66 years. the mean serum albumin levels were below 35 g/l in both sets of patients (mean ± s.d.: 23.5 ± 5.00 g/l for phenytoin, mean ± s.d.: 23.8 ± 5.9 g/l for valproic acid) (table 1). forty-five percent of patients on phenytoin were also receiving drugs known to interfere with its plasma binding or metabolism. for valproic acid, 28% of patients were concomitantly treated with drugs interfering with its plasma binding or metabolism. table 1: patient characteristics and total and predicted plasma concentrations of phenytoin and valproic acid in critically ill patients in the intensive care unit at windhoek central hospital (windhoek, namibia) from 2013 to 2019. the total plasma concentration of phenytoin recorded for 56 patients ranged from 0.1 μg/ml to 43.0 μg/ml, with an average of 12.6 ± 8.20 μg/ml (table 1). in 23 patients (41.1%), the phenytoin total plasma concentration was within the therapeutic range (10 μg/ml to 20 μg/ml) (figure 1). in 26 patients (46.4%), the plasma concentrations were subtherapeutic, that is, below 10 μg/ml, and in seven patients (12.5%) they were above 20 μg/ml. in 16 patients (28.6%), the predicted unbound plasma concentration of phenytoin was within the therapeutic range (1 μg/ml to 2 μg/ml). the unbound plasma concentration of phenytoin was subtherapeutic in eight patients (14.3%) and supratherapeutic in 32 patients (57.1%). figure 1: plasma concentrations of (a) total and (b) unbound phenytoin plasma concentration in 56 critically ill patients in the intensive care unit at windhoek central hospital (windhoek, namibia) from january 2013 to july 2019. the therapeutic range is indicated by dashed lines. for valproic acid, in 81 patients (87.1%), the total plasma concentration was below the therapeutic range of 50 μg/ml to 100 μg/ml (figure 2). binding of valproic acid to albumin in individual patients ranged from 9.0% to 39.0%. in 12 patients (12.9%), the total plasma concentration was within the therapeutic window, and no patients had a total plasma concentration of valproic acid above the therapeutic range. the average estimated unbound valproic acid plasma concentration was 24.0% (7.0% to 67.0%). in the majority of patients (n = 85, 91.4%), the unbound plasma concentration of valproic acid was within the therapeutic range (2.5 μg/ml to 20 μg/ml). in seven patients (7.5%), the unbound plasma concentration was below the therapeutic range, and in only one patient (1.1%) was it above the therapeutic range. figure 2: plasma concentrations of (a) total and (b) unbound valproic acid plasma concentration in 93 critically ill patients in the intensive care unit at windhoek central hospital (windhoek, namibia) from january 2013 to july 2019. the therapeutic range is indicated by dashed lines. the suggested therapeutic range for unbound valproic acid concentrations is quite wide because the lower and upper limits of therapeutic ranges reported in the literature differ substantially. discussion this retrospective study determined the plasma concentrations of phenytoin and valproic acid in critically ill patients at the icu of windhoek central hospital and found that the estimated unbound plasma concentration is a more accurate measure of the therapeutic plasma levels for the two drugs compared to total plasma concentration. using the total plasma concentration, most patients were within or below the phenytoin therapeutic range. in contrast, unbound plasma concentration showed that less than one-third of the patients were within the therapeutic range and over half were above. for valproic acid, total plasma concentrations placed most patients were below the therapeutic range. however, unbound plasma concentrations placed majority within the therapeutic range. noval et al. reported a 2-fold increase in critical care patients having phenytoin concentrations falling within the therapeutic range when unbound concentrations instead of total phenytoin concentrations were considered for dose adjustment.23 in our study, the percentage of patients within therapeutic concentrations using total plasma concentrations reduced from 41.1% to 28.6% when an unbound phenytoin concentration was used. thus, the use of total phenytoin concentrations suggests that dosing in these patients was at target therapeutic concentrations, suggesting no optimal patient dosage. the high variability in the binding of valproic acid to albumin observed in this study (9.0% – 39.0%) reflects reports in the literature, such as the 10.0% – 60.0% range reported in icu patients by lagneau et al.13 and the 15.0% – 89.0% range reported by riker et al.24 this high variability in plasma protein binding explains the marked difference in the percentage of patients having concentrations that were within the therapeutic range when total valproic acid was considered (12.9%) compared to when the unbound valproic acid concentrations were taken into account (91.4%). total valproate concentration is a poor predictor of the unbound drug concentration, even when correction is made for albumin.13 therapeutic drug monitoring of phenytoin and valproic acid by measuring unbound plasma concentrations is not widespread because of the additional time and cost implications. thus, some therapeutic drug monitoring services measure only the total drug plasma concentrations or estimate the unbound plasma drug concentrations. these equations, including those used in this study, estimate the unbound concentrations of phenytoin and valproic acid based on the patient’s measured total drug plasma concentration and serum albumin level.5,18,19,20,21,22 unfortunately, these equations are not very accurate and may underestimate the unbound plasma concentrations of phenytoin and valproic acid in critically ill patients.25,26,27,28,29,30 in addition, although the therapeutic plasma range for unbound phenytoin is relatively established (1 μg/ml – 2 μg/ml), various therapeutic ranges for the unbound plasma concentration of valproic acid have been proposed in the literature. however, more research is needed to determine the optimal unbound plasma concentration range for this anticonvulsant.31 furthermore, recent investigations on the predictive performance of these equations led to the conclusion that more complex, multivariate predictive equations may be required, which, in addition to the total drug plasma concentration and serum albumin level, considering the icu status of the patient, age, and blood urea nitrogen level.30,28 therapeutic drug monitoring of phenytoin and valproic acid should be based on unbound drug concentrations. the best way to monitor the unbound drug concentration would be by measuring it directly in ultrafiltrate obtained from serum to ensure that the accurate concentration of the free drug is determined and used to inform dosage adjustment. limitations the main limitation of this retrospective study is that the unbound plasma concentrations of phenytoin and valproic acid were not determined experimentally. however, because the albumin-based normalising equations that were used underestimate the patient’s unbound plasma concentrations of phenytoin and valproic acid, the discordance between total and unbound concentrations is less pronounced than it would be in reality. in addition, there was no follow-up to adjust the dosage regimen of these two drugs in the individual patients based on the observed total plasma concentrations. conclusion in conclusion, for therapeutic drug monitoring of phenytoin and valproic acid in critically ill patients, direct measurement of the unbound phenytoin and valproic acid concentration in plasma would be the best approach. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions b.s.s. supervised the study and reviewed the write-up of the manuscript. h.m. and l.i. collected the data and provided the draft manuscript. r.k.v. was the overall supervisor of the project and provided the final manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability data supporting the 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https://doi.org/10.1371/journal.pone.0238201. abstract introduction design and methods results conclusion acknowledgements references about the author(s) adrienne f.a. meyers national hiv & retrovirology laboratories, public health agency of canada, jc wilt infectious diseases research centre, winnipeg, canada department of medical microbiology and infectious diseases, university of manitoba, winnipeg, canada department of medical microbiology, university of nairobi, kenya michèle bergeron national hiv & retrovirology laboratories, public health agency of canada, jc wilt infectious diseases research centre, winnipeg, canada madhuri thakar national aids research institute, pune, india tao ding national hiv & retrovirology laboratories, public health agency of canada, jc wilt infectious diseases research centre, winnipeg, canada alexandre martel national hiv & retrovirology laboratories, public health agency of canada, jc wilt infectious diseases research centre, winnipeg, canada paul sandstrom national hiv & retrovirology laboratories, public health agency of canada, jc wilt infectious diseases research centre, winnipeg, canada department of medical microbiology, university of nairobi, kenya bharati mahajan national aids research institute, pune, india philip abraham national aids research institute, pune, india sandhya kabra national aids control organization, new delhi, india namita singh clinton foundation’s hiv/aids initiative, botswana trevor peter clinton foundation’s hiv/aids initiative, botswana terry b. ball national hiv & retrovirology laboratories, public health agency of canada, jc wilt infectious diseases research centre, winnipeg, canada department of medical microbiology and infectious diseases, university of manitoba, winnipeg, canada department of medical microbiology, university of nairobi, kenya department of immunology, university of manitoba, winnipeg, canada citation meyers afa, bergeron m, thakar m, et al. qasi: a collaboration for implementation of an independent quality assessment programme in india. afr j lab med. 2016;5(2), a442. http://dx.doi.org/10.4102/ajlm.v5i2.442 lessons from the field qasi: a collaboration for implementation of an independent quality assessment programme in india adrienne f.a. meyers, michèle bergeron, madhuri thakar, tao ding, alexandre martel, paul sandstrom, bharati mahajan, philip abraham, sandhya kabra, namita singh, trevor peter, terry b. ball received: 22 mar. 2016; accepted: 05 aug. 2016; published: 12 oct. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract objective: the hiv pandemic remains a significant global health concern. accurate determination of cd4+ t-cells in patient samples relies on reliable cd4 enumeration. the quality assessment and standardization programme for immunological measures relevant to hiv/aids (qasi) programme of the public health agency of canada provides clinical laboratories from resource-limited countries with a mechanism to evaluate the quality of cd4 testing and develop the implementation of an independent national external quality assessment (eqa) programme. this study describes how qasi helped develop the capacity for managing a sustainable national cd4 eqa programme in india. design: supported by the public health agency of canada and clinton foundation hiv/aids initiative, qasi engaged with the national aids control organization and the indian national aids research institute to assist in technology transfer in preparation for the implementation/management of an independent cd4 eqa programme. technology transfer training was provided to support corrective actions and to improve the quality of cd4 testing. inter-laboratory variation of eqa surveys between preand post-skill development was compared. results: prior to training, coefficient of variation values were 14.7% (mid-level cd4 count controls) and 39.0% (low-level). following training, variation was reduced to 10.3% for mid-level controls and 20.0% for low-level controls. conclusion: this training assisted the national aids control organization and the indian national aids research institute in identifying the information necessary for management of an eqa programme, and developed the foundation for india to provide corrective actions for sites with challenges in achieving reliable results for cd4 enumeration. this led to a demonstrable improvement in cd4 testing quality and illustrates how country-specific training significantly improved cd4 enumeration performance for better clinical management of hiv care in india. introduction infection by hiv and progression to death in the form of aids is responsible for significant morbidity and mortality worldwide, despite considerable efforts to prevent and/or treat the disease. recent figures from the world health organization estimate that 36.9 million people worldwide are living with hiv and more than two-thirds of these individuals are in resource-limited settings.1 while this number is an increase over the 2010 figures of 33.3 million, it represents the fact that more people are able to access and receive antiretroviral therapy (art) for their infections. according to unaids, 15.8 million people worldwide are accessing art.2 immune responses to hiv infection are largely mediated by cd4 t-lymphocytes – depletion of which reflects disease progression and aids.3,4,5 while the latest world health organization guidelines recommend treatment for all infected with hiv, irrespective of cd4 counts, many resource-limited countries have yet to adopt these recommendations due to constraints, and cd4 enumeration remains the hallmark for staging and monitoring patients on art.6 new targeted efforts aimed at the hiv epidemic are focused on the 90-90-90 initiative proposed by unaids that by 2020, 90% of individuals living with hiv will know their status, 90% of those infected with hiv will have access to ongoing art and 90% of those on art will achieve suppression of hiv infection.7 as such, it is critical to properly and rapidly detect hiv infection and responses to therapy. reliable cd4 measurements are critical to clinical decision-making in situations where viral load data is unavailable.8,9 the most common technology utilised for cd4 t-cell enumeration is flow cytometry. the complexity of this technology and the lack of a cd4 gold standard against which to measure accuracy can produce misleading results that affect medical decisions in resource-limited areas where infrastructure and levels of training required can be a challenge.10,11 to achieve accuracy and reproducibility of laboratory based testing, both internal and external quality control measures are essential.8,9,12 since 1996, the national laboratory for hiv immunology from the public health agency of canada has been providing the quality assessment and standardization programme for immunological measures relevant to hiv/aids (qasi) in an effort to assist resource-limited regions with cd4 testing.13 currently over 1400 laboratories from more than 50 countries are enrolled and actively participating in qasi. in addition to the provision of an external quality assessment (eqa) programme for relative and absolute cd4 enumeration, qasi also encompasses several elements that constitute a comprehensive quality management system. qasi has developed a framework to train and support implementation of sustainable quality assessment programmes in resource-limited countries. qasi provides free quality control panels; individual, national and global performance assessments; and supports capacity building activities toward quality improvement such as remedial action plans, biotechnology transfer workshops based on the ‘training-of-trainers’ model, and web-based platforms to assist with quality management activities at a national level. in 2005, the national aids control organization (naco) of the government of india joined qasi to help initiate an external quality assessment programme for cd4 count enumeration in india, with support from the clinton health access initiative. the indian national aids research institute (nari) in pune, india, being the apex laboratory in india, acted as a coordinator for the distribution of the qasi proficiency panel to the enrolled laboratories of the national hiv/aids programme. in september 2009, naco, nari and the clinton foundation engaged with qasi on technology transfer so that india could implement its own national cd4 eqa programme. a technology-transfer workshop was conducted by qasi to nari. additionally, naco and nari have also initiated focused training workshops for the cd4 testing laboratories enrolled as participants. this report describes how tailored and concerted intervention strategies can significantly improve laboratory performance at the national level, highlights the critical elements covered during training in preparation for launch of the indian national eqa program for cd4 enumeration, and provides insight to lessons learned during the process. design and methods cd4 eqa was initiated in india in 2005 with support from the clinton health access initiative. three times a year, nari distributed proficiency panels provided by qasi, consisting of two specimens, to all 219 enrolled cd4 laboratories of the indian national hiv/aids programme. the cd4 testing centres processed the samples as per their established procedures and reported the results back to qasi, with a copy to nari. qasi analysed the performance of all the laboratories and reported back to nari the individual, national, and global performance reports, as well as a set of recommendations for remedial action and follow-up where required. to ensure the sustainability and ongoing impact of this initiative, naco/nari engaged with qasi to assist in a series of technical consultations and discussions, culminating in a training-of-trainers workshop held in 2009. the goal of these meetings was for qasi to assist nari with planning procedures so that india could implement and manage an independent national cd4 eqa programme. technology transfer training qasi cd4 enumeration workshop: september 2009 the qasi cd4 technology transfer workshop was conducted in september 2009 at the nari in pune, india. six people from nari, including the laboratory-in-charges, technical and quality managers, and statisticians, participated in the workshop. additionally, three laboratory-in-charges from the national reference laboratory participated in the workshop. workshop objectives included skills transfer related to specific instruments, logistics of panel selection and procurement, policy development (eqa management team, participants, enrolment, standard operating procedures, panel testing, shipment, reporting, remedial action response), capacity building (planning and development of budget, identification of financial support), and training on the technical capacity, operation and management of the qasi web-based software application. qasi has the capacity to offer instrument-specific training for all cd4 technologies in use. the workshop was designed with this in mind so that all instruments in use throughout the country were included in the training modules, thus allowing the team to develop specific skills to evaluate and assist performance by participants in a precise and specialised manner. this training was also critical to the adaptation of the qasi eqa software to the india-specific management of the eqa programme, so as to enable an independent national cd4 eqa programme for naco’s hiv care and treatment programme. the design of the technology transfer workshop was established by first investigating potential sources of variation in cd4 enumeration within the country by means of examination of eqa results for trends and bias, followed by a comparison of national performance with global performance data. the workshop agenda was developed and approved based on country-specific details, including instruments used by participants, mechanisms of communication with participants, and modes of transportation for distribution of panels/collection of data. the training modules, which addressed internal and external quality control activities, included practical sessions targeting troubleshooting and quality management using a dedicated web application. in total, nine people with advanced instrument-specific knowledge were trained through problem-solving exercises and quality-management simulation activities using the qasi web platform. nacoand nari-designed laboratory technology training: november–december 2009 to assist in corrective actions and to improve the quality of cd4 testing centres, focused training sessions for individual cd4 testing laboratories were initiated by nari and naco in april 2009, which were continued after the technology transfer workshop delivered by qasi. eight training sessions were conducted with the collaboration of instrument manufacturers for more than 100 cd4 testing laboratories. a total of 100 technicians from the testing centres participated in the training. the agenda was tailor-made for facscalibur™, facscount™ and cyflow® counter users, who represented more than 98% of the enrolled cd4 testing laboratories in india. the workshop included hands-on equipment training, troubleshooting procedures and pipette calibration. the training module also included internal quality control procedures, information about participation in eqa and response to remedial action in the event of unsatisfactory performance. two additional sessions were conducted for the facscalibur users, specifically to address gating issues related to multiset™ analysis of eqa-stabilised blood samples. evaluation of performance pre-/post-initiation of training of technologists to study the impact of comprehensive training on cd4 enumeration abilities, national performance was evaluated based on the results generated from six consecutive participations in qasi surveys (qasi surveys 19 [october 2008] to survey 24 [june 2010]). each survey provided two controls of commercially-stabilised whole blood product for cd4 testing: low-level (100–150 cells/µl) and mid-level (450–700 cells/µl). the first three sessions took place prior to initiation of the technologist training, and the latter three took place after the training of all technologists from 100 laboratories was completed. national performance was evaluated against the global performance of qasi participants for each session examined. results pre-technology and targeted training transfer data from qasi surveys 19 to 21 (october 2008, february 2009 and june 2009) were used to assess pre-workshop and targeted training performance in india. in that period, more than 50 laboratories in india reporting absolute counts participated in qasi. the main cd4 technologies used were facscalibur (n = 25; 22%), facscount (n = 48; 43%) and cyflow counter (n = 39; 35%). as depicted in table 1, not all instruments were available for use in the qasi sessions for both preand post-technology training. reasons for this involved issues with the instruments (i.e., breakdown in machine function). initial observations of the submitted data showed that the inter-laboratory coefficient of variation (%cv) for both relative and absolute cd4 measurements in india was higher than values obtained by the global performance, especially with low-level cd4 count controls (figures 1 and 2). further analysis identified acute problems with specific technologies. the largest %cv levels of variation were identified by participants using the facscalibur, with 25.6% for mid-level cd4 count controls and 55.4% for low-level controls. cyflow counter measurements elicited 14.4% for mid-level controls and 26.3% variation for low-level controls. the best performance was achieved among the facscount users, who had %cv that were less than 8.5% for both levels of cd4 control samples. these data suggested the focus should be on providing remedial action for users of facscalibur and cyflow counter, due to the higher levels of variation with these two technologies in comparison with the facscount performance (table 1). figure 1: prevs post-training national and global cd4 absolute count performance. comparison of national and global performance across six consecutive surveys, reflecting the impact of technology transfer provided by both qasi and nari between surveys 21 and 22. pre-technology transfer reflects qasi sessions 19 through 21 (october 2008, february 2009, june 2009). qasi advanced workshop training was held in september 2009 at nari in pune, india. post-technology transfer reflects qasi sessions 22 through 24 (october 2009, february 2010, june 2010). group mean cd4 count, standard deviation (error bars) and %cv are illustrated for mid-level (a) and low-level (b) cd4 count controls. note the drop in %cv for india from 14.7% to 10.3% (a) and from 39.0% to 20.0% (b). figure 2: prevs post-transfer national and global cd4 percentage performance. comparison of national and global performance across six consecutive surveys reflecting the impact of technology transfer provided by both qasi and nari between surveys 21 and 22. pre-technology transfer reflects qasi sessions 19 through 21 (october 2008, february 2009, june 2009). qasi advanced workshop training was held in september 2009 at nari in pune, india. post-technology transfer reflects qasi sessions 22 through 24 (october 2009, february 2010, june 2010). group mean cd4 lymphocyte percentages, standard deviation (error bars) and %cv are illustrated for mid-level (a) and low-level (b) cd4 count controls. note the drop in %cv for india from 16.4% to 8.3% (a) and from 38.6% to 20.4% (b). table 1: performance of specific cd4 technologies in india for lowand mid-level cd4 count controls at preand post-technology transfer. in examining protocols utilised by sites equipped with facscalibur instruments, it was determined they were primarily relying on the use of the automated attractor multiset software (bd biosciences). in qasi’s experience, the attractor algorithm associated with the software can have a limited capacity to adapt to some stabilised whole blood products provided as eqa quality control panels.11,14,15 fluorescence intensities are significantly different in these specimens as compared with fresh whole blood samples. this characteristic is not, however, exclusive to stabilised controls, as patient samples may behave similarly due to drug therapy or exposure to environmentally hostile conditions during transit. both nari and qasi dedicated special attention to facscalibur users during the workshop and in the follow-up training period, emphasising the use of the manual analysis mode, where attractor regions are required to be readjusted in order to achieve accurate values. with respect to cyflow counter users, several laboratories were using a single cd4 antibody on a single-parameter histogram. training raised the importance of changing the cursor position of the gating strategy in order to accurately identify cd4 t-cell expression and reduce monocyte contaminants. the option of utilising a cd4 x side scatter heterogeneous algorithm was also introduced. these issues were then emphasised in the subsequent training workshops organised for the cd4 testing laboratory participants. post-technology and targeted training transfer post-technology transfer data analysis was based on qasi surveys 22 to 24 (october 2009, february 2010, june 2010). the analysis demonstrated that the %cv for both cd4 percentages and cd4 absolute counts improved following the training programme. the average %cv of reported cd4 percentages before the completion of all training workshops was nearly twice the average %cv of results obtained in surveys following the training; 16.4% versus 8.3% for the mid-level cd4 control and 38.6% versus 20.4% for the low-level control (figure 2). as inter-laboratory variation improved dramatically, so did accuracy, closing the gap between the global mean value and the national mean value. the same trend was observed with absolute cd4 measurements, with an important reduction in the %cv values following workshop training (figure 1). prior to training, mean %cv values were 14.7% for the mid-level cd4 controls and 39.0% for the low-level controls. following training, variation was reduced to 10.3% for mid-level and 20.0% for low-level controls. box 1: lessons learned. thus, both facscalibur and cyflow counter users showed dramatic improvement in their performance following the workshop and subsequent training. the combined training provided by both nari and qasi was successful in addressing countryand laboratory-specific issues, resulting in quality improvements in cd4 enumeration methodologies used by more than half of the participating indian laboratories and thus increasing their ability to provide better clinical and prognostic data for the management of hiv-infected patients. a dedicated web application was developed as an extension of the web platform application utilised by qasi for india in order to assist nari with pilot trials to pave the way for the eventual launch of their first independent national programme. in preparation for the launch, nari conducted six pilot surveys while still participating in qasi. this transitional phase allowed the indian eqa team to familiarise themselves with software functions, while also assessing and optimising all aspects of the logistics, such as selection and acquisition of quality control panels, identification and distribution of transport tubes, and policy development on subjects such as reporting and remedial action, so as to ensure a smooth transition during national eqa implementation. conclusion qasi has more than 20 years of experience in assisting the implementation of activities for quality improvement and best practice for quality assurance in developing countries and, as a part of its overall objective, qasi is dedicated to capacity building and training for countries wishing to develop and implement their own national or regional quality assessment programmes for cd4 enumeration. this report illustrates that specifically tailored workshops designed by qasi, and follow-up training provided by naco and nari, led to increased cd4 testing accuracy and provided a foundation for the development and implementation of an independent national quality assessment programme led by nari and naco in india. naco and nari launched the indian cd4 national programme in june 2011, and it has been operating independently since then. the importance of targeted training to diminish interand intra-laboratory variation in cell enumeration is well-established and demonstrates that training works. the joint efforts of qasi, naco, nari and the clinton health access initiative in conducting the technology transfer and specific training were extremely useful in improving laboratory performance and providing reliable results and, presumably, better and more effective patient care. in addition, the workshop training provided led to the development of a local eqa management team for the implementation of an independent national eqa programme for cd4 enumeration. the qasi workshop identified key areas to be considered for programme development, including mechanisms for financial sustainability, selection of an appropriate source material for proficiency panels, technical expertise in website management, data collection and analysis, reporting methods, and policies for provision of remedial action for those participants with unsatisfactory results. this report highlights a phased approach to launching independent eqa programmes for cd4 enumeration, which evolves from initial participation in an eqa programme to gradual stages of increased responsibility in providing services to a country or region until the eventual launch of a fully functional, independent national/regional programme. this model that qasi utilises includes tailored, country-specific training workshops that highlight the strengths and challenges a specific country faces as they prepare for programme development. with the gradual transition to independence, countries are able to properly develop methods and policies that will allow for sustainable programme provision to participants and develop their own unique training modules specific for participants in the region. in addition, the training and feedback provides early detection of systematic problems associated with reagents, instruments, or operations, provides objective evidence of testing quality, indicates areas that need improvement, and identifies ongoing training needs. this method helps to better identify the resources and/or needs for developing laboratory quality standards and eqas, and guides the organisations in providing support where significant gaps are identified. ultimately, the ability of a country to develop their own national eqa programme for hiv treatment and care is tightly linked to both improved diagnostics and clinical management of patients. our experience demonstrates the impact of effective interventions and the importance of collaboration and commitment of a national reference laboratory and partners to improving healthcare systems in a country. in conclusion, the data confirm that participation in an eqa programme results in better diagnostic accuracy, and that technology transfer around eqa programmes can be transferred reproducibly. acknowledgements competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. sources of support funded by the public health agency of canada and the national aids control organization of the government of india. authors’ contributions a.f.a.m., p.s., t.b.b., a.m., n.s. and t.p. made conceptual contributions. m.b. and m.t. were project leaders, t.d. and b.m. were responsible for experimental and project design. m.b., m.t., t.d., p.a. and s.k. led workshop sessions and evaluated results, performance and outcomes. references joint united nations programme on hiv/aids (unaids). aids by the numbers 2015 [document on the internet]. c2015 [cited 2016 mar 18]. available from: http://www.unaids.org/sites/default/files/media_asset/aids_by_the_numbers_2015_en.pdf unaids. fact sheet 2016 [page on the internet]. c2015 [cited 2016 mar 18]. available from: http://www.unaids.org/en/resources/campaigns/howaidschangedeverything/factsheet de wolf f, roos m, lange jm, et al. decline in cd4+ cell numbers reflects increase in hiv-1 replication. aids res hum retroviruses. 1988;4(6):433–440. http://dx.doi.org/10.1089/aid.1988.4.433 mellors jw, muñoz a, giorgi jv, et al. plasma viral load and cd4+ lymphocytes as prognostic markers of hiv-1 infection. ann intern med. 1997;126(12):946–954. o’brien wa, hartigan pm, daar es, et al. changes in plasma hiv rna levels and cd4+ lymphocyte counts predict both response to antiretroviral therapy and therapeutic failure. va cooperative study group on aids. ann intern med. 1997;126(12):939–945. world health organization. guidelines on when to start antiretroviral therapy and on pre-exposure prophylaxis for hiv. september 2015 [document on the internet]. c2015 [cited 2016 mar 18]. available from: http://apps.who.int/iris/bitstream/10665/186275/1/9789241509565_eng.pdf unaids. 90-90-90: an ambitious treatment target to help end the aids epidemic [document on the internet]. c2014 [cited 2016 mar 18]. available from: http://www.unaids.org/sites/default/files/media_asset/90-90-90_en_0.pdf mandy f, bergeron m, houle g, et al. impact of the international program for quality assessment and standardization for immunological measures relevant to hiv/aids: qasi. cytometry. 2002;50(2):111–116. glencross dk, aggett hm, stevens ws, et al. african regional external quality assessment for cd4 t-cell enumeration: development, outcomes, and performance of laboratories. cytometry b clin cytom. 2008;74 (suppl 1):s69–79. http://dx.doi.org/10.1002/cyto.b.20397 barnett d, walker b, landay a, et al. cd4 immunophenotyping in hiv infection. nat rev microbiol. 2008;6(11 suppl):s7–15. http://dx.doi.org/10.1038/nrmicro1998 crowe s, turnbull s, oelrichs r, et al. monitoring of human immunodeficiency virus infection in resource-constrained countries. clin infect dis. 2003;37(suppl 1):s25–35. barnett d, granger v, kraan j, et al. reduction of intraand interlaboratory variation in cd34+ stem cell enumeration using stable test material, standard protocols and targeted training. dk34 task force of the european working group of clinical cell analysis (ewgcca). br j haematol. 2000;108(4):784–792. bergeron m, ding t, houle g, et al. qasi, an international quality management system for cd4 t-cell enumeration focused to make a global difference. cytometry b clin cytom. 2010;78(1):41–48. http://dx.doi.org/10.1002/cyto.b.20487 ding t, bergeron m, seely p, et al. compatibility of stabilized whole blood products with cd4 technologies and their suitability for quality assessment programs. plos one. 9(8):e103391. http://dx.doi.org/10.1371/journal.pone.0103391.ecollection2014. diallo to, bergeron m, seely p, et al. automation for clinical cd4 t-cell enumeration, a desirable tool in the hands of skilled operators. cytometry b clin cytom. 2016 mar 15. http://dx.doi.org/10.1002/cyto.b.21370. [epub ahead of print] article information authors: mohammed abdel-maksoud1 rania abdel-khalek2 atef el-gendy2 rawia f. gamal3 hemmat m. abdelhady3 brent l. house1 affiliations: 1global disease detection and response program, us naval medical research unit, egypt 2bacterial and parasitic disease research program, us naval medical research unit, egypt 3faculty of agriculture, ain shams university, department of microbiology, egypt correspondence to: mohammed abdel-maksoud email: mohamed.abdelmaksoud.eg@med.navy.mil postal address: us naval medical research unit number 3, commanding officer, psc 452, box 5000, code 304 fpo ae 09835-0007 dates: received: 09 dec. 2013 accepted: 02 oct. 2014 published: 14 may 2015 how to cite this article: abdel-maksoud m, abdel-khalek r, el-gendy a, gamal rf, abdelhady hm, house bl. genetic characterisation of multidrug-resistant salmonella enterica serotypes isolated from poultry in cairo, egypt. afr j lab med. 2015;4(1), art. #158, 7 pages. http://dx.doi.org/10.4102/ajlm.v4i1.158 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. genetic characterisation of multidrug-resistant salmonella enterica serotypes isolated from poultry in cairo, egypt in this original research... open access • abstract • introduction • research methods and design    • isolation and identification of salmonella from poultry meat, egg and faecal samples    • serogrouping and serotyping of salmonella isolates    • antibiotic susceptibility testing and detection of extended-spectrum β-lactamase production    • detection of antimicrobial resistance genes    • detection and characterisation of integrons    • statistical analysis • results • discussion • limitations • conclusion • acknowledgements    • competing interests    • disclaimer    • copyright assignment statement    • authors’ contributions • references abstract top ↑ background: food-borne diseases pose serious health problems, affecting public health and economic development worldwide. methods: salmonella was isolated from samples of chicken parts, skin samples of whole chicken carcasses, raw egg yolks, eggshells and chicken faeces. resulting isolates were characterised by serogrouping, serotyping, antimicrobial susceptibility testing and detection of extended-spectrum β-lactamase (esbl) production. antibiotic resistance genes and integrons were identified by polymerase chain reaction (pcr). results: the detection rates of salmonella were 60%, 64% and 62% in chicken parts, skin, and faeces, respectively, whereas the egg yolks and eggshells were uniformly negative. salmonella kentucky and s. enteritidis serotypes comprised 43.6% and 2.6% of the isolates, respectively, whilst s. typhimurium was absent. variable resistance rates were observed against 16 antibiotics; 97% were resistant to sulfamethoxazole, 96% to nalidixic acid and tetracycline and 76% to ampicillin. multidrug resistance was detected in 82% (64/78) of the isolates and esbl production was detected in 8% (6/78). the β-lactamase blatem-1 gene was detected in 57.6% and blashv-1 in 6.8% of the isolates, whilst the blaoxa gene was absent. the sul1 gene was detected in 97.3% and the sul2 gene in 5.3% of the isolates. sixty-four of the 78 isolates (82%) were positive for the integrase gene (int i) from class 1 integrons, whilst int ii was absent. conclusion: this study reveals the presence of an alarming number of multidrug-resistant salmonella isolates in the local poultry markets in cairo. the high levels of drug resistance suggest an emerging problem that could impact negatively on efforts to prevent and treat poultry and poultry-transmitted human diseases in egypt. introduction top ↑ food-borne diseases caused by non-typhoid salmonella present an important public health problem that impacts significantly on the economy in many parts of the world. the main source of infection is food of animal origin, such as poultry, eggs, milk, beef and pork. in addition, fruits and vegetables have been implicated as vehicles for salmonella transmission.1 antibiotics are used extensively to prevent or treat microbial infections in veterinary medicine. microbial infections may be detected at various levels in animal products and disseminated into the environment when manure is applied to fields.2 in the last two decades, antimicrobial resistance has emerged quickly amongst salmonella isolates, creating a serious health hazard worldwide.1 although fluoroquinolones have recently been used as the drug of choice to treat gastrointestinal infections in humans, resistant strains have since emerged and have been associated with increased illness and death.3 whereas fluoroquinolones are contraindicated because of toxicity, cephalosporins have also been used to treat salmonellosis, particularly in children. however, resistance to this class of drugs appeared in 1992, mainly because of the emergence of extended-spectrum β-lactamases (esbls).4 esbls comprise rapidly evolving groups of β-lactamases, capable of hydrolysing (and thus inactivating) third-generation cephalosporins and aztreonam, which are inhibited by the β-lactamase inhibitor, clavulanic acid.5 the esbls are encoded frequently by genes located on r-plasmids, which often carry additional genes encoding resistance to other drug classes (e.g. fluoroquinolones and aminoglycosides).6 esbl-producing strains of bacteria are emerging worldwide, particularly amongst the enterobacteriaceae7 where the exchange of multidrug-resistant (mdr) plasmids between members of the family is common. this mdr can lead to severe limitations in treatment options for infection with these microorganisms, which are responsible for nearly half of all human infections.8 the presence of integron gene sequences has been identified as a primary method by which bacteria can acquire existing antimicrobial resistance genes. each integron sequence is unique in that it acts as a site-specific recombination system capable of capturing or excising novel genetic elements called ‘gene cassettes’. these gene cassettes are promotorless genes with a recombination site known as a 59-base element or attc located at the 3′ end of the gene. the gene cassettes code for a wide range of antimicrobial resistant determinants.9 only class 1 and 2 integrons have been detected in salmonella, with class 1 being the most predominant.10 in egypt, antibiotic resistance has been reported amongst human salmonella isolates, including salmonella enterica serovar typhi (s. typhi) and other diarrhoeagenic strains.11,12 however, with no national salmonella surveillance centre to provide reliable statistical data, little is known about food-borne salmonellosis in egypt. the present study was undertaken to determine the contamination rates of different salmonella serotypes in chicken eggs, raw chicken meat and related environmental samples in poultry markets in cairo, egypt; and to characterise the identified isolates by serotype, antimicrobial susceptibility testing (ast) profiles and esbl production. we also examined selected isolates to identify the presence of common antibiotic-resistance genes and integrons. research methods and design top ↑ isolation and identification of salmonella from poultry meat, egg and faecal samples a total of 165 samples were collected between december 2011 and may 2012 from 18 poultry markets (mostly street markets and retail shops that sell meat and live birds) distributed throughout 5 geographical locations in cairo governorate. the food samples were collected from 62 chicken meat parts (20 boneless breasts, 19 cloacae, 10 livers, 8 gizzards and 5 wings), 22 skin pieces from slaughtered birds, 30 raw egg yolks and shells from another 30 eggs. the faecal samples were collected from 21 separate chicken faeces specimens. the chicken parts and carcass samples were taken from different birds; all samples were cultured within 2 hours. the faecal samples were obtained from the same 18 poultry markets. salmonella strains were isolated and identified according to standard methods,13 and colonies that exhibited typical biochemical reactions were further confirmed as salmonella using the api 20e identification kit (biomerieux, craponne, france). serogrouping and serotyping of salmonella isolates biochemically identified salmonella isolates were serogrouped initially by slide agglutination using commercially-available salmonella o antiserum (difco laboratories, detroit, mi, united states). because of funding limitations, only serogroup b, c2 and d isolates were then serotyped for s. typhimurium, s. enteritidis and s. kentucky according to the kauffman white scheme,14 using salmonella h antisera (sifin, germany; statens, denmark). antibiotic susceptibility testing and detection of extended-spectrum β-lactamase production antimicrobial susceptibilities to tetracyclines (tetracycline), sulphonamides (sulfamethoxazole trimethoprim/sulphamethoxazole), quinolones (nalidixic acid), penicillins (ampicillin), penicillin/β -lactamase inhibitor combinations (ticarcillin/clavulanate, ampicillin/sulbactam), phenicols (chloramphenicol), fluoroqinolones (ciprofloxacin), aminoglycosides (streptomycin, gentamicin, amikacin), monobactams (aztreonam), cephalosporins (cefotaxime, ceftriaxone, ceftazidime, cefepime) and carbapenems (imipenem) were determined using kirby-bauer disc diffusion according to clinical and laboratory standards institute (clsi)15 guidelines. in addition, minimum inhibitory concentration (mic) was determined using e-test methods (ab biodisk, solana, sweden), also according to clsi guidelines. mdr salmonella was defined as any isolate that showed resistance to at least three different classes of antibiotics.11 screening for esbl production in salmonella isolates was done using the standard procedure of measuring the zones of inhibition surrounding cefotaxime and ceftazidime discs versus cefotaxime-clavulanic acid and ceftazidime-clavulanic acid discs, respectively. any isolate with a ≥ 5 mm increase in zone diameter for either antibiotic tested in combination with clavulanic acid, versus without clavulanic acid, was considered to be an esbl producer.15 the reference strains escherichia coli atcc 25922 and staphylococcus aureus atcc 25923 were used to verify the quality and accuracy of testing procedures. detection of antimicrobial resistance genes salmonella-isolate dna was purified using the dna-boiling method suggested by sambrook, fritsch and maniatis.16. the following genes implicated in antimicrobial resistance were detected by pcr amplification: for β-lactam resistance – blatem-1, blashv-1 and blaoxa-1; for sulphonamide resistance – sul1 and sul2. the primer sets and assay conditions used for amplification were as described previously.17,18 detection and characterisation of integrons the presence of class 1 and 2 integrase-coding genes (int i and int ii) were detected by pcr with specific primers.17,19 primers 5’-conserved segment (cs) and 3’-cs described by levesque,20 targeting the inserted gene cassette regions of class 1 integrons, were used to determine these regions. statistical analysis for statistical analyses to detect significant differences between antibiotic resistance rates, p values were determined by using student's t-test in microsoft® office excel 2010 (microsoft corp., redmond, wa, united states). results top ↑ salmonella isolates were recovered from 64 of the 165 samples collected, including 60% of chicken meat samples, 64% of chicken carcasses (skin) samples and 62% of chicken faeces samples. no salmonella was isolated from raw egg yolk or eggshell samples. serogroups identified amongst the salmonella isolates were b, c1, c2, and d. fifty-one samples yielded isolates from one serogroup and 12 samples yielded isolates from 2 serogroups, whilst one sample from skin yielded 3 serogroups. of the 78 salmonella isolates obtained, group c2 was the predominant group found in chicken meat (table 1). serotypes identified included s. kentucky (43.6%) and s. enteritidis (2.6%) (table 2). s. typhimurium was not identified. table 1: distribution of salmonella serogroups isolated from 144 poultry samples and 21 faecal samples collected between december 2011 and may 2012. table 2: distribution of serotypes for the 78 salmonella isolates recovered from poultry and faecal samples collected between december 2011 and may 2012. overall, there was a high level of antibiotic resistance found amongst the salmonella isolates (table 3). resistance was detected to 16 out of 18 antibiotics tested, whilst all salmonella isolates were susceptible to imipenem and cefepime. isolates demonstrated high levels of resistance to sulfamethoxazole (97%), nalidixic acid and tetracycline (96%), ampicillin (76%), ticarcillin/clavulanate (67%), chloramphenicol (56%) and ciprofloxacin (46%). multidrug resistance was observed amongst 82% (64/78) of the isolates, with 59 isolates (76%) resistant to more than 5 antibiotics. esbl production was detected in 8% (6/78), with all 6 being highly resistant to multiple antibiotics when compared to non-esbl producing strains. when compared with other salmonella serotypes, the s. kentucky isolates showed higher resistance rates to the majority of antibiotics tested, reaching statistical significance against ciprofloxacin and ticarcillin/clavulanate (p < 0.01). sixteen (46%) s. kentucky strains were resistant to at least 8 antibiotics. table 3: percent antibiotic resistance and mic range of s. kentucky, other salmonella serotypes and esbl-producing salmonella isolates from poultry meat and faecal samples. amongst the 75 strains resistant to nalidixic acid, 36 were resistant to the related ciprofloxacin (mic range of 4–12 μg/ml). imipenem showed the lowest mic values, followed by cefepime and amikacin, all of which were found to be effective against salmonella strains isolated from poultry in cairo. on the other hand, the highest mic values were obtained against streptomycin, followed by nalidixic acid and ampicillin (table 3). table 4 lists the resistance genes detected in the salmonella isolates. the most frequent β-lactam gene identified amongst ampicillin resistant isolates was blatem-1, detected in 57.6% of the isolates, followed by blashv-1 which was identified in 6.8%. the blaoxa-1 gene was not detected in any isolate in this study. regarding sulphonamide resistance genes, the presence of the sul1 gene was detected in 97.3% of the isolates and the sul2 gene in 5.3%. four isolates possessed both the sul1 and sul2 genes (table 4). table 4: antimicrobial resistance genes and the resistance phenotype of s. enterica strains isolated from poultry meat and faecal samples (n = 78). sixty-four of the 78 isolates (82%) were positive for the int i gene, whilst int ii was absent. the 5′and 3′-cs regions were identified in 46.8% of the int i positive isolates. eight types of class 1 integrons were detected for the salmonella spp. isolates, including the 1950 bp, 1550 bp, 1200 bp, 1100 bp, 1000 bp, 700 bp, a combination of 950 bp and 1200 bp and a combination of 1100 bp and 1550 bp integrons. five s. kentucky isolates resistant to ciprofloxacin carried the 1950 bp class 1 integron (table 5). table 5: characteristics of class 1 integron-carrying multidrug-resistant s. enterica strains isolated from poultry meat and faecal samples (n = 64). discussion top ↑ the objectives of this study were to determine the frequency of salmonella contamination of chicken eggs, meat and faeces in poultry markets in cairo, egypt and to identify prevalent serotypes and antibiotic susceptibility profiles, including esbl production. the results from this study revealed levels of salmonella-contamination in fresh chicken meat of 60% – 64%, which are higher than those previously reported in assiut, egypt from frozen chicken legs and fillet samples (36% – 52%);21 in senegal from chicken carcasses (32%);22 and in ethiopia from raw chicken meat and giblets (18%).23 in contrast to a study conducted by del cerro et al.24 which reported that faeces from chickens were positive for salmonella by culture in 39% of the samples tested, our study demonstrated a 62% positivity rate for salmonella isolation from faecal specimens. the high contamination rates seen in this study may, at least in part, be explained by the lack of hygienic slaughtering processes that occur commonly at small shops, away from the modern abattoirs available for mass slaughtering of poultry. at these shops, slaughtering is manual, rudimentary and may take place either indoors or outdoors. usually, one person provides all the labour, including live bird care, cleaning, slaughtering, de-feathering and evisceration, increasing the likelihood of cross-contamination amongst birds. in this study, serogroups b, c1 and c2 accounted for 97% of the isolates from chicken meat. a similar study performed in the pacific northwest, in the united states25 also found that serogroups b and c comprised the majority (95%) of all salmonella isolated from poultry and the poultry environment. a study in saudi arabia26 reported that 64% of the isolates from poultry and the poultry environment were from groups b and c. in the current study, there were only two isolates assigned to serogroup d, subsequently serotyped as s. enteritidis. in humans, s. enteritidis and s. typhimurium have been reported to be the two most prevalent salmonella serotypes in many regions of the world.26 in addition, a study in turkey demonstrated that s. enteritidis was the most prevalent salmonella serotype isolated from chicken meat.27 however, a study in senegal identified only 6 s. enteritidis serotypes out of 90 salmonella strains isolated.28 another study in sudan identified 2 chicken and 2 human origin s. kentucky strains resistant to both ciprofloxacin and norfloxacin out of 64 salmonella isolates studied.29 interestingly, studies in france,30 switzerland31 and the slovak republic32 have reported that infection with s. kentucky strains resistant to ciprofloxacin was associated with travel to egypt, morocco and other countries in the north african region. corroborating these studies, our results indicate that the most prevalent serotype in cairo, egypt is s. kentucky, with high rates of resistance to ciprofloxacin. s. enteritidis was isolated at a very low rate, and s. typhimurium was not detected at all. this is comparable to the results from senegal,27,29 where s. kentucky was also found to be the most prevalent serotype (30% of the total isolates). the observed low isolation rates for s. enteritidis and s. typhimurium in this study may result from replacement by other serotypes (54% were not serotyped in this study). the remarkably high rates of antibiotic resistance exhibited by salmonella strains from this study, particularly against sulfamethoxazole (97.4%), nalidixic acid (96.2%), tetracycline (96.2%), ampicillin (75.6%) and streptomycin (35.9%), are probably because of the early introduction and consequent widespread use of these antibiotics in veterinary and human medicine in egypt. the high resistance rates to nalidixic acid and ciprofloxacin are of particular note, since quinolones have been considered one of the last options for the treatment of mdr salmonella. all s. kentucky strains tested in this study were mdr and demonstrated significantly higher rates of resistance than other salmonella serotypes to ciprofloxacin (97% vs. 2.5%; p < 0.01) and ticarcillin/clavulanate (94% vs. 42.5%; p < 0.01). the correlation between antibiogram and serotype suggests poor infection control practices in the poultry production industry, which may facilitate the spread of these mdr s. kentucky strains. the most common mode of bacterial-acquired resistance to β-lactam antibiotics is the β -lactamase enzyme.34 in this study, 30 of 48 poultry meat isolates and 4 of 11 faeces isolates that were ampicillin resistant possessed the blatem gene. this result is in agreement with other findings.1,17 recently, esbl acquisition rates by salmonella, in particular, have arisen worldwide. in an earlier study conducted on salmonella isolates from poultry in egypt, 5% of the isolates belonging to serovar poona produced esbls.12 in this study 8% (n = 6) of salmonella isolates demonstrated esbl production, two of them belonging to serovar kentucky. the detection of an esbl phenotype in poultry meat and faecal samples in this study may indicate a lack of infectious disease barriers amongst clinics, humans and animals. in addition, approximately half of all antibiotics produced worldwide (many of which are used routinely in humans) are used in animals to prevent infection and consequently improve production.35 unfortunately, this leads to the development of resistant bacteria in animals that can infect humans directly or transfer antibiotic resistance genes to other human pathogens.36 sulphonamides are amongst the most commonly-used antibiotics for food animal production worldwide.37 these compounds are bacteriostatic antimicrobial drugs that act by means of competitive inhibition of the enzymes involved in the synthesis of tetrahydrofolic acid. sulphonamides compete with the structural analogue p-aminobenzoic acid binding to dihydropteroate synthetase (dhps), a catalytic enzyme in the folic acid biosynthesis pathway, thus inhibiting the formation of dihydrofolic acid.38 sulphonamide resistance in salmonella isolates has been attributed to the presence of an extra sul gene, which expresses an insensitive form of dhps. in this study, the presence of the sul1 gene was detected in 74 of 76 sulphonamide-resistant isolates. the pcr results were consistent with the antimicrobial susceptibility phenotypes; the sul1 and/or sul2 genes were detected in 97.4% of the sulphonamide-resistant salmonella isolates. other studies have found this gene to be present at moderate to high rates in retail meats and other foods.1,17,39 integrons are genetic elements that are able to recognise and capture mobile gene cassettes carrying the antibiotic resistance genes, which leads to mdr distribution and the subsequent limitation of treatment options for infectious diseases.40 in this study, pcr screening results of 78 salmonella isolates detected class 1 integrons in 62 (79.5%) isolates. very few studies have investigated class 1 integrons in salmonella isolates from human, poultry and faeces isolates in egypt. the presence of integrons was examined in 21 salmonella isolates from diseased broiler chickens in egypt where the researchers identified class 1 and class 2 integrons in 42.9% and 14.3% of the isolates, respectively.41 lower detection rates were obtained in a study of food isolates in germany (65%).1 a study by antunes, machad and peixe42 showed, in a large survey of 1183 salmonella isolates from various animal, human and food sources, that 75% carried class 1 integrons. class 1 integrons have been detected in s. kentucky.43 in our study, 5 isolates of s. kentucky carried the 1950 bp class 1 integron. multidrug resistance was observed amongst 82% (64/78) of the isolates, with 59 isolates (76%) being resistant to more than 5 antibiotics. our study indicates that 88.1% of the 59 mdr isolates harboured class 1 integrons, whilst none of the mdr isolates carried class 2 integrons. several groups have reported that integron-containing isolates are more antibiotic resistant than those isolates obtained from comparable patients which were lacking an integron.44 limitations top ↑ samples included in this study were collected from the cairo governorate during the six-month period from december 2011 to may 2012. thus, the results of this study may not be generalisable to other regions or seasons. more studies are needed on samples collected from the nile delta and upper egypt governorates and during different seasons. conclusion top ↑ in conclusion, this study demonstrated a relatively high prevalence of salmonella-contaminated poultry products, with s. kentucky the most prevalent serotype, in poultry markets in cairo, egypt. in addition, this study revealed significant mdr rates, particularly carried by s. kentucky serovar strains, against the β-lactam and fluoroquinolone (e.g., ciprofloxacin) classes of antibiotics. ultimately, these trends may limit treatment options and contribute to treatment failure and increased death rates. more comprehensive studies are needed to better determine the prevalence and antibiotic resistance patterns of salmonella-contaminated poultry meat and its products. more serotypes should be utilised in identification and be included in a national surveillance database to allow comparisons with findings within egypt and from other countries in the region. this surveillance should include antimicrobial susceptibility profiles to track the emergence and exacerbation of existing drug resistance amongst salmonella and other food-borne disease pathogens. acknowledgements top ↑ competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. disclaimer the views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the us department of the navy, the us department of defense, the us government or the egyptian ministry of health and population. copyright assignment statement the authors are employees of the us government. this work was prepared as part of their official duties. title 17 u.s.c. §105 provides that ‘copyright protection under this title is not available for any work of the united states government.’ title 17 u.s.c. §101 defines a us government work as a work prepared by a military service member or employee of the us government as part of that person's official duties. authors’ contributions m.a-m. (global disease detection and response program, us naval medical research unit) developed the concept, processed the samples and participated in writing the manuscript. b.l.h. (global disease detection and response program, us naval medical research unit), r.f.g. (ain shams university) and h.m.a. (ain shams university) developed the concept, analysed the results, wrote and reviewed the manuscript. r.a-k. and a.e.-g. (both bacterial and parasitic disease research program, us naval medical research unit) participated and supervised the molecular biology work and helped in analysing the results and writing the manuscript. references top ↑ miko a, pries k, schroeter a, et al. molecular mechanisms of resistance in multidrug-resistant serovars of salmonella enterica isolated from foods in germany. j antimicrob chemother. 2005;56(6):1025–1033. http://dx.doi.org/10.1093/jac/dki365 kümmerer k. resistance in the environment. j antimicrob chemother. 2004;54(2):311–320. http://dx.doi.org/10.1093/jac/dkh325 helms m, simonsen j, mølbak k. quinolone resistance is associated with increased risk of invasive illness or death during infection with salmonella serotype typhimurium. j infect dis. 2004;190(9):1652–1654. http://dx.doi.org/10.1086/424570 aarestrup fm, hasman h, olsen i, et al. international spread of blacmy-2mediated cephalosporin resistance in a multiresistant salmonella enterica serovar heidelberg 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abuja, nigeria correspondence to: ameh james email: ameh.james@research.usc.edu.au postal address: molecular engineering research laboratory, university of the sunshine coast, maroochydore dc, qld 4558, australia dates: received: 22 may 2013 accepted: 27 aug. 2015 published: 18 nov. 2015 how to cite this article: james a, ochei k, emenyonu n, lawson l. improved sensitivity, safety and laboratory turnaround time in the diagnosis of pulmonary tuberculosis by use of bleach sedimentation. afr j lab med. 2015;4(1), art. #117, 5 pages. http://dx.doi.org/10.4102/ajlm.v4i1.117 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. improved sensitivity, safety and laboratory turnaround time in the diagnosis of pulmonary tuberculosis by use of bleach sedimentation in this original research... open access • abstract • introduction • methods    • settings and patient recruitment    • sample collection    • direct microscopy    • bleach sedimentation    • microscopic examination and interpretation    • sputum decontamination (modified petroff method), culture and isolation of m. tuberculosis    • statistical analyses    • ethical considerations • results • discussion    • limitations    • recommendations    • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: inadequate diagnostic processes and human resources in laboratories contribute to a high burden of tuberculosis (tb) in lowand middle-income countries. direct smear microscopy is relied on for tb diagnosis; however, sensitivity rates vary. to improve sensitivity of direct microscopy, the researchers employed several approaches, including sputum digestion and concentration of acid-fast bacilli (afb), a technique which uses commercial bleach. objectives: this study compared methods used to diagnose active mycobacterium tuberculosis infections. methods: three sputum specimens were collected from each of 340 participants in abuja, nigeria, over two consecutive days. direct microscopy was performed on all specimens; following microscopy, one specimen from each patient was selected randomly for bleach sedimentation and one for lowenstein-jensen culture. results: direct microscopy produced 28.8% afb-positive results, whilst bleach sedimentation resulted in 30.3%. when compared with the cultures, 26.5% were afb true positive using direct microscopy and 27.1% using bleach sedimentation. whilst the specificity rate between these two methods was not statistically significant (p = 0.548), the sensitivity rate was significant (p = 0.004). conclusion: based on these results, bleach increases the sensitivity of microscopy compared with direct smear and has similar specificity. when diagnosing new cases of pulmonary tb, one bleach-digested smear is as sensitive as three direct smears, reducing waiting times for patients and ensuring the safety of laboratory technicians. introduction top ↑ pulmonary tuberculosis (tb) has a large impact on developing country populations, especially in sub-saharan africa where its burden has been increased by the rapid spread of human immunodeficiency virus (hiv). hiv infection impairs cell-mediated immunity, which provides an opportunity for the reactivation of tb, making individuals living with hiv more susceptible to this pathogen. furthermore, hiv can reduce the sputum positivity rate, leading to false sputum-smear-negative tb.1,2,3 tb can be caused by any member of the mycobacterium tuberculosis complex (m. tuberculosis, m. bovis, m. africanum, m. caprae, m. microti, m. cannettii and m. pinnipedi). the factors responsible for the high burden of tb in lowand middle-income countries (lmic) include poor diagnostic processes and inadequate human resources in laboratories. the most widely-available diagnostic test in these settings is direct smear microscopy (hereafter called direct microscopy), which is used for tb diagnosis. this test is cheap, simple and highly specific for mycobacterium tuberculosis.4 in 1995, through the directly observed therapy short (dots) course strategy, the world health organization (who) set a global target for 2005 to detect 70% of new smear-positive cases.5 unfortunately, this target has not been met, in part due to inaccurate diagnoses.6 several investigators reported varying sensitivity rates of direct microscopy, ranging from 20% – 60% in some settings to 80% in another setting.7,8,9,10,11,12,13, 14,15 this finding has led to the search for alternative techniques to improve the sensitivity rate of direct microscopy, resulting in the development of several methods to optimise the procedure. amongst these approaches is the sputum digestion and concentration of acid-fast bacilli (afb) using commercial bleach (sodium hypochlorite), a widely-available household commodity, instead of the standard sodium hydroxide (naoh) concentration method. the technique has been shown to improve the clarity of the smears and increase the yield of bacilli for easy detection.16 to achieve these benefits, investigators used centrifugation and sedimentation concentration methods. bleach centrifugation and sedimentation studies have been widely reported and reviewed by different authors to determine the suitability of the method for tb diagnosis in lmic.6,17,18 only a few reports that used lowenstein-jensen (lj) culture are available and none of the studies were conducted in nigeria.19,20 the only published study in nigeria utilised the bactec mgit 960 (beckton dickinson, franklin lakes, new jersey, united states), but because of a lack of required technology the researchers could not differentiate between the species of m. tuberculosis complex.21 furthermore, most published studies compared either bleach centrifugation or sedimentation with direct microscopy rather than with the gold standard of mycobacterial culture.13,22,23,24 additionally, in a recent review by cattamanchi et al., lack of validation of these studies has been challenged.18 as a result, this study was conducted to compare the method of bleach sedimentation for less than one hour with direct microscopy and lj culture. this study reports the sensitivity, specificity and positive and negative predictive values of bleach sedimentation and direct microscopy as compared to lj culture, the reference standard. methods top ↑ settings and patient recruitment the dots clinics in six different government-owned and managed health facilities across the federal capital territory (fct) in abuja, nigeria, referred 340 patients to the zankli medical centre. the referring health facilities included maitama district hospital, asokoro district hospital, wuse general hospital, gwagwalada specialist hospital, kubwa general hospital and gwarimpa general hospital. the participants, 192 men and 148 women aged between 10 and 64 years, were prospectively enrolled in the study between november 2004 and july 2005. the participants referred from the six sites were assessed for suspected pulmonary tb. participants who did not submit three specimens over a two-day period and participants receiving anti-tb treatment were excluded from the study. sample collection each of the 340 participants submitted three sputum specimens over two consecutive days. in total, 1020 specimens were collected. the first specimen was collected during the patients’ first visit to zankli medical centre, whilst the second was collected by the patients at their homes. patients were given instructions on how to collect an appropriate specimen for diagnosis of pulmonary tb; this process included taking the sample early in the morning before brushing the teeth. the third specimen was taken at the zankli medical centre when patients delivered their second specimen. the two specimens taken at the zankli medical centre were produced by patients in an open and well-ventilated area of the facility. laboratory technicians performed direct microscopy on all specimens collected and randomly selected one specimen for bleach sedimentation and one specimen for lj culturing. all diagnostic tests gave conclusive results for the 340 participants. direct microscopy smears (1 cm × 2 cm) were made from the purulent sputum, air-dried and heat-fixed on a hot plate at 85 °c for two to three minutes, then stained by the ziehl-neelsen (zn) method (1% filtered carbol-fuchsin and 0.1% malachite green or methylene blue). bleach sedimentation an equal volume of undiluted commercial bleach (5% sodium hypochlorite) was added to the remaining part of the specimen. the specimen cup was tightly closed and the contents were vigorously shaken by hand for about 20 seconds; the cup was then placed at an angle of 45 ° and remained, undisturbed, at room temperature (18 °c – 30 °c) for 30 minutes. the sediment was gently withdrawn using a disposable pasteur pipette and a drop of the deposit was transferred to a slide. this was used to make a smear of approximately 1 cm × 2 cm. the smear was air-dried, heat-fixed and stained using the zn method. microscopic examination and interpretation both the direct and bleach smears were read using the oil immersion lens (×100) of an ordinary light microscope by experienced microscopists who were blinded to the results. slides were read again in the case of discordant results. for both direct and bleach slides, positive and negative smears were defined according to the national tuberculosis and leprosy control program's afb grading system (table 1).7 a patient was reported smear-positive for tb if at least one to nine afb were seen in 100 high-power fields. hence, the study reported on the number of patients diagnosed with active m. tuberculosis infections. table 1: guide to acid-fast bacilli (afb) microscopy interpretation according to the national tuberculosis and leprosy control program's afb grading system.7 sputum decontamination (modified petroff method), culture and isolation of m. tuberculosis sputum for the lj culture technique was selected randomly from the participants’ three specimens. an equal volume of 4% sodium hydroxide was added to 5 ml of sputum in a 30 ml screw-cap tube. this tube was capped tightly and shaken to digest the sputum; thereafter, the tube stood at room temperature for 15 minutes with occasional shaking. the mixture was centrifuged at 3000 revolutions per minute for 15 minutes. the supernatant was carefully decanted, after which the deposit was resuspended with 15 ml of sterile normal saline and re-centrifuged at the same rate. the supernatant was removed and the tube sediment of the second centrifugation was inoculated on an lj agar slope and incubated at 37 °c ± 2 °c. for the first three days, the specimen was observed daily for signs of possible contamination. at weekly intervals over the following six to 10 weeks, the culture was examined regularly for the isolation of m. tuberculosis. positive and negative growth controls were always included, using wild strains of m. tuberculosis complex and sterile distilled water, respectively. the isolates were identified as mycobacterium species by the nitrate reduction test, catalase heat-labile test and zn smear method. statistical analyses the proportions (sensitivity, specificity and negative and positive predictive values) were calculated using standard definitions.25 the estimated proportions were then compared using the two-sample test of proportions for large samples (using the normal approximation to the binomial distribution) and estimating the confidence intervals in the process. in particular, an immediate form of the two-sample test of proportions was applied using the prtesti command in stata software version 11 (stata corp lp, college station, texas, united states). a p-value of < 0.05 was considered statistically significant. ethical considerations verbal informed consent was obtained from the participants and ethical approval granted by the ethical committee of the fct hospital management board and the zankli medical centre. results top ↑ of the 340 participants evaluated for tb, 28.8% were afb-positive using direct smear and 30.3% using bleach sedimentation (table 2). when compared with mycobacterial culture (figure 1), the gold standard, 26.5% of samples were true positive for afb using direct smear and 27.1% using bleach sedimentation (table 3). this comparison determined the sensitivity and specificity rates of the two methods (table 4). whilst the difference in the specificity rate between the two evaluated methods was not significantly significant (p = 0.548), the difference between sensitivity rates was significant (p = 0.004), indicating that bleach sedimentation is more sensitive. furthermore, unlike the difference between positive predictive values (p = 0.542), the difference in the negative predictive values was statistically significant (p = 0.003), demonstrating that the bleach sedimentation method more accurately identified the afb-negative participants who were not infected with m. tuberculosis, in contrast to those with false-negative results. figure 1: positive (a) and negative (b) lowenstein-jensen cultures for m. tuberculosis. table 2: results of ziehl-neelsen microscopy for acid-fast bacilli using both direct and bleach sedimentation-treated sputa, abuja, nigeria, november 2004 to july 2005. table 3: comparison of ziehl-neelsen microscopy for acid-fast bacilli using direct and bleach sedimentation-treated sputa with culture on lowenstein-jensen media following sodium hydroxide decontamination, abuja, nigeria, november 2004 to july 2005. table 4: diagnostic accuracy of direct microscopy and bleach sedimentation-treated sputa compared with lowenstein-jensen culture following sodium hydroxide decontamination, abuja, nigeria, november 2004 to july 2005. discussion top ↑ in this study, researchers found that bleach increased the sensitivity of microscopy compared with the direct smear and had similar specificity. this finding supports the outcomes reported in other studies that have evaluated bleach sedimentation whilst using culture as a reference standard.10,19,20 despite its drawbacks, direct microscopy remains the cornerstone of tb diagnosis in developing countries, particularly in sub-saharan africa. direct microscopy has low sensitivity, as described in a review of 14 studies.6 however, in this study, direct microscopy showed a relative increase in sensitivity. this finding cannot be extrapolated; however, as it was likely the result of the setting in which this study was conducted: a research laboratory with greater time resources than routine diagnostic laboratories, particularly government-owned or public health facilities. though not a problem in this study, an additional drawback of direct microscopy without bleach sedimentation is that it requires the submission of three specimens, which can result in increased dropout rates. many patients are unable to afford the cost of travelling to a health centre to submit one or multiple samples.26 therefore, the failure to attain the who's global target of 70% case detection is not surprising, given that the burden of tb is in developing countries where direct microscopy is still routinely used for diagnosis. the use of bleach sedimentation, requiring only one specimen, has several benefits, such as greatly reducing the workload of overextended laboratories in resource-poor settings and reducing the long turnaround time associated with both direct microscopy and the overnight sedimentation method employed in other studies.10,19 additional advantages associated with bleach sedimentation are safety, ease of manipulation and cost-effectiveness.21 significantly, bleach is readily available even in remote parts of the developing world, whereas sodium hydroxide, traditionally used in laboratories, may be difficult or even impossible to acquire. one study showed that the use of 3% bleach for 20 minutes completely sterilises sputum containing m. tuberculosis.27 however, a previous study found total sterilisation in only 93.6% of afb-positive sputa after treatment with 5% bleach for between 15 minutes and three hours.28 regardless, the use of household-strength bleach in processing sputa for afb microscopy has significant laboratory safety advantages, as it sterilises the majority of processed sputa, reducing technicians’ risk of exposure to afb. limitations this study was conducted in a controlled laboratory, unlike a typical public health laboratory where laboratorians are expected to meet a particular turnaround time. thus, this study was carefully carried out without any ‘time pressure’. it is suggested that a similar study should be performed in a typical routine diagnostic laboratory. recommendations this study recommends the use of bleach for sputum microscopy, as it helps to increase the sensitivity of the test and reduces the work load on the laboratory. it also offers protection for the laboratory personnel's against the bacteria. conclusion in conclusion, in settings with a high tb burden, bleach sedimentation may improve sensitivity and laboratory safety in the diagnosis of m. tuberculosis and reduce waiting periods for test results. evidence shows that one bleach-digested sputum smear may be more sensitive than three direct smears in the detection of new cases of pulmonary tb. this diagnostic test could increase the detection rate of new cases of tb in rural areas in developing countries. acknowledgements top ↑ we wish to thank the patients recruited for this study and james jafali (medical research council, the gambia) for providing statistical support without financial compensation. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions a.j. (family health international 360 and keystone laboratories international, diagnostic unit) performed the literature search, contributed to data analysis and interpretation and drafted the manuscript. k.o. (family health international 360) conceived and designed the study, acquired the samples and data and revised the manuscript. n.e. (zankli medical centre) contributed to the study design, acquired the samples and data and revised the manuscript. l.l. (zankli medical centre) contributed to the study design and revised the manuscript. all authors gave final approval of the version to be published and agree to be accountable for the accuracy and integrity of the work. references top ↑ world health organization. global tuberculosis control: surveillance, planning, financing. who report 2005 [homepage on the internet]. c2014 [updated 2014; cited 2014 jul 9]. available from: http://www.who.int/tb/publications/global_report/2005/en/;now available from: http://www.hst.org.za/sites/default/files/tb2005who.pdf. harries ad, maher d, nunn p. an approach to the problems of diagnosing and treating adult smear-negative pulmonary tuberculosis in high-hiv-prevalence settings in sub-saharan africa. bull world health organ. 1998;76(6):651–662. pmid: 10191561. cantwell mf, binkin nj. impact of hiv on tuberculosis in sub-saharan africa: a regional perspective. int j tuberc lung dis. 1997;1(3):205–214. pmid: 9432365. perkins md. new diagnostic tools for tuberculosis. int j tuberc lung dis. 2000;4(12 suppl 2):s182–s188. pmid: 11144551. dye c, hosseini m, watt c. did we reach the 2005 targets for tuberculosis control? bull world health organ. 2007;85(5):325–420. pmid: 17639221. steingart kr, ng v, henry m, et al. sputum processing methods to improve the sensitivity of smear microscopy for tuberculosis: a systematic review. lancet infect dis. 2006;6(10):664–674. pmid: 17008175, http://dx.doi.org/10.1016/s1473-3099(06)70602-8 james a, abba su, ibrahim a, et al. improving the case detection of pulmonary tuberculosis by bleach microscopy method in the north west of nigeria. jmld. 2013;4(3):34–37. angeby ka, alvarado-gálvez c, pineda-garcía l, et al. improved sputum microscopy for a more sensitive diagnosis of pulmonary tuberculosis. int j tuberc lung dis. 2000;4(7):684–687. pmid: 10907772. bruchfeld j, aderaye g, palme ib, et al. sputum concentration improves diagnosis of tuberculosis in a setting with a high prevalence of hiv. trans r soc 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ziehl-neelsen methods for tuberculosis diagnosis. west afr j med. 2005;24(4):316–320. pmid: 16483048. merid y, yassin ma, yamuah l, et al. validation of bleach-treated smears for the diagnosis of pulmonary tuberculosis. int j tuber lung dis. 2009;13(1):136–141. pmid: 19105892. lawson l, yassin ma, ramsay a, et al. microbiological validation of smear microscopy after sputum digestion with bleach; a step closer to a one-stop diagnosis of pulmonary tuberculosis. tuberculosis. 2006;86(1):34–40. pmid: 16263328, http://dx.doi.org/10.1016/j.tube.2005.06.003 gebre-selassie s. evaluation of the concentration sputum smear technique for the laboratory diagnosis of pulmonary tuberculosis. trop. doct. 2003;33(3):160–162. pmid: 12870603, http://dx.doi.org/10.1177/004947550303300313 miörner h, ganlöv g, yohannes z, et al. improved sensitivity of direct microscopy for acid-fast bacilli: sedimentation as an alternative to centrifugation for concentration of tubercle bacilli. j clin microbiol. 1996;34(12):3206–3207. pmid: 8940473. bonnet m, ramsay a, githui w, et al. bleach sedimentation: an opportunity to optimize smear microscopy for tuberculosis diagnosis in settings of high prevalence of hiv. clin infect dis. 2008;46(11):1710–1716. pmid: 18444789, http://dx.doi.org/10.1086/587891 drewe ja, tomlinson aj, walker nj, et al. diagnostic accuracy and optimal use of three tests for tuberculosis in live badgers. plos one. 2010;5(6):e11196. pmid: 20585404, http://dx.doi.org/10.1371/journal.pone.0011196 squire sb, belaye ak, kashoti a, et al. ‘lost’ smear-positive pulmonary tuberculosis cases: where are they and why did we lose them? int j tuberc lung dis. 2005;9(1):25–31. pmid: 15675546. chew r, calderón c, schumacher s, et al. evaluation of bleach-sedimentation for sterilising and concentrating mycobacterium tuberculosis in sputum specimens. bmc infect dis. 2011;11:269. pmid: 21985457, http://dx.doi.org/10.1186/1471-2334-11-269 githui wa, matu sw, tunge n, et al. biocidal effect of bleach on mycobacterium tuberculosis: a safety measure. int. j tuberc lung dis. 2007;11(7):798–802. pmid: 17609057. article information authors: maxwell waema1 naomi maina2 simon karanja1 beatrice gachie2 maina ngotho3 john kagira4 affiliations: 1institute of tropical medicine and infectious diseases, jomo kenyatta university of agriculture and technology, kenya2biochemistry department, jomo kenyatta university of agriculture and technology, kenya 3animal science department, institute of primate research, kenya 4department of land resources planning management, jomo kenyatta university of agriculture and technology, kenya correspondence to: john kagira postal address: jomo kenyatta university of agriculture and technology, po box 62000 – 00200 nairobi, kenya dates: received: 01 mar. 2013 accepted: 23 oct. 2013 published: 29 oct. 2014 how to cite this article: waema m, maina m, karanja s, gachie b, ngotho m, kagira j. development of a safer laboratory vervet monkey model for the study of human african trypanosomiasis. afr j lab med. 2013;3(1), art. #100, 6 pages. http://dx.doi.org/10.4102/ ajlm.v3i1.100 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. development of a safer laboratory vervet monkey model for the study of human african trypanosomiasis in this original research... open access • abstract • introduction • research methods and design    • trypanosomes    • drugs    • experimental animals    • experimental design    • clinical examination and sample collection    • laboratory analysis    • disease staging criteria    • statistical analysis    • ethical consideration • results    • clinical signs    • parasitaemia and cerebrospinal fluid parasitosis    • haematology profiles       • erythrocyte changes       • platelets       • leucocytes • trustworthiness    • reliability and validity of the research • discussion • limitations of the study    • recommendations • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: there are three subspecies of trypanosoma brucei: t. b. gambiense, t. b. rhodesiense and t. b. brucei. the first two are infectious to humans, whilst t. b. brucei is not. identifying an animal model of t. b. brucei that mimics human african trypanosomiasis (hat) would enable researchers to study hat without subjecting themselves to undue risks such as accidental infection.objectives: this study assessed the sequential clinical, parasitological and haematological changes in vervet monkeys infected with t. b. brucei. methods: three vervet monkeys were infected with a 104 inoculum of t. b. brucei (isolate gutat 1). late-stage disease was induced by subcurative treatment with diminazene aceturate 28 days post-infection. the animals were treated curatively with melarsoprol upon relapse. parasitaemia and clinical signs were monitored daily and, at weekly intervals, the monkeys’ blood and cerebrospinal fluid (csf) were sampled for haematology and parasitosis assessments, respectively. results: the first-peak parasitaemia was observed between seven and nine days post-infection. clinical signs associated with the disease included fever, dullness, pallor of mucous membranes, lymphadenopathy, splenomegaly and oedema. late-stage signs included stiffness of joints and lethargy. the monkeys developed a disease associated with microcytic hypochromic anaemia. there was an initial decline, followed by an increase, in total white blood cell counts from earlyto late-stage disease. trypanosomes were detected in the csf and there was a significant increase in white cell counts in the csf during late-stage disease. infected vervet monkeys displayed classical clinical symptoms, parasitological and haematological trends that were similar to monkeys infected with t.b. rhodesiense. conclusion: the t. b. brucei vervet monkey model can be used for studying hat without putting laboratory technicians and researchers at high risk of accidental infection. introduction top ↑ trypanosoma brucei (t. brucei) is a protozoan parasite, of which there are three subspecies: t. b. gambiense, t. b. rhodesiense and t. b. brucei. the first two subspecies are infectious to humans, causing human african trypanosomiasis (hat) (also known as sleeping sickness),1 whilst t. b. brucei only causes disease in animals. the disease has two recognised stages: the early (haemolymphatic) stage when parasites appear in the blood and the late (encephalitic) stage when the central nervous system is involved.t. b. rhodesiense is found in eastern and southern africa and causes an acute infection. the first clinical symptoms are observed a few weeks or months after initial infection – a result of the parasite invading the central nervous system (cns). t. b. gambiense is found in west and central africa and causes a chronic infection. the parasite can be present in the body for months or even years without causing severe symptoms. for the majority of patients infected with t. b. gambiense, the disease is already in an advanced stage by the time symptoms emerge, with parasites having affected the cns.1 hat remains a neglected disease of major health and socioeconomic consequence in sub-saharan africa,1 with an estimated 60 million people in 36 countries at risk.2 the management of the disease remains a major challenge due to poor diagnostics and treatment regimens, most of which have been used for decades. research and development of diagnostics and new drugs normally rely upon the use of laboratory animal models, principally mice and monkeys, which mimic human disease. six accidental, laboratory-acquired cases of t. b. rhodesiense have been reported.3,4,5 rather than identifying an animal model of a dangerous human pathogen, a safer option would be to identify a non-human pathogen that mimics hat. unlike t. b. rhodesiense, t. b. brucei is unable to infect humans, but mimics the pathogenesis of human-infective t. b. rhodesiense in infected animals.6 thus, an animal model of t. b. brucei that mimics hat would enable researchers to study the disease without subjecting themselves to undue risks. given that the vervet monkey disease model of t. b. rhodesiense bears a close relationship to hat,7 the authors hypothesised that infection with t. b. brucei could provide similar disease progression. these monkeys may provide an excellent opportunity to investigate controlled laboratory studies on serum and cerebrospinal fluid (csf) samples, which would also allow for identification of potential biomarkers of the disease stages. this study was designed to determine the clinical, parasitological and haematological profile of vervet monkeys infected with t. b. brucei and to determine whether a t. b. brucei model would be a suitable and safer alternative to the vervet monkey model of t. b. rhodesiense. research methods and design top ↑ trypanosomes the t. b. brucei gutat 1 isolate was used. the isolate was obtained from the international livestock research institute biobank, passaged three times in mice and preserved at the institute of primate research’s trypanosomes cryobank. the cryopreserved isolate was thawed and injected into donor swiss white mice for expansion. at peak parasitaemia, the mice were euthanased with co2 and their blood was harvested by cardiac puncture. the blood was serially diluted in phosphate saline glucose to a final concentration of 104 trypanosomes/ml. drugs the trypanocidal drugs used in this study were diminazene aceturate (pharma links, india) and melarsoprol (arsobal®, specia, france).diminazene aceturate is an aromatic diamidine used as treatment for livestock trypanosomiasis and is also effective against early-stage t. b. gambiense and t. b. rhodesiense infection.7 in monkey models of hat, the drug clears bloodstream parasites but is unable to clear parasites in the cns since it does not cross the blood-brain barrier. its mode of action involves interference with rna editing and trans-splicing and it inhibits adomet decarboxylase in trypanosomes, resulting in the reduction of spermidine content and the elevation of putrescine levels in the parasite. melarsoprol is a trivalent arsenical compound used for the treatment of late-stage sleeping sickness. the mode of action involves inhibition of trypanothione reductase in trypanosomes. melarsoprol can cross the blood-brain barrier and can thus clear trypanosomes that have crossed into the brain parenchyma. in hat monkey models, melarsoprol is used for curative treatment in late-stage disease.8 experimental animals five wild-caught vervet monkeys, each weighing 2–4 kg, were obtained from the animal unit colony of the institute of primate research. prior to experimentation, the monkeys underwent quarantine for 90 days, during which time they were screened for zoonotic diseases as well as ectoand endoparasites. because of the evidence of minor strongyle infections and mange infestations, they were treated with subcutaneous injections of ivermectin at dosages of 300 μg/kg for three days. the monkeys were housed in single 90 x 60 x 60 cm stainless-steel cages in a room maintained at temperatures in the range of 23–25 °c. they were fed twice daily with monkey cubes (goldstar feeds® ltd., nairobi, kenya), vegetables, green maize and bananas; water was provided ad libitum. experimental design three monkeys were infected intravenously with 1 ml of the suspension containing 104 trypanosomes of t. b. brucei (gutat 1), whilst the remaining two monkeys were kept as non-infected controls. the infected monkeys were monitored daily for parasitaemia as described in previous studies.7 at 28 days post-infection (dpi) the monkeys were treated subcuratively for three consecutive days using intramuscular diminazene aceturate at a dose rate of 5 mg/kg body weight. the monkeys were then monitored for parasitaemia and, on relapse (114 dpi), were treated curatively for four consecutive days with intravenous melarsoprol at a dose rate of 3.6 mg/kg body weight. after 180 days of monitoring, the monkeys were euthanased using euthatal (20% sodium pentobarbitone). clinical examination and sample collection the clinical status of the monkeys was monitored daily. at weekly intervals, the monkeys were anaesthetised with intramuscular injections of ketamine hydrochloride (ketamine®, rotexmedica, trittau, germany) at dosages of 10 mg/kg body weight and full physical examinations were conducted. furthermore, 2 ml of blood were collected weekly by venepuncture of the femoral vein and placed in tubes containing ethylenediaminetetraacetic acid (edta). csf was collected weekly by lumbar puncture. laboratory analysis immediately after blood collection, the haematological assays were performed using an ac3diff t coulter counter (miami, florida, usa). parasitaemia was scored using the method described by herbert and lumsden,8 which included the daily use of wet smear detection of microscopic parasites using the rapid matching method. to determine pathological effects, baseline biochemical values were compared to those post-infection. normal ranges are not generally used because of the variation found amongst vervet monkeys.10 disease staging criteria the stage of trypanosome infection was determined in accordance with world health organization criteria and as previously described.9,11 csf was collected by lumbar puncture and examined for the presence of trypanosomes and the number of white blood cells (wbcs). the wbcs were estimated by counting in a neubauer cell chamber. when cells were < 5 cells/µl, infection was classified as first stage. late-stage infection was diagnosed when trypanosomes were detected in the csf and the wbc count of the csf was > 5 cells/µl.12 statistical analysis data were managed using microsoft excel (microsoft us, version 2007). descriptive statistics and summary tables were employed for the initial description of the data and the results were displayed in excel charts. differences between the means were compared using the student’s t-test and were deemed to have statistical significance at p < 0.05. ethical consideration prior to commencement of the study, all protocols and procedures used were reviewed and approved by the institute of primate research institutional animal care and use committee (irc/19/10). results top ↑ clinical signs all the infected animals developed acute symptoms characteristic of rhodesian hat, including fever with a mean temperature of 40 °c, dullness, increased pulse and respiratory rates, pallor of the mucous membranes, enlarged superficial lymph nodes and spleen, raised hair coat, peri-orbital oedema and stiffness of joints. upon subcurative treatment with diminazene aceturate, most of the clinical signs of disease disappeared within 14 days. however, starting at 42 dpi, one monkey exhibited a general body weakness, sleepiness, ataxia and an arched back when sitting on the cage floor and was euthanased for humane reasons. the other two infected monkeys had relapses of parasitaemia from 114–119 dpi with clinical signs that included stiffness of joints and hind leg paralyses. these signs disappeared within 14 days after curative treatment with melarsoprol. parasitaemia and cerebrospinal fluid parasitosis all experimentally-infected animals had a prepatent period ranging from two to four days. the first parasitaemia peak of 107 trypanosomes per ml of blood occurred between 7–9 dpi (figure 1). treatment with diminazene aceturate resulted in clearance of the trypanosomes in the blood. in all three infected monkeys, the parasites relapsed, starting at 114 dpi. trypanosomes were detected in the csf of two monkeys on days 28 and 105 post-infection. treatment with melarsoprol at 119 dpi led to clearance of both the parasitaemia and csf parasitosis by 123 dpi. there was an increase in csf wbc counts during the late stage (figure 2). figure 1: mean daily parasitaemia of vervet monkeys infected with t. b. brucei gutat 1, indicating the point of subcurative treatment with diminazene aceturate (da) and curative treatment with melarsoprol (mel b). figure 2: white blood cell (wbc) count numbers in the cerebrospinal fluid of t. b. brucei-infected vervet monkeys, showing the point of subcurative treatment with diminazene aceturate (da) (28 dpi) and curative treatment with melarsoprol (mel b) (119 dpi). haematology profiles erythrocyte changes in the control animals, the mean total red blood cell (rbc) count was 5.4 x 106 cells/μl, packed cell volume (pcv) was 42% and haemoglobin (hb) was 12 g/dl. blood count parameters did not change significantly throughout the experimental period. however, by 28 dpi, the infected vervet monkeys had a decrease (p < 0.05) in the mean values for the rbc count, pcv and hb values to 4.7 x 106 ± 0.72 cells/μl, 27 ± 2.05% and 8.2 ± 0.8 g/dl, respectively (figure 3). after subcurative treatment, the levels increased gradually to those of pre-infection within 14 days post-diminazene aceturate treatment. blood count parameters decreased again at 98 dpi but recovered and attained the pre-infection levels within seven days of curative treatment with melarsoprol. figure 3: mean changes in red blood cell (rbc) counts, packed cell volume (pcv) and haemoglobin (hb) of vervet monkeys infected with t. b. brucei gutat 1, showing the point of subcurative treatment with diminazene aceturate (da) (28 dpi) and curative treatment with melarsoprol (mel b) (119 dpi). red cell distribution width (rdw) increased during early-stage infection and peaked (14.2 ± 1.5%) at 42 dpi. thereafter, rdw decreased during late-stage disease to normal levels (10.5 ± 0.6%) at 140 dpi. a decrease in mean corpuscular volume (mcv) was observed from the onset of infection until 7 dpi, after subcurative treatment with diminazene aceturate, thereafter returning to pre-infection levels (figure 4). mean corpuscular haemoglobin (mch) decreased 14 dpi from 23.5 to 22.5 pg, levelling off by 28 dpi. levels appeared stable after melarsoprol treatment (119 dpi), returning to pre-infection levels by 140 dpi (figure 5). figure 4: mean changes in red cell distribution width (rdw) and mean cell volume (mcv) of vervet monkeys infected with t. b. brucei gutat 1, showing the point of subcurative treatment with diminazene aceturate (da) (28 dpi) and curative treatment with melarsoprol (mel b) (119 dpi). figure 5: mean changes in mean corpuscular haemoglobin (mch) and mean corpuscular haemoglobin concentration (mchc) in vervet monkeys infected with t. b. brucei gutat 1, showing the point of subcurative treatment with diminazene aceturate (da) (28 dpi) and curative treatment with melarsoprol (mel b) (119 dpi). platelets platelet numbers declined after infection, reaching their lowest levels (58.5 × 10³ ± 0.15 cells/µl) at 21 dpi. levels increased after subcurative treatment with diminazene aceturate, returning to those of pre-infection by 56 dpi (figure 6). platelet counts decreased steadily for three weeks before trypanosome relapse in the blood (84 dpi), but stabilised to pre-infection levels (550 × 10³ cells/µl) after melarsoprol treatment (119 dpi). figure 6: changes in mean platelet numbers in vervet monkeys infected with t. b. brucei gutat 1, showing the point of subcurative treatment with diminazene aceturate (da) (28 dpi) and curative treatment with melarsoprol (mel b) (119 dpi). leucocytes in uninfected animals, the mean total wbc count ranged from 4.3 x 103 cells/μl to 5.7 x 103 cells/μl and did not change significantly throughout the experimental period (p < 0.05). . mean neutrophil counts ranged from 1.72 x 103 cells/µl to 2.12 x103 cells/µl, lymphocyte counts from 3.6 x103 cells/µl to 4.48 x 103 cells/ul and monocyte counts from 0.3 x 103 cells/µl to 0.26 x 103 cells/µl. none of these counts changed significantly throughout the experimental period (p < 0.05). in infected animals, total wbc counts declined during early stage infection (4.7 x 103 cells/µl to 2.5 x 103 cells/µl). after subcurative treatment with diminazene aceturate, wbc counts increased and were significantly higher at 84 dpi (7.7 x 103 ± 1.75 cell/µl, p < 0.05) than pre-infection levels (4.3 x 103 ± 1.18 cells/µl). thereafter, wbc counts declined to 3 x 103 cells/µl by 154 dpi. both lymphocyte and neutrophil counts followed a similar pattern (figure 7). changes in eosinophil and basophil counts were not significant during the course of the disease. monocyte counts dropped significantly from 0.2 x 103 cells/µl to 0.07 x 103 cells/µl between 0 and 14 dpi. they then peaked at 42 dpi (0.38 x 103 ± 0.04 cells/µl), after which there was a decline. a further increase in monocyte counts was noted, starting at 112 dpi with a peak at 126 dpi (0.3 x 103 ± 0.05 cells/µl) (figure 8). figure 7: mean changes in total white blood cells (wbc), lymphocyte and neutrophil counts of t. b. brucei gutat 1-infected vervet monkeys showing the point of subcurative treatment with diminazene aceturate (da) (28 dpi) and curative treatment with melarsoprol (mel b) (119 dpi). figure 8: mean changes in monocyte counts of t. b. brucei gutat 1-infected vervet monkeys, showing the point of subcurative treatment with diminazene aceturate (da) (28 dpi) and curative treatment with melarsoprol (mel b) (119 dpi). trustworthiness top ↑ to judge from the outcome of t. b. brucei infection in vervet monkeys, the observations obtained here appeared to compare well with those arising from t. b. rhodesiense and other related studies. reliability and validity of the research the experimental design of this study is reliable and valid. the procedures used in this research have been tested in other studies as cited in this article. discussion top ↑ this is the first study reporting the infection of vervet monkeys with t. b. brucei. human beings and some non-human primates have a trypanolytic factor, which prevents them from being infected with t. b. brucei and other livestock trypanosomes.13,14 results from this study suggest that vervet monkeys may lack trypanolytic factors, which have been reported to be present in other non-human primates, such as baboons and gorillas. the haptoglobin-related protein which has been demonstrated to have trypanolytic activity might be absent in the sera of vervet monkeys. in spite of the lack of human infection, t. b. brucei infections in rodents follow a similar pathogenesis to that of t. b. rhodesiense, thus rodents have been used as models for hat.8 some of the disadvantages of rodent hat models include phylogenetic distance and an inability to perform sequential monitoring of csf changes. these limitations can be addressed by using monkey models.in the current study, infected vervet monkeys displayed classical clinical symptoms, parasitological and haematological trends that were similar to monkeys infected with t. b. rhodesiense.15in addition, upon subcurative treatment with diminazene aceturate, disease relapse and re-emergence of blood parasitaemia took 85 days, which is similar to t. b. rhodesiense ketri 2537 infection.16 however, the duration was longer than that reported for t. b. rhodesiense ipr 001 infections.15 the differences noted may be due to variation in parasite virulence. anaemia gives a reliable indication of the disease status and productive performance of trypanosome-infected animals.17 disease in vervet monkeys tended to manifest with microcytic hypochromic anaemia during the early stages. this type of anaemia has been associated with iron deficiency that, in hat, might arise from lack of incorporation into red cell precursors – despite the presence of adequate iron storage – and inefficient recovery of iron from the phagocytosed erythrocytes.18,19 microcytic hypochromic anaemia has been described in vervet monkeys infected with t. b. rhodesiense.20 in this study, the severity of anaemia was greater in the acute stage of the disease (day 4 to 28 pi) than during the late stage (day 28 to 119 pi) and appeared to be associated with the level of parasitaemia. after curative treatment (119 dpi), all of the erythrocyte values recovered by 140 dpi because melarsoprol can clear parasites in all bodily compartments.21 there was a rapid decline in platelet counts in early-stage disease. low platelet counts could be indicative of toxic products, originating from the trypanosomes, which cause platelet destruction.18 splenic pooling of platelets and phagocytic removal of platelets by mononuclear cells has also been associated with thrombocytopaenia.19 severe progressive thrombocytopaenia has been reported in t. b. rhodesiense vervet monkey models and human cases of sleeping sickness.23,24 similarly to t. b. rhodesiense infections in vervet monkeys, lymphocytopoenia was noted in the early stage of the disease and lymphocytosis in the late stage.17 limitations of the study top ↑ only three animals were used in this study; however an extensive amount of data was obtained. parasitaemia was limited to the wet smear microscopic observation method only and, at very low parasitaemia, the method could have missed out early relapses. the characterisation of the model was also limited to haematological profiles and clinical examination. recommendations more extensive studies are needed to establish baseline levels of various biochemical parameters in vervet monkeys to aid in the determination of significant variations during infection/disease. haematological profiles need to be studied in shorter sampling intervals (e.g. daily sampling for the first week of infection) in order to discern properly any changes during the acute disease phase. conclusion top ↑ the clinical, parasitological and haematological observations obtained in this study compare well with those arising from t. b. rhodesiense; therefore, the vervet monkey t. b. brucei model may be used as substitute. this animal model may enable researchers and laboratory technicians to study hat without the high risk of accidental infection. acknowledgements top ↑ this project was funded jointly by the institute of primate research (ipr), jomo kenyatta university of agriculture and technology-research production and extension and foundation for innovative new diagnostics (find). we are grateful to the technical assistance provided by mr. ken waititu and mr tom adino of animal science department in institute of primate research. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions n.m. (jomo kenyatta university of agriculture and technology) was the project leader and in charge of animal welfare. j.k. (jomo kenyatta university of agriculture and technology) and n.m. were responsible for the experimental and project design. m.w. (jomo kenyatta university of agriculture and technology), k.j., n.m., s.k. (jomo kenyatta university of agriculture and technology) and b.g. (institute of primate research) performed the experiments, whilst m.w. and s.k. performed the data analysis. all the authors participated in the writing of the manuscript. references top ↑ 1. world health organization. trypanosomiasis, human african (sleeping sickness). fact sheet 259. geneva, switzerland: world health organization; 2006. 2. world health organization. african trypanosomiasis. world health organization fact sheet no.259; 2000, revised august 2006. http://www.who.int/mediacentre/factsheets/fs259/en/ 3. herwaldt bl. laboratory-acquired parasitic infections from accidental exposures. clin microbiol rev. 2001;14(4):659–688. http://dx.doi.org/10.1128/cmr.14.3.659-688.2001 4. robertson dhh, pickens s, lawson jh, et al. an accidental laboratory infection with african trypanosomes of a defined stock i. the clinical course of the infection. j infect. 1980;2(2):105–112. http://dx.doi.org/10.1016/s0163-4453(80)91084-1 5. herbert wj, parratt d, van meirvenne n, et al. an accidental 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in vitro on trypanosoma brucei brucei of plasma, high density lipoproteins, and apolipoprotein a-i from hosts both susceptible (cattle and sheep) and resistant (human and baboon) to infection. j lipid res. 1992;33(4):513–523. 14. pays e, vanhollebeke b. mutual self-defence: the trypanolytic factor story. microbes infect. 2008;10(9):985–989. http://dx.doi.org/10.1016/j.micinf.2008.07.020 15. gaithuma ak, karanja sm, ngotho m, et al. lipid metabolism and other metabolic changes in vervet monkeys experimentally infected with trypanosoma brucei rhodesiense. j med primatol. 2011;41(2):75–81. http://dx.doi.org/10.1111/j.1600-0684.2011.00523.x 16. ngotho m, maina n, kagira j, et al. il-10 is up regulated in early and transitional stage in vervet monkeys experimentally infected with trypanosoma brucei rhodesiense. parasitol int. 2006;55(4) 243–248. http://dx.doi.org/10.1016/j.parint.2006.06.004 17. ngotho m, kagira jm, kariuki c, et al. influence of trypanocidal therapy on the haematology of 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j biotechnol. 2007;6(18):2083–2086. 23. thuita jk, kagira jm, mwangangi d, et al. trypanosoma brucei rhodesiense transmitted by a single tsetse fly bite in vervet monkeys as a model of human african trypanosomiasis. plos negl trop dis. 2008;2(5):e238. http://dx.doi.org/10.1371/journal.pntd.0000238 24. chisi je, misiri h, zverev y, et al. anaemia in human african trypanosomiasis caused by trypanosoma brucei rhodesiense. east afr med j. 2004;81(10):505–508. http://dx.doi.org/10.4314/eamj.v81i10.9232 introduction human papillomavirus 35 in women of african ancestry urgent need for studies on the genomic epidemiology of human papillomavirus 35 in african women role of genomics in developing an improved multivalent vaccine that targets human papillomavirus 35 need for collaborative efforts in upscaling human papillomavirus 35 research in africa conclusion acknowledgements references about the author(s) sophia u. okeke department of medicine and surgery, faculty of clinical sciences, college of medicine, university of ibadan, ibadan, nigeria college research and innovation hub, university of ibadan, ibadan, nigeria citation okeke su. fighting cervical cancer in africa: taking a closer look at human papillomavirus 35. afr j lab med. 2024;13(1), a2243. https://doi.org/10.4102/ajlm.v13i1.2243 opinion paper fighting cervical cancer in africa: taking a closer look at human papillomavirus 35 sophia u. okeke received: 26 june 2023; accepted: 20 nov. 2023; published: 29 jan. 2024 copyright: © 2024. the author licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction recent findings show that global cancer mortality has fallen by 32% in the past 24 years.1 although this is good progress, it becomes evident, when examined in a geographical context, that africa still lags behind. while the continent has seen improvements in diagnostics, there is still much to be desired in aspects of cancer prevention and treatment. although 57% of all new cancer cases are recorded in lowand middle-income countries,1 cancer research output from these areas, including sub-saharan africa, remains low. furthermore, it is apparent that there are significant differences in the types of cancers constituting a burden across different regions of the world. while cervical cancer accounts for 3.3% of cancer-related deaths worldwide,2 24.55% of these deaths occur in sub-saharan africa.3 human papillomavirus 35 in women of african ancestry the steep decline in cervical cancer rates in countries with a high development index may be attributed to the implementation of suitable nationwide vaccination and screening programmes.4 just as there is a difference in cervical cancer burden, there is also a difference in the prevalence of high-risk human papillomavirus (hpv) strains. although hpv 16 and 18 account for 70% of hpv-positive cervical cancer cases,5 various epidemiological studies have shown that another high-risk hpv genotype, hpv 35, which is not covered by the available vaccines,6 accounts for a significant number of positive cases in sub-saharan africa.6,7,8 the largest hpv epidemiological study, conducted by the clinical genetics branch of the national cancer institute, united states, further established the strong association between hpv 35 and precancerous lesions in women of african ancestry.9 this study also reported a 10-fold increase in the prevalence of cervical intraepithelial neoplasia-3 among women of african ancestry infected with hpv 35 compared to white women.9 they also showed that certain single-nucleotide polymorphisms in the e7 oncogene were peculiar to african-american women.9 in contrast to other regions, hpv 35 is associated with between 4% and 10% of invasive cervical cancer cases in sub-saharan african women. various studies done across subpopulations in africa have reported high prevalence rates of hpv 35 in women with cervical intraepithelial neoplasia-3. mbulawa and colleagues, working in south africa in 2022, reported hpv 35 as the second-most dominant genotype detected in women with cervical intraepithelial neoplasia-3 (22.2%), and the fourth-most in those with cervical cancer (12.5%).6 a larger study that investigated cervical biopsy samples of 659 women from three african countries (ghana, nigeria, and south africa) also identified hpv 35 in 9.7% of cervical biopsies, making it the fourth-most prevalent hpv genotype in that cohort.7 other studies have observed a relationship between hpv 35 infections, hiv, and high-grade cervical intraepithelial lesions and have shown an increased variability in the l1 capsid (a major component of the current hpv vaccines) of hpv 35 in women living with hiv.8 another study conducted amongst zimbabwean women found a 13-base-pair insertion in the long control region of the hpv 35 genome relative to the reference hpv 35 genome from the united states.10 however, associations between these genetic variations and cervical dysplasia are yet to be described. human papillomavirus 35 has also been found to be the second-most prevalent and persistent hpv infection in hiv-positive women.11 a systematic review conducted in 2020 revealed that, amongst women in ethiopia, there was a marked difference in the prevalence of high-risk hpv strains depending on hiv status, with hpv 35 accounting for 10% of infections in hiv-positive individuals.12 interestingly, a systematic review in sub-saharan africa by okoye et al. in 2021 regarding prevalent hpv genotypes in hiv patients showed that hiv-positive women with hpv 35 were twice as likely to develop cervical cancer compared to those with hpv 16 infections.13 urgent need for studies on the genomic epidemiology of human papillomavirus 35 in african women these marked disparities observed amongst hpv in african women and the peculiarity of hpv 35 cannot continue to be neglected, as they have major implications for funding and prioritisation of hpv and cervical cancer research. despite its apparent association with a significant proportion of cervical dysplasia in african women, there is a paucity of studies on the genomics and biology of hpv 35. why does hpv 35 have a stronger predilection for african women? is there something in the genome of africans that increases the tissue tropism or persistent infectivity of hpv 35? what could be the role of hiv in hpv 35 infections? to address these lingering questions, the comprehensive genomic epidemiology of hpv infection among african women needs to be described. this is important, as the approved hpv vaccines do not cover hpv 35. advancements in high-throughput technologies and next-generation sequencing allow not just dna analysis but also analysis of genomic expression profiles in protein products, as well as the study of epigenomic patterns such as the heterogeneous methylation observed in high-risk hpv precancerous lesions.14 although genomic advances continue to bring us closer to the goal of precision medicine, africa is being left behind due to inadequate representation in the data being generated. despite africa having the most diverse population, only a small amount of genomic data from africa is available for decision-making. to achieve global health equity in cervical cancer care and prevention, genomics-informed research with adequate representation of africans is key. we must start generating genomic data in africa to allow sufficient evidence to drive the implementation of inclusive cancer policies. role of genomics in developing an improved multivalent vaccine that targets human papillomavirus 35 genomic technologies allowed coronavirus disease 2019 scientists to make significant progress, by sequencing the genomes of coronavirus disease 2019 strains and translating that knowledge to vaccine design. although some studies have shown that available vaccines offer cross-protection to other oncogenic hpv types, care must be taken in the interpretation of these studies due to the importance of various underlying factors such as hiv status and the occurrence of multiple-type hpv infections. a study conducted among participants in a gardasil vaccine trial back in 2007 showed that the quantity of antibodies required to neutralise hpv 45 (not covered by the vaccine) was about 10 to 100 times greater than that for hpv 18, even though they share the same phylogenetic origin.15 we need to conduct more specific studies on the effectiveness of these vaccines against hpv 35 and identify the basis for cross-protection and impact. this would help the scientific community establish the exact efficacy of the available hpv vaccines for african women. reverse vaccinology, which was applied in the development of the meningococcal vaccine,16 could be used in developing better hpv vaccine candidates. a thorough analysis of various hpv 35 sublineages would allow the detection of novel hpv antigens and inform the rapid development of effective multi-epitope vaccines. since host genetic factors may be responsible for the kind of hpv infections seen, studies should investigate candidate host genes involved in the immune response and genetic variations that may negatively impact hpv vaccination strategies. to further refine the available vaccines, the design and implementation of genome-wide association studies using data obtained across africa could help to identify certain high-risk loci in the human genome that mediate increased susceptibility to hpv 35 infections and to identify the precise genomic relationships between hpv 35 and hiv infections. need for collaborative efforts in upscaling human papillomavirus 35 research in africa as much as governments are saddled with providing basic services such as hpv vaccination and improving access to hpv screening to meet up with the global cervical cancer elimination target,17 we must always remember that research plays a major role in improving the effectiveness and quality of patient care. a recent study conducted of the genomes of 910 people of african descent showed that around 300 million base pairs of their genes and genomic regions were not present in the reference human genome.18 this poses a significant problem in the level of information we can harness about human diseases. previous phylogenetic analysis revealed that hpv 16, which is a close relative of hpv 35, originated from africa before dispersal into other populations.19 with the strong association of hpv 35 with cervical cancer in african women, there is a need for stakeholders to make efforts to drive research that would unearth the uniqueness and pathogenic mechanisms of hpv 35 among african women. generating the necessary scientific evidence would provide a strong basis for the implementation of policies enabling the coverage of hpv 35 by the available vaccines. currently, there is a dearth of trained scientists, necessary equipment, and funding to tackle some of the fundamental research questions surrounding hpv 35 carcinogenicity. national government research agencies, private corporations, foreign research foundations, and academic institutions need to come together to promote hpv 35 research. a good example of collaborations that have driven genomics-based research on the african continent is the h3africa consortium, an initiative of the united states’ national institutes of health, and wellcome sanger institute that has empowered thousands of scientists from gambia, nigeria, ethiopia, uganda, and botswana to produce quality research. one major discovery achieved through this scientific partnership was that, according to research on a study population from nigeria and ghana, africans tend to have a special variant of the gene encoding apolipoprotein l1, thus resulting in an increased risk of kidney disease.20 the east africa hpv consortium has also conducted research on various aspects of hpv, including delineating the immunohistochemical differences in cervical intraepithelial neoplasia-2 and -3 between hiv-negative and hiv-positive patients.21 these efforts should be emulated across other regions of sub-saharan africa. conclusion the lessons learnt from the coronavirus disease 2019 and hiv pandemics have been logical, precise, and unambiguous – for sustainable progress to be made, african scientists must be empowered to solve challenges in africa for africa. genomics studies are vital to elucidating the molecular epidemiology of hpv in africa, understanding the associated risk factors and carcinogenic potential of endemic genotypes like hpv 35, and designing effective vaccines to capture the hpv diversity within the african population. thus, there is an urgent need to improve genomics capacity in africa given its potential role in reducing the comparatively high burden of cervical cancer on the continent. to achieve equity, we must ensure an adequate representation of data from africa in the sequence data available to research scientists and industries. this can be achieved by funding genomic research in africa, building capacity, and raising awareness of these issues within the scientific community. acknowledgements i am grateful to my teacher, dr a.o. fowotade, for her immense support and scholarly guidance. i also want to express my gratitude to my friends and mentors, dr o.j. afolaranmi and dr k.f. okwunze, for their insightful comments and feedback on this article. competing interests the author declares that she has no financial or personal relationships that may have inappropriately influenced her in writing this article. author’s contributions s.u.o. is the sole author of this article. ethical considerations i confirm that ethical clearance was not needed for this article. this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the author and do not necessarily reflect the official policy or position of any affiliated agency of the author. references hamdi y, abdeljaoued-tej i, zatchi aa, et al. cancer in africa: the untold story. front oncol. 2021 apr 15;11:650117. https://doi.org/10.3389/fonc.2021.650117 sung h, ferlay j, siegel rl, et al. global cancer statistics 2020: globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries. ca cancer j clin. 2021 may;71(3):209–249. https://doi.org/10.3322/caac.21660 ngcobo n, jaca a, iwu-jaja cj, mavundza e. reflection: burden of cervical cancer in sub-saharan africa and progress with hpv vaccination. curr opin immunol. 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women in developing countries: systematic review and meta-analysis. virol j. 2020 nov 16;17(1):179. https://doi.org/10.1186/s12985-020-01448-1 okoye jo, ofodile ca, adeleke ok, obioma o. prevalence of high-risk hpv genotypes in sub-saharan africa according to hiv status: a 20-year systematic review. epidemiol health. 2021;43:e2021039. https://doi.org/10.4178/epih.e2021039 verlaat w, van leeuwen rw, novianti pw, et al. host-cell dna methylation patterns during high-risk hpv-induced carcinogenesis reveal a heterogeneous nature of cervical pre-cancer. epigenetics. 2018;13(7):769–778. https://doi.org/10.1080/15592294.2018.1507197 smith jf, brownlow m, brown m, et al. antibodies from women immunized with gardasil cross-neutralize hpv 45 pseudovirions. hum vaccin. 2007;3(4):109–115. https://doi.org/10.4161/hv.3.4.4058 masignani v, pizza m, moxon er. the development of a vaccine against meningococcus b using reverse vaccinology. front immunol. 2019;10:751. 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papillomavirus and cervical cancer in women living with hiv/aids. ann med. 2022 dec;54(1):1202–1211. https://doi.org/10.1080/07853890.2022.2067897 abstract introduction ethical considerations case presentation management and outcomes discussion acknowledgements references about the author(s) christoffel j. opperman division of medical microbiology, national health laboratory service, university of cape town, cape town, south africa gert j.k. marais division of medical virology, national health laboratory service, university of cape town and groote schuur hospital, cape town, south africa michelle naidoo division of medical virology, national health laboratory service, university of cape town and groote schuur hospital, cape town, south africa marvin hsiao division of medical virology, national health laboratory service, university of cape town and groote schuur hospital, cape town, south africa nazlee samodien division of medical microbiology, national health laboratory service, university of cape town, cape town, south africa citation opperman cj, marais gjk, naidoo m, hsiao m, samodien n,. response to a cluster of severe acute respiratory syndrome coronavirus 2 cases at a diagnostic laboratory. afr j lab med. 2020;9(1), a1307. https://doi.org/10.4102/ajlm.v9i1.1307 case study response to a cluster of severe acute respiratory syndrome coronavirus 2 cases at a diagnostic laboratory christoffel j. opperman, gert j.k. marais, michelle naidoo, marvin hsiao, nazlee samodien received: 18 june 2020; accepted: 06 july 2020; published: 25 aug. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract introduction: we report on the first documented cluster of coronavirus disease 2019 cases amongst diagnostic laboratory staff and outline some of the initial and ongoing steps that are being implemented to manage and prevent the spread of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) in our laboratory. case presentation: on 24 april 2020, three staff members of a tertiary diagnostic laboratory in groote schuur hospital, cape town, south africa, tested positive for sars-cov-2. within seven days, a further nine cases were identified, which suggested an outbreak and prompted a full investigation. management and outcome: a multifaceted strategic approach was adopted to halt the spread of sars-cov-2 in our laboratory. interventions focused on simultaneously establishing appropriate risk mitigation and stratification strategies through the upscaling of infection prevention and control measures, whilst minimising disruption to service delivery. conclusion: laboratory coronavirus disease 2019 outbreaks have the potential to cripple a laboratory’s testing capacity. contingency planning and risk assessments should occur early, and interventions should be modified according to each laboratory’s available resources and infrastructure. keywords: sars-cov-2; covid-19; diagnostic laboratory; outbreak; occupational exposure. introduction to our knowledge this is the first reported cluster of coronavirus disease 2019 (covid-19) cases in a laboratory in south africa. with transmission of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) firmly established in our communities, infection amongst laboratory staff and laboratory outbreaks pose a serious threat to both private and public pathology laboratories and have the potential to derail the already-strained pathology services. we outline some of the initial and ongoing steps that are being implemented in our laboratory to manage and prevent the spread of sars-cov-2. ethical considerations this work was done in accordance with all ethical standards for carrying out research, in an emergency situation, without direct contact with human or animal subjects. institutional (national health laboratory service) and departmental (department pathology, division microbiology,) approval was obtained. case presentation on 24 april 2020, three staff members at the national health laboratory service situated within groote schuur hospital, tested positive for sars-cov-2. within seven days, a further nine cases were identified, which suggested an outbreak and prompted a full investigation. this resulted in a temporary closure of affected sections of the laboratory and a significant disruption to laboratory services in the subsequent week. management and outcomes outbreak investigation strategy once the initial cluster was identified, a task team comprising pathologists and managers from multiple pathology disciplines was established to lead an outbreak investigation, whilst simultaneously upscaling infection prevention and control measures. the completion of a line list and analysis of the descriptive epidemiology allowed for the identification of laboratory-specific risk factors and transmission ‘hotspots’. screening for severe acute respiratory syndrome coronavirus 2 infection amongst staff members it is well described that asymptomatic and pre-symptomatic people can transmit sars-cov-2 effiently.1 thus, three days after the three initial confirmed cases, samples were collected from all staff members over a one-week period, regardless of exposure. a designated swabbing station was set up in the laboratory and staff were informed to collect their own swabs for reverse transcriptase polymerase chain reaction (rt-pcr) testing. a video on the correct technique for self-sampling was circulated prior to sample collection. this further limited potential exposure of laboratory staff, whilst reducing strain on hospital testing sites, and facilitated a shorter time to result, allowing for the resumption of clinical diagnostic services. currently, all laboratory staff are being screened daily with a temperature check and a simple symptom-based questionnaire to help identify cases early.2 response interventions and contact tracing all staff that tested positive for sars-cov-2 by rt-pcr were instructed to self-isolate at home and to return to work only after two weeks, if there was a resolution of clinical symptoms. this was in accordance with the south african covid-19 national guidelines.3 contact tracing was carried out immediately for all laboratory personnel who tested positive, to contain the spread of the virus. the contact tracing was limited to laboratory workers. expanded contact tracing, including family members, was conducted independently by the western cape department of health. asymptomatic staff with confirmed exposure to colleagues who tested positive by rt-pcr, were categorised as high-risk for covid-19 according to national guidelines.4 these asymptomatic employees were given special leave to quarantine for seven days and were required to submit a nasopharyngeal or mid-turbinate swab on day 8 for rt-pcr testing. they were to return to work if their rt-pcr test was negative but had to continue daily symptom self-checks until day 14 post exposure. in our laboratory, one staff member with known risk factors for severe covid-19 passed away, whilst the remaining 11 (this includes the first three cases) qualified to return to work after 14 days; 10 of the 11 were mildly symptomatic and one was asymptomatic.3,4 positive specimens have been stored with a view to conduct genomic sequencing and analysis. the resources to perform sequencing in real time were not available, although we did recognise that it would have been extremely helpful in deducing transmission dynamics.5 infection prevention and control measures reinforced in the laboratory laboratory infection prevention and control measures were communicated swiftly to all staff. the local pathology management team recommended several measures to combat intra-laboratory transmission of sars-cov-2. these included: wearing of appropriate masks for all staff as opposed to virology and specimen reception personnel only, frequent and thorough hand-washing and cleaning of surfaces, frequent sanitisation of hands with 70% alcohol-based solutions, correct usage of personal protective equipment in addition to masks in appropriate circumstances and practice of social distancing. to aid the implementation of the above-mentioned measures, masks and personal protective equipment were made available, and extra bottles of alcohol-based solutions were placed at the entry and exit points of the laboratory.6 risk mitigation strategies a shift system was implemented where amenable, to increase social distancing and to avoid having to isolate or quarantine the whole workforce if virus transmission continued. the need to acutely reduce the size of the on-site workforce in the sars-cov-2 diagnostic laboratory had to be weighed carefully against the inevitable increase in test turn-around time this would cause and the resultant public health implications for the community it serves. flexible solutions included staggering shifts and encouraging staff who could work from home to do so. in some departments, where feasible, senior staff over the age of 60 with risk factors7 for covid-19 were advised not to return to work during the initial phase of the outbreak. it took much deliberation to determine which of the high-risk employees could stay at home, because it had to be balanced against the consequences on service delivery in the context of a pandemic. extra personnel from other pathology departments within the national health laboratory service, as well as from affiliated departments at the university of cape town, were trained as backup sars-cov-2 testing staff. additional automated kit-based platforms requiring minimal molecular training to operate were also introduced. the purpose was to increase the operator pool and to avoid key-person dependency, which could potentially disrupt service delivery if any or all of the key persons were isolated or quarantined. testing platforms included the seegene allplex™ 2019-ncov assay (seegene, seoul, republic of korea) after in-house or nuclisens® easymag® nucleic acid extraction (biomérieux, marcy l’étoile, france) and the abbott realtime sars-cov-2 assay (abbott laboratories, abbott park, illinois, united states). assay data were analysed according to the manufacturers’ instructions. daily laboratory activities all academic activities were suspended and only essential on-site meetings that did not violate social distancing measures were held. areas where staff were less vigilant about infection prevention and control precautions, such as the tearoom, were identified and risks mitigated as far as possible. for the tea room, ipc measures included frequent reminders to practise social distancing, request that staff bring and clean their own crockery and cutlery, and limit on the number of staff members in the room at any given time to six people. in addition to the tearoom, high-touch areas, for example, communal telephones, scanners, keyboards, light switches and door handles, were also identified as high-risk areas. therefore, more frequent surface cleaning and disinfection was instituted. furthermore, carpooling by staff, especially from covid-19 ‘hot spots’ in the cape metropole, was discouraged and management provided masks for those commuting to and from work via public transport. maintaining service delivery sections of the laboratory (haematology and chemical pathology) needed to be closed briefly for decontamination during the outbreak. these samples were diverted to a neighbouring national health laboratory service facility to avoid a breakdown in service delivery. however, the sudden transfer of large volume of samples to an understaffed and unprepared laboratory did present challenges that delayed result turn-around times. in another attempt for the laboratory to meet with the increased sars-cov-2 testing demands, non-priority tests were suspended unless suitably justified by the clinician. communication staff were kept abreast of new covid-19-related laboratory information through regular small group or zoom (zoom video communications, san jose, california, united states) meetings. laboratory section-specific communication strategies, such as whatsapp (whatsapp, menlo park, california, united states) groups, were implemented. pathologists were also in regular telephonic contact with affected staff members who were in isolation to ensure that they had the necessary support during recovery. transparency and collaboration were a prominent feature of our response. in particular, the laboratory worked with groote schuur hospital and the provincial outbreak response team to minimise the service disruption and to ensure that the laboratory could be reopened both safely and timeously. discussion challenges and lessons learned the lack of a guideline specific for management of a sars-cov-2 laboratory outbreak posed a significant challenge in mobilising a quick outbreak response plan. although international guidelines were available and consulted, they were not applicable to our setting. the dynamic nature of the outbreak necessitated frequent revisions to staff scheduling rosters when certain employees had to be isolated or quarantined. this was not an easy task, especially because the skill levels amongst all staff were not equally matched. another difficulty was ensuring the safety of staff from sars-cov-2, whilst travelling to and from work. many relied on public transport and carpooling and had no alternative means of travel. lessons learned include the importance of early implementation of a symptom screening tool to detect cases earlier and to prevent spread within the laboratory. we realised the importance of developing contingency plans for any crisis causing laboratory closure in the future. had there been an existing plan, referral of large numbers of samples would probably have occurred faster and more smoothly, without impacting test turn-around time negatively. this outbreak emphasised the need for skills transfer amongst staff to avoid key person dependency issues. we need to institute training programmes that will allow staff to fulfil multiple roles, thereby making it less likely that we jeopardise testing. conclusion a sars-cov-2 outbreak in a diagnostic laboratory can cripple its testing capacity, particularly one that is already under strain. a multifaceted strategic approach was adopted to halt the spread of sars-cov-2 in our laboratory, whilst minimising service delivery disruptions. hopefully, our experiences serve to help other laboratories that find themselves in a similar situation. it is necessary for interventions to be modified based on each facility’s infrastructure and available resources. these recommendations should also be adjunctive to good laboratory practice principles. acknowledgements we would like to acknowledge all the staff in our laboratory who have worked tirelessly at the frontlines of this pandemic. competing interests the authors have no competing conflict of interest to declare with regards to the material discussed in the article. authors’ contributions all authors contributed equally to this work. sources of support the research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability statement data sharing is not applicable to this article, as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect an official policy or position of any affiliated agency of the authors. references gandhi m, yokoe ds, havlir dv. asymptomatic transmission, the achilles’ heel of current strategies to control covid-19. n engl j med. 2020;382(22):2158–2160. https://doi.org/10.1056/nejme2009758 world health organization. risk assessment and management of exposure of health care workers in the context of covid-19 interim guidance [homepage on the internet]. c2020 [cited 2020 may 14]. available from: https://apps.who.int/iris/bitstream/handle/10665/331496/who-2019-ncovhw_risk_assessment-2020.2-eng.pdf nicd clinical management of suspected or confirmed covid-19 disease version 3 [homepage on the internet]. c2020 [cited 2020 may 14]. available from: https://www.nicd.ac.za/wp-content/uploads/2020/03/clinical-management-of-suspected-or-acute-covid-19-version-3.pdf department of health, south africa. guidelines for quarantine and isolation in relation to covid-19 exposure and infection [homepage on the internet]. c2020 [cited 2020 may 14]. available from: https://www.nicd.ac.za/wp-content/uploads/2020/05/guidelines-for-quarantine-and-isolation-in-relation-to-covid-19.pdf nutman a, marchaim d. how to: molecular investigation of a hospital outbreak. clin microbiol infect. 2019;25(6):688–695. https://doi.org/10.1016/j.cmi.2018.09.017 department of health, south africa. covid-19 disease: infection prevention and control guidelines version 1 [homepage on the internet]. c2020 [cited 2020 may 14]. available from: http://www.health.gov.za/index.php/component/phocadownload/category/626 zhou f, yu t, du r, et al. clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study. lancet. 2020;395(10229):1054–1062. https://doi.org/10.1016/s0140-6736(20)30566-3 abstract introduction the caribbean region lqms-sip: implementation structure and stakeholders lqms-sip: implementation process discussion acknowledgements references about the author(s) george alemnji centers for disease control and prevention (cdc), caribbean regional office, bridgetown, barbados lisa edghill caribbean public health agency (carpha), port of spain, trinidad and tobago giselle guevara centers for disease control and prevention (cdc), caribbean regional office, bridgetown, barbados sacha wallace-sankarsingh caribbean public health agency (carpha), port of spain, trinidad and tobago rachel albalak centers for disease control and prevention (cdc), caribbean regional office, bridgetown, barbados sebastien cognat world health organisation (who), lyon, france john nkengasong us centers for disease control and prevention (cdc), atlanta, georgia, united states jean-marc gabastou pan american health organization (paho), lima, peru citation alemnji g, edghill l, guevara g, et al. development and implementation of the caribbean laboratory quality management systems stepwise improvement process (lqms-sip) towards accreditation. afr j lab med. 2017;6(1), a496. https://doi.org/10.4102/ajlm.v6i1.496 lessons from the field development and implementation of the caribbean laboratory quality management systems stepwise improvement process (lqms-sip) towards accreditation george alemnji, lisa edghill, giselle guevara, sacha wallace-sankarsingh, rachel albalak, sebastien cognat, john nkengasong, jean-marc gabastou received: 17 may 2016; accepted: 30 sept. 2016; published: 24 feb. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: implementing quality management systems and accrediting laboratories in the caribbean has been a challenge. objectives: we report the development of a stepwise process for quality systems improvement in the caribbean region. methods: the caribbean laboratory stakeholders met under a joint pan american health organization/us centers for disease control and prevention initiative and developed a user-friendly framework called ‘laboratory quality management system – stepwise improvement process (lqms-sip) towards accreditation’ to support countries in strengthening laboratory services through a stepwise approach toward fulfilling the iso 15189: 2012 requirements. results: this approach consists of a three-tiered framework. tier 1 represents the minimum requirements corresponding to the mandatory criteria for obtaining a licence from the ministry of health of the participating country. the next two tiers are quality improvement milestones that are achieved through the implementation of specific quality management system requirements. laboratories that meet the requirements of the three tiers will be encouraged to apply for accreditation. the caribbean regional organisation for standards and quality hosts the lqms-sip secretariat and will work with countries, including the ministry of health and stakeholders, including laboratory staff, to coordinate and implement lqms-sip activities. the caribbean public health agency will coordinate and advocate for the lqms-sip implementation. conclusion: this article presents the caribbean lqms-sip framework and describes how it will be implemented among various countries in the region to achieve quality improvement. introduction strengthening laboratory services and systems to meet the challenge of the growing burden of diseases of global public health importance has been the focus of discussions and recommendations by the international community.1,2,3 implementing laboratory quality management systems (qms) and attaining accreditation has been an important part of these discussions.4,5 laboratory accreditation to international standards verifies laboratories’ competence and ensures that they can provide evidence that test results are reliable and accurate to support patient care and public health practices.6,7 strengthening health systems, especially medical laboratories, in the caribbean has been a challenge. as a result, few public health laboratories are accredited. several country and regional efforts have been made to improve quality systems and achieve accreditation of the laboratories in the region. for example, the caribbean epidemiology centre/european union-funded project, carried out between 2002–2007, designed and implemented a 30-month blended accredited training programme on laboratory quality systems improvement that graduated 40 senior laboratory staff, initiated the caribbean laboratory accreditation scheme, and recommended that the international standard iso 15189 be the recognised standard in the caribbean for medical laboratories.8 despite these efforts, reports indicate that by 2008 only nine (5.2%) of the estimated 173 laboratories in the caribbean region were accredited. six of these were medical laboratories.9 all of the accredited laboratories were either private or faith-based health facility laboratories. thus, although more than 90% of the population utilises public or government laboratory services, none of these services were accredited. cognisant of these challenges, a joint collaboration between the pan american health organization and the us centers for disease control and prevention, together with other regional stakeholders in 2010, decided to develop the caribbean regional laboratory quality management system – stepwise improvement process (lqms-sip) towards accreditation framework to address laboratory quality challenges.10 this initiative aligns well with the world health organization (who) and the us centers for disease control and prevention joint conference on laboratory quality systems held in april 2008 in lyon, france, which recommended that ‘countries with limited resources consider taking a staged approach, where principal requirements for all are stated in the national laboratory standards as a minimum requirement, while more advanced and national reference laboratories are encouraged to aim at meeting internationally accepted standards such as iso 15189’.2 additionally, successful implementation of qms and achievement of accreditation using the stepwise approach have been documented in other regions such as the who regional office for africa’s (afro) stepwise laboratory improvement process towards accreditation (slipta),11,12 the thailand stepwise accreditation process,13 and the argentina scheme.14 in this article, we present the caribbean lqms-sip framework, developed following the above joint initiative. we also describe how the lqms-sip framework will be implemented among various stakeholders and countries to achieve a common goal. the caribbean region rich in culture and diverse ethnicities, the caribbean has the world’s largest collection of small island states, many of them independent nations. many of them depend almost solely on tourism as the main economic driver, resulting in a variety of different growth rates and per capita incomes. a disease outbreak can have catastrophic impacts on the social and economic development of these already fragile economies, which can lead to global isolation. evidence has shown that a priority for government is to ensure that the health sector, specifically medical laboratories, maintain a high capacity to provide reliable and timely information to protect public health and guarantee long-term sustainability of economic growth factors. the caribbean also has several cross-border regional organisations, unions, and groups that have been formed based on commonalities in culture, economy, religion, health specific needs, and the common good. these regional groupings have become very important as they leverage resources, reduce costs, and create a clear line of commitment and support to ensure that the smaller islands share and benefit from various opportunities and capacities found in the neighbouring larger islands. this is why a regional approach has been used in the development and implementation of the lqms-sip. lqms-sip: implementation structure and stakeholders the implementation of the lqms-sip is a joint effort by several stakeholders in the region, with each partner performing activities specific to its recognised role and providing expertise in the implementation process as required. specifically, the caribbean community regional organisation for standards and quality, which is the caribbean community centre for promoting efficiency and competitive production in goods and services through standardising and verifying quality, has hosted the secretariat of the lqms-sip since mid 2013. stakeholders agreed, at a regional consultation in 2014, that the management of the lqms-sip will be a mutual cooperation and collaboration among the two recognised caribbean national accreditation bodies and the national accreditation focal points, based within the bureau of standards of each country, and the caribbean regional organisation for standards and quality secretariat. the caribbean public health agency, mandated to serve as a coordination unit for the caribbean public health laboratory network, will continue this function to ensure coordination and collaboration of various stakeholders for a common interest. the ministries of health of various countries, recognised as the principal owners of the process, are expected to work closely with the above entities as they rollout and implement the lqms-sip in their various countries. other stakeholders including the pan american health organisation/who, the us centers for disease control and prevention, professional associations and non-government organisations will work to sustain functional public private partnerships to advance the course and ensure the sustainability of the lqms-sip.10 lqms-sip: implementation process eligibility, application, and enrolment all laboratories (both public and private) are eligible to participate in the caribbean lqms-sip. application and enrolment will follow guidelines of national laboratory regulatory systems in participating countries and the lqms-sip secretariat will receive and review all applications. a laboratory that applies to be evaluated for tier 1 or higher will be sent an enrolment letter containing the enrolment date, enrolment number, and proposed timeframe for scheduling an assessment. qualified assessors will then be selected based on the nature and type of laboratory. monitoring of the process, including competence and performance of the assessors, logistics, and communication with the ministry of health and/or laboratory to agree on suitable dates for the assessment, will be coordinated by the lqms-sip secretariat as outlined in figure 1.10 figure 1: flow chart of the caribbean laboratory quality management systems stepwise improvement process, showing the different steps for enrolment and participation of laboratories. checklist the assessment will be carried out using the caribbean lqms-sip checklist, which is on the iso 15189:2012 standard and is broken down into the two main sections: management and technical requirements. the checklist is further subdivided into 15 management and 10 technical clauses; hence, the same structure as the iso 15189 standard clause numbering system has been used to form the framework of the lqms-sip checklist categories (table 1). an update was also made to add safety requirements based on the iso 15190 standard, to ensure that each laboratory assessment gave a thorough review of the system. this checklist has a total of 104 requirements that have to be met by laboratories at the tier 3 level. table 1: checklist of the caribbean laboratory quality management systems stepwise improvement process, showing how laboratories will be scored following their quality improvements. the tiered structure following each assessment, laboratories that meet the specific qms requirements will be recognised according to the agreed three-tier (scoring) system (figure 2). the basic national mandatory requirement for licensing of laboratories by the ministry of health is equivalent to tier 1, while the quality improvement milestones that are acquired by fulfilling certain requirements of the iso 15189 standard correspond to tiers 2 and 3. once a laboratory has successfully attained a tier level (1 or 2), they will be expected to continuously improve the qms to meet the next level up at their follow up annual assessment. the national accreditation focal points nominated by each bureau of standards will play an important role in linking the laboratory staff to human and technical resources to effectively eliminate the identified non-conformances and improve their performance. laboratories attaining a tier 3 certification at any time, will be encouraged to enrol in an established (internationally-recognised) accreditation programme based on iso 15189: 2012 (figure 2).10 figure 2: caribbean laboratory quality management systems stepwise improvement process showing the stepwise levels of recognition of laboratories at enrolment and following quality improvement. mandatory tier 1 requirements the first tier will represent the minimum requirements corresponding to the mandatory criteria required for obtaining a national licence based on legislation enacted by the respective ministries of health (figure 2). ministries of health are expected and encouraged to establish their own national regulatory systems for the operation of clinical laboratories and to build capacity for licensing in-country. at present, there are countries in the region which do not have operational systems for granting licences to laboratories. these ministries of health will be encouraged to enrol their laboratories in the lqms-sip while they work on national regulations for recognising tier 1 as equivalent to a government license. those countries which already inspect and grant licenses will be encouraged to review the requirements for such and subsequently align it to the criteria for tier 1, since these are already based on the international standard. where appropriate, the ministries of health should appoint a national accreditation focal point to coordinate all activities in-country, including engagement with the lqms-sip secretariat. laboratory assessment and recognition process once the assessment is completed, the assessors will submit their preliminary report to the laboratory and the final assessment report to the lqms-sip secretariat. the laboratory will then be expected to respond to any gaps identified during the audit and to develop a customised corrective action plan within 30 days. laboratories will be given up to 90 days to provide evidence of satisfactory resolution of non-conformities and improvement of the qms. the assessors will review the non-conformances and produce a determination report. the advisory committee will be convened to review the determination report and recommend which tier level has been attained. the lqms-sip secretariat will then make a final determination on awarding a certificate to the laboratory (figure 1) for the relevant tier achievement. follow-up assessment and continuous quality improvement process laboratories that meet all the requirements of each tier will be issued a certificate of recognition that is valid for two years from the original date of issue. application for reassessment and renewal of the certificate should be submitted to the lqms-sip secretariat six months prior to the expiration of the existing certificate. laboratories that obtain the tier 3 recognition certificate are encouraged to apply for accreditation from a full member of the international laboratory accreditation cooperation. laboratories that are accredited to the iso standard, will be transitioned from the lqms-sip assessment register and their achievement will be recognised on the caribbean regional organisation for standards and quality website (http://www.crosq.org). discussion well-designed and effective qms operating in laboratories according to the requirements of an international standard such as iso 15189, which could eventually lead to accreditation, is necessary for the caribbean region. the benefits of accreditation include the release of quality assured results for patients, cost savings in laboratory management, improved turn-around times, client satisfaction and enhanced staff competency.6,4,7 furthermore, the international health regulations are an incentive for countries to develop, strengthen, and maintain the capacities of their laboratories to detect, assess, notify, and report health events to the who.1 since countries in the caribbean are signatory to this requirement, strengthening countries’ existing capacities for public health surveillance and quality laboratory services are critical for international health regulations implementation and continuous improvement of the health sector. quality systems improvement and accreditation of laboratories in the caribbean has been very slow to develop, with very few clinical laboratories achieving this milestone. this includes the lack of advocacy, weak laboratory infrastructure and workforce, inadequate financial resources, and the complexity of the entire accreditation process.8,9 however, there are indications that using the stepwise approach, together with other evidence-based laboratory improvement tools in implementing qms, could lead to more realistic results with minimal resources. for example, in 1994 the argentine biochemical foundation created an accreditation programme for clinical laboratories that used a stepwise approach, starting from minimum requirements up to higher standards, that allowed laboratories to slowly begin the process of quality improvement and eventually reach the desired result.14 in 2002, the latin american confederation for clinical biochemistry (confederación latinoamericana de bioquímica clínica) and the pan american health organisation/who developed a guideline for the development of medical laboratory accreditation tools in latin america and a course on quality management and good laboratory practices,15,16 which includes checklists for a gradual implementation. data from lesotho in africa show that using the who afro stepwise laboratory quality improvement programme,17 that also included the use of the strengthening laboratory management towards accreditation training programme and mentorship, resulted in tangible improvements in the development and implementation of quality systems in the enrolled laboratories.11 therefore, developing and using the stepwise-based caribbean lqms-sip for countries in the region has the potential to improve quality systems implementation and to result in eventual accreditation of more laboratories. an important component of the stepwise process for laboratory qms implementation towards accreditation is that it provides an opportunity for measuring laboratory progress and identifying and improving quality systems in real-time. it also provides an opportunity for recognising gains in competence through periodic assessments and award of recognition certificates to laboratories as they make progress toward fulfilling the requirements of the standard. this interactive approach makes the entire process more dynamic and encouraging and has the potential to inspire more laboratories in the region to initiate and continue the process of quality improvement toward accreditation. although the stepwise approach for quality systems improvement has proven to be very helpful, it is necessary to tailor it to the laboratory infrastructure and capacity of each region or specific country, as tools developed in different settings may not address the immediate laboratory needs of other environments. thailand developed a customised laboratory standard derived from multiple requirements contained in various international standards, but retained the most relevant and critical portions and made the standard applicable to thailand and its infrastructure. the requirements of this standard were affordable, measureable, adaptable, sustainable and effective, and resulted in many laboratories in the country being accredited.13 the who afro slipta programme,17 tailored to the needs of laboratories in africa, resulted in many laboratories being enrolled in the quality systems improvement and accreditation process.18 the caribbean lqms-sip was developed based on the needs and specific challenges of laboratories in the caribbean. there are a number of unique or innovative features of the caribbean scheme compared to systems that have been developed in other regions. the caribbean scheme, for example, has three tiers compared to the five-star rating of the who afro process. furthermore, the caribbean scheme has licensing/regulation as a baseline requirement of its tier 1, which is not common with the other systems. hence, this scheme is specifically designed to address laboratory regulatory and quality issues within the caribbean context. monitoring laboratory operations and ensuring compliance with national and international standards in many developing countries has been hampered by the lack of policies and regulations that empower governments to directly supervise laboratory operations.19,20 in some instances, this has resulted in using unqualified persons for laboratory operations, substandard testing, and release of unacceptable results meant for patient care and policy decision making.21,22 past review of legislation developed and implemented among select countries in the caribbean showed that there is still considerable work to be done toward establishing and instituting legislation for quality assurance in public health and clinical laboratories in many of these countries.23 as a result of this finding, it has been proposed that tier 1 of the lqms-sip be mandatory and correspond to the requirement for being granted a licence based on legislation enacted by participating countries. ministries of health are encouraged to establish their own regulatory systems for operation of medical laboratories and build capacity for licensing by the government. only laboratories – public or private – registered with the ministry of health and/or licenced in-country, will be allowed to enrol in the lqms-sip. hence, fulfilment of the requirement of tier 1 is considered an entry point for a laboratory to participate in the lqms-sip. this will minimise the number of incorrect medical laboratory practices in the caribbean. to ensure that this framework is supported at all levels, quality improvement and the accreditation process should be well articulated in national health laboratory policies and strategic plans. more advocacy and sensitisation campaigns among policy makers, ministry officials, laboratory management and staff on the benefits of implementing an effective and sustainable qms and attaining accreditation are needed. limitations of implementing lqms-sip in the caribbean the implementation of the stepwise process for quality systems improvement and accreditation is new in the caribbean. some challenges include the lack of understanding of the process, limited support from laboratory staff and policy makers, and inadequate human, financial, and infrastructure resources. it is recommended that laboratory stakeholders develop more advocacy and awareness strategies that target policy makers and laboratory staff as well as strategies to raise funds to support infrastructure upgrades and human capacity development to meet the basic requirements of lqms-sip. conclusion the caribbean lqms-sip framework is a three-tiered process developed to provide a user-friendly approach to implement qms towards laboratory accreditation. recognition certificates will be awarded as laboratories move from one tier to the next. laboratories that reach tier 3 will be encouraged to apply for accreditation from a recognised body. governments are urged to take greater responsibility and to ensure that structures are in place in-country for licensing of medical laboratories and for their enrolment and participation in the lqms-sip. there is evidence that the stepwise accreditation process provides an opportunity for measuring laboratory progress and for recognising gains in competence as well as providing opportunities for identifying and improving quality systems. hence, the lqms-sip has the potential to accelerate the process of quality systems improvement and laboratory accreditation in the region. box 1: lessons learned. acknowledgements this project was supported by the president’s emergency plan for aids relief through cooperative agreement number ps001426 from the us centers for disease control and prevention. we would like to acknowledge the contributions of all members of the caribbean lqms-sip technical working group (claudette brown, jamaica national agency for accreditation; eileen burke, caribbean epidemiology center; ellison floyd-tobas, trinidad & tobago laboratory accreditation service; floris gordon, african field epidemiology network; wendy kitson-piggott, caribbean med labs foundation; vernita maryat, caribbean community regional organisation for standards and quality; mary nagel, laboratoire de santé publique en haiti; marsha samaroo, ministry of health, republic of trinidad & tobago; david turgeon, us centers for disease control and prevention; and valerie wilson, caribbean med labs foundation). the who afro slipta process also played an important role in the development of this framework, since the who afro slipta guidance document was used as a template for conceptualising the lqms-sip. disclaimer the authors alone are responsible for the views expressed in this article and they do not necessarily represent the views, decisions or policies of the institutions with which they are affiliated. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. sources of support none. authors’ contributions g.a. was the project lead and wrote the manuscript. l.e. and g.g. provided technical input and data. s.w.-s. provided technical input. r.a., s.c., j.n. and j.-m.g. provided comments. all authors read and approved the final manuscript. references world health organization. international health regulations (2005) [document on the internet]. c2005 [cited 2012 dec 29]. available from: http://www.who.int/ihr/en/ world health organization. joint who-cdc conference on laboratory quality systems, lyon, april 2008 – joint statement and recommendations. wkly epidemiol rep. 2008;83(32):285–292. president’s emergency plan for aids relief. pepfar blueprint: creating an aids-free generation [document on the internet]. c2012 [cited 2016 jan 29]. available from www.pepfar.gov/documents/organisation/201386.pdf gershy-damet gm, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. https://doi.org/10.1309/ajcptuuc2v1wjqbm 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(2012) impact of mentorship on who-afro strengthening laboratory quality improvement process towards accreditation (slipta). afr j lab med. 2012;1(1), art. #6, 8 pages. https://doi.org/10.4102/ajlm.v1i1.6 mothabeng d, maruta t, mathabo l, et al. strengthening laboratory management toward accreditation: the lesotho experience. afr j lab med. 2012;1(1), art. #9, 7 pages. https://doi.org/10.4102/ajlm.v1i1.9 wattanasri n, manoroma w, viriyayudhagorn s. laboratory accreditation in thailand: a systemic approach. am j clin pathol. 2010;134(4):534–540. https://doi.org/10.1309/ajcpzyy19wmkmazt mazziotta d. accreditation of clinical laboratories in the latin-american region. clin biochem. 2009;42(4–5):309. https://doi.org/10.1016/j.clinbiochem.2008.09.026 latin american confederation for clinical biochemistry (colabiocli). guía para la elaboración de manuales de acreditación de laboratorios clínicos para américa latina, ops/hsp/hse-lab/06.2002 [document on the internet]. c2002 [cited 2016 dec 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the internet]. c2014 [cited 2016 dec 12]. available from: https://slmta.org/resources nkengasong j, birx d, sankalé j-l. challenges in developing laboratory capacity and infrastructure to support hiv/aids care programs. in: marlink rg, teitelman st (eds.). from the ground up: building comprehensive hiv/aids care programs in resource-limited settings. washington, dc: elizabeth glaser pediatric aids foundation; 2009. soenksen d. the “globalization” of the medical lab [document on the internet]. c2009 [cited 2016 dec 12]. available from: http://www.mlo-online.com/articles/200903/the-globalization-of-the-medical-lab.php. peeling rw, mabey d. point-of-care tests for diagnosing infections in the developing world. clin microbiol infect. 2010;16(8):1062–1069. https://doi.org/10.1111/j.1469-0691.2010.03279.x de kieviet w, frank e, stekel h. essentials of clinical laboratory management in developing regions [document on the internet]. c2008 [cited 2016 jan 29]. available from: http://www.ifcc.org/media/185572/2008%20-%20c-clm%20monograph.pdf pan american health organisation/world health organization. review and report on legislation developed/implemented for medical laboratories and public health laboratories in seven countries. paho/ecc/ecc/61.2/11.01; 2011. abstract introduction methods results discussion acknowledgements references about the author(s) timan t. eliya zankli research centre, bingham university, karu, nasarawa state, nigeria elvis e. isere zankli research centre, bingham university, karu, nasarawa state, nigeria bassey emmana zankli research centre, bingham university, karu, nasarawa state, nigeria chukwuebuka ugwu zankli research centre, bingham university, karu, nasarawa state, nigeria jonathan kushim zankli research centre, bingham university, karu, nasarawa state, nigeria precious ishaku zankli research centre, bingham university, karu, nasarawa state, nigeria aisha e. ibrahim department of biological sciences, faculty of sciences, bingham university, karu, nasarawa state, nigeria john s. bimba zankli research centre, bingham university, karu, nasarawa state, nigeria department of community medicine, bingham university, karu, nasarawa state, nigeria department of clinical sciences, liverpool school of tropical medicine, liverpool, united kingdom citation eliya tt, isere ee, emmana b, et al. impact of viral load on sample pooling for reverse-transcription polymerase chain reaction detection-based diagnosis of coronavirus disease 2019 in nigeria. afr j lab med. 2025;14(1), a2514. https://doi.org/10.4102/ajlm.v14i1.2514 original research impact of viral load on sample pooling for reverse-transcription polymerase chain reaction detection-based diagnosis of coronavirus disease 2019 in nigeria timan t. eliya, elvis e. isere, bassey emmana, chukwuebuka ugwu, jonathan kushim, precious ishaku, aisha e. ibrahim, john s. bimba received: 03 june 2024; accepted: 12 nov. 2024; published: 10 feb. 2025 copyright: © 2025. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the coronavirus disease 2019 (covid-19) pandemic strained diagnostic testing capacities globally, particularly in lowand middle-income countries like nigeria. reverse-transcription polymerase chain reaction (rt-pcr) remains the gold standard for covid-19 detection, but limited testing resources caused bottlenecks in nigeria’s response during the pandemic. sample pooling offers a cost-effective strategy to enhance testing capacity during future outbreaks. objective: this study determined the maximum number of covid-19 samples that can be pooled for rt-pcr testing in nigeria without compromising the detection sensitivity of a single positive sample. methods: a total of 1222 nasopharyngeal samples from symptomatic covid-19 patients in nasarawa state, nigeria, collected between march 2021 and august 2022, were retrieved from the laboratory biorepository and analysed from november 2022 to february 2023. these included five positive samples with cycle threshold (ct) values ranging from ≤ 20 to 40, and 1217 negative samples. positive samples were pooled with negative ones at increasing dilution ratios (1:4–1:64), to assess detection sensitivity on the genexpert platform. results: a positive sample with a ct value ≤ 25 could be pooled with up to 64 negative samples while maintaining a detectable positive result. however, samples with ct values of 36–40 could only be pooled with a maximum of eight negative samples. higher ct values reduced pooling effectiveness. conclusion: sample pooling is a feasible method for scaling up covid-19 rt-pcr testing in resource-limited settings like nigeria. the ct value is critical in determining optimal pool sizes for accurate detection. what this study adds: the findings provide critical guidelines for determining the optimal pool sizes based on ct values, aiding in effective covid-19 testing strategies. by optimising sample pooling based on viral load, health authorities can improve their response to future covid-19 outbreaks and similar public health emergencies. keywords: coronavirus disease 2019; pandemic; real-time polymerase chain reaction testing; cycle threshold; sample pooling; nigeria. introduction the coronavirus disease 2019 (covid-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2, created a global health crisis and drastically increased the demand for diagnostic testing.1,2 on 28 february 2020, the nigeria centre for disease control and prevention confirmed the first covid-19 case in nigeria.3,4 this was followed by rising daily cases and deaths across several states and the federal capital territory, including nasarawa state, which indicated widespread community transmission and unmet testing needs.5,6 as with many lowand middle-income countries, nigeria faced significant challenges in diagnostic testing during the pandemic because of limited resources. reverse-transcription polymerase chain reaction (rt-pcr), the gold standard for covid-19 diagnosis, was constrained by shortages of kits, reagents, and trained personnel.3 as of april 2020, nigeria had only 12 functional rt-pcr facilities (figure 1),3 which increased to 97 by january 2021, but these remained insufficient for a population of over 250 million.3,7 these limitations caused testing delays, hindered contact tracing, and underreported cases, weakening nigeria’s pandemic response.8 figure 1: laboratory testing capacity for coronavirus disease 2019 in nigeria as of 15 april 2020. sample pooling, a cost-effective diagnostic technique in which multiple samples were combined and tested together, emerged as a potential solution to these challenges.8,9,10,11,12,13,14,15,16,17,18,19 in this method, multiple patient samples were tested together as a single pool.14,15,16 if the pool tested negative, all samples were considered negative; if positive, each sample was tested individually to identify which samples were positive.17,18 this method reduced the number of tests required, conserved resources, reduced laboratory workload, and increased testing capacity.8,10,11 in countries like ghana, sample pooling proved successful during the pandemic.8,10,11 however, the effectiveness of pooling depended on several factors, including viral load, and determining the optimal number of samples per pool was crucial for ensuring pcr accuracy and efficiency.19 despite the global decline in covid-19 cases, as well as in nigeria, the threat of future pandemics and outbreaks due to covid-19 remains, especially with the emergence of different covid-19 variants, highlighting the importance of strengthening diagnostic systems.7 assessing the efficiency of sample pooling in nigeria, which was not widely implemented during the pandemic, could be crucial in preparedness for future outbreak response. this study aimed to determine the optimal number of covid-19 samples that could be pooled without missing a positive result in the nigeria setting, in order to enhance testing efficiency during any future covid-19 outbreak response in nigeria. methods ethical considerations ethical approval for this study was obtained from the federal capital territory health research ethics committee (reference no: fhrec/2024/01/48/04-03-24). a waiver for informed patient consent was granted, as stored laboratory samples were retrieved solely for analysis, with no additional clinical procedures involving patients. all personal information and patient identifiers were removed from the samples and study-specific numbers (identifiers) were assigned to each. these identifiers and outcome of the laboratory analysis were entered into an anonymised, password-protected excel spreadsheet (office 2016, microsoft, redmond, washington, united states), accessible only to the study investigators for data analysis. study area nigeria, the most populous country in africa, with over 250 million people,20 is organised into six distinct regional zones, each with unique ecological, climatic, and demographic characteristics.20,21 administratively, the country is divided into 36 states plus the federal capital territory, comprising 774 local government areas and 8812 wards.21 nasarawa state, situated in the north central zone, had a projected population of 3 079 710 in 2023.22 this state features a healthcare system of 1040 facilities, including 728 primary health centres, 18 secondary hospitals, 2 tertiary hospitals, and 292 private facilities.20,23 study specimens and laboratory analysis the samples for this study were aliquots stored in cryovials, retrieved from the zankli research centre molecular laboratory biorepository at bingham university, nasarawa state, nigeria. these included nasopharyngeal swab specimens in viral transport medium (mantacc viral transport media disposable sampling kit; huo-yan laboratory, shenzhen, china), collected from symptomatic patients suspected of covid-19 infection across communities in nasarawa state, nigeria, between march 2021 and august 2022. initially tested for covid-19 following nigeria centre for disease control and prevention guidelines and procedures,24 the samples were subsequently stored in an ultra-low freezer at –85 °c. the samples retrieved for this study comprised five positive samples and 1217 negative samples, each with documented cycle threshold (ct) values. the retrieved samples were analysed between november 2022 and february 2023. a repeat rt-pcr test was conducted to re-confirm the ct values for each sample before performing the pooled test procedure. these positive samples were tested individually by rt-pcr on cepheid’s genexpert® systems with the xpert® xpress sars-cov-2 (cepheid, sunnyvale, california, united states), and their ct values were grouped in the following ranges: ≤ 20, 21 ‒25, 26 ‒ 30, 31 ‒ 35, and 36 ‒ 40, to represent a decreasing viral load, with ‘≤ 20’ representing the highest and ’36‒40’ representing the lowest. similarly, 1217 samples confirmed to be negative through rt-pcr were pooled to create different pools of varying dilution factors. all procedures were conducted in a nuaire class ii 12469:2000 biological safety cabinet within the containment laboratory room by trained personnel. samples and pools were tested using cepheid’s genexpert® systems following the manufacturer’s instructions. each selected positive sample was pooled with negative samples at increasing dilution factors of 1:4 until the ct value of the pool with the highest dilution factor exceeded 40 while remaining positive. the ct values of each pool for each positive sample were recorded. for the first pool (1:4), 70 µl of the positive sample and 70 µl of each of the four (4) negative samples were transferred to a cryovial to obtain the 1:4 dilution factors. the contents were vortexed, transferred to a genexpert cartridge and loaded onto the genexpert platform according to the manufacturer’s instructions. the same procedure was used to constitute pools of 1:8, up through 1:64 for each of the five (5) positive samples. data management and analysis the ct values of the test outcomes from the rt-pcr analysis of pooled samples were entered into an anonymised, password-protected excel spreadsheet (office 2016, microsoft, redmond, washington, united states), and the data were analysed descriptively using charts and tables. samples with ct values ≤ 43 were considered positive for covid-19 infection, while samples with ct values > 43 were considered negative for covid-19 infection. results as shown in table 1 and figure 2, our investigation revealed that a single positive covid-19 sample with ct values ≤ 20 and 21–25 could be pooled with as many as 63 other samples to obtain a positive pool, which signifies that such a single positive sample is detectable in a pool of up to 1:64 negative samples. however, pooling covid-19 samples with a single positive covid-19 sample with ct values 26–30 can only be detected in a pool of 1:48 negative samples using rt-pcr, 31–35 as 1:24, and 36–40 as 1:8 (table 1 and figure 2). figure 2: distribution of cycle threshold values of a single positive sample across different pools of varying numbers of negative samples in nasarawa state, nigeria, from november 2022 to february 2023. table 1: positivity across different pools of varying dilution factors based on the cycle threshold value of a single positive sample among 4 to 64 negative samples in nasarawa state, nigeria, from november 2022 to february 2023. discussion this investigation showed that the ct value was a determining factor in choosing the maximum number of samples to be pooled for effective rt-pcr detection of covid-19. however, determining the maximum number of samples that can be pooled while still maintaining reliable and accurate test results is a critical challenge to consider. in our study, we discovered that a single positive sample with a presumed ct value of ≤ 25 could be pooled with up to 64 samples to obtain a positive pool, while a single presumed positive sample with a ct value > 40 could only be pooled with a maximum of 8 presumed negative samples to obtain a positive pool. a similar study carried out in israel in 2020 reported that using a pooled testing approach for standard rt-pcr for covid-19 detection with a single positive sample could detect a pool of up to 32 samples, with an estimated false-negative rate of 10%.20 a study from malaysia, evaluating the effectiveness of pooled sample testing for covid-19 rt-pcr, conducted in 2020 by lim et al.,1 utilised both retrospective and prospective samples. the findings indicated that pooled testing using volumes of 25 µl, 40 µl, 60 µl, and 100 µl produced test results comparable to individual testing, with positive cases detected consistently across pool sizes. the study defined a positive result as having a ct value of 38 or lower, as recommended in the world health organization-charité protocol.1 this finding is consistent with our research outcome, where a single positive sample with a ct value > 40 could only be pooled with a maximum of 8 negative samples to obtain a positive pool. our study revealed that the detection sensitivity of this method was similar to that of testing individual samples alone. similarly, wacharapluesadee et al.,25 in a 2020 study in thailand, found that pooling specimens did not reduce the sensitivity of detecting severe acute respiratory syndrome coronavirus 2 when the original specimen had a ct value below 35. however, for specimens with low viral loads (ct > 35), 13.3% (2 out of 15 pools) produced false-negative results. therefore, a high ct value indicates that pooling is not suitable for testing, and it is recommended to reduce the number of samples in the pool.25 the ct value is a measure of the amount of rt-pcr amplification required to detect a signal from the target nucleic acid in a sample.26 the ct value is inversely proportional to the amount of target nucleic acid present in the sample. the higher the ct value, the lower the amount of target nucleic acid in the sample.26 a ct value is considered positive for covid-19 when it is detectable within the test’s threshold, while undetectable values indicate a negative result for that test, as they fall below the test’s sensitivity range.26 notably, the maximum number of samples that can be pooled while still maintaining accurate and reliable results may vary depending on the prevalence of covid-19 in the population being tested, the assay used, and the quality of the samples.15,27,28,29,30,31 if the prevalence of covid-19 in the population is low, it may be necessary to reduce the number of samples in the pool to maintain accurate and reliable results; likewise, if the quality of the samples is poor, it may be necessary to reduce the number of samples in the pool to ensure accurate results.15,27,28,29 most importantly, determining the optimal pool size for rt-pcr testing for covid-19 detection requires consideration of several factors, including the presumed viral load of positive samples, the ct value, the sensitivity of the rt-pcr test, the prevalence of covid-19 in the population, and the cost and time efficiency of the testing process.7,15,27,28,29 while different studies have recommended different pool sizes, there is no one-size-fits-all approach, and the optimal pool size may vary depending on the context and resources available. therefore, a process of trial and error was necessary to determine the optimal pool size for rt-pcr testing in specific settings.15 however, this study provided a more detailed number of samples that can effectively be pooled for greater sensitivity in relation to the presumed ct values of the positive samples among the preceding batches of samples from the same location and period as shown table 1. additionally, yani et al.,32 in a study comparing rt-pcr ct values between individual and pooled severe acute respiratory syndrome coronavirus 2-infected nasopharyngeal swab specimens, conducted in indonesia in 2024, had reported no difference in the ct values between individual sample and pooled sample groups at all concentrations and for all pooled sizes, emphasising that a pooled rt-pcr testing strategy did not reduce the quality of individually measured rt-pcr ct values.32 limitations a limitation of this study is that we did not collect information on clinical data or symptoms; we believe that all patients may have been symptomatic with covid-19-related symptoms, as stated in the study protocol, as this was strictly monitored to eliminate protocol deviations. additionally, rt-pcr, like most other testing systems, could have misclassified some negative samples as positive and vice versa during the evaluation cycle. these errors might result from improper sample handling, deficient sample loading, or rna degradation during processing. however, to minimise such errors, we ensured strict adherence to all standard operating procedures for laboratory practices under the supervision of highly experienced senior laboratory scientists throughout the study implementation conclusion sample pooling for rt-pcr of covid-19 samples is a promising strategy that could increase testing capacity and overcome the limitations of current testing methods during a covid‒19 pandemic, especially in nigeria, which has a limited capacity for rt-pcr. however, sample pooling requires careful planning, coordination, and optimisation to ensure that it is both cost-effective and reliable. this research revealed that the presumed ct value is a determining factor in choosing the maximum number of samples to be pooled for effective rt-pcr detection of covid-19, and provides a guide for covid-19 sample pooling in nigeria for current and future outbreak responses. acknowledgements the authors sincerely appreciate the management of the zankli research centre molecular laboratory, bingham university, nasarawa state, nigeria, for their immense support toward conducting the study, as well as the staff members of the zankli research centre molecular laboratory, bingham university, nasarawa state, who were involved in sample processing during the study. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions t.t.e., e.e.i., c.u., j.s.b., b.e., and p.i. conceptualised the study and developed the methodology. the investigation was conducted by t.t.e., b.e., a.e.i., and p.i. data curation and formal analysis were performed by t.t.e. and j.k. the initial draft was prepared by t.t.e. and e.e.i., with study validation by c.u., and supervision by j.s.b. additionally, t.t.e., e.e.i., c.u., j.s.b., b.e., and p.i. reviewed and revised the article. t.t.e., e.e.i., j.s.b., b.e., c.u., j.k., p.i. and a.e.i. have read and approved the final version of the article. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability data sets analysed in this study are available from the corresponding author, e.e.i., upon reasonable request. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. the article does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the authors are responsible for this article’s results, findings, and content. references lim kl, johari na, wong st, et al. a novel strategy for community screening of sars-cov-2 (covid-19): sample pooling method. plos one. 2020;15(8):e0238417. https://doi.org/10.1371/journal.pone.0238417 libin pjk, 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https://doi.org/10.1002/jmv.26005 xu t, chen c, zhu z, cui m, chen c, dai h. clinical features and dynamics of viral load in imported and nonimported patients with covid-19. int j infect dis. 2020;94:68–71. https://doi.org/10.1016/j.ijid.2020.03.022 cabrera-alvargonzalez jj, larrañaga a, martinez j, et al. assessment of the effective sensitivity of sars-cov-2 sample pooling based on a large-scale screening experience: retrospective analysis. jmir public health surveill. 2024;10:e54503. https://doi.org/10.2196/54503 fagg j, beale r, futschik me, et al. swab pooling enables rapid expansion of high-throughput capacity for sars-cov-2 community testing. j clin virol. 2023;167:105574. https://doi.org/10.1016/j.jcv.2023.105574 filiatreau lm, zivich pn, edwards jk, mulholland ge, max r, westreich d. optimizing sars-cov-2 pooled testing strategies through differentiated pooling for distinct groups. am j epidemiol. 2023;192(2):246–256. https://doi.org/10.1093/aje/kwac178 paikrao vm, hassani us, nagdeo nv. comparative analysis of pooling methods for sars-cov-2 real-time polymerase chain reaction testing. biomed biotechnol res j. 2024;8(2):147–153. https://doi.org/10.4103/bbrj.bbrj_99_24 nazaryan i, pepanyan n, keshishyan a, petrosyan s, margaryan n, mnatsakanyan s. sars-cov-2 pooled testing methodology for pcr testing applied in private laboratory in armenia. adv infect dis. 2024;14(1):67–73. https://doi.org/10.4236/aid.2024.141006 yani h, yuan td, lubis ad, iswara lk, lubis ind. comparison of rt-pcr cycle threshold values between individual and pooled sars-cov-2 infected nasopharyngeal swab specimens. narra j. 2024;4(2):e765. http://doi.org/10.52225/narra.v4i2.765 article information authors: ruth mcnerney1 kimberly sollis1 rosanna w. peeling1 affiliations: 1london school of hygiene & tropical medicine, united kingdom correspondence to: ruth mcnerney postal address: london school of hygiene & tropical medicine, keppel street, london wc1e 7ht, united kingdom dates: received: 25 june 2013 accepted: 15 nov. 2013 published: 04 apr. 2014 how to cite this article: mcnerney r, sollis k, peeling rw. improving access to new diagnostics through harmonised regulation: priorities for action. afr j lab med. 2014;3(1), art. #123, 7 pages. http://dx.doi.org/10.4102/ ajlm.v3i1.123 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. improving access to new diagnostics through harmonised regulation: priorities for action in this improving access to new diagnostics through harmonised regulation: priorities for action ... open access • abstract • introduction    • regulating diagnostic devices    • risk classification of in vitro diagnostic medical devices    • harmonising regulation of diagnostic devices • a common submission dossier    • status quo    • action going forward • convergence in the auditing of quality systems    • status quo    • action going forward • reducing duplication in studies of clinical performance    • status quo    • action going forward • convergence in post-market surveillance    • status quo    • action going forward • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ a new generation of diagnostic tests is being developed for use at the point of care that could save lives and reduce the spread of infectious diseases through early detection and treatment. it is important that patients in developing countries have access to these products at affordable prices and without delay. regulation of medical products is intended to ensure safety and quality whilst balancing the need for timely access to beneficial new products. current regulatory oversight of diagnostic tests in developing countries is highly variable and weak regulation allows poor-quality tests to enter the market. however, inefficient or overzealous regulation results in unnecessary delays, increases costs and acts as a barrier to innovation and market entry. setting international standards and streamlining the regulatory process could reduce these barriers. four priority activities have been identified where convergence of standards and protocols or joint review of data would be advantageous: (1) adoption of a common registration file for pre-market approval; (2) convergence of quality standards for manufacturing site inspections; (3) use of common evaluation protocols, as well as joint review of data, to reduce unnecessary duplication of lengthy and costly clinical performance studies; and (4) use of networks of laboratories for post-market surveillance in order to monitor ongoing quality of diagnostic devices. the adoption and implementation of such measures in developing countries could accelerate access to new diagnostic tests that are safe and affordable. introduction top ↑ diagnostic tests make a major contribution to global health. they are needed in order to guide treatment decisions and ensure the appropriate use of medicines. they are also vital for screening for infections, such as hiv infection or syphilis, in asymptomatic individuals.1 such tests are often lifesaving, where delay or lack of access can result in deterioration of the patient’s health and lead to further complications. diagnostics are particularly important for the control of infectious diseases, where early detection allows intervention in order to prevent onward transmission.2 they may also be used to monitor the outcome of treatment. most diagnostic interventions utilise in vitro diagnostic medical devices (ivds) which test specimens obtained from a patient, such as blood or urine. ivds include a wide range of technologies, from rapid dip-stick strips for use at the point of care (poc) to sophisticated instrumentation for use in referral laboratories. access to diagnostic tests in developing countries is often limited by their availability, as patients frequently do not live within easy travelling distance of a well-equipped and functional laboratory.2,3 a new generation of diagnostic tests is being developed for use at the point of care that will not need a laboratory.4,5 prompt access to the new tests should be encouraged; if they are made available in developing countries at affordable prices, such tests could increase access to appropriate healthcare, thereby saving lives and reducing the spread of infectious diseases.4,5,6 regulating diagnostic devices regulation of medical products is intended to ensure safety and quality whilst ensuring that the public has timely access to beneficial new products. national regulatory authorities (nras) are usually mandated by law and, for a regulatory system to be effective, it should be enforceable in both the public and private healthcare sector. following impartial review, new tests that are considered substandard or unsafe are refused entry to the market, whereas satisfactory products are approved and registered. for regulatory purposes, ivds are classified as medical devices. in this context, ‘medical device’ means any instrument, apparatus, implement, machine, appliance, implant, reagent for in vitro use, software, material, or any other similar or related article intended by the manufacturer to be used alone or in combination, for human beings for one or more of the specific medical purpose(s): diagnosis, prevention, monitoring, treatment or alleviation of disease; diagnosis, monitoring, treatment, alleviation of or compensation for an injury; investigation, replacement, modification or support of the anatomy or of a physiological process; support or sustenance of life; control of conception; disinfection of medical devices; provision of information by means of in vitro examination of specimens derived from the human body.7 an ivd is defined as:medical device, whether used alone or in combination, intended by the manufacturer for the in vitro examination of specimens derived from the human body solely or principally to provide information for diagnostic, monitoring or compatibility purposes.7 most countries have a legal framework and a nominated body to regulate medicines, but regulation of medical devices in developing countries is less common.8 the demands on regulatory authorities for ivds differ from that of other medical products in that there are a very large number of ivds and, compared with drugs or vaccines, their market life is often short given the rapid pace of technology development.9 current regulatory oversight of ivds is variable8 and, in countries where there is no regulation, substandard and counterfeit tests may be sold openly.10 in countries that do regulate, approval for ivds is often costly, lengthy and, on occasion, lacking in transparency, thus regulation of diagnostics is currently seen as a barrier to innovation and access.9,11,12 in 2001, the pan american health organisation (paho), the regional office for the americas of the world health organization (who) and the united states food and drug administration (usfda) published a model regulatory programme for medical devices, with a set of guiding principles.13 these principles include the statement that: regulatory system should ensure that valuable new technologies are made available to the clinical community and to patients and consumers expeditiously while preventing unsafe or ineffective devices from reaching the market.13 they also state that regulatory decisions must be based on strong and clear science, free of external influences and, in addition, that countries instituting regulatory programmes should be cognisant of ongoing international efforts to harmonise activities. regulatory control of ivd has three components: • pre-market evaluation: to assess safety, performance, benefits and risks prior to approval to market the device. • marketing controls: to stipulate conditions under which devices can be offered for sale; identify who may use the device and under what conditions; and to avoid inappropriate marketing or misleading claims regarding test effectiveness. • post-marketing controls: to maintain vigilance and continued safety and quality of approved products that entails a method for information-sharing amongst users within the country and across national health authorities. there should be a means by which corrections can be made as well as a mechanism for removing substandard products that pose a risk to public health. risk classification of in vitro diagnostic medical devices the degree of regulatory control for a medical device should be proportionate to the risk posed by the product. unlike medicines or vaccines, ivds are not ingested by the patient, greatly reducing the potential for harm. thus the stringency of regulatory oversight required is related to the harm that a false positive or false negative test result may cause to either individual or public health. reagents used in diagnostics tests, such as microbiological stains or culture media, by themselves pose little risk to human or public health and are classed as low risk. high risk tests include those used to screen for infections such as hiv, where a false negative test result could lead to the individual not being given life-saving drugs and continuing to transmit the infection within the community. should the test be used to screen blood products, then recipients of that donation would be at risk of acquiring a life-threatening disease. such tests require more stringent control, including evidence of their performance as obtained through clinical studies. to guide the regulatory process, products are grouped or classified according to their risk of causing harm. in 2006, the global harmonization task force (ghtf), a voluntary partnership of stringent regulatory authorities and diagnostic companies, published recommendations regarding classification of medical devices. they suggested that each medical device be assigned to one of four classes based upon its intended use (table 1).14 classification provides a mechanism by which the cost and delay of registration are moderated according to the potential of a product to cause harm. manufacturers are required to provide less-substantial submission dossiers for products in risk group class a; whereas class d products require stringent conformity assessment, including evidence of performance in a clinical setting that is representative of the intended use. for devices to be used at the poc, studies should be conducted in the settings of intended use (clinic or outreach settings) with testing performed by local health providers. such studies are costly and, for some diseases, may require years to plan and execute. the classification system requires a shorter and less-costly route to pre-market approval for low-risk products. table 1: classification of medical devices with examples of diagnostic products. harmonising regulation of diagnostic devices whilst regulation of medical products is required in order to ensure their safety, it is essential that regulatory review processes do not obstruct or unnecessarily delay access to beneficial new products. it is recognised that the current lack of standardisation across national regulatory authorities and the lack of clarity surrounding the regulatory pathways presents an unnecessary burden on manufacturers and acts as a deterrent to marketing in countries where the financial returns may be modest.9 with the exception of the countries of the european union, where a harmonised system has been adopted, countries each have their own set of requirements. in some countries, transparency is lacking, with little information available about the regulatory process or the fees charged. several transnational initiatives are striving toward improved harmonisation of regulation of medical devices, including ivds (table 2). harmonisation requires the use of standardised terminology and definitions which are employed to classify the products under regulation. guidance on this topic was issued by the ghtf, which transitioned to the international medical device regulators forum (imdrf) in 2012. for ivds used in developing countries there are opportunities to streamline and harmonise activities where convergence of protocols and mutual recognition of other regulatory bodies could improve their safety and quality, accelerating access to new tests while simultaneously minimising the costs incurred. setting international standards and streamlining the regulatory process could reduce the regulatory burden and lower costs of new products. four priority areas for harmonisation have been recognised: (1) adoption of common registration requirements and submission dossiers for pre-market approval; (2) convergence of quality standards and mutual, or third party, recognition of audit activities; (3) rationalisation of clinical performance studies in order to avoid unnecessary duplication; and (4) building of transnational networks for post-market surveillance. each of the aforementioned areas of harmonisation is discussed in the following sections. the status quo and need for change are described, along with recommendations made for the way forward. the impact of the proposed harmonisation activities are summarised in table 3. table 2: organisations promoting regulatory harmonisation of medical devices or in vitro diagnostics. table 3: expected impact of harmonisation activities. a common submission dossier top ↑ companies seeking approval to market an ivd are required to supply a dossier to the appropriate nra describing the device and documenting evidence relating to the quality of manufacture, as well as the safety and stability of the components. in addition, those devices considered to be at risk of causing harm to either individual or public health also require evidence regarding the clinical performance of the device. the adoption of a standardised submission dossier template would promote efficiency within the regulatory review process and facilitate standardised training on good review practice using a common set of teaching materials. nras would retain independent review of the data, along with any decisions for approval based on national requirements as they pertain to their local population needs. the need for a common submission dossier was one of the top priorities recognised by the ghtf and was an activity already adopted by the asian harmonisation working party (ahwp).15 status quo with the exception of the european union, submission dossiers are unique to the country, with each nra utilising its own indicators, nomenclature and format in its own language.9,16 preparation of a submission dossier for regulatory approval is a substantial undertaking and the necessity of preparing individual dossiers and reformulating data for each nra is an unnecessary burden on diagnostic companies, causing overall increase to the cost of marketing a new product. in countries with weak economies, small populations or low prevalence of the condition or disease to be tested, the anticipated market may be insufficient to warrant the cost of registering the product. the problem is particularly acute for small companies with limited regulatory expertise or capacity. large companies are also not exempt from the burden that a submission dossier carries, as it represents a significant commitment of resources and time. a standardised template for submission dossiers would decrease the time and effort required of companies, improve the efficiency of regulatory reviews and reduce the cost of goods for diagnostic companies, resulting in a more affordable product and overall reduced time to market. action going forward following principles established by the ghtf, the ahwp has developed a common submission dossier template (csdt) for premarket submission of ivds. the dossier incorporates: (1) marketing history and, where appropriate, a risk/benefit assessment, any prior approvals and current regulatory status, as well as documentation to demonstrate conformity to the essential principles of the ghtf; (2) a product description which includes intended use and any warnings or precautions; (3) a summary of design verification and validation documents – these may include sensitivity, specificity, precision, stability, storage and controls – and, depending on the product classification, it may also include evidence from clinical performance studies; (4) device labelling, with instructions for use (including operating manual and user manual), patient information leaflet and promotional materials; (5) a summary of the risks identified and a description of how these risks have been controlled to an acceptable level; and (6) manufacturer information in order to identify manufacturing sites and to provide quality management system certification such as (iso) 13485:2003 and a description of the manufacturing process.15 a common dossier is to be piloted in africa and other regions using a poc test as an example. transparency of regulatory requirements and processes shall be promoted, preferably with documents available on line. good review practice will include the monitoring and publication of the time required for review. fees charged to companies should reflect the costs of the approval process and should not be seen as an opportunity for profit. harmonisation of submission dossier requirements and adoption of a common template will: (1) reduce the cost to manufacturers of registering a product, a cost that would ultimately be passed to the consumers of the test; (2) reduce delays in test registration; (3) reduce barriers to marketing in small economies; and (4) facilitate harmonised approval of diagnostic devices, ultimately reducing the burden on nras and the cost of national regulatory approval. harmonisation will require consensus on the fundamental principles of regulating diagnostic devices for health, but independent decision making shall be retained by the nra. convergence in the auditing of quality systems top ↑ regulatory oversight requires assurance that the manufacturer of the device conforms to a satisfactory quality management system. these quality management standards are universal and should not differ from country to country. the quality management system used by a manufacturer to control the quality of their product should be audited in order to provide assurance that safe and effective devices will be manufactured. the audit should make certain that specified minimum standards are met during manufacturing and provide impartial, reliable and objective evaluation of compliance with regulatory requirements. iso 13485:2003 is an international standard established for the manufacture of ivds.17 if satisfactory quality management is not practised by the manufacturers, corrective measures may be recommended. for ivds considered to have a risk of causing harm (to potential patients or users), assessment of adherence to satisfactory quality systems will include visiting the site of manufacture. audit teams must encompass a range of skills and expertise enabling them to assess the quality management system and to determine the effectiveness of its implementation. the range of skills includes understanding the regulations and standards applicable to the specific ivd submitted for approval, the intended use and associated risks of the device, as well as the knowledge to assess the design, manufacturing processes and technologies involved. convergence of quality standards and mutual or third-party recognition of inspection results could reduce duplication of efforts where teams representing different nras undertake independent audit of the same site, saving expenditure and reducing delays in regulatory approval. status quo regulatory audits and site visits are costly for companies, with each audit costing as much as usd 200 000 (personal communication). although costs are met in the first instance by the companies, they will ultimately be reflected in the price of the product and recouped from the end users. duplication of audits inflates the cost of bringing a product to market and results in unnecessary delays. nras that require manufacturing site inspections for every medical device sold often have long delays in approving products for the market. in their 2012 annual report, the brazilian national health surveillance agency (anvisa), reported a backlog of over 1000 products awaiting evaluation.18 nras in the developing world lack the expertise and capacity required in order to undertake audits, which ultimately results in delayed approval. action going forward minimum standards should be identified for quality management in the manufacture of poc diagnostics in addition to iso 13485:2003.17 to reduce duplication, competent authorities or organisations capable of undertaking inspections should be identified and mutual recognition or recognition of competent third-party quality management audits adopted. in addition, mechanisms for sharing information should be established. harmonisation of quality systems audits through convergence of standards and/or mutual recognition will make more efficient use of auditing resources and reduce the number of audits by different regulatory bodies for the same product, saving costs and reducing delays in new products reaching the market. use of standardised audit protocols and expert bodies will streamline auditing, leading to improved quality management and product quality. this will provide greater consistency and increased confidence in audits. enhanced consistency in audit practices and feedback provided to manufacturers regarding quality management will facilitate improvements in manufacturing practice. the ultimate beneficiaries will be patients and users of diagnostic devices, who will gain an increased assurance that medical devices placed on the market are safe and effective whilst simultaneously ensuring that the costs of implementing an effective system do not unnecessarily inflate the price. reducing duplication in studies of clinical performance top ↑ pre-market approval of those ivds considered to be high risk to individual or public health requires supporting evidence of the performance and operational characteristics of the device. for laboratory-based diagnostic tests, such evaluations are often conducted using well-characterised archived samples. if the new product is a poc test intended for use in decentralised health centres or dispensaries, evaluations of sensitivity, specificity, precision and ease of use should be conducted in the settings of intended use, with the test performed by the proposed end users.19 such studies need to be based on sufficient sample size with assurance of quality so as to allow informed decisions on the performance and utility of the device as to whether the probable benefits of the device outweigh the risks. data generated in clinical studies are presented to nras as part of the submission dossier for pre-market approval. the high cost of clinical trials and the length of time they require may result in higher cost of goods and significant delay in gaining access to new products that could potentially save lives.20 status quo the requirement of nras to have national trial data for approval has resulted in unnecessary duplication, where further studies provide little or no added scientific benefit.21,22 high costs and delays incurred during clinical studies are a deterrent for companies entering the market, particularly in smaller countries where financial returns may be modest.9,12 costs borne by the manufacturer are ultimately passed on to the consumer, making devices less affordable. since not every country has the capacity to conduct high quality studies in a timely manner, resulting from a lack of specialist facilities or limited access to appropriate patient groups, multi-country evaluations and nras coming together to review trial data and then making their own decisions for approval would be a more efficient approach. action going forward multi-country studies using a standardised protocol and joint review of trial data should be encouraged in order to shorten trial duration and reduce duplication. a clinical trials registry should be established to reduce unintentional duplication and promote maximal use of resources. clinical performance studies should be conducted by accredited laboratories and clinics with external oversight, so as to ensure conformity with international standards, including good laboratory and clinical practice.23,24,25 prior joint approval of clinical and laboratory protocols by institutional review boards will ensure quality and acceptance by representatives of regulatory authorities. reducing duplication of clinical trials will: (1) reduce the cost of accessing the market, making tests more affordable; (2) reduce delays in regulatory approval, accelerating access to new products; (3) reduce barriers to marketing in small economies; and (4) allow nras to maintain independent decision making for new products without requiring local trials. convergence in post-market surveillance top ↑ post-market surveillance ensures that products continue to meet expected safety and quality standards following approval by nras and an important component of regulatory oversight of diagnostic products for health.26 proactive post-market surveillance requires the systematic collection of data from laboratory studies in order to monitor test performance using a panel of appropriate reagents or, alternatively, through field testing using a panel of well-characterised samples. reactive surveillance requires manufacturers, or testers, to report problems voluntarily through an established reporting system. such activities are applicable to manufacturers, importers and distributors. there is a need for cross-border, regional and global sharing of adverse events that threaten safety of individuals or public health so as to accelerate information gathering and enable substandard products to be withdrawn more quickly. the national competent authorities report (ncar) is a membership-based system open to those countries with stringent regulatory authorities.27 the system incorporates post-market surveillance, vigilance and reporting of adverse events and is aimed at improving safety by reducing the likelihood of repeated adverse events. there are two levels of ncar participation. full participation involves national competent authorities with established national adverse event reporting programmes. being a full participant, a national authority receives both public and confidential or highly-sensitive information from other ncar participants. associate participants are a second tier of membership that receive public information, such as recall notices, safety and hazard alerts. status quo currently, there is limited capacity for post-market surveillance in much of the developing world. systems for post-market surveillance for diagnostic devices are not implemented globally26 and, in most developing countries, reporting and information sharing occurs on an ad hoc basis. in many countries, random quality checks, such as lot testing, are not performed and tests can enter the market without checks on their quality.21,28 most developing countries lack a feedback mechanism to provide manufacturers with information regarding the need for corrective action as well as a mechanism for removal of substandard tests that present a risk to public health. disruption of services for some priority diseases, such as hiv, has occurred where publicity regarding quality problems has led, in some countries, to discontinuation of use without replacement devices being available.29 action going forward a mix of proactive and reactive post-market surveillance activities should be encouraged. reporting should be mandatory in the case of a death, a serious injury that is life threatening or results in permanent damage or impairment of a body function, or a malfunction. to implement post-market surveillance for ivd medical devices in developing countries, regional networks of accredited laboratories should be established in order to undertake batch testing and monitor performance and safety. standardised protocols should be established and reference quality assurance materials shared. these practices would promote good practice and instil confidence in the system, in addition to facilitating the exchange of data. global and regional mechanisms should be established so as to facilitate investigation and procedural corrective actions or recalls for products found to be unsatisfactory. harmonisation of proactive activities, such as batch monitoring, would enhance safety by reducing reporting delays, allowing prompt action to be taken should unsafe products be reported. standardisation of protocols, test reagents and sample panels across geographic regions would assist surveillance whilst simultaneously minimising costs. national and transnational communication platforms are needed to simplify current procedures. networks of accredited laboratories should be established to facilitate mutual recognition of surveillance data. convergence of post-market surveillance activities will: (1) ensure that products continue to meet expected safety, performance and efficacy requirements following pre-market approval; (2) prevent substandard tests, or batches of tests, from entering the market; (3) enable corrective actions to be taken by manufacturers; and (4) facilitate removal of substandard tests from the market, ultimately reducing costs and maximising efficient use of resources by nras. conclusion top ↑ diagnostic tests can play a vital role in improving access to effective healthcare, but the current regulatory landscape in developing countries creates significant barriers to market entry and has become a deterrent to innovation. setting international standards and streamlining the regulatory process for in vitro diagnostic devices could diminish the regulatory burden. four areas where action could lower costs, reduce delays, and accelerate access to quality diagnostic products for health are: (1) adoption of common registration requirements and submission dossier for pre-market approval; (2) convergence of quality standards and mutual, or third-party, recognition of audit activities; (3) rationalisation of clinical performance studies in order to avoid unnecessary duplication; and (4) building transnational networks for post-market surveillance. acknowledgements top ↑ the authors received financial support from grand challenges canada. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions r.w.p. (london school of hygiene & tropical medicine) conceived the article; r.m. (london school of hygiene & tropical medicine) developed the first draft and all authors contributed to writing the article. references top ↑ 1. peeling rw, mabey d, herring a, et al. why do we need quality-assured diagnostic tests for sexually transmitted infections? nat rev microbiol. 2006;4(12):909–921. http://dx.doi.org/10.1038/nrmicro1555, pubmed pmid: 171090302. mabey d, peeling rw, ustianowski a, et al. diagnostics for the developing world. nat rev microbiol. 2004;2(3):231–240. http://dx.doi.org/10.1038/nrmicro841, pubmed pmid: 15083158 3. storla dg, yimer s, bjune ga. a systematic review of delay in the diagnosis and treatment of tuberculosis. bmc public health. 2008;8:15. http://dx.doi.org/10.1186/1471-2458-8-15, pubmed pmid: 18194573, pubmed central pmcid: 2265684 4. peeling rw, mabey d. point-of-care tests for diagnosing infections in the developing world. clin microbiol infect. 2010;16(8):1062–1069. http://dx.doi.org/10.1111/j.1469-0691.2010.03279.x, pubmed pmid: 20670288, 5. mcnerney r, daley p. towards a point-of-care test for active tuberculosis: obstacles and opportunities. nat rev microbiol. 2011;9(3):204–213. http://dx.doi.org/10.1038/nrmicro2521, pubmed pmid: 21326275 6. mabey d, peeling r, perkins m. rapid and simple point of care diagnostics for stis. sex transm infect. 2001;77(6):397–398. http://dx.doi.org/10.1136/sti.77.6.397, pubmed pmid: 11714933, pubmed central pmcid: 1744415 7. global harmonization task force. definition of the terms ‘medical device’ and ‘in vitro diagnostic (ivd) medical device’. ghtf/sg1/n071:2012; 2012. 8. special programme for research & training in tropical diseases (tdr). regulation of vitro diagnostics: a global perspective. find sa/tdr: geneva; 2002 [reproduced as annex to diagnostics for tuberculosis: global demand and market potential. geneva: world health organization; 2006]. 9. peeling r, mcnerney r. increasing access to diagnostics through technology transfer and local production. geneva: who; 2011. 10. dowdy dw, steingart kr, pai m. serological testing versus other strategies for diagnosis of active tuberculosis in india: a cost-effectiveness analysis. plos med. 2011;8(8):e1001074. http://dx.doi.org/10.1371/journal.pmed.1001074, pubmed pmid: 21857810, pubmed central pmcid: 3153451 11. macarthur g. global health diagnostics: research, development and regulation. london: the academy of medical sciences; 2009. isbn no: 1-903401-20-8 12. hoerr ra. regulatory uncertainty and the associated business risk for emerging technologies. j nanopart res. 2011;13(4):1513–1520. http://dx.doi.org/10.1007/s11051-011-0260-z 13. eccleston rc. a model regulatory program for medical devices: an international guide. washington, dc: world health organization and united states food and drug administration; 2001. 14. global harmonization task force. principles of in vitro diagnostic medical devices classification. ghtf/sg1/n452008. [document on the internet]. [c2012] [cited 22 january 2014]. available from: http://www.imdrf.org/docs/ghtf/final/sg1/technical-docs/ghtf-sg1-n77-2012-principles-medical-devices-classification-121102.pdf 15. global harmonization task force. summary technical documentation (sted) for demonstrating conformity to the essential principles of safety and performance of in vitro diagnostic medical devices. ghtf/sg1/n063:2011; 2011. 16. kramer db, xu s, kesselheim as. how does medical device regulation perform in the united states and the european union? a systematic review. plos med. 2012;9(7):e1001276. http://dx.doi.org/10.1371/journal.pmed.1001276, pubmed pmid: 22912563 17. praxiom research group limited. iso 13485 medical device standard translated into plain english. [page on the internet]. [c2004] [updated 2013 may 15; cited 2013 sep 03]. available from: http://www.praxiom.com/iso-13485.htm 18. agência nacional de vigilância sanitária. anvisa. anvisa annual report brazil; 2012. 19. banoo s, bell d, bossuyt p, et al. evaluation of diagnostic tests for infectious diseases: general principles. nat rev microbiol. 2010;8(12 suppl):s17–29. pubmed pmid: 21548184 20. cohen p. the changing regulatory environment of in vitro diagnostics: implications for sponsors, manufacturers and laboratory users. aus j med sci 2010;31(1):9–15. 21. peeling rw, smith pg, bossuyt pm. a guide for diagnostic evaluations. nat rev microbiol. 2006;4(9 suppl):s2–6. http://dx.doi.org/10.1038/nrmicro1568, pubmed pmid: 17003769 22. small pm, perkins md. more rigour needed in trials of new diagnostic agents for tuberculosis. lancet. 2000;356(9235):1048–1049. http://dx.doi.org/10.1016/s0140-6736(00)02724-0, pubmed pmid: 11009137 23. world health organization. handbook: good laboratory practice (glp): quality practices for regulated non-clinical research and development. geneva: world health organization; 2009. 24. world health organization. guidelines for good clinical practice (gcp) for trials on pharmaceutical products. geneva: world health organization; 1995. 25. bossuyt pm, reitsma jb, bruns de, et al. towards complete and accurate reporting of studies of diagnostic accuracy: the stard initiative. clin radiol. 2003;58(8):575–580. http://dx.doi.org/10.1016/s0009-9260(03)00258-7, pubmed pmid: 12887949 26. kramer db, tan yt, sato c, et al. postmarket surveillance of medical devices: a comparison of strategies in the us, eu, japan, and china. plos med. 2013;10(9):e1001519. http://dx.doi.org/10.1371/journal.pmed.1001519 27. global harmonization task force. application requirements for participation in the ghtf national competent authority report exchange program. ghtf/sg2/ n38r19:2009; 2009. 28. bell d, wongsrichanalai c, barnwell jw. ensuring quality and access for malaria diagnosis: how can it be achieved? nat rev microbiol. 2006;4(9 suppl):s7–20. http://dx.doi.org/10.1038/nrmicro1525, pubmed pmid: 17003770 29. africomnet. tanzania: search on for new supplier of hiv test kits. [page on the internet]. [c2012] [cited 2013 oct 28]. available from: http://www.africomnet.org/communication-resources/highlights/1904-tanzania-search-on-for-new-supplier-of-hiv-test-kits.html the impacts on health a global health emergency acknowledgements references about the author(s) kamran abbasi british medical journal, london, united kingdom parveen ali health sciences school, university of sheffield and doncaster and bassetlaw teaching hospitals, sheffield, united kingdom sheffield university interpersonal violence research group, sheffield, united kingdom virginia barbour medical journal of australia, brisbane, australia thomas benfield the danish medical journal, copenhagen, denmark kirsten bibbins department of medicine, university of california, san francisco, united states stephen hancocks british dental journal, london, united kingdom richard horton the lancet, london, united kingdom laurie laybourn-langton chatham house, exeter, united kingdom global systems institute, university of exeter, exeter, united kingdom robert mash division of family medicine and primary care, stellenbosch university, stellenbosch, south africa peush sahni department of gi surgery, all india institute of medical sciences, delhi, india wadeia m. sharief medical education & research department, dubai health authority, duba, united arab emirates emirates family medicine society, dubai, united arab emirates family medicine scientific council, arab board of health specialization, dubai, united arab emirates paul yonga ca medlynks medical centre and laboratory, nairobi, kenya east african medical journal, nairobi, kenya chris zielinski centre for global health, faculty of health and wellbeing, university of winchester, winchester, united kingdom world association of medical editors, winchester, united kingdom citation abbasi k, ali p, barbour v, et al. time to treat the climate and nature crisis as one indivisible global health emergency. afr j lab med. 2023;12(1), a2335. https://doi.org/10.4102/ajlm.v12i1.2335 editorial time to treat the climate and nature crisis as one indivisible global health emergency kamran abbasi, parveen ali, virginia barbour, thomas benfield, kirsten bibbins, stephen hancocks, richard horton, laurie laybourn-langton, robert mash, peush sahni, wadeia m. sharief, paul yonga, chris zielinski copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. over 200 health journals call on the united nations, political leaders, and health professionals to recognise that climate change and biodiversity loss are one indivisible crisis and must be tackled together to preserve health and avoid catastrophe. this overall environmental crisis is now so severe as to be a global health emergency. the world is currently responding to the climate crisis and the nature crisis as if they were separate challenges. this is a dangerous mistake. the 28th conference of the parties (cop) on climate change is about to be held in dubai while the 16th cop on biodiversity is due to be held in turkey in 2024. the research communities that provide the evidence for the two cops are unfortunately largely separate, but they were brought together for a workshop in 2020 when they concluded that ‘only by considering climate and biodiversity as parts of the same complex problem … can solutions be developed that avoid maladaptation and maximize the beneficial outcomes’.1 as the health world has recognised with the development of the concept of planetary health, the natural world is made up of one overall interdependent system. damage to one subsystem can create feedback that damages another – for example, drought, wildfires, floods and the other effects of rising global temperatures destroy plant life, and lead to soil erosion and so inhibit carbon storage, which means more global warming.2 climate change is set to overtake deforestation and other land-use change as the primary driver of nature loss.3 nature has a remarkable power to restore. for example, deforested land can revert to forest through natural regeneration, and marine phytoplankton, which act as natural carbon stores, turn over one billion tonnes of photosynthesising biomass every eight days.4 indigenous land and sea management has a particularly important role to play in regeneration and continuing care.5 restoring one subsystem can help another – for example, replenishing soil could help remove greenhouse gases from the atmosphere on a vast scale.6 but actions that may benefit one subsystem can harm another – for example, planting forests with one type of tree can remove carbon dioxide from the air but can damage the biodiversity that is fundamental to healthy ecosystems.7 the impacts on health human health is damaged directly by both the climate crisis, as the journals have described in previous editorials,8,9 and by the nature crisis.10 this indivisible planetary crisis will have major effects on health as a result of the disruption of social and economic systems – shortages of land, shelter, food and water, exacerbating poverty, which in turn will lead to mass migration and conflict. rising temperatures, extreme weather events, air pollution, and the spread of infectious diseases are some of the major health threats exacerbated by climate change.11 ‘without nature, we have nothing’, was un secretary-general antónio guterres’s blunt summary at the biodiversity cop in montreal last year.12 even if we could keep global warming below an increase of 1.5°c over pre-industrial levels, we could still cause catastrophic harm to health by destroying nature. access to clean water is fundamental to human health, yet pollution has damaged water quality, causing a rise in water-borne diseases.13 contamination of water on land can also have far-reaching effects on distant ecosystems when that water runs off into the ocean.14 good nutrition is underpinned by diversity in the variety of foods, but there has been a striking loss of genetic diversity in the food system. globally, about a fifth of people rely on wild species for food and their livelihoods.15 declines in wildlife are a major challenge for these populations, particularly in lowand middle-income countries. fish provide more than half of dietary protein in many african, south asian and small island nations, but ocean acidification has reduced the quality and quantity of seafood.16 changes in land use have forced tens of thousands of species into closer contact, increasing the exchange of pathogens and the emergence of new diseases and pandemics.17 people losing contact with the natural environment and the declining biodiversity have both been linked to increases in noncommunicable, autoimmune, and inflammatory diseases and metabolic, allergic and neuropsychiatric disorders.10,18 for indigenous people, caring for and connecting with nature is especially important for their health.19 nature has also been an important source of medicines, and thus reduced diversity also constrains the discovery of new medicines. communities are healthier if they have access to high-quality green spaces that help filter air pollution, reduce air and ground temperatures, and provide opportunities for physical activity.20 connection with nature reduces stress, loneliness and depression while promoting social interaction.21 these benefits are threatened by the continuing rise in urbanisation.22 finally, the health impacts of climate change and biodiversity loss will be experienced unequally between and within countries, with the most vulnerable communities often bearing the highest burden.10 linked to this, inequality is also arguably fuelling these environmental crises. environmental challenges and social/health inequities are challenges that share drivers and there are potential co-benefits of addressing them.10 a global health emergency in december 2022, the biodiversity cop agreed on the effective conservation and management of at least 30% of the world’s land, coastal areas, and oceans by 2030.23 industrialised countries agreed to mobilise $30 billion per year to support developing nations to do so.23 these agreements echo promises made at climate cops. yet many commitments made at cops have not been met. this has allowed ecosystems to be pushed further to the brink, greatly increasing the risk of arriving at ‘tipping points’, abrupt breakdowns in the functioning of nature.2,24 if these events were to occur, the impacts on health would be globally catastrophic. this risk, combined with the severe impacts on health already occurring, means that the world health organization should declare the indivisible climate and nature crisis as a global health emergency. the three pre-conditions for who to declare a situation to be a public health emergency of international concern25 are that it: (1) is serious, sudden, unusual or unexpected; (2) carries implications for public health beyond the affected state’s national border; and (3) may require immediate international action. climate change would appear to fulfil all of those conditions. while the accelerating climate change and loss of biodiversity are not sudden or unexpected, they are certainly serious and unusual. hence, we call for who to make this declaration before or at the 77th world health assembly in may 2024. tackling this emergency requires the cop processes to be harmonised. as a first step, the respective conventions must push for better integration of national climate plans with biodiversity equivalents.3 as the 2020 workshop that brought climate and nature scientists together concluded, ‘critical leverage points include exploring alternative visions of good quality of life, rethinking consumption and waste, shifting values related to the human-nature relationship, reducing inequalities, and promoting education and learning’.1 all of these would benefit health. health professionals must be powerful advocates for both restoring biodiversity and tackling climate change for the good of health. political leaders must recognise both the severe threats to health from the planetary crisis as well as the benefits that can flow to health from tackling the crisis.26 but first, we must recognise this crisis for what it is: a global health emergency. acknowledgements this editorial is being published simultaneously in multiple journals. for the full list of journals see: https://www.bmj.com/content/full-list-authors-and-signatories-climate-nature-emergency-editorial-october-2023. competing interests v.b. is an employee of the medical journal of australia and an unpaid committee member of wildlife queensland. t.b. receives unrestricted grants to his institution from gsk, novo nordisk foundation, simonsen foundation, lundbeck foundation, kai foundation, erik and susanna olesen’s charitable fund, pfizer, msd, and gilead sciences; is principal investigator for clinical trials funded by pfizer, boehringer ingelheim, gilead sciences, msd, roche, novartis, and kancera ab; is an advisory board member for gsk, pfizer, gilead sciences, msd, janssen, and astra zeneca; is a board member of pentabase; reports consulting fees from gsk and pfizer; reports honoraria for lectures from gsk, pfizer, gilead sciences, boehringer ingelheim, abbvie, and astra zeneca; and reports donation of trial medication (baricitinib) from eli lilly – all unrelated to the topic of this comment. l.l.-l. is a member of the advisory group to the uk climate change committee. r.m. reports a team grant to his institution stellenbosch university from vlir (belgium) to investigate primary healthcare and climate change in africa. s.h. reports honoraria for hosting webinars from procter & gamble oral-b unrelated to the topic of this comment. p.y. reports honoraria for lectures from biomérieux and pfizer; participation in a data safety monitoring board for us national heart, lung, and blood institute; has received covid-19 and influenza test kits from atea pharmaceuticals for a phase iii clinical trial for which he is a principal investigator; is a member the executive committee of the antimicrobial stewardship study group for the european society of clinical microbiology and infectious diseases — all of which are unrelated to the topic of this comment; and is editor-in-chief of the east african medical journal, which is the official medical journal of the kenya medical association. the other authors declare no competing interests. authors’ contributions l.l.-l. developed the idea of the editorial and led drafting along with c.z. all other authors contributed significantly to the editorial content. authors’ affiliated journals kamran abbasi, editor-in-chief, bmj; parveen ali, editor-in-chief, international nursing review; virginiabarbour, editor-in-chief, medical journal of australia; thomas benfield, editor-in-chief, danish medical journal; kirsten bibbins-domingo, editor-in-chief, jama; stephen hancocks, editor-in-chief, british dental journal; richard horton, editor-in-chief, the lancet; laurie laybourn-langton, university of exeter; robert mash, editor-in-chief, african journal of primary health care & family medicine; peush sahni, editor-in-chief, national medical journal of india; wadeia mohammad sharief, editor-in-chief, dubai medical journal; paul yonga, editor-in-chief, east african medical journal; chris zielinski, university of winchester. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support respective authors were paid by their employers. data availability data sharing is not applicable to this article, as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. it does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the authors are responsible for this article’s results, findings, and content. references otto-portner h, scholes b, agard j, et al. scientific outcome of the ipbes-ipcc co-sponsored workshop on biodiversity and climate change. ipbes secretariat: ipbes secretariat2021. https://doi.org/10.5281/zenodo.5101125 ripple wj, wolf c, lenton tm, et al. many risky feedback loops amplify the need for climate action. one earth. 2023;6(2):86–91. https://doi.org/10.1016/j.oneear.2023.01.004 european academies science advisory council. key messages from european science academies for unfccc cop26 and cbd cop15 [homepage on the internet]. 2021 [cited 2023 oct 01]. available from: 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water for all [homepage on the internet]. world health organization; 2022 [cited 2023 oct 01]. available from: https://www.who.int/publications/i/item/9789240060807 comeros-raynal mt, brodie j, bainbridge z, et al. catchment to sea connection: impacts of terrestrial run-off on benthic ecosystems in american samoa. mar pollut bull. 2021;169:112530. https://doi.org/10.1016/j.marpolbul.2021.112530 ipbes. assessment report on the sustainable use of wild species [homepage on the internet]. available from: https://www.ipbes.net/sustainable-use-assessment falkenberg lj, bellerby rgj, connell sd, et al. ocean acidification and human health. int j environ res public health. 2020;17(2):4563. https://doi.org/10.3390/ijerph17124563 dunne d. climate change ‘already’ raising risk of virus spread between mammals [homepage on the internet]. 2022 [cited 2023 mar 24]. available from: https://www.carbonbrief.org/climate-change-already-raising-risk-of-virus-spread-between-mammals/ altveş s, yildiz hk, vural hc. interaction of the microbiota with the human body in health and diseases. biosci microbiota food health. 2020;39(2):23–32. https://doi.org/10.12938/bmfh.19-023 schultz r, cairney s. caring for country and the health of aboriginal and torres strait islander australians. med j aust. 2017;207(1):8–10. https://doi.org/10.5694/mja16.00687 macguire f, mulcahy e, rossington b. the lancet countdown on health and climate change – policy brief for the uk [homepage on the internet]. 2022 [cited 2023 oct 01]. available from: https://s41874.pcdn.co/wp-content/uploads/lancet-countdown-2022-uk-policy-brief_en.pdf wong fy, yang l, yuen jwm, chang kkp, wong fky. assessing quality of life using whoqol-bref: a cross-sectional study on the association between quality of life and neighborhood environmental satisfaction, and the mediating effect of health-related behaviors. bmc public health. 2018;18:1113. https://doi.org/10.1186/s12889-018-5942-3 simkin rd, seto kc, mcdonald ri, jetz w. biodiversity impacts and conservation implications of urban land expansion projected to 2050. proc natl acad sci u s a. 2022;119:e2117297119. https://doi.org/10.1073/pnas.2117297119 secretariat of the convention on biological diversity. cop15: nations adopt four goals, 23 targets for 2030 in landmark un biodiversity agreement. in: convention on biological diversity [homepage on the internet]. 2022 [cited 2023 apr 21]. available from: https://www.cbd.int/article/cop15-cbd-press-release-final-19dec2022 armstrong mckay di, staal a, abrams jf, et al. exceeding 1.5°c global warming could trigger multiple climate tipping points. science. 2022;377:eabn7950. https://doi.org/10.1126/science.abn7950 who guidance for the use of annex 2 of the international health regulations (2005) [homepage on the internet]. world health organization. [cited 2023 oct 05]. available from: https://www.who.int/publications/m/item/who-guidance-for-the-use-of-annex-2-of-the-international-health-regulations-(2005) australian government department of health and aged care. consultation on australia’s first national health and climate strategy [homepage on the internet]. australian government department of health and aged care; 2023 [cited 2023 jul 26]. available from: https://www.health.gov.au/news/consultation-on-australias-first-national-health-and-climate-strategy hiv epidemiology in ethiopia hiv-related laboratory infrastructure in ethiopia for cd4, viral load and early infant diagnosis poc and quality assurance framework in ethiopia ethiopia’s experiences with implementing poc testing for cd4 and tuberculosis existing national quality assurance programmes challenges with poc testing acknowledgements references about the author(s) adisu kebede ethiopian public health institute, ethiopia yenew kebede centers for disease control and prevention, ethiopia laboratory branch, ethiopia adino desale ethiopian public health institute, ethiopia achamyeleh mulugeta ethiopian public health institute, ethiopia zelalem yaregal ethiopian public health institute, ethiopia atsbeha gebreegziabxier ethiopian public health institute, ethiopia yared tedla centers for disease control and prevention, ethiopia laboratory branch, ethiopia clement zeh centers for disease control and prevention, ethiopia laboratory branch, ethiopia gonfa ayana ethiopian public health institute, ethiopia citation kebede a, kebede y, desale a, et al. quality assurance for point-of-care testing: ethiopia’s experience. afr j lab med. 2016;5(2), a452. http://dx.doi.org/10.4102/ajlm.v5i2.452 country profile quality assurance for point-of-care testing: ethiopia’s experience adisu kebede, yenew kebede, adino desale, achamyeleh mulugeta, zelalem yaregal, atsbeha gebreegziabxier, yared tedla, clement zeh, gonfa ayana received: 30 mar. 2016; accepted: 11 aug. 2016; published: 17 oct. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. hiv epidemiology in ethiopia in 2015, there were an estimated 729 517 people living with hiv in ethiopia, including 95 094 children (aged 0–14 years), according to the 2015 estimation and projection package/spectrum modelling. hiv prevalence at the national level is 4.2% among urban populations and 0.6% rural populations; by gender, hiv prevalence is 1.52% among women and 1.0% among men.1 the hiv epidemic in ethiopia is primarily associated with proximity to major transport corridors and concentrated in urban areas, where prevalence is 5.2% in cities with a population above 50 000 people compared with 2.8% in smaller cities and 0.6% in rural areas.2 considerable progress has been made over the last decade in scaling up access to testing worldwide. the federal ministry of health of ethiopia adapted and started implementation of programmes to achieve the ambitious unaids 90-90-90 goal, whereby 90% of hiv-positive people are diagnosed by 2020, 90% of hiv-positive patients are on antiretroviral therapy, and 90% of hiv-positive patients achieve viral suppression.3,4 however, the biggest challenges to achieving the 90-90-90 goals in ethiopia are the first two goals. currently, 40% of infected people are unaware of their hiv status, with 12.1% of those identified as hiv-positive being neither in care nor on antiretroviral therapy. there is also very limited information available on viral suppression. therefore, closing these gaps would require some innovative approaches to find hiv-positive people unaware of their hiv status and link them to care and treatment, using both laboratory services and point-of-care (poc) testing.5 hiv-related laboratory infrastructure in ethiopia for cd4, viral load and early infant diagnosis the public health system in ethiopia has been decentralised through a tiered laboratory structure. the existing structure at the federal ministry of health enables the federal-level structures to monitor and ensure the effectiveness, efficiency, accessibility, equity and sustainability of health services, as well as engagement of local stakeholders (figure 1a). the ethiopian laboratory system comprises a network, including the national reference laboratory, regional reference laboratories, and hospital and health centre laboratories, in decreasing order of complexity and testing capacities (figure 1b). currently, there are 13 regional reference laboratories with diverse capacities, more than 300 hospital laboratories and 3500 health centre laboratories in the public-health sector. more than 4096 laboratories of various levels are associated with non-governmental organisations or private clinics and hospitals operating throughout the country.6 figure 1: health system and laboratory network: ethiopia’s referral linkage guideline. hiv-related testing is one of the most important laboratory services in the health sector. with the launch of a free antiretroviral therapy initiative by the government in january 2005, there has been an unprecedented effort to scale up antiretroviral therapy and provide access to hiv care to all hiv-positive individuals. currently, there are 10 centres providing dna pcr for early infant diagnosis of hiv, nine centres for hiv viral load testing using conventional methods and more than 300 centres providing cd4 testing services. the number of centres providing early infant diagnosis and viral load testing using conventional methods grew to 19 by the end of june 2016. poc and quality assurance framework in ethiopia the vancouver consensus statement sought the political will and resources needed to meet the new world health organization guidelines and related to the unaids 90-90-90 by 2020 goal.7 achieving the unaids goal by increased availability of hiv-associated poc testing devices will improve services. poc testing has been demonstrated to improve linkage to care among persons living with hiv.8 improving antiretroviral therapy coverage quickly and effectively to meet the 90-90-90 goals, by engaging persons living with hiv in care at earlier stages of disease, will require not only expansion of the clinic-based infrastructure with quality-assured poc testing, but also non-clinic-based testing with quality-assured poc testing.3 in 2013, the federal ministry of health and the ethiopian public health institute, with the involvement of stakeholders and partners, developed ethiopia’s national strategy for poc test selection, operational evaluation and implementation. as the technical arm of the ministry, the ethiopian public health institute is responsible for the selection, operational evaluation and implementation of new diagnostic technologies and is assisted by national technical working groups in the management of the processes. the generic national strategy document for new poc diagnostic technologies addresses the following important areas: product selection, evaluation, and implementation; supply chain and service/maintenance; quality assurance, connectivity (enables sending of results data from the archive of the alere pima™ analyser to a defined central server) and data management; monitoring and evaluation, training and certification of healthcare workers. the ethiopian poc testing strategy proposes the adoption of multiple technologies to encourage competition and enhance the deployment of multiple poc testing products in various healthcare settings where they can be effectively utilised. however, it also recommends limiting the number of products evaluated and approved for use in the country, in order to minimise the complexity of managing the supply chain, service and maintenance, training, and monitoring and evaluations. quality assurance activities have paramount importance for the effective utilisation of poc testing and must be planned strategically and executed correctly along with the expansion of poc testing, which will require strong support from the ministry of health and other stakeholders to all tiers of the national laboratory system (figure 2). figure 2: quality assurance framework. ethiopia’s experiences with implementing poc testing for cd4 and tuberculosis after completion of the alere pima cd4 evaluation by the ethiopian public health institute in different regions (unpublished data), 97 units were procured and distributed for scaling up cd4 testing services across the country. the placement of the pima platforms was guided by strict written criteria, which mainly considered the volume of cd4 testing at facilities. using this criterion helped effective and efficient placement and use of the devices. of the 97 pima platforms, 84 (87.0%) are participating in the oneworld accuracy external quality assurance (eqa) programme (health matrix, vancouver, canada). of these participants, 63 (75.0%) reported proficiency testing results for the second test event of the 2015 cycle (figure 3). among laboratories with scores, 75.4% scored 100.0% and root cause analysis and corrective action was undertaken and completed for laboratories with less than 100.0% to complete the quality assurance circle. nine laboratories did not respond within the deadline, and of the 12 laboratories excused, 8 were excused due to stock outs, one due to electric power outage and three due to specimen transportation issues. figure 3: responses from pima cd4 sites participating in oneworld accuracy test in 2015 (event 2). quality assurance implementation involved training of laboratory personnel at various levels. a five-day training-of-trainers training on pima technology was provided for regional laboratories, and site-level trainings were organised with certification of trainees. refresher trainings, including training on laboratory quality management systems, supply chain management and laboratory safety, were also provided. there are currently 104 genexpert® (cepheid) instruments being used for tuberculosis testing in ethiopia, but only 12 laboratories are participating in the united states centers for disease control and prevention, international laboratory branch eqa proficiency testing programme. ethiopia has limited experience with connectivity to monitor quality assurance for poc testing. currently, 70 of the 97 pima devices are connected. installation of the gxalert software application for connectivity on 37 genexpert devices is currently underway. for long-term sustainability, plans to build capacity for in-country proficiency testing production through technology transfer to ensure the availability of appropriate eqa methods and programmes for all poc testing sites continues to be an important strategy. existing national quality assurance programmes document standardisation as part of its quality assurance programme, ethiopia developed and standardised laboratory diagnosis of malaria, hiv and tuberculosis. in addition, poc implementation, poc testing for cd4 and tuberculosis, national eqa for malaria, guidelines for safety, a manual for sample collection, standard operating procedures for testing, and job aids, among others, were also developed. external quality assessment services in ethiopia, eqa services are being provided via three schemes (figure 4). in the international eqa scheme, the ethiopian public health institute uses testing materials purchased from international eqa service providers. currently, there are more than 285 laboratories participating in the oneworld accuracy eqa system. the national eqa scheme uses testing materials produced and maintained by the national reference laboratories for hiv, tuberculosis and malaria. the regional eqa scheme for the same programs is administered by the regional reference laboratories, which are responsible for the production and maintenance of the proficiency testing panels. they also coordinate, conduct and monitor blind rechecking of tests and site supervision within their respective regions. currently, there are more than 900 facilities with more than 3542 testing points enrolled in the regional eqa scheme for hiv. figure 4: ethiopian laboratories participating in the national eqa scheme (cryptococcus spp.), international eqa scheme from cdc atlanta (eid, hiv viral load and genexpert) and international eqa scheme from oneworld accuracy (other tests), march 2016. accreditation ethiopia adopted the stepwise laboratory quality improvement process towards accreditation in 2010 as a tool for laboratory quality improvement. currently, there are more than 109 laboratories (hospital, regional and national) enrolled in the stepwise laboratory quality improvement process towards accreditation programme. the ethiopian public health institute is in the process of assessing the laboratories to select best performers for eventual application for iso accreditation. recently, 53 laboratories were assessed with a group of certified auditors from the african society for laboratory medicine; 23 laboratories scored no stars, 11 laboratories scored 1 star, 13 laboratories scored 2 stars, five laboratories scored 3 stars, and one laboratory scored 4 stars. challenges with poc testing although ethiopia has accomplished a great deal in expansion of poc testing, some challenges have been noted. these include: the under-utilisation of pima and genexpert instruments; low eqa coverage for genexpert due to shortage of eqa panels; low response rates from proficiency testing participants; supply distribution issues; stock outs; and maintenance issues with prolonged downtime and transportation of devices to maintenance centres. lessons learnt and way forward implementation of poc testing has improved health service delivery through provision of laboratory services with shorter turnaround time. the national quality assurance programme will focus on implementing a comprehensive quality assurance programme for poc testing services that will include strengthening the laboratory–clinic interface, improving the supply chain system through collaboration with supply agencies, ensuring availability of service agreements for all new equipment and building capacity for in-country proficiency testing production through training on and implementation of the iso/iec 17043 standard. in collaboration with existing local courier systems, establishing a robust system for panel distribution and result feedback to reach all sites are major areas that will need to also be addressed. in addition, improving implementation of quality poc testing will include strengthening national capacity for ongoing evaluation and validation of new poc testing technologies by supporting the national product selection advisory group. ethiopia is committed to strengthening both national and regional laboratory capacity through networking with national reference laboratories across the african region for better data sharing that will improve upon systems for monitoring, evaluation and data management, including connectivity. acknowledgements we thank the regional laboratories capacity building directorate, ethiopian public health institute for their support so that this study to be conducted. we would also like to thank cdc ethiopia for financial and technical assistance. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. sources of support none. references ethiopian public health institute. report on the 2014 round antenatal care based sentinel hiv surveillance in ethiopia. addis ababa, ethiopia: ethiopian public health institute; 2015. ethiopian aids resource center. hiv/aids estimates and projections in ethiopia, 2011–2016 [document on the internet]. c2011 [cited 2013 dec 27]. available from: http://www.etharc.org/images/stories/downloads/hivaidsprevalenceestimate.pdf global aids response progress reporting. construction of core indicators for monitoring the 2011 united nations political declaration on hiv and aids [document on the internet]. c2014 [cited 2016 sep 13]. available from: http://www.unaids.org/sites/default/files/media_asset/garpr_2014_guidelines_en_0.pdf joint united nations programme on hiv/aids. 90-90-90: an ambitious treatment target to help end the aids epidemic. geneva, switzerland: unaids; 2014. ethiopian health and nutrition research institute. strategy for the implementation of point-of-care technologies in ethiopia. addis ababa, ethiopia; june 2013. the federal democratic republic of ethiopia ministry of health. health sector transformation plan (2015/16 – 2019/20). addis ababa, ethiopia; august 2015. international aids society conference. the vancouver consensus 2015 (paragraph 6) [page on the internet]. c2015 [cited 2016 mar 28]. available from: http://vancouverconsensus.org/ jani iv. pilot implementation of point-of-care cd4 counting in mozambique’s national health system [document on the internet]. c2011 [cited 2016 mar 28]. available from: http://tbevidence.org/wp-content/uploads/2011/11/33-i-jani-poc-pilot-implementation-in-mozambique.pdf references about the author(s) kamran abbasi british medical journal, london, united kingdom parveen ali international nursing review, sheffield, united kingdom virginia barbour medical journal of australia, brisbane, australia marion birch medact, university college london, london, united kingdom inga blum general practitioner, hamburg, germany peter doherty department of microbiology and immunology, university of melbourne, doherty institute, melbourne, australia andy haines london school of hygiene and tropical medicine, london, united kingdom ira helfand international physicians for the prevention of nuclear war, springfield, massachusetts, united states richard horton the lancet, london, united kingdom kati juva division of psychiatry, helsinki university central hospital, helsinki, finland jose f. lapena jr. department of otolaryngology, head and neck surgery, philippine general hospital, manila, philippines robert mash division of family medicine and primary care, stellenbosch university, stellenbosch, south africa olga mironova russian cardiology research and production complex, sechenov university, moscow, russian federation arun mitra indian doctors for peace and development, ludhiana, india carlos monteiro department of nutrition, school of public health, university of são paulo, são paolo, brazil elena n. naumova tufts university, boston, massachusetts, united states david onazi society of nigerian doctors for the welfare of mankind (sndwm), abuja, nigeria tilman ruff international physicians for the prevention of nuclear war, university of melbourne, melbourne, australia peush sahni all india institute of medical sciences (aiims), new delhi, india james tumwine school of medicine, kabale university, kampala, uganda carlos umaña costa rican ministry of health, san josé, costa rica paul yonga ca medlynks medical centre and laboratory, nairobi, kenya chris zielinski university of winchester and world association of medical editors, winchester, united kingdom citation abbasi k, ali p, barbour v, et al. ending nuclear weapons, before they end us. afr j lab med. 2025;14(1), a2847. https://doi.org/10.4102/ajlm.v14i1.2847 editorial ending nuclear weapons, before they end us kamran abbasi, parveen ali, virginia barbour, marion birch, inga blum, peter doherty, andy haines, ira helfand, richard horton, kati juva, jose f. lapena jr., robert mash, olga mironova, arun mitra, carlos monteiro, elena n. naumova, david onazi, tilman ruff, peush sahni, james tumwine, carlos umaña, paul yonga, chris zielinski copyright: © 2025. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. in may 2025, the world health assembly will vote on re-establishing a mandate for the world health organization to address the health consequences of nuclear weapons and war.1 health professionals and their associations should urge their governments to support such a mandate and support the new united nations comprehensive study on the effects of nuclear war. the first atomic bomb exploded in the new mexico desert 80 years ago, in july 1945. three weeks later, two relatively small (by today’s standards), tactical-size nuclear weapons unleashed a cataclysm of radioactive incineration on hiroshima and nagasaki. by the end of 1945, about 213 000 people were dead.2 tens of thousands more have died from late effects of the bombings. last december, nihon hidankyo, a movement that brings together atomic bomb survivors, was awarded the nobel peace prize for its ‘efforts to achieve a world free of nuclear weapons and for demonstrating through witness testimony that nuclear weapons must never be used again’.3 for the norwegian nobel committee, the award validated the most fundamental human right: the right to live. the committee warned that the menace of nuclear weapons is now more urgent than ever before. in the words of committee chair jørgen watne frydnes, [i]t is naive to believe our civilisation can survive a world order in which global security depends on nuclear weapons. the world is not meant to be a prison in which we await collective annihilation.4 he noted that our survival depended on keeping intact the ‘nuclear taboo’ (which stigmatises the use of nuclear weapons as morally unacceptable).5 the nuclear taboo gains strength from recognition of compelling evidence of the catastrophic humanitarian consequences of nuclear war, its severe global climatic and famine consequences, and the impossibility of any effective humanitarian response. this evidence contributed significantly to ending the cold war nuclear arms race.6,7 while the numbers of nuclear weapons are down to 12 331 now, from their 1986 peak of 70 300,8 this is still equivalent to 146 605 hiroshima bombs,9 and does not mean humanity is any safer.10 even a fraction of the current arsenal could decimate the biosphere in a severe mass extinction event. the global climate disruption caused by the smoke pouring from cities ignited by just 2% of the current arsenal could result in over two billion people starving.11 a worldwide nuclear arms race is underway. deployed nuclear weapons are increasing again, and china, india, north korea, pakistan, russia and the united kingdom are all enlarging their arsenals. an estimated 2100 nuclear warheads in france, russia, the united kingdom, the united states and, for the first time, also china, are on high alert, ready for launch within minutes.8 with disarmament in reverse, extensive nuclear modernisations underway, multiple arms control treaties abrogated without replacement, no disarmament negotiations in evidence, nuclear-armed russia and israel engaged in active wars involving repeated nuclear threats, russia and the united states deploying nuclear weapons to additional states, and widespread use of cyberwarfare, the risk of nuclear war is widely assessed to be greater than ever. this year, the doomsday clock was moved the closest to midnight since the clock’s founding in 1947.10 led by ireland and new zealand, in late 2024, the united nations general assembly voted overwhelmingly to establish a 21-member independent scientific panel to undertake a new comprehensive study on the effects of nuclear war,12 with its final report due in 2027. noting that ‘removing the threat of a nuclear war is the most acute and urgent task of the present day’, the panel has been tasked with examining the physical effects and societal consequences of a nuclear war on a local, regional and planetary scale. it will examine the climatic, environmental and radiological effects of nuclear war, and their impact on public health, global socioeconomic systems, agriculture and ecosystems. the resolution calls upon united nations agencies, including who, to support the panel’s work, including by ‘contributing expertise, commissioned studies, data and papers’. all united nations member states are encouraged to provide relevant information, scientific data and analyses; facilitate and host panel meetings, including regional meetings; and make budgetary or in-kind contributions. such an authoritative international assessment of evidence on the most acute existential threat to humankind and planetary health is long overdue. the last such report dates from 1989. it is shameful that france, the united kingdom and russia opposed this resolution.13 in 1983 and 1987,14 who convened an international committee of scientists and health experts to study the health effects of nuclear war. its landmark, authoritative reports were influential and an excellent example of who fulfilling its constitutional mandate ‘to act as the directing and coordinating authority on international health work’. in 1993, who produced an additional shorter report on the health and environmental effects of nuclear weapons, which included discussion of the production chain of nuclear weapons, including processing, testing and disposal.15 however, despite the world health assembly having mandated who to report periodically on relevant developments, no further work was undertaken and in 2020, who’s mandate on nuclear weapons and health lapsed. the marshall islands, samoa and vanuatu, supported by seven co-sponsoring states and international physicians for the prevention of nuclear war, are working to renew who’s mandate. they are seeking wide support for a resolution on the health effects of nuclear weapons/war at this year’s world health assembly in geneva on 19–27 may.1 the world health organization would then re-establish a programme of work on this most critical threat to health, and be able to lead strongly in providing the best health evidence to the united nations panel. health professionals are well aware how crucial accurate and up-to-date evidence is to making good decisions. we and our organisations should support such a renewed mandate by urging our national world health assembly delegates to vote in support and commit the modest funds needed to re-establish who’s work programme, especially now, as the organisation faces severe financial strain with the united states’ decision to withdraw its membership. our joint editorial in 202316 on reducing the risks of nuclear war and the role of health professionals, published in over 150 health journals worldwide, urged three immediate steps by nuclear-armed states and their allies: adopt a ‘no first use’ policy, take their nuclear weapons off hair-trigger alert, and pledge unequivocally that they will not use nuclear weapons in any current conflicts they are involved in. we also urged nuclear-armed states to work for a definitive end to the nuclear threat by urgently starting negotiations for a verifiable, timebound agreement to eliminate their nuclear arsenals, and called on all nations to join the treaty on the prohibition of nuclear weapons.17 it is an alarming failure of leadership that no progress has been made on these needed measures, nor on many other feasible steps away from the brink, acting on the obligation of all states to achieve nuclear disarmament. nine states jeopardise all humanity and the biosphere by claiming an exclusive right to wield the most destructive and inhumane weapons ever created. the world desperately needs the leaders of these states to freeze their arsenals, end the modernisation and development of new, more dangerous nuclear weapons, and ensure that new technology such as artificial intelligence can never trigger the launch of nuclear weapons. the united nations scientific panel and a renewed mandate for who’s work in this area can provide vital authoritative and up-to-date evidence for health and public education, evidence-based advocacy and policies, and the mobilised public concern needed to trigger decisive political leadership. this is a core health imperative for all of us. references world health organization. effects of nuclear weapons and war on health and health services [homepage on the internet]. eb156/conf./10, executive board. 2025 [cited 2025 may 04]. available from: https://apps.who.int/gb/ebwha/pdf_files/eb156/b156_conf10-en.pdf tomonaga m. the atomic bombings of hiroshima and nagasaki: a summary of the human consequences, 1945–2018, and lessons for homo sapiens to end the nuclear weapon age. j-pand. 2019;2(2):491–517. https://doi.org/10.1080/25751654.2019.1681226 nobelprize.org, 2024. the nobel peace prize [homepage on the internet]. 2024 [cited 2025 feb 25]. available from: https://www.nobelprize.org/prizes/peace/2024/summary/ award ceremony speech. nobel prize outreach [homepage on the internet]. 2025 [cited 2025 feb 25]. available from: https://www.nobelprize.org/prizes/peace/2024/ceremony-speech/ tannenwald n. the nuclear taboo: the united states and the normative basis of nuclear non-use. int organ. 1999;53(3):433–468. https://doi.org/10.1162/002081899550959 robock a, xia l, harrison cs, coupe j, toon ob, bardeen c. opinion: how fear of nuclear winter has helped save the world, so far. atmos chem phys. 2023;23(12):6691–6701. https://doi.org/10.5194/acp-23-6691-2023 helfand i, haines a, ruff t, kristensen h, lewis p, mian z. the growing threat of nuclear war and the role of the health community. world med j. 2016;62(3):86–94. kristensen h, korda m, johns e, knight m, kohn k. status of world nuclear forces. federation of american scientists [homepage on the internet]. 2025 [cited 2025 mar 18]. available form: https://fas.org/initiative/status-world-nuclear-forces/ norwegian people’s aid. nuclear weapons ban monitor 2024 [homepage on the internet]. 2025 [cited 2025 mar 25]. available from: https://banmonitor.org/ science and security board. closer than ever: it is now 89 seconds to midnight. 2025 doomsday clock statement [homepage on the internet]. bulletin of the atomic scientists. 2025 [cited 2025 mar 04]. available from: https://thebulletin.org/doomsday-clock/2025-statement/ xia l, robock a, scherrer k, et al. global food insecurity and famine from reduced crop, marine fishery and livestock production due to climate disruption from nuclear war soot injection. nat food. 2022;3:586–596. https://doi.org/10.1038/s43016-022-00573-0 united nations general assembly. nuclear war and scientific research [homepage on the internet]. a/c.1/79/l.39. 2024 [cited 2025 mar 04]. available from: https://reachingcriticalwill.org/images/documents/disarmament-fora/1com/1com24/resolutions/l39-.pdf united nations general assembly: 55th plenary meeting. item 98 a/79/408 dr xvii as a whole [homepage on the internet]. 2024 [cited 2025 mar 04]. available from: https://reachingcriticalwill.org/images/documents/disarmament-fora/1com/1com24/votes-ga/408drxvii.pdf world health organization. effects of nuclear war on health and health services [homepage on the internet]. 2nd ed. geneva: who; 1987 [cited 2025 mar 04]. available from: https://iris.who.int/handle/10665/39199 world health organization. health and environmental effects of nuclear weapons [homepage on the internet]. wha46/30. 1993 [cited 2025 mar 04]. available from: https://iris.who.int/bitstream/handle/10665/175987/wha46_30_eng.pdf?isallowed=y&sequence=1 abbasi k, ali p, barbour v, et al. reducing the risks of nuclear war. br med j. 2023;382:1682. https://doi.org/10.1136/bmj.p1682 united nations. treaty on the prohibition of nuclear weapons [homepage on the internet]. 2017 [cited 2025 mar 09]. available from: https://www.icanw.org/tpnw_full_text abstract introduction methods results discussion acknowledgements references about the author(s) naseem cassim department of haematology and molecular medicine, university of the witwatersrand, johannesburg, south africa national priority programme, national health laboratory service, johannesburg, south africa lindi m. coetzee department of haematology and molecular medicine, university of the witwatersrand, johannesburg, south africa national priority programme, national health laboratory service, johannesburg, south africa nelesh p. govender national institute for communicable diseases, division of the national health laboratory service (nhls), johannesburg, south africa faculty of health sciences, university of the witwatersrand, johannesburg, south africa deborah k. glencross department of haematology and molecular medicine, university of the witwatersrand, johannesburg, south africa national priority programme, national health laboratory service, johannesburg, south africa citation cassim n, coetzee lm, govender np, glencross dk. district and sub-district analysis of cryptococcal antigenaemia prevalence and specimen positivity in kwazulu-natal, south africa. afr j lab med. 2018;7(1), a757. https://doi.org/10.4102/ajlm.v7i1.757 original research district and sub-district analysis of cryptococcal antigenaemia prevalence and specimen positivity in kwazulu-natal, south africa naseem cassim, lindi m. coetzee, nelesh p. govender, deborah k. glencross received: 18 jan. 2018; accepted: 01 may 2018; published: 11 oct. 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: cryptococcal meningitis (cm) is a leading cause of mortality among hiv-positive south africans. reflex cryptococcal antigen (crag) testing of remnant plasma was offered as a pilot prior to implementation in october 2016 in kwazulu-natal province. the national reflex crag positivity was 5.4% compared to 7.3% for kwazulu-natal. objectives: the aim of this study was to interrogate crag positivity by health levels to identify hotspots. method: data for the period october 2016 to june 2017 were analysed. health district crag positivity and prevalence were calculated, with the latter using de-duplicated patient data. the district crag positivity and the number of crag-positive specimens per health facility were mapped using arcgis. for districts with the highest crag positivity, a sub-district crag positivity analysis was conducted. results: the provincial crag positivity was 7.6%. district crag positivity ranged from 5.7% (ugu) to 9.6% (umkhanyakude) with prevalence ranging from 5.5% (ugu) to 9.7% (umkhanyakude). the highest crag positivity was reported for the umkhanyakude (9.6%) and king cetswayo (9.5%) districts. in these two districts, crag positivity of 10% was noted in the umhlabuyalingana (10.0%), jozini (10.2%), umhlathuze (10.5%) and nkandla (10.8%) subdistricts. in these subdistricts, 135 crag-positive samples were reported for the ngwelezane hospital followed by 41 and 43 at the hlabisa and manguzi hospitals respectively. conclusion: cryptococcal antigen positivity was not uniformly distributed at either the district or sub-district levels, with identified facility hotspots in the umkhanyakude and king cetswayo districts. this study demonstrates the value of laboratory data to identify hotspots for planning programmatic interventions. introduction cryptococcal meningitis (cm) is a leading cause of mortality among hiv-positive persons in south africa.1 cryptococcal antigenaemia screening can identify persons at risk of developing cm (even among asymptomatic patients) to timeously initiate antifungal and antiretroviral treatment (art) for naive persons. cryptococcal antigen (crag) reflex testing of specimens with a cd4 count of ≤ 100 cells/µl was implemented across a network of cd4 testing laboratories in south africa.2 this followed the inclusion of crag screening in south africa’s national hiv guidelines in 2015, following recommendations by the world health organization (who).3,4 since the national implementation of reflex crag screening in south africa in october 2016, data collected by testing laboratories confirmed that nationally, about 10% of all cd4 samples tested had a count ≤ 100 cells/µl. these specimens were tested for crag using a lateral flow assay (lfa) (immuno mycologics, norman, oklahoma, united states). both the percentage of samples eligible for crag testing and the percentage of crag-positive results vary considerably across the nine provinces in south africa. data from a previously reported study showed a crag positivity of 2.4% in the northern cape versus 7.3% in kwazulu-natal.5 within kwazulu-natal, district crag positivity ranged from 6.2% in amajuba to 9.2% in king cetshwayo district, with the umkhanyakude district reporting the second highest crag positivity at 8.9%.5 kwazulu-natal is the second most populous province in south africa and has the highest hiv prevalence.6 there are 11 079 700 people living in the province representing 19.8% of the national population.7 the provincial hiv prevalence is 16.9% in 2016 compared to the national prevalence of 12.2%. the 2016 hiv infection rate in kwazulu-natal is 2.3% (compared to the national rate of 1.8%6) among 1 622 870 million hiv-seropositive individuals (15–49 years of age) living in the province. an estimated 1 129 314 hiv-positive individuals are on art with 69.5% coverage.6 reflex crag screening was offered for the first time in kwazulu-natal at the prince mshyeni memorial laboratory in july 2015 as part of a crag reflex testing pilot.8,9 by october 2016, all cd4 laboratories in kwazulu-natal offered reflex crag screening. there are limited published cm data available for kwazulu-natal as most reported studies investigated selected health facilities with small case numbers. a study conducted at an urban district hospital between 2011 and 2012 reported that of the 127 patients with confirmed cm, 65 were men (51.2%).10 in that study, cm affected predominantly the economically active population (mean age, 36 [±9.8] years).10 while 76% (n = 97) of patients knew their hiv status, but only 45% (n = 43) were on art.10 acute mortality was 55.9% (71/127) within 14 days of cm diagnosis highlighting the need for screening and preemptive treatment of subclinical cryptococcal disease before art initiation.10 a 2007 study conducted at the ngewlezane hospital reported that even in a setting where amphotericin b is available, the burden of cm deaths is particularly high in the immediate period after diagnosis (2.13 deaths per 100 person days).11 of the 186 patients enrolled in this cohort, 52 (28%) died within 14 days of diagnosis.11 in 2015 the national institute for communicable diseases reported 1745 laboratory-confirmed cases of cm from kwazulu-natal through its national network of clinical microbiology laboratories which participate in an active surveillance programme for pathogens of public health importance (germs-sa).12 given the higher-than-national average crag positivity in kwazulu-natal, the study reported here aimed to investigate crag positivity at the health district and sub-district levels with facility level investigation in the subdistricts with the highest reported positivity rates. methods ethical considerations ethics clearance for this work was obtained from the university of the witwatersrand (study approval number m1706108). this study was conducted in accordance with relevant national and international guidelines. this study involved the secondary analysis of laboratory test volumes data that do not contain any patient identifiers. no patient recruitment was necessary as routine laboratory data was used for the study. data data reported here include reflex crag testing subsequent to a confirmed cd4 count ≤ 100 cells/µl using automated laboratory information system rules that identify eligible samples to be tested, that is, these data exclude provider-initiated crag screening. owing to cd4 testing challenges at the church of scotland laboratory, all crag data from this laboratory were excluded for the purpose of this study. this laboratory serves the umzinyathi health district that contributes only 22% of crag samples tested in this district. the data analysis includes both crag positivity using specimen-level data as well as crag prevalence after the data set was de-duplicated. the de-duplication process involved using the corporate data warehouse (cdw)-assigned unique patient identifier that is determined through a probabilistic matching algorithm to identify if a patient had more than one crag test.13 the de-duplicated data were used to identify both the number of patients receiving a crag test as well as the number with one or more positive crag tests to determine prevalence.13 the de-duplicated data provide a more accurate analysis of crag positivity for a given population. data extraction cd4 and crag laboratory data were extracted from the national health laboratory service (nhls) cdw for the period october 2016 through to june 2017. data were filtered to include only health facilities within kwazulu-natal. the data extract included the absolute cd4 count (< 100 cells/µl) and crag result. additional data variables included the episode number, testing date, health facility location code and description, testing and referring laboratory names, date of birth, patient age (in years) and gender. the testing year and month were derived using the cd4 result review date. ethics clearance for all cdw data extraction was obtained through the university of the witwatersrand (m1706108). data was de-identified at the cdw. data analysis the cdw data extract were imported into a microsoft access table (washington, united states). queries were used to prepare the data for analysis. the cdw location code and description were mapped to the district health information system (dhis) health facility descriptions; for example, addington hospital was mapped to the dhis organisational unit (ou) 5 short description ‘addington hosp’. for each cdw location, in addition to the ou5 facility name, the dhis province (ou2), health district (ou3), health sub-district (ou4), ou type, latitude and longitude were added to the mapping table. where a cdw location description was not matched to the dhis list, it was marked as ‘excluded’ (e.g. empangeni prison). the ‘dhis ou_type’ was used to identify facility type, for example hospital (tertiary, regional and district), community health centre (chc), or primary health care clinic (phc). the mapping table was loaded on microsoft access and added to the cd4 and crag query. the dhis-mapped list was used to filter the data for legitimate health facilities. the list was also used to report data at the province, district and sub-district health care levels. the final data set was exported as a comma separated values (csv) file for analysis in stata se (texas, united states) and microsoft excel (washington, united states). provincial crag percentage positivity analysis the crag positivity for kwazulu-natal was reported as the proportion of crag-positive samples divided by all samples (with cd4 count ≤ 100 cells/µl) with the 95% confidence interval (ci) reported. the provincial crag positivity was analysed across age ranges, gender, facility types and cd4 test ranges. the crag positivity was also assessed monthly to identify changes or patterns over time. a sub-analysis was undertaken for the provincial crag-positive samples to determine cd4 test ranges by facility type, gender and age category. health district crag percentage positivity analysis crag positivity and prevalence was analysed for the 11 health districts in the province with the 95% ci reported (i.e. amajuba, ethekwini, harry gwala, ilembe, king cetshwayo, ugu, umgungundlovu, umkhanyakude, umzinyathi, uthukela and zululand). the total crag test volumes, number of positive crag samples and crag positivity was reported for each health district. both the district crag positivity and prevalence are reported with 95% ci. number of crag-positive samples by health facility arcgis was used to map the health district crag positivity as well as the number of crag-positive samples by health facility to identify where patients presented for care. the larger towns in the province were indicated on the map as reference points. the actual number of positive crag samples per health facility was reported in five categories: (1) 1–7, (2) 8–20, (3) 21–43, (4) 44–93 and (5) 94–148. shapefiles were obtained from the municipal demarcation board and the dhis spatial coordinates were applied for health facilities. district-level percentage of crag positivity was reported in four categories each allocated a different colour: (1) ≤ 5.7%, (2) 5.8% – 6.6%, (3) 6.7% – 8.1% and (4) 8.2% – 9.6%. category ranges were automatically assigned based on actual data. spatial analysis for two districts with the highest crag percentage positivity rate the two districts with the highest crag positivity were identified and the respective sub-district and health facility crag positivity was reported using arcgis. the two district shapefiles were merged using the arcmap data management toolbox. the percentage of crag-positive samples per health facility was reported using five categories: (1) 4.2% – 6.6%, (2) 6.7% – 8.7%, (3) 8.8% – 9.2% and (4) 9.3% – 10.87%. the actual number of positive crag samples per health facility was similarly reported in five categories: (1) 1–4, (2) 5–11, (3) 12–28, (4) 29–43 and (5) 44–135. category ranges were automatically assigned based on actual data. results provincial crag percentage positivity analysis for the reported period, 50 534 crag tests were performed (table 1) after exclusion of 586 samples from the church of scotland laboratory (1.2% of provincial volumes). for the entire province, a crag positivity of 7.6% (95% ci, 7.3% – 7.8%) was reported. a higher crag positivity of 8.0% (7.6% – 8.3%) was reported for males compared to 7.2% (6.8% – 7.5%) for females (p = 0.001). in the 16–19 years age group (n = 1415), the crag positivity was 8.2% (6.8% – 9.8%). a crag positivity of 8.4% was reported for both the 40–44 years (7.7% – 9.0%) and older than 49 years (7.7% – 9.1%) age groups. the lowest crag positivity was reported for the 15 years and younger group (4.9% [3.9% – 6.2%]) followed by the 20–25 years age group (5.7% [5.0% – 6.6%]). crag positivity was the highest for samples requested at hospitals at 10% (9.6% – 10.5%) compared to 6.5% (5.8% – 7.3%) and 5.7% (5.4% – 6.0%) for chcs and phcs respectively (p = 0.19). crag positivity was 12.4% (11.6% – 13.2%) for cd4 counts of ten cells/µl or lower. for the cd4 ranges of 11–29, 30–49 and ≥ 50 cells/µl, a crag positivity of 9.9% (9.4% – 10.5%), 7.4% (6.9% – 8.0%) and 5.2% (4.9% – 5.5%) was reported respectively (p = 0.21). table 1: kwazulu-natal provincial crag positivity descriptive statistics from october 2016 to june 2017. health district crag percentage positivity analysis crag positivity in the 11 districts ranged from 5.7% (ugu [4.9% – 6.5%]) to 9.6% (umkhanyakude [8.6% – 10.6%]) with an overall provincial crag positivity of 7.6% (7.3% – 7.8%) (table 2). for the 11 districts, 33.7% of crag samples were requested by health facilities in the ethekwini health district (n = 17 017). the highest crag positivity was reported by the umkhanyakude (9.6%), king cetswayo (9.5% [8.7% – 10.4%]) and zululand (8.1% [7.3% – 8.9%]) districts. the number of crag-positive samples per district ranged from 133 (harry gwala) to 1304 (ethekwini). following de-duplication, a provincial crag prevalence of 7.5% (7.3% – 7.8%) was reported. crag prevalence ranged from 5.5% (4.7% – 6.4%) to 9.7% (8.6% – 10.8%) for the ugu and umkhanyakude districts respectively. the highest crag prevalence was in the umkhanyakude (9.7%), king cetswayo (9.5% [9.0% – 10.8%]) and zululand (7.9% [7.1% – 8.8%]) districts. table 2: analysis of district crag positivity in kwazulu-natal between october 2016 and june 2017 for screened samples and de-duplicated patients de-duplicated patient data. number of crag-positive samples by health facility across the province, 472 health facilities requested a cd4 test for which a reflex crag test was performed where the count was ≤ 100 cells/µl. crag-positive tests per health facility ranged from 1 to 146 (figure 1). figure 1: spatial analysis of the number of positive crag samples by health facility reported across five categories a cluster of health facilities (n = 120) in close proximity with crag-positive results were noted in the ethekwini district (durban). the ethekwini district crag positivity was 7.7%. the number of crag-positive samples for this district ranged from one at smaller phc facilities such as merebank clinic to 146 at the prince mshiyeni hospital. two health facilities reported a crag positivity above 15%, that is, mahatma gandhi hospital (16.7%) and blue roof clinic (17.6%). smaller clusters of health facilities were noted in the umgungundlovu (pietermaritzburg), ilembe, king cetswayo (empangeni/richards bay), amajuba (newcastle) and ugu (margate) districts with district crag positivity results of 6.5%, 7.3%, 9.5%, 6.2% and 5.7%, respectively. three health facilities had between 94 and 146 crag-positive samples (i.e. vryheid, ngwelezana and prince mshiyeni memorial hospitals). there were 11 health facilities that had between 44 and 93 crag-positive samples identified, that is, rk khan, mahatma gandhi, madadeni, edendale, stanger, king edward, northdale, king dinuzulu, ladysmith and benedictine hospitals as well as the kwamashu poly chc. there were 27 health facilities with 21–43 crag-positive samples. health sub-district spatial analysis for two districts with the highest crag positivity rate as mentioned, the districts with the highest crag positivity rates were umkhanyakude (9.6%), and king cetswayo (9.5% [8.7% – 10.4%]). there are five subdistricts in the umkhanyakude district compared to six subdistricts in king cetswayo. across the two districts, crag positivity ranged from 4.2% (mthonjaneni) to 10.8% (nkandla) (figure 2). the jozini, umhlabuyalingana, umhlathuze and nkandla subdistricts were placed in the 9.3% – 10.8% category. their crag positivity rates were 10.2%, 10.0%, 10.5% and 10.8%, respectively. only the ngwelezana hospital in the umhlathuze sub-district was placed in 44–135 crag-positive sample category (value of 135). this was followed by four health facilities in the 29–43 crag-positive samples (i.e. hlabisa [43], manguzi [41], bethesda [36] and nkandla [34]). the majority of health facilities (n = 93) reported 28 or fewer crag-positive samples. figure 2: spatial analysis of the crag positivity rate by sub-district for the king cetshwayo and umkhanyakude health districts with the number of positive crag samples by health facility reported across five categories. discussion in this study, we describe a crag positivity of 7.6% [7.3% – 7.8%] for kwazulu-natal. this is the highest provincial crag positivity across south africa.5 a statistically significant higher crag positivity was reported for males (p < 0.05). the majority of crag samples (33.7%) in the province were requested by health facilities in the ethekwini health district. district crag positivity ranged from 5.5% in ugu to 9.7% for umkhanyakude. three districts reported a crag positivity ≥ 7.8% (i.e. umkhanyakude [9.7%], king cetswayo [9.5%] and zululand [7.9%]). within the umkhanyakude and king cetswayo districts, the sub-district crag positivity ranged from 4.2% to 10.8%. four subdistricts within these two districts reported a crag positivity between 9.3% and 10.8%. the proportion of cd4 samples with < 100 cells/µl for the 2014–2015 financial period was used to prioritise the implementation of crag screening at the provincial and district levels.5 across south africa, a proportion of cd4 <100 cells/µl of 9.69% was reported.5 the provincial proportion of cd4 samples ≤100 cells/µl ranged from 7.33% for kwazulu-natal to 11.82% for limpopo.5 in kwazulu-natal, the district proportion of cd4 samples ≤ 100 cells/µl ranged from 5.4% in umkhanyakude to 9.1% in the harry gwala district. consequently, this province was the last to implement crag screening. given the data reported in this study, it is clear that crag prevalence data would have been a better proxy for the order of implementation. unfortunately, at the time only the proportion of cd4 samples ≤ 100 cells/µl was available. kwazulu-natal has a population of over 11 million people with an hiv prevalence of 16.9% (national prevalence was 12.22%), equating to a hiv-seropositive population of 1.8 million.6,7 over a third (33.5%) of the kwazulu-natal population reside in the ethekwini health district (n = 3.7 million) followed by umgungundlovu (9.9%: 1.09 million) and king cetshwayo (8.8%: 971 thousand).14 34.9% of the population are under 14 years of age,7 with 36.7% (n = 4 065 052) in the 16 to 34 years of age group. overall, 71.6% of the provincial population is less than or equal to 34 years of age indicating a young population.7 the 2012 human sciences research council national household survey reported that most districts in the province had an hiv prevalence ranging from 16% to 22% with the exception of umkhanyakude, ilembe and ethekwini districts (ranging from 13% to 15%).15 an analysis of the hiv-treatment cascade in the province in 2016 revealed that 23% of the hiv-seropositive population did not know their status, that is of the 1.8 million hiv-positive people 1.63 million knew their hiv status.6 by 2016, 1.06 million people were on art (87% of hiv-positive people that know their status).6 johnson et al. assessed the provincial progress towards the 90–90–90 targets and reported a provincial art coverage of 62% compared to 57% nationally.16 to improve art coverage, the province recently adopted the universal test and treat (utt) strategy in which all hiv-seropositive individuals receive art regardless of cd4 count.6 the goal of the utt strategy is to reduce the incidence of hiv infections.6 the crag burden revealed in this study is possibly linked to the significant burden of patients still to be initiated on art or who disengage from hiv care. this study has demonstrated the inherent value of laboratory data to indicate how patients use and access services, to understand the burden of disease as evidenced in the study results and facilitate the identification of hotspots to guide programmatic interventions. this intrinsic value can be utilised at the national, provincial, district, sub-district and facility levels and even at the patient level and linked to service delivery or to a specific health programme. this is not possible with aggregate data systems. a good example is the results for action report that can deliver a list of babies that are hiv-positive to the district manager to ensure that they are linked to care.17 the data are compressed and password-protected to maintain confidentiality.17 to receive the results for action reports, healthcare workers are required to apply online using the cdw self-service portal.17 additionally, the laboratory data can provide indirect information about healthcare services that are being accessed by assessing what are being requested and from which health facility (based on geographic location, that is latitude and longitude). the use of spatial tools allows for analysis at a glance by presenting the information in a way that makes it easy to visualise hotspots and further identify how homogenous or heterogeneous crag positivity is, for example. aside from offering a broader public health perspective at the national level, due to linkage to specific geographical identifiers, the analysis enables drilling down to an individual health facility level to directly affect individual patient management or intervention. specifically, in this study we have illustrated, with the outcomes reported here, how specimen-level laboratory data can be used to assess a laboratory test, crag, and establish the positivity at the provincial, district, sub-district and even at health facility levels. there was no bias or predominant area with higher testing numbers; we have shown that crag screening coverage appears to be widely (and universally) spread across the province, with 473 health facilities linked to at least one positively identified crag sample. the information also suggests that the implementation of the national reflex crag testing programme is facilitating crag testing from all health facilities through a cd4 test request to their local laboratory. the analysis has further revealed that crag positivity is not uniformly distributed at either the district or sub-district-level, with some districts demonstrating a much higher positivity; to effect meaningful clinical and programmatic impact at facility level, further investigation is needed to assess the positivity and assess whether a similar pattern could be anticipated at facility level as well. in this study we also reported the number of crag-positive samples at the health facility level. this approach enables programme managers to identify health facility hotspots to focus programme or clinical interventions that would assist in the identification of patients who are more likely to have cryptococcal disease before they become sick. future directives and initiatives requiring coordination and collaboration by both the nhls and the national, regional and provincial departments of health are needed, to ensure that patients are linked to care. aside from the existing ‘results for action’ report of crag-positive patients that is issued at sub-district or facility level by the nhls for individual patient follow-up, a more generalised collated data report about the volumes of patient accessing specific services, and how sick these patients are, is necessary. this report could be used to ensure that the original aim of the crag screening initiative is realised and that there is meaningful infrastructure and systems streamlined so that the follow-up of hiv-seropositive patients who additionally require antifungal treatment when diagnosed with early cryptococcal disease, receive this treatment. limitations information about patient outcomes where a positive crag result was reported were not available. there is, however, currently a planned collaboration between the national institute for communicable diseases and the national department of health to undertake a prospective study. the purpose is to understand the outcomes of the sa crag screening initiative by following a crag-positive cohort from diagnosis to treatment. this will help to address some of the additional questions that cannot be answered solely with laboratory data. additional questions that can be answered may be the loss to follow up, whether patients positively identified through the screening initiative receive treatment and what their clinical outcomes are. all cd4 samples tested for crag at the church of scotland laboratory were excluded from this analysis. acknowledgements the authors would like to thank the nhls kwazulu-natal area and business managers, cd4 laboratory managers, cd4 training staff, and the information technology department and supply chain management, all of whom assisted with the implementation of cd4 laboratory-based reflexed crag screening in kwazulu-natal. we would also like to thank oriel mahlatsi and silence ndlovu from the national priority programme for cdw data extraction. we acknowledge the contribution and leadership of the national institute for communicable diseases which led the reflex crag screening pilot prior to national implementation within the nhls. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support none. authors’ contributions d.k.g. supervised the study by providing leadership and oversight. d.k.g. was also the project leader. n.c, n.p.g. and l.m.c. designed the study, developed the methodology and conducted the research. n.c. conducted the data analysis and prepared the maps. d.k.g and n.p.g. provided editorial comments and technical input. n.p.g. assisted with statistical analyses in stata. all authors contributed to the manuscript development. references adeyemi b, ross a. profile and mortality outcome of patients admitted with cryptococcal meningitis to an urban district hospital in kwazulu-natal, south africa. j int aids soc. 2014;17(4 suppl 3):19623. https://doi.org/10.7448/ias.17.4.19623 coetzee lm, cassim n, sriruttan c, mhlanga m, govender np, et al. (2018) cryptococcal antigen positivity combined with the percentage of hiv-seropositive samples with cd4 counts <100 cells/μl identifies districts in south africa with advanced burden of disease. plos one 13(6): e0198993. https://doi.org/10.1371/journal.pone.0198993 national department of health (ndoh). national consolidated guidelines for the prevention of mother-to-child transmission of hiv (pmtct) and the management of hiv in children, adolescents and adults [homepage on the internet]. pretoria, south africa; 2015 [cited 2017 april 12]. available from http://www.sahivsoc.org/files/art%20guidelines%2015052015.pdf world health organization (who). rapid advice: diagnosis, prevention and management of cryptococcal disease in hiv-infected adults, adolescents and children [homepage on the internet]. geneva, switzerland; 2011 [cited 2017 march 14]. available from www.who.int/hiv/pub/cryptococcal_disease2011/en/ coetzee lm, cassim n, sriruttan c, mhlanga m, govender np, glencross dk. national cryptococcal reflexed screening positivity rates and proportion of patients with cd4 counts < 100 cells/μl identifies districts for intensified programmatic support and fast tracking patients into care. 2018. unpublished manuscript. plos one. kwazulu-natal provincial department of health. universal test and treat: ‘a game changer in hiv prevention’ [homepage on the internet]. pietermaritzburg, south africa: kwazulu-natal provincial department of health; 2016 [cited 2017 october 6]. available from http://www.kznonline.gov.za/hivaids/councils/provincial-councils-on-aids/2016/universal%20test%20&%20treat%20presentation.pdf statistics south africa (stats sa). mid-year population estimates. pretoria, south africa: statistics south africa (stats sa); 2016. contract no.: p0302. govender np, chetty v, roy m, et al. phased implementation of screening for cryptococcal disease in south africa. s afr med j. 2012;102(12):914–917. https://doi.org/10.7196/samj.6228 govender np, chetty v, spencer d, et al., editors. cryptococcal screening in gauteng province, south africa: update from the first year of implenation, 2012–2013. xxi international aids conference; 2016 july; durban. adeyemi bo, ross a. profile and acute mortality outcome of patients admitted with cryptococcal meningitis to an urban district hospital in kwazulu-natal, south africa. s afr fam pract. 2015;57(2):131–135. https://doi.org/10.1080/20786190.2014.976962 lightowler jv, cooke gs, mutevedzi p, lessells rj, newell ml, dedicoat m. treatment of cryptococcal meningitis in kwazulu-natal, south africa. plos one. 2010;5(1):e8630. https://doi.org/10.1371/journal.pone.0008630 national institute for communicable diseases (nicd). germs-sa annual report [homepage on the internet]. johannesburg, south africa: national institute for communicable diseases (nicd); 2015 [cited 2017 january 2]. available from http://www.nicd.ac.za/assets/files/2015%20germs-sa%20ar.pdf the world bank. analysis of big data for better targeting of art adherence strategies: spatial clustering analysis of viral load suppression by south african province, district, sub-district and facility [homepage on the internet]. washington, dc: the world bank; 2015 [cited 2017 november 15]. available from http://documents.worldbank.org/curated/pt/922221474874783155/pdf/105322-revised-public-big-data-sa-report-april14-mar15-vnov16.pdf wikipedia. list of municipalities in kwazulu-natal [homepage on the internet]. wikipedia; 2017 [cited 2017 october 11]. available from https://en.wikipedia.org/wiki/list_of_municipalities_in_kwazulu-natal human sciences research council (hsrc). south african national hiv prevalence, incidence and behaviour survey [homepage on the internet]. pretoria, south africa: human sciences research council (hsrc); 2012 [cited 2017 october 11]. available from http://www.hsrc.ac.za/uploads/pagecontent/4565/sabssm%20iv%20leo%20final.pdf johnson lf, dorrington re, moolla h. progress towards the 2020 targets for hiv diagnosis and antiretroviral treatment in south africa. s afr j hiv med. 2017;18(1):a694. sherman g. eid hiv pcr results for action reports using laboratory data for postnatal follow-up of hiv-exposed infants [homepage on the internet]. national institute of communicable disease (nicd); 2017 [cited 2018 january 18]. available from http://www.saaids.co.za/presentations%20aids%202017/tuesday,%2013%20june%202017/hall%209/prof%20gayle%20sherman%20eid%20hiv%20pcr%20results%20for%20action%20reports.pdf article information authors: phoebe nzombe1 elizabeth t. luman2 edwin shumba1 douglas mangwanya3 raiva simbi3 peter h. kilmarx4 sibongile n. zimuto1 affiliations: 1zimbabwe national quality assurance programme (zinqap) trust, zimbabwe 2us centers for disease control and prevention (cdc), united states 3ministry of health and child welfare, zimbabwe 4us centers for disease control and prevention (cdc), zimbabwe correspondence to: phoebe nzombe postal address: zimbabwe national quality assurance programme, po box a1955, avondale, harare, zimbabwe dates: received: 05 sept. 2014 accepted: 15 sept. 2014 published: 03 nov. 2014 how to cite this article: nzombe p, luman et, shumba e, et al. maximising mentorship: variations in laboratory mentorship models implemented in zimbabwe. afr j lab med. 2014;3(2), art. #241, 8 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.241 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. maximising mentorship: variations in laboratory mentorship models implemented in zimbabwe in this original research... open access • abstract • introduction • research methods and design    • mentor recruitment and training    • mentorship models       • model 1: laboratory manager mentorship after slmta       • model 2: one week per month mentorship after slmta       • model 3: cyclical embedded mentorship after slmta       • model 4: cyclical embedded mentorship incorporated with slmta    • audits    • data analysis    • cost analysis • results    • cost of the mentorship models • discussion    • model 1: laboratory manager mentorship after slmta    • model 2: one week per month mentorship after slmta    • model 3: cyclical embedded mentorship after slmta    • model 4: cyclical embedded mentorship incorporated with slmta    • limitations of the study    • conclusion • acknowledgements    • competing interests    • authors’ contributions    • cdc disclaimer • references abstract top ↑ background: laboratory mentorship has proven to be an effective tool in building capacity and assisting laboratories in establishing quality management systems. the zimbabwean ministry of health and child welfare implemented four mentorship models in 19 laboratories in conjunction with the strengthening laboratory management toward accreditation (slmta) programme. objectives: this study outlines how the different models were implemented, cost involved per model and results achieved. methods: eleven of the laboratories had been trained previously in slmta (cohort i). they were assigned to one of three mentorship models based on programmatic considerations: laboratory manager mentorship (model 1, four laboratories); one week per month mentorship (model 2, four laboratories); and cyclical embedded mentorship (model 3, three laboratories). the remaining eight laboratories (cohort ii) were enrolled in cyclical embedded mentorship incorporated with slmta training (model 4). progress was evaluated using a standardised audit checklist. results: at slmta baseline, model 1–3 laboratories had a median score of 30%. after slmta, at mentorship baseline, they had a median score of 54%. at the post-mentorship audit they reached a median score of 75%. each of the three mentorship models for cohort i had similar median improvements from preto post-mentorship (17 percentage points for model 1, 23 for model 2 and 25 for model 3; p > 0.10 for each comparison). the eight model 4 laboratories had a median baseline score of 24%; after mentorship, their median score increased to 63%. median improvements from pre-slmta to post-mentorship were similar for all four models. conclusion: several mentorship models can be considered by countries depending on the available resources for their accreditation implementation plan. introduction top ↑ since its inception in 1980, the zimbabwean ministry of health and child welfare (mohcw) has been working to develop and manage the country’s healthcare delivery system, which faces challenges common to other healthcare systems in sub-saharan africa.1 zimbabwe’s situation was worsened by the effects of an economic crisis,2 which resulted in the exodus of highly-skilled health professionals to more stable countries, reduced funding for healthcare programmes and an inconsistent supply of basic resources.3 the zimbabwe national quality assurance programme (zinqap) is a non-profit organisation established in 1994 with a mandate to assist medical laboratories to attain and maintain a high standard of quality in their work. because high-grade laboratory services are the cornerstone of a well-functioning health delivery system, in 2010 the mohcw earmarked laboratory services for strengthening, developed a national laboratory strategic plan and embarked on implementation of quality management systems (qms). a cooperative agreement was signed between zinqap and the us centers for disease control and prevention (cdc) to assist the mohcw in the expansion and strengthening of laboratory technical capacity and quality nationwide. the strengthening laboratory management toward accreditation (slmta) programme was launched in 2009 by cdc and the world health organization’s regional office for africa (who afro) as a training curriculum for achieving immediate, measurable improvement in laboratories in resource-limited settings, using available resources.4 mohcw and zinqap adopted slmta as the tool to establish qms in zimbabwean laboratories. in january 2010, zinqap conducted baseline audits in 20 laboratories which included all the reference, central and provincial laboratories as well as some private laboratories. from these 20 laboratories, 11 were selected for the initial slmta cohort, based on their level in the tiered laboratory system, baseline audit scores, staff availability and geographic location. these 11 laboratories included reference (n = 2), central hospital (n = 4), private (n = 2), provincial (n = 2) and city council (n = 1) laboratories. the laboratory manager and the quality officer from each of the 11 laboratories were trained in the slmta three-workshop series. improvement projects were assigned to be completed in their laboratories following each workshop, and exit audits were conducted after the final workshop so as to determine the impact of the intervention. the entire process from baseline audit to exit audit lasted 22 months. despite marked improvements, laboratory managers believed that they could achieve greater results if they received assistance in setting up their qms for longer periods of time from a laboratory professional who was more experienced in qms establishment and implementation. reports from other countries such as lesotho and cameroon suggested that mentorship coupled with the slmta programme was an effective tool for achieving improvement in quality systems.5,6 the mohcw thus decided to implement a mentorship programme in the laboratories that had completed the slmta programme in an effort to continue improving quality management. several mentorship models were implemented based on available funds, resources and staff allocation. in march 2012, eight more laboratories were recruited in a second round of slmta (six district-level and two provincial); mentorship was incorporated into the programme for this cohort. this study examines the results achieved by the 19 laboratories after implementing four different mentorship models in order to determine their effectiveness, relative cost and lessons learnt. research methods and design top ↑ mentor recruitment and training zinqap established a training and mentorship department, which was mandated to assist medical laboratories in setting up quality systems for improved patient care and to help them work towards accreditation. zinqap was responsible for implementing the slmta and laboratory mentorship programmes with funding from the us president’s emergency plan for aids relief (pepfar). the department recruited five local mentors who were qualified medical laboratory scientists with a four-year university degree and who had at least five years’ experience working in a routine medical laboratory as well as some experience in implementing laboratory qms. a lead mentor, who was a slmta master trainer, was hired to coordinate the programme and to ensure that the mentors fulfilled their assignments. after recruitment, the mentors attended three training courses designed to equip them with the knowledge and skills needed to mentor laboratories in establishing qms: international organization for standardization (iso) 15189 requirements and internal auditing; slmta training-of-trainers; and mentorship (figure 1). figure 1: slmta and mentorship implementation in zimbabwe. mentorship models laboratories were assigned to one of four mentorship models. assignment was purposive, based on geographic location, performance of the laboratory managers, laboratory type and timing of slmta implementation. mentorship took place from march to october 2012 for all four models. model 1: laboratory manager mentorship after slmta the four cohort i laboratories with the strongest laboratory managers based on performance during slmta implementation were selected for mentorship model 1. these laboratory managers were trained in the three previously-described training courses. they were then tasked to mentor their own laboratory staff members toward accreditation. zinqap mentors were available to assist in the development of work plans and to provide clarification as needed during the scheduled monthly support visits, which lasted at least one full day each month, but were extended if the laboratory manager needed more assistance. model 2: one week per month mentorship after slmta the remaining private, reference and central laboratories from cohort i were assigned to mentorship model 2. one zinqap mentor was responsible for assisting these four laboratories and was on site in each laboratory for one week of every month. at the end of each mentorship week staff members were assigned tasks to complete during the three weeks of the mentor’s absence. model 3: cyclical embedded mentorship after slmta the three provincial laboratories in cohort i were each mentored by an embedded zinqap mentor who was based at the laboratory for six weeks, away for eight weeks and then back for another four weeks. the mentor assigned tasks for laboratory staff to complete between visits. model 4: cyclical embedded mentorship incorporated with slmta the eight laboratories in the second slmta cohort had mentorship combined with the slmta training. each mentor from model 3 above worked with two of the six district-level model 4 laboratories in their province in a cyclical manner. in cycle 1, after working with the provincial laboratory from model 3, these mentors then relocated to the first model 4 district laboratory for four weeks followed by the second district laboratory for another four weeks. more time was spent at the model 3 provincial laboratories because they have more sections and staff members than the district laboratories. in cycle 2, all laboratories were mentored for four weeks each. two new provincial laboratories were also added in cohort ii. these were mentored by an additional zinqap mentor who worked in each laboratory in the same cyclical manner as model 3 above. for cohort ii, slmta was delivered in a continuous, decentralised format, rather than in workshops. the mentors, who were also slmta trainers, conducted the first half of the slmta curriculum in cycle 1 and the second half in cycle 2. at least one activity was conducted each day and was attended by all laboratory staff, including general and administrative staff, in order to encourage cooperation. all activities were conducted in relation to the work plan. improvement projects, which emanated from specific activities, were assigned to staff responsible for that scope of work immediately after the daily session. for example, after conducting the ‘what is wrong with the storeroom?’ activity, the stores person was assigned to put the storeroom in order, with the mentor showing them how. other improvement projects, such as writing of procedures, were conducted as ongoing projects, continuing even after the mentor had left the laboratory. audits the cohort i baseline audits were conducted in january 2010 using the initial 2009 version of the who afro laboratory accreditation checklist. in 2012, the checklist was revised slightly and renamed the who afro stepwise laboratory quality improvement process towards accreditation (slipta) checklist,7 which was used for all subsequent audits. based on these audits, a laboratory’s progress is evaluated using a zero to five star rating, whereby 0% – 54% = zero stars, 55% – 64% = one star, 65% – 74% = two stars, 75% – 84% = three stars, 85% – 94% = four stars and 95% – 100% = five stars. for the 11 laboratories in cohort i, the slmta exit audit conducted in october 2011 was used as the baseline for assessing the impact of mentorship. the eight laboratories in cohort ii had their baseline audits conducted in march 2012. the post-mentorship audit to assess the mentorship programme in all 19 laboratories was conducted in october 2012 by two independent who-trained auditors from organisations based in uganda and botswana. data analysis differences between audit scores were calculated and tested using the student’s t-test with a significance level of p = 0.05. cost analysis a descriptive retrospective analysis evaluated the financial costs (cash expenditures) of implementing each of the four mentorship models. this was done through the use of an inventory of all resources and payments made for the programme.8 costs calculated included: the three training courses which were attended by the mentors; mentorship costs such as salaries, travel and lodging, internet access and laptops; and supervisory costs (table 1). this analysis was limited to the direct costs of providing mentorship and supervision only. other components of slmta implementation, such as slmta workshops, improvement projects and staff time needed to complete the programme were not included, nor was opportunity cost of the reduced time available for regular duties of the laboratory manager in model 1. costs were calculated on a per mentor basis, as well as per laboratory, factoring in the number of laboratories assigned to each mentor. amounts are reported in us dollars, which was the official currency of zimbabwe at the time of the study. table 1: cost of implementing mentorship in conjunction with strengthening laboratory medicine toward accreditation (slmta) programme for four mentorship models in zimbabwe. results top ↑ prior to implementing the slmta programme, cohort i laboratories had a median baseline score of 30% and none of the 11 laboratories had attained scores high enough to reach a one-star rating (figure 2). after slmta implementation, the laboratories improved to reach a median score of 54% and five of the laboratories attained at least one star. after the post-slmta mentorship programme the laboratories reached a median score of 75%, with two laboratories at four stars, five laboratories at three stars and four laboratories at two stars. each of the three mentorship models for cohort i (models 1–3) had similar improvements from preto post-mentorship, with a median improvement of 17 percentage points for model 1, 23 percentage points for model 2 and 25 percentage points for model 3 (p > 0.10 for each comparison). the eight cohort ii laboratories (model 4), which received mentorship along with on-site slmta training, had a median baseline score of 24%, with no laboratories reaching one star. after slmta/mentorship, one of the laboratories reached three stars, three laboratories reached two stars, three laboratories reached one star and the remaining laboratory was just one point away from achieving one star. the median scores of the laboratories increased 39 percentage points to 63%. these improvements were greater than those observed from the cohort i laboratories (models 1–3) at their slmta exit audit (median improvement 24 percentage points; p = 0.01), but similar to cohort i improvements from pre-slmta baseline to post-mentorship (median improvement 53 percentage points for model 1, 34 percentage points for model 2 and 40 percentage points for model 3; p > 0.10 for each comparison). figure 2: comparison of results based on four mentorship models implemented in zimbabwe. cost of the mentorship models the total cost per mentor for attending the three courses was $5486 (table 1). the other costs of mentorship varied by model, with total costs per mentor of $5486 for model 1, $19 042 for model 2 and $27 410 for models 3 and 4. after factoring in the number of laboratories assigned per mentor and the supervisory cost, the costs per laboratory ranged from $4761 for model 2 to $9137 for models 3 and 4. discussion top ↑ unlike other studies that have examined the combined effect of mentorship and slmta,9,10,11 the delayed implementation of mentorship in zimbabwe allowed us to evaluate the incremental impact of mentorship separate from slmta training. cohort i laboratories registered substantial improvement during slmta implementation, from a median baseline of 30% to a median exit audit of 54%. they then improved even further, to a median of 75% after mentorship. whilst it is not possible with our data to determine what the impact of mentorship would have been without first implementing the slmta programme, it is clear from these results that the addition of mentorship had a beneficial effect on the laboratories over and above the effect of slmta training alone. cohort ii, which implemented mentorship alongside slmta training, saw a similar total improvement, from 24% to 63%, suggesting that whether performed sequentially or simultaneously, mentorship can play an effective role in laboratory quality improvement. one laboratory in cohort i had regressed from slmta baseline to exit score; the two slmta participants reported that they were unsuccessful in getting the other laboratory personnel to work as a team in order to implement the improvement projects. after mentorship, their laboratory’s audit score more than doubled; mentorship was successful because all laboratory staff were mentored on site, so they all understood the process and were eager to play their part. the mentorship models showed similar improvements of approximately 17–25 percentage points for mentorship only and 39 percentage points for mentorship combined with slmta. we were not able to conclude that one model was better than the others – in part because of the small sample sizes, which limited statistical power, but also because laboratories were not assigned randomly to mentorship models but were selected based on the judgement of the programme leadership team. characteristics of the laboratories that were used to assign mentorship model (such as geographic location and effectiveness of laboratory managers) may also have affected laboratory performance. however, there were important lessons learned about the advantages and disadvantages of each model, as well as the situations in which they should be used (table 2). table 2: implementation of mentorship models in conjunction with the strengthening laboratory management toward accreditation (slmta) programme in zimbabwe. model 1: laboratory manager mentorship after slmta this model, which utilised existing laboratory managers as mentors, was targeted to laboratories which had excelled in slmta implementation and whose laboratory managers were strong and committed to implementing a qms. because of their established authoritative role as laboratory managers, these mentors were able to enforce the new quality culture in the laboratory. also, because they were full-time laboratory employees, they were able to provide mentorship on a continuous basis throughout the programme. perhaps the greatest advantage of this model is that it builds capacity at the grass-roots level, as these laboratories will benefit from having trained mentor–managers long after the programme has ended. on the other hand, these mentors had simultaneous management duties, including analysing patient results, attending hospital management meetings, representing the laboratory at regional and national meetings and authorising qms documents. whilst some of these duties could be delegated to deputies, these mentors were not able to devote all of their time to mentorship. this model may be best suited for situations in which there are strong laboratory managers who are supportive of setting up a qms and who can handle both mentorship and administrative duties. model 2: one week per month mentorship after slmta this model was implemented in four laboratories which were located within a 10 km radius of the mentor’s residence. the mentor worked with each laboratory for one week at a time, assigning tasks to be completed in the three weeks he was away. one benefit of this model is that it utilises full-time professional mentors, allowing them to focus completely on mentorship duties. also, laboratories have mentorship guidance each month, ensuring continuous progress. this was the least expensive mentorship model. this model is recommended for laboratories which are in close proximity to a qualified mentor who can commute daily to the laboratories. model 3: cyclical embedded mentorship after slmta the laboratories enrolled under this model were located in different provinces, requiring extensive travel for mentors. the main benefit of this model was that the laboratories had full-time mentors on site for long periods of time, allowing in-depth training and oversight of major improvement projects. however, they were also without their mentors for eight weeks between visits; problems arising may not have been identified and corrected for as long as two months. also, travel and lodging expenses were relatively expensive, as the greater distances required mentors to travel and lodge on site. this model is most appropriate for more remote laboratories needing long-term intensive mentorship in order to make substantive changes. model 4: cyclical embedded mentorship incorporated with slmta for our study, it was not possible to separate the effects of mentorship and of slmta training for model 4, as they were conducted simultaneously. standard slmta implementation includes mentorship between training sessions.4 several countries have utilised the embedded mentorship model incorporated with slmta training, with good results.11,12,13,14 our slmta training for cohort ii was conducted in a decentralised model rather than in centralised workshops; one study in cameroon also found decentralised training to be effective.12 in general, the strengths and weaknesses of model 4 are the same as those discussed for model 3. in addition, there are benefits associated with conducting concurrent mentorship and slmta training. logistically, it is easier to coordinate a single united programme, with everyone’s focus on laboratory quality. also, mentors worked through issues discussed at the previous training session, immediately reinforcing lessons learned in the classroom and assisting in translating those lessons to real-world problems in the laboratory. and because mentors are also trainers, there may be some cost sharing between the training and mentorship components of the programme, which was not evaluated in this study. limitations of the study this was a descriptive study based on a programmatic activity and, as such, is subject to several scientific limitations. firstly, the small number of laboratories in each mentorship model yielded low power for statistical testing, so the findings from the 19 study laboratories cannot be generalised to the > 200 laboratories nationwide. in addition, both absolute and relative costs will vary by setting based on contextual factors. for example, if the cost of mentor salaries or travel were substantially higher than in our programme, then model 1, which uses existing laboratory managers as mentors, could prove to be the least expensive option. secondly, representative sampling and randomised assignment to mentorship models were not done; laboratories were selected purposively based on convenience, programmatic needs and results of baseline audits. thirdly, model 4 results cannot be easily compared with the other three models. in our implementation, model 4 was used for the second cohort of slmta; these district and provincial laboratories had substantially lower baseline scores than did those in cohort i, which were predominantly central and referral laboratories. thus there was more room for improvement, as laboratories with lower baseline scores tend to have greater score increases than those with higher baseline scores.15 in addition, cohort ii laboratories were generally smaller, with fewer departments and staff members and offering a narrower menu of tests. these factors are advantageous in implementing qms because the mentor has fewer departments to work with; some tasks, such as writing standard operating procedures, are less complicated; and lower staff numbers promote easier team building. furthermore, lessons learned from the first cohort of slmta were incorporated into the second cohort, giving an additional advantage. on the other hand, the laboratories in cohort i had 34 months over which to make improvements, whilst those in cohort ii were measured after only seven months. conclusion based on our findings, we would recommend that countries include mentorship when implementing slmta for laboratory quality improvement. countries should carefully consider which mentorship model or models would be best suited to their individual situation. acknowledgements top ↑ the authors would like to acknowledge the laboratory managers and staff of the 19 laboratories which were enrolled in the slmta programme in zimbabwe for their commitment to implementing qms. this work was made possible through funding from pepfar and technical assistance from cdc’s zimbabwe office. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions p.n. (zinqap) implemented the programme and wrote the manuscript. e.t.l. (cdc, atlanta) assisted with analysis and interpretation of the data and provided substantial input to the writing of the manuscript. e.s. (zinqap) contributed to programme implementation and revision of the manuscript. d.m. (mohcw) provided high-level strategic direction for programme implementation and manuscript writing. r.s. (mohcw) provided high-level strategic direction for programme implementation and manuscript writing. p.h.k. (cdc, zimbabwe) provided high-level strategic direction for programme implementation and manuscript writing. s.n.z. (zinqap) was responsible for the overall oversight in project implementation and reviewed the manuscript. cdc disclaimer the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the cdc or the other organisations for which they work. references top ↑ 1.habte d, dussault g, dovlo d. challenges confronting the health workforce in sub-saharan africa. world hosp health serv. 2004;40(2):23–26, 40–41. 2.kapp, c. world report: zimbabwe’s humanitarian crisis worsens. lancet. 2009;373(9662):447. http://dx.doi.org/10.1016/s0140-6736(09)60151-3 3.truscott r. zimbabwe’s health challenges. bmj. 2009;338:b930. http://dx.doi.org/10.1136/bmj.b930 4.yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin pathol. 2010;134(3):401–409. http://dx.doi.org/10.1309/ajcpnbbl53fwuiqj 5.maruta t, rotz p, trevor p. setting up a structured laboratory mentoring programme. afr j lab med. 2013;2(1), art. #77, 7 pages. http://dx.doi.org/10.4102/ajlm.v2i1.77 6.maruta t, motebang d, mathabo l, et al. impact of mentorship on who-afro strengthening laboratory quality improvement process towards accreditation (slipta). afr j lab med. 2012;1(1), art. #6, 8 pages. http://dx.doi.org/10.4102/ajlm.v1i1.6 7.world health organization’s regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation in the african region (with checklist) [document on the internet]. c2012 [cited 2014 september 21]. available from: http://www.afro.who.int/en/clusters-a-programmes/hss/blood-safety-laboratories-a-health-technology/blt-highlights/3859-who-guide-for-the-stepwise-laboratory-improvement-process-towards-accreditation-in-the-african-region-with-checklist.html 8.shumba e, nzombe p, mbinda a, et al. weighing the costs: implementing the slmta programme in zimbabwe using internal versus external facilitators. afr j lab med. 2014;3(2), art. #248, 6 pages. http://dx.doi.org/10.4102/ajlm.v3i2.248 9.makokha ep, mwalili s, basiye fl, et al. using standard and institutional mentorship models to implement slmta in kenya. afr j lab med. 2014;3(2), art. #220, 8 pages. http://dx.doi.org/10.4102/ajlm.v3i2.220 10.mokobela ko, moatshe mt, modukanele m. accelerating the spread of laboratory quality improvement efforts in botswana. afr j lab med. 2014;3(2), art. #207, 6 pages. http://dx.doi.org/10.4102/ajlm.v3i2.207 11.audu ra, onubogu cc, nwokoye nn, et al. improving quality in national reference laboratories: the role of slmta and mentorship. afr j lab med. 2014;3(2), art. #200, 7 pages. http://dx.doi.org/10.4102/ajlm.v3i2.200 12.ndasi j, dimite l, mbome v, et al. decentralised facility-based training as an alternative model for slmta implementation: the cameroon experience. afr j lab med. 2014;3(2), art. #231, 6 pages. http://dx.doi.org/10.4102/ajlm.v3i2.231 13.nkwawir sc, batumani nn, maruta t, awasom cn. from grass to grace: how slmta revolutionised the bamenda regional hospital laboratory in cameroon. afr j lab med. 2014;3(2), art. #203, 6 pages. http://dx.doi.org/10.4102/ajlm.v3i2.203 14.guevara g, gordon f, irving y, et al. the impact of slmta in improving laboratory quality systems in the caribbean region. afr j lab med. 2014;3(2), art. #199, 9 pages. http://dx.doi.org/10.4102/ajlm.v3i2.199 15.yao k, luman et, slmta collaborating authors. evidence from 617 laboratories in 47 countries for slmta-driven improvement in quality management systems. afr j lab med. 2014;3(2), art. #262, 11 pages. http://dx.doi.org/10.4102/ajlm.v3i2.262 article information authors: elise schapkaitz1 dashini pillay2 affiliations: 1department of molecular medicine and haematology, charlotte maxeke johannesburg academic hospital, national health laboratory system complex and university of witwatersrand, south africa 2department of haematology, national health laboratory services and university of kwazulunatal, south africa correspondence to: elise schapkaitz email: elise.schapkaitz@nhls.ac.za postal address: po box 28985, sandringham 2131, south africa dates: received: 27 june 2014 accepted: 30 june 2015 published: 31 aug. 2015 how to cite this article: schapkaitz e., pillay d. prolonged storage-induced changes in haematology parameters referred for testing. afr j lab med. 2015;4(1), art. #208, 8 pages. http://dx.doi.org/10.4102/ajlm.v4i1.208 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. prolonged storage-induced changes in haematology parameters referred for testing in this original research... open access • abstract • introduction • research method and design    • ethical considerations    • materials and setting    • design       • blood sampling and laboratory methods       • laboratory testing       • analyses • results    • storage at room temperature    • storage at 4 °c – 8 °c • discussion • limitations of the study • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: referral of samples for the work-up of haematological disorders from remote laboratories can result in a delay in analysis. objective: the stability of the full blood count (fbc), differential count (diff), reticulocyte and peripheral blood smear (pbs) morphology during extended storage was evaluated. methods: forty blood samples stored in ethylenediaminetetraacetic acid (edta) were analysed on an advia® 120 haematology analyser. the samples (25% abnormal; 75% normal) were stored at room temperature (rt) and at 4 °c – 8 °c. analysis of samples stored at rt was performed every 12 hours for two days. analysis of samples stored at 4 °c – 8 °c was performed at 12 hours and subsequently every 24 hours for seven days. results: fbc parameters (red cell count, haemoglobin) and diff parameters (percentages of basophils, lymphocytes and monocytes) were stable for at least 48 hours when stored at rt. platelets were only stable for 12 hours and the white cell count was stable for 36 hours when stored at rt. storing samples at 4 °c – 8 °c significantly increased the stability of most parameters, in particular, mean cell volume and percentage of reticulocytes. however, diff parameters were associated with lower stability at 4 °c – 8 °c. pbs morphology was compromised prior to 12 hours whether stored at rt or at 4 °c – 8 °c. conclusion: this study provides evidence that blood samples stored in edta at 4 °c – 8 °c for seven days are suitable for testing on the advia® 120 analyser for the fbc and percentage of reticulocyte parameters. however, storage at 4 °c – 8 °c is not a solution for samples referred for diff and pbs morphology review. introduction top ↑ pre-analytical variables, such as storage time and temperature, affect the measurement of laboratory parameters collected in ethylenediaminetetraacetic acid (edta).1,2 laboratory staff need to be aware of the changes that occur during storage in their specific setting in order to decide whether to accept or reject samples that are too old to obtain reliable results. accurate measurement of full blood count (fbc), differential count (diff) and reticulocyte parameters, as well as peripheral blood smear (pbs) morphology, are essential for the correct interpretation of haematology results. it is recommended that traditional fbc parameters such as red cell count (rcc), white cell count (wcc), haemoglobin and platelet count be analysed 24 hours after sample collection when stored at room temperature (rt).3,4,5 however, parameters useful for diagnosis and monitoring of haematological disorders, such as mean cell volume (mcv), reticulocyte and pbs morphology, are unreliable after 12 hours.5 osmotic swelling of red cells during storage at rt affects volume-dependant variables and results in misclassification of a microcytic anaemia as normocytic and, similarly, a normocytic anaemia as macrocytic.6 reticulocytes mature into red cells after 24 hours in circulation. the clinical and laboratory standards institute (clsi) recommends that samples stored at rt should be analysed for reticulocytes within six hours of collection.7 it is further recommended that pbs for morphologic analysis be prepared within four hours, prior to the onset of edta-induced changes in red and white cell morphology.8,9,10 with centralisation of laboratory services, it is not always feasible to meet these deadlines. large academic laboratories are commonly faced with the scenario where a sample collected on friday is not received in the laboratory for analysis until monday morning. recent studies indicate that longer storage durations are acceptable when samples are stored at 4 °c – 8 °c.3,5,6,11,12 however, information on stability beyond 72 hours is limited.6 furthermore, these studies are small, specific to the haematology analyser, and the definition of stability used is not standardised. the aim of this study was to evaluate the stability of the fbc, diff, reticulocyte and pbs morphology during extended storage at rt and at 4 °c – 8 °c in order to determine laboratory criteria for storage time and temperature for specimens referred for the work-up of haematological disorders from remote laboratories. research method and design top ↑ ethical considerations the study was approved by the human research ethics committee of the university of the witwatersrand (m090688). all tests were done as part of routine diagnostic workups and no additional blood samples were taken from the participants for this study. materials and setting this study was conducted at the main haematology laboratory of the charlotte maxeke johannesburg academic hospital (cmjah), national health laboratory service complex, johannesburg, south africa. forty blood samples, representative of the patient population (25% abnormal and 75% normal specimens), that were left over after routine testing were selected from the haematology workload. only samples collected in edta (becton-dickinson, oxford, united kingdom) vials with adequate volume (> 4 ml) received within two hours of collection were included. samples with results that indicated partial aspiration were excluded from the final analysis. design blood sampling and laboratory methods blood samples for evaluation of fbc, diff and reticulocyte parameters were collected in k2edta tubes (1.5–2.2 mg of dipotassium edta dihydrate per millilitre of blood). the parameters were analysed with the laboratory's advia® 120 automated haematology analyser (siemens healthcare diagnostics, inc, tarrytown, new york, united states). cells were counted and sized by light scatter technology using white light for white cells and laser light for red cells and platelets. haemoglobin was measured by the conventional cyanmethaemoglobin method. the six-part differential analysis was performed in two channels. cells in the peroxidase channel were measured by peroxidase staining intensity and light scatter. cells in the basophil/lobularity channel were measured by dual laser light scatter, nuclear density and lobulation index. reticulocytes were stained with oxazine 750. the films were spread on the advia® autoslide slide maker and stained on the hema-tek® 2000 slide stainer (siemens healthcare diagnostics inc, tarrytown, new york, united states). laboratory testing the samples were analysed within two hours of collection (time zero) at rt. samples were aliquoted into two sets; one was stored at rt (18 °c – 24 °c) and and the other at 4 °c – 8 °c. analyses of samples stored at rt were performed after 12, 24, 36 and 48 hours of storage. analyses of samples stored at at 4 °c – 8 °c were performed after 12, 24, 36, 48, 72, 96, 120, 144 and 168 hours of storage. a manual diff was performed on pbs of five samples stored at rt and at 4 °c – 8 °c. reviews were performed at 12, 24, 36 and 48 hours. the pbs was first examined for the presence of edta-induced changes, including red cell spherocytes, echinocytes, sphero-echinocytes, increased rouleaux formation, degeneration of neutrophils and lobulation of lymphocyte nuclei,10 because these changes preclude an accurate manual diff. analyses data were captured from the analyser printouts on excel™ spreadsheets (microsoft office excel™ 2007, redmond, washington, united states) and analysed using statistica 9.1 software (statsoft, tulsa, oklahoma, united states). the mean percentage difference from the value at time zero was calculated and tabulated.13 stability of a parameter was defined in relation to the precision of the advia® 120 analytical method. acceptable limits were defined in accordance with the royal college of pathologists of australasia external quality assurance annual review for 2013.14 the coefficients of variation (% cv) for the parameters were as follows: wcc 3.4%, rcc 1.8%, haemoglobin 1.8%, haematocrit 2.4%, platelet count 2.4% and percentages of neutrophils 0.9%, lymphocytes 4.9%, monocytes 5.6%, eosinophils 16.7%, basophils 55% and reticulocytes 8.1%. a parameter was considered stable, when its difference was smaller than 1% cv for the assessed method.5 results top ↑ storage at room temperature the wcc was stable until 36 hours after collection and showed a significant decrease at 48 hours after collection (figure 1). a significant increase in the percentages of neutrophils and eosinophils was observed at 24 hours and 36 hours, respectively. red cell parameters including rcc, haemoglobin, mean cell haemoglobin (mch) and red cell distribution width (rdw) were stable for at least 48 hours after collection when stored at rt and were not significantly affected by storage temperature. in contrast, other rcc measurements, including haematocrit, mcv, mean cell haemoglobin content (mchc) and the percentage of reticulocytes, were not stable after storage at rt for 48 hours after collection. after rt storage for 24 hours, a significant increase in mcv, as well as a decrease in the percentage of reticulocytes, was observed. analysis of platelet stability showed platelets were stable for 12 hours and significantly decreased at 24 hours after collection. at rt storage, the stability of the mean platelet volume (mpv) was less than 12 hours as a result of artificial platelet swelling. at rt storage, the stability of the manual diff was also less than 12 hours. the percentages of neutrophils and monocytes showed significant increases, whereas percentages of lymphocytes and eosinophils showed significant decreases at 12 hours after collection. the slides examined contained too few basophils to obtain reliable results for basophil stability. edta-induced changes were noted at 24 hours after collection, which precluded a manual diff. figure 1: stability analysis (mean percent difference) at room temperature (18 °c – 24 °c) storage at 4 °c – 8 °c the wcc was stable at 4 °c – 8 °c until 48 hours after collection (figure 2). a significant decrease in the percentage of neutrophils was observed at 72 hours after collection. the percentages of eosinophils, basophils and monocytes were not stable when stored at 4 °c – 8 °c and showed significant increases at 12, 24 and 48 hours, respectively. compared with rt storage, we observed improved stability of rcc parameters when stored at 4 °c – 8 °c. haematocrit, mcv and mchc were stable until 168 hours when stored at 4 °c – 8 °c. the percentage of reticulocytes was stable until 120 hours after collection and showed significant decreases thereafter. platelets were stable until 96 hours after collection when stored at 4 °c – 8 °c. mpv showed a significant increase at 24 hours after collection. as a result of the presence of edta-induced changes prior to 12 hours after collection, a manual diff could not be performed. figure 2: stability analysis (mean percent difference) at 4 °c – 8 °c. discussion top ↑ routine tests such as the fbc, diff, reticulocyte and pbs morphology are commonly referred to the cmjah haematology laboratory as part of the diagnostic work-up for haematological disorders. in large academic laboratories, where aged samples make up a significant proportion of the workload, the storage time and temperature of samples must be taken into consideration. the findings of this study performed on edta samples add to the evidence that stability varies according to storage time and temperature. according to the findings of this study, fbc parameters, namely rcc, haemoglobin, mch and rdw, and diff parameters, namely percentages of basophils, lymphocytes and monocytes, were least affected by storage temperature and time and can be analysed until 48 hours after sample collection when stored at rt. it is recommended that traditional fbc parameters be analysed 24 hours after sample collection when stored at rt.3,4,5 however, in this study, platelets were only stable until 12 hours after collection when stored at rt. the stability of the wcc was also found to be shorter than other studies, which have recommended analysis up to 48 hours after collection when stored at rt.5,11,15 in this study, the wcc was stable only until 36 hours after collection when stored at rt. in this study, the mcv was stable until 12 hours after collection. imeri et al. found that mcv increased significantly after 4–10 hours, regardless of the haematology analyser;5 whereas other studies have indicated a longer stability of up to 24 hours for mcv at rt.3,11,15 these discordant results may be attributed to the different statistical methods used in these studies for the evaluation of stability, which limits accurate comparison. in this study, the percentage of reticulocytes was stable at rt until 12 hours after collection, which concurs with current recommendations.3,5 however, the stability of reticulocyte parameters has been found to be longer at rt on other haematology analysers, such as the coulter® lh 750, sysmex xe-2100™ and cell-dyn sapphire™.5,11 according to wiegand et al., the oxazine 750 on the advia® haematology analysers may not be sufficient to detect more mature reticulocytes.16 imeri et al. conducted a three-way comparison study of the coulter® lh750, sysmex xe-2100™ and advia® haematology analysers that further illustrated how the stability of many haematology parameters depends on the analytical method used.5 thus, the findings of this study are specific to advia® haematology analysers, which currently represent 60% of haematology analysers in south africa. therefore, the findings of this study have widespread local implications. however, clsi do recommend that ‘laboratories should assess fbc stability in their specific settings’.17 storage of samples at 4 °c – 8 °c for seven days increased the stability of most parameters. fbc parameters, namely wcc, platelet count, haematocrit, mcv and mchc, as well as diff parameters, namely percentages of neutrophils and reticulocytes, were more stable when stored at 4 °c – 8 °c. however, some diff parameters, namely percentages of eosinophils, basophils and monocytes, had lower stability. changes were present on pbs morphology prior to 12 hours after collection when stored at either rt or at 4 °c – 8 °c. this precluded assessment of dysplastic morphological features. it is currently recommended that pbs be prepared within a few hours for assessment of haematological disorders, in particular for the presence of dysplastic features.9,10,18 limitations of the study top ↑ a limitation of this study is that pbs morphology was not evaluated prior to 12 hours after collection (i.e., at four and six hours). as such, a manual differential could not be performed. furthermore, this study was performed under optimal conditions on inpatient samples that were received in the laboratory within two hours of collection. samples referred for testing are often subject to variation in temperature during collection and transport. conclusion top ↑ in conclusion, this study provides evidence regarding the viability of blood samples collected in edta vials and stored at rt and at 4 °c – 8 °c. samples that have been stored at 4 °c – 8 °c for seven days are suitable for testing on the advia® 120 analyser for fbc and reticulocyte parameters. however, this is not a solution for samples referred for diff or pbs morphology review. acknowledgements top ↑ we thank celeste mcpherson and the laboratory staff of the cmjah national health laboratory service complex for their technical assistance. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions e.s. (charlotte maxeke johannesburg academic hospital) was the author of the manuscript and also performed data entry and analysis. d.p. (national health laboratory services, university of kwazulu-natal) was responsible for data collection, data entry and manuscript review. references top ↑ queen e, ifeanyi oe, chinedum ok. the effect of storage on full blood count in different anticoagulant. iosr jdms. 2014;3(9):128–131. http://dx.doi.org/10.9790/0853-1392128131 guder wg. preanalytical factors and their influence on analytical quality specifications. scand j clin lab invest. 1999;59(7):545–549. http://dx.doi.org/10.1080/00365519950185328 bourner g, dhaliwal j, sumner j. performance evaluation of the latest fully automated hematology analyzers in a large, commercial laboratory setting: a 4-way, side-by-side study. lab hematol. 2005;11(4):285–297. http://dx.doi.org/10.1532/lh96.05036 cohle sd, saleem a, makkaoui de. effects of storage of blood on stability of hematologic parameters. am j clin pathol. 1981;76(1):67–69. imeri f, herklotz r, risch l, et al. stability of hematological analytes depends on the hematology analyser used: a stability study with bayer advia 120, beckman coulter lh 750 and sysmex xe 2100. clin chim acta. 2008;397(1–2):68–71. http://dx.doi.org/10.1016/j.cca.2008.07.018 ashenden m, clarke a, sharpe k, et al. stability of athlete passport parameters during extended storage. int j lab hematol. 2013;35(2):183–192. http://dx.doi.org/10.1111/ijlh.12014 national committee for clinical laboratory standards. methods for reticulocyte counting (flow cytometry and supravital dyes). approved guideline. h44-a. wayne, pa: nccls; 1997. vives-corrons jl, briggs c, simon-lopez r, et al. effect of edta-anticoagulated whole blood storage on cell morphology examination. a need for standardization. int j lab hematol. 2014;36(2):222–226. http://dx.doi.org/10.1111/ijlh.12170 zini g. stability of complete blood count parameters with storage: toward defined specifications for different diagnostic applications. int j lab hematol. 2014;36(2):111–113. http://dx.doi.org/10.1111/ijlh.12181 antwi-baffour s, quao e, kyeremeh r, et al. prolong storage of blood in edta has an effect on the morphology and osmotic fragility of erythrocytes. int j biomed sci eng. 2013;1(2):20–23. http://dx.doi.org/10.11648/j.ijbse.20130102.11 hedberg p, lehto t. aging stability of complete blood count and white blood cell differential parameters analyzed by abbott cell-dyn sapphire hematology analyzer. int j lab hematol. 2009;31(1):87–96. http://dx.doi.org/10.1111/j.1751-553x.2007.01009.x lippi g, salvagno gl, solero gp, et al. stability of blood cell counts, hematologic parameters and reticulocytes indexes on the advia a120 hematologic analyzer. j lab clin med. 2005;146(6):333–340. http://dx.doi.org/10.1016/j.lab.2005.08.004 international council for standardization in haematology. guidelines for the evaluation of blood cell analysers including those used for differential leucocyte and reticulocyte counting and cell marker applications. clin lab haematol. 1994;16(2):157–174. rcpa. rcpa quality assurance programs [homepage on the internet]. n.d. [cited 2015 apr 02]. available from: http://www.rcpaqap.com.au. gulati gl, hyland lj, kocher w, et al. changes in automated complete blood cell count and differential leukocyte count results induced by storage of blood at room temperature. arch pathol lab med. 2002;126(3):336–342. wiegand g, effenberger-klein a, weber r, et al. potential pitfalls of comparative measurements of reticulocytes with flow cytometry and microscopy in prematures and infants. clin chem lab med. 2004;42(10):1150–1154. http://dx.doi.org/10.1515/cclm.2004.234 clinical and laboratory standards institute. validation, verification, and quality assurance of automated hematology analyzers, approved standard – 2nd ed. h26-a2, vol. 30(14). wayne, pa: clsi; 2010. brunning rd, orazi a, germing u, et al. myelodysplastic syndromes/neoplasms, overview. in: swerdlow sh, campo e, harris nl, et al., editors. who classification of tumours of haematopoietic and lymphoid tissues.lyon: iarc, 2008; p. 88–93.  article information authors: katy yao1 talkmore maruta2 elizabeth t. luman1 john n. nkengasong1 affiliations: 1international laboratory branch, division of global hiv/aids, us centers for disease control and prevention (cdc), atlanta, united states 2african society for laboratory medicine (aslm), addis ababa, ethiopia correspondence to: katy yao postal address: 1600 clifton road, ms: g45, atlanta, ga 30329-4018, united states dates: received: 14 may 2014 accepted: 03 july 2014 published: 16 sept. 2014 republished: 07 nov. 2014 how to cite this article: yao k, maruta t, luman et, nkengasong jn. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. 2014;3(1), art. #194, 8 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.194 note: article republished with updated references relating the special issue. copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the slmta programme: transforming the laboratory landscape in developing countries in this lessons from the field... open access • abstract • introduction • key components • variations from the basic implementation model    • cameroon    • lesotho • capacity building for programme scale-up • additional considerations    • country commitment    • site selection    • human resources • experience from africa    • mozambique – country ownership and sustainability    • rwanda – data-driven advocacy    • cameroon – expanding quality past the laboratory    • zimbabwe – overcoming contextual challenges • slmta’s global reach and influence outside africa • lessons learned • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: efficient and reliable laboratory services are essential to effective and well-functioning health systems. laboratory managers play a critical role in ensuring the quality and timeliness of these services. however, few laboratory management programmes focus on the competencies required for the daily operations of a laboratory in resource-limited settings. this report provides a detailed description of an innovative laboratory management training tool called strengthening laboratory management toward accreditation (slmta) and highlights some challenges, achievements and lessons learned during the first five years of implementation (2009–2013) in developing countries. programme: slmta is a competency-based programme that uses a series of short courses and work-based learning projects to effect immediate and measurable laboratory improvement, while empowering laboratory managers to implement practical quality management systems to ensure better patient care. a slmta training programme spans from 12 to 18 months; after each workshop, participants implement improvement projects supported by regular supervisory visits or on-site mentoring. in order to assess strengths, weaknesses and progress made by the laboratory, audits are conducted using the world health organization’s regional office for africa (who afro) stepwise laboratory quality improvement process towards accreditation (slipta) checklist, which is based on international organization for standardization (iso) 15189 requirements. these internal audits are conducted at the beginning and end of the slmta training programme. conclusion: within five years, slmta had been implemented in 617 laboratories in 47 countries, transforming the laboratory landscape in developing countries. to our knowledge, slmta is the first programme that makes an explicit connection between the performance of specific management behaviours and routines and iso 15189 requirements. because of this close relationship, slmta is uniquely positioned to help laboratories seek accreditation to iso 15189. introduction top ↑ efficient and reliable laboratory services are essential to a functioning health system as high-quality laboratory testing plays a key role in patient care, surveillance and outbreak investigation.1 poor laboratory quality and its negative impact on healthcare systems have been documented for resource-limited settings, including sub-saharan africa (ssa).2,3,4,5 using the number of accredited laboratories as a quality metric, a 2013 survey showed that 37 out of the 49 countries in ssa had no medical laboratories accredited to any internationally-recognised standards. of the 380 accredited laboratories in that region, 91% were in south africa and only 17% were public health laboratories.6 in recent years, however, several landmark events have drawn attention to the poor state of public health laboratories and have pushed for strengthening of laboratory systems and networks.1,7 one of these events was the issuance of the world health organization (who)–lyon statement in 2008,8 which called for countries with limited resources to pursue practical quality management systems and to adopt a stepwise approach to quality improvement and accreditation.4,7 another was the 2009 launch of a laboratory management training programme called ‘strengthening laboratory management toward accreditation’ (slmta).1 effective management and leadership are critical to strengthening health systems and the scaling up of health service delivery.9 recently, many countries and partners have initiated efforts to enhance management of health programmes and service delivery in developing countries, with measurable success.10,11,12,13,14,15,16,17,18 most of these management capacity-building efforts focused on managers from hospitals, primary healthcare centers (such as family planning, mother–child health, etc.), or vertical public health programmes (such as tuberculosis [tb] and hiv). existing laboratory management capacity-building efforts have primarily targeted senior laboratory officials where the focus is on laboratory policy, system and network development, 19,20,21,22,23 as opposed to daily operations of individual laboratories. training programmes are needed to enable laboratory managers to use available resources (staff, budgets, supplies, equipment, buildings and information) efficiently for planning, implementation and evaluation of service delivery in order to meet patients’ and clinicians’ expectations and public health needs.24 the slmta programme was created in response to the observed need for structured laboratory management training and quality improvement by the us centers for disease control and prevention (cdc), in collaboration with the american society for clinical pathology, the clinton health access initiative, and the world health organization’s regional office for africa (who afro). slmta is a competency-based management training programme which uses a series of short didactic courses and work-based applied learning projects with the goal of achieving immediate and measurable laboratory improvements. it provides a practical approach to addressing everyday challenges using available resources. the slmta training curriculum and implementation method were pilot-tested in 15 laboratories in uganda from august 2008 to march 2009, yielding promising results.24 slmta was then officially launched in 2009, with implementation beginning in 2010. as of the end of 2013, slmta had been rolled out in 47 countries and 617 laboratories, and had improved enrolled laboratories an average of 23 percentage points after one round of slmta training in a pre/post study using the who afro stepwise laboratory quality improvement process towards accreditation (slipta) checklist.25 this report provides a detailed description of the slmta programme and highlights some challenges, achievements and lessons learned during its first five years of implementation (2009–2013) in developing countries. key components top ↑ the design of the slmta curriculum and its implementation exemplify what is known as ‘good practice’ in management competencies development.19,26 the slmta curriculum covers the 10 key competencies of a laboratory manager: productivity; work area; inventory; procurement; equipment maintenance; quality assurance; specimens; laboratory testing; test result reporting; and document and records control. a total of 66 tasks and job routines define effective laboratory management and constitute the learning objectives of the curriculum.24 a typical slmta training programme spans from 12 to 18 months (figure 1). training is conducted in a series of three workshops, each lasting three to four days, utilising 44 instructional activities27 and more than 100 job aids. each activity provides hands-on, practice-based learning experience for specific management tasks. the total training time is approximately 60 hours to teach all 44 activities. figure 1: standard slmta implementation process. after each workshop, participants implement improvement projects in their home laboratories. there are two types of improvement projects: complicated projects that require extensive planning and data collection before and after the change; and simpler ‘just do it’ types of projects that can be implemented immediately with minimal time and resources (box 1). implementation of improvement projects requires teamwork involving the entire laboratory staff, thus ensuring that the projects become part of the laboratory’s continuous improvement processes. participants are encouraged to implement locally-appropriate solutions using existing resources. during the home-based learning period after each workshop, participants are supported by periodic supervisory visits or on-site mentoring guided by standardised tools. this structured supervision and support component is critical to the success of the slmta programme. box 1: examples of improvement projects. the formal laboratory evaluation component is designed to identify weaknesses and areas that require improvement, measure success of the programme and indicate future goals for the laboratory. evaluations are based on who afro’s five-stage accreditation-preparedness scheme, called slipta, which recognises laboratories according to their level of compliance with the international organisation for standardization (iso) 15189 standard.1 under the slipta scheme, laboratories are audited using the slipta checklist, which includes 111 items divided into 12 sections (table 1) based on the 12 quality system essentials from the clinical and laboratory standards institute (clsi).28 after an audit, a laboratory receives a score out of 258 points in order to determine its star rating – from ‘0’ (0–141 points, < 55%) to ‘5’ (244–258 points, ≥ 95%).29 not all laboratories will pursue accreditation; regardless, the slipta scheme provides the roadmap and motivation for laboratories to make steady improvement in service delivery and patient care. slmta and slipta are closely linked. the slipta checklist provides the slmta programme with a means to identify gaps and benchmark progress. slmta, on the other hand, equips laboratory management with the ability to implement quality management systems in order to improve their performance on the slipta scale and eventually achieve formal accreditation status. to support this link, individual slipta checklist items are mapped to each of the 44 instructional activities in the slmta curriculum so that participants know exactly which management action will fulfill the requirements of any given checklist item. because of this close linkage between the slmta curriculum and the slipta checklist, in june 2012, after modification of the slipta checklist, the slmta curriculum underwent revisions to remap the revised checklist items to slmta instructional activities. each laboratory participating in slmta conducts an internal audit at the beginning (baseline) and the end (exit) of the programme using the slipta checklist. the difference between baseline and exit scores, as well as their respective star ratings, is calculated in order to quantify the effects of the programme on laboratory function and quality (figure 1). in addition to the slipta scores, laboratories demonstrate their progress through improvement project data such as turn-around time, sample rejection rate, stock out rate, customer satisfaction survey results and before-and-after photographs of physical changes. table 1: sections of the who afro slipta checklist and star ratings. variations from the basic implementation model top ↑ some countries have customised slmta delivery to fit their local context. two notable variations are cameroon and lesotho, which adapted their programmes to address local challenges and to enhance existing laboratory capacity-building efforts. despite the variations, both adaptations adhere to the critical requirement of implementing slmta as a process (a series of workshops with improvement projects and mentoring) rather than a single training event. cameroon most countries conduct the slmta training in a central location. this centralised model provides logistical convenience, particularly when many laboratories are enrolled in the same round, allowing the programme to train many laboratories at one time. it also enables personnel from various laboratories to interact and learn from each other. however, there are drawbacks, including, (1) high costs associated with renting a venue and travelling participants; (2) staff must be absent from their laboratories for prolonged periods because of travel between home and training locations; and (3) a limited number of staff can attend the course, creating a potential divide between those who are trained and those who are not. working with a very limited budget, cameroon decentralised the workshops and conducted facility-based training, with teams traveling to the laboratories in the programme to provide training on site. whilst this model required more time from the trainers, it enabled hospital management and clinicians to be involved in the training alongside laboratory management, facilitating advocacy. in addition, it allowed the course to be better tailored to the needs of the individual laboratories, with all discussions related to site-specific challenges and solutions.30 lesotho the schedule and frequency of trainings for the initial slmta round in lesotho were modified in order to match existing mentorship timetables.31,32 at the time that slmta was adopted, the country had already begun a structured mentorship programme with an embedded mentor. this mentor soon became certified as a slmta trainer so that he could enhance on-going mentoring efforts with the slmta programme. these laboratories received slmta training one day per week over two blocks of six weeks each, spaced six months apart. the total training time was the same as the standard three-workshop model. because of the availability of a full-time mentor, these laboratories received more intensive and frequent monitoring visits – a total of 12 visits versus the standard six – and were able to implement numerous improvement projects. capacity building for programme scale-up top ↑ in order to facilitate programme scale-up, a training-of-trainers approach was used to develop indigenous trainers, who in turn implement the slmta programme in-country.27 because the quality and integrity of the programme relies heavily on these local trainers, it is critical that they are competent and well qualified. to achieve that goal, the programme has established strict screening criteria in order to ensure that potential trainers have the necessary availability, motivation and commitment, along with a technical background. a formal training-of-trainers course was developed in which slmta master trainers teach both the curriculum content and also facilitation skills. this two-week course provides a demanding but supportive environment where participants conduct teach-back of assigned activities from the curriculum and immediately receive constructive feedback from master trainers in order to improve their facilitation skills and understanding of the content. to graduate, participants must fulfill several requirements: (1) 100% daily attendance, including group work sessions; (2) equal responsibility in the preparation and facilitation of teach-back assignments; (3) 100% completion of homework; and (4) endorsement by a master trainer. participants and their organisations also receive reports providing performance reviews and recommendations on specific roles that they are competent to play in programme implementation. timely, specific, behaviour-focused feedback is the cornerstone of training-of-trainers. as such, the master trainers’ ability to mentor the participants and provide constructive feedback determines the quality of trainers produced. the rapid expansion of the slmta programme has resulted in the demand for more master trainers who can train trainers. given the crucial role that master trainers play in developing competent trainers, they must be highly motivated and effective, their qualifications must be impeccable and their development and selection process rigorous. to be considered as a master trainer candidate, he or she must: (1) be a certified slmta trainer; (2) have conducted the entire slmta process; (3) have the availability and commitment needed to be a strong asset to the programme; and (4) be nominated by an existing master trainer. eligible candidates are invited to a training-of-trainers course, where they apprentice under existing master trainers whilst sharing the course workload equally .27 throughout the course, these candidates receive coaching and feedback on their performance from master trainers and their competence and commitment are assessed constantly. additional considerations top ↑ country commitment countries adopting the slmta programme are advised to fulfill certain pre-requisites to ensure success. firstly, they must have a national laboratory policy and strategic plan, along with a laboratory technical working group in order to drive the initiative forward. secondly, countries must ensure financial and political support for slmta and a commitment to improving laboratory quality at all levels: ministry of health, hospital management, laboratory management and laboratory staff. it is critical that slmta sites have dedicated quality assurance and safety officers. it is also important for participants to remain in the same job or organisation throughout the duration of the programme and to be allowed the time needed to participate in the programme. site selection site selection should be based on several factors, including facility infrastructure, staffing levels, impact on coverage of patient care, geographic considerations and demonstration of site commitment. the number of laboratories enrolled for each round of slmta (i.e., cohort) has varied by country – ranging from one each in angola and swaziland to 27 in malawi.25 countries have been advised to start small and scale up progressively. however, political pressure for broader impact and the desire for more laboratories to benefit from slmta may have resulted in some countries enrolling large numbers of laboratories. four countries (ethiopia, malawi, nigeria and uganda) have enrolled > 20 laboratories in the first or subsequent slmta cohorts.25 enrolling a large number of laboratories requires more human and logistical resources for the provision of sufficient site monitoring and support. in addition, it is essential that there is good communication and coordination amongst trainers and mentors so as to ensure consistency throughout the group. most countries have continued to enroll new laboratories in subsequent slmta cohorts.25 kenya to date has initiated six cohorts of slmta, enrolling a total of 50 laboratories and seven blood banks. lesotho, a small country with only 19 laboratories, has reached a high coverage of 18 (95%) laboratories over three cohorts of slmta. human resources countries vary in their capacity to rollout the slmta programme. implementation requires three primary cadres: trainers to teach the curriculum; auditors to perform the internal audits; and mentors to facilitate the improvement projects. regional and in-country slmta training-of-trainer workshops conducted during the past five years have steadily produced more local trainers.27 although the demand for slmta trainers still exceeds the supply, the deficiency is less severe than that of qualified auditors and mentors. using unqualified auditors may lead to inaccurate audit findings and missed non-conformities. this gap is being addressed slowly as many countries are seeking partners’ help with regard to scaling up auditor training. mentorship and site visits may be the most challenging aspect of implementation and are often overlooked in the initial programme planning. site visits require personnel time, transportation resources (fuel, vehicle, driver) and lodging and per diem if overnight stays are necessary. if this component is not scheduled and budgeted properly from the beginning, countries often struggle to provide the onsite support and supervision that are critical to the programme’s success. site visits are necessary in order to check the progress of the improvement projects, assess effectiveness of the previous workshops, troubleshoot site-specific issues and provide motivation and encouragement. site visits often involve meetings with top facility management to advocate support for the laboratory. the length of site visits has varied greatly between countries and even amongst laboratories within the same slmta cohort, ranging from half a day to three or more days at each site. the frequency and length of site visits should be considered carefully and planned according to the size and scope of testing activities in the laboratory. in addition, the level of quality at baseline and progress thereafter, as well as site staff’s experience with regard to implementing quality systems, should be considered. laboratories needing more support should receive longer or more frequent visits to enable them to make measurable improvements and sustain their motivation. the need for extensive but affordable site support has led countries such as cameroon,30 mozambique,33 swaziland and zimbabwe34 to establish structured mentorship programmes with full-time facility-based local mentors – a model spearheaded by lesotho.32,35 this model has well-defined goals for each mentoring engagement, extended contact time on site, defined periods when mentors are absent, consistent approaches across laboratories and measurement of progress using standardised tools. mentors may come from the laboratories they are assigned to mentor, from a local partner, or from outside the country. mentors receive training in slmta implementation, mentorship and auditing. because of their extended participation in the laboratories they are mentoring, they are able to gain knowledge of the rhythms, practices and personalities of the laboratory, enabling them to facilitate the necessary changes in attitudes and behaviours. other strategies have been used to provide the needed support for the slmta laboratories. in kenya, for example, select slmta hospital laboratories were paired, or ‘twinned’, with internationally-accredited research laboratories. the accredited laboratories mentored the slmta laboratories in quality management system implementation.36 experience from africa top ↑ slmta was launched in africa in 2009. by the end of 2013, it had been implemented in 23 countries on the continent with a total of 503 participating laboratories, which constituted 87% of all the slmta-enrolled laboratories in the world.25 as the continent that launched slmta, africa has demonstrated to the world that with ingenuity, innovation and determination, implementing quality management systems is possible, despite resource limitations. to date, four slmta-enrolled laboratories in africa have been accredited to iso 15189, whilst many more are making great progress in continuous quality improvement.25 in the sections below, we highlight the experiences of four african countries. mozambique – country ownership and sustainability to develop a self-sufficient quality programme, mozambique integrated slmta within the existing structure of the ministry of health laboratory system. a national laboratory quality technical working group was established and a dedicated coordinator hired. the ministry of health provided the vision and leadership in implementation and advocacy, coordinated and financed the programme with partner support and pressed for slmta activities to be included in provincial and hospital annual plans and budgets. decentralising programme management to the provincial level has enabled them to increase programme coverage and lower the costs.33 rwanda – data-driven advocacy as with many other countries, rwanda’s laboratories suffered from chronic service disruptions as a result of reagent stock-out and equipment breakdowns from lack of maintenance. an improvement project was assigned to the slmta-enrolled laboratories, which tracked the number of tests not performed because of stock-out and equipment breakdowns over a three-month period. they then calculated the funds required to purchase needed reagents and maintain equipment, along with the revenue that would have been generated from these tests, finding that the missed income was far greater than the cost of preventing stock-out and equipment breakdowns. this return on investment analysis persuaded hospital management to prioritise reagent supplies and to contract with manufacturers to provide regular maintenance services for the laboratory equipment.37 cameroon – expanding quality past the laboratory in cameroon, management at one hospital witnessed the transformation of its laboratory after slmta and undertook to extend the quality into other units of the hospital. they formed their own quality improvement teams, which have reported improved hospital cleanliness, reduced patient waiting times, greater patient satisfaction, development of new treatment protocols and increased recognition of the importance of patient safety. additionally, a reduction in infection rates and stillbirths, as well as an increase in the number of patients served and hospital revenue, have been observed.38 zimbabwe – overcoming contextual challenges zimbabwe has suffered economic crises in the past few decades, resulting in deterioration of the healthcare system and a shortage of human resources. participants in its two slmta cohorts have identified creative solutions to overcome the extensive logistic and resource challenges. for example, standard operating procedures were hand-written in exercise books, levy-jennings charts were plotted manually and a paper-based system was used where computerised laboratory information systems were not available. hospitals recognised the value of accreditation and prioritised budgets for equipment calibration, service contracts and staff vaccinations. funding from the us president’s emergency plan for aids relief (pepfar) supported the establishment of a training and mentorship department at the zimbabwe national quality assurance program trust in order to develop local capacity to support slmta programme rollout and continued quality improvement for laboratory services.39 slmta’s global reach and influence outside africa top ↑ the slmta-driven laboratory quality improvement achieved in africa has inspired countries in other regions to follow suit, even in the absence of a regional or national accreditation preparedness scheme such as who afro’s slipta. outside the continent of africa, 24 countries from the caribbean region, central and south america and southeast asia have adopted the slmta programme and have used the slipta checklist to measure gaps and the progress of enrolled laboratories. the caribbean region, comprising many island countries with diverse geography, people, size and economy, has implemented slmta in 12 countries.25 after completing the slmta programme, bahama’s national hiv reference laboratory was accredited and two other enrolled laboratories in the region are also seeking international accreditation.40 in southeast asia, impressive results have also been observed in cambodia and vietnam, where one provincial laboratory that tests clinical as well as food and environmental samples was accredited to iso 17025 in 2013.25 a desire to automate data collection, analyse and manage slipta audit data more efficiently and to enable real-time graphical display of actionable results at audited facilities led to the development of a multi-lingual electronic tool in vietnam.41 this tool has been shared with the global slmta community. in latin america, a partnership was forged where 14 military laboratories from eight countries in the region were enrolled in promela (programa de mejoramiento de laboratorios de las fuerzas armadas de latinoamérica), an overarching laboratory improvement programme using slmta as its principle training tool in addition to other practical laboratory training and biosafety and/or infection control training. the fact that two africa-based master trainers (one anglophone, one lusophone) came to assist in the first spanish-speaking training-of-trainers in latin america underscores the benefits of standardised training and highlights slmta’s true global nature and its far-reaching network across borders and continents. lessons learned top ↑ throughout the slmta rollout, countries have overcome many challenges such as attrition of slmta-trained staff, encouraging the entire laboratory to work as a team, engaging hospital management, and insufficient mentorship capacity. table 2 summarises the most common challenges and offers corresponding recommendations to help guide future implementation. despite the challenges, slmta has worked successfully by demonstrating that with resolve, commitment and ingenuity, laboratory teams in developing countries can improve their service delivery using existing limited resources. it also demonstrates that starting with small tangible improvements (‘low-hanging fruit’) and gradually building upon early successes can boost laboratory teams’ confidence and motivate them to tackle the harder issues. this strategy is similar to the ‘little steps’ approach42 that has been shown to be effective in sustaining healthcare quality improvement efforts in developing countries. table 2: common challenges and recommendations for slmta implementation. within a few years, slmta has demonstrated its transformative power, emerging as a flagship programme for laboratory system strengthening in pepfar-supported countries. a recent 2013 institute of medicine report43 recognised that improvement of laboratories under pepfar support and guidance has been a signature achievement. in addition, it states that: pepfar’s laboratory efforts have had a fundamental and substantial impact on laboratory capacity in countries. this laboratory infrastructure has been, and continues to be, leveraged to improve the functioning of countries’ entire health systems.43 as laboratories do not exist in a vacuum, there have been calls38,44 for the slmta model to be adapted for the clinical settings in developing countries, with a goal toward overall hospital accreditation. this will ensure the sustainability of laboratory improvements and accreditation, and boost the centrality of quality management systems in hospital facilities, resulting in better patient care. slmta implementation has been supported primarily with pepfar resources. to ensure its longevity and viability beyond pepfar, countries must work hard to integrate the slmta components into normal laboratory operations, decentralise programme planning and budgeting to the provincial or lower level, look for ways to be financially self-sufficient (such as charging enrollment fees for privately-owned laboratories) and incorporate the curriculum into pre-service education. conclusion top ↑ after five years of implementation, slmta has proven to be an effective programme for the strengthening of laboratory health systems, with a focus on building management capacity in order to achieve quality services for improved patient care. evidence to date has indicated widespread success of the programme in its ability to facilitate continuous quality improvement in the enrolled laboratories. slmta has the unique potential to help laboratories make progress through the slipta process, improve quality of services and subsequently achieve accreditation to iso 15189. acknowledgements top ↑ the authors would like to thank dr. barbara mckinney, anna murphy and philip rotz for their significant contribution in the development of the slmta toolkit. the authors also extend their gratitude to all the slmta implementers for their tireless effort in improving the quality of laboratory systems and patient care in resource-limited settings. this research has been supported by pepfar through the cdc. the findings and conclusions in this article are those of the authors and do not necessarily represent the views of the cdc. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. authors’ contributions k.y. (cdc) led the development of the slmta programme, oversaw its global implementation and wrote the manuscript. t.m. (aslm) played a key role in programme expansion and implementation and provided input to the manuscript. e.l. (cdc) provided substantial input to the writing of the manuscript. j.n. 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http://dx.doi.org/10.4102/ajlm.v2i1.77 36.makokha e, mwalili s, basiye f, et al. using institutional mentorship to roll out slmta in kenya. afr j lab med. 2014. in press. 37.nzabahimana i, sebasirimu s, gatabazi jb, et. al. innovative strategies for a successful slmta country program: the rwanda story. afr j lab med 2014. in press. 38.eno l, asong t, ngale e, et al. adapting the slmta programme for improved quality services in a regional hospital in cameroon. afr j lab med. 2014. in press. 39.simbi r. doing more with less: the zimbabwean story. paper presented at the first symposium on strengthening laboratory management toward accreditation (slmta) at the first international conference of the african society for laboratory medicine. cape town, south africa; 2012. 40. guevara g, gordon f, irving y, et al. the impact of slmta in improving laboratory quality systems in the caribbean region. afr j lab med. 2014. in press. 41.nguyen thi t, mckinney b, pierson a, et al. slipta e-tool improves laboratory audits in vietnam and cambodia. afr j lab med. 2014. in press. 42.umar n, litaker d, terris dd. toward more sustainable health care quality improvement in developing countries: the ‘little steps’ approach. qual manag health care. 2009 oct-dec;18(4):295-304. pubmed pmid: 19851237. 43.committee on the outcome and impact evaluation of global hiv/aids programs implemented under the lantos-hyde act of 2008; board on global health (bgh); board on children, youth, and families; institute of medicine. evaluation of pepfar. washington, dc: the national academies press; 2013. 44.mataranyika mn, beukes lk. view from the top: involvement of namibia�s health ministry in laboratory quality improvement and the building of a strong laboratory network. afr j lab med. 2014. in press. abstract introduction external quality assessment challenges for point-of-care testing proof of concept for an automated external quality assessment programme in zimbabwe acknowledgements references about the author(s) ben cheng international diagnostics centre, london school of hygiene & tropical medicine, london, united kingdom brad cunningham systemone, johannesburg, south africa debrah i. boeras international diagnostics centre, london school of hygiene & tropical medicine, london, united kingdom patron mafaune ministry of health and child care, harare, zimbabwe raiva simbi ministry of health and child care, harare, zimbabwe rosanna w. peeling international diagnostics centre, london school of hygiene & tropical medicine, london, united kingdom citation cheng b, cunningham b, boeras di, mafaune p, simbi r, peeling rw. data connectivity: a critical tool for external quality assessment. afr j lab med. 2016;5(2), a535. http://dx.doi.org/10.4102/ajlm.v5i2.535 lessons from the field data connectivity: a critical tool for external quality assessment ben cheng, brad cunningham, debrah i. boeras, patron mafaune, raiva simbi, rosanna w. peeling received: 12 july 2016; accepted: 31 aug. 2016; published: 17 oct. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract point-of-care (poc) tests have been useful in increasing access to testing and treatment monitoring for hiv. decentralising testing from laboratories to hundreds of sites around a country presents tremendous challenges in training and quality assurance. in order to address these concerns, companies are now either embedding connectivity in their new poc diagnostic instruments or providing some form of channel for electronic result exchange. these will allow automated key performance and operational metrics from devices in the field to a central database. setting up connectivity between these poc devices and a central database at the ministries of health will allow automated data transmission, creating an opportunity for real-time information on diagnostic instrument performance as well as the competency of the operator through external quality assessment. a pilot programme in zimbabwe shows that connectivity has significantly improve the turn-around time of external quality assessment result submissions and allow corrective actions to be provided in a timely manner. furthermore, by linking the data to existing supply chain management software, stock-outs can be minimised. as countries are looking forward to achieving the 90-90-90 targets for hiv, such innovative technologies can automate disease surveillance, improve the quality of testing and strengthen the efficiency of health systems. introduction diagnostic testing has traditionally been conducted in a laboratory setting, and requires highly-skilled operators working in strictly-controlled laboratory environments. the recent availability of point-of-care (poc) technologies allows for a shift from a centralised to a decentralised model of testing. such decentralisation of testing can offer a significant increase in access to much needed diagnostics for populations in peri-urban, rural and other hard to reach areas, which will be needed to achieve the unaids 90-90-90 target.1 furthermore, the scale up of quality poc testing can help strengthen health systems, especially in rural areas.2 the wide deployment of poc tests at hundreds or even thousands of sites raises a big challenge for ministries of health to track instruments and monitor the quality of the test and the testing. in order to address these concerns, numerous connectivity (connected diagnostic) initiatives and interventions have been undertaken in an attempt to ensure that the implementing institution is able to keep track of, at minimum, key performance and operational metrics for devices in the field. these initiatives have had mixed success in implementation due to a variety of reasons. companies are now either embedding connectivity in their new diagnostic instruments or providing some form of channel for electronic result exchange. zimbabwe, for example, has placed 346 alere pima™ poc cd4 devices in hard to reach areas throughout the country. at present, 114 of these instruments are connected via alere 3g modems to the clinton health access initiative’s in-country poclabs platform in conjunction with the national microbiology reference laboratory. the ministry of health has been receiving scant data from the devices, as is shown in the snapshot provided in table 1. this is likely due to a combination of reasons including: lack of awareness regarding sending of data, insufficient 2g/3g signal, sim cards with insufficient/expired airtime, and other sim issues. table 1: snapshot of reporting frequency from connected devices in zimbabwe, 2016. external quality assessment challenges for point-of-care testing external quality assessment (eqa) is a critical component of a laboratory quality assurance and management system.3 eqa for poc testing has the potential to provide a snapshot of quality, both in terms of the diagnostic instrument performance as well as the competency of the operator. the implementation of poc testing in rural and peri-urban settings, however, presents tremendous challenges in the organisation of an eqa programme. these challenges include: the scale of providing eqa samples to hundreds of sites within a country, requiring the preparation or procurement of a large number of proficiency testing panels and the means to distribute them. poc tests are typically operated by non-laboratory staff who are not familiar with a laboratory ‘culture’ of quality or eqa. poor response rates and delay in reporting weaken the value of eqa programmes. how can connectivity improve the quality of testing? connectivity for poc tests and other medical devices can be defined as an initiative that can improve patient flow, increase the quality of testing, and improve patient outcomes. connectivity should be viewed as a complementary approach to eqa. it can provide continuous quality monitoring, as all of the data generated by the instrument can be analysed as an average of a series of aggregated, non-unique events, whereas eqa is a series of controlled events, with known inputs and known outputs. connectivity can be used not only to monitor patient data such as diagnostic results and response to treatment, but it can also be used to capture operational data from the device and provide key performance and quality indicators. most poc instruments now include methods to exchange data electronically. this can be via a port (e.g., usb, ethernet, serial), or via embedded gsm/3g capabilities within the poc device. the diagnostics industry is beginning to take full advantage of these capabilities with a number of ecosystems being constructed around the consumption of this data. various stakeholders have distinct, and well-defined needs for the different data sets and subsets. broadly, the data can be categorised into clinical data, which includes patient-identifiable information, and operational data. operational data is defined as a subset of the clinical data which is de-identified. these systems allow ministries of health to receive information and derive intelligence, in real-time, from any testing facility throughout a country into one central database. this allows ministries of health to have daily monitoring of the performance of the devices, the test results and the competency of the operators from each site. furthermore, connectivity can help improve forecasting and prevent stock-outs based on the utilisation rate at each facility, as shown in figure 1. figure 1: using connectivity to monitor utilisation and predict stock-outs. operational data can be used by instrument manufacturers, ministries of health, funders, trainers, and supply chain managers. diagnostic data can be used for patient management by nurses, clinicians and epidemiologists and stored in a laboratory information system and/or a central data warehouse.4 these informatics systems are continually expanding and innovating features as the ecosystems become better defined. one example is the automated detection and transmission of eqa samples from diagnostic devices which is made available, in near-real time, to the eqa providers for analysis. existing point-of-care external quality assessment structure for the pima cd4 in zimbabwe historically, the pima cd4 eqa programme in zimbabwe requires a significant amount of hands-on time for both sample shipping, testing and reporting. the process, briefly, is as follows. eqa samples are shipped to a single, central, location for separation and distribution. this facility unpacks the samples and re-packages them for distribution by courier for delivery to the facility (figure 2). after the samples have been received by the testing facility, they are required to be completed before the scheduled deadline, and the results are reported, via different mechanisms, back to the central facility. figure 2: sample eqa set sent to sites in zimbabwe. once the eqa deadline closes, the central facility captures all of the received results, first on paper for central collection, and then via a web-form on the eqa provider’s informatics platform, for all participating sites. once the eqa results have been captured and submitted, the eqa provider is able to analyse the results and report these, with any corrective action suggestions, back to the central facility. at this point, the central facility is, again, responsible for separating the individual performance reports for the facilities and re-distributing these, along with any corrective actions, back to the participants. this whole process usually takes at least 12 weeks. for connected devices, it is clear that a much simpler and more efficient workflow can be leveraged to optimise the eqa process. poc tests connected to an informatics system capable of distinguishing eqa from routine samples via the sample id can dramatically help improve turnaround time for eqa results, thereby allowing for action to be taken if necessary. furthermore, there will be significantly fewer transcription errors and data analysis will be simplified. proof of concept for an automated external quality assessment programme in zimbabwe an automated eqa proof-of-concept pilot for cd4 counts was conducted in november 2015 in manicaland province in zimbabwe. several issues had to be resolved among different partners before the proof-of-concept testing could be initiated. a standardised barcoding format had to be agreed upon for use as the sample id for testing on the pima cd4 instrument. the barcode standardisation was necessary so that the informatics systems would be able to differentiate eqa samples from patient results to ensure that no patient data is transmitted beyond the clinical boundaries defined in these structures (figure 3). figure 3: steps involved in the automated eqa reporting proof-of-concept, manicaland province, zimbabwe, november 2015. alere, the developer of the pima poc cd4 device developed an ‘eqa only’ data-store and interface for its centralised data warehouse, data point. this allows for authorised, external systems to query data point for samples that have been identified as eqa samples from the barcode. for this pilot, the eqa provider, oneworld accuracy, developed an application program interface for their informatics system, oasys (oneworld accuracy system), which would automatically query data point every hour for any new data that has been identified as an eqa sample. all new data identified as eqa are then immediately received by oasys for storage and processing (both of these application program interfaces have now been included in the production version of both data point and oasys). the proof-of-concept study only involved one site, the marange rural health centre. three sets of eqa samples were delivered to marange and run by the operator over a three-week period. oneworld accuracy received the results from the eqa samples within 60 minutes of these tests being performed on the pima device, demonstrating that the automated system performed as expected. the next phase was to increase the number of sites to approximately 50 and eqa samples were distributed as part of the routinely-scheduled eqa assessment planned for the end of march 2016. due to the perceived impact of this intervention, a similar process is currently being established to automate the reporting of eqa samples (identified by a barcode) from the cepheid genexpert® platform to the relevant eqa provider informatics systems. the proof-of-concept testing for this took place in april 2016 at four sites in manicaland. post-proof of concept the proof-of-concept pilot highlighted the potential for improvement in turn-around time from testing to reporting of eqa results, as well as reducing the overall administrative burden of these events. it is recommended that some standard barcoding identification be implemented for all eqa samples for both laboratory and poc instruments. the implementation of a standardised barcode structure for the identification of eqa samples would allow any informatics system (operational or clinical), connected to any laboratory or poc instrument, to identify a sample as an eqa sample and transmit these results, in near-real time, to the relevant eqa provider for analysis and reporting. in the ideal case, the following workflow could become standard for eqa testing and reporting: the eqa provider sends out the sample to the test site. the test site receives the sample and tests the sample. the results are automatically uploaded to a server upon completion. the server identifies the result as an eqa result and forwards the result to the correct eqa provider. the eqa provider scores the result and generates the user report. this proposed workflow reduces the need for the paper transcription of results to be collected centrally, as well as the need for the collected results to be re-transcribed in an electronic format on the eqa provider’s informatics platform, thus reducing errors in both interpretation and transcription. way forward the automation of the collection and analysis of eqa results from laboratories, especially for poc instruments, can help countries: significantly improve the turn-around time of eqa result submissions and the impact of any prescribed corrective actions in a timely manner. reduce the transcription error rate and improve the quality of the resulting process. eliminate the time-consuming processes involved with eqa result collection, capture and re-distribution. reduce the cost and improve uptake of eqa programmes for both laboratories and poc facilities. additional expected outputs from this pilot are: feasibility and acceptability from eqa providers to adhere to a standardised barcode format. willingness for ministries of health to have this data (non-patient data) automatically shared between connected systems (and ecosystems) from the instrument to the eqa provider. definition of the role the instrument manufacturer can play in helping countries successfully implement and monitor eqa programmes via manufacturer platforms. conclusion connectivity has shown that it is possible for ministries of health to have up-to-the-hour information on testing and test results across the country. these results can also be accompanied by eqa results from laboratories, as well as each poc testing site across the country, so that corrective action and refresher training can be provided in a timely manner. these systems can also be twinned to supply chain management software to monitor supplies at each site, providing an automated system for alerts to avoid stock-outs. as countries are looking forward to achieving the 90-90-90 targets for hiv, such innovative technologies can automate surveillance, improve quality of testing and strengthen health systems. acknowledgements competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support none. authors’ contributions b.cu. and b.ch. conceived of and designed the study and drafted the manuscript. r.s. and p.m. contributed to the study design and revised the manuscript. r.w.p. and d.i.b. revised the manuscript. all authors gave final approval of the version to be published and agree to be accountable for the accuracy and integrity of the work. references joint united nations programme on hiv/aids (unaids). 90–90–90 – an ambitious treatment target to help end the aids epidemic [page on the internet]. c2014 [cited 2016 sep 23]. available from: http://www.unaids.org/en/resources/documents/2014/90-90-90 mabey d, sollis k, kelly h, et al. point-of-care tests to strengthen health systems and save newborn lives: the case of syphilis. plos med. 2012;9(6):e1001233. http://dx.doi.org/10.1371/journal.pmed.1001233 epub 2012 jun 12. fonjungo pn, osmanov s, kuritsky j, et al. ensuring quality: a key consideration in scaling-up hiv-related point-of-care testing programs. aids. 2016; 30(8):1317–1323. http://dx.doi.org/10.1097/qad.0000000000001031 [accessed 2016 jan 23 as epub ahead of print]. jani iv, sitoe ne, alfai er, et al. effect of point-of-care cd4 cell count tests on retention of patients and rates of antiretroviral therapy initiation in primary health clinics: an observational cohort study. lancet. 2011;378(9802):1572–1579. http://dx.doi.org/10.1016/s0140-6736(11)61052-0 epub 2011 sep 25. article information authors: elizabeth t. luman1 katy yao1 john n. nkengasong1 affiliations: 1international laboratory branch, division of global hiv/aids, center for global health, us centers for disease control and prevention, atlanta, georgia, united states correspondence to: elizabeth luman postal address: 1600 clifton road, atlanta, ga 30333, united states dates: received: 18 sept. 2014 accepted: 21 sept. 2014 published: 03 nov. 2014 how to cite this article: luman et, yao k, nkengasong jn. a comprehensive review of the slmta literature part 2: measuring success. afr j lab med. 2014;3(2), art. #276, 8 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.276 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. a comprehensive review of the slmta literature part 2: measuring success in this review article... open access • abstract • introduction • research methods and design • results and discussion    • literature search results    • global programme results    • quality system essentials meta-analysis    • official who afro slipta audits and accreditation    • service delivery indicators    • cost    • limitations to the study    • conclusion • acknowledgements    • competing interests    • authors’ contributions    • cdc disclaimer • references abstract top ↑ background: since its introduction in 2009, the strengthening laboratory management toward accreditation (slmta) programme has been implemented in 617 laboratories in 47 countries. objective: we completed a systematic review of the published literature on slmta. the review consists of two companion papers; this article examines quantitative evidence presented in the publications along with a meta-analysis of selected results. methods: we identified 28 published articles with data from slmta implementation. the slmta programme was evaluated through audits based on a standard checklist, which is divided into 12 sections corresponding to the 12 quality system essentials (qses). several basic service delivery indicators reported by programmes were also examined. results for various components of the programme were reviewed and summarised; a meta-analysis of qse results grouped by the three stages of the quality cycle was conducted for 126 laboratories in 12 countries. results: global programme data show improved quality in slmta laboratories in every country, with average improvements on audit scores of 25 percentage points. meta-analysis identified improvement management as the weakest stage, with internal audit (8%) and occurrence management (16%) showing the lowest scores. studies documented 19% – 95% reductions in turn-around times, 69% – 93% reductions in specimen rejection rates, 76% – 81% increases in clinician satisfaction rates, 67% – 85% improvements in external quality assessment results, 50% – 66% decreases in nonconformities and 67% increases in staff punctuality. conclusions: the wide array of results reported provides a comprehensive picture of the slmta programme overall, suggesting a substantive impact on provision of quality laboratory services and patient care. these comprehensive results establish a solid data-driven foundation for program improvement and further expansion. introduction top ↑ quality laboratory services are critical for ensuring optimal patient care and comprehensive public health response; however, laboratories in resource-poor countries have been one of the most neglected components of health systems.1 the strengthening laboratory management toward accreditation (slmta) programme was developed in an effort to improve the quality of laboratories throughout the developing world. it is a competency-based training programme designed to enable laboratories to implement practical quality management systems (qms) and encourage continuous quality improvement. since its introduction in 2009, the slmta programme has been implemented widely throughout africa, as well as in the caribbean, central and south america, and southeast asia.2 the primary focus of the programme thus far has been implementation and expansion; until recently, little attention has been paid to the systematic examination of programme results in order to guide programme improvement and decision making. this systematic literature review aims to compile existing results from evaluations of the slmta programme into a comprehensive report, in order to provide a broad view of the programme and to identify directions for the future. because of the large volume of information collected, the review has been published in two parts. in part 1, published separately, we present content analysis of qualitative findings and identified strategic directions for future priorities.3 in this companion paper, we compile the quantitative data presented in the publications, examine scores and indicators, and conduct a meta-analysis of selected results in order to establish a solid, data-driven foundation for programme improvement and to help guide future implementation. research methods and design top ↑ a comprehensive search of electronic bibliographic databases was performed, as described in part 1.3 we included all published and in-press studies that discussed the slmta programme. the standard slmta implementation model includes three workshops, each of which is followed by a period of several months for laboratories to implement improvement projects, usually with onsite support and mentorship.2 laboratories implementing the slmta programme are evaluated through audits based on the stepwise laboratory quality improvement process towards accreditation (slipta) checklist.4 audit scores are categorised into star ratings, with zero stars corresponding to a score of 0% – 54%, one star 55% – 64%, two stars 65% – 74%, three stars 75% – 84%, four stars 85% – 94%, and five stars 95% – 100%. the checklist items are divided into 12 sections that represent the 12 quality system essentials (qses) as defined by the clinical and laboratory standards institute (clsi).5 these qses can be grouped by stages of the quality cycle: resource management (equipment; facilities and safety; organisation and personnel; purchasing and inventory), process management (client management; documents and records; information management; process control and internal/external quality assessment) and improvement management (corrective action; internal audit; management reviews; occurrence management).6 to assess progress, baseline and exit audits are conducted before and after slmta implementation, respectively, using the slipta checklist. ‘surveillance’ audits are also often conducted after the exit audit in order to monitor continued improvement and assess sustainability. several studies provided scores by individual qses. we combined these data and conducted a meta-analysis in microsoft® excel 2013 so as to determine common areas of strength, weakness and improvement. for studies reporting only median or mean qse data for multiple laboratories, laboratory-level data were solicited from authors to further enhance the analysis. all cost estimates reported in local currency in published articles were converted into us dollars, based on the official exchange rate as of august 1, 2014. percent changes in indicator results were calculated from published results if not reported directly in the papers. results and discussion top ↑ literature search results we identified 28 published articles on the slmta programme2,7–33 (table 1). in total, these studies included detailed information on slmta implementation in 211 laboratories in 18 countries, as well as global summary data from all 617 laboratories in the 47 countries that have implemented slmta as of the end of 2013. table 1: characteristics of published slmta studies. global programme results data from all laboratories implementing the slmta programme were collated and summarised in a single paper describing the global results of the programme to date.33 in total, 617 laboratories in 47 countries on four continents have implemented slmta in 65 training cohorts, with nearly 2000 laboratory staff trained in the programme. most of the laboratories were at the district (38%), regional (27%) or national (18%) levels. the authors report that the starting level of laboratory quality in developing countries was very low, with 84% of slmta laboratories scoring below the one-star level at baseline. the 302 laboratories that had completed the programme had an average improvement of 25 percentage points; 70% achieved at least one star at exit audit and 22% of laboratories increased three or more star levels. estimates of the number of laboratory tests conducted by slmta laboratories suggested that the 617 laboratories enrolled in slmta conduct more than 100 million tests annually and that whilst only 16% of these tests were conducted by laboratories with at least one quality star before slmta, 68% were done by laboratories with at least one star after slmta implementation. that translates to approximately 58 million tests conducted by laboratories with little to no qms prior to slmta which now have at least a basic quality system in place.33 quality system essentials meta-analysis examining individual slipta checklist scores for each of the 12 qses enables laboratories to pinpoint strengths, weaknesses and areas of improvement. qse data have not been compiled systematically on a global scale. from the published papers, qse data were presented for 126 laboratories in 12 countries.8,11,12,14,15,18,20,21,22,25,26 individual studies reported substantial variability in highand low-scoring qses. for example, some laboratories scored 0% for five of the 12 qses at exit audit, whereas others scored 100% for the same five qses. at baseline, the weakest areas overall were in the improvement management stage of the quality cycle, including internal audit (5%), occurrence management (16%), corrective action (25%) and management reviews (29%) (figure 1). at an average of 20%, this stage scored less than half of the other two stages, namely, resource management (42%) and process management (40%). none of the 12 qses had mean baseline scores above 55%; the highest scores were in information management (51%), facilities and safety (47%), purchasing and inventory (42%) and process control and internal/external quality assessment (41%). at the exit audit, the four improvement management qses still showed the lowest scores, ranging from 32% – 50% (average 42%) (figure 1). the resource management and process management stages had higher scores ranging from 58% – 74% (average 65% for resource management and 63% for process management). the greatest improvements were in documents and records (34 percentage points), client management (29 percentage points), and facilities and safety (27 percentage points). each of the three stages had the same average improvement of 23 percentage points. figure 1: baseline and exit audit scores for quality system essentials grouped by quality cycle stage from 126 laboratories in 12 countries. based on results from five laboratories, maina et al. found that the laboratories with the greatest overall score increases had focused on internal audit and corrective action; they then hypothesised that an improvement in these areas may be a catalyst for overall improvement in other areas.14 meta-analysis results suggest that the corrective action qse may be the most predictive of overall improvement; laboratories in the top quartile of overall improvement outperformed those in the bottom quartile by 62 percentage points for the corrective action qse, compared to a median of 40 percentage points for the other qses. clsi defines corrective action as an ‘action to eliminate the (root) cause of a detected nonconformity or other undesirable situation’.34 in the slipta checklist, corrective action is assessed through four questions about how the laboratory deals with occurrence reports, nonconformities and discordant results.4 the international organization for standardization (iso) confirms the importance of corrective action, saying that ‘the corrective and preventive actions system is the most critical element for an efficient quality system’.35 additional work is needed to verify priority areas of improvement, as well as to delineate the set of essential improvement projects that will result in meaningful laboratory quality improvement. official who afro slipta audits and accreditation a july 2009 survey of accrediting body registers identified 340 accredited laboratories in sub-saharan africa; only 28 (8%) of these laboratories were located outside of south africa and nearly all were private, parastatal or donor-supported research facilities.36 by early 2013, little progress had been made, with 380 laboratories accredited in the region; only 35 (9%) laboratories outside of south africa were accredited and three quarters of the 49 countries in the region had no accredited laboratories.37 however, the impact of slmta is beginning to show; as of september 2014, six laboratories enrolled in slmta in kenya, the bahamas, vietnam and zimbabwe have been accredited, at a median of 31.5 months after starting the slmta programme.10,11,33 several laboratories have been recommended for accreditation or are in the process of application.11,18,20,28 ninety-seven slmta laboratories have received official who afro slipta audits conducted by representatives from the african society for laboratory medicine,33 including 11 laboratories in published reports included in this review.7,18,25,26,29 service delivery indicators in addition to audit scores, many of the studies reported improvements for indicators reflecting testing and customer and clinician satisfaction (table 2). three studies reported reductions in turnaround time for testing,10,20,28 with times decreasing by 19% – 95%. patient and clinician satisfaction were commonly measured using surveys. four studies showed relative improvements in patient satisfaction ranging from 30% to > 100%,9,10,25,28 although in one laboratory complaints from patients increased, possibly as a result of staff attrition.17 two studies reporting on clinician satisfaction found improvements of approximately 80%.17,28 indicators for laboratory management and overall functioning also showed improvements (table 2). one laboratory reported a 65% decrease in corrective actions,10 five laboratories in the caribbean region reported decreases in nonconformities of 50% – 66%11 and two laboratories showed improvements in external quality assessment results of 67% – 85%.10,17 in a kenyan laboratory, staff punctuality increased 67% and the need for equipment repairs decreased 63%.17 a botswana laboratory successfully reduced losses resulting from expired reagents from $18 000 in 2010 to $40 in 2013;28 and three studies showed reductions in specimen rejection rates of 69% – 93%.10,17,25 when slmta was adapted and implemented at a hospital in cameroon, patient wait times decreased 67% – 83%, infection rates and stillborn rates decreased (83% and 80%, respectively) and the number of patients and hospital revenue increased.9 table 2: health service indicators associated with slmta implementation as reported in published studies. table 2 (continues...): health service indicators associated with slmta implementation as reported in published studies. cost the reported costs per laboratory of implementing various components of slmta have varied widely (table 3). much of this variability is because of differences in what was included in the cost estimates, as well as location-specific factors, such the price of fuel, salary levels and distances to participating laboratories. the estimated cost of conducting the three-workshop slmta series has ranged from $1482 per laboratory in zimbabwe using local facilitators in a central location31 to $21 480 in cameroon using decentralised training.22 mentorship cost per laboratory has ranged from $5689 in zimbabwe30 to $24 000 in ghana.25 the cost of implementing improvement projects has ranged from $10 000 in ghana25 to $36 500 in a kenyan laboratory seeking accreditation.10 table 3: cost estimates of various components of slmta implementation as reported in published studies. three studies have compared the cost of various slmta implementation models. one study of 19 laboratories in zimbabwe found that mentorship and supervision costs for four different models were similar ($5689-$9601 per laboratory), recommending that ‘countries should carefully consider which mentorship model or models would be best suited to their individual situation’.30 another study in zimbabwe found that implementing slmta using local (in-country) facilitators is more expensive than external facilitators for the first slmta cohort because of the costs associated with conducting an in-country training-of-trainers; however, over the course of national scale-up in 120 laboratories, use of local facilitators would save the country nearly 50% ($580 000 vs. $322 000).31 a cameroonian study found that the cost per laboratory of centralised training was approximately the same as decentralised training ($21 122 vs. $21 480, respectively); centralised training required less trainer time, whilst decentralised training allowed more staff to participate.22 no published studies to date have reported a thorough examination of the cost of implementing the entire slmta programme, including each of the major components (training of mentors, trainers and auditors; conducting slmta workshops; mentorship, supervisory visits and implementation of improvement projects; and conducting audits). in addition, a more extensive cost-benefit analysis taking into consideration the value of laboratorians’ time (i.e., opportunity cost) to participate in the programme and implement changes in the laboratory along with tangible and intangible benefits of the programme is needed.31 limitations to the study this review is subject to several limitations. firstly, whilst 28 studies on slmta were identified and summarised, these reflect only 18 (38%) of the 47 countries and 211 (34%) of the 617 laboratories that have implemented the programme. their results may not be representative of the programme as a whole, or a comprehensive account of all laboratories’ experiences. secondly, whilst audit results were available for all laboratories because of the use of the slipta checklist, the other indicators presented here were available in few of the published studies; in addition, methodologies varied between the studies, limiting the ability to combine and compare results directly. authors of the studies published thus far also point out several limitations. firstly, the slmta programme as a whole is too young to allow an assessment of the long-term sustainability of results.14,33 secondly, all of the published studies were observational; several studies examining the effect of mentorship or training methodologies note that laboratories were not assigned randomly, but were rather selected purposively based on convenience or other programmatic considerations. thus there may have been other factors that could account for some of the differences.8,15,20,30 similarly, none of the studies included control laboratories upon which to base a comparison.22 thirdly, there is a lack of consistency in the qualifications of auditors; whilst the slipta checklist is designed to help standardise the audit process, some variability between auditors may remain.8,29 finally, several authors noted that their published studies are based on a small number of laboratories14,15,20,30 and some indicators were either not measured systematically9 or not measured at baseline.9,28 conclusion in their summary of global-level findings, yao et al. point out that ‘few [other] management and leadership development programmes have been implemented on a such a large scale with results-oriented outcome measures’.33 the wide array of results reported provides a comprehensive picture of the slmta programme overall, suggesting a substantive impact on provision of quality laboratory services and patient care. the full potential of the programme can be realised only if the lessons learned lead to informed action among laboratory workers, healthcare providers and policy makers toward the ultimate goal of providing quality patient care. acknowledgements top ↑ we would like to thank the lead authors of the in-press papers for allowing us to examine their results prior to publication, making it possible to publish this review simultaneously with their work: linda andiric, rosemary audu, laura eno, thomas gachuki, giselle guevara, tilahun hiwotu, adino lulie, robert maina, ernest makokha, talkmore maruta, phidelis maruti, jessina masamha, mary mataranyika, kelebeletse mokobela, juliana ndasi, thuong nguyen, bernard nkrumah, siyem nkwawir, michael noble, keoratile ntshambiwa, innocent nzabahimana, phoebe nzombe and edwin shumba. special thanks go to philip rotz, lee schroeder, bethanie rammer and penny smorenburg for their valuable feedback in manuscript revision. this research has been supported by the president’s emergency plan for aids relief (pepfar) through the cdc. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions e.t.l (cdc, atlanta) analysed the data and wrote the manuscript. k.y. 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organization for standardization. iso 9000 resources: corrective and preventive actions high level review [page on the internet]. c2014 [cited 2014 sep 18]. available from: http://www.iso9000resources.com/ba/corrective-preventive-actions.cfm 36.gershy-damet g, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. 37.schroeder lf, amukele t. medical laboratories in sub-saharan africa that meet international quality standards. am j clin pathol. 2014;141(6):791–795. abstract introduction methods results discussion acknowledgements references about the author(s) amah p.v. goran-kouacou laboratory of immunology and hematology, university hospital of cocody, abidjan, côte d’ivoire faculty of medicine, félix houphouët-boigny university, abidjan, côte d’ivoire oppong r. yéboah laboratory of immunology and hematology, university hospital of cocody, abidjan, côte d’ivoire faculty of medicine, félix houphouët-boigny university, abidjan, côte d’ivoire aya u.a. assi laboratory of immunology and hematology, university hospital of cocody, abidjan, côte d’ivoire faculty of medicine, félix houphouët-boigny university, abidjan, côte d’ivoire yida j. séri laboratory of immunology and hematology, university hospital of cocody, abidjan, côte d’ivoire faculty of medicine, félix houphouët-boigny university, abidjan, côte d’ivoire séry r. dassé laboratory of immunology and hematology, university hospital of cocody, abidjan, côte d’ivoire faculty of medicine, félix houphouët-boigny university, abidjan, côte d’ivoire citation goran-kouacou apv, yéboah or, assi aua, séri yj, dassé sr. antenatal toxoplasma gondii igg/igm seroprevalence at the university hospital of cocody. afr j lab med. 2025;14(1), a2846. https://doi.org/10.4102/ajlm.v14i1.2846 original research antenatal toxoplasma gondii igg/igm seroprevalence at the university hospital of cocody amah p.v. goran-kouacou, oppong r. yéboah, aya u.a. assi, yida j. séri, séry r. dassé received: 25 apr. 2025; accepted: 25 aug. 2025; published: 16 dec. 2025 copyright: © 2025. the author(s). licensee: aosis. this work is licensed under the creative commons attribution 4.0 international (cc by 4.0) license (https://creativecommons.org/licenses/by/4.0/). abstract background: toxoplasmosis is a parasitic zoonosis of major importance, particularly during pregnancy because of the risk of maternal–foetal transmission. objective: the aim of the study was to estimate the seroprevalence of toxoplasma gondii in pregnant women at the university hospital of cocody and to describe igg/igm serological profiles. methods: we conducted a retrospective cross-sectional study from april 2022 to march 2023 at the immunology laboratory of the university hospital of cocody. toxoplasma gondii-specific igg and igm antibodies were measured by electrochemiluminescence immunoassay and then interpreted according to serological profiles. chi-square test was used to assess the association between igm positivity and pregnancy trimester. results: out of 200 pregnant women, previous infection was observed in 45.0% (igg+/igm−), current infection in 4.0% (igg+/igm+), recent infection in 5.5% (igg−/igm+), and no infection in 45.5% (igg−/igm−). of the women, 83% were tested after the first trimester. the proportion of recent infections (igm+) was higher in the first trimester (17.6%) than in the second (8.2%) and third (7.4%) trimesters, with no statistically significant difference (p = 0.22). conclusion: the seroprevalence of toxoplasmosis remains high, with a non-negligible proportion of women presenting recent or current infection. late initiation of screening highlights the need for strengthened strategies, including early screening, targeted antenatal education and better access to diagnostic tools to reduce the risk of transmission. what this study adds: this study provides updated data on the seroprevalence of gestational toxoplasmosis in côte d’ivoire. thus, it reinforces the need for early screening and targeted health awareness campaigns. keywords: toxoplasmosis; pregnancy; seroprevalence; antenatal screening; immunodiagnosis; maternal–foetal transmission; côte d’ivoire. introduction toxoplasmosis is a common zoonotic infection caused by the protozoan toxoplasma gondii. humans are typically infected by ingesting oocysts that contaminate water or food, or by eating undercooked meat that contains tissue cysts.1,2 in immunocompetent individuals, the infection is often asymptomatic, but it can cause serious disease in immunocompromised people and during pregnancy.3,4 primary maternal infection exposes the foetus to vertical (maternal–foetal) transmission, and may lead to severe complications, including neurological and ocular damage, and even death in utero, especially in early infection.5,6 diagnosis relies mainly on serological measurement of t. gondii-specific igg and igm; igg-avidity testing and, where indicated, amniotic-fluid polymerase chain reaction (pcr) help to refine the timing of infection.7,8,9,10,11 in the absence of pre-existing igg, pregnant women remain susceptible throughout pregnancy. although serological screening is recommended as part of antenatal care in several countries, including côte d’ivoire, its implementation remains uneven, depending on the context and the resources available.12,13 in africa, toxoplasmosis seroprevalence, typically measured by anti-toxoplasma igg seropositivity, varies widely among pregnant women, with rates of 44.4% in cotonou,14 36.1% in rural benin,13 and 51% in morocco.15 in côte d’ivoire, data are scarce, with the most recent study in abidjan reporting a seroprevalence rate of 48%,16 underlining the high level of endemicity. however, these data appear outdated and require re-evaluation, particularly owing to the continued presence of environmental and dietary risk factors. this study was therefore designed to estimate the seroprevalence of toxoplasmosis and to describe igg/igm serological profiles in pregnant women attending the university hospital of cocody. methods ethical considerations the study was carried out with respect for the confidentiality of the data. the information used came exclusively from the laboratory registers and did not include any nominative elements. the study was performed in accordance with the ethical principles of biomedical research and received the approval of the institutional ethics committee (reference number: 042/mshp-cmu/chu-c/dms/rk/25) of the university hospital of cocody. the anonymity and confidentiality of the data were guaranteed. study design a retrospective cross-sectional study of serological data from pregnant women was conducted between april 2022 and march 2023. study location the study took place in the immunology laboratory at the university hospital of cocody. this laboratory is a reference centre for prenatal immunological examinations in the region. study population and selection criteria the study involved pregnant women who had undergone toxoplasma serology as part of their antenatal check-up or as a follow-up to a previous serology. patients for whom the following data were available were included: serological results (igg and igm), maternal age and gestational age. files with missing data were excluded. data collection data were collected from laboratory records using a standardised form. the variables collected included the patients’ age in years, gestational age in weeks of gestation, the reason for the request (routine screening or follow-up), and the results of specific igg and igm assays for t. gondii. in accordance with obstetric recommendations, the trimesters were divided as follows: first trimester (≤ 14 weeks of gestation), second trimester (15–27 weeks of gestation) and third trimester (≥ 28 weeks of gestation). data regarding any treatment and obstetric or neonatal outcomes are not recorded in laboratory registers and were therefore unavailable for analysis. laboratory tests igg and igm serologies were analysed by sandwich electrochemiluminescence immunoassay on a cobas 6000 e601 (roche diagnostics gmbh, mannheim, germany), using elecsys® toxo igg and igm reagents. the interpretation thresholds, according to the manufacturer’s recommendations, were as follows: specific igg was considered negative (< 1 iu/ml), equivocal (1 iu/ml – < 3 iu/ml), or positive (≥ 3 iu/ml); specific igm was considered negative (cut-off index: < 0.8), equivocal (cutt-off index: 0.8 – < 1.0), or positive (cut-off index: ≥ 1.0). serological interpretation based on igg and igm status, four serological profiles were defined: previous infection (igg+/igm–), current infection (igg+/igm+), recent infection (igg–/igm+), and no infection (igg–/igm–).17 equivocal results were interpreted according to the manufacturer’s instructions and were not used for prevalence calculations. data analysis data were entered and processed using microsoft excel 2016. statistical analyses were performed using spss version 26.0. descriptive analysis summarised the sociodemographic, obstetric and serological characteristics of the patients, with results presented in absolute numbers and percentages. comparative analysis was used to explore the association between igm positivity and the trimester of pregnancy, using pearson’s chi-squared (χ2) test; p < 0.05 was considered statistically significant. results general characteristics of the population data were analysed from 200 pregnant women aged 18–44 years, with a predominance in the 25–34 age group (44.0%), followed by those younger than 25 years (31.5%) (table 1). the majority were in the second trimester (42.5%), compared with 40.5% in the third and 17.0% in the first (table 1). table 1: distribution of pregnant women according to demographic, clinical and serological data, immunology laboratory, university hospital of cocody, abidjan, côte d’ivoire, april 2022 – march 2023. serological distribution based on t. gondii igg/igm results, four serological profiles were identified (table 2: previous infection, current infection, recent infection, and no infection). overall igg seroprevalence was 49.0% (45.0% igg+/igm−; 4.0% igg+/igm+). nineteen women (9.5%) had igm-positive profiles, indicating recent or current infection. igm positivity was most frequent in the first trimester (17.6%), followed by the second (8.2%) and third trimesters (7.4%) (table 3), with no statistically significant difference (χ2 = 2.99; df = 2; p = 0.22). table 2: distribution of immunological profiles based on igg and igm results for toxoplasma gondii, immunology laboratory, university hospital of cocody, abidjan, côte d’ivoire, april 2022 – march 2023. table 3: distribution of igm-positive profiles according to the age of pregnancy, immunology laboratory, university hospital of cocody, abidjan, côte d’ivoire, april 2022 – march 2023. reason for requesting serology toxoplasma serology was carried out as part of routine prenatal screening in 169 women (84.5%) and as part of the follow-up of patients at risk in 31 women (15.5%) (table 1). of the 169 screened, only 29 (17.2%) were tested in the first trimester, compared with 74 (43.8%) in the second and 66 (39.0%) in the third (table 4). table 4: distribution of requests for toxoplasma serology according to the age of pregnancy and the reason for carrying out the test, immunology laboratory, university hospital of cocody, abidjan, côte d’ivoire, april 2022 – march 2023. discussion the results of this study show an overall igg seroprevalence of 49.0% among pregnant women. this rate is comparable to those reported in côte d’ivoire in abidjan (48%),16 and in daloa (54.16%),18 as well as in benin in cotonou (44.4%),14 and in rural areas (36.1%),13 indicating sustained t. gondii endemicity in west africa. similar rates have also been observed in other tropical contexts, such as mayotte19 and morocco.15 at the individual level, previous infection (igg+/igm−) is generally considered reassuring for the current pregnancy. however, the significant proportion of women with positive igm (9.5%), including 5.5% with igg−/igm+ and 4.0% with igg+/igm+, is of concern. these serological profiles reflect recent or current infection, with a risk of foetal transmission, particularly during the first two trimesters.6,20 in the absence of an igg-avidity test, it remains difficult to distinguish acute infection, reinfection or persistence of igm.9,10 however, several authors recommend that, in cases of igm positivity, treatment with spiramycin be initiated early, accompanied by close ultrasound monitoring, even in the absence of confirmation by igg-avidity testing.12 furthermore, a substantial proportion of women with no infection (igg−/igm−; 45.5%) constitutes a susceptible group lacking protective igg. although exposure pathways were not assessed in this study, targeted antenatal counselling on food and environmental hygiene, including thorough cooking of meat, remains essential to prevent primary infection during pregnancy. in a highly endemic context such as côte d’ivoire,3,21 this risk must be considered actively. evidence indicates that the risk of vertical transmission rises with advancing gestation, from 10% – 15% in the first trimester to more than 60% in the third, although the most severe clinical forms of congenital toxoplasmosis are linked to early infections.22 cross-analysis between pregnancy trimesters and serological profiles showed that igm-positive cases occurred in all trimesters, with a higher proportion in the first (17.6%) than in the second (8.2%) and third (7.4%). although this difference did not reach statistical significance (χ2 = 2.99; df = 2; p = 0.22), the pattern is consistent with a higher risk of primary infection in early pregnancy, a critical period for the foetus. these results, although observed in the absence of ultrasound or neonatal confirmation, underscore the need for increased vigilance from the first trimester, including, where possible, amniotic-fluid pcr when a recent infection is suspected.11,12,23 our work is a seroprevalence study based on laboratory data; the assessment of clinical management and obstetric or neonatal outcomes was not part of the present analysis. furthermore, our study revealed late initiation of toxoplasmosis screening among pregnant women. only 17.2% were screened in the first trimester, compared with 43.8% and 39.0% in the second and third trimesters, respectively. this delay limits opportunities for early diagnosis and, where indicated, timely preventive measures. in côte d’ivoire, first trimester screening is recommended, but uptake is constrained by out-of-pocket payment, as the test is not covered routinely. this financial barrier delays early screening and supports consideration of measures to improve affordability, for example, through targeted fee exemptions or inclusion of toxoplasmosis serology in the essential antenatal-care package. the main limitation of this study lies in its retrospective design without longitudinal follow-up. seroconversions and pregnancy outcomes (confirmation of foetal infection or neonatal serology) could not be documented.24,25 in addition, clinical management data (e.g. treatment initiation) and obstetric or neonatal outcomes were not recorded in the laboratory registers and were therefore unavailable for analysis, which, together with the absence of igg-avidity testing and amniotic-fluid pcr, limits precise dating of infection phases and the clinical interpretation of the findings.11,12 although igg titres were recorded in the laboratory registers, they were not analysed here because their interpretation remains difficult without avidity testing; however, prior reports suggest that very high igg titres may indicate long-standing immunity.10,16 despite these limitations, our results support strengthening prenatal monitoring practices: the implementation of systematic screening from the first trimester, serological testing in cases of seronegativity or clinical doubt, and better interpretation of serological profiles. countries such as france have demonstrated that early treatment with spiramycin reduces the foetal transmission rate significantly from more than 30% to less than 5%.23 in côte d’ivoire, although national recommendations exist, their application remains inconsistent. it is therefore essential to train medical personnel in serological interpretation, to make igg-avidity and pcr testing available, to raise awareness among pregnant women about preventive measures, and to ensure access to specific treatments. conclusion gestational toxoplasmosis remains a concern in our setting. a notable proportion of pregnant women had serological evidence of recent or current infection. screening frequently begins after the first trimester, limiting opportunities for early diagnosis and timely preventive management. these findings support initiating screening in early pregnancy, strengthening targeted antenatal counselling on food and environmental hygiene, and improving access to confirmatory diagnostics (e.g. igg-avidity testing and, where indicated, amniotic-fluid pcr). acknowledgements the authors thank the entire staff of the immunology laboratory at the university hospital of cocody for their collaboration in data collection. competing interests the authors declare that they have no financial or personal conflicts of interest related to this article. authors’ contributions a.p.v.g.-k. designed the study, coordinated data collection, performed the statistical analysis, and wrote the article. o.r.y. and a.u.a.a. supervised the analysis of serological results, participated in the clinical interpretation, and critically reviewed the article. y.j.s. contributed to data collection and editing. s.r.d. scientifically validated the protocol and supervised the work. all authors, a.p.v.g.-k., o.r.y., a.u.a.a., y.j.s., and s.r.d. have read and approved the final version of the article. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the data that support the findings of this study are not openly available due to reasons of sensitivity and are available from the corresponding author, a.p.v.g.-k., upon reasonable request. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. the article does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the authors are responsible for the results, findings, and content of this article. references smith nc, goulart c, hayward ja, kupz a, miller cm, van dooren gg. control of human toxoplasmosis. int j parasitol. 2021;51(2–3):95–121. 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https://doi.org/10.1016/s0140-6736(04)16412-x robinson e, de valk h, villena i, le strat y, tourdjman m. national perinatal survey demonstrates a decreasing seroprevalence of toxoplasma gondii infection among pregnant women in france, 1995 to 2016: impact for screening policy. eurosurveillance. 2021;26(5):1900710. https://doi.org/10.2807/1560-7917.es.2021.26.5.1900710 ait hamou s, laboudi m. an analytical study on the awareness and practice relating toxoplasmosis among pregnant women in casablanca, morocco. bmc pub. health. 2021;21:1–9. https://doi.org/10.1186/s12889-021-10474-9 vaz rs, rauli p, mello rg, cardoso ma. congenital toxoplasmosis: a neglected disease? current brazilian public health policy. field act sci rep. 2011;3:1–9. abstract introduction methods results discussion acknowledgements references about the author(s) miguel j. teixeira ampath allergy clinic, pretoria, south africa vian pretorius department of internal medicine, chris hani baragwanath academic hospital, johannesburg, south africa robert c.j.g. hunt department of internal medicine, klerksdorp-tshepong complex academic hospital, klerksdorp, south africa sanam morar department of chemical pathology, faculty of health sciences, university of pretoria, pretoria, south africa department of chemical pathology, faculty of health sciences, national health laboratory service, tshwane academic division, pretoria, south africa jamie l. colloty department of microbiology, faculty of health sciences, national health laboratory service, tshwane academic division, pretoria, south africa department of medical microbiology, faculty of health sciences, university of pretoria, pretoria, south africa caleb m. radebe department of internal medicine, faculty of health sciences, potchefstroom hospital, potchefstroom, south africa rajen morar department of internal medicine, faculty of health sciences, school of medicine, university of the witwatersrand, johannesburg, south africa department of internal medicine, faculty of health sciences, charlotte maxeke johannesburg academic hospital, johannesburg, south africa citation teixeira mj, pretorius v, hunt rcjg, et al. a retrospective analysis of uropathogens isolated and antimicrobial susceptibility patterns at a regional hospital in north west province, south africa. afr j lab med. 2025;14(1), a2845. https://doi.org/10.4102/ajlm.v14i1.2845 original research a retrospective analysis of uropathogens isolated and antimicrobial susceptibility patterns at a regional hospital in north west province, south africa miguel j. teixeira, vian pretorius, robert c.j.g. hunt, sanam morar, jamie l. colloty, caleb m. radebe, rajen morar received: 23 apr. 2025; accepted: 16 sept. 2025; published: 30 nov. 2025 copyright: © 2025. the author(s). licensee: aosis. this work is licensed under the creative commons attribution 4.0 international (cc by 4.0) license (https://creativecommons.org/licenses/by/4.0/). abstract background: urinary tract infections are among the most common infections affecting the general population. their high incidence, as well as frequent antimicrobial use, contribute significantly to the development of antimicrobial resistance (amr). objective: to determine the profile and prevalence of uropathogens isolated from urine specimens at a regional hospital and assess susceptibility patterns to commonly used antimicrobials recommended by the national essential medicines list (neml). methods: this was a retrospective evaluation of laboratory reports for all urine specimens submitted between 01 january 2020 and 31 december 2023. results: the most frequently cultured organisms were escherichia coli (n = 1481; 42%); klebsiella pneumoniae (n = 568; 16%); enterococcus faecalis (n = 249; 7%); proteus mirabilis (n = 229; 7%), enterobacter cloacae (n = 137; 4%), and candida albicans (n = 119; 3%). escherichia coli maintained high sensitivity to antimicrobials such as nitrofurantoin (92.2%) and gentamicin (90.6%), whilst k. pneumoniae had decreased sensitivities of 40% and 77%, respectively. cumulative sensitivities of commonly used first-line antimicrobials showed low rates of susceptibility to ciprofloxacin (77%), nitrofurantoin (67%), and amoxicillin/clavulanate (68.7%). conclusion: escherichia coli was the most commonly identified isolate and remains sensitive to nitrofurantoin. it was, however, resistant to ciprofloxacin, amoxicillin/clavulanate, and trimethoprim sulfamethoxazole, as were all the other gram-negative organisms. these sensitivity patterns do not align with the antimicrobials recommended in the current neml guidelines, and highlight the need for targeted therapy and interventions. what this study adds: this retrospective analysis identifies predominant uropathogens’ updated antimicrobial susceptibility profiles, some of which misalign with neml guidelines. insights will guide targeted antimicrobial stewardship, empiric therapy, and local surveillance to curb amr. keywords: urinary tract infections; antimicrobial resistance; escherichia coli; nitrofurantoin; ciprofloxacin. introduction urinary tract infections (utis) represent a significant health concern globally, affecting an estimated 404.6 million individuals in 2019, and contributing to 5.2 million disability-adjusted life years.1 a systematic review of the aetiology and prevalence in sub-saharan africa found the prevalence to be 32.1%, with south africa recording the highest prevalence at 67.1%.2 women have a higher incidence compared with men; in their lifetime, approximately 50% of women will be diagnosed with a uti, with 20% – 40% experiencing recurrences.3 globally, the most common causative organisms of community-acquired utis are gram-negative bacteria, predominantly escherichia coli and klebsiella pneumoniae.4 the high incidence of a positive urinary culture frequently results in the prescription of an antimicrobial, which is often unnecessary, as in the case of asymptomatic bacteriuria. this has the effect of increasing the antimicrobial load in the environment, and exacerbation of the pandemic of antimicrobial resistance (amr), which has steadily increased internationally because of overand misuse of antimicrobials, both in humans and animals.5 concurrently, the development of novel antimicrobial therapies has lagged significantly behind the development of amr, highlighting the need for intensification of antimicrobial stewardship.5,6 a recent systematic analysis of the burden of amr has reported the alarming statistic of 4.95 million deaths worldwide related to amr in 2019 alone.7 the highest mortality rates were reported in western and sub-saharan africa, at approximately 27.3 deaths per 100 000 individuals. the need to implement changes that combat and reduce amr, both globally and in south africa, is therefore urgent.8 once infection has been confirmed, the use of antibiogram to guide treatment of susceptible organisms is recommended by the society for healthcare epidemiology of america, the infectious diseases society of america, and the clinical and laboratory standards institute (clsi). local susceptibility patterns should be determined such that appropriate empiric therapy can be utilised.9 this study aimed to determine the rate of culture positivity for uropathogens and the antimicrobial susceptibility patterns. this is to guide antimicrobial stewardship and prescribing practices at potchefstroom hospital; a regional hospital servicing the jb marks municipality and drainage area in the north west province, south africa. in addition, the results were compared with the national department of health’s standard treatment guidelines and national essential medicines list (neml)10 for empiric antimicrobial management of confirmed utis. the neml currently recommends the following for treatment of an uncomplicated uti10: gentamicin 160 mg intramuscular as a single dose fosfomycin 3 g po [per os, by mouth] as a single dose nitrofurantoin 100 mg po four times a day for 5 days additionally, the neml recommends the following for treatment of a complicated uti10: ciprofloxacin 500 mg po twice a day for 7–10 days for pregnant women, the following has been recommended10: fosfomycin 3 g po as a single dose nitrofurantoin 100 mg po four times a day for 5 days methods ethical considerations the research was approved by the human research ethics committee (medical) of the university of the witwatersrand (reference number: m230502 m230810-a-0011). additionally, institutional approval was granted by the potchefstroom hospital ethics committee. provincial approval was provided by the north west department of health, and national approval by the national department of health. informed consent was not deemed necessary because of the absence of identifiable patient data and information being provided by the national health laboratory service (nhls) academic affairs and research management systems for analysis. data confidentiality was maintained in accordance with the declaration of helsinki and the protection of personal information act of 2013. study design and setting the study was a retrospective analysis of urine microscopy, culture and sensitivity specimens submitted to the nhls at potchefstroom hospital. the nhls in south africa is a public entity providing diagnostic pathology services, research, and health surveillance, accredited by the south african national accreditation system under iso 15189 for medical laboratories. potchefstroom hospital is a state-funded, 335-bed regional healthcare facility in the kenneth kaunda district of south africa’s north west province, offering specialised inand outpatient care to patients in the surrounding area. inclusion and exclusion criteria the urine sample results included in the analysis were those submitted over 4 years between 01 january 2020 and 31 december 2023. all inand outpatients from whom urine for microscopy, culture and sensitivity was collected were included. samples that did not yield a positive microbial culture, cultures that did not have susceptibility testing, samples that had been rejected (for any reason) and cultures that exhibited mixed microbial growth were excluded. no formal prospective study sample size estimates were performed – all available isolates were included. sampling as this is a retrospective records review, the data were acquired from the nhls academic affairs and research management systems database, which functions as a central storage hub for all the primary information gathered by the nhls and its subsidiaries. once the access for the nhls academic affairs and research management systems data was granted, consultation with a data analyst and nhls collaborator was utilised to ensure the validity of the data. since the potchefstroom nhls does not have the capability to culture samples independently, the results of the urine microscopy, culture and sensitivity and subsequent primary data were collated into the nhls academic affairs and research management systems from several sources. the majority of urine samples (n = 3137) were sent to an nhls laboratory in mafikeng for performance of culture. the laboratory at tshepong hospital analysed a further 371 samples, and the remaining 53 samples were sent to nhls charlotte maxeke johannesburg academic hospital for processing and reporting. laboratory analysis the testing facility processed the samples according to their local standard operating procedure. macconkey agar and blood agar plates are inoculated and aerobically incubated for 24 h at 35 °c – 37 °c. the standard practice is that if a specimen collected by voiding or catheterisation shows no growth after overnight incubation, it is reported as no growth. the specimen is then removed from the incubator and stored at room temperature, should further tests be requested by the treating doctors. if growth is detected on the plate, the colonies are counted and the sample provisionally resulted. bacterial identification and antimicrobial susceptibility testing were carried out using the vitek® 2 automated system (biomérieux, marcy l’ètoile, rhône-alpes, france). all antimicrobial susceptibility results were interpreted using the clsi guidelines.11 the laboratory adheres to the standards set out in the clsi guidelines. compliance is ensured internally with numerous quality control programmes and externally with south african national accreditation system audit. statistical analysis the data were entered into stata, basic version 18.0 (statacorp llc, college station, texas, united states). variables included in the analysis were the date the sample was received, the department of sample origin, the organism cultured, and the corresponding antibiotic susceptibility results. the frequency of each organism cultured, and the susceptibility profile of commonly used antimicrobials, were determined. subsequently, the strength of association of antimicrobial efficacy against specific organisms was determined utilising the chi-squared test and fischer’s exact test (for n < 5), where a p-value < 0.05 was considered statistically significant. the clsi guidelines recommend that at least 30 isolates or organism-antibiotic combinations be available for inclusion into the report/analysis. as such, only the five most common organisms, namely e. coli, k. pneumoniae, proteus mirabilis, enterococcus faecalis, and enterococcus faecium, consistently reached the threshold of 30 samples when combined over the study period. the antimicrobials represented were selected based on current standard treatment guidelines, along with commonly used second-line agents. after assessing the susceptibility profile for each, the strength of the association was determined. finally, we described the trends in susceptibility of these organisms over the study period 2020–2023. results a total of 3561 urine cultures were identified during the 4-year period, 48 of which were excluded from the final analysis because of the presence of more than one organism cultured. therefore, a total of 3513 urine cultures were included in the data analysis. the number of positive urine cultures per year is depicted in figure 1. during the study period, the number of samples that were positive for an organism decreased from 1014 samples in 2020 to an average of 833 per year for the following 3 years (832 for 2021; 820 for 2022; and 847 for 2023) (figure 1). figure 1: number of positive urine cultures per year submitted from potchefstroom hospital, south africa, january 2020 – december 2023. as outlined in figure 2, the most frequently cultured organisms were e. coli (n = 1481; 42.2%), k. pneumoniae (n = 568; 16.2%), e. faecalis (n = 249; 7.1%), p. mirabilis (n = 229; 6.5%), enterobacter cloacae (n = 137; 3.9%), and candida albicans (n = 119; 3.4%). figure 2: spectrum of microorganisms isolated from urine samples submitted from potchefstroom hospital, south africa, january 2020 – december 2023. regarding first-line antimicrobials (table 1 and figure 3), the aminoglycosides (gentamicin) would have been appropriate for 87.8% of the cultured organisms (p < 0.001), except k. pneumoniae, which was susceptible in only 77.7% of isolates. more than 90% of all organisms isolated were susceptible to amikacin (p = 0.001). figure 3: antimicrobial susceptibility of the five most common uropathogens isolated at potchefstroom hospital, south africa, january 2020 – december 2023: (a) escherichia coli, (b) klebsiella pneumoniae, (c) enterococcus faecalis, (d) proteus mirabilis, and (e) enterobacter cloacae. table 1: antimicrobial susceptibility patterns of the most commonly isolated organisms in urine specimens at potchefstroom hospital, south africa, january 2020 – december 2023. the cumulative susceptibility to nitrofurantoin was 67.5% (p < 0.001). of the five most commonly isolated organisms, e. coli isolates were the only susceptible organism (92.2%). other isolates, such as k. pneumoniae (40%), e. cloacae (23.4%) p. mirabilis (1.4%), e. faecalis (0%), all had reduced susceptibility to nitrofurantoin. a similar trend of emerging resistance to ciprofloxacin was observed in four of the five most frequently isolated pathogens in this study; p. mirabilis remained the only persistently sensitive organism (92.5%), while reduced susceptibility was detected in e. cloacae (78.8%), k. pneumoniae (78.2%), e. faecalis (76.9%), and e. coli (74.3%). overall, organisms had a cumulative sensitivity of 77.2% (p < 0.001). individual sensitivity to trimethoprim sulfamethoxazole were as follows: e. cloacae (60.3%), p. mirabilis (54%), k. pneumoniae (51.5%), e. coli (36.7%), and e. faecalis (0%). cumulatively, sensitivity to trimethoprim sulfamethoxazole was 42.8% (p < 0.001). significant resistance to amoxicillin/clavulanic acid was present in e. cloacae (3.6% susceptible), k. pneumoniae (65.2% susceptible), and e. coli (79.6% susceptible), with 72.3% of all organisms isolated being susceptible (p < 0.001). there was a similar pattern with ampicillin, with an overall susceptibility of 30.6% of all the cultured organisms (p < 0.001). the susceptibility of individual organisms to amoxicillin/clavulanic acid was also greatly reduced, namely, k. pneumoniae (1.6%), e. cloacae (27.3%), e. coli (28.8%), and p. mirabilis (55.1%). ampicillin showed continued sensitivity of 93.5% for e. faecalis. for second-line antimicrobials, the cumulative susceptibility of the organisms to cefuroxime was 70.6% (p < 0.001), cefotaxime/ceftriaxone (79.5%; p < 0.001), and cefepime (77.9%; p < 0.001) (table 1). the thirdand fourth generation cephalosporins were effective against p. mirabilis, with sensitivities as high as 96% (third generation) and 95.2% (fourth generation). the sensitivity of e. coli to third generation cephalosporins was 82.2%, and to fourth generation, 81.8% and 66.4% (third generation) and 62.8% (fourth generation) for k. pneumoniae. similar rates of resistance were seen in e. cloacae, with susceptibilities of 77.6% (third generation) and 63% (fourth generation). the overall cumulative sensitivity to meropenem was 94.1% (p = 0.004), and 87.5% (p < 0.001) to imipenem. proteus mirabilis was only sensitive in 96.3% of cases to meropenem, and to imipenem in 21.4% of cases. e. coli, k. pneumoniae and e. cloacae had sensitivities of over 83% to bothimipenem and meropenem. the cumulative sensitivity to piperacillin–tazobactam was 87.1% (p = 0.017). escherichia coli was relatively sensitive, at 89.4%, as was p. mirabilis, at 96.4%, whereas k. pneumoniae (76.5%) and e. cloacae (72.7%) both had reduced sensitivity. candida albicans isolates were, as expected, relatively sensitive to fluconazole (92.9%; p < 0.001), amphotericin b (92.9%; p < 0.001) and voriconazole (94.9%; p < 0.001) (table 3). during the study period, pseudomonas aeruginosa was isolated on 33 occasions. susceptibility testing reached the threshold of 30 or more isolates for four antibiotics: amikacin (sensitivity 97%), gentamicin (sensitivity 50%), ciprofloxacin (sensitivity 76%), and piperacillin–tazobactam (sensitivity 94%) (table 2). acinetobacter baumannii was isolated 47 times, but only three antibiotics were tested in 30 or more isolates. these included ciprofloxacin (37% sensitivity), gentamicin (42% sensitivity), and piperacillin–tazobactam (28% sensitivity) (table 2). table 2: antimicrobial susceptibility patterns of pseudomonas aeruginosa and acinetobacter baumannii in urine specimens at potchefstroom hospital, south africa, january 2020 – december 2023. table 3: antimicrobial susceptibility patterns of candida albicans species in urine specimens at potchefstroom hospital, south africa, january 2020 – december 2023. there were significant changes in trend noted for second-, third-, and fourth generation cephalosporins, and nitrofurantoin over the study period (table 4). the organisms demonstrated an increase in sensitivity to cefepime, rising from 73.6% to 78.6% (p = 0.033). in contrast, sensitivity to cefotaxime/ceftriaxone and cefuroxime declined over the study period, decreasing from 79.5% to 75.6% (cefotaxime/ceftriaxone; p = 0.006) and from 68.7% to 63.9% (cefuroxime; p = 0.009). sensitivity to nitrofurantoin also improved over the study period, from 32.8% in 2020 to 37.2% by 2023 (p = 0.013). table 4: trend in cumulative antimicrobial susceptibility rates at potchefstroom hospital, south africa, january 2020 – december 2023. discussion the prevalence of the various uropathogens identified in this study aligns with findings from a recent investigation at rk khan hospital in kwazulu-natal, south africa, which employed a similar sample size and methodology. in this study, e. coli was the predominant pathogen, followed by k. pneumoniae and e. faecalis.12 the authors of the rk khan hospital study found that resistance rates to nitrofurantoin were low in e. coli, at 6.2%, and high in k. pneumoniae, at 61.3%.12 these figures reflect our findings. a multicentre local study conducted by lewis et al. in 2013 forms part of the basis of the neml, in which good cumulative sensitivity to nitrofurantoin (91.7%) was noted.13 the markedly reduced sensitivities of k. pneumoniae (40%), e. cloacae (23.4%), and p. mirabilis (1.4%) highlight emerging resistance and make nitrofurantoin less useful in our setting for empiric treatment. additionally, the clsi states that p. mirabilis is intrinsically resistant to nitrofurantoin, which reflects our results.14 given that resistance patterns tend to differ by geographical location, and the current recommendations by the neml are based partially on american data, we believe it was necessary to verify the neml recommendations for the use of gentamicin. the suggested utilisation of gentamicin in the guidelines was based on a systematic review, published in 2018, in arizona (united states) which showed that 94.5% of pathogens isolated from pooled studies were susceptible to gentamicin.15 at a local level, naidoo et al. also noted a lower cumulative susceptibility of 65% in k. pneumoniae.12 in this study, k. pneumoniae was sensitive to gentamicin in 77.7% of isolates, a 12.7% higher sensitivity rate when compared to naidoo et al. the use of gentamicin may still be an effective empiric antimicrobial in our setting, as this study revealed an 87.8% cumulative sensitivity overall. the infectious diseases society of america guidelines recommend that ciprofloxacin be used as empiric therapy for complicated utis, only if resistance rates are below 10%.16 lewis et al. noted that cumulative sensitivity to fluoroquinolones (94.1%) had been maintained; however, naidoo et al. published alarming ciprofloxacin sensitivity rates, with only 62% of e. coli and 70% of klebsiella spp. found to be sensitive.12,13 the authors highlighted that the reduced sensitivity would lead to high treatment failure rates with empiric ciprofloxacin use.12 another study conducted at 3 military hospital (bloemfontein) in 2011 also demonstrated that cultures in complicated utis showed a reduced sensitivity to ciprofloxacin (41%),17 and the authors recommended against its use at that hospital.17 ciprofloxacin has been reported to promote resistance through horizontal transmission of resistance genes. contributing collateral damage with higher resistance rates in other classes of antimicrobials.17,18 emerging resistance to ciprofloxacin was noted in four of the five common pathogens in the current study, specifically e. cloacae (79%), k. pneumoniae (78%), e. faecalis (77%) and e. coli (74%), with p. mirabilis remaining susceptible, at 92.5%. ciprofloxacin was once the preferred treatment for utis, but this is no longer the case in our setting. lewis et al. also reported reduced cumulative sensitivity rates of 82.8% to amoxicillin–clavulanate in 2013,13 and similarly, naidoo et al. found low sensitivity in both k. pneumoniae (58%) and e. coli (70%).12 a high level of resistance towards amoxicillin clavulanate, with reduced cumulative sensitivity of 72.3%, was also noted in our study, indicating that these agents may not be effective towards the two most prevalent uropathogens, namely e. coli and k. pneumoniae. whereas lewis et al. reported a high rate of sensitivity to cefuroxime (93.4%) in 2013,13 naidoo et al. showed that sensitivity to third-generation cephalosporins was only 74% in e. coli, and 58% in k. pneumoniae, which correlated with the reduced sensitivities in this study.12 the inherent resistance to cephalosporins found in e. faecalis has led the nhls to forgo susceptibility testing to cephalosporins.19 enterobacter cloacae has been reported to be intrinsically resistant to amoxicillin–clavulanate by the clsi, which is in line with our study (96.4%).14 furthermore, this organism is an ampc-producer and is therefore at high risk of developing ampc enzyme overproduction, subsequently leading to third-generation cephalosporin resistance.14 although we have variable sensitivities in our beta-lactams, clsi recommend against the use of third-generation cephalosporins and piperacillin–tazobactam because of the risk of treatment failure.14 naidoo et al. demonstrated meropenem sensitivities of 95% for k. pneumonia and 100% for e. coli.12 these findings mirrored the cumulative sensitivity of our study, of 94.1% overall. similarly, the authors reported the sensitivity to piperacillin–tazobactam to be 76% for k. pneumoniae and 95% for e. coli, which is consistent with our findings of 76.5% for k. pneumoniae and 89.4% for e. coli.12 there was a reduced cumulative sensitivity to trimethoprim–sulfamethoxazole at 42.8%. these figures were mirrored by lewis et al., with a cumulative sensitivity of 44.3%, and naidoo et al., with 39% e. coli and 52% k. pneumoniae susceptibility.12,13 the infectious diseases society of america guidelines recommend the use of trimethoprim–sulfamethoxazole for empiric therapy for uncomplicated utis only if local uropathogen resistance rates do not exceed 20%.16 in our setting, and considering sensitivities from other local studies, trimethoprim–sulfamethoxazole would unlikely be suitable as an empiric agent.12,15,17 fosfomycin susceptibility testing is not routinely performed by nhls north west, and thus no analysis was performed. future therapies include the possibility of using vaccines to prevent recurrent utis. a meta-analysis by mak et al. in 2024, showed limited evidence for this approach, but that more rigorous research is needed in order to determine the value of vaccines and oral bacterial lysates immunostimulants as an option for prophylactic treatment.20 we recommend that e. coli be considered as the culprit pathogen in both in-and outpatient utis and, in compliance with most international guidelines, nitrofurantoin should be used as first-line agent. this does not negate the need for culture and sensitivity to be performed, as well as de-escalation or escalation of therapy, as needed. if parenteral therapy is necessary, most organisms remain susceptible to gentamicin, and although we do not have data for fosfomycin at potchefstroom hospital, other local data support its use for empiric therapy. limitations this study was a retrospective analysis and some of the samples were processed at various sites which could have affected the results. while the study has a significant sample size, the data was collected from a single site and is only truly descriptive of the microbiome at potchefstroom hospital, and these findings may not be generalisable to other institutions or to the broader population. similarly, data were collected from patients and samples at a regional hospital and, therefore, may not be fully representative of the type of infections that would be encountered in the primary healthcare setting. furthermore, carbapenemase-resistant enterobacterales and extended spectrum beta-lactamase organisms were not analysed, as it was beyond the scope of our article. further research is recommended to evaluate the prevalence and morbidity of these organisms. being a laboratory-based study, demographic and clinical data were not collated and as such, we cannot make recommendations with regard to tailoring of antimicrobial therapy to specific clinical scenarios. in addition, it was not possible to differentiate between organism colonisation or contamination, asymptomatic bacteriuria, and true infection, and whether they were communityor hospital-acquired isolates. conclusion in this study, we have determined the prevalence of the most common uropathogens isolated from samples submitted to potchefstroom hospital. escherichia coli remains the most prevalent uropathogen; however, our findings show that e. coli and most other organisms isolated from urine samples at potchefstroom hospital were not susceptible to the first-line antimicrobial agents as suggested by the neml. ciprofloxacin is not recommended as the first-line agent for any uti at potchefstroom hospital, unless susceptibility is proven. this study also provides some recommendations for secondand third-line antimicrobials while awaiting susceptibility testing. in general, the other recommended first-line antimicrobial agents, gentamicin, nitrofurantoin, and fosfomycin are likely to treat most utis empirically. given the high rate of drug-resistant uropathogens, careful selection of empiric therapy is vital to ensure acceptable clinical outcomes and to prevent further development of amr. this study has provided an updated antibiogram for uropathogens isolated at potchefstroom hospital in the north west province. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. credit authorship contribution miguel j. teixeira: conceptualisation, data analysis, writing – review & editing. vian pretorius; project administration, data curation, resources, formal analysis, writing – original draft. robert c.j.g. hunt: data curation, writing original draft. sanam morar: data curation, writing original draft. jamie l. colloty: writing – review & editing. caleb m. radebe: writing – review & editing, supervision. rajen morar: writing – review & editing, supervision. all authors reviewed the article, contributed to the discussion of results, approved the final version for submission and publication, and take responsibility for the integrity of its findings. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors data availability the data that support the findings of this study are not openly available due to reasons of sensitivity and are available from the corresponding author, m.j.t. upon reasonable request. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. the article does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the authors are responsible for this article’s results, findings, and content. references zeng z, zhan j, zhang k, chen h, cheng s. global, regional, and national burden of urinary tract infections from 1990 to 2019: an analysis of the global burden of disease study 2019. world j urol. 2022;40(3):755–763. https://doi.org/10.1007/s00345-021-03913-0 mwang’onde bj, mchami ji. the aetiology and prevalence of urinary tract infections in sub-saharan africa: a systematic review. j health biol sci. 2022;10(1):1–7. https://doi.org/10.12662/2317-3076jhbs.v10i1.4501.p1-7.2022 fourie jl, claassen fm, myburgh jj. causative pathogens and antimicrobial resistance in community-acquired urinary tract infections in central south africa. s afr med j. 2021;111(2):124–128. https://doi.org/10.7196/samj.2021.v111i2.14905 paul r. state of the globe: rising antimicrobial 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by the infectious diseases society of america and the society for healthcare epidemiology of america. clin infect dis. 2016;62(10):e51–e77. https://doi.org/10.1093/cid/ciw118 south african national department of health. standard treatment guidelines and essential medicines list for south africa: hospital level adults [homepage on the internet]. 5th ed. 2019 [cited 2024 oct 10]. available from: https://www.gov.za/documents/standard-treatment-guidelines-and-essential-medicines-list-south-africa-hospital-level clinical and laboratory standards institute (clsi). m39: analysis and presentation of cumulative antimicrobial susceptibility test data. 5th ed. wayne, pa: clsi; 2022. naidoo a, kajee a, mvelase nr, swe-han ks. antimicrobial susceptibility of bacterial uropathogens in a south african regional hospital. afr j lab med. 2023;12(1): 1920. https://doi.org/10.4102/ajlm.v12i1.1920 lewis da, gumede lye, van der hoven la, et al. antimicrobial susceptibility of organisms causing community-acquired urinary tract infections in gauteng province, south africa. s afr med j. 2013;103(6):377–381. https://doi.org/10.7196/samj.6722 clinical and laboratory standards institute (clsi). performance standards for antimicrobial susceptibility testing. 35th ed. clsi supplement m100. wayne, pa: clsi; 2025. goodlet kj, benhalima fz, nailor md. a systematic review of single-dose aminoglycoside therapy for urinary tract infection: is it time to resurrect an old strategy? antimicrob agents chemother. 2019;63(1):e02165-18. https://doi.org/10.1128/aac.02165-18 gupta k, hooton tm, naber kg, et al. international clinical practice guidelines for the treatment of acute uncomplicated cystitis and pyelonephritis in women: a 2010 update by the infectious diseases society of america and the european society for microbiology and infectious diseases. clin infect dis. 2011;52(5):e103–e120. https://doi.org/10.1093/cid/ciq257 bosch fj, van vuuren c, joubert g. antimicrobial resistance patterns in outpatient urinary tract infections – the constant need to revise prescribing habits. s afr med j. 2011;101(5):328–331. https://doi.org/10.7196/samj.4346 dalhoff a. global fluoroquinolone resistance epidemiology and implications for clinical use. interdiscip perspect infect dis. 2012;2012:976273. https://doi.org/10.1155/2012/976273 spacek la. johns hopkins abx guide | enterococcus [homepage on the internet]. 2022 [cited 2024 oct 10]. available from: https://www.hopkinsguides.com/hopkins/view/johns_hopkins_abx_guide/540203/all/enterococcus?refer=true mak q, greig j, dasgupta p, malde s, raison n. bacterial vaccines for the management of recurrent urinary tract infections: a systematic review and meta-analysis. eur urol focus. 2024;10(5):761–769. https://doi.org10.1016/j.euf.2024.04.002 abstract introduction methods results discussion acknowledgements references about the author(s) abdulazeez a. anjorin department of microbiology, faculty of science, lagos state university, ojo, nigeria barakat a. adepoju department of microbiology, faculty of science, lagos state university, ojo, nigeria citation anjorin aa, adepoju ab. serologic evidence of seasonal influenza a and b in hiv patients on combined antiretroviral therapy in lagos, nigeria. afr j lab med. 2020;9(1), a1048. https://doi.org/10.4102/ajlm.v9i1.1048 original research serologic evidence of seasonal influenza a and b in hiv patients on combined antiretroviral therapy in lagos, nigeria abdulazeez a. anjorin, barakat a. adepoju received: 22 may 2019; accepted: 01 oct. 2020; published: 21 dec. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: influenza and hiv are endemic in nigeria but there is no epidemiological data on the co-infection of influenza a and b among hiv patients. objective: we investigated seasonal influenza a and b, and co-infection among hiv patients on combined antiretroviral therapy (cart) in lagos, nigeria. methods: in a prospective cross-sectional study, clear sera collected from 174 hiv-positive patients between august and september 2018 were analysed for immunoglobulin m-specific antibodies to seasonal influenza a subtypes h1n1 and h3n2, and influenza b by enzyme immunoassay. results: a total of 39.7% (69/174) of hiv patients were seropositive for influenza a or b viruses with 84.1% (58/69) being positive for influenza a, 13.04% (9/69) seropositive for both influenza a and b, and only 2.9% (2/69) positive for influenza b mono-infection. median age was 44 (mean 45, mode 40, range 18–74) years. the 41–50 years age group had the highest seroprevalence (39.1%; 27/69). seropositivity was highest among women (65.2%; 45/69). a total of 88.4% (61/69) of hiv patients seropositive for influenza a or b were on fixed dose cart, while 73.9% (51/69) were virologically suppressed. furthermore, 27.5% (19/69) were immunocompromised, of which 21.1% (4/19) were severely immunosuppressed (cluster of differentiation 4 < 200 cells/mm>3). conclusion: influenza a and b was prevalent among hiv patients on cart, which may predispose them to life-threatening complications. we recommend strong advocacy on the need to reduce the risk of exposure to influenza and for the provision of an influenza vaccine in nigeria. keywords: influenza a; influenza b; hiv co-infection; seroprevalence; lagos. introduction in humans, seasonal influenza viruses cause an annual 3–5 million illnesses and 290 000–650 000 deaths worldwide.1 the majority of the deaths and severe illness occur in lowand middle-income countries2 especially in africa where there is little or scanty information on epidemiological surveillance. influenza virus infection can be self-limiting and could vary in severity from asymptomatic to fatal disease arising from medical complications. these complications include pneumonia, meningitis and worsening of underlying medical conditions, mostly in immunocompromised individuals.1 on the other hand, hiv is a retrovirus that spreads through the body rapidly, causing damage to the immune system by attacking host cluster of differentiation 4 (cd4) cells with resultant aids.3 in 2017, hiv-related causes killed about 1 million people worldwide, having claimed over 35 million lives in the last three decades.4 according to the world health organization,4 as of 2018, 1.8 million newly infected individuals were added to the 36.9 million people living with hiv, with 25.7 million (> 66%) infected people living in the sub-saharan africa region.4,5 there are about 197 997 230 people living in nigeria, equivalent to 2.57% of the total world population (www.worldometers.info), with up to 3.2 million people living with hiv.6 this is the second-largest hiv epidemic after south africa and represents 9% of the total population living with hiv globally, among whom 1 million people are on antiretroviral therapy (art).6,7,8 lagos state with a total population of about 21 million has a hiv prevalence of 2.2%. influenza viral infection is a common cause of respiratory illness among hiv-infected persons in whom it can be more severe and prolonged.9 hiv has also been described as the most common underlying risk factor in patients with respiratory infection.10 influenza virus co-infections in hiv-infected patients cause a 4–8 times higher incidence of hospitalisation and death compared to non-hiv-infected individuals.11 the reduction in the number of cd4 t-cells due to infection with hiv causes immunodeficiency and thus higher susceptibility to complications of influenza in hiv patients.12 in general, the immunogenicity of influenza viral infections in hiv-infected patients is directly proportional to the cd4 cell count and correlates negatively to the viral load.13 studies have also shown that better immune health (i.e. higher cd4 cell counts and suppressed viral load of hiv patients on combined antiretroviral therapy [cart]) in comparison with immunocompromised patients made them less likely to experience prolonged shedding of influenza virus.9 however, a lack of epidemiological data on seasonal influenza in hiv-positive individuals is a major problem preventing the development of national policies on influenza preventive strategies in sub-saharan africa, including nigeria. in south africa, 44% of influenza positive patients having acute respiratory infections are hiv-infected.10 no data is available on influenza virus infection in hiv patients from nigeria and that underscores the importance of this study. serological assays usually complement epidemiological and clinical investigations in the detection and identification of influenza viruses. they measure antibodies developed in response to infection with antigenically novel and seasonal influenza viruses.14 the enzyme-linked immunosorbent assay method has been used in different studies for detecting serological evidence of the influenza virus in humans15,16,17 because it is apt, easy, fast, can be automated and is commercially available for large-scale sero survey, targeting specific antibodies against different subtypes of influenza virus. it is also affordable for sero epidemiology in poor and limited resource settings. this study was therefore designed to investigate seasonal influenza a and b virus infections in hiv patients on cart in lagos, nigeria. methods ethical considerations permission was sought from the head of the aids prevention initiative in nigeria (apin) clinic where the samples were collected after tendering the ethical approval (reference no.: lrec.06/10/1030) obtained from the health research and ethics committee of the lagos state university teaching hospital (lasuth). study area, population and design a prospective cross-sectional study was designed. outpatients attending the aids prevention initiative in nigeria (apin)-lagos university teaching hospital clinic who were previously laboratory confirmed to be hiv-positive were recruited for this study from august 2018 to september 2018. apin-lagos university teaching hospital clinic is a large university-based hiv clinic in lagos, nigeria, with about 15 000 patients currently enrolled in lagos. after explaining the concept of the study, a total of 174 hiv patients, including 165 on cart, who were unvaccinated for influenza and gave both oral and written informed consent, were bled by venepuncture. demographic and clinical data were appropriately collected with a designed questionnaire. results of further laboratory analyses including cd4 count, rna viral load and cart regimen were obtained from the patients’ clinical records. sample collection and treatment approximately 5 ml of whole blood samples were collected into sterile plain bottles. they were stored in sample coolers stacked with ice packs before being conveyed to the laboratory. the samples were centrifuged at 704 × g for 15 min to obtain clear sera that were aliquoted into labelled sterile plain cryovial tubes. the sera were then stored away at −30 °c until ready for serological analysis. laboratory analysis clear sera were analysed for the detection and quantitative determination of immunoglobulin m antibody specific for influenza virus by enzyme-linked immunoassay (demeditec diagnostics gmbh, kiel, germany) in the department of microbiology (virology research) laboratory, lagos state university, ojo. following manufacturer’s instructions, sufficient amounts of micro titre wells were prepared for the standards, controls and samples as well as for the substrate blank. the samples were diluted with ready-to-use sample diluent provided with the test kit in the ratio 1:100 (2 µl serum + 200 µl sample diluents). assay absorbance was read at a wavelength of 450 nanometres with an emax precision microplate reader (molecular devices, llc, san jose, california, united states). sample results were compared with the included standards and controls and were interpreted based on the assay standard curve as recommended by the manufacturer. it should however be noted that positive immunoglobulin m antibody specific for influenza virus implies a recent immunological reaction to circulating live influenza strains as a result of infection since nigeria does not currently practise influenza vaccination. statistical analysis raw data were entered into microsoft excel, version 2013 (microsoft corporation, redmond, washington, united states). descriptive statistics was performed while inferential statistics was analysed with chi-square at p less than 0.05 for statistical significance using graphpad prism version 8.0.1 (graphpad software inc., san diego, california, united states). results out of the 174 hiv-positive patients tested, 69/174 (39.7%) were seropositive for influenza a or b viruses, with 58/69 (84.1%) positive for influenza a, 2/69 (2.9%) for influenza b, and 9/69 (13.0%) for both influenza a and b (table 1). the median age of patients was 44, mean 45, mode 40, and range 18–74 years. seropositivity was higher in female patients (45/69; 65.2%) compared to male patients (17/69; 24.6%). a total of 51/69 (73.9%) of the patients were virologically suppressed with hiv rna under 400 copies/ml, and 19/69 (27.5%) were immunocompromised (cd4 < 400 cells/mm3). out of the immunocompromised patients, 4/19 (21.1%) were severely immunosuppressed (cd4 < 200 cells/mm3). 61/69 (88.4%) of hiv patients seropositive for influenza a or b were on fixed dose cart compared to those that were seronegative: 96/105 (91.4%) (p < 0.001). table 1: characteristics of hiv patients positive for influenza a and b viruses in 2018 in a university-based hiv clinic in lagos, nigeria. the most commonly prescribed cart used as a single-pill combination at the apin clinic included atazanavir, azidothymidine, efavirenz, lamivudine, lopinavir or ritonavir, nevirapine, and tenofovir (table 2). the majority of patients received a combined therapy of tenofovir, lamivudine and efavirenz (74/174; 42.5%) or azidothymidine, lamivudine and nevirapine (64/174; 36.8%). table 2: commonly prescribed combined antiretroviral therapy regimens in hiv patients positive for influenza virus immunoglobulin m antibodies in 2018 in a university-based hiv clinic in lagos, nigeria. seroprevalence was highest in patients aged 41–50 years (39.1%; 27/69), with no detections in the age groups 61–70 and 71–80 years (figure 1). figure 1: age (years) distribution of hiv patients seropositive to influenza in 2018 in a university-based hiv clinic in lagos, nigeria. discussion in this study, we described the recent infection of seasonal influenza a and b, and their co-infection, among hiv patients on cart in lagos, nigeria, with a high predominance of influenza a virus. while a high proportion of the patients were virologically suppressed, having hiv rna under 400 copies/ml, 27.5% of them were immunocompromised while 21.1% were severely immunosuppressed (cd4 < 200 cells/mm3). the hospital population-based study revealed an overall seasonal influenza serological prevalence of 39.7% (69/174) that will serve as a baseline for other studies in nigeria. we defined the seasonal influenza virus as any type or subtype of influenza virus including influenza a or b that are commonly circulating in our environment at any particular point in time as against a pandemic influenza virus. to our knowledge, this is the first study on clinical and sero-epidemiological prevalence of influenza viruses in hiv patients on cart in nigeria. the seroprevalence rate is comparable with the 31% serological response recorded in hiv-positive individuals in miami, united states.18 a significantly lower seroprevalence rate of 14.7% was recorded in taiwan.19 a higher prevalence rate of 71.6% for influenza-specific antibody responses in hiv patients was recorded in rome, italy.20 in africa, lower prevalence rates of 11% and 24.5% were also recorded in malawi and kenya.21,22 the disparity observed in the prevalence rates could be attributed to differences in many factors including sample size and the assay type used. our investigation further revealed that 84.1% (58/69) were immunoglobulin m seropositive for influenza a, indicating an epidemiological predominance of the influenza a over the influenza b virus during the study period in lagos, nigeria. in a study conducted in malawi,21 it was also reported that in low-resource settings with high hiv prevalence, there is a high prevalence of influenza illness and greater risk of hospitalisation among hiv-infected persons which supports the high prevalence rate of influenza a detected in this study. interestingly, 13.04% (9/69) of the hiv patients were seropositive for both influenza a and b viruses. co-infection of influenza a and b viruses in hiv patients may exacerbate the immunocompromised status by further impairing t-cell responses with resultant fatal consequences in such individuals. this position agrees with previous studies that influenza in hiv-infected persons leads to increased and prolonged hospitalisation, and an elevated risk of in-hospital death.9,11 furthermore, it is evident from previous reports that even mono-infection due to influenza a (h1n1) pandemic09 (now seasonal influenza virus) caused severe pneumonia leading to acute respiratory distress syndrome and multiple organ dysfunction associated with deaths.23,24 hence, simultaneous detection of influenza a and b in patients calls for serious public health intervention to ameliorate the menace of influenza co-infection especially in people living with hiv. our study demonstrated the highest 39.1% seroprevalence of influenza among hiv patients in the age group 41–50 years which disagrees with the united states centers for disease control and prevention report that persons older than 65 years are more likely to be at risk of the influenza virus.25 our observation is however supported by earlier findings in south africa in 2009 to 201111 and the united states in 201326 which reported that on account of hospitalisation and mortality rates, older adults do not have the highest rate of infection and do not represent the main contributors to local outbreaks. based on gender, a higher 65.2% seroprevalence was detected among women compared to 24.6% among men in our study, which is consistent with findings from malawi27 that reported higher seroprevalence among women. in contrast, a united states study recorded a higher seroprevalence in men than in women.9 a suggested reason for the higher seroprevalence among women could be the overwhelming burden of hiv that affects more women in sub-saharan africa making them more susceptible to influenza infection. of the 19/69 (27.5%) immunocompromised patients seropositive for the influenza virus, 4/19 (21.1%) were severely immunosuppressed (cd4 < 200 cells/mm3). however, the role of cd4 and its impact on influenza in hiv-positive patients is poorly understood, partly due to their heterogeneity and lack of epitope-specific systems.28 contrary to studies in south africa from 2009 to 201111 and 2012–2016,29 the prevalence of influenza in the current study was higher among individuals with a cd4 count over 400 compared to those with a lower cd4 count. however, it should be noted that the south african study11 reported influenza based on detection of virus nucleic acids in symptomatic patients whereas the current study detected with serology a fraction of patients who had asymptomatic influenza infections. similar to this study, a study in the united states from 2010 to 20119 recorded a higher prevalence of influenza in patients with a cd4 count over 200 cells/mm3 than those with less than 200 cells/mm3. one possible explanation for these observations is the traditionally accepted role of influenza-specific cd4 t-cells in providing help to b-cells for the production of high-quality antibodies.30 hence, depletion of cd4 t-cells prior to influenza virus infection may be responsible for the low prevalence observed. people with a viral load under 400 copies/ml (73.9%) had the highest seroprevalence of influenza virus. this agrees with a study in the united states between 2010 and 20119 that detected influenza virus in 85% of virologically suppressed (hiv rna < 400 copies/ml) patients. this indicates that reduced viraemia of hiv does not reduce the susceptibility of hiv patients to the influenza virus. among the 165 hiv patients on a fixed dose combination of some regularly prescribed cart including tenofovir, emtricitabine, azidothymidine, atazanavir, efavirenz, nevirapine, and lopinavir or ritonavir, 61 (37%) were seropositive for influenza a and b viruses. the direct relationship of cart on patients and their seropositivity to influenza is not well understood. nonetheless, our study demonstrated that hiv patients on cart were susceptible to recent infection with influenza viruses. in support of this finding, a systematic review done on co-infections and comorbidities of influenza in africa from 1900 to 20132 estimated an increased risk of influenza-associated mortality among hiv-positive individuals even after the widespread introduction of cart. also, a study in the united states from 2011 to 201218 concluded that regardless of influenza vaccination and control of viral load with cart, hiv-infected patients are at an elevated risk of acquiring seasonal influenza infection. however, in malawi, the introduction of highly active antiretroviral therapy has led to the reduction of influenza-related complications among hiv-infected patients.31 limitations one of the limitations of this study is that positive samples for the influenza virus by enzyme linked immunoassay could not be further tested by polymerase chain reaction due to the cost implication. also, serological evidence from different hiv treatment centres are necessary for representative data in nigeria. another limitation is that an hiv-uninfected control group was not included in the study for possible comparison of the prevalence rates of influenza. finally, it should be noted that the study was conducted over one influenza season and therefore the impact of seasonal changes in influenza strains was not studied. rather our study has provided sero-epidemiological data on the commonly circulating seasonal influenza virus in hiv patients. conclusion this study demonstrated using serology that hiv patients on cart are susceptible to both influenza a and b viruses. it however revealed low seroprevalence of influenza b mono-infection, and influenza a and b co-infections among hiv patients. viral co-infections may further impair the already compromised immune system of hiv patients. strong advocacy on the need to reduce risk of exposure to influenza, and for provision of an influenza vaccine in nigeria, is recommended to prevent or reduce the complications of viral co-infection. acknowledgements we sincerely acknowledge all the patients who volunteered to be part of this study. we acknowledge the lagos state university teaching hospital (lasuth) directorate of clinical services and training and the hospital research ethics committee for the approval of the study research proposal. we are grateful to the management and staff of apin-lagos university teaching hospital for the approval for sample collection. special thanks to all the laboratory technologists who supported the student’s research work. many thanks to all the senior faculty members who criticised and reviewed the initial manuscript. the abstract for this study was presented at the 52nd miami winter symposium 2019: evolving concepts in hiv & emerging viral infections, organised by elsevier, university of miami miller school of medicine, international union of biochemistry and molecular biology in florida, united states (27–30 january 2019). competing interests the authors have declared that no competing interest exists. authors’ contributions a.a.a. was responsible for experimental design and research supervision, data analysis, laboratory investigation and manuscript writing. b.a.a. was responsible for sample collection, laboratory investigation, data analysis and manuscript writing. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references who. influenza (seasonal) signs and symptoms [homepage on the internet]. geneva: who; 2019 [cited 2019 feb 06]. available from: 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years and older & influenza [homepage on the internet]. atlanta, ga: centers for disease control and prevention, national center for immunization and respiratory diseases (ncird); 2018 [cited 2019 feb 07]. available from: https://www.cdc.gov/flu/about/disease/65over.htm lambert nd, ovsyannikova ig, pankratz vs, jacobson rm, poland ga. understanding the immune response to seasonal influenza vaccination in older adults: a systems biology approach. exp rev vaccin. 2012;11(8):985–994. https://doi.org/10.1586/erv.12.61 ho a, mallewa j, peterson i, et al. epidemiology of severe acute respiratory illness and risk factors for influenza infection and clinical severity among adults in malawi, 2011–2013. am j trop med hyg. 2018;99(3):772–779. https://doi.org/10.4269/ajtmh.17-0905 sant aj, mcmichael a. revealing the role of cd4(+) t cells in viral immunity. j exp med. 2012;209(8):1391–1395. https://doi.org/10.1084/jem.20121517 tempia s, walaza s, moyes j, et al. attributable fraction of influenza virus detection to mild and severe respiratory illnesses in hiv-infected and hiv-uninfected patients, south africa, 2012–2016. emerg infect dis. 2017;23(7):1124. https://doi.org/10.3201/eid2307.161959 crotty s. follicular helper cd4 t cells (tfh). ann rev immunol. 2011;29:621–663. https://doi.org/10.1146/annurev-immunol-031210-101400 jambo kc, sepako e, glennie sj, et al. naturally-acquired influenza-specific cd4+ t-cell proliferative responses are impaired in hiv-infected african adults. plos one. 2012;7(6):e38628. https://doi.org/10.1371/journal.pone.0038628 abstract introduction methods results discussion acknowledgements references about the author(s) antony s. katayi department of medical laboratory, faculty of laboratory, bungoma county referral hospital, bungoma, kenya phidelis m. marabi department of medical laboratory sciences, school of health sciences, kisii university, kisii, kenya stanslaus k. musyoki department of medical laboratory sciences, school of health sciences, southeastern kenya university, kitui kenya citation katayi as, marabi pm, musyoki sk. feasibility analysis of the sicklecheck™ test kit for rapid screening of sickle cell disease at a county referral hospital in kenya. afr j lab med. 2025;14(1), a2739. https://doi.org/10.4102/ajlm.v14i1.2739 original research feasibility analysis of the sicklecheck™ test kit for rapid screening of sickle cell disease at a county referral hospital in kenya antony s. katayi, phidelis m. marabi, stanslaus k. musyoki received: 24 dec. 2024; accepted: 14 may 2025; published: 29 july 2025 copyright: © 2025. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the burden of sickle cell disease in western kenya is substantial; however, there is limited research on the effectiveness of rapid diagnostic tests for the condition. objective: this study evaluated the feasibility of using the sicklecheck™ rapid test kit for detecting sickle cell disease at bungoma county referral hospital, kenya. methods: a cross-sectional study was carried out between october 2023 and february 2024 and included both healthy children and children with a known haemoglobin phenotype. the sicklecheck™ rapid screening test was compared to bio-rad™ high-performance liquid chromatography, which served as the reference standard. sensitivity, specificity, positive predictive value, negative predictive value, and overall accuracy were calculated using medcalc™ statistical software. results: the study involved 194 children (98 girls and 96 boys), aged between 10 weeks and 15 years, with haemoglobin profiles sickle cell negative (n = 78), sickle cell trait (n = 21), and sickle cell disease (n = 95). the sicklecheck™ test demonstrated sensitivity, specificity, negative predictive value, and accuracy exceeding 97%, with a positive predictive value of 94.18% for haemoglobin a. it also effectively distinguished between normal (sensitivity 97.44%, specificity 99.14%), carrier (sensitivity 90.48%, specificity 98.27%), and disease (sensitivity 98.95%, specificity 98.99%) phenotypes. conclusion: based on the findings in this study, sicklecheck™ could be a reliable point-of-care diagnostic tool for sickle cell disease. the encouragement of healthcare facilities, especially in resource-limited settings, to adopt the sicklecheck™ rapid test for routine screening and diagnosis of sickle cell disease is recommended. what this study adds: this study highlights the diagnostic reliability of the sicklecheck™ rapid test in accurately identifying and differentiating sickle cell disease, trait, and normal haemoglobin phenotypes, reinforcing its potential role in strengthening early diagnosis efforts in clinical settings. keywords: sickle cell disease; rapid test; testing; accuracy; kenya. introduction sickle cell disease (scd) is a chronic, severely debilitating autosomal recessive disorder caused by a single-point mutation in the β-globin gene. under hypoxic conditions, sickle haemoglobin undergoes polymerisation, leading to the reversible deformation of red blood cells into a sickle shape.1 globally, scd is recognised as a major public health concern, with an estimated 300 000 to 400 000 infants born annually with the disease.2 the burden is heaviest in low-resource settings, where access to early diagnosis and effective care remains limited.3 sub-saharan africa accounts for approximately 75% of global scd births,4 and studies report that 50% to 80% of affected children in the region die before the age of five.2 screening of carriers in at-risk populations has been suggested since both parents as carriers carry a one in four likelihood of passing a homozygous offspring.5 the world health organization has recently called on governments and international organisations to implement strategies for precise documentation of morbidity and mortality. this effort aims to address haemoglobinopathies, which are becoming more prevalent in society, posing a growing challenge to healthcare systems.6 although several advances have been made in molecular diagnostics, their applicability in resource-constrained settings remains limited.7 there is a growing body of literature emphasising the need for simplified, accessible, and scalable testing options of scd, particularly in high-burden areas.8 there is a need for an affordable and efficient method for accurate and early detection, given the high prevalence of haemoglobin anomalies in malaria-endemic regions such as kenya.5 in kenya, scd and other haemoglobinopathies are highly prevalent, particularly in malaria-endemic regions such as western kenya and the coastal areas.5 recent data from bungoma county show a sickle cell trait prevalence of 14.28% among blood donors.9 although homozygous scd poses a significant burden in western kenya, studies on the effectiveness of rapid testing for the condition are scarce. this study assessed the practicality of using the sicklecheck™ test for rapid scd screening at bungoma county referral hospital, kenya. this study specifically examined the sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv), and accuracy of sicklecheck™ in comparison to bio-rad™ high-performance liquid chromatography (hplc), the reference method. methods ethical considerations the study received approval from the jaramogi oginga odinga teaching and referral hospital institutional scientific and ethical review committee (jootrh – iserc) (reference number iserc/jootrh/742/23). authorisation to conduct the research was granted by the national commission for science, technology, and innovation (reference number nacosti/p/23/29577). permission for data collection was obtained from bungoma county referral hospital (reference number bdh/s&e/11/1/[256]). the study’s objectives, procedures, potential risks, and benefits were clearly explained to both the participants and their parents or legal guardians. written informed consent was obtained from the parents or legal guardians of all participants. participation was entirely voluntary, and withdrawal from the study was permitted at any stage without penalty. to ensure data confidentiality, digital files were secured with password protection, while physical documents were stored in a locked and restricted-access location. no personally identifying information was collected or retained. study area the study took place at bungoma county referral hospital, bungoma, kenya, from 11 october 2023 to 22 february 2024. bungoma county referral hospital is a level six hospital situated in the administrative centre of bungoma county, western kenya. it serves as the primary referral facility for the county and its surrounding areas, providing healthcare to a population of 1 670 570 within a 2206.9 km2 region.10 each year, around 3000 children attend the paediatric clinic at bungoma county referral hospital, with scd affecting approximately 5% of them, as reported in the hospital’s records.11 sickle cell disease accounted for 7.8% of all paediatric admissions and was ranked among the top three causes of hospitalisation at bungoma county referral hospital in 2023.11 study design and population this study followed an analytical cross-sectional design. included were children who already had a known haemoglobin phenotype and were being monitored at the sickle cell clinic of bungoma county referral hospital in kenya, as well as children who were apparently healthy and visiting the maternal and child clinic of bungoma county referral hospital in kenya. sample size determination and sampling technique the sample size for the study was determined using fisher’s exact test,12 based on a previously reported prevalence of 14.8%.9 using this prevalence and standard parameters for statistical confidence and margin of error, the calculated sample size was approximately 194 participants, which was adopted for the study. a systematic random sampling method was employed, selecting every second child who met the inclusion criteria until the desired sample size was achieved. data collection recruitment and consent the positive cases consisted of children aged 10 weeks to 15 years attending the sickle cell clinic at bungoma county referral hospital with a known diagnosis of scd (hbss phenotype, i.e., homozygous for sickle cell haemoglobin) or sickle cell trait (hbas phenotype, i.e., heterozygous for sickle cell haemoglobin), confirmed by prior haemoglobin electrophoresis or hplc results. healthy children within the same age range attending the child welfare clinic for routine vaccinations were systematically selected as controls and presumed to be negative for scd. inclusion criteria for both groups included age between 10 weeks and 15 years, availability of haemoglobin profile for positive cases, and provision of informed consent by a parent or guardian. exclusion criteria included recent blood transfusion (within the last 3 months), current use of antibiotics or hydroxyurea, presence of acute illness, or lack of consent. participants were categorised into two groups, namely children with known scd and healthy children without the disease. parents or guardians received a detailed information sheet explaining the study objectives, participant selection process, procedures, potential risks and benefits, confidentiality measures, voluntary nature of participation, data sharing, and contact details for study-related inquiries. following parental consent, children were assigned a unique identification number and included in the sample collection process. sample collection for determination of the sickle cell phenotype, blood samples were collected from each participant. approximately 2 ml of whole blood was drawn into a vacuum collection tube containing ethylenediaminetetraacetic acid, gently inverted 8–9 times, and labelled with a unique participant identifier. the samples were promptly delivered to the laboratory. if not tested immediately, the samples were stored at 2 °c – 8 °c for up to 24 h. prior to testing, stored samples were allowed to reach room temperature and were mixed thoroughly, according to the manufacturer’s guidelines. laboratory results were recorded using the study’s standard data collection form. laboratory analysis the performance of the sicklecheck™ (zephyr biomedicals, a division of tulip diagnostics (p) ltd., goa, india) rapid test kit for detecting sickle cell phenotypes was evaluated in comparison to the bio-rad™ (bio-rad laboratories, marnes-la-coquette, france) hplc method. sickle cell phenotype testing using sicklecheck™ sicklecheck™ operates on the principle of antibody/antisera agglutination with the corresponding antigen in a competitive immune-chromatography format. it utilises nanogold particles as the agglutination detection agent. the conjugate pad contains two components: a monoclonal antibody for haemoglobin s (hbs) and a monoclonal antibody for haemoglobin a (hba), each conjugated to colloidal gold. the monoclonal antibody conjugated with colloidal gold, specific to either hbs or hba, binds to the corresponding antigens in the test specimen. as the specimen flows through the membrane assembly by capillary action, the antibody–antigen complex moves along the membrane. in the absence of hbs and hba on the membrane, the complex continues to the test regions (t1 and t2), where no band forms because of a lack of binding. the absence of colour bands at the test regions (t1 and t2) indicates the presence of the corresponding antigens (hbs or hba), respectively, in the specimen. in the presence of hbs and hba, the monoclonal antibody–colloidal gold conjugate moves along the membrane through capillary action, where it is captured by the respective antigen coated on the membrane (t1 & t2), resulting in the formation of colour bands. the appearance of colour bands at the test regions (t1 and t2) indicates that the specimen does not contain the corresponding antigens (hbs or hba). the membrane’s control region (c) is coated with goat anti-mouse immunoglobulin g (igg), which causes any unbound colloidal gold conjugates to continue traveling along the membrane and become immobilised. the control band validates the test results and serves as a procedural control. the components of the sicklecheck™ test kit were allowed to reach room temperature before use. the foil pouch was opened by tearing along the notch to remove the testing device. the desiccant pouch was checked to ensure it was blue; if colourless or pink, the test device was discarded and replaced. once opened, the device was labelled with the participant’s identity and used immediately. the device was placed on a flat surface, and the assay buffer vial was mixed and held upright to break off the tip. the buffer, which lyses red blood cells to release haemoglobin and ensures proper sample flow and reaction, was mixed with the specimen. two drops of this mixture were dispensed into the specimen port (s). visible coloured bands at test regions (t1 and t2) were observed over a 15-min period, depending on analyte concentration. tests were not read or interpreted after 15 min. test results were interpreted as follows: a result was deemed invalid if no band appeared in the control region (c), regardless of the presence of bands in the test regions (t1 or t2). a normal result was indicated by coloured bands in both the control (c) and test region t1. the presence of bands in the control (c) and test region t2 suggested scd or sickle cell in combination with other haemoglobinopathies. a band in the control region (c) with no bands in either test region (t1 or t2) was interpreted as sickle cell trait or its association with other haemoglobinopathies. the appearance of bands in the control (c), test region t1, and test region t2 indicated the presence of other haemoglobinopathies, thalassaemia, or a combination of haemoglobinopathies with thalassaemia. to ensure internal quality control, each test batch included positive and negative control samples with known hba, hbs, and mixed phenotypes, which were tested alongside participant samples. additionally, the laboratory participated in an external proficiency testing programme coordinated by a collaborating external quality assurance provider. sickle cell phenotype testing using high liquid performance chromatography the bio-rad™ hplc was used to separate and quantify haemoglobin variants in whole blood based on the cation exchange principle. to prepare the sample, 5 µl of ethylenediaminetetraacetic acid anticoagulated blood was mixed with 1 ml of haemolysis solution provided in the kit. the instrument was then loaded with the diluted samples, which were further processed with the appropriate haemolysing/wash buffer before being inserted into the analytical cartridge. a programmed buffer gradient with increasing ionic strength was pumped through the cartridge, separating haemoglobin fractions based on their ionic interactions with the cartridge material. these fractions passed through a flow cell, where absorbance was measured at 415 nm, with a secondary wavelength of 690 nm to eliminate background noise. the software generated a printed chromatogram showing all eluted haemoglobin fractions and their respective retention times. peaks were attributed to specific ‘windows’ based on retention times, with each peak corresponding to a haemoglobin component. if an unknown peak was observed, it was identified by its retention time. each analysis cycle took approximately 6.5 min, from sample loading to the final printed results. if the hbs peak was present without any detectable hba, the patient was diagnosed with scd (i.e., hbss phenotype). in contrast, the presence of both hba and hbs peaks, with hba being predominant, indicated sickle cell trait (i.e., hbas phenotype). commercially prepared bio-rad™ control samples, containing known haemoglobin variant compositions, were analysed daily alongside participant samples. the laboratory participated in an established external quality assurance programme coordinated by an accredited external body. as part of this programme, blinded proficiency testing samples were periodically analysed, and the results were submitted for assessment against established consensus values. data quality assurance a trained phlebotomist was responsible for collecting the blood samples, ensuring the correct volume was obtained to maintain data quality. the samples were processed and tested according to the standard operating procedures of bungoma county referral hospital’s laboratory. internal and external quality controls were consistently monitored and verified throughout the study. a second individual cross-checked the recorded results for accuracy. for enhanced validity and reliability, all statistical tests were performed using a z-score of 1.96 and a 95% confidence level. data analysis data were stored using microsoft excel (microsoft corporation, redmond, washington, united states), while medcalc™ statistical software (medcalc software ltd, ostend, belgium) was used to calculate the sensitivity, specificity, ppv, npv, and accuracy of sicklecheck™ in comparison to bio-rad™ hplc, which served as the reference method. results were presented in tables. the software computed sensitivity by dividing true positives by the total of true positives and false negatives, specificity by dividing true negatives by the sum of true negatives and false positives, ppv by dividing true positives by the sum of true positives and false positives, and npv by dividing true negatives by the sum of true negatives and false negatives. accuracy was calculated by multiplying sensitivity by the sum of prevalence and specificity, then multiplying by (1 – prevalence). results patient characteristics a total of 194 children were included in the analyses, comprising 90 boys (46%) and 104 girls (54%) (table 1). participants ranged in age from 10 weeks to 15 years. of these, 95 (49%) were confirmed to have scd (hbss phenotype), 21 (11%) were carriers of the sickle cell trait (hbas phenotype), and 78 (40%) were sickle cell negative (hbaa phenotype, i.e., homozygous for haemoglobin a). among the hbss group, 47 (49%) were boys and 48 (51%) were girls. of the children with the hbas trait, nine (43%) were boys and 12 (57%) were girls. among hbaa participants, 34 (44%) were boys and 44 (56%) were girls. table 1: basic demographic characteristics of the study population, kenya, 11 october 2023 – 22 february 2024. detection of phenotypes the sicklecheck™ assay correctly identified 98 out of 99 children with hba as true positives (99.0%), and 94 out of 95 children without hba as true negatives (98.9%). there was one false positive (1.1%) and one false negative (1.0%) for hba detection (table 2). for hbs detection, the test accurately identified 115 out of 116 children with hbs as true positives (99.1%), and 76 out of 78 without hbs as true negatives (97.4%). two false positives (2.6%) and one false negative (0.9%) were observed for hbs detection (table 2). the sicklecheck™ accurately identified 76 out of 78 children with hbaa as true positives (97.4%), and 115 out of 117 children without hbaa as true negatives (98.3%). there was one false positive (0.9%) and two false negatives (2.6%) for hbaa detection (table 3). for hbas detection, the test correctly identified 19 out of 21 children as true positives (90.5%), and 170 out of 173 children as true negatives (98.3%). three false positives (1.7%) and two false negatives (9.5%) were recorded for hbas detection (table 3). in detecting hbss, the test identified 94 out of 95 children as true positives (98.9%), and 98 out of 99 as true negatives (99.0%). there was one false positive (1.0%) and one false negative (1.1%) for hbss detection (table 3). table 2: sicklecheck™ detection of haemoglobin a and haemoglobin s compared to bio-rad™ high-performance liquid chromatography, kenya, 11 october 2023 – 22 february 2024. table 3: sicklecheck™ detection of haemoglobin phenotypes aa, as and ss compared to bio-rad™ high-performance liquid chromatography, kenya, 11 october 2023 – 22 february 2024. efficacy of sicklecheck™ the sicklecheck™ assay showed a sensitivity of 97.98%, specificity of 98.95%, and overall accuracy of 98.80% for hba detection (table 4). the ppv for hba was 94.18%, while the npv was 99.65%. for hbs detection, sensitivity was 99.14%, specificity was 97.44%, and overall accuracy was 97.69%. the ppv for hbs was 89.04%, and the npv was 99.85% (table 4). table 4: sensitivity and specificity of the sicklecheck™ assay for the detection of haemoglobin a and haemoglobin s, kenya, 11 october 2023 – 22 february 2024. sicklecheck™ showed a sensitivity of 97.44% for detecting hbaa, 90.47% for hbas, and 98.98% for hbss (table 5). specificity was 99.14% for hbaa, 98.27% for hbas, and 98.99% for hbss. the ppvs were 95.15% for hbaa, 90.06% for hbas, and 94.45% for hbss. negative predictive values were 99.55% for hbaa, 98.34% for hbas, and 99.82% for hbss. the overall accuracy was 98.89% for hbaa, 97.11% for hbas, and 98.98% for hbss (table 5). table 5: sensitivity and specificity of the sicklecheck™ assay for the detection of haemoglobin hbaa, hbas and hbss, kenya, 11 october 2023 – 22 february 2024. discussion the current study demonstrated that the sicklecheck™ assay achieved over 97% sensitivity, specificity, npv, and accuracy in detecting hba and hbs variants, as well as all relevant haemoglobin phenotypes (normal, carrier, and disease). the findings are consistent with a study conducted in ghana and the united states and published in 2019,13 which assessed the hemotypesc™ rapid testing method, reporting sensitivity and specificity above 99%, with an accuracy of 99.80% for detecting hba and 97.69% for hbs. similarly, the current results on the npv for hbs agree with the same study,13 which documented a npv of 99.8% for hbs. the present findings are also consistent with those of a study conducted in india and published in 2024,14 which evaluated a point-of-care rapid diagnostic test kit for screening scd. the study reported the following performance metrics: for hbaa, a sensitivity of 98.04%, specificity of 99.35%, ppv of 99.50%, and npv of 97.47%; for hbas, a sensitivity of 99.07%, specificity of 98.81%, ppv of 97.25%, and npv of 99.60%; and for hbss, a sensitivity of 97.92%, specificity of 100%, ppv of 100%, and npv of 99.60%. the current findings on accuracy are in agreement with a study conducted in the united states and published in 2016,15 that validated the diagnostic performance of the sickle scan™ test, reporting an overall accuracy of 99% at the bedside. the current specificity results also align with those reported by a study conducted in nigeria and published in 2019,16 which found that the hemotypesc™ test for sickle cell anaemia had a specificity of 99.9% under optimal field conditions. however, the current findings slightly differ in terms of sensitivity, which was somewhat lower at 93.4%. the current specificity findings also align with those of a study conducted in the united states and published in 2017,17 which developed and evaluated a competitive lateral flow assay, reporting a specificity of 100% for diagnosing sickle cell anaemia. however, while the current study reported a higher specificity, the united states study had a slightly lower specificity of 90.0%, indicating a modest difference in diagnostic accuracy. these findings also correspond with those of a study conducted at cincinnati children’s hospital medical center, united states, and published in 2016,18 which evaluated the effectiveness of a rapid, point-of-care lateral flow immunoassay for diagnosing scd. the study reported a sensitivity of 99.5% for hbs, and 98.3% for hba. however, the current results show slight differences in specificity, which were 92.5% for hbs, and 94.0% for hba. the present findings also contradict those of a study conducted in haiti and published in 2019,19 which reported a sensitivity of 90.0% for sickle scan™. the present findings also contradict those of a study conducted in mali and published in 2024 on the comparative performance of two rapid diagnostic tests (sickle scan™ and hemotypesc™).20 the study reported sensitivities of 96.10% and 95.22%, respectively, for detecting the hbas genotype, and 81.67% and 78.33%, respectively, for detecting the hbss genotype. the conflicting findings reported in some studies may be attributed to factors such as the variable interpretation of faint test bands,13 suboptimal environmental conditions during field testing,21 and reduced specificity in neonates because of high levels of foetal haemoglobin, which can interfere with antigen–antibody interactions in certain immunoassays.15 these discrepancies highlight the importance of considering context-specific variables such as age, environmental testing conditions, and the regional prevalence of haemoglobin variants when assessing the performance of rapid diagnostic tests. nevertheless, despite these occasional inconsistencies, the broader body of evidence, including the current study, strongly supports the effectiveness of lateral flow-based rapid diagnostics as reliable and scalable tools for screening scd, particularly in low-resource settings. while a few false positive and false negative results were noted in our study, such occurrences are not unusual and may arise from a combination of technical and biological factors. for instance, faint or borderline test bands in lateral flow immunoassays can complicate interpretation, potentially leading to misclassification.13,22 additionally, biological variability in haemoglobin expression, such as elevated levels of foetal haemoglobin in neonates, the presence of rare haemoglobin variants, or coexisting haemoglobinopathies, may influence the assay’s ability to accurately detect specific haemoglobin types.23 these factors can affect antibody binding or visual detection, contributing to diagnostic discrepancies.13 further investigation through larger, population-based studies is warranted to better understand and address these limitations. the sicklecheck™ test demonstrates high sensitivity, specificity, and accuracy for detecting scd and trait, comparable to other rapid point-of-care assays for scd identification. based on these results, the sicklecheck™ test is recommended as an effective rapid screening tool for haemoglobin variants and phenotypes. sicklecheck™ offers significant benefits for testing in situations requiring minimal blood volumes. for example, it is particularly effective for testing infants who cannot provide sufficient blood through venipuncture, as it requires only 10 µl of blood from a finger or heel stick. maintaining follow-up with patients after leaving healthcare facilities can be difficult, especially in rural areas. in many parts of rural africa, including kenya, haemoglobin screening is typically conducted at centralised hospitals or laboratories, necessitating the transfer of diagnostic specimens away from the point of care. this approach results in increased costs, logistical complexities, and delays in obtaining test results.13 rapid point-of-care screening methods, such as sicklecheck™, effectively address these challenges. sicklecheck™ does not rely on instruments, electricity, or refrigeration, requires minimal training for operators, and provides results through visual inspection within 15 min. these features make it a cost-effective, fast, and accurate diagnostic tool in low-resource settings with high rates of scd. with its substantial sensitivity and specificity, low sample volume requirements, independence from specialised equipment, ease of use, and rapid results, sicklecheck™ is a highly recommended tool for point-of-care screening of scd and traits. recommendations the study recommends that sicklecheck™ be adopted as a rapid point-of-care test for scd and trait because of its minimal sample requirement, lack of need for specialised equipment or electricity, ease of use, and quick result turnaround. limitations a limitation of our study was the inability to evaluate haemoglobin c (hbc) detection using the sicklecheck™ test, as no participants with this variant were identified. conclusion based on the findings in this study, sicklecheck™ could be a reliable point-of-care diagnostic tool for scd. acknowledgements dr makokha, ms anne oriko, and ms janet wakhungu contributed by assisting in the recruiting of participants. mr edwin samita assisted with the sample collection process. mr david mulongo and ms resi alili contributed by running the samples and data recording. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions a.s.k., p.m.m. and s.k.m. structured and participated in all aspects of the study. p.m.m. and s.k.m. oversaw the study. p.m.m. and a.s.k. performed data analysis and wrote the manuscript. s.k.m. evaluated the manuscript for logical contributions. all authors evaluated and approved the final manuscript for submission. sources of support this study received partial funding from tulip diagnostics (p) ltd. data availability the data sets collected and analysed during the current study are not publicly available because of ethical reviewing guidelines, but they are available from the corresponding author, a.s.k., upon reasonable request. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. it does not necessarily reflect the official policy or position of any affiliated institution, 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findings from a southwestern state in nigeria and implications for patient care in resource-poor settings of sub-saharan africa. glob pediatr health. 2021;8:2333794x211016789. https://doi.org/10.1177/2333794x211016789 abstract introduction methods results discussion acknowledgements references about the author(s) sanelisiwe t. duze department of clinical microbiology and infectious diseases, faculty of health sciences, school of pathology, university of the witwatersrand, johannesburg, south africa teena thomas department of clinical microbiology and infectious diseases, faculty of health sciences, school of pathology, university of the witwatersrand, johannesburg, south africa infection control services laboratory, national health laboratory services, charlotte maxeke academic hospital, johannesburg, south africa tshegofatso pelego infection control services laboratory, national health laboratory services, charlotte maxeke academic hospital, johannesburg, south africa sabelle jallow department of clinical microbiology and infectious diseases, faculty of health sciences, school of pathology, university of the witwatersrand, johannesburg, south africa centre for healthcare-associated infections, antimicrobial resistance and mycoses, national institute for communicable diseases, johannesburg, south africa olga perovic department of clinical microbiology and infectious diseases, faculty of health sciences, school of pathology, university of the witwatersrand, johannesburg, south africa centre for healthcare-associated infections, antimicrobial resistance and mycoses, national institute for communicable diseases, johannesburg, south africa adriano duse department of clinical microbiology and infectious diseases, faculty of health sciences, school of pathology, university of the witwatersrand, johannesburg, south africa infection control services laboratory, national health laboratory services, charlotte maxeke academic hospital, johannesburg, south africa citation duze st, thomas t, pelego t, et al. evaluation of xpert carba-r for detecting carbapenemase-producing organisms in south africa. afr j lab med. 2023;12(1), a1898. https://doi.org/10.4102/ajlm.v12i1.1898 note: additional supporting information may be found in the online version of this article as online supplementary 1. brief report evaluation of xpert carba-r for detecting carbapenemase-producing organisms in south africa sanelisiwe t. duze, teena thomas, tshegofatso pelego, sabelle jallow, olga perovic, adriano duse received: 24 mar. 2022; accepted: 15 sept. 2022; published: 05 jan. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract this study evaluated the performance of the xpert carba-r assay for detecting the five common carbapenemases in carbapenemase-producing organisms in johannesburg, south africa between april 2021 and september 2021. the assay demonstrated 98% sensitivity and 97% specificity. it was also able to detect all the carbapenemases in double carbapenemase producers, as well as carbapenemases in non-fermenter organisms. the xpert carba-r assay, therefore, allows the rapid (< 1 h) and accurate identification of the common carbapenemases in pure bacterial cultures and rectal swabs. this assay can aid in the timeous institution of appropriate treatment and infection prevention and control measures. keywords: enterobacterales; carbapenemase-producing enterobacterales; carbapenemase; xpert carba-r; rectal screening. introduction the rapid spread of carbapenemase-producing enterobacterales (cpe) is a major threat to public health worldwide.1 moreover, carbapenemases in enterobacterales reside on mobile genetic elements that facilitate their widespread dissemination to other bacterial species or genera, which can spread from person to person, particularly in hospital settings.2,3 carbapenemases have been classified into four classes (a, b, c, and d).4 within these classes, the most common carbapenemases are klebsiella pneumoniae carbapenemases (kpc) in class a, imipenemases (imp), verona integrated metallo-β-lactamases (vim) and new delhi metallo-β-lactamases (ndm) in class b, and the class d oxacilinases (oxa)-type enzymes (e.g. oxa-48 and its variants).2,4 in south africa, the most dominant carbapenemases are ndm and oxa-48 and its variants.5,6,7 a recent report by perovic et al. revealed an alarming increase in the prevalence of oxa-48 and its variants.7 this is concerning as oxa-48 and its variants can be missed by routine antimicrobial susceptibility testing as these isolates demonstrate low-level resistance to the carbapenems.8 therefore, rapid and accurate detection of carbapenemases through rectal screening of high-risk patients is crucial for infection prevention and control measures.3 automated molecular assays such as the xpert carba-r (cepheid, sunnyvale, california, united states) offer rapid detection of the five common carbapenemases.9 to our knowledge, no data has been published on the performance of the xpert carba-r assay in the african setting. to adopt this assay in our laboratories, local verification taking into account the local epidemiology and the prevalence of the disease is essential.10 therefore, this study aimed to evaluate the performance of the xpert carba-r assay on carbapenemase-producing organisms using cultured isolates and spiked rectal swab specimens in johannesburg, south africa. methods ethical considerations ethics clearance for this study was obtained from the university of witwatersrand ethics committee (study approval number: m210752). researchers involved in this study provided stool samples voluntarily, with verbal consent for which written documentation was kept. the stool samples were used as matrices for the preparation of spiked rectal swabs. to maintain confidentiality as well as protect researchers’ privacy, all data were anonymised and no personal identifiers were used. sample collection the study was conducted at the infection control laboratory services in johannesburg, south africa, between april 2021 and september 2021 using a collection of 95 previously characterised enterobacterales isolates. these included 66 cpe positive for either the blakpc, blandm, blavim, blaoxa-48, or blaimp-1 gene, and 29 non-cpe isolates. the 66 cpe were characterised using either the resist-4 o.k.n.v. kit (coris bioconcept, gembloux, belgium) or the lightmix® multiplex polymerase chain reaction (pcr) carbapenemase kit (tibmolbiol, berlin, germany) and the results were used as the reference genotypes. in addition, 10 uncharacterised non-fermenter gram-negative bacteria were included in the study to determine the performance of the assay in detecting carbapenemases in these organisms. xpert carba-r assay performance to determine the accuracy, sensitivity, and specificity of the xpert carba-r assay, the 95 bacterial cultures were processed according to the manufacturer’s instructions. briefly, bacterial suspensions corresponding to the 0.5 mcfarland turbidity standard (thermo fisher, johannesburg, south africa) were prepared from fresh cultures. thereafter, 10 ml of each suspension was transferred into a sample reagent vial, vortexed for 10 s, and 1.7 ml of the sample mixture was transferred into the sample chamber of the xpert cartridge for processing. due to challenges in obtaining clinical rectal swab specimens positive for cpe in the absence of an outbreak, rectal swabs spiked with the 95 characterised isolates were used. first, stool samples provided voluntarily by researchers involved in this study were screened for the presence of carbapenemases using the xpert carba-r assay as it can be performed directly from the stool and does not require culture. thereafter, for each of the 95 isolates, 10 ml of a 0.5 mcfarland bacterial suspension was added to 990 ml of the negative stool matrix and the mixture was seeded onto the sterile rectal swabs as per cepheid’s acceptance swab criteria, and then processed following the manufacturer’s instructions. a true positive was defined as when the expected carbapenemase genes were detected by the assay and a true negative was defined as when no carbapenemase gene was detected in non-cpe isolates and the cpe-negative rectal swabs. a discordant result was defined as an xpert carba-r assay result that was different from the reference genotype. isolates with discordant results were sent to the national institute for communicable diseases for whole-genome sequencing (wgs) to confirm the presence or absence of the targeted carbapenemase genes. for wgs, total genomic dna was extracted using the qiaamp mini kit (qiagen, germantown, maryland, united states). sequencing was performed using the nextseq instrument (illumina, san diego, california, united states) with 2 × 150 base pairs paired-end sequencing per flow cell and the paired-end reads were filtered for quality (q > 30 and length > 50 base pairs) using trim galore v0.6.2 (https://github.com/felixkrueger/trimgalore). de novo assembly was performed using spades v3.13 (https://github.com/kbaseapps/kb_spades). acquired and chromosomal antimicrobial resistance genes were determined using resfinder v4.1 (https://cge.cbs.dtu.dk/services/resfinder) and the comprehensive antibiotic resistance database (https://card.mcmaster.ca/). carbapenemases identified based on wgs data were considered as the final genotype for discordant isolates and used to determine the accuracy, sensitivity, and specificity of the assay. data were captured on microsoft excel (microsoft, redmond, washington, united states) and analysis was performed using the following equations: to determine sample stability, spiked rectal swabs were stored at room temperature and tested using the xpert carba-r assay on different days: day 0 (day of preparation), 1, 3, 5, and 7. the limit of detection of the assay was also determined by preparing serial dilutions up to 1.5 × 101 colony forming units (cfu)/ml from 0.5 mcfarland suspensions of cpe positive for blakpc, blandm, blavim, blaoxa-48, and blaimp-1. each dilution was tested using the xpert carba-r assay. lastly, the performance of the xpert carba-r assay was tested on non-fermenter organisms. carbapenemases detected in non-fermenter organisms were confirmed using the resist-4 o.k.n.v. kit (coris bioconcept, gembloux, belgium) and were subjected to wgs at the national institute for communicable diseases in johannesburg, south africa. these results were not included in the accuracy, sensitivity, and specificity calculations for the assay. results the xpert carba-r assay correctly identified all of the carbapenemases in 94% (61/66) of the tested isolates with a cycle threshold ranging between 20.6 and 27 for culture and between 24.5 and 30.6 for spiked rectal swabs. the assay further detected all double carbapenemase-producing isolates (blandm+blaoxa-48) and reported all 29 non-cpe isolates as negative for carbapenemases (table 1; online supplementary table 1). table 1: performance of xpert carba-r assay for the detection of carbapenemases in cultured bacterial isolates and spiked rectal swab specimens at the infection control laboratory services in johannesburg, south africa, april 2021 – september 2021. five isolates previously characterised as kpc-producing k. pneumoniae using the lightmix® multiplex pcr carbapenemase kit were negative for the blakpc gene on the xpert carba-r assay as well as on resist-4 o.k.n.v. three of these isolates were positive for the blandm gene while the other two isolates were positive for the blavim (isolate 42) and blaoxa-48 (isolate 43) genes. whole-genome sequencing confirmed the presence of blandm-1 and the absence of the blakpc in the three k. pneumoniae isolates; these isolates were thus true positives for blandm. whole-genome sequencing results also showed that isolate 42 was negative for the blakpc gene, and harbored blavim-1, which corroborated the results of the xpert carba-r assay. this isolate was considered a true positive for blavim. further, wgs detected the presence of the blakpc-2 gene in isolate 43, which was not detected by the xpert carba-r assay and resist-4 o.k.n.v kit. interestingly, the xpert carba-r assay and resist-4 o.k.n.v kit both detected the presence of the blaoxa-48 gene, which was not detected by wgs. isolate 43 was, therefore, considered a false negative for blakpc and a false positive for blaoxa-48 (table 2). table 2: investigation of five discordant results obtained using the xpert carba-r assay at the national institute for communicable diseases, johannesburg, south africa, april 2021 – september 2021. the overall accuracy of the xpert carba-r assay was 98%, with a sensitivity of 98% and specificity of 97%. for individual gene targets, the sensitivity and specificity were 100% for blandm, blavim, and blaimp-1. the sensitivity for blakpc was 89% while specificity was 100%, whereas for blaoxa-48, the sensitivity was 100% and the specificity was 97%. the limit of detection for the xpert carba-r assay was 1 × 101 cfu/ml for the blakpc, blavim, and blandm genes and 1 × 102 cfu/ml for the blaoxa-48 and blaimp genes. the spiked rectal swabs were still positive on day 7 when stored at room temperature and the assay was found to be 100% reproducible. the assay was also able to detect carbapenemase genes in non-fermenting gram-negative bacteria. two acinetobacter baumannii isolates were positive for blandm and one pseudomonas aeruginosa isolate was positive for blavim. whole-genome sequencing confirmed the presence of the blandm and blavim genes in these non-fermenter organisms (table 3). table 3: performance of xpert carba-r assay for the detection of carbapenemases in 10 non-fermenter organisms compared to the resist-4 o.k.n.v kit and whole-genome sequencing performed at the national institute for communicable diseases, johannesburg, south africa, april 2021 – september 2021. discussion in this evaluation study, the xpert carba-r assay demonstrated high performance with 98% sensitivity and 97% specificity when tested on cultured isolates on blood agar as well as on spiked rectal swabs. the results from this study are in agreement with the manufacturer’s information and corroborate results from other evaluation studies conducted in europe and the united states which demonstrated high sensitivity and specificity.11,12,13 the assay was able to detect double carbapenemases (blandm and blaoxa-48) in three k. pneumoniae and two enterobacter cloacae isolates. the occurrence of two or more carbapenemase resistance genes in cpe is not an unusual phenomenon and has been previously reported in strains of k. pneumoniae and escherichia coli isolated from polish tourists surviving the terrorist attack at the bardo museum in tunisia.14 more recently, the occurrence of blandm and blaoxa-48 carbapenemases was reported in an outbreak caused by k. pneumoniae in germany and in k. pneumoniae strains in greece.15,16 the xpert carba-r assay was also able to detect carbapenemases in three non-fermenters. this is in agreement with the manufacturer’s claim that the xpert carba-r assay can be performed on pure colonies of enterobacterales, acinetobacter baumannii, or pseudomonas aeruginosa. it is worth noting that the xpert carba-r assay has an intrinsic limitation in that it cannot detect carbapenemases that are not part of its test repertoire such as those detected by wgs in this study, including blaoxa-23, which is a potent carbapenemase that spreads via plasmid-mediated transfer and is predominantly found in clinical a. baumannii isolates.17 although routine screening is not currently recommended for non-fermenter organisms, their increasing prevalence raises questions of concern.18 further studies are necessary to understand the extent of carbapenemases in non-fermenter organisms and their role in the spread of these enzymes in healthcare settings. the limit of detection claims by cepheid for detecting the carbapenemases in rectal swabs ranges between 1 × 101 cfu/swab and 1 × 102 cfu/swab. furthermore, cepheid recommends that rectal swabs collected using the rectal specimen collection device (part number 900-0370) can be stored at room temperature for up to five days. these findings were corroborated by our verification. additionally, spiked rectal swabs remained positive for up to seven days when stored at room temperature. limitations this study had a few limitations. first, the performance of the assay on rectal specimens was tested using spiked rectal swabs instead of clinical specimens. second, the limit of detection was only tested on cultured isolates and not spiked rectal swabs. lastly, due to the low prevalence of imp-producing enterobacterales, only three isolates were used in the verification, and these included two non-fermenter organisms. conclusion we concur with other studies that the xpert carba-r assay offers a reliable and fast method for the detection of the five most common carbapenemases in carbapenemase-producing organisms. the assay can be performed on rectal swab specimens or pure bacterial cultures of carbapenem-nonsusceptible isolates and provides automated results in less than an hour. the assay can detect double carbapenemase producers as well as carbapenemases in non-fermenter organisms. the xpert carba-r assay can, therefore, aid in the timeous and appropriate institution of treatment and infection prevention and control measures. acknowledgements the authors would like to thank the staff at the infection control service laboratory unit and the centre for healthcare-associated infections, antimicrobial resistance and mycoses at the national institute for communicable diseases for providing us with the enterobacterales isolates used in this evaluation study. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions s.t.d. was responsible for the literature review, laboratory work, data analysis and manuscript completion. t.t. assisted with manuscript review and provided critical inputs. a.d. was responsible for the concept of this study and provided input in the manuscript. t.p. was responsible for testing the reproducibility of the assay. s.j. and o.p. assisted with wgs and analysis of wgs results. sources of support the xpert carba-r kits were donated by cepheid. the authors received no financial support for the research, authorship, or publication of this article. data availability raw data were generated at the infection control services laboratory of the national health laboratory services. derived data supporting the findings of this study are available from the corresponding author, s.t.d., on request. disclaimer the views expressed in this publication are those of the authors and do not directly represent the views of any support agencies. references sheu cc, chang yt, lin sy, chen yh, hsueh pr. infections caused by carbapenem-resistant enterobacteriaceae: an update on therapeutic options. front microbiol. 2019;10(80):1–13. https://doi.org/10.3389/fmicb.2019.00080 queenan am, bush k. carbapenemases: the versatile beta-lactamases. clin microbiol rev. 2007;20(3):440–458. https://doi.org/10.1128/cmr.00001-07 reyes ja, melano r, cárdenas pa, trueba g. mobile genetic elements associated with carbapenemase genes in south american enterobacterales. braz j infect dis. 2020;24(3):231–238. https://doi.org/10.1016/j.bjid.2020.03.002 ambler rp. the structure of beta-lactamases. philos trans r soc lond b biol sci. 1980;289(1036):321–331. https://doi.org/10.1098/rstb.1980.0049 thomas tsm, duse ag. epidemiology of 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dates: received: 06 may 2014 accepted: 03 nov. 2014 published: 14 may 2015 how to cite this article: hamel dj, sankalé jl, samuels jo, et al. building laboratory capacity to support hiv care in nigeria: harvard / apin pepfar, 2004–2012. afr j lab med. 2015;4(1), art. #190, 10 pages. http://dx.doi.org/10.4102/ajlm.v4i1.190 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. building laboratory capacity to support hiv care in nigeria: harvard/apin pepfar, 2004–2012 in this original article... open access • abstract • introduction • research methods and design    • clinic selection    • procurement of equipment    • laboratory modifications    • supply chain    • equipment maintenance    • data management    • laboratory trainings • results    • impact on health system strengthening    • training conferences    • electronic data management    • laboratory quality control and accreditation • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ introduction: from 2004–2012, the harvard/aids prevention initiative in nigeria, funded through the us president’s emergency plan for aids relief programme, scaled up hiv care and treatment services in nigeria. we describe the methodologies and collaborative processes developed to improve laboratory capacity significantly in a resource-limited setting. these methods were implemented at 35 clinic and laboratory locations. methods: systems were established and modified to optimise numerous laboratory processes. these included strategies for clinic selection and management, equipment and reagent procurement, supply chains, laboratory renovations, equipment maintenance, electronic data management, quality development programmes and trainings. results: over the eight-year programme, laboratories supported 160 000 patients receiving hiv care in nigeria, delivering over 2.5 million test results, including regular viral load quantitation. external quality assurance systems were established for cd4+ cell count enumeration, blood chemistries and viral load monitoring. laboratory equipment platforms were improved and standardised and use of point-of-care analysers was expanded. laboratory training workshops supported laboratories toward increasing staff skills and improving overall quality. participation in a world health organisation-led african laboratory quality improvement system resulted in significant gains in quality measures at five laboratories. conclusions: targeted implementation of laboratory development processes, during simultaneous scale-up of hiv treatment programmes in a resource-limited setting, can elicit meaningful gains in laboratory quality and capacity. systems to improve the physical laboratory environment, develop laboratory staff, create improvements to reduce costs and increase quality are available for future health and laboratory strengthening programmes. we hope that the strategies employed may inform and encourage the development of other laboratories in resource-limited settings. introduction top ↑ laboratories are fundamental and essential components of health systems, providing clinical staff and patients with test results that are the basis of disease diagnosis and treatment; yet, laboratories are often neglected by governments, development organisations and other stakeholders in plans to improve healthcare systems in developing countries. despite the scale-up of global health programmes in the last decade, sub-saharan africa continues to suffer the consequences of operating with some of the most poorly-equipped and under-resourced laboratories in the world.1 as such, by 2012, the us president’s emergency plan for aids relief (pepfar) blueprint, the world health organization (who) and the united nations millennium development goals (unmdg) each called for strengthened national laboratory systems as a critical component of scaling up hiv and tuberculosis (tb) prevention and treatment programmes.2,3 based on the nigerian national hiv sentinel surveillance surveys in 2005 and 2010, the national prevalence of hiv-1 has remained fairly stable at approximately 4%.4 the harvard school of public health received pepfar funds from 2004 to 2012 to support the development of prevention, care and treatment programmes in nigeria, botswana and tanzania. in nigeria, harvard partnered with the aids prevention initiative in nigeria (apin), an organisation developed through funding from the bill and melinda gates foundation from 2000–2006, to provide evidence-based hiv prevention in four states of the country. the harvard/apin pepfar programme was built upon this foundation of hiv prevention activities and initiated support of antiretroviral therapy (art) activities at six tertiary-level facilities in 2004; this expanded to 35 clinics and laboratories by 2009. to ensure sustainability, harvard helped to establish apin ltd./gte. as an independent, nigeria-based non-governmental organisation. beginning in 2009, and fully completed in february 2012, all harvard/apin pepfar programme activity was transitioned to apin management. from the beginning of the harvard/apin pepfar programme, it was determined that a fundamental component of the capacity-building efforts would be dedicated to laboratory infrastructure, with corresponding growth of logistics management for procuring supplies and laboratory staff training in order to ensure sustainability. in developing the programme frameworks and plans, we incorporated lessons learned from previously-developed art laboratories in both nigeria and senegal so as to elicit lasting gains in laboratory capacity and infrastructure.5 in this report, we describe the organisational framework that resulted in the establishment of and continuous quality improvements to laboratory capacity in nigeria over the eight years of the harvard/apin pepfar programme (2004–2012). we highlight the collaborative process, with details on specific strategies and methodologies, found to be essential for meaningful laboratory development in a resource-limited setting. research methods and design top ↑ our programme’s laboratories were organised with large tertiary facilities at the centre, providing support to secondary hospitals and associated primary health clinics using a hub-and-spoke model (figure 1). tertiary-level laboratories were associated with university teaching hospitals or research institutes with large hiv art programmes. secondary-level hospitals provided hiv serology, cd4+ cell count enumeration, haematology and clinical chemistry testing. they also had the capacity to store plasma samples for viral load (vl) testing, and dried blood spot (dbs) samples for early infant diagnosis, for up to two weeks before transport to an associated tertiary laboratory. primary health clinics were smaller health centres that provided basic care, performed hiv rapid testing, drew blood samples for testing to be done elsewhere and referred patients to the secondary or tertiary medical facilities. figure 1: illustration of hub-and-spoke laboratory organisation. clinic selection the selection of a clinic or hospital for development of laboratory capacity to support hiv care was a complex process and required accounting for a number of factors, including patient burden, existing infrastructure, prior collaborations, geographic proximity to other programme facilities and local politics. the harvard/apin pepfar programme both consulted and collaborated with in-country partners and funding organisations so as to identify candidate clinics. after a clinic was proposed, a detailed site visit was performed to survey the existing laboratory, clinical and personnel infrastructure. there were often substantial obstacles to laboratory development as a result of the poor existing infrastructure, such as the lack of running water or dependable electrical service. reliability of utilities was essential, as the programme’s protocol for laboratory testing was substantial, requiring electricity-driven instruments (table 1). table 1: antiretroviral therapy regimen testing schedule. procurement of equipment preliminary laboratory improvements began with a needs assessment and included ensuring reliable water and electricity supply, back-up generator, security and adequate air-conditioning capacity. whilst tertiary and secondary hospital laboratories had an existing patchwork of hiv diagnostics, clinical chemistry, haematology and cd4+ cell count analysers in place, the harvard/apin pepfar programme expanded and improved access to these critical technologies. in accordance with the who’s 2008 maputo declaration,6 we attempted to provide all laboratories with the same equipment manufacturer and models, supporting standardisation of platforms across sites. standardisation of laboratory equipment allowed for streamlined training and maintenance, eased acquisition of spare parts and reduced overall costs through higher-volume orders. the availability of in-country servicing along with predicted sustainability of manufacturers, vendors and platforms, were also important factors in selection criteria. for hiv testing, following the nigerian national rapid test algorithm guidelines at the time, the determine hiv rapid test (alere medical co., japan) was provided, followed by unigold (trinity biotech plc, ireland), with statpack (chembio diagnostic systems, medford, ny, united states) as the discordant result tiebreaker. if further hiv infection confirmation was required, western blot (immunetics, boston, ma, united states) was performed. immunologic monitoring of patients’ cd4+ cell counts was performed using the flow cytometry-based cyflow counter or cyflow ii (partec gmbh, munster, germany) platform. to monitor virologic treatment response, hiv vls were measured with the manual cobas amplicor hiv-1 monitor test, version 1.5 (roche diagnostics gmbh, mannheim, germany). eligibility for art and subsequent toxicity were evaluated using relevant blood chemistry assays (table 1) on the roche cobas c311, cobas c111 or equivalent. haematology monitoring was performed using the mindray bc-3200 (mindray medical ltd, shenzen, china) or equivalent. hiv-1 drug resistance was evaluated, when indicated, using the viroseq genotyping system version 2.0 (abbott molecular, des plaines, il, united states), with sequencing results being generated on the abi genetic analyser 3130xl (applied biosystems, foster city, ca, united states). laboratory modifications many laboratories required physical alterations to existing structures or reconfigurations to improve effective, logical sample processing. a laboratory’s ideal sample flow was established, beginning at the arrival bench, where samples were logged and separated as needed. sample aliquots were then sent to individual laboratory stations for routine testing, after which samples were moved to storage and to a final station where results were recorded and sent to data entry staff for entry to patients’ records. for more advanced testing, such as deoxyrobinucleic acid polymerase chain reaction (dna pcr), different steps of the assay protocol were performed in separate rooms, with access restricted to dedicated laboratory members in order to minimise risk of contamination. biosafety and fire preparedness procedures were reviewed and revised, and appropriate biohazard waste processing was ensured. security of laboratories was addressed through both physical and policy improvements, with signage, laboratory renovations and staff trainings that ensured the exclusion of non-essential staff from laboratory spaces. laboratory data were secured in locked locations with strict access controls and were maintained according to national standards. supply chain procurement processes were developed to maximise effective purchasing of equipment and consumables, from expanding use of non-cold chain reagents and regular meetings with in-country laboratory supply sales representatives to working with supply chain management systems (scms) and the clinton health access initiative (chai) so as to secure the necessary test kits. to store all materials for distribution to the sites, two warehouses were maintained – one in the south (lagos) and one in the centre (abuja) of the country. a programme logistics manager and head pharmacist were hired and trained, working together to organise and expedite distribution of supplies to the sites using programme vehicles with transport staff and/or by means of an express courier with a negotiated service contract. equipment maintenance maintenance of equipment is a critical aspect of ensuring strong laboratory infrastructure, particularly in a resource-limited setting. most laboratories had dedicated on-site engineers with varying levels of expertise. in addition, programme engineers were hired to travel to other sites for scheduled periodic preventive maintenance as well as specific repairs. retention of skilled engineers was a serious challenge and concern; accordingly, the programme made great efforts to build local capacity and allow flexible working hours. when soliciting quotes for large equipment purchases for the programme, every effort was made to include training for local engineers and application specialists. programme engineers also traveled to the united states, europe and elsewhere in africa for trainings for specific equipment maintenance on partec cyflow analysers (partec gmbh, munster, germany), nuaire laminar flow hoods (nuaire, inc., plymouth, mn, united states) and the roche cobas platform (roche gmbh, mannheim, germany). data management the harvard/apin pepfar data management team built an easy-to-use, electronic medical records system that allowed for consolidation of laboratory, clinical and pharmacy information using the filemaker pro platform (filemaker inc., santa clara, ca, united states). wherever possible, these key programme areas were linked by local computer networks within each site. database plug-ins or utility software tools were designed in order to import electronic laboratory results directly into databases when possible. every site also had dedicated data staff to maintain the electronic patient records. all databases were uploaded on a weekly basis to a secure server for compilation by the programme data team, for reporting and monitoring purposes. in addition, all laboratories were equipped with an internet-connected desktop computer for laboratory members to use for programme-related communication and a reference resource. laboratory trainings the larger tertiary laboratories carried much of the initial responsibility for training and mentoring their smaller secondary and primary satellite laboratories. programme satellite coordinators were the principal contact persons for all laboratory personnel and communicated problems needing attention to either local site management or up to programme management. laboratory quality conferences were held annually in-country, bringing members together from laboratories of every size. each conference typically had 70 to 90 attendees and were held in various locales within nigeria. this type of meeting was ideal for advancing overall laboratory quality, addressing changes to programme policy, developing consensus decisions and allowing smaller laboratory groups to interact closely with more experienced peers. results top ↑ in total, harvard/apin pepfar helped support and develop the infrastructure at 35 laboratories in nigeria. of the 18 major sites managed, 8 were tertiary and 10 were secondary laboratories. in addition, the nigerian federal ministry of health designated 7 as centres of excellence. all laboratories were housed in permanent buildings with electricity, back-up generators, running water and basic infrastructure; a number received substantial upgrades to ensure successful operation and future sustainability. notable examples of effective laboratory reorganisation were the infrastructure upgrades at the jos university teaching hospital (juth) tertiary laboratory (figure 2) and the logical workflow renovation of the molecular laboratory at the lagos university teaching hospital tertiary laboratory (figure 3). figure 2: infrastructure updates made to jos university teaching hospital in jos, plateau state. figure 3: diagram of the molecular biology laboratory renovations, produced in collaboration with us centers for disease control and prevention, for directional workflow at the lagos university teaching hospital, lagos state. all tertiary and secondary laboratory sites provided hiv serodiagnosis through rapid test technologies, automated haematology, clinical chemistry, laser-based cd4+ cell enumeration, vl quantitation and infant dna pcr diagnosis. the primary laboratories provided access to hiv rapid testing, haematology, clinical chemistry and cd4+ cell count enumeration. starting in late 2012, apin upgraded to automated vl equipment, using the cobas ampliprep/taqman hiv-1 test, version 2.0. all tertiary and secondary laboratories had the capacity for tb diagnosis with acid-fast bacilli (afb) staining and a subset of tertiary laboratories developed the infrastructure and capacity for the identification of multidrug-resistant tb (mdr-tb) through various assays, including genexpert (cepheid, sunnyvale, ca, united states) and genotype mtbdrplus (hain lifescience gmbh, nehren, germany). we introduced screening of select groups at two sites for mdr-tb using the mtbdrplus test and proposed using this test for expanded national surveillance to the national tb control programme.7 three tertiary laboratories had abi capillary sequencers (applied biosystems, foster city, ca, united states) for hiv drug resistance testing. in support of the national early infant diagnosis (eid) programme, with the support of chai and the us centers for disease control and prevention (cdc) office in nigeria, laboratories with pcr testing capacity were also able to provide eid testing of dbs samples. stock rooms were also improved with greater security mechanisms, such as bars on all doors and windows, sturdy shelving, stock cards and clear ordering and restocking procedures. by leveraging the high volume of regular laboratory tests required by the programme, contracts were secured for significantly reduced reagent costs from most vendors. by moving from a manual dynabeads (life technologies, grand island, ny, united states) method for cd4+ cell count enumeration, to automated partec cyflow platforms, test costs were reduced initially from us$22.00/test to us$5.00/test, to a cost in 2012 of under us$2.00/test. the cost of routine chemistry tests, such as alanine aminotransferase (alt) and creatinine, dropped with the implementation of automated platforms from over us$1.00/test to approximately us$0.29/test. vl test costs using manual roche amplicor kits were initially us$33.00/test, but by maintaining a high volume of tests over time and migrating to the automated roche cobas platform, costs were reduced to us$14.00/test by 2012. starting in 2010, harvard began shifting laboratory logistics responsibilities to apin. existing vendors began to bill apin directly, and supply chains were modified to increase local procurement of consumables and test kits. changing import regulations, supplier stock-outs and local strikes necessitated occasional aid from an established non-profit organisation that could provide additional mechanisms for import and customs clearance. the achievements of the eight-year laboratory scale-up in the harvard/apin pepfar programme have been significant. from 2004–2012, harvard/apin supported laboratories were able to provide haematology, chemistry, cd4+ cell count enumeration and vl results for over 2.5 million samples (table 2, figure 4). the collaboration for eid testing expanded rapidly in nigeria, with a greater than 10-fold increase in capacity from 2007 to 2008, when over 9000 hiv exposed infants were tested. table 2: number of tests performed annually by harvard/apin pepfar laboratories. figure 4: cumulative laboratory tests performed by harvard/apin pepfar laboratories. impact on health system strengthening beyond improvements of specific laboratory services to support the ongoing art programme, the training and mentorship activities expanded the goal of providing the highest quality of overall healthcare. the programme sought to extend beyond art delivery and to integrate quality processes and equipment benefits throughout the hospitals and clinics. for example, the purchase of portable tb-diagnostic x-ray equipment became available for use by other hospital departments. training of programme staff also benefited the overall institution’s laboratory capacity-building efforts, as most programme staff also had roles in the hospitals’ non-hiv laboratories. generally, only two to three persons from each laboratory were invited to attend the annual group trainings; subsequently, conference attendees provided step-down training in order to transfer new information to the entire local laboratory team. this also incentivised laboratory managers to maintain high levels of performance, helping to ensure leadership positions for their teams in future trainings. we found the utilisation of training-of-trainer and step-down training methods to be a very cost-effective system of providing training across programme laboratories and allowed for dissemination of training across local laboratory systems. training conferences centralised laboratory conferences provided programme management the opportunity to address the laboratory staff teams directly and to acknowledge and commend their hard work. these central workshops allowed programme staff continual assurance of standardisation across programme laboratories. over time, it was realised that workshop participation could be strengthened through administration of preand post-tests encompassing major topics. additionally, these meetings offered laboratory teams an opportunity to connect with others that were conducting similar work and allowed for generation of a network that could provide local troubleshooting and support. programme-wide laboratory trainings were attended by 211 laboratory staff over 59 training days; and programme management provided direct laboratory mentorship training over 526 days. in-country teams also developed trainings for more targeted topics such as equipment maintenance, new laboratory platform initiation and accreditation preparedness, training 159 laboratory staff over 84 training days. these numbers do not account for the step-down training days or for retraining sessions that took place upon return to individual laboratory sites. if programme management identified a laboratory with specific concerns, a site-specific training visit was organised. these localised trainings addressed a wide array of issues, from laboratory staff reorganisation, to launching a trial of a new diagnostic point-of-care platform, to troubleshooting an assay performing out of range. coordination was sought with clinical and pharmacy training teams so as to extend laboratory topics to their trainings and to apprise laboratory members of any updates to other programme areas that could have an impact on laboratory functions, such as the introduction of new drugs or drug regimens with a specific toxicity concern. internal and external trainings for laboratory engineers resulted in reduced service calls to factory technicians and less equipment downtime for sites.8 properly-functioning equipment ensured that test kits were consumed in a timely manner, prior to expiration, constituting another cost-saving goal. additionally, because of both interest and observed need, workshops were held to assist laboratory and research staff with grant writing and publication skill building. electronic data management significant gains were achieved in both electronic data capture and improving delivery of laboratory results to clinical staff and patients. electronic data capture decreased the opportunity of transcription errors and allowed laboratory staff more time for laboratory activities. laboratory result turnaround times were reduced by an average of two days through the use of electronic data, compared with the prior method that consisted of only handwritten logs and manual transcription. an advantage of creating a programme-specific database system was that it offered great flexibility, allowing data managers to revise clinical chemistry results to be reported in units that conformed to international standards, or the ability to introduce modifications rapidly as laboratory technologies evolved. for example, the databases were adapted to produce pop-up flags for critical values, such as low haemoglobin or elevated liver or renal enzymes. another unique electronic tool was the ‘viral load utility’, created to convert analyser data into standardised test results for direct database import. additionally, the database system was flexible enough to allow for transfer of data to national forms and provide aggregate reporting, when the federal ministry of health developed new registers and aggregate reporting forms. a major innovation by the harvard/apin pepfar laboratory and data teams was the design and implementation of an electronic database for both compiling patient laboratory information and then distributing reports over local networks to clinic and pharmacy locations. this ‘treatment response utility’ tool was developed primarily to provide medical staff with a comprehensive picture of a patient’s treatment profile over time and to transfer a greater number of laboratory results presented in a more useful format to the clinical decision-making team (figure 5). figure 5: example of a patient history in the treatment response utility. the utility provides a graphical layout that displays a timeline with vl, cd4+ cell count and drug pick-up data, as well as links to a more detailed clinical history for a particular patient. the visual snapshot aids the physicians in communicating and educating the patient on the positive health effects of arv drug adherence. the tool also assists clinicians in detecting failure of a drug regimen, assisting in a more rapid switch to a new drug regimen, or intervening for patients struggling with adherence. laboratory quality control and accreditation at the onset of the harvard/apin pepfar programme, very few laboratories had quality management systems in place. as laboratories became equipped and staff were trained, data quality processes and laboratory quality assurance systems were instituted. in-country external quality assurance (eqa) was scaled up, with distribution of standardised controls for vl testing. harvard/apin pepfar-supported laboratories subscribed to eqa programmes distributed by the college of american pathologists for blood chemistry and viral markers, as well as the united kingdom national external quality assessment service for cd4+ cell count monitoring. in addition, eqa for eid through dbs testing was conducted through the international laboratory branch of the cdc’s division of global hiv/aids. an eqa programme was not available for the viroseq drug resistance genotyping; however, genotyping and sequence analysis were verified at harvard. whilst being costly, the international eqa programmes were a requirement of laboratory accreditation programmes, and resulted in marked improvements in reported results for all laboratories by the second year of subscription. since 2009, addressing long-term sustainability, the harvard/apin pepfar laboratories have partnered with the nigerian national external quality assessment laboratory, managed by the medical laboratory science council of nigeria, to expand an accredited national eqa programme in lieu of international subscriptions. the slmta programme was launched in kigali, rwanda in 2009 by the who regional office for africa (who afro), the cdc, the chai and the american society for clinical pathology in an effort to promote accessible pathways to accreditation in sub-saharan africa.1 six laboratories from the harvard/apin programme were selected for inclusion in the initial slmta rollout in 2010. these initial laboratories were nominated by the government of nigeria and cdc’s office in nigeria and achieved marked improvements from 2010 to 2012 (table 3). table 3: slmta assessment update of harvard/apin pepfar-supported labs. six tertiary laboratories enrolled in the initial slmta rollout in 2010 and achieved exit scores of five stars (one laboratory) and four stars (five laboratories) on a five-star scale. the one five-star laboratory has also been iso 9001 certified and plans are in place to move additional secondary laboratory sites into future slmta quality assessment programmes.9 this programme has served as a springboard, not just for the initial laboratories enrolled, but also for all laboratories in the harvard/apin programme, to focus on the who afro assessment scheme and to make dramatic improvements to laboratory quality processes. discussion top ↑ quality laboratory services have become a foundation of the harvard/apin pepfar programmes in nigeria and capacity has grown to include automated clinical chemistries and haematology for monitoring art toxicity at 24 laboratories, pcr-based vl monitoring and eid at 10 laboratories and capillary-based genetic sequencing for hiv drug-resistance mutations at 3 laboratories. over the course of the programme, the number of patients supported with hiv care rose from 2439 in 2004 to 159 897 by 2012 and our programme laboratories provided over 2.5 million laboratory test results for these patients. in addition, many sites achieved documented improvements in quality services as they moved through the slmta programme toward accreditation. many of the outcomes detailed in this article originated from major international investments to expand global health programmes in the developing world (eg. pepfar). such scale-ups could represent an opportunity to apply proven methods and novel approaches to effect meaningful improvement in local laboratory capacities. sustainability of all laboratory improvement endeavours must be considered carefully, with attention being given to the realities of economic constraints, transitions to in-country support and management and integration with national strategic plans. it is critical to build a solid foundation of local laboratory leadership that can maintain improvements independently when international teams depart. the authors believe that without the described improvements to laboratory capacity and quality, the growth and achievements of the harvard / apin pepfar programme could not have been attained. various processes of the 12 quality system essentials that we used to scale up laboratory activities were effective. specifically, using multiple training methods worked well in ensuring sufficient numbers of trained laboratory staff at each site along with maintenance of high-quality, standardised services throughout the programme sites. the system of centralised procurement and supply distribution allowed for efficient monitoring of supply use and reduction in costs through bulk ordering. by implementing an electronic medical record system, we ensured increased use of data by clinical staff for improved patient care. furthermore, laboratory teams elevated the overall quality of care at the sites by providing data readily accessed by the electronic treatment response utility. the training efforts also resulted in personnel that were able to develop and maintain laboratories worthy of international accreditation. additionally, the laboratory scale-up and training efforts had many indirect effects. one major impact of the laboratory scale-up efforts was concomitant health system strengthening across the hospital settings in which our hiv programmes were located. other groups have documented similarly that the pepfar scale-up and integration within existing healthcare systems has improved the linkage of hiv and tb care10 and increased the number of in-hospital births in resource-limited settings.11 in addition, as a result of pepfar-associated training efforts, various programme-affiliated laboratory researchers from nigeria have been successful in gaining fogarty fellowships (fogarty international center, nih, bethesda, md, united states) to spend several months working on research projects in the harvard research laboratories in boston, ma, united states. the harvard/apin pepfar team has published research in peer-reviewed journals supporting the cost effectiveness and patient benefit from regular vl and drug resistance monitoring.12,13 finally, harvard worked with apin to expand testing capabilities by distribution of point-of-care equipment (including the partec cyflow minipoc) and are embarking on a new programme to test a point-of-care technology for measuring vls in an african resource limited setting.14 conclusion top ↑ in this article we have provided an overview of methods that may be useful in the development and support of a sustainable laboratory infrastructure, whilst simultaneously developing quality processes through a quality management system model and building upon the existing physical and human capital in a resource-limited setting such as nigeria. looking forward, as pepfar’s reorganisation of management by implementing partners occurs, apin endeavours to apply these strategies to strengthen laboratories at the hospitals it inherits and hopes that new partners at ceded locations continue to do the same. we hope that many of these lessons learned and strategies employed may assist and encourage the development of other laboratories in resource-limited settings. acknowledgements top ↑ the authors gratefully acknowledge the patients and the hard work and dedication of all the staff at collaborating clinics, hospitals, and laboratories. this work was funded, in part, by the us department of health and human services, health resources and services administration (u51ha02522). the contents are solely the responsibility of the authors and do not represent the official views of the funding institution. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions d.j.h. (harvard school of public health) conceived of the evaluation; conducted laboratory trainings and site visits; ordered equipment; and wrote the manuscript. j-l.s. (globomics) provided senior laboratory and programme development direction; contributed to the outline of the evaluation; edited the manuscript; and performed site visits. j.o.s (aids prevention initiative in nigeria ltd) assisted in the conception for the evaluation; served as apin laboratory programme director; consulted for new site additions; and conducted site visits. a.d.s. (cdc malawi) provided senior leadership; contributed to the outline of the evaluation; initiated and developed the laboratory quality programme, eqa management and who afro assessment processes; performed laboratory trainings and site visits; and advised on new equipment platforms. b.c. (harvard school of public health) contributed to the outline of the evaluation; edited the manuscript; developed molecular laboratory capacity in nigeria; and developed numerous laboratory databases, including the treatment response utility. e.o. (aids prevention initiative in nigeria ltd) contributed to the outline of the evaluation; performed site visits and laboratory trainings; and served as the primary liaison for the who afro laboratory accreditation processes. s.t.m. (harvard school of public health) contributed to the outline of the evaluation; reviewed and edited all versions of the manuscript; consulted on all site and laboratory activities; and oversaw collection and quality of all electronic laboratory data and reporting. p.o. (aids prevention initiative in nigeria ltd) provided medical leadership on all laboratory and programme activity in nigeria; contributed to the outline of the evaluation and reviewed and edited all versions of the manuscript; and contributed to laboratory trainings and performed site visits. p.j.k. (harvard school of public health) conceived of the evaluation; contributed to the outline of the evaluation; reviewed and edited all versions of the manuscript; provided senior leadership on all laboratory and programme activities; advised on laboratory expansion and development; and contributed to laboratory trainings. all authors read and approved the final manuscript. references top ↑ gershy-damet gm, rotz p, cross d, et al. the world health organization, african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. http://dx.doi.org/10.1309/ajcptuuc2v1wjqbm department of state. office of global aids coordinator. pepfar blueprint: creating an aids-free generation; 2012. http://www.pepfar.gov/documents/organization/201386.pdf. accessed december 2014. world health organization. guidance for development of national laboratory strategic plans. geneva: who; 2010. nigeria federal ministry of health. technical report: 2010 national hiv sero-prevalence sentinel survey. department of public health, abuja, nigeria; 2011. http://www.nigeria-aids.org/documents/2010_national%20hiv%20sero%20prevalence%20sentinel%20survey.pdf. accessed december 2014. kanki pj. the challenge and response in senegal. in: kanki p, marlink r (editors). a line drawn in the sand: responses to the aids treatment crisis in africa. cambridge, ma: harvard center for population and development studies, 2009; pp. 65–93. world health organization. the maputo declaration on strengthening of laboratory systems [document on the internet]. c2008. http://www.who.int/diagnostics_laboratory/maputo-declaration_2008.pdf. accessed december 2014. dinic l, akande p, idigbe eo, et al. genetic determinants of drug-resistant tuberculosis among hiv-infected patients in nigeria. j clin microbiol. 2012;50(9):2905–2909. http://dx.doi.org/10.1128/jcm.00982-12 fonjungo pn, kebede y, messele t, et al. laboratory equipment maintenance: a critical bottleneck for strengthening health systems in sub-saharan africa. j public health policy. 2012;33(1):34-45. http://dx.doi.org/10.1057/jphp.2011.57 guzel o, guner el. iso 15189 accreditation: reqirements for quality competence of medical laboratories, experience of a laboratory i. clin biochem. 2009;42(4–5); 274–278. http://dx.doi.org/10.1016/j.clinbiochem.2008.09.011 howard aa, gasana m, getahun m, et al. pepfar support for the scaling up of collaborative tb/hiv activities. j acquir immune defic syndr. 2012;60(suppl 3): s136–s144. http://dx.doi.org/10.1097/qai.0b013e31825cfe8e kruk me, jakubowski a, rabkin m, et al. pepfar programs linked to more deliveries in health facilities by african women who are not infected with hiv. health aff (millwood). 2012;31(7):1478–1488. http://dx.doi.org/10.1377/hlthaff.2012.0197 rawizza he, chaplin b, meloni st, et al. accumulation of protease mutations among patients failing second-line antiretroviral therapy and response to salvage therapy in nigeria. plos one. 2013;8(9):e73582. http://dx.doi.org/10.1371/journal.pone.0073582 rawizza he, chaplin b, meloni st, et al. immunologic criteria are poor predictors of virologic outcome: implications for hiv treatment monitoring in resource-limited settings. clin infect dis. 2011;53(12):1283–1290. http://dx.doi.org/10.1093/cid/cir729 lee hh, dineva ma, chua yl, et al. simple amplification-based assay: a nucleic acid-based point-of-care platform for hiv-1 testing. j infect dis. 2010;201(suppl 1): s65–s72. http://dx.doi.org/10.1086/650385 article information authors: tilahun m. hiwotu1 gonfa ayana1 achamyeleh mulugeta1 getachew b. kassa1 yenew kebede2 peter f. fonjungo2,3 gudeta tibesso1 adino desale1 adisu kebede1 wondwossen kassa1 tesfaye mekonnen1 katy yao3 elizabeth t. luman3 amha kebede1 mary k. linde4 affiliations: 1ethiopian public health institute (ephi), addis ababa, ethiopia 2us centers for disease control and prevention (cdc), addis ababa, ethiopia 3international laboratory branch, division of global hiv/aids, us centers for disease control and prevention (cdc), atlanta, united states 4the american society for clinical pathology (ascp), chicago, united states correspondence to: achamyeleh mulugeta postal address: po box 1242, addis ababa, ethiopia dates: received: 21 aug. 2014 accepted: 15 sept. 2014 published: 03 nov. 2014 how to cite this article: hiwotu tm, ayana g, mulugeta a, et al. laboratory system strengthening and quality improvement in ethiopia. afr j lab med. 2014;3(2), art. #228, 6 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.228 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. laboratory system strengthening and quality improvement in ethiopia in this original research... open access • abstract • introduction • research methods and design    • participating laboratories    • slmta implementation    • improvement projects    • supportive site visits    • measuring improvements • results • discussion    • recommendations    • conclusion • acknowledgements    • competing interests    • authors’ contributions    • cdc disclaimer • references abstract top ↑ background: in 2010, a national laboratory strategic plan was set forth in ethiopia to strengthen laboratory quality systems and set the stage for laboratory accreditation. as a result, the strengthening laboratory management toward accreditation (slmta) programme was initiated in 45 ethiopian laboratories. objectives: this article discusses the implementation of the programme, the findings from the evaluation process and key challenges. methods: the 45 laboratories were divided into two consecutive cohorts and staff from each laboratory participated in slmta training and improvement projects. the average amount of supportive supervision conducted in the laboratories was 68 hours for cohort i and two hours for cohort ii. baseline and exit audits were conducted in 44 of the laboratories and percent compliance was determined using a checklist with scores divided into zeroto five-star rating levels. results: improvements, ranging from < 1 to 51 percentage points, were noted in 42 laboratories, whilst decreases were recorded in two. the average scores at the baseline and exit audits were 40% and 58% for cohort i (p < 0.01); and 42% and 53% for cohort ii (p < 0.01), respectively. the p-value for difference between cohorts was 0.07. at the exit audit, 61% of the first and 48% of the second cohort laboratories achieved an increase in star rating. poor awareness, lack of harmonisation with other facility activities and the absence of a quality manual were challenges identified. conclusion: improvements resulting from slmta implementation are encouraging. continuous advocacy at all levels of the health system is needed to ensure involvement of stakeholders and integration with other improvement initiatives and routine activities. introduction top ↑ medical laboratories form the backbone of health systems, as test results are critical for diagnosing diseases, guiding treatment, determining drug resistance and identifying diseases of public health significance through surveillance.1,2,3 an integrated, tiered, functional and sustainable laboratory system is necessary in order to address these health system needs.4despite their importance, laboratories are often under-resourced, resulting in inadequate infrastructure, poorly-trained personnel and lack of standardisation.1,2,5,6 likewise, laboratory services in ethiopia received little attention until recent years. funds and improved testing technologies were made available to laboratories after the hiv pandemic burdened medical facilities; however, several challenges to implementing laboratory improvement remain, such as the lack of adequately-trained personnel, clearly-defined responsibilities and well-established organisation.7 in response to these challenges, laboratory assessments were conducted on a national scale within ethiopia so as to elucidate specific deficiencies; as a result, corrective measures were proposed. amongst these were milestones of developing the first laboratory strategic plan in 2005 and mandating the ethiopian public health institute (ephi) to lead laboratory programmes nationwide.7 the first laboratory strategic plan (2005 to 2008) was focused primarily on hiv, which accounted for the bulk of both testing and funding. this plan was used as a roadmap for implementing laboratory improvement programmes in ethiopia. the second strategic plan (2009 to 2013) was developed to encompass integrated laboratory services.7 however, because of reforms within the ethiopian health sector, the second strategic plan was revised in 2010 (2010 to 2015), to focus on three goals: establishing and strengthening laboratory quality systems, laboratory capacity building and laboratory accreditation.8 the strengthening laboratory management toward accreditation (slmta) programme was selected in order to advance laboratory quality improvement and to expedite the accreditation preparation process. slmta is a task-based, hands-on training programme aimed at effecting tangible laboratory improvements in developing countries.9 it includes a series of three workshops that are supplemented by assigned improvement projects and supportive site visits or mentoring.10 to evaluate its effect, an audit is performed before (baseline) and after (exit) slmta implementation using the world health organization regional office for africa’s (who afro) stepwise laboratory quality improvement process towards accreditation (slipta) checklist.11 slipta is an accreditation preparation framework that consists of an incremental recognition system awarding a range of zero to five stars, in contrast to the pass or fail score of traditional accreditation schemes. this stepwise approach recognises where a laboratory stands currently and encourages continual improvement through positive reinforcement by rewarding progress at each step.3,4 laboratories in the public health system of ethiopia are divided into four tiers: peripheral, hospital, regional reference and national reference laboratories.7,12 ephi has national reference (multi-purpose) and research-only laboratories. its nine national reference laboratories, each with different specialties, provide research, diagnostic, training and external quality assessment services. as per the administrative hierarchy, the higher-level laboratories support and oversee the next lower-level laboratories. for example, ephi oversees the 12 regional reference laboratories, which oversee the 135 hospital laboratories, which, in turn, oversee health post/centre laboratories. however, in the case of new initiatives such as slmta, ephi supports hospital laboratories until the regional reference laboratories are prepared to take over the task. this article evaluates the implementation and impact of the slmta programme in 45 laboratories in ethiopia, as well as the role of supportive site visits and integration of selected improvement projects. research methods and design top ↑ participating laboratories a total of 45 laboratories were enrolled for participation in the slmta programme between 2010 and 2012 in order to facilitate quality improvement and to accelerate stepwise accreditation preparation. laboratories were selected based on tier level, type, testing volume, geographical distribution and the operational area of the four american university-affiliated partners that participated in the audits and supportive site visits. six national reference laboratories, seven regional reference laboratories and 32 public hospital laboratories were selected. for ease of implementation, laboratories were divided into two cohorts. national and regional laboratories were included in the first cohort from june 2010 to october 2011, along with 11 of the hospital laboratories; the remaining 21 hospital laboratories were included in the second cohort from january 2011 to may 2012 (table 1). table 1: timeline of slmta implementation in ethiopia. slmta implementation the standard slmta model of three workshops was employed.10 the laboratory manager and quality officer, who are responsible for leading their staff and establishing a sustainable system for quality-assured laboratory services, were invited from each participating laboratory to attend the slmta training organised by ephi in collaboration with the american society for clinical pathology (ascp). a total of 60 people were trained in each cohort. trainings were facilitated by graduates of a two-week training-of-trainers course conducted by the african centre for integrated laboratory training in johannesburg, south africa, in collaboration with the us centers for disease control and prevention (cdc).13 improvement projects improvement projects were assigned based on the common gaps identified during the baseline audit and the components of the quality management system addressed during the specific training. examples of areas in which improvement projects were conducted include customer satisfaction, external quality assessment, turnaround time and equipment utilisation rate. these improvement projects were expected to be implemented before the next training, or the exit audit in the case of improvement projects following the third workshop. progress was reported to and monitored by ephi. additionally, participants were instructed to identify and assign more improvement projects to other staff members for execution. supportive site visits supervisors trained in mentorship and supervisory skills were tasked to provide supportive site visits. visits were scheduled approximately three weeks after each workshop. the plan required supervisors to spend two to three days in each slmta-enrolled laboratory. during this time, they conducted a site visit using prepared checklists that specifically focused on assigned improvement projects. for cohort i, the supportive site visits were led by two or three supervisors; one from ephi, one from an american university-affiliated partner and one (for three-person teams) from a regional reference laboratory. the four university partners were: columbia university’s international center for aids care and treatment program in ethiopia (icap-ethiopia); the university of washington’s international training and education center on hiv in ethiopia (itech-ethiopia); the johns hopkins university technical support for the ethiopian hiv/aids initiative (jhu-tsehai); and the university of california, san diego’s technical assistance programme for hiv prevention, care and treatment in ethiopia (ucsd-ethiopia). the university partners were assigned to specific regions covering the entire country. an average of 68 (range 24 to 80) hours of joint supportive site visits were provided to each of the first cohort laboratories. supportive site visits for cohort ii did not take place as planned. the responsibility for providing supportive site visits was handed over to supervisors from the regional reference laboratories. because of budget issues, no site visits occurred after the second or third workshop; total site visit time averaged two (range 0 to 24) hours per laboratory. measuring improvements audits were conducted before (baseline) and after (exit) slmta implementation using the slipta checklist. the pool of auditors comprised the experts who had conducted the supportive site visits. audits were performed over two or three days by a group of two to three individuals who had not conducted supportive site visits or previous audits in the respective laboratory. the audit checklist is based on 12 quality system components. star levels were granted based on total audit scores; one, two, three, four and five stars were awarded for scores reaching 55%, 65%, 75%, 85% and 95%, respectively. one of the first cohort laboratories was unwilling to have an exit audit; thus results are presented for 44 of the 45 laboratories. audit data were analysed using microsoft® excel 2013. a paired t-test was used to compare baseline and exit results; a t-test for unequal variances was used to compare score changes between the two cohorts (f-test showed unequal variances, p < 0.01). major success factors and challenges encountered during implementation of slmta were identified by focus group discussions. results top ↑ overall, for the 44 laboratories that participated in both the baseline and exit audits, there was a 15 percentage point average increase in slipta audit score, with 41% as the average baseline score and 56% the average exit score. for cohort i, average scores increased from 40% at baseline to 58% at exit audit (p < 0.01). the average scores of cohort ii increased from 42% at baseline to 53% at exit audit (p < 0.01) (figure 1). figure 1: slipta baseline and exit audit scores of the two slmta cohorts. baseline scores ranged from 24% to 60% whereas exit scores ranged from 25% to 86%. forty-two of the 44 laboratories (95%) improved their scores (from < 1 percentage point to 51 percentage points). a higher mean improvement was observed in cohort i laboratories (18 percentage points) compared with cohort ii laboratories (11 percentage points), although the difference was of borderline statistical significance (p = 0.07). five laboratories in cohort i showed an improvement of 30–51 percentage points, whilst no laboratories in cohort ii achieved greater than a 30 percentage point improvement. however, decreases were also observed in two laboratories in cohort i (table 2). table 2: improvement of laboratory slipta scores. during the baseline audit, all laboratories in the first and all but one of the laboratories in the second cohort were at the zero-star level (scored < 55%). at the exit audit, 61% (n = 14/23) of the first and 48% (n = 10/21) of the second cohort laboratories attained one to four stars (figure 2). figure 2: change in slipta star levels from baseline to exit audit.* results from the 12 quality system components of the slipta audit checklist are presented in figure 3. at both the baseline and exit audits, lower scores were observed for internal audit (6% baseline and 18% exit), occurrence management and process improvement (14% and 29%), corrective action (31% and 41%) and management reviews (32% and 44%). relatively higher scores were shown in client management and customer service (42% and 59%), organisation and personnel (44% and 59%), information management (58% and 63%) and facilities and safety (51% and 71%). the greatest improvements were seen in the areas of documents and records (32 percentage points), facilities and safety (22 percentage points) and client management and customer service (17 percentage points). smaller improvements were observed for information management (five percentage points), equipment (six percentage points) and process control and internal and external quality assessment (nine percentage points). figure 3: percent compliance across the 12 quality system components of the 44 audited laboratories before (baseline) and after (exit) the implementation of slmta. in focus group discussions, laboratory managers and quality officers pointed out several challenges to successful implementation of improvement projects. these challenges included: (1) poor awareness of the programme by upper management and regional health bureaus (i.e., slmta was not budgeted); (2) lack of harmonisation with other hospital improvement programmes; (3) inadequate awareness of quality management systems and insufficient commitment amongst non-slmta-trained staff; (4) high workload relative to available staff; (5) lack of quality manuals prescribing laboratory policies and procedures; and (6) inadequate supportive site visits. detailed results from these focus group discussions are reported by lulie et al.14 discussion top ↑ prior to the introduction of slmta, several trainings and quality improvement initiatives had been implemented in hospitals and laboratories in ethiopia, but little improvement was noted. by contrast, ethiopian public health laboratories achieved remarkable improvements in quality systems after implementation of the slmta programme. similar rapid and tangible improvements resulting from slmta training have been reported by other countries.15,16whilst similar training curricula and improvement projects were given to the two cohorts, a lower level of supportive site visits was administered to the second cohort. results for the first cohort surpassed that of the second cohort, with nearly twice the improvement. although not conclusive, these results suggest a positive effect of site visits. site visits offer an opportunity to assess progress and enforce application of skills learned in the workshops. these visits often involve meetings with upper management and other staff members in order to elicit their support, which in turn motivates the implementers. our data suggest that supportive site visits were critical with regard to reinforcing the knowledge and motivation offered during the training in order to achieve the expected behavioural changes required for quality improvement. on-site mentorship may also be used in a similar capacity; for example, it was found that after 10 weeks of dedicated on-site mentoring, laboratories in lesotho demonstrated significant improvements.15 at baseline, participating laboratories displayed deficient quality management systems in many areas, as evidenced by all but one laboratory earning a zero-star rating. a poor laboratory quality management system facilitates erroneous or delayed results, which in turn can lead to poor or adverse patient outcomes.17 in ethiopia, slmta implementation, combined with intensive supportive site visits, has proven to be a promising tool for the improvement of quality management systems. participating laboratories were found to be weaker in some quality system components than others. specifically, internal audit, occurrence management and process improvement, corrective action and management reviews scored the lowest at both the baseline and exit audits. similar results have been reported elsewhere.16these weaker components are critical for continuous improvement of the overall quality system. therefore, focused attention must be paid so that greater and sustainable advancements can be made. several barriers to the effective implementation of quality systems were identified during the focus group discussions. for example, an accreditation preparation budget was not included in the annual plans of the regional health bureaus, hospitals or laboratories. this omission prevented adequate supportive site visits for cohort ii laboratories. it was also pointed out that promoting the importance of accreditation and the need for improving laboratory quality management systems is necessary for enhancing acceptance and ownership at all levels. such promotion should be conducted prior to and during slmta implementation so as to ensure adequate support throughout the established healthcare structure and management teams.118 hospital system and process reforms are underway in public health facilities throughout ethiopia, with a goal of improving efficiency and effectiveness (i.e., high quality, low cost and rapid services), thereby increasing customer satisfaction.14,19,20 strengthening laboratory quality management and moving toward accreditation should be considered an integral part of this process. failure to implement laboratory quality management systems in harmony with other initiatives misses a valuable opportunity and could hinder the pace of progress and long-term sustainability of health system reforms and improvements.18 at the same time, the lack of laboratory quality manuals, guidelines, policies and procedures that provide clear and concrete directions and instructions posed a challenge during implementation. it was difficult for laboratories to execute quality improvement tasks consistently in the absence of these guiding materials. improvements were found to vary significantly amongst laboratories of each cohort; two laboratory exit scores showed a decrease from baseline, whilst the remaining 42 laboratory exit scores reflected an increase of up to 51 percentage points. the challenges identified during the trainings and focus group discussions could be contributing factors for this variation. further collection and analysis of data throughout the implementation phase could enable programme leaders to identify and redress impeding factors. recommendations improvements resulting from slmta implementation are encouraging and expanding slmta to other laboratories may help to improve quality management of laboratories for a better healthcare system. the evaluation suggests that supportive site visits are important for effective implementation of slmta and quality management systems. availing a clear plan of supportive site visits and gaining upper management ownership in participating laboratories is needed in order to obtain the maximum benefit of slmta. conclusion continuous advocacy at all levels of the health system will foster involvement of stakeholders and facilitate the integration of slmta with other hospital improvement initiatives, allowing slmta to benefit the system on a wider scale. acknowledgements top ↑ the authors would like to thank the regional health bureaus, laboratory mangers and quality officers of participating laboratories for their assistance in leading their laboratories toward a sustainable system for quality assured laboratory services. we also thank ascp for their dedication to strengthening laboratory quality systems in ethiopia. the authors would also like to acknowledge the contributions of icap-ethiopia, itech-ethiopia, jhu-tsehai and ucsd-ethiopia for their support in implementing slmta and strengthening laboratory systems in ethiopia. this research has been supported by the us president’s emergency plan for aids relief (pepfar) through the cdc. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions t.m.h (ephi) served as a focal person for the programme and a trainer, analysed the data and wrote the manuscript. g.a (ephi) played a key role in the design and implementation of the programme and reviewed the manuscript. a.m. (ephi) took part in providing supportive supervision, participated in data collection and reviewed the manuscript. g.b.k.(ephi) played a key role in organising training sessions, provided supportive site visits and revised the manuscript. y.k.(cdc, ethiopia) and p.f.f. (cdc, ethiopia; cdc, atlanta) played a key role in co-designing the programme, coordinated partners’ contributions and reviewed the manuscript. g.t.(ephi) led and supervised the design and implementation of the programme. a.d.(ephi), adisu k. (ephi), w.k.(ephi) and t.m.(ephi) took part in training, provided supportive site visits and were involved in data collection. k.y. (cdc, atlanta) and e.t.l.(cdc, atlanta) played a key role in analysing the data and made extensive revisions to the manuscript. amha k.(ephi) provided overall guidance for the programme development and oversaw its implementation. m.k.l.(ascp) led the organisation of the trainings and made extensive revisions to the manuscript. cdc disclaimer the findings and conclusions in this article are those of the authors and do not necessarily represent the official position of the cdc. references top ↑ 1. panteghini m. the future of laboratory medicine: understanding the new pressures. clin biochem rev. 2004;25(4):207–215.2. wians fh. clinical laboratory tests: which, why, and what do the results mean? lab medicine. 2009;40:105–113. http://dx.doi.org/10.1309/lm4o4l0hhutwwudd 3. gershy-damet gm, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. http://dx.doi.org/10.1309/ajcptuuc2v1wjqbm 4. nkengasong jn. a shifting paradigm in strengthening laboratory health systems for global health: acting now, acting collectively, but acting diifferently. am j clin pathol. 2010;134(3):359–360. http://dx.doi.org/10.1309/ajcpy5asuejyq5rk 5. vitoria m, granich r, gilks cf, et al. the global fight against hiv/aids, tuberculosis, and malaria: current status and future perspectives. am j clin pathol. 2009;131(6):844–848. http://dx.doi.org/10.1309/ajcp5xhdb1pnaeyt 6. nkengasong jn, nsubuga p, nwanyanwu o, et al. laboratory systems and services are critical in global health: time to end the neglect? am j clin pathol. 2010;134(3):368–373. http://dx.doi.org/10.1309/ajcpmpsinq9brmu6 7. ethiopian health and nutrition research institute. master plan for the public health laboratory system in ethiopia, second edition (2009–2013). addis ababa: ehnri; 2009. 8. ethiopian health and nutrition research institute. five year, balanced score card-based strategic plan (2010–2015). addis ababa: ehnri; 2010. 9. yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin pathol. 2010;134(3):401–409. http://dx.doi.org/10.1309/ajcpnbbl53fwuiqj 10. yao k, maruta t, luman et, nkengasong jn. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. 2014;3(2), art. #194, 8 pages. http://dx.doi.org/10.4102/ajlm.v3i1.194 11. world health organization’s regional office for africa. who afro stepwise laboratory quality improvement process towards accreditation (slipta) checklist [document on the internet]. c2012 [cited 2014 may 31]. available from: http://www.afro.who.int/en/clusters-a-programmes/hss/blood-safety-laboratories-a-health-technology/blt-highlights/3859-who-guide-for-the-stepwise-laboratory-improvement-process-towards-accreditation-in-the-african-region-with-checklist.html 12. world health organization’s regional office for africa (who afro). consultation on technical and operational recommendations for clinical laboratory testing harmonization and standardization. maputo: who afro; 2008. 13. maruta t, yao k, ndlovu n, moyo s. training-of-trainers: a strategy to build country capacity for slmta expansion and sustainability. afr j lab med. 2014;3(2), art. #196, 7 pages. http://dx.doi.org/10.4102/ajlm.v3i2.196 14. lulie ad, hiwotu tm, mulugeta a, et al. perceptions and attitudes toward slmta amongst laboratory and hospital professionals in ethiopia. afr j lab med. 2014;3(2), art. #233. http://dx.doi.org/10.4102/ajlm.v3i2.233 15. mothabeng d, maruta t, lebina m, et al. strengthening laboratory management towards accreditation: the lesotho experience. afr j lab med. 2012;1(1), art. #9, 7 pages. http://dx. doi: 10.4102/ajlm.v1i1.9 16. luman et, yao k, nkengasong jn. a comprehensive review of the slmta literature part 2: measuring success. afr j lab med. 2014;3(2), art. #276, 8 pages. http://dx.doi.org/10.4102/ajlm.v3i2.276 17. peter tf, rotz pd, blair dh, et al. impact of laboratory accreditation on patient care and the health system. am j clin pathol. 2010;134(4):550–555. http://dx.doi.org/10.1309/ajcph1skq1hnwghf 18. silva p. guidelines for establishment of accreditation of health laboratories [document on the internet]. c2007 [cited 2014 may 31]. available from: http://cedoc.cies.edu.ni/general/world%20health%20organization%20hiv-ai%20(d)2/pdfs/publications_sea-hlm-394.pdf 19. bradley e, hartwig ka, rowe la, et al. hospital quality improvement in ethiopia: a partnership-mentoring model. int j qual health care. 2008;20(6):392–399. http://dx.doi.org/10.1093/intqhc/mzn042 20. federal democratic republic of ethiopia, ministry of health. annual performance report of hsdp-iii [document on the internet]. c2009. [cited 2014 oct 03]. available from: http://www.moh.gov.et/documents/26765/28899/annual+perfomance+report+2009/a6c0fcff-d2f5-4c2b-b9d1-1eac2c0a7531;jsessionid=b39c317151aa1d3cf8153a46ce48faa4?version=1.1 abstract background description of the intervention lessons learnt recommendations acknowledgements references about the author(s) alban zohoun department of hematology, faculty of health sciences, national university hospital center hubert koutoukou maga, cotonou, benin army teaching hospital, university hospital center, cotonou, benin tatiana b. agbodandé department of hematology, faculty of health sciences, national university hospital center hubert koutoukou maga, cotonou, benin angélique kpadé army teaching hospital, university hospital center, cotonou, benin raliatou o. goga army teaching hospital, university hospital center, cotonou, benin rené gainsi army teaching hospital, university hospital center, cotonou, benin paul balè army teaching hospital, university hospital center, cotonou, benin bibata m. sambo army teaching hospital, university hospital center, cotonou, benin remi charlebois global scientific solutions for health (gsshealth), baltimore, maryland, united states rachel crane global scientific solutions for health (gsshealth), baltimore, maryland, united states michele merkel global scientific solutions for health (gsshealth), baltimore, maryland, united states ludovic anani department of hematology, faculty of health sciences, national university hospital center hubert koutoukou maga, cotonou, benin ekaterina milgotina global scientific solutions for health (gsshealth), baltimore, maryland, united states citation zohoun a, agbodandé tb, kpadé a, et al. from benchmarking to best practices: lessons from the laboratory quality improvement programme at the military teaching hospital in cotonou, benin. afr j lab med. 2021;10(1), a1057. https://doi.org/10.4102/ajlm.v10i1.1057 lessons from the field from benchmarking to best practices: lessons from the laboratory quality improvement programme at the military teaching hospital in cotonou, benin alban zohoun, tatiana b. agbodandé, angélique kpadé, raliatou o. goga, rené gainsi, paul balè, bibata m. sambo, remi charlebois, rachel crane, michele merkel, ludovic anani, ekaterina milgotina received: 06 june 2019; accepted: 14 sept. 2020; published: 11 feb. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: in 2015, the army teaching hospital–university teaching hospital (hia-chu [hôpital d’instruction des armées de cotonou centre hospitalier et universitaire]) laboratory in benin launched a quality improvement programme in alignment with the world health organization regional office for africa’s stepwise laboratory improvement process towards accreditation (slipta). among the sub-saharan african laboratories that have used slipta, few have been francophone countries, and fewer have belonged to a military health system. the purpose of this article was to outline the strategy, implementation, outcomes and military-specific challenges of the hia-chu laboratory quality improvement programme from 2015 to 2018. intervention: the strategy for the quality improvement programme included: external baseline slipta evaluation, creation of work plan based on slipta results, execution of improvement projects guided by work plan, assurance of accountability via regular meetings, training of personnel to improve personnel competencies, development of external stakeholder relationships for sustainability and external follow-up post-slipta evaluation. lessons learnt: over a period of 3 years, the hia-chu laboratory improved its slipta score by 29% through a quality improvement process guided by work plan implementation, quality management system documentation, introduction of new proficiency testing and internal quality control programmes, and enhancement of personnel competencies in technical and quality management through training. recommendations: the programme has yielded achievements, but consistent improvement efforts are necessary to address programme challenges and ensure continual increases in slipta scores. despite successes, military-specific challenges such as the high mobility of personnel have hindered programme progress. the authors recommend that further implementation research data be shared from programmes using slipta in under-represented settings such as military health systems. keywords: laboratory medicine; stepwise laboratory improvement process towards accreditation; slipta; quality improvement; laboratory quality improvement; military laboratory; quality assurance; slmta; strengthening laboratory management toward accreditation. background the benin army health service provides healthcare to army personnel, their families and civilians across benin. the benin army health service is involved in disaster response and supports the beninese public health system, with approximately 75% – 80% of its services provided to the civilian population. the army teaching hospital–university teaching hospital (hia-chu [hôpital d’instruction des armées de cotonou centre hospitalier et universitaire]) of the benin army health service in cotonou is a reference health centre known for the quality of its services and training. the hia-chu laboratory plays a leading role in the public healthcare continuum by providing diagnosis, screening, and initial and follow-up treatment as well as preventative care.1 therefore, the hia-chu laboratory prioritises quality, in that it promotes the accuracy, reliability and timeliness of results. in recent years, there has been an emphasis on improving medical laboratory services globally. health priorities have been set forth by public declarations, such as the international health regulations and the maputo declaration (2008), in which signatories pledged to address and strengthen laboratory and health services.2 the international standards organization (iso) 15198:2012 standard, ‘medical laboratories–particular requirements for quality and competence’, serves as a standard for the laboratory quality management system (qms) – a formalised system that outlines required structures and functions for medical laboratories. progress made on the iso 15189 standard can help a laboratory prepare for accreditation through a recognised agency; this allows the laboratory to demonstrate to clients, partners and staff that it has attained a high level of technical competence, thus instilling confidence in stakeholders on the accuracy and reliability of its results. in sub-saharan africa, the clinical laboratory remains a weak link in the healthcare chain.2,3 though progress has been made, most clinical laboratories in sub-saharan africa remain under-equipped, under-funded and far from attaining international norms and standards. few laboratories are accredited to international quality standards, and most of these internationally accredited laboratories are based in south africa.4 quality assurance, which comprises qms including the existence of a quality manual, use of internal quality controls (iqc) and participation in external quality assessment (eqa), is poorly implemented and often unavailable in many laboratories in sub-saharan africa.5,6,7 in the absence of quality assurance, there is the risk of laboratory errors, which could adversely impact patient care. to alleviate these challenges and support awareness of the importance of achieving quality standards in medical laboratories in sub-saharan africa, the world health organization regional office for africa developed in 2009 a phased laboratory quality management evaluation system called the stepwise laboratory quality improvement process toward accreditation (slipta).8 the african society of laboratory medicine serves as the secretariat of the world health organization regional office for africa slipta programme. the slipta framework guides improvement of performance, measures and evaluates the progress of laboratories towards iso 15189 international accreditation and awards a certificate of recognition from zero to five-star ratings.8,9 the hia-chu in cotonou, benin, has a staff consisting of military and civilian personnel and includes a clinical pathologist, 12 medical laboratory scientists and support staff. the services provided include medical fitness evaluation, disease prevention, treatment and care, and teaching and research in medical, biological, pharmaceutical, paramedical, odontological and veterinary specialities. laboratory services at hia-chu are versatile and the laboratory manages, on average, 20 000 patient files and 50 000 tests per year. in january 2015, the hia-chu laboratory service initiated, for the first time, a quality improvement (qi) programme in alignment with the slipta framework. the qi programme goal was to improve laboratory services related to disease diagnosis and monitoring via the implementation of qms using slipta framework. the qi programme aimed to provide training for laboratory personnel on quality management concepts and practices, monitor laboratory quality and adherence to quality systems, conduct laboratory test method validation and provide proficiency testing panels for hiv, tuberculosis and other critical tests – successful implementation of the qi programme ensures that laboratory stakeholders and end-users have confidence in laboratory data which informs clinical decisions and optimises patient care. the hia-chu laboratory qi programme was launched through a collaboration with the us department of defense hiv/aids prevention program (dhapp). as of 2015, the benin army health service already had a long-standing collaboration with dhapp, which had been funding laboratory equipment and reagents, specifically to support hiv diagnosis and treatment monitoring. over the years, both parties recognised that laboratory tests and equipment alone are not sufficient to demonstrate test result quality; tests and equipment must be accompanied by laboratory quality practices implemented by properly trained and motivated personnel. to address this recognised need, dhapp began supporting the benin army health service in the execution of the hia-chu laboratory qi programme in 2015, with implementation assistance provided by the united states-based global health company global scientific solutions for health (gsshealth). the purpose of this article is to report on the implementation of the qi programme at the hia-chu laboratory from 2015 to 2018. description of the intervention baseline slipta evaluation and work plan creation the laboratory qi programme began in january 2015 with initial programme planning among stakeholders (hia-chu leadership, dhapp, gsshealth) and the facilitation of a comprehensive baseline evaluation of the hia-chu laboratory by gsshealth using the world health organization regional office for africa slipta checklist, version 2:2015. the baseline slipta evaluation allowed the assessment of the 12 quality management sections: organisation and personnel, management reviews, process control and internal quality assessment and eqa, information management, corrective action, equipment, purchasing and inventory, documents and records, occurrence management and process improvement, internal audit, client management and customer service, and facilities and safety. following the evaluation, leaders from hia-chu, dhapp and gsshealth convened for a collaborative session to review findings, identify priority qi areas and develop a tailored qi approach for each priority area. a tailored qi approach was developed to strengthen hiv testing processes – the focus of the funding agency dhapp; it covered the identification of key quality indicators to track and improve upon over time and the development and execution of the qi work plan. the key indicators identified to measure laboratory improvement based on ease of collection and relevance to the work plan included: slipta: percentage improvement in total and by section. eqa: percentage score in pt programme. iqc: accuracy of quality control results. personnel training: percentage change in theoretical test scores for training workshops. laboratory documentation: number of documents created and adequately implemented to standardise laboratory practices and formalise the commitment to the qms. the laboratory-set targets were: continual improvement in the slipta score through the establishment and implementation of improvement projects tracked by work plans, participation in eqa and pt and improvement of performance where the score is less than 100%, participation in iqc activities and improvement of processes in case of discrepancies between expected and obtained results, strengthening of laboratory quality management processes guided by well defined documentation, and the advancement of laboratory personnel competencies through training and mentorship workshops. the hia-chu laboratory supervisor facilitated work plan development in collaboration with partners (dhapp and gsshealth) and laboratory staff. the work plan included high-level qi goals, specific objectives for each goal and details of specific, measurable, achievable, realistic, and timely, or ‘smart’, tasks and deliverables to meet set targets within defined timeframes (see figure 1). figure 1: example of a quality improvement work plan (first page) developed by the army teaching hospital–university teaching hospital laboratory, benin, 2016–2017. the work plan was progressively implemented and updated through improvement project execution, progress monitoring, ongoing data collection and review, and continual alignment with the funder’s priorities. staff were engaged in the work plan implementation through their appointments to specific qi projects with oversight by the laboratory supervisor, and through continuous engagement in recurring collaborative work plan update meetings to promote accountability. quality improvement activities conducted in the context of the work plan included: nomination of new qms personnel roles (quality manager, biosafety manager), targeted laboratory personnel training and mentorship, implementation of eqa and iqc, definition of essential qms processes and development and use of associated documentation, and coordination of a follow-up slipta evaluation to measure progress. lessons learnt from the baseline slipta evaluation, the identified priority issues, which correspond to low-scoring slipta sections, include the following: the lack of a quality manual or safety manual, the lack of standard operating procedures for all processes and technical procedures, the lack of pt and quality control for tests, the lack of equipment maintenance and repair logs, the lack of temperature monitoring processes, and the lack of mistakes or error logs. the initiation and implementation of a qi programme at the hia-chu laboratory yielded numerous positive changes as perceived by hia-chu hospital leadership, laboratory management and staff, and partners (dhapp and gsshealth). positive changes included the establishment of a laboratory quality management team, achievement of designated qi projects resulting in slipta score improvements, the improvement of iqc and eqa test result accuracy, the development and implementation of over 50 standard operating procedures, and the strengthening of personnel competencies through targeted qms training and mentorship. throughout the years of the qi programme, support from funding partner dhapp and implementing partner gsshealth ensured continuous evaluation of processes via the internal and external evaluations and the update of work plans and associated qi projects. staff training and work plan updating the engagement of laboratory staff was key to the establishment and implementation of the laboratory qi work plan at hia-chu. to encourage staff commitment to the qi approach and ensure staff in-depth orientation to quality management concepts and testing processes, the laboratory qi approach prioritised staff training and mentorship. over 50 technicians participated in training workshops on qms co-organised and co-facilitated by the benin military health system and gsshealth. all training sessions consisted of didactic and interactive sessions and tests were administered preand post-training to evaluate knowledge change among the trainees. the median change in test score from pre-training to post-training for all the trainees increased from 15% to 40% (figure 2). improvements in participants’ theory test results demonstrated improved understanding of qms and technical concepts and facilitated personnel participation in qi projects. figure 2: preand post-training workshop theoretical test scores of military laboratory staff from quality management (left panel) and biosafety (right panel) workshops, benin, 2015–2018. box plots illustrate the distribution of scores among trainees (red diamonds for pre-training and yellow diamonds for post-training) and grey horizontal lines with data labels indicate median score across all the trainees. the interquartile range shows how the data are dispersed by dividing the data into quartiles (depicted by the dark and grey horizontal lines). (a) qms training: test scores, (b) bs&s training: test scores. in the first year of the programme, two quality workshops were held for laboratory staff with gsshealth facilitators. the objectives of the workshops were to improve the competency of laboratory staff in the areas of quality assurance, qms and hiv and tuberculosis testing. these topic areas were priorities for the laboratory director and dhapp – the funder, whose focus is the prevention and control of hiv and hiv comorbidities. the first workshop took place in april 2015 with a focus on quality management concepts for equipment management, document writing and eqa. staff received guidance and mentorship on hiv rapid testing. a second workshop took place in september 2015, with a focus to increase staff competency on laboratory supply chain and stock management. at the end of the first year, a follow-up slipta evaluation was conducted. in 2016, the qi programme continued with an updated work plan, and laboratory staff participated in a national biosafety and biosecurity workshop hosted by the ministry of defense and the ministry of health. the second year concluded with a poster presentation by the hia-chu laboratory supervisor on the quality programme at the african society of laboratory medicine international conference in december 2016. in 2017, the laboratory quality team updated their quality work plan, worked with laboratory personnel on qi projects, implemented the use of quality controls for cd4 testing, and organised an internal slipta evaluation. in april 2017, one laboratory staff member from hia-chu was sponsored to attend a multi-country, 5-day training workshop in senegal. the workshop covered essential aspects of laboratory quality management, including document management and standard operating procedure writing; equipment management and maintenance; error occurrence management, prevention and corrective action, and quality indicators. finally, a follow-up external slipta evaluation was conducted in october 2017 to re-evaluate the laboratory system, measure progress since the previous external slipta evaluation, and update the qi programme. as the qi programme moved into its third year in 2018, the laboratory again updated its work plan, based on the previous evaluation, and revised and updated key documentation including the laboratory quality manual and biosafety manual. in april 2018, a joint ministry of defense and ministry of health laboratory biosafety and quality management workshop was organised, to increase staff competency on principles and iso standards for biosafety and biosecurity. through its ongoing commitment to training personnel in both technical and quality management topics, the laboratory has observed significant improvements in staff competencies and performance. for example, the slipta sections in which the laboratory achieved the most significant quality improvements correspond to the quality management topics covered during training workshops. data from the qi programme showed that post-training, personnel knowledge was improved and retained (figure 2). additionally, in 2015, staff undertook training on hiv rapid testing processes; thereafter, their hiv pt scores reached 100%. document creation and implementation the laboratory document management system has been developed gradually over time and has included the creation and implementation of a quality manual, a biosafety manual, a sample collection manual and more than 50 standard operating procedures, technical instructions, forms and logs (e.g. temperature monitoring logs, corrective and preventive action forms). the laboratory supervisor and designated quality manager took the lead in developing documents, executing a document management process, and introducing new documents into circulation in the laboratory. laboratory personnel were oriented to new documents and procedures during regular laboratory meetings, facilitating the understanding and proper use of new documents. the availability of procedures and other documents helped laboratory management ensure that personnel observed standardised processes across the pre-analytical, analytical and post-analytical phases, and simplified the training of new personnel. furthermore, to ensure that only correct and updated documents were available in the laboratory, processes to control document revision, approval and version release were instituted by the laboratory supervisor and quality team. external quality assessment and internal quality control results in september 2015, the hia-chu laboratory enrolled and participated in an annual pt programme for hiv serology with commercial eqa provider thistle quality assurance, and later with the benin ministry of health, to promote sustainability. during the four eqa events for hiv serology, nine technicians participated, and pt scores of 100% were obtained. no corrective action was needed to improve proficiency; laboratory personnel were nonetheless oriented on root-cause analysis, and corrective and preventive action in case of future instances of lower eqa scores. in 2018, the hia-chu laboratory participated in two commercial haematology eqa programmes with human quality assessment services. the laboratory results for the first haematology eqa test were in the acceptable range for all analytes except for monocytes or mid cells, due to a reporting error. as a corrective action, the laboratory established a system for the supervised recording of results, in which the laboratory section supervisor verifies the accuracy of test results entered manually by technicians, after which results are entered into a computer by a secretary and printed for final validation. this process has helped reduce transcription errors. the hia-chu also began an iqc programme in biochemistry for the first time, in which the laboratory used high, normal and low commercial controls on equipment daily. when control values are outside the acceptable range, the laboratory staff will conduct a root-cause investigation and analysis. elements to be investigated include reagents (conservation, expiration, etc.), room temperature, quality of distilled water, quality of the control product, etc. if the issue persists after the above steps, the equipment is then recalibrated. all corrective actions and measures taken are documented and recorded. improvement of slipta score overall improvement of the laboratory’s quality system was demonstrated by a 29% increase in slipta score, from 16% at baseline evaluation in 2015 to 45% at the follow-up evaluation in 2017 (see figure 3). figure 3: performance of the army teaching hospital–university teaching hospital laboratory in each section of the stepwise laboratory improvement process towards accreditation checklist, benin, 2016-2018. baseline evaluation, september 2015; follow-up evaluation 1, january 2016; follow-up evaluation 2, october 2017. since 2018, reliance on the ministry of health national hiv reference laboratory for the administration of an hiv serology pt programme has allowed the hia-chu laboratory and other military laboratories to ensure a more sustainable and affordable pt option. several important factors can explain the successes achieved during the qi programme. the laboratory leadership and staff benefited from the strong support of the hospital management, thus underpinning laboratory staff motivation to sustain quality improvements. also, the implementing partner provided support in the execution of work plans, drafting of procedures, funding of training visits and organisation of regular teleconferences to track progress. the implementing partner provided sound advice for the appropriation of the quality approach and the implementation of improvement steps. recommendations although slipta is an adaptable structure and was used to guide the hia-chu laboratory’s quality successes, its formal implementation in francophone african countries and military contexts has been limited.8 military health systems face unique challenges that impact their laboratory qi efforts; the challenges and associated responses of hia-chu laboratory may be relevant to other military laboratory programmes or to non-military laboratories that have limited funds to invest in qi efforts. regardless of improvements made, qi is a never-ending process and the sustainability of gains is not guaranteed.10 despite the quality advancements made over the past several years, challenges remain, requiring corrective action to ensure the efficacy of the ongoing programme.11,12,13 the issue of the high mobility of military personnel has made it difficult to fully and consistently integrate staff into quality laboratory operations and maintain the laboratory qms. as a result of high staff turnover – a key facet of the military system – all members of the starting laboratory quality team are no longer in service at the hia-chu laboratory in cotonou. continuous staff turnover in part explains the slowdown in progress observed between the 2016 and 2017 follow-up evaluations. staff mobility has also negatively impacted the timelines for the execution of planned activities such as management reviews, internal evaluations and inventory processes, for all of which personnel must be properly trained and oriented to ensure their execution per slipta and iso 15189 requirements. lesson learned the implementation of a qi approach supported by improved documentation and record-keeping has increased staff and end-user confidence in the reliability and reproducibility of laboratory test results. support from the hospital hierarchy is essential to ensure the necessary time and resources are allocated to quality improvement processes. for a qi initiative to be effective and sustainable, all laboratory personnel should be engaged in the process, with quality management roles well defined. staff mobility in military settings renders the ongoing implementation of a qi programme more challenging; military sites should take into account the need to engage all laboratory personnel in a qi mindset and should plan backup qi roles in case of personnel deployment. although the laboratory qi programme has incorporated training workshops, the programme would benefit from the establishment of a structured framework for continous personnel training allowing the laboratory to maintain a pool of trained and dedicated personnel. going forward, the ambition of hia-chu laboratory is to continue advancing in laboratory quality using the slipta guideline, to ultimately achieve a five-star slipta rating and become internationally accredited to the iso 15189 standard. the hia-chu laboratory also has the support of the benin military health system authorities to expand the qi programme to multiple laboratory sites, prioritise the accreditation of clinical laboratories, and to integrate accreditation programmes into health sector policy and development programmes. to date, the hia-chu laboratory has expanded the qi programme to military clinical laboratories in ouidah, porto-novo and parakou. military laboratory leaders envision the further expansion of the qi programme to additional military sites using a network approach. acknowledgements we hereby thank the united states department of defense hiv/aids prevention program for sponsoring the quality programme of the hia-chu laboratory. competing interests the authors have declared that no competing interest exists. authors’ contributions a.z. is the initiator of the study and wrote the manuscript. t.b.a., a.k., r.o.g., r.g., p.b., b.m.s., l.a., r. charlebois, r. crane, m.m. and e.m. reviewed, commented on and contributed to its improvement. ethical considerations the advice of an ethics committee was not required. the article is an account of an ongoing process and does not involve an investigation or technical manipulation. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references saliou p. le laboratoire en zone tropicale. bull soc pathol exot. 2006;99(5):409–417. https://doi.org/10.3185/pathexo2822 revitalised laboratory declaration to forge a path for action [homepage on the internet]. aslm; 2015 [cited 2019 dec 26]. available from: https://aslm.org/news-article/revitalised-laboratory-declaration-to-forge-a-path-for-action/ petti ca, polage cr, quinn tc. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006;42(3):377–382. https://doi.org/10.1086/499363 beyanga m, gerwing-adima l, jackson k, et al. implementation of the laboratory quality management system (iso 15189): experience from bugando medical centre clinical laboratory – mwanza, tanzania. afr j lab med. 2018;7(1):a657. https://doi.org/10.4102/ajlm.v7i1.657 mboup s, gershy-damet gm, touré kane c, bélec l. le défi de la formation dans le domaine du laboratoire de biologie clinique en afrique. med sante trop. 2014;24:237–240. https://doi.org/10.1684/mst.2014.0349 deom a. qualité et biologie dans quelque pays européens et américains. spec biol. 1996;15(78):17–23. gershy-damet gm, rotz p, cross d. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin patho. 2010;134(3):393–400. https://doi.org/10.1309/ajcptuuc2v1wjqbm ndihokubwayo jb, maruta t, ndlovu n, et al. implementation of the world health organization regional office for africa stepwise laboratory quality improvement process towards accreditation. afr j lab med. 2016;5(1):a280. https://doi.org/10.4102/ajlm.v5i1.280 mothabeng d, maruta t, lebina m, lewis k, wanyoike j, mengstu y. strengthening laboratory management towards accreditation: the lesotho experience. afr j lab med. 2012;1(1):a9. https://doi.org/10.4102/ajlm.v1i1.9 andiric lr, massambu cg. one laboratory’s progress toward accreditation in tanzania. afr j lab med. 2014;3(2):a202. https://doi.org/10.4102/ajlm.v3i2.202 yao k, luman et, slmta collaborating authors. evidence from 617 laboratories in 47 countries for slmta-driven improvement in quality management systems. afr j lab med. 2014;3(3):a262. https://doi.org/10.4102/ajlm.v3i2.262 bouchet b. iso 15189: 2012: quels changements pour les laboratoires africains? afr j lab med. 2015;4(1):a181. https://doi.org/10.4102/ajlm.v4i1.181 guevara g, gordon f, irving y, et al. the impact of slmta in improving laboratory quality systems in the caribbean region. afr j lab med. 2014;3(2):a199. https://doi.org/10.4102/ajlm.v3i2.199 abstract introduction methods results discussion acknowledgements references about the author(s) astha goyal department of biochemistry, faculty of medicine and health sciences, sgt university, gurugram, india sanjiv k. bansal department of biochemistry, faculty of medicine and health sciences, sgt university, gurugram, india shikhar ganjoo department of dermatology, faculty of medicine and health sciences, sgt university, gurugram, india citation goyal a, bansal sk, ganjoo s. oxidative stress in acne vulgaris of varying severities in india. afr j lab med. 2025;14(1), a2912. https://doi.org/10.4102/ajlm.v14i1.2912 original research oxidative stress in acne vulgaris of varying severities in india astha goyal, sanjiv k. bansal, shikhar ganjoo received: 20 june 2025; accepted: 16 oct. 2025; published: 29 nov. 2025 copyright: © 2025. the author(s). licensee: aosis. this work is licensed under the creative commons attribution 4.0 international (cc by 4.0) license (https://creativecommons.org/licenses/by/4.0/). abstract background: acne vulgaris is a common inflammatory skin disease with a multifactorial pathogenesis. although the pathophysiology of acne has been linked to free radical-mediated responses, it is still unknown how these reactions trigger the disease’s progression. objective: this study aimed to evaluate the status and role of oxidative stress in patients with acne vulgaris, and to find a potential correlation with varying clinical severities. methods: an observational cross-sectional study was conducted amongst 200 subjects (50 in each grade of acne vulgaris: grades 1, 2, 3, and 4) and 50 controls aged 18 to 45 years, between 01 july 2023 and 30 june 2024 at sgt university, faculty of medicine and health sciences, gurugram, india. the analysis included superoxide dismutase (sod), catalase (cat), glutathione (gsh) and malondialdehyde (mda) by using enzyme-linked immunosorbent assay kits. a student’s t-test and pearson’s correlation were applied, and a p-value of < 0.05 is considered statistically significant. results: the present study included 50 healthy controls: 29 women and 21 men, with a mean age of 25.80 ± 5.45 years, while the mean age of the cases was 26.24 ± 6.56 years, with 121 women and 79 men. acne patients had considerably higher serum mda levels than healthy controls (p < 0.001). malondialdehyde was highest in grade 4 patients (10.61 nmol/ml ± 1.75 nmol/ml), while lowest mda levels were observed in grade 1 acne (5.83 nmol/ml ± 0.60 nmol/ml). superoxide dismutase, cat and gsh activities were significantly lower in acne patients in comparison to healthy controls (p < 0.001). the levels of sod, cat and gsh decreased as the severity of disease increased, indicative of antioxidant defence system damage. conclusion: these findings provide credence to the link between acne and oxidative stress, which suggests that oxidative stress markers could be helpful in assessing the course of acne, and in outlining the patho-physiological support of the condition. what this study adds: the study suggests that levels of sod, cat, gsh and mda differ significantly in patients with acne vulgaris, adding region-specific evidence from the indian population, addressing a gap in the literature on oxidative stress in dermatological conditions in this demographic. keywords: acne vulgaris; biomarkers; lipid peroxidation; oxidative stress; superoxide dismutase. introduction acne vulgaris (av) is a chronic skin disease with an inflammatory condition caused by changes in pilosebaceous units. it is by far the most common of the several cutaneous disorders that affect people.1 it is common, not only in teenagers, but also in the adult population. the global burden of disease estimates that 85% of young adults aged between 12 and 25 years suffer from acne, which can occur in individuals of any age, race, or sex or gender.2,3 approximately 20% of the individuals who develop severe acne end up with scarring. while white people are more likely to have mild acne, asian and african people are more likely to acquire severe forms. generally, hyperpigmentation is more common in darker-skinned individuals.4 four key interrelated concepts of pathogenesis that lead to the formation of av are: increased sebaceous gland activity, keratinisation of the middle part of the infundibulum, bacterial colonisation, and inflammation of the follicle.5 the most common clinical signs of av include papules, pustules, nodules, cysts, open and closed comedones, and sometimes, scarring.6 the genetic component of acne has been supported by numerous studies. acne risk can be up to three times higher for those with afflicted first-degree family members than for those who do not have a family history.7 despite studies, no single primary cause has been identified and the aetiology and pathogenesis of acne remain unclear.8,9 it has been suggested that one of the pathogenic conditions in av is the generation of ros (reactive oxygen species) by neutrophils, resulting in tissue injury and inhibition of ros production can be beneficial from a therapeutic standpoint.10,11 the ros system includes free radicals such as the superoxide ion radical, the hydroxyl radical, and the nitric oxide radical, as well as non-radicals such as hydrogen peroxide and ozone. these have been linked to processes of mutagenesis, tumorigenesis, inflammatory responses, and senescence.12,13 acne is characterised by changes in sebum composition, and neutrophil-produced ros contribute to follicular wall irritation and destruction, which in turn fuels the inflammatory course of acne.14 moreover, the reactions between lipidic structures and ros can result in the peroxidation of polyunsaturated fatty acid and the production of toxic compounds such as malondialdehyde (mda), which can be used as markers to assess oxidative damage in cells.15 enzymatic and non-enzymatic antioxidant defence mechanisms are both present in human cells. as the first line of defence against oxygen-derived free radicals, the sod–cat (superoxide dismutase–catalase) system is a key enzymatic system that catalyses the dismutation of superoxide into hydrogen peroxide, which subsequently converts into water by catalase. this process regulates the production of ros and is therefore essential for preserving a proper cellular redox balance.16 oxidative stress can result from changes in this natural balance, which can be brought on by increased ros generation and/or decreased antioxidant levels.17,18 a few studies have reported the increased oxidative stress in av.19,20 however, the disturbance in oxidants–antioxidant systems in the disease progression has not been studied extensively in the indian regional setting. therefore, we measured the levels of biochemical indicators of oxidative stress in patients of av, and compared their relationship to the severity of disease activity (grades 1, 2, 3, and 4) in the indian population. our results not only support an association between oxidative stress and acne; they show that it may be used as a biomarker index for evaluating the activity of the disease and to monitor the course of treatment. methods ethical considerations the study was carried out in compliance with the declaration of helsinki and approved by the institution ethics committee of sgt university, faculty of medicine and health sciences, with vide letter number iec/fmhs/md/ms/2023-29 dated 03/06/2023. written and informed consent was obtained after explaining the purpose and details of the study to all of the subjects. protocol numbers were assigned for reference, and participant personal information was kept private and used only for research. data security measures included password protection of the computer and data storage in a safe physical location. study design and settings this observational cross-sectional study was carried out in the department of biochemistry of the faculty of medicine and health sciences at sgt university, gurugram, india, from 01 july 2023 to 30 june 2024. sample size acne severity was assessed by a single experienced dermatologist using a simple grading system by tutakne and chari,21 to ensure consistency and avoid interrater variability: grade 1: comedones, occasional papules grade 2: papules, comedones, few pustules grade 3: prominent pustules, nodules and abscesses grade 4: mainly cysts, abscesses, widespread scarring by keeping the confidence level at 95% (1.96), prevalence (85%),22 and maximum allowance of error (5%) in the cochran’s formula (n = z2pq/e2), a sample size of 196 subjects was established. we rounded up to 200 to account for potential exclusions. therefore, 200 subjects (50 in each grade of av: grades 1, 2, 3, and 4) and 50 healthy controls were selected. for sub-group comparison, a post-hoc power calculation was performed in acne grades. by keeping alpha = 0.05, power (1-β) comes as: for sod, large effect size (cohen’s d = 1.14) and power = 1.0. malondialdehyde and gsh (glutathione) comparisons demonstrated high power, whereas cat comparisons had smaller effect sizes and slightly lower power (0.85). eligibility criteria the cross-sectional study included patients with av, who did not receive any topical or systemic treatment in the previous four weeks, were aged between 18 and 45 years, were from both genders, and were willing to participate. however, the study excluded patients on immune-suppressive therapy such as corticosteroids, regular analgaesic intake, and hormonal therapy; patients with any systemic disease (diabetes mellitus, liver disease, thyroid disorders, cardiovascular events, known cases of malignancy); patients with any dermatological disorder other than av; patients taking any vitamin supplementation; pregnant and lactating women; and smoking and/or alcohol abusers. the information on social demographics and disease duration was collected by directly interviewing study participants in the local language using a semi-structured questionnaire. sample collection and equipment using aseptic technique, 5 ml of venous blood was taken in dry sterile red top (serum) vacutainers from each participant after gaining written consent. serum was separated by centrifugation at 3500 revolutions per minute (rpm) for 10 min. serum sod, cat, gsh and mda were measured using the erba lisa scan em analyser (erba diagnostics mannheim gmbh, mannheim, germany) via an enzyme-linked immunosorbent assay kit. calibration and quality control of this analyser were performed before the samples were run. the manufacturer’s instructions for the machine and the reagents were strictly followed. standard curves were generated for each biomarker (sod, cat, gsh, mda) using the calibrators provided in the enzyme-linked immunosorbent assay kits, and sample concentrations were calculated from these curves. to reduce the analytical variation, all samples were analysed using the same lots of reagent kits, and intra-assay coefficient of variation values remained within acceptable limits. (table 1). table 1: different parameters with method, cut-off range and coefficient of variation, gurugram, india, from 01 july 2023 to 30 june 2024. statistical analysis data and various parameters were analysed on ibm® spss® statistics (spss®) version 24 (ibm corporation, armonk, new york, united states). the shapiro-wilk test was applied for assessing the normal distribution of the dataset and was found to be normally distributed. mean and standard deviation of all parameters were calculated. a student’s t-test (unpaired) was used to compare two groups while a one-way analysis of variance test was applied to compare the various parameters in three or more groups. a post-hoc test was conducted to determine pair-wise differences between groups. pearson’s correlation coefficient (r-value) was applied to find the correlation. the p < 0.05 was considered as statistically significant, and p < 0.001, highly significant. results general characteristics of study population the present study included 50 healthy controls: 29 women and 21 men with a mean age of 25.80 ± 5.45 years. two hundred patients with av—121 females and 79 males—were also enrolled (figure 1). the mean age and gender distribution of each grade of av is expressed in table 2, with p = 0.0007. the distribution of patients according to duration of acne is shown in table 3. over 31.4% of the women had struggled with av for 2–5 years, while a higher number of men (50.6%) had acne for a similar time period, followed by 34.2% who had acne for more than five years (p < 0.0001). figure 1: a bar graph showing number of male and female participants in control and acne patients, gurugram, india, from 01 july 2023 to 30 june 2024. table 2: demographic data of controls and cases, gurugram, india, from 01 july 2023 to 30 june 2024. table 3: disease duration of acne vulgaris patients, gurugram, india, from 01 july 2023 to 30 june 2024. table 4 shows a comparison of sod, cat, gsh and mda levels between controls and severity grades of acne. the serum levels of sod were lowest in severe cases (grade 4) and highest in mild cases (grade 1), when compared with controls with a mean ± standard deviation of 5.68 u/ml ± 1.95 u/ml, 11.71 u/ml ± 2.12 u/ml, and 89.39 u/ml ± 9.91 u/ml (p < 0.0001). similarly, the serum levels of cat and gsh were markedly decreased in grade 4 acne patients versus individuals in any other grades. meanwhile, a comparison of mda levels across the four grades revealed a substantial difference. malondialdehyde levels were significantly lower in grade 1, and higher in grades 3 and 4 (p < 0.0001). post-hoc pair-wise comparisons between acne severity grades revealed significant differences for most oxidative stress markers. superoxide dismutase levels showed highly significant reductions across all grade comparisons (p ≤ 0.0001). catalase levels were comparable between grades 1 and 2 (p = 0.0703), but significantly lower from grade 2 onwards (p ≤ 0.0001). glutathione levels demonstrated an early decline, with a significant difference observed between grades 1 and 2 (p = 0.0161), and highly significant differences in subsequent grade comparisons (p < 0.0001). malondialdehyde levels were significantly elevated across all inter-grade comparisons (p < 0.0001), confirming progressive oxidative damage with increasing acne severity (table 5). table 4: oxidative biomarkers levels in controls and acne patients of varying severity, gurugram, india, from 01 july 2023 to 30 june 2024. table 5: post-hoc multiple comparisons of oxidative stress markers between acne severity grades, gurugram, india, from 01 july 2023 to 30 june 2024. to adjust for potential confounding variables and to explore the combined effect of oxidative stress markers, a multiple linear regression analysis was performed. acne severity was used as the dependent variable, with serum levels of sod, cat, gsh, mda, and patient age as independent predictor variables. as shown in table 6, mda was a significant positive predictor (b = 0.232, p < 0.0001), while sod (b = −0.125, p < 0.0001), cat (b = −1.920, p = 0.0004), and gsh (b = −0.839, p < 0.0001) were significant negative predictors. age was not a significant predictor (p = 0.311). overall, these findings indicate that oxidative stress markers independently contribute to acne severity, with higher oxidative damage (mda) and lower antioxidants (sod, cat, gsh) predicting more severe disease. table 6: multiple linear regression analysis of oxidative stress markers and age as predictors of acne severity, gurugram, india, from 01 july 2023 to 30 june 2024. table 7, table 8, table 9, table 10, figure 2, figure 3, figure 4 and figure 5 displays the correlation between acne severity and levels of oxidative and antioxidative biomarkers. a significant negative correlation was observed between mda levels and cat activity in the serum of grade 4 acne patients (r = –0.403, p = 0.0037). however, no significant correlation was found in the other acne subgroups. figure 2: correlation between oxidative stress biomarker and antioxidants in grade 1 acne patients, gurugram, india, from 01 july 2023 to 30 june 2024. correlation between (a) mda and sod (non-significant, p = 0.3491, r = −0.1352); (b) mda and cat (non-significant, p = 0.9753, r = 0.0044); and (c) mda and gsh (non-significant, p = 0.6738, r = −0.0610). figure 3: correlation between oxidative stress biomarker and antioxidants in grade 2 acne patients, gurugram, india, from 01 july 2023 to 30 june 2024. correlation between (a) mda and sod (non-significant, p = 0.9943, r = 0.0010); (b) mda and cat (non-significant, p = 0.4299, r = 0.1141); and (c) mda and gsh (non-significant, p = 0.7104, r = 0.0538). figure 4: correlation between oxidative stress biomarker and antioxidants in grade 3 acne patients, gurugram, india, from 01 july 2023 to 30 june 2024. correlation between (a) mda and sod (non-significant, p = 0.4306, r = 0.1140); (b) mda and cat (non-significant, p = 0.2106, r = 0.1802); and (c) mda and gsh (non-significant, p = 0.6938, r = −0.0570). figure 5: correlation between oxidative stress biomarker and antioxidants in grade 4 acne patients, gurugram, india, from 01 july 2023 to 30 june 2024. correlation between (a) mda and sod (non-significant, p = 0.8880, r = 0.0204); (b) mda and cat (significant, p = 0.0037, r = −0.4035); and (c) mda and gsh (non-significant, p = 0.4202, r = 0.1166). table 7: correlation analysis of oxidative markers in grade 1 acne patients, gurugram, india, from 01 july 2023 to 30 june 2024. table 8: correlation analysis of oxidative markers in grade 2 acne patients, gurugram, india, from 01 july 2023 to 30 june 2024. table 9: correlation analysis of oxidative markers in grade 3 acne patients, gurugram, india, from 01 july 2023 to 30 june 2024. table 10: correlation analysis of oxidative markers in grade 4 acne patients, gurugram, india, from 01 july 2023 to 30 june 2024. discussion the current study’s findings showed that lipid peroxide levels in patients of av were much higher than those of healthy controls. on the other hand, the activities of serum sod and cat as well as the levels of gsh were statistically reduced in acne patients. prior research has demonstrated that in inflammatory diseases, the level of lipid peroxidation increases. furthermore, mda is thought to be a sign of oxidative stress within cells.27 therefore, the elevated serum mda levels in this study could be the consequence of cellular damage caused by ros. these findings imply that acne is mediated by the increased ros production, which can be linked to a decrease in antioxidant enzyme levels, such as sod or cat. it has been noted that the sod-cat system increases or decreases or goes in two different directions in a variety of disorders.15 on the other hand, there are some studies that do not agree with the current study. according to a study by mahmoud et al.,28 higher levels of sod and mda were found in acne patients than in controls (p < 0.001).28 in their study, 37 patients were divided into two groups. group i (the patient group) included 27 patients (21 women and six men) with acne; and group ii (the control group) included 10 age-matched and sex-matched healthy individuals as controls; there were eight women and two men. similarly, in another study conducted by el garem et al., 50 acne patients and 20 healthy controls were compared, and results showed higher sod in acne patients, which was statistically non-significant.14 a study conducted by arican, kurutas, and sasmaz, who investigated 43 acne patients, including 30 women and 13 men, and 46 healthy controls, including 28 women and 18 men, came up with the result of elevated sod and mda in patients compared to the control (p < 0.001).20 they proposed that the imbalance between oxidants and antioxidants in the cells caused oxidative stress, which in turn led to an increase in sod levels. additionally, our results showed a correlation between the higher levels of oxidative stress biomarkers and the severity of acne patients. patients with grade 4 acne had noticeably higher serum mda levels than those in patients with grade 1, grade 2, or grade 3 acne. moreover, sod–cat activity and gsh levels were significantly decreased in grade 4 patients compared to the levels of patients with other grades of acne. the increase in sod activities in grade 1 acne patients indicates that the antioxidant defence system is responding appropriately to oxidative stress, which keeps mda levels within the normal range. this adaptive response protects the cells from the harmful products of free radical reactions. membrane lipids and other tissue lipids may peroxide as a result of the increased production of ros, which can start the lipid peroxidation chain reaction, when the disease process becomes more severe and the protective mechanism is no longer sufficient. this is shown with the decreased sod activities and increased mda levels observed in patients with grade 2 and grade 3 acne. in patients with grade 4 acne, sod activities were significantly decreased, and the substantial increase in mda levels suggests that an increase in ros production has outpaced the antioxidant capability. contrary to the current findings, basak et al. did not find any correlation between acne severity and antioxidant enzymes levels or mda in the leukocytes.11 according to them, this proved that while each patient’s oxidant and/or antioxidant balance may be impacted to a certain degree, the total number of factors or agents involved in each case could not affect the enzyme levels. we reasoned that this disagreement was most likely caused by the samples used for measuring oxidative biomarkers coming from different sources. the levels of these markers do not indicate the origin of their production in plasma.29 in the present study, data showed that patients with grade 4 acne had a strong negative association between their cat and mda levels (figure 5b), whereas patients with other grades did not show this correlation. furthermore, regardless of the severity of the condition, no discernible relationship between sod activity, gsh levels, and mda levels was found in acne patients. these results have unequivocally shown that oxidative stress contributes to acne and may be a significant factor in its aetiology and progression.30 however, the origin of these enzymes cannot be ascertained. whether aberrant antioxidant enzymes are the famous ‘egg or chicken dilemma’ in acne is still up for debate. it is likely that these alterations are a result of cutaneous inflammation, such as acne, rather than the cause. limitations of the study the limitations of the study were as follows: the study design is cross-sectional, which limits the ability to establish a causal relationship between biomarkers and acne severity. the study does not account for lifestyle and environmental factors, such as diet, stress, pollution exposure, and genetic predisposition, all of which can influence oxidative stress levels. the effects of oxidative stress markers after treatment with antioxidants were not measured in this study. future scope future research should involve lesion-level assays–such as quantification of squalene peroxides in sebum, punch biopsy, or scraping of skin surface by a dermatologist for correlating serum and local cutaneous oxidative stress directly. conclusion according to the present results, statistically high levels of mda in patients with av is evidence of the existence of oxidative stress which can cause cell injury and inflammation. furthermore, low levels of sod, cat and gsh enzyme in acne clearly indicate that the antioxidant defence system is damaged, irrespective of the degree of severity, and thereby antioxidant drugs can be indicated for the treatment of acne. however, it is still unclear whether oxidative stress is a cause or an outcome of inflammation. combining antioxidants drugs with other treatment modalities of acne might be beneficial in treating patients, especially those with inflammatory acne lesions, or their effects may be mitigated. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions astha goyal conceived and designed the study, collected and analysed the data and drafted the manuscript. dr. sanjiv k. bansal reviewed and revised the final article and contributed to the visualization of the manuscript. dr. shikhar ganjoo contributed to the study methodology, and assisted in manuscript editing. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the data that support the findings of this study are available on request from the corresponding author, astha goyal upon reasonable request. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. the article does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the authors are responsible for this article’s results, findings, and content. references dabash d, salahat h, awawdeh s, et al. prevalence of acne and its impact on quality of life and practices regarding self-treatment among medical students. sci rep. 2024;14(1): 4351. https://doi.org/10.1038/s41598-024-55094-6 febyan wk. acne vulgaris in adults: a brief review on diagnosis and management. int j res rev. 2020;7(5):246–252. costa cs, bagatin e, yang z, et al. systemic pharmacological treatments for acne: an overview of systematic reviews (protocol). cochrane database syst rev. 2021;2021(11):1. https://doi.org/10.1002/14651858.cd014917 özçelik s, kulaç i̇, yazıcı m, öcal e. distribution of childhood skin diseases according to age and gender, a single institution experience. turk archiv pediatr. 2018;53(2):105. https://doi.org/10.5152/turkpediatriars.2018.6431 baldwin h, farberg a, frey c, et al. unmet needs in the management of acne vulgaris: a consensus statement. j drugs dermatol. 2023;22(6):582–587. https://doi.org/10.36849/jdd.7587 assiri a, alameer m, zogel b, et al. prevalence of acne vulgaris and its association with lifestyle among the general population of jazan city, saudi arabia: a cross-sectional study. int j med dev countr. 2023;7(10):1305–1312. https://doi.org/10.24911/ijmdc.51-1683495519 sutaria ah, masood s, saleh hm, schlessinger j. acne vulgaris. treasure island, fl: statpearls publishing; 2023. simpson rc, grindlay dj, williams hc. what’s new in acne? an analysis of systematic reviews and clinically significant trials published in 2010–11. clin exp dermatol. 2011;36(8):840–844. https://doi.org/10.1111/j.1365-2230.2011.04218.x johnson ba, nunley jr. use of systemic agents in the treatment of acne vulgaris. am fam phys. 2000;62(8):1823–1830. akamatsu h, horio t. the possible role of reactive oxygen species generated by neutrophils in mediating acne inflammation. dermatology. 1998;196(1):82–85. https://doi.org/10.1159/000017876 basak py, gultekin fa, kilinc i, delibas n. the effect of benzoyl peroxide and benzoyl peroxide/erythromycin combination on the antioxidative defence system in papulopustular acne. eur j dermatol. 2002;12(1):53–57. bickers dr, athar m. oxidative stress in the pathogenesis of skin disease. j investig dermatol. 2006;126(12):2565–2575. https://doi.org/10.1038/sj.jid.5700340 kardeh s, ashkani-esfahani s, alizadeh am. paradoxical action of reactive oxygen species in creation and therapy of cancer. eur j pharmacol. 2014;735:150–168. https://doi.org/10.1016/j.ejphar.2014.04.023 el garem yf, ahmed ra, ragab ma, abouzeid aa. study of oxidative stress in different clinical severities of acne vulgaris. egypt j dermat venerol. 2014;34(1):53–57. https://doi.org/10.4103/1110-6530.137313 del rio d, stewart aj, pellegrini n. a review of recent studies on malondialdehyde as toxic molecule and biological marker of oxidative stress. nutr metab cardiovasc dis. 2005;15(4):316–328. https://doi.org/10.1016/j.numecd.2005.05.003 matés jm, pérez-gómez c, de castro in. antioxidant enzymes and human diseases. clin biochem. 1999;32(8):595–603. https://doi.org/10.1016/s0009-9120(99)00075-2 farag rm, ahmed mb, el-gendy ma, mohamed is. evaluation of oxidative stress and apoptosis in breast cancer. egypt j biochem mol biol. 2009;27(2):63–82. https://doi.org/10.4314/ejbmb.v27i2.49254 pugliese pt. the skin’s antioxidant systems. dermatol nurs. 1998;10(6):401–402. https://doi.org/10.1016/s0950-7051(98)00033-1 sarici g, cinar s, armutcu f, altınyazar c, koca r, tekin ns. oxidative stress in acne vulgaris. j eur acad dermatol venereol. 2010;24(7):763–767. https://doi.org/10.1111/j.1468-3083.2009.03505.x arican o, kurutas eb, sasmaz s. oxidative stress in patients with acne vulgaris. mediators inflamm. 2005;2005(6):380–384. https://doi.org/10.1155/mi.2005.380 tutakne ma, chari kv. acne, rosacea and perioral dermatitis. in: valia rg, valia ar, editors. iadvl textbook and atlas of dermatology. 2nd ed. vol. 2. mumbai: bhalani publishing house, 2003; p. 689–710. agarwal d, mathur dk, paliwal v. psychological morbidity in young adults with acne vulgaris: a hospital-based study. j clin diagn res. 2023;17(2):wc01–wc04. https://doi.org/10.7860/jcdr/2023/61797.17483 mybiosource. human superoxide dismutase (sod) elisa kit [homepage on the internet]. catalog no. mbs005068. research use only. san diego, ca: mybiosource; 2020 [cited n.d.]. available from: https://www.mybiosource.com/sod-human-elisa-kits/superoxide-dismutase/5068 abbexa. human catalase (cat) elisa kit (catalogue no. abx572370) [product datasheet] [homepage on the internet]. abbexa ltd; 2022 [cited n.d.]. available from: https://www.abbexa.com/human-catalase-cat-elisa-kit?srsltid=afmboopps1-& abbexa. human glutathione (gsh) elisa kit (catalogue no. abx573700) [product datasheet] [homepage on the internet]. abbexa ltd; 2022 [cited n.d.]. available from: https://www.abbexa.com/human-glutathione-gsh-elisa-kit mybiosource. human malondialdehyde (mda) elisa kit (catalog no. mbs263626) [product manual] [homepage on the internet]. mybiosource; 2021 [cited n.d.]. available from: https://www.mybiosource.com/mda-elisa-kits/human/263626 maccarrone m, catani mv, iraci s, melino g, agrò af. a survey of reactive oxygen species and their role in dermatology. j eur acad dermatol venereol. 1997;8(3):185–202. https://doi.org/10.1111/j.1468-3083.1997.tb00478.x mahmoud a, gaber mae, dawood aae. oxidants and antioxidants role in acne vulgaris. menoufia med. j. 2014;27(2):465–468. https://doi.org/10.4103/1110-2098.141728 kurutas eb, arican o, sasmaz s. superoxide dismutase and myeloperoxidase activities in polymorphonuclear leukocytes in acne vulgaris. acta dermatovenerol alp panonica adriat, 2005;14:39–42. akamatsu h, horio t, hattori k. increased hydrogen peroxide generation by neutrophils from patients with acne inflammation. int j dermatol. 2003;42(5):366–369. https://doi.org/10.1046/j.1365-4362.2003.01540.x abstract introduction methods results discussion acknowledgements references about the author(s) amos marume department of infection prevention and control, school of laboratory medicine and medical sciences, college of health sciences, university of kwazulu-natal, durban, south africa paraclinical department, faculty of veterinary sciences, university of zimbabwe, harare, zimbabwe theresa chimponda department of biochemistry, faculty of science, university of zimbabwe, harare, zimbabwe arthur vengesai department of biochemistry, faculty of science, university of zimbabwe, harare, zimbabwe caroline mushayi department of biochemistry, faculty of science, university of zimbabwe, harare, zimbabwe jaclyn mann department of infection prevention and control, school of laboratory medicine and medical sciences, college of health sciences, university of kwazulu-natal, durban, south africa takafira mduluza department of infection prevention and control, school of laboratory medicine and medical sciences, college of health sciences, university of kwazulu-natal, durban, south africa department of biochemistry, faculty of science, university of zimbabwe, harare, zimbabwe citation marume a, chimponda t, vengesai a, mushayi c, mann j, mduluza t. effects of tnf-α and il-10-819 t>c single nucleotide polymorphisms on urogenital schistosomiasis in preschool children in zimbabwe. afr j lab med. 2021;10(1), a1138. https://doi.org/10.4102/ajlm.v10i1.1138 original research effects of tnf-α and il-10-819 t>c single nucleotide polymorphisms on urogenital schistosomiasis in preschool children in zimbabwe amos marume, theresa chimponda, arthur vengesai, caroline mushayi, jaclyn mann, takafira mduluza received: 22 nov. 2019; accepted: 28 jan. 2021; published: 29 apr. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: knowledge gaps exist between host genetic factors and susceptibility to schistosomiasis. objective: this study determined cytokine levels and single nucleotide polymorphisms of tumour necrosis factor (tnf)-α (rs1800629) and interleukin (il)-10 (rs1800871) and their possible impact on susceptibility to schistosomiasis in preschool-age children in the madziva area of shamva district, mashonaland central province, zimbabwe. methods: urogenital schistosomiasis was diagnosed using the urine filtration method, while a sandwich enzyme-linked immunosorbent assay was used for cytokine level determination. the survey was done in august 2015 and reinfection levels post treatment were assessed at 3, 6 and 12 months. amplification refractory mutation system polymerase chain reaction with visualisation on 2% agarose gel electrophoresis was used for genotyping. results: schistosomiasis prevalence was found to be 10.5% (59/563). reinfections were detected in only six children at 3 months and only one was reinfected at 12 months. there were no significant differences in tnf-α-308 g/a allele or genotype frequencies between the schistosoma haematobium infected participants (p = 0.360) and uninfected participants (p = 0.279). however, no children with the il-10-819 tt genotype had schistosomiasis. the tnf-α gg genotype corresponded with significantly lower tnf-α levels when compared with the ga or aa genotypes (p < 0.001), and tnf-α levels were significantly lower in infected children compared to uninfected children (p < 0.001). conclusion: higher tnf-α levels and lower il-10 levels are potentially protective against schistosomiasis infection. the il-10-819 tt genotype is potentially protective against infection through its association with lower il-10 levels. keywords: schistosoma haematobium; polymorphisms; cytokines; susceptibility; protective immunity. introduction preschool children from poor and endemic regions like sub-saharan africa are at high risk of schistosomiasis due to poor hygiene and sanitation, conditions that facilitate schistosoma thriving. in these endemic regions, people living in both rural and urban centres use open water sources infested with intermediate snails harbouring the human host infectious stage of schistosoma.1 most of the people who are at risk of schistosomiasis worldwide have low awareness levels of the disease.2,3 urogenital schistosomiasis has been described as a significant public health threat for preschool-age children (≤ 5 years); often they have higher infection burdens, and show serious morbidity and pathology relative to school-age children.4 thus preschool children in endemic areas are regarded as a high-risk group needing inclusion in mass drug administration programmes.1 praziquantel treatment, despite some few known side effects, is acceptable, safe and effective in all age ranges; treatment leads to 91% reduction in prevalence as well as 93% decrease in infection intensity.4 praziquantel is known to remove the immunosuppressive effects of adult worms, and leads to the expression of worm antigens as a result of dying worm degeneration.5 thus, praziquantel aids in immunity development. most endemic regions of zimbabwe have prevalence rates that range from moderate to high risk according to world health organization guidelines.6,7 this qualifies those regions for mass praziquantel administration at least once every two years.7 morbidity and mortality of schistosomiasis is well recognised to be a consequence of many host immune factors.5 people in endemic areas are known to develop protective immunity against reinfection, which offers hope for possible vaccines.4 the immunology of schistosomiasis is therefore of great significance in the management, control and possible eradication of infection. research, control strategies and treatment guidelines of schistosomiasis in children under five years of age are reported to have lagged behind those in other age groups.5 significant knowledge gaps exist for the preschool-age group in relation to prevalence, morbidity, mortality, immunology, immunopathology, treatment effectiveness and safety, and impact of host genetic factors. tumour necrosis factor (tfn)-α and interleukin (il)-10 have been associated with susceptibility and other key immune responses in many diseases including those caused by helminthes.8,9,10 we therefore assessed cytokine levels as well as cytokine genotypes in preschool children at risk of schistosomiasis. methods ethical considerations the study was registered and ethically approved by zimbabwe’s ethics board for bio-medical research (the medical research council of zimbabwe – mrcz/a/1710). in addition, community leaders, the provincial medical director and the district medical officer also granted permission to conduct the study in madziva, shamva. the children agreed to participate in the study and their parents or guardians signed informed consent forms allowing their participation. the children and parents were educated on the aims, risks and benefits of the study. participation was voluntary and participants were free to withdraw from the study at any time. study population and sampling the survey of urogenital schistosomiasis prevalence was conducted among 563 children aged between one year and five years through collection of urine specimens. genotyping was done on 268 children who were willing and able to provide adequate blood. convenience sampling was used, and the sample was several times larger than the 50 children per centre estimate used in other studies.6 the children were permanent residents of the madziva area in shamva district, mashonaland central province, zimbabwe. the area was selected for this study based on its high annual rainfall, a condition conducive for the schistosoma species and which could lead to high schistosomiasis endemicity.6 the study was a cross-sectional study, which was part of an ongoing longitudinal study on urogenital schistosomiasis for the 1–5 year age group (see funding section below). participants infected with schistosoma mansoni were excluded from this study.11 the survey was done in august 2015 and reinfection levels post treatment were assessed at 3, 6 and 12 months. detection of schistosoma haematobium schistosoma haematobium was diagnosed by the microscopic examination of urine specimens for the presence of parasite eggs using the urine filtration technique.12 in brief, urine specimens were collected from willing participants for three consecutive days. for diagnosis, 10 ml of urine was filtered through a nitrile filter membrane. the membrane was stained with iodine and examined under a light microscope. the same procedure was repeated on three consecutive days in order to prevent misdiagnosis due to day-to-day variation of egg excretion.13 if positive, the number of eggs in the entire sample of 10 ml of urine was counted and recorded as light infection if 1–10, moderate infection if 11–49 or heavy infection if more than 50 eggs. participants who were s. haematobium positive were treated with a single dose of praziquantel (40 mg per kilogram of body weight). bread and orange juice were given as supplementary food in order to enhance absorption as well as to alleviate the nauseating effects of praziquantel. stool samples were collected once for s. mansoni diagnosis. urine collection procedures and the diagnosis were repeated at 3, 6 and 12 months to assess reinfection levels post treatment with praziquantel. blood sample collection approximately 5ml of blood was collected into ethylenediamine tetraacetic acid tubes. the tubes were centrifuged at 3000 revolutions per minute for 10 min using a rotofix centrifuge. plasma was collected and used to measure the levels of systemic cytokines. host dna was extracted from whole blood for genotyping using the qiagen flexigene dna extraction kit (qiagen flexigene® dna handbook, qiagen, hilden, germany). genotyping the tnf-α (rs18006290) and il-10-819 t>c (rs1800871) polymorphisms were determined using amplification refractory mutation system polymerase chain reaction. the primers used were from inqaba biotechnology (pretoria, south africa) (table 1). primers specific for wild type genotype (1 µl) and mutant genotype (1 µl) were separately mixed with 1 µl (10 mm) generic primer, 0.5 µl (10 mm) forward and 0.5 µl (10 mm) reverse internal control primers (human growth hormone), 12.5 µl quick load taq 2x master mix (new england biolabs, ipswich, massachusetts, united states) and 4.5 µl of sterile, nuclease-free water (new england biolabs). five µl of the template dna was added to the master mixes and loaded onto a pxe 0.2 thermocycler (thermo electron corporation, waltham, massachusetts, united states). the forward and reverse internal control primer pair sequences were 5’-gccttcccaaccattccctta-3’ and 5’tcacggatttctgttgtgtttc -3’. il-10-819 t>c alleles were amplified using the following conditions: 1 min denaturation step at 95 °c; 10 cycles of 15 s at 95 °c, 50 s at 65 °c, and 40 s at 72 °c; and 20 cycles of 20 s at 95 °c, 50 s at 59 °c and 30 s at 72 °c, followed by cooling at 4 °c. tnf-α polymorphisms were amplified using the following conditions: 1 min denaturation step at 95 °c; 10 cycles of 15 s at 95 °c, 45 s at 60 °c, and 35 s at 72 °c; 25 cycles of 20 s at 95 °c, 45 s at 60 °c, and 45 s at 72 °c, followed by cooling at 4 °c. amplification refractory mutation system polymerase chain reaction amplicons were analysed by electrophoresis on 2% agarose gel stained with ethidium bromide. the gels were viewed on an ultraviolet trans-illuminator to determine the presence or absence of the cytokine gene polymorphisms. table 1: primers used in detecting tnf-α (rs18006290) and il-10-819 t>c (rs1800871) polymorphisms among s. haematobium infected and uninfected preschool children from shamva district, mashonaland central province, zimbabwe, september 2018. tnf-α and il-10 determination the levels of the cytokines were measured by an indirect enzyme-linked immunosorbent assay using mabtech 3510-1h-6 kits (mabtech ab, büro deutschland, germany) according to the manufacturer’s instructions. the assay was performed in duplicate, and the data were averaged. statistical analysis genotype frequencies of the tnf-α (rs1800629) and il-10-819 t>c (rs1800871) between s. haematobium infected and uninfected participants were compared by a chi-square test. the hardy-weinberg equilibrium was assessed for tnf-α (rs1800629) and il-10-819 t>c (rs1800871) s. haematobium infected and uninfected participants groups by chi-square. genotype data were analysed with the software international business machines corporation statistical package for social sciences for windows, version 19.0. (international business machines corporation, armonk, new york, united states). unpaired t-tests were used to compare cytokine levels between infected and uninfected participants and between different genotypes, and analysis of variance was used to compare cytokine levels between light, moderate and heavy infection groups. p-values less than 0.05 were considered statistically significant. results prevalence of s. haematobium infection there was no significant difference (p = 0.823) between boys (n = 294) and girls (n = 269) recruited. the prevalence of s. haematobium infection was 10.5% (59/563), with an even distribution between boys (50.8%) and girls (49.2%) (p = 0.82). tnf-α and il-10 genotypes are not associated with infection status or intensity all genotypes of rs1800629 and rs1800871 were represented in the population and, consistent with previous literature, the tnf-α-308 ga genotype and il-10 ct genotype were the most common. none of the genotypes studied showed significant differences between the infected and uninfected groups (table 2). it was found that the rs1800629 single nucleotide polymorphism was in line with the hardy-weinberg equilibrium in uninfected participants. however, the rs1800871 single nucleotide polymorphism was not in line with the hardy-weinberg equilibrium in both s. haematobium infected and uninfected participants. table 2: genotype distributions of tnf-α (rs1800629) and il-10-819 t>c (rs1800871) between s. haematobium infected and uninfected preschool children from madziwa, shamva district, zimbabwe, september 2018. when the data were stratified by infection intensities, there was no significant difference between the infected and uninfected groups (table 3). interestingly, 10 children had the il-10 tt genotype (associated with low production of il-10) and none of them were infected, raising the hypothesis that the tt genotype might be protective against s. haematobium, although this was not statistically significant. table 3: genotype distributions of tnf-α (rs1800629) and il-10-819 t>c (rs1800871) grouped according to s. haematobium infection intensity in preschool children from madziwa, shamva district, zimbabwe, september 2018. cytokine levels were associated with cytokine genotypes and infection the tnf-α gg genotype corresponded with significantly lower tnf-α levels when compared with the ga or aa genotypes (figure 1a) (p < 0.001), and tnf-α levels were significantly lower in infected children compared to uninfected children (figure 1b). the il-10 tt genotype corresponded with significantly lower il-10 levels when compared with cc (figure 1c). il-10 levels were significantly lower in uninfected children compared to infected children (figure 1d) (unpaired t-test; p < 0.001). figure 1: genotypes, cytokine levels and infection status in preschool children from madziwa, shamva district, zimbabwe, september 2018. (a) genotypes and tnf-α production (mean level for aa is 179.3 ± 31.2 pg/ml n = 34, ga is 176.8 ± 23.8 pg/ml n = 98 and gg is 131.6 ± 36 pg/ml n = 14); (b) tnf-α levels against infection intensity (239 ± 71.6 pg/ml n = 23 and 210 ± 17.6 pg/ml n = 286); (c) genotypes against il-10 plasma levels (cc – 528.5 pg/ml, ct – 323.8 pg/ml and tt – 309.4 pg/ml) (d) il-10 plasma levels against infection status (452.5 pg/ml and 376.4 pg/ml). reinfection reinfections at three months post treatment were detected in 10.2% (6/59) of children who were previously infected. at six months there were no reinfections observed and only one child was reinfected after one year post treatment. discussion the study describes the prevalence of urogenital schistosomiasis in preschool children and the impact of single nucleotide polymorphisms of tnf-α-308 g/a and il-10-819 t>c gene promoter regions on cytokine levels, susceptibility to s. haematobium infections and treatment outcomes. the results highlight and support existing evidence on schistosomiasis prevalence levels (10.5%) in children aged between one and five years and also support the possible effectiveness of mass drug administrations and immune development facilitation effects of praziquantel treatment as only six were reinfected one year post treatment. the study showed that cytokine levels were different depending on the individual’s genotype: the il-10-819 cc genotype corresponded with the highest levels of il-10, while the ct and tt genotypes corresponded with moderate and low levels of il-10, and the tnf-α-308 aa genotype corresponded with the highest, ga with moderate to high and gg with low tnf-α levels. a major finding was that tnf-α and il-10 levels might predict susceptibility to s. haematobium infection, with higher tnf-α and lower il-10 levels appearing to confer protection. interestingly, all 10 children who had the il-10 tt genotype (associated with low production of il-10) were uninfected, suggesting that this genotype may confer a protective effect, although this was not supported by statistical tests. higher levels of il-10 have also previously been linked with possible s. haematobium susceptibility.14 il-10 has immunomodulatory effects which may explain the susceptibility of individuals with high il-10 levels to schistosome infections. higher levels of il-10 have also been demonstrated to be protective against serious morbidity.14,15 our study suggests that higher levels of the pro-inflammatory cytokine (tnf-α) could aid in immunity development against schistosomes and that the intensity of infection was somewhat linked to tnf-α levels as those with moderate levels of tnf-α had a light infection (1–10 eggs/10 ml of urine) and those with low levels had moderate infection (11–49 eggs/10 ml), although this was not statistically significant. this supports the hypothesis that tnf-α is also helpful in limiting the proliferation of the parasite. the allele or genotype frequencies were as expected in africans: il-10 819 ct genotype frequencies were above 50% and tnf-α-308 ga genotype frequencies were above 80%.16,17 the relationship between cytokine levels and genotypes was also consistent with that in previous studies.18 il-10-819 cc genotype corresponded to high levels of plasma il-10, ct to moderate, and tt to low il-10 levels. for tnf-α-308, aa genotype corresponded to high, ga corresponded to moderate to high and gg corresponded to low plasma levels of tnf-α.18 il-10 promoter snps, –1082g/a, –819c/t and –592c/a, are involved in differential il-10 gene transcription and hence differential il-10 production.18 the findings highlighted a general lack of association between any genotype and urogenital schistosomiasis; however, the data pointed to a possible protective effect of il-10-819 tt. given that actual cytokine levels, rather than the snps studied here, show an association with susceptibility to s. haematobium infection, the findings highlight complexities that may exist in the expression of those genes and that additional snps may play an important role in the expression of these cytokines. although no significant association was demonstrated in this study, il-10 haplotypes gcc and gta were previously linked to high to moderate il-10 production and were demonstrated to be associated with schistosomiasis.18 this may highlight the need for a larger sample size to demonstrate the suggested link between genotypes and susceptibility or the need to identify and study other polymorphisms that affect expression of il-10 and tnf-α. peripheral blood mononuclear cells from subjects with schistosomiasis have been shown to produce eightfold greater levels of egg antigen-driven tnf-α, leading to a 99-fold greater mean tnf-α to il-10 ratio, relative to negative controls.15 some studies have established a positive relationship between infection intensity and tnf-α production.19,20 il-10 elevation in schistosome infections has also been demonstrated, with some authors suggesting both tnf-α and il-10 as possible biomarkers in schistosomiasis.19,20,21 thus the high levels of il-10 in infected individuals could have been a result of the infection rather than the cause of infection. however, there are previous reports that high levels of il-10 predispose to reinfection8,9,10 and we found that no individuals with the il-10 tt genotype (which was also associated with lower il-10 levels) were infected. taken together, these observations support the hypothesis that the association between il-10 levels and schistosomiasis in our study is due to higher il-10 levels predisposing to infection and lower levels conferring protection. some studies detected elevated levels of tnf-α in infected individuals relative to uninfected controls especially in acute schistosomiasis.22 the findings of this study however contradict those observations as low levels of tnf-α were associated with infection. according to the findings of this study, higher levels of tnf-α and lower levels of il-10 appear to be protective against s. haematobium. in this study most children did not get reinfected after 3, 6 and 12 months, suggesting the possible efficacy of praziquantel mass drug administrations in elimination of schistosomiasis. the few that were reinfected fall into the category of moderate to high production of tnf-α (individuals with the tnf-α ga and aa genotypes), which appears contradictory to our observation that high levels of tnf-α are negatively associated with infection. the effects of praziquantel treatment on immunological profiles and immunity to reinfection were reported to be similar in all age groups of children.4 however, tnf-α was shown to play a complex role in propagating the intramammalian stages of the schistosome life cycle – an effect that helps schistosomes survive in the host.23 this might explain the observed few reinfections. alternatively, some of the ‘reinfections’ might represent treatment failure cases, particularly since 5 of the 6 cases were observed at three months post treatment. some studies suggest that il-10 blocks the development of immunity after praziquantel treatment, thus reinfection is better prevented when praziquantel treatment is administered in individuals with low or blocked il-10.24 the reinfections could also be explained by the fact that the participants were inhabitants of an endemic area, and that praziquantel administrations were not coupled with other key interventions like killing of intermediate hosts and provision of safe water. thus, the study could not conclusively explain the reinfections. the low reinfection levels over the 12-month period could help support sustained community-based praziquantel administrations. such efforts can be reasonably expected to significantly reduce the schistosome burdens in endemic areas. the prevalence of 10.5%, although still high, may suggest effectiveness of prior mass drug administration and health education efforts as it is below 20.8% (nationally reported urogenital schistosomiasis prevalence in 2012).25 in 2011, the province (mashonaland central) had an overall prevalence of 26.1% (23.46–28.90) and shamva was also categorised among the top districts with heavy intensities (53.2%) of s. haematobium infection.6 more efforts like snail control, preventative chemotherapy and improvement of hygiene and sanitation are still required as these have at some point sustained prevalence below 10% in areas like hippo valley sugar estates, zimbabwe.26 the observed lack of differences in prevalence of schistosomiasis in boys and girls is in contrast to what was established by major surveys conducted in zimbabwe previously.6,27 in those primary school surveys, girls consistently had a lower prevalence of infection compared to their male counterparts. the absence of differences between boys and girls can be attributed to many, often overlapping, activities or chores like swimming, washing and fetching water from unprotected sources. the major factor is also that the water contact of preschool children is parent or guardian related, so no differences could be expected. gender differences in activities often start to vary at around age six, thus similar prevalence of infection is expected in children under six years of age and gender differences may emerge thereafter.28 limitations the prevalence of schistosomiasis was low and could have limited the ability to highlight an association of any of the studied genotypes with susceptibility to s. haematobium. although the sample was larger than the recommended 50 participants per centre, resources limited the ability to have more participants which could have helped in establishing the rare phenomenon. the results could also have been affected by the fact that we had a few individuals who had heavy infection intensities. some of the recruited children were not willing or able to provide adequate blood for genotyping. future studies are recommended to include many cytokines and all possible snps known to influence cytokine gene expression. conclusion prevalence of urogenital schistosomiasis among boys and girls was indistinguishable statistically. ga was the most frequent tnf-α-308 genotype. aa, ga and gg genotypes were associated with a high, moderate to high and low production of tnf-α, respectively. tnf-α-308 g/a and il-10-819 t>c polymorphisms were not significantly associated with susceptibility to s. haematobium infection, although no individuals with the il-10 tt genotype (n = 10), which is associated with lower il-10 levels, were infected in this study. higher tnf-α and lower il-10 levels were negatively associated with infection and may confer protection against schistosomiasis. praziquantel helped in reducing reinfection levels of schistosomiasis in preschool children staying in an endemic area. the findings of this study highlight the need for continued efforts (mass drug administrations, better sanitation and hygiene and health education and promotion) to reduce the prevalence of schistosomiasis to zero among children in endemic areas of zimbabwe. additionally, there is the need for immune-based interventions that encourage tnf-α but limit il-10 production as this will promote immunity against s. haematobium. acknowledgements we would like to thank the community of madziva, shamva district, for their participation and support in the study. we also thank the biochemistry staff at the university of zimbabwe and the infectious disease group for their assistance in the fieldwork. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions a.m., t.c., a.v., c.m. and t.m. developed the field study design, conducted field and sampling work and immunoassays and analysed the data. t.m. and j.m. supervised the work. all authors contributed to the manuscript. sources of support funding for this research was received from malaria training and research capacity building in southern africa (nih/fic2d43tw001587-06a2), wellcome trust and thrasher medical fund, schistosomiasis control initiative, united nations children’s fund and the world health organization. data availability the data used to support the findings of this study are available from the corresponding author upon request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references dabo a, badawi hm, bary b, et al. urinary schistosomiasis among preschool-aged children in sahelian rural communities in mali. parasit vectors. 2022;4:1–7. https://doi.org/10.1186/1756-3305-4-21 midzi n, mtapuri-zinyowera s, mutsaka mj, et al. impact of school based health education on knowledge, attitude and practice of grade three primary school children in zimbabwe. community med heal educ. 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department of pathology, aga khan university hospital, nairobi, kenya kenneth fleming green templeton college, university of oxford, oxford, united kingdom citation mudenda v, malyangu e, sayed s, fleming k. addressing the shortage of pathologists in africa: creation of a mmed programme in pathology in zambia. afr j lab med. 2020;9(1), a974 https://doi.org/10.4102/ajlm.v9i1.974 lessons from the field addressing the shortage of pathologists in africa: creation of a mmed programme in pathology in zambia victor mudenda, evans malyangu, shahin sayed, kenneth fleming received: 20 jan. 2019; accepted: 04 feb. 2020; published: 03 june 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: with approximately one pathologist for one million people compared to ratios of approximately 1 to 25 000 in the united states and united kingdom, there is a severe shortage of pathologists in much of africa. the situation is particularly severe in zambia, where, in 2009, the ratio was 1 to 1.4 million. objective: to address this, a postgraduate master of medicine (mmed) training programme was launched in lusaka in 2011. methods: the process and most significant challenges and lessons learned were documented, as they may be of value to other countries facing similar challenges. results: since 2011, four zambian pathologists have graduated, doubling the number of indigenous pathologists in the country. currently 10 students are in training. the most significant problem was issues arising from the split responsibilities of the ministries of health and of education and the most important lesson learned was the crucial need for broad local ownership and commitment. conclusion: successfully addressing the shortage of local pathologists by creating country-specific, postgraduate mmed training programmes, even in situations of restricted resources, is feasible. however, having access to and support from the shared resources, expertise and knowledge of a regional college of pathologists would be a major advantage. keywords: pathologist shortage; africa; postgraduate mmed; zambia; college of pathologists of east central and southern africa (copecsa). introduction although hard data are sparse, it has become increasingly recognised over the last decade that there is grossly inadequate capacity in pathology and laboratory medicine in many lowand middle-income countries (lmics), especially in sub-saharan africa, and that, even where present, standards are highly variable.1 addressing this issue will require multi-year and multifaceted approaches, especially given both the increasing population and the increasing burden of non-communicable diseases facing many lmics.2 zambia has a population of around 17 million. in 2000, it was one of the poorest countries in the world, but over the next decade, rapid growth raised it into the lower middle-income bracket (https://data.worldbank.org/country). it has a high infectious disease burden, but it has also seen a rise in non-communicable diseases, such as ischaemic heart disease and cancer (especially hiv-related cancers) (http://www.healthdata.org/zambia). given the shortage of doctors and other healthcare workers needed to face this double healthcare challenge, in 2009, both the 5th national development plan and the ministry of health national strategic plan3 identified human resource development as critical to meeting the united nations millennium development goals for health (https://www.un.org/millenniumgoals/). a particular problem was the shortage of pathologists, with the resultant lack of medical input into laboratory diagnosis and management of disease. pathologists are medically qualified specialists in all the clinical laboratory disciplines – haematology, microbiology, clinical chemistry, immunology, anatomic pathology, etc. while non-medical laboratory scientists can also provide laboratory services, they do not do so in anatomic pathology. anatomic pathology is concerned with diagnosis based on morphology of the tissues, using cytology, microscopy and autopsy. as tissue diagnosis is the gold standard for many diseases, including all cancers, and many infectious and non-infectious diseases of, for example, liver, kidney, gastro-intestinal tract, muscle and nerve, it is crucial for accurate therapy and management. it also reduces errors and resultant health costs and improves accuracy of health data for planning and budgeting (health management and financing). the shortage was partly due to the general lack of financing for medical education, but also reflected the lack of local faculty to provide the specialist training. in 2009, there were fewer than 10 anatomic pathologists working in zambia (of whom only 4 were zambian), a population ratio of 1 to 1.4 million. for comparison, in the united kingdom, there are about 1800 anatomic pathologists for a population of about 66 million, a population ratio of 1 to 36 000. this deficit meant the majority of the population had very limited access to pathology expertise. to illustrate this, in 2009, the main pathology department in the country, based in the university teaching hospital in lusaka, population around 1.7 million, had around 5000 anatomic pathology cases per year. in contrast, oxford, united kingdom, which serves an immediate population of around 750 000 and a wider population of around 2.5 million (incorporating five district general hospitals), has around 55 000 anatomic pathology cases per year (personal information, k.f.). in addition, prior to 2007, cancer therapy had been largely provided in south africa or zimbabwe. however, the opening of the cancer diseases hospital in lusaka in 2007 made provision of appropriate local anatomic pathology services crucial. furthermore, since 2004, the university of zambia had trained an increasing number of laboratory scientists, but without a parallel increase in pathologists. because there was no local pathologist training programme, small numbers of pathologists had been trained abroad (e.g. united kingdom, south africa), but the great majority had not returned to zambia. accordingly, in 2009, in recognition of the need to increase clinical input into laboratory diagnoses and address the shortfall in anatomic pathology, the ministry of health identified the local training of pathologists as one of its priorities. to do this, it was decided to set up a master of medicine (mmed) degree programme in pathology at the school of medicine at university of zambia. the salary costs of the trainees and of the local trainers and some local infrastructure would be covered by the university and hospital. but given the shortage of specialist staff, equipment and other infrastructure, assistance to set up and run the programme was sought from the tropical health education trust, london. a grant from the department for international development, united kingdom, to the tropical health education trust (thet) funded the recruitment and travel of external trainers, infrastructure, such as microscopes, books, modest refurbishment of space and the costs of the external placements of the students. as there are very few publications describing the creation of pathologist training programmes in africa,4,5,6 and as many other lmics have a similar lack of pathologists as in zambia, exploring the problems the zambia mmed faced and overcame, and describing the lessons learned from this process, may act as a roadmap for such countries. furthermore, the launch of this programme in 2011 coincided with the launch of the college of pathologists for east, central and southern africa (copecsa) (see accompanying article7). as many of the issues identified in zambia illustrate the potential role for such colleges to assist lmics in improving the quantity and quality of training in relevant specialties, this article also illustrates how the zambia mmed could have benefited from the prior existence of copecsa. intervention creation of the master of medicine programme in 2010, a needs assessment was conducted in lusaka to analyse the current situation, determine the magnitude of the need and plan the development of a mmed programme in pathology. the assessment took into account the major concerns of the public and other stakeholders and health workers, especially clinicians. to do this there were internal interviews with lecturers and students at university of zambia and key stakeholders including clinicians and ministry of health staff, meetings within other clinical departments across the school, and input from a small number of united kingdom pathologists. throughout this process, there was considerable discussion about duration and content of the curriculum. proposals ranged from a one-year or two-year multidisciplinary programme involving haematology, microbiology, clinical biochemistry and anatomic pathology, to a four-year mono-specialty course in cell pathology, with variants in between. the argument for the former was that as the greatest need was for service provision in all the disciplines of pathology, particularly outside the capital city, producing significant numbers of multi-specialty staff as quickly as possible should therefore be the priority. these could be either medical or non-medical staff. conversely, it was argued that as the ‘clinical pathology’ specialties of microbiology, biochemistry and haematology already had some modest provision through scientists with bachelor of science (bsc), master of science (msc), or doctor of philosophy (phd) degrees, the absolute crucial need was for medically qualified anatomic pathologists. as the ministry of health had ordained that the objective of the programme was to produce pathologists who would direct provincial hospital laboratories, with overall responsibility for all aspects of pathology, but with personal responsibility for anatomic pathology, eventually the university and ministry agreed that a four-year course, predominantly focused on anatomic pathology, but with some involvement in haematology, microbiology and clinical biochemistry, would be adopted. the process of discussion culminated in a two-day meeting in 2010 for the definition of the aims and objectives of the course, development of a curriculum, determination of the modes of teaching and agreement on the modes of evaluation of the students (see figure 1). to encourage retention, students would be bonded for the duration of their training, that is, the student would continue to work for the government for a length of time equivalent to the time they received government support. furthermore, appointment to consultant posts would follow from successful completion of the full programme. lastly, it was anticipated that the new consultants would have a significant leadership role in the future development of pathology in zambia. the programme would be deemed successful if at least two to three students graduated each year, were retained within the public sector in zambia and assisted with the training of future students. figure 1: programme map for development of mmed programme, zambia, 2010–2016. recruitment of students to recruit students, the school of medicine placed an advert in one of the local newspapers for zambian doctors interested in pursuing mmed training in pathology at the university teaching hospital, lusaka. the main criteria for acceptability were the possession of a basic medical degree at the university of zambia (or equivalent), registration with the health professionals council of zambia, and completion of at least the intern year and one year as a senior house officer, or equivalent, in country of previous practice. applicants who satisfied these criteria were interviewed by the course tutors to assess and confirm commitment before selection. programme the programme, exams and assessments were conducted under the school of medicine, university of zambia, regulations for postgraduate degrees. the students had to complete five courses over the four-year programme (figure 1). progress from one course to the next was dependent on passing the end-of-course exam or assessment. the university of zambia requires external examiners for end-of-year exams in year 1 and for the final exams in year 4; professor dhiren govender from the university of cape town, south africa, and professor martin hales from the university of witwatersrand, johannesburg, south africa, filled this role 2011–2013 and 2014–2016. students had a weekly programme of tutorials alongside practical sessions and group teaching, the latter taking place around the microscope in the pathology department in the university teaching hospital, lusaka. the great majority of the teaching was done by local pathologists, but some sub-speciality teaching was provided by teachers visiting from the united kingdom for approximately two weeks (e.g. liver, gastroenterological and urological disease and forensic pathology). these teachers were all volunteers, recruited on the basis of being long-standing consultants with experience of teaching trainee united kingdom staff. it was hoped to involve trainee staff from the united kingdom, but this proved not possible (see section ‘lesson learned’). sessions on general skills such as leadership and management, ethics and professionalism (as provided for all the university’s mmed students) were interspersed throughout the programme. a review of the curriculum and programme was undertaken in 2015, which confirmed that the programme was successful and that only minor changes to timing were needed. external clinical placements clinical placements outside zambia in the third year of tuition were an integral part of the programme. they provided trainees with the opportunity to experience techniques and specialisms with limited availability in zambia – liver, renal, neuro-muscular and bone and joint biopsies and excisions, specialist cytology and autopsy cases and specialist techniques like immune histochemistry and molecular biology. in 2015 and 2016, the clinical placements took place in the toronto general hospital, toronto, canada, under professor runjan chetty, and to the aga khan university hospital in nairobi, kenya, under dr shahin sayed and dr zahir moloo. through these placements, the trainees acquired new clinical and service development skills. the experience enhanced not only their clinical practice, but also their personal development, as they were exposed to new ways of working in different environments. in addition, the clinical placements encouraged regional collaboration through the exchange of ideas, of lessons learned and through exchange and sharing of resources. they also gave trainees opportunities to attend international conferences while abroad. costs we do not have access to the costs incurred by the zambian government, university of zambia and the university teaching hospital, lusaka, but the costs covered by the thet grant from 2009 until the end of 2015 are shown in table 1. table 1: tropical health education trust costs for development of mmed programme in zambia, 2010–2016. training outcomes after a further one-year preparation period, the mmed in pathology was launched in 2011 with four recruits. the first final exam was held in october 2015 and three out of the four candidates passed. in october 2016, another one of two candidates passed. thus, in the first two cycles of the programme, the number of local pathologists in zambia had increased from 4 to 8. all these graduates are currently (2018) working within the university teaching hospital, lusaka. two have joined the medical school as lecturers and one is working under the ministry of home affairs as a forensic pathologist. he is currently training in toronto, canada, on a year’s forensic pathology fellowship. the fourth graduate has remained in the employ of the ministry of health. currently (2018) there are two students in year 4, five in year 3, one in year 2, and two in year 1 (zambia graduates about 100 medical students per year). lessons learned challenges as one would expect in setting up a new postgraduate programme anywhere, there were a number of challenges and many lessons were learned. however, there were a small number of key challenges (box 1). one of the most significant, somewhat unexpected and difficult to address, was the differing perspectives of the ministry of health and of the ministry of education. this became especially apparent on issues of funding where trying to identify whether funding for any particular need arose from a service demand or an educational one. assigning an activity to one or the other was often impossible. indeed, the apprenticeship nature of pathology (and most medical) education, where a student is taught while performing routine service, means that trying to separate the costs of the two activities is, by its very nature, impossible. this meant that some activities were on the boundaries of the ministry of education and the ministry of health, with neither willing to fund the activity. this ministerial split responsibility occasionally also contributed to difficulties in identifying an appropriate decision-maker. on these occasions, decisions on key issues were passed back and forth between ministries and sometimes never completely resolved. this split was also replicated at hospital and university level and even within the pathology department. box 1: main challenges in developing a mmed programme in zambia, 2010–2016. given the constraints on funding, resources were inevitably another challenge. the biggest constraint was provision of space for the students for private study. funding for significant new building was primarily the responsibility of the university and the zambian government and became a prime example of the divergence of opinion between the respective ministries. even modest refurbishment became difficult as the student numbers increased with each year’s recruits. obtaining books, journal subscriptions and microscopes, although requiring considerable effort, was less difficult due to the generosity of charities and well-established international organisations dedicated to providing educational support to lmics. funding the visits of the external visitors was also largely not an issue as the thet grant covered these costs. funding the clinical placements was initially well covered by the thet grant, but as numbers increased this became more difficult. in addition, inevitably there were costs associated with the placement that had not been anticipated. for example, in the original budget, we did not allow for fees for the host departments of the clinical placements, necessitating some last-minute budgetary reorganisation (deferred spending and cuts). similarly accommodation for the trainees abroad proved more expensive than anticipated. the overall spending on the thet grant over the six years was £221 718.62. accordingly, the cost of each graduate in the first five years of the programme was £55 429.00 ($71 464.00 at current rates). the two biggest costs were the clinical placements and the overheads for the administration of the programme. most of the rest of the costs were travel and the curriculum review undertaken in 2015. given that these components had substantial overseas involvement, this is perhaps not surprising. in the future, with much less overseas involvement, the costs per graduate will diminish substantially. similarly, recruitment of more students in each year will reduce the cost per student. an unexpected issue was the difficulties of united kingdom staff obtaining time off work to visit and teach. employers proved very reluctant to view such activity as good for their institution, despite the argument that the returning staff would likely be refreshed with broader horizons. most visitors had to use holiday entitlement, which inevitably reduced the flexibility and numbers of possible contributors. similarly, it proved unexpectedly impossible to recruit trainees from the united kingdom. originally, we had intended to recruit united kingdom pathology trainees towards the end of their five-year training programme. it was hoped that, having been through a similar experience recently, they might have a greater insight into the challenges faced by the new zambian trainees. in addition, like their senior colleagues, it was hoped that the experience would broaden their horizons and benefit their later careers. the recruitment difficulty appeared to be related to constraints within the postgraduate training programmes that had been recently introduced in the united kingdom where time out from the highly structured programme requires official permission, including formal approval that the external environment is appropriate for training. while this is not unreasonable, it introduced a layer of complexity and bureaucracy which may have deterred some potential participants. another factor may have been the fact that not all the time spent in lusaka would have been recognised as equivalent to training time in the united kingdom, resulting in some extension in the duration of trainee’s overall training. perhaps not unexpectedly, dealing with the bureaucracy of obtaining visas and registration with the local medical board for the zambia students while on their clinical placements took more time than anticipated. recommendations firstly, to overcome the inevitable problems of starting a new training programme, it is crucial to have both broad ownership and commitment (box 2). this must be a programme owned and desired by the local staff, university and the national government. the latter two organisations are crucial as resolving issues in a national programme cannot be dealt with by an external or overseas organisation. it is also necessary to have a clear leader and champion who can deal with the issues as they arise. this may seem obvious, but it bears emphasis as implementation of the programme would simply have been impossible otherwise. box 2: programme development recommendations. secondly, adequate funding is obviously crucial, but it also needs to be flexible and relatively easily increased – if only modestly. starting a new programme means that underestimates in budgeting will probably be made, either due to unfamiliarity with costs or because unexpected needs arise – for example, the need to find fees for the clinical placements. budgetary flexibility is needed to address these unexpected costs. knowledge of the numerous international charities and organisations that provide free or low-cost access to educational materials is invaluable. a collated list of such organisations would be ideal. thirdly, in a hybrid programme with both local and external participants, the local leadership needs to be matched with an external leader and champion. the challenges of distance and resource constraints mean there needs to be a clear external individual who is willing and able to take on the challenges. this person needs to be a pathologist. for example, dealing with the problems of identifying external visitors would have been almost impossible via a committee or a non-pathologist. similarly, it is difficult to imagine identifying and getting the collaboration of external departments and examiners as a non-pathologist. additionally, it is necessary to have effective external administration. dealing with several funding bodies, ensuring compliance with appropriate financial and other regulations, identifying external teachers and organising their participation over several years cannot be done without effective administration. fourthly, in setting up any new programme, there are always problems and regular (e.g. weekly) effective communication between participants is vital. this is particularly true in a hybrid programme such as the zambian one. when the programme was initiated, even email was somewhat intermittent, proving frustrating. this has improved greatly in recent years with better telecommunication methods such as skype. it is also necessary to have regular, if infrequent, face-to-face meetings. these are not so much to solve problems (although annual review of progress and appropriate modification to the programme is best done face-to-face) but to provide opportunities for participants to develop relationships and build trust. lastly, it is crucial to recognise that initiating such a hybrid programme is not a short-term commitment. to allow the programme time enough to stabilise requires at least 5 years and perhaps up to 10 years. this is particularly important to ensuring appropriate continuity of funding and external support. conversely, it also means ensuring that there is a jointly agreed exit strategy for the external support to allow the local participants sufficient time to prepare for independent ownership. discussion of the various issues discussed above, two stand out. the first is that the single most difficult and intractable issue was getting the ministry of health and the ministry of education to agree on which would fund activities and infrastructure that had both educational and service aspects. such a split between university and hospital perspectives is not unique to zambia. it is found in most countries where medical education and service delivery are the responsibility of different authorities and is similarly contentious. however, where resources are severely constrained, the tensions are magnified. given this, what are possible solutions? once a programme is running, there is no obvious solution to this problem other than repeated discussions with the key individuals – this is where strong local leadership is vital, as identifying such individuals needs local insight. in retrospect, a discussion of the issue during the preliminary stages of setting up the programme, resulting in an agreed allocation of responsibilities, would have been the most appropriate solution. for instance, agreeing during the planning phase to create a joint fund which could have then been used by the programme organisers to fund such combined activities and infrastructure would have saved considerable time, effort and angst. whatever the approach, the key is to have the issue clearly identified and addressed early in the planning phase. the second issue is that in many low-resource regions, it is very difficult, if not impossible, for any one country to set up and sustain a fully comprehensive training programme. for example, in any one country, there may just not be the necessary number of trainers or the required case mix. the creation of the zambia mmed in pathology would have been impossible without sharing resources between countries, in this case, between zambia, the united kingdom, canada, kenya and south africa. however, the use of trainers from the united kingdom and of placements in canada markedly increased the costs of the programme. this is where a regional body, such as a college like copecsa, could have a key role – especially where such institutions are seen as dedicated to standards and independent of any particular country or government policy. as a transnational organisation, copecsa could have identified, for example, sites with specialist staff and equipment necessary for specific investigations. it could then have facilitated the short-term transfer of trainees who need training in such topics to this site – this would be analogous to, but much cheaper than, the external clinical placements to toronto of the zambia programme. similarly, having a copecsa regional core curriculum would have been of major benefit in the creation of the zambia mmed, saving time and effort. lastly a regional body such as copecsa could also have reduced the burden of designing and undertaking examinations by recruiting regional examiners and sharing assessment procedures. a general benefit of such activity would be to ensure high and equivalent standards across the region, perhaps leading in due course to trans-regional recognition of qualifications. conclusion the creation of a postgraduate training programme in pathology in countries with limited local resources has many challenges but taking a hybrid approach involving the sharing of resources with other countries is a pragmatic and feasible solution. it is likely that such hybrid programmes are more achievable and sustainable if the countries involved are part of a regional grouping and are facilitated by an independent body such as copecsa. acknowledgements we are grateful to many colleagues who helped devise the curriculum, taught in the programme and mentored residents. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions all authors contributed to the design and implementation of the programme and also contributed to and approved the final version of this article. ethical considerations the article describes the development and implementation of a teaching course at the university of zambia. there are no ethical issues. sources of support we are grateful to the tropical health education trust, london, united kingdom, for funding and for administrative support, and to the british division of the international academy of pathology and the association of clinical pathology, united kingdom, for funding. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views expressed in the submitted article are those of the authors and not an official position of their institutions or funders. references wilson ml, fleming ka, kuti ma, et al. access to pathology and laboratory medicine services: a crucial gap. lancet. 2018;391(10133):1927–1938. https://doi.org/s01406736(18)304586 sayed s, cherniak w, tan sy, et al. improving pathology and laboratory medicine in low income and middle income countries: roadmap to solutions. lancet. 2018;391(10133):1939–1952. https://doi.org/s01406736(18)304598 republic of zambia. national health strategic plan 2006–2010 [homepage on the internet]. ministry of health. [cited 2019 oct 26]. available from: http://www.zukhwa.ed.ac.uk/sites/default/files/alth_strategic_plan_revised_july_20__2006_1_.pdf busisiwe cm, mwesigwa s, mboowa g, et al. the collaborative african genomics network training program: a trainee perspective on training the next generation of african scientists. genet med. 2017;19:826–833. https://doi.org/10.1038/gim.2016.177 zulfu aa, abass sk, awadalla h, et al. assessment of pathology trainees’ satisfaction: results of a survey from sudan. j public health emerg 2018;2:29–35 http://doi.org/10.21037/jphe.2018.10.01 nelson am, hale m, diomande mij-m, et al. training the next generation of african pathologists. clin lab med. 2018;38:37–51. https://doi.org/10.1016/j.cll.2017.10.004 sayed s, mutasa r, kaaya e, et al. establishing copecsa – the regional ecsa college of pathology. afr j lab med. 2020;9(2):a979. https://doi.org/10.4102/ajlm.v9i2.979 introduction background: prospects for laboratory-based surveillance in africa and novel opportunities why whole genome sequencing surveillance? acknowledgements references about the author(s) hajo grundmann institute for infection prevention and hospital epidemiology, medical centre, university of freiburg, freiburg, germany hellen gelband centre for global health research, university of toronto, ontario, canada global public health consulting, takoma park, maryland, united states citation grundmann h, gelband h. antimicrobial resistance surveillance with whole genome sequencing in africa: it’s (about) time. afr j lab med. 2018;7(2), a761. https://doi.org/10.4102/ajlm.v7i2.761 opinion paper antimicrobial resistance surveillance with whole genome sequencing in africa: it’s (about) time hajo grundmann, hellen gelband received: 25 jan. 2018; accepted: 12 june 2018; published: 06 dec. 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction the current move to establish a global system for antimicrobial resistance (amr) surveillance was born of the need for reliable data that would allow estimating the impact of amr on public health and the economy, strengthen the evidence for acting, and point toward appropriate interventions. surveillance represents an important component of the global action plan on amr adopted by the 68th world health assembly and embodied in the world health organization’s developing global antimicrobial resistance surveillance system (glass).1 glass is partly predicated on reproducing programmes that have been successful in europe and elsewhere, following a longstanding and often successful tradition of transferring public health strategies from richer to poorer countries. the main aim is to generate datasets for trend analysis and benchmarking across nations. this is an important and useful purpose, mainly to guide policy decisions and allocate resources where and when predictable threats to public health need to be addressed. at the same time, it should not be seen as a reason to delay the introduction of another layer that is already being added in europe2,3 whole genome sequencing (wgs) enrich our understanding of amr evolution and spread and contribute practical information for local and national infection control and clinical guidance. our view is that wgs-based surveillance could and should be developed now in africa in parallel, and may actually produce informative results more quickly until the conventional approach renders authoritative and comparable data. our rationale is explained in this article. background: prospects for laboratory-based surveillance in africa and novel opportunities the most accomplished example of repurposing routine laboratory susceptibility results for amr surveillance and the basic model for who’s system is the european antimicrobial resistance surveillance network (ears-net). when it was conceived, it faced big challenges, ranging from scepticism that laboratory data were comparable across countries to doubts about the usefulness of comparing data from countries with vastly different healthcare systems and diagnostic practices.4 in the end, ears-net has robust participation across all european union member states and has become a strong advocacy tool for antibiotic policy. the idea of ears-net was inspired by a ready supply of relatively reliable results from antibiotic susceptibility tests that were conducted routinely in the course of patient care. using the data for surveillance added value to their original purpose to guide patient treatment without incurring additional laboratory costs. collection and analysis is not free, but the cost is small in comparison to the sunk laboratory costs. these aggregated laboratory data expressed as the proportion of isolates resistant to specific antibiotics are useful for broad policy purposes, particularly when paired with antibiotic consumption data which can be collected in europe. together, these complementary surveillance inputs provide ecological level evidence that antibiotic consumption is causally related to amr prevalence.5 however, even together, these information streams do not support the development of targeted amr transmission and infection control strategies. current concepts such as the onehealth approach embody the idea of an increasingly connected world and emphasise the importance of controlling antibiotic exposure in all microbial habitats humans, animals and the environment. it has become clear that the dissemination of amr is strongly associated with the expansion of highly adapted bacterial lineages (‘high-risk clones’) or successful and transferrable genetic elements. it is therefore crucial for surveillance systems to capture the transmission dynamics at more informative epidemiological scales as well as across ecological interfaces. crucial to understanding transmission dynamics driven by ecological constraints, for example drug pressure, migration, trade, climate change, etc. are sampling strategies that would provide snapshots of microbial populations at different points in time. wgs of bacterial pathogens has already proven useful for epidemiological surveillance, outbreak detection and infection control.6,7,8,9 as its use becomes more widespread, the need for standard sampling techniques, standard operating procedures and standardised analysis will increase in importance. more well-trained personnel both in the laboratory and bioinformatics aspects will be needed as well as, online tools and software that will facilitate the routine analysis of data.10 where does that lead us in pushing forward with amr surveillance in africa in 2018? regarding what we are considering ‘conventional’ laboratory-based surveillance, an important question is whether the laboratory results being produced routinely would, if aggregated, provide useful policy guidance. a 2016 evaluation of the world bank-supported east african public health laboratory networking project, which includes about 30 clinical laboratories in five countries (kenya, tanzania, uganda, rwanda, and burundi), demonstrated shortfalls in adequately trained personnel, as well as stockouts of key material resources.11 these lead to shortcomings in the utilisation of diagnostic services caused by long turn-around times and a perceived lack of clinical relevance for therapeutic choice. clearly, under these circumstances, current investigation habits will lead to an overestimation of resistance and will not be able to provide the data needed to inform local or national amr containment strategies. why whole genome sequencing surveillance? we argue that wgs could provide a level of scrutiny elusive to conventional amr surveillance systems. what wgs offers, is the ability to appraise the evolutionary blueprints that reveal the genetic composition of currently extant pathogens. tremendous insights into multi-drug resistant typhoid one of africa’s most important pathogens have already been gained through wgs, and the need to continue tracking typhoid infection patterns will not diminish any time soon.10 thus, wgs data would provide an insight into the genomic population structure and pinpoint clones of public health importance irrespective of the representativeness of the original sample. high-risk clones can be readily identified by their clonal relatedness, abundance, geographic clustering as well as by their genetic contents, that is, virulence genes, antibiotic resistance genes and the like. in this way, even biased samples would allow the discovery of emerging lineages, transmission and spread, and would provide invaluable benefits for understanding the origin and management of epidemics. whole genome sequencing generates biologically meaningful, robust and portable data. their relevance is increasing over time as national and international datasets grow and provide historical, evolutionary, ecological and epidemiological contexts with increasing granularity. it is therefore high time for african health systems to consider the necessary steps toward the collection and analysis of samples from patients, livestock and wild animals, and the environment for analysis by wgs. the expertise and some facilities for wgs already exist in several countries in sub-saharan africa, and we would propose that the enabled laboratories themselves devise a plan for a pilot programme. this could be kicked off by a foundation-funded brainstorming meeting bringing together africa’s leading genome scientists who represent the institutions with existing capacity. the initial project could be in one country with sampling in different areas over the course of a year, or something more ambitious. the vision should encompass all infectious agents with public health relevance including susceptible bacteria, viruses and parasites threatening the health of humans and animals. the programme would require external funding and possibly collaboration from scientists engaged in similar activities in europe or elsewhere. in addition to strengthening the science and producing information of continent-wide relevance, a coordinated continent-wide effort will be better able than small efforts to avoid certain pitfalls, have greater bargaining power with equipment and supply companies, and enjoy sharing of scarce human resources, for example bioinformaticians. it is particularly important, given the periodic improvements in technology (and the continuous declines in cost), that a sustainable plan for equipment updating be in place. it is, in 2018, however, feasible and reasonable to begin this effort and that it be africa led. conclusion surveillance using wgs can provide direct insight into the evolution and spread of antimicrobial resistant pathogens and complements traditional laboratory-based surveillance. the scientific capacity and equipment are already present in several african countries to conduct wgs-based surveillance, and there is no reason to delay utilising it and every reason to launch the effort without delay. acknowledgements competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support none. authors’ contributions the authors developed the idea for the paper jointly. h.g. (university of freiburg) drew heavily on his personal experience with amr surveillance in europe and africa. h.g. (university of toronto; and global public health consulting) drew on her previous experience leading the global antibiotic resistance partnership in africa and asia. the authors each wrote sections of the article. references european centre for disease prevention and control. ecdc study protocol for genomic-based surveillance of carbapenemresistant and/or colistin-resistant enterobacteriaceae at the eu level. ecdc: stockholm; 2017. ecdc. ecdc roadmap for integration of molecular and genomic typing into european-level surveillance and epidemic preparedness. ecdc: stockholm; 2016. bax r, bywater r, cornaglia g, et al. surveillance of antimicrobial resistance – what, how and whither? clin microbiol infect. 2001;7(6):316–325. https://doi.org/10.1046/j.1198-743x.2001.00239.x ford l, carter gp, wang q, et al. incorporating whole-genome sequencing into public health surveillance: lessons from prospective sequencing of salmonella typhimurium in australia. foodborne pathog dis. 2018;15(3):161–167. https://doi.org/10.1089/fpd.2017.2352 donker t, reuter s, scriberras j, et al. population genetic structuring of methicillin-resistant staphylococcus aureus clone emrsa-15 within uk reflects patient referral patterns. microb genomics [serial online]. 2017;3:1–12. available from: http://mgen.microbiologyresearch.org/content/journal/mgen/10.1099/mgen.0.000113 aanensen dm, feil ej, holden mtg, et al. whole-genome sequencing for routine pathogen surveillance in public health: a population snapshot of invasive staphylococcus aureus in europe. mbio. 2016;7(3):e00444–16. https://doi.org/10.1128/mbio.00444-16 coll f, harrison em, toleman ms, et al. longitudinal genomic surveillance of mrsa in the uk reveals transmission patterns in hospitals and the community. sci transl med. 2017;9(413). https://doi.org/10.1126/scitranslmed.aak9745 argimón s, abudahab k, goater rje, et al. microreact: visualizing and sharing data for genomic epidemiology and phylogeography. microb genomics [serial online]. 2016;2(11). available from: http://www.microbiologyresearch.org/content/journal/mgen/10.1099/mgen.0.000093 world bank. drug-resistant infections: a threat to our conomic future. world bank rep [serial online]. 2016; september:1–132. available from: www.worldbank.org wong vk, baker s, pickard dj, et al. phylogeographical analysis of the dominant multidrug-resistant h58 clade of salmonella typhi identifies interand intracontinental transmission events. nat genet [serial online]. 2015;47:632. available from: http://dx.doi.org/10.1038/ng.3281 gelband h, okeke in, aboderin ao, et al. east africa public health laboratory networking project: strengthening the role of laboratories in tracking antimicrobial drug resistance in east africa. final report to the world bank. cddep: washington, dc; 2016. available from: https://cddep.org/wp-content/uploads/2017/06/wb_report_32-1.pdf abstract introduction research method and design results discussion trustworthiness acknowledgements references about the author(s) feyisayo jegede family health international 360 (fhi360), department of laboratory services, abuja, nigeria henry a. mbah labtrail global, smyrna, delaware, united states ado dakata department of haematology, bayero university/aminu kano teaching hospital, kano, nigeria dalhatu h. gwarzo department of haematology, bayero university/aminu kano teaching hospital, kano, nigeria surajudeen a. abdulrahman family health international 360 (fhi360), department of laboratory services, abuja, nigeria aisha kuliya-gwarzo department of haematology, bayero university/aminu kano teaching hospital, kano, nigeria citation jegede f, mbah ha, dakata a, et al. evaluating laboratory request forms submitted to haematology and blood transfusion departments at a hospital in northwest nigeria. afr j lab med. 2016;5(1), a381. http://dx.doi.org/10.4102/ajlm.v5i1.381 original research evaluating laboratory request forms submitted to haematology and blood transfusion departments at a hospital in northwest nigeria feyisayo jegede, henry a. mbah, ado dakata, dalhatu h. gwarzo, surajudeen a. abdulrahman, aisha kuliya-gwarzo received: 20 oct. 2015; accepted: 04 feb. 2016; published: 12 may 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the laboratory request form (lrf) is a communication link between laboratories, requesting physicians and users of laboratory services. inadequate information or errors arising from the process of filling out lrfs can significantly impact the quality of laboratory results and, ultimately, patient outcomes. objective: we assessed routinely-submitted lrfs to determine the degree of correctness, completeness and consistency. methods: lrfs submitted to the department of haematology (dh) and blood transfusion services (bts) of aminu kano teaching hospital in kano, nigeria, between october 2014 and december 2014, were evaluated for completion of all items on the forms. performance in four quality indicator domains, including patient identifiers, test request details, laboratory details and physician details, was derived as a composite percentage. results: of the 2084 lrfs evaluated, 999 were from dh and 1085 from bts. overall, lrf completeness was 89.5% for dh and 81.2% for bts. information on patient name, patient location and laboratory number were 100% complete for dh, whereas only patient name was 100% complete for bts. incomplete information was mostly encountered on bts forms for physician’s signature (60.8%) and signature of laboratory receiver (63.5%). none of the dh and only 9.4% of bts lrfs met all quality indicator indices. conclusion: the level of completion of lrfs from these two departments was suboptimal. this underscores the need to review and redesign the lrf, improve on training and communication between laboratory and clinical staff and review specimen rejection practices. introduction efficient laboratory service remains a foundation of modern healthcare systems. laboratory testing is an essential part of the clinical decision-making process, because it provides the majority of critical information required for making timely and informed decisions for patient care.1 relationships with laboratories and utilisation of laboratory services by physicians and other stakeholders in the healthcare system occur mainly through the use of laboratory request forms (lrfs) for two-way communication. requesting physicians may not fully utilize this important communication medium.2 inadequate information or errors arising from the process of filling out lrfs can have a significant impact on the quality of laboratory outputs and, ultimately, on patient safety.3,4 the notion of the brain-to-brain loop for laboratory diagnostics was first introduced by george lundberg over 30 years ago.5 the first step in this loop model involves the selection of appropriate laboratory tests in the brain of the physician, which is then communicated through the lrf. this is followed by numerous intermediary steps, such as identification of the patient, specimen collection and specimen handling; and then by the actual specimen analysis in the laboratory. the last steps involve the release of test results, either manually or electronically, for the physician’s review and reaction to the laboratory information, the interpretation of the results and the implementation of appropriate clinical action.5 traditionally, laboratory practice is divided into three phases (pre-analytical, analytical and post-analytical).6 evidence shows that the majority of laboratory errors (50% – 70%) occur during the pre-analytical phase and involve the handling of the lrf.7 errors occurring during the analytical phase average less than 10%,8 whereas errors occurring during the the post-analytical phase average about 15%.3 the preand post-analytical phases lie outside of the control of the laboratory, but contribute approximately 93% of total laboratory errors across the entire testing process.9,10,11,12 the most frequent pre-analytical errors as compiled by lippi12 are: missing sample and/or test request; wrong/missing identification; in vitro haemolysis; undue clotting; wrong container; and contamination from infusion route. other errors include: insufficient sample; inappropriate blood-to-anticoagulant ratio; insufficient mixing of the sample; or inappropriate transport and storage conditions. insufficient information or omission of information on the lrf may lead to laboratory errors,13 as well as make result interpretation difficult and delay communications with the requesting physician, moreso in patients with life-threatening medical conditions. misidentification of either the patient or the requested test have also been encountered frequently.14 the lrf not only provides information about the laboratory test being requested, but is also used to communicate results back to physicians and patients. the standard lrf contains demographic data and other information, such as location of the patient, laboratory information, physician’s name and signature, telephone number of the requesting physician, amongst others. pre-analytical errors, such as the absence of important clinical information on lrf, can have serious effects on patient care by causing post-analytical errors, such as inappropriate interpretative comments.15 the majority of errors occurring during the pre-analytical phase are a result of individual or system design defects.16 the pre-analytical phase should be prioritised so as to reduce errors across the entire laboratory testing process.16 in australia, planned interventions and sustained improvements in compliance with standards resulted in an immediate reduction in the proportion of incomplete lrfs, from 43% to 2%.14 in developed countries, laboratory quality management systems have been institutionalised, with functional and robust monitoring systems in place to detect and minimise errors before they occur at any phase in the laboratory work flow. unfortunately, the converse is true in most laboratories in developing countries, such as nigeria.17,18 in these countries, the focus tends to be on the analytical phase of the work flow without consideration of other factors or variables beyond the control of the laboratory. in nigeria, there are few studies on the handling of lrfs or the impact of the lrf on the pre-analytical phase of laboratory process. the objective of this study was to assess routinely-submitted lrfs for correctness, completeness and consistency and to evaluate the contributions of the lrf to quality service delivery. research method and design ethical considerations the study protocol was reviewed and approved by the aminu kano teaching hospital ethical committee (reference number: akth/mac/sub/12a-3/vi/1330) in line with the international standard of research requirement. team members were trained to retain patient confidentiality and patient names were not collected as part of the data set. study design and setting this was a retrospective, cross-sectional, descriptive study. all lrfs submitted to the department of haematology (dh) and the blood transfusion services (bts) of aminu kano teaching hospital, kano, nigeria, from october 2014 to december 2014 were reviewed systematically and evaluated for completeness, correctness and consistency. selection of dh and bts lrfs was based on presumed availability of lrfs and the high workload of these departments. aminu kano teaching hospital is a tertiary health institution located in kano state in northwestern nigeria. it is a 600-bed hospital which serves as the referral centre to kano and other neighbouring states, including katsina, jigawa and bauchi. data collection and analysis hard copies of both inpatient and outpatient lrfs received for routine laboratory investigations were reviewed and evaluated for the purposes of this study. data from the dh and bts were extracted manually from the lrfs and entered into an excel file (microsoft, redmond, washington, united states), then collated, cleaned and reviewed before analysis using the statistical package for social scientists (spss; version 21/2012; ibm, armonk, new york, united states). a score of 1 was used to indicate complete and correctly-filled information, whereas a score of 0 was recorded when any item was missing. a frequency distribution table was created to summarise the data collected. data were analysed and categorised into groups of quality indicators (qi), based on international federation of clinical chemistry-working group (ifcc-wg) guidelines.19 the qis used were: (1) patient identifiers (name, age, sex and unit number); (2) appropriateness of test request (request date and specimen type); (3) availability and completeness of laboratory details (eg. laboratory number and reference range); and (4) availability and completeness of physician’s details (doctor’s name and signature, name of consultant, phone and fax number). in this setting, ‘consultants’ head a medical team and are the most experienced senior clinicians. results a total of 2084 lrfs were evaluated, comprising 999 from dh and 1085 from bts. dh forms requested a total of 12 data elements, whereas bts forms requested a total of 18 data elements. department of haematology overall, 89.5% of dh forms were filled out completely (table 1). of all the required information on the lrf, only patient name, location within the hospital (ward) and laboratory number were filled out both completely and correctly for all patients. patient age, sex, request date, unit number, specimen type and clinical information were both available and correctly filled out for over 98% of the forms. of the 244 (24.4%) lrfs with incomplete information for either the doctor’s name and signature or the consultant’s name, a greater number of forms (n = 145; 14.5%) were missing the consultant’s name as compared with those missing the physician’s name and signature (n = 99; 9.9%). the current dh lrf does not request the phone number of the requesting physician, the time of specimen collection, the signature of the laboratory supervisor/manager to validate the patient results or the reference range. table 1: completeness of laboratory request forms submitted to department of haematology of aminu kano teaching hospital, kano, nigeria, from october 2014 to december 2014 (n = 999). blood transfusion service overall, 81.2% of bts lrfs were filled out completely (table 2), which was lower than for the dh. of all the variables expected to be completed on the lrfs, only patient name was available and completed on all forms examined. conversely, none of the bts forms had time of request completed. as observed with the dh forms, patient age was completed on 1071 forms (98.8%) and sex was completed on 1056 forms (97.4%). for 62 forms (5.7%), hospital/unit number was not indicated. for test request information, clinical information/diagnosis was completed on 877 forms (80.9%), whereas 1000 forms (92.2%) had information on specimen type completed. similarly, date of request was completed on 1005 forms (92.7%). for other lrf details, number of units of blood requested was complete on 1013 forms (93.4%), type of product requested on 866 forms (79.8%), and degree of urgency on 811 forms (74.7%). the physician’s name was missing on 124 forms (11.4%) and the physician’s signature on 660 forms (60.8%), whereas 271 forms (25%) were missing the supervising consultant’s name. all but one (0.1%) of the bts forms had laboratory number completed. almost all forms (n = 1082; 99.7%) had the date of sample collection completed, whereas only 396 (36.5%) had the signature of the laboratory receptionist (receiver) completed. the current bts form does not request the phone number of the requesting physician or the time of the request. table 2: completeness of laboratory request forms submitted to blood transfusion services of aminu kano teaching hospital, kano, nigeria, from october 2014 to december 2014 (n = 1085). major quality indicators overall, the most frequently occurring data quality gap identified on dh forms was completion of laboratory details (n = 0), followed by physician’s details, which were complete on 762 of the forms examined (76.3%; table 3). patient identifiers were available and complete on 968 dh forms (96.9%), and 991 forms (99.2%) had relevant fields for test request completed. the most frequently observed quality gap on bts forms was completion of physician’s details (n = 349; 32.2%), followed by completion of laboratory details (n = 396; 36.5%) and test request details (n = 522; 48.1%). the least commonly occurring data quality gap on bts forms was completion of patient identifiers (n = 1043; 96.1%). none of the dh request forms and only 102 bts forms (9.4%) examined met all of the qis analysed in this study. the majority of bts forms (n = 983; 90.6%) met one or more qi requirements. table 3: completeness of laboratory request forms submitted to department of haematology (n = 999) and blood transfusion services (n = 1085) of aminu kano teaching hospital, kano, nigeria, from october 2014 to december 2014: analysis based on major quality indicators according to international federation of clinical chemistry working group.19 discussion the study revealed that of the 12 required pieces of information on lrfs from the dh, only three (patient’s name, location within the hospital [ward] and laboratory number) were filled out both completely and correctly for all patients. for lrfs from the bts, of 18 pieces of required information, only patient name was filled out both completely and correctly for all patients. the most commonly incomplete item on dh forms was the specimen receiver’s signature, whereas for bts forms, specimen receiver’s signature and doctor’s signature were commonly incomplete. this study demonstrated that a higher proportion of lrfs from the dh were completed compared with lrfs from the bts (89.5% for dh vs 81.2% for bts). these findings are in agreement with a proportion of 84% completion previously reported from a similar study conducted in ile-ife, nigeria.20 however, our finding is in sharp contrast with a proportion of 1.73% for lrf completion reported from a similar study conducted in lagos, nigeria.21 in our study, the name of the requesting physician was completed on most dh and bts forms (90.1% for dh; 88.6% for bts). unfortunately, because of the design of the lrf, the contact details of the requesting physician, which may be needed for follow-up, are not requested. various studies conducted in south africa have reported comparable (89.6%)22 or a lower (65.2%)15 proportions for missing physician details and contact information. consultant name was well documented, with complete information on 75% of bts forms and 85.5% of dh forms. however, these proportions are lower than an ile-ife study reporting 96.6% completion of consultant-in-charge information.20 an australian study reported that 43% of forms lacked complete information; missing items included physician’s name and pager number(s).14 one reason for this variation in our setting may be attributed to work pressure on junior physicians and improper orientation regarding the impact of incomplete lrfs on the quality of patient care. this training is done by senior physicians without collaboration with laboratory professionals. in nigeria, it is not uncommon for physicians to be reluctant to follow guidance from medical laboratory professionals because of the prevailing power differential between physicians and other health care providers.23 healthcare workers’ attitudes18,24 toward the completion of lrfs cannot be overlooked, following reports of poor documentation of laboratory processes. in nigeria, it is common for staff to consider such documentation as unnecessary paperwork and an extra burden.18,24 our study reported that 99.8% of dh forms and 80.9% of bts forms had clinical diagnosis details completed. this is consistent with a similar study conducted in ile-ife, nigeria, which reported 93.2% completion of clinical diagnosis details.20 however, our findings contrast with the 65.9% completion of clinical details reported in lagos, nigeria,21 77% completion reported at nepal university teaching hospital25 and 22.7% completion reported at ghana tertiary hospital.13 furthermore, a study of lrfs conducted in cape town, south africa, reported that 20.8% had no diagnosis information and 25.3% had diagnosis information given in an abbreviated form.15 in an indian study, diagnosis was not indicated on 61.20% of forms.26 unfortunately in these cases, critical results found by the laboratory for 17.30% of the patients could not be communicated to them by the physicians because of incomplete forms.26 in our study, about 98% of dh and bts forms had complete information for patient age and sex, which is comparable to 86.4% completion for age and 99.8% completion for sex reported from the ile-ife, nigeria study.20 our findings are higher than the lagos, nigeria study, which reported 68% completion for patient age,21 as well as the much lower completion reported for patient age (25.6%) and sex (32.7%) in a ghanaian study.13 both our report and the ile-ife study20 found that the only well-completed parameter on the lrfs was patient information. the design of the request form may itself be a contributing factor to eliciting completion of some desired information, as patient demographic characteristics are displayed prominently at the beginning of each form. however, in addition to patient information, the lagos study found that the referring physician’s name was the most completed information (99%),21 which is better than our findings of 90.1% for dh forms and 88.6% for bts forms. we found that documentation of specimen type was better for dh (99.7%) compared with bts (92.2%). this is close to the 89.9% reported in the ile-ife, nigeria study.20 however, our finding contrasts with the much lower ~12% reported at the north indian neuropsychiatry institute.26 it is worth noting that only 4.3% of dh forms appropriately captured the signature of the laboratory receptionist. reception of samples from inpatients is usually in bulk; as such, the receptionist may be overwhelmed with work and therefore not able to individually sign all lrfs. other contributing factors may be: lack of proper training; and commitment to utilise standard operating procedures and guidelines in the laboratory. more importantly, information on result verification by the laboratory supervisor/manager before release was unavailable, as the lrfs examined in this study did not request this information. in general, none of the dh or bts forms examined in this study met all of the ifcc-wg qis.19 considering the frequency of omission of very vital information on both departments’ lrfs, including physician contact details, laboratory details, and test request, we suggest that both lrfs be redesigned to meet international standards. limitations one of the major limitation of this study is that the opinion of the healthcare workers involved in completing the lrfs was not sought. this would have given more insight into the reasons for the incomplete items. another limitation of this study is its design and the differences in the two lrfs. the dh form has a total of 12 items, whereas the bts form has 18 items. hence, comparing the quality and completion of the two forms for the different sections should be interpreted with caution in the light of these design variations. in addition, we did not classify the lrfs in terms of inpatient or outpatient, routine or emergency service. hence, the impact of the missing information on care of critical patients could not be assessed. recommendations this study demonstrates that the currently-used lrf for both dh and bts should be reviewed and redesigned. the redesign should include: the physician’s phone number, time of the sample collection, time of the request, signature of the laboratory supervisor (to validate results) and the biological reference range interval in line with iso 15189 requirements and standards.27 biological reference ranges serve as guidance for the proper interpretation of laboratory test results. there is a need to develop a laboratory quality manual, guidelines and standard operating procedures, especially for sample rejection practices, as well as details on utilisation and completion of lrfs. basic components of laboratory processing with an emphasis on the pre-analytical stage of laboratory work flow should be prominent in the orientation training of all new house officers, residents and other users of laboratory services. there is a need to revive and sustain joint physician-laboratory conferences and review meetings to share knowledge, strengthen communication and foster feedback for quality improvement. periodic comprehensive laboratory audits with an emphasis on lrf evaluation could be beneficial in comparing baseline information with post-evaluation data for continuous quality improvement efforts. extension of similar assessment of the lrf currently being used in other departments at aminu kano teaching hospital (microbiology, chemical pathology and histopathology) could also create an opportunity for improvement in the quality of laboratory outputs and, ultimately, on patient care. conclusion overall completion of lrfs submitted to dh was higher compared with those submitted to bts; however, completion of bts lrfs was higher when assessed according to qis. this study highlights the level of incompleteness of routinely-submitted lrfs and points out certain expected and vital pieces of information that were completely missing. this underscores the need to redesign the lrf, provide capacity building, strengthen communication between laboratory staff and physicians and enforce specimen rejection practices. trustworthiness the findings reported in this article reflect the outcome of work done on lrfs by our research team members who participated in the research design and excusion, collection, analysis of data and report writing. reliability the findings of the research presented in this report were based on a review of lrfs submitted routinely to the dh and bts of aminu kano teaching hospital in kano, nigeria. based on the study design, the findings are specific and limited to amino kano teaching hospital, nigeria. validity the findings, outcomes and recommendations from this study may be of benefit to developing countries, such as nigeria. in addition to the percentage performance reported, data were also subjected to four composite qis to evaluate the most important qis expected on lrfs. importantly, the findings and outcomes of this study will form a baseline for comparison in future and the practical recommendations offered will help aminu kano teaching hospital to make informed decisions about re-designing the assessed lrfs and to stimulate review of other lrfs in other sections of the hospital. acknowledgements the authors sincerely appreciate the aminu kano teaching hospital chief medical director, the ethical committee and staff of dh and bts, especially the laboratory staff, for the support and conducive atmosphere provided during data collection for the successful conduct of this study. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. the views expressed here are those of the authors and do not necessarily reflect those of aminu kano teaching hospital, kano or fhi-360. authors’ contributions f.j. conceptualised the study, performed the literature search and was involved in the writing of the manuscript. s.a.a. contributed to study conceptualisation and writing of the manuscript and performed statistical analysis. h.a.m. was the research team lead, co-conceptualised the study and critically reviewed the manuscript. a.k.-g. contributed to the literature search, conceptual contributions and writing of the manuscript. d.h.g. performed some of the experiments and contributed to the writing of the manuscript. a.d. performed the experiments, as well as being involved in data collection and writing of the manuscript. references harrison jp, mcdowell gm. the role of laboratory information systems in healthcare quality improvement. int j health care qual assur. 2008;21(7):679–691. levinson w, lesser cs, epstein rm. developing physician communication skills for patient-centered care. health affairs. 2010;29(7):1310–1318. http://dx.doi.org/10.1377/hlthaff.2009.0450 goswami b, singh b, chawla r, et al. evaluation of errors in a clinical laboratory: a one-year experience. clin chem lab med. 2010;48(1):63–66. http://dx.doi.org/10.1515/cclm.2010.006 ogbaini-emovon e, ojide ck, mordi rm, et al. inadequate information in laboratory test requisition in a tertiary hospital in benin city, nigeria. ann biomed sci. 2013;12(2):6–13. plebani m, favaloro ej, lippi g. patient safety and quality in laboratory and hemostasis testing: a renewed loop? semin thromb hemost. 2012;38(6):553–558. http://dx.doi.org/10.1055/s-0032-1315960 plebani m. errors in laboratory medicine and patient safety: the road ahead. clin chem lab med. 2007;45(6):700–707. http://dx.doi.org/10.1515/cclm.2007.170 plebani m, sciacovelli l, aita a, et al. harmonization of pre-analytical quality indicators. biochemia medica. 2014;24(1):105–113. http://dx.doi.org/10.11613/bm.2014.012 sonntag o. analytical interferences and analytical quality. clin chim acta. 2009;404(1):37–40. http://dx.doi.org/10.1016/j.cca.2009.03.031 singla p, parkash aa, bhattacharjee j. preanalytical error occurrence rate in clinical chemistry laboratory of a public hospital in india. clin lab. 2011;57(9–10):749–752. plebani m. the detection and prevention of errors in laboratory medicine. ann clin biochem. 2010;47(2):101–110. http://dx.doi.org/10.1258/acb.2009.009222 simundic am, lippi g. preanalytical phase—a continuous challenge for laboratory professionals. biochem medica (zagreb). 2012;22(2):145–149. lippi g, chance jj, church s, et al. preanalytical quality improvement: from dream to reality. clin chem lab med. 2011;49(7):1113–1126. http://dx.doi.org/10.1515/cclm.2011.600 olayemi e, asiamah-broni r. evaluation of request forms submitted to the haematology laboratory in a ghanaian tertiary hospital. pan afr med j. 2011;8(1):33. http://dx.doi.org/10.4314/pamj.v8i1.71148 burnett l, chesher d, mudaliar y. improving the quality of information on pathology request forms. ann clin biochem. 2004;41(1):53–56. http://dx.doi.org/10.1258/000456304322664708 zemlin ae, nutt l, burgess lj, et al. potential for medical error: incorrectly completed request forms for thyroid function tests limit pathologists’ advice to clinicians. s afr med j. 2009;99(9):668–671. da rin g. pre-analytical workstations: a tool for reducing laboratory errors. clin chim acta. 2009;404(1):68–74. http://dx.doi.org/10.1016/j.cca.2009.03.024 mbah h, ojo e, ameh j, et al. piloting laboratory quality system management in six health facilities in nigeria. plos one. 2014;9(12):e116185. http://dx.doi.org/10.1371/journal.pone.0116185 jegede fe, mbah ha, yakubu tn, et al. laboratory quality audit in 25 anti-retroviral therapy facilities in north west of nigeria. open j clin diagnostics. 2014;4:193–204. http://dx.doi.org/10.4236/ojcd.2014.44028[pdf] solberg he. the ifcc recommendation on estimation of reference intervals. the refval program. clin chem lab med. 2004;42(7):710–714. http://dx.doi.org/10.1515/cclm.2004.121 adegoke oa, idowu aa, jeje oa. incomplete laboratory 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et al. evaluation of pre-analytical errors: inadequacies in the completion of laboratory requisition forms. j nepal assoc med lab sci. 2012;11(1):43–49. chhillar n, khurana s, agarwal r, et al. effect of pre-analytical errors on quality of laboratory medicine at a neuropsychiatry institute in north india. indian j clin biochem. 2011;26(1):46–49. http://dx.doi.org/10.1007/s12291-010-0082-2 international organization for standardization. iso 15189:2012: medical laboratories. requirements for quality and competence. geneva: international organization for standardization; 2012. introduction artificial intelligence: an overview in the diagnostic context the role of artificial intelligence in diagnostics in africa challenges and risks in artificial intelligence-enabled diagnostics ethical and equity considerations for african contexts references about the author(s) talkmore maruta department of programs, african society for laboratory medicine, addis ababa, ethiopia citation maruta t. the role of artificial intelligence in diagnostics: a new frontier for laboratory medicine in africa. afr j lab med. 2025;14(1), a2952. https://doi.org/10.4102/ajlm.v14i1.2952 editorial the role of artificial intelligence in diagnostics: a new frontier for laboratory medicine in africa talkmore maruta copyright: © 2025. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction the past decade has witnessed an unprecedented convergence of data science and healthcare, catalysed by advances in artificial intelligence (ai) and machine learning. laboratory medicine has not been spared, with ai transforming and improving laboratory diagnostics’ efficiency, accuracy, and speed. the capabilities of ai algorithms, to analyse large data sets, automate tasks, and improve diagnostic accuracy, lead to faster turnaround times, reduced human error, and more personalised patient care. for the african continent, where diagnostic capacity remains uneven and often strained, based on the lance report,1 the integration of ai presents both a transformative opportunity and a complex challenge. this editorial explores the evolving role of ai in the practice of laboratory medicine and diagnostics, and its potential to close health equity gaps while ensuring ethical, sustainable, and contextually appropriate adoption across africa. artificial intelligence: an overview in the diagnostic context hussein abbass defines ai simply as ‘the automation of cognition’.2 in essence, ai is a science that helps machines find a solution to complex problems in a more humanlike fashion. this generally involves borrowing characteristics from human intelligence and applying them as algorithms in a computer-friendly way. in diagnostics, ai systems are trained on large data sets to identify patterns in clinical, molecular, or laboratory data. it has found its application in molecular and genomic diagnostics, laboratory-based diagnostics, for example interpretation of microscopy, haematology, or clinical chemistry results, point-of-care and wearable diagnostics, and clinical decision support systems. for africa, where the ratio of diagnostic professionals to patients is significantly lower than global averages, ai can help fill critical human resource and infrastructure gaps, support task-shifting, and accelerate access to timely and accurate diagnoses.3 the role of artificial intelligence in diagnostics in africa addressing diagnostic delays and errors one of the most persistent barriers to effective healthcare in africa is delayed or missed diagnosis. artificial intelligence-enabled diagnostic tools can assist in rapidly analysing laboratory results, flagging abnormalities, and suggesting possible diagnoses. automated digital pathology platforms can analyse tissue biopsies or blood smears with speed and precision, potentially reducing diagnostic turnaround times from days to minutes. enhancing diagnostic access in underserved regions with 47% of the global population having little to no access to diagnostics, particularly in africa’s rural or resource-limited areas, access to trained laboratory professionals or specialised services, such as pathologists, may be non-existent. artificial intelligence-powered mobile platforms and point-of-care devices can extend diagnostic capabilities to the frontlines, allowing community health workers to leverage advanced tools for screening diseases such as tuberculosis, malaria, or cervical cancer. supporting laboratory workflow efficiency workflow is central to efficiency and incorporation of ai into laboratory processes improves efficiency in workflow, quality control, inventory management, and test interpretation. mckinsey and harvard reported that ai can save the healthcare industry us$360 billion a year, because of improved efficiencies.4 automated image analysis for haematology (e.g. white blood cell classification), microbiology (e.g. culture plate reading), and clinical chemistry (e.g. flagging abnormal trends) can alleviate the workload of laboratory professionals and improve consistency in result interpretation. integrating multimodal data for complex diagnoses complex diseases, such as cancer or antimicrobial resistance, require integration of clinical, laboratory, imaging, and molecular data. artificial intelligence systems are uniquely suited to synthesise these diverse data streams to support differential diagnoses and personalised treatment decisions. many countries in africa have begun to realise the advantages of ai in diagnostics. south africa and zambia are using computer-aided detection of tuberculosis using chest x-rays and algorithms such as cad4tb,5 automated malaria detection from giemsa-stained blood films is under evaluation in kenya and ghana, aiming to improve diagnostic accuracy and reduce inter-observer variability,6 and predictive algorithms for antimicrobial resistance patterns, using laboratory surveillance data, can assist in early identification of resistance trends and guide empirical therapy.7 in addition to these specific examples, ai has been incorporated into laboratory information management systems to automate interpretation and generation of laboratory reports. modules can also be built into the equipment test menus for reagents stock management and staff productivity. these examples underscore the continent’s growing engagement with ai in diagnostics, although they remain largely pilot-driven and limited in scale. figure 1 highlights the role of ai in diagnostics. figure 1: the role of artificial intelligence in diagnostics. challenges and risks in artificial intelligence-enabled diagnostics while the benefits of ai in diagnostics are substantial, several challenges must be addressed to ensure equitable, effective, and responsible deployment in africa. data quality and representativeness artificial intelligence algorithms are only as good as the data used to train them. most ai diagnostic models are developed using data sets from high-income countries, which may not generalise to african populations because of differences in disease prevalence, comorbidities, and health system contexts. there is an urgent need to generate and curate high-quality, diverse, and representative data sets from african settings to train contextually relevant models. infrastructure and digital divide reliable electricity, internet connectivity, and computational infrastructure are preconditions for implementing ai tools. many laboratories and health facilities across africa lack these basics. without parallel investments in infrastructure, ai technologies risk exacerbating existing health disparities. ethical and regulatory concerns artificial intelligence systems raise complex ethical questions around data privacy, consent, accountability, and transparency. in the diagnostic context, who is responsible when an ai system makes a wrong diagnosis? what safeguards exist to prevent bias in decision-making? african governments must urgently develop regulatory frameworks to govern ai in healthcare, drawing on global best practices but tailored to local contexts. capacity building and workforce integration the adoption of ai in diagnostics requires a workforce that is not only ai-aware but ai-capable. laboratory scientists, pathologists, and clinicians must be trained to understand how ai works, how to critically interpret its outputs, and how to integrate it into clinical workflows. additionally, new roles, such as health data scientists and clinical ai specialists, must be cultivated within africa’s health education systems. job replacements although specialised skills such as laboratory scientist and pathologist are still below set global standards, most african countries are still battling with very high unemployment levels. from that angle, ai maybe seen as an option that will result in job replacements for those few that are currently employed. ethical and equity considerations for african contexts artificial intelligence holds the potential to democratise access to diagnostics, but only if its development and deployment are guided by principles of equity, inclusion, and justice. africa must avoid becoming merely a consumer of externally developed technologies. instead, investments must be directed towards homegrown innovation ecosystems, enabling african researchers, institutions, and entrepreneurs to co-create ai solutions that reflect the continent’s diverse needs. artificial intelligence systems must be transparent and explainable. the ‘black box’ nature of ai – wherein users may not understand how decisions are made – can undermine trust in diagnostic processes. emphasis must be placed on developing explainable ai models, especially in life-critical scenarios. artificial intelligence is rapidly reshaping the landscape of diagnostics, offering unprecedented opportunities to enhance laboratory medicine in africa. from improving accuracy and turnaround times to extending diagnostic reach in underserved regions, ai holds immense promise for addressing the continent’s persistent health system gaps. however, realising this potential requires more than just technological adoption – it demands intentional investment in infrastructure, ethical frameworks, local innovation, and capacity building. as african countries navigate this transformative frontier, a people-centred, inclusive, and context-aware approach will be essential to ensure that ai serves as a catalyst for equitable, high-quality healthcare rather than a source of further disparity. the future of diagnostics in africa lies not just in smarter machines, but in smarter systems that empower professionals, protect patients, and promote health for all. references fleming ka, horton s, wilson ml, et al. the lancet commission on diagnostics: transforming access to diagnostics. lancet. 2021;398(10315):1997–2050. https://doi.org/10.1016/s0140-6736(21)00673-5 hussein abbass. editorial: what is artificial intelligence?. ieee trans artif intell. 2020;2(2):94–95. https://doi.org/10.1109/tai.2021.3096243 mills a, atun r, rasheed ma, et al. artificial intelligence and the future of global health. lancet. 2022;400(10360):1533–1536. https://doi.org/10.1016/s0140-6736(22)01795-0 agrawal a, lacob m, liebovitz d. the economic potential of generative ai: the next productivity frontier. mckinsey & company; 2023 [cited 2025 aug 01]. available from: https://www.mckinsey.com/business-functions/mckinsey-digital/our-insights/the-economic-potential-of-generative-ai-the-next-productivity-frontier muyoyeta m, maduskar p, moyo m, et al. the sensitivity and specificity of using a computer aided diagnosis program for automatically scoring chest x-rays of presumptive tb patients compared with xpert mtb/rif in lusaka zambia. plos one. 2014;9(4):e93757. https://doi.org/10.1371/journal.pone.0093757 yang f, poostchi m, yu h, et al. deep learning for smartphone-based malaria parasite detection in thick blood smears. ieee j biomed health inform. 2020;24(5):1427–1438. https://doi.org/10.1109/jbhi.2019.2939121 kumburu hh, sonda t, mmbaga bt, alifrangis m, mshana se. using artificial intelligence to predict antimicrobial resistance and improve empirical treatment. lancet digit health. 2019;1(6):e253–261. keen ce. preparing for ai in clinical laboratories [homepage on the internet]. healthcare in europe. 2024 [cited 2025 aug 01]. available from: https://healthcare-in-europe.com/en/news/preparing-ai-clinical-laboratory.html article information authors: innocent nzabahimana1 sabin sebasirimu1 john b. gatabazi2 emmanuel ruzindana1 claver kayobotsi3 mary k. linde4 jean b. mazarati1 edouard ntagwabira1 janvier serumondo1 georges a. dahourou5 wangeci gatei5 claude m. muvunyi1 affiliations: 1rwanda biomedical center/national reference laboratory, rwanda2rwanda military hospital, rwanda 3single project implementation unit (spiu)/ministry of health, kenya 4american society for clinical pathology (ascp), united states 5us centers for disease control and prevention (cdc), rwanda correspondence to: innocent nzabahimana postal address: po box 4668, kigali, rwanda dates: received: 25 july 2014 accepted: 11 sept. 2014 published: 03 nov. 2014 how to cite this article: nzabahimana i, sebasirimu s, gatabazi jb, et al. innovative strategies for a successful slmta country programme: the rwanda story. afr j lab med. 2014;3(2), art. #217, 6 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.217 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. innovative strategies for a successful slmta country programme: the rwanda story in this original research... open access • abstract • introduction • research method and design    • slmta sites and training    • audits    • mentorship and performance-based financing • results    • cohort i    • cohort ii    • cohort iii    • national reference laboratory    • performance-based financing • discussion    • conclusion • acknowledgements    • competing interests    • authors’ contributions    • attribution and disclaimer • references abstract top ↑ background: in 2009, to improve the performance of laboratories and strengthen healthcare systems, the world health organization regional office for africa (who afro) and partners launched two initiatives: a laboratory quality improvement programme called strengthening laboratory management toward accreditation (slmta), and what is now called the stepwise laboratory quality improvement process towards accreditation (slipta).objectives: this study describes the achievements of rwandan laboratories four years after the introduction of slmta in the country, using the slipta scoring system to measure laboratory progress. methods: three cohorts of five laboratories each were enrolled in the slmta programme in 2010, 2011 and 2013. the cohorts used slmta workshops, improvement projects, mentorship and quarterly performance-based financing incentives to accelerate laboratory quality improvement. baseline, exit and follow-up audits were conducted over a two-year period from the time of enrolment. audit scores were used to categorise laboratory quality on a scale of zero (< 55%) to five (95% – 100%) stars. results: at baseline, 14 of the 15 laboratories received zero stars with the remaining laboratory receiving a two-star rating. at exit, five laboratories received one star, six received two stars and four received three stars. at the follow-up audit conducted in the first two cohorts approximately one year after exit, one laboratory scored two stars, five laboratories earned three stars and four laboratories, including the national reference laboratory, achieved four stars. conclusion: rwandan laboratories enrolled in slmta showed improvement in quality management systems. sustaining the gains and further expansion of the slmta programme to meet country targets will require continued programme strengthening. introduction top ↑ reliable laboratory services are vital to a high-quality healthcare system; thus, investing in laboratory quality improvement is not only valuable, but essential.1 despite a multitude of efforts to strengthen laboratories through infrastructure and human resource development, laboratory quality remains a challenge in resource-poor settings.2,3accreditation is a critical measure of a laboratory’s quality level, as recognised by a series of international conventions, which called for accreditation to be part of laboratory-strengthening efforts in low-income countries.4,5,6,7 in order to help address deficiencies in the system, two initiatives were launched concurrently in kigali, rwanda in july 2009 by the world health organization’s regional office for africa (who afro) and partners.4 these were: strengthening laboratory management toward accreditation (slmta), an innovative training and mentoring programme designed to facilitate the implementation of laboratory quality management systems in resource-limited settings;8 and an incremental laboratory accreditation preparation process, which later became known as the stepwise laboratory quality improvement process towards accreditation (slipta).9 rwanda has a tiered laboratory system, funded through the ministry of health, which consists of the national reference laboratory (nrl) overseeing the entire laboratory network, four central referral laboratories, 43 district hospital laboratories and approximately 500 health centre laboratories. the nrl and five of the district hospital laboratories receive additional funding as part of the east african public health laboratory network (eaphln), a world bank project aimed at controlling epidemics by strengthening laboratory capacity in five east african countries. to date, rwanda has enrolled 15 laboratories (three cohorts of five each) in the slmta programme. the ministry of health aims to eventually enrol all national, central and district hospital laboratories, a total of 48 countrywide, in the accreditation preparation process.9 this study describes the achievements of the first three cohorts of the slmta programme and shares their experiences and lessons learned four years after the launch of the programme in rwanda. research method and design top ↑ slmta sites and training in january 2010, the rwandan ministry of health enrolled its nrl, three central referral laboratories (centre hospitalier universitaire de kigali [chuk], centre hospitalier universitaire de butare [chub] and king faisal hospital [kfh]), as well as one military hospital, kanombe military hospital (kmh), into the first cohort of slmta (cohort i). twenty-three participants were trained: three from chuk (one laboratory manager and two laboratory technologists), three from chub (one laboratory manager and two laboratory technologists), three from kmh (one medical doctor in charge of paediatrics and two laboratory technologists), three from kfh (one laboratory manager and two heads of units) and 11 from the nrl (two laboratory managers and nine heads of different sections). during the nine-month programme, participants attended three slmta workshops and implemented assigned improvement projects.the second slmta cohort (cohort ii) began in november 2011 with the five district hospital laboratories funded by the eaphln project: byumba, gihundwe, gisenyi, kibungo and nyagatare. the training included 14 participants from these laboratories, three participants each from four laboratories (one lab manager, one quality officer and one safety officer) and two from nyagatare hospital laboratory (one lab manager and one safety officer). in addition, six staff members from cohort i laboratories participated (four from the nrl, one from chuk and one from chub) because of a need to replace slmta-trained staff lost due to turnover and transfers. in march 2013, five additional district hospital laboratories (bushenge, kibagabaga, ruhango, ruhengeri and rwamagana) were enrolled in cohort iii. each laboratory provided three participants: one laboratory manager, one quality officer and one safety officer. in addition to these 15 participants, laboratories from previous cohorts sent 11 participants (five from the nrl, two from kmh, one from chuk, one from chub, one from kibungo and one from nyagatare), again to replace trained staff who had left. audits to evaluate progress, audits were conducted for all three cohorts using the slipta checklist, before (baseline) and after (exit) slmta workshops. depending on the audit scores, laboratories were awarded zero to five stars. a rating of zero stars was given for a score of < 55% (0–141 points ), one star for 55% – 64% (142–166 points), two stars for 65% – 74% (167–192 points ), three stars for 75% – 84% (193–218 points), four stars for 85% – 94% (219–243 points) and five stars for ≥ 95% (244–258 points).10 follow-up audits (performed from three to 18 months after the exit audits) were conducted for cohorts i and ii, but follow-up audits for cohort iii laboratories had not yet been completed at the time of the writing of this article. cohort i laboratories received one follow-up audit, with the exception of nrl, which had four. in cohort ii, byumba, gihundwe and gisenyi each had two follow-up audits, whereas gihundwe and nyagatare had three.all audits for cohort i were conducted by consultants from the american society for clinical pathology (ascp). ascp consultants teamed with rwanda slmta facilitators to conduct baseline and exit audits for cohorts ii and iii, whilst eaphln auditors conducted follow-up audits for cohort ii. the ministry of health selected two high-performing laboratories from cohort ii for official slipta audit by the african society for laboratory medicine (aslm), namely, nyagatare hospital laboratory and gihundwe hospital laboratory. mentorship and performance-based financing seventeen local mentors with advanced diplomas or bachelor’s degrees received a two-day training in-country in march 2012 in order to facilitate the implementation of quality management systems in the laboratories. they were tasked with helping slmta participants in the implementation of improvement projects, in reviewing lessons learned during workshops and in closing gaps identified during the audits. these local mentors visited each laboratory for five days following each workshop. additionally, for cohort ii, mentors (two from rwanda, one from uganda) with master’s degrees in microbiology spent two weeks per month in the laboratories from may 2012 to december 2013, overlapping with slmta implementation.cohort ii laboratories also implemented performance-based financing, the first time such a model had been used with slmta. the performance-based financing model is a contractual approach stipulating that services and purchasing activities performed by health providers must be of good quality and compliant with standards. linking financial incentives for the facility with results is designed to motivate healthcare providers to provide health services according to the qualities required by national norms and standards. a payment amount of $15 000 was allocated on a quarterly basis to each cohort ii laboratory with a score of 100% on the slipta checklist. the incentive was discounted based on the slipta audit score for laboratories not achieving a score of a 100%. for example, if a laboratory received a score of 70% on the slipta checklist, it would receive a payment of 70% of $15 000, or $10 500. to incentivise continuous quality improvement, performance-based financing allowances were withheld if the laboratory’s slipta score dropped by ≥ 3 percentage points from its previous score or resulted in a lower star rating. the laboratory could use this incentive money to buy commodities and conduct post-audit activities, gap analysis, workshops and employee-recognition activities. results top ↑ cohort i at the baseline audit for cohort i, four laboratories had zero stars (chub, chuk, nrl, kmh) and one laboratory (kfh) was at two stars (figure 1a, table 1). kfh is a private hospital laboratory and had been pursuing hospital accreditation actively for three years prior to enrolment in slmta. at the exit audit, one laboratory (kmh) received one star, two laboratories received two stars (chub, nrl) and two laboratories received three stars (chuk, kfh). there was marked improvement in all laboratories, with median scores increasing from 43% to 73%. at the follow-up audit, one year after the exit audit, two laboratories earned three stars (chub, kfh) and three laboratories achieved four stars (chuk, nrl, kmh). table 1: cohort-level audit scores. figure 1: progress of slmta cohorts i (a), ii (b) and iii (c) in rwanda based on slipta checklist scores. cohort ii in cohort ii, all laboratories received zero stars at the baseline audit (figure 1b, table 1). at the exit audit, three laboratories received two stars (gihundwe, gisenyi, kibungo) and two laboratories received three stars (byumba, nyagatare). median scores increased from 28% at baseline to 70% at the exit audit. at the first follow-up audit, three months after exit, one laboratory was at one star (kibungo), three laboratories had earned three stars (byumba, gisenyi, nyagatare) and one had earned four stars (gihundwe). at the official slipta audit conducted by aslm in july 2013, five months after the first follow-up audit, nyagatare hospital laboratory was awarded two stars and gihundwe hospital laboratory three stars. scores were somewhat lower (three percentage points for nyagatare hospital laboratory and eight for gihundwe hospital laboratory) than those received at the first follow-up audit. a second follow-up audit in november 2013 resulted in similar scores to the first follow-up, with the exception of kibungo hospital laboratory, whose score increased 20 percentage points to 80% (figure 1b). cohort iii at the baseline audit for cohort iii, all five district hospital laboratories had zero-star ratings (figure 1c, table 1). at the exit audit, four laboratories received one star (kibagabaga, ruhango, ruhengeri, rwamagana) and one laboratory received two stars (bushenge). median scores increased from 32% at baseline to 56% at exit. national reference laboratory the nrl participated in six audits during the period of 2010 to 2013. the laboratory showed marked, though unsteady, improvements from 43% at baseline to 86% at the fourth follow-up audit nearly four years later. at the first follow-up audit in november 2011, the nrl received two stars, a score similar to that awarded at the exit audit six months earlier. at the second follow-up audit in may 2012, the nrl earned four stars, but at the third follow-up audit in february 2013, the nrl decreased slightly to a three-star rating (figure 2). figure 2: progress of the national reference laboratory (nrl) from 2010–2013, based on stepwise laboratory quality improvement toward accreditation (slipta) checklist scores. performance-based financing performance-based financing incentives of $75 000 were planned to be awarded to the five laboratories in cohort ii for each quarter. the maximum amount received in a quarter was $13 050 by gihundwe laboratory which scored 87% at their first follow-up audit. two laboratories (nyagatare and kibungo) were not awarded incentives for one quarter because of a drop in star levels. discussion top ↑ results of this study show substantial improvement in laboratories enrolled in slmta since 2010, as shown by star rating results. all but one of the 15 laboratories had a zero-star rating at the baseline audit, suggesting very low levels of quality management. at the conclusion of the slmta training programme, every laboratory had achieved at least one star, with four laboratories obtaining three or more stars. furthermore, laboratories continued to improve after the end of the slmta programme, with nine of the 10 laboratories conducting follow-up audits achieving three or more stars.establishing a stepwise approach in order to guide laboratories in a gradual improvement process, as well as offering evaluations that demonstrate progress at each level, is a dynamic way of implementing quality laboratory standards in developing countries.11 improvements resulting from slmta implementation have been observed elsewhere; however, rwanda’s results are somewhat higher than what is typically found. for example, amongst 321 laboratories worldwide that have completed the slmta training, nearly one third (29%) remained at zero stars after slmta implementation, with a mean score increase of 23 percentage points, compared with rwanda’s results of all laboratories achieving at least one star and a median improvement of 34 percentage points.12 for system-wide improvement, the rwandan government encourages collecting and using laboratory data for advocacy; programmatic data are now used in developing policies aimed at improving quality services. for example, a cross-cutting problem in many laboratories in rwanda has been service interruptions as a result of stockouts and equipment breakdowns. to address this problem, chuk conducted an improvement project between its second and third slmta workshops which focused on calculating the financial impact of service interruption. from july to september 2010, stockouts and equipment breakdowns prevented the laboratory from performing 6486 tests, which were referred to private laboratories. the laboratory estimated that, if performed, the tests would have generated revenue of $14 308. in contrast, the funds needed to purchase the necessary reagents and maintain equipment were estimated at $5711, resulting in a net loss of $8597 in potential income to the hospital. after reviewing these findings, hospital senior management agreed to purchase a back-up clinical chemistry analyser and signed a maintenance agreement with laboratory equipment manufacturers with the aim of ensuring continuity of laboratory services. sustainability is a critical issue for slmta and other improvement programmes. data from cohorts i and ii show that not only were the gains achieved through slmta implementation sustained a year after completion of the training programme, but they continued to increase a median of 10 additional percentage points. the kmh laboratory in cohort i showed the greatest post-slmta improvement, with scores increasing from 56% (1 star) at the exit audit to 90% (4 stars) one year later. this laboratory had the lowest baseline score amongst all laboratories in rwanda’s slmta programme to date, yet has now earned the highest follow-up score in the country’s programme. staff at kmh attributed this remarkable achievement to high levels of commitment, team work and hospital management support of and direct involvement in the quality improvement effort. the kmh staff’s pride in their accomplishments is highlighted by the fact that in may 2012 they changed their name from kanombe military hospital, which was linked to their military camp, to rwanda military hospital (rmh). they also began to expand their testing capacity by introducing new services, including molecular biology, enzyme-linked immunosorbent assays and systematic bacteriology culture, as well as building a new laboratory infrastructure in their preparation to transition into a referral hospital. overall, cohort ii showed the greatest improvement of the three cohorts, with a median improvement of 38 percentage points from baseline to exit and an additional 11 percentage points a year later (table 1). several factors may help to explain these successes. firstly, these laboratories received additional funding from the world bank’s eaphln in order to support improvement projects and other elements of quality management systems, including building infrastructure and purchasing back-up equipment and safety items such as first aid kits, spill kits and eye wash stations. secondly, these laboratories had the benefit of extensive on-site expert mentorship to assist with improvement projects and programme implementation. however, cohort ii was not without challenges. for example, nyagatare hospital laboratory, which was one of the two laboratories audited by aslm, lost their quality officer (september 2012) and laboratory manager (august 2013); despite sending replacements to be trained along with cohort iii laboratories, their scores declined steadily after the exit audit, dropping from 78% at exit to 72% at the second follow-up audit 11 months later (figure 1b). cohort ii also implemented an innovative performance-based financing incentive system. performance-based financing has been used by many development organisations to ensure greater accountability and to improve the efficiency of funded programmes.13 haiti was the first low-income country in which health service providers were remunerated according to their performance.14 in cambodia, performance-based financing was applied to the public sector; despite promising results, however, it did not materialise into a national policy.15 rwanda has been on the cutting edge of this approach, implementing performance-based financing in several sectors since 2002.16,17,18 nrl staff participated extensively in all three cohorts, as this laboratory is expected to provide leadership and guidance on quality management systems for rwanda’s entire laboratory network. also, as part of the eaphln, the nrl was in a unique position to monitor the progress and challenges of slmta implementation in the network laboratories. multiple factors may have contributed to variability in audit scores for nrl. as the country’s only national reference laboratory, the nrl provides a large proportion of services and routine testing in the country. this creates a heavy and fluctuating workload for the staff and the staff may not consistently prioritise quality improvement activities. variability in scores could also reflect the senior management’s lack of focus on the accreditation preparation process. to overcome these challenges, there has been renewed commitment by senior management to focus on strengthening the laboratory systems at the nrl. in march 2013, a laboratory technical working group was launched with an accreditation subcommittee. the nrl is also undertaking extensive decentralisation to reduce routine testing and workloads, enrolling in external quality assessment programmes and supporting mentorship in all sections of the laboratory. the rwanda ministry of health is forging ahead with its goal of implementing slmta in the remaining district hospital laboratories and ensuring that laboratories sustain momentum after programme completion by integrating continuous improvement into routine management. conclusion in rwanda, laboratories enrolled in the slmta programme demonstrated measurable improvements. performance-based financing, intensive monitoring and supplementary financial resources may have contributed to gains in cohort ii laboratories. strengthening of an effective laboratory technical working group is needed to oversee the accreditation preparation process, mobilise resources and further develop the plan outlined by the ministry of health for long-term sustainability of quality laboratory systems. expanding the use of performance-based financing to incentivise the quality improvement process in rwanda may contribute to accreditation readiness. acknowledgements top ↑ we are grateful to drs. katy yao and elizabeth luman from the division of global hiv/aids, center for global health, cdc, for their inspirational discussion and assistance in manuscript writing. we acknowledge dr. david mcalister for his technical support in initiating rwanda’s laboratory accreditation process. we also acknowledge global scientific solutions for health for manuscript edits and technical support, the rwanda ministry of health for launching and supporting the laboratory accreditation process, as well as the ascp and cdc’s rwanda office for funding and technical support to the rwanda slmta programme. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions i.n. (rwanda biomedical center/national reference laboratory) and w.g. (cdc, rwanda) were the study leaders. j.b.m., i.n., e.n., c.m.m. and s.s. (all rwanda biomedical center/national reference laboratory), as well as w.g., conceived and designed the study. m.k.l. (ascp), j.b.g. (rwanda military hospital) and c.k. (spiu/ministry of health, kenya), as well as e.r., i.n., s.s. and j.s. (all rwanda biomedical center/national reference laboratory) collected the data. i.n., w.g. and g.a.d. (cdc, rwanda) analysed the data; and i.n., c.m.m., e.n., w.g. and j.b.g. wrote the manuscript. attribution and disclaimer this manuscript was supported by the cdc. the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the cdc. references top ↑ 1.olmsted ss, moore rc, meili hc, et al. strengthening laboratory systems in resource-limited settings. am j clin pathol. 2010;134(3):374–380. http://dx.doi.org/10.1309/ajcpdqosb7qr5glr2.ravishankar n, gubbins p, cooley rj, et al. financing of global health: tracking development assistance for health from 1990 to 2007. lancet. 2009;373(9681):2113–2124. http://dx.doi.org/10.1016/s0140-6736(09)60881-3 3.nkengasong jn, mesele t, orloff s, et al. critical role of developing national strategic plans as a guide to strengthen laboratory health systems in resource-poor settings. am j clin pathol. 2009;131(6):852–857. http://dx.doi.org/10.1309/ajcpc51blobbpakc 4.nordling l. african disease labs to get health check [published online 2009 jul 27]. nature. 5.world health organization. the maputo declaration on strengthening of laboratory systems [document on the internet]. c2008 [cited 2013 jan 02]. available from: http://www.who.int/diagnostics_laboratory/maputo-declaration_2008.pdf 6.world health organization. joint who – cdc conference on health laboratory quality systems. who/hse/ihr/lyo/2008.3 [document on the internet]. c2008 [cited 2013 jan 03]. available from: http://www.who.int/ihr/lyon/report20080409.pdf 7.world health organization regional office for africa. who report calls for strengthening public health laboratories. yaoundé, 02 september 2008 [page on the internet]. c2008 [cited 2014 jul 21]. available from: http://www.afro.who.int/en/media-centre/pressreleases/item/679-who-report-calls 8.yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin pathol. 2010;134(3):401–409. http://dx.doi.org/10.1309/ajcpnbbl53fwuiqj 9.government of rwanda, ministry of health. third health sector strategic plan july 2012 – june 2018. final version [document on the internet]. c2011 [cited 2012 june 30]. available from: http://www.moh.gov.rw/fileadmin/templates/docs/hssp_iii_final_version.pdf 10.world health organization regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation in the african region (with checklist) [document on the internet]. c2011 [cited 2013 may 31]. available from: http://www.afro.who.int/index.php?option=com_docman&task=doc_download&gid=8642&itemid=2593 11.maruta t, motebang d, wanyoike j, et al. impact of mentorship on who-afro strengthening laboratory quality improvement process towards accreditation (slipta). afr j lab med. 2012;1(1), art. #6, 8 pages. 12.yao k, maruta t, luman et, et al. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. 2014;3(2), art. #194, 8 pages. http://dx.doi.org/10.4102/ajlm.v3i1.194 13.world bank. world development report 2004: making services work for poor people. washington, dc: the world bank; 2004. 14.eichler r, auxila p, antoine u, et al. performance-based incentives for health: six years of results from supply-side programs in haiti. cgd working paper #121. washington, dc: center for global development; 2007. 15.soeters r, griffiths f. improving government health services through contract management: a case from cambodia. health policy plan. 2003;18(1):74–83. http://dx.doi.org/10.1093/heapol/18.1.74 16.meessen b, musango l, kashala jp, et al. reviewing institutions of rural health centres: the performance initiative in butare, rwanda. trop med int health. 2006;11(8):1303–1317. http://dx.doi.org/10.1111/j.1365-3156.2006.01680.x 17.rusa l, schneidman m, fritsche g, et al. rwanda: performance-based financing in the public sector. in: center for global development. performance incentives for global health: potential and pitfalls. baltimore, md: brookings institution press, 2009; pp. 189–214. 18.basinga p, gertler pj, binagwaho a, et al. paying primary health care centers for performance in rwanda. policy research working paper wps 5190. washington, dc: the world bank; 2010. abstract introduction methods results discussion conclusion acknowledgements references about the author(s) lungile ndlovu department of biomedical science, faculty of applied and health sciences, mangosuthu university of technology, durban, south africa nokukhanya thembane department of biomedical science, faculty of applied and health sciences, mangosuthu university of technology, durban, south africa ziningi n. jaya department of biomedical science, faculty of applied and health sciences, mangosuthu university of technology, durban, south africa citation ndlovu l, thembane n, jaya zn. comparative analysis of inflammatory markers in hiv-positive individuals on antiretroviral therapy versus hiv-negative individuals in south africa. afr j lab med. 2025;14(1), a2756. https://doi.org/10.4102/ajlm.v14i1.2756 original research comparative analysis of inflammatory markers in hiv-positive individuals on antiretroviral therapy versus hiv-negative individuals in south africa lungile ndlovu, nokukhanya thembane, ziningi n. jaya received: 10 jan. 2025; accepted: 31 july 2025; published: 26 jan. 2025 copyright: © 2025. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: hiv is associated with chronic inflammation and immune activation, which can persist even in individuals on antiretroviral therapy (art), increasing the risk of cardiovascular disease, neurocognitive disorders, and other inflammatory conditions. objective: this study comparatively investigates inflammatory markers among hiv-positive individuals receiving art with those in hiv-negative individuals. methods: we analysed retrospective laboratory results, including viral load, c-reactive protein (crp), and d-dimer, from 275 individuals (aged 0–39 years) treated at a tertiary hospital in south africa. the study period commenced on 02 december 2023 and ended on 28 october 2024. spearman’s rank correlation was used to evaluate relationships among demographic factors, viral load, and inflammatory markers. results: findings revealed that hiv-positive individuals had significantly higher crp levels (r = 0.140, p = 0.010), indicating persistent inflammation despite art. d-dimer levels remained high within normal ranges across the sample, suggesting a generally low thrombotic risk, though elevated in a small subset of hiv-positive individuals. it also revealed that crp levels were notably higher among male patients (r = 0.133, p = 0.014) compared to female patients. the age group with the highest inflammatory markers, such as crp, were young adults (18–39 years old). distribution results show the predominant gender being female (n = 211; 76.7%) versus male (n = 64; 23.3%). conclusion: this study highlights the need for tailored strategies to manage inflammation and reduce cardiovascular risks in hiv-positive individuals, especially young adults and male patients. what this study adds: this study provides insight into specific inflammatory markers that are reduced or elevated in people living with hiv. it also assessed how art influences the immune response in hiv-positive individuals, particularly in terms of inflammation. this can help in developing targeted therapies and monitoring disease progression. keywords: inflammatory markers; antiretroviral therapy; art; hiv-positive individuals; hiv-negative individuals; comparative analysis; immune response. introduction kwazulu-natal, particularly durban, has the highest hiv prevalence in south africa, with approximately 27.3% of the population living with hiv.1,2 of these, 61.8% are on antiretroviral therapy (art), and 58.6% of art users achieve viral suppression.3 chronic immune activation and inflammation, which are central to hiv, contribute to complications such as neurocognitive disorders, coronary artery disease, liver/kidney issues, metabolic syndrome, osteoporosis, and certain cancers.4 this poses significant concerns for the south african department of health, as hiv is an inflammatory disease.5 inflammation in hiv-positive individuals is indicated by elevated levels of biomarkers such as interleukin-6 (il-6), c-reactive protein (crp), and d-dimer.6 raised crp levels are linked to inflammation and a higher risk of cardiovascular disease,7 while increased il-6 levels are associated with heart disease and cognitive decline.8 elevated d-dimer levels signal enhanced coagulation and inflammation, increasing the risk of cardiovascular events and venous thromboembolism.9 antiretroviral therapy has reduced hiv-related mortality and may lower inflammation, but also introduces other complications.10 a key consideration in managing hiv treatment is therapeutic drug monitoring, which can assess art adherence and drug efficacy.11 therapeutic drug monitoring is usually integrated to track art drug concentrations in patients, and to determine how these levels correlate with inflammatory marker profiles (il-6, crp, d-dimer). this provides insight into whether suboptimal art concentrations contribute to ongoing inflammation or inadequate viral suppression. while inflammatory markers in hiv-positive individuals have been studied, direct comparisons with hiv-negative individuals are limited.12 thus, this study aims to address this gap by comparing il-6, crp, and d-dimer levels in both groups, helping to evaluate the effectiveness of art in normalising inflammation. monitoring these markers is essential for assessing treatment effectiveness and preventing complications, ultimately improving the health outcomes and quality of life for people living with hiv. this study aimed to investigate the distribution of inflammatory markers – crp and d-dimer – and viral load among hiv-positive individuals in durban, kwazulu-natal, and to explore potential correlations between these biomarkers. kwazulu-natal’s high hiv prevalence highlights ongoing challenges in managing hiv, particularly persistent inflammation, which contributes to complications such as cardiovascular diseases and neurocognitive decline. methods ethical considerations ethical approval for this study was granted by the mangosuthu university of technology research ethics committee (reference number: rd5/28/2024). retrospective laboratory data were obtained from the national health laboratory service academic affairs and research management system. patient details were anonymised to ensure confidentiality; no names, identity numbers, or any personal information was disclosed. data were securely stored in a password-protected microsoft excel file, accessible only to the research team. study design and setting this study, conducted between 02 december 2023 and 28 october 2024, used a retrospective cohort design, in which retrospective laboratory results were utilised from an accredited laboratory based at a tertiary hospital in durban, kwazulu-natal. kwazulu-natal is a region with one of the highest hiv prevalence rates in south africa. the study analysed retrospective inflammatory marker laboratory results of people living with hiv and hiv-negative patients tested between january 2020 and december 2023. the data included inflammatory markers such as crp, interleukins, and d-dimer. data collection and processing an excel spreadsheet containing anonymised laboratory records was received. after obtaining the downloaded laboratory data from electronic records, a process of data cleaning and organisation was performed. initially a total of 204 131 results were obtained for the requested period. the data set also included data not related to the study, leading to its removal and exclusion for the current study. after data cleaning, which saw the removal of duplicate patient results, and parameters ineligible for inclusion because of missing values, including interleukins, only 275 records were eligible for the study. of these, 162 were hiv positive and 113 were hiv negative. additionally, a new column was created from an existing variable, namely viral load, using the amputation technique to handle the missing data. the final analysis focused on comparing changes in inflammatory markers, such as crp and d-dimer, with percentages, between hiv-positive individuals on art and hiv-negative individuals. although this study utilised retrospective laboratory results, to the researcher’s knowledge the analysis of d-dimer as an inflammatory marker was conducted using sysmex cs-2100 automated blood coagulation analyser. the cobas procedure for assessing crp involves an immunoturbidimetric assay, a highly sensitive method used to quantify crp levels in blood samples. statistical analysis the data were organised by gender, age, and inflammatory markers. various charts were generated to visually represent the distribution of age, gender, and inflammatory markers such as crp and d-dimer, with percentages in both hiv-positive and hiv-negative patients. descriptive statistics such as mean, standard deviation, and mode, were calculated using microsoft excel. spearman’s rank correlation coefficient was applied to assess the relationship between inflammatory markers and hiv status. statistical analysis was conducted using descriptive tables and spearman’s rank correlation for assessing the relationships between variables. the results of this analysis were then used to evaluate the hypothesis, ‘there is a significant positive correlation between viral load, crp, and d-dimer levels in hiv-positive individuals art compared to hiv-negative individuals’, and determine whether it could be rejected or supported. results demographic distribution a total of 275 patient records were included in the study with 98.2% (n = 270) aged 18–39 years and 1.8% aged < 17 years. the majority of results included were from female patients (n = 211; 76.7%), and 23.3% (n = 64) from male patients (table 1). table 1: demographic distribution of study participants by age and gender in south africa between january 2020 and december 2023. distribution of hiv infection by age group and gender among tested patients a total of 113 (41%) results were hiv negative, while 162 (59%) were hiv positive. among the 275 tested individuals, a higher prevalence of hiv was observed in the 18–39 years age group (158 positive cases; 57%), and among female patients (112 positive cases; 40%). the majority of participants aged < 17 years of age tested negative for hiv, with only four cases (1.5%) testing positive (table 2). table 2: distribution of hiv infection by age group and gender among tested patients in south africa tested between january 2020 and december 2023. distribution of viral load among tested patients by age group and gender table 3 shows the distribution of viral load among tested patients, categorised by age group and gender. it highlights the prevalence of undetectable viral loads, acceptable levels, and the need for monitoring or urgent medical attention across different subgroups. table 3: distribution of viral load among tested patients by age group and gender in south africa tested between january 2020 and december 2023. distribution of normal and high c-reactive protein levels by age group and gender table 4 shows the distribution of normal and elevated crp levels across different age groups and genders. normal range used for children, adult male and female patients is less than 10.0 mg/l. the data reveal that individuals aged < 17 years predominantly had normal crp levels, while the 18–39 years group had a nearly equal distribution of normal and high crp levels. additionally, female patients had a higher proportion of normal crp levels, whereas male patients exhibited a greater incidence of elevated crp levels. table 4: distribution of normal and high c-reactive protein levels by age group and gender in south africa tested between january 2020 and december 2023. distribution of d-dimer levels by age group and gender table 5 shows the distribution of normal and elevated d-dimer levels across age groups and genders. reference normal range used was less than 0.50 mg/l. most participants, regardless of age or gender, exhibited normal d-dimer levels, with only a small proportion showing elevated levels. notably, all individuals in the < 17 years age group had normal levels, and elevated levels were rare among both female and male patients. table 5: correlation among age, viral load, c-reactive protein, and d-dimer levels in south africa tested january 2020 to december 2023. spearman’s rank correlation coefficients for age, viral load, c-reactive protein, and d-dimer levels a weak negative correlation between age and viral load was observed, which was not statistically significant. however, a significant positive correlation was found between viral load and crp (r = 0.140, p = 0.010), suggesting that higher viral loads may be linked to elevated crp levels. additionally, crp and d-dimer levels exhibited a significant negative correlation (r = −0.133, p = 0.014), indicating that higher crp levels might be associated with lower d-dimer levels (table 6). table 6: spearman’s rank correlation coefficients for age, viral load, c-reactive protein, and d-dimer levels (n = 275). south africa, tested january 2020 to december 2023. discussion this study aimed to investigate the distribution of inflammatory markers (including crp and d-dimer), and viral load, among hiv-positive individuals in durban, kwazulu-natal, and to explore potential correlations between these biomarkers. the study highlights that despite being on art, there is a risk of elevated inflammatory markers (notably crp). this suggests a need for better strategies to manage inflammation and reduce health risks.4,5 viral load and inflammation the findings from this study demonstrate that, despite the majority of participants having low viral loads, a significant proportion of hiv-positive individuals still exhibited elevated crp and d-dimer levels, suggesting ongoing inflammation despite adherence to art.13 this observation is consistent with previous research highlighting the association of higher viral loads with increased immune activation and inflammation.6 a noteworthy finding in our study was the positive correlation between viral load and crp levels (r = 0.140, p = 0.010), which aligns with prior research conducted in france (2012)10 and china (2023),7 and that suggests persistent immune activation in hiv-positive individuals, even under art conditions. this finding supports the hypothesis that suboptimal viral suppression may contribute to sustained inflammation, which has been linked to various hiv-associated complications, including cardiovascular diseases and neurocognitive disorders.7,8,14 in contrast, the weak correlation observed between age and viral load, as well as age and crp levels, suggests that other factors, such as art adherence, immune status, and co-morbidities, may play a more prominent role in modulating viral load and inflammation in this cohort.15,16 the lack of a strong association between age and these biomarkers reflects the growing understanding that chronic inflammation in hiv-positive individuals can persist, regardless of age, particularly if viral suppression is not optimal.10,17,18 this finding highlights the importance of considering not only age but also other variables when assessing inflammation and the potential for hiv-related complications. c-reactive protein and d-dimer levels the data from this study provide insights into the relationship between crp, d-dimer, and viral load in hiv-positive individuals. elevated crp levels, indicative of systemic inflammation, were associated with higher viral loads, suggesting a link between hiv replication and immune activation. the negative correlation between crp and d-dimer (r = −0.133, p = 0.014) is intriguing, as it contrasts with the typical positive association between inflammation and coagulation markers, such as d-dimer, in hiv.9 however, such findings are contradictory to findings of other studies, which have found a positive correlation between crp and d-dimer and hiv-positive patients compared to negative controls.19,20,21 this inverse relationship may reflect a more complex inflammatory profile, influenced by factors such as art efficacy, treatment duration, and individual variations in immune and coagulation responses. while art effectively reduces viral load and improves health outcomes, it may not fully resolve inflammation, particularly in cases of suboptimal viral suppression. some studies suggest art reduces crp levels, but its effect on d-dimer remains more variable.10 gender differences our study also revealed notable gender-based differences in inflammatory markers, with female patients in the cohort exhibiting higher levels of normal crp and lower rates of elevated crp compared to male patients. this finding aligns with existing literature, which suggests that women tend to exhibit higher levels of immune activation and inflammatory responses than men.12,22 this gender-based disparity could be because of biological differences in immune function, as well as sociocultural factors that influence healthcare access and art adherence. on the other hand, elevated d-dimer levels were more frequently observed in male patients, which is consistent with findings in a study conducted in italy23 and a systematic review conducted in 2021,9 indicating that hiv-positive men may be at higher risk for thrombosis and other cardiovascular complications compared to women. this could suggest gender-specific risks for cardiovascular and thrombotic events among hiv-positive individuals, which may require tailored interventions to mitigate these risks. implications for antiretroviral therapy and inflammation management the persistence of elevated crp and d-dimer levels despite art use is a critical finding in this study. although art has revolutionised the management of hiv by reducing mortality and improving quality of life, it may not fully address the underlying inflammation that persists, even in individuals with viral suppression.10,24,25 these findings call attention to the need for ongoing monitoring of inflammatory markers, such as crp and d-dimer, as part of routine clinical care for people living with hiv. such monitoring could help identify individuals who are not experiencing adequate viral suppression, or those who may require adjustments to their art regimen. additionally, the results emphasise the potential value of therapeutic drug monitoring in the clinical practice of hiv management in different populations. therapeutic drug monitoring could be used to assess art adherence and its efficacy. recommendations future research should explore the mechanisms driving inflammation in hiv-positive individuals, the role of personalised art regimens in managing persistent inflammation, and the impact of art on long-term health outcomes such as cardiovascular disease and cognitive decline. future researchers should be more age specific and should include the length of arv treatment. by advancing our understanding of the relationship between inflammation, hiv, and art, this research has the potential to guide more targeted interventions aimed at improving the health and quality of life of people living with hiv, particularly in high-prevalence regions such as kwazulu-natal. limitations of the study the study had limitations. the relatively small sample size of 275 participants may limit the generalisability of the findings. a larger sample would provide more robust conclusions, especially when analysing demographic subgroups such as age and gender. the study focused solely on crp and d-dimer as indicators of inflammation, which limits the findings. including other markers, such as il-6 or tumour necrosis factor-alpha, could offer a more comprehensive view of inflammation in hiv-positive individuals. conducting the study in a single location may limit the applicability of the findings to other populations. including different populations in other laboratories in south africa would enhance the external validity of the results. conclusion in summary, this study highlights the complex interplay between viral load, inflammatory markers (crp and d-dimer), and art in hiv-positive individuals. while art effectively suppresses the virus, it does not always normalise inflammation, as evidenced by the elevated crp and d-dimer levels observed in this cohort. these findings suggest that hiv-related inflammation is a persistent issue, even in individuals with viral suppression. given the potential link between chronic inflammation and hiv-related complications, ongoing monitoring of inflammatory biomarkers is essential for improving the management of hiv and reducing the risk of associated comorbidities. acknowledgements the authors would like to express their gratitude to the collaborative partnership to the department of biomedical science at mangosuthu university of technology and the national health laboratory service for enabling and supporting this research. this article is partially based on l.n.’s thesis entitled ‘exploring the inflammatory markers of individuals receiving antiretroviral treatment for human immunodeficiency virus compared to hiv negative individuals’ towards the bachelor of health sciences: medical laboratory science degree in the department of biomedical sciences, mangosuthu university of technology, south africa, on 15 november 2024, with dr z.n. jaya as supervisor and ms n. thembane as co-supervisor. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions l.n. collected the data. z.n.j. and n.t. were involved in conceptualising and supervising the work. l.n. cleaned and analysed the data and wrote the draft manuscript. z.n.j. and n.t. supervised all stages of the research, reviewed the draft manuscript, and provided general supervision and mentorship. sources of support the authors received no financial support for the research, authorship, and/or publication of this article. data availability the authors confirm that the data supporting the findings of this study are available within the article. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. the article does not necessarily reflect the official 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authors: kundai moyo1 carol porter1 ben chilima2 reuben mwenda3 mark kabue1 lutho zungu3 abdoulaye sarr4 affiliations: 1howard university, technical assistance project, lilongwe, malawi 2preventive health services, ministry of health, lilongwe, malawi 3health technical support services (diagnostics), lilongwe, malawi 4senior laboratory advisor, centers for disease control and prevention, lilongwe, malawi correspondence to: kundai moyo email: kundayyi@yahoo.co.uk postal address: po box 31400, lilongwe, malawi dates: received: 09 dec. 2014 accepted: 15 aug. 2015 published: 18 nov. 2015 how to cite this article: moyo k, porter c, chilima b, mwenda r, kabue m, zungu l, et al. use of laboratory test results in patient management by clinicians in malawi. afr j lab med. 2015;4(1), art. #277, 8 pages. http://dx.doi.org/10.4102/ajlm.v4i1.277 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. use of laboratory test results in patient management by clinicians in malawi in this original research... open access • abstract • introduction • methods    • study population    • sampling    • data collection    • data analyses    • ethical considerations • results    • demographic characteristics    • patterns of ordering and use of laboratory tests    • factors associated with use of laboratory tests    • laboratory interaction and desired improvements    • clinician observations    • key informant interviews • discussion    • limitations    • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: malawi has a high burden of infectious disease. the expansion of programmes targeting these diseases requires a strong laboratory infrastructure to support both diagnosis and treatment. objectives: to assess the use of laboratory test results in patient management and to determine the requirements for improving laboratory services. methods: a cross-sectional study was conducted in 2012 to survey practising clinicians. two hospitals were purposively selected for observations of clinicians ordering laboratory tests. twelve management-level key informants were interviewed. descriptive statistics were conducted. results: a total of 242 clinicians were identified and 216 (89%) were interviewed. of these, 189 (87%) reported doubting laboratory test results at some point. clinicians most often doubted the quality of haematology (67%), followed by malaria (53%) and cd4 (22%) test results. a total of 151 (70%) clinicians reported using laboratory tests results in patient management. use of laboratory test results at all times in patient management varied by the type of health facility (p < 0.001). ninety-one percent of clinicians reported that laboratories required infrastructure improvement. during 97 observations of clinicians’ use of laboratory test results, 80 tests were ordered, and 73 (91%) of these were used in patient management. key informants reported that the quality of laboratory services was good and useful, but that services were often unavailable. conclusion: gaps in the public laboratory system were evident. key recommendations to enhance the use of laboratory test results in patient management were to strengthen the supply chain, reduce turn-around times, improve the test menu and improve the laboratory infrastructure. introduction top ↑ malawi has a population of 15 million people1 and the major disease burden is caused by hiv, malaria and tuberculosis. according to the 2010 malawi demographic and health survey,2 hiv prevalence was 11% amongst adults aged 15–49 years. almost six million malaria cases are reported annually and contribute to 40% of hospitalisations in children aged five years or younger and 34% of outpatient cases across all age groups.3 in the malaria indicator survey conducted in 2012, the prevalence of malaria diagnosed by slide microscopy was 28% nationally.3 to address these challenges, malawi set up the essential health package (ehp), which was designed to reach citizens at all levels and was focused on these high burden diseases. the ehp provides free health services at the point of delivery in an effort to ensure the accessibility of healthcare to all, including the poor.4 according to the world health organization (who), ehps are set up to ensure that limited resources are concentrated on interventions that provide the best value to achieve efficiency, equity, political empowerment, accountability and effectiveness.5 malawi's essential medical laboratory services form an integral part of the ehp for providing basic laboratory services. the medical laboratory policy6 states in part that ‘the profile of essential laboratory tests shall be standardized and provided for at each level of care and based on the level of support required for the ehp, public health importance, clinical importance, cost and affordability, suitability to the working environment and the level of expertise’. in order to ensure the effectiveness and efficiency of the ehp, resources are directed toward proper diagnosis and management of diseases through either eradication or reduction in prevalence, whilst at the same time ensuring equitable access to health services. in this article, ‘patient management’ refers to the interaction between clinicians and patients. clinicians often request laboratory tests as part of the decision-making process, expecting the results to provide answers to the condition of a particular patient for proper management.7 according to wians,8 clinicians can request a laboratory test for the following reasons: ruling a disease in or out, monitoring of therapy, screening for congenital diseases and researching the pathology of a disease. a study by sturm9 showed that use of laboratory tests as the basis for prescribing antimicrobial drugs had better patient outcomes compared with basing the diagnostic decision on clinical presentation only. however, some clinicians often do not use laboratory tests. in an observational study conducted at ntcheu district hospital in malawi, laboratory tests were requested for only 68% of cases that required them and only 73% – 79% of the laboratory results were used appropriately. this means that laboratory tests are not requested for all cases requiring one, and, for tests that are requested, not all influence patient management.10 in kenya, a study was conducted to investigate reasons why clinicians failed to use laboratory test results in patient management. the most common reasons were lack of time for clinicians to order tests, lack of trust in the test results and long turn-around times for receiving tests results.11 a similar study conducted in ghana identified the following potential barriers for using laboratory test results: limited availability of laboratory services, lack of quality of testing, challenges in the delivery of results to clinicians, lack of time for clinicians to order tests, and negative attitudes and behaviours toward laboratory services.12 it is important that healthcare providers ensure optimal use of resources for the benefit of their clients. therefore, determining and addressing the factors that deter clinicians from using laboratory tests for patient management will ensure that laboratory services are improved to meet clinicians’ expectations and enable clinicians to use laboratory tests in patient management more effectively. the malawi ministry of health (moh) and its partners are committed to strengthening laboratory services through a five-year national laboratory strategy. coverage and utilisation of laboratory services in patient management has not been assessed in malawi on a large scale. this study investigates the factors associated with the use of laboratory test results by clinicians working in public health facilities in malawi. the study also aimed to determine the requirements for improving laboratory services to enhance usage of laboratory test results in patient management. methods top ↑ study population this cross-sectional study collected primary data from practising clinicians across all 75 of malawi's public hospitals with laboratory facilities from october to november 2012. a clinician was defined as a medical doctor, clinical officer, or medical assistant who was authorised to request laboratory tests. the key informants targeted in this survey were moh programme managers from the departments of clinical services, nursing, hiv and aids, as well as representatives from moh partners (clinton health access initiative [chai], the christian health association of malawi [cham] and the laboratory capacity consortium [lcc]). sampling all four of malawi's central hospitals were included in the study. in each region, the district or cham hospitals were selected by stratified systematic sampling. all of the district hospitals in the region were listed in alphabetical order and 30% of the total number of hospitals with laboratories were systematically selected. this was repeated for the cham facilities. fifty clinicians from each of the central hospitals and 10 from each of the district and cham facilities were asked to participate. the following 22 sites were selected: all central hospitals from each of the three regions (n = 4). 30% of district hospitals in each region (n = 9). 30% of cham hospitals in each region (n = 9). a list of key informants was purposively compiled from laboratory managers, laboratory partners and programme managers from four moh departments (hiv, tuberculosis, malaria and clinical services). all key informants were asked to participate in the interviews by providing their perceptions of laboratory services and the utilisation of laboratory tests in public hospitals. the results of this survey were used to validate the findings of the clinician survey. data collection data were collected from clinicians using a questionnaire, which was administered through face-to-face interviews by trained research assistants. the questionnaire solicited demographic information (age, facility, qualifications and years of experience) and information on factors that influence a clinician's decision to request and use laboratory test results. the study focused on the following tests: cd4, glucose, haemoglobin (hb) or full blood count (fbc), malaria microscopy, tuberculosis microscopy, bacteriology microscopy, culture and sensitivity. an interview guide that contained closedand open-ended questions was used during the key informant interviews. observations of clinicians’ use of laboratory services were conducted at malamulo mission and mzimba district hospitals. a data collection form adapted from mepham et al.10 was used. without interfering with routine decision-making processes, an independent researcher, who was a qualified practising clinician recruited as a research assistant, observed clinicians conducting consultations with patients and healthcare workers for at least one week in the wards and outpatient departments at the two facilities. for each test ordered, the clinician-researcher noted whether the correct laboratory tests were requested; whether the appropriate specimens were collected and processed by the laboratory; and whether the test result(s) were used in patient management. data analyses themes were identified for qualitative data and analysed initially using descriptive statistics. the chi-square test was conducted to determine factors associated with use of laboratory tests. p-values less than 0.05 were considered to be statistically significant. all analyses were conducted using stata™ version 11.0 (statacorp, college station, texas, united states). ethical considerations ethical clearance for this study was granted by the national health sciences research committee (nhsrc) of malawi in june 2011 and the us centers for disease control and prevention, center for global health, office of the associate director for science/laboratory science in august 2012 as an activity not including human subjects research. after the purpose of the study, procedures, risks and benefits and participants’ rights were explained, all participants were asked for written consent before a questionnaire was administered. oral consent to observe consultations was obtained from both the clinician and patient for the clinic observations. names of participants and other personal identifiers were not documented; unlinked and anonymous codes were used on the questionnaires. results top ↑ a total of 242 potentially eligible clinicians were targeted for interviews based on the most current list of practising clinicians at the 22 health facilities selected in 2012. of the 242 clinicians targeted, 216 (89%) were interviewed, 21 (9%) refused to participate and five (2%) were unavailable. demographic characteristics table 1 summarises the demographic characteristics of the clinicians who participated. men comprised 167 of the 216 respondents (77%). the mean age of respondents was 32 years and the modal age category was the 25–29 age-group, comprising 68 (31%) respondents. the median years of service as a clinician was four (interquartile range: 2–8). the distribution of the type of institutions where the respondents were working was as follows: central/referral hospitals (n = 100; 46%), district hospitals (n = 88; 41%) and cham hospitals (n = 28; 13%). a majority of respondents were clinical officers (n = 132; 61%), 21% (n = 45) were medical assistants, 10% (n = 21) were medical doctors and 8% (n = 17) were in other categories that included nurses and specialist clinicians. the majority (n = 202; 94%) of the clinicians were trained in malawi, and about one-third (n = 76; 36%) had been in the work-force for less than three years. table 1: characteristics of clinicians in malawi reporting use of laboratory test results, october–november 2012. patterns of ordering and use of laboratory tests table 2 describes clinicians’ ordering and use of laboratory results and their levels of confidence in the results. a majority (n = 151; 70%) of respondents indicated that all tests ordered were used for patient management, whilst 30% (n = 64) indicated that tests results were used sometimes. most of the 216 respondents (n = 189; 87%) reported the occasional questioning of laboratory results for any specific reason and only a small proportion (n = 23; 11%) had never questioned laboratory results. respondents indicated that they would request a repeat test if they doubted the initial laboratory results. the results of hb/fbc tests were the most commonly doubted, with 67% (n = 140) of clinicians reporting they would request a repeat test. approximately half requested repeat malaria testing (n = 110; 53%), whilst the cd4 count test had the least frequency (n = 41; 22%) of repeat requests. these findings were further supported by the level of confidence clinicians reported in certain test results; clinicians had the most confidence in glucose test results (n = 105; 52%) and the least confidence in bacteriology (n = 51; 30%) test results. table 2: ordering and use of laboratory results by clinicians in malawi, october–november 2012. table 3 summarises the reasons reported by clinicians for requesting repeat tests. the main reason for ordering a repeat test was because the clinical presentation was not consistent with the laboratory test result. other reasons reported included: doubting laboratory expertise, especially for cd4 count results (n = 9; 22%) and malaria results (n = 20; 18%). glucose (n = 9; 24%) and tuberculosis microscopy (n = 12; 23%) tests had the highest proportion of clinicians who indicated that they requested repeat tests just to check the quality of the laboratory results. table 3: reasons given by clinicians in malawi for repeating tests, october-november 2012. factors associated with use of laboratory tests table 4 summarises the clinicians’ responses to questions about factors that influenced use of laboratory tests and situations in which they did not use results from the six laboratory tests investigated. reasons varied by the type of test. the most common reasons across the tests were unavailability of reagents and the perception that history taking was more important than laboratory tests. table 4: scenarios in which clinicians in malawi did not use laboratory test results, october-november 2012. table 5 summarises factors associated with use of laboratory test results. a higher proportion of clinicians working in central hospitals responded that they used laboratory test results all of the time compared with clinicians working in cham or district hospitals. medical doctors were more likely to use laboratory test results all of the time. in addition, clinicians who never queried laboratory results, clinicians who interacted with the laboratory some of the time and clinicians who noted gaps in infrastructure were more likely to use the laboratory results all of the time. the type of facility that clinicians worked in was the only factor significantly associated with using laboratory test results all of the time (p< 0.001). table 5: factors associated with use of laboratory results by clinicians in malawi, october-november 2012. laboratory interaction and desired improvements a total of 201 (93%) clinicians reported interacting with laboratory staff either all of the time (n = 135; 63%) or some of the time (n = 66; 30%) (table 6). reasons for interacting with laboratory staff varied and included a combination of clinic meetings, consultation visits and being friends with laboratory personnel (data not shown). table 6: laboratory interactions and desired improvements, in malawi, october-november 2012. a total of 190 clinicians (91%) reported that the laboratories at their facilities required infrastructural improvements. specifically, 53% (n = 139) indicated that additional equipment was required and 35% (n = 93) said that the laboratory in their health facility should be expanded. the most frequently mentioned area of needed improvement was shorter turn-around times for test results (n = 63; 22%), followed by the need for consistent availability of reagents and supplies (n = 55; 19%) to ensure that laboratory services were uninterrupted. a small proportion indicated the need for laboratories to offer tests in addition to those currently available (n = 47; 16%). whereas some clinicians indicated need for additional laboratory personnel (n = 41; 14%), only 4% (n = 13) indicated the need for qualified, skilled or specialist laboratory personnel. clinician observations a total of 97 clinician observations were completed, during which 80 (83%) tests were ordered, mainly for patients in the medical, outpatient department and paediatric wards (table 7). all ordered tests were returned to the original wards. the final diagnosis and patient management were compared to the laboratory test results and 73 (91%) were noted as having been used by the clinicians in making a diagnosis or planning patient management. all laboratory tests ordered in the paediatric and surgical wards were used by the clinicians in making a diagnosis or planning patient management, whereas clinicians in other wards did not use all results. table 7: clinician observations, malawi, october-november 2012. key informant interviews twelve key informants were interviewed (table 8). the key informants reported that laboratory services were very useful and that the quality was good, but services were often unavailable and hence underutilised. key recommendations were to strengthen laboratory services, supply chain and equipment maintenance services to ensure uninterrupted services. table 8: key informants’ emerging themes, malawi, october-november 2012.† discussion top ↑ in this study, it was observed that 9 in 10 laboratory tests were being used to inform patient management decisions and that 7 in 10 clinicians reported always using the results of requested laboratory tests in patient management. this is higher than previously reported in malawi by mepham et al. in 2002, where 64% of clinicians were using laboratory results to inform clinical decisions.10 one possible explanation for the differences observed in these two studies might be attributed to laboratory strengthening efforts by malawi's moh and partners in increasing human resources and building a strong laboratory infrastructure over the past decade. it is also noteworthy that the previous study was conducted at a single health facility, whereas the current study included 22 health facilities and 216 clinicians across the country, thus increasing its representativeness. in this study, we found that the vast majority of clinicians often questioned laboratory results, which led them to request repeat tests. malaria and hb/fbc tests were repeated most often; the main reason for requesting repeat tests was that the clinical presentation was not consistent with the laboratory results. a study in ghana12 had similar findings on the high reliance of clinical symptoms in diagnosing malaria and found that clinicians’ perception of laboratory services was the main factor influencing use of laboratory test results. in another study,13 malaria and hb tests had the least accurate results amongst surveyed laboratories. the observations in malawi could be attributed to use of non-laboratory personnel to perform these quantitative and semi-quantitative tests in point-of-care settings, although further investigation would be required to establish such an association. clinicians provided scenarios in which laboratory tests were ordered but results not used in diagnosis and patient management. the main theme from the scenarios demonstrates the importance that clinicians place on obtaining a detailed patient history instead of making clinical decisions primarily based on laboratory results. the malawi standard treatment guidelines 2009 states that ‘[g]ood therapeutics depends on accurate diagnosis, based on thorough history-taking, necessary careful physical examination and, if required, supporting laboratory testing’.14 these treatment guidelines allow the clinicians to determine whether laboratory testing is required. this finding is similar to petti et al.'s15 observation that the inadequacy of laboratory capacity to perform certain tests and assure the quality of those tests promotes the perception amongst clinicians that laboratory tests are not necessary for patient management. another emerging theme was the continuous unavailability of reagents, forcing clinicians to use clinical management instead of laboratory test results, even after reagents are available. a laboratory assessment conducted in 2010 across the 42 laboratories in malawi showed that whilst central hospitals had 100% availability of reagents and consumables, district hospitals had 83% availability of reagents for hiv rapid tests, 71% for cd4 tests and 54% for haematology tests.16 cham facilities had 93% availability of reagents and consumables for rapid hiv tests, 43% for cd4 tests and 86% for haematology tests. the supply chain systems in resource-limited settings face challenges in forecasting, procurement, distribution and inventory management. in order for clinicians to use laboratory tests, these services need to be available consistently. clinicians who used laboratory test results all the time were more likely to work in central hospitals, be medical doctors by profession, never question laboratory test results, interact with laboratory staff some of the time, and not report the need for infrastructure improvement. however, working in a central hospital was the only factor significantly associated with the use of laboratory test results (p< 0.001). a national laboratory assessment conducted in 2010,16 found that central hospitals were well-stocked with haematology, hiv and cd4 count test reagents and consumables compared with other health facilities. thus, consistent availability of reagents may be an influencing factor in the use of laboratory results by clinicians. another finding from this study was the frequent interaction of clinicians and laboratory staff, suggesting that there is a good working relationship between the two cadres. a high proportion of clinicians reported interacting with laboratory staff on a daily basis, with only 3 in 10 clinicians interacting some of the time. this high level of interaction provides an opportunity for improvements in service delivery to meet clinicians’ expectations and increase the likelihood that they will use laboratory test results in patient management. these interactions can also provide an avenue for clinicians and laboratory staff to work together to improve the laboratory-clinic interface at the time when health systems strengthening is a priority to the malawi government and partners. clinicians in our study suggested infrastructural and service improvements in the laboratories at their health facilities. additional equipment and expansion of laboratories were the most common suggestions for infrastructural improvements. the close interaction between clinicians and laboratory staff might mean that they were aware of the challenges their laboratories faced. the earlier laboratory assessment17 showed that only central hospitals offered laboratory tests according to a tiered test menu. clinicians may have suggested that the need for additional equipment would address the test menu gap. in addition, turn-around time, availability of reagents and additional tests were suggested areas of needed improvement at laboratories in their health facilities. these suggestions are similar to the findings of petti et al.15 that suggest barriers to effective use of laboratories in healthcare in africa are related to the lack of laboratory consumables, essential equipment and logistical support. availability of laboratory results in a shorter period would allow clinicians to make more comprehensive treatment decisions. unavailability or delays in receiving laboratory results has the potential to prompt clinicians to use clinical diagnosis only versus in combination with laboratory diagnosis. the clinician consultation observations showed that a majority of clinicians requested laboratory tests, although not all tests were used for decision making in patient management. these findings were corroborated by the clinician interviews, which indicated that whilst there are challenges in requesting laboratory tests, there are also challenges in using the results. reasons for not using laboratory test results could be attributed to varying interpretations of malawi's treatment guidelines, trust in laboratory quality, availability of reagents, or a laboratory's robustness in providing results within an acceptable timeframe. the surveyed key informants’ top emerging themes were to strengthen laboratories to expand the test menu, supply chain and quality systems. the key informants’ suggestions validated the survey findings that the availability of the test menu at the health facility level influenced clinicians to use the laboratory tests. availability of reagents would ensure consistent availability of laboratory services for clinicians. strengthening of the laboratory quality systems would build confidence for clinicians to use the results of all laboratory tests ordered. availability of reagents as a factor associated with the use of laboratory test results in patient management would seem to be beyond the scope of possible interventions at the health facility level, because reagent procurement is done centrally. however, other factors, such as confidence in the test and need for better infrastructure are within the scope of interventions that could be undertaken at the health facility level. considering that the majority of the clinicians who participated in this survey were stationed in central or referral hospitals where laboratories offered an expanded test menu, these findings provide a fairly accurate picture of the use of laboratory test results in situations where the required tests are offered. it would be a reasonable strategy to include the value of laboratory investigations in clinicians’ pre-service training and reinforce this at the health facility level through routine advocacy by the laboratory staff. a high proportion of clinicians suggested the need for expansion, additional human resources and expertise, as well as for additional equipment. in malawi, a national assessment of laboratory services conducted in 201016 showed that district-level laboratories had a lower proportion of qualified staff compared with central hospital laboratories (32% and 46%, respectively). a third of the district laboratories had adequate space, although microbiology and biochemistry equipment were not available in most district laboratories.16 these might be some of the reasons clinicians suggested the improvements they did. these gaps are currently being addressed through the national laboratory strategic plan (2010–2014)17 and the who accreditation process, strengthening laboratory management toward accreditation (slmta).18 as clinicians increase utilisation of the laboratory results, expectations will continue to rise in terms expanded test menus, shorter turn-around times, good quality and interpretation of results.19 in view of this, it will be necessary for laboratories in malawi to continuously re-evaluate ways of improving the laboratory-clinic interface in order to ensure test results get to the patients efficiently and laboratory services are used more frequently in the management of patient care and treatment. limitations one limitation of this study is that the clinician consultation observations were conducted at only two hospitals. further, the presence of an observer might have influenced clinicians’ behaviour with regard to laboratory test requests and use of test results. the use of a single observer per site might have introduced some degree of subjectivity. in addition, interviews relied on self-reported information and are thus subject to bias. clinician responses may not have been an accurate representation of laboratory test request and result usage habits, as they might have been biased towards what they know they were supposed to do as opposed to what they actually do. conclusion this study highlights the patterns and possible factors and barriers related to the use of laboratory test results by clinicians in public health facilities in malawi. it also highlights the challenges inherent in the laboratory-clinic interface in malawi and points to opportunities for improvement. limited test menus, longer turn-around times, poor quality, low confidence and inconsistent availability of reagents were the major gaps that were identified in the public laboratory tier system in malawi from the clinicians’ perspective. these gaps can deter utilisation of laboratory services by clinicians in public health facilities in malawi. a lesson learnt from this study is the need for continuous solicitation of feedback from users of laboratory services in order to improve these services. the key recommendations from this study are to strengthen the laboratory infrastructure, supply chain and quality management systems in order to restore confidence and increase utilisation of laboratory tests in patient management. a follow-up study would provide a platform for evaluating malawi's national laboratory strategic plan and impact on the use of laboratory tests by clinicians. acknowledgements top ↑ we are grateful to the leadership and staff at the 22 hospitals selected across the country, laboratory partners in malawi (laboratory capacity consortium, clinton health access initiative) and the following departments of the ministry of health: clinical services, malaria and tuberculosis programmes. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions k.m., c.p. and m.k. (howard university) were responsible for conception, design, proposal writing, data analysis, interpretation and manuscript writing. b.c. (ministry of health, malawi) and r.m. (health technical support services) were responsible for proposal and article approval. b.c., r.m., l.z. (health technical support services) and a.s. (centers for disease control and prevention, malawi) were responsible for critically reviewing the data collection tools and final approval of the published version. references top ↑ the world bank. malawi [page on the internet]. n.d. [cited 2013 jan 22]. available from: http://data.worldbank.org/country/malawi national statistical office (nso) [malawi] & icf macro. malawi demographic and health survey 2010. calverton, maryland: nso & icf macro; 2011. national malaria control programme (nmcp) [malawi] and icf international. malawi: malaria indicator survey (mis) 2012. lilongwe, malawi and calverton, maryland, usa: nmcp and icf international; 2012. lawson m, mazengera s, nkhoma-mbawa f, et al. malawi essential health services campaign. for all campaign: country case study. oxford, uk: oxfam international; 2008. world health organization. essential health packages: what are they for? what do they change? who service delivery seminar series. draft technical brief no. 2 [document on the internet]. c2008 [cited 2010 oct 21]. available from: http://www.who.int/healthsystems/topics/delivery/technical_brief_ehp.pdf ministry of health. malawi medical laboratory policy. lilongwe: moh; 2007. price cp. evidence-based laboratory medicine: supporting decision-making. clin chem. 2000;46(8):1041–1050. wians fh. clinical laboratory tests: which, why, and what do the results mean? labmedicine. 2009;40(2):105–113. sturm aw. rational use of antimicrobial agents and diagnostic microbiology facilities. j antimicrob chemother. 1988;22(2):257–260. http://dx.doi.org/10.1093/jac/22.2.257 mepham so, squire sb, chisuwo l, et al. utilisation of laboratory services by health workers in a district hospital in malawi. j clin pathol. 2009;62(10):935–938. http://dx.doi.org/10.1136/jcp.2009.069062 mengo, d. evidence based laboratory medicine: do doctors use laboratory results prior treatment of patients? poster session presented at: 29th world congress of biomedical laboratory science, june 6–10, nairobi, kenya; 2010. polage cr, bedu-addo g, owusu-ofori a, et al. laboratory use in ghana: physician perception and practice. am j trop med hyg. 2006;75(3):526–531. bates i, bekoe v & asamoa-adu a. improving the accuracy of malaria-related laboratory tests in ghana. 2004;3:38. ministry of health. malawi standard treatment guidelines. 4th edition. malawi: ministry of health; 2009. petti ca, polage cr, quinn tc, et al. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006;42(3):377–382. http://dx.doi.org/10.1086/499363 ministry of health. national laboratory assessment: baseline report. malawi: ministry of health; 2010 (unpublished report). ministry of health. 5-year national laboratory strategic plan. malawi: ministry of health; 2010 gershy-damet gm, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. http://dx.doi.org/10.1309/ajcptuuc2v1wjqbm dubois c, swartz v. a vision for change: the expanding role of clinical diagnosis is evident. information technology series [page on the internet]. c2011 [cited 2011 nov 12]. available from: http://laboratory-manager.advancedweb.com/archives/article-archives/a-vision-for-change.aspx article information authors: coosje j. tuijn1 elizabeth msoka2 declare l. mushi3 marion sumari-de boer2 jaffu chilongola2,3 ankie van den broek4 affiliations: 1royal tropical institute (kit) biomedical research, amsterdam, the netherlands2kilimanjaro clinical research institute (kcri), moshi, tanzania 3kilimanjaro christian medical university college, tumaini university makumira, moshi, tanzania 4royal tropical institute (kit) health, amsterdam, the netherlands correspondence to: coosje tuijn postal address: médecins sans frontières plantage middenlaan 14, 1018 dd amsterdam, the netherlands dates: received: 10 july 2013 accepted: 25 feb. 2014 published: 23 july 2014 how to cite this article: tuijn cj, msoka e, mushi dl, sumari-de boer m, chilongola j, van den broek a. the interface between clinicians and laboratory staff: a field study in northern tanzania. afr j lab med. 2014;3(1), art. #126, 7 pages. http://dx.doi.org/10.4102/ ajlm.v3i1.126 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the interface between clinicians and laboratory staff: a field study in northern tanzania in this original research... open access • abstract • introduction • research method and design    • study design    • study population    • sample size, sampling procedures and data collection    • data analysis • ethical considerations • trustworthiness • results    • participants    • factors influencing the interface       • organisational factors    • personal factors • discussion • limitations of this study • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: strengthening the communication and professional relationships between clinicians and laboratory workers is essential in order to positively change clinicians’ attitudes about the reliability of diagnostic tests, enhancing the use of laboratory diagnostics and, ultimately, improving patient care. we developed an analytical framework to gain insight into the factors that influence communication amongst health professionals. objective: to explore whether the interaction between clinicians and laboratory workers influences the use of laboratory test results in clinical decision making. methods: four health facilities in northern tanzania were selected using convenience sampling, whereas study participants were selected using purposive sampling. the quantitative and qualitative data collection methods included self-administered questionnaires; semi-structured, individual interviews; in-depth, individual interviews; and/or focus group discussions with clinicians and laboratory workers. thematic content analyses were performed on qualitative data based on the framework. descriptive statistical analyses of quantitative data were conducted using microsoft excel. results: contact between clinicians and laboratory professionals is seldom institutionalised and collaboration is rare. the clinicians believe collaboration with laboratory staff is a challenge because of the gap in education levels. laboratory workers’ education levels are often lower than their positions require, leading to clinicians’ lack of respect for and confidence in laboratory professionals, which compromises the laboratory staff’s motivation. conclusions: hospital managers, clinicians and laboratory workers need to recognise the critical and complementary roles each professional plays and the importance of addressing the gap between them. field application of the framework proved successful, justifying the expansion of this study to a larger geographical area to include additional healthcare institutions. introduction top ↑ medical laboratories play a significant role in the diagnosis, monitoring and treatment of diseases; yet the efficacy of the information they provide may be questioned because of several factors, including the capacity of the laboratory workforce, the laboratory infrastructure and the availability of equipment and materials, especially in low-income countries. whilst improving the quality of laboratories is a solution, it does not always result in proper execution of tests.1,2,3 other obstacles that must also be considered are the cultural beliefs of the patients, attrition of healthcare workers, physicians’ attitudes and inadequate supplies of consumables.4medical laboratory services offer essential information for diagnoses and/or treatment plans. the communication and interactions between laboratory and clinical health workers can influence physicians’ request behaviour and treatment interventions. previous studies have shown that lack of communication is a barrier to effective healthcare.5,6,7,8,9improved communication between clinicians and laboratory workers is essential to changing clinicians’ attitudes about the reliability of diagnostic tests, possibly leading to increased use of laboratory diagnostics and, ultimately, improving patient care.5 this interface between clinicians and laboratory health workers is complex; the two groups may communicate face-to-face or by request and result forms, phone calls, text messages, e-mails or computerised forms. the factors that influence the mode of communication and shape the relationship between these two professional groups require further exploration. for this reason we constructed an analytical framework based on existing literature.10,11,12,13,14,15after further literature searches, analysis of guidelines for laboratories and discussion with experts, a conceptual model was developed.16 the model addresses the phases where clinicians and laboratory workers interact; the organisational and personal factors affecting their interface; and the socio-political, economic and cultural environment within which the health facility operates. the objective of this study was to demonstrate and test the analytical framework and to gain insight into the relationship between clinicians and laboratory workers and into the factors that influence their interface, with the intention of later scaling up the study using a calculated sample size. the analytical framework includes three phases of communication (pre-analytical, analytical, post-analytical) during which clinicians and laboratory workers interact (tables 1a and 1b).15 the testing process starts with a clinician ordering a test and sample collection, known as the pre-analytical phase. during the analytical phase, the sample is processed and analysed by laboratory staff. the post-analytical phase includes transfer of results from the laboratory back to the clinician. each phase consists of organisational factors, subdivided into ‘identity’ and ‘management’, as well as personal factors, subdivided into ‘individual’ and ‘professional’. the primary aim of the study was to explore whether the interaction between clinicians and laboratory workers influences the use of laboratory test results in clinical decision making. by means of the framework quantitative and qualitative tools were designed. the results of this study provide information on the importance of the interface between clinicians and laboratory workers and may form a basis for larger studies in the future. the implications of our findings are useful for health institutions in any country. table 1a: analytical framework. this framework was developed to test our conceptual model,16 and displays the organisational and personal factors playing a role during the three phases where clinicians and laboratory workers interact: pre-analytical, analytical and post-analytical. table 1b: analytical framework. this framework was developed to test our conceptual model,16 and displays the three phases where clinicians and laboratory workers interact: pre-analytical, analytical and post-analytical. each phase consists of organisational and personal factors (table 1a). research method and design top ↑ study design this was an exploratory study, employing both quantitative and qualitative methods. its purpose was to use tools to test the analytical framework and to better understand the factors that influence the interaction between clinical and laboratory workers. most participants took part in a focus group discussion (fgd) immediately following completion of an anonymous, self-administered questionnaire (saq). if there were fewer than three participants for a fgd, in-depth, individual interviews were conducted. semi-structured, individual interviews were used for hospital directors and heads of departments. fgds and in-depth, individual interviews followed the same format and covered the same topics. the assessment of the data collection tools was done at a private, not-for-profit, faith-based, district hospital, where a group of clinicians and laboratory staff were invited to assess the tools. study population the study population included hospital directors, heads of clinical and laboratory departments, clinicians and laboratory staff. staff came from three categories of health facilities: private, government; faith-based not-for-profit; and private for-profit. four hospitals participated in the study: a non-government referral hospital with 450 beds; a private not-for-profit hospital with 150 beds; a government regional hospital with 300 beds; and a private for-profit health centre with 50 beds. sample size, sampling procedures and data collection study sites were selected using convenience sampling, whereas study participants were selected using purposive sampling. as this was a pilot study, we did not determine a sample size. we contacted the sites and received verbal consent from the hospital directors for their participation. the interviewed staff members all signed written informed consent forms. initially, a total of 48 staff members were asked to participate, including six clinicians and six laboratory workers from each of the four sites. however, at the time the study was conducted, only 35 staff members were present: 18 clinicians and 17 laboratory personnel. amongst the latter were laboratory assistants and attendants who often perform the routine tests, namely, those who interacted most with clinicians (tables 2a and 2b). saqs were used to collect data from clinicians and laboratory staff at each hospital. after filling in the saqs, staff members participated in either an fgd or an in-depth, individual interview, allowing for the opportunity to elaborate on the saqs and further share their views. interviews were carried out with hospital directors and heads of departments. all interviews and fgds supplemented the saqs and were conducted in kiswahili for laboratory staff and in english for clinicians. interviews were recorded via tape recorder or note taking. as outlined in the study protocol, fgds involved six to 12 clinicians and three to nine laboratory staff members. these numbers were predetermined and agreed upon by the study team. data analysis data were collected within a period of five working days at the end of november 2011 and analysis was carried out throughout 2012. the individual interviews and fgds were transcribed, translated and analysed by a social scientist and a research nurse. qualitative data were analysed independently and manually, using a thematic framework approach involving data familiarisation, coding and development and categorisation of themes. coding of collected qualitative data was driven by the developed framework (tables 1a and 1b), whereby common words were sorted together, following an inductive method of code-creation. once a theme was identified and reviewed, categorisation and corresponding codes were developed to sort and organise the data. after reading through the data, the two independent researchers discussed the codes and themes until they agreed on each one. quotations were used to support and clarify the information provided using an editing analysis style. descriptive statistical analysis of quantitative data from the structured questionnaires were carried out using microsoft excel. ethical considerations top ↑ ethical clearance was obtained from the kilimanjaro christian medical university college research and the ethical review committee of tumaini university, makumira (research ethical clearance certificate number 448 from research proposal 467). verbal and written consent for the saqs, interviews and fgds was provided by study participants. confidentiality was assured at all stages of this study, through the use of coding for sites and participants. no names or data can be traced back to individual participants. trustworthiness top ↑ the results of this study are based on actual findings as described in the research method and design section. qualitative research (fgds and in-depth interviews) were complementary to results obtained from the saq. the experimental design of this exploratory study is reliable and valid and the procedures of qualitative and quantitative research used in this study are according to standardised methods, as laid out by varkevisser, pathmanathan and brownlee.17 results top ↑ participants pre-testing of the data collection tools, firstly, through discussions with clinical and laboratory staff at a referral hospital and secondly, by group discussions at a district hospital, allowed researchers to strengthen and modify the data collection methods. in total, 35 questionnaires were administered to 18 clinicians and 17 laboratory staff members, an estimated one-third of the official staff number, according to the heads of departments. factors influencing the interface organisational factors management factors: the analysis of the saqs showed that 11 of the 18 clinicians (61.1%) and eight of the 17 laboratory staff (47.1%) were aware of the availability of rules and guidelines for requesting tests and reporting results. of those remaining, one clinician (5.5%) and nine laboratory staff (52.9%) said there were no guidelines, whilst six clinicians (33.3%) did not know whether or not guidelines existed. these findings were also evident in the fgds. those who were aware that there are guidelines in place noted that there is little time to adhere to them because of staffing shortages and an insufficient supply of reagents. other factors that the study group cited as impacting on communication in relation to management of the organisation included the clinicians’ doubts about laboratory test results and uncertainty as to whether standard operational procedures are followed as well as the awareness of the persons to whom clinicians and laboratory staff report (figure 1 and figure 2). furthermore, the lack of competent and highly-educated laboratory staff was cited by clinicians as being a barrier to effective communication; in many health facilities, only laboratory attendants and assistants are present to perform tests and they sometimes lack the communication skills of a more highly-educated laboratory technician. in fgds with laboratory workers, it was noted that clinicians do not always use the test results with which they are provided. patients sometimes ask clinicians to prescribe treatment straight away as the waiting time to be tested in the laboratory can be very long. clinicians sometimes agree to this request in order to save time. laboratory staff also mentioned that nurses and sometimes patients play a role in the contact between the clinicians and laboratory staff. in some health facilities, nurses collect the sample request from the wards and are responsible for transfer of results from the laboratory back to the clinician. this supports the findings of the quantitative data analysis. figure 1: factors related to the management of the organisation, response of clinicians. figure 2: factors related to the management of the organisation, response of laboratory staff. identity: ten (55.5%) of the 18 clinicians and nine (52.9%) of the 17 laboratory workers involved in the survey worked in a referral hospital. others worked in a faith-based, public or private, for-profit facility or in a general laboratory or health facility. personal factors within the personal factors, individual and professional subfactors (qualitative data: quotes) were supportive of and in agreement with the quantitative data. individual factors: the personal factors of the clinicians (table 2a) and laboratory staff (table 2b) investigated in this study were ethnicity, religion, age and professional qualifications. data are displayed by gender, position in the organisation and the last time a course or workshop was attended. table 2a: the demographic distribution related to the personal identities of the clinicians participating in this field study. table 2b: the demographic distribution related to the personal identities of the laboratory workers participating in this study. professional factors: laboratory staff members indicated that clinicians regularly devalue their services. whilst seven of the 17 laboratory staff members (41.1%) believed that clinicians understand what laboratory workers do and six (35.3%) believed that clinicians know how to interpret the results when making clinical decisions, three (17.6%) noted that clinicians do not wait for laboratory test results before starting treatment and one (5.8%) noted that test ordering is not always specific. furthermore, in the fgds, nearly all of the laboratory staff members from public and faith-based organisations expressed that they lack recognition from clinicians, a sentiment also expressed by staff from the private health facility as being a contributing factor for their lack of motivation. further playing a part in the complicated relationship between clinicians and laboratory staff is the perceived frequency of use of test results for clinical decision making. only six clinicians (33.3%) and three laboratory workers (17.6%) claimed that test results are often used in clinical decision making. one laboratory staff member (5.8%) believed that clinicians never use the test results (figure 3 and figure 4). figure 3: professional factors. the perceptions of clinicians regarding the frequency with which test results are used for making clinical decisions. figure 4: professional factors. the perceptions of laboratory workers regarding the frequency with which test results are used for making clinical decisions. six of the 18 clinicians (33.3%) do not always trust the laboratory results; 10 (55.5%) mentioned that the waiting time for test results is too long; and eight (44.4%) were not satisfied with the type of tests that can be performed and also believed that the reporting of test results is not done properly. four clinicians (22.2%) believed that the quality of laboratory services was weak or substandard. several times during the fgds, laboratory workers noted that lack of equipment contributes to poor quality output. laboratory staff in the private hospitals pointed out the issue of lack of reagents and mentioned that expired reagents may be in use, further compromising clinicians’ confidence in the laboratory test results. in spite of the complicated relationship between clinicians and laboratory workers, a majority in both groups must interact on a daily basis (figure 5 and figure 6). yet, when grievances arise, the two groups use different avenues to address them. when laboratory staff members have problems with clinicians, they often discuss them within their own professional group; however, when a clinician has a complaint, he or she will often approach the individual laboratory worker or laboratory manager. whilst issues like these may be discussed broadly in staff meetings at most hospitals, the majority of those surveyed at the private not-for-profit hospital pointed out that the department was so small that organising meetings to discuss problems seemed unnecessary. figure 5: the frequency of professional contact between clinicians and laboratory workers. eight of 16 clinicians reported having daily interactions with laboratory workers. figure 6: the frequency of professional contact between clinicians and laboratory workers. eleven of 14 laboratory workers reported having daily professional contact with clinicians. poor reporting was identified as being a factor that contributed to inadequate communication between clinicians and laboratory staff. in some cases, either the handwriting was misinterpreted, or test requests or test results were incomplete. in addition, it was noted that communication only occurs between the groups when the need arises. whilst most staff members noted that communication and positive interactions between laboratory workers and clinicians are crucial, there is no managerial support, formalised system or motivation to maintain regular meetings or contact between clinicians and laboratory staff. discussion top ↑ the main objectives of this study were to test the analytical framework; to gain a better understanding of the factors that influence the interface between clinicians and laboratory health workers; and to investigate the impact of the use of laboratory test results on the way clinicians and laboratory workers interact to deliver effective and improved healthcare. our research results show that the roles of laboratory workers within the organisation are not determined by education levels, but by availability. according to clinicians, differences in levels of education lead to a lack of trust between clinicians and laboratory staff, impacting negatively on their collaboration and communication and creating a climate of distrust. poor communication between clinicians and laboratory staff further causes hostility when clinicians request a large number of tests, unaware of the high workload of the frequently understaffed laboratory. in all three phases of communication where clinicians and laboratory workers interact (pre-analytical phase: ordering of tests, sample collection; analytical phase: sample processing and analysis; post-analytical phase: results transfer), clinicians discuss their complaints and grievances with the laboratory staff more often than vice versa, suggesting that hierarchy plays a role in the dynamic between the two groups. despite their different perceptions of a variety of issues, the groups agreed that clinicians were sometimes reluctant to use test results for clinical decision making. all in all, the issue of ineffective communication between clinicians and laboratory staff on patient care and worker dissatisfaction remains largely unresolved, providing a major source of frustration for staff and resulting in inefficiency in expected outputs.567this study has increased the understanding of the interface between clinicians and laboratory workers and highlighted its importance in improving the quality of patient care. scaling up data collection in a larger group of health facilities is essential with regard to quantifying our findings. this will enable hospital managers to make suggestions for improvements, such as refresher training courses that cover communication skills, as well as involving clinical and laboratory staff, nurses and patients. the research may also motivate clinicians and laboratory managers to pay more attention to the international organization for standardization (iso)18 stipulations regarding communication. it is hoped that the findings from this study and similar future studies will, ultimately, improve the quality of patient care and communication between clinical and laboratory staff.19 limitations of this study top ↑ clinicians were sometimes rushed during the interview, as patients were waiting for assistance. only those staff who were on duty participated at the time of this study, which may have biased our results toward the perspectives of laboratory staff with lower education levels, since many more highly-educated staff were out in the field, in training, on holiday or had resigned. the perspectives of nurses and patients were not included in this study. laboratory staff often had difficulties in filling in the english saq. some questions were not clear and allowed multiple answers, which were adjusted for in the analysis. there were inconsistencies between the questionnaires for clinicians and those for laboratory workers. the outcome of our study was based mainly on qualitative data; the quantitative components were limited. in addition, the sample size of this study was too small to draw firm conclusions. as this was a pilot study, mainly descriptive statistical methods were used. this may limit the interpretation of the data presented, but it does provide valuable information on the interface between clinicians and laboratory workers and on the effectiveness of the framework to assess this interface. these insights may be used for future studies. conclusion top ↑ by combining quantitative and qualitative information, some insight emerged regarding the relationship between clinicians and laboratory workers and the perspectives that contribute to their sometimes problematic interactions. this explorative study has given us additional information on factors that influence the interface between clinicians and laboratory workers and shown the effectiveness of the analytical framework. the findings and discussions also provided information for the improvement of our analytical framework, indicating the need for inclusion of nurses and patients in future studies. the findings from this study underscore the relevance of the subject: the daily struggle of hospital managers, clinicians and laboratory workers to recognise the critical role each plays in providing efficient and reliable healthcare. performing this field study has provided information on the complexities of the interface between clinicians and laboratory staff and its impact on clinician decision making. the results justify expanding this study to a larger geographical area to include more health institutions. acknowledgements top ↑ we would like to thank all the staff of the hospital and clinic sites for their time and cooperation. a special thanks goes to the translators and data clerks at kilimanjaro clinical research institute (kcri). we would also like to acknowledge marjolein dieleman of the royal tropical institute (kit) health for her valuable input and critical assessment of this article. kind thanks to linda oskam (kit biomedical research) for her preliminary reading of the manuscript. this study would not have been possible without core funding from kcri and kit. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. authors’ contributions a.vdb. (royal tropical institute [kit]), c.j.t. (royal tropical institute [kit]), biomedical research), d.l.m. (kilimanjaro christian medical university college), e.m. (kilimanjaro clinical research institute) and m.s-db. (kilimanjaro clinical research institute) set up the study design and developed the tools. a.vdb., c.j.t., d.l.m., e.m., j.c. (kilimanjaro christian medical university college; kilimanjaro clinical research institute) and m.s-db. performed the fieldwork. d.l.m., e.m. and m.s-db. carried out the qualitative data analysis, a.vdb. and c.j.t. analysed the quantitative data, c.j.t. wrote the report and all authors contributed to and approved the final manuscript. references top ↑ 1.cohen gm. access to diagnostics in support of hiv/aids and tuberculosis treatment in developing countries. aids. 2007;21(suppl 4):s81–s87. http://dx.doi.org/10.1097/01.aids.0000279710.47298.5c 2.ishengoma dr, rwegoshora rt, mdira ky, et al. health laboratories in the tanga region of tanzania: the quality of diagnostic services for malaria and other communicable diseases. ann trop med parasitol. 2009;103(5):441–453. http://dx.doi.org/10.1179/136485909x451726 3. nkengasong jn. a shifting paradigm in strengthening laboratory health systems for global health: acting now, acting collectively, but acting differently. am j clin pathol. 2010;134(3):359–360. http://dx.doi.org/10.1309/ajcpy5asuejyq5rk 4. polage cr, bedu-addo g, owusu-ofori a, et al. laboratory use in ghana: physician perception and practice. am j trop med hyg. 2006;75(3):526–531. 5.petti ca, polage cr, quinn tc, et al. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006;42(3):377–382. http://dx.doi.org/10.1086/499363 6. chilundo b, sundby j, aanestad m. analysing the quality of routine malaria data in mozambique. malaria journal. 2004;3:3. http://dx.doi.org/10.1186/1475-2875-3-3 7. leshabari mt, muhondwa ep, mwangu ma, et al. motivation of health care workers in tanzania: a case study of muhumbili national hospital. east afr j public health. 2008;5(1):32–37. http://dx.doi.org/10.4314/eajph.v5i1.38974 8. manongi rn, marchant tc, bygbjerg ic. improving motivation among primary health care workers in tanzania: a health worker perspective. hum resour health. 2006;4:6. http://dx.doi.org/10.1186/1478-4491-4-6 9. garcia p, hughes j, carcamo c, et al. training pharmacy workers in recognition, management, and prevention of stds: district-randomized controlled trial. bull world health organ. 2003;81(11):806–814. 10.carter j, müller-stöver i, östensen h, et al. good clinical diagnostic practice: a guide for clinicians in developing countries to the clinical diagnosis of disease and to making proper use of clinical diagnostic services. world health organization regional office for the eastern mediterranean: cairo; 2005. isbn: 978-92-9021-393-2. available from: applications.emro.who.int/dsaf/dsa236.pdf‎ 11.world health organization. strategic approach for the strengthening of laboratory services for tuberculosis control, 2006–2009 [document on the internet]. 2006 [cited 2014 mar 30]. available from: http://apps.who.int/iris/bitstream/10665/69303/1/who_htm_tb_2006.364_eng.pdf?ua=1 12.butao d, chafulumira f, felling b, et al. malawi: laboratory services and supply chain assessment. usaid/deliver project: arlington, va; 2009. 13.mepham so, squire sb, chisuwo l, et al. utilisation of laboratory services by health workers in a district hospital in malawi. j clin pathol. 2009;62(10):935–938. http://dx.doi.org/10.1136/jcp.2009.069062 14.may ta, clancy m, critchfield j, et al. reducing unnecessary inpatient laboratory testing in a teaching hospital. am j clin pathol. 2006;126(2):200–206. http://dx.doi.org/10.1309/wp59ym73l6cegx2f 15.plebani m. exploring the iceberg of errors in laboratory medicine. clin chim acta. 2009;404(1):16–23. http://dx.doi.org/10.1016/j.cca.2009.03.022 16.van den broek a, tuijn cj, van ’t klooster l, et al. understanding the interface between clinical and laboratory staff. afr j lab med. 2014;3(1), art. in press. 17.varkevisser cm, pathmanathan i, brownlee a. designing and conducting health systems research projects. volume 1: proposal development and fieldwork. kit publishers: amsterdam; international development research centre (idrc); who regional office for africa. isbn 90 6832 148 x. 18.international organization for standardization. iso15189:2007. medical laboratories – particular requirements for quality and competence [page on the internet]. 2007 [cited 2014 mar 30]. available from: http://www.iso.org/iso/catalogue_detail?csnumber=42641 19. carter jy, lema oe, wangai mw, et al. laboratory testing improves diagnosis and treatment outcomes in primary health care facilities. afr j lab med. 2012;1(1), art. #8, 6 pages. http://dx.doi.org/10.4102/ajlm.v1i1.8 abstract introduction methods results discussion acknowledgements references about the author(s) olumuyiwa b. salu department of medical microbiology and parasitology, college of medicine, university of lagos, lagos, nigeria centre for human and zoonotic virology, central research laboratory, college of medicine, university of lagos, lagos, nigeria ayorinde b. james centre for human and zoonotic virology, central research laboratory, college of medicine, university of lagos, lagos, nigeria department of biochemistry, college of medicine, university of lagos, lagos, nigeria honoré s. bankolé department of applied microbiology and pharmacology of natural sciences, university of abomey-calavi, abomey-calavi, benin, benin national laboratory of public health, ministry of health, benin, benin jijoho m. agbla national laboratory of public health, ministry of health, benin, benin magloire da silva national laboratory of public health, ministry of health, benin, benin fernand gbaguidi national laboratory of public health, ministry of health, benin, benin department of pharmacognosy and pharmaceutical organic chemistry, university of abomey-calavi, abomey-calavi, benin christian f. loko de la pharmacie, du medicament et des explorations diagnostiques, ministére de la santé, cotonou, benin sunday a. omilabu department of medical microbiology and parasitology, college of medicine, university of lagos, lagos, nigeria centre for human and zoonotic virology, central research laboratory, college of medicine, university of lagos, lagos, nigeria citation salu ob, james ab, bankolé hs, et al. molecular confirmation and phylogeny of lassa fever virus in benin republic 2014–2016. afr j lab med. 2019;8(1), a803. https://doi.org/10.4102/ajlm.v8i1.803 original research molecular confirmation and phylogeny of lassa fever virus in benin republic 2014–2016 olumuyiwa b. salu, ayorinde b. james, honoré s. bankolé, jijoho m. agbla, magloire da silva, fernand gbaguidi, christian f. loko, sunday a. omilabu received: 09 mar. 2018; accepted: 19 feb. 2019; published: 22 aug. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the changing epidemiology of the lassa virus from endemic areas to other parts of west africa has been reported. however, there have been no documented lassa fever transmission chains in the benin republic. two outbreaks of lassa fever (november 2014 and january 2016) in the benin republic were characterised by a high number of deaths (more than 50%) among 27 confirmed and other unconfirmed cases. objectives: we report the detection, confirmation and relatedness of the lassa virus strains from the benin republic with other isolates within the west african sub-region. methods: a total of 70 blood samples (16 from 2014 and 54 from 2016) from suspected cases with signs and symptoms suggestive of viral haemorrhagic fever were received for molecular analysis at the centre for human and zoonotic virology, college of medicine, university of lagos and the lagos university teaching hospital. with the detection of the lassa virus rna by reverse transcriptase polymerase chain reaction, sequencing and phylogenetic analyses were performed using the sanger dideoxy sequencing technology platform and the mega6 software. results: s segments of the lassa virus rna genome were detected in 5 (7.1%) of the 70 samples analysed. sequencing and a phylogenetic tree construction confirmed that the strain of lassa virus had close relationships with strains previously isolated from nigeria. conclusion: we confirmed the presence of the lassa virus in the benin republic, with 2 strains having molecular epidemiological links with lineage i and ii strains from nigeria. to reduce the likelihood of outbreaks, there is a need for heightened awareness and strengthened surveillance systems about lassa fever, particularly in the sub-region. keywords: lassa virus; phylogeny; surveillance; benin republic; west africa. introduction lassa fever is an acute and often fatal viral haemorrhagic disease caused by an arenavirus called lassa virus (lasv), an enveloped bi-segmented negative-sensed single-stranded rna virus, endemic in parts of west africa. the illness has caused morbidity of about 300 000 persons and has an estimated death rate of 5000 per annum in west africa.1,2,3 the original lasv strain was isolated in 1969 from a missionary nurse involved in a nosocomial transmission chain from an obstetrical to patient residing in lassa village, maiduguri, now borno state, in nigeria, who travelled and sought treatment at the evangel hospital in jos, plateau state, for a septic abortion.4,5 in 1974, the reservoir host of lasv was identified: the multimammate rat mastomys natalensis, which is a peri-domestic rodent predominantly found across the sub-saharan africa region.6,7 the incubation period of lassa fever ranges between 5 and 21 days with an average of about 10 days.6 transmission is mainly by the ingestion of food or materials contaminated with feces or urine of infected rodents. however, human-to-human transmission of the virus in hospital settings or within the community expands the spread during epidemics.2,8 additionally, global travel, international trading and related commitments are also efficient routes of transmission for highly infectious pathogens, particularly those causing lassa fever and other viral haemorrhagic fevers (vhfs). by these routes, the movement of infectious agents from endemic countries to new places is a more probable event than has been documented.9,10,11,12,13 recently, lasv was discovered to be maintained by multiple rodent reservoirs other than mastomys natalensis.1 the isolation of the virus from the forest dwelling hylomyscus pamfi and ubiquitous mastomys erythroleucus highlights emerging evidence regarding the complexity of the virus’ ecology. the findings add to the growing probability of the emergence of lasv in new environments other than where it currently exists in the west african region.1 this is cause for concern regarding the changing epidemiology of lasv within the african continent. in the benin republic, two outbreaks of lassa fever (november 2014 and january 2016) were characterised by a high number of deaths (>50%) among confirmed cases.7,14 we report the detection, confirmation and phylogenetic relationship of lasv from these outbreaks using reverse transcription polymerase chain reaction (rt-pcr) targeting the 5` region of the s segment of the rna genome and sanger sequencing of partial fragments of the s segment of the rna genome. methods ethical considerations no ethics approval was obtained. this investigation was performed as part of the lassa fever public health response in the benin republic and nigeria. it was not considered to be research on human subjects, as documented in otto et al.15 all data were completely anonymised before analysis. setting in 2014, a lassa fever outbreak was reported for the first time in the tanguiéta and cobly communes, atakora department, north-west benin republic. the chain of infection stemmed from a woman who died from lassa fever 2 days after the delivery of a baby girl. the baby girl took ill 2 weeks after birth and was cared for at hôpital de saint jean de dieu, where the outbreak was enhanced by nosocomial transmission and eventual transmission within the community. within a period of 2 weeks (15 october to 04 november), 16 suspected cases with signs and symptoms of vhf and two laboratory confirmed cases, with a case fatality of 56.3%, were recorded in the country in 2014. this included the deaths of four personnel at the healthcare facility with signs and symptoms of vhf. due to the high case fatality rate within a short period of time, an alarm for the possible outbreak of the ebola virus was sounded by the health authorities in the benin republic. however, all samples were negative for the ebola virus; lasv was detected instead. a resurgence of the epidemic was witnessed in several districts in the central and eastern regions of the benin republic in 2016 with reports of 54 suspected cases with signs and symptoms of vhf and 16 laboratory confirmed cases, with a case fatality of about 50%. during this outbreak, the communes of tchaourou (borgou department) and djougou (donga department) along the nigerian border were the most affected areas. since the benin republic had never identified a case of vhf, blood samples collected from the people who died and suspected cases were sent to a specialised laboratory, the centre for human and zoonotic virology, college of medicine, university of lagos and the lagos university teaching hospital in lagos, nigeria, for vhf investigation. specimen transportation, handling and processing blood samples collected from different individuals with signs and symptoms of vhf (16 cases during the 2014 outbreak and 54 cases during the 2016 outbreak) were cold-chain-transported in triple-level packaging to the centre for human and zoonotic virology and lagos university teaching hospital via the benin republic ministry of health and the world health organization. universal sample and handling precautions were carried out as recommended by the united states centers for disease control and prevention.16 all specimen transport containers were disinfected with 10% hypochlorite solution in an airtight glove box. viral agents in specimen aliquots (undiluted and 1:10 dilution) were inactivated in a guanidinium-thiocyanate-based lysis buffer at room temperature for 10 min. nucleic acid extraction and reverse transcriptase-polymerase chain reaction the viral nucleic acid from inactivated sample aliquots (undiluted and 1:10 dilution) were extracted using a mini spin column rna extraction kit by qiagen (qiagen, germantown, maryland, united states) in a class iia biological safety cabinet according to the manufacturer’s instructions. after the extraction of viral nucleic acid, s segment of the rna genome, 3` non-coding region and 5` non-coding region of the nucleic acid of lasv (according to olschlager et al.17), dengue virus (according to drosten et al.18) and yellow fever virus (using in-house primers) were amplified in discrete rt-pcrs with primers as listed in table 1. separate reaction mixtures for lassa, dengue and yellow fever viruses were prepared and cycled as described in the one-step rt-pcr kit by ambionagpath-id protocol (applied biosystems, foster city, california, united states). the reaction was performed using the 9700 applied biosystems thermocycler with the following temperature profile: 50 °c for 30 min and 95 °c for 5 min, followed by 35 cycles of 95 °c for 30 s, 55 °c for 30 s, and 72°c for 30 s with a final extension of 72 °c for 5 min. subsequently, pcr amplicons were subjected to 1.5% agarose gel electrophoresis with 1x sybr® safe dna gel staining dye (invitrogen, carlsbad, california, united states) for 30 min at 120 v/400ma and images of amplicon bands under uv light were taken with a biodocanalyze 2.0 (biometra, goettingen, germany). table 1: primers used for lassa, dengue and yellow fever investigation, lagos, nigeria, march 2018. the positive control used for lassa assays were previously detected lassa samples from irrua, edo state, nigeria with accession number gu481078 nig 08-a47 2008 irrua, while those for dengue and yfv assays were both tissue cultured inactivated samples all from the virology unit laboratory of the bernhard nocht institute of tropical medicine, hamburg, germany through our collaborations. sanger sequencing and phylogenetic analysis the specific amplicon band size (320 bp) for lasv was purified using the jena bioscience gel extraction kit (jena, germany). purified pcr products were sequenced using 3130xl applied biosystems genetic analyzer at genewiz laboratories in south plainfield, new jersey, united states. sequence data in fasta format of the s segment of the rna genome of other submitted or published lasv genome sequences particularly from nigeria, liberia and sierra leone were downloaded from the national center for biotechnology information. downloaded sequences were aligned using the muscle tool of mega6 software.19,20 the tamura 3-parameter (t92) model was used to deduce the phylogeny of the strains. a phylogenetic tree was constructed using the maximum-likelihood method. evolutionary rate differences among sites (+g, parameter = 0.5024) were determined with a discrete gamma distribution and the evolutionary invariability allowed for some sites was estimated to be +i (36.3524% sites)21 using the variation rate model. the consistency of each node on the phylogenetic tree was verified by bootstrapping with 1000 replicates. results reverse transcriptase-polymerase chain reaction amplification and agarose gel analysis of the lassa, yellow fever and dengue viruses among the 70 samples, 5 (7.1%) were positive for lasv, while none (0%) was positive for both yellow fever and dengue viruses. the expected amplicon band size of approximately 320 base pairs (bp) of the s segment of the rna genome for lasv was detected by the agarose gel electrophoresis analysis17 (figure 1). the detected band size of the lasv amplicons was on par with the positive lasv controls and no band was observed in the negative control lane on the gel picture (pcr-grade water) (figure 1). however, none of the expected band sizes (~405 bp) and (~75 bp) were detected for yellow fever or dengue18 viruses (figures 2 and 3). figure 1: reverse transcription polymerase chain reaction detection of s gene fragment of the lassa virus, lagos, nigeria, march 2018. the gel lanes represent neat (undiluted, n) and 1:10 dilutions (d) of the rna extracts used. three nigerian outbreak samples representing lanes s1–s3 (accession numbers: mf317933-35) were run alongside benin republic outbreak samples (s4–s5). rnase/dnase free water was used as a negative extraction control (-ve ctrl) while a 2008 outbreak positive sample (gu481078_nig_08-a47_2008_irrua) was used as a positive control (+ve ctrl). figure 2: reverse transcription polymerase chain reaction detection of dengue virus, lagos, nigeria, march 2018. the gel lanes represent neat (undiluted, n) and 1:10 dilutions (d) of the rna extracts used. three nigerian outbreak samples representing lanes s1–s3 (accession numbers: mf317933-35) were run alongside benin republic outbreak samples (s4–s5). rnase/dnase free water was used as negative extraction control (-ve ctrl) while a tissue culture inactivated sample of dengue virus from the virology unit laboratory of the bernhard nocht institute of tropical medicine, germany was used as a positive control (+ve ctrl). figure 3: reverse transcription polymerase chain reaction detection of the yellow fever virus, lagos, nigeria, march 2018. the gel lanes represent neat (undiluted, n) and 1:10 dilutions (d) of the rna extracts used. three nigerian outbreak samples representing lanes s1–s3 (accession numbers: mf317933-35) were run alongside benin republic outbreak samples (s4–s5). rnase/dnase free water was used as a negative extraction control (-ve ctrl) while a tissue culture inactivated sample of 17d yellow fever strain from the virology unit laboratory of the bernhard nocht institute of tropical medicine, germany, was used as a positive control (+ve ctrl). sequencing and phylogenetic analysis of the s segment of the lassa virus rna genome sequence data of the s segment of the rna genome of lasv were obtained for 2/5 (40%) of the positive samples. the generated nucleotide sequences of the s segment of the rna genome of the lassa strains from the benin republic showed relatedness with documented lasv strains particularly from nigeria. phylogenetic analysis of the sequences of the 2 lasv strains from the benin republic with submitted sequences in the genbank database showed that each of the strains were closely related to lineage i that covers the 1969 lassa lp strain and lineage ii which covers strains from lagos, the eastern states of nigeria such as the onitsha strain in 1974, abakaliki, irrua, the middle belt and a few northeast central states in nigeria as shown in figure 4. figure 4: molecular phylogenetic analysis of the s gene segment of benin republic lassa sequences in comparison with selected lassa sequences from nigeria, liberia and sierra leone by the maximum likelihood method, lagos, nigeria, march 2018. the numbers at the nodes are bootstrap values. isolates in green boxes are from the benin republic. the isolate in the red box is the positive control of the assay. lineage i covers the 1969 lassa lp strain while lineage ii covers strains from lagos, the eastern states of nigeria such as the onitsha strain in 1974, abakaliki, irrua, the middle belt and the few northeast central states in nigeria; lineage iii covers the nig-csf-jos 2000 strain, the western states and northwestern states strains such as ga392 in zaria, nigeria; lineages iv/v cover the josiah strain of sierra leone, and the liberian strain. discussion this study confirms the presence of the lassa virus in the benin republic, with 2 isolates having molecular epidemiological links with lineage-ii strains from nigeria. large-scale outbreaks of lassa fever have been reported from nigeria since 2015 with a frequent and widening geographical spread.22 neighbouring countries are also at risk, because the types of rodents that harbour and spread the virus are found throughout the west african sub-region.3,6,7 the lassa fever virus has been endemic in nigeria, sierra leone and liberia for decades; however, proven and imported cases have been reported in ghana, the central african republic, guinea, cote d’ivoire, senegal, mali and togo.22 the benin republic reported the first few cases of confirmed lassa fever as evident from our laboratory findings during the 2014–2016 outbreaks. it is in no doubt that lassa fever is becoming a regular global health burden with immense impact on most of west africa’s communities.23 the emergence of the virus in new communities may be attributed to changes in socio-ecological and climatic conditions, global travel and improved surveillance using molecular biology tools for rapid detection of viral nucleic acids. due to the proximity of the benin republic to nigeria and reports of several lassa fever outbreaks in nigeria since 1969, it was expected that a case of lassa fever should have been reported in the benin republic earlier than now due to fluidic inter-border travel between the two countries. furthermore, reports have shown that migration of the lassa virus strains out of nigeria over a long period of time might have contributed to the increased diversity of lasv, with non-nigerian strains exhibiting improved codon adaptation to the human host, greater viral loads, and increased case fatality rates.24,25 our phylogenetic analysis of the partial sequences of the glycoprotein region of the lassa fever virus shows that the 2014 benin republic nucleic acid sequence clustered with the lily pinneo strain of 1969, whereas the 2016 sequence clustered with the 1974 onitsha strain. our findings indicate that the virus may have existed in the mastomys sp. reservoir in the benin republic, which did not affect humans until the 2014 outbreak, highlighting the possibility of the emergence of the virus in the benin republic which is possibly not a case of inter-border travel. however, cross border transmission from nigeria to the benin republic and vice versa is also still a possibility. thus, more surveillance studies are required particularly among rodents in the benin republic for a better understanding of the molecular epidemiology of the lassa fever virus in the country. conclusion despite the findings from this study, it is still envisioned that the likelihood of movement of vhfs, particularly lasv, from endemic into non-endemic countries within and beyond the west african sub-region remains a possibility. increased awareness and surveillance are effective tools in curbing the menace of these agents. thus, laboratory infrastructure, appropriate facilities, technical proficiency and investigation capacity must be improved for a positive impact on our surveillance mechanisms, diagnosis and identification of infections, clinical case management and the development of new approaches to control lassa fever outbreaks in the sub-region. acknowledgements the authors would like to acknowledge and thank all staff members who contributed to the collection and analysis of samples in the laboratory, especially the laboratory scientists mrs mr orenolu, mr ra anyanwu and mrs ma abdullah of the virology research laboratory, central research laboratory, college of medicine, university of lagos. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions o.b.s., a.b.j., and s.a.o. were responsible for the conceptualisation and design of the project, performed part of the laboratory work, analysed the data and wrote the manuscript. h.s.b., j.m.a., m.d.s., f.g. and c.f.l. all made conceptual contributions and assisted in writing and preparing the manuscript. sources of support this work was supported by the european union support for evd (2014), bernhard nocht institute for tropical medicine, germany and virology unit laboratory, central research laboratory, college of medicine, university of lagos, lagos, nigeria. data availability statement the data are not publicly available due to (restrictions e.g. containing information that could compromise the privacy of research participants). the data that support the findings of this study are available from the corresponding author, upon reasonable request. disclaimer responsibility for the information and views set out in this publication lies entirely with the authors. references mccormick jb, fisher-hoch sp. lassa fever. curr top microbiol immunol. 2002;262:75–109. https://doi.org/10.1007/978-3-642-56029-3_4 yun ne, walker dh. pathogenesis of lassa fever. viruses. 2012;4:2031–2048. https://doi.org/10.3390/v4102031 olayemi a, cadar d, magassouba n, et al. new hosts 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cagliani r, et al. evolutionary analysis of old world arenaviruses reveals a major adaptive contribution of the viral polymerase. mol ecol. 2017;26:5173–5188. https://doi.org/10.1111/mec.14282 andersen kg, shapiro bj, matranga cb, et al. clinical sequencing uncovers origins and evolution of lassa virus. cell. 2015;162(4):738–750. https://doi.org/10.1016/j.cell.2015.07.020 abstract introduction methods results discussion acknowledgements references about the author(s) sophia rossouw south african medical research council bioinformatics unit, south african national bioinformatics institute, university of the western cape, cape town, south africa hocine bendou south african medical research council bioinformatics unit, south african national bioinformatics institute, university of the western cape, cape town, south africa liam bell centre for proteomic and genomic research, observatory, cape town, south africa jonathan rigby department of anatomical pathology, national health laboratory service, tygerberg hospital, stellenbosch university, cape town, south africa alan christoffels south african medical research council bioinformatics unit, south african national bioinformatics institute, university of the western cape, cape town, south africa citation rossouw s, bendou h, bell l, rigby j, christoffels a. effect of polyethylene glycol 20 000 on protein extraction efficiency of formalin-fixed paraffin-embedded tissues in south africa. afr j lab med. 2021;10(1), a1122. https://doi.org/10.4102/ajlm.v10i1.1122 note: additional supporting information may be found in the online version of this article as supplementary documents s1, s2, tables s1 and s2 and figures s1 and s2. original research effect of polyethylene glycol 20 000 on protein extraction efficiency of formalin-fixed paraffin-embedded tissues in south africa sophia rossouw, hocine bendou, liam bell, jonathan rigby, alan christoffels received: 25 nov. 2019; accepted: 08 sept. 2021; published: 17 dec. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: optimal protocols for efficient and reproducible protein extraction from formalin-fixed paraffin-embedded (ffpe) tissues are not yet standardised and new techniques are continually developed and improved. the effect of polyethylene glycol (peg) 20 000 on protein extraction efficiency has not been evaluated using human ffpe colorectal cancer tissues and there is no consensus on the protein extraction solution required for efficient, reproducible extraction. objective: the impact of peg 20 000 on protein extraction efficiency, reproducibility and protein selection bias was evaluated using ffpe colonic tissue via liquid chromatography tandem mass spectrometry analysis. methods: this study was conducted from august 2017 to july 2019 using human ffpe colorectal carcinoma tissues from the anatomical pathology department at tygerberg hospital in south africa. samples were analysed via label-free liquid chromatography tandem mass spectrometry to determine the impact of using peg 20 000 in the protein extraction solution. data were assessed regarding peptide and protein identifications, method efficiency, reproducibility, protein characteristics and organisation relating to gene ontology categories. results: polyethylene glycol 20 000 exclusion increased peptides and proteins identifications and the method was more reproducible compared to the samples processed with peg 20 000. however, no differences were observed with regard to protein selection bias. we found that higher protein concentrations (> 10 µg) compromised the function of peg. conclusion: this study indicates that protocols generating high protein yields from human ffpe tissues would benefit from the exclusion of peg 20 000 in the protein extraction solution. keywords: mass spectrometry; formalin-fixed paraffin-embedded proteomics; archival tissue; protein extraction; polyethylene glycol 20 000; sp3-on-bead-digestion. introduction archival formalin-fixed paraffin-embedded (ffpe) tissue repositories are valuable resources for clinical proteomic studies; such repositories may include retrospective as well as protein biomarker discovery and validation studies.1,2,3 these repositories are often composed of a large variety of patient biopsy tissues, which are accompanied by their associated clinical metadata, in the form of patient medical records. the wealth of information stored in these archival ffpe tissue repositories, together with the easily accessible ffpe samples, has generated improved methods for ffpe tissue analysis in the context of genomic, proteomic and immunohistochemical studies.1,2,3 the development and standardisation of ffpe sample processing for mass spectrometry (ms)-based analysis to determine changes (or similarities) in the proteome composition of tumour versus healthy tissues is of great interest to clinical and translational research.4,5 part of this process involves using an optimal and efficient protein extraction buffer to generate reproducible results. studies have found that experimental factors, such as protein extraction buffer, ph, detergents, denaturants and temperature, play important roles in the final attainable protein yield from ffpe tissues.3,6 other factors to consider include limited availability of clinical specimens and therefore a limited amount of starting material (tissue) available for optimising a protein extraction procedure. this places limitations on the choice of proteomics workflows (including protein extraction, protein sample enrichment, fractionation and digestion) that can be used to generate samples of suitable quality for high sensitivity liquid chromatography (lc) tandem ms analysis.2,7 additional challenges faced in ffpe proteomics studies, which cannot be remedied after the fact, are pre-analytical factors that affect protein extraction efficiency and often produce variable protein yields. these may include tissue ischemic time, the composition of the fixative, fixation time (duration or range of formalin-fixation times used), as well as block age and storage conditions.2,4,8 during the protein extraction process, the effect of the formaldehyde fixation chemistry on the tissues poses another challenge to overcome. due to extensive formaldehyde cross-linking between molecules, accurate and efficient protein extraction from ffpe tissues is difficult. it requires specific sample processing techniques to allow for complete breakage of cross-linking bonds, which in turn allows for proper trypsin digestion.2,9,10,11 for this reason multiple strategies have been employed, including the use of denaturants, detergents, precipitants and antigen retrieval. however, several aspects of the formaldehyde-protein interactions remain unresolved and are the focus of continued research in the ffpe proteomics field. we have previously studied the effects of ffpe block age on the quality and quantity of protein extracted from ffpe tissues and also evaluated protein purification methods using lc-ms/ms analysis.12 however, the optimal protein extraction buffer components were not investigated. therefore, of interest to this study are the effects of polyethylene glycol (peg), specifically peg 20 000, on protein extraction efficiency of human ffpe tissues using lc-ms/ms analysis, as there is no current consensus with regard to peg usage and advantages for human ffpe tissue proteomics. polyethylene glycol, a high molecular weight synthetic polymer, reduces non-specific protein adsorption to surfaces, such as experimental plastic-ware (micropipette tips and microcentrifuge tubes), thereby preventing subsequent protein loss.3,13 polyethylene glycols also precipitate proteins through a steric exclusion mechanism, whereby they occupy most of the space in solution, thus concentrating the proteins until they exceed solubility and precipitate.14,15,16,17 therefore, subsequent centrifugation may pellet the precipitated proteins17 and these may be lost in the sample pellets (after clarifying the protein lysates and removal of the supernatants for analysis). polyethylene glycol also causes interference and ion signal suppression in downstream lc-ms/ms analysis, if it is not completely removed from the sample analysed.5,13 removal of high concentrations of pegs is challenging and peg carry-over into sample fractions and lc columns is a huge problem.14,18 however, due to its advantages and available techniques to remove peg before lc-ms/ms analysis, it is often used for protein extraction of ffpe tissues.3,13 to our knowledge, however, peg efficacy with regard to protein extraction of human ffpe tissues has not been fully evaluated yet. polyethylene glycol can vary in polymer size, and for this study peg 20 000 was chosen, because it is the most extensively used form in ffpe tissue proteomics; subsequently all references to peg in this article are to the 20 000 form. the aim of this study was to evaluate the effects of peg within the protein extraction buffer using label-free lc-ms/ms analysis of manually micro-dissected ffpe human colorectal carcinoma (crc) resection samples. the sample pellets were also tested for residual protein, which was not extracted in the whole cell protein lysates (wcpls). methods ethical considerations ethics clearance was obtained from the health research ethics committee of stellenbosch university (ethics reference number: s17/10/203) and biomedical science research ethics committee of the university of the western cape (ethics reference number: bm17/7/15). all patient specimens were anonymised before being archived for long-term storage and before they were accessed for the study. patient consent was not required since it was a retrospective study using archival tissues. formalin-fixed paraffin-embedded human colorectal carcinoma samples this study conducted from august 2017 to july 2019, included retrospectively chosen human colorectal resection specimens acquired from the department of anatomical pathology at tygerberg hospital in western cape, south africa. the specimens were preserved as ffpe blocks when the tissue was resected and archived between january 2016 and december 2017. due to retrospective collection of the samples, the exact pre-analytical factors, such as the handling, fixation times and conditions, and storage conditions, were unknown and could not be accounted for. table 1 shows the details of the three patient cases selected. table 1: details of the three ffpe patient cases selected for analysis at the south african national bioinformatics institute, university of the western cape, bellville, south africa, from august 2017 to july 2019. to ensure tissue quality and comparability, a pathologist reviewed the patient tissue sections after haematoxylin and eosin staining to select only specimens that had carcinomas with more than 90% viable tumour nuclei (figure 1). patient samples were also classified and diagnosed with low-grade or high-grade colorectal carcinoma after haematoxylin and eosin staining. figure 1: colon adenocarcinoma tissue specimens analysed at the south african national bioinformatics institute, university of the western cape, bellville, south africa, from august 2017 to july 2019. microscopic images of haematoxylin and eosin-stained colon tissue sections of patient resection specimens analysed in this study: patient 1 (a), patient 2 (b) and patient 3 (c) at 100× magnification. protein extraction and purification to overcome the effects of formaldehyde cross-linking, we opted to combine protein extraction techniques that employed the use of antigen retrieval, strong detergent concentration, as well as a synthetic polymer for protein precipitation (peg 20 000). for protein purification before lc-ms/ms analysis, we used the single-pot solid-phase-enhanced sample preparation (sp3)19,20 method, which ensures minimal sample loss during processing and was also found to be highly sensitive, therefore requiring less starting material (tissue).12,19,20 the equivalence of 23 mm3 of manually micro-dissected ffpe tumour tissue was cut and processed for each patient case (figure 2). protein was extracted using a solution that consisted of 50 mm of ammonium bicarbonate (ph 8.0) and 2% sodium dodecyl sulphate (sds) and either with or without the addition of 0.5% peg. to further determine protein extraction buffer efficiency, the sample pellets were also assessed for residual proteins that were not extracted in the initial extraction. in total, 12 samples were analysed, including the wcpls as well as the sample pellets (figure 2). figure 2: summarised workflow and experimental design followed at the south african national bioinformatics institute, university of the western cape, bellville, south africa, from august 2017 to july 2019. ffpe colorectal carcinoma tissue from three patients were cut at 25 µm thickness and tumour areas were manually micro-dissected for analysis. from each patient ffpe block, 4 tissue sections (each 25 µm thick and equivalent to approximately 23 mm3 tissue) were used per experimental sample. protein extraction buffer, with or without the addition of peg, was used to extract protein. sample pellets were analysed for residual protein by further protein extraction (using 4% sds), followed by protein quantification and subsequent sample processing (for lc-ms/ms analysis) by the hilic/sp3 method. wcpls from each patient were quantified and processed by the hilic/sp3 sample preparation method, followed by ms analysis. the mass spectra generated were then analysed during the data analysis phase. the method used for sample processing, protein extraction and protein yield quantification was modified from the protocols used by scicchitano21 and wiśniewski22 and previously described in more detail by rossouw.12 both wcpls and sample pellets were subsequently processed by the hydrophilic interaction liquid chromatography (hilic)/sp3 magnetic bead digestion method,19 before lc-ms/ms analysis (figure 2). the magresyn® (resyn biosciences, edenvale, gauteng, south africa) hilic/sp3 method (using on-bead digestion) was used for protein purification and tryptic digestion (peptide generation) prior to lc-ms/ms analysis. the method was modified from the protocol used by hughes19 and previously described in more detail by rossouw.12 mass spectrometry analysis mass spectrometry analysis of each sample’s peptides was performed using the q-exactive quadrupole-orbitrap (thermo fisher scientific, waltham, massachusetts, united states), which was coupled with a dionex ultimate 3000 nano-uplc system as described before by rossouw.12 using xcaliburtm (version 4.2) (thermo fisher, waltham, massachusetts, united states), spectral data was collected in a data-dependent manner and details are shown in supplementary document – table s1. identification of peptides and proteins the raw spectral data were converted into ‘mascot generic format’ (matrix science, london, united kingdom), which is a standard format used for tandem ms data that converts the raw data into a simpler format for subsequent database searches, using msconvert (proteowizard, palo alto, california, united states).23 x!tandem (version 2015.12.15.2)24 (the global proteome machine organization, winnipeg, manitoba, canada), ms amanda (version 2.0.0.9706)25 (protein chemistry facility imp/imba/gmi, vienna, austria), and ms-gf+ (version 2018.04.09)26 (pacific northwest national laboratory, richland, washington, united states) were used to identify peak lists from ms/ms spectra.12 searchgui (version 3.3.3)27 (computational omics and systems biology group, ghent university, gent-zwijnaarde, belgium) was used to allow for simultaneous searches. a concatenated target-decoy28 version of the homo sapiens (73101, > 99.9%), sus scrofa (1, < 0.1%) complement of the uniprotkb29 reference proteome (up000005640; 9606-homo sapiens) (version downloaded on 29/10/2018) was used for protein identifications. searchgui generated the decoy sequences. the identification settings are shown in table 2 and the certificate of analysis lists all algorithms settings used and validation thresholds (supplementary document 3 – file s1). peptideshaker (version 1.16.31)30 (computational omics and systems biology group, ghent university, gent-zwijnaarde, belgium) was used to infer peptide and protein identifications from spectrum identification data and validated at 1% false discovery rate estimated using the decoy hit distribution. d-score31 and phosphors score32 (threshold of 95.0 as implemented in the compomics utilities package33) were used to score post-translational modification localisations. adequate or acceptable reproducibility or reliability, as it pertains to the results (including figure 3a and 3c), was defined as the observable extent (measured, for example, as the standard deviation) of stability within measured data points when measurements are repeated under similar experimental conditions. figure 3: numbers of identified peptides and proteins from wcpls and pellets, compared between different protein extraction buffers with or without addition of peg at the south african national bioinformatics institute, university of the western cape, bellville, south africa, from august 2017 to july 2019. (a) box and whiskers plot showing the number of peptides identified (for all three patient samples) per condition – pellet with peg (4% sds), pellet without peg (4% sds), wcpl with peg (2% sds), wcpl without peg (2% sds). (b) venn diagram depicting the distribution of identified peptides (for all three patient cases) among all conditions. (c) box and whiskers plot showing the number of proteins identified (for all three patient samples) per condition. (d) venn diagram depicting the distribution of identified proteins (individual and protein groups) (for all three patient cases) among all conditions. (–peg) refers to protein extracted without peg and (+peg) refers to protein extracted with peg. red boxplots refer to pellet samples extracted with peg; purple boxplots refer to pellet samples extracted without peg; blue boxplots refer to wcpl samples extracted with peg; green boxplots refer to wcpl samples extracted without peg. table 2: peptide and protein identification settings at the south african national bioinformatics institute, university of the western cape, bellville, south africa, from august 2017 to july 2019. data analysis data were analysed and graphically visualised and displayed using pandas, numpy and matplotlib python packages (python software foundation, wilmington, delaware, united states), as well as microsoft excel (microsoft corporation, redmond, washington, united states). the merged lists of either peptide sequences or protein accession numbers (individual as well as protein groups) identified in each sample group and experimental condition were processed using venny (version 2.1.0)34 (bioinfogp service, universidad autónoma de madrid, madrid, spain), which calculated and visually displayed the percentage overlap. to determine the qualitative reproducibility of each experimental condition, the peptide identification overlap (supplementary document – figure s1) was computed using the peptide sequences identified for each sample from the data set (regardless of peptide abundance). from these results, the physicochemical properties of the peptides (unique as well as shared) for all conditions were assessed for each patient (supplementary document – file s2). spectrum counting abundance indexes were estimated using the normalised spectrum abundance factor35 as generated by the peptideshaker software.27,36 the normalised spectrum abundance factor values were normalised to facilitate comparisons and then used to calculate the pearson’s correlation coefficient, for each pair of experimental conditions compared with regard to differential protein abundance, to determine the level of correlation between samples (supplementary document – figure s2). propas (version 1.1)37 (state key laboratory of proteomics, beijing institute of radiation medicine, beijing, china) was used to calculate the physicochemical properties (hydropathicity [kyte and doolittle scale],38 molecular weight and isoelectric point) of identified peptides for each sample analysed. sample physicochemical characteristics were assessed and visualised using box and whisker plots. gene ontology (go) analysis was performed using protein annotations retrieved from ensembl (www.ensembl.org) and goslim uniprotkb-goa (www.ebi.ac.uk/goa) with hypergeometric testing to determine go term significance. the protein extraction buffers’ (with or without addition of peg) protein selection bias, as well as residual proteins of the sample pellets, was assessed with regard to subcellular localisation, using go analysis. results from the go annotation were visualised with a bar plot showing percentages of proteins belonging to each go term and their location. the percentages of missed cleavages for all samples were calculated and graphically visualised and displayed using pandas, numpy and matplotlib python packages (python software foundation, wilmington, delaware, united states). results effect of polyethylene glycol on peptide and protein identification we processed the ffpe colonic resection tumour tissues of three patients (diagnosed as indicated in table 1). for all three patients, non-fractionated lc-ms/ms analysis showed that overall, the wcpls extracted with 2% sds and peg had lower numbers of identifications at both the peptide and protein levels (validated peptides = 6840 [± 588 standard deviation {s.d.}]) and validated proteins = 2302 (± 127 s.d.) (figure 3a and figure 3c). on the other hand, the wcpls extracted without peg showed higher numbers of identifications (validated peptides = 7058 [± 649 s.d.] and validated proteins = 2314 [± 230 s.d.]) with adequate reproducibility (figure 3a and figure 3c). the pellet samples extracted with 4% sds showed higher overall variability at both peptide and protein levels. however, the numbers of peptide and protein identifications were high for pellets extracted without peg (validated peptides = 5999 [± 2176 s.d.] and validated proteins = 1893 [± 555 s.d.]) and for pellets extracted with peg (validated peptides = 4778 [± 1764 s.d.] and validated proteins = 1564 [± 456 s.d.]) (figure 3a and figure 3c). for overlap calculated from merged lists of peptide sequences, 27.1% of identified peptides were shared or overlapped between all the experimental conditions (figure 3b). lower percentages of unique peptides were identified for the pellets (8.7% without peg and 3.7% with peg), compared to the wcpls (10.3% without peg and 11.8% with peg). for overlap calculated from merged lists of protein accession numbers (individual as well as protein groups), 38% of identified proteins were shared or overlapped between all the experimental conditions. lower percentages of unique proteins were identified for the pellets (5.1% without peg and 3.5% with peg), compared to the wcpls (9.4% with and without peg). no substantial differences were observed for the physicochemical properties of the peptides for each patient (supplementary document – file s2). all experimental conditions yielded comparable relative protein abundances, indicating that protein extraction with and without peg did not introduce a substantial observable bias with regard to proteome composition. evaluation of protein physicochemical properties and go analysis the hydropathicity scales of all identified peptides generated from each experimental condition were similar (figure 4a). the majority of proteins extracted (with and without peg) and processed via the hilic/sp3 method were hydrophilic, since the average hydropathicities of all samples were negative (in accordance with the kyte and doolittle scale38 and as described by farias39). some differences were observed between pellet samples and wcpls (extracted with and without peg). the pellet samples seemed slightly more hydrophobic or neutral (closer to 0) in nature compared to the wcpls. however, neither the addition nor omission of peg from the protein extraction buffer affected or showed a substantial hydropathicity preference or selection bias with regard to extracted peptides. the molecular weight ranges (majority > 1000 dalton [da] and < 2000 da) (figure 4b), as well as isoelectric point (pi) ranges (majority above pi 4 and below pi 7) (figure 4c) of identified peptides were overall similar for all samples and experimental groups compared. figure 4: physicochemical properties of peptides extracted under the different experimental conditions at the south african national bioinformatics institute, university of the western cape, bellville, south africa, from august 2017 to july 2019. (a) hydropathicity was based on kyte and doolittle’s (1982) grand average of hydropathy (gravy) scoring matrix. (b) molecular weight. (c) isolectric point (pi). (–peg) refers to protein extracted without peg and (+peg) refers to protein extracted with peg. red boxplots refer to pellet samples extracted with peg; purple boxplots refer to pellet samples extracted without peg; blue boxplots refer to wcpl samples extracted with peg; green boxplots refer to wcpl samples extracted without peg. overall, similar go functional annotation profiles were obtained for all samples (figure 5). the majority of proteins were preferentially extracted from the cytoplasm (< 90%), organelles (> 90%), intracellular region (> 90%) and extracellular region (> 60%). figure 5: gene ontology annotation profiles according to subcellular localisation for proteins identified from all samples and conditions at the south african national bioinformatics institute, university of the western cape, bellville, south africa, from august 2017 to july 2019. the average percentages of occurrence of go terms for all three patients (per experimental group) are displayed with error bars showing standard deviation (significance level = 0.05). (–peg) refers to protein extracted without peg and (+peg) refers to protein extracted with peg. red bar graphs refer to pellet samples extracted with peg; purple bar graphs refer to pellet samples extracted without peg; blue bar graphs refer to wcpl samples extracted with peg; green bar graphs refer to wcpl samples extracted without peg. assessment of sample preparation method reproducibility and trypsin digestion efficiency all samples had a majority (> 80%) of fully cleaved peptides (0 missed cleavages), with approximately < 20% peptides with 1 missed cleavage, and approximately < 5% peptides with 2 missed cleavages (figure 6). in addition, the hilic/sp3 sample preparation method shows a similar range of missed cleavages in all samples and experimental conditions analysed. figure 6: numbers of missed cleavages for all samples at the south african national bioinformatics institute, university of the western cape, bellville, south africa, from august 2017 to july 2019. for each sample, the percentage of missed cleavages is shown. (–peg) refers to protein extracted without peg and (+peg) refers to protein extracted with peg. discussion in this present study, the samples processed using peg in the protein extraction buffer had overall lower peptide and protein identifications. using hela cells, wiśniewski13 found that the addition of peg to the protein extraction buffer improves protein extraction efficiency of samples that contained sub-microgram to microgram amounts of protein. however, peg’s ability to improve protein extraction efficiency was compromised when processing cell lysates that contained more than 10 µg of protein. furthermore, shen3 found that the addition of peg to ffpe rat tissues, which contain > 10 µg protein, failed to increase the amount of peptide and protein identifications. as our study extracted protein in the range of approximately 400 µg – 900 µg per sample (supplementary document – table s2), it would explain why peg’s extraction efficiency was compromised and resulted in lower overall peptide and protein identifications. the number of peptide (6840–7058) and protein (2302–2314) identifications reported here for the wcpls fall within the range of previously published studies and are higher than those reported by sprung40 (approximately 400–500 protein groups identified for triplicate samples). craven41 identified between 1335 and 1945 proteins on average for four biological replicates, as well as bronsert4 who identified between 3850 and 4210 peptides and between 765 and 1003 proteins on average for five biological replicates. on the other hand, wiśniewski13 identified more than 6000 proteins (extracted using peg) from the analysis of three ffpe colon cancer patient samples and they also reported higher identifications elsewhere using peptide fractionation.22,42 overall, the standard deviations reported here for the wcpl samples fall within approximately 10% of the sample means. nel43 reported similar or higher variances between triplicate technical replicates of a bacterial cell culture, as did wiśniewski13 for human cell lines. however, sample variance and standard deviations were not explicitly reported for the aforementioned ffpe clinical sample studies. our results indicate that the majority of proteins were extracted in the initial wcpls. therefore, the extraction buffer containing 2% sds and the extraction protocol used was sufficiently efficient to extract the majority of proteins from the patient samples; the main differences occurred due to the addition of peg to the extraction solution. tanca1 used technical replicates only for their study and found a similar variance in peptide identification overlap, ranging from as low as 26.6% for all experimental conditions to 32.6% overlap between one set of replicates from the same tissue block (patient). our results showed similar levels of overlap between biological replicates of different tissue blocks (patients), excluding the pellet samples (which were not the main focus of the study). in addition, shared or common peptides and proteins between the pellet samples and wcpls are due to soluble fraction or liquid (containing protein) remaining trapped within the sample pellets, after protein extraction and homogenate clarification (by centrifugation).44 furthermore, the unique peptides of the pellet samples may also, in part, be attributed by the higher sds concentration (4% sds) used for extraction, since other studies have found greater protein extraction efficiency by using higher sds concentrations.4,41,42,45 trypsin digestion efficiency influences the molecular weight of peptides.46 however, all samples in this current study were subjected to the same digestion protocol. therefore, our results show that the addition or omission of peg to the protein extraction buffer did not affect end-result molecular weight distributions, nor were there any significant differences in molecular weight distributions of residual proteins from the pellets. overall, neither the addition nor omission of peg to the protein extraction solution had any selection bias with regard to extracted proteins’ physicochemical properties. similar results were observed by hughes19 and moggridge.47 after processing protein extracts using the hilic/sp3 method, they found no obvious bias with regard to the molecular mass, isoelectric point or average relative hydropathicity of resultant isolated peptides. in addition, go analysis did not indicate any bias with regard to protein enrichment either. the hilic/sp3 protocol also generated low percentages of missed cleavages across all samples, indicating that the workflow was sufficiently reproducible and efficient at removing any interfering chemicals (such as peg and sds). batth,48 hughes49 and moggridge47 have also demonstrated the sensitivity, reproducibility and efficiency of the hilic/sp3 sample preparation method in removing sample contaminants for optimal recovery of peptides for lc-ms/ms analysis. limitations the current study had access to tissue samples that were not limited with regard to sample volumes and concentrations required for ms analysis compared to, for example, limited samples such as fine needle biopsies. therefore, it was neither feasible nor cost-beneficial for us to determine the effects of peg at < 10 µg protein, since this was not compatible with the material we had available, and did not fall within the scope of the present study or studies stemming from it.12 conclusion using ffpe human colorectal cancer resection tissue, we demonstrated that the addition of 0.5% peg to protein extraction buffer resulted in overall lower peptide and protein identifications, compared to buffer without the addition of peg. in addition, protein samples extracted without peg showed higher reproducibility, and the addition of peg to the protein extraction buffer generated lower percentages of unique peptides remaining in the sample pellets. by expanding on previous studies that only analysed ffpe animal tissues and human cells, we have demonstrated that high protein concentrations (> 10 µg) from ffpe human colon tissue also compromises the function of peg. the data from this study, together with our recently published selection of protein purification protocols for different ffpe block ages,12 should provide pathologists with an optimised methodological approach to exploit the use of archival ffpe tissue blocks. acknowledgements we thank prof. gerhard walzl for making available laboratory bench space at the stellenbosch university immunology group, and mrs andrea gutschmidt at the stellenbosch university immunology group for her technical assistance and support during the use of their laboratory space. we also thank mr charles gelderbloem and mr yunus kippie at the university of the western cape’s biotechnology and pharmacology departments for their technical assistance and support during the project. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions s.r., j.r. and a.c. designed the project. s.r. collected all samples, did the protein extraction work and drafted the first version of the manuscript. a.c. provided the funding for the project. l.b. performed the mass spectrometry experiment. h.b. assisted with the data analysis. the manuscript was written through contributions from all authors. all authors have given approval to the final version of the manuscript. sources of support this work was supported by the south african research chairs initiative of the department of science and innovation and national research foundation of south africa (grant uid 64751) and the south african medical research council. data availability mass spectrometry data (with identification results) were deposited to the pride archive (http://www.ebi.ac.uk/pride/archive/) via the pride partner repository with the identifiers pxd014419 and 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2018 [cited 2020 may 11]. available from: https://www.biorxiv.org/content/early/2018/10/25/447904 hughes cs, moggridge s, müller t, et al. single-pot, solid-phase-enhanced sample preparation for proteomics experiments. nat protoc. 2018;14(1):1–18. https://doi.org/10.1038/s41596-018-0082-x abstract introduction methods results discussion acknowledgements references about the author(s) sarishna singh national health laboratory service tygerberg academic laboratory, division of medical microbiology, tygerberg hospital, tygerberg, south africa division of medical microbiology, department of pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa mae newton-foot national health laboratory service tygerberg academic laboratory, division of medical microbiology, tygerberg hospital, tygerberg, south africa division of medical microbiology, department of pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa pieter nel national health laboratory service tygerberg academic laboratory, division of medical microbiology, tygerberg hospital, tygerberg, south africa division of medical microbiology, department of pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa colette pienaar national health laboratory service tygerberg academic laboratory, division of medical microbiology, tygerberg hospital, tygerberg, south africa division of medical microbiology, department of pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa citation singh s, newton-foot m, nel p, pienaar c. comparison of commercial assays and two-step approach to detect clostridioides difficile in south africa. afr j lab med. 2022;11(1), a1809. https://doi.org/10.4102/ajlm.v11i1.1809 note: additional supporting information may be found in the online version of this article as online supplementary material. original research comparison of commercial assays and two-step approach to detect clostridioides difficile in south africa sarishna singh, mae newton-foot, pieter nel, colette pienaar received: 03 dec. 2021; accepted: 26 may 2022; published: 29 sept. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: clostridioides difficile is the number one cause of hospital-acquired diarrhoea. accurate diagnosis of c. difficile is of utmost importance as it guides patient management and infection control practices. studies evaluating the performance of commercially available nucleic acid amplification tests (naats) versus algorithms are lacking in resource-limited settings. objective: this study assessed the performance of three commercially available tests and a two-step approach for the diagnosis of c. difficile infection using toxigenic culture (tc) as the gold standard. methods: two hundred and twenty-three non-duplicate loose stool samples were submitted to the national health laboratory service microbiology laboratory at tygerberg hospital, cape town, south africa, from october 2017 to october 2018. the samples were tested in parallel using the c. diff quik chek complete enzyme immunoassay (eia) and two naats (xpert c. difficile and bd max cdiff), and the results were compared to tc. the performance of a two-step approach consisting of the c. diff quik chek complete followed by the xpert c. difficile was also determined. results: of 223 faecal specimens tested, 37 (16.6%) were tc-positive. the sensitivity and specificity of the c. diff quik chek complete were 54.1% and 98.9%; xpert c. difficile, 86.4% and 96.8%; bd max cdiff, 89.2% and 96.8%; and two-step approach, 89.2% and 96.2%. conclusion: the c. diff quik chek complete, in a two-step approach with the xpert c. difficile, performed similarly to the naats on their own and offer advantages in terms of cost and workflow in low-resource settings. keywords: clostridioides difficile; clostridium difficile; xpert; bd max; quik chek; toxigenic culture. introduction clostridioides (clostridium) difficile is an anaerobic gram-positive rod capable of forming endospores and producing toxins. c. difficile infection is associated with an asymptomatic carrier state, self-limiting diarrhoea, pseudomembranous colitis, and toxic megacolon which can be fatal.1 c. difficile infection accounts for up to 20% of nosocomial diarrhoea cases worldwide.2 in south africa, there is very little published data regarding the incidence and prevalence of c. difficile; a previous study in patients with diarrhoea reported the prevalence to be 16%.3 in the past two decades, c. difficile has also emerged as a cause of community-acquired diarrhoea.2,4 most c. difficile strains produce two major toxins, namely toxin a, an enterotoxin, encoded by the tcda gene, and toxin b, a cytotoxin, encoded by the tcdb gene. c. difficile infection associated with increased severity and mortality has been reported in north america and europe.5 the c. difficile strain responsible for these outbreaks, the hypervirulent 027/nap1/bi, is characterised by a deletion in the tcdc gene (a negative regulator of tcda and tcdb expression), resulting in increased production of toxin a and toxin b.6 certain strains of c. difficile may produce a binary toxin, c. difficile transferase, encoded by cdta and cdtb. data regarding binary toxin conflict, with some studies suggesting its significance is unclear whereas others have shown that it may be associated with a higher mortality rate.7,8,9 accurate diagnosis of c. difficile infection is essential as it guides patient management and infection control practices. the two diagnostic reference standards for the diagnosis of c. difficile are toxigenic culture (tc) and the cell culture neutralisation assay; however, these are labour-intensive and have extended turnaround times of 2–3 days.10 current guidelines recommend a two-step approach: an enzyme immunoassay (eia), followed by a molecular test to increase the diagnostic yield.11 this approach, compared to toxin eia only, is much more sensitive. algorithm-based testing is recommended to optimise the positive predicative value of laboratory results.11 commercial eias utilise monoclonal antibodies to detect glutamate dehydrogenase (gdh), an antigen common to all c. difficile strains irrespective of toxin production, and toxin a and/or toxin b (tox a/b). the c. diff quik chek complete eia detects gdh as well as tox a/b. commercial nucleic acid amplification tests (naats) are usually real-time polymerase chain reaction (pcr) assays which target tcdb. in this study, the performance of c. diff quik chek complete (quik chek) (alere techlab, blacksburg, virginia, united states) and two commercial naats, the xpert c. difficile (xpert) (cepheid, sunnyvale, california, united states) and bd max cdiff (bdm) (becton dickinson, san jose, california, united states) were compared to tc. the performance of a two-step approach consisting of the quik chek and xpert, that is, the current standard of care (soc) at our institution, was also evaluated. methods ethical considerations ethics approval was obtained from the health research ethics committee of stellenbosch university, cape town, south africa (reference number s17/03/064). a waiver of informed consent was obtained as no patient identifiers were published and no invasive procedures were performed as a result of this study. only the results of the current soc tests were reported to physicians. after the results were recorded, a study number was assigned to the specimen and all patient identifiers were removed. study design this was a prospective diagnostic test accuracy study comparing three different assays, namely the quik chek, xpert and bdm, to tc (reference method) for the detection of toxigenic c. difficile in faecal samples. the performance of the current soc (a two-step approach consisting of the quik chek and xpert), at our institution was also compared to tc. a composite reference standard (crs) analysis was performed to account for limitations in tc as a reference method (supplementary material).12 the crs composite positive was defined as tc-positive or positive by two commercial assays in a tc-negative sample. sample size tygerberg hospital near cape town, south africa, is a 1380-bed tertiary hospital which delivers specialist services to approximately half the population of the western cape province (total population 6.2 million). the microbiology laboratory of the national health laboratory service at tygerberg hospital performs c. difficile testing on patient samples from tygerberg hospital as well as peripheral hospitals and clinics within the tygerberg drainage area. assuming a c. difficile prevalence of 16% based on previous studies, and using a 95% confidence interval with a 5% error rate on both sides, a sample size of 207 was calculated using the open epi sample size calculator (g dean & km sullivan, atlanta, georgia, united states).13 sample processing non-duplicate loose stool samples (defined as taking the shape of the container) from adult and paediatric patients older than two years of age submitted to the national health laboratory service microbiology laboratory at tygerberg hospital from october 2017 to october 2018 for routine c. difficile testing were tested in parallel with the four assays. none of the samples was frozen and thawed prior to testing. in the rare event that samples could not be tested on the day of collection, they were kept at 2 °c – 8 °c and processed within 48 h of collection. the quik chek eia was performed as per instructions provided by the manufacturer and read by multiple laboratory technologists as part of their routine daily work. the results were interpreted as follows: gdh-positive and tox a/b-positive samples were regarded as positive, gdh-negative and tox a/b-negative samples were regarded as negative, and gdh-positive and tox a/b-negative samples were regarded as negative. the xpert naat was performed as per the instructions of the manufacturer. the test was interpreted as positive for toxigenic c. difficile if the cytotoxin gene (tcdb) was detected within the valid cycle threshold range and above the minimum endpoint setting, and as toxigenic c. difficile negative if the tcdb gene was not detected, provided the sample processing control and probe check controls met the manufacturer’s requirements. testing was repeated on any samples with invalid or error results due to failure of the sample processing control or probe check controls. the bdm naat was performed by following the instructions of the manufacturer. test results were automatically interpreted by the bdm instrument. positive, negative and unresolved results were based on the target’s and sample processing control’s amplification status. the test was interpreted as toxigenic c. difficile positive if the cytotoxin gene (tcdb) was detected within the valid cycle threshold range and above the minimum endpoint setting, and as toxigenic c. difficile negative if the tcdb gene was not detected, provided the sample processing and probe check controls met the manufacturer’s requirements. indeterminate and incomplete results are obtained when the bdm system fails. any unresolved, indeterminate and incomplete samples were repeated. the two-step algorithm was performed as follows: the quik chek was done and samples that were gdh-positive and tox a/b-positive were regarded as positive; gdh-negative and tox a/b-negative samples were regarded as negative. samples that were gdh-positive and tox a/b-negative were interpreted in conjunction with the xpert results to establish if toxin genes were present or absent. toxigenic culture was used as the reference method and performed by culturing c. difficile from stool samples on chromid c.diff agar (biomérieux, marcy l’etoile, france), a differential and selective medium, followed by testing for the organism’s ability to produce toxin by pcr.14,15,16 a swab was dipped into the stool sample and inoculated onto the agar medium. the inoculum was streaked to enhance the recovery of single colonies. the plates were placed in a jar with an anaerobic sachet, anaeropack-anaero (mitsubishi gas chemical company, inc., tokyo, japan) and incubated at 35 ºc. after 48 h of incubation, the plates were examined for grey and black colonies; if none were present, the culture was considered negative for c. difficile. a multiplex pcr was performed on a streak of grey and black colonies to detect the c. difficile-specific tpi gene, as well as the tcda and tcdb genes.17 cultures that were pcr-positive for the tpi and tcdb genes were considered tc-positive, while those that were pcr-negative for the tpi or tcdb genes were considered tc-negative. in humans, the tcda gene has been reported in tcdb-positive c. difficile strains only.18 statistical analysis sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) were calculated for each assay against both tc and the crs using epicalc 2000 version 1.00 software (brixton books, london, england). results a total of 223 samples were included in the study. of these, 37 (16.6%) were tc-positive and 19/37 (51.4%) were also positive from all three commercial assays. the soc positivity rate was 17.9% (40/223). quik chek was gdh-pos in 45/223 (20.2%) samples. twenty-two of the 45 samples (48.9%) were tox a/b-pos, of which 20/22 (91%) were also tc-positive. of the 23 gdh-pos samples that were tox a/b-neg, 11 (47.8%) were tc-positive (table 1). table 1: performance characteristics of diagnostic assays in comparison with toxigenic culture on stool samples submitted to the national health laboratory service microbiology laboratory at tygerberg hospital, cape town, south africa, from october 2017 to october 2018.1 thirty-eight of the 223 stools (17%) tested positive by xpert and 32/38 (84.2%) were confirmed as positive by tc. none of the samples was positive for the binary toxin or epidemic 027/nap1/bi strain. of the 185 xpert-negative stools, 180 (97.3%) were confirmed as negative by tc, while 5/185 (2.7%) xpert-negative samples were tc-positive (table 1). the bdm detected 39/223 (17.5%) positive stool samples; 33/39 (84.6%) were confirmed by tc. one hundred and eighty of the 184 bdm-negative stools (97.8%) were confirmed as negative using tc, while the other 4/184 (2.2%) were tc-positive. the soc two-step approach detected 40 positive stool samples, 33 (82.5%) of which were confirmed by tc. of the 183 soc-negative stool samples, 179 (97.8%) were confirmed as negative by tc (table 1). the quik chek performed poorly while the xpert and bdm and the two-step approach had similar sensitivities, specificities, ppv and npv when compared to tc. results were also compared to a crs (supplementary table 1). the xpert, bdm and two-step approach showed higher sensitivities, specificities and ppv in comparison with the crs, but the npv of all the assays were similar. discussion there is a lack of consensus regarding the optimal diagnostic c. difficile laboratory assays. high-quality evidence for the best diagnostic testing strategy is scarce and researchers rarely use either of the two accepted reference standards, that is, cell culture neutralisation assay or tc, for assessment of diagnostic accuracy, but rather their own laboratory-defined crs criteria. in this study, we determined the diagnostic accuracy of three different commercial assays for the detection of toxigenic c. difficile in stools compared to both tc and a crs to account for any limitations of the tc. in this study, the sensivitity of the quik chek was 54.1%, xpert was 86.4%, bdm was 89.2% and the two-step approach was 89.2%. the specificity for quik chek was 98.9%, for xpert was 96.8%, for bdm was 96.8% and for the two-step approach was 96.2%. the ppv of the quik chek was 90.9%, xpert was 84.2%, bdm was 84.6% and the two-step approach was 82.5%. the npv for quik chek was 91.5%, xpert was 97.3%, bdm was 97.8%, and the two-step approach was 97.2%. clostridioides difficile toxin eias lack sensitivity. all c. difficile contain the gdh antigen, whether toxin genes are present or absent. glutamate dehydrogenase eias have high sensitivities but poor specificities; therefore, an additional test (most commonly a toxin assay) must be performed. the gdh eia is the first test performed in a two-step or three-step approach where a gdh-positive result is followed by a toxin assay or a naat to detect toxin genes.11 our findings for the quik chek showed a sensitivity, specificity, ppv and npv of 54.1%, 98.9%, 90.9% and 91.5%, respectively. these findings were similar to a study conducted in 2012 in kuwait comparing the xpert, quik chek and tc, which found the sensitivity, specificity, ppv and npv of the quik chek to be 53.85%, 100%, 100% and 98.51%, respectively, when using a crs defined as two tests being in agreement.19 a 2009–2018 study conducted by chung and lee in korea compared the diagnostic performance of the quik chek to xpert as a reference test, with sensitivity, specificity, ppv and npv of 55.4%, 100.0%, 100.0% and 80.0%, respectively.20 however, they had a higher prevalence of c. difficile infection (35.9%) than our study population (16.6%), which could explain the difference in ppv (100.0% vs 90.9%).20 in contrast, a study conducted in 2013–2014 by seo et al. in korea, found a lower sensitivity of 45.7% when using either tc or the combination of quik chek and xpert as a reference standard.21 nucleic acid amplification tests that target chromosomal toxin genes show high sensitivity and specificity, provide rapid results, and are amenable to both batch and on-demand testing.22 reported estimates of sensitivity for naats range from 77% to 100% when compared to tc. specificity ranges from 83% to 100% in comparison to tc.23 nucleic acid amplification tests may detect asymptomatic carriage due to possible non-expression of the toxin encoding gene and therefore the clinical specificity may be lower than reported.24 in their meta-analysis conducted in 2019, kraft et al. reported an estimated sensitivity of 94% and a specificity of 97% when naats were compared to either cell culture neutralisation assay or tc or both in studies where it was specifically stated that stools were only included if conforming to the shape of the container.10 in this study, the xpert had a sensitivity, specificity, ppv and npv of 86.4%, 96.8%, 84.2% and 97.3% respectively, and the bdm performed similarly showing a sensitivity, specificity, ppv and npv of 89.2%, 96.8%, 84.6% and 97.8%, respectively. a study by yoo et al. in korea found a sensitivity of 82.8% for xpert and 81.6% for bdm when compared to tc. they attributed the difference in sensitivity between the two tests to a freeze-thaw cycle before the bdm testing.25 in a study conducted in germany, dalpke et al. found a sensitivity, specificity, ppv and npv of 97.3%, 97.9%, 90.0% and 99.5% for the xpert and 90.5%, 97.9%, 89.3% and 98.1% for the bdm.26 in a study conducted 2014–2017 in south africa demonstrated the impact of diagnostic methods on the diagnosis of c. difficile infection, nomlomo et al. found a 15.9% positivity rate when using an algorithm approach (consisting of eia followed by pcr) versus 11.4% and 21.1% when using a toxin eia and pcr.27 however, neither of the two accepted references tests was performed as the comparator test in this study. we showed a 16.6% tc positivity rate which is very similar to nomlomo et al.’s finding using the algorithm approach.27 similar to the sensitivity of 89.2% and specificity of 96.2% for the two-step algorithm approach in our study, kraft et al.’s meta-analysis found a sensitivity of 89.0% and specificity of 99.0% when comparing gdh/toxin/naat algorithms to the tc or cell culture neutralisation assay.10 in contrast, the seo et al. study found a higher sensitivity, specificity, ppv and npv of 94.0%, 100.0%, 100.0% and 100.0% for the two-step approach, which could be attributed to the crs in this study being either tc-positive or a combination of xpertand quik chek-positive.21 our algorithm performed similarly to the xpert or the bdm alone in terms of sensitivity, specificity, ppv and npv. the xpert, bdm and two-step algorithm performed similarly, with overlapping confidence intervals when compared to tc and crs. from these findings it is evident that tc is a robust reference test for the statistical measures of sensitivity, specificity, ppv and npv for the xpert, bdm and two-step approach. limitations limitations of our study include the use of stool sampless conforming to the shape of the container as a surrogate for diarrhoea, as well as not excluding other causes of diarrhoea. in addition, we did not collect pre-analytical data such as prior or current antibiotic use or determine any other risk factors for c. difficile. post-analytical patient outcome data was also not collected. the sensitivity of lateral flow assays is limited by the dissociation constant of the antibody–antigen conjugate and by user interpretation of the colorimetric read-out. the tc method used in this study did not include heat-shock treatment prior to the inoculation of samples onto media, which may have improved the detection of c. difficile. conclusion the xpert and bdm assays and two-step approach performed similarly in detecting toxigenic c. difficile in faecal samples. quik chek cannot be used on its own for the diagnosis of c. difficile due to its poor sensitivity but the soc two-step approach using quik chek followed by xpert showed a similar sensitivity and ppv compared to molecular testing alone. the continued use of the current two-step approach in a resource-limited setting such as south africa is recommended as it is rapid, easy to perform and reduces cost without compromising diagnostic accuracy. acknowledgements the authors would like to thank bd diagnostics for partly funding the bdm kits and placement of the bdm instrument, dr motlatji maloba from the division of medical microbiology, university of the free state and national health laboratory service, bloemfontein, south africa, for her guidance with the study protocol design, brian kullin from the department of molecular and cell biology, university of cape town, south africa, for providing the toxigenic c. difficile control strains, and the biostatistics unit, division of epidemiology and biostatistics, faculty of medicine and health sciences, stellenbosch university, south africa, for their assistance with the statistical analysis of data. competing interests bd diagnostics provided the bdm instrument for the duration of the study and partially sponsored the bdm kits used in the study; however, bd diagnostics was not involved in the study design, collection, analysis and interpretation of data, writing of the article, or in the decision to submit the article for publication. authors’ contributions s.s. was involved in the conceptualisation, methodology, validation, investigation, data curation, original draft preparation and funding acquisition. m.n.-f. contributed to the conceptualisation, methodology, software, validation, data curation, reviewing and editing, supervision and project administration. p.n. and c.p. assisted with the methodology, reviewing and editing, supervision and project administration while c.p. also contributed to the visualisation. sources of support this research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. data availability raw data were generated at the national health laboratory service. derived data supporting the findings of this study are available from the corresponding author, s.s., on request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references burnham ca, carroll kc. diagnosis of clostridium difficile infection: an ongoing conundrum for clinicians and for clinical laboratories. clin microbiol rev. 2013;26(3):604–630. https://doi.org/10.1128/cmr.00016-13 depestel dd, aronoff dm. epidemiology of clostridium difficile infection. j pharm pract. 2013;26(5):464–475. https://doi.org/10.1177/0897190013499521 kullin b, meggersee r, d’alton j, et al. prevalence of gastrointestinal pathogenic bacteria in patients with diarrhoea attending groote schuur hospital, cape town, south africa. s afr med j. 2015;105(2):121–125. https://doi.org/10.7196/samj.8654 abdullatif vn, noymer a. clostridium difficile infection: an emerging cause of death in the twenty-first century. biodemography soc 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2015;83(1):7–10. https://doi.org/10.1016/j.diagmicrobio.2015.05.005 dalpke ah, hofko m, zorn m, zimmermann s. evaluation of the fully automated bd max cdiff and xpert c. difficile assays for direct detection of clostridium difficile in stool specimens. j clin microbiol. 2013;51(6):1906–1908. https://doi.org/10.1128/jcm.00344-13 nomlomo e, nana t. the impact of diagnostic methods on the diagnosis of clostridiodes difficile infection. s afr med j. 2020;110(2):135–139. https://doi.org/10.7196/samj.2020.v110i2.13684 abstract introduction methods results discussion acknowledgements references about the author(s) katherine e. hodkinson department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa institution of national health laboratory service, johannesburg, south africa nikki bouwer department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa institution of national health laboratory service, johannesburg, south africa jenifer vaughan department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa institution of national health laboratory service, johannesburg, south africa citation hodkinson ke, bouwer n, vaughan j. south african study of blast phase chronic myeloid leukaemia: a poor prognostic outlook. afr j lab med. 2022;11(1), 1578. https://doi.org/10.4102/ajlm.v11i1.1578 original research south african study of blast phase chronic myeloid leukaemia: a poor prognostic outlook katherine e. hodkinson, nikki bouwer, jenifer vaughan received: 11 mar. 2021; accepted: 22 feb. 2022; published: 31 may 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: chronic myeloid leukaemia (cml) is a haematological malignancy characterised by the translocation t(9;22)(q34;q11.2), resulting in a constitutively active tyrosine kinase. globally, overall survival of blast crisis phase (bc) cml is one year. newer tyrosine kinase inhibitors and allogeneic stem cell transplantation offer remission; however, refractory and relapsed disease remain the biggest challenges. objective: in south africa, literature is lacking on bc-cml. this study aimed to determine the disease characteristics and overall survival in south africa. methods: this retrospective, laboratory-based study reviewed all new bc-cml diagnoses via flow cytometry at charlotte maxeke johannesburg academic hospital in johannesburg, south africa, between april 2016 and october 2019. bc-cml was defined as the presence of > 20% blasts with a cml history or the bcr-abl1 fusion gene (p210/p190) in the appropriate clinical or pathological context. survival outcomes were inferred from clinical and laboratory data. results: twenty-two new cases of bc-cml were diagnosed (median age: 34 years). there were 20 (91%) cases with the fusion transcripts p210 and two (9%) cases with p190 bcrabl1. for blast lineage, 14 cases were myeloid (63.6%), six were lymphoid (27.3%), and two were ambiguous (9.1%). there was a 72.7% mortality (16 cases); sepsis, refractory and relapsed disease were the major causes. patients who achieved remission had lower blast percentages, simple karyotypes, and a trend towards higher white cell and platelet counts at presentation. conclusion: optimised management of early-stage cml, prevention and aggressive management of sepsis, with advocation for newer therapies are needed to improve the overall survival of bc-cml in south africa. keywords: chronic myeloid leukaemia; blast phase; major molecular route abnormalities; south africa; p210 bcrabl1 fusion transcript; p190 bcrabl1 fusion transcript; responses by quantitative reverse transcriptase-polymerase chain reaction; karyotype. introduction chronic myeloid leukaemia (cml) is a haematological malignancy that arises within a pluripotent haemopoietic stem cell.1,2,3 the defining molecular aberration is a translocation involving the long arms of chromosome 9 and 22 (t[9;22][q34;q11.2]) which results in a chimeric breakpoint cluster region-abelson murine leukaemia viral oncogene homolog 1 (bcr-abl1) fusion gene encoding for a constitutively active tyrosine kinase.4,5,6,7 this protein drives the expansion and survival of the clone through the proliferation of the myeloid compartment, anti-apoptotic properties and reduced adherence to the bone marrow niche.8,9,10 chronic myeloid leukaemia is a bior tri-phasic disease that classically progresses through chronic, accelerated, and blast phases, as defined by the world health organization and european leukaemianet criteria.11,12,13,14,15 tyrosine kinase inhibitors (tkis) were the first targeted therapies developed for cml. the introduction of the first-generation tkis as the mainstay therapy in 1998 halted the natural progression of the disease. this has allowed patients to maintain chronic phase disease with the achievement of morphological, clinical, and molecular remission targets. since the introduction of second and third-generation tkis, there has been advancement in both the speed and depth of molecular remission.16 in the united states, the overall survival (os) in chronic phase disease is nearly equivalent to that of the general population when response milestones are achieved.17,18 however, disease progression to accelerated or blast phase still occurs in this era of effective targeted therapy, as a result of drug resistance, cessation of therapy resulting from drug intolerance or side effects, poor compliance, altered metabolism, and drug interactions. in contrast, the diagnosis of chronic myeloid leukaemia in blast crises phase (bc) cml has been associated with a low median os of 12 months in the united states. combination therapy with tkis and intensive chemotherapy, consolidated with an allogeneic haemopoietic stem cell transplant (allosct), offers patients the most favourable outcomes.19 in africa, access to second or third generation tkis and allosct is limited, and there is a paucity of data on the prevalence and outcomes of bc-cml. this study aimed to describe the disease and patient characteristics as well as the os of bc-cml diagnosed in the state sector setting in johannesburg, south africa. methods ethical considerations ethics clearance was obtained from the university of the witwatersrand human ethics committee (protocol number m150160). the study was a retrospective analysis of laboratory results obtained from the laboratory information system (lis) (trakcare, intersystems, cambridge, massachusetts, united states) and patient consent was not a requisite. patient privacy was ensured through the use of unique study numbers, with the link to patient identifying information kept in a separate, password-protected excel spreadsheet. study design this study included all new cases of acute leukaemia diagnosed by flow cytometry at the charlotte maxeke johannesburg academic hospital (cmjah) over 42 months, between 08 april 2016 and 16 october 2019. these cases were identified from the flow cytometry register with the largest proportion of cases received from cmjah, chris hani baragwanath academic hospital (chbah) and helen joseph hospital. the lis was used to collect available clinical data, demographics, laboratory results, treatment responses, os, and cause of death. the results were collated on an excel spreadsheet (microsoft corporation, santa rosa, california, united states) and cases of bc-cml were extracted. inclusion of cases into the bc-cml category required a preceding history of cml or presence of the bcr-abl1 fusion gene (p210/ p190) in the context of splenomegaly, peripheral smear features of cml (leucocytosis, left shift, myelocyte peak, eosinophilia and basophilia) and major molecular route abnormalities on conventional cytogenetics (ccy). statistical analysis the data were analysed using stata software, version 14.1 (statistics/data analysis, statacorp llc, college station, texas, united states). continuous data were reported as the median (interquartile ranges [iqr]) and categorical data as percentages and frequencies. the two-sample wilcoxon rank-sum (mann-whitney) test was used to compare continuous variables. statistical significance was accepted at a two-sided p-value of 0.05. results twenty-two cases of bc-cml were diagnosed by flow cytometry in the reviewed period between 2016 and 2019. the rate of blastic transformation from chronic and accelerated phase cml could not be determined as the prevalence of cml was unknown for the referring centres. patient demographics and treatment history the median age was 34 years (iqr: 21 years) with two patients aged younger than 18 years, and a slight predominance of male patients, 57% (n = 12). hiv testing was performed in less than half (n = 20) of the patients with a 50% positivity (table 1). table 1: demographic and laboratory information for patients with cml in blast phase, johannesburg, south africa, 08 april 2016 to 16 october 2019. information on the use of tki therapy was limited to that documented on the lis. imatinib mesylate therapy was reported in one patient; dasatinib in three; and nilotinib in two. none of the patients had been managed with a trial of treatment-free remission (tfr). molecular and immunophenotypic results twenty cases had p210 bcr-abl1 fusion transcripts, and two cases had p190 bcr-abl1. for blast lineage in bc-cml, 14 cases (63.6%) were myeloid, six cases (27.3%) were lymphoid and two cases (9.1%) were ambiguous. of the lymphoid cases, approximately two-thirds (n = 4) were of b lineage. the p210 case lineages were myeloid in 13 cases; t in one case, b in four cases; and ambiguous in two cases. the p190 cases were of myeloid and t lineage, with the latter relapsing as an ambiguous lineage acute leukaemia (t/myeloid). additional clonal chromosomal abnormalities were detected on ccy in 11 of the 16 individuals with successful karyotypes. complex karyotypes and trisomy 8 were the most frequently observed, with five ccy demonstrating multiple additional clonal chromosomal abnormalities (figure 1). one patient bore the t315i mutation at the time of diagnosis of bc-cml with a history of treatment failure on both imatinib mesylate and nilotinib. figure 1: frequency of additional clonal chromosomal abnormalities in patients with cml in blast phase (n = 16) johannesburg, south africa, 08 april 2016 to 16 october 2019. cytogenetics analysis was available for 16 patients. additional clonal chromosomal abnormalities was identified in 11 patients. five of the karyotypes demonstrated multiple additional clonal chromosomal abnormalities. trisomy 19 and isochromosome 17q were always present with a trisomy 8. patient outcomes there was a 72.7% (16 cases) mortality with a median os of 7 months (iqr: 13.6 months) (figure 2). the details of the therapeutic management of each bc-cml was not available. five deaths occurred in the first month with sepsis the predominant cause in 80% (four cases). the remaining 11 deaths occurred at a median of 6.7 months (iqr: 4.5 months) with two-thirds of the deaths attributed to relapsed and/or refractory disease, and one-third to sepsis. patients with the p190 bcr-abl1 fusion transcript survived a median of 6.1 months (iqr: 3.3 months) with relapsed disease as cause of death. figure 2: overall survival of patients with cml in blast phase johannesburg, south africa, 08 april 2016 to 16 october 2019, excluding one patient, who was lost to follow-up. at the time of the study, remission with reversion to chronic phase cml (based on laboratory parameters) was achieved in 22.7% (five cases) with a median remission time of 25.6 months (iqr: 12.5 months). the disease characteristics in the remission group included a dominance of myeloid lineage (80%, four cases) with a second philadelphia (ph) chromosome being the only additional abnormality present in one of the three patients with successful ccy. the molecular responses by quantitative reverse transcriptase-polymerase chain reaction were extremely varied from undetectable transcript levels to no molecular response (figure 3). one patient was lost to follow-up at one year with achievement of a major cytogenetic response at this time point. figure 3: cumulative quantitative reverse transcriptase-polymerase chain reaction results for patients in remission, from time of diagnosis of cml in blast phase (n = 5), johannesburg, south africa, 08 april 2016 to 16 october 2019. the following were the demographics of patients 1 to 5; patient 1: 51-year-old female; patient 2: 26-year-old hiv-positive male; patient 3: 19-year-old hiv-negative female; patient 4: 54-year-old male; patient 5: 37-year-old male. all patients had a p210 bc-cmlrabl1 transcript, the blast lineage was t in patient 2 and myeloid in the remainder. johannesburg, south africa, 08 april 2016 to16 october 2019. the median peripheral blood blast percentage at diagnosis was significantly higher in patients who died, 56% (16 cases) versus those in remission 18% (five cases) (p = 0.03). conversely, the white cell and platelet counts at diagnosis were lower among patients who died, but the difference was not statistically significant (white cell count for deceased patients: median = 37.3 (iqr: 175.4) versus for patients in remission: median, 144.2 (iqr: 367.4, p = 0.52) and the platelet count for deceased patients: median, 31 (iqr: 177) versus for patients in remission: median, 101 (iqr: 324, p = 0.54) (table 1). discussion this retrospective laboratory report review determined the disease charatersistics and overall survival of bc-cml in south africa. the median age of bc-cml in our study was significantly lower than the 61 years reported internationally and corresponds with the younger age at diagnosis of cml in our setting.20,21,22 this finding may be reflective of south african demographics, which are characterised by a young age structure of the population or alternatively this may be the result of unknown environmental exposures or unique genetic/biological characteristics. consequently, this young age at diagnosis has a financial impact upon the public health sector, in particular for chronic phase cml. the cost resulting from the earlier initiation, and thus additional years on tki therapy, is substantial. the risk factors for the development of cml remain largely unknown with acute radiation exposure and rare cases of possible familial predisposition reported.19 to our knowledge there were no cases of familial cml in our study nor have there been published cases from south africa. progression from chronic phase cml to bc-cml could not be determined from our study as a result of the paucity of local prevalence data. indirectly, the rates of transformation at the chbah appear high as compared with international reports (~2%), with a yearly number of ~3 new adult cases of bc-cml, in a background of ~12 new cases of cml per year.20,23 plausible reasons for higher rates of transformation include the development of tki resistance in the initial stages of cml as a result of poor compliance or treatment interruptions, and late presentation of patients to medical facilities resulting in more advanced stage disease. the lineage distribution of bc-cml in this study is similar to that reported in the literature with myeloid lineage accounting for around ~52% to 67% and lymphoid for around ~19% to 35%.24,25 the proportion of ambiguous lineage cases in our setting was similar to that seen in studies performed in taiwan and mexico, reported at ~12% to 14%.24,25 for the lymphoid lineage, an expected ratio of b to t cases was present in our study.24,25 this suggests a similar disease biology and accordingly supports the use of international treatment approaches/guidelines for bc-cml, with the ability to compare treatment and survival endpoints. the p210 bcr-abl1 fusion (e13a1 [b2a2]/e14a2 [b3a2]) is the most common transcript seen in cml. co-expression of p210 with p190 (e1a2) is described, but a very rare phenomenon. rarer still is an isolated p190 bcr-abl1 fusion which has been described in 1% of cml. this latter group is associated with poor response to tki therapy and disease progression in greater than 80% of the group.26 these characteristics likely explain the higher than expected number of p190 bc-cml cases in this study (9.1%). the median (iqr) os in these cases is reported at ~13 months (8–40), a figure which is double that seen in our study. the reason for this difference likely reflects a lack of alternative or novel treatment options for relapsed/refractory disease in the south african public health sector.27 given the high risk of this subgroup of patients, close monitoring of the therapeutic response is imperative (especially during the early phases of cml), with early intervention should the response be lost.26 high-risk cytogenetic abnormalities in cml include a complex karyotype and major molecular route abnormalities which are markers of disease acceleration. this is associated with more rapid disease progression, treatment resistance or suboptimal treatment response.28 the major molecular route abnormalities include a second philadelphia chromosome, isochromosome i (17q), trisomy 8, and 19.29 unsurprisingly, a high percentage of the bc-cml cases with existing ccy bore these abnormalities. as a result of the low number of cases in remission and with ccy, correlations between molecular characteristics and outcomes could not be made. however, a second philadelphia chromosome was the only additional chromosomal abnormality present. notably, in ~30% of cases, no sample was submitted for ccy or the procedure was unsuccessful. ccy is critical to prognostication, detection of clonal evolution, monitoring treatment response and directing therapeutic decisions. practically, in such cases, peripheral blood may serve as an easily accessible, alternative source for ccy. cognisance of this would help to limit the number of cases missing valuable molecular information. notably, but not unexpectedly, alternative markers of high-risk cml were present in this cohort; these included peripheral blood blast percentages of > 10%, p190 bcr-abl1 transcripts and platelet counts of < 102 × 10^9/l.19,30 the tkis for the treatment of chronic phase cml at the time of this study included: the first-generation tki, imatinib mesylate and second-generation tki, dasatinib (on a compassionate basis) and nilotinib. currently, in the south african state sector, imatinib mesylate remains the first-line therapy together with hydroxyurea (for cytoreduction) in chronic phase cml. nilotinib is the second line tki and is used in situations of treatment failure. dasatinib is no longer available on compassionate grounds. importantly, comparable response rates and survival outcomes have been demonstrated for these second-generation tkis.31 despite a minimal risk of worsening renal dysfunction and bleeding in those with pre-existing renal dysfunction, treated with nilotinib or dasatinib, both agents can be used in the majority of patients with mild to moderate renal dysfunction as well as those with mild liver dysfunction.32 in the situation of a t315i mutation or treatment resistance to two sequential tkis, motivation can be made for the potent third-generation tki, ponatinib. with life expectancies nearing that of the general population and the associated chronicity of treatment, the practice of tfr was introduced. this refers to the discontinuation of tki therapy with a durable off-treatment molecular response. treatment-free remission aims to limit drug toxicities and adverse effects, reduce the financial burden of therapy and prevent teratogenicity. several guidelines have been developed to identify patients eligible for tfr; the criteria include age ≥ 18 years, the presence of typical bcr-abl1 transcripts, tki therapy used for a minimum of five years, the achievement of a deep molecular response (dmr) ≤ 4.5, a sustained dmr of ≤ 4–4.5 for ≥ 2–3 years, no prior history of accelerated or bc-cml and the ability to perform frequent (monthly initially) responses by quantitative reverse transcriptase-polymerase chain reaction testing which is both sensitive (minimum mr 4.5) and delivered within a rapid turnaround time.33,34,35 given these requirements, tfr is not commonplace in the south african public sector, because it requires frequent follow-ups and timeous molecular results. the reported success of tfr is about 40% to 60%, with the possibility to regain a dmr in those who fail if intervention is prompt.36 thus future studies should aim to identify factors that can predict a successful tfr. the median os in our cohort was almost half of that reported in a united states-based study (7 vs 12 months), driven by a substantial proportion of patients with early deaths (defined as deaths within the first month of diagnosis).19 these were predominantly sepsis-related, highlighting the need for improved and optimised supportive care for these patients. in our setting, bc-cml is treated with high dose imatinib mesylate and combination chemotherapy, the latter determined by the blast lineage.37 additionally, intrathecal chemotherapy is initiated in patients with lymphoid bc-cml. at the time of the study, allosct was not routinely available at any of the treatment centres in this cohort, further driving the poor outcomes.38,39,40,41 reports suggest that allosct gives the best chance of long-term remission. however, in a minority, there could be a return to pre-transplantation chronic phase disease.37,42 globally, the outcomes of bc-cml are poor and novel treatment approaches are needed. experience in the united states with dasatinib monotherapy showed a median os of 11.8 months in myeloid and and 5.3 months in lymphoid bc-cml.43 the combination of hyper-cvad (hyperfractionated cyclophosphamide, vincristine sulfate, doxorubicin hydrochloride, and dexamethasone) with dasatinib in lymphoid bc-cml resulted in a 5-year os rate approaching 60%, which was further improved to 88% when followed by allosct in the first remission.40,44 use of the selective bcl2 inhibitor venetoclax has shown synergistic effect when used in combination with dasatinib in heavily pre-treated patients with bc-cml (myeloid), with a median os of 10.9 months (with particular benefit noted in those with a dominant bc-cmlr-abl1 clone).45 unfortunately, these novel agents and combination therapies are not available within the south african state sector and allosct is not uniformly accessible. there is a great need to advocate for these new and novel approaches to improve the currently dismal outcome of this disease in our setting.37 a separate challenge faced for clinicians in the management of cml is the need to screen and monitor for the development of cardovascular events and second malignancies.46 there is a significantly increased incidence of cardiovascular and arteriothrombotic events (incidence ratio 95% confidence interval: for cardiovascular of 8.6 [7.6–9.8], and for arteriothrombotic of 1.7 [1.2–2.2]) in patients using second and third-generation tkis (except bosutinib) and in individuals with pre-existing cardiovascular risk factors.46,47 in regard to second malignancies, a large united states-based study reported a slightly increased risk of cml (overall standardised incidence ratio of 1.204, 95% confidence interval: 1.108–1.301, except breast cancer) and a higher risk of death in this group.44 the most common sites of involvement included the digestive, skin, and urinary systems. the reason for this increased incidence is unknown but may reflect a patient-specific risk of malignancy (genetic/exposure).46 given these findings it is imperative to identify and aggressively manage cardiovascular risk factors at baseline to prevent morbidity and mortality. screening for second malignancies poses more of a challenge as a result of the varied sites of potential involvement and the costs involved in routine radiological screening and endoscopies. realistically in our setting, the clinical history and findings are used as a screening tool to identify patients that require further work-up for second malignancies. at present, the most effective management of bc-cml is its prevention. this is through the achievement of rapid and deeper molecular responses in chronic and accelerated disease, identification of high-risk individuals with close monitoring (laboratory, clinical) and timeous therapy changes in non-responders. more advocay for new and novel treatment approaches in our setting is necessary. limitations the limitations in this study include the paucity of clinical information and reliable outcome data as a result of the retrospective design. cases without flow cytometry, especially isolated extramedullary transformation, were missed. furthermore, molecular information, namely ccy or responses by quantitative reverse transcriptase-polymerase chain reaction, was not available for a notable proportion of the patients, preventing an accurate picture of the molecular landscape of bc-cml. lastly, given the rarity of this entity, factors which affect outcomes are difficult to determine. conclusion a laboratory-based retrospective study of bc-cml was performed in a public health sector setting in south africa. the results showed that bc-cml occurs at a younger age in adults as compared with international studies. this corresponds to the younger age of chronic phase cml seen in our setting, a finding which may reflect the country demographics, different environmental exposures or genetic predispositions. disease characteristics were similar when compared with other reports of bc-cml, supporting the use of international treatment protocols and guidelines. there was a dismal overall survival of 7 months, with sepsis and relapsed refractory disease being the major contributors, highlighting the need to advocate for new and novel therapies. acknowledgements we would like to acknowledge all the staff members of the following units for their invaluable contributions towards leukaemia diagnosis and management in johannesburg: the flow cytometry laboratory at the cmjah, the somatic cell genetics unit at the cmjah, the morphology units of the cmjah, the chbah and helen joseph hospital, the medical oncology unit at the cmjah, the paediatric oncology units at the cmjah and chbah, and the clinical haematology unit at the chbah. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions k.e.h., n.b. and j.v. were involved in the conceptualisation and methodology, k.e.h. and j.v. in data collection, k.e.h. in data analysis and drafting the initial manuscript, n.b. and j.v. in editing and all authors discussed the results and contributed to the final manuscript. sources of support this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. data availability the authors confirm that the data supporting the findings of this study are available within the article. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references fialkow pj, jacobson rj, papayannopoulou t. chronic myelocytic leukemia: clonal origin in a stem cell common to the granulocyte, erythrocyte, platelet and monocyte/macrophage. am j med. 1977;63(1):125–130. 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https://doi.org/10.1002/cncr.33539 maiti a, franquiz mj, ravandi f, et al. venetoclax and bcr-abl tyrosine kinase inhibitor combinations: outcome in patients with philadelphia chromosome-positive advanced myeloid leukemias. acta haematol. 2020;143(6):567–573. https://doi.org/10.1159/000506346 sasaki k, kantarjian hm, o’brien s, et al. incidence of second malignancies in patients with chronic myeloid leukemia in the era of tyrosine kinase inhibitors. int j hematol. 2019;109(5):545–552. https://doi.org/10.1007/s12185-019-02620-2 jain p, kantarjian h, boddu pc, et al. analysis of cardiovascular and arteriothrombotic adverse events in chronic-phase cml patients after frontline tkis. blood adv. 2019;3(6):851–861. https://doi.org/10.1182/bloodadvances.2018025874 abstract introduction methods results discussion acknowledgements references about the author(s) howard newman national health laboratory service, virology, port elizabeth, south africa department of pathology, division of medical virology, stellenbosch university, cape town, south africa faculty of health sciences, nelson mandela university, port elizabeth, south africa donald tshabalala department of paediatrics, nelson mandela academic hospital, mthatha, south africa department of paediatrics, walter sisulu university, mthatha, south africa sikhumbuzo mabunda department of public health, walter sisulu university, mthatha, south africa mpumalanga department of health, nelspruit, south africa nokwazi nkosi department of pathology, division of medical virology, stellenbosch university, cape town, south africa national health laboratory service, tygerberg academic hospital, cape town, south africa candice carelson national health laboratory service, virology, port elizabeth, south africa citation newman h, tshabalala d, mabunda s, et al. rapid testing for respiratory syncytial virus in a resource-limited paediatric intensive care setting. afr j lab med. 2020;9(1), a1084. https://doi.org/10.4102/ajlm.v9i1.1084 brief report rapid testing for respiratory syncytial virus in a resource-limited paediatric intensive care setting howard newman, donald tshabalala, sikhumbuzo mabunda, nokwazi nkosi, candice carelson received: 27 aug. 2019; accepted: 12 aug. 2020; published: 08 dec. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract we analysed the performance characteristics of the respiratory syncytial virus lateral flow rapid antigen assay in use when compared to a multiplex polymerase chain reaction for detection of respiratory viruses. the study was conducted at a tertiary paediatric hospital in port elizabeth, south africa, from 01 january 2017 to 31 december 2018. we found the clinical sensitivity (36.8%) of the rapid test to be too low for routine diagnostic use. knowledge of assay performance characteristics of rapid tests are important for appropriate interpretation of rapid test results. keywords: respiratory syncytial virus; rapid antigen tests; respiratory viruses; respiratory multiplex polymerase chain reaction; assay performance characteristics. introduction respiratory syncytial virus (rsv) infection is a common cause of casualty visits and hospital admissions in infants.1,2,3,4 in 2005, a meta-analysis reported that between 66 000 and 160 000 children under age 5 years died of rsv infection worldwide.4 during the south african rsv season, 50% – 60% of all respiratory admissions in children are due to rsv.5 data from the 2016 pneumonia surveillance programme in south africa showed that 17% of enrolled patients tested positive for rsv, with a case fatality rate of less than 1%.6 rsv is highly contagious and numerous hospital outbreaks have been reported in multiple age groups,7 thus necessitating appropriate infection prevention and control measures. for timeous initiation of appropriate infection prevention and control measures, rapid laboratory confirmation of rsv is essential. options for laboratory testing include virus isolation, rapid antigen tests, direct fluorescent antibody tests and molecular methods such as polymerase chain reaction (pcr).8,9 rapid assays utilising antigen capture technology, that can be performed in less than 30 min, are available.10 numerous commercial assays exist, ranging in sensitivity from approximately 50% to 96% in children, while the majority of assays have specificity above 97%.1,11,12,13,14,15,16 the sensitivity of rapid antigen assays for rsv is affected by several factors; decreased sensitivity was noted in patients with a prolonged duration of symptoms at the time of testing, infection with subtype b, and older age.13,14,15,16 nucleic acid amplification tests have been shown to be superior to classical methods such as virus isolation and direct fluorescent antibody testing.17,18 not only is pcr more sensitive and specific than conventional methods, but it is also more amenable to ‘multiplexing’, thus allowing for the simultaneous detection of a panel of common respiratory viruses.19 one limitation of molecular testing is its potentially prohibitive cost. this is of particular concern in resource-limited settings. in addition, the laboratory turn-around time may not be rapid enough to aid clinical decision making, especially when samples have to be referred to distant laboratories.20 the commercial rapid antigen rsv assay used in the virology laboratory of the national health laboratory service in port elizabeth, south africa, is the rsv respi-strip by coris bioconcept (gembloux, belgium). it has a reported sensitivity of approximately 80%, with a specificity of greater than 95%.16 when compared to direct fluorescent antibody tests, the rsv respi-strip was found to be 92% sensitive and 98% specific.21 if the reported sensitivity of the assay of 80% is accurate, while not ideal, this may still be a clinically useful test in resource-limited settings, since positive cases would be detected 80% of the time, allowing for patient cohorting and isolation, and thereby limiting nosocomial transmission.7 for respiratory multiplex pcr testing, the virology laboratory in port elizabeth refers specimens to another national health laboratory service virology laboratory at tygerberg hospital in cape town, south africa. this laboratory makes use of the seeplex® rv16 assay by seegene (seoul, south korea). the sensitivity and specificity for the individual viruses varies. for rsv, the sensitivity and specificity is reported to be above 90% when compared with singleplex or duplex pcr.17 since patients admitted to our paediatric intensive care unit (icu) for suspected lower respiratory tract infection are all tested for rsv with a rapid antigen assay, in addition to testing for a panel of common respiratory viruses (including rsv subtypes a and b) by pcr, we compared the two assays to determine the sensitivity and specificity of the rsv respi-strip assay. we additionally sought to describe the common respiratory viruses detected in our icu setting. methods ethical considerations this study received ethical clearance from the human research committee of the faculty of health sciences, walter sisulu university (reference 043/2018). study design this was a descriptive, retrospective cross-sectional study analysing laboratory reports for rsv rapid antigen and multiplex pcr tests for respiratory viruses. results from the rsv rapid antigen assay were compared to results from the multiplex pcr assay, thus allowing for the calculation of performance characteristics of the rapid antigen assay. factors associated with false-negative rsv rapid antigen results were analysed. sample selection the study population comprised all paediatric patients admitted to icu at dora nginza hospital in port elizabeth, eastern cape province, south africa, who had respiratory samples taken for laboratory investigation of viral infections from 1 january 2017 to 31 december 2018. rapid testing for rsv is performed locally by the virology laboratory of the national health laboratory service in port elizabeth, south africa, with the nasopharyngeal aspirates being the specimen matrix. in addition, remnant specimen is referred to another virology laboratory (cape town, south africa) within the national health laboratory service, for multiplex pcr. this allowed for retrospective analysis of laboratory data. data analysis all variables were captured and coded in microsoft excel 2013 (microsoft corporation, seattle, washington, united states) and exported to stata 14.1 for analysis (stata corp lp, college station, texas, united states). the distribution of age in days (numerical variable) was explored using the shapiro wilk test, and age was further converted into a categorical variable in months. clinical sensitivity and specificity of the rsv respi-strip rapid antigen assay was calculated by comparison against the multiplex pcr (seeplex® rv16). categorical variables are presented using frequency tables, percentages and graphs. bivariate logistic regression models were used to determine associations of false rsv rapid antigen results with risk factors such as age, sex, hiv co-infection, rsv subtype, and co-infections with other viruses or bacteria. the odds ratio was the relative measure of association used. the 95% confidence interval was used to estimate the precision of estimates. the level of statistical significance was set at 5% (p-value ≤ 0.05). results test reports from a total of 152 participants were obtained and included in the study. eighty-one of the participants (53.3%) were female, and 121 were under the age of one year. table 1 shows all the demographic characteristics considered during this study. table 1: demographic characteristics of the study population from dora nginza hospital, port elizabeth, south africa, 2017–2018. results from the multiplex pcr assay show that rhinovirus was the most common virus detected (n = 55), followed by adenovirus (n = 30), rsv (n = 19) and enterovirus (n = 17), with the remainder of the viruses occurring less commonly (figure 1). respiratory syncytial virus subtype a was detected much more frequently than subtype b. figure 1: prevalence of the various respiratory viruses detected in paediatric patients from dora nginza hospital, port elizabeth, south africa, 2017–2018. when comparing the rsv rapid antigen assay to the multiplex pcr assay, the former was found to be 36.8% sensitive (table 2). table 2: retrospective analysis of laboratory data from doran nginza hospital, port elizabeth, south africa, 2017–2018. when analysing factors associated with false-negative rsv rapid antigen results, only viral–viral co-infection and infection with rsv subtype a were statistically significant associations (table 3). table 3: factors associated with false-negative respiratory syncytial virus rapid antigen results from paediatric patients at dora nginza hospital, port elizabeth, south africa, 2017–2018. discussion to the best of our knowledge, this is the first study comparing a rsv rapid antigen assay to a multiplex pcr assay that includes rsv as a target. in addition, it is also the first study to report on the prevalence of the common respiratory viruses found in our local paediatric icu. we found the clinical sensitivity of the rsv rapid antigen assay (rsv respi-strip by coris bioconcept) to be 36.84%; rhinovirus was the most commonly detected virus on the respiratory multiplex pcr assay. although the majority of respiratory admissions worldwide are due to rsv,5 the most common viruses detected in our setting were rhinovirus (n = 55) and adenovirus (n = 30), followed by rsv (n = 19) and enterovirus (n = 17). the main aim of this study was to determine the performance characteristics of the rsv rapid antigen assay in use (rsv respi-strip) by comparison to the respiratory multiplex pcr assay (seeplex® rv16 assay), which includes rsv as a target. the sensitivity of the rsv rapid antigen assay was found to be 36.84% (confidence interval: 16.29% – 61.64%). even at the upper end of the confidence interval, this sensitivity is too low for routine diagnostic use. while the specificity of above 90% may be sufficient for a rapid antigen assay, the low sensitivity would have resulted in many cases being undiagnosed, thus defeating the main purpose of using a rapid test, which is to allow for early institution of infection prevention and control measures to mitigate against the well-known nosocomial outbreak risk.7 potential reasons for the discrepancy in performance of this rapid test in our setting compared to the manufacturer’s claims are manifold. rapid antigen assays are generally less sensitive than pcr. this can be compounded by low viral loads in the clinical samples, as may be the case with upper respiratory tract infections only. it was beyond the scope of this study to differentiate patients based on severity of disease. the relatively small sample size may also account for the discrepancy in clinical sensitivity. the bivariate logistic regression analysis revealed a statistically significant association between false-negative rsv rapid antigen tests and viral co-infection, and infection with rsv subtype a. in contrast, a previous study showed that infection with rsv subtype b was associated with decreased sensitivity of rapid antigen assays.15 cytomegalovirus and hiv positivity were more likely to be associated with false rsv rapid antigen results, as well as more likely to be associated with false-negative results specifically. neither of these associations was statistically significant. limitations the main limitation of this study is the small sample size as a result of resource constraints, resulting in wider-than-ideal confidence intervals in the calculation of clinical sensitivity of the rsv rapid antigen test. however, even at the upper limit of the confidence intervals, the main conclusion that the sensitivity and positive predictive value are not acceptable for routine diagnostic use, still holds. conclusion this study highlights the importance of ongoing monitoring of newly introduced assays, as verification experiments may not always determine whether an assay will perform optimally in real-world conditions. based on the results of this study, the rsv rapid antigen assay in use (rsv respi-strip) was discontinued due to an unacceptably high rate of false results. in conclusion, we have demonstrated the common respiratory viruses found in our local paediatric icu setting. in addition, we report on the poor performance of the rsv rapid antigen assay in use and the potential factors associated with false results, with only viral co-infections and infection with rsv subtype a being statistically significant. clinicians should have an idea of the sensitivity and specificity of the rapid tests in use to allow for the appropriate interpretation of results. acknowledgements we would like to express our gratitude to all the staff at the virology laboratory of the national health laboratory service, port elizabeth, south africa, who assisted in collection of data. competing interests the authors have declared that no competing interests exist. authors’ contributions h.n. initiated the study. d.t., s.m., n.n. and c.c. assisted in producing the protocol. d.t. oversaw the ethics application. c.c. and h.n. handled the data collection. s.m. performed the data analysis. h.n., d.t., s.m., n.n. and c.c. wrote and revised the manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references chartrand c, tremblay n, renaud c, papenburg j. diagnostic accuracy of rapid antigen detection tests for respiratory syncytial virus infection: systematic review and meta-analysis. j clin microbiol. 2015;53(12):3738–3749. https://doi.org/10.1128/jcm.01816-15 green rj, zar hj, jeena pm, madhi sa, lewis h. south african guideline for the diagnosis, management and prevention of acute viral bronchiolitis in children. s afr med j. 2010;100(5):320–325. https://doi.org/10.7196/samj.4016 madhi sa, cutland cl, downs s, et al. burden of respiratory syncytial virus infection in south african human immunodeficiency virus (hiv)-infected and hiv-uninfected pregnant and postpartum women: a longitudinal cohort study. clin infect dis. 2018;66(11):1658–1665. https://doi.org/10.1093/cid/cix1088 nair h, nokes dj, gessner bd, et al. global 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children. j pediatr. 2013;163(3):911–913. https://doi.org/10.1016/j.jpeds.2013.03.067 blyth cc, booy r, dwyer de. point of care testing: diagnosis outside the virology laboratory. in: stephenson jr, warnes a, editors. diagnostic virology protocols. methods in molecular biology, vol. 665. new jersey: humana press, 2011; pp. 415–433. https://doi.org/10.1007/978-1-60761-817-1 pillet s, lardeux m, dina j, et al. comparative evaluation of six commercialized multiplex pcr kits for the diagnosis of respiratory infections. plos one. 2013;8(8):e72174. https://doi.org/10.1371/journal.pone.0072174 gharabaghi f, hawan a, drews sj, richardson se. evaluation of multiple commercial molecular and conventional diagnostic assays for the detection of respiratory viruses in children. clin microbiol infect. 2011;17(12):1900–1906. https://doi.org/10.1111/j.1469-0691.2011.03529.x somerville lk, ratnamohan vm, dwyer de, kok j. molecular diagnosis of respiratory viruses. pathology. 2015;47(3):243–249. https://doi.org/10.1097/pat.0000000000000240 mills jm, harper j, broomfield d, templeton ke. rapid testing for respiratory syncytial virus in a paediatric emergency department: benefits for infection control and bed management. j hosp infect. 2011;77(3):248–251. https://doi.org/10.1016/j.jhin.2010.11.019 gregson d, lloyd t, buchan s, church d. comparison of the rsv respi-strip with direct fluorescent-antigen detection for diagnosis of respiratory syncytial virus infection in pediatric patients. j clin microbiol. 2005;43(11):5782–5783. https://doi.org/10.1128/jcm.43.11.5782-5783.2005 abstract introduction methods results discussion acknowledgements references about the author(s) fred maate department of pathology and microbiology, adult and emergency hospital, university teaching hospitals, lusaka, zambia department of pathology and microbiology, school of medicine, university of zambia, lusaka, zambia peter julius department of pathology and microbiology, school of medicine, university of zambia, lusaka, zambia stepfanie siyumbwa department of pathology and microbiology, adult and emergency hospital, university teaching hospitals, lusaka, zambia leeya pinder department of obstetrics and gynecology, university of washington, seattle, washington, united states trevor kaile department of pathology and microbiology, school of medicine, university of zambia, lusaka, zambia mulindi mwanahamuntu department of obstetrics and gynaecology, women and newborn hospital, university teaching hospitals, lusaka, zambia groesbeck parham department of obstetrics and gynecology, university of north carolina at chapel hill, chapel hill, north carolina, united states citation maate f, julius p, siyumbwa s, et al. high-risk human papillomavirus-associated vulvar neoplasia among women living with human immunodeficiency virus in zambia. afr j lab med. 2022;11(1), a1563. https://doi.org/10.4102/ajlm.v11i1.1563 original research high-risk human papillomavirus-associated vulvar neoplasia among women living with human immunodeficiency virus in zambia fred maate, peter julius, stepfanie siyumbwa, leeya pinder, trevor kaile, mulindi mwanahamuntu, groesbeck parham received: 21 feb. 2021; accepted: 24 feb. 2022; published: 12 may 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: globally, women living with hiv have a higher risk of vulvar neoplasia than hiv-negative women. vulvar neoplasia among women living with hiv has not previously been characterised in zambia. objective: this study determined the clinical and pathologic features of vulvar neoplasia among women living with hiv at the university teaching hospital, lusaka, zambia. methods: we conducted a cross-sectional study of vulvar lesions among 53 women living with hiv who presented with vulvar lesions between july 2017 and february 2018. the study assessed clinical and histological characteristics and prevalence of high-risk human papillomavirus (hrhpv). results: twenty-one patients were diagnosed with vulvar squamous cell carcinoma (vscc), 20 with usual vulvar intraepithelial neoplasm (uvin), and the rest with either benign lesions or non-neoplastic lesions (nnl). participants’ mean age was 40 years. patients with vscc were significantly older than those with nnl (mean (s.d.): 43 (21) vs 33 (10), p = 0.004). the prevalence of hrhpv was 88.9% in vscc patients and 100.0% in high-grade squamous intraepithelial lesion patients. hpv16 was the most common (52.6%) genotype. the clinical features of neoplasia were similar to those of nnl. conclusion: vscc was significantly more common among women aged ≥ 40 years. hrhpv in vscc and high-grade squamous intraepithelial lesions was high. women with vulvar lesions, especially those aged > 40 years, should be evaluated for vulvar cancer. young girls should be vaccinated to prevent vulvar cancer. keywords: vulvar neoplasia; vulvar cancer; human papillomavirus virus; hiv; zambia. introduction vulvar intraepithelial neoplasia (vin) is a precursor of vulvar squamous cell carcinoma (vscc).1 two types of vin have been described: differentiated vin and usual vulvar intraepithelial neoplasia (uvin). usual vin occurs in young women and is associated with high-risk human papillomavirus (hrhpv) infection.2,3 differentiated vin occurs in older women and is not associated with hrhpv4 but is associated with chronic inflammatory disorders such as lichen sclerosus and squamous cell hyperplasia.5,6 by definition, hrhpvs are those that are associated with cervical and anogenital cancers, including vulvar cancer.7 in 2005, the international agency for research on cancer defined 13 genotypes of hpv as high-risk/carcinogenic; these include genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68.8 other genotypes not related to cancer, such as hpv6 and 11, are said to be low-risk, and these are known to cause benign exophytic lesions such as condyloma acuminatum.9 vscc is the most common histologic type of vulvar cancer. it accounts for 95% of malignant tumours of the vulva.2,10 two forms of vscc have been established, including the hrhpv-associated warty and basaloid vscc and the non-hrhpv-associated keratinising vscc.11,12 warty and basaloid vscc arise from uvin, while keratinising vscc arises from differentiated vin.5 globally, vulvar cancer is more common among elderly women above 60 years.13,14,15 however, over the last two decades, its incidence has increased, particularly among young women, and this is attributed to infection with hpv.16,17,18,19 in zambia, vulvar cancer was the second most common gynaecologic malignancy in 2018.20 between 2007 and 2014, the age-standardised incidence rate of the disease was 1.5 per 100 000 women.21 also, it was reported recently that the disease peaked in the reproductive age group (20–40 years old).21 there is substantial evidence that hiv positivity is associated with hpv infection; one in every four women with vulvar cancer in zambia was hiv-positive.21,22,23 however, little is known about the clinical and pathological presentation of vulvar cancer and other vulvar lesions in zambia, particularly among women living with hiv. this study aimed to compare the clinicopathologic features and risk factors of vulvar neoplasia with those of non-neoplastic lesions (nnl) among women living with hiv. it also aimed to determine the prevalence of hrhpv infection among vulvar neoplasia patients. this information would provide etiological and clinical context to vulvar neoplasia in zambia and identify areas for further research and any challenges associated with vulvar neoplasia. methods ethical considerations this study was approved by the university of zambia biomedical research ethics committee (reference: 015-12-16), the national health research authority, and the university of north carolina at chapel hill institutional review board (reference: irb: 16-3422). permission to conduct the study was obtained from the management of the university teaching hospitals. participants provided written informed consent to participate in the study. to maintain confidentiality, no identifying information was collected and participant data were de-identified using unique codes before storage in a password-protected computer accessed only by the principal investigator. data from participants who withdrew from the study were completely removed. study design and setting this was a cross-sectional hospital-based clinical and laboratory study conducted between july 2017 and february 2018 at the university teaching hospital’s women and new born hospital, lusaka, zambia. the hospital is the country’s largest public tertiary hospital for obstetric, gynaecologic, and neonatal care. women attending the university teaching hospital’s gynaecologic clinic between july 2017 and february 2018 were recruited. the sample size was determined using the fisher formula: z2 p (1 − p)/d2 based on an assumed prevalence (p) of 3.6%, a z score of 1.96 and a margin of error (d) of ±5%. women who were aged 18 years or older, presented with a vulvar lesion and were documented as hiv-positive were included in the study. women who were unwilling to undergo a punch biopsy and had a previous diagnosis of vulvar cancer and a history of bleeding disorder were excluded. a total of 56 women were approached, of which 53 were recruited for the study; two were ineligible and one did not consent. demographic information, frequency of selected risk factors (smoking, age at first sexual intercourse, number of lifetime sexual partners, alcohol use, and oral contraceptive use), and a comprehensive clinical history were obtained using a structured questionnaire. cd4 count tests were conducted or results obtained from patients’ medical records if the test was done within the last six months. clinical examinations of the vulva were conducted and the characteristics of the vulvar lesions were recorded. the sizes of the vulvar lesions were determined using a measuring tape. biopsy and histopathological analysis tissues for histologic diagnosis were collected using keyes’ punch biopsy forceps under local anaesthesia and aseptic conditions. the tissues were preserved in 10% neutral buffered formalin and transported to the pathology laboratory of the university teaching hospital’s adult and emergency hospital. the tissues were processed in the laboratory according to standard protocols, after which the histologic evaluation was conducted by three pathologists. based on the classification of the international society for the study of vulvovaginal disease (2015),24 the lesions were classified as nnl, condylomata acuminatum, uvin, or vscc. nnl types included acute non-specific inflammation, chronic non-specific inflammation and herpes simplex ulcer. usual vin types included low-grade squamous intraepithelial lesion (lsil) and high-grade squamous intraepithelial lesion (hsil). the pathologists diagnosed the vulvar lesions as lsil if the dysplastic cellular changes were limited to the lower third of the epithelium, and hsil if the dysplastic cellular changes involved up to or greater than the upper two-thirds of the epithelium. detection of high-risk human papillomavirus punch biopsies for the polymerase chain reaction test were collected during the collection of tissue for histology. each tissue was preserved in a 1.5 ml microcentrifuge tube containing qiagen buffer atl (qiagen, germantown, maryland, united states) and stored in a refrigerator at –80 °c. samples were allowed to defrost at room temperature, mixed with 20 µl of proteinase k, and then incubated and vortexed to ensure a complete mixture. total dna was extracted from the pre-lysed samples using the nuclisens® easymag™ automated extraction machine (biomerieux, inc, marcy i’etoile, france) according to the manufacturer’s protocol. the elution volume was 100 µl. polymerase chain reaction for the detection of high-risk hpv was carried out using the xpert® hpv platform (cepheid, sunnyvale, california, united states). to do this, 100 µl of each extracted sample was mixed with 900 µl of the thin prep solution (hologic, marlborough, massachusetts, united states), allowed to homogenise, and then transferred into a cartridge. the cartridge was loaded into the gene xpert pcr machine (cepheid, sunnyvale, california, united states) and run according to the manufacturer’s protocol. results were exported to a disc and transferred to a computer. xpert hpv detects the presence of high-risk hpv and the following hpv genotypes: hpv16, hpv18_45, and other hpvs, including p3 (hpv31, hpv33, hpv35, hpv52, hpv58), p4 (hpv51, hpv59), and p5 (hpv39, hpv56, hpv66, hpv68). statistical analysis all data management was done using excel (microsoft corporation, redmond, washington, united states) and data analyses were carried out using statistical package for social sciences version 22 (international business machines corporation, armonk new york, united states). clinical features (symptoms, and signs), demographic features and risk factors of uvin and vscc were compared with those of nnl using chi-square or fisher’s exact tests of association. we excluded condyloma acuminatum from the comparative analysis because of its benign nature. the continuous variables (age, age at first sexual intercourse, duration of antiretroviral therapy [art], number of lifetime sexual partners, and duration of symptoms in years) were tested for normality using the shapiro-wilk test. the means of the continuous variables that were normally distributed (age [p = 0.969] and duration of art [p = 0.070]) were compared using levine’s t-test between the diagnostic categories vscc and nnl, as well as between uvin and nnl. medians and interquartile ranges were reported for variables (number of lifetime sexual partners and duration of symptoms) that were not normally distributed. the median duration of symptoms and the median number of lifetime sexual partners were compared using mood’s median test between the diagnostic categories vscc and nnl, as well as between uvin and nnl. results histologic diagnoses histologic types of vulvar lesions included vscc (21/53, 39.6%), uvin (20/53, 37.7%), nnl (9/53, 17.0%) and condyloma acuminatum (3/53, 5.7%). vscc followed by uvin were the sole types of cancer and premalignant disease identified (figure 1). two types of uvin were reported: hsil (14/20, 70.0%) and lsil (6/20, 30.0%). the types of nnl included chronic non-specific inflammation (7/9, 77.8%), acute inflammation (1/9, 11.1%), and herpes simplex ulcer (1/9, 11.1%) (figure 2). data from the three participants with condyloma acuminatum were excluded from downstream analyses. the vscc cases were either warty (19/21, 90.5%) or basaloid types (2/21, 9.5%). no keratinising squamous cell carcinoma was observed. among the warty vscc, two cases (2/19, 10.5%) were moderately differentiated (grade 2) and 17 (17/19, 89.5%) were poorly differentiated (grade 3). on the other hand, all of the basaloid (2/2, 100.0%) vsccs were poorly differentiated (grade 3). among the vscc cases, 16 (76.2%) had concurrent hsil, 19 (90.5%) had a high ki-67 index (30% or more), one (4.8%) was positive for p53, one (4.8%) had vascular invasion, and one (4.8%) had concurrent herpes simplex viral cytopathic effects. figure 1: histological diagnosis of vulvar lesions from women attending the university teaching hospital’s gynaecologic clinic, zambia, 2018. figure 2: p53 and ki-67 immunostaining photomicrographs of warty vulvar squamous cell carcinoma and basaloid vulvar squamous cell carcinoma among women attending the university teaching hospital, zambia, 2018. (a) warty vulvar squamous cell carcinoma: nests of malignant squamous cells are noted surrounded by a desmoplastic stroma. (b) basaloid vulvar squamous cell carcinoma: sheets of cells with hyperchromatic nuclei and scant to no cytoplasm are seen with overlying atypical surface epithelium. (c) p53 immunostaining in warty vulvar squamous cell carcinoma. focal staining is noted. (d) ki-67 stain. a diffuse nuclear staining pattern is noted. demographic characteristics of participants with vscc and uvin compared to those with nnl approximately three-quarters (39/50, 78.0%) of the study participants were residents of lusaka; 21/50 (42.0%) were married and 32/50 (64.0%) were employed (table 1). there was no difference in employment (uvin vs nnl: p = 0.205; vscc vs nnl: p = 0.443) or marital status (uvin vs nnl: p = 0.439; vscc vs nnl: p = 0.389) between the study participants with nnl and participants with uvin or vscc. over half (27/50, 54.0%) were aged between 20 years and 39 years, and 46.0% (23/50) were 40 years or older. the mean age of the participants was 40 years (range: 23–63 years). vscc was significantly more common among participants aged ≥ 40 years (p = 0.014) than among participants aged between 20 and 39 years. the mean age was also significantly higher among participants with vscc (43 vs 33 years, p = 0.004) compared to those with nnl. there was no difference between the mean ages of participants with uvin and nnl (uvin: 40 years vs nnl: 33 years, p = 0.110). the majority (23/50, 46.0%) of participants reported primary level as their highest level of education; however, the tests of association could not be performed as the assumptions for χ2 and fisher’s exact tests were not met. table 1: characteristics of participants diagnosed with usual uvin, vscc and nnl at the university teaching hospital’s gynaecologic clinic, zambia, 2018. risk factors among participants with vscc and uvin compared to those with nnl there was no association between the selected risk factors and the diagnosis of vscc or uvin: history of alcohol intake (uvin vs nnl: p = 0.675; vscc vs nnl: p = 0.681), oral contraceptive intake (uvin vs nnl: p = 0.568; vscc vs nnl: 1.000), cigarette smoking (uvin vs nnl: p = 0.532; vscc vs nnl: 0.517). the median age at first sexual intercourse was significantly lower among participants with uvin than those with nnl (uvin: 18 years vs nnl: 19 years; p = 0.048). only a few (3/50, 6.0%) participants reported smoking. the median number of lifetime sexual partners reported among all participants was four (95% confidence interval [ci]: 2–6); four partners (95% ci: 2–6) among participants with vscc, four partners (95% ci: 2–6) among participants with uvin and three partners (95% ci: 2–4) among participants with nnl. hiv characteristics and history of cervical cancer screening all participants diagnosed with uvin (20/20, 100.0%) and vscc (21/21, 100.0%) were on art, while seven (7/9; 77.8%) of those diagnosed with nnl were on art. the majority of the participants (39/50, 78.0%) had been on art for longer than one year (mean: 7 ± 4 years). the cd4 count distribution was approximately normal (shapiro-wilk test: 0.952 [degree of freedom{df} = 46], p = 0.055). the overall mean cd4 count was 387.2 ± 237.2 cells/µl. the average cd4 count was 414.0 ± 233.1 cells/µl among participants with vscc, 359.5 ± 225.7 cells/µl among participants with uvin, and 381.6 ± 294.1 cells/µl among participants with nnl. there was no significant difference in mean cd4 counts between participants with nnl and those with uvin (t = –0.210, p = 0.835) or vscc (t = 0.312, p = 0.757). in terms of history of cervical cancer screening, there was no difference between uvin and nnl (13/20 (65.0%) vs 7/9 (77.8%); p = 0.694) or between vscc and nnl (13/21 [61.9%] vs 13/20 [65.0%]; p = 1.000). among those who were screened for cervical cancer, two of them reported that they had been diagnosed with cervical cancer (one with uvin and the other with vscc). symptoms of participants with uvin, vscc and nnl approximately three-quarters (37/50, 74.0%) of the participants had had vulvar symptoms for at least 1 year. although a larger proportion of the participants with vscc (17/21, 80.9%; p = 0.082) and uvin (16/20, 80.0%; p = 0.088) had symptoms for ≥ 1 year compared to participants with nnl (4/9, 44.4%), these differences were not statistically significant. among all the participants, the most frequently reported first symptom was itching (23/50, 46.0%). the frequency of itching as a first symptom was similar across all groups of participants (uvin: 9/20, 45.0%; nnl: 4/9, 44.4%; vscc: 10/21, 47.6%). the overall most common symptoms reported among the study participants were itching (44/50, 88.0%) and pain (39/50, 78.0%). there was no significant association between the diagnostic groups and symptoms experienced. location, sizes and types of lesions overall, most lesions involved the labia majora (42/50, 84.0%), followed by the labia minora (36/50, 72.0%) and clitoris (14/50, 28.0%). the labia majora was affected in all women with nnl (9/9, 100%), 90.0% (19/21) of women with vscc, and 70.0% (14/20) of women with uvin. the vagina, perineum and anus were also involved in participants with uvin and vscc. overall, the majority of participants (41/50, 82.0%) had multifocal rather than unifocal lesions. all nnl were multifocal; comparatively, 18 (80.0%) women with uvin, and 16 (76.2%) women with vscc had multifocal lesions. the majority of the participants (42/50, 84%) had vulvar lesion sizes > 2 cm. nineteen (19/21; 90.5%) women with vscc had lesions > 2 cm compared to eight (88.9%) women with nnl, and 15 (75.0%) women with uvin. the overall most common type of vulvar lesion was the warty type (17/50, 34.0%), followed by the fungating (12/50, 24.0%), smooth exophytic (10/50, 20.0%), ulcerated (8/50, 16.0%) and flat (3/50, 6.0%) types. among women with uvin, 11/20 (55.0%) lesions were warty, and 3/20 (15.0%) were flat; smooth exophytic, fungating, and ulcerated lesions each accounted for 2/20 (10.0%) of the cases. on the other hand, the most common type of vulvar lesion among participants with vscc was the fungating type (9/21, 42.9%), followed by the smooth exophytic lesion (6/21, 28.6%); warty and ulcerated lesions each accounted for 14.3% (3/21). prevalence of hrhpv infection among participants with neoplastic lesions among the samples collected for hrhpv dna analysis, we were able to extract sufficient dna from 38 tissue samples; these included samples from 17 patients with uvin, 18 with vscc and three with condylomata acuminatum. the prevalence of any type of hrhpv among all participants was 81.6% (31/38); 82.4% (14/17) among women with uvin (1/4, 25% in lsil, and 13/13, 100% in hsil) and 88.9% (16/18) among women with vscc (table 2). the most prevalent genotype of hrhpv among all participants was hpv16 (20/38, 52.6%), followed by p3 (13/38, 34.2%) (table 3). only hpv16, p3 and p5 genotypes were present in hsil; only hpv16 was detected in lsil cases. the hpv genotypes that were detected in vscc cases were hpv16, hpv18_45, p3 and p5. hpv16 occurred as a co-infection with other hpvs (p3 and p5) in both uvin (hsil) and vscc. table 2: prevalence of high-risk human papillomavirus among women with condyloma acuminatum, uvin (lsil, hsil) and vscc at the university teaching hospital’s gynaecologic clinic, zambia, 2018. table 3: frequency of high-risk human papillomavirus genotypes among women with condylomata acuminatum, uvin and vscc at the university teaching hospital’s gynaecologic clinic, zambia, 2018. discussion to the best of our knowledge, this is the first study to describe vulvar lesions and to compare vulvar neoplasia with nnl among any cohort of women living with hiv at any hospital in zambia. most of the studies in other african countries largely focused on vulvar cancer or hpv.22,25 among the cohort of women living with hiv in our study, both neoplastic (condylomata acuminatum, uvin, and vscc) and nnl were detected. this study showed that vscc cannot be distinguished from nnl on account of clinical features, although vscc was associated with women aged ≥ 40 years. the prevalence of hrhpv in both vscc and uvin (accounted for by hsil) was high. vscc was significantly more common among women aged ≥ 40 years compared to nnl. similarly, a study in tunisia found that vulvar cancer was more common in older women (86.9% of patients were more than 55 years old) although none of the patients was hiv-positive.26 clinicians should therefore evaluate and biopsy the vulva in women who present with vulvar symptoms, especially those who are older than 40 years. the ages of women affected with uvin in our study were similar to the ages reported in other studies for hsil.27 this study demonstrated that there was no difference in age between women with uvin and those with nnl. our study supports the view that vulvar cancer cannot be differentiated from nnl based on clinical evaluation alone.26 vulvar pruritus, pain, and other gross features of vulvar lesions are not cancer-specific symptoms. this similarity of vulvar cancer signs and symptoms with those of nnl has been cited as a possible reason for the delay in the diagnosis of gynaecologic malignancies by clinicians.28,29,30 it is therefore imperative that all vulvar lesions are biopsied as soon as possible, especially among women living with hiv. although they have been identified as risk factors of vulvar cancer, the smoking of cigarettes, alcohol use, use of oral contraceptives, and early sexual debut were not associated with vscc or uvin in our cohort of participants.31,32 also, the finding that 90% of patients with vscc had been on art for over a year supports previous reports that art does not affect the regression of lowor high-grade lesions and has no effect on the prevention or elimination of hpv infection.33,34,35,36 in our study, the mean cd4 count among women with vscc was above the aids diagnosis threshold: 200 cells/µl.37 in contrast, in a 2014 study in botswana, 35% of women living with hiv with vulvar cancer had cd4 counts below 200 cells/µl (range: 68–854).22 in another study in ethiopia in 2018, the range of cd4 counts was above the aids diagnosis threshold (230 cells/µl – 600 cells/µl).27 our study was unique in that it showed that there was no association between cd4 count and the diagnosis of vscc. this study supports previous findings that itching is the most frequent symptom of uvin and vscc7,38 and demonstrated that vscc presents with multifocal growth patterns.39,40,41 the finding of concurrent hsil and a high ki-67 proliferation index with a diffuse pattern among vscc was indicative of a bad prognosis.42 however, an evaluation of the correlation between patients’ clinical outcomes and these prognostic factors was beyond the scope of this study. although polymerase chain reaction showed that hrhpv was present in the biopsy tissue, it did not prove that hrhpv was incorporated into the host genome. nevertheless, the pcr findings were supported by the histologic examination which detected uvin, as well as warty and basaloid vscc, all of which are known to be associated with hrhpv.5 the high prevalence of hrhpv in vscc and hsil in our cohort of women living with hiv is premised on the extensively described interactions between hpv infection and hiv.43,44,45,46 on one hand, women who are infected with hpv have a higher risk of infection with hiv.36 on the other hand, hiv-positive persons are at higher risk of developing hpv infection through various mechanisms. one of these mechanisms involves the secretion of a viral transactivator protein and gp120 by hiv-infected cells, which disrupt epithelial tight junctions and enhance intercellular penetration of the hpv to the target cells.36 because of defective immunity in the host, hpv evades the host immunity, and the suppressed immunity further enhances increased hpv viral loads, infection with multiple hpv types, reactivation of latent hpv infection, and persistence of hpv-associated disease. the prevalence of hrhpv in vscc in our study (88.9%) was higher than the prevalence reported in botswana (82.9%).19 worldwide, the prevalence of hpv in vscc varied from 15.0% to 79.0%.5 the prevalence of hpv16 in vscc in this study was low (44.4%)47 compared to that reported by a study in botswana (82.9%) but similar to the prevalence reported worldwide (29.3% – 50.0%).22,48,49,50 we reported a prevalence of 44.4% for the group p3 genotypes (hpv31, 33, 35, 52 and 58) among patients with vscc. this was lower than the prevalence rate of hpv31 (51.4%) and higher than the prevalence rate of hpv33 (11.4%) reported in a botswanan study; it was also higher than the prevalence rates reported worldwide (0.8% – 1.7% for hpv31 and 3.3% – 3.5% for hpv33).48,49 a further evaluation of the exact genotypes that are prevalent in zambian patients is imperative. the prevalence of hpv18/45 (5.6%) among patients with vscc in our study is difficult to compare with other regional studies as such studies measured the prevalence rates of hpv18 and hpv45 separately. for example, the botswanan study in 2014 reported a 37.1% hpv18 prevalence and an 8.6% hpv45 prevalence.22 in this study, the prevalence of hrhpv was high (100%) among hsil cases and low among lsil cases (25.0%); the overall prevalence of hrhpv among uvin cases was 82.4%. this was in keeping with previous studies worldwide that showed that hpv prevalence varied from 52.0% to 100.0% in uvin.5 the 64.7% prevalence of hpv16 among uvin cases in this study is lower than the 74.6% prevalence reported worldwide among uvin cases.5,51 the presence of hpv group p3 and the absence of hpv18/45 among hsil cases suggests that hpv31, 33, 35, 52 and 58 may have a higher pathogenic role in causing hsil than hpv18/45. vaccination against hpv is effective in preventing hpv-related cancer. for instance, in scotland, there were statistically significant reductions in all grades of cervical intraepithelial neoplasia. the vaccine was estimated to be ≥ 80% effective in preventing cervical intraepithelial lesions when administered routinely to girls aged 12–13 years in the targeted population.52 different types of hpv vaccines are available, including the quadrivalent hpv vaccine, gardasil® 9 (sanofi pasteur msd)/silgard® (merck sharp & dohme), and the bivalent hpv vaccine, cervarix® (glaxosmithkline biologicals). in europe, these vaccines were indicated for the prevention of cervical, vulvar and vaginal premalignant lesions and cervical cancer related to hpv16/18. the quadrivalent hpv vaccine was also indicated against premalignant anal lesions and anal cancer and protected against low-risk hpv6/11, which are responsible for about 90% of genital warts.53 zambia initially introduced the hpv vaccine on a pilot basis in 2013 and by 2020, the country had integrated it into the routine immunisation programme using a system that offers two doses of the quadrivalent hpv vaccine.54 based on the preliminary findings of our study, more research needs to be conducted to determine the specific genotypes of hrhpv that are prevalent in the population affected by either cervical or vulvar cancer to inform current vaccination efforts. limitations even though university teaching hospital is zambia’s highest level referral centre for the treatment of major ailments, a multicentre study would have represented the country’s population better. the responses given by the participants may have been negatively influenced by their recall bias. one downside with our methodology is that genexpert reports pooled hpv genotypes and hence we could not report the specific genotypes in the p3, p4 or p5 groups. furthermore, we did not evaluate viral load, which is considered a better marker of immunologic status. also, we did not successfully determine viral dna incorporation into the host genome using either p16 immunohistochemistry or in situ hybridisation. conclusion vscc was associated with women aged above 40 years. the prevalence of hrhpv in vscc and hsil was high. we recommend that women living with hiv, especially women aged above 40 years, should have a close clinical follow-up with routine evaluation of the vulva and biopsy of all lesions. we recommend a multicentre study to further evaluate vulvar neoplastic lesions in zambia and determine other genotypes other than hpv16 to inform current vaccination efforts. human papillomavirus vaccination of adolescent girls should be supported as a public health approach to the elimination of vulvar neoplasia. acknowledgements we acknowledge the support of the university of north carolina global projects zambia team and the university of zambia, school of medicine. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. author’s contributions all the authors contributed to the conceptualisation of the article. f.m. wrote the initial draft, while the rest of the authors reviewed and revised it. f.m., p.j., l.p., m.m. and g.p. conducted the investigations. formal analysis was conducted by all authors. l.p., t.k., m.m., p.j. and g.p. provided supervision. s.s conducted statistical analysis and review of the article. f.m. was the project administrator and co-principal investigator with g.p. sources of support this study was partly funded through the university of north carolina at chapel hill, johns hopkins university, morehouse school of medicine, and tulane university fogarty global health fellowship program (grant number r25 tw009340) financial support. data availability data supporting the findings of this study are available from the corresponding author, f.m., on request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessary reflect the official policy or position of the affiliated agency of the authors. references dasgupta s, ewing-graham pc, swagemakers sma, et al. precursor lesions of vulvar squamous cell carcinoma – histology and biomarkers: a systematic review. crit rev oncol hematol. 2020;147:102866. https://doi.org/10.1016/j.critrevonc.2020.102866 alkatout i, schubert m, weigel mt, mundhenke c, günther v. vulvar cancer : epidemiology, 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jj, dominiak-felden g, et al. estimation of the epidemiological burden of hpv-related anogenital cancers, precancerous lesions, and genital warts in women and men in europe: potential additional benefit of a nine-valent second generation hpv vaccine compared to first generation hpv vaccines. papillomavirus res. 2015;1:90–100. https://doi.org/10.1016/j.pvr.2015.06.003 who. costing the national strategic plan on prevention and control of cervical cancer: zambia, 2019–2023. 2020. hiv situation in senegal laboratory infrastructure in senegal quality assurance framework lessons learned and challenges quality assurance for poc acknowledgements references about the author(s) mouhamed a.s. mbengue laboratory of bacteriology and virology, cheikh anta diop university, chu aristide le dantec, dakar, senegal moussa sarr laboratory of bacteriology and virology, cheikh anta diop university, chu aristide le dantec, dakar, senegal westat, rockville, maryland, united states papa a. diaw laboratory of bacteriology and virology, cheikh anta diop university, chu aristide le dantec, dakar, senegal papa a.n. diall national committee for the control of aids, dakar, senegal maimouna d. toure laboratory of bacteriology and virology, cheikh anta diop university, chu aristide le dantec, dakar, senegal ndeye f.f.n. faye division for the control of aids and stds, ministry of health, dakar, senegal bousso gueye laboratory of bacteriology and virology, cheikh anta diop university, chu aristide le dantec, dakar, senegal ndeye c.t. kane laboratory of bacteriology and virology, cheikh anta diop university, chu aristide le dantec, dakar, senegal souleymane mboup laboratory of bacteriology and virology, cheikh anta diop university, chu aristide le dantec, dakar, senegal citation mbengue mas, sarr m, diaw pa, et al. establishing a national laboratory quality system for hiv diagnosis and monitoring in resource-limited settings: experience from senegal. afr j lab med. 2016;5(2), a440. http://dx.doi.org/10.4102/ajlm.v5i2.440 country profile establishing a national laboratory quality system for hiv diagnosis and monitoring in resource-limited settings: experience from senegal mouhamed a.s. mbengue, moussa sarr, papa a. diaw, papa a.n. diall, maimouna d. toure, ndeye f.f.n. faye, bousso gueye, ndeye c.t. kane, souleymane mboup received: 19 mar. 2016; accepted: 12 aug. 2016; published: 17 oct. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. hiv situation in senegal senegal is a western african country, with a population of around 14 million people and 14 administrative regions. based on the 2013 census, the total population of the country is estimated at 14 799 859 inhabitants, with a median population age of 18 years old.1 senegal, has a broad-based, pyramid-shaped age structure, which is characteristic of a population with a very high proportion of children and young people (table 1). in 2013, the crude birth rate was 37.2 per thousand, with a sex ratio at birth of around 105 men per 100 women. additionally, approximately 43% of the population is under 15 years of age, with an infant mortality rate of 47 deaths per 1000 births, and an overall risk of dying between birth and five years estimated at 72 deaths per 1000. the life expectancy at birth was 64 years in 2013.1 table 1: key hiv and health statistics in senegal. senegal has a concentrated hiv epidemic with high prevalence rates in most-at-risk groups, including commercial sex workers, but low prevalence rates in the general population in most regions (figure 1). based on the 2010–2011 senegal demographic and health survey, the global prevalence of hiv amongst women and men aged 15–49 years is 0.7%.2 more women are infected with hiv than men; the sex ratio is typically around 60 men per 100 women infected. however, the hiv seroprevalence is higher among vulnerable populations, with rates at 18.5% among sex workers, 17.8% among men having sex with men, and 9.4% among injection drug users.3,4 senegal has adopted the world health organization–joint united nations programme on hiv/aids recommended 90-90-90 targets.5 the adoption of this strategy means that the country is expected, by 2020, to have 90% of its population living with hiv diagnosed, 90% of all those diagnosed receiving sustained hiv treatment, and 90% of those receiving antiretroviral therapy having suppressed viral load measures.5 to achieve these outcomes, having good clinical laboratory services for diagnosis and follow-up will be critical.6 more specifically, investments will be needed to improve laboratory infrastructure, and to facilitate the access and availability of routine viral load and early infant diagnosis (eid) measures through the implementation of point-of-care (poc) diagnostic platforms along with an efficient and sustainable quality assurance programme. figure 1: hiv prevalence rate per region in senegal. laboratory infrastructure in senegal diagnostic laboratories in senegal operate within a three-tiered laboratory system: (1) district and peripheral health centre level laboratories; (2) regional level laboratories; and (3) central and hospital level laboratories (table 2). the organisation of the laboratory system is part of the overall health system structure and is placed under the leadership of the ministry of health. there is a bureau of laboratories within the ministry of health that is responsible for the implementation of policies defined by the government concerning the functioning and organisation of clinical laboratories. this office also promotes good clinical laboratory practice, not only for public medical laboratories, but also for private laboratories within the country. table 2: existing technologies or platforms hiv diagnosis, cd4 count, and viral load monitoring at each level of the tiered health system in senegal. the laboratory of bacteriology and virology (lbv) at cheikh anta diop university (cadu), which is located at le dantec hospital in the capital city, has been recognised by health authorities as the hiv national reference laboratory since 1986. in partnership with the ministry of health and the national aids programme, lbv/cadu is responsible for evaluating and validating diagnostic testing technologies, and technologies for cd4 count, viral load and eid. additionally, lbv/cadu ensures the supervision of other laboratories through a national external quality assessment (eqa) programme, through distribution of regular proficiency testing panels, collection of data for analysis, and follow-up and corrective actions in partnership with the ministry of health, the national committee for the control of aids and the division for the control of aids and sexually transmitted diseases. at the central and reference laboratory level, the hiv viral load, eid, cd4-count monitoring and hiv-diagnosis capabilities of lbv/cadu are described in table 2. at the regional and hospital laboratories level, these infrastructures are capable of performing hiv rapid diagnostic test confirmation, cd4-count measures and, at some of them, viral load and eid testing. however, the vast majority of the laboratory activities are conducted at the district level and peripheral health centre level. the capabilities for the district/peripheral health centre-level laboratories include: hiv rapid testing, cd4 count and collection of dried-blood specimens for eid. there are also few private laboratories operating at different levels of the health system. quality assurance framework quality assurance is the backbone of quality laboratory performance7 and proficiency testing is one of the major components of a quality assurance programme. in senegal, laboratory infrastructure is relatively well developed at the national and regional levels. however, issues such as inadequate infrastructure, shortage of qualified staff, lack of equipment, limited quality assurance and control procedures are frequently seen among district and peripheral health centre laboratories. the maintenance of a quality management system, including quality assurance, is crucial for having and providing good and reliable laboratory services.7,8,9 lbv/cadu has been working recently with the us centers for disease control and prevention to strengthen laboratory systems in africa and senegal though the implementation of quality management systems. the lbv/cadu-hosted afriqualab has been created for that purpose. afriqualab aims to improve laboratory quality in africa through regular organisation and distribution of proficiency testing panels across the african continent, including in francophone countries. afriqualab works in partnership with the us centers for disease control and prevention, westat and one world accuracy, a private canadian-based organisation specialising in eqa. during 2015, there were 174 participating laboratories from 28 countries, of which eight laboratories were from senegal. the proficiency testing organized by afriqualab is mostly free and focused on hiv-related proficiency testing panels, including hiv serology and flow cytometry. through technology transfer from the us centers for disease control and prevention, the programme is also offering free eqa for eid using dried-blood specimens and for hiv viral load using dried-tube specimens. currently, more than 60 laboratories are participating in this eqa sub-programme for eid using dried-blood specimens and for hiv viral load using dried-tube specimens in africa, including eight laboratories in senegal. additionally, afriqualab is also involved in an eqa programme focusing only on cd4 count technologies within senegal, with the support of the national aids programme and the public health agency of canada, an international programme for quality assessment and standardisation for immunological measures relevant to hiv. afriqualab also offers other panels, such as haematology, biochemistry, hepatitis b and c and mycobacteria, to participating labs using a fee-based service structure. lbv/cadu has made major improvements to its own quality management system and has recently achieved international organization for standardization 15189 accreditation of its medical laboratories. additionally, within senegal and at the national level, the ministry of health has recently released a national strategic plan that will guide the quality management system across the medical laboratory services and system in the country. lessons learned and challenges laboratories participating in the eqa programme for hiv rapid testing and cd4 (including poc) received feedback and, based on their results, have implemented corrective actions, as needed, to improve the quality of services, and were encouraged to participate in a laboratory network for continuous improvement. these outcomes are positive steps toward the implementation of quality management systems. the feedback received from the eqa programme has been used as an opportunity for participating laboratories to be more vigilant in many aspects of their work; for example, with respect to the expiration date for reagents, implementation of corrective actions, and on-site training for staff. the quality assurance programme and the external quality control activities must be followed up by systems-strengthening activities, such as staff training at all levels. we found that there is a need for more communication between the national reference laboratories and the bureau of laboratories, located at the ministry of health, regarding the type of corrective actions and support to be provided from the central/national level to regional and peripheral laboratories. there is also a need to provide incentives such as certificates of participation or achievement to successfully performing labs. quality assurance for poc currently, all 24 sites in-country with poc diagnostic technologies, including poc for cd4, are participating in an eqa programme organised by lbv. since 2004, lbv, with the support of the national aids programme, has provided voluntary, free-of-charge eqa of hiv rapid testing to assess the performance of laboratories conducting hiv diagnostics in dakar and other regions. currently, the eqa programme for hiv rapid testing uses dried-tube specimens. the proficiency testing panels consist of four specimens (two negatives, one hiv-1, one hiv-2) distributed at ambient temperature to participants. the results from the participants are sent to the national reference laboratory via email or cell-phone text messaging. costing and cost-effectiveness of hiv poc testing with regard to the 90-90-90 goals, the affordability of viral load testing and cd4 count measures is a key factor in plans to expand hiv laboratory services and scaling antiretroviral therapy across senegal. the country will need to put in place an affordable and sustainable strategy to reinforce the capacity of the public laboratories as well as to advocate for a strong political commitment from the ministry of health and sponsors. in partnership with the national aids programme, lbv/cadu is planning to estimate the costing and to undertake a cost-effectiveness study of implementing a nationwide hiv poc testing system, for cd4 count and viral load testing. the costing for, and cost-effectiveness of, a related quality assurance programme will be also taken into account. a model of costing for hiv poc testing, including quality assurance activities is currently under review with the international diagnostics centre of the london school of hygiene and tropical medicine. acknowledgements the authors are grateful to the staff of all sites participating in related eqa programmes. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. sources of support none. references agence nationale de la statistique et de la démographie. recensement général de la population et de l’habitat, de l’agriculture et de l’elevage. senegal: ansd; 2014. agence national de la statistique et de la démographie. enquête démographique et de santé et à indicateurs multiples 2010–2012. senegal: ansd; 2012. comité national de lutte contre le sida. rapport national de la riposte vih/sida 2014. yaoundé, cameroun: cnls; 2015. leprêtre a, ba i, lacombe k, et al. prevalence and behavioural risks for hiv and hcv infections in a population of drug users of dakar, senegal: the anrs 12243 udsen study. j int aids soc. 2015;18:19888. http://dx.doi.org/10.7448/ias.18.1.19888. ecollection 2015. joint united nations programme on hiv/aids. global aids response progress reporting 2015. geneva, switzerland: who press; 2015. nguyen s, ramos a, chang j, et al. monitoring the quality of hiv-1 viral load testing through a proficiency testing program using dried tube specimens in resource-limited settings. j clin microbiol. 2015;53(4):1129–1136. http://dx.doi.org/10.1128/jcm.02780-14. epub 2015 jan 21. garcia a, subbarao s, zhang g, et al. impact of proficiency testing program for laboratories conducting early diagnosis of hiv-1 infection in infants in lowto middle-income countries. j clin microbiol. 2014;52(3):773–780. http://dx.doi.org/10.1128/jcm.03097-13. epub 2013 dec 18. pai np, vadnais c, denkinger c, et al. point-of-care testing for infectious diseases: diversity, complexity, and barriers in lowand middle-income countries. plos one. 2012;9(9):e1001306. http://dx.doi.org/10.1371/journal.pmed.1001306. epub 2012 sep 4. miller wg, jones grd, horowitz gl, et al. proficiency testing/external quality assessment: current challenges and future directions. clin chem. 2011;57(12):1670–1680. http://dx.doi.org/10.1373/clinchem.2011.168641. epub 2011 sep 30. abstract introduction methods results discussion acknowledgements references about the author(s) zhiyong zhou international laboratory branch, division of global hiv & tb, center for global health, centers for disease control and prevention, atlanta, georgia, united states kevin tang biotechnology core facility branch, division of scientific resources, centers for disease control and prevention, atlanta, georgia, united states guoqing zhang international laboratory branch, division of global hiv & tb, center for global health, centers for disease control and prevention, atlanta, georgia, united states nellie wadonda-kabondo department of preventive health, ministry of health, lilongwe, malawi kundai moyo department of preventive health, ministry of health, lilongwe, malawi lori a. rowe biotechnology core facility branch, division of scientific resources, centers for disease control and prevention, atlanta, georgia, united states joshua r. devos international laboratory branch, division of global hiv & tb, center for global health, centers for disease control and prevention, atlanta, georgia, united states nick wagar international laboratory branch, division of global hiv & tb, center for global health, centers for disease control and prevention, atlanta, georgia, united states du-ping zheng international laboratory branch, division of global hiv & tb, center for global health, centers for disease control and prevention, atlanta, georgia, united states hongxiong guo international laboratory branch, division of global hiv & tb, center for global health, centers for disease control and prevention, atlanta, georgia, united states john nkengasong international laboratory branch, division of global hiv & tb, center for global health, centers for disease control and prevention, atlanta, georgia, united states mike frace biotechnology core facility branch, division of scientific resources, centers for disease control and prevention, atlanta, georgia, united states scott sammons biotechnology core facility branch, division of scientific resources, centers for disease control and prevention, atlanta, georgia, united states chunfu yang international laboratory branch, division of global hiv & tb, center for global health, centers for disease control and prevention, atlanta, georgia, united states citation zhou z, tang k, zhang g, et al. detection of minority drug resistant mutations in malawian hiv-1 subtype c-positive patients initiating and on first-line antiretroviral therapy. afr j lab med. 2018;7(1), a708. https://doi.org/10.4102/ajlm.v7i1.708 original research detection of minority drug resistant mutations in malawian hiv-1 subtype c-positive patients initiating and on first-line antiretroviral therapy zhiyong zhou, kevin tang, guoqing zhang, nellie wadonda-kabondo, kundai moyo, lori a. rowe, joshua r. devos, nick wagar, du-ping zheng, hongxiong guo, john nkengasong, mike frace, scott sammons, chunfu yang received: 13 oct. 2017; accepted: 01 feb. 2018; published: 30 may 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: minority drug resistance mutations (drms) that are often missed by sanger sequencing are clinically significant, as they can cause virologic failure in individuals treated with antiretroviral therapy (art) drugs. objective: this study aimed to estimate the prevalence of minor drms among patients enrolled in a malawi hiv drug resistance monitoring survey at baseline and at one year after initiation of art. methods: forty-one plasma specimens collected from hiv-1 subtype c-positive patients and seven clonal control samples were analysed using ultra-deep sequencing technology. results: deep sequencing identified all 72 drms detected by sanger sequencing at the level of ≥20% and 79 additional minority drms at the level of < 20% from the 41 malawian clinical specimens. overall, drms were detected in 85% of pre-art and 90.5% of virologic failure patients by deep sequencing. among pre-art patients, deep sequencing identified a statistically significant higher prevalence of drms to nucleoside reverse transcriptase inhibitors (nrtis) compared with sanger sequencing. the difference was mainly due to the high prevalence of minority k65r and m184i mutations. most virologic failure patients harboured drms against both nrtis and non-nucleoside reverse transcriptase inhibitors (nnrtis). these minority drms contributed to the increased or enhanced virologic failures in these patients. conclusion: the results revealed the presence of minority drms to nrtis and nnrtis in specimens collected at baseline and virologic failure time points. these minority drms not only increased resistance levels to nrtis and nnrtis for the prescribed art, but also expanded resistance to additional major first-line art drugs. this study suggested that drug resistance testing that uses more sensitive technologies, is needed in this setting. introduction rapid scale-up of antiretroviral therapy (art) over the past decade has remarkably reduced the mortality and morbidity of hiv-positive patients and decreased hiv transmission. seventeen million hiv-1-positive patients around the world were receiving art by the end of 2015.1 however, the scale-up of art in resource-limited settings without adequate treatment monitoring has raised concern about the development of hiv drug resistance. the quasi-species nature of hiv-1 makes the detection of drug resistant mutations (drms) more difficult, because the commonly-used sanger sequencing for drug resistance testing is incapable of detecting these drug resistant hiv variants at a level of less than 20% of the viral population.2,3,4,5 minority drug resistant variants (also known as low-frequency mutants) that are not detected by sanger sequencing are clinically important, as they can cause virologic failure in patients treated with art for the first time.6,7,8,9 recent studies have demonstrated that particular drug resistant hiv mutant viruses are clinically significant at a level of 1% of the viral population, as the minority variants can replicate quickly and become the predominant viral population through the selective pressure of art drugs, leading to treatment failure.9,10 however, in the absence of drug pressure in treatment-naïve patients, the stability of transmitted drms is different.11 for instance, a transmitted m184v mutation can quickly revert to wild-type due to diminished viral fitness.12 in patients on art, minority drms may persist for months or years during and post-art.13,14,15 these minority drms, not detected by sanger sequencing, present a need for more sensitive methods to detect the minority drms in a clinical sample. deep sequencing or next-generation sequencing technologies are extensively used to examine hiv viral diversity and minority drug resistant variants. next-generation sequencing is a highly sensitive and high-throughput sequencing platform. it can detect hiv variants that make up 0.05% to 1% of viral populations.16,17,18,19,20,21 as part of hiv drug resistance surveillance by the malawi ministry of health, a prospective cohort study to monitor art outcomes and drug resistance development was conducted among patients from art initiation to one year later. in this 2008 art patient monitoring survey, 6.1% of the patients on art for 12–15 months harboured drug resistant hiv.22 the most common non-nucleoside reverse transcriptase inhibitor (nnrti) mutations were k103n (58.1%), y181c (41.9%) and g190a (6.5%), and the most frequent nucleoside reverse transcriptase inhibitor (nrti) mutation was m184v (61.3%). the drms conferring resistance against nnrti at baseline were associated with drms detected at 12–15 months on art.22 the present study aimed to evaluate parallel tagged deep sequencing primers on clinical samples and to investigate the profile of minority drms and their association with virologic failure in the same malawi art monitoring cohort. methods ethical considerations the study protocol was approved by the national health sciences research committee of malawi institutional review board (#1001). the use of de-identified data and drug resistance testing using sanger sequencing and roche 454 deep sequencing at the centers for disease control and prevention’s (cdc) global hiv drug resistance laboratory, was determined to be non-human subjects research under cdc protocol #6501 by the office of the associate director for science at the center for global health, cdc, atlanta, georgia, united states. clinical samples between february and june 2008, hiv-1-positive patients aged 15 years or older, who initiated first-line art at four art clinics following the malawi art guidelines, were enrolled. patients were treated with a first-line regimen combination of stavudine, lamivudine and nevirapine, or an alternative first-line regimen of stavudine to zidovudine substitutions in case of toxicity. plasma specimens were collected before art initiation and at 12–15 months on art for viral load and hiv drug resistance testing.22 in the present study, we selected plasma specimens that had enough volume available to evaluate the assay. these were 20 samples collected from participants before art initiation with viral loads ranging from 10 471 to 2 041 738 copies/ml and 21 samples collected from art patients at virologic failure (defined as viral load ≥1000 copies/ml) after 12–15 months on art (viral load ranging from 1738 to 776 247 copies/ml). in addition, six plasmid clones and one mixed clone containing 1% mutant (2495 copies/µl) under the background of a wild-type clone were prepared and used to verify sequence accuracy in this study. all of these plasmid clones were derived from the malawian cohort samples. all plasmids were constructed using topotm vectors in escherichia coli (thermo fisher scientific, carlsbad, california, united states). the six wild-type plasmid clones contained the hiv-1 pol gene without any drms, and the mutant clone contained drms at codons 103, 181, 184 and 190 of the hiv-1 pol gene. viral ribonucleic acid extraction and reverse transcriptase polymerase chain reaction rna was extracted from plasma specimens using the automated abbott™ sample preparation system (m2000sp) (abbott laboratories. abbott park, illinois, united states). the copy number of hiv-1 rna was measured using rt-qpcr on the m2000rt real time analyzer (abbott laboratories. abbott park, illinois, united states).22 the viral rna was subjected to one-step rt-pcr amplification as described previously.11 parallel deep sequencing degenerate primers, capable of amplifying multiple hiv-1 group m subtypes, were designed based on the hiv-1 pol gene sequences (www.hiv.lanl.gov) (table 1). six overlapping primer sets (forward and reverse) were used for bidirectional coverage of protease amino acids 6 to 99 and reverse transcriptase amino acids 1 to 251. the size of the assembled gene fragment was 1035 base pairs. these six primers, tailed with roche 454 adaptor and multiplex identifier sequences (tags), were synthesised at the cdc biotechnology core facility. for pcr amplification, a 50µl reaction contained 1× accuprime pcr buffer ii (thermo fisher scientific, carlsbad, california, united states), 0.5 u accuprime taq high fidelity (thermo fisher scientific, carlsbad, california, united states), 13.5 µl water, 0.3 µm, each forward and reverse primer, and 2 µl dna. all reactions were performed in 9700 thermal cyclers (applied biosystems, austin, texas, united states) under the following program: 95°c for 10 minutes (min); five cycles of 94°c for 20 seconds (s), 48°c for 20 s, and 72°c for 1 min for annealing the primers with unique molecular tags followed by 35 cycles of 94°c for 30 s, 60°c for 20 s, 72°c for 30 s; and one cycle of 72°c for 5 min. the reaction products were confirmed by 1% agarose gel electrophoresis. the amplicons were purified using the agencourt ampure xp beads (beckman coulter, beverly, massachusetts, united states) or qiagen gel purification kits (qiagen, germantown, maryland, united states) and then quantified using the quant-it picogreen dsdna kit (thermo fisher scientific, carlsbad, california, united states). each sample had its own unique tag sequences for its six amplicons. six barcoded samples were pooled and sequenced in one region. a total of eight regions were used for 48 samples on a plate. pcr amplicons from six samples were pooled in equal amounts, and amplified in water-in-oil emulsion pcr at the cdc biotechnology core facility. amplicons of 41 field samples and seven plasmid dna samples were sequenced on the 454 platform (gs-flx, roche applied science, indianapolis, indiana, united states). table 1: hiv-1 primers designed and used for amplifying pol gene amplicons with the deep sequencing method. deep sequencing analysis sequence files generated by roche 454 deep sequencing were analysed using gs amplicon variant analyzer pipeline from roche applied science (indianapolis, indiana, united states). the deep sequencing analysis process in the present study included quality restriction for base call setting at 60 for signal intensity. all amplicon reads of alignment and single nucleotide polymorphism (snp), calling against the hxb2 reference sequence, were evaluated with a quality score ≥ 25 and read length ≥ 220 base pairs. sequence accuracy of roche 454 runs was evaluated using the sequences generated by sanger sequencing of the seven plasmid clones. the minority variant was defined as a snp detected at > 0.68% and < 20% of the frequency of mutations. for drm analysis, mutations were called and grouped based on international aids society (ias)-usa 2011 recommendations.23 additional mutations for those samples collected before art initiation were analysed based on the 2009 world health organization surveillance drm list.24 standard sanger sequencing the standard genotyping of hiv drug resistance was performed on all plasma samples using an in-house population-based sequencing assay.11 raw sequencing data were analysed using the customised recall software, v.2.24 (provided by dr. richard harrigan from british columbia’s center for excellence in hiv/aids research, vancouver, canada).25 the mixed mutation calling threshold was set at ≥ 20% of the main peak. the drms were analysed as stated above for parallel deep sequencing results. statistical analysis all statistical analysis was performed using spss statistics v19 (spss inc., chicago, illinois, united states). the wilcoxon signed-rank test was used to analyse the statistical differences in the number of drms detected between sanger sequencing and deep sequencing. fisher’s exact test was used to compare the prevalence of hiv drug resistance by these two methods. a p-value of < 0.05 was considered statistically significant. results parallel deep sequencing coverage and estimate of sequence errors the gs-flx deep sequencing of a single run yielded 246 849 raw sequence reads, of which 242 246 (98.1%) reads passed the quality restrictions with a mean read length of 270 nucleotides, while 4603 (1.9%) low-quality sequences were removed from the analysis pipeline. on average, 5490 reads per sample were obtained (range from 420 to 5673 reads). from the 1% mixed clone, k103n was not detected within the 520 sequence reads; y181c and m184v were detected at the level of 1.03% with 1335 reads, while g190a was detected at 0.97% with 785 reads. the mean error rate plus two standard deviations was 0.43 from the six control plasmid clones. sequence errors mostly occurred at the overlapping areas of amplicons in the rt gene. there was only one snp showing an error rate of 0.68% at codon 17 of the pr gene in a polyg region (ggggggca, nucleotide 35, figure 1). this error at pr codon 17 was not in the drm position according to ias and stanford hiv database definitions. another high-error site was codons 63 of the rt gene (nucleotides 470 and 471, figure 1), with a 0.61% error rate. based on these background errors for each nucleotide position, the frequency > 0.68% error rate was used as the threshold for true snps when evaluating the minority drms in the clinical samples. figure 1: error frequency of 454 deep sequencing on the six plasmid control clones at each nucleotide position from amplified amplicons. the x axis is the nucleotide sequence location in protease and reverse transcriptase genes; the y axis is the percentage of single nucleotide polymorphism error. comparative analysis of drug resistance mutations detected by parallel deep sequencing and sanger sequencing barcoded deep sequencing primers amplified all amplicons from the clinical samples. roche gs-flx deep sequencing identified all 72 of the drms that had been detected by sanger sequencing at a level of ≥ 20% from the 41 clinical malawian samples. additionally, a total of 79 drms were exclusively detected by deep sequencing. thus, sanger sequencing missed 52.3% of drms at a level of < 20% in these clinical samples. the differences in the numbers of drms detected by these two sequencing approaches were significant against nrtis (p = 0.004) and nnrtis (p = 0.0001). further, the cohen’s effect size value (d = 0.91 and 0.79) suggests a moderate to high practical significance. no significant difference was found in detection of protease inhibitor (pi) mutations (p = 0.083) (figure 2). figure 2: comparison of mean number of hiv-1 drug resistant mutations detected by sanger and deep sequencing methods. the vertical bars present mean number of mutations ± standard error; p < 0.05 was considered statistically significant. prevalence of minority drug resistance mutations in baseline samples before antiretroviral therapy initiation overall, the frequency of drms increased by deep sequencing with minority drms being detected in 17 (85%) of the 20 pre-art samples by deep sequencing. minority nrti were detected in 10 (50%) of these samples and nnrti mutations in 14 (70%), while minority pi mutations were found in three (15%) of the 20 samples. in addition, the number of drms detected by deep sequencing was higher than sanger sequencing. this increase of drm numbers detected by deep sequencing was statistically significant for nrti (2/20 vs 13/20, p = 0.015), but not for nnrti (9/20 vs 14/20, p = 0.13) or pi (0/20 vs 3/20, p = 0.235) when compared to sanger sequencing. the most common minority nrti mutation was k65r (6 of 20), followed by m184i (2 of 20, table 2). the snp frequency for k65r ranged from 0.7% to 2.5% of the sequence reads in these six patients and both mutant alleles of agg and aga were detected in these hiv-1 subtype c-positive samples. the common minority nnrti mutations detected were v106m, v179t and g190a (3/20 each), followed by e138k and y181c (2/20 each), and k101p, k103n, v108i, e138a, v179d, a190e and h221y (1/20 each). the minority pi mutations detected in three of the samples were m46i (2/20) and d30n (1/20) (table 2). table 2: number of drug resistance mutations detected by two sequencing methods against pis, nrtis and nnrtis from plasma specimens collected from patients before art initiation and at virologic failure† prevalence of minority drug resistance mutations in patients experiencing virologic failure after antiretroviral therapy overall, minority drms were detected by deep sequencing in 90.5% (19/21) of the patients failing art. of the 21 malawian samples, minority nrti mutations were detected in 8 (38%) samples, while minority nnrti mutations were detected in 14 (66.7%) samples (table 2). minority pi mutations were only detected in 3 of 21 (14.3%) samples. however, the increased number of minority mutations in individual patients for nrti or nnrti in art-failing patients was not statistically significant (nrti: 12/21 vs 14/21, p > 0.05; or nnrti: 18/21 vs 19/21, p > 0.05) when compared to sanger sequencing. among the minority nrti mutations, m184i was detected in three samples, and m184v and k65r in two samples respectively. other minority mutations including m41l, a62v, d67n, t69d, v75m, t215f and k219e were found in one sample each. among the minority nnrti mutations, v108i, v179t were detected in four samples and g190a, h221y and k101e were detected in three samples. other nnrti mutations, such as v90i, k103n, v106a/m, e138a, v179e, y181c, y188c/l and g190e were also found in one or two samples. clinical impact of minority drug resistance mutations detected by deep sequencing on virologic failure to study the impact of minority drms detected by deep sequencing on the clinical outcome of patients on art, we compared drug resistance levels (genotype susceptibility score) or expansion of drug resistance to additional drugs or drug classes from those 21 art-failure patients against nrtis and nnrtis using the stanford hiv drug resistance database tool. among 19 patients with the additional minority drms detected, we found that seven (36.8%) of the patients had enhanced resistance levels to nrtis. more importantly, three of the seven patients had gained lowto high-level resistance against tenofovir (table 3), a key component of the current world health organization-recommended firstand second-line regimens.26 fourteen (73.7%) out of the 19 patients also had an enhanced resistance level to nnrtis and 13 of these 14 patients had intensified or expanded resistance profiles against the second generation of nnrtis (etravirine and rilpivirine) (table 4). furthermore, the drug resistance profile analyses revealed an intermediateor high-level resistance to the relevant first-line regimens (stavudine, lamivudine and nevirapine or zidovudine, lamivudine and nevirapine) that are prescribed to these malawian patients and which might explain the virologic failures these patients experienced. table 3: comparison of nrti drug resistance mutations detected from plasma specimens collected from patients with virologic failure after 12–15 months on art by sanger sequencing and deep sequencing and their impacts on nrti susceptibility. discussion the present study has shown that massively parallel deep sequencing is capable of detecting minority hiv-1 variants from hiv-1 subtype c clinical samples. the higher prevalence of minority drms in pre-art malawian patients to nrtis by deep sequencing was statistically significant compared to sanger sequencing. the minority mutation profile revealed that the increased minority drms were associated with enhanced dr levels in virologically failing patients. this study was designed to evaluate parallel tagged deep sequencing in detection of minority drms in samples collected from malawi. we successfully amplified all 41 plasma specimens collected from the patients and seven plasmid dna samples that generated 242 246 sequencing reads using the degenerate primers designed for hiv-1 group m subtypes and circulating recombinant forms. our results not only showed 100% concordance of drms detected by sanger sequencing and deep sequencing, but deep sequencing also detected over 50% minority drms in these clinical samples. at the lower detection level of 0.68%, set by the current study, minority drms were detected in a majority of samples collected from patients before art initiation and at virologic failure. although we only analysed hiv-1 subtype c-positive malawian samples in this study, the primers were designed for all relevant hiv-1 group m subtypes and circulating recombinant forms. in fact, we were able to amplify subtype b, b/c, f and g samples using these primers which were confirmed by gel electrophoresis and successfully detect 5 of 5 subtype b samples using deep sequencing methods.27 previous studies reported the use of parallel tagged deep sequencing methods in detecting low levels of hiv-1 subtype b variants.18,20,21,28,29 a study by dudley et al.20 evaluated a 454 gs junior sequencer by multiplexing 48 samples collected from hiv-1 subtype b-positive individuals and obtained a sequencing success rate of 93% and an error rate of 0.71%. our study using gs-flx with multiplexing on 48 samples collected from hiv-1 subtype c-positive patients showed a 100% amplification rate and 0.265% mean error rate. many studies have reported that the mean error rate of pyrosequencing techniques can be down to 0.05% to 1%.17,19,21,30 the error rates for deep sequencing not only affect the accuracy of base calling, but also impact the sensitivity of minority variant detection. it has been reported that factors, such as the input number of template molecules, sequence primers, amplicon length, nucleotide sequences, pcr errors and operational procedures, might contribute to deep sequencing assay sensitivity and accuracy.17,31,32,33 in addition, error rates are nucleotide position-dependent and roche 454 deep sequencing methods are prone to have more errors at the homopolymeric regions.17,31 in the present study, cross-over errors of major drms between samples were not found. to balance the detection sensitivity with detection accuracy, we set up the base calling threshold for low-frequency mutations at > 0.68% (mean error rate + 2 standard deviations) which was calculated based on the error rates of individual nucleotide positions of six control plasmid samples. in the present study, the k103n mutation was not detected from the mixed clone at 1% of minority variant level of control plasmids from 520 reads. this was likely due to not having enough reads amplified for this mixed plasmid as a previous study demonstrated that at least 1950 reads are required for detecting a minority variant for k103n mutations at about the 1% level.17 one limitation of our study was that the primer pair for amplifying amplicons containing codon 103 of rt gene was not optimal for the depth of coverage. some minority k103n mutations could have been missed in this study. thus, the number of sequence reads obtained for each nucleotide position and errors at the homopolymeric regions played an important role in the depth of the next-generation sequencing. our results from samples collected from patients failing art and initiating art demonstrate that deep sequencing has the added benefit of detecting low-frequency mutations in this malawian cohort. overall, deep sequencing detected significantly more drms than sanger sequencing. of those specimens collected from patients initiating art, more drms against nrti were detected by deep sequencing. among the minority drms, detected by 454 deep sequencing, k65r and m184i were the most common and may compromise the effectiveness of both firstand second-line drugs used according to the malawi art guidelines. the k65r mutation can confer resistance to stavudine and cross-resistance to lamivudine, abacavir, emtricitabine and tenofovir,23,34,35 and is more frequently identified in hiv-1 subtype c.30,35,36 similar to previous studies, the k65r mutation was seen in both treatment-naïve patients and patients failing art in this cohort. several studies have reported that increased presence of k65r mutations is caused by pyrosequencing errors or by the nucleotide template of subtype c viruses (such as the ata sequence at codon 63 of the rt gene).30,32,37 even though no errors at codon 65 of the rt gene were found by deep sequencing in the current study, we did find a relatively higher error rate at codon 63 of the rt gene in the subtype c plasmid sequences. however, we did not find higher error rates for k65r compared with other drm sites in these patients. the m184i mutations were only detected at low frequencies by deep sequencing in pre-art patients and patients with treatment failure. m184i was considered to be a transient mutation before being replaced by m184v.19,38,39 no detectable levels of m184v mutations were found in pre-art samples using deep sequencing, but m184v mutation was detected in over three-quarters of samples from patients failing art. taken together, the higher nrti resistance mutations of m184v and k65r in patients failing art were more likely acquired by selective drug pressure in this malawian cohort treated with a regimen containing stavudine and lamivudine.22 in this study, most samples collected from virologic failure patients had detectable drms to nnrti by both sanger sequencing and deep sequencing. the mutations k101e, k103n, v106a/m, v179d/t, y181c, g190a/e and h221y to nnrti were the most common minority mutations detected in these patients. virus with k101e/y181c/g190a and other mutations could increase levels of resistance to nevirapine 893-fold.40 the h221y mutation could also impact clinical outcomes as y181c/h221y along with the k103n or k101q mutations could increase resistance levels to nevirapine over 100-fold (k103n) or 3000-fold (k101q).41 the drug resistance profile generated by deep sequencing revealed that these mutations were associated with the first-line regimen (stavudine, lamivudine and nevirapine). the drms to nnrti in pre-art samples were also relatively high in these patients and were likely due to single-dose nevirapine used in the prevention of mother-to-child transmission program in malawi.22 however, our results could not rule out the presence of transmitted drug resistance to nnrti in these pre-art patients. although drms against pis were not detected using sanger sequencing, they were detected by deep sequencing in this cohort. m46i/l is considered a major pi mutation and would increase drug resistance levels to pis along with other mutations.34,42 because no pi drugs were used in the first-line art in this cohort, these pi mutations were likely natural polymorphisms of hiv-1. the natural polymorphism of m46i has been reported to have a replicative advantage for subtype b,43 while the impact of m46i/l natural polymorphisms on the development of drug resistance in patients is unknown. as malawi has started to use lopinavir/ritonavir for second-line regimens,44 the emergence of drms to pis should be closely monitored. evidence is lacking in understanding the real clinical impact of minority drms. clinical trials are needed to accurately evaluate the clinical consequences of these drms. however, our results indicate that minority mutations detected by barcoded deep sequencing show an increased or expanded level of resistance to nrtis and nnrtis (see mutation scores in tables 3 and 4). for instance, increased m184iv/i mutations would reduce susceptibility to lamivudine and emtricitabine (scores from 0 to 60, from susceptible to high-level of resistance). k65r+ m184v/i would reduce susceptibility to tenofovir and didanosine from low-level (scores from 0 to 60) to high-level resistance (scores from 15 to 75). additionally, other individual thymidine-analog mutations showed an intermediate or high level of resistance to malawi’s first-line regimens (stavudine, lamivudine and nevirapine). these low-frequency mutations detected by the barcoded parallel sequencing added significant values to the resistant reservoir in the hiv-positive population. all mutation data, both majority and minority mutations, can be used by doctors or policy makers as a reference when changing or revising treatment therapy for patients with virologic failure or country first-line regimens in malawi. table 4: comparison of nnrti drug resistance mutations detected from plasma specimens collected from patients with virologic failure after 12–15 months on art by sanger sequencing and deep sequencing and their impacts on nnrti susceptibility. limitations this study had its limitations. first, the sample size was small due to the availability of remnant samples and budget constraints. a statistically-appropriate sample size should be used for population-level estimations of drms in order to make a meaningful statement. second, although significantly increased minority drms were observed in the samples collected from pre-art and patients with virologic failure using roche 454 barcoded deep sequencing, lack of proper plasmid mutant k65r control in the test might have compromised the accuracy of calculating the k65r mutation rate. third, some methods in the current study could not be applied further as a result of the 454 platform and technologies being discontinued due to high cost and errors at some homopolymeric regions. however, the designed primers and barcoded strategy in the current study could be applied to other deep sequencing platforms for hiv drug resistance testing or studies. conclusion in conclusion, our study confirmed that barcoded parallel deep sequencing technology is capable of detecting minority drms from clinical patient samples. these minority drms not only increased resistance levels to the antiretroviral drugs that are being prescribed, but they also expanded resistance to additional major first-line antiretroviral drugs such as tenofovir. the minority drms detected by deep sequencing may be helpful for selecting the optimal regimens for patients initiating art and for patients who fail first-line regimens. acknowledgements this work was supported by the president’s emergency plan for aids relief (pepfar) through the united states centers for disease control and prevention. the findings and conclusions in this paper are those of the authors and do not necessarily represent the official position of the centers for disease control and prevention. all authors read and approved the final manuscript. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support none. authors’ contributions c.y. and z.z. conceived and designed the current study. l.a.r. and z.z. performed the deep sequencing experiments. k.t. and g.z. performed deep sequencing analysis and statistical analysis. n.w.-k. and k.m. participated in a prospective cohort study to monitor art outcomes and drug resistance development in malawi, including sample collection, data collection and management. j.r.d., n.w. and h.g. performed sanger sequencing; d.-p.z., m.f. and s.s. participated in sequencing data collection and management; and j.n. supervised and supported the study. z.z. and c.y. wrote the manuscript. references usaid. 17 million people with access to antiretroviral therapy [page on the internet]. c2016 [cited 2016 jun 22]. available from: http://www.who.int/hiv/mediacentre/news/global-aids-update-2016-news/en/ gunthard hf, wong jk, ignacio cc, et al. comparative performance of high-density oligonucleotide sequencing and dideoxynucleotide sequencing of hiv type 1 pol from clinical samples. aids res hum 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mutations and hiv-i subtypes among newly-diagnosed drug-naive persons visiting a voluntary testing and counselling centre in northeastern south africa. j health popul nutr. 2011;29(4):303–309. https://doi.org/10.3329/jhpn.v29i4.8444 manosuthi w, thongyen s, nilkamhang s, et al. hiv-1 drug resistance-associated mutations among antiretroviral-naive thai patients with chronic hiv-1 infection. j med virol. 2013;85(2):194–199. https://doi.org/10.1002/jmv.23452 hosseinipour mc, van oosterhout jj, weigel r, et al. the public health approach to identify antiretroviral therapy failure: high-level nucleoside reverse transcriptase inhibitor resistance among malawians failing first-line antiretroviral therapy. aids. 2009;23(9):1127–1134. https://doi.org/10.1097/qad.0b013e32832ac34e abstract introduction methods results discussion acknowledgements references about the author(s) mireille b. kalou international laboratory branch, division of global hiv and tuberculosis, center for global health, us centers for disease control and prevention, atlanta, georgia, united states arnold castro laboratory reference and research branch, division of std prevention, national center for hiv/aids, viral hepatitis, std and tb prevention, us centers for disease control and prevention, atlanta, georgia, united states amy watson international laboratory branch, division of global hiv and tuberculosis, center for global health, us centers for disease control and prevention, atlanta, georgia, united states heather jost laboratory reference and research branch, division of std prevention, national center for hiv/aids, viral hepatitis, std and tb prevention, us centers for disease control and prevention, atlanta, georgia, united states stacy clay international laboratory branch, division of global hiv and tuberculosis, center for global health, us centers for disease control and prevention, atlanta, georgia, united states ye tun office of the associate director of laboratory science, center for global health, us centers for disease control and prevention, atlanta, georgia, united states cheng chen laboratory reference and research branch, division of std prevention, national center for hiv/aids, viral hepatitis, std and tb prevention, us centers for disease control and prevention, atlanta, georgia, united states kevin karem laboratory reference and research branch, division of std prevention, national center for hiv/aids, viral hepatitis, std and tb prevention, us centers for disease control and prevention, atlanta, georgia, united states john n. nkengasong international laboratory branch, division of global hiv and tuberculosis, center for global health, us centers for disease control and prevention, atlanta, georgia, united states ronald ballard office of the associate director of laboratory science, center for global health, us centers for disease control and prevention, atlanta, georgia, united states bharat parekh international laboratory branch, division of global hiv and tuberculosis, center for global health, us centers for disease control and prevention, atlanta, georgia, united states citation kalou mb, castro a, watson a, et al. laboratory evaluation of the chembio dual path platform hiv-syphilis assay. afr j lab med. 2016;5(1), a433. http://dx.doi.org/10.4102/ajlm.v5i1.433 original research laboratory evaluation of the chembio dual path platform hiv-syphilis assay mireille b. kalou, arnold castro, amy watson, heather jost, stacy clay, ye tun, cheng chen, kevin karem, john n. nkengasong, ronald ballard, bharat parekh received: 08 mar. 2016; accepted: 28 june 2016; published: 15 sept. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: use of rapid diagnostic tests for hiv and syphilis has increased remarkably in the last decade. as new rapid diagnostic tests become available, there is a continuous need to assess their performance and operational characteristics prior to use in clinical settings. objectives: in this study, we evaluated the performance of the chembio dual path platform (dpp®) hiv–syphilis assay to accurately diagnose hiv, syphilis, and hiv/syphilis co-infection. method: in 2013, 990 serum samples from the georgia public health laboratory in atlanta, georgia, united states were characterised for hiv and syphilis and used to evaluate the platform. hiv reference testing combined third-generation enzyme immunoassay and western blot, whereas reference testing for syphilis was conducted by the treponema pallidum passive particle agglutination method and the trepsure assay. we assessed the sensitivity and specificity of the dpp assay on this panel by comparing results with the hiv and syphilis reference testing algorithms. results: for hiv, sensitivity was 99.8% and specificity was 98.4%; for syphilis, sensitivity was 98.8% and specificity was 99.4%. of the 348 co-infected sera, 344 (98.9%) were detected accurately by the dpp assay, but 11 specimens had false-positive results (9 hiv and 2 syphilis) due to weak reactivity. conclusion: in this evaluation, the chembio dpp hiv–syphilis assay had high sensitivity and specificity for detecting both hiv and treponemal antibodies. our results indicate that this assay could have a significant impact on the simultaneous screening of hiv and syphilis using a single test device for high-risk populations or pregnant women needing timely care and treatment. introduction both syphilis and hiv infections can cause significant morbidity and mortality and are important public health concerns, especially in resource-limited settings (rls). while the number of hiv-positive individuals continues to decline, in 2013, the joint united nations program on hiv/aids (unaids) estimated that 32.6 million people were still living with hiv in lowand middle income countries.1 similarly, approximately 90% of new syphilis cases globally occur in low-income countries where sexually-transmitted hiv is also a major public health problem.2 syphilis is common among individuals with hiv, and the risk of acquiring hiv is estimated to increase exponentially when syphilis is present.3,4 the world health organization (who) has estimated that more than 12 million new cases of adult syphilis occur worldwide each year, and the disease can be transmitted congenitally, affecting 500 000 or more infants annually.5 among pregnant women, the transmission of hiv and syphilis infections to their unborn infants can result in serious adverse pregnancy outcomes, such as premature delivery, low birth weight, congenital anomalies and perinatal death.6,7,8 who and unicef guidelines on essential maternal and child health services provide recommendations that all pregnant women have a laboratory profile including testing for hiv and syphilis.9 however, in rls, most women receive their maternal and child health services at the lowest level of the tiered health systems, with very limited laboratory capacity.10 over the past decade, the development of single infectious disease rapid diagnostics has allowed detection and treatment to take place on-site, even in low-level health facilities that lack basic public laboratory infrastructure.9,10 unfortunately, sexually-transmitted infections (stis) are often not viewed as a public health priority in many rls. sti surveillance, prevention and treatment programmes are generally poorly resourced and staffed. however, as technology advances, a single device capable of screening multiple diseases could increase the uptake of syphilis testing, especially in rls, where syphilis infection often remains undiagnosed because routine testing is not part of the national guidelines. as global efforts continue to scale-up programmes for screening, treatment and prevention of both hiv and syphilis, the use of accurate rapid diagnostic tests (rdts) remains a reliable and cost-effective tool for rls.8,11,12 rdts allow access to testing in geographic areas where laboratory services are limited and can be performed by staff with minimal training, such as antenatal care settings, tuberculosis clinics, and clinics serving hard-to-reach populations.13,14,15,16,17 the use of rdts also decreases the turnaround time and the overall cost of the testing, which could contribute significantly to the uptake of testing and the acceptance by countries with limited resources. the availability of rdts designed to screen individual infectious diseases, including hiv and syphilis, has increased remarkably in the past decade.11,12,18,19 recently, the multiplexing of rdts has also been developed to address operational challenges around confirmatory testing, turnaround time and specimen volume required to perform multiple tests.20,21,22 with the introduction of integrated approaches, such as the making pregnancy safer initiative and dual elimination of mother-to-child transmission of hiv and syphilis, more national programmes are advocating for combined screening and treatment of these two diseases.23 to support these programmes, a single rdt device to screen simultaneously for both hiv and syphilis using finger-prick blood is vital in order to be cost-effective, increase coverage and manage supply chain challenges.24 therefore, the availability of a quality dual rapid test for hiv and syphilis would greatly strengthen the prevention and control programmes that target the most at-risk populations and mother-to-child transmission of hiv and syphilis. the aim of this study was to evaluate the performance characteristics of the chembio dual path platform (dpp)® hiv–syphilis assay (chembio, medford, new york, united states; hereafter termed dpp hiv–syphilis assay) and to determine its eligibility for inclusion in the us agency for international development procurement waiver list for rdts intended for use in countries supported by the united states president’s emergency plan for aids relief (pepfar). methods ethical considerations this study was conducted under an existing protocol which was submitted for human subjects review and approval at the us centers for disease control and prevention (cdc). the specimens used in this evaluation were obtained as part of an existing material transfer agreement with the laboratory reference and research branch, division of std prevention, cdc. consent was sought according to the georgia public health laboratory policy. specimen characterisation and reference testing algorithm in 2013, we prospectively collected 1006 sera from the georgia public health laboratory in atlanta, georgia, united states. the specimens, normally discarded, were delinked from personal identifiers or any other demographic information (i.e., age, gender, etc.) and unique cdc identifiers were assigned. specimens were stored at −70 °c until ready for testing. these sera were characterised and constituted the evaluation panel. the serum panel was initially characterised for syphilis by qualitative treponema pallidum passive particle agglutination (tppa; fujirebio diagnostics, inc., malvern, pennsylvania, united states), after which all tppa-positive specimens were confirmed by trepsure (trinity biotech, jamestown, new york, united states) testing. additionally, all 1006 specimens were screened for hiv using a us fda-approved enzyme immunoassay (eia; genetic systems hiv-1/hiv-2 plus o eia, bio-rad, hercules, california, united states). all reactive specimens by the eia were further confirmed by a us fda-approved western blot (wb) assay (cambridge biotech hiv-1 western blot, cambridge biotech corporation, rockville, maryland, united states). all specimens with incomplete hiv and/or syphilis test results, including specimens with hiv wb indeterminate results, were excluded from the evaluation. chembio dpp hiv–syphilis assay performance characteristics the dpp hiv–syphilis assay is a single-use immune-chromatographic rapid screening test for the detection of specific antibodies against hiv types 1 and 2 (hiv 1/2) and t. pallidum, with either finger-stick whole blood, venous whole blood, serum, or plasma samples. two trained operators independently performed and interpreted the assay according to the manufacturer’s instructions, and the test results were recorded on separate sheets. any visible band in the positive region was considered as a positive result for hiv and/or syphilis, irrespective of the strength of the band. performance characteristics of the dpp hiv–syphilis assay were determined by comparing the assay with the hiv and syphilis gold standards as outlined above. the eligibility criteria for inclusion of chembio dpp hiv-syphilis assay in the usaid waiver procurement list of rdts were sensitivity > 99% and specificity > 98% for the hiv line and sensitivity 94% and specificity > 95% for syphilis line. inter-reader and inter-lot variability assessment to assess the consistency of the assay’s performance, three different test lots were evaluated using dilution panels totaling 100 individual specimens for each biomarker. the panels were created from a five-fold serial dilution using 10 hiv-positive and 10 syphilis-positive sera with strong reactivity prepared in pooled negative plasma (seracare life science, gaithersburg, maryland, united states). the end-point detection limits of each lot were determined by comparing the performance of the reference lot (lot #1) against lot #2 and lot #3. the reference lot was used to establish the sensitivities and specificities of the assay. the performance of each lot was considered acceptable, if the lot was within at least one dilution end-point titre from the reference lot, with an overall agreement of ≥ 90%.25 if a lot differed by > 10% from the reference lot then it was considered unacceptable. due to the subjectivity in interpretation of the rdt results, we also assessed inter-reader variability of the assay by independently comparing the results interpreted and recorded by a total of three technicians, including the two technicians who performed the testing. assessment of operational characteristics the rapid scale-up of most hiv and syphilis testing programmes involves the use of rdts, most often performed by health professionals with a wide range of expertise in a variety of settings. therefore, it is crucial to assess key operational characteristics, such as the ease of use, number of steps, storage conditions and ease of interpretation, which might impact the large-scale implementation of the dpp hiv-syphilis assay in rls. for quality assurance purposes, two different technicians performed testing of 10 test devices at a time, to allow for sufficient reading time. each day, a set of positive and negative controls for hiv and syphilis was run by each technician prior to testing the evaluation specimens. the test results were recorded on a worksheet by the technician performing testing and verified by a second technician. data analysis all test results were entered independently by two technicians into a microsoft excel spreadsheet (microsoft corporation, redmond, washington, united states). the spreadsheets were reviewed for accuracy and merged prior to data analysis. the analysis included the calculation of the performance characteristics of the dpp hiv–syphilis assay compared to the hiv and syphilis predicate testing results (i.e., sensitivities, specificities, their corresponding 95% confidence intervals [cis] and kappa values). the kappa values were computed using stata software (statacorp lp, college station, texas, united states). we also determined the ability of the dpp hiv–syphilis assay to accurately identify hiv only, syphilis only, and co-infected (both hivand syphilis-positive) specimens. results panel characterisation data of the 1006 serum samples tested by the reference algorithms (eia/wb for hiv and tppa/trepsure for syphilis), 16 (1.6%) specimens with incomplete/invalid predicate hiv and/or syphilis testing results were excluded from the evaluation. thus, 990 (98.4%) fully characterised sera for both biomarkers were included in the final analysis. of the 990 sera, 79 (7.9%) were hiv-positive, 299 (30.2%) were syphilis-positive, 348 (35.2%) were both hivand syphilis-positive, and 264 (26.7%) were negative for both syphilis and hiv (table 1). table 1: composition of the evaluation panel characterised for hiv and syphilis in 2013 (n = 990). performance characteristics of the dpp hiv-syphilis assay of the 427 samples confirmed to be hiv-positive by the hiv eia/wb reference algorithm, only one sample was identified as hiv-negative by the dpp hiv–syphilis assay, resulting in a sensitivity of 99.8% (95% ci: 98.7% – 100%) (table 2). similarly, of the 563 samples confirmed to be hiv-negative by the hiv reference algorithm, 554 samples were identified as hiv-negative by the dpp hiv–syphilis assay. thus, the specificity of the assay was 98.4% (95% ci: 97.0% – 99.3%) for the hiv component. table 2: performance characteristics of the chembio dpp hiv-syphilis assay when compared to hiv and syphilis reference testing algorithms (n = 990). of the 647 samples confirmed to be syphilis-positive by the tppa/trepsure reference algorithm, 639 were identified as syphilis-positive by the dpp hiv-syphilis assay, resulting in a sensitivity of 98.8% (95% ci: 97.6% – 99.5%). only two of the samples confirmed to be syphilis-negative by the syphilis reference algorithm were identified as positive by the dpp hiv-syphilis assay, resulting in a specificity of 99.4% (95% ci: 97.9% – 99.9%) (table 2). of the 990 sera, 20 (2.0%) were discordant between the dpp hiv–syphilis assay and the predicate testing for both hiv and syphilis. these 20 specimens included 11 false positives (9 hiv and 2 syphilis) and nine false negatives (1 hiv and 8 syphilis). the 11 false positives were repeated using the dpp hiv-syphilis assay and the results did not change. the kappa-values indicated high agreement between the dpp hiv-syphilis assay and the reference testing algorithms (kappa-value: 0.98 for both methods). a comparison of the dpp hiv–syphilis assay results with the reference testing data showed that 344 (98.9%) of the 348 dually-reactive samples were confirmed by the reference methods (table 3). however, the remaining four specimens (1.1%) identified as dually-reactive by the reference testing were identified as positive for syphilis by the dpp hiv–syphilis assay, but negative for hiv. no invalid results were observed during the evaluation. table 3: comparison of chembio dpp hiv–syphilis assay results and hiv and syphilis reference testing (n = 990). inter-lot and inter-operator variability compared to the reference lot, the performance of lot #2 differed by 3% for hiv and 6% for syphilis, whereas lot #3 differed by 4% for hiv and 3% for syphilis. in addition, there was high consistency in the interpretation of the dpp hiv–syphilis assay results for both hiv (96%) and syphilis (91%) among three different technicians. both inter-lot and inter-operator variability were considered acceptable because both were less than 10%. operational characteristics of the dpp hiv-syphilis assay the test device included two ports and two buffer vials, which could potentially lead to confusion with the pre-dilution step of the blood in buffer and possible mix-ups at the additional steps. the product evaluated also did not include a specimen application device (i.e., disposable micropipette). some level of complexity in the interpretation of the results of this three-line test required appropriate training. however, the run time (15–20 minutes per sample), long shelf life and individual packaging added value to the high performance of the assay. discussion the dpp hiv-syphilis assay displayed high sensitivity and specificity for detecting hiv and syphilis in co-infected individuals and met the minimum requirements for inclusion in the usaid procurement waiver list, thus making it accessible to pepfar supportive countries that may consider including it to their current national hiv testing algorithms. the high agreement between the dpp hiv-syphilis assay and the reference testing algorithms demonstrates the ability of the dual rapid test to accurately identify individuals co-infected with syphilis and hiv. while previous studies have suggested that the interpretation of serological assays for syphilis can be challenging in hiv-positive patients,26 our evaluation demonstrates that the dpp hiv-syphilis assay can accurately detect syphilis in hiv-positive individuals. we observed eight false-negative syphilis results with the dpp hiv-syphilis assay. these findings are comparable with previous studies which have found that, while uncommon, false-negative syphilis results may occur among hiv-positive individuals when using serological tests such as the quantitative rapid plasma reagin test and the tppa assay, especially during the late stages of the disease.27 similarly, the nine false-positive hiv results identified by the dpp hiv-syphilis assay could be attributed to serological cross-reactivity or non-specific immune reactivity, sometimes observed with hiv rapid tests.28 reassuringly, there were only two false-positive syphilis results identified by the dpp hiv-syphilis assay, demonstrating the high specificity of the test for syphilis detection, as has been reported previously.20 with the massive roll out of rdts in most rls, the need for a laboratory-based confirmatory test for hiv and syphilis is no longer required to initiate treatment. the recently-released who guidelines on hiv testing services recommends that countries and programmes implement the retesting strategy for verification purposes.29 as countries adopt these recommendations, all patients with an initial hiv-positive result will be retested by a different healthcare worker at the treatment centre prior to initiating care and treatment, minimizing the turnaround time for returning results to the referring clinic which would occur if the confirmatory testing was conducted in a laboratory setting.30,31 although there has been concern that healthcare providers who typically work alone in settings offering integrated services may be unable to efficiently perform several individual rapid diagnostics within a single visit, it is important to highlight that with the increased focus on task-sharing strategies to address staffing issues, healthcare professionals routinely perform multi-test algorithms to screen hiv patients, in addition to other duties. thus, the inclusion of a multi-disease single test into the current hiv multi-test algorithm may help decrease work load and increase the uptake of syphilis testing.32 screening multiple diseases with a single test device such as the dpp hiv-syphilis assay provides the opportunity to potentially strengthen health systems. the main operational considerations should be training on the different rtds as they are introduced, and robust monitoring of providers’ performance through the implementation of quality assurance measures, such as use of standard operating procedures or job aides, proficiency testing panels and proper documentation of the test results in a standardised logbook or register that can be reviewed to identify issues and provide corrective actions. the multiplex rdts might help address issues observed in vertical programmes related to the cost of testing, quality assurance, and the integration of training to providers on use of the tests.33 while this evaluation did not include a cost analysis to determine the cost-effectiveness of the dpp hiv-syphilis assay, the current unit price, estimated at us $1.20 – $1.50, is comparable to that of other rapid tests commonly used by national hiv programmes and could be substituted for some of the screening tests combined in more cost-effective algorithms for both hiv and syphilis diagnosis.24 moreover, in settings where test stock outs are recurrent because of the lack of proper supply chain management systems, the dpp hiv-syphilis assay may be a suitable alternative. the dpp hiv-syphilis assay has the potential to screen for hiv and syphilis infections with a single test device in settings such as antenatal care and sexually-transmitted disease clinics in rls where perinatal hiv and congenital syphilis are significant contributors to morbidity and mortality. the presence of stis such as syphilis increases the risk of transmission of hiv.34 failure to diagnose and immediately treat or provide appropriate care for a pregnant woman, her partner, and the infant may result in serious complications, ranging from foetal wastage, neonatal and infant infections, and premature death.35 in addition, the dpp hiv-syphilis assay may have applications both domestically and in international settings with limited on-site laboratory capacity and/or where loss to follow-up is high. moreover, in settings where access to hiv testing is prohibitive due to stigma, a combined diagnostics method could lessen stigmatisation and increase access to testing.28,29 recommendations operationally, the dpp hiv-syphilis assay, considered to be a rapid test, requires a pre-dilution step, use of second buffer and multiple steps, which may add some level of complexity for providers with limited laboratory expertise. moreover, the presence of three lines, one for control, a second for syphilis and a third for hiv, can lead to misinterpretation of results by less-trained individuals. therefore, it would be important to ensure adequate training is provided prior to its use in the field. appropriate labeling, for example colour coding of sample and buffer ports and matching buffer bottles, and careful interpretation of results with clear job aides to avoid mix-ups, may assist in ensuring that no errors are made. in addition, the use of standardised registers to monitor ongoing agreement between tests, enrollment of testers in an external quality assessment programme and close supervision and monitoring, are strongly recommended prior to routine roll out. similar to recent publications on dual hiv-syphilis rdts, this study was a laboratory-based evaluation using a panel of well-characterised and archived serum samples.36 in the same study, all three dual hiv-syphilis rdts exhibited high sensitivity and specify when performed in a laboratory setting and by trained personnel. limitations this evaluation did not determine the performance of rdts outside the laboratory, by staff with limited training, or using fresh specimens obtained by fingerpick. as such, additional field evaluations will be needed. the findings of this evaluation were primarily based on a serum panel obtained from the local georgia public health laboratory, which may not necessarily be representative of samples from other geographic locations with varied rates of hiv/syphilis infections and antibody profiles. nor does our study sample represent populations from countries with limited resources who will likely be tested by rdts. thus, there is a need for further field evaluations prior to the broader use of the dpp hiv-syphilis assay in clinical settings. conclusion this laboratory evaluation suggests that the chembio dpp® hiv–syphilis assay could be a suitable screening method for hiv and syphilis using the same device. this test was deemed eligible for inclusion in the usaid procurement waiver list for rdts intended for use in pepfar supported countries. moreover, it could improve the acceptability and increase the uptake of testing and treatment to accelerate elimination of mother-to-child transmission of syphilis and hiv. in addition, for high-risk populations, it could potentially increase uptake of testing, linkage to early care and treatment, and play an important role in syphilis control. in most instances, rdts are reliable for screening hiv and syphilis; however, it is important to remember that misclassifications due to cross-reactivity or atypical immune response may occur. with the adoption of a task-sharing approach to address staff shortages in rls, it will be important to develop a simple and practical job aides and emphasise hands-on training of healthcare providers in order to ensure that manufacturer testing procedures and national testing guidelines are followed so as to minimise operational errors. moreover, further field evaluations should be conducted to assess the feasibility and acceptability of the dpp hiv–syphilis assay among healthcare workers and to determine its cost-effectiveness when included in the routine hiv testing algorithms. acknowledgements we thank mr. javan esfandiari from chembio for providing, free of charge, the chembio dpp® hiv–syphilis assay test kits for this evaluation, and dr elizabeth a. franko from the georgia public health laboratory for supplying the serum samples. competing interests the authors declare that they have no financial or personal relationship that may have inappropriately influenced them in writing this article. sources of support this evaluation was supported in part by the president’s emergency plan for aids relief (pepfar) through the cdc. authors’ contributions m.b.k., a.c., y.t., c.c. and b.p. made substantial contributions to the design of the study, analysis, and interpretation of the data. m.b.k. and a.c. were both the project leaders, and a.c. acquired all the serum samples used for this evaluation. a.w., h.j. and s.c. performed all the laboratory testing, and m.b.k. performed the statistical analyses. m.b.k. and a.c. developed the draft manuscript for publication. both c.c. and y.t. revised the draft critically for important intellectual content. j.n.n., b.p., r.b. and k.k. provided the final approval of the version to be published. m.b.k. and a.c. agreed to be accountable 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antenatal syphilis screening in burkina faso. pan afr med j. 2014;17 suppl 1:12–16. http://dx.doi.org/10.11604/pamjs.supp.2014.17.1.3423 ward h, ronn m. contribution of sexually transmitted infections to the sexual transmission of hiv. curr opin hiv aids. 2010;5(4):305–310. http://dx.doi.org/10.1097/coh.0b013e32833a8844 yeganeh n, watts hd, camarca m, et al. syphilis in hiv-infected mothers and infants: results from the nichd/hptn 040 study. pediatr infect dis j. 2015;34(3):e52–57. http://dx.doi.org/10.1097/inf.0000000000000578 bristow cc, adu-sarkodie y, ondondo ro, et al. multisite laboratory evaluation of a dual human immunodeficiency virus (hiv)/syphilis point-of-care rapid test for simultaneous detection of hiv and syphilis infection. open forum infect dis. 2014;1(1):ofu015. http://dx.doi.org/10.1093/ofid/ofu015 abstract introduction research method and design results discussion acknowledgements references about the author(s) sharana mahomed national health laboratory services, inkosi albert luthuli central hospital, durban, kwazulu natal, south africa department of medical microbiology, university of kwazulu-natal, durban, south africa nomonde r. dlamini-mvelase national health laboratory services, inkosi albert luthuli central hospital, durban, kwazulu natal, south africa department of medical microbiology, university of kwazulu-natal, durban, south africa moses dlamini national health laboratory services, inkosi albert luthuli central hospital, durban, kwazulu natal, south africa koleka mlisana national health laboratory services, inkosi albert luthuli central hospital, durban, kwazulu natal, south africa department of medical microbiology, university of kwazulu-natal, durban, south africa citation mahomed s, dlamini-mvelase nr, dlamini m, et al. failure of bactec™ mgit 960™ to detect mycobacterium tuberculosis complex within a 42-day incubation period. afr j lab med. 2017;6(1),a537, https://doi.org/10.4102/ajlm.v6i1.537 brief reports failure of bactec™ mgit 960™ to detect mycobacterium tuberculosis complex within a 42-day incubation period sharana mahomed, nomonde r. dlamini-mvelase, moses dlamini, koleka mlisana received: 24 jul. 2016; accepted: 19 oct. 2016; published: 26 apr. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract for the optimal recovery of mycobacterium tuberculosis from the bactec™ mycobacterium growth indicator tube 960™ system, an incubation period of 42–56 days is recommended by the manufacturer. due to logistical reasons, it is common practice to follow an incubation period of 42 days. we undertook a retrospective study to document positive mycobacterium growth indicator tube cultures beyond the 42-day incubation period. in total, 98/110 (89%) were positive for m. tuberculosis complex. this alerted us to m. tuberculosis growth detection failure at 42 days. introduction tuberculosis remains an important cause of morbidity and mortality, especially in developing countries. recent molecular methods, in particular the cepheid genexpert™, have revolutionised the rapid diagnosis of tuberculosis due to their high sensitivity, even in smear-negative sputum samples. although culture-based methods take longer, they remain an integral part of the laboratory diagnosis of tuberculosis. the clinical and laboratory standards institute recommends that both solidand liquid-based culture methods be used to maximise the recovery of mycobacterium tuberculosis.1 in comparison to solid-based culture methods, liquid-based culture methods have the advantage of providing more rapid results and, thus, are commonly utilised in tuberculosis diagnostic laboratories. previously, the bactec™ 460tb™ system (becton dickinson diagnostic systems, sparks, maryland, united states) was widely used for the diagnosis of tuberculosis, but due to the disadvantage of containing a radioactive-labelled substrate, it was replaced by the non-radiometric bactec™ mycobacterium growth indicator tube (mgit) 960™ system. this system can be used for the detection of mycobacteria in all types of clinical specimens, except blood. it contains a liquid culture medium (modified middlebrook 7h9 broth), a growth supplement and the antimicrobials polymyxin, amphotericin, nalidixic acid, trimethoprim and azlocillin, to prevent growth of contaminants. a fluorescent compound is embedded in silicone at the bottom of the 16x100 mm tube. the fluorescence indicator is initially quenched by large amounts of dissolved oxygen. as organisms grow and respire, they consume the oxygen, which allows the compound to fluoresce.2 for the optimal recovery of m. tuberculosis, an incubation period of 42–56 days is recommended by the manufacturer. in addition, the manufacturer recommends that a visual check be performed on all ‘instrument negative’ mgit tubes. if the tube is shown to contain small granular clumps or appears non-homogeneously turbid, an acid-fast bacilli (afb) stain should be performed. if the afb smear is positive, then the tube should be regarded as a presumptive positive. although these recommendations are clearly stated by the manufacturer, due to logistical and practical reasons, it is common practice to follow an incubation period of 42 days in most tuberculosis laboratories. studies have illustrated that the sensitivity of the system increases by prolonging the incubation period beyond 42 days.3 in our laboratory, which is located in a busy tuberculosis referral hospital and processes 12 000 mgit tubes a month, we follow an incubation period of 42 days. mgit tubes are incubated until the instrument flags them positive. after 42 days, the instrument flags the tubes negative if there is no growth. all mgit tubes that are negative at 42 days are removed from the system. however, it was brought to our attention that mgit tubes were, in fact being left in the system beyond 42 days during public holidays and over weekends. more importantly, it was noted that some of these mgit tubes flagged positive after 42 days. ziehl-neelson (zn) staining revealed the presence of afb with and without cording. these afb-positive mgit tubes with positive cording were thereafter sent for a mtbdr+ line probe assay (hain lifescience gmbh, nehren, germany), which confirmed the presence of m. tuberculosis complex. this alerted us to the possibility that the system failed to detect the growth of m. tuberculosis at 42 days. we therefore undertook a study to document our experience regarding mgit tube cultures that became positive beyond the usual 42-day incubation period. research method and design ethical considerations blanket ethical approval for this laboratory-based study was obtained from the biomedical research ethics committee, university of kwazulu-natal, south africa. the confidentiality of the data was maintained throughout the study. study setting this study was conducted at the national health laboratory services, provincial tb reference laboratory, based at the inkosi albert luthuli central hospital. this laboratory currently performs all culture and culture-based phenotypic drug susceptibility testing for the province of kwazulu-natal, south africa. a retrospective study for the period 01 january 2014 to 31 march 2015 (15 months) was performed. bd epicenter™ (becton, dickinson and company, franklin lakes, new jersey, united states) software was utilised to specifically look at all mgit tubes that flagged positive after the period of 42 days. this software provides advanced data management for all bd microbiology systems. the basic configuration of the software provides all the communication and reporting capabilities optimised for supporting bd bactec™ and bd bactec mgit™ systems. zn staining was performed on all positive mgit tubes to determine whether afb was present and to document cording characteristics. mgit tubes were presumed to be positive for m. tuberculosis if: (1) afb were present (straight or slightly curved rods), (2) a beaded appearance was observed due to cording characteristics, and (3) staining was pink on a blue background. in order to discriminate between m. tuberculosis and mycobacteria other than tuberculosis, testing for antigens to the mpt64 protein (mpt) and line-probe hybridisation assays were performed on all isolates that were afb-positive. we used the sd bioline tb ag mpt 64 rapid® (standard diagnostics, seoul, korea) immunochromatographic commercial assay, which uses monoclonal antibodies against the mpt64 antigen for confirmation of m. tuberculosis isolates. as part of the routine workflow, all m. tuberculosis-positive isolates were subjected to line probe assay testing (hain lifescience gmbh, nehren, germany) for anti-tuberculosis drug susceptibility, as per standard operating procedures. late contaminants were defined as isolates that flagged positive on the mgit system but were afb-negative with zn staining. mycobacteria other than tuberculosis were identified as isolates that were afb-positive with zn staining but mpt/line-probe assay-negative. isolates that were afb-positive, exhibited cording and were mpt-positive were considered to be positive for m. tuberculosis complex. results a total of 20 914 mgit tubes flagged positive during this period. of these, 159 flagged positive after 42 days (0.8%). a total of 49/159 (31%) were negative on zn staining and were regarded as late contaminants, and 110/159 (69%) were positive on zn staining. a total of 12/110 afb-positive isolates (11%) exhibited no cording, underwent mpt antigen testing, were found to be negative, and were thus identified as mycobacteria other than tuberculosis. a total of 98/110 (89%) exhibited cording, were mpt-positive, and were therefore identified as positive for m. tuberculosis complex. only 78/98 isolates were submitted for line probe assay testing, which further identified all 78 isolates as m. tuberculosis complex. of these 78 isolates, 37/78 (47.4%) were sensitive to both isoniazid and rifampicin, 7/78 (9%) were resistant only to rifampicin, 33/78 (42.3%) were resistant to both isoniazid and rifampicin (i.e., multi-drug resistant) and 1/78 (1.3%) was identified as extensively drug resistant. discussion a number of studies have reported that the bactec mgit 960 system is efficient, rapid, reliable, safe and fully automated, with the added advantage of continuous monitoring of the culture tubes.3,4,5,6,7,8,9 although most studies have confirmed that the bactec mgit 960 has a high sensitivity for recovery of mycobacteria, it has been reported that the instrument detection system may occasionally fail to detect mycobacterial growth at the end of a 42-day incubation period.4,5,10 this has been observed with mycobacteria other than tuberculosis isolates, with m. xenopi being highlighted specifically.4,10,11 this failure to detect growth at 42 days was attributed to the granular growth pattern of the organism, which created less surface contact, keeping oxygen consumption below the detection threshold. the small bacterial load, slower metabolism, and biochemical and thermophilic characteristics of these isolates were also considered as possible contributing factors.10 failure of the bactec mgit 960 to specifically detect m. tuberculosis complex isolates within a 42-day incubation period has not previously been reported. the findings of our study are important in that of the 20 914 mgits processed by our laboratory, 159 (0.8%) were flagged positive after 42 days and were identified purely by a failure to rigidly follow the laboratory protocol. although this represents a small percentage overall, 110/159 mgits were afb-positive. more importantly, 89% of these (98/110) were subsequently identified as positive for m. tuberculosis complex, of which 52.6% (41/78) represented drug-resistant m. tuberculosis. time to detection of positive growth depends on several factors. the number of viable afb inoculated into a mgit tube, the type of species of mycobacteria, the specimen type and the treatment status of the patient must all be taken into consideration. in order to avoid false negatives, the manufacturer refers to troubleshooting procedures in the manual.12 incubation temperatures, decontamination procedures, centrifugation, the use of antibiotics (polymyxin, amphotericin, nalidixic acid, trimethoprim and azlocillin) and procedure checks need to be rigorously carried out in order to avoid missed detection. this study therefore highlights the importance of following laboratory protocols and manufacturer guidelines. limitations this study was purely laboratory-based and all data were accessed via the mgit and epicenter software. we did not have access to patient information for the resistant samples; thus, the treatment status of these patients, which could possibly be related to the delayed positivity, could not be ascertained. conclusion these findings present a challenge for routine, clinical m. tuberculosis laboratories regarding the dogma of incubating tuberculosis cultures for 42 days only. a possible solution to avoid missing m. tuberculosis complex, including resistant strains, would be to prolong the incubation period beyond 42 days. however, this would prove impractical in busy tuberculosis laboratories, due to space constraints and the increased number of mgit tubes that would be required. a more practical option would be to routinely visually examine all negative mgit tubes at 42 days as recommended by the manufacturer. if colony-like clumps are visible at the bottom of the tube, then these mgits should be further analysed. an afb smear should first be performed and, if found to be afb-positive, the sample should be reported as presumptive positive and sent for further testing according to the laboratory’s protocol. troubleshooting guidelines, quality control for the reagents and products used in the isolation, as well as the actual test procedures, are critically important for mycobacteriology laboratories. this study serves to highlight the possibility of missed m. tuberculosis due to growth detection failure. if there is a high suspicion of tuberculosis and the result is negative, this must be communicated to the laboratory. it is thus imperative to maintain good communication between laboratories and clinicians. good history taking and clinical judgement guide the laboratory and thus cannot be over-emphasised. acknowledgements the authors would like to acknowledge the staff at the provincial tb reference laboratory. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. sources of support none. authors’ contributions k.m. was the project leader, and s.m. and m.d. were responsible for the experimental and project designs. m.d. performed most of the experiments. s.m. was responsible for the writing of the manuscript and n.r.d.-m. made contributions. references clinical and laboratory standards institute. performance standards for antimicrobial susceptibility testing; twenty-fifth informational supplement. clsi document m100-s25 (isbn 1-56238-989-0 [print]; isbn 1-56238-990-4 [electronic]). wayne, pa: clsi; 2015. tortoli e, benedetti m, fontanelli a, et al. evaluation of automated bactec mgit 960 system for testing susceptibility of mycobacterium tuberculosis to four major antituberculous drugs: comparison with the radiometric bactec 460tb method and the agar plate method of proportion. j clin microbiol. 2002;40(2):607–610. https://doi.org/10.1128/jcm.40.2.607-610.2002 rohner p, ninet b, metral c, et al. evaluation of the mb/bact system and comparison to the bactec 460 system and solid media for isolation of mycobacteria from clinical specimens. j clin microbiol. 1997;35(12):3127–3131. idigoras p, beristain x, iturzaeta a, et al. comparison of the automated nonradiometric bactec mgit 960 system with löwenstein-jensen, coletsos, and middlebrook 7h11 solid media for recovery of mycobacteria. eur j clin microbiol infect dis. 2000;19(5):350–354. scarparo c, piccoli p, rigon a, et al. evaluation of the bactec mgit 960 in comparison with bactec 460 tb for detection and recovery of mycobacteria from clinical specimens. diagn microbiol infect dis. 2002;44(2):157–161. hanna ba, ebrahimzadeh a, elliott lb, et al., multicenter evaluation of the bactec mgit 960 system for recovery of mycobacteria. j clin microbiol. 1999;37(3):748–752. hillemann d, richter e, rüsch-gerdes s. use of the bactec mycobacteria growth indicator tube 960 automated system for recovery of mycobacteria from 9,558 extrapulmonary specimens, including urine samples. j clin microbiol. 2006;44(11):4014–4017. https://doi.org/10.1128/jcm.00829-06 leitritz l, schubert s, bücherl b, et al. evaluation of bactec mgit 960 and bactec 460tb systems for recovery of mycobacteria from clinical specimens of a university hospital with low incidence of tuberculosis. j clin microbiol. 2001;39(10):3764–3767. https://doi.org/10.1128/jcm.39.10.3764-3767.2001 tortoli e, cichero p, piersimoni c, et al. use of bactec mgit 960 for recovery of mycobacteria from clinical specimens: multicenter study. j clin microbiol. 1999;37(11):3578–3582. piersimoni c, nista d, bornigia s, et al. unreliable detection of mycobacterium xenopi by the nonradiometric bactec mgit 960 culture system. j clin microbiol. 2009;47(3): 804–806. https://doi.org/10.1128/jcm.01444-08 peña ja, ferraro mj, hoffman cg, et al. growth detection failures by the nonradiometric bactec mgit 960 mycobacterial culture system. j clin microbiol. 2012;50(6):2092–2095. https://doi.org/10.1128/jcm.00108 siddiqi sh, rüsch-gerdes s. mgit™ procedure manual for bactec™ mgit 960™ tb system. geneva, switzerland: find diagnostics; 2006. abstract introduction intervention outcomes discussion acknowledgements references about the author(s) annelies w. mesman department of global health and social medicine, harvard medical school, boston, massachusetts, united states partners in health, boston, massachusetts, united states musa bangura partners in health, boston, massachusetts, united states sahr m. kanawa ministry of health and sanitation, koidu, sierra leone joseph s. gassimu partners in health, boston, massachusetts, united states kerry l. dierberg partners in health, boston, massachusetts, united states division of infectious diseases and immunology, new york university, new york, new york, united states mohamed m. sheku ministry of health and sanitation, koidu, sierra leone j. daniel orozco partners in health, boston, massachusetts, united states regan h. marsh department of global health and social medicine, harvard medical school, boston, massachusetts, united states partners in health, boston, massachusetts, united states department of emergency medicine, brigham and women’s hospital, boston, massachusetts, united states citation mesman aw, bangura m, kanawa sm, et al. a comprehensive district-level laboratory intervention after the ebola epidemic in sierra leone. afr j lab med. 2019;8(1), a885. https://doi.org/10.4102/ajlm.v8i1.885 lessons from the field a comprehensive district-level laboratory intervention after the ebola epidemic in sierra leone annelies w. mesman, musa bangura, sahr m. kanawa, joseph s. gassimu, kerry l. dierberg, mohamed m. sheku, j. daniel orozco, regan h. marsh received: 06 aug. 2018; accepted: 18 mar. 2019; published: 22 oct. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the 2014–2016 ebola outbreak exposed the poor laboratory systems in sierra leone. immense needs were recognised across all areas, from facilities, diagnostic capacity, supplies, trained personnel to quality assurance mechanisms. objective: we aimed to describe the first year of a comprehensive intervention, which started in 2015, in a public hospital’s general laboratory serving a population of over 500 000 in a rural district. methods: the intervention focused on (1) supporting local authorities and healthcare workers in policy implementation and developing procedures to enhance access to services, (2) addressing gaps by investing in infrastructure, supplies, and equipment, (3) development of quality assurance mechanisms via mentorship, bench-side training, and the introduction of quality control and information systems. all work was performed alongside counterparts from the ministry of health and sanitation. results: we observed a strong increase in patient visits and inpatient and outpatient testing volumes. novel techniques and procedures were taken up well by staff, leading to improved and expanded service and safety, laying foundations for further improvements. conclusion: this comprehensive approach was successful and the results suggest an increase in trust from patients and healthcare workers. keywords: sierra leone; intervention; district hospital; diagnostics; service expansion; mentorship. introduction laboratories are an often neglected yet crucial part of a functioning health system. laboratory services are required for clinical diagnosis, patient care, treatment and monitoring and have an important public health role in surveillance of disease and antimicrobial resistance. however, many low-income countries lack adequate laboratory services at the community, district and even national levels, hence laboratories have been referred to as the ‘achilles heel’ in efforts to combat disease.1 the world health organization (who) has included laboratory services in the ‘basic minimum package’ for health interventions in low-resource settings,2 and it is recommended to invest in integrated laboratory services, rather than disease-specific programmes.3 however, despite these recommendations, laboratories receive little attention in most health intervention programmes.4 laboratory capacity building is often restricted to short training or equipment donations (many times, equipment is obsolete or being phased out of facilities in high-income countries).5,6 health professionals in low-resource settings are familiar with the sight of unused or broken machines scattered throughout hospital grounds.5,6 because a trained workforce and quality assurance mechanisms, such as the routine maintenance of equipment, are often lacking, patients and clinicians often mistrust diagnostic services.4,7,8,9,10 in sierra leone most people live below the poverty line, life expectancy and adult literacy in 2016 were as low as 51.3 years and 48.1% respectively while the maternal mortality rate of 1360 per 100 000 live births – was among the highest in the world.11 the national public health system has been devastated by a series of events including an 11-year civil war from 1991 to 2002, and the 2014–2016 ebola virus disease (evd) outbreak, during which many health workers lost their lives.12,13 the ebola outbreak exposed a fragile health system, with weak diagnostic networks in the region. public laboratories were not equipped with facilities for evd diagnostics or adequate sample referral systems, leading to long delays in care, which contributed to higher mortality for both non-evd and evd patients.14,15,16 this situation changed only when international organisations and governments offered temporary, deployable laboratory support. in june–july 2015, after the peak of evd transmission, the sierra leone ministry of health and sanitation (mohs) conducted a comprehensive evaluation of the national laboratory system, using an adapted who laboratory assessment tool,17,18 which was funded by the united kingdom department for international development. three hundred and fifteen (315) laboratories, ranging from community health centre laboratories to all district and regional hospital level laboratories, were assessed. the study identified gaps in systems, supplies, infrastructure and quality, including insufficient numbers of adequately trained personnel to respond to outbreaks. of 20 public hospitals, 11 (55%) had no reliable water supply, 16 (80%) lacked 24-h electricity, and five (25%) had no freezer space. regarding testing resources, 16 (80%) offered no blood biochemistry tests, and only three (15%) had electrolytes and liver function tests. standard operating procedures for available tests were absent at 16 (80%) sites. the majority (83%) of the facilities had no guidelines for specimen referral. additionally, while acknowledging the needs, the report stressed limited government resources available to invest in laboratory services.18 at the invitation of the sierra leonean government, the non-profit organisation partners in health (pih) started working in sierra leone during the ebola outbreak in 2014. by 2015, pih shifted its focus from emergency response and committed to providing long-term comprehensive support at the koidu government hospital (kgh), the district hospital in kono. the intervention in the district laboratory was initiated in october 2015, a few months after the mohs laboratory assessment. here, we describe the first year of this programme and summarise initial outcomes, as a replicable model for laboratory strengthening in low-resource settings. intervention ethical considerations no identifiable patient data were collected for this study. baseline setting kono is a district in eastern sierra leone (506 867 population). rich in diamond mines, kono was the epicentre of the civil war. there were 301 confirmed evd infections in the district.19 in october 2015, kgh, serving the entire district population, had unreliable water and electricity, insufficient numbers of trained staff, and was underused by the community. in the hospital laboratory, electronic equipment and computers were not used. supplies of diagnostic tests, consumables, and reagents from the national level were unreliable. standard operating procedures were largely absent, unknown, or poorly implemented, leading to unsafe situations for staff and patients. patient fees were applied (at times inconsistently) per individual test. the kgh laboratory was staffed by a total of 15 technicians and technician assistants, of whom several were not paid or had no laboratory training. this baseline capacity was reflected in the 2015 mohs national laboratory assessment, in which the overall score for the laboratory, based on availability of supplies, equipment, and quality assurance was 30%, ranking kgh among the five lowest scoring districts.18 during the ebola outbreak, patients with suspected evd were quarantined in a holding unit on the hospital grounds. after receiving a positive or negative test result, they were referred to an ebola treatment centre or the kgh wards. koidu government hospital laboratory staff collected blood samples from patients in the holding unit, and received oral swabs from deceased individuals in the district. referral of all specimens to a public health england-supported laboratory in the region was well organised and strictly regulated. in contrast, referral systems for other samples (i.e. for testing or surveillance of other diseases) was poor. external quality assurance mechanisms, such as reading tuberculosis microscopy slides at the national level, were hampered by the outbreak, and for biosafety reasons referral of sputum samples for culture or drug sensitivity testing at the supranational laboratory was on hold. program objective and strategy partners in health worked alongside local mohs counterparts to support their laboratory strategy and accompany them in the improvement process. partners in health’s health system strengthening model – also referred to as ‘stuff, staff, space, and systems’ – incorporates investments in materials, human resources, facilities, and systems.20 this comprehensive approach was applied to our laboratory programme, aiming to improve quality from preto post-analytical stages of diagnostics, while meeting national strategies and offering the services described in mohs policies.15,18 in the first year of the intervention, the programme focused on the most crucial or attainable issues (listed below), while laying a foundation for continuous and structural improvements. supporting local health authorities on policy implementation to enhance access to services the laboratory diagnostic menu was revised to align with sierra leone’s national guidelines for district level services and who policies (table 1). new systems and tests were introduced gradually throughout the year to ensure proper time for training and adjustment to novel modalities for both laboratory staff and clinicians. to improve access to services for patients, a flat laboratory fee (5000.00 sll [sierra leonean leone] or $0.70 [united states dollars]) for outpatients was introduced. inpatient laboratory services were free of charge. in collaboration with the hospital’s hiv and tuberculosis clinics, referral systems were revised to (1) ensure proper and rapid transport of specimens from community clinics to the laboratory for diagnostic testing and treatment monitoring and (2) refer patients with positive test results from the laboratory to the hiv or tuberculosis clinic for further testing and counselling. as the hospital laboratory relied on smear microscopy for tuberculosis diagnosis, a referral system with a nearby pih-supported outpatient clinic was set up for molecular testing for tuberculosis for patients suspected to have multidrug-resistant or extrapulmonary tuberculosis. community health workers were given a central role in hiv and tuberculosis referral processes. all community health workers at kgh were trained in hiv and tuberculosis care and equipped to guide patients through the hospital system, accompanying them to and from the laboratory. additionally, community health workers coordinated collection and transport of sputum samples for tuberculosis testing from patients in the community to the laboratory. to improve inpatient care, routine blood draws were introduced in all wards in the mornings, and the results were disseminated in the afternoon. table 1: diagnostic testing menu for koidu government hospital central laboratory, sierra leone, 2016. addressing gaps in the system infrastructure: utility improvements were made at the hospital, including the laboratory, ensuring water and electricity were available 24 hours per day seven days per week. minor infrastructure and repair work was completed at the laboratory building. the laboratory areas were reorganised, and a separate space for phlebotomy and sample collection was built to improve both patient and sample flow, as well as safety and privacy. air conditioning was installed to protect equipment, as well as fans (to increase air circulation) and uv lights, two recommended interventions to reduce the risk of tuberculosis transmission. procurement and supplies: a procurement and cold-chain supply system was established to fill gaps and support transport of mohs supplies. procurement was organised in biannual international orders and quarterly regional and local orders to ensure uninterrupted availability of consumables and reagents and to reduce stock-outs and expiration. refrigerators and freezers were bought and installed to expand cold storage capacity in the laboratory, hospital and district warehouses. replenishment of supplies to the laboratory from a central warehouse took place on a weekly basis. equipment: to improve quality and expand diagnostic services, novel equipment was introduced. technicians from respective companies installed and provided training on larger equipment. smaller equipment was introduced by the laboratory manager: electronic light-emitting diode (led) microscopes for tuberculosis and malaria diagnosis, as well as urine and stool microbiological diagnostics electrical centrifuges for separation of plasma and serum and sedimentation of urine to replace hand centrifuges haematology analyser (20 parameter) for complete blood counts biochemistry analyser, semi-automated point-of-care testing equipment for electrolytes, haemoglobin and blood glucose. supporting operations and improving quality assurance management and education. partners in health recruited a laboratory manager, who worked with the mohs laboratory superintendent on supervision and management. provision of ongoing mentorship and training to improve test performance were essential parts of the programme. in addition to didactic training for the entire team, individual technicians received bench-side training to use novel equipment during multiple weeks of mentorship. quality control and assurance: for every new test and procedure, a standard operating procedure was developed to standardise the quality of testing and biosafety. daily, weekly and monthly controls, maintenance and calibration were implemented for all novel equipment according to manufacturers’ guidelines; contracts and service agreements were obtained for all relevant equipment to cover regional or international technical support services and warranty. daily temperature monitoring was introduced for all cold storage space in the laboratory and warehouses. data management and information systems: to improve documentation and registration, forms when needed were updated, and standardised registration for all requests and results was introduced in the laboratory. the laboratory received monitoring and evaluation support to develop data collection tools and continuously monitor operations, including inventory and consumption rates. by ensuring provision of test and consumption data to national programmes, including the national aids secretariat and national aids control programme, national tuberculosis control programme, and the malaria control programme, sufficient hiv, tuberculosis and malaria testing materials were supplied from national stores for these global fund-supported programmes. partners in health filled any remaining supply gaps. outcomes increased patient and testing volumes between november 2015 and october 2016, the volume of individual outpatient test requests increased 5.8 times over baseline, from 246 to 1428 per month (figure 1). during this timeframe, pih’s health system programming primarily supported inpatient and outpatient hiv and tuberculosis care, but was not involved in general outpatient services. therefore, this increase in outpatient visits (including from surrounding districts) suggests increased trust in the health system and kgh among national staff and local community members, as evidenced by increasing test requests and larger patient volumes, and potentially reflects expanded service delivery. figure 1: patient request volume by month, koidu government hospital central laboratory, sierra leone, november 2015–2016. individual patient test requests per month separated for inpatient (black triangles) and outpatient (open squares) departments. after improving infrastructure and preparing the laboratory and technicians for new equipment, semi-automated haematology and biochemistry analysers were installed in may 2016. during the first six months after introduction, this new equipment enabled the laboratory to perform a total of 224 liver and renal function tests for hiv and tuberculosis patient monitoring and inpatient care, as well as 1352 complete blood counts for inpatient and outpatient care (figure 2). overall, the programme and, specifically, the introduction of new machines boosted staff morale. introducing new procedures and tests required a moderate amount of initial cost and training, but was absorbed relatively easily by the national mohs laboratory team. figure 2: introduction of haematology and biochemistry services, koidu government hospital central laboratory, sierra leone, 2016. (a) the haematology laboratory before introduction of equipment, with a broken incubator for storage (left) and (b) after installation of air conditioning and equipment, with a technician turning on the haematology machine (right). (c) number of haematology (closed squares) and biochemistry (open squares) samples received for testing during the first six months of introducing the new equipment. below the x-axis is indicated when various biochemistry tests were introduced. to illustrate the effect of our intervention on diagnostics of hiv, tuberculosis and malaria – which are all national and who priorities – testing volumes are presented in figure 3.these numbers reflect volumes within the laboratory and exclude point-of-care hiv and malaria tests performed by clinicians and support staff in the wards and hiv clinic. hiv: prior to the intervention, almost all hiv tests for outpatients were performed at the hiv clinic on the hospital grounds and on the wards for inpatients. the laboratory performed only eight hiv tests in november 2015; this number increased to 251 in october a year later. this result was achieved by training technicians in testing procedures, patient confidentiality, and improving communication with clinicians, hiv clinic staff, and community health workers. this change gave the hiv clinic staff more time to focus on other tasks, and increased access to testing services for outpatients, without a required visit to the hiv clinic. tuberculosis: repair and ongoing maintenance of a mycobacterium tuberculosis molecular testing platform and supply of cartridges located at our partner outpatient health clinic enabled the testing of 66 specimens (for patients suspected of having multidrug-resistant tuberculosis or extrapulmonary tuberculosis) during the first six months of this system. malaria: throughout the year, the laboratory tested 9501 patients for malaria, with a peak in volume (and positivity rate, data not shown) in the rainy season from may to september. to support the increase in volume, pih supplied rapid diagnostic tests in addition to slide tests to reduce the burden on staff. figure 3: testing volumes of hiv, tuberculosis and malaria by month, koidu government hospital central laboratory, sierra leone, november 2015–2016. number of tests performed per month for hiv (open triangles, left y-axis), tuberculosis (afb, closed squares, left y-axis) and malaria (closed circles, on right y-axis). discussion as a result of this programme, the diagnostic capacity at kgh was expanded and improved and an increase in patient visits was recorded. this suggests that an improvement in environment, organisation and access to diagnostics allowed for more trust and satisfaction with the system by patients and clinicians, as has been shown by others.8,9 this programme had several limitations. all operational and clinical support was part of a hospital-wide health-strengthening programme. these combined efforts had the immeasurable effect of improving service delivery and trust among patients, community and hospital staff, likely contributing to the increase in laboratory utilisation as well. therefore, we cannot observe our outcomes in isolation from other interventions. secondly, our data demonstrate improved access to laboratory services, as more patients visited the laboratory, but do not quantify improved quality or how access to these services changed clinical practice and outcomes. a laboratory satisfaction survey would provide better information about perceived laboratory services. in a mohs 2016 progress report (personal communication), the laboratory score increased from 65% to 87% for availability of tests, supplies, and standard operating procedures. this indicates that improvement of laboratory services was recognised by the government. unfortunately, this report was not published nationally and the structure differed from the 2015 comprehensive assessment and hence cannot be compared with other districts or the 30% score in the previous report. lastly, this score is not synonymous with quality services; for example, the kgh laboratory lost credits in 2016 after replacing the haemoglobin colour scale with an automated analyser, because only the colour scale was provided by mohs and included in the assessment. it is the mission of pih to strengthen public health systems to ensure the access of health services to people living in resource-limited settings. investing in public sector laboratory services though a comprehensive approach has proven effective in other countries.21 in rural haiti and rwanda, multidrug-resistant tuberculosis and pathology departments are in operation or being built. these established ‘beacon facilities’ serve the poorest populations, function as teaching sites, and are considered a model for health service provision on national and regional levels.20 while partnering with governments is central to rebuilding public health infrastructure, it may also limit certain programmatic decisions. for example, pih aims to offer free health care for all, but the sierra leone public system and many other health services in low-resource countries are based on cost recovery. we could therefore only lower the costs per patient for laboratory services, not eliminate these costs for all patients. although our staff would not deny anyone care, and some patients received free care according to national policy (those under the age of 5 years, pregnant and lactating women, evd survivors, and patients receiving hiv or tuberculosis care), over 50% of the population lives on less than $2.00 per day11 and these fees may still pose a barrier to many for accessing health services. to remove these barriers and rebuild health systems, advocacy and collaboration with national programmes are needed. these efforts are also required regarding testing policies. there are discrepancies between national guidelines, who recommendations, and accepted standards for tests used in high-income countries. for many low-income countries, cheap testing methods for diseases such as hiv and tuberculosis are considered acceptable22 and on many occasions point-of-care tests are used to replace instead of complement more accurate or sensitive tests.4 more sensitive procedures, such as nucleic acid amplification tests and microbiological culture, in sierra leone and many other countries, are limited to reference laboratories, which cannot serve the total population. the lack of logistics, supply system, referral networks and facilities are considered major bottlenecks to introduce novel diagnostics,23 creating a vicious cycle and preventing the introduction of new technologies. very recently, following calls from experts,24 who developed the first version of an ‘essential diagnostics list’25 – 40 years after the release of the ‘essential medicines list’, which has been updated every 2 years since 1977. furthermore, who, the united states centers for disease control and prevention and the association of public health laboratories developed a global laboratory leadership programme.26 all of these steps may contribute to a new commitment for better quality testing globally. the intervention described here is still ongoing as pih continues to support the mohs staff in the laboratory and other clinical service delivery. to improve the quality of diagnostic services, pih has recruited a quality officer to support the laboratory manager. the next steps of the programme include, but are not limited to, enrolment of tests into (national) external quality assurance systems, strengthening the referral system, introduction of microbiological culture assays and equipment for automated tuberculosis and hiv viral load detection, eventually leading to improved quality services, emergency preparedness, and first steps towards accreditation.15 in many ways, kgh is representative of an extremely underserved district hospital and we offer this example of our model to demonstrate how services can be reliably and sustainably strengthened in similar contexts. acknowledgements the authors acknowledge all laboratory and blood bank staff in the kono district and are grateful to the staff of partners in health for their dedication to health equity in sierra leone. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this paper. authors’ contributions a.w.m. conceived of and wrote the manuscript, analysed the data and implemented the project; m.b. and s.m.k. were involved in project implementation, analysis and interpretation of data; j.s.g. performed data collection and analysis; k.l.d. and m.m.s. provided programmatic direction and support; j.d.o. provided technical laboratory support; r.h.m. supervised the intervention and provided support. all authors critically reviewed the manuscript and approved the final version. sources of support the intervention was funded by partners in health. a.w.m. is funded by the harvard medical school department of global health and social medicine. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references berkelman rg, cassell s, specter m. the “achilles heel” of global efforts to combat infectious diseases. clin infect dis. 2006;42(10):1503. https://doi.org/10.1086/504494 tarimo e. essential health service packages – current concerns. division of analysis, research and assessment. world health organization. geneva 1997. parsons lm, somoskovi a, lee e, et al. global health: integrating national laboratory health systems and services in resource-limited settings. afr j lab med. 2012;1(1), art. #11:1–5. https://doi.org/10.4102/ajlm.v1i1.11 engel n, wachter k, pai m, et al. addressing the challenges of diagnostics demand and supply: insights from an online global health discussion platform. bmj glob health. 2016;1:e000132. https://doi.org/10.1136/bmjgh-2016-000132 perry l, malkin r. effectiveness of medical equipment donations to improve health systems: how much medical equipment is broken in the developing world? med biol eng comput. 2011;49:719–722. https://doi.org/10.1007/s11517-011-0786-3 bauserman m, hailey c, gado j, et al. determining the utility and durability of medical equipment donated to a rural clinic in a low-income country. int health. 2015;7:262–265. https://doi.org/10.1093/inthealth/ihu091 nkengasong jn, birx d. quality matters in strengthening global laboratory medicine. afr j lab med. 2014;3(2), art. #239:1–4. https://doi.org/10.4102/ajlm.v3i2.239 mfinanga sg, kahwa a, kimaro g, et al. dissatisfaction with the laboratory services in conducting hiv related testing among public and private medical personnel in tanzania. bmc health serv res. 2008;8:4–9. https://doi.org/10.1186/1472-6963-8-171 mfinanga sg, kahwa a, kimaro g, et al. patient’s dissatisfaction with the public and private laboratory services in conducting hiv related testing in tanzania. bmc health serv res. 2008;8:1–5. https://doi.org/10.1186/1472-6963-8-167 chaw ps, schlinkmann km, raupach-rosin h, et al. knowledge, attitude and practice of gambian health practitioners towards antibiotic prescribing and microbiological testing: a crosssectional survey. trans roy soc trop med hyg. 2017;111:117–124. https://doi.org/10.1093/trstmh/trx027 united nations development programme. human development report 2016. united nations development programme. canada; 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(2013). cox h, nicol mp. tuberculosis eradication: renewed commitment and global investment required. lancet infect dis. 2017;3099:10–11. mcnerney r. diagnostics for developing countries. diagnostics (basel). 2015;5:200–209. https://doi.org/10.3390/diagnostics5020200 schroeder lf, guarner j, elbireer a, castle pe, amukele tk. time for a model list of essential diagnostics. n engl j med. 2016;374:2511–2514. https://doi.org/10.1056/nejmp1602825 world health organization. model list of essential in vitro diagnostics. world health organization. geneva. 2018. albetkova a, et al. critical gaps in laboratory leadership to meet global health security goals. bull. world health organ. 2017;95:0–1. https://doi.org/10.2471/blt.17.195883 reviewer acknowledgement open accesshttp://www.ajlmonline.org page 1 of 1 we would like to take this opportunity to thank all of those who provided scientific and logistical support for this issue of the african journal of laboratory medicine: should names have 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miguel e. quiñones-mateu milijaona radrianarivelojosia zilma rey jean louis sankale tom shinnick gajendran sivakumar john sorkin sanon souleymane andrew thaiyah david turgeon musau wakabongo diane waku larry westerman burton w. wilcke patty wilkins volume 2, number 1 volume 3, number 1 aaron o. aboderin michael aidoo umubieyi alaine amal alla william ampofo linda de gouveia stephania k. deme alpha diallo dennis ellenberger ifeoma enweani nwadiuto e. esiobu jessie n. githanga esther de gourville jean m. heraud john kagira edward kamau phyllis kanki reba kanungo samoel khamadi segundo r. leon henry s. limula john f. nahabedian jack nyamongo bryan nyary beldinah r. ochola brenda okech eric opiyo wellington oyibo julius oyugi milijaona radrianarivelojosia zilma rey abdoulaye sarr ritu shrivastava andrew thaiyah john thuita musau wakabongo larry westerman abstract introduction methods results discussion acknowledgements references about the author(s) arielle g. hernandez division of hematology, cincinnati children’s hospital medical center, cincinnati, ohio, united states global health center, cincinnati children’s hospital medical center, cincinnati, ohio, united states department of epidemiology, human genetics and environmental sciences, school of public health, university of texas health science center, houston, texas, united states charles kiyaga central public health laboratories, ministry of health, kampala, uganda thad a. howard division of hematology, cincinnati children’s hospital medical center, cincinnati, ohio, united states global health center, cincinnati children’s hospital medical center, cincinnati, ohio, united states isaac ssewanyana central public health laboratories, ministry of health, kampala, uganda grace ndeezi department of paediatrics and child health, makerere university college of health sciences, kampala, uganda jane r. aceng ministry of health, kampala, uganda russell e. ware division of hematology, cincinnati children’s hospital medical center, cincinnati, ohio, united states global health center, cincinnati children’s hospital medical center, cincinnati, ohio, united states department of pediatrics, university of cincinnati college of medicine, cincinnati, ohio, united states citation hernandez ag, kiyaga c, howard ta, et al. operational analysis of the national sickle cell screening programme in the republic of uganda. afr j lab med. 2021;10(1), a1303. https://doi.org/10.4102/ajlm.v10i1.1303 original research operational analysis of the national sickle cell screening programme in the republic of uganda arielle g. hernandez, charles kiyaga, thad a. howard, isaac ssewanyana, grace ndeezi, jane r. aceng, russell e. ware received: 16 june 2020; accepted: 13 apr. 2021; published: 12 aug. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: sickle cell anaemia is a common global life-threatening haematological disorder. most affected births occur in sub-saharan africa where children usually go undiagnosed and die early in life. uganda’s national sickle cell screening programme was developed in response to a 2014 sickle cell surveillance study that documented a high disease prevalence. objective: this study describes the temporal and financial aspects of uganda’s 2014–2019 sickle cell screening programme. methods: national sickle cell screening data from uganda’s central public health laboratories were used to calculate turn-around times (tats) from sample collection to delivery, testing, and result reporting for blood samples collected from february 2014 to march 2019. the parameters affecting specific tats were assessed. the exact programme expenditures were analysed to determine cost per test and per positive sickle cell disease case detected. results: a total of 278 651 samples were analysed. the median tat from sample collection to laboratory receipt was 8 days (interquartile range [iqr]: 6–12), receipt to testing was 3 days (iqr: 1–7), and testing to result reporting was 6 days (iqr: 3–12). altogether, the sample continuum averaged 16 days (iqr: 11–24). lower level healthcare facilities were associated with longer sample delivery tats. calendar months (january and december) and larger sample volumes impacted testing and result reporting tats. the cost per test was $4.46 (united states dollars [usd]) and $483.74 usd per positive case detected. conclusion: uganda’s sickle cell screening programme is efficient and cost-effective. universal newborn screening is the best strategy for detecting sickle cell anaemia in uganda. keywords: uganda; sickle cell disease; newborn screening; turn-around time; cost-effectiveness. introduction sickle cell anaemia is a monogenic haematological disorder that manifests as a devastating systemic disease with high morbidity and early mortality. the pathophysiology begins in infancy with acute and life-threatening complications, which presents as increased susceptibility to infections, chronic haemolytic anaemia, and painful vaso-occlusive events.1,2 sickle cell anaemia is the most prevalent haemoglobinopathy that impacts more than 20 million people worldwide, with an estimated 312 302 babies born with the disease each year.3 over 75% of the world’s annual sickle cell anaemia births occur in sub-saharan africa where resources for providing early detection and care are most constrained,3 leading to the deaths of more than 500 affected children per day.4 the world health organization and other international groups have begun to recognise sickle cell anaemia as a major public health concern around the globe, but particularly in sub-saharan africa where there is a lack of government programmes to address this overwhelming and growing burden of disease.5,6,7,8 these organisations have identified the need to implement affordable evidence-based strategies that can be sustainably woven into existing healthcare systems, highlighting newborn screening as a priority intervention.6 evidence from both high-resource and limited-resource countries have shown that newborn screening for sickle cell anaemia can significantly decrease morbidity and mortality by enabling early initiation of penicillin prophylaxis and pneumococcal immunisations as primary prevention.9,10,11,12 however, the accurate diagnosis of sickle cell anaemia in limited-resource settings still faces major challenges related to high equipment and reagent costs, education and training of healthcare providers, and appropriate medical interventions. newborn sickle cell screening requires efficient laboratory methodologies and infrastructure for an early and accurate diagnosis to reduce preventable deaths among children born with sickle cell disease and to support the ultimate goal of universal screening of all babies across sub-saharan africa. the uganda sickle surveillance study (us3) commenced in 2014 using a simple but innovative approach to screen children for the sickle cell trait and disease on a national level.13 residual dried blood spot cards collected across the country as part of the early infant hiv detection programme were tested. isoelectric focusing (ief) was conducted on about 100 000 samples over a cross-section of one year and found an overall high prevalence of sickle cell trait at 13.1% and disease at 0.7%, but with a disproportionate distribution across the country. broader sickle cell screening has been initiated since 2015, strategically focused on the highest burden districts. to expedite early detection and facilitate linkage to care for affected infants, it is crucial to identify where delays in diagnosis occur. here, we describe a detailed operational analysis of the temporal and financial aspects of sickle cell screening in uganda. we documented the turn-around times (tats) for sickle cell screening, starting from sample collection to arrival, testing, and result reporting at the national centralised laboratory. to examine the cost implications of integrated sickle cell screening, we calculated the cost per test, based on exact expenditures from the us3, and made estimates of the cost-effectiveness of sickle cell screening in uganda. methods ethical considerations the initial us3 research proposal was approved by the school of medicine research ethics committee at makerere university (no. 2012-138) in kampala, uganda, on 14 august 2012, with continuous annual renewal through august 2021. approval from the uganda national council of science and technology was also completed. the proposal included an objective to conduct a cost-effectiveness analysis of the sample collection and testing process. as described in the us3 manuscript,13 the uganda ministry of health waived informed consent for all samples tested as part of us3. data integrity and patient privacy were assured through the use of de-identified specimen numbers maintained in a secured database. integrated screening us3 was conducted from february 2014 to march 2015 to determine the prevalence of sickle cell trait and disease in uganda. a foundational goal of the study was building local sickle cell laboratory capacity within the ministry of health’s existing centralised laboratory infrastructure and determining the feasibility of the scale-up of newborn screening to the national level. at the central public health laboratories (cphl) in kampala, uganda, a partnership with cincinnati children’s hospital medical center led to the construction of a new sickle cell laboratory that was outfitted with ief equipment, and local personnel were recruited and trained on a standardised haemoglobin electrophoresis protocol. as previously described for the early infant hiv detection programme, blood was collected from hiv-exposed infants and young children across the country using standard dried blood spot cards.14 each health facility populated a dispatch form with demographic information and testing requests. samples were transferred to the cphl via the national sample transport system.14 in the us3, all samples requesting hiv testing were also queued for sickle cell testing; results were entered into the centralised database and disseminated back to health facilities and onward to caretakers. following the us3, the dispatch form included an option for sickle cell testing only. turn-around time analysis the cphl database collects laboratory data on all samples received and processed; data for this analysis were abstracted for children aged 0–24 months who underwent routine sickle cell screening over five years, from 01 february 2014 to 31 march 2019. inbound tat was defined as the total number of days between sample collection at health facilities and the result dispatch from the cphl back to healthcare providers. three phases were defined within inbound tat: (1) sample delivery phase tat is the time between sample collection and receipt at the cphl, (2) sample testing phase tat is the time between receipt at the cphl and date of testing, and (3) sample result reporting phase tat is the time between testing and result dispatch from the cphl. outbound tat, defined as the number of days between result dispatch and result receipt or subsequent action by the healthcare providers, was not assessed in this analysis because those data points are not currently collected by the cphl. median tats were calculated for inbound tat and its three phases: sample delivery, testing, and result reporting using stata statistical software (statacorp llc. 2019. release 16. college station, texas, united states). the tat calculation included weekends and holidays because the cphl operates 24 hours a day, 7 days a week, 365 days a year. summary statistics included medians, 25% and 75% interquartile ranges (iqr), and frequencies. programme year 1 was the period of us3, followed by years 2 to 5 for every 12 months through march 2019. there were two distinct cohorts for sickle cell screening, based on how the sickle cell testing service was requested. the sickle/hiv co-testing cohort included all samples in the database that had both a sickle cell result and an hiv result. the sickle specific testing cohort included all samples that had a sickle cell result only.15 phase-specific parameters were determined for sample delivery, testing, and result reporting tats to assess different parts of the inbound sample continuum. for the sample delivery phase tat, parameters included geographical region, health facility level, programme year, collection month, and testing cohort. for the sample testing phase tat, parameters were programme year, testing month, and testing cohort. for the sample result reporting phase tat, parameters were programme year, dispatch month, and testing cohort. analysis of collection month was conducted using only year 5 data (01 april 2018 to 31 march 2019) to better understand recent temporal trends in tat. although there is no numerical value for laboratory efficiency or success, tat is considered an indicator of laboratory performance, where the shorter the tat the more efficient a laboratory is in producing results promptly. cost analysis the cost analysis of sickle cell screening was conducted by using microsoft excel (microsoft, corp., redmond, washington, united states) included the cost per test by ief and cost per positive case detected. costs were assessed by detailed analysis of all us3 expenditures over 13 months (01 february 2014 to 31 march 2015), using actual procurement documents and vendor price sheets for ief equipment, reagents, and consumables, plus cphl labour costs, including salary and other employment costs. costs that were shared with other cphl programmes and the health facilities were not included, such as the dried blood spot kits, sample transportation, local healthcare provider salaries, and screening-related training. expenses incurred by cincinnati children’s hospital medical center for travel and initial training of cphl sickle cell laboratory personnel were also excluded. cost for equipment, reagents, and consumables was reported in united states dollars (usd) according to actual costs using out-of-country and in-country vendors. for labour costs, annual salaries were expressed in uganda shillings and converted to usd using the purchasing power parity exchange rate.16 the national sickle cell disease prevalence data for 2014–2019 and the calculated cost per test were used to generate the cost per sickle cell disease case detected by region within uganda. results sickle cell testing turn-around time between 01 february 2014, and 31 march 2019, a total of 324 356 samples were collected and tested at the cphl. samples were excluded from analysis if they were outside the study date range, beyond 24 months of age, or collected and tested outside of routine sickle cell screening. the number of samples included in the final analysis was 278 651. in the subsequent analysis, only samples with present and plausible dates for collection, receipt, testing, or result dispatch were included depending on the tat being calculated. accordingly, there were 265 766 samples included in the inbound tat calculation, 276 521 samples for sample delivery tat, 263 417 samples for sample testing tat, and 267 325 samples for sample result reporting tat. the overall median inbound tat, from sample collection to result dispatch, was 16 days (iqr: 11–24) (table 1). most of the samples (n = 228 684, ~86%) did not exceed an inbound tat of one month. programme year 1 had the largest sample volume (n = 90 872), followed by almost 70 000 samples in year 4. the south western region had the shortest median inbound tat of 13 days (iqr: 9–20), while the mid-western region had the longest tat of 18 days (iqr: 12–26). the sickle cell/hiv co-testing cohort had a slightly shorter inbound tat (15 days) than the sickle specific cohort (17 days). table 1: characteristics of sickle cell samples tested in uganda from 01 february 2014 to 31 march 2019. for the sample delivery phase, the median tat was 8 days (iqr: 6–12). parameters affecting sample delivery tat included the region of the requesting hospital and the level of healthcare provided (table 2). the kampala region, where the cphl is located, had the shortest median sample delivery time of 6 days (iqr: 4–9), while six regions had a median sample delivery time of 9 days (table 2). regional referral hospitals had a shorter median sample delivery tat of 7 days (iqr: 5–9) compared to the lower level health centres ii, iii, and iv. the collection month of december 2018 had the longest median tat of 10 days (iqr: 5–18) compared to other collection months. table 2: sample delivery phase turn-around time for sickle cell samples collected in uganda, by parameter, february 2014 to march 2019. once the sample arrived at the cphl, the median sample testing tat was 3 days (iqr: 1–7) and for sample result reporting tat, it was 6 days (iqr: 3–12). for sample testing phase tat, programme year 4 had the longest median tat of 7 days (iqr: 4–14) (table 3). median sample testing and sample result reporting tats were highest in the months of december 2018 and january 2019 (table 3 and table 4). the two testing cohorts had similar median sample testing and sample result reporting tats. table 3: sample testing phase turn-around time for sickle cell samples collected in uganda, by parameter, february 2014 to march 2019. table 4: sample result reporting phase turn-around time for sickle cell samples collected in uganda, by parameter, february 2014 to march 2019. sickle cell screening costs inputs included in the cost analyses were exact direct expenditures for equipment, reagents, consumables, and labour for 99 243 us3 samples tested by ief from a dried blood spot at the cphl over 13 months (table 5). the total equipment costs were annualised and calculated at $0.94 usd per test. reagent costs and consumables were calculated at $1.04 usd and $0.15 usd per test. the annual salary for laboratory personnel included gross pay, national social security fund 10% monthly tax, customary ‘13th month’ end-of-year compensation, and medical insurance per standard ministry of health employee contracts. labour costs were $2.33 usd per test, for an overall cost per test of $4.46 usd. personnel costs made up more than half of the cost per test total at 52%, followed by reagents (23%), equipment (21%), and consumables (3%). table 5: costs for sickle cell screening and testing for 99 243 children in uganda from the us3 project over 13 months, february 2014 to march 2015. in uganda, the highest burden of sickle cell disease is observed in the east central (1.6%) and mid-northern (1.5%) regions (table 6). using the calculated total cost per test and national prevalence data, the cost per sickle cell disease case detected was $483.74 usd (table 6). when stratified by region, however, the cost per positive case detected ranged widely from $278.07 usd in the east central region to $2607.19 usd in the south western region. table 6: cost per positive sickle cell disease case in uganda, by region from february 2014 to march 2019. discussion for any newborn screening programme, reduction of sample tat is an important intervention to ensure that poor health outcomes or preventable deaths are not the results of laboratory or operational delays. identification of the specific barriers related to longer tat and implementation of measures to mitigate those causes are essential to strengthen screening programmes. in this first detailed operational analysis of tat for sickle cell screening in uganda, the total duration between sample collection at the health facility to the dispatch of results from the cphl averaged 16 days (figure 1). multiple parameters affected this time interval, including the health facility level, programme year, collection month, and testing cohort. however, the vast majority (~86%) had results sent by cphl back to the local healthcare facilities within one month of receipt (table 1), which we propose as the maximal tat threshold before putting sickle cell disease patients at risk by delaying diagnosis and treatment. penicillin prophylaxis and pneumococcal immunisations are expected to be administered by three months of age, and these tat results are sufficient to meet those goals.9,10,11,12 figure 1: uganda central public health laboratories sample continuum and turn-around time phases, with results shown as the median in days (25% – 75% iqr) from february 2014 to march 2019. for the uganda sickle cell screening programme, sample delivery accounted for most of the inbound tat (figure 1). lower health facilities likely have greater barriers to timely delivery, such as delayed collection of their samples for transport (table 2). within each district, hub motorbike riders perform a weekly pickup from the more rural locations of health centres ii, iii, and iv in comparison to regional referral hospitals or district hospitals that experience daily pickup. it is also possible that samples are not readily available for transport at the time the hub motorbike riders come for collection, or some facilities are missed due to inclement weather or mechanical problems. to address these issues, a twice-weekly sample pickup with validation measures, such as timestamps, should be put into place. this should also be supplemented with ongoing training regarding sample preparation and the importance of timely sample transport for all personnel involved in the process. after sample receipt at the cphl, sample testing was relatively rapid with an average of only 3 days (figure 1). this phase involves the routing of samples for sickle cell testing to the laboratory, punching and eluting of dried blood spots, testing by ief by highly trained personnel, and entering of results into database. based on the short tat, this internal process can be deemed efficient. however, when looking at the parameter of the programme year, it does appear that longer tat in this phase is associated with periods of increased sample volumes (table 3). this may be the cause of reagent stock out or point to the need for more cross-training of cphl personnel on the ief procedure to support the sickle cell laboratory. as the programme expands toward the eventual goal of universal newborn screening, more human resources will be necessary to handle the increased number of samples. following sample testing, result reporting took an average of 6 days (figure 1). altogether, the time that samples spent at the cphl was prolonged. delays with the dispatch of results could be sample retesting or stock out of printing supplies. it was also observed that the testing and dispatch months of december 2018 and january 2019 had distinctly longer tats (table 4). this may be linked to personnel leave due to the holidays, thus increasing or staggering support in the sickle cell laboratory during this time could help avoid lengthy delays. in a previous study by kiyaga et al. at the uganda cphl regarding tat of early infant hiv detection screening, the average sample delivery tat was 12 days, sample processing was 2 days, and the overall average tat from sample collection to reporting to the patient was 26 days.14 as part of this study, short message service printers were piloted to allow test results to be transmitted immediately to healthcare facilities after testing, which further reduced the overall tat to 14 days.14 in the united states, the national newborn screening and global resource center reported newborn bloodspot screening tat to be within 10 to 14 days from sample collection, with considerable variation by state.17 to overcome these differences in state programmes, the united states department of health and human services advisory committee on heritable disorders in newborns and children recently set out updated newborn screening timeliness goals specifying that tests should be completed within 7 days of birth.18 in our study, we found that tat for sickle cell screening was shorter in all inbound phases compared to what was previously reported for uganda’s early infant hiv detection programme, and exhibited overall improved time efficiency for the entire cphl sample continuum (median 16 days vs 26 days). the current programme is approaching a comparable timeframe to that of the long-standing united states newborn sickle cell screening programme and will likely continue to evolve and improve over time. information on outbound tat, which includes time to family notification and matriculation to care, could not be examined in this study, because those data are currently not collected by the cphl. however, with greater visibility and a push toward newborn screening in sub-saharan african countries, steered by the new american society of hematology african screening consortium,19 outbound tat and individual patient follow-up data will be coveted information. we predict that tat will greatly impact healthcare provider and patient utilisation of sickle cell screening, as well as overall satisfaction with the programme in uganda. most importantly, prolonged tat very likely postpones life-saving treatments for patients with the disease, if the diagnosis is not established and communicated promptly. also, delayed result reporting can lead to affected infants being lost to follow-up and may require a replacement test. improving tat helps ensure that caretakers collect their results on time and minimises the need for inefficient and expensive repeat testing. ultimately, making improvements in all tat phases for sickle cell screening is essential to optimise infant health outcomes, cost-effectiveness, and screening satisfaction for healthcare providers and patients in uganda. a major strength of this analysis was the complete national representative long-term data from the cphls centralised database, which allowed us to investigate previously undocumented parameters that affect sickle cell testing tat in uganda. limitations include the access to only inbound tat data, which limits our ability to connect our findings to patient indicators such as notification and receipt of sickle cell results, followed by care and management of affected patients. another limitation is the lack of recommendations for sickle cell testing tat by the uganda ministry of health, other regional programmes, or by international bodies such as the world health organization for a comparison of efficiency. however, our data provided the first detailed time analysis of sickle cell screening in sub-saharan africa, to enable monitoring and evaluation of future interventions and other programmes. analysis of the direct costs of a sickle cell screening programme in uganda showed that the cost per test by ief using dried blood spots was $4.46 usd. in angola, the cost per infant screened in a newborn screening programme that used ief was $15.36 usd, or $7.42 usd when considering just the inputs of laboratory personnel, reagents, consumables, and equipment as in our study.20 the cost of the screening test for sickle cell disease performed by ief in the united states was $2.29 usd and approximately $4.50 usd (reported as £3.51) in the united kingdom.21,22 the united states and united kingdom have addressed the cost‐effectiveness of newborn screening for sickle cell disease for universal and targeted programmes. both nations have justified the greater cost incurred by screening every newborn for sickle cell disease, because it identifies all infants with the disease, prevents more deaths, and has demonstrated better outcomes for patients.21,22,23 in africa, modelling simulations have found universal newborn sickle cell screening to be extremely cost-effective, especially in countries with a high disease prevalence of 0.2% – 0.5%.24,25 yet, no federal newborn sickle cell screening currently exists in any country in sub-saharan africa, despite the well-evidenced economic and humanitarian basis for such programmes. in uganda, the estimated costs per sickle cell disease case detected among the 10 regions varied from $278.07 usd in the east central to nine times that in the south western region ($2607.19 usd) (table 6). these results show that newborn screening in regions with low sickle cell disease prevalence would result in a higher cost per positive case detected compared with screening focused in high-burden areas. however, because uganda is a country with a high overall disease prevalence of 0.9%,26 universal newborn screening would still be the most economical and impactful public health strategy for sickle cell disease across the entire country. limitations this analysis only considered costs and numbers of cases detected for partial cost-effectiveness analysis. more extensive studies with patient follow-up data will be vital to provide evidence that screening is effective in reducing sickle cell morbidity and mortality, the benefits and harms of screening, and the long-term cost-effectiveness of a newborn sickle cell screening programme in uganda. conclusion this study provides a contemporary and detailed description of the time and costs of sickle cell screening in uganda. analysis of the different phases of tat highlights areas for improvement to reduce the number of samples with excessive delays, and to strengthen the overall integrated cphl sample continuum. the lack of sickle cell testing guidelines limits our ability to compare these results to screening and care standards; however, this study documents that the cphl centralised database can be used to accurately monitor and manage tat for sickle cell screening and can identify factors affecting tat at different phases to prompt targeted improvements. this study also shows that sickle cell testing by ief is provided at under $5.00 usd, and the strategy of universal newborn screening is cost-effective to save and improve the lives of thousands of individuals with sickle cell disease in uganda. acknowledgements we are very thankful to the uganda ministry of health leadership and the central public health laboratories personnel for their contribution and dedication to the sickle cell screening programme. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions a.g.h., c.k., t.a.h., i.s., g.n., j.r.a. and r.e.w. substantially contributed to the design and implementation of the research, to the analysis of the results, and to the writing of the manuscript. sources of support this work was financially supported by the cincinnati children’s research foundation. data availability these data are not publicly available. disclaimer the 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cell disease in 47 countries in sub-saharan africa: a cost-effectiveness analysis. bmc health serv res. 2016;16:304. https://doi.org/10.1186/s12913-016-1572-6 davies sc, cronin e, gill m, greengross p, hickman m, normand c. screening for sickle cell disease and thalassaemia: a systematic review with supplementary research. health technol assess. 2000;4(3):i–v, 1–99. review. https://doi.org/10.3310/hta4030 kiyaga c, hernandez ag, ssewanyana i, et al. sickle cell screening in uganda: high burden, human immunodeficiency virus comorbidity, and genetic modifiers. pediatr blood cancer. 2019;66(8):e27807. https://doi.org/10.1002/pbc.27807 article information authors: linda r. andiric1 charles g. massambu2 affiliations: 1globalhealth, american society for clinical pathology (ascp), chicago, united states 2ministry of health and social welfare, tanzania correspondence to: linda andiric postal address: globalhealth, american society for clinical pathology (ascp), 33 west monroe street, suite 1600, chicago, united states dates: received: 22 may 2014 accepted: 08 aug. 2014 published: 03 nov. 2014 how to cite this article: andiric lr, massambu cg. one laboratory’s progress toward accreditation in tanzania. afr j lab med. 2014;3(2), art. #202, 4 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.202 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. one laboratory’s progress toward accreditation in tanzania in this lessons from the field... open access • abstract • introduction • programme implementation • results • discussion • acknowledgements    • competing interests    • authors’ contributions    • disclosure statement • references abstract top ↑ introduction: the amana regional hospital laboratory in tanzania was selected, along with 11 other regional and district laboratories, to participate in a pilot programme for laboratory quality improvement using the strengthening laboratory management toward accreditation (slmta) training programme. programme implementation: the slmta programme entailed hands-on learning, improvement projects between and after a three-workshop series, supervisory visits from an oversight team and an expert laboratory mentor to facilitate and coach the process. audits were conducted at baseline, exit (approximately one year after baseline) and follow-up (seven months after exit) using the stepwise laboratory quality improvement process towards accreditation (slipta) checklist. quality stars (zero to five) were awarded based on audit scores. results: with a dedicated staff and strong leadership from laboratory management, amana laboratory implemented processes, policies and procedures recommended as elements of best laboratory practices. the laboratory improved from zero stars (36%) at baseline to successfully achieving three stars (81%) at exit. this was the highest score achieved by the 12 laboratories in the programme (the median exit score amongst the other laboratories was 58%). seven months after completion of the programme, the laboratory regressed to one star (62%). discussion: as the slmta improvement programme progressed, amana laboratory’s positive attitude and hard work prevailed. with the assistance of a mentor and the support of the facility’s management a strong foundation of good practices was established. although not all improvements were maintained after the conclusion of the programme and the laboratory dropped to a one-star rating, the laboratory remained at a higher level than most laboratories in the programme. introduction top ↑ until recently, laboratories in africa were largely ignored, distrusted and negated so that, for example, fewer than 50% of patients treated for malaria actually had laboratory confirmation of the disease.1 given the fact that in the united states as many as 94% of all patients’ treatment and diagnoses are based on laboratory data for confirmation,2 laboratory utilisation in africa was, by comparison, undervalued, underused and limited in its capacity. clinicians often rationalised that laboratory tests were an unnecessary additional cost because diagnoses and treatment protocols were based solely on the physician’s clinical judgement. if or when laboratory data were available, they were perceived as unreliable. if test results were received and were contradictory to clinical impressions, those test results were frequently ignored and only the clinical indicators were considered.3 financial resources from funding organisations and programmes such as the us president’s emergency plan for aids relief (pepfar) focused on the prevention and care of infectious diseases such as hiv, malaria and tuberculosis.4 because control of these infectious diseases is dependent upon accurate laboratory data, laboratory improvement became a priority. in july 2009 in kigali, rwanda, the world health organization’s regional office for africa (who afro), in partnership with the us centers for disease control and prevention (cdc), the clinton health access initiative and the american society for clinical pathology (ascp), launched the strengthening laboratory management toward accreditation (slmta) training programme,5 along with a stepwise accreditation preparation scheme that provides a five-step assessment method rather than a pass-fail one.6 in july 2010, the ministry of health and social welfare (mohsw) of tanzania began a pilot programme for the improvement of the country’s medical laboratory services using the slmta programme. twelve hospitals, comprising six regional and six district laboratories, were enrolled in slmta as tanzania’s first cohort. the minimum criteria for selection into the programme, as set by the country’s laboratory task force, were sufficient and qualified staff, a recently remodelled infrastructure to include sufficient space and utilities to carry out quality testing, and a basic knowledge of quality management by prior attendance in a quality management systems (qms) workshop. participation in an external quality assessment (eqa) programme was also preferred. this article describes amana regional hospital laboratory’s success in the improvement of its practices using the slmta programme and examines possible factors contributing to this success. programme implementation top ↑ amana regional hospital is located in dar es salaam city centre, tanzania. in 2010, the newly-remodelled hospital laboratory was both spacious and well-endowed with modern laboratory equipment. there were 23 staff members, including phlebotomists and cleaners. the laboratory was maintained neatly and was well organised. when the tanzanian mohsw notified amana laboratory that it had been selected to participate in the slmta programme, the laboratory manager was aware of international organization for standardization (iso) 15189, but he and his staff had no previous knowledge of slmta or the who afro accreditation preparation scheme. the improvement programme for amana laboratory and the other 11 selected facilities began in july 2010 with a baseline audit by three teams of auditors. two team members were trained south african national accreditation system (sanas) auditors, two were ascp slmta facilitators (one of whom was also a college of american pathologists [cap] inspector) and three others were slmta facilitators from the cdc office in tanzania. the audits were conducted using the who afro stepwise laboratory quality improvement process towards accreditation (slipta) checklist, which is based on iso 15189. with this checklist, recognition is given for progressive improvement; the lowest level of recognition (one star) requires a minimum of 55% of the total score possible on the checklist, two stars require 65% – 74%, three stars require 75% – 84%, four stars require 85% – 94% and five stars require ≥ 95% compliance. at the highest level (five stars), a laboratory is considered ready to seek accreditation to iso 15189 standard by any international accreditation agency.6 following the baseline audits, the slmta programme consisted of three five-day workshops where participants (laboratory managers and quality officers of the selected facilities) were taught the skills they needed in order to improve the quality of their laboratory services. each participant received a laboratory management framework document5 that defined the tasks laboratory managers must perform in order to meet the standards for best laboratory practices as set forth by the iso 15189 standard. a copy of the slipta checklist and a slmta toolkit with modules defining best laboratory practices were also given. the three workshops were held with three-month intervals between them so that skills taught in the workshop could be applied in the participants’ home laboratories. at the conclusion of each workshop, improvement projects were assigned that had either been taught during the previous workshop or were selected to address the common nonconformities found at the baseline audits. after completion of the programme in august 2011, exit audits were conducted in the 12 laboratories by the same team that conducted the baseline audits. in february 2012, seven months later, an official who afro slipta audit was conducted in amana laboratory by mohsw on behalf of the african society for laboratory medicine. results top ↑ the median baseline score for the 12 laboratories was 31% (figure 1). amana laboratory scored 36% at the baseline audit, well below the 55% needed for a one-star rating. during the audit debrief, it was clear that this low score was both a surprise and a disappointment to the amana laboratory staff. the laboratory manager, quality officer and mentor, an experienced tanzanian laboratory professional with good laboratory practices assigned to assist amana laboratory in its implementation of the improvement projects, responded positively, however, with determination to resolve the underlying quality problems in the laboratory. by the exit audit, improvements were seen in all of the participating laboratories, with a median exit score of 62% (figure 1). amana laboratory earned a score of 81% at the exit audit, corresponding to a three-star recognition level, which was the highest score in the cohort. much had been accomplished. for example, a quality manual for the laboratory had been developed by the quality officer in collaboration with the laboratory manager, which documented policies on how the laboratory would ensure quality in every aspect of service delivery. each policy was either described in detail in the quality manual or was referenced to a full standard operating procedure (sop) denoted by both the document title and control number. however, despite this exemplary improvement, seven months later in the official who afro slipta audit, the laboratory had regressed to one star (62%). figure 1: baseline and exit audit scores for the 12 laboratories in the first slmta cohort in tanzania, including slipta audit score for laboratory 12, the amana laboratory. discussion top ↑ at the exit audit, all 12 laboratories in tanzania’s first slmta cohort showed improvements in slipta scores, with five sites achieving a two-star recognition level, one achieving one-star recognition and five achieving no stars but showing improvement. amana laboratory achieved three stars and the highest score in the cohort, standing out as an example of the remarkable progress that can be achieved within a single year. when asked about the important factors that contributed to the successful outcome, the laboratory manager responded that the programme, in its entirety, contained all the essentials that were important for success. firstly, the tools and job aids taught and practised in the slmta programme, including the laboratory management framework and audit checklist, provided the crucial ‘how-to’ guidance for accomplishing best laboratory practices. secondly, there were supportive visits by the tanzanian slmta supervisory team that provided guidance for the implementation of improvement projects and clarified some translation misunderstandings that resulted from the workshops being taught in english rather than the native kiswahili. thirdly, encouragement from the facility medical officer and the regional medical officer was important with regard to supporting the efforts of the laboratory as they implemented improvements. fourthly, the assigned laboratory mentor assisted in all aspects of implementing the improvements and reinforced the skills learned from the slmta curriculum. finally, the laboratory staff’s team spirit, involvement and commitment to achieveing quality were crucial in meeting the goals of the programme. whilst all 12 laboratories in the cohort underwent the same training and supportive supervisory visits, it is possible that varied levels of support from facility medical officers contributed to differences in outcomes. in addition, amana laboratory’s relatively greater improvement could be attributed to laboratory leadership; amana’s laboratory manager made an effort to inspire a team spirit within his staff which, in turn, may have motivated them to strive for achievement of the improvement goals. also important was maintaining an open mind and positive attitude, despite the low baseline audit score, as amana’s laboratory management team approached the slmta improvement projects. they took the slmta curriculum seriously and were diligent in its implementation. they believed in the overall goal and the value of this initiative for the improvement of laboratory service and, by example to their staff, dedicated themselves to the extra hours and hard work required. the establishment of these essential elements ensured that the slmta curriculum was utilised to its fullest potential; the supervisory team was welcomed; and the mentor was well-received, enabling him to assist and to carry out his assignment. despite amana laboratory staff’s remarkable success during the programme, they were not able to maintain the improvements after the programme ended. seven months later, the score had dropped to the one-star level. as is often the case, once the focus on the programme ended, the laboratory found it difficult to maintain and update records and reviews. amana laboratory has learned that quality improvement is a continuous process and that, whilst progress may not always be steady, they are committed to continue to both improve and implement their quality systems. acknowledgements top ↑ the authors wish to acknowledge the support of the ascp, the cdc, the mohsw of tanzania and the slmta workshop facilitators. the authors are also grateful for the excellent assistance provided by the cdc editorial staff and the excellent peer review that was received to make this article better. competing interests the authors declare that they have no financial or personal relationship(s), which may have inappropriately influenced them in writing this article. authors’ contributions l.r.a. (globalhealth, ascp) is a technical consultant for ascp, a master trainer for slmta in tanzania and co-authored this article. c.g.m. (mohsw, tanzania) provided data on laboratory audit outcomes and co-authored this article. disclosure statement this report was made possible through support provided by the cdc, under the terms of cooperative agreement number 5u2gps001285-03. the opinions herein are the opinions of the author and do not necessarily reflect the views of the cdc. references top ↑ 1.reyburn h, mbata r, drakeley c, et al. overdiagnosis of malaria in patient with severe febrile illness in tanzania: a prospective study. bmj. 2004:329:1212–1217. http://dx.doi.org/10.1136/bmj.38251.658229.55 2.forsman rw. the value of the laboratory professional in the continuum of care. clin leadersh manag rev. 2002;16(6):370–373. 3.petti ca, polage cr, quinn tc, et al. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006;42(3):377–382. http://dx.doi.org/10.1086/499363 4.masanza mm, nqobili n, mukanga d, et al. laboratory capacity building for the international health regulations (ihr[2005]) in resource-poor countries: the experience of the african field epidemiology network (afenet). bmc public health. 2010;10(suppl 1):s8. http://dx.doi.org/10.1186/1471-2458-10-s1-s8 5.yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin pathol. 2010;134(3):401–409. http://dx.doi.org/10.1309/ajcpnbbl53fwuiqj 6.gershy-damet gm, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. http://dx.doi.org/10.1309/ ajcptuuc2v1wjqbm article information authors: keoratile ntshambiwa1 winnie ntabe-jagwer1 chandapiwa kefilwe1 fredrick samuel1 sikhulile moyo2 affiliations: 1sekgoma memorial hospital, ministry of health, botswana2botswana-harvard aids institute partnership and botswana-harvard hiv reference laboratory, princess marina hospital, botswana correspondence to: keoratile ntshambiwa postal address: po box 11299, palapye, botswana dates: received: 27 june 2014 accepted: 15 sept. 2014 published: 03 nov. 2014 how to cite this article: ntshambiwa k, ntabe-jagwer w, kefilwe c, samuel f, moyo s. translating a national laboratory strategic plan into action through slmta in a district hospital laboratory in botswana. afr j lab med. 2014;3(2), art. #209, 5 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.209 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. translating a national laboratory strategic plan into action through slmta in a district hospital laboratory in botswana in this original research... open access • abstract • introduction • research methods and design    • improvement project 1: reducing sample turnaround time    • improvement project 2: increasing patient and clinician satisfaction    • improvement project 3: ‘6s’    • improvement project 4: improving specimen management and document control • results • discussion    • limitations of the study    • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: the ministry of health (moh) of botswana adopted strengthening laboratory management toward accreditation (slmta), a structured quality improvement programme, as a key tool for the implementation of quality management systems in its public health laboratories. coupled with focused mentorship, this programme aimed to help moh achieve the goals of the national laboratory strategic plan to provide quality and timely clinical diagnoses.objectives: this article describes the impact of implementing slmta in sekgoma memorial hospital laboratory (smhl) in serowe, botswana. methods: slmta implementation in smhl included trainings, improvement projects, site visits and focused mentorship. to measure progress, audits using the stepwise laboratory quality improvement process towards accreditation (slipta) checklist were conducted at baseline and exit of the programme, with scores corresponding to a zeroto five-star scale. turnaround times, customer satisfaction, and several other health service indicators were tracked. results: the laboratory scored 53% (zero stars) at the baseline audit and 80% (three stars) at exit. nearly three years later, the laboratory scored 85% (four stars) in an official audit conducted by the african society for laboratory medicine. turnaround times became shorter after slmta implementation, with reductions ranging 19% to 52%; overall patient satisfaction increased from 56% to 73%; and clinician satisfaction increased from 41% to 72%. improvements in inventory management led to decreases in discarded reagents, reducing losses from us $18 000 in 2011 to $40 in 2013. conclusion: the slmta programme contributed to enhanced performance of the laboratory, which in turn yielded potential positive impacts for patient care at the hospital. introduction top ↑ laboratory medicine plays a critical role in healthcare and public health,1,2,3 and expanding laboratory capacity has been a focus of funding partners such as the us president’s emergency plan for aids relief (pepfar).4 implementation of laboratory quality management systems (qms) and accreditation to international standards contribute to increased accuracy of diagnoses and improvement in patient management.3 although considerable resources have been invested in recent years for the improvement of laboratory systems in resource-limited settings, lack of access to reliable laboratory testing still remains a barrier to effective healthcare and treatment for diseases, including tuberculosis, hiv and malaria.5,6,7 amongst the numerous challenges identified are the lack of national standards and policies specifically addressing qms.2,3 in 2008, the world health organization’s (who) maputo declaration8 called for the adoption of national laboratory strategic plans; nkengasong et al. concurred, arguing that such plans are one of the essential requirements for laboratory capacity building.9the ministry of health (moh) in botswana recognises the need for a collaborative and coordinated effort to raise the standards of its national laboratories. however, over the years there has been slow progress in implementing qms. previous training of healthcare workers has focused on general management topics rather than identifying tangible tasks to bring about change, making the training difficult to apply in the laboratory.10 in 2009, the moh developed a health sector laboratory strategic plan for 2009–2014, in which laboratory accreditation was a stated goal: to have four district-level laboratories accredited by 2013 and two national-level laboratories accredited by 2014. it became apparent that a radical shift in strategy would be required in order to realise these ambitious goals. to spearhead the moh’s national laboratory strategic plan, strengthening laboratory management toward accreditation (slmta), a structured quality improvement programme, was selected to assist in the development of qms within healthcare facilities.10,11 since its launch in 2009, slmta has demonstrated success in many countries12because of its task-oriented measureable design. sekgoma memorial hospital laboratory (smhl), a district laboratory of the sekgoma memorial hospital, was selected by the moh as one of eight laboratories to participate in the first slmta training cohort. sekgoma memorial hospital is a 350-bed facility located in serowe, a trade and commerce centre located in central district in eastern botswana. smhl has eight units: chemistry, haematology, microbiology, cd4, viral load, blood bank, serology and reception. patient samples for laboratory testing originate from the hospital wards, from 18 clinics within the hospital’s catchment area, and from outpatient services. on average, approximately 100 samples are received daily for chemistry, cd4 and haematology testing; 150 samples for viral load testing; and 30 samples for microbiology testing. this article describes the implementation and results of the slmta training and mentorship programme at smhl. research methods and design top ↑ the botswana slmta training programme was conducted in accordance with the standard slmta implementation model.111 training consisted of a series of three workshops, each lasting four days, held in august 2010, november 2010 and february 2011. attendees from smhl included the laboratory manager, the quality officer and two section supervisors. at each workshop, attendees selected improvement projects to implement in the laboratory based on the slmta modules presented. two supervisory visits of one day each were conducted by the three slmta trainers following each workshop.programme performance was measured by audits conducted before (baseline, july 2010) and after (exit, november 2011) slmta implementation using the who’s regional office for africa (afro) stepwise laboratory quality improvement process towards accreditation (slipta) checklist.113 slipta is an accreditation preparation framework, which measures and rewards the incremental progress of implementing qms requirements using a comprehensive audit checklist addressing the 12 quality system essentials of the laboratory.114 the slipta checklist and scoring system rate laboratory quality on a scale of zero to five stars; a rating of five stars signifies that the laboratory is ready to seek international accreditation. in-country auditors trained by a global healthcare public foundation, an independent external contractor based in kenya, conducted the baseline and exit audits. in august 2014, smhl received an official who afro slipta audit by the african society for laboratory medicine. from april to june 2011, smhl received focused mentorship from the botswana bureau of standards (bobs) to augment the slmta training. the bobs mentorship programme consisted of monthly visits, each lasting one week, during the three-month mentoring period. the visits included training on the international organization for standardization (iso) 15189 standard and qms documentation, with a focus on the management requirements of iso 15189. all laboratory staff members participated in the training sessions, whilst key staff members received additional mentorship on essential documentation of the qms. the laboratory developed and implemented four overarching improvement projects to address deficiencies identified during the baseline audit. a plan-do-check-act (pdca) cycle was implemented in order to facilitate continuous quality improvements, with past results driving future activities. progress of improvement projects was monitored using audit items selected from the slipta checklist, which were tracked and recorded. below we describe the improvement projects implemented: reducing sample turnaround time; increasing customer satisfaction; the ‘6s’ project; and improving specimen management and documentation. improvement project 1: reducing sample turnaround time in clinical laboratories, turnaround time is the total elapsed time from when a test is ordered to when the results are verified and released. a target turnaround time of two hours was established for haematology, chemistry and cerebrospinal fluid (csf) tests; a target of one hour was established for pregnancy tests. to assess this improvement project, data were obtained from the laboratory computer system, the integrated patient management system, on a monthly basis. average turnaround time for tests was analysed for two 6-month periods: april to september 2011 and october 2011 to march 2012. turnaround time data from before slmta implementation were not available. improvement project 2: increasing patient and clinician satisfaction customer satisfaction surveys are mandated by moh guidelines. separate questionnaires were administered for both patients and clinicians once a year in 2011, 2012 and 2013. the questionnaires utilised a rating system of ‘poor’, ‘fair’, ‘good’ and ‘very good’, with the latter two responses interpreted as ‘satisfied’ for purposes of analysis. the patient questionnaire asked patients to evaluate the laboratory reception, the staff members’ ability to answer questions, staff behaviours and attitudes, staff availability, the waiting time for test results, and overall satisfaction. patient questionnaires were distributed to patients every morning for a period of one month at the end of each year in 2011, 2012 and 2013. the clinician questionnaire asked staff to rate hospital services, turnaround times, and overall satisfaction; evaluate the attitudes of laboratory staff; and suggest ways to respond to enquiries. twenty doctors and nurses in each of the 10 hospital wards completed the clinician questionnaires for each year of evaluation. improvement project 3: ‘6s’ the slmta team implemented ‘6s’ (sorting, straightening, shining, standardising, sustaining and safety) in order to improve storage space and reduce expired products. an action plan was developed for each issue identified. wastage generated from discarded products as a result of expiry was tracked and their value calculated for fiscal years (fy) 2011 and 2013. improvement project 4: improving specimen management and document control to improve specimen management, missing and deficient essential documents were identified based on the slipta checklist and iso 15189 requirements. standard operating procedures (sops) were created and revised, including system procedures, safety procedures and technical procedures; staff were then trained on the relevant sops. signs with directions were placed in all testing areas so as to facilitate movement of staff and clients throughout the facility. selected hospital drivers attended a half-day workshop of presentations and simulation exercises, where they were trained on safe specimen handling, proper transportation and timely result distribution. results top ↑ the laboratory audit score improved from zero stars (53%) at baseline in july 2010 to three stars (80%) at the exit audit in november 2011. at the official who afro slipta audit in august 2014, the laboratory received four stars (85%).turnaround times decreased for all tests monitored: 19% reduction for haematology tests, 44% for chemistry tests, 30% for csf analyses and 52% for pregnancy tests (figure 1). pre-set targets of one hour for pregnancy tests and two hours for the remaining tests were all met. figure 1: overall patient satisfaction increased from 56% in 2011 to 85% in 2012, then decreased to 73% in 2013 (figure 2a). this pattern was consistent in each of the individual questions. clinician satisfaction increased steadily from 41% in 2011 to 64% in 2012 and 72% in 2013 (figure 2b), with steady improvements in all areas. figure 2a: figure 2b: the ‘6s’ improvement project led to the reorganisation of workstations and removal of unused items, which resulted in the creation of space and the alleviation of staff overcrowding. storage shelves were labelled with contents and the storeroom was cleaned and reorganised; all expired items were removed. systematic tracking of reagent expiration dates and improved inventory management led to a decrease in discarded reagents, with a subsequent drop in laboratory losses from p128 000 (approximately $18 000) in fy2011 to p280 (approximately $40) in fy2013. improved sample management and documentation revealed problems with specimen collection, especially a high sample rejection rate caused by poor phlebotomy. as a result, a specimen collection manual was developed and distributed to all clinics and wards in the hospital and guidance was provided. a total of 154 sops were developed and implemented, including management and technical documents for specimen receipt and results dispatch. long turnaround times during lunch hours were reduced by implementing an improved mid-day staff coverage system. a sign-in book for sample delivery was initiated in order to reduce delays in the transport of samples from the laboratory reception to the benches. all urgent samples were tagged with a white sticker on the cap to make their priority visible. discussion top ↑ implementation of slmta and targeted mentorship enabled smhl to develop an effective qms, including mechanisms to monitor critical aspects of laboratory service in pre-analytical, analytical and post-analytical processes. this resulted in rapid and measurable changes as evidenced by the jump from zero stars at the baseline audit to three stars at the exit audit. these improvements were sustained and further increased, with the laboratory reaching four stars at the official who slipta audit nearly three years later.reduction of turnaround time and improved customer satisfaction after completion of the slmta programme also indicated positive and sustained impact on patient care. the decrease in turnaround time for haematology occurred despite an increase in testing volume; this was because of a combination of reduced equipment downtime as a result of effective preventive maintenance of analysers and reduced stock-outs from the introduction of an effective inventory management system. slmta training enabled the laboratory staff to take on the daily challenges of increased volume that could otherwise strain the laboratory system. one important lesson learned was that staff motivation and management buy-in are critical for the success of the slmta programme. involving management in the early stages of slmta planning and periodic structured updates facilitated their understanding of qms and built consensus on the collaborative role of each department in achieving the goals of the national laboratory strategic plan. productive and frequent discussions between clinicians and laboratory staff paved the road to success and became a vehicle for translating strategic plans to action. the slmta programme brought about a cultural shift at smhl by providing the staff with a step-by-step process to break down tasks into actionable items with the common goal of providing high-quality laboratory services. the focus on continuous quality improvement ensures that the gains are maintained in a practical way, with active involvement of management and clinicians in identifying and addressing remaining challenges. staff motivation was also noted as a key driver to implementing qms. for instance, immediate changes in the organisation and utilisation of space as a result of the ‘6s’ project improved staff morale, largely because staff members were involved directly in the results-oriented process. this immediate transformation encouraged and motivated the staff to find other solutions using existing resources. improvement projects that were identified and planned during the slmta training stimulated creativity, ownership and action amongst laboratory and hospital staff; and were a great asset to the quality improvement programmes at smhl. it was noted by management that the more the staff were trained on slmta, the more enthusiastic and confident they became, thus enhancing the working relationship between staff and clients. in addition to slipta scores and stars, monitoring quality indicators such as turnaround time and customer satisfaction was valuable for assessing the laboratory’s success and identifying areas needing improvement. for example, customer satisfaction surveys alerted laboratory staff to declining patient satisfaction with waiting times from 2012 to 2013. after brainstorming sessions, the team concluded that this decline may have been caused by a failure to communicate to patients about the newly-introduced practice of prioritising services to patients needing urgent assistance such as pregnant women, the elderly and the disabled. armed with results from the survey, laboratory staff were able to address the problem head-on, by improving communication with patients to ensure that they understood the purpose of the new triage system. limitations of the study we acknowledge some limitations associated with this work. primarily, data for turnaround time and customer satisfaction were not collected before slmta implementation, preventing a pre-/post-slmta comparison. thus, our results are likely an underestimate of the true effect of slmta implementation. conclusion the botswana moh has made great strides in its drive toward continuous quality improvement in public-sector laboratories by adopting the slmta programme and focused mentorship as tools to help translate the national laboratory strategic plan into action. whilst tuberculosis, hiv, malaria and other diseases remain a challenge in botswana, the achievement of quality, timely and accurate laboratory results will play an important role in reducing the incidence of infections and improving treatment outcomes. at smhl, the slmta programme has been essential for the strengthening of laboratory management systems and has helped lay a firm foundation for further advancements in patient care. acknowledgements top ↑ we would like to thank all laboratory staff members of smhl, the slmta master trainers and management of serowe district health management team, all of whom contributed to the success story of smhl. we acknowledge and appreciate the moh clinical services for guidance and assistance, as well as for selecting smhl for participation in the first cohort. we would also like to acknowledge support from the chief medical laboratory scientist, mr david matema. in addition, we would like to thank the cdc botswana office for their unwavering support and contribution to smhl. we also acknowledge the bobs and moh for their guidance and assistance. we acknowledge the slmta master trainers and in-country slipta auditors for their enthusiasm and objectivity, and for raising the standards of implementation of qms in botswana. we would also like to acknowledge drs katy yao and elizabeth luman, along with global scientific solutions for health, for their contribution and support during the preparation of this manuscript. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions k.n., w.n-j., c.k. and f.s. (all sekgoma memorial hospital) implemented the slmta programme and wrote the manuscript. s.m. (princess marina hospital) provided technical writing support and analysis and contributed to writing of the manuscript. references top ↑ 1. hay burgess dc, wasserman j, dahl ca. global health diagnostics. nature. 2006;444(suppl 1):1–2. http://dx.doi.org/10.1038/nature054402. nkengasong jn. strengthening laboratory services and systems in resource-poor countries. am j clin pathol. 2009;131:774. http://dx.doi.org/10.1309/ajcp8gyx8ktkdatz 3. peter tf, rotz pd, blair dh, et al. impact of laboratory accreditation on patient care and the health system. am j clin pathol. 2010;134(4):550–555. http://dx.doi.org/10.1309/ajcph1skq1hnwghf 4. the president’s emergency plan for aids relief. fy 2014 country operational plan (cop) guidance. version 2 [document on the internet]. c2013 [cited 2013 dec 15]. available from: http://www.pepfar.gov/documents/organization/217765.pdf 5. petti ca, polage cr, quinn tc, et al. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006;42(3):377–382. http://dx.doi.org/10.1086/499363 6. abimiku ag. building laboratory infrastructure to support scale-up of hiv/aids treatment, care, and prevention: in-country experience. am j clin pathol. 2009;131(6):875–886. http://dx.doi.org/10.1309/ajcpelmg6gx6rqsm 7. birx d, de souza m, nkengasong jn. laboratory challenges in the scaling up of hiv, tb, and malaria programs: the interaction of health and laboratory systems, clinical research, and service delivery. am j clin pathol. 2009;131(6):849–851. http://dx.doi.org/10.1309/ajcpgh89qdswfons 8. world health organization. the maputo declaration on strengthening of laboratory systems [document on the internet]. c2008 [cited 2014 sep 08]. available from: http://www.who.int/diagnostics_laboratory/maputo-declaration_2008.pdf 9. nkengasong jn, mesele t, orloff s, et al. critical role of developing national strategic plans as a guide to strengthen laboratory health systems in resource-poor settings. am j clin pathol. 2009;131(6):852–857. http://dx.doi.org/10.1309/ajcpc51blobbpakc 10. yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin pathol. 2010;134(3):401–409. http://dx.doi.org/10.1309/ajcpnbbl53fwuiqj 11. yao k, maruta t, luman et, nkengasong jn. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. 2014;3(2), art. #194, 8 pages. http://dx.doi.org/10.4102/ajlm.v3i2.194 12. yao k, luman et, slmta collaborating authors. evidence from 617 laboratories in 47 countries for slmta-driven improvement in quality management systems. afr j lab med. 2014;3(2), art. #262, 11 pages. http://dx.doi.org/10.4102/ajlm.v3i2.262 13. world health organization’s regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation in the african region (with checklist) [document on the internet]. c2013 [cited 2014 may 27]. available from: http://www.afro.who.int/en/clusters-a-programmes/hss/blood-safety-laboratories-a-health-technology/blt-highlights/3859-who-guide-for-the-stepwise-laboratory-improvement-process-towards-accreditation-in-the-african-region-with-checklist.html 14. clinical and laboratory standards institute. application of a quality management system model for laboratory services; approved guidelines – third edition. clsi document gp26-a3 [isbn 1-56238-553-4]. wayne, pa: clinical and laboratory standards institute; 2004. abstract introduction methods results discussion acknowledgements references about the author(s) samson m. haumba university research co., llc, mbabane, eswatini mitsuru toda epidemic intelligence service (eis), division of scientific education and professional development, center for surveillance, epidemiology, and laboratory services (csels), centers for disease control and prevention (cdc), atlanta, georgia, united states mycotic diseases branch (mdb), division of foodborne, waterborne, and environmental disease (dfwed), national center for emerging and zoonotic infectious diseases (ncezid), centers for disease control and prevention (cdc), atlanta, georgia, united states rossana jeffries university research co., llc, mbabane, eswatini peter ehrenkranz global development, bill and melinda gates foundation, seattle, washington, united states munyaradzi pasipamire ministry of health eswatini national aids programme (enap), mbabane, eswatini trong ao centers for disease control and prevention (cdc), mbabane, eswatini nomthandazo lukhele ministry of health eswatini national aids programme (enap), mbabane, eswatini sikhathele mazibuko ministry of health eswatini national aids programme (enap), mbabane, eswatini mandzisi mkhontfo university research co., llc, mbabane, eswatini rachel m. smith mycotic diseases branch (mdb), division of foodborne, waterborne, and environmental disease (dfwed), national center for emerging and zoonotic infectious diseases (ncezid), centers for disease control and prevention (cdc), atlanta, georgia, united states tom chiller mycotic diseases branch (mdb), division of foodborne, waterborne, and environmental disease (dfwed), national center for emerging and zoonotic infectious diseases (ncezid), centers for disease control and prevention (cdc), atlanta, georgia, united states citation haumba sm, toda m, jeffries r, et al. prevalence of cryptococcal antigen (crag) among hiv-positive patients in eswatini, 2014–2015. afr j lab med. 2020;9(1), a933 https://doi.org/10.4102/ajlm.v9i1.933 original research prevalence of cryptococcal antigen (crag) among hiv-positive patients in eswatini, 2014–2015 samson m. haumba, mitsuru toda, rossana jeffries, peter ehrenkranz, munyaradzi pasipamire, trong ao, nomthandazo lukhele, sikhathele mazibuko, mandzisi mkhontfo, rachel m. smith, tom chiller received: 05 nov. 2018; accepted: 07 apr. 2020; published: 29 july 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: cryptococcal meningitis is a leading cause of death amongst people living with hiv. however, routine cryptococcal antigen (crag) screening was not in the national guidelines in eswatini. objectives: a cross-sectional study was conducted between august 2014 and march 2015 to examine crag prevalence at mbabane government hospital in eswatini. methods: we collected urine and whole blood from antiretroviral-therapy-naïve patients with hiv and a cluster of differentiation 4 (cd4) counts < 200 cells/mm3 for plasma and urine crag lateral flow assay (lfa) screening at the national hiv reference laboratory. two cd4 cut-off points were used to estimate crag prevalence: cd4 < 100 and < 200 cells/mm3. sensitivity and specificity of urine crag lfa was compared to plasma crag lfa. results: plasma crag prevalence was 4% (8/182, 95% confidence interval [ci]: 2–8) amongst patients with cd4 counts of < 200 cells/mm3, and 8% (8/102, 95% ci: 3–15) amongst patients with cd4 counts of < 100 cells/mm3. urine crag lfa had a sensitivity of 100% (95% ci: 59–100) and a specificity of 80% (95% ci: 72–86) compared with plasma crag lfa tests for patients with cd4 < 200 cells/mm3. forty-three per cent of 99 patients with cd4 < 100 were at world health organization clinical stages i or ii. conclusion: the prevalence of crag in eswatini was higher than the current global estimate of 6% amongst hiv-positive people with cd4 < 100 cell/mm3, indicating the importance of initiating a national screening programme. mechanisms for crag testing, training, reporting, and drug and commodity supply issues are important considerations before national implementation. keywords: cryptococcal antigenaemia screening; prevalence; people living with hiv; cryptococcal meningitis; advanced hiv disease package; eswatini. introduction cryptococcosis, caused by cryptococcus neoformans or cryptococcus gattii, is an invasive and life-threatening fungal infection, often affecting immunocompromised patients. cryptococcal meningitis, a type of cryptococcosis affecting the brain meninges, is one of the leading opportunistic infections and causes of death amongst people living with hiv. it especially affects patients with advanced hiv who have a cluster of differentiation 4 (cd4) count < 200 cells/mm3 or who are at stages iii or iv of the world health organization (who) hiv infection clinical stages.1 in 2014, globally, the cryptococcal antigenaemia prevalence was estimated at 6% (278 000) amongst patients with cd4 < 100 cells/mm3, with approximately 223 100 cryptococcal meningitis cases occurring annually.2 in the same year, 2014, the annual deaths due to cryptococcal meningitis were estimated at 181 100, with 75% (135 900) of deaths occurring in sub-saharan africa.2 the who recommends targeted screening of hiv-positive patients to enable early detection and pre-emptive treatment of cryptococcal infection.1 pre-emptive antifungal therapy can prevent cryptococcal meningitis-related mortality and morbidity in cryptococcal antigen (crag)-positive antiretroviral therapy (art)-naïve patients.3 the crag lateral flow assay (lfa) is an affordable,4 cost-effective,5 and simple point-of-care assay for crag testing in blood and cerebrospinal fluid (csf). crag can be detected a median of three weeks before clinical evidence of cryptococcal meningitis.6 the prevalence of hiv in eswatini (formerly swaziland) is amongst the highest in the world.7 despite the high hiv prevalence and known risk of cryptococcal meningitis, crag prevalence amongst people with advanced hiv disease, or cd4 < 200 cells/mm3, is unknown in eswatini. at the time of this study, routine crag screening was not included in the national guidelines and its clinical utility was unknown. we conducted a cross-sectional study to examine crag prevalence at a national hospital in eswatini. we also compared sensitivity and specificity of urine crag lfa relative to plasma crag lfa. methods ethical considerations the swaziland scientific and ethics committee (mh/599c/ fwa 000 15267), and the centers for disease control and prevention institutional review board reviewed and approved the study protocol (cgh hsr tracking #2014-139). study nurses obtained verbal informed consent from all participants. study location and population a cross-sectional study was implemented at mbabane government hospital, a national hospital, in eswatini. mbabane government hospital is a public hospital with a 500 bed capacity. it is the largest hiv treatment and care centre in eswatini and serves the population of the hhohho region (320 651), as well as the population of the nation at large (1 093 2381 million).8 we assessed eligibility of art-naïve adults ≥ 18 years old in the study who attended the voluntary counseling and testing clinic, as well as hospitalised patients, by determining their cd4 levels using alere pimatm (abbott laboratories, chicago, illinois, united states). for hiv-positive patients with cd4 ≤ 350 cells/mm3 by the alere pimatm cd4 test, a confirmatory test was completed using bd facscaliburtm (beckton dickinson, san jose, california, united states) flow cytometry. patients with cd4 < 200 cells/mm3 on bd facscaliburtm were enrolled in the study. enrolment occurred from 18 august 2014 to 19 march 2015. we excluded pregnant women, patients with a previous diagnosis or treatment for cryptococcal meningitis, and patients who had ever received fluconazole for < 5 days before study enrolment. blood and urine samples were obtained during the patient’s first visit to the hiv testing clinic or soon after hiv testing, if an inpatient. plasma and urine crag screening were conducted using a lfa (immy, norman, oklahoma, united states) at the hospital laboratory for patients both with and without signs of cryptococcal meningitis. data collection trained study nurses and research assistants conducted data collection. study nurses and research assistants gave a communication leaflet providing basic information on cryptococcus spp. and hiv infection as well as the objectives of the study to eligible patients. after the informed consent process, data on patient demographic characteristics including age, sex, marital status, area of residence (rural or urban), level of education, and the clinical stage of hiv infection9 were collected. clinical procedures at enrolment, study nurses assessed patients’ clinical signs and symptoms of cryptococcal meningitis such as fever, headache, neck stiffness, altered mental status, photophobia, nausea, night sweats, cough, vomiting, shortness of breath, skin papules, or kerning sign. patients with positive plasma crag results and signs and symptoms of cryptococcal meningitis underwent a lumbar puncture as part of routine practice. csf samples obtained from the lumbar puncture were collected for crag lfa to diagnose cryptococcal meningitis. patients diagnosed with cryptococcal meningitis were treated according to a standard of care based on who guidelines,3 which included amphotericin b (0.7 mg/kg/day) and fluconazole (800 mg) for 2 weeks followed by 400 mg of fluconazole for 8 weeks and then maintained at 200 mg for secondary prophylaxis. patients who refused a lumbar puncture but had positive plasma crag results were offered pre-emptive therapy which followed who recommendations of fluconazole 800 mg/day for the first 2 weeks, followed by 8 weeks of 400 mg/day of fluconazole daily, and a 200 mg/day fluconazole maintenance dosage.3 the south african clinical guidelines (2013) were followed for patients with positive plasma crag and negative csf crag results.10 art was delayed for 2 weeks to decrease the risk of immune reconstitution inflammatory syndrome, and they were prescribed pre-emptive fluconazole oral treatment to prevent the development of meningeal infection.10 because of resource constraints, patients’ cryptococcal infections were not verified using x-ray or other imaging technology, and we did not follow the individual patients’ clinical course to collect outcome measures for the purposes of this study. laboratory procedures all blood and urine specimens were packaged according to who standards. study nurses collected fresh urine samples and recorded urine crag lfa positive results. in addition, study nurses collected whole blood in ethylene diamine tetra acetic acid-treated test tubes and stored it at room temperature. the blood was centrifuged for 5 min at 300 revolutions per minute to obtain plasma samples on the same day that blood was collected. plasma and urine samples were stored at 2 °c – 8 °c for up to 72 h. cd4 retesting was completed using facscaliburtm and research assistants conducted crag testing at the laboratory the next day using crag lfa on samples from patients with cd4 < 200 cells/mm3. in addition, research assistants performed daily positive and negative control lfa testing as well as lot-to-lot testing to ensure the quality of reagents. research assistants communicated all laboratory results to physicians for clinical management, and clinicians informed the participants of their results. sensitivity and specificity analyses we compared urine and plasma crag lfa results using plasma lfa as the gold standard. records were retained for study purposes and not for clinical diagnosis or treatment purposes, and our specificity and sensitivity analyses did not take into account clinical or radiological findings. analyses data were double entered using epidata (epidata association, odense, denmark). the descriptive analyses and calculation of wilson scores on 95% confidence intervals (ci) on the crag prevalence were conducted using stata 15 (statacorp, college station, texas, united states). results of 313 patients initially screened for eligibility, 58% (182/313) had cd4 counts < 200 cells/mm3 by facscaliburtm (figure 1). of the 182 eligible patients, 79% (144/182) were from the outpatient voluntary counseling and testing clinic and 21% (38/182) were from the inpatient ward (table 1). figure 1: study flow for cryptococcal screening at mbabane government hospital, eswatini, august 2014 to march 2015. table 1: characteristics of patients enrolled in the crag screening study of patients with a cd4 count < 200 cells/mm3, eswatini, august 2014 to march 2015. of the 182 patients with cd4 counts < 200 cells/mm3 by facscaliburtm, 42% (76/182) were women, the median age of the participants was 35 years (interquartile range: 30–42)], and 61% (111/182) were from urban areas (table 1). most (86%, 156/182) patients were from the hhohho region, 10% (18/182) from manzini, 3% (5/189) from shiselweni, and 2% (3/182) from lubombo. almost half of the patients (47%, 86/182) did not complete primary school or did not receive any schooling, 41% (75/182) completed primary school, and 12% (21/182) completed secondary school or higher. most (62%, 113/182) patients were single and never married, 34% (61/182) were married, and 4% (8/182) were widowed, divorced, or separated. more than half of the patients (54%, 99/182) were at who clinical stages i or ii, 40% (73/182) at stage iii, and 5% (10/182) at stage iv. of the 182 patients with cd4 count < 200 cells/mm3, 56% (102/182) had a cd4 count < 100 cells/mm3. forty-three percent (44/102) of these were at who clinical stages i or ii and 57% (58/102) at stages iii or iv. of patients with cd4 counts 100 cells/mm3 – 199 cells/mm3, 69% (55/80) were at who clinical stages i or ii and 31% (25/80) at stages iii or iv. cryptococcal antigen lateral prevalence the plasma crag prevalence was 8% (8/102, 95% ci: 3–15) amongst patients with cd4 counts < 100 cells/mm3. when considering the higher cut-off point of a cd4 count < 200 cells/mm3, the plasma crag prevalence decreased to 4% (8/182, 95% ci: 2–8) (figure 1). all eight plasma crag-positive patients had cd4 cell counts < 100 cells/mm3 and none had cd4 cell counts in the 100 cells/mm3 – 199 cells/mm3 range (prevalence 4% [8/182], 95% ci: 2–8). of the patients at the voluntary counseling and testing clinic, 47% (68/144) had cd4 counts 100 cells/mm3 – 199 cells/mm3, and 53% (76/144) patients had cd4 counts < 100 cells/mm3. of the 76, 5% (4/76), were plasma crag-positive. three out of the four patients who were plasma crag-positive were csf crag negative; one patient did not complete a lumbar puncture. of the patients at the inpatient ward, 32% (12/38) had cd4 counts 100 cells/mm3 – 199 cells/mm3 and 68% (26/38) had cd4 counts < 100 cells/mm3. fifteen percent (4/26) of patients, all with cd4 counts < 100 cells/mm3, were plasma crag-positive. of the four patients who were plasma crag-positive at the inpatient ward, one patient was csf crag-positive and the other three patients were csf crag negative (figure 1). sensitivity and specificity of urine cryptococcal antigen lateral flow assay urine samples were collected from 168 patients; 82% (137/168) from the voluntary counseling and testing clinic and 18% (31/168) from the inpatient ward. forty-six percent (77/168) of patients had cd4 counts of 100 cells/mm3 – 199 cells/mm3 and 54% (91/168) had cd4 counts < 100 cells/mm3 by facscaliburtm. urine crag-positive prevalence was 25% (23/91) amongst patients with cd4 counts < 100 cells/mm3 and 22% (17/77) amongst patients with cd4 counts 100 cells/mm3 – 199 cells/mm3. urine crag lfa had a sensitivity of 100% (95% ci: 59–100) and a specificity of 80% (95% ci: 72–86) compared with plasma crag lfa for patients with cd4 < 200 cells/mm3. there were seven true positives, 33 false positives (i.e., positive on urine crag lfa but negative on plasma crag lfa), 128 true negatives, and zero false negatives (i.e., negatives on urine crag lfa but positive on plasma crag lfa) (table 2). table 2: sensitivity and specificity of urine cryptococcal antigen lateral flow assay compared with plasma cryptococcal antigen lateral flow assay for patients with a cd4 count < 200 cells/mm3, eswatini, august 2014 to march 2015. for patients with < 100 cells/mm3, sensitivity of the urine crag lfa compared with plasma crag lfa was 100% (95% ci: 59–100), and specificity was 81% (95% ci: 71–89). there were seven true positives, 16 false positives (i.e., positive on urine crag lfa but negative on plasma crag lfa), 68 true negatives, and zero false negatives (i.e., negatives on urine crag lfa but positive on plasma crag lfa) (table 3). table 3: sensitivity and specificity of urine cryptococcal antigen lateral flow assay compared with the plasma cryptococcal antigen lateral flow assay for patients with a cd4 count < 100 cells/mm3, eswatini, august 2014 to march 2015. discussion we conducted a cross-sectional study on crag prevalence at a national reference hospital in eswatini. the study showed that patients with cd4 < 100 cells/mm3 at the national reference hospital had a crag prevalence of 8%, which is higher than the 2014 global estimate of 6%2 and similar to the prevalence found in uganda5 and south africa.11 this study is relevant in the context of recent who guidelines on managing advanced hiv disease, rapid initiation of art,1,9 and the current recommendation to include crag screening in national guidelines in eswatini12 and in other countries.13 combining crag screening and early treatment for cryptococcal infection are cost-effective interventions compared with standard care to prevent morbidity and mortality from cryptococcal meningitis amongst immunocompromised people living with hiv.5,11,14,15,16,17 most importantly, crag screening has the potential to improve hiv outcomes by reducing morbidity and mortality. the ‘reduction of early mortality among hiv-infected subjects starting antiretroviral therapy’ (remstart) trial in tanzania and zambia showed crag screening, pre-emptive treatment, and community support that led to a 28% reduction in mortality amongst patients with advanced hiv disease,18 and the ‘reduction of early mortality in hiv-infected african adults and children starting antiretroviral therapy’ (reality) trial in kenya, malawi, uganda, and zimbabwe showed that an enhanced prophylaxis package reduced mortality by 27%.19 cd4 cell counts are essential in determining the timing of art initiation for patients with advanced hiv disease, because early art initiation could lead to worse outcomes for crag-positive patients.20 current who recommendations include crag screening for people living with hiv at cd4 cell counts < 100 cells/mm3, and offering pre-emptive antifungal therapy before initiating art.1 in the era of ‘90-90-90’21 and ‘test and start’ strategies where rapid art initiation is promoted, there is potential risk in offering art to patients presenting with advanced hiv disease with low cd4 cell counts or who clinical stages iii or iv22 who are at high risk of opportunistic infection.23 screening these patients for undiagnosed cryptococcus infection can prevent life-threatening immune reconstitution inflammatory syndrome.26 yet, assessment of a clinical stage without a cd4 count is not sufficient. in our study, 43% of patients with cd4 < 100 cells/mm3 were classified as who clinical stages i or ii. these patients would not have been screened for crag in the absence of cd4 cell count testing. although studies suggest that screening may be considered at cd4 < 200 cells/mm3,24 our study suggests that selecting for patients with cd4 < 100 cells/mm3 might be a priority, since all plasma crag-positive patients had cd4 < 100 cells/mm3. since crag-positive patients were found at both outpatient (prevalence: 5%) and inpatient (prevalence: 15%) facilities, cd4-directed crag screening should be recommended for patients presenting in either setting. the sensitivity and specificity of urine crag compared with plasma crag testing were similar to the finding of a tanzanian study.25 those findings showed that urine crag testing could be useful in ruling out disease, if the result is negative, but may not be a reliable stand-alone screening tool because of the prevalence of false-positive results. the eswatini ministry of health revised its hiv guidelines in 2018 and included crag screening for adults with cd4 cell < 100 cells/mm3 or children with cd4 counts at less than 25% of total lymphocytes,12 as well as pre-emptive treatment as a package for advanced disease management to reduce morbity and mortality.26,27,28 education of patients is important in order to inform them that cryptococcal meningitis is not, as some swazi patients refer to it, the ‘headache that kills’, but rather a preventable and treatable disease (personal communication with patients). before rollout, training of the clinical and laboratory workforce on crag testing and reporting, and ensuring adequate supplies of antifungal prophylaxis, treatment (fluconazole, flucytocine, and amphotericin b), lumbar puncture kits and crag lfa kits are essential. moreover, joint ownership of the crag screening program by the swaziland national hiv/aids programme and swaziland health laboratory services have been established in-country, which could help accelerate buy-in from the clinical and laboratory workforces and successfully roll out the national crag screening programme. limitations there are several limitations to this study. firstly, the study was conducted in one hospital, thus it is not a nationally-representative sample. however, this hospital is a national reference hospital that captures a large number of patients who are crag-positive. secondly, the study only assessed art-naïve patients, whereas patients failing art may also have cd4 cell counts < 100 cell/mm3 and become at risk of cryptococcal infection. in addition, the cross-sectional study design and small sample size limited our ability to measure or compare patient outcomes. further studies to examine patient treatment and outcome measures, and address crag prevalence among art-experienced patients, are warranted. finally, sensitivity and specificity analyses of urine crag lfa were evaluated compared with plasma lfa and did not take into account radiological or clinical findings. conclusion this study indicated that the crag prevalence at a national hospital in eswatini was higher than the global crag estimates of 6% amongst hiv-positive people with cd4 < 100 cell/mm3. our findings support the current eswatini national cryptococcal screening and treatment guidelines. issues of drug and commodity supply and training mechanisms for crag testing, monitoring, evaluation and reporting are important considerations, before national implementation of plasma crag lfa screening. trustworthiness the following measures were undertaken to ensure trustworthiness: cryptococcal antigen lateral flow assay quality control: laboratory technologists at the national reference laboratory conducted quality control testing for the crag lfa reagents on a weekly basis. a positive and negative control lfa test as well as lot-to-lot testing were performed to ensure the quality of the reagents. the reagents were discarded when the positive or negative controls did not yield the expected results, and a new set of lfa reagents were requested from the laboratory supervisor and tested prior to further patient testing for crag. training of clinicians and study personnel: the cdc-eswatini and cdc-atlanta study teams, who were experienced in crag screening, trained the research assistants, clinicians and laboratory personnel involved in the care of patients. the training of clinicians and study personnel covered the following topics: basic information about cryptococcal disease, including early and disseminated infection explanation of the rationale behind the implementation of this screening programme review of the algorithm for implementation and associated study forms and documents review of procedure for sending samples to the national reference laboratory and obtaining results role-playing using different clinical scenarios in order to ensure proper adherence to the algorithm and flow of patients instructions and practice in performing and interpreting the lfa crag test review of the procedure for sending csf crag results to hiv care centre staff reviewing standard operating procedures relevant to the laboratory staff study protocol data collection tools research ethics quality assurance. acknowledgements we would like to thank the eswatini ministry of health, the mbabane government hospital voluntary counseling and testing clinic, laboratory, and inpatient wards for facilitating the study and all of the patients and healthcare workers who participated in this study. we thank charmaine khudzie mlambo, lydia mpango and marianne calnan for being part of the team that conceptualised this study, kelly rose-clarke and greg greene for training data collectors, thokozani maseko and mulungie mwembo for enrolling participants and collecting specimens, dan gama for facilitating reagent support from the african society for laboratory medicine, and nombuso ntshalintshali for testing specimens in the laboratory. we are grateful to brendan r. jackson, alexander jordan, and juliana da silva for providing valuable feedback to the draft manuscript. competing interests authors declare that they have no competing interests. authors’ contributions s.m.h. (project team leader), p.e., t.a. and t.c. conceived the study. r.j. (overall study implementation coordinator), s.m.h., m.p., t.a., n.l., s.m. and r.m.s. oversaw implementation of the study. m.t. and r.j. performed the analysis, and m.t., s.m.h., m.m. and r.j. wrote the original draft. all authors read and approved the final manuscript. source of support the study was made possible by the generous support of the american people through the united states president’s emergency plan for aids relief through the united states centers for disease control and prevention under the provision of hiv/aids and tb related laboratory support and technical assistance project in eswatini managed by university research co., llc (cooperative agreement number 1u2g/ps001896 and principal investigator – dr. neeraj kak). study reagents were procured through the african society for laboratory medicine. data availability statement the data that support the findings of this study are available from the corresponding author, [s.m.h.], upon reasonable request. disclaimer the findings and conclusion in this report are those of the authors and do not necessarily represent the official position of the centers for disease control and prevention. references world health organization. guidelines for the diagnosis, prevention, and management of cryptococcal disease in hiv-infected adults, adolescents and children, march 2018: supplement to the 2016 consolidated guidelines of the use of antiretroviral drugs for treating and preventing hiv infection [homepage on the internet]. 2018 [cited 2018 may 18]. available from: http://www.who.int/hiv/pub/guidelines/cryptococcal-disease/en/ rajasingham r, smith rm, park bj, et al. global burden of disease of hiv-associated cryptococcal meningitis: an updated analysis. lancet infect dis. 2017;17(8):873–881. https://doi.org/10.1016/s1473-3099(17)30243-8 world health organization. rapid advice: diagnosis, prevention and management of cryptococcal disease in hiv-infected adults, adolescents and children [homepage on the 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https://doi.org/10.1016/s0140-6736(15)60164-7 hakim j, musiime v, szubert aj, et al. enhanced prophylaxis plus antiretroviral therapy for advanced hiv infection in africa. new engl j med. 2017;377(3):233–245. https://doi.org/10.1056/nejmoa1615822 abassi m, rhein j, meya db, et al, editors. cryptococcal disease in the era of “test and treat”: is there cause for concern? open forum infectious diseases. oxford university press us; 2017. joint united nations programme on hiv/aids (unaids). 90-90-90 an ambitious treatment target to help end the aids epidemic [homepage on the internet]. 2014 [cited 18 may 2019]. available from: http://www.unaids.org/sites/default/files/media_asset/90-90-90_en_0.pdf ford n, meintjes g, calmy a, et al. managing advanced hiv disease in a public health approach. clin infect dis. 2018;66(suppl_2):s106–s110. denning dw. minimizing fungal disease deaths will allow the unaids target of reducing annual aids deaths below 500 000 by 2020 to be realized. phil trans royal soc b: biol sci. 2016;371(1709):20150468. https://doi.org/10.1098/rstb.2015.0468 ford n, shubber z, jarvis jn, et al. cd4 cell count threshold for cryptococcal antigen screening of hiv-infected individuals: a systematic review and meta-analysis. clin infect dis. 2018;66(suppl_2):s152–s159. https://doi.org/10.1093/cid/cix1143 magambo ka, kalluvya se, kapoor sw, et al. utility of urine and serum lateral flow assays to determine the prevalence and predictors of cryptococcal antigenemia in hiv-positive outpatients beginning antiretroviral therapy in mwanza, tanzania. j int aids soc. 2014;17(1):19040. https://doi.org/10.7448/ias.17.1.19040 scriven je, lalloo dg, meintjes g. changing epidemiology of hiv-associated cryptococcosis in sub-saharan africa. lancet infect dis. 2016;16(8):891–892. https://doi.org/10.1016/s1473-3099(16)30145-1 williamson pr. the relentless march of cryptococcal meningitis. lancet infect dis. 2017;17(8):790–791. https://doi.org/10.1016/s1473-3099(17)30245-1 who. rapid advice: diagnosis, prevention and management of cryptococcal disease in hiv-infected adults, adolescents and children [homepage on the internet]. 2011 [cited 2019 may 17]. available from: https://apps.who.int/iris/bitstream/handle/10665/44786/9789241502979_eng.pdf?sequence=1 abstract introduction laboratory structures laboratory services laboratory workforce development laboratory system acknowledgements references about the author(s) stephen b. kennedy incident management system, emergency operations center, ministry of health, monrovia, liberia partnership for research on ebola virus in liberia, liberia-us clinical research partnership program, first floor, john f. kennedy medical center, monrovia, liberia john b. dogba national reference laboratory, ministry of health, charlesville, margibi county, liberia christine l. wasunna partnership for research on ebola virus in liberia, liberia-us clinical research partnership program, first floor, john f. kennedy medical center, monrovia, liberia philip sahr partnership for research on ebola virus in liberia, liberia-us clinical research partnership program, first floor, john f. kennedy medical center, monrovia, liberia national reference laboratory, ministry of health, charlesville, margibi county, liberia candace b. eastman africabio enterprises, inc., payne avenue, sinkor, monrovia fatorma k. bolay partnership for research on ebola virus in liberia, liberia-us clinical research partnership program, first floor, john f. kennedy medical center, monrovia, liberia liberia institute for biomedical research, ministry of health, charlesville, margibi county, liberia national research ethics board, partnership for research on ebola virus in liberia, first floor, john f. kennedy medical center, monrovia, liberia gloria t. mason national research ethics board, partnership for research on ebola virus in liberia, first floor, john f. kennedy medical center, monrovia, liberia mark w.s. kieh partnership for research on ebola virus in liberia, liberia-us clinical research partnership program, first floor, john f. kennedy medical center, monrovia, liberia citation kennedy sb, dogba jb, wasunna cl, et al. pre-ebola virus disease laboratory system and related challenges in liberia. afr j lab med. 2016;5(3), a508. http://dx.doi.org/10.4102/ajlm.v5i3.508 lessons from the field pre-ebola virus disease laboratory system and related challenges in liberia stephen b. kennedy, john b. dogba, christine l. wasunna, philip sahr, candace b. eastman, fatorma k. bolay, gloria t. mason, mark w.s. kieh received: 20 june 2016; accepted: 15 aug. 2016; published: 31 oct. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract prior to the ebola virus disease outbreak in liberia, the laboratory system was duplicative, fragmented and minimally coordinated. the national reference laboratory was conceptualised to address the existing challenges by promoting the implementation of effective and sustainable laboratory services in liberia. however, in a resource-limited environment such as liberia, progress regarding the rebuilding of the health system can be relatively slow, while efforts to sustain the transient gains remain a key challenge for the ministry of health. in this paper, we describe the pre-ebola virus disease laboratory system in liberia and its prevailing efforts to address future emerging infectious diseases, as well as current infectious diseases, all of which are exacerbated by poverty. we conclude that laboratory and diagnostic services in liberia have encountered numerous challenges regarding its efforts to strengthen the healthcare delivery system. these challenges include limited trained human resource capacity, inadequate infrastructure, and a lack of coordination. as with most countries in sub-saharan africa, when comparing urban and rural settings, diagnostic and clinical services are generally skewed toward urban health facilities and private, faith-based health facilities. we recommend that structured policy be directed at these challenges for national institutions to develop guidelines to improve, strengthen and sustain diagnostic and curative laboratory services to effectively address current infectious diseases and prepare for future emerging and re-emerging infectious diseases. introduction the primary focus regarding the implementation of effective and sustainable laboratory services in liberia was accentuated with the conceptualisation of the national reference laboratory (nrl) in 2008.1 initially, the nrl was formulated, with an agenda for future expansion, as a single-room facility housed within the national drug service at the john f. kennedy memorial hospital, a tertiary referral hospital in monrovia. with prevailing viral infectious diseases as its initial core, its primary laboratory diagnostic capacity was limited to measles, rubella and yellow fever. at the time, samples for lassa fever were collected, packaged and transported for diagnosis at the lassa fever laboratory in kenema, sierra leone. with support from international partners, basic equipment and human resource development aimed at expanding the capacity of the nrl were secured, and the laboratory was subsequently relocated in 2009 to the compound of the liberia institute for biomedical research in charlesville, lower margibi county. afterward, a programme known as the national diagnostic unit (ndu) was established. liberia has gradually been rebuilding its healthcare delivery system after 16 years of devastating civil conflicts which erupted in december 1989, destroyed the country’s health infrastructure2 and subsequently culminated with a drastic brain drain as the result of the migration of trained liberians to politically-stable countries. in a resource-limited environment such as liberia, progress regarding the rebuilding of the health system has been relatively slow and sustaining the gains from transient spikes in improved health indicators remain a major challenge. accordingly, laboratory-based service delivery in liberia has been basically defined in several key policy documents1,2,3 of the ministry of health that established the minimum threshold for trained human resources development, laboratory infrastructure, disease surveillance and laboratory diagnostic capacities at each level of the healthcare delivery system in the country. laboratory structures liberia’s laboratory system was initially classified into a three-tiered health system structure consisting of the: (1) national; (2) county (main political sub-divisions); and (3) peripheral (health centres and clinics) levels.1,2,3 the national level comprises the nrl and ndu; the county level involves the referral hospitals in the political subdivisions of the country; and the peripheral level accounts for the district and community health facilities. with the development of the country’s national decentralization policy2,4 for the regionalisation of services within the country, geared toward the improvement of services in the disenfranchised rural areas, a fourth level of the laboratory system was incorporated; thereby, allowing for (1) national; (2) regional; (3) county; and (4) peripheral levels. accordingly, the nrl and ndu are strategically situated within the national level. the regional tier consists of five regional laboratories based on the decentralisation of the country, namely: (1) southeast a; (2) southeast b; (3) north central; (4) south central; and (5) western. the county and peripheral tiered systems are basically the same levels as in the prior three-tiered approach. laboratory services in general, there are two main categories of laboratory services in liberia. the first is the clinical laboratory services designated for clinical specimen testing for diagnostic purposes and the second is public health laboratory services directed at disease surveillance. according to its mandate, the nrl has a four-fold responsibility: (1) perform diagnostic testing services on specimens for diseases with epidemic potential that are under surveillance, especially those that are immediately notifiable to the world health organization, now inclusive of ebola virus disease (evd), as required by the international health regulations; (2) oversee the network of public laboratory structures within the country and the provision of services through quality assurance, training, monitoring and supportive supervision; (3) assess new and available technologies; and (4) provide technical advice to the ministry of health. similar to the nrl, the ndu conducts quality assurance, monitoring and evaluation, and training. furthermore, it coordinates procurement processes for the distribution of laboratory commodities for special programmes supported by the global funds, such as hiv, aids, malaria and tuberculosis programmes. when constructed, the regional laboratories will support the nrl in its surveillance testing (i.e., its mandate for public health laboratory services), as well as the county-level coordination of the activities of the laboratory system. in addition, they will lend diagnostic support to the laboratories at the county hospitals. these structures are responsible solely for clinical specimen testing for diagnostic purposes. test categories include, among others, chemistry, haematology and parasitology. apart from the traditional structures indicated for service delivery, certain programs of the ministry of health are also involved in cross-cutting testing activities (laboratory clinical diagnosis and public health surveillance purposes). these programmes include: (1) the national aids control program; (2) the national malaria control program; (3) the national tuberculosis & leprosy control program; and (4) the national blood safety program. laboratory equipment, reagents and supplies for the operations of the various laboratory structures are either procured by the ministry of health or donated by international partners. the operation of the procurement activities is performed by two organisations: (1) the national drug service; and (2) the global fund, which supports the hiv, tuberculosis and malaria programmes. both of these key operational processes (procurement and donations) for the effective functionality of the laboratory system in liberia exist without defined ‘maintenance and/or service contracts’. laboratory workforce development there are two accredited faith-based and one publicly-owned laboratory training institutions in liberia. these are: (1) the mother patern college of health sciences in monrovia, montserrado county; (2) the phebe school of nursing in suacoco, bong county; and (3) the tubman national institute of medical arts in monrovia. the qualifications offered by these training institutions, based on a duration of training that ranges from 2–4 years, includes diploma in laboratory science (tubman national institute of medical arts and phebe school of nursing); associate of science and bachelor of science in laboratory technology by the mother patern college of health sciences. laboratory system the status of the existing laboratory structure and service delivery system in liberia is characterised by numerous challenges, which were uncovered during the evd outbreak in liberia that became a major public health problem in the country and the sub-region.5,6 poor health infrastructure, lack of logistics during the beginning of the outbreak, and an inadequate number of trained staff in public health disease surveillance and laboratory diagnostic response in emergency situations, were key factors leading to the delay in response to the outbreak. these pre-evd laboratory challenges, among others, included: lack of integration between public health surveillance and laboratory diagnostic services, resulting in fragmented service delivery. lack of a well-defined organisational structure with clearly-defined objectives, including existing duplications of roles and responsibilities within the tiered laboratory systems to support an effective leadership and management system. lack of adequate financial resources for infrastructure support and operational costs. inadequately trained laboratory workforce, including inadequate deployment across the country. unavailability of maintenance and service contracts for procured and/or donated laboratory equipment and supplies to sustain the effective operation of the laboratory infrastructure. lack of adequate biomedical equipment technicians to maintain and repair laboratory equipment. unstable electric power supply and inadequate water supply for laboratory service utilisation. lack of effective laboratory information management system for coordination, operations, and the efficient functionality of the various tiered systems. lack of a well-organised quality management scheme despite these challenges, efforts were directed at the development of a national laboratory policy to prioritise the delivery of laboratory services within the health system.7 it was aimed at addressing the challenges associated with limited diagnostic services, which subject patients to inappropriate treatment, chronic illnesses, high out-of-pocket expenditures on healthcare, loss of income and, ultimately, loss of confidence in health services. to mitigate the challenges, the proposed policies targeted the following areas: organisation: strengthen the legal and regulatory framework, as well as the technical and administrative organisational structures, for the delivery of laboratory services nationwide. test selection and referral linkage: ensure that appropriate tests are performed at the appropriate facility level in a decentralised network and the provision of oversight to establish appropriate communication structure and processes in order to create a referral linkage or network between facilities. human resources: ensure the presence of certified laboratory personnel in all public and private laboratories, regulate through periodic certification and develop curriculum standards to support training institutions. quality assurance: ensure that laboratories are certified according to defined standards with established systems for independent certification. infrastructure: provide safe and adequate infrastructure standards for all public and private laboratories at each level of care and ensure, through certification, that laboratories have standard operating procedures and required infrastructure for laboratory safety and operation. equipment: ensure that all laboratories have properly working equipment with both preventative and curative maintenance, and enforce standardisation such that all equipment and supplies meet national specifications and are maintained according to manufacture guidance. supply chain management: support an uninterrupted distribution of reagents and supplies to all laboratories, and ensure that an effective system is in place to select, quantify, procure, transport, store, distribute and keep records of all equipment, reagents, and supplies. national blood safety: ensure a safe supply of blood for transfusions and promote voluntary blood donations. biosafety: provide a safe environment and ensure laboratory personnel adhere to safety protocols and quality assurance standards, including laboratory safety for protection of professional staff, public and the environment. financial resources: allocate resources to sustain quality laboratory services. research and development: identify major laboratory research priorities and promote the culture for operational research, and evaluate and introduce new innovative technologies into the laboratory system. collaboration: improve laboratory services through public and private partnerships, as well as support for national and international collaborations. ethics: ensure a professional code of conduct and client confidentiality. information system: design a laboratory information system that is fully integrated for information flow between laboratories within a referral network. policy implementation: monitor and evaluate the efficiency and effectiveness of the laboratory system, as well as its sustainability. policy regulation: ensure that laboratory guidelines are updated regularly and in compliance with internationally-accepted standards. recommendations we recommend that a joint public–private partnership team be established to conduct a comprehensive technical assessment of the laboratory system within the country and provide appropriate recommendations to combat emerging infectious diseases. enhancing the laboratories’ capacity for research requires: the availability of essential equipment and appliances; development of relevant and sustained human capacity through continuing education, on-the-job training and mentorship; formulation and adherence to guidelines and standard operating procedures, and compliance with good (clinical) laboratory practice and other international standards, such as iso 15189. the evd outbreak in liberia has necessitated improved laboratory capacity to respond to an epidemic of emerging infectious pathogens as well as support for the development of a suitable research platform during such public health emergencies. accordingly, we propose a framework to strengthen the laboratory system of the country based on a preparedness strategy to mitigate future outbreaks of emerging and re-emerging infectious diseases (see figure 1). figure 1: proposed mechanism for strengthening the laboratory system in liberia. conclusion laboratory and diagnostic services in liberia have encountered numerous challenges regarding their contributions toward the strengthening of the healthcare delivery system in liberia. these challenges are a consequence of limited trained human resource capacity, inadequate infrastructure, and lack of coordination among the four tiers. diagnostic services are skewed in urban health facilities as compared to those in rural communities, and also skewed in favour of private and faith-based health facilities when compared to public health facilities. these disparities are markedly observed throughout the country and significantly impact patient care and emergency situations. the lack of in-country diagnostic laboratories for the diagnosis of viral haemorrhagic fevers, such as lassa fever and evd, prior to the evd outbreak, confirmed the limitations and challenges the country faced with regard to diagnostics services. prior to the evd outbreak, suspected lassa fever specimens were transported to sierra leone for diagnostic confirmation, which significantly increased the duration of the reporting and turnaround time for case confirmation, especially in a lassa fever-endemic country, such as liberia. limited training of laboratory personnel in molecular diagnostic techniques, and laboratory response in a health emergency, were also a major challenge. disease surveillance on the national level has been limited to a paper-based system. protracted turnaround time and delay in reporting has been a key diagnostic and service delivery challenge in most areas, predominantly in rural settings. surveillance of diseases has been limited to those in humans and not in animals. in the recent evd outbreak, active case findings were primarily based on the assumptions of humans as the portals for sporadic outbreaks or re-emerging infections. evd being a zoonotic disease, its surveillance in animals such as bats, monkeys and other primates should also receive adequate attention from the laboratory diagnostic and public health surveillance systems. furthermore, hunting communities should also be considered as sources of information gathering and active case findings. as with our west african neighbours and other countries in sub-saharan africa, liberians have close cultural ties and extended families across the porous borders. cultural practices, such as eating bush meat (including monkeys, bats, gorillas, etc.), are common cultural norms which could expose communities to zoonotic-associated diseases, such as evd.8 religious practices such as bathing the dead, wake-keeping for the deceased, gathering in groups and rituals for worship, and secret societies for men and women regarding cultural initiation, also predispose communities by creating favourable portals and suitable enabling environments for the transmission of emerging infectious diseases. for these reasons, the development and availability of diagnostic platforms remain paramount for rapid detection and control of emerging and re-emerging diseases. acknowledgements the authors acknowledge the contributions of the incident management system of liberia’s ministry of health, as well as international organisations, in strengthening the laboratory system in liberia. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support none. authors’ contributions the first draft of the manuscript was written by s.b.k., j.b.d., c.l.w. and p.s., and reviewed for scholarly contents by c.b.e., f.k.b., g.t.m. and m.w.s.k. laboratory resources and materials were provided by s.b.k., p.s., c.b.e., f.k.b. and g.t.m. suggestions for modifications were made by j.b.d., c.l.w., p.s., c.b.e., f.k.b., g.t.m. and m.w.s.k., and subsequently incorporated by s.b.k. the final version of the manuscript was approved by all authors. references ministry of health and social welfare, republic of liberia. establishment of a national reference laboratory, june 2008. monrovia, liberia; 2008. ministry of health and social welfare, republic of liberia. national health and social welfare policy, 2011–2021 [document on the internet]. c2011 [cited 2016 oct 08]. available from: http://www.nationalplanningcycles.org/sites/default/files/country_docs/liberia/ndp_liberia.pdf ministry of health and social welfare, republic of liberia. national diagnostics unit: strategic plan, 2011–2013. monrovia, liberia; 2011. united nations development programme. liberia decentralization support program (ldsp) [page on the internet]. c2012 [cited 2016 oct 08]. available from: http://www.lr.undp.org/content/liberia/en/home/operations/projects/democratic_governance/the-liberia-decentralization-support-program--ldsp-.html kennedy sb, neaton jd, lane hc, et al. implementation of an ebola virus disease vaccine clinical trial during the ebola epidemic in liberia: design, procedures, and challenges. clin trials. 2016;13(1):49–56. http://dx.doi.org/10.1177/1740774515621037 massaquoi mb, kennedy sb, tegli jk, et al. fostering collaboration on post-ebola clinical research in liberia. lancet glob health. 2016;4(4):e239. http://dx.doi.org/10.1016/s2214-109x(15)00323-x ministry of health and social welfare, republic of liberia. national laboratory policy. monrovia, liberia; 2011. wood jl, leach m, waldman l, et al. a framework for the study of zoonotic disease emergence and its drivers: spillover of bat pathogens as a case study. philos trans r soc lond biol sci. 2012;367(1604):2881–92. http://dx.doi.org/10.1098/rstb.2012.0228 abstract introduction methods results discussion acknowledgements references about the author(s) deborah k. glencross department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa department of molecular medicine and haematology, charlotte maxeke academic hospital, national health laboratory service, johannesburg, south africa leanne swart department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa department of molecular medicine and haematology, charlotte maxeke academic hospital, national health laboratory service, johannesburg, south africa melanie pretorius department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa department of molecular medicine and haematology, charlotte maxeke academic hospital, national health laboratory service, johannesburg, south africa denise lawrie department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa department of molecular medicine and haematology, charlotte maxeke academic hospital, national health laboratory service, johannesburg, south africa citation glencross dk, swart l, pretorius m, lawrie d. evaluation of fixed-panel, multicolour clearllab 10c at an academic flow cytometry laboratory in johannesburg, south africa. afr j lab med. 2022;11(1), a1458. https://doi.org/10.4102/ajlm.v11i1.1458 original research evaluation of fixed-panel, multicolour clearllab 10c at an academic flow cytometry laboratory in johannesburg, south africa deborah k. glencross, leanne swart, melanie pretorius, denise lawrie received: 19 nov. 2020; accepted: 30 mar. 2022; published: 15 july 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: flow cytometric immunophenotyping is well established for the diagnosis of haematological neoplasms. new commercially available systems offer fixed, pre-aliquoted multi-parameter analysis to simplify sample preparation and standardise data analysis. objective: the beckman coulter (bc) clearllab™ 10c (4-tube) system was evaluated against an existing laboratory developed test (ldt). methods: peripheral blood and bone marrow aspirates (n = 101), tested between august 2019 and november 2019 at an academic pathology laboratory in johannesburg, south africa, were analysed. following daily instrument quality control, samples were prepared for ldt (using > 20 2–4-colour in-house panels and an extensive liquid monoclonal reagent repertoire) or clearllab 10c, and respectively analysed using in-house protocols on a becton dickinson facscalibur, or manufacturer-directed protocols on a bc navios. becton dickinson paint-a-gate or bc kaluza c software facilitated data interpretation. diagnostic accuracy (concordance) was established by calculating sensitivity and specificity outcomes. results: excellent agreement (clinical diagnostic concordance) with 100% specificity and sensitivity was established between ldt and clearllab 10c in 67 patients with a haematological neoplasm and 34 participants with no haematological disease. similar acceptable diagnostic concordance (97%) was noted when comparing clearllab 10c to clinicopathological outcomes. additionally, the clearllab 10c panels, analysed with kaluza c software, enabled simultaneous discrimination of disease and concurrent background myeloid and lymphoid haematological populations, including assessing stages of maturation or sub-populations. conclusion: clearllab 10c panels provide excellent agreement to existing ldts and may reliably be used for immunophenotyping of haematological neoplasms, simplifying and standardising sample preparation and data acquisition. keywords: clearllab 10c; immunophenotyping; fixed-panel; standardisation; multicolour; leukaemia; lymphoma; diagnostics. introduction flow cytometric immunophenotyping is an established methodology for the diagnosis of haematological neoplasms1,2,3,4,5,6 outlined in the world health organization classification of haemopoietic and lymphoid tissues.7,8 published protocols for sophisticated multicolour flow cytometry systems have markedly expanded the scope of routine immunophenotypic testing and improved diagnostic capabilities, allowing for simultaneous multi-parameter examination and documentation of specific neoplastic disease immunophenotypes alongside normal haematological development.9,10,11,12,13,14 rare event populations, however small, are likewise identified to reveal evidence of minimal residual disease.13,15,16 in addition, harmonised approaches, including standardised multi-parameter marker staining, instrument setup and data collection, and automated gating, can reduce test variability, streamline analysis for immunophenotyping and ensure reproducible outcomes between and within centres.17,18 leading experienced and knowledgeable groups have published specific panels and protocols for multi-parametric flow cytometry immunophenotyping method setups, panel selection and data analysis,9,19,20 irrespective of the flow cytometer used. however, multi-parametric flow cytometry is not straightforward18,19,20,21,22,23,24; the setup is complex, requiring expertise in flow cytometry techniques.20,25 therefore, many testing facilities opt to retain their in-house-developed predicate testing methods to meet their local service needs.26 to meet these challenges, beckman coulter (bc) launched a united states food and drug administration-approved, standardised, fixed multicolour clearllab reagent system for leukaemia and lymphoma immunophenotyping in 2019.27,28 the four 10-colour marker panels facilitate the detection of most acute leukaemias and lymphomas. when used with a bc navios instrument, the system offers the advantages of multi-parametric analysis but with simplified pre-prepared marker panels and standardised instrument setup and quality control.28 this study aimed to evaluate the clearllab™ 10c system (including the b-cell, t-cell, m1 and m2 diagnostic tubes)27,28 against our existing predicate method (utilising > 20 in-house, 2–4-colour fluorescence panels and a vast liquid monoclonal reagent repertoire) in our busy, academic flow cytometry laboratory. the laboratory offers leukaemia and lymphoma immunophenotyping services at the national health laboratory service, johannesburg, south africa. a secondary aim of this study was to compare the clearllab 10c system against other routinely employed clinicopathological diagnostic methods used in our site for detecting haematological neoplasms, including morphological and/ or histological review. methods ethical considerations ethics clearance for this study was obtained from the university of the witwatersrand ethics committee (hrec) (m1704129). the hrec waived patient consent as validation for clearllab 10c testing utilised remnant samples previously submitted for routine diagnostic testing. all flow cytometric data analysed were de-identified; data were therefore effectively anonymised, and no patient identifiers were used. study design and site this prospective observational cohort study29 was performed at a national referral flow cytometry laboratory at the charlotte maxeke johannesburg academic national health laboratory service flow cytometry laboratory, johannesburg, south africa. the unit is an academic testing facility with a 300-sample monthly workload, referred from four large sister academic hospitals, other district-level facilities, and other regional centres across the national network. in addition, the laboratory participates in the united kingdom national external quality assessment scheme (sheffield, united kingdom) proficiency testing scheme for leukaemia immunophenotyping (part 1) and leukaemia diagnostic interpretation (part 2).30 specimens bodily specimens, including bone marrow aspirates, peripheral blood, cerebrospinal and pleural fluid, and lymph node tissue, referred to the national health laboratory service in johannesburg, south africa, for leukaemia immunophenotyping by local sister hospitals were included in the study. after diagnostic testing with the laboratory developed test (ldt) was completed, the same sample was tested using the index method (clearllab 10c), provided that sufficient prepared cell concentrate for at least 4 aliquots of concentrated sample, or at least 1 ml of the whole sample, remained. all specimens for clearllab 10c evaluation were selected per the bethesda guidelines.2 the guideline explicitly outlines the appropriate use of flow cytometry for patients with clinically suspected oncological haematolymphoid neoplasms or patients with previously diagnosed oncological haematolymphoid disease enrolled for treatment. samples with no pertinent history or clinical suspicion of haematological disease, older than 24 h, visibly haemolysed or clotted, with insufficient volume at receipt of the sample, or insufficient remnant sample to undertake index testing, were excluded from the study. one hundred and one remnant specimens (from 36 children and 65 adults from any ethnic or racial background) were processed for clearllab 10c from august through november 2019: 82 bone marrow aspirate samples and 15 peripheral blood samples were collected into dipotassium ethylenediaminetetraacetic acid. three bodily fluid samples (two cerebrospinal and one pleural fluid) were submitted; a single lymph node biopsy was collected into saline. sample preparation and cell concentrates for this study, the existing predicate method is the ldt; samples were analysed on the becton dickinson (bd) facscalibur (san hose, california, united states). the index method is the clearllab 10c panel; samples were analysed on a beckman navios flow cytometer (bc, miami, florida, united states). all sample cell concentrates were prepared for flow cytometric analysis according to current local standard operating procedures, irrespective of reagents used. two to four 500 µl aliquots of blood or bone marrow per patient were prepared depending on the initial white blood cell count. the aliquots of the sample were placed in conical centrifuge tubes to which 14.5 ml of 0.899% solution of ammonium chloride (nh4cl) containing 0.084% sodium bicarbonate and 0.0037% ethylenediaminetetraacetic acid (merck, darmstadt, germany) was added to lyse the red blood cells. this was allowed to stand for 15 min, followed by a short 3-min spin at 3000 rpm. a further 5-min incubation time in the nh4cl solution, followed by a 3-min spin, was performed if the red cells were macroscopically visible on the cell pellet. samples were subsequently washed three times with a commercial phosphate-buffered saline (pbs) solution at ph 7.3 ± 2 (oxoid ltd, basingstoke, united kingdom) containing 0.09% sodium azide (merck, darmstadt, germany) and 0.2% bovine serum albumin (biowest, nuaille, france). cell pellets were resuspended with 1 ml pbs. white cell count was obtained for each sample using 100 µl of the cell concentrate and 100 µl of bc flowcount™31,32 beads (bc, miami, florida, united states) in a 1 ml pbs solution on the navios flow cytometer. the volume of cell concentrate estimated to contain ~1 × 106 cells per aliquot was calculated, and this predetermined cell concentrate volume was added to each labelled marker tube of the ldt panel first. remnant cell concentrates, or cell concentrates harvested from whole remnant samples, were aliquoted into the clearllab tubes. each sample aliquot was incubated in the dark at room temperature (22 °c) for 15 min, followed by a final wash in pbs. the cell pellet was reconstituted with 700 µl of pbs and immediately run on either the facscalibur flow cytometer (bd biosciences, san jose, california, united states) for the ldt or the navios flow cytometer (bc, miami, florida, united states) for the clearllab samples. whole samples (remaining after cell concentrate preparation) were stored on the bench at room temperature (~22 °c) during testing; after analysis was completed, remnants of both the whole sample and the prepared samples were refrigerated at 4 °c. flow cytometer quality control and immunophenotyping laboratory developed test method before march 2020, the ldt used the bd facscalibur™ flow cytometer. daily quality control for the ldt on the bd facscalibur included assessment of background contamination, carryover, acquisition and analysis of manufacturer-recommended 3-colour and apc calibrite beads (bd biosciences, san jose, california, united states) and acquisition and analysis of immunotroltm process control (bc inc., brea, california, united states), using four monoclonal antibodies namely cd45 (percp), cd3 (apc), cd14 (fitc) and cd13 (pe). liquid monoclonals were used according to the manufacturer specifications and manually pipetted individually to constitute 2–4-colour assembled panels using varying combinations of cell markers chosen by attending pathologists according to the merits of the patient’s presenting case history (table 1). the patients’ samples were acquired on bd facscalibur™ using cellquest™ software (bd biosciences, san jose, california, united states); the instrument typically stops counting at 5000 events. in the event of a paucicellular sample, the tube would run for the maximum time permissible ~300 s, to ensure as many cells as possible could be counted, but consequently with total variable events counted. listmode (.fcs) data files were analysed and interpreted by consultant hematopathologists using bd paint-a-gate™ software (bd biosciences, san jose, california, united states). table 1: summary of the laboratory developed test panels and monoclonal antibody combinations and respective fluorochromes used during routine diagnostic workup at the charlotte maxeke johannesburg academic laboratory in johannesburg, south africa, august 2019 – november 2019. clearllab 10c panel method before starting the study, bc provided training for the navios instrument, including application setup, control runs and panel acquisition. colour compensation for clearllab testing was performed according to manufacturer specifications.28 before commencing the validation study, an initial reproducibility study was performed (data not shown), utilising both the normal and abnormal controls (10 for each of the respective clearllab tubes; see table 2 for details of markers included in the clearllab tubes). the assay values obtained were within the published expected ranges of the package inserts; the reproducibility of the process controls met the manufacturer’s percentage coefficient of variation (%cv) of ≤ 10% for all targeted populations and repeatability of ≤ 20%cv for all targeted populations.33 daily internal quality control for the navios flow cytometer included a daily background count (with locally established target values of < 100 events/100 s) and daily carryover (locally established target value < 1% carryover). daily flow check pro (target hpcv < 2.0% for fl1-fl5, < 3% for fl7-fl8 and < 4% for fl9-fl10) was used to verify optical alignment and fluidics (bc, lismeehan, ireland) on the navios, as well as standardise the optical and fluorescence settings (flow-set pro, bc, lismeehan, ireland). clearllab ‘normal’ and ‘abnormal’ process control cells (bc, lismeehan, ireland) were used to verify sample processing, acquisition and analysis against established package insert values and assess repeatability over time (data not shown). weekly reproducibility was ensured through locally established target values for the bead count rate31,32 and cell counts.33 the sensitivity and specificity of the clearllab were expected to match or exceed the published bc clearllab 10c acceptance limits of > 70% sensitivity and > 80%, specificity. table 2: reagents used with the clearllab 10c panels verified at the charlotte maxeke johannesburg academic laboratory in johannesburg, south africa, august 2019 – november 2019. samples were prepared according to manufacturer specifications but with a modification; the iotest3 lysing solution was replaced with an nh4cl solution. following red cell lysis and wash steps, prepared ~106 cell aliquots were added to each of the clearllab™ 10c b, t, m1 and m2 tubes (table 2). after a 15-min incubation in the dark, tubes were again washed. all samples were acquired on a bc navios™ flow cytometer. at minimum, 50 000 to 100 000 events were acquired on all samples. data files were analysed offline by trained attending hematopathologists using bc kaluza c™ software (bc, miami, florida, united states). assessment of immunophenotype immunophenotypes were established using appropriately applied, pathologist-defined, sub-population gates that were unique to each case analysed. for the ldt data, a combination of light scatter and cd45 or cd19 gating was used to identify target cell (neoplastic) populations. for the clearllab 10c, initial gating included isolation of singlets followed by primary gating on cd45 positive events, with secondary gating applied to the identified target population. the specific antigen expression was documented for both the haematological neoplasm present as well as the normal haematological populations in the background (as an internal control of marker expression), based on the expression of all the available markers (antigens) across the four panels included in the clearllab 10c panel set. the specific presence or absence of antigens, and the respective specific intensity of staining of each antigen, was used to establish the diagnosis of specific subtypes of leukaemia or lymphoma per the world health organization’s classification of haemopoietic and lymphoid tissues7,8. also, the clinical history, other laboratory parameters (e.g. full blood count and white blood cells differential counts), and bone marrow aspirate morphology were taken into account. statistical analysis data were collected and collated into microsoft excel (redmond, washington, united states) spreadsheets for data analysis. sensitivity outcomes were calculated; the clinical accuracy of the qualitative flow cytometric results (concordance of diagnosis as normal or abnormal) between the ldt and clearllab 10c outcomes were assessed for samples tested, and a contingency sensitivity (as the percentage of patients with malignancy) and specificity (the percentage of patients with no malignancy) table was constructed. in addition, the positive predictive value, as the probability of malignant outcomes with an abnormal immunophenotype detected, as well as negative predictive value as the probability of non-malignant outcomes in a patient with no disease, were also calculated. thereafter, the clearllab results of each patient were compared to their clinicopathological or haematological diagnosis based on a similar approach by hedley et al.28 to demonstrate the objectivity of a comprehensive clearllab 10c diagnostic approach. clinical concordance was achieved using the categories of ‘haematologically malignant’ or ‘haematologically non-malignant’, as suggested by the presence or absence of an abnormal immunophenotype. in this comparative analysis, both false positives and negatives, and true positives and negatives, as well as both positive predictive value and negative predictive value outcomes, were reported using clearllab outcomes as the objective standard. results daily quality control the daily quality control results over the study period confirmed that daily background counts were consistently < 100 events in 100 s, carryover < 1% and flow check pro results acceptable with hpcv < 2 for fl1 to fl5, hpcv < 3 for fl6 to fl8 and < 4 for fl9 and fl10 for the days that the patient samples were tested; flow-set pro also confirmed acceptable fluorescence stability, with %cvs < 5% for the blue and violet laser (fl1–fl5 and fl9–fl10) and < 8% for the red laser (fl6–fl8). further weekly cd4 reproducibility studies on the navios confirmed that the flow count rate and absolute counts had a %cv < 5% for the duration of the validation. precision and accuracy of clearllab controls were in agreement with the manufacturer’s claims for repeatability and accuracy of clearllab control cells (i.e. control cells assay values fell within the package insert limits and %cvs were consistently < 10%). comparing clearllab 10c against predicate laboratory developed test one hundred and one bodily specimens from 36 children and 65 adults, irrespective of ethnic or racial background, were enrolled into the study. these included: 82 bone marrow aspirates, 15 peripheral blood samples, one pleural and two cerebrospinal fluids, and one lymph node biopsy. sixty seven of the 101 cases tested had haematological malignancy, while 34 cases had no evidence of malignancy despite a clinical suspicion (figure 1). among three bodily fluid samples tested, two cerebrospinal fluid samples showed evidence of tumour infiltration, one with t-cell acute lymphoblastic leukaemia, and another infiltrated with acute myeloid leukaemia; a separate (third) pleural fluid sample showed infiltration by follicular lymphoma, confirmed with cd10 expression and other markers suggesting b-cell maturity. the lymph node biopsy had a large t-cell lymphoproliferative disorder; additional ldt testing revealed an anaplastic large cell lymphoma (confirmed with cd30 expression). a diagnostic concordance of 100% was noted between the clearllab outcomes and the ldt, revealing identical diagnostic outcomes (figure 2). these findings exceeded the manufacturer acceptance limits of > 70% sensitivity and >80% specificity for the clearllab testing system. all markers met the manufacturer’s specifications and claims, specifically those not typically included in first-line ldt testing, including cd16, cd38, cd56, cd64, cd123, cd200 and tcrγδ. cases tested and reviewed using clearllab 10c were shown to have essentially similar immunophenotypes observed with the use of the ldt, leading to the same overall diagnostic outcome. in certain categories, however, diagnostic specificity matched diagnostic outcomes exactly, that is, there was 100% concordance of measured markers. such examples include cd19/cd5 dual positivity for diagnosis of chronic lymphocytic leukaemia or cd19/cd10+ dual positivity for a common acute lymphoblastic leukaemia. although overall diagnostic concordance was 100%, the four compact clearllab panels offered timely simultaneous, collated, and concise interpretation for both tumour and normal background populations. this improved efficiency was difficult to achieve, during interpretation and analysis of the predicate ldt, where limited 2–4-colour marker combinations had to be interpreted across in at least 10–12, but often as many as 20, separate panels. figure 1: comparison of diagnostic immunophenotypic outcomes of cases tested by laboratory developed test and clearllab 10c at the charlotte maxeke johannesburg academic laboratory in johannesburg, south africa, august 2019 – november 2019. figure 2: part i: comparison of diagnostic immunophenotypic outcomes of cases tested by the predicate laboratory developed test and clearllab 10c at the charlotte maxeke johannesburg academic laboratory in johannesburg, south africa, during the period from august 2019 – november 2019. comparing clearllab 10c results with clinicopathological outcomes the overall diagnostic sensitivity and specificity were comparable for the outcomes reported on corresponding clinicopathological bone marrow aspirate or trephine findings (figure 3). the clearllab 10c system reliably identified and excluded all diseases and, in two cases, detected the presence of a b-cell lymphoproliferative neoplasm that was not detected morphologically on the corresponding bone marrow aspirate. figure 3: part 2: contingency table34 comparing the clearllab 10c test method (as an objective standard) versus clinicopathological outcomes reported at the charlotte maxeke johannesburg academic laboratory in johannesburg, south africa, august 2019 – november 2019. discussion this study aimed to verify the performance of a new commercially available, fixed pre-titrated multicolour flow cytometric panel system, the clearllab 10c, against an existing ldt that required multiple manual tube setups (often exceeding 10–12 tubes per patient) and required manual titration and separate addition of reagents from a large repertoire of liquid monoclonal antibodies. this study has revealed that the clearllab 10c system reliably enables full discovery of most leukaemias and lymphomas, with full concordance to our existing in-house ldt panels. excellent concordance was also reported against corresponding clinical-pathological outcomes in a subsequent analysis that looked at clearllab 10c outcomes against reported morphological findings. it was expected, however, that the clearllab 10c system would have broadly similar clinical outcomes to that generated by the ldt, as both systems used flow cytometry and the same markers to derive outcomes. the multicolour capability of the clearllab system versus 2–4-colour panel ldt is what sets the two methods apart; compact access to 27 markers in just four panels, with trackable markers between analyses, is a game-changer, certainly for our site, and a notable improvement over our previous approach to first-line immunophenotypic workup. specifically, the concise multi-parameter format of the m1, m2, b and t-cell tubes, especially with additional markers that were not included in a typical first-line investigation in our laboratory ldt, including cd16, cd38, cd56, cd64, cd200, cd123 and tcrγδ, has enabled more precise and detailed immunophenotypic insights. myelomonocytic markers in the m1 tube (cd11b, cd14, cd16, cd64 and hla-dr) or myeloid markers (cd13, cd15, cd33, cd117 and hla-dr) in the m2 tube proved to be a distinct advantage over the ldt to identify and distinguish monocyte from granulocyte lineage, discern all stages of maturation, and identify asynchronous myelomonocytic maturation, including characterising immature forms and blasts. the combination of cd11b, cd13, cd14 and cd16, notably with cd64, facilitated a clear distinction of monocytic maturation and monocyte subsets while also providing insights into granulocytic differentiation. the inclusion of cd16 was especially useful to define both early and late maturation of granulocytes; clustered bright cd16, cd11b and cd13 associated with weak cd10 expression allowed for ease of identification of neutrophils which was helpful in discerning haemodilution of a bone marrow sample. the b and t-cell tubes also enabled simultaneous distinction of lymphoid subsets and provided concise immunophenotypic information about normal or aberrant lymphoid maturation stages. coincident aberrant loss or asynchronous gain of specific markers was easier to discern using the clearllab 10-c panels; in contrast to our 2–4-colour ldt, multiple markers included together in a single clearllab panel provided clearer evidence of simultaneous antigen losses or gains of markers. the specific combination of cd45, cd19, cd10, cd20, cd34 and cd200 in the b-cell tube, with hla-dr, cd38 and cd19 in the m2 tube, usually reserved for specific disease-profiled ldt panels in our previous practice, has also markedly improved our ability to discriminate early normal b-cell precursors from later mature polyclonal b-cells in first-line workup; cd38-bright expressing, cd45 negative plasma cells are also easily recognised upfront. further, we have found that cd123, along with cd19, cd34, cd38 and hla-dr, identifies b-cells in the m2 tube, which is especially valuable to discern and document discordant expression in normal and abnormal precursor b-cells.35,36 concurrent assessment of most t-cell markers in the t-cell tube was valuable for discerning both immature and mature t-cell lymphoproliferative disorders. natural killer or cytotoxic cells were easily identified with cd7, cd56 or tcrγδ in the t-cell tube and confirmed with expression of cd16 and cd7 in the m1 tube. from a marker reproducibility perspective, and in stark contrast to our ldt method, the primary backbone markers, cd45 and cd34, included as all four tubes with additional secondary backbone markers, namely cd5, cd7, cd10, cd13, cd38 and hla-dr, each repeated across at least two tubes, proved to be enormously helpful to track populations between the four clearllab analyses. another distinct advantage was that substantively fewer cells were needed to achieve a relatively extensive workup in a paucicellular sample using clearllab. often during ldt use, our site would be unable to complete full immunophenotypic workup in paucicellular samples, especially in paediatric and bodily fluid samples like cerebrospinal fluid. careful consideration was previously given to implementing the established state-of-the-art euroflow system9,20,37 that is widely used in europe. however, the complex euroflow demands manual assembly of 8-colour marker panels21,38, uses a repertoire of monoclonal antibodies and requires multiple titrations and pipetting protocols. these pose a significant barrier to use in our relatively skills-scarce environment, considering the technical effort needed and potential for error during the dispensing of monoclonal reagent encountered in our existing 2–4-colour ldt. another factor that hindered the implementation of euroflow was the unit’s heavy workload. the unit is a 24-h cd4 laboratory processing up to 12 000 samples per month39,40; the technical staff managing the cd4 and hiv immunology bench also support the leukaemia bench. thus, although the staff are competent to run the cd4 services using pre-titrated and pipetted reagents and standardised, automated testing procedures,40,41 they are not primarily trained in flow cytometry. therefore, from a technical perspective, the compact, pre-titrated clearllab 10c fixed tube reagents27,28 with automated procedures and standardised instrument setup, allows our laboratory to undertake sophisticated multicolour panel testing confidently while still addressing local challenges. implementation can significantly reduce leukaemia sample preparation time and improve the efficiency and quality of leukaemia testing in our site. the difficulties faced in our centre are not unique though. flow cytometry outcomes vary even in far better-resourced sites.25 despite that testing is undertaken by dedicated and trained flow cytometry laboratory personnel or that there is prescribed standardisation of multicolour methods, deviation from standardised protocols for instrument setup and colour compensation,25,42 as well as differences in sample preparation, gating strategies and data interpretation, are reported.25 the clearllab 10c system can enable our centre to overcome such challenges as it is implemented with standardised flow cytometer multicolour instrument setup and daily prescribed quality control, effectively providing predetermined, user-independent operation in the navios flow cytometers. the capability for standardised kaluza c™ hierarchical immunophenotypic analysis of four fixed tubes can also harmonise consistent gating approaches and reporting strategies between reporting pathologists, which is especially important for standardised disease interpretation. commercial lyophilised panels, like the clearllab 10c system evaluated in this study, are also expected to considerably reduce the technical effort and time needed for sample preparation and analysis. for example, the implementation of clearllab will drastically reduce sample preparation and data acquisition time for our first-line investigation of patient samples and reduce the number of (variable) ldt monoclonal reagent panels prepared from around 20 (table 1) to just four standardised panels (see table 2) with an estimated reduction in sample preparation from 1.5 h to ~30 min. a notable advantage of the clearllab tubes is that antibodies are pre-titrated so that no monoclonal reagents will be added; this is in striking contrast to our previous ldt practice, where up to 40 or more individual monoclonals had to be pipetted into multiple tubes, in varying combinations with the potential of multiple errors (see table 1). our previous large inventory of liquid-reagent monoclonal antibodies also required considerable staff time to manage procurement and stock-taking. pre-dispensed reagent obviates the need for time-consuming flow cytometric titration experiments as well. limitations firstly, while there was 100% concordance between the ldt and clearllab outcomes, only 101 case reviews were included. secondly, although the clearllab instrument auto-setup makes managing daily quality control and review of complicated multicolour analyses much quicker, easier and standardised, the auto-setup itself still requires training of operators. attending pathologists also need training and are required to familiarise themselves with kaluza software and patterns of normal and aberrant haematological population maturation. thirdly, the clearllab 10c m1, m2, t and b-cell panels allow for reasonably extensive discovery of most leukaemias, but there are currently no supplementary ‘second-line’ clearllab investigative panels available7,8 to discern markers needed for full diagnoses; for example, cytoplasmic studies to discern myeloperoxidase or nuclear terminal deoxynucleotidyl transferase (tdt) expression, cd41, cd42b and cd61 for platelet marker investigation, or extended b-cell lymphoproliferative workup, including amongst others cd11c, cd23, cd30, cd43, cd79b and cd123 amongst others, is still performed in our laboratory to enable the world health organization classification of haematological disease. some commercially produced, pre-dispensed, fixed panels marketed for in vitro rare event use27,38,43,44,45,46,47,48 could fill the gap. such products could potentially be modified to extend the diagnostic repertoire of the clearllab 10c and provide for multicolour second-line immunophenotypic investigation, especially for b-cell lymphoproliferative disorders and plasma cell dyscrasias, but this needs further study. conclusion in this study, there was excellent concordance between our ldt and the multicolour bc clearllab 10c panels, with 100% sensitivity and specificity recorded against existing ldt methods. there was also excellent concordance of clearllab 10c reporting to documented clinicopathological outcomes. the clearllab 10c panels, with manufacturer-standardised setup for colour compensation, appropriate quality control and data acquisition on a navios flow cytometer, provides concise and comprehensive multi-immunophenotyping for typical leukaemias and lymphomas encountered during routine service delivery while substantially simplifying and standardising sample preparation and data acquisition. acknowledgements we would like to thank the leukaemia immunophenotyping laboratory staff, in particular mrs merriam machaba for preparation of samples used for this evaluation study. competing interests the authors declare they have no financial or personal relationships that may have inappropriately influenced the writing of this article. authors’ contributions d.k.g. led the study and compiled the article for publication. l.s. and m.p. analysed the cases and recorded the data for analysis comparison. d.k.g. undertook reporting of the validation outcome. d.l. and d.k.g. oversaw technical aspects and the setup of new protocols on the flow cytometer. all authors discussed the study findings. d.k.g. wrote and finalised the final draft. all authors reviewed and contributed to the final manuscript. sources of support funding for this study was made possible with research incentive funds accrued to d.k.g., awarded to her by the university of the witwatersrand. data availability additional data not represented in this article are available from the corresponding author, d.k.g., upon reasonable request. disclaimer the views and opinions expressed in this article 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of minimal residual disease in b cell acute lymphoblastic leukemia using an eight-color tube with dried antibody reagents. cytometry b clin cytom. 2019;96(2):158–163. https://doi.org/10.1002/cyto.b.21766 abstract background on laboratory systems in africa determinants of remaining gaps in national laboratory network capabilities current normative standards for national medical laboratory networks aim design of the scorecard selection of core capabilities and components development of indicators and definition of capability levels purpose of the labnet scorecard assessment scoring system and options for data analysis assessment process validation of the labnet scorecard: lessons learnt anticipated utilisation and benefit of the labnet scorecard assessment perspectives and conclusions acknowledgements references about the author(s) pascale ondoa amsterdam institute for global health and development (aighd), department of global health, academic medical center, amsterdam, the netherlands tjeerd datema royal tropical institute biomedical research (kit), amsterdam, the netherlands datos, amsterdam, the netherlands mah-sere keita-sow african society of laboratory medicine (aslm), addis ababa, ethiopia jean-bosco ndihokubwayo world health organization regional office for africa, brazzaville, republic of congo jocelyn isadore association of public health laboratories, silver spring, maryland, united states linda oskam royal tropical institute biomedical research (kit), amsterdam, the netherlands datos, amsterdam, the netherlands john nkengasong division of global hiv and tb, international laboratory branch, us centers for disease control and prevention, atlanta, georgia, united states kim lewis association of public health laboratories, silver spring, maryland, united states citation ondoa p, datema t, keita-sow m-s, et al. a new matrix for scoring the functionality of national laboratory networks in africa: introducing the labnet scorecard. afr j lab med. 2016;5(3), a498. http://dx.doi.org/10.4102/ajlm.v5i3.498 lessons from the field a new matrix for scoring the functionality of national laboratory networks in africa: introducing the labnet scorecard pascale ondoa, tjeerd datema, mah-sere keita-sow, jean-bosco ndihokubwayo, jocelyn isadore, linda oskam, john nkengasong, kim lewis received: 30 may 2016; accepted: 06 aug. 2016; published: 31 oct. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: functional national laboratory networks and systems are indispensable to the achievement of global health security targets according to the international health regulations. the lack of indicators to measure the functionality of national laboratory network has limited the efficiency of past and current interventions to enhance laboratory capacity in resource-limited-settings. scorecard for laboratory networks: we have developed a matrix for the assessment of national laboratory network functionality and progress thereof, with support from the african society of laboratory medicine and the association of public health laboratories. the laboratory network (labnet) scorecard was designed to: (1) measure the status of nine overarching core capabilities of laboratory network required to achieve global health security targets, as recommended by the main normative standards; (2) complement the world health organization joint external evaluation tool for the assessment of health system preparedness to international health regulations (2005) by providing detailed information on laboratory systems; and (3) serve as a clear roadmap to guide the stepwise implementation of laboratory capability to prevent, detect and act upon infectious threats. conclusions: the application of the labnet scorecard under the coordination of the african society of laboratory medicine and the association of public health laboratories could contribute to the design, monitoring and evaluation of upcoming global health security agenda-supported laboratory capacity building programmes in sub saharan-africa and other resource-limited settings, and inform the development of national laboratory policies and strategic plans. endorsement by the world health organization regional office for africa is foreseen. background on laboratory systems in africa in spite of current funding and partnership opportunities to strengthen laboratory capacity in resource-limited settings, poor access to quality laboratory testing continues to lead to misdiagnosis, inappropriate treatment,1 increased morbidity and mortality, and an inability to determine the true prevalence of diseases.2 unreliable test results generate mistrust in laboratory services by clinical staff, aggravating the misuse of drugs such as antibiotics and causing undue cost to the patients.3 in addition, delays in laboratory testing confirmations complicate the public health response and impede the control of epidemic diseases.4,5,6 a national laboratory network can be defined as a collaborative group including all (public, private and private not-for-profit) medical laboratories within a country. the networks are typically organised in three to five-tiered structures of testing facilities operating under common principles and procedures with tier-specific roles, responsibilities and functions.7 the national laboratory network supports the entire health system in accessible, high-quality and efficient testing for individual patient care and public health needs.7 peter et al. highlight that the proper functioning of the national laboratory networks requires central management and direction through policies, regulatory oversight and coordination of operational functions,8 but these components are missing in many networks. because the laboratory is a key component of the health system and is recognised as one of the six essential core functions of public health identified by the us centres for disease control and prevention, for which strengthening would have the widest influence on the health system,9 it is essential to aid countries to build laboratory capacity to detect, report and respond to public health events. in the early 2000s, the combined efforts of global health partners (us centres for disease control and prevention, clinton health access initiative, global alliance for vaccines and immunization, global fund), national and international institutions (world health organization [who] and governments) have led to several ambitious programmes to strengthen laboratory systems in sub-saharan africa. this momentum is illustrated by the numerous landmark meetings on laboratory policy that took place between 2008 and 2012,10,11 and which provided a framework for laboratory system strengthening and development in sub-saharan africa. other key accomplishments are the creation of the world health organization regional office for africa based stepwise laboratory improvement towards accreditation (slipta),10 the development of the strengthening laboratory management toward accreditation (slmta)12 training programme, and the launch of the african society of laboratory medicine (aslm), three important initiatives for the improvement of laboratory systems in africa.11 in the same period, substantial funding has been dedicated to integrating laboratory services, developing laboratory policy and strategic planning for tiered laboratory networks, implementing quality system improvement schemes, and increasing the laboratory workforce. despite the efforts to strengthen laboratory systems and networks and the remarkable level of resources made available, serious gaps remain. such insufficiencies include shortages in skilled and trained professionals, inadequate and poorly-maintained infrastructures and equipment, inconsistent supply of reagents and consumables, lack of clear national policies and poor leadership. in practice, laboratory diagnostics are unavailable for most fevers13 and accurate laboratory results are lacking to support evidence-based treatment of diseases, surveillance and outbreak investigations. these deficiencies were dramatically exposed during the recent ebola epidemic, which claimed 11 323 victims in sierra leone, guinea and liberia,14 and underscore the lack of preparedness of many countries to detect, respond to and prevent health threats. in the context of increasing international travel and trade and of emerging and re-emerging infectious diseases, dysfunctional laboratory systems and networks of africa constitute a serious barriers to reducing mortality and morbidity and to achieving international regulation (ihr) 2005 goals for international and collective response to outbreaks. failure to address the chronic weaknesses of african laboratory systems represents an impediment to achieve several of the united nations 2030 sustainable development goals.15 determinants of remaining gaps in national laboratory network capabilities the causes of persisting deficiencies of the laboratory systems and network are multifactorial and include: the influence of vertical programmes;16 insufficient strengthening of general national laboratory networks;8 poorly-implemented laboratory policies and improvement strategies;17 and a lack of clear indicators to measure the status of laboratory systems and networks18,19 in current standardised tools available to assess laboratories. these tools are either facility-oriented (e.g., who laboratory assessment tool facility assessment,20 who slipta21) and/or address individual aspects of laboratory system,22 and/or are narrowed to one disease,23 and/or are not specific to laboratories,24 and/or measure absence of key components without sufficiently characterising the discrete levels of laboratory network capabilities to guide performance improvement initiatives.25 furthermore, system or network standards may not be adequately defined to set actionable objectives.25 the inability to assess benchmarks of laboratory systems and networks performance hides the reality of the problem, weakens advocacy efforts and makes progress impossible to measure. collectively, these observations call for the development of a novel standardised framework capable of capturing metrics relevant to the laboratory network performance and measuring progress toward a more comprehensive set of laboratory systems standards. current normative standards for national medical laboratory networks normative standards, directives and recommendations guiding the development of national laboratory systems and networks include: requirements for public, international and global health;26 access to primary healthcare; disease control at the human, animal and ecosystem interface;27,28 surveillance of diseases;29 and directives for sustainable, integrated and quality laboratory services.30 in addition, the recently-launched global health security agenda (ghsa) provides additional resources and recommendations to accelerate the implementation of the ihr 2005, especially in terms of infectious disease control. the ghsa objectives specifically underscore national laboratory systems and laboratory-based disease surveillance systems, comprehensively cover essential public health and clinical functions and highlight the need for medical laboratory services to collaborate closely with other sectors within the ministry of health and other agencies.31 essential laboratory requirements per key normative standards and regulation are provided in table 1. table 1: components and targets (stage 5) per core capabilities. conceivably, the degree of availability of resources (material, human and technical) and capacity to conduct the necessary improvements toward all key normative standards correlates with the degree of the national laboratory network’s capability to carry out essential public health and clinical functions. aim ghsa-supported programmes strive to support the implementation of health security preparedness as a way to comprehensively strengthen laboratory capacity for all essential public health and clinical functions.31 an important step to further advance public health laboratory systems toward health security is to conduct objective evaluations of each country’s national laboratory network preparedness.32 such assessments serve as a basis to develop targeted enhancement plans and to measure progress of the national laboratory network capability to support the prevention, detection and response to health threats. to such end, the african society for laboratory medicine (aslm) commissioned the development of a standardised methodology to assess progress and changes in national laboratory networks’ performance toward global health security targets. the assessment tool was designed through a collaborative effort between the amsterdam institute for global health and development, the association of public health laboratories (aphl) and the royal tropical institute – biomedical research of the netherlands (currently datos). terms of reference: the labnet scorecard the desired features for a standardised tool to assess national laboratory network functionality against current normative standards include: the overarching assessment of a full set of core capabilities required to achieve all key laboratory standards of global health security. clear indicators describing the maturation of each core capability according to a linear scale of increasing functionality. a practical roadmap of what needs to be implemented for the country to achieve next levels of functionality for each core capability. this could serve as the basis to define targeted improvement strategies at regional or at country level that can leverage advancement toward global health security targets and/or the development of well-informed national laboratory policies and plans. the possibility to provide a quick visual representation of national laboratory networks functionality within a country and across countries, which can serve advocacy efforts toward high level policymakers. the capability maturation model (cmm)33 was selected as the most appropriate format to design the national laboratory network assessment tool. cmm is a structured and sequential approach to evaluation34 which provides a matrix describing an evolutionary path from ad hoc, chaotic processes to mature, disciplined processes. the generic framework can be applied to the assessment of any system35,36 with a scorecard describing five levels of capability, generally used to evaluate system maturity.34 design of the scorecard the scorecard for national laboratory network functionality (hereafter referred to as the labnet scorecard) was designed as a cmm and began with: (1) the definition of the laboratory network core capabilities necessary to conduct clinical and public health functions supporting global health security targets; and (2) the description of the maturation stages for each capability in such a way that improvements at each stage provide the foundation on which to build improvements undertaken at the next stage. hence, the scorecard can identify deficiencies and guide advancement of the laboratory network. core capabilities are typically described by one to four criteria or components against which indicators are developed. selection of core capabilities and components core capabilities were defined as the overarching functions of the country national laboratory network to detect, assess, notify and respond to health events in accordance with the main normative standards guiding the development of national laboratory network – ihr; ghsa; the who guidance to establish national health laboratory systems;37 the who integrated disease surveillance and response;38 the who global strategy for the containment of antimicrobial resistance (amr);39 and the who regional guide for establishing laboratory-based surveillance of amr40 – under the one health concept27 and following the recommendations of the maputo declaration for strengthening laboratory systems in resource-poor settings.30 fifty essential functions described in at least two normative standards or guiding documents were listed and grouped into nine main themes, which served as core capabilities to be assessed for the national laboratory network cmm. one to three components best describing each core capability were drawn from the list of most recurrent functions described in normative documents. the nine core capabilities identified across the main relevant guidelines, their corresponding components and targets are shown in table 1. development of indicators and definition of capability levels indicators characterising the functionality of each component were developed in the form of questions, to measure the maturity of the system along three key dimensions: inputs: including infrastructure, commodities, technology, equipment, people, legislation, policies, and finances. processes: including networking, procedures for processes, implementation, expansion, coverage, quality and integration of laboratory network services. measurement: including targets, indicators, output, outcomes, cost-efficiency, data reporting systems, data collection and data use to improve the quality of the laboratory network. the definition of capability levels in relation to input, process and measurement is provided in figure 1. given the anticipated weaknesses of laboratory networks in some areas, a stage 0 was incorporated to match situations in which no key attributes are present in a given core capability. in order to obtain sufficient granularity, each component was broken down in to one to four questions with responses grading from 0 to 5, describing the situation along the capability maturation scale. the measurements are mostly descriptive and qualitative, but some incorporate a quantitative dimension. the example of core capability 3 (coverage and rapid response) and its associated components and indicators broken down into questions with graded responses is shown in figure 2. figure 1: maturation stages proposed for each core capability (or function) of the national laboratory network. figure 2: example organisation of core capabilities, components and indicators on labnet scorecard. purpose of the labnet scorecard assessment the labnet scorecard assesses a country’s national laboratory network functionality to support the implementation of the ihr 2005 and to reach the ghsa targets of preventing, detecting and responding to infectious disease threats. a regional and international evaluation process is foreseen in ghsa target countries in africa and asia, under the coordination of aslm and aphl, two organisations mandated to implement the ghsa, as a way to accelerate the achievement of the ihr 2005. the labnet scorecard assessment is voluntary and is applied through a series of external evaluations that will allow a country to measure the progress of the national laboratory network towards the health security targets. the first evaluation will provide a baseline measurement of the national laboratory network capabilities and guide the design and implementation of laboratory strengthening initiatives. subsequent evaluation(s) will measure progress made and ensure improvements in capacity are sustained. scoring system and options for data analysis based on the current country situation, each question receives a unique score from 0 (key attribute(s) completely absent) to 5 (key attribute(s) compliant with international standards), describing the level of maturity. key definitions and rationale behind the questions are incorporated into the scorecard to facilitate the scoring process. without achievement of all attributes at prior capability level, the national laboratory network cannot achieve the next level. the responses should be supported by documentation whenever possible. the score attributed to each indicator can be used in two ways, as described below. percentage-based scoring system a ratio can be calculated dividing the total number of points scored for all questions within a core capability by the maximum possible score (total number of questions x 5). the percentages are useful in estimating the overall degree of advancement of each core capability toward the standards (figure 3a). however, the percentages provide little information on the extent to which the various components within a core capability mature and work together towards a functional laboratory network. figure 3: summary of country results using percentages and color-codes. color-scoring system of component and core capabilities according to the cmm methodology, up to two additional layers of scores can be calculated for each of the component and the core capabilities. each component can be assigned a score, which corresponds to the lowest score achieved by any question within that component. this tends to bring the overall results down the scale of maturity, while allowing the identification of critical weaknesses that prevent the system from achieving higher stages. similarly, each core capability can be assigned a score, which corresponds to the lowest score achieved by any component within that core capability (figure 3b). any core capability or component with a low score offers the opportunity for drilling down to identify specific component(s) and/or specific question(s) which have not been adequately addressed by the country and require follow-up actions. color-coded graphs allow for a straightforward visual representation of scoring (figures 3b and 3c). assessment process in order to ensure the optimal standardisation of the assessment process, a pool of francophone and anglophone assessors from africa and the united states were trained to use the labnet scorecards in a joint aslm and aphl effort and a detailed scorecard instruction manual was developed (supplementary material). the first stage of the evaluation is the country self-reporting for all the indicators across the nine core capabilities of the labnet scorecard. a national committee led by the country contact point for the national laboratory network and including a wide representation of stakeholders under the one health concept will complete the self-evaluation. a list of typical committee members is provided in box 1. box 1: list of national committee members. a labnet scorecard evaluation team comprising three international experts, fluent in the country language, reviews the information prior to a one-week visit to the country. the visit is comprised of an initial two-day workshop during which the evaluation team members review and validate the pre-filled data with the national committee, gather information explaining the scores and collect documented evidence. during the next three days, the evaluation team visits representative laboratory facilities at each tier level. the visits also include key departments, such as the national health data unit and the central procurement and distribution facility for laboratory consumables. site visits aim to verify information provided at the central level and are not structured laboratory facility assessments. after completing the country visit, the evaluation team drafts a report to identify scores levels for each indicator, and to describe the status of each component and core capability. based on the explanations provided to justify the scores assigned to each indicator, the report also includes a root-cause analysis, supporting the evidence-based selection of strategies with highest leverage potential for comprehensive laboratory improvement. upon validation by aslm and/or aphl, the report is shared with the host country and with other relevant stakeholders, as permitted by the country. validation of the labnet scorecard: lessons learnt following the initial development phase, the labnet scorecard underwent revision, consolidation and validation, scrutinising the relevance, order of the questions, logic of scores, and clarity of phrasing. in total, more than 40 persons provided input on the labnet scorecard during: the freetown meeting organised by aslm during a breakout session attended by 25 experts (freetown, sierra leone, october 2015); a meeting of aphl us senior laboratory experts on system assessment of national laboratory network (silver spring, md, us, february 2016); and a meeting of 16 african senior laboratory facility and laboratory system assessors (dakar, senegal, february 2016). the feasibility of using the revised and consolidated version of the labnet scorecard was tested through pilot assessments in uganda (december 2015) and tanzania (february 2016). the key lessons learnt were multifold: getting country buy-in is crucial. a series of interactions between the host country and the implementing coordinating partners needs to take place before the assessment so that the importance of the evaluation and its complementarities with other ongoing assessments is clearly understood. this preparatory work also guides the country in collecting all necessary evidence supporting the scoring beforehand. the labnet scorecard assessment is a pedagogic tool to introduce system-thinking to stakeholders not previously familiar with the process. highlighting root-cause of interrelated problems helped the countries to identify single interventions capable of advancing the status of several component and/or core capabilities. for instance, the creation of a department of laboratory directly under the authority of the ministry of health was identified as one of the most effective strategies to leverage improvements regarding governance, network coordination, financing, legislation and policy pertaining to the laboratory sector. metrics including the private laboratory sector were difficult to measure in practice and were kept to the strict minimum in the final version of the labnet scorecard. the inclusion of sections dedicated to ‘priority diseases’ (core capability 9) and ‘laboratory network coverage and rapid response’ (core capability 3) bind the evaluation framework together through their cross-cutting relationship with all other laboratory key functions. anticipated utilisation and benefit of the labnet scorecard assessment an important outcome of the regional ghsa consultation for laboratory strengthening in october 2015 was the recognition that adequate laboratory network capability to operate is required to support the ghsa and achieve compliance to ihr requirements.7,41 hence, future enhancement strategies should yield measurable improvement of the laboratory network functionality. the labnet scorecard assessment was specifically designed to fulfill this need. it can be used as a stand-alone assessment tool but preferably in conjunction with the who joint external evaluation tool.42 the latter option will allow the collection of in-depth information on national laboratory network capabilities, in the context of a larger assessment of country preparedness for health security. additionally, the labnet scorecard assessment offers a good starting point to combine in-depth evaluation of more specific laboratory aspects, such as amr surveillance capability22 or biosafety/biosecurity,43 at either system or facility level. gaps and weaknesses identified during the assessment will allow countries to prioritise areas with most urgent needs, and serve as the basis for the formulation of tailored plans for laboratory capacity strengthening under the ghsa framework. the success story of the slipta/slmta programmes11,12 illustrates how tailored laboratory improvement plans based on outcomes from rounds of standardised assessments, can lead to measurable advancement toward laboratory (quality) standards. similar to the approach described in the who ‘better lab for better health’ initiative in eastern europe and central asia,44 the data collected during the labnet scorecard assessment will inform the development or revision of the national laboratory policy and/or strategic plan. the standardised assessment process will allow monitoring the progress of individual countries over time (as part of the monitoring and evaluation process). the comparison of data from different countries will facilitate the implementation of regional capacity building programmes according to an economy of scale approach. labnet scorecard assessment reports include clear visual representations of the status of each core capabilities at the country level or at the regional level, highlighting critical weaknesses for which additional resources are required. these figures can be used by laboratory system managers when advocating to their governments or to international stakeholders for the improvement of laboratory systems. perspectives and conclusions we have developed a new matrix for the comprehensive evaluation of national laboratory functionality. the labnet scorecard complements the who joint external evaluation tool42 for health system-wide assessments by providing laboratory-specific information with a high level of granularity. following the principle that ‘what gets measured, gets done’, we propose that the labnet scorecard can be one of the essential pieces of a phased approach to implementing/strengthening laboratory capability and capacity to assure access to quality clinical and public health functions, in support of the achievement of global health security goals the ghsa initiative31 is a unique opportunity for countries in africa and elsewhere, to access resources including professional services of aslm and aphl to overcome the barriers to strengthening laboratory networks. box 2: lessons learnt. country buy-in is an essential prerequisite to adoption of the labnet scorecard and can be facilitated by the endorsement of who regional offices. we propose that the labnet scorecard be applied under the coordination and the leadership and normative organisation of the who and its regional offices, through international laboratory professional organisations such as aslm and aphl. such a framework can ensure efficiency and economy of scale of the intervention, in a similar fashion to the slipta/slmta approach for implementing laboratory quality management systems in africa.12 the possibility of countries using the labnet scorecard as a self-evaluation tool to keep track of their own progress and guide their capacity building efforts is another additional benefit of this tool. acknowledgements the authors are grateful to patricia riley (cdc) for sharing her experience on the application of the capability maturity model and to trevor peter for providing the initial guidance for the design of the scorecard. we thank sebastien cognat, virginie dolmazon and maagdi saman (world health organization global capacity alert and response) for discussing gaps of national laboratory network assessment in the context of the ihr 2005. we thank reshma kakkar and david mills (aphl), benjamin park and rachel smith (cdc) for their input to the design of the indicators for laboratory information management system and antimicrobial resistance (amr) and sally liska (aphl) for her help in developing the scorecard manual. we thank lucy maryogo-robinson and ralph timperi for reviewing the manuscript (aphl). we are indebted to steven aisu (ministry of health, uganda) and fausta mosha (ministry of health, tanzania) for facilitating the piloting of the labnet scorecard in their respective country, and to ahmad iyane sow (moh, senegal) for hosting the training of assessors in dakar, senegal. sources of support this work was funded through the us centers for disease control and prevention cooperative agreement ‘building laboratory capacity to 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[document on the internet]. c2016 [cited 2016 mar 30]. available from: http://www.who.int/ihr/publications/who_hse_gcr_2016_2/en/ association of public health laboratories. a biosafety checklist: developing a culture of biosafety [document on the internet]. c2015 [cited 2016 jul 27]. available from: http://www.aphl.org/aboutaphl/publications/documents/id_biosafetychecklist_42015.pdf brown c, zwetyenga j, berdieva m, et al. new policy-formulation methodology paves the way for sustainable laboratory systems in europe. who panorama. 2015;1(1):41–47. abstract introduction methods results discussion acknowledgements references about the author(s) phidelis m. marabi department of medical laboratory sciences, faculty of health sciences, kisii university, kisii, kenya department of biomedical sciences, faculty of public health and community development, maseno university, maseno, kenya stanslaus k. musyoki department of medical laboratory sciences, faculty of health sciences, south eastern kenya university, kitui, kenya fred monari department of mathematics and actuarial science, faculty of pure applied sciences, kisii university, kisii, kenya paul m. kosiyo department of medical laboratory sciences, faculty of medicine, maseno university, maseno, kenya collins ouma department of biomedical sciences, faculty of public health and community development, maseno university, maseno, kenya citation marabi pm, musyoki sk, monari f, kosiyo pm, ouma c. association between coagulation indicators and menorrhagia among women in kenya. afr j lab med. 2024;13(1), a2438. https://doi.org/10.4102/ajlm.v13i1.2438 original research association between coagulation indicators and menorrhagia among women in kenya phidelis m. marabi, stanslaus k. musyoki, fred monari, paul m. kosiyo, collins ouma received: 26 feb. 2024; accepted: 15 july 2024; published: 17 sept. 2024 copyright: © 2024. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: despite the significant burden of menorrhagia (bleeding > 80 ml every menstrual cycle) among women in western kenya, it remains unknown whether coagulation disorders are an important underlying cause of this condition in the region. objective: this study assessed differences in coagulation profiles, associations between menorrhagia and coagulation profiles and compared morphological features of platelets among women attending bungoma county referral hospital in kenya. methods: a comparative cross-sectional study of women with and without menorrhagia, aged 18–45 years, was performed between december 2022 and september 2023. sociodemographic factors, prothrombin time (pt), activated partial thromboplastin time, thrombin time, fibrinogen, international normalised ratio (inr), and platelet count were compared between groups, and associations with menorrhagia were assessed. prothrombin time and inr levels above normal references were deemed increased. results: a total of 428 (214 per group) women were included. family history of bleeding disorders (p < 0.0001) was more frequent in menorrhagic than in non-menorrhagic women. additionally, menorrhagic women had high pt (p < 0.0001) and high inr (p < 0.0001) levels. menorrhagia was significantly associated with an increased pt (odds ratio = 2.129, 95% confidence interval = 1.658–2.734; p < 0.0001) and increased inr (odds ratio = 7.479, 95% confidence interval = 3.094–18.080; p < 0.0001). conclusion: in this population in western kenya, menorrhagia was associated with a family history of bleeding disorders, increased pt, and increased inr. routine assessment of the coagulation profile and family history of bleeding disorders is crucial for diagnosing and managing menorrhagia. what this study adds: our findings suggest that menorrhagic and non-menorrhagic women differ in terms of pt and inr, which may be predictive of menorrhagia. keywords: menorrhagia; coagulation; profile; women; kenya. introduction menorrhagia is defined as heavy menstrual bleeding that exceeds 80 ml per cycle.1 it is a common disorder that, on average, affects 30% of women.2 having heavy periods significantly decreases women’s quality of life, requires time away from work, involves surgical intervention, including hysterectomy, and ultimately has a substantial cost impact on the healthcare system.3 menorrhagia is a diagnosable ailment that needs to be treated to improve the quality of life of affected women,4 however, a significant number of menorrhagia cases are classified as idiopathic. research has shown that when women are dissatisfied with their normal therapy options, they frequently choose surgery, such as hysterectomy.5 these women might benefit from a specialised method of diagnosis and treatment that emphasises pertinent coagulation parameters. a series of biochemical processes, known as the coagulation pathway, results in hemostasis.6 this complex pathway promotes healing and prevents spontaneous bleeding.7 an international expert panel in obstetrics, gynaecology, and haematology made recommendations on the best ways to manage menorrhagia in women without a known bleeding disorder.8 the proposals call for a laboratory assessment of complete blood counts, platelet functions, and coagulation profiles in menorrhagia patients; however, more than 61% of patients presenting with menorrhagia are still categorised as having a non-structural disorder without discernible coagulopathy as the cause of their condition.9 menorrhagia may be caused by haemostatic disorders in as many as 17% – 20% of cases, according to a recent study in the united states (us).10 nonetheless, coagulopathies are infrequently regarded as the cause of menorrhagia. a comprehensive history and clinical suspicion of an underlying bleeding issue in women who present with menorrhagia can help with the early identification and effective management of antepartum/postpartum haemorrhage.11 if there is no evident cause for menorrhagia, the coagulation profiles of all women should be checked. coagulation problems are relatively common – 1% of the general population is affected, and such problems can affect as many as 5% of gynaecological patients.12 according to multiple studies conducted in high income countries, von willebrand disease is the most common hereditary blood disorder that causes menorrhagia.13 in addition, previous research has shown that platelet function disorders are a major cause of heavy menstrual bleeding in adolescents.14 we hypothesise that derangements in coagulation profiles that affect the integrity of the intrinsic, extrinsic and common pathways in the coagulation cascade may be associated with menorrhagia. the explanations for coagulation problems and platelet dysfunction, which are some of the underlying causes of menorrhagia in women, are not well understood in kenya. furthermore, despite the substantial burden of menorrhagia (35.3%) in western kenya,15 and menorrhagia affecting approximately 3% of all gynaecological patients who visit the bungoma county referral hospital (bcrh) clinic,16 the basic coagulation parameters of menorrhagic women compared to non-menorrhagic women are unknown. therefore, this study set out to determine the differences in selected coagulation parameters between menorrhagic and non-menorrhagic women. in addition, the study investigated the associations between coagulation parameters and menorrhagia and the morphological characteristics of platelets from menorrhagic and non-menorrhagic women at bcrh, kenya. methods ethical considerations the study was approved by the maseno university scientific and ethical review committee (reference number muserc/01166/22), and the authority to carry out the research was granted by the national commission for science, technology, and innovation (reference number nacosti/p/22/22573). bungoma county referral hospital (reference number bdh/tp/b/vol2) provided permission to perform the study. each participant provided written informed consent after a brief explanation of the study. measures taken to secure data included a password on the computer used and protected physical space. protocol numbers were assigned, and personal information was collected from the participants in a confidential manner and utilised solely for the purpose of the study. study area the study was conducted at bcrh in bungoma county, in western kenya, between december 2022 and september 2023. bungoma county referral hospital is a level 5 hospital located within the headquarters of bungoma county and is the main referral health facility for bungoma county and its soundings, serving a population of 1 670 570 across an area of 2206.9 km2.17 every year, approximately 6000 patients visit the bcrh gynaecological clinic. study design and population this study used a comparative cross-sectional design. the sample size formula for a quantitative comparative survey18 was used to calculate the number of study participants. the sample size was 428 (214 per group). a systematic random sampling procedure was adopted, with every second person satisfying the inclusion criteria being selected until the sample size was reached. inclusion criteria the study recruited adult women between the reproductive ages of 18 years and 45 years. the study included women who visited the clinic with complaints of menorrhagia. the comparison group consisted of healthy women picked at random. a qualified gynaecologist determined menorrhagia in each patient and established the clinical diagnosis based on the definition of menorrhagia as menstruation at regular cycle intervals but with excess flow (more than 80 ml of blood loss per cycle or requiring more frequent than 2 h changes of hygiene products) and/or duration (> 7 days).19 exclusion criteria women who experienced menorrhagia due to known causes, such as uterine fibroids or intrauterine contraception, were excluded from the study. women with other known disorders that may alter coagulation parameters were excluded. women who were found to be using any anticoagulants, as determined by a thorough clinical history by qualified clinicians, were also ineligible. patients who had taken sylate or tranexamic acid within the previous 3 months, as well as other drugs known to manage bleeding difficulties, were ineligible. sample and data collection using a screening procedure as used before,20 participants were classified into menorrhagic and non-menorrhagic women. a structured questionnaire was used to collect sociodemographic and clinical information, such as age, residence, parity, occupation, literacy, family history of bleeding disorders, history of epistaxis or gum bleeding, bleeding complications after tooth extraction, postoperative extended bleeding, and prolonged bleeding after delivery or miscarriage. the phlebotomy order of drawing blood using evacuated tubes was blood collection in sodium citrate, followed by blood collection in ethylenediaminetetraacetic acid. for the coagulation profile, whole blood was collected from the participant’s arm by a qualified phlebotomist and put in a vacuum tube containing 3.2% sodium citrate (becton, dickinson and company corporate, franklin lakes, new jersey, us), to ensure a 9:1 dilution of blood to the anticoagulant for prothrombin time (pt), international normalised ratio (inr), activated partial thromboplastin time (aptt), thrombin time (tt) and fibrinogen tests. thereafter, the participant’s distinctive identifying number tube was affixed to the tube. the sample(s) were delivered to the laboratory immediately after collection and spun for 5 min at 4000 rpm to ensure that the samples were platelet poor within 4 h. the separated samples were stored at 2°c – 8°c for 24 h if not tested immediately. the lyophilised coagulation test control materials (human gesellschaft fur biochemica and diagnostica mbh, wiesbaden, germany) were reconstituted following the manufacturer’s instructions. for platelets count and morphology, whole blood was collected in a vacuum tube with ethylenediaminetetraacetic acid (becton, dickinson and company corporate, franklin lakes, new jersey, us), immediately inverted gently 8–9 times, and labeled with a unique study number. thin blood films for performing reflex testing when the analyser results were flagged and determining the platelet cell morphology were immediately prepared and fixed using absolute ethanol (sigma-aldrich, st. louis, missouri, us). the lyophilised control materials (human gesellschaft fur biochemica and diagnostica mbh, wiesbaden, germany) were brought to room temperature and mixed well according to the manufacturer’s instructions before testing. laboratory analyses coagulation profile parameters, including pt, aptt, tt, fibrinogen, and the inr, were tested using a humaclot junior semiautomated coagulation analyser (human gesellschaft fur biochemica and diagnostica mbh, wiesbaden, germany) as used previously.21 the analyser uses the photometric principle in which fibrin increases sample turbidity, which the photometer detects. after mixing the reagents with plasma, fibrinogen is converted into fibrin and coagulates. thus, the optical density of the test sample changes, and the analyser can detect the coagulation endpoint.22 the fibrinogen assay was a quantitative clot-based test that assessed the rate of fibrinogen to fibrin conversion in a diluted sample in the presence of excess thrombin. because the fibrinogen content is rate-limiting under these conditions, the clotting time is utilised to assess the concentration of the fibrinogen.23 for interpretation purposes, the reference ranges for the coagulation profile of adult women were adopted from the bcrh laboratory as follows: pt: 11.0–12.5 s; aptt: 30–40 s; tt: 15–19 s; fibrinogen: 200–400 mg/dl; and inr: 1.1 or less. the samples were examined at the same time as both abnormal and normal control plasma. a celtac f, mek-8222 k analyser (nihon kohden corporation, tokyo, japan), as described previously,24 was used to determine the thrombocyte count. this analyser measures thrombocytes by the electrical resistance sensing technique. for interpretation purposes, the reference ranges for the platelet count of adult women were adopted from the bcrh laboratory as follows: 150–450 × 109/l. low, normal, and high levels of control materials were analysed before the samples were tested. the morphology of the thrombocytes was evaluated using leishman-stained blood films and olympustm optical light compound microscopy (olympus global, tokyo, japan), as described previously,25 and the size, morphology, and granulation of the platelets were evaluated by a qualified medical laboratory technologist. data analysis microsoft excel sheets (microsoft corporation, redmond, washington, us) were used to store the data. the statistical package for social science (spss v.26) (ibm corporation, armonk, new york, us) was used for the analysis. the shapiro-wilk test was used to check for normality, and histograms and box plots were generated to verify the normality of the distribution of the data. the chi-square test was used to analyse categorical descriptive data that included residence, parity, occupation, family history of bleeding disorders, history of bleeding or gum bleeding, history of extended bleeding following tooth extraction, postoperative extended bleeding and history of prolonged bleeding following delivery or miscarriage. the mann‒whitney u test was used to compare coagulation profiles between menorrhagic and non-menorrhagic women, as the data were not normally distributed. since the current study included a categorical dependent variable, binary logistic regression was used to predict the association between menorrhagia and coagulation parameters. the platelet morphological characteristics are presented as frequencies and percentages. the frequency, median, and dispersion of the descriptive statistics were shown. for all analyses, statistical significance was established at p ≤ 0.05. results demographic characteristics of the study population a total of 428 adult women between the ages of 18 years and 45 years (median [interquartile range]: 33.5 [11.0] years) participated in the study (table 1). there were 214 menorrhagic patients and an equal number of non-menorrhagic healthy women. participants did not differ with respect to place of residence in urban or rural location (p = 0.139), parity (p = 0.390), occupation (p = 0.846), history of epistaxis or gum bleeding (p = 0.499), and history of prolonged bleeding following delivery or miscarriage (p = 0.123) (table 1). menorrhagic women (n = 12/214; 5.6%) were more likely than non-menorrhagic women (n = 0/214; 0%) to have a family history of bleeding disorders (p < 0.001) (table 1). table 1: demographic characteristics of the study participants at bungoma county referral hospital, kenya, between december 2022 and september 2023. comparison of coagulation profiles between menorrhagic and non-menorrhagic women menorrhagic women (n = 38/214; 17.8%) were more likely to have increased pt than non-menorrhagic women (n = 3/214; 1.4%) (p < 0.0001) (table 2). menorrhagic women (n = 22/214; 10.3%) were more likely to have increased inr than non-menorrhagic women (n = 2/214; 0.9%) (p < 0.0001) (table 2). both menorrhagic and non-menorrhagic women showed no difference in terms of aptt (p = 0.106), tt (p = 0.176), fibrinogen (p = 0.082) and platelet count (p = 0.101) (table 2). table 2: comparison of coagulation profiles between menorrhagic and non-menorrhagic women, bungoma county referral, kenya, between december 2022 and september 2023. association between coagulation profile and menorrhagia menorrhagia was associated with increased pt (odds ratio [or] = 2.129, 95% confidence interval [ci] = 1.658 – 2.734; p < 0.0001) and increased inr (or = 7.479, 95% ci = 3.094–18.080; p < 0.0001) (table 3). menorrhagia was not associated with aptt (or = 1.064, 95% ci = 0.976–1.161; p = 0.158), tt (or = 1.129, 95% ci = 0.961–1.325; p = 0.139), fibrinogen (or = 0.996, 95% ci = 0.992–1.000; p = 0.062), or platelet count (or = 0.998, 95% ci = 0.994–1.002; p = 0.349) (table 3). table 3: association between menorrhagia and abnormal coagulation profile, bungoma county referral hospital, kenya, between december 2022 and september 2023. comparison of morphological characteristics of platelets between menorrhagic and non-menorrhagic women in both menorrhagic and non-menorrhagic women, there were no morphologic differences in the platelets. discussion in this study, menorrhagic women were more likely to have a family history of bleeding disorders than non-menorrhagic women. menorrhagic women had statistically significant increases in pts and inrs compared with non-menorrhagic women, and menorrhagia had a statistically significant association with increased pt and inr. the demographic characteristics of the study participants were comparable between the two groups in terms of age, place of residence, parity, occupation, and literacy; however, menorrhagic women had no family history of bleeding disorders, while 5.6% of the menorrhagic women had a bleeding history of bleeding disorders. due to the age range in this study, the median age of 34 years for menorrhagic women compared to 33 years of non-menorrhagic women was higher than those obtained in previous studies conducted in sudan, india and turkey.26,27,28 the residential areas of menopausal women (rural or urban) were not substantially different from those of non-menorrhagic women in the present study, which was similar to the findings of a study performed in tanzania and published in 2023.29 the current study found that the majority of menorrhagic women had previously had a child, but no statistically significant difference in parity between menorrhagic and non-menorrhagic women. the current study revealed that a considerable number of menorrhagic women were informally employed, and there was no statistically significant difference from the findings for non-menorrhagic women. these findings are consistent with research performed in tanzania and published in 2023,29 which showed that 52.5% of menorrhagic women work as peasants. the present study found that whereas menorrhagic women were more likely to have a family history of bleeding disorders, almost three times as many menorrhagic women did not have a family history of bleeding disorders. none of the non-menorrhagic women had a family history of bleeding disorders. these findings are consistent with studies performed in egypt, published in march 2014,30 sudan, published in december 2015,26 and sweden, published in 2006.31 previous studies have shown a substantial association between idiopathic menorrhagia and a family history of heavy menstrual bleeding, suggesting that familial menorrhagia is an inherited feature.28 a family history of spontaneous or induced bleeding may increase the possibility of congenital bleeding, but multiple drugs can increase the risk of bleeding.32 however, some previous studies have demonstrated that one-third of menstrual bleeding problem diagnoses have no documented family history and can be caused by a random gene mutation.33 the high number of menorrhagic women without a family history of bleeding disorders can be attributed to the fact that a diagnosis of bleeding problems can develop completely unexpectedly due to a genetic mutation, which still needs further exploration. in addition, some people may be unaware of a bleeding issue in their family if other relatives are undiagnosed or have different symptoms. in light of the present findings, it is recommended that a thorough assessment of the family history of bleeding in menorrhagic women be performed routinely. furthermore, women in this category should be evaluated for the possibility of having a genetic mutation of the f5 gene which has been found to cause factor v leiden deficiency.34 the present study revealed that a history of epistaxis or gum bleeding in menorrhagic women was comparable to that in non-menorrhagic women; however, 0.9% of menorrhagic women had a history of epistaxis or gum bleeding. additionally, the study showed that the history of prolonged bleeding following delivery or miscarriage in menorrhagic women and non-menorrhagic women was comparable; however, 1.9% of menorrhagic women had a history of prolonged bleeding following delivery or miscarriage. these findings agree with studies performed in south sudan, published in december 2015,26 and turkey, published in june 2013,28 which demonstrated that some patients had a history of additional bleeding issues in addition to presenting with menorrhagia. these findings could be attributed to the fact that previous studies have suggested that familial menorrhagia is a hereditary characteristic.28 in light of the findings of the current study, a history of bleeding disorders other than menorrhagia, comprising epistaxis, gum bleeding, and severe bleeding after delivery or miscarriage, should be evaluated extensively in women presenting with menorrhagia. in the current investigation, the pt and inr were considerably greater in menorrhagic women than in non-menorrhagic women and were associated with menorrhagia, but the aptt was comparable between the two groups and was not associated with menorrhagia. this was consistent with the results of a research done in sudan, published in december, 2015,26 which demonstrated higher pt and inr in menorrhagic women than in non-menorrhagic women. these findings, however, contradicted those of a study undertaken in india, published in january 2011,27 which revealed pt to be normal in all except one patient. these findings also contradicted a study conducted in the us, published in 2013,14 which showed that pt levels were comparable between menorrhagic and non-menorrhagic women. the prolonged pt and inr and their association with menorrhagia in the current study could be attributed to the fact that women in the mid-luteal phase are more hypercoagulable than women in the early follicular phase.35 most of those previous investigations associated pt with adenomyosis,36 but not directly with menorrhagia. however, prolonged pt and normal aptt may reflect vitamin k deficiency caused by malnutrition, biliary obstruction, malabsorption syndromes, antibiotic use, or liver disease, which lowers clotting factor synthesis.37 the pt test evaluates the extrinsic coagulation pathway (also known as the tissue factor pathway), which comprises factors vii, x, v, and ii, while the aptt evaluates the intrinsic pathway (also known as the amplification pathway or contact system), which consists of factors xii, xi, ix, viii, x, v, and ii.38 it is further postulated that if the aptt is normal, then a prolonged pt could be due to factor vii deficiency.38 in light of the present findings, it is recommended that pt and the inr, together with other coagulation factors, be routinely evaluated in menorrhagic women. in the present study, tt, fibrinogen and platelet count were comparable in menorrhagic and non-menorrhagic women and were not associated with menorrhagia. patients with dysfibrinogenaemia can also experience combined haemorrhagic and thrombotic tendencies.39 dysfibrinogenaemia is a clotting disorder characterised by a range of structural defects in the fibrinogen molecule that result in aberrant fibrinogen function. it can be hereditary or acquired.40 in female patients with hereditary afibrinogenaemia, repeated severe intra-abdominal haemorrhages due to rupture of the graafian follicle during ovulation have been reported.41 patients with acquired dysfibrinogenaemia frequently have no history of bleeding or clotting, and a family history of dysfibrinogenaemia does not predict haematologic problems.41 clinical symptoms of acquired dysfibrinogenaemia range from acute bleeding or thrombosis to chronic thromboembolic pulmonary hypertension and renal amyloidosis. menorrhagia, postoperative bleeding, epistaxis, postoperative wound dehiscence, deficient wound healing, gingival bleeding, haemorrhage, and mild soft-tissue haemorrhage are all possible presenting signs and symptoms.42 despite the current data indicating comparable platelet counts in menorrhagic and nonmenorrhagic women, it is important to highlight that platelet count, size, and morphology are usually normal in individuals with glanzmann thrombasthaenia, a hereditary qualitative condition that affects platelet function through failure of aggregation and others hereditary conditions.43 in the present study, neither large/giant platelets nor hyper-granular or agranular platelets were found in menorrhagic or non-menorrhagic women. despite these findings, bernard–soulier syndrome can be linked to uncontrollable menorrhagia.44 overall, the pt, inr, and family history of bleeding disorders, among other parameters, should be evaluated in menorrhagic women as soon as possible to better understand the coagulation profile and diagnose this life-threatening condition so that prompt treatment can be provided. a more accurate classification of menorrhagia would most likely result in the identification of a broader range of characteristics that could indicate haemostatic diseases or other aetiologies. recommendations the study recommends that women who experience heavy menstrual bleeding have a coagulation profile, as well as a family history of bleeding and history of bleeding disorders other than menorrhagia assessed. this approach allows for a more accurate diagnosis of menorrhagia based on the identified coagulation hallmark indicators. limitations the study was limited in that we did not assess von willebrand factor, which is known to affect coagulation. furthermore, the study did not perform platelet aggregometry as platelet function test to measure platelet response or aggregation to a panel of antagonists such as collagen, and did not measure vitamin k-dependent factors due to a lack of equipment at the laboratory and financial constraints. a longitudinal study with a larger sample size could help to close the knowledge gap in determining and interpreting such an association. conclusion in this study, a family history of bleeding disorders was present in some menorrhagic but not in non-menorrhagic women. prothrombin time and inr were both increased among menorrhagic compared with non-menorrhagic women, and were associated with menorrhagia. thus, family history of bleeding disorders, pt and the inr, which are coagulation parameters, may contribute to the aetiology of menorrhagia. acknowledgements leonard lusenaka contributed to the recruitment of the participants. thomas wekesa contributed by performing the sample collection exercise. antony sangolo and emmanuel khisa assisted in the operation of the samples and the data collection. the authors acknowledges the following preprint.45 competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions p.m.m., s.k.m., p.m.k., and c.o. planned the study and participated in all areas. s.k.m., p.m.k., and c.o. oversaw the study. p.m.m. and f.m. conducted the data analysis and developed the manuscript. s.k.m., p.m.k., and c.o. reviewed the manuscript for philosophical insights. p.m.m., s.k.m., p.m.k., c.o. and f.m. reviewed the final manuscript and approved it for submission. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability the data that support the findings of this study are not openly available due to ethical reviewing guidelines and are available from the 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2002;126(4):499–505. https://doi.org/10.5858/2002-126-0499-ldod wang y, zhao l, feng x, et al. two-pronged treatment of hemoperitoneum and abnormal uterine bleeding in an adolescent girl with congenital fibrinogen deficiency. front med. 2020;7:181. https://doi.org/10.3389/fmed.2020.00181 tziomalos k, vakalopoulou s, perifanis v, garipidou v. treatment of congenital fibrinogen deficiency: overview and recent findings. vasc health risk manag. 2009;5:843–848. https://doi.org/10.2147/vhrm.s5305 rajpurkar m, o’brien sh, haamid fw, cooper dl, gunawardena s, chitlur m. heavy menstrual bleeding as a common presenting symptom of rare platelet disorders: illustrative case examples. j pediatr adolesc gynecol. 2016;29(6):537–541. https://doi.org/10.1016/j.jpag.2016.02.002 bhadra d, chakraborty s. bernard–soulier syndrome (bss) with uncontrollable menorrhagia. asian j transfus sci. 2020;14(1):93–95. https://doi.org/10.4103/ajts.ajts_61_18 maruti pm, stanslaus km, fred m, paul k, collins o. coagulation hallmark indicators of menorrhagia in a comparative study between menorrhagic and healthy women attending bungoma county referral hospital in kenya. research square. 2024. https://doi.org/10.21203/rs.3.rs-3944100/v1 introduction infectious diseases why pathogen genomics translating pathogen genomic data obstacles to be considered way forward acknowledgements references about the author(s) lamech m. mwapagha department of biology, chemistry and physics, faculty of health, natural resources and applied sciences, namibia university of science and technology, windhoek, namibia citation mwapagha lm. why pathogen genomics is crucial in africa’s public health. afr j lab med. 2023;12(1), a2166. https://doi.org/10.4102/ajlm.v12i1.2166 opinion paper why pathogen genomics is crucial in africa’s public health lamech m. mwapagha received: 19 jan. 2023; accepted: 04 aug. 2023; published: 22 sept. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction pathogens are bacteria, parasites, fungi and viruses that cause diseases by evading the body’s immune system. hiv, bacillus anthracis, ebola virus, zika virus, severe acute respiratory syndrome coronavirus 2 and mpox virus are some of the pathogens that cause serious diseases. thus, understanding the genetic information of these pathogens could play a significant role in curbing the spread of infectious diseases. the traditional and standard pathogen detection methods rely on identifying previously known agents associated with a specific disease, with most conventional laboratory assays having low pathogen detection efficiency and involving time-consuming tasks.1,2 despite being considered the gold standard in pathogen identification, these traditional methods are still incapable of detecting causative agents quickly and precisely, delaying infection diagnosis and complicating disease surveillance.3 the introduction of next-generation sequencing (ngs) in pathogen identification is critical in preventing and treating infectious diseases by allowing pathogen genomes to be determined rapidly and precisely.4 however, for africa to reap the benefits of pathogen genomics, genomic research findings must be effectively translated into public health benefits. infectious diseases infectious diseases remain the leading cause of death in africa. diseases such as malaria, hiv/aids, cholera, tuberculosis, meningitis, hepatitis, schistosomiasis, lymphatic filariasis, sleeping sickness, ebola, coronavirus disease 2019 (covid-19), mpox and other well-known existing, emerging, and re-emerging diseases are causing suffering and mortality to a large population in many developing countries. these have become a significant burden on public health and detrimental to african and global economies.5 the recent global covid-19 pandemic demonstrated how unprepared the world was and how it remains vulnerable for the next major health crisis.6,7 other previously known microorganisms have recently resurfaced (such as ebola and mpox) and caused widespread outbreaks in various african regions. disease re-emergence now requires africa to devise and implement new approaches to dealing with current and future infectious disease outbreaks to benefit public health. why pathogen genomics as high-throughput ngs technologies improve, genome-sequencing costs significantly fall.8,9 high-throughput technologies and bioinformatics can thus shed light on disease transmission, virulence, and antimicrobial resistance. next-generation sequencing technology has resulted in many new opportunities for public health, allowing researchers to examine pathogen genomes in greater depth. pathogen genomics is already in use in many public health applications, such as precision infection identification and diagnosis (parasites, hiv and tuberculosis), disease surveillance (covid-19, influenza, salmonellosis, and extraintestinal illness), antimicrobial resistance monitoring (in candida auris and streptococcal pathogens), outbreak investigations (salmonella spp. and mycobacterium tuberculosis), and in developing and evaluating interventions, such as vaccines (covid-19 and polio vaccines). translating pathogen genomic data clinical ngs has been used in oncology to identify genomic abnormalities linked to cancer, such as indels, copy number variants, and single-nucleotide variants.10 clinical ngs has also led in the identification of biomarkers for cancer diagnosis and treatment, with notable examples being the use of personalised ngs panels for adult acute myeloid leukaemia and lung cancer diagnosis.11,12 in pathogen identification and diagnosis, targeted metagenomic ngs panels target pathogens associated with specific diseases, such as respiratory and gastrointestinal ailments.13 genomics data has also played a critical role in detecting unexpected or previously undiagnosed pathogens, tracking evolution, tracing local chains of transmission, and investigating the origins of epidemics such as ebola, malaria, influenza and covid-19.14,15 these data, in turn, facilitated disease diagnosis and the development of effective treatment for various diseases globally. pathogen genomic data is increasingly used in africa to meet the public health demands for disease surveillance, prevention, and control of new illnesses. the ebola virus outbreak in west africa (2013–2016) and the democratic republic of the congo (2018–2020), the 2018 lassa virus outbreak in nigeria, the discovery of the severe acute respiratory syndrome coronavirus 2 omicron variant in botswana and south africa, and the epidemiological and evolutionary dynamics of respiratory syncytial virus viral populations in kilifi, kenya, are some noteworthy examples.16 thus, pathogen genomic data combined with epidemiological data has played a crucial role in advancing our understanding of transmission dynamics, as well as the efficacy of diagnostic tests and vaccine regimens for preventing future outbreaks on the african continent. obstacles to be considered despite the enormous potential of pathogen genomics in addressing public health issues, africa has yet to capitalise fully on these benefits due to various challenges.17 nonetheless, several factors must be considered before africa fully implements pathogen genomics. among these factors are: next-generation sequencing must be cost-effective compared to conventional methods, that is, personalised medicine, laboratory equipment upgrades, computer resources, and training. laboratory and analytical capabilities in clinical and public health systems must be adaptable to enable the rapid development of ngs technologies and platforms. systems should integrate pathogen genome sequences, bioinformatics, and epidemiology to facilitate knowledge exchange on disease surveillance, outbreak, and control. choosing the right sequencing platform and bioinformatics analysis tools is critical, because it depends on sequencing objectives and requires accuracy, efficiency, and cost trade-offs that may not be universally applicable to all infectious diseases and public health laboratories. ethical, legal, and social frameworks addressing access discrimination18 to precision medicine, data sharing, protection, and management in collaboration with national, regional, and global partners for infectious disease surveillance, prevention and control. way forward pathogen genomics has great potential to improve public health in africa by allowing for more effective disease surveillance, outbreak containment, and control investigations to inform vaccine development. a number of factors must be considered for africa to reap these benefits. firstly, infrastructure must be built and new technologies must be introduced and integrated into existing infrastructure (computational and storage systems) to support the rapid change in genomics technology. secondly, capacity building to address the scarcity of african scientists with genomic and computational expertise is needed to translate pathogen genomics into public health systems. thirdly, collaboration with various public health policymakers (national public health institutes, academic institutions, ministries of health, and non-governmental organisations) should be encouraged to strengthen public health research capacity on disease surveillance and control. fourthly, establish public health biobanks to aid our understanding of genetic susceptibility and support healthcare research strategies for infectious disease surveillance, diagnosis, and prognosis.19,20,21 fifthly, all players should embrace guiding principles and standards for pathogen genomic data sharing that are ethical, egalitarian, efficient, and effective to inform public health policy and response.8,9,22,23 africa is on the right track, having established programmes supporting pathogen genomic initiatives. for example, the africa centres for disease control and prevention through the africa pathogen genomics initiative and the african academy of sciences are programmes that promote scientific and technological development in africa while supporting public health pathogen genomics and bioinformatics. furthermore, in addition to equipping laboratories with high throughput genomic sequencing equipment, the africa pathogen genomics initiative is working to improve laboratory staff technical capacity by collaborating with key partners (african centre of excellence for genomics of infectious diseases, kenya medical research institute, international livestock research institute-kenya, south african national bioinformatics institute, national institute for communicable diseases, kwazulu-natal research innovation and sequencing platform/centre for epidemic response & innovation, institut pasteur de dakar, and institut national de la recherche biomédicale) engaged in genomic sequencing and bioinformatics to facilitate training, knowledge-sharing, and technical support. other programmes, such as the african field epidemiology network and the african society for laboratory medicine, also strengthen public health laboratory networks and systems throughout africa by increasing public health capacity, monitoring disease outbreaks, and promoting the one health approach. although advances in ngs have brought us closer to a complete understanding of infectious diseases, much work remains. the undeniable value of ngs, particularly for rapid and precise pathogen identification, can not be overemphasised. if science is to progress, it must delineate the diverse array of microorganism sequences and improve genomic tools and methodologies. overall, if africa is to reap the public health benefits of these novel approaches to disease surveillance, prevention, and control, all public health policymakers and global partners (national public health institutes, ministries of health, academic institutions, non-governmental organsiations, industry, government, private sector, philanthropists, and funding agencies) must be involved from the outset of all discussions involving the integration of pathogen genomics into public health benefits. acknowledgements competing interests the author declares that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. author’s contributions l.m.m. is the sole author of this article. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the author and do not necessarily reflect the official policy or position of any affiliated agency of the author. references huang j, jiang e, yang d, et al. metagenomic next-generation sequencing versus traditional pathogen detection in the diagnosis of peripheral pulmonary infectious lesions. infect drug resist. 2020;13:567. https://doi.org/10.2147/idr.s235182 buchan bw, ledeboer na. emerging technologies for the clinical microbiology laboratory. clin microbiol rev. 2014 oct;27(4):783–822. https://doi.org/10.1128/cmr.00003-14 yang y, zhu x, sun y, qian k, liu z. comparison of next-generation sequencing with traditional methods for pathogen detection in cases of lower respiratory tract infection at a community hospital in eastern china. medicine. 2022 dec 23;101(51):e32423. https://doi.org/10.1097/md.0000000000032423 waddington c, carey me, boinett cj, higginson e, veeraraghavan b, baker s. exploiting 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8fhi360, vietnam 9french department, hanoi university of science and technology, hanoi, vietnam correspondence to: kyle bond postal address: 1600 clifton road, atlanta, ga 30329-4018, united states dates: received: 06 sept. 2014 accepted: 22 sept. 2014 published: 03 nov. 2014 how to cite this article: nguyen tt, mckinney b, pierson a, luong kn, et al. slipta e-tool improves laboratory audit process in vietnam and cambodia. afr j lab med. 2014;3(2), art. #219, 5 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.219 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. slipta e-tool improves laboratory audit process in vietnam and cambodia in this lessons from the field... open access • abstract • introduction • research methods and design    • development of the e-tool • results • discussion • acknowledgements    • competing interests    • authors’ contributions    • cdc disclaimer • references abstract top ↑ background: the stepwise laboratory quality improvement process towards accreditation (slipta) checklist is used worldwide to drive quality improvement in laboratories in developing countries and to assess the effectiveness of interventions such as the strengthening laboratory management toward accreditation (slmta) programme. however, the paper-based format of the checklist makes administration cumbersome and limits timely analysis and communication of results.development of e-tool: in early 2012, the slmta team in vietnam developed an electronic slipta checklist tool. the e-tool was pilot tested in vietnam in mid-2012 and revised. it was used during slmta implementation in vietnam and cambodia in 2012 and 2013 and further revised based on auditors’ feedback about usability. outcomes: the slipta e-tool enabled rapid turn-around of audit results, reduced workload and language barriers and facilitated analysis of national results. benefits of the e-tool will be magnified with in-country scale-up of laboratory quality improvement efforts and potential expansion to other countries. introduction top ↑ the stepwise laboratory quality improvement process towards accreditation (slipta) checklist was established by the world health organization’s regional office for africa (who afro) and partners to assess the level of a laboratory’s compliance with the international organization for standardisation (iso) 15189 standard.1 this checklist is the standardised tool used to assess the effectiveness of the strengthening laboratory management toward accreditation (slmta) training programme2 through audits at the start (baseline) and end (exit) of the programme. audits are conducted using the paper-based slipta checklist containing 111 major questions (and numerous, related sub-questions) divided into 12 sections; as of 2012 only the english version was available. to date, the checklist has been used in 617 laboratories implementing slmta in 47 countries.3 data collected using the slipta checklist reveal the current state of a laboratory’s quality management systems and identify gaps in the 12 quality system essential areas defined by the clinical and laboratory standards institute.4 audit results are used to develop action plans and guide the selection of quality improvement activities for the slmta programme so as to help laboratories move toward national or international accreditation.the slmta programme in vietnam and cambodia began in 2010 when representatives from each country participated in a two-week training-of-trainers workshop. after stakeholder engagement, governmental approval and, in the case of vietnam, planning with international partners, baseline audits were conducted in seven laboratories in cambodia in june of 2011 using the standard paper-based slipta checklist. auditors and implementing leadership from vietnam noted that using the paper-based checklist created several challenges. cumbersome audit and administrative processes meant that auditors were required to bring blank paper-based checklists and, in the case of exit or follow-up audits, previously-completed checklists to review prior recommendations and scores. scores for all 111 questions had to be calculated and/or graphed manually for each report in each round of audits. additionally, communication opportunities were lost; data analysis was inefficient and delayed; reports were not standardised; and there were difficulties in combining data for regionalor country-level management. as a result of the observed difficulties, prior to performing the baseline audits in vietnam, ministry of health (moh) laboratorians supporting slmta in vietnam set out to develop an electronic tool (e-tool) for collection and use of audit data. this paper describes the development of the slipta checklist e-tool and discusses its benefits for slmta implementation. research methods and design top ↑ development of the e-tool development of the slipta checklist e-tool began with analysis of the current audit workflow. next, experts from the moh and us centers for disease control and prevention’s vietnam office (cdc-vietnam) gathered background information about the structure, scoring strategy and other aspects of the slipta checklist from auditors and other stakeholders. the tuberculosis electronic laboratory assessment tool (tb-lat), a microsoft® excel-based e-tool developed previously by integrated quality laboratory services and used globally for tuberculosis assessment,5 was modified by the slmta management team within the vietnam hospital administration system so as to incorporate the slipta checklist. the structure of the checklist was retained. during the development of the e-tool, input was sought from the moh and cdc-vietnam laboratorians working as slmta trainers and auditors in vietnam.the vietnam slmta team pilot tested the e-tool in early 2012 in parallel with the paper-based checklist in a provincial public health laboratory. the e-tool was then used for the baseline audits of a mix of 12 public health, hospital and hiv laboratories in vietnam in may 2012. the tool was further enhanced in an iterative process of feedback and improvement. exit audits of the first round of slmta-supported laboratories in both cambodia (january 2013) and vietnam (june to july 2013) provided additional opportunities to refine the tool and validate macro formulae. results top ↑ the baseline audit in cambodia using the paper-based slipta checklist had several limitations. audits required at least one-and-a-half days, including two hours to manually fill out the 44-page slipta checklist and calculate site scores in order to create the reports. although audit teams gave verbal reports at the individual laboratory summation conferences immediately following the audits, final written reports to the laboratories were compiled remotely after the audit and delivered by mentors at their next visit, sometimes delayed as long as two weeks. because the existing paper-based checklist is in a pdf or microsoft® word format, multi-site data were not easily manipulated, reducing the analytical value. additionally, the country team scanned and saved all final paper documents, requiring significant administrative time and costs for electronic data storage.the e-tool6 addresses several issues related to ease of use during the audit process and solutions for data management (table 1). drop-down response selections enable automatic scoring, easily accounting for non-applicable questions. additional features of the electronic format include: automatic linkage of information; pre-programmed calculations to compare baseline and exit audits; and pre-set data check limits so as to help improve scoring accuracy. all comments entered in each of the 12 sections are automatically pulled to the end of the report to complete the recommendation section. summary pages visually highlight the accomplishments and remaining gaps to be addressed, utilising clear colour-coded graphs. table 1: challenges of the paper-based slipta checklist, and solutions provided by the slipta e-tool. in addition to improved functionality of data at the laboratory-level, the e-tool improves usability for country-level programme management. the ability to merge data easily from multiple laboratories affords an accurate analysis with robust statistical indicators, including means and medians, minimum and maximum values, interquartile ranges, standard deviations of means and coefficients of variation at both indicator and section levels. compiling results electronically for multiple laboratories allows the remaining gaps to be addressed by appropriate improvement projects in the slmta programme and shared with moh leadership (figure 1). figure 1: example of e-tool data output for baseline (row 1) and exit audit (other rows) for section 1 (documents and records) compiled from 12 laboratories in vietnam, 2013. data collected from all laboratories can be collated and used to assess countrywide gaps and initiate corrective actions. figure 2 presents a sample e-tool report, which combines results from all 12 participating laboratories in vietnam. baseline audits showed major gaps in four sections: internal audit (0%), corrective action (0%), occurrence management and process improvement (0%) and documents and records (22%) (figure 2a). section-level reports break out details of each question. for example, the section 1 issues pertained predominantly to the development of manuals and standard operating procedures (sops) (figure 2b). additional training was conducted on sops for the laboratory system, lectures on development of manuals and internal audit were included in the third slmta workshop and improvement projects related to these gaps were organised. exit audit results showed substantial increases in the scores of these sections (100%, 57%, 87% and 88%, respectively) (figure 2a), as well as in the subsection scores for section 1 (figure 2c). the clear visual summary of results provided by the e-tool report facilitated timely development of an action plan to address the issues. figure 2: visualised results for 12 laboratories in vietnam using the slipta e-tool. (2a) spider plot of combined baseline (may 2012) and exit (june to july 2013) audit median data for all 12 laboratory sites. (2b) computer screen shot of baseline audit data for section 1 (documents and records) of the slipta checklist with bar-chart results. colours indicate results that are acceptable (green), unacceptable (red) or partially compliant (yellow). (2c) computer screen shot of exit audit data for section 1 (documents and records) of the slipta checklist with bar-chart results. discussion top ↑ the newly-developed slipta e-tool improved the audit process, enhanced communication of audit results and ensured timely usability of the data collected in vietnam and cambodia.7 the e-tool received positive feedback from in-country auditors for reducing workload. because auditors are highly-trained limited resources, they must be utilised as efficiently and effectively as possible. vietnam and cambodia have only 10 to 12 qualified auditors in each country. as laboratory quality improvement scales up to include the more than 1700 public laboratories in vietnam8 and 82 public laboratories in cambodia,9 increasing the efficiency of the audit process will become even more critical.the ability to leave a one-page printed summary in vietnamese for each laboratory was a substantial improvement over the paper tool. the communication benefits of providing same-day, on-site language-appropriate results to laboratorians and administrators encourage engagement from leadership in the improvement process. furthermore, presenting reliable results in a graphical format leads to better understanding of problems and use of data for improvement. as with the tb-lat e-tool, this first version of the slipta e-tool has capacity for data exporting; however, some minor issues have been identified when transferring data between various versions of microsoft® excel. improvements to the tool should continue, based on feedback from end-users. in addition, studies are needed to provide a formal evaluation of the tool, including time and cost savings, improved accuracy and user preferences. potential benefits of the e-tool expand beyond improved auditing. the tool incorporates auditing instructions for each slipta question, which will enhance learning and consistency for laboratories performing internal audits. furthermore, as additional auditors are trained, the e-tool can be used for evaluation and validation of their competency and standardisation of audit criteria. moreover, when unlocked, the electronic checklist can be customised and expanded to address additional laboratoryor countryspecific data needs. the slipta checklist e-tool is now available for use by other countries and can easily be customised as needed; for example, optional languages can be added for local settings. widespread use of the tool will allow development of a database for slmta programme managers at the global level in order to evaluate systematic gaps in laboratory quality and to enhance overall planning, implementation and sharing of results. acknowledgements top ↑ the authors would like to extend their sincere thanks to dr katy yao, cdc, for her strong support and invaluable contribution to and review of the design of the slipta e-tool, as well as to dr elizabeth luman, cdc, for her patience and professional guidance in development of the manuscript. finally, the authors would like to thank all end-users of the slipta e-tool for their invaluable feedback and contribution.this project has been supported by the us president’s emergency plan for aids relief (pepfar) through the cdc under the terms of cooperative agreement project number ps001928 (vietnam administration for medical services) and ps001285 (american society for clinical pathology). competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions t.t.n. (nihe) led the design of the e-tool, analysis/ interpretation of data and led the manuscript writing/ revision. b.m. (mayo clinic) participated in implementation of the audit, interpretation of data and supported the writing and revision of the manuscript. a.p. (iqls) led the design and review of the manuscript. k.n.l. (vams) was responsible for implementation of the slmta programme in vietnam and supported introduction of the e-tool. q.t.h. (cdc, vietnam), s.m. (usaid, indonesia), c.q.n. (fhi360, vietnam) and k.t.n. (hust, vietnam) participated in the design and review of the e-tool. h.m.c. (samhsa) was responsible for coordination of the implementation work and participated in the design of the e-tool. k.b.b. (cdc, vietnam) directed the e-tool introduction and supported the writing of the manuscript, as well as being responsible for the programme implementation and revision of the manuscript. cdc disclaimer the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the cdc. references top ↑ 1. world health organization’s regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation in the african region [document on the internet]. c2013 [cited 2014 sep 20]. available from: http://www.afro.who.int/en/clusters-a-programmes/hss/blood-safety-laboratories-a-health-technology/blt-highlights/3859-who-guide-for-the-stepwise-laboratory-improvement-process-towards-accreditation-in-the-african-region-with-checklist.html2.yao k, maruta t, luman et, et al. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. 2014;3(1), art. #194, 8 pages. http://dx.doi.org/10.4102/ajlm.v3i1.194 3.yao k, luman et, slmta collaborating authors. evidence from 617 laboratories in 47 countries for slmta-driven improvement in quality management systems. afr j lab med. 2014;3(2), art. #262, 11 pages. http://dx.doi.org/10.4102/ajlm.v3i2.262 4.clinical and laboratory standards institute. application of a quality management system model for laboratory services; approved guidelines – third edition. [clsi document gp26-a3]. clinical and laboratory standards institute; 2004. 5.integrated quality laboratory services (iqls). tb-lat tuberculosis electronic laboratory assessment tool [document on the internet]. c2013 [cited 2014 apr 28]. available from: http://www.iqls.net/docs/tb-lat_presentation.pdf 6.thuong nt, pierson a, hoang qtt, et al. vietnam slmta e-tool [document on the internet]. c2013 [cited 2014 apr 28]. available from: http://aslm.org/aslm2012/images/docs/speaker_presentations/sunday_2_dec/symposium_overview_day1/2b_-_vietnam_slmta_e-tool.pdf 7.carvalho hm. slmta in vietnam – the roadmap for laboratory quality. vietnam: us centers for disease control and prevention; 2012 (unpublished). 8.ministry of health of vietnam. laboratory qms in vietnam – roads towards improvement. report presented at the symposium on quality management for medical laboratories. bangkok, thailand; 2011 march 17–18. 9.kingdom of cambodia, ministry of health. appendix 17: complementary package of activities for referral hospitals 2006–2010. in: national guidelines on complementary package of activities for referral hospital development from 2006 to 2010. 2nd ed., 2006; p. 128–130. abstract introduction methods results discussion acknowledgements references about the author(s) naseem cassim department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa national priority programme, national health laboratory service, johannesburg, south africa lindi m. coetzee department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa national priority programme, national health laboratory service, johannesburg, south africa abel l. makuraj national priority programme, national health laboratory service, johannesburg, south africa wendy s. stevens department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa national priority programme, national health laboratory service, johannesburg, south africa deborah k. glencross department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa national priority programme, national health laboratory service, johannesburg, south africa citation cassim n, coetzee lm, makuraj al, stevens ws, glencross dk. establishing the cost of xpert mtb/rif mobile testing in high-burden peri-mining communities in south africa. afr j lab med. 2021;10(1), a1229 https://doi.org/10.4102/ajlm.v10i1.1229 original research establishing the cost of xpert mtb/rif mobile testing in high-burden peri-mining communities in south africa naseem cassim, lindi m. coetzee, abel l. makuraj, wendy s. stevens, deborah k. glencross received: 16 sept. 2020; accepted: 16 july 2021; published: 30 nov. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: globally, tuberculosis remains a major cause of mortality, with an estimated 1.3 million deaths per annum. the xpert mtb/rif assay is used as the initial diagnostic test in the tuberculosis diagnostic algorithm. to extend the national tuberculosis testing programme in south africa, mobile units fitted with the genexpert equipment were introduced to high-burden peri-mining communities. objective: this study sought to assess the cost of mobile testing compared to traditional laboratory-based testing in a peri-mining community setting. methods: actual cost data for mobile and laboratory-based xpert mtb/rif testing from 2018 were analysed using a bottom-up ingredients-based approach to establish the annual equivalent cost and the cost per result. historical cost data were obtained from supplier quotations and the local enterprise resource planning system. costs were obtained in rand and reported in united states dollars (usd). results: the mobile units performed 4866 tests with an overall cost per result of $49.16. staffing accounted for 30.7% of this cost, while reagents and laboratory equipment accounted for 20.7% and 20.8%. the cost per result of traditional laboratory-based testing was $15.44 us dollars (usd). the cost for identifying a tuberculosis-positive result using mobile testing was $439.58 usd per case, compared to $164.95 usd with laboratory-based testing. conclusion: mobile testing is substantially more expensive than traditional laboratory services but offers benefits for rapid tuberculosis case detection and same-day antiretroviral therapy initiation. mobile tuberculosis testing should however be reserved for high-burden communities with limited access to laboratory testing where immediate intervention can benefit patient outcomes. keywords: genexpert test, tuberculosis screening, mobile testing, costing. introduction globally, tuberculosis is one of the top 10 causes of mortality.1 in 2017, tuberculosis infected about 10 million individuals and accounted for an estimated 1.3 million deaths among hiv-negative people, with an additional 300 000 deaths among people living with hiv.1 the epidemiology of tuberculosis varies widely between countries. in 2017, the tuberculosis incidence in most high-income countries was under 10 tuberculosis cases per 100 000 population compared to between 150 and 400 tuberculosis cases per 100 000 population in most of the top 30 high-burden countries.1,2 countries such as south africa (567), mozambique (551) and the philippines (554) reported over 500 cases per 100 000 population.1 as reported by the world health organization, there were 227 224 new cases of tuberculosis in south africa in 2017. although 322 000 cases of active tuberculosis were diagnosed in 2017 in south africa, only 65% of the cases were bacteriologically confirmed, with a treatment coverage of 68% (95% confidence interval [ci]: 51–96).3 clinically, a patient is suspected of having tuberculosis based on the following symptoms: persistent cough of 2 weeks or more, persistent cough of any duration for hiv-positive individuals, fever for over 2 weeks, night sweats, and unexplained weight loss (≥ 1.5 kg within 1 month).4 tuberculosis can present with different symptoms and atypical radiologic findings, and the pathological diagnosis has historically been based on acid-fast bacilli smear microscopy and bacteriological culture.5 the latter has a higher sensitivity for diagnosing and confirming active tuberculosis than acid-fast bacilli smear microscopy.5 the development of polymerase chain reaction tuberculosis assays has improved tuberculosis diagnosis and facilitates early treatment initiation by significantly reducing the time to result to 2 h, compared to 6 months for bacteriological culture.6 in south africa, the xpert mtb/rif polymerase chain reaction assay (cepheid, california, united states) is used routinely for tuberculosis diagnosis using patient sputum. test results, which determine the therapeutic intervention and management in line with the diagnostic algorithm, are returned within two days.7 tuberculosis incidence rates globally are especially high in the mining sector. in gold mines around the world, an estimated 3000 per 100 000 population are infected.8 in south africa, the mining sector accounted for 7.5% of the national gross domestic product, employing 495 592 workers in 2014.9 mining activities and environments are associated with a high risk of hiv and tuberculosis transmission and the migration of miners to their place of work is known to disrupt tuberculosis detection and care.10,11 given the higher rates of tuberculosis transmission in mines, it is anticipated that the communities where miners live, the so-called peri-mining communities, would also have higher tuberculosis incidence rates. due to the higher burden of disease among miners, a framework to address tuberculosis in the mining sector was developed for the southern african development community in 2014.11 in march 2015, a comprehensive tuberculosis campaign targeted at inmates in correctional services prisons, mine workers and peri-mining communities was launched in south africa under the banner ‘ending sa [south africa] tuberculosis epidemic: accelerating the response in key populations’.12 in response to this call and through the support of the global fund, the national health laboratory service and its clinical partner, the aurum institute, introduced a funded mobile genexpert testing facility to improve tuberculosis diagnosis in peri-mining communities.13 this initiative aimed to increase resources to deal with three of the world’s most devastating diseases (hiv and aids, tuberculosis and malaria) by focusing on the areas of greatest need.13 mobile testing was targeted at communities with a high burden of disease (high tuberculosis or hiv prevalence) and little or no access to laboratory testing facilities. these included remote areas of the north west and limpopo provinces in south africa between 2016 and 2019.13 the step-by-step approach to introducing mobile testing included identification of testing needs, execution of a feasibility study, procurement of funding, conducting of the necessary steps and processes to prepare for testing (setup of vehicles and equipment), assay verification, training, competency assessment, identification of measurable outcomes for monitoring, and commencement of testing. various studies have demonstrated that mobile testing is feasible, improves access to diagnostics, and may improve linkage to care and decrease time to treatment.14,15,16,17 a local study has reported that linkage to tuberculosis treatment was not associated with either sex or service type (mobile versus stand-alone), but older patients were less likely to be linked to tuberculosis treatment.15 mobile testing for hiv, tuberculosis and, more recently, severe acute respiratory syndrome coronavirus 2 can bring diagnostics to where it is needed in high-burden or outbreak communities.18 as previously reported in a local study to evaluate mobile versus traditional laboratory cd4 testing, mobile diagnostics could be substantially more expensive.19 mobile testing is not widely used in south africa, with its use limited to pilot projects or funded studies. however, it should be possible to integrate mobile testing as part of a national tiered laboratory network to extend services20 and absorb the higher cost of mobile testing into the national laboratory expenditure allocations. there is limited local data on the cost to provide mobile xpert mtb/rif testing in high-burden communities. only one local study reported that the cost to detect one tuberculosis case was $1117.00 united states dollars (usd)based on 1385 patients enrolled.16 the paucity of local data for mobile tuberculosis testing highlights the need for a comprehensive costing study, which could inform the modalities of providing these services and identify scenarios that are best suited for on-site testing. the objective of this study was to determine the cost per result and cost per positive result of mobile xpert mtb/rif testing and to compare it to the cost of traditional laboratory-based testing. methods ethical considerations ethics clearance was obtained from the university of the witwatersrand (reference number: m160978). our study did not contain any patient identifiers. no patient consent was required. context the national health laboratory service implemented mobile testing in three high-tuberculosis-burden districts in south africa (kenneth kaunda, north west, waterberg, limpopo, and sekhukhune, limpopo). traditional laboratory-based testing was conducted at the potchefstroom laboratory, a clinical pathology district laboratory offering a basic repertoire of testing, including tuberculosis testing, in the kenneth kaunda district. costing methodology the costing analysis was undertaken using microsoft excel (redmond, washington, united states).21 a bottom-up costing approach was used to determine the cost per result from a provider perspective; all costs are reported for the national health laboratory service as the provider of mobile tuberculosis testing. all costs (excluding value-added tax) were obtained in south african rand and reported in united states dollars, with an exchange rate of r14.4838 south african rand (zar) to the dollar.22 the main outcome of interest was the cost per result. the ingredients-based costing approach established annual equivalent costs (aec) for the following categories of mobile testing: staff (medical technologist and driver), reagents, external quality assurance, vehicle purchase, vehicle operations, laboratory equipment, and coordinator costs to manage testing. for the costing of the traditional laboratory-based xpert mtb/rif testing, we reported the following cost categories: staff (medical technologist), reagents, external quality assurance, laboratory equipment, courier logistics, and coordinator costs to manage testing. all laboratory equipment was purchased outright. for traditional laboratory testing, a placement agreement includes the costs for regular maintenance and servicing of the analyser. all data are reported for the 2018 calendar year. the consolidated health economic evaluation reporting standards checklist was used in the preparation of the manuscript.23 for laboratory equipment costing, useful life, which refers to the projected lifespan of depreciable equipment, was set at seven years, with a discount rate of 4%. for the calculation of staff costs, we determined the full-time equivalent hours (the number of hours worked by an employee divided by the number of hours worked by a full-time employee) based on the amount of time employees were assigned to mobile testing and multiplied this by the annual cost to company salary scales to determine the aec. reagent and test consumable costs were obtained from quotations received from the oracle enterprise resource planning system used by the national health laboratory service, and the aec was determined using annual test volumes.24 for external quality assurance, the frequency of panel testing and the number of samples prepared were used to calculate the aec per site, that is, panels were sent out quarterly, with three samples per instrument. the aecs for vehicle purchase, vehicle operations, laboratory equipment and the coordinator costs were also determined and are described in more detail below. start-up costs were defined as all aecs associated with the purchase of the mobile vehicle and laboratory equipment. the total cost per result minus the contribution of start-up costs was also determined. we reported the cost per positive result (the cost to find one tuberculosis-positive case) for both mobile and laboratory tuberculosis testing. this was calculated as the aec divided by the number of tuberculosis-positive results. for mobile testing, it was also possible to use the clinical outcomes data to estimate the diagnostic cost per tuberculosis-positive patient, as well as the cost per patient initiated on treatment (calculated as aec divided by the number of people that received treatment). mobile xpert mtb/rif costing the costs for the initial start-up of the mobile service were determined and included the costs for the purchase of the vehicles, modifications made to the mobile units (benches, air conditioning), and purchase and placement of equipment on the mobile units. the mobile units were equipped with genexpert platform instruments (cepheid, sunnyvale, california, united states). this is an automated real-time polymerase chain reaction test for the simultaneous detection of tuberculosis and rifampicin resistance.25 four genexpert instruments, as well as one computer per analyser, were placed in each mobile unit for a combined daily testing capacity of 64 samples. operational vehicle costs were included in the cost per result and comprised maintenance, fuel, repairs, and annual licensing costs. additional operational costs included costs for procurement of reagents, consumables, specimen collection and quality control materials (internal and external schemes), as well as other miscellaneous costs such as for printing of results. each mobile testing unit required a driver and a medical technologist. the percentage of time spent offering mobile testing was used for full-time equivalent calculations, ranging from 40% to 80%. the cost to company salary for a coordinator was calculated using historical expenditure data. the aecs for travel, office setup, miscellaneous costs and coordinator costs were also determined (total aec divided by the number of mobile testing sites). the test volumes and number of positive results for each mobile unit were reported using bar charts, with the total cost per result presented as a line chart on the secondary y-axis. the cost per result without start-up costs and the cost per kilometre were also reported. the number of site visits and kilometres travelled were indicated as text on the charts. for the three mobile units, we reported the correlation between the cost per result and distance travelled. laboratory-based xpert mtb/rif comparative costing as a comparator, the cost per result was determined for traditional laboratory-based xpert mtb/rif testing. initial laboratory setup included the installation of the four genexpert systems (cepheid, sunnyvale, california, united states) (capacity of 64 samples per day), an air conditioner, a level two biosafety hood and a vortex mixer. operational costs included costs to procure reagents, consumables, specimen collection materials, quality control materials (internal and external), printer cartridges and paper. the assumptions for these operational costs were similar to those for mobile testing. the staff complement required to perform mobile testing included a medical technologist and a laboratory manager, who provided minimal supervision. the technologists performed other testing in addition to xpert mtb/rif. the costs of the business management unit (coordinator costs) in the north west province were determined and included the following personnel: business manager, secretary, quality assurance coordinator, human resources officers, training staff, and other support staff. to determine the coordinator costs per result, the aec was divided by the annual test volume for the province. for the courier costs, the annual expenditure for the laboratory was used. results the three mobile units performed 4866 tuberculosis tests, of which the majority were performed by mobile unit 1 (68.7%). the mobile units covered a total distance of 64 605 km, with mobile units 3 and 1 contributing 73.7% of all travel. a total of 258 healthcare facilities were visited, evenly distributed between the three units. there were 544 tuberculosis-positive samples reported, with an overall tuberculosis positivity of 11.2%. the tuberculosis positivity was 9.6% for mobile unit 1, 16.6% for mobile unit 2, and 10.7% for mobile unit 3. for the period reported, 11 603 tests were done at the potchefstroom laboratory, of which 1086 were positive (9.4%). mobile testing costs the overall cost per result for mobile testing was $49.16 usd with an aec of $239 130.00 usd (table 1). without the start-up costs, the overall cost per result decreased to $31.11 usd. a breakdown of cost contributors showed that staff accounted for 30.7%, primarily due to the cost per result ($11.69 usd; 23.8%) of the medical technologist performing the test. reagents accounted for 20.7% ($10.16 usd), while vehicle operation costs made up 3.6% ($1.76 usd) of the overall cost per result. specimen collection and external quality control only contributed 0.5% ($0.27 usd) to the final cost per result. the aec for reagents, staffing and laboratory equipment made up 72.2% of the total cost. the start-up costs, which comprised the costs to purchase the mobile vehicle and laboratory equipment, accounted for 36.7% ($87 804.00 usd) of the total cost of mobile testing. these initial costs need to be considered when mobile units are rolled out without links to an established laboratory network or testing programme. the cost per result for the three mobile units ranged from $30.22 usd to $154.31 usd. without the start-up costs, the cost per result ranged from $21.47 usd to $95.06 usd (figure 1). figure 1: number of tuberculosis tests performed (dark blue bars) by mobile xpert mtb/rif testing units in high-burden peri-mining communities in south africa, 2018. positive results (red bars) are reported on the primary y-axis. on the secondary y-axis, the green line indicates the total cost per result in usd, the purple line indicates the total cost per result less start-up costs, and the orange line indicates the cost per kilometre travelled. the number of site visits for testing and the total distance travelled for those visits are indicated as text for each mobile unit. table 1: comparison of cost per result between mobile xpert mtb/rif testing in high-burden peri-mining communities and traditional laboratory-based xpert mtb/rif testing offered at a laboratory in the kenneth kaunda district in south africa, 2018. effect of distance travelled on the cost per result the three mobile units covered distances of 21 766 km, 16 985 km and 25 854 km. the estimated overall cost per kilometre was $2.34 usd, with mobile unit 2 accounting for the highest cost per kilometre ($8.91 usd). the number of health clinics visited by the mobile units ranged from 79 to 90 clinics. the correlation between the cost per result and distance travelled was not statistically significant (p = 0.053), with a perfect negative correlation reported (−1.0000). cost per positive tuberculosis result the aec for offering mobile testing was $239 130.78 usd to produce 4866 results. there were 544 positive results (11.2%), with 300 patients documented as having received tuberculosis treatment (55.1%). the cost to find one positive tuberculosis case using mobile testing was $439.58 usd and the cost of initiating a positive patient on treatment was $797.10 usd (table 1). comparative costing analysis the overall cost per result for laboratory-based xpert mtb/rif testing was $15.44 usd (table 1). equipment for laboratory testing is procured through a national tender process, that is, there are no costs for installation and maintenance of adequate testing platforms. reagent costs were similar to that of mobile testing and accounted for 65.8% of the total cost per result. staff costs contributed $1.62 usd (10.5%) to the cost per result. for specimen collection materials, the cost per result was $0.34 usd (2.2%); for test consumables, the cost was $1.61 usd (10.4%); for external quality assurance, the cost was $0.02 usd (0.1%); for laboratory equipment, the cost was $1.37 usd (8.9%); for the coordinator, the cost was $0.06 usd (0.4%). the courier costs contributed $0.26 usd (1.7%) to the total cost per result. the aec for laboratory-based testing was $179 132.08 usd to produce 11 603 results. the cost to find one positive tuberculosis case was $164.95 usd. unfortunately, the number of patients with a laboratory test result who received tuberculosis treatment was not available. discussion mobile diagnostics for high burden diseases such as tuberculosis can provide significant public health and epidemiological value in regions where individuals do not have easy access to laboratory facilities. overall, the average cost per result for all three mobile units was $49.16 usd. however, the cost per result ranged from $30.22 usd to $154.31 usd, highlighting differences in how and where mobile testing was offered. the biggest contributors to cost differences were test volumes and distance travelled. for example, mobile unit 1 performed the most testing with short travel distances and reported the lowest cost per result. in contrast, mobile unit 3 served a very remote area with longer travel times and had the highest cost per result. staff, reagents, laboratory equipment and vehicle purchase contributed 88.1% of the total cost per result. this indicates that the majority of costs associated with mobile testing are not flexible, and suggests that the cost of mobile testing could only be reduced by increasing test volumes, reducing input costs or widening the test repertoire. test volumes could be increased by identifying clinical settings with higher test volumes that would maximise the use of mobile testing. test volumes are however limited by the daily throughput of the testing platform and space on the mobile units for multiple units of the test platforms. negotiations with suppliers could result in lower reagent and consumable pricing. in addition, by adding mobile testing to the existing traditional laboratory national tenders, the placement agreement for reagents and analysers could be extended to mobile testing. the higher test volumes would lower the unit costs of the traditional laboratory supply chain management agreements and, by extension, benefit mobile testing. various point-of-care platforms with a very small footprint could be used to offer additional routine haematology and chemical pathology mobile testing.26 these could be used to facilitate the fast-tracking of antiretroviral therapy for patients with tuberculosis and hiv.27 a wide range of tuberculosis positivity rates were reported for the three mobile units in this study. this highlights the importance of identifying high-burden settings with high tuberculosis prevalence for effective deployment of mobile testing. the reported cost to find a single tuberculosis-positive case would vary substantially based on the setting where testing is offered. offering mobile testing in high-burden areas with a large population would substantially reduce the overall diagnostic cost and simultaneously offer immediate access to treatment. the higher cost of mobile testing should be weighed against the impact of earlier diagnosis, improved coverage, same-day treatment and care, as well as reduced loss to follow-up.17,28,29,30,31 mobile testing as an extension of laboratory testing could also see its higher costs offset by high volume laboratory testing, as bulk testing is still reserved for the laboratory service. the findings of this study confirmed that mobile testing is 3.2 times more expensive than conventional laboratory testing on the same genexpert testing platform. some of the reasons for the higher cost per result for mobile testing include lower test volumes, lost time due to travel to the health facility, and the impact of the clinical workflow on sample collection. an earlier study conducted to determine the cost of providing mobile cd4 testing in pixley ka seme in the northern cape of south africa also reported a substantially higher cost for mobile testing versus laboratory testing.19 in such remote areas, the cost of mobile testing should be weighed against improving sample collection and distribution routes to the nearest testing laboratory. for mobile tuberculosis testing, scenarios should be identified that match the increased costs of mobile testing with improved patient outcomes such as rapid tuberculosis case identification and same-day antiretroviral therapy initiation. a clinical outcome study should be embedded within any future mobile testing to assess the impact on patient outcomes. similarly, detailed cost-effectiveness studies are needed to provide evidence of how mobile tuberculosis testing can save lives and fully realise the potential of targeting high-risk groups. limitations this study used actual costs from the 2018 calendar year that would be more accurate than a desktop exercise. however, some staffing estimates are based on the typical number of days of mobile testing and this could have underestimated the costs. more so, the costs reported are based on the clinical referral of patients for testing. in a different clinical scenario with higher patient volumes, the costs could be very different. there are several assumptions made for this costing analysis that could have affected the reported cost per result. the number of xpert platforms in each mobile unit, the level and type of staff employed (technologist versus technician), full-time equivalent assumptions, and the exclusion of some costs, such as overheads, would affect the reported cost per result. conclusion this study reported that mobile tuberculosis testing is more expensive than traditional laboratory testing. however, mobile testing holds the potential to offer rapid tuberculosis case detection and improve coverage and diagnostics in communities with a high burden of disease. furthermore, mobiles could be dovetailed to be used to deliver same-day antiretroviral therapy initiation. further cost-effectiveness studies are needed using the patient outcome data reported. acknowledgements the authors thank the staff that operated the mobile units. we also wish to thank the global fund for making this project possible and the aurum institute (clinical partner). competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions n.c. and l.m.c. designed the study, developed the methodology and conducted the research. n.c. conducted the costing analysis. a.l.m. provided the data required for the costing analysis. d.k.g. and w.s.s. provided editorial comments and technical input. d.k.g. supervised the study by providing leadership and oversight as the project leader. all authors reviewed the results and contributed to the manuscript development. sources of support no funding was obtained for this study. the global fund to fight aids, tuberculosis and malaria covered the cost of mobile testing in the peri-mining communities (zaf-c-ndoh [national department of health]). data availability the authors do not have permission to share the data used for this study. disclaimer the authors declare that the views expressed in the submitted article are our own and not the official position of any institution or funder. references world health organization (who). global tuberculosis report [homepage on the internet]. geneva: world health organization; 2018 [cited 2019 jul 29]. available from: https://www.who.int/tb/publications/global_report/en/ world health organization (who). use of high burden country lists for tb by who in the post-2015 era: summary [homepage on the internet]. geneva: world health organization; 2015 [cited 2019 jul 29]. available from: https://www.who.int/tb/publications/global_report/high_tb_burdencountrylists2016-2020.pdf world health organization (who). south africa: tuberculosis profile [homepage on the internet]. geneva: world health organization; 2017 [cited 2019 jul 29]. available from: https://extranet.who.int/sree/reports?op=replet&name=/who_hq_reports/g2/prod/ext/tbcountryprofile&iso2=za&outtype=pdf national department of health (ndoh). national tuberculosis management guidelines [homepage on the internet]. pretoria: national department of health; 2014 [cited 2019 aug 01]. available from: http://www.tbonline.info/media/uploads/documents/ntcp_adult_tb-guidelines-27.5.2014.pdf ryu yj. diagnosis of pulmonary tuberculosis: recent advances and diagnostic algorithms. tuberc respir dis (seoul). 2015;78(2):64–71. https://doi.org.10.4046/trd.2015.78.2.64 singer-leshinsky s. pulmonary tuberculosis: improving diagnosis and management. jaapa. 2016;29(2):20–25. https://doi.org.10.1097/01.jaa.0000476207.96819.a7 national department of health (ndoh). national consolidated guidelines for the prevention of mother-to-child transmission of hiv (pmtct) and the management of hiv in children, adolescents and adults [homepage on the internet]. pretoria, 2015 [cited 2019 jun 10]; p. 128. available from: http://www.health.gov.za/index.php/2014-03-17-09-09-38/policies-and-guidelines/category/230-2015p?download=937:national-art-guidelines-2015final chang st, chihota vn, fielding kl, et al. small contribution of gold mines to the ongoing tuberculosis epidemic in south africa: a modeling-based study. bmc med. 2018;16(1):52. https://doi.org.10.1186/s12916-018-1037-3 u.s. department of the interior, u.s. geological survey. 2014 minerals yearbook [homepage on the internet]. u.s. department of the interior u.s. geological survey; 2017 [cited 2019 aug 01]. available from: https://s3-us-west-2.amazonaws.com/prd-wret/assets/palladium/production/mineral-pubs/country/2014/myb3-2014-sf.pdf stuckler d, basu s, mckee m, lurie m. mining and risk of tuberculosis in sub-saharan africa. am j public health. 2011;101(3):524–530. https://doi.org.10.2105/ajph.2009.175646 churchyard gj, mametja ld, mvusi l, et al. tuberculosis control in south africa: successes, challenges and recommendations. s afr med j. 2014;104(3):244–248. https://doi.org.10.7196/samj.7689 south african government. ending south africa’s tb epidemic: accelerating our response in key populations [homepage on the internet]. pretoria: south african government; 2015 [cited 2019 aug 01]. available from: https://www.gov.za/world-tb-day-2015 national department of health (ndoh). global fund grants operations manual for national department of health [homepage on the internet]. pretoria: national department of health; 2016 [cited 2019 aug 01]. available from: https://www.theglobalfund.org/media/6586/oig_gf-oig-17-014_report_en.pdf sloot r, glenshaw mt, van niekerk m, meehan s-a. rapid point-of-care cd4 testing at mobile units and linkage to hiv care: an evaluation of community-based mobile hiv testing services in south africa. bmc public health. 2020;20(1):528. https://doi.org.10.1186/s12889-020-08643-3 meehan s-a, sloot r, draper hr, naidoo p, burger r, beyers n. factors associated with linkage to hiv care and tb treatment at community-based hiv testing services in cape town, south africa. plos one. 2018;13(4):e0195208. https://doi.org.10.1371/journal.pone.0195208 kranzer k, lawn sd, meyer-rath g, et al. feasibility, yield, and cost of active tuberculosis case finding linked to a mobile hiv service in cape town, south africa: a cross-sectional study. plos med. 2012;9(8):e1001281. https://doi.org.10.1371/journal.pmed.1001281 bassett iv, forman ls, govere s, et al. test and treat tb: a pilot trial of genexpert mtb/rif screening on a mobile hiv testing unit in south africa. bmc infect dis. 2019;19(1):110. https://doi.org.10.1186/s12879-019-3738-4 abdool karim ss. the south african response to the pandemic. n engl j med. 2020;382(24):e95. https://doi.org.10.1056/nejmc2014960 coetzee lm, cassim n, glencross dk. a cost analyses of mobile laboratory cd4 testing in a national health insurance (nhi) pilot site [homepage on the internet]. cape town: african society for laboratory medicine; 2012 [cited 2020 dec 12]. available from: https://www.researchgate.net/publication/267651724_a_cost_analyses_of_mobile_laboratory_cd4_testing_in_a_national_health_insurance_nhi_pilot_site glencross dk, coetzee lm, cassim n. an integrated tiered service delivery model (itsdm) based on local cd4 testing demands can improve turn-around times and save costs whilst ensuring accessible and scalable cd4 services across a national programme. plos one. 2014;9(12):e114727. https://doi.org.10.1371/journal.pone.0114727 microsoft corporation. office 365. redmond, wa: microsoft corporation, 2019. standard bank of south africa (sbsa). forex closing indication rates for 24 april 2019 as at 16:08 [homepage on the internet]. johannesburg: standard bank of south africa; 2019 [cited 2019 apr 24]. available from: https://ws15.standardbank.co.za/finsnapshot/getforexservlet husereau d, drummond m, petrou s, et al. consolidated health economic evaluation reporting standards (cheers) statement. int j technol assess health care. 2013;29(2):117–122. https://doi.org.10.1017/s0266462313000160 national health laboratory service (nhls). annual report 2017/18 [homepage on the internet]. johannesburg: national health laboratory service; 2018 [cited 2019 oct 08]. available from: https://nationalgovernment.co.za/entity_annual/1714/2018-national-health-laboratory-service-(nhls)-annual-report.pdf schnippel k, meyer-rath g, long l, et al. scaling up xpert mtb/rif technology: the costs of laboratoryvs. clinic-based roll-out in south africa. trop med int health. 2012;17(9):1142–1151. https://doi.org.10.1111/j.1365-3156.2012.03028.x martin cl. i-stat – combining chemistry and haematology in poct. clin biochem rev. 2010;31(3):81–84. cassim n, coetzee lm, stevens ws, glencross dk. addressing antiretroviral therapy-related diagnostic coverage gaps across south africa using a programmatic approach. afr j lab med. 2018;7(1):681. https://doi.org.10.4102/ajlm.v7i1.681 govindasamy d, van schaik n, kranzer k, wood r, mathews c, bekker lg. linkage to hiv care from a mobile testing unit in south africa by different cd4 count strata. j acquir immune defic syndr. 2011;58(3):344–352. https://doi.org.10.1097/qai.0b013e31822e0c4c grolla a, jones s, kobinger g, et al. flexibility of mobile laboratory unit in support of patient management during the 2007 ebola-zaire outbreak in the democratic republic of congo. zoonoses public health. 2012;59 suppl 2:151–157. https://doi.org.10.1111/j.1863-2378.2012.01477.x larson ba, schnippel k, ndibongo b, et al. rapid point-of-care cd4 testing at mobile hiv testing sites to increase linkage to care: an evaluation of a pilot program in south africa. j acquir immune defic syndr. 2012;61(2):e13–e17. https://doi.org.10.1097/qai.0b013e31825eec60 van schaik n, kranzer k, wood r, bekker lg. earlier hiv diagnosis – are mobile services the answer? s afr med j. 2010;100(10):671–674. https://doi.org.10.7196/samj.4162 abstract introduction methods results discussion acknowledgements references about the author(s) kafui c. kouassi unity of external quality assessement, division of laboratories, ministry of health and public hygiene, lomé, togo medical and biological analysis-biochemistry, higher school of biological and food techniques, university of lomé, lomé, togo améyo m. dorkenoo division of laboratories, ministry of health and public hygiene, lomé, togo department of health sciences, faculty of health sciences, university of lomé, lomé, togo komivi gbada division of laboratories, ministry of health and public hygiene, lomé, togo lomé commune regional hospital, ministry of health and public hygiene, lomé, togo yaovi-gameli afanyibo division of laboratories, ministry of health and public hygiene, lomé, togo national institute of hygene, ministry of health and public hygiene, lomé, togo minogblon têko division of laboratories, ministry of health and public hygiene, lomé, togo bè secondary hospital, ministry of health and public hygiene, lomé, togo adjane koura division of laboratories – resaolab, ministry of health and public hygiene, lomé, togo citation kouassi kc, dorkenoo am, gbada k, afanyibo y-g, têko m, koura a. the togo national proficiency test pilot programme for basic clinical chemistry tests. afr j lab med. 2022;11(1), a1565. https://doi.org/10.4102/ajlm.v11i1.1565 original research the togo national proficiency test pilot programme for basic clinical chemistry tests kafui c. kouassi, améyo m. dorkenoo, komivi gbada, yaovi-gameli afanyibo, minogblon têko, adjane koura received: 24 feb. 2021; accepted: 09 mar. 2022; published: 24 june 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: a national proficiency test (pt) programme is not currently implemented in most low-income countries. however, participation in such pt programmes assists improves test performance and result accuracy. objective: this study assessed how well 11 government hospital laboratories performed 18 basic clinical chemistry tests and identified areas needing improvement. methods: a cross-sectional study was carried out by the division of laboratories of the ministry of health of togo from 01 july 2016 to 31 december 2016. the test performance was evaluated using panels provided by one world accuracy, canada (vancouver). the clinical laboratory improvement amendments criteria were used in evaluating the laboratories, and their success rates were compared with the world health organization regional office for africa’s target of 80%. results: the overall rate of acceptable results at the laboratories was over 80% for glucose, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, alkaline phosphatase and triglycerides tests. the laboratories using fully automated spectrophotometers had an acceptable results rate of 89% (p = 0.001). the overall performance of the laboratories by cycles varied from 71% to 82%. conclusion: this national pt programme identified the tests, which laboratories must improve their performance (urea, creatinine, uric acid, bilirubin, cholesterol, total protein, calcium, magnesium, phosphorus). it demonstrated the need for the use of routine appropriate internal quality control in all laboratories. the proficiency test programme should be extended to all clinical laboratories and target all biology disciplines. keywords: quality control; biochemistry; laboratory; performance; togo. introduction clinical laboratory test results are for screening, diagnosis, prognosis, therapeutic monitoring of chronic pathologies and epidemiology surveillance.1 these results are more reliable when internal quality control (iqc) and external quality assessment (eqa) are implemented in the clinical laboratories. a proficiency test (pt) is one form of eqa that uses pre-established criteria to evaluate the performance of a participating laboratory (pl) compared with other laboratories.2 the inter-laboratory comparison, audit, and accreditation culture has not yet taken root in low-income countries, such as togo, with a gross national income per capita of $1035 or less. a 2013 survey reported that more than 90% of african countries had no accredited laboratories meeting the international organization for standardization standard 15189 (iso 15189), the international quality standard for clinical laboratories.3,4 clinical biochemistry tests remain predominant in the management of pathologies, whether resulting from transmissible or non-transmissible diseases.5,6 it is therefore essential that the results of these clinical chemistry tests are accurate. the participation of government hospital laboratories in a national pt programme as required by the iso 15189 standard could help to ensure accurate test results.7 in togo, the clinical diagnostics laboratories face many challenges, the most important of which are: obtaining market authorisation from distributors of in vitro diagnostic medical devices; implementing appropriate quality assurance processes, including iqc, and participating in a national or private eqa (pt) programme; and getting the iso 15189 accreditation (particularly for the national reference laboratories). since 2012, only three clinical laboratories in togo have been iso 15189:2012 accredited, two within the public health institute by the west african accreditation system and one private laboratory by the french accreditation committee.8,9 after a successful pt feasibility assessment in 2012 involving 11 clinical laboratories across the lomé municipalities, in 2016, the division of laboratories, ministry of health, togo, implemented a national pt programme.10 the resaolab project (west african network of clinical laboratories) implemented the pt programme, supported by fondation mérieux.11 in total, the health system of togo is composed of 179 government hospital laboratories organised in a tiered system, depending on their capabilities. a pilot phase of this programme was initiated the same year and involved 11 government hospital laboratories representing the central, intermediate and peripheral laboratory levels. the study aimed to assess how well these 11 government hospital laboratories performed 18 basic clinical chemistry tests and to identify areas where improvement may be required. methods ethical considerations this study did not involve human subjects or animal research. this pt programme is part of regular assessment activities of the ministry of health of togo and does not require any particular ethical consideration. a unique pt biological material was obtained from the canadian company one world accuracy and sent to all participating laboratories. the pt programme is part of the ministry of health’s regular assessment activities and does not require a particular ethical consideration. more so, the pt activity did not violate the current ethical considerations of the declaration of helsinki. study design a cross-sectional assessment was carried out from 01 july 2016 to 31 december 2016. the programme comprised four cycles at the rate of one cycle per month from august to november 2016. the study was conducted in three steps: (1) identification and training of participating laboratories (pls); (2) selection of tests, reception of samples at the central level, and dispatch of samples to pls; (3) collection of pt results and data evaluation. participating laboratories and personnel training eleven government hospital laboratories (representing 32% of 34 laboratories that routinely perform the 18 basic clinical chemistry tests), were enrolled in the pt programme. these 34 government hospital laboratories represent 19% of all the government hospital laboratories spread across the six health regions in togo. the pls were purposefully selected to ensure that all three levels of the health system were represented: three university hospital laboratories for the central level; six regional hospital laboratories for the intermediate level; and two district hospital laboratories for the peripheral level. these pls were randomly anonymised by numbering them 1–11. none of these pls had iso/international electrotechnical commission (iec) 15189 accreditation. a unique pt panel was obtained from oneworld accuracy® (owa) located in vancouver, british columbia, canada, and shipped by fedex® expedition services. following arrival and customs clearance, the package was immediately sent to the institut national d’hygiène, the national public health reference laboratory in togo, which is iso 15189 accredited. at institut national d’hygiène, sample integrity and adherence to temperature requirements (2 °c – 8 °c) were confirmed using a calibrated thermometer. these samples were stored for up to 24 h at 2 °c – 8 °c at the institut national d’hygiène and then sent to the pls for immediate testing upon reception. samples were transported refrigerated in individual insulated containers to each pl. the maximum transit time was 12 hours for the furthest pl. two laboratory technicians from each pl were trained by two members of the coordinating team of the national pt programme of the division of laboratories, ministry of health of togo. the training was provided by members with experience in the areas of quality management, statistical analysis, and biochemical analysis. the pl technicians were trained on pt principles under iso/iec 17043, iqc and the inter-laboratory comparison requirements in iso/iec 15189, and owa® pt pl guidelines for demonstration.2,7,12 training on the use of the oneworld accuracy system software platform (collaboration secretariat of oneworld accuracy group, vancouver, british columbia, canada), for setting measurement units, methods, equipment and test result submission, was also provided. laboratory tests and samples the 18 basic clinical chemistry tests and serum analytes selected by the ministry of health division of laboratories for this pt programme were: urea, blood glucose, creatinine, uric acid, alanine aminotransferase (enzyme commission [ec] number: 2.6.1.2 [international union of biochemistry and molecular biology, https://iubmb.qmul.ac.uk]), aspartate aminotransferase (ec 2.6.1.1), gamma-glutamyl transferase (ec 2.3.2.2), alkaline phosphatase (ec 3.1.3.1), total bilirubin, direct bilirubin, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, total protein, calcium, magnesium, and phosphorus. these tests were performed by the technicians of the 11 pls using commercially purchased reagents and iqc was used where available. analytical methods used for each test were recorded. the 11 pls used the same methods but not the same vendor kits for the following tests: urea (urease), blood glucose (glucose oxidase-hydrogen peroxide), creatinine (alkaline picrate), uric acid (uricase-hydrogen peroxide), alanine aminotransferase (international federation of clinical chemistry and laboratory medicine method without pyridoxal-5-phosphate cofactor), aspartate aminotransferase (international federation of clinical chemistry and laboratory medicine method without pyridoxal-5-phosphate cofactor), gamma-glutamyl transferase (carboxy–gamma-glutamyl-p-nitroanilide), alkaline phosphatase (p-nitrophenyl phosphate-diethanolamine), cholesterol (cholesterol oxidase-hydrogen peroxide), high-density lipoprotein cholesterol (phosphotungstic acid), low-density lipoprotein cholesterol (calculated by friedewald’s formula), triglycerides (glycerol phosphate oxidase-hydrogen peroxide), total protein (biuret), phosphorus (phosphomolybdic by ultraviolet spectrophotometry) and direct bilirubin (diazosulfanilic acid). for the three remaining tests (total bilirubin, calcium and magnesium), two different methods (a and b) were used by the pls (table 1). table 1: clinical chemistry tests with two different methods used in togo from july 2016 to december 2016. calibration traceability information was stated in the package inserts and all assays were traceable to an appropriate international reference standard or method. tests were performed by a fully automated or semi-automated spectrophotometer, depending on the analyser available in each pl (table 2). table 2: spectrophotometers and brands of analysers and reagents used by the participating laboratories in togo from july 2016 to december 2016. the sample analysed by each pl consisted of a lyophilised multiparametric serum supplied by owa. in the four cycles of this pilot programme, the pls received pt samples of different ranges in each cycle. five millilitre vials of philco water 5® brand distilled water (philco pharma carsten, grosshansdorf, germany) was provided for the reconstitution of samples using a volumetric micropipette. proficiency test data collection participating laboratories were instructed to test each sample in the same manner as patient samples. results were documented and sent to owa for analysis, with the final pt reports typically being received from owa within 15 days of sample receipt in togo. a whatsapp group (whatsapp llc, menlo park, california, united states) including all 11 pls and staff from the coordinating team was created to monitor the timely submission of results and pt reports. this whatsapp group was also used to discuss the implementation of corrective actions when a result was unacceptable or when a pl had issues with continuing the programme. the corrective actions were tracked using a ‘non-conformity management sheet’. data evaluation and performances criteria the pls were divided into two main groups to determine and compare their pt performance: one group of pls used fully automated spectrophotometers and the second group used semi-automated spectrophotometers and volumetric micropipettes. in addition, the performance of labs that used iqc was compared with that of labs that did not, evaluating the impact of iqc. the performance measurements included the overall performance of pls by cycle, the performance of the pls in each test across the four cycles, the performance of labs using automated or semi-automated spectrophotometers, and the adherence to the iqc process as identified through the whatsapp group discussions. the evaluation criteria were based on the clinical laboratory improvement amendments (clia) acceptable limits. this acceptable limit corresponded to the owa peer group mean ± (allowable total error) defined by clia.13 the total error of all the 18 tests was given in plus or minus percentage (± %) except for urea and calcium, which were expressed as an absolute value.2,14 the pt reports were qualified as acceptable when the results of the test provided by the pl were within the acceptability limits. for tests where there were no clia criteria, such as gamma-glutamyl transferase and direct bilirubin, owa used the peer group mean ± 2 s.d. (standard deviation) to determine the acceptability limits. allowable error rates were determined by owa for each test and stated on the pt reports for each pl during each cycle. statistical data analysis results of all pls were sent both to pls and directly to the ministry of health division of laboratories by owa in a microsoft excel file (microsoft, redmond, washington, united states), with the quantitative results of the pls identified as compliant or non-compliant. the pt results of each pl, with the assigned values of each test, were also checked by the pt coordinating team as recommended in the iso 13528 guideline.15 the data were collated and analysed using epi-info software version 3.5.3 (2011, centers for disease control and prevention, atlanta, georgia, united states). the calculation of the number of compliant results for a test or an identified group was used to determine the compliance rates in percentage (%). the performance rates of identified groups were compared using the uncorrected chi-square test or fisher’s exact test where appropriate. the same statistical method was used in comparing the number and percentage of acceptable results between two successive cycles. the target score of acceptable results was 80% as recommended by the world health organization regional office for africa.16 a cycle participation rate of 100% was expected from all pls. the p-value significance level was < 0.05. results laboratory participation rate each pl submitted results after performing all 18 tests. a cycle participation rate of 100% was obtained by nine pls (82%). the participation rate for the two remaining pls was 50% for pl4 and 25% for pl9. overall analytical performance of participating laboratories in performing tests seventy-six per cent of 775 results were acceptable. the performance scores for urea and direct bilirubin tests were less than 60%. the pls had a performance score above 80% for blood glucose, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transferase, alkaline phosphatase and triglycerides (figure 1). figure 1: analytical performance score of participating laboratories for each test performed in togo from july 2016 to december 2016. comparing the overall analytical performance between cycles assessing the overall performance level by cycle provides a measurement for the rate of improvement. the performance score increased from 71% to 81% (table 3). eight corrective actions were implemented at least once during the four cycles: providing a refrigerator thermometer to monitor the cold chain for better preservation of reagents and samples; planning a daily temperature measurement and performing root cause analysis for any observed deviations relating to out-of-range temperatures; purchasing new refrigerators suitable for laboratory use; calibrating micropipettes; performing periodic preventive maintenance on instruments and corrective maintenance when required; running iqc after each maintenance and re-calibrating instruments when necessary; implementing the use of iqc periodically; and analysing the results of the iqc using a levey-jennings chart. table 3: comparison of participating laboratories’ performance between cycles in togo from july 2016 to december 2016. comparing participating laboratories’ performance based on the use of internal quality control out of the 11 pls, four (36%) used control sera before performing each test, but none of the sites calculated uncertainty. during cycles 2, 3 and 4, the acceptable results rate of pls using iqc serum was significantly higher than for those not using iqc (table 4). table 4: comparison of participating laboratories’ performance based on the use of internal quality control in togo from july 2016 to december 2016. rate of acceptable results based on the spectrophotometer category five out of 11 pls used a fully automated spectrophotometer. three hundred and thirteen (89%) out of 352 results produced were acceptable, against 292 (69%) acceptable results (p = 0.001) in the six pls that used semi-automated spectrophotometers. discussion this study assessed how well 11 government hospital laboratories performed 18 basic clinical chemistry tests and identified areas needing improvement may be required. this assessment focused on the most common clinical chemistry laboratory tests and therefore a logical starting point for national pt activities.17 in togo, eqa initiatives have only been possible with external funding, and are currently available for tuberculosis, malaria and hiv testing.18 on 12 august 2015, the togo ministry of health issued ministerial decree n°115/2015/msps/cab/sg, which formally adopted iso/iec 15189:2012 as the quality management system standards to be met by every clinical laboratory in the country. article 4 of this decree stipulates that: ‘directors of government hospitals and heads of clinical laboratories are required to comply with the standard requirements laid down in iso 15189’. consequently, they must start planning and budgeting for pt activities as part of the national funding priorities to ensure a sustainable pt programme. planning and budgetting is important and should be improved for a sustainable national pt programme.5 the low participation rate of pl9 in this study was caused by the breakdown of their spectrophotometer. three months was insufficient for pl9 to perform the appropriate corrective action and to participate in subsequent cycles. in addition, pl4 suffered reagentstock-outs for six tests, resulting in low participation. the scenario demonstrates the challenge of pls in low-income countries to implement corrective actions in a timely fashion, even when root causes are identified.17,19 this challenge in the present pilot study could in part be mitigated by including at least a three months delay between pt cycles. insufficient delay between cycles could also explain the lack of significant improvement between cycles. implementing corrective maintenance and servicing actions for the spectrophotometers and other equipment needs a high level of commitment from the hospital authorities in charge of clinical laboratories, as recommended by iso/iec 15189 standards.7 this commitment should be manifested through the provision of resources to ensure the availability of reagents and equipment with efficient maintenance services.19,20 in addition, a quality management system to monitor overall laboratory quality, including equipment repair, maintenance and calibration, should be implemented as required by the iso/iec 15189 regulations.16,19 the routine use of iqc by pls will also enhance laboratory performance, as shown in this study. the use of iqc material at concentrations equal to or close to clinical decision values results in improved test performance and validates reported results.7 the laboratories should consider using independent third-party control materials in place of or in addition to reagent or instrument manufacturer iqc materials. the regular review of iqc data to identify acceptable and unacceptable results as well as trends is a good indicator for measuring laboratory performance, and helps to detect performance trends that may indicate problems in the analytical system.7,20 the study results showed that pls using semi-automated spectrophotometers obtained a significantly lower rate of acceptable results than those using fully automated systems. one likely source of this additional error with semi-automated spectrophotometers is the use of micropipettes to measure sera and reagents. these volumetric micropipettes were not calibrated by an iso/iec 17025 accredited laboratory and so may not be dispensing accurate volumes. measurement instruments should be subject to initial reference calibration and regular recalibrations to monitor performance.21 also, pls using semi-automated spectrophotometers performed poorly because they failed to maintain correct assay temperature or assay incubation period and used test tubes that may not have been washed properly.22 the lower performance in urea testing was also documented in a similar study in ethiopia that used the same owa pt panel as used in this study. the performance rate for urea testing in 12 ethiopian laboratories over six cycles was 21% lower than those obtained in the present study.23 the suboptimal performance of the pls for the urea test in this assessment could be attributed to the infrequent urea calibration when using different batches of reagents. also, the failure to maintain consistent assay temperature might have contributed to the poor urea testing performance.22 generally, proficiency testing evaluation is often done by a specific instrument group or analytical method used. the pls studied utilised multiple small instruments and multiple reagent kits that are not likely to fit into a specific peer group. this could lead to poorer pt performance for urea and other tests, because the mean method utilised for comparison may not be optimal.2,24 the low-performance scores for both total and direct bilirubin can be attributed to multiple factors, such as failure to maintain consistent assay temperature to which the diazo reaction is sensitive, failure to maintain reagent cold chain, and failure to protect calibrators and specimens from exposure to light, as bilirubin is light sensitive.25 limitations the major limitations of this study are the use of multiple instruments and reagent kits by pls. other limitations of the study include the few numbers of pls because of limited funds, and the insufficiently spaced pt cycles that did not allow for corrective actions to be implemented before the subsequent pt cycle. in a future study, the impact of the implementing iso/iec 15189 requirements on pls’ performance will be evaluated with the possibility of benchmarking between the central, intermediate and peripheral health levels. conclusion this study identified areas for improvement for a national pt programme and also demonstrated the value of such work in togo. it also identified some tests (urea, creatinine, uric acid, bilirubins, cholesterols, otal protein, calcium, magnesium, phosphorus) for which laboratories must improve their performance. it showed that the use of fully automated spectrophotometers is more likely to lead to reliable test results and demonstrated the need for the use of routine appropriate iqc in all laboratories. it emphasised the necessity to plan cycles with sufficient delay for implementing sustainable corrective actions. the national pt programme should be extended to all clinical laboratories in togo with three cycles per year and should also target all clinical laboratory disciplines. acknowledgements the authors would like to thank fondation mérieux and their partners for funding the resaolab project, togo’s ministry of health, dr katawa gnatoulma, and the members of participating laboratories. competing interests the authors declare that they have no financial or personal relationship that may have inappropriately influenced them in writing this article. authors’ contributions k.c.k. and a.m.d. conceived of the presented idea; k.c.k. developed methods and followed up the field activities; k.g. and m.t. verified the analytical methods and helped participating laboratories with results submission; y-g.a. wrote the manuscript with support from a.m.d. and k.c.k.; a.k. helped supervise the project under a.m.d.’s supervision. all authors discussed the results and contributed to the final manuscript. sources of support this work was supported by fondation mérieux and partners, who funded resaolab project (phase 2). data availability the authors confirm that the data supporting the findings of this study are available within the article. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated 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[cited 2020 jul 29]. available from: https://www.soacwaas.org/internal-documents.html kouassi k, fétéké l, assignon s, dorkenoo a, napo-koura g. external quality assessment in clinical biochemistry laboratories: pilot study in 11 laboratories of lomé. ann biol clin. 2015;73(2):165–175. https://doi.org/10.1684/abc.2015.1026 fondation mérieux. report of 4th steering commitee of resaolab at dakar [homepage on the internet]. 2017. [cited 2020 feb 04]. available from: https://www.resaolab.org/wp-content/uploads/2018/05/4e-resaolab-comite-de-pilotage-senegal-2017-compte-rendu.pdf one worldaccuracy. one world accuracy® pt participating laboratory guidelines for demonstration [homepage on the internet]. [cited 2018 oct 24]. available from: https://demo.oneworldaccuracy.com/healthmetrx/secure/home.do;jsessionid=cadb0995a1e394ec622cdc7d4186fe8a.node7?method=prepare westgard j. clia requirements for analytical quality [homepage on the internet]. westgard. 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[cited 2020 mar 02]. available from: https://www.westgard.com/2019-clia-changes.htm international standardization organization. iso 13528:2015 – statistical methods for use in proficiency testing by inter-laboratory comparison. geneva; 2015. world health organization regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation in the african region (slipta) [homepage on the internet]. brazzaville: who-afro; 2015 [cited 2020 mar 02]. available from: https://www.afro.who.int/sites/default/files/2017-06/slipta-checkist0711.pdf krleza jl, celap i, tanaskovic jv. external quality assessment in laboratory medicine: external quality assessment in croatia: problems, challenges, and specific circumstances. biochem med. 2017;27(1):86–92. https://doi.org/10.11613/bm.2017.011 carter jy. external quality assessment in laboratory medicine: external quality assessment in resource–limited countries. biochem med. 2017;27(1):97–109. https://doi.org/10.11613/bm.2017.013 john nn, katy y, philip o. laboratory medicine in low-income and middle-income countries: progress and challenges. lancet. 2018 391(10133):1873–1875. https://doi.org/10.1016/s0140-6736(18)30308-8 kaseha wm, manyilizu wb. variations on external quality assessment performance of public medical laboratories: the effect of strengthening laboratory management towards. acta sci med sci. 2019;33:1–10. https://doi.org/10.31080/asms.2019.03.0419 lothar s, robert iw, craig mj, linda t, stephane m. outline of the calibration and measurement hierarchy in laboratory medecine – quality policy and definitions wg2-p-00 [homepage on the internet]. [cited 2020 feb 13]. available from: https://www.bipm.org/en/committees/jc/jctlm-wg2/wg2_quality-manual.html bertrand s. biochimie clinique. instruments et techniques de laboratoire. diagnostics médico-chirugicaux. 2nd ed. paris: maloine; 1985. ashebir g, abay s. proficiency test feedback utilization at government hospitals laboratory, addis ababa, ethiopia. j med diagn methods. 2017;6:1–8. https://doi.org/10.4172/2168-9784.1000258 kristensen gbb, meijer p. external quality assessment in laboratory medicine: interpretation of eqa results and eqa-based trouble shooting. biochem med. 2017;27(1):49–62. https://doi.org/10.11613/bm.2017.007 westwood a. the analysis of bilirubin in serum. ann clin biochem. 1991;28(2):119–130. https://doi.org/10.1177/000456329102800202 abstract introduction methods results discussion acknowledgements references about the author(s) chikwelu l. obi school of science and technology, sefako makgatho health sciences university, pretoria, south africa joshua o. olowoyo department of health sciences and the water school, florida gulf coast university, fort myers, florida, united states department of biology and environmental sciences, school of science and technology, sefako makgatho health sciences university, pretoria, south africa thembinkosi d. malevu department of physics, school of science and technology, sefako makgatho health sciences university, pretoria, south africa department of physics, school of physical and chemical sciences, north-west university, mahikeng, south africa liziwe l. mugivhisa department of biology and environmental sciences, school of science and technology, sefako makgatho health sciences university, pretoria, south africa taurai hungwe department of computer science and information technology, school of science and technology, sefako makgatho health sciences university, pretoria, south africa modupe o. ogunrombi department of clinical pharmacology and therapeutics, school of medicine, sefako makgatho health sciences university, pretoria, south africa nqobile m. mkolo department of biology and environmental sciences, school of science and technology, sefako makgatho health sciences university, pretoria, south africa citation obi cl, olowoyo jo, malevu td, et al. impact of artificial intelligence and digital technology-based diagnostic tools for communicable and non-communicable diseases in africa. afr j lab med. 2024;13(1), a2516. https://doi.org/10.4102/ajlm.v13i1.2516 note: special collection: the article is a contribution to the themed collection titled ‘transforming african laboratory diagnostic systems through digital technology and artificial intelligence’. review article impact of artificial intelligence and digital technology-based diagnostic tools for communicable and non-communicable diseases in africa chikwelu l. obi, joshua o. olowoyo, thembinkosi d. malevu, liziwe l. mugivhisa, taurai hungwe, modupe o. ogunrombi, nqobile m. mkolo received: 03 june 2024; accepted: 18 sept. 2024; published: 21 nov. 2024 copyright: © 2024. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: artificial intelligence (ai) and digital technology, as advanced human-created tools, are influencing the healthcare sector. aim: this review provides a comprehensive and structured exploration of the opportunities presented by ai and digital technology to laboratory diagnostics and management of communicable and non-communicable diseases in africa. methods: the study employed the preferred reporting items for systematic reviews, meta-analyses guidelines and bibliometric analysis as its methodological approach. peer-reviewed publications from 2000 to 2024 were retrieved from pubmed®, web of science™ and google scholar databases. results: the study incorporated a total of 1563 peer-reviewed scientific documents and, after filtration, 37 were utilised for systematic review. the findings revealed that ai and digital technology play a key role in patient management, quality assurance and laboratory operations, including healthcare decision-making, disease monitoring and prognosis. metadata reflected the disproportionate research outputs distribution across africa. in relation to non-communicable diseases, egypt, south africa, and morocco lead in cardiovascular, diabetes and cancer research. representing communicable diseases research, algeria, egypt, and south africa were prominent in hiv/aids research. south africa, nigeria, ghana, and egypt lead in malaria and tuberculosis research. conclusion: facilitation of widespread adoption of ai and digital technology in laboratory diagnostics across africa is critical for maximising patient benefits. it is recommended that governments in africa allocate more funding for infrastructure and research on ai to serve as a catalyst for innovation. what this study adds: this review provides a comprehensive and context-specific analysis of ai’s application in african healthcare. keywords: artificial intelligence; diagnostic laboratories; communicable diseases; non-communicable diseases; machine learning; deep learning; internet of things. introduction the fourth industrial revolution epitomises the current state of emerging technologies, including how the internet of things, which includes devices that can communicate, sense, and exchange data with other devices and systems through the internet, and artificial intelligence (ai) are influencing the ways we undertake different endeavours of life.1 it unravels the implications for skills development in different sectors, including healthcare. it also affords opportunities to reimagine and re-invent ways of teaching and learning in africa. the fourth industrial revolution includes digital technology and ai, gene sequencing, quantum computing, nanotechnology, and big data analytics, all of which are of significance in laboratory diagnostics.2 big data involves large volume, high speed, and multiplicity of information processing for better insights, decision-making and automation.2 quantum computing is useful in healthcare, including machine learning (ml) methods to diagnose diseases.1 artificial intelligence is an emerging area in science and technology that may soon change every aspect of human life.3 kayembe and nel define ai as computer systems that can complete complex functions related to human intelligence.1 artificial intelligence is the use of technologies that provide alternatives to human functions by allowing machines to perform effective functions that were previously carried out by humans by mimicking human action.3 from the foregoing, ai could thus be seen as a subset of digital transformation to perform different tasks that require human intelligence using computing devices such as robots.4 it is evident from the literature that ai alters the way humans perform their functions and leads to increased productivity in various sectors.5 sub-fields of ai include: machine learning, which involves pattern discovery and evaluation from data sets, leading to machines’ performance improvement as the machines learn; deep learning, a sub-field of ml, comprising neural networks to facilitate ml in order for machines to formulate their own decisions; natural language processing, a way that computers unearth data on human language to formulate decisions; and computer vision, which enables computers to acquire information and understanding from images or videos.6 apart from what has been discussed earlier, ai technologies also encompass rule-based systems, robotic processing automation, clinical decision support systems, and generative adversarial networks, among others.6 even though ai is a rapidly growing field all over the world, the adoption rate varies between developed and developing countries.7 in some countries, arguments against the use of ai include ethical use, which may violate the rights of citizens and cause loss of employment, especially for those with low skills.8 however, it has been estimated that ai will proliferate in many industries and contribute over 15.7 trillion united states dollars to the world economy by 2030.9 in 2023, business sectors in the united states, according to a report by sahni and carrus,10 are rapidly accepting the use of ai, except for the health sector, which has a low adoption rate of about 5%, even though ai is estimated to lead to 5% – 10% cost savings. in europe, the council of europe’s ai convention 2023–2024 used a human rights-based approach and reported that ai could be beneficial if formulated properly.9 in africa, ai is currently infiltrating the african system through various technologies.3,6,7 in the early 1950s to 1970s, ai was based on machine development for inferences and decisions. during the ‘ai winter’ period (1970s to 2000s), funding was reduced, and fewer developments were achieved. from the late 2000s to 2020s, there was a seminal advancement in ai and digitalised medicine became more readily available; this was prompted by improved computer hardware and software programs.6 for example, through applications of deep learning in kenya, healthcare services are now available to people without visiting hospitals or medical doctors, and in nigeria, zenvus, an electronic sensor, provides soil component information to farmers.3,6 south africa has witnessed appreciable growth in ai research and application, especially in relation to power utilities and tax compliance.11 also in south africa, the mukuru mobile phone application is assisting immigrants to send money home to their loved ones without physically visiting a bank for such transactions.11 in 2020, okolo et al. also noted that the implementation of ai in african laboratories, particularly in low-resource contexts, is a growing area of interest.12 the 2023 report of manson13 showed that ai has been included in some fields of medicine and laboratories in africa. manson also noted that ai can support knowledge-based treatment planning in the field of radiotherapy, although prior knowledge and inclusion in curricula for training and education may be important.13 the application of meaningful medical ai has been shown to occur more in developed nations in comparison to what is found in africa.8 to address this, the united nations has indicated that there is a need for all stakeholders to be brought together to deliberate on how ai can be used for the provision of critical services in the public sector so that the sustainable development goals can be achieved.8 the introduction of ai in world health organization african regions is still emerging and there is a lack of studies or research undertaken due to inadequate resources, lack of infrastructure, and lack of knowledge about ai among healthcare practitioners compared to those in better-resourced, high-income countries.14 the use of information and communications technology through management systems employing mobile tools and technologies has become the most relevant emerging trend in the monitoring of non-communicable diseases in africa due to their portability, low cost, continuous connectivity, personalised effects, and ease of use.8 nevertheless, there has been limited literature on the use of key enabling technologies and ai in sub-saharan african countries and there is still a need for further studies to show whether ai may still be crucial in the expansion of aids for diagnosis or advancing signals from the use of affordable sensors which can be produced easily.15 within the healthcare sector in africa, the use of ai is growing, though at a lower speed when compared to other regions of the world.16 in african countries, the dissemination of information on health and other areas is done via the news media, involving television and radio services using modern technology such as cell phones or other media.8,16 despite this slow development in some countries of sub-saharan africa, ai has been used in some instances to carry out various duties, which include the prediction of diseases and outbreaks, determining the extent of a disease, performing homecare and distance monitoring of patients in some settings.17 in nigeria, mali, senegal, and burkina faso, among others, scientists have developed models to monitor and predict the spread of malaria using ml.18 in south africa and zambia, according to a 2021 report by yadav,19 ai has been used to monitor and survey the epidemiology of tuberculosis, and hiv aids. in 2020, onu et al. also reported that in nigeria, a program called ‘ubenwa’ used ai to improve the diagnosis of birth asphyxia in low-income and rural areas of the country.20 one of the greatest challenges for most african countries is the current episode of brain drain, which is not limited to the health sector only. most countries in africa lose qualified healthcare personnel to wealthier countries, both within and outside the region. the use of ai in the african health sector may thus assist in filling these gaps by providing improved diagnosis, treatment, and disease monitoring programmes, especially in rural areas.21 however, there are several drawbacks in the adoption of ai in developing countries, which may include data availability, analysis, and infrastructure.22 in addition, the state of laboratories and hospitals, level of education, shortage of skills, and development of appropriate software may impact the application of ai, thus leading to an increased burden of communicable and non-communicable diseases.21,22 non-communicable diseases are diseases that are typically chronic and not directly transmitted from one person to another; they include cardiovascular disease, diabetes, and cancer.23 communicable diseases are infectious diseases which spread by contact with contaminated surfaces, bodily fluids, blood products, insect bites, or through the air.23,24 in africa, 31.4 million people die annually from non-communicable diseases, including cancer, diabetes and cardiovascular disease, with 71% of deaths in the 30–70 years age range.24 the three most prevalent communicable diseases in africa are hiv/aids, malaria and tuberculosis, and are responsible for nearly 80% of the total infectious disease burden, claiming more than 6 million people per year in africa.23,24 according to oronti,25 recently in 2024, there has been a need for the world to move away from responses to the coronavirus disease 2019 pandemic and to address the other communicable and non-communicable diseases which account for most of the expenses associated with healthcare and mortality. the united nations general assembly has proposed the sustainable development goal 3.4 target to decrease by one-third the untimely deaths from communicable and non-communicable diseases which have become a global major challenge in the past decade.26 by 2030, investments in digital health technologies, such as telemedicine, devices that can be worn, and ai, should be used to offer support to accomplish the united nations sustainable development goal for health in africa.26 one of the key aspects of this review is to highlight the current state, potential and challenges of ai and digital technology in african laboratories and hospitals, especially in the diagnosis and treatment of communicable and non-communicable diseases in the past 24 years. an overview of the healthcare system is germane to the subject matter. significance of the review the novelty of this review lies in its comprehensive and context-specific analysis of ai’s application in african healthcare. it provides a holistic overview of current technological implementations and potential benefits, with a particular focus on the unique infrastructural and socio-economic conditions of african countries. moreover, it also provides the current state of ai and digital technology, and their impact in healthcare and laboratory diagnostics of communicable and non-communicable diseases in africa. by addressing ethical considerations such as equitable access, the review underscores the necessity of robust regulatory frameworks. additionally, it offers practical, actionable recommendations for governments, educational institutions, and healthcare organisations, promoting targeted investments and initiatives to enhance ai adoption. the review also highlights significant gaps in research, setting an agenda for future efforts and encouraging collaborative research between african and international partners. this focus on reducing disparities and fostering digital inclusion aims to ensure sustainable health improvements and drive innovation in ai applications for healthcare across the continent. aim and objectives the aim is to provide a comprehensive and structured exploration of the opportunities presented by ai and digital technology to laboratory diagnostics and the management of communicable and non-communicable diseases in africa. to achieve this aim, the study will systematically identify, narrate and analyse the various opportunities of ai and digital technology in healthcare and laboratory diagnostics in africa. additionally, it will evaluate the current state of ai and digital technology in healthcare and laboratory diagnostics in africa, identify challenges, and make recommendations. methods the preferred reporting items for systematic reviews and meta-analyses standards and bibliometric analysis were employed in the study.27 this was to ensure transparency, consistency and controlled or structured reproducible review processes. inclusion and exclusion criteria, and a thorough search strategy, formed part of the methods. qualitative thematic analysis was employed for a comprehensive literature assessment on ai’s prospects for laboratory diagnostics of communicable and non-communicable diseases, including narratives on salient themes, concepts, or patterns among the chosen studies. retrieval of evidence and data analysis the researchers utilised databases of pubmed® (united states national library of medicine, bethesda, maryland, united states [https://pubmed.ncbi.nlm.nih.gov], web of science™ core collection database (clarivate analysis, boston, massachusetts, united states [https://clarivate.com/products/scientific-and-academic-research/research-discovery-and-workflow-solutions/webofscience-platform/web-of-science-core-collection]) and google scholar (https://scholar.google.com) in july 2024, to find peer-reviewed articles that had been written about ai and digital technology in african healthcare. different kinds of research articles and study designs were included in this search. however, the selection of the communicable and non-communicable diseases was based on prevalence in africa. as mentioned in the foregoing section, the three most prevalent communicable diseases in africa are hiv/aids, malaria and tuberculosis.23,24 we also based our selection on non-communicable diseases such as cancer, cardiovascular disease and diabetes, which are the most prevalent non-infectious diseases in africa.23,24 the designated diseases were integrated interchangeably with the keywords ‘artificial intelligence’ or ‘deep learn’ or ‘machine learning’ or ‘neural network’ or ‘compu intelligent’ or ‘robot’ for assessing the impact of ai and digital technology on communicable and non-communicable diseases in africa. we limited the publication years from 2000 to 2024. we excluded non-english language articles that were not focused on healthcare in africa. extracted data were synthesised thematically to identify patterns, trends, and key insights related to the opportunities presented by ai and digital technology-based diagnostic tools for communicable and non-communicable diseases in africa. clusters that reflected commonalities and areas of focus were identified. emerging trends and patterns within the selected literature were noted. moreover, graphpad prism v. 10.2.3 (graphpad, la jolla, california, united states [https://www.graphpad.com/features]) was utilised for statistical analyses. the pearson correlation was calculated to identify disproportionate national scientific and socio-economic characteristics, association between publication output and gross domestic expenditures for research and development (gerd), number of research funding agents, and gross domestic product (gdp). variables were expressed as percentage and mean ± standard deviation. results current state and opportunities of artificial intelligence and digital technology comprehending the present status of ai and digital technology research in the african healthcare sector is crucial for creating ai solutions that are customised to the unique circumstances found on the continent, such as the high incidence of infectious diseases and resource scarcity. with the integration of ai and digital technologies, the evidence from the perused pubmed®, web of science™ and google scholar databases suggests that the african healthcare landscape is undergoing an early-stage revolutionary shift. these improvements are most noticeable in laboratory settings, where ai is helping to improve tailored treatment, surveillance for communicable and non-communicable diseases, and diagnostic competencies. figure 1 displays the medical uses of ai domains and subdomains in africa. figure 1: illustration of artificial intelligence and digital technology, and their importance in an african laboratory environment. artificial intelligence has several applications in the sector of digital healthcare. the study incorporated a total of 1563 peer-reviewed scientific documents focusing on research of ai and digital healthcare for communicable and non-communicable diseases that are most prevalent in africa; after filtration, only 37 were utilised for systematic review (figure 2). table 1 depicts various opportunities of ai and digital technology in healthcare and laboratory diagnostics in africa. in reference to non-communicable diseases (figure 3), the three most dominant african countries with high ai and digital technology related to cardiovascular disease research productivity are south africa (29.73%), morocco (27.02%), and egypt (21.62%). in the case of the research field of ai and digital technology related to diabetes and cancer diseases, the same three countries remained dominant. egypt (25.00% for diabetes; 43.28% for cancer), was ranked as a high-producing african country in these research fields of ai and digital technology related to diabetes and cancer diseases. morocco (18.75% for diabetes; 15.35% for cancer) ranked second, and south africa (12.50% for diabetes; 14.92% for cancer) ranked third. figure 2: preferred reporting items for systematic reviews and meta-analyses flow diagram for evaluating the impact of artificial intelligence and digital technology-based diagnostic tools for communicable and non-communicable diseases in africa. figure 3: overall research status performance of artificial intelligence and digital technology in healthcare and laboratory diagnostics of non-communicable diseases in different african countries: (a) cardiovascular disease, (b) cancer and (c) diabetes. table 1: classification of various opportunities of artificial intelligence and digital technology in african healthcare and laboratory diagnostics. regarding communicable diseases (figure 4), the countries that dominate in the research field of ai and digital technology related to hiv/aids diseases are algeria (31.06%, ranked first), egypt (26.71%, ranked second), and south africa (20.71%, ranked third). however, south africa was ranked as the highest-producing african country in the research field of ai and digital technology related to tuberculosis (50.00%) and malaria (35.29%), and nigeria ranked second (tuberculosis, 16.21%, and malaria, 20%). morocco ranked third in the fields of ai and digital technology related to tuberculosis (12.16%), and ghana ranked third in the fields related to malaria (7.05%). figure 4: overall research status performance of artificial intelligence and digital technology in healthcare and laboratory diagnostics of communicable diseases in different african countries: (a) hiv/aids, (b) malaria and (c) tuberculosis. artificial intelligence and digital technology for non-communicable diseases in africa cardiovascular disease one of the digital technologies that is common in sub-saharan countries is the mobile phone, which is used by almost 45% of the population.28 the technology of mobile phones, in particular the use of phone calls, mobile applications and short message service (sms), has been the most reported use of a variety of technology applications for dealing with hypertension in africa.28 even though there has been more interest in the use of mobile phones for healthcare purposes in children, maternal health and infectious diseases in sub-saharan countries, there has been an escalating interest in the use of mobile phones in non-communicable diseases. according to a study by stokes et al. in 2022, the strategy of using mobile communications technology in sub-saharan countries and africa as a whole has been shown to have a positive impact on the control of hypertension that is related to cardiovascular diseases through adherence to treatment and health knowledge.29 in addition, there was an indication of positive perceptions by the stakeholders on the management and prevention of hypertension. in cape town, south africa, an intervention using an automated system with either interactive or informational sms text messages was used for remote delivery of reminders for pick-up of medication, support for medication adherence, education related to cardiovascular diseases, and clinic appointments.30 in this study, a low-cost system was based on a commonly available mobile technology to deliver the sms-based intervention for the management of communication with the patients.30 the sms system, together with voice mail reminder messages, was found to be more applicable to health education, adherence to taking medication, healthy diets, and promotion of physical exercises than to early prognosis, diagnosis, and detection.31 a school of thought believes that the general application of the sms intervention was found to be doubtful and inapplicable to every sector of the african population due to low literacy levels that are still prevalent in many parts of africa.15 hence, there was a need to reassess or further engage these innovative ways of providing new focus or other combinations with the sms. the use of sms was also shown to enhance the positive behaviour of patients, even though this was not shown to have contributed to reductions or improvements in the control of hypertension, which may result in an enlarged heart.15 however, the use of techniques such as ml and ai have been considered to offer superior and innovative ways for providing efficient and low-cost ways of diagnosing, monitoring and managing non-communicable diseases related to hypertension and cardiovascular diseases.28,32 it was hence recommended that other avenues that could be employed to combat high literacy and lack of specific knowledge were applications that use graphical illustrations and pictures and prudent design principles. in addition, ml, three-dimensional printing technologies and ai could be employed successfully through elaborate research that was based on quality and evidence-based results in the health sector in africa.28,32,33,34 one of the mhealth tools that has been used in africa for intervention to monitor blood pressure in ghana, included the use of a bluetooth-enabled blood pressure device together with a smartphone application to monitor the readings of blood pressure and the intake of medications under the guidance of a nurse.31 in this study, there was a positive attitude and satisfaction with the intervention by the majority of the participants who had suffered a stroke, with some of the participants even stating that they regretted not having been exposed to the intervention earlier. the technologies in mhealth have the potential to achieve a greater implementation to assuage the burden of non-communicable diseases in africa.28 according to opoku et al. in 2017, the impact of mhealth interventions in sub-saharan african countries to improve healthcare and treatment through access to specialised services which were previously unavailable in a remote manner, had become increasingly beneficial.35 according to stephani36 in 2016, it is not possible as yet to conclude on the efficiency of mhealth interventions due to a limited number of studies and vast variations in the reported outcomes and heterogeneity of the mhealth interventions evaluated. hence, there is a need for further research to understand the particular effects of different types of mhealth interventions on a variety of people with non-communicable diseases in lowand middle-income countries.36,37 diabetes in rwanda, screening that was supported by ai increased the opportunity for the provision of immediate counselling and health education on eye care for diabetic patients who required referral.38 these referrals contributed to enhanced adherence in grading of diabetic retinopathy when compared to cases where there was delayed communication of results that were done and graded by healthcare workers.38,39 as a result, ai screening was shown to provide a crucial benefit in promoting adherence to the recommended management of diabetic eye care in sub-saharan africa.40,41,42,43 a similar study on the importance of ai diabetic retinopathy screening software in combination with a cheap hand-held fundus camera, to cater for the needs of patients with diabetes in a hospital, was carried out in malawi.44 results of the study showed that the ai software was able to pickup images that did not satisfy the quality standards for the precise prediction of diabetic retinopathy grading.44 the ai model using deep learning showed comparable outcomes with humans in the detection of prevalence of referable diabetic retinopathy and associated risk factors in the screening programme of a population in zambia.45 the ai model was shown to produce clinically suitable performance in the detection of diabetic retinopathy that could be referred.45 cancer the application of ai in the development of reliable tools for cancer outcome predictions in africa is a fairly new attempt.46 nevertheless, the advancement and endorsement of the technical aspects of ai models related to cancer in africa are on the whole satisfactory; this may be due to the available knowledge of people with adequate skills to undertake ideal model selection, pre-processing of data, validation, tuning of data, and deployment of data.47,48,49,50,51,52,53 however, the models that have been developed have not yet been evaluated for their efficiency and impact in assistive automated clinical risk stratification and decision-making. there is an expectation that there will be an integration of digital health technologies and ai-based predictions to alleviate the challenges of diminishing facilities, manpower, and lack of access to healthcare that are being experienced in the management and diagnosis of cancer in africa.45 the third most common cancer contributing to death is colorectal cancer in sub-saharan africa, with south africa having the highest incidence.53 supervised ml algorithms used together with statistical algorithms have been shown to be able to offer crucial predictive factors for recurrences of colorectal cancer, survival of the patients, and enhanced interpretation of colorectal cancer globally. the importance of ml is that it can be used for the extrapolation of the data that are collected externally from the hospitals, because the variables that regulate the outcome are as important as the pre-hospital detection of colorectal cancer patients and the treatment prescribed by clinicians.53 in 2022, a study by joseph et al. showed that the use of a handcrafted approach for the use of algorithms, such as deep neural network classifiers and feature extractors, enhanced performance in multi-classification of breast cancer in nigeria, and that data augmentation played the main role in improving the accuracy of the classification of breast cancer using histopathological images.54 artificial intelligence and digital technology for communicable diseases in africa hiv/aids in 2010, singh and mars investigated ml application for the prediction of future cd4 cell count change in south africa, since cd4 cell count is a preferred surrogate marker and assists clinicians and other health practitioners with the management of the infection as well as with the allocation of the resources needed.55 it was reported that close monitoring of the progression of hiv infection by counting cd4 cells is vital to its effective management. they built a support vector machine classification model that predicted the degree of cd4 cell count change.55 the model took as input the genome, current viral load, and number of weeks from baseline cd4 cell count, and predicted the range of cd4 cell count change. the model produced an accuracy of 83%. this pilot project shows that a change in cd4 cell count may be accurately predicted using ml on genotype, viral load, and time. moreover, there are several mobile health interventions for people living with hiv: cell phone-delivered reminders and inspirational messages to boost clinic attendance and antiretroviral therapy adherence, laboratory test delivery, and behaviour modification messages are some of the most common.56,57,58 tuberculosis machine learning is often used together with signal processing methods to automate communicable disease diagnoses. most interventions for diagnoses with the use of ai in lowand middle-income countries reported high specificity, sensitivity and accuracy to comparator diagnostic tools. machine learning assists medical practitioners in diagnosing tuberculosis and malaria with expert systems.59 in 2020, peiffer-smadja60 reported the impact of current ai tools over a range of clinical outcomes such as the diagnosis of tuberculosis or surgical site infections, indicating sensitivity and specificity. the ai tool predicted a bacterial infection in individuals who were not identified by clinicians as having an infection on admission, but were diagnosed later with a bacterial infection.60 moreover, ai and digital technologies are currently being used for drug discovery for several research studies.61,62,63 malaria in 2023, silka et al. presented a novel convolutional neural network architecture for the detection of malaria from blood samples with a 99.68% accuracy.64 the novel convolutional neural network accurately classified infected and uninfected samples with high specificity and sensitivity. an analysis of model performance on different subtypes of malaria was performed and the implications of the findings for the use of deep learning in infectious disease diagnosis was determined.16,17,64 moreover, there are several studies indicating the benefits of in silico research, including capacity to screen a huge number of drug candidates in a comparatively short length of time, which saves time and money when compared to traditional drug development methods.65,66,67 identification of national scientific and socio-economic characteristics challenges non-communicable diseases in africa a pearson correlation was performed to determine if there is a correlation between variable of publication outputs and variable of awarded funding or with gerd as a share of gdp (figure 5). cardiovascular disease research metric data exhibited a positive, but not significant, correlation (r = 0.5, p = 0.17) between variables of publication outputs (11.11 ± 8.25) and awarded funds (11.11 ± 26.26) even with the gerd as a share of gdp variable (0.56 ± 0.25; r = 0.5, p = 0.158). egypt and south africa are among the countries that are awarded more funding. morocco, ethiopia, kenya, and ghana did not declare their awarded funding (figure 5a, b). figure 5: correlational data of non-communicable disease research in africa. trends of research publications output, awarded funds for (a) cardiovascular disease, (c) diabetes and (d) cancer, and gross domestic expenditures for research and development as a share of gross domestic product for (b) cardiovascular disease, (d) diabetes and (f) cancer. however, there was no significant positive correlation when metric data of diabetic disease research were utilised (r = 0.32, p = 0.394), between variables of publication outputs (11.11 ± 8.3) and awarded funds (11.11 ± 15.79). similar findings were achieved, where variables of gerd as a share of gdp (0.56 ± 0.25) exhibited association with publication outputs (r = 0.35, p = 0.359). although south africa was awarded more funding (50%) compared to other african countries, the country had fewer publication outputs compared to egypt and morocco which were awarded less funding of 15.38% (egypt) and 7.69% (morocco). algeria, tunisia, and sierra leone did not declare their awarded funding (figure 5c, d). cancer disease research metric data exhibited a significant correlation (r = 0.92, p = 0.001) between publication outputs (6.67 ± 11.41) and awarded funds (6.67 ± 12.22). there was also a significant positive association (r = 0.53, p = 0.044) between publication and gerd as a share of gdp (0.44 ± 0.26). egypt, morocco, and south africa are among the top three countries with high research output productivity and more awarded funding. tunisia, ethiopia, sudan, botswana, libya, sierra leone, tanzania, and zambia did not declare their awarded funding (figure 5e, f). communicable diseases in africa pearson correlation showed that there was a significant correlation (r = 0.79, p = 0.001) between publication outputs (4.55 ± 9.14) and awarded funds (4.55 ± 10.36), when metric data of hiv/aids research was utilised. moreover, there was a significant positive association (r = 0.63, p = 0.002) between variables of publication outputs and gerd as a share of gdp (0.29 ± 0.28). egypt (30.49%) and south africa (40.32%) were awarded a greater amount of funding compared to other african countries. however, these countries had fewer publication outputs compared to algeria, which was awarded less funding of 7.54%. namibia did not declare its awarded funding (figure 6a, b). figure 6: correlational data of communicable diseases research in africa. trends of research publications output, awarded funds for (a) hiv/aids, (c) tuberculosis and (e) malaria, and gross domestic expenditures for research and development as a share of gross domestic product for (b) hiv/aids, (d) tuberculosis and (f) malaria. metric data for tuberculosis research also exhibited a significant correlation (r = 0.69, p = 0.001) between publication outputs (3.70 ± 7.20) and awarded funds (3.70 ± 10.89). moreover, there is no significant positive association (r = 0.365, p = 0.258) between publication and gerd as a share of gdp (0.27 ± 0.27). even though south africa received more funding, of approximately 54.25% compared to other african countries, it had fewer publication outputs compared to algeria and egypt which were granted less funding of 4.91% (algeria) and 19.85% (egypt). ghana managed to publish research outputs despite the non-declared awarded funding (figure 6c, d). metric data for malaria research also exhibited a significant positive correlation (r = 0.86, p = 0.001) between variables of publication outputs (4.55 ± 8.85) and awarded funds (4.55 ± 14.16). however, there was no positive association (r = 0.35, p = 0.359) between variables of publication outputs and gerd as a share of gdp variable (0.32 ± 0.28). south africa had more publication outputs and greater funding compared to other african countries. chad, côte d’ivoire and democratic republic of the congo did not declare any awarded funding (figure 6e, f). discussion the application of ai is changing the complex nature of healthcare due to its role in revolutionising healthcare systems, thereby enhancing healthcare delivery and patient satisfaction.68 researchers have noted that the use of ai in the african health sector is still at the infant stage, which may necessitate a continuous monitoring process to identify potential risks and hazards associated with the use of ai.69,70 this view can be consolidated with our findings, where the research outputs evidence suggests that the african healthcare landscape is undergoing an early-stage revolutionary shift in terms of integrating ai and digital technologies. the application of ai and digital technologies can improve treatment effectiveness and efficiency through various data and identification processes.68,71,72 since ai is a technology that relies on the reduction of human involvement in healthcare delivery, speeding up the process of summarising data and assisting in identifying common factors in a problem.73,74,75 artificial intelligence may reduce the over-reliance on manpower, especially in countries where there are shortages of both skilled and unskilled workforce.72 from the patient’s perspective, ai can assist with data collection and management. this will ease the burden of repeated hospital visits on the part of patients and may also provide warning on issues of drug overdose, drug-to-drug interactions, and any unforeseen hazards.73,76,77,78 there are, however, some risks associated with the use of ai.79,80,81,82,83 a major consideration in the use of ai in laboratory diagnostics and healthcare in general are inequality or poverty, and income distribution in africa.84,85 our results reflected the disproportionate research outputs distribution across africa for three most prevalent communicable and non-communicable diseases in africa. in terms of non-communicable diseases research, south africa, egypt, and morocco contribute 78.38% of the entire continent’s total cardiovascular disease research, 60% for diabetes research, and 73.56% cancer research, while for the communicable diseases research field, algeria, south africa, nigeria, and morocco contribute 86.11% towards the field of hiv/aids research, and 79.72% towards tuberculosis research. south africa, nigeria, and ghana contribute 62.35% for the total research outputs in the malaria research field. these disproportionate research outputs can be elucidated by a several factors, including the reality that both south africa and egypt are rated as the top two countries with the highest gdp on the continent.86 besides, south africa, morocco, and egypt are still statistically recognised as the three african countries with the highest gerd, which is approximately between 6.2 and 8.86 billion united states dollars.87 although, nigeria and ghana are rated in the top 16 african countries in terms of gerd, the results revealed that nigeria contributes 54.25% towards the communicable diseases research field and ghana contributes 21.82% for the non-communicable diseases research field. there is a notion that only rich people will be able to afford new technologies for healthcare, while the poor may trail behind, eventually creating social unrest. as a matter of fact, the overall success of the use of ai to foster healthcare, including laboratory diagnostics, should be viewed in terms of the impact on people at various socio-economic levels, and their intercultural perspectives.84 other challenges regarding the use of new technologies include inadequate funding for healthcare, exposure of healthcare workers to changing technologies, fostering self-learning and discovery, promoting imaginative and critical thinking, and systemic change management. the analysed data of this study depict special consideration to a clear correlating trajectory of africa’s research outputs and obtained research funds. the designation of a superior share of south african local funding may possibly be rationalised given that in the year 2003, the south african government assigned a new funding system that is grounded in the research output of tertiary institutions.87 moreover, south africa spends approximately 1% of its gdp on research and development, as recommended by the african union.87,88 accordingly, the mapping of ai and digital health research in africa can play an important role in providing strategic guidance to governments. this can assist the governments to prioritise investments in digital health technology according to the particularities of each country in establishing sustainable economic development and health returns.84,85 hence, digital infrastructure should be in place to sustain the health sector and economy in most african countries. recommendations based on the results of this review, the following recommendations are made to guide future ai implementation in laboratory diagnostics in africa. to facilitate the widespread adoption of ai and digital technology in laboratory diagnostics across africa, a multifaceted approach is recommended. firstly, streamlining of the curriculum of universities in africa, especially in medical schools, to align with the era of ai, digital technology, and the changing landscape of innovations and rapidly emerging technologies. this educational reform will empower future healthcare professionals with the requisite skills to effectively utilise these technologies in diagnostics. healthcare practitioners should be taught the need to adapt readily to emerging situations by learning, unlearning and re-learning in order to catch up with the momentum of developments in the field. secondly, concerted public awareness campaigns are essential to educate communities about the benefits and applications of ai in laboratory diagnostics, dispelling misconceptions and fostering trust. policy formulators and decision-makers, including executive and legislative arms of governments in africa, should make substantial improvements in healthcare delivery systems and infrastructural facilities to support ai laboratory diagnostics. thirdly, substantial investments in infrastructure are imperative to support ai implementation, addressing challenges such as power outages and inadequate internet connectivity. additionally, comprehensive capacity-building programmes should be established to enhance the proficiency of healthcare professionals in utilising ai and digital tools for diagnostics, potentially through collaborations with international organisations and technology firms. fourthly, african academic and research institutes and academics must develop self-governing sources of funding for federal and philanthropic gestures. this will enable africans to introduce their special research agenda which will permit less reliance on international research funding agents. fifthly, african countries must utilise a full 1% of gdp for gerd. lastly, fostering collaborative research endeavours among researchers, healthcare practitioners, and technology experts is crucial for evaluating the effectiveness and feasibility of ai applications within the african context, thus informing evidence-based policies and strategies for scaling up ai adoption in healthcare. by implementing these recommendations, africa can overcome existing barriers and realise the full potential of ai in improving laboratory diagnostics and enhancing healthcare delivery across the continent. conclusion it is concluded that while ai and digital technology are useful in the laboratory diagnosis of communicable and non-communicable diseases in africa, the paucity of data and practices on such use shows that the method of diagnosis may not be widespread, most likely as a result of inadequate resources in several countries on the continent, especially in rural and peri-urban areas. machine learning, alongside signal processing methods and mobile communication strategies, is often used to automate the diagnosis of communicable diseases. screening involving the use of ai has been demonstrated to be beneficial in enhancing adherence to the management of non-communicable diseases such as diabetic retinopathy. it is also concluded that algeria, egypt, south africa, and morocco were the leading african countries in terms of published data, where the use of ai in the diagnosis of non-communicable and non-communicable diseases, such as cardiovascular, diabetes and cancer, as well as hiv/aids, have been extensively researched. in relation to malaria and tuberculosis, data revealed that south africa, nigeria, ghana, and morocco were the dominant countries in the integration of ai to the diagnosis, management, and adherence to medication of the designated diseases. however, there is still room for improvement for acquiring enough funding. as it is flagged as a major research outputs drawback among african countries. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions c.l.o. conceived the idea. c.l.o, j.o.o., t.d.m., l.l.m., t.h., m.o.o., and n.m.m. collected and analysed the data, drafted the article, reviewed, revised and agreed to the published version of the article. ethical considerations this article does not contain any studies involving human participants performed by any of the authors. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability in this study, the data sources used are available in google scholar, web of science, and pubmed databases. further inquiries can be directed to the corresponding author, n.m.m. disclaimer the views and opinions expressed in this 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med. 2021;4(1):125. https://doi.org/10.1038/s41746-021-00487-4 penprase be. the fourth industrial revolution and higher education. in: gleason nw, editor. higher education in the era of the fourth industrial revolution. singapore: palgrave macmillan, 2018; p. 207–229. galal s. value of gross domestic expenditure on r&d in africa 2020–2022, by country [homepage on the internet]. 2023 [cited 2024 aug 15]. available from: https://www.statista.com/ dst. department of science and innovation on south africa’s expenditure on research and development [homepage on the internet]. 2009 [cited 2024 aug 15]. available from: http://www.gov.za/ united nations (un). economic commission for africa towards achieving the african union’s recommendation of expenditure of 1% of gdp on research and development [homepage on the internet]. addis ababa; 2018 [cited 2024 aug 15]. available from: https://hdl.handle.net/10855/24306 abstract introduction methods results discussion acknowledgements references about the author(s) talkmore maruta laboratory department, african centres for disease control and prevention, lusaka, zambia sikhulile moyo laboratory department, botswana harvard aids institute partnership, gaborone, botswana citation maruta t, moyo s. impact of pre-covid-19 epidemic preparedness on the trajectory of the pandemic in african countries. afr j lab med. 2022;11(1), a1571. https://doi.org/10.4102/ajlm.v11i1.1571 original research impact of pre-covid-19 epidemic preparedness on the trajectory of the pandemic in african countries talkmore maruta, sikhulile moyo received: 28 feb. 2021; accepted: 02 feb. 2022; published: 31 mar. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the novel coronavirus disease 2019 (covid-19), declared a pandemic by the world health organization (who) in march 2020, has taught us about the importance of epidemic preparedness. objective: we analysed the pre-covid-19 preparedness of sub-saharan african countries and how this may have influenced the trajectory of covid-19 cases. methods: the who joint external evaluation (jee) tool and the global health security (ghs) index were used to determine the epidemic preparedness of countries in the who african region. the relationship between pre-covid-19 preparedness and the reported number of cases per million people was evaluated over the first 120 days of the first reported case in each country, between february 2020 and september 2020. results: the overall performance of the 42 countries was 40% in the 19 jee core capacities and 32% in the six ghs index indicators. at day 1, the mean number of cases per million population was significantly higher among countries rated as ‘prepared’ in the jee legislation, policy and finance (p = 0.03), ports of entry (p = 0.001), and international health regulation coordination, communication and advocacy (p = 0.03) categories. at day 90, countries rated as ‘prepared’ in the national laboratory systems (p = 0.05) and real-time surveillance (p = 0.04) jee categories had statistically significantly fewer cases per million population. conclusion: this analysis highlights the importance of building capacity for pandemic preparedness in africa. the who african region was not adequately prepared for the covid-19 pandemic as measured by the who jee tool and the ghs index. keywords: covid-19; preparedness; response; world health organization joint external evaluation; global health security index. introduction the rapid spread of the novel coronavirus disease 2019 (covid-19) prompted the world health organization (who) to declare it as a public health emergency of international concern in january 2020 and a global pandemic on 11 march 2020.1,2 the first covid-19 case in africa was reported by egypt on 14 february, and by 14 may, all african countries had reported at least one covid-19 case.3 by june 2020, 152 442 covid-19 cases and 4334 deaths (case fatality rate: 3%) had been reported by the 54 african countries.4 african countries instituted various public health and social measures to curb the transmission and allow them to prepare for the pandemic.5 although the coronavirus was novel, the emergency of its spread exposed the status of countries’ preparedness for threats posed by epidemics, disasters, and other events of public health concern. countries are expected to invest resources to limit the impact of disease outbreaks and natural disasters.6 epidemic preparedness is a measure of the capacity of countries to detect, report, and respond to outbreaks. to effectively respond to outbreaks, countries need to set up robust systems for surveillance and outbreak investigation with capabilities to rapidly identify, characterise, and track emerging infectious diseases.6 for a coordinated response, these systems need to function within strong national public health systems linked to an effective international system.6 the 2014 ebola outbreak in west africa infected 28 000 and killed 10 000 people in the three affected countries of guinea, liberia, and sierra leone, re-emphasising that epidemics can emerge unexpectedly and unprepared countries are a threat to all.7 the estimated combined loss was $2.8 billion in gross domestic product.7 this outbreak prompted globally coordinated efforts to ensure that countries strengthen their capacities as a way of ensuring that threats are limited and contained within borders when they occur. the international health regulations of 2005 (ihr 2005), among other innovations, was a means to ensure that countries are obliged to develop minimum capacities, defined as core public health capacities.8 due to the limited implementation of ihr 2005, especially in africa, the who developed the integrated disease surveillance and response (idsr) to improve surveillance by linking community, health facility, district, and national levels in a streamlined manner using a one health approach.9 however, by the 2012 deadline, only 58% of the signatories had developed national plans to meet ihr 2005 core capacity requirements, with as few as 10% indicating full implementation of the requirements. further to that, the global health security agenda was launched in 2014 to address the limited implementation of global commitments to building capacities for preparedness for epidemics and other events of public health concern using a multisectoral approach that includes the human, agriculture, animal, security, finance, border control, education, and research sectors.10 the impact of epidemics is well understood by governments.11 in addition to loss of lives, the cost to the broader economic and social sectors is a notable impact of epidemics, with epidemic response costlier than investing in preparedness. despite all this, the argument to invest in preparedness has not been won by many governments. ten years after the abuja declaration, where leaders of african union countries pledged to allocate at least 15% of their annual budget to the health sector, only 26 countries had increased the proportion of total government expenditures for health, with only one, tanzania, having achieved the 15% target.11 within that health budget, pandemic preparedness is often overlooked in favour of more immediate and visible curative demands.12 the covid-19 pandemic once again tested the epidemic preparedness of countries and the global community. in this report, we review the pre-covid-19 epidemic preparedness of countries in the who african region and its impact on how the virus spread, as well as the strength and effectiveness of the initial responses by countries and the global community. lessons learnt can be useful for the inevitable future pandemics. methods ethical considerations the study did not involve human or animal subjects and therefore no ethical approval or clearance was required. study population data from the who joint external evaluations (jee) conducted between 2016 and 2019 and the global health security (ghs) index conducted in 2019 were used to determine the levels of preparedness of countries in the who african region. forty-two countries from the who african region with complete scores for both the who jee and ghs index were included in the analysis. data on the reported number of cases per million people from day 1 (date of the first reported case; range: 28 february 2020 – 15 may 2020) to day 120 (120 days from the date of the first reported case; range: 26 june 2020 – 11 september 2020) in each of the 42 countries was also obtained and analysed. preparedness data sources world health organization jee the who jee is a process established by the who to assess countries’ capacities to prevent, detect, and rapidly respond to public health risks.13 the jee has two levels of assessment: an initial self-evaluation by the host country using local experts from all relevant sectors and an in-country evaluation conducted by an external team made up of multisectorial subject matter experts and peer countries. the process measures country-specific preparedness for events of public health concern across 19 technical areas, as well as progress made towards achieving ihr 2005 targets.13 the scored jee tool contains a set of questions to collect data on the status of implementation of the 19 technical areas, which are divided into 4 areas, namely prevent, detect, respond, and other ihr-related hazards (table 1).14 each question is scored from 1, indicating no evidence of implementation, to 5, indicating full implementation. table 1: sections and technical areas of the who jee tool. global health security index the ghs index is a project of the nuclear threat initiative and the johns hopkins center for health security that provides a recurring measure of the international capability for preventing, detecting, and rapidly responding to epidemic and pandemic threats.15 the ghs index tracks health security and related capabilities of the 195 countries that are signatories to the ihr 2005 guidelines and uses a framework with 140 questions organised into six categories, 34 indicators, and 85 sub-indicators (table 2). the ghs index was considered as an additional measure of countries’ preparedness as it also includes an assessment of the robustness of the broader healthcare system, national political and socio-economic risks, and adherence to international norms. table 2: categories, indicators, and sub-indicators of the ghs index. each country is assigned an overall score between 0% and 100% as a weighted sum of the six categories. to allow for comparisons, each category is normalised based on the sums of its indicators and sub-indicators. the ‘our world in data’ programme the ‘our world in data’ is a collaborative programme between the university of oxford and the global change data laboratory that, among others, has been collecting data on several areas related to the progress of the covid-19 pandemic across the globe.16 data on the number of daily confirmed covid-19 cases per million people from day 1 (date of the first reported case in each country) to day 120 (120 days from the date of the first reported case in each country) of the epidemic was collected from the our world in data database. countries’ responses during the first 120 days of the pandemic were taken as a reflection of their existing pre-covid-19 capacities and preparedness. the stringency index, which is tracked by ‘our world in data’, is also reported. the stringency index is a composite measure calculated from nine response indicators, including school closures, workplace closures, cancellation of public events, restrictions on public gatherings, closures of public transport, stay-at-home requirements, public information campaigns, restrictions on internal movements, and international travel controls and travel bans. strictness is measured on a scale of 0–100, with 100 being the strictest. determination of preparedness the determination of pre-covid-19 preparedness was based on the who jee and ghs indices. the who jee evaluations were conducted between 2016 and 2019. the total scores from each of the 19 technical areas assessed were converted to percentage scores. being prepared was defined as obtaining a 50% or greater score within the technical areas and overall. using data on related capabilities from the ghs index conducted in 2019, the weighted scores for each of the six categories were used to measure performance in each of the categories. the overall score was used as a measure of each country’s preparedness, where overall score = ∑ category scores, and category score = ∑ weighted indicator scores. the ghs index scoring system was adopted to rate the countries as having low scores (0.0% – 33.3%), moderate scores (33.4% – 66.6%) or high scores (66.7% – 100.0%). a concordance analysis, using cohen’s kappa, was used to determine the agreement between the who jee and the ghs index, with an agreement of 0.61 ≤ κ ≤ 0.80 considered as ‘substantial’.17 kendall’s coefficient of concordance was also calculated for concordance between the ratings using the jee and ghs index measurement scales. a kendall’s coefficient of concordance > 0.5 with an associated p < 0.05 was considered a strong agreement. stata® version 16 (statacorp llc, college station, texas, united states) was used for statistical analysis. p < 0.05 was considered statistically significant. student t-tests were used to compare the mean number of cases per million population by performance in the jee categories or ghs indices rating. results forty-two (89%) of the 47 member states of the who african region (who/afro) were included; the other five (11%) had no jee and ghs index data. of the 19 jee core capacities, only four (21%) categories, including immunisation, laboratory systems, real-time surveillance, and workforce development, had a mean score of at least 50%. the overall performance of the 42 countries in the 19 core capacities was below 50% (mean: 40%; standard deviation [s.d.]: 9) (table 3). table 3: performance of the 42 who african region member states in the 19 core capacities of the jees conducted between 2016 and 2019. in the ghs index evaluation, the overall performance of the 42 countries was below 50% in all the six categories of prevention, detection and reporting, rapid response, status of health systems, compliance with international norms and standards for biosafety and biosecurity, and risk and vulnerability of the country system (table 4). the overall performance was also low (mean: 32; s.d.: 7.1). table 4: performance of the 42 who african region member states in the ghs index evaluation conducted in 2019. using the jee-based ratings (not prepared: 0.0% – 49.0%, prepared: 50.0% – 100.0%), overall, the 42 countries were rated as ‘prepared’ in only five of the 19 technical areas, including immunisation (95.0%), real-time surveillance (81.0%), laboratory systems (62.0%), workforce development (60.0%), and reporting (55.0%). using the ghs index scoring system (low score: 0.0% – 33.3%; moderate score: 33.4% – 66.6%; high score: 66.7% – 100.0%), ≥ 50.0% of the countries had medium scores in three categories, including detection and reporting (n = 21; 50%), risk environment (n = 29; 70.0%), and compliance with international norms (n = 38; 90.0%). few member states had high scores, and these were in the risk environment (n = 1; 3.0%), compliance to international norms (n = 2; 5.0%), and detection and reporting (n = 2; 5.0%) categories. notably, no country had a low score for compliance with international norms; all had either high (n = 2) or moderate (n = 40) scores. there was a strong overall agreement between the jee and ghs index percentages (kendall’s coefficient of concordance = 0.7; p = 0.003). the rate of increase in the number of covid-19 cases from day 1 to day 90 was 14.3 cases per million people per week (n = 41, s.d.: 21.1, range: 0.6–114.0) and from day 90 to day 120 was 49.8 cases per million population per week (n = 42, s.d.: 106.8, range: 98.6–577.1). the stringency index, calculated as the mean score of the response indicators of school closures, workplace closures, public events, use of public transport, lockdowns, and internal and international travel, was evaluated. each indicator, taking a value between 0 and 100 (100 = strictest), increased from an average of 29.3% at day 1 to 64.1% by day 90, and slightly reduced to 60.1% by day 120 (figure 1). figure 1: average stringency index of 42 who african region countries at day 1 (28 february 2020 – 15 may 2020); and day 120 (120 days from the date of the first reported case; range: 26 june 2020 – 11 september 2020). there were no significant differences in reported cases per million people on day 1, day 90, and day 120 by performance rating in all the ghs index categories (table 5). at day 1, the mean number of covid-19 cases per million population was significantly higher among countries rated as ‘prepared’ based on overall jee scores compared to countries rated as ‘not prepared’ (p = 0.01) (table 6). the mean number of covid-19 cases per million population at day 1 was also significantly higher in the countries rated as ‘prepared’ in the following jee core capacities: legislation, policy and finance (3.0 vs 0.2; p = 0.03), international health regulation coordination, communication and advocacy (2.9 vs 0.3; p = 0.03), food safety (3.4 vs 0.2; p = 0.01), and ports of entry (4.8 vs 0.2; p = 0.001). conversely, at day 90, the mean number of covid-19 cases per million population was significantly higher in the countries rated as ‘not prepared’ in the core competencies of national laboratory systems (298.3 vs 121.5; p = 0.05) and availability of real-time surveillance (376.8 vs 145.3; p = 0.04). however, the mean number of covid-19 cases per million population was still significantly higher in the countries rated as ‘prepared’ in the jee immunisation category compared to the countries rated as ‘not prepared’ (1589.8 vs 579.4; p = 0.01). at day 120, the mean number of covid-19 cases per million population was significantly higher in the ‘not prepared’ countries in the jee immunisation category (1370.2 vs 370.3; p = 0.04) but higher in the ‘prepared’ countries in the jee preparedness (1167.0 vs 359.7; p = 0.04) and ports of entry (1046.1 vs 330.9; p = 0.02) categories. table 5: mean number of cases per million population and the performance of the 42 who african region countries in the ghs index evaluations conducted in 2019. table 6: mean number of cases per million population and performance of the 42 who african region countries in the jee conducted between 2016 and 2019. discussion at the onset of the pandemic in who/afro member countries on 14 february 2020, 44 countries had conducted and published their jee reports as an indicator of preparedness of their systems for any event of public health concern. although the 42 countries considered in this review scored below 50% on average, indicating a lack of preparedness, there were notable areas where systems were in place. these included structures for immunisation, laboratory systems, surveillance, and workforce development. over the years, many african countries have conducted and built systems for immunisation of children under five years, leading to successes like the elimination of the wild poliovirus.18 in addition, numerous outbreaks of cholera, ebola, influenza, rift valley fever and other endemic diseases like malaria may have contributed to the observed existence of surveillance systems. since 2009, who/afro, in collaboration with the africa society for laboratory medicine and other partners, has developed and implemented the strengthening laboratory management towards accreditation training programme and the strengthening laboratory quality improvement process towards accreditation programme across africa.19,20 this may have contributed to the preparedness of the laboratory systems as reported by the jee. nevertheless, who/afro countries were generally unprepared for a global pandemic as determined by both the who jee and ghs index assessments. there was a lack of preparedness in key core capacities such as legislation, coordination, preparedness, emergency response, medical countermeasures, risk communication, and ports of entry. in the early phases of the epidemic, all reported cases were imported through the various ports of entry. most countries did not have legislation to empower governments to institute some of the public health measures needed to control the epidemic, resulting in delays and court challenges in some instances.21 it took time to mobilise all the coordination mechanisms required, especially the activation of the emergency operation centres and the establishment of covid-19 task teams, resulting in disaggregated responses in the early days. although the concept of a rapid response team was known and, in some cases, documented, these had not been tested at the scale needed and the anxiety associated with the disease delayed the full activation and deployment of the rapid response teams. there are two additional areas measured by the ghs index that provide additional measures of preparedness. these include the country’s vulnerability to biological threats and the overall risk environment, as well as the sufficiency and robustness of health systems to treat the sick and protect health workers. the ghs index suggests that health systems in who/afro countries are underdeveloped, with the 42 countries having their lowest scores in the health systems category among the six indicators of preparedness. deficiencies identified in the health systems by the ghs index include poor infrastructure, lack of dedicated finance from fiscus, and lack of documented commitment to prioritising healthcare services for healthcare workers who participate in a public health response, among others.15 the ghs index also showed that who/afro did not have a conducive environment in terms of political systems and government effectiveness in dealing with epidemics, as indicated by the low score in the risk environment category.15 a sizeable number of countries in the region have political and security risks, including civil wars, political and economic instabilities, and other cross-boundary disputes that could undermine national capabilities to counter threats. the ebola epidemic in west africa was an example where accessibility to affected areas was hampered by civil strife.22 did the level of readiness of who/afro member countries as depicted by the who jee and ghs index have a bearing on how the epidemic spread to and within africa? the authors examined the onset of the epidemic and its progression from day 1, the date of the first reported case in each country, to day 120. this was done because, after 120 days, the status or progression of the epidemic may have been affected by the rapid changes adopted by the countries following the realisation of its possible social and economic impacts. preparedness, as measured by the ghs index indicators, was found to have no significant impact on the mean number of cases per million population from the onset of the epidemic until day 120. however, at the onset of the pandemic (day 1), the mean number of cases was statistically significantly higher among countries rated as ‘prepared’ compared to countries rated as ‘not prepared’ based on overall jee scores and scores in the food safety, ports of entry, legislation, policy and finance, and international health regulation jee core capacities. with an average overall jee score of 40%, the poor status of overall preparedness of countries in the region seems to have had an impact on the onset and spread of the disease in the region. coordination, legislation, and advocacy are key to mounting a response in the event of a threat like the covid-19 pandemic. all initial cases in the region were imported through the various ports of entry, which, besides the airports, are largely very porous. the designated ports of entry were not adequately prepared, with no designated screening and isolation infrastructure or adequate and appropriately trained personnel resources to deal with covid-19.23 this calls for the strengthening of early warning systems similar to the who global influenza surveillance and response system.24 as the epidemic progressed, countries began to mobilise resources and set coordination structures, including the activation of emergency operation centres and the establishment of covid-19 coordination mechanisms like task force teams that were reporting to the highest offices in the country. implementation of public health measures, including lockdowns, was the most immediate response for most countries. this is reflected in the reported increase in stringency index from 29.3% at the onset to 64.1% by day 90. early in the pandemic, the who established the identification of cases through laboratory testing as central to the response.25 consequently, most countries prioritised localising diagnostic capacity and, with support from the who and the africa centres for disease control and prevention, the number of countries able to confirm covid-19 increased from 2, when the first case was reported in africa on 14 february 2020, to 24 countries able to confirm severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection by 20 february 2020.26 this supports the observed higher rating of the laboratory system capacity in most countries 120 days into the pandemic. limitations the findings of the study may be limited by inconsistent reporting in the early days of the pandemic as countries were establishing various capacities. through external funding, many countries made rapid changes within the first few months of detecting the first cases. this may have altered the relationship between baseline preparedness rating and the mean number of cases at day 90 and day 120 after the first case. we did not have enough data to adjust for these rapid interventions. despite the weaknesses of the data collection tools, the data generated using the jee and ghs index was critical in drawing international attention to the critical areas for building the capacity of nations to prevent, detect, and respond to epidemic threats. conclusion this analysis highlights the importance of building capacity for pandemic preparedness and response at multiple levels. at the onset of the covid-19 pandemic in february 2020, who/afro member countries were not adequately prepared as measured by the who jee and the ghs index. countries’ ratings in the legislation, policy and finance, and ihr coordination, communication and advocacy who jee categories, as well as in the health systems ghs index category, were not optimal. given all the lessons learnt during the covid-19 pandemic, including the rapid global spread and emergence of variants of concern, critical areas that predict the successful handling of epidemics need to be assessed. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions t.m. initiated the study and prepared the first draft of the manuscript. t.m. and s.m. analysed the data and reviewed and revised the manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the data that support the findings of this study are available on request from the corresponding author, t.m. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the affiliated agency of the authors. references word health organization emergency committee. statement on the second meeting of the international health regulations (2005) emergency committee regarding the outbreak of novel coronavirus (covid-19) [homepage on the internet]. geneva: who; 2020 [cited 2020 feb 1]. available from: https://www.who.int/news-room/detail/30-01-2020-statement-on-the-second-meeting-of-the-international-health-regulations-(2005)-emergency-committee-regarding-the-outbreak-of-novel-coronavirus-(covid-19) world health organization. director general’s opening remarks at the media briefing on covid-19 [homepage on the internet]. 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[cited 2020 apr 01]. available from: https://apps.who.int/iris/bitstream/handle/10665/331509/who-covid-19-lab_testing-2020.1-eng.pdf?sequence=1&isallowed=y reliefweb. news and press release – 20 february 2020 [homepage on the internet]. [cited 2020 apr 08]. available from: https://reliefweb.int/report/world/more-20-african-countries-can-now-test-coronavirus-disease maruta t, kebede y. the evolution of sars-cov-2 testing in africa: observations from the first 1 million cases. south afr j pub health. 2020;4(4):106–110. abstract introduction methods results discussion acknowledgements references about the author(s) noutin f. michodigni department of molecular biology and biotechnology, pan african university institute for basic sciences technology and innovation (pausti), nairobi, kenya department of reproductive health and biology, institute of primate research (ipr), nairobi, kenya atunga nyachieo department of reproductive health and biology, institute of primate research (ipr), nairobi, kenya juliah k. akhwale department of zoology, school of biological sciences, jomo kenyatta university of agriculture and technology (jkuat), nairobi, kenya gabriel magoma department of molecular biology and biotechnology, pan african university institute for basic sciences technology and innovation (pausti), nairobi, kenya department of biochemistry, college of health sciences, jomo kenyatta university of agriculture and technology (jkuat), nairobi, kenya abdoul-salam ouédraogo department of medical microbiology laboratories, souro-sanou teaching hospital, bobo-dioulasso, burkina faso andrew n. kimang’a department of medical microbiology, college of health sciences, jomo kenyatta university of agriculture and technology (jkuat), nairobi, kenya citation michodigni nf, nyachieo a, akhwale jk, magoma g, ouédraogo a-s, kimang’a an. formulation of phage cocktails and evaluation of their interaction with antibiotics in inhibiting carbapenemase-producing klebsiella pneumoniae in vitro in kenya. afr j lab med. 2022;11(1), a1803. https://doi.org/10.4102/ajlm.v11i1.1803 note: additional supporting information may be found in the online version of this article as online supplementary document 1. original research formulation of phage cocktails and evaluation of their interaction with antibiotics in inhibiting carbapenemase-producing klebsiella pneumoniae in vitro in kenya noutin f. michodigni, atunga nyachieo, juliah k. akhwale, gabriel magoma, abdoul-salam ouédraogo, andrew n. kimang’a received: 28 nov. 2021; accepted: 12 may 2022; published: 18 july 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the development of alternative control measures, such as phage therapy or adjunctive therapy, is urgently needed to manage the dissemination of carbapenemase-producing klebsiella pneumoniae. objective: this study aimed to evaluate the therapeutic potential of formulated phage cocktails and their interaction with select antibiotics in inhibiting the growth of carbapenemase-producing k. pneumoniae clinical isolate in vitro in kenya. methods: the study was conducted from february 2021 to october 2021 at the institute of primate research, nairobi, kenya. phage cocktails were formulated based on the morphology and biological properties of precipitated klebsiella phages. the efficacy of individual bacteriophages and phage cocktails as well as their combination with antibiotics were determined for their inhibitory activity on carbapenemase-producing k. pneumoniae (kp20). results: the precipitated bacteriophages were members of myoviridae, siphoviridae and podoviridae. regarding the evaluation of the phage cocktails, the absorbances at 600 nm of the bacterial culture treated with the two-phage cocktail (2φ ma) ranged from 0.173 to 0.246 at 16 h and 20 h whereas it peaked from 2.116 to 2.190 for the positive control. moreover, the results of the adjunctive therapy showed that the optical density at 600 nm of the bacterial culture treated with 2φ ma was 0.186 at 24 h post-incubation time while it was 0.099 with the bacterial culture treated with imipenem in combination with 2φ ma. conclusion: this study demonstrated that the two-phage cocktail in combination with imipenem was able to synergistically delay the increase in carbapenemase-producing k. pneumoniae growth in vitro. keywords: phage cocktail; antibiotics; klebsiella pneumoniae; carbapenemase; absorbances. introduction the development of alternative control measures is urgently needed to manage the dissemination of carbapenemase-producing (cp) klebsiella pneumoniae because of the decrease in the effectiveness of antibiotic drugs.1,2 bacteriophage therapy has, therefore, been proposed as an alternative strategy in controlling multidrug-resistant (mdr) bacterial infections, including mdr k. pneumoniae clinical isolates.3,4 some of the beneficial effects of bacteriophages over antibiotics include their abundance, host specificity and exponential replication.5 however, the narrowness of phage host range and the ever emergence of novel pathogen variants will at minimum represent some limitations for phage therapy.6,7 this challenge can be managed by formulating phage cocktails that contain different phages infecting one species or by combining phages with antibiotics, which may result in a broad spectrum of activity.8 the characterisation of therapeutic phages in terms of their biology and bactericidal activity is mandatory before any preclinical trials because the application of non-characterised lytic bacteriophages may cause undesirable virulence as an adaptive response to bacteriophage infection.5,9 in other words, the development of effective strategies, such as phage growth parameters, host range spectrum, the presence of lytic proteins, formulation of phage cocktail and phage synergistic interaction with antibiotic to enhance phage efficacy, is a key prerequisite for optimal treatment of mdr infections caused by k. pneumoniae. several studies have reported the advantages and drawbacks of phage cocktails in treating mdr bacteria.10,11 unfortunately, bacteriophages employed as cocktail or adjunctive therapy have not been well investigated in the african continent to effectively control mdr bacterial infections.12 this study aimed to evaluate the therapeutic potential of formulated phage cocktails and their interaction with select antibiotics in inhibiting the growth of cp k. pneumonia clinical isolate in vitro in kenya. methods ethical considerations this study was not human or animal research. however, clearance was issued by the nairobi city water & sewerage company limited (ncwsc/hr/trg.14/vol.8/14/mmm/ak) for collection of waste water samples. bacterial isolate collection, phage isolation and purification the study was conducted from february 2021 to october 2021 in the phage biology research laboratory at the institute of primate research, nairobi, kenya. the clinical isolate of cp k. pneumoniae (kp20) was obtained from the stock of the recently published data in kenya.13 bacteriophages were isolated from waste water samples of dandora estate sewage treatment works (ruai) and rongai effluent, nairobi, kenya. they were purified from waste water samples through spot assay and double-layer plaque method as described, previously.14,15 determination of minimum inhibitory concentrations for kp20 the minimum inhibitory concentrations of a panel of antibiotics including carbapenems for kp20 were determined using vitek 2 systems version 9.2 and ast-xn05 (bar code 1481424403205844) card (biomerieux, inc., hazelwood, missouri, united states), according to the manufacturer’s instructions. the detailed procedure is described in online supplementary method 1. bacteriophage propagation and precipitation phage lysate (≥ 108 pfu/ml or 109 pfu/ml) was propagated in large volume, concentrated and cleaned up in the presence of sodium chloride and polyethylene glycol as described elsewhere.14,16 morphological characterisation of precipitated bacteriophages the morphological characterisation of the precipitated klebsiella phages was conducted at the university of leicester, united kingdom. negative staining with 1% (weight/volume) uranyl acetate on 3 mm carbon-coated copper grids of the precipitated klebsiella phages was conducted at the university of leicester and the bacteriophages were visualised with a transmission electron microscope (jeol uk ltd., welwyn garden city, united kingdom).17 bacteriophages were then classified according to their morphology.18 bacteriophage host range determination a spot assay was conducted to determine the host range of precipitated bacteriophages and phage cocktails as described by kutter.19 bacterial strains tested in our study included reference strains and mdr bacterial strains reported by michodigni et al.13 and described in online supplementary methods 2. one-step growth experiment a one-step growth experiment for determination of latent period, and burst size of precipitated bacteriophages, was performed as described elsewhere, with some modifications.20 exponential-growth-phase culture of cp k. pneumoniae (≈ 2.98 × 108 cfu/ml) and phage precipitate (≈ 109–1010 pfu/ml) were mixed with bacterial suspension to obtain a multiplicity of infection (moi) of 0.1.14 the suspension was incubated at 37 °c in a shaking incubator for 10 min at 120 rotations per minute following with centrifugation. the pellet was then suspended in 7 ml of fresh tryptic soy broth and incubated at 37 °c in a shaking incubator at 125 rotations per minute. at 3-min intervals, from 0 min to 36 min, 500 µl of the suspension was taken and phage titres were estimated.20,21 formulation of phage cocktails and evaluation of their in vitro activity on carbapenemase-producing klebsiella pneumoniae three klebsiella phages, designated as cprsa, cprsb and esbla, were used for the formulation of two-phage cocktail (2φ ma) and three-phage cocktail (3φ mb). the two-phage cocktail comprised cprsa and cprsb while the three-phage cocktail consisted of cprsa, cprsb and esbla. bacteriophage cocktails consisting of two and three tested phage lysates were formulated by combining in equal ratios of 1:1 and 1:1:1. the evaluation of the phage cocktails’ effectiveness was carried out based on the method described by merabishvili with some modifications.22 two-phage (2φ ma) and three-phage (3φ mb) cocktails were composed and their ability to inhibit the growth of kp20 at its exponential-growth-phase was evaluated. subsequently, the efficacy of two individual bacteriophages, designated as klebsiella phage cprsa and cprsb at moi 1.0, 0.1 and 0.001, was also investigated. only broth media (tryptic soy broth) and individual bacteriophages represented the negative controls. bacterial culture with a final resulting concentration of ≈ 2.98 × 106 cfu/ml served as positive controls. the od600 of the host bacterium was measured every 4 h for 24 h, and finally at 48 h, to assess the inhibitory activity of bacterial growth by both individual phages and phage cocktails. the different absorbances obtained at 600 nm were compared to the ones of the positive control sample. the different concentrations of individual bacteriophages and phage cocktails are summarised in online supplementary table 1. the experiment was performed in duplicate. table 1: minimum inhibitory concentrations of a panel of antibiotic and carbapenemase produced to kp20, nairobi, kenya, february 2021 – october 2021. evaluation of inhibitory activity of phage cocktails in combination with antibiotics on carbapenemase-producing klebsiella pneumoniae in vitro the inhibitory activity of the phage cocktail alone, and in combination with imipenem or tigecycline was assessed on kp20 at its optimal exponential growth (od600 ≈ 0.6). the two antibiotics meropenem and tigecycline (glentham life sciences ltd. wiltshire, united kingdom) were tested in combination with 2φ ma and 3φ mb. the phage cocktails 2φ ma and 3φ mb with optimal concentrations (moi 0.1 and 0.001) were used in this experiment in combination with the single and double of the minimum inhibitory concentrations of imipenem and tigecycline. sterile broth media (tryptic soy broth), bacterial culture treated with antibiotics, and bacteriophage cocktails represented the negative controls. bacterial culture with a final resulting concentration of ≈ 2.98 × 108 cfu/ml served as a positive control sample. the growth of the host bacterium was monitored at 3-h intervals, between 0 h and 24 h, and at 48 h of incubation by measuring od600. the optical densities obtained at 600 nm were compared to the ones of positive and negative control samples. the different concentrations of phage cocktails and antibiotics are indicated in online supplementary table 2. the experiment was performed in duplicate. table 2: spectrum of activity of individual klebsiella phages isolated from ruai and rongai in february 2021 (cprsa, cprsb and esbla) and phage cocktail (2φ ma and 3φ mb) on reference strains and different bacterial species. data analysis a two-tailed t-test was performed to determine the significance levels of phage cocktails in combination with select antibiotics using graphpad prism version 9.2.0. (graphpad software, san diego, california, united states). a p < 0.05 was considered as significantly different. results minimum inhibitory concentrations of antibiotics and detected carbapenemase genes the minimum inhibitory concentrations of imipenem and tigecycline to kp20 were 4 µg/ml and 1 µg/ml (table 1). we previously reported the presence of extended-spectrum beta-lactamase (esbl) genes (blatem and blaoxa) and carbapenemase genes (blandm-1, blaimp).13 the esbl and carbapenemase genes produced by kp20 are indicated in table 1. morphological characteristics of precipitated klebsiella phages a transmission electron microscope showed that the bacteriophages included in this cocktail were all tailed phages (figure 1). among the three imaged bacteriophages, two were myoviruses (cprsa and cprsb) and one was a podovirus (esbla). figure 1: transmission electron microscope images of the precipitated klebsiella bacteriophages isolated from ruai and rongai in february 2021. (a) and (b) bacteriophages belonging to the family of myoviridae with contractile tails consisting of a sheath and a central tube (cprsa and cprsb); (c) and family of podoviridae short tail (esbla). precipitated klebsiella phages growth characteristics the burst sizes of the precipitated phages in the cocktail were 610 (1.812 × 1011 ÷ 2.98 × 108), 1 (2.294 × 108 ÷ 2.98 × 108) and 10 (3.123 × 109 ÷ 2.98 × 108) for klebsiella phage cprsa, cprsb, esbla. regarding their latent period, klebsiella phages cprsa and cprsb had the same latent period (9 min) and the one of esbla was 18 min as shown in online supplementary figure 1. host range of klebsiella phage lysates klebsiella phages cprsa, cprsb and esbla were used for the formulation of the two-phage cocktail (2φ ma) and the three-phage cocktail (3φ mb). the two-phage cocktail comprised cprsa and cprsb while the three-phage cocktail consisted of cprsa, cprsb and esbla. the two-phage cocktail was the combination of two bacteriophages belonging to the family myoviridae while the three-phage cocktail was the combination of 2φ ma and one belonging to the family podoviridae. the results of the host range analysis showed that both the individual phages and phage cocktails (2φ ma and 3φ mb) were active on bacterial strains belonging to klebsiella species. the three carbapenem-resistant k. pneumoniae were susceptible to only one individual phage, the klebsiella phage cprsa. the two-phage preparations were also active on the three cp k. pneumoniae strains, in addition to k2, which was described as an extended-spectrum beta-lactamase producer in our previous study. inhibitory activity of individual klebsiella phages and phage cocktails on carbapenemase-producing klebsiella pneumoniae culture the ability of the mixture of two phages (ma) and three phages (3φ mb) to inhibit the growth of kp20 culture at an absorbance of ≈ 0.3 was determined at three different multiplicities of infection (moi 1.0, 0.1 and 0.001). it was observed in our study that there was an increase in cp k. pneumoniae growth to the individual phage cprsb at its three different doses and to the phage cprsa at mois 0.1 and 0.001 after 8 h and 16 h of incubation (figure 2a). the two-phage combination (2φ ma) was able to control the rapid growth of cp k. pneumoniae after 16 h of incubation and up to 24 h (figure 2b). this bactericidal activity of the phage cocktail was observed at mois 0.1 and 0.001 whereas the three-phage cocktail (3φ mb) could slightly control the increase in cp k. pneumoniae growth after 16 h and at mois 1.0 and 0.001. at mois 0.1 and 0.001, the dose response of the two-phage cocktail was not significantly different in the ability to inhibit the bacterial growth at this incubation time (p = 0.105). at the most effective phage dose (moi: 0.001), the absorbances at 600 nm of the bacterial culture treated with the individual phage cprsa, and the two-phage cocktail (2φ ma), increased from 0.019 to 0.515, and 0.173 to 0.246 at 16 h and 20 h, while it rose from 2.116 to 2.190 for the positive control during the same interval of time (figure 2b). for the most active individual phage (cprsa) and phage cocktail (2φ ma), the dose responses were not significant (p = 0.12) at moi 0.1 after 16 h of treatment time (table 3). significant difference (p = 0.01) was observed between the two treatments at moi 0.001. figure 2: efficacy of phage cocktails in inhibiting the growth of carbapenem-resistant k. pneumoniae (kp20), nairobi, kenya, february 2021 – october 2021. optical density of kp20 cultures (≈ 106 cfu/ml) infected with (a) individual phages cprsa and cprsb at moi 1.0, 0.1 and 0.001 (b) the 2-phage cocktail (2φ ma at moi 1.0, 0.1 and 0.001) or 3-phage cocktail (3φ mb at moi 1.0, 0.1 and 0.001). the digits 1, 2 and 3 coming next after the id of the phages represents moi 1.0, 0.1 and 0.001. the most effective phages were the individual phage cprsa and phage cocktail 2φ ma at moi 0.1 (cprsa2 & 2φ ma2) and 0.001 (cprsa3 & 2φ ma3). table 3: statistical significance between the concentrations of individual phages (cprsa and cprsb) and phage cocktails (2φ ma and 3φ mb), nairobi, kenya, february 2021 – october 2021. interactive properties between the phage cocktails and antibiotics combinations in inhibiting the growth of carbapenemase-producing klebsiella pneumoniae in vitro the in vitro studies showed that the same phage cocktails (2φ ma and 3φ mb) maintained their bactericidal activity after 24 h of incubation at the lowest phage concentration (moi 0.001) (figure 3a). at different timepoints of 18 h and 24 h, the optical density at 600 nm of the bacterial culture treated with 2φ ma decreased from 0.217 to 0.186 whereas it increased from 0.226 to 0.269 for bacterial culture treated with 3φ mb. the results of the adjunctive treatment (phage and antibiotic combination therapy) experiments revealed that the absorbance of the bacterial culture treated with the combination of imipenem and two-phage cocktail (imp2 + 2φ ma3) decreased from 0.256 (21 h) to 0.099 (24 h) at the lowest phage concentration (figure 3b). when the phage cocktails were combined with tigecycline, the od600 decreased from 0.294 (21 h) to 0.189 (24 h) for bacterial culture treated with tigecycline and the two-phage preparation at the lowest phage concentration, moi 0001 (tg1 + 2φ ma3) while it increased slightly from 0.207 (21 h) to 0.331 (24 h) in the case of bacterial culture treated with tigecycline and the three-phage preparation (tg1 + 3φ mb3). the absorbances of the bacterial culture treated with tigecycline was 0.249 (21 h) and 0.314 (24 h) while it ranged from 1.212 (21 h) to 1.245 (24 h) for the bacterial culture treated with imipenem. the positive control sample exhibited an optical density of 1.166 (21 h) and 1.270 (24 h). the level of significance between the bacterial culture treated with 2φ ma and the bacterial culture treated with imp2 + 2φ ma3 after 21 h of incubation are shown in table 4. the results of bacterial cultures treated with phage cocktail alone, and in combination with imipenem, were statistically significant compared with the result of bacterial culture treated with only imipenem (p = 0.02). no statistical difference was observed between the results of the bacterial culture treated with a combination of phage and antibiotic and those of bacterial culture treated with tigecycline alone. a significant difference (p = 0.04) was observed between the bacterial culture treated with 2φ ma at moi 0.001 and the adjunctive therapy (bacterial culture treated with imp2 + 2φ ma3). table 4: level of significance between bacterial culture treated with two-phage cocktail and the bacterial culture treated with two-phage cocktail and imipenem combination, nairobi, kenya, february 2021 – october 2021. figure 3: bactericidal activity of klebsiella phage cocktail alone or in combination with antibiotics, nairobi, kenya, february 2021 – october 2021. (a) carbapenemase-producing k. pneumoniae culture (od600≈0.6) treated with phage cocktail 2φ ma and 3φ mb at moi 0.1 or 0.001, imipenem (imp, mic = 4 µg/ml), and tigecycline (tg, mic = 1µg/ml), separately. imp2 and tg2 were considered as double the volume of each antibiotic; (b) carbapenemase-producing k. pneumoniae culture treated with phage cocktail + imipenem or phage cocktail + tigecycline. imp2 + 2φ ma3 was the treatment of kp20 culture with the double volume of imipenem combined with two-phage cocktail at moi 0.001, and tg1 + 3φ mb2 was the treatment of kp20 culture with the single volume of tigecycline combined with the three-phage cocktail at moi 0.1. discussion this study aimed to evaluate the therapeutic potential of formulated phage cocktails and their interaction with select antibiotics in inhibiting the growth of the kp20 clinical isolate. on one hand, the phenotypic results revealed that the precipitated phages obtained in this study had lytic activity with high titers and they were related to tailed phages. the latter are generally divided into three families including myoviridae with contractile tails consisting of a sheath and a central tube (25% of tailed phages), siphoviridae with long and noncontractile tails (61%), and podoviridae having short tails (14%).23 klebsiella tailed phages identified in our study belonged to the family of myoviridae and podoviridae.24 previous studies reported similar observations regarding the morphological characters of klebsiella phages.25 on the other hand, this study showed that one of the precipitated phages (cprsa) had the shortest latency period (9 min) and high burst size (610). our findings were supported by horváth et al. in 2020. the authors reported that their isolated klebsiella phage had a relatively short latency period of 18 min and its burst size was ~220 phage particles per infected bacteria.20 indeed, a high burst size is key to achieve a productive adsorption and replication of bacteriophages and to reach the benefits that phages could have in comparison with antibiotics while a short latency period is recommended from a phage therapy perspective.26 both the individual bacteriophages and phage cocktails were not susceptible to the reference bacterial strains tested in this study and, hence, displayed high specificity for their host bacteria, which were kp20 clinical isolates. previous studies on host ranges of klebsiella phages reported similar results.27,28,29 the major consequence of phage host specificity is that it demands an appropriate diagnosis of the bacteria involved in the infection before initiation of phage treatment.7,30 nevertheless, this narrowness of phage host range to the strains of the same bacterial species could limit its lytic activity on microbiota.7,30 the phage host range is indeed affected by a number of factors including the absence of required accessory proteins for phage adsorption, restriction-modification and clustered regularly interspaced short palindromic repeats (crispr) systems.31,32 despite their narrow host range, bacteriophages with lytic activity may still be useful in phage therapy and the use of bacteriophages as cocktails for adjunctive treatment can represent a highly attractive strategy.33 besides the choice of bacteriophages with different bacterial cell wall receptor recognition sites in formulating a phage cocktail, the most important criterion for successful phage cocktail preparation also includes the compatibility of bacteriophages in mixtures.22 therefore, our study demonstrated that the mixture of two phages (2φ ma) was able to significantly delay the resurgence of bacterial cells in culture, as compared to the application of individual phage or the mixture of three phages (3φ mb) (p = 0.02). our result was in line with the previous data published on phage cocktail efficacy.11 this synergistic activity of two-phage cocktail over the mixture of three phages in our study suggested that the individual phages might have employed different receptors to adsorb to the bacterial cells and, hence, effectively inhibited the bacterial growth. some authors have reported that one phage in their two-phage cocktail used an outer membrane protein (ompc) as a receptor, and another one employed a lipopolysaccharide component as its receptor to effectively control bacterial resistance.34,35 moreover, the mixture of many phages may be less effective in the absence of identification of specific bacteriophage receptors because the same phages might share the same receptors and interfere with one another.36,37 the adjunctive treatment (imp2 + 2φ ma3) significantly inhibited the growth of kp20 in vitro compared to the two-phage treatment alone (p = 0.04). at moi 0.001, the two-phage cocktail combined with imipenem had significantly lysed the bacterial cell compared to the two-phage cocktail. this statistical difference might be related to the beneficial effect of bacteriophage treatment in adjunctive therapy. our finding contradicted the study of pacios et al., who indicated the inability of phage-imipenem combination to kill imipenem-resistant isolate harbouring oxa-245 b-lactamase.38 this divergence might be related to the use of a single lytic phage in adjunctive treatment instead of phage cocktail. furthermore, an antibacterial effect was also observed between the phage cocktails in combination with the most effective antibiotic (tigecycline) without significant difference (p = 0.99). a number of studies have reported the positive effect of bacteriophages in combination with antibiotics.39 this synergistic activity between phage cocktail in combination with imipenem might have been due to the sensitivity of the bacterial strains to the select antibiotic after bacteriophage action. indeed, it was reported in a previous study that phage-resistant bacterial strains are more susceptible to antibiotics and the rate of their growth is slower in comparison with wild bacterial strains.40 our study also demonstrated the efficacy of phage cocktail in the absence of phage receptor analysis in formulating phage cocktails. interestingly, this study pointed out the repurposing of imipenem using phage cocktail therapy, and further encourages the repurposing of the efficacy of food and drug administration-approved antibiotics for acceptance of phage therapy worldwide, especially in africa. limitations the limitation of this study was the absence of genomics, which could enable us to further conclude on the taxonomic classification of the bacteriophages. the conclusions were made based on a single carbapenem-resistant strain, which was also a limiting factor in our study due to the high host specificity of phages, and the diverse k. pneumoniae strain types and resistance mechanisms which may affect the utility of the adjunctive therapy. the potential impact of phages on the normal microbiota was not considered in the current study, especially given that the phage cocktails might have activity against k. pneumoniae strains other than those that are carbapenem-resistant, and e. coli strains. conclusion this current study revealed the presence of lytic tailed klebsiella phages belonging to the family myoviridae, siphoviridae and podoviridae in nairobi sewage systems with relatively short latent periods and optimal burst sizes, indicating their therapeutic potential in composing phage cocktails and synergistic interaction in combination with non-sensitive antibiotic (imipenem) against carbapenem-resistant k. pneumoniae clinical isolate in vitro. acknowledgements we are grateful to the pan african university under the african union for financial support. we are thankful to ms ivy mutai, angela juma, mercy munini and mr dennis kotty for their collaboration on phage isolation. our gratitude to dr jessica sacher of the phage directory for having facilitated the transmission electron microscope analysis. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions n.f.m., a.n., j.k.a., g.m. and a.n.k. conceived and designed the study. n.f.m. conducted the laboratory experiments, data analysis and drafted the original manuscript. n.f.m., a.n., j.k.a., g.m. and a.-s.o. reviewed and approved the final manuscript for submission. sources of support this research project was supported by the pan african university. data availability all data are available from the corresponding author, n.f.m., upon request. disclaimer the views and opinions expressed in this article are only those of the authors. references tacconelli e, carrara e, savoldi a, et al. discovery, research, and development of new antibiotics: the who priority list of antibiotic-resistant bacteria and tuberculosis. lancet infect dis. 2018;18(3):318–327. https://doi.org/10.1016/s1473-3099(17)30753-3 ssekatawa k, byarugaba dk, wampande e, ejobi f. a systematic 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bacteriophages occurring during bacteriophage therapy. viruses. 2018;10(7):351. https://doi.org/10.3390/v10070351 abstract introduction methods results discussion acknowledgements references about the author(s) tshiphiri senamela national health laboratory services, pretoria, south africa department of haematology, faculty of health sciences, university of pretoria, pretoria, south africa marleen kock national health laboratory services, pretoria, south africa department of haematology, faculty of health sciences, university of pretoria, pretoria, south africa piet becker department of haematology, faculty of health sciences, university of pretoria, pretoria, south africa joachim j.c. potgieter national health laboratory services, pretoria, south africa department of haematology, faculty of health sciences, university of pretoria, pretoria, south africa citation senamela t, kock m, becker p, et al. detection of the janus kinase 2 v617f mutation using a locked nucleic-acid, real-time polymerase chain reaction assay. afr j lab med. 2018;7(1), a658. https://doi.org/10.4102/ajlm.v7i1.658 brief report detection of the janus kinase 2 v617f mutation using a locked nucleic-acid, real-time polymerase chain reaction assay tshiphiri senamela, marleen kock, piet becker, joachim j.c. potgieter received: 30 jun. 2017; accepted: 17 oct. 2017; published: 31 jan. 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract the purpose of this study was to develop a real time polymerase chain reaction (pcr) assay for the detection of the jak2 v617f mutation that could be used in diagnostic laboratories. sanger sequencing and a newly developed locked nucleic-acid, real-time pcr assay were used to detect the jak2 v617f mutation. there was 100% agreement between the sequencing and pcr analysis. both assays were able to detect the mutation in all 24 of the 60 test specimens harbouring the mutation. introduction myeloproliferative neoplasms (mpns) are a heterogeneous group of clonal disorders that result from mutational transformation of a haematopoietic stem cell.1 this transformation of haematopoietic stem cells causes uncontrolled proliferation of myeloid cell lines leading to overproduction of both mature and immature blood cells.1 prior to 2005, no underlying genetic abnormality had been associated with classic breakpoint cluster region-abelson leukaemia virus negative mpns. in 2005, a mutation at base 1849 in exon 14 of the jak2 gene on chromosome 9 was discovered in patients with polycythaema vera, essential thrombocythaemia and primary myelofibrosis.2,3,4 this somatic point mutation causes a substitution of guanine by thymine and the amino acid is changed from valine to phenylalanine in codon 617 of the jak2 protein.2 this discovery has improved understanding of the pathophysiology of mpns and has renewed interest and research in mpn biology and genetics.5,6 the ability to demonstrate the presence of this mutation has not only simplified the diagnosis of mpn but also ensured greater diagnostic accuracy.5 the identification of this mutation has also led to the development of therapies targeted at inhibition of the jak2 kinase.7,8 sensitive and specific assays are required for the detection of the jak2 v617f mutation.9 molecular assays with high sensitivity and specificity should be offered by diagnostic laboratories for this purpose.9 several different commercial and in-house assays that offer different sensitivity and specificity levels have been developed.9 direct sequencing is a gold standard for mutation analysis but is limited by low sensitivity and high cost.10 in-house assays that are currently used include allele-specific polymerase chain reaction or amplification refractory mutation system, direct sequencing, polymerase chain reaction (pcr) with restriction fragment length polymorphism (pcr-rflp) and real-time pcr.9,11,12 diagnostic laboratories may find it challenging to select the most appropriate methodology to detect the jak2 mutation.13 however, the use of a reliable, quick and sensitive assay that is able to detect 1% of the mutant allele in a wild-type background is recommended.13 the aim of this study was to develop a real-time pcr assay for the detection of the jak2 v617f mutation for implementation in a routine clinical laboratory. methods ethical considerations the study received approval from the university of pretoria faculty of health sciences research ethics committee (s32/2012) for using sample remnants for further analysis. leftover blood samples were collected anonymously from a tshwane tertiary hospital for dna extraction and pcr assay. all samples were used solely for the detection of the jak2 v617f mutation. samples collected and study site this study used specimens of whole blood collected in edta (n = 60) that were submitted by clinicians from steve biko academic hospital to be evaluated for the presence of the jak2 v617f mutation in patients suspected to have mpns. the study was conducted between 01 october 2013 and 30 september 2015. genomic dna genomic dna (gdna) was extracted from 0.2 ml of peripheral blood using the genelute™ blood genomic dna kit (sigma aldrich, st. louis, missouri, united states) according to the manufacturer’s instructions. the concentration of dna was confirmed by the nanodrop 2000c uv spectrophotometer (thermo scientific, waltham, massachusetts, united states) to obtain gdna with a concentration of 1.6 to 1.9 µg/ml. extracted dna was stored at –20°c until analysis. sequencing of amplified products primers for the detection of the single nucleotide polymorphism in codon 617 were designed based on the known dna sequence of the jak2 gene (genbank® accession number ng_009904.1) using the primerquest software (integrated dna technologies, coralville, iowa, united states) (table 1). the product of interest was amplified using the dna engine peltier thermocycler (bio rad, münchen, germany) under the following conditions: initial denaturation at 94°c for 10 minutes, followed by 35 cycles of amplification with denaturation at 94°c for 30 seconds, annealing at 52°c for 30 seconds and extension at 72°c for 45 seconds. sanger sequencing was performed by inqaba biotechnical industries (pretoria, south africa) in both directions on all 60 amplified products. the clc main workbench software program v6.0 (clcbio, waltham, massachusetts, united states) was used to analyse the sequences against the ncbi reference sequence ng_009904. table 1: polymerase chain reaction primer and probe sequences used to detect the jak2 v617f mutation (5′ → 3). locked nucleic-acid, real-time polymerase chain reaction assay wild-type and mutant probes were designed to be complementary to the wild-type and mutant nucleotide sequences (table 1). the real-time pcr assay was performed using the cepheid smartcycler ii platform (cepheid, maurens-scopont, france). the reaction mixture contained 0.4 µm each of the forward and reverse primers, 0.4 µm of mutant and 0.05 µm of wild-type probes, 2x qiagen quantinovatm probe pcr master mix (qiagen, hilden, germany) and < 100 ng template gdna. in an attempt to optimise the assay’s performance, the wild-type probe concentration was adjusted in a separate experiment by making a serial dilution of the wild-type probe in nuclease-free water and assessing its performance. the concentration of the wild-type probe that gave optimal results was found to be 0.05 µm. this concentration of wild-type probe was used together with 0.4 µm mutant probe in the pcr assay with satisfactory results. polymerase chain reaction was performed under the following conditions: initial denaturation at 95°c for two minutes, followed by 40 cycles of amplification with denaturation at 95°c for 10 seconds, then combined annealing and extension at 60°c for 30 seconds. genomic gblocks® gene fragments (integrated dna technologies, coralville, iowa, united states) consisting of mutant and wild-type sequences each together with no template control were included with each run. the analytical sensitivity of the locked nucleic-acid (lna) real-time pcr assay was determined using wild-type dna mixed with that of homozygous mutant dna in various concentrations (100% mutant, 50% mutant, 20% mutant, 10% mutant, 5% mutant, 2% mutant, 1% mutant and 0.1% mutant). the dilutions were prepared fresh and subjected to pcr in three replicate experiments to ensure reproducibility of the assay. the limit of detection was defined as the lowest dilution of mutant dna in which all three replicates resulted in positive amplification.14 data and statistical analysis the real-time pcr assay results were compared to the sequencing assay results to assess the assay’s usefulness for allelic discrimination of the jak2 v617f mutation. the results were used to assess the agreement between the two assays using cohen’s kappa for inter-rater agreement to assess the agreement between methods using interpretation cut-offs: κ > 0.75 excellent agreement, κ = 0.4–0.75 good agreement and κ < 0.4 poor agreement.15 results both sequencing and the lna real-time pcr assay detected the mutation in the same 24 samples (40%) and with κ = 1 (i.e. perfect agreement). during the experiment, it was noted that the standard 1:1 wild-type to mutant probe ratio did not provide optimal results. the ratio of wild-type to mutant probe that provided optimal results was 1:8 (figures 1 and 2 show how a heterozygous sample amplifies when the 1:1 and 1:8 ratios of mutant to wild-type probes were used). the limit of detection of this assay is 0.1% in a wild-type dna background (figure 3). figure 1: an amplification curve showing results with 0.4 µm wild-type and 0.4 µm mutant probes. mutated allele curve with no markers, wild-type allele curve with crosses. figure 2: an amplification curve showing results with 0.05 µm wild-type and 0.4 µm mutant probes. mutated allele curve with no markers, wild-type allele curve with crosses. figure 3: amplification curves showing the analytical sensitivity of one sample. green: 100% homozygous mutant dna; red: 20% mutant with 80% wild-type dna; blue: 0.1% mutant dna in 99.9% of wild-type dna. discussion in this study, an lna probe-based, real-time pcr assay was developed and evaluated for its ability to detect the jak2 v617f mutation. an lna is an analogue of nucleotides that contains an internal 2′-o, 4′-c methylene bridge, which locks the ribose ring into a c3′-endo conformation.16 introduction of lnas into probes increases the thermal stability of the probe by + 3°c to + 8°c per modification and allows binding to complementary target sequences with high affinity.16,17,18 in this study, the wild-type probe contained six lnas and the mutant probe seven lnas. increasing the number of lnas in the mutant probe was necessary to increase the melting temperature (tm) of the probe to greater than that of the primers and thus increase the specificity of the probe. the wild-type probe binds with high affinity, thus reducing the intensity of the mutant probe as it is competing with the mutant probe in this multiplexed assay. both sequencing and lna real-time pcr assays detected the mutation in 40% of the samples. the agreement between the real-time pcr assay and sequencing was 100%. consistent positive amplification was obtained down to the 0.1% dilution (figure 3). the limit of detection is determined experimentally by preparing serial dilutions of mutant dna in a wild-type dna background and analysing each dilution point in a six-fold run.14 the last dilution where all six replicates give a positive and specific amplification is considered to be the limit of detection.14 the acceptable sensitivity of a qualitative test should be equal to or below 20%.14 however, it is recommended that assays for the detection of the jak2 v617f mutation in a clinical setting should have an analytical sensitivity of at least 1% to ensure that more than 90% of cases are detected.13 our newly designed lna probe, real-time pcr assay detects up to 0.1% of mutant dna in a wild-type dna background using gdna in contrast to an earlier assay with an analytical sensitivity of 2% using complementary dna.19 this may be an improvement on previously published probes. with the good analytical sensitivity of this assay, it is considered suitable for use in a diagnostic laboratory and constitutes a good screening tool. however, it should be noted that an assay with this sensitivity may be prone to give more false positives in a diagnostic setting.13 limitations to assess the robustness of a real-time pcr assay different experimental conditions, such as annealing temperatures, the use of different instruments and operators should be introduced.14 this aspect of assay evaluation was not explored in this study. however, to show that the assay was able to perform equally well on another platform, the dna samples were analysed on the qiagen rotor-gene q 2plex system (germany). similar results were obtained to that seen using the cepheid smartcycler ii system. conclusion the developed lna probe, real-time pcr assay is a suitable diagnostic method for detecting the jak2 v617f mutation in a clinical setting. it has good sensitivity, is easy to set up and has rapid turn-around times. acknowledgements we would like to thank the national health laboratory service research trust for funding this study with a pathology research development grant. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support national health laboratory service research trust pathology research development grant. authors’ contributions j.j.c.p., m.k. and t.s. were responsible for the experimental and project design. t.s. performed the experiments. p.b. and m.k. made conceptual contributions. p.b. performed the calculations. j.j.c.p. and t.s. wrote the paper. j.j.c.p. was responsible for funding acquisition. all authors read and approved the final manuscript. references titmarsh gj, duncombe as, mcmullin mf, et al. how common are myeloproliferative neoplasms? a systematic review and meta-analysis. amj. 2014;89(6):581–587. https://doi.org/10.1002/ajh.23690 baxter ej, scott lm, campbell pj, et al. acquired mutation of the tyrosine kinase jak2 in human myeloproliferative disorders. lancet. 2005;365(9464):1054–1061. https://doi.org/10.1016/s0140-6736(05)74230-6 kralovics r, passamonti f, buser as, et al. a gain-of-function mutation of jak2 in myeloproliferative disorders. n engl j med. 2005;352(17):1779–1790. https://doi.org/10.1056/nejmoa051113 levine rl, wadleigh m, cools j, et al. activating mutation in the tyrosine kinase jak2 in 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this article: mothabeng d, maruta t, lebina m, lewis k, wanyoike j, mengstu y. strengthening laboratory management towards accreditation: the lesotho experience. afr j lab med. 2012;1(1), art. #9, 7 pages. http://dx.doi.org/10.4102/ ajlm.v1i1.9 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. strengthening laboratory management towards accreditation: the lesotho experience in this original research... open access • abstract • introduction • methods    • baseline assessments    • improvement projects    • follow-up visits • results    • improvement project outcomes • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ introduction: the lesotho ministry of health and social welfare’s (mohsw) 5-year strategic plan, as well as their national laboratory policy and yearly operational plans, directly addresses issues of accreditation, indicating their commitment to fulfilling their mandate. as such, the mohsw adopted the world health organization regional headquarters for africa's stepwise laboratory quality improvement toward accreditation (who–afro–slipta) process and subsequently rolled out the strengthening laboratory management towards accreditation (slmta) programme across the whole country, becoming the first african country to do so.methods: slmta in lesotho was implemented in two cohorts. twelve and nineteen laboratory supervisors and quality officers were enrolled in cohort 1 and cohort 2, respectively. these 31 participants represented 18 of the 19 laboratories nationwide. for the purposes of this programme, the queen elizabeth ii (qe ii) central laboratory had its seven sections of haematology, blood bank, cytology, blood transfusion, microbiology, tuberculosis laboratory and chemistry assessed as separate sections. performance was tracked using the who–afro-slipta checklist, with assessments carried out at baseline and at the end of slmta. two methods were used to implement slmta: the traditional ‘three workshops’ approach and twinning slmta with mentorship. the latter, with intensive follow-up visits, was concluded in 9 months and the former in 11 months. a standard data collection tool was used for site visits. results: of the 31 participants across both cohorts, 25 (81%) graduated (9 from cohort 1 and 16 from cohort 2). at baseline, all but one laboratory attained a rating of zero stars, with the exception attaining one star. at the final assessment, 7 of the 25 laboratories examined at baseline were still at a rating of zero stars, whilst 8 attained one star, 5 attained two stars and 4 attained three stars. none scored above three stars. the highest percentage improvement for any laboratory was 51%, whereas the least improved dropped by 6% when compared to its baseline assessment. the most improved areas were corrective actions (34%) and documents and records (32%). process improvement demonstrated the least improvement (10%). conclusion: the slmta programme had an immediate, measurable and positive impact on laboratories in lesotho. this success was possible because of the leadership and ownership of the programme by the mohsw, as well as the coordination of partner support. introduction top ↑ the lesotho ministry of health and social welfare (mohsw), through its laboratory services directorate, is committed to the provision of essential services as part of the national health-care delivery to all basotho people. these services include comprehensive diagnostic testing of all prevalent major infections, for example, hiv and tb, monitoring of patient treatment, drug resistance testing and surveillance studies that inform policymaking decisions and major health reform.1the laboratory system in lesotho is structured in three tiers: referral, regional and district laboratories. the central laboratory at the queen elizabeth ii (qe ii) hospital in maseru serves as the national reference laboratory. there are two regional laboratories. the other 16 laboratories are at the district level, with 7 of these managed by the mohsw, 1 by the military and 7 by the christian health association of lesotho. the final district laboratory is owned by the partners in health, anon-governmental organisation. there has been a significant and progressive increase in demand for laboratory services in lesotho, with the qe ii central laboratory testing 114 114 specimens in 2006, compared to 16 250 in 2003, a 600% increase.2 the mohsw realised that the increased demand for testing had to be matched with high quality testing. accreditation was identified as one means of assuring continuous quality testing services.3 the mohsw and its laboratory partners have put in place a number of key pillars for launching their bid to accredit the public health and clinical laboratories successfully. these include a national laboratory policy, a 5-year national strategic plan, the appointment of a laboratory director and a national quality assurance unit (qau) headed by a national quality manager who is supported by key national quality officers. the qau is a unit created by laboratory services to manage the quality improvement initiatives for the entire laboratory network. in the strategic plan, objective 2.3 directly addresses these initiatives, aiming ‘to strengthen quality assurance of laboratory services and have a mechanism for attaining international accreditation defined’.3 these clearly stated objectives have been translated into yearly operational plans for the last 3 years,2 culminating, in 2011, in the generation of a ground breaking yearly operational plan that aimed to have seven laboratories declared ready for the world health organization regional headquarters for africa stepwise laboratory quality improvement toward accreditation (who–afro–slipta) process by the 3rd quarter of that year.2 this target was achieved but, unfortunately, the who–afro–slipta office was not ready to accept applications by that time. the qau was therefore established to ensure continued support for these quality improvement efforts. the strengthening laboratory management towards accreditation (slmta) programme was launched concurrently with the stepwise who–afro–slipta process in kigali, rwanda in 2009.4 slmta, a task-based curriculum, assists countries in the training of laboratory managers to implement the quality management system requirements of the who–afro–slipta process, with the aim of granting them eventual international accreditation.5 the mohsw of lesotho immediately embraced the slmta programme soon after engaging in the trainers’ workshopheld at the african centre of integrated laboratory training in johannesburg, south africa in november 2009. this programme was implemented at an opportune time in lesotho, because the country had already embarked on laboratory improvements through a number of policies and critical documents such as the national laboratory policy and 5-year strategic plan. a number of critical officers, namely the laboratory director, quality manager, national safety officer, national training officer, as well as programmes such as mentorship, were also in place. the slmta programme in lesotho was coordinated by the mohsw with the slmta coordinator facilitating as one of the quality officers within the qau. a number of laboratory partners, namely the association of public health laboratories (aphl), the centers for disease control and prevention (cdc) and the clinton health access initiative (chai), have provided technical and logistical support to the programme. the slmta programme has been part of the laboratory services’ yearly operational plans for the past 2 years.2 this paper describes the experience of lesotho in implementing the slmta programme. the purpose is to share this experience and the lessons learned from it with other countries that are implementing, or planning to implement, the slmta programme. the analysis of the programme in this paper will also inform present programme activities in lesotho, as plans for the training of more cohorts across the country are already underway. methods top ↑ the slmta programme in lesotho was implemented in two cohorts, with cohort 1 comprising 12 participants. these 12 participants selected from 4 district laboratories, 7 qe ii central laboratory sections (chemistry, haematology, cytology, microbiology, blood bank, blood transfusion and the tb laboratory) and 1 quality officer from the qau. all four participants from district laboratories were laboratory managers, whilst three of the seven from the qe ii central laboratory were section supervisors and the other four were section level quality officers (table 1). cohort 1 enrolled only laboratories that were located within the vicinity of maseru, the location of the training venue. table 1: profile of cohort 1 and cohort 2 participants in the strengthening laboratory management towards accreditation (slmta) programme in lesotho. figure 1: schematic of the strengthening laboratory management towards accreditation (slmta) cohort 1 rollout. figure 2: strengthening laboratory management towards accreditation (slmta) cohort 2 implementation model. cohort 1 did not follow the three workshop series recommended for slmta; instead, slmta was twinned with mentorship, which was already under way. seven of the participants in this cohort came from the qe ii central laboratory, where the mentor, who was also the slmta facilitator, was conducting the second round of mentorship at that time. the other four were based within an 80 km radius of the central laboratory, whilst the participant from the qau was based within the mohsw headquarters, the training venue.instead of the recommended 4–5 day workshops, the slmta modules were delivered 1 day per week on a friday over two blocks of 6 weeks each. the two blocks were spaced 3 months apart. in total, the 1-day workshops over 12 weeks matched the 12 days of the recommended 4-day workshops, after the three-workshop series was completed (figure 1). more intensive follow-ups were feasible because of the proximity of all participants. each of the participants had one follow-up visit a week, to a total of 12 visits each over a 9-month period. the intensity of the follow-up visits allowed the participants to complete the recommended three improvement projects over the course of 9 months. the increased supervisory visits also allowed most of the participants to have more than one project running at a time; for example, if one was waiting for supply requisition documents from procurement, another project could be initiated on sample rejections. cohort 2 followed the recommended slmta three workshops approach (figure 2), with three certified slmta facilitators affiliated with the mohsw, aphl and chai. a total of 16 district laboratories and 3 qe ii central laboratory sections (each with 1 participant per laboratory, or per section, enrolled) were part of the slmta cohort 2 (table 1). only one laboratory, quithing district laboratory, did not attend the training because the communication for workshop attendance did not arrive on time. an implementation plan was developed before the programme started to ensure that the slmta programme for cohort 2 had specific, fixed dates of activities spanning the entire 12 months. these were adhered to 98% of the time; that is, only one of the planned activities did not take place on the assigned date. the last activity not met was the assessment by who for the slipta star status recognition, as the structures for such assessments were not in place by march 2010 (table 2). baseline assessments baseline assessments were conducted by the three slmta facilitators using the who–afro–slipta checklist. two facilitators assessed six laboratories each, whilst one assessed seven. training on the use of the checklist was conducted for two of the facilitators by the other facilitator who was a who trained assessor. the who–afro–slipta checklist provides a quantitative measure of adherence to accreditation requirements for quality and competency. the scored checklist (totalling 250) allows for the rating of a laboratory’s quality improvement status by using a zero–five star rating, calculated as follows: 0–137 = zero stars, 138–160 = one star, 161–185 = two stars, 186–211 = three stars, 212–236 = four stars, and 237–250 = five stars. improvement projects baseline assessments were conducted by the three slmta facilitators using the who–afro–slipta checklist. two facilitators assessed six laboratories each, whilst one assessed seven. training on the use of the checklist was conducted for two of the facilitators by the other facilitator who was a who trained assessor. the who–afro–slipta checklist provides a quantitative measure of adherence to accreditation requirements for quality and competency. the scored checklist (totalling 250) allows for the rating of a laboratory’s quality improvement status by using a zero–five star rating, calculated as follows: 0–137 = zero stars, 138–160 = one star, 161–185 = two stars, 186–211 = three stars, 212–236 = four stars, and 237–250 = five stars. figure 3: performance of all laboratories at baseline and final assessments conducted in january 2010 and january 2011, respectively, using the world health organization regional headquarters for africa stepwise laboratory quality improvement toward accreditation (who–afro–slipta) checklist. figure 4: example of an improvement project (from cohort 2) on improving performance and reviewing the internal quality control for cd4 the cd4 section using a cyflow analyser, indicating (a) the baseline data from february 2010 and (b) the final data at project completion in june 2010. follow-up visits for cohort 2, each of the three facilitators was assigned laboratories which they followed throughout the duration of the programme. each facilitator followed up with both the laboratories and the qe ii central laboratory sections they had assessed at baseline. this allowed for relationship building between the facilitator and the laboratory. during each visit, the facilitator made an appointment with the hospital management to explain the vision of the laboratory directorate of accreditation, as well as the slmta process. the need to support the laboratory was also emphasised.a slmta follow-up visit assessment tool was developed and implemented for cohort 2 to standardise follow-up visits and guide the facilitator on areas that needed to be covered during the site visit. these included the provision of supervision on the improvement project, follow-up on the implementation of activities taught during the last workshop by determining the uptake of slmta tools, the tracking of quality indicators and the implementation of a set of agreed compulsory activities. compulsory activities were considered to be the ‘must do’ and easy-to-implement activities that did not constitute an improvement project, for example, a duty roster, an equipment master list, a team meeting and the use of a management calendar. in addition, the tool required the facilitator to document coaching provided to the slmta participant or other staff with regard to the improvement project, as well as in relation to other areas of laboratory improvement. each visit lasted one full working day. all reports from visits were submitted to the slmta coordinator at the mohsw, who ensured that the participating laboratories received copies of their progress reports. figure 5: average performance of all laboratories across the 12 sections, as measured by the world health organization regional headquarters for africa stepwise laboratory quality improvement toward accreditation (who–afro– slipta) checklist. table 2: lesotho strengthening laboratory management towards accreditation (slmta) cohort 2 implementation plan, developed before the start of the programme. table 3: list of improvement projects carried out by cohort 1 and cohort 2 participants in the strengthening laboratory management towards accreditation (slmta) programme in lesotho. results top ↑ a total of 12 participants were enrolled for cohort 1 (table 1), of whom 3 (25%) did not graduate. of the nine who graduated, four were from each of the four district laboratories, four where affiliated with the qe ii central laboratory sections of cytology, haematology, microbiology and blood transfusion, and one was from the qau (table 1). of the three who did not graduate, one was transferred during the course of the training and could not continue, whilst the other two did not meet the requirements of 100% attendance and the completion of three improvement projects.for cohort 2, 19 participants were enrolled (table 1), of whom 16 (84%) graduated. three did not graduate because of reasons ranging from non-completion of three improvement projects and attending less than three workshops, to one participant being placed under disciplinary suspension. a total of 25 participants successfully completed the slmta programmes for cohorts 1 and 2. these 25 were from 18 of the 19 laboratories of lesotho. as mentioned above, the nineteenth laboratory, quithin, missed the first workshop as a consequence of miscommunication of training dates. for assessment purposes, the qe ii central laboratory had its seven sections of haematology, blood bank, cytology, blood transfusion, microbiology, tb laboratory and chemistry classified as separate sections; hence, the 25 assessments results in figure 3. improvement project outcomes for cohort 1, the participant from the qau received the award for the best improvement projects. the selection was based on the overall impact that the participant’s three projects had on the entire laboratory service of lesotho. the winner’s first project resulted in the upgrade and implementation of the current document control system for the entire network. their second project improved the tracking of equipment down-time and the reporting of equipment breakdown for all contracted equipment within the government laboratory network, whilst their third project designed a mechanism of providing assessment for laboratories that miss the external quality assurance deadlines for various reasons.in cohort 2, the butha buthe district laboratory received the award for the best improvement project, which investigated the improvement of documentation in internal quality control for its cd4 section. the difference between baseline and final data collected between february and june 2010 for the butha buthe improvement project is illustrated in figure 4. the overall performance of laboratories over the entire slmta period improved over time (figure 3). of the 25 laboratories, 24 (96%) demonstrated an improvement over the 12-month period. one section at queen ii central (laboratory 13, figure 3) was not assessed post-slmta because of miscommunication with the laboratory manager, which resulted in the assessor being unable to access the laboratory. the most improved laboratory demonstrated an increase of 51% from its baseline to final assessments, whilst the laboratory that performed the least demonstrated a 6% drop between these two assessments. the average performance of the 25 laboratories across the 12 sections of the who–afro–slipta checklist is illustrated in figure 5. the most improved areas – measured by the difference between baseline and final percentage score for each of the 12 sections – were corrective actions (34%), documents and records (32%), and customer service (29%). the process improvement category demonstrated the least improvement (10%). the star rating of the laboratories using the who–afro–slipta checklist star rating is reflected in table 3. at baseline, all but one laboratory had zero stars; yet, by the end of slmta, 17 (68%) of the laboratories had achieved a star status. of that 17, 8 (32%) had one star, 5 (20%) had two stars, and 4 (16%) had three stars, gained over a 12-month period of slmta (table 4). table 4: star rating of all laboratories between baseline and final assessments using the world health organization regional headquarters for africa stepwise laboratory quality improvement toward accreditation (who–afro–slipta) checklist. discussion top ↑ tracking of performance data by using the who–afro–slipta checklist showed that only one (14%) of the 25 enrolled laboratories had at least a star one status rating at baseline out of a possible 5 stars after this word. however, by january 2011, 17 (68%) had achieved a star rating, with four of the laboratories reaching three-star status. the results indicate that there was a measurable improvement over the 12-month period of slmta.one laboratory (laboratory 20, figure 3) had a negative improvement of 6% because of an unexpected staff departure: the supervisor, who was in the slmta programme, was transferred to another duty station. then, of the two technologists from laboratory 20 who remained, one went on extended maternity leave. even though systems could have been implemented, sustainment would have been difficult with only one out of three possible staff members in place. in addition, the tebellong district laboratory (laboratory 10, figure 3) had the smallest percentage of positive improvement, namely 1%, because of the unexpected withdrawal of an slmta participant. the participant had to be withdrawn from slmta because of his disciplinary suspension from the hospital and, as such, he could not attend workshops 2 and 3. tebellong had a staff complement of only two technologists. replacement processes are carried out centrally at the mohsw public services department and the process takes at least 6 months. laboratories, in general, were weaker in some areas than others. in particular, internal audits, management reviews, corrective actions and process improvement, showed the lowest average scores. these areas have been strengthened in slmta cohort 3, currently in progress, as well as in the on-going mentorship programme. one of the strongest pillars of success of slmta in lesotho was the strong commitment shown by the ministry of health laboratory services to the slmta programme. the ownership and the strong leadership of the directorate of laboratory services and its coordination of technical support by laboratory partners made the slmta successful in lesotho. the high level of dedication demonstrated by slmta participants created tremendous enthusiasm within the laboratories, as observed by the three facilitators during the supervisory visits and workshop training. this also might have contributed to improvements, despite ever-increasing workloads. planning the entire slmta programme from the start helped to ensure that the programme was completed on time with few logistical problems. the entire 12-month programme for cohort 2 was designed in january 2010, with fixed dates of baseline assessments, all three workshops, six follow-up visits and the final assessments decided upon at that time. this was critical, because slmta is a long and continuous process and therefore chances of disruptions are high. in this phase of slmta, 98% of the planned activities were met within the agreed timetable. coordination by the mohsw was central to the success of slmta and functioned as a means of strengthening local capacity building within the qau. findings from the slmta assessments and site visits informed the qau on priority areas. standardising the supervisory visits and the meetings of hospital management for support was also critical for the improvement efforts. the mohsw and its partners involved in the slmta as facilitators had to have dedicated time for the programme. the technical support and effective coordination of activities with the mohsw by its partners (chai, aphl and cdc) also played a pivotal role in the rollout of the slmta programme. conclusion top ↑ the slmta programme in lesotho resulted in immediate, measurable laboratory improvements shown by all but one laboratory. with this performance, seven have been prepared and are ready for application to the who–afro–slipta process.4 the seven selected are those with the highest who–afro–slipta checklist marks from the slmta final assessments conducted by facilitators. as part of the preparation, these seven laboratories have already been assessed by who–afro–slipta trained assessors who were invited to lesotho in february 2011.the lesotho experience demonstrated that if slmta is planned and executed appropriately with the minimum of six follow-up visits and the three improvement projects, it will become an effective programme. thus, it is clear that the improvement projects and follow-up visits are the two critical pillars of the slmta programme. however, the programme did face some challenges. from the perception of the participants to the process, it was indicated that improvement projects consumed a lot of time and could not be carried out during the course of their normal working day. in some instances, participants had to request to be relieved from their routine work to work on the improvement projects. only a few of the laboratories had computers for typing their projects and for drafting quality documents. participants also felt that the time of 3 months allocated for improvement projects was too short. furthermore, as a result of the strict criteria for slmta participation to graduation, there is a risk that participants may not be able to fulfil all criteria. this could result in the exclusion of laboratories from the slmta programme and a missed opportunity for laboratory improvement. this is another good reason to continue to roll out the slmta programme, as this will allow for continuous and sustainable laboratory quality improvement. this is the approach that lesotho has taken by continuing the slmta training of more cohorts comprising different participants from the same laboratories. acknowledgements top ↑ we would like to thank all laboratory supervisors who actively participated in the: slmta programme and management who supported the implementation of slmta. the financial and technical support provided by the cdc, aphl and chai, is acknowledged. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. authors’ contributions d.m. (ministry of health and social welfare) was the project leader as director of laboratory services, lesotho. t.m. (clinton health access initiative) was an slmta facilitator for both cohort 1 and 2 and wrote the manuscript. m.l. (ministry of health and social welfare) and k.l. (association of public health laboratories) were slmta facilitators for cohort 1. jw (clinton health access initiative), as the country director of chai, and y.m. (centers for disease control and prevention), as the cdc laboratory advisor, were the project coordinators of this project. m.l., k.l. and y.m. reviewed the manuscript. references top ↑ 1. lesotho ministry of health and social welfare. laboratory services national policy, october 2008. maseru. unpublished.2. lesotho ministry of health and social welfare. laboratory services national yearly operational plan, 2009/2010 to 2010/2011.maseru. unpublished. 3. lesotho ministry of health and social welfare. laboratory services national strategic plan, 2008/2009 to 2012/2013. maseru. unpublished. 4. world health organization. [who representative’s office for rwanda] [press release]. kigali: who; 2009 jul 27. french. 5. yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries. an innovative training approach to accelerate laboratory accreditation. am j clin pathol. 2010;134(3):401–409. http://dx.doi.org/10.1309/ajcpnbbl53fwuiqj, pmid:20716796 abstract introduction methods results discussion acknowledgements references about the author(s) victor n. fondoh bamenda regional hospital laboratory, regional hospital bamenda, cameroon department of medical laboratory sciences, school of health and medical sciences, catholic university of cameroon, bamenda, cameroon department of health economics policy and management, faculty of business management, catholic university of cameroon, bamenda, cameroon nobert ndzenjempuh department of medical laboratory sciences, school of health and medical sciences, catholic university of cameroon, bamenda, cameroon tamunjoh stella department of medical laboratory sciences, school of health and medical sciences, catholic university of cameroon, bamenda, cameroon richard m. fondoh north-west regional fund for health promotion, bamenda, cameroon charles n. awasom department of anatomy, school of health and medical science, catholic university of cameroon, bamenda, cameroon rebecca enow-tanjong department of medical laboratory sciences, school of health and medical sciences, catholic university of cameroon, bamenda, cameroon egbe p. egbengu department of medicine and surgery, school of health and medical science, catholic university of cameroon, bamenda, cameroon robert leke department of medicine and surgery, school of health and medical science, catholic university of cameroon, bamenda, cameroon njini f.n. rose regional hospital bamenda, bamenda, cameroon denis nsame regional hospital bamenda, bamenda, cameroon citation fondoh vn, ndzenjempuh n, stella t, et al. prevalence of alpha and beta haemolysin among blood group o donors in bamenda, cameroon. afr j lab med. 2022;11(1), a1432. https://doi.org/10.4102/ajlm.v11i1.1432 original research prevalence of alpha and beta haemolysin among blood group o donors in bamenda, cameroon victor n. fondoh, nobert ndzenjempuh, tamunjoh stella, richard m. fondoh, charles n. awasom, rebecca enow-tanjong, egbe p. egbengu, robert leke, njini f.n. rose, denis nsame received: 25 oct. 2020; accepted: 03 feb. 2022; published: 19 apr. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the occurrence of high titres of alpha (anti-a) and beta (anti-b) haemolysin immunoglobulin g antibodies in blood causes haemolysis during blood transfusion from a group o donor, commonly and inappropriately known as the ‘universal blood donor’, to a group a, b or ab recipient. surprisingly, haemolysin testing is not routinely done during blood transfusion services in bamenda, cameroon. objective: this study aimed to determine the prevalence of haemolysin among blood group ‘o’ donors at the regional hospital bamenda blood bank, bamenda, cameroon. methods: this was a cross-sectional descriptive study carried out between june and september 2020 at the regional hospital bamenda blood bank, bamenda, cameroon. blood group o donors who were free from transfusion-transmissible infections were selected systematically and serially and their serum tested for the presence of haemolysin. haemolysin titres were determined, and titres ≥ 8 were considered significant. the associations between haemolysin prevalence and age group, gender and rhesus d blood group were determined using the chi-square test. results: the prevalence of haemolysin among the 480 study participants was 52.1% and significant haemolysin titres were detected in 18.5%. there was no association between haemolysin and gender, age group or the rhesus d blood group. conclusion: the prevalence of significant titres of haemolysin among participants in this study was high. there is the need to test for haemolysin in blood group o donors to prevent the potential risk to blood group a, b, and ab recipients and to provide safer blood for transfusion. keywords: prevalence; haemolysin; immunoglobulin; blood group o; donors; bamenda; cameroon. introduction the occurrence of alpha (anti-a) and beta (anti-b) immunoglobulin m antibodies in the absence of corresponding red blood cell antigens is a significant feature of the abo blood group system in individuals.1 when blood transfusion is done without consideration for abo compatibility, these naturally occurring antibodies are a potential cause of dangerous haemolysis in recipients.2 although these antibodies react optimally at 4 °c, they may also cause haemolysis at 37 °c. also, some blood group o, and sometimes blood group a2, individuals may develop alpha and beta antibodies of the immunoglobulin g class that react optimally at 37 °c. these antibodies are commonly referred to as haemolysins and are more dangerous compared to naturally occurring haemolysins.2 in cases of non-iso group-compatible abo transfusion, these antibodies can trigger the complete complement cascade, leading to haemolytic reactions.2 several studies have reported complications in patients who were transfused with non-identical blood groups, including disseminated intravascular coagulation,3,4 hepatic and renal failure leading to death,3,5 paleness, jaundice, fever, ecchymosis and generalised exfoliation of the skin,6 significant reduction in packed cell volume,7 hyperbilirubinaemia,8 varying degrees of haemoglobinaemia, and intravascular agglutination.9 several studies have advocated that the transfusion of group o blood to a, b, or ab recipients be discontinued due to the high prevalence of alpha and beta haemolysins among blood group o donors.10,11,12 despite this concern, the practice is yet to be discontinued. several studies have postulated that these alpha and beta antibodies originated as products of immunisation from allogenic stimulation (due to transfer of antigen by red cells from pregnancy with an abo-incompatible foetus, incompatible blood transfusion, tissue transplant, etc.) and heterogeneous stimulation (due to vaccination, serotherapy, and inoculation from vectors such as blood-sucking insects and certain pharmaceutical preparations contaminated with alphaand beta-like antigens, etc.).13,14,15 due to the high demand for and shortage of allogenic blood in most developing countries, including cameroon, as well as the relative difficulty in getting abo group-compatible blood during emergencies, blood group o, which is often inappropriately referred to as the ‘universal blood donor’, has been increasingly transfused to non-group o recipients.16 besides, there is evidence that blood group o is the most abundant group in the abo blood system, with a prevalence of 51.1% reported in a study in cameroon.17 blood transfusion is an essential medical practice that replenishes lost blood or blood products in the recipient. the transfused blood should be as safe as possible to ensure maximum benefit to the recipients. thus, there is the need to strictly adhere to standard screening protocols to achieve safe blood transfusion. unfortunately, this practice is limited in the developing world due to inefficient blood banking systems and scarcity of screening facilities. besides, the standard protocol for compatibility testing to ensure safe blood has been omitted or abbreviated by many screening services. this standard compatibility testing protocol18 requires that all group o blood intended for transfusion to group a, b, or ab recipients should be screened for the presence of high titres of alpha and beta haemolysins and that only haemolysin-free blood should be reserved for blood group a, b, or ab recipients, while haemolysin-positive blood should be reserved for group o recipients only.13 several countries, including côte d’ivoire, have included a haemolysin test as part of the standard protocol in their national blood transfusion programmes.18 surprisingly, this practice is yet to be implemented in blood bank facilities in bamenda or cameroon in general. hence, this study aimed to determine and demonstrate the presence of significant titres of alpha and beta haemolysins among blood group o donors at the regional hospital bamenda blood bank (rhbbb), bamenda, cameroon, to guide the implementation of a policy to include haemolysin testing in the protocol for compatibility testing in cameroon. methods ethical considerations administrative authorisation to carry out this work was provided by the catholic university of cameroon, bamenda, north-west regional delegation of public health, bamenda, and regional hospital bamenda. ethical clearance was provided by the institutional review board of the regional hospital bamenda (irb number: 211/app/rdph/rhb/irb). participants provided written informed consent and were free to withdraw from the study at any time. the participants’ data were coded by assigning numbers to identify the participants instead of names. the anonymity of participants and their data were ensured by storing the data on password-protected computers and in locked file cabinets accessible only to the study staff and researchers. study area this research was carried out in bamenda at the rhbbb, bamenda, a unit at the regional hospital bamenda. the rhbbb receives approximately 5400 blood donors and issues about 4200 safe pints of blood yearly. it has a standard blood transfusion service and is enrolled in a certification programme with the safe blood for africa foundation. it is also the largest blood transfusion centre in the north-west region and provides transfusion services to the region and beyond. research design this was a descriptive, cross-sectional study conducted between june 2020 and september 2020 at the rhbbb. the sample size was calculated based on a previous study conducted in 2011 in eastern nigeria16 that reported an overall haemolysin prevalence of 55.4%. a minimum of 385 participants was required for this study. blood donors arriving at the reception area of the rhbbb undergo routine screening to determine physical fitness to donate blood using a standard questionnaire validated and provided by the rhbbb quality team. this routine screening selects individuals who had not donated blood and had no history of sexually transmitted diseases in the three months preceding the blood donation, were free from non-communicable diseases such as diabetes and hypertension, had not been vaccinated in the last four months, had not taken medication for at least one week, and had not smoked on the day of the donation or taken alcohol in the last 24 h. women who were pregnant, breastfeeding, or menstruating or expecting their menses within one week were excluded. in addition, only donors who weighed greater than 50 kg, were between the ages of 18 and 60 years (women) or 18 and 65 years (men) and had blood pressures between 100 mmhg and 140 mmhg over 60 mmhg – 100 mmhg and temperatures between 36 °c and 37.5 °c were endorsed as fit for blood donation. as part of the routine protocol for screening donors to obtain safe blood in the blood bank, abo and rhesus d blood group tests and transfusion transmittable infection (tti) tests were done on samples from all donors. abo and rhesus blood groups were determined using the procedure described by dacie and lewis19 using the blood in the ethylenediaminetetraacetic acid tube. the tti test was done using the blood in the plain tube. tti testing included the following: hiv test using the hiv-1/2 ag/ab combo determine (alere medical co., ltd, matsuhidai, matsudo-shi, chiba-ken, japan) as the first-line test and oraquick (orasure technologies, inc., bethlehem, pennsylvania, united states) as the second-line test; hepatitis b and hepatitis c virus tests using the diaspot diagnostic kit (diaspot diagnostics, jawa barat, indonesia); syphilis test using the rapid plasma reagin (rpr)-carbon slide agglutination assay (cypress diagnostics, langdorp, belgium) and treponema pallidum haemagglutination assay (omega diagnostic, alva, scotland, united kingdom); and malaria test using the carestartth malaria pf/pan (hrp2/pldh)ag combo rdt (accessbio, somerset, new jersey, united states). as part of the donation process, blood samples were collected into two tubes – one in a plain tube and the other in an ethylenediaminetetraacetic acid tube from donors already screened using the questionnaire as fit for blood donation. the screened donors were systematically and serially contacted to participate in the study. only donors who were blood group o, free from all the ttis, and who consented to be part of the study were included. a standard data collection format was used to collect information on the age, blood groups and gender of the study participants. haemolysin test and titration blood specimens collected in the plain tubes (used for tti screening) were used to determine haemolysin titres within 24 h of specimen collection. briefly, the sample was allowed to clot for about 45 min and then centrifuged to separate the serum. the serum was then tested for the presence of haemolysins.11,16 zero point 5 mililetres of the serum was placed in three test tubes labelled ‘a’, ‘b’ and ‘o’, and 0.5 ml of 5% blood group a, b, or o washed red cells suspended in physiological saline was added to each tube. the blood group o red cells were used as a negative control. the setup was incubated at 37 °c for 2 h and centrifuged afterwards. the supernatant was then examined macroscopically (in bright light) and microscopically for the presence of haemolysins. the degree of haemolysis was graded as 1+ for traces of haemolysis, 2+ for partial (greater than 50% but not complete) haemolysis, 3+ for complete haemolysis, and negative when no haemolysis was observed.11,20,21,22 all sera positive for haemolysis were titrated to quantify the degree of haemolysis.11,20,21 0.5 ml of the sera was diluted twofold with physiological saline to a titre of 526, and 0.5 ml of 5% washed red cells of the respective positive sera was added. the setup was incubated for 2 h and observed for haemolysis macroscopically and microscopically. the reciprocal of the serum dilution in the last tube with haemolysis was considered as the titre.11,20,21 statistical analysis collected data were entered into microsoft excel 2010 (microsoft corporation, redmond, washington, united states) and double-checked for errors by a second person. all analyses were done using statistical package for social sciences version 20 (ibm corp., chicago, illinois, united states). haemolysin prevalence was determined as the proportion of participants whose blood samples were positive for haemolysin (alpha, beta or both alpha and beta haemolysin). haemolysin titres equal to or greater than 8 were considered significant. the prevalence of significant titres was also determined as the proportion of participants with significant haemolysin titres. associations between haemolysin prevalence and age group, gender and rhesus d blood group were determined using chi-square test, and p-values < 0.05 were considered as statistically significant. results in total, 1161 blood donors presented to the rhbbb for blood donation between june 2020 and september 2020, 820 of whom were screened for physical fitness. of the 820 donors, 812 were classified as physically fit based on the applied standard questionnaire prepared by the rhbbb, and all the 812 donors consented to participate in the study. out of the screened 812 donors, 493 were blood group o, 480 of whom were free from ttis and were included to participate in the study (figure 1). figure 1: selection of study participants among blood group o donors at the rhbbb, cameroon, june 2020 – september 2020. participants were aged between 18 years and 55 years and comprised 383 (79.8%) men and 97 (20.2%) women (table 1). four hundred and sixty-three (96.5%) of the participants were rhesus d positive and 17 (3.5%) were rhesus d negative. of the 480 blood group o donors tested for haemolysins, 230 were negative while 250 were positive, giving a haemolysin prevalence of 52.1%. haemolysins were detected in 204 (53.3%) men and 46 (47.4%) women. the single participant aged ≥ 55 years was positive for haemolysin. in the other age groups, haemolysin prevalence was highest among participants aged between 45 and 54 years (28/48; 58.3%), followed by participants aged 18–24 years (86/156; 55.1%), 35–44 years (44/87; 50.6%) and 25–34 years (91/188; 48.4%). there was no association between haemolysin production and gender (p = 0.304), age group (p = 0.501) or rhesus d positivity (p = 0.628). two hundred and forty (240) of the 463 rhesus d-positive participants (51.8%) were positive for haemolysin while 10 (58.8%) of the 17 rhesus d-negative participants were positive for haemolysin. table 1: association between haemolysin production and gender, age group or rhesus d positivity of blood group o donors at the rhbbb, cameroon, june 2020 – september 2020. of the 250 participants positive for haemolysin (alpha, beta or both), 105 (42.0%) were positive for only alpha haemolysin, 69 (27.6%) were positive for only beta haemolysin, and 76 (30.4%) were positive for both alpha and beta haemolysins (figure 2). haemolysins from the 181 participants positive for alpha haemolysin showed trace haemolysis (47; 26.0%), partial haemolysis (95; 52.5%) and complete haemolysis (39; 21.5%) (table 2). of the haemolysins from the 145 participants positive for beta haemolysin, 58 (40.0%) showed trace haemolysis, 58 (40.0%) showed partial haemolysis, and 29 (20.0%) showed complete haemolysis. the highest observed haemolysin titre was 32 and was detected in five (2.8%) alpha haemolysin-positive participants and three (2.1%) beta haemolysin-positive participants. eighty-nine (89; 18.5%) participants presented with significant haemolysin titres (table 3), of which 45 (50.6%) were alpha haemolysin-positive only, 16 (18.0%) were beta haemolysin-positive only, and 14 (15.7%) were positive for both alpha and beta haemolysin (figure 3). figure 2: the prevalence of alpha and beta haemolysins among haemolysin-positive blood group o donors at the rhbbb, cameroon, june 2020 – september 2020. figure 3: prevalence of significant titres of alpha and beta haemolysins among blood group o donors at the rhbbb, cameroon, june 2020 – september 2020. table 2: degree of haemolysis of alpha and beta haemolysins from blood group o donors at the rhbbb, cameroon, june 2020–september 2020. table 3: titres of alpha and beta haemolysins among blood group o donors at the rhbbb, cameroon, june 2020–september 2020. discussion this study was carried out to determine the prevalence and titres of haemolysin among blood group o donors at the rhbbb. our study found a high haemolysin prevalence of 52.1% among the study population. significant haemolysin titres (defined as titres ≥ 8) were also detected in a high proportion (18.5%) of participants. this high prevalence may be attributed to immunisation arising from exposure to mosquito bites and parasitic infections of the gastrointestinal system.22 high prevalence rates of malaria23 and gastrointestinal parasites24 have been reported in bamenda, north-west region, cameroon.25 the high prevalence of haemolysin in this study is comparable to that (52.8%) reported by a study in abakaliki, nigeria, in 2014.25 lower haemolysin prevalence rates have been reported by studies among healthy blood donors in south india (10.8% in 2019),26 abidjan, côte d’lvoire18 (35.1% in 2016), lagos, nigeria10 (30.3% in 2015), bauchi, nigeria27 (22.2% in 2015), anambra, nigeria28 (16.06% in 2015), tunisia14 (6.6% in 2008), ilorin, nigeria11 (23.2% in 2001), and nigeria12 (30.6% in 1990). a higher prevalence of 69.0% was reported in a 2012 study on healthy blood donors in bangkok, thailand.29 these differences in prevalence rates may be due to the admixture of blood of immigrants as a result of intermarriages,20 variations in serum-cell ratios,30 or differences in geographical location,26 particularly due to the differences in the degree of exposure to gastrointestinal parasites22 and mosquitoes.13,14,15 it has been reported that higher serum-cell ratios increase the tendency for red cell lysis.30 the alpha haemolysin prevalence in this study (53.6%) was higher than that reported in a study conducted in 2010 in southeast nigeria that reported a prevalence of 10.3% for alpha haemolysin.20 in contrast, the observed prevalence rates of beta haemolysin, and both alpha and beta haemolysins in this study were lower than that reported in the same study (8.3% vs 12.6% for beta haemolysin, and 15.8% vs 32.5% for both alpha and beta haemolysins).20 alpha haemolysins were more prevalent in our study compared to beta haemolysin, which is consistent with the findings of a study conducted in 201511 in lagos, nigeria, but different from the findings of another study conducted in 2001 in ilorin, nigeria, that observed a higher prevalence of beta haemolysin compared to alpha haemolysin.11 the reasons for these variations may either be genetic or environment-induced.31 the absence of associations between haemolysin production and gender, age group or rhesus d blood group of blood group o donors in our study is consistent with the findings from previous studies.4,10,11,14,18,31,32 the prevalence of significant haemolysin titres (titres ≥ 8) in our study is noteworthy, considering the evidence that titres above this threshold can significantly cause haemolysis in vivo.33 this may be because parasitic infections such as malaria are endemic in the study area. there is evidence that the malaria parasite can stimulate the production of haemolysin.22 our observation is lower than those reported by studies conducted among blood group o donors in lagos, nigeria, in 2015 (18.6%)10 and ilorin, nigeria, in 2021 (31.7%).11 this may be due to differences in geographical location26 and the degree of exposure to gastrointestinal parasites22 and mosquitoes.13,14,15 group o blood should not be transfused to blood group a, b, or ab recipients except when such blood is tested and determined to be free of haemolysins. the haemolysin test should be included in the protocol for screening and compatibility testing of blood donors in the national blood transfusion programme of cameroon. this can be achieved through the collaborative efforts of the government, the ministry of public health of cameroon, the national blood transfusion programme of cameroon, as well as staff of the rhb and rhbbb. training for haemolysin testing should be conducted at the national level. furthermore, more studies should be carried out in other localities in cameroon to determine the haemolysin prevalence or presence of significant titres of haemolysin among blood group o donors. limitations due to limited resources, we only used the visual titration method and could not carry out the spectrophotometric or gel methods for the quantification of red blood cell lysis; this could have influenced the detection of lysis. however, our findings were compared only with studies that used the visual method. conclusion alpha and beta haemolysins are prevalent and exist in significant titres among blood group o donors in bamenda, cameroon. thus, there is an urgent need for public health intervention. considering the frequent practice of non-iso group-compatible abo transfusion, there is a need to routinely test for the presence of haemolysins in blood donors to prevent the potential risk to recipients and to provide safer blood for maximum benefits to the recipient. acknowledgements we acknowledge all the participants who gave their blood for this study. thanks to the management of the department of medical laboratory science, faculty of health and medical science, and the department of health economics, policy and management, faculty of business management – catholic university of cameroon-bamenda for academic support during this study. lastly, we appreciate the management and staff of the rhbbb for their wonderful support during the study. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions v.n.f. (study lead) designed the model and the computational framework, conceived, and planned the experiments and finalised the writing of the manuscript. t.s. and v.n.f. were responsible for supervision of the findings. v.n.f., r.m.f and c.n.a. were responsible for statistical analysis. v.n.f. and n.n. were responsible for specimen collection, performing the experiments and data collection. t.s., n.n., r.m.f. c.n.a., r.e-t., e.p.e., r.l, n.f.n.r. and d.n. reviewed and edited the manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability the data supporting the findings of this study are available within the article. data are also available on request from the corresponding author, v.n.f. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references 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saphire d, rudolph n, hackleman s, stone w. the effect of age on the level of human abo blood group antibodies. aging clin exp res. 1993;5(3):177–184. https://doi.org/10.1007/bf03324152 article information authors: peter n. fonjungo1 mulu girma2 zenebe melaku3 teferi mekonen1 amilcar tanuri3 bereket hailegiorgis3 belete tegbaru2 yohannes mengistu1 aytenew ashenafi4 wubshet mamo5 tesfay abreha3 gudetta tibesso2 artur ramos6 gonfa ayana2 richard freeman7 john n. nkengasong6 solomon zewdu4 yenew kebede1 almaz abebe2 thomas a. kenyon1 tsehaynesh messele2 affiliations: 1center for disease control and prevention (cdc), addis ababa, ethiopia2ethiopian health and nutrition research institute (ehnri), addis ababa, ethiopia 3icap, columbia university, addis ababa, ethiopia 4john hopkins university tsehai program, addis ababa, ethiopia 5university of washington, itech program, addis ababa, ethiopia 6center for global health, centers for disease control and prevention (cdc), atlanta, usa 7clinton hiv/aids access initiative (chai), addis ababa, ethiopia correspondence to: peter fonjungo postal address: po box 1014, entoto road, addis ababa, ethiopia dates: received: 20 mar. 2012 accepted: 05 oct. 2012 published: 22 may 2013 how to cite this article: how to cite this article: fonjungo pn, girma m, melaku z, et al. field expansion of dna polymerase chain reaction for early infant diagnosis of hiv-1: the ethiopian experience. afr j lab med. 2013;2(1), art. #31, 7 pages. http://dx.doi.org/10.4102/ ajlm.v2i1.31 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. field expansion of dna polymerase chain reaction for early infant diagnosis of hiv-1: the ethiopian experience in this lessons from the field... open access • abstract • introduction • materials and methods    • project area    • approach to project implementation • results    • laboratory infrastructure and training    • scale-up of laboratory services    • quality of dbs dna pcr testing • ethical considerations • discussion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: early diagnosis of infants infected with hiv (eid) and early initiation of treatment significantly reduces the rate of disease progression and mortality. one of the challenges to identification of hiv-1-infected infants is availability and/or access to quality molecular laboratory facilities which perform molecular virologic assays suitable for accurate identification of the hiv status of infants.method: we conducted a joint site assessment and designed laboratories for the expansion of dna polymerase chain reaction (pcr) testing based on dried blood spot (dbs) for eid in six regions of ethiopia. training of appropriate laboratory technologists and development of required documentation including standard operating procedures (sops) was carried out. the impact of the expansion of eid laboratories was assessed by the number of tests performed as well as the turn-around time. results: dna pcr for eid was introduced in 2008 in six regions. from april 2006 to april 2008, a total of 2848 infants had been tested centrally at the ethiopian health and nutrition research institute (ehnri) in addis ababa, and which was then the only laboratory with the capability to perform eid; 546 (19.2%) of the samples were positive. by november 2010, ehnri and the six laboratories had tested an additional 16 985 hiv-exposed infants, of which 1915 (11.3%) were positive. the median turn-around time for test results was 14 days (range 14−21 days). conclusion: expansion of hiv dna pcr testing facilities that can provide quality and reliable results is feasible in resource-limited settings. regular supervision and monitoring for quality assurance of these laboratories is essential to maintain accuracy of testing. introduction top ↑ the joint united nations programme on hiv and/or aids (unaids) has estimated that in 2009 between 230 000 and 510 000 children under the age of 15 years became infected with hiv worldwide, with over 90% of these new infections occurring in sub-saharan africa, and mainly through mother-to-child-transmission.1 disease progression is aggressive in the first months of life in infants who acquired the infection in utero or around the time of delivery. if left untreated, almost half of these children will die before turning two years of age,2 and 75% by the age of five years. most of these deaths in children with hiv could have been avoided through early infant diagnosis (eid) and provision of effective care and treatment. interventions like the use of antiretroviral (art) drugs by infected pregnant women, safe delivery practices and safe infant feeding have helped reduce the risk of transmission to infants (from 40% to 5−10%).3,4,5,6 despite the availability of these life-saving interventions, some exposed infants still get infected. in 2003, art services were initiated in ethiopia and by 2005 free art services started with the support of the president’s emergency plan for aids relief (pepfar) and the global fund to fight aids, tuberculosis and malaria (gfatm).7 furthermore, these services have been rolled out to regional health facilities at both hospital and health centre levels. the total number of patients on art as of february 2010 was estimated at 179 183,8 including 10 496 children. one of the major challenges to expansion of art to children is limited or inadequate hiv diagnostic capacity. early identification of infants infected with hiv followed by prompt art treatment can help reduce morbidity and mortality. the need for early initiation of treatment in infants infected with hiv was emphasised in a south african cohort study that showed that 55% and 85% of hiv-infected infants had their cd4 cell count reduced to less than 25% by three and six months after birth, respectively.9 another study conducted in south africa showed that eid and early initiation of antiretroviral therapy helped to reduce mortality by 76% and hiv progression by 75%.10 other studies have shown improved outcomes when early art treatment is started in children.11,12 thus, laboratory diagnosis plays a critical role in increasing access of hiv-exposed infants to testing, and eventual timely and effective treatment and care. in adults, hiv is diagnosed by testing for the presence of antibodies to hiv in blood. in infants, antibody testing is inadequate, as persistent passively acquired maternal antibodies in the infant may yield false-positive results for up to 18 months or longer.13,14 it is important to provide accurate diagnostic services for identification of infants infected with hiv. because of its high sensitivity and specificity, dna polymerase chain reaction (pcr) has been widely used for diagnosis of hiv amongst exposed infants.15,16,17,18 the technology allows for pcr to be performed using a small spot from a dried blood spot (dbs) sample, as well as identification of infection from birth.19 additionally, this molecular test has been successfully established in several resource-limited settings to increase access to treatment.20,21,22,23 ethiopia has a tiered laboratory network, which is a hierarchical or ladder-like system with the national reference laboratory at the top followed by regional, referral and/or specialised hospital laboratories, then district and health centre laboratories. the tiered laboratory network reflects the structure through which health services are delivered to the population, and is under the coordination of the ethiopian health and nutrition research institute (enhri), which is the technical arm of the ministry of health, with responsibility and oversight of the country’s public health laboratories. ehnri has developed a comprehensive national laboratory master plan for integrated diseases. it has been used to coordinate and guide partners and stakeholders in the implementation of strategic laboratory objectives. the importance of the master plan in health system strengthening has previously been described.24 until october 2007, ehnri, which hosts ethiopia’s national hiv reference laboratory, was the only facility performing dna pcr for early infant diagnosis. ehnri was able to serve the entire country with the implementation of a sample referral system which allowed for dbs to be transported and tested, and returned results to the different regions of the country. ethiopia consists of about 1 127 127 square kilometers of land area, ranking it tenth in africa in terms of land size.25 the challenge was to scale up laboratory capacity for dna pcr in selected regions of ethiopia in order to provide better and more adequate coverage. here we report on the experience of expansion of dna pcr testing capabilities for early infant diagnosis to six different sites in ethiopia with a substantial increase in the numbers of hiv-exposed infants tested. materials and methods top ↑ project area the project was carried out in ethiopia, a country with a large land area situated in the horn of africa and divided into nine administrative regions and two metropolitan city administrations, addis ababa and dire dawa. hiv prevalence is estimated at 2.1%.26 there are an estimated 64 813 children under the age of 14 years living with hiv and/or aids, 75 420 hiv positive pregnant women per year with an estimated 14 138 new infections occurring in children annually.26 the decentralisation of dna pcr laboratories for eid was envisaged for six different sites to cover a greater geographic population. approach to project implementation in 2006, the national implementation plan for eid of hiv infection was developed by ehnri with the support of the us centers for disease control and prevention (cdc) and its partner, columbia university’s international center for aids care and treatment program (icap). the dbs-based dna pcr technology using the amplicor hiv-1 monitor v1.5 manual assay (roche diagnostics, indianapolis indiana, usa) was evaluated and eid services established at the national hiv reference laboratory of ehnri. dna pcr testing of dbs specimens at the national hiv reference laboratory started in april 2006. because of the vastness of the country and the time taken to transport dbs samples to the only eid laboratory at ehnri, there was the need to decentralise dna pcr testing for eid to the regions. in 2007, a joint assessment between cdc ethiopia, ehnri and university partners was conducted on the facilities in the identified regions. prior to this, a site assessment checklist was developed and used to assess potential sites. the joint site visit assessed state of infrastructure, personnel capacity, water, electricity and inventory, and provided recommendations. technical inputs were provided in the design of the laboratories, including physical separations of pre-pcr and post-pcr rooms. university partners (john hopkins, columbia university and university of washington) refurbished the laboratories in six regions for carrying out dna pcr for eid. cdc ethiopia and ehnri developed and purchased a comprehensive list of laboratory equipment, reagents and consumables. all sites had generators to serve as back-up for power outages. standard operating procedures were developed and distributed to all six sites. training materials were developed by ehnri in collaboration with cdc ethiopia and icap-columbia university and used at each site. at least two laboratory technologists were trained per site on dna pcr for eid using dbs. following training, laboratory technologists were independently assessed for accurately performing the test. two methods were used for assessing staff competency: the direct observational method was employed where the technologists were closely observed by a mentor as they performed assays independently per the sops and corrective actions provided where needed. additionally, each technologist was provided with six dbs of known results (blinded dbs specimen) and asked to perform assays unsupervised. as part of maintaining quality of testing in each laboratory, yearly refresher trainings were conducted, followed by challenging trainees with blind dbs specimens. additional trainings were provided for selected facilities based on the needs identified. also, to control for quality of testing in the laboratory, any two or more consecutive enzyme-linked immunosorbent assay (elisa) wells samples that were positive were each repeated from the same dbs card sample on a different day. ehnri served as a quality assurance laboratory for retesting the first 100 dbs specimens for each of the newly established sites. a comprehensive eid supply list was developed and laboratory technologists were trained in inventory management to ensure availability of supplies and thus avoid disruption of services as a result of shortage of one or several commodities. dna pcr testing started at the addis ababa regional laboratory in april 2008, and at the mekelle, adama and harar regional laboratories in may 2008, whilst at bahir dar and hawassa it started in june 2008. analysis of dbs tested for all sites was done up to november 2010. all six laboratories were enrolled in the cdc atlanta external quality assessment programme to monitor laboratory performance for dna pcr testing. proficiency testing (pt) panels were sent three times per year to each testing site. significant stages in the development and expansion of laboratory services for dna pcr for eid are presented here (figure 1). figure 1: milestones in expansion of laboratory services for early hiv infant diagnosis in ethiopia. in 2010, the postal service was introduced to transport dbs samples from referring sites to eid laboratories as well as delivering results back to referring sites. prior to initiation of sample referral using the postal service, a memorandum of understanding was signed between the ethiopian postal services enterprise and ehnri that included 565 facilities referring infant dbs samples. postal workers were trained in transportation of dbs, filling in of dbs samples transport log forms indicating date and time of pick up and drop off from dbs referring sites and eid laboratories, respectively. before the implementation of postal services in 2010, dbs samples and results were transported by an assigned facility staff member and/or university partners.the laboratory turn-around time was established with the help of the eid laboratory logbook. each dbs sample collected from an infant was accompanied by an eid request form from the referring site. the laboratory technologists recorded in the eid laboratory log book the date the dbs sample was received at the laboratory. when eid results are ready, the date the results are released or returned from the laboratory is recorded and signed for by a university partner or postal worker for the return of results to the referring site. results top ↑ laboratory infrastructure and training the six regional laboratories identified for expansion were strategically located around the country and included (1) bahir dar for the amhara region, (2) mekelle for tigray region, (3) hawassa for the southern nations, nationalities and people’s (snnp) region, (4) adama for the oromia region, (5) harar for the harari and (6) somali regions and addis ababa for addis ababa city administration (figure 2). by june 2008, all six identified regional reference laboratories had been completely renovated and well equipped with the instruments, software and laboratory consumables required for performing hiv-1 dna pcr. figure 2: regionalisation of early infant diagnosis. the laboratory technologists were trained in laboratory biosafety, dbs specimen reception and/or rejection using criteria developed by cdc,27 accurately performing and interpreting test results, and reporting results back to referral sites. a total of 12 laboratory technologists (2 per site) were successfully trained and certified competent on-site for hiv-1 dna pcr testing. the laboratory technologists each scored 100% in their competency assessment when challenged with six blind specimens. that is, they correctly interpreted test kit controls, in-house controls, as well as all blind dbs specimens. the modest number of laboratory technologists trained was aimed at ensuring that testing services would be minimally disrupted should one technologist be absent. sample referral testing documentation and mechanisms to enable specimens to be transferred from referring sites to laboratories performing dna pcr as well as reporting results back to referring sites were fully established. scale-up of laboratory services prior to april 2008, only prior to decentralisation the national hiv reference laboratory at ehnri, located in addis ababa, was performing dna pcr for eid (figure 2). from 2006 to april 2008, the national hiv reference laboratory had tested a total of 2848 infants’ dbs samples, of which 546 (19.2%) were positive and the results were sent back to referring sites (figure 3). the dbs of hiv-exposed infants from prevention of mother-to-child transmission services and diagnostic specimens for clinical symptoms were tested. the results obtained for all laboratories that tested and provided infant dbs results up to november 2010 are shown in figure 3. of the 2209 tested for bahir dar, 278 (12.6%) were positive; 330 (7.5%) of 4369 were positive for addis ababa; 123 (10.1%) of 1222 were positive for hawassa; 594 (13.1%) of 4525 positive for adama; 117 (11.6%) of 1007 tested positive for harar; and 214 (13.9%) of 1542 tested positive for mekelle (figure 3). following decentralisation, ehnri tested 2110 specimens, of which 259 (12.3%) were positive (figure 3, ehnri). figure 3: proportion of dried blood spot (dbs) tested positive by site. although the number of specimens tested at ehnri declined, ehnri was still serving as a quality assurance laboratory for the newly established sites and testing specimens received from non-governmental organisation facilities, military and police hospitals, afar, gambella and benishangul gumuz regions. of the total 19 832 dbs specimens tested from all testing sites between april 2006 and november 2010, 2461 (12.4%) were positive. at the end of 2007, 26 clinical sites were collecting dbs samples. a total of 565 active facilities are currently sending infant samples to these testing laboratories. together with the establishment of these new collection sites, there was a substantial increase in the number of samples tested following decentralisation of laboratory services from 2008 and each year thereafter (figure 4). figure 4: the total number of dried blood spot (dbs) specimens tested and the number positive by year between 2006 and 2010. before decentralisation (april 2006 to march 2008), all the dbs samples were tested only at ehnri laboratory and the median turn-around time was 13 days (range 1−59 days). after decentralisation (april 2008 to november 2010), the median turn-around time at ehnri was 12 days (range 1-63 days). after decentralisation, the other six regional laboratories in the country had a median turn-around time of 14 days (range 14−21 days). quality of dbs dna pcr testing in addition to routinely conducting cdc atlanta ’in-house’ dbs controls and test kit controls to validate performance of assays, all laboratories performed satisfactorily by scoring 100% in all pt sessions with the cdc pt panel programme. the quality assurance results on the first 100 dbs specimens for each of the newly established sites was concordance between results of ehnri and those of the newly established laboratories. the repeat testing of any two or more consecutive positive samples showed concordance between the initial and the repeated test results. ethical considerations top ↑ because this was a programme impact assessment of expansion of dna pcr laboratory for eid, no ethical approval was required. discussion top ↑ in this project, we have demonstrated the feasibility of laboratory expansion of dna pcr testing for eid in a resource-limited setting. following the expansion of testing facilities and the establishment of new collection sites, there was an increase in the number of infant dbs samples tested. early diagnosis of hiv-infected infants can enable faster access to treatment and, in combination with other indicators, can contribute to monitoring the success in programmes for the prevention of mother-to-child transmission. hiv dna pcr is recommended as the gold standard for virologic diagnosis of hiv in infants. however, issues with the high expense associated with establishing a virologic testing laboratory, technical complexities as well as the need to ensure good quality control and assurance in a resource-constrained setting have been raised. this has led to the establishment of a few facilities in centralised areas in resource-limited countries.the commitment of the government and partners was key to the rapid and successful regional expansion of eid molecular technology. the expansion of the laboratory diagnostics services is in line with one of the strategic objectives of the master plan developed by ehnri. this further demonstrates the importance of developing a master plan with clear strategic objectives that serves as a rallying and coordinating tool for implementing laboratory programmes amongst several laboratory stakeholders as well as evaluation of the implementation of the master plan. there was also the need to quickly establish good laboratory practice in these new facilities and to maintain quality of testing, considering the technical complexities of such molecular methods and the fact that infants may only have the opportunity for a single hiv test to determine their hiv infection status. proper on-site training and competency assessment coupled with yearly refresher training and regular joint supportive site visits by ehnri and partners have been important to maintaining quality testing. this has also been reflected in the satisfactory performance (100%) of all the facilities in cdc external quality assessment through pt panels. following decentralisation of dna pcr testing sites and scaling up of the number of collection sites, we observed a significant increase in the number of infants tested with dbs without an increase in the turn-around time from when specimens are received at the laboratory to return of results from the laboratory. with decentralisation, we observed that turn-around time of results to referring sites was a median of 14 days. this reflects a strong and functional sample and results referral system for expediting results. however, because of the low number of dbs specimens and the need for batch testing, it still took harar and hawassa up to three weeks to return results. who guidelines recommend that hiv virologic tests be used to diagnose hiv infection in infants and children up to the age of 18 months.29 additionally, the guidelines emphasise post-natal follow up of infants with unknown or uncertain hiv exposure in order to have their hiv exposure status ascertained and that hiv-infected infants be put immediately on art. although 90% of children living with hiv acquired their infection through mother-to-child transmission, relatively few have access to testing, treatment and care.30 there are several challenges to implementing early treatment for hiv-infected infants, including the diagnosis of hiv-infected infants, lack of rapid diagnostic capabilities for infants, weak sample referral testing systems, under-developed quality controls, limited trained personnel, inadequate clinical systems to follow up with patients, lack of integration amongst health services, and exogenous social factors. addressing these challenges is likely to improve and maintain quality of testing and lead to increase uptake of hiv-infected infants to receiving treatment. direct comparison of the number of dbs tested at the different sites is difficult. the number of infants tested at the new facilities varied greatly, with bahir dar, addis ababa and adama each having tested about two or more times as many as hawassa or harar. the observed difference could partly be explained by the population size and hiv prevalence of these regions, as well as the robustness of their pmtct programme and clinical practices. of the nine regions, bahir dar and adama in amhara and oromiya regions, respectively, account for 23% and 36%, respectively, of the general population. also, the hiv prevalence of the two regions is 2.7% and 1.5% for amhara and oromiya, respectively.26 the city of addis ababa site also showed an increase in dbs testing but accounts for only 3% of the population. however, the city of addis ababa has a relatively higher hiv prevalence of 7.5%.26 by contrast, hawassa in the snnp region, which accounts for about 20% of the population, tested only 1222 dbs specimens. together with the harar and mekelle sites, they tested fewer dbs specimens. these low numbers could be due to challenges with coordination of pediatric hiv care and treatment, pmtct, and laboratory services in these regions. despite the successful scale-up of laboratory diagnostic services, significant challenges still persist and pose a problem for the provision of more comprehensive services for diagnosis and treatment of children. for example, the need for proper integration or linkages between programmes (e.g., laboratory and clinicians), identification of pregnant women, enrolling and scaling-up of the pmtct programme. furthermore, it has been difficult to determine the number of infants testing positive who received their test results and who received treatment and care. it is important to put in place a system for tracking dbs-positive results to ensure those infants receive treatment as well as for negative pcr results, to ensure that uninfected infants who continue to be exposed to hiv receive a final diagnosis. similarly, improving the system of follow-up for infants and their caretakers is critical to avoid delays in initiating treatment. the rapid scale-up of laboratory services to perform dna pcr for eid and treatment of infected children is an essential component to child survival and successful outcomes of pmtct programmes. proper joint planning, implementation, commitment and coordination involving stakeholders have made rapid scale-up possible. continuous monitoring of these new sites is essential, and adhering to all aspects of quality would ensure that accurate results are provided for prompt intervention. acknowledgements top ↑ the authors thank the regional health bureau heads, regional laboratory heads and laboratory technnologists from bahir dar regional laboratory (amhara region), mekelle regional laboratory (tigray region), hawassa regional laboratory (snnp), adama regional laboratory (oromiya region), harar regional laboratory (harari and somali regions) and addis ababa regional laboratory (addis ababa city administration) for their helpful assistance in facilitating and ensuring infrastructure renovations and trainings were carried out properly and in a timely manner. we are grateful to jelaludin ahmed for providing the map of ethiopia. competing interests the authors declare that they have no financial or personal relationship(s), which may have inappropriately influenced them in writing this article. authors’ contributions p.n.f. (cdc ethiopia), t.m. (ehnri), z.m. (icap, columbia university), y.k. (cdc ethiopia), g.t. (ehnri), m.g. (ehnri) and y.m. (cdc ethiopia) conceived and designed the study. a.t. (icap, columbia university), b.h. (icap, columbia university), a.r. (cdc atlanta) and b.t. (ehnri) validated dbs technology for eid at ehnri hiv reference laboratory. p.n.f. (cdc ethiopia), t.m. (cdc ethiopia), y.k. (cdc ethiopia), b.t. (ehnri), y.m. (cdc ethiopia), b.h. (icap, columbia university), g.a. (ehnri), r.f. (chai ethiopia), a.a. (ehnri) coordinated implementation and rollout of the program centrally. t.m. (cdc ethiopia), m.g. (ehnri), p.n.f. (cdc ethiopia), b.h. (icap, columbia university), r.f. (chai ethiopia), a.a. (john hopkins university tsehai program) and s.z. (john hopkins university tsehai program) were involved in rollout of dna pcr laboratories in addis ababa and hawassa. t.m. (cdc ethiopia), m.g. (ehnri), b.h. (icap, columbia university), r.f. (chai ethiopia), t.a. (icap, columbia university) and z.m. (icap, columbia university) were involved in rollout of dna pcr laboratories in adama and harar. t.m. (cdc ethiopia), m.g. (ehnri), p.n.f. (cdc ethiopia), b.h. (icap, columbia university), r.f. (chai ethiopia), w.m. (university of washington, itech program) were involved in rollout of dna pcr laboratories in mekelle and bahir dar. t.a.k. (cdc ethiopia) and j.n.n. (cdc atlanta) contributed original ideas, ongoing discussions on all drafts. all co-authors provided critical feedbacks on all drafts. references top ↑ 1. unaids. unaids report on the global aids epidemic 2010. [unaids global aids report web site]. 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[cited 2012 sep 19] available at: http://www.who.int/hiv/pub/paediatric/framework_2008/en/index.html abstract introduction methods results discussion acknowledgements references about the author(s) ts’aletseng m. siimane department of environmental health, faculty of health sciences, national university of lesotho, roma, maseru, lesotho motlatsi e. nts’ihlele department of environmental health, faculty of health sciences, national university of lesotho, roma, maseru, lesotho citation siimane tm, nts’ihlele me. healthcare waste management knowledge, attitudes and practices of laboratory workers at a regional hospital, lesotho. afr j lab med. 2024;13(1), a2485. https://doi.org/10.4102/ajlm.v13i1.2485 original research healthcare waste management knowledge, attitudes and practices of laboratory workers at a regional hospital, lesotho ts’aletseng m. siimane, motlatsi e. nts’ihlele received: 07 june 2024; accepted: 02 oct. 2024; published: 06 dec. 2024 copyright: © 2024. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: safe management of healthcare waste (hw) safeguards laboratory biosafety and biosecurity. knowledge and attitudes influence hw practices, presenting a need for evidence of the current status. objective: this study assessed the knowledge, attitudes and practice of laboratory workers towards waste management at a regional hospital laboratory in lesotho. methods: the study was conducted from march 2023 to june 2023 using a mixed-methods descriptive case study design. the entire population (n = 30) of technical and non-technical laboratory workers and generated waste were sampled. a structured questionnaire and an observational checklist were used to collect data. waste generation was assessed by weighing and measuring waste volumes. data were analysed using descriptive statistics. results: all respondents (26/26; 100%) can define hw and (3/3) laboratory assistants (100%) gave correct responses for three questions, namely: risk associated with hw, waste container colour-coding, and disposal requirements. knowledge on waste management responsibilities ranged between 0% (0/4) for cleaners and 54.5% (6/11) among laboratory technicians. attitudes were mainly positive, and practices conformed in part to standard operating procedures. infectious solid waste comprised 77% of solid hw, while 63% of chemical liquid waste emanated from the full blood count area. conclusion: knowledge exists among workers and attitudes are predominantly positive; however, some unsafe practices continue, thus knowledge is not fully translated to safe practices. regular training and measuring and recording of hw were recommended. what this study adds: the study contributes understanding of the status of hw knowledge, attitudes and management practices, highlighting the need for compliance monitoring. keywords: healthcare waste management; knowledge; attitudes; practices; biosafety; biosecurity. introduction healthcare waste (hw) is defined as all waste generated within healthcare facilities, research centres, and laboratories related to medical procedures.1 medical laboratories, in particular, generate various categories of hw including general, infectious, pathological, sharps, and chemical waste. healthcare waste generation rates are predominantly lower in developing countries than in developed countries, and a steady global increment of the hw production is observed worldwide, partly due to improved access to healthcare services.2,3,4 the risk posed by hw such as laboratory-acquired infections, injuries, toxicity, and cancers from carcinogenic chemical substances should be mitigated in the interest of biosafety. equally important, poor practices may provide opportunity for threats to access pathogens and toxins, thus hampering biosecurity. safe management requires a robust system with built capacities and the importance of staff knowledge, attitudes, and practice (kap) cannot be overemphasised in such systems. developed countries deal with the fine-tuning of their already established systems, while developing countries are faced with the difficulties of setting up systems that work,5 and are burdened with huge national debt, lowering their possibility of investment in improved healthcare waste management (hwm) technologies and best practices. developing countries need to establish affordable systems.5 annual hw generation is predicted to increase in the coming years.6 african countries generate between 0.53 kg/bed/day and 1.53 kg/bed/day, while asian countries show ranges between 0.5 kg/bed/day and 5.3 kg/bed/day. developed countries in north america and europe have generation rates of 1.7 kg/bed/day – 8.4 kg/bed/day.7 among the factors influencing generation rates, higher gross domestic product and increased access to health services are associated with higher generation rates.3 the trends indicating an increase in waste generation imply laboratory personnel have more interactions with hw and thus will need thorough risk assessment followed by relevant mitigation and monitoring of hwm system performance. adequate knowledge (and positive attitudes) about hw risks can go a long way in influencing the methods of handling waste, and the practice of safety and protecting public health and the environment.8 various studies3,9,10,11 have indicated that generally there is an increased awareness among health professionals about the hw hazards and appropriate management techniques. an abundance of developing countries, however, still face inadequate knowledge and awareness as a result of inadequate training and a lack of safe systems for practising safe hwm.12 it remains important to note that there are still areas of lower to no knowledge, including the dangers of inhalation or skin exposure of chemicals, and reporting of incidents such as needle-stick injuries.2 furthermore, even some developed countries have been facing challenges with regard to making the hospital employee’s behaviour and actions more sustainable. an intervention study has shown that a training programme brings significant increases in preand post-intervention scores among health workers.13 mehta et al.14 stated that training has also been observed to bring about improved attitude and practice scores among the trained, compared to untrained, personnel.14 other studies15,16,17 showed inadequate or low knowledge could not be ignored, since some5 associate low knowledge with risky practices. laboratory workers are among the personnel with good knowledge regarding colour coding and waste segregation at source.8 in terms of attitudes, significant and positive correlations were found between knowledge and attitude, knowledge and practice, and practice and attitude.18 in addition, findings by some studies14,19 identified that more than 90% of the participants in their studies had a positive attitude towards hwm. it is imperative, therefore, that further studies correlate the attitudes to practice; however, adequate knowledge of hwm is one of the strategies that was found to impact positively on attitudes.13 with varying levels of investments made to improve hwm, a gamut of studies8,20,21,22 show unsatisfactory and unsafe practices which jeopardise biosafety and biosecurity. countries should therefore aim at dealing with factors such as the high cost of training, lack of policies, failure of planning at national and facility levels, poor law enforcement from regulatory bodies, and lack of appropriate waste management responsibilities and utilities, which are barriers to safe and secure hwm practice.5,23,24 the management of hw has been of major concern because of potentially high risks to human health and the environment.25 waste has historically not been managed safely; however, there have been positive strides in some countries such as bahrain.5 advancements in different countries have led to the development of policies, legislation, standards, and guidelines to mitigate the impact of hw, but there is a question of implementation which calls for analysis on the kap of hwm systems. medical and diagnostic laboratories are mandated to maintain biosafety and biosecurity in their operations in an effort to align with national and international legislation and treaties. lesotho developed the national hwm policy (2010),26 integrated waste management strategy (2022),27 hazardous (healthcare) waste management regulations (2012),28 and hwm guidelines, procedures and tools (2012).29 training is done at various intervals for cadres such as incinerator operators, environmental health practitioners, laboratory staff, hospital administrators, and others. all health facilities have access to incineration. irrespective of these developments, 17% of nationally available incinerators are malfunctioning, and only 39% of health facilities were segregating according to the basic three-bin system. the majority of health facilities do not have hwm plans. since the promulgation of the lesotho hazardous (healthcare) waste management regulations (2012),28 no such study has been published that interrogates the kaps on hwm in lesotho with a focus on the laboratory setting, especially following a series of trainings offered on the subject. this study therefore aimed to assess the kap of laboratory staff in a regional hospital laboratory in lesotho. to the best knowledge of the researchers, no such study has been published, and it is against this background that the study was undertaken. it is anticipated that the findings herein will go a long way toward informing hwm practices in the specified laboratory, and will serve as a benchmark for improving practice in other laboratories. methods ethical considerations the study was subjected to review by the institutional review board of the faculty of health sciences, national university of university of lesotho (nul/en/2023). ethical clearance was also sought from the ministry of health’s research ethics committee (id55-2023). to access the study site, permission to conduct the study was received from the management of the hospital and the laboratory. care was taken to gain written informed consent from participants. privacy was ensured by obtaining written consent and allowing participants control over their information through a self-administered questionnaire. for anonymity, participants did not disclose their names on the questionnaire and confidentiality was guaranteed through storing data in a locked space and their freedom to participate and withdraw from the study was explained prior to signing consent forms. study design and setting a descriptive cross-sectional study design was used to assess kap and to undertake hazardous waste characterisation, with the variables of concern being kap and masses and volumes of waste generated. for kap assessment, the total population sampling method was selected for respondents (n = 30), including technical laboratory workers, sample transporters, cleaners, and data clerks. a survey was conducted using a self-administered semi-structured questionnaire and an observational checklist. the questionnaire and checklist were created by the researchers and approved by the institutional review board and the laboratory head/manager prior to data collection. the questionnaire was distributed to consenting respondents and collected data pertaining to kaps. the observational checklist recorded staff practices vis-à-vis the laboratory’s standard operating procedure (sop) for waste management. a total of 20 observations were made on a convenience sampling basis on six technical staff on duty during three shifts and two cleaners during two shifts to assess the implementation of hwm. to enumerate waste generation, the waste stream was defined as all solid and liquid waste resulting from the processing of specimens. all waste generated on weekdays between the dates 16 may 2023 and 26 may 2023 was included in the study. an electronic balance was used to weigh the masses of solid waste, and estimation of chemical liquid waste volumes was undertaken using containers of known volume. the laboratory is located approximately 105 km north-west of the capital, maseru, and serves as a regional laboratory for referred specimens from nearby hospitals and selected health centres in the region. the laboratory includes a reception and six sections focusing on tuberculosis and coronavirus disease 2019 (covid-19), molecular diagnostics, full blood count, cd4, microbiology, and clinical chemistry; all sections, including the reception, were included in the study assessments. on average, 13 625 specimens are processed monthly. combustible risk waste emanating from tests is managed onsite through autoclaving and incineration, chemical waste is exported from the facility to the national reference laboratories for stockpiling and treatment, while general waste is collected and disposed of at the district’s designated disposal area by a contracted private company. data analysis for qualitative data, a category scheme was devised and data coded according to the categories. descriptive statistics were then computed from the coded data and quantitative data using microsoft excel (2019) (microsoft corporation, redmond, california, united states). frequencies and percentages were used to express the data, from which a discussion was developed. observations on kap were made among the different categories of staff in the laboratory. results profile of respondents the target population was n = 30 and a high response rate of 86.7% (26/30) was achieved through the consenting participants. most respondents were women (53.8%), and 50% were aged 30–39 years (table 1). education level ranged from primary certificate to university postgraduate studies, where the majority (34.6%) had completed high school and college level. work experience ranged from less than a year (15.4%) to a maximum of 20 or more years (11.5%), and the highest proportion of staff had 3–4 years of experience (26.9%). table 1: socio-demographic characteristics of self-administered questionnaire respondents at a regional hospital, lesotho, march 2023 to june 2023. knowledge of staff on healthcare waste management all respondents (26/26; 100%) knew what hw is (figure 1). in addition, all laboratory assistants (100%) answered three questions correctly, namely: the risk associated with hw, the colour coding used for segregating, and the requirements for hw disposal. findings revealed that for three questions regarding what hw is, container colour coding, and disposal requirements, all 4 cleaners (100%) answered correctly, thus performing better than laboratory technicians, data clerks, and sample transporters on the latter two questions. a crucial finding also was that the question most poorly performed on by all cadres was regarding who is responsible for hwm in the laboratory, ranging from 0% among cleaners (0/4) to 55% (6/11) among laboratory technicians. figure 1: healthcare waste management knowledge among laboratory workers at a regional hospital, lesotho, march 2023 to june 2023. included in the study were 11 laboratory technologists, 3 laboratory assistants, 5 sample transporters, 3 data clerks, and 4 cleaners. attitudes of staff on healthcare waste management ninety-six per cent (25/26) believe that segregation is key to hwm, while all 26 respondents (100%) agree on the importance of training to hwm (figure 2). a positive attitude portrayed by 84% (22/26) of the respondents was that hw is best managed with teamwork. in addition, more respondents (69.2%; 18/26) felt that cleaners should not be allowed to handle all infectious waste on work benches. there was a tie at 50% (13/26) regarding whether hwm is or is not a time-consuming phenomenon. it was also discovered that 65.4% (17/25) of the respondents felt that hwm is an extra burden. figure 2: laboratory workers attitudes towards healthcare waste management at a regional hospital, lesotho, march 2023 to june 2023. practices of staff on healthcare waste management the laboratory has a waste management sop which is in version 2 and was last reviewed on 07 february 2022 (table 2). from the records, all technical and non-technical staff had signed that they had read and understood the sop. table 2: summary findings of healthcare waste management practices among laboratory staff at a regional hospital, lesotho, march 2023 to june 2023. waste generated through laboratory practices out of seven (n = 7) days of segregation observations, the findings showed that the microbiology section did not comply with segregation requirements at all (0/7 days), while the tuberculosis and covid-19 section complied for 4 out of 7 days (table 3). table 3: healthcare waste segregation compliance by work area at a regional hospital, lesotho, 16 may 2023 to 26 may 2023. findings revealed that the laboratory does not routinely quantify and record the amounts of waste generated. for solid waste, the findings reveal that the category of hw generated the most is infectious waste (77%), while the lowest generated was sharps waste (7%) (figure 3a). healthcare general waste constituted 9% of the waste stream. the major infectious waste generating areas were the cd4, molecular, and tuberculosis and covid-19 areas. figure 3: characteristics of solid and liquid waste at a regional hospital, lesotho, 16 may 2023 to 26 may 2023. (a) solid healthcare waste and (b) liquid chemical healthcare waste not disposed to sewer. the majority of the chemical liquid waste that was not directly disposed of via the drain was generated by the full blood count area (63%), and the least emanated from the molecular section (6%) (figure 3b). discussion this study, conducted between march 2023 and may 2023 at the regional hospital in lesotho, found that laboratory workers have knowledge about hwm, and that workers’ attitudes towards hwm are predominantly positive. despite the findings, some unsafe hw practices remain. demographic data show that the majority of participants were women (53.8%), which is in line with the country’s demographic profile, where women in the labour force constitute 53.5% in urban settlements.30 love et al.,31 in a study conducted in the united states in 2022, concluded that a high proportion of women on scientific teams contributes to successful team collaboration, although it is not understood why this is the case or what proportion of women impacts team success. it is unclear whether the dominance of women in this setting influences kaps in managing waste. in general, it is known that the knowledge of workers will influence the attitudes and practices towards hwm.32 participants who had no to low knowledge on who is responsible for hwm were the cleaning staff, sample transporters, and laboratory assistants. the lowest percentages regarding who is responsible for hwm were obtained from laboratory technicians (54.54%) and data clerks (33.33%). the lack of knowledge may lead to inefficiency of work, as duplication of efforts could lead to omission of work and lack of accountability. this aligns with the 2011 findings of a study undertaken in by mathur et al.8 in india, attesting that inadequate and inappropriate knowledge of handling hw have serious health consequences and a significant impact on the environment. all cleaners (100%) answered correctly for the three questions regarding defining hw, container colour coding, and disposal requirements, thus performing better than laboratory technicians, data clerks, and sample transporters for these questions. cleaners may therefore be further empowered as part of the laboratory’s support system to strengthen biosafety and biosecurity and continuous training can benefit all cadres in line with el-gilhany et al.,13 whose study conducted at the mansoura university, egypt, in 2015–2016 showed that training brings significant improvement in waste management. all participants (100%) had positive attitudes towards hwm training, and they were willing to take part in such trainings. the majority (88.5%) had a positive attitude towards managing hw as a team. the positive attitude could be harnessed by the laboratory to continue to champion improved hwm as demonstrated in a study conducted in south africa in 2016 by olaifa et al.,25 showing that there is a positive correlation between attitude and practice. on the other hand, there was high negativity (65.4%) of participants who found hwm as an extra burden proving the reluctance of participants to address the waste they generate. in their 2015 study in pakistan, khan et al.11 revealed similar findings among physicians and paramedic staff, identifying hwm as a financial burden. the microbiology section indicated that it generated infectious waste only. there were no records indicating that pathological waste such as culture plates were autoclaved. this presents a biosafety and biosecurity challenge since it becomes impossible for the laboratory to account for the destruction of pathogens using validated methods as required by chartier et al.1 and the united nations environment programme.33 this could be attributed to the finding that some workers consider waste management to be a burden. in 2020, a study by endris et al.34 established that medical laboratories in addis ababa, ethiopia, properly performed waste treatment procedures. in 2011 in india, mohankumar and kottaiveeran35 observed the process of segregation, collection, transport, storage, and final disposal of infectious waste being done in compliance with the standard procedures. this is contrary to what was observed in some laboratory sections in this study, where requirements for segregation and containerisation of sharps waste were not performed according to the sops and national regulation. these practices expose waste handlers to sharps-related injuries. in consideration of the high levels of knowledge of containerisation among laboratory technologists (90.9%) and laboratory assistants (100%), this knowledge is not fully translated into practice, since some waste is contained in inappropriate colour containers. this differs slightly from the findings of where the majority (81.6%) of laboratory workers knew segregation requirements, and even more of them (91.8%) showed safe practices.15 risk assessment and audits can be used to track such practices, and generally trainings can be followed up with studies that assess the effectiveness of training (on-the-job). the laboratory generated more healthcare risk waste (91%) than healthcare general waste (9%). of the healthcare risk waste, infectious waste was highest at 77%, and sharps waste the lowest (7%). this tallies with the 2018 findings of mazloomi et al.36 in iran, where infectious waste formed the majority, and sharps waste the minority. most chemical liquid waste was generated by the full blood count area. the fact that the laboratory does not routinely quantify their waste is contrary to radha et al.,37 whose 2009 study in india asserted that waste generated in a healthcare setting should be quantified to facilitate safe waste management systems. waste quantification also can assist in adequate procurement and tracking of the use of waste containers. in addition, it guides the access to treatment facilities. the laboratory should consider quantifying and recording healthcare risk waste. the full blood count area generated the majority (63%) of the liquid chemical waste, with the lowest amount emanating from the molecular area (6%). the chemical liquid waste is drained into an impermeable underground conservancy tank which is emptied by trained and authorised personnel. the tank contents are secured from unauthorised access, contrary to the findings of ali et al.2 in their 2017 review, who establish that the majority of developing countries discard chemical waste from the laboratories into public sewers without any treatment. this act shows that the laboratory is ensuring biosafety and biosecurity precautions towards liquid waste generated. it was observed that full hepatitis immunisation status was not achieved by the majority of workers (67.8%), which differs from the findings of soyamet al.19 in their study conducted in india in 2011, where a significant proportion of workers (64.5%) were vaccinated. this practice compromises the occupational health and safety of laboratory personnel, and those exposed may develop laboratory-acquired infections. training intervention on viral hepatitis has been shown to increase hepatitis vaccine uptake among laboratory staff and should be implemented.13 it was not clear as to when and how often temporary storage area was emptied of laboratory waste. historically, hw was well secured and locked until recently, where the storage key was lost and the area left unlocked. in addition, the storage area has no signage to control entry, affirming some of the challenges of hwm as reviewed in 2017 by ali et al.2 these conditions expose the waste to unauthorised persons and present a biosecurity risk. the study revealed that there are good hwm practices; however, some unsafe practices were also observed. even though a clear and up-to-date sop on hwm was available (review date 07 february 2022), there are some practices that are not conformed to. this is in line with the findings of the ministry of health.38 additionally, knowledge and skills gaps that hamper sop implementation could still exist among staff and should be identified and addressed. limitations responses for the questionnaire were self-reported and could bring bias. furthermore, because of the short period for monitoring waste generation, the picture of waste generation gained cannot be generalised over longer periods of operation. lastly, inferential statistics could have improved the researchers’ understanding of the dynamics at play between kaps. conclusion the laboratory has oversight mechanisms in place for engaging in safe hwm. while knowledge may exist among workers and attitudes may be positive, these do not always translate into the best practices for compliance with national regulation and sops. therefore, improvement is needed to enhance biosafety and biosecurity. the study findings have been used to inform action such as refresher trainings, which are tied to monitoring and training impact assessment activities. further investigation is needed to understand the factors leading to what hampers and promotes the observed attitudes and practices. as the laboratory continues to implement programmes that promote a culture of safety, performance monitoring should also be strengthened. acknowledgements the authors would like to pass their gratitude to all staff members in the hospital laboratory for their participation in providing valuable data that brought this study to fruition. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions t.m.s. and m.e.n. conceptualised and designed the study. t.m.s. supervised the project, reviewed the data collection tools, reviewed and curated the data, and wrote the article. m.e.n. developed the data collection tool, collected and analysed the data, and contributed to the review and finalisation of the article. funding for the study was sourced by m.e.n. sources of support this research received no research grant from any funding agency in the public, commercial or not-for-profit sectors. data availability the authors confirm that the data supporting the findings from this study are available within this article. raw data supporting this evaluation’s findings are available from the corresponding author, t.m.s., upon reasonable request. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. the article does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the authors are responsible for this article’s results, findings, and content. references chartier y, emmanuel j, pieper u, et al. safe management of wastes from health-care activities. 2nd ed. geneva: world health organization; 2014. ali m, wang w, chaudhry n, geng y. hospital waste management in developing 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discussion acknowledgements references about the author(s) winnie mwanza zambia aids related tuberculosis (zambart) project, school of medicine, university of zambia, lusaka, zambia deborah milimo zambia aids related tuberculosis (zambart) project, school of medicine, university of zambia, lusaka, zambia maureen m. chilufya zambia aids related tuberculosis (zambart) project, school of medicine, university of zambia, lusaka, zambia nkatya kasese zambia aids related tuberculosis (zambart) project, school of medicine, university of zambia, lusaka, zambia maina c. lengwe zambia aids related tuberculosis (zambart) project, school of medicine, university of zambia, lusaka, zambia stembiso munkondya zambia aids related tuberculosis (zambart) project, school of medicine, university of zambia, lusaka, zambia petra de haas zambia aids related tuberculosis (zambart) project, school of medicine, university of zambia, lusaka, zambia department of infectious and tropical diseases, london school of hygiene and tropical medicine, bloomsburg, london, united kingdom helen ayles zambia aids related tuberculosis (zambart) project, school of medicine, university of zambia, lusaka, zambia department of infectious and tropical diseases, london school of hygiene and tropical medicine, bloomsburg, london, united kingdom monde muyoyeta zambia aids related tuberculosis (zambart) project, school of medicine, university of zambia, lusaka, zambia citation mwanza w, milimo d, chilufya mm, et al. diagnosis of rifampicin-resistant tuberculosis: discordant results by diagnostic methods. afr j lab med. 2018;7(2), a806. https://doi.org/10.4102/ajlm.v7i2.806 brief report diagnosis of rifampicin-resistant tuberculosis: discordant results by diagnostic methods winnie mwanza, deborah milimo, maureen m. chilufya, nkatya kasese, maina c. lengwe, stembiso munkondya, petra de haas, helen ayles, monde muyoyeta received: 09 apr. 2018; accepted: 26 sept. 2018; published: 06 dec. 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract the performance of the xpert© mtb/rif and mtbdrplus assays for the detection of rifampicin resistant mycobacterium tuberculosis was compared to culture-based drug susceptibility testing in 30 specimens with rifampicin-resistant and rifampicin-indeterminate xpert mtb/rif results collected between march 2012 and march 2014. xpert mtb/rif and mtbdrplus were 100% sensitive and 100% concordant for rifampicin resistance detection, but 3 of 13 samples (23%) positive for rifampicin resistance on xpert mtb/rif and mtbdrplus were negative for rifampicin resistance on mycobacteria growth indicator tube drug susceptibility testing. specificity was 72% for xpert mtb/rif and 80% for mtbdrplus. positive predictive value for xpert mtb/rif for multidrug resistant tuberculosis was 47.8% for new patients and 77.8% for previously treated patients; negative predictive value was 100% for both new and previously treated patients. the discordant rifampicin resistance test results indicate a need to fully characterise circulating rifampicin resistant mycobacterium tuberculosis strains in zambia and to inform the development of guidelines for decision-making in relation to diagnosis of drug-resistant tuberculosis. introduction management and control of multidrug resistant (mdr) tuberculosis, tuberculosis that is resistant to both isoniazid and rifampicin, relies on timely and correct diagnosis. the world health organization has endorsed the xpert®mtb/rif (cepheid sunnyvale, ca, united states) and genotype mtbdrplus (hain life sciences, nehren, germany) tests for rapid diagnosis of mdr tuberculosis.1,2 for the xpert mtb/rif test, the current recommendation is to treat any rifampicin-resistant individual as a case of mdr tuberculosis until proven otherwise by subsequent drug susceptibility testing, after which treatment is to be tailored or individualised accordingly. the combination of xpert mtb/rif and mtbdrplus test results would be a powerful tool to achieve prompt confirmation of diagnosis of mdr tuberculosis. these genotypic methods, however, do not detect resistance conferred by mutations outside of the targeted gene region.3,4,5 we report comparative observations made in a pilot study on the performance of xpert mtb/rif, mtbdrplus and mycobacteria growth indicator tube (mgit) drug susceptibility testing methods for detection of rifampicin resistance in lusaka, zambia, at the zambart central laboratory (zcl). methods ethical considerations the study was approved by the university of zambia biomedical research ethics committee (ref. no. 003-12-11) and all patients were requested to give written informed consent before participating in the study. the written informed consent was administered by study staff, and participants were given an opportunity to discuss the study before signing the informed consent form. to maintain confidentiality, each participant was allocated a unique study number that was used on all data collection tools, as well as for labelling of laboratory specimens. no names were used on any data collection tool. informed consent forms and all study data were kept in secure cabinets with access limited to the principal investigator and the data manager. paper-based case report forms were captured into secured, password-protected electronic databases. study design the study was conducted between march 2012 and march 2014 on samples collected during an evaluation of xpert mtb/rif use in primary health care facilities in lusaka, zambia.6,7 samples were collected from consecutive patients presenting to the primary health care facility who had rifampicin-resistant and rifampicin-indeterminate xpert mtb/rif results for confirmation of rifampicin resistance and for quality assurance in the zcl. samples were transported to the central laboratory on the same day of collection or the following day and no preservatives were added. at the zcl, the samples were decontaminated using the naoh/nalc method (2% naoh for 20 minutes) and 0.5 ml of the pellet was inoculated into each of two manual mgit tubes for primary isolation and identification of m. tuberculosis.; 0.5 ml was used to repeat the xpert mtb/rif test, and two drops were used to prepare a concentrated smear for fluorescent microscopy. 1.5 ml of the xpert mtb/rif sample reagent was added to 0.5 ml of the re-suspended pellet and xpert mtb/rif testing was performed as per standard procedure described by the manufacture. acid-fast bacilli positive cultures were identified using the capillia tuberculosis neo assay (tauns laboratories, inc., shizuoka, japan) to confirm presence of mycobacterium tuberculosis complex.8 confirmed m. tuberculosis complex isolates were tested for drug sensitivity using the becton dickinson manual mgit sire (streptomycin, isoniazid, rifampicin, ethambutol; becton dickinson, bergen county, new jersey, united states) drug susceptibility testing method and the mtbdrplus assay (version 1; hain life sciences, nehren, germany). the drug concentrations used were 1.0 µg/ml streptomycin, 0.1 µg/ml isoniazid, 1.0 µg/ml rifampicin, and 5.0 µg/ml ethambutol. the mgit sire tubes were read daily as per the manufacturer’s recommendation. to perform the mtbdrplus assay, 1 ml of broth from an m. tuberculosis complex culture-positive mgit tube was used for dna extraction using the genolyse method. briefly, 1 ml of culture broth was centrifuged, the pellet was incubated for 5 minutes at 95°c in lysis buffer after which a neutralisation buffer was added. subsequent steps for polymerase chain reaction and hybridisation were followed as per standard procedure for mtbdrplus testing.9 all procedures included positive and negative controls. the data were analysed using stata version 13 (statacorp llc, college station, texas, united states). an initial descriptive analysis was done to determine the distribution of results, then the xpert mtb/rif results obtained at the primary health care setting were compared with the results obtained at the central laboratory. further analysis was done to compare the performance of xpert mtb/rif, mtbdrplus and mgit m. tuberculosis drug susceptibility testing and to determine the sensitivity and specificity of xpert mtb/rif and mtbdrplus compared to the gold standard mgit culture for drug susceptibility testing. results during the study period, 1070 patients had m. tuberculosis complex detected by the xpert mtb/rif test. of these, 28 (2.6%) were positive for rifampicin resistance and 15 (1.4%) had indeterminate rifampicin resistance results. of the 43 patients eligible for inclusion in the study (i.e. had rifampicin-resistant or rifampicin-indeterminate xpert mtb/rif results), only 30 submitted samples for further testing at the central laboratory (table 1). of these, 21 (70%) were positive for both m. tuberculosis and rifampicin resistance, and nine (30%) were positive for m. tuberculosis but indeterminate for rifampicin resistance. on repeat xpert mtb/rif testing at the central laboratory, of the 21 patients initially positive for rifampicin resistance, 17 (81%) were again positive for rifampicin resistance, three (14%) were negative for rifampicin resistance and one (5%) tested negative for m. tuberculosis. table 1: comparison of xpert mtb/rif testing at the primary health care facility peripheral laboratory to xpert mtb/rif testing at the central laboratory, zambia, march 2012 and march 2014. of the nine patients initially indeterminate for rifampicin resistance, five (56%) tested negative for rifampicin resistance and four (44%) tested negative for m. tuberculosis on repeat xpert mtb/rif testing (table 1). these four (44%) samples negative for m. tuberculosis on repeat xpert mtb/rif testing were m. tuberculosis positive but rifampicin resistance negative when tested by both mtbdrplus and mgit drug susceptibility testing (tables 2–3). xpert mtb/rif and mtbdrplus were 100% concordant for detection of rifampicin resistance (table 2). when compared to mgit drug susceptibility testing, the sensitivity of xpert mtb/rif and mtbdrplus for detection of rifampicin resistance was 100%, while specificity was 72% for xpert mtb/rif and 80% for mtbdrplus. three patients had positive rifampicin resistance results on both xpert mtb/rif and mtbdrplus (table 2), but rifampicin resistance was not detected with mgit drug susceptibility testing (tables 3 and 4). on xpert mtb/rif all three samples showed no signal for probe d and on mtbdrplus, all were missing the wild-type 7 probe and showed no mutation probe (results not shown). these three isolates were also resistant to isoniazid on both mgit drug susceptibility testing and mtbdrplus (tables 3 and 4). table 2: comparison of repeat xpert mtb/rif and mtbdrplus test results. table 3: comparison of repeat xpert mtb/rif and mycobacterium growth indicator tube mycobacterium tuberculosis drug susceptibility testing. table 4: comparison of mtbdrplus and mycobacterium growth indicator tube mycobacterium tuberculosis drug susceptibility testing. the overall positive predictive values and negative predictive values for mdr tuberculosis diagnosis for xpert mtb/rif compared to mgit drug susceptibility testing were 62.5% and 100% and compared to mtbdrplus were 81.2% and 100% (results not shown). for previously treated patients, the xpert mtb/rif positive predictive value was 77.8% and negative predictive value was 100%; for new patients, positive predictive value was 47.8% and negative predictive value was 100%. the mtbdrplus positive predictive value was 78.6% and negative predictive value was 100% compared to mgit. discussion indeterminate rifampicin resistance results can be resolved by repeating the test, as shown in our study. of all the samples that were initially indeterminate for rifampicin resistance, none tested positive on repeat testing. an indeterminate rifampicin resistance xpert mtb/rif result maybe due to low dna quantity in the sample; this was supported by the high proportion of samples that were initially indeterminate that did not detect m. tuberculosis on repeat testing with xpert mtb/rif but were positive on m. tuberculosis culture. for prediction of mdr tuberculosis, our findings confirm that xpert mtb/rif has a low positive predictive value for mdr tuberculosis in low-prevalence mdr tuberculosis settings like zambia, which has an estimated mdr prevalence of 4.2%.10 depending on xpert mtb/rif alone for diagnosis of mdr tuberculosis in our setting would lead to subjecting a significant proportion of patients to unnecessary second-line treatment.1 the performance of xpert mtb/rif and mtbdrplus were similar, which was expected since both are genotypic tests. the sensitivity of xpert mtb/rif for detection of rifampicin resistance was within the range of what has been reported by others, whereas the specificity of 73% was lower.11 the prevalence of silent rpob gene mutations that do not confer phenotypic resistance determines the specificity of genotypic tests, if using a phenotypic test as a gold standard. while we were limited by a lack of capacity to sequence the three samples that had results suggesting false rifampicin resistance, other studies have reported false xpert mtb/rif or mtbdrplus rifampicin resistance caused by silent mutations in the rpob gene.12,13,14,15,16 further, these three isolates were also resistant to isoniazid on both mgit drug susceptibility testing and mtbdrplus, consistent with the widely observed fact that in most cases, silent mutations in the rpob gene are associated with mutations in the katg gene or promoter of the inha gene.17,18 probe d in the xpert mtb/rif and the wild-type 7 probe in mtbdrplus both target the same region of the rpob gene, codon 526, which is one of the regions with known mutations that confer resistance to rifampicin. detection of mutations in this region by both xpert mtb/rif and mtbdrplus shows strong evidence of the accuracy of our genotypic results. however, mgit drug susceptibility testing has been shown to produce false susceptibility results for strains with minimal inhibitory concentrations for rifampicin close to the cut-off value of 1 µg/ml used in mgit drug susceptibility testing, or strains that have sub-critical minimal inhibitory concentrations for rifampicin.19 however, it is unlikely that the discordant results in our study were due to false susceptibility results for the mgit drug susceptibility testing. to resolve these discrepant results, sequencing is required to determine the presence of silent mutations. however, lack of access to sequencing services was a limitation in our study. this study was also limited by its small sample size but provides some insights into the comparable performance of xpert mtb/rif, mtbdrplus and culture drug susceptibility testing culture for the diagnosis of drug-resistant tuberculosis. there is an urgent need for zambia to perform a full identification and classification of rpob mutations and investigate minimal inhibitory concentrations for rifampicin, so as to optimise national guidelines for diagnosis of drug-resistant tuberculosis. a thorough investigation of the performance of xpert mtb/rif and mtbdrplus for diagnosis of rifampicin resistance and prediction of mdr tuberculosis in the zambian setting is recommended to avoid inappropriate treatment. whole genome sequencing capacity is required to fully characterise circulating rifampicin-resistant m. tuberculosis strains. improvements are needed to make these genotypic tests function as stand-alone tests, as they offer the best prospects for early and accurate diagnosis for tuberculosis and mdr tuberculosis. acknowledgements the authors would like to acknowledge the ministry of health of zambia for permission to conduct the study and lusaka district health staff at the facilities where this study was conducted. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support this study was a sub-study of the xpert evaluation study funded by the stop tb partnership/tb reach wave 2: t9-370-114. authors’ contributions m.m. and h.a. conceptualised the study. d.m. and n.k. designed the databases. m.c.l., m.m.c., s.m., m.m., d.m. and w.m. performed the experiments. m.m. analysed the data. w.m. and m.m. wrote the manuscript. p.d.h. critically reviewed the manuscript. all authors reviewed and approved the final version of the manuscript. references world health organization. policy statement: automated real-time nucleic acid amplification technology for rapid and simultaneous detection of tuberculosis and rifampicin resistance. geneva: who; 2011. world health organization. policy statement. molecular line probe assays for rapid screening of patients at risk of multidrug resistant tuberculosis (mdr-tb) [homepage on the internet]. [cited 2008 june 27]. available from: http://www.who.int/tb/features_archive/policy_statement.pdf siu gk, zhang y, lau tck, et al. mutations outside the rifampicin resistance-determining region associated with rifampicin resistance in mycobacterium tuberculosis. j antimicrob chemother. 2011;66(4):730–733. https://doi.org/10.1093/jac/dkq519 jamieson fb, guthrie jl, neemuchwala a, et al. profiling of rpob mutations and mics for rifampin and rifabutin in mycobacterium tuberculosis. j clin microbiol. 2014;52(6):2157–2162. https://doi.org/10.1128/jcm.00691-14 horng yt, j w-y, chen y-y, et al. molecular analysis of codon 548 in the rpob gene involved in mycobacterium tuberculosis resistance to rifampin. antimicrob agents chemother. 2015;59(3):1542–1548. https://doi.org/10.1128/aac.04374-14 muyoyeta m, moyo m, kasese n, et al. implementation research to inform the use of xpert mtb/rif in primary health care facilities in high tb and hiv settings in resource constrained settings. plos one. 2015;10(6):e0126376. https://doi.org/10.1371/journal.pone.0126376 muyoyeta m, et al. sensitivity, specificity, and reproducibility of the capilia tb-neo assay. j clin microbiol. 2013;51(12):4237–4239. https://doi.org/10.1128/jcm.02441-13 muyoyeta m, mwanza wc, kasese n, et al. evaluation of the capilia tb assay for culture confirmation of mycobacterium tuberculosis infections in zambia and south africa. j clin microbiol. 2010;48(10):3773–3775. https://doi.org/10.1128/jcm.01688-09 hain life sciences. genotype mtbdrplus: instructions for use. nehren, germany: hain life sciences. kapata n, chanda-kapata p, bates m, et al. multidrug-resistant tb in zambia: review of national data from 2000 to 2011. trop med int health. 2013;18(11):1386–1391. https://doi.org/10.1111/tmi.12183 steingart kr, schiller i, horne dj, et al. xpert(r) mtb/rif assay for pulmonary tuberculosis and rifampicin resistance in adults. cochrane database syst rev. 2014;1:cd009593. marlowe em, novak-weekley sm, cumpio j, et al. evaluation of the cepheid xpert mtb/rif assay for direct detection of mycobacterium tuberculosis complex in respiratory specimens. j clin microbiol. 2011;49(4):1621–1623. https://doi.org/10.1128/jcm.02214-10 claessens j, mathys v, derdelinckx i, saegeman v. case report of a false positive result of the xpert(r) mtb/rif assay for rifampicin resistance in mycobacterium tuberculosis complex. acta clin belg. 2017 jun;72(3):195–197. doi: 10.1179/2295333715y.0000000072. mathys v, van de vyvere m, de droogh e, soetaert k, groenen g. false-positive rifampicin resistance on xpert(r) mtb/rif caused by a silent mutation in the rpob gene. int j tuberc lung dis. 2014;18(10):1255–1257. https://doi.org/10.5588/ijtld.14.0297 alonso m, palacios jj, herrantz m, et al. isolation of mycobacterium tuberculosis strains with a silent mutation in rpob leading to potential misassignment of resistance category. j clin microbiol. 2011;49(7):2688–2690. https://doi.org/10.1128/jcm.00659-11 williamson da, basu i, bower j, freeman jt, henderson g, roberts sa. an evaluation of the xpert mtb/rif assay and detection of false-positive rifampicin resistance in mycobacterium tuberculosis. diagn microbiol infect dis. 2012;74(2):207–209. https://doi.org/10.1016/j.diagmicrobio.2012.06.013 ocheretina o, byrt e, mabou m-m, et al. false-positive rifampin resistant results with xpert mtb/rif version 4 assay in clinical samples with a low bacterial load. diagn microbiol infect dis. 2016;85(1):53–55. https://doi.org/10.1016/j.diagmicrobio.2016.01.009 n’guessan k, assi js, ouassa t, et al. assessment of the genotype mtbdrplus assay for rifampin and isoniazid resistance detection on sputum samples in cote d’ivoire. eur j microbiol immunol (bp). 2014;4(3):166–173. https://doi.org/10.1556/eujmi-d-14-00014 ocheretina o, escuyer ve, mabou m-m, et al. correlation between genotypic and phenotypic testing for resistance to rifampin in mycobacterium tuberculosis clinical isolates in haiti: investigation of cases with discrepant susceptibility results. plos one. 2014;9(3):e90569. https://doi.org/10.1371/journal.pone.0090569 abstract introduction methods results discussion acknowledgements references about the author(s) nomonde r. mvelase department of medical microbiology, kwazulu-natal academic complex, national health laboratory service, durban, south africa school of laboratory medicine and medical sciences, college of health sciences, university of kwazulu-natal, durban, south africa lindiwe p. cele department of public health, epidemiology and biostatistics unit, sefako makgatho health sciences university, pretoria, south africa ravesh singh department of medical microbiology, kwazulu-natal academic complex, national health laboratory service, durban, south africa school of laboratory medicine and medical sciences, college of health sciences, university of kwazulu-natal, durban, south africa yeshnee naidoo kwazulu-natal research innovation and sequencing platform, nelson r mandela school of medicine, university of kwazulu-natal, durban, south africa centre for epidemic response and innovation, school of data science and computational thinking, stellenbosch university, stellenbosch, south africa jennifer giandhari kwazulu-natal research innovation and sequencing platform, nelson r mandela school of medicine, university of kwazulu-natal, durban, south africa eduan wilkinson kwazulu-natal research innovation and sequencing platform, nelson r mandela school of medicine, university of kwazulu-natal, durban, south africa centre for epidemic response and innovation, school of data science and computational thinking, stellenbosch university, stellenbosch, south africa tulio de oliveira kwazulu-natal research innovation and sequencing platform, nelson r mandela school of medicine, university of kwazulu-natal, durban, south africa centre for epidemic response and innovation, school of data science and computational thinking, stellenbosch university, stellenbosch, south africa centre for the aids programme of research in south africa, university of kwazulu-natal, durban, south africa khine swe swe-han department of medical microbiology, kwazulu-natal academic complex, national health laboratory service, durban, south africa school of laboratory medicine and medical sciences, college of health sciences, university of kwazulu-natal, durban, south africa koleka p. mlisana department of medical microbiology, kwazulu-natal academic complex, national health laboratory service, durban, south africa school of laboratory medicine and medical sciences, college of health sciences, university of kwazulu-natal, durban, south africa centre for the aids programme of research in south africa, university of kwazulu-natal, durban, south africa citation mvelase nr, cele lp, singh r, et al. consequences of rpob mutations missed by the genotype mtbdrplus assay in a programmatic setting in south africa. afr j lab med. 2023;12(1), a1975. https://doi.org/10.4102/ajlm.v12i1.1975 original research consequences of rpob mutations missed by the genotype mtbdrplus assay in a programmatic setting in south africa nomonde r. mvelase, lindiwe p. cele, ravesh singh, yeshnee naidoo, jennifer giandhari, eduan wilkinson, tulio de oliveira, khine swe swe-han, koleka p. mlisana received: 06 june 2022; accepted: 24 oct. 2022; published: 06 feb. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: rifampicin resistance missed by commercial rapid molecular assays but detected by phenotypic assays may lead to discordant susceptibility results and affect patient management. objective: this study was conducted to evaluate the causes of rifampicin resistance missed by the genotype mtbdrplus and its impact on the programmatic management of tuberculosis in kwazulu-natal, south africa. methods: we analysed routine tuberculosis programme data from january 2014 to december 2014 on isolates showing rifampicin susceptibility on the genotype mtbdrplus assay but resistance on the phenotypic agar proportion method. whole-genome sequencing was performed on a subset of these isolates. results: out of 505 patients with isoniazid mono-resistant tuberculosis on the mtbdrplus, 145 (28.7%) isolates showed both isoniazid and rifampicin resistance on the phenotypic assay. the mean time from mtbdrplus results to initiation of drug-resistant tuberculosis therapy was 93.7 days. 65.7% of the patients had received previous tuberculosis treatment. the most common mutations detected in the 36 sequenced isolates were i491f (16; 44.4%) and l452p (12; 33.3%). among the 36 isolates, resistance to other anti-tuberculosis drugs was 69.4% for pyrazinamide, 83.3% for ethambutol, 69.4% for streptomycin, and 50% for ethionamide. conclusion: missed rifampicin resistance was mostly due to the i491f mutation located outside the mtbdrplus detection area and the l452p mutation, which was not included in the initial version 2 of the mtbdrplus. this led to substantial delays in the initiation of appropriate therapy. the previous tuberculosis treatment history and the high level of resistance to other anti-tuberculosis drugs suggest an accumulation of resistance. keywords: tuberculosis; rifampicin resistance; rpob mutations; mtbdrplus; discordance. introduction access to reliable, rapid and automated nucleic acid amplification tests remains one of the key factors in fulfilling the early tuberculosis diagnosis and universal access to drug susceptibility testing (dst) requirement of the world health organization (who) end tb strategy.1 the past decade has seen the development of many molecular tests, some of which have been endorsed by the who. the genotype mtbdrplus assay was the first to be endorsed by the who in 2008, followed by the xpert mtb/rif (xpert) in 2010.2,3 because of its ease of use, the xpert has been implemented in many settings for the initial diagnosis of tuberculosis and detection of rifampicin resistance. both the mtbdrplus and the xpert detect rifampicin resistance by identifying mutations in the 81-base pair region of the rpob gene, which spans codon 426–452 in the mycobacterium tuberculosis numbering system and codon 507–533 in the escherichia coli numbering system.4 this region is also called the rifampicin resistance-determining region (rrdr), as most of the rifampicin resistance-conferring mutations are found in this region.5 additionally, the mtbdrplus also detects resistance against isoniazid by identifying mutations in the katg gene and the promoter region of the inha gene. due to the limitations of the xpert and the mtbdrplus assays, phenotypic methods remain the gold standard for tuberculosis dst. both assays demonstrate variable performance in detecting heteroresistance and do not detect rpob gene mutations outside the rrdr.6 until recently, mutations outside the rrdr were believed to only account for less than 5% of overall rifampicin resistance.7,8 however, in a national drug resistance survey conducted in eswatini between 2009 and 2010, 30% of multidrug-resistant tuberculosis (mdr-tb: resistant to rifampicin and isoniazid) isolates carried the i491f rpob gene mutation located outside the rrdr.9 this caused concerns, especially in neighbouring countries like south africa, because while this mutation is rare globally, it might be more common in certain geographical settings. a subsequent study conducted in the northern provinces of south africa showed that 15% of isoniazid mono-resistant strains carried the i491 mutation, meaning they were mdr-tb strains.10 the same study also revealed that strains carrying this mutation may be driving outbreaks of mdr-tb in eswatini and south africa.10 rapid molecular tests that only detect mutations in the rrdr may fail to detect rifampicin resistance in patients with tuberculosis caused by strains carrying mutations outside the rrdr, and this may lead to inappropriate management, resulting in resistance selection, accumulation of resistance, treatment failure and increased transmission. in settings where molecular and phenotypic rifampicin dst are performed concurrently, discordant results often occur, especially with liquid culture-based assays.11,12 given the fact that rifampicin is the key determinant of the choice of a treatment regimen, the hesitancy caused by discordant results may also affect the decision to start appropriate treatment in the affected patients. often, an attempt is made to confirm a discordant result by either repeating the test or using another confirmatory assay (if available), thus causing further delay in initiating appropriate therapy. because the kwazulu-natal province accounts for almost 30% of south africa’s drug-resistant tuberculosis (dr-tb) cases, and since eswatini forms part of its northern border, we conducted this study in kwazulu-natal, south africa, to determine why phenotypically resistant isolates were reported as rifampicin susceptible on the mtbdrplus.13 considering the dearth of information on the clinical management of patients with rifampicin-discordant tuberculosis results globally, we also report on the programmatic management of these patients in our setting. methods ethical considerations ethics approval was obtained from the university of kwazulu-natal biomedical research ethics council (be267/18). individual patient consent was not required as only routine programmatic data was accessed; however, permission was obtained from the provincial department of health. for anonymity, patients’ names were only used for data collection and were not used during analysis. study design and setting the study was conducted in the kwazulu-natal province in south africa. the province has the second-highest population in the country with more than 11 million people. there are 11 districts in the province and one mdr-tb treatment facility per district. in health facilities in the kwazulu-natal province, the initial diagnosis of tuberculosis and rifampicin resistance is routinely done using the xpert (xpert mtb/rif, cepheid, sunnyvale, california, united states) in all patients suspected of tuberculosis disease. the xpert was previously used but was later replaced by its successor, the xpert mtb/rif ultra (xpert ultra, cepheid, sunnyvale, california, united states), in 2017. for patients with rifampicin-susceptible tuberculosis, no further dst is performed, and they are treated using first-line tuberculosis therapy. in patients with rifampicin-resistant tuberculosis on the xpert (ultra), a second sample is taken for culture and dst. other indications for tuberculosis culture include treatment failure and paucibacillary tuberculosis that shows a negative result on the xpert (ultra). during the study period between january 2014 and december 2014, the automated bactec mycobacteria growth indicator tube 960 system (becton dickinson, sparks, maryland, united states) was used for m. tuberculosis culture, and initial dst was done on all positive cultures using the mtbdrplus version 2 assay (hain lifescience, nehren, germany) to confirm rifampicin resistance and test for isoniazid resistance. the mtbdrplus assay uses dna strip technology where the strip contains both wild-type probes and mutation probes for the commonly occurring mutations (s450l, h455y, h455d, and d435v for rifampicin). the labelled polymerase chain reaction products from an amplified target are hybridised with specific probes immobilised on a strip (reverse hybridisation). resistance is reported when there is a lack of binding to the wild-type probe with or without binding to a mutation probe.14 isolates that were resistant to either rifampicin or isoniazid on the mtbdrplus assay were further tested for resistance to critical concentrations of isoniazid (0.2 µg/ml), rifampicin (1 µg/ml), ofloxacin (2 µg/ml), streptomycin (2 µg/ml), and kanamycin (5 µg/ml) using the 1% agar proportion method on middlebrook 7h10 agar (becton dickinson, sparks, maryland, united states).15 the simultaneous performance of molecular and phenotypic rifampicin dst allowed the detection of discordance between these two tests. laboratory analysis routine clinical isolates from specimens received at the inkosi albert luthuli central hospital laboratory of the kwazulu-natal province between january 2014 and december 2014 were used for this study. isolates were selected if they showed rifampicin susceptibility on the mtbdrplus but were rifampicin resistant on the 1% agar proportion method on middlebrook 7h10 agar at a critical rifampicin concentration of 1 µg/ml. the isolates from 2014 were chosen because simultaneous molecular and phenotypic rifampicin dst was performed during this time but was subsequently stopped. the selected isolates were then stored at –70 °c and later used for this study. of the isolates that had discordant rifampicin results, 36 were randomly selected for further evaluation using whole-genome sequencing. whole-genome sequencing stored isolates were grown on 7h11 middlebrook agar for over three weeks. genomic dna was extracted from isolates using the quick-dna™ miniprep kit (zymo research, irvine, california, united states). the concentration of dna was determined using the qubit dsdna hs assay kit (invitrogen, carlsbad, california united states). a minimum of 2 ng/µl dna was used for library preparation. libraries were prepared using the nextera dna library preparation kit and nextera cd index kit (illumina, san diego, california united states) according to the manufacturer’s protocol. each library was pooled and diluted to an equimolar concentration of 4 nm followed by denaturation and dilution to the final loading concentration. the library was spiked with 1% phix, which serves as an internal control to account for low-diversity libraries, and run on an illumina miseq platform (illumina, san diego, california, united states) using the miseq v2 500 cycle reagent kit (illumina, san diego, california, united states). drug resistance and strain-type profiles were determined using the tbprofiler pipeline (http:\\tbdr.lshtm.ac.uk\).16 mutations were called out at 100× depth of coverage. clinical data patients with discordant rifampicin susceptibility results were identified from the laboratory. further laboratory results (phenotypic dst, hiv status, cd4 count, and viral load results) were obtained from the laboratory information system. treatment data was obtained from the electronic drug-resistant tuberculosis treatment register of the kwazulu-natal provincial department of health. treatment outcomes were defined according to the who definitions.17 data analysis the data were captured into an excel file (microsoft corp., redmond, washington, united states) and cleaned and coded before being imported into stata version 13 (statacorp, college station, texas, united states) for statistical analysis. patient names and ages were used to remove duplicate entries. descriptive analysis was conducted on data for all patients with rifampicin-discordant tuberculosis results, as well as those selected for whole-genome sequencing. categorical variables such as sex, hiv status, previous tuberculosis treatment, as well as the xpert, phenotypic dst and mtbdrplus results, were presented as proportions and percentages. continuous variables such as age, cd4 count, and the time taken to treatment initiation were presented as means with standard deviation. a bivariate analysis was conducted using the two-sample t-test to compare the mean time taken to treatment initiation between the xpert-susceptible and xpert-resistant results, and a p-value of < 0.05 was considered indicative of statistical significance. results in 2014, out of 12 279 m. tuberculosis complex cases detected using the mtbdrplus assay, 505 (4.1%) were isoniazid mono-resistant. from the 505 isoniazid mono-resistant cases, 145 (28.7%) were mdr-tb based on the phenotypic 1% agar proportion method (i.e., had discordant rifampicin dst results). the median age of the patients with discordant rifampicin dst results was 33.8 years, and 52.4% were male (table 1). table 1: patient characteristics of isolates with rpob gene mutations missed by the genotype mtbdrplus assay at inkosi albert luthuli central hospital laboratory in kwazulu-natal, south africa between january 2014 and december 2014. microbiology results out of the 145 isolates with discordant rifampicin dst results, phenotypic dst showed that 79 (54.5%) were mdr-tb plus streptomycin resistant, 43 (29.7%) were mdr-tb, 10 (6.9%) were mdr-tb plus resistance to a fluoroquinolone, seven (4.8%) were mdr-tb plus resistance to a fluoroquinolone and any second-line injectable agent, five (3.5%) were mdr-tb plus resistance to a second-line injectable agent, and one (0.7%) was rifampicin mono-resistant. xpert results were available for 97 (66.9%) of the 145 patients. of these, 37 (38.1%) were rifampicin resistant, and 60 (61.9%) were susceptible. treatment details patient records were found for 108 (74.5%) of the 145 isolates on the dr-tb treatment register. of these, 71 (65.7%) patients had a previous tuberculosis treatment history. sixty-seven (62.0%) patients had favourable treatment outcomes (cured and treatment completed) and the average treatment duration was 18.4 months. seventy-six (70.4%) patients on the dr-tb treatment register had an xpert result; 34 of these were rifampicin resistant and 42 were rifampicin susceptible (table 2). the mean time to dr-tb treatment initiation was 32.7 days for patients with rifampicin-resistant xpert results, and 186.6 days for patients with rifampicin-susceptible xpert results. the difference in the time to treatment between the two groups was statistically significant (p < 0.001). table 2: time from availability of results to initiation of drug-resistant tuberculosis treatment in patients with m. tuberculosis isolates with discordant rifampicin susceptibility results in kwazulu-natal, south africa between january 2014 and december 2014. when calculating the mean time to results in reference to the mtbdrplus assay and phenotypic dst results, the 34 xpert rifampicin resistant cases were removed. additionally, in two other patients initiated on treatment, the date of initiation was not recorded. of the remaining 72 patients, 11 patients started treatment before mtbdrplus results became available, while 61 started treatment after a mean of 93.7 days from the availability of results. similarly, for phenotypic dst, 49 of the 72 patients started treatment prior to the availability of results while 23 started treatment after (mean 19.8 days) results were available. whole-genome sequencing results out of the 36 isolates whose whole genomes were sequenced, 19 (52.8%) had single rpob mutations outside the rrdr, namely i491f (16 isolates; 44.4%), v170f (2; 5.6%) and p483l (1; 2.8%) (figure 1). there were 14 (38.9%) isolates with single rpob mutations located within the rrdr, namely 12 isolates (33.3%) with l452p mutation and two isolates (5.6%) with s450l mutation. the three remaining isolates (8.3%) had double rpob mutations, including one isolate with a combination of the s450l mutation (located within the rrdr) and t400a mutation (located outside the rrdr), and two isolates with double mutations located within the rrdr – one with d435g and l452p mutations and another one with d435y and l452p mutations. figure 1: whole-genome sequencing results of 36 isolates with rpob gene mutations missed by the genotype mtbdrplus assay at inkosi albert luthuli central hospital laboratory in kwazulu-natal, south africa, between january 2014 and december 2014. the most common isoniazid resistance-conferring mutation was the s315t mutation, (29 isolates; 80.6%). this was followed by the inha promoter region mutation t-8a (15 isolates; 41.7%), which was found together with the katg mutation in all isolates. besides resistance to rifampicin and isoniazid, most isolates were also resistant to other first-line drugs, including pyrazinamide (25 isolates; 69.4%), ethambutol (30; 83.3%), and streptomycin (25; 69.4%). some isolates were also resistant to second-line drugs, including the second-line injectable agents (three isolates; 8.3%), fluoroquinolones (six; 16.7%), and ethionamide (18; 50.0%). one isolate had an insertion in the mmpr5 gene, which is associated with bedaquiline and clofazimine resistance. among the 16 isolates with an i491f rpob mutation, one belonged to sub-lineage 2.2.1, while the remaining 15 isolates belonged to three distinct sub-lineages of lineage 4. of these 15 isolates, three isolates each had unique mutation patterns, while 12 clustered into two groups based on mutation patterns. one group of six isolates belonging to the 4.4.1.1 sub-lineage carried katg s315t, pnca h51d, embb m306i, and rpsl k43r, which confer resistance to isoniazid, pyrazinamide, ethambutol, and streptomycin. another group of six isolates belonging to sub-lineage 4.3.3 had mutations conferring isoniazid (inha [fabg1 8t > a] plus katg s315t), pyrazinamide (pnca g132a), ethambutol (embb m306v), streptomycin (gidb 130bp deletion), and ethionamide (etha 11a > g) resistance. the 12 isolates carrying a single l452p mutation also belonged to lineage 4. among the 12 isolates, there were two clusters with common resistance-conferring mutations. the first group consisted of eight isolates belonging to sub-lineage 4.3.3 and had the inha fabg1c.-8t > a plus katg s315t isoniazid resistance-conferring mutations, as well as the pnca 456_457 c insertion (pyrazinamide resistance), embb m306v (ethambutol resistance), etha_11a > g (ethionamide resistance), and the gidb 130bp deletion (streptomycin resistance) mutations. the second group consisted of three isolates belonging to sub-lineage 4.4.1.1.1 and carrying katg s315t and embb m306i mutations for isoniazid and ethambutol resistance. discussion in this study, almost 29% of the isoniazid mono-resistant tuberculosis cases detected using the mtbdrplus assay were mdr-tb cases. this led to significant delays in the initiation of dr-tb treatment. the main cause of rifampicin resistance missed by the mtbdrplus assay was the presence of mutations outside the rrdr (mainly i491f), as well as the l452p rpob mutation. mutations outside the rrdr are not detected by the currently used who-endorsed rapid molecular assays, while the l452p mutations were missed by the previous version of the mtbdrplus assay. importantly, isolates carrying these mutations were also resistant to other first-line anti-tuberculosis drugs whose resistance is not routinely tested in tuberculosis patients globally, and the isolates also clustered into distinct groups with unique mutation profiles. the rpob l452p mutation was left out of the earlier version (version 2, released in 2011) of the mtbdrplus assay as it was thought to be clinically insignificant.18 this was later corrected in an updated version of the assay launched in 2014.18,19 at the time of this study, the older version 2 was still in use, hence the discordant rifampicin results between the mtbdrplus assay and the phenotypic assay in isolates harbouring this mutation. the mtbdrplus assay may also miss heteroresistance. one belgian study from 2019 found that the limit of detection of rifampicin heteroresistance was 5% – 10%.6 this may explain the other rrdr mutations missed by the mtbdrplus assay in this study. notably, among isolates that had an xpert result and had the l452p mutation as detected by whole-genome sequencing, the xpert assay detected rifampicin resistance. mutations outside the rrdr were found in just over half (19/36) of isolates with discordant results that were tested using whole-genome sequencing. if we assume that this proportion is representative of the whole 145 samples with discordant results (i.e. 52.7% of all discordant results are due to mutations outside the rrdr), this will equate to 76 out of 145 discordant isolates. this means that about 15% (76/505) of isoniazid mono-resistant cases had mutations outside the rrdr. this is the same prevalence found by makhado et al. from pretoria, south africa, when they screened isoniazid mono-resistant cases for the i491f mutation in clinical samples collected between 2013 and 2016.10 unlike makhado et al., who used molecular methods to screen for the i491f mutation, we used the 1% agar proportion method to test for rifampicin resistance missed by the mtbdrplus assay. phenotypic methods, especially liquid-based methods, can fail to detect rifampicin resistance caused by the i491f mutation. in a 2019 study conducted in belgium by torrea et al., the agar proportion method detected rifampicin resistance in 75% of isolates with i491f that was missed by the mycobacteria growth indicator tube dst.12 it is therefore likely that the occurrence of these mutations is more frequent than what we found in this study. while the overall prevalence of i491f mutation among tuberculosis patients is reportedly low, in patients with isoniazid resistance, the prevalence is high.9,10,20 the who defines universal access to dst as performing rapid dst for at least rifampicin in all patients with bacteriologically confirmed tuberculosis plus additional dst for at least fluoroquinolones and second-line injectable agents in patients with rifampicin resistance.1 the use of xpert as an entry point to tuberculosis care without investigating isoniazid resistance would prove disastrous for patients infected with m. tuberculosis strains that have mutations outside the area of detection and are resistant to all other first-line drugs. recent studies conducted between 2015 and 2017 have shown that isoniazid resistance generally develops before rifampicin resistance.21,22 notwithstanding the importance of testing for rifampicin resistance, the neglect of isoniazid testing leads to inappropriate therapy, treatment failure and accumulation of resistance in patients with initial isoniazid resistance.23 we therefore propose an algorithm to optimise dr-tb detection (figure 2). we submit that the initial dst should include both isoniazid and rifampicin. importantly, if resistance is found to any of these two drugs, it should trigger further dst of other first-line and second-line drugs that will be used for treatment. moreover, an attempt should be made to look for the i491f mutation in isolates from patients with isoniazid mono-resistant tuberculosis as this mutation may be missed by both phenotypic and genotypic dst methods that are routinely used for the detection of rifampicin resistance. figure 2: proposed algorithm for the diagnosis of drug-resistant tuberculosis. the largest global cluster of extensively drug-resistant tuberculosis that was ever reported was from tugela ferry in kwazulu-natal in 2005 and it was caused by a strain named f15/lam4/kzn.24 a study conducted by pillay et al. using m. tuberculosis isolates collected between 1994 and 2002 showed how this extensively drug-resistant strain accumulated resistance over time under a tuberculosis programme that lacked appropriate dst.25 patients received inappropriate therapy, thus allowing the selection and spread of resistant strains and leading to treatment failure with dire consequences, especially among patients who were also hiv-positive.24 with the current tuberculosis diagnostic algorithm that only tests for rifampicin resistance, we find ourselves in a similar circumstance that calls for swift action if we are to avoid the same unfortunate outcome. targeted next-generation sequencing can overcome some of the challenges of rapid molecular assays and phenotypic dst by allowing the rapid detection of rpob mutations outside the rrdr and additional mutations conferring resistance to other anti-tuberculosis drugs, including those that are difficult to test by phenotypic methods (e.g. pyrazinamide). however, the cost, skill levels and expertise required to perform next-generation sequencing and interpret its results remain the prohibiting factors limiting the implementation of this technology, especially in high-burdened, low-resource countries where it is needed the most.26 therefore, in many countries, including south africa, next-generation sequencing remains confined to the reference and research laboratories. although molecular tests have decreased the time to tuberculosis dst results from weeks, using the previous phenotypic tests, to hours and days, discordant results may reverse this benefit. given the fact that mtbdrplus results in this study showed rifampicin-susceptible m. tuberculosis, appropriate treatment (dr-tb treatment) could not be initiated until phenotypic dst results showing rifampicin resistance became available. even so, due to the inferiority of second-line tuberculosis treatment compared to the standard first-line treatment, clinicians may be reluctant to change patient treatment based on a discordant result. the patients in this study often had multiple results, showing that the clinicians sought more evidence before committing patients to dr-tb treatment regimens. as shown in this study, there were significant delays in the initiation of dr-tb treatment, which devalues the benefits of rapid molecular tests. it was alarming to find such high levels of resistance to other first-line drugs (pyrazinamide and ethambutol), as well as ethionamide and streptomycin. phenotypic dst for pyrazinamide and ethambutol is not routinely performed in many settings because of poor reproducibility and reliability.27,28,29 in the south african setting where xpert (ultra) is used for the initial diagnosis of tuberculosis and no further dst is performed for rifampicin-susceptible cases, these patients would be treated with first-line therapy. in fact, given the high number of patients with a previous tuberculosis treatment history and current indications for performing m. tuberculosis culture, phenotypic dst was probably performed for these patients because they had already failed tuberculosis therapy. the presence of resistance to streptomycin suggests that these patients may have failed a few rounds of tuberculosis therapy because streptomycin was previously used as part of a standard re-treatment regimen in patients who had failed first-line therapy. in south africa, this regimen was stopped after the rollout of xpert, which allowed universal testing of all tuberculosis patients. the rollout of xpert was completed towards the end of 2013. isolates in this study belonged predominantly to lineage 4, which is known to predominate among dr-tb cases in the kwazulu-natal province.30 most of the isolates clustered based on the i491f and l452p rpob mutations, with each cluster carrying a unique set of mutations conferring resistance against isoniazid, pyrazinamide, ethambutol, streptomycin, or ethionamide. this suggests that these highly resistant strains may have been spreading undetected in the community. furthermore, lineage 4.4.1.1 strains with rpob i491f, katg s315t, pnca h51d, embb m306i, and rpsl k43r mutations have been linked to an outbreak that originated in eswatini and later spread to south africa.10 limitations this study reports old data on m. tuberculosis isolates from 2014. however, we examined this period because this was when both phenotypic and genotypic rifampicin dst were performed simultaneously in our setting. moreover, the tuberculosis diagnostic algorithm has not changed since then, although phenotypic rifampicin dst was subsequently stopped. another limitation of this study was our use of phenotypic dst to select isolates with possible mutations outside the rrdr instead of using molecular screening. this may have underestimated the prevalence of isolates with these mutations as some of them remain susceptible on the phenotypic assay. due to limited resources, we sequenced only a subset of the isolates with discordant results. the data from the susceptible tuberculosis treatment register was not available to compare with that on the dr-tb register to determine if patients not listed on the dr-tb register were treated with first-line tuberculosis therapy. finally, the study was performed in one province of south africa so the findings may not apply to other regions. nonetheless, this province has the highest prevalence of dr-tb cases in the country and similar findings have been reported in the northern provinces. conclusion the presence of highly drug-resistant m. tuberculosis strains with mutations missed by the routine rapid molecular assays highlights the need for the revision of the who definition of universal access to dst so that tuberculosis diagnostic algorithms include testing for both isoniazid and rifampicin in all patients with bacteriologically confirmed tuberculosis. the recent endorsement of the xpert mtb/xdr by the who for detection of isoniazid, fluoroquinolone and second-line injectable agent resistance in xpert (ultra)-confirmed tuberculosis cases provides an opportunity to close the gap in isoniazid testing.1 the i491f mutation remains the most commonly detected mutation outside the rrdr and its frequent occurrence in isoniazid-resistant cases calls for its inclusion in assays that detect rifampicin resistance. this codon is not too far away from the rrdr, so current assays can be upgraded to include it to avoid the use of inappropriate therapy, prevent the accumulation of resistance, and reduce community spread. acknowledgements the authors would like to thank the national health laboratory service tuberculosis laboratory staff at the inkosi albert luthuli central hospital who performed the routine clinical work for the province of kwazulu-natal. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions n.r.m. and k.p.m. were involved in the original conceptualisation of the study as well as obtaining the study funding. n.r.m. also carried out laboratory work, data acquisition and analysis, project administration and writing of the manuscript. l.p.c. performed the data analysis and writing of the manuscript. r.s. was involved in performing laboratory work. y.n. and j.g. performed whole-genome sequencing. e.w. did the whole-genome sequencing analysis. t.d.o. supervised the overall whole-genome sequencing work. k.s.s.-h. assisted with the writing and reviewing of the manuscript and k.p.m. was the overall supervisor of the project including the writing of the manuscript. sources of support this work was supported by the university capacity development programme of the university of kwazulu-natal and the national health laboratory service research trust. data availability all data generated from this study are available on request from the corresponding author, n.r.m. disclaimer the views expressed in this manuscript are those of the authors and not an official position of the institutions involved or any funders. references who. who operational handbook on tuberculosis. module 3: diagnosis – rapid diagnostics for tuberculosis detention, 2021 update. geneva: world health organization; 2021. who. molecular line probe assays for rapid screening of patients at risk of multidrug-resistant tuberculosis (mdr-tb). geneva: world health organization; 2008. who. automated real-time nucleic acid amplification technology for rapid and simultaneous detection of tuberculosis and rifampicin resistance: xpert mtb/rif system. policy statement. geneva: world health organization; 2011. andre e, goeminne l, cabibbe a, et al. consensus numbering system for the rifampicin resistance-associated rpob gene mutations in pathogenic 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distributions and pnca gene sequencing. antimicrob agents chemother. 2012;56(3):1253–1257. https://doi.org/10.1128/aac.05894-11 gandhi nr, brust jcm, moodley p, et al. minimal diversity of drug-resistant mycobacterium tuberculosis strains, south africa. emerg infect dis. 2014;20(3):426–433. https://doi.org/10.3201/eid2003.131083 article information authors: michael a. noble1 robert martin2 jean-bosco ndihokubwayo3 affiliations: 1program office for laboratory quality management, university of british columbia, canada 2department of global health, university of washington, united states 3world health organization regional office for africa, republic of the congo how to cite this article: noble ma, martin r, ndihokubwayo j-b. making great strides in medical laboratory quality. afr j lab med. 2014;3(2), art. #256, 2 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.256 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. making great strides in medical laboratory quality in this editorial... open access • content • references content top ↑ whilst the observation of blood and urine as a commentary of illness and disease can be traced back to hippocrates as early as 300 bc, the true roots and foundations of the modern medical laboratory as a vital investigation process to better understand pathology and diagnosis were established in the late-19th and early-20th centuries.1 modern day laboratory tests have become the cornerstone for objective data collection to assist, affirm and document diagnoses rather than depending on anecdotal and subjective opinion. use of highly-crafted optical lenses made microscopic examination of urine, sputum, blood and spinal fluid achievable. microbiology techniques for blood and sputum culture made the diagnosis of tuberculosis, diphtheria and typhoid both possible and documentable. examination for bilirubinaemia and abnormal glucose levels also became feasible. the first hospital laboratories were established in london (guys hospital) and baltimore (johns hopkins hospital)1 and, by the early 20th century, laboratories began to become a permanent part of the infrastructure of hospitals.also in the early part of the 20th century, professional organisations were emerging as self-regulating groups that addressed the competencies of laboratory professionals. however, by the 1940s, fw sunderman and w belk were concerned because the evidence and experience in united states laboratories indicated that physicians could not trust laboratory test results. in their key study2 they demonstrated that interlaboratory testing of identical samples resulted in considerable variability of test performance and results. this was the origin of proficiency testing as a valuable quality measure that ultimately led to the development of quality control, laboratory inspections and accreditation bodies across the united states, canada, europe and australia. fortunately, the foundations of the quality movement had already been developed by giants such as walter shewhart, w. edwards deming and others, in their studies of statistical design. medical laboratory quality control and the use of the levey-jennings chart3,4 were based upon the studies of shewhart. over the next 60 years, many countries saw continued growth and improvement in the medical laboratory based upon the tenets of quality and improvements in accreditation processes. lessons were learned from a variety of sources. deming5 and juran6 promoted quality measures as they assisted the post-war rebirth of japanese industry. the military developed tools to ensure that suppliers would deliver high quality, consistent and reliable provisions.6 standards documents, in particular the international organization for standardization (iso) 9001 (quality management) and its seed documents (http://www.iso.org/iso/home/store/catalogue_tc/catalogue_detail.htm?csnumber=46486), led the way to internationalising quality as we know it today. these efforts led to further refinements for testing laboratories and medical laboratories alike. arguably the most significant foundational document for medical laboratory quality improvement in the modern era has been the publication of iso 15189:2003, 2007 and 2012: medical laboratories – requirements for quality and competence.7 errors and challenges continued to occur, with perhaps the greatest challenge of the modern era being the recognition that containment of hiv would require even greater strides in healthcare improvement and enhancement of medical laboratory quality.8 great efforts by the us president’s emergency plan for aids relief (pepfar)9 programme, the bill and melinda gates foundation, the clinton health access initiative and the world health organization (who) pointed the way for laboratory improvement in every part of the world. it is through those processes and the active energy of the who’s regional office for africa (afro) that we see the growth and successes of quality management system programmes such as stepwise laboratory quality improvement process towards accreditation (slipta) and strengthening laboratory management toward accreditation (slmta). time has demonstrated that the path to improvement is not always a straight line; there are periods of success and periods of fallback. laboratory improvement must be experienced as a slow and gradual process based upon building a culture of quality, increasing knowledge and a goal of continual improvement. the study of risk management teaches us that we cannot predict all errors; but through the active practice of quality processes we can detect errors earlier and reduce the repetition of the same error time and time again. it is through the persistence of personal interest and action that we sustain quality efforts and have confidence in successful outcomes for the overall process of laboratory investigation. the authors of the excellent manuscripts in this special issue of the african journal of laboratory medicine, entitled transforming the quality of laboratory medicine through the strengthening laboratory management toward accreditation program, attest to the great strides that are being made through the progressive stepwise approach to adopting and embracing quality measures. but to them we put forward this challenge: whilst it is a great achievement to reach a level of success where the requirements of accreditation are met, the true accomplishment is reaching the point where that level of quality is an everyday practice and expectation, and the laboratory is ‘accreditation-ready’ over and over. when the inevitable slips and mistakes occur in laboratories that are accreditation-ready, the processes of error detection, correction and improvement, and progress back to quality, must occur quickly, smoothly and sustainably.10 references top ↑ 1.1. berger d. a brief history of medical diagnosis and the birth of the clinical laboratory. part 1 – ancient times through the 19th century. mlo med lab obs. 1999;31(7):28–30, 32, 34–40.1.2. belk wp, sunderman fw. a survey of the accuracy of chemical analyses in clinical laboratories. am j clin pathol. 1947;17(11):853–861. 1.3. levey s, jennings er. the use of control charts in the clinical laboratory. am j clin pathol. 1950;20(11):1059–1066. 1.4. levey s, koenig s. quality control standards for analysis of sodium, potassium, and calcium in urine. am j clin pathol. 1958;30(5):404–406. 1.5. walton m. the deming management method. new york, ny: perigee books; 1986. 1.6. juran jm. a history of managing for quality: the evolution, trends, and future directions of managing for quality. milwaukee, wi: asqc quality press; 1995. 1.7. noble ma. making progress in implementing quality in canadian medical laboratories. clin biochem. 2013;46(13–14):1194. http://dx.doi.org/10.1016/j.clinbiochem.2013.04.025 1.8. mfinanga sg, kahwa a, kimaro g, et al. dissatisfaction with the laboratory services in conducting hiv related testing among public and private medical personnel in tanzania. bmc health serv res. 2008;8:171. http://dx.doi.org/10.1186/1472-6963-8-171 1.9. woodcock s, fine g, mcclure k, et al. the role of standards and training in preparing for accreditation. am j clin pathol. 2010;134(3):388–392. http://dx.doi.org/10.1309/ajcp03tfpbkeyynt 1.10. noble ma. quality management in the medical laboratory. keynote presentation. association for pathologists in eastern, central, and southern africa (apecsa) arusha tanzania; august 2006. abstract introduction tb slmta development results from tb slmta implementation workshops discussion acknowledgements references about the author(s) heidi albert foundation for innovative new diagnostics (find), cape town, south africa andre trollip foundation for innovative new diagnostics (find), cape town, south africa donatelle erni foundation for innovative new diagnostics (find), geneva, switzerland kekeletso kao foundation for innovative new diagnostics (find), geneva, switzerland citation albert h, trollip a, erni d, et al. developing a customised approach for strengthening tuberculosis laboratory quality management systems toward accreditation. afr j lab med. 2017;6(2), a576. https://doi.org/10.4102/ajlm.v6i2.576 lessons from the field developing a customised approach for strengthening tuberculosis laboratory quality management systems toward accreditation heidi albert, andre trollip, donatelle erni, kekeletso kao received: 04 oct. 2016; accepted: 22 nov. 2016; published: 31 mar. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: quality-assured tuberculosis laboratory services are critical to achieve global and national goals for tuberculosis prevention and care. implementation of a quality management system (qms) in laboratories leads to improved quality of diagnostic tests and better patient care. the strengthening laboratory management toward accreditation (slmta) programme has led to measurable improvements in the qms of clinical laboratories. however, progress in tuberculosis laboratories has been slower, which may be attributed to the need for a structured tuberculosis-specific approach to implementing qms. we describe the development and early implementation of the strengthening tuberculosis laboratory management toward accreditation (tb slmta) programme. development: the tb slmta curriculum was developed by customizing the slmta curriculum to include specific tools, job aids and supplementary materials specific to the tuberculosis laboratory. the tb slmta harmonized checklist was developed from the world health organisation regional office for africa stepwise laboratory quality improvement process towards accreditation checklist, and incorporated tuberculosis-specific requirements from the global laboratory initiative stepwise process towards tuberculosis laboratory accreditation online tool. implementation: four regional training-of-trainers workshops have been conducted since 2013. the tb slmta programme has been rolled out in 37 tuberculosis laboratories in 10 countries using the workshop approach in 32 laboratories in five countries and the facility-based approach in five tuberculosis laboratories in five countries. conclusion: lessons learnt from early implementation of tb slmta suggest that a structured training and mentoring programme can build a foundation towards further quality improvement in tuberculosis laboratories. structured mentoring, and institutionalisation of qms into country programmes, is needed to support tuberculosis laboratories to achieve accreditation. introduction the world health organization’s (who) end tb strategy calls for an end to the global tuberculosis epidemic. it aims to reduce deaths by 95% and new tuberculosis cases by 90% and to ensure that no family is burdened with catastrophic expenses due to tuberculosis by 2025.1 despite the fall in global tuberculosis mortality by 47% since 1990, the disease still claimed more than 1.5 million lives in 2014.2 a cascade of events, including poor screening, failure to link screened patients to diagnostic services, and failure to link diagnosed patients to treatment, means that many people die from tuberculosis due to delayed diagnosis and treatment initiation.3 quality-assured laboratory services are critical for the provision of timely, accurate and reliable results to support diagnosis, drug-resistance testing, treatment monitoring and surveillance of disease. weak laboratory systems result in high levels of laboratory error that impact patient care and undermine the confidence healthcare providers have in laboratory services.4 in recent years, the focus on improving laboratory quality management systems (qms), and assuring the quality of laboratory services by working toward national or international laboratory accreditation has intensified.5 accreditation is the formal recognition of implementation of a qms that adheres to international standards and has been shown to improve the quality of healthcare for patients through reduction in testing errors.6 the strengthening laboratory management toward accreditation (slmta) programme was developed by the united states centers for disease control and prevention in collaboration with the american society for clinical pathology, the clinton health access initiative, and the who regional office for africa to promote immediate and measurable quality improvement in laboratories in developing countries. slmta is a programme that may be used to prepare laboratories for accreditation.7 since its launch in kigali, rwanda in 2009, slmta has been implemented in 47 countries (23 in africa), with 617 laboratories already enrolled. eighteen per cent of the enrolled laboratories are at the national level and most (98%) are providing hiv-related services.8 only four national tuberculosis reference laboratories (ntrls) in africa have achieved international accreditation to date,9,10 and only six ntrls have undergone a formal stepwise laboratory quality improvement process towards accreditation (slipta) audit by the african society for laboratory medicine (t. mekonen, personal communication). accredited ntrls are better equipped to support the national tuberculosis laboratory network and also provide reliable support to their national tuberculosis control and treatment programmes.11 since 2007, the foundation for innovative new diagnostics (find) has worked with ministries of health to introduce new diagnostic technologies to improve the diagnosis of tuberculosis, detection of drug resistance12 and upgrading of facilities.13,14,15,16 although technical capacity to conduct new tests can be developed within a relatively short time frame, persistent challenges to providing quality results in a consistent manner often remain, many of which are linked to laboratory quality system weaknesses. in 2011, through funding from the united states president’s emergency plan for aids relief, find was involved in implementation of the slmta programme in clinical laboratories in dominican republic. measurable improvement was observed in cohorts of laboratories participating in the programme. however, tuberculosis laboratories were not included in this programme. concurrently, the global laboratory initiative (gli) was developing its stepwise process towards tuberculosis laboratory accreditation online tool.17 this tool provided online resources and a framework consisting of four phases, but did not have training materials or an implementation plan to enable adoption by tuberculosis laboratories. tuberculosis laboratories, particularly at the central or regional-level, have separate facilities from other clinical laboratories. they have different requirements for biosafety and quality assurance, and have often been excluded from accreditation efforts. recognising the unique needs of tuberculosis laboratories, find developed a comprehensive approach to tuberculosis laboratory strengthening based on the existing slmta approach and incorporating aspects of the gli stepwise process towards tuberculosis laboratory accreditation online tool. in this article, we describe the development of the tuberculosis strengthening laboratory management toward accreditation (tb slmta) programme and challenges experienced during early implementation in 10 countries. we also reflect on approaches that will ensure continued quality improvement to reach accreditation and institutionalisation of the programme. tb slmta development customisation of training materials in 2012, find conducted a review of the slmta materials, and customised the content for tuberculosis laboratories based on available tuberculosis resources (either developed internally by find or by other organisations). this customisation included the development of specific tools, job aids and supplementary materials for the implementation of a qms in the tuberculosis laboratory (table 1), but kept the overall structure of the slmta curriculum. customisation included major changes to the content of the slmta facilities and safety and quality assurance modules (the focus was changed from the quantitative testing in slmta to the qualitative and semi-quantitative testing relevant to the tuberculosis laboratory). the slmta laboratory testing and test result reporting modules were combined and an auditing module was introduced. tuberculosis laboratory-specific tools, examples and scenarios were introduced throughout all modules in the training. the tb slmta harmonized checklist was also introduced as part of the programme. the tb slmta curriculum was piloted in cape town in april 2013 in a shortened training-of-trainers (tot) workshop led by slmta master trainers and with experienced tuberculosis laboratory specialists as participants. following the pilot workshop, some changes were made to the training materials (e.g. organisation and cross-referencing of tools, adjustment of training notes for clarity, and editing errors) and the tb slmta harmonised checklist was revised. subsequent review and revision of the tb slmta curriculum has been conducted to keep the content current with an updated gli tool (version 2.0, 2013) and who regional office for africa slipta (2015) tool. a review of the tb slmta curriculum was conducted in 2015 due to experience that improvement projects did not necessarily target the highest priority non-conformities. based on feedback from previous trainings, minor changes were also made to the cross-cutting, facilities and safety and quality assurance modules. tb slmta harmonized checklist the tb slmta harmonized checklist18 is based on the who regional office for africa slipta checklist (2007),19 and incorporates tuberculosis laboratory-specific requirements as provided in the gli stepwise process towards tuberculosis laboratory accreditation tool, which were inserted as sub-clauses in the slipta checklist. the tb slmta harmonized checklist is used to assess the qms of the tuberculosis laboratory prior to enrolment in the programme (baseline assessment) and after programme completion (exit assessment). the differences between the scores obtained overall and for each section, are a measure of the impact of the programme. assessors evaluate the laboratory operations as per checklist items, scoring the assessment and documenting their findings in detail. table 1: comparison of slmta and tb slmta programme components. the pilot version of the tb slmta harmonized checklist20 had additional scores allocated to the tuberculosis-specific clauses. a revised checklist (tb slmta harmonized checklist v1.0), which maintained the original slipta scoring system,21 was used in the tb slmta roll-out. recognition is given using a five-star grading system, with the following scores corresponding to the indicated number of stars: zero stars (0–142 points; < 55%), one star (143–165 points; 55–64%), two stars (166–191 points; 65%–74%), three stars (192–217 points; 75%–84%), four stars (218–243 points; 85%–94%) and five stars (244–258 points; ≥ 95%). the tb slmta harmonized checklist 1.0 was recently revised in keeping with slipta v2:2015, and the additional clauses of international organization for standardization 15189:2012. the questions added pertain to risk assessment, laboratory information system, contingency planning and safety. the tb slmta harmonized checklist v1.0 is available in english and spanish. the tb slmta harmonized checklist v2.1 is available in english and russian.16 implementation of tb slmta implementation of the tb slmta programme starts with the initial engagement with the ministry of health on the programme scope and expected outputs, as well as commitments required from the country (figure 1). during this planning phase, the country selects the participating tuberculosis laboratories, the model of implementation, the trainees to attend the tot and the tb slmta participants who will attend the in-country training. countries selects two or three participants per laboratory to attend the in-country tb slmta training. typically, participants include the laboratory manager, quality officer and one technician. after graduation from the tot, the certified trainers implement the programme in the country. baseline and exit assessments are conducted with the tb slmta harmonized checklist v1.0 by trainers or slipta-trained assessors with tuberculosis laboratory experience. in-country national or regional trainings are conducted over a period of 12–15 months. between trainings, participants work on improvement projects supervised by the tb slmta mentors. post-tb slmta activities are conducted in the laboratories under supervision of the mentors before an external assessment determines the readiness for accreditation. training-of-trainers workshop the tb slmta tots are conducted by slmta master trainers, and are based on teach-back methodology.22 this practice-based training approach requires trainees to play the roles of both trainer and participant as they teach the curriculum at the same time as they are learning the content. the tots provide trainees with an introduction to the tb slmta materials, practice in delivering the content and receiving feedback on their performance. the ratio of trainees to master trainers is a maximum of eight to one. to certify as trainers, trainees must demonstrate knowledge of tb slmta curriculum and proficiency in delivering training. trainees that find teach-back challenging and do not show a good understanding of the materials graduate as one-one coaches. they can facilitate rollout in their laboratory, but are not certified to train others. mentors are trainers who support the in-country training participants during the implementation phases between trainings. during mentoring visits to the laboratory, they supervise the participants as they implement the improvement projects, and provide resources (e.g. standard operating procedures) to implement what was taught in the training in the tuberculosis laboratory. the fundamentals of mentoring are modelled during the tot. trainees, who are certified as trainers, and who show an aptitude for mentoring are selected by the master trainers to perform mentoring in their countries. mentoring in tb slmta builds on the relationship established between trainer and participant, and seeks to support programme implementation in the laboratory. master trainers support the certified trainers and mentors during their first national or regional training, and where possible provide at least one interim visit to support mentoring. trainers under supervision receive additional support from the master trainers during the workshop and, if assessed as proficient, can then graduate as trainers. figure 1: diagrammatic representation of the tb slmta programme from initiation to accreditation the tots are intensive and highly interactive hence good language skills and a working knowledge of qms concepts is required. based on this observation and challenges experienced in conducting a tot with participants with varying levels of english fluency, a mandatory online training was introduced prior to the tot, based on the who laboratory quality management system: handbook,23 to ensure that trainees have a basic understanding of qms principles. in addition, trainees whose first language is not english are required to successfully complete an online language competency training before registration for the tot. models for implementation two models have been adopted for implementation of tb slmta: workshop approach: where several tuberculosis laboratories are available in-country (or in cases where more than one country conducts centralised trainings), the three-workshop approach can be used. three five-day regional workshops are conducted by trainers, approximately three months apart. facility-based approach: where there is only one tuberculosis laboratory in the country being enrolled in tb slmta the facility-based approach may be used. the facility-based approach follows the same tb slmta curriculum, with training sessions split into three blocks over 12 months. factors affecting choice of implementation model include funding, number of laboratories participating in tb slmta, and availability of staff. tb slmta is targeted for implementation in tuberculosis laboratories at the national or referral level. these laboratories are conducting advanced tuberculosis testing, and generally have separate facilities from general laboratories. laboratories conducting tuberculosis testing on lower levels of the healthcare system are not targeted with this training. however, this does not preclude the use of tb slmta resources to guide them, especially those related to safety and quality assurance. improvement projects and mentoring improvement projects are broad-based activities that address weaknesses in the qms. topics for improvement projects are chosen from subjects covered in the trainings. as with slmta (table 1), each participant is required to complete two improvement projects between trainings. the ‘just do it’ project (e.g. maintaining personnel files) is implemented as a group by all the participants from the laboratory. the ‘complex’ project, which requires extensive planning and before-and-after data collection, is chosen with assistance from the certified trainers. ideally, the laboratory management is included in the decision of the topic and scope (if laboratory managers are not participants), to ensure management engagement and allocation of time and resources to complete the projects. the projects are implemented by the participants, but should involve the entire laboratory staff. participants present their findings at national or regional workshops or on a day set aside by the laboratory (facility-based approach). find found that often the choice of improvement projects does not reflect the priority gaps of the laboratory. in 2015, find adopted a more stringent criterion for improvement project selection. under the guidance of the certified trainers, each participant completes two improvement projects between trainings; both are ‘complex’ and require extensive planning and data collection. the first project is based on the subjects covered in the trainings. for example, training 1 (quality indicators and facilities and safety), training 2 (equipment, purchasing and inventory, and quality assurance) and training 3 (documents and records, client management and customer service, and specimen management) (table 2). the second project addresses the weaknesses identified during the baseline assessment. these non-conformities are split between the participants and a different section of the tb slmta harmonized checklist is covered between trainings. tb slmta uses a short-term mentoring model instead of the embedded model encouraged by slmta. mentoring visits are conducted by the trainers over two or three days. each facility receives two visits between each workshop. the outcomes of the mentoring visits and, in particular, the progress with improvement projects, is monitored by the mentors for each laboratory, and any necessary support provided. standardised data collection tools are used to record the findings of mentorship visits. table 2: examples and types of improvement projects implemented in the tb slmta programme. figure 2: implementation of tb slmta in 37 tuberculosis laboratories in 10 countries since 2013. results from tb slmta implementation workshops since 2013, four regional tots have been conducted in lesotho, vietnam, south africa and moldova. seventy trainees from 27 counties have been trained, and 59 are certified as trainers (including trainers under supervision), of which four participants are from who supranational reference laboratories that provide tuberculosis laboratory technical support to countries. twenty-six trainers are currently active in the tb slmta programme. currently there are three master trainers. one master trainer, based in the african region, graduated after conducting a round of tb slmta, and we expect two more graduates in the coming year (one in the african region and one in south east asia) for a total of six master trainers. the tb slmta programme has been rolled out in 37 tuberculosis laboratories in 10 countries (figure 2). national or regional tb slmta trainings using the workshop approach were conducted in 32 laboratories in five countries (dominican republic, ethiopia, lesotho, tanzania and vietnam). the facility-based approach has been used in one regional tuberculosis laboratory in cameroon. the instructional phase is complete in these laboratories, but is ongoing in the four ntrls in eastern europe (armenia, azerbaijan, belarus and moldova). baseline and exit assessment scores for 18 laboratories in four countries (cameroon, ethiopia, lesotho and tanzania) were available for analysis and are summarised in table 3. at baseline, six of the eighteen laboratories had a zero-star rating, three had a one-star rating, seven had a two-star rating and two laboratories had a four-star rating. no laboratories had threeor five-star ratings at baseline assessment. at exit, two laboratories remained at zero stars, two were rated at one-star, four laboratories were rated at two stars, seven were rated at three-stars and three laboratories were rated at four-stars. the impact of tb slmta, as well as the individual country experiences will be addressed in separate publications. find developed an online biosafety training programme in 2014,24 and tb slmta participants in tanzania and lesotho were enrolled in this training to complement the basic biosafety module of the tb slmta programme. this task-based online training was implemented in conjunction with biosafety improvements projects following workshop 1. active participation for this extended time of the in-country training is a challenge for trainers and participants alike. in our cohort, 21 participants (lesotho, 1; dominican republic, 8; ethiopia, 7; tanzania, 3; vietnam, 2) were unable to complete the compulsory trainings and improvement projects due to personal or job-related reasons. although in most cases, additional participants from the same laboratory meant that the laboratory was not excluded from continuing the programme, one regional tuberculosis laboratory in tanzania was not able to complete the programme as both participants were unable to finish the training. discussion tuberculosis laboratories are an essential element of tuberculosis prevention and care, providing testing for diagnosis, surveillance and treatment monitoring that can be accessible at all levels of the healthcare system. the tb slmta programme provides tuberculosis laboratories with customised support to accelerate the process of strengthening their qms towards accreditation. there is an urgent need to expand the programme, as only 21 ntrls (43%) on the african continent have received slmta training and only four ntrls have reached accreditation. although 44% of ntrls report implementing a qms, the extent of implementation is not known.25 table 3: ‘stars’ at baseline and exit for 18 tuberculosis laboratories in four countries completing the tb slmta programme (2013–2016).† there were a number of challenges to implementing the tb slmta programme in the initial cohort of laboratories. the lack of experienced assessors was a challenge in some countries. slipta-trained assessors with experience in tuberculosis testing were used to supplement certified tb slmta trainers. however, limited hands-on time spent with the tb slmta harmonized checklist during the tb slmta tot, and slipta trained assessors who are unfamiliar with implementing the tuberculosis laboratory specific clauses, may lead to inflated scoring during these assessments. while laboratories enrolled in the tb slmta programme may use the who regional office for africa slipta checklist, the additional components from gli included in the tb slmta harmonized checklist v1.0 enable technical assessment alongside assessment of international organization for standardization components. in instances where management had not been fully engaged in the tb slmta implementation, participants struggled to complete the improvement projects. it is therefore critical to actively engage upper management, both at the facility level and at the national ministry of health, to ensure their commitment to the programme. institutionalisation of qms into country programmes will be needed to support tuberculosis laboratories in achieving accreditation. training and quality improvement activities may be seen as extra workload, especially in settings where staff shortage and high workload are existing challenges. furthermore, trainers and mentors, who were critical components of the programme, are required to support the programme in addition to their usual duties. this may put additional strain on the laboratory as other staff are required to cover their workstations during their absence. in addition to senior level engagement of the ministry of health, qms activities being conducted by various implementing partners and donors should be coordinated centrally to ensure synergy to avoid duplication of effort and the risk of confusion and wastage of resources. we found multiple partners conducting overlapping activities related to qms without clear coordination to ensure cost-efficiency and maximum impact from available resources. partners should seek active collaboration on qms activities, harmonisation of approaches and contributions of various groups, under the leadership and coordination of the ministry of health. the tots are highly interactive, and some trainees whose first language is not english find the training challenging. introduction of language proficiency and an introduction to qms online training in 2014, helped ensure that trainees in the tots were successfully certified as trainers. however, this approach limits potential trainees. in 2016, find conducted a tot in english, with real-time russian translation (using a tuberculosis laboratory specialist as translator). all the trainees passed, suggesting that the model can be expanded to non-english speaking countries using translated materials (including the tb slmta harmonized checklist) and real-time translation. careful considerations must be given to the translator, with preference given to those who have an insight into laboratory testing or qms. further analysis of this approach is required. master trainers are certified after successful supervision of the roll-out of the tb slmta programme in a country. to facilitate the expansion of the tb slmta programme, there is a need for more master trainers, particularly those that can train in languages other than english. as noted earlier, find recently adopted a more stringent criterion for improvement project selection. a focus on the weaknesses identified in baseline assessment, in particular quality indicator and quality control monitoring and safety in the tuberculosis laboratory, has the potential to improve the impact of the tb slmta programme. as the cohort of tuberculosis laboratories that have used this strategy increases, the impact will be measured. mentoring of laboratories was found to be an important component to successful implementation of slmta. embedded mentorship has proven to result in measurable improvement in the qms in many countries, including lesotho, zimbabwe, kenya and nigeria.26,27,28,29 in tb slmta, certified trainers mentor participants during site visits and remotely between workshops. this short-term mentoring model is cost-effective, scalable and sustainable, and is well suited to the workshop approach of implementation used in our cohort. ongoing structured mentoring of the tuberculosis laboratories that obtained four-star ratings at tb slmta exit assessment is being conducted in preparation for accreditation. the tb slmta programme is currently focused on tuberculosis laboratories with the capacity to perform advanced diagnostics such as culture and drug susceptibility testing. tuberculosis laboratories on the lower level of the healthcare system may consider integration into current slmta activities. in addition, if feasible, countries should consider sharing mentoring and assessments between programmes. these cost-cutting approaches have an added benefit of integrating services and present opportunities for knowledge sharing and will encourage sustainability and institutionalisation of qms. limitations this study is subject to a number of limitations. firstly, none of the tb slmta laboratories have reached accreditation yet, and we are thus reporting on intermediate measures of quality improvement leading to the ultimate target of accreditation. secondly, quality improvement from three stars to five stars (which is considered equivalent to accreditation readiness) is challenging.30 thirdly, the role of mentorship in this final phase is still to be determined. finally, in this article we have not addressed the costs of tb slmta. a cost estimation exercise is being undertaken. we do not expect the costs to differ substantially from costs of the slmta programme as reported by others.31 conclusions tb slmta is a structured training and mentoring programme that is customised to meet the needs of tuberculosis laboratories in implementing a qms in tuberculosis laboratories in resource-limited settings within a reasonably short time frame building a foundation toward further quality improvement toward achieving accreditation. expansion of this programme is an urgent priority to address the need for accreditation of tuberculosis laboratories on the african continent and beyond. box 1: lessons learned acknowledgements the findings and conclusions in this publication are those of the authors and do not necessarily represent the official position of the cdc. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. sources of support we are grateful to the united states president’s emergency plan for aids relief through the united states centers for disease control and prevention (3u2gps002746), expandtb, unitaid, uk aid, aus aid, and the who for funding support. authors’ contributions h.a., a.t. and k.k. contributed to development and implementation of the programme, data analysis, preparation and critical review of the manuscript. d.e. contributed to the data analysis. all authors agreed with the content of the manuscript. references world health organization. who end tb strategy. global strategy and targets for tuberculosis prevention, care and control after 2015 [page on the internet]. c2015 [cited 2016 aug 22]. available from: http://who.int/tb/post2015_strategy/en/ world health organization. global tuberculosis report 2015, 20th ed [document on the internet]. c2015 [cited 2016 aug 22]. available from: http://apps.who.int/iris/bitstream/10665/191102/1/9789241565059_eng.pdf kuznetsov vn, grjibovski am, mariandyshev ao, et al. two vicious circles contributing to a diagnostic delay for tuberculosis patients in arkhangelsk. emerg health threats j. 2014;7:24909. https://doi.org/10.3402/ehtj.v7.24909 alemnji ga, zeh c, yao k, et al. strengthening national health laboratories in sub-saharan africa: a decade of remarkable progress. trop med int health. 2014;19(4):450–458. https://doi.org/10.1111/tmi.12269 gershy-damet gm, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. https://doi.org/10.1309/ajcptuuc2v1wjqbm peter tf, rotz pd, blair dh, et al. impact of laboratory accreditation on patient care and the health system. am j clin pathol. 2010;134(4):550–555. https://doi.org/10.1309/ajcph1skq1hnwghf yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin pathol. 2010;134(3):401–409. https://doi.org/10.1309/ajcpnbbl53fwuiqj yao k, luman et, slmta collaborating authors. evidence from 617 laboratories in 47 countries for slmta-driven improvement in quality management systems. afr j lab med: 2014;3(2):262. https://doi.org/10.4102/ajlm.v3i2.262 south african national accreditation system. directory of accredited facilities [page on the internet]. c2015 [cited 2016 aug 22]. available from: http://home.sanas.co.za/?page_id=38 instituto português de acreditação (ipac). directório de entidades acreditadas [page on the internet]. n.d. [cited 2016 jan 19]. available from: http://www.ipac.pt/pesquisa/acredita.asp ridderhof jc, van deun a, kam km, et al. roles of laboratories and laboratory systems in effective tuberculosis programmes. bull world health organ. 2007;85(5):354–359. https://doi.org/10.2471/06.039081 raizada n, sachdeva ks, chauhan ds, et al. a multi-site validation in india of the line probe assay for the rapid diagnosis of multi-drug resistant tuberculosis directly from sputum specimens. plos one. 2014;9(2):e88626. https://doi.org/10.1371/journal.pone.0088626 raizada n, sachdeva ks, sreenivas a, et al. feasibility of decentralised deployment of xpert mtb/rif test at lower level of health system in india. plos one. 2014;9(2):e89301. https://doi.org/10.1371/journal.pone.0089301 albert h, bwanga f, mukkada s, et al. rapid screening of mdr-tb using molecular line probe assay is feasible in uganda. bmc infect dis. 2010;10:41. https://doi.org/10.1186/1471-2334-10-41 albert h, manabe y, lukyamuzi g, et al. performance of three led-based fluorescence microscopy systems for detection of tuberculosis in uganda. plos one. 2010;5(12):e15206. https://doi.org/10.1371/journal.pone.0015206 paramasivan cn, lee e, kao k, et al. experience establishing tuberculosis laboratory capacity in a developing country setting. int j tuberc lung dis. 2010;14(1):59–64. global laboratory initiative. gli stepwise process towards tb laboratory accreditation [page on the internet]. n.d. [cited 2016 aug 22]. available from: http://www.gliquality.org foundation for innovative new diagnostics. tb slmta harmonized checklist. [cited 2017 jan 19]. available from: http://www.finddx.org/wp-content/uploads/2016/07/new-tb-harmonized-checklist-v2.1-2-2016.pdf african society for laboratory medicine. who afro slipta checklist (2007). [cited 2017 jan 19]. available from: http://www.afro.who.int/en/downloads/cat_view/1501-english/787-blood-safety.html maruta t, albert h, hove p, et al. harmonizing quality improvement of tb laboratories with generic accreditation initiatives. conference proceedings: aslm conference, cape town, south africa 2012. [cited 2017 jan 19]. available from: http://citeweb.info/20121448643 albert h. strengthening laboratory management toward accreditation programme: transforming the lab landscape in developing countries and customisation for labs. conference proceedings: 45th union world conference on lung health, barcelona, spain, 2014 [link on the internet]. c2014 [cited 2016 august 22]. available from: http://html5.slideonline.eu/event/14union maruta t, yao k, ndlovu n, et al. training-of-trainers: a strategy to build country capacity for slmta expansion and sustainability. afr j lab med. 2014;3(2):196. https://doi.org/10.4102/ajlm.v3i2.196 world health organization. laboratory quality management system: handbook [document on the internet]. c2011 [cited 2016 august 22]. available from: http://www.who.int/ihr/publications/lqms/en/ foundation for innovative new diagnostics. find online training [homepage on the internet]. n.d. [cited 2016 august 22]. available from: http://finddiagnostics-training.org/moodle/ albert h, iragena j, kao k, et al. implementation of quality management systems and progress towards accreditation of national tuberculosis reference laboratories in africa. in press. maruta t, motebang d, mathabo l, et al. impact of mentorship on who-afro strengthening laboratory quality improvement process towards accreditation (slipta). afr j lab med. 2012;1(1):8 pages. https://doi.org/10.4102/ajlm.v1i1.6 nzombe p, luman et, shumba e, et al. maximising mentorship: variations in laboratory mentorship models implemented in zimbabwe. afr j lab med. 2014;3(2):8 pages. https://doi.org/10.4102/ajlm.v3i2.241 makokha ep, mwalili s, basiye fl, et al. using standard and institutional mentorship models to implement slmta in kenya. afr j lab med. 2014;3(2):8 pages. https://doi.org/10.4102/ajlm.v3i2.220 maruta t, rotz p, peter t. setting up a structured laboratory mentoring programme. afr j lab med. 2013;2(1):7 pages. https://doi.org/10.4102/ajlm.v2i1.77 ndihokubwayo j-b, maruta t, ndlovu n, et al. implementation of the world health organization regional office for africa stepwise laboratory quality improvement process towards accreditation. afr j lab med. 2016;5(1):a280. https://doi.org/10.4102/ajlm.v5i1.280 shumba e, nzombe p, mbinda a, et al. weighing the costs: implementing the slmta programme in zimbabwe using internal versus external facilitators. afr j lab med. 2014;3(2):6 pages. https://doi.org/10.4102/ajlm.v3i2.248 abstract introduction ethical considerations case presentation management and outcomes discussion acknowledgements references about the author(s) mpho l. sikhosana south african field epidemiology training programme, national institute for communicable diseases, division of national health laboratory services, johannesburg, south africa asma salloo department of critical care, chris hani baragwanath academic hospital, soweto, south africa department of critical care, university of witwatersrand, johannesburg, south africa monica birkhead centre for emerging zoonotic and parasitic diseases, national institute for communicable diseases, division of national health laboratory services, johannesburg, south africa kerrigan mccarthy division for public health surveillance and response, national institute for communicable diseases, division of national health laboratory services, johannesburg, south africa citation sikhosana ml, salloo a, birkhead m, mccarthy k. atypical presentation of herpes simplex virus type 1 infection in paediatric burns patients in a large tertiary hospital, south africa. afr j lab med. 2019;8(1), a916. https://doi.org/10.4102/ajlm.v8i1.916 case study atypical presentation of herpes simplex virus type 1 infection in paediatric burns patients in a large tertiary hospital, south africa mpho l. sikhosana, asma salloo, monica birkhead, kerrigan mccarthy received: 03 oct. 2018; accepted: 03 sept. 2019; published: 23 oct. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract introduction: herpes simplex virus has been reported in the literature to commonly complicate burn wounds. however, there is paucity of such data in the south african setting. case presentation: eight paediatric burns patients with ages ranging between 10 months and 5 years presented with a febrile maculopapular rash illness in a paediatric ward of a large south african tertiary hospital. the rash became vesicular in three cases, involving the limbs and face. varicella was suspected. management and outcome: medical records of suspected cases were reviewed. blood, vesicular fluid and scab samples were collected. electron microscopy of vesicular fluid revealed herpes virus particles. laboratory testing confirmed herpes simplex virus type 1. conclusion: herpes simplex virus type 1 infection can present atypically in burns patients. keywords: herpes simplex virus type 1; burns; paediatrics; south africa. introduction prevention of infection is an important part of managing burns patients. prophylactic administration of antibiotics or antivirals is not routine, with treatment warranted only in patients in whom infection is highly suspected or proven by laboratory testing.1,2,3,4 differences in the clinical presentation of skin infections in burns patients are not always apparent; however, infectious and non-infectious causes must be included in the differential diagnosis.1,5 viral infections by members of the herpesviridae family, including herpes simplex virus type 1 (hsv1), cytomegalovirus and varicella-zoster virus, have been found to occur commonly in severely burnt patients.4,5,6 these infections can either be primary or due to reactivation of a latent virus. over 3700 million people between 0 and 49 years have been estimated to be latently infected with hsv1, with africa being one of the most affected regions globally.7 as such, of all the herpesviruses, hsv1 is the most frequently reported virus that complicates burns, whereas varicella-zoster virus infections occur rarely.4 herpes simplex virus type 1 infections present as a febrile illness 1 to 3 weeks following extensive, full-thickness burns injuries.5,6 the infection frequently occurs in patients with burns to the head or neck. when associated with burn wounds, the lesions typically begin as clustered vesicles or vesicular pustules within or around the wound margins, with subsequent impaired wound healing.5,6 herpes simplex virus type 1 lesions can resemble those of pox viruses, with the latter also having been identified in burns patients.8,9 cytomegalovirus infections have not been shown to cause severe complications or increase mortality in burns patients.6 however, the presence of both primary and reactivation cytomegalovirus infections in severely burned children has been recorded previously.6 underlying herpes viral infections can promote bacterial infections, resulting in prolonged hospitalisation, need for mechanical ventilation, delayed recovery and higher mortality rates.1,4,10,11 herpes viral infections in burns patients have not been described in the south african setting. however, due to the contagious nature of these infections, there are implications for infection prevention and control practices, particularly in the sub-population of immunosuppressed burns patients. in addition, possible complications such as hsv1-associated encephalitis make knowledge on the management of these infections important. ethical considerations due to the initial presentation as a febrile maculopapular rash illness, the cluster was initially investigated as a possible measles outbreak. all outbreak investigations, which would include history taking (in this case, from the parents in light of the patients’ ages), patient examination as well as sample collection, that are conducted by the national institute for communicable diseases have ethics clearance from the human research ethics committee of the university of the witwatersrand, south africa (m160667, 2016–2020). in terms of this ethics clearance, patient consent is not required and any patient specimen collected is anonymised case presentation during july 2017, seven paediatric burns patients between the ages of 10 months and 5 years presented with a maculopapular rash at a tertiary hospital in gauteng, south africa. four of these patients were female. the rash was associated with both fever and coryza in four of the cases. the characteristics of the cases involved in this cluster are shown in table 1. the cluster was reported to the national institute for communicable diseases. due to a concurrent measles outbreak in the province, measles was initially suspected. the rash subsequently evolved and became vesicular in two of the cases, affecting the limbs and hands in one of the cases (figure 1). contemporaneously, an eighth patient presented with a vesicular rash on the trunk and on both upper and lower extremities bilaterally. of note is that this patient did not initially present with a maculopapular rash. varicella-zoster became a differential diagnosis. as children are not routinely immunised against varicella in south africa’s public health sector, the cost, availability and resource utilisation of prophylactic varicella immunoglobulins for the cases posed a number of challenges.6 the natural history in the cases that developed the vesicular lesions was also atypical of the varicella-zoster infection. there was therefore a possibility of administering the immunoglobulins unnecessarily. other possible diagnoses that were considered included enterovirus and pox infections. an investigation was initiated by the national institute for communicable diseases in order to establish the cause of the illness. a composite case definition that was used to identify other cases in the ward included any patient who was admitted to the unit during july 2017, presenting with a maculopapular or vesicular rash, with or without fever or coryza. the medical records of suspected cases were reviewed using a pre-designed case investigation form. cases were also examined for any residual or new symptoms. further information on the history of the illness was also obtained from the caregivers of some of the cases. blood samples were collected for serological and molecular testing using the following assays: siemens enzygnost® measles igm and rubella igm eia (siemens, marburg, germany), liaison® vzv igm clia (diasorin, saluggia, italy), herpeselect® hsv-1 eia (focus diagnostics, cypress, california, united states), genesig® herpes simplex virus type 1 and 2 real-time polymerase chain reaction kit (primerdesign ltd, southampton, hampshire, united kingdom). vesicular fluid and scab samples were collected and submitted to the national institute for communicable diseases for transmission electron microscopy using negative staining.9 figure 1: vesicular lesions on two of the cases, gauteng, south africa, july 2017. (a, b) vesicular lesions seen on one of the cases that initially presented with a maculopapular rash. (c, d) vesicular lesions on the third case. this patient was not part of the initial cluster with a maculopapular rash. table 1: characteristics of the cases in the febrile rash illness at the tertiary hospital in gauteng, south africa, july 2017. measles, rubella and varicella infections were excluded based on negative serological testing. viral particles consistent with those of the herpesviridae family were seen on electron microscopy of the vesicular fluid, while no virions were detected in the scab specimen (figure 2). based on these electron microscopy findings, further laboratory testing for herpes viruses was conducted, and hsv1 was confirmed based on serological and molecular testing. the medical record review of other patients in the ward identified two other cases with a documented vesicular rash. a timeline showing the sequence of events in this investigation is shown in figure 3. figure 2: transmission electron microscopy of negatively-stained virions in vesicular fluid, gauteng, south africa, july 2017. (a) and (b) show typical herpesviridae particles with an icosahedral nucleocapsid (2) surrounded by a loose envelope (1). during collection and processing of vesicular fluids, the fragile envelope frequently ruptures to release the nucleocapsid, which can be seen to be composed of characteristic cylindrical capsomers (inset b). figure 3: sequence of events during the investigation, gauteng, south africa, july 2017. management and outcomes due to the initial differential diagnosis of measles, cases were given vitamin a, and measles vaccine was administered to all ward contacts below the age of 5 years. all of the cases recovered completely without any complications. discussion the presentation of hsv1 infection in this cluster was atypical for several reasons. firstly, hsv infections are usually associated with more extensive burns.4,5,6 all of the cases in this cluster had partial thickness burn wounds that were ≤ 20% of the total body surface area. secondly, unlike the commonly described distribution of the vesicular rash within or around the wound margins, the lesions occurred peripherally from the burn wounds in all except one of the cases. the vesicular rash was also distributed on the limbs and torso in some of the cases, with only one case having orolabial involvement. the fact that no virions were observed in the scab using transmission electron microscopy is in keeping with the presentation of herpes viruses. this finding also aided in the exclusion of pox viruses, as the latter are typically found in the crusted lesions. pox viruses are also more robust and long-lived compared to herpes viruses which have more delicate and pleomorphic envelopes.9 the incidence of hsv1 infections have been estimated to occur more commonly in the 0–4 year age group in both boys and girls in africa.7 due to the spatial and temporal association of the cases, primary nosocomial infection is the likely mode of transmission. however, due to a high hsv1 seroprevalence in our study setting, the reactivation of latent hsv1 is also a likely possibility. hsv is transmitted through contact with vesicular lesions, and this was the most likely mode of transmission in this cluster. this highlights the importance of adhering to standard precautions, particularly in burns patients in whom the protective skin barrier has been compromised. we postulate that the communal dressing room, where most of the cases had their wounds dressed prior to the onset of the illness, was most likely where transmission occurred. limitations limitations of this outbreak investigation included delayed identification of two probable cases found on the medical records review. as their vesicular lesions had already healed, appropriate samples were not available for laboratory confirmation of hsv1. in addition, comparative sequencing in the three cases with laboratory-confirmed hsv1 could not be conducted due to unavailability of residual specimens. conclusion although hsv1 infection in burns patients has been described in other international settings, a literature review showed that the illness has not been well described in the south african setting. treatment of laboratory-confirmed hsv infection includes intravenous acyclovir at 5–10 mg/kg 8 hourly for 10–14 days, with or without the addition of topical acyclovir.4,5,6 this management was not prescribed for the cases in this cluster, but there were subsequently no complications in any of the patients. prevention of the nosocomial spread of hsv1 includes isolating infected patients and avoiding the use of communal facilities by cases until the vesicular lesions have healed. surgery to infected wounds should also be deferred until vesicles have resolved. acknowledgements we would like to thank the laboratory staff of the national health laboratory service and lancet laboratories for providing the serological and molecular testing. competing interests the authors have none to declare. authors’ contributions all authors contributed to the intellectual content of the article as well as in drafting and reviewing the manuscript. m.l.s. and k.m. conceptualised the project. m.l.s. performed the field epidemiology. m.b. performed the electron microscopy. all authors (m.l.s., a.s., m.b., k.m.) made contributions to the acquisition, analysis and interpretation of data for the work, and contributed to the initial draft of the article. m.l.s. and m.b. prepared the final manuscript that was reviewed by all authors. sources of support this investigation was funded by the national institute for communicable diseases, division of the national health laboratory service. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references church d, elsayed s, reid o, winston b, lindsay r. burn wound infections. clin microbiol rev. 2006;19(2):403–434. https://doi.org/10.1128/cmr.19.2.403-434.2006 legrand m, guttormsen ab, berger mm. ten tips for managing critically ill burn patients: follow the rastafari! intensive care med. 2015;41(6):1107–1109. https://doi.org/10.1007/s00134-014-3627-7 lundy jb, chung kk, pamplin jc, ainsworth cr, jeng jc, friedman bc. update on severe burn management for the intensivist. j intensive care med. 2016;31(8):499–510. https://doi.org/10.1177/0885066615592346 wurzer p, cole mr, clayton rp, et al. herpesviridae infections in severely burned children. burns. 2017;43(5):987–992. https://doi.org/10.1016/j.burns.2017.01.032 haik j, weissman o, stavrou d, et al. is prophylactic acyclovir treatment warranted for prevention of herpes simplex virus infections in facial burns? a review of the literature. j burn care res. 2011;32(3):358–362. https://doi.org/10.1097/bcr.0b013e318217f6de wurzer p, guillory a, parvizi d, et al. human herpes viruses in burns patients: a systematic review. burns. 2017;43(1):25–33. https://doi.org/10.1016/j.burns.2016.02.003 looker kj, magaret as, may mt, et al. global and regional estimates of prevalent and incident herpes simplex virus type 1 infections in 2012. plos one. 2015;10(10):e0140765. https://doi.org/10.1371/journal.pone.0140765 hsu ch, rokini gr, aghazadeh n, et al. unique presentation of orf virus infection in a thermal-burn patient after receiving an autologous skin graft. j infect dis. 2016;214(8):1171–1174. https://doi.org/10.1093/infdis/jiw307 gelderblom hr, madeley d. rapid viral diagnosis of orthopoxviruses by electron microscopy: optional or a must? viruses. 2018;10(4):142. https://doi.org/10.3390/v10040142 jugmohan b, loveland j, doedens l, moore rl, welthagen a, westgarth-taylor cj. mortality in paediatric burns victims: a retrospective review from 2009 to 2012 in a single centre. s afr med j. 2016;106(2):189–192. https://doi.org/10.7196/samj.2016.v106i2.8942 öncül o, öksüz s, acar a, et al. nosocomial infection characteristics in a burn intensive care unit: analysis of an eleven-year active surveillance. burns. 2014;40(5):835–841. https://doi.org/10.1016/j.burns.2013.11.003 abstract introduction methods results discussion acknowledgements references about the author(s) babafela b. awosile texas tech university school of veterinary medicine, amarillo, texas, united states michael agbaje department of veterinary microbiology, college of veterinary medicine, federal university of agriculture, abeokuta, nigeria oluwawemimo adebowale department of veterinary public health and preventive medicine, college of veterinary medicine, federal university of agriculture, abeokuta, nigeria olugbenga kehinde department of veterinary public health and preventive medicine, college of veterinary medicine, federal university of agriculture, abeokuta, nigeria ezekiel omoshaba department of veterinary microbiology, college of veterinary medicine, federal university of agriculture, abeokuta, nigeria citation awosile bb, agbaje m, adebowale o, kehinde o, omoshaba e. beta-lactamase resistance genes in enterobacteriaceae from nigeria. afr j lab med. 2022;11(1), a1371. https://doi.org/10.4102/ajlm.v11i1.1371 review article beta-lactamase resistance genes in enterobacteriaceae from nigeria babafela b. awosile, michael agbaje, oluwawemimo adebowale, olugbenga kehinde, ezekiel omoshaba received: 26 aug. 2020; accepted: 25 aug. 2021; published: 22 feb. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: beta-lactamase genes are one of the most important groups of antimicrobial resistance genes in human and animal health. therefore, continuous surveillance of this group of resistance genes is needed for a better understanding of the local epidemiology within a country and global dissemination. aim: this review was carried out to identify different beta-lactamase resistance genes reported in published literature from nigeria. methods: systematic review and meta-analysis was carried out on eligible nigerian articles retrieved from electronic literature searches of pubmed®, african journals online, and google scholar published between january 1990 and december 2019. the preferred reporting items for systematic reviews and meta-analyses method was adopted to facilitate clarity and transparency in reporting review findings. results: fifty-seven articles were included. all beta-lactamases reported were detected from gram-negative bacteria, particularly from enterobacteriaceae. thirty-six different beta-lactamase genes were reported in nigeria. these genes belong to the narrow-spectrum, ampc, extended-spectrum and carbapenemase beta-lactamase resistance genes. the pooled proportion estimate of extended-spectrum beta-lactamase genes in nigeria was 31% (95% confidence interval [ci]: 26% – 36%, p < 0.0001), while the estimate of the blactx-m-15 gene in nigeria was 46% (95% ci: 36% – 57%, p < 0.0001). the proportion estimate of ampc genes was 32% (95% ci: 11% – 52%, p < 0.001), while the estimate for carbapenemases was 8% (95% ci: 5% – 12%, p < 0.001). conclusion: this study provides information on beta-lactamase distribution in nigeria. this is necessary for a better understanding of molecular epidemiology of clinically important beta-lactamases, especially the extended-spectrum beta-lactamases and carbapenemases in nigeria. keywords: antimicrobial resistance; beta-lactamase gene; nigeria; review; epidemiology. introduction beta-lactam antimicrobials are one of the most important groups of antimicrobial drugs used in both human and animal health. antimicrobials such as extended-spectrum cephalosporins and carbapenems have been categorised by the world health organization as ‘last resort’ and ‘critically important antimicrobials’ because antimicrobial alternatives for treating last resort antimicrobial resistant bacteria is limited.1 however, resistance to last resort antimicrobials is occurring rapidly on a global scale.2 most resistance to beta-lactams in enterobacteriaceae is mainly due to the production of beta-lactamases, which are often encoded either on the chromosome or the plasmid.3,4 the production of beta-lactamases such as extended-spectrum beta-lactamases (esbls), ampc beta-lactamases, and carbapenemase beta-lactamases have increasingly been detected globally in food and companion animals, wildlife, humans, and the environment.4,5 the dissemination of several beta-lactamase genes across different resistant bacterial populations from different hosts and environments illustrates that antimicrobial resistance (amr) is a one health challenge.6 beta-lactamase production in enterobacteriaceae is a public health concern due to the possibility of therapeutic failure, serious consequences for infection control and increased risk of morbidity and mortality in animals and humans.7 the predominant esbl genes encountered are blactx-m, blatem, and blashv. the prevalent ampc beta-lactamase is blacmy-2, while for carbapenemases, blaoxa-48 and blandm-1 have been reported globally.5 although beta-lactamase genes are globally disseminated, they are not equally prevalent among human and animal bacteria. also, the occurrence and prevalence of these resistance genes varies across different geographic regions. for instance, while blactx-m-15 is widely disseminated and has been reported in almost every region of the world, ampc blacmy-2 has been mostly encountered in north america in both animal and human hosts.8 therefore, there is a need for continuous surveillance of beta-lactamase resistance genes to better understand the local and global epidemiology of these genes. while detailed national amr information exists for high-income european and north american countries through integrative surveillance, this is often lacking in most lowand middle-income african countries including nigeria. nigeria’s amr surveillance is in its infancy; thus, the generation of amr data through the systematic review of published literature is still a useful tool that can give a glimpse of the amr situation in nigeria. this systematic review was carried out to identify the different beta-lactamase resistance genes reported in published nigerian literature, to describe the distribution of these genes between animal, human and environmental settings, and to estimate the proportion of the different beta-lactamase resistance genes in nigeria. this systematic review was conducted per the preferred reporting items for systematic reviews and meta-analyses checklist.9 methods literature search and data sources the literature search was conducted in pubmed®, google scholar and african journals online (ajol) electronic databases using a combination of boolean operators (and, or) and predefined keywords. we used the following terms for our search: beta-lactamases and nigeria or beta-lactamase resistance genes and nigeria, blactx-m and nigeria, blatem and nigeria, blashv and nigeria, blaoxa and nigeria, carbapenemases and nigeria, ampc beta-lactamase resistance and nigeria, and esbl resistance and nigeria. the search was limited to publications between january 1990 to december 2019. the primary aim of the review was to determine the distribution and different types of beta-lactamase genes circulating in nigeria across the health sectors. title screenings of the articles were done using the following eligibility criteria: (1) the study location must be nigeria and (2) the study must have reported the detection of any type of beta-lactamase genes. afterwards, an abstract review was done to determine the relevance of each article to the review’s objectives and purpose. for an article to be included it must report the phenotypic antimicrobial susceptibility testing method used and the molecular techniques used to detect beta-lactamase resistance genes. furthermore, a supplementary literature search was done by reviewing the references of eligible articles. studies that reported beta-lactamase production based on phenotypic synergy test without molecular beta-lactamase gene detection were excluded from the review. included articles were then sorted into qualitative and quantitative categories. all articles that reported the molecular detection of at least one beta-lactamase resistance gene were included in the qualitative, while studies with extractable data on the proportions of different types of beta-lactamase resistance genes from animals, humans and the environment were further considered for quantitative meta-analysis. data extraction and analysis the data were extracted into a microsoft office excel 2010 spreadsheet (microsoft corporation, redmond, washington, united states). for each eligible study, data extracted included: first author’s details, publication year, sample type (animal faeces, retail meat products, human clinical samples, environmental samples), sample source (animal, human or environment) and study location or geopolitical zone. also, the number and type of bacteria isolated, the beta-lactamase gene detected, the number of isolates phenotypically positive for the beta-lactamase production and the number of bacterial isolates genotypically positive for beta-lactamase genes were recorded. the antimicrobial susceptibility testing method (disc diffusion, broth microdilution, agar dilution, e-test or automated methods) and the beta-lactamase genotyping and phenotyping methods were noted. the proportion (with 95% confidence interval [ci]) of each beta-lactamase gene as reported for each study was calculated by dividing the number of bacteria positive for the beta-lactamase gene by the total number of bacteria positive phenotypically or the total number of bacteria isolated depending on the data available. random effects meta-analysis was used to calculate the pooled (weighted) proportions for the different types of the beta-lactamase groups with 95% cis. the analysis was done to allow for any heterogeneity between studies. studies reporting a low number of bacterial isolates (< 10 isolates) were not included in the meta-analysis. the pooled prevalence and each study’s estimates were presented using forest plot. the i2 statistic (a measure of inconsistency) was used to assess the variation between studies due to heterogeneity. a value of 0% means there was no observed heterogeneity; increasing values indicate increasing heterogeneity. the i2 statistic with values 25% or less were subjectively considered as low, 26% – 50% as moderate, and higher than 50% as substantial heterogeneity. subgroup analysis was performed to account for potential sources of heterogeneity between studies. a separate meta-analysis was carried out for each of the dominant beta-lactamase groups. statistical significance (p) was set at 0.05 while statistical analysis was carried out using stata se version 15.0 (college station, texas, united states). ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. results study characteristics systematic search from three electronic databases identified 567 articles (197 from pubmed®, 30 from ajol and 340 from google scholar) (figure 1). a total of 510 articles were excluded based on the selection criteria (figure 1). fifty-seven articles were included in the qualitative review, of which 11 were animal, 38 human and 8 environmental studies. for the animal studies, isolates were from poultry, pigs, cattle, pigeons and ducks. however, all the human studies were hospital-based, with the beta-lactamases reported from clinical samples collected within the hospitals in nigeria. for the environmental studies, samples were collected from the beach, river, wastewater, and sources of drinking water. figure 1: flow diagram summarising the process of literature search and selection. the majority of the studies were carried out in south west nigeria (n = 36), while the rest were carried out in the other regions including south east (n = 7), north central (n = 6), north east (n = 3), north west (n = 1) and south south (n = 2). sixteen (n = 16) studies used the broth microdilution method to determine the minimum inhibitory concentrations; other methods used for minimum inhibitory concentrations determination included e-test (n = 3) and vitek-2 (n = 2). however, the disc diffusion method (n = 36) was mainly used for the antimicrobial susceptibility of bacteria. forty-three of the 57 studies reported the use of phenotypic screening methods for beta-lactamase production; this included a modified hodge test and carba test (n = 6) for carbapenemases production as well as the double-disc synergy test (n = 37) for other beta-lactamase production. five different genotypic methods including polymerase chain reaction (pcr), gene sequencing, whole genome sequencing (wgs), isoelectric point and restriction fragment length polymorphism (rflp) were reported by the studies. forty-four studies used pcr alone (n = 44), eight studies used pcr and gene sequencing, two used pcr with an isoelectric point, one used pcr and whole-genome sequencing, one used pcr with rflp while only one study used whole-genome sequencing alone for the genotypic detection of various beta-lactamase resistance genes. among the 57 studies reviewed, the majority of beta-lactamases were detected in enterobacteriaceae (n = 54), followed by acinetobacter baumannii (n = 2) and vibrio spp (n = 1). one health distribution of beta-lactamase resistance genes in nigeria thirty-six different beta-lactamase genes were detected and reported in the 57 studies (table 1). seventeen genes were detected in animals, 28 in humans, and 12 in the environment. these genes belong to the ampc,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24 extended-spectrum,10,11,12,13,14,15,17,19,20,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56 narrow-spectrum,13,14,15,16,19,24,32,35,37,43,57,58,59 and carbapenemase beta-lactamase resistance genes.19,22,59,60,61,62,63,64,65,66 eight genes (blactx-m-1, blactx-m-14, bladha, blages-1, blaoxa-1, blaoxa-2, blatem-1 and blaveb-1) were found in animals and humans; five genes (blandm-1, blashv-1, blashv-2, blashv-11, and blashv-12) were common to both humans and the environment while none of the genes was unique to both animals and the environment (table 1). four genes, namely blaampc, blacmy, blatem-1 and the international pandemic blactx-m-15, were reportedly detected from animals, humans and the environment. no carbapenemase gene was reported in animals but seven beta-lactamase genes (blaacc, blaact-5, blacmy-2, blactx-m-27, blactx-m-55, blaecb, and blafox-1) were unique to animals alone, 12 (blactx-m-2, blakpc, blandm-5, blaoxa-10, blaoxa-23, blaoxa-48, blaoxa-181, blashv-28, blashv-112, blatem-2, blavim-1, and blavim-2) were unique to humans, while only two genes (blavim-5 and blaz) were unique to the environment. table 1: distribution of beta-lactamase resistance genes between animals, humans and environmental settings in nigeria (1900–2019). proportion estimates of extended-spectrum beta-lactamase genes in nigeria thirty-two studies were included in the meta-analysis for the generation of the overall pooled estimate of esbl. the overall pooled proportion of esbl was 31% (95% ci: 26% – 36%, p < 0.001). the overall between-study heterogeneity was significant and substantial (i2 = 97.87 %, p < 0.001). between the studies, the proportions of esbl genes range from 1% to 95% (figure 2). the overall proportion estimate of esbls in human-based studies was 35% (95% ci: 27% – 43%, p < 0.001), in animal studies was 25% (95% ci: 17% – 33%, p < 0.001) and in environmental-based studies was 22% (95% ci: 0% – 44%, p = 0.06). a separate meta-analysis was conducted to determine the proportion estimate of blactx-m-15 producing enterobacteriaceae in nigeria (figure 3); 17 studies were included in the quantitative analysis. the overall pooled proportion was 46% (95% ci: 36% – 57%), the unexplained between-study heterogeneity was significant and substantial (i2 = 99.04%, p < 0.001). the proportion estimate of blactx-m-15 gene from human-based studies was 47% (95% ci: 25% – 69%), for the animal studies it was 47% (95% ci: 27% – 67%) and for the environmental-based studies, it was 41% (95% ci: 33% – 50%). between subgroups, heterogeneity was non-significant (p = 0.812). however, the within-group heterogeneity for both human and animal studies was significant and substantial (i2 = 99%, p < 0.001). figure 2: subgroup analysis and forest plot of proportion estimates of extended-spectrum beta-lactamases for animal (a), human (b) and environmental settings (c) in nigeria (1990–2019). figure 3: subgroup analysis and forest plot of proportion estimates of blactx-m-15 extended-spectrum beta-lactamase in nigeria (1990–2019). proportion estimates of ampc and carbapenemase beta-lactamase genes in nigeria based on 13 studies, the overall pooled proportion of ampc beta-lactamases was 32% (95% ci: 11% – 52%, p < 0.001), with the overall between-study heterogeneity significant and substantial (i2 99.15 %, p < 0.001). however, the proportions of ampc beta-lactamases reported from the studies range from 2% to 88% (figure 4). the proportion of ampc beta-lactamases was higher in humans at 37% (95% ci: 4% – 70%, p = 0.03) than in the environment at 20% (95% ci: 14% – 25%, p < 0.001) and animals at 26% (95% ci: 0% – 64%, p = 0.20). figure 4: subgroup analysis and forest plot of proportion estimates of ampc beta-lactamases for human, animal and environmental settings in nigeria (1990–2019). six studies were included in the carbapenemase pooled proportion estimation (figure 5). the overall pooled proportion of carbapenemases was 8% (95% ci: 5% – 12%, p < 0.001). between the studies, the proportions of carbapenemase beta-lactamases ranged from 1% to 48%, while the overall between-study heterogeneity was significant (i2 = 87.6 %, p < 0.001). the proportion of carbapenemases for the environment was 15% (95% ci: 8% – 22%, p < 0.001) and was higher than that observed in human studies (6%; 95% ci: 3% – 10%, p < 0.001). between-study heterogeneity was mostly due to studies from human setting. figure 5: subgroup analysis and forest plot of proportion estimates of carbapenemase beta-lactamases for human and environmental settings in nigeria (1990–2019). discussion in africa, data on amr are often limited due to the lack of sustainable integrated national amr surveillance programmes. the lack of systematically collected data has impeded the proper understanding of resistance to critically important antimicrobials such as beta-lactam drugs in africa compared to the high-income countries. this study identified the different types, distribution and proportion estimates of beta-lactamase resistance genes in nigeria contributing to the national, continental and global molecular epidemiology of beta-lactamases. it provides data that can support ongoing efforts of integrative surveillance programmes and policies for the mitigation of amr within nigeria. from this review, beta-lactamases were mainly detected in the enterobacteriaceae bacteria family. this higher detection in enterobacteriaceae may be skewed because the studies included in this review targeted this bacterial family. also, bacteria of the enterobacteriaceae family are ubiquitous in nature, causing different infections (particularly escherichia coli) in both humans and animals and can readily be maintained in the environment. also, the emergence and occurrences of amr due to beta-lactamases in enterobacteriaceae are driven mainly by ease of acquisition of amr genes and the rapid dissemination of resistance determinants by enterobacteriaceae to other pathogenic and non-pathogenic bacteria.2,67 among the 57 studies, 75% reported a double-disc synergy test for the phenotypic detection of beta-lactamases before the genotypic method. this complies with the clinical laboratory standard institute guideline for the screening and detection of beta-lactamases in bacteria. the pcr method was mostly reported; this may be due to ease of access and reduced cost compared with other advanced techniques such as wgs.68 while the wgs method is commonly used in high income countries for research and surveillance programmes, the use of wgs in beta-lactamase studies in nigeria is still limited. only two studies reported the use of wgs for the detection of beta-lactamase genes. this may be due to the limited access to the necessary equipment, expertise, and bioinformatics skill in nigeria. the majority of the articles published were from southern nigeria and from human health studies. therefore, beta-lactamases reported in this systematic review may not reflect the true geographic distribution of beta-lactamases genes in nigeria. however, beta-lactamases were detected more in isolates from human settings, consistent with what has been reported from a similar south african review.68 in this review, 36 different types of beta-lactamases had been detected and reported in nigeria. these beta-lactamases included the clinically important types such as esbls, ampc, and carbapenemases that are commonly responsible for treatment failures in both human and veterinary settings.69,70 while previous reviews within africa focused mainly on systematic reviews of esbl in enterobacteriaceae,71,72,73 this study was conducted to capture as many beta-lactamases detected in nigeria as possible beyond esbls. this is because esbls, the ampc-type, and the carbapenemases remain the most clinically challenging beta-lactamase resistance gene family in both human and animal health. antibiotic resistance is recognised as a one health challenge because of the dissemination of important resistant bacteria and genes among humans, animals and the environment at a global scale.74 this review showed that of the 36 beta-lactamases reported in nigeria, some of the genes detected in nigeria were reported from more than one setting. between human and environmental sources, five different beta-lactamases were reported while between animal and human sources, eight different genes were reported. this finding further highlights a one health amr transmission. also, it reveals how the environment, including food-animal production systems, could serve as reservoirs of essential amr genes, driving transmission and colonisation as well as infection of clinically important beta-lactamase producing bacteria in humans.6 epidemiologically, all the beta-lactamases detected in nigeria have been reported from other parts of the world. five different types of the ampc beta-lactamase group (blaacc, blaact, blacmy-2, bladha, and blafox-1) were reported in nigeria to date based on this review. while blacmy-2 is the most important ampc-type and has the broadest geographic spread, based on this review this gene was not common in nigeria compared to other countries such as the united states and canada.8 within africa, there are limited reports on ampc beta-lactamases. however, blacmy-2 and bladha have been reported in algeria,71 while blaacc, bladha, and blafox-1 were reported in uganda.75 no carbapenemase gene was reported in animals in nigeria based on this review; however, carbapenemases have been commonly reported from wildlife, food-producing animals and companion animals from other countries.70,76 the lack of reports of carbapenemase from animal settings may be due to the lack of research or surveillance in this regard and not necessarily the absence of carbapenemase genes in animals from nigeria. all the epidemiologically important carbapenemases including blakpc, blandm-1, blaoxa-23, blaoxa-48, blaoxa-181, blavim-1, blavim-2 and blavim-5 reported in nigeria were mostly from the human setting. these carbapenemases have been reported in many african countries including south africa, gabon, angola, senegal, kenya, tanzania, morocco, algeria, tunisia, libya and egypt.77,78 in most cases, blandm-1 and blaoxa-48 are the commonly reported carbapenemases. these carbapenemases are known to be prevalent in south asian countries, particularly the indian sub-continent; the trend in africa may indicate that the global dissemination of carbapenemase-producing enterobacteriaceae has reached the african continent. among the esbls, five different groups were reported including blactx-m, blages, blaoxa, blashv and blaveb with blactx-m the most commonly reported. during the last decade, blactx-m–type enzymes have spread globally, becoming the most common esbl in enterobacteriaceae from both humans and animals.79,80 among the different types of blactx-m reported in nigeria, blactx-m-1, blactx-m-2, blactx-m-14 and blactx-m-55 are known to be commonly detected in food animals.4,8 however, the internationally disseminated blactx-m-15 has been associated with the e. coli serotype o25:h4 (st131), causing both community and hospital-acquired human infections.79,81 blactx-m-15 was also the only esbl commonly reported in human, animal and environmental settings in this review. this revealed that blactx-m-15 is ubiquitous and prevalent in all environments with possible anthropozoonotic and zooanthroponotic transmissions. blactx-m-15 has also been commonly reported from other regions of africa,71 which may suggest blactx-m-15 is a predominant esbl in africa similar to what has been reported in the united states, europe and asia.82 blashv esbls, in particular, blashv-12 and blashv-2, reported in nigeria have also been frequently detected in europe and north america.83 however, the globally disseminated blatem esbls, that is, blatem-10 and blatem-52, were not reported in nigeria; narrow-spectrum blatem-1 coding for ampicillin resistance was common. from the meta-analysis, subgroup analyses provide some explanation for the between-study heterogeneity and also the pooled proportion of esbls based on the one health distribution. the proportion estimate of esbls in nigeria was 32.00% compared to 2.03% of ampc and 8.00% of carbapenemases. this is unsurprising: even though ampc has been found worldwide and carbapenemases are increasingly being reported, both ampc and carbapenemases are less prevalent than esbl globally,5 and this is consistent with the finding of this review. the proportion estimate of 29.00% for ampc in the human setting in this review is slightly higher than the 28.30% estimate from egypt,84 but lower than 34.00% from canada85 and 39.60% from uganda.75 the proportion estimate of 8.00% for carbapenemases is comparable to what has been reported from other african countries.77 the lower proportion of carbapenemases reported in nigeria is encouraging considering the importance of carbapenems as last resort antimicrobials for treating cases of esbl-producing bacterial infections. while carbapenems resistance is emerging globally at a rapid rate, surveillance and prudent use practices of carbapenems will monitor and minimise multidrug resistant bacterial infections at the national level. the proportion estimate of 32.00% for esbl in human, animal and environmental settings from this review is higher than the 22.60% reported by a similar study from tanzania.73 the proportion estimate of 35.00% for esbls in the human setting is comparable to proportions previously reported for different countries within africa.72,71 for blactx-m, the proportion estimate of 34.00% for the blactx-m gene in nigeria was lower compared to 56.70% reported in iran86 and 69.00% from a previous similar systematic review.87 however, the proportion estimate of 45.00% for blactx-m-15 in nigeria was lower than 78.00% reported from both tanzania73 and sudan.88 none of the articles reviewed reported any risk factors associated with the occurrence of beta-lactamase resistance genes in nigeria; however, the occurrences and proportion estimates of clinically significant beta-lactamases reported maybe due to the uncontrolled and indiscriminate use of antimicrobials as well as the lack of active infection control programmes in most animal and human settings. in nigeria, animal and human antimicrobials can readily be purchased from both pharmaceutical and non-pharmaceutical stores without prescriptions.89 this is a cause for concern because indiscriminate use of antimicrobials drives resistance; also, poor hygienic practices in both community and hospital environments facilitates the spread and transmission of important multidrug resistant bacteria. in addition, extended-spectrum cephalosporins and carbapenems have been designated as critically important antimicrobials by the world health organization with limited alternatives in the case of treatment failure.1 lastly, infections with bacteria producing esbl or ampc or carbapenemase may result in prolonged hospitalisation, higher treatment costs, delays in the initiation of timely and adequate antimicrobial therapy, and increased risk of morbidity and mortality.7 therefore, resistance to clinically important beta-lactamases is a significant threat to public health and collaborative efforts. limitations this review is not without limitations. the literature search was limited to the pubmed®, google scholar and ajol electronic databases; therefore, some studies may have been omitted in this review. also, information on risk factors associated with beta-lactamase resistance was not available. this information is necessary for better explanation of the beta-lactam resistance observed in nigeria and a better understanding of the epidemiology of beta-lactamase resistance genes in nigeria. conclusion this review has provided information on the beta-lactamases distribution in nigeria. thirty-six different beta-lactamases have been reported in nigeria with blactx-m-15 commonly distributed in animals, humans and the environment consistent with the reports from other african countries. carbapenemases are most common in human settings and have not been reported in animals yet. the information provided on beta-lactamase resistance genes is necessary for better understanding of the national and global molecular epidemiology of clinically important beta-lactamase genes, especially ampc, esbls and carbapenemases. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions b.b.a., m.a., o.a., o.k. and e.o. conceptualised the research and equally carried out the research and the writing of the 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https://doi.org/10.4102/ojvr.v82i1.816 abstract introduction methods results discussion acknowledgements references about the author(s) samson e. olerimi department of medical biochemistry, faculty of basic medical sciences, ambrose alli university, ekpoma, nigeria school of biomedical sciences, nottingham trent university, nottingham, united kingdom ehitare i. ekhoye department of physiology, faculty of basic medical sciences, edo state university, uzairue, nigeria oriasotie s. enaiho department of medical biochemistry, faculty of basic medical sciences, ambrose alli university, ekpoma, nigeria school of biomedical sciences, nottingham trent university, nottingham, united kingdom alexander olerimi department of mathematics, faculty of natural sciences, ambrose alli university, ekpoma, nigeria citation olerimi se, ekhoye ei, enaiho os, olerimi a. selected micronutrient status of school-aged children at risk of schistosoma haematobium infection in suburban communities of nigeria. afr j lab med. 2023;12(1), a2034. https://doi.org/10.4102/ajlm.v12i1.2034 original research selected micronutrient status of school-aged children at risk of schistosoma haematobium infection in suburban communities of nigeria samson e. olerimi, ehitare i. ekhoye, oriasotie s. enaiho, alexander olerimi received: 22 july 2022; accepted: 08 mar. 2023; published: 31 may 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the parasite schistosoma haematobium causes urogenital schistosomiasis, a chronic infectious disease that occurs mainly among school-age children. objective: the prevalence of s. haematobium infection and level of intensity relative to age, gender and status of selected serum micronutrients among school-age children were investigated in suburban communities in bekwarra, nigeria. methods: this cross-sectional school-based study randomly recruited 353 children aged between 4 and 16 years from five elementary schools between june 2019 and december 2019. we gathered socio-demographic data about each child using a semi-structured questionnaire. blood samples were collected for micronutrient analysis and urine samples were collected for assessment of s. haematobium infection. results: a total of 57 (16.15%) school-age children were infected with s. haematobium. girls (n = 34; 9.63%) were more frequently infected than boys (n = 23; 6.52%). infection was most frequent among children aged 8–11 years (n = 32; 23.19%) and was significantly associated with age (p = 0.022) and gender (p < 0.001). serum levels of iron, calcium, copper and zinc among infected children were significantly lower than those of non-infected children. intensity of infection was negatively associated with iron (r = −0.21), calcium (r = −0.24), copper (r = −0.61; p < 0.001) and zinc (r = −0.41; p < 0.002). conclusion: this study showed that s. haematobium infection adversely impacted the micronutrient status of school-age children in suburban nigeria. measures to lower the prevalence of schistosomiasis among school-age children, including efficient drug distribution, education campaigns and community engagement, are necessary. what this study adds: this research emphasises the significance of implementing infection prevention and control interventions to mitigate the transmission and prevalence of schistosomiasis among school age children. keywords: micronutrients; schistosoma haematobium; schistosomiasis; school-age children; zinc. introduction micronutrients are essential in health and development. the spread of helminth infestations and micronutrient deficiencies overlaps in tropical and subtropical regions.1,2 schistosomiasis, also known as bilharzia, is a neglected tropical disease caused by trematodes classified in the genus schistosoma.3 it manifests as intestinal and/or urinary disease in the gastrointestinal or genitourinary tract.4 global estimates in 2019 suggest that schistosomes infect 240 million people, while about 700 million persons are at risk annually.5 most countries have successfully reduced schistosomiasis spread and infection rates; however, a large burden of infection persists in africa, especially in tropical regions.6,7 nigeria has one of the highest prevalences of schistosomiasis in sub-saharan africa,8,9 with over 101 million people at risk and an estimated 29 million people infected, of which 16 million are children.10 schistosoma haematobium, the most extensively distributed schistosoma species in nigeria, is found mainly in the country’s southern regions.11 schistosomiasis is associated with poverty, lack of or insufficient clean water, poor sanitation, and hygiene.12 it has been extensively documented in nigeria that schistosomiasis mortality and morbidity are highest among children of school-age.13,14 schistosomiasis is most prevalent among school-age children.15 it is transmitted by the bulinus snail, which is the intermediate host for s. haematobium.16 the disease spread among school-age children aged 10–15 years may primarily be attributable to frequent contact with organism-contaminated water in endemic areas.17 parasite infection negatively impacts the nutritional status of children by inducing loss of appetite and increasing nutrient waste owing to blood loss, vomiting, and diarrhoea.18 micronutrients, such as copper, zinc, iron, and calcium, are essential for the maintenance of life. micronutrient deficiencies of iron, copper, manganese and zinc exacerbate schistosoma spp. infections and other parasitic ailments associated with haemorrhage.19 these micronutrients have numerous roles in the complex enzyme systems that perform various biological processes. the trace element zinc has catalytic, structural and regulatory functions,20 and its deficiency is responsible for 20% of all child deaths worldwide.21 symptoms of zinc deficiency include chronic diarrhoea, coeliac disease, inflammatory bowel disease, ileostomy, alcoholic cirrhosis, and haemolytic anaemia.22 copper protects cells from free-radical damage by acting as an antioxidant adjuvant.23 copper deficiency is uncommon; however, it has been reported in preterm neonates, cowmilk-fed infants, and infants recovering from diarrhoea-caused malnutrition.24 copper deficiency results in anaemia, neutropenia, growth retardation, altered glucose and lipid metabolism, and higher infection rates.25 iron is involved in the formation of haemoglobin, myoglobin and catalase.26 its deficiency is believed to affect around a quarter of the world’s population. infants aged 4–24 months, school-age children, women and girls, adolescents, and pregnant and breastfeeding mothers are the most affected.27 calcium is essential for many biological activities, such as bone and tooth structure, signal transmission, muscle contraction, enzyme regulation, and blood coagulation.28 there is evidence that appropriate calcium intake is vital during childhood and adolescence, and that adequate calcium during these life stages is crucial.29 calcium depletion is associated with rickets and osteomalacia, pre-eclampsia, osteoporosis, and preterm delivery, particularly in developing countries.30 schistosomiasis is a serious disease that mostly affects children, especially in impoverished communities with limited health care and a lack of access to safe drinking water. malnutrition promotes susceptibility to infection due to lower appetite and increased nutritional requirements, resulting in a vicious cycle that can have adverse effects on the nutrition status of children and adolescents. the effects of s. haematobium on school-age children have been established in sub-saharan africa and some regions of nigeria. data on the impact of schistosoma spp. on the micronutrient levels of school children in bekwarra are sparse. the current estimate of the population of bekwarra local government area (lga) is 134 108 as of 2017, using the 2017 annual growth rate of 2.4% in the cross river state. the bekwarra lga is a typical rural area with a tropical climate, characterised by a hot, dry season (december–february) and a cooler, wet season (april–november). most communities in bekwarra lga are rural settlements that are encircled by a multitude of freshwater ecosystems, streams and rivers. residents depend mostly on rainfall, wells, rivers and streams for their water needs. thus, there is the possibility that in these riverine settlements the s. haematobium vector, the bulinus snail, is endemic. moreso, people’s reliance on streams and rivers for consumption and household water may increase their exposure to s. haematobium infection. this study is thus aimed to investigate the serum levels of selected micronutrients in school-age children with urinary schistosomiasis in bekwarra lga, cross river state, nigeria. methods ethical considerations ethical clearance to conduct this study was obtained from the cross river ministry of health research ethics committee (no. crsmoh/rp/rec/2017/806). the teachers helped to interview the participants. they also assisted the research staff in communicating the research objectives to participants. during the daily weekday child pick-up, each legal guardian and/or parent whose children verbally assented to participate was contacted separately. guardians and/or parents were informed verbally, either in english or the local dialect of the research’s objectives and protocols, and subsequently administered an informed consent form that included items that were discussed with them. the consent forms given were either returned immediately or at the next day’s drop-off. only school-age children with verbal and written agreement from their legal guardian and/or parent were included in the study. each participant’s information was handled in strict confidentiality. all the questionnaires and results were coded (participants did not write their names on the questionnaires and all responses were typed into a password-locked software program) to meet the principle of confidentiality. parents and/or guardians whose children tested positive for s. haematobium were notified and advised to take their children to the primary health centres, bekwarra, for treatment. a follow-up by the research team through the headmaster or head-mistress and class teachers of the affected school ensured that all infected school-age children were treated successfully. study design and areas adopting a systematic random-sampling approach, this cross-sectional study was carried out in five separate primary schools in the communities of nyanya, abouchichie, otupuru, and ukpah in bekwarra lga, cross river state, nigeria for 6 months, from june 2019 to december 2019. study sampling the sample size was determined using the single population proportion formula. based on earlier data on schistosomiasis in communities within bekwarra lga,31 the sample size was computed with a prevalence of 29.5%. the sample size was 325 with a precision of 0.05 (5%). the statistical power utilised was 95%. therefore, children within the age range of 4–16 years attending primary schools within bekwarra lga, cross river state, who in the preceding 6 months had not received any anti-helminth treatment, were selected from the communities under focus. a response rate of 98.06% was achieved with the participation of 353 school-age children out of the original target population of 360. all the participants had lived in their respective communities for the past 2 years. all participants who completed the questionnaire and delivered their samples of urine for examination were selected for this study. menstruating girls were exempted. collection of samples and data questionnaire administration a structured questionnaire was administered to each child by pre-trained research assistants who were well supervised. the questionnaire contained sections that requested information on aspects of socio-demography. parasitology survey a 10 ml urine sample was collected from each child between 10:00 and 14:00 in a designated private area of the school to assess the presence of a high s. haematobium egg load, using the sedimentation quantitative technique.32 then, 0.2 ml of 37% formalin was added to preserve the urine sample, which was then transported to a dedicated facility (a few metres from the site of collection) within the school in a cooler with ice packs, for examination to determine the presence and parasitic load of s. haematobium. the urine samples were centrifuged at 5000 revolutions per minute for 5 min. the supernatant obtained after the centrifugation was then discarded, leaving only the sediment. the sediment was then placed on a cleaned glass slide, then covered with a coverslip. these slides were microscopically observed using a × 40 objective lens for s. haematobium eggs, characterised by a terminal spine. the presence of s. haematobium eggs indicates a positive sample and was expressed as the number of eggs per 10 ml of the sample. the parasitic load of s. haematobium was categorised as mild (< 50 eggs/10 ml of urine) or heavy (≥ 50 eggs/10 ml of urine).33 a few drops of saponin solution were applied to samples with visible haematuria to enhance clarity for microscopy.34 serum micronutrient assay participants’ blood specimens were collected to determine micronutrient levels. trace element-free syringes, stainless steel needles, and special trace element tubes made of polypropylene (becton dickinson, franklin lakes, new jersey, united states) were used. all tubes used were stored in cool, dark boxes (0 °c – 4 °c). the separation of sera from blood cells was done by centrifugation at 4000 revolutions per minute for 10 min at 4 °c, within 4 h of sample collection. aliquots of sera obtained were stored at –70 °c until analysis. serum samples were diluted with sterile distilled water at a 1:6 ratio. an atomic absorption spectrophotometer (varian aa 100, victoria, australia) was used to measure the activity of serum zinc (at 213.9 nm), copper (324.8 nm), iron (510 nm) and calcium (422.7 nm),35,36 following previously published procedures.37 the results were then expressed in mg/dl for calcium, and μg/dl for copper, iron and zinc. statistical analysis data analysis was done using the statistical package for social sciences software version 23.0 (spss, inc., chicago, illinois, united states). a one-sample kolmogorov-smirnov test was adopted to measure data normality distribution. all micronutrient values in the serum were of normal distribution. the means for participants’ serum micronutrients levels were compared using one-way analysis of variance, followed by the least significant difference post hoc test for comparison between groups. chi-square and fisher’s exact tests were used to determine the relationship between infection patterns and age and gender. the correlation of two continuous variables was determined using pearson’s test. statistical significance was accepted as p-values less than 0.005 and 0.001. results this study enrolled 353 school-age children within the age range of 4–16 years (table 1). they consist of 223 (63.17%) boys and 130 (36.83%) girls. the overall prevalence was 16.15%. a total of 43 (12.18%) children had a mild infection, while 14 (3.97%) had a heavy infection; of this group with heavy infection, 2 (1.43%) had visible urinary haematuria. s. haematobium prevalence was highest in children 8–10 years old (n = 32, 23.19%), and lowest in children 14–16 years old (n = 4, 7.28%). there was a significant association between the infection rate and the age of the children (p = 0.022). in addition, the highest rate of s. haematobium infection was observed in girls (n = 34, 26.15%), while boys had an infection rate of 23 (10.31%). a significant association was observed between the gender and infection rate (p < 0.001). table 1: demographics and schistosoma haematobium infection among school-age children from bekwarra communities, nigeria, june 2019 – december 2019. the mean calcium serum level was significantly (p = 0.001) lower in heavily-infected children when compared to children without infection. serum concentrations of iron (p < 0.001), copper and zinc were significantly lower in children with mild s. haematobium infection compared with children without infection. furthermore, there were significant reductions in serum concentrations of iron (p < 0.001), zinc (p < 0.001) and copper (p < 0.002) in children with heavy s. haematobium infection when compared to children without infection. bivariate correlation analysis showed a significant inverse correlation between the number of s. haematobium eggs/10 ml in urine and the levels of serum copper (r = −0.61, p < 0.001) and zinc (r = −0.41, p < 0.002). also, an inverse correlation was found between the number of s. haematobium eggs/10 ml in urine and serum calcium (r = −0.24) and iron (r = −0.21) levels, but the correlations were not statistically significant (table 2). table 2: serum levels of selected micronutrients in school-age children with s. haematobium infection from bekwarra communities, nigeria, june 2019 – december 2019. discussion this study provides baseline epidemiological data on urogenital schistosomiasis in school-age children in the bekwarra villages of cross river, nigeria. our findings suggest that urogenital schistosomiasis is a prevailing parasitic infection in our study setting, similar to reports of it being a significant cause of morbidity in nigerian children in some riverine communities.38,39 overall, the prevalence in our study area is 16.15%, which is almost double the national nigerian average of 8.1% recorded in 2012.11 our results are consistent with previous findings from numerous endemic regions in nigeria 40,41 and other endemic countries such as tanzania, angola, malawi, and burkina faso.42,43,44,45 however, the severity and incidence of infection in our study are lower compared to data from several regions of nigeria and other sub-saharan countries.46,47,48 these studies had a larger participant size, consequently increasing the proportion of exposed and infected participants. the age group with the highest burden of infection in this study is those children younger than 11 years old, and the infection prevalence peaked at 8–11 years, similar to reports by olalubi and olukunle.49 our observation is most likely because children within this age range are frequently involved in water-contact activities (e.g., swimming, animal watering, and washing), and perhaps, this age group may not have been included in the recent campaign and distribution of preventive chemotherapy conducted in cross river state.50 the study showed that boys were frequently exposed to s. haematobium risk factors; nonetheless, infection prevalence was greater in girls. the higher prevalence in girls might be because the girls may routinely engage in house chores and associated activities, such as dish and cloth washing, fetching water from the stream for cleaning, bathing, and other domestic purposes, as well as the inherent anatomical differences between girls and boys.51 the greater prevalence rate found in girls compared to boys is comparable with previous findings for s. haematobium infection in uganda52 and nigeria,53 where female subjects had a higher infectious burden than males. however, the findings of this study contrast those of other comparable studies conducted in mauritania,54 senegal,55 benin and ethiopia.56 the distinction may be explained by differences in sociocultural elements, where boys are more likely to engage in water-contact activities including swimming and bathing, fishing, farming, and watering cattle. parasitic infections can cause nutrient loss, leading to poor nutritional status, which increases susceptibility to infection, creating a vicious cycle.57 we observed a negative correlation between the intensity of s. haematobium infection and micronutrient status. this observation indicates that the concentrations of calcium, iron, copper and zinc diminish as the infection intensity increases in school children. although the report of osazuwa et al.58 confirmed our observed negative correlation between iron and s. haematobium infection, previous studies found no correlation between micronutrient status for calcium, copper and zinc with helminth infection.59,60,61,62 thus, the explicit impact of parasitic infection on nutrient loss remains unclear. limitations confounders, such as other schistosoma species and microorganisms, were not examined; however, they might have influenced the data distribution in the research. the inability to analyse the snail host’s water sources and objectively quantify school-age children’s water-contact activities may restrict the interpretation of findings. another limitation of the study was the difficulty in obtaining guardian permission due to superstitious concerns regarding the use of participants’ urine samples. conclusion this study found a relatively low incidence of urinary schistosomiasis infection. providing potable water in the study communities will reduce exposure to infected water, thereby reducing transmission. there were significant differences established between sex and age and urinary schistosomiasis infectious diseases. these findings showed that urogenital schistosomiasis causes considerable morbidity and may be one of the contributing reasons to malnutrition in school-age children residing in bekwarra lga, cross river state. routine mass treatment will decrease morbidity and gradually eradicate the disease in the communities. therefore, there is a need for continual disease evaluation, provision of basic health education, implementation of broad-based health care policy, and development of socioeconomic infrastructures including providing safe and clean water for drinking through government agencies and cooperating organisation collaborations. these are essential in endemic areas to reduce infection transmission. acknowledgements the authors thank school teachers in bekwarra local government area, nigeria, who assisted in participant recruitment. appreciation goes to mrs. nneka vivien olerimi who also contributed in both advisory and manuscript review roles. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions e.i.e. wrote the original draft. e.i.e. and s.e.o. co-designed the study, and managed the literature search. s.e.o. vetted the manuscript, and supervised the laboratory analysis. a.o. managed the statistical analyses. s.e.o. and o.s.e. administered the questionnaire, managed specimen collection and carried out the laboratory analysis. all authors read and approved the final manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability the datasets are not publicly available but are available from the corresponding author, e.i.e., upon reasonable request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references utzinger j, becker sl, lieshout l van, dam gj van, knopp s. new diagnostic tools in schistosomiasis. clin microbiol infect 2015;21(6):529–542. https://doi.org/10.1016/j.cmi.2015.03.014 idris oa, wintola oa, afolayan aj. helminthiases; prevalence, transmission, host-parasite interactions, resistance to common synthetic drugs and treatment. heliyon 2019;5(1):e01161. https://doi.org/10.1016/j.heliyon.2019.e01161 colley dg, bustinduy al, secor we, 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introduction methods results discussion acknowledgements references about the author(s) jazira tumusiime department of medical laboratory science, faculty of medicine, mbarara university of science and technology, mbarara, uganda charles n. bagenda department of medical laboratory science, faculty of medicine, mbarara university of science and technology, mbarara, uganda uganda institute of allied health and management sciences, kampala, uganda daniel nzaramba department of medical laboratory science, faculty of medicine, mbarara university of science and technology, mbarara, uganda bashir ruzige department of emergency medicine, faculty of medicine, mbarara university of science and technology, mbarara, uganda elastus ssemwanga department of medical laboratory technology, faculty of medicine, mayanja memorial medical training institute, mbarara, uganda frank ssedyabane department of medical laboratory science, faculty of medicine, mbarara university of science and technology, mbarara, uganda winnie r. muyindike department of internal medicine, faculty of medicine, mbarara university of science and technology, mbarara, uganda ritah kiconco department of medical laboratory science, faculty of medicine, mbarara university of science and technology, mbarara, uganda department of biochemistry, school of health sciences, soroti university, soroti, uganda simon p. rugera department of medical laboratory science, faculty of medicine, mbarara university of science and technology, mbarara, uganda citation tumusiime j, bagenda cn, nzaramba d, et al. vitamin d deficiency and its associated factors among hiv patients at mbarara city health centre iv, south-western uganda. afr j lab med. 2025;14(1), a2957. https://doi.org/10.4102/ajlm.v14i1.2957 original research vitamin d deficiency and its associated factors among hiv patients at mbarara city health centre iv, south-western uganda jazira tumusiime, charles n. bagenda, daniel nzaramba, bashir ruzige, elastus ssemwanga, frank ssedyabane, winnie r. muyindike, ritah kiconco, simon p. rugera received: 01 aug. 2025; accepted: 30 sept. 2025; published: 10 dec. 2025 copyright: © 2025. the authors. licensee: aosis. this work is licensed under the creative commons attribution 4.0 international (cc by 4.0) license (https://creativecommons.org/licenses/by/4.0/). abstract background: hiv is a global health challenge. despite increased life expectancy because of antiretroviral therapy, vitamin d deficiency (vdd) remains widespread among hiv-positive individuals. factors such as poor nutrition, limited sunlight exposure, and certain medications, contribute to high vdd rates. despite this, the magnitude of this problem is not well documented in our setting. objective: the purpose of this study was to determine the prevalence and factors associated with vdd among hiv patients in south-western uganda. methods: this cross-sectional study at mbarara city health centre iv involved 218 randomly selected hiv patients on antiretroviral therapy. data were collected through structured questionnaires, medical records review, and blood samples for biochemical analysis. vdd was defined as serum levels < 25 ng/ml. levels of ˃ 5 mg/l for c-reactive protein (crp) were considered elevated. body mass index (bmi) was categorised as < 25 kg/m2 normal, 25–29.9 kg/m2 overweight, and ≥ 30 kg/m2 for obese. statistical analysis included descriptive statistics and logistic regression to assess factors associated with vdd. results: the median age of the participants was 38 years (interquartile range 30–45) with most being female (163, 74.8%). of the 218 study participants, 66 had vitamin d levels < 25 ng/ml, giving a prevalence of vdd of 30.3% (95% ci: 24.51% – 36.73%). being overweight, obese, and having a crp level of > 5 mg/l, were significantly associated with vdd. conclusion: the study found a 30.3% prevalence of vdd among hiv-positive individuals at mbarara city health centre iv. key associated factors included higher bmi and elevated crp levels. what this study adds: this study highlights the significant burden of vdd among hiv-positive individuals in a ugandan setting, with a prevalence of 30.3%. it identifies obesity and elevated crp levels as key risk factors, emphasising the need for integrated nutritional and inflammatory monitoring in hiv care. keywords: vitamin d; human immunodeficiency virus; deficiency; inflammation; obesity; nutrition; uganda; body mass index. introduction hiv infection is an intercontinental health challenge affecting millions of people. according to the world health organization, by the end of 2022, there were 39.0 million (33.1–45.7 million) hiv-positive individuals worldwide.1 since the introduction of antiretroviral therapy (art), people with hiv have a considerably greater life expectancy.2 however, this prolonged survival has brought to the forefront a range of health-related issues, which could result from prolonged use of art or other factors.3 among these health issues, vitamin d deficiency (vdd) was identified as a neglected and upcoming epidemic among people living with hiv.4 vitamin d is an important component needed for many physiological processes, such as hormone regulation, bone health, and immunological function.5 it also plays a role in calcium phosphate metabolism.6 an optimal vitamin d level increases muscle strength and improves bone mineral density, lowering the possibility of falls and fractures. in hiv patients, optimal vitamin d levels are essential for maintaining a healthy immune function and improving overall health outcomes.4 however, vdd is a common issue worldwide, affecting different populations, including individuals living with hiv. a review article reported that the prevalence of vitamin d insufficiency is overly high within those living with hiv around the world, with approximately 70% to 85% of patients experiencing insufficient levels.7 this high prevalence is as a result of a complex interplay of factors that could be specific to the virus and its treatment, as well as general lifestyle and environmental conditions.8 studies by manion et al.,9 escota, cross and powderly,10 and lambert et al.11 reported factors such as poor nutrition, limited sunlight exposure, impaired absorption of vitamin d in the gut, and increased inflammation, to be linked to vitamin d insufficiency in hiv-positive individuals. additionally, the use of art in hiv treatment has been affiliated with further reductions in vitamin d levels. furthermore, certain antiretroviral medications, such as efavirenz and tenofovir, have been linked to both lower vitamin d levels and elevated parathyroid hormone levels.12,13 in east africa, a study at aga khan university hospital, nairobi indicated a high deficiency of vitamin d, with a prevalence of 39%, among people living with hiv.14 a study done in kampala, uganda, among hiv patients at the infectious disease institute in makerere, found a vdd prevalence of 17%.15 tuberculosis also remains one of the most common opportunistic infections among people living with hiv, and hiv/tuberculosis co-infection has been associated with vdd. a meta-analysis study conducted in 2024 found that the prevalence of vdd was higher in the hiv-tuberculosis group than in the hiv group, addressing this deficiency in co-infected populations.16 people living with hiv often face dietary challenges, stemming from factors such as side effects of medications, limited access to a balanced diet, and socioeconomic constraints.17 this may further contribute to suboptimal intake of vitamin d through diet. also, environmental and lifestyle factors, including geographic location, limited sun exposure, and co-infections, can exacerbate the risk of vdd in these individuals.5 in uganda, south-western uganda in particular, there is little information on the prevalence of vdd among hiv patients, yet information from other countries has indicated a high prevalence.7 at mbarara city health centre iv, vitamin d is not routinely assessed in people living with hiv, despite the fact that it has a functional art clinic, with approximately 5000 active art patients (verbal communication from the clinic in-charge). the clinic receives about 700 patients per week, the majority of whom are women of advanced age, making them prone to vdd. therefore, the goal of this research is to determine the prevalence and the factors that are associated with vdd among hiv-infected patients at mbarara city health centre iv, south-western uganda. methods ethical considerations approval was sought from the research ethics committee of mbarara university of science and technology (reference number: must-2024-1609) before patients were involved. clearance was obtained from the mbarara city town clerk through the city health officer and the director of mbarara city health centre iv to access the antiretroviral therapy clinic and the laboratory. written informed consent was also obtained from the participants, and all data were kept private and anonymous by assigning each participant a unique identification code. direct feedback was given to physicians in-charge of patients who were found with vdd, for immediate management. study design, site and population this study used a cross-sectional observational study design. this study was conducted at mbarara city health centre iv, a government health facility which is located in kamukuzi division, mbarara city. it has a functional art clinic, with approximately 5000 active art patients. the study population was hiv patients who are currently receiving art at mbarara city health centre iv. we used simple random sampling to recruit participants from the art clinic. a thorough explanation was given, and those patients who volunteered to take part and gave consent were recruited into the study. eligibility criteria hiv-positive patients who were 18 years old and above, had been on art for a minimum of 6 months, who gave both verbal and written consent, and who were willing to provide a blood sample for biochemical analysis, were included. participants who were critically ill and those who were already on vitamin d supplementation were excluded. sample size calculation the kish and leslie sample size formula of 1965 was used, using 17%, a prevalence that was obtained by a study that was carried out among hiv patients at the infectious disease institute in kampala.15 therefore, this study included a minimum of 217 participants. study procedure participants were randomly selected, and those that fulfilled these research inclusion criteria were enrolled on each of the art clinic days of the week. the data were collected from 13 august 2024 to 23 august 2024. the principal researcher and the research assistants approached patients at the art clinic each day of data collection to explain the goal of the study. prior to being interviewed by the research assistants using a structured questionnaire that recorded social demographic, behavioural, clinical, and hiv/art-related factors, patients who consented to participate in the study were asked to sign an informed consent form. the participant was then requested to provide a blood sample to be used for laboratory vitamin d, c-reactive protein (crp), and lipid profile measurement. the samples collected from the facility main laboratory were centrifuged to separate cells and serum, which was stored in the laboratory fridge at 2 °c – 8 °c before analysis, either in the evening of each day of blood sample collection or the following day. the sample was then stored long term in the freezer at -20 °c. this procedure was repeated until the required sample size was achieved. data management data collection data were obtained using a researcher-administered structured questionnaire to obtain information on sociodemographics, lifestyle factors, and participants’ knowledge regarding vdd. the clinic records of the eligible individuals were consulted for information regarding hiv disease-related factors. the time of art initiation, the recent viral load, the current art regimen, and the length of time on that art are among the data that were gathered. a 5 ml venous blood sample was obtained from every study participant in a red-top vacutainer bottle, and it was used to analyse the serum vitamin d levels, crp levels, triglycerides, high density lipoprotein (hdl), low density lipoprotein (ldl), and total cholesterol concentrations. blood specimen collection and analysis venous blood (5 ml) was extracted into a red top blood collection vacutainer from each participant according to world health organization standard operating procedures. blood was centrifuged within an hour of collection at 3000 rpm for 10 min, and serum was collected into cryovials and stored at 2 °c – 8 °c before the biochemical analysis and long-term storage at –20 °c. laboratory assessment of the biochemical tests the serum vitamin d (25-hydroxyvitamin d) was measured using the cobas e 411 analyser (roche diagnostics gmbh, mannheim, germany). serum samples were transferred into tubes, placed in sample racks, and analysed using the cobas e 411 fully automated immunoassay analyser, with all procedures carried out in line with its instructions for use. the reagent used was elecsys vitamin d total iii, and calibration was done using the calibrator, calset vitamin d total iii. the serum crp, triglycerides, hdl cholesterol levels, ldl cholesterol levels, and total cholesterol levels were measured using the cobas c 111 chemistry analyser (roche diagnostics gmbh, mannheim, germany). the serum samples were carefully transferred into sample tubes. the sample tubes with serum were loaded into sample racks; sample information was entered, and the fully automated testing was started. biochemical analysis was done using the respective reagents, namely: crp4, tina-quant crp iv for crp, trigl, triglycerides for triglycerides, chol2, cholesterol gen.2 for total cholesterol, hdlc4, hdl cholesterol gen.4 for hdl cholesterol, and ldlc3, cholesterol gen.3 for ldl cholesterol. dyslipidaemia was defined based on standard cutoffs: total cholesterol ≥ 5.2 mmol/l, hdl cholesterol < 1 mmol/l for men or < 1.3 mmol/l for women, triglycerides ≥ 1.7 mmol/l, and ldl cholesterol ≥ 3.4 mmol/l. the calibration and quality control of this chemistry analyser was performed using the respective calibrators (c.f.a.s proteins for crp, and c.f.a.s lipids for the lipid parameters) before the biochemical analysis was done. the manufacturer’s instructions for the machines and the reagents were strictly followed. the analysis for 25-hydroxyvitamin d was performed since it is the most abundant form in blood and has a longer half-life, making it a more reliable indicator.18 the analysers automatically handle all reagent and sample pipetting, incubation, photometric measurements, and computations. they can run any combination of tests on up to 80 samples per work plan. anthropometric measurements all participants underwent anthropometric measurements following world health organization guidelines. weight was measured without shoes using a calibrated seca scale (seca gmbh & co. kg, hamburg, germany), accurate to 0.5 kg. height was recorded with a stadiometer (seca gmbh & co. kg, hamburg, germany) to the nearest 0.5 cm while participants stood upright. body mass index (bmi) was then derived by diving weight in kilograms by height in square metres. data storage completed consent forms and questionnaires were kept under lock and key, in order to maintain confidentiality of the participants’ information. data were coded, entered into microsoft excel 2019 (microsoft corporation, redmond, california, united states), and stored on flash disks and an external drive. these were also kept under lock and key. data analysis data were entered into microsoft excel 2019 and exported to stata 17.0 (statacorp llc, college station, texas, united states) for cleaning and analysis. all the studied variables were compared by vdd of the participants at a univariate level to test for statistical difference between two groups, that is, hiv-infected persons with vdd, and those without vdd. continuous variables were tested for normality using the shapiro-wilk test. we used mean and standard deviation to summarise the continuous variables which are normally distributed, while those that are not normally distributed were summarised using median and interquartile range. continuous variable distribution was then compared using student’s t-test for means and wilcoxon rank sum test for medians. frequencies and proportions were used to describe categorical variables. the significance of categorical variables’ association with vdd was tested using the chi-square test and fisher’s exact test for those which had a frequency below 5. the proportion of patients with vdd and its 95% confidence interval (95%ci) was determined by dividing the total number of participants with vdd by the total number of study participants; this was expressed as a percentage. logistic regression analysis was used to assess the factors associated with vdd, which was the dependent binary variable. all independent variables were compared with vdd at a bivariate level. the associations were measured using odds ratio and their 95% ci, and a p-value. the variables that were clinically or statistically significant with a p-value ≤ 0.2 at this level were assessed in the multivariate model to adjust for confounding. in the final multivariate model, a p-value ≤ 0.05 were considered significant. results characteristics of study participants the study comprised 218 participants, with a median age of 38 years (interquartile range 30–45). the distribution across age groups was nearly uniform, with 80 (36.7%) participants in each of the 31–40 years and over 40 years age groups. the majority of participants were women (163, 74.8%), and educational attainment was predominantly at the primary level (107, 49.1%), with only 13 (6.0%) having completed tertiary education. most participants were married or cohabiting (141, 64.7%) and employed (157, 72.0%), with a significant proportion earning less than ugx100000 (115, 52.8%). participants’ characteristics were grouped by the presence of vdd. a significant difference was observed with bmi, where a higher proportion of individuals with vdd had a bmi of 25 kg/m2 or more, as well as crp levels, with a significant trend towards higher crp levels in participants with vdd (p < 0.05). no significant differences were found in median age, sex, marital status, or employment status between those with and without vdd. other factors, such as smoking, alcohol consumption, physical activity, and biochemical variables, such as total cholesterol, hdl cholesterol, and ldl cholesterol levels, also did not show significant differences between the two groups (p > 0.05) (table 1, table 2 and table 3). table 1: sociodemographic characteristics of study participants stratified by vitamin d deficiency (n = 218), at mbarara city health centre iv, from 13 august 2024 to 23 august 2024. table 2: lifestyle and illness-related characteristics of study participants stratified by vitamin d deficiency (n = 218), at mbarara city health centre iv, from 13 august 2024 to 23 august 2024. table 3: biochemical tests results characteristics of study participants stratified by vitamin d deficiency (n = 218), at mbarara city health centre iv, from 13 august 2024 to 23 august 2024. prevalence of vdd among hiv patients at mbarara city health centre iv out of the 218 study participants, 66 had vitamin d levels < 25 ng/ml, giving an overall prevalence of vdd of 30.3%, with a 95%ci of 24.51% – 36.73%, as indicated in figure 1. figure 1: prevalence of vitamin d deficiency among the study participants, at mbarara city health centre iv, from 13 august 2024 to 23 august 2024 (n = 218). factors associated with vdd among hiv patients at mbarara city health centre iv after adjustment for confounding effects at multivariate logistic regression analysis, having a bmi of 25–29.9 kg/m2 (adjusted odds ratio [aor]: 2.43; 95%ci:1.18–5.04; p-value: 0.016), having a bmi of > 30 kg/m2 (aor: 2.96; 95%ci: 1.18–7.38; p-value: 0.020), and having a crp level of > 5 mg/l (aor: 2.29 (95%ci: 1.04–5.03; p-value: 0.039), were significantly associated with vdd (table 4). table 4: factors associated with vitamin d deficiency among the study participants at mbarara city health centre iv, from 13 august 2024 to 23 august 2024. discussion prevalence of vdd among hiv patients at mbarara city health centre iv the prevalence of vdd in this study was found to be 30.3% among hiv-positive persons. this prevalence is relatively lower than what has been reported in various studies globally, but still highlights a significant public health concern, as vdd is recognised as a global health issue, with widespread prevalence both in developed and developing countries.7 the relatively lower prevalence of vdd in our study could possibly be supported by the fact that this was a relatively young population (median age of 38 years), and young people are known to synthesise and produce more vitamin d than older ones, as they have more levels of 7-dehydrocholestrol.19 in comparison to global data, our study’s prevalence of 30.3% is markedly lower than that from a review study that was found to range from 70.3% to 83.7% after grading it by country.7 however, the same study found the ugandan prevalence among hiv patients to be at 35% – 45%, which is relatively similar to our findings. the difference in these prevalences is attributed to the differences in race, ethnicity and lifestyle factors, such as diet, between our population and other countries’ data that were analysed in this study. our prevalence is also lower than the 60% – 77% vdd prevalence reported in hiv-positive patients in the united states whose study population was only hiv-positive men,20 as well as the 55.6% prevalence found among hiv-positive individuals in france.13 this difference can be attributed to the variations in the study population, considering that race is a key player in vitamin d status.21 our findings also contrast with those from studies conducted in regions such as lahore, pakistan, where a staggering 76% of patients were found to be vitamin d deficient, and 9.4% had severe deficiency.22 similarly, a study conducted on a cohort of 2044 hiv patients in brussels, belgium, reported an exceptionally high prevalence of 89.2% for vdd.23 the lower prevalence observed could potentially be linked to differences in the extent of urbanisation, dietary patterns, and levels of outdoor physical activity in south-western uganda compared to more industrialised settings. their findings are also in line with the eurosida study that documented an 83% prevalence of vdd.24 this study randomly selected 2000 participants from within the eurosida study, and their samples were analysed for vitamin d. this very high prevalence of vdd could be because the majority of the participants were sampled during winter, where the sunshine exposure was minimal. the prevalence we observed aligns more closely with the data from east africa, where a kenyan study reported 6.3% of patients as vitamin d deficient, with 29.7% experiencing insufficiency.25 this similarity in prevalence could be because the kenyan population is closer to our study population in various aspects, including skin complexion, sun exposure, and diet, which are the major sources of vitamin d.26 our study’s findings are also relatively higher than another ugandan study conducted among hiv-positive children, which reported a relatively low prevalence of vdd, at 3%, and insufficiency, at 13%.27 this difference could be because during infancy and preschool years, children tend to spend more time taking part in outdoor activities, hence get more sunshine exposure compared to the adult population.28 a study that was conducted by the infectious disease institute in uganda found a baseline prevalence of 17% among hiv-infected adults, which was significantly lower than our findings.15 these lower prevalence rates in uganda might reflect regional differences in sun exposure (uganda being a sun-rich country that is crossed by the equator), dietary habits, or other environmental and genetic factors influencing vitamin d metabolism. while the prevalence of vdd in our study is lower compared to other regions, it remains a significant concern given the role of vitamin d in immune function and bone health, especially in hiv-positive individuals who may already be at a higher risk for comorbidities.4,5,6 factors associated with vdd among hiv patients at mbarara city health centre iv our study found that higher bmi categories were significantly associated with vdd among hiv-positive individuals. specifically, individuals with a bmi of 25–29.9 kg/m2 had an aor of 2.43, indicating more than double the likelihood of vdd compared to those with a normal bmi. additionally, individuals with a bmi greater than 30 kg/m2 had an even higher aor of 2.96, suggesting nearly a threefold increased risk of vdd. these findings align with previous research, indicating that both being overweight and obesity are linked to an increased risk of vdd in the context of hiv infection.29,30,31 these studies focused on the factors associated with vdd in a population that is similar to the current study, that is hiv patients. this relationship may be explained by the fact that vitamin d, being fat-soluble, is sequestered in adipose tissue, leading to lower circulating levels of the vitamin in individuals with higher body fat percentages.32 contrary to our study, a study carried out among hiv-infected and hiv-uninfected individuals found that lower bmi was associated with vdd.33 this contradiction can be attributed to the difference in the geographical location of the participants since it only included those from europe, the middle and east of asia, america, and australia. our study also identified a significant association between vdd and elevated crp levels (> 5 mg/l aor: 2.29) in hiv-positive individuals, indicating that vdd is linked to increased inflammation. this finding is consistent with existing research, which has shown that vdd in hiv patients is associated with greater inflammation and immune dysfunction.7,33 the study by mansueto et al.7 is a review article, and it focused on various markers of inflammation, including crp. it also suggested that there seems to be a considerable overlap in the outcomes associated with vdd and chronic inflammation, in both hiv-infected and -uninfected individuals. our results align closely with those of manion et al.,9 who found that hiv-infected individuals with vdd had higher levels of inflammatory markers such as il-6, tnf-α, hscrp, and d-dimer. ansemant et al.34 also reported a similar link between vdd and increased immune activation in hiv patients. these consistent findings across different studies suggest that the relationship between vdd and heightened inflammation is a common occurrence in hiv-positive populations. this relationship was also suggested by a study by shepherd et al.,35 who found a significant association with vdd and il-6, although there was no association with crp (contrary to our study). this contradiction can be attributed to a difference in the ethnicity and geographical location, since the study involved participants from various countries, all of which were european countries. however, a study by hoffman et al.36 did not find an association between vdd and markers of inflammation. this difference could be attributed to the fact that their study only enrolled participants on art with documented hiv-1 rna < 200 copies/ml, and also analysed samples that had been stored at participant’s baseline visit. study limitations this was a cross-sectional study, therefore the cause-effect relationship between the exposure variables; overweight, obesity, and elevated crp with the outcome variable vdd remain unclear. recommendations we recommend routine screening for vdd among hiv-infected persons with elevated crp and bmi. nutritional support, including vitamin d supplementation and dietary counselling, could be integrated into hiv care programmes to address vdd. a prospective study should be conducted to further investigate the risk of developing vdd among hiv patients that are overweight, obese, and those with elevated crp levels. conclusion the study found a 30.3% prevalence of vdd among hiv-positive individuals at mbarara city health centre iv. key associated factors included higher bmi and elevated inflammation (crp levels). our study underscores the importance of addressing both obesity and inflammation in the management of hiv-infected persons. acknowledgements the authors would like to thank the director, the in-charge art clinic, the laboratory staff and the art clinic staff at mbarara city health centre iv for their technical support throughout the period of data collection. this article is based on research originally conducted as part of jazira tumusiime’s master’s thesis titled ‘vitamin d deficiency and its associated factors among hiv patients at mbarara city health centre iv, south western uganda’ submitted to the faculty of medicine, mbarara university of science and technology, in 2025. the thesis is currently unpublished and not publicly available. the thesis was supervised by frank ssedyabane, winnie r. muyindike, ritah kiconco, and simon p. rugera. the manuscript has been revised and adapted for journal publication. the author confirms that the content has not been previously published or disseminated and complies with ethical standards for original publication. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions j.t., c.n.b., d.n., e.s., b.r., f.s., w.r.m., r.k., and s.p.r. contributed equally to the conceptualisation, writing, and editing of the article, and share first authorship. all authors contributed to the article, discussed the results, and approved the final version for submission and publication. sources of support this research received no specific grant from and funding agency in the public, commercial or not-for-profit-sectors. data availability the data that support the findings of this study are not openly available due to reasons of sensitivity and are available from the corresponding author, j.t., upon reasonable request. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. the article does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the authors are 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se. vitamin d status and associated factors among hiv-infected children and adolescents on antiretroviral therapy in kampala, uganda. plos one. 2021;16(6):e0253689. https://doi.org/10.1371/journal.pone.0253689 antonucci r, locci c, clemente mg, chicconi e, antonucci l. vitamin d deficiency in childhood: old lessons and current challenges. j pediatr endocrinol metab. 2018;31(3):247–260. https://doi.org/10.1515/jpem-2017-0391 canuto jmp, canuto vmp, lima mhad, et al. risk factors associated with hypovitaminosis d in hiv/aids-infected adults. arch endocrinol metab. 2015;59:34–41. https://doi.org/10.1590/2359-3997000000007 crutchley rd, gathe jr j, mayberry c, trieu a, abughosh s, garey kw. risk factors for vitamin d deficiency in hiv-infected patients in the south central united states. aids res. 2012;28(5):454–459. https://doi.org/10.1089/aid.2011.0025 mueller nj, fux ca, ledergerber b, et al. high prevalence of severe vitamin d deficiency in combined antiretroviral therapy-naive and successfully treated swiss hiv patients. aids. 2010;24(8):1127–1134. https://doi.org/10.1097/qad.0b013e328337b161 jawad ih, baiee ha. prevalence of vitamin d deficiency and its correlate with overweight and obesity in community-dwelling old adults. med j babylon. 2020;17(1):36–40. https://doi.org/10.4103/mjbl.mjbl_98_19 wang y, huang x, wu y, et al. increased risk of vitamin d deficiency among hiv-infected individuals: a systematic review and meta-analysis. front nutr. 2021;8:722032. https://doi.org/10.3389/fnut.2021.722032 ansemant t, mahy s, piroth c, et al. severe hypovitaminosis d correlates with increased inflammatory markers in hiv infected patients. bmc infect dis. 2013;13:1–7. https://doi.org/10.1186/1471-2334-13-7 shepherd l, souberbielle j-c, bastard j-p, et al. prognostic value of vitamin d level for all-cause mortality, and association with inflammatory markers, in hiv-infected persons. j infect dis. 2014;210(2):234–243. https://doi.org/10.1093/infdis/jiu074 hoffman rm, lake je, wilhalme hm, tseng c-h, currier js. vitamin d levels and markers of inflammation and metabolism in hiv-infected individuals on suppressive antiretroviral therapy. aids res. 2016;32(3):247–254. https://doi.org/10.1089/aid.2015.0200 abstract introduction slipta implementation preliminary outcomes discussion acknowledgements references about the author(s) jean-bosco ndihokubwayo world health organization, regional office for africa, brazzaville, congo talkmore maruta african society for laboratory medicine, addis ababa, ethiopia nqobile ndlovu african society for laboratory medicine, addis ababa, ethiopia sikhulile moyo botswana-harvard aids institute partnerships, gaborone, botswana ali ahmed yahaya world health organization, regional office for africa, brazzaville, congo sheick oumar coulibaly world health organization, regional office for africa, brazzaville, congo francis kasolo world health organization, regional office for africa, brazzaville, congo david turgeon united states centers for disease control and prevention, center for global health, division of global hiv and tuberculosis, atlanta, georgia, united states angelii p. abrol united states centers for disease control and prevention, center for global health, division of global hiv and tuberculosis, atlanta, georgia, united states citation ndihokubwayo j-b, maruta t, ndlovu n, et al. implementation of the world health organization regional office for africa stepwise laboratory quality improvement process towards accreditation. afr j lab med. 2016;5(1), a280. http://dx.doi.org/10.4102/ajlm.v5i1.280 lessons from the field implementation of the world health organization regional office for africa stepwise laboratory quality improvement process towards accreditation jean-bosco ndihokubwayo, talkmore maruta, nqobile ndlovu, sikhulile moyo, ali ahmed yahaya, sheick oumar coulibaly, francis kasolo, david turgeon, angelii p. abrol received: 04 nov. 2014; accepted: 03 dec. 2015; published: 20 may 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the increase in disease burden has continued to weigh upon health systems in africa. the role of the laboratory has become increasingly critical in the improvement of health for diagnosis, management and treatment of diseases. in response, the world health organization regional office for africa (who afro) and its partners created the who afro stepwise laboratory (quality) improvement process towards accreditation (slipta) program. slipta implementation process: who afro defined a governance structure with roles and responsibilities for six main stakeholders. laboratories were evaluated by auditors trained and certified by the african society for laboratory medicine. laboratory performance was measured using the who afro slipta scoring checklist and recognition certificates rated with 1–5 stars were issued. preliminary results: by march 2015, 27 of the 47 (57%) who afro member states had appointed a slipta focal point and 14 ministers of health had endorsed slipta as the desired programme for continuous quality improvement. ninety-eight auditors from 17 african countries, competent in the portuguese (3), french (12) and english (83) languages, were trained and certified. the mean score for the 159 laboratories audited between may 2013 and march 2015 was 69% (median 70%; sd 11.5; interquartile range 62–77). of these audited laboratories, 70% achieved 55% compliance or higher (2 or more stars) and 1% scored at least 95% (5 stars). the lowest scoring sections of the who afro slipta checklist were sections 6 (internal audit) and 10 (corrective action), which both had mean scores below 50%. conclusion: the who afro slipta is a process that countries with limited resources can adopt for effective implementation of quality management systems. political commitment, ownership and investment in continuous quality improvement are integral components of the process. introduction the burden of disease, especially infectious disease, continues to weigh heavily upon health systems in africa, where diseases such as hiv, malaria, tuberculosis, acute respiratory infections and diarrhoea continue to have high mortality rates.1 a high burden of disease is devastating in developing economies where it adversely affects all components of societal development, including income, health, and education. the role of the laboratory is increasingly recognised as critical in the overall improvement of countries’ health systems.2 medical and public health laboratories play a central role in the continuum of healthcare from diagnosis to treatment, management of patients and surveillance of diseases.2,3,4 without a definitive diagnosis from the laboratory, treatment is often based on syndromic patient management, in which symptoms that are often unspecific are used for decision-making, potentially leading to incorrect treatment.5 this can often lead to extended hospital stays, unnecessary admissions, loss of quality of life, deaths, irrational use of antimicrobial drugs and economic burden on families.6 realising the central role of the laboratory, increased funding from global health partners, such as the united nations’ global fund to fight hiv, tuberculosis and malaria, the united states president’s emergency fund for aids relief, the world bank and the bill & melinda gates foundation, has targeted the strengthening of laboratory systems.2,3,4 to capture this demonstrated impact of laboratories on overall patient outcomes, satisfaction levels, hospital stays and cost of care, reliable information in the form of quality laboratory results is required. to produce quality results consistently, laboratories need to implement and maintain continuous quality improvement systems that ensure policies, procedures, organisational and technical requirements are established as part of their quality management system.7 attaining accreditation is one means of independently confirming the existence of a quality management system4 and that the laboratory has the competence to perform high-quality testing.8 the number of laboratories in africa with internationally-recognised accreditation status has been very low, with figures below 400 reported as of 2014.9,10 although the exact total number of medical laboratories in africa is not known, uganda alone had 1234 government laboratories in 2007.9 this is a clear indication of how few laboratories in africa have successfully implemented quality management systems with the goal of international accreditation. in 2014, schroeder et al. reported 380 accredited laboratories in sub-saharan africa, 345 (91%) of which were located in south africa and 37 of the 49 of these countries had no laboratories accredited to international standards as of may 2013.10 countries in the african region have long recognised the need to strengthen laboratory systems, dating back to the maputo declaration in 2008.11 over the following years, the world health organization (who), in collaboration with its member states, made further commitments to remove identified barriers linked to poor laboratory systems, including poor quality management systems. this notably included the september 2008 resolution afr/rc58/r2 on public health laboratory strengthening adopted during the 58th session of the who regional committee in yaoundé, cameroon12 and the 2008 joint who-united states centers for disease control and prevention (cdc) conference on laboratory quality systems in lyon, france, where implementation of laboratory quality management systems in a staged approach was first recognised and endorsed by partners.13 recognising the need for a strong laboratory system in the efforts to maximise gains made through investments in health systems, these commitments by the governments of africa were critical in solidifying the much-needed political commitment to drive the agenda for laboratory system strengthening. a key recommendation from the 2008 joint who-cdc meeting was for the who to develop potential models of accreditation preparedness for adaptation and use by member states.13 the who regional office for africa (who afro), the united states cdc, the clinton health access initiative, the american society for clinical pathology and other partners launched the first version of the who afro stepwise accreditation preparedness process in kigali, rwanda, in 2009.14 in 2011, in nairobi, kenya, it was renamed the who afro stepwise laboratory (quality) improvement process towards accreditation (slipta).15 slipta is a framework for a stepwise quality improvement process for clinical, medical and public health laboratories in developing countries aimed at achieving the requirements of the international organization for standardization (iso) 15189 clinical laboratory standard.15 the policy guidance and checklist were published by who afro in july 2013, in english, french and portuguese.15 the revised slipta checklist replaced the who afro accreditation checklist and was expanded to include 111 items worth a total of 258 points. the policy document provides guidance for implementation and describes the key elements, governance structure and key stakeholder roles and responsibilities. it defined key terms such as standard, certification, accreditation, standardisation bodies – and their respective applicability – and broadened ‘accreditation bodies’ to include national, regional and international bodies. the term ‘assessment’ was replaced by ‘audit’ to differentiate the stepwise recognition process from an assessment conducted by an accrediting body. the intent of slipta was to embed the continuous improvement culture in laboratories whilst providing an avenue for laboratories to eventually meet the standards required for national, regional, or international accreditation.15 concurrently, the strengthening laboratory management toward accreditation (slmta) programme was launched. slmta is a hands-on, activity-based training programme designed to support the implementation of quality management systems in laboratories seeking recognition through slipta. endorsed by the ministerial call for action at the 1st international african society for laboratory medicine (aslm) conference in december 2012,16 the aslm2020: strategies and vision to strengthen public health laboratory medicine in africa recognised the challenges in public health laboratories and inspired the region to achieve excellence in laboratories.17 six ministers of health initially endorsed this call for action. it emphasised aslm’s role in addressing these challenges through collaborative partnerships with governments, national, regional and international partners and organisations, the private sector and other stakeholders. the aslm2020 goal for laboratory accreditation includes enrolment of 2500 laboratories in slipta and 250 public laboratories accredited by international standards by the year 2020.17 in this article, we describe the implementation of the who afro slipta programme and progress to date in 18 countries. governance structure the who afro slipta guidance document defines the slipta governance structure with six main stakeholders15 (figure 1) and who afro’s role of setting the norms and standards and implementing programmes through partnerships. the independent evaluating group, established through a memorandum of understanding with who afro, implemented slipta by first identifying a secretariat and second, establishing a slipta independent advisory group, comprising regional and international experts in laboratory quality management systems and accreditation who oversee the coordination and implementation of the process. figure 1: the who afro slipta governance structure. the secretariat, identified as aslm in 2011, coordinates the implementation of slipta in close collaboration with ministries of health, cdc and other regional and local development partners.15 the secretariat works with the who afro slipta focal points. slipta implementation implementation of slipta was defined by who afro in its slipta guidance document of 2011,15 which provided the policy direction for all stakeholders and the tools to be used in the application, audit and recognition processes, as described below. country slipta focal points the guidance document provided for the nomination of a national slipta focal point by each member state. in october 2012, who afro issued a memorandum endorsing the slipta policy, checklist technical documents and implementation of slipta in the region and requested member states to designate a national focal point for who afro slipta. it stated that slipta would be aligned with the maputo declaration, resolution afr/rc58/r2 issued in yaoundé, the initial launch of slipta in kigali and the millennium development goals.18 the country slipta focal points play a governmental support role by coordinating all in-country slipta implementation activities. they oversee the application process for laboratories enrolling in slipta and work with laboratories, aslm and local implementing partners to organise audits and to implement post-audit improvement plans. in collaboration with aslm, slipta focal points also oversee the selection, training and certification of in-country slipta auditors. by march 2015, 27 of the 47 who afro member states had nominated a national slipta focal point, including algeria, angola, burkina faso, burundi, tanzania, mozambique, south africa, uganda, kenya, zambia, malawi, botswana, namibia, cameroon, senegal, côte d’ivoire, chad, swaziland, lesotho, nigeria, mali, zimbabwe, cape verde, são tomé and principe, gambia, benin and togo. the who afro slipta checklist the who afro slipta scoring checklist was revised in 2011, two years after the initial launch of the programme, and is based on international standards, including the iso 15189:2007 standard19 and the clinical laboratory standards institute guideline gp26-a3.20 the checklist has 111 items organised into 12 quality systems essentials with a maximum score of 258 points (table 1), allowing for quantitative measurement of progress in the implementation and adherence to accreditation requirements for quality and competency. the checklist was revised and published by who afro in 2015 to align with the iso 15189:2012 standard. table 1: the who afro slipta checklist covering the 12 quality system essentials, the points allocated to each section and the overall star rating system. the scoring checklist allows for laboratory performance to be rated using a scale of 0 to 5 stars with each star level associated with the laboratory’s total score on the audit as follows: 0–142 points = 0 stars, 143–165 points = 1 star, 166–191 points = 2 stars; 192–217 points = 3 stars, 218–243 points = 4 stars and 244–258 points = 5 stars. at the end of the audit, each laboratory that achieves a star level of one star or higher is issued a corresponding star level certificate that is valid for two years (table 1). progress up the star scale denotes increased compliance with the who afro slipta checklist aligned with international standards. laboratory enrolment the country slipta focal point coordinates the application process with aslm for enrolment into slipta and is expected to prioritise laboratories as defined by their country’s strategic plans and tiered laboratory network. minimum criteria for enrolment for a laboratory includes at least one star (at least 143 points) achieved from an internal audit, a completed application form, an approved quality manual or equivalent, an organisational structure diagram or organogram and an application fee. laboratory applications denote the type of laboratory as public, private or research and the level of the laboratory as national, reference, regional or district, depending on the country. all applications submitted through the country slipta focal point to the aslm secretariat are reviewed and approved for enrolment if all criteria are met. audit logistics, such as selection and deployment of auditors, are coordinated by aslm. auditor training, selection and deployment the who afro slipta audits are conducted by aslm-certified auditors. auditor training consists of a five-day standardised training curriculum plus a practicum, which comprises the successful completion of three to five slipta audits under the observation of a lead auditor. the lead auditor evaluates the auditor trainee’s competency based on standard evaluation criteria that include: rating the trainee on his/her understanding of the who afro slipta process, auditing skills; and understanding of the who afro slipta checklist and the iso 15189 standard. three master aslm trainers, initially trained by who afro and the southern african development community accreditation service, provided the initial training and certification of auditors. subsequently, capacity to conduct auditor training has been built by other collaborating partners, including the national health services of south africa, a global healthcare public foundation of uganda and individual master trainers from the ministries of health. each audit team, led by a lead auditor, comprises at least two certified auditors, depending on the size and scope of the testing of the laboratory. team composition is guided by careful review of the laboratory application documents to make sure the selected audit team competencies match the laboratory’s scope of testing. when multiple laboratory teams are deployed concurrently in a country, aslm appoints an overall lead auditor to coordinate all audits and submission of audit reports to the aslm secretariat. laboratory audits the standard audit process starts with an opening meeting, the audit on day one and a feedback meeting on day two. the slipta audit follows a horizontal approach whereby the auditors review quality and technical records, ask questions of the laboratory staff and observe laboratory practices on the day of the audit. unique to slipta, the auditor has two key responsibilities: (1) identification of areas of non-conformity clearly documented using objective evidence and, more importantly, (2) providing on-site technical assistance and recommendations for closing identified gaps, differentiating it from a standard accreditation assessment. at the end of the audit, usually on day one, the audit teams discuss their findings, agree on scores and develop a draft audit report with documented non-conformities and recommendations. the lead auditor submits the draft audit report to the laboratory for review prior to the debrief meeting on day two. the final report is agreed upon by the laboratory and the audit team by the end of the debrief meeting before submission to the aslm secretariat. the final report is supported by a form agreeing to the report content and signed by the laboratory representative. key to the slipta process is advocacy for laboratory system strengthening in the target countries. built into the slipta audit process are opportunities for advocating for the laboratories where each audit team must meet hospital management before and after each audit. depending on country context, this may also include provincial or district management teams. when multiple laboratories are audited, debrief is held with high-level ministry of health officials, who country representatives, laboratory directorate, in-country implementation partners, laboratory associations, and managers of audited laboratories. during the debrief session, the auditors provide feedback on the overall performance of the audited laboratories and advocate for implementation of corrective actions identified during the audit. star recognition star determination and the issuance of the certificate of recognition is recommended by the independent advisory group based on the audit reports. after review, the independent advisory group recommends the star level to the aslm secretariat, who issues the certificate of recognition; these certificates are valid for two years. the laboratory is expected to apply for another audit six months before the expiration of the current recognition certificate as part of the re-audit process. at this point, the laboratory, country focal point and local implementing partners would have worked to address the gaps identified in the previous audit. it is through this process of incremental implementation of quality requirements that the laboratory progresses over the 0to 5-star rating until it can be recommended to apply for international iso 15189 accreditation. preliminary outcomes by march 2015, 27 of 47 (57%) who afro member states had identified and appointed a slipta focal point to coordinate their in-country slipta activities. at the aslm 2012 bi-annual conference in december 2012, six ministers of health endorsed slipta as the framework for strengthening laboratory services in their respective countries;16 an additional eight ministers of health have endorsed slipta since then, for a total of 14 ministerial endorsements. a total of 98 auditors from 17 african countries, with competencies in the portuguese (3), french (12) and english (83) languages, have been trained and certified and conducted the audits (table 2). between may 2013 and march 2015, 159 laboratories from 18 of the 27 african countries with slipta focal points were audited (figure 2). of these 159 laboratories, 145 (91%) achieved at least one star, the majority achieved two stars (57 laboratories) and 2 (1%) received five stars on the slipta checklist (figure 3). figure 2: map of the 18 who afro member states that were slipta-audited from may 2013 to march 2015. figure 3: distribution of stars across the 159 slipta-audited laboratories, may 2013 to march 2015. table 2: summary of laboratories audited through the slipta process and certified slipta auditors by country, may 2013 to march 2015. the mean score for all 159 laboratories was 69% (median, 70%; sd, 11.5; interquartile range [iqr], 62–77) (table 3). slipta checklist sections 4 (client management), 9 (information management) and 12 (facilities and safety) had mean scores of at least 80%, whereas for checklist sections 6 (internal audit) and 10 (corrective action), the mean scores were below 50%. the greatest variability, as shown by the iqr, was observed for checklist sections 2 (management reviews; iqr, 41–71), 6 (internal audit; iqr, 20–60) and 11 (occurrence management; iqr, 33–92). table 3: summary of performance of the 159 laboratories across 12 sections of the who afro slipta checklist. two slipta-audited laboratories, the national tuberculosis reference laboratory in maputo, mozambique and the princess marina hospital laboratory in gaborone, botswana, have since attained international iso 15189 accreditation. four laboratories in cameroon have since applied to the south africa national accreditation system and await assessment. discussion based on these preliminary results, the who afro slipta programme has been met with positive responses regionally, as observed by its rapid expansion over the past three years of implementation and endorsement by ministers of health. this is the first large-scale, regional, standardised approach of its kind to assess laboratories’ preparedness toward international accreditation standards using an external auditing process, with the premise that a star score in one country would be comparable to that of another country across the continent. the audit findings can be used as a roadmap for countries to achieve accreditation through measurable steps of continuous quality improvement by pinpointing specific areas for improvement as part of strategic planning. tools, such as slmta, can be used to improve the identified areas where corrective actions are required, as part of the laboratory’s continuous quality improvement. similar stepwise accreditation preparedness approaches have been adopted in other regions globally to encourage continuous laboratory quality improvement. these include the accreditation program of clinical laboratories established by argentina in 1994;21 the thailand medical technology council national accreditation program of 200222 and the korean laboratory accreditation program of 1999.23 in all cases, there was significant progress in the implementation of quality management systems toward accreditation.21,22,23 similarly, slipta was adopted to address quality improvement incrementally toward achieving accreditation requirements in the resource-limited settings of africa. it recognises and rewards efforts made toward meeting accreditation requirements as measured by the achieved percentage of compliance and star levels. this regional approach should transition into national governance as countries develop their national capacity and establish their own accreditation bodies, similar to those in argentina, thailand and korea. on average, laboratories performed poorly (mean: < 50%) on the internal audit and corrective action sections of the slipta checklist. these two quality system essentials are linked, in that they form part of the continual improvement activities inherent in a quality management system. for all areas identified as non-conforming, a root cause analysis followed by corrective actions and preventative actions must be performed. hence, if laboratories do not perform well in the process of non-conformity identification, root cause analysis, preventative actions and monitoring of the effectiveness of corrective and preventative actions, they are not likely to perform well on the internal audit process. on the other hand, performance in the areas of client management, inventory control and information management were all above 50%, indicating better progress. notably, almost all laboratories had conducted a customer survey, which forms the basis for understanding the needs of its clients. the who afro slipta checklist primarily evaluates how inventory is managed once received in the laboratory with a few questions that evaluate the entire procurement system. for laboratories that did not have an electronic-based information system, a paper-based system was always available, which the iso 15189 standard recognises as acceptable; hence, the better performance in this area. the results also showed that countries are at very different levels of capacity for implementation of the slipta process. in particular, the francophone and lusophone countries are not well covered. this could be because most of the countries covered by the slipta audits to date have benefitted from programmes such as the united states president’s emergency plan for aids relief. generally, at the time of the audits, most of the laboratories had not surpassed the 3-star rating and only two laboratories had achieved five stars. this implies that although the slipta process has been widely accepted by most countries and notable improvements made, the majority of laboratories have yet to reach international accreditation readiness. to reach their goals, country strategic planning with committed resources for infrastructure, human resources and training in laboratory quality improvement and structured laboratory mentoring are all part of continuous quality improvement and accreditation preparedness. recommendations based on the preliminary audit results from 159 laboratories over three years, the majority of laboratories reached two stars. the adoption of the slipta scheme is a viable option for countries with limited resources to guide their path of accreditation preparedness. slipta audit findings equip countries to identify and address specific areas of improvement in the laboratories. this will empower the countries to make informed decisions as part of national planning through smart investments. to support the scale-up of slipta, advocacy and coordination is critical; all 47 member states should have a slipta focal point to guide implementation of the national slipta strategy as part of a continuous improvement process. member states are encouraged to integrate slipta and resources for implementation into their national laboratory policies and strategic plans, invest sustained financial and human resources to strengthen the laboratory system and the country’s auditing capacity and strengthen the countries’ national capacity and governance to oversee slipta under a national accreditation body. we recommend further analysis on the possible barriers to the implementation of quality management systems in africa, whether countries have used the slpita audit findings to improve their laboratories and how slipta has helped improve the laboratories from initial audit to follow-up audits. the internal audit and corrective actions quality system essentials need further review to better understand the challenges laboratories face, for example, which specific checklist questions are laboratories failing to address. an understanding of the profile of laboratories and their performance would assist in understanding the greater variability observed in sections 2 (management reviews) and 11 (occurrence management). conclusion based on the results, the who afro slipta is a process that countries with limited resources can adopt for effective implementation of quality management systems. political commitment, ownership and investment in continuous quality improvement are integral components of the process. acknowledgements the authors would like to acknowledge dr tsehaynesh messele, chief executive officer of aslm at the time of implementation of the slipta programme; teferi mekonen, a member of aslm’s technical staff who implemented the slipta programme; and the auditors. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. sources of support implementation of the slipta programme was made possible by the support of the united states centers for disease control and prevention, aslm grant number: 1u2ggh000710-01: ‘development of laboratory network and society to implement a quality system’ (principal investigator: dr tsehaynesh messele, chief executive officer, aslm). authors’ contributions j.-b.n. contributed to the policy development of who afro slipta and the programme’s implementation framework. t.m. and n.n. both contributed to programme implementation and coordination. s.m. contributed to the data analysis. a.a.y., s.o.c. and f.k. revised the manuscript. d.t. contributed to programme implementation. a.p.a. contributed to the policy development of who afro slipta and the programme implementation framework and 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al. the implementation and effects of a clinical laboratory accreditation program in korea from 1999 to 2006. korean j lab med. 2009;29(2):163–170. http://dx.doi.org/10.3343/kjlm.2009.29.2.163 article information authors: fayefori m. abbiyesuku1 augustine n. agbakwuru1 olatunbosun o. olawale1 affiliations: 1department of chemical pathology, university college hospital, ibadan nigeria correspondence to: augustine agbakwuru postal address: po box 1684, sabo yaba, lagos nigeria dates: received: 26 apr. 2012 accepted: 09 apr. 2013 published: 24 july 2013 how to cite this article: abbiyesuku fm, agbakwuru an, olawale oo. the association between sex hormone-binding globulin and type 2 diabetes in nigerian men. afr j lab med. 2013;2(1), art. #44, 5 pages. http://dx.doi.org/10.4102/ ajlm.v2i1.44 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the association between sex hormone-binding globulin and type 2 diabetes in nigerian men in this original research... open access • abstract • introduction • research method and design    • participants    • collection of blood and clinical data    • laboratory analysis    • statistical analysis • results • ethical considerations    • potential benefits and hazards    • recruitment procedures    • informed consent    • data protection • trustworthiness    • reliability • discussion • limitations of the study    • recommendations • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: epidemiological studies have shown that sex hormone-binding globulin (shbg) has a role in glucose homeostasis in both men and women. however, a prospective study on japanese-american subjects concluded that shbg was not a significant risk factor in either men or women, suggesting ethnic differences. we were not aware of any evaluation of shbg in subjects of african ancestry.objectives: we investigated the association between shbg and insulin resistance in type 2 diabetic diabetic men in a hospital in nigeria. method: forty-eight male subjects with type 2 diabetes and 20 non-diabetic male subjects were recruited in this cross-sectional hospital-based study by the convenient sampling method. height and circumferences around the waist and hip were measured to the nearest 0.5 cm and the waist–hip ratio was calculated from this measurement. weight was measured and body mass index was calculated. fasting plasma glucose concentration was measured by the glucose oxidase method with a between-run coefficient of variation of 3%. insulin and shbg were measured by means of enzyme-linked immunosorbent assay (elisa). results: there was a statistically-significant difference between test results for the diabetic and non-diabetic patients. the mean shbg concentration was higher in the non-diabetic group (42.2 nmol/l) than the diabetic group (30.5 nmol/l). a significant inverse association between insulin resistance and shbg was observed (r = 0.353, p < 0.015). conclusion: this study supported earlier observations that a significant inverse correlation exists between shbg and insulin resistance and provides evidence that the relationship may extend to type 2 diabetic men of african ancestry in nigeria. introduction top ↑ diabetes mellitus has been recognised as a disease for more than 3500 years, since its description in the eber papyrus in 1552bc.1 surviving texts from the ancient greeks, chinese, indians, and persians also refer to the disease, which was subsequently named diabetes mellitus. it is defined as a metabolic disorder of multiple aetiologies, characterised by chronic hyperglycaemia with disturbance of carbohydrate, fat, protein, water and electrolyte metabolism which result from defects in insulin secretion, insulin action or both.2 much of the morbidity and mortality burden of diabetes mellitus is due to the acute and chronic complications that often arise. these complications include damage to various organs, especially the eyes, kidneys, nerves, heart and blood vessels. in nigeria, 3.9 million people have diabetes mellitus, representing an estimated prevalence rate of 4.04%.3 although laboratory tests to screen for and diagnose diabetes mellitus are widely available, the disease is still underdiagnosed. approximately 25% of people with newly-diagnosed diabetes mellitus already have microvascular disease, suggesting that they had the disease for four to seven years by the time of diagnosis.4,5because there are no unique biological markers and few symptoms in the early years of the disease, the diagnosis of diabetes mellitus is based exclusively on hyperglycaemia the consequence of a disrupted carbohydrate metabolism. the tests for early diagnosis of diabetes mellitus are limited to direct and indirect tests of hyperglycaemia namely, serum glucose and glycated protein (hba1c), respectively. serum glucose testing is affected by short-term lifestyle changes, lower reproducibility and sample processing6 (e.g. during the oral glucose tolerance test, incretin effect on the ingested glucose cannot be accounted for, neither can it be standardised, which affects reproducibility). the use of hba1c for screening for and diagnosing diabetes mellitus also has its limitations, as it is only able to reflect glycaemic control in retrospect and is also affected by haemolytic anaemia.7,8,9,10,11,12,13,14 it therefore follows that a new test that can identify type 2 diabetes mellitus earlier, with greater sensitivity and specificity, will be desirable. an emerging alternative is sex hormone-binding globulin (shbg). sex hormone-binding globulin, once considered to be merely a way for the body to store extra sex hormones, now appears to be a player in a number of biological functions. there is evidence showing a more direct role for shbg through an intracellular signaling cascade mediated by a membrane-bound shbg receptor.15,16 delineation of the biological effect of shbg signaling has lagged behind our understanding of the biochemical analysis of its camp signaling pathway; however, epidemiological studies on subjects of european ancestry have reported an inverse association between low circulating shbg and type 2 diabetes.17,18 this means that those with higher serum levels of shbg are protected from developing type 2 diabetes. genetic studies have defined a causal relationship between shbg and type 2 diabetes mellitus. genetic studies have defined a causal relationship between shbg and type 2 diabetes mellitus in subjects of european ancestry. in a prospective study on japanesein subjects of european ancestry. in a prospective study on japanese-american subjects who were followed for 3 years, however, shbg did not correlate with insulin resistance in either men or women,19 suggesting ethnic differences. there is a paucity of data on the relationship of shbg with type 2 diabetes mellitus in subjects of african ancestry. this study investigated the association between circulating shbg and type 2 diabetes in in a group of nigerian men. research method and design top ↑ participants this cross-sectional study was conducted at university college hospital, ibadan, oyo state, nigeria. the formula zα2ε2/d2 was used to determine the sample size, where zα is the standard normal deviate of α at 5%, ε is the standard deviation of 9.11 as defined by a previous study17 and d is the level of precision. substituting 1.96 for zα and 3 for d in the formula gives a sample size of 34.5. adding a non-response rate of 10% brings the required sample population to approximately 40. forty-eight male participants with type 2 diabetes mellitus and 20 controls, were recruited from the general outpatient department, metabolic research clinic and medical outpatient clinics in university college hospital by the convenient sampling. all adult men diagnosed with type 2 diabetes mellitus who attended the clinics during 2011 were invited to participate. the response rate was 62%. none were turned away, which led to a sample size that was greater than initially called for. these were nigerians residing in ibadan, the western part of nigeria. exclusion criteria included injured and very sick, those on medication such as insulin, steroids, anticonvulsants, beta blockers and thiazides. informed consent was obtained and the study lasted for the year of 2011. collection of blood and clinical data seven millilitres of fasting venous blood were drawn from the antecubital vein between 08h00–09h00 and aliquoted into fluoride oxalate tubes for glucose and plain tubes for hormonal (insulin and shbg) assays. the plain tube bloods were allowed to clot for 30 minutes and the sera were collected after centrifugation at 4500 rpm for five minutes, then stored at 80 °c for four months before analysis. waist (iliac crest) and hip (widest region at the tronchanter level) circumference were measured to the nearest 0.5 cm and from these measurements, the waist–hip ratio (whr) was calculated. weight was measured in kilograms using a weighing balance (medical laboratory scientific england). a stadiometer rgz 160 (medical laboratory scientific england) was used to measure height, from which body mass index (bmi) was calculated (kg/m2). biodata and self-reported drug history were also documented. laboratory analysis plasma glucose concentration was measured by the glucose oxidase method, using a pus 2018g biochemistry analyzer (beijng perlong new technology and co ltd, china) with a between-run coefficient of variation of 3% (glucose concentration of 4.94 mmol/l at 10 replicates). insulin and shbg concentration were measured using enzyme-linked immunosorbent assay (elisa) kits (genwaybio, san diego, ca), according to the kit protocol. briefly, these are solid phase enzyme-amplified heterogeneous immunoassays performed on microtiter plates.20 elisa technology uses monoclonal antibody (mab) directed against distinct epitopes. calibrators and samples react with the capture monoclonal antibody (mab 1) coated on the microtiter well and with monoclonal antibody (mab 2) labeled with horseradish peroxidase. after an incubation period allowing the formation of a sandwich (mab 1 target mab 2 horseradish peroxidase, where target, in this case, is human insulin or shbg), the microtiter plate is washed to remove unbound enzyme-labeled antibody. a chromogenic solution (tetramethylbenzydine) is added and incubated. the reaction is stopped and the absorbance, which is directly proportional to the insulin or shbg concentration, is measured. a calibration curve is plotted using 5-parameter logistic software (www.readerfit.com) and the insulin or shbg concentration of the samples is interpolated from the calibration curve. the insulin elisa kit had an interassay coefficient of variation of 13% (kit controls for insulin concentration set at 25 µu/ml [control 1] and 80 µu/ml [control 2] at 5 replicates) and 0.3% cross-reactivity with proinsulin; and the shbg kit had an interassay coefficient of variation of 16% (shbg concentration = 50 nmol/l at 9 replicates). fasting insulin and glucose values were used to calculate insulin sensitivity, b-cell function and insulin resistance using the mathematical equation described by jonathan levy et al. in 1998,21 as calculated using homa-2 calculator software (www.dtu.ox.ac.ukm). statistical analysis the data were log transformed to enhance compliance with normality assumptions. one observed value for shbg was treated as an outlier using the dixon test.22 the dixon test uses the ratio d/r, where d is the absolute difference between an extreme observation (large or small) and the next largest and r is the range between the largest and smallest observations, including extremes. the cut-off value for the ratio (d/r) is 1/3; that is to say, if the observed value for d is equal to or greater than one third of the range r, the observed value is deleted. mean and standard deviation (sd) were calculated for anthropometric characteristics and laboratory-measured and -derived data (age, bmi, whr, fasting glucose, fasting insulin, shbg, insulin resistance, insulin sensitivity and pancreatic beta-cell function). simple linear regression and mann-whitney u-tests were also performed. the statistical package for social science 16.0 software was used for the computation. results top ↑ the mean age was similar for the diabetics (62, sd = 11) and the controls [non-diabetics] (60, sd = 14) (table 1). twenty-five (52.5%) of the diabetic subjects had a normal bmi whilst 14 (25%) were overweight, 8 (20%) were class i obese and 1 (2.5%) were class ii obese. twenty-seven subjects (56.3%) had a normal whr of < 1 and within this group, 17 (63%) had a normal bmi. in the control group, 10 (50%) subjects had normal bmi, 6 (30%) were overweight and 4 (20%) were class i obese. whr was < 1 except for 3 (15%) that were > 1. within the abnormal whr control group, 2 (10%) were overweight and 1 (5%) class i obese. table 1: descriptive characteristics. there was no local reference range for shbg so the kit manufacturer’s reference interval of 15–100 nmol/l was used to interpret the results. there was a statistically-significant difference between the means of shbg concentration in the controls and the diabetic group (mann-whitney u = 307, p = 0.026, 2-tailed). in the diabetic group, the shbg level of 36 (75%) subjects was within the reference range, whilst 11 (23%) had high values (above the upper limit of the reference interval) and only one subject had an abnormally low value (below the lower limit). five (25%) subjects in the control group had shbg levels above the upper limit of the reference interval whilst the others were within the reference range. the mean fasting insulin concentration was 13.64 μu/ml in the diabetic group and 12.34 μu/ml in the controls, with no statistically-significant difference being observed. forty (83%) subjects had fasting insulin concentrations within the kit manufacturer’s reference range of 5–19 µu/ml, one had a result that fell below the lower limit of the reference interval and seven (15%) had values above the upper limit. two (10%) subjects in the control group had insulin concentration above the reference interval whilst 18 (90%) were within the interval. a weak (though statistically significant) inverse association (r = 0.353, p < 0.015) was observed between insulin resistance and shbg (figure 1), whereas a positive association (r = 0.356, p < 0.014) was observed between age and circulating shbg (figure 2). there was no significant correlation between shbg and fasting insulin (r = 0.264, p < 0.073) or between shbg and fasting glucose (r = 0.236, p < 0.110). there was no significant correlation between shbg and bmi (r = 0.189, p < 0.203) or between shbg and whr (r = 0.126, p < 0.397). similarly, ir did not correlate significantly with insulin resistance (r = 0.043, p < 0.772) or with whr (r = 0.068, p < 0.646). figure 1: scatter plot showing the relationship between insulin resistance and levels of sex hormone-binding globulin. figure 2: scatter plot showing the relationship between age and levels of sex hormone-bindin. ethical considerations top ↑ ethical approval was given by the institute for advanced medical research and training, university of ibadan, nigeria, with ui/uch ec registration number: nhrec/05/01/2008a. potential benefits and haz there was no potential physical, psychological or disclosure danger to the subjects except the pain of the needle-prick when the blood samples were taken. recruitment procedures participation was voluntary and subjects were free to withdraw at any time. this entitlement lapsed on 15 december 2011, at the conclusion of the study. informed consent informed written consent was signed by each participant. data protection each participant was assigned a number which was known only to him and the investigator. these numbers were used for blood sample collection so the technologist did not know whose sample was being analysed. trustworthiness top ↑ reliability all assays were performed in duplicate. glucose estimation by the glucose oxidase method had a between-run coefficient of variation of 3% (glucose concentration of 4.94 mmol/l at 10 replicates). the insulin estimation had an interassay coefficient of variation of 13% (kit controls for insulin concentration set at 25 µu/ml [control 1] and 80 µu/ml [control 2] at 5 replicates) and 0.3% cross-reactivity with proinsulin; and the shbg kit had an interassay coefficient of variation of 16% (shbg concentration = 50 nmol/l at 9 replicates). discussion top ↑ there are four important findings that can be drawn from this study:1. we found a weak inverse correlation between the serum concentration of shbg and insulin resistance amongst our group of nigerian men with type 2 diabetes. 2. we identified a statistically-significant difference between the concentration of shbg in diabetic and non-diabetic men. 3. there was no significant correlation between insulin resistance and bmi/whr amongst type 2 diabetic men in this study. 4. shbg did not correlate meaningfully with fasting insulin and fasting glucose amongst these subjects. there is a paucity of studies on the relationship between shbg and type 2 diabetes in men of african descent. in the 1990s birkeland et al.17 observed a direct correlation between insulin sensitivity and shbg levels in men of european ancestry which was independent of obesity and abdominal fat accumulation. they estimated insulin sensitivity using the hyperinsulinaemic euglycaemic glucose clamp (the gold standard test) and fat distribution with bmi and whr. haffner et al.,18 in their nested case control study, observed a consistent inverse correlation between shbg concentration at baseline and the subsequent development of diabetes in men. the findings in this study are in agreement with their findings, in that we also found an inverse correlation between shbg and insulin resistance. association does not mean causality, but two recently-published studies using a mendelian randomisation approach provided strong evidence that circulating shbg is involved directly in the pathogenesis of type 2 diabetes in both men and women.24,25 these studies described the effect of genotypes of gene-encoding shbg on plasma levels of sbhg and the risk of development of type 2 diabetes. in these nested case control studies, gene analysis of the single nucleotide polymorphisms (snps), rs1799941, rs6257 and rs6259, coding for circulating shbg, was performed in men and women with type 2 diabetes and matched with controls, indicating that low levels of shbg are a strong predictor for the development of type 2 diabetes mellitus. since genetic studies are a powerful epidemiological tool, their findings may now help to more deeply address the complex and indubitably important role of circulating shbg in the natural history of type 2 diabetes mellitus. the current study was able to replicate some of the previously-mentioned findings in subjects. circulating shbg had an inverse correlation with insulin resistance. surprisingly, no significant association between insulin resistance and bmi/whr was observed, even though they are strong predictors for the risk of developing type 2 diabetes,26,27,28 probably because of the complex interaction of genetics, environment and lifestyle choices in the aetiology of the disease. it was also observed that there was no significant association between body fat distribution (bmi and whr) and circulating shbg, which is in agreement with earlier observations that circulating levels of shbg are determined by liver fat and not total body fat.29 insulin decreases the hepatic production of sex hormone-binding globulin in vitro,30 meaning that inhibition of insulin release increases plasma levels of sex hormone-binding globulin in humans.31 hyperinsulinaemia induced by insulin resistance probably causes low levels of shbg in insulin-resistant conditions such as obesity, type 2 diabetes and polycystic ovary syndrome. interestingly, no correlation between circulating shbg and fasting insulinaemia did not reach statistical significance at the 0.05 level. the findings in this study argue against the widely-held explanation that hyperinsulinaemia is responsible for a decline in circulating shbg in humans.31 the positive correlation between age and shbg levels supports earlier finding that shbg levels increase once people are in their sixties.32,33 this implies that the elderly should be protected from the development of type 2 diabetes mellitus but, paradoxically, age is a risk factor for development of the disease and screening is recommended from the age of 45 years.34 this would seem to argue against this observation, although the explanation could be that those that eventually develop type 2 diabetes mellitus are genetically disposed toward having low circulating shbg levels, as was suggested by ding et al.25 the statistical difference between the shbg concentration in the two groups suggests that shbg estimation could be useful in the management of type 2 diabetes. taken together, these findings indicate that a significant inverse association exists between circulating shbg and insulin resistance. limitations of the study top ↑ measurement of insulin resistance was one of the recognised difficulties in this study. insulin resistance was calculated using an indirect measurement (a surrogate index using the homa 2 calculator) which has a significant but imperfect correlation (r = 0.88, p < 0.0001)35 with the direct gold-standard measurement (the hyperinsulinaemic euglycaemic clamp method) of insulin resistance. bmi and whr were used as proxies for a more direct assessment of overall adiposity. recommendations evidence for the underlying mechanism behind the association between shbg levels and insulin resistance is yet to be elucidated fully. despite the association between shbg and type 2 diabetes, further studies to evaluate changes in sex hormone-binding globulin concentration as glycaemic control improves, as well as delineation of the underlying mechanism regarding how shbg affects glucose homeostasis, are necessary as they may provide novel targets for diabetes prevention and management. conclusion top ↑ this study supports earlier observations that a significant, though weak, inverse correlation exists between shbg and insulin resistance and extends to type 2 diabetic nigerian men. further studies to delineate the underlying mechanism behind the association between shbg and type 2 diabetes mellitus are advocated as they may provide a novel target for prevention and management of type 2 diabetes mellitus. acknowledgements top ↑ special thanks to dr elizabeth luman and her team for their editing assistance. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions f.m.a. ( university college hospital), a.n.a. (university college hospital), o.o.o. (university college hospital), confirmed they have contributed equally to the intellectual content of this paper and have met the following three requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the article. references top ↑ 1. carpenter s, rigard m, barile m, et al. an interlinear transliteration and english translation of portions of the ebers papyrus. new york: annadale-on-hudson; 1998.2. world health organization. definition, diagnosis and classification of diabetes mellitus and its complications. [page on internet]. [c2006] [cited 2011 jan 30]. available from http://apps.who.int/iris/bitsteam/10665/66040/1/who_ncd_ncs_99.2.pdf 3. international diabetes federation. idf diabetes atlas. 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[page on internet] [c2006] [cited 2011 aug 30]. available from http://www.who.int/bmi/index.jsp?intropage=intro13.html 24. perry jr, weedon mn, langenberg c, et al. genetic evidence that raised sex hormone binding globulin (shbg) levels reduce the risk of type 2 diabetes. hum mol genet. 2010;19(3):535–544. http://dx.doi.org/10.1093/hmg/ddp522, pmid:19933169, pmcid:2798726 25. ding el, song y, manson je, et al. sex hormone-binding globulin and risk of type 2 diabetes in women and men. n engl j med. 2009;361(12):1152–1163. http://dx.doi.org/10.1056/nejmoa0804381, pmid:19657112, pmcid:2774225 26. american diabetes association. standard of medical care in diabetes–2010. diabetes care. 2010;33(suppl 1):s11–s61. http://dx.doi.org/10.2337/dc10-s011, pmid:20042772, pmcid:2797382 27. haffner sm, stern mp, mitchell bd. incidence of type ii diabetes in mexican americans predicted by fasting insulin and glucose levels, obesity, and body fat distribution. diabetes. 1990;39(3):283–288. http://dx.doi.org/10.2337/diabetes.39.3.283, pmid:2407581 28. lundgren h, bengtsson c, blohme g, et al. adiposity and adipose tissue distribution in relation to incidence of diabetes in women: results from a prospective population study in gothenburg, sweden. int j obes. 1989;13(4):413–423. pmid:2793297 29. selva dm, hogeveen kn, innis sm, et al. monosaccharide-induced lipogenesis regulates the human hepatic sex hormone-binding globulin gene. j clin invest. 2007;117(12):3979–3987. pmid:17992261, pmcid:2066187 30. plymate sr, matej la, jones re, et al. inhibition of sex hormone binding globulin production in the human hepatoma (hep g2) cell line by insulin and prolactin. j clin endocrinol metab. 1988;67(3):460–464. http://dx.doi.org/10.1210/jcem-67-3-460, pmid:2842359 31. nestler je, powers lp, matt dw, et al. a direct effect of hyperinsulinemia on serum sex hormone-binding globulin levels in obese women with the polycystic ovary syndrome. j clin endocrinol metab. 1991;72(1):83–89. http://dx.doi.org/10.1210/jcem-72-1-83, pmid:1898744 32. anderson dc. sex-hormone-binding globulin. clin endocrinol (oxf.). 1974;3(1):69–96. http://dx.doi.org/10.1111/j.1365-2265.1974.tb03298.x 33. vermeulen a. physiology of the testosterone-binding globulin in man. ann n y acad sci. 1988;538:103–111. http://dx.doi.org/10.1111/j.1749-6632.1988.tb48855.x, pmid:3056178 34. sacks db, arnold m, bakris gl, et al. guidelines and recommendations for laboratory analysis in the diagnosis and management of diabetes mellitus. diabetes care. 2011; 34(6):e61–99. http://dx.doi.org/10.2337/dc11-9998, pmid:21617108, pmcid:3114322 35. wallace tm, levy cj, matthew rd. use and abuse of homa modeling. diabetes care. 2004;27(6):1487–1495. http://dx.doi.org/10.2337/diacare.27.6.1487, pmid:15161807 abstract introduction methods results discussion acknowledgements references about the author(s) rucita severaj discipline of chemical pathology, faculty of laboratory medicine, national health laboratory service and university of kwazulu-natal, durban, south africa verena gounden discipline of chemical pathology, faculty of laboratory medicine, national health laboratory service and university of kwazulu-natal, durban, south africa citation severaj r, gounden v. effect of covid-19 lockdown on glycated haemoglobin testing and utilisation in kwazulu-natal, south africa. afr j lab med. 2024;13(1), a2302. https://doi.org/10.4102/ajlm.v13i1.2302 note: additional supporting information may be found in the online version of this article as online supplementary document 1. original research effect of covid-19 lockdown on glycated haemoglobin testing and utilisation in kwazulu-natal, south africa rucita severaj, verena gounden received: 27 aug. 2023; accepted: 27 feb. 2024; published: 13 may 2024 copyright: © 2024. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: diabetic monitoring and treatment guidelines are easily accessible, but compliance is poor in kwazulu-natal. the coronavirus disease 2019 (covid-19) pandemic had a devastating impact on diabetic healthcare, both directly and through public health interventions. objective: this study aimed to close the gaps in knowledge concerning glycated haemoglobin (hba1c) test utilisation and how this was affected by the covid-19 lockdown in kwazulu-natal. methods: we reviewed hba1c test volumes and results from public health facilities in the 11 health districts in kwazulu-natal, south africa. we compared testing trends and glycaemic control between two 10-month study periods before (march 2019 – december 2019) and during (march 2020 – december 2020) the covid-19 pandemic. results: the number of hba1c tests performed decreased 6.1% during the pandemic period, with 173 760 hba1c tests performed in 2019 and 163 236 hba1c tests performed in 2020. there was a statistically significant increase in the average hba1c level during the pandemic (mean hba1c level in the pre-pandemic period: 70.5 mmol/mol [8.6%] versus mean hba1c level in the pandemic period: 72.7 mmol/mol [8.8%]; p-value < 0.001). of patients with suboptimal hba1c results (83 421 in 2019, 83 259 in 2020), 11 656 (14.0%) in 2019 and 10 086 (12.1%) in 2020 had more than one hba1c test performed during the study period. conclusion: the covid-19 pandemic impacted glycaemic monitoring in kwazulu-natal with lower hba1c test volumes and worse glycaemic control seen during the pandemic. hba1c testing practices are not in keeping with recommended guidelines. what this study adds: the study demonstrates that the covid-19 pandemic impacted hba1c utilisation in kwazulu-natal. importantly, hba1c testing practices in kwazulu-natal are not in keeping with society for endocrinology, metabolism and diabetes of south africa guidelines regarding the monitoring of diabetic patients, and this requires more attention for future diabetic healthcare interventions. keywords: diabetes; glycated haemoglobin; hba1c; glycaemic control; covid-19; south africa. introduction diabetes mellitus poses a major public health concern worldwide, especially in developing countries where diabetes prevalence is increasing at an alarming rate.1 in 2020, the international diabetes federation estimated that over 4.5 million adult south africans were living with diabetes.2 of greater concern is that an estimated 66.7% of diabetes cases in sub-saharan africa are undiagnosed, the highest of any other region in the world.3 kwazulu-natal is south africa’s second most populous province, with an estimated population of 11.5 million (19% of the south african population) people.4 the burden of diabetes mellitus in kwazulu-natal is increasing, and poor glycaemic control is a major contributor to this.5,6 the society for endocrinology, metabolism and diabetes of south africa’s 2017 guidelines define optimal glycaemic control as glycated haemoglobin (hba1c) levels < 53 mmol/mol (< 7.0%), which is in keeping with global recommendations.3 only 8.3% of diabetic patients attending a regional hospital in kwazulu-natal and 5.3% of diabetic patients managed at a central hospital in kwazulu-natal achieved optimal glycaemic control.7,8 hba1c testing is the standard of care for the monitoring of treatment and glycaemic control in diabetic patients, both globally and within south africa.3,9 hba1c is preferred as it reflects the average plasma glucose over the previous 8 weeks to 12 weeks and can be performed at any time of the day, not requiring special preparations such as fasting.10 the society for endocrinology, metabolism and diabetes of south africa’s 2017 guidelines recommend six-monthly hba1c testing in patients with stable glycaemic control and threeto six-monthly hba1c testing in those who have yet to achieve optimal control.3 compliance with diabetic monitoring and treatment guidelines in public health institutions in kwazulu-natal is generally poor, as demonstrated by a study performed in a regional hospital in 2010 which found that monitoring and treatment guidelines were not followed in up to 80% of patients with poor glycaemic control,11 possibly due to lack of continuity of care and deficiencies of knowledge regarding recommended guidelines. this is problematic, because reduced hba1c testing frequency is significantly associated with poor diabetes control,12 whilst adequate monitoring of diabetes patients is known to improve glycaemic control.13 the coronavirus disease 2019 (covid-19) pandemic had a devastating effect on healthcare globally, both directly and indirectly. in addition to causing severe illness and resulting in the deaths of millions of people throughout the world, responses to the pandemic have had far-reaching consequences on the health and well-being of patients. these indirect effects occurred due to government responses to the pandemic, including lockdowns and the lifestyle changes associated with this, changed priorities of medical and surgical procedures, cessation of screening programmes, altering medical facilities to covid-19 centres, public fear of seeking healthcare, and reduced referrals.14 a world health organization survey found that disruption to healthcare service, especially primary healthcare, was greatest among lower-income countries where overburdened healthcare systems, high rates of both communicable and non-communicable diseases, and poor socioeconomic determinants allowed the pandemic to exacerbate pre-existing vulnerabilities.15,16 in south africa, lockdown measures were implemented based on ‘alert’ levels, with the most restrictive measures seen in march 2020 – august 2020. these measures directly affected ambulatory clinic visitations and the management of many chronic diseases. health services relating to reproductive health, maternal and child health, hiv and tuberculosis, and diabetes and hypertension were the hardest hit because of decreased outpatient visits during this period.17,18 several studies have investigated the effects of the covid-19 lockdown on hba1c testing and glycaemic control, with highly variable results. studies performed in india and turkey reported up to 70% reduction in hba1c testing volumes for outpatients during the lockdown, with an associated increase in hba1c levels.19,20,21 others studies performed in denmark and italy found no significant difference in glycaemic control during the lockdown periods in their respective countries,22,23 and other studies from the united kingdom and spain even demonstrated improved glycaemic control during lockdown periods.24,25 improved control was often seen in developed, higher-income countries where telemedicine, home hba1c monitoring, and mobile phlebotomy units are available and easily accessible.26,27 only a few studies have investigated hba1c utilisation in diabetic patients managed at public health facilities in kwazulu-natal and how this relates to glycaemic control and established practice guidelines. most of the published studies were performed at an institutional level, with little information at the provincial or district levels. in addition, no local studies have investigated the effect of the covid-19 lockdown on hba1c testing and glycaemic control. this information is essential for public health planning and policy development, especially in light of increased demands on smaller healthcare budgets. this study thus attempts to close the gaps in knowledge concerning hba1c testing and glycaemic control, and how these parameters have been affected by the covid-19 lockdown in kwazulu-natal. methods ethical considerations ethics approval for this study was obtained from the university of kwazulu-natal, biomedical research ethics committee (brec/00004109/2022). approval to conduct research was also obtained from the kwazulu-natal department of health (kz_202206_024). the requirement for patient consent was waived by the ethics committee. data were collected on a password-protected computer accessible by only the primary investigator. patients were identified by unique patient identification numbers which did not contain identifiable patient data and their identities were not revealed. data collection hba1c data from all national health laboratory service laboratories in the province of kwazulu natal were extracted from the national health laboratory service central data warehouse. the 11 health districts included were amajuba, ethekwini, harry gwala, ilembe, king cetshayo, ugu, umgungundlovu, umkhanyakude, umzinyathi, uthukela and zululand. data from two 10-month study periods were included. period 1 (01 march 2019 to 31 december 2019) was the pre-pandemic period, and period 2 (01 march 2020 to 31 december 2020) was during the pandemic. two methods were used to analyse hba1c across all laboratories: ion exchange high performance liquid chromatography and immunoassay. all methods were national glycohemoglobin standardization program traceable, and there was harmonisation between methods allowing for comparison of results from different laboratories. variables extracted included demographic information, specifically the age and sex of the patient, district and facility name, clinical diagnosis, date reviewed, hba1c result (percentage) and hba1c text which provided information regarding less than (<) or greater than (>) hba1c results and hba1c variants. all patients with at least one hba1c result within the study periods were included. results with a lower than detectable value, unfeasible results, and hba1c tests where a result was not provided, such as in the case of interfering haemoglobin variants, were excluded. based on their infrastructure and development, the ethekwini, ilembe, umgungundlovu and ugu districts were classified as urban, and the other districts were classified as rural. ‘strict’ lockdown levels were defined as all alert levels greater than alert level 1. data analysis data analysis was carried out using microsoft excel 2016 (microsoft corporation, redmond, washington, united states). we determined the cumulative total number of hba1c tests conducted during each study period, both as an overall aggregate and separately for each of the 11 health districts in kwazulu-natal. we determined the impact of the pandemic by comparing the testing volumes, average hba1c levels, and the percentage of abnormal hba1c results (defined as hba1c levels > 53 mmol/mol [> 7.0%]) in both period 1 and period 2. we also plotted the number of hba1c test results that fell within different hba1c ranges, and compared pre-pandemic results with results from during the pandemic. statistical significance was determined with the use of a student t-test for the average hba1c level, and a chi squared test for the percentage of abnormal hba1c results. a p-value of < 0.05 was considered significant. to gauge whether hba1c utilisation was in keeping with local guidelines, we looked at the total number of patients who had repeat hba1c tests performed within the study periods. we also looked at the number of patients who had a single hba1c test that was performed within the first 4 months (march – june) of a study period. these data were extracted using unique patient identification numbers. we also looked particularly at the number of patients with suboptimal hba1c levels, which we defined as an hba1c > 58.5 mmol/mol (> 7.5%), who had repeat hba1c tests performed within the study periods. results a total of 174 076 hba1c tests were performed between march 2019 and december 2019 compared to 163 626 tests performed during the same period in 2020 (figure 1). of these, 307 tests conducted in 2019 and 390 conducted in 2020 were excluded. excluded results included 109 lower than detectable results in 2019 and 209 in 2020, 198 interfering hba1c variants in 2019 and 176 in 2020, and 5 unfeasible results in 2019 and 9 in 2020. unfeasible hba1c results included results of 0.0% and 1065.0%. in total, 173 769 hba1c test results for 2019 and 163 236 results for 2020 were included in this study. figure 1: inclusion criteria applied to glycated haemoglobin (hba1c) test results of all patients attending public health facilities in kwazulu-natal, south africa, 01 march 2019 – 31 december 2019 and 01 march 2020 – 31 december 2020. approximately two-thirds (67%) of the results were from female patients, and over half of the results (55%) were from patients aged between 50 years and 70 years (table 1). notably, the demographic distribution was similar in the pre-pandemic and pandemic periods. table 1: demographic data of patients attending public health facilities in kwazulu-natal, south africa, 01 march 2019 – 31 december 2019 and 01 march 2020 – 31 december 2020. a reduction in hba1c tests performed (6.1%) was seen in the pandemic period compared to the pre-pandemic period (table 2). most of the hba1c tests (50% in 2019 and 43% in 2020) came from the major metropolitan area of ethekwini, followed by umgungundlovu and ilembe. these three health districts were also the only ones to have had fewer hba1c tests performed during the pandemic compared to the pre-pandemic period. the other eight health districts had more hba1c tests in 2020. a significant increase in average hba1c of 2.2 mmol/mol (0.2%) was seen during the pandemic. nine of the 11 health districts demonstrated a higher average hba1c in 2020 compared to 2019. harry gwala district showed a decrease in average hba1c, but this was minimal (0.2%). table 2: number of glycated haemoglobin (hba1c) tests and average hba1c, and percent difference† of all patients attending public health facilities in kwazulu-natal, south africa, 01 march 2019 – 31 december 2019 and 01 march 2020 – 31 december 2020. the distribution of hba1c results by hba1c value range demonstrated fewer hba1c results in the low ranges (below 85.8 mmol/mol; 10%) in 2020 compared to the same period in 2019 (figure 2). conversely, there were more hba1c results in the high ranges (especially above 96.7 mmol/mol; 11%) in 2020 compared to 2019. figure 2: distribution of glycated haemoglobin (hba1c) results according to hba1c range of all patients attending public health facilities in kwazulu-natal, south africa, 01 march 2019 – 31 december 2019 and 01 march 2020 – 31 december 2020. there were much lower test volumes in the first few months of the pandemic (march, april, may, july and august 2020) compared to the same period in 2019 (figure 3). this trend was reversed during the pandemic, with higher test volumes recorded in september 2020, november 2020, and december 2020 compared to the same months in 2019. the hba1c test volume per month for each district followed a similar pattern as described for the total volumes (online supplementary figure 1). a significant increase in the percentage of abnormal hba1c results is seen during the pandemic for the months april 2020 – november 2020 (table 3). the highest difference in the percentage of abnormal hba1c results were seen in may 2020 – july 2020, the period following the strictest lockdown levels, alert levels 5 and 4. the percentage of abnormal results recorded per month in each district did not show much variation between the pre-pandemic and pandemic periods, (online supplementary table 1). five of the health districts had more abnormal results in 2020 than 2019 (online supplementary figure 2). figure 3: volume of glycated haemoglobin (hba1c) tests and percentage of abnormal results per month for all patients attending public health facilities in kwazulu-natal, south africa, 01 march 2019 – 31 december 2019 and 01 march 2020 – 31 december 2020. abnormal was defined as hba1c levels > 53 mmol/mol (> 7.0%). table 3: volume of glycated haemoglobin (hba1c) tests and percentage of abnormal results per month for all patients attending public health facilities in kwazulu-natal, south africa, 01 march 2019 – 31 december 2019 and 01 march 2020 – 31 december 2020. hba1c tests were conducted for 155 985 patients in 2019 and 148 447 patients in 2020 (figure 4). of these, only 15 359 (9.8%) patients in 2019 and 13 085 (8.8%) in 2020 had more than one hba1c test performed within the 10-month study period. suboptimal hba1c levels were reported for 83 421 (53.5%) patients in 2019 and 83 259 (56.0%) patients in 2020. of those patients, only 11 656 (14.0%) in 2019 and 10 086 (12.1%) in 2020 had more than one hba1c test performed. there were 54 235 patients in 2019 and 49 125 patients in 2020 who only had a single hba1c test performed within the study period and this test was within the first 4 months of the study period. two hba1c tests per patient (13 358/15 359 patients in 2019 and 11 582/13 085 patients in 2020) was the most common test frequency for patients who had multiple hba1c tests conducted (online supplementary table 2). figure 4: glycated haemoglobin (hba1c) testing frequency among patients attending public health facilities in kwazulu-natal, south africa, 01 march 2019 – 31 december 2019 and 01 march 2020 – 31 december 2020. suboptimal hba1c level was defined as > 58.5 mmol mol (> 7.5%). discussion this study demonstrated that the covid-19 pandemic did impact hba1c utilisation in patients attending public health facilities. lower hba1c test volumes were recorded during the pandemic in 2020 than during the same period pre-pandemic in 2019. a significant increase in the average hba1c level was seen during the pandemic as well as significantly higher abnormal hba1c results, particularly during periods of strict lockdown, when hba1c test volumes were at their lowest. it was also demonstrated that pre-pandemic and pandemic hba1c testing practices were not in keeping with established international and national guidelines regarding the monitoring of glycaemic control in diabetic patients. fewer hba1c tests are being performed on sub-optimally controlled diabetic patients than should be as per the guidelines. several studies have been conducted globally, particularly in high-income countries, to investigate the impact of the covid-19 pandemic on diabetes care. studies performed in the united kingdom in 2021 and canada in 2020, found that monthly hba1c requests during the pandemic dropped to 7.9% – 18.1% of pre-pandemic volumes and patients had a 28% lower probability of undergoing an hba1c test during the pandemic.28,29 studies conducted among north american patients in 2020 and 2021, respectively, found that hba1c testing frequency during the pandemic reduced by as much as 66%,30 and was at 75% – 85% of the rate of prior years’.31 the findings of our study mirrored these, where a reduction of hba1c test volumes was seen during the pandemic compared to the same period in 2019. however, we found this reduction to be more subtle, at 6.1%, compared to reductions of between 50% and 70% reported by these studies from higher-income settings.28,29,30,31 interestingly, reduced hba1c test volumes were only noted in two of the 11 kwazulu-natal health districts – ethekwini, the metropolitan (−17.5%) and ilembe (−14.8%). these districts have a relatively higher level of urban development in terms of setting and infrastructure. in the other more rural districts, increases in hba1c test volumes by as much as 33.4% (umzinyathi) were reported during the pandemic. although such increases have not been reported in other studies around the world, the paucity of studies performed in rural populations is a likely contributing factor. another consideration is that our study was performed using data from the public health service in kwazulu-natal, where remote forms of healthcare such as telemedicine were not available to the study population. the inability to consult remotely with healthcare practitioners meant that diabetic patients in kwazulu-natal continued with only in-person health consults. for the majority of patients from these rural and semi-urban areas, there would have been no alternatives to access healthcare and medication without face-to-face visits to the primary care clinics or hospitals. thus, the frequency of patient visits would not have been significantly decreased by the pandemic in these districts. whilst awareness by clinicians regarding the poorer outcomes of diabetic individuals infected by covid-19 may have increased the frequency of hba1c testing, this is not substantiated by the testing volumes across all the districts. the most notable decline in hba1c test volume was recorded between april 2020 and august 2020, during which the strictest levels of lockdown were in place in south africa. hba1c test volumes recovered in september 2020, and exceeded 2019 volumes in november 2020 and december 2020. this finding concurs with studies conducted in the united states, canada and spain between 2020 and 2021.19,31,32 despite the consistent evidence of decreased hba1c testing during the pandemic, studies have demonstrated variable effects of this on glycaemic control. studies conducted in china in 2022 and taiwan in 2021 found no significant difference in hba1c results between the pandemic and pre-pandemic periods.33,34 remarkably, a study conducted in spain in 2021 instead reported improved glycaemic control during the pandemic, and this was supported by two systematic reviews and meta-analyses performed in the united kingdom and germany in 2021.35,36,37 in contrast to the findings of these studies, we found a significant increase in the average hba1c result during the pandemic of 2.2 mmol/mol (0.2%) overall and in nine of the districts, indicating poorer glycaemic control during the pandemic where decreased numbers of hba1c tests were being conducted. notably, the harry gwala district, which had an increase of 31.8% in hba1c tests performed during the pandemic, also demonstrated a significantly improved pandemic average hba1c result. this study also found that there were more patients with high-range hba1c levels during the pandemic than before the pandemic, an indication of poorer glycaemic control in sub-optimally controlled patients. this may suggest lack of prioritisation of poorly controlled diabetic patients during the pandemic similar to the findings of holland et al. from the united kingdom in 2021.28 considerably fewer studies have investigated the effect of the pandemic on glycaemic control in diabetic patients in lowand middle-income settings. most of those performed were concerned with poor health outcomes in diabetic patients with covid-19 and had small sample sizes and cohorts of admitted patients during the pandemic.38,39,40,41 a study performed in a lower-middle income setting in south asia in 2020 demonstrated a decrease in mean hba1c level by 0.3% during the pandemic and found improved hba1c results in 37.6% of participants.42 a south african study performed in gauteng province in 2021, found similar findings to our study, where they showed decreased hba1c testing volumes during the covid-19 lockdown periods and, while it reported variability in hba1c results and glycaemic control between these pre-pandemic and pandemic periods, the significance of this was not determined.40 a smaller study which included only 28 participants, conducted in 2020 in the western cape, south africa reported a sharp decline in hba1c test requests compared to the same period the year before; however, they did not quantify this decline or explore how this related to glycaemic control.41 while our study demonstrated similar decreased hba1c test volumes during the pandemic as described by these three studies, the finding of a significantly increased average hba1c result between pre-pandemic and pandemic periods is novel in this middle-income setting. our study also demonstrated that significantly more abnormal hba1c results were recorded from april 2020 to august 2020, during the stricter lockdown periods, compared to pre-pandemic values from the same period in 2019. this relates to the same period where the most notable decreases in hba1c testing are seen. this increase in abnormal hba1c results during these ‘hard’ lockdown periods may be related to both the indirect effects of the pandemic, such as the reduced access to healthcare, and reduced glycaemic monitoring. it is also possibly a consequence of the direct effects of covid-19 on patients living with diabetes; however, without access to comorbidity data, we were unable to test this hypothesis. the latest guidelines published in the journal of endocrinology, metabolism and diabetes of south africa in 2017 recommend 6-monthly hba1c testing in patients with stable control and 3-monthly testing in those with suboptimal glycaemic control.3 the proportion of patients who had multiple hba1c tests performed was alarmingly low in both the pre-pandemic period (9.8%) and the pandemic period (8.8%). notably, 54 235 patients (35.0% of all patients) in 2019 and 49 125 patients (33.0% of all patients) in 2020 had an hba1c performed within the first 4 months of their respective study periods, yet did not have a repeat hba1c performed. this is a clear indication that guidelines are not being followed appropriately. of the small number of patients who had multiple hba1c tests performed, the overwhelming majority had two hba1c tests done, which may be appropriate in well-controlled patients. however, considering that over 50% of patients (53.5% in 2019 and 56.0% in 2020) included in each study period had suboptimal hba1c levels (defined as hba1c > 7.5%), this low frequency of hba1c testing is more noteworthy. the low rate of follow-up hba1c tests being performed overall is of great concern and is a likely contributor to the high burden of diabetes morbidity and mortality experienced in south africa. this finding highlights a crucial gap in the management of diabetic patients in kwazulu-natal and identifies an area of focus for future interventions to improve diabetic care in the province and country. the strength of our study lies in its uniqueness, as studies of this magnitude investigating hba1c utilisation in lowand middle-income settings are very few. no such study has also included results from two 10-month periods. the 10-month pandemic period chosen for this study included the period when south africa implemented strict lockdown measures as well as some months after the lockdown was lifted, thus allowing us to review the effect of the lockdown on hba1c testing practices. additionally, the study included both paediatric and adult populations in whom hba1c testing is equally important. this study also analysed the data per health district in the province, thus providing insights into hba1c utilisation in each province during the pandemic and allowing us to discriminate between urban and rural populations. limitations one limitation of this study is that we considered a single indicator of glycaemic control, hba1c. hba1c is accepted as the preferred test for the monitoring of glycaemic control in diabetic patients and because we were looking at a large number of results over 20 months, we chose to analyse a single and robust indicator. another limitation of the study is the lack of clinical information available to us due to the method of data collection used. as such we were unable to differentiate hba1c tests performed for diagnosis or monitoring. conclusion hba1c utilisation in kwazulu-natal health districts decreased during the covid-19 pandemic when public health interventions restricted access to routine medical care. this decrease, however, was much lower than seen in higher-income settings, where remote healthcare services such as telemedicine are accessible. the decreased hba1c testing during the pandemic was a possible contributor to significant changes in glycaemic control seen. most concerning, hba1c utilisation in general is not in keeping with established diabetic monitoring guidelines. acknowledgements the authors would like to acknowledge the national health laboratory services laboratory information system (trak care) central data warehouse for the data retrieval. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions v.g. conceived the study. r.s. and v.g. researched the literature. r.s. was involved in gaining ethical approval. r.s. and v.g. were involved in protocol development and data analysis. r.s. wrote the first draft of the manuscript. r.s. and v.g. reviewed and edited the manuscript and approved the final version of the manuscript. sources of support the authors received no financial support for the research, authorship, or publication of this article. data availability raw data were extracted from the national health laboratory service central data warehouse. testing data may be made available on request from the corresponding author, r.s. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. it does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the authors are responsible for this article’s results, findings, and content. references sifunda s, mbewu ad, mabaso m, et al. prevalence and psychosocial correlates of diabetes mellitus in south africa: results from the south african national health and nutrition examination survey (sanhanes-1). int j environ res public health. 2023;20:5798. https://doi.org/10.3390/ijerph20105798 international diabetes federation. international diabetes federation: diabetes in south africa (2021). 2021 [cited 2023 dec 02]. available from: https://idf.org/our-network/regions-and-members/africa/members/south-africa/ amod a, dave j, mohammed n, et al. semdsa 2017 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during the sars-cov-2 pandemic on the management of diabetes in a northern gauteng region of south africa. pan afr med j. 2023;45:129. 20230718. https://doi.org/10.11604/pamj.2023.45.129.39255 delobelle pa, abbas m, datay i, et al. non-communicable disease care and management in two sites of the cape town metro during the first wave of covid-19: a rapid appraisal. afr j prim health care fam med. 2022;14(1):e1–e7. https://doi.org/10.4102/phcfm.v14i1.3215 dissanayake h, soysa p, samarathunga t, et al. impact of covid-19 lockdown on people living with diabetes: experience from a low-middle income country in south asia. prim care diabetes. 2022;16(1):127–134. https://doi.org/10.1016/j.pcd.2021.12.003 abstract introduction methods results discussion acknowledgements references about the author(s) vuyolwethu fadana school of pathology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa department pathology, national health laboratory services, johannesburg, south africa teena thomas school of pathology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa infectious control services laboratory, national health laboratory services, johannesburg, south africa nina von knorring school of pathology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa mycobacteriology referral laboratory, national health laboratory service, johannesburg, south africa citation fadana v, thomas t, von knorring n. retrospective analysis of vitek®2 performance compared to manual broth micro-dilution for colistin susceptibility testing of acinetobacter baumannii complex isolates in south africa. afr j lab med. 2022;11(1), a1597. https://doi.org/10.4102/ajlm.v11i1.1597 brief report retrospective analysis of vitek®2 performance compared to manual broth micro-dilution for colistin susceptibility testing of acinetobacter baumanniicomplex isolates in south africa vuyolwethu fadana, teena thomas, nina von knorring received: 29 mar. 2021; accepted: 27 oct. 2021; published: 28 feb. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract the manual broth micro-dilution (mbmd) is the recommended reference method for colistin minimum inhibitory concentration determination; however, it is not as readily available in south africa as the vitek®2. this retrospective study compared the performance of vitek®2 against mbmd in determining the colistin minimum inhibitory concentration of 337 extensively drug-resistant acinetobacter baumannii complex isolates. vitek®2 yielded a categorical agreement of 89%, an essential agreement of 56%, a major error rate of 8% and a very major error rate of 55%. the vitek®2 is not an alternative to mbmd for colistin susceptibility testing. keywords: acinetobacter; colistin; broth micro-dilution; vitek®2; antimicrobial susceptibility testing. introduction the increasing antimicrobial resistance in the acinetobacter baumannii complex, and the recently observed resistance to the polymyxin antibiotic (colistin), demands timeous identification and antimicrobial susceptibility profiling of causal pathogens to ensure timely and appropriate antimicrobial therapy decisions.1,2 different laboratory methods for the assessment of colistin susceptibility testing among a. baumannii isolates have been assessed.3 since 2016, the reference colistin susceptibility testing method recommended by the clinical and laboratory standards institute and the european committee on antimicrobial susceptibility testing is the international organization for standardization (iso) broth micro-dilution (bmd) method (iso-20776).4 implementing manual bmd (mbmd) is currently not feasible in the routine microbiology laboratory due to its laborious nature.5 the vitek®2 (biomerieux inc., marcy l’étoile, france) is an automated microbial identification and antimicrobial susceptibility testing (ast) platform. most public and private south african laboratories use the vitek®2, making it an attractive alternative to the mbmd. however, studies comparing vitek®2 and the mbmd method have reported discordant colistin susceptibility results. dafopoulou et al. compared the mbmd with polysorbate-80, vitek®2, etest, agar dilution, and the minimum inhibitory concentration (mic) methods for the colistin susceptibility testing of 51 klebsiella pneumoniae and 20 a. baumannii clinical isolates.6 eighteen of the a. baumannii isolates were colistin-resistant by mbmd, and vitek®2 categorically agreed in 85% and essentially agreed in 90% of the a. baumannii isolates. there were no ‘very major errors’ (vme) reported. these findings were similar to those obtained by lo-ten-foe et al.7 piewngam and kiratisin also observed a low vme rate of 0.7% when testing 290 a. baumannii isolates.8 these findings suggest that vitek®2 could be a viable alternative to the mbmd. in contrast to these results, vourli et al. reported unacceptably high vme rates for phoenix100 and vitek®2 against the mbmd (41.4% and 37.9%).9 additionally, in 2017 biomerieux retracted the use of vitek®2 for colistin testing owing to the clinical and laboratory standards institute-european committee on antimicrobial susceptibility testing recommendations and an in-house observed performance issue: high vme rate against agar dilution and mbmd methods.10 due to these contradictory findings in the literature, further research into this area was warranted. this study aimed to compare the performance of vitek®2 colistin susceptibility testing to mbmd for clinical extensively drug-resistant (xdr) a. baumannii complex isolates at charlotte maxeke johannesburg academic hospital in johannesburg, south africa. methods ethical considerations ethical approval was obtained from the university of the witwatersrand human research ethics committee (clearance certificate number m191048 med 19-10-043). data were anonymised before analysis to maintain patient confidentiality. patient consent was not required. approval to utilise patient data was obtained from the chief executive officer of charlotte maxeke johannesburg academic hospital. data collection this was a descriptive, retrospective analysis of the a. baumannii complex isolated from charlotte maxeke johannesburg academic hospital inpatients between 01 january 2017 and 30 june 2019. the xdr a. baumannii complex isolates were resistant to one or more agents in all but one or two categories of antibiotics. microbiological data, including the sample type, vitek®2 and mbmd colistin mic results, were extracted from the corporate data warehouse, a division of the national health laboratory service (nhls). isolate identification and ast microbiology services within the hospital are provided by the nhls. the identification of isolates within the institution was performed using either the vitek®2 (biomerieux inc., marcy l’étoile, france) gram-negative identification (gn id) card or matrix-assisted laser desorption ionisation-time of flight mass spectrometry. these methods are unable to differentiate species within the a. baumannii complex. routine ast was performed using the kirby-bauer disc diffusion susceptibility method or vitek®2 ast-n256 card. isolates that had ast by the former method were excluded from the study. quality control strains and pure cultures of test isolates were used for the vitek®2 ast. isolates were further tested using mbmd when colistin therapy was considered, that is, for clinically significant xdr a. baumannii complex isolates. manual bmd was performed by trained personnel with appropriate controls according to the iso-20776 recommendation. isolates that were colistin-resistant by mbmd were then sent to a reference laboratory for mcr 1-5 testing (data not shown). data analysis all xdr a. baumannii complex isolates cultured between 01 january 2017 and 30 june 2019 were analysed. no patient admission data was available to discriminate between community-acquired and hospital-acquired infections. isolates that were obtained from outpatient departments or without the ward specified were excluded. duplicate patient samples, such as blood cultures collected within two weeks and other sample types collected within one month of the initial sample, were excluded. intravenous central venous catheter tips were not included as they were processed in a separate laboratory using a different automated ast platform. only isolates with both vitek®2 and mbmd colistin susceptibility results were included. the performance of vitek®2 colistin susceptibility testing was determined by evaluating the categorical and essential agreements and the major and vme rates in comparison to the mbmd colistin susceptibility testing method, according to the food and drug administration (fda) recommendations.11 microsoft® excel 2016 (microsoft corporation, redmond, washington, united states) was used for data analysis. the following equations were employed to assess agreement and error rates: categorical agreement = (number of isolates correctly classified by vitek®2 as either colistin susceptible or resistant in comparison to mbmd ÷ total number of isolates tested) × 100     [eqn 1] essential agreement = (number of isolates within one doubling dilutions of the mbmd mic on vitek®2 ÷ total number of isolates tested) × 100     [eqn 2] major error rate = (number of falsely resistant isolates on vitek®2 ÷ number of susceptible isolates by mbmd) × 100     [eqn 3] very major error rate = (number of falsely susceptible isolates by vitek®2 ÷ number of resistant isolates by mbmd) × 100     [eqn 4] results data for 523 isolates were obtained from all specimen types that harboured xdr a. baumannii complex and were submitted for colistin mbmd. after appropriate exclusions, 337 (64%) isolates had both vitek®2 and mbmd mic results (figure 1). figure 1: isolate selection and inclusion for vitek®2 colistin susceptibility assessment. of the 337 isolates with both vitek®2 and mbmd colistin susceptibility results, 20 (6%) were resistant to colistin by mbmd. the highest proportion of colistin-resistant acinetobacter was isolated from tracheal aspirates and swabs (figure 2). figure 2: distribution of colistin-resistant and susceptible xdr a. baumannii complex isolates by sample type (n = 337). vitek®2 was found to have a categorical agreement of 89% (300/337) and an essential agreement of 56% (190/337) with mbmd. the vme rate was 55% (11/20) and the major error rate was 8% (26/317) (figure 3). figure 3: colistin minimum inhibitory concentration (µg/ml) results for xdr a. baumannii complex isolates by both vitek®2 and manual broth micro-dilution. discussion colistin susceptibility testing by mbmd according to iso-20776 is difficult to implement in routine microbiology laboratories. due to financial constraints and the technical competencies required, only one nhls laboratory can offer mbmd in south africa. this is likely to impair patient care due to delays in turnaround times of results. in contrast, vitek®2 is available in most nhls microbiology laboratories and serves as an alternative. however, our study demonstrates unacceptable performance, with 11 of 20 (55%) colistin-resistant isolates being falsely susceptible by vitek®2. the vitek®2 also falsely reported some isolates with mbmd colistin mic of > 64 µg/ml as susceptible. this has the potential to result in inappropriate antimicrobial therapy and adverse patient outcomes. this extremely high vme contrast with previous studies mentioned earlier. however, those studies included fewer a. baumannii isolates compared to our study. in addition to the unacceptable vme rate, the categorical agreement, essential agreement and the major error rates with vitek®2 were also unacceptable according to the fda requirements for an ast testing platform.11 attempts to make mbmd more readily available and easier to implement until other testing options become available are required. matuschek et al. evaluated five recently developed commercial bmd systems – sempa1 (sensititre™ custom plate [thermo fisher scientific, east grinstead, united kingdom], micronaut-s and micronaut mic-strip [merlin diagnostika gmbh, bornheim, germany], sensitest™ [liofilchem, roseto degli abruzzi, italy] and umic [biocentric, bandol, france]).12 these were evaluated against mbmd using various gram-negative organisms including 22 acinetobacter spp isolates.12 they demonstrated overall better performance compared to our findings with vitek®2. the majority of the platforms had acceptable categorical agreement and essential agreement (> 90%), with significantly lower error rates than obtained in this study. interestingly, there were no vmes detected in the a. baumannii isolates. however, this study tested fewer isolates than our study. these methods may offer an alternative to mbmd and further research on their performance is required. limitations only a small number of colistin-resistant isolates were obtained for the study. analysis of larger numbers of resistant isolates with wider mic distribution is required to confirm our finding of high vmes. conclusion based on the results of this study, vitek®2 is not an alternative for mbmd for colistin ast in our setting. further studies are required to determine if the commercially available colistin bmd methods are a cost-effective option with acceptable analytical performance. additionally, the semi-automated platforms such as vitek®2 should be better optimised for colistin ast. acknowledgements the co-authors played an invaluable role in completion of this work. gratitude also goes to the employees of the nhls who ensured that the data used for this study were of high quality. competing interests the authors declare that there were no competing interests, financial or otherwise, that may have affected the writing of this article. authors’ contributions v.f. conceptualised the idea of the manuscript, was the primary author and collated co-authors’ feedback. t.t. contributed to the write-up of the article and provided critical feedback. n.v.k. contributed to the write-up of the article and provided critical feedback. sources of support the authors received no funding for the research or write-up of this study. data availability the data that support the findings made in this study can be made available from the corresponding author, v.f., on request. disclaimer the views expressed in this study are those of the authors and are not an official position of the university of the witwatersrand. references almasaudi sb. acinetobacter spp. as nosocomial pathogens: epidemiology and resistance features. saudi j biol sci. 2018 mar 1;25(3):586–596. https://doi.org/10.1016/j.sjbs.2016.009 ahmed ss, alp e, hopman j, voss a. global epidemiology on colistin-resistant acinetobacter baumannii. j infect dis ther. 2016;4(4):287. https://doi.org/10.4172/2332-0877.1000287 bakthavatchalam y, pragasam a, biswas i, veeraraghavan b. polymyxin susceptibility testing, interpretative breakpoints and resistance mechanisms: an update. j glob antimicrob resist. 2018;12:124–136. https://doi.org/10.1016/j.jgar.2017.09.011 clsi-eucast polymyxin breakpoints working group. recommendations for mic determination of colistin (polymyxin e) [homepage on the internet]. eucast; 2016 [cited 2020 feb 03]. available from: http://www.eucast.org/fileadmin/src/media/pdfs/eucast_files/general_documents/recommendations_for_mic_determination_of_colistin_march_2016.pdf poirel l, jayol a, nordmann p. polymyxins: antibacterial activity, susceptibility testing, and resistance mechanisms encoded by plasmids or chromosomes. clin microbiol rev. 2017 apr 1;30(2):557–596. https://doi.org/10.1128/cmr.00064-16 dafopoulou k, zarkotou o, dimitroulia e, et al. comparative evaluation of colistin susceptibility testing methods among carbapenem-nonsusceptible klebsiella pneumoniae and acinetobacter baumannii clinical isolates. antimicrob agents chemother. 2015 aug 1;59(8):4625–4630. https://doi.org/10.1128/aac.00868-15 lo-ten-foe jr, de smet am, diederen bm, kluytmans ja, van keulen ph. comparative evaluation of the vitek 2, disk diffusion, etest, broth microdilution, and agar dilution susceptibility testing methods for colistin in clinical isolates, including heteroresistant enterobacter cloacae and acinetobacter baumannii strains. antimicrob agents chemother. 2007 jul 23;51(10):3726–3730. https://doi.org/10.1128/aac.01406-06 piewngam p, kiratisin p. comparative assessment of antimicrobial susceptibility testing for tigecycline and colistin against acinetobacter baumannii clinical isolates, including multidrug-resistant isolates. int j antimicrob agents. 2014 nov 1;44(5):396–401. https://doi.org/10.1016/j.ijantimicag.2014.06.014 vourli s, dafopoulou k, vrioni g, tsakris a, pournaras s. evaluation of two automated systems for colistin susceptibility testing of carbapenem-resistant acinetobacter baumannii clinical isolates. j antimicrob chemother. 2017 jun 12;72(9):2528–2530. https://doi.org/10.1093/jac/dkx186 biomerieux. urgent product correction notice [homepage on the internet]. [cited 2020 mar 26]. available from: https://www.bfarm.de/shareddocs/kundeninfos/en/08/2017/04963-17_kundeninfo_en.pdf?__blob=publicationfile&v=1 us food and drug administration. class ii special controls guidance document: antimicrobial susceptibility test (ast) systems; guidance for industry and fda. rockville, md: us food and drug administration; 2003. matuschek e, åhman j, webster c, kahlmeter g. antimicrobial susceptibility testing of colistin–evaluation of seven commercial mic products against standard broth microdilution for escherichia coli, klebsiella pneumoniae, pseudomonas aeruginosa, and acinetobacter spp. clin microbiol infect. 2018 aug 1;24(8):865–870. https://doi.org/10.1016/j.cmi.2017.11.020 abstract introduction methods results discussion conclusion acknowledgements references about the author(s) oluwalana t. oyekale department of medical microbiology and parasitology, faculty of medicine and health sciences, afe babalola university, ado ekiti, nigeria department of medical microbiology and parasitology, federal teaching hospital, ido-ekiti, nigeria bola o. ojo department of medical microbiology and parasitology, federal teaching hospital, ido-ekiti, nigeria adewale t. olajide department of surgery, faculty of medicine and health sciences, afe babalola university, ado ekiti, nigeria department of surgery, federal teaching hospital, ido-ekiti, nigeria oluwatoyin i. oyekale department of radiology, federal teaching hospital, ido-ekiti, nigeria citation oyekale ot, ojo bo, olajide at, oyekale oi. bacteriological profile and antibiogram of blood culture isolates from bloodstream infections in a rural tertiary hospital in nigeria. afr j lab med. 2022;11(1), a1807. https://doi.org/10.4102/ajlm.v11i1.1807 original research bacteriological profile and antibiogram of blood culture isolates from bloodstream infections in a rural tertiary hospital in nigeria oluwalana t. oyekale, bola o. ojo, adewale t. olajide, oluwatoyin i. oyekale received: 01 dec. 2021; accepted: 25 may 2022; published: 24 aug. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: bloodstream infections (bsis) are a cause of significant morbidity and mortality requiring urgent antibiotic treatment. however, there is widespread antibiotic-resistance from the bacterial causes, necessitating regular surveillance for drug-resistant bacteria and their antibiograms. objective: this study isolated and identified various bacterial causes of bsis, determined their antibiotic susceptibility patterns, and determined the best empirical treatment for cases of bsi in the setting. methods: a cross-sectional study was carried out at the federal teaching hospital, ido-ekiti, nigeria between june 2020 and february 2021 on 177 blood culture samples from cases of bsi. identification of isolated bacteria and antibiotic susceptibility testing of the isolates were carried out following the standard protocol. results: culture positivity in this study was 19.2%. no significant difference was seen in culture positivity between male and female participants (p = 0.97). gram-negative enteric bacteria were predominantly isolated (67.6%), including escherichia coli (29.4%) and klebsiella aerogenes (20.6%). staphylococcus aureus was the most common gram-positive bacterium isolated (23.5%). three (37.5%) s. aureus isolates were methicillin-resistant. all isolates were sensitive to meropenem, and 97.1% were sensitive to imipenem; other sensitivity patterns were: ceftazidime (85.3%), ciprofloxacin (79.4%), ofloxacin (79.4%), and gentamicin (76.5%). there was low sensitivity to ampicillin (32.4%) and cotrimoxazole (38.2%). all gram-positive isolates, including methicillin-resistant s. aureus, were sensitive to vancomycin. conclusion: regular surveillance of isolate sensitivity patterns, formulation of hospital antibiotic policies based on existing data and compliance with treatment guidelines will promote rational antibiotic use and reduce resistance among bacteria. keywords: antibiotic-resistance; antibiotic sensitivity; bacterial isolates; blood culture; bloodstream infections. introduction bloodstream infections (bsis), which range from self-limiting bacteraemia to an outright life-threatening septicaemia, are some of the most common healthcare-associated infections globally.1 bacteraemia is simply described as the presence of viable bacteria in the blood, while septicaemia connotes systemic manifestations caused by bacteria or their toxins in blood. septicaemia constitutes a significant cause of morbidity and mortality, requiring prompt assessment, diagnosis, and antibiotic treatment. bloodstream infections account for up to 9% – 11% of hospital-acquired infections in the developed countries of europe and the united states, while higher prevalence of up to 19% has been recorded from lowand middle-income countries of the world.2,3 a european study had estimated that bsi patients spend an additional 6.0–11.5 days in hospital compared to other patients and the cost of management associated with bsi ranges from $8000 united states dollars (usd) to $56 000 usd.4 the risk factors for bsis include the use of healthcare devices such as: peripheral and central venous catheters on patients; age (elderly patient, neonates); and premorbid medical conditions of patients, such as diabetes mellitus, malignancies, renal failure, burns, and prior hospitalisation.5 the mortality rate from bloodstream infections ranges from 4.0% to 41.5% depending on severity, age, sex, and other risk factors. infections due to antibiotic-resistant strains of bacteria present with a significantly higher morbidity and mortality.6 blood culture remains the gold standard in the laboratory diagnosis and identification of bloodstream pathogens; however, bacteria are not isolated in many cases of bsi.7 blood culture positivity rates among patients with bsi in developing countries range from 9.2% to 44.0%. there is a paucity of data from nigeria on bsi as a result of a poor surveillance system for the associated pathogens, thus depriving us of any useful antibiotic policy or treatment guidelines for these infections.8,9 the few studies carried out in nigeria on bsis were mainly among neonates and younger children. ogunkunle et al., iregbu et al., and uzodima et al., all from nigeria, reported blood culture positivity rates of 19.0%, 22.0% and 35.0%, respectively, among suspected cases of bsi.10,11,12 numerous bacteria have been associated with causation of bsis including gram-negative bacteria: escherichia coli, pseudomonas species (spp.), klebsiella spp., serratia spp., salmonella spp. and enterobacter spp.; and gram-positive bacteria: staphylococcus spp., streptococcus spp. and enterococcus spp.13,14 however, recent findings suggested an upsurge in bsis caused by multidrug-resistant bacteria, including the members of the enterobacteriaceae family and other gram-negative bacteria, such as klebsiella spp., pseudomonas spp., acinetobacter spp. and citrobacter spp., most of which are extended spectrum beta-lactamase (esbl) producers, and also some gram-positive bacteria, including methicillin-resistant staphylococcus aureus (mrsa) and the vancomycin-resistant enterococci.15,16 the carbapenem antibiotics remain the antibiotic agents of choice in the management of emerging esbl-producing gram-negative bacteria, while vancomycin is the mainstay in treatment of mrsa.17 however, of particular concern is the recent emergence of increasing resistance of some gram-negative bacteria to carbapenems through the production of enzymes, carbapenemases.18 this trend of multidrug resistance, especially among the gram-negative bacteria causing bsis, has created a very serious therapeutic dilemma, especially in the management of intensive care unit patients, since it leads to fewer therapeutic options, use of more expensive drugs, increased hospital stay, and increased morbidity and mortality.19 bearing in mind this trend of antibiotic-resistant bacterial agents of bsis and the known fact that antibiotic-resistance patterns vary with geographical locations, regular surveillance and documentation of blood culture isolates and their antibiogram is imperative in formulating an antibiotic policy and identifying the best empirical antibiotic therapeutic options for different scenarios of bsis in each hospital environment.20 this will encourage ‘rational use’ of antibiotics and reduce the tendency towards increasing antibiotic-resistance. there have been random reports of multiple antibiotic-resistant isolates causing a therapeutic dilemma among patients with bsis in our hospital with occasional attendant case fatalities in the past years. also, there was no existing antibiotic policy or treatment guideline for bsi management in our centre, as there was poor or non-existent surveillance for these multiple antibiotic-resistant pathogens. these factors necessitated this study. the objectives of this study are: to isolate and identify different bacterial causes of bsis; to determine the antibiotic susceptibility patterns of isolated bacteria; and to suggest the best empirical treatment of bsis in different scenarios in the hospital. methods ethical considerations ethical approval (protocol number: erc/2020/10/16/431a) for the study was obtained from the human research and ethics committee, federal teaching hospital, ido-ekiti, nigeria. written informed consent was also obtained from all participants prior to inclusion in the study. for underage participants, consent was sought and obtained from the parent/guardian. participants were de-identified by encoding their names with research numbers determined based on the units from which participants were recruited. study design and hospital setting this cross-sectional study was conducted at the department of medical microbiology and parasitology of federal teaching hospital, ido-ekiti, a rural southwestern nigerian teaching hospital. the hospital is a tertiary health facility which serves as a referral centre to other primary and secondary healthcare facilities in ekiti state, nigeria. it is a 290-bed hospital with many modern facilities for healthcare. study population and sampling method using a simple random sampling technique, a total of 177 clinically diagnosed bsi patients were recruited into the study, thus a total of 177 blood culture samples from different hospital units were received and investigated at the medical microbiology laboratory of the hospital between june 2020 and february 2021. patients’ clinical history and other relevant details were recorded in a predesigned form. blood sample collection and processing a set of two venous blood samples from two different sites were collected 30 minutes apart from each participant with suspected bsi, following strict aseptic precautions before commencement of antibiotic treatment. each set consisted of 8 ml – 10 ml of venous blood from adults, 2 ml of venous blood from neonates and 2 ml – 5 ml from other paediatric patients. blood samples were immediately inoculated into bact/alert® (biomérieux, inc., durham, north carolina, united states) aerobic blood culture bottles (adult or paediatric bottles, as necessary). these bottles were immediately incubated in a bact/alert® 3d automated blood culture analyser (biomérieux, inc., durham, north caroline, united states). all bact/alert-positive broths were immediately brought out for subculture onto blood agar (oxoid, wade road, basingstoke, united kingdom) and macconkey agar (oxoid, basingstoke, united kingdom). for those bottles without positive signals, blind subculture onto blood agar and macconkey agar was done on days 2, 5 and 7 of incubation. the inoculated blood agar and macconkey agar plates were incubated at 37 °c for 18 hours – 24 hours. any bacterial growth on agar plates was identified using colonial morphology, gram staining, and conventional biochemical tests using standard laboratory protocols.21 antibiotic susceptibility testing of isolated bacteria was performed on mueller-hinton agar (oxoid, basingstoke, united kingdom) using the modified kirby-bauer disc diffusion method. the results were interpreted as sensitive or resistant using the guidelines of the clinical and laboratory standard institute.22 the following antibiotics were tested on all identified isolates: imipenem (10 μg), meropenem (10 μg), ampicillin/sulbactam (20/10 μg), gentamicin (10 μg), ciprofloxacin (5 μg), ofloxacin (5 μg), cefriaxone (30 μg), ceftazidime (30 μg), amoxycillin/clavulanate (20/10 μg), cefixime (5 μg), and ampicillin (10 μg). all antibiotic discs were from oxoidtm (oxoid, wade road, basingstoke, united kingdom). all gram-negative bacteria isolates were tested for esbl production by the double disc synergy test using ceftazidime (30 μg) and ceftazidime/clavulanate (30/10 μg) discs, while the cefoxitin disc diffusion method was used to identify mrsa.22 all s. aureus isolated, including mrsa and coagulase-negative s. aureus (cons), were tested against vancomycin using etest® (biomérieux, inc., durham, north carolina, united states). inclusion and exclusion criteria all blood samples of patients with suspected bsi without a history of antibiotic medication prior to sample collection were included, while blood samples of bsi patients with a history of antibiotic medication prior to sample collection were excluded. data entry and analysis data were entered into microsoft excel 2017 (microsoft corporation, redmond, washington, united states) and data analysis was done using the statistical package for social sciences version 20.0 (ibm corp., armonk, new york, united states). results were presented in tables and expressed as frequencies and percentages. association between variables was tested using chi-square and/or fisher’s exact tests, as appropriate. statistical significance was accepted at p ≤ 0.05. results the age range of participants was 4 days to 87 years (mean: 23.29 ± 26.58 years) (table 1). of the 177 suspected cases of sepsis and other bloodstream-related infections that were investigated, 102 (57.6%) of the patients were male, and 75 (42.4%) were female. there were 27 (26.5%) male patients and 15 (20.0%) female patients below the age of one month (neonates), and 15 (14.7%) male patients and 23 (30.7%) female patients older than 50 years. table 1: age distribution of bloodstream infection study participants in relation to gender at the federal teaching hospital, ido-ekiti, nigeria, between june 2020 and february 2021. only 34 (19.2%) of the total blood culture broths were culture-positive for bacteria (table 2). culture positivity was highest among neonates (aged < 1 month; 23.8%) and lowest among patients aged 6–17 years (12.9%). culture positivity was higher among female patients (20.0%) compared to male patients (9.5%) aged 6–17 years, but no significant difference was seen (p = 0.57). no significant difference was seen in the overall isolation rate between male and female patients (p = 0.97). table 2: frequency of culture-positive broths by age group among bloodstream infection cases at the federal teaching hospital, ido-ekiti, nigeria, between june 2020 and february 2021. the most commonly isolated bacteria were e. coli (10/34, 29.4%), s. aureus (8/34, 23.5%), and k. aerogenes (7/34, 20.6%) (table 3). gram-negative bacteria species were the most commonly isolated (23/34 isolates, 67.6%) (table 4). gram-negative bacteria were isolated at a higher rate among neonates < 1 month (8/42 neonates, 19.0%) compared to other participants older than one month (15/135 participants, 11.1%) but no significant difference was seen in the rate of isolation (p = 0.18). the isolation rate was higher among neonates (10/42 neonates, 23.8%) compared to other participants older than one month (24/135 participants, 17.8%), but no significant difference was found in the isolation rate (p = 0.39). table 3: frequency of isolates from culture-positive broths among bloodstream infection cases by age group at the federal teaching hospital, ido-ekiti, nigeria, between june 2020 and february 2021. table 4: comparison of gram-positive and gram-negative isolates from bloodstream infection cases between neonates and other participants at the federal teaching hospital, ido-ekiti , nigeria, between june 2020 and february 2021. all (100.0%) of the isolates were sensitive to meropenem, 33/34 isolates (97.1%) were sensitive to imipenem, 29/34 isolates (85.3%) were sensitive to ceftazidime, 27/34 isolates (79.4%) were sensitive to ciprofloxacin and ofloxacin, and 26/34 isolates (76.5%) were sensitive to gentamicin (table 5). very poor sensitivity was observed for ampicillin (11/34 isolates, 32.4%) and cotrimoxazole (13/34 isolates, 38.2%). none of the gram-negative enteric bacteria isolated were esbl producers. three (37.5%) of the eight s. aureus isolated were mrsa. all of the isolated cons (3/3 isolates, 100%) and s. aureus (8/8 isolates, 100%), including the mrsa, were sensitive to vancomycin. table 5: antibiotic susceptibility pattern of isolates from bloodstream infection cases at the federal teaching hospital, ido-ekiti, nigeria, between june 2020 and february 2021. discussion the low culture positivity rate of 19.2% seen in this study is similar to findings in some previous studies. deku et al. reported a culture positivity rate of 13.1% in ghana and gupta et al. reported 16.5% in north india.23,24 lower rates have been reported in other studies: khanal et al. and gohel et al. reported a culture positivity rate of 10.3% and 9.2%, respectively, from bsi cases in india.8,25 a higher rate of 44.0% has been reported by khanal et al. in another prospective study on patients with infective endocarditis in india.9 most similar studies conducted in nigeria and africa focused mainly on neonatal and childhood bsis, and data on adult bsis is scanty. of those studies on neonatal bsis, some have reported similar culture positivity rates to the 23.8% seen among neonates in this study: ogunkunle et al. (19.0%) and iregbu et al. (22.0%), both in nigeria, while higher isolation rates were reported by sorsa in ethiopia (29.3%), uzodimma et al. in nigeria (35.0%) and el-din et al. in egypt (40.7%).10,11,12,26,27 factors determining rate of culture positivity include the methods or technique used in isolating bacteria, volume and the number of blood samples collected for the culture; also in addition, prior use of antibiotics before sample collection will affect the likelihood of culture positivity.28 in this study, adequate volumes of blood samples were collected before the commencement of antibiotic treatment, and patients with history of antibiotic medication prior to sample collection were excluded from the study, yet, prior self-medication at home or use of antibiotics at peripheral health facilities before transfer to our centre could not be totally ruled out. this coupled with the fact that some of these patients clinically diagnosed as bsi may actually have been suffering from other conditions mimicking bsi (and not bsi), thus accounting for the low culture positivity seen. gram-negative enteric bacteria (67.6%) were the predominant isolates in this study, of which e. coli (29.4%) was the most commonly isolated. staphylococcus aureus (23.5%) and cons (8.8%) were the only gram-positive bacteria isolated. similar findings have been reported by earlier studies where gram-negative bacteria were the predominant isolates from cases of bsi.24,28 however, some studies have reported s. aureus or cons as the most commonly isolated bacterial pathogen from cases of bsi.15,23,25,29,30,31 generally, there is wide variability in the pathogens isolated from cases of bsi in different settings. gram-positive bacteria were the most common cause of sepsis prior to the advent of antibiotics in the 1950s, but gram-negative bacteria became the most predominant after the introduction of antibiotics from the 1960s to 1980s. however, from the 1980s, gram-positive bacteria, most commonly staphylococcus spp., were thought to cause more than 50% of cases of sepsis.32,33 in hospital patients, there is a higher chance of hospital-selected gram-negative bacteria causing bsis due to the instruments and procedures carried out on these patients. this may account for the preponderance of gram-negative bacteria isolates in this study. predominant isolation of gram-negative enteric bacteria among neonates in this study contrasted with some previous studies on neonatal bsis: ogunkunle et al., sorsa and el-din et al. all reported gram-positive bacteria as the most commonly isolated pathogens from cases of neonatal bsis.10,26,27 this contrasting result may be because of the small number of neonates examined in this study compared to those other studies that focused mainly on neonates. iregbu et al., however, reported isolation of gram-positive and gram-negative bacteria in equal proportion from cases of neonatal bsis in abuja, nigeria.11 all of the cons and s. aureus, including the three mrsa isolated in this study, were sensitive to vancomycin. resistance of cons and mrsa to vancomycin is of immense challenge to the treatment of diseases caused by these strains because of associated persistent infections, vancomycin treatment failure and a generally poor clinical outcome. all of the isolates in this study were sensitive to meropenem, and 97.1% were sensitive to imipenem. these carbapenems represent the last resort, in antibiotic treatment, for most facilities in the less-developed world, where newer antibiotics are out of reach of the majority, especially for esbl-producing gram-negative bacteria.17 favourable sensitivity patterns were also seen in this study to other antibiotics, including ceftazidime (85.3%), ofloxacin (79.4%), ciprofloxacin (79.4%), and gentamicin (76.5%). the poor sensitivity pattern seen with these isolates to cotrimoxazole (32.4%), ampicillin (38.2%), amoxycillin-clavulanate (64.7%), ampicillin-sulbactam (67.6%) and ceftriaxone (67.6%) is undoubtedly a result of irrational antibiotic use. some of these drugs are regularly abused by patients even without prescription while others are indiscriminately used in our health facilities, resulting in the generation of resistance to these antibiotics. formulation of antibiotic policy on bsis from this data and compliance with treatment guidelines are of paramount importance, not only to save patients’ lives, but also to reduce further resistance generated by bacteria to more antibiotics. for the empirical treatment of bsis in this setting, a combination of ceftazidime with gentamicin is recommended in children less than 16 years of age, while a combination of ciprofloxacin/ofloxacin with gentamicin is recommended in the older age-groups. these three antibiotics demonstrated favourable sensitivity patterns against most bacteria isolated in this study; their combination in treatment of bsis will not only produce a synergistic effect, but will also reduce the rate at which isolates develop resistance to individual antibiotics. meropenem and imipenem should be reserved for cases not amenable to the aforementioned combination therapy. it is advisable that meropenem or imipenem should also be used in combination with other classes of antibiotics with a good sensitivity profile to the isolated pathogen, such as gentamicin, to reduce the speed at which bacteria generate resistance to these valuable drugs. in cases of bsi due to mrsa, vancomycin is the recommended treatment of choice in this setting. limitations the method of antibiotic sensitivity testing employed for all antibiotics (except vancomycin) tested against isolates in this study was limited to disc diffusion sensitivity testing. additional determination of the minimum inhibitory concentration of the different antibiotics which would have supplied quantitative data on the susceptibility pattern of isolates to these antibiotics was desired, but the study was limited by supply of funds. similarly, due to limited funding, we were unable to carry out the molecular characterisation of the resistant genes among the mrsa isolated. moreover, this study involved only our centre, which may have limited the relevance of data generated as regards antibiotic policy formulation and determination of treatment guideline for managing bsis throughout nigeria and other african countries, a multicentre study is desirable in future. conclusion gram-negative bacteria were predominantly isolated from cases of bsi. isolates demonstrated good sensitivity to meropenem, imipenem, ceftazidime, ciprofloxacin/ofloxacin, and gentamicin. regular surveillance of isolate sensitivity patterns, formulation of hospital antibiotic policies based on existing data, and compliance with treatment guidelines will promote rational antibiotic use and reduce resistance generation among bacteria. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions o.t.o. was responsible for conceptualisation of the study, design, definition of intellectual content, manuscript preparation, data collection, statistical analysis, and manuscript review. b.o.o. and a.t.o. were involved in data collection, literature search, experimental studies, data acquisition, manuscript, editing, and manuscript review. o.i.o. was involved in the literature search, data acquisition, manuscript editing, and manuscript review. sources of support the study was funded by all four authors. data availability the raw data from the study are available upon request from the corresponding author, o.t.o. disclaimer the views and opinions expressed in the article are those of the authors and do not necessarily reflect the critical policy or position of any affiliated agency or authors. references diekema dj, beekman se, chapin kc, morel ka, munson e, doern gv. epidemiology and outcome of nosocomial and community onset 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https://doi.org/10.1128/jcm.01555-07 reddy ea, shaw av, crump ja. community acquired bloodstream infections in africa: a systematic review and meta-analysis. lancet infect dis. 2010;10(6):417–432. https://doi.org/10.1016/s1473-3099(10)70072-4 mia ar, zerin t. antibiogram of blood culture isolates of patients from a hospital in dhaka, bangladesh. matrix sci med. 2020;4(1):1–5. https://doi.org/10.4103/mtsm.mtsm_4_19 sangita km, tomar r, saha nk. bacteriological profile and antibiogram of blood culture isolates from a tertiary care hospital. int j med sci innov res [serial online]. 2019;4(6):187–192. available from: http://www.ijmsir.com polat g, ugan ra, cadirci e, halici z. sepsis and septic shock: current treatment strategies and new approaches. eurasian j med. 2017;49(1):53–58. https://doi.org/10.5152/eurasianjmed.2017.17062 martins gs. sepsis, severe sepsis and septic shock: changes in incidence, pathogens and outcomes. expert rev anti infect ther. 2012;10(6):701–706. https://doi.org/10.1586/eri.12.50 abstract diagnostic landscape in the developing world ten years of building capacity to assure quality acknowledgements references about the author(s) debrah i. boeras london school of hygiene and tropical medicine, london, united kingdom rosanna w. peeling london school of hygiene and tropical medicine, london, united kingdom citation boeras di, peeling rw. external quality assurance for hiv point-of-care testing in africa: a collaborative country-partner approach to strengthen diagnostic services. afr j lab med. 2016;5(2), a556. http://dx.doi.org/10.4102/ajlm.v5i2.556 lessons from the field external quality assurance for hiv point-of-care testing in africa: a collaborative country-partner approach to strengthen diagnostic services debrah i. boeras, rosanna w. peeling received: 31 aug. 2016; accepted: 31 aug. 2016; published: 17 oct. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract it is important to consider the role of diagnostics and the critical need for quality diagnostics services in resource-limited settings. accurate diagnostic tests play a key role in patient management and the prevention and control of most infectious diseases. as countries plan for implementation of hiv early infant diagnosis and viral load point-of-care testing, the london school of hygiene & tropical medicine has worked with countries and partners with an interest in external quality assurance to support quality point-of-care testing on the continent. through a series of collaborative consultations and workshops, the london school of hygiene & tropical medicine has gathered lessons learned, tools, and resources and developed quality assurance models that will support point-of-care testing. the london school of hygiene & tropical medicine is committed to the continued advancement of laboratory diagnostics in africa and quality laboratory services and point-of-care testing. diagnostic landscape in the developing world accurate diagnostic tests play a key role in patient management and the prevention and control of most infectious diseases (table 1). early diagnosis and treatment not only reduces the risk of developing long-term complications in the patient, but for diseases such as tuberculosis, sexually-transmitted infections and hiv, prompt treatment also reduces further transmission to other members of the community. table 1: role of diagnostic tests in patient management and disease control. in many developing countries, laboratory services are often limited to major urban centres. in settings where access to diagnostic laboratory services is limited, the world health organization (who) recommends the use of a syndromic approach to clinical management, where patients presenting with a particular syndrome are treated for all the major causes of the syndrome. although these algorithms are inexpensive and easy to use, especially in primary healthcare settings, a major disadvantage is over-treatment and the potential for development of antibiotic resistance. syndromic management also does not allow contact tracing to prevent onward transmission, public health surveillance and interventions. even when laboratory services are available, there are often problems with the quality of the services due to lack of resources, supply logistics and trained personnel. these problems lead to physicians not trusting laboratory results, which in turn leads to further neglect of laboratory services. with the more recent joint united nations programme on hiv/aids 90-90-90 targets to identify 90% of those hiv infected, provide treatment to 90% of those hiv infected, and ensure viral suppression for 90% of those on treatment, diagnostics near the point of care will play a pivotal role for expanding hiv diagnosis and monitoring.1 lack of international and national regulatory standards for approval of diagnostics national regulatory processes for drugs provide safeguards for the safety and efficacy of drugs used in a country. most countries have a process for reviewing the evidence from drug trials to support the introduction of new drugs, and this has done much to improve the quality of therapeutics used in developing countries. unfortunately, apart from tests used for blood banking, regulatory standards are often lacking for diagnostic tests, especially those targeting diseases that are uncommon in industrialised countries. as a result, diagnostic tests are often sold in most of the developing world, without any formal evaluation of their performance and effectiveness. ten years of building capacity to assure quality in a review article by petti et al. in 2006, the authors described laboratory medicine as a barrier to effective healthcare, with misdiagnosis occurring often.2 clinicians must rely on alternative diagnostics in their clinical practice due to the lack of available and affordable diagnostics. this article elicited numerous responses, acknowledging diagnostic insufficiencies in africa, and even referring to laboratory systems and diagnostics as the achilles heel of global health.3,4 as of 10 years ago, we understood the need for sustainable laboratory infrastructures to support the diagnosis of infectious disease. since 2008, there have been several landmark events to advance laboratory diagnostics in africa: 2008: january – the maputo declaration for strengthening laboratory health systems;5 april – the lyon statement on the need for developing countries to establish practical quality management systems;6 and september – the yaoundé resolution issued by the who regional office for africa (who afro) to strengthen public health laboratories in the who african region;7 july 2009: several african countries, donors, who and implementing partners met in kigali, rwanda, to launch the stepwise laboratory quality improvement process towards accreditation (slipta), the who afro accreditation scheme to assist laboratories in obtaining internationally-recognised accreditation standards8 (iso – international organization for standardization; cap – college of american pathologists; sanas – south african national accreditation system; sadcas – southern african development community accreditation service); 2010: the kampala statement called for the creation of the african society for laboratory medicine;9 2011: launch of the african society for laboratory medicine as a pan-african professional body working to advocate for the critical role and needs of laboratory medicine and networks throughout africa. the african society for laboratory medicine addresses these challenges by working collaboratively with governments, local and international organisations, implementing partners, and the private sector to achieve the following goals by 2020: strengthening laboratory workforce by training and certifying laboratory professionals and clinicians through standardised frameworks; transforming laboratory testing quality by enrolling laboratories in quality improvement programmes to achieve accreditation by international standards; developing strong, harmonised regulatory systems for diagnostic products as defined by the global harmonization taskforce; and building a network of national public health reference laboratories to improve early disease detection and collaborative research. 2015: who/us centers for disease control and prevention point-of-care (poc) handbook;10 2016: 27 of the 47 (57%) who afro member states had appointed a slipta focal point and 14 ministers of health had endorsed slipta as the desired programme for continuous quality improvement. 159 laboratories were audited between may 2013 and march 2015, with a 69% mean score. of those audited, 70% achieved 55% compliance or higher (2 or more stars) and 1% scored at least 95% (5 stars). the lowest scoring sections of the who afro slipta checklist were sections 6 (internal audit) and 10 (corrective action), which both had mean scores below 50%.8 figure 1 shows the scoring system (stars) as outlined by the slipta inspection checklist. figure 1: stepwise laboratory quality improvement process towards accreditation (slipta) inspection checklist score corresponds to the number of stars awarded to a laboratory. this progress is very encouraging, but there are new challenges with the introduction of poc testing: poc testing is performed and results interpreted by non-lab personnel with no expertise or experience in doing any form of testing; poc devices and cartridges are transported all over the country without temperature control; poc devices and cartridges stored at remote sites often have no temperature control. the devices may be dusty or exposed to extreme humidity. all of these affect the test validity; many poc tests lack ‘on-board’ controls, meaning that there is no quality control for the testing process; central or national laboratories often do not have sufficient capacity to supervise all poc testing or institute external quality assessment (eqa) programmes for every poc site. a collaborative approach to assure quality in consultation with the ministry of health and representatives from 10 countries with an interest in external quality assurance, the london school of hygiene and tropical medicine, through funding from unitaid, worked with partners and quality assurance experts to develop strategies and initiatives to support countries in scaling up quality poc testing. countries shared lessons learned and possible planning for quality poc testing. quality assurance models for point-of-care testing while poc has the potential to provide faster results for quicker clinical decisions, a major concern with decentralising diagnostics and laboratory services is the lack of oversight and ensuring quality standards. from the consultations, several models were captured for organising poc testing in africa (figure 2). figure 2: quality assurance models for point-of-care testing integrated within existing laboratory networks. quality assurance/external quality assurance consultations for point-of-care testing initial country consultations with unicef these consultations found a large need for quality assurance planning and policies, especially around poc testing. regional country consultations (east and west africa) this set the stage for our eqa work, as we found that most countries were enrolled in some sort of eqa programme and were also supporting quality assurance activities. countries shared lessons and tools and identified major gaps in their quality assurance programmes. us centers for disease control and prevention, atlanta, external quality assessment workshops these brought together eqa providers as a think tank for global proficiency testing schemes, eqa programmes, and capacity building. external quality assessment country pilot this piloted full coverage of eqa for cd4 poc in one country, which helped the country map the poc landscape and identify gaps and challenges (e.g. broken instruments, supply chain management issues). external quality assessment/connectivity pilot this programme explored how to monitor quality assurance of poc testing. it identified numerous gaps as well as promising opportunities with connectivity and automated eqa reporting. costing workshops countries shared costing information and were trained by modellers and economists to perform costing exercises. this information has formed the basis for costing quality assurance as a percentage of the total cost of a poc test. this information can be used for advocacy and budget planning, as well as business plan development. technical external quality assessment workshops a series of technical workshops to provide global coverage for hiv cd4, eid, and viral load external quality assurance, including proficiency testing schemes, and to transition eqa programmes to the continent through technology transfer trainings and capacity-building discussions. these workshops will also support regional eqa centres and south-to-south collaborations. conclusions and way forward diagnostics have become central to patient management and disease surveillance/control in the developing world. as laboratory testing is pushed to more remote areas, ensuring accurate diagnostic results as well as sustainable laboratory services becomes a greater challenge to systems that are already struggling. the london school of hygiene & tropical medicine is working to continue the advancement of laboratory diagnostics in africa, working with african countries and partners with an interest in eqa to support quality poc testing on the continent. through a series of collaborative consultations and workshops, the london school of hygiene and tropical medicine has gathered lessons learned, tools and resources, and developed quality assurance models that will support poc testing. it is important to consider the role of diagnostics and the critical need for quality diagnostics services in the more remote resource-limited settings. clinicians and patients will increasingly depend on poc testing for improved health outcomes. at the same time, these new diagnostics should be seen as opportunities to improve the health of the community with surveillance and disease control programmes. acknowledgements open review: this manuscript underwent open peer review by adrienne meyers and ben cheng. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support none. authors’ contributions d.i.b. and r.w.p. both contributed equally to writing and revising the manuscript. references unaids. 90–90–90 – an ambitious treatment target to help end the aids epidemic [document on the internet]. c2014 [cited 2016 sep 24]. http://www.unaids.org/en/resources/documents/2014/90-90-90 petti ca, polage cr, quinn tc, et al. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006;42(3):377–382. epub 2005 dec 20. okeke in. diagnostic insufficiency in africa. clin infect dis. 2006;42(10):1501–1503. http://dx.doi.org/10.1086/503308 berkelman r, cassell g, specter s, et al. the “achilles heel” of global efforts to combat infectious diseases. clin infect dis. 2006;42(10):1503–1504. http://dx.doi.org/10.1086/504494 world health organization’s regional office for africa. the maputo declaration on strengthening of laboratory systems [document on the internet]. c2008 [cited 2013 jan 02]. available from: http://www.who.int/diagnostics_laboratory/maputo-declaration_2008.pdf world health organization. joint who–cdc conference on health laboratory quality systems. who/hse/ihr/lyo/2008.3 [document on the internet]. c2008 [cited 2013 jan 03]. available from: http://www.who.int/ihr/lyon/report20080409.pdf gershy-damet gm, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. http://dx.doi.org/10.1309/ajcptuuc2v1wjqbm yao k, luman et, slmta collaborating authors. evidence from 617 laboratories in 47 countries for slmta-driven improvement in quality management systems. afr j lab med. 2014;3(2), art. #262, 11 pages. http://dx.doi.org/10.4102/ajlm.v3i2.262 african field epidemiology network (afenet). kampala statement for the creation of the african society for laboratory medicine. kampala, uganda: afenet; 2010. world health oranization (who)/us centers for disease control and prevention. improving the quality of hiv-related point-of-care testing: ensuring the reliability and accuracy of test results. geneva, switzerland: who; 2015. abstract introduction methods conclusion acknowledgements references about the author(s) ethan j. gantana department of pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa department of haematology, national health laboratory service, cape town, south africa ernest musekwa department of pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa department of haematology, national health laboratory service, cape town, south africa zivanai c. chapanduka department of pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa department of haematology, national health laboratory service, cape town, south africa citation gantana ej, musekwa e, chapanduka zc. advances in estimating plasma cells in bone marrow: a comprehensive method review. afr j lab med. 2024;13(1), a2381. https://doi.org/10.4102/ajlm.v13i1.2381 note: special collection: transforming african lds with dt and ai. review article advances in estimating plasma cells in bone marrow: a comprehensive method review ethan j. gantana, ernest musekwa, zivanai c. chapanduka received: 30 nov. 2023; accepted: 25 mar. 2024; published: 11 july 2024 copyright: © 2024. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract the quantitation of plasma cells in bone marrow (bm) is crucial for diagnosing and classifying plasma cell neoplasms. various methods, including romanowsky-stained bm aspirates (bma), immunohistochemistry, flow cytometry, and radiological imaging, have been explored. however, challenges such as patchy infiltration and sample haemodilution can impact the reliability of bm plasma cell percentage estimates. bone marrow plasma cell percentage varies across methods, with immunohistochemically stained biopsies consistently yielding higher values than romanowsky-stained bma or flow cytometry alone. cd138 or mum1 immunohistochemistry and artificial intelligence image analysis on whole-slide images are emerging as promising tools for accurate plasma cell identification and quantification. radiological imaging, particularly with advanced technologies like dual-energy computed tomography and radiomics, shows potential for multiple myeloma diagnosis, although standardisation remains a challenge. molecular techniques, such as allele-specific oligonucleotide quantitative polymerase chain reaction and next-generation sequencing, offer insights into clonality and measurable residual disease. while no consensus exists on a gold standard method for bm plasma cell quantitation, cd138-stained biopsies are favoured for accurate estimation and play a pivotal role in diagnosing and assessing multiple myeloma treatment responses. combining multiple methods, such as bma, bm biopsy, and flow cytometry, enhances accuracy of diagnosis and classification of plasma cell neoplasms. the quest for a gold standard requires ongoing research and collaboration to refine existing methods. furthermore, the rise of digital pathology is anticipated to reshape laboratory medicine and the role of pathologists in the digital era. what this study adds: this article adds a comprehensive review and comparison of different methods for plasma cell estimation in the bone marrow, highlighting their strengths and limitations. the goal is to contribute valuable insights that can guide the selection of optimal techniques for accurate plasma cell estimation. keywords: plasma cell neoplasms; multiple myeloma; digital pathology; artificial intelligence; haematology; pathology. introduction plasma cell neoplasms encompass a wide variety of disorders that result from the clonal expansion of terminally differentiated b cells (plasma cells), typically secreting a single homogeneous monoclonal immunoglobulin.1 the differentiation between some of these disorders and the monitoring of plasma cell (pc) neoplasms depends on the quantitation of clonal pc in the bone marrow (bm) and the use of biochemical and haematological parameters and imaging techniques.1,2,3,4 quantitation of pc in bm samples has historically relied on manual methods such as microscopy-based cell counting on bm aspirates and immunohistochemically stained trephine biopsies. the surface marker cd138 is sensitive and specific for pc, thus cd138 immunohistochemistry is commonly used for estimating pc in bm biopsies. this practice is supported by the world health organization.1,5,6 however, various alternative approaches have emerged, each with their strengths and limitations. as the field continues to evolve, it is essential to critically review and evaluate the available methods for estimating pc in bm. currently, there is no acceptable reference method for estimating pc in bm, although many methods have been developed and evaluated for accuracy and reproducibility. this method review aims to comprehensively evaluate existing techniques for the quantitation of pc in bm samples. by examining the principles, procedures, advantages, and challenges associated with the various methods, we aim to provide a comprehensive understanding of the options available to clinicians, researchers, pathologists, and haematologists. in this review the authors examine the factors that affect the reliability of plasma cell estimation and share thoughts on the promising role of new technologies. in addition, the challenges of standardisation, clinical relevance and future directions will stimulate discussion and encourage collaboration in this field. we hope to contribute to the refinement of pc estimation methods and their integration into routine clinical practice. methods based on the recommendations by levac et al., colquhoun et al. and the methods of arksey and o’malley, we conducted a literature search for a narrative (scoping) review article.7,8,9 the four main electronic databases (pubmed, google scholar, cochrane library and mendeley) were searched to map the existing landscape of methods for estimating bone marrow plasma cell percentage (bmpc%), and to compare and summarise these methods to provide suggestions for the use and further development of appropriate and cost-effective methods for estimating bmpc%, particularly in resource-limited settings. a summary table (table 1) was created to summarise the data from the literature review and to present the different methods, as well as their advantages and disadvantages, in a clear format. the search was conducted from inception to 31 november 2023 to find relevant articles published in peer-reviewed journals. the search strategy consisted of a combination of keywords and medical subject heading terms related to the topic of interest, such as ‘multiple myeloma’, ‘bone marrow plasma cells’, ‘plasma cell estimation’, etc. articles that were irrelevant, did not contain a methods section, and were not written in english were excluded. all relevant articles, literature sources and citations were included in the reference list and duplicates were removed. table 1: summary of various methods used for the estimation of bone marrow plasma cell percentages, highlighting their advantages and disadvantages. pre-analytical factors affecting plasma cell estimation bone marrow aspiration and processing for accurate visualisation the first millilitre of aspirated bm represents pure bm elements. the subsequent sampling becomes significantly blood diluted (haemodilution).10,11 ancillary testing including flow cytometry and cytogenetics require additional drawing of bm aspirate (bma). the first and second draw of bma yield discrepant results, with the second draw of bma smears significantly underrepresenting the pc count. this remains important in the evaluation of newly diagnosed multiple myeloma (mm) patients.12 bone marrow aspirate smears are considered the least manipulated sample. therefore, the numeric discrepancy between the morphological assessment and the flow cytometric assessment cannot be attributed solely to technical or methodological deficiencies. note that the same degree of discrepancy is not seen with other haematological malignancies such as acute myeloid leukaemia.13 bone marrow aspirate smears are usually stained with romanowsky-type stains, which are used either in combination or individually. the staining of bm films may be adversely affected by any of the pre-analytical processes.11 plasma cells on romanowsky-stained bma are recognised by their distinctive nuclear and cytoplasmic features.14 bone marrow trephine biopsy specimens and selection of appropriate antibodies the common problem with myeloma trephine samples is inadequate or fragmented cores. the biopsy may be inadequate if the same bone puncture site is used for aspiration and biopsy.15 multiple myeloma trephine biopsy should ideally contain five to six intertrabecular spaces that provide a reasonable probability of detecting focal bm lesions.14 bone marrow trephine biopsy specimens are usually fixed in formalin, embedded in paraffin, and stained with haematoxylin and eosin.11 identifying plasma cells on haematoxylin and eosin preparations is relatively easy for the experienced microscopist. however, pc are more easily distinguished from other nucleated cells by immunohistochemistry using antibodies targeting antigens such as cd138 or mum1. these antibodies are specific to pc in the bm; however, soft tissue tumours such as melanoma, osteosarcoma and osteoblastoma may also stain positive for cd138 on immunohistochemistry.5,16 sections of clotted bma may be used in cases where it is difficult to obtain a trephine biopsy. the bma is allowed to clot in a syringe. the clot is then carefully removed, placed in a fixative such as formalin, and processed in the same way as a trephine biopsy, except that no decalcification is required.11 current methods for estimating plasma cells in bone marrow manual cell counting on bone marrow aspirate smears cellular trails of romanowsky-stained bm aspirates are used for differential counts under the light microscope, but can be misleading if pc do not ‘fall out’ from the bm particles or are not aspirated due to the patchy nature of mm. plasma cell are counted as a proportion of all other nucleated haematopoietic cells in a 200–500 cell differential to estimate the percentage of pc in the bm.11 bone marrow aspirate smears often underestimate the percentage of pc compared to bm biopsies, leading to a lack of consensus on the appropriateness of smears for determining the percentage of pc in bm.17,18,19,20,21,22,23 in a study conducted in the united states in 2007, al-quran et al. evaluated cd138-stained bm specimens and showed poor intraobserver concordance with an intraclass correlation coefficient of 0.55 for one observer and an intraclass correlation coefficient of 0.47 for another between pc quantitation on bm aspirate smears and cd138 immunohistochemistry.19 smith and elnawawi, in a study conducted in the united states in 2008, found that aspirate smear pc counts were considerably lower (mean 10.6; standard deviation [s.d.] ± 13.1) than the counts by other methods such as overview estimates (mean 19.2; s.d. ± 20.4) and manual counts on cd138 immunohistochemistry (mean 23.1; s.d. ± 17.7), and attributed this to an under-representation of pc in aspirate smears.18 despite this percentage discrepancy, one study found a significant linear correlation between bmpc% in aspirate enumeration and cd138 immunohistochemistry (r = 0.71 [pearson correlation coefficient]), which suggests the possibility of extrapolating the true bmpc% from the manual count on bma smears.23 morphological assessment of bma smears has limitations, including the assessment of tumour burden and the determination of pc clonality. it is therefore best used in combination with immunohistochemistry and with or without multiparameter flow cytometry (mfc) when detection of clonality with immunohistochemistry is difficult, especially in the context of post-therapeutic hypocellular bm samples or a low tumour burden.1,3,4,14,24 the world health organization allows the bmpc% to be determined either by manual counting of the aspirate or by cd138 immunohistochemistry. it is recommended to consider the highest pc percentage of aspirate or trephine if there is a discrepancy between the two methods.1,3 multiparameter flow cytometry on bone marrow aspirate specimens the literature supports the use of mfc in the diagnosis and follow-up of mm, particularly regarding establishing the clonal nature of the pc.25,26,27 multiparameter flow cytometry has been shown to provide more prognostic information for overall survival than morphological assessment in patients with mm.25 the use of mfc to quantify pc in bm is more controversial. it has been shown that the percentage yield of pc is consistently lower with flow cytometric methods than with morphological assessment of smears from bma.12,13,22,25,27,28,29 this discrepancy is thought to be because of pc fragility, differences in sample quality, dilution with peripheral blood (haemodilution) or clotted specimens and cellular processing techniques used in flow cytometry that reduce the pc count. in contrast to this, a 2017 study by matsue et al. in japan showed that the bmpc% on aspirate smear counts correlated with flow cytometry (r = 0.93), but bma smear and flow cytometry significantly underestimated the pc percentage compared to bm biopsy or clot stained with cd138 immunohistochemistry.30 another study showed that even though a discrepancy was seen with morphological enumeration average (60%) in mm patients compared to flow cytometry (20%), this discrepancy was not found between morphological and flow cytometry assessment in acute myeloid leukaemia samples.13 based on these findings, the discrepancy regarding mm cannot be attributed solely to technical or methodological problems. it may stem from unique interaction between malignant pc and the bm niche they occupy, including bone surfaces, basement membranes, and lipid-enriched spicules.13 furthermore, patchy marrow infiltration, hypoplastic or fibrotic marrows may cause the underestimation of pc percentages. with the concern of the significant discrepancy between morphological assessment and flow cytometry pc percentage estimation, frebet et al., in a study in france in 2011, developed an mfc protocol that uses specific antibody combinations to include pc (cd138+/cd38+) and haematopoietic precursors (cd45+/cd117+/cd34+), and exclude erythroblasts and debris (cd36+/cd45–), to calculate a plasma cell:precursor ratio.29 a plasma cell:precursor ratio threshold of 2 was validated as a discriminative tool to differentiate between monoclonal gammopathy of undetermined significance and smouldering mm with high specificity (84%) and sensitivity (81%).29 immunohistochemistry on bone marrow biopsy trephine specimens cd138 (syndecan-1) is a cell surface, sulphate-rich proteoglycan adhesion molecule which is expressed in the late stages of b-cell differentiation and on pc. cd138 is a highly sensitive and specific marker for bmpcs, making it useful in identification and enumeration of bmpc.5,6 cd138 staining has the advantage of improved pc identification compared to haematoxylin and eosin-stained sections. the estimation of pc in bm biopsies using cd138 immunohistochemistry, as suggested by the world health organization, is generally performed using a semi-quantitative overview estimation method.1 in their 2008 study, smith and elnawawi proposed an alternative to overview estimation by performing a manual count on a single selected field of view at high magnification (40x objective). the manual count (mean 23.1; s.d. ± 17.7) correlated better with computer-assisted image cytometry (mean 21.4; ± 10.7 s.d.) than the overview estimates (mean 19.2; s.d. ± 20.4), on 44 cd138-stained core biopsy specimens.18 importantly, the s.d. of selected field counts in this study and the overview estimation are high, and may be clinically relevant for the diagnosis and evaluation of treatment response of pc neoplasms. a single area was selected for the manual counting method of pc estimation, even though mm is a patchy disease and shows considerable variation in bm infiltration. in their 2017 study in japan, matsue et al. counted more cells (500–2500) in 2–5 representative microscopic fields at low magnification in cd138-stained bm clot and biopsy sections. they showed significant correlation between the marrow clot and biopsy sections (r = 0.96); however, quantification by bma smear (bmpc% = 3.7 [interquartile range {iqr}: 1.2–10]) and flow cytometry (odds ratio = 2.4 [iqr: 0.8–6.2]) underestimated pc percentage compared to bm biopsy (odds ratio = 13.3 [iqr: 6.7–36.2]) and bma clot (odds ratio = 12.8 [iqr: 6.8–31.9]).30 samples with a nodular or diffuse pattern of the pc and cells with golgi staining or non-specific cytoplasmic staining were excluded from the study. in their 2023 publication in south africa, gantana et al. evaluated a counting method in which reviewers selected three representative biopsy areas with low, intermediate, and high pc densities, followed by a manual count in each of these areas and averaging the bmpc% for each sample. this study showed superior interobserver concordance with the manual counting method at low pc tumour burden compared to an overview estimation method (intraclass correlation coefficient = 0.105).31 many studies show significantly higher numbers of bmpc% on cd138-stained immunohistochemistry biopsies compared to bma.12,18,19,21 suggested reasons for this discrepancy are the patchy nature of mm, haemodilute bma, and poor sample quality. for these reasons, cd138 immunohistochemistry is regarded as the most sensitive method for pc estimation. immunohistochemistry determination of pc percentage is valuable for diagnosis and monitoring treatment response, notably the complete and stringent complete response criteria as defined by the international myeloma working group (imwg).3 furthermore, bm trephine pc percentages have shown to correlate with corresponding end organ disorders, quantitative fluorescence in situ hybridisation results, and overall survival in patients with a low bma pc percentage.24 molecular techniques molecular techniques are based on the amplification of unique variable, diversity, joining rearrangements of the immunoglobulin heavy chain variable (ighv) gene on chromosome 14, which are specific for each pc clone and can be used for identification by amplification of the variable, diversity, joining sequence. the light chain genes lack the d segment, which reduces the sensitivity of detecting the pc clone.32 patient-specific ighv gene rearrangements are amplified and sequenced from ighv gene-specific complementary dna to determine the clone-specific variable gene sequence.32,33 this should be done at diagnosis, to design primers for real-time polymerase chain reaction (pcr) quantification for determination of the level of tumour contamination in a sample after chemoimmunotherapy and stem-cell transplants to be used for follow-up monitoring. the sense primers are usually derived from the second complementarity-determining region and the antisense primer from the highly hypervariable third complementarity-determining region in the ighv gene to be used for the amplification of the sequence of interest during follow-up.34,35,36 molecular techniques such as allele-specific oligonucleotide quantitative pcr (aso-qpcr) and next-generation sequencing (ngs) are used during follow-up for the quantification of clonal plasma cells to evaluate treatment response and detect measurable residual disease (mrd) rather than quantification at diagnosis.4 the use of aso-qpcr for the detection of these specific variable, diversity, joining sequences correlates well with mfc in mrd quantitation (r = 0.881, p < 0.001), but its application was limited to 42% of cases in the study due to lack of clonality detection, unsuccessful sequencing and suboptimal aso performance.37 this complicates the practicality of using only molecular techniques for this indication. radiological imaging techniques radiological imaging plays a significant role in the diagnosis and management of mm as approximately 90% of mm patients develop bone disease.38 radiological imaging may suggest diagnosis, assess possible bone complications, and determine response to treatment or disease progression.1,4,39 radiographic examination (skeletal survey) to identify lytic bone lesions has been replaced by more sensitive alternatives such as whole body, low-dose computed tomography (ct), 18f fluorodeoxyglucose (18f-fdg) positron emission tomography/ct (pet/ct) scanning, and magnetic resonance imaging (mri). in under-resourced countries, skeletal surveys are still widely used due to the lack of availability of superior modalities especially for detection of osteolytic lesions in the spine and pelvis.40,41,42,43,44 positron emission tomography/ct and mri can be used to assess tumour burden and disease activity.45,46 positron emission tomography/ct uses a surrogate measure of intracellular glucose metabolism to distinguish between metabolically active and inactive sites of proliferating cells, while mri examines the water and fat composition of tissues.47 magnetic resonance imaging is therefore more sensitive for the detection of diffuse infiltration and pet/ct is better for the assessment of focal lesions, especially when these lesions are outside the field of view of mri.44 the patchy and focal nature of mm infiltration means that disease burden measured on a random bm biopsy may not be representative. furthermore, seven focal lesions on spinal mri and more than three lesions and extramedullary disease on pet/ct have been shown to be prognostically significant, and it is therefore recommended that the absolute number of focal lesions should be reported.39,47,48,49,50 a south african study in patients at a tertiary hospital showed good concordance between representative bm biopsy samples and 18f-fdg pet/ct image analysis. however, this did not reliably predict pc infiltration of < 10% or diffuse non-hypermetabolic marrow involvement on imaging.51 this study further emphasised the patchy nature of the disease where patients with irregular bm infiltration may result in the underestimation of pc infiltration.51 based on these results, the use of pet/ct imaging can avoid the need for repeated bm biopsies in most patients with diffuse and irregular bm uptake, but cannot replace bm biopsy to determine bmpc%. imaging will continue to play a role in assessing myeloma-defining events, providing prognostic information, and evaluating response to treatment by providing complementary information.4,39 emerging technologies and innovations digital pathology digital pathology, with or without incorporation of artificial intelligence (ai), is growing in popularity while presenting both challenges and opportunities for the future. careful consideration is required when introducing this new area of medicine to patient care. digitisation of immunostained bm biopsy sections can be done by taking still images with a microscope camera or dynamic images with whole slide imaging (wsi) systems. images can be analysed manually or using ai. herein, we review several studies that have explored the use of these innovations in estimating bmpc%. computer-assisted image analysis (cia) for estimating bmpcs in bm biopsies dates to the early 2000s. computer-assisted image analysis was shown to be superior to simple visual estimation of cd138 immunohistochemistry, due to elimination of subjectivity. in a swiss study by went et al. in 2006, cia was performed using image processing software (ks 300, carl zeiss ag, feldbach, switzerland) after digitising selected fields with a light microscope and colour video camera.52 this study showed minimal interobserver and intraobserver variability (determined by spearman’s rank correlation coefficient) between two reviewers using cia.49 a study by stifter et al. in croatia in 2010 used similar image processing software (alphelys spot browser 2.4.4., plaisir, france) for cia after digitisation using a similar method showed high reproducibility.24 the cia evaluated the relative area of overall positive staining rather than a calculated percentage of individually positively stained cells. computer-assisted image analysis is therefore significantly influenced by background staining, large atypical pc, and pc clusters.21,49 technology has improved so that an entire biopsy slide can be digitised with wsi scanners, and more advanced software programmes than those used in cia and ai are used to analyse the images.50,51,53 the wsi, used together with an image analysis tool and established algorithms (asperio; positive pixel count v9, vista, california, united states), has shown good correlation with visual estimates and counts by experienced pathologists. however, it is often influenced by the intensity of the staining, leading to an overestimation of bmpc%.50 the overestimation is due to a ‘halo’ effect in cases with strong positivity, but in these cases wsi still showed good correlation (r = 0.857) with visual estimates.53 another study also claimed that automated digital enumeration of the entire, immunohistochemically stained (cd138 and mum1) biopsy can accurately determine pc burden, irrespective of pattern or extent of disease.54 it also showed that manual visual assessment (a 500-cell manual count in ‘representative regions’) tends to overestimate pc burden.54 the use of an open-source digital image analysis tool, qupath, was validated by baranova et al., in a study published in canada in 2021, for the automation of pc quantification in bm biopsies from patients with mm.55 they accurately and reliably estimated bmpc% on cd138-stained slides with good correlation between manual counting and software image analysis (pearson’s r = 0.96, p < 0.001).56 the study used an aperio scanscope xt scanner (aperio technologies, california, united states) for digitisation of the biopsies to generate wsi for analysis.56 similar studies were done by fu et al. in canada and the united states in 2022, and by lomas et al. in the united kingdom in 2022, using wsi.57,58 whole slide imaging may be used to overcome the considerable variability in cellularity and distribution of plasma cells in bm biopsies by providing images of the full length of the biopsy. whole slide imaging scanners are not readily available in resource-limited settings, hence alternative means of digitising and analysing biopsies are still required. using the ai of qupath, gantana et al., in their south african study published in 2023, counted cd138-positive pc in bm biopsies and showed that digital analysis is superior to both a manual counting method and a previously reported overview estimation method, regardless of the tumour burden of the sample.59 in this study, digitisation of representative biopsy areas with low, intermediate and high pc densities was done with a standard microscope and camera rather than wsi. fu et al. developed a web application that utilises their developed convolutional neural network, which allows pathologists to upload single images from microscope cameras to obtain a percentage of plasma cells in real time based on the cd138-stained plasma cells on that image.57 these methods may be more accessible to low-resource settings than using wsi. the advantage of digital image analysis is therefore considerable. it may be incorporated into routine use, but slides need to be of equal quality. this requires standardisation of biopsy processing protocols.52 one study showed that all pre-analytical aspects affect the appearance of tissue sections and their suitability for digital pathology and therefore recommended visually checking the pc estimates and adjusting both parameters and thresholds accordingly.60 the college of american pathologists recently released an updated guideline for the validation of wsi for diagnostic purposes.61 artificial intelligence the accurate determination of bmpcs plays a central role in the diagnosis, prognosis, the choice of therapy and the monitoring of pc neoplasms and is therefore essential. artificial intelligence, in combination with methods such as flow cytometry and ngs, can provide information to predict risk status and potentially change the diagnostic criteria and risk stratification for mm. in their 2022 publication in france, clichet et al. used an ai-based gradient-boosting machine algorithm to develop a decision tree for determining the risk of mm in a cohort of pc neoplasms. they used mfc (10-colour single tube) parameters including pc %, pathological to normal pc ratio, total pc to haematogone ratio, total pc to progenitor cd117+ ratio, mean fluorescence intensity of cd38 and cd27 from pathological pc, and mean fluorescence intensity of cd27 from normal cells at diagnosis. the ai-based decision tree showed a sensitivity of 94.2%, a specificity of 93.7% and an independent validation success rate of 91.0% without misclassification between monoclonal gammopathy of undetermined significance and smouldering multiple myeloma.62 this online diagnostic tool is currently freely available and could be used for cases with diagnostic dilemmas in pc neoplasms. yenamandra et al., in their study published in the united states in 2021, used data at the time of diagnosis which included variables such as patient age, white blood cell count, percentage of pc in the bm on the myelogram, high-risk cytogenetic alterations, and ngs. these data were used to create neural networks with which they developed a predictor for the risk status of mm, which has the potential to predict high-risk patients.63 the limitation is lack of estimated bmpc% in the bm biopsy sections. with picture archiving and communications systems as the standard in many tertiary institutions, radiology has a significant potential for radiomics, the use of pattern recognition for the extraction of quantitative descriptors from imaging data.64 radiomics and computational algorithms may potentially be used for quantitative imaging in mm patients by assessing different bone lesions in pet/ct imaging and mri.65 this is an interesting and dynamic field, in early phase of development.65,66 however, this depends on the availability of technology in some tertiary facilities with pet/ct and mri technologies. estimation of plasma cells in bone marrow for measurable residual disease there are several strategies for mrd detection in myeloma, including mfc, qpcr, and high-throughput sequencing. the two currently validated mrd methods recommended by the imwg are next-generation flow cytometry and ngs. next-generation flow refers to the 8-colour 2-tube flow cytometry method developed by the euroflow consortium, which uses specific combinations of antibodies to identify and cross-reference neoplastic pc. it has a sensitivity of 10-6. in contrast, conventional 4–8 colour flow cytometry methods can only detect neoplastic pc with a sensitivity of 10-4.67,68 next-generation flow cytometry multiparameter flow cytometry is a sensitive and rapid approach to assessing treatment efficacy with confirmed prognostic value that predicts progression-free and overall survival independent of categorical response assessment and patient biology.4,37,68,69,70 the imwg defined ‘flow mrd negative’ as the absence of phenotypically aberrant clonal pc detected by bm next-generation flow cytometry with a minimum sensitivity of 1 in 105 nucleated cells. the same minimum sensitivity is proposed for the ‘sequencing mrd negative’ state detected by ngs.4 the threshold used to distinguish between mrd positive and mrd negative may vary from sample to sample, and may change as both technologies and treatments improve. the international clinical cytometry society proposed a consensus guideline for the use of flow cytometry for mrd. the international clinical cytometry society recommends the use of at least five initial gating parameters (cd38, cd138, cd45, forward scatter and side scatter) within the same aliquot to accurately identify the total pc compartment and to specify the limit of detection, with a limit of detection of 0.001% (requiring a total number of 3 × 106 cells for analysis) and ideally a limit of quantification of 0.001% (requiring a total number of 5 × 106 cells for analysis).67 the international clinical cytometry society recommendation is to report limit of detection and limit of quantification for each case based on the number of events below the detection limit and the number of events.71 furthermore, cd9, cd56, cd27, cd81 and cd117 should be analysed in combination with the initial gating parameters for detection of mrd in mm.72 another method, the memorial sloan kettering cancer centre 10-colour single tube method, was compared with the imwg-recommended euroflow 8-colour 2-tube method and showed a high degree of agreement (r2 = 0.97) with the residual disease burden detected with abnormal pc.73 it is noteworthy that a bm aspirate sample containing 10 x 106 cells is required for consistent disease detection, rapid processing avoids cell degeneration, and the use of therapeutic monoclonal antibodies such as daratumumab can lead to false-negative mrd results. in addition to sample processing problems, immunophenotype in mm has been shown to be unstable, and many patients (41%) change immunophenotype, which can complicate interpretation.74 in an attempt to overcome the potential loss of typical gating markers (e.g., cd38 and cd138) after treatment with monoclonal antibodies, the members of the signalling lymphocyte activation molecules family, cd229 and cd319, have been identified as new gating markers for pc in flow cytometric immunophenotyping, particularly in relapsed or resistant cases of mm.75,76,77 the cd229 marker has a strong and homogenous expression in pc while cd138 expression showed significant variability.75,76 the new markers may prove to be useful additions for gating clonal pc, estimating pc in bm and assessing mrd in relapsed and resistant mm. it makes sense for pathologists to familiarise themselves with these novel markers and mrd methods as monoclonal antibody therapies such as anti-cd38 become more readily available in resource-limited settings. next-generation sequencing the use of ngs, as with aso-qpcr, is based on the identification and amplification of the sequence of interest, usually in the variable regions of the igh gene on chromosome 14. however, for the follow-up of patients using pcr-based methods, a patient-specific primer must be developed from the diagnostic sequence. with ngs, these regions are amplified and sequenced with specific primers that are not patient specific.36 for this reason, among others, aso-pcr has been largely replaced by ngs methods because patient specific probes are not as reliable in identifying clonal igh after somatic hypermutation compared to ngs during follow-up.36,37,78,79 this is an indication of the superior applicability of ngs over the need to develop patient-specific primers. next-generation sequencing is not entirely free from the effects of somatic hypermutation as it uses consensus primers for clonality detection and subsequent mrd analysis. cost and poor availability are disadvantages of ngs, especially in resource-limited settings. in addition, due to the labour-intensive nature and difficulties in interpreting the results of ngs, a high level of expertise is required. but it remains a useful instrument because it is very sensitive and has been sufficiently standardised for general use. summary of reviewed methods for plasma cell estimation the quantitation of bmpc% on romanowsky-stained bma alone may misrepresent the accurate count of pc due to the patchy nature of infiltration and sample haemodilution. bone marrow pc percentage is consistently lower on bma as compared to immunohistochemistry-stained bm biopsies. flow cytometry alone yields consistently lower results compared to bma myelograms and are not readily available in resource-constrained settings. flow cytometry is, however, useful for determination of pc clonality, mrd, and risk stratification of mm. a combination of bma, bm biopsy, and flow cytometry assessment of pc yield superior results compared to using each sample on its own. cd138 or mum1 immunohistochemistry improves pc identification compared to haematoxylin and eosin and other stains. manual counting in representative areas of cd138-stained biopsies has been shown to improve reproducibility, compared to overview estimation. use of ai image analysis on wsi or static images may currently be the best method for bmpc estimation. guidelines for the standardisation and validation of digital techniques have been published and using static images in combination with open-source software for image analysis will be possible in situations with limited resources. radiological imaging is crucial for the diagnosis and treatment of mm. introduction of new technologies such as double-energy ct and radiomics as standard practice may necessitate changes to imwg guidelines. these techniques are still in the early stages of development for the assessment of patients with mm and are an exciting and rapidly evolving field. further development will be required before radiological imaging can replace bm biopsy for bmpc quantitation. allele-specific oligonucleotide-qpcr and ngs are commonly used to quantify clonal pc in the follow-up of treatment response and detection of mrd. allele-specific oligonucleotide-qpcr correlates well with mfc for mrd quantification, but has limitations in clonality detection and sequencing, which affects its applicability. next-generation sequencing has replaced aso-qpcr because it is more sensitive and does not require patient-specific primers for mrd monitoring, but using either molecular technique for this purpose can be challenging. conclusion diagnosis and classification of pc neoplasms depends on accurate quantification of pc in bm biopsy. although there is no consensus on the optimal method for estimating pc in bm, cd138-stained biopsies are the preferred sample for this estimation and remain invaluable for diagnosis and assessment of response. no sample or method should be used in isolation, as they are better used in combination for this estimation, and laboratories without advanced techniques should continue to use the best methods available to them. in our quest for a gold standard method, this will require continued research and collaboration to expand and improve the currently published methods. finally, digital pathology is likely to redefine the way we practise laboratory medicine and the role of the pathologist in this new digital age, and limited resources need not necessarily be a limiting factor. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions e.j.g. conceptualised the manuscript, wrote the original draft and participated in the review and editing. z.c.c. and e.m. were involved in the preparation of subsequent drafts and in the review and editing of the manuscript. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability 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https://doi.org/10.1182/blood-2009-03-213280 gebreslassie ks, bassa fc, chapanduka zc, warwick jm. the relationship between bone marrow involvement on 18f-fdg pet/ct and bone marrow biopsy in patients with multiple myeloma and other plasma cell neoplasms. s afr j oncol. 2022;6:197. https://doi.org/10.4102/sajo.v6i0.197 went p, mayer s, oberholzer n, dirnhofer s. plasma cell quantification in bone marrow by computer-assisted image analysis. histol histopathol. 2006; 21(9):951–956. https://doi.org/10.14670/hh-21.951 keller a, laziuk k, hammer r, kovalenko m. estimation of plasma cells by cd138-labeled tissue sections is superior to standard bone marrow differential aspirate smears and whole slide image analysis. am j clin pathol. 2016;146(suppl_1):164. https://doi.org/10.1093/ajcp/aqw151.025 malherbe jaj, fuller ka, mirzai b, augustson bm, erber wn. automated digital enumeration of plasma cells in bone marrow trephine biopsies of multiple myeloma. j clin pathol. 2022;75(1):50–57. https://doi.org/10.1136/jclinpath-2020-207066 bankhead p, loughrey mb, fernández ja, et al. qupath: open source software for digital pathology image analysis. sci rep. 2017;7(1):16878. https://doi.org/10.1101/099796 baranova k, tran c, plantinga p, sangle n. evaluation of an open-source machine-learning tool to quantify bone marrow plasma cells. j clin pathol. 2021;74(7):462–468. https://doi.org/10.1136/jclinpath-2021-207524 fu f, guenther a, sakhdari a, mckee td, xia d. deep learning accurately quantifies plasma cell percentages on cd138-stained bone marrow samples. j pathol inform. 2022;13:100011. https://doi.org/10.1016/j.jpi.2022.100011 lomas oc, royston d, ryou h, et al. artificial intelligence-based quantitative and topological analysis of plasma cell burden: a new tool for the assessment of myeloma. blood. 2022;140(supplement 1):12603–12604. https://doi.org/10.1182/blood-2022-158525 gantana ej, nell e, musekwa e, et al. evaluation of a new technique using artificial intelligence 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inform. 2021;12(1):35. https://doi.org/10.4103/jpi.jpi_26_21 kaplan kj, rao lkf. digital pathology: historical perspectives, current concepts and future applications. springer, london, 2016, chapter 1, introduction; p. 1–3 tagliafico as, dominietto a, belgioia l, campi c, schenone d, piana m. quantitative imaging and radiomics in multiple myeloma: a potential opportunity? medicina (lithuania). 2021;57(2):94. https://doi.org/10.3390/medicina57020094 wennmann m, murray jm. potential of radiomics and artificial intelligence in myeloma imaging: development of automatic, comprehensive, objective skeletal analyses from whole-body imaging data. radiologe. 2022;62:44–50. https://doi.org/10.1007/s00117-021-00940-1 arroz m, came n, lin p, et al. consensus guidelines on plasma cell myeloma minimal residual disease analysis and reporting. cytometry b clin cytom. 2016;90(1):31–39. https://doi.org/10.1002/cyto.b.21228 flores-montero j, sanoja-flores l, et al. next generation flow for highly sensitive and standardized detection of minimal residual disease in multiple myeloma. leukemia. 2017;31(10):2094–2103. https://doi.org/10.1038/leu.2017.29 paiva b, gutiérrez nc, rosiñol l, et al. high-risk cytogenetics and persistent minimal residual disease by multiparameter flow cytometry predict unsustained complete response after autologous stem cell transplantation in multiple myeloma. blood. 2012;119(3):687–691. https://doi.org/10.1182/blood-2011-07-370460 lahuerta jj, paiva b, vidriales mb, et al. depth of response in multiple myeloma: a pooled analysis of three pethema/gem clinical trials. j clin oncol. 2017;35(25):2900–2912. https://doi.org/10.1200/jco.2016.69.2517 rawstron ac, böttcher s, letestu r, et al. improving efficiency and sensitivity: european research initiative in cll (eric) update on the international harmonised approach for flow cytometric residual disease monitoring in cll. leukemia. 2013;27(1):142–149. https://doi.org/10.1038/leu.2012.216 stetler-stevenson m, paiva b, stoolman l, et al. consensus guidelines for myeloma minimal residual disease sample staining and data acquisition. cytometry b clin cytom. 2016;90(1):26–30. https://doi.org/10.1002/cyto.b.21249 roshal m, flores-montero ja, gao q, et al. mrd detection in multiple myeloma: comparison between mskcc 10-color single-tube and euroflow 8-color 2-tube methods. blood adv. 2017;1(12):728–732. https://doi.org/10.1182/bloodadvances.2016003715 cao w, goolsby cl, nelson bp, singhal s, mehta j, peterson lac. instability of immunophenotype in plasma cell myeloma. am j clin pathol. 2008;129(6):926–933. https://doi.org/10.1309/8uvf7yq1d4d4etqv ghogale s, tauro w, priyadarshini a, et al. slam family member ‘cd229’: a novel gating marker for plasma cells in flow cytometric immunophenotyping (fci) of multiple myeloma (mm). clin lymphoma myeloma leuk. 2017;17(1):e19. https://doi.org/10.1016/j.clml.2017.03.032 tembhare pr, ghogale s, tauro w, et al. evaluation of cd229 as a new alternative plasma cell gating marker in the flow cytometric immunophenotyping of monoclonal gammopathies. cytometry b clin cytom. 2018;94(3):509–519. https://doi.org/10.1002/cyto.b.21619 sriram h, ghogale s, subramanian pg, et al. evaluation of cd319 (slamf7) as a novel gating marker for plasma cells in flow cytometric immunophenotyping of multiple myeloma. clin lymphoma myeloma leuk. 2019;19(10):e153. https://doi.org/10.1016/j.clml.2019.09.255 bakkus mhc, bouko y, samson d, et al. post-transplantation tumour load in bone marrow, as assessed by quantitative aso-pcr, is a prognostic parameter in multiple myeloma. br j haematol. 2004;126(5):665–674. https://doi.org/10.1111/j.1365-2141.2004.05120.x ladetto m, brüggemann m, monitillo l, et al. next-generation sequencing and real-time quantitative pcr for minimal residual disease detection in b-cell disorders. leukemia. 2014;28(6):1299–1307. https://doi.org/10.1038/leu.2013.375 article information authors: modisa s. motswaledi1 rosinah sekgwama2 ishmael kasvosve1 affiliations: 1faculty of health sciences, department of medical laboratory sciences, university of botswana, botswana2jwaneng mine hospital, jwaneng, botswana correspondence to: modisa motswaledi postal address: private bag ub 00712, gaborone, botswana dates: received: 27 july 2013 accepted: 17 july 2014 published: 16 oct. 2014 how to cite this article: motswaledi ms, sekgwama r, kasvosve i. tuberculosis alters pancreatic enzymes in the absence of pancreatitis. afr j lab med. 2014;3(1), art. #129, 5 pages. http://dx.doi.org/10.4102/ ajlm.v3i1.129 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. tuberculosis alters pancreatic enzymes in the absence of pancreatitis in this original research... open access • abstract • introduction • research method and design    • ethical considerations • results • discussion    • limitations of the study    • recommendations    • conclusion • acknowledgements    • competing interests    • author contributions    • trustworthiness    • reliability and validity • references abstract top ↑ background: lipases and phospholipases are thought to contribute to the pathogenesis of mycobacterium tuberculosis (mtb) infection. genes coding for lipases, phospholipases and amylase are present in mtb, enabling the bacteria to produce these enzymes.objective: to compare serum lipase and amylase activity levels in patients with tuberculosis (tb) against those of healthy controls. methods: serum lipase and amylase activity levels were measured in 99 patients and 143 healthy controls using the vitros 250 chemistry analyser. reference ranges for serum lipase and amylase were 23–300 u/l and 30–110 u/l, respectively. results: lipase was higher in patients with mtb than in controls (81.5 iu/l versus 66.5 iu/l, p = 0.006). similarly, amylase was higher in the mtb patient group (76 iu/l versus 60 iu/l, p < 0.001). the pearson correlation coefficient for lipase versus amylase (r) was higher in the controls (r = 0.351, p < 0.0001) compared with mtb patients (r = 0.217, p = 0.035). amongst mtb patients, lipase activity correlated positively with erythrocyte sedimentation rate (esr) (r = 0.263, p = 0.013), but not with haemoglobin concentration or treatment duration. a weak inverse correlation was noted between esr and treatment duration (r = -0.222, p = 0.028). conclusion: pancreatic enzyme levels differ between mtb patients and normal controls; however, this difference still lies within the normal range. the concomitant increase of lipase with esr, an inflammatory marker, could conceivably suggest a causal relationship. further research is necessary to characterise mtb-derived enzymes for diagnostic and therapeutic utility. introduction top ↑ serum lipase and amylase activity levels are used traditionally in the diagnosis of pancreatitis. however, in recent years, many investigators have demonstrated the production of pancreatic enzymes in infectious diseases that do not necessarily involve the pancreas.1,2enzymes with lipolytic activity have been implicated as the cause of cytopathic effects on macrophages through lysis of host cell membrane lipids2,3,4,5 and have been suggested as potential biomarkers for active tuberculosis (tb) infection.6 the identification of genes in mycobacterium tuberculosis (mtb) coding for these enzymes has further corroborated the case for their involvement in the pathogenesis of tb.2,7,8,9,10,11,12the purpose of this research was therefore to investigate whether serum activity levels of lipase are elevated in patients with mtb infection. serum amylase was included in order to exclude lipase elevations resulting from pancreatitis, when activity of both enzymes would be expected to be high. it was hypothesised that if lipase enzymatic activity is altered during tb infection, such activity should have a positive correlation with erythrocyte sedimentation rate (esr), a marker of active inflammation, but should diminish with treatment duration. on the other hand, the resolution of infection after treatment was expected to result in an improvement of the haemoglobin (hb) level, which is thus a negative correlation. lipase production has been demonstrated in different microorganisms, including staphylococcus aureus.13 a lipase that inhibits the phagocytic activity of alveolar macrophages has been described in pseudomonas cetacean,14 whilst yet another lipolytic enzyme has been shown to be important for the penetration of endocervical epithelial cells in neisseria gonorrhoeae infection.15 similarly, yersinia enterocolitica’s phospholipase a has been demonstrated to contribute to bacterial pathogenesis in mouse models.16 mtb is known to possess genes coding for several lipases, including phospholipases a2, c and d.3,4,6,9 these reports suggest a new role of pancreatic enzymes in non-pancreatic, infectious disorders. phospholipases are particularly important in bacterial interactions with host cell membranes which provide the substrate for such enzymes, leading to cell lysis.3 cell lysis as a result of phospholipase activity could be important for explaining some forms of tissue necrosis, anaemia and low blood cell counts observed in many infectious diseases, including tb.5,17 the accumulation of membrane lipid components in the mtb caseum such as triacylglycerides, cholesterols and lactosylceramides provides further evidence that mtb possesses the enzymes necessary for degrading membrane lipids.12 in this connection, bakala n’goma et al. demonstrated that phospholipase c from mtb induced cytopathic effects on murine macrophages.3 lee et al. described hydrolysis of cell membrane lipid bi-layers that was triggered by excessive numbers of mycobacteria per cell and postulated that this is the mycobacterium’s escape mechanism from cells that can no longer support the bacterial load.5 in fact, deb et al. had demonstrated previously that mycobacterial lipy, also referred to as rv3097c, was activated under hypoxic conditions and was responsible for the degradation of the caseum-stored triacylglycerides in the face of limited nutrients.10 mtb synthesises as well as acquires lipids from its host cell membranes. these lipids are then utilised as energy sources during the reactivation phase by employing lipolytic enzymes.2,6 various lipolytic enzymes have been ascribed to mtb, including those that hydrolyse monoacylglycerides, triacylglycerides, lipids and phospholipids.3,4 brust et al. demonstrated that patients with active mtb infection produce immunoglobulin g antibodies against certain mycobacterial lipases (lipy, rv0183, rv1984c and rv3452).6 however, such antibodies were not detected in bacille calmette guerin (bcg)-vaccinated individuals, suggesting that they were elicited by active live mycobacteria. côtes and colleagues identified and characterised a monoacylglycerol lipase that was exported to the extracellular medium, indicating the possibility of mycobacterial enzymes acting at sites remote from the focus of infection.9 the findings of brust et al. also documented the presence of lipolytic enzymes in mtb and reported that rev3452 exhibited phospholipase a2 activity with the ability to lyse macrophages.6 mtb therefore possesses the ability to hydrolyse phospholipids and lysophospholipids at virtually any position using enzymes such as lipy (a triacylglycerol hydrolase),18 rv3452 (a phospholipase a2),4 rv1984c (acting on medium-length chain carboxylic esters and monoacylglycerol),4 rv0183 (diacyl and triacylglycerols),2 rv1399c (soluble triacylglycerol and vinyl esters).7 dhouib et al., working with a recombinant protein from mycobacterium smegmatis exhibiting lipase activity (msmeg_0220), showed that the protein had extensive amino acid sequence identity with the mycobacterial lipase rv0183.19 a mutation of this gene impaired the ability of the bacterium to utilise mono-olein for growth and increased its susceptibility to rifampicin, but imparted more resistance against isoniazid. an understanding of the role of lipases in mycobacterial infections may therefore provide insight into their interactions with chemotherapeutic agents. amylase, on the other hand, is an enzyme almost exclusively devoted to the digestive system. however, the product of the gene rv1326c from mtb exhibits both the glycogen branching and α-amylase activities.11 a study by villena et al., involving 40 neoplastic lesions and 26 benign conditions, found that tb was the most frequent non-neoplastic cause of a high serum amylase. moreover, high levels of serum amylase correlated with worse survival.1 the function of amylase in mycobacterial metabolism remains to be unravelled. research method and design top ↑ patients were recruited at various tb treatment facilities by explaining the purpose and procedure of the study. written, informed consent (in both setswana and english) was obtained from each consenting individual 18 years and older. participants were informed of their right to withdraw from the study at any time without fear of victimisation or compromised care. parental permission was obtained prior to obtaining samples from persons under the age of 21 years. ninety-nine patients of all ages with mtb, who were receiving standard anti-tb treatment at 12 clinics in botswana, were studied. testing was done at jwaneng mine hospital.all patients in the study were diagnosed with tb and were at various stages of treatment, including those who were commencing treatment, those who had been on treatment for some time and those being managed for relapses of pulmonary or extrapulmonary tb. the patients selected for this study were neither being managed for renal insufficiency nor was there any clinical evidence of inflammatory conditions other than tb. written informed consent was obtained from the patients. for children aged less than 18 years, permission was obtained from their parents and/or guardians. demographic data and other data needed for the research were retrieved from patients’ medical records. control samples were obtained from the national blood transfusion service. these were selected on the basis of having undergone stringent health screening and been found to be free of any signs of acute or chronic diseases to ensure the safety of their blood for transfusion. all samples with an enzyme activity level four times the upper limit of normal or more, for either of the enzymes under study (lipase or amylase), were excluded. levels in this range defined pancreatitis. none of the patients included in the computation had amylase or lipase activity levels elevated sufficiently to suggest pancreatitis defined in this manner. ethical considerations permission to carry out research was obtained from the university of botswana’s institutional review board, the botswana ministry of health (permit no. ppme13/18/1 us v [184]), district health management teams (ah 10/7 i [79] plus gaborone dhmt letter dated 17/01/2012) and the jwaneng district health management team (jdhmt pf 58). demographic data were entered in a password-protected computer and no details of names or personal identifiers were used. access to data was limited to the researchers only.venous blood was collected following standard safety and operating procedures. for the serum lipase and amylase assays, 3 ml – 5 ml of blood was collected in plain tubes. for the esr and full blood count assays, 3 ml – 5 ml of blood was collected in tri-potassium ethylene diamine tetra acetic acid (k3edta) tubes. for transportation, samples were stored in a cooler box with ice packs and processed on the day of collection. blood in plain tubes was centrifuged at 3500 rpm for 5 minutes and the serum was transferred into micro vials, stored at 2–8 °c in a refrigerator and processed the following day in accordance with the manufacturer’s (vitros chemistry products, ortho clinical diagnostics, rochester, ny) recommendation that lipase and amylase activity levels are stable for 3 weeks at refrigerator temperatures.20,21 serum lipase and amylase activity levels were measured using the vitros 250 chemistry analyser (ortho clinical diagnostics, rochester, ny) using reagents, control sera and test protocols supplied by the manufacturer. only lipase and amylase were measured in the control group. haemoglobin concentration was measured in samples from mtb patients, using the sysmex® xt2000i (sysmex® asia pacific, woodlands, singapore) and esr was determined using the westergren method.22 in this study, pancreatitis was defined as an amylase activity level at least four times the upper limit of normal (≥ 440 iu/l) or lipase at least five times the upper limit of normal (≥ 1500 iu/l).23 statistical analysis was performed using ibm spss version 20 statistical software (ibm corporation, somers, ny). the independent samples mann-whitney u test was used to test for differences in lipase and amylase activity level between patients with mtb and controls and pearson correlation analysis was used to determine relationships between lipase, amylase, esr, haemoglobin concentrations and treatment duration in the patient and control groups. for purposes of calculations, esr values above 150 mm/hr and amylase values below the detection level (< 31 iu/l) were excluded and not used in any of the calculations. treatment duration data were rounded to the nearest whole month. results top ↑ serum lipase and amylase activity levels were measured in 99 mtb-infected individuals and 143 uninfected controls. the results of one patient with tb were excluded from the analysis since their lipase and amylase levels were consistent with pancreatitis as defined above.23 lipase levels were higher (81.5 iu/l) in mtb patients than in the controls (66.5 iu/l), p = 0.006, as were amylase levels (patients 76 iu/l versus controls 60 iu/l, p < 0.001). the enzyme level data distribution was non-gaussian, hence the choice for using the median rather than the conventional mean. the results are summarised in table 1. table 1: comparison of lipase and amylase activity levels in patients with tuberculosis versus normal controls. in the controls, lipase and amylase levels correlated significantly (pearson correlation coefficient r = 0.351, p < 0.0001). this relationship, although still present in the mtb patient group, was weaker (r = 0.217, p = 0.035). as expected, esr correlated negatively with both hb (r = -0.420, p < 0.001) and treatment duration (r = -0.222, p = 0.028). the mean values (standard deviation) for other parameters were: esr = 66 (40) mm/hr; hb = 11.8 (2.5) g/dl; and treatment duration = 3.1 (2) months. lipase levels in the mtb patient group correlated positively with esr, a surrogate test for inflammation. amylase levels in patients did not correlate with any of the parameters measured. the results are summarised in table 2. table 2: lipase and amylase pearson correlation statistics in mtb patients versus other parameters. discussion top ↑ we measured lipase and amylase activity levels in mtb-infected individuals and normal controls. lipase and amylase activity levels were significantly higher in the mtb patient group. moreover, lipase correlated with severity of inflammation as estimated by the esr, leading us to infer that lipase is conceivably causally related to the underlying inflammatory process.the serum lipase levels in normal controls correlated with those of amylase, suggesting a common pancreatic source. although the lipase-amylase relationship was weak, it was clearly stronger in the control group than among mtb patients. the weakening of this correlation in infected individuals could suggest production of the enzymes at independent, non-pancreatic sites in mtb where genes are regulated by varying or independent physiological parameters. mycobacterial lipase rv1399c, for example, is a hormone-sensitive hydrolase, whilst production of r3097c is only stimulated under hypoxic and nutrient-deficient conditions.10 in addition, the bacterial load, which differs from one individual to another, may also influence the degree to which released mycobacterial enzymes affect the total enzyme activity level. this could be complicated further by the presence of antibodies against mycobacterial lipases in infected individuals.24 it remains to be shown by further enquiry whether the enzyme elevations in our study represent de novo mycobacterial activity rather than pancreatitis or the effect of anti-tb drugs.25,26 in this study, the lipase and amylase levels were far below those that define pancreatitis, thus technically excluding pancreatic inflammation. moreover, treatment duration with anti-tb drugs did not correlate with lipase or amylase activity levels. it should be noted, however, that anti-tb drugs could either elevate the enzymes if they caused pancreatitis, or diminish mtb-derived enzymes if treatment were successful. patients with tb typically present with an elevated esr and anaemia.27the esr is affected by both the anaemia and the underlying inflammation.28 the anaemia of chronic infection is generally considered to be a result of ferrokinetics as regulated by the balance between hepcidin and ferroportin.29 although trending negative as expected, the relationship between lipase and hb was not significant in this study. our results suggest that lipase should be investigated as a possible contributor to haemolysis, anaemia and elevated esr in the light of other reports of phospholytic activity in patients with tb, which is known to undermine the integrity of cell membranes.3,9,16,30 limitations of the study the assays used in this study have not been validated for mtb enzymes. however, mycobacterial lipases are diverse, including several lipases with mono-acyl-, di-acyland tri-acyl-glycerol hydrolase activity, as well as multiple phospholipases which should, in principle, be able to act on the triglycerides used as a substrate in this assay. the use of months rather than days to measure the treatment duration may have decreased the sensitivity of the study with regard to making distinctions between new entrants and those who had already undergone treatment for some days. we recommend that future studies include collection of baseline (pre-treatment) samples for each patient, with subsequent or serial samples taken for appropriate statistical comparison. recommendations further investigations are necessary to explain the unknown role of amylase in tuberculosis. furthermore, future research must focus on distinguishing mycobacterial enzymes from those of pancreatic origin so as to improve the interpretation of pancreatic enzyme levels. conclusion our findings suggest that lipase and amylase activity levels are elevated mildly in patients with tb. the cause of this elevation is not known. lipase enzyme activity levels appeared to increase with increasing esr, a marker of inflammation, which in turn diminished with duration of treatment, which suggests a possible effect of mycobacteria on lipase levels. although amylase enzyme levels were elevated in patients, there was no correlation with any of the parameters measured and no explanation could be found for this phenomenon, thus the role of amylase in tb remains enigmatic.our method, however, did not distinguish between the various enzymes with lipolytic activity. therefore, development of specific assays for mycobacteria-specific lipases and amylases is recommended and may yield definitive tools for the diagnosis and monitoring of mycobacteria-related diseases. acknowledgements top ↑ this investigation was supported by a student grant from the government of botswana, ministry of health. gratitude is also extended to the gaborone, lobatse and jwaneng district health management teams for support through nursing and laboratory staff. gratitude is also extended to the ministry of health for granting the required permissions and to the participants for their kind assistance. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. author contributions m.s.m. (university of botswana) was responsible for the project conception and project supervision, as well as writing the paper. r.s. (jwaneng mine hospital) performed experiments and co-wrote the paper. i.k. (university of botswana) analysed the data and provided editorial oversight. trustworthiness the authors attest to the trustworthiness of these findings and declare that observations were reported as measured or observed. reliability and validity all procedures in this report included routine, tested and validated medical laboratory procedures. analysers were calibrated according to manufacturer’s instructions and routine quality assurance procedures, such as the use of normal and abnormal controls, were followed. references top ↑ 1. villena v, pérez v, pozo f, et al. amylase levels in pleural effusions: a consecutive unselected series of 841 patients. chest. 2002;121(2):470–474. http://dx.doi.org/10.1378/chest.121.2.4702.côtes k, bakala n'goma jc, dhouib r, et al. lipolytic enzymes in mycobacterium tuberculosis. appl microbiol biotechnol. 2008;78(5):741–749. http://dx.doi.org/10.1007/s00253-008-1397-2 3.bakala n'goma jc, schué m, carrière f, et al. evidence for the cytotoxic effects 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slides. lipase. ver 2.0, pub. no. mp2-72. rochester, ny, 2002. 22.clinical and laboratory standards institute. reference and selected procedure for the erythrocyte sedimentation rate (esr) test. approved standard – 4th ed. volume 20 number 27. h2-a4. wayne, pa: clsi, 2000. 23.panteghini m, bais r, solinge ww. enzymes. in: burtis ca, ashwood er, bruns de, editors. tietz textbook of clinical chemistry and molecular diagnostics. 4th ed. st louis, mo: elsevier-saunders. pp. 597–643. 24.brust b, lecoufle m, tuaillon e, et al. mycobacterium tuberculosis lipolytic enzymes as potential biomarkers for the diagnosis of active tuberculosis. plos one. 2011;6(9):e25078. http://dx.doi.org/10.1371/journal.pone.0025078 25.chan ty. isoniazid and rifampicin rarely cause acute pancreatitis in patients with tuberculosis. int j clin pharmacol ther. 1999;37(2):109. 26.sementsov i. acute pancreatitis in patients with pulmonary tuberculosis. article in russian. probl tuberk. 2000;2:33–35. 27.nwankwo e, kwaru a, ofulu a, et al. haematological changes in tuberculosis in kano, nigeria. j med lab sci. 2007;14(2):35–39. http://dx.doi.org/10.4314/jmls.v14i2.35326 28.motswaledi ms, kasvosve i. iron deficiency anemia and its complications. in: dincer y, editor. iron deficiency and its complications. new york: nova science publishers, inc., 2011; pp. 121–130. 29.weiss g, goodnough lt. anemia of chronic disease. n engl j med. 2005;352(10):1011–1023. http://dx.doi.org/10.1056/nejmra041809 30.johansen ka, gill re, vasil ml. biochemical and molecular analysis of phospholipase c and phospholipase d activity in mycobacteria. infect immun. 1996;64(8):3259–3266. abstract background methods results and discussion acknowledgements references about the author(s) daniel m. desalegn ethiopia public health institute, addis ababa, ethiopia addis ababa public health research and emergency management core process, addis ababa city administration health bureau, addis ababa, ethiopia addis ketema district health center laboratory, addis ketema, addis ababa, ethiopia boja d. taddese addis ababa public health research and emergency management core process, addis ababa city administration health bureau, addis ababa, ethiopia nebiyou yemanebrhane ethiopia public health institute, addis ababa, ethiopia mulye s. getahun addis ketema district health center laboratory, addis ketema, addis ababa, ethiopia kumera t. kitila ethiopia public health institute, addis ababa, ethiopia addis ababa public health research and emergency management core process, addis ababa city administration health bureau, addis ababa, ethiopia tariku t. dinku addis ababa public health research and emergency management core process, addis ababa city administration health bureau, addis ababa, ethiopia kassahun d. asferie addis ababa public health research and emergency management core process, addis ababa city administration health bureau, addis ababa, ethiopia elizabeth a. wolde addis ketema district health center laboratory, addis ketema, addis ababa, ethiopia gemechis b. tura addis ketema district health center laboratory, addis ketema, addis ababa, ethiopia tilahun b. mersha addis ketema district health center laboratory, addis ketema, addis ababa, ethiopia alemayhu w. rorissa addis ketema district health center laboratory, addis ketema, addis ababa, ethiopia daniel d. wondimagegnehu ethiopia public health institute, addis ababa, ethiopia tinsae k. hailu addis ababa public health research and emergency management core process, addis ababa city administration health bureau, addis ababa, ethiopia abrham t. bika addis ababa public health research and emergency management core process, addis ababa city administration health bureau, addis ababa, ethiopia citation desalegn dm, taddese bd, yemanebrhane n, et al. medical laboratory accreditation in a resource-limited district health centre laboratory, addis ababa, ethiopia. afr j lab med. 2019;8(1), a793. https://doi.org/10.4102/ajlm.v8i1.793 lessons from the field medical laboratory accreditation in a resource-limited district health centre laboratory, addis ababa, ethiopia daniel m. desalegn, boja d. taddese, nebiyou yemanebrhane, mulye s. getahun, kumera t. kitila, tariku t. dinku, kassahun d. asferie, elizabeth a. wolde, gemechis b. tura, tilahun b. mersha, alemayhu w. rorissa, daniel d. wondimagegnehu, tinsae k. hailu, abrham t. bika received: 19 feb. 2018; accepted: 05 feb. 2019; published: 19 sept. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: improving the quality of medical laboratory services is a high priority in many countries. however, quality management systems for laboratories in resource-limited settings are often inadequate. objectives: this article shares the experiences, benefits and challenges of the laboratory journey towards accreditation in a primary healthcare laboratory in addis ababa, ethiopia. methods: a retrospective review of laboratory records in addis ketema health center was conducted from 2012 to 2015. the study was supplemented by observations from some of the authors of this article who worked in the laboratory. results: the laboratory journey towards accreditation began with a baseline assessment in 2012 using the world health organization african region stepwise laboratory quality improvement process towards accreditation; the baseline score was 78 points (0 stars). after mentorship support, the laboratory improved to 198 points (3 stars) in 2013 and 249 points (5 stars) in 2014. the laboratory scaled up to international organization for standardization 15189 requirements and received limited-scope accreditation for tuberculosis sputum microscopy and hematology tests in 2015. after adopting and implementing the standards, steady improvement was observed in the reliability of the laboratory services. lack of resources was the major challenge the laboratory encountered. conclusion: even though a remarkable quality performance improvement was observed over the entire process, inadequate skilled personnel was the major challenge identified in the road towards accreditation. therefore, an appropriate, workload-based staffing structure should be developed to improve and sustain medical laboratory quality standards in resource-limited settings. keywords: laboratory accreditation; iso 15189; world health organization african region; stepwise laboratory accreditation preparedness scheme. background in developing countries, the expansion of laboratory diagnostic infrastructure has increased to the extent of meeting the needs of evidence-based treatment.1 along with investing in accessibility, simultaneous improvements in the quality of laboratory services are needed to ensure the quality of health services.1,2 laboratory service in resource-limited settings is very weak and quality assurance system practices are either very weak or do not exist at all.3 laboratory testing errors can mislead clinicians and cause serious harm to patients, but the errors can be reduced by establishing a comprehensive laboratory quality management system.1,4 the implementation of a laboratory quality management system enables the healthcare system to provide reliable services and strengthens the overall quality of patient care. moreover, it yields long-term benefits in the quality of healthcare, optimises the expenditure of healthcare resources as well as supporting health policy.5 poor infrastructure, low human resource capacity and inappropriate technologies are notable challenges to the implementation of clinical laboratory international quality standards in resource-limited regions. to address this challenge, the world health organization african region (who-afro) established a stepwise approach in developing countries.6,7 laboratories that demonstrate outstanding performance in the who-afro process are accelerated toward international organization for standardization (iso) 15189 clinical laboratory accreditation.6,8 in many developed countries, accreditation of medical laboratories has been established for several decades.9 however, few laboratories have been accredited to date in ethiopia. the who-afro stepwise laboratory quality improvement process towards accreditation (slipta) was launched in ethiopia in 2009 in order to strengthen the quality of laboratory services.10 laboratories in the public health system of ethiopia are divided into four tiers: district/health centre laboratories, hospital laboratories, regional reference laboratories and national reference laboratories.10 the addis ketema district health center laboratory is one of the public health facilities under the addis ababa city administration health bureau. the laboratory has implemented the slipta standards, and its performance improved from a baseline of 0 stars in 2012 to 5 stars in 2015. this outstanding performance on the who-afro checklist encouraged the health centre management and laboratory staff to apply for accreditation to iso 15189:2012, a standard with particular requirements for quality and competence for medical laboratories. this article shares the experiences, benefits and challenges of implementing the who-afro slipta process and iso 15189 clinical laboratory accreditation in a primary healthcare laboratory in addis ababa, ethiopia. methods study setting and context a retrospective review of existing data for who-afro slipta external audit reports and quality indicators at addis ketema district health center laboratory from 2012 to 2015 was conducted. the health centre was established in 1963 under the addis ababa city administration health bureau. in addis ababa, healthcare facility expansion has improved physical access to health services with an emphasis on primary healthcare units, resulting in the potential for an estimated 100% health service coverage of the city.11 the city has 47 hospitals, 573 private clinics (204 higher-, 226 medium-, 143 lower-level clinics) and 100 government health centres.12,13 regarding clinical laboratory accreditation practices at the time of this assessment, only four hospitals, two regional laboratories and the addis ketema district health center laboratory had received limited-scope accreditation to the iso 15189 laboratory standards. the study was supplemented by the observation and experience of some of the authors of this article who worked in the laboratory as experts or managers throughout the accreditation process. data on the laboratory quality improvement process and key laboratory quality indicators, including the who-afro slipta external audit findings, were collected by using a data extraction form. the who-afro external audit reports were assessed on the basis of the slipta checklist scoring system which weighted marks out of a total of 258 points. the checklist star rating was as follows: 0–142 points: 0 stars, 143–165 points: 1 star, 166–191points: 2 stars, 192–217 points: 3 stars, 218–243 points: 4 stars and 244–258 points: 5 stars.14 all collected data were checked for completeness, transcribed, coded, categorised and analyzed to indicate the impact of implementing quality standards on the laboratory services improvements. operational definitions district health centre a district health centre is a health facility that functions as a basic first-line unit and that provides primary healthcare services. quality indicators a quality indicator is an objective measure that potentially evaluates all performance improvements in laboratory services based on iso 15189 standards (turn-around time, external laboratory assessment, customer satisfaction, laboratory service interruption, equipment downtime, specimen rejection and laboratory supplies stock level). results and discussion the road toward laboratory accreditation the laboratory journey towards accreditation began with a baseline assessment in 2012 using the who-afro slipta checklist; the baseline audit result was 78 points (0 stars). major gaps identified at baseline were poor laboratory infrastructure, inconsistent electric power supply, lack of laboratory supplies and knowledge gaps on the standards. in addition, the human power at the laboratory was insufficient to handle the additional workload that resulted from the implementation of the quality management system. this additional work, such as extensive paperwork for adopting laboratory policies, standard operating procedures, manuals, guidelines and other daily task records, was not considered part of the ‘daily tasks’ used to determine the number of laboratory professionals assigned to the health centre. the laboratory staff and management discussed the gaps identified at the baseline and developed a comprehensive and goal-oriented action plan. the laboratory organised sensitisation meetings and mobilised resources from government and partners for renovation, equipment servicing, laboratory supplies and external quality assessment (eqa) materials. the laboratory rooms were renovated and a backup power supply (generator) was purchased using funds obtained from the addis ketema sub-city health offices. for the implementation of a laboratory information system, computers were donated by technical support for the ethiopia hiv/aids initiative, which is an international non-governmental organization at the johns hopkins bloomberg school of public health, johns hopkins university, baltimore, maryland, united states. even though an appropriate, workload-based staffing structure was not developed in ethiopia to implement the laboratory quality standards at the health centre level, the health centre’s management hired two additional laboratory professionals after getting permission from the civil services agency of addis ababa to minimise the workload on laboratory staff. training on the standards and laboratory mentoring services was requested from the addis ababa city administration health bureau, the ethiopian public health institute and other partners. in addition, the german society for international collaboration covered the fee for eqa materials. after a year, the laboratory was enrolled in a national eqa scheme, which is one of the criteria for laboratory accreditation. after one year of mentorship support, the laboratory improved from 78 points (0 stars) on the slipta audit in 2012, to 198 points (3 stars) in 2013, 249 points (5 stars) in 2014 and 251 points (5 stars) in 2015 (figure 1). figure 1: who-afro slipta score trend at addis ketema district heath center laboratory, 2012 – 2015. this outstanding performance of the laboratory on the who-afro checklist encouraged us to seek international accreditation. preparation for accreditation required the involvement and commitment of the entire staff, organisation and partners. we began requesting consultation services from the german society for international collaboration, one of the international organisations working in ethiopia, and the addis ababa city administration health bureau. they provided consultancy services and coaching on how to implement iso 15189 accreditation requirements. the full scope of clinical laboratory accreditation was difficult to implement in our setting due to limited resources and inadequate experience. therefore, based on the availability of internal quality control materials, eqa schemes and other resources in the study setting, the laboratory management and the consultancy organisation selected tuberculosis direct sputum smear microscopy and hematology tests for accreditation. the organisational structure of the laboratory started with the laboratory head, who directly reports to the health centre management, followed by the quality officer, supervisor and ends with the signatory of each section. the laboratory management worked to ensure the responsibility, authority and interrelationships as they were defined and documented in the quality manual. we established different teams and gave them the responsibility for adopting the standard. different teams coordinated on different activities in the areas that needed improvement. the document and record team developed and adopted various documents, including the manual, job-aids and technical and managerial standard operating procedures. the laboratory management customised the laboratory quality policy, staff competency assessment guideline and incidence reporting format. other teams and personnel were assigned as required for the laboratory quality standard, including an internal audit team, customer officer, quality officer, safety officer, and logistics officer. all levels of the laboratory management were tasked with ensuring continued workflow and communication among the staff, so that complaints could be quickly identified, addressed and resolved. the laboratory staff attended regular weekly meetings to assess progress in every unit and any challenges encountered. in addition, the laboratory had monthly meetings with the health facility’s upper management and clinicians, which facilitated an open forum for presenting problems and opportunities for improvement. in addition, a computer-based laboratory information system was adapted locally. it serves as the interface between laboratory testing services and out-patient department services. the system receives laboratory requests from out-patient departments, releases results to clinicians and is used to monitor all laboratory quality indicators. moreover, in order to take corrective action, the laboratory information system notifies the laboratory of any deviation from predefined quality standards, including panic results and unreleased test results within the defined turn-around time. to check the sustainability of the implementation of the systems, the laboratory routinely monitored all quality indicators, including turn-around time, sample rejection rate, daily quality control, refrigerator and room temperature monitoring, supply management, equipment performance and internal and external complaints. external quality assessment performance, customer surveys and safety audit findings were evaluated quarterly. personnel competency assessment and internal audit findings were reviewed twice a year. the monitoring process helped to regulate the entire laboratory process from the pre-analytical to the post-analytical phases. the laboratory management established different agreements with other laboratories in the region for inter-laboratory comparison, referral and backup services to ensure quality services and reduce service interruptions. following all these preparations, the laboratory requested iso 15189 certification from the ethiopian national accreditation offices. on 19 november 2015, 3 years after commencing the journey, the laboratory received limited-scope accreditation for tuberculosis direct sputum smear microscopy and hematology tests15 and became the first iso 15189-accredited district health centre laboratory in ethiopia. the lesson learned from this partial clinical laboratory accreditation may encourage the organisation to scale up to the full scope of accreditation. challenges on the road toward accreditation lack of internal quality control materials and other reagents, inconsistent electric power supply and a limited number of trained laboratory personnel were the major challenges the laboratory encountered and had to overcome. in addition, equipment service and calibration were challenging to procure at minimal cost in the study setting. this problem was due to the limited availability of qualified engineers, high maintenance costs and lack of equipment and spare parts. this led to extended equipment downtime, service interruptions and delays in service delivery, which resulted in increased complaints from clinicians and customers. lack of funds for renovation of existing structures, inadequate laboratory rooms and unsuitable laboratory design were additional challenges in the accreditation process. to implement the standards, we performed major alterations to the daily laboratory operation, which created an additional workload and required resources; owing to these problems, some staff and management members did not accept the standard at the beginning. however, regular training, the inclusion of additional staff, partners’ support and pertinent discussion on the benefits of the standards led to increased understanding and cooperation. in summary, continual training encouraged members of staff to read about the standards and share experiences with quality management. moreover, supplies and equipment service agreements with local vendors, government and partners’ support, leadership and staff commitment were crucial to overcome the challenges. accreditation benefits on adopting the standards, steady improvement was observed in the reliability, reproducibility, traceability and uniformity of laboratory services. we established effective inventory management systems that enabled us to forecast laboratory supplies, reduce reagent wastage and service interruptions due to stock-out. as a result, for 18 consecutive months from march 2014 to august 2015, there were no interruptions to tuberculosis direct sputum smear microscopy and hematology testing services. overall, laboratory diagnostic service interruptions significantly decreased from 2013 to 2015 (figure 2). figure 2: trends in laboratory service interruption, addis ababa, ethiopia, 2013 – 2015. implementation of the standards aided profound improvement in eqa performance. tuberculosis direct sputum smear microscopy slide test performance (blinded, rechecking) improved from 97.9% to 100% and hematology proficiency improved from 84.2% to 100% (table 1). these improvements were due to increased staff competence, which enabled the laboratory to establish a reliable system that provides accurate, reliable and quality service delivery. table 1: tuberculosis direct sputum smear microscopy external quality assessment and hematology proficiency test performance, addis ababa, 2013 2015. the laboratory scheduled the assessment of quality indicators which helped in timely identification of system weaknesses and rapid resolution of identified problems. it assisted in rapid detection of ineffective systems and led to taking corrective actions. the laboratory quality indicator results improved after 2013. on average turn-around time declined from 6 h to 55 min for hematology tests and 3 days to 1 day for tuberculosis direct sputum smear microscopy tests. mean equipment downtime declined from one month to one and a half days, stock-out of supplies declined from 11 days to zero days. higher compliance with sample collection guidelines and sample acceptance and rejection criteria were accompanied by a reduction of sample rejection rates from 3.7% in 2013, to 2.1% in 2014 and 0.43% in 2015. the scheduled assessment of quality indicators benefited the laboratory in tracing errors and complaints. consequently, overall customer satisfaction for all services increased from 63% in 2013 to 87% in 2015 (table 2). table 2: laboratory quality indicators performance, addis ababa, ethiopia, 2013 2015. conclusion the lesson learned from our experience is that clinical laboratory accreditation in a resource-limited primary healthcare laboratory is achievable when the system is implemented using a stepwise approach, such as scaling up from who-afro slipta to a partial-scope accreditation, and scaling up to the full scope of accreditation. however, limited resources and inadequately skilled laboratory personnel were the major challenges identified in our journey towards accreditation. therefore, considering the quality standards, an appropriate, workload-based staffing structure and resources, as well as the well-coordinated commitment of the entire staff, organisation, mentors and stakeholders, must be sought to adopt and sustain medical laboratory quality standards in a resource-limited setting. lessons learned acknowledgements first and foremost, we would like to thank our almighty god for the successful completion of the study. secondly, we would like to express our heartfelt gratitude to addis ketema district health center laboratory staff and management whose participation made this possible. we would also like to extend our gratitude to the addis ababa city administration health bureau, the addis ababa health research and laboratory service version process, the ethiopian public health institute, the addis ketema sub-city health office, the ethiopian national accreditation offices, the german society for international collaboration and alert hospital for their unyielding cooperation. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions d.m.d. and a.t.b. designed the study. d.m.d. led data collection and writing up of the results and the manuscript. all authors participated in data collection, analysis, interpretation and critically evaluated and approved the manuscript. sources of support none. data availability statement the data is available from the corresponding author on reasonable request. disclaimer the views and opinions expressed in this article are those of the author(s) and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references peter tf, rotz pd, blair dh, khine a-a, freeman rr, murtagh mm. impact of laboratory accreditation on patient care and the health aystem. am j clin pathol. 2010;134(4):550–555. https://doi.org/10.1309/ajcph1skq1hnwghf petti ca, polage cr, quinn tc, ronald ar, sande ma. laboratory medicine in africa: a barrier to effective 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[cited 2017 jun 21]. available from: http://www.enao-eth.org/facilities.php?type=medical+laboratories. abstract introduction research method and design results discussion acknowledgements references about the author(s) nokukhanya mdlalose department of virology, national health laboratory service, university of kwazulu-natal, south africa raveen parboosing department of virology, national health laboratory service, university of kwazulu-natal, south africa pravi moodley department of virology, national health laboratory service, university of kwazulu-natal, south africa citation mdlalose n, parboosing r, moodley p. the prevalence of hepatitis b virus infection in hiv-positive and hiv-negative infants: kwazulu-natal, south africa. afr j lab med. 2016;5(1), a283. http://dx.doi.org/10.4102/ajlm.v5i1.283 original research the prevalence of hepatitis b virus infection in hiv-positive and hiv-negative infants: kwazulu-natal, south africa nokukhanya mdlalose, raveen parboosing, pravi moodley received: 18 nov. 2014; accepted: 24 nov. 2015; published: 31 mar. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the prevalence of hepatitis b virus (hbv) amongst south african infants and children has been reported in the pre-hiv era. despite the reported high prevalence of hiv in the general population of south africa, the rate of hiv/hbv co-infection amongst infants and children remains poorly reported. objectives: we describe the prevalence of hbv infection amongst hiv-positive and hiv-negative infants by molecular methods of diagnosis using dried blood spot samples. methods: this retrospective cross-sectional study was conducted between july 2011 and december 2011 in an academic referral laboratory offering viral diagnostic services to the entire kwazulu-natal province of south africa. a total of 322 study samples were collected from discarded residual dried blood spot samples following routine infant diagnosis of hiv. equal proportions of hiv-positive and hiv-negative infant specimens were studied. statistical differences in the prevalence of hbv between the hiv-positive and hiv-negative samples were calculated using the pearson chi-square test, and a p-value < 0.05 was considered statistically significant. further testing for hbv dna using a nested polymerase chain reaction method was performed. results: the overall prevalence of hbv was 10%. in the hiv-positive group, 21 of 161 infants tested positive for hbv compared with 12 of 161 hiv-negative infants who tested positive for hbv. the proportion of infants infected with hbv was marginally higher amongst hiv-positive infants (13.0%; 95% ci 6.8–19.9) compared with hiv-negative infants (7.5%; 95% ci 2.5–13.7; p = 0.098), though not statistically significant. conclusion: the finding of a 10% hbv prevalence in this infant cohort is clinically significant. the non-statistically significant difference in hbv prevalence between the hiv-positive and hiv-negative infants suggests that high prevalence of hbv infection in children may be a problem independent of hiv. introduction hepatitis b virus (hbv) infection remains a major health care problem throughout the world. of the two billion people infected with the virus, more than 350 million are chronic carriers of hbv, and approximately 600 000 die annually from hbv-related complications, including hepatocellular carcinoma.1 expanded vaccination initiatives have been associated with a decline in global hbv prevalence, but national and sub-national prevalence data are still required to monitor disease burden continuously and guide vaccine policies.2 south africa introduced compulsory infant vaccination against hbv as part of an expanded programme on immunisation in 1995.3 pre-immunisation seroprevalence of hbv infection amongst children from the eastern cape province of south africa was reported to be 10.4% in a study conducted between 1995 and 1996.4 a study conducted in the limpopo province showed that the vaccination programme had a positive impact, and there was evidence of elimination of hbv surface antigen carriage amongst children aged 5 years or younger.5 however, another south african study suggested that the response to hbv vaccination amongst hiv-positive infants may be sub-optimal.6 there is a growing body of evidence showing that the prevalence of hbv is significantly higher amongst hiv-positive individuals, mainly because of shared risk factors.7,8,9,10 despite the generally reported high prevalence of hiv and hbv, there is still no clear government policy in south africa for screening or active case finding for hbv amongst high-risk individuals or pregnant women.9 the general prevalence of hbv amongst pregnant women is under-reported, with a retrospective cross-sectional study in the western cape province of south africa reporting a regional seroprevalence of 3.4% amongst hiv-positive women and 2.9% amongst hiv-negative women.11 a study in the kwazulu-natal province of south africa reported a 5.3% hbv prevalence and a 3.1% hiv/hbv co-infection prevalence amongst pregnant women in a province where antenatal hiv prevalence was reported to be 37.4% in 2012.12 the prevalence of hbv amongst south african infants and children was widely reported in the pre-hiv era. vardas et al. reported the overall prevalence of hbv in the eastern cape to be 10.4%, ranging from 8.9% in the 0–6-months age group to 15.7% in the 61–72-months age group between 1995 and 1996.4 another, earlier study in kwazulu-natal province reported the prevalence of hbv in urban, rural and institutionalised children to be 10%, 18.5% and 25.1%, respectively.13 in the era of the south african hiv epidemic, a study conducted in the western cape in 2011 found the prevalence of hbv amongst hiv-exposed infants to be 0.4%.14 the rate of hiv/hbv co-infection amongst infants and children remains poorly reported compared with reports of high exposure to hbv in hiv-infected pregnant women.9 the concept of using whole blood or plasma blotted onto a filter paper to screen for neonatal metabolic disorders was introduced in scotland by guthrie et al. in 1963.15 the use of dried blood spot (dbs) samples for the detection of hiv, as well as the stability of nucleic acids in this sample collection method, is well described.16,17 dbs samples have been shown not only to be reliable and convenient, but also to have improved access to laboratory testing in developing countries.18 the stability of markers for the testing of other infectious diseases, including hbv, on dbs samples is well documented.19,20 the objective of this study was to describe the prevalence of hbv infection amongst hiv-positive and hiv-negative infants (aged between 6 weeks and 18 months) tested for early-infant diagnosis of hiv in kwazulu-natal by detecting hbv dna using polymerase chain reaction (pcr) in dbs samples. we also evaluated whether the prevalence of hbv in this population group was influenced by hiv co-infection. research method and design ethical considerations ethical approval was obtained from the biomedical research and ethics committee (brec) of the university of kwazulu-natal (be243/010). the samples were sequentially selected from those discarded and anonymised. study population and design this retrospective cross-sectional study was conducted in the department of virology at inkosi albert luthuli central hospital, located in the city of durban, kwazulu-natal, south africa. the department of virology is an academic referral laboratory that offers diagnostic virology services to the entire kwazulu-natal province and is a laboratory of the national health laboratory service. approximately 9000 dbs samples are received by the laboratory per month for routine diagnostic detection of hiv using a commercial kit, namely the hiv dna pcr cobas® ampliprep/cobas taqman® hiv-1 qualitative test (roche molecular systems, inc., branchburg, new jersey, united states). the study samples were randomly selected from residual samples scheduled to be discarded after testing for early-infant diagnosis of hiv. samples with a full blood spot from infants aged 18 months or younger were included for hbv testing. samples with inadequate blood spots or from patients aged older than 18 months were excluded. once the hiv results and age were recorded, the samples were anonymised and given unique study numbers. the study samples were collected over a six-month period (july 2011 to december 2011). the samples were stored in individual plastic packets with desiccant at room temperature until tested for hbv using the nested pcr method. the sample size for the number of hiv-positive and hiv-negative samples was calculated using an hiv prevalence of 29.3%, as reported in the south african annual national surveillance in antenatal clinics report of 2008.12 the sample size of 322 was based on an estimated 250 000 live births in kwazulu-natal21 and allowed for a confidence interval (ci) of 95% for a population epidemiological survey, as calculated using epi info™, version 3.5.1 (centers for disease control and prevention, atlanta, georgia, united states). equal proportions of hiv-positive and hiv-negative infant specimens were included. laboratory methods a qualitative in-house nested pcr method22 was used for the detection of hbv dna in the dbs samples. the primers (table 1) targeted the surface gene of the hbv genome. the first outer set of primers was used to amplify the entire coding region for the large-surface antigen gene of the hbv genome, which incorporated the middle-surface antigen and the small-surface antigen. the nested primers amplified the pres2 region and most of the surface antigen. details of this method have been published previously.23 table 1: pcr primers used for detection of hbv dna. nucleic acid extraction was achieved using the magna pure lc™ dna isolation kit iii on the roche magna pure™ lc instrument (roche molecular diagnostics, mannheim, germany). a pre-extraction step was performed24 as a modification to the manufacturer’s protocol for dbs extraction. a whole-blood spot containing approximately 50 µl of dried blood was cut from the filter paper and added into a 300 µl mixture of lysis buffer with proteinase k and elution buffer in a 1-ml microtube. the tubes were incubated on a heating block for 10 min at 65 °c and another 10 min at 95 °c, then centrifuged at 1500 g for 3 minutes. a 200-µl lysate was then transferred for automated nucleic acid extraction. amplification was carried out using the genamp 9700™ thermocycler (applied biosystems™, life technologies, carlsbad, california, united states). the master mix reagents for both pcr rounds were prepared in a separate clean laboratory and kept on an ice block to prevent nonspecific reactions and minimise the formation of primer dimers. the first-round cycling programme had 36 cycles and dilution of the first-round pcr products and addition to the nested master mix were performed in a biosafety cabinet to minimise the inherent contamination risk of the nested pcr method. each run contained one negative and one positive control. owing to the efficacy of the nested pcr, the first-round products were diluted in a 1:2 ratio to minimise background on the agarose gel and increase efficacy of the pcr. after a further 36 cycles of nested pcr, a fragment of approximately 780 bp was visualised using 1% agarose gel electrophoresis. a 100-bp dna molecular weight marker xiv (roche molecular diagnostics, mannheim, germany) was used to identify the desired amplicon size. data analysis and statistical methods data were analysed using spss®, version 22 (ibm® inc., armonk, new york, united states). statistical differences in the prevalence of hbv between the hiv-positive and hiv-negative samples were calculated using the pearson chi-square test. further analysis of the influence of sex and age on the prevalence of hbv was conducted using stratified analysis. a p-value of < 0.05 was regarded as statistically significant. cis were calculated by bootstrapping, using the default options in spss®. results amongst the 161 samples from hiv-positive infants and 161 samples from hiv-negative infants, the overall prevalence of hbv was 10% (table 2). amongst the hiv-positive infants, 21 tested positive for hbv compared with 12 hiv-negative infants who tested positive for hbv. the proportion of hbv infection amongst hiv-positive infants was found to be marginally higher than amongst hiv-negative infants (13.0%, 95% ci: 6.8–19.9 for hiv-positive infants vs 7.5%, 95% ci: 2.5–13.7 for hiv-negative infants; p = 0.098). the observed difference in hbv infection between boys and girls (table 3) was not found to be statistically significant (p = 0.33). further analysis by age categories showed approximately equal proportions of hbv infection amongst all categories for infants aged 52 weeks or younger, with the exception that all samples from infants aged 53 weeks or older were negative for hbv (table 4). the chi-squared test showed that there were no differences in hbv infection across all age categories (p = 0.73). table 2: prevalence of hbv infection amongst hiv-positive and hiv-negative infants, kwazulu-natal, south africa (july 2011 to december 2011).† table 3: hbv infection amongst hiv-positive and hiv-negative infants stratified by sex, kwazulu-natal, south africa (july 2011 to december 2011). table 4: hbv infection amongst hiv-positive and hiv-negative infants stratified by age, kwazulu-natal, south africa (july 2011 to december 2011).† discussion from this cohort of samples referred to an academic laboratory for routine early-infant diagnosis of hiv, we found a 10% prevalence of hbv infection for a sub-population of infants of the kwazulu-natal province of south africa. the world health organization (who) categorises sub-saharan africa as highly endemic for hbv, with a prevalence of more than 8%.25 exposure to hbv in different regions of the african continent is variable, as evidenced by the prevalence of antibodies to the hbv core antibody (anti-hbc). anti-hbc prevalence in western africa has been reported to exceed 85%, whereas the prevalence in eastern africa is reported to range from 65% to 85%, with rural areas showing a higher prevalence than urban areas.26 a systematic review of global epidemiology of hbv over a 27-year period (1980–2007) showed a decrease in hbv prevalence in southern africa, from around 7% in 1990 to just under 4% in 2005.2 south africa has also been shown to have a variable hbv seroprevalence between the different provinces, with rural south african regions being reported to have a higher prevalence than their urban counterparts.27 in the pre-immunisation era, hepatitis b surface antigenaemia was reported to range from 10% amongst urban children to 18.5% amongst rural children in kwazulu-natal.13 a retrospective database analysis conducted for a nine-year period (2002 to 2010) in a reference laboratory in kwazulu-natal estimated the overall seroprevalence of hepatitis b surface antigen in all age groups to be 12.05%.27 our finding of a 10% prevalence in this infant cohort is clinically significant. although previously hbv prevalence has been mostly reported using serological markers, the use of hbv dna as a screening tool can identify even occult infections. occult hbv infection is defined as the presence of hbv dna without hepatitis b surface antigen outside the acute-phase window period. most hbv infections in endemic areas, including sub-saharan africa, are thought to be due to transmissions in the perinatal period and early childhood.28,29 the high prevalence of hbv infection amongst infants in this study affirmed this assertion. studies on the natural progression of hbv infection have shown that perinatally acquired infections progress to chronic hepatitis b, which is worrying for vaccination efforts.31 our study specifically considered hbv rates amongst hiv-positive and hiv-negative infants and therefore differs from previous studies conducted in the pre-hiv epidemic era.4,13 further, in the hiv era, higher prevalences of hbv have been reported amongst hiv-positive adults7,8,9,10 owing to shared routes of transmission. however, the observed prevalence of hbv amongst hiv-positive infants in our study was not statistically significant compared with hiv-negative infants, suggesting that high hbv infection rates may be independent of hiv in infants. similar findings have been reported by studies of pregnant women in south africa.11,30 analysis of data following age stratification showed an equivalent distribution of hbv infection, with almost 10% of infections observed amongst infants aged 6 weeks or younger, suggesting congenital infection in this sub-group. routine screening of pregnant women for hbv infection is still not policy in the south african public health care sector. the recommended treatment for infants exposed to hbv in utero is the administration of the hbv vaccine and hbv-specific immunoglobulin at birth.32 the availability of hbv vaccine and hbv-specific immunoglobulin for passive active immunisation at birth remains a challenge in most south african obstetric units.33 the who recommends hbv vaccination within 24 hours of birth. providing this birth dose is feasible within the south african immunisation schedule, as other vaccines are currently given at birth. we propose that the birth dose of hbv vaccination, which has been shown to reduce early perinatal transmission,34 should be considered as part of the expanded programme on immunisation in south africa. limitations of the study testing was conducted on residual samples that were referred for early-infant diagnosis of hiv, which may have created a bias towards infants who had been exposed to hiv in utero, impacting the generalizability of our findings to the broader population of all infants in kwazulu-natal province of south africa. in addition, no follow-up was conducted to confirm chronic hbv infection. finally, the sample size of our study was relatively small limiting the statistical power to detect differences between hiv-positive and hiv-negative infants. conclusion this study found a clinically significant prevalence of hbv infection (10%) amongst a sample of hiv-positive and hiv-negative infants in kwazulu-natal, south africa. the difference in hbv prevalence between the two groups of infants was not statistically significant, suggesting that hbv infection amongst children in this region may be a problem independent of hiv. acknowledgements competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. sources of support the authors would like to thank the national health laboratory service research trust for funding this research. authors’ contributions n.m. was the project leader and was responsible for performing all laboratory tests and drafting the manuscript. r.p. was responsible for the study design and statistical analysis. p.m. was involved during study conceptualisation and review of the manuscript. all authors are affiliated with the national health laboratory service at the inkosi albert luthuli central hospital, university of kwazulu-natal, south africa. references lavanchy d. hepatitis b virus epidemiology, disease burden, treatment, and current and emerging prevention and control measures. j viral hepat. 2004;11(2):97–107. http://dx.doi.org/10.1046/j.1365-2893.2003.00487.x ott jj, stevens ga, groeger j, wiersma st. global epidemiology of hepatitis b virus infection: new estimates of age-specific hbsag seroprevalence and 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laboratory, botswana correspondence to: talkmore maruta postal address: po box 354, harare, zimbabwe dates: received: 18 may 2014 accepted: 02 july 2014 published: 16 sept. 2014 republished: 07 nov. 2014 how to cite this article: maruta t, yao k, ndlovu n, moyo s. training-of-trainers: a strategy to build country capacity for slmta expansion and sustainability. afr j lab med. 2014;3(2), art. #196, 7 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.196 note: article republished with updated references relating the special issue. copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. training-of-trainers: a strategy to build country capacity for slmta expansion and sustainability in this lessons from the field... open access • abstract • introduction • research method and design    • teachback methodology    • the slmta trainer’s toolkit    • slmta training-of-trainers workshop structure    • final participant assessment    • training-of-trainers programme scale-up    • development of the master trainer cadre    • training-of-trainers programme evaluation • results • discussion    • next steps • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: the strengthening laboratory management toward accreditation (slmta) programme uses a training-of-trainers (tot) model to build capacity for programme scale-up. the tot strategy is designed to maximise utilisation of its graduates whilst minimising inconsistencies and ensuring high programme quality during global expansion.objectives: to describe the slmta tot programme approach. methods: the two-week training, led by carefully selected and trained master trainers, enables effective and authentic implementation of the curriculum by its graduates. the teachback methodology used allows participants to practise teaching the curriculum whilst learning its content. a trainer’s toolkit provides all the materials necessary for teaching and must be followed faithfully during training. two surveys were conducted to assess the effectiveness of the tot strategy: one sent to 316 tot graduates in 25 countries and the other sent to the programme leaders in 10 countries. results: by the end of 2013, 433 slmta trainers had been trained who, in turn, taught more than 1900 people to implement slmta in 617 laboratories in 47 countries. ninety-seven percent of the 433 tot graduates and 87% of the 38 master trainers are based in developing countries. ninety-two per cent of the graduates have been utilised at least once in programme implementation and, as of august 2013, 87% of them were still actively involved in programme activities. ninety-seven per cent of the graduates stated that the tot workshop prepared them well for training or other programme tasks. conclusion: the slmta tot strategy is effective in building local capacity for global programme expansion whilst maintaining programme quality. introduction top ↑ the current drive toward laboratory quality improvement and accreditation in resource-limited settings will be hard to sustain without building local capacity. launched in 2009, the strengthening laboratory management toward accreditation (slmta) programme has revolutionised the ability of government medical laboratories in low-resource settings to implement quality management systems and pursue international accreditation.1,2 slmta is an innovative management training programme that employs a series of workshops and improvement projects to realise immediate and measurable improvement.1 within five years of its launch, slmta has been implemented in 617 laboratories from 47 countries in africa, southeast asia, the caribbean and latin america and has been used to train more than 1900 people.2 its rapid expansion is attributed in part to its effective local capacity-building effort using a training-of-trainers (tot) strategy.tot has been applied across many disciplines including education, healthcare, health promotion and disease prevention to provide would-be trainers with the necessary knowledge and skills for training others.3,4 its popularity is because of its cost-effectiveness and the potential for rapid expansion of local capacity.5 additionally, the development and utilisation of local trainers ensure that the curriculum content is culturally relevant and applicable.6 despite these benefits, there are challenges associated with the tot approach. one is the concern over the ‘wastage’ or low utilisation rate of tot graduates; some studies have reported that 30% − 50% of tot trainees did not deliver any training after the tot.5,6 there are also issues with ‘fidelity of implementation’ – the dilution and distortion of core messages and information as the programme is cascaded downstream because trainers fail to adhere to the curriculum and training protocols;5,6 and also perhaps because documentation of the curriculum does not provide sufficient details to ensure standardisation. furthermore, qualifications of tot participants may be inadequate if their selection does not take into account future availability to train and their defined roles in the programme, in addition to technical competency. great variations of tot methodology have been described, ranging from didactic presentations to group discussions and role-play.7 some tot workshops put equal emphasis on curriculum content learning and facilitation skills development whilst others focus exclusively on the former. although there is no consensus regarding the ideal tot model,4 there has been an increasing trend from traditional passive teaching methods toward active methods that involve the students in both the teaching and learning process.8 in this paper, we describe a teachback-based tot model for building in-country and regional capacity in the implementation of laboratory quality management systems and, specifically, the slmta programme. we discuss how the slmta tot strategy ensures high utilisation of tot graduates and maintains the fidelity of implementation whilst facilitating the rapid scale-up and sustainability of the programme. research method and design top ↑ teachback methodology the slmta tot uses the teachback methodology developed by dr gordon pask in 1975.9 teachback has been recognised as an effective method for educating and assessing learning for patients in clinical practice10 and has been adapted to the teaching of training skills to trainers as well. this intensive practice-based training approach requires participants to play the roles of both trainer and participant: they must teach the curriculum at the same time as they are learning the curriculum content. the slmta tot is organised into three phases: (1) tot facilitators, also called master trainers, teach the curriculum whilst modeling the learner-centred, interactive facilitation skills; (2) participants learn the curriculum content and practise teaching it back to other participants; and (3) master trainers provide feedback on the teachback performance.11feedback is the cornerstone of teachback methodology and follows a distinct protocol. it is given immediately at the end of each teachback session after the participants have had a chance to assess their own performance. praise is always offered first, followed by suggestions for improvement. effective feedback is positive, encouraging and specific, building upon each participant’s strengths. comments must be constructive, focusing on areas that can be improved and behaviours that can be changed. master trainers use standard feedback forms to guide them on what to look for during the teachback. participants receive their feedback both orally and on the written feedback forms.11 the slmta trainer’s toolkit the slmta programme provides a training curriculum that embodies the principle of ‘learning by doing’; lectures are limited to only 20% of the total training time whilst 80% is demonstration, discussion, role play, simulation and hands-on practice. the curriculum comprises 44 activities, which teach participants to use more than 100 tools and job aids in performing 66 management tasks and routines. it requires a facilitation style that engages the participants actively in the learning process. each tot participant receives a trainer’s toolkit containing all the information necessary to teach the 44 slmta activities. in addition to the handouts, tools, worksheets and job aids, the toolkit provides detailed preparation instructions and a step-by-step teaching protocol, grounded in adult learning principles, for each activity. during the tot, master trainers and participants are both required to adhere to the protocols prescribed in each activity. the only deviation allowed is their personal examples and stories for illustrating key concepts and creativity in designing the visual aids. this comprehensive toolkit, coupled with the rigorous teachback process, serves to preserve the fidelity of the programme as the participants deliver the training back home. slmta training-of-trainers workshop structure the slmta tot teaches participants to deliver the slmta curriculum effectively and to implement the programme appropriately. the tot workshop lasts two weeks. because of its intensive performance-based nature, the participant-to-facilitator ratio must not exceed eight to one; a typical tot workshop has at least three master trainers for a maximum of 24 participants.to ensure return on investment and a high utilisation rate of the tot graduates, countries are asked to screen their participants carefully using set criteria. ideal candidates are those who have participated in the slmta process and successfully implemented improvement projects in their laboratories. additional qualifications include: (1) availability to train; (2) defined role to implement the programme as designated by the country’s ministry of health; (3) evidence of motivation; (4) excellent training and communication skills; (5) technical laboratory experience in a clinical setting; and (6) proven ability to manage a laboratory successfully. it is critical to balance participants’ need to learn the unconventional curriculum from the master trainers first hand, with sufficient time to practise teaching each other. the 44 slmta activities are thus presented differently, depending on their level of complexity. master trainers typically teach the 25 most difficult activities so as to ensure fidelity, whilst describing (instead of teaching) the 10 least complicated activities to the participants. sixteen activities are assigned to participants to teach back. some of these activities are so important that they are taught initially by master trainers and then assigned to participants for teachback. table 1 lists the 44 activities in the curriculum and how they are presented during the tot. table 1: workshop activities and how they are presented. the 35 activities taught or described by master trainers occur in plenary sessions. the teachback sessions, on the other hand, occur initially in small breakout groups in order to foster a nurturing and less intimidating environment. each breakout group typically contains eight people, who are then further divided into four teams of two. these teams become teachback partners and remain together throughout the workshop for their teachback assignments. each breakout group is led by a master trainer who serves as a mentor and coach for the group. the 16 teachback activities are assigned evenly to the four teams within each breakout group: each team is responsible for teaching back a total of four activities. the first three are done within their small breakout groups and the fourth is done in plenary sessions to give participants the experience of teaching in a large-group setting. to allow time for preparation, teachback does not begin until the end of the first week of the two-week tot workshop. specific times are allocated for coaching, usually at the end of each day. coaching sessions last 30–60 minutes and allow teams to discuss their upcoming teachback assignments with their master trainers. additional coaching sessions are available by appointment with master trainers. teams then prepare for their assignments, often late into the evening. final participant assessment in addition to the feedback given immediately after each teachback presentation, master trainers meet with individual participants privately at the end of the tot and provide assessment orally on their overall performance. a written report is also submitted to the participants and their organisations following the tot. irrespective of the assessment, all participants who meet the 100% participation requirements are allowed to graduate from the course. training-of-trainers programme scale-up beginning in 2009, slmta tots have been conducted at the african centre for integrated laboratory training (acilt) in johannesburg, south africa, enrolling participants from multiple countries. as the slmta programme spreads deeper within countries, more local trainers and implementers are needed. the acilt-based tot, which admits only two to four participants from each country, is no longer sufficient. to meet the demand, 12 countries and/or regions to date (botswana, dominican republic, ethiopia, ghana, kenya, mozambique, nigeria, rwanda, south africa, tanzania, vietnam/cambodia and zimbabwe) have hosted their own tot workshops. prerequisites for an in-country tot include completion of at least one round of the slmta process (three slmta workshops with supervisory visits and audits), as well as a sound country roll-out plan that details how the increased capacity will be used effectively. development of the master trainer cadre slmta’s tot strategy hinges on the availability of master trainers. with the expansion of the tot programmes, the need for master trainers, who facilitate the tot workshops and mentor future trainers, has escalated. starting in 2011, an effort was made to increase the number of master trainers with a focus on building in-country capacity.master trainers have extensive experience in training, coaching and feedback as well as in implementing laboratory quality systems. they play a critical role in ensuring programme fidelity in the rapid scale-up of slmta around the world. there is a stringent selection and grooming process to create these master trainers. to be eligible for consideration, a candidate must be a certified slmta trainer and have implemented at least one round of slmta roll-out in-country. this includes teaching the entire series of slmta workshops, mentoring laboratories to implement improvement projects and conducting follow-up visits after each workshop. the candidate must be available and must be released by their employer to conduct the two-week long tot. in addition, they must be engaged in all preparation tasks leading to the workshop. finally, this candidate must be nominated by a master trainer and approved by his or her country’s slmta programme coordinator or office before being invited to a tot for apprenticeship. master trainer candidates shoulder equal amounts of responsibility in the tot under the watchful eye of master trainers. they participate fully in the pre-tot planning sessions and serve as lead facilitators for activities assigned to them. they coach the teams to prepare for teachback and provide feedback afterwards. candidates must be prepared to re-teach activities whenever a team fails to deliver their teachback activity effectively. they also provide input to the final assessment of each participant’s performance (both oral and written). in turn, they receive coaching and feedback from the master trainers throughout the tot. at the end of the workshop, master trainers provide feedback and recommendations. because of the apprenticeship model used to develop new master trainers, each tot workshop can accommodate up to three master trainer candidates, with each master trainer mentoring one candidate. training-of-trainers programme evaluation an online survey was conducted in august 2013 in order to assess the effectiveness of the tot strategy on building country capacity for programme scale-up. it included both openand closed-ended questions to collect data on tot graduates’ utilisation rates and on how well the tot prepared them for programme implementation using a four-point likert scale (extremely well; well; not sure; and not at all). the survey was sent to all 316 slmta tot graduates from 25 countries who attended one of the 14 regional or in-country tot workshops held between november 2009 and march 2013. one hundred and seventeen more recent tot graduates were not surveyed because they would not yet have had enough time to deliver any slmta training.to verify the tot graduates’ survey data, a second survey was sent to the programme leaders in all 10 countries that had held a local tot before march 2013 and therefore had large trainer populations. these countries’ slmta programme leaders were asked to provide data on the number of trainers that are still involved in slmta activities. results top ↑ between november 2009 and november 2013, 8 regional and 11 local tot workshops have been conducted, yielding a total of 433 trainers (figure 1). to expand the programme in non-english speaking countries, vietnam, mozambique and the dominican republic each hosted a tot dedicated to producing vietnamese-, portugueseand spanish-speaking trainers, respectively. to address the shortage of francophone trainers, two french-language tots are being planned by cameroon and cote d’ivoire. figure 1: cumulative number of slmta trainers, master trainers and tots 2009-2013. figure 2: global distribution of slmta master trainers. before 2011, there were only two master trainers, both based in the united states. as of the end of 2013, there were a total of 38 master trainers: 27 in africa (71%), seven (18%) in the americas (including two in latin america) and four in southeast asia (11%) (figures 1 and 2). the language portfolio of the master trainers now includes french (n = 5), portuguese (n = 5), vietnamese (n = 4) and spanish (n = 2), in addition to english. of the 316 tot graduates from november 2009 to march 2013, 195 (62%) returned the survey. of the respondents, 160 (82%) have delivered at least one slmta training. an additional 19 (10%) are still involved in slmta programme activities such as mentoring and coordination, yielding a 92% utilisation rate. for the remaining 8% (n = 15) of the tot graduates, reasons for non-involvement included being too busy, not being chosen to train, not being released by supervisors and changing jobs (figure 3). figure 3: results from the tot graduates survey. of the 160 respondents who had facilitated slmta training(s), 59% (n = 94) stated that the tot prepared them extremely well and 36% (n = 58) well. the remaining 5% (n = 8) of the respondents were not sure about the effectiveness of the tot, viewed it as being not at all helpful, or did not respond. the 19 respondents who had been involved only in non-training aspects of programme implementation stated that the tot had prepared them extremely well (n = 13; 69%) or well (n = 6; 31%). all 10 country programme leaders returned the survey. current involvement rates of their local tot graduates (as of august 2013) ranged from 62% to 100%, with an overall 87% (n = 197) of tot graduates still being involved in slmta activities (table 2). table 2: results from the country programme leader survey. discussion top ↑ since slmta tot began in 2009, the slmta programme has expanded rapidly1,2 and many countries have rolled out the programme independently with little or no outside assistance. evidence suggests that the slmta tot strategy has been effective in building local capacity to accelerate programme expansion in multiple regions of the world,2 with 97% of the tot graduates and 87% of the master trainers based in developing countries. today, these cadres of master trainers, trainers and slmta-trained laboratory managers serve as the local champions of laboratory quality and activists in a global network that has kept the slmta ‘grassroots movement’ going.high utilisation rates of the tot graduates have been observed, at 92%. however, the relatively low response rate (62%) on the part of the tot graduates may limit the generalisation of this observation. the low response rate can most likely be attributed to the short survey period, which lasted only two weeks, as well as the fact that an online survey was used which required access to the internet. in the second survey, however, all 10 countries’ programme leaders responded and they reported an average 87% utilisation rate, which is similar to the results from the first survey. fidelity of implementation is also critical as the programme expands and the cadre of trainers multiplies. the quality of training is more difficult to evaluate and was not measured directly in this study. however, evidence suggests that there has been no deterioration of implementation quality with programme expansion, as average audit improvement scores for laboratories implementing slmta in 2011–2013 were the same as those for laboratories implementing slmta during its initial year (2010), at 24%.2 additionally, tot participants reported overwhelmingly that the training was effective in preparing them to implement the programme. several key elements have been built into the slmta tot in an effort to ensure continued quality: (1) the slmta training toolkit comprehensively standardises training protocols and content, assisting trainers to teach the information consistently; (2) a highly structured and demanding tot programme ensures that trainers are fully capable and confident in their training abilities; (3) a prescribed intensive implementation process fosters commitment, understanding and collaboration; and (4) a rigorous process is used for selecting and grooming master trainers, who are the gatekeepers for the quality of future trainers. a collaborative south-to-south network has emerged. eighty-seven per cent of the 38 master trainers reside in 15 developing countries across africa, southeast asia and latin america. these highly-skilled master trainers are often invited to conduct tot workshops in countries other than their own. notably, the depth of the african master trainer pool allowed the export of two master trainers to assist latin america when it held its first regional slmta tot in the summer of 2013. furthermore, most in-country tots provide a few slots for participants from other countries. this fosters networking and experience sharing, as well as assisting countries who are not ready to conduct their own tot but do not want to wait for the next available tot at acilt. there are many examples where countries with slmta experience are aiding those without sufficient training capacity: rwandan trainers travelled to burundi to deliver workshops; namibian participants went to zimbabwe to be trained; the dominican republic enrolled participants from six central american countries alongside its own people; and the african field epidemiology network, headquartered in uganda, delivered slmta training in the caribbean region. the globally-standardised slmta curriculum and common implementation process facilitate cross-border and cross-region technical assistance. next steps casual observers of the programme have noted that the current geographic distribution of the master trainers does not reflect the size and maturity of country programmes (table 3). of the seven countries with the largest number of laboratories enrolled in slmta,2 three (ethiopia, uganda and tanzania) have no master trainers, whilst kenya, with the most rounds of implementation (six), has only one master trainer. lacking master trainers does not necessarily correspond to the quality of a country’s programme, since slmta is implemented by trainers rather than master trainers; nevertheless, going forward it seems prudent that these countries should be encouraged to build a pool of local master trainers. this will not only reduce costs for future tots by eliminating the need to bring in international master trainers, but will also motivate in-country trainers to be involved in more programme activities so they may be eligible to become master trainers. table 3: number of master trainers in countries with the most enrolled slmta laboratories. conclusion top ↑ slmta has helped transform the laboratory landscape in resource-limited countries worldwide.1 through the careful execution of a deliberate tot strategy, the programme has focused on building local capacity to accelerate the breadth and depth of programme spread. rapid programme scale-up has been achieved with continued high quality results,2 using a strategy of centralised and in-country tots, in-country workshops and implementation at the laboratory level. strict maintenance of training and qualification standards has been key to the success of the strategy. the growing pool of motivated and skilled local implementers will be critical with regard to sustaining the on-going quality improvement and accreditation drives in resource-limited settings.skills acquired through the slmta tot, such as programme planning, mentoring and training facilitation, will reach beyond slmta to make participants become more effective managers and mentors. moreover, it has been suggested that slmta be ‘adapted for clinical settings in developing countries, with a goal towards overall hospital accreditation’.1 based on the success of the slmta tot model, we would recommend that other programmes consider adopting the model to build local capacity for rapid programme expansion whilst maintaining programme quality. acknowledgements top ↑ the authors would like to thank dr barbara mckinney, anna murphy and philip rotz for their significant contribution in the development of the slmta toolkit and the design of the slmta tot workshop. the authors also extend their gratitude to all the slmta master trainers and trainers for their tireless effort in building country capacity with regard to implementing the slmta programme. thanks also go to acilt staff members for their on-going support for workshop logistics and preparations.this research has been supported by the president’s emergency plan for aids relief (pepfar) through the us centers for disease control and prevention. the findings and conclusions in this paper are those of the authors and do not necessarily represent the views of the us centers for disease control and prevention. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. authors’ contributions t.m. (clinton health access initiative) implemented the programme and wrote the manuscript. k.y. (us centers for diseases control and prevention) designed and implemented the slmta tot training programme, collected and analyzed the data and substantially revised the manuscript. n.n. (african field epidemiology network) implemented the programme and reviewed the manuscript. s.m. (botswana–harvard hiv reference laboratory), reviewed the article and assisted in development of the data analysis plan. references top ↑ 1.yao k, luman et. slmta collaborating authors. evidence from 617 laboratories in 47 countries for slmta-driven improvement in quality management systems. afr j lab med. 2014. in press.2.yao k, maruta t, luman et, nkengasong jn. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. 2014. in press. 3.usaid. training of trainers manual: conflict transformation and peacebuilding in rwanda [document on the internet]. c2008 [cited 2013 april 01]. available from: http://pdf.usaid.gov/pdf_docs/pnadm806.pdf 4.ray ml, wilson mm, wandersman a, et al. using a training-of-trainers approach and proactive technical assistance to bring evidence based programs to scale: an operationalization of the interactive systems framework’s support system. am j community psychol. 2012;50(3–4):415–427. http://dx.doi.org/10.1007/s10464-012-9526-6 5.unicef. evaluation of unicef learning strategy to strengthen staff competencies for humanitarian response 2002–2004. new york: united nations children’s fund; 2005. 6.hiner ca, mandel bg, weaver mr, et al. effectiveness of a training-of-trainers model in a hiv counseling and testing program in the caribbean region. hum resour health. 2009;7:11. available from: http://dx.doi.org/10.1186/1478-4491-7-11 http://www.human-resources-health.com/content/7/1/11 7.pearce j, mann mk, jones c, et al. the most effective way of delivering a train-the-trainers program: a systematic review. j contin educ health prof. 2012;32(3):215–226. http://dx.doi.org/10.1002/chp.21148 8.yolsal n, bulut a, karabey s, et al. development of training of trainers programmes and evaluation of their effectiveness in istanbul, turkey. med teach. 2003;25(3):319–324. http://dx.doi.org/10.1080/0142159031000092779 9.pask g. the cybernetics of evolutionary process and of self-organising systems. 3rd intern conf on cybernetics. namur: gauthier-villars; 1961. 10.white m, garbez r, carroll m, et al. is ’teach-back’ associated with knowledge retention and hospital readmission in hospitalized heart failure patients? j cardiovasc nurse. 2013;28(2):137–146. http://dx.doi.org/10.1097/jcn.0b013e31824987bd 11.us centers for disease control and prevention, national center for hiv, std and tb prevention, global aids program training team. train-up with teachback! [dvd] produced in partnership with international training and education center on hiv (i-tech) and caribbean hiv aids regional training network (chart). abstract introduction methods discussion acknowledgements references about the author(s) clinton van der westhuizen division of medical microbiology, department of pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa department of medical microbiology, tygerberg hospital, national health laboratory service, cape town, south africa mae newton-foot division of medical microbiology, department of pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa department of medical microbiology, tygerberg hospital, national health laboratory service, cape town, south africa pieter nel division of medical microbiology, department of pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa department of medical microbiology, tygerberg hospital, national health laboratory service, cape town, south africa citation van der westhuizen c, newton-foot m, nel p. performance comparison of three commercial multiplex molecular panels for respiratory viruses at a south african academic hospital. afr j lab med. 2024;13(1), a2415. https://doi.org/10.4102/ajlm.v13i1.2415 note: additional supporting information may be found in the online version of this article as online supplementary document 1. original research performance comparison of three commercial multiplex molecular panels for respiratory viruses at a south african academic hospital clinton van der westhuizen, mae newton-foot, pieter nel received: 24 jan. 2024; accepted: 29 may 2024; published: 20 aug. 2024 copyright: © 2024. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: respiratory infections are a major contributor to hospital admissions. identification of respiratory pathogens by means of conventional culture and serology methods remains challenging. multiplex molecular assays are an appealing alternative that endeavours to be rapid, more accurate and less arduous. objective: the study aimed to compare the clinical performance of three commercial multiplex molecular assays for respiratory viruses. methods: forty-eight respiratory specimens obtained from patients at tygerberg hospital in the western cape province of south africa were studied. these specimens were collected between may 2020 and august 2020. the results of the seegene anyplex™ ii rv16, filmarray® respiratory 2.1 plus panel (farp), and qiastat-dx® respiratory sars-cov-2 panel (qrp) were analysed based on the overlapping targets. a composite reference standard was applied to provide a standard reference for comparison. results: the overall sensitivity of the seegene anyplex™ ii rv16 was 96.6% (57/59), the farp 98.2% (56/57) and the qrp 80.7% (46/57). the overall specificities were 99.8% (660/661), 99.0% (704/711) and 99.7% (709/711), respectively. the qrp failed to detect coronaviruses and parainfluenza viruses in 41.7% (5/12) and 28.6% (4/14) of positive specimens, respectively, while the farp produced the lowest target specificity of 88.4% (38/43) for rhinovirus/enterovirus. conclusion: the overall specificity of all three platforms was comparable; however, the sensitivity of the qrp was inferior to that of the arv and farp. what this study adds: this study adds to the body of performance characteristics described for respiratory multiplex panels, especially in the african context where molecular diagnostics for infectious diseases are gaining momentum. keywords: composite reference standard; multiplex reverse transcription polymerase chain reaction; molecular panels; respiratory viruses; anyplex™ ii rv16; filmarray® respiratory 2.1 plus panel; qiastat-dx® respiratory sars-cov-2 panel. introduction respiratory infections are a major contributor to hospital admissions and result in considerable morbidity and mortality.1,2 published literature denotes viruses and bacteria as the main aetiologies of such disease, and also emphasises the value of laboratory confirmation of specific causative pathogens.3,4 however, isolating and identifying the vast spectrum of infective microbes by means of conventional testing methods can be arduous.5,6 adopting an approach of syndromic testing has thus garnered momentum since the first such test was approved by the food and drug administration of the united states in 2009.7 multiplex molecular assays offer this attractive diagnostic alternative, proving to be quicker and less labour-intensive, while being able to detect multiple targets simultaneously.8 the ability to identify respiratory pathogens both rapidly and accurately relates to multiple potential benefits, including decreased length of hospital stay, improved antimicrobial stewardship and seasonal outbreak surveillance.4,7,9,10,11 however, an important limitation to nucleic acid assay tests is the clinical relevance of the results – a positive result reflects the presence of an infective virus or its antigens, but it does not distinguish between present or past infection.10 other potential drawbacks include false positive (fp) results due to cross-reactivity or nonspecific amplification, false negative (fn) results caused by preferential amplification of one target over another, and the high cost of commercial products.10,11 the routine use of multiplex polymerase chain reaction (pcr) platforms for the detection of respiratory pathogens in south africa, and similarly in africa, is uncertain, and there is a paucity of published research with little local data that contribute to diagnostic and treatment guidelines.12 the performance of a nucleic acid test must be evaluated before it is implemented for clinical use. the advent of coronavirus disease 2019, caused by the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2), has placed reference standards that are used to validate multiplex molecular assays under scrutiny.13,14 the composite reference standard (crs) has been proposed as a reasonable method for this purpose, especially when no gold standard is available, and several imperfect tests are accessible to serve as references.15 a crs combines multiple independent testing methods or approaches to establish the most accurate and reliable result for a particular measurement or diagnosis. it minimises the limitations and biases that may be present in any single individual method, and ensures reproducibility of results.16,17 however, as the sensitivity of the crs increases with more component tests, the specificity may reciprocally decrease, which can lead to accuracy estimates for the index test that are biased.17 tygerberg hospital, an academic hospital in south africa, employed the anyplex™ ii rv16 (arv) at the time of this study to detect respiratory viruses. patients that presented with upper respiratory symptomatology were investigated by means of this commercial assay to diagnose viral pathogens. however, both the biofire® filmarray® respiratory panel 2.1 plus (farp) and the qiastat-dx® respiratory sars-cov-2 panel (qrp) were already in use in the private health sector. these commercially available kits promised quicker turn-around times, with the addition of detecting bacterial pathogens. other multiplex assays, for example xtag® respiratory pathogen panel, esensor® respiratory viral panel and verigene® respiratory virus plus nucleic acid test, are not commonly used in the south african laboratory landscape. implementation of molecular systems for clinical diagnosis has expanded considerably and, although the local epidemiology of respiratory pathogens has been described, published data regarding the evaluation of multiplex molecular assays for respiratory specimens in sub-saharan africa remain limited.18,19,20 this study aimed to compare the clinical performance of the arv, farp and qrp on respiratory specimens in a tertiary hospital setting in south africa by applying a crs. methods ethical considerations ethical approval for this study was obtained from the health research ethics committee of stellenbosch university with study approval number, s22/03/040_sub-study n20/04/047. a waiver of informed consent was obtained as routine patient results were anonymised and no further clinical procedures involving patients were performed for this study. depersonalisation of data was done for the evaluation by allocating unique identifiers to the specimens. these identifiers were used to generate an anonymised entry into an electronic database. no personal details or laboratory information system indicators were captured on the database. study design this study performed the farp and qrp on stored residual clinical respiratory specimens from an academic hospital. the arv results were obtained from medical records containing previous routine testing of the same specimens at the hospital. to allow for the interpretation of sensitivity and specificity of each platform, a crs was utilised to construct a reference standard for comparison between the three assays. study assays the arv is developed by seegene® (seoul, south korea) and utilises tagging oligonucleotide cleavage and extension technology for simultaneous detection of melt curves from 16 viral targets.21 pre-extraction and pre-reverse transcription are required, but it makes use of common real-time pcr platforms. a range of respiratory samples including sputum, bronchoalveolar lavage and tracheal aspirates, can be processed, and a final result is available within 2 h. the farp by biomérieux® biofire® (marcy-l’étoile, france) can detect 23 targets (19 viral and four bacterial) and makes use of melt curve-based reverse transcription pcr (rt-pcr) within a closed automated system.22 respiratory specimens are inoculated in a single-use pouch which integrates nucleic acid extraction and a two-step multiplex rt-pcr to produce a result in 45 min. the qrp from qiagen® (hilden, germany) not only detects 19 viruses and 3 bacteria, but also provides semi-quantitative cycle threshold values.23 the diagcore® technology includes silica membrane-based nucleic acid extraction and highly sensitive rt-pcr. it has been designed to process dry nasopharyngeal swab specimens as well as transport liquid specimens by means of a single-use cartridge. this automated platform has a turn-around time of 69 min. a few differences regarding the arv, farp and qrp targets must be noted (online supplementary table 1). firstly, the arv only detects viral targets, it does not subtype influenza a (h1/h1-2009/h3) and does not test for coronavirus hku1, middle east respiratory syndrome coronavirus or sars-cov-2. comparison of these viral targets in the other two assays, as well as the bacterial targets (bordetella pertussis, bordetella parapertussis, chlamydophila pneumoniae, legionella pneumophila and mycoplasma pneumoniae), is thus not possible. only the arv can subtype respiratory syncytial virus into a or b and distinguish rhinovirus and enterovirus. the farp does not detect bocavirus or l. pneumophila. lastly, the qrp does not test for middle east respiratory syndrome coronavirus, b. parapertussis or c. pneumoniae. study specimens and processing forty-eight respiratory specimens (32 nasopharyngeal aspirates and 16 nasopharyngeal swabs in universal transport media) were included in this study based on results obtained through testing on the arv. a maximum of approximately 50 tests could be considered for the study as resources for the farp and qrp assays were limited. this routine testing occurred during 01 may 2020 and 31 august 2020 on patients that presented with upper respiratory illness to tygerberg hospital which is situated in the western cape province of south africa. these clinical specimens were selected to only include targets that were represented across all three assays. residual specimens were stored at −80 °c in microcentrifuge tubes after arv testing. these specimens were thawed within a period of 12 months between 01 april 2021 and 30 may 2021 to be concurrently run on the farp and qrp. nucleic acid extraction for the arv was done via the biomérieux® nuclisens® easymag® system (marcy-l’étoile, france). the arv rt-pcr was performed on the bio-rad® cfx96™ thermocycler (redmond, washington, united states). a test was considered valid if the amplicon was interpretable and the controls passed for that run. farp and qrp processing were performed according to the manufacturers’ guidelines. a test was considered valid if it completed without error and the internal control passed. it remained valid in the instance where the internal control failed but a target was detected. results interpretation where assays were unable to report subtype, identification to an appropriate group was considered acceptable – for example, influenza a h1 (farp or qrp) was comparable to influenza a with no subtype (arv). where assays were able to distinguish between viruses, identification to an appropriate group was considered acceptable – for example, human rhinovirus (arv) was comparable to rhinovirus/enterovirus (farp and qrp). thawed specimens that failed to demonstrate targets on the farp and qrp that were detected by the arv were immediately re-run on the arv, provided that these targets were used for comparative analysis. this allowed for parallel testing to account for nucleic acid degradation, specimen contamination, and errors in specimen labelling and/or storage. the repeat arv results superseded the initial arv results for analysis. data analysis a crs was applied to the targets that were comparable across the assays. therefore, each target that overlapped across all three assays was included in the crs for that particular specimen. a true positive constituted agreement of two or more of the three assays. a true negative described no detection of targets by two or more of the three assays. an fp reflected a target detected by one assay, but a negative composite reference result. an fn indicated a target not detected by one assay, in contrast to a positive composite reference result. the overall sensitivity and specificity of each platform were calculated based on the comparison of only overlapping targets according to the composite reference established. according to the acceptance criteria of the south african national accreditation system, both sensitivity and specificity must exceed 90% to meet their standard for molecular testing;24 thus, ≥ 90% was used to indicate acceptable performance. if comparison of a specific target was not possible, it was excluded from analysis. this included coronavirus hku1, middle east respiratory syndrome coronavirus, sars-cov-2, bocavirus, and all bacterial pathogens. as such, these targets could not form part of the crs. mcnemar’s chi-squared test was used to determine significant difference between the performance of an assay for a specific target and the crs; a p-value of ≤ 0.05 was used to indicate significance. results valid runs were obtained for all 48 specimens on both the farp and qrp. one specimen failed initial testing on the qrp but was successful with an immediate repeat run, and another specimen had a qrp internal control failure but was deemed valid, as parainfluenza virus 3 was detected. thirteen specimens (27.1%) were positive for targets on the arv that were not detected by either the farp or qrp. these underwent repeat arv testing and only two demonstrated loss of detectable targets (adenovirus and enterovirus, respectively) in comparison to the initial arv result. as all three platforms failed to detect these targets, it was concluded that nucleic acid degradation was the most likely reason. a composite reference was generated for all 48 specimens (table 1). the overall sensitivity of the arv (96.6%) and farp (98.2%) was comparable. however, the qrp had a lower sensitivity of 80.7%. the three platforms had similar specificities ranging from 99.0% to 99.8% (table 2). table 1: seegene anyplex™ ii rv16, biofire® filmarray® respiratory 2.1 plus panel, and qiagen® qiastat-dx® respiratory sars-cov-2 panel targets detected, and composite reference generated for 48 respiratory samples collected in south africa during 2021 and 2022. table 2: performance of the seegene anyplex™ ii rv16, biofire® filmarray® respiratory 2.1 plus panel, and qiagen® qiastat-dx® respiratory sars-cov-2 panel tested on 48 respiratory samples collected in south africa during 2021 and 2022. results of 27 specimens (56.3%) were in consensus across all three assays, while the remaining specimens produced discordant results that amounted to 14 fn and 10 fp results. the qrp contributed 11 fn results, of which five involved the coronaviruses and four the parainfluenza viruses. target-specific sensitivities for coronaviruses 229e, oc43 and nl63 were equal to or less than 60.0%. five of the seven fp results on the farp were due to the rhinovirus/enterovirus target, which translated to a target specificity of 88.4%. however, a mcnemar’s chi-squared test showed no statistically significant impact on farp performance (p = 0.07). the sensitivity and specificity for both influenza a and influenza b viruses were 100.0%. all seven specimens with a composite reference for influenza a were subtyped by the farp and qrp, apart from one where the farp subtyped it as h1-2009, but the qrp failed to subtype the detected influenza a target. discussion this study evinces reliable accuracy of the arv and farp when compared to the crs, but calls attention to the sensitivity of the qrp. it adds to the body of performance characteristics described for respiratory molecular panels and is, to the authors’ knowledge, the first comparison of the latest versions of these assays in africa. the specimens used for this study were selected to include a diverse range of comparable targets to best assess the performance of the platforms. the overall accuracy of the arv and farp were found to be comparable, but the qrp demonstrated lower sensitivity. the high overall sensitivity (98.2%) achieved by the farp contrasts with the overall sensitivity of 84.5% with other platforms that was found in a comparative study in 2012 from north carolina in the united states of america, which also demonstrated significantly lower sensitivity (57.1%) for the detection of adenovirus.4 our study revealed a 100.0% sensitivity for adenovirus, whereas both the arv and qrp showed sensitivities of 83.3% for this target. it was suggested that certain adenovirus serotypes were missed by the previous iterations of the farp, but since the implementation of version 1.7 (current version is 2.1 plus), retrospective and prospective studies have shown improved adenovirus sensitivity as demonstrated in 2013 at a paediatric department in texas, united states of america.25 five fp results were identified for the farp, which involved the detection of the rhinovirus/enterovirus target. this generated the lowest target-specific specificity (88.4%) for any platform, and even though this was not statistically significant in comparison to the crs (p = 0.07), the possible impact of the small study sample should be highlighted. the lack of statistical difference between the farp and crs specificities seems substantiated as no literature was found in support of high fp rates for this target. the arv achieved the best overall specificity compared to the other two platforms, while its overall sensitivity was also acceptable as per south african national accreditation system criteria. a previous study from south korea recommended in 2013 that the sensitivity for the detection of human rhinovirus (88.8%) required improvement.26 however, this was not found in our study, since the arv sensitivity (and specificity) for this target was 100.0%. a peculiar finding in one specimen was the detection of coronavirus oc43 by the arv, but coronavirus hku1 by both the farp and qrp. according to the composite reference methodology, coronavirus hku1 would have been the standard reference. however, this target was excluded from analysis as the arv assay does not detect hku1. this apparent misidentification was noted by another comparative study from 2018 that was conducted in a general hospital of singapore, but not described in further detail.5 the study further commented that distinguishing coronavirus subtypes may be clinically irrelevant as they were historically accepted to cause mild disease and were not monitored for circulation in the population.5 however, this perception has been challenged in the last two decades by the identification of risk groups for severe disease27 and, more recently, refuted by the spectrum of pathology caused by sars-cov-2 during the coronavirus disease 2019 pandemic.28 although nucleic acid integrity was a concern due to the frozen storage of specimens after routine arv runs, only two from a total of 58 positive targets (3.4%) failed detection in the repeat runs, and thus seemed to have degraded. it was noted though that of the seven bocavirus targets that were detected by the arv, only two were detected by the qrp. these specimens were not all retested by the arv. it remains unclear whether the qrp has a lower sensitivity for bocavirus, or whether failed detection was due to nucleic acid degradation. contrary to previous evaluation studies of the qrp in germany (2020) and france (2021), its overall sensitivity in this study (80.7%) did not meet south african national accreditation system acceptance criteria.6,28 this was particularly evident among the coronavirus, and to a lesser extent, the parainfluenza virus targets. although one would be inclined to label these targets as problematic based on this study, the few numbers of specimens analysed during this study remains a point to scrutinise. whether these pathogens contribute to severe illness, as mentioned previously, is another consideration. notably, the arv and farp yielded no fn results for the coronavirus targets. the other qrp fn results were for adenovirus and respiratory syncytial virus. the two fp results of the qrp included parainfluenza virus 1 and respiratory syncytial virus. it was noted that the cycle thresholds values were 33.0 for parainfluenza virus 1 and 31.4 for respiratory syncytial virus. these are higher than the median cycle thresholds value of the true positive results (25.7). this implies that the arv and farp may have missed these targets and that more extensive discrepancy testing could have been of benefit. as the purpose of these assays is detecting pathogens, the major concern of the qrp sensitivity noted in this study cannot be disregarded. but it would be remiss not to recommend that validations of such assays with an appropriate cohort number should be conducted in each laboratory to ensure accurate results. limitations the sample size of this study was limited by the number of kits sponsored by the manufacturers due to supply challenges during the coronavirus disease 2019 pandemic. processing of both routine and thawed specimens occurred which might confound comparison of targets, especially where nucleic acid degradation could have transpired. even though care was taken to account for specimen integrity, it was not within the scope of this study to resolve discrepancies definitively. additionally, as the specimens constituted a selected population (and not a sample of a particular population), the sensitivities and specificities were precise and therefore confidence intervals were not applicable. important targets that were not assessed include sars-cov-2 and the bacterial pathogens – exclusion was due to the routine assay (arv) not being able to detect these targets, and making use of single plex assays to detect them was beyond the scope of this research. as most pathogens were studied in small numbers, extrapolation of the performances of the assays should not be strictly applied. conclusion as multiplex molecular platforms are gaining popularity within clinical diagnostics, rigorous verification of their performance should be underscored. this study demonstrated comparable sensitivity and specificity of the arv and farp using a crs on overlapping targets of stored respiratory specimens. although the qrp produced comparable specificity, its sensitivity was inferior. a more extensive prospective study is required to assess additional targets over a larger sample size. acknowledgements the authors acknowledge prof. tonya esterhuizen from the division of epidemiology and biostatistics, department of global health, at stellenbosch university, who assisted with statistical calculations and interpretations. we also pass our gratitude to ms mathilda claassen from medical virology national health laboratory service, tygerberg hospital, and the staff from medical microbiology national health laboratory service, tygerberg hospital, for their assistance with sample processing. competing interests the authors disclosed that farp and qrp instruments were provided by biomérieux® biofire® and qiagen®, respectively, for the duration of the study. kits and reagents used in this study were also sponsored by both manufacturers. neither biomérieux® biofire® nor qiagen® was involved in the study design, collection, analysis, and interpretation of data, or the writing of the article. authors’ contributions c.v.d.w. was responsible for the conceptualisation, methodology, writing of the original draft, formal analysis, data curation and editing. p.n. was responsible for the conceptualisation, writing review and supervision. m.n.-f. was responsible for the conceptualisation, writing review and supervision. sources of support biomérieux® biofire® and qiagen® sponsored the instruments, kits and reagents used in this study. the authors received no other financial support for the research and/or authorship of this article. data availability the authors confirm that the data supporting the findings of this study are available within the article or its supplementary materials. raw data were generated by the national health laboratory service at tygerberg hospital and are available from the corresponding author, p.n., on request. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. it does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the authors are responsible for this article’s results, findings, and content. references templeton ke. why diagnose respiratory viral infection? j clin virol. 2007;40 (suppl 1): s2–s4. 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evaluation and implementation of filmarray version 1.7 for improved detection of adenovirus respiratory tract infection. j clin microbiol. 2013;51(12):4036–4039. https://doi.org/10.1128/jcm.02546-13 kim hk, oh sh, yun ka, sung h, kim mn. comparison of anyplex ii rv16 with the xtag respiratory viral panel and seeplex rv15 for detection of respiratory viruses. j clin microbiol. 2013;51(4):1137–1141. https://doi.org/10.1128/jcm.02958-12 principi n, bosis s, esposito s. effects of coronavirus infections in children. emerg infect dis. 2010;16(2):183–188. https://doi.org/10.3201/eid1602.090469 lebourgeois s, storto a, gout b, et al. performance evaluation of the qiastat-dx respiratory sars-cov-2 panel. int j infect dis. 2021;107:179–181. https://doi.org/10.1016/j.ijid.2021.04.066 abstract introduction methods results discussion acknowledgements references about the author(s) anthony m. smith centre for enteric diseases, national institute for communicable diseases, johannesburg, south africa department of medical microbiology, faculty of health sciences, university of pretoria, pretoria, south africa phuti sekwadi centre for enteric diseases, national institute for communicable diseases, johannesburg, south africa hlengiwe m. ngomane centre for enteric diseases, national institute for communicable diseases, johannesburg, south africa bolele disenyeng centre for enteric diseases, national institute for communicable diseases, johannesburg, south africa linda k. erasmus centre for enteric diseases, national institute for communicable diseases, johannesburg, south africa juno thomas centre for enteric diseases, national institute for communicable diseases, johannesburg, south africa dineo bogoshi centre for enteric diseases, national institute for communicable diseases, johannesburg, south africa shannon l. smouse centre for enteric diseases, national institute for communicable diseases, johannesburg, south africa nomsa p. tau centre for enteric diseases, national institute for communicable diseases, johannesburg, south africa citation smith am, sekwadi p, ngomane hm, et al. whole-genome sequencing for surveillance of salmonella at a public health institution in south africa. afr j lab med. 2025;14(1), a2900. https://doi.org/10.4102/ajlm.v14i1.2900 original research whole-genome sequencing for surveillance of salmonella at a public health institution in south africa anthony m. smith, phuti sekwadi, hlengiwe m. ngomane, bolele disenyeng, linda k. erasmus, juno thomas, dineo bogoshi, shannon l. smouse, nomsa p. tau received: 12 june 2025; accepted: 11 oct. 2025; published: 09 dec. 2025 copyright: © 2025. the authors. licensee: aosis. this work is licensed under the creative commons attribution 4.0 international (cc by 4.0) license (https://creativecommons.org/licenses/by/4.0/). abstract background: whole-genome sequencing (wgs) is transforming communicable disease surveillance globally. the national institute for communicable diseases, south africa, participates in national laboratory-based surveillance for human isolates of salmonella. objective: this study was to investigate human salmonella isolates from south africa, 2020–2023, using wgs analysis. methods: wgs was performed using illumina nextseq technology. data were analysed using multiple bioinformatics tools, including those available at the center for genomic epidemiology, pathogenwatch and enterobase. data analysis allowed for identification and characterisation of isolates. core-genome multilocus sequence typing was used to investigate the phylogeny of isolates. results: of the 8006 isolates of salmonella that were analysed using wgs, 130 distinctive serovars and subspecies were identified. salmonella enterica serovar enteritidis (salmonella enteritidis) (4271/8006; 53.3%) and salmonella typhimurium (1430/8006; 17.9%) were the most prevalent serovars, accounting for 71.2% of all isolates. this was followed by salmonella typhi (482/8006; 6.0%). sixteen per cent (1288/8006) of isolates showed the presence of antimicrobial resistance (amr) determinants associated with ≥ 2 classes of antimicrobials. salmonella isangi (167/8006; 2.1%) showed the highest prevalence of amr, with most isolates (159/167; 95.2%) showing amr determinants associated with ≥ 7 classes of antimicrobials. core-genome multilocus sequence typing was used to confirm several suspected clusters and outbreaks and identified additional cryptic or unreported clusters and outbreaks. investigation of clusters and outbreaks mostly involved salmonella enteritidis and salmonella typhi. conclusion: the implementation of wgs has enabled genomic surveillance of salmonella, which allows for enhanced characterisation and amr determination of isolates and identification of clusters and outbreaks, which informs targeted public health investigation and response. what this study adds: this study describes the population structure of salmonella isolated from humans in south africa and hugely contributes to the available salmonella wgs data from africa. keywords: salmonella; whole-genome sequencing; genomics; surveillance; outbreak; cluster; south africa; africa; public health. introduction salmonella remains a major cause of human disease worldwide, particularly in developing countries, where they are a leading cause of morbidity and mortality.1,2 in africa, salmonella disease is largely associated with non-invasive gastrointestinal infections; however, a sizable amount of the disease is also caused by invasive infections. in africa, invasive salmonella infections are associated with both typhoidal and non-typhoidal salmonella.3,4 surveillance and laboratory characterisation of salmonella is important to monitor prevalence and trends in salmonella infections. recent trends in public health microbiology have shown an evolution towards whole-genome sequencing (wgs) as the methodology of choice for laboratory investigation of infectious diseases. globally, many public health institutions have transitioned to wgs as their primary methodology for characterisation of bacterial pathogens, and this has included salmonella.5,6,7 the world health organization has endorsed genomics and wgs approaches to investigate various communicable diseases.8,9 the national institute for communicable diseases (nicd) (https://www.nicd.ac.za/) is a national public health institute for south africa, providing disease surveillance, specialised diagnostic services, outbreak response, public health research, and capacity building to support the government’s response to communicable disease threats. the centre for enteric diseases (ced), nicd, performs surveillance on pathogens associated with diarrhoea and enteric fever, and is involved with investigation and response to enteric disease outbreaks. the ced also provides specialised reference laboratory testing for enteric bacteria and viruses. in addition, the ced plays a part in national laboratory-based surveillance for human isolates of salmonella,10 whereby isolates of salmonella are received from more than 200 public and private clinical microbiology laboratories throughout south africa. suspected or laboratory-confirmed cases of enteric fever and clinical laboratory identifications of salmonella isolates are ‘notifiable medical conditions’ in south africa and it is thus mandatory for these to be reported to the department of health.11 the ced performs routine wgs on all salmonella isolates received. whole-genome sequencing data are analysed to confirm the identification of isolates with respect to genus, species and serovar, and further characterise the isolates with respect to multilocus sequence typing (mlst) and presence of antimicrobial resistance (amr) determinants. furthermore, wgs data is also submitted to the public enterobase platform (https://enterobase.warwick.ac.uk/species/index/senterica),12 where data are further interrogated using core-genome mlst (cgmlst) to investigate for clusters of genetically related cases (predictive of possible outbreaks) and to complement epidemiological investigation of outbreaks. before implementation of wgs, presumptive salmonella isolates were identified using traditional phenotypic microbiological methods, which included vitek identification and serotyping performed according to the white-kauffmann-le minor scheme.13,14 this would have been followed by molecular subtyping of isolates, on selected isolates only (mostly associated with outbreak investigations), using methods such as pulsed-field gel electrophoresis analysis and multiple-locus variable-number tandem-repeats analysis.13,14,15,16 in 2020, ced implemented wgs for routine surveillance of clinical isolates of salmonella. we now present the results of this wgs implementation and summarise key findings following analysis of isolates from 2020 to 2023. we report on the number of isolates sequenced, predominant serovars and subspecies identified, significant strains identified, notable amr profiles identified, clusters identified, and outbreaks investigated. methods ethical considerations ethical approval to perform surveillance activities and laboratory analysis on clinical isolates of salmonella was obtained from the human research ethics committee of the university of the witwatersrand, johannesburg, south africa (protocol reference numbers: m160667, m1809107, m210752, m230985). databases where patient data are stored are password protected and the patient identifiers were removed from genomic data shared at public repositories. surveillance for clinical isolates of salmonella in south africa this project started on 01 january 2020 and ended on 31 december 2023. the nicd is a national public health institute for south africa, providing disease surveillance, specialised diagnostic services, outbreak response, public health research, and capacity building to support the government’s response to communicable disease threats. the ced plays a part in national laboratory-based surveillance for human isolates of salmonella. isolates were received from more than 200 public and private clinical microbiology laboratories throughout the country. after salmonella identification at laboratories, isolates were usually received at the ced within 1–4 weeks. following receipt at the ced, isolates were immediately processed for wgs analysis (as described below), a process which is usually completed within 2–3 weeks. metadata and epidemiological investigation salmonella isolates were received with information related to basic metadata, including details of the patient, place of residence and specimen collection date; data all obtained from laboratory request forms. in some situations, such as cases of enteric fever, cases associated with outbreak investigations and cases from some enhanced surveillance sites; patients were followed up to obtain more detailed information and case investigation forms were completed. clinical laboratory identifications of salmonella isolates are ‘notifiable medical conditions’ in south africa, so it is mandatory for these to be reported to the department of health. receipt of bacterial cultures and phenotypic characterisation the ced received and processed isolates using methodology as previously described.17,18 methodology is briefly described as follows. following receipt of presumptive isolates on dorset-egg transport media (diagnostic media products, national health laboratory service, johannesburg, south africa), isolates are sub-cultured onto 5% blood agar (diagnostic media products) to check for viability and purity, following which the isolates are processed to extract genomic dna for wgs analysis. if there is suspicion that a culture is not a salmonella, then that culture will be further investigated using standard phenotypic microbiological identification and serotyping methodologies, including vitek-2 identification (biomérieux, marcy-l’étoile, france) and serotyping completed as per the white-kauffmann-le minor scheme. when required, antimicrobial (ampicillin, ciprofloxacin, ceftriaxone, azithromycin) susceptibility testing was achieved via the etest methodology (biomérieux). interpretation of susceptibility data was performed as per the clinical and laboratory standards institute guidelines.19 genomic dna extraction and whole-genome sequencing of bacteria genomic dna was extracted from bacteria using either the qiagen qiaamp dna mini kit (qiagen, hilden, germany) or the invitrogen purelink microbiome dna purification kit (invitrogen, waltham, massachusetts, united states). whole-genome sequencing was performed by the nicd sequencing core facility (scf). from 2020 to 2023, wgs was performed using various models of illumina equipment (illumina, san diego, california, united states), including illumina miseq, illumina nextseq 550 and illumina nextseq 1000. dna libraries were prepared using various illumina kits, including the nextera xt dna library preparation kit, the nextera dna flex library preparation kit and the illumina dna prep kit. sequencing included paired-end sequencing runs, including ~80 times coverage. analysis of whole-genome sequencing data the ced performed analysis of wgs data using methodology as previously described.17,18 methodology is briefly described as follows. illumina data were processed and investigated with the jekesa bioinformatics pipeline (https://github.com/stanikae/jekesa), which includes several analysis tools. default options were set for all tools, unless otherwise mentioned. quality control and filtering of reads were performed with fastqc version 0.11.9 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and trimgalore version 0.6.2 set at a minimum phred quality score of 30 and minimum read length of 50 bp. identification of species and detection to closest reference were accomplished using bactinspector version 0.1.3 (https://gitlab.com/antunderwood/bactinspector). checking for contamination was accomplished with confindr version 0.7.4 (https://github.com/olc-bioinformatics/confindr) and kraken2 version 2.0.8-beta (https://github.com/derrickwood/kraken2/releases). de novo assembly was accomplished using skesa version 2.3.0 (https://github.com/ncbi/skesa), followed by optimisation of the assemblies with shovill version 1.1.0 (https://github.com/tseemann/shovill), with depth set to 100 and minimum contig length set to 200. assessment of assembly metrics were performed with quast version 5.0.2 using pubmlst typing schemes (https://pubmlst.org/). identification of amr determinants was accomplished with resfinder version 4.1 (https://www.genomicepidemiology.org/services/) and ncbi amrfinder version 3.11.26.20 prediction of salmonella serovars was accomplished with seqsero2 version 1.1.0 (https://denglab.info/seqsero2) and sistr version 1.1.2 (https://github.com/phac-nml/sistr_cmd). investigation of wgs data was further accomplished at enterobase (http://enterobase.warwick.ac.uk/species/index/senterica). raw sequencing data were submitted to enterobase, where the data were quality checked, assembled and analysed via multiple tools, to provide information concerning salmonella serovar, amr determinants, mlst and cgmlst. the phylogeny of isolates was explored with the enterobase cgmlst tool incorporating the ‘cgmlst v2 + hiercc v1’ scheme, which performs an analysis on 3002 core genes of salmonella. the phylogeny and genetic relatedness of isolates were visualised with a grapetree-generated minimum spanning tree using the ‘mstree v2’ algorithm.21 for cluster detection, we followed the following steps. once a grapetree was produced, the settings/operators of the tool were set to ‘collapse branches’ at a value of ‘5’, which ensued that isolates showing ≤ 5 allelic differences were collapsed together into a ‘cluster’. our cluster definition was ≥ 3 isolates showing ≤ 5 allelic differences, as found by the above actions, following cgmlst analysis and creation of a grapetree. for all salmonella serovars (except s. enterica serovar enteritidis [salmonella enteritidis]), we defined a cluster of isolates at a ≤ 5-allele difference threshold, to denote high genetic relatedness among isolates and identify cases likely associated with a common cause (epidemiological link). salmonella enteritidis is a highly clonal serovar, so in order to refine cluster identification for this serovar to obtain the most epidemiological informative clusters, we lowered the cluster definition threshold to a 0-allele difference. clusters were assigned (associated with) enterobase cgmlst hierarchical cluster level 5 identifying numbers (hierarchical cluster level 5 is where isolates are clustered at five allele differences). data availability sequencing data were uploaded to the public enterobase platform (http://enterobase.warwick.ac.uk/species/index/senterica) and are freely available to access at this platform. data are also available at the european nucleotide archive under the project accession numbers prjeb39002, prjeb39546, and prjeb39988. results turnaround time to whole-genome sequencing results and costing of whole-genome sequencing following genomic dna extraction from bacteria, we typically batch samples and submit weekly (usually on a friday) to our scf. the turnaround time to completion of wgs at our scf is ~10 working days. for urgent sequencing, such as for outbreak investigations, sequencing can be fast-tracked, resulting in decreased turnaround times (3–5 working days). in general, for routine sequencing, the turnaround time from receipt of a culture at the ced laboratory to completion of analysis of wgs data, is ~15 working days. the costs to perform illumina wgs have steadily decreased year on year. in january 2020, our cost to perform illumina wgs (paired-end sequencing, at ~80 times coverage) on a single salmonella isolate was ~r2510 south african rand (zar) as compared to ~r1210 zar in december 2023 (r0.055 zar to united states dollar conversion rate, on 10 march 2025). so, wgs has become more affordable with time. sharing of whole-genome sequencing data and public health action all wgs data were uploaded and shared at the enterobase salmonella database (http://enterobase.warwick.ac.uk/species/index/senterica). data shared at enterobase are immediately made available publicly to benefit the global public health and research community. enterobase also auto-uploads data to the sequence read archive, following which project and sample accession numbers are assigned to isolate data. as of 10 march, 2025, enterobase ranked south africa as country number one with respect to the number of salmonella genome submissions from africa, and seventh with respect to global country submissions. as required, salmonella wgs data was presented and discussed at the nicd weekly ‘communicable diseases meetings’. these meetings include representatives from the nicd outbreak response unit, all nicd centres, and epidemiologists from all provinces across south africa. matters discussed included: any interesting findings related to routine disease surveillance activities, trends in disease notifications and reporting, reports of disease clusters, outbreak investigations, and new/emerging cases of diseases. centre for enteric disease will report on any significant findings related to analysis of salmonella wgs data, including clusters identified and outbreak investigations. as required, representatives of our outbreak response unit will communicate with our department of health on all relevant matters. so, the chain of custody for reporting wgs data for public health action is: nicd centre > nicd outbreak response unit > department of health > public notification (as required), and further sharing of information. top (most common) eight salmonella serovars and subspecies in south africa from 2020 to 2023, 8006 isolates of salmonella were analysed using wgs. one hundred and thirty distinctive salmonella serovars or subspecies were identified (figure 1). salmonella enteritidis (4271/8006; 53.3%) and salmonella typhimurium (1430/8006; 17.9%) were the most prevalent serovars, accounting for 71.2% of all isolates. the following serovars or subspecies completed our top (most common) eight: salmonella typhi (482/8006; 6.0%), s. enterica subspecies salamae (279/8006; 3.5%), salmonella isangi (167/8006; 2.1%), salmonella dublin (114/8006; 1.4%), salmonella muenchen (108/8006; 1.3%), and salmonella infantis (98/8006; 1.2%). for salmonella typhi, 414/482 (85.9%) were of the h58 haplotype (genotype 4.3.1) strain (figure 2). for salmonella typhimurium, 269/1430 (18.8%) isolates were of the st313 variant; while 109/1430 (7.6%) isolates were of the monophasic variant (1,4,[5],12:i:-). figure 1: minimum spanning tree created using cgmlst data for salmonella isolates (n = 8006), south africa, 2020–2023. the spherical nodes represent isolates. the larger the spherical node, the more isolates which are indicated. the figure legend lists salmonella serovars and subspecies identified, in order from highest to lowest number of isolates. figure 2: minimum spanning tree created using cgmlst data for salmonella typhi isolates (n = 482), south africa, 2020–2023. the spherical nodes represent isolates. the larger the spherical node, the more isolates which are indicated. the number of segments within a spherical node is representative of the number of isolates. the number values between adjoining nodes specify the number of allele differences between connecting nodes (isolates). the figure legend lists the year of isolation. the cluster of h58 haplotype (genotype 4.3.1) strains is indicated. notable clusters and outbreak investigations table 1 provides a summary of notable salmonella clusters and outbreaks investigated in south africa from 2020 to 2023. these investigations included the following serovars: salmonella enteritidis, salmonella typhi, salmonella typhimurium, salmonella isangi, salmonella panama, salmonella vejle, salmonella newport, and salmonella muenchen. most investigations were associated with salmonella enteritidis and salmonella typhi. figure 3 shows some clusters investigated within the background of other circulating isolates, for salmonella enteritidis, and figure 4 shows the same for salmonella typhi. figure 3: snapshot from a minimum spanning tree created using cgmlst data for salmonella enteritidis isolates, south africa, 2020–2023. the spherical nodes represent isolates. the larger the spherical node, the more isolates which are indicated. the number of segments within a spherical node is representative of the number of isolates. the number values between adjoining nodes specify the number of allele differences between connecting nodes (isolates). the figure legend points to some notable clusters investigated, of which details are described in table 1. figure 4: minimum spanning tree created using cgmlst data for salmonella typhi isolates (n = 68) sourced from the western cape province of south africa, 2020–2021. the spherical nodes represent isolates. isolates showing ≤ 5 allelic differences, are collapsed together into a single spherical node. the larger the spherical node, the more isolates which are indicated. the number of segments within a spherical node is representative of the number of isolates. the number values between adjoining nodes specify the number of allele differences between connecting nodes (isolates). the figure legend lists the year of isolation. some notable clusters investigated are indicated, of which details are described in table 1. table 1: notable salmonella clusters and outbreaks investigated in south africa, 2020–2023. antimicrobial resistance determinants for amr determinants, data were reported as per analysis at the enterobase salmonella database where the ncbi amrfinder version 3.11.26 tool20 is used to report on the following amr classes: aminoglycoside, penicillin, extended-spectrum beta-lactamase (esbl), carbapenemase, colistin, fosfomycin, macrolide, phenicol, quinolone, sulfonamide, tetracycline, and trimethoprim. sixteen per cent (1288/8006) of isolates showed the presence of amr determinants associated with ≥ 2 classes of antimicrobials. among our top (most common) eight serovars or subspecies, salmonella enteritidis showed the lowest prevalence of amr, while salmonella isangi showed the highest prevalence of amr (table 2). most salmonella isangi (159/167; 95.2%) showed amr determinants associated with ≥ 7 classes of antimicrobials, including esbl genes (blaoxa-1, blaoxa-10, blactx-m-15, blatem-63, bladha). for salmonella typhimurium st313 (n = 269), only 37/269 (13.8%) were associated with amr determinants, while most (232/269; 86.2%) were pan-susceptible (figure 5). figure 5: minimum spanning tree created using cgmlst data for salmonella typhimurium st313 isolates (n = 269), south africa, 2020–2023. the circular nodes represent isolates. the larger the circular node, the more isolates which are indicated. the number of segments within a circular node is representative of the number of isolates. the number values between adjoining nodes specify the number of allele differences between connecting nodes (isolates). the legend points to isolates with or without antimicrobial resistance determinants. table 2: antimicrobial resistance determinants associated with the top (most common) eight salmonella serovars and sub-species in south africa, 2020–2023. for the period of this research (2020 to 2023), we identified 297 cases of esbl-positive salmonella, which showed a variety of esbl genes including: blactx-m variants, blacmy variants, bladha, blaoxa-1, blaoxa-10, blatem-63, and blashv-2. most esbl-positive salmonella (162/297; 54.5%) were associated with salmonella isangi. for carbapenemase-positive salmonella, we identified 22 cases, which included: eight isolates of salmonella isangi (housing either the blandm-1 gene or blaoxa-181 gene), four isolates of salmonella enteritidis (housing either the blaoxa-48 gene or blaoxa-181 gene), four isolates of salmonella typhimurium (housing the blaoxa-48 gene), two isolates of salmonella montevideo (housing the blaoxa-48 gene), one isolate of salmonella gallinarum (housing the blaoxa-48 gene), one isolate of salmonella virchow (housing the blaoxa-48 gene), one isolate of salmonella muenster (housing the blaoxa-48 gene), and one isolate of s. enterica subspecies salamae (housing the blaoxa-181 gene). during this same period of research, we also identified five cases of extensively drug-resistant salmonella typhi, of which two cases had confirmed travel history to pakistan. three isolates included resistome: blatem-1, blactx-m-15, cata1, sul1, sul2, dfra7, qnrs1, gyra s83f; while two isolates included resistome: blatem-1, blactx-m-15, cata1, sul1, dfra7, qnrs1, gyra s83f. discussion the ced, nicd, is a member of the regional pulsenet africa laboratory network (https://www.pulsenetafrica.org/), which forms part of the pulsenet international network (http://www.pulsenetinternational.org/), a global molecular subtyping network for foodborne disease surveillance. the ced has always followed standardised molecular subtyping methodologies as suggested by pulsenet international, of which in years gone by, the suggested primary methodology was pulsed-field gel electrophoresis analysis. the ced has published extensively on the use of these older (traditional) molecular subtyping methodologies for routine surveillance activities and for investigation of outbreaks involving enteric bacterial pathogens, which have included the use of pulsed-field gel electrophoresis analysis,15,22 multiple-locus variable-number tandem-repeats analysis,14,16 and mlst (using sanger sequencing of polymerase chain reaction-amplified genes).13,14 in late 2015, ced took the first step towards the use of wgs for analysis of enteric pathogens. this coincided with the establishment of the nicd scf facility equipped with illumina miseq next-generation sequencing equipment. our first wgs activities investigated a cluster of listeria monocytogenes cases reported from the western cape province, south africa, 2015. this analysis was timely, as the steering committee of the pulsenet international network was in discussions to start with implementation of wgs, of which the vision of the network for implementation of wgs was later published in 2017.23 in 2020, ced terminated the use of all older (traditional) molecular subtyping methodologies (pulsed-field gel electrophoresis and multiple-locus variable-number tandem-repeats analysis), and implemented the use of wgs analysis for routine surveillance and analysis of all clinical isolates of enteric bacterial pathogens, including the salmonella. this was needed in order to align with trends in public health microbiology showing the evolution towards wgs as the primary methodology for laboratory investigation of infectious disease. globally, many public health institutions and reference laboratories have transitioned to wgs as their primary methodology for characterisation of bacterial pathogens.5,6,7 we experienced very few challenges with our implementation of wgs. the reasons probably have been that we implemented well-established and well-validated methodologies, and we were supported by a well-established and well-equipped scf with dedicated core staff, including bioinformatics support. whole-genome sequencing all starts with a good quality dna extraction from bacteria. we used good quality dna extraction kits to produce quality in our dna extractions. this then eliminated almost all further problems in downstream sequencing steps, including library preparation. the quality of our sequence data outputs were mostly excellent. on rare occasions, we would encounter assembled data which failed minimum quality thresholds. the quality metrics for our assembled data include an n50 value that must be > 20 kb and number of contigs that must be < 300. for assembled data that fail quality checks, the sample is subjected to a repeated round of sequence analysis, and this usually corrects the quality issue. we rarely encountered contamination problems in our analysis of sequence data. if contamination was encountered, then a repeat dna extraction on a new pure culture and a new sequence analysis solved the problem. on rare occasions, species identification tools would 100% identify sequence data as a non-salmonella species, and in these situations, presumptive laboratory identifications would be updated to reflect the wgs identification. importantly, the implementation of any new laboratory testing methodology must be accompanied by validation data and other checks associated with good laboratory practice. as such, our wgs analysis is accredited as per the international iso 15189 standard, and we are regularly audited by the south african national accreditation system, the official laboratory accreditation body of south africa. validation of our wgs analysis is continuously affirmed by annual participation in two external wgs quality assessment schemes, one managed by the national institute for public health and the environment, the netherlands, in collaboration with the european centre for disease control,24 and the other managed by the national food institute, technical university of denmark.25 in the last 5 years, next-generation sequencing technology has advanced rapidly, resulting in shorter turnaround times to wgs results and higher accuracy of sequencing data. in parallel, costs of next-generation sequencing and wgs have also decreased dramatically over recent years, making the technology more affordable and cost-effective for use in public health laboratories. year-on-year decrease in wgs costs has also been noted by ced, nicd, where the cost to perform illumina wgs on a single salmonella isolate in january 2020 as compared to the cost in december 2023 decreased by 51.8%. in south africa, the costs associated with illumina sequencing is generally more affordable as compared to many other african countries (anecdotal evidence). to some extent, this affordability in south africa can probably be attributed to the presence of an official illumina product distributor in the country, namely separations (https://separations.co.za/). this not only ensures the affordability of reagents and consumables but also ensures rapid order and delivery of said reagents and consumables, timely maintenance and repair of illumina equipment, and overall good customer support. of the 8006 salmonella isolates analysed using wgs, 130 distinctive salmonella serovars and subspecies were identified (figure 1). salmonella enteritidis and salmonella typhimurium (5701/8006; 71.2%) were the most prevalent, which aligns with global trends.5,26,27 the following serovars or subspecies completed our top (most common) eight: salmonella typhi, s. enterica subspecies salamae, salmonella isangi, salmonella dublin, salmonella muenchen, and salmonella infantis. for salmonella enteritidis, this serovar was associated with the majority of our cluster and outbreak investigations (table 1), and was mostly associated with a low prevalence of amr (table 2). among salmonella typhimurium, the st313 variant was commonly encountered (269/1430; 18.8%). the st313 variants are known to be highly associated with salmonella bloodstream infections in africa.28 our reported st313 variants were mostly (232/269; 86.2%) pan-susceptible (figure 5), which was an interesting finding, considering that literature reports st313 variants as typically multidrug-resistant.28 for salmonella typhimurium, 109/1430 (7.6%) of our isolates were of the monophasic variant, of which this variant has emerged globally to become an important pandemic variant with increasing amr,27,29 and increasingly associated with foodborne disease outbreaks.30 for salmonella typhi, most (414/482; 85.9%) of our isolates were of the h58 haplotype (genotype 4.3.1) strain (figure 2). the h58 haplotype is a globally dominant variant of salmonella typhi and commonly associated with amr.31 we were able to identify the h58 haplotype using the enterobase cgmlst hierarchical cluster assignment tool, where hc50:202 is known to be indicative of the h58 haplotype. among our salmonella typhi h58 haplotype strains, most (411/414; 99.3%) showed amr determinants associated with ≥ 4 classes of antimicrobials, commonly including the following resistome: blatem-1b, cata1, sul1, sul2, dfra7. salmonella typhi was often associated with our cluster and outbreak investigations (table 1). a notable investigation involved salmonella typhi cases in 2020 to 2022, associated with an outbreak among illegal gold miners, likely resulting from the consumption of contaminated groundwater while working in a gold mine shaft (table 1).32 salmonella enterica subspecies salamae (279/8006; 3.5%) was our fourth most prevalent salmonella serovar/subspecies. the s. enterica subspecies salamae isolates were genetically diverse, with no evidence to suggest any clonal spread (data not shown). our s. enterica subspecies salamae data were a surprise finding, considering that they are mostly reported from environmental and animal (mostly cold-blooded animals like reptiles) sources, and are generally considered less pathogenic for humans.33 the prevalence of this subspecies in south africa has previously not been reported. historically, before the use of wgs analysis, we would have a large contingent of salmonella isolates reported as ‘salmonella species’, because the traditional serotyping methodologies (using antisera) were sometimes inconclusive in making a call on subspecies or serovar. now with the use of wgs analysis tools, salmonella characterisation is more complete and more accurate, and is now able to more accurately identify s. enterica subspecies salamae. these isolates were mostly cultured from stool specimens of patients. unfortunately, no further information was available about these cases, as no follow-up investigations were conducted for these. also, we are not able to speculate on any possible environmental or zoonotic source for this subspecies, as no further investigations were conducted. salmonella isangi (167/8006; 2.1%) was our fifth most prevalent salmonella serovar or subspecies. salmonella isangi represents an emerging pathogen in south africa. however, globally, salmonella isangi is an uncommon serovar. very few (n = 409) salmonella isangi isolates have been reported in the enterobase database (as of 27 march 2025), with most of the cases (203/409; 49.6%) reported from south africa. among all the serovars or subspecies in south africa, salmonella isangi showed the highest prevalence of amr (table 2). most salmonella isangi (159/167; 95.2%) showed amr determinants associated with ≥ 7 classes of antimicrobials, including esbl genes (blaoxa-1, blaoxa-10, blactx-m-15, blatem-63, bladha). globally (including south africa), salmonella isangi are typically multidrug-resistant, and are often associated with hospital outbreaks.34,35,36,37 there is a pressing need for studies to identify the reservoir and transmission pathway for salmonella isangi, as this serotype is very capable of acquiring and retaining extensive drug resistance, and once introduced into the hospital environment, it appears to happily thrive and cause lengthy hospital outbreaks.35 salmonella dublin (114/8006; 1.4%) was our sixth most prevalent salmonella serovar or subspecies. globally, salmonella dublin is a relatively uncommon cause of human infections. salmonella dublin is host-adapted to cattle, so is most prevalent in cattle and cow’s raw milk cheese. countries that produce large volumes of cheese (such as france) often show an increased prevalence of salmonella dublin.38,39 salmonella muenchen (108/8006; 1.3%) was our seventh most prevalent salmonella serovar or subspecies. this prevalence aligns with a global reported prevalence of 1.2%, where globally, salmonella muenchen is listed as the 13th most prevalent salmonella serovar.27 salmonella muenchen is a relatively uncommon cause of human infections globally and there are also very few documented reports of outbreaks associated with salmonella muenchen. salmonella infantis (98/8006; 1.2%) was our eighth most prevalent salmonella serovar or subspecies. salmonella infantis is currently perhaps the biggest mover and shaker among the global salmonella population, gaining increased global prevalence over recent years.40 salmonella infantis has become the fourth most prevalent salmonella serovar causing human infections among european union member countries.41 salmonella infantis is among the most frequently isolated salmonella serovar in poultry in europe and the united states.41 globally, salmonella infantis is currently listed as the third most prevalent salmonella serovar, with a global reported salmonella prevalence of 6.6%.27 interestingly, the population structure of south african salmonella infantis has been shown to differ substantially from salmonella infantis isolated elsewhere globally.42 conclusion the implementation of wgs for routine surveillance of clinical isolates of salmonella in south africa has seen a significant increase in the critical mass of salmonella genomic data now available from the african continent. south africa is currently ranked country number one with respect to the number of salmonella genome submissions from africa, and seventh with respect to global country submissions. large wgs data sets, methodically generated over long time periods, provide essential information for: detailed and enhanced characterisation of bacterial strains (pathogens), molecular epidemiological investigations, early detection of clusters of disease, outbreak investigations, investigating for new and emerging strains, investigating for new or unusual amr profiles, tracking the spread of strains, data for development of treatment strategies and vaccine development, and data for monitoring the effect of treatment interventions and vaccine rollout. whole-genome sequencing data not only provide value ‘in the now’ but are ‘the gift that keeps on giving’, as data can be further and repeatedly investigated by multiple parties, be that for research purposes or public health activities, all to assist with investigation and containment of future public health threats. acknowledgements the authors would like to thank all participants of the nicd germs-sa laboratory surveillance network for submission of clinical isolates of salmonella species to the nicd. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions a.m.s. contributed towards project conceptualisation, funding acquisition, project administration, project supervision, data analysis, data curation, writing the original draft of the article, and is the corresponding author for the project. p.s., l.k.e., and j.t. contributed towards data analysis, review, and editing of the article. h.m.n., b.d., d.b., s.l.s., and n.p.t. contributed towards laboratory analysis, data analysis, data curation, review, and editing of the article. funding information this work was supported by the seqafrica project which is funded by the department of health and social care’s fleming fund using united kingdom aid. the views expressed in this publication are those of the authors and not necessarily those of the united kingdom department of health and social care or its management agent, mott macdonald. data availability the data that support the findings of this study are uploaded to the public enterobase platform (http://enterobase.warwick.ac.uk/species/index/senterica) and are freely available at this platform. in addition, sequencing data are deposited in the european nucleotide archive under the project accession numbers prjeb39002, prjeb39546 and prjeb39988. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. the article does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the authors are responsible for this article’s results, findings, and content. references smith si, seriki a, ajayi a. typhoidal and non-typhoidal salmonella infections in africa. eur j clin microbiol infect dis. 2016;35(12):1913–1922. https://doi.org/10.1007/s10096-016-2760-3 martin lb, tack b, marchello cs, et al. vaccine value profile for invasive non-typhoidal salmonella disease. vaccine. 2024;42(19 suppl 1):s101–s124. https://doi.org/10.1016/j.vaccine.2024.04.045 crump ja, nyirenda ts, kalonji lm, et al. nontyphoidal salmonella invasive disease: challenges and solutions. open forum infect dis. 2023;10(suppl 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https://doi.org/10.2807/1560-7917.es.2017.22.23.30544 european centre for disease prevention and control. thirteenth external quality assessment for salmonella typing [homepage on the internet]. stockholm: ecdc; 2024 [cited 2024 oct 31]. available from: https://www.ecdc.europa.eu/en/publications-data/thirteenth-external-quality-assessment-salmonella-typing dtu genomic. genomic proficiency test 2023 [homepage on the internet]. 2023 [cited 2024 oct 31]. available from: https://www.globalsurveillance.eu/projects/genomic-proficiency-test-2023 alikhan nf, zhou z, sergeant mj, achtman m. a genomic overview of the population structure of salmonella. plos genet. 2018;14(4):e1007261. https://doi.org/10.1371/journal.pgen.1007261 deng x, li s, xu t, et al. salmonella serotypes in the genomic era: simplified salmonella serotype interpretation from dna sequence data. appl environ microbiol. 2025;91(3):e0260024. van puyvelde s, de block t, sridhar s, et al. a genomic appraisal of invasive salmonella typhimurium and associated antibiotic resistance in sub-saharan africa. nat commun. 2023;14(1): 6392. https://doi.org/10.1038/s41467-023-41152-6 sun h, wan y, du p, bai l. the epidemiology of monophasic salmonella typhimurium. foodborne pathog dis. 2020;17(2):87–97. https://doi.org/10.1089/fpd.2019.2676 larkin l, pardos de la gandara m, hoban a, et al. investigation of an international outbreak of multidrug-resistant monophasic salmonella typhimurium associated with chocolate products, eu/eea and united kingdom, february to april 2022. euro surveill. 2022;27(15):2200314. https://doi.org/10.2807/1560-7917.es.2022.27.15.2200314 carey me, thi nguyen tn, tran dhn, et al. the origins of haplotype 58 (h58) salmonella enterica serovar typhi. commun biol. 2024;7(1):775. https://doi.org/10.1038/s42003-024-06451-8 sekwadi p, smith am, maruma w, et al. a prolonged outbreak of enteric fever associated with illegal miners in the city of matlosana, south africa, november 2020-september 2022. open forum infect dis. 2024;11(3):ofae118. https://doi.org/10.1093/ofid/ofae118 lamas a, miranda jm, regal p, vázquez b, franco cm, cepeda a. a comprehensive review of non-enterica subspecies of salmonella enterica. microbiol res. 2018;206:60–73. https://doi.org/10.1016/j.micres.2017.09.010 dos santos amp, panzenhagen p, et al. genomic characterization of salmonella isangi: a global perspective of a rare serovar. antibiotics. 2023;12(8): 1309. https://doi.org/10.3390/antibiotics12081309 national institute for communicable diseases. communicable diseases communique, august 2022 [homepage on the internet]. 2022 [cited 2025 feb 14]. available from: https://www.nicd.ac.za/wp-content/uploads/2022/08/310822-nicd-monthly-communique-aug-nw5.pdf suleyman g, tibbetts r, perri mb, et al. nosocomial outbreak of a novel extended-spectrum β-lactamase salmonella enterica serotype isangi among surgical patients. infect control hosp epidemiol. 2016;37(8):954–961. https://doi.org/10.1017/ice.2016.85 wadula j, von ga, kilner d, et al. nosocomial outbreak of extended-spectrum beta-lactamase-producing salmonella isangi in pediatric wards. pediatr infect dis j. 2006;25(9):843–844. https://doi.org/10.1097/01.inf.0000233543.78070.a2 de sousa violante m, podeur g, michel v, et al. a retrospective and regional approach assessing the genomic diversity of salmonella dublin. nar genom bioinform. 2022;4(3):lqac047. https://doi.org/10.1093/nargab/lqac047 velasquez-munoz a, castro-vargas r, cullens-nobis fm, mani r, abuelo a. review: salmonella dublin in dairy cattle. front vet sci. 2023;10:1331767. https://doi.org/10.3389/fvets.2023.1331767 mattock j, chattaway ma, hartman h, et al. a one health perspective on salmonella enterica serovar infantis, an emerging human multidrug-resistant pathogen. emerg infect dis. 2024;30(4):701–710. https://doi.org/10.3201/eid3004.231031 alba p, leekitcharoenphon p, carfora v, et al. molecular epidemiology of salmonella infantis in europe: insights into the success of the bacterial host and its parasitic pesi-like megaplasmid. microb genom. 2020;6(5):e000365. https://doi.org/10.1099/mgen.0.000365 mattock j, smith am, keddy kh, et al. genetic characterization of salmonella infantis from south africa, 2004–2016. access microbiol. 2022;4(7):acmi000371. https://doi.org/10.1099/acmi.0.000371 brümmer b, smith am, modise m, et al. whole genome sequencing assisted outbreak investigation of salmonella enteritidis, at a hospital in south africa, september 2022. access microbiol. 2024;6(11):000835.v000833. https://doi.org/10.1099/acmi.0.000835.v3 abstract introduction methods results discussion acknowledgements references about the author(s) reola haripersadh department of haematology, national health laboratory services, inkosi albert luthuli central hospital, durban, south africa department of haematology, school of laboratory medicine, university of kwazulu-natal, durban, south africa dashini pillay department of haematology, national health laboratory services, inkosi albert luthuli central hospital, durban, south africa department of haematology, school of laboratory medicine, university of kwazulu-natal, durban, south africa nadine rapiti department of haematology, national health laboratory services, inkosi albert luthuli central hospital, durban, south africa department of haematology, school of laboratory medicine, university of kwazulu-natal, durban, south africa citation haripersadh r, pillay d, rapiti n. impact of rapid centrifugation on routine coagulation assays in south africa. afr j lab med. 2022;11(1), a1901. https://doi.org/10.4102/ajlm.v11i1.1901 original research impact of rapid centrifugation on routine coagulation assays in south africa reola haripersadh, dashini pillay, nadine rapiti received: 24 mar. 2022; accepted: 10 aug. 2022; published: 28 nov. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the recommendation for coagulation blood samples is to centrifuge at 4000 revolutions per minute (rpm) for 15 min to produce platelet-poor plasma before analysis. rapid centrifugation, defined as centrifuging samples at higher speeds for shorter durations, could potentially reduce turn-around times (tat), provided sample integrity is maintained. objective: this study assessed the impact of rapid centrifugation on routine coagulation assay results. methods: blood samples were collected from volunteers at inkosi albert luthuli central hospital and king edward viii hospital, durban, kwazulu-natal, south africa, from september to november 2021. samples were centrifuged using method a, the current standard (4000 rpm/15 min), method b (4000 rpm/10 min), method c (5000 rpm/10 min) and method d (5000 rpm/5 min). platelet count, prothrombin time, activated partial thromboplastin time, thrombin time (tt), fibrinogen and d-dimer levels were analysed and results from methods b, c and d compared to reference method a. results: platelet-poor plasma was obtained from all samples (n = 60) using methods a and b, and from 33/60 (55%) samples using methods c and d. differences between method a and methods c and d for normal prothrombin time, normal d-dimer and abnormal tt results were statistically significant. prothrombin time results correlated strongly across all methods, while tt and d-dimer results correlated poorly. activated partial thromboplastin time and fibrinogen results showed no significant differences across all methods. conclusion: rapid centrifugation at 4000 rpm/10 min (method b) showed results consistent with the reference method. this method could potentially reduce the overall tat for routine coagulation assays. keywords: rapid centrifugation; coagulation assays; platelet-poor plasma; pre-analytic; clinical and laboratory standards institute guidelines; haemostasis. introduction laboratories are expected to provide accurate and reliable results within defined turn-around times (tats) to facilitate the diagnosis, management and prognostication of patients.1,2 there are ongoing attempts to improve tats as this will directly impact patient management.3,4 coagulation assays are some of the most commonly ordered urgent and routine investigations.1,2 the recommended sample preparation method for coagulation testing is the centrifugation of whole blood to obtain platelet-poor plasma (ppp), defined as plasma with a platelet count of < 10 × 109/l, which minimises interference by the platelet phospholipid surface, thereby preventing the activation of clotting factors.5 obtaining ppp from whole blood requires the application of specific centrifugal forces over a given period.6,7 minimal centrifugation times for routine coagulation assays vary across laboratories, often based on local observations.8 the clinical and laboratory standards institute guidelines recommend that whole blood collected in tri-sodium citrate tubes be centrifuged at 4000 revolutions per minute (rpm) for 15 min at room temperature to produce ppp.9 strategies such as rapid centrifugation that are designed to reduce specimen processing times are being pursued as they could potentially reduce tat in laboratories, eliminate the need for batch processing of samples and free up resources.10 as centrifugation time can be a bottleneck in coagulation testing, there is a need to determine the impact of rapid centrifugation on the laboratory workflow and tat.1,7,9 several studies have shown that centrifuging samples at higher speeds for shorter durations to obtain ppp has no significant effect on sample integrity and the accuracy of coagulation assay results.11,12,13,14 the primary goal of this study was to assess the impact of rapid centrifugation on the accuracy of routine coagulation test results. methods ethical considerations the study was approved by the biomedical research ethics committee of the university of kwazulu-natal, south africa (brec/00002366/2021). informed consent was obtained from each study participant and a unique study number was allocated to samples to ensure anonymity. the research data was stored electronically on password-protected devices and was only accessible by the researchers. study design and setting this study was conducted at the inkosi albert luthuli central hospital and king edward viii hospital, durban, kwazulu-natal, south africa, from september 2021 to november 2021. patients older than 18 years were included in the study. samples were excluded if there were insufficient blood volumes, or if clots, fibrin strands, or haemolysis were observed. sixty-two participants were included in the study (table 1). two samples were excluded from the data analysis due to tube breakage and one sample was treated as an outlier due to an abnormally high d-dimer result (35.2 mg/l) for method a.15 from each participant, venous blood samples were collected into four 3.2% sodium citrate tubes (bd vacutainer 0.109 m/3.2 citrate, becton dickinson, new jersey, united states) labelled a, b, c and d, each representing one of the four centrifugation methods. blood samples were collected and transported to the laboratory at room temperature (20 °c – 25 °c). table 1: demographics and clinical diagnoses of study participants, inkosi albert luthuli central hospital and king edward viii hospital, durban, kwazulu-natal, south africa, september 2021 – november 2021. centrifugation samples were centrifuged using two table-top consul 22r (ortoaltresa®, madrid, spain) instruments. the first instrument (instrument 1) had a maximum rotor speed of 4000 rpm and required a 5 ml tube, while the second instrument (instrument 2) had a maximum rotor speed of 9000 rpm and required a 50 ml tube (table 2). a pilot experiment revealed that decanting the sample from the 5 ml citrate tube into the larger 50 ml tube before centrifugation resulted in a very thin supernatant plasma layer that was difficult to remove with a pipette. placing the 5 ml citrate tube directly into the 50 ml tube resulted in the risk of tube breakage, therefore, cotton wool was used as a buffer between the two tubes. samples were centrifuged within 4 h of phlebotomy. table 2: speed and time specifications for centrifugation methods, inkosi albert luthuli central hospital and king edward viii hospital, durban, kwazulu-natal, south africa, september 2021 – november 2021. method a was the clinical and laboratory standards institute-recommended reference method for centrifugation in the laboratory (4000 rpm for 15 min). methods b (4000 rpm/10 min), c (5000 rpm/10 min) and d (5000 rpm/5 min) were compared to method a.9,16,17,18 a literature search confirmed that centrifuging samples at 4000 rpm for 5 min did not produce ppp and therefore this method was excluded from the study.19,20 samples for methods a and b were centrifuged on instrument 1, while samples for methods c and d were centrifuged on instrument 2. the supernatant plasma was transferred to an empty tube using a plastic pipette.16,21 all samples were tested on the sysmex® cs5100 (siemens healthcare gmbh, marburg, germany) automated coagulation analyser with reagents from siemens healthcare products gmbh (marburg, germany). the reagents included innovin® (prothrombin time [pt] – clotting assay); actin® fsl (activated partial thromboplastin time [aptt] – clotting assay); test thrombin® (thrombin time [tt] – clotting assay); dade® thrombin reagent (fibrinogen – clotting assay) and innovance® (d-dimer – immunoassay). the study samples were only analysed when the commercial controls were within the predetermined limits.16,22 an automated platelet count was performed on every sample using the sysmex® xn 3000 automated haematology analyser (siemens healthcare gmbh, marburg, germany) by flow cytometry based on principles of light scattering. this was done to assess the percentage of samples that produced a platelet count of < 10 × 109/l in each of the four methods. data analysis capturing of results, statistical tests and construction of bland altman plots were done using microsoft® excel 2016 (microsoft®, redmond, washington, united states). the ep evaluator software version 8 (informer technologies inc, los angeles, california, united states) and stata version 17 (statacorp®, college station, texas, united states) were used for statistical analysis. for descriptive statistics, numerical data were summarised as means, medians, standard deviations or percentages.23,24 the quality of the data was assessed using an acceptable correlation coefficient (r) value of > 0.975. the correlation coefficient (r) was used to assess the linear relationship between the reference method and methods b, c and d.25,26 the paired student t-test was used to assess the statistical significance of differences between samples. the level of statistical significance was set at a p-value < 0.05. percentage variations were compared with the current desirable quality specifications for bias and were derived from westgard biological variation.20,26 results platelet-poor plasma was produced in all samples (n = 60) centrifuged at 4000 rpm for 15 and 10 min (methods a and b) (table 3). for methods c and d, ppp was produced in 55% (33/60) of the samples, and 45% (27/60) of the samples had a platelet count between 11 and 90 × 109/l. table 3: platelet counts, prothrombin time, activated partial thromboplastin time, thrombin time, fibrinogen and d-dimer levels using four centrifugation methods, inkosi albert luthuli central hospital and king edward viii hospital, durban, kwazulu-natal, south africa, september 2021 – november 2021. forty-nine (49/60; 82%) samples had a normal pt level (method a: mean [seconds] = 10.71 s, median [seconds] = 10.6 s; method b: mean = 10.69 s, median = 10.5 s; method c: mean = 10.62 s, median = 10.5 s; method d: mean = 10.62 s, median = 10.5 s). eleven (11/60; 18%) samples had an abnormal prothrombin result (method a: mean = 19.26 s, median = 15.6 s; method b: mean = 19.22 s, median = 15.1 s; method c: mean = 19.25 s, median = 15.5 s; method d: mean = 19.30 s, median = 15.4 s). there were statistically significant differences for method c (p = 0.002) and method d (p = 0.005) in the normal pt group, with results being lower than the pt results for methods a and b. despite there being statistically significant differences in the normal pt results for methods c and d, the pt results obtained using all three test methods correlated strongly with results obtained using the reference method (r ≥ 0.99) (figure 1). figure 1: comparison of prothrombin time results between the reference centrifugation method (method a) and methods b, c and d, inkosi albert luthuli central hospital and king edward viii hospital, durban, kwazulu-natal, south africa, september 2021 – november 2021. linear regressions for: (a) method a vs method b, (b) method a vs method c, (c) method a vs method d. thirty-eight (38/60; 63%) of the samples had normal aptt values (method a: mean [seconds] = 28.26 s, median [seconds] = 27.9 s; method b: mean = 27.97 s, median = 27.6 s; method c: mean = 28.02 s, median = 27.7 s; method d: mean = 28.16 s, median = 27.7 s). twenty-two (22/60; 37%) samples had abnormal aptt results (method a: mean = 37.48 s, median = 24.1 s; method b: mean = 37.27 s, median = 24.4 s; method c: mean 37.44 s, median = 24.3 s; method d: mean = 37.19 s, median = 24.2 s). there were no statistically significant differences in the aptt results obtained using method a versus methods b, c and d; aptt results from the three processing methods strongly correlated with the results of the reference method (r > 0.975). forty-six (46/60; 77%) samples had normal tt levels (method a: mean [seconds] = 17.47 s, median [seconds] = 17.5 s; method b: mean = 16.69 s, median = 17.3 s; method c: mean = 17.37 s, median = 17.3 s; method d: mean = 17.34 s, median = 17.5 s). fourteen (14/60; 23%) had abnormal tt levels (method a: mean = 16.72 s, median = 15.6 s; method b: mean = 16.69 s, median = 15.6 s; method c: mean = 16.57 s, median = 15.5 s; method d: mean = 16.46 s, median = 15.6 s). abnormal tt levels were significantly lower among samples analysed using method d compared to methods a, b and c. all three methods correlated poorly (r = 0.92–0.93) with the reference method (figure 2). figure 2: comparison of thrombin time results between the reference centrifugation method (method a) and methods b, c and d, inkosi albert luthuli central hospital and king edward viii hospital, durban, kwazulu-natal, south africa, september 2021 – november 2021. linear regressions for: (a) method a vs method b, (b) method a vs method c, (c) method a vs method d. forty-seven (47/60; 78%) samples had normal fibrinogen levels (method a: mean [g/l] = 3.41, median [g/l] = 3.63; method b: mean = 3.46, median = 3.61; method c: mean = 3.52, median = 3.64; method d: mean = 3.45, median = 3.57). thirteen (13/60; 22%) samples had abnormal fibrinogen levels (method a: mean = 5.28, median = 5.31; method b: mean = 5.18, median = 5.43; method c: mean = 5.44, median = 5.60; method d: mean = 5.37, median = 5.40). the results of all three test methods correlated strongly with the reference method. nineteen (19/59; 32%) samples had normal d-dimer levels (method a: mean [mg/l] = 0.20, median [mg/l] = 0.19; method b: mean = 0.27, median = 0.19; method c: mean = 0.21, median = 0.19; method d: mean = 0.21, median = 0.20). forty (40/59; 62%) samples had abnormal d-dimer levels (method a: mean = 1.5, median = 0.58; method b: mean = 1.49, median = 0.57; method c: mean = 1.44, median = 0.56; method d: mean = 1.47, median = 0.60). d-dimer levels were significantly lower for methods c (p = 0.04) and d (p = 0.02) when compared to method a. method d correlated strongly with the reference method, while methods b and c correlated poorly with the reference method (r < 0.975) (figure 3). a single sample result for method a (35.2 mg/l) had an extreme outlying value when compared with the results from methods b, c and d (< 0.36 mg/l). figure 3: comparisons of d-dimer results between the reference centrifugation (method a) and methods b, c and d, inkosi albert luthuli central hospital and king edward viii hospital, durban, kwazulu-natal, south africa, september 2021 – november 2021. linear regressions for: (a) method a vs method b, (b) method a vs method c, (c) method a vs method d. discussion our study found that centrifuging samples at 4000 rpm for 10 min yielded ppp in 100% of samples compared to centrifugation at 5000 rpm for 10 min and 5 min, which only yielded ppp in 55% of samples. the higher centrifugation speed caused an increase in platelet count in 45% of cases. this study has shown that method b (4000 rpm/10 min) is superior to methods c and d as it did not have a significant impact on the coagulation assay results. hence, method b could be an alternate method of processing samples for coagulation tests. a study by barnes et al showed that 10 min was the minimum centrifugation time required to consistently meet the recommendations for ppp.27 this differed from a previous study by sultan et al., which reported that the majority of the 46 samples tested produced ppp when centrifuged at 5000 rpm for 5 min.12 although ppp is recommended for coagulation testing, studies suggest that coagulation test results are not affected by platelet counts of > 10 × 109/l and that samples with platelet counts of up to 199 × 109/l produce similar results to samples with ppp.28 in our study, platelet counts differed significantly between the samples centrifuged at 4000 rpm (method a and b) and those processed at 5000 rpm (method c and d); however, there were no statistically significant differences between method a and methods b, c and d in results for the aptt and fibrinogen assays, as well as for the abnormal pt, normal tt and abnormal d-dimer assays. this is similar to the findings of previous studies conducted in 2002 and 2011.19,27 statistically significant differences were observed in the normal pt and normal d-dimer assay results when centrifuged at 5000 rpm compared to the reference method. abnormal tt levels were significantly lower when measured with method d compared to method a; however, this was not the case for method c which was also a 5000-rpm method. although the results for normal pt were found to be statistically significantly different between the reference method and methods c and d, the differences in the actual mean, median and standard deviation values between the groups were minimal. when combined, the normal and abnormal pt results showed a good correlation between the different methods (r = 0.99) and these results are similar to those discovered in a previous study by azlin et al.19 there were statistically significant differences in the abnormal tt (method d) and normal d-dimer values (methods c and d) when compared to the reference method; however, there was minimal variation in the mean, standard deviation and median values. furthermore, the tt results showed poor correlation (r = 0.92–0.93) for all three methods when compared to the reference method. these findings suggest that the variation of the raw data may not be clinically significant as the clinical management of patients in our setting would not be altered. the aptt assay is usually more sensitive to platelet contamination than the pt assay.19 this was not observed in our study as the results of the aptt and fibrinogen assays showed no significant differences and correlated strongly between the test methods and the reference method. a single d-dimer result from method a had an outlying value when compared with the results from methods b, c and d. it was confirmed that the sample was collected following standard practice guidelines, labelled correctly and processed using the standard operating procedure of the laboratory.29 processing errors can occur during the pre-analytical phase8,30; however, a transcription error was excluded in this case. owing to limited sample availability and repeat sampling not being possible, method a could not be rerun.30,31 our study results encourage further research on rapid centrifugation of coagulation samples to verify the reliability of the results and explore the potential benefits it could have in a clinical laboratory setting. these findings could have practical applications and serve as a basis for additional research to establish local centrifugation protocols in laboratories.19 limitations the results of this study (sample size = 60) need to be validated with a larger case-control study. a larger number of healthy individuals (controls) should be included. the pt, tt and fibrinogen assays had low abnormal sample numbers ranging from 20% to 30% and the tt results did not reflect extreme abnormal ranges. participants were recruited on a voluntary basis; therefore, some assays showed a bias in the normal-to-abnormal ratios. conclusion this study demonstrates that method b is superior to methods c and d as it produced results that were most consistent with those obtained using the reference method. methods c and d produced statistically significant differences in results for the pt, tt and d-dimer assays. we show that the centrifugation of whole blood samples in 5 ml citrate tubes at 4000 rpm for 10 min is suitable for routine coagulation testing. this rapid centrifugation method provides consistent and reliable results and could potentially reduce the overall tat. these findings may assist experts in revising the current recommendations for the centrifugation of coagulation samples. acknowledgements the authors wish to thank: rookaya mahomed – senior technologist, national health laboratory services inkosi albert luthuli central hospital, for the processing of the study samples; catherine connolly – statistician university of kwazulu-natal, for assisting with the statistical analysis of the results; national health laboratory services haematology laboratory, inkosi albert luthuli central hospital, staff for the technical assistance; inkosi albert luthuli central hospital and king edward viii hospital: department of clinical haematology staff for their assistance during the sample collection process. competing interests the authors wish to declare that they have no personal or financial interests that may have had an influence on the writing of this article. authors’ contributions r.h. conceived and presented the idea of the study to d.p. r.h. and d.p. developed the theory, methodology and protocol for this study. n.r. reviewed the work and advised on the study design and implementation of the research. d.p. supervised and n.r. co-supervised the project. all authors contributed to the analysis of the results and to the writing of the final manuscript. sources of support the authors acknowledge the receipt of financial support for the research from the national health laboratory services department of haematology, inkosi albert luthuli central hospital. data availability the data generated during the study is available on request from the corresponding author, r.h., due to privacy/ethical restrictions. disclaimer the views expressed in this article are those of the authors and not of the institution (university of kwazulu-natal) or the funder (national health laboratory services). references rajashekar r, krishnamurthy v, murthy doreswamy s. does platelet contamination really affect the coagulation tests? a prospective study. int j hematol res. 2016;2(3):155–159. https://doi.org/10.17554/j.issn.2409-3548.2016.02.38 gürsoy o, tür fç, aksay e, et al. the impact of coagulation testing on patient management in the emergency department. acta medica mediterr. 2015;31(3):597–602. khan ih, savarimuthu s, leung mst, harky a. the need to 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[cited 2021 jun 19]. available from: http://www.bd.com/vacutainer/faqs bt lab systems. convert g-force (rcf) to rpm for lab centrifuges [homepage on the internet]. geno technology inc; 2018 [cited 2020 jul 20]. available from: https://blog.btlabsystems.com/blog/centrifuge-g-force azlin i, hafiza a, rz a, rk a, nh h. effect of higher centrifugation speed and shortened centrifugation time on prothrombin and activated thromboplastin time. med health. 2011;6(1):68–72. lippi g, salvagno gl, montagnana m, manzato f, guidi gc. influence of the centrifuge time of primary plasma tubes on routine coagulation testing. blood coagul fibrinol. 2007;18(5):525–528. https://doi.org/10.1097/mbc.0b013e3281eec945 hinz ar, boehm m. technical guide on efficient sample preparation for clinical diagnosis [homepage on the internet]. vol. 1, no. 1. waltham, ma: thermo fisher scientific inc., 2016; p. 1–8 [cited 2020 jul 20]. available from: https://assets.thermofisher.com/tfs-assets/led/application-notes/clinical-centrifuge-sample-prep-application-note-ancfgclinicalbench.pdf suchsland j, friedrich n, grotevendt a, et al. optimizing centrifugation of coagulation samples in laboratory automation. clin chem lab med. 2014;52(8):1187–1191. https://doi.org/10.1515/cclm-2014-0038 morgan cj, aban i. methods for evaluating the agreement between diagnostic tests. j nucl cardiol. 2016;23(3):511–513. https://doi.org/10.1007/s12350-015-0175-7 briggs c, culp n, davis b, d’onofrio g, zini g, machin sj. icsh guidelines for the evaluation of blood cell analysers including those used for differential leucocyte and reticulocyte counting. int j lab hematol. 2014;36(6):613–627. https://doi.org/10.1111/ijlh.12201 swartz m, hanes h, shim j, stevens p, sholander a. hematology validation plan. equ52-01. baltimore, md: smile, 2020; p. 1–6. westgard j. desirable biological variation database specifications [homepage on the internet]. 2019 [cited 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acknowledgements references appendix 1 appendix 2 about the author(s) anne n. mbuthia kenya methodist university, nairobi, kenya eunice m. mwangi kenya methodist university, nairobi, kenya musa o. ong’ombe kenya methodist university, nairobi, kenya citation mbuthia an, mwangi em, ong’ombe mo. organisational management of hospital blood transfusion services in nairobi county, kenya: evidence of implementation. afr j lab med. 2019;8(1), a676. https://doi.org/10.4102/ajlm.v8i1.676 original research organisational management of hospital blood transfusion services in nairobi county, kenya: evidence of implementation anne n. mbuthia, eunice m. mwangi, musa o. ong’ombe received: 11 sept. 2017; accepted: 19 dec. 2018; published: 26 aug. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the world health organization in 2002 recommended implementation of a quality system for national blood programmes to ensure adequate and safe blood products to patients. key elements of the quality system include organisational management, standards, documentation, training and assessment. objectives: the aim of this study was to describe the extent to which organisational management, which is the first element of a quality system, has been implemented in hospitals in nairobi county, kenya. methods: a descriptive, cross-sectional study design was used. sixty health workers were selected as respondents from 15 hospitals that provide blood transfusion services in nairobi county. the data collection period was from june to august 2015 and the data were analysed in 2016. results: faith-based hospitals had the lowest level of organisational management implementation (33.3%), private hospitals had 42.5%, whereas government hospitals had the highest implementation (60%). the extent of implementation was based on performance of the senior management team, overall rated by the respondents at 40.1%, establishment of hospital transfusion committees in nine (60%) of the hospitals and appointment of key staff – quality officers in three (20%) hospitals and blood transfusion specialists in six (40%) hospitals. these key staff were instrumental in steering the quality system and ensuring sound blood transfusion practices. conclusion: the implementation of quality management systems in hospital blood transfusion services can be improved through commitment from senior management teams, who should provide the necessary resources for employment of key staff and establish and empower hospital transfusion committees to guide the blood transfusion services. keywords: blood transfusion; quality system; organisational management; quality management system. introduction blood transfusion services are a key life-saving function in every health system. scarcity of blood poses serious challenges, which sometimes hinder the ability to improve health outcomes.1,2 this is further compounded by wastage, inappropriate utilisation or storage and failure to maintain the cold chain. availability of safe blood is a recurrent challenge in hospital blood transfusion services in kenya. this is because of high demand for blood, especially for emergency obstetric care.3 the issue of scarcity is further compounded by challenges such as poor documentation, inadequate knowledge regarding the use of blood components and lack of haemovigilance tools.4 these shortcomings negatively affect the overall quality of service and contribute to wastage and inappropriate usage. comprehensive quality management systems serve two main purposes within blood transfusion services. firstly, they ensure the services and blood components are safe, adequate and effective.2 secondly, they provide data on adverse events for the national haemovigilance system.1 to achieve this, a robust quality management system is necessary at individual hospital blood transfusion services. to improve blood transfusion services, the world health organization (who) recommends adoption of a quality system that has five key elements: organisational management, implementation of quality standards, documentation, training of staff and regular assessment.5 there are three key areas of organisational management in blood transfusion services: a well-defined organisational structure that describes obligation, authority and responsibility, good planning for quality and consistency in service delivery, and leaders perfecting a culture that integrates quality into all activities.6 the senior management team ensures quality and consistency in service delivery by establishing a multidisciplinary hospital blood transfusion committee, appointing quality officers and blood transfusion specialists who are key members of the transfusion committee and providing resources to run the service. in kenya, the responsibilities of a quality officer include haemovigilance to monitor trends of adverse reactions and hospital response, track blood utilisation, evaluate hospital transfusion practice, and prepare monthly reports for the transfusion committee and regional blood transfusion centre (rbtc).7 blood transfusion specialists support blood management best practices, which eliminate, reduce or optimise blood transfusions to improve health outcomes.8 other key personnel include the blood bank manager who directs and controls operations, and the operational staff (laboratory technologists, nurses and clinicians) who offer direct services to patients. the primary aim of this study was to assess the implementation of organisational management in the hospital blood transfusion services within nairobi county, following who recommendations. the study further explored the level of implementation by private, faith-based and government hospitals and the role played by clinicians, nurses, laboratory technologists and managers (figure 1). figure 1: blood transfusion chain. methods ethical considerations ethical clearance to carry out the study was sought from the kenya methodist university – scientific and ethics review committee (ethical approval number, hsm-3791-27115). permission for data collection was also sought from individual hospital authorities. the hospital authorities gave verbal permission for their staff to participate. no patients’ data were used in the study. the individual respondents or hospital staff were required to complete an informed consent form prior to participation in the study. study design a cross-sectional descriptive study was conducted. data were collected over 3 months, from june to august 2015. questionnaires (appendix 1) and first-hand observation guided by a checklist (appendix 2) were used to collect the study data. the tools were developed and administered by the researcher. the hospitals selected were registered under the kenya medical practitioners and dentists board or the ministry of health as the regulatory bodies and recognised by the kenya national blood transfusion service and regional society for blood transfusion kenya. the selected facilities had a bed capacity of more than 20, so that they experienced sufficient demand for blood components to make installing a fully fledged quality management system in the hospital’s blood transfusion service cost-effective. out of 25 hospitals in nairobi county, 18 met the inclusion criteria. the researcher visited each of the 18 health facilities selected and requested the participation of the institution. permission was sought from the hospital authorities. an introduction letter was used when the administrator could not be reached. once authorised, the researcher was introduced to one key staff member (manager) involved in blood transfusion services, who served as the contact person. a tour of the facility was provided, during which the self-administered questionnaires were issued to the respondents. a written informed consent form explaining the need, purpose, benefit and process of participation was filled out by the respondents before completing the questionnaires. the observation checklist was filled out by the researcher during the hospital visits. healthcare workers were the primary respondents. the respondents were selected through stratified random sampling and grouped into four categories: clinicians, nurses, medical laboratory technologists and managers involved in blood transfusion service. the respondents had at least 2 years of work experience in the blood transfusion service of the hospital. the inclusion of different cadres of staff captured the organisational management of the entire transfusion chain. the first respondent was the manager in charge of the blood transfusion service. the remaining respondents were selected randomly. overall organisational management was assessed by questionnaire items in four main areas: senior management team, hospital transfusion committee (htc), quality officers and blood bank specialists. senior management team (smt) involvement was assessed via five specific yes-or-no questions. the respondents were asked if the smt reviewed blood transfusion policies, ensured that staff held regular meetings, had appointed a quality officer, had appointed a blood transfusion consultant and reviewed audit reports. confirmation of the presence of a multidisciplinary htc in each health facility was sought from the respective blood transfusion manager. the presence of a file for the htc with minutes of meetings held was considered sufficient proof. respondents were asked about the functionality and visibility of the committee within the hospital, and they indicated the performance of the committee for monitoring blood utilisation, development and promotion of the use of guidelines and carrying out audits of the processes within the service. the quality officer and the blood transfusion specialists are key staff in blood transfusion service responsible for implementing the quality system and promoting best practices within the transfusion chain.7,8 respondents were asked if these two designated staff were employed within their institutions. statistical analyses data were coded and entered into a microsoft 2013 excel spreadsheet (microsoft corp., redmond, washington, united states). data analysis was carried out using spss version 23 (ibm corp., armonk, new york, united states). qualitative data were analysed using content and thematic analysis. results nairobi county hospital blood transfusion service of the 18 hospitals selected, 15 (83%) hospitals agreed to participate in the study. these hospitals were government, private and faith-based organisations, with five hospitals from each category. in total, 60 randomly selected health professionals from the 15 hospitals participated as respondents (100% response rate). a total of 33 (55%) respondents had worked in the blood transfusion services (bts) for 4 years or fewer, and only two clinicians had served more than 5 years in their current station (table 1). table 1: profiles of the respondents, nairobi county, kenya, june–august 2015. senior management team involvement in blood transfusion service the overall performance of the senior management team was below average. a total of 27 respondents (45%) indicated that the senior management team met annually to review blood bank policies (table 2). having a protocol for staff meetings received the highest score (31 respondents, 52%), and review of audit reports received the lowest (14 respondents, 23.3%). table 2: participants’ responses on extent of implementation of organisation management, in hospitals of nairobi county, kenya. hospital transfusion committee hospital transfusion committees were present in nine participating hospitals (60%) (table 2). all five government hospitals had established an htc as opposed to only two private hospitals and two faith-based hospitals. the most commonly implemented roles of the htcs were monitoring use of blood (22 respondents, 37%) and establishing guidelines (22 respondents, 37%). the least commonly implemented function of an htc was audit of the service (15 respondents, 25%). this was further corroborated by the observation that only four (26.7%) of the hospitals had records that showed that audits were done. quality officers twelve respondents (20%) reported that their hospital had a quality officer within the hospital blood transfusion service in nairobi county (table 2). records of audit activities by the quality officer in the laboratory and the clinical area provided evidence that three (20%) of the hospitals had a designated quality officer. blood transfusion specialists twenty-four health professionals (40%) indicated that their transfusion service had a clinician employed in the capacity of a blood transfusion specialist, who was responsible for providing expertise in therapeutic modalities and promoting best practice in transfusion medicine (table 2). records from the htc meetings further indicated that 6 hospitals (40%) had a clearly designated staff member in this position. overall, based on how the respondents from each of the three types of hospital scored their bts, organisation management implementation in bts in nairobi county was below average on the four areas assessed: smt involvement (24 respondents, 40%), performance of the htc (23 respondents, 38%), and appointment of blood transfusion specialists (24 respondents, 40%) and quality officers (12 respondents, 20%). faith-based hospitals had the lowest level of implementation at 33.3%, private hospitals were at 42.5%, whereas government hospitals had the highest level of implementation at 60%. the performance of the government hospitals was boosted by close supervision and interaction with the rbtc. discussion blood transfusion is a multistep, multidisciplinary service and involves different cadres of staff. this study was conducted in hospitals to explore the entire transfusion service chain. in this study more than half of the staff had served for less than 4 years in their current stations. employee retention is important to create a pool of knowledgeable staff to serve in the htc and as blood transfusion managers, quality officers or transfusion specialists. this study explored the implementation of organisational management as a foundational component of the quality system. the main pillar of this element was the involvement of senior management team in the blood transfusion service, which ensures the service obtains the necessary resources for appointment of key staff and supports other activities such as the establishment of an htc. the senior management team scored poorly on the review of audit findings. this indicates that organisational management may not be able to incorporate audit findings to improve the service. audits check blood wastage and monitor utilisation practices, blood storage and transfusion procedures. failing to act on audit reports may lead to an inadequate or unsafe blood supply. lack of tangible commitment from the hospital management is likely to cause poor implementation of recommendations in blood transfusion service.9 the establishment of htcs in government hospitals was attributed to their closer interaction with the rbtc. in kenya, there are six rbtcs; they are branches of the kenya national blood transfusion service and are mandated by the government to collect, process and distribute blood to all hospitals and provide oversight of transfusion services. this emphasises the role of the rbtc in providing administrative support to institutions to help them set up transfusion committees. hospital blood transfusion committees have also been established in other countries, such as brazil, where they are present in 63.4% of blood transfusion services.10 in a survey conducted in the netherlands in 2012, all 76 hospitals were found to have htcs.11 this study found that although the hospitals had established htcs, they were not fully functional. low performance on audits indicates that the hospitals may not implement measures to replenish blood bank stocks in good time. audits may provide the htc with information on blood bank stocks, wastage or other failures within the transfusion chain. other studies have shown that without adequate support from senior management, resources and substantial authority, these committees cannot positively impact transfusion practice.12 according to the who, the role of an htc is to ensure availability of blood components, audit transfusion practice, implement the national policy and monitor utilisation of blood in the hospital.13 the presence of a full-time quality officer in the blood transfusion service was very infrequent. this staff member is responsible for promoting adherence to best practices throughout the transfusion chain, implementing the htc and senior management team decisions and ensuring that blood components are safe and services are adequate. the quality officer can be a nurse, a laboratory technologist or a clinician with sufficient experience in blood transfusion and quality management. failure to appoint quality officers implies that problems that contribute towards blood shortage (such as wastage, misuse or adverse events) are not promptly captured, investigated and corrected through clearly defined guidelines. although there have been mixed findings on employment of quality officers, with some developed countries also performing below average in this area, the vital role they play is not in question. in the united states, 42.9% of healthcare facilities had employed full-time quality assurance staff to investigate transfusion-related adverse reactions.14 in the 2012 dutch national survey, all 76 hospitals sampled had appointed a quality officer to oversee transfusion safety.11 less than half of the respondents indicated that they had a blood transfusion specialist in their institution. this specialist is a clinician with adequate experience and knowledge in blood transfusion who provides therapeutic advice, supports blood management and implements best practices, such as autologous transfusion and other strategies that eliminate, reduce or optimise blood transfusions.15 this shows that most of the hospitals in nairobi do not have clearly defined guidelines to promote blood management and best practices in transfusion. blood management is a multidisciplinary approach to improve patient clinical outcomes, while also reducing the need for allogeneic blood. this may include measures to prevent anaemia, such as iron supplementation to improve haemoglobin levels, and enhancement of coagulation function to limit bleeding in preoperative patients; other best practices include intraoperative blood recovery and use of fibrin sealants to reduce bleeding.16 these measures decrease and may eliminate the need for transfusion, thereby reducing wastage and inappropriate use of blood. a study conducted in england showed that 83% of hospitals had employed lead specialists for transfusion following the implementation of recommendations for ‘better blood transfusion’.9 in general, nairobi has not fully implemented the organisational management element despite who recommendations.5 the hospitals need strong involvement of senior management teams in transfusion service, effective transfusion committees and appointment of key staff (quality officers and transfusion specialists) in order to build a robust quality system. through the quality system, the hospitals can then incorporate alternatives to donor blood, reduce wastage and maximise available blood components to benefit patients. the alternatives to allogeneic blood transfusion include autologous donations and intraoperative blood salvage.16 this would contribute significantly to safety, adequacy and effectiveness of the service. robust blood transfusion services would significantly strengthen the healthcare system. limitations as this study was carried out in nairobi county, the findings cannot be generalised to the entire country. nairobi, as the capital city, has closer supervision from the rbtc and the kenya national blood transfusion service. the study involved healthcare organisations categorised as hospitals in the kenya e-health database. other institutions such as dispensaries or clinics offering blood transfusion services were not included. recommendations the senior management teams should establish and empower htcs to monitor utilisation and availability of blood components, audit transfusion practices and implement the national blood transfusion policy in the hospital. hospital blood transfusion services should appoint key staff: quality officers and blood transfusion specialists. these two will maintain the quality system, promote transfusion best practices, investigate transfusion-related adverse reactions and support blood management practices, which eliminate, reduce and optimise blood transfusions. the rbtc should extend oversight and increase interaction with the private and faith-based hospitals to help establish strong htcs in these facilities. further research is needed to highlight the extent of implementation of the other who quality elements in nairobi county. conclusion the smts in nairobi county need to play a more visible role in strengthening the quality system in the blood transfusion service. the htc, quality officers and blood transfusion specialists, where available, were not performing optimally. hence, their roles in promoting transfusion best practices, blood management techniques to eliminate the need for transfusion and components preparation to maximise available units may not be realised. essentially, nairobi has not fully implemented the organisational management element to strengthen the blood transfusion service in its hospitals despite the who recommendation in 2002 for implementation of the quality management system. trustworthiness the data included in this study were collected and analysed by the authors. the results obtained and recommendations made are based on the information generated from the data collected. there were no updated or additional data added after the end of the data collection period. reliability piloting of the questionnaire and observation checklist was done at the african inland church kijabe hospital. this is a faith-based hospital outside nairobi county. this selection was made to avoid inadvertent sensitisation of the respondents who sometimes take up locum positions in neighbouring hospitals. the pre-test exercise and results were used to refine the research instruments. validity the study used both questionnaires and observation for triangulation of the results. the information provided by the respondents was verified by first-hand observation made by the researcher. acknowledgements the authors thank kenya methodist university for supervision, ms carol nyambura for contributions to the design of the project and dr julius kahuthia for contributions to data management. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions a.n.m. conceived and designed the project and wrote the paper. e.m.m. and m.o.o. were research supervisors. source of support none. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references mamaye. factsheet on malawi’s blood services [homepage on the internet]. 2015 [cited 2015 sep]. available from: 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bethesda, ma: aabb; 2011. appendix 1 appendix 2 abstract introduction methods results discussion acknowledgements references about the author(s) abdul k. el karaaoui department of pathology and laboratory medicine, faculty of medicine, american university of beirut, beirut, lebanon nada assaf department of pathology and laboratory medicine, faculty of medicine, american university of beirut, beirut, lebanon citation el karaaoui ak, assaf n. using the slipta checklist to assess laboratory readiness for joint commission international accreditation. afr j lab med. 2023;12(1), a2044. https://doi.org/10.4102/ajlm.v12i1.2044 original research using the slipta checklist to assess laboratory readiness for joint commission international accreditation abdul k. el karaaoui, nada assaf received: 28 july 2022; accepted: 13 dec. 2022; published: 15 mar. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the stepwise laboratory improvement process towards accreditation (slipta) helps prepare laboratories in lowand middle-income countries to achieve international accreditation aligned with the iso 15189:2012 standards. accreditation by the joint commission international (jci) is among the highest sought by hospitals worldwide. while the readiness of laboratories with a five-star slipta score to undergo iso 15189:2012 accreditation was recently assessed, the compliance of the slipta checklist with jci is still unknown. objective: the study evaluated the slipta checklist’s utility in assessing laboratories to meet the jci standards. methods: we conducted a detailed gap analysis between slipta and jci laboratory standards from january 2021 to january 2022. we cross-matched the jci standard requirements to slipta clauses and categorised each standard into ‘met’, ‘partially met’, and ‘not met’. we highlighted similarities, discrepancies, and improvement areas. results: a total of 109 jci standards were included. the slipta checklist completely met 61 standards, partially met four, but did not meet 44. the unmet jci standards focused on the quality planning, control, and improvement sections. healthcare organisation management and quality control processes, including selecting an accredited reference laboratory, collecting quality management data, creating of post-analytical policies and procedures, and validating monitoring systems, constitute the basis of this preparation. conclusion: the slipta checklist covers major quality management system elements of the jci standards for laboratories. however, some components should be addressed to assure readiness for jci accreditation. what this study adds: this study identified additional areas not covered by the slipta checklist that are required for jci accreditation. keywords: standard culture; joint commission international; laboratory accreditation; lowand middle-income countries. introduction medical laboratories play a pivotal role in maintaining the healthcare chain in a nation. laboratory services are crucial for disease prevention, diagnosis, surveillance, and treatment.1,2 in developed countries, up to 70% of clinical decisions depend on laboratory test results.3,4 as such, maintaining quality laboratory services is essential for the efficient functioning of the healthcare system. accreditation is an external quality review that evaluates an organisation’s conformity with a predefined set of standards to receive formal recognition of compliance by an authorised external body.5 as a primary goal, laboratory accreditation aims to improve patient safety and the standard of care.6 in addition, accredited laboratories show superior test reliability, better operational efficiency, and higher competence.7 with increased outbreaks of diseases such as hiv, tuberculosis, and the recently emerging coronavirus disease 19 in lowand middle-income countries (lmics), there is increased attention on strengthening healthcare systems in limited-resource areas.8 in 2009, the world health organization regional office for africa, in collaboration with the african society for laboratory medicine, the united states center for disease control and prevention and african countries consorted on the creation of a stepwise laboratory improvement process towards accreditation (slipta) to implement quality management systems (qms) and organise quality improvement processes in lmic laboratories.9 the slipta programme consists of a checklist questionnaire linked to a five-star recognition depending on the level of compliance ranging from ≤ 55% (0 stars) to ≥ 95% (5 stars).8,9 it is an interim recognition, and laboratories that achieve a slipta five-star rating are encouraged to apply for international accreditations such as iso 15189:2012 and joint commission international (jci).8 founded in 1951, jci is today the largest international accreditation body for hospitals.11 the jci laboratory accreditation standards cover all laboratory needs for continual improvement within a quality management environment.11 it also addresses essential laboratory managerial and clinical functions while focusing on patient safety.11 because jci is a hospital accreditation scheme, laboratory jci clauses are clinically oriented. therefore, it is complementary to iso 15189 standards, which are process based. as an increasing number of hospitals worldwide are seeking jci accreditation, laboratories (including lmic laboratories) might undergo on-site evaluation by the jci during their hospital accreditation inspection. the suitability of the latest version of slipta to guide laboratories towards an iso 15189:2012 accreditation was recently assessed by datema et al.8 however, the readiness of slipta five-star laboratories to undergo jci accreditation is yet undetermined. the aim of this study was to evaluate the slipta checklist’s usefulness in assessing laboratories to meet jci standards. methods ethical considerations the local institutional review board (human research protection program at the american university of beirut) deemed the study exempt from review. this study did not involve human or non-human participants and did not require participant consent or data protections. study design this study was conducted at the college of american pathologists accredited department of pathology and laboratory medicine at the american university of beirut medical center, lebanon. the american university of beirut medical center is a jci-accredited tertiary healthcare facility. the assessors (the manuscript’s two authors) were selected based on their experience in the different accreditation processes and their certification as auditors for select certification bodies. the latest versions of slipta (version 2:2015) and jci standards for laboratories (3rd edition; january 2017) were retrieved from the world health organization regional office for africa and jci websites9,10,11. data analysis after thoroughly reviewing the jci and slipta standards, the authors performed a gap assessment separately between january 2021 and january 2022. first, each jci clause was compared to its counterpart in the slipta checklist and categorised as a ‘met’, ‘partially met’, or ‘not met’. ‘met’ jci clauses were addressed and totally covered by the slipta checklist, ‘partially met’ standards were addressed but not all elements were covered by the checklist, while ‘not met’ standards were not addressed. percentages were calculated using a spreadsheet, microsoft excel (microsoft, redmond, washington, united states). we then compared each author’s results, discussed areas of discrepancies, and issued a unified consensual analysis. the jci standards were divided into the three categories of the juran trilogy model (quality planning, quality control and quality improvement) as similarly done by juran.12 the results of our scoring system were used to determine the categories with the highest percentage of gaps. the slipta comprises 12 sections, each corresponding to a single qms element, which can be grouped into three categories: ‘quality planning’, ‘quality control’ and ‘quality improvement’, according to the juran trilogy model.12 the jci booklet comprises three general divisions, ‘accreditation participation requirements’, ‘patient-centred standards’, and ‘healthcare organisation management standards’, subdivided into seven sections. of the 152 jci standards, 109 were reviewed and compared to their counterparts in the slipta checklist. forty-three standards in sections 1 and 7 of jci were excluded from this review. section 1 targets accreditation participation requirements. this section is specific to jci and includes general principles such as the timely submission of accurate and complete data to the jci committee. section 1, including its 12 clauses, was excluded from the gap analysis due to lacking a counterpart. section 7 includes standards covering speciality quality control process (qcp) (qcp. 2 – qcp. 10.1 and qcp. 26 – qcp. 27.5: 31 clauses), which details requirements for single laboratory speciality sections. because slipta views hospital laboratories as a single unit, it lacks speciality-specific questions, and the corresponding standards (20%; 31/152) were excluded from the analysis. results the slipta v2:2015 addresses 60% of the 109 jci standards reviewed (56%; 61/109 wholly met and 4%; 4/109 partially met) but does not meet 40% (44/109) (table 1). among ‘partially met’ standards, 75% (3/4 standards) belong to the quality planning category and 25% (1/4) to quality improvement (table 2). among the ‘not met’ jci clauses, 11% (5/44) are quality planning clauses, 82% belong to quality control (36/44) and 7% (3/44) to quality improvement. table 1: gap analysis of joint commission international and stepwise laboratory improvement process towards accreditation, january 2021 – january 2022. table 2: distribution of non-conformities between joint commission international clauses and the stepwise laboratory quality improvement process towards accreditation checklist based on the categories of the juran trilogy, january 2021 – january 2022. gaps were noted in standards related to the organisation’s vision and mission statement, the accreditation of referral laboratories, ongoing communication with stakeholders, and quality monitoring. in terms of quality control, the validation of monitoring systems before implementation was also missing in the slipta checklist. slipta checklist coverage of joint commission international standards joint commission international section 2: patient safety goals this section focuses on patient safety goals and includes correct patient identification, effective communication of critical results, and hand hygiene standards. section 8 of the slipta (process control) addresses all the requirements for preanalytical quality checks, including patient identification, sample collection and reception. the first section of the slipta covers effective reporting of critical results, including adequate documentation and record keeping, while the facilities and biosafety section cover the hand hygiene standards. in addition, the slipta standards require proper staff training on hand hygiene practices (section 3.6 d). joint commission international section 3: healthcare organisation management the jci section 3 consists of healthcare organisation management standards, for which the initial step is the design of a leadership organisational chart and the determination of the laboratory’s mission and vision. the slipta checklist enquires about the presence of an organisational chart and a narrative description (section 3.2) of the different internal and external relationships within the laboratory. however, no points are assigned to developing a vision and mission statement. the following organisation management requirement is the appointment of a qualified laboratory manager, a clause fulfilled in the slipta checklist. despite the presence of a section in the slipta checklist addressing client management and customer service, there is no requirement for a qualified individual to oversee these procedures. in the jci, management is also responsible for service organisation and continuous communication with stakeholders; however, this is uncovered by the slipta checklist. resource planning is addressed in section 2.2 of the slipta checklist as part of the output review by leadership. the following section in jci regulates the contracts and services with reference laboratories, namely the presence of standard operating procedures to define the process for selecting and approving contracts and services by the leadership (governance, leadership and direction [gld] [gld.2.2]), the need for proof of accreditation or certification (gld.2.2.1), and performance monitoring (gld.2.2.2). while the slipta sets detailed requirements for the choice of referral laboratories and their continuous monitoring, along with the responsibility of the laboratory director in these tasks, it does not require checking the accreditation status of referral laboratories. the slipta checklist views the laboratory as a single unit rather than different laboratory speciality sections. thus, unlike the jci, it does not address standards for blood bank (gld.2.3) and point-of-care services (gld.2.4). nevertheless, the slipta standard 3.3, section 3, requires a laboratory director with adequate competency to supervise all laboratory workstations, which by extension includes blood bank and point-of-care services, as required by the jci gld.2.3 and gld2.4. in the jci, gld.3, 3.1 and 3.2 regulate effective interand intra-laboratory communication and efficient determination of clients’ needs; these are also detailed in slipta. the next jci section defines the different aspects of planning and coordination of a quality management programme, including the responsibilities of the leadership in developing the programme (gld.4) and implementing activities (gld.4.3), along with the required criteria for a well-functioning programme (gld.4.1) and its monitoring by a qualified individual (gld.4.2). the slipta checklist conforms to the jci requirements for quality management design. however, it does not completely cover quality management monitoring. for example, the jci quality management monitoring the leadership to determining processes to be measured (gld.4.5) and standards required to identify critical measures for clinical (gld.4.5.1) and managerial (gld4.5.2) structures, all of which slipta does not address. in addition, the analysis of measurement data requires skilled individuals (gld.5), an internal process to validate data (gld.6) and an appropriate investigation of off-target trends (gld.7). these criteria are met in the slipta checklist sections 1.5, 6.1 and 11.1 that determine the need for effective auditing, including personnel, procedures, and trend verification tools. the joint commission internationalgld.8 to 12.1 state principles for well-functioning safety and quality insurance programmes, including, but not limited to, the presence of an ongoing programme (gld.9), and the responsibility of the leaders to create, implement (gld.8), provide resources (gld.11) and monitor ongoing programmes (gld.10) while maintaining a safety culture throughout the laboratory (gld.12 and gld.12.1). laboratories with a five-star slipta accreditation are considered compliant with the safety and quality insurance programmes set by jci. joint commission international section 4: information management section 4 of the jci focuses on information management. it states the need for the presence of written policies and procedures addressing all testing phases (preanalytical: [management of information {moi}] moi.1, 2, 2.1, 2.2; analytical: moi.3; post-analytical: moi.4) and storage (moi.5), as well as their implementation (moi.1.1). it also requires the definition of a turnaround time (moi.4.1). the slipta checklist requires the presence of written policies and procedures (section 1.2) and their implementation (section 1.2). therefore, slipta meets all standards for all testing phases (preanalytical: sections 1.5, 8.4; analytical: sections 1.5, 8.7; post-analytical: section 1.5), including setting turnaround time and storage requirements for records and specimens. joint commission international section 5: staff qualifications and education section 5 standards are related to staff qualification and education. the first standard ([staff qualification and education {sqe}] sqe.1) mentions the role of laboratory leaders in defining the education and skills required for staff members, which is met by slipta section 3.3 on the role of laboratory directors. section 3 of slipta complies with the second standard in jci section 5, which discusses the training and experience required for supervisors and other leaders, licensure and registration of pathologists, and the need for defined responsibilities in the job descriptions. the next jci standard focuses on staff orientation, ongoing education, and yearly competency assessment needed to maintain and improve staff skills and knowledge (sqe.2, 2.1 and 3); the slipta sections 3.3, 3.6, and 3.7 meet these requirements. also, the slipta section 3.5. mentions the jci standard sqe.3.1 (maintenance of documented personnel information for every staff member) requirements. finally, the last standard in this section requires the laboratory to provide health services to all staff (sqe.4) and is met by the slipta checklist. joint commission international section 6: facility management laboratory facility management constitutes the focus of section 6. the first standard targets the responsibilities of laboratory leaders in providing the basic facilities and their facility’s compliance with laws and regulations. this requirement is met by slipta section 1.5 on accommodation and environmental conditions. the next standards state the requirement for sufficient storage space ([facility management and safety {fms}] fms.2.1) and proper record keeping (fms.2.2). resources for this goal should be provided by the laboratory leadership (fms.2). storage requirements, record keeping, and leadership roles are covered by the slipta sections 12.6, 1.5 (records control and sections) and 12.1. the jcistandards fms.3 to 4.1.1 mention the different aspects for efficient and effective operation of all utility systems and equipment. these aspects include inspection, testing of critical operating components, maintenance, and availability of emergency backup for critical utilities. interestingly, slipta section 1.5 on laboratory equipment and section 5 on equipment give a more detailed description of proper equipment protocol due to the question format checklist. the jci standard fms.4.2 on the requirement for a defined process for validating and maintaining computer software and laboratory information is met by slipta sections 9.9 and 9.10. the standards for periodic evaluation, labelling and documentation of reagents are met in slipta sections 7.4 (inventory control) and 8.8 (reagents acceptance testing). finally, section 12 in slipta (12.8–12.13) covers all safety aspects required by the last jci section 6 standard on safety (fms.5), including the management of hazardous materials, waste (fms.6 to 6.3), and fire (fms.7 and 7.1). joint commission international section 7: quality control the last jci section deals with quality control (qc) standards for all testing areas. it includes establishing qc processes for the different test methods, external and internal qc, verification of results accuracy, and methods comparison. it also targets new instrument validation and instrument monitoring systems implementation. the qc processes are addressed through several slipta sections with high compliance with jci clauses. however, the slipta checklist does not have questions on the validation of all monitoring systems before their implementation, as required by jci. discussion the level of compliance of slipta with jci requirements appears to be lower than its compliance with iso 15189, as determined by datema et al.8 this observation is because the slipta checklist is primarily based on the iso 15189 and the clinical and laboratory standards institute guideline qms01-a4 clauses in 2009.9 the slipta checklist and jci standards are structured differently. in addition to its checklist format, slipta is more detailed and extensive than jci, with specific instructions. the structure difference is because of their respective perspectives and objectives. the slipta checklist leans towards operations rather than management and targets laboratories with newly implemented qms. on the other hand, the jci accreditation standards are more outcome based than process based. they focus on the role of the laboratory leadership and management structure in achieving the desired quality outcomes, resource management, qc processes, the laboratory environment and development. the jci thus targets healthcare organisations with a particular quality background to achieve higher excellence. the jci booklet contains general standards addressing the laboratory as a single unit (similar to the slipta checklist). this approach is important to cover essential laboratory qms elements. in addition, requirements related to specific sections within the laboratory (blood bank, haematology, chemistry) are delineated. quality managers of laboratories undergoing jci evaluation might benefit from ensuring that section-specific requirements are met as they constitute up to 20% of jci standards. our analysis also reveals that the slipta quality planning and control sections contain the highest number of gaps when compared to jci (table 2). as an example, the definition of the organisation’s mission and vision, which slipta does not address, is crucial in the initial steps of the establishment of any qms. similarly, the role of leadership in service and resource planning and the implementation of culture safety should be emphasised in slipta. also, in the quality planning section, the safety programme should be improved in future versions, focusing on reducing infection risks and handling radioactive material. essential qc elements are missing in the slipta checklist. the relationship with external (referral) laboratories should be regulated. another important requirement for jci is the validation of qc systems before their implementation. the post-analytical testing phase should receive special focus for the improvement of the next versions, with the appropriate policies, procedures, and controls. in terms of quality improvement, our analysis reveals a need for the improvement of the managerial decisions of improvement activities and the associated data collection for quality measurement to comply with jci. however, the slipta checklist is skewed towards quality planning and qc sections, with quality improvement having the lowest number of allocated points.8 thus laboratories might focus on quality planning and qc clauses while neglecting quality improvement, which is a critical element in jci accreditation.8 limitations this study reviewed the slpita and jci checklists; thus, limitations are due to possible human errors during the review. each of the manuscript’s authors performed a full review independently to minimise errors. conclusion this article encourages all laboratories in lmics seeking a qms programme to start by implementing slipta, as per the world health organization regional office for africa recommendations. laboratories with high slipta scores seeking jci accreditation might benefit from addressing the following elements: selecting an accredited reference laboratory, collecting data for qm, creating post-analytical policies and procedures, and validating monitoring systems used in qc. because gap analysis is the initial step of any accreditation transition process, this analysis constitutes a useful guide for laboratory quality managers and directors in lmics planning to undergo the jci accreditation process as part of their healthcare organisations. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions n.a, and a.k.e.k. performed data collection and analysis, n.a. and a.k.e.k. wrote the manuscript. n.a. and a.k.e.k. reviewed drafts and approved the final version of the manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability data supporting the findings of this study are available from the corresponding author, n.a., on request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references datema ta, oskam l, van beers sm, klatser pr. critical review of the stepwise laboratory improvement process towards accreditation (slipta): suggestions for harmonisation, implementation and improvement. trop med int health. 2012;17(3):361–367. https://doi.org/10.1111/j.1365-3156.2011.02917.x nkengasong jn. strengthening laboratory services and systems in resource-poor countries. am j clin pathol. 2012;131(6):774. https://doi.org/10.1309/ajcp8gyx8ktkdatz forsman rw. why is the laboratory an afterthought for managed care organisations? clin chem. 1996;42(5):813–816. https://doi.org/10.1093/clinchem/42.5.813 centers for medicare & medicaid services. medicare and medicaid statistical supplement [homepage on the internet]. baltimore: centers for medicare & medicaid services [cited 2022 feb 03]. available from: https://www.cms.gov/research-statistics-data-and-systems/statistics-trends-and-reports/archives/mmss international organization for standardization. iso 15189:2012 medical laboratories – requirements for quality and competence [homepage on the internet]. geneva: international organization for standardization [cited 2022 feb 03]. available from: https://www.iso.org/standard/56115.html zima t. accreditation of medical laboratories – system, process, benefits for labs. j med biochem. 2017;36(3):231–237. https://doi.org/10.1515/jomb-2017-0025 peter tf, rotz pd, blair dh, khine aa, freeman rr, murtagh mm. impact of laboratory accreditation on patient care and the health system. am j clin pathol. 2010;134(4):550–555. https://doi.org/10.1309/ajcph1skq1hnwghf datema tam, oskam l, broerse jew, klatser pr. review of the stepwise laboratory quality improvement process towards accreditation (slipta) version 2:2015. afr j lab med. 2020;9(1):1068. https://doi.org/10.4102/ajlm.v9i1.1068 world health organisation, who regional office for africa. stepwise laboratory quality improvement process towards accreditation (slipta checklist version 2:2015 for clinical and public health laboratories) [homepage on the internet]. [cited 2022 feb 03]. available from: https://apps.who.int/iris/bitstream/handle/10665/204423/slipta-checkist0711.pdf?sequence=1&isallowed=y african society for laboratory medicine. slipta audited laboratories distribution map [homepage on the internet]. ethiopia:. african society for laboratory medicine. addis ababa. [cited 2022 feb 03]. available from: https://aslm.org/resource/slipta-map/ joint commission international. joint commission international accreditation standards for laboratories [homepage on the internet]. illinois: joint commission international. [cited 2022 feb 03]. available from: https://www.jointcommissioninternational.org/en/ juran jm. juran on leadership for quality – an executive handbook. new york, ny: collier macmillan. article information authors: jessie githang’a1 nina hurwitz2 affiliations: 1department of human pathology, university of nairobi, kenya2department of pathology, university of basel, switzerland correspondence to: jessie githang’a postal address: po box 19601-00202, nairobi, kenya dates: received: 27 sep. 2012 accepted: 20 aug. 2013 published: 27 nov. 2013 how to cite this article: githang’a j, hurwitz n. haematopathology diagnosis: role of multidisciplinary training workshops. afr j lab med. 2013;2(1), art. #82, 3 pages. http://dx.doi.org/10.4102/ ajlm.v2i1.82 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. haematopathology diagnosis: role of multidisciplinary training workshops in this opinion papers... open access • introduction    • background    • scope       • format of workshops       • objectives       • emphasis on communication       • training sessions       • identification of areas for future study and training • conclusion • acknowledgements    • competing interests    • author's contributions • references introduction top ↑ background cancer is an increasingly-major health problem in developing countries and the director-general of the world health organization (who) has stated that ‘if no action is taken, deaths from cancer in the developing world are forecast to grow to 6.7 million in 2015 and 8.9 million in 2030’.1 accurate pathologic diagnosis is a key factor in proper management of cancers, yet it has been largely under-resourced with little attention paid to the upgrading of facilities or personnel development.2,3 haematopathology, a complex discipline combining pathology and haematology, is particularly affected by these shortcomings. multidisciplinary training workshops that focus on achievable, accessible and relatively-inexpensive methods and techniques are an important approach to improving the diagnosis of haematolymphoid neoplasms in resource-limited settings. scope format of workshops multidisciplinary haematopathology training workshops must be held at a centre that has suitable facilities and sufficiently-trained personnel. it is important to include peripheral centres in training programmes since pathologists and technologists from less-accessible health facilities may be isolated and might have limited opportunities for further learning. in addition, experts from other institutions, who may be from the same country or region or from abroad, can be included. for example, regional training programmes may be held in adjacent countries and the expertise of the visiting trainers could then be utilised in multiple centres. centralised training has been used successfully in east africa by the international network for cancer treatment and research and the east african division of the international academy of pathology. two such workshops were held in september 2011 in nairobi, kenya and dar es salaam, tanzania under the auspices of the international network for cancer treatment and research. the multidisciplinary haematopathology training workshops brought together clinicians, oncologists, haematologists, pathologists and medical laboratory technologists for training in haematopathology diagnosis. centralised programmes can be made more cost-effective by the involvement of suppliers who are willing to donate or loan equipment and reagents or provide them at a discounted rate. objectives prior to a workshop, its organisers should agree upon and set out clear objectives, identify the training priority areas and key issues to be tackled, the strategies to be used and the expected deliverables and outcomes. this will make the workshop more focused, with clear outcomes that are appropriate for all the attendees. an example of such planning is shown in table 1. table 1: examples of training priority areas, key issues, strategies and the deliverables and/or outcomes of a haematopathology training workshop. emphasis on communication during training workshops, multidisciplinary plenary sessions for oncologists, pathologists, haematologists and technologists can create a unique forum for sharing experiences as well as for laying emphasis on the interdisciplinary nature of cancer diagnosis (figure 1). use of a multidisciplinary model contrasts with programmes in which the training is held separately for different specialties, where pathologists may never meet with their clinical counterparts and medical laboratory technologists never meet the pathologists or haematologists. all cadres need to communicate more with each other. emphasis must be placed upon the importance of clinical inputs that are often not available to the pathologist or haematologist. for example, a lymph node biopsy may be reported on by a histopathologist whilst the bone marrow specimen of the same patient is looked at by the haematologist. separate analyses could result in the two specialists sending independent reports. correlation of the reports from the two samples is important in the clinical decision-making process. bringing together different specialists in the multidisciplinary workshop is an invaluable way of fostering interdisciplinary working relationships and highlighting the critical roles of each cadre. improved attitudes between the different cadres can thus be nurtured. figure 1: steps involved in obtaining histologic diagnosis, highlighting the interdisciplinary nature diagnostic process. training sessions haematopathology training workshops should include individualised training sessions targeting different specialties. these sessions should focus on the delivery of both knowledge and practical skills that can be applied by the participants once they return to their places of work. the workshops that target medical laboratory technologists should address diverse procedures and hands-on training in critical and sometimes basic areas such as fixation, preparation and storage of high-quality histology, cytology and bone marrow specimens. the role of cytochemistry as a simple and relatively-inexpensive technique used in the diagnosis of acute leukaemias should be emphasised. paying close attention to quality control in practical sessions will ensure that technologists are aware of the crucial role of quality in the provision of an accurate diagnosis on the part of the pathologist or haematologist. identification of areas for future study and training it is necessary to identify the major training gaps in the field so that appropriate workshops can be developed. haematologists in their specialist track, for example, would learn aspects of bone marrow examination and interpretation. conferences targeting pathologists and haematologists should focus on specific areas of need, for example lymph node diagnosis using traditional light microscopy as well as immunohistochemistry. the latter is an invaluable technique in lymph node pathology. understanding immunohistochemical reactions in lymph nodes and the pitfalls with regard to interpretation of results, as well as how immunohistochemistry is best used in resource-limited settings, are important aspects to be delivered in such training sessions.4,5 the rationale for the selection of available immunological markers must be discussed. workshops impress upon pathologists and haematologists the need to rationalise the indiscriminate use of multiple markers that are not required for accurate diagnosis. in addition, new frameworks for understanding cancer can be discussed. current knowledge, for example the world health organization’s classification of haematolymphoid neoplasms, can be provided.6 the standardisation of lymph node pathology classification, as well the development of strategies to use available resources to apply the who classification, could provide a stimulating discussion. interactive training methods geared to the adult learner can provide useful, interesting and practical examples for learning, as do case examples that the participants bring from their centres. tutorials may also be of benefit, especially those that include informal discussions on topics such as laboratory management, the improvement of diagnostic services and the importance of standardisation in reports. an important topic for discussion is the role of telepathology in improving the quality of diagnoses and providing education, particularly in areas that have inadequate numbers of pathologists and/or haematologists.7,8,9 conclusion top ↑ standards of haematopathology in sub-saharan africa can be raised through multidisciplinary training workshops so as to effect the delivery of knowledge and practical skills as well as positive attitudes. emphasis should be placed on attainable goals to ensure that the training is relevant and applicable to the attendees when they are back at their practice. a direct consequence of the workshop held in nairobi, kenya was the creation of the kenyan national pathology forum on ipath8 (http://www.ipath-network.com/inctr) for mutual consultation on difficult cases by kenyan experts and members of the international network for cancer treatment and research international faculty. in addition, ipath coordinates an internet forum where technologists can seek advice on issues arising in their daily routine and supports the establishment of newly-acquired techniques in laboratories. advice is given by senior kenyan technologists as well as by an international group of senior technologists. acknowledgements top ↑ thanks to prof. prasanna kumar, psg institute of medical sciences & research, coimbatore, india for her valuable comments and editing of the manuscript; the local organising faculty at the university of nairobi, kenya and muhimbili university of health and allied sciences, dar es salaam, tanzania; the international faculty who were involved in conducting the training workshops; and mr amani shabani, director of labulax supplies ltd, nairobi and merck pharmaceutics, darmstadt germany for supplies received. financial support was received from the international academy of pathology, the international cancer technology transfer fellowships and the national cancer institute office of hiv and aids malignancy. competing interests the authors declare that they have no personal or financial relationship(s) which may have inappropriately influence them in writing this article. author’s contributions j.g. (university of nairobi) and n.h. (university of basel) co-authored this opinion piece based on their experiences in initiating, organising and conducting multidisciplinary workshops in low-income settings. references top ↑ 1. chan m. cancer in developing countries: facing the challenge. address at the iaea scientific forum 2010 (video message) geneva, switzerland 21 september 2010. 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[page on internet]. c2012 [cited 2013 may 25]. available from inctr-news.wikidot.com/pathpoint-conference-2012 8. inctr news. the inctr program for on-line consultation and continuing education using ipath. [page on internet]. c2012 [cited 2013 may 25]. available from inctr-news.wikidot.com/apecsa 9. malami sa. recent advances in telepathology in the developing world. in: g graschew, editor. advances in telemedicine: applications in various medical disciplines and geographical regions. rijeka, croatia: intech, 2011; p. 279–297. [available from www.intechopen.com/download/pdf/14332] diagnostic systems not diagnostics the hidden burdens of technology what is diagnosis for? acknowledgements references about the author(s) rashid ansumana school of community health sciences, njala university, bo, sierra leone fatmata bah kings sierra leone partnership, king’s centre for global health and health partnerships, freetown, sierra leone kan biao sierra leone-china friendship biological safety laboratory, chinese center for disease control and prevention, beijing, china national institute for communicable disease control and prevention, beijing, china doris harding public health laboratories, sierra leone ministry of health and sanitation, freetown, sierra leone mohamed b. jalloh department of community health, faculty of clinical sciences, college of medicine and allied health sciences, university of sierra leone, freetown, sierra leone ann h. kelly global health and social medicine, kings college london, london, united kingdom francess koker kings sierra leone partnership, king’s centre for global health and health partnerships, freetown, sierra leone zikan koroma sierra leone-china friendship biological safety laboratory, chinese center for disease control and prevention, beijing, china clinical laboratories and national coordinator biobanking and biosecurity, sierra leone ministry of health and sanitation, freetown, sierra leone mambu momoh kenema government hospital, viral hemorrhagic fever consortium, kenema, sierra leone school of nursing and medical laboratory sciences, eastern polytechnic, kenema, sierra leone mohamed h. rogers college of medicine and allied health sciences, university of sierra leone, freetown, sierra leone james rogers laboratory technical working group, sierra leone ministry of health and sanitation, freetown, sierra leone alice street school of social and political sciences, university of edinburgh, edinburgh, united kingdom eva vernooij school of social and political sciences, university of edinburgh, edinburgh, united kingdom isatta wurie college of medicine and allied health sciences, university of sierra leone, freetown, sierra leone citation ansumana r, bah, f, biao k, et al. building diagnostic systems in sierra leone: the role of point-of-care devices in laboratory strengthening. afr j lab med. 2020;9(2), a1029. https://doi.org/10.4102/ajlm.v9i2.1029 opinion paper building diagnostic systems in sierra leone: the role of point-of-care devices in laboratory strengthening rashid ansumana, fatmata bah, kan biao, doris harding, mohamed b. jalloh, ann h. kelly, francess koker, zikan koroma, mambu momoh, mohamed h. rogers, james rogers, alice street, eva vernooij, isatta wurie received: 12 apr. 2019; accepted: 14 jan. 2020; published: 01 apr. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the 2014–2016 ebola virus disease (evd) outbreak highlighted the vital importance of investing in west africa’s laboratory infrastructure and systems. in the absence of facilities capable of handling highly infectious pathogens, the national response across the region was hamstrung by costly delays in case identification and blind spots in epidemiological surveillance. the rapid development of evd diagnostic tools that could speed up testing and be used at or near the point of care became a public health priority. to expedite their deployment, a series of cross-sectional research and development initiatives were launched including innovative financing mechanisms, data-sharing platforms, public-private partnerships and accelerated regulatory pathways.1 a number of novel evd diagnostics were trialled in sierra leone.2,3,4,5,6,7 guided by a national testing algorithm, rapid diagnostic tests (rdts) such as reebov (corgenix, broomfield, colorado, united states) and the oraquick (orasure technologies, bethlehem, pennsylvania, united states), intended for use at the primary care level, and automated real-time reverse transcription polymerase chain reaction (rt-pcr), primarily run on the benchtop, underwent rapid validation and performance evaluation through the world health organization’s emergency use and assessment listing mechanism.8,9,10,11 the reebov test was temporarily deployed in a field setting in sierra leone and another rapid test, the dstl evd rapid diagnostic antigen test, developed by the defence science technology laboratory in the united kingdom, was validated in hospital and primary care settings.6 however, biosafety concerns, related to challenges in procuring, distributing and providing training for the use of personal protective equipment to safely draw, handle and dispose of blood samples suspected of being infected with ebola virus, in addition to imperfect sensitivity and specificity, meant rdts were rarely used outside the laboratory. instead, they were deployed at laboratories in key areas of the country alongside automated rt-pcr machines, such as genexpert (cepheid, ab, sunnyvale, california, united states) and biofire film array (biofire diagnostics, salt lake city, utah, united states), that could quickly confirm their results. in addition to commercially available platforms, international public health agencies and non-governmental organisations brought their own in-house assays, some of which came to provide a point of reference for field evaluations of novel tests.12 collectively, these advances in rapid and proximate point-of-care diagnostic capacity helped bring the outbreak to an end and became the cornerstone of sierra leone’s enhanced surveillance plan, intended to sustain a ‘resilient zero’ number of cases.13 in the aftermath of ebola, some of these devices and machines were left in clinical and surveillance laboratories as part of the post-ebola effort to improve disease detection and response capabilities. an rt-pcr machine in the biosafety level 3 laboratory supported by the china center for disease control and prevention (china cdc) currently serves the freetown area, while a genexpert machine for point-of-care testing supports upcountry evd surveillance in the bo and makeni government laboratories. while important for future outbreak response, the availability of evd diagnostics is clearly only a minor part of public health emergency resilience.14 the handover of epidemic preparedness from international partners to national institutions offers a unique opportunity to think through the broader, system-level needs for the accurate and rapid diagnosis of infectious diseases. critically, more attention is needed to grasp how national strategic plans for strengthening laboratory infrastructure can be best adapted to and advanced by the 2024 global health security agenda.15 global health and policy debates, largely dominated by institutions in europe and the united states, have focused on the development of new diagnostic technologies appropriate for resource-poor settings, which generally means that tests should be affordable, easy to use, rapid, and available at the point of care.16,17,18 less prominent in these discussions are the voices of experts in laboratory medicine from the countries for which the rapid tests are designed. the experience of sierra leonean laboratory medicine experts, both during and after the outbreak, offers a valuable, under-recognised and much-needed perspective on the role of point-of-care tests and diagnostic innovation in global health. on 04 march 2019, a multidisciplinary group of policymakers, biomedical and social scientists and local experts in the field of laboratory medicine convened to discuss the role of point-of-care diagnostic devices in outbreaks and their integration with healthcare infrastructure in sierra leone. the meeting was organised by the diadev research project, funded by the european research council under the horizon 2020 framework, which investigates the development of point-of-care diagnostic devices in global health and their role in transforming health systems in resource-constrained settings (www.diadev.eu). the meeting featured short presentations as well as a panel discussion with sierra leonean laboratory scientists and health workers, who shared their first-hand experience using rapid diagnostic tests during the ebola outbreak. three key insights from the day are highlighted here as disruptive and constructive contributions to debates about diagnostic futures in africa. diagnostic systems not diagnostics point-of-care devices are often championed as solutions for places with weak or no laboratory infrastructure. for evd alone, there are up to 27 assays at different stages of development or use, including 9 antigen-based rapid tests and 18 rt-pcr assays for evd.19 but the lower sensitivity of point-of-care devices means they are often integrated into complex algorithms that require confirmatory testing prior to treatment, which necessitates a wider public health laboratory network. moreover, even in the case of tests approved for stand-alone use, routine quality assurance systems require proficiency training and regular cross-checking of samples by a reference laboratory. lessons from sierra leone show that effective use of point-of-care devices also depends on the existence of regulatory capacity to safeguard the quality of point-of-care devices. many rdts on the sierra leonean market are of questionable quality and have not passed through the pharmacy board, the agency that regulates medicines and diagnostics. representatives of the ministry of health and sanitation underscored that, while the central public health reference laboratory is mandated with overseeing quality assurance and post-market validation of diagnostic tests for specific diseases such as hiv, there is a need to expand regulatory and quality assurance systems for all diagnostics, especially those for the national priority special pathogens. one current priority for the central public health reference laboratory is to improve the post-market regulatory control by re-introducing lot verification testing of diagnostic devices before and after they are distributed to clinical settings. enhancing these systems will curtail the supply of low-standard tests and also empower national clinical and public health systems to demand more rigorously tested and, arguably, superior products from international vendors. moreover, further investigation is needed into how these devices are being used in clinical practice and their capacity to improve patient care. for example, in the country’s main referral hospital, rapid tests for hepatitis b, helicobacter pylori and urine analysis, are used inside the clinical laboratory, but routine reporting systems in the laboratory mean results are only given back to clinicians the next day, even when the test result is ready earlier, impacting the ability of point-of-care tests to reduce result turn-around times. the migration of rapid diagnostic technologies into clinical laboratories also poses the risk that technologies designed to provide preliminary clinical diagnosis in places without laboratories actually replace and diminish existing laboratory capacity. point-of-care tests do not present an alternative to investment in central laboratories, which remain essential for quality assurance, confirmatory testing and research.20 particularly among patient populations likely to be afflicted by more than one disease, the availability of a comprehensive suite of basic laboratory tests located at a primary care level, as outlined by the world health organization’s recently published essential diagnostics list,21 is critical to the establishment of person-centred healthcare. rather than asking whether we should invest in laboratory strengthening or point-of-care tests, we need to look at how we can best build diagnostic systems and what role new diagnostic technologies might play in strengthening those systems. the world health organization framework for health systems strengthening has drawn critical attention to the six technical ‘building blocks’ of health systems: service delivery, health workforce, information, medical products, financing, and leadership and governance.22 all these areas are fundamental to the operation of a diagnostic system, whether this means training the health workforce in the use of new diagnostic devices, ensuring health information systems are aligned with the data from new diagnostic devices, securing supply systems to ensure reagents and other equipment are at hand, or building quality assurance systems that connect peripheral health facilities to central laboratories. but while the world health organization framework focuses on technical elements, our on-the-ground experience of laboratory medicine in sierra leone emphasises the relationships between people, technologies and infrastructure that enable diagnostic systems to work. these relationships are social and political, as well as technical, and their understanding requires attention to the interaction between local and global normative frameworks and value systems in addition to formal management structures.23 building on the work of social scientists working in this area, we propose that systems thinking entails a shift away from a focus on diagnostic technologies to understanding the way relationships between people, technologies and infrastructure unfold within everyday diagnostic processes and practices.24 the hidden burdens of technology recent global health policy discussions about diagnosis in lowand middle-income countries have focused on the need to incentivise markets for new diagnostic technologies. point-of-care tests that can be sold as affordable products are often championed as simple and easy-to-use solutions for places with limited infrastructure. but the sierra leonean experience suggests that new technologies also place a significant un-costed burden on the health system and the people who work in it, reinforcing the existing findings from social science research that point-of-care tests in africa have unexpected, intensive infrastructure requirements.25,26 contributors to the workshop noted that the burden is most sharply felt in supply chains, especially around the need for a constant and reliable supply of reagents, and transportation costs for confirmatory testing as part of quality assurance. in a recent measles outbreak in sierra leone in 2018, limited resources for specimen transportation to the reference laboratory from districts, provided through the national surveillance programme, and the lack of a full complement of reagents for analysis, hampered timely diagnosis. in this instance, preliminary diagnosis with a point-of-care test may have provided a stopgap for a quicker response. however, the 2012–2013 cholera outbreak in sierra leone showed that point-of-care tests often lack the required sensitivity, with only 10% of cases confirmed positive by conventional culture in the bacteriology laboratory, resulting from weak international and national regulatory frameworks safeguarding the quality of cholera rdts.27 point-of-care tests with very high sensitivity and specificity need to be coupled with confirmatory testing infrastructure and proper coordination between government institutions with oversight roles to ensure that the right types of kits are used for routine diagnosis. beyond the compromises in accuracy that point-of-care test designs often entail, every new diagnostic device and each iteration of platforms already in use requires a retraining of health workers and laboratory staff, creating heavy logistical, administrative and financial costs on public health institutions. these expenditures are compounded by those associated with the routine operations of the laboratory, including the purchasing of equipment, reagents, storage of large volumes of tests that require refrigeration, everyday maintenance and, critically, management of non-biodegradable waste, generated by point-of-care tests such as the cartridges used in genexpert machines or the test cassettes holding reagent strips for viral hepatitis tests. in the past, these institutional overhead costs have been mainly supported by donors as part of short-term research budgets. to support efforts to improve quality-assured diagnostic operations, a clearer understanding is needed of the hidden costs of diagnostics and where they fit in the broader financial landscape of national laboratory infrastructure and systems. what is diagnosis for? diagnostic testing is not a medical intervention, but a means of generating information for evidence-based medical practice. the question we must ask is: what can be done with this information in this place, with these resources, by these people? the point of diagnosis is not just to know what disease someone has, but to be able to act on that knowledge – whether in terms of public health measures or therapeutic intervention. as the panellists in a round table discussion dedicated to reflections on the use of point-of-care tests during the evd outbreak made clear, detecting the presence of a pathogen is just one component in a larger set of testing needs. diagnosing evd is key to public health measures, such as the isolation of patients, safe burial of bodies and contact tracing, but for clinicians it is only the starting point for therapeutic intervention. when it comes to care, other tests, such as liver function or electrolytes are arguably more important and depend on a generalised laboratory capacity. for example, point-of-care bedside analysers, such as the i-stat (abbott, lake forest, illinois, united states), which can help monitor patients in the red zone of the treatment unit, were crucial to saving lives during the outbreak but received far less attention than ebola diagnostics. intermittent power outages can affect the validity of standard laboratory tests, such as blood culture, with dangerous implications for clinical outcomes during routine care practices but also poses a significant challenge for clinicians working during the outbreak. finally, as we look ahead towards building a sustainable healthcare laboratory system in sierra leone, we need to link the question of diagnostic use to that of diagnostic value, or ‘what worth does a specific diagnostic test have for the particular health system?’ at a cost-effectiveness level, answering this question might mean calculating in the hidden burdens that new technologies generate, including workload burdens involved in using tests and reporting results, burdens on patients to travel for testing, and burdens on the health system to provide training, quality assurance, regulation, procurement and supply, and waste disposal systems. some point-of-care tests may reduce the cost of care in wealthy countries but are a burden in resource-constrained countries such as sierra leone.28 for example, point-of-care pcr tests, such as the biofire film array system and genexpert machines, require the use of expensive cartridges that deter their routine use for testing; the cost of a biofire gastro-intestinal panel ($155.00) is higher than the minimum monthly wage in sierra leone.28 calculations of value would caution against the hasty introduction of new, more advanced testing devices for particular diseases, when local capacity for testing already exists. for instance, investing in simple and affordable technology such as ammonia solution for assessment of haemoglobin using a colorimeter may offer better value for money than handheld point-of-care haemoglobin meters with costly cuvettes (e.g. hemocue, ängelholm, sweden), which also require the training of laboratory staff and placement in coordinated systems. as another example, while biosequencing may be a compelling orientation for research on emerging infections, for clinical use in a resource-poor context, its running costs are plainly prohibitive. the value of a diagnostic test cannot be determined merely by the accuracy of the test or the global health priority of the pathogen but on the basis of local needs and consideration of the test’s clinical and operational benefits. the work that the viral hemorrhagic fever consortium did in partnering with a local institution in the design and development of an rdt for lassa fever virus is an example of how local priorities can be built into innovation processes from the outset and such local institutional partnerships are to be encouraged in the development of future diagnostic systems in african countries.29 a novel diagnostic test’s value, moreover, is not the same as its value for money. beyond a bottom-line economic analysis, the adjudication of diagnostic value requires attention to the everyday lives and work of patients, clinicians, nurses, laboratory technicians, surveillance officers and public health officials. conclusion point-of-care tests are never introduced in a vacuum. ebola brought visibility to the need for improved diagnostic systems in lowand middle-income countries, but even countries severely lacking in laboratory infrastructure have pre-existing and highly specific diagnostic needs and capacities. sierra leone had a national medical laboratory policy and five-year strategic plan in place when the ebola outbreak occurred. while the renewed focus on global health security strengthening and, by extension, laboratory system improvement, is welcome, it is critical that the national tools and plans put in place are aligned to any new diagnostic devices or laboratory strengthening initiatives and allow for national priorities to be addressed. point-of-care diagnostic devices are often championed for their ability to work anywhere, but technologies are never autonomous from the systems in which they are used. diagnostic innovation needs to start from existing diagnostic systems and national policies and plans. this requires the involvement of social science research in understanding the local context into which point-of-care testing devices are introduced and in identifying local priorities for strengthening diagnostic systems. global health research and development should be directed at making diagnostic systems become workable in their own right, rather than finding ways that an ever-increasing range of individual technologies can be best accommodated. post-ebola, sierra leone is focused on improving the resilience of healthcare delivery.30 this goal will require building a diagnostic system that can prepare for and respond to ‘health shocks’, such as outbreaks and natural disasters, and also withstand the chronic stresses that accompany long-term resource constraints.31 a systems approach that encompasses both emergency and long-term timeframes needs to be present at the outset of the diagnostic development process, not only at the point at which new technologies are deployed. most importantly, diagnostic futures need to be designed with the input of the people who work in and use them and they need to incorporate the insight that local experts have gained on the front line of global health innovation. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions a.s. was the project leader, co-wrote the manuscript and led responses to reviewers and the redrafting for publication. e.v. and a.h.k. organised the workshop on which this opinion piece is based, gave workshop presentations and co-wrote the manuscript. i.w., r.a. and m.b.j. provided conceptual contributions, gave workshop presentations on which the manuscript is based, and made significant critical revisions to the manuscript. f.b., k.b., d.h., f.k., z.k., m.m., m.h.r. and j.r. provided conceptual contributions and gave workshop presentations on which the manuscript is based. ethical considerations ethical approval for research on which this manuscript is based was granted by the research and research ethics integrity committee, school of social and political science, university of edinburgh on 14 september 2016 and by the office of the sierra leone ethics and scientific review committee on 27 september 2018. sources of support this project has received funding from the european research council under the european union’s horizon 2020 research and innovation programme under grant agreement no. 715450. the kings sierra leone partnership provided support in hosting and organising the workshop on which this opinion piece is based. ann h. kelly is funded by the national institute of health research (nihr) global health research unit on health system strengthening in sub-saharan africa, king’s college london (ghru 16/136/54) using aid from the uk government. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views expressed in this publication are those of the authors and do not directly represent the views of any funding or support agencies. references world health organization. emergency use and assessment listing procedure (eual) for candidate in vitro diagnostics (ivds) [homepage on the internet]. 2014; p. 1–9. available from: https://www.who.int/diagnostics_laboratory/eual/emergency/en/%0a biava m, colavita f, marzorati a, et al. evaluation of a rapid and sensitive rt-qpcr assay for the detection of ebola virus. j virol methods. 2018;252(november 2017):70–74. https://doi.org/10.1016/j.jviromet.2017.11.009 broadhurst mj, kelly jd, miller a, et al. reebov antigen rapid test kit for point-of-care and laboratory-based testing for ebola virus disease: a 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systems: a proposal for a resilience index. bmj [serial online]. 2017;357(may):1–8. https://doi.org/10.1136/bmj.j2323 article information authors: atef m. el-gendy1 momtaz o. wasfy1 adel m. mansour1 buhari oyofo1 marwa m. yousry2 john d. klena1,3 affiliations: 1united states naval medical research, egypt2central public health laboratories, egypt 3united states centers for disease control and prevention, china correspondence to: atef el gendy postal address: united states naval medical research unit no.3, psc 452 box 5000, fpo, ae, 09835-0007, usa dates: received: 02 apr. 2012 accepted: 25 oct. 2012 published: 26 july 2013 how to cite this article: el-gendy am, wasfy mo, mansour am, oyofo b, yousry mm, klena jd. heterogeneity of campylobacter species isolated from serial stool specimens of egyptian children using pulsed field gel electrophoresis. afr j lab med. 2013;2(1), art. #34, 9 pages. http://dx.doi.org/10.4102/ ajlm.v2i1.34 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. heterogeneity of campylobacter species isolated from serial stool specimens of egyptian children using pulsed field gel electrophoresis in this original research... open access • abstract • introduction • research method and design    • campylobacter isolates    • antibiotic susceptibility    • pulsed-field gel electrophoresis • results    • exposure determined by pulsed-field gel electrophoresis analysis    • recovery of pre-diarrhoeal to diarrhoeal isolates    • recovery of diarrhoea to shedding isolates    • only active diarrhoea observed • ethical considerations • discussion • acknowledgments    • competing interest    • authors’ contributions    • copyright assignment statement • references abstract top ↑ background: the genus campylobacter spp. is a common cause of human acute bacterial enteritis and travellers’ diarrhoea worldwide. objective: to determine whether multiple serial isolations of campylobacter spp., when obtained from a single child, represented the same or a different organism. methods: in a birth cohort study conducted in egypt, numerous children showed serial isolations of campylobacter spp. of these, 13 children were selected from different households based on the successive isolation of six or more campylobacter isolates from stool samples. results: eighty isolates were recovered and identified as either campylobacter coli (n = 25) or campylobacter jejuni (n = 55). pulsed-field gel electrophoresis (pfge) revealed the presence of 38 unique c. jejuni and 24 c. coli profiles at a similarity level of ≥ 90%. although no serially-identical isolates were detected in six children, others demonstrated at least one identical couple of isolates; all identified serially between one to six weeks. two children demonstrated > 80% similar couples of isolates that appeared seven months apart. pfge could be a useful tool for differentiating reinfection, relapse and convalescent excretion phases. conclusion: our data suggest that campylobacter infection in children is a complex process; children are exposed to multiple species in endemic environments and strains of the same bacterium appear to be shed serially between one to six weeks after the first exposure. isolates that persisted for longer periods were relatively less similar, as shown from the results of this study. introduction top ↑ the genus campylobacter comprises a group of closely-related gram-negative bacteria which primarily colonise the gastrointestinal tracts of a wide variety of host species.1 campylobacter jejuni and campylobacter coli are common causes of human acute bacterial enteritis worldwide.2 in egypt, the disease causes severe problems, with both acute and chronic clinical manifestations in infants aged less than two years, and is a known cause of travellers’ diarrhoea.3,4 investigations have been complicated by the growth requirements of the bacteria, differences in pathogenic mechanisms and the possibility for molecular variability, constituting a substantial challenge for many scientific disciplines.5 pulsed-field gel electrophoresis (pfge) has been used for the molecular subtyping of campylobacter species and is capable of intraspecies differentiation.6 in developed countries, most campylobacter infections result from a unique exposure; reports of repeated infections are uncommon. the purpose of this study was to assess whether multiple serial isolations of campylobacter spp., when obtained from a single child, represented the same or a different organism. we used pfge analysis on a unique set of campylobacter spp. isolates collected during a birth cohort study performed in the nile delta region of egypt to answer this question. participating children had diarrhoeal and non-diarrhoeal stool samples collected over a two-year period and the microbiological content of these samples was investigated. we selected a set of children who had a minimum of six successive campylobacter spp. isolations during this time, and we examined these isolates by pfge to determine if the isolates were the same species and the same pulsetype, based on smai restriction enzyme digestion.7 research method and design top ↑ campylobacter isolates isolates used in this study were collected from children participating in a birth cohort study conducted in a rural district of the nile delta, egypt, between january 2004 and april 2007. overall, 13 children showing six or more campylobacter isolates with at least one diarrhoeal episode over a period of two years were chosen randomly for this study. the children resided in five different villages and had a median age of eight months (range 3–14 months) at first isolation of campylobacter spp., and none were bottle-fed. stool samples were generally collected every other week if no diarrhoea was reported. when diarrhoea was reported, two rectal swabs and a stool specimen were obtained. campylobacter colonies were recovered from skirrow’s medium and confirmed by colonial morphology and gram stain, as well as oxidase and catalase activity.8,9 speciation was performed using hippurate hydrolysis (positive for c. jejuni and negative for c. coli isolates)8,10 and multiplex polymerase chain reaction (mpcr) based on the nucleotide sequence of the lipid a gene, lpxa.11 antibiotic susceptibility antibiotic susceptibility measurements were made using the epsilometer test (etest) (ab biodisk, solna, sweden) according to the manufacturer’s instructions. etests were used to determine the minimum inhibition concentrations (mic) of isolates against ciprofloxacin and erythromycin. suspensions of the organisms were prepared in 5 ml of mueller-hinton broth and adjusted to a turbidity of 1 mcfarland standard.12,13 the antibiotic susceptibility tests were performed from the adjusted inocula.9 standardised bacterial suspensions were used to inoculate mueller-hinton agar supplemented with 5% defibrinated sheep blood. excess moisture was allowed to be absorbed for about 10 to 15 minutes. when the surface of each plate had dried, etest strips were placed aseptically on the plates (using sterile forceps). the plates were incubated within 15 minutes of applying the etest strips in microaerophilic conditions (5% o2, 7.5% co2, 7.5% h2, 80% n2) by using campypak microaerophilic system envelopes (becton, dickinson and company, loveton circle sparks, md, usa) at 37 °c for 48 hours. mics were read directly from the etest strip at the point where the elliptical zone of inhibition intersects the mic scale on the strip. quality control strains (c. jejuni atcc 33291 and c. coli atcc 33559) were tested exactly the same as the clinical isolates14. mics and disk-diffusion breakpoints for antibiotic resistance and susceptibility of campylobacter isolates were determined according to table 1 as described previously.14 table 1: breakpoints for antibiotic susceptibility and resistance. figure 1: smal macrorestriction profiling of c. jejuni isolates by pulsed-field gel electrophoresis. pulsed-field gel electrophoresis intact deoxyribonucleic acid (dna) was prepared from isolates from each child and digested as described previously7 using the u.s. centers for disease control and prevention (cdc) pulsenet protocol for foodborne disease surveillance7 using a chef mapper (bio-rad, ca, usa). agarose gels were stained with ethidium bromide (50 µg/ml) and dna band patterns were viewed by ultraviolet (uv) illumination. gel images were captured and analysed using the bionumerics software package (version 5.10; applied maths, austin, tx, usa). similarity between pulsetypes was calculated using the dice coefficient with a 2% tolerance for the band migration distance and clustering was performed using the complete linkage method (furthest neighbour) that considers the distance between any two given clusters as being the maximum distance between these clusters to reveal straggly clusters or outliers. this analysis method is considered to be a more strict interpretation of banding patterns than the unweighted pair group method using arithmetic averages (upgma).7 results top ↑ we examined 87 campylobacter single-colony isolates collected from 13 children; 12 children showing six separate campylobacter isolations, whilst 15 campylobacter isolates were recovered from the remaining child over a period of 13 months. of these isolates, seven were mixed c. jejuni and c. coli and were eliminated from further analysis.amongst the 13 children, nine demonstrated diarrhoea due to c. jejuni (child 1, 2, 3, 4, 5, 7, 8, 9, 13), three due to c. coli only (child 6, 10, 12), and one child had episodes resulting from infection with both campylobacter species (child 11) (figures 1–5). the number of diarrhoeal episodes varied amongst the children, with six showing a single episode (child 1, 2, 5, 6, 8, 12) and three showing two episodes (child 4, 7, 10), whilst two had three episodes (child 9, 13) or even four (child 3, 11). although no serially-identical isolates were detected in six children (child 1, 2, 4, 6, 8, 10), others demonstrated at least one identical couple of isolates (child 3, 5, 7, 9, 11, 12, 13), all identified at one to six week intervals from each other. two children demonstrated ≥ 80% similar couples of isolates (child 6, 8) that appeared seven months and one year apart, respectively. child 7, 9, 11 and 12 had only five unique isolates, since each had one identical serial isolate that was recovered after one to six weeks. only three children had more than one pair of indistinguishable campylobacter isolates associated with their stools (child 3, 5, 13). confirmation of campylobacter spp. isolates was conducted with the lpxa mpcr; 55 c. jejuni and 25 c. coli species were identified. comparing phenotypic and genetic methods, seven c. jejuni isolates (8%) failed to hydrolyse hippurate and would have been misclassified using phenotypic methods alone. most diarrhoeal episodes were due to c. jejuni (19/24, 79%); all six campylobacter isolates recovered from two children (child 3, 9) were c. jejuni and at least one c. jejuni isolate was recovered from the remaining 11 children, who also had at least one c. coli isolate recovered, although no child was exclusively positive for only c. coli. excluding the seven mixed infections, 86% (69/80) of the campylobacter isolates were resistant to ciprofloxacin (cip), an antibiotic often selected for treatment of campylobacteriosis.15 only 5% (n = 3/55) of the c. jejuni isolates were susceptible to ciprofloxacin; however 32% (n = 8/25) of c. coli were susceptible. all isolates were susceptible to erythromycin. table 2 shows the results of the susceptibility testing. table 2: susceptibility testing of campylobacter isolates. figure 2: smal macrorestriction profiling of c. coli isolates by pulsed-field gel electrophoresis. because stool samples were collected from children routinely over a two-year period, we were able to look at whether a single campylobacter isolate persisted in a child, or if that child was colonised (infected) by multiple isolates. we utilised the discriminatory power of pfge to assess the relationship of isolates recovered from each child (figures 1–5), as well as to provide an overall picture of the campylobacter population associated with the children (figures 1 and 2). we grouped the children into four categories, namely, (1) children exposed to multiple species and strains of campylobacter spp., (2) pre-diarrhoeal to diarrhoeal, (3) diarrhoeal-shedding and (4) active infection only. exposure determined by pulsed-field gel electrophoresis analysis using pfge at ≥ 90% similarity level, it was found that six children had six unique (dissimilar) campylobacter isolates, (for example, the six isolates recovered from child 1) (isolates 1–6; figure 3) were unique. the first isolate was recovered when child 1 was eight months old and the last at 16 months. child 1 was colonised by both c. jejuni (n = 3) and c. coli (n = 3) during this eight-month period and all of the isolates had distinguishable smai pfge profiles. only isolate 6 (c. jejuni) was associated with diarrhoea. the same pattern was observed in child 2, 4 and 8. child 6 and 10 showed the same pattern, but demonstrated diarrhoea due to c. coli. the data suggest that children are exposed to a wide range of strains of c. coli and c. jejuni.in child 2, diarrhoea was associated with only one c. jejuni isolate (isolate 12) that was cip-resistant and 64% similar to a forerunner (isolate 11), but appeared three months earlier. all c. coli isolates were recovered from that child without symptoms at one to three month intervals and exhibited 28% – 64% similarity. in child 6 (figure 3), diarrhoea was associated with a single c. coli isolate (isolate 32) that was 83.3% similar to another (isolate 31) that had been identified four days earlier and was 21.1% similar to a third (isolate 33) recovered 10 days later. child 8 (figure 4) demonstrated one diarrhoeal episode due to c. jejuni (isolate 44), whilst disease in child 10 was associated with one c. coli isolate (isolate 58) that was 32% similar to another c. coli successor (isolate 59) isolated 28 days later. recovery of pre-diarrhoeal to diarrhoeal isolates of the six isolates recovered from child 7 (figure 4), two (isolate 38, 40) were associated with diarrhoea. both isolates were c. jejuni and were recovered just over two weeks apart (24 july 2005 and 10 august 2005); however, these isolates were readily distinguishable by pfge analysis (figure 4). a third c. jejuni isolate (isolate 39), recovered on 7 august 2005 from a routine (non-diarrhoeal) sample, was indistinguishable from isolate 40. the scenario suggested by the pfge patterns is that after infection by isolate 38, child 7 was re-colonised by a new strain of campylobacter (isolate 39) that was also able to cause an infection that resulted in diarrhoea (recovery of isolate 40). prior to and after these c. jejuni infections, c. coli was recovered from child 7 (isolate 37, 10 july 2005 and isolate 41, 4 september 2005, respectively). interestingly, the final isolate from child 7 was a mixed infection (isolate 42, 18 september 2005). it is unclear from the pfge profile whether this mixed infection was two previously-documented isolates, new isolates, or a combination of old and new isolates. pfge results from child 7 (figure 4) indicate a complicated microbiological milieu, since six campylobacter isolates were recovered from this child over a three-month span. the first, isolate 37 (c. coli), was not associated with diarrhoea, but isolate 38 (c. jejuni), recovered 14 days later, was. recovery of diarrhoea to shedding isolates for child 5, three c. jejuni and three c. coli isolates were recovered over a 12-month period (figure 3). after initial recovery of a c. jejuni isolate (isolate 25) that was not associated with diarrhoea, a second distinguishable c. jejuni isolate (isolate 26), associated with diarrhoea, was recovered three months later. isolate 26 had an indistinguishable pulsetype from isolate 27, a non-diarrhoea-associated c. jejuni recovered one week later. one distinguishable c. jejuni and three distinct c. coli isolates were recovered prior to and after the campylobacter-associated diarrhoea caused by isolate 26. in addition, the recovery of indistinguishable isolates 29 and 30 two weeks apart (figure 3) highlights the possibility of carriage of campylobacter spp. for extended periods of time, although not necessarily associated with diarrhoea. a similar story is presented by child 13, from whom 15 campylobacter isolates were recovered. this child demonstrated 11 serial isolates of c. jejuni (figure 5). of these, two identical strains (isolate 74, 75) were recovered at an interval of two weeks, but did not cause diarrhoea. two months later, two diarrhoeal episodes occurred as a result of infection by an identical couple of isolates (isolate 78, 81) that persisted for four weeks without diarrhoea (isolate 82, 83). three months later, two identical strains (isolate 84, 85) persisted for two weeks with 41.4% similarity to the preceding two clusters (isolate 74, 75, 78, 81, 82, 83). child 13 continued to shed isolate 81 for nearly a month (along with recovery of indistinguishable isolates 82 and 83 up until 24 november 2005), after which time we no longer recovered this pulsetype. the data from these two children suggest that c. jejuni can be shed for at least one week and for as long as six weeks after active infection. in a campylobacter-endemic environment, it also appears that multiple colonisation events, involving different species and strains, can take place during this time. child 12 continued to shed c. jejuni isolate 67 for nearly six weeks (recovery of indistinguishable isolates 67 and 69, 24 march 2005); however, this child only demonstrated diarrhoea resulting from c. coli infection (isolate 71), recovered 9 months later. figure 3: smal macrorestriction profiling of campylobacter spp. isolates from (a) child 1, (b) child 2, (c) child 3, (d) child 4, (e) child 5, (f) child 6. only active diarrhoea observed child 3 (figure 3) had five c. jejuni isolates associated with diarrhoea (isolate 14, 15, 16, 17, 18), with two pairs (isolates 14 and 15, 16 and 17) being indistinguishable from each other by pfge analysis. in each case, the respective pairs were recovered less than a week apart (11 september 2005 and 18 september 2005; 21 september 2005 and 25 september 2005). c. jejuni was also recovered prior to the first diarrhoeal episode (isolate 13, 13 july 2005), but this isolate was distinguishable from all of the other recovered isolates from child 3. additionally, isolate 18, recovered after isolate 17 (12 october 2005), was also associated with diarrhoea. isolate 18 was also distinct from all other child 3 isolates.likewise, the remaining two children (child 9, 11) had multiple isolates associated with diarrhoea and whilst we were able to recover campylobacter spp. after these diarrhoeal episodes, we could not recover an isolate with a pulsetype that was distinguishable from the diarrhoeal cause. in child 11, a single diarrhoeal episode resulted in the recovery of two indistinguishable c. jejuni isolates (isolate 64, 65; figure 4). however, these isolates were distinct from two campylobacter-associated diarrhoea isolates (isolate 62, 63), both of which were c. coli. pfge analysis also suggests that isolates 62 and 63 were distinct; indicating that child 11 had three consecutive campylobacter-associated diarrhoeas involving two species and three unique isolates. whilst a final c. jejuni was recovered from child 11 after the diarrhoea caused by isolates 64 and 65, this isolate had a unique pfge pattern. child 9 was unique in that only c. jejuni (n = 6) was recovered from this child who showed three successive episodes (isolates 50–52; cip-resistant). ethical considerations top ↑ this study was supported by the u.s. department of defense (dod) global emerging infections system (geis), dod 31934 namru3. 2003. ethical considerations and informed consent forms were reviewed and approved by both the institutional review board (irb) of the us naval medical research unit no. 3 and the egyptian ministry of health in compliance with all federal regulations governing the protection of human subjects (irb protocol no.145, step d, titled: ‘natural immunity to etec infections in egyptian infants and children’. voluntary participation was adopted, with freedom to withdraw. no major lapses were observed. informed consent was obtained from parents or legal guardians of minors. no hazards were entailed in subject participation, but patients, physicians and the community could benefit from the results with regard to disease treatment, diagnosis and control. before sending samples to the laboratory, the privacy and confidentiality of the participating subjects were maintained by removing any direct personal identifiers and replacing them with codes (letters and numbers), not derived from or related to the personal information. the original identifiers were maintained securely and only traced back to the source at the physician’s discretion for treatment or control purposes. figure 4: smal macrorestriction profiling of campylobacter spp. isolates from (a) child 7, (b) child 8, (c) child 9, (d) child 10, (e) child 11, (f) child 12. figure 5: smal macrorestriction profiling of campylobacter spp. isolates from child 13. discussion top ↑ worldwide, thermotolerant campylobacter species are amongst the most common causative agents of gastroenteritis.11,16 the main sources of infection include the consumption of unpasteurised milk, undercooked poultry or beef and contaminated water.17 in a two-year cohort study conducted in egypt, many children showed serial isolations of campylobacter spp., with at least one diarrhoeal episode (range=1–4 episodes per child), which may reflect the varying degrees of pathogenicity amongst campylobacter isolates as well as the limited role of cross-protective mechanisms. this investigation attempted to explore whether these isolates represented the same or different infections. previous studies have demonstrated age-related immunity, with a decrease in the case-to-infection ratio, absence of symptoms and shorter convalescent-phase excretion.18,19 this may or may not be supported by the observation that the enrolled children had a median age of eight months (range 3 months – 14 months) at first isolation of campylobacter, whilst the study had lasted for two years. the species of all campylobacter isolates were confirmed by the lpxa mpcr. we found that seven c. jejuni isolates detected by this method were negative using the hippurate hydrolysis test, which may be due to the variable expression levels of the n-benzoylglycine amidohydrolase (hippuricase) gene.20 this finding supports the use of a genetic-based approach for the rapid and accurate identification of thermotolerant campylobacter.8,21 c. jejuni isolates were the most commonly-recovered campylobacters22,23 and were most frequently associated with diarrhoeal episodes (79%) amongst the 13 children. however, c. coli was responsible for diarrhoea in only three children, suggesting that although not as dominant as c. jejuni, the opportunity for environmental exposure to c. coli is significant in children in the nile delta. globally, c. jejuni has been reported to cause more than 90% of campylobacter infections,22,24 regardless of the severity of the clinical picture.18 to define the relationship amongst the campylobacter isolates more accurately overall and from each individual child, isolates were pulsetyped using a protocol developed by pulsenet international that is specific for campylobacter testing.7 the restriction enzyme, smai, was used since other options have been shown to provide less reproducible results.25 analysing the profiles obtained per child, it appears that identical c. jejuni isolates (96% – 100%) were recovered frequently during diarrhoea episodes (as in child 5, 9 and 12) for intervals ranging between one and six weeks, whilst c. coli isolates were identical in only one child (child 5), within two weeks. however, most c. coli isolates were distinguishable, with 50% – 67% pfge band similarity, when recovered at periods from two weeks to seven months. assuming that an episode of diarrhoea may last for an average of one week,26 isolation of an identical strain after longer periods without diarrhoea may be regarded as being a convalescent-phase excretion.27,28,29 for instance, in child 13, indistinguishable c. jejuni isolates were recovered up to nine weeks after a diarrhoeal episode (isolates 78 and 81–83). this is longer than reported earlier,3 which marked the presence of convalescent-phase excretion for only four weeks. however, at least one other study reported the shedding of campylobacter isolates for more than nine months post-infection.27 in our study, child 6 showed isolates with > 83% similarity that were identified seven months post-infection. reinfection with c. jejuni spp. was observed in child 3, 4, 7 and 9, who developed diarrhoeal episodes in association with weakly-related isolates (< 33%, 42%, 31%, and 36% band-matching, respectively) that appeared after variable periods of shedding, ranging from three days to 18 months. child 11 was similar, but four consecutive diarrhoeal episodes occurred due to both c. jejuni and c. coli isolates (36% similarity), with one to two week intervals between each. in child 13, two identical c. jejuni cip-resistant isolates were recovered from diarrhoeal episodes that took place nine weeks apart (september 2005 and 24 november), which may be regarded as recrudescence. this observation may be attributed to the lack of an adaptive immune response or resistance to antibiotic treatment.30,31 prolonged campylobacter diarrhoeal attacks have been reported to last for about 12 days only and were associated with cip-resistant campylobacter.32 three children (child 3, 7, 9) showed isolates identical to those causing diarrhoea, but at an earlier time before disease (10 days, three days and one week, respectively), which may be explained by the presence of a colonisation phase during the incubation period.33 it is also possible that pre-shedding of distinguishable campylobacter strains is an in vivo predisposition through mutations at multiple sites (13 contingency loci) to augment bacterial virulence and speed up adaptation to the host.34,35 for instance, our pfge results from child 7 show that two indistinguishable c. jejuni isolates (isolates 39 and 40) were distinct from an earlier isolate (isolate 38), suggesting that isolate 38 was rapidly replaced or dominated by the more invasive c. jejuni isolate 39, which resulted eventually in a second diarrhoeal episode. overall, this study identified 80 campylobacter spp. isolates from 13 children showing six serial isolations during a two-year study. the bacteria comprised 38 unique smai-pfge profiles of c. jejuni and 24 profiles of c. coli at a similarity value of > 90% as described previously.36 this supports an immense genotypic diversity amongst campylobacter species, an observation that has been attributed to the occurrence of interand intra-strain genetic exchanges during infection.28 some children showed reinfection after variable periods from the first episode, whilst relapse was noted after seven weeks. additional studies using multiple isolates from the same subject are required to advance our understanding of campylobacter infection and immunity. our data suggest that campylobacter infection in children in endemic environments is a complex process; when children are exposed to multiple species and strains of campylobacter, the same bacterium appears to be shed serially for between one to six weeks after the first exposure. isolates that persisted for longer periods were relatively less similar as shown from the results of this study. further detailed evaluation of the epidemiology and the isolates associated with diarrhoea and from non-diarrhoeal periods will be necessary in order to determine risk factors and mechanisms to prevent or control re-exposure of children in these settings. acknowledgments top ↑ we are highly appreciative of the expertise of mr abdel hakam abdel fattah in the recovery and identification of campylobacter spp. isolates. also, we value the support of dr rania abdel khalek in analysing the pfge results. competing interest the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. authors’ contributions a.m.g. (namru-3) carried out microbiology and molecular studies and wrote the manuscript. m.o.w. (namru-3) performed data analysis, results interpretation and helped in the writing of the manuscript. a.m.m. (namru-3) coordinated the selection of study subjects and obtained the consent forms and samples. b.a.o. (namru-3) performed critical revisions of the manuscript, made conceptual contributions and provided final approval for publication. m.m.y. (central public health laboratories) participated in most of the laboratory experiments and j.d.k. (namru-3) was the project leader. copyright assignment statement el-gendy a, wasfy m, mansour a and oyofo b are employees of the u.s. government. this work was prepared as part of our official duties. title 17 u.s.c. §105 provides that ‘copyright protection under this title is not available for any work of the united states government.’ title 17 u.s.c. §101 defines a u.s. government work as a work prepared by a military service member or employee of the u.s. government as part of that person’s official duties. references top ↑ 1. blaser mj. epidemiologic and clinical features of campylobacter jejuni infections. j infect dis. 1997;176 suppl 2:s103–105. http://dx.doi.org/10.1086/513780, pmid:9396691 2. wasfy mo, oyofo ba, david jc, et al. isolation and antibiotic susceptibility of salmonella, shigella, and campylobacter from acute enteric infections in egypt. j health popul nutr. 2000;18(1):33–38. pmid:11014768 3. rao mr, naficy ab, savarino sj, et al. pathogenicity and convalescent excretion of campylobacter in rural egyptian children. am j epidemiol. 2001;154(2):166–173. http://dx.doi.org/10.1093/aje/154.2.166, pmid:11447051 4. putnam sd, frenck rw, riddle ms, et al. antimicrobial susceptibility trends in campylobacter jejuni and campylobacter coli isolated from a rural egyptian pediatric population with diarrhea. diagn microbiol infect dis. 2003;47(4):601–608. http://dx.doi.org/10.1016/s0732-8893(03)00154-8 5. praakle-amin k, roasto m, korkeala h, et al. pfge genotyping and antimicrobial susceptibility of campylobacter in retail poultry meat in estonia. int j food microbiol. 2007;114(1):105–112. http://dx.doi.org/10.1016/j.ijfoodmicro.2006.10.034, pmid:17182145 6. ono k, kurazono t, niwa h, et al. comparison of three methods for epidemiological typing of campylobacter jejuni and c. coli. curr microbiol. 2003;47 (5):364–371. http://dx.doi.org/10.1007/s00284-002-4037-6, pmid:14669910 7. ribot em, fitzgerald c, kubota k, et al. rapid pulsed-field gel electrophoresis protocol for subtyping of campylobacter jejuni. j clin microbiol. 2001;39(5): 1889–1894. http://dx.doi.org/10.1128/jcm.39.5.1889-1894.2001, pmid:11326009, pmcid:88044 8. nakari um, puhakka a, siitonen a. correct identification and discrimination between campylobacter jejuni and c. coli by a standardized hippurate test and species-specific polymerase chain reaction. eur j clin microbiol infect dis. 2008;27(7): 513–518. http://dx.doi.org/10.1007/s10096-008-0467-9, pmid:18317822 9. saenz y, zarazaga m, lantero m, et al. antibiotic resistance in campylobacter strains isolated from animals, foods, and humans in spain in 1997–1998. antimicrob agents chemother. 2000;44(2):267–271. http://dx.doi.org/10.1128/aac.44.2.267-271.2000, pmid:10639348, pmcid:89669 10. morris gk, el sherbeeny mr, patton cm, et al. comparison of four hippurate hydrolysis methods for identification of thermophilic campylobacter spp. j clin microbiol. 1985;22(5):714–718. pmid:3902875, pmcid:268512 11. klena jd, parker ct, knibb k, et al. differentiation of campylobacter coli, campylobacter jejuni, campylobacter lari, and campylobacter upsaliensis by a multiplex pcr developed from the nucleotide sequence of the lipid a gene lpxa. j clin microbiol. 2004;42(12):5549–5557. http://dx.doi.org/10.1128/jcm.42.12.5549-5557.2004, pmid:15583280, pmcid:535264 12. ge b, bodeis s, walker rd, et al. comparison of the etest and agar dilution for in vitro antimicrobial susceptibility testing of campylobacter. j antimicrob chemother. 2002;50(4):487–494. http://dx.doi.org/10.1093/jac/dkf162, pmid:12356792 13. luangtongkum t, morishita ty, el-tayeb ab, et al. comparison of antimicrobial susceptibility testing of campylobacter spp. by the agar dilution and the agar disk diffusion methods. j clin microbiol. 2007;45(2):590–594. http://dx.doi.org/ 10.1128/jcm.00986-06, pmid:17122005, pmcid:1829028 14. gaudreau c, girouard y, ringuette l, et al. comparison of disk diffusion and agar dilution methods for erythromycin and ciprofloxacin susceptibility testing of campylobacter jejuni subsp. jejuni. antimicrob agents chemother. 2007; 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accepted: 07 aug. 2020; published: 23 sept. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction the coronavirus disease 2019 (covid-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) resulted in 7 145 539 confirmed cases and 408 025 deaths by 03 june 2020.1 understandably, almost all attention has been on covid-19, which continues to take lives and stretch healthcare systems around the world. a critical issue receiving much less attention during this pandemic is the effect it could have on antimicrobial resistance (amr). with no proven therapy for covid-19, prescribers are more likely to use antibiotics indiscriminately for treatment and prevention of presumed bacterial co-infections. in high-income settings, where bacterial cultures can be done on a timely basis, such antibiotic regimens can be stopped within 48 hours. however, in africa, unneeded empiric antibiotic regimens are likely to be continued for a longer period of time due to lack of bacterial culture and antimicrobial susceptibility testing (ast) capabilities. in addition, the widespread use of hand sanitizers and antimicrobial soaps could exacerbate amr among healthcare workers and the general population. in this article, we discuss how the covid-19 pandemic could affect the already precarious amr situation in africa and discuss ways to minimise the effects on public health. antimicrobial resistance, a global menace antimicrobial resistance occurs when microbes change and render antimicrobial agents ineffective. this phenomenon is fuelled by overuse and misuse of antimicrobial agents in humans and animals. antimicrobial resistance leads to treatment failures, prolongs hospital stays, worsens clinical outcomes and makes surgical procedures and chemotherapy risky and unsafe. with increasing antibiotic resistance and few new antimicrobial agents in the production pipeline, it is imperative to monitor the epidemiology of bacteria species, especially respiratory pathogens to inform treatment decisions2,3 in the era of covid-19. it has been estimated that amr could lead to 10 million deaths by 2050 if nothing is done about the menace, and most of the deaths are likely to occur in asia and africa.4 actions such as infection prevention and control, antibiotic stewardships, capacity building (laboratory infrastructure and personnel) and surveillance are necessary to ameliorate the impact of amr. to support a global action plan on amr, the world health organization has developed the global antimicrobial resistance surveillance system to provide a standardised approach for collection, analysis and sharing of data related to amr to inform decision-making at national and international levels; surveillance of amr bacteria in humans and animals across the globe is key in tackling the problem of amr.5 unfortunately, this is hampered by the limited data from africa due to limited diagnostic microbiology infrastructure on the continent.6 coronavirus disease 2019, secondary bacterial infection and antimicrobial resistance viral respiratory infections can be complicated with secondary bacterial infections, commonly with streptococcus pneumoniae, haemophilus influenzae and staphylococcus aureus, resulting in increased severity and mortality.7 the majority of deaths from the 1918 influenza pandemic resulted from secondary bacterial infections.8 during the 2009 swine influenza pandemic, there was an increase in hospital pneumonia cases as a result of secondary bacterial pneumonia, which resulted in 29% – 55% of deaths.9 in a recent study on the outcome of covid-19 patients from the wuhan region of china, half of the non-survivors had secondary infections.10 of note, the majority (95%) of the patients in the study were given antibiotics.10 azithromycin, a broad-spectrum macrolide antibiotic (in combination with hydroxychloroquine) has become a common treatment for covid-19 patients in several parts of the world including africa11,12 without much evidence to support its use. it is worth noting that the world health organization has warned against indiscriminate use of antibiotics during covid-19 treatment.2 while antibiotics may not affect covid-19 directly, they are agents administered to prevent or treat secondary bacterial infections in covid-19 patients. therefore, the swell in the numbers of covid-19 patients could lead to the inappropriate use of antibiotics and subsequent selection of amr bacteria. in previous epidemics, there were indications of an increase in methicillin resistant s. aureus infections in hospitals, which was linked to heavy use of antimicrobial agents.13,14 it is therefore possible that covid-19 patients could also be battling with amr bacteria, in addition to the virus, resulting in poor patient outcomes. urgent studies are needed to define the bacteria aetiologies and amr bacteria that contribute most to mortality in covid-19 patients. coronavirus disease 2019, the environment and antimicrobial resistance from the onset of the pandemic, the general public has been constantly directed to regularly wash hands with soap and water, and use hand sanitizers. although such practices will help improve hygiene standards and reduce the spread of covid-19, they could have negative effects on amr. disinfectants and antimicrobial soaps contain biocides or antimicrobials. an increase in use of these agents may lead to an increase in their concentration in waste water and receiving water bodies; this may have a potentially negative impact, because the elevated concentrations may lead to selection of amr bacteria, posing a health risk to persons exposed to such environments.15 more research in africa is needed to determine how the widespread use of antimicrobial sanitizers and soaps affects the microbiome and contributes to amr. antimicrobial resistance in africa in the era of coronavirus disease 2019 the world health organization reports that there is limited data on amr prevalence from africa due to limited laboratory capacity and surveillance networks.6,16 a survey indicated that very few countries in africa have functional national surveillance systems for amr of common bacterial infections from community and hospitalised patients.17 an external quality assessment report indicated poor performance of ast in several african countries.18 quality assurance in ast is key for reporting and implementation of the global antimicrobial resistance surveillance system19; laboratories in africa have to be supported with the needed investments for better performance. with the paucity of data on amr, realistic evidence on how amr may compromise first-line empirical treatment in common bacterial infections is lacking on the continent.16 in the absence of microbiology and amr data, prescribers often manage clinical symptoms rather than specific bacteria,16 a situation that may fuel inappropriate use of antimicrobials and emergence of resistant microbes. in ghana, like most african countries, the majority of microbiology laboratories do not have the capacity to perform culture and ast tests.20 for the few laboratories that are able to do culture and ast, performing such tests in a standard way is a challenge often due to a lack of reference strains and up-to-date standard interpretation guidelines. in addition, culture of bacteria and ast are often not requested by clinicians, because of cost to patients and the long turn-around time that often render the results useless for patient care. altogether, data on amr bacteria to guide treatment decisions at local and national levels are scarce. newman et al.20 conducted the first nationwide amr surveillance in ghana between 2002 and 2003 and found bacteria species resistant to ceftriaxone (6.3%) and ciprofloxacin (11%).21 opintan et al. (2015), followed up with another nationwide survey between june 2014 and november 2014 and observed that > 50% of the commonly isolated bacteria species were resistant to third-generation cephalosporins and fluoroquinolones. in this study, the majority of the gram-negative bacteria species recovered were positive for extended spectrum beta lactamase; these organisms are resistant to a wide range of antimicrobial agents.22 in another study, a new delhi metallo-beta-lactamase-producing escherichia coli strain resistant to meropenem and belonging to st410 was detected in a urine sample from a hospitalised patient in the northern part of ghana. in this first report, the detected plasmid co-carried other resistance genes.23 this is disturbing, mainly because these organisms are resistant to the antimicrobials used as last-line treatment for severe bacterial infections.24 among female patients who presented with vaginal discharge, dysuria, intermenstrual bleeding or abdominal pain and men who presented with urethral discharge or dysuria, gonococcal isolates recovered from samples collected from five healthcare centres were resistant to tetracycline (100%), benzylpenicillin (91%) and ciprofloxacin (82%). one isolate resistant to cefixime (mic: 0.75 µg/ml) belonged to st1407, a pandemic resistant clone.25 a total of 520 methicillin susceptible s. aureus and 30 methicillin resistant s. aureus isolates were recovered from 1219 nasal swabs (community and hospital carriers) and 916 clinical isolates (blood, skin and soft tissues, wounds) in other studies; methicillin resistant s. aureus isolates detected belonged to global epidemic clones, including usa300.26,27,28,29 our review of more than 20 articles on amr from ghana for this write-up revealed that resistance of bacteria species (recovered from human, food and animals) to commonly used antimicrobial agents such as ampicillin, tetracycline, chloramphenicol and trimethoprim sulfamethoxazole was common. the situation is not different from other parts of the continent.16 the majority of amr data in ghana are from pockets of studies focusing on particular bacteria species recovered in select hospitals or research institutions. they therefore may not reflect the national amr situation and could be an underestimation of the actual magnitude of the amr problem in a country where self-medication is rampant and antimicrobial agents are often available without prescription.30 ghana is gradually gathering momentum to get to a stage of having a functional national system to monitor amr. the antimicrobial use and resistance policy and the national action plan on amr, which provide strategies and plans to guide amr data generation for evidence-based interventions at local, national and international levels, were launched by the president of ghana on 11 april 2018. the national action plan was fashioned along the lines of the objectives of the global action plan on amr.5 ghana also has a national amr working group; the group meets regularly to deliberate on amr issues in the country. to strengthen knowledge and evidence base through surveillance and research, ghana received the first fleming country grant, which is currently supporting a government-led system of collecting, analysing and reporting of amr and antimicrobial use data from humans and animals from 11 sentinel sites. these data will provide a national amr picture to inform treatment decisions at the national level. plans are underway to begin a pilot surveillance study. the prevalence and mortality rate of covid-19 in africa is lower compared to that of europe, america and asia. however, the rapid spread of the virus is another call to strengthen the laboratory diagnostic capacity in africa to perform standard antimicrobial susceptibility testing, especially of relevant respiratory pathogens to inform treatment decisions in healthcare facilities. importantly, ast data need to be collected yearly to support empiric treatment in hospitals. continuous surveillance is required on the african continent and across the globe to understand the epidemiology of amr pathogens, provide the needed data to guide and inform treatment decisions and policies and monitor resistance trends and emergence of new clones. in all of these actions, the role of networks and collaborations in enhancing the success of various interventions cannot be overemphasised. ownership and sustainability plans by governments and local authorities are key to maintaining the successes for continuous amr surveillance activities with local and international partners. capacity building (infrastructure and personnel) is a must-have and must be continuously improved to fight amr in ghana and africa. going forward one cannot blame the african prescribers for throwing the ‘kitchen sink’ of antibiotics at sick and dying covid-19 or other virally infected patients. rather, these practices could be reduced significantly, if physicians were empowered to order cultures and obtain results in a timely manner. doctors faced with negative culture results are more likely to stop antibiotics than if no results are available. with the necessary political will, african governments and academics can do a few things in line with the five strategic objectives of the global action plan on combating amr during the current pandemic and beyond. firstly, there is a need to raise awareness of amr among personnel in human and animal health, and agriculture, as well as among consumers, to ensure a proper understanding of amr pathogens and the impact of amr across sectors. there is an urgent need to build laboratory capacity to generate the required microbiology data through surveillance and research to inform treatment decisions, especially in urban centres where resistant organisms are often abundant. evidence-based prescribing and dispensing should be the way to go to optimise antimicrobial use. more automated systems like the genexpert platform used for tuberculosis could be repurposed for organisms like methicillin-resistant s. aureus. in addition to phenotypic methods used in the detection of resistance, genomic tools such as whole genome sequencing can be utilised to generate extensive data to expand our knowledge on the changing epidemiology of amr bacteria. during the covid-19 pandemic, urgent studies are needed to document the bacterial organisms responsible for co-infections at the local level to guide empiric therapy. due to the widespread use of hand sanitizers and antimicrobial soaps, research is needed to study healthcare workers and others for changes in the skin flora that they carry, which could be transmitted to vulnerable patients. healthcare institutions without antibiotic stewardship programmes must take steps to institute measures for rational antibiotic use. antibiotic stewardship optimises institutional antibiotic use and reduces the selection and spread of amr; similarly, infection prevention needs strengthening across the board to limit the spread of resistant bacteria. finally, the need for increased investment by african governments to drive development of new diagnostic tools, novel antimicrobial agents and vaccines cannot be over-emphasised. lessons and conclusion the amr menace has been described as a problem that knows no borders; resistant bacteria can be found in humans, animals, food and the environment. the current pandemic shows that we remain susceptible to infections for which we have no therapeutic options. the experience from covid-19 therefore should be another reminder of the life-threatening consequences of amr microbes, and the need for rapid capacity building (infrastructure and human resources) for surveillance of resistant bugs on the african continent. acknowledgements the authors of this manuscript are grateful to all other authors whose works were cited. competing interests the authors have declared that no competing interests exist. authors’ contributions b.e. 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2013;1(4):189–193. https://doi.org/10.1016/j.jgar.2013.05.006 donkor es, tetteh-quarcoo pb, nartey p, agyeman io. self-medication practices with antibiotics among tertiary level students in accra, ghana: a cross-sectional study. int j environ res public health. 2012;9(10):3519–3529. https://doi.org/10.3390/ijerph9103519 abstract introduction methods results discussion acknowledgements references about the author(s) tjeerd a.m. datema datos b.v., leiden, the netherlands linda oskam datos b.v., leiden, the netherlands jacqueline e.w. broerse department of science communication, faculty of science, vrije universiteit amsterdam, amsterdam, the netherlands paul r. klatser athena institute, faculty of science, vrije universiteit amsterdam, amsterdam, the netherlands citation datema tam, oskam l, broerse jew, klatser pr. review of the stepwise laboratory quality improvement process towards accreditation (slipta) version 2:2015. afr j lab med. 2020;9(1), a1068. https://doi.org/10.4102/ajlm.v9i1.1068 original research review of the stepwise laboratory quality improvement process towards accreditation (slipta) version 2:2015 tjeerd a.m. datema, linda oskam, jacqueline e.w. broerse, paul r. klatser received: 18 july 2019; accepted: 19 aug. 2020; published: 28 oct. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: in 2011 the stepwise laboratory quality improvement process towards accreditation (slipta) was launched, aimed at strengthening the quality and competence of african clinical, public health and reference laboratories. we reviewed the first version of the slipta checklist in 2011. the continued development and publication of a new version of the international organization for standardization (iso) 15189 standard demands a renewed review. objective: this study aimed to determine the suitability of slipta in guiding laboratories towards iso 15189:2012 compliance and accreditation and provide recommendations for further slipta improvement. methods: the study was conducted between september 2018 and april 2019. coverage of iso 15189:2012 by slipta checklist version 2:2015 was determined and the point distribution of the scoring system over the different sections of the slipta checklist was re-investigated. these findings were compared with the review of the first version of the slipta checklist (based on iso 15189:2007) and with findings published on slipta implementation and roll-out. results: the coverage of iso 15189 by the slipta checklist has increased, even though iso 15189:012 is more extensive than iso 15189:2007. the point distribution is still skewed towards sections related to quality planning rather than quality control and improvement. although to date 314 laboratories have been assessed, barriers for laboratories to participate in slipta are high. sustainability of slipta results is insufficiently studied. conclusion: slipta checklist version 2:2015 has improved compared to earlier versions. we recommend increasing accessibility for laboratories to participate and increasing guidance for iso-based quality management system implementation. keywords: accreditation; iso 15189; laboratory; slipta; slmta; quality assurance; total quality management. introduction in 2011 the stepwise laboratory quality improvement process towards accreditation (slipta) was launched, aimed at strengthening quality and competence of clinical, public health and reference laboratories in the african region.1,2,3,4 the launch of slipta was the result of a series of events. in 2008 the world health organization (who) and the united states centers for disease control and prevention (cdc) organised a conference on laboratory quality systems. one of the recommendations stated that laboratories in resource-limited settings should consider taking a staged approach towards implementation of a quality management system (qms).5,6 in the following years, several resolutions on laboratory strengthening were drafted for the african region.7,8 in 2009, the who regional office for africa (who-afro), in collaboration with cdc and other partners, launched the who-afro laboratory accreditation process and the strengthening laboratory management towards accreditation (slmta) training and mentoring programme.9,10,11,12 in 2011, the who-afro accreditation process was renamed slipta.4,10,13 in 2012, who-afro designated the african society for laboratory medicine (aslm) as the slipta secretariat.4,12,14 the aslm established slipta’s implementation structure consisting of4,14: who-afro slipta focal point, responsible for mobilisation of resources, providing guidance on content and implementation, and reviewing and updating slipta. ministry of health slipta focal point, responsible for -in-country promotion of laboratory improvement through slipta. the ministry of health develops an implementation plan and prioritises slipta applicant laboratories and allocates financial and human resources for slipta implementation. aslm-certified slipta auditors, responsible for conducting audits and providing advice to auditees. slipta independent advisory group, enrolling laboratories into the slipta programme, organising audits and making a final decision on laboratory recognition through awarding a star rating based on audit reports (varying from zero to five stars) (table 1). star ratings are valid for a two-year period.12,14 table 1: stepwise laboratory quality improvement process towards accreditation checklist compliance levels versus star ratings. the aslm aims to enrol 2500 laboratories in slipta and have 250 public laboratories accredited to international standards by 2020.12 not all laboratories can voluntarily participate in slipta because ministries of health are encouraged to select laboratories in phases, considering tiered laboratory networks and giving precedence to laboratories that have already completed laboratory quality improvement training. eligibility criteria include a slipta self-audit score of 55% or higher, participation in proficiency testing schemes or alternative methods in the past 6 months and having conducted internal audits and a management review in the past 12 months. the laboratory should also have documented its qms.14 upon enrolment a laboratory is audited to determine its initial star rating. laboratories are expected to work towards the next star. laboratories that achieve a five-star rating are encouraged to apply for iso 15189 accreditation.14 key in the slipta programme is the slipta checklist, which is primarily based on iso 15189 and, to a lesser extent, clinical and laboratory standards institute (clsi) guideline qms01-a4.3,15 because laboratories with a five-star rating are encouraged to apply for accreditation, it is important to obtain insight into the coverage of iso 15189 requirements by the slipta checklist. this determines how much of the iso 15189 requirements still must be addressed before full compliance with iso 15189 is achieved. in 2011, the authors reviewed the first version of the slipta checklist (published in 2009), referred to as the alpha version,16 and determined coverage of iso 15189:2007.1 in that same year slipta was revised and the slipta alpha version became slipta checklist v1.0.11,12 because a new version of the iso 15189 standard was published in 2012, slipta checklist v2, based on iso 15189:2012, was published in 2015.3,12 the current study determines coverage of iso 15189:2012 by slipta checklist v2:2015 and re-investigates the point division over the different sections of the slipta checklist to determine the relative weight of qms elements in slipta. we also reviewed published slipta implementation data. this article informs potential users about slipta’s suitability to guide laboratories towards iso 15189:2012 compliance and accreditation and provides recommendations for further improvement of slipta. methods ethical considerations ethical clearance was not required for this study. study design the study was conducted between september 2018 and april 2019. the methodology used in this study was adapted from datema et al. 2011.1 the first analysis determined the slipta checklist’s coverage of iso 15189:2012 by linking each question of slipta checklist v2:2015 to iso 15189:2012 clauses. the second analysis provides insight into the point distribution of the scoring system over the different slipta checklist sections. the slipta checklist is divided into 12 sections corresponding with 12 qms elements. for each section, points can be scored, the total of which determines the number of stars awarded. excel 2016 (microsoft, redmond, washington, united states) was used to analyse and compare the number of points that can be scored per section. results were compared with results of the review of the slipta checklist alpha version.1 in datema et al. 2011, the 12 sections of the slipta checklist were divided over the categories ‘resource management’, ‘process management’ and ‘improvement management’. in this article we renamed these categories to ‘quality planning’, ‘quality control’ and ‘quality improvement’, in line with the juran trilogy (table 2).17 the overall intention of each slipta checklist section led the categorisation process, which was identical to the review of the alpha version of the slipta checklist.1 hence, the overall aim of the sections assigned to the juran category quality planning is to ensure quality of work before it is started, that is, before work can be conducted in a quality-assured way, proper organisation and functioning of equipment, purchasing and inventory management processes, good facilities and competent personnel are needed. as such, with the implementation of these elements the laboratory is ‘planning for quality’, justifying the decision for assigning these sections to the juran category quality planning. similarly, the primary, shared objective of the sections on process control, information management, documents and records, and client management is to control quality of work while it is being conducted. therefore, these sections were assigned to the juran category quality control. the sections on management reviews, evaluation and audits, occurrence or incident management and process improvement, and identification of non-conformities, corrective and preventive actions all share the common goal of continuously improving the quality of laboratory work. therefore, these sections were assigned to the juran category quality improvement. table 2: distribution of stepwise laboratory quality improvement process towards accreditation checklist v2:2015 sections over the different categories of the juran trilogy. a pubmed search was conducted on 06 march 2019 to gather literature on outcomes of slipta implementation and roll-out. the search terms were ‘slipta’ or ‘stepwise laboratory improvement process towards accreditation’ and the search yielded 29 hits. after primary and secondary selection based on title and abstract, and identification of additional reports through snowballing, a total of 23 articles were identified. upon further scrutiny 12 papers were excluded because they did not report findings on slipta implementation, roll-out, effectiveness or sustainability per se. finally, 11 papers were included. results changes to stepwise laboratory quality improvement process towards accreditation checklist v2:2015 compared with the alpha version structurally, slipta checklist v2:2015 is very similar to the alpha version. the checklist is still divided over 12 sections based on the quality system essential structure developed by the clsi.15 a notable change is the addition of one very detailed question on the presence and content of 36 specific standard operating procedures (sops). iso 15189:2012 coverage the slipta checklist v2:2015 addresses 82% of iso 15189:2012 clauses, of which 35% are fully addressed, 47% addressed partially and 18% are not addressed at all. this is an improvement compared to the alpha version, which wholly or partially covered 52% of iso 15189:2007 clauses. in some areas slipta checklist v2:2015 is more detailed and prescriptive compared to iso 15189 requirements, whereas in other areas iso 15189 requirements are more extensive than the slipta checklist. point distribution and relative weight (importance) of quality management system elements the total number of points in the slipta checklist v2:2015 has increased from 250 to 275. in most sections a higher number of points can be scored compared to the alpha version. however, the relative weight of each section has remained similar due to the increase in the total number of points (see figure 1). figure 1: relative point distribution of the scoring system over the different sections of stepwise laboratory quality improvement process towards accreditation checklist alpha version and version 2:2015. when the point distribution of the slipta checklist v2:2015 scoring system was analysed using the juran trilogy model, points were still heavily skewed towards quality planning (45% of the weight) and quality control (33%). quality improvement received the lowest number of points (22%) (figure 2). figure 2: relative point distribution of the scoring system of stepwise laboratory quality improvement process towards accreditation checklist alpha version and version 2:2015 over the different categories of the juran trilogy. outcomes of stepwise laboratory quality improvement process towards accreditation roll-out up to 24 april 2019, 314 laboratories had been audited in 20 countries, which is still far below the ambition to enrol 2500 laboratories by 2020 (table 3).18 the percentage of laboratories per star rating is shown in figure 3. the distribution is still in line with data published by ndihokubwayo et al. in 2016, and andiric et al. in 2018.12,19 figure 3: percentage of laboratories per star rating, of a total of 314 laboratories, retrieved from stepwise laboratory quality improvement process towards accreditation database on 24 april 2019.18 table 3: number of laboratories per star level per country on 24 april 2019.18 although many papers have been published on the slmta training and mentoring programme, in which the slipta checklist was used to measure progress, papers evaluating the slipta initiative per se are scarce. the paper by ndihokubwayo et al. (2016) is the only paper summarising slipta implementation and lessons learned.12 two additional papers were found that present slipta implementation findings, but these also combined slipta with additional assistance.20,21 both studies indicated that combining slipta with mentoring has a positive effect on qms implementation, although neither control laboratories nor findings on sustainability of this model were included.20,21 ndihokubwayo et al. found that slipta laboratories performed most poorly on the internal audit and corrective action sections.12 this finding is corroborated by other studies.22,23,24,25,26,27,28 they further state that advocacy for laboratory strengthening is key to the slipta process (as it is owned by the ministry of health) and that particularly francophone and lusophone countries are not well covered. an explanation for the latter finding might be that slipta was initially implemented through the united states president’s emergency plan for aids relief, which is oriented towards anglophone countries.12 discussion slipta checklist v2:2015 has improved compared to the alpha version. even though iso 15189:2012 is more extensive than iso 15189:2007, iso 15189 coverage has increased, decreasing the gap that still needs to be bridged by 5-star laboratories aiming for iso 15189 accreditation. however, the gap is still considerable: only 35% of iso 15189 clauses are fully covered, leaving 47% partially covered and 18% not covered at all. compliance with some of these requirements may be reached as part of the continuous improvement process which 5-star laboratories may have already partially implemented. the main point for slipta improvement remains the absence of prioritisation of qms implementation activities. there are no conditions for the different star ratings (other than the star rating thresholds) that encourage laboratories to implement the qms in a specific, rational manner, indicating that qms implementation guidance remains limited. most slipta checklist v2:2015 questions are supplemented with short notes including examples, but neither a stepwise plan nor a detailed explanation of implementation of requirements is provided, showing that slipta remains primarily an assessment checklist. slipta auditors may provide advice on implementation during assessments4,12 but this might come late, for laboratories may first try to implement a qms before being assessed as laboratories must score at least 55% in self-assessment to meet eligibility criteria for enrolment.14 currently, most points can be scored in slipta checklist sections related to quality planning, followed by quality control. the least number of points can be scored on quality improvement. this creates an imbalance. implementing a qms is a ‘systems approach’: all qms elements work together to create a sustainable system that can deliver quality-assured laboratory services and continuously improve itself. when one qms element is not (correctly) functioning, quality assurance of overall laboratory services and continuous quality improvement may be compromised. therefore, one could argue that slipta should award an equal number of points for each section. on the other hand, the skewed point distribution may point laboratories in the right direction by encouraging them to address sections related to quality planning first because of the high number of points that can be scored in this category, as was also argued by datema et al. in 2011 (although no evidence was found in literature supporting this hypothesis).1 however, this is counterbalanced by the higher amount of work required for implementing the sections related to quality planning. another improvement opportunity for the slipta checklist is the imbalance in the effort required to earn points per question. for example, for question 1.5 one needs to develop 36 sops to earn five points, whereas by developing a list of documents used in the laboratory (question 1.4), making sure that sops are accessible to staff (question 1.6), and indicating date of authorisation, location and date of discontinuation on each sop (question 1.8) one can score a total of six points. writing 36 sops obviously requires considerably more effort. stepwise laboratory quality improvement process towards accreditation implementation and roll-out a strong point is that slipta requires the ministry of health to play an active role; government commitment has been shown to be key to success in both slipta21 and slmta.11,12,20,24,25,27,29,30,31,32,33,34,35 also, slipta can be well combined with other guidance methods for laboratory accreditation as is evident from many studies on slmta implementation.20,22,27,29,30,33,34,35,36 a major downside is the indirect accessibility of the slipta programme: laboratories are selected by the ministry of health for participation. only 27 of the 47 who-afro member states have established a slipta focal point within their ministry of health. moreover, the programme has a language bias towards anglophone countries.12,18,28 also, up to april 2019, 314 laboratories had been assessed, which is low considering that kampala, the capital of uganda, alone counts 954 laboratories18,37 and that aslm aimed to include 2500 laboratories by 2020.12 recommendations for improvement the level of guidance provided by slipta for qms implementation could be increased by making better use of the star rating system. currently, stars are simply awarded based on the number of points scored, regardless of the section these points are scored in. setting certain benchmarks and conditions for the different star ratings may improve guidance. an example could be the definition of key questions that have to be implemented for each star rating. this may help laboratories in using a rational approach towards qms implementation. it may also assist laboratories in lower tiers of laboratory networks, for which iso 15189:2012 is not (yet) feasible, to implement a basic yet functional qms. the phased approach incorporated in the who laboratory quality stepwise implementation (lqsi) tool as well as the tiered approach of the laboratory quality management system stepwise improvement process (lqms-sip) used in the caribbean region could serve as models in assigning key questions to the different star ratings, which would also contribute to harmonisation of slipta with these laboratory strengthening tools and initiatives.38,39,40 sustainability is a challenge for laboratory strengthening efforts. literature on slipta implementation does not provide sufficient clarity on sustainability. slipta assessments, like accreditation assessments in general, are snapshot measurements of laboratory compliance, creating the risk that the efforts may weaken after an assessment, as was also witnessed in slmta evaluations.11,24,32 a possible measure to decrease this risk is announcing assessment visits only shortly before they are scheduled, leaving just enough time for a laboratory to prepare logistics but not for correction of elements that would otherwise not have been corrected, yielding a more representative view of the daily practice. another measure is the adoption of a point scoring system that awards negative points for questions that are not in place anymore compared to the previous assessment. this emphasises the importance of quality assurance and may be an extra driver for the laboratory to ensure continued compliance. in the case of slipta, both measures could be adopted without requiring major revisions as it would primarily require amendment of the audit scoring section of the slipta checklist. it should be noted that slipta checklist v2:2015 already indicates that audit scores should be based on laboratory performance during the 12 months preceding the slipta audit, which is an encouragement for laboratories to maintain compliance.3 the last recommendations relate to implementation and roll-out of slipta: increasing the accessibility of slipta by increasing the capacity, among others through ensuring the presence of slipta focal points at each ministry of health and training of more slipta auditors. this should include training of more slipta auditors fluent in portuguese and french to increase accessibility for laboratories in lusophone and francophone countries. limitations the analysis using the juran trilogy model was limited to categorisation of overall slipta sections. although the authors are aware that the same analysis can be conducted at the individual question level, the decision was made to categorise based on the overall intention of each slipta section and, therefore, categorise each slipta checklist section as a whole as described in the methods section. this was also required to enable comparison with the review of the alpha version of the checklist. this study is a desk-based review. ideally, the findings of this study should be triangulated through an observational study monitoring slipta implementation with a sufficiently large sample size. this would enable substantiation of the findings and may reveal additional opportunities for improvement of the slipta checklist and programme. conclusion slipta checklist v2:2015 has improved compared to the alpha version. suggestions for improvement are mainly related to the point scoring system, including the designation of key questions to specific star ratings to improve the level of guidance for implementation of a qms. the lqsi tool and lqms-sip could serve as examples, leading to harmonisation of slipta with these tools. recommendations for enhanced slipta roll-out include increasing accessibility by translation into french and portuguese, training more auditors, and increasing capacity for participation. acknowledgements competing interests the authors have declared that no competing interests exist. authors’ contributions t.a.m.d. designed and performed the review and wrote the manuscript. l.o., j.e.w.b. and p.r.k. critically reviewed the analysis and the manuscript. all authors approved the present version of the manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability statement data are available upon request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references datema tam, oskam l, van beers sm, klatser pr. critical review of the stepwise laboratory improvement process towards accreditation (slipta): suggestions for harmonization, implementation and improvement. trop med int health. 2012;17(3):361–367. https://doi.org/10.1111/j.1365-3156.2011.02917.x world health organization regional office for africa. laboratory accreditation checklist. brazzaville: who-afro; 2009. world health organization regional office for africa. 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[cited 2019 jul 17]. available from: https://extranet.who.int/lqsi abstract introduction methods results discussion acknowledgements references about the author(s) robert k. langat kenya aids vaccine initiative, institute of clinical research, university of nairobi, nairobi, kenyainternational aids vaccine initiative (iavi), human immunology laboratory, imperial college, london, united kingdom bashir farah kenya aids vaccine initiative, institute of clinical research, university of nairobi, nairobi, kenya jackton indangasi kenya aids vaccine initiative, institute of clinical research, university of nairobi, nairobi, kenya simon ogola kenya aids vaccine initiative, institute of clinical research, university of nairobi, nairobi, kenya gloria omosa-manyonyi kenya aids vaccine initiative, institute of clinical research, university of nairobi, nairobi, kenya omu anzala kenya aids vaccine initiative, institute of clinical research, university of nairobi, nairobi, kenya jean bizimana projet san francisco, kigali, rwanda emmanuel tekirya projet san francisco, kigali, rwanda caroline ngetsa kenya medical research institute centre for geographical medicine research coast, mombasa, kenya moses silwamba zambia emory hiv research project, lusaka, zambia enoch muyanja ugandan virus research institute-iavi, entebbe, uganda paramesh chetty international aids vaccine initiative, johannesburg, south africa maureen jangano clinical laboratory services, johannesburg, south africa nancy hills school of medicine, university of california, san francisco, california, united states jill gilmour international aids vaccine initiative (iavi), human immunology laboratory, imperial college, london, united kingdom len dally emmes corporation, rockville, maryland, united states josephine h. cox clinical trials program, vaccine research center, national institutes of health, bethesda, maryland, united states peter hayes international aids vaccine initiative (iavi), human immunology laboratory, imperial college, london, united kingdom citation langat rk, farah b, indangasi j, et al. performance of international aids vaccine initiative african clinical research laboratories in standardised elispot and peripheral blood mononuclear cell processing in support of hiv vaccine clinical trials. afr j lab med. 2021;10(1), a1056. https://doi.org/10.4102/ajlm.v10i1.1056 note: additional supporting information may be found in the online version of this article as supplementary document 1. original research performance of international aids vaccine initiative african clinical research laboratories in standardised elispot and peripheral blood mononuclear cell processing in support of hiv vaccine clinical trials robert k. langat, bashir farah, jackton indangasi, simon ogola, gloria omosa-manyonyi, omu anzala, jean bizimana, emmanuel tekirya, caroline ngetsa, moses silwamba, enoch muyanja, paramesh chetty, maureen jangano, nancy hills, jill gilmour, len dally, josephine h. cox, peter hayes received: 06 june 2019; accepted: 23 sept. 2020; published: 17 feb. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: standardisation of procedures for performing cellular functional assays across laboratories participating in multicentre clinical trials is key for generating comparable and reliable data. objective: this article describes the performance of accredited laboratories in africa and europe on testing done in support of clinical trials. methods: for enzyme-linked immunospot assay (elispot) proficiency, characterised peripheral blood mononuclear cells (pbmcs) obtained from 48 hiv-negative blood donors in johannesburg, south africa, were sent to participating laboratories between february 2010 and february 2014. the pbmcs were tested for responses against cytomegalovirus, epstein barr and influenza peptide pools in a total of 1751 assays. in a separate study, a total of 1297 pbmc samples isolated from healthy hiv-negative participants in clinical trials of two prophylactic hiv vaccine candidates in kenya, uganda, rwanda and zambia were analysed for cell viability, cell yield and cell recovery from frozen pbmcs. results: most (99%) of the 1751 elispot proficiency assays had data within acceptable ranges with low responses to mock stimuli. no significant statistical difference were observed in elispot responses at the five laboratories actively conducting immunological analyses. of the 1297 clinical trial pbmcs processed, 94% had cell viability above 90% and 96% had cell yield above 0.7 million per ml of blood in freshly isolated cells. all parameters were within the predefined acceptance criteria. conclusion: we demonstrate that multiple laboratories can generate reliable, accurate and comparable data by using standardised procedures, having regular training, having regular equipment maintenance and using centrally sourced reagents. keywords: pbmc processing; peripheral blood mononuclear cells; elispot; clinical trials; good clinical laboratory practice; proficiency testing. introduction clinical trials related to hiv, malaria and tuberculosis have been conducted in africa for many years.1,2,3,4,5,6 to harmonise the immunological data generated from these trials, laboratories responsible for clinical sample processing must establish standardised procedures to meet international conference on harmonization good clinical laboratory practice (gclp) and the world health organization guidelines for collecting, processing, storing and performing of functional assays of samples such as enzyme-linked immunospot assay (elispot), which is used to assess the immunogenicity of vaccine candidates. the international aids vaccine initiative (iavi) has partnered with local institutions and established good clinical laboratory practice (gclp)-compliant laboratories across africa, europe and india to conduct safety and immunogenicity assessments in support of clinical trials of hiv vaccine candidates.7,8 these laboratories are equipped to process and store samples for later testing and can perform elispot and flow cytometry immunological assays. to-date iavi has conducted over 20 phase 1 hiv vaccine trials (https://www.iavi.org/our-work/clinical-epidemiology-research/clinical-research-centres), most of them in africa.9.10,11,12 to ensure uniformity of data, iavi sponsored a central laboratory – the iavi human immunology laboratory (hil) – based at imperial college london to provide standardised operating procedures, technical training, critical assay reagents and long-term storage of samples. additionally, iavi and partners have built the capacity of local personnel both professionally and academically, through technical training, mentoring and funding for investigator-initiated research projects within sites. the iavi gclp-accredited laboratories have been used as reference laboratories by local and international research organisations for training and storage of samples on a short-term basis. cellular functional assays have been used for assessing the immune response to many vaccines.13 in this article, we focus on the interferon-gamma (ifn-γ) elispot assay and report on the results from the elispot proficiency scheme. ifn-γ elispot is a standard assay for measuring the immunogenicity of vaccine candidates such as for hiv and tuberculosis13 and has been utilised by many research groups. although the elispot assay has been used in many clinical trials, it is prone to inherent variability within samples and between operators within and across laboratories.14 these discrepancies could be attributed to different reagents and equipment being used, inadequate training of personnel, lack of proficiency testing schemes and lack of quality management systems. therefore, there is an urgent need to standardise this assay to generate accurate and reliable data across multiple laboratories. previously, it was shown that multiple laboratories reported varied responses in elispot proficiency testing.15 there have been measures put in place to address this shortcoming and recent reports on elispot proficiency have shown incredibly improved results with concordant results between multiple gclp-accredited laboratories.16,17,18 the assessment of ifn-γ producing t-cells in vaccinees by elispot is a standard measure for determining a vaccinee’s immune response to hiv-1 vaccine candidates. in the field of human t-cell immunology, ifn-γ has been widely used as a measure of cd4 and cd8 activation after stimulation with various peptides and ifn-γ is easily detected by elispot. samples showing ifn-γ t-cell responses to test peptides are further assayed for other cytokine responses and immunological functions. it is worth noting that most of the laboratories performing end-point ifn-γ elispot either for proficiency testing or clinical trial schemes are based either in europe or the united states,16,19 which is disproportionate to where the burden of hiv/aids, infectious diseases and re-emerging ‘orphan’ tropical infectious diseases are predominant.20 there is now an increased focus on conducting vaccine trials where the pandemic is most severe; thus, networks of laboratories to support large clinical trials need to be developed in sub-saharan africa. on this front, iavi with its partners pioneered the gclp accreditation of laboratories in africa. for external quality assurance, these gclp-accredited laboratories were also enrolled in an elispot proficiency scheme. the gclp-accredited laboratories now conduct safety and immunogenicity assays such as elispot and flow cytometry for the assessment of hiv vaccine candidates and are fully equipped to collect, process and store samples for later testing. methods ethical considerations the study protocol was approved by the ethics committees of kenyatta national hospital, university of nairobi, kenya (reference numbers p81/3/2010 & p298/7/2011), the uganda virus research institute, entebbe, uganda (gc/127/10/08/31 & gc/127/11/09/12), projet san francisco (psf), kigali, rwanda (006/rnec/2011), zambia emory hiv research project, university of zambia (008-03-10), emory university (rec-270606-013) and south african national blood service (irb00041163) and was reviewed by the responsible regulatory authorities in each country. each participant provided written informed consent before undertaking any study procedures. at the south african national blood service, blood donors signed informed consent after the procedure was explained to them. blood collected from these donors was not labelled with donor names, but blood bag identifiers. the blood bag was further labelled at clinical laboratory services (cls) with a project number linked to the blood bag on the laboratory information management system. each participant in the hiv-1 clinical trial study provided written informed consent before blood samples were collected. the samples were de-identified and labelled with a study number. participating laboratories six of the seven participating laboratories were iavi-supported laboratories: (1) kenya aids vaccine initiative-institute of clinical research (kavi-icr) university of nairobi, nairobi, kenya, (2) iavi human immunology laboratory, imperial college london, united kingdom, (3) uganda virus research institute (uvri), entebbe, uganda, (4) kenya medical research institute centre for geographical medicine research coast (kemri-cgmrc), kilifi, kenya, (5) zambia emory hiv research project (zehrp), lusaka, zambia, and (6) projet san francisco (psf), kigali, rwanda. clinical laboratory services, witwatersrand university, johannesburg, south africa, is the only laboratory not supported by iavi but contracted to coordinate the sourcing, testing and shipping of peripheral blood mononuclear cells (pbmcs) for elispot proficiency testing. clinical laboratory services is accredited for both iso 15189 and gclp and followed the same operating procedures as the iavi-sponsored laboratories. clinical laboratory services also participated in elispot proficiency testing and is included in this report. laboratory preparation process of establishing clinical trial laboratories under good clinical laboratory practice guidelines comprehensive training programmes, calibrated and maintained equipment and quality control measures were integral in establishing iavi’s sponsored laboratories (table 1) and are described in detail in supplementary document 1. table 1: summary of the process of establishing a clinical trial laboratory under good clinical laboratory practice guidelines. to minimise potential failures in the iavi laboratories, quality control systems and operating procedures were put in place and corrective actions were instituted whenever a laboratory encountered a technical or assay failure to prevent future re-occurrence. two laboratories (hil and cls) were designated to provide laboratory support and quality control management as these two laboratories were based in ideal locations to support a global clinical trial programme. both locations are major international hubs in europe and africa, with direct flights to and from the iavi-supported laboratories, thereby reducing time and cost to transport samples as well as reduce damage risk to samples while in transit. elispot proficiency panel design international aids vaccine initiative gclp laboratories were enrolled in an ifn-γ elispot proficiency scheme coordinated by cls, south africa. clinical laboratory services sourced buffy coat blood pack samples from the south african national blood service, then isolated and cryopreserved pbmcs. frozen pbmc samples were thawed and assessed for cell viability, cell recovery and performance in elispot. samples with poor viable cell recovery or performance in elispot were excluded. pbmc samples with 50 to 100 vials were selected to provide laboratories with identical sets of pbmc samples (six pbmc samples per set) to allow testing of the same pbmc set over 6 to 12 months (that is 7 labs over 6 months would require 42 vials of each pbmc sample; 12 months would require 84 vials). frozen pbmc were maintained within a liquid nitrogen vapour phase freezer repository at cls until distribution to the laboratories participating in elispot proficiency. four laboratories – actively involved in clinical trials and performing cellular functional assays – conducted elispot monthly (kavi-icr, uvri, psf-kigali and hil), while three laboratories – not involved in clinical trials – conducted elispot quarterly (kemri-cgmrc, zehrp and cls). pbmcs were tested against two peptide pools; (1) 32 8–10-mer peptides representing an immunodominant cluster of differentiation 8+ (cd8+) t-cell epitopes from cytomegalovirus, epstein barr virus and influenza virus (anaspec inc., california, united states),21 and (2) 138 15-mer peptides overlapping by 11 amino acids spanning the human cytomegalovirus pp65 protein. these peptides were chosen because the majority of the population have pre-exposed immunity against these viruses and will have detectable t-cell response to these peptides. phytohaemagglutinin (pha-l; sigma l4144, sigma, poole, dorset, united kingdom) and dimethyl sulfoxide (diluted in culture medium) were used as positive and negative controls respectively. sample processing and elispot assay were performed according to the minimal information about t-cell assays guidelines.22 source of proficiency peripheral blood mononuclear cells and clinical trial samples peripheral blood mononuclear cells used in elispot proficiency testing were obtained from the buffy coat (south african national blood service, johannesburg, south africa) by ficoll-paque gradient centrifugation. blood donors were screened for hiv, hepatitis b, and syphilis. briefly, whole blood was diluted with sterile phosphate-buffered saline (pbs, sigma-aldrich. st. louis, missouri, united states) at a ratio of 1:1 and layered gently over 20 ml ficoll density gradient (ficoll-hypaque premium; ge healthcare, uppsala, sweden) at a ratio of 2:1 in a 50 ml falcon tube (greiner bio-one, stonehouse, united kingdom). the tubes were centrifuged at 750 × g for 25 min at room temperature with the brakes off. after centrifugation, the plasma component was aspirated using a sterile pasteur pipette (alpha laboratories, eastleigh, united kingdom) into a waste container containing bleach. the pbmc component was then removed into a new sterile 50 ml tube and washed two times with 20 ml pbs by centrifugation at 400 × g for 10 min with the brakes on. after washing, the cells were resuspended in 10 ml of roswell park memorial institute (rpmi) 1640 medium supplemented with 10% calf serum, 1 u/ml penicillin, 1 μg/ml streptomycin, and 300 µg/ml l-glutamine. peripheral blood mononuclear cells obtained from clinical trial participants was isolated from heparinised blood by density gradient centrifugation using histopaque 1077 (h8889, sigma). briefly, 20 ml of blood was layered onto 20 ml of histopaque in a 50 ml tube (falcon 357522, sarstedt, nümbrecht, germany) using a sterile serological pipette (falcon, sarstedt). the blood was then centrifuged at 400 × g for 40 min at room temperature with brakes off. after centrifugation, the upper plasma fraction was aspirated to within 1.5 to 2 cm of the pbmc band located at the interface between the yellow plasma fraction and the clear fluid below the pbmc band. the cells were washed in hank’s balanced salt solution without ca+ or mg+ with phenol red (sigma-aldrich) by centrifugation at 500 × g for 10 min at room temperature (1st wash) with brakes on. after centrifugation, the supernatant fluids were discarded into the waste container, the cells were resuspended in hank’s balanced salt solution and the tubes were centrifuged again at 400 × g for 10 min (second wash). after centrifugation, the supernatant fluids were discarded into the waste container and cells were resuspended in hank’s balanced salt solution and centrifuged at 400 × g cells for 10 min at room temperature with brakes on (third wash). after centrifugation, the supernatant fluids were discarded, and cells were resuspended in hank’s balanced salt solution for counting. in a separate study, pbmc from clinical trial samples were obtained from heparinised blood from a healthy hiv-negative placebo and vaccine recipients participating in clinical trials of two prophylactic hiv vaccine candidates at kavi-icr, uvri-iavi, psf and zehrp.10,11 these four laboratories participated in hiv clinical trials and the pbmc data were available for analysis. these pbmcs were processed as described above and had to meet the following predefined acceptance criteria: (1) processing of pbmc within 6 h from blood draw to cryopreservation, (2) cell viability above 90% and cell yield greater than 0.7 × 106 pbmc per ml of blood for freshly isolated pbmc, (3) cell viability above 80% for frozen, thawed and overnight rested pbmc. after isolation, cells were counted using a vi-cell xr automated cell counter (beckman coulter, united kingdom) and cryopreserved in 1 ml foetal calf serum containing 10% dimethyl sulfoxide in nalgene system 100tm cryogenic tubes (thermofisher scientific, new york, united states) at a final concentration of 10–15 ×106 pbmc per ml and transferred to a rate-controlled freezer (planer, sunbury-on-thames, united kingdom). this system cools the cells by a temperature decrease of 1°c per min from +4 °c to –80 °c followed by a 10 °c per min decrease until the temperature of the pbmcs was –120 °c after which the cells were transferred to vapour phase liquid nitrogen (ln) for long-term storage. pbmcs for elispot proficiency testing were cryopreserved in the same manner. shipment of proficiency peripheral blood mononuclear cells cryopreserved pbmcs were shipped to the participating laboratories as non-infectious human specimens –biological substances, category b and un337, packed in compliance with the international air transport association (iata) packing instruction 650. before shipping, cls notified the receiving laboratories of the pbmc proficiency panels’ shipping itinerary so that they can be ready to appropriately store the samples upon receipt. the proficiency pbmcs were shipped on a temperature-controlled dry shipper (mve jencons, united kingdom). dry shippers were calibrated for 7 days to ensure they are in good condition for shipment of pbmcs. briefly, on day 1, empty dry shippers were weighed and filled with ln to the brim and left overnight to adsorb. the next day, excess ln was decanted; the weight and temperature of the shipper were recorded. for the next 5 days and at the same time as the second day, the weight and temperature of the dry shipper were measured and recorded to determine the weight and temperature loss. for each dry shipper to pass calibration, its average weight and temperature loss in 24 h over the 5 days should be no more than 0.66kg (manufactureres specification is 0.6kg + 10%) and less than –190 °c. a day before shipment, the calibrated dry shippers were filled with ln and left overnight. the next day, the excess ln was decanted after which the weight and temperature of the shipper were recorded. the pbmcs were loaded onto a pre-cooled canister and placed into the shipper. the shippers were fitted with temperature loggers which were activated only after the samples were loaded to monitor the temperature of pbmcs while in transit. once the paperwork was completed the dry shippers were collected by the iata certified shippers (world courier). upon arrival at the laboratory, the loggers were removed and temperature data was downloaded and recorded. quality checks were then done for the pbmcs against the shipment manifest that accompanied the samples and samples cryopreserved in the ln tank until use. ifn-γ elispot assay setup of ifn-γ elispot assay: to minimise variations and allow investigation of any technical issue resulting from reagent use, although no such reagent issues were noted, the following measures were taken. all laboratories used the same batch of calf serum, which was pre-tested for performance in pbmc isolation, cryopreservation, cell recovery from frozen pbmc, and pha-l and cytomegalovirus acceptable response elispot assay. also, the catalogue number, lot number and expiry date of all reagents used in each assay batch were recorded, including elispot plates, which were obtained in batches of at least 50 plates. thawing and recovery of pbmcs: the cryopreserved pbmc panel was thawed and rested overnight in culture media. briefly, pbmcs were removed from the ln tank and transported to the laboratory in dry ice. they were immediately immersed in a 37 °c water bath and left until a small amount of ice of the freezing media remained. the cells were aspirated into a 10 ml rpmi medium supplemented with 10% calf serum, 1 mm l-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 1 mm sodium pyruvate and 0.5 mm hepes (all supplements were diluted in rpmi and purchased from sigma-aldrich). the tube was centrifuged at 250 × g for 10 min room temperature, after which the cell supernatant was discarded. the cells were resuspended in 4 ml rpmi medium supplemented with 20% calf serum and plated in two wells of a 24-well plate at a concentration of 2.5 × 106 pbmcs per ml. the inoculated plates were incubated overnight. the next day, cells were washed in culture media and counted. plate treatment: the 96-well filter plates (multiscreen hts ip msip4510; millipore, united kingdom) to be used for the assay were treated with ethanol. briefly, 50 µl of 70% ethanol was added to the wells and let to stand for 2 min. the wells were then washed three times with 200 µl sterile pbs. plate coating: after the ethanol treatment, each well was coated with 100 µl of 10 µg/ml pbs-diluted anti-human ifn-γ antibody (clone 1-d1k; 1 mg/ml; mabtech, sweden). the plates were then incubated at 4 oc overnight. plate blocking: the next day, plates were washed three times with 200 μl pbs and blocked with 200 μl rpmi medium supplemented with 10% calf serum per well at 37 °c for at least 2 h. after 2 h of incubation, the plates were removed and the medium was aspirated and discarded. addition of antigen and inoculation of cells: 100 µl of: (1) cell medium containing 2.25 µg/ml each of cytomegalovirus, epstein-barr virus and influenza virus (cef) and cytomegalovirus peptide was added to the reaction; (2) mock culture (dimethyl sulfoxide + culture medium) was added to the mock wells (medium negative control); (3) cell medium containing 15 µg/ml pha-l was added to the assay positive control wells and (4) cell medium containing 2.25 µg/ml cytomegalovirus peptide into the cell-free (peptide negative control) wells. pbmcs were added to all the wells except the cytomegalovirus cell-free control well at a density of 200 000 cells per 50 µl culture media. assay was done in quadruplicate. the plates were then moved to the incubator for 16 h – 24 h. addition of detection antibody: the next day, the plates were washed six times with 200 µl pbs supplemented with 0.05% tween (pbs/t) using an automated washer (bio-tek instruments inc., winooski, vermont, united states). detection was done by adding 100 μl 1 μg/ml pbs-diluted biotinylated anti-human ifn-γ antibody (clone 7-b6-1, 1 mg/ml) (mabtech, sweden) to the plates. the plates were then incubated at room temperature for 2 h. the plates were then washed six times as above after which 100 µl of peroxidase avidin-biotin complex (vector laboratories, burlingame, california, united states) was added to each well and incubated at room temperature for an hour. after incubation, plates were washed three times with 200 µl pbs/t followed by another triple wash with 200 µl pbs. spot development: 100 µl of 3-amino-9-ethyl carbazole substrate solution (vector laboratories, burlingame, california, united states) was added to the plates. the 3-amino-9-ethyl carbazole substrate solution was obtained by dissolving 3-amino-9-ethyl carbazole tablets in 2.5 ml dmf (vwr international, united states) after which the solution was diluted in 47 ml sterile tissue culture water supplemented with 280 µl 2m acetic acid, 180 µl 2m sodium acetate and 25 µl hydrogen peroxide (all from sigma-aldrich, st. louis, missouri, united states). the plates were then incubated at room temperature for 4 min. the reaction was stopped by running the plates over gentle-running tap water. the underdrain of the plates was removed, and plates left to dry overnight in the dark. imaging and analysis of elispot plates: all of the participating laboratories in this study used the same elispot reader system (autoimmun diagnostika, germany) with software version 4.0. it is a computer-based system for the semi-automatic interpretation of 96-well elispot plates. the system had the following settings for ifn-γ: well(s): a1 h12; count settings: ifn-γ; algorithm: v.3.2.x; intensity min: 15; size min: 72; and emphasis: small. this plate reader system is regularly maintained by use of a master lot plate supplied alongside the reader system, which contains artificial spots designed to test the performance of the reader system. as part of the internal quality control programme, each laboratory read this control plate once a week to assess the performance of the elispot reader using predefined spot parameters. the developed plates were imaged on the aid elispot reader system. the elispot data were expressed as the numbers of spot-forming cells (sfc) per million pbmc (supplementary document 1 figure 2). the acceptance criteria for each assay are: (1) the average sfc in the mock wells (peptide and pha free) must be less than 50 sfc per 106 pbmc; (2) the average sfc in the negative antigen control wells (cell free) must be less than 5 sfc per well; (3) the average sfc in the assay positive control wells (peptide free + pha) must be more than 50 sfc per 106 pbmc in the positive control pha. the plates were read and raw sfc data were submitted to hil for evaluation by a senior scientist and results shared on the access restricted cls website. statistical analyses this study aimed to compare the assay results obtained from the analysis of the same (frozen) pbmc samples provided to seven laboratories. we analysed the inter-lab and inter-operator variability and investigated cell recovery, viability and processing time. outcome measures include the recovery and viability rates of frozen pbmcs, and elispot counts for mock, cytomegalovirus and cef stimuli. for uniformity of data, cell recovery data after thawing and resting overnight from one clinical trial with pbmcs frozen at 15 million cells per ml/vial were normalised to 10 m cells per ml. for each sample, four replicate elispot plate wells per peptide pool were assayed and the arithmetic mean was used for analysis. results based on fewer than 4 replicate counts were assumed to be less accurate and excluded from the analysis. a total of 1751 assays were performed of which 50 were excluded (i.e. about 2.9%). for peptide pool repeated measures, the poisson regression model was fit to background-subtracted (except mock) elispot counts, with counts from the same volunteer assumed to be correlated. the resulting least-squares parameter estimates are presented together with their 95% confidence intervals adjusted for multiple comparisons using the bonferroni method. each model included volunteer, laboratory and month as covariates. pair-wise comparisons between laboratories are shown as the ratio of the least-squares estimates of the mean count with corresponding adjusted (bonferroni) 95% confidence intervals. statistical significance was defined as a 95% confidence interval for the ratio that excludes unity (i.e. entirely above or below the value 1). figures 1, 4 and 5, and supplementary document 1 figure 1 were generated by graph pad prism software version 8.3.0 (graphpad software, san diego, california, united states), while the rest of the figures and statistical analyses were performed using sas version 9.3 (sas institute, cary, north carolina, united states). results elispot testing performance across seven laboratories almost all (1733/1751; 99%) of the elispot proficiency assays performed had data within acceptable ranges with low responses to mock stimuli within the acceptance criteria of less than 50 sfc per million cells across the seven laboratories over time (figure 1). a small fraction of these (18/1751; 0.01%) pbmcs had mock responses over 50 sfc per million cells; however, on average replicates per donor, all mock responses were below 50 sfc per million cells in all the panels. all the five laboratories actively conducting immunological analyses in support of iavi-sponsored clinical trials performed similarly in elispot testing with comparable data in elispot counts against cytomegalovirus and cef peptides (figure 1). the elispot counts were background-subtracted (except mock). the covariates in the model were sample, laboratory and month. across the seven laboratories, the geometric mean elispot counts (sfc per 106 pbmc) for mock stimuli were 6–10 (supplementary document 1 table 1), 289–438 for cef stimuli (supplementary document 1 table 2) and 172–266 for cytomegalovirus (supplementary document 1 table 3). we observed significant differences in mock counts across the laboratories with cls mock counts estimated to be 1.73 times higher than zehrp and at psf which were 0.78 times lower than uvri. zambia emory hiv research project tends to have lower mock counts than all other laboratories (supplementary document 1 table 1; p < 0.001). figure 1: distribution of elispot responses across laboratories in kenya, uganda, rwanda, zambia, south africa and the united kingdom, 2010–2014. responses against mock, cytomegalovirus and cytomegalovirus, epstein-barr virus, and influenza virus stimuli from a panel of six peripheral blood mononuclear cells tested over 6 months, (a) first 24 months and (b) second 24 months. box plots represent the quartiles, horizontal line the median and whiskers the maximum and minimum values. each point represents average spot-forming cells per 106 peripheral blood mononuclear cells from replicates per donor at each laboratory. the laboratories are color-coded as follows: kenya aids vaccine initiative-institute of clinical research (red); uganda virus research institute (blue); projet san francisco (green); zambia emory hiv research project (purple); kenya medical research institute centre for geographical medicine research coast (yellow); clinical laboratory services (cyan); human immunology laboratory (black). when we compared the elispot responses against cef peptide pools across laboratories, kemri-cgmrc had significantly higher counts than other laboratories (p = 0.004, figure 2 and supplementary document 1 table 2). when data from kemri-cgmrc are excluded, the overall difference between laboratories is not statistically significant (p = 0.11, supplementary document 1 table 2). the same trend was seen in elispot responses against the cytomegalovirus stimulus where kemri-cgmrc again had significantly higher counts than other laboratories (p = 0.012, figure 2 and supplementary document 1 table 3). even after excluding data from kemri-cgmrc, the overall difference between laboratories is still statistically significant because of lower counts in zehrp than in cls and kavi-icr (p = 0.033, supplementary document 1 table 3). figure 2: comparison of elispot responses across laboratories in kenya, uganda, rwanda, zambia, south africa and the united kingdom, 2010–2014. the graphs show which site pairs are significantly different (blue lines) and which are not (red lines). for each comparison, a line segment, centred at the least-squares-means in the pair, is drawn. the segment length corresponds to the projected width of a confidence interval for the least-squares mean difference. each line corresponds to the pair of labs with reference lines that cross at the midpoint. shown here are the pair-wise least-squares means and their statistical significance, on a natural log scale, for mock, cytomegalovirus, epstein-barr virus, and influenza virus and cytomegalovirus stimuli. differences for alpha = 0.05 (bonferroni adjustment); red line denotes not significant while blue line denotes significant. (a) mock, (b) cytomegalovirus, epstein-barr virus, and influenza virus and (c) cytomegalovirus. inter-operator analysis the performance of three operators from kavi-icr in elispot testing during the study period was analysed. pbmcs from 12 volunteers were analysed by the three operators on a rotational basis with each operator conducting the same set of samples at monthly time points. elispot counts were obtained for mock and background-subtracted cytomegalovirus and cef responses and the data analysed using the repeated measures poisson regression model and comparison-adjusted against the sample, operator and month. the geometric mean elispot counts for mock were 9–12, 368–393 for cef and 538–598 for cytomegalovirus stimuli (supplementary document 1 table 4). we found no significant difference in elispot performance between the three operators (figure 3 and supplementary document 1 table 4). figure 3: comparison of inter-operator elispot responses from three operators at kenya aids vaccine initiative-institute of clinical research, kenya, 2010–2014. the graphs show which operators are significantly different (blue lines) and which are not (red lines). shown here are the pair-wise least-squares means and their statistical significance, on a natural log scale, for mock, cytomegalovirus, epstein-barr virus, and influenza virus and cytomegalovirus stimuli. for each comparison, a line segment, centred at the least-squares means in the pair, is drawn. the length of the segment corresponds to the projected width of a confidence interval for the least-squares mean difference. segments that fail to cross the 45° reference line correspond to significant least-squares mean differences. none of the pairs of operators is significantly different (all lines cross the 45° reference line). differences for alpha = 0.05 (bonferroni adjustment); red line denotes not significant while blue line denotes significant. (a) mock, (b) cytomegalovirus, epstein-barr virus, and influenza virus and (c) cytomegalovirus. performance of four laboratories in pbmc processing a total of 1297 pbmcs isolated from clinical trial samples at the four aforementioned laboratories supporting two iavi-sponsored hiv clinical trials were analysed for cell viability, recovery and cell yield per ml of blood. of the 1297 pbmcs processed, 1220 (94%) freshly isolated pbmcs had viability above 90% with a median of 95% (range 81% – 100%) and those with viability below 90% had a median of 88% (range 81% – 90%) (figure 4a). over 96% (1249/1297) of these samples had cell yields greater than 0.7 × 106 pbmc per ml blood, all within the predefined acceptability criteria (figure 4b). there were a few samples that had low cell yield ranging from 0.13 to 0.56 × 106 pbmc per ml blood (figure 4b). a fraction of these samples (1205/1297; 93%) including those with cell yield below 0.7 × 106 pbmc per ml blood were thawed and tested for elispot performance and almost all (1196/1205; 99%) pbmcs had viability above 80% following thaw and overnight rest (within acceptability criteria) and only 9 pbmcs were below 80% (range 66% – 78%) (figure 4c). the cell recovery for these samples was above 6.0 × 106 pbmc per vial (data were normalised to 10 m cells (figure 4d); samples from the clinical trials analysed in this study were frozen at 10 m cells per ml except one trial where samples were frozen at 15 m cells per ml per vial at which the data were normalised to 10 m cells per ml for uniformity of data. in elispot testing, all the samples were functional with over 95% having mock responses under 50 sfc per 106 pbmc, pha over 1000 sfc per 106 pbmc and a range of cytomegalovirus responses. figure 4: performance of laboratories in peripheral blood mononuclear cell (pbmc) processing, kenya, uganda, rwanda, zambia, 2010–2014. (a) the percentage cell viability of freshly isolated pbmc, (b) cell yield per ml of blood, (c) percentage cell viability from frozen pbmcs, (d) cell recovery of frozen pbmc (pbmcs were cryopreserved at a final concentration of 10–15 m cells per ml; here data were normalised to 10 m cells), and (e) duration of pbmc processing (in hours). each dot represents a sample and the horizontal line represents the median with interquartile range. the long horizontal line shows the acceptance cut-off. the duration of pbmc processing from blood draw to cryopreservation has been shown to affect the integrity of cells.23,24,25 in this study, we analysed the processing time of pbmc (in hours) and report that nearly all the samples were processed within 6 h with only 6% (81/1297) processed beyond 6 h (range 6.1–9.5 h) (figure 4e and supplementary document 1 figure 1). to assess the integrity of samples processed past 6 h, we analysed the pbmc viability and cell yield from freshly isolated pbmcs and later analysed the viability and cell recovery from thawed frozen pbmcs. we found the delays did not affect the pbmcs’ integrity in all samples; one had cell viability above 90% and a cell yield greater than 0.7 × 106 pbmc per ml blood (both parameters within acceptable range) (supplementary document 1 figure 1). only one sample had a slightly lower cell yield of 0.57 × 106 per ml blood and cell viability of 98%. post pbmc freezing cell viability ranged from 93% to 100% and cell recovery was above 6 × 106 pbmc per vial in 71 of 81 (87%) samples (supplementary document 1 figure 1). we further tested these samples in an elispot assay to assess their cell functionality and all samples performed well with the mock responses under 50 sfc per 106 pbmc, pha responses greater than 1000 sfc per 106 pbmc and a typical range of cytomegalovirus responses. these data are similar to what was seen from samples processed within 6 h (supplementary document 1 figure 1). discussion we compared data generated in multiple laboratories over time to assess their performance in the processing of pbmc samples and elispot testing. some of these laboratories supported clinical trials of hiv prophylactic vaccine candidates and to standardise their cell functionality assays and harmonise procedures to achieve reliable and accurate data, they were enrolled in an elispot proficiency scheme as part of external quality assurance. all laboratories performed well in elispot proficiency testing with data comparable across laboratories; however, there were a few sporadic outliers in the data. these outliers were expected considering the large number of data points analysed: sporadic outliers would be expected even from experienced and competent laboratories. in this study, we saw comparable elispot data from five out of seven laboratories. these five laboratories all supported clinical trials except one: cls which performed elispot regularly. since cls was the laboratory responsible for sourcing and qualifying the pbmcs for the elispot proficiency scheme, they were expected to perform well in elispot testing. in the two laboratories that did not support clinical trials and routine elispot testing, we saw significant differences in elispot proficiency data compared to other laboratories. it is worth mentioning that these laboratories performed elispot testing quarterly and would be less experienced compared to other laboratories. to identify the root cause of this data discrepancy, corrective action was initiated at these two laboratories. the staff were retrained and assessed for competency. also, improvement measures were put in place such as continuous training and monitoring of their performance in elispot testing in subsequent rounds of proficiency testing panels. as a requirement of the gclp programme, at any given time, there should be more than one person processing the clinical trial samples to ensure accuracy and reliability of data. likewise, in elispot proficiency testing, different operators at each laboratory fully trained in the required procedures conducted elispot proficiency assays on a rotational basis as determined by laboratory management. the influence of the operator on the variability of results is a known factor as shown by janetzki and colleagues;14 here we analysed the performance of three operators in elispot proficiency testing in one laboratory which maintained the same staff throughout the study period. it was not possible to analyse inter-operator variability in all laboratories as many sites experienced frequent staff turnover during the study period. from the analysis, there was no significant difference in data generated by the three operators over the study period. this demonstrates that with regular training and competency assessment, reliable, accurate and comparable data can be obtained notwithstanding the inherent operator variability. sample integrity is critical for achieving accurate and reliable results in clinical trials. in a multicentre trial, processes for sample processing need to be harmonised to generate comparable data. we assessed the processing of pbmcs in four of seven laboratories, focusing on five areas: sample collection, isolation of pbmc, cryopreservation, thawing of frozen pbmc, and performance in elispot testing of clinical trial samples. first, all laboratories were required to process samples to cryopreservation within 6 h of a blood draw. this is to ensure that pbmcs obtained are of good quality as it has been documented by olson and colleagues that a delay in the processing of pbmc of more than 8 h may reduce cell viability and compromise cell functionality.26 we found most samples were processed within 6 h with few samples outside of this time frame. the few samples were processed outside this time frame due to reasons such as a delay in delivery to the laboratory, as some clinics were some distance from the laboratory, batching of sample processing and a backlog of samples. to mitigate these, corrective and preventative actions were initiated to prevent such recurrence which included prioritising the delivery of samples from the clinics, processing samples promptly as they arrive in the laboratory and cryopreserving pbmc immediately after processing to reduce any backlog that may have arisen from batching of cryopreservation. these measures initiated were monitored monthly. although some samples were processed beyond the expected time frame, their integrity was not compromised as cell viability, cell yield and recovery after thawing were indistinguishable from other samples processed within the stipulated time. additionally, these samples were tested for functionality in downstream assays such as elispot and they performed well, again with indistinguishable results compared with samples processed within the stipulated time. in conclusion, we have shown that samples processed beyond 6 h and up to 9 h from blood draw to cryopreservation are still viable and functional, similar to findings from other groups.24,25 in assessments of pbmc isolation, our focus was on cell viability and cell yield. we found that the majority of pbmc samples isolated in all four laboratories supporting clinical trials met the predefined acceptance criteria for viability and cell yield with few outliers seen across the laboratories. in most cases, samples obtained in a multicentre clinical trial are shipped to a central laboratory either for long-term storage or cellular functionality testing. therefore, proper cryopreservation after pbmc isolation and shipping is critical in preserving cell integrity and functionality.27 clinical trial samples isolated at four laboratories were cryopreserved at the local freezing repository until shipped to a central laboratory. upon request, the samples were shipped to the central laboratory according to the iata guidelines for long-term storage. the majority of thawed samples were viable with cell viability and recovery within the acceptable limits and thus demonstrated the competency of the laboratories in cryopreservation and shipping of samples. additionally, thawed samples performed well in elispot testing with responses within the expected ranges. to maintain high standards and produce reliable and comparable data, these laboratories were audited regularly for gclp compliance either by internal or external independent auditors. the audit covers areas such as operating procedure development and documentation, elispot and flow cytometry testing proficiency schemes and the data management system. all laboratories were audited annually by an external auditor for gclp accreditation. limitations the main limitation of this study was the variation in the operators who performed elispot testing at the laboratories. there was a frequent turnover of personnel in most of the laboratories during the study period which made it difficult to assess the inter-operator variability within sites. to assess the operator effect on data variability, data were analysed from only one laboratory which had three operators who consistently performed elispot testing over the 4 years on a rotational basis. though we saw no significant difference in data generated between these three operators, this result does not entirely represent what could be seen across all seven laboratories. conclusion in conclusion, we have demonstrated the capabilities of multiple laboratories in africa and europe in processing clinical trial samples to high standards and performing cell functionality assays. furthermore, we have shown that multiple laboratories can generate reliable, accurate and comparable data by using standardised procedures, having regular training, regular equipment maintenance and using centrally sourced reagents. these efforts and the elispot proficiency programme continue across the network of iavi-sponsored laboratories supporting clinical trials. therefore, we highly recommend the approach taken by the iavi gclp-accredited laboratories to produce such data to any donor, sponsor or research institution who may plan to conduct clinical trials in the region. acknowledgements we wish to acknowledge the support from the university of california, san francisco’s international traineeships in aids prevention studies, us nimh r25 mh064712, under which this manuscript was written. we thank university of california san francisco faculty staff for helpful discussions, matt price and kathy crisafi of iavi for manuscript review and laboratory technicians for experimental assistance. we also thank all iavi clinical research centres’ principal investigators for overseeing the laboratories. the contents of this manuscript are the responsibility of the authors and do not necessarily reflect the views of united states agency for international development or the united states government. competing interests the authors have declared that no competing interests exist. authors’ contributions r.k.l. performed the experiments and wrote the manuscript. j.i., s.o., e.t., c.n., m.s. and e.m., performed the laboratory experiments. m.j. isolated and qualified the proficiency pbmcs used in this study and also performed the laboratory experiments. n.h. and l.d. performed the statistical analysis. b.f., j.b., o.a., p.c., g.o.-m. and j.g. designed the study. j.h.c. and p.h. designed the experiments, interpreted results and critically reviewed the manuscript. sources of support this work was funded in part by iavi and made possible by the support of the united states agency for international development and other donors. the full list of iavi donors is available at www.iavi.org. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated 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for hiv vaccine trials. j immunol methods. 2007;322(1–2):57–69. https://doi.org/10.1016/j.jim.2007.02.003 olson wc, smolkin me, farris em, et al. shipping blood to a central laboratory in multicenter clinical trials: effect of ambient temperature on specimen temperature, and effects of temperature on mononuclear cell yield, viability and immunologic function. j transl med. 2011;9:26. https://doi.org/10.1186/1479-5876-9-26 smith jg, joseph hr, green t, et al. establishing acceptance criteria for cell-mediated-immunity assays using frozen peripheral blood mononuclear cells stored under optimal and suboptimal conditions. clin vaccine immunol. 2007;14(5):527–537. https://doi.org/10.1128/cvi.00435-06 abstract background description of the intervention lessons learnt recommendations acknowledgements references about the author(s) john n. waitumbi kenya medical research institute (kemri)/united states army medical research directorate-africa, basic science laboratory, kisumu field station, kisumu, kenya esther omuseni kenya medical research institute (kemri)/united states army medical research directorate-africa, basic science laboratory, kisumu field station, kisumu, kenya josphat nyataya kenya medical research institute (kemri)/united states army medical research directorate-africa, basic science laboratory, kisumu field station, kisumu, kenya clement masakhwe kenya medical research institute (kemri)/united states army medical research directorate-africa, basic science laboratory, kisumu field station, kisumu, kenya faith sigei kenya medical research institute (kemri)/united states army medical research directorate-africa, basic science laboratory, kisumu field station, kisumu, kenya allan lemtudo kenya medical research institute (kemri)/united states army medical research directorate-africa, basic science laboratory, kisumu field station, kisumu, kenya george awinda kenya medical research institute (kemri)/united states army medical research directorate-africa, basic science laboratory, kisumu field station, kisumu, kenya eric muthanje department of biological sciences, university of embu, embu, kenya brian andika department of molecular biology and bioinformatics, jomo kenyatta, university of agriculture and technology, juja, kenya rachel githii kenya medical research institute (kemri)/united states army medical research directorate-africa, basic science laboratory, kisumu field station, kisumu, kenya rehema liyai kenya medical research institute (kemri)/united states army medical research directorate-africa, basic science laboratory, kisumu field station, kisumu, kenya gathii kimita kenya medical research institute (kemri)/united states army medical research directorate-africa, basic science laboratory, kisumu field station, kisumu, kenya beth mutai kenya medical research institute (kemri)/united states army medical research directorate-africa, basic science laboratory, kisumu field station, kisumu, kenya citation waitumbi jn, omuseni e, nyataya j, et al. covid-19 mass testing and sequencing: experiences from a laboratory in western kenya. afr j lab med. 2022;11(1), a1737. https://doi.org/10.4102/ajlm.v11i1.1737 lessons from the field covid-19 mass testing and sequencing: experiences from a laboratory in western kenya john n. waitumbi, esther omuseni, josphat nyataya, clement masakhwe, faith sigei, allan lemtudo, george awinda, eric muthanje, brian andika, rachel githii, rehema liyai, gathii kimita, beth mutai received: 21 sept. 2021; accepted: 21 apr. 2022; published: 22 july 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the basic science laboratory (bsl) of the kenya medical research institute/walter reed project in kisumu, kenya addressed mass testing challenges posed by the emergent coronavirus disease 2019 (covid-19) in an environment of global supply shortages. before covid-19, the bsl had adequate resources for disease surveillance and was therefore designated as one of the testing centres for covid-19. intervention: by april 2020, the bsl had developed stringent safety procedures for receiving and mass testing potentially infectious nasal specimens. to accommodate increased demand, bsl personnel worked in units: nucleic acid extraction, polymerase chain reaction, and data and quality assurance checks. the bsl adopted procedures for tracking sample integrity and minimising cross-contamination. lessons learnt: between may 2020 and january 2022, the bsl tested 63 542 samples, of which 5375 (8.59%) were positive for covid-19; 1034 genomes were generated by whole genome sequencing and deposited in the global initiative on sharing all influenza data database to aid global tracking of viral lineages. at the height of the pandemic (august and november 2020, april and august 2021 and january 2022), the bsl was testing more than 500 samples daily, compared to 150 per month prior to covid-19. an important lesson from the covid-19 pandemic was the discovery of untapped resilience within bsl personnel that allowed adaptability when the situation demanded. strict safety procedures and quality management that are often difficult to maintain became routine. recommendations: a fundamental lesson to embrace is that there is no ‘one-size-fits-all’ approach and adaptability is the key to success. keywords: covid-19; coronavirus; sars-cov-2; nasal swab; nasopharyngeal swabs; mass testing; genome sequencing. background the first case of coronavirus disease 2019 (covid-19) was traced to a kenyan citizen who arrived in nairobi from the united states on 5th march 2020.1 severe acute respiratory syndrome coronavirus 2 (sars-cov-2) was confirmed in the index case on friday the 13th of march. kenyans are not known to have paraskevidekatriaphobia (“friday the 13th” superstition), but the coincidence did not escape notice. through active contact tracing, two contacts who sat next to the index case enroute to nairobi tested positive 2 days later. since then, as in the rest of the world, covid-19 infections have risen dramatically, appearing in waves,2 to date at a count of five (figure 1). surveillance for the virus and containment measures evolved over time, from original contact tracing, isolation and obligatory testing into mandatory masking, social distancing, lockdown, curfew, social gathering ban, restrictions on local and international flights, and closing of country borders. these strict measures were eventually relaxed and at the time of writing (april 2022), the kenyan ministry of health (moh) had removed the masking requirement. figure 1: daily new confirmed covid-19 cases in kenya from march 2020 to march 2022. by early may 2020, it was clear that long-distance truck drivers were transporting the virus across the borders.3 mandatory mass testing and certification for absence of the sars-cov-2 became a requirement for all cross-border truck drivers. testing could not cope with the demand and long queues of trucks held at the border crossing points were common (figure 2). figure 2: long queues of trucks at malaba, kenya–uganda border on 29 april 2020 as drivers waited to be certified free of covid-19. prior to covid-19, the kenya medical research institute (kemri)/walter reed project, kisumu field station laboratory conducted surveillance for pathogens associated with acute febrile illness and outbreak response, and provided basic science support for clinical trials in western kenya. the kenyan government, through the moh, responded to the covid-19 pandemic by designating multiple laboratories, including the basic science laboratory (bsl) of the kenya medical research institute/walter reed project in kisumu as covid-19 testing centres that would provide public health support in combatting the pandemic. this report covers may 2020 to january 2022 and aims to document how the bsl rose to the challenges of sars-cov-2 diagnostics dictated by increased testing demand amidst global supply shortages. description of the intervention ethical considerations the work reported here was conducted as part of public health support to combat the covid-19 outbreak in kenya. a human subject research protocol review was therefore not required. specimen reception, decontamination, anonymization, and testing upon designation in may 2020 as a covid-19 testing centre, the bsl immediately embarked on developing standard operating procedures to guide specimen reception, decontamination of primary and secondary sample containers, anonymisation, risk mitigation, testing and quality assurance procedures, including algorithms of test result interpretation, re-testing of all inconclusive results and reporting of validated results to the moh. an important part of the standard operating procedure was daily decontamination of workspaces with 5% chemgene (medimark scientific limited, kent, england), before and after use and in case of spillage. surfaces decontaminated included workbenches, thermocyclers, nucleic acid extraction robots, centrifuges and vortexes), pipettes, bio-safety cabinets and door handles. to track down potential contamination, all surfaces listed above were swabbed every monday with a disposable sampler (qingdao yongqiang huashang medical technology co., ltd., danyang, china) and tested for sars-cov-2. all surfaces had to have a negative test before nasal sample testing commenced. this procedure was repeated whenever contamination was identified or suspected. semi-automated extractions of rna from nasal and oropharyngeal samples were performed using either the kingfisher flex (thermo fisher scientific inc., waltham, ma, united states) that can perform 96 extractions per hour and or magpurix evo® 24 (zinexts life science, taipei, taiwan) that performs 24 extractions per hour. we also braced for manual extractions, in case the semi-automated kits ran out. for polymerase chain reaction (pcr) testing, the laboratory had the abi 7300 and 7500 systems (applied biosystems, foster city, california, united states) that can perform 96 tests per 2-h run and the magnetic induction cycler (bio molecular systems, queensland, australia) that performs 48 tests in a 2-h run. samples came from different parts of the country and were transported in cool boxes. our estimated maximum output per 24 h was 750 tests. supplies for rna extractions and testing were provided by the moh. because of global shortages of sequencing reagents, the sequencing efforts at the bsl was supported by the united states department of defense global emerging infectious surveillance programme. all kits (extractions and pcr) were validated against known positive samples before use. for quality assurance, external quality assessment samples were provided by the kemri (nairobi, kenya), national public health laboratory (nairobi, kenya), one world accuracy (vancouver, canada) and thistle, johannesburg (south africa). strict procedures were implemented for entry into the laboratory and weekly testing of personnel for sars-cov-2 was required. entry to the laboratory required a covid-19 wellness self-assessment. personnel were asked not to report to work if they had any of the following symptoms: fever ≥ 37.8 °c, sore throat, flu-like symptoms, direct contact or taking care of a covid-19 patient, direct or accidental unprotected contact with samples suspected to contain sars-cov-2. to encourage compliance, absence of work on suspicion of having contracted covid-19 was considered administrative leave and was not deducted from employees’ leave days. lessons learnt strict personnel procedures prevented disease spread in the laboratory over the 21-month testing period (may 2020 to january 2022), seven out of 11 laboratory personnel tested positive for covid-19. these mitigating interventions were considered successful and considering the slow spread of the infection over the testing period, it is unlikely that the infections originated from inside the bsl. initial travel restrictions and lockdowns slowed virus spread, but impacted sars-cov-2 supplies because initial sample accrual was based on active case detection in each county, zero samples were brought to the laboratory in march 2020 or april 2020. in the 4th week of may 2020, we received a batch of 452 respiratory samples collected at the port of busia from truck drivers. of the 452, only seven (1.6%) were positive for sars-cov-2. from june 2020 onwards, the sample sources diversified to include the community, truck drivers, hospitals and military personnel. from may 2020 to january 2022, five waves of covid-19 were discernible (figure 3). the first wave started picking up steam in the first week of june 2020 at 3.0%, reached peak level by mid-july 2020 at 13.2% and thereafter declined steadily, remaining at 3.0% till end of september 2020. the second wave was discernable from the first week of october 2020 at 9.0%, reached peak level of 32.8% by end of that month, and thereafter declined steadily to 1.0% by the end of january 2021. probably fueled by the christmas and new year festivities, wave three emerged suddenly in nairobi and its environs from the second week of february 2021 at 14.2%. although the wave had burned out in nairobi by june 2021, it was still going on in western kenya. wave four emerged while wave three was still in progress. therefore, there was no clear separation between these two waves and by the time wave four started in july 2021, infection rates from wave three were above 5%. wave four did not die off until november 2021, making it the longest wave in kenya. unlike wave four, wave five emerged stealthily in december 2021, spread quickly, and died off as quickly as it had emerged by january 2022, making it the shortest covid-19 wave in kenya. since then, infection rates have been minimal, forcing the moh to reconsider the mandatory requirement for masking. figure 3: five waves of covid-19 in the samples tested at the basic science laboratory, kenya medical research institute/walter reed project, kisumu field station, may 2020 – january 2022. waves caused by variants of concern had a higher viral load real-time pcr cycle threshold (ct) values for the sars-cov-2 orf1ab gene in samples obtained during the five waves were used as surrogates for tracking viral load (figure 4). samples tested during the third, fourth and fifth waves had significantly higher lower mean ct values (28.439 ± 0.304, 28.013 ± 0.251 and 26.033 ± 0.225 standard error of the mean, respectively) compared to wave one (30.303 ± 0.256 standard error of the mean) and wave two (30.890 ± 0.296 standard error of the mean, p < 0.05), indicating a higher viral load. from our sequence data, wave three was dominated by the alpha variant of concern (originally identified in the united kingdom), four by delta (originally identified in india) and five by omicron (originally identified in south africa).4 these variants of concern have been associated with higher viral loads and/or doubling time.5,6,7 we recognise that there are caveats in relying on ct values, but as suggested by hay et al., at a population level, ct values can be used to explain the trajectory of the covid-19 epidemic.8 figure 4: scatter plot showing the viral load as determined from cycle threshold values for the five waves tested at the basic science laboratory, kenya medical research institute/walter reed project, kisumu field station, may 2020 – january 2022. increased test demand put a huge strain on testing reagents and supplies the automated extraction kits were the first to be depleted, and resupply was erratic due to banning of flights in and out of the country, coupled with increased global demand and competition. in addition, because infection rates in kenya, and indeed in africa, were still very low compared to global rates, kenya was at the bottom of the ladder on supply priorities. the laboratory resorted to manual extraction using kits supplied by moh, philanthropists and different embassies. because of the increased testing demands and to save on resources and time, a sample pooling strategy was adopted. this was done when the infection rates were not more than 5%. creation of process flow units reduced errors and increased efficiency at the height of testing, the laboratory was receiving over 500 respiratory samples per day. laboratory personnel organised themselves into work units consisting of extraction, pcr, and data with quality control (qc) checks at various stages (figure 5). the extraction team was located in the ‘dirty room’ and donned complete body suits and facemasks. they were responsible for sample receipt and disinfection, sorting and assigning processing ids, sample pooling and the nucleic acid extractions. work units helped in two main areas. first, they reduced error rates, because teammates could qc each other. second, because of increased workload that required working late and during weekends, shifts were easier to organise without too much disruption of sample processing and analysis. figure 5: laboratory personnel work units consisting of extraction, pcr, and data with quality control checks at various stages of sample processing at the basic science laboratory, kenya medical research institute/walter reed project, kisumu field station, may 2020 – january 2022. the pcr team worked in the ‘clean room’ equipped with a level 2 biosafety cabinet where pcr master mixes and extracted nucleic acids were added before being loaded in thermocyclers that were located in a separate room. this separation further reduced the chances of amplicon cross contamination. three personnel were designated to perform the pcr, with at least two working together at any given time. the pcr teams anticipated completion of rna extractions to allow the pcr process to commence immediately. real-time pcr amplification took 2 h. once complete, amplification output was analysed, quality assurance checked and interpreted. samples in the negative pools were reported as negative. samples in the positive pools were re-extracted individually and re-tested. each real-time pcr reaction included the human rnase p gene (rnase p) to check for sample integrity. a total of 64 643 samples were tested in the reporting period, of which 5376 (8.3%) were positive for sars-cov-2. resampling was requested in 668 (1.4%) samples because of poor sample quality. of the 5376 positive samples, 1034 with ct values ≤ 33 were sequenced on an illumina miseq (illumina, san diego, california, united states). recommendations a great source of pride for any laboratory contemplating sars-cov-2 testing in support of the covid-19 pandemic control is the realisation that the effort is part of what the world health organization refers to as critical preparedness, readiness and response actions that save lives.9 a fundamental lesson to embrace is that there is no ‘one-size-fits-all’ approach, and that adaptation of traditional workflows and processes are crucial. given the covid-19 diagnostic demand amidst the global shortfall in supplies, the bsl would not have been able to meet the testing requests without adopting the specimen pooling strategy. with these realisations, the bsl was able to quickly adapt to increased testing demand dictated by an emergent new disease. of the 323 272 covid-19 confirmed cases in kenya, bsl contributed 5376 (1.7%) positive samples. the nasopharyngeal swab collection centres did a commendable job of ensuring sample integrity, despite the long distance to the testing laboratory. acknowledgements the kenyan ministry of health provided extraction and pcr kits. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions j.w. conceptualised, wrote the first draft of the manuscript, provided the resources, and obtained funding; e.o. performed the assays, analysed the data, organised the assay workflow for qualitative polymerase chain reaction, was in charge of quality assurance/quality control, and reviewed and edited the manuscript; j.n. performed the assays, analysed the data, was in charge of assay workflow, assisted in plotting graphs, and reviewed and edited the manuscript; c.m. performed the assays, analysed the data, assisted with the qualitative polymerase chain reaction assay workflow, assisted in plotting graphs, and reviewed and edited the manuscript; f.s. performed the assays, analysed the data, assisted in quality assurance and quality control, and reviewed and edited the manuscript; a.l. performed the assays, analysed the data, supervised the nucleic acid extractions workflow, and reviewed and edited the manuscript; g.a. performed the assays, assisted in the nucleic acid extractions workflow, and reviewed and edited the manuscript; e.m., b.a. and r.l. performed the assays, assisted in nucleic acid extractions, and analysed the data; r.g. was in charge of data curation and capture of associated metadata; g.k. analysed the data, reviewed and edited the manuscript, and assisted in assay validation; b.m. ensured that all staff had appropriate personal protective equipment and that all biosafety cabinets were calibrated and safe to use, supervised all aspects of the assays, and revised and edited the manuscript. sources of support sars-cov-2 sequencing and personnel costs were funded by the armed forces health surveillance division (afhsd) and its global emerging infections surveillance and research branch (promis p0095_21_ky, 2021) (silver springs, maryland, united states). the funders had no role in data collection and analysis, the decision to publish, or preparation of the manuscript. data availability all data are included in the manuscript. disclaimer material has been reviewed by the walter reed army institute of research. there is no objection to its publication. the opinions or assertions contained herein are the private views of the authors, and they are not to be construed as official, or as reflecting true views of the department of the army or the department of defense. references ministry of health. first case of coronavirus disease confirmed in kenya [homepage on the internet]. 2020 [cited 13 march 2020]. available from: https://www.health.go.ke/first-case-of-coronavirus-disease-confirmed-in-kenya/ hasell j, mathieu e, beltekian d, et al. a cross-country database of covid-19 testing. sci data. 2020;7:345. https://doi.org/10.1038/s41597-020-00688-8 bugembe d, kayiwa j, phan m, et al. main routes of entry and genomic diversity of sars-cov-2, uganda. emerg infect dis. 2020;26(10):2411–2415. https://doi.org/10.3201/eid2610.202575 who. tracking sars-cov-2 [homepage on the internet]. 2022 [cited 2022 june]. available from: https://www.who.int/activities/tracking-sars-cov-2-variants?msclkid=c2729b29d10811eca952b5e97b4da00a kidd m, richter a, best a, et al. s-variant sars-cov-2 lineage b1.1.7 is associated with significantly higher viral loads in samples tested by thermo fisher taqpath rt-qpcr. j infect dis. 2021;223(10):1666–1670. https://doi.org/10.1093/infdis/jiab082 hill kj, dewar r, templeton k. a multiregional evaluation of ct values in sars-cov-2 voc-20dec-01 variant. j infect dis. 2021;224(5):927–928. https://doi.org/10.1093/infdis/jiab303 karim ssa, karim qa. omicron sars-cov-2 variant: a new chapter in the covid-19 pandemic. lancet. 2021;398(10317):2126–2128. https://doi.org/10.1016/s0140-6736(21)02758-6 hay ja, kennedy-shaffer l, kanjilal s, et al. estimating epidemiologic dynamics from cross-sectional viral load distributions. sci. 2021;373(6552):eabh0635. https://doi.org/10.1126/science.abh0635 critical preparedness, readiness and response actions for covid-19 [homepage on the internet]. world health organization; 2021 [cited 27 may 2021]. available from: https://www.who.int/emergencies/diseases/novel-coronavirus-2019/technical-guidance/critical-preparedness-readiness-and-response-actions-for-covid-19 abstract introduction methods results discussion acknowledgements references about the author(s) vicky cuylaerts institute of tropical medicine, department of clinical sciences, sti reference laboratory, antwerp, belgium irith de baetselier institute of tropical medicine, department of clinical sciences, sti reference laboratory, antwerp, belgium claude m. muvunyi college of medicine and health sciences, university of rwanda, kigali, rwanda lambert mwambarange legacy clinics and diagnostics, kigali, rwanda hilde smet institute of tropical medicine, department of clinical sciences, sti reference laboratory, antwerp, belgium john rusine national reference laboratory, ministry of health, kigali, rwanda viateur musengamana rinda ubuzima, kigali, rwanda janneke van de wijgert department of clinical infection, microbiology and immunology, institute of infection and global health, university of liverpool, liverpool, united kingdom tania crucitti institute of tropical medicine, department of clinical sciences, sti reference laboratory, antwerp, belgium citation cuylaerts v, de baetselier i, muvunyi cm, et al. implementation and evaluation of the presto combined qualitative real-time assay for chlamydia trachomatis and neisseria gonorrhoeae in rwanda. afr j lab med. 2019;8(1), a739. https://doi.org/10.4102/ajlm.v8i1.739 original research implementation and evaluation of the presto combined qualitative real-time assay for chlamydia trachomatis and neisseria gonorrhoeae in rwanda vicky cuylaerts, irith de baetselier, claude m. muvunyi, lambert mwambarange, hilde smet, john rusine, viateur musengamana, janneke van de wijgert, tania crucitti received: 19 dec. 2017; accepted: 05 oct. 2018; published: 18 apr. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the presto combined qualitative real-time assay for chlamydia trachomatis and neisseria gonorrhoeae (presto ct/ng pcr assay) is appealing for developing countries, because it can be used with multiple dna extraction methods and polymerase chain reaction (pcr) platforms. objectives: the objective of the study was to implement and evaluate the presto ct/ng pcr assay at the national reference laboratory (nrl) in kigali, rwanda, where no real-time pcr assays for the detection of c. trachomatis or n. gonorrhoeae were available. methods: the presto ct/ng pcr assay was first evaluated at the institute of tropical medicine (itm) in antwerp, belgium. next, nrl laboratory technicians were trained to use the assay on their abi prism 7500 real-time pcr instrument and their competencies were assessed prior to trial initiation. during the trial, endocervical swabs were tested at the nrl, with bi-monthly external quality control testing monitored by the itm. the final nrl results were evaluated against extended gold standard testing at the itm, consisting of the abbott m2000 realtime system with confirmation of positive results by an in-house real-time pcr assay for c. trachomatis or n. gonorrhoeae. results: of the 192 samples analysed using the presto assay at the nrl, 16 samples tested positive for c. trachomatis and 17 tested positive for n. gonorrhoeae; four of these were infected with both. the sensitivity and specificity of the presto assay were 93.3% (95% confidence interval [ci]: 68.1% – 99.8%) and 99.4% (95% ci: 96.8% – 100%) for c. trachomatis and 100% (95% ci: 76.8% – 100%) and 98.8% (95% ci: 95.8% – 99.9%) for n. gonorrhoeae. conclusion: c. trachomatis and n. gonorrhoeae testing with the presto assay was feasible in kigali, rwanda, and good performance was achieved. keywords: qpcr; chlamydia trachomatis; neisseria gonorrhoeae. introduction chlamydia trachomatis and neisseria gonorrhoeae infections are the most common sexually transmitted bacterial infections worldwide.1 both are frequently asymptomatic in women, which increases the risk of undiagnosed and untreated infections and of onward transmission. untreated infections may lead to serious complications such as endometritis, salpingitis, pelvic inflammatory disease, chronic pelvic pain, ectopic pregnancy and infertility.1,2 in order to treat c. trachomatis and n. gonorrhoeae infections adequately, they need to be detected early and accurately.3 highly sensitive and specific nucleic acid amplification assays to identify c. trachomatis and n. gonorrhoeae infections are commercially available.4,5 as compared to culture, the detection rates with these assays are higher. in addition, they do not require stringent sample transport conditions, because viable organisms are not necessary and they are less cumbersome compared to, for example, chlamydia intracellular culture. over the past decades, dual detection systems for c. trachomatis and n. gonorrhoeae based on polymerase chain reaction (pcr) have been developed. among them is the presto combined qualitative real-time assay for c. trachomatis and n. gonorrhoeae (presto ct/ng pcr assay; goffin molecular technologies, houten, the netherlands). the presto ct/ng pcr assay is characterised by its flexibility in use of sample types (with or without transport medium), dna can be isolated using any system and it can be implemented on different real-time pcr amplification platforms.6,7 in addition, it provides faster results and considerably reduces the risk of contamination by amplicons compared to end-point pcr. the assay includes an internal amplification control (iac) to monitor both nucleic acid extraction and amplification efficiency. clinical samples may contain inhibitory substances which are not always reliably removed during sample preparation. the specifically designed iac will not only detect pcr inhibition but also inefficient dna isolation from each individual sample. the iac consists of an inactivated escherichia coli containing a modified genomic dna fragment with primer binding sites identical to the c. trachomatis and n. gonorrhoeae sequences. detection of the amplified sequence, which is different from the c. trachomatis and n. gonorrhoeae amplicon fragments is done by a probe sequence with a different reporter dye. the presto assay also includes a c. trachomatis and a n. gonorrhoeae selector. these selectors facilitate the detection of concurrent infections, especially when the high concentration of one infection (or target) masks the presence of the other. in other words, they are meant to select for either c. trachomatis or n. gonorrhoeae in a clinical sample with a strong positive c. trachomatis or n. gonorrhoeae result. the selector blocks the highly concentrated target present in the specimen and enhances the amplification of the other not-blocked target, if present. another concern that has arisen in recent years is the detection of the c. trachomatis swedish variant strain. this is a c. trachomatis variant with a deletion in the cryptic plasmid.8 amplification assays targeting the cryptic plasmid may therefore miss the detection of the c. trachomatis swedish variant.8 according to the manufacturer, the presto assay detects the c. trachomatis swedish variant strain. real-time pcr assays for the detection of c. trachomatis and n. gonorrhoeae were not available at the national reference laboratory (nrl) in kigali, rwanda, in 2013. we installed the presto assay at the nrl in the context of a clinical trial (ring plus study) funded by the european & developing countries clinical trials partnership.9,10 we report here on the procedures followed for implementation of the presto assay and its performance at the nrl. methods ethical considerations the evaluation of the presto assay was embedded in the clinical trial (ring plus study; clinicaltrials.gov nct01796613), which was conducted at rinda ubuzima research center, kigali, rwanda, from may 2013 until march 2014. the clinical trial protocol has been published elsewhere.9 the protocol and all study documents were reviewed and approved by the institutional review board of the institute of tropical medicine (itm) (864/13), the ethics committee of the university hospital of antwerp (13/7/85), the rwanda national ethics committee (122/rnec/2014), the national health research committee (nhrc/2013/prot/0054) and the rwandan ministry of health (20/2774/phis/me&r/2013). all participants were between 18 and 35 years old and provided written informed consent prior to participation in the trial. evaluation at the institute of tropical medicine, antwerp, belgium prior to the start of the clinical trial in rwanda, the presto assay was evaluated at the itm in antwerp, belgium. a 1:10 dilution series of a c. trachomatis serovar l2 and a n. gonorrhoeae strain in diluted phosphate buffered saline (dpbs) was tested using the presto assay, as well as an extended gold standard consisting of the abbott m2000 realtime system (lake forest, illinois, united states) with confirmation of positive results by in-house real-time pcr assays for c. trachomatis and n. gonorrhoeae. the added value of the selectors in the presto assay was evaluated on five aliquots containing n. gonorrhoeae strains in dpbs in a final concentration of 3 × 105 colony forming units (cfu) per pcr reaction and five aliquots of c. trachomatis serovar l2 strains diluted in dpbs in a final concentration of 9 × 104 elementary bodies (eb) per pcr reaction. in addition, the aliquots were spiked with a 10-fold dilution series of low concentrations of c. trachomatis l2 (range: 102 – 10-2 eb per pcr reaction) and n. gonorrhoeae (range: 3 × 102 – 3 × 10-2 cfu per pcr reaction). the c. trachomatis selector was added to the highly concentrated n. gonorrhoeae-positive suspensions (3 × 105 cfu per pcr) and the n. gonorrhoeae selector was added to the highly concentrated c. trachomatis-positive suspensions (9 ×******* 104 eb per pcr). a quality control for molecular diagnostics (qcmd) panel, consisting of 10 samples for c. trachomatis (qcmd 2011) and 10 samples for n. gonorrhoeae (qcmd 2010), was also tested with the presto assay and the extended gold standard. finally, the analytical specificity for n. gonorrhoeae was evaluated by testing eight different non-gonococcal neisseria species: n. meningitidis, n. lactamica, n. pharingis, n. animalis, n. caviae, n. ovis, n. subflava and n. perflava. specimen collection, processing and transport several sexually transmitted infection assays were performed at the clinical trial’s baseline visit,10 including the presto ct/ng pcr assay on endocervical swabs (flocked swabs, copan technologies srl, brescia, italy) that were collected by a study physician during a speculum examination. the dry swabs were eluted in the on-site rinda ubuzima laboratory by adding 1.2 ml dpbs directly onto the swabs. the dpbs was prepared by dissolving 0.96 g phosphate buffered saline (pbs) and 7.65 g nacl in 1l molecular biology water (mbw) and sterilised by filtration. after vortexing the swabs for 15 seconds, two aliquots of 550 µl were prepared: one was stored at 2 °c – 8 °c until transportation in a temperature-controlled cool box with cooling elements to nrl within 1 week of sample collection (or at -20 °c in case transportation was delayed) and the other at -20 °c until transportation in a temperature-controlled dry shipper to the itm. the presto assay was performed at the nrl within two working days after sample receipt according to the manufacturer’s instructions. training and external quality control itm and nrl staff wrote a standard operating procedure together, and hands-on training was conducted by itm staff in the nrl laboratory. the competence of the trained nrl technicians was evaluated using a blind specimen panel consisting of a positive c. trachomatis sample, a positive n. gonorrhoeae sample, a sample positive for both c. trachomatis and n. gonorrhoeae and a negative sample. external quality control panels were provided by the itm on a bi-monthly basis throughout the study. each panel contained a strong and a weak-positive c. trachomatis sample, a strong and a weak-positive n. gonorrhoeae sample, a sample concurrently infected with both c. trachomatis and n. gonorrhoeae and a sample negative for both c. trachomatis and n. gonorrhoeae. all samples with the exception of the weak-positive samples were expected to be correctly identified by the nrl. weak-positive samples were allowed to be missed: they were included in the panel to encourage the nrl to excel in their competence. chlamydia trachomatis and neisseria gonorrhoeae testing using the presto assay the presto assay targets the c. trachomatis cryptic plasmid and the opa gene of n. gonorrhoeae. assay procedures were performed in physically separated rooms including a sample preparation room, a dna-free area, and an amplification room. the qiaamp dna mini kit (qiagen, hilden, germany) was used for the dna extraction of 400 µl of aliquot, following the buccal swab spin protocol. before extraction, 5 µl of the iac was added to every sample tube. after adding dna of samples and controls to the master mix, the reaction plate was loaded into the abi prism 7500 instrument (applied biosystems™, foster city, california, united states) with the following settings: fixed threshold, 0.01; activation polymerase, 30 sec at 95 °c; number of cycles, 40; denaturation, 3 sec at 95 °c; annealing, extension and exonuclease activity, 30 sec at 60 °c. if a positive signal with cycle threshold (ct) value lower than 35 was obtained for either c. trachomatis or n. gonorrhoeae, the pcr was repeated with 1 µl of a selector for n. gonorrhoeae or c. trachomatis and 9 µl of dna to check for the presence of the other pathogen. specimens were considered negative when the ct value of the specimen was greater than 40 and iac was 37 or less, positive when the ct value of the specimen was 35 or less for c. trachomatis or n. gonorrhoeae with any ct value of the iac, and equivocal when the ct value of the specimen was between 35 and 40 and a ct value of the iac was less than 40. specimens were considered inhibited when the ct value of the specimen was greater than 40 and the ct value of the iac was greater than 37. the assay kit contained two positive and one negative control for test-run validation which were interpreted according to the manufacturer’s instructions (cg 160501, rev05, 10/2011). extended gold standard at the itm, dna extraction and subsequent amplification was performed using the abbott m2000 real time system (lake forest, illinois, united states) according to the manufacturer’s instructions. c. trachomatis-positive samples were confirmed with an in-house real-time pcr based on the publication by chen et al.11 and n. gonorrhoeae-positive samples were confirmed with an in-house real-time pcr based on the publication by hopkins et al.12 confirmation of the samples was carried out on the same extracts as those used on the abbott system and amplification was performed on the rotor-gene 6000 (qiagen, hilden, germany). statistical methods for both c. trachomatis and n. gonorrhoeae, samples were defined as true positive if identified positive with both the abbott assay and the in-house real-time pcr. samples were defined as true negative when found to be negative in the abbott assay or when a positive result could not be confirmed by the in-house real-time pcr. prevalence at baseline was calculated for c. trachomatis and n. gonorrhoeae based on the extended gold standard. sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) of the presto assay were calculated at baseline with the extended gold standard as reference. equivocal results in the presto assay were considered as negative in the above mentioned calculations. wilson binomial 95% confidence intervals (ci) were calculated for the sensitivity, specificity, ppv and npv. results evaluation at the institute of tropical medicine evaluation of the presto assay against the extended gold standard at the itm showed that the analytical sensitivities of the presto assay were in the same order of magnitude (100 eb/ml for c. trachomatis, 330 cfu/ml for n. gonorrhoeae versus 86 eb/ml for c. trachomatis, 280 cfu/ml for n. gonorrhoeae). the analytical sensitivity of the presto pcr increased by 2 log10 or 100 fold when a c. trachomatis selector was added to the strong-positive n. gonorrhoeae suspensions. the ct value of the iac decreased, reducing the inhibition. no difference in sensitivity was obtained by using a n. gonorrhoeae selector on strong-positive c. trachomatis suspensions, although the ct value of the iac also decreased. no effect of the use of selectors was obtained when strong c. trachomatis-positive clinical samples were spiked with low concentrations of n. gonorrhoeae or when strong n. gonorrhoeae-positive clinical samples were spiked with low concentrations of c. trachomatis. the qcmd panel included eight positive and two negative c. trachomatis samples. six samples tested c. trachomatis-positive by the presto assay. of the two low-positive c. trachomatis samples that were not detected by the presto assay, one was also missed using the extended gold standard. the qcmd panel included a c. trachomatis swedish variant, which was successfully detected with the presto assay. the qcmd panel for n. gonorrhoeae included seven positive and three negative n. gonorrhoeae samples, of which one was neisseria cinerea. six samples tested positive in the presto assay. one sample was not tested due to insufficient volume to perform the test. the three n. gonorrhoeae-negative samples tested negative in the presto assay. no cross reactivity was detected with the other eight neisseria species. implementation of the presto assay at the national reference laboratory the results of the blind specimen panel used for competency check were 100% concordant. the bi-monthly external quality control panels also produced satisfactory results: two c. trachomatis weak-positive samples were missed with the presto assay, and one n. gonorrhoeae-positive sample result was equivocal. clinical trial results baseline samples from 185 women were tested for c. trachomatis and n. gonorrhoeae by the presto assay. additional samples from seven women were tested at follow-up visits. the results are summarised in table 1. table 1: presto ct/ng pcr assay results, national reference laboratory, kigali, rwanda, may 2013 -march 2014. all study samples (n = 192) were retested according to the extended gold standard algorithm at the itm. two samples were excluded from data analysis as they contained pcr inhibitors: one sample was inhibited in the presto assay and one sample was inhibited in the abbott assay. the presto assay missed one c. trachomatis-positive sample, although this was equivocal in the assay. it falsely detected three n. gonorrhoeae and one c. trachomatis. the latter c. trachomatis sample was positive on the abbott assay but could not be confirmed using the in-house real-time pcr (table 2). table 2: cross-tabulation of the presto ct/ng pcr assay and extended gold standard chlamydia trachomatis and neisseria gonorrhoeae results for chlamydia trachomatis national reference laboratory, kigali, rwanda, may 2013 -march 2014. at baseline, the overall prevalence was 8.1% for c. trachomatis and 7.0% for n. gonorrhoeae. the sensitivity, specificity, ppv and npv of the presto assay are summarised in table 3. table 3: diagnostic characteristics of the presto assay national reference laboratory, kigali, rwanda, may 2013 – march 2014. discussion we successfully implemented the presto combined qualitative real-time assay for chlamydia trachomatis and neisseria gonorrhoeae at the nrl, kigali, rwanda. the implementation was a stepwise process covering hands-on training and writing of standard operating procedures. the competency of the laboratory technicians in the execution of the presto assay was confirmed through the 100% concordant results obtained for the testing of the blind specimen panel. the testing of the bi-monthly external quality control panels allowed us to monitor and advise the nrl on testing for c. trachomatis and n. gonorrhoeae using the presto assay. in the end, we were able to successfully install the presto assay at the nrl and to guarantee the quality of the results during the clinical trial. in addition, the quality control procedures and retesting of samples at itm may contribute to the method validation that is required for laboratory accreditation. the nrl may also use a similar implementation approach in the event the amplification testing for c. trachomatis and n. gonorrhoeae should be used in other settings such as for diagnosis or for research or clinical trials. the analytical sensitivity of the presto assay for c. trachomatis and n. gonorrhoeae was comparable to that of the extended gold standard. previous versions of pcr assays for n. gonorrhoeae showed cross reactivity and amplification of non-gonorrhoeae neisseria species,13 which increased the ratio of false-positive n. gonorrhoeae detection and resulted in unnecessary treatment. superfluous antibiotic use increases the risk of untreatable n. gonorrhoeae due to multidrug resistance and should be maximally prevented.14 in the brief evaluation study of the presto assay at the itm, none of the non-gonorrhoeae neisseria species was amplified by the presto assay, confirming its analytical specificity. however, the ppv for n. gonorrhoeae detection using the presto assay was under 90%, which necessitates a confirmation test for n. gonorrhoeae as recommended by the international union against sexually transmitted infections (iusti) guidelines.15 ppvs are influenced by prevalence and specificity of the assay, and in this study the lower ppv was mainly caused by two n. gonorrhoeae false-positive samples. the testing for c. trachomatis and n. gonorrhoeae described in this manuscript was part of a clinical trial which was conducted according to good clinical and laboratory practice requirements.9 the delivered results were quality assured, and the traceability and transparency of the test procedures and handlings within the laboratory were guaranteed. the obtained and reported sensitivity and specificity reflect the potentially achievable performance of the assay in a quality-assured environment within the nrl in kigali, rwanda. our study population was characterised by a high prevalence of both c. trachomatis and n. gonorrhoeae. almost one quarter of the infections were dual infections, highlighting the need for a dual detection system in this setting. the advantages of a dual detection system are staff time and assay cost reduction while two detection results are delivered. notwithstanding this study’s small sample size, the presto assay’s sensitivity and specificity for c. trachomatis detection was in line with two previous studies.6,7 we cannot explain the lower specificity found for n. gonorrhoeae detection; we did not obtain cross reactions with the non-gonorrhoeae neisseria species, and we therefore advise that a possible lower specificity should be confirmed in future studies. a total of eight equivocal results (4.2%) were found during this small evaluation. in clinical practice, clinicians should be instructed to collect another sample for retesting. the result will be reported as equivocal in the event of a second equivocal result and conclusion regarding the diagnosis cannot be drawn, or as positive or negative, if the second test result is positive or negative. to date no reports have been found on the current presence of the c. trachomatis swedish variant strain in rwanda or africa, but it has become a global requirement that newly designed pcr assays for c. trachomatis detection also detect the c. trachomatis swedish variant.8 although the c. trachomatis target of the presto assay lies within the c. trachomatis cryptic plasmid, the manufacturer states that the c. trachomatis swedish variant will be detected. this statement was confirmed by testing a qcmd panel which included a sample with the c. trachomatis swedish variant. the laboratory and etiological diagnosis of c. trachomatis and n. gonorrhoeae is scarcely available and problematic in resource-poor settings. to date many settings have adopted the syndrome-based management approach.16 however, limitations of the vaginal discharge algorithm, and in particular the management of gonococcal and chlamydia infections, has been recognised.17,18 the approach guides the diagnosis of c. trachomatis and n. gonorrhoeae based on symptoms and signs.14,19 per definition, asymptomatic infections are not recognised and not treated; on the other hand, symptomatic treatment increases the risk of overtreatment.20 although attempts have been made to develop rapid diagnostic assays for the detection of c. trachomatis and n. gonorrhoeae, none, except one point-of-care test, the cepheid genexpert ct/ng assay, is sufficiently accurate to be useful in an etiological diagnosis.21,22 the results of our study and feedback from the nrl staff suggest that the presto assay can be used in a molecular laboratory within a resource-limited context. the performance of the assay is comparable to other commercial amplification assays and has the advantage that it can be run on any amplification platform available in standard molecular laboratories, thus avoiding extra investment costs. at the time of this evaluation, we paid €17.00 per sample including all consumables and reagents. however, the manufacturer recently revised the presto kit prices; the current cost per sample should be around €10 inclusive of consumables and reagents. the genexpert is the only molecular assay for c. trachomatis and n. gonorrhoeae that may outcompete the presto assay in developing country settings. it is faster, but not as flexible, and it requires its own genexpert equipment, which is widely used in developing countries’ laboratories for tuberculosis diagnostic testing. still, the reagent for the genexpert ct/ng assay is more expensive compared to the presto assay. limitations an initial evaluation of the presto assay was done in belgium at the itm. ideally, this should have been performed in rwanda, the country of use. however, at that time a pcr assay for the detection of c. trachomatis and n. gonorrhoeae was not applied at the nrl in kigali, and well-characterised reference, clinical or quality control panels were not available. we acknowledge that the inability to initially evaluate the presto assay in rwanda is a limitation of the study. conclusions in conclusion, testing for c. trachomatis and n. gonorrhoeae with the presto assay was easily implemented and feasible in kigali, rwanda. overall, a good performance of the assay was achieved, but our results suggest that positive presto n. gonorrhoeae results may need confirmation using a nucleic acid amplification assay with a different amplification target. further field evaluations are recommended to confirm our findings. acknowledgements we would like to thank all women who participated in the ring plus study and all the ring plus staff and laboratory technicians of national reference laboratory and institute of tropical medicine for their dedicated collaboration. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. sources of support the main study was funded by the european & developing countries clinical trials partnership through a project entitled ‘preparing for clinical trials with vaginal rings that protect women from hiv and unintended pregnancy’ (grant code sp.2011.41304.043), the university of liverpool and the institute of tropical medicine. authors’ contributions v.c. and t.c. wrote the first draft of the manuscript. i.d.b., c.m.m., j.r. and j.v.d.w. revised and edited the text. h.s. designed and performed the evaluation at institute of tropical medicine. v.c. provided the hands-on training at the nrl and supervised the elaboration of the standard operating procedures and quality control procedures. l.m. and v.m. generated the data. all authors revised and approved the present version of the manuscript. references unemo m, bradshaw cs, hocking js, et al. sexually transmitted infections: challenges ahead. lancet infect dis. 2017;17(8):e235–e279. https://doi.org/10.1016/s1473-3099(17)30310-9 tsevat dg, wiesenfeld hc, parks c, peipert jf. sexually transmitted diseases and infertility. am j obstet gynecol. 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author(s) natasha gous global health, systemone, llc, johannesburg, south africa alaine u. nyaruhirira management sciences for health, pretoria, south africa bradford cunningham strategic initiatives, systemone, llc, johannesburg, south africa chris macek business development, systemone, llc, northampton, massachusetts, united states citation gous n, nyaruhirira au, cunningham b, macek c. driving the usage of tuberculosis diagnostic data through capacity building in lowand middle-income countries. afr j lab med. 2020;9(2), a1092. https://doi.org/10.4102/ajlm.v9i2.1092 lessons from the field driving the usage of tuberculosis diagnostic data through capacity building in lowand middle-income countries natasha gous, alaine u. nyaruhirira, bradford cunningham, chris macek received: 04 sept. 2019; accepted: 12 aug. 2020; published: 18 nov. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: connectivity platforms collect a wealth of data from connected genexpert instruments, with the potential to provide valuable insights into the burden of disease and effectiveness of tuberculosis programmes. the challenge faced by many countries is a lack of training, analytical skills, and resources required to understand and translate this data into patient management and programme improvement. objective: we describe a novel training programme, the tuberculosis data fellowship, designed to build capacity in lowand middleincome countries for tuberculosis data analytics. methods: the programme consisted of classroom and remote training plus mentorship over a 12-month period. the focus was on skills development in tableau software, followed by training in exploration, analysis, and interpretation of genexpert tuberculosis data across five key programme areas: patient services, programme monitoring, quality of testing, inventory management, and disease burden. results: the programme was piloted in six countries (bangladesh, ethiopia, ghana, malawi, mozambique) in july 2018 and nigeria in september 2018; 20 participants completed the training. a number of key outputs have been achieved, such as improved instrument utilisation rates, decreased error rates, and improved instrument management. conclusion: the training programme empowers local tuberculosis programme staff to discover and fix critical inefficiencies, provides high-level technical and operational support to the tuberculosis programme, and provides a platform for continued sharing of insights and best practices between countries. it supports the notion that connectivity can increase efficiencies and clinical benefits with better data for decision making, if coupled with commensurate capacity building in data analysis and interpretation. keywords: tuberculosis; genexpert; diagnostic data; monitoring and evaluation; data analysis; programmatic. introduction tuberculosis has been declared a global public health emergency. an estimated one-third of the world’s population is infected with tuberculosis; 10 million people developed tuberculosis disease in 2017 alone,1 a number that may be underestimated due to under-reporting and lack of reliable data. the widespread implementation of the xpert® mtb/rif assay (cepheid, sunnyvale, california, united states) for detection of mycobacterium tuberculosis and rifampicin resistance as a first-line tuberculosis diagnostic, has been hailed as the most significant advancement in decades, becoming the first molecular assay to provide a tuberculosis and first-line drug resistance diagnosis in just 2 hours. following widespread adoption of this technology, the world health organization’s agenda for action on digital health for the end tuberculosis strategy called for 100% of all sites using rapid tuberculosis diagnostic instruments to be connected by 2020,2 becoming the first to recognise the role of digital health in the fight against tuberculosis. over the past 2 to 3 years, numerous countries have begun adopting connectivity platforms to help monitor and manage their genexpert fleet by collecting the vast amounts of rich clinical diagnostic and operational data produced by the instrument. rarely before has such a rich data resource been both produced by a diagnostic instrument and been made available via connectivity platforms, at scale, for analysis. as yet, it remains largely untapped.3 if these data can be analysed, interpreted and translated into appropriate recommendations and actions, they have the potential to provide significant transformative impact on the management and effectiveness of infectious disease programmes worldwide. gxalert® (systemone, llc, northampton, massachusetts, united states) is currently collecting data from genexpert platforms in 43 countries running cepheid’s xpert mtb/rif assay. gxalert is a connectivity platform that integrates directly with diagnostic instruments to collect and send a digital copy of test results and associated instrument metadata to an in-country or gxalert server. from there, results can be sent and accessed via short message service and email alerts, microsoft excel (microsoft corp, redmond, washington, united states) reports and web dashboards. the type of data being collected includes not only the diagnostic result, but also information on when and where the test was run and by whom, demographic information about the patient (through an application called gxconnect), reagent lot numbers, probe data, cycle thresholds as well as instrument operational data such as instrument failures, inventory consumption and instrument downtime. from these data, critical insights can be gained or inferred about the tuberculosis programme and can help shed light on both clinical and operational return on investment. data can also provide useful information on testing coverage, disease status and trends, circulating strains and drug resistance profiles, instrument utilisation rates, training needs, supply chain, inventory, and quality of the testing programme.4 but there is a challenge: even though countries now collect this type of data in large volumes, it is a new arena for them. most high-disease burden countries lack the tools, resources and expertise required to analyse, understand and translate these data into improved programme and patient outcomes. a recent study by the foundation for innovative diagnostics (find), found that despite large investments by donors to implement electronic data management systems, there is limited usage of the data to improve service delivery, mainly due to a lack of understanding and awareness of what data means.5 as a result, tuberculosis programmes are accumulating but not using the data being collected to drive decision making. this becomes apparent when one considers the various challenges still hindering tuberculosis programmes today, including gross under-utilisation of instruments,3,6,7,8 high unsuccessful test and error rates (loss of tests),9,10 cartridge stock-outs, instrument breakdowns, and lack of adequate module replacements and maintenance of instruments.11 there is a dire need to build capacity in health data analytical skills amongst staff within national tuberculosis programmes (ntps) in order to bolster the usage of data. to address this need, we designed a novel training programme to develop the expertise and skills required for the analysis and understanding of connected diagnostic data. the tuberculosis data fellowship programme the tb data fellowship (tdf) programme was initiated in 2018 through a joint collaboration between systemone and management sciences for health, with the support of the tableau foundation. designed to build the foundation for sustainable in-country capacity, the programme aimed to enhance the understanding of tuberculosis data and its translation into actionable outputs. achieving these goals required a new cadre of healthcare worker to be trained, one with the ability to understand and interpret the vast amounts of diagnostic and operational data being collected through connected diagnostic systems. for the pilot programme, staff from the ntps, national tuberculosis reference laboratories and ministries of health from several countries using the gxalert connectivity platform were invited to apply. countries invited included bangladesh, ethiopia, ghana, malawi, mozambique and nigeria. the selection criteria for the programme included a minimum of 2 years’ work experience in the field of tuberculosis, and more specifically the genexpert tuberculosis programme, and at least 6 months of work experience with gxalert software. participants also had to have experience working in excel. informatics infrastructure the programme leveraged the existing connectivity infrastructure, gxalert, to gain access to live xpert mtb/rif data. in addition, each participant was provided with a tableau desktop and tableau online licence (tableau, seattle, washington, united states). tableau is a powerful data visualisation and analytics software package that is specifically aimed at helping people understand large amounts of data through the creation of structured storyboards, dashboards and visual representations. systemone developed the server architecture to enable participants to extract live country data from gxalert and to import it into tableau (figure 1). this allowed participants to safely interact with data, enforce the necessary patient privacy and country-specific data permissions, and generate visualisations to allow them to share this with the ntp, neighbouring disease programmes, the ministry of health or donors, via tableau online (figure 1). figure 1: general informatics infrastructure and data flow for tb data fellowship programme. live xpert mtb/rif data from each country is collected via gxalert and stored on a in-country or private cloud hosted gxalert server (depending on country preference). each data fellow is able to download an extract of their own country data to tableau desktop in order to create various graph-like visualisations and basic analytics. once analysis is complete, data fellows could choose to publish a subset of these visualisations, unlinked to the data source, via a community folder on tableau online, allowing them to share insights, ideas and graphics with other data fellows, the ministry of health or ntp. training structure two pilot training programmes were undertaken: the first accepted participants from five countries supported by the global challenge tb project, funded by the united states agency for international development. the first countries were bangladesh (n = 2), ethiopia (n = 2), mozambique (n = 2), ghana (n = 1) and malawi (n = 1). the second programme accepted participants from nigeria only (n = 12). the training lasted 12 months and was structured in two parts: a 1-week in-person classroom training followed by an 11-month remote training and mentorship. classroom training the in-person classroom training consisted of a 1-week intensive centralised training, the first of which was held in johannesburg, south africa from 9–13 july 2018, and the second in abuja, nigeria from 24–28 september 2018. during the first three days of each session, participants were trained extensively on tableau desktop v2018.1 software (tableau, seattle, washington, united states). this was followed by two days of training on how to integrate and interpret gxalert tuberculosis data in tableau, perform basic data analysis, and prepare visualisations to improve the interpretation and reporting of tuberculosis data. remote training and mentorship for 11 months following the classroom training, participants received monthly training and mentorship remotely via 2-hour skype sessions as two separate groups, depending on which classroom training session they attended. systemone designed these sessions to allow in-depth data analysis and development of visualisations to improve understanding, with a focus on the development of data-driven recommendations for programmatic improvement. to promote a platform for sharing of data, insights and best practices, each month participants were required to complete assignments and publish their developed visualisations and insights on the tableau online server for the entire group to see. this encouraged collaboration among members of the groups. focus areas of the training through the 12-month programme, participants were taught how to understand genexpert tuberculosis diagnostic and operational data being collected by the gxalert platform and to translate this data into insights about their respective tuberculosis programmes (table 1). they identified programme gaps and appropriate intervention needs in different programme areas (table 1), while learning how to lower operating costs (by reducing supervision frequency and troubleshooting services), improve the quality of the programme and manage the programme more effectively. table 1: key topics covered during the 2018 tb data fellowship training. key insights from the programme the training has yielded new country-level insights and programme improvements due to improved data use and decision making as demonstrated by numerous technical reports, conference abstracts and presentations. for example, in bangladesh, participants have used data about instrument utilisation rates to influence the ntp to improve referral mechanisms for underperforming sites and further optimise genexpert placement within their ntp to better meet testing demand.12 these insights have also helped the ntp plan for future placement of additional genexpert machines. in ethiopia, analysis of instrument utilisation and subsequent proactive monitoring have led to an improvement in utilisation, from 28% to 75%.13 such dramatic improvements can translate immediately into programme return on investment – whereby tuberculosis programmes can make existing resources go much farther than expected and deploy resources more effectively when receiving future grants or allocating domestic budgets. by teaching participants how to monitor unreportable test rates or the number of tests resulting in errors, no results and invalid results, programme efficiency and response speed can be improved. unreportable or unsuccessful tests do not provide a clinically valid result to the patient and thus need to be repeated. besides the cost in ‘lost’ cartridges, when one considers that the actual cost per test performed has been estimated at $23.00 (united states dollors [usd]) and the cost per diagnosis at $99.00 usd,14 unsuccessful tests represent a significant cost to the health system. the majority of unsuccessful tests are due to error results and can, to a large extent, be corrected. unfortunately, countries seldom know how to interpret error codes to inform appropriate corrective actions. the tdf helped participants categorise error codes according to their suspected sources and, through doing this, identify the most frequent types of errors to troubleshoot while pinpointing the sites needing supervision and follow-up. this real-time support is less costly compared to conventional monitoring, which requires a person to visit sites to troubleshoot issues, without any understanding of which issues pertain to which sites. across all participating countries, the majority of errors were user or technical errors. these errors are associated with incorrect specimen processing or volumes added to the cartridge.15 for example, in ghana, up to 67% of error results were identified as user related, and this insight has led to the introduction of refresher training and targeted supervision for sites.16 the same issue was identified in nigeria and bangladesh, where both programmes have managed to reduce their national error rates due to targeted supervision, refresher training for laboratory staff and regular feedback to laboratories aimed at addressing the high incidence of these user related errors.12,17 another focus area of the tdf training that has led to significant programmatic improvement is the monitoring of testing fleet and instrument downtime. a challenge faced by many tuberculosis programmes is that genexpert instruments are often located at remote facilities, leading to delays in maintenance and replacement of broken modules. by monitoring trends in how instruments report in real-time through connectivity tools, the bangladesh ntp are now identifying directly when modules are down or instruments require calibration. through this real-time monitoring of instrument performance, bangladesh has managed to reduce instrument maintenance turn-around time from anywhere between 5 and 14 months to just 2 weeks, and is now also maintaining 90% module functionality.12 ethical considerations ethical clearance was not required for this study. discussion various connectivity solutions exist to collect diagnostic data, some of which have already been adopted widely. connectivity tools can play a major role in addressing many of the challenges that tuberculosis programmes face by facilitating the central collection and aggregation of diagnostic instrument data so that it can be analysed. however, the introduction of connectivity tools is not sufficient to ensure improved programme management. well-functioning health systems need to utilise this data at all levels in order to drive evidence-based decisions and interventions to improve the quality of care provided.18 to our knowledge, the tdf programme is the first of its kind to build capacity and resources in lowand middle-income countries for the analysis of tuberculosis data collected from connected diagnostics. to address sustainability, the programme provided participants with the much-needed tools required to drive data analytics, counting on participants to lead the ongoing analysis of tuberculosis-related health data from the national genexpert programme in their respective countries. we chose tableau software as a data analytics and visualisation tool, because it enables users to explore, manipulate and create visual representations of large amounts of data in order to produce insights as well as to communicate those insights to a broader audience. we leveraged an existing connectivity footprint, namely the gxalert system (systemone, llc, northampton, massachusetts, united states), to gain access to genexpert tuberculosis data within each respective country, but the programme is translatable to any connectivity platform collecting tuberculosis genexpert data. while the initial pilot programme focused on tuberculosis, the intent is to expand into related disease streams and diagnostics within the ministry of health, such as hiv. by providing an online tool (tableau online) where participants could post their visualisations and insights, the programme also provides a platform whereby countries can share best practices and help to create value. the tdf programme has already seen rapid development and analysis of country key performance indicators leading to immediate publications, programme engagements and strengthening.12,13,16,17 by using the data to recognise programme gaps and identify needs, issues and priorities, participants have been equipped to help develop their national strategies, address challenges and inform data-driven decision making. conclusion the programme empowers local ministry of health, ntp and national tuberculosis reference laboratory staff to lead the analysis of tuberculosis-related data, discover and fix critical inefficiencies, and provide high-level technical and operational support to tuberculosis programmes. through data-driven, actionable recommendations, the tdf helps to strengthen, improve and complement ntps in lowand middle-income countries and, ultimately, improve healthcare delivery. acknowledgements the authors wish to acknowledge and thank neal myrick and jason schumacher from the tableau foundation for their technical and financial support of the programme, sarah hinrichsen from tableau and iwan rÿnders from moyo business advisory, south africa for providing the tableau training. barbara k. timmons edited the article. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions n.g. was the project leader, and n.g., a.u.n. and b.c. were responsible for project design. n.g. wrote the article, and a.u.n., b.c. and c.m. contributed to the conceptualisation, design, development and editing of this article. sources of support the tableau foundation funded this study under award number r007 tableau foundation. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the offifical policy or position of any affiliated agency of the authors. references world health organization. tuberculosis. key facts [homepage on the internet]. c2018 [updated 2018 sept 18; cited 2019 aug 14]. available from: https://www.who.int/news-room/fact-sheets/detail/tuberculosis world health organization. digital health for the end tb strategy: an agenda for action. geneva: who press; 2015. albert h, nathavitharana rr, isaacs c, et al. development, roll-out and impact of xpert mtb/rif for tuberculosis: what lessons have we learnt and how can we do better? eur respir j. 2016;48(2):516–525. https://doi.org/10.1183/13993003.00543-2016 gous n, boeras di, cheng b, et al. the impact of digital technologies on point-of-care diagnostics in resource-limited settings. expert rev mol diagn. 2018;18(4):385–397. https://doi.org/10.1080/14737159.2018.1460205 foundation for innovative diagnostics (find). case study: be data driven: find’s actionable diagnostics data for improved tb care (add for tb) initiative [homepage on the internet]. c2018 [cited 2019 july 10]. available from: https://digitalprinciples.org/wp-content/uploads/find-case-study.pdf creswell j, codlin aj, andre e, et al. results from early programmatic implementation of xpert mtb/rif testing in nine countries. bmc infect dis. 2014;14:2. https://doi.org/10.1186/1471-2334-14-2 karamagi e, nturo j, donggo p, et al. using quality improvement to improve the utilisation of genexpert testing at five lab hubs in northern uganda. bmj open qual. 2017;6(2):e000201. ndlovu z, fajardo e, mbofana e, et al. multidisease testing for hiv and tb using the genexpert platform: a feasibility study in rural zimbabwe. plos one. 2018;13(3):e0193577. gounder a, gounder s, reid sa. evaluation of the implementation of the xpert® mtb/rif assay in fiji. public health action. 2014;4(3):179–183. https://doi.org/10.5588/pha.14.0025 gidado m, nwokoye n, nwadike p, et al. unsuccessful xpert® mtb/rif results: the nigerian experience. public health action. 2018;8(1):2–6. https://doi.org/10.5588/pha.17.0080 joshi b, lestari t, graham sm, et al. the implementation of xpert mtb/rif assay for diagnosis of tuberculosis in nepal: a mixed-methods analysis. plos one. 2018;13(8):e0201731. hossain st, imtiaz es, modak pk, et al. gxalert for real-time monitoring management and strengthening of remote genexpert network in bangladesh [homepage on the internet]. technical brief. c2018 [updated 2018 sept 21; cited 2019 july 10]. available from: https://www.msh.org/resources/gxalert-for-real-time-management-and-strengthening-of-remote-genexpert-network-in mengesha e, nyaruhirira a, scholten j, et al. the experience of innovative specimen transportation and genexpert expansion in ethiopia. presented at: 49th union world conference on lung health; 2018 oct 24–27; the hague. unitaid. unitaid end-of-project evaluation: tb genexpert: scaling up access to contempory diagnostics for tb. geneva: dalberg; 2017. cepheid. improving your experience with xpert mtb/rif [homepage on the internet]. c2012 [updated may 2012; cited 2019 aug 12]. available from: https://www.ghdonline.org/uploads/improving_your_experience_of_xpert_mtb_rif.pdf kudzawu s. genexpert error codes: an evaluation of their definitions and its implications on program strenghtening efforts. accepted to:50th union world conference on lung health; 2019 oct 30–nov 2; hyderabad. agbaiyero kj, emeka e, kuye o. benefits of using technology supported gxalert in managing genexpert high error rates in nigeria. presented at: 49th union world conference on lung health; 2018 oct 24–27; the hague. wagenaar bh, hirschhorn lr, henley c, et al. data-driven quality improvement in low-and middle-income country health systems: lessons from seven years of implementation experience across mozambique, rwanda, and zambia. bmc health serv res. 2017;17(suppl 3):830. https://doi.org/10.1186/s12913-017-2661-x article information authors: orji bassey1 kyle bond1 adebayo adedeji2 odafen oke1 ado abubakar1 kachiro yakubu2 tapdiyel jelpe1 ezekiel akintunde3 patrick ikani4 adeniyi ogundiran5 ali onoja6 issa kawu2 gabriel ikwulono2 idris saliu7 okey nwanyawu1 varough deyde1 affiliations: 1us centers for disease control and prevention (cdc), nigeria 2federal ministry of health (fmoh), nigeria 3us department of defense (dod), nigeria 4global hiv aids initiative in nigeria (ghain), nigeria 5world health organization (who), nigeria 6african health project (ahp), nigeria 7safe blood for africa foundation (sbfaf), nigeria correspondence to: varough deyde email: che5@cdc.gov postal address: 1140 prospect street, 2nd floor, bldg 3, pretoria, south africa dates: received: 20 aug. 2014 accepted: 14 feb. 2015 published: 29 may 2015 how to cite this article: bassey o, bond k, adedeji a, et al. evaluation of nine hiv rapid test kits to develop a national hiv testing algorithm in nigeria. afr j lab med. 2015;4(1), art. #224, 17 pages. http://dx.doi.org/10.4102/ajlm.v4i1.224 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. evaluation of nine hiv rapid test kits to develop a national hiv testing algorithm in nigeria in this original research... open access • abstract • introduction • research method and design    • strategy, sampling and testing    • test kit selection and characteristics    • source and size of specimens    • testing procedure    • reference testing/gold standard    • quality control reference laboratory testing    • data collection, management and analysis    • cost estimations    • ethical considerations • results    • sensitivity and specificity of the evaluated individual test kits    • kits dropped from consideration    • accuracy of testing algorithms    • cost estimates for the algorithms    • proposed test algorithms • discussion    • limitations of the study    • conclusion • trustworthiness    • reliability    • validity • acknowledgements    • competing interests    • authors’ contributions • references • appendix 1 • appendix 2 • appendix 3 • appendix 4 abstract top ↑ background: non-cold chain-dependent hiv rapid testing has been adopted in many resource-constrained nations as a strategy for reaching out to populations. hiv rapid test kits (rtks) have the advantage of ease of use, low operational cost and short turnaround times. before 2005, different rtks had been used in nigeria without formal evaluation. between 2005 and 2007, a study was conducted to formally evaluate a number of rtks and construct hiv testing algorithms. objectives: the objectives of this study were to assess and select hiv rtks and develop national testing algorithms. method: nine rtks were evaluated using 528 well-characterised plasma samples. these comprised 198 hiv-positive specimens (37.5%) and 330 hiv-negative specimens (62.5%), collected nationally. sensitivity and specificity were calculated with 95% confidence intervals for all nine rtks singly and for serial and parallel combinations of six rtks; and relative costs were estimated. results: six of the nine rtks met the selection criteria, including minimum sensitivity and specificity (both ≥ 99.0%) requirements. there were no significant differences in sensitivities or specificities of rtks in the serial and parallel algorithms, but the cost of rtks in parallel algorithms was twice that in serial algorithms. consequently, three serial algorithms, comprising four test kits (bunditm, determinetm, stat-pak® and uni-goldtm) with 100.0% sensitivity and 99.1% – 100.0% specificity, were recommended and adopted as national interim testing algorithms in 2007. conclusion: this evaluation provides the first evidence for reliable combinations of rtks for hiv testing in nigeria. however, these rtks need further evaluation in the field (phase ii) to re-validate their performance. introduction top ↑ nigeria is the tenth most populous country in the world and the most populous country in africa, with an estimated population of 162.3 million.1 the first hiv case in nigeria was reported in 1986.2 this stimulated interest in the screening of various populations in nigeria for hiv. the national hiv sero-prevalence sentinel survey amongst populations of pregnant women attending antenatal clinics (anc) commenced in nigeria in 1991 and has since become a biennial activity. the trend of hiv infection amongst this anc population since the commencement showed a steady increase − 1.8% (1991), 3.8% (1993), 4.5% (1995, 1996), 5.4% (1999), to a high of 5.8% in 2001 – before declining to 5.0% in 2003 and then stabilising subsequently at 4.4% in 2005, 4.6% in 2008 and 4.1% in 2010.3 nigeria has a generalised hiv epidemic – each of the 36 states and the federal capital territory has over 1.0% hiv prevalence4 – and an estimated 3.5 million people are infected with the virus in the country. there are about 0.4 million estimated new infections per year, 1.5 million persons requiring antiretroviral therapy and an estimated 2.2 million total aids orphans currently living in the country.5,6 in 2005, the nigeria national action committee on aids (naca) strategic framework set out to provide antiretrovirals (arvs) to 80.0% of adults and children with advanced hiv infection and to 80.0% of hiv-positive pregnant women, all by 2010. the implications of these efforts entail screening several million people for hiv infection. a 2005 survey of types of rapid test kits (rtks) used in facilities participating in anc in two of the six geopolitical zones of nigeria revealed 19 different brands ranging from cold chain-dependent to non-cold chain-dependent (adedeji aa, personal communication, march 2005). the lack of coordinated use of hiv rtks also resulted in some discrepancies observed in results from the same sample within a health facility or at different health facilities, thereby making it difficult to provide centralised quality assurance or a post-marketing validation programme in-country (adedeji aa, personal communication, march 2005). as a result of these problems, the nigerian government saw the need to adopt the use of non-cold chain-dependent hiv rtks for hiv testing. hiv rapid testing remains a key entry point to hiv prevention, treatment, care and support in resource-limited settings.7 its main advantages include the relative ease of use, low cost and faster turn-around time over enzyme immunoassays (eias) and western blot (wb) assays. with an hiv rapid testing strategy, increased awareness of hiv status amongst many groups who would otherwise have been unaware of their status has been achieved.8,9,10 providing quality-assured and accurate rapid hiv serological testing is critical in the early diagnosis and timely counselling of hiv-infected people for referral to care and treatment as well as prevention of mother-to-child transmission and monitoring of hiv prevalence in the population.7,9 hiv rapid testing also readily provides access to and enhances hiv counselling and testing in hard-to-reach rural populations,11,12 as well as in hard-to-reach, high-risk target populations, such as men who have sex with men.10 high-risk groups with acute hiv infection in nigeria have previously been characterised by use of a combination of rapid hiv testing in mobile units and laboratory-based specimens pooling for nucleic acid amplification testing.13 to date, several african countries have conducted evaluation studies and implemented rapid hiv testing as a tool for fighting the hiv epidemic. these studies have demonstrated that the use of rapid testing can be an important part of the overall hiv testing strategies in resource-limited settings, where cold storage capacity, reliable power, efficient transportation and sufficient numbers of skilled laboratorians may not be readily available.14,15,16,17,18,19,20 a number of sub-saharan african countries follow the world health organization (who) 2009 guidelines21,22 on the use of hiv antibody detection tests, where the recommended test algorithm includes a sensitive enzyme-linked immunosorbent assay (elisa) as a screening test, followed by a confirmatory test done on all positive samples using wb.23,24,25,26 recent studies have shown that diagnostic algorithms based on two or more serological tests are dependable and significantly lower the rate of recurrence of false positivity, thereby minimising misdiagnosis.26,27 recently, the use of rapid testing combined with elisa has increased significantly in africa and asia and tends to replace the use of wb assays.26,28,29,30,31,32 accurate hiv diagnosis in resource-limited settings, as is the case in most regions of nigeria, can be affected by emergence of new hiv subtypes and recombinant forms, hence the importance of occasionally assessing and selecting the best-performing serological assays before their wide-scale usage within the country.26,33,34,35 the goal of this evaluation was to assess and select non-cold chain-dependent hiv rtks for the development of evidence based national testing algorithms based on key criteria such as performance, ease of use and cost, amongst others. it also sought to develop a list of highly-sensitive and specific hiv rtks with documented good performance to serve as alternative algorithms in times of stock-outs of the rtks included in the primary algorithms. the present evaluation allowed the identification and recommendation of three national interim algorithms for hiv rapid testing in the country. to our knowledge, these recommendations are still implemented by the federal ministry of health (fmoh) and a second, field evaluation, phase has been conducted, although the results are not yet available. the methodology applied by the present evaluation could be used by other countries planning to develop hiv testing algorithms. research method and design top ↑ strategy, sampling and testing in august 2005, a multi-agency working group was set up by the government of nigeria for the evaluation of hiv rtks. the working group included participants from the fmoh and other organisations, specifically, the national aids and stis control program (nascp), naca, the national agency for food, drug administration and control (nafdac), the national institute for pharmaceutical research and development (niprd), the who, the centers for disease control and prevention – global aids program (cdc-gap) and other partners implementing the us president's emergency plan for aids relief (pepfar) programme in nigeria, who had international experience in rtk evaluations. test kit selection and characteristics the hiv rtks used in this evaluation were chosen based on the following who 2001 and 2009 recommended criteria: (1) stability within the climate in the country and not cold chain-dependent; (2) ability to test whole blood; (3) easy to use and interpret; (4) low test price (≤ us$3.20); (5) ability of manufacturers to produce and provide adequate numbers of testing kits to meet the needs of testing programmes in the country; (6) prior experience and validation – documented performance in the country and other african countries; (7) ability to detect hiv-1, hiv-2 and hiv type o subtypes; (8) ability to detect both igg and igm antibodies in order to reduce the window period; (9) do not require additional equipment to run tests or read results; (10) packaging of test kits not excessively bulky; (11) long shelf life (at least one year) and robust; and (12) test results provided in 30 minutes or less.21,22 in addition to the criteria above, test kits were selected based on their sensitivity and specificity when used singly and in combination using the minimum sensitivity and specificity (both ≥ 99.0%) criteria.17,36 the criteria were ranked in order of importance and relevance to the nigerian context. a total of nine test kits were selected for the evaluation (table 1). all the tests studied in this evaluation are qualitative tests for the detection of antibodies to hiv-1 and hiv-2 and use immunochromatographic technology. table 1: characteristics of hiv rapid tests included in this evaluation. source and size of specimens specimens were collected from sites in five geopolitical zones of nigeria between 2005 and 2006. ten health facilities were originally planned to contribute specimens for this evaluation; however, because of logistical challenges, specimens from only five facilities were used for the study. these sites still provide a good representation of the population. patient identification information was removed from all specimens and only hiv sero-status was reported. all specimens included in this study were unlinked and anonymised before inclusion and no blood specimen was drawn solely for the purpose of this validation. the specimen panel used for this evaluation was prepared from two sources. the first was existing sample archives (leftover plasma or serum collected routinely for diagnostic purposes) in hiv testing laboratories at federally administrated teaching hospitals. the second was the remaining samples from a joint cdc/university of maryland hiv sero-conversion project. the following specimen acceptance or rejection criteria were put in place to ensure that specimens of high quality were used in this evaluation: (1) properly collected, no haemolysis; (2) properly processed, no obvious signs of fungal or bacterial contamination/growth; (3) properly stored, freshly collected, at 20 °c, not stored for longer than two months at the collection sites; (4) clear hiv eia sero-status, positive or negative. hiv-positive specimens had to contain high titres of hiv-specific antibody and an eia signal-to-cutoff ratio of 3.0 or higher. hiv-negative specimens had to have eia results comparable to that of the kit negative control; and (5) adequate specimen volume (at least 3.0 ml). all specimens were treated and prepared based on the cdc/who guidelines.15,16 specimens were then given new identification numbers, logged into a database and divided into about three aliquots (volume permitting) to avoid repeated freeze-thaw cycles that may affect antibody titres. the aliquots were stored at -20 °c for a maximum of two months until being characterised and used in the evaluation. to avoid several freeze-thaw cycles, aliquots were kept in a refrigerator whilst in use during the validation period. approximately 200 hiv-positive and 200 hiv-negative specimens are needed to provide 95% confidence intervals of less than ± 2.0% for both the estimated sensitivity and specificity. thus, to meet the minimum acceptable test characteristics of the hiv rapid test as stated above, the final panel sample contained 528 specimens, of which 198 (37.5%) were hiv-1 positive and 330 (62.5%) were hiv negative. testing procedure all specimens were assigned new identification numbers between 1 and 528, then ordered by their reactivity (positive or negative) and randomised to allow for blinded testing. ten skilled and experienced laboratorians working on the serology bench at the sites that contributed the specimens for the evaluation were recruited and were then provided with background information on the evaluation, refresher training on good laboratory practice and an orientation to the data entry forms. job aids were provided for each rtk and each test was demonstrated. under the supervision of cdc and nascp laboratory staff, the laboratorians practised on control specimens prior to evaluating the panel. testing of each assay was implemented using the specimens according to the manufacturer's instructions for each individual test kit. laboratorians worked in pairs; each pair evaluated approximately 100 specimens over a half-day period per test product. specimen sets were rotated between the testers. each test result was read independently by two individuals. all the laboratorians then completed a questionnaire concerning various aspects of the rtk they had just evaluated (see appendices). the laboratorians appraised each rtk based on the following criteria: ease of running and reading test results, including ease of reading the reaction line; ease of interpreting the test results; ease of learning the test procedure; overall ease of running the assay; packaging size; and waste generation. this was done in an effort to capture information, in addition to accuracy, which is also critical in identifying tests for an algorithm. reference testing/gold standard all specimens were fully characterised using standardised reference testing (gold standard): two third-generation eias, plus wb for all eia-reactive specimens. all specimens with discordant eia and wb results were excluded from the panel. specimens with indeterminate wb results were also excluded. the two eias selected for this validation, namely, vironostika® hiv uniform ii plus o (biomerieux, france) and genscreen® 1/2 version 2 (bio-rad, usa), were both third-generation eias targeting both igg and igm of hiv-1 and -2, plus type o antibodies using recombinant antigens covering all group m, hiv-1 subtypes a-h. both assays have been widely used throughout africa12,18,19,25 and have consistently produced reliable data and detected hiv-specific antibodies. an antibody-only test is the most appropriate for comparison with hiv antibody-detecting rapid tests. the wb kit selected was new lav-blot i (bio-rad). all reference testing was conducted as per the manufacturer's instructions. quality control reference laboratory testing all laboratory work associated with this evaluation was carried out at the asokoro training laboratory, located at the asokoro general hospital in abuja. this work included specimen characterisation, storage and the evaluation exercise. this institute of human virology, nigeria (ihvn)-supported site was selected for the following reasons: current status as a national hiv laboratory training facility; central location within federal capital territory; constant electrical power; ongoing external quality assurance/laboratory monitoring programme; appropriate infrastructure for reference testing (eia equipment); and adequate specimen storage space. data collection, management and analysis all test results were collected on paper forms and entered into a spreadsheet database (microsoft® excel™) for analysis. access to the project databases was limited to only key project staff through password-protected computers and all paper forms were kept in locked filing cabinets. during the data analysis, the sensitivity and specificity of each rtk were calculated by comparing the rtk results with reference results derived from eia/wb testing. cost estimations the supply chain management system (scms) was established in nigeria in 2007 following the who hiv test kit bulk procurement scheme established in 1989, which is aimed at facilitating access to high-quality test kits at a low cost through an easy purchasing procedure. the scms coordinates pooled procurement systems for hiv arvs and rtks and provided pricing information for the analysis as negotiated with the manufacturers and/or companies or their agents.37,38 each of the rtks under consideration was evaluated in both parallel and serial testing algorithms and anticipated costs for each algorithm were determined in us dollars based on the negotiated scms price. for the parallel testing algorithms, the price of each screening rtk was added to that of the confirmatory (i.e., second) rtk. the cost of the tie-breaker rtk was not included, since the frequency of use of a tie breaker is low (at most, 1.8% of the time). for the serial algorithms, the full price of the screening rtk was added to the price of the confirmatory rtk at 10.0% hiv prevalence (since the second test would only be used to confirm positive test results), plus the price of the tie breaker when needed at an hiv prevalence of 10.0%. ethical considerations the protocol for this evaluation was developed following the who's regional office for africa (who afro) guidelines21 and received ethical approval from the niprd, nigeria and institutional review board (irb) as well as the cdc irb (approval dated 03.21.2006). results top ↑ sensitivity and specificity of the evaluated individual test kits the sensitivity and specificity results were calculated for each individual test (table 2). all nine tests performed well in this evaluation, as indicated by high sensitivity and specificity values. the sensitivity value for seven of the nine tests was 100.0%, indicating that none of these tests produced false-negative results. two tests, first response® and instantchektm, had lower sensitivities (98.9% and 96.9%, respectively). specificity varied slightly between the tests, ranging from 96.0% to 100.0%; oraquick® and stat-pak® were each 100.0% specific. table 2: sensitivity and specificity calculations for the nine selected rapid test kits. kits dropped from consideration figure 1 shows the kit selection process and results. after the initial performance of each individual kit was tested, three of the nine kits (instantchektm, first response® and oraquick®) were removed from further consideration. both instantchektm and first response® ere excluded because of their performance (sensitivity and specificity) and the complexity of the result interpretation. oraquick® was dropped because of its cost and short shelf-life. figure 1: process for selecting hiv rapid test kits for development of interim national hiv testing algorithms. commercial kits available in nigeria were selected for evaluation singly based on who guidelines (step 1). of the nine kits, six were retained for inclusion in the algorithm testing exercise and three were dropped (step 2). serial and parallel testing algorithms were assessed for performance (sensitivity and specificity), cost and the country context (step 3). serial algorithms using four kits were selected and recommended as interim national guidelines in 2007 (step 4). figure 2 presents a summary of findings from the questionnaire on rtk characteristics administered to the laboratorians who performed the testing. figure 2: results from questionnaires administered to laboratorians conducting the evaluation. respondents were asked to rate all nine kits based on the following criteria: ease of reading the reaction line (panel a); ease of interpreting the test results (panel b); ease of learning how to perform the test (panel c); and overall ease of using and running the kit (panel d). scores ranged from very easy (1) to very difficult (5) for this set of four questions; panels a–d represent average scores. respondents were also asked about the size of the packaging (panel e), with scores ranging from 1 (very bulky) to 5 (very compact); and about quantity of waste generated (panel f), with scores ranging from 1 (a lot of waste) to 5 (minimal waste). panels e-f represent average scores. accuracy of testing algorithms in diagnostic settings, rtks are used in testing algorithms, not as individual tests. one major advantage of evaluating rtks using a single, well-characterised specimen panel is that sensitivity and specificity can be calculated for all possible combinations of tests. this was completed for both parallel and serial algorithms (box 1, appendix 3 and 4). box 1: cost (us $), sensitivity and specificity of parallel and serial testing algorithms†. all possible parallel algorithms had a sensitivity of 100.0%, which indicates that none of the specimens in this panel had a false-negative result with more than one test product (appendix 3). specificity was also high, ranging from 99.1% to 100.0%. this represents from zero to three false-positive results for each algorithm. over one-third (n = 24/60) of the possible test combinations had the highest possible (100.0%) sensitivity and specificity. for all 120 possible serial algorithms, sensitivity was 100.0% (appendix 4). specificity ranged from 99.1% to 100.0%. over half (n = 67) of the proposed algorithms had 100.0% sensitivity and specificity. this included five of the eight algorithms utilising the two rtks currently in wide use in nigeria (determinetm and stat-pak®). the number of specimens (out of 528) requiring a tie-breaker test because of discordant results between the first two tests is also reported for serial and parallel algorithms (appendix 3 and 4). eight of 30 serial algorithms (two test combinations) did not require the use of a tie-breaker test. other combinations, for both algorithm types, ranged from one to 10, representing at most 2.0% of specimens. cost estimates for the algorithms the cost for the serial testing algorithms was found to range from us$0.70 to us$1.90, whilst parallel algorithms ranged from us$1.40 to us$3.30 (table 3, appendix 3 and 4). in general, the cost for the serial testing algorithms was about half the cost of the parallel testing algorithms, since the latter require two tests to be run on all clients, even the 90.0% of clients who are hiv negative. table 3: national interim serial hiv rapid testing algorithm implemented in 2007. proposed test algorithms determinetm and stat-pak®, both of which have been evaluated and used widely internationally, have also been used widely in the nigerian hiv programme since the 2001 and 2006 anc surveys, respectively. tremendous investment has been made in training large numbers of laboratory staff, including the adaptation of the training package for both test kits for use in nigeria. determinetm has also been used widely throughout africa. in this evaluation, both tests had high sensitivity and specificity individually and in the serial algorithms. determinetm, with its high sensitivity (100.0%), is strongest as a screening test and was recommended as the first test in any proposed algorithm. determinetm was not recommended as a confirmatory test because of its lower specificity (97.8%). use as a tie-breaker was only recommended in the event that stat-pak® is not available. uni-goldtm has also been used widely internationally and performed well in this evaluation. in light of the fact that larger numbers of tests will soon be available in nigeria to support hiv diagnostic testing programmes, it was also included in the interim national hiv rapid testing algorithm. based on its performance and the need for ongoing quality assurance, adequacy of supply of kits and the development of a track record, it was recommended that bunditm be included as a tie-breaker test. this would allow for continued monitoring of this new product. based on the above findings, the construction of the three interim serial testing algorithms was based on four of the six rapid hiv test kits, namely determinetm, stat-pak®, uni-goldtm and bunditm (table 3). discussion top ↑ the expansion of hiv prevention and care services in resource-constrained settings comes with great challenges regarding how to maintain quality-assured and accurate hiv testing as the number of hiv testing facilities increases.21 in addition, there are challenges associated with the quest for alternative, less expensive and efficient rapid hiv testing strategies, devoid of the supplemental confirmatory testing using the expensive wb assay and capable of retaining a high level of sensitivity in the face of the divergent hiv-1 subtypes dominating most sub-saharan african countries.14,15,16,17,32,33 this is even further complicated by the move to decentralise hiv testing by involving fewer skilled and experienced laboratory/non-laboratory personnel.17,32 this study evaluated nine hiv rtks using double eias as the reference test and wb as a supplemental confirmatory test for eia-concordant reactive specimens. this serves as a gold-standard testing method for this evaluation and is comparable to the methods adopted in similar studies.29,32,39,40 all of these studies were in line with the cdc/who afro guidelines17 for hiv testing technologies in africa. of the nine rtks selected for the evaluation, three (oraquick®, instantchektm and first response®) were dropped because of short shelf-life, poor performance, cost or complexity following the who phase 1 hiv rtk evaluation criteria. the remaining six rtks (bunditm, determinetm, double-check goldtm, stat-pak®, surecheck® and uni-goldtm) were then subjected to parallel and serial testing algorithms in several possible combinations, resulting in combinations with high levels of sensitivity and specificity, as well as a high accuracy for diagnosing hiv infection. sixty possible parallel algorithms had costs ≤ us$3.20, had a sensitivity of 100.0% – indicating that no-false negative results were obtained with the panel of specimens – and had specificities ranging from 99.1% – 100.0%. this represents zero to three false-positive results for each of the algorithms. it was also observed that over one-third (n = 24/60) of the possible test combinations had the highest possible (100.0%) sensitivity and specificity. of note, the remaining 60 possible parallel combinations of the rtks were not presented in this report because of their higher cost (> $3.20). similarly, of the 120 possible serial algorithms, sensitivity was 100%, whilst specificity ranged from 99.1% – 100.0%. additional analysis revealed that over half (n = 67/120) of the possible serial algorithms had 100.0% sensitivity and specificity, indicating that none of the panel specimens showed a false-negative or false-positive result with more than one test product. a comparative analysis of the performance characteristics between the parallel and serial testing algorithms revealed no differences in accuracy regarding individual performance in diagnosing hiv infection. similar comparative analyses of performance of combinations of elisas and rtks in parallel or serial testing algorithms have shown that these combinations can also produce accurate results for hiv infections diagnosis.17,27 however, a comparative cost analysis between the two testing strategies showed a substantial difference, as the cost of carrying out a parallel testing algorithm is twice as expensive as the cost of a serial testing algorithm. these findings are comparable to those previously reported.14,17 besides sensitivity, specificity and the cost of the testing algorithms, other important factors were also considered before making a choice of assay for the national testing algorithm. over the years, the fmoh, through its hiv/aids division in its efforts to implement national programmes has also significantly invested in terms of training the laboratorians and non-laboratorians involved in hiv testing using the stat-pak® and determinetm hiv rtks. this shows that both test kits are both commerciallyand readily available and had wide-scale use in nigeria. furthermore, based on laboratorians’ evaluation and rating of the nine rapid test kits using questionnaires and the test selection criteria recommended by the who, determinetm was identified as the most compact test kit, allowing for less expensive transport and generating the least waste, thereby alleviating concerns about biohazard waste disposal at testing sites, whilst stat-pak® ranked high in terms of readability of the reaction line and result interpretation. cost-wise, a serial testing algorithm comprising determinetm and stat-pak® was found to be inexpensive, as it costs less than one us dollar. the cost of the serial testing algorithm is vital, considering the expected testing targets and the large size of the voluntary counselling and testing programme in nigeria. also rated highly was uni-goldtm, which is known to be used widely internationally. bunditm, on the other hand, was included in the serial algorithm as a tie-breaker, because it is assembled locally and readily available, in addition to its high performance in the evaluation. the ease and convenience of performing the assay were also considered as in previous, similar studies.17 based on the above findings and criteria, nigeria adopted the serial hiv testing algorithm as an interim national testing algorithm (table 3). similar considerations and decision strategies were also adopted in a comparable study conducted in 11 african countries.17 the six non-cold chain-dependent test kits (bunditm, determinetm, double-check goldtm, stat-pak®, surecheck® and uni-goldtm) performed well in this laboratory-based evaluation, both as individual tests and in serial testing algorithms. data from this initial evaluation suggest that any combination of these six rtks would perform well in a three-test, serial algorithm and that the tests with the highest sensitivity, such as determinetm and uni-goldtm, should be used as the screening test, whereas those with highest specificity, such as stat-pak®, should be used for confirmation. limitations of the study the present evaluation is not without limitations. first of all, this evaluation was limited to stored frozen plasma specimens and oral fluid was not collected for evaluation. as a result, the comparative advantage of using test kits with oral fluid or fresh specimens could not be evaluated. another limitation was the variability in performance of some of the test kits in the hands of different testing personnel; this phenomenon has also been observed previously.14,15,16,17 in addition, the sensitivity of these test kits/testing algorithms is not well established and may differ based on hiv-1 subtypes, given the great genetic diversity of hiv-1 in africa.17 as a result, the who developed guidelines to help country-based evaluation and implementation of rapid hiv testing.17 considering these limitations, a formal hiv test kit performance evaluation should be an ongoing process that starts before testing implementation and continues after testing processes have been implemented in the field. as a result, since this evaluation provided data on laboratory-based validation, the selected rtks should be field tested (phase ii) in varying combinations before a final national testing algorithm is selected. furthermore, it is critical to ensure that the hiv test algorithms currently in place and future ones be monitored continuously through a quality-assurance programme (phase iii) developed within nigeria. this quality-assurance programme should have the capacity to rapidly identify and correct testing problems related to the selected test kits and use of those kits in algorithms. finally, it is important to note that, at the time of preparing the present manuscript, field monitoring had revealed a performance issue with bunditm and the kit was removed from the algorithm in 2008. only three kits thus remain in use (determinetm, uni-goldtm and stat-pak®). the second phase (field-based evaluation) was conducted in 2012, however, the results are not yet available (adedeji aa, personal communication, june 2012). conclusion three hiv testing algorithms with high sensitivity, specificity and accuracy to diagnose hiv infections were identified and recommended for use as interim national algorithms. these hiv testing algorithms provide a cheaper and more efficient alternative to wb supplemental confirmatory testing. the results of this analysis showed further that serial testing algorithms are not only sensitive and specific, but also less expensive. finally, the present evaluation provides the first evidence-based and reliable combination of hiv test kits in nigeria. it is important that a field, ‘point-of-care testing’ evaluation is conducted and the findings used to inform future decisions on what test kits to use in the country for accurate hiv testing. trustworthiness top ↑ the current report reflects the findings observed by the technical working group, those who performed the testing, as well as the team that analysed the data. reliability the results of the experiments presented in this report were obtained using specimens collected in nigeria and these results have been confirmed using who-recommended gold standard testing procedures for evaluating hiv rapid test kits. however, the methods of the evaluation can be applied in other countries. validity the development and recommendation of an interim hiv rapid testing algorithm in nigeria demonstrated the study's success in achieving its goal. not only were the test kits evaluated based on gold standard methods and procedures, but also the outcome of the study were scientific evidence-based recommendations that allowed the government of nigeria to make informed decisions on what kits to use in their hiv testing programmes. acknowledgements top ↑ this publication was made possible by support from pepfar through the department of health and human services, cdc, division of global hiv/aids. the findings and conclusions in this manuscript are those of the authors and do not necessarily represent the official position of the cdc. the authors are deeply grateful to other members of phase i laboratory evaluation working group for their immense involvement and supportive supervision during the rapid testing phase and contributions to the initial report writing phase. members of this group include: hiv/aids division, fmoh (a. akinsete, a. lawanson, o. salawu, t.o. adonye, a. uwah, mrs g.m. bassey, f. simon); naca (a. ikpeazu); nafdac (c.u. anyakora); who (a.h. fagbami); institute of human virology, nigeria (a. abubakar, b. okelade, j. jugu, p.j. nwadike); ghain (i.g. audu); national blood transfusion service (a. abayomi, s.m. aminu); national institute for pharmaceutical research and development (o. dauda); cbge university of jos (m. njoku); nigeria ministry of defense – emergency plan implementation committee (epic) (l. ukachukwu); university of abuja teaching hospital, gwagwalada (m. rubainu); university college hospital, ibadan (d. olaleye); university of port harcourt (s. esiet); usman danfodio univserity teaching hospital (d.b. idowu); aminu kano teaching hospital (h. takalmawa); university of benin teaching hospital (f. agbontaen); university of maiduguiri teaching hospital (m. anietie); asokoro general hospital (e. kayode); plateau state specialist hospital virology research center (p. amangam); nnamdi azikiwe university teaching hospital (k. stephen); university of nigeria, teaching hospital (a. abah). we want to especially acknowledge the following subject matter experts from the cdc in atlanta for reviewing and editing the manuscript: dr bharat parekh, de anindya, and taiwo abimbola. we also appreciate terrell peggy for coordinating these efforts. in addition, we thank the cdc nigeria staff: dr ahmed mukhtar, dr obinna ogbanufe, dr solomon odafe, dr mustapha bello, dr bertrand odume, dr ibrahim jahun and mr raphael akpan for their review and discussions of the manuscript. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions o.b. (cdc-nigeria) performed some of the experiments and wrote the manuscript. a.a., k.y., i.k. (all fmoh-nigeria) and a.o. (who-nigeria) co-led the project and experimental design. t.j., a.a., o.o. (all cdc-nigeria), p.i. (ghain), g.i. (fmoh), i.s. (sbfaf), a.o. (ahp) and e.a. (dod) made conceptual contributions and performed the experiments. k.b., v.d. and o.n. 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2005. world health organization. who hiv test kit – bulk procurement scheme. geneva: who; 2002. wilkinson d, wilkinson n, lombard c, et al. on-site hiv testing in resource-poor settings: is one rapid test enough? aids. 1997;11(3):377–381. http://dx.doi.org/10.1097/00002030-199703110-00016 delaney kp, branson bm, uniyal a, et al. evaluation of the performance characteristics of 6 rapid hiv antibody tests. clin infect dis. 2011;52(2):257–263. http://dx.doi.org/10.1093/cid/ciq068 appendix 1 top ↑   data collection sheet appendix 2 top ↑   testers’ ratings of rapid test kits (rtks) during laboratory evaluation appendix 3 top ↑ table 1-a3: parallel algorithms. appendix 4 top ↑ table 1-a4: serial algorithms. abstract introduction methods results discussion acknowledgements references about the author(s) bavinash pillay department of internal medicine, faculty of health sciences, university of the witwatersrand, johannesburg, south africa sarah a. van blydenstein department of internal medicine, faculty of health sciences, university of the witwatersrand, johannesburg, south africa department of internal medicine, faculty of health sciences, chris hani baragwanath hospital, johannesburg, south africa shahed omar school of clinical medicine, department of critical care, faculty of health sciences, university of the witwatersrand, johannesburg, south africa department of critical care, faculty of health sciences, chris hani baragwanath hospital, johannesburg, south africa citation pillay b, van blydenstein sa, omar s. thromboelastography in covid-19 patients: an observational study in the south african context. afr j lab med. 2025;14(1), a2681. https://doi.org/10.4102/ajlm.v14i1.2681 note: additional supporting information is available for download alongside the online version of this article as online supplementary document 1. original research thromboelastography in covid-19 patients: an observational study in the south african context bavinash pillay, sarah a. van blydenstein, shahed omar received: 05 nov. 2024; accepted: 28 jan. 2025; published: 26 june 2025 copyright: © 2025. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: coronavirus disease 2019 (covid-19) increases the risk of venous thromboembolism, requiring monitoring of low molecular weight heparin (lmwh) via a time-consuming, costly and often unavailable test – anti-factor xa (anti-xa). an affordable, rapid point-of-care alternative, the thromboelastogram, is available, but performance comparisons to anti-xa are lacking. objective: this study evaluated the relationship between anti-xa and thromboelastogram in patients with covid-19 receiving lmwh. methods: this was a retrospective study of patients with covid-19 receiving lmwh at chris hani baragwanath academic hospital, johannesburg, south africa, between november 2020 and january 2021. blood samples tested with thromboelastogram and anti-xa were drawn at three timepoints (one prior to and two after administration of lmwh). thromboelastogram parameters comprised reaction time (r-time; onset of testing to the start of clot formation), kinetics time (k-time; start of clot formation until the clot reached 20 mm), and thromboelastogram coagulation index (overall coagulation status of whole blood). results: forty-two patients with covid-19 (15 male and 27 female) met the study criteria. there was a statistically significant, low to moderate correlation (spearman’s correlation coefficient [rs 0.43, p = 0.014]) between anti-xa and thromboelastogram coagulation index. a statistically significant moderate correlation (rs 0.52, p = 0.002) between anti-xa and r-time, and a statistically significant low correlation (rs 0.35, p = 0.049) between anti-xa and k-time, were found. all correlations were 48 h post admission. conclusion: thromboelastogram coagulation index, r-times and k-times had a statistically significant association with anti-xa levels in patients with covid-19. further research is required regarding their clinical utility. what this study adds: thromboelastograms may represent a more cost-effective and accessible option to the conventional anti-xa test in patients receiving lmwh. however, future research with larger sample sizes, varying disease profiles, and severity of illness is required. keywords: thromboelastogram; low molecular weight heparin; heparin; coronavirus disease 2019; viscoelastic methods; coagulopathy. introduction coronavirus disease 2019 (covid-19)-infected patients are known to be at higher risk of venous thromboembolism (vte).1 an accurate incidence rate of vte in covid-19-positive patients remains unknown, ranging from 4.8% to 85.0%.2 autopsies of 12 patients with severe acute respiratory syndrome coronavirus 2 (sars-cov-2), revealed a vte incidence of 58.0%.3 in an observational study of 150 patients admitted to the intensive care unit in france, pulmonary emboli occurred in 20.6% of patients.3 a cohort of 184 patients admitted to intensive care units in the netherlands showed that pulmonary emboli occurred in 33.3% of all patients suffering from sars-cov-2 acute respiratory distress syndrome.3 the incidence of pulmonary embolism was found to be twice as high in patients with sars-cov-2 compared to patients with influenza. the pathophysiology of vte in covid-19 is multifactorial. the infection is associated with an immune-mediated exaggerated inflammatory response, namely a cytokine storm including tumour necrosis factor-alpha, interleukin-8, and interleukin-6.4 other contributory factors include endothelial injury, abnormalities in gas exchange and lung compliance, as well as haemodynamic changes.2 owing to the high occurrence of vte in patients with sars-cov-2, the international society of thrombosis and haemostasis recommends that all clinicians should employ a high level of suspicion in the diagnostic work-up of deep venous thrombosis and pulmonary embolism.5 emphasis is placed on both prophylactic and therapeutic dosed anticoagulation.5 the current anticoagulant of choice in the prevention of vte amongst hospitalised covid-19 infected patients is low molecular weight heparin (lmwh). monitoring of lmwh has been evaluated through assessing anti-factor xa (anti-xa), through a measure of factor xa enzyme (xa) inhibition.6 anti-xa assessment has been found to improve the dosage regulation of lmwh in the treatment and prevention of vte amongst sars-cov2 infected patients.7,8,9 a study by trunfio et al.10 revealed that prompt correction of lmwh dosages based on an initial anti-xa level measurement reduced sars-cov-2 related mortality. in addition to the current gold standard of anti-xa levels, current tests for monitoring lmwh include activated partial thromboplastin time and thrombin generation.11 anti-xa assays are not without limitations. importantly, these assays are not widely available and often need to be referred to reference laboratories, which prolongs turnaround time, reducing their clinical utility. drawbacks with anti-xa level monitoring include the additional cost of testing with estimates as high as 55 united states dollars (usd; approximately 500 south african rand [zar] – 1000 zar, or approximately 27 usd – 55 usd) per patient per test.6 pre-analytic factors which may impair the validity of anti-xa levels include poor blood sampling technique, delays of two or more hours in sample analysis, inadequate centrifugation, as well as haemolysis. repeated tests may be required to establish a trend in anti-xa levels, further increasing costs. biologic limitations include antithrombin deficiency, increased binding, and inactivation of heparin-binding proteins (as a result of infection and inflammation), obesity (linked to inadequate levels of lmwh because of the increased volume of distribution) and impaired renal function (decreased elimination of lmwh).12 the prolonged turnaround time required to obtain a result from overburdened laboratories is problematic. the meticulous timing that each test needs to adhere to may be challenging in many settings.10 thromboelastography is a point-of-care non-invasive in vivo test carried out in a specialised machine known as a thromboelastograph.12 thromboelastograms identify and measure dynamic changes in coagulation, identifying where abnormalities in coagulopathy are found. thromboelastogram parameters consist of a reaction time (r-time), kinetics time (k-time), alpha angle, maximum amplitude (ma), and lysis at 30 min (a30).12 this is performed at the point of care, limiting the delay expected in laboratory-processed testing.13 there may be overall cost benefits to using thromboelastograms.14 according to a retrospective study of 32 sars-cov-2 infected patients, thromboelastogram analysis has proven to be useful for both screening for hypercoagulability and vte, and also for determining appropriate dosing of anticoagulation therapy.15 a systematic review published by the international society of thrombosis and haemostasis, assessing 153 articles, including 841 patients, revealed a good thromboelastogram detection rate of hypercoagulability associated with sars-cov-2.16 currently, there is a paucity of data exploring the relationship between thromboelastograms, anti-xa levels and lmwh. furthermore, there has yet to be defined a specified time period after the administration of lmwh, when thromboelastography is to be done.17 owing to the potential advantages of thromboelastograms which may address some limitations of anti-xa testing, this study aimed to assess the correlation between thromboelastograms and d-dimers, and thromobelastograms and anti-xa levels. the primary objective was to determine if there is an association between thromboelastogram coagulation index (tci) and anti-xa levels among covid-19 positive patients. secondary outcomes were to determine if there was a correlation between anti-xa levels and thromboelastogram parameters, namely the r-time, k-time, and alpha angle. methods ethical considerations ethical clearance was obtained from the university of the witwatersrand human research ethics committee (reference number: m230570). written consent was obtained from each participant when collecting data directly from patients and adding to a database for the study. measures taken to ensure confidentiality included obtaining informed consent, anonymising data, strict access controls to data, secure storage of data on the redcap® electronic data capture system, clear confidentiality policies for all researchers involved and the signing of a confidentiality agreement. the necessary ethical clearance documentation has been provided. setting the study was a retrospective cross-sectional observational study at chris hani baragwanath academic hospital covid-19 and intensive care unit wards, between november 2020 and january 2021. chris hani baragwanath academic hospital is a tertiary hospital located in johannesburg, gauteng, south africa. patient inclusion and exclusion criteria patients included in the study had reverse transcription polymerase chain reaction-confirmed sars-cov-2 infection, were ≥ 18 years old, and had severe sars-cov-2 disease. severe disease was defined as oxygen saturation < 93% with a respiratory rate ≥ 25 breaths per min, requiring supplemental oxygen support without the need for invasive or non-invasive ventilation. critical illness was defined as hypoxaemia and the need for additional ventilatory support, in the form of non-invasive or invasive ventilation. exclusion criteria for patients were: a prolonged activated partial thromboplastin time of > 50 s; patients receiving chronic anticoagulation therapy (including, but not limited to, aspirin, warfarin, clopidogrel, lmwh such as enoxaparin and direct-acting oral anticoagulants such as rivaroxiban); patients with prior vte, currently receiving quinine or a derivative thereof; known thrombotic thrombocytopenia purpura or thrombotic thrombocytopenia purpura-like disease; known haemoglobinopathies; pregnancy; oestrogen replacement therapy; and patients not receiving anticoagulation acutely or having any contraindication to anticoagulation. sample size to achieve a correlation coefficient of 0.5 with 80% confidence and precision of approximately 10% between thromboelastograms and anti-xa levels, as shown previously by tekkesin et al.,18 a sample size of 40 was required. we collected an additional 5% (2 samples) to allow for test failures, thereby arriving at 42 samples in total. data collection data collection was carried out by the primary investigator and supervisors. a database was created as part of a phd for one of the supervisors, with data being collected from 01 november 2020 to 31 january 2021. the data were collected by both the primary investigator and the supervisors during this period and entered directly into the redcap® electronic data capture tool.19 the data were then exported into statistica® version 13.3 (tibco software inc., santa clara, california, united states) for data interpretation. data extraction from this database was conducted from 01 march 2021 to 31 may 2021. sociodemographic data were collected directly from patients, including age, gender and ethnicity. whole venous blood samples were drawn directly from patients from the antecubital vein using a tourniquet and syringe for thromboelastogram, d-dimer, and anti-xa. these blood samples were drawn on admission (prior to enoxaparin administration), 48 h post admission (3 h after enoxaparin administration); and at clinical resolution (defined as resolution of hypoxia) or day 10 of illness. thromboelastograms were processed within 5 min of whole blood being taken, and the corresponding sample was taken to the laboratory for anti-xa testing within the specified 5 min as per manufacturer’s instructions. the teg® 6s machine (haemonetics®, boston, massachusetts, united states) was then used by filling a droplet of blood and a droplet of reagent into a cartridge which is then processed. the result was displayed and printed revealing an r-time, k-time, alpha angle, ma, and tci. only results from the standard kaolin thromboelastograms were considered. blood samples for d-dimer and anti-xa tests were stored in citrate tubes (becton-dickinson, oxford, uk). d-dimer, and anti-xa tests were done by the chris hani baragwanath academic hospital national health laboratory service haematology department, accredited by the south african national accreditation system (sanas), iso 15189. d-dimers were measured using the d-dimer plus assay (siemens healthcare diagnostics products gmbh, marburg, germany). the test requires a latex-agglutination test which is then processed using an automated quantitative turbidimetric d-dimer assay. anti-xa levels were measured using the sta®-liquid anti xa assay (diagnostica stago pty ltd, doncaster, australia). the recommended target reference ranges for adults on treatment is 0.6 iu/ml – 1.0 iu/ml, and 0.2 iu/ml – 0.6 iu/ml for prophylaxis. reaction time evaluates the time from coagulation cascade initiation to fibrin generation and clot propagation. it reflects activity of the coagulation cascade. a shorter r-time reflects hypercoagulability. it is calculated from the beginning of the test to the first detectable clot formation (2 mm). the normal reference range is 5 min – 10 min.20 kinetics time determines the rate at which a clot is formed and is measured from the beginning of clotting to the proper formation of a clot (20 mm). it measures fibrin deposition and cross-linking. the normal reference range is 1 min – 2 min.20 the alpha angle determines the speed of clot growth and strengthening. it is the angle between r-time and a line from the time of clotting initiation to the point of maximal clot formation speed. the normal angle is 53°–72°. when decreased, it suggests a deficiency in fibrinogen.20,21 maximum amplitude refers to the maximal amplitude of the thromboelastograph curve; the normal range is 50 mm – 70 mm.20 lysis at 30 min depicts the speed of fibrinolysis and shows the percentage of reduction 30 min after ma. the normal range is 0% – 8%.20 the tci assesses overall coagulation status, and the formula (equation 1) is as follows: the normal tci range is within −3.0 and +3.0 (3 standard deviations from the mean of zero). hypercoagulability is a tci greater than +3.0, and coagulopathy is a tci less than −3.0.20 statistical analysis data were collected and managed using redcap®19 electronic data capture tools hosted at the university of the witwatersrand, and statistical analyses was performed using statsoft, inc. data analysis system, statistica version 13.3 (www.statsoft.com; tibco software inc., santa clara, california, united states).spearman’s correlation was used to determine if there was an association between anticoagulation test parameters. student’s t-test and mann whitney u test were used to determine the relationship between the dosing groups of enoxaparin and coagulation tests. a multiple linear regression model was performed (online supplementary table 1) using the thromboelastogram parameters (r-time, k-time, alpha angle, ma and a30) to predict anti-xa levels (table 1). only r-time, k-time and a30 were included in the final model, as p-values were ≤ 0.2 (a 20% probability that the correlation was a result of chance was considered acceptable). alpha angle was omitted, as this is dependent on k-time. only r-time was significantly associated with anti-xa level, with a p-value of < 0.05. table 1: regression summary of thromboelastogram results and anti-xa levels of patients at 48 h post admission (n = 42), chris hani baragwanath hospital, johannesburg, gauteng, south africa, 01 november 2020 to 31 january 2021. results baseline characteristics forty-two participants were enrolled into the study (figure 1). a total of 22 patients required a prophylactic dose (≤ 40 mg/day) of enoxaparin (52.38%), while 20 patients were treated with a therapeutic dose (> 40 mg/day) of enoxaparin (47.61%) (table 2). fifteen men and 27 women, with ages ranging from 33 years to 74 years, met the entry and exit criteria, enrolling them into the study. patients with prior vte, bleeding disorders, or on any anticoagulants, were excluded from the study to ensure a population which would not be biased towards bleeding or thrombosis. figure 1: study flow diagram of records reviewed of patients enrolled into the study between 01 november 2020 and 31 january 2021 at chris hani baragwanath hospital, johannesburg, gauteng, south africa. table 2: baseline patient characteristics (n = 42). anti-xa levels and thromboelastogram coagulation index there was a statistically significant low to moderate correlation between anti-xa levels and tci, using spearman’s correlation = 0.43 (p = 0.014) among patients admitted for suspected covid-19 pneumonia (figure 2). figure 2: correlation between anti-factor xa and thromboelastogram coagulation index of the 42 patients enrolled between 01 november 2020 and 31 january 2021 at chris hani baragwanath hospital, johannesburg, gauteng, south africa. anti-xa and thromboelastogram parameters there was a statistically significant moderate correlation between anti-xa levels and r-time, with spearman’s correlation = 0.52, p = 0.002 (figure 3). figure 3: correlation between anti-factor xa and reaction time of the 42 patients enrolled between 01 november 2020 and 31 january 2021 at chris hani baragwanath hospital, johannesburg, gauteng, south africa. there was a statistically significant low correlation between anti-xa levels and k-time. with spearman’s correlation = 0.35, p = 0.049 (figure 4). figure 4: correlation between anti-factor xa and kinetics time of the 42 patients enrolled between 01 november 2020 to 31 january 2021 at chris hani baragwanath hospital, johannesburg, gauteng, south africa. although the k-time and alpha angle are mathematically correlated, we did not find a statistical correlation between anti-xa levels and alpha angle. there were also no significant correlations between d-dimer and thromboelastogram parameters. spearman rank order correlations revealed no marked correlations between thromboelastogram and d-dimers, at a significance of p < 0.05. enoxaparin dose, thromboelastogram parameters, and anti-xa levels only anti-xa levels were significantly associated with the dose of enoxaparin, while amongst the thromboelastogram parameters, lysis time at 30 min was significantly associated with the dose of enoxaparin (online supplementary table 2). discussion the main finding of our study demonstrated an association between thromboelastogram parameters and anti-xa levels 48 h after admission and anticoagulation with enoxaparin. using tci, we found that higher anti-xa levels were associated with higher tci values. this does not necessarily mean that tci can be used as a surrogate marker for anti-xa levels; it merely indicates that patients with higher thrombotic risk (higher tci values) were correctly put onto therapeutic lmwh. the mean tci of our sample was 5.67 (5.14–6.27). this is significantly higher than the normal range of −3.0 to +3.0, indicating hypercoagulability. this is likely indicative of the hypercoagulability associated with covid-19 and the need for lmwh. tekkesin et al.,18 in a study conducted in turkey in 2015, report all tci values in their sample to be below 0.0 following administration of enoxaparin. this is a large discrepancy which could be explained by various factors, ranging from severity of illness of participants to enoxaparin preparations, and requires further investigation. tekkesin et al.18 found a correlation of 0.38 between tci and anti-xa levels 24 h post initiation of anticoagulation in turkey. the tci values, as expected, predictably dropped at higher anti-xa levels. in our study, higher anti-xa levels appear to either be associated with higher tcis or there appears to be no association at all. our study population comprised a group of patients with a higher pro-coagulable phenotype; patients were not excluded on the basis of comorbidities and organ dysfunction. our data included subgroups with both prophylactic and therapeutic enoxaparin dosing. these differences may account for the variance in correlation between the two studies. our study measured tci and anti-xa levels at 48 h post initial enoxaparin dosing. buckley et al., in the united states, 2021, looked at a similar population to ours (covid-19), and also found a weak correlation (r = 0.30) between tci and anti-xa levels.22 the important differences in their study was that therapeutic anticoagulation was targeted using a continuous enoxaparin infusion for more than 24 h at the discretion of the treating physician, rather than radiologically confirmed vte. no standardised anticoagulation protocol was used among different patients in the population, rather enoxaparin infusion rates were altered depending on anti-xa levels. the differences in population characteristics may allude to the reasoning for our differences in results, such as: all patients in their study were admitted to the intensive care unit as opposed to 52% in our study, and almost 60% of their population was male compared to 35% in our data. we had a predominantly black african population, while less than 10% of their study population was black. finally, median sequential organ failure assessment scores differed, with 5 versus 3 in our study data.22 we found a significant correlation between anti-xa levels and r-time at 48 h, where higher anti-xa levels appear to coincide with longer r-time. but this does not mean r-times can be used as a surrogate for anti-xa levels. tekkesin et al. found a correlation between anti-xa levels and r-time, which was only significant at 4 h post first dose of anticoagulation and normalised by 12 h.18 this discrepant finding between our study and that of tekkesin may be attributed to the underlying disease in our studies. tekkesin et al. had a patient population who underwent surgical orthopaedic intervention, which, as postulated by bunescu et al.23 in london, 2002, releases granular factor from platelets, which has an impact on reaction time. granular factors are released during surgery but then end at or shortly after completion of surgical procedures. this may be one of the mechanisms of rapid r-time normalisation in the study done by tekkesin et al.,18 as opposed to ongoing pro-coagulopathic processes in covid-19. klein et al.17 (united states, 2000) found a significant correlation between r-time and anti-xa levels in a population of 24 patients undergoing orthopaedic surgery. contrary to tekkesin et al., the significance was maintained at both peak and trough levels, questioning the platelet granular factor theory.17 white et al. (australia, 2011) found no correlation between anti-xa levels and thromboelastogram r-time (p = 0.38).24 their population of 50 patients were admitted to a coronary care unit and received therapeutic dosed enoxaparin (1 mg/kg twice daily); anti-xa levels and thromboelastograms were taken after 48 h post first dose of anticoagulation. of note, the non-significant correlation between anti-xa levels and r-times may be attributed to sampling errors (mean time from dosing of enoxaparin to thromboelastogram measurement was 4.17 h ± 0.5 h), as this study failed to find a correlation between enoxaparin dose and anti-xa levels. despite all patients receiving therapeutic dosed enoxaparin, a cutoff of 100 mg/dose was implemented in patients weighing more than 100 kg in this study. white et al. had not factored in the decline in creatinine clearance as a result of age, which would impact on enoxaparin clearance.24 buckley et al. found a weak but significant correlation between thromboelastogram r-time and anti-xa level;22 however, the differences in this study compared to ours may be because of the nature of enoxaparin administration (continuous infusion vs daily and twice-daily dosing). artang et al. (denmark, 2009) conducted a study using seven healthy male volunteers who were injected subcutaneously with dalteparin (lmwh) 120 iu/kg.25 reaction time was then compared to anti-xa levels at baseline, then at 2, 4, 5 and 24 h post dalteparin administration. at 24 h, a strong correlation of r = 0.82 was found. however, multiple differences are found in this study when compared to ours; most importantly, the lmwh used was not enoxaparin but rather dalteparin. alongside this, the study consisted of seven male participants who were all healthy and did not suffer from the same disease process as our population (covid-19). thromboelastogram kinetics time and alpha angle we found a statistically significant low correlation between anti-xa levels and k-time (r = 0.35), which is different from the study by buckley et al.,22 who found no relationship between anti-xa levels and k-time (r = 0.08). this was likely because we measured the k-time at peak enoxaparin dose effect (3 h post dose) compared to the continuous infusion, which may not have achieved the optimal anti-xa level. furthermore, differences in our study population, especially regarding disease profile, may be contributory as previously mentioned.22 we failed to detect a statistically significant relationship between anti-xa levels and ma, which is similar to the findings of buckley et al.22 these findings are not surprising, as much as 80% of the ma is contributed to by platelet activity,26 which is not affected by enoxaparin. our study found no statistically significant correlations between d-dimer and thromboelastogram parameters. this has also been found by chandel et al. in a study of 24 patients with severe covid-19 pneumonia.27 their study found a discordant or unpredictable relationship between clot burden and d-dimer levels. this may explain the lack of relationship between d-dimer, anti-xa levels and thromboelastogram parameters.27 although we found a statistically significant difference in a30 percentages between prophylactic and therapeutic dose enoxaparin, there was no clinically significant difference. this finding is confirmed by lloyd-donald et al. (australia, 2020) who failed to find any clinically significant relationship in a30.28 however, our finding is an interesting one, and may require further investigation on the effect of enoxaparin on fibrinolysis. limitations our study had several limitations which need to be considered. firstly, we included a limited sample size, which is open to large variations in precision. secondly, although we had two dosing strategies of enoxaparin, the exact dose in the therapeutic dosed group was not considered for the analysis. the data are also limited by use of a single lmwh medication (enoxaparin) and are likely not to apply exactly to other anticoagulants. the nature of a single centre study limits the generalisability of the findings. however, given the limited published data available, our study findings are not insignificant, given that they contribute the second largest published sample sized study. conclusion we have demonstrated that the tci (comprising r-time, k-time, and alpha angle) is statistically significantly correlated with anti-xa levels in this study sample receiving lmwh. in particular, tci values appear to increase as anti-xa levels increase. it would be useful to see if this finding is replicated in other studies and, if so, why. our findings should be interpreted with reserve and do not mean that tci can replace anti-xa testing. anti-xa levels were associated with the dose of lmwh (as expected), with lysis time at 30 min being the thromboelastogram parameter associated with lmwh dose (an interesting finding worth further investigation regarding the relationship between lmwh and fibrinolysis). the possibility should also be considered that the high tci values obtained in patients with higher anti-xa levels are merely a reflection of the hypercoagulability present in these patients (who are correctly, it would seem, treated with higher doses of enoxaparin), and that these high tci values have no direct relationship with the measured anti-xa levels at all, but rather with the particular patient group receiving higher enoxaparin doses. this study serves as a basis for future research with larger sample sizes, including patients with varying disease profiles and severity of illness. further studies should focus on threshold values for r-time and other thromboelastogram indices that indicate specific anticoagulation targets. acknowledgements this article is partially based on the b.p’s thesis entitled ‘thromboelastography in covid-19 patients: an observational study in the south african context’ towards the degree of masters in medicine in the department of internal medicine, university of the witwatersrand, south africa on 20 december 2024, with supervisors dr s.a. van blydenstein and professor s. omar. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. teg® 6s cartridges were provided by qiagen. qiagen had no role in the study design, the collection, analysis, and interpretation of data, in writing of the report, or in the decision to submit the article for publication. authors’ contributions b.p., s.a.v.b., and s.o. contributed to study conception and design. s.o. and b.p. contributed to analysis and interpretation of results. b.p. and s.a.v.b. were responsible for the preparation of the draft manuscript. b.p., s.a.v.b., and s.o. reviewed the results and approved the final version of the article. sources of support teg® 6s cartridges were provided by qiagen. data availability the data that support the findings of this study are not openly available because of reasons of sensitivity (human data) and are available from the corresponding author, b.p., upon reasonable request. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. the article does not necessarily reflect the official policy or position of any affiliated 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anticoagulation resulting from low molecular weight heparin: a comparison with anti-xa concentrations. anesth analg. 2000;91(5):1091–1095. https://doi.org/10.1097/00000539-200011000-00009 tekkesin n, tekkesin m, kaso g. thromboelastography for the monitoring of the antithrombotic effect of low-molecular-weight heparin after major orthopedic surgery. anatol j cardiol. 2015;15(11):932–937. https://doi.org/10.5152/akd.2014.5723 harris pa, taylor r, minor bl, elliott v, fernandez m, o’neal l, et al. the redcap consortium: building an international community of software platform partners. j biomed inform. 2019 jul;95:103208. whitton tp, healy wj. review of thromboelastography (teg): medical and surgical applications. ther adv pulm crit care med. 2023;18:29768675231208426. https://doi.org/10.1177/29768675231208426 shaydakov me, sigmon df, blebea j. thromboelastography [homepage on the internet]. treasure island: statpearls publishing; 2025 [cited 2025 jan 21]. available from: http://www.ncbi.nlm.nih.gov/books/nbk537061/ buckley ms, benanti ge, gilbert b, meckel j, dzierba al, maclaren r. correlation between heparin anti-xa activity and thromboelastography in adult critically ill covid-19 patients. pharmacotherapy. 2023;43(8):795–803. https://doi.org/10.1002/phar.2829 bunescu a, widman j, lenkei r, menyes p, levin k, egberg n. increases in circulating levels of monocyte-platelet and neutrophil-platelet complexes following hip arthroplasty. clin sci (lond). 2002;102(3):279–286. https://doi.org/10.1042/cs1020279 white h, sosnowski k, bird r, jones m, solano c. the utility of thromboelastography in monitoring low molecular weight heparin therapy in the coronary care unit. blood coagul fibrinolysis. 2012;23(4):304–310. https://doi.org/10.1097/mbc.0b013e32835274c0 artang r, frandsen nj, nielsen jd. application of basic and composite thrombelastography parameters in monitoring of the antithrombotic effect of the low molecular weight heparin dalteparin: an in vivo study. thromb j. 2009;7:14. https://doi.org/10.1186/1477-9560-7-14 harr jn, moore ee, chin tl, et al. platelets are dominant contributors to hypercoagulability after injury. j trauma acute care surg. 2013;74(3):756–762; discussion 762–765. https://doi.org/10.1097/ta.0b013e3182826d7e chandel a, patolia s, looby m, bade n, khangoora v, king cs. association of d-dimer and fibrinogen with hypercoagulability in covid-19 requiring extracorporeal membrane oxygenation. j intensive care med. 2021;36(6):689–695. https://doi.org/10.1177/0885066621997039 lloyd-donald p, vasudevan a, angus p. comparison of thromboelastography and conventional coagulation tests in patients with severe liver disease. clin appl thromb hemost. 2020;26:107602962092591. https://doi.org/10.1177/1076029620925915 article information authors: caroline mangare1 amos mbugua1 peter maturi2 jamila rajab2 rainer blasczyk3 hans-gert heuft3 affiliations: 1jomo kenyatta university of agriculture and technology, nairobi, kenya 2kenyatta national hospital/university of nairobi, department of hematology and blood transfusion, nairobi, kenya 3institute for transfusion medicine, hannover medical school, hannover, germany correspondence to: caroline mangare email: caroleunice2000@gmail.com postal address: 54128 00200 nairobi, kenya dates: received: 28 jan. 2015 accepted: 15 aug. 2015 published: 25 sept. 2015 how to cite this article: mangare c, mbugua a, maturi p, et al. red cell alloand autoimmunisation in transfused sickle cell and cancer patients in kenyatta national hospital, nairobi, kenya. afr j lab med. 2015;4(1), art. #297, 7 pages. http://dx.doi.org/10.4102/ajlm.v4i1.297 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. red cell alloand autoimmunisation in transfused sickle cell and cancer patients in kenyatta national hospital, nairobi, kenya in this original research... open access • abstract • introduction • methods    • setting and design    • data and sample collection    • laboratory investigations    • immunohaematological testing    • statistical analyses    • ethical considerations • results    • patient data    • serological results    • rbc alloantibody identification    • red cell autoimmunisation    • comparison of combined rbc alloand autoimmunisation in sickle cell versus cancer patients    • healthy donor phenotypes • discussion    • limitations    • conclusion • trustworthiness    • reliability and validity • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: currently, no data are available on the prevalence of red blood cell (rbc) antibody formation amongst kenyan patients with multiple transfusion needs, such as patients with sickle cell disease (scd) or haematological malignancies (hm) and solid (sm) malignancies. objectives: we determined the prevalence and specificities of rbc alloantibodies and autoantibodies in two patient groups with recurrent transfusion demands at kenyatta national hospital, nairobi, kenya. method: between february and august 2014, 300 samples from scd, hm and sm patients were collected and screened for alloantibodies. samples from 51 healthy blood donors were screened for irregular antibodies and phenotyped. results: amongst the 228 patients with viable samples (scd, n = 137; hm, n = 48; sm, n = 43), the median transfusion frequency was two to three events per group, 38 (16.7%) were rbc immunised and 32 (14.0%) had a positive direct antiglobulin test. we identified specific alloantibodies in six patients (2.6%). four of these six were scd patients (2.9%) who had specific rbc alloantibodies (anti-cw, anti-m, anti-cob, anti-s); amongst hm patients one had anti-k and one had anti-lea. rbc autoantibody prevalence was 3.1% (7/228). amongst the healthy blood donors, the ror, ccd.ee and r2r, ccd.ee phenotypes accounted for 82% of the rhesus phenotypes and all were kell negative. conclusion: the numbers of transfusions and the rates of rbc alloantibodies are low and the most important rbc alloantibody-inducing blood group antigens are relatively homogeneously distributed in this population. a general change in the kenyatta national hospital pre-transfusion test regimen is thus not necessary. the current transfusion practice should be reconsidered if transfusion frequencies increase in the future. introduction top ↑ blood transfusion constitutes an important supportive modality in the management of patients with sickle cell disease (scd) and cancer, because of longer periods of treatment and increased survival rates. scd is the most prevalent haematologic genetic disease in kenya1 and cancer is an increasingly important challenge for the kenyan public health system.2 red blood cell (rbc) alloand autoimmunisation often develop as a result of transfusions with allogeneic blood and occur because of the response of recipients’ immune systems to foreign rbc antigens from donors.3 some of the facets involved in these immunological reactions are: recipient age; sex; history of pregnancy; number of blood units transfused; and diagnosisand treatment-related impairment to the recipient's immune system.4,5 rbc alloimmunisation is associated with clinical complications, such as morbidity resulting from acute and delayed haemolytic transfusion reactions. the former can mimic a sickle cell crisis. furthermore, alloimmunisation creates difficulties for laboratories, including expensive and time-consuming laboratory workups to determine compatible blood, especially for cases with multiple alloantibodies (alloab). alloabs can become undetectable over time and/or be boostered as an anamnestic response after another transfusion. rbc alloab against incompatible rbc in an allogeneic bone marrow transplant may require procedures for rbc reduction.6,7 the development of alloab has been associated with that of autoantibodies (autoab),8,9 which can shorten the lifespan of recipients’ own rbcs and/or transfused rbcs and potentially cause haemolysis. because of this, these patients may require several transfusions and may need interventions, such as drugs to suppress the immune system and/or splenectomy.9 these challenges need to be considered when handling patients who are likely to be transfusion-dependent, as well as those who could benefit from haematopoietic stem cell transplantations. decreasing the risk of rbc alloimmunisation by implementing strategies to avoid allogeneic blood transfusions (e.g., erythropoietin administration in cancer patients) or extensive phenotypic matching of rbc blood group antigens, such as the rhesus, kell, duffy, kidd and mns blood groups, has been advocated previously.10,11 however, this is costly and impractical in many health settings, particularly in developing countries. studies conducted on the frequency of rbc alloimmunisation in different patient populations have reported rates of 1% to 6% in occasionally transfused patients and up to 30% in poly-transfused patients.12 in europe and the united states, alloimmunisation rates of 5% to 36% have been reported amongst transfused scd patients.13 currently, there are minimal data from africa regarding transfusion-dependent rbc allo-/autoimmunisation. the few existing studies have reported varied results. a ugandan study3 recently reported an rbc alloimmunisation prevalence rate of 6.1% amongst 428 scd patients. an investigation conducted in egypt amongst 42 scd patients reported an alloimmunisation rate of 21.4%,14 whereas amongst 130 tunisian thalassaemia patients, rbc alloimmunisation was 7.7% and 40% of these patients developed rbc autoantibodies.15 in a study of 108 ugandan patients with malignancies, alloimmunisation was reported at a frequency of 8.3%.16 there are no data on the prevalence of rbc alloab/autoab formation amongst kenyan patients, where pretransfusion testing is limited to abo/rhesus d group typing and crossmatching only. as there is no routine pre-transfusion rbc antibody screening or identification, this study sought to determine the prevalence and specificities of rbc alloabs and autoabs amongst two different groups of transfusion recipients at kenyatta national hospital (knh), nairobi, kenya. in addition, we screened samples from blood donors for irregular antibodies and phenotyped them for abo, rhesus and kell antigens in order to determine whether there is alloimmunisation in the general population served by knh. methods top ↑ setting and design using a cross-sectional design, scd, haematological malignancy (hm) and solid malignancy (sm) patients attending haematology and oncology clinics at knh were approached between february and august 2014 and invited to participate in the study. to be eligible for the study, participants had to be knh patients with scd, hm or sm who had received at least one allogeneic blood transfusion; 300 patients met the inclusion criteria. samples from 51 healthy blood donors of african ancestry from knh's blood bank were obtained for limited rbc antigen phenotyping. the kenya national blood transfusion policy defines the criteria for healthy donors as those who are aged 18–65 years; weigh more than 50 kg; have a minimum haemoglobin of 12 g/dl; have normal blood pressure (systolic 120–129 mmhg, diastolic 80–89 mmhg) and a pulse rate of 60–100 beats per minute.17 data and sample collection after obtaining informed consent, 2–4 ml of blood was drawn from patients into ethylenediaminetetraacetic acid tubes for laboratory investigations. patients’ notes were reviewed for: demographic characteristics; recipient age; sex; diagnosis; history of pregnancy; and transfusion history and indications. the number of blood components, units transfused and transfusion episodes were recorded. healthy blood donor samples were collected from donors who gave consent and met the healthy donor criteria. documentation of patient ethnicity is a routine requirement in kenyan medical records or clinical data. laboratory investigations plasma and rbcs were separated within two hours after collection and the plasma was frozen whilst the red cells were stored at 2 °c – 6 °c. rbcs were preserved by adding a drop of citrate phosphate dextrose anticoagulant. the samples were then shipped on dry ice at a controlled temperature to the institute for transfusion medicine at hannover medical school, hannover, germany for immunohaematological analysis. immunohaematological testing the bio-rad id gel card system (diamed-id®; bio-rad laboratories, diamed gmbh, cressier, switzerland) was used with both untreated and papain-treated rbc reagents. plasma samples were screened for the presence of rbc alloab by use of a standard three-cell panel of reagent group o rbcs using nacl gel cards at room temperature for alloabs with low thermal range and low ionic strength saline (liss) gel cards (liss/coombs) at 37 °c for warm-reacting alloabs. for samples that showed agglutination, subsequent antibody identification was carried out with at least one 11-cell group o rbc panel (usually bio-rad, switzerland). in instances without immediate determination of alloab specificity, additional cell panels (e.g., an 11-cell [grifols inc., los angeles, california, united states] and/or a 16-cell panel [sanquin, plesmanlaan, amsterdam, netherlands]) were used. if a patient's plasma sample showed agglutination of reagent screening cells, an autocontrol was also performed by reacting the patient's rbcs with his or her own plasma. positive autocontrols were further evaluated by means of a poly-/monospecific direct antiglobulin test (dat). dat was performed using monoclonal gel cards consisting of anti-igg, anti-iga, anti-igm, anti-c3c and anti-c3d. from samples that were positive with at least one of these antiglobulins, an acid eluate was prepared. the eluate was screened using the standard three-cell panel. those that were positive were then analysed for specificity using 11-cell panels containing these antigens: d, c, e, c, e, k, k, fya, fyb, jka, jkb, lea, leb, p1, m, n, s and s. in addition, donor blood was screened for irregular antibodies using a panel of three screening cells and then phenotyped for abo, rhesus (c, c, d, e, e) and kell antigens. plasma samples of 40 blood donors were screened for rbc alloab. patients were considered to be alloimmunised if antibodies to one or more rbc antigens could be identified, whilst autocontrol and dat screening remained negative. patients with a positive autocontrol, a positive dat and a reactive acid eluate were considered to be sensitised to have autoabs to rbcs. in cases with a positive autocontrol and a positive dat, but a non-reactive acid eluate, a non-specific loading of the rbc surface with immunoglobulins was assumed. ‘immune’ antibodies are formed after immunisation through pregnancy and/or previous transfusions. ‘naturally-occurring’ antibodies are formed as a result of exposure to environmental agents similar to red cell antigens, such as bacteria. statistical analyses statistical software packages were used: excel 5.0 (microsoft, redmond, california, united states 1993) for data management and statistical package for the social sciences 12.0 (spss inc., chicago, illinois, united states 2003) was used for analysis. student's t-test was used for variables with normal distribution. categorical variables of possible associations between rbc alloimmunisation and sex, units of blood transfusion, diagnosis of scd or solid and haematological malignancy were compared using the chi-squared test. groups were assumed to differ significantly when the probability level was less than 0.05. ethical considerations ethical approval was obtained from knh/university of nairobi ethics review committee. both oral and written informed consent was obtained from patients or their guardians. donors completed a questionnaire provided by the blood bank services and signed a consent form. results top ↑ patient data of the samples from 300 patients who met the inclusion criteria, 72 samples could not be evaluated for the following reasons: insufficient sample because of leakage during shipment (n = 40); samples breaking in the centrifuge whilst processing (n = 20); and lack of proper labeling (n = 12). a total of 228 samples were analysed, including 137 from scd patients, 48 from hm patients and 43 from sm patients, with a median number of two to three transfusions per group (table 1). of these, 117 (51.3%) were women, of whom 22 (18.8%) had a history of pregnancy. overall, the mean age at the time of blood transfusion was aged 17.2 years (range: 1–93). cancer patients, in particular sm patients, were significantly older than scd and hm patients (p < 0.001). indeed, the majority of patients were children aged 16 years or younger (n = 159; 70%); 14% were aged three years or younger (figures 1 and 2). there were no significant differences in the female to male ratios between the groups. figure 1: transfused sickle cell disease patients by age, sex and seropositivity at kenyatta national hospital, nairobi, kenya, 2014. figure 2: transfused cancer patients by age, sex and seropositivity at kenyatta national hospital, nairobi, kenya, 2014. table 1: characteristics of transfused sickle cell disease and cancer patients at kenyatta national hospital, nairobi, kenya, 2014. patients received abo⁄rhesus d compatible and non-leucocyte-depleted whole blood units (n = 532), packed rbc transfusions (n = 85) and platelet transfusions (n = 68), totaling 685 units of blood in 593 transfusion events (i.e., 1 transfusion unit per transfusion episode or a mean of 2.7 transfusion units per patient). of the scd patients, 90% were transfused because of severe anaemia – haemoglobin less than 5–6 g/dl according to world health organization guidelines.18 transfusion in malignancy patients was mainly a result of anaemia caused by intensive chemotherapy, by the disease process and/or surgical interventions. all cancer patients were receiving chemotherapy at the time of enrollment into the study. hm patients had the following types of malignancies: acute lymphoblastic leukaemia (n = 15), chronic lymphoblastic leukaemia (n = 10), hodgkin's lymphoma (n = 9), multiple myeloma (n = 6), acute myeloid leukaemia (n = 5) and chronic myeloid leukaemia (n = 3). sm patients had the following types of malignancies: abdominal tumour (n = 1), bladder (n = 2), breast (n = 8), cervical (n = 4), colon (n = 3), hepatocellular (n = 2), mouth (n = 1), nasopharyngeal (n = 1), pancreatic (n = 2), prostate (n = 4), rectum (n = 4), rhabdomyosarcoma (n = 4), sinonasal tumour (n = 1), squamous cell carcinoma (n = 2) and stomach cancer (n = 4). the p-value for the number of blood units transfused was 0.004, which was statistically significant as cancer patients received more transfusions. serological results the overall prevalence of rbc immunisation was 16.7%, with 38 of the 228 patients testing positive for antibody screening. the prevalence of rbc immunisation amongst scd patients was 20.4% (28 of 137 patients) and amongst malignancy patients, 11.0% (dat+, n = 8 plus alloab+, n = 2; altogether 10 out of 91 patients). rbc alloantibody identification only 38 patients were positive for the antibody screening, and rbc alloantibodies were detected in only 6 of 228 patients (2.6%) (table 2). the rate of alloab formation amongst scd patients was 2.9% (4 of 137) and 4.2% (2 of 48) amongst hm patients, whereas the prevalence amongst sm patients for alloab identification was 0. the specificities of the alloabs from the scd patients were anti-cw, anti-s, anti-cob (probably immune in nature) and anti-m (probably naturally occurring). in addition, there was one anti-k (immune) and one anti-lea (natural) in the two hm patients, whereas the sm group showed no rbc alloimmunisation. the rate of alloimmunisation was 6.14% for men versus 8.33% for women; the difference was not statistically significant (p = 0.25). table 2: serological results of transfused sickle cell disease and cancer patients at kenyatta national hospital, nairobi, kenya, 2014. red cell autoimmunisation of the 228 patients, 32 patients (14.0%) presented a positive dat (table 2). fifty per cent of these patients (16 of 32) were positive for anti-igg alone, whereas 18.8% (6 of 32) showed reactions to anti-igg plus anti-c3c or c3d. out of the subset of 21 igg-positive patients, the acid eluate was reactive in seven, thereby indicating a true rbc autoab prevalence of 3.1% for this population of patients (7 of 228) and 33.3% (7 of 32) amongst the dat-positive patients. rbc autoab prevalence was 5.1% (7 of 137) amongst scd patients, whereas there were no rbc autoabs amongst patients with malignancies. moreover, we observed a few cases (3 of 32) with isolated igm or iga reactivity. the majority (24 of 32) of the dat-positive reactions with anti-igm and anti-iga were observed in the scd group. eighteen per cent (24 of 137) of the scd group were dat positive compared with 8.8% (8 of 91) in the hm/sm group. comparison of combined rbc alloand autoimmunisation in sickle cell versus cancer patients the prevalence of rbc immunisation (demonstration of an immune alloab and a positive dat) amongst scd patients was 19.7% (27 of 137) versus 9.9% (9 of 91). immune alloabs were found in 2.2% (3 of 137) of the scd patients versus 1.1% (1 of 91) of the patients with malignancies. with one exception (polyspecific in eluate, but auto-anti-e in serum), these autoabs showed polyspecificity only. we also performed a comparison for demographic and transfusion variables between patients with and without serological reactivity (table 3). we did not find a significant link between patients’ sex, age or number of units of blood transfused and the positivity of the antibody screening. table 3: characteristics of immunised and non-immunised transfused sickle cell and cancer patients at kenyatta national hospital, 2014. healthy donor phenotypes amongst the blood donor samples, there were no serological peculiarities. fifty-one donors were phenotyped for the rhesus antigens c/c, d, e/e and for the antigen k (table 4). of these, 29 donors (57%) showed the rh phenotype ccd.ee, the other phenotypes were ccd.ee (n = 13), ccd.ee (n = 5), ccddee (n = 2) and single cases of ccddee (n = 1) and ccd.de (n = 1). none of the 51 donors were kell positive. table 4: rhesus and kell phenotypes amongst 51 healthy kenyan blood donors at kenyatta national hospital, 2014. discussion top ↑ the risk of alloimmunisation is a concern that needs to be addressed and managed, especially amongst patients requiring multiple blood transfusions, such as those with scd and malignances. this investigation sought to determine the magnitude of rbc immunisation and to identify antibodies amongst two transfused patient groups. in this study, we detected a significant proportion of patients with some degree of rbc autoimmunisation, as shown by a positive dat in 14.0% of the patients, seven of whom had true rbc autoantibodies. eighteen per cent of scd patients were dat positive compared with 8.8% of hm/sm patients. in contrast, rbc alloab formation was low, at only 2.6%. moreover, the specificities of the demonstrated alloabs do not occur often in daily laboratory results. anti-cw and anti-s are comparatively rare rhand mns antibodies, respectively; and anti-cob is a very rare rbc alloab specificity of the colton system. the one example of anti-k that we detected was the only common rbc alloab specificity. other common rbc alloab specificities, such as anti-d, anti-e, anti-c, anti-c, belonging to the rhesus system, or those of the kell system (other than anti-k), the duffy or the kidd blood group systems, were not found in our study population. in this study, the frequency of alloimmunisation across all patients was determined to be 2.6%; and the rate of alloab formation was 2.2% amongst patients with malignancies and 2.9% amongst scd patients. there may be several reasons for these unexpected findings. firstly, the total numbers of transfusions and the numbers of transfusion events were low, never exceeding the mean values of three transfusion events per patient. this was particularly true for the scd patients, who are known to be at high risk for rbc alloab formation.3,10,19 however, scd patients showed the lowest values for transfused rbc units per patient and transfusion events per patient in our study. the rbc alloimmunisation rate of 2.9% amongst our scd patients is comparable to a study in a jamaican cohort,19 where the rate was 2.6% amongst 115 transfused scd patients and 1.6% amongst the total number of 190 patients. however, this rate differs considerably from that reported in a ugandan study of 428 scd patients, where the prevalence rate was 6.1%.3 although the mean number of transfusions was three blood units in all of these studies, 21 of the 26 alloimmunised patients in the ugandan study had received up to 10 blood units. this is marginally higher than the maximum number (n = 8) of transfusions observed in our study. our scd patients received a mean of 2.4 units of transfusions, hm patients received 4.7 units and sm patients received 3.0 units. therefore, all groups were exposed to minimal antigenic challenge. numerous studies have reported that the rate of rbc alloimmunisation increases with the number of transfusions.3,10,19,20,21,22 this could explain the low alloimmunisation rate amongst our study participants compared with their counterparts in developed countries who received more transfusions. secondly, many other studies have reported higher percentages of rbc alloimmunisation in haemoglo­binopathies, such as scd or thalassaemia, including uganda3 (scd, alloab 6.1% amongst 428 patients), tunisia21 (scd and thalassaemia, alloab 7.8% amongst 309 patients), italy23 (thalassaemia, alloab 5% amongst 1435 patients) and brazil24 (scd, alloab 9.9% amongst 828 patients). these studies included a significantly higher number of patients; thus, the relatively low number of patients in our study might be a second limiting factor. the low rates in our study also differ from studies conducted in populations where there is high heterogeneity between donors and patients. in a study by rosse et al.,10 involving 1814 scd patients with an rbc alloimmunisation rate of 18.6%, the donors were of european-american ancestry and the scd patients were of african-american ancestry. thirdly, patients in our study were predominantly children aged 16 years or younger (n = 159; 70%), 14% were aged ≤ 3 years. studies of paediatric patients have reported lower rbc alloimmunisation rates. aygun et al.25 and sarnaik et al.21 concluded that children with scd who were hypertransfused had a lower frequency of alloimmunisation as compared with adults.21 another study involving 167 paediatric and 62 adult scd patients supported this observation, where the rates of alloand autoimmunisation in children and adults were 29% and 8%; 47% and 9.7%, respectively.26 other authors advocate that transfusion started when patients are young (aged 1–3 years) may induce immune tolerance against alloimmunisation.9 the fact that 14% of our study patients were aged ≤ 3 years could have contributed to the low rate of rbc alloab formation that we observed. fourth, the prevalence of rbc alloimmunisation amongst our cancer patients was low (2.2%), with only two hm patients and no alloabs amongst sm patients. this is lower than that in the ugandan study, where the rate was 8.3% amongst cancer patients. shahida et al.22 studied 150 cancer patients who had at least five transfusions and found the prevalence rate of alloabs to be 6%. in a study by seyfried and walewska11 of 1502 multi-transfused patients, the overall incidence of alloabs was 5.7%, with the lowest rate found amongst patients with lymphoproliferative syndromes (1.8%).11 of note, all the cancer patients in our study were undergoing chemotherapy at the time of transfusion. it has been observed that patients with progressive malignancies undergoing intensive chemotherapy tend to have a low antibody formation response to foreign antigens.26,27,28 finally, a majority (57%) of our donor population expressed the rh formula of ccd.ee, which could partly explain why we did not find rbc alloantibodies directed against highly immunogenic antigens such as d or e. a study by badjie et al.,29 conducted amongst 800 donors from various ethnic groups, found the prevalence of the ccd.ee phenotype to be 81.9% in east africa and a study by baby et al.30 found a prevalence of 67.9% in west africa (mali). these results suggest that a large proportion of donors – exceeding 50% – and transfusion recipients in africa share equal rh phenotypes, so that rh antibodies may be less frequently induced than in other parts of the world. this view is also supported by the low numbers for the ‘rr’ (rhesus-d negative) phenotype amongst our donor group (only 4%). this phenomenon might be also true for kell antibody formation, as we found no kell positive individuals amongst our donors. it has been reported that more than 98% of black africans are kell negative.30,31 we found a positive dat in 32 (14.0%) patients, with a subgroup of seven igg warm autoabs, which can induce significant clinical autoimmune haemolysis. we did not seek information about the presence of autoimmune haemolytic anaemia in these patients, because this can be clinically asymptomatic and the reaction can be masked by the severity of the underlying disease and lack of adequate post-transfusion records. limitations it has been reported that 25% of alloantibodies become undetectable within a median of 10 months of follow-up, which may lead to the underestimation of the prevalence of antibodies formed.10,32 this can result in a patient receiving rbcs and consequently experiencing a secondary immune response that may compromise the benefit of the following transfusion.33 because this was a cross-sectional study, some rbc alloantibodies might have been missed, since they have been reported to disappear with time.32,34 other factors that might also be responsible for the disparity in results include: the fact that the majority of the study patients were children; low mean of transfused units; inability to meet optimal transfusion needs for these patient groups; and the frequency of testing. conclusion in this study, we observed a low rate of rbc alloimmunisation amongst both scd and cancer patients. the low numbers of transfusions and transfusion events that are currently being applied at knh and the relatively homogeneous distribution of rh-/k-rbc alloantigens amongst kenyan donors provide an explanation for the low alloab frequency amongst kenyan transfusion recipients. at the current stage of the kenya health care system, routine antibody screenings or extended rbc antigen matching do not seem to be justified, as the relatively homogenous rbc alloantigen distribution of kenyan blood donors provides at least some protection from immune rbc alloab formation. however, with improvements in health care, more scd and haemato-oncology patients are likely to receive a more intensive transfusion treatment, which could lead to an increased risk of rbc alloimmunisation. therefore, further development of the healthcare system in kenya will require a thorough reconsideration of the pretransfusion laboratory practice, in particular, if transfusion frequencies increase and/or donor groups change. trustworthiness top ↑ this study reflects the findings obtained from laboratory testing and analysis as observed by the technical group. reliability and validity the experimental design and procedures used in this study are reliable and valid as they have been used previously in other studies, most of which are cited in this article. the results of the experiments in this article were obtained using specimens collected in various clinics at kenyatta national hospital, kenya and were analysed using standard procedures in hannover, germany. acknowledgements top ↑ the authors thank the staff of the hematology and oncology clinics and blood transfusion unit at knh, kenya for their help in recruiting patients and donors respectively. the authors also thank knh research and programs department for their monetary support. the authors thank the staff at the institute for transfusion medicine, hannover medical school, hannover, germany, for providing laboratory reagents, space and technical and medical advice. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions c.m. (jomo kenyatta university of agriculture and technology) was the project leader, developed the proposal, obtained ethical clearance for the study, collected and shipped specimens, performed most of the experiments and developed the manuscript. a.m. (jomo kenyatta university of agriculture and technology) made conceptual contributions. p.m. and j.r. (kenyatta national hospital/university of nairobi) were responsible for experimental and project designs. h.-g.h. and r.b. (hannover medical school) provided laboratory space and reagents and performed some of the experiments. all authors were responsible for polishing and approving the final manuscript. references top ↑ aluoch jr, aluoch lh. survey of sickle disease in kenya. trop geogr med. 1993;45(1):18–21. strother rm, asirwa fc, busakhala nb, et al. the evolution of comprehensive cancer care in western kenya. journal of cancer policy. 2013;1(1–2): e25–e30. http://dx.doi.org/10.1016/j.jcpo.2013.04.001 natukunda b, schonewille h, ndugwa c, et al. red blood cell alloimmunization in sickle cell disease patients in uganda. transfusion. 2010;50(1):20–25. http://dx.doi.org/10.1111/j.1537-2995.2009.02435.x klein hg, anstee dj. immunology of red cells. in: klein hg, anstee dj, editors. mollison's blood transfusion in clinical medicine. 11th ed. oxford: blackwell publishing, 2005; p. 48–113. http://dx.doi.org/10.1111/j.1537-2995.2007.01433.x bauer mp, wiersum-osselton j, schipperus m, et al. clinical predictors of alloimmunization after red blood cell transfusion. transfusion. 2007;47(11): 2066–2071. http://dx.doi.org/10.1111/j.1537-2995.2007.01433.x de la rubia j, arriaga f, andreu r, et al. development of non-abo rbc alloantibodies in patients undergoing allogeneic hpc transplantation. is abo incompatibility a predisposing factor? transfusion. 2001;41(1):106–110. http://dx.doi.org/10.1046/j.1537-2995.2001.41010106.x franchini m, gandini g, aprili g. non-abo red blood cell alloantibodies following allogeneic hematopoietic stem cell transplantation. bone marrow transplant. 2004;33(12):1169–1172. http://dx.doi.org/10.1038/sj.bmt.1704524 ahrens n, pruss a, mayer b, et al. association between alloantibody specificity and autoantibodies to red blood cells. transfusion. 2008;48(1):20–24. singer st, wu v, mignacca r, et al. alloimmunization and erythrocyte autoimmunization in transfusion-dependent thalassemia patients of predominantly asian descent. blood. 2000;96(10):3369–3373. rosse wf, gallagher d, kinney tr, et al. transfusion and alloimmunization in sickle cell disease. blood. 1990;76(7):1431–1437. seyfried h, walewska i. analysis of immune response to red blood cell antigens in multitransfused patients with different diseases. mater med pol. 1990;22(1): 21–25. norol f, nadjahi j, bachir d, et al. 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[frequency of red blood cell alloimmunization in polytransfused patients at the university teaching hospital of point g, bamako, mali] article in french. transfus clin biol. 2010;17(4):218–222. http://dx.doi.org/10.1016/j.tracli.2010.06.026 m’baya b, mfune t, mogombo e, et al. the prevalence of red-cell antigens and antibodies in malawi. transfus med. 2010;20(3):196–199. http://dx.doi.org/10.1111/j.1365-3148.2009.00985.x schonewille h, haak hl, van zijl am: rbc antibody persistence. transfusion. 2000;40(9):1127–1131. http://dx.doi.org/10.1046/j.1537-2995.2000.40091127.x natukunda b. red blood cell alloimmunization and antigen matching in sickle cell disease – the african perspective. isbt science series. 2012;7(1):129–133. http://dx.doi.org/10.1111/j.1751-2824.2012.01572.x reverberi r. the persistence of red cell alloantibodies. blood transfus. 2008; 6(4):225–234. abstract introduction methods results discussion acknowledgements references about the author(s) tarsizio chikaonda department of molecular medicine and haematology, faculty of health sciences, school of pathology, university of the witwatersrand, johannesburg, south africa university of north carolina project, lilongwe, malawi nelson nguluwe university of north carolina project, lilongwe, malawi brian barnett university of north carolina project, lilongwe, malawi runa h. gokhale university of north carolina project, lilongwe, malawi robert krysiak university of north carolina project, lilongwe, malawi isaac thengolose university of north carolina project, lilongwe, malawi nora e. rosenberg university of north carolina project, lilongwe, malawi christopher stanley university of north carolina project, lilongwe, malawi james mpunga malawi national tuberculosis programme, lilongwe, malawi irving f. hoffman university of north carolina project, lilongwe, malawi university of north carolina at chapel hill, chapel hill, north carolina, united states mina hosseinipour university of north carolina project, lilongwe, malawi university of north carolina at chapel hill, chapel hill, north carolina, united states lesley scott department of molecular medicine and haematology, faculty of health sciences, school of pathology, university of the witwatersrand, johannesburg, south africa wendy stevens department of molecular medicine and haematology, faculty of health sciences, school of pathology, university of the witwatersrand, johannesburg, south africa citation chikaonda t, nguluwe n, barnett b, et al. performance of xpert® mtb/rif among tuberculosis outpatients in lilongwe, malawi. afr j lab med. 2017;6(2), a464. https://doi.org/10.4102/ajlm.v6i2.464 original research performance of xpert® mtb/rif among tuberculosis outpatients in lilongwe, malawi tarsizio chikaonda, nelson nguluwe, brian barnett, runa h. gokhale, robert krysiak, isaac thengolose, nora e. rosenberg, christopher stanley, james mpunga, irving f. hoffman, mina hosseinipour, lesley scott, wendy stevens received: 08 apr. 2016; accepted: 13 oct. 2016; published: 31 mar. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: xpert® mtb/rif is a molecular test for the detection of mycobacterium tuberculosis and rifampicin resistance. it is considered to be a great advance over smear microscopy and culture. however, there is very little information regarding the performance characteristics of xpert mtb/rif in malawi. objective: we aimed to evaluate the performance of xpert mtb/rif in a malawian setting. methods: stored sputum pellets were processed on xpert mtb/rif between june 2012 and may 2014. results were compared to mycobacteria growth indicator tube and löwenstein-jensen cultures, led fluorescent microscopy and genotype® mtbdrplus assay. rifampicin resistance was confirmed by dna sequencing. results: of the 348 specimens with valid xpert mtb/rif results, 129/348 (37%) were smear-positive and 198/348 (57%) were culture-positive. xpert mtb/rif demonstrated a sensitivity of 93.8% (95% ci 89.4% – 96.8%) and specificity of 97.4% (95% ci 93.5% – 99.3%), with a positive predictive value of 97.8% (95% ci 94.6% – 99.4%) and a negative predictive value of 92.6% (95% ci 87.4% – 96.1%). xpert mtb/rif correctly identified 185/186 (99.5%) rifampicin-sensitive and 2/2 (100%) rifampicin-resistant m. tuberculosis strains. mutations were not detected by sequencing in one isolate which was rifampicin resistant on xpert mtb/rif but sensitive on mtbdrplus. four non-tuberculous mycobacteria grew from four smear-negative specimens, namely, m. avium (n = 1) and m. intracellulare (n = 3). no cross-reactivity was observed with any of the non-tuberculous mycobacteria when using xpert mtb/rif. conclusion: when fully implemented, xpert mtb/rif may have an impact on patient care in malawi. the increased diagnostic yield of xpert mtb/rif over smear microscopy can increase laboratory-confirmed tuberculosis detection and ensure that treatment is given to appropriate individuals or groups. introduction tuberculosis remains a major health challenge that has worsened with the emergence of multi-drug resistant (mdr) tuberculosis strains that are resistant to rifampicin and isoniazid. globally, the impact of tuberculosis is significant, with an annual estimate of 9.6 million tuberculosis cases and over 1.5 million deaths due to tuberculosis in 2014.1 preliminary data show that the prevalence of tuberculosis in malawi is 286/100 000, which is higher than previous estimates by the world health organization.2 diagnosis of tuberculosis continues to be a major challenge in malawi due to the widespread use of diagnostics with poor sensitivity, such as sputum smear microscopy. although more sensitive than smear microscopy, tuberculosis culture, when available, can take days to weeks before a result is available. due to limitations of both smear and culture, pulmonary tuberculosis is often diagnosed late or presumptively. low utilisation of laboratory confirmation and widespread use of empirical treatment can either lead to true tuberculosis cases being missed or to the initiation of tuberculosis treatment in people without the disease.1 malawi has a high hiv burden, with an estimated one million hiv-positive people. the major cause of morbidity and mortality of people living with hiv is tuberculosis. it is estimated that of all tuberculosis cases, 41% are smear-negative and 64% are hiv co-infected.3 diagnosis of tuberculosis may be delayed or missed in hiv-positive, smear-negative tuberculosis patients. as such, it is imperative to rapidly diagnose and treat tuberculosis cases in people living with hiv.3,4 introduction of simple and rapid diagnostic methods, such as the xpert® mtb/rif (cepheid, sunnyvale, california, united states), could benefit smear-negative patients in countries like malawi, where culture is limited to the national tuberculosis reference laboratory. currently, the malawi national tuberculosis programme (ntp) is using xpert mtb/rif to increase tuberculosis case detection and to detect rifampicin resistance as a proxy for mdr tuberculosis followed by culture (liquid and solid) and drug-susceptibility testing (dst) as confirmatory tests.2 dst is not routinely performed in malawi, with the exception of retreatment cases and individuals at risk for mdr tuberculosis.3 this can lead to the development of under-reporting of drug resistance, unnecessary suffering, additional costs for patients, and treatment with suboptimal regimens.5 consequently, this might increase the spread of drug-resistant tuberculosis in the population.1 the xpert mtb/rif can both detect tuberculosis and identify rifampicin resistance in a single, rapid assay,6,7 and is reported as having excellent sensitivity and specificity in detecting mycobacterium tuberculosis and rifampicin resistance.6,8 some recent evaluations have demonstrated that xpert mtb/rif accurately detects 72.5% of smear-negative and 98.2% of smear-positive cases, and rifampicin resistance is detected with a specificity of 100% and sensitivity of 99.1%.6,9 the test has an overall pooled sensitivity of 90.4% (95% ci 89.2% – 91.4%), which is much higher than smear microscopy but is similar to that of solid culture in detecting tuberculosis.1,7 among smear-positive tuberculosis samples, xpert mtb/rif has a pooled sensitivity of 98.7% (95% ci 98.0% – 99.2%), but has a substantially lower sensitivity of 75% (95% ci 72.0% – 77.8%) among smear-negative tuberculosis samples. the overall pooled sensitivity in diagnosing for rifampicin-resistant tuberculosis is 94.1% (95% ci 91.6% – 96.0%) and 97.0% (95% ci 96% – 97.7) pooled specificity.7 furthermore, use of xpert mtb/rif has been linked to improved diagnosis, resulting in early appropriate treatment.10 the malawi ntp recommended rolling out xpert mtb/rif in 2012 to increase detection of tuberculosis, especially in sputum smear-negative and hiv-positive individuals. while xpert mtb/rif has been approved for clinical use, its optimal use in the clinical algorithm for malawi has not been established. there are currently over 50 genexpert® instruments distributed across 40 of the 315 public and private diagnostic centres (laboratories) with acid-fast bacilli testing capacity in the country.2 xpert mtb/rif’s performance is not known in the adult outpatient population in malawi. in this study, we evaluated the performance of xpert mtb/rif in detecting m. tuberculosis complex and determining resistance to rifampicin among both hiv-positive and hiv-negative outpatients at bwaila hospital in lilongwe, malawi. methods ethical considerations approvals for this study were granted by the university of the witwatersrand human research ethics committee (m120256), national health sciences research committee (nhsrc) in malawi (nhsrc # 999) and the university of north carolina (chapel hill) institutional review board (cid 1211). laboratory methodologies we used frozen stored sputum pellets (n = 351) obtained from an observational cohort study which collected 702 samples between april 2011 and july 2012 at bwaila in lilongwe, malawi. the study was looking at the prevalence of drug-resistant tuberculosis among adult outpatients (≥ 18 years) with laboratory-confirmed or clinically-diagnosed tuberculosis registering for tuberculosis treatment at this hiv/tuberculosis clinic.11 pellets were selected at random for use in this study without any special criteria to eliminate bias. testing of these pellets using xpert mtb/rif started in june 2012 at the university of north carolina (unc) project laboratory, lilongwe, malawi. pellets were re-suspended in 1.5 ml phosphate buffer and an aliquot of 0.5 ml was processed on the xpert mtb/rif assay. in the primary study, all sample processes were performed at the unc project laboratory. in brief, sputum smears were prepared and stained with auramine-o stain. smear microscopy was performed using led fluorescent microscopy. mycobacterial cultures (reference standard) were performed using both bactec mycobacteria growth indicator tube media (liquid) and löwenstein-jensen slants (solid). the reference standard was considered positive if there was growth of m. tuberculosis on either of the media and negative if both media were negative. drug susceptibility was investigated using the genotype® mtbdrplus assay (version 2) (hain lifesciences gmbh, nehren, germany). data collected from the above processes were included with data in this evaluation. xpert mtb/rif samples for xpert mtb/rif testing were prepared as per manufacturer’s instructions. sample reagent was added to 500 µl of the re-suspended pellet in the ratio of 3:1 (sample reagent:specimen) as described previously.12 results were available within two hours of sample loading. dna extraction genomic bacterial dna was extracted using a genolyse® kit (hain lifescience gmbh, nehren, germany) from mycobacteria growth indicator tube liquid media by transferring 1.0 ml to a sarstedt micro-centrifuge tube. the tube was centrifuged for 15 minutes at 14 000 rpm. the supernatant was carefully removed and the pellet was re-suspended in 100 µl lysis buffer, vortexed thoroughly and incubated for five minutes at 95°c in a heating block. at the end of the five-minute incubation, tubes were briefly centrifuged to remove condensation. neutralization buffer (100 µl) was added to each tube, vortexed for five seconds and then centrifuged for five minutes at 14 000 rpm. supernatant was transferred to a new tube and the pellet discarded. samples were stored at -80°c for use as dna template. line probe assays the genotype® mtbdrplus (version 2) (hain lifesciences gmbh, nehren, germany) was performed on positive culture isolates according to manufacturer’s instructions to identify m. tuberculosis complex and to determine drug susceptibility to rifampicin and isoniazid as described previously.13 genotype® cm (hain lifesciences gmbh, nehren, germany) was used to identify other common mycobacterium species in samples which tested negative for m. tuberculosis complex on genotype mtbdrplus. pcr and dna sequencing for the rifampicin resistance determining region determination of eligibility for dna sequencing was based on detection of resistance to rifampicin by xpert mtb/rif. all eligible m. tuberculosis strains were shipped to the hiv genotyping laboratory (university of the witwatersrand) and were processed as described below. dna amplification amplification of the 450 bp rpob gene fragment which included the rifampicin resistance determining region was carried out by using forward primer rpobf2 (5’-gag ggt cag acc acg atg ac-3’) and reverse primer rpobr2 (5’-gag ccg atc aga ccg atg t-3’) in a geneamp pcr system 9700 thermocycler (applied biosystems, foster city, california, united states). the total volume of the reaction mix was 50 µl, which contained 5 µl of 10x high fidelity buffer, 1 µl of 10 mm dntp mix, 2 µl of 50 mm mgso4, 1 µl of each primer, 0.2 µl of taq hifi (invitrogen, carlsbad, california, united states), 36.8 µl of molecular grade water and 3 µl of genomic dna. amplification conditions were set as follows: 94°c for 2 minutes (initial denaturation); followed by 35 cycles of 94°c for 30 seconds, 55°c for 30 seconds, 68°c for 40 seconds; followed by a 10-minute final elongation at 68°c. amplified products were visualised on a 1% agarose gel after staining with ethidium bromide. sequencing pcr products, after purification by genejet pcr purification kit (thermo scientific, waltham, massachusetts, united states), were subjected to dna sequencing by automated dna sequencer (abi 3700, applied biosystems, foster city, california, united states). pcr sequencing was carried out with a bigdye terminator v3.1 sequencing kit according to manufacturer’s instructions, using forward primer rpobs (5’-gca gac gtt gat caa cat cc-3’) and reverse primer rpobr2 (5’-gag ccg atc aga ccg atg t-3’). the resulting sequences were analysed using sequencher software v4.8 (genecodes corporation, ann arbor, michigan, united states). statistical analysis all data were double entered into a microsoft excel spreadsheet (microsoft, redmond, washington, united states). discrepancies were checked against source records for completeness and consistency. data analyses were done in stata version 12 (statacorp, college station, texas, united states). the following statistics were calculated: sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv) and 95% confidence intervals (ci). mycobacterium culture was considered the reference standard. results a total of 351 sputum pellets were processed both on culture and xpert mtb/rif. however, only 348 sputum pellets had valid xpert mtb/rif results. the remaining three samples were not included in the final analysis due to an error (n = 1) and an invalid result (n = 2) on xpert mtb/rif. of the 348 sputum pellets, 200 (57%) were from hiv-positive individuals, and 148 (43%) were hiv-negative. among all samples, 219/348 (63%) were smear-negative and 129/348 (37%) were smear-positive. as shown in table 1, 70/219 (32%) of smear-negative and 128/129 (99.2%) of smear-positive samples were culture positive. table 1: comparison of xpert mtb/rif and culture diagnostic test results in relation to smear microscopy for outpatients at a single hospital in lilongwe, malawi (2011–2012). xpert mtb/rif detected m. tuberculosis in 58/219 (27%) of smear-negative and 127/129 (98.5%) smear-positive cases. xpert mtb/rif correctly detected m. tuberculosis in 181/198 (91.4%) of the culture-positive samples. of the remaining 17 samples, 1/198 (0.5%) was positive while 16/198 (8.1%) were negative for acid-fast bacilli on smear microscopy. a total of 150 samples were culture-negative regardless of their smear status. among the 150 culture-negative samples, m. tuberculosis was detected by xpert mtb/rif in four (2.7%) samples which were smear-negative on microscopy. xpert mtb/rif did not detect m. tuberculosis in 12/70 (17%) of smear-negative, culture-positive samples. we further observed that 145/219 smear-negatives were culture negative and xpert mtb/rif negative. out of these, 93/145 (64%) were from hiv-positive individuals. four non-tuberculous mycobacteria grew from a total of four smear-negative specimens: m. avium (n = 1) and m. intracellulare (n = 3). no cross reactivity was observed with any of the non-tuberculous mycobacteria when using xpert mtb/rif (table 1). when compared to culture, the overall sensitivity for xpert mtb/rif was 93.8% (95% ci 89.4% – 96.8%) and specificity was 97.4% (95% ci 93.5% – 99.3%), with ppv of 97.8% (95% ci 94.6% – 99.4%) and npv of 92.6% (95% ci 87.4% – 96.1%). among smear-negative individuals, the overall sensitivity of xpert mtb/rif was 81.8% (95% ci 70.4% – 90.2%) and specificity was 97.4% (95% ci 93.4% – 99.3%), with ppv of 93.1% (95% ci 83.3% – 98.1%) and npv of 92.5% (95% ci 87.3% – 96.1%) (table 2). table 2: sensitivity, specificity, positive predictive value and negative predictive value of smear microscopy and xpert mtb/rif at 95% ci compared with sputum culture for outpatients at bwaila hospital, lilongwe, malawi (2011–2012). stratified by hiv status, sensitivity for smear microscopy was 78.9% (95% ci 69.4% – 86.6%) among hiv-negative individuals, with a specificity of 100% (95% ci 93.3% – 100%). among hiv-positive individuals, sensitivity was 51.5% (95% ci 41.4% – 61.4%) and specificity was 99.0% (95% ci 94.4% – 100%). the sensitivity (90%; 95% ci 82.4% – 95.1%) and specificity (96%; 95% ci 90.1% – 98.9%) of xpert mtb/rif were lower in hiv-positive individuals, with ppv of 95.7% (95% ci 89.5% – 98.8%) and npv of 90.6% (95% ci 83.3% – 95.4%). this was compared to hiv-negative participants, which showed a sensitivity of 97.9% (95% ci 92.5% – 99.7%) and specificity of 100% (95% ci 93.4% – 100%), with ppv of 100% (95% ci 96.1% – 100%) and npv of 96.4% (95% ci 87.7% – 99.6%). among smear-negative, hiv-positive individuals, the sensitivity of xpert mtb/rif was 78.7% (95% ci 64.3% – 89.3%) with a specificity of 96% (95% ci 90% – 98.9%) (table 2). dst using genotype mtbdrplus was performed on 188/348 tuberculosis strains. when compared to genotype mtbdrplus, xpert mtb/rif correctly identified 185 of 186 (99.5%) rifampicin-sensitive m. tuberculosis and 2/2 (100%) rifampicin-resistant m. tuberculosis. a single point mutation was detected in each of the two rifampicin-resistant strains (s531l and d516v). it was observed that for both strains, the corresponding xpert mtb/rif probes gave cycle threshold (ct) value = 0 and were also detected as resistant to rifampicin by genotype mtbdrplus line probe assay. one (0.5%) smear-negative, culture-positive tuberculosis strain was resistant on xpert mtb/rif but sensitive on genotype mtbdrplus (table 3). a delayed amplification on probe b was observed in this strain, with a ct value of 24.4. further testing on the isolate using dna sequencing revealed no rpob gene mutation. table 3: rifampicin susceptibility results (n=188) from both genotype mtbdrplus and xpert mtb/rif for outpatients at bwaila hospital, lilongwe, malawi (2011–2012). discussion in the present study, the xpert mtb/rif assay detected 58 more tuberculosis cases than smear microscopy. the paucibacillary nature of samples cultured from tuberculosis/hiv co-infected individuals revealed a more serious inability to detect tuberculosis using smear microscopy than was the case for xpert mtb/rif. among hiv-positive individuals registering for tuberculosis treatment, a relatively larger percentage of those who were smear-negative, were also xpert mtb/rif and culture negative, and thus had no laboratory confirmation of disease. overall analysis revealed expected results using the xpert mtb/rif assay, proving more sensitive and specific than fluorescent microscopy in detecting m. tuberculosis when using culture as the reference standard and there was no overlap of the confidence intervals. the sensitivity of xpert mtb/rif was lower in hiv-positive than in hiv-negative individuals. the xpert mtb/rif correctly distinguished m. tuberculosis from non-tuberculous mycobacteria. among the non-tuberculous mycobacteria, three were isolated from hiv-positive individuals, and one was from an hiv-negative individual. findings from our study are in agreement with studies by rahman et al.14 and barnard et al.15 who demonstrated an overall agreement of 92.4% and 100%, respectively, between xpert mtb/rif and mtbdrplus for the detection of rifampicin susceptibility. other studies have shown results in which rifampicin resistance was detected with a specificity of 100% and sensitivity of 99.1%.16,17 steingart et al. demonstrated a pooled sensitivity of 94% and a specificity of 98% for the detection of rifampicin resistance.18 the sensitivity of xpert mtb/rif (93.8%) observed in this study is in contrast to dorman et al. and geleta et al., who reported 62.6 % in south africa and 65.5% in ethiopia when compared with culture.19,20 the possible explanation for this discrepancy could be differences in study design, considering that the current study used processed pellets while the other studies performed their evaluations using unprocessed sputum. prior studies have shown greater accuracy of xpert mtb/rif as compared to smear microscopy6 and sensitivities of xpert mtb/rif ranging from 78% to 100% for smear-positive/culture-positive samples.21,22 since xpert mtb/rif identified 58/219 (26.5%) of smear-negative samples, it can therefore assist in rapid detection of tuberculosis cases among smear-negatives and has the potential to impact patient care, although a small percentage (17%, 12/70) of the smear-negative/culture-positive cases was missed by xpert mtb/rif. in malawi, smear-negative adult tuberculosis patients have poor treatment outcomes with high death rates, largely due to concurrent hiv infection.23 it is of particular interest to note that 64% of smear-negative samples which did not grow on culture and where m. tuberculosis was not detected by xpert mtb/rif, were from hiv-positive individuals. this may be interpreted as significant incorrect diagnosis of tuberculosis with a likelihood of treating people for tuberculosis who do not have the disease. it is therefore of paramount importance that steps should be taken to improve tuberculosis diagnosis to avoid exposing non-tuberculosis patients to the adverse effects of the tuberculosis medication. misdiagnosis of tuberculosis can be a contributing factor to the high death rate observed in hiv-positive individuals globally.1 due to the increased diagnostic yield and short turn-around time of xpert mtb/rif as compared to culture, xpert mtb/rif offers an opportunity to improve the diagnosis of tuberculosis, both in hiv-positive and hiv-negative individuals, if used as a first diagnostic for all presumptive tuberculosis cases in malawi, considering the high proportion of misdiagnoses observed in this study. our results are comparable to other studies which have shown that xpert mtb/rif increased tuberculosis case detection by almost 31%, despite its low sensitivity with smear-negative samples.20 another study demonstrated that 89% of rifampicin-resistant tuberculosis patients started second-line treatment when xpert mtb/rif was introduced and that the average time to start second-line treatment was reduced from 1 – 1.5 months by culture to one week with xpert mtb/rif.23 the current study does not highlight this for malawi since xpert mtb/rif testing was performed retrospectively and the initial study did not have a long-term follow-up on patients, as it was only concentrated on diagnostics. results in this study show that xpert mtb/rif detected primary rifampicin resistance in 3/188 (1.6%) specimens. dna sequencing of the rifampicin resistance determining region of the rpob gene confirmed two mutations (s531l and d516v) and further helped to resolve the discordant result, since no mutation was detected. mixed infection with multiple m. tuberculosis strains was excluded in this strain, given the wild-type rpob gene sequence and no observed underlying peaks on the dna sequence chromatogram. prior studies demonstrated that dna sequencing resolves discrepancies in favour of xpert mtb/rif,6 but this was not the case in our study; a false rifampicin resistance was most likely. in malawi, the xpert mtb/rif is widely used to identify mdr tuberculosis patients and to initiate mdr tuberculosis treatment until culture and dst results are obtained. malawi has a low mdr tuberculosis prevalence; as such the expected low ppv of xpert mtb/rif could contribute to false-positive results and would require a second xpert mtb/rif test as per current international recommendations.2 false resistance to rifampicin on xpert mtb/rif has been reported previously, although in some of the studies hetero-resistance could not be excluded, because neither cartridge amplicons nor unprocessed sputum were sequenced,17,25 which was also the case in our study. we performed dna sequencing on isolates from culture, whereas xpert mtb/rif was performed on sputum pellets. theron et al.22 detected mutations in 5/6 cases which were rifampicin resistant on xpert mtb/rif but were susceptible on phenotypic dst. marlowe et al.26 further investigated a sample which was susceptible on phenotypic dst and no mutation was detected in dna sequencing but was repeatedly resistant to rifampicin on xpert mtb/rif. investigations carried out in these studies show how difficult it is to distinguish true-positive rifampicin resistance from false positives in clinical practice. also, the samples run on xpert mtb/rif might have included both dead and live bacteria since the specimens were taken directly from pellets, whereas for dna sequencing, dna extraction was performed after observed growth on culture (live bacteria). it was demonstrated previously that xpert mtb/rif has a pooled sensitivity of 94.1% (95% ci 91.6% – 96.0%) and pooled specificity of 97.0% (95% ci 96.0% – 97.7%) in diagnosing rifampicin resistance.7 in the present study, the sensitivity, specificity, ppv and npv were not calculated due to the small number of rifampicin-resistant isolates identified. the low rifampicin resistance detected in this study is similar to results obtained by glynn et al., which identified 3/373 samples as resistant to rifampicin in karonga, in the northern region of malawi.27 low rifampicin resistance detection among incident cases observed in this study reflects well on the prevalence of rifampicin-resistant tuberculosis strains in communities surrounding bwaila. however, it is debatable whether xpert mtb/rif results alone can be used for making a decision to start on second-line treatment in outpatients detected with resistance to rifampicin in malawi, considering the number of isoniazid mono-resistances detected in previous studies.11 it is therefore crucial to test tuberculosis strains for resistance to isoniazid as well, and to confirm rifampicin resistance detected by xpert mtb/rif before switching to second-line treatment. the success of the tuberculosis-control programme could be measured by the level of anti-tuberculosis drug resistance in a community. consequently, the level of drug resistance gives future indications of suitable drug regimens.27,28 the malawi ntp has phased in plans within the context of national strategic plan for tuberculosis to expand and maintain xpert mtb/rif as a primary test for active case finding at high-volume antiretroviral therapy services and among high risk and vulnerable populations. this will consequently lead to improved tuberculosis diagnosis and initiation of appropriate treatment to patients, thereby reducing the spread of tuberculosis and/or transmission of resistant strains in the country. additionally, xpert mtb/rif offers to help in reducing the time lost in the diagnostic pathway and potential loss of patients at each step of the pathway.2 as part of the scale-up plan for the genexpert technology, the malawi ntp plans to assess the concordance between phenotypic and genotypic technologies in order to inform interpretation of single rifampicin-resistance results. systematic confirmation of mdr tuberculosis status of rifampicin-resistant patients diagnosed with xpert mtb/rif is lacking in the country.2 findings from the current study form a bank of data which can be beneficial to the ntp in its assessment of the xpert mtb/rif assay. limitations with our limited sample size from a single outpatient location, the observed results may not apply across the country due to differences in the prevalence of hiv in rural districts. the relatively small number of patients with drug-resistant tuberculosis limits the ability to evaluate comparative performance of xpert mtb/rif. we recommend that future studies extend to other sites with an overall sample size and/or focus on evaluating xpert mtb/rif on raw sputum, since is it known that testing frozen stored specimens may influence results.29 in the current algorithm for malawi, a residual specimen from the second sample of presumptive tuberculosis patients is tested on xpert mtb/rif if both samples are smear-negative. xpert mtb/rif is also performed on samples from all patients suspected of having mdr tuberculosis and retreatment cases, but not on new smear-positive cases.3 conclusion our results demonstrate that xpert mtb/rif has the potential to increase the number of patients initiated early on appropriate treatment, given that additional tuberculosis cases were identified among smear-negative/culture-confirmed cases, one of which was resistant to rifampicin. it is expected that the use of xpert mtb/rif will offer a greater opportunity in increasing tuberculosis case detection among high-risk and vulnerable groups in malawi where sputum smear microscopy is still widely used. we believe that results obtained by xpert mtb/rif may facilitate a proactive approach to tuberculosis by enhancing decisions towards treatment and ensuring that the tuberculosis programme is not treating people who do not have tuberculosis but are obviously sick. when this is achieved, it is anticipated that there will be a reduction in the burden of tuberculosis among people living with hiv. acknowledgements the authors thank the laboratory staff at unc project and the hiv genotyping laboratory at the university of the witwatersrand for assisting with the study. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. source of support this project was funded by the national commission for science and technology (malawi) through the health research capacity strengthening initiative (hrcsi). additional funds were made available by the unc project (malawi), unc fogarty aitrp (d43 tw001039), unc centre for aids research (p30 ai50410) and malawi hiv implementation research scientist program (d43 tw010060). authors’ contributions t.c. conceptualised the study. b.b. and r.h.g. oversaw patient enrolment in the primary study. t.c., n.n. and i.t. performed the laboratory experiments. m.h., l.s., w.s., r.k., j.m. and i.f.h. oversaw the operational aspects of the study. t.c., n.e.r. and c.s. conducted the data analysis, and t.c. drafted the manuscript. all authors revised the manuscript and approved the final draft. references world health organization. global tuberculosis report 2015. geneva, switzerland: who; c2015 [cited 2016 jan 27]. available form: http://apps.who.int/iris/bitstream/10665/191102/1/9789241565059_eng.pdf malawi national tuberculosis programme. tuberculosis control programme: national strategic plan 2015 to 2020. lilongwe, malawi: ministry of health; 2015 malawi national tuberculosis programme. national tuberculosis control programme manual. 7th ed. lilongwe, malawi: ministry of health; 2012. rachow a, zumla a, heinrich n, et al. rapid and accurate detection of mycobacterium tuberculosis in sputum samples by cepheid xpert mtb/rif assay—a clinical validation study. plos one. 2011;6(6):e20458. https://doi.org/10.1371/journal.pone.0020458 van rie a, page-shipp l, scott l, et al. xpert® mtb/rif for point-of-care diagnosis of tb in high-hiv burden, resource-limited countries: hype or hope? expert rev mol diagn. 2010;10(7):937–946. https://doi.org/10.1586/erm.10.67 boehme c, nabeta p, hillemann d, et al. rapid molecular detection of tuberculsis and rifampin resistance. n engl j med. 2010;363(11):1005–1015. https://doi.org/10.1056/nejmoa0907847 chang k, lu w, wang j, et al. rapid and effective diagnosis of tuberculosis and rifampicin resistance with xpert mtb/rif assay: a meta-analysis. j infect. 2012;64(6):580–588. https://doi.org/10.1016/j.jinf.2012.02.012 world health organization. automated real-time nucleic acid amplification technology for rapid and simultaneous detection of tuberculosis and rifampicin resistance: xpert mtb/rif system: policy statement. geneva, switzerland: who; c2011 [cited 2011 nov 07]. available from: http://apps.who.int/iris/bitstream/10665/44586/1/9789241501545_eng.pdf world health organization. rapid implementation of the xpert mtb/rif diagnostic test: technical and operational ‘how-to’ practical considerations. who/htm/tb/2011.2. geneva, switzerland: who; c 2011 [cited 2011 nov 07]. available from: http://apps.who.int/iris/bitstream/10665/44593/1/9789241501569_eng hanrahan cf, clouse k, bassett j, et al. the patient impact of point-of-care vs. laboratory placement of xpert® mtb/rif. int j tuberc lung dis. 2015 jul;19(7):811–816. https://doi.org/10.5588/ijtld.15.0013 barnett b, gokhale rh, krysiak r, et al. prevalence of drug resistant tb among outpatients at an hiv/tb clinic in lilongwe, malawi. trans r soc trop med hyg. 2015;109(12):763–768. https://doi.org/10.1093/trstmh/trv092 nicol mp, workman l, isaacs w, et al. accuracy of the xpert mtb/rif test for the diagnosis of pulmonary tuberculosis in children admitted to hospital in cape town, south africa: a descriptive study. lancet infect dis. 2011;11(11):819–824. https://doi.org/10.1016/s1473-3099(11)70167-0 hillemann d, rüsch-gerdes s, richter e. evaluation of the genotype mtbdrplus assay for rifampin and isoniazid susceptibility testing of mycobacterium tuberculosis strains and clinical specimens. j clin microbiol. 2007;45(8):2635–2640. https://doi.org/10.1128/jcm.00521-07 rahman a, sahrin m, afrin s, et al. comparison of xpert mtb/rif assay and genotype mtbdrplus dna probes for detection of mutations associated with rifampicin resistance in mycobacterium tuberculosis. plos one. 2016;11(4):e0152694. https://doi.org/10.1371/journal.pone.0152694 barnard m, gey van pittius nc, van helden pd, et al. the diagnostic performance of the genotype mtbdrplus version 2 line probe assay is equivalent to that of the xpert mtb/rif assay. j clin microbiol. 2012;50(11):3712–3716. https://doi.org/10.1128/jcm.01958-12 kwak n, choi sm, lee j, et al. diagnostic accuracy and turnaround time of the xpert mtb/rif assay in routine clinical practice. plos one. 2013;8(10):e77456. https://doi.org/10.1371/journal.pone.0077456 boehme cc, nicol mp, nabeta p, et al. feasibility, diagnostic accuracy, and effectiveness of decentralised use of the xpert mtb/rif test for diagnosis of tuberculosis and multidrug resistance: a multicentre implementation study. lancet. 2011;377(9776):1495–1505. https://doi.org/10.1016/s0140-6736(11)60438-8 steingart k, schiller i, horne dj, et al. xpert® mtb / rif assay for pulmonary tuberculosis and rifampicin resistance in adults. cochrane database syst rev. 2014;1:cd009593. https://doi.org/10.1002/14651858.cd009593.pub3 dorman se, chihota vn, lewis jj, et al. performance characteristics of the cepheid xpert mtb/rif test in a tuberculosis prevalence survey. plos one. 2012;7(8):e43307. https://doi.org/10.1371/journal.pone.0043307 geleta da, megerssa yc, gudeta an, et al. xpert mtb/rif assay for diagnosis of pulmonary tuberculosis in sputum specimens in remote health care facility. bmc microbiol. 2015;15:220. https://doi.org/10.1186/s12866-015-0566-6 scott le, mccarthy k, gous n, et al. comparison of xpert mtb/rif with other nucleic acid technologies for diagnosing pulmonary tuberculosis in a high hiv prevalence setting: a prospective study. plos med. 2011;8(7):1–11. https://doi.org/10.1371/journal.pmed.1001061 theron g, peter j, van zyl-smit r, et al. evaluation of the xpert mtb/rif assay for the diagnosis of pulmonary tuberculosis in a high hiv prevalence setting. am j respir crit care med. 2011;184(1):132–140. https://doi.org/10.1164/rccm.201101-0056oc hargreaves nj, kadzakumanja o, whitty cjm, et al. ‘smear-negative’ pulmonary tuberculosis in a dots programme: poor outcomes in an area of high hiv seroprevalence. int j tuberc lung dis. 2001;5(9):847–854. van kampen sc, tursynbayeva a, koptleuova a, et al. effect of introducing xpert mtb/rif to test and treat individuals at risk of multidrug-resistant tuberculosis in kazakhstan: a prospective cohort study. plos one. 2015;10(7):e0132514. https://doi.org/10.1371/journal.pone.0132514 van rie a, mellet k, john m, et al. false-positive rifampicin resistance on xpert® mtb/rif: case report and clinical implications. int j tuberc lung dis. 2013;16(2):206–208. https://doi.org/10.5588/ijtld.11.0395 marlowe em, novak-weekley sm, cumpio j, et al. evaluation of the cepheid xpert mtb/rif assay for direct detection of mycobacterium tuberculosis complex from respiratory specimens. j clin microbiol. 2011;49(4):1621–1623. https://doi.org/10.1128/jcm.02214-10 glynn jr, jenkins pa, fine pem, et al. patterns of initial and acquired antituberculosis drug resistance in karonga district, malawi. lancet. 1995;345(8954):907–910. mitchison da. drug resistance in mycobacteria. br med bull. 1984;40:84–90. carriquiry g, otero l, gonzález-lagos e, et al. a diagnostic accuracy study of xpert® mtb/rif in hiv-positive patients with high clinical suspicion of pulmonary tuberculosis in lima, peru. plos one. 2012;7(9):e44626. https://doi.org/10.1371/journal.pone.0044626 abstract background description of the intervention lessons learnt recommendations acknowledgements references about the author(s) iriagbonse i. osaigbovo department of medical microbiology, school of medicine, college of medical sciences, university of benin, benin city, nigeriadepartment of medical microbiology, university of benin teaching hospital, benin city, nigeria isaac o. igbarumah molecular virology laboratory, university of benin teaching hospital, benin city, nigeria ekene b. muoebonam institute of lassa fever research and control, irrua specialist teaching hospital, irrua, nigeria darlington e. obaseki department of anatomic pathology, school of medicine, college of medical sciences, university of benin, benin city, nigeriadepartment of anatomic pathology, university of benin teaching hospital, benin city, nigeria citation osaigbovo ii, igbarumah io, muoebonam eb, obaseki de. setting up a molecular diagnostic laboratory for sars-cov-2 testing: experience of a single centre in a resource-constrained setting. afr j lab med. 2021;10(1), a1326. https://doi.org/10.4102/ajlm.v10i1.1326 lessons from the field setting up a molecular diagnostic laboratory for sars-cov-2 testing: experience of a single centre in a resource-constrained setting iriagbonse i. osaigbovo, isaac o. igbarumah, ekene b. muoebonam, darlington e. obaseki received: 08 july 2020; accepted: 04 feb. 2021; published: 30 mar. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: molecular detection of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is at the forefront of the global response to the coronavirus disease 2019 (covid-19) pandemic. however, molecular diagnostic capabilities are poorly developed in many african countries. efforts by the nigeria centre for disease control and other public health agencies to scale up facilities for molecular testing across the continent are well documented, but there are few accounts from the laboratories at the frontline. intervention: as part of an institutional response to the covid-19 pandemic, the university of benin teaching hospital, benin city, nigeria, signed a memorandum of understanding with a world bank-supported institution to obtain a non-proprietary testing platform, renovated an existing molecular virology laboratory and validated the test process to make sars-cov-2 testing readily available for decision-making by frontline health workers. these efforts resulted in the university of benin teaching hospital’s inclusion in the nigeria centre for disease control covid-19 molecular laboratory network. the laboratory achieved a turnover of 12 123 tests within 7 months of operation. challenges faced and dealt with include incompatible equipment, limited skilled manpower, unstable (unreliable) electric power supply, disrupted procurement and supply chain, and significant overhead costs. lessons learnt: molecular diagnostic capability is essential in laboratory preparedness for pandemic response and can be achieved by establishing collaborative networks in low-resource settings. recommendations: molecular diagnostic capabilities attained during the covid-19 pandemic should be maintained by governmental support of the local biotechnology sector, collaboration with partners and stakeholders and the expansion of diagnostics to include other diseases of public health importance. keywords: covid-19; coronavirus disease 2019; laboratory; nigeria; molecular diagnosis; sars-cov-2; severe acute respiratory syndrome coronavirus 2; ubth; university of benin teaching hospital; resource-constrained. background coronavirus disease 2019 (covid-19) caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) was first described following an outbreak of atypical pneumonia in china in late 2019.1 it has since escalated into a global pandemic with over 36 million cases and more than a million deaths worldwide as of 10 october 2020.2 laboratory testing to detect, isolate and treat cases is key to the containment of the virus; establishing widespread diagnostic capacity has been a strategy of successful response in countries like south korea.3 to date, molecular methods, particularly real-time reverse transcriptase polymerase chain reaction (pcr), remain the mainstay of diagnostic testing.4 molecular diagnostic capability is in short supply in many parts of sub-saharan africa. in nigeria, available molecular diagnostic capabilities arose from vertical disease programme models for hiv and lassa fever in the 2000s.5,6,7 since the 2014 west african ebola outbreak, which emphasised the need for strong public health systems, the nigeria centre for disease control (ncdc) has gradually developed a network of laboratories capable of molecular diagnosis of infections of public health importance.8,9 this enabled nigeria to establish diagnostic capacity for sars-cov-2 testing in three laboratories within one month of the report of covid-19 cases from china and before the detection of the first case in sub-saharan africa on 27 february 2020.10,11 by leveraging already existing infrastructure for hiv, lassa fever and drug-resistant tuberculosis, the number of public and private laboratories able to test for sars-cov-2 has been gradually and consistently scaled up to 90 laboratories distributed across all 36 states and the federal capital territory of nigeria as of 08 october 2020 (figure 1a). the tremendous efforts by ncdc and other public health agencies across africa to scale up testing are well documented in the scientific literature.11 it is desirable that laboratory testing for sars-cov-2 is made available close to the ‘frontline’ of the pandemic, that is, within hospitals whenever feasible and safe, as this enables clinical teams to make critical decisions about inpatient management.12 these decisions include whether to isolate patients, thereby preventing the nosocomial spread of covid-19, and how to allocate scarce personal protective equipment (ppe). however, there is a paucity of accounts from laboratories at the frontline of testing and diagnosis. figure 1: covid-19 laboratory network in nigeria and edo state, 2020. (a) map of nigeria showing nigeria centre for disease control covid-19 molecular laboratory network (public laboratories only). (b) map of edo state showing local government areas and the locations of university of benin teaching hospital molecular virology laboratory and the institute of lassa fever research and control, irrua specialist teaching hospital. two public laboratories in the ncdc network for covid-19 testing are domiciled in edo state, nigeria (figure 1b). one of these, the institute of lassa fever research and control (ilfrc), irrua specialist teaching hospital, is a pioneer laboratory conscripted early in the fight against covid-19. the other, located at the university of benin teaching hospital (ubth) and included in the network on 10 may 2020, was set up because the management envisioned frontline diagnostic testing as a critical aspect of institutional response to the pandemic. this article describes the steps taken to set up testing for sars-cov-2 in the ubth. site description the ubth is an 850-bed tertiary hospital located in the south-south geopolitical zone of nigeria. it serves edo state which has a landmass of 17 802 square kilometres and neighbouring states delta, kogi, anambra and ondo, providing both primary and referral heathcare services. located in the state capital benin city, the institution is at the forefront of the state’s response to combating the pandemic.13 as of 31 december 2020, 2870 cases of laboratory-confirmed covid-19 have been reported in edo state.14 the molecular virology laboratory in ubth is a biosafety level 2 containment facility. it was set up in 2005 and moved to its current location in 2011 with the support of the institute of human virology of nigeria to offer early infant diagnosis and viral load detection services for the united states president’s emergency plan for aids relief aids care treatment in nigeria programme in the state.5 this partnership provided training in pcr diagnostic testing for laboratory scientists as well as laboratory equipment including a conventional pcr machine (later replaced with the roche cobas® taqman 96, a proprietary real-time pcr machine not compatible with currently accessible sars-cov-2 assays). other important equipment and items that were already in place in the laboratory include biosafety cabinets, fixed angle micro-centrifuge, bench centrifuge, heating blocks, freezers, an uninterrupted power supply and back-up generator. besides hiv testing, the laboratory also recently joined the ncdc network for yellow fever and measles testing following a favourable performance in a laboratory audit exercise conducted in december 2019. the staff of six consists of three laboratory scientists, one laboratory technician and two data clerks. description of the intervention ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. planning and preparation memorandum of understanding and polymerase chain reaction machine setup to expedite the commencement of testing while sourcing funds to purchase a new pcr machine, the hospital collaborated with the centre for excellence in reproductive health innovation, university of benin, nigeria. the centre for excellence in reproductive health innovation is one of the several world bank-funded centres for excellence in african higher education institutions initiated to promote homegrown and regional research networks. the collaboration with centre for excellence in reproductive health innovation was made official by signing a memorandum of understanding on 15 april 2020. this memorandum of understanding clearly states the purpose of the agreement, which is to jointly operate a covid-19 testing unit in ubth. the areas and scope of cooperation (including equipment, personnel and biosafety, provision of consumables and data collection for research purposes), implementation and the mode of handling intellectual property that may arise from the collaboration were also spelt out. following the signing of the memorandum of understanding, a new magnetic induction cycler real-time dx48 pcr instrument (biomolecular systems, upper coomera, queensland, australia) belonging to the centre for excellence in reproductive health innovation was transferred to the ubth molecular laboratory to conduct sars-cov-2 testing. the machine is an open (non-proprietary) platform compatible for use with many assays (figure 2). figure 2: magnetic induction cycler real-time dx48 pcr instrument obtained from centre for excellence in reproductive health innovation, edo state, nigeria, 2020. renovation of existing laboratory physical infrastructure the existing laboratory was ill-designed for molecular testing of highly infectious diseases like sars-cov-2. clean and dirty areas were not demarcated, that is, staff had to take the specimens through the master mix preparation area to get to the extraction room. the laboratory was thus physically reconfigured and renovated to demarcate the dirty (red) zone from clean areas to comply with a strictly unidirectional flow of movement. the cleanroom or section for mastermix preparation room, extraction room with a biosafety cabinet and centrifuge, and amplification and detection room for real-time detection of nucleic acid (figure 3) were each equipped with a supply of ppe, multi-channel pipette and other equipment and consumables to prevent contamination. for increased biosafety, an exit room for doffing ppe and showering was created to ensure a strictly unidirectional movement and prevent contamination of administrative and office areas. a well-demarcated sample reception area was also created. figure 3: amplification and detection room for real-time detection of nucleic acid, edo state, nigeria, 2020. online mentorship and training to gain proficiency in the use of the machine, the chief scientist, other laboratory scientists and staff in the laboratory underwent hands-on training which, due to the lock-down situation in many states and ban on interstate travel in nigeria, was done via online video conferencing. an experienced molecular biologist, conversant with the magnetic induction cycler testing platform and sars-cov-2 molecular testing provided the training. sessions included the development of standard operating protocols and job aids specific for sars-cov-2 testing, biosafety protocols applicable to the virus and appropriate waste disposal. manpower reinforcement staff strength was reinforced by redeploying two scientists from the general medical microbiology laboratory and two data clerks. this scaled up the number of staff to 10: five laboratory scientists, one laboratory technician and four data clerks. the new staff received orientation and training for their specific tasks. specifically, the laboratory technician is responsible for specimen reception and registration, the laboratory scientists carry out the various stages of reverse transcription-pcr and run quality control checks, while the data clerks are responsible for uploading data from the case investigation forms and test results onto online surveillance platforms. stockpiling of personal protective equipment personal protective equipment items and stock were increased to ensure adequate protection of laboratory staff. personal protective equipment items included gloves, gowns, goggles, footwear, face shields, face masks and respirators. evaluation and validation by nigeria centre for disease control site visit by officials of institute of lassa fever research and control as a regional reference laboratory for covid-19 testing, ilfrc officials represented ncdc for the laboratory assessment visit using a checklist designed by the ncdc. areas of assessment included biosafety checks, availability of ppe, a waste management plan, staff strength, equipment and stock management. on-site mentorship, training and evaluation an experienced molecular scientist from the ilfrc was deployed to provide technical support to the laboratory staff in workflow optimisation, infection prevention and control and handling of laboratory data. the ubth laboratory scientists were also evaluated for competence in conducting the test using the national algorithm, laboratory safety, environmental cleaning and decontamination protocols. validation equipment validation was done using commercial standards and controls (liferiver, shanghai zj biotech, shanghai, china), while assay runs were validated by inter-laboratory comparison with ilfrc. duplicate samples were collected from all suspected cases in the facility: one sample was sent to ilfrc while the other was processed and analysed by the on-site laboratory. inter-laboratory concordance was measured and independent testing was commenced when 100% concordance was achieved in 40 samples. operations following a successful validation process and on the recommendation of the site evaluators from ilfrc, the ubth molecular laboratory was included in the ncdc sars-cov-2 testing network on 10 may 2020. the testing protocol begins with inactivation of each sample, typically a nasopharyngeal swab and oropharyngeal swab in the same tube of viral transport medium, in a biosafety cabinet (figure 4) using an external lysis buffer reagent, which accompanies the commercial rna extraction kit. the sample is then moved to the rna extraction room where rna is extracted manually using an rna isolation kit (shanghai zj biotech, shanghai, china). five microlitres (μl) of the extracted rna is then added to 20 μl of prepared master mix (composed of 19 μl supermix and 1 μl enzyme mix) in the reaction tube. polymerase chain reaction amplification was achieved using a novel coronavirus real-time multiplex reverse transcription-pcr kit (liferiver, shanghai zj biotech, shanghai, china) on a magnetic induction cycler real-time dx48 pcr instrument (biomolecular systems, upper coomera, queensland, australia). results were interpreted based on the detection of the envelope (e), nucleocapsid (n) and open reading frame 1ab (orf1ab) genes. for a test to be considered positive, at least two of these genes, which must include the orf1ab, must have been detected at the recommended cycle threshold (ct) of less than 41. figure 4: sample inactivation room with a biosafety cabinet, edo state, nigeria, 2020. due to the open nature of the test platform, we were able to use other commercial rna extraction and detection kits, for instance daan gene (da an gene co. limited of sun yat-sen university, guangzhou, china) was used when the liferiver kit (liferiver, shanghai zj biotech, shanghai, china) was not available. the real-time outbreak and epidemic surveillance software surveillance outbreak response management and analysis system, employed by ncdc, was installed in the laboratory information management system and data clerks were trained by ncdc officials on test data imputation. quality assurance was emphasised along the entire testing pathway. pre-analytical conditions were addressed by a clinical pathologist closely liaising with clinical staff on proper specimen collection, handling and transport. quality control was ensured in each run by the use of appropriate positive, negative and internal controls. equally, data were collected and analysed to monitor key performance indicators such as turnaround time, positivity and specimen rejection rates. results in seven months of operation, the laboratory has performed 12 123 assays on ubth patient samples, referral samples from other health facilities and community samples from the edo state government-driven community screening and testing efforts. before the inclusion of the laboratory into the covid-19 testing network, all samples from ubth had to be sent in a once-daily trip to ilfrc which is over 100 km away and the result turnaround time was 3–4 days. the availability of on-site sars-cov-2 testing has shortened the turnaround time to 1–2 days as sample transportation-related delays in test turnaround time have been eliminated. the shortened sample collection-to-test result turnaround time expedites crucial patient care decision-making such as treatment initiation, triaging of patients in emergency departments, allocation of scarce ppe and other actions that prevent nosocomial spread. the renovations carried out in the laboratory improved the workflow and minimised the risks of contamination by ensuring that laboratory staff only move from clean areas to the dirty area (amplification area containing nucleic acids). challenges establishing sars-cov-2 testing amid the covid-19 pandemic has been problematic even in the most developed countries.15,16 general challenges include a slowdown in global manufacturing of sampling materials like swabs and viral transport medium, difficulties in shipping and procurement of commercial testing kits, contamination of molecular diagnostic reagents, lack of performance data for covid-19 testing kits that have been approved for emergency use, lack of positive control materials for covid-19 testing and shortage of ppe, among others. these obstacles are, expectedly, more pronounced in resource-poor settings, which also have their unique challenges.17 currently, sars-cov-2 testing in nigerian public health facilities is offered at no cost to individual patients and clients. although reagents and consumables are provided by ncdc, the supply is often irregular and testing institutions bear the brunt of significant overhead running costs. to mitigate the impact of delays in supply of testing reagents, ample lead time is given in making requests for reagents to prevent stock-outs. in addition, the dedicated 30 kilovolt-amperes (kva) back-up generator had to be replaced with a 60 kva to cope with the unstable electric power supply to prevent disruptions in testing and ensure proper storage of samples (table 1). table 1: challenges of setting up of sars-cov-2 testing in university of benin teaching hospital and mitigating actions taken, edo state, nigeria, 2020. lessons learnt the experience of setting up the molecular laboratory during the covid-19 pandemic emphasised the need for laboratory preparedness as a major aspect of institutional response to infectious disease outbreaks and other emergencies. molecular diagnostic capability is key to this laboratory preparedness and can be achieved by establishing collaborative networks in low-resource settings. firstly, ubth was able to obtain a platform for testing by signing a collaborative memorandum of understanding with a funded research centre in the university. secondly, despite travel restrictions, the laboratory staff were able to get the laboratory running via remote mentoring using web conferencing technology. the mentorship and guidance from the reference laboratory in the state was also invaluable particularly on the laboratory design, on-site evaluation and testing capacity validation. another lesson learnt is the value of non-proprietary molecular testing platforms that allow for flexibility and capacity to scale up in emergencies. this became especially evident when there were difficulties in accessing a particular commercial assay as the flexibility of the platform allowed us to easily switch to other available assay kits. recommendations the covid-19 pandemic has demonstrated the essential role of diagnostics in the control of infectious diseases and sparked renewed interest in molecular technologies in low-resource settings, including nigeria. we recommend that the capabilities attained are sustained by governmental support of the local biotechnology sector, continued surveillance for sars-cov-2 beyond the pandemic period, fostering collaboration and broadening the scope to include other diseases of public health importance. we expand on these recommendations below. it is hoped that the rekindled interest in molecular diagnostics will bring about a burgeoning biotechnology sector including a network of biomedical engineering services and local sources for laboratory reagents and consumables. these are necessary for sustaining efforts at the institutional healthcare level. indeed, this is already occurring in nigeria; for instance, the viral transport medium required for the shipment of samples that was being imported at the start of the pandemic is now locally produced and supplied by the national veterinary research institute in vom, jos. laboratories with the capacity to develop and validate quality in-house assays should be supported to reduce the over-reliance on external commercial sources. if sars-cov-2 becomes endemic in the population, surveillance for covid-19 will need to be maintained. low complexity, rapid point of care molecular testing platforms already in use in many hospitals can be leveraged to carry out smaller-scale testing and surveillance in the post-pandemic period when prevalence rates are likely to be lower. for instance, the xpress sars-cov-2 test cartridge can be used in the genexpert® (cepheid, sunnyvale, california, united states) machine that is already in use for diagnosis of tuberculosis in many hospitals. costs can be further lowered and throughput increased by deploying a pooled sample testing strategy after validating.18 there are no guarantees that government support for supplies of reagents, consumables and other logistics will last beyond the pandemic period. sustaining operations in a molecular laboratory is capital intensive and it is pertinent to consider how this will be achieved in the future. as observed in several other contexts, capital investments in laboratory equipment are difficult to recoup especially where test volumes are low and overhead maintenance costs are high.5,19 collaboration with an institution that had external funding was pivotal to the sars-cov-2 testing initiative in ubth and collaboration will be what sustains capabilities attained going forward. coordinated and goal-directed partnerships with national and regional stakeholders, research institutes, global donor agencies, international governing bodies and biomedical industries are required.19 also, the trained workforce will need to be retained utilising incentives and new hands will need to be recruited. while maintaining surveillance for sars-cov-2, laboratories, including ubth, will need to broaden their focus to include other diseases of public health importance that can attract collaboration from both ncdc and external donor agencies invested in global health. lassa fever, a disease endemic to many parts of nigeria, is an attractive candidate. the world health organization also prescribes molecular diagnosis for some other relevant infectious diseases like sexually transmitted diseases and dengue fever in the essential diagnostics list currently in its second edition.20 additionally, the covid-19 outbreak has impressed the need to better define the epidemiology of severe acute respiratory illnesses of viral aetiology. these viruses are best detected by molecular means and it will be worthwhile attaining the tools necessary to differentiate them from the novel coronavirus.21 in conclusion, the successful setup of sars-cov-2 testing in ubth was predicated on collaborative efforts, established quality management systems culture and innovativeness. whatever the future focus of the molecular laboratory, it is pertinent that molecular diagnostic infrastructure is kept patent as part of both the laboratory and institutional preparedness plans. this will ensure swift mobilisation to deal with infectious disease crises that will almost inevitably arise in the future. acknowledgements the authors acknowledge the centre for excellence in reproductive health innovation and the centre leader, professor friday okonofua, for providing the magnetic induction cycler real-time dx48 polymerase chain reaction instrument used in this project, the nigeria centre for disease control and its director-general, dr chikwe ihekweazu, for expediting activation of the laboratory, the chief medical director of irrua specialist teaching hospital, professor sylvanus okogbenin, and the director of the institute of lassa fever research and control, irrua specialist teaching hospital, dr ephraim ogbaini-emovon, for assessing the laboratory and contributing to its design. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions i.i.o., i.o.i., e.b.m. and d.e.o. contributed equally to the design and implementation of the research, to the analysis of the results and to the writing of the manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability data sharing does not apply to this article as no new data were created or analysed. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or 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[cited 2021 jan 12]. available from: https://covid19.ncdc.gov.ng/progression/ babiker a, myers cw, hill ce, guarner j. sars-cov-2 testing: trials and tribulations. am j clin pathol. 2020;153(6):706–708. https://doi.org/10.1093/ajcp/aqaa052 mögling r, meijer a, berginc n, et al. delayed laboratory response to covid-19 caused by molecular diagnostic contamination. emerg infect dis. 2020;26(8):1944–1946. https://doi.org/10.3201/eid2608.201843 kobia f, gitaka j. covid-19: are africa’s diagnostic challenges blunting response effectiveness. aas open res. 2020;3:4. https://doi.org/10.12688/aasopenres.13061.1 becker mg, taylor t, kiazyk s, cabiles dr, meyers af, sandstorm pa. recommendations for sample pooling on the cepheid genexpert system using the cepheid xpert xpress sars-cov-2 assay. biorxiv preprint. in press 2020. https://doi.org/10.1101/2020.05.14.097287 zhang hl, omondi mw, musyoka am, et al. challenges of maintaining good clinical laboratory practices in low-resource settings: a health program evaluation framework case study from east africa. am j clin pathol. 2016;146(2):199–206. https://doi.org/10.1093/ajcp/aqw083 world health organization 2018. world health organization model list of essential in vitro diagnostics [homepage on the internet]. 2nd ed. geneva: world health organization [cited 2020 jun 14]. available from: https://www.who.int/medical_devices/publications/second_who_model_list_of_essential_in_vitro_diagnostics/en/ somerville lk, ratnamohan vm, dwyer de, kok j. molecular diagnosis of respiratory viruses. pathology. 2015;47(3):243–249. https://doi.org/10.1097/pat.0000000000000240 abstract introduction methods results discussion acknowledgements references about the author(s) rodgers n. demba school of health sciences, kisii university, kisii, kenya institute of tropical and infectious diseases, university of nairobi, nairobi, kenya sylviah m. aradi department of internal medicine, university of nairobi, nairobi, kenya matilu mwau center for infectious and parasitic diseases control research, kenya medical research institute, busia, kenya walter o. mwanda institute of tropical and infectious diseases, university of nairobi, nairobi, kenya citation demba rn, aradi sm, mwau m, mwanda wo. kaposi’s sarcoma-associated herpesvirus protein orf75 among hiv-1 patients in kenya. afr j lab med. 2020;9(1), a939. https://doi.org/10.4102/ajlm.v9i1.939 original research kaposi’s sarcoma-associated herpesvirus protein orf75 among hiv-1 patients in kenya rodgers n. demba, sylviah m. aradi, matilu mwau, walter o. mwanda received: 01 dec. 2018; accepted: 15 may 2020; published: 25 aug. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: histology is used to identify kaposi’s sarcoma (ks) in countries with low resources to fund healthcare costs. approximately 95% of ks cases can be detected using a polymerase chain reaction. objective: to determine the presence of the open reading frame 75 (orf75) gene associated with kaposi’s sarcoma herpes virus among hiv-1/aids patients and to describe morphological presentations of ks. methods: this was a retrospective, descriptive study of archived tissue blocks collected from 2013 to 2016. haematoxylin and eosin staining was used to identify ks. deoxyribonucleic acid from archived tissue blocks was extracted and a nested polymerase chain reaction was used to detect the orf75 gene. results: all 81 cases in this study had been diagnosed as hiv-1 positive, of which 68 had hallmark features of ks in the histology report and 13 had features suggestive of ks (‘ks-like’). microscopic identification of ks by haematoxylin and eosin staining was considered a significant indicator of ks herpes virus orf75 gene positivity (p = 0.002). the orf75 gene was detected in 60.5% (49/81) of tissue blocks; 27.2% were men (22/81) and 33.3% were women (27/81). the orf75 gene was observed to be present in up to 15.4% (2/13) of the cases reported to have ks-like features. conclusion: following the initial diagnosis of ks by histology, the orf75 gene was fur-ther detected from both cases that had hallmark features of ks as well as among cases with ks-like fea-tures. keywords: human herpes virus 8; kaposi’s sarcoma; histology; nested pcr; orf75 gene. introduction kaposi sarcoma (ks) is a tumor formed from blood vessels; it later shows lesions on the skin or organs of hiv-positive people.1 all forms of ks are caused by kaposi’s sarcoma herpesvirus (kshv), also known as human herpesvirus 8 (hhv-8).2,3 the genome of hhv-8 contains a minimum of 100 open reading frames (orf), of which 4 to 75 are known to be unique to herpesvirus.4 the kshv genome encodes more than 84 proteins that play a role in viral replication and host-virus interaction.5 the replication cycle of kshv entails latent and lytic phases. during the lytic cycle, the orf75 genes are expressed resulting in the manifestation of ks.6,7 the orf75 gene product has been proven to aid in lytic replication and enhancement of virus pathogenesis in host cells.8 kaposi’s sarcoma is listed among the defining malignancies of hiv/aids.9,10,11 the dis-tinct feature of hiv-associated ks is that it might affect the lymph nodes, gastrointestinal tract, lungs or liv-er.12,13 despite the fact that saliva is the main route by which kshv is transmitted,14 hhv-8 has been isolated from other body fluids.14,15,16 the main route of hhv-8 transmission to the opposite sex is through sexual relations.17 the pathogenesis of ks presents as an abnormal neoangiogenesis, proliferation of cancer cells and inflammation of endothelial cells.18 a classic ks lesion manifests various features ranging from maculopapular to nodular or plaque-like and, in most cases, is pain-less.12,19,20,21 in sub-saharan africa, kshv is endemic and approximately 84% of worldwide cases of ks occur in this region.22 since ks is common among hiv/aids pa-tients,13 early detection of kshv is essential in disease monitoring.22 the sensitivity of diagnostic tests for detection of kshv depends on the sample selected for analysis.23 for example, biopsies obtained from patients with hiv/aids-ks were found to yield better results compared to using peripheral blood mononuclear cells from the same patient.23 identification of ks in tissue biopsies by use of histological staining techniques should not be underestimated.24 in tissue biopsies, microscopic examination involves identification of proliferated spindle cells and oedema.25 clinical diagnoses of ks have been shown to have limited predictive value.26 the use of molecular techniques such as polymerase chain reaction (pcr) permits the detection of the hhv-8 gene even for patients who present with early vascular lesions that histological techniques might miss.27 the use of pcr in the diagnosis of ks can detect approximately 95% of cases.28 the hhv-8 dna has been successfully amplified using nested pcr previously.29,30 this study was aimed at determining the presence of the orf75 gene linked to kshv among hiv-1/aids patients. in addition, the objective of this current study was to describe the morphological presentations of ks among the studied cases. methods the present study only included patients aged 18 years and older. data on clinical information that was useful for this study were extracted from the registry records with the help of the data clerk. the following data were obtained from the registry records: sex, age, hiv-1 status, if patient was on antiretroviral or highly active antiretroviral therapy treatment, anatomic location of ks lesions, number of ks lesions, distribution of ks lesions, cluster of differentiation 4 cell count and histology diagnosis. ethical considerations study approval number p682/11/2014 was assigned by kenyatta national hospital/university of nairobi ethics and research committee. study design a cross-sectional, descriptive, hospital-based study was used. formalin-fixed, paraffin-embedded tissue blocks were retrieved from archives following histological reports of the patients who were diagnosed with ks or ks-like disease between 2013 and 2016. a consecutive sampling technique was used to select the archived tissue blocks from thematic unit of anatomic pathology, department of human pathology, college of health sciences, university of nairobi, and department of laboratory medicine, cytology section, kenyatta national hospital. for this study, a total of 81 tissue blocks were selected and analysed. a rotary microtome was used to section the formalin-fixed, paraffin-embedded blocks. a different blade was used for every formalin-fixed, paraffin-embedded block so as to avoid carry-over of genetic material. once a block was cut, the microtomes surface was decontaminated using dnazaptm pcr dna degradation solution (catalog number: am9890; thermo fisher scientific, waltham, massachusetts, united states). each tissue section was cut to 10 µm thick. the tissue sections were processed for haematoxylin and eosin staining and a qualified pathologist reported on the results. deoxyribonucleic acid extraction and polymerase chain reaction isolation of dna from tissue sections was done using a generead dna ffpe kit (qiagen, hilden, germany). the extraction kit removes paraffin and reverses formalin cross-links from tissue before dna is bound to the qiaampminelute column (qiagen, hilden, germany). the eluted dna is then ready to be used for nested pcr to detect the orf75 gene in hhv-8. a taq pcr core kit (catalog number: 201223; qiagen, hilden, germany) was used to detect the orf75 gene. the set of primers used were; orf75 product size 895 bp forward ks 1000 5′cggttcggtggcatacaggc3′; reverse ks 1034 5′ctgactacagagggtgtccccg3′.31 orf75 product size 804 bp forward ks 2000 5′ggaaacagggtgctgtg3′; reverse ks 2034 5′catggcctacgacgtcac3′.32 the cycling conditions of the pcr for the targeted ks regions were similar and consisted of 30 cycles of: initial denaturation at 94 °c for 3 minutes, denaturation at 94 °c for 1 min, annealing at 63 °c for 1 min, extension at 72 °c for 1 min and final extension at 72 °c for 10 min. amplified pcr products were analysed by electrophoresis on a 1% agarose gel containing ethidium bromide (1 µl/ml of agarose solution) and were visualised under ultraviolet light alongside a 1 kilobase (kb) deoxyribonucleic acid (dna) ladder. for a positive control, a known case of ks was used. the ribonuclease-free water was used as a negative control. statistical analysis the data were analysed using statistical package for social sciences version 21 (spss inc binghamton, new york, united states); the relationship between the orf75 gene and clinical characteristics were tested by using chi-square and t-tests. a p-value of less than 0.05 was considered to be statistically significant. odds ratios in a cross-sectional study are known as prevalence odds ratios and were used as a measure of association.33 results of the 81 tissue samples included in the study, 43.2% (35/81) were from women and 56.8% (46/81) were from men (table 1). all of the 81 cases studied had been diagnosed with hiv-1 implying that they were living with the virus. in addition, it was observed that none of the cases had a cluster of differentiation 4 cell count above 350 cells/mm3. among the 81 cases, the orf75 gene was detected in 49 cases (60.5%); 27.2% (22/81) were women and 33.3% (27/81) were men. among cases positive for the orf75 gene, 4.1% (2/49) were never on any form of antiretroviral therapy and 95.9% (47/49) were on antiretroviral therapy. no statistically significant association was found between the presence of the orf75 gene and sex, antiretroviral treatment status, number of ks lesions or distribution of the ks lesions (all p-values > 0.05). table 1: crude prevalence odds ratio and 95% confidence intervals for patient characteristics and presence of the kshv orf75 gene, nairobi, kenya, 2013–2016. age the mean age of patients with tissue blocks positive for the orf75 gene was 41 years (standard deviation = 9.2; maximum age, 66 years; minimum age, 19 years). detection of the orf75 gene was most common in the 30–39 years age group (n = 21; 42.9%). age had a statistically significant association with orf75 gene positivity (prevalence odds ratio: 1.05; 95% confidence interval: 1.00–1.11, p = 0.047). kaposi sarcoma morphology and distribution of lesions in the histology report, 68 cases had hallmark features of ks, whereas 13 cases had features suggestive of ks (ks-like). the types of ks morphology identified included patchy, nodular, plaque and ks-like (figure 1). the morphological distribution of ks was as follows: 61.7% (50/81) was nodular, 16% (13/81) was patchy, and 22% (18/81) were plaques. among the cases that were positive for the orf75 gene, 75.51% (37/49) was nodular, 4.08% (2/49) patchy, and 20.41% (10/49) were plaques. figure 1: morphological descriptions of kaposi’s sarcoma and ka-posi’s sarcoma-like cases, obtained from university of nairobi and kenyatta national hospital, nairobi, kenya, 2013–2016. microscopic identification by haematoxylin and eosin staining, x20. (a) patchy, (b) nodular, (c) plaque and (d) ks-like. the total number of ks cases diagnosed by histology was 68 (84%) and 13 cases (16%) had ks-like features (table 1). among the 49 cases with the orf75 gene, 47 (95.9%) showed hallmark features of ks and 2 (4.1%) had ks-like features with microscopic examination. there was an association between microscopic identification of ks by histology and the presence of the orf75 gene (prevalence odds ratio = 12.3; 95% confidence interval = 12.51 – 60.49; p = 0.002) (table 1). the amplified orf75 genes of hhv-8 were identified by 1% agarose gel electrophoresis (figure 2). figure 2: polymerase chain reaction agarose gel electrophoresis results of ka-posi’s sarcoma herpes virus orf75 gene. cases obtained from university of nairobi and kenyatta national hospital, nairobi, kenya, 2013–2016. discussion retrieved clinical data revealed that all of the tissue blocks retrieved in the present study were collected from patients who had been diagnosed with hiv-1. these patients might have developed ks lesions due to immunosup-pression or because they were immunocomprised due to increased viral load that impaired their immune system. other studies have also associated ks as an hiv/aids-defining illness.9,13,24,34,35,36 the findings of this study revealed that men were more prone to development of ks: 56.8% (46/81) compared with women 43.2% (35/81). this observation is concordant with others who also noted more frequent development of ks among men.37,38,39,40 there is a lack of consensus as to why all forms of ks are more common among men than women.41,42 we hypothesise that gender-related factors such as hormones might influence the development of ks lesions. the results of this study showing ks pre-ponderance among men was consistent with the country’s published data on the distribution of malignancy cases as captured in the national cancer control strategy, 2017.43 kaposi’s sarcoma immune reconstitution occurs when a portion of aids-ks cases responds to the introduction of combined antiretroviral therapy with disease advancement.44,45,46 in this study, ks lesions manifested among patients despite the fact that 77 (95.1%) were on antiretroviral treat-ment. contrary to other findings that antiretroviral therapy alone can result in the resolution of ks,47,48 in our study, being on antiretroviral treatment did not have a statistically significant association with the presence of ks (p = 0.66). this finding is in agreement with another study that stated that there has been continued diagnosis of ks in hiv-positive patients, despite the availability of highly active antiretroviral therapy.49 other studies have stated that patients infected with aids-associated ks respond to combined antiretroviral therapy by 50% depending on geographical location and severity of the presentation, thereby resulting in immune reconstitution and hiv suppression.50,51,52 in the current study, these manifestations of ks could be attributed to the weakening of the immune system by hiv-1. this study found patchy, plaque and nodular morphological presentations of ks. the morphological appearance of ks shows progression from plaques to nodular form and fungiform.1 kaposi sarcoma lesions are known to progress from asymptomatic to macule, papule, plaque and nodule forms.53 the findings of this study revealed that the ks lesions were disseminated in different body regions, including the lower limbs, upper limbs, genitalia, eyelids, palate, oral cavity and trunk (chest and back). in another study, fatality was witnessed in hiv-positive patients who had ks lesions manifested in the gastrointestinal tract, lungs and lymph nodes.54 the decision in this study to use tissue biopsy for detection of the orf75 gene of hhv-8 is in agreement with another study that supported the use of tumor biopsies as suitable for viral dna identification due to high viral load as opposed to the use of blood.30 further to that, nested pcr has been used successfully to assess the prevalence of hhv-8 among hiv-positive patients in brazil.27 a tissue biopsy excised from a ks lesion has been shown to have high viral load; hence, biopsies are the ideal sample for the detection of kshv dna.55 the detection of the orf75 gene implies that this gene was present in 49 (60.5%) of the studied cases. strength and limitations our study used the haematoxylin and eosin staining technique and the nested pcr method for detection of the orf75 genes of the kshv. however, hhv-8 immunohistochemical biopsy has been demonstrated to be the ‘gold standard’ for ks diagnosis.56 cases in the present study had a dark skin pigmentation. in another study of dark-skinned patients, ks had been confirmed to mimic a number of non-ks-like dermatological conditions.56 in another study, it was observed that it is difficult to identify ks in dark-skinned individuals, who presented with violaceous skin lesions.56 the use of the pcr technique in the detection of kshv has been shown to give the utmost specificity compared to the use of tests that determine exposure to infection.28 in addition, the pcr technique can detect approximately 95% of all ks cases.28 however, the cost associated with the use of pcr is quite high, which would limit the clinical application of hhv-8 dna detection in resource-limited facili-ties.28 implications and recommendations the present study considered microscopic detection of ks by haematoxylin and eosin as a significant indicator of kshv orf75 gene positivity. it therefore recommends the use of both clinical diagnosis and routine microscopy in the diagnosis of ks in resource-limited facilities. however, among individuals with dark skin pigmentation, there is the need to employ the use of a robust diagnostic technique to ascertain the true causative agent. conclusion the presence of the orf75 gene of kshv among immunosuppressed patients due to hiv-1 was successfully detected. following the initial diagnosis of ks by histology, the orf75 gene was further detected from both cases that had the hallmark features ks and those that had ks-like features. microscopic detection of ks by haematoxylin and eosin should be considered a significant indicator of kshv orf75 gene positivity. acknowledgements the authors of this manuscript would like to extend their gratitude to kenyatta national hospital and university of nairobi for allowing them to use their archived tissue blocks. competing interests the authors declare no conflict of interest. authors’ contributions r.n.d., s.m.a., m.m. and w.o.m. critically revised the manuscript for important intellectual content. r.n.d., s.m.a. and w.o.m. drafted the manuscript. r.n.d. and w.o.m. conceptualised and designed the study and ana-lysed and interpreted the data. r.n.d. and s.m.a. acquired the data. sources of support this study was funded by the principal investigator (r.n.d.) as a fulfilment for the award of a postgraduate de-gree. data availability statement 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china. viruses. 2014;6(12):4800–4810. https://doi.org/10.3390/v6124800 van bogaert lj. clinicopathological proficiency in the diagnosis of kaposi’s sarcoma. isrn aids. 2012(5); article #565463, 7 pages doi:10.5402/2012/565463 abstract introduction methods results discussion acknowledgements references about the author(s) ernest p. makokha laboratory services branch, division of global hiv & tb, united states centers for disease control and prevention, nairobi, kenya raphael o. ondondo laboratory services branch, division of global hiv & tb, united states centers for disease control and prevention, nairobi, kenya daniel k. kimani laboratory services branch, division of global hiv & tb, united states centers for disease control and prevention, nairobi, kenya thomas gachuki national hiv reference laboratory, division of public health laboratories, ministry of health, nairobi, kenya frank basiye laboratory services branch, division of global hiv & tb, united states centers for disease control and prevention, nairobi, kenya mercy njeru laboratory services branch, division of global hiv & tb, united states centers for disease control and prevention, nairobi, kenya muthoni junghae laboratory services branch, division of global hiv & tb, united states centers for disease control and prevention, nairobi, kenya marie downer laboratory services branch, division of global hiv & tb, united states centers for disease control and prevention, nairobi, kenya mamo umuro national hiv reference laboratory, division of public health laboratories, ministry of health, nairobi, kenya margaret mburu laboratory services branch, division of global hiv & tb, united states centers for disease control and prevention, nairobi, kenya jane mwangi laboratory services branch, division of global hiv & tb, united states centers for disease control and prevention, nairobi, kenya citation makokha ep, ondondo ro, kimani dk, et al. enhancing accreditation outcomes for medical laboratories on the strengthening laboratory management toward accreditation programme in kenya via a rapid results initiative. afr j lab med. 2022;11(1), a1614. https://doi.org/10.4102/ajlm.v11i1.1614 original research enhancing accreditation outcomes for medical laboratories on the strengthening laboratory management toward accreditation programme in kenya via a rapid results initiative ernest p. makokha, raphael o. ondondo, daniel k. kimani, thomas gachuki, frank basiye, mercy njeru, muthoni junghae, marie downer, mamo umuro, margaret mburu, jane mwangi received: 06 may 2021; accepted: 04 mar. 2022; published: 31 may 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: since 2010, kenya has used slipta to prepare and improve quality management systems in medical laboratories to achieve iso 15189 accreditation. however, less than 10% of enrolled laboratories had done so in the initial seven years of slmta implementation. objective: we described kenya’s experience in accelerating medical laboratories on slmta to attain iso 15189 accreditation. methods: from march 2017 to july 2017, an aggressive top-down approach through high-level management stakeholder engagement for buy-in, needs-based expedited slipta mentorship and on-site support as a rapid results initiative (rri) was implemented in 39 laboratories whose quality improvement process had stagnated for 2–7 years. in july 2017, slipta baseline and exit audit average scores on quality essential elements were compared to assess performance. results: after rri, laboratories achieving greater than a 2-star slmta rating increased significantly from 15 (38%) at baseline to 33 (85%) (p < 0.001). overall, 34/39 (87%) laboratories received iso 15189 accreditation within two years of rri, leading to a 330% increase in the number of accredited laboratories in kenya. the most improved of the 12 quality system essentials were equipment management (mean increase 95% ci: 5.31 ± 1.89) and facilities and biosafety (mean increase [95% ci: 4.05 ± 1.78]) (both: p < 0.0001). information management and corrective action management remained the most challenging to improve, despite rri interventions. conclusion: high-level advocacy and targeted mentorship through rri dramatically improved laboratory accreditation in kenya. similar approaches of strengthening slipta implementation could improve slmta outcomes in other countries with similar challenges. keywords: rapid results initiative; accreditation; quality systems essentials; slipta; slmta; e-slipta checklist. introduction in the recent past, lowand middle-income countries have witnessed improved access to quality-assured laboratory diagnosis, leading to better patient outcomes.1 one of the key drivers for this improvement was the implementation of the twin strengthening laboratory management toward accreditation (slmta) task-based training programme and the world health organization’s (who) stepwise laboratory quality improvement process towards accreditation (slipta), a stepwise accreditation preparedness programme.2,3 used in combination, the slmta programme, the slipta checklist and related processes enabled laboratories in lowand middle-income countries to implement quality systems improvements and prepare them for accreditation: a formal recognition of the implementation of laboratory quality systems and services that adhere to international standards.4 international standards organization (iso) 15189 is the standard of choice for accreditation of clinical laboratories, as it promotes the delivery of reliable results for patient safety and care, as well as reliable data for public health interventions.5 kenya adopted the slmta programme in 2010 to guide laboratory quality improvements prior to accreditation by the national accreditation body, kenya national accreditation service. the slmta programme was designed to span between 12 and 18 months, to include three workshops spaced between quality management systems (qms) implementation, and to have participants implement improvement projects after each workshop, supported by regular supervisory mentorship visits or on-site mentoring. audits are conducted at the beginning and end of the training using the slipta checklist to assess strengths, weaknesses, and progress made.6 based on the assessments, laboratories are scored from 0 to 5 stars. laboratories scoring 3 stars and above are eligible to apply to accreditation institutions to commence the formal accreditation assessment process. by february 2020, after ten years of slmta implementation, 52 countries, 23 in africa, had implemented the slmta programme in more than 1000 laboratories with 220 accredited to iso 15189.2 kenya was well funded and equipped with a system for implementing, managing, and monitoring laboratory activities, and by 2017 had implemented slipta in more than 140 laboratories. in accordance with the who guide for slipta implementation,7 the kenya ministry of health slipta focal office prioritised laboratories, coordinated and guided this process. despite measurable improvements recorded by the laboratories, less than 10% had achieved iso 15189 accreditation, with most stagnating at 2–3 slipta stars.8 audu et al.9 describe experiences in nigeria where laboratories implementing slmta with expert on-site mentorship improved faster and steadier, achieving a score of 5 stars. hospital management buy-in and strong laboratory staff camaraderie were found to be essential for the positive changes observed during slmta implementation in botswana.10 further, a study conducted in ethiopia on perceptions and attitudes toward slmta among laboratory and hospital professionals recommended additional training and staff advocacy workshops for medical directors and hospitals’ upper management in a bid to address concerns about the duration of the process and resource allocation.11 a qualitative study carried out in rwanda to better understand the reasons and context underlying decrease in laboratory performance during slmta implementation identified various factors, notably lack of coordination, ownership by the laboratory workforce and insufficient stakeholders’ communication, as contributors to low performance.12 therefore, to accelerate the pace of medical laboratory accreditation in kenya, the united states centers for disease control and prevention in collaboration with the ministry of health and other stakeholders embarked on a rapid results initiative (rri) to re-energise the kenya’s laboratory quality improvement process through slipta. historically, kenya has used a 100-day rri aggressive programming approach to attain accelerated achievement of targets that had stagnated or made slow progress. the rri approach was applied in the hiv programme to improve uptake of the prevention of mother-to-child transmission programme and male involvement13,14 and uptake of voluntary medical male circumcision.15 this approach also has been implemented in other sectors of the kenyan government.16 this project sought to fast-track through an aggressive rri, the achievement of improved laboratory qms and increase iso 15189 accreditation among medical laboratories whose quality management improvements had stagnated based on slipta assessment after more than two years of slmta implementation. in this article, we describe kenya’s experience with accelerating medical laboratories on slmta towards attaining iso 15189 accreditation. methods ethical considerations we conducted this rri project as part of ongoing pepfar-supported initiatives in kenya to improve laboratory testing quality to support access to quality laboratory services for optimal care and treatment of persons with hiv and tuberculosis. data collection and analysis were part of routine laboratory strengthening programmes approved by the kenya ministry of health. the study was approved by the kenyatta national hospital – university of nairobi ethical review committee (knh-uon#: p723/10/2018). this project was further reviewed by the united states cdc, center for global health, associate director for science in accordance with cdc human research protection procedures and was determined to be non-research (cgh-hsr#: 2018-355). rapid results initiative taskforce in march 2017, the ministry of health in kenya set up a rri taskforce to review and advise on kenya’s progress in implementing quality systems improvement through slipta, among laboratories enrolled in the slmta programme since 2010. the rri taskforce included representatives from the united states president’s emergency plan for aids relief (pepfar), ministry of health and the slipta focal office, centers for disease control and prevention and pepfar implementing partners, with extensive knowledge and experience in the stepwise laboratory qms strengthening initiatives in kenya. as part of the deliverables, the rri taskforce reviewed all 140 slmta-implementing laboratories and selected 39 that had stagnated in the slmta programme for more than 2 years without achieving accreditation to iso 15189 standard. to accelerate progress towards accreditation of these laboratories that had gone through the full cycle of standard slmta without success, the taskforce provided the following as part of the rri process: high-level advocacy, upper management and facility stakeholder engagements, rri on-site support and mentorship, training, laboratory audits and rri technical assistance visits. high-level advocacy, upper management and facility stakeholder engagements the rri taskforce convened two-tier engagement meetings with stakeholders and health facility personnel in march 2017. the first tier of meetings were at national and county-level and entailed advocacy engagement workshops that drew attendees from national and county laboratory leadership: ministry of health division of laboratory services, kenya national accreditation service, county directors of health, county medical laboratory coordinators and pepfar laboratory programme implementing partners. this was done to gain consensus and commitment for support from the stakeholders as a top-down approach. the second tier of meetings were held in several regions bringing together top county-level laboratory leadership, upper health facility management, and laboratory leadership from the targeted laboratories. typically, attendees of the regional meetings included county directors of health, hospitals’ medical superintendents, county medical laboratory coordinators, facility laboratory managers, laboratory equipment maintenance staff, human resource officers, procurement officers and laboratory quality assurance officers. at each regional meeting, the rri taskforce discussed the key steps for the rri process and need to provide additional resources to address gaps identified. each regional meeting also served as an official kick-off for the rri process at the selected facilities. rapid results initiative on-site support and mentorship for all the laboratories on rri, mentorship was targeted based on slipta baseline audit findings, gaps were rapidly addressed following the slmta process but in shortened timelines, because the laboratories had already gone through the standard slmta training. the aggressive mentorship entailed a review and in some cases revision of the basic quality documents such as the quality manual, standard operating procedures, and test methods. these documents were then used to guide the rest of the rri process to raise the laboratory performance on the 12 quality system essentials (qses) which include: documents & records (qse 1), management reviews (qse 2), organization & personnel (qse 3), client management & customer service (qse 4), equipment (qse 5), evaluation and audits (qse 6), purchasing & inventory (qse 7), process control (qse 8), information management (qse 9), identification of non conformities, corrective and preventive actions (qse 10), occurrence/incident management & process improvement (qse 11) and facilities and biosafety (qse 12). trainings targeted slmta-driven qms improvement trainings were conducted at either central or facility-level based on cross-cutting needs among laboratories on the rri (table 1). trainings conducted at each site were recommended by laboratory quality systems experts informed by the gaps identified during the baseline assessments and internal audits. the trainings were facilitated by slmta-trained trainers and experienced laboratory mentors drawn from the ministry of health and implementing partners. the trainings entailed a mix of didactic instructions and hands-on demonstrations in the laboratories. table 1: trainings and qse-based mentorship interventions used to fast-track implementation of quality management system and monitoring by slipta in 39 laboratories in kenya, 2017–2019. laboratory audits two external audits, baseline in april 2017 and exit in july 2017, were conducted at each laboratory using the e-slipta checklist (adapted from the official who afro slipta checklist6), which was loaded on tablets with open data kit (get odk inc., san diego, california, united states). for this initiative, utility of e-slipta checklist was to enhance rapid transmission and analysis of audit data. the short turnaround time of two days for audit report summaries achieved using the e-slipta checklist expeditiously completed the communication loop quickly providing information on identified gaps for targeted slipta mentorship towards their fast-tracked resolution. each audit lasted one day and was conducted by experienced laboratory auditors who had been trained on using the e-slipta checklist. the laboratory audit team reviewed quality and technical records, observed laboratory practices and discussed qms practices with the laboratory staff. the auditors documented areas of non-conformity and, per the slipta guidance, provided on-site technical assistance and recommendations for mitigating identified gaps. results of the laboratory audits were recorded for each of the 12 sections of the checklist, which covers the 12 qses (clsi gp 26-a317). all audits and laboratory rating classifications were performed as per the who afro slipta checklist.6 the checklist has 334 questions for a total of 275 possible points. at the end of each audit, the scores were analysed and presented to each individual laboratory on follow-up technical visits. the laboratories were tasked to perform internal audit at rri mid-term and as needed following resolution of most nonconformities identified at previous audit. the mid-term internal audit was used by the rri taskforce to conduct additional facility technical visits for both the hospital management and the laboratory. laboratories that scored ≥ 2 stars at exit audit were recommended for the 6-month post-rri mentorship period in phase 3. laboratories scoring slipta ≥ 3 stars at any time during the rri process were recommended to initiate the kenya national accreditation service iso 15189 accreditation application process. kenya national accreditation service accreditation was achieved in phase 3 (the post-rri period) following iso 15189 accreditation assessments and closure of assessment gaps. rapid results initiative technical assistance visits members of the rri taskforce conducted technical site visits to the target laboratories following baseline audits and provided on-site technical assistance and recommendations for closing identified gaps. first came meetings with laboratory staff, mentors and pepfar implementing partners providing moral support and technical details for resolving highlighted slipta audit gaps. second were the technical site visit meetings with top management which utilised the audit report scores to advocate for support to the laboratory from the health facility and county administration and management offices. advocacy to the upper management was focused on addressing deficiencies related to management review meetings, infrastructure, and resource mobilisation aimed at meeting quality-related gaps in the laboratories. additionally, the pepfar partners working at those facilities provided one-off support for nonconformities that required additional financing beyond what the facility and county management teams were able to resolve. period of implementation the rri process was implemented in three phases: phase 1: this was a pre-rri planning and activation period of 2 months (april 2017 – june 2017) that entailed high-level advocacy, and national and county stakeholder engagement, national and county-level trainings, external and internal audit refresher trainings for slmta auditors, and deploying the e-slipta checklist. phase 2: this was the aggressive, facility-level rri implementation period of 100 days (july 2017 – october 2017) that entailed facility-level advocacy and hospital management engagement, laboratory qms trainings, targeted laboratory mentorships and laboratory audits using the e-slipta checklist. baseline and exit external audits were conducted at the beginning and at the end of this phase. the rri process implementation support, aimed at closing gaps identified by slipta audits, was standardised across all 39 laboratories. phase 3: this was the post-rri period of six months (october 2017 – march 2018) that entailed follow-up ongoing mentorship to close gaps and nonconformities identified by rri exit audits and 12 months (april 2018 – march 2019) of monitoring achievement of accreditation to iso 15189 as the outcome of interest for eligible facilities. data analysis to describe qms performance of laboratories participating in the rri, proportions of laboratories at different star ratings before and after rri interventions were computed based on the two external rri audits conducted at baseline and at exit. average qse point audits scores and respective percentage scores were calculated and presented as means with standard deviation (s.d.) and as percentages with their 95% confidence intervals (ci). to describe improvements in the laboratory qms, qse-specific mean point scores between baseline and exit audits were compared using a paired t-test to assess changes in performance. radar charts (spider graphs) of average baseline and exit qse percentage scores were plotted to demonstrate relative improvement in qse performance following rri interventions. to tease out some of the challenging qses that did not make significant improvements despite the rri process, qse performance was compared between laboratories that moved upwards by 2 or more stars from baseline audit star rating to exit audit star rating (i.e. made a stepwise move upwards of ≥ 2 star steps; e.g. from 0 stars at baseline to 2 stars at exit or 1 star at baseline to 3 stars at exit and so on) and those that did not (moved upwards < 2 star steps). average percentage score changes or mean point score increases between baseline and exit audits with p-value < 0.05 were considered statistically significant. analysis was performed using stata statistical software, version 12.0 (stata corp., college station, texas, united states). results overall laboratory stepwise laboratory improvement process towards accreditation star rating performance all of the 39 laboratories targeted for rri process were in urban settings. of these, 32 (82%) were ministry of health-owned, while seven (18%) were affiliated to faith-based organisations (fbos). all 39 laboratories enrolled in the rri completed the intensive mentorship sessions including scheduled baseline and exit external audits (table 1). at baseline audit, most laboratories (24/39, 62%) were below 2 stars, eight (21%) were rated at 2 stars, and only seven (18%) were at ≥ 3 stars. at the exit audit, only six (15%) laboratories scored below 2 stars, 21 (54%) were at 2 stars, and 12 (31%) achieved ≥ 3 stars (figure 1). therefore, the number of laboratories that scored ≥ 2 stars rating increased significantly from 15 (38%) at baseline to 33 (85%) at rri exit (p < 0.001). there was no significant difference in performance between ministry of health-owned and fbo-affiliated laboratories, of which 75% and 71% achieved ≥ 2 stars rating. overall, 34 (87%) laboratories achieved the desired goal of iso 15189 accreditation in less than two years of the rri process. this resulted in a 330% increase in the number of iso-accredited laboratories (from 15 laboratories in june 2017 to 49 laboratories in june 2019) through pepfar funding in kenya. figure 1: laboratory star rating before and after rapid results initiative implementation of strengthening laboratory management toward accreditation in 39 laboratories in kenya, 2017–2019. mean changes in quality system essentials scores during rri the performance of the 12 qses before and after the rri is displayed in table 2. of the 35 possible points assigned to equipment management (qse 5) on the who slipta checklist, the mean (s.d.) baseline score for the 39 rri laboratories was 21.97 (4.31) and increased to 26.28 (4.07) at exit. this qse experienced the most significant mean increase of 5.31 ± 1.89 (p < 0.0001). facilities and biosafety (qse 12) with a mean increase of 4.05 ± 1.78 (p < 0.0001) and management reviews (qse 2) with a mean increase of 3.18 ± 1.77 (p < 0.001) were the second and third most impacted qses by the rri interventions (table 2). client management & customer service (qse 4) (p = 0.0224) and occurrence/incident management & process improvement (qse 11) (p = 0.0277) also had significant mean point increases. although evaluation and audits (qse 6) had the second-highest percent increase in performance, the mean point increase from baseline to exit of 1.51 ± 2.24 for this qse was not statistically significant (p = 0.1581). table 2: quality system essentials score changes following rri for implementation of qms through slipta in 39 laboratories in kenya, 2017–2019. quality system essential performance-related laboratory star improvement of the 12 qses, management reviews (qse 2), evaluation and audits (qse 6), information management (qse 9) and, identification of nonconformities, corrective and preventive actions (qse 10) were overall the most challenging to improve toward the maximum achievable respective scores, barely crossing the 50.0% mark after rri interventions. the baseline average percentage scores were 30.0% for qse 2, 43.0% for qse 6, 54.0% for qse 9, and 47.0% for qse 10, compared to 52.0%, 51.0%, 54.0%, and 43.0% at rri exit (figure 2a). to understand qses that were most problematic despite the rri efforts, we assessed qse level performance for 13 (33.0%) of the 39 laboratories that improved by 2-star steps compared to the other 26 (67.0%) laboratories that did not (i.e. moved upwards < 2-star steps) as shown in figure 2b and 2c. in the first stratum of these 13 laboratories that attained ≥ 2-star step increases, the average percentage scores for all qses increased substantially following rri interventions, except for qse 10, which only had minimal improvement from 47.0% at baseline to 49.0% at exit (figure 2b). in these 13 laboratories, qse 2 and qse 6 were the most improved with an increase in performance from 20.3% (95% ci: 5.2% – 50.1%) to 76.9% (95% ci: 47.3% – 93.5%) and 28.7% (95.0% ci: 10.1% – 57.1%) to 73.3% (95.0% ci: 44.8% – 91.1%), despite being among the most challenging qses to improve when all 39 laboratories were pooled together. qse 9 and qse 10 remained the most difficult to improve across the 13 laboratories included in this stratum (figure 2b), barely crossing the 50.0% mark. conversely, among the 26 laboratories in the second stratum, qse 2, qse 6, and qse 10 had the least improvement in performance, barely crossing the 40.0% mark after rri interventions. therefore, these three qses in addition to qse 9 remained the most challenging among the 26 laboratories that increased < 2-star step despite rri interventions (figure 2c). although overall star rating performance for the second stratum of 26 laboratories was not substantial, several qses improved due to the rri interventions (figure 2c). qse 4, qse 5, qse 11, and qse 12 had the highest increase in average percentage scores, ranging from 27.0% to 33.0%. figure 2: average percent score for quality system essentials performance before and after 100-day rapid results initiative for laboratories to fast-track stepwise laboratory improvement towards accreditation in 39 laboratories in kenya, 2017–2019. (a) all laboratories (n = 39); (b) laboratories with ≥ 2-step star increase (n = 13); (c) laboratories with < 2-step star increase (n = 26). discussion our findings confirm that aggressive high-level advocacy and management buy-in, targeted mentorship and site-level support implemented in 100 days followed by an additional 6-month ongoing mentorship resulted in a 330% increase in number of iso 15189 accredited medical laboratories in kenya. to our knowledge, our report is the first to describe expedited improvement of laboratory quality systems for laboratories on the slmta programme through rri. several factors can be cited for the rapid improvement of laboratories that implemented rri. the qms intervention trainings that were conducted at facility and centralised levels renewed the stakeholders’ interest in the iso 15189 accreditation and motivated the laboratory staff to ‘sprint toward accreditation’. the rri was implemented by a coalition of laboratory experts in quality systems implementation that ensured inter-mentor strengths across the 12 qses, while maintaining accountability and focus on targeted interventions. further, by leveraging on the strengths of each mentor, this arrangement provided the much-needed capacity to rapidly execute the rri process in 39 laboratories spread across the country using a relatively small team. during the implementation process, mentors provided laboratory performance and non-conformance data to upper management for action, ensuring continuous communication with upper management. this in turn provided commitment and resources needed for laboratory staff to address emerging challenges within a short time. our experience accords with the available reports on the critical role of laboratory mentorship and advocacy in helping laboratories achieve accreditation goals during the slipta process.18,19 an additional success factor for this initiative was the adoption of kenya’s version of the e-slipta checklist to audit laboratories. the e-slipta checklist provided an interactive dashboard for identifying gaps and improving laboratory performance in real time. this simplified the feedback of audit findings to upper hospital management for prompt action.20 we found that of the 12 qses monitored during the rri initiative, improvement in qse 2 (management reviews), qse 6 (evaluation and audits), and qse 10 (identification of nonconformities, corrective and preventive actions) were critical for any laboratory to demonstrate a 2-star improvement after 100 days. this observation is consistent with previous findings that identified upper management support for laboratory improvement, adequate audit skills and planning as critical factors for high audit scores and corrective actions necessary for laboratories to achieve accreditation.21,22,23 the rri taskforce addressed these obstacles before and during rri implementation. initial trainings and mentorships served to plan and build staff audit skills while sharing of audits findings through e-slipta ensured timely implementation of corrective and preventive actions. a significant number (67%) of the laboratories that implemented rri recorded a less than 2-step star improvement after the rri, with some stagnating at the 1-star level or below. poor performance and stagnation in some laboratories were linked to three qses: management reviews (qse 2), evaluation and audits (qse 6), and identification of nonconformities, corrective and preventive actions (qse 11). these results were unexpected, because the rri interventions had been designed primarily to address these qses based on previous observations of most laboratories initially progressing rapidly toward meeting the slmta goals but stagnating between 2 and 3 stars, while others regress to 1 or 0 star (unpublished work; makokha et al.). in addition, most of the staff in the rri-targeted laboratories were experienced in implementing laboratory qms and had received refresher training in corrective action, occurrence management, and internal audits as part of this project. a low score in information management (qse 9) among the laboratories that recorded less than 2-star improvements after the rri process was unexpected because all the laboratories implementing rri had functional laboratory information systems. however, qse 9 was flagged as a stagnating factor, because the 100 days within which the rri was implemented were inadequate to address it. limitations this project had several limitations. although implementation of the rri process achieved tangible improvements in qms and overall quality within a short time, these results were based on a small cohort of laboratories that had previously received slmta trainings and implemented several qse improvements. the rri was implemented in a group of laboratories with cumulative experience of both success and stagnation in improving quality through slmta. another limitation is that we did not analyse the effect of laboratory staff levels and their experience with the rri process and outcomes. hence, these results cannot be easily generalised, because previous experience may have facilitated rapid improvements in some of the laboratories. conclusion with the right advocacy strategy, mentorship teamwork, leadership buy-in, and effective communication, implementation of laboratory qms improvement through slmta and assessed by slipta could benefit from rri catalysing accreditation preparedness for medical laboratories. engaging facility management and county leadership throughout the process of laboratory accreditation is key to closing nonconformities identified during mentorship. the rri led to several carry-over effects well beyond the 100 days and increased the number of laboratories achieving accreditation to the iso 15189 standard in kenya. acknowledgements we acknowledge the following institutions for their participation in the consensus-building engagements with both stakeholders and facility management as well as implementation of the rri process including on-site mentorship and laboratory auditing: african medical research foundation – health africa, association of public health laboratories, clinical and laboratory standards institute, global implementation solutions, fhi360, kenya aids vaccine initiative, university of maryland baltimore, and becton-dickinson-president’s emergency plan for aids relief (pepfar) public private partnership. we thank the county governments through their county medical laboratory coordinators who provided critical links between the implementing partners and county leadership. many thanks to the university of maryland, baltimore data team who assisted with daily data collection and analysis through the e-slipta tool. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions e.p.m. contributed towards project conception and design, initial drafting of the article, collection and assembly of data, manuscript submission and approvals. r.o.o. contributed towards drafting of the original article, data validation, data analysis, data visualisation and graphics, review and editing of the article. d.k.k. contributed towards drafting of the original article, technical and logistical support, and interpretation of the data. m.j., f.b., m.m., m.d. and m.n. contributed towards revision of the article for important technical and intellectual content. m.u. and t.g. provided administrative, technical, or logistic support for the project. in addition, they provided study laboratories and documents. j.m. contributed towards critical review and editing of the article, and provided administrative support, supervision, and resources. sources of support this project was supported by the united states pepfar through the centers for disease control and prevention (cdc) under the terms of cdc-rfa-gh15-1544 cooperative agreements: gh001358, gh001374, gh001388, gh001371 and ps13-1366 coag# gh001114 data availability the data used and analysed for this article are available and may be obtained from the corresponding author, e.p.m. disclaimer the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the funding agencies. references nkengasong jn, yao k, onyebujoh p. laboratory medicine in low-income and middle-income countries: progress and challenges. lancet. 2018. https://doi.org/10.1016/s0140-6736(18)30310-6 slmta. strengthening laboratory management towards accreditation [homepage on the internet]. 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balinda ronald ballard lawrence barker drew baughman kapila bhowan waqo boru hayat caidi walter campos manhattan charurat anicet georges dahourou tjeerd datema joshua dawurung esther de gourville linda de gouveia alpha diallo katie distelhorst frances pouch downes robert downing peter drotman mignon du plessis ifeoma enweani nwadiuto esiobu benson estambale eric fevre peter fonjungo fengxiang gao jessie githanga sheba gitta barbara miriam goldsmith jean-michel heraud brad hersh duncan hooker koichi ishikawa ilesh jani jeanne jordan jessica justman john kagira edward kamau phyllis kanki 73 reba kanungo samuel kariuki luc kestens olen kew samoel khamadi makobetsa khati stella kiambi paul klatser keith klugman stephania koblavi-deme jessee kwiek william lali segundo leon henry limula christophe longuet elizabeth luman julius lutwama eliguis lyamuya david mabey jemal ali mahdi naomi maina barbara marston sharon martin talkmore maruta charles massambu souleymane mboup karen mcclure seema 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tchongwe kevin tetteh andrew thaiyah oyewale tomori daniel tullo david turgeon nzovu ulenga kathleen victoir anne von gottberg diane waku-kouomou ndadilnasiya endie waziri burton wilcke daniel asrat woldetsadik nicole wolter chunfu yang clement zeh should names have inadvertently been excluded from this list the publisher apologises and undertakes to amend the exclusion in the next issue. ajlm african journal of laboratory medicine in an effort to facilitate the selection of appropriate peer reviewers for the african journal of laboratory medicine, we ask that you take a moment to update your electronic portfolio on www. ajlmonline.org for our files, allowing us better access to your areas of interest and expertise, in order to match reviewers with submitted manuscripts. if you would like to become a reviewer, please visit the journal website and register as a reviewer. to access your details on the website, you will need to follow these steps: 1. log into the online journal at http://www. ajlmonline.org 2. in your ‘user home’ [http://www.ajlmonline. org/index.php/ajlm/ user] select ‘edit my profile’ under the heading ‘my account’ and insert all relevant details, bio statement and reviewing interest. 3. it is good practice as reviewer to update your personal details regularly to ensure contact with you throughout your professional term as reviewer to the african journal of laboratory medicine. please do not hesitate to contact me if you require assistance in performing this task. suzanne taylor submissions@ajlmonline. org tel: +27 (0)21 975 2602 fax: +27 (0)21 975 4635 acknowledgements references about the author(s) farouk a. umaru independent laboratory supply chain and quality assurance consultant, washington, district of columbia, united states citation umaru fa. united states foreign aid freeze: an urgent call to action for support for african national laboratory programmes. afr j lab med. 2025;14(1), a2794. https://doi.org/10.4102/ajlm.v14i1.2794 opinion paper united states foreign aid freeze: an urgent call to action for support for african national laboratory programmes farouk a. umaru received: 19 feb. 2025; accepted: 18 apr. 2025; published: 16 may 2025 copyright: © 2025. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the united states government, under the trump administration, imposed a freeze on foreign aid by means of an executive order on 20 january 2025 (eo 14169. reevaluation and realigning united states foreign aid), initiating a 90-day pause on all foreign aid programmes. the united states agency for international development, established under the foreign aid act of 1961 (22 usc ch. 32: foreign assistance),1 is an independent agency responsible for implementing united states foreign programmes and managing congressionally allocated funds. the president’s emergency plan for aids relief is the largest aid initiative managed by the united states agency for international development, and is supported by the united states centers for disease control and prevention (cdc), department of defense, and state department, significantly enhancing laboratory capacity in clinical diagnosis, disease surveillance and pandemic preparedness. the dismantling of the united states agency for international development’s operations will significantly affect laboratory services in many countries that rely on its health programmes. this opinion presents key historical contributions of the president’s emergency plan for aids relief in strengthening the diagnostics landscape in africa and makes the case that, if immediate action is not taken, the continent is on the verge of losing this progress, thereby reversing decades of success: procurement and supply chain of laboratory supplies and technologies: for nearly 20 years, united states agency for international development implementing partners such as john snow inc. and chemonics international have led the president’s emergency plan for aids relief project in over 50 countries, delivering over $10 billion in health commodities, including hiv viral load tests, rapid diagnostic tests for hiv, tuberculosis, and malaria, antiretrovirals, and diagnostics equipment such as cepheid genexpert, abbott mpima, roche cobas instruments, hologic panthers, abbott m2000, and abbott malinity systems (these are distributed by united states companies significantly impacted by the trump administration’s america first agenda). standardisation of diagnostic services: efforts by the united states cdc, the african society for laboratory medicine, africa cdc and partners have led to more than 400 laboratories receiving iso 15189 and iso 17025 accreditation,2 improving the quality of laboratory services on the continent. united states foreign aid programmes supported the procurement and technical assistance to these laboratories. disease surveillance and outbreak preparedness: the global health security programme, along with africa cdc, the african society for laboratory medicine, and the world health organization, has enhanced public health laboratory infrastructure in numerous countries, training thousands of staffers for disease detection and response, and assisting in the global surveillance and response to outbreaks that could arrive in the united states. harmonisation of tiered laboratory functions: over 10 african countries have restructured laboratories into a tiered network, aligning with the 2008 maputo declaration, optimising test menus, and staffing for efficiency and cost savings.3 the united states agency for international development has developed diagnostics networks optimisation strategies, supporting the restructuring of laboratory networks into an efficient and responsive turn-around time service delivery.4 private sector participation in laboratory services: the united states agency for international development has included private sector manufacturers, suppliers, and vendors in laboratory management, reducing risks of equipment downtime, stockouts, and expiration. the united states agency for international development negotiated all-inclusive agreements and service level contracts, and engaged local companies in last-mile vendor management for storage and distribution of essential laboratory supplies, improving access to site-level laboratory services. access to hiv viral load tests. achieving the joint united nations programme on hiv and aids 95-95-95 goals (95% people living with hiv know their status, 95% of people who know their status initiate treatment, and 95% on treatment are virally suppressed) includes millions of people to be tested and virally suppressed.5 over 50 million viral load test results have been delivered to 58 countries, accelerating countries’ progress towards the 95% targets of viral suppression, aiming to control the hiv pandemic by 2030.6 controlling hiv/aids-related child mortality: the early infant diagnostics service was expanded with modern multiplex technologies as a result of president emergency plan for aids relief programme funding, allowing access to early detection and prevention of mother-to-child transmission. human resources for health: preand in-service training, curriculum reviews, and employment of expert personnel have strengthened national laboratory programmes and filled human capital gaps. local economic growth and stability: grants, sample distribution contracts, and technical assistance have created stable jobs for youth and supported local businesses. given the uncertainty surrounding the future of united states foreign aid, it is imperative for african leaders to take immediate action by mobilising resources to advance the continent’s laboratory programmes. key considerations for immediate and sustainable laboratory programme resolutions are provided below: establish resilience funding: african leaders and advisors should immediately mobilise resources to advance laboratory programmes across the continent. this includes setting up a laboratory resilience fund structure, modelled after the global fund or the joint united nations programme on hiv and aids, to allow local and international philanthropies and other bodies to contribute towards laboratory programmes. increase domestic allocation: african governments must increase financial commitments to diagnostics laboratory infrastructure, specifically clinical diagnosis, disease detection, surveillance, and response to clinical and emerging pandemics. engage private sector providers: africa cdc, the african society for laboratory medicine, and related entities should engage the private sector in providing affordable essential testing services in countries where the private sector thrives. in addition, governments should incentivise private laboratory service providers through tax holidays for affordable services and support local manufacturing and supply chain infrastructure. partner with international companies: collaborate with international companies with bases in the united states, such as abbott, becton dickinson, hologic, cepheid and roche to continue with the current leasing and maintenance of service level agreements, and to encourage technology transfer for local production of diagnostics and pharmaceuticals, including patent waivers. foster local innovation: promote entrepreneurship within educational systems by providing incentives for science and technology startups, creating innovation hubs, and offering low-interest financing for emerging small businesses in laboratory technologies. acknowledgements competing interests the author declares that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. author’s contributions f.a.u. is exclusively responsible for conception of, drafting, reviewing and finalising the article. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support the author received no financial support for the research, authorship, and/or publication of this article. data availability data used for this manuscript are open source and readily available online. specific programme numbers were extracted from the global health supply chain procurement and supply management website and fully acknowledged. disclaimer the views and opinions expressed in this article are those of the author and are the product of professional research. the article does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, government, or that of the publisher. the author is responsible for this article’s results, findings, and content. references united states congress. title 22 foreign relations and intercourse, chapter 32, foreign assistance [homepage on the internet]. c1961 [updated 2025 jan 20; cited 2025 apr 30]. available from: https://uscode.house.gov/view.xhtml?path=/prelim@title22/chapter32&edition=prelim centers for disease control and prevention. slmta laboratories that have achieved accreditation [homepage on the internet]. c2024 [updated 2024 sep 30; cited 2025 feb 19]. available from: https://www.slmta.org/accredited-labs/ williams j, umaru f, edgil d, kuritsky j. progress in harmonizing tiered hiv laboratory systems: challenges and opportunities in 8 african countries. glob health sci pract. 2016;4(3):467–480. https://doi.org/10.9745/ghsp-d-16-00004 ondoa p, ndlovu n, keita ms, massinga-loembe m, kebede y, odhiambo c, et al. preparing national tiered laboratory systems and networks to advance diagnostics in africa and meet the continent’s health agenda: insights into priority areas for improvement. afr j lab med. 2020; 9(2):1103. https://doi.org/10.4102/ajlm.v9i2.1103 united nations programme on hiv and aids. 2025 aids targets – putting people living with hiv and communities at risk at the center. [homepage on the internet]. c2025. [updated 2025 jan 1; cited 2025 apr 30]. available from: https://aidstargets2025.unaids.org/#section-targets global health supply chain. global health supply chain-procurement and supply management fiscal year 2024 q4 by the numbers [homepage on the internet]. c2024 [updated 2024 nov 15; cited 2025 feb 19]. available from: https://www.ghsupplychain.org/node/2228 abstract introduction case presentation management and outcomes discussion acknowledgements references about the author(s) olumuyiwa b. salu department of medical microbiology and parasitology, college of medicine, university of lagos, lagos, nigeria central research laboratory, college of medicine, university of lagos, lagos, nigeria ayorinde b. james department of biochemistry, college of medicine, university of lagos, lagos, nigeria central research laboratory, college of medicine, university of lagos, lagos, nigeria bamidele o. oke department of medical microbiology and parasitology, college of medicine, university of lagos, lagos, nigeria mercy r. orenolu central research laboratory, college of medicine, university of lagos, lagos, nigeria roosevelt a. anyanwu central research laboratory, college of medicine, university of lagos, lagos, nigeria maryam a. abdullah central research laboratory, college of medicine, university of lagos, lagos, nigeria christian happi african center of excellence for genomics of infectious diseases, redeemers university, ede, osun state, nigeria department of biological sciences, college of natural sciences, redeemers university, ede, osun state, nigeria jide idris honourable commissioner for health, lagos state ministry of health, alausa, ikeja, lagos, nigeria ismail a. abdus-salam epidemiology unit, directorate of disease control, lagos state ministry of health, alausa, ikeja, lagos, nigeria abdul-salam nasidi nigeria center for disease control, federal ministry of health, abuja, nigeria folashade t. ogunsola department of medical microbiology and parasitology, college of medicine, university of lagos, lagos, nigeria central research laboratory, college of medicine, university of lagos, lagos, nigeria oyewale tomori nigerian academy of science, lagos, nigeria sunday a. omilabu department of medical microbiology and parasitology, college of medicine, university of lagos, lagos, nigeria central research laboratory, college of medicine, university of lagos, lagos, nigeria citation salu ob, james ab, oke bo, et al. biosafety level-2 laboratory diagnosis of zaire ebola virus disease imported from liberia to nigeria. afr j lab med. 2016;5(1), a468. http://dx.doi.org/10.4102/ajlm.v5i1.468 case studies biosafety level-2 laboratory diagnosis of zaire ebola virus disease imported from liberia to nigeria olumuyiwa b. salu, ayorinde b. james, bamidele o. oke, mercy r. orenolu, roosevelt a. anyanwu, maryam a. abdullah, christian happi, jide idris, ismail a. abdus-salam, abdul-salam nasidi, folashade t. ogunsola, oyewale tomori, sunday a. omilabu received: 14 apr. 2016; accepted: 09 july 2016; published: 17 oct. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract introduction: global travel is an efficient route of transmission for highly infectious pathogens and increases the chances of such pathogens moving from high disease-endemic areas to new regions. we describe the rapid and safe identification of the first imported case of ebola virus disease in a traveler to lagos, nigeria, using conventional reverse transcription polymerase chain reaction (rt-pcr) in a biosafety level (bsl)-2 facility. case presentation: on 20 july 2014, a traveler arrived from liberia at lagos international airport and was admitted to a private hospital in lagos, with clinical suspicion of ebola virus disease. methodology and outcome: blood and urine specimens were collected, transported to the virology unit laboratory at the college of medicine, university of lagos, and processed under stringent biosafety conditions for viral rna extraction. rt-pcr was set-up to query the ebola, lassa and dengue fever viruses. amplicons for pan-filoviruses were detected as 300 bp bands on a 1.5% agarose gel image; there were no detectable bands for lassa and dengue viral rna. nucleotide blast and phylogenetic analysis of sequence data of the rna-dependent rna polymerase (l) gene confirmed the sequence to be zaire ebolavirus (ebov/hsap/nga/2014/lib-nig 01072014; genbank: km251803.1). conclusion: our bsl-2 facility in lagos, nigeria, was able to safely detect ebola virus disease using molecular techniques, supporting the reliability of molecular detection of highly infectious viral pathogens under stringent safety guidelines in bsl-2 laboratories. this is a significant lesson for the many under-facilitated laboratories in resource-limited settings, as is predominantly found in sub-saharan africa. introduction ebola virus is the causative agent of ebola virus disease (evd), previously known as ebola haemorrhagic fever.1 the genus ebolavirus belongs to the filoviridae family, alongside marburgvirus and the newly-identified genus cuevavirus.1 the ebolavirus genus consists of five different species, including zaire ebolavirus, sudan ebolavirus, bundibugyo ebolavirus, tai forest ebolavirus and reston ebolavirus.2 the first three have been associated with major disease outbreaks in west africa, whereas tai forest ebolavirus has one reported case of human infection in an individual who had contact with an infected chimpanzee from the tai forest in ivory coast and reston ebolavirus has been reported in the asia-pacific region, but with no recorded cases of human infection.2,3,4 members of the family filoviridae in the order of mononegavirales are all non-segmented negative-stranded rna viruses. their genomic rna contains filoviral genes that are organised in the following linear order: 3’-(leader)-np-vp35-vp40-gp-vp30-vp24-l-(trailer)-5’.5,6 on 17 july 2014, the ministries of health in liberia, guinea and sierra leone, in collaboration with the world health organization, announced a cumulative total of 1048 suspected and 745 laboratory-confirmed cases of evd, with 632 (60.3%) deaths7. blood and urine specimens were sent to the virology unit laboratory of the central research laboratory, college of medicine (cmul)-university of lagos and lagos university teaching hospital (luth). this report presents the laboratory methods used, including conventional reverse transcription polymerase chain reaction (pcr), to diagnose the first imported case of evd in nigeria in a biosafety level (bsl)-2 facility. case presentation a 40-year-old liberian man, who had been living in elwa, monrovia, travelled by air through accra (ghana) and lomé (togo) to lagos, nigeria. upon arrival at lagos international airport on 20 july 2014, he collapsed and was taken to a private hospital in the obalende area of lagos, nigeria (figure 1), where he was admitted. he presented with high fever, lymphadenopathy and sore throat and was treated for malaria and typhoid fever until he later developed diarrhoea, vomiting and microscopic haematuria. the culmination of the clinical presentation, the patient’s non-response to treatment for malaria and typhoid, as well as his epidemiological link to liberia, raised the suspicion for evd. on 22 july 2014, blood samples in edta tubes and urine samples were collected and sent for viral investigations to the cmul-luth virology unit laboratory. aliquots of the specimens were sent in parallel to the african center of excellence for genomics of infectious diseases (acegid) at redeemers university in ogun state, nigeria. figure 1: travel route of index case with ebola virus disease from liberia to lagos, nigeria. lower inset shows the closest health care facility to the airport (lasuth), the private health care facility where the index case was admitted (private hospital), the laboratory where the diagnosis was made (virology lab, cmul/luth) and the genomics centre where the corroborative diagnosis and nucleotide sequencing was done (acegid). map was plotted with gps coordinates using arc gis 10.1. ethical considerations no ethics approval was required for the diagnosis of an infection of great public health importance and which was part of the normal course of management of any infected patient. management and outcomes specimen handling and processing due to the fact that no bsl-3 or bsl-4 facilities were available to handle the patient’s specimens, all samples received were handled in a bsl-2 facility with extreme care and in line with us centers for disease control and prevention (cdc) safety guidelines.8 personal protective equipment (ppe) was worn in accordance with the ‘donning and doffing of ppe’ protocol set out by the cdc.8 sample carrier boxes were sprayed with 10% hypochlorite solution on a disposable laboratory absorbent mat in a biosafety class iia cabinet. specimen containers were removed from the carrier box and wrapped immediately with a laboratory paper towel soaked in 10% hypochlorite in order to disinfect any possible spillage around the specimen container. an identical process was used for removal of samples from the container box. outer gloves were sprayed with 10% hypochlorite solution every time hands were withdrawn from the safety cabinet and then disposed of in double-jacketed bio-hazard waste bags if they were soiled during sample handling or at the end of the procedure. aliquots of specimens used for viral assays were immediately inactivated in a guanidinium-thiocyanate lysis buffer for viral nucleic acid extraction (qiagen, germantown, maryland, united states). viral rna isolation and rt-pcr for viral rna extraction, neat (undiluted), 1:10 and 1:100 dilutions in sterile phosphate-buffered saline specimens of both blood and urine were processed using a qiagen rna extraction kit (qiagen, germantown, maryland, united states). segments of the l-gene of the ebola virus, the s-gene of the lassa virus and the 3’ non-coding region of the dengue fever virus were amplified in singleplex pcrs using the primers listed in table 1. separate master mixes for each queried virus were prepared and cycled as described in the ambionagpath-id one-step rt-pcr kit (applied biosystems, foster city, california, united states) protocol. subsequently, pcr amplicons were subjected to 1.5% agarose gel electrophoresis with 1x sybr® safe dna gel staining dye (invitrogen, carlsbad, california, united states) at 120v for 30 minutes and images were taken with a biodocanalyze 2.0 (biometra, goettingen, germany). table 1: primers used in the laboratory investigation of viral haemorrhagic diseases. ebov l-gene sequencing and phylogenetic analysis pcr amplicons on the agarose gel were purified using an agarose gel extraction kit (jena bioscience, jena, germany). purified non-infectious pcr products, packaged and transported using triple-level packaging, were sequenced using the filo a2.3 primer on the sanger dideoxy sequencing technology platform with an applied biosystems 3130xl genetic analyser at jena bioscience in jena, germany. a sequence trace file was used for blast analysis in genbank.9 the rna-dependent rna polymerase (l) gene, partial coding sequence (cds) for ebov/hsap/nga/2014/lib-nig 01072014 was deposited in the national center for biotechnology information (ncbi) with the accession number km251803.1. a set of 30 different filovirus genomes were selected from ncbi’s genbank. the fasta format of the l-gene region of all the selected genomes was downloaded and aligned with the km251803.1 sequence using the muscle tool of mega-6 software9,10 (figure 2). the tamura 3 parameter (t92) was found to be the best maximum likelihood model to infer the phylogenetic tree. a discrete gamma distribution was used to model evolutionary rate differences among sites (+g, parameter = 0.8048) and the rate variation model allowed for some sites to be evolutionarily invariable ([+i], 36.3524% sites).11 the reliability of each node on the phylogenetic tree was tested by bootstrapping with 1000 replicates. figure 2: agarose gel image showing pcr amplicon bands of the specimens. the ~300 bp band is the target band size for filoviruses. rt-pcr detection of queried haemorrhagic viruses investigations carried out on viral rna purified from the blood and urine specimens of the patient revealed the presence of the expected amplicon size (300 bp) for filoviruses (figure 2). however, no signals were observed for lassa or dengue fever viruses. the filovirus amplicons were still detected in blood specimens with logscale dilutions of 1 (1:10) and 2 (1:100), whereas in urine samples the signal was no longer detectable at a log 3 dilution. phylogenetic analysis of rna-dependent rna-polymerase l-gene partial cds phylogenetic analysis of the l-gene partial cds indicated that the virus evolved from the guinea and mano river isolates of the 2014 outbreak with a bootstraping value of 87% (figure 3). other isolates represented on the phylogenetic tree confirmed the evolutionary relationships between the different filoviruses. figure 3: molecular phylogenetic analysis of the l-gene segment of ebov/hsap/nga/2014/lib-nig 01072014 in comparison with selected filoviruses sequences by the maximum likelihood method. sequences are labelled using the ictv consensus nomenclature for variants of the filoviridae family with their corresponding genebank accession numbers in parenthesis. discussion by using molecular techniques and following cdc safety guidelines, our bsl-2 laboratory was able to safely identify the ebola virus in blood and urine samples. the clinical symptoms of the index case were initially linked to malaria and typhoid fever, until the patient began to haemorrhage, which led to a differential diagnosis of lassa, ebola or dengue fever. the time lapse between hospital admission, malaria/typhoid treatment and the clinical suspicion of evd was two days and led to the exposure of 72 persons at the airport and the hospital.12 this relatively quick diagnosis of the disease once the specimens reached the virology laboratories at cmul-luth and acegid was significant in limiting the extent of the outbreak, unlike the situation in guinea where the infection spread undetected for months, leading to the catastrophic epidemic in the west african sub-region.13 partial genomic sequence analysis data identified zaire ebolavirus as the aetiological agent of the disease (genbank accession: km251803.1). phylogenetic analysis of the rna-dependent rna polymerase (l) gene, partial cds showed a clustering (87%) with the zaire ebolavirus sequences originating from gueckedou and mano river in 2014. this finding corroborates the clinical suspicion and the travel history of the index case to nigeria. according to kuhn et al., the ebola virus responsible for the west african outbreak is the makona variant.14 the name ‘makona’ originated from the makona river which is central to the epicentre of the 2014 outbreak.14 many african countries, governments and research institutions are inadequately equipped or trained in diagnostics, active surveillance and reporting of highly infectious diseases.3 however, a number of centres such as ours are now being equipped to train scientists and scale up surveillance activities in the event of another outbreak. although we had no previous experience in diagnosing ebola virus, our involvement in the diagnosis of lassa virus, which is endemic in nigeria, led to the development of infrastructure for its molecular diagnosis under stringent biosafety procedures. because of collaborations with the bernard notch institute for tropical infections in germany, our laboratory had acquired sets of primers for diagnosis and surveillance of ebola, lassa, crimean-congo, rift valley, dengue and yellow fever viruses over the past 14 years.15 during that time, our laboratory grew to serve various hospital diagnostics and outbreak investigations in nigeria with funds provided by research granting bodies such as laboratoire national de santé institute, crp-santé, luxembourg, and the german research council, germany. thus, in collaboration with the bernard notch institute for tropical infections, we have been in the forefront of viral haemorrhagic fever investigations and surveillance in nigeria. limitations molecular detection using pcr is the most sensitive method for viral diagnostics. the use of chaotropic lysis buffer for the isolation of viral nucleic acids from the highly contagious specimens was a key step, because it rendered specimens non-infectious and thus safe for processing in a bsl-2 laboratory. however, despite our success, it should be noted that manipulations of highly pathogenic viruses such as ebola cannot be attempted in a bsl-2 laboratory. manipulations, such as culturing live ebola viruses, must be restricted to bsl-4 and bsl-3-enhanced laboratories, because of the higher risk of contracting ebola virus when incidents occur.16,17,18,19 thus, in our case the sequencing of the detected ebola virus was done at jena bioscience in germany. conclusion one of the major lessons learnt from this outbreak is that that there is an urgent need to build capability for rapid detection of and response to disease outbreaks in resource-limited countries, especially in west africa where the health systems are very weak. this should serve as a wake-up call, not only for african governments but also to the world, that investment in laboratory infrastructure and improvements in laboratory capabilities, as well as building capacity for disease surveillance, infection control and biosafety, is critical so that these countries do not constitute the weak links in the ongoing fight against infectious diseases. acknowledgements the authors are grateful to and salute the courage of the late dr ameyo stella adadevoh (first consultant hospital). her courage in restraining the index patient and promptly contacting the authorities led to the prompt diagnosis of the virus and initiated the chain of events that ended the outbreak in nigeria with very few casualties. we are also grateful to the management and staff of first consultant hospital, obalende, lagos, lagos state ministry of health, nigerian centre for disease control / federal ministry of health and all those that assisted to curtail the spread of the ebola virus in nigeria. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. sources of support our laboratory is a national reference laboratory for viral haemorraghic fevers and a world health organization’s regional office for africa reference laboratory for evd. this project was supported by the federal ministry of health and the world health organization’s regional office for africa. authors’ contributions s.a.o. was the laboratory director and team lead. o.b.s., a.b.j. and b.o.o. were responsible for experimental and project design, analysis of data and writing the manuscript. m.r.o., r.a.a. and m.a.a. performed most of the experimental analysis. c.h. made conceptual contributions, performed some experimental analysis and assisted in preparing the manuscript. j.i., i.a.a-s., a-s.n., f.t.o. and o.t. all made conceptual contributions and assisted in preparing the manuscript. references negredo a, palacios g, vázquez-morón s, et al. discovery of an ebolavirus-like filovirus in europe. plospathog. 2011;7(10):e1002304. formenty p, hatz c, le guenno b, et al. human infection due to ebola virus, subtype côte d’ivoire: clinical and biologic presentation. j infect dis. 1999;179(suppl 1):s48–s53. tambo e, ugwu ec, ngoyang jy. need of surveillance response systems to combat ebola outbreaks and other emerging infectious diseases in african countries. infect dis poverty. 2014;3:29. world health organization. ebola virus disease, west africa – update [page on the internet] c2014 [cited 2014 may 8]. available from: http://www.who.int/csr/don/2014_05_08_ebola/en/ volchkov ve, volchkova va, chepurnov aa, et al. characterization of the l-gene and 5’trailer region of ebola virus. j gen virol. 1999;80(2):355–362. feldmann h, geisbert tw. ebola haemorrhagic fever. lancet. 2011;377(9768):849–862. world health organization. ebola virus disease, west africa – update [page on the internet] c2014 [cited 2014 jul 19]. available from: http://www.who.int/csr/don/2014_07_19_ebola/en/ centers for disease control and prevention. guidance for the selection and use of personal protective equipment (ppe) in healthcare settings [document on the internet]. c2007 [viewed 2014 jul 24]. available from: http://www.cdc.gov/ncidod/dhqp/pdf/ppe/ppeslides6-29-04.pdf [updated url 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viruses. 2014;6(11):4760–4799. drosten c, göttig s, schilling s, et al. rapid detection and quantification of rna of ebola and marburg viruses, lassa virus, crimean-congo hemorrhagic fever virus, rift valley fever virus, dengue virus, and yellow fever virus by real-time reverse transcription-pcr. j clin microbiol. 2002;40(7):2323–2330. günther s, feldmann h, geisbert tw, et al. management of accidental exposure to ebola virus in the biosafety level 4 laboratory, hamburg, germany. j infect dis. 2011;204(suppl 3);s785–s790. anonymous. russian scientist dies after ebola lab accident. science. 2004;304(5675):1225. emond r, evans b, bowen e, et al. a case of ebola virus infection. br med j. 1977;2(6086);541–544. kortepeter mg, martin jw, rusnak jm, et al. managing potential laboratory exposure to ebola virus by using a patient biocontainment care unit. emerg infect dis. 2008;14(6):881–887. esri. arcgis 10.1 simplifies sharing of geographic information: new tools and infrastructure extend the reach of gis throughout organizations (press release on the internet). c2015 [viewed 2012 jun 15]. available from: http://www.esri.com/news/releases/12-2qtr/arcgis-101-simplifies-sharing-of-geographic-information.html abstract introduction methods results discussion acknowledgements references about the author(s) ameerah davids department of pathology, division of chemical pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa department of pathology, division of chemical pathology, national health laboratory service, cape town, south africa annalise e. zemlin department of pathology, division of chemical pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa department of pathology, division of chemical pathology, national health laboratory service, cape town, south africa elsie c. kruger department of pathology, division of chemical pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa department of pathology, division of chemical pathology, national health laboratory service, cape town, south africa citation davids a, zemlin ae, kruger ec. challenges with communication of critical laboratory results in a resource-limited setting in south africa. afr j lab med. 2024;13(1), a2457. https://doi.org/10.4102/ajlm.v13i1.2457 original research challenges with communication of critical laboratory results in a resource-limited setting in south africa ameerah davids, annalise e. zemlin, elsie c. kruger received: 21 mar. 2024; accepted: 30 july 2024; published: 23 sept. 2024 copyright: © 2024. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: critical laboratory results are test results suggesting a patient is in immediate danger unless treatment is administered promptly. there is a paucity of studies in sub-saharan africa on clinicians’ utility of these results and affected patients’ outcomes. in our resource-limited setting in south africa, we rely on telephonic communication to convey critical results. objective: the aim of this study was to determine the average time for clinicians to acknowledge these results on the laboratory information system and to determine the outcome of affected patients. methods: a retrospective descriptive audit at tygerberg academic hospital was conducted between 01 october 2021 and 31 march 2022. critical results and the time of acknowledgement by clinicians on the laboratory information system were obtained from inpatients and outpatients. one hundred and twenty inpatient critical results were randomly selected for a folder review to determine patient outcome. results: overall, 2514 critical results were reported, and 63 results were excluded. the remaining 2451 results were obtained from 1346 patients. the majority (94.5%) of results were obtained from inpatients, where 1681 (68.6%) were acknowledged within 24 h. the folder audit of 120 patients determined that 40 (33.3%) patients demised. in 82 (68.3%) patients, communication of a critical result did not alter clinical management. conclusion: critical laboratory results are crucial to patient care. this study found that approximately one-third of critical laboratory results were not used within 24 h. engaging clinicians in current practice and implementing a means of improved communication of critical results is required. what this study adds: the study adds to the evidence of challenges experienced with communicating critical results to clinicians which could impact patient care. this is especially true in resource-limited settings; clinicians need to be made aware of the importance of these results, and communication modes need to be improved. keywords: laboratory critical results; patient outcome; patient safety; post-post analytical; communication introduction patient management is highly dependent on laboratory data, where already in 1996, forsman claimed that approximately 70% of clinical decisions regarding diagnosis, treatment or prevention are based on laboratory results.1 therefore, exploring the bridge between laboratory and clinical medicine and its impact on patient outcomes is crucial. a critical laboratory result was first described by lundberg in 1972, and is defined as a laboratory test result that suggests a patient is in imminent danger unless appropriate intervention is given timeously.2 critical result reporting is a parameter required for laboratory accreditation, where the international organization for standardization (iso 15189:2022) states that a clinician or another authorised staff member must promptly be notified of critical results.3 the ‘brain-to-brain loop’, introduced by lundberg in 1981, describes the nine processes involved in generating a laboratory result.4,5 these nine steps include test ordering, collection, identification, transportation, separation, analysis, reporting, interpretation, and action. the post-post analytical phase of laboratory testing consists of receiving the result, interpreting the test and, lastly, using the information for patient management by the clinician.5,6 laboratory testing plays a vital role in patient care, and lundberg later proposed a 10th step to the brain-to-brain loop – namely, patient outcome.6 critical result reporting is one aspect of the post-analytical phase that may impact patient outcomes. globally, critical values are often institute specific and determined by expert committees that include laboratory staff and clinicians.7,8 in south africa, the national health laboratory service (nhls) is the largest provider of clinical laboratory services and is the primary laboratory service used for public healthcare facilities.9 within the nhls, the chemical pathology expert committee determines the critical chemistry result values, which are nationally harmonised and regularly reviewed. patient safety is a crucial criterion used by regulatory bodies, and in 2009 the world health organization world alliance for patient safety listed communication of critical test results as a potential solution.10 despite the importance of these results regarding patient safety, issues related to their reporting and management are concerning. this includes the measures for evaluating and monitoring the outcomes of critical results which occur outside of the laboratory’s control.11 there is currently a paucity of data on the impact of critical laboratory results in the south african setting, where most public hospitals have limited information technology infrastructure. at tygerberg academic hospital (tah), clinicians complete physical request forms for laboratory testing and patient results are accessed via a web-based laboratory information system (lis). although critical results are not automatically communicated on the web system, a short message service (sms) can be sent to clinicians’ cell phones to alert them of critical results. unfortunately, this often does not occur due to certain limitations. clinicians must register to access this system using their unique medical registration numbers which can in turn be used to track activity across the platform. incomplete laboratory request forms lacking a registration number have no contact details assigned via lis, preventing an sms from being sent. additionally, no registered contact number of the requesting clinician may be listed. therefore, the tah chemistry laboratory relies on telephonic communication with wards to notify clinicians of critical results, which is challenging. the aim of this study was to determine the average time for critical results to be acknowledged by clinicians on trakcare® and to review the outcome of patients with these results. due to the critical nature of these results, rapid acknowledgement within approximately 4 h was expected. methods ethical considerations this study was approved by the stellenbosch university ethics review board of the faculty of medicine and health sciences (reference number: s22/07/139) and was performed in accordance with the declaration of helsinki. a waiver of informed consent was obtained for retrospective data collection. patient data were anonymised to maintain confidentiality and were stored on password-protected devices. only the investigators of the study had access to the data. study site this retrospective audit was conducted at the chemical pathology laboratory of the nhls at tah, cape town, south africa. the nhls laboratory delivers a 24-h service to tah, public secondary hospitals and primary healthcare facilities and receives approximately 120 000 chemistry samples monthly. tygerberg academic hospital comprises 1384 beds across various disciplines, including adult and paediatric intensive care, high-care and general care units, and offers outpatient services. additionally, it serves as the teaching hospital for the faculty of medicine and health sciences, stellenbosch university. critical results are communicated telephonically to both inpatient and outpatient departments from the nhls laboratory by qualified pathologists, registrars, and medical technologists. data collection all chemistry critical results for inpatients and outpatients at tah between 01 october 2021 and 31 march 2022 were obtained from the nhls central data warehouse. the data were extracted from the lis (intersystems trakcare® lab enterprise, cambridge, massachusetts, united states) into microsoft office excel (microsoft corporation, redmond, washington, united states). the data obtained included patient age, sex, requesting ward, critical laboratory result, critical result reporting time (time the critical result was communicated), time of clinician acknowledgement, and the requesting clinician’s registration status on trakcare®. the results with an automatic sms sent were obtained from the lis, which automatically records whether this was successfully sent to the clinician, as well as time to acknowledgement obtained as described above. in addition, turn-around times were calculated and captured. turn-around time in the laboratory is defined as the time from sample registration to result verification, in line with standard practice. results viewed within 24 h were used for statistical analysis as we expected outpatient results to have a prolonged time, being communicated during business hours only (8:00 – 16:00). thus, results viewed after 24 h were excluded from statistical analyses. tygerberg academic hospital chemistry laboratory has no quality indicators for the time taken for clinicians to acknowledge critical results on trakcare®. folder review for patient outcome the critical inpatient results were randomised and 120 of these were selected for a folder review. clinical information was obtained from patient records, which were accessed using tah’s enterprise content manager system. the records were used to extract data regarding patient diagnosis, the use of point-of-care testing devices, whether patient management was changed once critical results were acknowledged, and patient outcomes. critical results acknowledged after 24 h were excluded from the folder review. data analysis after extraction, the data were analysed using excel® and statistical package for social sciences version 27 (ibm corp., armonk, new york, united states), in consultation with the biostatistics division at the university of stellenbosch. a mann-whitney u test was performed to evaluate whether there was a difference in the time taken to acknowledge a critical result between telephonic communication compared to sms. a p-value < 0.05 was considered statistically significant. critical results acknowledged on the trakcare® within 24 h of being communicated were used to calculate the median time to view results (in minutes). results all critical results during the study period there were 2514 critical results. sixty-three results were excluded from the analysis; 13 results were spurious, and 50 results were registered while the laboratory system was offline, and clinicians were unable to access the lis to view results. the remaining 2451 critical results were obtained from 1346 patients (figure 1). figure 1: critical results obtained over 6 months at tygerberg academic hospital, south africa, october 2021 to march 2022. the sex was equally distributed amongst the study population: 680 female patients (50.5%) and 664 male patients (49.3%). table 1 depicts the age distribution of the patients, where 69.7% of results were obtained from patients aged 18 years and older. table 1: demographic of patients with critical results at tygerberg academic hospital, south africa, october 2021 to march 2022. most critical results (n = 2316, 94.5%) were from inpatients. in 69 (2.8%) samples, the patient’s location was not recorded on the laboratory request form. these results had the longest inpatient laboratory turn-around time (262 min) but did not have the longest time to clinician confirmation compared to the other wards. of the 2451 included critical results, 68.6% (1681/2451) were viewed within 24 h; the median time to acknowledgement ranged from 80 to 198 min across the different wards. the paediatric intensive care unit and high-care unit had the lowest median time for reviewing critical results (table 2). the time taken from samples being registered in our laboratory to the result being communicated was up to 4 h in certain inpatient wards (figure 2). figure 2: time distribution from requesting wards at tygerberg academic hospital, south africa, october 2021 to march 2022. table 2: critical results from requesting wards at tygerberg academic hospital, south africa, october 2021 to march 2022. during the study period, 565 samples (23.1%) had a registered clinician. however, only 256 (10.4%) of the samples had an automatic sms sent, and the remainder (n = 2195) were communicated telephonically. there was no significant difference between sms communication compared to a telephone call with regard to the time taken to acknowledge on trakcare® (p = 0.454). folder review results one hundred and twenty (120) electronic patient records were evaluated. there was an equal distribution between the sexes: 59 (49.2%) women and 61 (50.8%) men. two-thirds of patients (n = 80) were above 18 years of age. in 68.3% of the patients, no new management occurred after critical results were communicated by the laboratory (table 3). forty (33.3%) patients demised, where 31 (77.5%) were above the age of 50 years and 7 (17.5%) were under the age of 1 year. derangements in potassium were found in half of the patients who demised; 14 patients (35.0%) had critical hyperkalaemia and 6 (15.0%) a critical hypokalaemia. twelve patients (30%) had sodium derangements, where 9 patients (22.5%) had a critical hypernatraemia and 3 patients (7.5%) had a critical hyponatraemia. of note, during the folder audit, 65 (54.2%) clinical decisions were based on point-of-care testing results. point-of-care devices utilised included blood gas analysers (n = 52, 80%), glucometers (n = 5, 7.69%), and total cutaneous bilirubin meters (n = 8, 12.3%) (table 3). table 3: randomised folder audit of patients with critical results at tygerberg academic hospital, south africa, october 2021 to march 2022. discussion in this study of time to clinician acknowledgement of critical laboratory results, we found that only two-thirds of these results were acknowledged within 24 h. thus, the efficacy of telephonic communication, the method by which critical laboratory results are typically communicated at tah, is unclear. despite trakcare® sending an automatic sms for critical results, we found there was no significant difference in the time to acknowledge these results compared to telephonic communication. further investigation into the reasons for clinicians not being registered may improve this method of communication in our setting. directly informing the primary treating on-call clinician of a critical result may be a more effective method; however, this may be challenging if the requesting clinician is not on duty and unavailable to accept the result.7 this is particularly challenging in the outpatient setting, where important results can only be communicated during business hours. further engagement with these stakeholders may offer additional insight into improving communication of these results. the time for clinician acknowledgement within 24 h ranged between 80 and 198 min across the different wards. a study in boston, united states, reviewed 37 503 critical results reported from their core laboratory over a year and found that the average time to convey a critical result to the clinician or patient location was 22 min.12 this study found that factors associated with delayed reporting times included testing performed in outpatient departments (where staff are only available during office hours), as well as incomplete test request forms that lack the patient location or treating clinician details.12 a previous study at tah laboratory assessing incomplete laboratory request forms and their impact on critical results had similar findings to our study.13 in this study, 2550 paper-based request forms were analysed; 61.2% had no clinician contact details, 4.9% had incomplete ward information, and a critical result was not communicated in 19.9% of cases.13 the present study found that 2.8% of critical results had no ward allocation, with these results having the longest inpatient laboratory turn-around time to communication. however, it did not have the longest time to clinician acknowledgement compared to the other wards. this may be attributed to the laboratory staff contacting the hospital’s central directory to determine the patient’s location and the on-call clinician’s contact details, which allows direct communication with the clinician. at tah, critical results are often not relayed directly to the treating clinician. a previous audit of critical result reporting at tah found that treating clinicians received 12.7% of critical results and the remainder were given to ward staff such as nurses or clerks.14 a study by howantiz et al. found that only 12.2% of critical results were received by clinicians, and they further reported that only 20.8% of nursing supervisors thought critical result lists were helpful, compared to 94.9% of clinicians.15 this gap in the knowledge of the impact of critical results may contribute to the delay in clinicians acknowledging these results. it also serves as a possible avenue for educational intervention in this regard. a retrospective study by kuperman et al. reviewed the time for inpatients to be treated for critical results and found that the median time to intervention was 2.5 h.16 they reported that an important reason for the delay in intervention was that the primary clinician did not receive the information promptly.16 in our study, we found that only 23.1% of samples had a registered clinician and our laboratory often relies on the ward to communicate a result. tygerberg academic hospital laboratory does not have direct access to the clinician on call and obtaining this information from the hospital’s central directory is time consuming. schapkaitz et al. carried out a survey investigating critical value policies for haematology tests in private intensive care units in south africa; 42.9% preferred the requesting clinician be contacted with the result and 28.6% stated that the on-call doctor should be contacted.17 directly informing the primary treating or on-call clinician of a critical result may be a more effective method; however, this may be challenging if the requesting clinician is not on duty and is unavailable to accept the result.7 alarmingly, a third of the patients in our folder audit demised. thirty-five per cent had a critical hyperkalaemia result. a similar finding was reported by kuo et al. in 2021 in a study conducted in taiwan, where critically high potassium results were reviewed and a mortality of 34% was found.18 additionally, we found that 30% of patients who demised had a critical sodium result, with hypernatraemia being more prominent (22.5%). a study in the united states in 2007 by howanitz et al. evaluated critical sodium values in serum and whole blood over 6 months and found a mortality rate of 48% in those with critical hypernatraemia and 19% in those with critical hyponatraemia.19 the high mortality reported and reflected in these studies demonstrates the link between critical results and poor patient outcomes. this highlights the importance of regular auditing and improvement of critical results reporting from the laboratory. clinician engagement and education may play a significant role in improving outcomes by ensuring that timeous management is initiated based on critical results. despite the high mortality in our study, we found that communication of critical laboratory results did not alter the management in over two-thirds of patients. this may be due to clinicians initiating empiric management while awaiting laboratory results, as we found the average time from samples being registered to being called from our laboratory ranged from 71 to 363 min. of note, 54.2% of patients had a confirmatory point-of-care test performed where the clinician had already instituted management for the affected critical result. these devices are helpful in the clinical setting as they provide rapid availability of results. however, they carry limitations which clinicians may not be aware of. these include insufficient training of personnel utilising these instruments, a lack of quality control testing, and failure to partake in external quality control programmes.20,21 in our setting, point-of-care testing devices do not fall under the management of the central laboratory and results do not transmit to the lis, thus the results of these tests were not added to our audit. although the international federation of clinical chemistry and laboratory medicine has established quality indicators for critical result reporting,22 our laboratory does not have these indicators in place. recommendations a survey will be sent to clinicians in the hospital to gain insight into the current practice of critical result reporting and the results will be used to develop a standard operating procedure and quality indicators. additionally, a presentation on critical results will be provided for hospital staff, which will aim to educate them on their importance. after these interventions, a re-audit will be performed to determine their impact. limitations patient data, collected from reviewing electronic patient records, may not have been accurately completed during the patient admission. the assumption was made that laboratory request forms, which were not manually checked during this study, were correctly captured onto the lis. the patient’s location was based on the data captured on the lis and may not reflect patient relocation to a different level of care before the critical result was available. although different information systems are available for result notification, south africa is a developing country with limited access to this type of infrastructure in the public healthcare setting. the lack of formal documentation for the time auxiliary staff communicated critical laboratory results to clinicians made it challenging to accurately reflect the clinician’s response time once alerted. point-of-care testing is not under the management of our central laboratory and the results of these tests are not communicated to the lis and have not been included in this audit. lastly, downtime of trakcare® may have contributed to a delay in the time clinicians took to review results. conclusion to our knowledge, this is one of the few studies from africa reviewing the use of critical values and the impact on patient care. our study demonstrates the significance of timeous critical review verification on patient management. we will utilise our study findings to develop quality indicators for critical result reporting. the high mortality we reported, which is echoed in other studies, shows the need for collaboration between the laboratory and clinicians, and highlights the importance of establishing and maintaining this relationship to improve patient outcomes. acknowledgements the authors thank tonya esterhuizen, biostatistics unit, faculty of medicine and health sciences, stellenbosch university for the statistical analysis. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions a.d., e.c.k. and a.e.z. conceptualised the study. a.d. collected and analysed the study data and wrote the first draft of the article e.c.k. and a.e.z. reviewed study findings and assisted with editing of the manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the data that support the findings of this study are not publicly available. however, the data can be made available upon reasonable request from the corresponding author, a.d. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. it does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the authors are responsible for this article’s results, findings, and content. references forsman rw. why is the laboratory an afterthought for managed care organizations? clin 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[homepage on the internet]. 2024 [cited 2024 aug 08]. available from: https://nationalgovernment.co.za/units/view/251/national-health-laboratory-service-nhls campbell ca, horvath ar. harmonization of critical result management in laboratory medicine. clin chim acta. 2014;432:135–147. https://doi.org/10.1016/j.cca.2013.11.004 plebani m. harmonization in laboratory medicine: the complete picture. clin chem lab med. 2013;51(4):741–751. https://doi.org/10.1515/cclm-2013-0075 dighe as, rao a, coakley ab, lewandrowski kb. analysis of laboratory critical value reporting at a large academic medical center. am j clin pathol. 2006;125(5):758–764. https://doi.org/10.1309/r53xvc2u5ch6tng8 nutt l, zemlin ae, erasmus rt. incomplete laboratory request forms: the extent and impact on critical results at a tertiary hospital in south africa. ann clin biochem. 2008;45(5):463–466. https://doi.org/10.1258/acb.2008.007252 rensburg ma, nutt l, zemlin ae, erasmus rt. an audit on the reporting of critical results in a tertiary institute. ann clin biochem. 2009;46(2):162–164. https://doi.org/10.1258/acb.2008.008182 howanitz pj, steindel sj, heard nv. laboratory critical values policies and procedures: a college of american pathologists q-probes study in 623 institutions. arch pathol lab med. 2002;126(6):663–669. https://doi.org/10.5858/2002-126-0663-lcvpap kuperman gj, boyle d, jha a, et al. how promptly are inpatients treated for critical laboratory results? j am med inform assoc. 1998;5(1):112–119. https://doi.org/10.1136/jamia.1998.0050112 schapkaitz e, levy b. critical limits for urgent clinician notification at south african intensive care units. int j lab hematol. 2015;37(5):620–625. https://doi.org/10.1111/ijlh.12373 kuo wh, you hl, huang wt, et al. incidence, characteristics and outcomes among inpatient, outpatient and emergency department with reported high critical serum potassium values. clin chem lab med. 2021;59(7):1231–1237. https://doi.org/10.1515/cclm-2020-1476 howanitz jh, howanitz pj. evaluation of serum and whole blood sodium critical values. am j clin pathol. 2007;127(1):56–59. https://doi.org/10.1309/q3y27qqlel19340a plebani m, lippi g. point of care testing: evolving scenarios and innovative perspectives. clin chem lab med. 2014;52(3):309–311. https://doi.org/10.1515/cclm-2013-0654 plebani m. does poct reduce the risk of error in laboratory testing? clin chim acta. 2009;404(1):59–64. https://doi.org/10.1016/j.cca.2009.03.014 sciacovelli l, lippi g, sumarac z, et al. quality indicators in laboratory medicine: the status of the progress of ifcc working group ‘laboratory errors and patient safety’ project. clin chem lab med. 2017;55(3):348–357. https://doi.org/10.1515/cclm-2016-0929 references about the author(s) amy piatek bureau for global health, united states agency for international development, washington, dc, united states citation piatek a. tuberculosis diagnostic networks: moving beyond the laboratory to end tuberculosis in africa. afr j lab med. 2017;6(2), a608. https://doi.org/10.4102/ajlm.v6i2.608 editorial tuberculosis diagnostic networks: moving beyond the laboratory to end tuberculosis in africa amy piatek copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. tuberculosis is a curable disease when diagnosed early and treated with an appropriate drug regimen, yet 750 000 africans die unnecessarily every year from tuberculosis. we know that when a person with tuberculosis is treated in africa, 81% will be cured – and even those infected with hiv are cured most of the time. tragically, less than half of all africans with tuberculosis are diagnosed and started on treatment. this vastly low tuberculosis case detection contributes to unacceptably high rates of death from tuberculosis in africa. deaths attributed to tuberculosis, among both hiv-negative and hiv-positive people in africa, are higher than any region in the world – more than twice the global rate among hiv-negative people, and five times higher among people with hiv. the need for more accurate, faster technologies to diagnose tuberculosis in sub-saharan africa is more urgent than ever before. in 2015, 2.7 million africans fell ill with tuberculosis (30% of whom were infected with hiv); however, only 48% sought and received a diagnosis and were reported to national tuberculosis programmes. consequently, over 1.4 million people were undiagnosed and likely transmitted tuberculosis to their families and communities for weeks and months, until their probable death. of these undiagnosed cases, more than 80 000 have multi-drug resistant tuberculosis. although undiagnosed tuberculosis is a small proportion of the overall missing cases, increasing prevalence of multi-drug resistant tuberculosis will eventually diminish africa’s current successes toward stopping the tuberculosis epidemic.1 even if all africans with presumptive tuberculosis or multidrug-resistant tuberculosis accessed a diagnostic facility, the predominant testing method using smear microscopy will accurately detect tuberculosis in 20% to 80% of tuberculosis cases, depending on the report and method used.2 since smear microscopy can only detect tuberculosis in specimens with sufficient bacillary load and does not identify resistance to anti-tuberculosis drugs, the method is of very limited utility for diagnosing hiv-associated tuberculosis3 and is of no use in diagnosing a person with drug-resistant tuberculosis. other methods to diagnose active tuberculosis and drug-resistant tuberculosis are available, including culture-based tools for diagnosis and drug susceptibility testing, nucleic acid amplification technologies and sequencing methods, lateral flow-based rapid diagnostic tests, and detection of volatile organic compounds. the success of a tuberculosis testing method not only relies on the test’s diagnostic accuracy but on whether or not it is placed in the context of a comprehensive and functional tuberculosis diagnostic network. even if african national tuberculosis programmes have access to the full menu of new and old testing methods, significant improvements in tuberculosis case detection will not happen if underlying systems that make up the diagnostic network are absent. a comprehensive and high-quality tuberculosis diagnostic network is one where tests are accessible, accurate, adaptable, and rapid: accessibility: the network is based on a patient-centred approach that consists of the appropriate diagnostic tests (based on disease epidemiology) that are available where people seek care (e.g., all levels of the system in the public or private sector). accuracy: the network provides quality-assured results that give clinicians, laboratorians and the community confidence to diagnose tuberculosis and multi-drug resistant tuberculosis and place a person on appropriate treatment. adaptability: the network provides a strong platform to support all diagnostic tests currently used by a national tuberculosis programme and tests that will be introduced in the future. rapidity: the network consists of tests that produce results quickly (hours instead of days) – or supports a network to reduce the turn-around time from specimen collection to reporting of results to initiation of treatment for longer duration tests. to reach an optimal diagnostic network, immediate investments will be needed in several supportive areas such as specimen transport and referral mechanisms; electronic linkages between diagnostic and clinical facilities; laboratory, programme and clinical staff capacity building; commodity and logistics management; biosafety policies and procedures; and continuous quality improvement schemes. the african region will need to continue to develop and implement supportive policies and initiatives for the development of tuberculosis diagnostic networks and systems strengthening. one such initiative is the global laboratory initiative for africa (gli africa), established in 2014 to support countries to achieve quality assured, accessible and sustainable tuberculosis laboratory services in the african region. gli africa assists countries through its network of national tuberculosis programmes and laboratories, multilateral agencies, development and technical partners, public health institutions and other key stakeholders to improve tuberculosis laboratory and diagnostic services through several areas of support including: restructuring the laboratory network to a patient-centred, coordinated diagnostic network; narrowing the gap between policy and implementation; framing continuous quality improvement within the diagnostic network; establishing the diagnostic-clinical interface; implementing biosafety and biosecurity in the network and building bioengineering capacity; and building capacity and mechanisms for responsive technical assistance. this issue of the african journal of laboratory medicine explores how countries are optimising current diagnostics, introducing and scaling up modern technologies, and thinking through how to improve the many health system factors that are preventing the successful uptake and use of many of the available diagnostics. while africa and the entire global community continues to need developers interested in creating the next best diagnostic, countries must adapt and improve current systems to maximise the use of existing technologies. no african should die of tuberculosis because they cannot access a fast and reliable test that leads them to life-saving treatment. references world health organization. global tuberculosis report 2016 [who/htm/tb/2016.13]. geneva, switzerland: who; 2016. steingart kr, ramsay a, pai m. optimizing sputum smear microscopy for the diagnosis of pulmonary tuberculosis. expert rev anti infect ther. 2007;5(3):327–331. https://doi.org/10.1586/14787210.5.3.327 barnes pf, bloch ab, davidson pt, et al. tuberculosis in patients with human immunodeficiency virus infection. n engl j med. 1991;324(23):1644–1650. https://doi.org/10.1056/nejm199106063242307 abstract introduction research method and design results discussion limitations acknowledgements references about the author(s) kerusha govender department of virology, inkosi albert luthuli central hospital, national health laboratory service, durban, kwazulu-natal, south africa department of virology, school of laboratory medicine and medical sciences, university of kwazulu-natal, south africa raveen parboosing department of virology, inkosi albert luthuli central hospital, national health laboratory service, durban, kwazulu-natal, south africa department of virology, school of laboratory medicine and medical sciences, university of kwazulu-natal, south africa ntombizandile siyaca department of virology, inkosi albert luthuli central hospital, national health laboratory service, durban, kwazulu-natal, south africa pravikrishnen moodley department of virology, inkosi albert luthuli central hospital, national health laboratory service, durban, kwazulu-natal, south africa department of virology, school of laboratory medicine and medical sciences, university of kwazulu-natal, south africa citation govender k, parboosing r, siyaca n, moodley p. dried blood spot specimen quality and validation of a new pre-analytical processing method for qualitative hiv-1 pcr, kwazulu-natal, south africa. afr j lab med. 2016;5(1), art. #349, 6 pages. http://dx.doi.org/10.4102/ajlm.v5i1.349 original research dried blood spot specimen quality and validation of a new pre-analytical processing method for qualitative hiv-1 pcr, kwazulu-natal, south africa kerusha govender, raveen parboosing, ntombizandile siyaca, pravikrishnen moodley received: 03 aug. 2015; accepted: 12 dec. 2015; published: 25 feb. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: poor quality dried blood spot (dbs) specimens are usually rejected by virology laboratories, affecting early infant diagnosis of hiv. the practice of combining two incompletely-filled dbs in one specimen preparation tube during pre-analytical specimen processing (i.e., the two-spot method) has been implemented to reduce the number of specimens being rejected for insufficient volume. objectives: this study analysed laboratory data to describe the quality of dbs specimens and the use of the two-spot method over a one-year period, then validated the two-spot method against the standard (one-spot) method. methods: data on hiv-1 pcr test requests submitted in 2014 to the department of virology at inkosi albert luthuli central hospital in kwazulu-natal province, south africa were analysed to describe reasons for specimen rejection, as well as results of the two-spot method. the accuracy, lower limit of detection and precision of the two-spot method were assessed. results: of the 88 481 specimens received, 3.7% were rejected for pre-analytical problems. of those, 48.9% were rejected as a result of insufficient specimen volume. two health facilities had significantly more specimen rejections than other facilities. the two-spot method prevented 10 504 specimen rejections. the pearson correlation coefficient comparing the standard to the two-spot method was 0.997. conclusions: the two-spot method was comparable with the standard method of pre-analytical specimen processing. two health facilities were identified for targeted retraining on specimen quality. the two-spot method of dbs specimen processing can be used as an adjunct to retraining, to reduce the number of specimens rejected and improve linkage to care. introduction early diagnosis and treatment of hiv-positive infants significantly reduce hiv disease progression and mortality. early infant diagnosis of hiv-positive infants is a critical step in the timely initiation of antiretroviral therapy (art).1,2 despite these substantial benefits, achieving early infant diagnosis is challenging in resource-limited settings. problems in the pre-analytical stages of specimen collection and processing lead to poor quality specimens and laboratory rejections.3 a meta-analysis of studies between 2001 and 2012 showed that 33.9% of hiv-exposed infants in sub-saharan africa are lost to follow up by three months of age.4 early infant diagnosis in south africa occurs within a prevention of mother-to-child transmission continuum of care. qualitative hiv-1 dna and rna pcr testing is done on dried blood spot (dbs) specimens collected from hiv-exposed infants between six weeks and 18 months of age, using the cobas® ampliprep/cobas® taqman® (cap/ctm) hiv-1 qualitative test (roche® molecular systems, inc., branchburg, new jersey, united states). the current south african national laboratory guideline allows for testing of one completely-filled dbs per assay.5 there have been significant successes in the prevention of mother-to-child transmission programme. however, challenges have been identified in south africa. only 70.4% of hiv-exposed south african infants were tested for hiv by two months of age in 2011, reflecting a lack of implementation of policies in the field.6 the department of virology (dov) in kwazulu-natal province in south africa provides early infant diagnosis services for the province. laboratory data from kwazulu-natal show a reduction in mother-to-child transmission of hiv which reflects national trends.7 however, there is a challenge of identifying and linking every hiv-exposed infant to care, which contributes to a significant number of hiv-positive infants not commencing art in a timely manner.8 laboratory specimen rejections are an essential part of a laboratory quality management system, allowing the technologist to optimise accuracy of results. however, this practice has also been shown to adversely impact patient care as it can lead to abandonment of the test request.9,10 specimen rejection may play a role in the failure to link hiv-exposed infants to care. the challenges with dbs specimens in our setting have not been described. in the standard method of pre-analytical specimen processing, one complete dbs is inserted into one specimen preparation tube. at the dov, the current practice for dbs with blood spots that are smaller than the recommended size is to use two incomplete dbs and combine them in one specimen preparation tube during pre-analytical specimen processing (i.e., the two-spot method). specimen preparation is followed by pcr testing for hiv-1 using the cap/ctm analyser. the two-spot method is based on the hypothesis that the quality of these specimens may be adversely affected by the small volume of blood they contain. by maximising the amount of blood eluted from each specimen, the correct volume for the pcr can be obtained and a good quality laboratory result can be produced. neither the two-spot method nor the impact of this method in reducing laboratory rejections have been described in the scientific literature. the objectives of this study were to analyse dov laboratory data to describe trends in dbs specimen quality, particularly relating to specimens with insufficient volume. in addition, we sought to describe the use of the two-spot method in routine hiv pcr diagnosis. finally, we performed laboratory experiments to validate the two-spot method. research method and design ethical considerations the university of kwazulu-natal biomedical research ethics committee (brec) approved this research under the approval classes bca-143/09 and bca 256/010. setting the study was conducted in the dov at inkosi albert luthuli central hospital in kwazulu-natal province, south africa. the dov is an iso 15189:2007 accredited laboratory within an academic quaternary-level hospital. the dov does early infant diagnostic testing of dbs using hiv-1 pcr on the cap/ctm analyser for all hiv-exposed infants in public health facilities in kwazulu-natal province. during the study period from january 2014 to december 2014, the south african antiretroviral treatment guidelines11 recommended that all hiv-exposed infants have an hiv-1 pcr test at age six weeks, at six weeks post-cessation of breastfeeding and at any age less than 18 months that the child may be symptomatic (after age 18 months, serology was used for hiv diagnosis). paediatric guidelines recommend testing infants at the six-week immunisation visit and, for those who test positive, referral for confirmation of hiv infection and initiation of art. infants testing negative should be followed up and retested at age 18 months and/or after cessation of breastfeeding.11,12 laboratory database search we searched our laboratory database for reports on routine hiv-1 pcr test requests that were rejected during 2014. the database is managed with trakcare lab software (intersystems corporation, cambridge, massachusetts, united states) and the data were downloaded and stored in microsoft excel 2010 files (microsoft corporation, redmond, washington, united states). the data extracted included specimen numbers, reasons for specimen rejections and patient demographics, including the health facility where the dbs was collected (each facility was assigned a two-letter code). we also extracted the results for all hiv-1 cap/ctm pcr routine diagnostic tests conducted during 2014. laboratory experiment to validate the two-spot method specimen preparation residual edta blood specimens from previous successful, routine diagnostic hiv-1 pcr tests were selected and anonymised using a number allocated for this experiment. specimens with insufficient volume and specimens that were collected more than one day prior to testing were excluded. blood samples from 20 hiv-negative and 20 hiv-positive specimens were selected for the validation study.13 blood was pipetted onto pre-labelled ss906 filter paper cards by filling one circle completely (so that it contained 70 µl of blood), then filling two circles incompletely (so that one contained 30 µl and the other 40 µl of blood). examples of sufficient and insufficient specimens are shown in figure 1. the original specimen tubes were discarded as per routine biohazard waste disposal procedures. the blood spots were allowed to air-dry at room temperature. the sizes of the dbs were measured using calipers and recorded, then manually cut for hiv-1 pcr testing. figure 1: example filter paper card with sufficient and insufficient blood spots. validation experiment the complete (70 µl) dbs were processed using the standard (one-spot) method: each blood spot was inserted into a specimen preparation tube with lysis buffer, which had 1000 µl of specimen pre-extraction reagent (30 mm sodium citrate dehydrate, 42.5% guanidine thiocyanate, 4% polydocanol and 2% dithiothreitol) added to each tube. the tube was then subjected to thermomixing in a thermomixer comfort (eppendorf, hamburg, germany) at 56 °c for 10 minutes at 1000 rpm in order to elute the dbs. the two incomplete (30 µl and 40 µl) dbs were then combined in a single specimen preparation tube and processed using the above method. for both sample types, the dbs were pushed to one side when inserted into the specimen preparation tube, as per the standard procedure. both types of dbs were then tested in parallel over a period of five days for hiv-1 nucleic acids using the cap/ctm as per manufacturer’s instructions. briefly, the specimen preparation tubes were transferred to specimen racks, then loaded into the cap/ctm analyser, which is an automated closed system with combined extraction, real-time pcr amplification and detection allowing for reduced contamination. known low-positive and negative controls were included with every batch. the analyser software automatically determined the validity of the run, then determined the presence of hiv-1 nucleic acids based on the ct value, namely, the pcr cycle at which the signal indicated amplification. an hiv standard (world health organization 3rd hiv-1 international standard national institute for biological standards and control code: 10/152) was used to perform a dilution series and establish a lower limit of detection for the hiv-1 pcr test when using the two-spot method. replicates of the hiv standard were tested to establish precision.13 statistical methods statistical analyses were performed using ibm spss statistics for windows, version 22.0 (ibm corp., armonk, new york, united states). for the analysis of the laboratory database search, the frequency of each reason for specimen rejection was determined and for each health facility, a percentage of tests rejected as a result of pre-analytical problems was determined out of the total number of hiv-1 pcr tests requested. the hiv-1 pcr results of specimens processed pre-analytically using the standard method were then compared with specimens processed using the two-spot method. the chi-squared test was used to compare categorical variables. a p-value of < 0.05 was regarded as statistically significant. for the analysis of the laboratory experiment to validate the two-spot method, measures of sensitivity and specificity were used to describe the accuracy of the method. a scatter plot of ct values was used to compare the two methods. precision was determined by calculating the standard deviation and coefficient of variance of replicates. a probit regression analysis was used to determine the lower limit of detection of the assay. results laboratory database search the analysis of database records for 2014 revealed that 88 481 specimens were sent to dov for hiv-1 pcr testing, of which 3.8% were rejected or had no results. ninety-one (0.1%) test requests had no results as a result of analytical problems and 3276 (3.7%) were rejected because of pre-analytical problems (table 1). table 1: dried blood spot specimens received in 2014 at the department of virology laboratory at inkosi albert luthuli central hospital, kwazulu-natal province, south africa. insufficient specimen volume comprised 48.9% of the total number of rejections resulting from pre-analytical problems (table 2). other reasons for rejections included: inadequate information supplied on the test request form; incorrect clinical indication for the test; and poor specimen quality, such as incorrect specimen type, specimens that were too old for analysis and specimen cards that were expired. table 2: dried blood spots rejected because of pre-analytical problems in 2014 at the department of virology laboratory at inkosi albert luthuli central hospital, kwazulu-natal province, south africa. the data on the percentage of requests that were rejected per health facility were normally distributed (figure 2). the mean of the percentages of tests rejected for all health facilities in kwazulu-natal was 3.6%. two facilities (eh and gf, names blinded for publication purposes) were more than two standard deviations away from the mean, with rejection rates of 9.7% for eh and 7.6% for gf. figure 2: percentage of hiv-1 pcr requests rejected because of pre-analytical factors by health facilities in kwazulu-natal province, south africa, in 2014. each health facility is represented by a two-letter code. the analysis included data from 88 481 dried blood spot specimens. the horizontal line at 3.57% indicates the mean percentage of rejected requests for all facilities. the horizontal lines at 7.43% and -0.29% indicate two standard deviations from the mean. for all hiv-1 pcr test results in 2014 using the cap/ctm assay, there was an overall indeterminate rate of 1.7% (table 3). there were 10 307 valid positive and negative test results using the two-spot method. the two-spot method had an indeterminate rate of 1.9%. table 3: results of specimens tested by standard method and two-spot method in 2014 at the department of virology laboratory at inkosi albert luthuli central hospital, kwazulu-natal province, south africa. validation of the two-spot method the average diameter of the 30 µl dbs was 8.99 mm (95% confidence interval: 8.89–9.12 mm) and for the 40 µl dbs was 10.08 mm (95% confidence interval: 9.95–10.21 mm). a comparison of the qualitative results of patient specimens showed 100% agreement in accuracy between the two methods. the comparison of the ct values for the standard method versus the two-spot method showed that the pearson correlation coefficient was 0.997 (figure 3). in the precision study, 19 replicates of a specimen with a known concentration yielded a coefficient of variance of 0.0078. the probit regression analysis of the dilution series showed that 95% of replicates were detected at 3.6 log iu/ml (95% confidence interval: 2.9–4.925 log iu/ml). figure 3: validation experiment correlations between ct values for the standard (one-spot) and two-spot specimen processing methods. twenty positive and 20 negative specimens were processed by both the two-spot and the standard one-spot methods of pre-analytical specimen processing. the scatterplot shows the ct values obtained during real-time pcr amplification of the positive specimens. an overlaid line of linearity and pearson’s correlation coefficient of 0.997 shows the correlation between the ct values for each of the specimen processing methods. discussion this study is the first to describe the reasons for laboratory rejections of dbs specimens being submitted for hiv-1 pcr testing in kwazulu-natal province. almost 4% of dbs specimens that were submitted to the laboratory were rejected because of pre-analytical problems. our analysis showed that 48.9% of pre-analytical problems resulted from insufficient specimen volume and we identified two health facilities with high specimen rejection rates. insufficient dbs volume has been shown to affect the yield of hiv-1 nucleic acids14 and these specimens are therefore rejected by laboratories. others have also described challenges in the field with specimen collection and processing, as a result of specimen quality factors such as mislabeling or insufficient volume. rates of specimen rejection vary considerably and there is a paucity of data describing the frequency of each specimen quality issue.15,16 data such as specimen quality should be analysed in order to review the performance of hiv-1 pcr testing in the field and to standardise dbs specimen collection methods, storage and testing.14 this study has shown that analysing specimen quality can identify health facilities which require targeted interventions, such as retraining of healthcare workers. the prevention of mother-to-child transmission program in kwazulu-natal has shown significant success, largely because of the implementation of testing pregnant women and starting them on art,7 but problems have been described in the follow-up of mothers and infants.8,17 it has been shown that delaying hiv diagnosis in hiv-exposed infants results in a failure to link infants to hiv care, significant loss to follow up and mortality.18 at immunisation clinics in kwazulu-natal, nurses perform the specimen collection, whereas in hospital wards and clinics, some specimens are submitted by attending doctors. it has been shown that intense training and mentoring of nurses significantly improves the uptake of infant hiv testing and the overall quality of care.19 however, this needs to be expanded and implemented on a much larger scale.20,21 the need to expand nurse training will increase with the planned implementation of hiv-1 dna pcr testing of exposed neonates at birth.22 as this is being newly implemented, healthcare workers in labour wards and nurseries may not be as experienced with dbs specimen collection as those in immunisation clinics. we have described an innovative two-spot method for pre-analytical specimen processing that can reduce the number of dbs specimen rejections resulting from insufficient specimen volume. this method has been implemented in dov for approximately three years and is used as an adjunct to ongoing telephonic and electronic communication and training of nurses in the public healthcare sector. personal communications with other laboratories indicate that the two-spot method is being used in other hiv-1 pcr testing laboratories in south africa. however, to our knowledge the two-spot method has not yet been described in the scientific literature. the specimens processed using the two-spot method in 2014 yielded over 10 000 additional results from specimens that would otherwise have been rejected. furthermore, the proportion of specimens processed using the two-spot method with indeterminate results was comparable with that of all specimens processed in 2014. the validation study found that the two-spot method correlated well with the standard method and had good precision. the lower limit of detection of the hiv-1 pcr test when using the two-spot method was 3.6 log iu/ml, which is comparable to the 2.7 log iu/ml stated on the package insert for the standard method. future studies comparing the detection limits between the two methods may be required. limitations a major limitation with respect to analytical sensitivity is that the test is being used in infants within a prevention of mother to child transmission programme, most of whom have been exposed to art. the presence of these drugs has been stated as a limitation by the manufacturer on the hiv-1 pcr package insert. therefore, field evaluation of detection limits of the test may be required in a significantly art-exposed patient population such as south africa.23 another limitation of our study is that the volumes of 30 µl and 40 µl used in the validation study were selected in an attempt to simulate the real-world situation. however, a range of volumes could be present in dbs specimens in the field, for example, 20 µl and 50 µl. a third limitation is that the proportion of positive results using the two-spot method was significantly lower compared with the standard one-spot method. we speculate that this difference may be because of inconsistent selection of specimens for two-spot processing. practical application of the two-spot method would require an objective assessment of the dbs specimen to reduce the subjectivity of specimen processing in the laboratory. mean circle diameters for 30 µl and 40 µl spots can be provided to laboratory assistants on a template in order to demonstrate acceptable specimen volumes and aid the selection of patient specimens which are suitable for the two-spot method. we are in the process of implementing the use of such a template based on the findings of this study. a follow-up database analysis will be required to assess whether this intervention is effective. laboratories that plan to use or are currently using the two-spot method should consider this cautionary measure. conclusion in conclusion, we describe real-world problems with dbs specimen quality, which may have major implications for the management of hiv-infected infants in a resource-limited setting. the laboratory database can be used to monitor specimen quality and identify specific health facilities for interventions such as retraining of healthcare workers. as an adjunct to training, the two-spot method of specimen processing may be used to help reduce the number of specimens rejected. this method can potentially be applied to other hiv-exposed infant populations in resource-limited settings to reduce the need for patients having to repeat specimen collection and improve linkage to care. acknowledgements the authors are grateful to the patients of kwazulu-natal whose data were used for this study and to the laboratory staff of dov who conduct the hiv-1 pcr testing for the infants in the province. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. sources of support this research was supported by the department of virology, national health laboratory service, kwazulu-natal, south africa. authors’ contributions k.g. (national health laboratory service and university of kwazulu-natal) was the project leader. k.g. and p.m. (national health laboratory service and university of kwazulu-natal) were responsible for the project design; k.g. and n.s. (national health laboratory service) performed the laboratory work; and k.g. and r.p. 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korf division of chemical pathology, faculty of health sciences, stellenbosch university, cape town, south africa division of chemical pathology, national health laboratory service, tygerberg hospital, cape town, south africa division of chemical pathology, faculty of health sciences, university of cape town, cape town, south africa division of chemical pathology, national health laboratory service, groote schuur hospital, cape town, south africa jody rusch division of chemical pathology, faculty of health sciences, university of cape town, cape town, south africa division of chemical pathology, national health laboratory service, groote schuur hospital, cape town, south africa aye aye khine division of chemical pathology, national health laboratory service, green point laboratory, cape town, south africa nalene strauss division of chemical pathology, national health laboratory service, green point laboratory, cape town, south africa lourens jacobsz division of chemical pathology, national health laboratory service, green point laboratory, cape town, south africa annalise e. zemlin division of chemical pathology, faculty of health sciences, stellenbosch university, cape town, south africa division of chemical pathology, national health laboratory service, tygerberg hospital, cape town, south africa helena vreede division of chemical pathology, faculty of health sciences, university of cape town, cape town, south africa division of chemical pathology, national health laboratory service, groote schuur hospital, cape town, south africa citation korf m, rusch j, khine aa, et al. mitigating the risk of tube shortages: a blood collection tube validation study conducted in south africa. afr j lab med. 2025;14(1), a2628. https://doi.org/10.4102/ajlm.v14i1.2628 note: additional supporting information is available for download alongside the online version of this article as online supplementary document 1. original research mitigating the risk of tube shortages: a blood collection tube validation study conducted in south africa marizna korf, jody rusch, aye aye khine, nalene strauss, lourens jacobsz, annalise e. zemlin, helena vreede received: 23 sept. 2024; accepted: 19 dec. 2024; published: 23 apr. 2025 copyright: © 2025. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the national health laboratory service was using becton dickinson (bd) blood drawing tubes and, in 2021, the supplier notified customers of supply challenges, indicating a risk of global shortages. objective: this study aimed to validate candidate blood collection tubes from four brands (vacucare, vacuette®, vacutest®, and v-tube™) compared to bd vacutainer® tubes in three national health laboratory service laboratories in cape town, south africa. methods: blood was collected from 300 healthy volunteers between october 2021 and november 2021. the technical validation assessed 11 quality indicators, with a sigma metric greater than 4 deemed acceptable. usability feedback was gathered from phlebotomists. the clinical validation estimated differences in results across 52 clinical chemistry tests, using desirable bias specified by the european federation of clinical chemistry and laboratory medicine biological variation database, or ricos, as acceptance criteria. analysis was performed on roche cobas® 6000 and diasorin liaison® xl analysers. results: all vacucare tubes exhibited sigma metrics above 4, indicating excellent performance. vacuette® and v-tube™ were not uncapped by all roche pre-analytical systems. vacutest® caps had rigid rubber, making it more challenging to puncture and detach the tube, which resulted in needle displacement. both vacucare and v-tube™ were reported as user-friendly. all candidate tube analytes showed acceptable clinical performance. conclusion: vacucare, vacuette®, vacutest® and v-tube™ are viable alternatives to bd vacutainer®. however, based on the results obtained from the technical validation, vacucare was identified as the most suitable interim replacement for bd vacutainer® during the shortage. what this study adds: this study addresses a gap in the literature on tube validation and provides valuable insights for clinical laboratories considering a replacement. it also presents an alternative approach to technical validation by utilising sigma metrics. keywords: blood specimen collections; pre-analytical phase; risk management; validation study; south africa. introduction blood collection tube technology has advanced, with the use of complex materials and additives designed to enhance convenience and improve turnaround time. these tubes are an important, and often under-recognised, pre-analytical consideration in clinical laboratories, since these devices are used to analyse patient samples on complex state-of-the-art equipment. the tube type, its additives, and analyte stability under various storage conditions, can potentially contribute to significant error. results of laboratory tests are critical to patient care, and precision and accuracy are essential. manufacturers and clinical laboratories should, therefore, validate the technical and clinical performance of new blood collection products.1,2 proper tube validation is crucial to prevent the financial and human consequences related to inaccurate testing. these include unnecessary testing, delayed results, and compromised patient care. several factors, including supply chain disruptions driven by transportation delays, constrained supplies, and increased costs, as well as the increased demand during the coronavirus disease 2019 pandemic, imposed unprecedented challenges on the entire healthcare industry. as a result, becton, dickinson and company (bd, uk limited, berkshire, united kingdom), a prominent manufacturer of blood collection tubes, notified customers of supply challenges with the risk of global product shortages in 2021.3 following this notice, the national health laboratory service of south africa, which is mandated by government to provide clinical laboratory services to over 80% of the population, sent a notice to healthcare workers with guidance on the efficient use of blood collection tubes. clinicians and phlebotomists were urged to refrain from collecting spare tubes, since a properly filled blood collection tube will be sufficient for all tests requested, in most cases. clinicians were reminded to first review patients’ results before ordering more tests, to avoid unnecessary testing and re-testing, and add-on requests were encouraged. additionally, legible completion of request forms and good phlebotomy technique were advised to prevent unnecessary rejections. although the current blood tube shortage has been resolved, these measures should continue to be practised, promoting environmental stewardship and sustainable laboratory practices. to mitigate tube shortage risk, the research team conducted a comprehensive blood collection tube validation study. this was considered a validation study in accordance with the international organization for standardization 9000:2005 specifications, which define validation as the ‘confirmation, through the provision of objective evidence … that the requirements … for a specific intended use or application have been fulfilled’.4 additionally, the terminology aligns with that used by the european federation of clinical chemistry and laboratory medicine (eflm) working group for pre-analytical phase in their opinion paper on validating blood collection tubes in clinical laboratories.5 while the validation protocol followed eflm working group for pre-analytical phase guidance, the study’s unique circumstances involved a global tube shortage. unlike the usual transitions to new tubes following the awarding of a new tender, the urgent aim was to identify and validate alternative options to mitigate potential supply risks for the national health laboratory service. blood collection tubes from four brands (vacucare, vacuette®, vacutest®, and v-tube™ [table 1]) were validated to identify the closest match to the bd tubes’ performance, with the intention of returning to bd tubes once supply issues were resolved. table 1: manufacturer details and lot numbers of blood collection tubes used during the study at all three testing sites in cape town, south africa, october 2021 to november 2021. methods ethical considerations the study received full approval from the health research ethics committee of stellenbosch university (reference no.: n21/09/095), cape town, south africa, on 12 november 2021. the study was conducted in compliance with the 2013 world medical association declaration of helsinki. written informed consent was obtained from all volunteers. each participant was assigned a unique identifier number during blood collection, which was used for labelling the tubes, registering the samples on the laboratory information system, and during data analysis. only the principal investigator held the key to reidentify participants, allowing the research team to share their blood results with them. participants a total of 300 healthy male and female adult volunteers participated in this blood collection tube validation study. the study was conducted across three laboratories in cape town, south africa, from october 2021 to november 2021 (figure 1).6 investigators sent recruitment invitations to division heads, who then forwarded them to national health laboratory service staff and healthcare workers. additional participants, including friends and relatives of staff members, joined the study through word-of-mouth awareness. the number of 300 volunteers was selected to ensure an adequate number of sample pairs for the clinical validation and to limit the number of tubes collected per participant to a maximum of five tubes each for candidate and comparative tubes (figure 1). exclusion criteria: anaemia or receiving treatment for anaemia or iron deficiency, autoimmune diseases, hereditary haematological disorders, bleeding disorders or anticoagulation treatment, heart conditions with low cardiac output, confirmed or possible pregnancy, and being unwell at the time of the study. the exclusion criteria were included in the recruitment invitations, allowing volunteers to self-screen and only enrol if they met the eligibility requirements. figure 1: this flow diagram illustrates the participation of volunteers across three study sites in cape town, south africa, from october 2021 to november 2021. the blocks highlighted in grey indicate tubes collected by the same phlebotomy team. only tubes marked with† or‡ were included in the reported study. the other tubes were used for haematology6 and therapeutic drug monitoring validations, which are not reported in the current study. study design the design of the study was based on the 2016 opinion paper by the eflm working group for pre-analytical phase and the clinical and laboratory standards institute gp34a guideline.1,5 technical and clinical validations were conducted. ‘comparative tubes’ refer to the tubes currently used in the laboratory (bd), while ‘candidate tubes’ are those under evaluation as alternatives. details of comparative and candidate tube manufacturers are summarised in table 1. the v-tube™ glucose tube was excluded as it contained ethylenediaminetetraacetic acid, which was incompatible with certain glucose methods used in the national health laboratory service. lithium heparin tubes were only included in the technical validation, as troponin t was the only test performed on lithium heparin plasma, and its results were expected to fall below the measuring range within the healthy study population. sample collection venous blood samples were collected by experienced phlebotomists using a blood collection device (evacuated tube holder) and 21-gauge needle (or 22-gauge butterfly needle) following clinical and laboratory standards institute gp41 guidelines.7 the same team performed phlebotomy for most of the study, in close proximity to the laboratories (figure 1). beckton dickinson devices were used for both comparative and candidate tubes. phlebotomy procedures were standardised bilaterally. comparative tubes were filled from one arm, and candidate tubes from the other, with consistent sides but alternating order. blood was drawn first from the arm that the phlebotomist assessed as having more challenging venous access, based on their evaluation of vein characteristics such as size, depth, or visibility. this approach helped determine the appropriate device and the number of tubes that could be collected. tube combinations varied across sites, adhering to the specified draw order. samples were processed per manufacturers’ instructions and tested immediately or stored under conditions that ensured analyte stability. all analyses were completed within 3 days of collection. technical validation pathology registrars and experienced laboratory technologists assessed 11 quality indicators (qis) during various study stages (figure 2). during the pre-check, pack and blood collection tube details, including lot number, expiry date, draw volume, and fill line presence were reviewed. the ‘filling distance’, measured as the distance between the top of the fill line and the bottom of the tube, was used to identify underfilling, defined as less than 90% of the filling distance. each tube pack was assigned a reference number recorded during phlebotomy. figure 2: process flow of technical validation of blood collection tubes with quality indicators checked at each stage – cape town, south africa, october 2021 to november 2021. while the clinical and laboratory standards institute gp34a guideline recommends visual haemolysis assessment, this method is unreliable.8,9,10 haemolysis was instead measured automatically using roche analysers (roche diagnostics gmbh, mannheim, germany), which determine the haemolysis index through absorbance measurements of diluted samples at 570 nm and 600 nm.11 to improve accuracy, a quantitative haemolysis index of 30 mg/dl or lower was considered acceptable. this threshold was chosen because several routine chemistry analytes, including potassium, lactate dehydrogenase, and conjugated bilirubin, begin to be affected around this level.12,13,14 tubes showing underfilling or in vitro haemolysis during the technical validation were excluded if phlebotomy difficulties were indicated on the phlebotomy checklist. after qi evaluation, the compatibility of all candidate tubes with the pre-analytical systems at the testing sites, namely the modular pre-analytics system (roche diagnostics gmbh, mannheim, germany) and probenverteiltechnik cobas® p 612 pre-analytical system (roche diagnostics gmbh, mannheim, germany), was assessed. phlebotomists provided feedback on their user experience. the global tube shortage limited the study from including the recommended 120 candidate and comparative tubes for the technical validation.5 clinical validation the clinical validation aimed to determine whether the tube introduced bias, increasing total error and potentially having a clinically significant effect on patient results. the clinical and laboratory standards institute ep09 guideline recommends 100 sample pairs for validation and 40 for verification.15 however, because of the tube shortage and other resource constraints, results from routine tests conducted at both groote schuur hospital and tygerberg hospital were combined, yielding a total of 50 sample pairs. additionally, 25 sample pairs from selected routine tests and specialised tests conducted only at a single study site were included. the choice of analytes was limited by the healthy study population recruited. analytes expected to have values below the measuring range in healthy participants, such as direct bilirubin, c-reactive protein, free prostate specific antigen, growth hormone, progesterone, and oestradiol, were excluded. in addition, lactate dehydrogenase and aspartate aminotransferase are affected by minor degrees of haemolysis and were also excluded. thirty analytes were measured on cobas® c501 (roche diagnostics gmbh, mannheim, germany) analysers: sodium, potassium, chloride, urea, creatinine, glucose, haemoglobin a1c, calcium, magnesium, phosphate, uric acid, total protein, albumin, total bilirubin, alanine transaminase, alkaline phosphatase, gamma glutamyltransferase, creatine kinase, lipase, total cholesterol, triglycerides, high-density lipoprotein cholesterol, haptoglobin, complement 3 (c3), complement 4 (c4), immunoglobulin g, immunoglobulin a, immunoglobulin m, iron, and transferrin. serum kappa and lambda free light chains were measured with freelite® assays (the binding site group ltd©, birmingham, united kingdom) on cobas® c501. sixteen analytes were measured on cobas® e601 (roche diagnostics gmbh, mannheim, germany) analysers: ferritin, vitamin b12, serum folate, prostate specific antigen, thyroid stimulating hormone, free thyroxine, free triiodothyronine, cortisol, follicle stimulating hormone, luteinising hormone, testosterone, sex hormone binding globulin, insulin, prolactin, parathyroid hormone, and 25-hydroxy vitamin d. renin, aldosterone, insulin-like growth factor 1, and c-peptide were measured on the diasorin liaison® xl (diasorin, saluggia, italy) analyser. most analytes were tested on serum, except for parathyroid hormone and renin which were tested on k2edta plasma, glucose tested on nafl/kox plasma, and haemoglobin a1c tested on k2edta whole blood. the analytical coefficient of variation for each analyte was determined from 1 year’s quality control data and have been provided to demonstrate performance, and the potential contribution of coefficient of variation to total error in this study. statistical analysis technical validation data were captured and analysed in microsoft© excel® (microsoft corporation, redmond, washington, united states). the sigma metric was used as a performance metric to assess the technical quality of blood collection tubes, by calculating the defect rate across all 11 qis. each qi per tube was treated as a defect opportunity. total observed defects were converted into defects per million opportunities using an online calculator.16 to adhere to typical performance standards observed in industry processes, a minimum sigma metric of 4 (6210 defects per million opportunities) was deemed to be the acceptable minimum threshold for the candidate tube.16 additionally, the sigma metric of the candidate tube was required to be equal to or greater than the sigma metric of the comparative tube. clinical validation data were analysed using ep evaluatortm (data innovations llc, south burlington, vermont, united states) and excel®. passing-bablok regression was used to assess correlation, with acceptability based on the 95% confidence interval (ci) for the slope including 1 (no proportional difference) and the intercept including 0 (no systematic difference).17 given the anticipated strong correlation between comparisons of the same analyte, the correlation coefficient (r) was likely to be highly influenced by the range of measurement results; r > 0.95 was deemed acceptable.18 bland-altman plots were used to assess differences between the tubes, with normality evaluated via histograms in ep evaluator. for analytes with non-normal distribution, pairwise (walsh) averages of the differences were calculated in a trial version of minitab® (minitab, lcc, state college, pennsylvania, united states) with subsequent determination of the hodges-lehmann point estimator (median) and tukey two-sided cis.15 average percentage bias was deemed acceptable if it was at or below the desirable bias calculated from the eflm biological variation database19 or ricos criteria.20 for analytes tested at two laboratories, bias exceeding the desirable bias was acceptable if the 95% ci included the bias specification.15 analytes that failed both passing-bablok regression and bland-altman analysis underwent total error calculation (% total error = % bias + 1.65*cva), compared against eflm biological variation database or ricos total allowable error. if the % total error exceeded the total allowable error, the clinical significance of the differences rendered the tube unsuitable for the intended purpose. outlier detection used the ‘extreme studentized deviate’ method in ep evaluator.15 results technical validation during the pre-check, no major physical defects were observed in any candidate or comparative tubes. all candidate tubes were ‘conformité européenne’ marked, with expiry dates, lot numbers, and clear fill lines. no tubes were excluded during the pre-check. the primary reasons for exclusion in assessing the remaining technical qis were primarily due to missing data and underfilling caused by difficult phlebotomy. no haemolysed samples were attributed to phlebotomy difficulties (table 2 and table 3). table 2: the number of defects observed and defect opportunities for 11 quality indicators assessed during technical validation – cape town, south africa, october 2021. vacucare tubes achieved sigma metrics of 4 or higher across all comparisons. in contrast, bd vacutainer® ss had sigma metrics below 4 in three of four comparisons (bd vs. vacucare, vacutest®, and v-tube™), mainly due to underfilled tubes in the latter two comparisons. vacuette® tubes also fell below a sigma metric of 4, primarily due to cap leakage after needle withdrawal. for vacutest ss and lithium heparin tubes, haemolysis and underfilling contributed to their sigma metrics falling below 4. the modular pre-analytics system could not uncap vacuette® tubes without hardware modifications, which were unavailable because of the manufacturer’s discontinuation of the modular pre-analytics system. similarly, the probenverteiltechnik cobas® p 612 pre-analytical system could not uncap v-tube™ tubes, and no application settings were available for achieving compatibility. phlebotomists reported usability concerns, noting that the rigid rubber in vacutest® caps made tubes difficult to puncture and detach from collection devices, often causing needle displacement during the procedure. vacuette® tube caps were shorter in length compared to bd vacutainer®, resulting in increased angular movement within the collection device. in contrast, vacucare and v-tube™ tubes were user-friendly. based on sigma metrics, pre-analytical systems compatibility, and usability, vacuette®, vacutest® and v-tube™ were disqualified. clinical validation the clinical validation results are detailed in the online supplementary table 1, online supplementary table 2, online supplementary table 3, and online supplementary table 4. the study aimed to include 50 or 25 sample pairs per comparison; however, the total sample size was occasionally reduced because of missing results or samples. additionally, outliers were identified and excluded using ep evaluator. for v-tube™, 25 of 50 c3 pairs tested at groote schuur hospital were excluded due to a significant positive shift in internal quality control during the run. table 3: the number of defects observed and defect opportunities for 11 quality indicators assessed during technical validation – cape town, south africa, november 2021. most analytes had correlation coefficients above 0.95, except sodium, potassium, and magnesium, which consistently fell below 0.95 across all comparisons. calcium, total protein, and albumin displayed coefficients below 0.95 in most comparisons, except with vacutest®. passing-bablok regression and bland-altman analysis indicated acceptable performance for all analytes except for aldosterone on v-tube™. passing-bablok regression for aldosterone on v-tube™ showed a slope of 0.927 (95% ci: 0.874, 0.961) and intercept of −13.18 (95% ci: −25.170, –6.060). the mean bias was −15.63 (95% ci: −19.70, −11.55), with a total error of 30%, below the eflm biological variation database limit of 42.8%, deeming the difference clinically insignificant. discussion in response to an impending bd vacutainer® tube shortage, technical and clinical validations were conducted to evaluate alternative tubes. a modified approach reduced the number of tubes required while maintaining rigorous standards. a key feature was the application of sigma metrics to assess technical qis, offering an objective measure of their quality. the technical validation proved critical, as all tubes met standards in the clinical validation phase. sigma metrics are often used to monitor qis in the extra-analytical phases, with a six sigma level corresponding to a defect rate of 3.4 defects per million opportunities. while sigma metrics often evaluate the process quality, they can also be used to assess product performance. by treating each qi per tube as a defect opportunity, a sigma metric for each blood collection tube was determined. this approach also addressed the global shortage of blood collection tubes, which prevented inclusion of the full 120-tube sample size recommended in the eflm opinion paper.5 few publications address the technical validation of blood collection tubes. to the authors’ knowledge, this is the first study to compare vacutest™ and bd vacutainer® tubes. during technical validation, vacutest ss failed to meet the minimum sigma metric of 4 due to underfilling and haemolysis but outperformed bd vacutainer® ss tubes overall. notably, underfilled bd vacutainer® ss tubes were traced to a single lot (1151158), identified through reference numbers assigned during the pre-check process. the eflm opinion paper recommends calculating percentage differences between candidate and comparative tubes for each qi, but poorly performing comparative tubes may distort results.5 this was similarly noted in a 2019 turkish study, where 4 candidate tubes and 16 comparative tubes (bd vacutainer ss) were found to be underfilled.21 using sigma metrics with a pre-set acceptance threshold allows independent evaluation of candidate tubes, reducing reliance on comparative tube performance. to the best of the authors’ knowledge, this study provides the first evaluation of vacucare tubes compared to bd vacutainer®. however, a 2009 korean study assessed tubes from zhejiang gongdong medical technology co., ltd., likely using similar manufacturing technology across all their blood collection tubes.22 however, that study did not incorporate a technical evaluation, and it relied on clinical laboratory improvement amendments of 1988 limits instead of biological variation data, finding no significant differences between the tubes. vacuette® and bd vacutainer® serum tubes were compared in a 2011 brazilian study by lima-oliveira et al. using a roche cobas® c501.23 like the current study, clinical validation acceptance criteria were based on desirable bias, derived from biological variation. while the previous study found a clinically significant mean difference of 4.9% for magnesium, the current study demonstrated a mean difference of -0.69% (95% ci: -1.64, 0.25), well below the desirable bias of 1.62%. a 2014 study by bowen et. al. in the united states assessed vacuette® and bd vacutainer® serum tubes against glass tubes lacking clot activators, separator gels, and surfactants that could potentially impact results.24 both vacuette® and bd vacutainer® tubes showed no statistically or clinically significant differences in cortisol results using the siemens immulite™ 1000 analyser. the current study found a mean difference of 1.90% (95% ci: -2.00, 5.90) for cortisol, in a direct comparison between vacuette® and bd vacutainer® serum tubes, further supporting comparable performance. two korean studies (2013, 2018) performed comparisons between bd vacutainer® and v-tube™ tubes for routine analytes in healthy volunteers and patients. one study showed statistically significant differences for sodium, chloride, alkaline phosphatase, high-density lipoprotein cholesterol, whereas the other study showed differences for creatinine, high-density lipoprotein cholesterol, iron, lipase, magnesium, potassium, total cholesterol, and uric acid.25,26 yet, these findings were not deemed clinically significant in the context of clinical laboratory improvement amendments of 1988 limits. in the current study, most analytes showed strong correlation coefficients (r > 0.95), but sodium, potassium, and magnesium consistently fell below this threshold. similarly, calcium, total protein, and albumin displayed correlation coefficients below 0.95 in all comparisons, except for vacutest®. poor correlation does not necessarily indicate poor agreement between tubes, but may reflect the limited range of results in the healthy study population.18,27 while combining blood collection tubes and devices from different manufacturers is discouraged,5 the unique study circumstances required using bd devices for all candidate tubes. sigma metrics for vacuette® tubes consistently fell below 4 because of cap leakage from the puncture sites after needle withdrawal, posing safety concerns. however, this issue did not affect the clinical validation. given these findings, it is advised against combining bd devices with vacuette® tubes. strengths the strength of this validation study lies in its comprehensive assessment of analytes using vacucare, vacuette®, vacutest®, and v-tube™ compared to bd vacutainer® tubes. it is among the few studies reporting on the technical validation of blood collection tubes and introduces a novel assessment approach. furthermore, it demonstrates no clinically significant differences in specialised tests between comparative and candidate tubes, using desirable bias specified by the eflm biological variation database or ricos as acceptance criteria. limitations a key limitation was the inclusion of only healthy adult volunteers, which excluded several analytes due to low levels in this population and resulted in a narrow testing range for several analytes. conclusion the technical validation component, combined with pre-analytical system compatibility considerations, was essential and led to the exclusion of certain candidate tubes. comprehensive technical validation should precede the clinical validation. vacucare, vacuette®, vacutest® and v-tube™ are viable alternatives to bd vacutainer®. however, based on technical validation results, vacucare was identified as the most suitable interim replacement for bd vacutainer® during the shortage. acknowledgements m.k. and j.r. are joint first authors. haematopathology co-investigators: dr jenique bailly, prof. zivanai chapanduka, dr michael linström, dr erica-mari nell, dr diana oelofse, prof. jessica opie. phlebotomists: aqeela abrahams, jehaan brown, nokuzola gemi, rayaan isaacs, marcellino johannes, asiphe ngqandu, boraro sehoshe. assistants: gadija abdullah, shanaaz arendse, morné bezuidenhout, wendy blose, sylvester chabunya, benito dekela, cameron francis, thando gcingca, kulsoem kasker, thobile khanyile, daleen kriel, gail lawrence, mariam mahomed, lwando mampunye, abugyer mosavel, thembi mthembu, igsaan noordien, david richardson, eva rienhardt, kagiso seakamela, bianca southon, ashuelita thompson, dieter van der westhuizen, sigrid vollmer, laetitia walters, owen wiese. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions m.k., j.r., a.a.k., n.s., l.j., a.e.z., and h.v. made substantial contributions to the concept and design of the study. m.k., j.r., a.a.k., n.s., l.j., a.e.z., and h.v. were involved in protocol development. m.k. was involved in obtaining ethical approval. m.k., j.r. and h.v. were involved in data analysis. m.k. and j.r. wrote the first draft of the article. m.k., j.r., a.a.k., n.s., l.j., a.e.z., and h.v. reviewed and edited the article and approved the final version of the article. sources of support the national health laboratory service funded the analysis and supply of bd vacutainer blood collection tubes for the validation. ilex south africa (pty) ltd (gauteng, south africa) supplied v-tube™, lasec (cape town, south africa) supplied vacuette®, reliable diagnostic supplies (pty) ltd (gauteng, south africa) supplied vacutest® and synbiolab (gauteng, south africa) supplied vacucare™ blood collection tubes for the validation. data availability data supporting the findings of this study are available from the corresponding author, m.k., upon request. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. the article does not necessarily reflect 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https://doi.org/10.1016/j.clinbiochem.2013.11.003 won ej, shin m-g, jang m-j, et al. comparison between v-tubes and bd vacutainer tubes for use in laboratory tests. lab med online. 2013;3:145–154. https://doi.org/10.3343/lmo.2013.3.3.145 jo sj, kim ys, lee s-g, et al. evaluation of the vacuum blood collection tube, v-tube, a domestic product for clinical chemistry and thyroid hormone assays. lab med qual assur. 2018;40(4):211–222. https://doi.org/10.15263/jlmqa.2018.40.4.211 bland jm, altman dg. measuring agreement in method comparison studies. stat methods med res 1999;8(2):135–160. https://doi.org/10.1177/096228029900800204 about the author(s) marli van heerden national health laboratory service, johannesburg, south africa faculty of health sciences, charlotte maxeke johannesburg academic hospital, university of the witwatersrand, johannesburg, south africa jaya a. george national health laboratory service, johannesburg, south africa faculty of health sciences, charlotte maxeke johannesburg academic hospital, university of the witwatersrand, johannesburg, south africa siyabonga khoza national health laboratory service, johannesburg, south africa faculty of health sciences, charlotte maxeke johannesburg academic hospital, university of the witwatersrand, johannesburg, south africa citation van heerden m, george ja, khoza s. corrigendum: the application of sigma metrics in the laboratory to assess quality control processes in south africa. afr j lab med. 2023;12(1), a1996. https://doi.org/10.4102/ajlm.v12i1.1996 note: doi of original article published: https://doi.org/10.4102/ajlm.v11i1.1344 correction corrigendum: the application of sigma metrics in the laboratory to assess quality control processes in south africa marli van heerden, jaya a. george, siyabonga khoza published: 15 may 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. in the published article, van heerden m, george ja, khoza s. the application of sigma metrics in the laboratory to assess quality control processes in south africa. afr j lab med. 2022;11(1):a1344. https://doi.org/10.4102/ajlm.v11i1.1344, there was an error. the incorrect sigma metric calculation was stated. the correction has now been made on page 2 in the methods section, under data collection and analysis, paragraph 4 and should read: the original paragraph: sigma metrics were calculated for each analyte on two levels as follows: the revised and updated paragraph: sigma metrics were calculated for each analyte on two levels as follows: the authors apologise for this error. the correction does not change the study’s findings of significance or overall interpretation of the study’s results or the scientific conclusions of the article in any way. abstract introduction methods results discussion acknowledgements references about the author(s) noah obeng-nkrumah department of medical laboratory science, school of biomedical and allied health sciences, university of ghana, accra, ghana gloria d. tawiah-abrokwa department of medical laboratory science, school of biomedical and allied health sciences, university of ghana, accra, ghana enid owusu department of medical laboratory science, school of biomedical and allied health sciences, university of ghana, accra, ghana francisca duah department of medical laboratory science, school of biomedical and allied health sciences, university of ghana, accra, ghana daniel oduro-mensah department of biochemistry, cell and molecular biology, school of biological sciences, university of ghana, accra, ghana paul kwao department of medical laboratory science, school of biomedical and allied health sciences, university of ghana, accra, ghana bako evariste tenkodogo university center, thomas sankara university, ouagadougou, burkina faso appiah-korang labi department of medical microbiology, university of ghana medical school, university of ghana, accra, ghana citation obeng-nkrumah n, tawiah-abrokwa gd, owusu e, et al. role of ctx-m-15 gene in spread of extended-spectrum beta-lactamases among immunocompetent patients in ghana. afr j lab med. 2023;12(1), a2135. https://doi.org/10.4102/ajlm.v12i1.2135 note: additional supporting information may be found in the online version of this article as online supplementary document 1. original research role of ctx-m-15 gene in spread of extended-spectrum beta-lactamases among immunocompetent patients in ghana noah obeng-nkrumah, gloria d. tawiah-abrokwa, enid owusu, francisca duah, daniel oduro-mensah, paul kwao, bako evariste, appiah-korang labi received: 29 nov. 2022; accepted: 11 aug. 2023; published: 20 nov. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: patients with faecal carriage of extended-spectrum beta-lactamases (esbl)-producing enterobacterales serve as reservoirs and sources of dissemination and infection. objective: this report examined immunocompetent patients for faecal carriage of esbl-producing enterobacterales in a district care hospital setting in ghana. methods: between march 2019 and may 2020, cross-sectional sampling was performed to enrol patients and conduct questionnaire-structured interviews for factors that predispose patients to esbl faecal carriage. faecal samples from study patients were quantified for esbl-producing enterobacterales. the esbl genes were characterised by polymerase chain reaction and sequencing. results: the overall proportion of esbl faecal carriage was 35.5% (n = 38/107). the blactx-m gene, mostly ctx-m-15, was detected in 89.5% (n = 34/38) of the esbl-producing isolates. the other esbl types included blashv (n = 3) and blaoxa (n = 1). the ctx-m-15-positive isolates, when present in a faecal sample compared to the non-esbl-ctx-m-15 isolates, constituted the predominant faecal enterobacterales, with significantly higher colony counts than all other enterobacteria in that sample. in multivariate regression, independent risk factors for faecal carriage of esbl-producing enterobacterales were hospitalisation in the past year, infections since admission, use of antibiotics in the past 6 weeks, and admission from another hospital. conclusion: the study found that ctx-m-15-producing isolates were the predominant faecal enterobacterales, and that further investigations are needed to determine the reasons behind this dominance. what this study adds: the ctx-m-15-producing isolates dominance in this study shows the misuse and abuse of antibiotics in an african medical facility and indicates the potential role of immunity in controlling esbl spread, which is to be investigated further. keywords: extended-spectrum beta-lactamases; prevalence; inpatient; faecal carriage; enterobacterales; ghana. introduction the presence of extended-spectrum beta-lactamases (esbl) in enterobacterales remains the chief resistance mechanism against beta-lactam antibiotics. the esbl-producing organisms may be widespread in ghana. about 40% of the escherichia coli and klebsiella pneumoniae isolates causing infection in the nation’s largest tertiary care facility were found to be esblproducing.1 similar prevalence rates have been reported elsewhere across the country. infections with esbl-producing enterobacterales increase the risk of antibiotic treatment failure, morbidity and mortality, length of hospital stay, and cost of hospitalisation.2,3 prior intestinal colonisation with esbl-producing isolates has been identified as a significant risk factor for esbl infections.4,5 these colonised patients remain reservoirs and serve as revolving doors for the spread of resistant pathogens between the community and hospital.4,5 this phenomenon increases the hospital’s potential as a repository of multidrug-resistant pathogens, with consequential increases in hospital-acquired infections and its associated economic burden to the patient, hospital and country.2,3 despite this menace, few studies have reported on the intestinal carriage of esbls in africa,6,7,8,9,10,11,12,13,14 particularly in ghana.15 many advances for reducing the esbl menace are proposed in the literature. one such approach involves reducing the effect of patient characteristics that may predispose them to intestinal colonisation with esbls.16,17 an important factor that predisposes patients to esbl intestinal colonisation is the misuse and abuse of antibiotics.2,18 this is more pronounced in immunosuppressed patients where antibiotics are systematically used as prophylaxis to prevent infections. however, there is a knowledge gap about correlations between immunosuppression, immunocompetence, and esbl intestinal colonisation. most studies on esbl colonisation have looked at all patients together, without separating them into groups based on their immune status.4,6,7,8,9,10,11,12,13,14,15 this current study focuses specifically on the occurrence of intestinal colonisation with esbl-producing enterobacterales among immunocompetent patients, with particular emphasis on the quantification of esbl-producing isolates and genotypes of esbls. risk factors for intestinal colonisation with esbls were also analysed. the overarching goal of the current study is to improve our understanding of esbl faecal colonisation by analysing immunocompetent patients exclusively. the present study is part of a larger explorative study on immunocompetence and esbl intestinal colonisation. by focusing on this specific group, we hope to provide valuable insights into the dynamics of esbl colonisation in a subset of patients who have intact immunity. such knowledge will assist in the development of targeted interventions and strategies to control the spread of esbl-producing bacteria, improve patient outcomes, and optimise antibiotic usage within this specific patient population. methods ethical considerations the study was approved by the ethics and protocol review committee of the school of biomedical and allied health sciences, university of ghana (approval number: sbahs-md./10551508/aa/5a/2016-2017). participation in the study was entirely voluntary, and patients were under no obligation to take part. participating patients provided written informed consent before their enrolment in the study. interviews were done at the respondent’s bedside to ensure privacy. faecal specimens were assigned arbitrary numbers. data were aggregated and randomised with unique identification numbers so that no individual could be linked to the study information. study design and setting this was a cross-sectional study to document the presence of intestinal carriage with esbl-producing enterobacterales. faecal specimens were collected, and a questionnaire-based data collection tool (online supplementary file 1) was used to conduct interviews at the time of sampling to evaluate patient characteristics that predispose patients to intestinal colonisation with esbl-producing isolates. the study was conducted from march 2019 through may 2020 at achimota district hospital, accra, ghana. the hospital is a 100-bed-capacity primary care government facility with no intensive care units. study participants and sampling before the commencement of the study, appropriate permissions were sought from the hospital authorities. on day 1 of the survey, all patients hospitalised for ≥ 2 days at the achimota district hospital were considered potential study participants. an android app called ‘random generator’ (apps n blue, amman, jordan) was used to randomly select hospitalised patients to be invited to participate in the study. we assigned each patient a number and entered them into the application. the application generated a random list of numbers, and we approached patients in the order listed. if a patient declined to participate, we moved on to the next patient on the list. on day 2, the patients previously contacted were requested to join the study. those who agreed to participate were asked to provide informed consent. participant’s medical records were examined with the help of an attending physician to identify patients who were immunocompetent, based on the definitions published by public health, england, on immunisation against infectious diseases.19 patients were considered immunocompetent, in consultations with their attending physician, if they had no major organ or bone marrow transplant, no diagnosis of hiv, no severe combined immunodeficiency, no wiskott-aldrich syndrome, and no record of prescription of an immunosuppressive drug or steroid in the month before the sampling date. data collection tool for study participants, a structured data collection instrument (online supplementary file 1) was administered to collect demographic, clinical, lifestyle and hospitalisation history information. the tool was adapted from similar studies on esbl faecal carriage risk factors reported in the literature.2,16,18 we collected data from multiple sources including medical records, direct interactions with the patients, and discussions with their attending doctor. the collected data included patients’ demographics (e.g., age, gender, employment status, educational level), which were primarily obtained from medical records. we determined the duration of hospital stays until the survey day through direct patient interactions. information regarding the type of hospitalisation (hospitalised directly from home or transferred from another health facility) was gathered from discussions with the attending doctors. the number of patients in the participants’ ward at the time of recruitment was determined by investigators. patient lifestyle characteristics, including the use of hand sanitiser at least once in the past three months, travel outside ghana in the past year, travel outside the home in the past year, and access to pipe-borne water (treated municipal water source) in the household were collected through patient interviews. information regarding toilet facilities in the household and animal contact in the past three months was also obtained through direct interactions with patients. data on patient hospitalisation history, including hospitalisation in the past year, use of medication that affects intestinal flora, use of antibiotics in the past six months, and bacterial infections since admission, were extracted from the patients’ medical records, discussions with attending doctors, and, where applicable, interactions with patients. culture and identification patients were given sterile stool containers and instructed to self-collect and submit at least 1 g of stool sample. participant faecal samples were transported on ice at 0 °c to the microbiology laboratory. at the laboratory, 1 g of each faecal specimen was vortexed in 10 ml of 0.9% sterile saline. ten-fold serial dilutions of each suspension were prepared at 10−1 to 10−4. the serial dilutions were cultured onto statens serum institut enteric age (ssi diagnostica, hillerød, denmark) by mixing 1 ml of each dilution with 24 ml of molten statens serum institut agar (at 52 °c) and incubating aerobically at 35 °c – 37 °c for 18 h – 24 h. the statens serum institut enteric medium combines selective properties with growth differentiation for direct isolation and rapid diagnosis of enterobacterales. quantification was performed by counting growing colonies per enterobacterium morphotype and estimating the number of colony-forming units per gram (cfu/g) of faecal sample. the number of enterobacterales harboured by each faecal specimen in cfu/g was calculated as the number of colonies per morphotype × 101 (for first faecal dilution) × 10(dilution factor). each enterobacterales morphotype was subcultured onto agar for purity. phenotypic determination of extended-spectrum beta-lactamases production each colony morphotype of enterobacterales was examined for esbl by the combined disk synergy assay. this test was performed using cefpodoxime (30 µg), ceftazidime (30 µg), cefotaxime (30 µg) and cefepime antibiotic discs (30 µg), with and without clavulanate (10 µg), on cation balanced mueller-hinton agar. according to the clinical and laboratory standards institute interpretative guideline reference,20 the study isolates that demonstrated clavulanic acid effect defined by an increase in zone diameter greater than 5 mm for at least one test antibiotic were considered esbl producers. klebsiella pneumoniae atcc 700603 (becton dickinson, berkshire, united kingdom) was used as a positive control for esbl production. escherichia coli atcc 25922 (becton dickinson, berkshire, united kingdom) was used as a negative control. only isolates with esbl producer phenotypes were identified to the species level using the biochemical test kit minibact-e diagnostic (ssi diagnostica, hillerød, denmark), according to the manufacturer’s guidelines. multiplex polymerase chain reaction for extended-spectrum beta-lactamases genes bacterial template dna for polymerase chain reaction (pcr) was obtained by the boiling suspension method using distilled water as resuspension buffer.21 for the detection of esbl genes, primers for the sulfhydryl variable active site enzyme (shv), temoniera enzyme (tem), oxacillinase 2 and 10 enzymes (oxa-2, oxa-10), and the cefotaxime-munich (ctx-m) groups 1, 2, and 9 enzymes were used (online supplementary file 2). each pcr mixture was a total of 24.5 µl, constituted as 2 µl template dna, 12.5 µl of 2 × multiplex mastermix (inqaba biotec, pretoria, south africa), 2.5 µl of 10 × reverse or forward primer each, and 5 µl of dnase/rnase free water (inqaba, pretoria, south africa). the pcr conditions15,22 were initial denaturation at 94 °c for 15 min followed by 30 cycles of amplification for tem, shv, oxa-1 and oxa-2 and 27 cycles of amplification for ctx-m group 1, 2, 9 consisting of 30 sec at 94 °c, 1 min at the appropriate annealing temperature for the specific primer (online supplementary file 1) and 1 min at 72 °c for primer extension, followed by 10 min at 72 °c for the final extension. the pcr products were analysed on 2% agarose gel (seakem®gtg®agarose, lonza, basel, switzerland) alongside a generuler 100 base pairs dna ladder plus marker (fermentas, leon-rot, germany). nucleotide sequencing all pcr products of tem, shv, ctx-m-1, ctx-m-2, ctx-m-8 and ctx-m-9 were sequenced to characterise specific genotypes of esbl. the pcr products were first purified using nucleofast 96 pcr plates (macherey-nagel gmbh & co., düren, germany). the purified products, together with additional internal primers for ctx-m-1, ctx-m-9, shv, and tem (online supplementary file 2), were submitted on ice to inqaba biotec (pretoria, south africa) for nucleotide sequencing. the sequenced contigs were analysed using codoncode aligner version 10.0 (codoncode corporation, centerville, massachusetts, united states) and the aligned nucleotide sequences were compared by blast (https://blast.ncbi.nlm.nih.gov/blast.cgi) with sequences in the the national center for biotechnology information database (https://www.ncbi.nlm.nih.gov/). all tem and shv beta-lactamase sequences were compared to wild-type e. coli tem-1 and shv-1 sequences (gen bank accession number af427133.1 and af148850, respectively, at http://www.lahey.org/studies). statistical analysis data were analysed using statistical package for social sciences version 16 (spss inc., chicago, illinois, united states, 2007). comparisons between categorical data were conducted with χ2 or fisher’s exact tests. continuous data were compared using a student’s t-test. point estimates of statistical significance were indicated with two-tailed p < 0.05. univariate analyses were computed with an odds ratio (or) with a 95% confidence interval (ci); variables with p < 0.05 were analysed in multivariate logistic regression models to determine independent associated predictor variables(s). the predictive accuracy of the models was evaluated by hosmer and lemeshow goodness-of-fit test (http://vassarstats.net/) with p < 0.05 suggesting that the model predicts accurately on average. the area under the receiver operating characteristic curve > 0.7 was used to analyse the discriminatory capability of esbl faecal carriage versus their respective controls. results demographics of study patients a total of 107 hospitalised patients with the age range of 13–38 years were enrolled (figure 1). the mean age (with standard deviation) of the participants was 39.4 ± 13.8 (95% ci: 42–37). based on medical records, 42 (39.2%) study patients were men and 65 (60.7%) were women. of the 107 patients, 72 (67.3%) were admitted from home and 35 (32.7%) from another hospital (n = 35, 32.7%). overall, 77 (72.0%) patients were employed, 6 (5.6%) had no formal education, and 64 (59.8%) had travelled outside ghana in the past year. of the 107 patients, 75 (70.1%) had had a previous hospitalisation in the past year, 61 (57.0%) had no pipe-borne water in their household, and 72 (43.0%) had no toilet facilities in their household. all 107 study participants submitted faecal specimens for laboratory investigations. figure 1: design of study of faecal carriage of extended-spectrum beta-lactamases-producing enterobacterales in accra, ghana, march 2019 to may 2020. enterobacterales isolates cultured from faecal samples a total of 676 enterobacterales isolates were found in 107 faecal samples (table 1). out of these, 83 (77.5%) samples contained enterobacterales that were resistant to either the third-generation cephalosporins (cefpodoxime, cefotaxime, ceftazidime) or the fourth-generation cephalosporin (cefepime), or both. these resistant isolates were referred to as third/fourth-generation cephalosporin-resistant isolates and included different species: e. coli (n = 37), k. pneumoniae (n = 12), other klebsiella species (n = 10), citrobacter species (n = 9), proteus species (n = 4), providencia species (n = 4), and morganella species (n = 4). among the third/fourth-generation cephalosporin-resistant isolates, 38 exhibited the esbl producer phenotype, with e. coli (n = 31, 44.5%) and k. pneumoniae (n = 7, 8.4%) being the most common. among these 38 isolates, 37 carried one or more esbl genes. overall, the prevalence of intestinal colonisation with esbl-producing e. coli or k. pneumoniae was 35.5% (n = 38/107). table 1: enterobacterales cultured from faecal samples in accra, ghana, march 2019 to may 2020. extended-spectrum beta-lactamases gene type all isolates with esbl phenotype harboured at least one corresponding esbl gene type, except for one e. coli strain (which harboured a blatem-13 but no identifiable tem, ctx-m, shv or oxa esbl gene). nucleotide sequence analysis of ctx-m, shv, tem and oxa genes are shown in table 2. overall, 23 (54%) isolates harboured only one esbl gene, 8 (25%) had two esbl genes, while 6 (54%) combined an esbl plus a broad-spectrum beta-lactamase gene. eighteen different esbl gene sequences (12 ctx-m types; 3 shv types; 2 tem types; and 1 oxa type) were identified either alone (n = 12) or in various combinations (n = 6). the most predominant esbl gene type was blactx-m-15 (n = 12), followed by blactx-m-2 (n = 4) and blactx-m-14 (n = 4). overall, 34 (91.8%) of the 37 esbl sequenced genes were blactx-m (21.6%, n = 8/37), which were found in various combinations with blashv or blatem or blaoxa esbls. the shv esbls were blashv-40, blashv-86, and blashv-2a; these were identified among only k. pneumoniae isolates. the tem esbls were blatem-3 and blatem-15. the only oxa esbl identified was blaoxa-2. table 2: specific types of extended-spectrum beta-lactamases gene sequences in escherichia coli and klebsiella pneumoniae in accra, ghana, march 2019 to may 2020. faecal load of extended-spectrum beta-lactamases producers the faecal concentration of esbl-positive e. coli and k. pneumoniae varied considerably by the type of esbl gene present (table 3). for example, among patients (n = 11) colonised by a ctx-m-15-positive e. coli and/or k. pneumoniae, the esbl producers comprised > 80.0% of the total enterobacterales counts per gram of the faecal sample (mean ± standard deviation [s.d.]: 190 × 104 ± 27 × 104 cfu/g; interquartile range [iqr]: 175 × 104 – 205 × 104 cfu/g). in contrast, the faecal concentration of esbl-negative enterobacterales was ≤ 50 × 104 cfu/g per patient (for all 11 patients: mean ± s.d.: 38 × 104 ± 6 × 104 cfu/g; iqr: 34 × 104 – 43 × 104 cfu/g). we then compared the faecal load of enterobacterales among the 27 patients with non-ctx-m-15 esbl colonisation. within this cohort, the esbl-negative enterobacterales constituted the predominant (> 80%) faecal isolates (for all 27 patients: mean ± s.d.: 134 × 104 cfu/g; iqr: 46 × 104 – 200 × 104 cfu/g). the mean faecal concentration of non-ctx-m-15 esbl-positive e. coli and/or k. pneumoniae in all 27 patients was 22 × 104 ± 21 × 104 cfu/g with an iqr of 10 × 104 – 24 × 104 cfu/g. the total faecal concentration of the non-ctx-m-15 e. coli and k. pneumoniae comprised 16.4% of the overall faecal concentration of the enterobacterales among the patients with non-ctx-m-15 esbl colonisation. table 3: comparison of the faecal concentration (colony-forming units/g) of extended-spectrum beta-lactamase-positive and all extended-spectrum beta-lactamase-negative enterobacterales bacteria in faecal samples of 38 extended-spectrum beta-lactamase faecal carriers in accra, ghana, march 2019 to may 2020. univariate comparison of patients’ characteristics compared to those admitted from home, patients who were admitted from another hospital had a statistically significant higher likelihood to have faecal carriage with esbl-producing e. coli or klebsiella pneumoniae (or: 3.5; 95% ci: 1.5–8.4; p = 0.003) (table 4). patients with a history of hospitalisation in the preceding year were frequently faecal carriers of esbl producers (or: 3.6; 95% ci: 1.5–8.6; p = 0.003). faecal carriage of esbl producers had a statistically significant association with patients who had acquired an infection since admission (or: 5.2; 95% ci: 2.2–12.3; p < 0.001). lifestyle patterns that had a statistically significant association with faecal carriage of esbl producers included chronic alcohol use (or: 12.7; 95% ci: 4.9–32.8; p < 0.001) and animal contact in the preceding 3 months (or: 3.7; 95% ci: 1.5–8.5; p < 0.002). diarrhoea (or: 0.1; 95% ci: 0.02–0.16; p < 0.001) or use of hand sanitiser in preceding three months (or: 0.4; 95% ci: 0.1–2.0; p < 0.04) indicated a protective effect. table 4: univariate comparison of the risk factors among patients with and without esbl-positive faecal carriage in accra, ghana, march 2019 to may 2020. multivariable logistic regression for independent risk factors use of antibiotics in the previous 6 months was the strongest predictor for faecal carriage of esbl producers (adjusted or: 3.4; 95% ci: 1.5–10.5; p = 0.0001) (table 5). patients with faecal carriage of esbl producers had used antibiotics 3.4 times more during the preceding six months than patients without faecal carriage of esbl producers (table 5). other independent risk factors were the acquisition of infection(s) since admission (adjusted or: 3.2; 95% ci: 1.9–7.8; p = 0.002), hospitalisation in the preceding year (adjusted or: 1.5; 95% ci: 1.2–3.6; p = 0.04), and admission from another hospital (adjusted or: 1.6; 95% ci: 1.1–4.3; p = 0.003). table 5: independent risk factors for faecal carriage with extended-spectrum beta-lactamase-producing enterobacterales among study participants using multivariate logistic regression analysis in accra, ghana, march 2019 to may 2020. discussion in this present study, we examined the occurrence of faecal carriage of esbl-producing enterobacterales recovered from immunocompetent patients in a district hospital setting in ghana. the overall prevalence of faecal carriage by esbl-producing isolates was 35.5%. the ctx-m genes, mostly blactx-m-15, were the predominant esbl genotype. when ctx-m-15-producing isolates occurred in a faecal specimen, these isolates were the predominant faecal enterobacterales. in this study, independent risk factors for esbl faecal carriage included previous hospitalisation for the past year, admission from another hospital, infections since admission, and use of antibiotics during the past six weeks. studies on esbl faecal carriage among hospitalised patients is globally prevalent but there is little data on the role of immunity in this subject matter. available studies on esbl faecal colonisation have not clearly distinguished between immunocompetent and immunosuppressed patients. what is not clear in the literature is whether esbl intestinal colonisation patterns differ in patients with varying immune statuses. although answers to this conundrum are beyond the purview of this study, the findings of this work compared to other studies may offer some preliminary insights. according to a meta-analysis review conducted by bezabih in 2021,23 the combined prevalence of esbl-producing e. coli intestinal carriage in apparently healthy individuals across african communities was estimated to be 21.4% (95% ci: 12.7% – 30.1%). in contrast, our current study found a higher prevalence of 35.5% for all enterobacterales among hospitalised patients. this suggests that the risk of esbl carriage may be greater among hospitalised individuals compared to the general population in community settings. however, while the pooled prevalence provides a useful estimate of esbl carriage across africa, individual studies may reveal variations and provide more localised data because the prevalence of esbl carriage varies widely between different countries, regions, and populations. for example, the esbl faecal carriage prevalence in our current study is significantly lower than the esbl levels reported for other patient hosts in ghana by obeng-nkrumah et al. in 2013 (49.3%; n = 148/300),1 feglo et al. in 2013 (48.4%, n = 77/159),24 feglo and adu-sarkodie in 2016 (57.8%, n = 234/405),25 falgenhauer et al. in 2019 (61%, n = 33/54),26 donkor et al. in 2022 (44.6%, n = 145/334),27 and mahazu et al. in 2022 (62.2%, n = 102/164).28 it is worth noting that variations in study populations can also influence esbl carriage rates. for example, the proportion of esbl faecal carriage reported in our work is lower than that cited for similar populations in comparative reviews6,17,18 spanning several regions across africa, including uganda in 2023 (61%),29 ghana in 2022 (53.1%),30 kenya in 2022 (44%),31 niger in 2022 (92.4%),32 malawi in 2021 (47.2%),33 central african republic in 2016 (59%),10 morocco in 2014 (42.9%),14 and cameroon in 2013 (55.3%).11 in all these studies, the reports were silent on whether the study patients were immunocompetent or immunosuppressed. indeed, there are limited data on esbl faecal carriage versus immunity and the paucity of data hinders the ability to compare our findings. nonetheless, our report provides much-needed data on esbl carriage rates in immunocompetent patients and serves as a valuable foundation for future studies aiming to compare data and explore the impact of immunity on esbl carriage rates. this will help us to better understand the factors that contribute to esbl carriage and to develop strategies for preventing and treating esbl infections. a noteworthy finding in this current study was the fact that whenever ctx-m-15 esbl-positive isolates occurred in a faecal sample, these isolates constituted the predominant faecal bacteria compared to all other enterobacteria. in such instances, the ctx-m-15-producing isolates constituted over 80% of the total faecal enterobacteria. these patients with ctx-m-15 faecal carriage comprise high-density esbl shedders and may be of public health significance in the dissemination of multidrug-resistant bacteria. reports on ctx-m esbls, predominantly ctx-m-15, are increasingly becoming common worldwide. in the past decade, various studies conducted across africa7,17,18 and various global settings34,35 consistently indicate that the ctx-m gene is the most prevalent among esbl genes. overall, ctx-m enzymes represented 91.8% of the esbls in our current study. this observation mirrors the current trend observed across africa. in ghana, a recent study was conducted across different communities to examine faecal samples from 736 healthy community residents in order to assess the occurrence of esbl-producing enterobacterales. the results showed that over 47% of the participants carried esbl producers in their faecal specimens. furthermore, 99.4% (n = 354/356) of the isolates with esbl phenotype carried the blactx-m gene, predominantly ctx-m-15. a possible explanation for the dominance of ctx-m genes could be its ability to localise on large plasmids and co-harbour other resistant genes such as blaampc, quinolone-resistance genes or methylase-affecting aminoglycosides.23 the ctx-m-15-borne plasmids in particular have been reported to have a high conjugation frequency and are thus more frequently disseminated to other enterobacterales species.24,25 the ctx-m genes are transmissible by conjugation with high transfer frequencies of 10−7 to 10−2 per donor cell. perhaps the few tem, shv and oxa esbl types identified in this study point to the fact that ctx-m genes are fast replacing other esbl types.26 overall, our report broaches relevant conversations on the possible role of immunity to limiting the spread of esbl. will a dominant population of colonising non-esbl enterobacterales limit the intestinal occurrence and significance of ctx-m-15? does the immune status of patients play a role in intestinal colonisation by ctx-m-15 and other esbl types? further investigations to ascertain the reasons behind such dominance is warranted. this report is the first of our series to compare esbl carriage between immunocompetent and immunosuppressed patients. in multivariable logistics analysis, this study identified antibiotic use in the past 6 months as the highest predictor of esbl faecal carriage. several other studies have linked prior antibiotic use within the past 4 or 12 months to an increased possibility of faecal esbl colonisation.2,5,15,16,18 in our univariate comparisons, patients with esbl faecal carriage were significantly associated with an acquired infection in the preceding 3 months. the analysis also showed that antibiotic use in the past 3 weeks among patients with esbl faecal carriage was about four-fold higher compared to that of patients without esbl faecal carriage. some studies have even suggested that the more current the antibiotic use, the greater the risk of faecal carriage with an esbl-positive enterobacteria.5,17,18 these observations are not surprising given that esbls and many other antibiotic-resistant mechanisms evolved as a consequence of the misuse and abuse of antibiotics. limitations there are some potential limitations of this study that should be discussed briefly. bacterial clonal relatedness was not investigated in this work. such investigations would have enabled the study to compare various esbl faecal enterobacteria to determine the extent to which these isolates are being disseminated. another noteworthy limitation is the rather few study patients. a more large-scale survey is much more likely to be with little bias for high-at-risk esbl faecal carriers. conclusion the findings of this study suggest that faecal carriage of esbl-producing enterobacterales is a significant public health concern in ghana. the high prevalence of esbl-producing bacteria, the association with antibiotic use, and the identification of ctx-m-15 genes as a potential driver of multidrug-resistant bacteria dissemination in immunocompetent patients are all concerning trends. these findings highlight the need for increased surveillance of esbl-producing bacteria in ghana and other parts of africa. further research is needed to explore the relationship between esbl faecal carriage and immunity, understand the dominance of ctx-m genes, and address the consequences of antibiotic misuse. more research is also needed to better understand the factors that contribute to esbl faecal carriage and to develop effective interventions to prevent the spread of these bacteria. acknowledgements the authors are grateful to all the patients from whom the records were obtained and to the staff of the wards of achimota hospital for their support. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions n.o.-n. and a.-k.l. conceived of the study, participated in the study design, coordination, and collation of laboratory data, performed the statistical analysis, and helped to draft the article. d.o.-m., e.o. and b.e. participated in the study’s design and coordination and helped to draft the article. g.d.t.-a., f.d. and p.k. participated in the study’s design, coordination, data collection, and collation of laboratory data, and helped to draft the article. all authors read and approved the final article. sources of support this research has been supported by the university of ghana – carnegie building a new generation of academics in africa project with funding from the carnegie corporation of new york (grant number: ug-ba/pd-016/2017-2018, principal investigator n.o.-n). data availability the data are available from the corresponding author, a.-k.l., upon reasonable request. disclaimer the views and opinions expressed in 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in niger due to the spread of blacctx-m-15 gene in multiple commensal clones [homepage on the internet]. 2022 [cited 2023 jun 03]. available from: https://papers.ssrn.com/abstract=4099407 onduru og, mkakosya rs, rumisha sf, aboud s. carriage prevalence of extended-spectrum β-lactamase producing enterobacterales in outpatients attending community health centers in blantyre, malawi. trop med infect dis. 2021;6(4):179. https://doi.org/10.3390/tropicalmed6040179 castanheira m, simner pj, bradford pa. extended-spectrum β-lactamases: an update on their characteristics, epidemiology and detection. jac antimicrob resist. 2021;3(3):dlab092. https://doi.org/10.1093/jacamr/dlab092 bevan er, jones am, hawkey pm. global epidemiology of ctx-m β-lactamases: temporal and geographical shifts in genotype. j antimicrob chemother. 2017;72(8):2145–2155. https://doi.org/10.1093/jac/dkx146 reviewer acknowledgementpage 1 of 1 http://www.ajlmonline.org open access the editorial team of the african journal of 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sanchez david jahbiuk sambian willie sang sandra p. santander shahin sayed candice sher-locketz neeraj sidharthan aminder singh vipul singh anthony m. smith aliyah sohani arvinda sooka paul d. stamper a.p. sunjaya paul a. tambyah ajaykumar k. thirumala leopold tientcheu ralph timperi kwasi torpey florette treurnicht obioma uchendu kinglsley n. ukwaja johannes van pelt lara vojnov john n. waitumbi juliana maira watanabe pinhata brooke weckselblatt everlyne wesangula larry westerman toni whistler shannon whitmer katy yao dorothy yeboah-manu li xiaoqin zhang acknowledgement to reviewers http://www.ajlmonline.org https://ajlmonline.org https://ajlmonline.org https://ajlmonline.org/index.php/ajlm/user https://ajlmonline.org/index.php/ajlm/user mailto:publishing@aosis.co.za abstract introduction methods results discussion acknowledgements references about the author(s) nokuthula nhlapo department of chemical pathology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa doreen jacob department of chemical pathology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa department of chemical pathology, national health laboratory service, johannesburg, south africa siyabonga khoza department of chemical pathology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa department of chemical pathology, national health laboratory service, johannesburg, south africa mpho r. maphayi department of chemical pathology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa department of chemical pathology, national health laboratory service, johannesburg, south africa citation nhlapo n, jacob d, khoza s, maphayi mr. comparison of second and third-generation parathyroid hormone assays at a tertiary hospital in south africa. afr j lab med. 2025;14(1), a2700. https://doi.org/10.4102/ajlm.v14i1.2700 note: additional supporting information may be found in the online version of this article as supplementary document 1. original research comparison of second and third-generation parathyroid hormone assays at a tertiary hospital in south africa nokuthula nhlapo, doreen jacob, siyabonga khoza, mpho r. maphayi received: 22 nov. 2024; accepted: 24 june 2025; published: 30 aug. 2025 copyright: © 2025. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: parathyroid hormone (pth) measurement is key for diagnosing parathyroid disorders, and for management of chronic kidney disease. available pth assays include second (intact pth) and third (pth 1–84) generations. data comparing interchangeable use are insufficient. objective: the objective of this study was to compare intact and 1–84 pth assays to determine the difference in analytical performance and impact on clinical interpretation. methods: a method comparison was done on residual samples with pth requests (06 april 2022 – 21 september 2022) from a tertiary hospital in south africa. parathyroid hormone was measured using both intact pth and 1–84 pth assays. clinical performance was compared in the diagnosis of hypoand hyperparathyroidism, and in pre-dialysis and dialysis chronic kidney disease patients. results: among 481 samples, intact pth had a higher median concentration than pth 1–84 (9.85 pmol/l vs. 8.51 pmol/l, p < 0.0001), but the two showed good correlation (r = 0.994, p < 0.0001). regression analysis revealed systematic (intercept = 0.887 pmol/l [95% confidence interval: 0.788 – 1.005]) and proportional differences (slope = 0.713 pmol/l, [95% confidence interval: 0.703 – 0.723]), with increased deviations at higher concentrations. the average bias was 18.5%, exceeding allowable limits. among the 276 patients (170 women, 106 men, age range: 18–89 years) included in the clinical study, interpretation was unchanged. conclusion: a bias was observed between the pth assays, indicating that they should not be used interchangeably. however, no changes in clinical interpretation were observed when one assay was used over the other. what this study adds: the study confirms the recommendation by kidney disease: improving global outcomes for the use of assay-specific upper limit of normal instead of generic cut-off in dialysis patients. this study further highlights the need for standardisation of pth assays. keywords: parathyroid hormone; second-generation pth assays (intact pth); third-generation pth assays (pth 1–84); chronic kidney disease; dialysis; method comparison; bias, estimated glomerular filtration rate; hyperparathyroidism. introduction chronic kidney disease (ckd) is a global health problem with a prevalence of 5% – 10%.1 in africa, ckd presents a major public health concern, particularly against a background of limited resources.2 such patients have an increased risk of ckd-mineral and bone disorder (ckd-mbd), which is associated with high morbidity and mortality.1 bone histomorphometry is the gold standard in the assessment of renal osteodystrophy, which reflects the skeletal facet of ckd-mbd. however, it is invasive, expensive, and requires expertise not readily available, thus measurement of circulating biomarkers, including parathyroid hormone (pth), offers an attractive non-invasive alternative.1,3 changes in pth concentrations contribute to the bone abnormalities observed in ckd-mbd.4 this makes its measurement essential in the management of ckd-mbd. parathyroid hormone regulates calcium and phosphate homeostasis and bone remodelling,4 and determination thereof is crucial in the diagnostic workup of hypocalcaemia, hypercalcaemia, and in the perioperative assessment in thyroid or parathyroid surgery.1,4 therefore, accurate and reliable pth assays are needed for proper patient management. parathyroid hormone is composed of a single polypeptide produced exclusively by the chief cells of parathyroid glands, with a molecular weight of ~9.5 kda.5 it is sequentially cleaved at the amino terminal end to yield the mature biologically active 84 amino acid molecule pth (1–84), which consists of two main parts: the amino (n) terminus, made up of the first 34 amino acids, and the carboxyl (c) terminus, that contains the remaining 50 amino acids. as a result of rapid proteolytic cleavage in circulation, pth exists as intact pth (1–84) and various fragments, that is, n-terminal, mid-region and c-terminal pth, the most abundant being 7–84 pth.6 although the plasma half-life of pth (1–84) is between 2 and 4 min, c-terminal pth fragments have a longer half-life of several hours because of renal clearance.6,7 the latter may account for more than 20% of circulating pth in normal kidney function individuals and up to 45% in patients with renal failure.6 this heterogeneity of pth in circulation has made its measurement challenging. parathyroid hormone measurements have numerous pitfalls. currently, no reference method exists, and although the use of a single recognised international standard, for example, world health organization pth 95/646, has been proposed, it has not been fully implemented, and pth measurement remains unstandardised.8,9 the radioimmunoassay was a first-generation competitive pth assay that utilised a single polyclonal antibody generated against epitopes in the midor c-terminus of the pth peptide.7 the radioimmunoassays had several limitations, including cross reactivity with the biologically inactive c-fragments, which are highest in ckd patients because of decreased renal clearance.10,11 to overcome this issue, the immunometric secondand third-generation pth assays were developed. the second-generation pth assay is a non-competitive sandwich immunoassay that makes use of two antibodies, generated against the mid-region n-terminal and c-terminal amino acids. it was assumed that it detected the biologically active pth (1–84) and avoided cross reactivity with c-fragments, and thus was referred to as the ‘intact’ pth assay.11,12 however, subsequent studies revealed that these assays overestimated values in patients with kidney failure compared to pth values in normal subjects because of the accumulation of n-truncated pth fragments, the most abundant of which is 7–84 pth.13,14 the third-generation pth assays use a capture antibody against the c-terminal amino acids similar to the second-generation. however, the detection antibody is directed against epitopes in the first four amino acids of the n-terminus, which avoids cross reactivity with 7–84 pth fragments.7,11 third-generation assays were termed bio-intact (bio-pth) or whole pth assays, as they were more specific for pth (1–84).12 second-generation assays have provided the basis for the kidney disease: improving global outcomes (kdigo) clinical guidelines on management of ckd-mbd.12,15 significant inter-method variability among second-generation assays has been shown, thus affecting comparability of results.3 taking this into consideration, the recent kdigo guidelines proposed utilisation of multiples of 2–9-fold of the upper limit of normal for a given pth assay, instead of absolute values in ckd patients on dialysis. in patients not on dialysis, the kdigo guidelines state that the target pth concentration is unknown and thus recommend monitoring trends rather than a single value.15 research has shown that third-generation assays produce results that are 50% – 60% lower in ckd patients, and 15% lower in non-ckd patients, when compared to second-generation assays.10,12 third-generation assays are more specific to bioactive pth than second-generation, and should in theory be more accurate in the evaluation of pth status. yet, there is still dispute as to which assay is preferable and whether third-generation assays offer improved clinical value.5 second-generation assays are readily available, extensively researched and form part of current clinical guidelines.12 in contrast, there is insufficient research for the third-generation to be universally adopted in clinical practice.12 in our setting, the national health laboratory service services two major academic hospitals, namely chris hani baragwanath academic hospital and charlotte maxeke johannesburg academic hospital (cmjah), which utilise the secondand third-generation pth assays, respectively, from the same manufacturer with similar reference intervals. there is considerable movement of patients and blood samples between the two hospitals, which may affect pth interpretation and clinical decision-making depending on the assay used. the aim of this study was to compare the analytical performance of secondand third-generation pth assays and their impact on clinical interpretation of hypoand hyperparathyroidism, and in pre-dialysis and dialysis ckd based on current kdigo guidelines. methods ethical considerations this study has been approved by the human research ethics committee (medical) of the university of the witwatersrand and performed in line with the declaration of helsinki. the study made use of remnant patient samples on which routine pth measurement was requested. ethics clearance (clearance certificate number m210475) was obtained for use of remnant samples and informed consent was not required. all patient data obtained were fully anonymised and de-identified before accession. study design this cross-sectional study was conducted between 06 april 2022 and 21 september 2022, in two parts. the first part was a method comparison study conducted on residual patient samples from both inand outpatients on which a pth was requested at cmjah national health laboratory service. these samples were tested on both secondand third-generation pth assays consecutively. the second part assessed clinical interpretation of the assays’ results among a subset of the same group of patients, using data from the laboratory information system. sample size calculations for a method comparison study, a minimum of 40 samples is required to achieve statistical significance as per clinical laboratory standard institute ep09-a3 guideline.16 however, more than 100 are recommended to improve statistical power. all pth samples received during the study period were considered for the study and included if they met the criteria. for clinical interpretation, the equation 117 was used to derive the sample size (n): utilising a prevalence of 6.4% in south africa for ckd; z denotes the z-score of 1.96 that corresponds to the confidence interval (ci) of 95%, p is expected prevalence (6.4%) and d is precision, which reflects the effect size and was estimated to be 5%.17,18 a minimum of 92 samples from ckd patients were required to demonstrate a difference between the two assays. method comparison samples were collected in bd vacutainer® ethylenediamine-tetraacetic acid tubes™ (becton dickinson and company, franklin lakes, new jersey, united states) by clinicians as part of the routine clinical care. samples of sufficient volume and within recommended stability period of 48 h when stored at 2 °c – 8 °c were included. the samples were initially analysed using the third-generation roche th 1–84 assay on the roche cobas® e602 analyser (roche diagnostics, mannheim, germany), the routine assay at cmjah. the second-generation intact pth assay was validated on the same analyser for the study at cmjah laboratory, replicating the assay used at chris hani baragwanath academic hospital. parathyroid hormone is unstable in blood samples; to maintain sample stability, method comparison was performed at the same site. after analysing the samples on the third-generation pth assay, they were immediately re-analysed using the newly validated second-generation roche intact pth assay. laboratory analysis a verification study for the second-generation pth assay was performed according to the clinical laboratory standard institute ep15-a3 guideline.19 this was done because the second-generation pth assay was not validated on the roche cobas® e602 analyser at cmjah. assay performance was within the acceptable limits of the manufacturer’s claims with intra-assay variability of 0.9%, and inter-assay variability of 2.2%. the pth assays’ analytical performance was compared as per the clinical laboratory standard institute ep 09-a3 guideline.16 analyses of samples for pth were performed on an e602 module of a cobas® 8000 system (roche diagnostics, mannheim, germany). the analytical characteristics of the pth assays used are depicted in online supplementary table 1.20,21 biochemical parameters including creatinine, total calcium and phosphate were measured using standard chemistry methods on the roche cobas® 8000 analyser e702 module. table 1: demographic and biochemical data for the patient samples used in clinical interpretation, johannesburg, south africa, 06 april 2022 to 21 september 2022. clinical interpretation the same data set used in the method comparison study was considered for the clinical performance study, and those that met criteria were included. the clinical performance of the two assays was assessed for their performance in the diagnosis of hypoparathyroidism and hyperparathyroidism and in ckd patients, including those on dialysis. data were collected from the national health laboratory service trakcaretm (intersystems, cambridge, massachusetts, united states) laboratory information system during the study period (06 april 2022 and 21 september 2022) and included: age, gender, calcium, phosphate, creatinine, and 25 (oh) vitamin d. the glomerular filtration rate (gfr) was estimated using the chronic kidney disease epidemiology collaboration 2009 equation without the race factor.22 samples within storage stability limits, those with all required biochemical data and patients 18 years old and above were included for the clinical interpretation (online supplementary figure 5b). statistical analysis the data obtained were captured onto microsoft excel® (microsoft, seattle, washington, united states) and analysed using medcalc®, version 200218 (medcalc software, ostend, belgium). the shapiro-wilks test was used to assess for normality. data were demonstrated to be not normally distributed and expressed as median and interquartile range. categorical data were expressed as proportion and percentage. the wilcoxon test was used to compare pth concentrations between the two assays. a mann-whitney test was used to compare the median age between the ckd and non-ckd groups. intact pth was used as the comparative method (x) and pth 1–84 as the test method (y). passing-bablok regression was used to assess method agreement, and the bland-altman difference plot was used to determine the magnitude of bias (observed differences) between the two methods. the observed inter-assay bias was compared with desirable analytical performance specifications (bias ±7.1%) for pth derived from the european federation of clinical chemistry and laboratory medicine biological variation database.23 the mcnemar test was used to determine if there were significant differences in the classification of patients and if the clinical interpretation changes when one assay is used over the other. a p-value of < 0.05 was considered statistically significant. chronic kidney disease patients on dialysis were classified as above, below, or within the pth target range of 2–9 times the upper limit of normal as recommended by the kdigo guidelines, using the two pth assays. a kappa statistic was used to assess the performance of the classification using both assays, with a value of ≥ 0.60 representing acceptable agreement.24 results method comparison a total of 505 residual patient samples were initially identified for the study. however, 24 samples were excluded: 17 had results exceeding the measuring range of the elecsys pth 1–84 assay and seven had exceeded the stability period. thus, 481 samples were analysed and utilised for the method comparison study as shown in online supplementary figure 5a. the median pth concentration for the third-generation assay was lower, at 8.51 pmol/l (interquartile range: 4.30 – 25.5) compared to the second-generation median of 9.85 pmol/l (interquartile range: 4.53 – 33.58), and the difference was statistically significant (p < 0.0001). the regression analysis equation was calculated as y = 0.713x + 0.887 (figure 1). there was good correlation between the two methods, with a coefficient correlation (r) = 0.994 and p < 0.0001. the intercept was calculated at 0.887 pmol/l (95% ci: 0.788 – 1.005) and the slope at 0.713 pmol/l (95% ci: 0.703 – 0.723), indicating that there were systematic and proportional differences between the two methods. figure 1: distribution of results across all measured concentrations comparing intact parathyroid hormone and parathyroid hormone 1–84 assays (y = 0.713x + 0.887; n = 481), johannesburg, south africa, 06 april to 21 september 2022. the bland-altman analysis showed an average overall difference between the pth assays of 9.00 pmol/l (18.5%, p < 0.0001) among all samples analysed, with increased deviations observed at higher pth concentrations (figure 2 and figure 3). the bias in the entire ckd group was 27.2%, and 25.3% in the pre-dialysis ckd group (p < 0.0001). the dialysis group, however, demonstrated the highest bias of 35.2% (p < 0.0001). this is in stark contrast to the difference observed within the non-ckd group with estimated gfr (egfr) > 60 ml/min/1.73 m2, which was demonstrated to be 5.7% (p < 0.0001) (online supplementary figures 1–4). figure 2: absolute differences in parathyroid hormone (pth) results across concentrations levels comparing intact pth and pth 1–84 assays, johannesburg, south africa, 06 april 2022 to 21 september 2022. figure 3: percentage difference in parathyroid hormone (pth) results across concentration levels comparing intact pth and pth 1–84 assays, johannesburg, south africa, 06 april 2022 to 21 september 2022. clinical interpretation the clinical performance of the two assays was assessed in the diagnosis of hypoparathyroidism and hyperparathyroidism and in ckd patients, including those on dialysis. the same 505 residual samples considered for the method comparison study were reviewed for the clinical interpretation arm of the study. sixteen (16) were excluded because they exceeded the stability period and had missing demographics, 110 were from patients with an age less than 18 years old, and 103 had missing biochemical data. a total of 276 patients (170 women, 106 men, age range: 18 – 89 years) were included in the clinical interpretation part of the study as depicted in the flow diagram (online supplementary figure 5b). the overall median age was 52 years with the majority being women (62%). the median age for ckd patients was lower than the non-ckd; however, the difference was not statistically significant (p = 0.1032) (table 1). vitamin d results were not included, as very few patients had results available on the laboratory information system. the mcnemar test did not demonstrate any statistically significant differences in clinical interpretation, as shown by p-values above 0.05 in the detection of low, normal, and high pth using both assays. in both the ckd and dialysis group, no statistically significant (p = 0.500) differences in interpretation were found using mcnemar, as is shown in table 2. table 2: differences in clinical interpretation using manufacturer reference interval, johannesburg, south africa, 06 april 2022 to 21 september 2022. there were 29 patients with ckd on dialysis. classification according to kdigo guidelines revealed minimal discordance between the two pth assays, with a weighted ҡ coefficient of 0.797 (0.594–1.000) (table 3). table 3: classification according to kidney disease: improving global outcomes guidelines for intact parathyroid hormone and parathyroid hormone 1–84 assays, johannesburg, south africa, 06 april 2022 to 21 september 2022. discussion the study conducted at a tertiary hospital in johannesburg, south africa, between 06 april 2022 and 21 september 2022 found that there was a difference (inter-assay bias) between the secondand third-generation pth assays. however, there was no clinically significant change in the classification of hypoparathyroidism, primary hyperparathyroidism, and secondary hyperparathyroidism in pre-dialysis and dialysis ckd patients when one assay was used over the other. with regard to analytical performance, the methods were strongly correlated. however, as the percentage mean bias surpassed the european federation of clinical chemistry and laboratory medicine biological variation goal, this implies that these assays are neither interchangeable nor comparable. our study showed that for the entire patient group, the third-generation pth assay produced results that were, on average, approximately 20% lower than the second-generation assay, and even lower in ckd (27%) and dialysis (35%) patients. this is in keeping with other reported research.3,14,15,25,26,27 studies by hashim et al. conducted in a malaysian population in 2020 and o’flaherty et al. published in 2013 from the united kingdom, both using roche pth assays, found that third-generation assays were lower by 30% and 40% compared to second-generation assays, respectively.28,29 in contrast, in the non-ckd group with egfr above 60 ml/min/1.73 m2, the observed bias was 5.7%. this indicates that in patients with normal gfr, there are minimal differences between secondand third-generation assays, which is in keeping with other reported studies conducted in france and malaysia.26,28 although the observed differences between the two pth assays were higher in both the pre-dialysis and dialysis ckd groups, there was no change in interpretation of secondary hyperparathyroidism with either assay. the kdigo guidelines do not state the target pth range in pre-dialysis ckd patients.15 furthermore, we did not find studies that assessed the impact on clinical interpretation in this group. there is a paucity of studies that have compared secondand third-generation pth assays in dialysis patients using the kdigo classification of 2–9 times the upper limit of normal.26,30 in the dialysis group, using the manufacturer’s upper limit of normal, classification using kdigo did not demonstrate statistically significant discordance between the two assays. this study was limited by a small sample size of confirmed dialysis patients which reflects the limited dialysis resources in our country. however, our finding contrasted with a few published studies, including that by dupuy et al. conducted in france and published in 2018, which demonstrated a high percentage of discordant results in dialysis patients, with a ҡ coefficient of < 0.20.26 in addition, a study by beko et al., published in 2012 from hungary, found that using the manufacturer’s upper limit of normal, the clinical classification was altered in up to 23% of dialysis patients because of the switch from secondto third-generation pth assay.30 presence of discordance is important, as pth concentrations are used in the management of ckd-mbd. hence, the use of secondover third-generation pth assays may result in misclassification, leading to inadequate management. there have been various attempts to reduce the discrepancies between the secondand third-generation pth assays in dialysis patients, by applying a correction factor or deriving an equation to allow interconversion between the two assays.31 a study by wójtowicz et al. conducted in 2016 in poland suggested the use of 1–84/7–84 pth ratio to report pth results to minimise potential misdiagnosis of ckd-bmd in ckd patients.31 however, further validation studies are needed, and these efforts are not currently adopted by clinical guidelines. overestimation of pth by second-generation assays because of pth fragment cross reactivity is of negligible consequence in patients with primary hyperparathyroidism. nonetheless, the question regarding whether third-generation pth assays offer superior diagnostic efficacy remains. in our study, there were no differences in interpretation between the two assays with regard to diagnosis of primary hyperparathyroidism. this is consistent with other published studies: boudou et al. from france,32 and bonansea et al. from brazil.33 taking into consideration published evidence, the recent international guideline on evaluation and management of primary hyperparathyroidism recommend the use of either secondor third-generation pth assays.34 the international federation of clinical chemistry and laboratory medicine committee for bone metabolism has established a working group for the purpose of creating pth standardisation to make the results comparable among different assays.35 in the absence of a reference measurement method, the international federation of clinical chemistry and laboratory medicine has recommended the use of the world health organization standard, but this has not been completely implemented because of a lack of commutability.9,10 even though second-generation pth assays are the most utilised assays, third-generation pth assays offer the best hope for harmonisation of pth because the majority are traceable to the world health organization standard.35 to the best of our knowledge, this is the first study in africa to evaluate the differences in analytical performance and impact on clinical interpretation of secondand third-generation pth assays. limitations our study had some limitations. firstly, we had a small sample size of patients on dialysis. secondly, not all patients had laboratory results of tests used in the evaluation of ckd, for example urine albumin. finally, the study was based on information available on the laboratory information system, with limited clinical information. conclusion the secondand third-generation pth assays correlate very well, though there was a bias observed between the two methods which increases with rising pth concentrations that prevail in ckd patients. however, using the manufacturer’s assay-specific reference intervals and kdigo guideline on pth classification, there were no major changes in clinical interpretation when either assay was used. thus, the decision to use thirdover second-generation pth should take all of this into account. we recommend that assays not be used interchangeably. therefore, as stated by the manufacturer and in accordance with the kdigo recommendations, clinical laboratories should state the generation of pth assay employed and inform clinicians of any change in clinical methods to facilitate appropriate interpretation of pth results. acknowledgements we would like to acknowledge all the national health laboratory services technical staff at charlotte maxeke johannesburg academic hospital for their assistance in analysing samples. this article is partially based on the n.n.’s thesis entitled ‘comparison of second and third generation parathyroid hormone (pth) assays performed at charlotte maxeke johannesburg academic hospital – are they fit for purpose?’ towards the degree of master of medicine in the department of chemical pathology, university of the witwatersrand, south africa, november, 2023, with supervisors m.m. and d.j. it is available here: https://hdl.handle.net/10539/44544. competing interests the author reported that they received funding from national health laboratory service k-funding and university of the witwatersrand research grants, which may be affected by the research reported in the enclosed publication. the author has disclosed those interests fully and has implemented an approved plan for managing any potential conflicts arising from their involvement. the terms of these funding arrangements have been reviewed and approved by the affiliated university in accordance with its policy on objectivity in research. authors’ contributions n.n. collected and analysed the samples, conducted data analysis, and drafted the first manuscript. d.j. and s.k. supervised the study, and were involved in project management, data analysis, and reviewing and editing of the article. m.r.m. conceptualised the study, and was involved in methodology, supervision, funding acquisition, and reviewing and editing of the article. sources of support national health laboratory service k-funding and university of the witwatersrand research grants provided the funds for study consumables. data availability the data that support the findings of this study are available on request from the corresponding author, m.r.m. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional 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references about the author(s) john b. kalule faculty of health sciences, university of cape town, cape town, south africa karen h. keddy center for enteric disease research unit, national institute for communicable diseases, johannesburg, south africa anthony smith center for enteric disease research unit, national institute for communicable diseases, johannesburg, south africa mark p. nicol faculty of health sciences, university of cape town, cape town, south africa lourens robberts faculty of health sciences, university of cape town, cape town, south africa citation kalule jb, keddy kh, smith a, nicol mp, robberts l. development of a real-time pcr assay and comparison to chromagartm stec to screen for shiga toxin-producing escherichia coli in stool, cape town, south africa. afr j lab med. 2017;6(1), a609. https://doi.org/10.4102/ajlm.v6i1.609 original research development of a real-time pcr assay and comparison to chromagartm stec to screen for shiga toxin-producing escherichia coli in stool, cape town, south africa john b. kalule, karen h. keddy, anthony smith, mark p. nicol, lourens robberts received: 28 jan. 2017; accepted: 19 sept. 2017; published: 14 dec. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract introduction: shiga toxin-producing escherichia coli (stec) is an emerging infectious pathogen which could lead to haemolytic uremic syndrome. even though previous studies have compared the performance of chromagartmstec to real-time polymerase chain reaction (pcr) in europe, no study has been done to assess its performance on african isolates. objectives: this project aimed to validate and test an in-house-developed duplex real-time pcr and use it as a reference standard to determine the performance of chromagartmstec on african isolates from diarrhoeic stool samples. methods: this study evaluated stec diagnostic technology on african isolates. an in-house-developed duplex real-time pcr assay for detection of stx1 and stx2 was validated and tested on diarrhoeic stool samples and then used as a reference standard to assess the performance of chromagartmstec. real-time pcr was used to screen for stx in tryptic soy broth and the suspected stec isolates, while conventional pcr was used to detect the other virulence genes possessed by the isolates. results: the real-time pcr limit of detection was 5.3 target copies/μl of broth. the mean melting temperature on melt-curve analysis for detection of stx1 was 58.2 °c and for stx2 was 65.3 °c. of 226 specimens screened, real-time pcr detected stx in 14 specimens (6.2%, 95% confidence interval = 3.43% – 10.18%). the sensitivity, specificity, negative predictive value and positive predictive value of the chromagartmstec were 33.3%, 77.4%, 95.3% and 11.3%. conclusions: the in-house developed real-time pcr assay is a sensitive and specific option for laboratory detection of stec as compared to chromagartmstec in this setting. introduction globally, foodand water-borne outbreaks of both o157 and non-o157 shiga toxin-producing escherichia coli (stec) have been successfully detected due to the availability of good baseline data and effective active laboratory-based surveillance systems.1,2,3,4 early detection of outbreaks is important to minimise morbidity, mortality and associated economic losses.5 there is a lack of good baseline data on stec in africa, which can be attributed to a lack of laboratory resources and the surveillance strategy employed. stec has been implicated in outbreaks of bloody diarrhoea in sub-saharan countries;6,7,8 however, these have been difficult to track and manage due to laboratory weakness.9,10 furthermore, typical haemolytic uremic syndrome, which is overwhelmingly caused by stec, was reported as the leading contributor to acute renal failure in paediatric patients at a south african academic hospital.11 even though several studies have evaluated the performance of chromagartmstec by comparison to molecular and antigen detection methods in developed countries,12,13 no study has so far evaluated its performance in africa. this is necessary, especially given that there are geographical differences in characteristics of stec that are dependent on the index of suspicion for the different stec serotypes and on the availability of suitable laboratory methods to detect them.14 in many south african (and african) laboratories, stool specimens are not routinely tested for stec, although physicians may request tests specific for e. coli serotype o157:h7, if it is clinically suspected. testing is based on the non-sorbitol fermenting property using sorbitol macconkey and only on request by a physician. this practice is of concern, is misleading and underestimates the real magnitude of stec, since not all serotype o157 strains are non-sorbitol fermenting (o157: nm), and over 470 non-o157 serotypes have been attributed to clinical disease.15 laboratory capacity for molecular detection is increasingly available in african countries and may, in some cases, be simpler than culture-based detection. this project, therefore, aimed to validate and test diarrhoeic stool samples by using an in-house developed duplex real-time polymerase chain reaction (pcr) and use it as a reference standard to determine the performance of chromagartmstec on african isolates. the duplex assay was used to screen tryptic soy broth (tsb) for stx following overnight stool enrichment, and conventional pcr was used to screen for the other diarrhoeagenic e. coli virulence genes. diarrhoeagenic e. coli were serotyped, and stx-positive isolates were tested for shiga toxin production using immunochromatography. methods ethical approval this study was approved by the ethics and research committee of the faculty of health sciences at the university of cape town (hrec ref: 014/2015). study design this study validated an in-house-developed duplex real-time pcr assay for detection of stx1 and stx2. the assay was then tested on diarrhoeic stool samples at a tertiary referral hospital and was used as a reference standard to assess the performance of a commercial chromogenic medium (chromagartmstec) for stec screening (figure 1). figure 1: summary of methods used in this study. target plasmid preparation the real-time pcr previously described by grys et al.16 was used to amplify stx1 and stx2 gene targets from a stec o157:h7 nctc control strain (c4193-1) with both stx1 (subtype 1a) and stx2 (subtype 2a). pcr amplicon size was confirmed visually by agarose gel detection (~208 bp for stx1 and ~204 bp for stx2) before confirmation by sequencing using the big dye® terminator v3.1 cycle sequencing kit (life technologies corporation, carlsbad, california, united states). we used primers 1a and 2a (table 1) for unidirectional sanger sequencing of the amplicons. resultant sequences were then trimmed and submitted for blast analysis against the ncbi database and confirming stx1 or stx2 target sequences in comparison to o157:h7 edl933 (ncbi reference: nc_002655.2).17 purified amplicons (mini elute gel extraction kit, qiagen, madrid, spain) were cloned using clonejet pcr cloning kit (thermofisher scientific, austin, texas, united states) into a pjet 1.2/blunt vector using the sticky end cloning protocol and transfected into the jm109 competent cells by calcium chloride transformation. plasmids containing stx1 and stx2 were separately extracted using a genopure plasmid maxi kit (roche life sciences, rotkreuz, switzerland) and quantified by spectrophotometry. to verify successful preparation purified plasmids were subjected to pcr amplification using primers 1a and 1b for stx1 and 2a and 2b for stx2 with amplicon size visually confirmed by agarose gel detection and subsequent sequence analysis. plasmid quantification was determined spectrophotometrically employing the biodrop-μlite (isogen life science, b.v, veldzigt, netherlands). the a260 was used to calculate the plasmid concentration expressed as the number of molecules/μl. table 1: primers and probes used for real-time polymerase chain reaction. polymerase chain reaction assay validation to assess the potential for pcr cross-reactivity and assess the analytical specificity of the hybridisation probe-based real-time pcr described by grys et al.,16 the primer and probe sequences were subjected to blast analysis on the ncbi database. the pcr reaction was optimised for use on the lightcycler®480 instrument ii (roche life sciences, rotkreuz, switzerland) employing the lightcycler® 480 probes master mastermix (roche life sciences, rotkreuz, switzerland) with modification to the thermal cycling conditions for amplification consisting of denaturation at 95 °c for 10 min followed by 45 cycles of 95 °c for 5 s, 56 °c for 5 s and 72 °c for 15 s. a positive amplification signal was defined as an increase in fluorescence signal that crossed the threshold before 30 cycles. amplicon identity was determined using the melt-curve analysis program of 95 °c for 30 s, 40 °c for 60 s and 85 °c for 5 s with continuous fluorescence acquisition. the multi-color hybprobe detection format was used for analysis, combining the red 610, red 640 and fam filter pairs (lightcycler®480 instrument ii manual, roche life sciences, rotkreuz, switzerland). the resulting amplicon size was visualised using agarose gel electrophoresis and subjected to dna sequencing and blast alignment to reference stx1a and stx2a sequences (nc_002655.2). to mimic the sample matrix for sensitivity determination, tsb was inoculated with a pea-size amount of stool (from a single donor) shown to be stx-negative by pcr. to this inoculated broth 1 ml of plasmid stock (5.3*106 copies/μl) containing both stx1 and stx2 was added and serially diluted eight times in 9 ml of tsb, to a lowest dilution of 1:108 (53 plasmid copies/ml). nucleic acid extraction was performed on 200 μl broth employing the magnapure lc instrument (roche diagnostics, rotkreuz, switzerland) to yield 100 μl of extract. initially, real-time pcr was performed in triplicate using a template from each of the eight dilutions to estimate a limit of detection (lod). subsequently, real-time pcr was performed in eight replicates on the dilution with the estimated lod, as well as one dilution above and one dilution below the estimate. the lod was defined as the lowest plasmid concentration spiked into tsb, before nucleic acid extraction, yielding a positive signal, as described above in all eight replicates. nucleic acid extractions from stec subtypes 1d (reference strain mh1813, genbank accession no. ay170851), 2b (reference strain eh250, genbank accession no. af043627), 2c (reference strain 031, genbank accession no. l11079), 2d (reference strain c165-02, genbank accession no. dq059012), 2e (reference strain s1191, genbank accession no. m21534), 2f (reference strain t4/97, genbank accession no. aj010730) and 2g (reference strain 7v, genbank accession no. ay286000) were also subjected to pcr amplification to assess impact of strain variation on detection. the reproducibility of melting temperature assessment for stx1 and stx2 differentiation was determined by testing 24 replicates of tsb spiked with cloned stx1 and stx2 plasmids. to further assess the reproducibility of melting temperature across the subtypes, three stx1 subtypes and seven stx2 subtypes were tested similarly. clinical specimen testing between september 2014 and may 2015, we collected same day residual stool after routine testing from 226 consecutive stool specimens (the stool samples were transported in a temperature regulated box and processed within 12 h of collection) from the national health laboratory services located at the groote schuur hospital in cape town, south africa, a tertiary care academic teaching laboratory affiliated with the university of cape town. this tertiary academic hospital serves the greater cape town area. a pea-sized stool sample was inoculated in 90 ml of tsb and vortexed before incubation at 37 °c for 18 h. two hundred microlitres of broth were subsequently extracted employing the magna pure lc total nucleic acid isolation kit (roche diagnostics, rotkreuz, switzerland) using the total variable elution volume protocol and following the manufacturer’s manual (version 14) to yield 100 μl of nucleic acid extract. in addition, chromagartmstec (chromagar microbiology, paris, france) was inoculated with a loop full of overnight inoculated broth and incubated at 37 °c for 18 h. bright mauve colonies (up to five mauve colonies were picked per sample, depending on the number of mauve colonies formed) were sub-cultured onto macconkey agar with crystal violet, sorbitol macconkey agar, and 2% blood agar (green point media, national health laboratory service, albertynshof, south africa). e. coli was presumptively identified as lactose-positive, oxidase-negative, spot indole-positive and pyrrolidonyl arylamidase (pyr)-negative with confirmatory identification using vitek 2 (biomerieux, inc., durham, north carolina, united states). isolate characterisation isolates yielding mauve colonies on chromagartmstec and presumptively identified as e. coli were subjected to stx characterisation employing the real-time pcr assay characterised herein. other diarrhoeic e. coli virulence genes, including the fimbrial adhesion gene for diffusely adherent e. coli, the anti-aggregation protein transporter gene for enteroaggregative e. coli, heat-stable and heat-labile enterotoxin genes of enterotoxigenic e. coli, the intimin coding gene eae for enteropathogenic e. coli (epec) and the bundle-forming pili gene for the typical epec, were determined using standard gel-based pcr as previously described using primers as shown in table 2.18 table 2: primers used for detection of virulence genes by conventional polymerase chain reaction. to confirm shiga toxin production among stx-positive isolates, the immunocard stat!® ehec (meridian biosciences, inc., cincinnati, ohio, united states) was used to detect shiga toxin 1 and 2 (by employing immunochromatography with toxin-directed monoclonal antibodies labelled with red-coloured gold particles). all mauve isolates found to carry virulence genes were serotyped at the centre for enteric diseases, national institute of communicable disease, johannesburg, by employing antisera (statens serum institut, copenhagen, denmark) and the detection of somatic o-antigens as previously described.19,20 h-antigen serotyping was not undertaken. statistical analysis data on the possession of virulence genes, cultural characteristics on different media, serotypes and shiga toxin production was entered in microsoft office excel 2010 (microsoft corp.,redmond, washington, united states) and then exported to epiinfotm 7.1.5.2 (centers for disease control and prevention, atlanta, georgia, united states) for analysis. using the lightcycler 480 ii software, efficiency of the in-house real-time pcr assay was determined. the amplification curves and the melting peaks were used to differentiate between stx1 and stx2. results real-time polymerase chain reaction validation the blast analysis of the primers and probe sequence specificity yielded no significant homology to non-stx targets (data not shown). real-time pcr amplicons generated were confirmed as 208 bp for stx1 and 204 bp for stx2 (figure 2). sequencing and blast analysis confirmed the identity of both stx1 and stx2 amplicons. the serially diluted plasmid-stool-tsb was successfully amplified in 8/8 replicates in the sixth dilution, whereas the seventh dilution yielded an amplification signal in 3/8 replicates, yielding a lod of 5.3 target copies/μl of broth. all other stx subtypes investigated (stx1a, stx1b, stx2a, stx2b, stx2c, stx2d, stx2e, stx2f, and stx2g) were successfully amplified by this assay (data not shown). stx1 and stx2 were successfully distinguished by a melting temperature of 58.2 °c (sd = 0.033) and 65.3 °c (sd = 0.037) (figures 3–6). the tm for stx2 subtypes 2a, 2b, 2c, 2d, 2e, 2f and 2g were the same at 65.3 °c (sd = 0.037, 0.041, 0.035, 0.039, 0.034, 0.033 and 0.032, respectively), whereas that of 1d was 44.7 °c (sd = 0.042). the efficiency of the assay was 1.99 as calculated from the amplification curves generated using the light cycler® 480 software. the duplex assay detected both targets in the same run, and these could be differentiated by the melt curve with two distinct peaks at 58.2 °c for stx1 and 65.3 °c for stx2 (figure 7). figure 2: polymerase chain reaction amplicons of stx1 and stx2 after cloning. figure 3: amplification curves for the validated duplex real-time polymerase chain reaction for detection of the stx gene figure 4: melting peaks for stx1 – peak a (subtypes other than 1d – 55.7 °c) and stx2 – peak b (65.3 °c) detected in the same run. figure 5: melting peaks for optimised duplex real-time polymerase chain reaction showing stx2 peak b (65.3°c). figure 6: melting peaks for the detection of stx1d using the duplex real-time polymerase chain reaction assay. figure 7: amplification curves and melting peaks obtained on screening tryptic soy broth using the in-house optimised real-time polymerase chain reaction assay. (a) amplification curves. (b) melting peaks for the duplex real-time polymerase chain reaction assay. clinical specimens of the 226 specimens screened, real-time pcr detected shiga toxin genes in 14 samples (6.2%), comprising 8 stx1, 5 stx2 and 1 specimen containing both stx1 and stx2. chromagartmstec yielded mauve colonies from 23.45% (53/226) of the stool broth cultures (figure 8). figure 8: summary of isolate characterisation results. performance of chromagartmstec of the 53 mauve isolates, 48 were negative for stx genes using the validated real-time pcr assay. of the 14 broths that were positive on pcr, nine did not yield any mauve colonies on chromagartmstec culture. isolate characteristics forty-four (83%) of the 53 mauve colonies fermented lactose on macconkey agar with crystal violet. eleven (25%) of the 44 lactose fermenters were non-sorbitol-fermenting. real-time pcr on the 44 e. coli confirmed the presence of stx genes in five (11%), whereas 39 were negative for the stx gene. real-time pcr was not done on the nine non-lactose fermenting isolates as these were found not to be e. coli on biochemical testing. four of the five stx positive e. coli colonies were also positive in the real-time pcr broth assay. of the 39 stx-negative e. coli, only four (12.5%) carried eae genes, whereas four possessed aat genes. of the four eae positive isolates, two also had the bfp genes and were typical epec. the other two eae positive isolates did not possess the bfp genes and were classified as atypical epec. the four enteroaggregative stx-negative isolates all belonged to e. coli serotype o104. all the typical epec belonged to serotype o55, whereas one of the two atypical epec belonged to serotype o101. the atypical epec serotype o101 was from stx2 positive broth. the other atypical epec isolate was untypeable. no diffusely adherent e. coli, enteroinvasive e. coli or enterotoxigenic e. coli were detected. none of the 53 e. coli isolates that were screened by immunochromatography was positive for shiga toxins. for the chromagartmstec, sensitivity was 33.3%, specificity was 77.4%, negative predictive value was 95% and positive predictive value was 9.4% (table 3). table 3: performance of chromagartmstec compared with real-time polymerase chain reaction assay. discussion we validated the use of a previously described duplex real-time pcr assay with modification able to detect and differentiate stx1 (melting temperature = 58.2 °c) and stx2 (melting temperature = 65.3 °c) from overnight broth enrichment with a lod of 5.3 target copies/μl broth. this assay was able to detect both stx1 and stx2 in the same run, thus potentially reducing process turn-around time in a busy laboratory setting. timely reporting of stec infections is important, because use of certain antibiotics is contraindicated in stec infections. compared to the validated duplex real-time pcr, chromagartmstec showed a sensitivity of 33%, specificity of 77.4%, negative predictive value of 9.4% and positive predictive value of 95% for detection of stec in stool following tsb enrichment. of the 53 mauve isolates, 48 were negative for stx genes on use of the validated real-time pcr, whereas nine of the 14 pcr-positive broths did not yield any mauve colonies when cultured on chromagartmstec. reasons for the poor performance of this medium in relation to the in-house developed duplex real-time pcr include the following: (1) delays in reporting of diarrhoea cases to a tertiary hospital (where samples were collected) may have led to loss of stx genes; stec numbers are sharply reduced in stool after one week of illness, and the shiga toxin genes might be lost by the bacteria.21 (2) chromagartmstec selects for tellurite resistant strains but misses the tellurite susceptible stec whose prevalence in this setting is not known. for a chromogenic medium to be considered for routine screening purposes, it must have high specificity so as not to waste scarce laboratory resources on false positives. the false positivity rate in this setting (48/53 [90.6%]) is higher than has previously been reported in europe (16.3% reported by gouali et al., 2013 and 18.3% by wylie et al., 2013).12,22 similar studies to evaluate this medium were done in canada, finland, and germany, all of which reported high sensitivities for stec serotypes o26, o111, o121, o145, o118, and o157.13,22,23 the specificity of 77.4% noted in this study was low compared to values (between 95.8% and 98.9%) reported in similar studies done in europe. the difference in sensitivity and specificity could be explained by the differences in the patient characteristics (whether they present with haemolytic uremic syndrome and or bloody diarrhoea or not). unlike the studies in europe, this study did not focus on only patients with haemolytic uremic syndrome or bloody diarrhoea. additionally, the distribution of tellurite resistant stec (which is targeted by chromagartmstec) in this setting is not known. the prevalence of stx genes in stool samples (6.2%) was lower than the 9% previously reported by kullin et al., 2015.24 among the 53 isolates that formed mauve colonies on chromagartmstec, five were stec (two serotype o26 and others non-typeable), four were enteroaggregative e. coli (serotype o104), two were atypical epec (serotype o101 and non-typeable) and two were typical epec (serotype o55). serotypes o26 and o104 are among the top six stec serotypes globally.25,26 chromagartmstec is intended for stec culture; however, we detected other diarrhoeic e. coli pathotypes using this medium. the other pathotypes detected might have been hybrid strains that lost the stx genes or hybrid strains whose stx genes could not be detected using the primers employed in this study. notably, bacteriophages carrying the stx genes are very quickly lost both in vivo and in vitro,27 and not all stx primers can detect all the stx gene variants.28 limitations not all stec are tellurite resistant and may have been missed on chromagartmstec culture. this study only focused on the strains that formed mauve colonies. the stool samples in this study were collected between september 2014 and may 2015, and therefore the results of this study as regards prevalence of stec may not reflect the entire year or areas of south africa other than cape town. conclusions the in-house developed real-time pcr assay is a sensitive and specific alternative to the currently used diagnostic strategy. due to the high false positivity rate, chromagartmstec can only be used as an adjunt to a more sensitive and specific assay such as real-time pcr. trustworthiness to the best of our knowledge, the findings of this study can be used as per the scope of the study and in light of the study limitations as clearly pointed out. reliability we confirm that the experiments conducted in this study will yield the same results during repeated trials using the same reagents and detection platforms. validity to the best of our knowledge, the findings of this study, as obtained using the methods we employed, are valid for the study area and season. the in-house developed real-time pcr may, however, be adopted in other laboratories in developing countries. acknowledgements we acknowledge the center for enteric disease research unit, national institute of communicable disease (sandringham, johannesburg, south africa) for serotyping of the e. coli isolates. we also acknowledge the national research foundation, south africa, the acp-rise scholarship fund and the adb-hest fund. competing interests the authors declare that there are no financial or personal relationships that may have had influence in the writing of this article. sources of support funds for this study were provided by the national research foundation, south africa, the adb-hest scholarship fund and the acp-rise scholarship fund. authors’ contributions j.b.k. participated in processing the samples, data analysis and writing of the manuscript. k.h.k. participated 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9, no 1 | a823 | 10 september 2020 original research fluorescence microscopy for the diagnosis of smear-negative pulmonary tuberculosis in ethiopia gemeda abebe, dossegnaw aragaw, mulualem tadesse african journal of laboratory medicine | vol 9, no 1 | a810 | 28 september 2020 original research using systematized nomenclature of medicine clinical term codes to assign histological findings for prostate biopsies in the gauteng province, south africa: lessons learnt naseem cassim, ahsan ahmad, reubina wadee, jaya a. george, deborah k. glencross african journal of laboratory medicine | vol 9, no 1 | a909 | 28 september 2020 original research development of dried tube specimens for xpert mtb/rif proficiency testing kyle degruy, katherine klein, zilma rey, patricia hall, andrea kim, heather alexander african journal of laboratory medicine | vol 9, no 1 | a1166 | 29 september 2020 original research endometrial sampling at an academic hospital in south africa: histological findings, lessons learnt and interesting surprises reena d. mohanlal african journal of laboratory medicine | vol 9, no 1 | a1038 | 29 september 2020 original research serological detection of brucellosis among febrile, malaria-negative children and domesticated dogs in an urban african setting john b. kalule, joseph tomusange, teddy namatovu african journal of laboratory medicine | vol 9, no 1 | a864 | 30 september 2020 original research review of the stepwise laboratory quality improvement process towards accreditation (slipta) version 2:2015 tjeerd a.m. datema, linda oskam, jacqueline e.w. broerse, paul r. klatser african journal of laboratory medicine | vol 9, no 1 | a1068 | 28 october 2020 original research postmortem diagnosis of covid-19: antemortem challenges of three cases at the 37 military hospital, accra, ghana seth a. attoh, frederick hobenu, lawrence edusei, kwasi agyemanbediako, clement t. laryea, edward o. nyarko, michael k. amedi, richard h. asmah, edward asumanu, mary mcaddy, anthony maison, godwin nyarko, raymond d. fatchu, kafui akakpo african journal of laboratory medicine | vol 9, no 1 | a1290 | 03 november 2020 56 62 67 73 82 90 97 103 110 page i of iii table of contents i editorial african laboratory medicine in the time of covid-19 iruka n. okeke african journal of laboratory medicine | vol 9, no 1 | a1447 | 21 december 2020 original research diagnostic outcomes of bone marrow aspirate and trephine biopsies performed at a hospital in kwazulu-natal, south africa wanda s. tshabalala, somasundram pillay, douglas p.k. wilson african journal of laboratory medicine | vol 9, no 1 | a1028 | 25 february 2020 original research evaluation of corrective actions of feedback from clinicians on clinical laboratory services at bamenda regional hospital laboratory, cameroon victor n. fondoh, charles n. awasom, rebecca enow-tanjong, richard m. fondoh, patrick njukeng, judith shang, julianna ndasi, moses samje, claris n. muluh, thompson n. kinge african journal of laboratory medicine | vol 9, no 1 | a843 | 23 march 2020 original research clinical staff knowledge and awareness of point-of-care-testing best practices at tygerberg hospital, south africa thumeka p. jalavu, megan rensburg, rajiv erasmus african journal of laboratory medicine | vol 9, no 1 | a853 | 16 july 2020 original research evaluation of the ogawa-kudoh method for tuberculosis isolation in two health units in mozambique carla m. madeira, khalide i. azam, daisy n. sato, celso khosa, nilesh bhatt, sofia o. viegas african journal of laboratory medicine | vol 9, no 1 | a929 | 20 july 2020 original research prevalence of cryptococcal antigen (crag) among hiv-positive patients in eswatini, 2014–2015 samson m. haumba, mitsuru toda, rossana jeffries, peter ehrenkranz, munyaradzi pasipamire, trong ao, nomthandazo lukhele, sikhathele mazibuko, mandzisi mkhontfo, rachel m. smith, tom chiller african journal of laboratory medicine | vol 9, no 1 | a933 | 29 july 2020 original research detecting tuberculosis in pregnant and postpartum women in eswatini munyaradzi pasipamire, edward broughton, mandzisi mkhontfo, gugu maphalala, batsabile simelane-vilane, samson haumba african journal of laboratory medicine | vol 9, no 1 | a837 | 30 july 2020 original research prevalence of urinary schistosomiasis amongst primary school children in ikwo and ohaukwu communities of ebonyi state, nigeria nse o. umoh, chimezie f. nwamini, nyoho j. inyang, anthony n. umo, victor u. usanga, amos nworie, michael o. elom, boniface n. ukwah african journal of laboratory medicine | vol 9, no 1 | a812 | 24 august 2020 original research prevalence and aetiology of moderate and severe thrombocytopenia in a tertiary and quaternary centre in kwazulu-natal ayanda g.p. jali, bongani b. nkambule african journal of laboratory medicine | vol 9, no 1 | a799 | 24 august 2020 1 4 10 17 25 30 37 46 51 vol 9, no 1 (2020) issn: 2225-2002 (print) | issn: 2225-2010 (online)african journal of laboratory medicine http://www.ajlmonline.org open access table of contents ii original research adult and paediatric haematology and clinical chemistry laboratory reference limits for liberia mark w. kieh, sarah m. browne, greg a. grandits, julie blie, jestina w. doe-anderson, marie l. hoover, bionca davis, cavan s. reilly, james d. neaton, h. clifford lane, stephen b. kennedy african journal of laboratory medicine | vol 9, no 1 | a1080 | 25 november 2020 original research infant hiv diagnosis and turn-around time for testing in malawi, 2015 hammad ali, peter minchella, geoffrey chipungu, evelyn kim, james kandulu, dalitso midiani, andrea kim, mahesh swaminathan, steve gutreuter, john nkengasong, daniel singer african journal of laboratory medicine | vol 9, no 1 | a904 | 26 november 2020 original research a global proficiency testing programme for xpert® mtb/rif using dried tube specimens, 2013–2015 katherine klein, kyle degruy, zilma rey, patricia hall, andrea kim, steve gutreuter, heather alexander african journal of laboratory medicine | vol 9, no 1 | a1167 | 27 november 2020 original research prevalence and antifungal susceptibility of gastrointestinal candidiasis among diabetic patients: a cross-sectional study anthony p. oyom, emmanuel okello, victoria acam, christine aramo, bashir mwambi, john c. okiria, caesar oyet african journal of laboratory medicine | vol 9, no 1 | a997 | 10 december 2020 original research evaluation of ceftriaxone-sulbactam-disodium edetate adjuvant combination against multi-drug resistant gram-negative organisms shilpi gupta, mahadevan kumar, shelinder p.s. shergill, kundan tandel african journal of laboratory medicine | vol 9, no 1 | a991 | 10 december 2020 original research validation of phase for deriving n-acetyltransferase 2 haplotypes in the western cape mixed ancestry population celeste swart, surita meldau, chad m. centner, adrian d. marais, fierdoz omar african journal of laboratory medicine | vol 9, no 1 | a988 | 17 december 2020 original research sensitivity and specificity of mean platelet volume as a laboratory marker for irritable bowel syndrome: can it be added to rome criteria? mostafa vaghari-tabari, soheila moein, durdi qujeq, mehrdad kashifard, haydeh alaoddolehei, karimollah hajian-tilaki african journal of laboratory medicine | vol 9, no 1 | a1001 | 21 december 2020 original research serologic evidence of seasonal influenza a and b in hiv patients on combined antiretroviral therapy in lagos, nigeria abdulazeez a. anjorin, barakat a. adepoju african journal of laboratory medicine | vol 9, no 1 | a1048 | 21 december 2020 original research haematological indices of sickle cell patients with chronic leg ulcers on compression therapy oluwatoyin a. babalola, ayodele ogunkeyede, abayomi b. odetunde, foluke fasola, anthony a. oni, chinedum p. babalola, adeyinka g. falusi african journal of laboratory medicine | vol 9, no 1 | a1037 | 21 december 2020 118 126 133 141 148 154 161 166 172 original research categorising specimen referral delays for cd4 testing: how interlaboratory distances and travel times impact turn-around time across a national laboratory service in south africa naseem cassim, lindi m. coetzee, deborah k. glencross african journal of laboratory medicine | vol 9, no 1 | a1120 | 21 december 2020 original research weekly laboratory turn-around time identifies poor performance masked by aggregated reporting lindi-marie coetzee, naseem cassim, deborah k. glencross african journal of laboratory medicine | vol 9, no 1 | a1102 | 21 december 2020 original research haematological reference intervals for healthy adults in bamenda, cameroon victor n. fondoh, richard m. fondoh, charles n. awasom, pefoule l. edith, winlove a. ntungwen, bong roland, rebeca enow-tanjong, patrick njukeng, judith shang, egbe p. egbengu, talkmore maruta, akazong etheline, robert leke, ayuk leo, denis nsame african journal of laboratory medicine | vol 9, no 1 | a1193 | 21 december 2020 original research biochemical changes in whole blood stored for transfusion at bungoma county referral hospital, kenya phidelis m. marabi, stanslaus musyoki, angela amayo african journal of laboratory medicine | vol 9, no 1 | a1182 | 21 december 2020 lessons from the field h3africa partnerships to empower clinical research sites to generate highquality biological samples talishiea croxton, ndidi agala, emmanuel jonathan, olasinbo balogun, petronilla j. ozumba, enzenwa onyemata, shefiya lawal, manmak mamven, samuel ajayi, sylvia e. melikam, mayowa owolabi, bruce ovbiagele, dwomoa adu, akinlolu ojo, christine m. beiswanger, alash’le abimiku african journal of laboratory medicine | vol 9, no 1 | a935 | 18 march 2020 lessons from the field addressing the shortage of pathologists in africa: creation of a mmed programme in pathology in zambia victor mudenda, evans malyangu, shahin sayed, kenneth fleming african journal of laboratory medicine | vol 9, no 1 | a974 | 03 june 2020 lessons from the field establishing the college of pathologists of east, central and southern africa – the regional east central and southern africa college of pathology shahin sayed, rudo mutasa, ephata kaaya, victor mudenda, erasmus rajiv, edda vuhahula, jamilla rajab, robert lukande, edwin walong, angela mutuku, kenneth fleming african journal of laboratory medicine | vol 9, no 1 | a979 | 03 june 2020 lessons from the field laboratory organisation and management of sars-cov-2 infection in niger, west africa abdourahamane yacouba, adamou lagaré, daouada alhousseini maiga, halimatou moumouni sambo, sani ousmane, zelika hamidou harouna, boubacar marou, maman k. sanoussi, balki aoula, ali amadou, hassane boureima, saidou amatagas, abdoulaye ousmane, eric adehossi, saidou mamadou african journal of laboratory medicine | vol 9, no 1 | a1308 | 21 december 2020 180 187 195 203 208 217 224 232 page ii of iii http://www.ajlmonline.org open access table of contents iii brief report herpes simplex virus-2 infections in pregnant women from south africa: evaluation of the immunoflow rapid test shanthie govender, lungile mbambo, makandwe nyirenda, motshedisi sebitloane, nathlee abbai african journal of laboratory medicine | vol 9, no 1 | a854 | 31 august 2020 brief report rapid testing for respiratory syncytial virus in a resource-limited paediatric intensive care setting howard newman, donald tshabalala, sikhumbuzo mabunda, nokwazi nkosi, candice carelson african journal of laboratory medicine | vol 9, no 1 | a1084 | 08 december 2020 brief report critical success factors for vietnamese laboratories striving to implement quality management systems cathy robinson, james johnson, katy yao, hien bui african journal of laboratory medicine | vol 9, no 1 | a937 | 18 december 2020 case study post-procedural bacillus cereus septic arthritis in a patient with systemic lupus erythematosus barend mitton, roxanne rule, nontombi mbelle, wesley van hougenhouck-tulleken, mohamed said african journal of laboratory medicine | vol 9, no 1 | a1119 | 20 august 2020 case study response to a cluster of severe acute respiratory syndrome coronavirus 2 cases at a diagnostic laboratory christoffel j. opperman, gert j.k. marais, michelle naidoo, marvin hsiao, nazlee samodien african journal of laboratory medicine | vol 9, no 1 | a1307 | 25 august 2020 case study brucellosis – laboratory workers’ nightmare come true: a case study lebogang skosana, farzana ismail, nontombi mbelle, mohamed said african journal of laboratory medicine | vol 9, no 1 | a1114 | 29 september 2020 review article the network approach to laboratory procurement and supply chain management: addressing the system issues to enhance hiv viral load scale-up jason williams, dianna edgil, matthew wattleworth, clement ndongmo, joel kuritsky african journal of laboratory medicine | vol 9, no 1 | a1022 | 13 august 2020 review article covid-19 rapid diagnostic test could contain transmission in lowand middle-income countries adesola olalekan, bamidele iwalokun, oluwabukola m. akinloye, olayiwola popoola, titilola a. samuel, oluyemi akinloye african journal of laboratory medicine | vol 9, no 1 | a1255 | 30 september 2020 237 242 247 251 255 259 263 272 opinion paper scaling up testing for covid-19 in africa: responding to the pandemic in ways that strengthen health systems farouk a. umaru african journal of laboratory medicine | vol 9, no 1 | a1244 | 14 may 2020 opinion paper establishing diagnostic training programs in resource-poor settings: the case of sierra leone lance d. presser african journal of laboratory medicine | vol 9, no 1 | a889 | 15 june 2020 opinion paper covid-19 pandemic and antimicrobial resistance: another call to strengthen laboratory diagnostic capacity in africa beverly egyir, noah obeng-nkrumah, george b. kyei african journal of laboratory medicine | vol 9, no 1 | a1302 | 23 september 2020 opinion paper how can clinical immunology laboratories contribute to the management of severe covid-19 cases in limited resource contexts? brahim admou, abdelhamid hachimi, mohammed abdenasser samkaoui african journal of laboratory medicine | vol 9, no 1 | a1282 | 02 november 2020 opinion paper covid-19 during malaria transmission season in africa and why we should be prepared: an example from senegal khadim diongue, mamadou a. diallo african journal of laboratory medicine | vol 9, no 1 | a1332 | 18 november 2020 opinion paper will long-read sequencing technologies replace short-read sequencing technologies in the next 10 years? boluwatife a. adewale african journal of laboratory medicine | vol 9, no 1 | a1340 | 26 november 2020 scientific letter inflammatory markers as predictors of mortality in covid-19 infection awadia a. ahmeidi, ashraf musa, hind s. ahmed, adel a. elahmar, ryhana b. goota, ibtihal a. ahmed, abdelhakam h. ali, mushal allam, mozan o. hassan african journal of laboratory medicine | vol 9, no 1 | a1298 | 21 december 2020 reviewer acknowledgement african journal of laboratory medicine | vol 9, no 1 | a1445 | 21 december 2020 280 282 287 291 294 297 302 304 page iii of iii health system and hiv epidemiology in mozambique laboratory infrastructure and hiv-related testing in mozambique framework for quality assurance of diagnostics in mozambique lessons learnt from existing quality assurance programmes national quality assurance programme for point-of-care testing the way forward references about the author(s) patrina chongo national institute of health, maputo, mozambique nádia sitoe national institute of health, maputo, mozambique sofia viegas national institute of health, maputo, mozambique isabel pinto national medical care, ministry of health, maputo, mozambique admiro macave national medical care, ministry of health, maputo, mozambique fernando sitoe national medical care, ministry of health, maputo, mozambique adolfo vubil national institute of health, maputo, mozambique nédio mabunda national institute of health, maputo, mozambique bindiya meggi national institute of health, maputo, mozambique eduardo s. gudo national institute of health, maputo, mozambique ilesh v. jani national institute of health, maputo, mozambique citation chongo p, sitoe n, viegas s, et al. quality assurance for point-of-care testing in mozambique’s national health service. afr j lab med. 2016;5(2), a445. http://dx.doi.org/10.4102/ajlm.v5i2.445 country profile quality assurance for point-of-care testing in mozambique’s national health service patrina chongo, nádia sitoe, sofia viegas, isabel pinto, admiro macave, fernando sitoe, adolfo vubil, nédio mabunda, bindiya meggi, eduardo s. gudo, ilesh v. jani received: 24 mar. 2016; accepted: 11 aug. 2016; published: 17 oct. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. health system and hiv epidemiology in mozambique medical care in mozambique is mostly provided through the national health service of the ministry of health. all primary healthcare and hiv-related services are provided free of charge. there are over 1500 public sector health facilities in mozambique and most of these are primary healthcare centres. although all hospitals have a laboratory, only a quarter of the health centres have a formal laboratory. in this context, point-of-care (poc) testing and syndromic management of diseases play an important role in the health system. both communicable and non-communicable diseases are prevalent in the mozambican population. mozambique has a population of 28 million and is among the nine countries with the highest hiv prevalence in the world.1 hiv prevalence in the country among people aged 15–49 years is 11.5%, ranging from 3.7% in the niassa province in the north to 25.1% in the gaza province in the south.2,3 hiv prevalence is higher among women (13.1%) than among men (9.2%), and higher in urban areas (15.9%) compared with rural areas (9.2%).2,3 among children aged between 0 and 11 years, hiv prevalence is 1.4%, and 2.3% in those younger than one year.2,3 it is estimated that 102 new infections in children occur daily in mozambique (ministry of health, unpublished data). demographic impact studies show that an estimated 1.6 million mozambicans were living with hiv in 2009.2,3 laboratory infrastructure and hiv-related testing in mozambique the public laboratory system encompasses 344 laboratories located in institutes, hospitals, health centres and non-governmental organisations. most of these laboratories are directly managed by institutions affiliated with the ministry of health. two branches within the ministry of health coordinate the laboratory network. firstly, the central laboratory department, which is part of the directorate of medical services, is in charge of medical laboratories within all health facilities, assuring human resources, procurement of major equipment, consumables and reagents, and equipment maintenance (figure 1). secondly, the national institute of health (instituto nacional de saúde; ins) houses the national reference laboratories and provides technical support to the laboratory network in terms of technology assessment, on-the job training, specialised testing, and external quality assessment. figure 1: governance structure of mozambique’s public laboratory network. hiv testing is performed in laboratories and at the poc within several departments of health facilities by using a serial algorithm of two rapid tests to detect antibodies against hiv. hiv testing is also widely used in voluntary counselling and testing facilities. from a handful of settings that tested for hiv in the late 1990s, today thousands of testing points exist nationwide. annually, around six million hiv serological rapid assays are performed in mozambique.1 cd4 t-cell counting in mozambique was initially done using conventional laboratory-based flow cytometers. in 2003, three laboratories were providing cd4 testing for the whole country. currently, 201 cd4 testing devices exist in the country. of these, 45 are laboratory-based technologies, 19 are full flow cytometers and 26 are dedicated bench-top cytometers. the remaining 156 instruments are of the poc type and are placed in various settings within and outside laboratories, including 13 located in mobile clinics. annually, around 250 000 cd4 t-cell enumerations are performed in mozambique (cd4 eqa ins data). early infant diagnosis (eid) was first performed in mozambique, using manual pcr assays, at the ins reference laboratory in 2005. currently, eid takes place in five laboratories, one in the north, two in the centre and two in the south of the country, using high-throughput pcr methods on dried blood-spot specimens. collection of dried blood-spots is done in 1136 health facilities nationwide, and collected samples are sent to one of the five network laboratories. results of testing are sent back mainly using mobile phone technology to printers located at the health facility level (www.portaldpi.com). annually, 60 000 exposed children undergo eid testing in mozambique, and viral load testing for treatment monitoring is currently being established within eid laboratories.1 poc technologies for eid and viral load testing are under evaluation, with implementation planned during 2016. framework for quality assurance of diagnostics in mozambique a national laboratory policy document has been drafted and awaits formal approval by the government. this policy sets clear responsibilities related to quality assurance at the different levels of the health system. currently, quality assurance activities in mozambique include: strengthening internal management and internal quality control: this is done mainly through the implementation of the strengthening laboratory management towards accreditation (slmta) tool.4,5,6 since its inception in mozambique in 2011, 32 laboratories have benefited from this programme. external quality assessment (eqa): the eqa programme in mozambique started in pilot form, sending out proficiency panels for cd4 t-cell enumeration in 2005. currently, the eqa programme covers 451 sites and includes: (i) proficiency panels for 12 schemes (figure 2), which, with the exception of cd4 counting, genexpert® and bacteriology, are prepared in mozambique. for cd4 counting, ins and qasi (public health agency of canada) are planning a training activity to build national capacity for proficiency panel preparation; (ii) blind re-checking for tuberculosis smear and malaria smear microscopy; and (iii) data analysis collected from cd4 counting poc devices using mobile phone technology. this activity is done in addition to the proficiency testing described in (i) above. figure 2: schemes for external quality assessment (eqa) in mozambique, 2016. site visits are carried out at all sites that have low performance on two consecutive panels. follow-up visits may be carried out to monitor implementation of corrective measures. regulatory approval systems for new diagnostics are currently being established and designed based on existing platforms for drugs and vaccines. most current policies do not differentiate laboratory-based from poc technologies. lessons learnt from existing quality assurance programmes slmta tool implementation the following lessons were learned from implementing slmta in mozambique: encouraging the entire laboratory to work as a team has guaranteed better results and increased confidence in the programme. reporting slmta activities to the hospital management has motivated local leaders to monitor the implementation of quality systems in clinical laboratories. implementation of the slmta tool has shown better results when done together with mentorship from nationally-recognised leaders. accreditation at the highest standards is possible for public medical laboratories in africa, even under challenging environments. in 2015, the national tuberculosis reference laboratory of the ins received iso 15189 accreditation by the instituto português para acreditação for fluorescence smear microscopy and for culture in liquid and solid media assays. external quality assessment the release of a periodic overall report, where sites are not identifiable, but which shows the performance of the laboratory network, is important with regard to creating awareness about the quality of diagnostic testing. presentation of annual certificates to participating sites increases confidence in the programme and boosts the willingness of sites to participate in subsequent rounds. in addition, eqa results improve the confidence of results that are produced by poc tests and allow the identification of sites and operators with performance problems. site visits positively impact the performance of proficiency testing, and strategies to establish the capacity to produce proficiency panels at the national level have allowed the country to increase the coverage and efficiency of eqa programmes. a further observation is that innovations, such as the use of mobile phone connectivity on diagnostic platforms, are critical for assuring the quality of the next generation of poc assays. national quality assurance programme for point-of-care testing the laboratory network in mozambique is a hybrid and dynamic environment where both laboratory-based and poc assays co-exist with complementary roles. therefore, in this context, and given the paucity of resources, the quality of poc testing is not the target of a distinct programme. current quality assurance activities cover both laboratory-based and poc assays. nevertheless, specific operational features of poc tests demand focused solutions within the programme. for example, training of lay counsellors for hiv testing and of nurses for poc cd4 testing has required the establishment of specific training packages and appropriate supervision checklists. mozambique developed a guideline for a poc implementation, where participation in an eqa programme is mandatory and must be part of the process. with the rapid increase in the implementation of technology, specifically for cd4 testing, the eqa provider faced problems with fulfilling all sites and is still facing problems adding more sites. based on that, it has been challenging for the programme to start preparing in-country panels to cover these sites. the main eqa activities in place in mozambique include managing proficiency testing panels, building capacity for producing panels in-country, and performing corrective actions (table 1). table 1: mozambique external quality assessment activities in place, 2016. the way forward the health system is constantly subjected to external forces such as demographic changes, epidemiological transitions, scientific discoveries, technological innovations and public health breakthroughs. the demands for poc testing to improve access will continue to increase, thus the capacity to produce proficiency panels in africa will allow quality assurance programmes to keep up with the demand of poc testing. the laboratory network and associated quality assurance programmes must remain in readiness for the challenges facing our continent. references joint united nations programme on hiv/aids (unaids). fact sheet 2015. geneva, switzerland: unaids; 2015. instituto nacional de saúde. inquérito nacional de prevalência, riscos comportamentais e informação sobre o hiv e sida em moçambique 2009. calverton, md: icf macro; 2010. instituto nacional de saúde, us centers for disease control and prevention, university of california san francisco, et al. relatório final: inquérito integrado, biológico e comportamental entre mulheres trabalhadoras de sexo, moçambique 2011–2012. san francisco, ca: ucsf; 2013. yao k, maruta t, luman et, et al. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. 2014;3(1), art. #194, 8 pages. http://dx.doi.org/10.4102/ajlm.v3i2.194 yao k, luman et, slmta collaborating authors. evidence from 617 laboratories in 47 countries for slmta-driven improvement in quality management systems. afr j lab med. 2014;3(2), art. #262, 11 pages. http://dx.doi.org/10.4102/ajlm.v3i2.262 maruta t, yao k, ndlovu n, et al. training-of-trainers: a strategy to build country capacity for slmta expansion and sustainability. afr j lab med. 2014;3(2), art. #196, 7 pages. http://dx.doi.org/10.4102/ajlm.v3i2.196 abstract introduction methods results discussion acknowledgements references about the author(s) lucius c. imoh department of chemical pathology, faculty of medical sciences, university of jos, jos, plateau state, nigeria idris y. mohammed department of chemical pathology & immunology, college of health sciences, bayero university and aminu kano teaching hospital, kano, kano state, nigeria ifeyinwa d. nnakenyi department of chemical pathology, faculty of medical sciences, university of nigeria nsukka and university of nigeria teaching hospital, enugu, enugu state, nigeria ephraim u. egbuagha department of pathology, clinix healthcare ltd, lagos, lagos state, nigeria tomisin m. adaja department of chemical pathology, federal medical centre, owo, ondo state, nigeria chinelo p. onyenekwu department of chemical pathology, ben carson snr school of medicine, babcock university and babcock university teaching hospital, ilishan-remo, ogun state, nigeria citation imoh lc, mohammed iy, nnakenyi id, egbuagha eu, adaja tm, onyenekwu cp. critical values notification: a nationwide survey of practices among clinical laboratories across nigeria. afr j lab med. 2023;12(1), a2249. https://doi.org/10.4102/ajlm.v12i1.2249 original research critical values notification: a nationwide survey of practices among clinical laboratories across nigeria lucius c. imoh, idris y. mohammed, ifeyinwa d. nnakenyi, ephraim u. egbuagha, tomisin m. adaja, chinelo p. onyenekwu received: 04 july 2023; accepted: 12 oct. 2023; published: 15 dec. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: critical value notification (cvn) entails notifying doctors or other laboratory users of aberrant laboratory results that threaten the patient’s life and of any values for which reporting delays could negatively impact the patient’s health. critical value notification practices in clinical laboratories in nigeria and sub-saharan africa are largely unknown. objective: we conducted a nationwide survey to obtain baseline information on cvn practice by nigeria’s laboratories. methods: this cross-sectional study was conducted among purposively selected secondaryand tertiary-tier, public and private clinical laboratories across northern and southern nigeria between october 2015 and december 2015. consenting senior laboratory staff completed and returned a structured questionnaire, that gathered data on respondents’ demographics, designations, and institutional characteristics and practices regarding cvn. results: one hundred and thirty-four laboratories responded to the questionnaires. only 69 (51.5 %) laboratories practised cvn; only 23 (33.3%) had existing written policies guiding the practice. most (43; 62.3%) laboratories use similar critical values (cvs) for adult and paediatric populations. most laboratories (27; 39.1%) obtained their cvs by combining published literature and local opinions from stakeholders. physical dispatch (42; 60.9%) followed by telephone calls (38; 55.1%) were the most common means of notification. private laboratories, compared with public hospital laboratories, were likelier to have separate paediatric cv lists (p = 0.019) and practise telephone notifications (p < 0.001). conclusion: critical value notification practices vary and are often suboptimal in many clinical laboratories in nigeria, which is exacerbated by the absence of guiding policies and national recommendations for post-analytical procedures. what this study adds: this study provides baseline information on cvn practice by nigeria’s laboratories. the study explores the causes of practice variations that can serve as a foundation for enhancing critical reporting and post-analytical services, particularly in clinical laboratories in sub-saharan africa. keywords: critical values; critical value reporting; critical value notification; post-analytical services, laboratory quality management. introduction laboratory results are essential for optimal patient care. prompt release and transmission of crucial test results significantly impacts healthcare decisions and patient outcomes.1,2,3,4 a critical value in laboratory practice generally refers to a laboratory result that suggests a potentially life-threatening condition needing prompt and appropriate medical intervention. as defined by lundberg, the critical value is a result that is so highly abnormal that it is deemed potentially lethal or likely to cause considerable morbidity and necessitates immediate intervention.5,6 the expression has been used interchangeably with alternative terms such as ‘panic value’ and ‘alarm value’.5 many laboratories have long focused on improving pre-analytical processes, and the use of automation and improved analytical systems is helping to produce more accurate and timely results.7 however, standardisation of post-analytical processes, such as critical value reporting, is more challenging and potentially prone to human mistakes or violations, with untoward implications for patient safety.7,8 the importance of timely delivery of critical values (cvs) is underscored by the fact that reporting critical results is a vital quality indicator for the post-analytical phase of the total testing process. additionally, accreditation and regulatory authorities demand that laboratories have policies in place to communicate critical results to ensure patient safety.9,10,11,12 there are few or no established standards or guidelines for reporting cvs in many countries and regions, and crucial results-reporting practices vary widely around the globe.7,9 there are many reasons for this heterogeneity in practice, including differences in patient demographics, staffing levels, instrumentation, and institutional structures.13 from published literature, some of the areas of considerable differences in cvs reporting practices include the policy framework for critical value notification (cvn), selection of cvs, the source(s) for determining critical limits, acceptable timeframe for delivering cvs, repeat testing for cvs and which results to report.7,11 other issues relating to the communication of cvs include means of reporting, to whom to report, and how to handle cases of unreachable caregivers.7,14,15,16,17 nigeria has no known standard practices for cvn. any efforts at harmonising cvn would logically begin with assessing current practices and the underlying basis for such practices across laboratories. therefore, we surveyed cvn for routine clinical chemistry and haematology analytes to obtain baseline information on the practice of cvn in nigeria. methods ethical considerations ethical approval was obtained from the human research ethical committee of the jos university teaching hospital (juth/dcs/adm/127/xxv/152). the respondents gave their written informed consent, and confidentiality was maintained per the declaration of helsinki’s ethical standards for medical research involving human beings. this was done by the allocation of unique and anonymised study codes. the research data were stored electronically on password-protected devices accessible only by the researchers. study design and duration this cross-sectional nationwide survey was conducted between october 2015 to december 2015 among secondaryand tertiary-tier clinical laboratories. these include laboratories of public hospitals such as general/district secondary care hospitals and laboratories of teaching hospitals that service several peripheral hospitals. the study also involved laboratories of private clinics and hospitals, faith-based health institutions, and commercial laboratories. these laboratories provide routine chemistry tests such as glucose, bilirubin, urea, creatinine, electrolytes, urine, triglycerides, cholesterols, liver enzymes, hormones and tumour markers, as well as basic haematology tests, such as haematocrit, complete blood count and clotting profile. the study involved a total sampling of consenting laboratories in 25 purposively selected states. these states had a large distribution of secondaryand tertiary-tier (including public and private) clinical laboratories across the six geopolitical zones in the north and south of nigeria. the federal capital territory (abuja) and 24 of the 36 states in the country were surveyed. a structured questionnaire was given to consenting laboratories and completed by any senior laboratory staff deemed competent by the head of the laboratory to respond to the questionnaires (one person per laboratory). according to the scheme of service, these laboratory staff include pathologists, pathologists-in-training (resident doctors), laboratory directors, and senior laboratory scientists or technologists. the questionnaire gathered data on demographics, respondents’ designations, and the type and speciality of laboratory or health institutions. we also looked into data on the existence of and practices surrounding cvns, and the handling of cvs. the questionnaire addressed the cvn policy framework, which refers to documented management commitment, processes and procedures for handling cvs in this manuscript. the responders were invited to select each item applicable to a specific question and provide more information if necessary. the self-administered questionnaire was pre-tested using responses from three laboratory specialists who were knowledgeable about the subject and were not part of the study respondents to ensure the validity and reliability of the collected data. the pre-test explored how well the questionnaire represents a theoretical concept of the study. areas of the questionnaire that could be misinterpreted were changed or left out of the study’s questions, for instance the clarification of the institution’s designation as government or public versus private or commercial and the level of health institution (primary, secondary, or tertiary). furthermore, as a result of pre-testing the questionnaire, it became necessary to allow the respondents to select all options that applied to a specific question and to provide additional information whenever necessary. ambiguous or loosely used laboratory terms were avoided, or a term definition was provided, for instance the definition of cv policy as described above and the term ‘read-back policy’. the respondents were also encouraged to provide a copy of their laboratory’s critical values for sighting. statistical analysis microsoft excel® version 15.0 (microsoft corp. 2013, redmond, washington, united states) was used to compile the data before being exported for analysis into statistical product and service solutions version 23.0 software (ibm corp. 2015, armonk, new york, united states). charts, frequency tables, counts, and percentages were used to present descriptive data. the chi-square or fisher’s exact test was used for inferential statistics to test associations, as appropriate. a p-value of 0.05 or lower was considered significant. results one hundred and thirty-four (n = 134) laboratories returned their questionnaires from a total of 185 laboratories surveyed, giving a response rate of 72.4%. forty-five (33.6%) were in northern nigeria and 89 (66.4%) in southern nigeria; 81 laboratories (60.4%) served government/public hospitals, and 118 (88.1%) were situated in tertiary centres (figure 1). figure 1: general characteristics of 134 surveyed laboratories across 25 states in nigeria, october 2015 to december 2015. critical value notification operating policy and critical value limits list sixty-nine laboratories (51.5 %) practise cvn, while 65 (48.5%) do not practise cvn. among the cvn-practising laboratories, only 23 (33.3%) have an existing written cvn policy (table 1). also, the actual list of cv limits was sighted in only 26 (37.7%) laboratories that reported practising cvn. forty-three (62.3%) laboratories used the same cvs for both adult and paediatric populations. twenty-seven (39.1%) laboratories obtained their cvs by combining published literature and local opinions from stakeholders, such as clinical and laboratory staff. table 1: aspects of critical value notification practices in 134 surveyed laboratories across 25 states in nigeria, october 2015 to december 2015. analytical handling of critical values among the laboratories that practise cvn, most (n = 66; 95.7%) repeated the assay for results with cv, mostly once (37.7%). nearly three-quarters of the laboratories (n = 51; 73.9%) had no policy to repeat tests with cvs. among the cvn-practising laboratories that repeated testing, 18 (27.3%) laboratories reported the average of the repeated results; 9 (13.6%) laboratories reported the most severe cvs generated, while 8 (12.1%) reported the least severe cvs (table 2). table 2: analytical handling of critical values in 69 laboratories practising critical value notification across 25 states in nigeria, october 2015 to december 2015. post-analytic management of critical value notification senior laboratory staff (pathologists, pathologists in-training, laboratory managers and senior laboratory scientists) take responsibility for notifying critical results in most cases (n = 49; 71.0%), while cvn is the responsibility of the technologist on the bench in 32 (46.4%) laboratories (table 3). the most common means of notification was physical dispatch (n = 42; 60.9%), followed by telephone calls (n = 38; 55.1%) and short message service (n = 8; 12.6%). nine (23.7%) laboratories with phone call notification practised a read-back policy, while 29 (76.3%) did not. thirty-four (49.3%) laboratories notified the doctor or nurse directly involved in the patient’s care (table 3). only 18 laboratories (26.1%) have mechanisms to deal with situations where the patient’s caregiver is unreachable, while 51 (73.9%) laboratories do not. in 40 (58%) laboratories, there were no specifications on the time limit to notify a caregiver of a cv. other time limits include: ‘within 10 min’ (n = 2; 3.0%), ‘within 30 min’ (n = 6; 8.7%), ‘within 60 min’ (n = 3; 4.4%), ‘within 24 min’ (n = 2; 3.0%), or no response (n = 16; 23.2%). table 3: post-analytic management of critical values in 69 laboratories practising critical value notification across 25 states in nigeria, october 2015 to december 2015. factors associated with the practise of critical value notification the practice of cvn was not related to whether the laboratory was a private establishment or a public hospital laboratory (p = 0.39) nor the level of healthcare they serviced (secondary or tertiary), p = 0.89 (table 4). private laboratories were likelier to have separate paediatric cv lists than public hospital laboratories (p = 0.02) and to practise telephone notification than public or government hospital laboratories (p < 0.001). however, the practice of telephone notification was not related to the level of healthcare service, secondary or tertiary (p = 1.00). table 4: factors associated with the practice of critical value notification of 134 surveyed laboratories across 25 states in nigeria, october 2015 to december 2015. discussion this survey showed remarkable variability in the policy and procedures of cvn across clinical laboratories in nigeria, consistent with the findings in previously published surveys on cvs.7,18,19,20,21 nearly half of the centres we studied did not consistently conduct cvn, and nearly two-thirds of those that did had neither a defined list of critical limits nor an existing guideline to govern cvn. this finding is worrisome, considering the importance of management of cvs in patient safety. moreover, most of the centres surveyed serviced secondary and tertiary centres where specialist cases are managed, and a greater emphasis on timely results for prompt action is indicated. although it is estimated that cvs comprise just about 2% of all laboratory results, laboratories are tasked with tracking this crucial part of the post-analytical stage.18,20 even though the survey’s scope did not include the reasons for not using cvn, it is conceivable that some laboratory personnel may be unaware of the value of cvn. our study’s findings differ from those of a survey conducted in croatia, where less than 1% of the laboratories did not practise cvn.18 other surveys reported between 2003 and 2011 in many high-income countries also show much higher rates.19,20 however, our result is consistent with reports in the last decade in some southeast asian and african countries, where substantial gaps in cvn practices were observed.21,22,23,24 clinical laboratories are encouraged to provide a list of cvs for their users.13,14 our findings show that the cv list was not readily available in many laboratories. there is a conspicuous unmet need for cvn, and laboratory professionals should lead in strengthening this aspect of the laboratory-clinical interface.19 the australian consensus documents recommendations also specify that laboratories should, whenever possible, give separate lists for cvs to account for unique differences in patient groups and clinical contexts. the stratification may be by age, category of wards, inpatient or outpatient status.7 for many analytes, paediatric cvs differ considerably from adult values. due to ongoing adaptations to extra-uterine environments, newborns and young children are especially prone to greater risk when alterations in biochemical composition in the body are not addressed promptly. advisedly, it becomes imperative for critical limits to provide early enough warning to enable prompt action. in the united states, national surveys linked paediatric cvn with improved outcomes.25,26 most laboratories that participated in the survey used similar critical values for paediatric and adult populations. the lack of separate lists for adult and paediatric cvs was more prominent in public hospitals than in private laboratories. this lack of separate lists for adult and paediatric cvs may reflect a limited understanding of cvn practice and a lack of input from clinicians and other relevant stakeholders. many professional organisations’ cv requirements rely heavily on published literature; however, clinicians are encouraged to contribute their professional experience.7 the findings in this study indicate that most cvn-practising laboratories assign critical limits to chosen analytes by coalescence of local stakeholder viewpoints, including those of clinical and laboratory staff and published literature. nevertheless, several laboratories exploited published works without consensus from clinical stakeholders. however, most of the laboratories in our analysis lacked literature to support their critical limits. schapkaitz et al. reported in 2014 that most laboratories in a survey of 36 clinical laboratories in south africa have cv policies derived from local clinical opinion.27 the heterogeneity in the sourcing of cvs and the guiding policies that were observed in this study may impede the harmonisation of cv lists in nigerian laboratories and has been previously noted as a challenge to cv management.18 automatic repetition for critically abnormal results is common practice in clinical laboratories.7 almost all of the laboratories in our survey repeated cvs before notification. indeed, some authors have demonstrated that, at most, there is only a modest advantage of repeat tests with cvs over a single run.28,29 besides, there are concerns about the impact of delayed results due to repeat testing on managing patients with critically abnormal results. given that many laboratories lack the high-precision autoanalysers available in high-income countries, the advantage of repeat testing for abnormally crucial results may be lost in resource-constrained settings like nigeria. to address the concern of turnaround time, laboratories should set up procedures whereby cvs are relayed immediately to the clinicians as preliminary results, while informing them of the final confirmatory result after repeat testing. this process will allow clinicians to make quick clinical judgments to determine if the test result is consistent with the patient’s condition and to prepare for the right course of action when the result is affirmed.7 we observed considerable variability in handling repeated cvs as most laboratories had no policy for handling repeat-tested cvs. after repeat testing, most laboratories reported the mean of the cvs, while others reported the most critical of the values generated or the least critical of the values. since most laboratories do not have guiding policies, the decision was left at the discretion of individual laboratory operators. although there is no consensus on which result to report, this decision may ultimately affect the frequency of test errors.7 therefore, laboratories should determine the results to report based on the peculiarities of their analytical systems and audits based on patient outcomes. our survey also shows that, most commonly, senior laboratory staff, such as pathologists, pathologists-in-training, laboratory managers and senior laboratory scientists, reported cvs. in some laboratories, however, cvn is the bench technologist’s responsibility. in a similar study in egypt, the hospital laboratory physician bore the responsibility for cvn, followed by the laboratory technician.24 it is likely that the reporting of critical values is related to the cadre of staff available in the laboratory and their numerical strength. clinical laboratories in nigeria, like many health institutions, are often understaffed.30,31 there may not be enough senior laboratory personnel to effectively cover all shifts. for effective cvn in the nigerian laboratory setting, it is, therefore, imperative that all cadres of laboratory staff be suitably trained to recognise, authenticate and handle cvs. the need for adequate training, defined responsibilities, and procedures to notify cvs should been emphasised.7,12 the most common means of notification was physical dispatch. almost half of the cvn-practising laboratories did not practise telephone notification. the definition of cvs requires prompt notification; thus, it is unlikely that physical dispatch would be able to achieve this. telephone reporting is the cornerstone of rapid communication of results in laboratories in developed countries. in a croatian survey, more than 90% of the laboratories practise telephone reporting, and in an egyptian survey, the means of cvn was mainly by telephone.18,24 in the index survey, private laboratories were more likely than public or government hospital laboratories to use telephone notification. since the majority of private laboratories are likely to view this as a crucial commercial service strategy to maintain their competitive edge, this may not come as a surprise. on the other hand, telephone service is not consistently available in many public hospital laboratories. moreover, missing contact details for persons to inform in the event of a cv may be a plausible explanation.32 in many government hospitals, electronic medical records are unavailable, and test orders are placed manually. the hand-written paper requisition forms have only the name of the requesting physician or consultant in charge, with usually no provisions for telephone contacts in case of a cv. many public hospitals in lowto middle-income countries grapple with the challenge of unreliable power supply, which may affect the use of technology in cvn.32 therefore, in developing procedures for cvn in limited resource settings, this challenge must be considered and addressed. laboratorians must liaise with clinicians and hospital management to emphasise the need to provide information for a reliable contact for cvn. timely reporting of critically abnormal results is central to effective cv management. in our survey, more than half of the laboratories did not have notification time limits for cvs. about a quarter of the laboratories reported cvs within 30 min of generating results, similar to a 2014 croatian national survey finding.18 however, a 2019 study in indonesia reported that about 99% of cvn was done within 7 min.33 most laboratories notify the doctor or nurse directly involved in the patient’s care, similar to a kuwait report in 2022, where ordering physicians and nurses were the most likely to receive a cv.34 however, most laboratories do not have mechanisms to deal with situations where the patient’s caregiver is unreachable, undermining the essence of notification of cvs for optimal patient care. many guidelines recommend that laboratories develop structured algorithms describing actions to sustain communication when caregivers are unreachable. alternative caregivers have been suggested in case of repeated failures; however, this will entail that the laboratory personnel are regularly furnished with contact information of the ordering clinicians and their surrogates.7,35,36 limitations the number of respondents for the pre-test may not have been adequate, given the eventual sample size of the study. there may have been some selection bias since the question was completed by any senior laboratory staff deemed competent by the head of the laboratory to respond to the questions. the data on how long the responding staff had worked in the institution were not obtained. therefore, some personnel may not be conversant with the cvn policies. furthermore, advanced statistical analysis was not done to explore the reasons behind the practices of cvn by the laboratories surveyed, such as links with accreditation status. however, we have been able to describe the current practices across biochemical clinical laboratories across nigeria. conclusion our research has demonstrated that cvn practices are widely variable and often suboptimal in many clinical laboratories in nigeria, which may be attributed to the absence of policies and defined procedures for cvn. the lack of national cvn recommendations or guidelines may exacerbate this situation. training and capacity building in setting up, monitoring, and auditing cvns are required to significantly enhance the practice of cvns in nigeria. this study’s findings have established a baseline for cvn that can be used to create a national consensus for cvn in nigerian laboratories. acknowledgements the authors appreciate the study participants for their participation in this study and mrs joy a. imoh for helping with statistical analysis. the authors also acknowledge prof. rajiv t. erasmus and prof. annalise e. zemlin and the international federation of clinical chemistry and laboratory medicine for their mentorship in laboratory quality management through the international federation of clinical chemistry and laboratory medicine professional management exchange programme, which partly inspired conducting this study. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions c.p.o. served as the lead investigator and coordinator of the study. c.p.o., l.c.i., i.y.m., i.d.n., e.u.e. and t.m.a. contributed to study conception, design and data acquisition. c.p.o. and l.c.i. contributed to data analysis and interpretation, and writing of the article; and c.p.o., l.c.i., i.y.m., i.d.n., e.u.e. and t.m.a. contributed to editing, reviewing and final approval of the article. all authors read and approved the final version of the manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the data sets used and analysed during the current study are available from the corresponding author, l.c.i., on reasonable request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references callen j, georgiou a, li j, westbrook ji. the impact for patient outcomes of failure to follow up on test results. how can we do better? ejifcc. 2015;26(1):38–46. dahm mr, georgiou a, westbrook ji, 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https://www.rcpath.org/resourcelibrary/out-of-hours-reporting-of-laboratory-results-requiring-urgent-clinical-action-to-primary-care--2010.html singh h, vij ms. eight recommendations for policies for communicating abnormal test results. joint comm j qual patient saf. 2010;36(5):226–232. https://doi.org/10.1016/s1553-7250(10)36037-5 acknowledgements references about the author(s) noah fongwen international diagnostics centre, london school of hygiene and tropical medicine, london, united kingdom debi boeras global health impact group, atlanta, georgia, united states rosanna w. peeling international diagnostics centre, london school of hygiene and tropical medicine, london, united kingdom timothy amukele department of pathology, johns hopkins school of medicine, johns hopkins university, baltimore, maryland, united states citation fongwen n, boeras d, peeling rw, amukele t. connected diagnostics systems: the future of disease control in africa. afr j lab med. 2020;9(2), a1365. https://doi.org/10.4102/ajlm.v9i2.1365 editorial connected diagnostics systems: the future of disease control in africa noah fongwen, debi boeras, rosanna w. peeling, timothy amukele copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the coronavirus disease 2019 (covid-19) pandemic has ushered us into a new era of global public health urgency, with diagnostics and laboratory medicine at its centre.1 to take advantage of this focus on diagnostics to leapfrog some of the barriers in lowand middle-income countries, we must first understand what is feasible and effective in our setting. this african journal of laboratory medicine’s special issue on the ‘future of diagnostics’ offers us the opportunity to examine innovations in diagnostics that are already afoot in africa. these innovations cover all three phases of testing and include a discussion of open (rather than closed) reagent systems for molecular testing, a tool for assessing the diagnostic capacity of a country, nigeria’s experience establishing laboratory networks, tuberculosis data analytics, and mobile testing, reporting and surveillance – among others. the global competition for covid-19 reagents and molecular tests has taught us that african communities must strive for self-reliance; this self-reliance starts long before testing in the laboratory begins.2 three articles in this issue address the need for changes outside the laboratory to support a better future for diagnostics. the article by emperador et al. encourages the design and deployment of open reagent systems for molecular testing rather than the closed ones we have now.3 they also discuss some ways to address the changes in reimbursement and ownership that will be needed to support such a system.3 the article by ondoa et al. presents the initial africa-wide roll-out of a powerful new tool, the joint external evaluation tool, which allows a country-wide comprehensive evaluation of diagnostic laboratory gaps and capabilities.4 finally, the article by naidoo and ihekweazu describes how nigeria built a national referral laboratory and established a country-wide specimen referral network to support national priority diseases including yellow fever, lassa fever, monkeypox, cerebrospinal meningitis, cholera, influenza and other enteric pathogens.5 networks of expert diagnostic laboratory sites for diseases of epidemic potential need to be established in a pan-african manner, so that specimens can be tested efficiently and reliably bio-banked to support local development and evaluation of new tests.6 the ability to develop and manufacture diagnostics in africa is the future of disease control in africa.7 the rapid spread of covid-19 means that countries need real-time smart data systems for evidence-based decisions on public health measures and travel restrictions – as illustrated in three articles in this issue. first, cassim et al. report on the use of an interactive dashboard at a high-volume laboratory that, when coupled with good management, dramatically improved the percentage of turn-around times within the expected range from 10% to 90%.8,9 gous and macek describe a new in-person and remote training programme, the tb data fellowship, which builds a tableau-based tuberculosis analytics capacity in lowand middle-income countries.10 rapid advances in data connectivity and digital computing, coupled with increased access to internet coverage and portable communications such as mobile phones in africa, provide us with a window of opportunity to adapt these technologies to strengthen diagnostic systems for the surveillance of diseases of epidemic potential, as well as for hiv, tuberculosis and neglected tropical diseases. a well-connected diagnostic system will cause a seismic shift in the way disease control is tackled in africa. mobile phones and other mobile surveillance systems present some examples of changes in the status quo. in senegal, epidemiological and diagnostic data on influenza-like illnesses from sentinel sites were collected and reported by phone through encrypted short message service to the ministry of health.11 peaks in the incidence of febrile syndromes were detected and the specific cause identified, leading to early notification of public health officials at the ministry of health.11 fall et al. report in this issue on a successful pilot evaluation of the use of a mobile van equipped with a biosafety laboratory (complete with internal negative pressure chambers) that was deployed for outbreak investigations.12 in tanzania, the control of neglected tropical diseases has taken a new leap forward by using mobile phones in early reporting of potential rabies cases, thus increasing compliance with post-exposure prophylaxis.13 in south africa, researchers in the africa health research institute have developed a user-friendly mobile phone diagnostic test for hiv that uses a camera and an online app that allows for self-testing and interpretation of results.14 whether this will translate to an increase in linkage to care is still being studied,15 but such an application can provide useful state-of-the art data on the transmission dynamics of hiv within communities, and has the potential to accelerate elimination efforts for hiv and other infectious diseases like syphilis and hepatitis. in malawi, the ministry of health has partnered with village reach to establish a nation-wide health information hotline called health centre by phone.16 such innovation has been critical in disseminating useful health information and advice to the lowest level of the health system and dispelling myths and rumours during the covid-19 pandemic. this special issue has highlighted that africa is a continent of qualified laboratory professionals and innovators. as new diagnostics are being developed on the continent, test developers should consider data connectivity as an essential component of any final product. connectivity of new and existing point-of-care diagnostics should be fully functional and integrated with laboratory services to form connected diagnostic systems that will support disease surveillance and outbreak responses. mobile phones are ubiquitous. we use them to access massive amounts of information and trust them with our mobile banking. they are powerful pocket computers and we should consider how we can use them to take healthcare to the last mile. the time to consider how we should harness this wealth of technology for the future of disease control in africa is now. acknowledgements competing interests the authors have declared that no competing interests exist. authors’ contributions all authors contributed equally to this work. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references lippi g, plebani m. the critical role of laboratory medicine during coronavirus disease 2019 (covid-19) and other viral outbreaks. clin chem lab med. 2020;58(7):1063–1069. https://doi.org/10.1515/cclm-2020-0240 nkengasong j. let africa into the market for covid-19 diagnostics. nature. 2020;580:565. https://doi.org/10.1038/d41586-020-01265-0 emperador dm, mazzola lt, kelly-cirino cd. an open source molecular diagnostic platform approach for outbreak and epidemic preparedness [lessons from the field]. afr j lab med. 2020;9(2):a1017. https://doi.org/10.4102/ajlm.v9i2.1017 ondoa p, ndlovu n, keita m-s, et al. preparing national tiered laboratory systems and networks to advance diagnostics in africa to meet the continent’s health agenda: insights into priority areas for improvement. afr j lab med. 2020;9(2):a1103. https://doi.org/10.4102/ajlm.v9i2.1103 naidoo d, ihekweazu c. nigeria’s efforts to strengthen laboratory diagnostics – why access to reliable and affordable diagnostics is key to building resilient laboratory systems. afr j lab med. 2020;9(2):1019. https://doi.org/10.4102/ajlm.v9i2.1019 peeling rw, boeras d, wilder-smith a, sall a, nkengasong j. need for sustainable biobanking networks for covid-19 and other diseases of epidemic potential. lancet infect dis. 2020;3099(20):1–6. https://doi.org/10.1016/s1473-3099(20)30461-8 organization for economic cooperation and development. africa’s response to covid-19: what roles for trade manufacturing and intellectual-property [homepage on the internet] [cited 2020 jun 23]. available from: http://www.oecd.org/coronavirus/policy-responses/africa-s-response-to-covid-19-what-roles-for-trade-manufacturing-and-intellectual-property-73d0dfaf/ cassim n, tepper me, coetzee lm, glencross dk. timely delivery of laboratory efficiency information, part i: developing an interactive turnaround time dashboard at a high-volume laboratory. afr j lab med. 2020;9(2):a947. https://doi.org/10.4102/ajlm.v9i2.947 cassim n, coetzee lm, tepper me, perelson l, glencross dk. timely delivery of laboratory efficiency information, part ii: assessing the impact of a turn-around time dashboard at a high-volume laboratory. afr j lab med. 2020;9(2):a948. https://doi.org/10.4102/ajlm.v9i2.948 gous n, nyaruhirira au, cunningham b, macek c. driving the usage of tb diagnostic data through capacity building in lowand middle-income countries [lessons from the field]. afr j lab med. 2020;9(2):a1092. https://doi.org/10.4102/ajlm.v9i2.1092 dia n, sarr fd, thiam d, et al. influenza-like illnesses in senegal: not only focus on influenza viruses. plos one. 2014;9(3):e93227. https://doi.org/10.1371/journal.pone.0093227 fall c, cappuyns a, faye o, et al. field evaluation of a mobile biosafety laboratory in senegal to strengthen rapid disease outbreak response and monitoring. afr j lab med. 2020;9(2):a1041. https://doi.org/10.4102/ajlm.v9i2.1041 mtema z, changalucha j, cleaveland s, et al. mobile phones as surveillance tools: implementing and evaluating a large-scale intersectoral surveillance system for rabies in tanzania. plos med. 2016;13(4):1–12. https://doi.org/10.1371/journal.pmed.1002002 i-sense united kingdom. using mobile technology to test and treat those hardest hit by hiv [homepage on the internet] [cited 2016 jun]. available from: https://www.i-sense.org.uk/news/using-mobile-technologies-test-and-treat-those-hardest-hit-hiv venter w, coleman j, chan vl, et al. improving linkage to hiv care through mobile phone apps: randomized controlled trial. jmir mhealth uhealth. 2018;6(7):e155. https://doi.org/10.2196/mhealth.8376 blauvelt c, west m, maxim l, et al. scaling up a health and nutrition hotline in malawi: the benefits of multisectoral collaboration. bmj. 2018;363:k4590. https://doi.org/10.1136/bmj.k4590 references about the author(s) iruka n. okeke department of pharmaceutical microbiology, faculty of pharmacy, university of ibadan, ibadan, nigeria citation okeke in. towards a fiercely urgent expansion of laboratory medicine in africa. afr j lab med. 2021;10(1), a1785. https://doi.org/10.4102/ajlm.v10i1.1785 editorial towards a fiercely urgent expansion of laboratory medicine in africa iruka n. okeke copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the african journal of laboratory medicine (ajlm) has published its 10th annual volume! our journal was established in 2011 on the tails of rapid and unprecedented growth of laboratory medicine in africa that began in the current century.1 the journal’s first nine volumes published articles covering virtually all scientific, social science and practice specialties in, or studying, laboratory medicine. the ajlm has also proudly curated six forward-looking special issues focused on the strengthening laboratory management towards accreditation programme (2014),2 quality assurance in point-of-care testing for hiv (2016),3 the role of the laboratory in global health security (2016),4 strengthening tuberculosis diagnostic networks in africa (2017),5 african laboratories in antimicrobial resistance surveillance (2018)6 and the future of diagnostics (2020).7 it has been especially rewarding to steer our journal in the last four years of its first decade and to simultaneously watch the reputational quality my predecessors established maintained, while growing what is now the pre-eminent laboratory medicine journal on the african continent. working during a pandemic has made the last two extraordinarily productive years doubly strenuous, for us and all other biomedical science editorial offices. at the end of 2020, our valiant, but inaccurate, assessment was that our essential pandemic contributions might be temporary.8 one year later, it is clear that the battle against coronavirus disease 2019 (covid-19) will continue for some time to come and, therefore, how we operate now must become part of our future. it is perhaps fitting that i reflect on the fact that, while the scope of our journal is very broad, 8 of 32 articles published in volume 10, excluding this editorial, relate to the pandemic. covid-19 articles make roughly a quarter of our pages; this reflects the enormous effort that laboratory medicine researchers and practitioners have put into the response. it serves as one of many records of the heavy burden the pandemic has placed on the african continent, which has made giant strides in developing laboratory medicine in the last decade. there are 24 non-covid-19 articles in volume 10 (2021), while the number of such articles published pre-pandemic in 2018 and 2019 was 17 and 29. thus, we have added pandemic scientific publishing onto our usual, and growing, ajlm output. nonetheless, we have not diverted all our focus towards the response to a single disease. for centuries, africa has been fighting infections without necessary laboratory medicine support;9 a new journal was born to document the african laboratory’s role a decade ago,1 but this substantial upsurge in published papers – just one reading of diagnostic progress – happened now. therefore, in a sense, at least some of our growth was pandemic-driven. the pandemic also forces us to accept that there is some predictability that new diseases of unpredictable nature could emerge in the future, and that scientific knowledge and laboratory capacities must be extended to meet them. a more recent, but equally pressing, rise in non-communicable disease prevalence adds to the essentiality of, and demand for, effective laboratory medicine and improved health systems, which can offer needed laboratory support for high-quality care. for all of these reasons, we should expect our journal to grow geometrically in the next decade. our editors, readers and authors must keep pace with the rising magnitude and increased diversity of laboratory operations that support clinical care. no doubt, growth is essential, but how quickly does it need to take place? synthetic evidence has shown that most patients in africa still do not have access to needed diagnostics for managing their illnesses,10 despite advances in laboratory medicine on the continent in the past two decades. even for severe acute respiratory syndrome coronavirus 2 (sars-cov-2), where we have seen phenomenal growth and a truly impressive scale of testing and laboratory-led surveillance,11,12 access to laboratory medicine for covid-19 on the african continent is still suboptimal for public health and patient management. the experts that author and read our articles are working frenetically to close the gap. the answer to the question of ‘how quickly?’ must be ‘very’. the response needed to close africa’s diagnostic gaps is not only critically important, it is fiercely urgent. the value of health systems, and the will to maintain them, will become recognisable only when the best evidence, often from laboratories, forms the basis for patient treatment and management.13 moreso, every missed or mistreated illness due to diagnostic and laboratory insufficiency is a prolonged or, often, fatal illness. once the need for any change becomes apparent, harder pushes are needed to make change happen and sometimes events coincide to produce a landscape that will help ensure that the effort invested will field results. the pandemic, although a time of great difficulty, has created such a time for laboratory medicine. we must leverage current impetus for better laboratory diagnostics to avert future disaster. one of the oft-quoted speeches of the united states’ martin luther king jr enjoins us to push when opportunity stands in front of emergency: we are now faced with the fact that tomorrow is today. we are confronted with the fierce urgency of now. in this unfolding conundrum of life and history, there ‘is’ such a thing as being too late. this is no time for apathy or complacency. this is a time for vigorous and positive action.14 as our journal, and the african society for laboratory medicine that owns it, move into their second decades, we must consider the need to grow not only expansively, but also quickly and purposefully as part of a multipronged, continent-wide and locally proclaimed effort to enhance health for africans.15 we must just as fiercely move from the motivating rhetoric of another place and era to now, and we must hold our selves, as well as others, to account.16 this is particularly true because the current pandemic has shown us that rapid but rigorous growth is possible in laboratory medicine and powerful tools exist to help get us there.17,18 we have now seen that the price of ‘rapid’ growth is insignificant compared to the cost of not growing responsively is far responsively. we must, therefore, plan to scale up laboratory medicine accordingly. references luman e. the african journal of laboratory medicine – advancing laboratory medicine and science in africa. afr j lab med. 2012;1(1):1. https://doi.org/10.4102/ajlm.v1i1.86 noble ma, martin r, ndihokubwayo j-b. making great strides in medical laboratory quality. afr j lab med. 2016;5(2):1–2. murtagh mm. quality assurance for point-of-care diagnostic testing: it is not negotiable. afr j lab med. 2016;5(2):1–2. https://doi.org/10.4102/ajlm.v5i2.554 peter t, keita m-s, nkengasong j. building laboratory capacity to combat disease outbreaks in africa. afr j lab med. 2016;5(3):1–2. https://doi.org/10.4102/ajlm.v5i3.579 piatek a. tuberculosis diagnostic networks: moving beyond the laboratory to end tuberculosis in africa. afr j lab med. 2017;6(2):1–2. https://doi.org/10.4102/ajlm.v6i2.608 kariuki s, keddy kh, antonio m, okeke in. antimicrobial resistance surveillance in africa: successes, gaps and a roadmap for the future. afr j lab med. 2018;7(2):1–2. https://doi.org/10.4102/ajlm.v7i2.924 fongwen n, boeras d, peeling rw, amukele t. connected diagnostics systems: the future of disease control in africa. afr j lab med. 2020;9(2):a1365. https://doi.org/10.4102/ajlm.v9i2.1365 okeke in. african laboratory medicine in the time of covid-19. afr j lab med. 2020;9(1):1–3. https://doi.org/10.4102/ajlm.v9i1.1447 okeke in. divining without seeds: the case for strengthening laboratory medicine in africa. ithaca, ny: ilr/cornell university press, 2011; p. 222. fleming ka, horton s, wilson ml, et al. the lancet commission on diagnostics: transforming access to diagnostics. lancet. 2021, p. 1–54. https://doi.org/10.1016/s0140-6736(21)00673-5 kapata n, ihekweazu c, ntoumi f, et al. is africa prepared for tackling the covid-19 (sars-cov-2) epidemic. lessons from past outbreaks, ongoing pan-african public health efforts, and implications for the future. int j infect dis. 2020;93:233–236. https://doi.org/10.1016/j.ijid.2020.02.049 wilkinson e, giovanetti m, tegally h, et al. a year of genomic surveillance reveals how the sars-cov-2 pandemic unfolded in africa. science. 2021;374(6566):423–431. https://doi.org/10.1126/science.abj4336 ondoa p, oskam l, loembe mm, okeke in. transforming access to diagnostics: how to turn good intentions into action? lancet. 2021. https://doi.org/10.1016/s0140-6736(21)02182-6 king ml, washington jm. a testament of hope: the essential writings of martin luther king, jr. new york: harper & row; 1986, p. 231–244. nkengasong jn, tessema sk. africa needs a new public health order to tackle infectious disease threats. cell. 2020;183(2):296–300. https://doi.org/10.1016/j.cell.2020.09.041 sirleaf ej, clark h. achieving vaccination justice: a call for global cooperation. plos global public health. 2021;1(10):e0000036. https://doi.org/10.1371/journal.pgph.0000036 okeke in, ihekweazu c. the importance of molecular diagnostics for infectious diseases in low-resource settings. nat rev microbiol. 2021;19:547–548. https://doi.org/10.1038/s41579-021-00598-5 gous nm, onyebujoh pc, abimiku a, macek c, takle j. the role of connected diagnostics in strengthening regional, national and continental african disease surveillance. afr j lab med. 2018;7(2):775. https://doi.org/10.4102/ajlm.v7i2.775 africa has suffered disproportionately although it has done little to cause the crisis the fight against the climate crisis needs all hands on deck acknowledgements references about the author(s) lukoye atwoli department of internal medicine, medical college east africa, the aga khan university, nairobi, kenya brain and mind institute, the aga khan university, nairobi, kenya gregory e. erhabor department of medicine, college of health sciences, obafemi awolowo university, ile-ife, nigeria chest unit, obafemi awolowo university teaching hospital, ile-ife, nigeria aiah a. gbakima department of technical and higher education, government of sierra leone, freetown, sierra leone abraham haileamlak college of public health and medical sciences, jimma university, jimma, ethiopia jean-marie kayembe ntumba department of pneumology, faculty of medicine, university of kinshasa, kinshasa, the democratic republic of the congo james kigera department of human anatomy, college of health sciences, university of nairobi, nairobi, kenya laurie laybourn-langton department of sustainability accelerator, chatham house, london, united kingdom robert mash division of family medicine and primary care, faculty of medicine and health sciences, stellenbosch university, cape town, south africa joy muhia centre for global mental health, london school of hygiene and tropical medicine, london, united kingdom fhumulani m. mulaudzi department of nursing science, faculty of health sciences, university of pretoria, pretoria, south africa david ofori-adjei department of medicine and therapeutics, university of ghana, accra, ghana friday okonofua department of obstetrics and gynecology, university of medical sciences, ondo, nigeria arash rashidian department of science, information and dissemination, eastern mediterranean regional office, world health organization, cairo, egypt maha el-adawy department of health protection and promotion, eastern mediterranean regional office, world health organization, cairo, egypt siaka sidibé faculty of medicine and odonto-stomatology, university of sciences, techniques and technology of bamako, bamako, mali abdelmadjid snouber faculty of medicine, university of oran 1, es sénia, algeria james tumwine department of paediatrics and child health, school of medicine, kabale university, kabale, uganda sahar yassien mohammad faculty of nursing, ain shams university, cairo, egypt paul yonga ca medlynks clinic and laboratory, nairobi, kenya nairobi fountain projects and research office (fopro), fountain health care hospital, eldoret, kenya lilia zakhama faculty of medicine of tunis, university of tunis el manar, tunis, tunisia department of cardiology, security forces hospital, la marsa, tunisia chris zielinski centre for global health, faculty of health and wellbeing, university of winchester, winchester, united kingdom citation atwoli l, erhabor ge, gbakima aa, et al. cop27 climate change conference: urgent action needed for africa and the world. afr j lab med. 2022;11(1), a2080. https://doi.org/10.4102/ajlm.v11i1.2080 editorial cop27 climate change conference: urgent action needed for africa and the world lukoye atwoli, gregory e. erhabor, aiah a. gbakima, abraham haileamlak, jean-marie kayembe ntumba, james kigera, laurie laybourn-langton, robert mash, joy muhia, fhumulani m. mulaudzi, david ofori-adjei, friday okonofua, arash rashidian, maha el-adawy, siaka sidibé, abdelmadjid snouber, james tumwine, sahar yassien mohammad, paul yonga, lilia zakhama, chris zielinski copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. wealthy nations must step up support for africa and vulnerable countries in addressing past, present and future impacts of climate change the 2022 report of the intergovernmental panel on climate change (ipcc) paints a dark picture of the future of life on earth, characterised by ecosystem collapse, species extinction, and climate hazards such as heatwaves and floods.1 these are all linked to physical and mental health problems, with direct and indirect consequences of increased morbidity and mortality. to avoid these catastrophic health effects across all regions of the globe, there is broad agreement – as 231 health journals argued together in 2021 – that the rise in global temperature must be limited to less than 1.5°c compared with pre-industrial levels. while the paris agreement of 2015 outlines a global action framework that incorporates providing climate finance to developing countries, this support has yet to materialise.2 cop27 is the fifth conference of the parties (cop) to be organised in africa since its inception in 1995. ahead of this meeting, we – as health journal editors from across the continent – call for urgent action to ensure it is the cop that finally delivers climate justice for africa and vulnerable countries. this is essential not just for the health of those countries, but for the health of the whole world. africa has suffered disproportionately although it has done little to cause the crisis the climate crisis has had an impact on the environmental and social determinants of health across africa, leading to devastating health effects.3 impacts on health can result directly from environmental shocks and indirectly through socially mediated effects.4 climate change-related risks in africa include flooding, drought, heatwaves, reduced food production, and reduced labour productivity.5 droughts in sub-saharan africa have tripled between 1970–1979 and 2010–2019.6 in 2018, devastating cyclones impacted 2.2 million people in malawi, mozambique and zimbabwe.6 in west and central africa, severe flooding resulted in mortality and forced migration from loss of shelter, cultivated land, and livestock.7 changes in vector ecology brought about by floods and damage to environmental hygiene has led to increases in diseases across sub-saharan africa, with rises in malaria, dengue fever, lassa fever, rift valley fever, lyme disease, ebola virus, west nile virus and other infections.8,9 rising sea levels reduce water quality, leading to water-borne diseases, including diarrhoeal diseases, a leading cause of mortality in africa.8 extreme weather damages water and food supply, increasing food insecurity and malnutrition, which causes 1.7 million deaths annually in africa.10 according to the food and agriculture organization of the united nations, malnutrition has increased by almost 50% since 2012, owing to the central role agriculture plays in african economies.11 environmental shocks and their knock-on effects also cause severe harm to mental health.12 in all, it is estimated that the climate crisis has destroyed a fifth of the gross domestic product (gdp) of the countries most vulnerable to climate shocks.13 the damage to africa should be of supreme concern to all nations. this is partly for moral reasons. it is highly unjust that the most impacted nations have contributed the least to global cumulative emissions, which are driving the climate crisis and its increasingly severe effects. north america and europe have contributed 62% of carbon dioxide emissions since the industrial revolution, whereas africa has contributed only 3%.14 the fight against the climate crisis needs all hands on deck yet it is not just for moral reasons that all nations should be concerned for africa. the acute and chronic impacts of the climate crisis create problems like poverty, infectious disease, forced migration, and conflict that spread through globalised systems.6,15 these knock-on impacts affect all nations. covid-19 served as a wake-up call to these global dynamics and it is no coincidence that health professionals have been active in identifying and responding to the consequences of growing systemic risks to health. but the lessons of the covid-19 pandemic should not be limited to pandemic risk.16,17 instead, it is imperative that the suffering of frontline nations, including those in africa, be the core consideration at cop27: in an interconnected world, leaving countries to the mercy of environmental shocks creates instability that has severe consequences for all nations. the primary focus of climate summits remains to rapidly reduce emissions so that global temperature rises are kept to below 1.5 °c. this will limit the harm. but, for africa and other vulnerable regions, this harm is already severe. achieving the promised target of providing $100bn of climate finance a year is now globally critical if we are to forestall the systemic risks of leaving societies in crisis. this can be done by ensuring these resources focus on increasing resilience to the existing and inevitable future impacts of the climate crisis, as well as on supporting vulnerable nations to reduce their greenhouse gas emissions: a parity of esteem between adaptation and mitigation. these resources should come through grants not loans, and be urgently scaled up before the current review period of 2025. they must put health system resilience at the forefront, as the compounding crises caused by the climate crisis often manifest in acute health problems. financing adaptation will be more cost-effective than relying on disaster relief. some progress has been made on adaptation in africa and around the world, including early warning systems and infrastructure to defend against extremes. but frontline nations are not compensated for impacts from a crisis they did not cause. this is not only unfair, but also drives the spiral of global destabilisation, as nations pour money into responding to disasters, but can no longer afford to pay for greater resilience or to reduce the root problem through emissions reductions. a financing facility for loss and damage must now be introduced, providing additional resources beyond those given for mitigation and adaptation. this must go beyond the failures of cop26 where the suggestion of such a facility was downgraded to ‘a dialogue’.18 the climate crisis is a product of global inaction, and comes at great cost not only to disproportionately impacted african countries, but to the whole world. africa is united with other frontline regions in urging wealthy nations to finally step up, if for no other reason than that the crises in africa will sooner rather than later spread and engulf all corners of the globe, by which time it may be too late to effectively respond. if so far they have failed to be persuaded by moral arguments, then hopefully their self-interest will now prevail. acknowledgements this editorial is being published simultaneously in multiple journals. for the full list of journals see: https://www.bmj.com/content/full-list-authors-and-signatories-climate-emergency-editorial-october-2022. competing interests we have read and understood the bmj policy on declaration of interests and declare the following roles and relationships: j.k. is the ex-officio, president and secretary of the kenya orthopedic association, and the editor in chief of annals of african surgery. r.m. received a research grant on climate change and primary health care in africa with university ghent, funded by vlir (flemish interuniversity network). p.y. received payment for a cme lecture on antimicrobial use in the intensive care unit (icu) on behalf of novartis, for an educational event on antimicrobial stewardship on behalf of biomerieux, and for a cme lecture on adult vaccination updates on behalf of pfizer. p.y. also serves on the dsmb of the nhlbi-funded strengths trial focusing on hypertension and has served as a chair on a pfizer advisory board on pneumococcal vaccinations in adults. payment is received for both. c.z. received payment from the uk health alliance on climate change as senior advisor on the international journals project. j.m. is an unpaid board member on the international working group for health systems strengthening and employee of london school of hygiene & tropical medicine. doa has involvement with the inovio pharmaceuticals phase 1b vaccine trial and the glico healthcare ltd. the authors declare no further conflicts of interest beyond those inherent in the editorial roles listed above. authors’ contributions l.l-l. developed the idea of the editorial and led drafting along with c.z. j.m. contributed with l.a.’s guidance. all other authors contributed significantly to the editorial content. authors’ affiliated journals lukoye atwoli, editor-in-chief, east african medical journal; gregory e. erhabor, editor-in-chief, west african journal of medicine; aiah a. gbakima, editor-in-chief, sierra leone journal of biomedical research; abraham haileamlak, editor-in-chief, ethiopian jo urnal of health sciences; jean-marie kayembe ntumba, chief editor, annales africaines de medecine; james kigera, editor-in-chief, annals of african surgery; laurie laybourn-langton, university of exeter; bob mash, editor-in-chief, african journal of primar y health care & family medicine; joy muhia, london school of medicine and tropical hygiene; fhumulani mavis mulaudzi, editor-in-chief, curationis; david ofori-adjei, editor-in-chief, ghana medical journal; friday okonofua, editor-in-chief, african journal of reproductive health; arash rashidian, executive editor, and maha el-adawy, director of health promotion, eastern mediterranean health journal; siaka sidibé, director of publication, mali médical; abdelmadjid snouber, managing editor, journal de la facul té de médecine d’oran; james tumwine, editor-in-chief, african health sciences; mohammad sahar yassien, editor-in-chief, evidence-based nursing research; paul yonga, managing editor, east african medical journal; lilia zakhama, editor-in-chief, la tunisie médicale; chris zielinski, university of winchester. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. funding information respective authors were paid by their employers. chris zielinski’s time was funded by the uk health alliance on climate change. data availability data sharing is not applicable to this article, as no new data were created or analysed in this study. disclaimer the views and opinions in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references ipcc. climate change 2022: impacts, adaptation and vulnerability. working group ii contribution to the ipcc sixth assessment report. cambridge, uk and new york, ny: cambridge university press; 2022. un. the paris agreement [homepage on the internet]. united nations; 2022 [cited 2022 sep 12]. available from: https://www.un.org/en/climatechange/paris-agreement climate investment funds. climate change and health in sub-saharan africa: the case of uganda. climate investment funds; 2020. who. strengthening health resilience to climate change. geneva: world health organization; 2016. trisos ch, adelekan io, totin e, et al. africa [homepage on the internet]. in: climate change 2022: impacts, adaptation, and vulnerability. cambridge, uk and new york, ny: cambridge university press; 2022 [cited 2022 sep 26]. available from: https://www.ipcc.ch/report/ar6/wg2/ world bank. climate change adaptation and economic transformation in sub-saharan africa. washington, dc: world bank; 2021. opoku sk, leal filho w, hubert f, adejumo o. climate change and health preparedness in africa: analysing trends in six african countries. int j environ res public health. 2021;18(9):4672. https://doi.org/10.3390/ijerph18094672 evans m, munslow b. climate change, health, and conflict in africa’s arc of instability. perspectives in public health. 2021;141(6):338–341. https://doi.org/10.1177/17579139211058299 anugwom e.e. reflections on climate change and public health in africa in an era of global pandemic. in: stawicki sp, papadimos tj, galwankar sc, miller ac, firstenberg ms, editors. contemporary developments and perspectives in international health security volume 2 [homepage on the internet]. london: intechopen; 2021 [cited 2022 sep 12]. available from: https://www.intechopen.com/chapters/76312 african climate policy centre. climate change and health in africa: issues and options [homepage on the internet]. african climate policy centre; 2013 [cited 2022 sep 12]. available from: https://archive.uneca.org/sites/default/files/publicationfiles/policy_brief_12_climate_change_and_health_in_africa_issues_and_options.pdf un. climate change is an increasing threat to africa [homepage on the internet]. 2020 [cited 2022 sep 12]. available from: https://unfccc.int/news/climate-change-is-an-increasing-threat-to-africa atwoli l, muhia j, merali z. mental health and climate change in africa. bjpsych international. 2022;19(4):86–89. https://doi.org/10.1192/bji.2022.14 vulnerable twenty group. climate vulnerable economies loss report. switzerland: vulnerable twenty group; 2020. ritchie h. who has contributed most to global co2 emissions? our world in data [homepage on the internet]. 2019 [cited 2022 sep 12]. available from: https://ourworldindata.org/contributed-most-global-co2 bilotta n, botti f. paving the way for greener central banks. current trends and future developments around the globe. rome: edizioni nuova cultura for istituto affari internazionali (iai); 2022. who. cop26 special report on climate change and health: the health argument for climate action. geneva: world health organization; 2021. al-mandhari a, al-yousfi a, malkawi m, el-adawy m. ‘our planet, our health’: saving lives, promoting health and attaining well-being by protecting the planet – the eastern mediterranean perspectives. east mediterr health j. 2022;28(4):247–248. https://doi.org/10.26719/2022.28.4.247 evans s, gabbatiss j, mcsweeney r, et al. cop26: key outcomes agreed at the un climate talks in glasgow. carbon brief [homepage on the internet]. 2021 [cited 2022 sep 12]. available from: https://www.carbonbrief.org/cop26-key-outcomes-agreed-at-the-un-climate-talks-in-glasgow/ abstract background description of the intervention lessons learnt recommendations acknowledgements references about the author(s) anafi mataka african society for laboratory medicine, addis ababa, ethiopia esther a.j. tumbare elizabeth glaser pediatric aids foundation (egpaf), maseru, lesotho tsietso motsoane laboratory services, ministry of health, maseru, lesotho david holtzman partners in health, maseru, lesotho monkoe leqheka laboratory services, ministry of health, maseru, lesotho kolisang phatsoane laboratory services, ministry of health, maseru, lesotho emma sacks school of public health, george washington university, washington, district of columbia, united states anthony isavwa elizabeth glaser pediatric aids foundation (egpaf), nairobi, kenya appolinaire tiam elizabeth glaser pediatric aids foundation (egpaf), washington, district of columbia, united states how to cite this article: mataka a, tumbare eaj, motsoane t, et al. strategic site selection for placement of hiv early infant diagnosis point-of-care technology within a national diagnostic network in lesotho. afr j lab med. 2021;10(1), a1156. https://doi.org/10.4102/ajlm.v10i1.1156 lessons from the field strategic site selection for placement of hiv early infant diagnosis point-of-care technology within a national diagnostic network in lesotho anafi mataka, esther a.j. tumbare, tsietso motsoane, david holtzman, monkoe leqheka, kolisang phatsoane, emma sacks, anthony isavwa, appolinaire tiam received: 23 dec. 2019; accepted: 28 may. 2021; published: 24 aug. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: new technologies for rapid point-of-care (poc) diagnostic tests hold great potential for improving the health outcomes of hiv-exposed infants. poc testing for hiv early infant diagnosis (eid) was introduced in lesotho in late 2016. here we highlight critical requirements for selecting routine poc eid sites to ensure a sustainable and optimised eid diagnostic network. intervention: lesotho introduced poc eid in a phased approach that included assessments of national databases to identify sites with high test volumes, the creation of local networks of sites to potentially increase access to poc eid, and a standardised capacity assessment to determine site readiness. potential site networks comprising ‘hub’ testing sites and ‘spoke’ specimen referring sites were created. lessons learnt: after determining optimal placement, a total of 29 testing facilities were selected for placement of poc eid to potentially increase access to 189 facilities through the use of a hub-and-spoke model. site capacity assessments identified vital human resources and infrastructure capacity gaps that needed to be addressed before introducing poc eid and informed appropriate poc platform selection. recommendations: poc placement involves more than just purchasing the testing platforms. considering the relatively small proportion of sites that can be eligible for placement of a poc platform, utilising a hub-and-spoke model can maximise the number of health facilities served by a poc platform while reducing the necessary capacity building and infrastructure investments to fewer sites. keywords: hiv early infant diagnosis; point-of-care; increased health access; site selection. background in lesotho, a country with a high burden of hiv infection of nearly one in every four pregnant women and adults aged between 15 years and 49 years,1 antenatal care and hiv treatment for women and children are delivered at three levels: primary, secondary and tertiary. at the primary healthcare level are health centres, health posts and all community-level outreaches. district hospitals serve as next level referral facilities for all health centres in the district and refer cases to a national referral hospital at the tertiary level. laboratory services are structured similarly. however, in 2016 only one laboratory, the national reference laboratory (nrl) in the capital city, processed dried blood spot (dbs) samples for hiv early infant diagnosis (eid) testing, and this was done using conventional complex laboratory equipment. the dbs samples were transported from various health facilities all over the country to the nrl using a motorcycle-based national sample transport system. one of the main challenges with this centralised eid system was long turn-around times (tats) – typically between 30 days and 90 days – for returning results to local health centres and caregivers.2,3 reasons for the long tats include delays at the clinics before specimens are dispatched to the district hospital laboratory and delays in sending samples from the district hospital laboratory to the nrl. also, at the nrl, waiting to batch samples before analysis to allow sufficient numbers for full test runs on the instruments added to the long tat. further delays occur during the return of paper-based results to clinics through the same sample transport system before finally reaching caregivers, who may not have telephone access to facilitate quicker result communication or live far from facilities. to strengthen this centralised system, the ministry of health has implemented mechanisms such as routine training on dbs collection to ensure the quality of specimens and reduce rejection rates and provision of more motorcycles to increase the pick-up frequency of dbs and other clinical samples from facilities; however, many challenges remain. new technologies for rapid, point-of-care (poc) molecular diagnostic tests hold great potential for improving timely management of infants with hiv infection by eliminating delays in the return of results and enabling rapid initiation of antiretroviral therapy.4,5 lesotho embraced poc eid testing in 2016, making it one of the first countries to introduce the technology into routine clinical settings. the poc platforms were initially thought to be particularly useful for hard-to-reach areas, where delays in result return were often the longest. however, given the large numbers of primary and secondary health facilities (255 such facilities in lesotho) and the limited number of platforms that were available due to funding constraints, not all health facilities or service delivery points could receive poc eid platforms. hence, strategic placement of the poc eid platforms was necessary. before 2016, poc eid testing was primarily introduced in controlled environments such as research and pilot projects in sub-saharan africa.5,6,7 many poc platforms for various diagnostic tests have been deployed in healthcare settings. however, few implementers have tried to describe and explain the actual real-life process for introducing and integrating a new poc into a national health system for routine use. a better understanding of how the introduction of new poc technologies is implemented could help us anticipate possible challenges and identify elements needed for successful implementation in other limited-resource settings. this article aims to share lesotho’s experiences of a systematic approach to identifying and assessing the readiness of potential poc eid sites and to highlight the critical requirements for selecting sites to integrate routine poc eid into a sustainable and optimised eid diagnostic network. description of the intervention ethical considerations the approval to conduct site selection and capacity assessments was obtained from the lesotho ministry of health research and ethics committee (approval number id 29-2016, dated 13 january 2016). setting the processes of selecting sites eligible for poc eid testing, assessing site capacity and needs, and designing the poc eid network were carried out for all the ten districts of lesotho in the year 2016. the geography of lesotho’s highly mountainous kingdom poses many challenges to providing high-quality healthcare because of the difficulties to reach the remote facilities, especially those high up the mountains. the site selection was carried out in two main phases: an initial desk analysis to determine potential sites that could access poc eid and capacity assessments to determine the readiness for implementation of poc testing. desk analysis to determine potential health facilities eligible for poc eid two data sources were used in the desk analysis, namely the 2014 national population projections (unpublished) for catchment areas of health facilities (n = 158) in rural, peri-urban and urban settings and the ministry of health’s laboratory information system. data on the expected volume of eid tests in each site were extracted; these data were based on the highest number of expected pregnancies from hiv-positive women, the expected number of hiv-exposed infants, or historical dna polymerase chain reaction tests. data was also collected on the availability of onsite antiretroviral therapy or paediatric antiretroviral therapy services, facilitating the ability to use results immediately in a real poc setup. the ministry of health’s laboratory information system was queried in march 2016 for all eid test samples collected from hiv-exposed infants in the 255 facilities nationwide in the previous year (january 2015 to december 2015). the data were disaggregated to total samples per site in a year. to get testing rates per day, the annual test volume was divided by 12 months, following which the result was divided by 22, which is the number of working days per month. a threshold of 0.5 tests per day (at least one test every two days) was set as the minimum test volume for sites that would receive a poc platform. this threshold was regarded as sufficient to maintain operator competency and provided a throughput of tests high enough to justify the cost of the poc platforms. using a hub-and-spoke model, several health facilities with low test volumes were combined with other low-volume sites or with sites with higher test volumes to form local testing networks that reached or exceeded the threshold of 0.5 tests per day. in this model, the sites were grouped around a central location (hub) with a testing platform that would serve the surrounding health facilities (spokes). criteria for eligibility of a spoke site included proximity to the nearest potential poc hub site and the existence of a sample transport system to a nearby possible hub site. proximity was defined as facilities that were, on average, no further than 60 km from the possible testing site. spoke sites are required to send samples using sample transport carriers to a nearby hub site that provides onsite poc eid testing service, thus eligibility was also based on the ability to send samples in ethylenediaminetetraacetic acid-treated capillary tubes according to laboratory and manufacturers’ standards. the latter recommends specimens to be carried within 24 h before testing if kept at ambient temperature and within three days if kept and transported between 2 °c and 8 °c. these hubs and spokes already existed as part of the national sample transport system; hence, no additional transport or new routes were created. eligible sites were then scheduled for site capacity assessments to determine their readiness for poc eid introduction. the first 15 sites assessed were located in rural, peri-urban and urban settings across all the 10 districts. the remaining 14 testing sites were also scheduled to undergo capacity assessments later and were placed in a ‘scale-up’ phase of the implementation. site capacity assessments of selected poc eid sites capacity assessments were conducted using a standardised checklist adapted from the stepwise process for improving the quality of hiv-related point-of-care testing checklist version 2.0, 16 september 2014.8 the stepwise process for improving the quality of hiv-related point-of-care testing checklist attempts to harmonise with international regulations to evaluate sites consistently. the adapted tool has eight domains and 49 questions. each domain had questions on how well the facility performs specific tasks. based on the findings, each of the eight domains was given a weighted score. the total for all the domains was computed to arrive at a numerical percentage score. site readiness level, ranging from level 0 to level 4, was determined based on these scores. level 0 (0% – 40%) sites were those that needed improvement in all areas, level 1 (40% – 59%) sites required improvement in specific areas, while level 2 (60% – 79%) sites were partially eligible. level 3 (80% – 89%) sites were close to pilot site capacity but needed some upgrades or improvements, while level 4 (90% or higher) sites were fully eligible for selection as pilot sites. data management and analysis descriptive analyses (percentage scores by checklist domain for each health facility and median scores per checklist domain) were carried out using microsoft excel (microsoft corporation, redmond, washington, united states). statistical comparisons between facilities that underwent capacity assessments were not conducted due to the small sample size (n = 15). data on historical laboratory eid test volumes were exported into an excel database to calculate the test rate per site and aggregated test volumes for each potential local testing network. lessons learnt this article presents the steps taken before the introduction of poc platforms in lesotho, including health facility pre-selection, data analysis aimed at maximising the use of available poc platforms, and the determination of site readiness for poc eid. desk analysis to increase access to poc eid testing important considerations for setting up poc eid testing at a health facility include sufficient test volumes of at least one test every two days and the availability of hiv treatment services or referral treatment, including paediatrics, at the health facility or within a set distance to allow quick referrals. the geographic locations are also an important consideration as tats for delivery of eid results from the centralised laboratory may be prolonged in hard-to-reach areas and affect the ability to monitor the performance of eid testing. most of the sites assessed had test volumes that were too low to justify the placement of an instrument. creating local testing networks to increase testing volumes can improve access in underserved areas that are very far from the nrl. we considered all facilities with records of having collected samples for polymerase chain reaction from infants in the previous year. it has been demonstrated that some areas in hard-to-reach regions have challenges sending samples to the national laboratories and often have very long tats from sample collection to return of results.3 thus, in developing an optimised network map for both conventional and poc eid testing, poc testing should be considered not only as a tool for hard-to-reach areas but also to reduce tat from sample collection to return of results. the availability of systems for sample transport to the hubs with poc platforms would mean that although these sites would not get the eid result on the same day, they would get them in a few days since most of the sites were serviced by the sample transport system at least twice a week. this would be a potentially significant improvement compared to conventional eid when specimens are processed in the conventional laboratory, where results often took quite long (usually months) to reach the caregiver for several reasons.3 these poc sites would also take some of the burden off conventional laboratories that are overloaded with viral load (vl) testing and improve tat. following the desk analysis, in which a total of 255 sites were analysed, poc eid was determined to be potentially suitable for introduction into 29 testing sites. through the use of the hub-and-spoke model, it was found that access to poc eid could be increased 6.5-fold from 29 to 189 sites; five of the 29 sites were stand-alone testing sites with no associated spoke sites and 24 were testing hubs that were designated to receive samples from an additional 160 spoke sites (table 1). thus, in total, 189 sites were selected for possible access to poc eid based on the hub-and-spoke model, most (88%; 166/189) of which had fewer than one test every two days (< 0.5 eid/day). the remaining 66 of the 255 sites would continue to be served by the conventional system because of their proximity to the nrl and the sample transport network. table 1: outcomes of the desk analysis for site selection and mapping for placement of hiv point-of-care early infant diagnosis using a hub-and-spoke model in lesotho, 2016. during the assessment of each spoke site, it was essential to assess distance and accessibility to the nearby potential testing site in terms of how samples could be transported in ethylenediaminetetraacetic acid-treated capillary tubes according to laboratory and manufacturers’ standards. for the optimal quality of samples, the facility should be within 60 min of the hub site by normal transport modes. where this is not possible, a dbs sample becomes ideal; this is in place in many countries. however, the challenge of long tats for results delivery still exists. therefore, governments need to strengthen systems to improve the value of using alternative sample types for eid and vl.9 creating a local hub-and-spoke network contributes to strengthening the overall network by potentially reducing tat and placing less burden on the conventional system that could use the space for vl testing. due to the high workload at the nrl, which had a backlog, eid was run only 1–2 days per week. the platforms selected for potential poc facilities, namely the cepheid genexpert (gx-vi) four-module instrument (cepheid, sunnyvale, california, united states) and the m-pima (abbott laboratories, chicago, illinois, united states), formerly the alere q, are capable of conducting other tests beyond hiv eid. for example, both can do hiv vl testing, while the cepheid genexpert can also test for other infections such as tuberculosis, hepatitis, human papillomavirus and more.10 however, at the time of selection, there was no evidence of the feasibility of poc testing for hiv vl monitoring. since these were new, it was decided to start testing for eid and not perform integrated testing. point-of-care site capacity assessments a total of 15 sites with the highest test volumes were assessed for capacity to conduct poc eid testing. of the 15 facilities assessed, only one facility was fully eligible (level 4) for implementation of poc eid (table 2). the majority (n = 14) were not immediately ready to implement new poc eid testing and required structural upgrades, including process-related improvements. the required infrastructural improvements included providing room and tables for poc platforms and shelves to store poc commodities, providing fridge thermometers, and installing air conditioners and power inverters or backup for some sites. required process-related improvements included improving site-level stock management through training, integrating standardised forms to document patient and specimen information, as well as printing and distributing eid testing algorithms, job aids, logbooks, and poc eid testing forms. documentation of standard operating procedures, safety practices and data management mechanisms also needed to be improved in all facilities (table 3). also, competent poc platform operators were required to be present or recruited in all facilities. across all health facilities, the highest median scores were obtained in integrating poc services into hiv care (100%), which evaluated the prior experience in poc testing for any disease, such as rapid testing for hiv and malaria. this was followed by the domains on quality control (92%) and safety practices (92%). the lowest median scores were obtained in the supplies, reagents and equipment management domain (57%) and the personnel training, competency and certification domain (67%) (figure 1). figure 1: median performance scores per stepwise process for improving the quality of hiv-related point-of-care testing checklist domain of potential point-of-care early infant diagnosis health facilities (n = 15) in lesotho, february 2016. table 2: readiness of selected health facilities in lesotho (n = 15) in 2016 to introduce hiv point-of-care early infant diagnosis based on a standardised tool adapted from the stepwise process for improving the quality of hiv-related point-of-care testing checklist version 2.0. 9/16/2014. table 3: common gaps identified during the site capacity assessments of health facilities (n = 15) in lesotho, 2016. there was adequate space in all the 15 facilities where poc testing equipment could be placed, although not all were designated for poc. all facilities had reliable paediatric hiv counselling and treatment services at the facility or within a reasonable distance. however, despite the prior experience of conducting poc tests for rapid hiv or any other disease, there were several common gaps across all health facilities. several sites had inadequate security or lacked room temperature monitoring at the poc testing or storage areas for commodities. the facilities also had poor documentation, did not sufficiently manage the supplies of already existing poc tests and lacked storage capacity for poc commodities (table 3). before the introduction of poc eid in these sites, room temperature monitoring thermometers, security padlocks, tables, and cabinets to store poc commodities had to be procured. in addition to equipment-related training, all poc testing personnel were trained on supplies and commodities management, safety, pre-testing, and post-testing procedures, including result management and poc eid use in the existing national eid algorithm. since poc eid was new in the country, a testing algorithm for use at poc sites was developed and provided to facilities as a reference guide. the algorithm followed the same testing interval as the existing national algorithm. still, it specified how to handle test results, given that tests are available on the same day, which was not the case with the current centralised system. another critical consideration was the duration of the steps taken to introduce poc testing. the site selection process took three months, while the procurement, including site upgrades, took nearly four months to complete. it is therefore essential to take into account the time lag, including procurement lead times for commodities. where possible, it is best to consider procuring materials for upgrades locally to reduce lead times associated with external procurement. the procurement of poc eid platforms was planned to suit each site’s needs-based infrastructure, availability of reliable electricity, dynamics of daily patient testing volumes, and other characteristics as determined on the findings of the physical capacity assessments. the selection was informed by a side-by-side analysis, using a tool12 that compared three platforms based on various characteristics such as maximum throughput, storage temperature of reagents, power requirements, type of sample (whole blood or capillary), time to result, waste management needs and regulatory approvals. many of these elements were assessed during the capacity assessment. the cepheid genexpert four-module instrument and a laptop computer were placed in 16 sites, and the m-pima, formerly the alere q, was found to be suitable for 13 of the sites. a phased rollout plan was developed, which involved the implementation of five sites (three hubs and two stand-alone sites) in the first phase, the implementation of a further ten sites after 6 months with the modification of appropriate steps based on lessons learned in the first phase, and the implementation of 14 additional sites in a final scale-up phase after 12 months. at the time of writing, a total of 23 hubs with 131 spokes and six stand-alone sites had been implemented. we used historical testing rates to estimate expected test volumes. one limitation of this method is that we could not fully guarantee the same test volumes in future because other variables such as population movements and birth rates could change. while the results of the site selection and network mapping process showed that poc eid could potentially increase access to testing, a post evaluation is needed to verify this potential impact in this setting. recent data from the multi-country evaluation showed that poc eid resulted in better outcomes for hiv-exposed and hiv-positive infants. the data, which included lesotho, showed that compared to conventional laboratory testing,12 there was a five-fold increase in the proportion of infants receiving eid results within 30 days of the eid test and the number of infants initiated on antiretroviral therapy within 2 months more than doubled. given the many steps involved in introducing poc testing in a country and the data requirements to make evidence-based decisions, all key stakeholders, must be identified and consulted. stakeholders’ participation should be coordinated, preferably by the directorate of laboratories or equivalent leadership for diagnostics in a country. such stakeholders include development partners who may also have plans to roll out poc in specific areas, other ministries such as the information ministry to advise on data connectivity, and environmental units to guide waste management. clinicians and key technical working groups such as the prevention of mother to child transmission and the laboratory technical working groups are also important stakeholders in their advisory roles for the ministry of health. supply chain and procurement units need to be looped in early to avoid unnecessary delays for procured materials. recommendations our site selection process suggests that placement of poc eid platforms at well-prepared sites within diagnostic networks in limited-resource settings can improve access and that optimal introduction of poc eid transcends mere poc platform procurement. for pre-selected potential sites meeting all preliminary criteria, more detailed site capacity assessments of human resources and infrastructure capacity are required. in the case of many sites being ineligible for a poc platform based on insufficient testing volumes, and considering that the vast majority of them require some level of capacity building or infrastructure upgrade, a hub-and-spoke model can be adopted to potentially increase access to poc eid. acknowledgements the authors wish to thank the poc eid teams in lesotho and the elizabeth glaser pediatric aids foundation headquarters, the ministry of health in lesotho, and the laboratory and health worker staff at all participating facilities. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions a.m. drafted the manuscript, contributed to the data analysis and revised the manuscript. e.a.j.t. and d.h. contributed to the study design and data analysis and revised the manuscript, and collected data. m.l. and k.p. collected data and revised the manuscript. t.m., e.s. and a.i. analysed and interpreted the data and revised the manuscript. a.t. contributed to the design of the study and revised the manuscript. all authors gave final approval of the version to be published and agree to be accountable for the accuracy and integrity of the work. sources of support the introduction of poc eid in lesotho was made possible through funding from unitaid, ‘introduction of point-of-care early infant diagnosis in decentralized settings: creating a market for affordable, effective, and equitable hiv testing of exposed infants’. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and 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[cited 2021 feb 16]. available from: https://www.cepheid.com/en/tests bianchi f, cohn j, sacks e, et al. evaluation of a routine point-of-care intervention for early infant diagnosis of hiv: an observational study in eight african countries. lancet hiv. 2019;6(6):e373–e381. https://doi.org/10.1016/s2352-3018(19)30033-5 abstract introduction methods results discussion acknowledgements references about the author(s) francina kaindjee-tjituka directorate of special programs, ministry of health and social services, windhoek, namibia souleymane sawadogo us centers for disease control and prevention, windhoek, namibia graham mutandi us centers for disease control and prevention, windhoek, namibia andrew d. maher us centers for disease control and prevention, windhoek, namibia natanael salomo us centers for disease control and prevention, windhoek, namibia claudia mbapaha us centers for disease control and prevention, windhoek, namibia marytha neo us centers for disease control and prevention, windhoek, namibia anita beukes us centers for disease control and prevention, windhoek, namibia justice gweshe directorate of special programs, ministry of health and social services, windhoek, namibia alexinah muadinohamba directorate of special programs, ministry of health and social services, windhoek, namibia david w. lowrance directorate of special programs, ministry of health and social services, windhoek, namibia citation kaindjee-tjituka f, sawadogo s, mutandi g, et al. task-shifting point-of-care cd4+ testing to lay health workers in hiv care and treatment services in namibia. afr j lab med. 2017;6(1), a643. https://doi.org/10.4102/ajlm.v6i1.643 lessons from the field task-shifting point-of-care cd4+ testing to lay health workers in hiv care and treatment services in namibia francina kaindjee-tjituka, souleymane sawadogo, graham mutandi, andrew d. maher, natanael salomo, claudia mbapaha, marytha neo, anita beukes, justice gweshe, alexinah muadinohamba, david w. lowrance received: 24 may 2017; accepted: 02 july 2017; published: 18 sept. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract introduction: access to cd4+ testing remains a common barrier to early initiation of antiretroviral therapy among persons living with hiv/aids in lowand middle-income countries. the feasibility of task-shifting of point-of-care (poc) cd4+ testing to lay health workers in namibia has not been evaluated. methods: from july to august 2011, pima cd4+ analysers were used to improve access to cd4+ testing at 10 selected public health facilities in namibia. poc pima cd4+ testing was performed by nurses or lay health workers. venous blood samples were collected from 10% of patients and sent to centralised laboratories for cd4+ testing with standard methods. outcomes for poc pima cd4+ testing and patient receipt of results were compared between nurses and lay health workers and between the poc method and standard laboratory cd4+ testing methods. results: overall, 1429 patients received a pima cd4+ test; 500 (35.0%) tests were performed by nurses and 929 (65.0%) were performed by lay health workers. when pima cd4+ testing was performed by a nurse or a lay health worker, 93.2% and 95.2% of results were valid (p = 0.1); 95.6% and 98.1% of results were received by the patient (p = 0.007); 96.2% and 94.0% of results were received by the patient on the same day (p = 0.08). overall, 97.2% of pima cd4+ results were received by patients, compared to 55.4% of standard laboratory cd4+ results (p < 0.001). conclusions: poc cd4+ testing was feasible and effective when task-shifted to lay health workers. rollout of poc cd4+ testing via task-shifting can improve access to cd4+ testing and retention in care between hiv diagnosis and antiretroviral therapy initiation in lowand middle-income countries. introduction in 2012, united nations programme on hiv/aids (unaids) reported that only about 8 million of the 15 million people in lowand middle-income countries (lmic) in need of antiretroviral therapy (art) were receiving art by the end of 2011.1 in the lmic of sub-saharan africa, many barriers exist to early initiation of art among eligible hiv-positive patients. of these barriers, limited access to cd4+ testing for determination of art eligibility is one of the most common.2,3,4,5,6,7 a recent systematic review of 28 published studies examining the retention of patients in sub-saharan africa in hiv care between testing and treatment found that the median proportion of patients retained between hiv diagnosis and receipt of cd4+ test results was 59% (range: 35% – 88%).8 implementation of rapid, point-of-care (poc) cd4+ testing in hiv care and treatment (hct) settings has been shown to increase the number of patients who undergo and receive the results of cd4+ testing, and has the potential to improve retention between hiv diagnosis and art initiation.9 the pima® poc cd4+ analyser (alere) has been validated in the field against standard-of-care, laboratory-based platforms10,11,12,13 and was listed as a world health organization (who) prequalified diagnostic in november 2011.14 recent field-based studies in mozambique and south africa demonstrated that poc testing with the pima cd4+ enabled clinics to rapidly stage patients on-site after enrolment into care, which reduced opportunities for pre-treatment loss to follow-up.15,16 poc cd4 testing has also been successfully deployed in mobile, community-based and household settings in south africa.17,18 task-shifting to meet human resources for health needs is recommended by who.19 because poc cd4+ testing using the pima cd4+ analyser does not require a laboratory and is relatively easy to perform, the task of performing cd4+ tests in the hct setting could potentially be shifted from nurses and laboratory technicians to non-professional or lay health workers. in namibia, lay health workers in the ‘community counsellors’ cadre have been trained and certified by the namibia ministry of health and social services (mohss) to provide health facility-based hiv testing and counselling. roll-out and scale-up of poc cd4+ testing in hct settings could follow a similar model. we assessed the feasibility of poc cd4+ testing performed by nurses or lay health workers and the effectiveness of poc cd4+ testing in reducing result turn-around time (tat) and increasing the proportion of patients who receive their cd4+ test results. methods ethical considerations prior to implementation, the study protocol was approved by the research committee of the namibia institute of pathology and the us centers for disease control and prevention. the analysis of the data collected for this study was approved as ‘non-research’ by the us centers for disease control and prevention and the office of the permanent secretary of the mohss of namibia. informed patient consent is not required for receipt of routine services (including cd4 count testing) performed at mohss facilities and no personally identifiable information was available to the researchers; thus, written informed consent was not collected from patients. study design in july 2011, the namibia mohss began a multi-phased rollout of poc cd4+ testing using the pima cd4+ analyser in select public sector hct clinics throughout the country. prior to the first phase of the rollout of poc cd4+ testing, nurses and lay health workers received theoretical and practical training in use of the pima analyser. trainees were required to pass a written examination designed by the device manufacturer (alere, waltham, massachusetts, united states) prior to performing testing in this study. from july 2011 to august 2011, pima cd4+ poc tests were used for routine cd4+ testing for both baseline assessment of art eligibility and routine art monitoring among adult patients (age ≥ 15 years) receiving hct services at 10 public clinics scattered across namibia selected purposively by virtue of having an on-site laboratory. lay health worker and nurse testers were also purposively selected for testing during this study by virtue of availability at these 10 clinics. testing procedures all patients eligible to receive standard-of-care cd4 testing at the participating clinics during the study period were consecutively sampled. pima cd4+ testing was performed by trained nurses or lay health workers and both cadres were represented at all 10 sites. patient management actions including initiation of art, referral for pre-art follow-up and requisition of further laboratory tests were initiated by clinicians based on the pima cd4+ result of each patient. for the purpose of comparing some cd4+ results, tat and patient receipt of results for pima cd4+ and standard testing methods, venous blood was collected by nurses from approximately 10% of participants and sent to the laboratory for standard cd4+ testing on either the epics xl or fc500 (beckman-coulter, brea, california, united states); results were returned to the clinic upon availability. data collection the following data were recorded by nurses or lay health workers in study-specific registers: location, date and time of specimen collection, testing cadre, validity (i.e. ‘valid’ or ‘invalid’) of each pima cd4+ test result, time of pima cd4+ test result availability, time of test result disclosure to the patient, pima cd4+ result and patient demographic information, including age, sex and reason for cd4+ testing (i.e. art eligibility assessment or art monitoring). a questionnaire was sent to all nurses and lay health workers by the study staff to rate their experience with using the pima cd4+ test towards the end of the study period. the rating categories ranged from very easy, easy, fair, difficult to very difficult. the times of specimen collection and patient receipt of results for standard laboratory cd4+ testing were also collected. patient management actions initiated by clinicians following receipt of poc cd4+ results were recorded. analysis basic patient demographic and clinical characteristics were calculated and stratified by sex and reason for cd4+ testing. the feasibility of task-shifting poc cd4+ testing to lay health workers was assessed by calculating the following for both nurses and lay health workers: percentage of valid pima cd4+ test results produced, percentage of pima cd4+ test results received by patients overall and received by patients on the same day as testing, median pima cd4+ result tat and percentage of eligible patients (i.e. pima cd4+ result ≤ 350 cells/µl) who initiated art at the same or next clinic visit. all percentages were calculated at the 95% confidence level. two-proportion z-tests and pearson’s chi-square or the mann-whitney test (for median result tat only), were used to test for significant differences between the tester cadres. the effectiveness of poc cd4+ testing was assessed by calculating the percentage of patients who received their pima cd4+ or standard laboratory cd4+ results and the median and interquartile range (iqr) for the tat for both cd4+ testing methods. two-proportion z-tests and pearson’s chi-square or the wilcoxon matched-pairs test (for median tat results), were used to test for significant differences between the pima cd4+ testing and standard laboratory testing methods. the wilcoxon test, which is non-parametric, was used due to the asymmetrical distribution of cd4+ tat results. results overall, 1429 patients (68% women) received a pima cd4+ test during the study period (table 1). of these, 407 patients (28.5%) received pima cd4+ tests for an art eligibility assessment and 1022 patients (71.5%) received pima cd4+ tests for art monitoring. pima results ranged from 11–641 cells/µl overall. the median pima cd4+ result among patients tested for an art eligibility assessment was 233 cells/µl (iqr: 128–365) among women and 202 cells/µl (iqr: 82–363) among men. based on their pima cd4+ results, 69.5% of patients were eligible to initiate art (i.e. ≤ 350 cells). the median pima cd4+ result among patients tested for art monitoring was 338 cells/µl (iqr: 221–462) among women and 218 cells/µl (iqr: 142–358) among men. table 1: demographic and clinical characteristics of patients who received point-of-care pima cd4+ tests performed by nurses or lay health workers, namibia, july–august 2011. of the 1429 pima cd4+ tests performed, 500 (35.0%) were performed by nurses and 929 (65.0%) by lay health workers (table 2). sixty-one per cent (61%) of testers were lay health workers. results for nurses and lay health workers were similar for the validity of pima cd4+ test results and for receipt of results by patients on the same day as testing. a statistically significant difference between cadres was found for overall receipt of pima cd4+ results by the patient (95.6% for nurses, 98.1% for lay health workers; p = 0.007). the median result tat was 20 min when performed by nurses and 21 min when performed by lay health workers (p = 0.39). among the 283 patients who were eligible to initiate art based on the results of their pima cd4+ test, data on patient management action taken either at the same visit when pima cd4+ testing was performed or at the next follow-up visit were available for 226 (79.9%). among these, 7.7% who were tested by nurses and 5.6% who were tested by lay health workers (p = 0.5) were documented as initiating art at the same eligibility assessment visit or at the next follow-up visit. the majority of both nurses and lay health workers rated the pima cd4+ test favourably for ease of use. table 2: comparison of outcomes for point-of-care pima cd4+ testing performed by nurses or by lay health workers, namibia, july–august 2011. among patients who received a pima cd4+ test, 1389 (97.2%) patients received their test results, of whom 1317 (92.2%) received their results on the same day (table 3). of the 128 specimens collected for cd4+ testing by standard laboratory methods, 71 (55.4%) of results had been received by patients by the end of the study period. the median tat for pima cd4+ test results was 20 min (iqr: 18–27), whereas the median tat for standard laboratory cd4+ test results was 4 days (iqr: 2–8). table 3: comparison of outcomes for point-of-care pima cd4+ testing with outcomes for standard laboratory cd4+ testing method, namibia, july–august 2011. discussion this study demonstrated that poc cd4+ testing was feasible and effective when performed by lay health workers. whether performed by nurses or lay health workers, poc cd4+ testing reduced tats for laboratory results by a median of 4 days compared to tats for standard laboratory methods and greatly increased receipt of cd4+ test results by patients. with more than 95% of patients receiving same-day cd4+ results, the potential for attrition between hiv diagnosis and art initiation was greatly reduced. when compared to the 59% median for the percentage of patients who were retained between hiv diagnosis and receipt of cd4+ test results in rosen and fox’s systematic review,8 our results indicate that retention between hiv diagnosis and receipt of cd4+ results could be greatly improved by task-shifting poc cd4+ testing to nurses and lay health workers. almost all nurses and lay health workers in our pilot rated the poc pima cd4+ favourably on ease-of-use questionnaires; thus, this pilot provides additional evidence supporting the feasibility of task-shifting poc cd4+ testing to nurses and lay health workers in this environment. extensive evidence supports such task-shifting in hct settings in lmic in sub-saharan africa.20,21,22,23,24,25,26,27 in several countries in sub-saharan africa that are affected by a generalised hiv epidemic and a critical shortage of skilled health care providers, scale-up of voluntary hiv testing and counselling has been facilitated by the utilisation of non-professional or lay health worker cadres.28,29 results from the current study show that additional efficiencies can be gained by task-shifting poc cd4+ testing to lay health workers. although current who recommendations for expanding access to poc cd4+ testing only recommend that poc diagnostics (with the exception of hiv rapid tests) be performed by first-tier clinical staff, our results support the idea that task-shifting of poc cd4+ testing and other diagnostics to lay health workers is appropriate. in our study, poc cd4+ testing greatly increased the percentage of patients who received their cd4+ test results. however, among patients eligible to initiate art based on their pima cd4+ results, fewer than 10% actually initiated art at the baseline study visit or the next follow-up visit. among these, 7.7% who received pima cd4+ results from tests performed by nurses and 5.6% (p = 0.5) who received results from lay health workers initiated art at the same eligibility assessment visit or at the next follow-up visit. the possible reasons for the slightly higher percentage who initiated art soon after a pima cd4+ test done by a nurse rather than a lay health worker are not clear. however, this finding may indicate that nurses who conduct the test and review the results are more likely to initiate the patient on art themselves, thereby eliminating the potential barrier of referral to a doctor for art initiation. in contrast, lay health workers are not qualified to interpret cd4 results, nor can they initiate patients on art. they can only refer patients for review of results and clinical management, steps which create a potential bottleneck for art initiation. at the time this study was conducted in 2011, the namibia guidelines30 recommended routine baseline tests for everyone prior to initiation of art. this has since slightly changed with the 2014 national guidelines31 which adopted the who 2013 guideline recommendation32 of same-day art initiation for some eligible patients. while still recommending baseline tests for all patients, the namibia guidelines now recommend that all hiv-positive pregnant and breastfeeding women should be initiated on art on the same day that art eligibility is established, even in the absence of baseline results. this indicates that, apart from pregnant and breastfeeding women for whom baseline tests are now not mandatory to initiate art, poc cd4+ testing alone may not remove all barriers to early initiation of art and opportunities for pre-treatment loss to follow-up may persist without additional steps. similarly, low rates of early art initiation following receipt of pima cd4+ results were observed by jani et al.15 in their studies of pima cd4+ in mozambique. one contributing factor may be that additional diagnostics for the purpose of determining appropriate first-line regimens, including serum creatinine testing for contraindication to tenofovir-containing regimens33,34 and alanine transaminase or hepatitis b virus serology for contraindication to nevirapine-containing regimens, are recommended, and may be performed over several visits before art is initiated. in namibia, and in most lmic, these tests are typically performed through standard laboratory-based methods in centrally located laboratories away from the poc so that, like standard laboratory cd4+ testing, patients must return at a later date to receive their results. poc diagnostics for hepatitis b virus,35 alanine transaminase,36,37 serum creatinine38 and other tests are available and should be evaluated alongside poc cd4+ testing to further reduce opportunities for pre-treatment loss resulting from sub-optimal access to the diagnostics used to determine art eligibility and appropriate first-line regimens. limitations several factors may limit the external validity of results from this study. selection bias may have been introduced by purposive selection of clinics that have an on-site laboratory. clinics with a robust infrastructure may have had better trained or more skilled staff than clinics with weaker infrastructure. selection bias may also have been introduced by purposive selection of testers. lay health workers and nurses were selected by virtue of their availability at the selected clinics during the study period. the lay health worker to nurse ratio in public clinics across namibia may not reflect the lay health worker to nurse ratio in our study. finally, we did not assess the accuracy of pima cd4+ results in relation to the standard laboratory-based platforms, nor did we compare accuracy of testing between lay health workers and nurses. although the accuracy of cd4+ testing is paramount for determining eligibility and effective monitoring, the investigators felt that the accuracy of the pima analyser had been sufficiently validated in previously published studies. therefore, assessing the accuracy of pima cd4+ testing was not included among the objectives or measures of this study. conclusion in this study, we demonstrated that poc cd4+ testing was feasible and effective when performed by lay health workers and nurses within an hct service delivery setting, leading to high levels of patient receipt of results and greatly improved tats for cd4+ results. by removing one of the major sources of pre-treatment loss to follow-up, poc cd4+ testing has great potential to increase early art initiation. however, additional diagnostic barriers, particularly the ancillary tests required to determine the appropriate first-line regimen after determination of art eligibility, need to be removed in order to further increase linkage to treatment. task-shifted poc cd4+ testing should be more widely implemented as an important tool to both increase access to treatment and improve the efficiency of art services. acknowledgements the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the centers for disease control and prevention or the agency for toxic substances and disease registry. competing interest we declare that we have no financial or personal relationships that may have inappropriately influenced us in writing this article. sources of support this research was supported by the president’s emergency plan for aids relief (pepfar) through the centers for disease control and prevention under the terms of award #ups001094a. author contributions s.s., j.g. and g.m. conceived the study and drafted the manuscript. f.k.-t., n.s., m.n., c.m. and a.b. conducted the pilot and collected the data. a.d.m. performed the data analysis. a.m. and d.w.l. provided resources and oversaw the study. references unaids. report on the global aids epidemic [homepage on the internet]. 2012. 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[cited 2015 jun 15]. available from: http://nhscep.useconnect.co.uk/cepproducts/catalogue.aspx abstract introduction case presentation management and outcomes discussion acknowledgements references about the author(s) lebogang skosana department of medical microbiology, tshwane academic division, national health laboratory services, pretoria, south africa farzana ismail centre for tuberculosis, national institute of communicable diseases, johannesburg, south africa nontombi mbelle department of medical microbiology, tshwane academic division, national health laboratory services, pretoria, south africa mohamed said department of medical microbiology, tshwane academic division, national health laboratory services, pretoria, south africa citation skosana l, ismail f, mbelle n, said m. brucellosis – laboratory workers’ nightmare come true: a case study. afr j lab med. 2020;9(1), a1114. https://doi.org/10.4102/ajlm.v9i1.1114 case study brucellosis – laboratory workers’ nightmare come true: a case study lebogang skosana, farzana ismail, nontombi mbelle, mohamed said received: 22 oct. 2019; accepted: 25 june 2020; published: 29 sept. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract introduction: brucella spp. are rarely encountered organisms in the medical microbiology laboratory and, when encountered, can cause concern in laboratory workers. laboratory personnel may in fact develop serious disease as a result of this exposure. this case highlights shortcomings in recognition of brucella spp. from a patient presenting atypically as well as the follow-up and management of an infected patient. case presentation: the patient was an 8-year-old boy from a rural area of south africa who presented to an academic hospital with a bladder mass and history of enuresis in september 2016. brucella melitensis was isolated from a blood culture submitted to the laboratory. the child was subsequently treated for brucellosis in november 2016. management and outcome: the source of infection in the patient was traced to consumption of unpasteurised milk from a local farmer. the patient was treated with doxycycline 100 mg twice daily and rifampicin 600 mg daily for 6 weeks and completed treatment, however he was not followed up at our hospital. the laboratory personnel, however, did not handle the specimen as a biosafety level 3 pathogen as this organism is not commonly encountered; they were provided with prophylaxis for brucellosis (rifampicin and doxycycline). conclusion: brucella spp. is a dangerous pathogen, easily capable of causing significant exposure in an unsuspecting and unprepared laboratory. the case discusses the management of brucellosis in the infected patient as well as the management of laboratory exposure to brucella spp. our case also describes the public health response to a case of brucellosis. keywords: brucellosis; laboratory exposure; prophylaxis; public health; microbiology. introduction brucellosis is a zoonotic disease with a worldwide distribution that is caused by the brucella genus, which are gram-negative bacteria.1 there are approximately 500 000 brucellosis cases reported worldwide annually, however the figure may be much higher as the disease is under-reported, largely because of its non-specific signs and symptoms.2 transmission to humans is through direct contact with animal reservoirs and/or through consumption of infected milk and milk products.1,3 the most common clinical presentation is undulant fever, malaise and arthralgia, after an incubation period ranging from four weeks to several months.1 seven brucella species are potentially pathogenic to humans and each species has a preferred animal host.1,3,4 these include b. abortus (cattle), b. melitensis (sheep, goats), b. suis (swine), b. canis (dogs), b. ovis (sheep), b. ceti (cetaceans), b. pinnipedialis (pinnipeds) and b. inopinata (unknown host).1,3,4 brucellosis, particularly that caused by b. melitensis, is among the most frequently reported laboratory-acquired infections resulting from accidental or inadvertent exposure during aerosolisation procedures in the laboratory.3 this case aims to illustrate the holistic response required when a case of human brucellosis is encountered. ethical considerations this case study received ethical approval from the university of pretoria research ethics committee (ethics number: 412/2017). the patient’s mother was contacted and gave consent to publish by signing a consent form. case presentation the routine processing of blood culture specimens at the tshwane academic division microbiology laboratory (pretoria, south africa) includes removal of positive blood culture bottles from the bact/alert® (biomérieux, marcy-l’étoile, france) system and performing a gram stain, followed by direct sensitivity testing on those positive bottles. the bottle contents are then sub-cultured onto blood, chocolate and macconkey agar plates routinely. cultured plates are preliminarily examined after 10–12 hours of incubation and the chocolate plate is examined for any growth. if pure growth is noted, the plate is sent for identification using the vitek® 2 automated system (biomérieux, marcy-l’étoile france). in this case, the blood culture was taken from an 8-year-old boy from rural mpumalanga, south africa who was admitted to the steve biko academic hospital on 23 september 2016 and was being investigated for a bladder mass and a 2-year history of enuresis (figure 1). for this case isolate, the chocolate plate had pure growth of fine grey colonies after 14 h of incubation. this isolate was sent for vitek 2 for identification and was identified as b. melitensis. once this identification was noted, all further processing was done in a biological safety cabinet class 2 using biosafety level 3 precautions, as recommended by the united states centers for disease control and prevention.5 a gram stain from the culture plates revealed gram-negative coccobacilli which were catalase and oxidase positive and indole negative which was in keeping with a preliminary identification of brucella species. the positive b. melitensis culture was sent to the national institute of communicable diseases in sandringham, south africa on 24 september 2016, where the identification of the organism was confirmed using matrix-assisted laser desorption/ionisation time of flight (maldi-tof) mass spectrometry. the instrument used in this case was the vitek ms (biomérieux, marcy-l’étoile, france), instrument software version 1.5.0.4, myla version 4.5.1, knowledge base (database) version 3.2 (biomérieux, marcy-l’étoile, france). figure 1: significant timelines in the series of events related to the brucellosis case at steve biko hospital (pretoria, south africa) from september to november 2016. management and outcomes the blood culture was taken as part of ‘routine’ investigations as the child had no symptoms or clinical signs which may have led one to consider brucellosis as a possible differential diagnosis. the child was discharged once the blood samples were obtained. the outbreak response unit of the national institute of communicable diseases was alerted to the case on 25 september 2016 and began further investigations and follow-up of affected individuals and possible contacts. it was discovered that the child’s family had been consuming unpasteurised cow’s milk from a local farmer for many years. this farmer’s cattle were tested in late october/early november 2016 using standard south african veterinary testing guidelines.6 sixty-eight cows were tested and 13 were found to be positive for b. melitensis, using the rose bengal test, complement fixation test and serum agglutination test, all of which are serology-based tests. the affected cattle were then isolated, sent to an abattoir and slaughtered as stipulated in the control programme of brucellosis in animals of south africa – animal diseases act (act no. 35 of 1984 ss. 9.1, 9.2, 11).6 the farmer was instructed to send his milk to another local farm for pasteurisation until this farmer was able to pasteurise effectively on his own farm. the patient was treated with doxycycline 100 mg twice daily and rifampicin 600 mg daily for six weeks. he commenced treatment in the first week of november 2016. the treatment was initiated by the clinicians in his hometown and it was uncertain why he was started on the treatment six weeks following the initial blood culture result. soon after commencing with treatment, the patient reported side effects which included abdominal pain and vomiting. these were managed symptomatically, and the patient was able to complete treatment. the affected patient’s family was tested serologically for brucellosis and were found to be negative. prophylaxis was given to those family members who consented to receive it. the united states centers for disease control and prevention’s brucellosis laboratory exposure risk stratification tool was used to screen for exposed staff and staff exposures were stratified as minimal risk, low risk and high risk using these guidelines.5 twenty-one of seventy-two staff members were identified, who had either directly handled the specimen on an open bench or were within 1.5 metres of the person who handled the specimen on an open bench, thus rendering them at high risk for acquiring brucellosis.5 baseline serology was performed on all potentially exposed staff members and standard prophylaxis was provided by the employer. this comprised of rifampicin 600 mg oral daily for three weeks as well as doxycycline 100 mg twice daily for three weeks.5 one staff member was pregnant (in her second trimester) at the time, and subsequently received trimethroprim-sulfamethoxazole (160/800 mg) twice daily for three weeks.5 exposed staff were advised to undergo follow-up serology at 0, 6, 12, 18 and 24 weeks post-exposure.5 weekly symptom watch forms were completed and daily fever self-checks were done for six weeks as recommended by the centers for disease control and prevention and the world health organization.5,6 none of the staff members reported any symptoms for the duration of the symptom check. discussion the transmission of b. melitensis to humans could be through direct contact with infected cattle or the products of abortion of the infected cattle through breaks in the skin as well as through the consumption of unpasteurised milk and milk products.7 transmission can also occur as a result of laboratory exposure to the organism.7 brucella spp. have an infective dose of 10–100 organisms and are often aerosolised during routine laboratory processing of microbiology specimens on an open bench.5 this processing could include performing routine tests on the bench, such as the catalase, oxidase test and indole tests, as well as performing antimicrobial susceptibility testing. these procedures could lead to exposure and possible infection with the organism and can be prevented by ensuring that all processing of brucella spp. isolates is done in an appropriate biosafety cabinet employing biosafety level 3 precautions.5,6 once our laboratory had a preliminary identification of brucella spp., a decision was taken not to manipulate the culture further but to send the isolate to a reference lab with biosafety level 3 facilities for further processing. even though the gold standard for definitive diagnosis of brucella spp. is a positive culture, there is often a delay to final identification because of a number of factors: low index of suspicion, misidentification and unfamiliarity with the organism. the process of identifying exposed individuals and informing them of their risk of developing brucellosis caused much panic among laboratory personnel, and counselling may have been inadequate when addressing fears in the workplace. three staff members are known to have defaulted treatment because of a low perceived risk of acquiring brucellosis and also because of the intolerable side-effects of prophylactic drugs. results of serological testing did not show any significant rise in antibody titres which could have suggested acute infection. the laboratory had no standard operating procedure or policy for the management of possible exposure to a harmful organism before this case and were subsequently prompted to implement these. although b. melitensis mainly affects goats and sheep, cattle may also be infected as a result of indirect contact with goats and sheep.8 furthermore, the systems used in this case may have misidentified the species of brucella.8 the vitek® 2 automated system gram-negative card can only identify the species b. melitensis, while the vitek® maldi-tof ms (biomérieux, marcy-l’étoile, france) was found to correctly identify brucella spp. to the genus level with less correction for species-level identification.8 maldi-tof ms is a reliable method of species-level identification, provided that the brucella spp. reference library used in the database is regularly updated.9 the strengths of this case were the rapid follow-up of exposed laboratory workers and their thorough assessment and management, which managed to contain any potential laboratory outbreak of brucellosis. a limitation of this study was the lack of follow-up on the patient’s clinical progress by the microbiology laboratory. this could be attributed to the outbreak investigation unit having taken over the follow-up of the case, as directed by the public health response protocol. conclusion in conclusion, this case highlights the occupational exposure to b. melitensis as well as the shortcomings associated with the laboratory management thereof. brucella melitensis is a dangerous pathogen, easily capable of causing significant exposure in an unsuspecting and unprepared laboratory. laboratories must ensure that staff are frequently reminded of risks of exposure to such organisms. institutional policy documents should be updated and reviewed regularly, especially with regard to occupational hazards. collaboration between different sectors (eg. agriculture, health) is needed to ensure adequate surveillance and control efforts. acknowledgements competing interests the authors have declared that no competing interests exist. authors’ contributions l.s. collated data and wrote up the manuscript; f.i. collated data; n.m. collated data; and m.s. collated data and proofread the manuscript. all authors read and approved the final manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references bennett j, dolin r, blaser m. brucellosis (brucella species). in: bennett je, dolin r, blaser mj, editors. mandell, douglas, and bennett’s principles and practice of infectious diseases. philadelphia, pa: elsevier saunders; 2015. p. 2584–2585. godfroid j, al dahouk s, pappas g, et al. a ‘one health’ surveillance and control of brucellosis in developing countries: moving away from improvisation. comp immunol microbiol infect dis. 2013;36(3):241–248. https://doi.org/10.1016/j.cimid.2012.09.001 traxler rm, lehman mw, bosserman ea, guerra ma, smith tl. a literature review of laboratory-acquired brucellosis. j clin microbiol. 2013;51(9):3055–3062. https://doi.org/10.1128/jcm.00135-13 hull n, schumaker b. comparisons of brucellosis between human and veterinary medicine. infect ecol epidemiol. 2018;8(1):1500646. https://doi.org/10.1080/20008686.2018.1500846 us centers for disease control and prevention. brucellosis reference guide. assessing laboratory risk level and pep. atlanta, ga: us cdc; 2012. chisi ls, marageni y, naidoo p, zulu g, akol gw, van heerden h. an evaluation of serological tests in the diagnosis of bovine brucellosis in naturally infected cattle in kwazulu-natal province in south africa. j s afr vet assoc. 2017;88(1):e1–e7. https://doi.org/10.4102/jsava.v88i0.1381 corbel mj. brucellosis in humans and animals. geneva, switzerland: world health organization in collaboration with the food and agriculture organization of the united nations and world organisation for animal health; 2006. ferreira l, vega castaño s, sánchez-juanes f, gonzález-cabrero s, menegotto f, orduña-domingo a. identification of brucella by maldi-tof mass spectrometry. fast and reliable identification from agar plates and blood cultures. plos one. 2010;5(12):e14235. https://doi.org/10.1371/journal.pone.0014235 lista f, reubsaet f, santis r, et al. reliable identification at the species level of brucella isolates with maldi-tof-ms. bmc microbiol. 2011;11(1):267. https://doi.org/10.1186/1471-2180-11-267 introduction conclusion acknowledgements references about the author(s) tivani p. mashamba-thompson department of public health medicine, school of nursing and public health, university of kwazulu-natal, durban, south africa benn sartorius department of public health medicine, school of nursing and public health, university of kwazulu-natal, durban, south africa fred c.j. stevens department of educational development & research, faculty of health, medicine and life sciences, maastricht university, maastricht, netherlands paul k. drain departments of global health, medicine, and epidemiology, university of washington, seattle, washington, united states department of surgery, massachusetts general hospital, harvard medical school, boston, massachusetts, united states citation mashamba-thompson tp, sartorius b, stevens fcj, drain pk. experiential bloom’s taxonomy learning framework for point-of-care diagnostics training of primary healthcare workers. afr j lab med. 2016;5(1), a449. http://dx.doi.org/10.4102/ajlm.v5i1.449 opinion paper experiential bloom’s taxonomy learning framework for point-of-care diagnostics training of primary healthcare workers tivani p. mashamba-thompson, benn sartorius, fred c.j. stevens, paul k. drain received: 27 mar. 2016; accepted: 03 aug. 2016; published: 30 sept. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction the delivery of accessible, affordable and equitable primary healthcare (phc) is a key focus in many resource-limited settings. one strategy that has been used to improve health access and healthcare equity in rural and resource-limited settings is the use of point-of-care (poc) diagnostics in phc clinics. poc testing is defined as: pathology testing performed in a clinical setting at the time of patient consultation, generating a result that is used to make an immediate informed clinical decision that contributes to an improved health outcome for the patient.1 although it relies on clinical, non-laboratory staff and frontline workers, such as nurses, to perform diagnostic testing, poc testing is seen as one of the ways to improve affordability, access and equity in rural areas. a primary advantage of poc diagnostics is that the completion of the test and treatment cycle in the same encounter is conducive to retention in care and patient outcomes.2 the advent of poc diagnostics in rural and resource-limited settings brings hope of improving health outcomes, particularly in hiv-epidemic countries. prompt access to these diagnostics, in resource-limited settings with a high burden of disease, poor access to quality healthcare facilities and lack of laboratory infrastructure, is strongly encouraged.3,4 this will, however, demand task-shifting and the delegation of diagnostic services from laboratory-trained staff to non-laboratory-trained staff in phc clinics. these changes could have serious consequences with regard to the quality of testing, especially as disease burden and, hence, testing volumes increase. in this article, we discuss the significance of quality assurance as it pertains to poc diagnostic testing in phc clinics. in addition, we suggest a training/retraining strategy for phc nurses to ensure both the quality of poc diagnostic testing and the sustainability of quality control procedures in phc clinics, by improving the proficiency and retention of skilled nurses. concerns related to quality of point-of-care diagnostic services the need for good quality assurance programmes to ensure accuracy of poc devices used to inform appropriate patient care in resource-limited settings has been demonstrated.3 quality assurance programmes should include the following: running of internal quality control samples on every test day; regular calibration of the instruments; participation in external quality assessment schemes; adherence to standard operating procedures; and test operator competency checks.5 research has also demonstrated poor regulatory control standards for the diagnosis of sexually-transmitted infections, in both publicand private-sector venues, within lowand middle-income countries.6 the entire process of poc diagnostics, from the pre-analytic through the analytic and post-analytic phases, has hidden reliability risks, including false-positive and false-negative test results, that can lead to gross medical errors. a false-positive result, such as an incorrect hiv-positive diagnosis in a person who is not infected with hiv, can lead to devastating consequences whereby patients may have to start antiretroviral treatment and change their lifestyle, which in turn may have a negative impact on their employment status and family relationships. a false-negative test result caused by a test’s failure to detect hiv antibodies or antigens in an hiv-infected person could lead to an hiv-negative diagnosis in an hiv-positive patient. this could have devastating consequences for the patient, particularly if their condition is at a stage where antiretroviral treatment is needed. it also increases the risk of infection of others, if the patient is sexually active. adequate phc clinics must have the resources to enable appropriate storage and performance of diagnostic tests, as well as appropriate training of staff. the implementation of quality assurance practices is critical in ensuring the suitable administration of poc diagnostic tests and correct interpretation of their results.7 inadequate human resources, due to high turnover rates of skilled nursing staff, are an ongoing problem in some high disease-burden countries such as south africa, particularly at the rural phc level.8,9 in addition, the competency of the diagnostic test user is one of the factors that affects the reliability of poc diagnostic services.10 it has been noted that there is a need for healthcare providers to be educated on the appropriate use of poc diagnostics, as well as a need for incorporation of poc diagnostics training into medical and nursing curricula as part of continual professional development.11 importance of competency of point-of-care diagnostic test users on service delivery in recent years, there has been a significant increase in publicand private-sector investment in hiv and tuberculosis poc diagnostics.12 however, the complexity of the assay that can be used in a given setting is determined by the degree of infrastructure required for the assay platform.13 the competency of the user of poc diagnostic tests affects the reliability of the poc diagnostic services delivered in phc clinics.14,15 thus, the requirement not only for reliable electricity and climate-controlled testing rooms, but also for the staff skills and competencies required for more complex diagnostics, means that the majority of patients in resource-limited settings do not have access to the more complex assays at the community and phc level.14 this potentially undermines the impact of poc diagnostics on patient outcomes. a strategy for improving the quality of primary healthcare clinic-based point-of-care diagnostic services despite the need for improving the availability of essential poc diagnostics in settings that lack laboratory infrastructure, such as rural phc clinics,3,4 the shifting of the performance of diagnostic services to less-qualified and less-experienced staff could have serious consequences on the quality of testing, especially as disease burden and testing volumes increase. there is a need for a well-structured poc diagnostic training programme for all frontline health workers, particularly for those working in rural and resource-limited settings. the overall aim of such a training programme would be to supplement current education and training resources for healthcare workers with a specialised poc diagnostics training curriculum. as stated above, since the competency of the healthcare professionals performing diagnostic poc testing influences the reliability of such services, we suggest a training strategy to equip frontline phc workers with appropriate diagnostic knowledge and skills. this will help to ensure the quality, reliability and sustainability of poc diagnostic services. such training on the appropriate use of poc diagnostics needs to be incorporated into the medical and nursing curricula as part of continual professional development. in order to provide a real opportunity for ensuring the effectiveness and sustainability of such a programme, an appropriate educational strategy is essential. for example, a training programme that incorporates practical experiential learning is most desirable. implementation of experiential learning for a point-of-care diagnostic training programme experiential learning, which is defined as a client-focused, supported learning approach,16 would help to engage users of poc diagnostics by inclusion of the following elements: action, reflection, and transfer to gain competency. we suggest incorporating a framework borrowed from bloom’s taxonomy17 to guide the objectives of an experiential learning programme and to ensure quality of poc diagnostics services provided in phc clinics (table 1). this approach would provide phc workers with a practical, cost-effective, on-site learning opportunity. furthermore, it would enable direct exposure to poc diagnostic service delivery, rather than a traditional passive learning approach, which offers no exposure to service delivery. table 1: objectives for point-of-care diagnostics experiential learning training programme for primary healthcare clinic workers using bloom’s taxonomy. however, due to the ever-changing nature of healthcare services and associated technologies, implementation of an experiential learning strategy alone will not guarantee continual quality service delivery and retention of skilled staff. therefore, complementary strategies are needed, including continual mentorship, monitoring and evaluation to provide professional oversight to trained users of poc diagnostics. professional oversight of staff trained to provide point-of-care diagnostic services to ensure the retention of the acquired knowledge and skills by the trained healthcare workers, we suggest an expert-driven process for professional oversight of trained phc-based poc diagnostics users, using the following four strategies:18 (a) regular monitoring of the learner’s competence on diagnostics through audits conducted by experienced poc diagnostic-competent healthcare workers with experience in phc clinics; (b) evaluation of the competency of trained poc diagnostics users by poc diagnostic-competent staff; (c) retraining of previously-trained users with poor competency and new users replacing an existing user; and (d) mentorship of trained poc diagnostics users by phc clinic workers with competence and experience in poc diagnostics. figure 1 depicts a framework for an experiential learning programme for continual, quality delivery of poc diagnostics services by phc-based healthcare workers. figure 1: a bloom’s taxonomy guided framework for implementing point-of-care diagnostics experiential learning programme to improve point-of-care testing proficiency and staff retention in primary healthcare. appropriate implementation of the proposed programme will likely result in staff empowerment and provide an incentive to improve staff retention in phc clinics. successful implementation of the programme would require incorporation of a community-based, participatory research approach prior to and during implementation.19 this research approach would involve all stakeholders: researchers and healthcare workers, phc clinic pathology service providers and policy makers. such engagement should help invest stakeholders in team building and sharing of resources, open discussion and sharing of ideas and expertise to ensure continual quality delivery and effectiveness of the programme. an ‘enforcement’ approach to implementation is unlikely to achieve results.20 rather, an approach developed through mutual consent and agreement is much more likely to be effective in the long term.20 conclusion delivery of high quality poc diagnostic services requires highly competent and dedicated staff. a special focus on well-structured poc diagnostic training strategies for improving staff competency in performance and quality maintenance for poc diagnostic services is urgently needed to ensure the highest quality diagnostic services. we recommend a guided framework, based on a bloom’s taxonomy approach, for implementing a poc diagnostics experiential learning programme to improve poc testing proficiency and staff retention in phc workers. stakeholder engagement, consensus building and collaboration will be crucial to ensuring appropriate implementation and sustainability of the proposed programme. acknowledgements the authors wish to thank rowan mark thompson for assistance with technical graphics. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support this project was supported by research project number be484/14. authors’ contributions t.p.m-t., f.c.j.s., b.s. and p.k.d. conceptualised the study, and t.p.m-t. wrote the first draft. t.p.m-t was the principal investigator for the project, p.k.d. and b.s. were supervisors, and f.c.j.s was a collaborator. all authors contributed to the writing and editing of the report and approved the final version. references price cp, john as, 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developing countries. diagnostics. 2015;5(2):200–209. http://dx.doi.org/10.3390/diagnostics5020200 world health organization. consolidated guidelines on hiv testing services. geneva, switzerland: who; 2015. moodley d, moodley p, ndabandaba t, et al. reliability of hiv rapid tests is user dependent. s afr med j. 2008;98(9):707–709. yeo cp, ngo a, ng wy, et al. assessing performance of i-stat at the point of care in the emergency room. proceedings of singapore healthcare. 2011;20(3):157–161. beard c, wilson jp. the power of experiential learning: a handbook for trainers and educators: london, uk: kogan page; 2002. marzano rj, kendall js. the new taxonomy of educational objectives. thousand oaks, ca: corwin press; 2006. kolb ay, kolb da. learning styles and learning spaces: enhancing experiential learning in higher education. acad manag learn edu. 2005;4(2):193–212. horowitz cr, robinson m, seifer s. community-based participatory research from the margin to the mainstream: are researchers prepared? circulation. 2009;119(19):2633–2642. http://dx.doi.org/10.1161/circulationaha.107.729863 eldredge lkb, markham cm, ruiter rac, et al. planning health promotion programs: an intervention mapping approach. 4th ed. san francisco, ca: jossey-bass; 2016. sources of support references about the author(s) samuel kariuki kenya medical research institute, nairobi, kenya karen h. keddy faculty of health sciences, university of the witwatersrand, johannesburg, south africa martin antonio world health organization collaborating centre for new vaccines surveillance, medical research council unit, at london school of hygiene & tropical medicine, banjul, the gambia division of microbiology & immunity, warwick medical school, university of warwick, warwick, united kingdom iruka n. okeke department of pharmaceutical microbiology, university of ibadan, ibadan, nigeria citation kariuki s, keddy kh, antonio m, okeke in. antimicrobial resistance surveillance in africa: successes, gaps and a roadmap for the future. afr j lab med. 2018;7(2), a924. https://doi.org/10.4102/ajlm.v7i2.924 editorial antimicrobial resistance surveillance in africa: successes, gaps and a roadmap for the future samuel kariuki, karen h. keddy, martin antonio, iruka n. okeke copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. since the world health organization (who) launched the global antimicrobial resistance surveillance system (glass) in 2015,1 there has been rapidly growing awareness among many african countries that they need to be doing more to combat antimicrobial resistance (amr). the africa centres for disease control and prevention (cdc) was officially inaugurated in january 20172 and will support countries commencing surveillance for serious infectious disease threats in africa, including resistance. review of the recent who glass report suggests that, while certain nations do have some surveillance systems in place, very few countries in africa currently conduct effective routine surveillance.1 as editors of this special edition, our experience confirms this observation. nonetheless, we were encouraged by the range and scope of articles we received when the call for this special edition went out. the subjects covered include multi-drug resistant tuberculosis,3,4,5 which remains a critical public health threat on the african continent, nosocomial infections,6,7 including those due to clostridium difficile8, laboratory methods and technical issues,9,10 including external quality assessment11 and molecular methods,3,12,13 and malaria.14 community-acquired resistance was underrepresented, although there was a major review on amr in vibrio cholerae o1.15 the articles included opinion pieces, general and systematic reviews and original research. what we missed was more original research with a wider range of country submissions and better regional representation, more articles looking at one health, veterinary medicine and amr, more studies with data on amr in community-acquired pathogens and amr trend data by country, including bacteria, fungi and parasitic infections, and better detail on antiretroviral resistance in hiv infection. we are pleased to find that the special issue’s call for papers increased the overall frequency of submissions in this area and so subsequent regular issues of the journal are likely to continue the exposition of amr surveillance in africa that this issue has initiated. omissions notwithstanding, what is represented in this issue is a great and promising start: glass and the africa cdc are both still in their infancy and no country can be expected to begin with comprehensive surveillance. what we have seen and present in this issue is an architect’s rough sketch of where we can go as a continent. readers should understand that surveillance for amr, irrespective of the pathogen or the setting, is not about the academic exercise of data collection but is a practical and powerful tool that is critical in defining optimal interventions both in local health settings and for what has become a global health crisis. when amr surveillance data are published in a journal, compared with submitting national data to a supra-governmental organisation, these data need not be fully comprehensive or subscribe to a predetermined list of pathogens or antimicrobials. the articles in this special edition hence provide a valuable snapshot of what can be done in africa, rather than a comprehensive analysis of the amr surveillance in africa. readers of this special edition, which emphasised the need for planning and progress as well as amr data, should be able to see beyond the amr data published here to what is being achieved by their continental neighbours, recognising that they too have the potential to do similar work. remembering that imitation is the highest form of flattery, we can use the experience of our neighbours, start small and grow our surveillance systems. there are new resources that we can access through the who1 and africa cdc2,16 to support us. the responsibility rests with us to find the will and the way to take surveillance for amr to the next phase of effective implementation. we all concede that there are numerous challenges we face in our attempt to implement effective and sustainable amr surveillance programmes in africa. these challenges range from inadequate resources and weak supply chains for consumables for microbiological laboratory procedures, lack of human resources well trained in amr surveillance and poor or lack of commitment by facility management to embrace amr as a healthcare issue. where facilities do exist, under-utilisation is common. however, despite the lack of infrastructure and other resources required to perform optimal surveillance for amr, most laboratories in africa can still generate, collate and disseminate quality data, albeit with some ingenuity. if there is a lack of samples for systematic evaluation of amr trends, mutually beneficial partnerships between centres responsible for coordinating surveillance and local hospitals or healthcare facilities can jumpstart focused surveillance on specific organisms. such centres can then gradually expand the surveillance to include more pathogens and more facilities. indeed, we could perform systematic sentinel surveillance so as to generate reliable, accurate and quality-controlled data, using the minimal resources typically available in our settings, for implementation of infection control and prevention programmes. similarly, livestock-serving veterinary laboratories could target a component of the meat value chain and purposively sample within their capacity, for example, one in every 10 meat swabs in the local abattoir for amr testing and surveillance. this effort can additionally be embedded in the public health requirement for ensuring food safety. quality routine culture and susceptibility testing can easily and cost-effectively provide high-quality surveillance data. however, as omar et al.,3 smith,12 gellband13 and mohammed et al.15 all demonstrate, additional valuable information can be gleaned from surveillance systems that use whole genome sequencing and this is something that developing systems should consider. for quality assurance, which is a hallmark of a standard surveillance system, it will be important for laboratories to be registered and participate in an external quality assessment scheme operating to internationally recognised standards, some of which are supported by who and other partners and are therefore potentially more affordable for laboratories in our african setting. implementation of national action plans for amr surveillance from a one health approach will be critical in ensuring sustainability of the collaborative, multi-sectoral, and trans-disciplinary efforts at the local, regional and national level. leveraging resources that can be jointly deployed both in-country and across national borders for sample collection, laboratory analysis, and data capture, analysis and dissemination, will optimise the use of resources to achieve the desired goals, providing evidence for action to prevent and contain amr. on funding amr surveillance activities and for a sustainable amr surveillance programme, it will be critical that budget commitments made by national governments to support the national action plan activities are adequately maintained. this too will require some ingenuity so as to demonstrate that data from amr surveillance actually impact on delivery of healthcare, food security and sustenance of a healthy environment. thus, governments are constantly reminded that good surveillance systems are actually cost efficient, particularly in the long term. we need to work with health economists to deliver data on those economic costs associated with a lack of action on the amr menace in our countries in a language that policymakers will easily understand. in the meantime, we will need to utilise the resources currently available through various initiatives, such as the tripartite united nations agencies (who, the food and agriculture organization and the world organization for animal health), as well as other partners who have specific interests in promoting and supporting one health amr surveillance in africa. the outlines of two successful routes to national surveillance, ibrahim et al.17 and opintan18, showcase how donor funds were leveraged to support local planning and initiatives in ethiopia and ghana. in making the most of external initiatives and opportunities, however, we must remember that these will only serve as shortand medium-term solutions – we have to own the process of fully implementing our national action plans, using our own resources, if amr surveillance and containment will be sustainable, and serve our principal national goals and objectives. sources of support this special issue was supported by the bill and melinda gates foundation (opp1178631) and by the centre for disease dynamics, economics and policy. references who. global antimicrobial resistance surveillance system (glass) report: early implementation 2016–2017. geneva: who; 2017. amukele t. africa cdc: establishing integrated surveillance and laboratory networks for rapid disease detection and response, control, prevention, and clinical care in africa. afr j lab med. 2017;6:a638. https://doi.org/10.4102/ajlm.v6i1.638 omar sv, joseph l, said hm, et al. whole genome sequencing for drug resistance determination of mycobacterium tuberculosis. afr j lab med. in press. ismail n. ending drug resistant tuberculosis in africa. afr j lab med. 2018;781. otokunofor k. prevalence of multidrug resistant mycobacterium tuberculosis in port harcourt, nigeria. afr j lab med. 2018;7(1), a805. irek e. a systematic review of healthcare-associated infections in africa: an antimicrobial resistance perspective. afr j lab med. 2018;7(1), a796. singh-moodley a, ismail h, perovic o. an overview of antimicrobial resistance surveillance among healthcare-associated pathogens in south africa. afr j lab med. 2018;7(1), a741. kullin br, reid s, abratt v. c. difficile in patients attending tb hospitals in cape town, 2014–2015. afr j lab med. 2018; 7(1), a846. gutierrez c. is suboptimal incubation temperature in laboratories impairing antibiotic resistance monitoring? afr j lab med. 2018; 7(1), a789. mwanza w, milimo d, chilufya mm, et al. diagnosis of rifampicin resistant tuberculosis – discordant results by diagnostic methods. afr j lab med. 2018;7(1), a806. perovic o, yahaya aa, viljoen c, et al. trends in quality of antimicrobial susceptibility testing at national public health laboratories in africa, 2011–16. afr j lab med. in press. smith am. molecular epidemiology of outbreaks due to multidrug-resistant enteric bacterial pathogens in sub-saharan africa. afr j lab med. in press. gelband h. amr surveillance with whole genome sequencing in africa: it’s (about) time. afr j lab med. 2018;7(1), a761. ouologuem dt, kone co, fofana b, et al. differential infectivity of gametocytes after artemisinin-based combination therapy of uncomplicated falciparum malaria. afr j lab med. 2018; 7(1), a784. mohammed y, aboderin ao, okeke in, olayinka at. antimicrobial resistance of vibrio cholerae from sub-saharan africa: a systematic review. afr j lab med. 2018;7(1), a778. varma jk, oppong-otoo j, ondoa p, et al. africa cdc’s framework to control antimicrobial resistance in africa. afr j lab med. 2018;7(1), a830. ibrahim ra, teshal am, dinku sf, et al. antimicrobial resistance surveillance in ethiopia: implementation experiences and lessons learned. afr j lab med. 2018;7(1), a770. opintan ja. leveraging donor support to develop a national antimicrobial resistance policy and action plan: ghana’s success story. afr j lab med. 2018;7(1), a825. article information authors: juliana ndasi1 laura dimite2 victor mbome3 charles awasom4 elive ngale1 sidney akuro1 ewane leonard1 omotayo bolu2 terence asong2 patrick njukeng1 judith shang2 affiliations: 1global health systems solutions, cameroon 2us centers for disease control and prevention (cdc), cameroon 3buea regional hospital, south west region, cameroon 4bamenda regional hospital, north west region, cameroon correspondence to: juliana ndasi postal address: po box 732, limbe, cameroon dates: received: 22 aug. 2014 accepted: 15 sept. 2014 published: 03 nov. 2104 how to cite this article: ndasi j, dimite l, mbome v, et al. decentralised facility-based training as an alternative model for slmta implementation: the cameroon experience. afr j lab med. 2014;3(2), art. #231, 6 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.231 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. decentralised facility-based training as an alternative model for slmta implementation: the cameroon experience in this original research... open access • abstract • introduction • research methods and design    • selection of laboratories    • preparation    • slmta implementation and supplemental training    • slmta implementation and supplemental training • results • discussion    • limitations of the study    • conclusion • acknowledgements    • competing interests    • authors’ contributions    • cdc disclaimer • references abstract top ↑ background: the strengthening laboratory management toward accreditation (slmta) programme is designed to build institutional capacity to help strengthen the tiered laboratory system. most countries implement the slmta three-workshop series using a centralised model, whereby participants from several laboratories travel to one location to be trained together. objectives: we assessed the effectiveness and cost of conducting slmta training in a decentralised manner as compared to centralised training. methods: slmta was implemented in five pilot laboratories in cameroon between october 2010 and october 2012 by means of a series of workshops, laboratory improvement projects and on-site mentorship. the first workshop was conducted in the traditional centralised approach. the second and third workshops were decentralised, delivered on-site at each of the five enrolled laboratories. progress was monitored by repeated audits using the stepwise laboratory quality improvement process towards accreditation (slipta) checklist. results: audit scores for all laboratories improved steadily through the course of the programme. median improvement was 11 percentage points after the first (centralised) training and an additional 24 percentage points after the second (decentralised) training. the estimated per-laboratory cost of the two training models was approximately the same at us$21 000. however, in the decentralised model approximately five times as many staff members were trained, although it also required five times the amount of trainer time. conclusion: decentralised slmta training was effective in improving laboratory quality and should be considered as an alternative to centralised training. introduction top ↑ international organization for standardization (iso) 15189 accreditation is viewed worldwide as the gold-standard mark of competence for clinical laboratories. however, the process of achieving international accreditation is labour-intensive, complex and expensive, making it challenging even for the best-resourced laboratories.1 these difficulties are magnified in resource-limited settings, where laboratories struggle to maintain staff levels and competence, basic infrastructure, equipment and supplies.1,2,3,4,5 as a result, few laboratories in sub-saharan africa are accredited and no laboratory in cameroon has been accredited to international standards.6 large-scale public health programmes, such as the us president’s emergency plan for aids relief (pepfar), have highlighted gaps in laboratory services, emphasising the urgent need for quality improvement.7 the world health organization’s regional office for africa (who afro) has responded by launching the stepwise laboratory quality improvement process towards accreditation (slipta) scheme, which is a phased approach to quality improvement.8 training and mentoring in laboratory management have been identified as being critical for the implementation of quality management systems (qms).9 however, many training programmes fail to result in measurable changes in laboratory practices because they focus more on theory and generic management topics than on practical aspects that can lead to direct implementation. they also lack follow-up with trainees to assist with application of knowledge into practice.10 the strengthening laboratory management toward accreditation (slmta) programme is an innovative, taskand competency-based training programme that aims to address deficiencies in laboratory quality through a series of workshops, improvement projects and mentoring.11 the slmta three-workshop series is typically conducted in a central location that is logistically convenient for all laboratories in the training cohort. centralised training allows many laboratories to be trained simultaneously and provides opportunities for laboratory networking and inter-facility knowledge-sharing. however, a centralised model can be expensive because of venue hiring and participant travel. some have argued that decentralised training can be more sensitive to the needs of the trainees and tied to specific organisational or project goals, as trainers are able to respond rapidly to the needs of the audience and revise the training approach based on their feedback.12 in addition, decentralised training has been shown to improve relationships between local and central authorities and to increase institutional capacity.13,14 in an effort to improve laboratory quality, cameroon began slmta implementation in 2010, with a first cohort of five laboratories. the initial training workshop was conducted in a centralised location. for the remaining two workshops, the programme shifted to a decentralised model, with facility-based training. the objective of this study is to compare the results and cost of decentralised training versus centralised training for the establishment of a qms in five laboratories in cameroon. research methods and design top ↑ selection of laboratories in 2009, four public hospital laboratories were selected by the ministry of public health, cameroon to enrol in the slmta programme: buea regional hospital laboratory (burhl), bamenda regional hospital laboratory (brhl), laquintinie hospital laboratory douala (lhld) and the yaoundé central hospital laboratory (ychl). in august 2010, a private laboratory, laboratoire d’analyses médicales du centre (lamc), was added to the four selected public laboratories in order to improve the link between the public and private sectors, which is essential for building sustainable national laboratory systems in resource-limited countries3 (table 1). table 1: laboratories included in cameroon’s first cohort of the strengthening laboratory management toward accreditation programme. preparation global health systems solutions (ghss) – a local implementing partner – and the cameroon office of the us centers for disease control and prevention (cdc) led slmta implementation. in preparation, all technical staff members from ghss and the laboratory team of cdc-cameroon underwent several training courses: (1) good clinical laboratory practice, provided by the south african national accreditation system (sanas); (2) use of iso 15189 in internal audits and laboratory assessments toward accreditation, provided by sanas; (3) slmta training, provided by cdc-cameroon staff; and (4) laboratory mentorship, provided by a clinton health access initiative (chai) mentor. to build capacity at the five selected slmta laboratories and to ensure sustainability, two employees from each laboratory were appointed as on-site mentors and trained for five days by a chai mentor on qms, mentoring techniques, the 12 quality system essentials (qses), iso 15189 and conducting laboratory audits using the slipta checklist. slmta implementation and supplemental training in october 2010, five participants from each of the five selected laboratories travelled to a central location in mutengene, south west region, cameroon, for the first five-day slmta training workshop. the second and third trainings of five days each were held on-site in each laboratory, from february to march 2011 and june to july 2011, respectively. a catch-up training was provided to personnel who missed the initial centralised training. the number of personnel trained per site ranged from 12 to 24 persons, including laboratory managers and clinicians. in addition to slmta training, the following centralised supplemental training courses were conducted for two employees from each laboratory: laboratory biosafety and biosecurity; development of standard operating procedures; internal audit; and use of a basic laboratory information system. most of these supplemental trainings were done at the end of the three slmta training workshops. a mentorship model that embeds a mentor within the daily routine of a laboratory for an extended period with a defined engagement schedule was used in these laboratories. these embedded mentors provided on-site coaching and guided the laboratories toward international accreditation by ensuring the implementation of improvement projects. in addition to embedded mentors, visiting mentors conducted two site visits following each workshop. laboratory improvement projects are an integral part of the slmta programme, and were assigned to participants after each workshop. in subsequent workshops, participants presented their improvement projects and shared results and lessons learned. these sessions offered an opportunity for participants to learn from each other and facilitated the formation of a peer-learning network. evaluation the slipta checklist was used to evaluate the laboratories’ progress, strengths and weaknesses. this checklist contains 12 sections (a total of 111 items) for a total of 258 points.15 slipta checklist scores are categorised into star levels, with < 55% corresponding to zero stars, 55% – 64% one star, 65% – 74% two stars, 75% – 84% three stars, 85% – 94% four stars and 95% – 100% five stars. a ghss staff member trained by who afro as an auditor conducted baseline audits of the four public laboratories between november and december 2009, and the fifth, private laboratory in august 2010. who afro-trained in-country auditors conducted four intermediate audits, just before the second and third slmta training workshops and after the third slmta workshop, in order to evaluate progress, identify gaps and develop action plans to close existing gaps. costs in us dollars to implement slmta training workshops were estimated for the centralised and decentralised models. for centralised training, we assumed that five people per laboratory would attend the three workshops. for decentralised training, we assumed that 24 participants would attend each on-site workshop. for both models, we assumed four trainers would be needed and that each workshop would last five days. costs included lodging, per diem, land transport to the training venue, training materials for all participants, food and venue hiring (for centralised training only). we did not include salary or time missed from work for participants or trainers, nor other components of slmta implementation such as improvement projects, mentorship and audits, which would not be affected by training location. trainer days were estimated for each model based on one travel day and five training days per workshop. results top ↑ at baseline audit, the five laboratories scored a median of 23%, all at zero stars. median scores increased steadily to 34% at the first intermediate audit, 58% at the second, 66% at the third and 68% at the fourth; they remained at 68% for the exit audit. thus, there was a median total improvement of 45 percentage points. after 24 months of the slmta programme, two laboratories attained one star, two attained two stars and one attained three stars based on the audit scores. lamc had the largest improvement of 69 percentage points (figure 1). figure 1: performance of five cameroon laboratories over 24 months of slmta implementation as measured by the slipta checklist. the median improvement from the first slmta training to the second (intermediate audits 1 and 2) was 11 percentage points. after the first decentralised training, median scores improved an additional 24 percentage points. from the final training to exit, median scores improved 10 more percentage points. there was substantial variability in the timing of improvements. for example, lamc had their greatest improvement between the baseline and first intermediate audit, whilst ychl had their greatest improvement between the second intermediate audit and exit; lhld’s score decreased slightly from the second intermediate audit to exit (figure 1). all five laboratories improved their scores in each of the 12 qses. internal audit had the highest percentage average improvement (61 percentage points), followed by corrective action (55 percentage points) and documents and records (53 percentage points). improvements from the baseline audit for five of the qses were greatest after the first slmta training, whilst seven of the qses improved most after the second slmta training (figure 2). figure 2: average performance of the five laboratories measured at baseline, first and second intermediate, and exit audits. average marks are expressed as a percentage of the total for each of the 12 quality system essentials sections of the slipta checklist. the estimated cost of the workshop portion of slmta implementation for the two models is presented in table 2. with the centralised training model, it would cost approximately $105 610 to hold the three slmta workshops for 25 participants from five laboratories ($21 122 per laboratory). if the workshops were decentralised and conducted in-house in each laboratory, the total cost would be approximately $107 400 to train 120 participants from the five laboratories ($21 480 per laboratory). centralised training would require 72 trainer days; decentralised training would require 360 trainer days. table 2: estimated cost in us dollars (usd) of conducting centralised versus decentralised slmta training workshops. discussion top ↑ laboratory scores improved steadily throughout the two-year programme, with all laboratories reaching at least the one-star level. improvements after the decentralised second slmta workshop were twice as large as those after the centralised first workshop. the total estimated cost of the centralised and decentralised training models was about the same. however, in the decentralised model, approximately five times as many staff were trained as compared with the centralised model, whilst the centralised model required one-fifth the amount of trainer time as the decentralised model. decentralised workshops allowed more staff to participate in the training, facilitating shared understanding of the importance of quality improvement and the plan to achieve it. hospital managers and clinicians were able to participate in the training alongside laboratory managers, improving clinician–laboratory interactions and providing them an opportunity to understand the potential for long-term improvement. on-site training enabled the use of familiar facilities to conduct interactive activities; slmta concepts could easily be shared amongst laboratory staff and any site-specific non-compliance could be discussed during the workshops. finally, on-site workshops allowed the course to be tailored to the needs of the individual laboratories, with all workshop discussions related to site-specific challenges and solutions. on the other hand, centralised training fosters communication between laboratories, helping to build important networks. participants in centralised training can learn from the experiences of other laboratories and get feedback on what did and did not work for them. two critical aspects to consider when implementing slmta are cost and manpower. in this study, we found that centralised and decentralised training cost roughly the same amount, at approximately $21 000 per laboratory. savings made in the decentralised model for reduced costs for per diem, lodging, transport and venue hire were offset by the increased cost of trainers, training materials and food for the expanded group of participants. however, the two models have important consequences regarding manpower. experience from other countries implementing slmta has suggested that staff attrition, especially through reassignment to other laboratories within the ministry of health system or to employment in private laboratories, is one of the critical challenges facing sustainability of results after slmta completion.16 when only a few staff members from each laboratory are trained, their departure has a pronounced effect on institutional memory and new staff must receive intensive training in order to continue the qms work. in the decentralised model, the majority of laboratory staff are trained to implement qms, reducing the impact of attrition of a few trained staff members. on the other hand, decentralised training requires far more trainer time, as the full series of workshops is conducted at each laboratory. the shortage of qualified trainers throughout africa has been well noted.17 usually, countries use trainers who are borrowed from their normal laboratory duties for the slmta training weeks. but when those weeks increase geometrically from three per cohort to three per laboratory, the feasibility of borrowing trainers is questionable. globally, the median cohort size for slmta training has been 10 laboratories, with cohorts ranging from one to 27 laboratories.18 the logistical and manpower issues associated with decentralised training could quickly escalate in larger programmes, such that national laboratory programmes may need to consider adding staff dedicated to implementing slmta if decentralised training is desired. several challenges were faced in the implementation of the slmta programme in cameroon. the first challenge was that governmental bureaucracy caused delays in project implementation. this problem was exacerbated by the lack of a national laboratory strategic plan to define overall goals and stakeholders. this plan has now been developed and is pending adoption. another major challenge was the lack of personnel trained and skilled in quality laboratory practices in selected facilities and insufficient numbers of local slmta trainers. this challenge is being resolved by recent training of three more slmta trainers and a plan to conduct in-country slmta training-of-trainers in the near future. the concept of qms was entirely new to most laboratory staff in the selected facilities where a culture of quality has been lacking. as the staff undertook the training modules and understood the benefits of quality improvement, they became more cooperative and committed. finally, in a system without biomedical engineers, there were difficulties with equipment maintenance. most of the laboratory equipment is not available in the cameroonian markets; this, coupled with the high cost of import duties in cameroon, made equipment procurement and maintenance very costly. this challenge is being addressed with an on-going improvement project on equipment maintenance and calibration. limitations of the study whilst the greater median improvement in scores after decentralised training suggests that it may have been more effective than centralised training, these results should be viewed in light of study limitations. most importantly, this was an observational study of the natural progress of a programme; thus, there were no control laboratories on which to base a comparison. the difference in changes over time could be as a result of several factors, including timing of specific improvement projects undertaken after each workshop and variability in mentorship support. furthermore, the pattern was not consistent amongst all five laboratories in the cohort; whilst three laboratories improved more after the second training than after the first, lamc had its greatest improvement after the first and ychl after the third. the immediate improvement in lamc could possibly be because it is a private laboratory with few administrative bottlenecks that are common in larger public health facilities. additional operational studies randomising cohorts to centralised versus decentralised training would provide more solid evidence of the relative effectiveness of these strategies. conclusion quality laboratory systems are essential for providing patient care and global health. a competency-based programme such as slmta can assist public health laboratories in resource-limited settings to improve the quality of their services. the success of any programme depends on its sustainability. the lack of a national laboratory strategic plan, along with inadequate government funds and the absence of policies for equipment procurement and maintenance were major challenges faced by laboratories in cameroon and other resource-limited settings and may continue even in the post-accreditation period. training of facility-based mentors will help ensure continuous quality improvement, sustainability and country ownership. although the challenges were many, slmta implementation successfully improved laboratory quality, ensuring better laboratory services and patient care. whether to conduct slmta trainings using a centralised or decentralised model will depend on situation-specific factors; however, decentralised training should be considered to widen the reach of the training within the laboratories. cameroon intends to use decentralised training for future slmta cohorts. acknowledgements top ↑ this programme was financed with funds from pepfar through a cooperative agreement with cdc. it was implemented in collaboration with cdc’s office in cameroon, with technical support from cdc’s division of global hiv/aids laboratory team. we thank the minister of public health for the guidance and commitment to facilitating the implementation of these activities. we appreciate the collaborations of ministry staff, especially the regional delegates, directors of hospitals and all the laboratory staff that worked closely together in this programme. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions j.n. (global health systems solutions) was the project coordinator. l.d. (cdc, cameroon) was the activity manager for this project. v.m. (buea regional hospital) and c.a. (bamenda regional hospital) are hospital directors and were implementers of the project. e.n., s.a. and e.l. (all global health systems solutions) were laboratory mentors for this project. o.b. (cdc, cameroon) facilitated implementation of the programme by advocating for it to the ministry of health. t.a. (cdc, cameroon) was a laboratory coach and mentor. p.n (global health systems solutions) was principal investigator for this project. j.s. (cdc, cameroon) was the project officer. cdc disclaimer the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the cdc. references top ↑ 1.olmsted ss, moore m, melli rc, et al. strengthening laboratory systems in resource-limited settings. am j clin pathol 2010;134(3):374–380. http://dx.doi.org/10.1309/ajcpdqosb7qr5glr 2.nkengasong jn. a shifting paradigm in strengthening laboratory health systems for global health: acting now, acting collectively, but acting differently. am j clin pathol. 2010;134(3):359–360. http://dx.doi.org/10.1309/ajcpy5asuejyq5rk 3.nkengasong jn, nsubuga p, nwanyanwu n, et al. laboratory systems and services are critical in global health: time to end the neglect? am j clin pathol. 2010;134(3):368–373. http://dx.doi.org/10.1309/ajcpmpsinq9brmu6 4.justman je, stephania kd, amiliar t, et al. developing laboratory systems and infrastructure for hiv scale-up: a tool for health systems strengthening in resource-limited settings. j acquir immune defic syndr. 2009;52(suppl 1):s30–s33. http://dx.doi.org/10.1097/qai.0b013e3181bbc9f5 5.gershy-damet gm, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. http://dx.doi.org/10.1309/ajcptuuc2v1wjqbm 6.schroeder l, amukele t. medical laboratories in sub-saharan africa that meet international quality standards. am j clinpathol. 2014;141(6):791–795. http://dx.doi.org/10.1309/ajcpq5ktkagsscfn 7.the president’s emergency plan for aids relief. planning and reporting: next generation indicators reference guide. version1.1 [document on the internet]. c2009 [cited 2013 mar]. available from: http://www.pepfar.gov/documents/organization/81097.pdf 8.world health organization. kigali host the launch of a program to accelerate national laboratory service capacity building towards accreditation in the african region [document on the internet]. c2009 [cited 2014 oct 05]. available from: http://www.who.int/hiv/amds/diagnostics/amds_kigali_pr_lab.pdf 9.yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin pathol. 2010;134(3):401–409. http://dx.doi.org/10.1309/ajcpnbbl53fwuiqj 10.mothabeng d, maruta t, lebina m, et al. strengthening laboratory management towards accreditation: the lesotho experience. afr j lab med. 2012;1(1), art. #9, 7 pages. 11.yao k, maruta t, luman et, nkengasong jn. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. 2014;3(1), art. #194, 8 pages. http://dx.doi.org/10.4102/ajlm.v3i1.194 12.oakes k. grand central training, part two: industrial experts provide their thoughts on moving to centralized training [document on the internet]. c2005 [cited 2014 mar 14]. available from: http://www.astdcascadia.org/conference/2005/pdf_documents/oakes_article_2.pdf 13.lawrence sh. centralization and decentralization: the compunications connection [document on the internet]. c1983 [cited 2013 mar]. available from: http://www.pirp.harvard.edu/pubs_pdf/lawrenc/lawrenc-i83-2.pdf 14.fida office of evaluation. evaluación del programa en el país [country program evaluation]. report no. 2322-ar [document on the internet]. c2010 [cited 2014 oct 14]. available from: http://www.ifad.org/evaluation/public_html/eksyst/doc/country/pl/argentina/argentina_cpe.pdf 15.world health organization’s regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation in the african region (with checklist) [document on the internet]. c2012 [cited 2013 jun]. available from: http://www.afro.who.int/en/clusters-a-programmes/hss/blood-safety-laboratories -a-health-technology/blt-highlights/ 3859-who-guide-for-the-stepwise-laboratory-improvement-process-towards-accreditationin-the-african-region-with-checklist.html 16.luman et, yao k, nkengasong jn. a comprehensive review of the slmta literature part 1: content analysis and future priorities. afr j lab med. 2014;3(2), art. #265, 11 pages. http://dx.doi.org/10.4102/ajlm.v3i2.265 17.maruta t, yao k, ndlovu n, moyo s. training-of-trainers: a strategy to build country capacity for slmta expansion and sustainability. afr j lab med. 2014;3(2), art. #196, 7 pages. http://dx.doi.org/10.4102/ajlm.v3i2.196 18.yao k, luman et, slmta collaborating authors. evidence from 617 laboratories in 47 countries for slmta-driven improvement in quality management systems. afr j lab med. 2014;3(2), art. #262, 11 pages. http://dx.doi.org/10.4102/ajlm.v3i2.262 abstract introduction prioritising antimicrobial resistance in ethiopia national antimicrobial resistance surveillance system challenges and lessons learned from implementation conclusion acknowledgements references about the author(s) rajiha a. ibrahim ethiopian public health institute, addis ababa, ethiopia amete m. teshal ethiopian public health institute, addis ababa, ethiopia surafel f. dinku ethiopian public health institute, addis ababa, ethiopia negga a. abera ethiopian public health institute, addis ababa, ethiopia abebe a. negeri ethiopian public health institute, addis ababa, ethiopia feven g. desta ethiopian public health institute, addis ababa, ethiopia eyasu t. seyum ethiopian public health institute, addis ababa, ethiopia adugna w. gemeda ethiopian public health institute, addis ababa, ethiopia wubshet m. keficho american society for microbiology, addis ababa, ethiopia citation ibrahim ra, teshal am, dinku sf, et al. antimicrobial resistance surveillance in ethiopia: implementation experiences and lessons learned. afr j lab med. 2018;7(2), a770. https://doi.org/10.4102/ajlm.v7i2.770 lessons from the field antimicrobial resistance surveillance in ethiopia: implementation experiences and lessons learned rajiha a. ibrahim, amete m. teshal, surafel f. dinku, negga a. abera, abebe a. negeri, feven g. desta, eyasu t. seyum, adugna w. gemeda, wubshet m. keficho received: 30 jan. 2018; accepted: 22 june 2018; published: 06 dec. 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract introduction: antimicrobial resistance (amr) poses a global threat. high levels of amr to commonly used antibiotics have been reported in east africa. a situation analysis of amr in ethiopia also indicated high resistance levels. to prevent and contain amr, ethiopia established a national surveillance network. objectives: this article describes the steps taken to prioritise amr and establish the national antimicrobial resistance surveillance system in ethiopia, as well as present the challenges and lessons learned through implementation. methods: in april 2017, ethiopia had developed and approved the national amr surveillance plan for laboratory-based amr surveillance. the world health organization recommendations and ethiopias’s current microbiology capacity were used to prioritise organisms for reporting. the surveillance system is comprised of a network linking the national reference laboratory with surveillance sentinel sites. roll-out of the amr surveillance network occurred in three phases in order to ensure successful implementation. results: electronic capture and transmission of data, supply chain for the microbiology laboratory and communication problems were challenges observed after implementation started. support from ethiopian public health institute focal persons for data entry, regular scheduled communication establishment and procurement of supplies by the american society for microbiology were some of the measures taken to address the challenges. conclusion: ethiopia has demonstrated that setting up amr surveillance in lower resource settings is possible with strong leadership and stakeholder engagement. introduction across the globe, the emergence of antimicrobial resistance (amr) is threatening the effective and successful treatment of infectious diseases. drug resistance proliferates due to the improper use of drugs, poor regulation of antibiotics, limited antimicrobial stewardship, poor prescribing habits and non-compliance with prescription.1 mutation of bacteria genomes by different mechanisms may lead to drug-resistant strains and antimicrobial use or misuse provides a selective advantage to resistant variants. these, in turn, lead to more common treatment failure, which can result in increased morbidity and mortality, prolonged illness, premature death and other worsened clinical outcomes.2 globally, it is estimated that 700 000 people die every year from drug resistance in common bacterial infections, hiv and malaria. this number is believed to be underestimated due to poor reporting and surveillance.2 in addition, amr puts a financial burden on resource-limited countries.3 studies from east africa have reported high levels of amr to commonly used antibiotics with gram-negatives showing 50% – 100% resistance to ampicillin and trimethoprim or sulfamethoxazole (for instance in uganda, 100% resistance in escherichia coli has been observed). the extent and burden of this resistance, however, is not being monitored through established ongoing surveillance systems.4 in 2009, concerned about the increasing prevalence of amr globally, the ethiopian drug administration and control authority, in collaboration with management sciences for health or strengthening pharmaceutical system, conducted a situation analysis, the antimicrobial use, resistance, and containment baseline survey to understand the status of resistance and trends in the use of antimicrobial drugs in ethiopia.5 the survey estimated changes in resistance of a variety of pathogens, including streptococcus pneumoniae, salmonella spp. and staphylococcus aureus between 1996 and 2000 in ethiopia. s. pneumoniae showed an increase in resistance to erythromycin from 0% in 1996 to 19.2% in 2000. the survey also found organisms with a high level of multidrug resistance. shigella dysenteriae showed an overall resistance of 31.8%, 43.8%, 81% and 89.5% to chloramphenicol, trimethoprim or sulfamethoxazole, ampicillin and tetracycline respectively.5 other studies conducted across ethiopia also indicated increasing rates of resistance in e. coli, shigella spp., salmonella spp. and s. aureus to commonly prescribed antibiotics such as ampicillin, amoxicillin, penicillin, tetracycline and trimethoprim or sulfamethoxazole.6,7 in addition to increased morbidity and mortality, amr is an increasing threat to global health security with potential economic, social and political ramifications.8 to promote global health security as an international priority, the global health security agenda (ghsa) was launched in 2014 as an effort by nations, international organisations and civil society to accelerate progress toward a world safe and secure from infectious disease threats. the prevention and containment of amr was identified as one of the priority initiatives under ghsa.9 to contain the spread of amr and maintain the usefulness of antimicrobial agents for the future, a focus on laboratory diagnostics, surveillance, stewardship and regulation is required in countries. in particular, laboratory-based surveillance that detects resistance patterns and monitors their spread is needed to advance a country’s understanding of its amr burden and illuminate areas to target.1 in response to the findings of the 2009 situation analysis, the federal democratic republic of ethiopia began prioritising efforts to detect and combat amr in the country and in 2016, with support from the ghsa, established a national surveillance network to detect amr. the purpose of this article is to describe the steps taken to establish the national antimicrobial resistance surveillance system in ethiopia and present the challenges and lessons learned through implementation. prioritising antimicrobial resistance in ethiopia following the 2009 situation analysis, ethiopia began to focus its efforts on disseminating information about amr to the community and establishing a national strategy and action plan to curb the rising resistance. the national advisory committee on antimicrobial resistance prevention and containment, a multi-disciplinary body to govern and oversee the development and implementation of the national strategy, was established in 2011 with representation from both the human and animal health sectors of the government. by august of that year, the national strategic framework for prevention and containment of antimicrobial resistance had been developed and endorsed.10 the amr national proposal was also developed in 2014. however, it was not implemented due to limited resources for implementation. in 2015, with financial and technical support from the united states centers for disease control and prevention (cdc) through the ghsa, various multi-sector partners collaborated to produce a revised 5-year strategy for the prevention and containment of antimicrobial resistance for ethiopia (2015–2020). the updated strategy prioritised promotion of optimal use of antimicrobials in human and animal health through effective stewardship practices, and strengthening the knowledge and evidence on antimicrobial use and resistance through one health surveillance and research.11 national antimicrobial resistance surveillance system the development and implementation of the ethiopia amr surveillance plan was a national effort led by the ethiopian public health institute (ephi) under the federal ministry of health and supported by cdc, the american society for microbiology (asm) as well as ohio state university’s global one health initiative. in august 2016, ephi held a 3-day workshop to kick off discussions around amr surveillance to address the need for a ‘surveillance system that captures the emergence of resistance, trends, its spread and utilization of antimicrobial agents in different settings’. the workshop served as an opportunity to both sensitise key stakeholders about the importance of amr and discuss and agree on priorities and methods for surveillance implementation. staff from ephi, stakeholders and other decision-makers used guidance materials from the world health organization’s (who) global amr surveillance system (glass) to inform discussions and decision-making around the selection of sites, organisms and specimens to prioritise for reporting and data management methods.12 by april 2017, ethiopia had developed and approved the national amr surveillance plan.13 the objective of the plan is to establish a national surveillance network capable of detecting priority amr pathogens, analysing and reporting data, characterising resistance and generating evidence to inform the implementation of targeted prevention and control programmes.13 the plan outlines the activities needed to implement a national amr surveillance system, the approach for data management and reporting, and the roles and responsibilities of clinical and laboratory stakeholders. in addition to who glass recommendations, selection of organisms and specimens to prioritise for reporting took into consideration pathogen prevalence and the current microbiology capacity of laboratories in ethiopia. ethiopia chose to focus on surveillance of e. coli, klebsiella pneumoniae, and s. aureus obtained from urine and wound specimens and all carbapenem-resistant organisms, regardless of specimen type (table 1). it is expected that, over time, as the surveillance system is strengthened, the number of pathogens and specimen types targeted will increase. table 1: priority surveillance pathogens by specimen for inclusion in ethiopia amr surveillance. ethiopia’s surveillance system is structured to connect sentinel surveillance sites to the national reference laboratory at ephi. each surveillance site includes a laboratory that is either affiliated with or located within a hospital. currently, the surveillance network includes a total of 16 sentinel surveillance sites located across four regions and one city administration. roll-out of the amr surveillance network is occurring in three phases in order to ensure successful implementation and to effectively prepare sites for sample collection, diagnostic testing, data management and reporting. in order to prioritise sites for roll-out, ephi, with assistance from asm, assessed laboratories across ethiopia in late 2016. a standardised assessment tool was used to better understand the conditions and capacities of potential amr surveillance sites. in the first phase of implementation, 4 of the 16 sites have been targeted to participate. the national reference laboratory at ephi is coordinating and overseeing all surveillance activities. focal persons from ephi have been assigned to each of the initial four sites to support implementation. implementation began with personnel training for clinicians and laboratory staff. asm supported ephi to train laboratory staff from the initial four surveillance sites in basic microbiology, antibiotic susceptibility testing and data management. a model of laboratory mentorship has been put in place. the four sites have begun receiving hands-on mentorship during which experienced laboratorians from asm and ephi work alongside the staff from the respective surveillance sites to ensure procedures are understood, followed and refined. to ensure that quality laboratory data is generated, all sites have been enrolled in an external quality assessment programme to evaluate diagnostic and reporting ability. as the quality of specimens sent for testing also affects laboratory data quality, ohio state university’s global one health initiative conducted training for clinical and laboratory staff on proper methods for clinical specimen collection. in addition, the training aimed at introducing the purpose and goals of amr surveillance and broadly conveying the importance of stewardship, infection prevention and control in the context of amr surveillance. the four sentinel sites receiving hands-on mentorship are currently involved in active surveillance. as part of surveillance, specimens, sent to the sentinel site laboratories, undergo routine culture and antibiotic susceptibility testing (figure 1). laboratory results, including measurement of the zone of inhibition, are then entered into an electronic database and sent to ephi monthly. if a culture is positive for a priority pathogen (table 1), the corresponding isolate will be shipped to the national reference laboratory for confirmatory testing. at ephi, the data on pathogen prevalence and susceptibility will be analysed by using whonet software, published and shared with the sentinel surveillance sites and regional reference laboratories, and eventually, entered into who glass. figure 1: diagram of amr surveillance data flow within the ethiopia national antimicrobial resistance surveillance system. challenges and lessons learned from implementation implementation and roll-out of amr surveillance has started according to plan. after the fourth month of implementation, an early evaluation was conducted with support from cdc subject matter experts. the evaluation identified a number of challenges that ephi is now taking strides to address. a primary challenge has been the integration of electronic data capture for amr surveillance into the normal work and laboratory processes at the sentinel surveillance sites. one reason for this is that none of the sites has an established electronic laboratory information management system in use for microbiology and thus staff are not accustomed to inputting data electronically as a regular activity. as an effect of incorporating electronic entry of amr results into their normal workflow, microbiology staff at some of the sites have reported slower turnaround times for getting laboratory results back to the ordering physicians. frequent microbiology staff turnover at the sites has also made electronic data entry challenging, as fewer staff are available to run culture and input data. laboratory capacity building through on-site mentorship at the surveillance sites and provision of necessary supplies has proved a useful method for ensuring sites are producing quality data. the use of focal persons from ephi in monitoring progress at each surveillance site has been crucial for identifying problems and supply needs, and for facilitating corrective action on laboratory practices and data reporting. prior to the evaluation, focal persons from ephi were visiting sites irregularly and thus creating gaps in communication. communication between the ephi focal persons and surveillance sites has since been improved by establishing weekly calls and arranging monthly site visits for the focal persons at ephi to work on capacity building and quality improvement activities. local procurement of quality microbiology supplies is a challenge in ethiopia. in the meantime, asm purchased the needed amr supplies for ephi and the sentinel surveillance sites. for long-term sustainability, ephi has begun working with the pharmaceutical fund and supplies agency, ethiopia’s central procurement agency, to ensure adequate, quality supplies are available in future. conclusion ethiopia has committed to join global partners in the detection and prevention of amr. in a region where amr data is under-represented and often lacking, ethiopia has made great strides in the establishment its national antimicrobial resistance surveillance system to properly understand and address the prevailing problem in the country.14 ethiopia has proven that surveillance in lower resource settings is possible given strong leadership and stakeholder engagement during the planning and implementation phases of surveillance. oversight of sentinel sites through constant communication and provision of sustainable mentorship to achieve quality data is a core element for ensuring surveillance is successful. the next steps for amr surveillance in ethiopia will be continuous strengthening of active surveillance sites. as capacity of the sites is strengthened, gradual engagement of additional sites will take place. this will enable the country to fulfil its goal of establishing a national amr surveillance network that can provide quality data to further inform policies on antimicrobial prescribing and purchasing, improve the delivery of care and treatment in ethiopia and, ultimately, reduce morbidity and mortality due to microbial infections. acknowledgements the authors would like to acknowledge the united states centers for disease control and prevention, atlanta for technical and financial support. we would also like to thank the american society for microbiology for technical support. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support the surveillance was funded by the united states centers for disease control and prevention, atlanta. authors’ contributions r.a.i. was the surveillance coordinator. a.m.t., s.f.d. and n.a.a. made conceptual contributions and participated in the write-up. a.a.n. contributed to the write-up. f.g.d. made conceptual contributions. e.t.s. contributed to the write-up and revision. a.w.g. made conceptual contributions and w.m.k. contributed to the write-up. references bosco jn, ahmed ay, tamer ad, et al. antimicrobial resistance in the african region: issues, challenges and actions proposed. african health monitor. 2013;16. o’neill j. tackling drug-resistant infections globally: final report and recommendations. the review on antimicrobial resistance 2016. https://amr-review.org/sites/default/files/160518_final%20paper_with%20cover.pdf seale ac, hutchison c, fernandes s, et al. supporting surveillance capacity for antimicrobial resistance: laboratory capacity strengthening for drug resistant infections in low and middle-income countries. wellcome open res. 2017;2:91. https://doi.org/10.12688/wellcomeopenres.12523.1 ampaire l, muhindo a, orikiriza p, mwanga-amumpaire j, bebell l, boum y. a review of antimicrobial resistance in east africa. afr j lab med. 2016;5(1):1–6. https://ajlmonline.org/index.php/ajlm/article/view/432 antimicrobials use, resistance and containment baseline survey syntheses of findings. addis ababa: daca, msh/sps; 2009. moges f, endris m, mulu a, et al. the growing challenges of antibacterial drug resistance in ethiopia. j glob antimicrob resist. 2014;2(3):148–154. https://doi.org/10.1016/j.jgar.2014.02.004 seboxa t, amogne w, abebe w, et al. high mortality from blood stream infection in addis ababa, ethiopia, is due to antimicrobial resistance. plos one. 2015;10:12. https://doi.org/10.1371/journal.pone.0144944 howell l. global risks insight report. geneva: world economic forum; 2013. sililanukee p. global health security agenda – ghsa [homepage on the internet]. ministry of social affairs and health, finland. available from: http://stm.fi/en/international-cooperation/ghsa national strategic framework for prevention and containment of antimicrobial resistance, 2011. national strategic framework for prevention and containment of antimicrobial resistance, 2015. world health organization. global antimicrobial resistance surveillance system. manual for early implementation, 2015. http://www.who.int/antimicrobial-resistance/publications/surveillance-system-manual/en/ ethiopia antimicrobial resistance surveillance plan, 2017. the surveillance of antimicrobial resistance using public health laboratory-based sentinel sites in ethiopia 2016–2020. https://ephi.gov.et/images/pictures/download2010/ethiopia-amr-surveillance-plan_final.pdf world health organization. antimicrobial resistance: global report on surveillance. 2014. http://www.who.int/drugresistance/documents/surveillancereport/en/ abstract introduction methods results discussion acknowledgements references about the author(s) annie zhang department of microbiology, college of arts and sciences, the ohio state university, columbus, ohio, united states enoch jumbe child legacy international, msundwe, lilongwe, malawi robert krysiak department of infectious diseases, school of medicine, university of north carolina project, tidziwe centre, lilongwe, malawi sabeen sidiki department of microbial infection and immunity, college of medicine, the ohio state university, columbus, ohio, united states holden v. kelley department of microbial infection and immunity, college of medicine, the ohio state university, columbus, ohio, united states elly k. chemey child legacy international, msundwe, lilongwe, malawi chancy kamba district tuberculosis control office, ministry of health, lilongwe, malawi victor mwapasa department of community health, college of medicine, blantyre, malawi juan i. garcía department of pediatrics, obstetrics, gynecology and preventive medicine, autonomous university of barcelona, barcelona, spain alison norris college of public health, the ohio state university, columbus, ohio, united states xueliang j. pan center for biostatistics, college of medicine, the ohio state university, columbus, ohio, united states carlton evans the wellcome centre for clinical tropical medicine, imperial college of london, london, united kingdom department of microbiology, cayetano heredia university, lima, peru shu-hua wang division of infectious diseases, department of internal medicine, college of medicine, the ohio state university, columbus, ohio, united states jesse j. kwiek department of microbiology, college of arts and sciences, the ohio state university, columbus, ohio, united states jordi b. torrelles department of microbial infection and immunity, college of medicine, the ohio state university, columbus, ohio, united states citation zhang a, jumbe e, krysiak r, sidiki s, kelley hv, chemey ek, et al. low-cost diagnostic test for susceptible and drug-resistant tuberculosis in rural malawi. afr j lab med. 2018;7(1), a690. https://doi.org/10.4102/ajlm.v7i1.690 original research low-cost diagnostic test for susceptible and drug-resistant tuberculosis in rural malawi annie zhang, enoch jumbe, robert krysiak, sabeen sidiki, holden v. kelley, elly k. chemey, chancy kamba, victor mwapasa, juan i. garcía, alison norris, xueliang j. pan, carlton evans, shu-hua wang, jesse j. kwiek, jordi b. torrelles received: 07 oct. 2017; accepted: 29 jan. 2018; published: 04 june 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: rural settings where molecular tuberculosis diagnostics are not currently available need easy-to-use tests that do not require additional processing or equipment. while acid-fast bacilli (afb) smear is the most common and often only tuberculosis diagnosis test performed in rural settings, it is labour intensive, has less-than-ideal sensitivity, and cannot assess tuberculosis drug susceptibility patterns. objective: the objective of this study was to determine the feasibility of a multidrug-resistant (mdr) or extensively drug-resistant (xdr)-tuberculosis coloured agar-based culture test (tuberculosis cx-test), which can detect mycobacterium tuberculosis growth and evaluate for drug susceptibility to isoniazid, rifampicin and a fluoroquinolone (i.e. ciprofloxacin) in approximately 14 days. method: in this study, 101 participants were enrolled who presented to a rural health clinic in central malawi. they were suspected of having active pulmonary tuberculosis. participants provided demographic and clinical data and submitted sputum samples for tuberculosis testing using the afb smear and tuberculosis cx-test. results: the results showed a high level of concordance between the afb smear (12 positive) and tuberculosis cx-test (13 positive); only one sample presented discordant results, with the molecular genexpert mtb/rif® test confirming the tuberculosis cx-test results. the average time to a positive tuberculosis cx-test was 10 days. of the positive samples, the tuberculosis cx-test detected no cases of drug resistance, which was later confirmed by the genexpert mtb/rif®. conclusion: these findings demonstrate that the tuberculosis cx-test could be a reliable low-cost diagnostic method for active pulmonary tuberculosis in high tuberculosis burden rural areas. introduction in 2017, the world health organization (who) estimated that 4000 people die of tuberculosis every day. malawi is among the 20 countries with a who-defined ‘high’ tuberculosis and hiv burden: the country reported 15 737 new and relapsed cases in 2015.1 of these cases, only 6% were tested with rapid tuberculosis diagnostics at the time of diagnosis (by genexpert mtb/rif®, cepheid, sunnyvale, california, united states).2 most, 75%, were diagnosed as pulmonary tuberculosis by clinical symptoms; 58% of these had confirmatory culture and only 47% were provided with tuberculosis treatment. the recently-reported tuberculosis incidence rate in malawi is 193 per 100 000 people per year, with 53% of cases occurring in people that are also hiv-positive.1 who data for malawi estimates that 0.75% of new cases and 6.4% of previously-treated cases are multidrug-resistant (mdr)-tuberculosis1; however, real numbers may be higher due to current limited drug susceptibility testing in the country. with the adoption of the ‘sustainable development goals’ and the ‘end tb strategy’ in late 2015, worldwide efforts to end the global tuberculosis epidemic are ambitious and require new advancements in tuberculosis diagnostics. while acid-fast bacilli (afb) smear is the most common tuberculosis diagnostic method in malawi and many other low-income and high tuberculosis-burden countries around the globe, it is labour intensive, has less-than-ideal sensitivity, and cannot be used to assess tuberculosis drug susceptibility. access to tuberculosis diagnostics has improved in recent years but still remains limited. in malawi, there are approximately two smear and microscopy facilities per 100 000 people and one laboratory capable of performing tuberculosis drug susceptibility tests per 10 million people.3 culture-based diagnostic methods remain the gold standard for drug susceptibility testing, but can take up to 86 days to yield results.4 in 2010, the who recommended genexpert mtb/rif® as an initial tuberculosis diagnostic test. the genexpert mtb/rif® is a polymerase chain reaction-based test that takes less than two hours to perform and simultaneously detect mycobacterium tuberculosis and rifampicin resistance in the tested sample. in this test, rifampicin resistance is used as a surrogate marker for mdr-tuberculosis, which is defined as resistance to both isoniazid and rifampicin. despite its sensitivity and utility, the genexpert mtb/rif® implementation in high tuberculosis burden areas is cost prohibitive (approximately $18 per sample in malawi), requires expensive instrumentation with weekly maintenance and monthly calibration, a sustained power source, and laboratory technicians with specialised training.5 these drawbacks render it currently inaccessible to most areas in high tuberculosis-burden countries. there is a need for a simple, inexpensive tuberculosis diagnostic test that can be performed in rural health facilities, where access to molecular tuberculosis diagnostics may not be feasible. the mdr/xdr-tuberculosis coloured agar-based test (tuberculosis cx-test) is a non-commercial, thin-layer agar-based tuberculosis culture method capable of simultaneously detecting m. tuberculosis and tuberculosis drug susceptibility in approximately 14 days. the tuberculosis cx-test contains four quadrants: one quadrant detects m. tuberculosis growth and the other three quadrants detect resistance to isoniazid, rifampicin and a fluoroquinolone (i.e. ciprofloxacin). the tuberculosis cx-test is simple to use: expectorated sputum is mixed with disinfectant, the mixture is cultured onto the tuberculosis cx-test, and colonies are enumerated after incubation. in a study completed in a research laboratory setting using 197 archived m. tuberculosis clinical isolates, the tuberculosis cx-test detected drug resistance with 98% sensitivity for isoniazid, rifampicin, and ciprofloxacin and 99% for mdr-tuberculosis, compared to drug susceptibility testing results using a liquid culture method.6 specificities reported for isoniazid were 100% (95% ci 82–100), 88% (95% ci 69–97) for rifampicin, 91% (95% ci 83–96) ciprofloxacin and 90% (95% ci 74–98) for mdr-tuberculosis.6,7 a systematic review of three studies assessing the thin-layer agar-based assay technique found a pooled sensitivity of 100% (95% ci 97–100) and pooled specificity of 100% (95% ci 99–100) for the detection of rifampicin; and a pooled sensitivity of 100% (95% ci 91–100) and pooled specificity of 100% (95% ci 99–100) for the detection of isoniazid.8,9,10,11 the tuberculosis cx-test has also been shown to be highly specific in identifying m. tuberculosis from atypical mycobacteria.12 although the tuberculosis cx-test has been shown to be accurate in research laboratories, its performance in the field and in clinical settings in high tuberculosis-burden areas has yet to be characterised. in this study, we sought to determine the feasibility of the tuberculosis cx-test to diagnose active pulmonary tuberculosis and patterns of tuberculosis drug susceptibility to isoniazid, rifampicin, and ciprofloxacin in a rural malawian health clinic using direct sputum specimens, where currently only afb smear is performed and no routine cultures are sent for confirmation or drug susceptibility testing. methods ethical considerations institutional review board approval was obtained from the ohio state university (study number 2014h0381) and the malawi college of medicine research and ethics committee (study number p.09/14/1627). all participants were adults and were enrolled in the study using a written consent form. tuberculosis cx-test results were for research only and were not used for diagnosis and they did not influence treatment outcomes. following the malawian ministry of health recommendations, genexpert mtb/rif® positive samples were to be retested by the malawian national tuberculosis control program before patients were notified. study population participants were recruited from the child legacy international-mcguire wellness center in msundwe, malawi. child legacy international serves a catchment area of 68 rural villages (~18 000 people) in the central region of malawi.13,14,15,16,17 participants were limited to patients suspected of having active pulmonary tuberculosis disease based on clinical symptoms (i.e. fever, chills, night sweats, shortness of breath, chest pain, cough ≥ 2 weeks, loss of weight, fatigue), who were ≥ 18 years of age, capable of providing informed written consent, and able to provide sputum. participants answered a survey regarding demographics (i.e. age, sex) and clinical history (i.e. hiv status, tuberculosis clinical symptoms, previous diagnosis and treatment). hiv status and testing were verified by health passport or offered on-site to all participants, along with preand post-hiv counselling; hiv testing was not required for participation in this study. participants were recruited over the course of 11 months during the period of november 2015 to october 2016. tuberculosis cx-test preparation the tuberculosis cx-tests were prepared according to published methods as previously described6 in different batches in research laboratories at the ohio state university in the united states. tuberculosis cx-test quality control per each batch was confirmed in an ohio state university biosafety level 3 laboratory using verified susceptible, mono-isoniazid resistant, mono-rifampicin resistant and mdr m. tuberculosis clinical isolates provided by the ohio department of health state laboratory. upon tuberculosis cx-test quality control confirmation, tuberculosis cx-test batches were transported to malawi and properly stored at 4 °c until used within four months of being made. tuberculosis cx-testing sputum samples were collected, numerically coded (de-identified) and equally divided by pipetting for (1) afb smear, (2) tuberculosis cx-test, and (3) genexpert mtb/rif® testing (in the case of discordant results). sputa for afb smear and tuberculosis cx-tests were processed immediately after collection, whereas samples for genexpert mtb/rif® testing were stored at −20 °c until used. for the afb smear, standard procedures dictated by the malawi ministry of health were followed for sputum collection, ziehl-neelsen technique for afb staining, microscopy, and smear grading.18 during ziehl-neelsen staining, sputum was applied to a slide and heat fixed. the slide was submerged into carbol fuchsin, heated to dry, and rinsed with water. next, slides were submerged in a 3% solution of hydrochloric acid (de-staining step), briefly washed with water, and then counterstained with methylene blue.19 for the tuberculosis cx-test (figure 1), one-third of the collected sputum was added to a 50 ml tube containing twice the volume of disinfectant (stock solution: 2 g tri-sodium phosphate, 0.05 g ammonium sulphate, 0.005 g magnesium sulphate, 0.0025 g ferric ammonium citrate, 10 ml sterile water, and 0.01 ml red food coloring, all mixed by manual shaking). two drops of the sputum/disinfectant mixture (1:2, v/v) were then plated directly onto each quadrant of the tuberculosis cx-test.6 the tuberculosis cx-test was then incubated at 37 °c for 28–42 days and checked three times per week for bacterial growth. colonies from each quadrant were counted during each check. the presence of m. tuberculosis was microscopically defined as the presence of the typical rough colony on the detection quadrant, using a low-resolution (20x) microscope (clear quadrant). drug resistance was defined by the detection of growth on each of the specific quadrants: isoniazid (yellow quadrant), rifampicin (green quadrant), and ciprofloxacin (blue quadrant). the drug concentration in each quadrant was as follows: isoniazid (0.2 µg/ml), rifampicin (1 µg/ml) and ciprofloxacin (2 µg/ml). as a precautionary measure, all tuberculosis cx-tests were kept in the incubator for an additional seven days before being discarded. figure 1: the tuberculosis cx-test. for discordant results between the afb smear and the tuberculosis cx-test, coded sputum samples stored at −20 °c were transported to lilongwe and tested with genexpert mtb/rif® by the university of north carolina project at lilongwe, as described previously.1 moreover, approximately an additional 10% of samples tested by afb smear and the tuberculosis cx-test were selected randomly and also analysed by genexpert mtb/rif® to confirm the obtained results. statistical analysis descriptive data regarding participant demographics and clinical characteristics were collected and summary statistics were compiled. the wilcoxon rank-sum test was used to test age association with tuberculosis diagnosis. fisher’s exact test was used to identify clinical characteristics significantly associated with a positive tuberculosis diagnosis. data analysis was performed using jmp software (version 11; sas [https://www.jmp.com/en_us/home.html]). results a total of 101 participants were enrolled in the study. of these, five participants were excluded due to tuberculosis cx-plate contamination or the plate drying out. the average participant age was 47 years (sd = 17) (table 1). a large percentage of participants had been already tested for hiv (97%) at enrolment. of these, 10 (10.4%) stated they were hiv-positive, 82 (85.4%) stated they were hiv-negative, while the remaining 4 (4.2%) did not know their status or had never been tested. the majority (88.5%) had never been tested previously for tuberculosis, while eleven (11.5%) had previous tuberculosis testing but of these only eight (8.3%) were diagnosed with active pulmonary tuberculosis. of the 10 hiv-positive participants, only two (20%) reported they were taking antiretroviral treatment. no participant reported taking tuberculosis medication at the time of enrolment. the participants’ most commonly-reported symptoms included dry and persistent cough (97%), weight loss (71%), fatigue (63%), night sweats (55%), fever (54%), chills (39%), and bloody sputum (30%). none of these symptoms was found to be significantly associated with a positive tuberculosis diagnosis. table 1: participant demographics and clinical characteristics. of the 96 participants, 12 (12.5%) were positive by both afb smear and the tuberculosis cx-test, and 83 (86.5%) were negative by both afb smear and the tuberculosis cx-test (table 2). one sample was found to have discordant results between afb smear (negative) and the tuberculosis cx-test (positive). in this case, the genexpert mtb/rif® revealed a positive result for the presence of m. tuberculosis in sputum, in agreement with the tuberculosis cx-test. the tuberculosis cx-test was also comparable to the afb smear (99% agreement on the diagnosis results), and positively identified a tuberculosis diagnosis, while afb smear yielded a single false negative result. the time to obtain a positive tuberculosis cx-test result ranged between 7 and 14 days (mean = 10, sd = ±2.33) (figure 2). the tuberculosis cx-test detected no cases of drug resistance; however, late growth was observed in the antibiotic quadrants of three plates. late growth is defined as growth in any of the antibiotic quadrants that takes place after growth in the detection quadrant is observed. this growth is not indicative of drug resistance. the genexpert mtb/rif® testing of nine positive samples that were sent for verification confirmed the presence of m. tuberculosis and no rifampicin drug resistance, including the three samples that contained late growth in the rifampicin quadrant. among patients with a positive tuberculosis diagnosis, three (23%) stated they were also hiv-positive. younger age (18 to 39-years old) was significantly associated with a positive tuberculosis diagnosis (z = −2.2, p < 0.03). figure 2: time to positive tuberculosis cx-test. table 2: contingency table of acid-fast bacilli smear microscopy vs. tuberculosis cx-test. discussion our findings demonstrate that the tuberculosis cx-test can be implemented in rural health clinics in a low-income and high tuberculosis-burden setting, with diagnosis sensitivity comparable to afb smear. the sensitivity of afb staining is dependent on the presence of high bacterial load in the patient’s sputum and the technical skills of the microscopist. one study estimated that afb smear microscopy fails to detect m. tuberculosis in a third of patients who are later diagnosed by culture, and that afb smear microscopy is particularly insensitive in high-risk tuberculosis populations including children and hiv-positive individuals.5 moreover, in areas with high incidence of nontuberculous mycobacteria infections, the afb smear cannot distinguish clearly between nontuberculous mycobacteria and m. tuberculosis. however, studies that used the tuberculosis cx-test demonstrated that this test is able to differentiate m. tuberculosis from nontuberculous mycobacteria infections with > 99.6% specificity.6 in this study, there was high concordance between afb staining and the tuberculosis cx-test, with the latter offering higher sensitivity over afb staining. altogether, our results suggest that the tuberculosis cx-test accurately detects drug susceptible tuberculosis in low-income and high tuberculosis-burden settings. a previous study of 702 patients at a hiv and tuberculosis treatment clinic in lilongwe found a low (1.4%) prevalence of drug-resistant tuberculosis, with only 10 cases of culture-confirmed isoniazid resistance, and one case (0.1%) each for rifampicin resistance and mdrtuberculosis.20 our results also confirmed the lack of drug-resistant tuberculosis among participants in this study and thus we are unable to report on the tuberculosis cx-test’s ability to identify drug-resistant m. tuberculosis. of a subset of positive samples (10%) sent for the genexpert mtb/rif® testing, the tuberculosis cx-test was in agreement with the genexpert mtb/rif®, in that the samples were susceptible to rifampicin. none of the participants who stated that they had taken first-line anti-tuberculosis drugs for susceptible tuberculosis had failed therapy, further suggesting the absence of clinically-significant drug-resistant tuberculosis. although there are several commercially-available molecular tuberculosis diagnostic tests on the market,21 including the genexpert mtb/rif®, which has exceptional sensitivity, specificity, and minimal time to results, genexpert tests are currently cost prohibitive in many low-income countries, and especially in countries with a high tuberculosis burden. as a result, the long-term implementation of such diagnostic tests requires ongoing financial support from governments or nongovernmental organisations such as the who, the united states agency for international development, and the world bank, among others. limitations of the 96 valid tests carried out in this study, three (3%) showed late growth in the rifampicin quadrant several days after growth was detected in the detection quadrant; results from the genexpert mtb/rif® test indicated that the three samples were all drug susceptible. there are several possible explanations for the late growth in the tuberculosis cx-test in the drug-containing quadrants days after growth was observed in the detection quadrant. this may be as a result of either a spontaneous mutation allowing bacteria to grow in the drug-containing quadrant, or could be due to breakdown of drugs after a certain period of time. alternatively, the drug concentration in the quadrants with late growth could be suboptimal (thereby giving a bacteriostatic effect instead of a bactericidal effect), or samples could contain bacteria that are heteroresistant. indeed, tuberculosis patients may harbour both drug susceptible and -resistant m. tuberculosis strains. although these are minor possibilities, these point out the importance of reading drug resistance results on the same day that the growth is observed in the detection quadrant (drug susceptible) of the tuberculosis cx-test. in the case that growth is observed in the drug-containing quadrants within seven days after being observed in the detection quadrant, the late growth will need to be further confirmed by genexpert mtb/rif® testing or other molecular techniques to rule out heteroresistance or a false negative result. other limitations include the small sample size and that the same personnel tabulated both the afb smear and tuberculosis cx-test. findings presented herein showed good correlation to afb smear results; however, afb microscopy (the only method used in the clinic where this study was performed) has been shown to have poor sensitivity or specificity when compared to either genexpert mtb/rif® or liquid culture (mycobacteria growth indicator tube [mgit]) tests. importantly, the time to detection of a positive tuberculosis cx-test was between 7 and 14 days, which is at least comparable to the time to detection for positive mgit results for m. tuberculosis cultures. finally, a correlation of smear grades to time to detection of the tuberculosis cx-test would also be useful in providing further comparators of performance. recommendations to determine the accuracy of diagnosis of tuberculosis drug resistance, the tuberculosis cx-test needs to be tested in countries with a high prevalence of drug resistance, where it can be compared to culture-confirmed drug susceptibility testing and genexpert mtb/rif®. conclusions a tuberculosis test that is easy-to-use and inexpensive does not require expensive equipment and highly-specialised staff, and is relatively quick to detect drug resistance (~14 days vs. 86 days), is currently in demand in many low-resource, high tuberculosis-burden countries. the tuberculosis cx-test, with a current production cost of $2.00 usd, could be the necessary tool to fill this gap. while our findings support the accuracy of the tuberculosis cx-test in detecting active pulmonary tuberculosis, a larger study focusing on the ability to detect drug susceptibility is needed before this test could be evaluated for implementation as a front-line drug susceptibility test. a comparison of the tuberculosis cx-test to either genexpert mtb/rif® or mgit culture would also be useful and is recommended to show the cx-test as an alternative to either of these platforms. in a setting of low drug resistance, this test is less useful than afb smear, which is cheaper and less labour intensive. thus, we hypothesise that the greatest utility for the tuberculosis cx-test is an environment with higher prevalence of mdr or xdr tuberculosis. acknowledgements we thank the malawian clients for their willingness to participate in this study. we thank the directors of child legacy international, mr. and mrs. jeff and karen rogers, for access to laboratory space to perform the afb staining and the tuberculosis cx-test. we also thank the university of north carolina project-malawi for performing the genexpert mtb/rif® testing. we would also like to thank the district tuberculosis control office, ministry of health in lilongwe, malawi, and the department of community health, college of medicine in blantyre, malawi for facilitating this study. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support this study was partially funded by the ohio state university public health preparedness for infectious diseases program (osu-phpid), osu office of international affairs, and osu college of medicine funds to j.j.k. and j.b.t. authors’ contributions a.z. analysed and interpreted the data and drafted the first version of the manuscript. e.j. acquired the samples, performed the experiments, contributed to data analysis and revised the manuscript. r.k. performed experiments. s.s. and h.v.k. performed the experiments and revised the manuscript. e.k.c., c.k., v.m., j.i.g., a.n., x.j.p., c.e. and s.-h.w. conceived of and designed the study and revised the manuscript. j.j.k. and j.b.t. conceived of and designed the study, performed data analysis and interpretation, and wrote the final version of the manuscript and were both co-senior authors in this manuscript. references world health organization. 2015 malawi tuberculosis profile [homepage on the internet]. c2015 [cited 2017 jul 10]. available from: https://extranet.who.int/sree/reports?op=replet&name=/who_hq_reports/g2/prod/ext/tbcountryprofile&iso2=mw&outtype=pdf cepheid. xpert mtb/rif [homepage on the internet]. c2015 [cited 2015 mar 16]. available from: http://www.cepheid.com/us/cepheid-solutions/clinical-ivd-tests/critical-infectious-diseases/xpert-mtb-rif world health organization. 2014 malawi tuberculosis profile. geneva, switzerland: world health organization; 2014. centers for disease control and prevention. introduction to the core curriculum on tuberculosis: what the clinician should know. atlanta, ga: cdc stacks public health publications; 2013, p. 92–94. evans ca. genexpert--a game-changer for tuberculosis control? plos med. 2011;8:e1001064. https://doi.org/10.1371/journal.pmed.1001064 toit k, mitchell s, balabanova y, et al. the colour test for drug susceptibility testing of mycobacterium tuberculosis strains. int j tuberc lung dis. 2012;16(8):1113–1118. https://doi.org/10.5588/ijtld.11.0609 herrera b, gilman rh, grandjean l, et al. optimization of tb field testing: in transit sputum decontamination & culture on colorimetric selective media for tb diagnosis & drug-susceptibility testing [homepage on the internet]. abstract symposium on tb field diagnostics ‘dying for a test’. cape town, south africa: medecins sans frontieres; c2007. p. 15–16 [cited 2017 aug 21]. available from: https://www.msfaccess.org/sites/default/files/msf_assets/tb/docs/tb_event_dyingforatest_eng_2007.pdf minion j, leung e, menzies d, et al. microscopic-observation drug susceptibility and thin layer agar assays for the detection of drug resistant tuberculosis: a systematic review and meta-analysis. lancet infect dis. 2010;10(10):688–698. https://doi.org/10.1016/s1473-3099(10)70165-1 martin a, paasch f, von groll a, et al. thin-layer agar for detection of resistance to rifampicin, ofloxacin and kanamycin in mycobacterium tuberculosis isolates. int j tuberc lung dis. 2009;13(10):1301–1304. schaberg t, reichert b, schülin t, et al. rapid drug susceptibility testing of mycobacterium tuberculosis using conventional solid media. eur respir j. 1995;8:1688–1693. https://doi.org/10.1183/09031936.95.08101688 robledo j, mejia gi, paniagua l, et al. rapid detection of rifampicin and isoniazid resistance in mycobacterium tuberculosis by the direct thin-layer agar method. int j tuberc lung dis. 2008;12(12):1482–1484. ramos maguina es, osorio ce, valencia tr, et al. the specificity of the mdr/xdr-tb colour test for 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intervention. j cancer educ. 2017;32(1):35–42. https://doi.org/10.1007/s13187-015-0953-6 rao n, esber a, turner a, et al. the impact of joint partner decision making on obstetric choices and outcomes among malawian women. int j gynaecol obstet. 2016;135(1):61–64. https://doi.org/10.1016/j.ijgo.2016.03.019 malawi ministry of health. malawi national tuberculosis control programme manual [homepage on the internet]. c2012 [cited 2016 apr 06]. available from: https://pdf.usaid.gov/pdf_docs/pa00kgdd.pdf ellis rc, zabrowarny la. safer staining method for acid fast bacilli. j clin pathol. 1993;46(6):559–560. https://doi.org/10.1136/jcp.46.6.559 barnett b, gokhale rh, krysiak r, et al. prevalence of drug resistant tb among outpatients at an hiv/tb clinic in lilongwe, malawi. trans r soc trop med hyg. 2015;109(12):763–768. https://doi.org/10.1093/trstmh/trv092 drobniewski f, nikolayevskyy v, balabanova y, et al. diagnosis of tuberculosis and drug resistance: what can new tools bring us. int j tuberc lung dis. 2012;16(7):860–870. https://doi.org/10.5588/ijtld.12.0180 abstract introduction materials and methods results discussion acknowledgements references about the author(s) phidelis m. marabi department of health, bungoma county referral hospital, bungoma, kenya school of health sciences, kisii university, kisii, kenya stanslaus musyoki school of health sciences, kisii university, kisii, kenya angela amayo department of human pathology, faculty of health sciences, university of nairobi, nairobi, kenya citation marabi pm, musyoki s, amayo a. biochemical changes in whole blood stored for transfusion at bungoma county referral hospital, kenya. afr j lab med. 2020;9(1), a1182. https://doi.org/10.4102/ajlm.v9i1.1182 original research biochemical changes in whole blood stored for transfusion at bungoma county referral hospital, kenya phidelis m. marabi, stanslaus musyoki, angela amayo received: 27 jan. 2020; accepted: 14 oct. 2020; published: 21 dec. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: during storage, transfusion blood may undergo a series of biochemical changes that could pose risks to patients when used. it is important therefore to monitor biochemical changes that may reduce survival or function of stored blood cells. objective: this study assessed biochemical changes in whole blood stored for transfusion at bungoma county referral hospital in the western region of kenya between february 2019 and august 2019. methods: a prospective study design involving 20 randomly selected donor blood units in citrate phosphate dextrose adenine anticoagulant was employed. biochemical changes were evaluated for 35 days. potassium and sodium levels were tested using the humalyte plus5 analyser. blood ph level was estimated using the hanna ph meter. results: at the end of the 35 days of storage under blood bank conditions, the mean potassium level significantly increased from 7.31 mmol/l at baseline to 20.14 mmol/l at week 5 (p < 0.0001), and the mean sodium level significantly decreased from 150.72 mmol/l at baseline to 121.56 mmol/l at week 5 (p < 0.0001). the ph level decreased insignificantly from 7.48 at baseline to 7.38 at the end of week 1 (p = 0.0757) but decreased significantly to 6.15 at the end of week 5 (p < 0.0001). conclusion: potassium increased and sodium concentrations decreased significantly from the first week of blood storage. the ph decreased significantly from the second week of storage. therefore, aged blood should be avoided to circumvent potential adverse outcomes from biochemical changes and stored blood should be tested before use. keywords: blood; transfusion; biochemical changes; storage; kenya. introduction biochemical variations take place during transfusion blood reservations, affecting their function.1 for many years, researchers have investigated the biochemical variations that take place in blood reserved before transfusions. more recently, clinical investigations have proposed that blood units reserved for extended periods (frequently detailed as 14–21 days) might be detrimental to the recipients, causing morbidity and mortality.2 potassium is the prominent intracellular cation with sodium being the prominent extracellular cation. maintenance of the circulation of potassium and sodium between the intracellular and the extracellular environments depends on a handful of homeostatic mechanisms. literature discloses that potassium efflux, a renowned effect of prolonged red blood cell reservation, causes vesicle creation and may have a significant effect on the transfusion outcome.3 studies have also shown a steady decrease in ph throughout the blood storage period which renders the cell membrane too rigid and predisposes the cells to lysis.4 blood reservation improves access to blood for transfusions. however, questions arise regarding the toxicity and value of such blood reserved for extended periods. in various medical retrospective investigations, authors propose that transfusion of reserved blood may cause harm, particularly in severely ill patients such as patients with severe myocardial infarction conditions.2 they detected an elevated incidence of death, morbidity, infections, renal and lung breakdown, swelling, and thrombosis in patients who received extensively reserved blood compared to those who received fresh blood5; however, the causes of these remain unknown. these adverse effects may be attributed to the inability of red blood cells in reserved blood to transport oxygen as a result of the reduced concentration of adenosine triphosphate and 2, 3-diphosphoglycerate.4 also, cytokines, enzymes, and ions such as potassium and calcium from white blood cells in stored blood may modify red blood cells and adversely affect transfused recipients. these modifications affect an assortment of membrane molecules involved with adhesion, oxygen delivery and complement modulation.6 the storage of blood for extended periods can potentially result in the manipulation of the membrane exterior of red blood cells, and consequently their biological functions.6 according to records of bungoma county referral hospital in bungoma, kenya, the transfusion rate averages 300 transfusions monthly and there has been an increase in blood reactions in the patients that receive a blood transfusion. a monthly average of 2% – 3% of patients react to transfused blood, especially aged blood, which is blood that has been stored for over 20 days. despite the high levels of post-reaction after transfusions in bungoma county referral hospital and the risk of toxicity to patients due to leakage of blood chemicals, the biochemical changes in blood stored for transfusions have not been studied and remain unknown. therefore, this study aimed to assess the biochemical changes in whole blood stored for transfusions at bungoma county referral hospital. materials and methods ethical considerations the study was cleared by the ethical committee of jaramogi oginga teaching and referral hospital (#erc.ib/vol.1/454) and authority to carry out the research was issued by the national commission for science, technology and innovation (#nacosti/p/19/32125/27143). written informed consent was obtained from each donor after a brief explanation of what the study is about. participation in the study was voluntary. donor details were kept confidential by the exclusion of all forms of identification on the data collection tool, and filled data tools were stored physically under lock and digitally with restricted password access on a computer. the donors were informed that their blood units, if selected for the study, were not transfused to patients. study area the study was carried out between february 2019 and august 2019 at bungoma county referral hospital, which is located in the western region of kenya. this was a good study area because adverse reactions among the patients who receive a blood transfusion was common. the hospital also has an accredited laboratory that is well equipped with biochemistry equipment required for the study. the hospital has a blood donation centre which collects on average 600 blood units monthly. study design this was a prospective study involving the collection of blood from healthy volunteers into blood bags containing citrate phosphate dextrose adenine anticoagulant. all donated blood samples were reserved at temperatures between 2 °c and 6 °c and analysed at baseline and then at the ends of week one, two, three, four and five. the whole units of blood were used in the study to ensure that all storage conditions for blood meant for transfusions were met. study population the sample size for the research was determined by yamane taro’s formulae for a finite population.7 a sample size of 20 blood units was adopted for the study. a simple random sampling technique was used in this study where every 10th sample was selected for the study in order to eliminate bias. data collection sample collection and analysis all blood units were collected according to blood transfusion donor guidelines as described by the world health organization.8 at baseline, samples were immediately separated from blood units collected from the volunteer donors to test for potassium, podium and ph levels. blood units were then stored at blood bank conditions of 2 °c – 6 °c for 35 days with intermittent sampling at 7-day intervals to test for potassium and sodium levels. potassium and sodium levels were tested using a humalyte plus5 analyser (human diagnostics worldwide, wiesbaden, germany) while ph was estimated using a hanna ph meter (scientific instrumentation, woonsocket, rhode island, united states). briefly, the plasma supernatant was aseptically transferred from blood units to plain test tubes and brought to the right temperature as per the manufacturer’s instructions before being fed into the machine for automated analysis of potassium and sodium. the instrument measures the electrode potentials, and the data are processed by the microprocessor to obtain the concentration of a given ion. the normal range for potassium concentration is between 3.5 mmol/l and 5.5 mmol/l and between 145 mmol/l and 155 mmol/l for sodium concentration. aliquots of the whole blood units were taken for ph determination at the study points using the automated hanna ph meter, with results displayed on the screen. the normal range for blood ph is 7.35–7.45. all laboratory results were recorded on a standard data collection form developed for this study. quality assurance of the data to ensure the quality of data collected, pre-donation requirements (such as ensuring that donors had a haemoglobin level of 12.5 g/dl and above, weighed 50 kg and above, had no transfusion in the past 12 months, had not donated blood in the past 3 months, had no history and signs of malignancy, had no signs and symptoms of sickle cell disease, had no signs and symptoms of polycythaemia rubra vera and had no history of haemophilia and other coagulation disorders) were followed and only blood units that met these criteria were used. a qualified phlebotomist collected the blood samples, ensuring that the right quantity of between 450 ml and 500 ml was collected. the pints were transported and stored as per blood transfusion guidelines at 2 °c – 6 °c. sample aliquots at the various study points were brought to optimum temperature as per the manufacturer’s instructions before testing. samples were analysed in duplicate for each sample and an average computed to ensure accuracy. both external and internal quality controls for ph, sodium and potassium tests were verified and ensured during the study. recorded results were verified by a second analyst to ensure accuracy. the laboratory is enrolled in the human quality assessment services external quality assurance scheme for chemistry scope including ph, sodium and potassium. the chemistry scope is also accredited by the kenya accreditation services which further assured the quality of data collected. data management and analysis the data were stored in microsoft excel (microsoft corporation, redmond, washington, united states). data analysis was done using the statistical package for the social sciences (spss v.23) (ibm corporation, chicago, illinois, united states). descriptive statistics (frequencies, mean and standard deviation) were used to describe the data. the trends of the biochemical changes were shown using line plots. analysis of variance was used to establish if there were significant biochemical changes in transfusion blood at baseline and each study point compared to normal reference intervals for 35 days of storage. findings were considered significant at a p-value less than 0.05. tukey’s honest significant difference test was used to collate all feasible pairs of means. results potassium (k+) levels increased progressively from 7.31 mmol/l to 20.14 mmol/l, while sodium (na+) levels and ph respectively demonstrated corresponding decrease from 150.72 mmol/l to 121.56 mmol/l and from 7.48 to 6.15 as storage period progressed (figure 1). figure 1: potassium levels demonstrated an increasing trend, while sodium levels and ph demonstrated a decreasing trend as storage period progressed, february 2019 to august 2019. this study observed significant variance in potassium levels at baseline compared to the normal reference interval median of 4.5 mmol/l. potassium levels also increased significantly throughout the blood storage period compared to the baseline mean of 7.31 mmol/l (table 1). there was no significant variance in sodium concentrations at the baseline compared to the normal reference interval median of 150 mmol/l; however, a significant decrease in sodium concentration was observed throughout the blood storage period compared to the baseline. ph levels demonstrated no significant variance at baseline compared to the normal reference interval median of 7.4 and no significant variance at the end of week 1 (day 7) compared to baseline. ph levels at weeks 2, 3, 4 and 5 however decreased significantly from 7.31 at baseline (table 1). table 1: biochemical changes in blood throughout the blood storage period at bungoma county referral hospital, kenya, february 2019 to august 2019. discussion the current study sought to evaluate the biochemical changes in whole blood for transfusion stored at blood bank conditions. at the end of the 35 days of blood storage at blood bank conditions, the potassium levels significantly increased while sodium levels significantly decreased over time. on the other hand, the ph levels significantly decreased over time; however, the change was insignificant at week 2 of storage. potassium levels had a significant variation from normal reference at the baseline while sodium concentrations and ph had insignificant variance from normal reference intervals. regardless of the advantages of blood transfusion in clinical care (such as to treat anaemia, prepare patients for a surgical process, and limit manifestations of blood depletion), the procedure may cause harm to patients.9 there are various deleterious after-effects of blood transfusion that could occur despite cautious laboratory methods in handling and cross-matching the donors and the recipient. the undesired effects may be attributed to changes in the usual micro-environment or biochemistry of the blood cells in the course of blood storage. the present study has found that there are biochemical variations in blood during the 35 days storage period. potassium ions increased significantly after one week and further increased significantly through to 35 days of storage. this result compares with previous studies in portugal,10 uganda,9 india11 and nigeria12 which demonstrated that potassium significantly increased throughout the storage period. the current study observed an increase of plasma potassium level to 10.59 mmol/l within the first week. the increase continued to 20.14 mmol/l at the end of the fifth week which was equivalent to an average of 12.83 mmol/l rise after 35 days of storage. this indicates that blood transfusion after the first week of storage may not be safe for patients due to the potassium levels. in the course of blood reservations, there is a steady but continuous efflux of potassium ions from cells into the encircling plasma.12 during critical kidney disorder, even minute amounts of potassium ion variations are potentially threatening, and relatively fresh or washed red cells are recommended. potassium ions efflux is recognised as a secondary metabolic change that occurs due to cooling.12 the rapid effusion of potassium ions from cells into encircling plasma possibly accounts for the radical elevation in potassium ion concentration in this study. based on the findings of the present study, monitoring of potassium ion concentration during storage of blood for transfusion is recommended to improve blood safety. the current study observed that a decrease in sodium levels was significant after 2 weeks (14 days) and further decreased significantly through to 35 days of storage. this result compares with previous studies in india, portugal and ghana which demonstrated that sodium levels decreased throughout the storage period.10,11,13 our results, however, contrast a study done in india which demonstrated that sodium did not show any significant changes due to storage time14 as well as another study done in nigeria in which reservation period was not established to influence sodium concentration.12 the reference range for plasma sodium level is 145 mmol/l – 155 mmol/l. the findings of this study indicate that within the first week of storage, sodium levels in blood could fall below the normal range. blood stored at blood bank conditions slows down cellular metabolism and energy demand which permits blood to be reserved for 35–42 days. this makes the sodium-potassium pump defective and, as a result, permits potassium ions to depart the cell and sodium ions to move into the cells through the semipermeable membrane.10 therefore, these significant changes may affect patients that may be affected by low levels of sodium such as an increased chance of such a recipient being prone to oedema especially in patients with low sodium intake or those experiencing diarrhoea.15 in light of the findings from the present study, monitoring of sodium ion concentration during storage of blood for transfusion with the goal to improve blood safety is recommended. this study reported a significant ph decrease after 14 days of storage which further decreased significantly through to 35 days of storage. this result compares with previous studies in uganda, india and ghana which demonstrated that the ph of reserved blood lessened during the storage period.9,10 the reference range for blood ph is 7.35–7.45; this study reported a decrease in blood ph from 7.48 to 6.15 which was equivalent to an average drop of 1.33 after 35 days of storage. this indicates that the ph decreased far below the normal range by the end of 35 days. according to oyet et al.,9 the increase in protons due to accumulation of lactate after 14 days of storage causes the ph level to decrease and subsequently changes glycolytic metabolism. the elevated lactate level with the reciprocal descent in ph exerts ravaging consequences on blood receivers, principally those who may be given several units of blood in a short period. this diminishes blood potency and predisposes blood receivers to undesired transfusion-associated morbidity and mortality.9 the depletion of diphosphoglycerate and diminished glycolytic action are also linked to lessening ph.12 this study recommends that transfusion of blood reserved for more than 1 week should be exercised with caution. taking into account the findings from the present study, ph level monitoring during blood storage especially after the first week of storage with the focus on improving blood safety is recommended. overall, together with other parameters, biochemical changes in stored blood for blood transfusion should be properly monitored especially in consideration of the patient to be transfused. because of the increase in potassium level with storage, caution is required to ascertain the storage duration of blood to be transfused, volume to be transfused at a time, and rate of transfusion to decrease hyperkalaemia-linked blood transfusion problems. transfusing hyperkalaemic blood, however, may have little effect if the recipient’s kidneys are working normally and the sodium-potassium adenosine triphosphate pump is also working efficiently. however, this is a significant concern in recipients with renal failure. recommendations the study recommends the monitoring of biochemical changes and the use of fresh blood to avoid the negative effect of changes in stored blood especially to patients who suffer from renal failure and myocardial infarction. limitations in the current study, other biochemical parameters such as iron, lactate, calcium, and so on, which could have an impact on the viability of blood were not assessed. conclusion this study showed that variations in biochemical parameters occur during the storage of blood despite the use of adequate storage conditions, and may cause risks to patients during transfusion. acknowledgements mr thomas khisa wekesa contributed by assisting in the donors’ recruitment and blood donation. ms joan wasike, ms beatrice lungai, mr antony sangolo and ms julia wasike contributed by assisting in the running of the samples and the data collection. competing interests the authors have declared that no competing interests exist. authors’ contributions all authors contributed equally to this work. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references yalcin o, ortiz d, tsai ag, johnson pc, cabrales p. microhemodynamic aberrations created by transfusion of stored blood. transfusion. 2014;54(4):1015–1027. https://doi.org/10.1111/trf.12361 roback jd. vascular effects of the red blood cell storage lesion. hematology am soc hematol educ program. 2011;2011:475–479. https://doi.org/10.1182/asheducation-2011.1.475 burger p, kostova e, bloem e, et al. potassium leakage primes stored erythrocytes for phosphatidylserine exposure and shedding of pro-coagulant vesicles. br j haematol. 2013;160(3):377–386. https://doi.org/10.1111/bjh.12133 d’alessandro a, gevi fzl. red blood cell metabolism under prolonged anaerobic storage. mol biosyst. 2013;9(6):1196–1209. https://doi.org/10.1039/c3mb25575a hod ea, spitalnik sl. harmful effects of transfusion of older stored red blood cells: iron and inflammation. transfusion. 2011;51(4):881–885. https://doi.org/10.1111/j.1537-2995.2011.03096.x adams f, bellairs g, bird ar, oguntibeju oo. biochemical storage lesions occurring in nonirradiated and irradiated red blood cells. biomed res int. 2015; 102015:968302. https://doi.org/10.1155/2015/968302 israel gd. determining sample size. fact sheet peod-6. orlando, fl: florida coop extension service; 1992. world health organization. blood donor selection: guidelines on assessing donor suitability for blood donation. geneva: who;2012. available from https://apps.who.int/iris/handle/10665/76724 oyet c, okongo b, onyuthi ra, muwanguzi e. biochemical changes in stored donor units: implications on the efficacy of blood transfusion. j blood med. 2018; 9:111–115. https://doi.org/10.2147/jbm.s163651 nogueira d, rocha s, abreu e, costa e s-sa. biochemical and cellular changes in leukocyte-depleted red blood cells stored for transfusion. transfus med hemother. 2015;42(1):46–51. https://doi.org/10.1159/000370140 verma m, dahiya k, malik d, et al. effect of blood storage on complete biochemistry. j blood disord transfus. 2015;6:6. https://doi.org/10.4172/2155-9864.1000329 adias tc, moore-igwe b, jermeiah za. storage related haematological and biochemical changes of cpda-1 whole blood in a resource limited setting. j blood disord transfus. 2012;3(3):124. opoku-okrah c, acquah bk, dogbe ee. changes in potassium and sodium concentrations in stored blood. pan afr med j. 2015;20:236. https://doi.org/10.11604/pamj.2015.20.236.5851 upadya ub, seema h, motakapalli k. effect of blood storage on biochemical parameters assessed at periodic intervals in cpda1 blood bags. j clin diagnostic res. 2018;12(10):ec05–ec07. https://doi.org/10.7860/jcdr/2018/37223.12139 metheny nm. fluid and electrolyte balance: nursing considerations. 5th edn. sudbury, ma: jones & bartlett learning; 2012. abstract introduction methods results discussion acknowledgements references about the author(s) prenika jaglal department of medical microbiology, national health laboratory services, school of laboratory medicine and medical science, university of kwazulu-natal, durban, south africa melendhran pillay department of medical microbiology/virology, national health laboratory service, durban, south africa koleka mlisana department of medical microbiology, national health laboratory services, school of laboratory medicine and medical science, university of kwazulu-natal, durban, south africa citation jaglal p, pillay m, mlisana k. resazurin microtitre plate assay and sensititre® mycotb for detection of mycobacterium tuberculosis resistance in a high tuberculosis resistance setting. afr j lab med. 2019;8(1), a840. https://doi.org/10.4102/ajlm.v8i1.840 original research resazurin microtitre plate assay and sensititre® mycotb for detection of mycobacterium tuberculosis resistance in a high tuberculosis resistance setting prenika jaglal, melendhran pillay, koleka mlisana received: 30 may 2018; accepted: 13 mar. 2019; published: 13 dec. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: rapid diagnosis of drug-resistant mycobacterium tuberculosis is a challenge in low-income countries. phenotypic drug susceptibility testing using sensititre® mycotb assay and the resazurin microtitre plate assay (rema) are relatively new innovative methods to determine drug susceptibility. objectives: this study aimed to determine the performance of the sensititre and rema for m. tuberculosis drug susceptibility testing in a high-volume tuberculosis reference laboratory. methods: a laboratory-based study was performed at the inkosi albert luthuli central hospital tuberculosis laboratory from january 2014 to june 2015. the sensititre® mycotb plate and rema were compared to the gold standard agar proportion method (apm) using 134 stored isolates. results: agreement between the sensititre® mycotb plate and apm was observed with 98% sensitivity, 82% specificity, 94% positive and 93% negative predictive values of the sensititre® mycotb assay for the detection of rifampicin resistance and 97%, 96%, 99% and 88% for isoniazid resistance. good categorical agreement between the rema and the apm was observed among isolates with 89% sensitivity, 68% specificity, 89% positive and 68% negative predictive value for the detection of rifampicin resistance and 95%, 96%, 99% and 81% for isoniazid resistance. results for the second-line drugs showed elevated minimum inhibitory concentrations for multidrug-resistant and extensively drug-resistant tuberculosis isolates. conclusion: the rema and sensititre® mycotb plate are attractive alternatives to the gold standard apm for the phenotypic detection of m. tuberculosis drug resistance. keywords: mycobacterium tuberculosis; agar proportion method; multidrug-resistant tuberculosis; extensively drug-resistant tuberculosis; sensititre® mycotb assay; resazurin microtitre plate assay. introduction multidrug-resistant tuberculosis has been declared a public health crisis by the world health organization (who).1 the global burden of tuberculosis remains enormous with an incidence of 10 million new cases and a mortality of 1.3 million attributed to the disease worldwide in 2017.1 the 2018 who global tuberculosis report documented new tuberculosis cases, which included an additional 300 000 tuberculosis cases among hiv-positive tuberculosis patients.1 south africa is among the top 20 high-tuberculosis-burden countries worldwide and was previously ranked third following india and china.1,2 multidrug-resistant (mdr) tuberculosis is steadily increasing in south africa; cases doubled from 7350 in 2007 to 14 000 in 2017.1,3 a form of tuberculosis known as extensively drug-resistant (xdr) tuberculosis has been reported in 92 countries. south africa has reported 73% of the global xdr cases following the historical outbreak of xdr tuberculosis in tugela ferry.4 the standard tuberculosis treatment regimen lasts for a minimum of 6 months. treatments regimens for mdr tuberculosis (resistance to isoniazid and rifampicin) and xdr tuberculosis (resistance to any of the injectable drugs including fluoroquinolone, plus mdr tuberculosis),5,6 have an extended treatment duration of 18–24 months with harmful second-line anti-tuberculosis drugs. the who has recently proposed a 9–12-month shortened regimen duration for mdr tuberculosis, with moxifloxacin replacing gatifloxacin (used in the original bangladesh regimen).7,8 the conventional, culture-based phenotypic method is recognized as the gold standard for confirmation of disease and tuberculosis drug susceptibility testing (dst). the agar proportion method (apm) compares growth of mycobacterium colonies on drug-free and drug-containing mediums, where growth in a particular antibiotic-containing medium determines resistance. it is a low-cost method requiring no special equipment, but with a turnaround time of up to 6 weeks.9,10 bactec mycobacterium growth indicator tubes (mgit) 960 liquid culture system (becton dickinson) is expensive and prone to contamination but has the advantage of a rapid turnaround time.2 the who has endorsed the genexpert mtb/rif ultra system, which is a real-time polymerase chain reaction-based assay for tuberculosis diagnosis and detection of rifampicin resistance, yielding results within 2 hours. the genotype mtbdr plus identifies common mutations for rifampicin and isoniazid.1,11 although molecular methods are rapid, they require costly equipment and staff expertise, and cannot detect resistant strains caused by unidentified mutations.11 an innovative colorimetric assay has been designed using redox indicators to detect tuberculosis cell viability by a simple colour change. the resazurin microtitre plate assay (rema) uses resazurin salts in a liquid culture medium.12,13 this nontoxic compound incorporates into living cells and is reduced to the fluorescent molecule resorufin via a reduction and oxidation reaction.14 visible colour change of the reagent from blue to pink demonstrates cell viability and therefore drug resistance. this technique has been applied for high throughput screening and determining minimum inhibitory concentrations (mic) of anti-tuberculosis drugs.14,15 another advance in determining the phenotypic susceptibility of m. tuberculosis is the sensititre® mycotb (mycotb trek diagnostics) plate method. the sensititre® mycotb plate method is a novel 96-well microtitre plate broth microdilution dst method incorporating drug concentrations of 12 anti-tuberculosis drugs for mic determination.16 the drugs included consist of both first-line (rifampicin, isoniazid and ethambutol) as well as second-line drugs (moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, streptomycin, amikacin, cycloserine, ethionamide and kanamycin) existing as lyophilised forms in microtitre wells. a 7–21-day period of incubation is needed to observe culture growth noted as turbidity or cellular deposits at the base of a well.14,16 the aim of this study was to determine the performance of the sensititre® mycotb plate and rema as potential tools for tuberculosis dst in a high tuberculosis-burden reference laboratory. this entailed comparison of the rema and sensititre assay using the apm as a gold standard in order to evaluate turnaround times, sensitivity, specificity, and positive and negative predictive values. methods ethical considerations the biomedical ethics research committee (university of kwazulu-natal) granted ethical approval for the use of stored study isolates (reference number be 268/12). processing and culture of sputum specimens previously stored m. tuberculosis strains isolated from sputum samples received at the tuberculosis laboratory based at the inkosi albert luthuli central hospital, durban, south africa, from january 2014 to june 2015 were used in this study. briefly, sputum samples were digested and decontaminated using the n-acetyl-l-cysteine–naoh-sodium citrate (nalc–naoh, 2% naoh final concentration) method and cultured in mycobacterium growth indicator tubes (mgit). positive cultures were confirmed for the presence of m. tuberculosis using the ziehl neelsen or mpt64 antigen assay (sd bioline, gyeonggi-do, south korea). mdr and xdr m. tuberculosis isolates were routinely stored in the laboratory. one hundred and fifty stored m. tuberculosis isolates were subcultured onto middlebrook 7h11 agar and incubated at 37 °c for 21 days. a total of 134 isolates had confluent growth and were used in the study. drug susceptibility testing by the agar proportion method drug susceptibility of tuberculosis-positive cultures was determined using the indirect apm on middlebrook 7h10 agar2,16 for firstand second-line anti-tuberculosis drugs (rifampicin, isoniazid, kanamycin, moxifloxacin and capreomycin). one hundred microlitres from a positive mgit was inoculated into each quadrant of the middlebrook 7h10 dst agar plate and incubated for 21 days. growth of more than 1% on the antibiotic-containing quadrant when compared to the antibiotic-free growth control was regarded as resistant to the corresponding antibiotic. a susceptible growth control isolate, h37rv (american type culture collection, 25618), was used in the study. resazurin microtitre plate assay the susceptibility of mdr, xdr and sensitive tuberculosis isolates were evaluated against firstand second-line anti-tuberculosis drugs by the colorimetric rema method for the 134 isolates. one hundred microlitres of middlebrook 7h9 (m7h9) broth was aseptically prepared and dispensed carefully into each of the wells of a flat-bottomed, 96-well microtitre plate with lid (lasec, midrand, south africa). the anti-tuberculosis drugs that were tested using the rema method included rifampicin, isoniazid, kanamycin, moxifloxacin and capreomycin. working solutions of the drugs were initially prepared (four times the final concentration) in m7h9 broth supplemented with 0.5% glycerol, 0.1% casitone and 10% oadc (oleic acid, albumin, dextrose and catalase). one hundred microlitres of the working drug concentrations (isoniazid 1.0 µg/ml, rifampicin 8.0 µg/ml, kanamycin 10.0 µg/ml, capreomycin 8.0 µg/ml and moxifloxacin 2.0 µg/ml) were added to the wells containing middlebrook 7h9 broth. the anti-tuberculosis drugs were then further serially diluted twofold to a final concentration consisting of isoniazid (0.03 µg/ml), rifampicin (0.25 µg/ml), kanamycin (2.5 µg/ml), capreomycin (1.0 µg/ml) and moxifloxacin (0.06 µg/ml). an inoculum turbidity of mcfarland standard number 1 was prepared from middlebrook 7h11 (m7h11) agar, diluted in m7h9 (1:10) broth and thereafter added (100 µl) to each of the drug-free and drug-containing wells.10,12 a sterile control and a growth control for each isolate were also included. to prevent evaporation during incubation, sterile m7h9 broth was added to all perimeter wells. the plate was incubated at 37 °c after being sealed in a plastic bag. a working solution of resazurin salt (30 µl of 0.02% concentration) was inoculated after 8 days of incubation into each microtitre well.10 after overnight incubation, plates were then read the next day, a total of 9 days turnaround time for results interpretation. a colour change from blue to pink denoted a positive reaction (reduction of resazurin to resorufin) confirming drug resistance due to m. tuberculosis cell viability.10 sensititre® mycotb assay setting up the microtitre plates and their interpretation were performed according to manufacturer’s instructions. colonies from a culture plate (m7h11) were emulsified in a glass tube containing saline, tween and glass beads until a 0.5 mcfarland standard was obtained. one hundred microlitres of the suspension was inoculated into each well. the plates were then incubated at 37 °c. drug concentrations present in each plate were: 4 µg/ml isoniazid, 16 µg/ml rifampicin, 32 µg/ml ethambutol, 40 µg/ml ethionamide, 40 µg/ml kanamycin, 32 µg/ml ofloxacin, 64 µg/ml para-aminosalicyclic acid, 16 µg/ml rifabutin, 32 µg/ml streptomycin, 256 µg/ml cycloserine, 16 µg/ml amikacin and 8.0 µg/ml moxifloixacin. observations of the plates were made from day 7 to 10 for the presence of turbidity or cellular material confirming growth of m. tuberculosis and therefore resistance.14,16 time to results was calculated as the number of days from plate inoculation (day 0) to plate reading with visible growth (day 7–10). interpretation of results currently, there are no mic-interpretative breakpoints for the broth microdilution assays when testing m. tuberculosis isolates on sensititre plates; accordingly, the endpoint was determined as the first well that did not contain any growth. these antimicrobial drug mic results were recorded on a data sheet per isolate tested. the reproducibility of sensititre® mycotb assay by duplicate testing of the isolates was not performed for conditional (or categorical) agreement between the sensititre® mycotb method and apm on initial testing. in this study, for interpretation of the sensititre data, true resistance by sensititre® mycotb was established by comparing the sensititre mic to the apm critical concentration. this means that an isolate was considered resistant if the sensititre mic was greater than the apm critical concentration and therefore truly susceptible if it was less than or equal to the apm critical concentration. for the rema method, resistance to each drug was determined when there was growth in wells above the apm critical concentrations of 0.25 µg/ml (isoniazid), 1.0 µg/ml (rifampicin), 2.0 µg/ml (moxifloxacin), 4.0 µg/ml (capreomycin) and 5.0 µg/ml (kanamycin).18 rema plates were therefore interpreted categorically based on the calorimetric reaction. the rema and sensititre plates were interpreted by two readers who were blinded to the apm results. the two readers were in agreement with all plates read and therefore there was no discordance in interpretation. results the 134 isolates utilised comprised clinically derived xdr (n = 65), mdr (n = 28), pre-xdr (n = 3), isoniazid mono-resistant (n = 15), rifampicin mono-resistant (n = 4) and susceptible (n = 19) according to the gold standard. the rema and sensititre® mycotb results for all 134 (100%) clinical isolates were tested and interpreted. the time to results for the sensititre® mycotb method was as early as 7 days, with more reliable results being produced within 10 days. of the 100 rifampicin-resistant isolates by apm, 98 were resistant by sensititre assay. of these 98 isolates, 9 had an mic of 1 µg/ml, 7 had an mic of 2 µg/ml, 16 had an mic of 4 µg/ml, 24 had an mic of 8 µg/ml, 4 had an mic of 16 µg/ml and 38 had an mic above 16 µg/ml. refer to figure 1 for rifampicin mic distributions using the sensititre® mycotb. figure 1: distribution of isolates by minimum inhibitory concentration for isoniazid and rifampicin, inkosi albert luthuli central hospital, durban, south africa, january 2014 – june 2015. (a) distribution of rif-resistant and -susceptible isolates as determined by apm according to their mic as determined by the sensititre® mycotb assay. (b) distribution of inh-resistant and -susceptible isolates as determined by apm according to their mic as determined by the sensititre® mycotb assay. of the 16 isolates that were rifampicin-resistant by the sensititre® mycotb having mic of 4 µg/ml, 75% (12/16) were xdr, 12.5% (2/16) mdr and 12.5% (2/16) mono-resistant to rifampicin by the apm. of the 24 isolates that were resistant to rifampicin by the sensititre® mycotb with mic of 8 µg/ml, 67% (16/24) were xdr, 8% (2/24) pre-xdr and 25% (6/24) were mdr by the apm. the rema correctly detected rifampicin resistance in all seven isolates with an mic of 2 µg/ml by the sensititre® mycotb (43% [3/7] xdr, 43% [3/7] mdr and 14% [1/7] mono-resistant to rifampicin by apm) with an mic of 2 µg/ml by the sensititre® mycotb. the rema confirmed rifampicin resistance in all of the 38 isolates with an mic above 16 μg/ml by the sensititre® mycotb (68% [26/38] xdr and 32% [12/38] mdr) by apm. the overall sensitivity, specificity, positive and negative predictive values of the sensititre® mycotb plate for rifampicin-resistance detection were found to be 98%, 82%, 94% and 93% (table 1). the rema displayed a sensitivity, specificity, positive and negative predictive values of 89%, 68%, 89% and 68% for the detection of rifampicin resistance (table 2). table 1: comparative performance of the sensititre® mycotb assay and agar proportion method on multidrug-resistant and extensively drug-resistant tuberculosis isolates, inkosi albert luthuli central hospital, durban, south africa, january 2014 – june 2015. table 2: comparative performance of the resazurin microtitre plate assay and agar proportion method on multidrug-resistant and extensively drug-resistant tuberculosis isolates, inkosi albert luthuli central hospital, durban, south africa, january 2014 – june 2015. of the 111 isoniazid-resistant isolates by apm, 108 were resistant by the sensititre assay. of these 108 isolates, 7 had an mic of 1 µg/ml, 9 had an mic of 2 µg/ml, 39 had an mic of 4 µg/ml, 52 had an mic of greater than 4 µg/ml and 1 had an mic above 8 µg/ml. the seven isolates that were isoniazid-resistant by the sensititre® mycotb with an mic of 1 µg/ml (72% mdr and 28% mono-resistant to isoniazid by apm) were also confirmed resistant by the rema assay. nine isolates resistant to isoniazid by the sensititre® mycotb with an mic of 2 µg/ml comprised clinically-derived xdr (n = 6), mdr (n = 2) and pre-xdr (n = 1). resistance to isoniazid by the rema method was noted in all 9 isolates. of the 52 isoniazid-resistant isolates with an mic of over 4 µg/ml by the sensititre® mycotb (figure 1), 25 were xdr, 23 mdr and 4 were mono-resistant to isoniazid by the apm. the rema assay confirmed isoniazid resistance in 51 of the 52 (98%) isolates. refer to figure 1 for isoniazid mic distributions using the sensititre® mycotb. the sensititre® mycotb assay sensitivity, specificity, positive and negative predictive values for the detection of isoniazid resistance were found to be 97%, 96%, 99% and 88% (table 1). the rema showed sensitivity, specificity, positive and negative predictive values of 95%, 96%, 99% and 81% for the detection of isoniazid resistance (table 2). sensititre® mycotb testing for the detection of resistance among the mdr and xdr isolates for rifampicin, isoniazid, ofloxacin and kanamycin correlated well with the apm (table 1). discrepancies between apm and rema were observed with moxifloxacin susceptible isolates (apm) where 12 isolates were falsely designated as resistant by rema (table 2). the overall sensitivity, specificity, positive and negative predictive values of the sensititre® mycotb plate for moxifloxacin-resistance detection were found to be 91%, 98%, 91% and 96% (table 1). the rema assay showed sensitivity, specificity, positive and negative predictive values of 91%, 78%, 64% and 96% for the detection of moxifloxacin resistance (table 2). the overall sensitivity, specificity, positive and negative predictive values of the sensititre® mycotb assay for ofloxacin resistance detection were found to be 100%, 97%, 97% and 100% (table 1). the sensitivity of detection for ofloxacin resistance was not assessed by the rema as the drug could not be procured due to limited funds. the overall sensitivity, specificity, positive and negative predictive values of the sensititre® mycotb plate for kanamycin resistance detection were found to be 92%, 85%, 86% and 92% (table 1). the rema assay showed sensitivity, specificity, positive and negative predictive values of 83%, 94%, 93% and 85% for kanamycin resistance detection (table 2). refer to figure 3 for kanamycin mic distributions using the sensititre® mycotb. good accuracy with regard to resistance to the first-line anti-tuberculosis drugs was observed with the sensititre method as compared to the rema (tables 1 and 2). figure 2: distribution of isolates by minimum inhibitory concentration for ofloxacin and moxifloxacin, inkosi albert luthuli central hospital, durban, south africa, january 2014 – june 2015. (a) distribution of oflox-resistant and -susceptible isolates as determined by apm according to their mic as determined by the sensititre® mycotb assay. (b) distribution of mox-resistant and susceptible isolates as determined by apm according to their mic as determined by the sensititre® mycotb assay. figure 3: distribution of isolates by minimum inhibitory concentration for kanamycin, inkosi albert luthuli central hospital, durban, south africa, january 2014 – june 2015. distribution of kana-resistant and -susceptible isolates as determined by apm according to their mic as determined by the sensititre® mycotb assay. the performance of the rema for the detection of capreomycin resistance was determined. all of the 62 capreomycin-resistant isolates by the rema were confirmed to be xdr tuberculosis by the apm (table 2). capreomycin was not part of the drug panel included in the sensititre® mycotb assay, therefore its performance could not be assessed. the levels of resistance among the mdr and xdr tuberculosis isolates to the additional firstand second-line antibiotics were further assessed using the sensititre® mycotb assay using established critical concentrations (figures 4 and 5). resistance to rifabutin was observed in 93.5% (58/62) of xdr tuberculosis isolates with the majority 27% (17/62) showing an mic of 16 µg/ml (figure 4) at a critical concentration of 0.5 µg/ml. in contrast to this, 80% (20/25) of mdr isolates were confirmed to be susceptible to rifabutin with an mic of 0.5 µg/ml (figure 5). figure 4: distribution of minimum inhibitory concentrations (µg/ml) for additional firstand second-line antibiotics using the sensititre plate method for clinical, extensively drug-resistant tuberculosis isolates, inkosi albert luthuli central hospital, durban, south africa, january 2014 – june 2015. (a) str (60 isolates), (b) rfb (62 isolates), (c) pas (61 isolates), (d) cyc (61 isolates), (e) ami (62 isolates), (f) eth (62 isolates) and (g) emb (62 isolates). figure 5: distribution of minimum inhibitory concentrations (µg/ml) for additional firstand second-line antibiotics using the sensititre plate method for clinical multidrug-resistant tuberculosis isolates. (a) str (25 isolates), (b) rfb (25 isolates), (c) pas (25 isolates), (d) cyc (25 isolates), (e) ami (25 isolates), (f) eth (25 isolates) and (g) emb (25 isolates), inkosi albert luthuli central hospital, durban, south africa, january 2014 – june 2015. most (58/61, 95%) of the xdr isolates displayed resistance to para-aminosalicyclic acid (figure 4). at an mic of 1 µg/ml, 76% (19/25) of mdr isolates displayed susceptibility to para-aminosalicyclic acid (figure 5). resistance to amikacin was observed in 46 of the 62 (74%) xdr isolates (figure 4). most (59/62, 95%) xdr isolates were resistant to ethambutol. conversely, 91% (21/23) mdr isolates were susceptible to ethambutol (figure 5). resistance to amikacin was observed in 46 of the 62 (74%) xdr isolates; the majority (n = 23) of amikacin-resistant isolates had an mic of greater than 16 µg/ml (figure 4). in contrast to this, all of the 25 mdr isolates tested were susceptible to amikacin with a large proportion 76% (19/25) showing an mic of 0.25 µg/ml (figure 5). more than half (37/62, 60%) of xdr isolates were resistant to ethionamide with an mic of 40 µg/ml or more (figure 4). among the ethionamide susceptible isolates, 92% (23/25) were mdr with 83% (19/23) showing an mic of 0.6 µg/ml (figure 5). resistance to streptomycin was observed in 90% (54/60) of xdr isolates with a large proportion of the streptomycin-resistant isolates, 40% (22/54), showing an mic of more than 32 µg/ml (figure 4). a susceptible streptomycin mic of 0.5 µg/ml was observed in 19 of the 25 mdr isolates (figure 5). most xdr isolates (54/61, 80%) and 12% (3/25) of mdr isolates had mics for cycloserine of 32 µg/ml or more (critical concentration of 30 µg/ml; resistant by apm) (figures 4 and 5). discussion the objective of tuberculosis dst is to determine resistant strains that are prognostic of treatment failure and relapse. the sensititre® mycotb assay provides a faster method of testing firstand second-line tuberculosis drugs and displayed 99.3% concordance with the agar proportion method in prior studies.19 in our study, the percentage agreements between sensititre plate and apm for resistant isoniazid (97%), rifampicin (98%), moxifloxacin (91%) and ofloxacin (100%) were found in similar studies; however, kanamycin had the lowest categorical agreement with the apm (92%).19 discrepant moxifloxacin (apm susceptible) isolates could be due to the subjectivity of the microtitre plate reading. a discrepant analysis would be useful to clarify discordant results, for example, repeat testing of isolates and ruling out technical errors. according to the who, current critical concentrations of all anti-tuberculosis drugs will be reviewed.1 as phenotypic dst is required for the determination of moxifloxacin susceptibility (who recommendation due to poor concordance of genotype mtbdrsl with apm), this might prove problematic if the sensititre® mycotb assay is used.1 the distribution of mic with regard to second-line tuberculosis antimicrobials among mdr and xdr isolates was clearly evident as rising mic values with the increasingly mdr strains. resistance to capreomycin has been documented in kwazulu-natal in approximately 90% of xdr isolates of newly diagnosed individuals.17,19 both ethambutol and pyrazinamide resistance has been reported as exceeding 60%, the first-line drugs composing the bangladesh regimen, as described in previous studies.8 the sensititre® mycotb plate rapidly produced results in comparison to the apm, however, it produced 44% (15/34) false rifampicin-resistant isolates (sensitive by apm). the treatment implication would include patients who were mismanaged with prolonged, toxic second-line therapy in an mdr facility. the rema assay had a relatively short turnaround time of 9 days.21 delays in results retrieval and therefore suitable treatment options for resistant tuberculosis may result in the further selection of resistant m. tuberculosis, morbidity and mortality in patients afflicted with the disease. advantages of the rema format are that it is faster, low cost, easy to interpret and does not need special equipment.21 a disadvantage of this method is the potential for aerosolisation since the plates utilise a liquid medium resulting in a biosafety hazard.15 in our study, rema proved to be labour-intensive, requiring individual drug and dye preparation, as well as tedious microtitre plate inoculation. the current study showed the sensitivity and specificity of the rema are comparable to that of the apm for isoniazid, rifampicin, capreomycin, moxifloxacin and kanamycin to be comparable to previous studies.21 agreement between rema and the apm was over 90% for resistance testing among mdr and xdr isolates (isoniazid 95%, capreomycin 93%, moxifloxacin 91%). limitations a limitation with the apm is that the test provides single set critical concentrations and not clear-cut mic results for each drug in comparison to the sensititre® mycotb plate method. minimum inhibitory concentration values obtained from using the sensititre® mycotb plate could possibly be a prospective guide to establishing definite mic breakpoint values for anti-tuberculosis drugs in the future as there are no interpretive mic breakpoints that correlate with critical concentrations. a lack of apm results for most of the second-line antimicrobials meant that the performance of the sensititre® mycotb method in conjunction with the apm could only be calculated and assessed against a limited number of antimicrobials. results for second-line drugs did show elevated mics for mdr and xdr isolates with the majority of xdr isolates being resistant. conclusion the sensititre® mycotb assay is a desirable alternative method compared to the apm for tuberculosis dst. simultaneous firstand second-line antimicrobial testing eradicates the need to set up and maintain antimicrobial drug solutions (rema). the rema is ideal for use in resource-poor settings due to its low cost and lack of instrumentation.12 in comparison to the apm, which has a short shelf life, the sensititre® mycotb mic plate can last for up to 2 years at room temperature. the sensititre plate may be used together with rapid molecular tests immediately targeting firstand second-line drug testing in scenarios of mdr and xdr tuberculosis.18 little information is available to date on a worldwide, regional and local scale on the use of the sensititre plate. the comparative performance of sensititre® mycotb assay to apm did show much discordance in this study and therefore cannot be recommended as a replacement of the current gold standard. in future, more studies will have to be performed to determine anti-tuberculous drug mic interpretive breakpoints using wild type and non-wild type isolates, as well as discordant analysis, in order to increase its use in high-burden areas both locally and nationally, especially in the province of kwazulu-natal. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions p.j. was the principal investigator. m.p. was the scientist involved in study methodology and design. k.m. was the supervisor and was responsible for revision of the manuscript. sources of support this study was supported by a grant that was obtained from the university of kwazulu-natal by dr prenika jaglal as a principal investigator for research purposes, cs28 cost centre. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references world health organization [home page on the internet], global tuberculosis report 2018. accessed 18 september 2018, from: https://www.who.int/tb/publications/global_report/en/. parsons linda m, somoskovi a, gutierrez c. laboratory diagnosis of tuberculosis in resource-poor countries: challenges and opportunities. clin microbiol rev. 2011;24(2):314–350. https://doi.org/10.1128/cmr.00059-10 africa millennium development goals 6: combat hiv/aids, malaria and other diseases 2015/statistics south africa [homepage on the internet]. millenium development goals. 2015. accessed 15 november 2016, from: www.mdgmonitor.org/mdg-6-combat-hiv-aids-malaria-and-other-diseases. brust jcm, gandhi nr, carrara h. high treatment failure and default rates for patients with multidrug-resistant tuberculosis in kwazulu-natal, south africa, 2000–2003. int j tuberc lung dis 2010;14(4):413–419. martin a, camacho m, portaels f. resazurin microtitre assay ilate testing of mycobacterium tuberculosis susceptibilities to second-line drugs: rapid, simple and inexpensive method. j antimicrob agents chemother. 2003;47(11):3616–3619. https://doi.org/10.1128/aac.47.11.3616-3619.2003 yajko dm, madej jj, lancaster mv. colorimetric method for determining mics of antimicrobial agents for mycobacterium tuberculosis. j clin microbiol. 1995;33(9):2324–2327. world global tb report [homepage on the internet]. world health organization; 2016. accessed 12 october 2016, from: www.who.int/tb/publications/global_report/gtbr. sotgiu g, tiberib s, centisc r. applicability of the shorter ‘bangladesh regimen’ in high multidrug-resistant tuberculosis settings. int j infect dis. 2016;56:190–193. montoro e, lemus d, echemendia m. comparative evaluation of the nitrate reduction assay, the mtt test, and the resazurin microtitre assay for the drug susceptibility testing of clinical isolates of mycobacterium tuberculosis. j antimicrob agents chemother. 2005;55(february):500–505. https://doi.org/10.1093/jac/dki023 palomino jc, martin a, camacho m. resazurin microtitre assay plate: simple and inexpensive methods for detection of drug resistance in mycobacterium tuberculosis. j antimicrob agents chemother. 2002(46):2720–2722. nichols mp. new developments in the laboratory diagnosis of tuberculosis. contin med educ. 2010;6(june):246–251. martin a, morcillo c, lemus d. multicenter study of mtt and resazurin assays for testing susceptibility to first-line anti-tuberculosis drugs. int j tuberc lung dis. 2005;9(8):901–906. franzblau sg, witzig rs, mclaughlin jc. rapid, low-technology mic determination with clinical mycobacterium tuberculosis by using the microplate alamar blue assay. j clin microbiol. 1998;36(2):362–366. hall l, jude kp, clark sl. antimicrobial susceptibility testing of mycobacterium tuberculosis complex for first and second line drugs by broth bilution in a microtiter plate format. j vis exp 2011;52(11):3094. coban ay, cehan cc, bilgan k. rapid susceptibility test for mycobacterium tuberculosis to isoniazid and rifampicin with resazurin method in screw cap tubes. j chemother. 2006;18(2):140–143. https://doi.org/10.1179/joc.2006.18.2.140 abuali mm, katariwala a, labombardi vj. a comparison of the sensititre mycotb panel and the agar proportion method for drug susceptibility testing of mycobacterium tuberculosis. eur j clin microbiol infect dis. 2012;31(5):835–839. https://doi.org/10.1007/s10096-011-1382-z o’donnell mr, pillay m, pillay m. primary capreomycin resistance is common and associated with early mortality in patients with extensively drug-resistant tuberculosis in kwazulu-natal, south africa. j acquir immune defic syndr. 2015;69(5):536–543. https://doi.org/10.1097/qai.0000000000000650 lee j, armstrong dt, ssengooba w, park. sensititre mycotb mic plate for testing mycobacterium tuberculosis susceptibility to firstand second-line drugs. j antimicrob agents chemother. 2014;51(8):11–15. https://doi.org/10.1128/aac.01209-13 abdel-rahman sm, abdel-latif w, kholeif h, erfan d. evaluation of sensititre® mycotb panel for the susceptibility testing of mycobacterium tuberculosis to first and second lines anti-tuberculosis drugs. j clin med diagn. 2016;6(1):13–19. parrish nm, carroll kc. role of the clinical mycobacteriology laboratory in diagnosis and management of tuberculosis in low-prevalence settings. j clin microbiol. 2011;49(3):772–776. https://doi.org/10.1128/jcm.02451-10 khalifa ra, nasser ms, gomaa aa. resazurin microtiter assay plate method for detection of susceptibility of multidrug resistant mycobacterium tuberculosis to second-line anti-tuberculosis drugs. egyptian j chest dis tuberc. 2013;62(2):241–247. https://doi.org/10.1016/j.ejcdt.2013.05.008 conclusion acknowledgements references about the author(s) olayinka s. ilesanmi department of community medicine, college of medicine, university of ibadan, oyo state, nigeria department of community medicine, university college hospital, ibadan, oyo state, nigeria aanuoluwapo a. afolabi department of community medicine, college of medicine, university of ibadan, oyo state, nigeria citation ilesanmi os, afolabi aa. biobanking of covid-19 specimens during the pandemic: the need for enhanced biosafety. afr j lab med. 2021;10(1), a1379 https://doi.org/10.4102/ajlm.v10i1.1379 scientific letter biobanking of covid-19 specimens during the pandemic: the need for enhanced biosafety olayinka s. ilesanmi, aanuoluwapo a. afolabi received: 10 sept. 2020; accepted: 18 june 2021; published: 21 oct. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. dear editor, biobanking holds promising benefits particularly for improving the understanding of specific diseases and illnesses,1 as evidenced for the zika virus disease. research using biobanked blood samples helped resolve the ‘dengue-like syndrome’ misunderstanding associated with the zika virus. secondly, it provided comprehensive knowledge on the possibility for vertical transmission of the zika virus between mother and child, as well as transmission via sexual relationships and blood transfusions.2 biobanked biological samples could be kept for indefinite periods, allowing for long-term retrospective research in the future. the disease control opportunities that abound in biobanking can only be activated through efficient biobank structures. biobanks receive, process, store, and use available biological samples for research purposes to improve healthcare and reduce the incidence of disease occurrence. biobanks are essential elements in the epidemiological surveillance of diseases and are often integrated into normal laboratory practices. biobank staff are often trained on the rudiments of ethical guidelines for sample collection and handling and infection prevention and control measures. the efficiency of biobanks is influenced by both internal and external factors. internal factors could include the choice of specimen storage, information management, and communication technologies.4 external factors include ethical issues such as informed consent to use provided samples, conflict of interests of stakeholders (e.g. scientists, biobank administrators, sample donors, and commercial organisations), as well as commercialisation and profit distribution issues. inefficient handling and storage of specimens, information management systems, and communication modalities between sample handling and storage units of biobanks, as well as the commercialisation of samples, will reduce the efficiency of biobanks. this letter aimed to describe strategies for maintaining efficiency and safety in the coronavirus disease 2019 (covid-19) biobanks. safety guidelines have been developed by the governments of many countries for covid-19 biobanks.5,6 these guidelines mandate general laboratory practices and standard biosafety precautions but barely dwell on the sustenance of safety protocols.7 laboratory safety could be maintained by regular training of biobank staff on covid-19 guidelines and standard operating procedures, particularly specimen handling, risk assessments, emergency operating procedures, and overall safety precautions.8 this will ensure that laboratory managers and staff remain updated and maintain good laboratory practice. the use of personal protective equipment, such as disposable latex gloves, face shields, and laboratory gowns, is required for the safety of covid-19 biobank staff.8 while preventing staff infection, personal protective equipment also reduces the risk of specimen contamination. donning and doffing of personal protective equipment by biobank staff should be done in restricted areas, and proper handwashing when exiting the laboratory should be practised. individual covid-19 samples must be handled as biohazardous material to prevent the infection of biobank staff. when covid-19 samples are collected in designated laboratories, they are to be packaged and transferred to a biobank at 2 °c to –8 °c or frozen at –70 °c in a viral medium from the source laboratory8 and samples are to be transferred within 5 days of collection to a biobank. affiliation with the nearest biobank is advised to allow for prompt transfer of collected covid-19 samples. laboratory infrastructure may need to be reviewed to promote the efficiency of covid-19 biobanks. the decontamination of specimen containers should be done before removing each specimen from the safety cabinet. in biosafety level-2 laboratories, routine testing should be automated and limited to inactivated specimens.8 tasks involving the culture of severe acute respiratory syndrome coronavirus 2 must be carried out in biosafety level-3 laboratories.9 all examinations of covid-19 specimens should be conducted in a certified class ii biological safety cabinet with a high-efficiency particulate air filter, which should be used alongside a high-efficiency particulate air-filtered incubator for culture samples obtained from suspected covid-19 cases.9 inactivated specimens should be stored in biosafety level-2 or higher safety laboratories and sample integrity must be ensured. periodic internal and external quality assurance assessment of laboratories handling covid-19 biospecimens should be conducted to validate the generated results by biobanks. to achieve this, partnerships with public or private organisations with an excellent external quality assurance track record should be instituted. internal quality assurance could be commenced and promoted through the establishment of a quality assurance unit within each biobank. similarly, regular quality assurance training, reporting, and appraisal should be instituted. the implementation of biosecurity measures should be implemented at all stages from sampling to labelling, tracking, and handling of covid-19 specimens and results.7 due to the highly infectious nature of covid-19 samples, they could be targeted as weapons of bioterrorism, and biobank staff could be used to gain access to these samples. thus, regulations such as a material transfer agreement similar to those used in middle east respiratory syndrome coronavirus biobanks should be developed. this will guard against the use of collected covid-19 samples for personal, self-motivated purposes. also, ethical considerations such as privacy, fairness, and beneficence must be considered when sharing covid-19 specimen-related data among donors, researchers, and institutions. biobanked specimens could be used to conduct population-wide genetic sequencing research, which can identify severe acute respiratory syndrome coronavirus 2 mutation patterns among the population. presently, covid-19 vaccines such as moderna, astrazeneca, pfizer, and johnson & johnson are being rolled out globally.4 however, many countries have reported that these vaccines are inadequate to cover the variants circulating within their population.10 hence, prioritisation of certain populations may be needed during this period. therefore, biobanking could provide essential information to influence the distribution of covid-19 vaccines. conclusion biobanks provide an opportunity through which insights can be gained into the diagnosis of diseases. they are also important for providing long-term storage of biological samples. while carrying out their roles, biobank staff could be at risk of covid-19 infection. to prevent such occurrences, adherence to infection prevention and control measures should be promoted in biobanks. in addition, regular internal and external quality assurance should be ensured in all biobanks. to sustain biobanks, measures such as multisectoral collaboration are needed to ensure that the financial needs of biobanks are met. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions o.s.i. conceptualised the study. a.a.a. drafted the manuscript. all authors provided critical feedback and helped shape the manuscript. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support the authors received no financial support for the research, authorship, or publication of this article. data availability data sharing does not apply to this article, as no new data were created or analysed in this study. disclaimer the views expressed in this manuscript are those of the authors and are not an official position of any institution. references amin l, hashim h, mahadi z, ismail k. determinants of the willingness to participate in biobanking among malaysian stakeholders in the klang valley. mc med res methodol. 2018;18:163. https://doi.org/10.1186/s12874-018-0619-2 madhav n, oppenheim b, gallivan m, et al. pandemics: risks, impacts, and mitigation. in: jamison dt, gelband h, horton s, et al., editors. disease control priorities: improving health and reducing poverty. 3rd ed. [homepage on the internet]. washington, dc: the international bank for reconstruction and development/the world bank; 2017. chapter 17. available from: https://www.ncbi.nlm.nih.gov/books/nbk525302/ henderson ge, cadigan rj, edwards tp, et al. characterizing biobank organizations in the u.s.: results from a national survey. genome med. 2013;5(1):3. https://doi.org/10.1186/gm407 sargsyan k, jaksa b, hartl g, et al. risk management in biobanks. in: j. rocha, s. oliveira 7 c. capinha, editors, risk management and assessment. london: intechopen. https://doi.org/10.5772/intechopen.91463 peeling rw, boeras d, wilder-smith a, sall a, nkengasong j. need for sustainable biobanking networks for covid-19 and other diseases of epidemic potential. lancet infect dis. 2020;20(10):e268–e273. https://doi.org/10.1016/s1473-3099(20)30461-8 guidance covid-19: a safe handling and processing for samples in laboratories [homepage on the internet]. gov.uk (online). [cited 2020 sept 09]. available from: https://www.gov.uk/government/publications/wuhan-novel-coronavirus-guidance-for-clinical-diagnostic-laboratories/wuhan-novel-coronavirus-handling-and-processing-of-laboratory-specimens cloudlims. covid-19 clinical data management using lims [homepage on the internet]. 2020. [cited 2020 sept 10]. available from: https://www.cloudlims.com/blog/covid-19-clinical-data-management-using-lims-html who. laboratory testing for coronavirus disease (covid-19) in suspected human cases [homepage on the internet]. world health organization; 2020. [cited 2020 sept 09]. available from: https://apps.who.int/iris/handle/10665/331501 yang j-r, liu m-t, huang h-i, teng h-j, chen j-h, li s-y. building the national sars-cov-2 laboratory diagnostic capacity in taiwan. health secur. 2020;18(5):383–391. https://doi.org/10.1089/hs.2020.0056 scroll. in. coronavirus crises: covid-19: vaccine production and distribution has exposed – and intensified – global inequality [homepage on the internet]. [cited 2021 may 04]. available from: https://scroll.in/article/992328/covid-19-vaccine-production-and-distribution-has-exposed-and-intensified-global-inequality abstract introduction methods results discussion acknowledgements references about the author(s) joseph anejo-okopi department of microbiology, university of jos, jos, nigeria aids prevention initiative in nigeria, jos university teaching hospital, jos, nigeria edith okeke department of internal medicine, university of jos, jos university teaching hospital, jos, nigeria pantong m. davwar department of internal medicine, university of jos, jos university teaching hospital, jos, nigeria chika onwuamah center for human virology and genomics nigeria institute of medical research, lagos, nigeria harris onywera institute of infectious disease and molecular medicine, university of cape town, cape town, south africa division of medical virology, department of pathology, university of cape town, cape town, south africa research, innovations, and academics unit, tunacare services health providers limited, nairobi, kenya patience omaiye department of internal medicine, university of jos, jos university teaching hospital, jos, nigeria mary duguru department of internal medicine, university of jos, jos university teaching hospital, jos, nigeria ocheme j. okojokwu department of microbiology, university of jos, jos, nigeria otobo i. ujah department of community and family health, college of public health, university of south florida, tampa, florida, united states bulus jonathan department of family medicine, plateau state specialist hospital, jos, nigeria chima a. george department of family medicine, bingham university teaching hospital, jos, nigeria ramyil s. crown department of medical microbiology and parasitology, bingham university teaching hospital, jos, nigeria fiyaktu b. yakubu department of chemical pathology, jos university teaching hospital, jos, nigeria judith o. sokei center for human virology and genomics nigeria institute of medical research, lagos, nigeria leona c. okoli center for human virology and genomics nigeria institute of medical research, lagos, nigeria onyemocho audu department of epidemiology and community health, benue state university, makurdi, nigeria seth c. inzaule department of hiv and global hepatitis program, world health organization, geneva, switzerland isaac o. abah department of pharmacology, university of jos, jos university teaching hospital, jos, nigeria patricia agaba aids prevention initiative in nigeria, jos university teaching hospital, jos, nigeria department of family medicine, university of jos, jos university teaching hospital, jos, nigeria oche o. agbaji department of internal medicine, university of jos, jos university teaching hospital, jos, nigeria atiene s. sagay department of obstetrics and gynaecology, university of jos, jos university teaching hospital, jos, nigeria claudia hawkins department of medicine, feinberg school of medicine, northwestern university, chicago, illinois, united states citation anejo-okopi j, okeke e, davwar pm, et al. molecular detection of hepatitis b virus genotype e with immune escape mutations in chronic hepatitis b patients on long-term antiviral therapy in jos, nigeria. afr j lab med. 2022;11(1), a1677. https://doi.org/10.4102/ajlm.v11i1.1677 original research molecular detection of hepatitis b virus genotype e with immune escape mutations in chronic hepatitis b patients on long-term antiviral therapy in jos, nigeria joseph anejo-okopi, edith okeke, pantong m. davwar, chika onwuamah, harris onywera, patience omaiye, mary duguru, ocheme j. okojokwu, otobo i. ujah, bulus jonathan, chima a. george, ramyil s. crown, fiyaktu b. yakubu, judith o. sokei, leona c. okoli, onyemocho audu, seth c. inzaule, isaac o. abah, patricia agaba, oche o. agbaji, atiene s. sagay, claudia hawkins received: 19 july 2021; accepted: 28 apr. 2022; published: 18 oct. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: previous studies in nigeria have reported the presence of hepatitis b virus (hbv) genotype e and the availability of immune escape mutants. there is a paucity of data on chronic patients on long-term antiviral therapy for hbv infection. objective: this study assessed hbv genotypes and drug resistance variants among patients with chronic hbv infection receiving tenofovir in jos, nigeria. methods: this cross-sectional study consecutively enrolled 101 patients (51 with hiv/hbv co-infection and 50 with hbv infection only) on antiviral therapy from february 2018 to may 2019 at four hospitals in jos, nigeria. dna quantification of hbv was performed on all samples; 30 samples with detectable viral load were selected for genotyping using sanger sequencing by targeting the full-length sequences of reverse transcriptase gene of the hbv genome. phylogenetic analysis was performed with reference sequences from genbank. escape mutant and drug resistance analysis were performed using hbv drug resistance interpretation and geno2pheno. results: only 30 (29.7%) of the 101 study participants had detectable hbv dna. of these, six (20.0%) isolates were successfully amplified and sequenced. the identified genotype was e, including escape mutations l127r (16.7%) and g145a (16.7%). conclusion: this study revealed exclusive dominance of genotype e in nigeria. the s gene mutations g145a and l271r are known to be associated with modified antigenicity and impaired serologic assays, which may cause false negatives in the detection of anti-hbv surface antigen. the presence of mutants that are associated with vaccine immune escape may also have diagnostic and vaccine immune response implications. keywords: hepatitis b virus (hbv); genotyping; antiviral drug resistance; chronic hepatitis b. introduction hepatitis b virus (hbv) infection is a public health problem worldwide, with an estimated global infection of 2 billion, 257 million of whom are chronically infected.1,2 hepatitis b virus infection causes about 880 000 deaths yearly due to cirrhosis and hepatocellular carcinoma (hcc).3 the prevalence of hcc is rapidly increasing with most cases being attributable to chronic hepatitis b. the disease is endemic in sub-saharan africa, affecting more than 8.0% of the population, most of whom are infected in early childhood.3 individual infections may resolve, become sub-clinical, chronic or result in liver damage. sub-saharan africa has one of the highest hbv-related liver cancer rates in the world.4,5 hepatitis b virus infection increases the risk of developing liver cirrhosis and hcc by 25.0% to 40.0%.6 hepatitis b virus prevalence in nigeria varies among different populations (ranging between 9.2% and 18.0%), including hbv/hiv co-infected population.7,8,9 there is mounting evidence for heterogeneity of hbv treatment outcomes and vaccination efficacy attributed to the variations in the distribution of hbv genotypes.10 hepatitis b virus has been characterised into 10 genotypes: a–i4,11 and j which was recently identified in a single case in japan.12 genotypes a, d and e circulate in africa, with the latter genotype prevailing in western africa,4,13 including nigeria.14,15,16,17,18 looking at nigeria’s huge population and cross-border movement, it is important that the molecular characterisation of hbv genotypes and prevalence of drug resistance mutations is determined for better management and epidemiological purposes. the investigation of hbv genotype is becoming more important due to its role in the pathogenesis and treatment response in both hbv-mono and hbv/hiv co-infected patients and the wide availability of antiviral therapy.19 equally, drug resistance is a major clinical concern impacting clinical outcomes. studies have reported the impact of some mutations that showed significant role of viral persistence on liver disease, immune escape and resistance to antiviral therapy.20,21 the reverse transcriptase encoded by the pol gene lacks effective proofreading ability, which has implications in hbv therapy.22 however, the use of low genetic barrier drugs such as lamivudine has contributed to the development of a drug-associated vaccine-escape mutant which is becoming a growing health concern. this mutant alters envelope antigen that permits the virus to escape the neutralisation antibody to hepatitis b surface antigen.21 this called for the need of drugs with higher genetic barriers to resistance than lamivudine or telbivudine.19 however, there is a paucity of knowledge regarding the hbv genotypes and resistant strains in chronic adult patients on long-term antiviral therapy. we therefore characterised hbv in chronic patients on tenofovir to identify circulating genotypes and drug resistance variants in nigeria. methods ethical considerations the study protocol was approved by the four study sites research ethics committees, namely: jos university teaching hospital, faith alive foundation, bingham university teaching hospital and plateau state specialist hospital (study approval numbers: juth/adm/11/0517, fa/0112f, buth/ec/01-25-18b and plashrec/12018). after explaining the study objectives and procedures to the participants, a written informed consent was obtained from each patient before data and sample collection. the patients’ data were coded and reported without divulging personal health information. all other aspects of the study were conducted in accordance with the ethical standards of the helsinki declaration. study design and population we conducted a cross-sectional study between february 2018 and may 2019 at jos university teaching hospital, plateau state specialist hospital, faith alive foundation and bingham university teaching hospital, all in jos metropolis, nigeria. the 101 consecutively enrolled adult patients, including 51 patients co-infected with hiv, were confirmed to have chronic hbv infection after testing positive for hepatitus b surface antigen antibodies. for hbv sero-diagnosis, 5 ml of blood sample was collected, centrifuged at 2500 revolutions per minute for 1 min, and 1 ml of plasma was used for testing hbsag by third-generation enzyme-linked immunosorbent assay (monolisa, bio-rad, paris, france) according to manufacturer’s instruction; a positive hbsag result indicates current infection. all participants were on antiviral therapy (hbv/hiv: lamivudine/tenofovir combination, hbv-mono: tenofovir alone) ≥ 12 months. sample preparation the 5 ml of blood samples collected were centrifuged, and the sera separated, aliquoted, and stored at –80 °c until use. the hbv dna detection and quantification were performed using automated cobas® ampliprep taqman hbv test version 2.0 (roche diagnostics international ag, rotkreuz, switzerland). a total of 30 samples with detectable viral load were selected for dna sequencing reactions. hepatitis b virus dna extraction dna extraction, amplification and direct sequencing of the full-length hbsag and the reverse transcriptase genes were performed at center for human virology and genomics, nigeria institute of medical research, lagos, nigeria. viral dna was extracted from 200 µl of plasma samples using the zr viral dna kittm (zymo research, irvine, california, united states), according to the manufacturer’s protocol. primers and nested polymerase chain reaction universal primers from previous study were used for detection of hbv genotypes,23 which were synthesised by inqaba biotech (pretoria, south africa). two outer primer sets were used. primer set 251f (5ʹ-gga tgt gtc tgc ggc gtt t-3ʹ) and 1797r (5ʹ-gac cca caa ttc ktt gac ata ctt tcc-3ʹ) were used for the initial amplification, and 251f (5ʹ-cga acc act gaa caa atg gc-3ʹ) and 1190r (5ʹ-tca cca tat tct tgg gaa caa ga-3ʹ) used for the second amplification, as previously described.23 these primer sets were designed to generate overlapping fragments, and polymerase chain reaction (pcr) was performed using platinum™ superfi™ pcr master mix (thermofisher, waltham, massachusetts, united states). each 25.0 µl reaction mixture contained 12.5 µl of 2× premix, 1.25 µl of each 10.0 µm primer, 5.0 µl of pcr water and 5.0 µl dna template for first pcr while for the second pcr, 3.0 µl of first pcr product was used. both pcr were carried out in applied biosystems miniamp plus thermal cycler (thermofisher, waltham, massachusetts, united states) with the following cycling conditions: 95 °c for 3 min followed by 35 cycles of 97 °c for 30 s, 54 °c for 60 s and 72 °c for 60 s for the first round of pcr while 31 cycles were done for the second round of pcr. each round of pcr was followed by a final extension of 72 °c for 10 min. amplified products were verified using 1% agarose gel stained with gelred® (biotium, fremont, california, united states) on a cyfox gel documentation system (sysmex-partec, münster, germany). the amplified products were purified using exosap-it (thermofisher, waltham, massachusetts, united states) and quantified using dsdna high-sensitivity (thermofisher, waltham, massachusetts, united states) reagent on the qubit-4tm (thermofisher, waltham, massachusetts, united states). samples with dna concentration ≥ 5 ng/µl and gel bands of appropriate size were selected for further processing. purification and sanger sequencing verified nested pcr products were purified using exosap-ittm pcr product cleanup reagent (thermofisher, waltham, massachusetts, united states) at 37 °c for 15 min (to digest excess primers and deoxynucleoside triphosphates), and 80 °c for 15 min (to inactivate the enzymes) according to manufacturer’s protocol. the sequencing reaction mixture contained 1.0 µl of bigdye® terminator version 3.1 ready reaction mix (applied biosystems, foster city, california, united states), 4.0 µl of 5× sequencing buffer, 2.0 µl of template, 11.0 µl of double distilled water, and 2.0 µl of 10.0 µm 251f and 1190r primers. cycle sequencing was performed using 251f and 1190r primers in a miniamp plus thermal cycler with the following program: 25 cycles of 96 °c for 10 s, 50 °c for 5 s and 60 °c for 4 min. the cycle sequencing product was purified using the bigdye xterminator® solution (applied biosystems, waltham, massachusetts, united states) followed by the capillary electrophoresis on the 3130xl genetic analyzer using a 50 cm capillary array, and pop-7 polymer (applied biosystems, waltham, massachusetts, united states). all the generated sequences were analysed and assembled using clc genomic workbench version 8.0.3 (clc bio, aarhus, denmark) and then subjected to ncbi nucleotide blast (https://blast.ncbi.nlm.nih.gov/blast.cgi) for quality check. in this study, a chromatogram was considered of good quality if (1) at least 80% of total amplicon length was sequenced and (2) the noise-to-signal ratio was estimated to be < 5%. phylogenetic analysis of hepatitis b virus sequences to infer the evolutionary relationship between the six hbv nucleotide sequences in our study and hbv sequences from different parts of the world, we used molecular evolutionary genetics analysis (mega) x version 10.1.7 (mega software, dortmund, germany).24 first, a total 102 sequences (including a sequence from a chimpanzee) representing the hbv genotypes from different parts of the world were mined from the ncbi genbank nucleotide database (https://www.ncbi.nlm.nih.gov/nucleotide/). the sequence from the chimpanzee was used as an out-group. the generated nucleotide sequence data were deposited in the genbank database under the accession numbers on236588–on236593. these sequences, together with the six from our cohort, were aligned using multiple sequence comparison by log-expectation (muscle) software,25 which is a sequence alignment option in mega. the multiple sequence alignment was trimmed to 846-base pairs, followed by determination of the dna model that best describes the nucleotide substitution pattern. this was performed using the maximum likelihood (ml) statistical method. the general time reversible (gtr) model with discrete gamma distribution (+g = 0.51, with 5 rate categories) and some invariant evolutionary sites (+i = 35.51% sites) was chosen as the best model, based on its low bayesian information criterion score (18 980). for the phylogeny reconstruction the ml statistical method was used with a bootstrap of 1000 replicates and the sub tree-pruning-regrafting (extensive; spr level 5) as the ml heuristic method. moreover, we used the option of ‘moderate branch length filter’ to enable a semi-stringent exhaustive optimisation of the branch lengths of the phylogenetic trees. initial trees for the heuristic search were generated using neighbor-joining and bionj algorithms and the phylogenetic tree with superior log likelihood value selected. phylogenetic trees were produced in newick format employing interactive tree of life interface (https://itol.embl.de/tree/). to determine the genetic diversity of the six hbv sequences, we performed an alignment analysis using mega.24 the overall mean (average) and maximum distance were used as proxies for genetic diversity. assessment of hepatitis b virus drug resistance mutations the obtained sequences of overlapping surface (s) and pol gene were translated to the protein sequences and aligned with the references in bioedit version 7.1.3.0.26 escape mutant analysis and drug resistance analysis were performed using hbv drug resistance interpretation and geno2pheno (hbv) version 2.0 (https://hbv.geno2pheno.org/index.php). it is a program that searches for homology between the input sequence and other dna sequences in the existing stored database for drug resistance and surface gene mutations. genotype assignments were confirmed using the genbank blast and hbvseq tools from the hiv drug resistance database.26,27 statistical analysis the obtained data were analysed using statistical package for social science software (version 19.0; ibm corp., armonk, new york, united states) and expressed as medians (with interquartile ranges [iqr]) for continuous variables, and as counts and percentages for discrete variables. chi-squared test was used for discrete data. results thirty (29.7%) of the 101 had detectable hbv dna. of the 30 patients, 11 (36.7%) were co-infected with hiv while 19 (63.3%) had hbv-mono infection. seventeen (56.7%) were female; the mean age was 41 years. the proportion of patients with hbv dna copies/ml of < 20 copies/ml was 22/30 (73.3%); 6/19 (31.6%) of hbv-mono and 2/11 (18.2%) of hiv/hbv had 20 copies/ml – 20 000 copies/ml. median aspartate aminotransferase u/l was 28 (23.8–36.0) for hiv/hbv co-infected patients and 27 (iqr: 19–32) for those with hbv-mono infection. median alanine aminotransferase was 26.1 (iqr: 19.8–35.5) for those with hiv/hbv dual infection and 27 (iqr: 20–41) for those with hbv-mono infection while platelet level was 259 (iqr: 198.3–298.8) for those with hiv/hbv dual infection and 195 (168–257) for those with hbv-mono infection. phylogeny and genetic diversity of the hepatitis b virus sequences all the generated sequences were from the hbv-mono infected patients. the genotypes of the six hbv nucleotide sequences were inferred from the phylogenetic tree (figure 1), which showed that all the sequences were hbv genotype e. all the six sequences clustered with hbv genotype e, most of which were from the west african region. figure 1: phylogenetic maximum likelihood circular consensus tree with branch node statistics as viewed using interactive tree of life (https://itol.embl.de/tree/). hepatitis b virus drug resistance report according to the geno2pheno (hbv) 2.0 report, all the six hbv genotype e sequences were susceptible to lamivudine, adefovir, entecavir, tenofovir disoproxil fumarate, and telbivudine. two (33.3%) of the six genotypes, all from hbv-mono infected individuals, had escape mutations, the sh2 domain-containing adapter protein b protein of the surface-gene. one sample had l127r escape mutation while the other had g145a at a position first described as vaccine escape mutation. these mutations are amino acid substitutions within the major hydrophilic region called the ‘a’ determinant (124–147). discussion our findings showed a predominance of genotype e consistent with previous studies from nigeria and other west african regions.4,14,16,17,18,28,29,30 we however found that the genotypes had a lower diversity compared to a previous report,14 although other studies have equally observed a low genetic diversity of hbv genotype e in west africa.18,31 a possible explanation may be due to natural selection, drug pressure and hyperendemicity of hbv genotype e infection in other west african countries.28 the ease of transmission and epidemiological pattern of hbv have been linked to genotype variability, as well as clinical virological parameters. the pathogenicity of hbv has been shown to vary with genotype and certain mutations have been associated with genotype diversity that influences disease outcome, hcc development, diagnostic testing and treatment outcomes.28 studies have shown a higher severity of liver disease progression and hcc in patients infected with c and d genotypes than those infected with a or b genotypes.32,33 although the clinical implications of genotype e, the predominant genotype in our study, have not been well reported, it is speculated that patients with genotype e have better liver disease prognosis than other genotypes.29 larger studies are needed to provide further evidence on good clinical prognosis for patients infected with genotype e. nonetheless, these findings suggest the need for adoption of individualised genotypic testing, which may play a key role in guiding future treatment strategies to combat the hbv disease. in the interim, early treatment is warranted to ensure good hbv prognosis. as reported in other studies, we did not find any tenofovir or underlying lamivudine-associated mutations including the rta194t that was earlier reported to be resistant to tenofovir.34,35 this suggests that tenofovir-containing and monotherapy regimen is still effective against the virus in our setting. however, we observed vaccine escape mutations (l127r and sg145a) in two (33.3%) of the six genotypes. the finding of sg145a is consistent with findings from nigeria,16 ghana36 and gabon.37 these hbsag vaccine escape mutants may have arisen as a result of specific selection such as the host immune system due to vaccination or antiviral selective pressure that truncates the production of hbsag resulting in low plasma hbv dna levels.38 although we did not take the history of patients’ vaccination, but all the patients were on antiviral therapy (≥ 12 months), including mono-lamivudine or lamivudine-containing regimen before switching to tenofovir, and this may suggest a possible reason for emergence of drug-associated potential vaccine-escape mutants (sg145a) in the study patients. similarly, mutations arising in the s orf region because of inherent or selective pressure by antiviral drugs can end up in the emergence of virus escape mutants in the adjacent s region with subsequent lack of response to hbv vaccine. the presence of this mutant over time could lead to partial replicative capacity of resistant hbv variant, and lack of response to hbv vaccine.39 this mutation may be due to poor adherence to antiviral therapy and the use of drugs with a low genetic barrier to resistance such as lamivudine, which was the major drug used in nigeria before switching to tenofovir among hbv-mono infected patients. this mutation sg145 confers immune escape competence on the virus,40,41 which has negative impact on humoral immune response to hbv vaccine. however, a recent study has shown that the risk of transmission of hbv infection is low in a vaccinated individual.42 similarly, the presence of this mutation in immune-compromised patients could be responsible for hbv reactivation among persons previous immune to hepatitus b surface antigen antibodies, suggesting immune escape mutant.43 also, this mutation among others can be responsible for virus detection failure in most routine screening tests.44 this is because an assay used for hbv screening may give false-negative results if the test kit was not designed to detect mutants in the ‘a’ determinant. this is common in the clinical setting where the patients have hbsag negative result, yet will have classical symptoms and, if tested for hbv dna, will have positive high hbv dna result. the detection of some of these emerging mutants has become a major challenge to commercially available immunoassays. the l127r escape mutation in the ‘a’ determinant region observed in our study has been associated with hbv antigenicity, diagnostics and immunogenicity.45 overall, l127r mutation was mostly associated with patients who had experienced lamivudine treatment in other african countries.36,37 the long-term impact of hbsag vaccine escape mutations on the natural history of chronic hbv remains unclear. however, it has been hypothesised that the presence of stop codons may favour the production of a truncated hbsag (despite low serum hbv dna), which accumulates in the endoplasmic reticulum and induces oxidative stress to enhance cell proliferation.38 this may also explain the transmission of hbv genotype e infection even in vaccinated individuals. studies on the hbv genotypes, immune escape and antiviral drug resistance mutations in nigeria are scarce. this is the first published study, to the best of our knowledge, that characterised hbv genotypes and drug resistance in chronic hepatitis b patients (hbv/hiv co-infected and hbv-mono infected patients) on long-term antiviral therapy (tenofovir) in nigeria. however, one recent study assessed the effect of hbv mutations with liver severity in hiv/hbv co-infected antiretroviral therapy-naïve patients in nigeria.27 in this study, hbv mutations were independently associated with liver disease severity, but the effect declined after antiretroviral therapy initiation. our study however, highlighted the need to assess hbv mutations due to the potential link with disease severity. limitations there are limitations to this study that need to be highlighted. it is a cross-sectional study with a small sample size, and low amplification and sequencing rate, yielding only 20.0% of the intended samples. many factors could be responsible for this. for example, several freeze-thawing cycles and poor storage due to frequent power outage could have affected the sample integrity, resulting in decreased hbv dna quantity. other causes include intrinsic reagent-related issues and impact of hbv/e on the sensitivity of hbsag assays due to surface antigen gene mutations located outside the sequenced region.46 also, there is a growing concern that the accumulation of escape mutations may lead to detection failure of hbsag using the available commercial diagnostic assays. this in turn may lead to a false-negative hbv result. moreover, this has an impact on risk of transmission, including rendering the vaccination ineffective due to undiagnosed mutant carriers. conclusion our study confirmed the dominance of genotype e, and it has great public health significance, given that the protective vaccine currently in use by the national vaccination programme is from the hbv-a2 strain. additionally, no patients had hbv drug resistant mutations, suggesting that the use of tenofovir is an effective treatment in the management of hbv infection in both chronic hbv/hiv co-infected and hbv-mono infected patients. the study also showed the presence of mutation associated with immune escape mutant in chronic hbv-infected patients on long-term antiviral therapy. therefore, further larger study is required to understand the dynamics of immune escape mutant on hbv diagnosis, and impact of genotypes on treatment outcomes. acknowledgements we thank the patients who participated in this study. we also thank the staff and management of jos university teaching hospital, faith alive foundation hospital, bingham university teaching hospital, and plateau state specialist hospital for their support during patients’ enrolment and sample collection. finally, we thank the of the management and staff of center for human virology and genomics, and nigeria institute of medical research, lagos, nigeria, for providing the laboratory space and equipment during the analysis. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions j.a.-o., e.o. and c.h. were involved in conceptualisation, methodology, funding acquisition, project administration, investigation, writing original draft preparation and supervision. a.s.s. and o.a. assisted with funding acquisition, supervision, project administration, resources, writing, reviewing and editing. h.o. and c.o. performed data curation, assisted with software and contributed to the formal analysis, validation, preparation of the original draft and validation. p.m.d., p.o. and m.d. were responsible for investigation, project administration, resources, writing, reviewing and editing. all authors discussed the results, contributed to and approved the final manuscript. sources of support research reported in this publication was partly supported by the fogarty international center and national institute of mental health, of the united states national institutes of health under award number d43 tw010543. data availability the data sets generated with unique identifiers and analysed during the study are available on request from the 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garcia-samaniego j, et al. selection of hepatitis b virus (hbv) vaccine escape mutants in hbv-infected and hbv/hiv-coinfected patients failing antiretroviral drugs with anti-hbv activity. j acquir immune defic syndr. 2007;46(3):279–282. https://doi.org/10.1097/qai.0b013e318154bd89 salpini r, colagrossi l, bellocchi mc, et al. hepatitis b surface antigen genetic elements critical for immune escape correlate with hepatitis b virus reactivation upon immunosuppression. hepatology. 2015;61(3):823–833. https://doi.org/10.1002/hep.27604 cao gw. clinical relevance and public health significance of hepatitis b virus genomic variations. world j gastroenterol. 2009;15(46):5761–5769. https://doi.org/10.3748/wjg.15.5761 zeid wma, ramadan di, shemis ma. prevalence of mutations within major hydrophilic region of hepatitis b virus and their correlation with genotypes among chronically infected patients in egypt. arab j gastroenterol. 2016;17(1):34–40. https://doi.org/10.1016/j.ajg.2016.03.001 launay o, masurel j, rosenberg ra, et al. high levels of serum hepatitis b virus dna in patients with ‘anti-hbc alone’: role of hbsag mutants. j viral hepat. 2011;8(10):721–729. https://doi.org/10.1111/j.1365-2893.2011.01482.x abstract introduction methods results discussion trustworthiness acknowledgements references about the author(s) daniel rhodes clinical affairs, beckman coulter immunotech, marseille, france guislaine carcelain immunology laboratory, assistance publique hopitaux de paris, paris, france mike keeney lawson health research institute, london health sciences centre and st. joseph’s health care, victoria hospital, london, ontario, canada christophe parizot department of immunology, university hospital, paris, france dominika benjamins london health sciences centre, london, ontario, canada laurine genesta biomnis laboratory, lyon, france jin zhang life science flow cytometry, beckman coulter incorporated, miami, florida, united states justin rohrbach clinical affairs, beckman coulter incorporated, miami, florida, united states denise lawrie national health laboratory service, charlotte maxeke johannesburg academic hospital, johannesburg, south africa deborah k. glencross faculty of health sciences, university of the witwatersrand, johannesburg, south africa national health laboratory services, johannesburg, south africa citation rhodes d, carcelain g, keeney m, et al. assessment of the aquios flow cytometer – an automated sample preparation system for cd4 lymphocyte panleucogating enumeration. afr j lab med. 2019;8(1), a804. https://doi.org/10.4102/ajlm.v8i1.804 original research assessment of the aquios flow cytometer – an automated sample preparation system for cd4 lymphocyte panleucogating enumeration daniel rhodes, guislaine carcelain, mike keeney, christophe parizot, dominika benjamins, laurine genesta, jin zhang, justin rohrbach, denise lawrie, deborah k. glencross received: 13 mar. 2018; accepted: 18 mar. 2019; published: 05 dec. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: flow cytometry has been the approach of choice for enumerating and documenting cd4-cell decline in hiv monitoring. beckman coulter has developed a single platform test for cd4+ t-cell lymphocyte count and percentage using panleucogating (plg) technology on the automated aquios flow cytometer (aquios plg). objectives: this study compared the performance of aquios plg with the flowcare plg method and performed a reference interval for comparison with those previously published. methods: the study was conducted between november 2014 and march 2015 at 5 different centres located in canada; paris, france; lyon, france; the united states; and south africa. two-hundred and forty samples from hiv-positive adult and paediatric patients were used to compare the performances of aquios plg and flowcare plg on a fc500 flow cytometer (flowcare plg) in determining cd4+ absolute count and percentage. a reference interval was determined using 155 samples from healthy, non-hiv adults. workflow was investigated testing 440 samples over 5 days. results: mean absolute and relative count bias between aquios plg and flowcare plg was −41 cells/µl and −7.8%. upward and downward misclassification at various cd4 thresholds was ≤ 2.4% and ≤ 11.1%. the 95% reference interval (2.5th – 97.5th) for the cd4+ count was 453–1534 cells/µl and the percentage was 30.5% – 63.4%. the workflow showed an average number of hiv samples tested as 17.5 per hour or 122.5 per 8-hour shift for one technician, including passing quality controls. conclusion: the aquios plg merges desirable aspects from conventional flow cytometer systems (high throughput, precision and accuracy, external quality assessment compatibility) with low technical operating skill requirements for automated, single platform systems. keywords: hiv; cluster of differentiation 4, cd4 enumeration; panleucogating. introduction flow cytometry has been the system of choice for cd4 lymphocyte enumeration and documentation of the decline of cd4 t-cells associated with immunosuppression and lowered counts in hiv-positive patients.1,2,3 many diverse cd4 systems that offer solutions to improve and ensure the quality of testing and improve access to testing technologies have been described over the last 25 years.4,5 in resource-limited settings, there are many instances where laboratory infrastructure is a limiting factor. however, flow cytometric systems and simpler technologies (such as point-of-care technologies), when used in a tiered laboratory approach, can offer a solution.6,7,8,9,10 in such an approach, primary centres offer simplified testing and refer testing for flow cytometry analysis to secondary or tertiary centres.11,12,13 despite the relative technical complexity, flow cytometry systems, particularly those that require less technical expertise, have been implemented with success in some national programmes.11,12 the suitability of proposed instrumentation must be assessed in the context of the destination laboratory. concerns such as the level of technical skill required for operation (ease of use, training, and automation), daily sample load and turn-around time requirements, external quality assessment programme compatibility and quality control reagent availability, supplier availability and support, transit requirements, infrastructure, and cost per test should be considered.11,14 the panleucogating (plg) cd4 counting method12,15,16 incorporates a simple gating strategy with only cd45 and cd4 to enumerate cd4 lymphocytes. quality assessment programmes reported improved performance of plg cd4 counting and revealed better quality in both the intraand inter-laboratory reported percent coefficient of variation outcomes.12,17 decreased costs of the simplified system also played an important role in addressing some of the aforementioned concerns.16,18 this method was adopted as the predicate method by the south african national health laboratory service (nhls) in 2004. the nhls programme had grown to 35 laboratories by 2007,12 reaching 60 networked cd4 laboratories by 201411 in a tiered system utilising either beckman coulter fc500 (beckman coulter, inc., miami, florida, united states) or xl (beckman coulter, inc., miami, florida, united states) instruments according to service workload requirements.11 a single platform volumetric flow cytometer (aquios, beckman coulter, inc., miami, florida, united states) was recently developed that utilises a conventional cd4 gating method based on cd45 and cd3 with both cd4 and cd8 for cd4-positive and cd8-positive t-cell lymphocyte counts and percentages. this system was updated in 2013-2014 using bead-based counting for use with the current south african laboratory network plg predicate13,14. the aquios system is fully automated from sample preparation to flow cytometry analysis. it allows for operator independent loading and testing for multiple samples. it has pre-configured panels or protocols that are not modifiable by the user, enabling standardised testing. in line with the tiered model adopted by the national health laboratory service, plg testing on the aquios system (aquios plg) was proposed as the system to replace aging and redundant fc500 and xl flow cytometers operational within the south african network, as well as extend its use into small laboratories that offered basic clinical pathology but no cd4 services.19 the objective of this study was to compare the performance of aquios plg with traditional plg cd4 methods generated on the fc500 instrument at a local south african site, as well as established cd4 reference centre sites in high-income countries. additionally, normal samples were collected and these data were used to calculate a reference interval to establish whether normal counts generated by aquios plg matched other published reference intervals.20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42 methods ethical considerations all sites had ethics committee or internal review board approval for the collection of samples or use of leftover samples and use of minimal demographic data of age and gender or a waiver was in place for use of these samples. use of leftover samples for research purposes was agreed to at the time of routine laboratory blood draw. the following ethical clearances were in place: south africa (identification number: m121020); france (lyon; identification number: ac-2013-1808); canada (identification number: 09763e); and the united states (identification number: 10259-05). in france (paris), consent was given at the time of routine blood draw for the use of leftover samples for research under a waiver according to current french legislation (loi jardé, n°2012-300). patients providing samples for the reference interval study signed a consent form. specimens method comparison patient samples were obtained from incoming routine laboratory specimens for cd4 testing between november 2014 and march 2015 from four different centres located in canada; paris, france; lyon, france and south africa. a total of 270 samples from hiv-positive adult and paediatric patients were tested, ranging in age from 2 months to 77 years. thirty samples (22 adults, 8 children) were excluded for the following reasons: cd4 < 20 cells/µl (the instrument’s lower limit of quantitation) (18 samples), operator error with manual flowcount addition (fc500) (4 samples), short blood draw (2 samples), reliability quality control failure (fc500) (2 samples), clot (1 sample), clog (1 sample), insufficient lymphocytes (1 sample) and duplicate patient (1 sample). testing was performed in duplicate, with replicate 1 used for analysis, except for two samples, for which replicate 2 was used due to system error for replicate 1 (1 high count rate and 1 clog). two hundred and forty samples were included in the final analysis with specimen ages at time of testing ranging from 20 to 71 hours from collection. reference interval in healthy adults participants were enrolled between december 2014 and february 2015 from three centres located in canada; paris, france and the united states. samples were obtained from: 1) healthy, non-hiv patient donors who were either hospitalised patients or outpatients with no haematological disease upon final diagnosis (canada); 2) leftover samples from healthy, non-hiv volunteers donating blood to the etablissement francais du sang (paris, france); and 3) self-reported healthy, non-hiv participants enrolled through the internal donor programme at beckman coulter (miami, florida, united states). participants with cd4 < 300 cells/µl were excluded so as not to include participants with potential idiopathic cd4+ lymphopenia.20,21 a total of 173 participant samples from healthy adults aged 18–65 years, with normal complete blood counts and differential were tested. eighteen samples were excluded for the following reasons: haematologic diagnosis (10 samples), lymphopenia (3 samples), participants < 18 years of age (2 samples), lymphocytosis (1 sample), time of collection missing (1 sample) and cd4 < 300 cells/µl (1 sample). one hundred and fifty-five samples were included in the final analysis, all tested within 24 hours of collection. workflow samples were obtained from incoming routine laboratory samples for cd4 testing at one centre in south africa over five days in february 2015. a total of 440 participant samples were tested over five days. laboratory prior to each day’s testing, stabilised blood products immuno-trol cells (normal cd4 count) (beckman coulter, inc., miami, florida, united states) and immuno-trol low cells (beckman coulter, inc., miami, florida, united states) passed their assay requirements as quality control material. samples were collected into ethylenediaminetetraacetic acid vacutainers and tested in duplicate on both instruments for method comparison and only on aquios for reference interval. the plg gating strategy, used for both instruments, is detailed in figure 1. all reagents were supplied by beckman coulter, inc., hialeah, florida, united states and immunotech, marseille, france. figure 1: cd4 t-cell enumeration by panleucogating with aquios flow cytometer, canada; paris, france; lyon, france; and south africa, november 2014 to march 2015. (a) cd45 versus side scatter plot is used to identify total leukocytes (cd45-positive); (b) all events gated in the cd45-positive region (pan-leucogate) are used to plot cd4 versus side scatter to identify cd4-positive lymph cells (cd4-positive count/μl). (c) lymphs gate from a is used to plot the cd4 versus side scatter to calculate the cd4 percentage of lymphoid cells (cd4-positive lymph percent). aquios instrument cd4+ counts and percentages were determined using aquios plg, as per manufacturer’s instructions for use. each site had an instrument and all required reagents. fc-500 instrument comparator testing was done using flowcare plg cd4 reagent on the beckman coulter fc-500 mcl flow cytometer (flowcare plg) per the manufacturer’s instructions for use. all required reagents were provided. a tq-prep™ workstation (beckman coulter, inc., miami, florida, united states) was used for red cell lysing. sites either used a prepplus™ 2 workstation (beckman coulter, inc., miami, florida, united states) for specimen preparation or manual preparation in place of instrumentation. statistical analysis clinical and laboratory standards institute guidelines ep09-a2 and ep28-a343,44 for method comparison43 and reference interval determination44 were followed. replicate 1 was used for analysis, with replicate 2 used for the resolution of replicate 1 discrepancies. for method comparison, the number and percentage for sex, adult or paediatric specimens, mean age, cd4 count and percentage and cd4 count range were calculated. aquios plg and flowcare plg results were analysed using deming regression to estimate bias at the clinically relevant cd4-positive levels of 50, 100, 200, 350 and 500 cells/µl. weighted deming regression was used for count because the variability (scatter) of the data depended on the range of measurements, while simple deming regression was used for percentages. the coefficient of determination, r2 (pearson correlation squared), was used to measure the overall correlation between the two methods. bland-altman analysis45 was used to calculate the mean and median difference between methods. mean and median relative bias expressed as percent was also calculated. mean and median absolute difference and relative difference were calculated by cd4 subgroups ≤ 200, 201–1000, and > 1000 cells/µl and ≤ 350, 351–1000, and > 1000 cells/µl, where 200 cells/µl and 350 cells/µl represent antiretroviral treatment (art) thresholds and 1000 cells/µl was used to control for variability of paediatric samples. upward and downward misclassification probabilities at art thresholds of 100, 200, 350 and 500 cells/µl were determined (the method has been described elsewhere46). upward misclassification represents the percentage of additional patients who would fall above a defined threshold with the new test, whereas downward misclassification represents the percentage of additional patients that would fall below this threshold. mean percent similarity with a standard deviation (sd) and coefficient of variance was also calculated (the method has been described elsewhere47). for the reference interval, demographic characteristics were calculated (number and percentage for sex and mean ± sd for age). mean, sd, median, and range for both cd4-positive count and cd4-positive percentage were calculated by sex and for total participants. the 95% (2.5th – 97.5th) reference interval was determined using non-parametric methods. statistical differences in variables sex, adult or paediatric patient status were determined by t-test for the means and non-parametric mann whitney u test for the medians. for workflow analysis, time to first result, average sample results per hour, and sample results per 8-hour shift were calculated. statistical analysis was performed using microsoft excel (microsoft corporation, redmond, washington, united states) with analyse-it. results method comparison two hundred and forty specimens were included in this method comparison study, 92 (38.7%) were obtained from female participants (mean age 35.2 years) and 146 (61.3%) were from male participants (mean age 42.6 years); 202 (84.2%) were adults and 38 (15.8%) were children. mean cd4 count and percentage were not significantly different by sex (females: 339 cells/µl and 22.65%, males: 359 cells/µl and 23.47%; p > 0.05), but were by adult versus paediatric participants (adults: 361 cells/µl and 22.20%, children: 948 cells/µl and 32.28%; p < 0.001). the cd4 count range showed a higher minimum–maximum for paediatric specimens (46–2645 cells/µl) than adult specimens (24–1278 cells/µl). cd4 count for all specimens showed an r2 = 0.992 and mean bias of −41 cells/µl between aquios plg and flowcare plg (figure 2a). mean relative bias was −7.8%. of 12 samples with a cd4 count above 1250 cells/µl, 11 were from paediatric participants. for cd4 percentage, r2 was 0.994 and there was an average bias of −0.16% (figure 2b). because the mean cd4 count between adults and children was significantly different, adult specimens were analysed separately, showing an r2 = 0.992 and average bias of −25 cells/µl (figure 2c). mean relative bias was −6.8%. figure 2: deming regression and bland-altman for aquios panleucogating versus flowcare panleucogating, canada; paris, france; lyon, france; and south africa, november 2014 to march 2015. (a) absolute cd4 count in cells/μl for adults and children, (b) percentage of cd4 for adults and children, (c) absolute cd4 count in cells/μl for adults only. bias analysis by cd4 subgroups based on art thresholds of 200 cells per µl and 350 cells per µl showed relatively consistent median relative bias for samples below and above the respective thresholds: −7.8% and −8.1%, and −7.9% and −8.1% (table 1). table 1: cd4 count absolute and relative bias between aquios panleucogating and flowcare panleucogating overall, by cd4 subgroup and at clinically relevant cd4 levels, canada; paris, france; lyon, france; and south africa, november 2014 to march 2015. upward misclassification ranged from 0.0 to 2.4% and downward misclassification ranged from 1.5 to 11.1%, depending on the threshold (table 2). mean percent similarity (sd, coefficient of variance) was 96.1% (6.1%, 6.3%) for the absolute cd4 count and 99.5% (3.9%, 4.0%) for the cd4 percentage. table 2: misclassification percentages at various cd4 count thresholds, canada; paris, france; lyon, france; and south africa, november 2014 to march 2015. reference interval in healthy adults demographic characteristics for the 155 samples included in the reference interval analysis showed the proportions of female participants were paris, france, 0.73; canada, 0.59; united states (0.39). the mean overall ages were 40 years (canada); 45 years (united states); the mean female-to-male ages were 36 versus 45 years (canada) and 52 versus 41 years (united states). specific age data for samples from paris, france were not available. the mean cd4-positive count ± sd was higher in canada (1009 ± 239 cells/µl) compared to both the united states (866 ± 221 cells/µl) and paris, france (802 ± 273 cells/µl). similar results were seen for the mean cd4-positive count by sex at each site. the 95% reference intervals for both absolute cd4-positive counts and percentages by site were similar and overlapped. the mean cd4 absolute count and percentage for female and male participants were not different statistically (p = 0.61 and 0.48, respectively) (table 3). the overall mean cd4 positive count ± sd was 888 ± 255 cells/µl and mean cd4-positive percentage ± sd was 46.82 ± 7.86%. the 95% reference interval (2.5th – 97.5th) for cd4-positive count and percentage was 453–1534 cells/µl and 30.5% – 63.4% (table 4). recent, previously established reference interval results for cd4 absolute count and percentage from healthy, non-hiv individuals from different parts of the world using differing instrument platforms show consistent results (table 5). table 3: demographic characteristics and means, medians, and ranges for cd4 and percentages for a healthy adult reference interval, canada; paris, france; and united states, december 2014 to february 2015. table 4: 95% reference interval (2.5th – 97.5th) for apparently healthy adults, canada; paris, france; and united states, december 2014 to february 2015. table 5: comparison of overall normal reference intervals (2.5th – 97.5th) for cd4 lymphocytes in hiv-negative adults, canada; paris, france; lyon, france; and south africa, november 2014 to march 2015. as per protocol analysis that is presented in the aquios plg test instructions for use, six participant samples from paris were excluded. these samples had cd4 levels of 300–500 cell/µl. as per protocol, samples with cd4 levels < 500 cells/µl required an in-house medical monitor review of the complete blood count with differential to confirm haematological normal status and qualify for inclusion. the complete blood count with differential results were not provided for samples from the french site, so for the per protocol analysis, those with cd4 < 500 cells/µl were excluded, as it was not possible to exclude the presence of idiopathic cd4-positive t-lymphocytopenia. for this manuscript analysis, these samples with a cd4 ≥ 300 −500 cells/µl were included for the following reasons: 1) the french samples had a confirmed haematologically normal complete blood count with differential performed at the etablissement francais du sang (although results were not available to beckman), and 2) the centers for disease control does not consider a decreased cd4 level in healthy, non-hiv patients significant unless < 300 cells/µl.17,18 as expected, the inclusion of these samples decreased the lower reference interval value from 532 cells/µl to 453 cells/µl. the upper reference interval value was not affected and thus remained unchanged. workflow a total of 440 samples were tested over 5 days (table 6). the average time to the first result was 39.2 minutes. the average number of samples processed per hour was 17.5 or 122.5 for an 8-hour shift, minus 1 hour for start-up, quality control testing and shut-down. technician hands-on time required one hour, including start-up, quality control testing, sample testing and shut-down. table 6: high volume workflow results with aquios panleucogating, south africa, february 2015. discussion in this study, we compared plg on the aquios flow cytometer versus plg cd4 counts generated by fc500 instruments. additionally, cd4 counts generated from normal individuals were used to establish and gain insights into the reference interval of the cd4 counts of patients tested by aquios plg with respect to other published reference intervals. the method comparison of aquios plg to flowcare plg showed a mean absolute count bias of −41 cells/µl and a mean relative bias of −7.8% including both adult and paediatric hiv samples. for adults only, a mean absolute count bias of −25 cells/µl with a mean relative bias of −6.8% was observed, similar to outcomes noted in separate evaluations.48 the slight negative bias in our study appears to be largely platform related and not related to the gating strategy used. where different gating strategies (aquios tetra, with primary cd45 bright and cd3 gating to define cd4 lymphocytes and aquios plg which relies only on cd45 total and cd4/ss to discriminate monocytes) were applied on the same platform, a bias of just 10 cells/µl with an r2 of 0.996 (unpublished data) was noted. this finding is similar to the slight positive bias previously reported where bead-based counting versus volumetric-based counting comparison was performed.6 while the mean and median absolute count difference increases with the cd4 level, the relative difference remained stable (around 8% – 11%) across the entire range of samples tested, even at counts above 1000 cells/µl (where clinical importance is less). paediatric patients are known to have higher cd4 absolute levels and higher variability in these counts than adults,49 and this was seen in our study as well, where mean and maximum cd4-positive counts were higher for paediatrics than adults. misclassification probability measures are used to determine the likelihood that a patient’s result for a new test compared to a reference test will be classified above or below a defined threshold used in clinical decision-making50,51 these measures provide a more direct interpretation for health policy and management decision making with regard to potential financial and healthcare impacts from implementation of new instrumentation.51 misclassification results for cd4 should be interpreted for regions where world health organization guidelines52 for universal testing and treatment are not applied and where varying treatment thresholds still exist. our results indicate that the introduction of aquios plg may result in ≤ 2.4% upward and ≤ 11.1% downward misclassification. the cd4-positive absolute count mean and 95% (2.5th – 97.5th) reference interval among healthy, non-hiv adults from our study were consistent with previously established reference intervals in other studies20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42 on healthy individuals from different parts of the world (table 5). this consistency of reference intervals established the equivalency of plg gating methods in the context of reference intervals where typically conventional gating strategies were used. reference intervals enable meaningful interpretation of a patient’s laboratory results. it is recommended that laboratories establish local reference ranges for clinical use44,53 due to known differences relating to location, race or ethnicity, sex, age, disease burden and drug intake.21,22,27,28,29,33,36,38,39,42,49,54 in many countries, especially lowand middle-income countries, resources frequently limit local reference range development24 and there are few published reference interval datasets. interpretation of reporting is largely based on reference ranges published from high-income, industrialised areas of north america and europe.24 our results, however, indicate that cd4 results across multiple geographies, instruments and platforms are quite consistent, including results for cd4 obtained by plg on the aquios. the circumstances and conditions for cd4 testing in laboratories vary in lowand middle-income countries. challenges faced vary and include a basic lack of infrastructure such as an unstable electricity supply, a lack of cold storage facilities to a paucity of skills.55 the burden of hiv disease may also frequently dictate high workload volumes in certain countries.11,12 manual sample preparation requires multiple pipetting, which in turn increases pipetting errors. however, automated sample preparation lowers percent coefficients of variance through the reduction of human pipetting errors.12,56 the simplified plg cd4 method12,16 is the predicate system of the south african programme, and it combines the reliable bead-based testing technology8 and a gating strategy.8,12,16,57 plg on the aquios platform is regarded as a suitable candidate for this programme to replace older aging equipment currently used12,48 it is envisaged that its user-independent and on-board sample preparation features could improve testing outcomes in small laboratory sites with fewer staff or less technical flow cytometry expertise. thus, this enables them to provide local cd4 services even with workload equivalents of up to 100 samples per day and improve local service delivery turn-around times in more remote parts of south africa.11,19 the workflow showed a medium-high throughput level. lower throughput (12 samples per hour or 96 samples per shift tested) was seen in a previous workflow study performed in the same laboratory using manual preparation and analysis on a fc-500 flow cytometer, and, 6 hours of hands-on technician time were required.58 the aquios plg daily workflow includes available quality control material and the system works with stabilised blood products, making it compatible with external quality assessment programmes. this allows for ongoing monitoring of intraand inter-laboratory precision which is important for quality management of large-scale country-wide programmes.11,12 the system tracks quality control results and alerts user and technical support staff of deviations or trending. as a single platform system with on-board sample preparation, no additional laboratory equipment was needed. lastly, it is important to briefly discuss the relevance of cd4 counting in the face of recent world health organization guidelines52 which recommend that all patients who are hiv-positive start art, irrespective of their cd4 counts. for the past 30 years, medical personnel caring for hiv patients have used cd4 counts as prognostic indicators of disease progress3 or death.2 also, the cd4 count is used to determine eligibility for initiating art, managing and treating opportunistic infections and monitoring the patient’s response to art.59 it is widely agreed that hiv viral load testing is the optimal assay to monitor the response to art or determine treatment failure.60,61 however, in light of the documented worldwide number of individuals with advanced hiv disease61,62,63 and lack of funding and infrastructure for routine viral load testing in lowto middle-income geographies, cd4 counts will continue to play an important role in managing hiv patients in terms of: stratifying long-term risk, fast-tracking onto art outside the standard of care64 and identifying patients with immunological or clinical failure.60,65,66 the high number of individuals with advanced hiv disease also dictates that cd4 counts should play an important role in identifying patients at risk of opportunistic infections, such as cryptococcal disease, prophylactic treatment for tuberculosis and pneumocystis pneumonia. limitations our study utilised samples mainly from high-income, industrialised or urban areas, and such as may not completely represent samples found in an entirely african population. conclusion the aquios plg merges desirable aspects from conventional flow cytometer systems (high throughput, precision and accuracy, external quality assessment compatibility) with low technical skill requirements for automated, single platform systems. trustworthiness the findings of these studies should be used per the scope of the study and with regard to the indicated limitations. the results review and release on aquios should be performed by a qualified professional. acknowledgements the authors would like to thank the technicians at each testing site for their detailed and dedicated work: paris, france: isabelle mauger and virginie merere; lyon, france: sophie tronchet, karin dumoulin, audrey milhau and laura dinier; united states: nhuan ha; south africa: keshendree moodley. testing in canada was done by dominika benjamins. thank you to karen lo and robert magari for support on the statistical analysis. competing interests d.k.g. is an employee of the south african national health laboratory service and is the named inventor of a family of patents, including ep 1 405 073 and us 7 670 793, covering the plg/cd4 method of establishing cd4 counts in a sample. this patent family is wholly owned by the national health laboratory service and is exclusively licensed to beckman coulter. m.k. is a consultant for beckman coulter. d.r., j.r. and j.z. are beckman coulter employees. no other authors have declared competing interests. authors’ contributions all authors contributed equally to concept, design, data acquisition, critical review of the article for intellectual content, and approval of the final version to be submitted. sources of support this study was supported and funded by beckman coulter, inc. (miami, florida, united states) which provided reagents, instruments, and supplies. data availability statement all relevant data are within the article. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references prince he, lesar wj. simultaneous determination of absolute total lymphocyte and cd4+ lymphocyte levels in peripheral blood by flow cytometry. am j clin pathol. 1989;92(2):206–209. https://doi.org/10.1093/ajcp/92.2.206 pedersen c, gerstoft j, tauris p, et al. trends in survival of danish aids patients from 1981 to 1989. aids. 1990;4(11):1111–1116. https://doi.org/10.1097/00002030-199011000-00009 lee ca, phillips an, elford j, janossy g, griffiths p, kernoff p. progression of hiv disease in a haemophilic cohort followed for 11 years and the effect of treatment. bmj. 1991;303(6810):1093–1096. https://doi.org/10.1136/bmj.303.6810.1093 mandy 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national consolidated guidelines for the prevention of mother-to-child transmission of hiv (pmtct) and the management of hiv in children, adolescents and adults [homepage on the internet]. pretoria, south africa: department of health, republic of south africa; 2015 [cited 2018 dec 15]. available from: https://sahivsoc.org/files/art%20guidelines%2015052015.pdf ford n, meintjes g, vitoria m, greene g, chiller t. the evolving role of cd4 cell counts in hiv care. curr opin hiv aids. 2017;12(2):123–128. https://doi.org/10.1097/coh.0000000000000348 moorhouse m, conradie f, venter f. what is the role of cd4 count in a large public health antiretroviral programme? s afr j hiv med. 2016;17(1):446. https://doi.org/10.4102/sajhivmed.v17i1.446 introduction human resources technology upgrades and investments funding and financing strengthening policy recommendations conclusion acknowledgements references about the author(s) symon f. nayupe laboratory department, kamuzu university of health sciences private clinic, blantyre, malawi patrick mbulaje center for the development of people, lilongwe, malawi steven munharo montfort hospital, chikwawa diocese, nchalo, chikwawa, malawi parth patel department of health systems and policy, faculty of health systems, kamuzu university of health sciences, blantyre, malawi don e. lucero-prisno iii department of global health and development, london school of hygiene and tropical medicine, london, united kingdom citation nayupe sf, mbulaje p, munharo s, patel p, lucero-prisno de. medical laboratory practice in malawi – current status. afr j lab med. 2023;12(1), a1921. https://doi.org/10.4102/ajlm.v12i1.1921 opinion paper medical laboratory practice in malawi – current status symon f. nayupe, patrick mbulaje, steven munharo, parth patel, don e. lucero-prisno received: 13 apr. 2022; accepted: 26 sept. 2022; published: 06 jan. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction medical practice has evolved over the past years from symptom-based clinical diagnoses to evidence-based diagnoses demanding clinical laboratory investigations. clinical experts at the mayo clinic in the united states estimated that almost 70% of patient management decisions rely on laboratory diagnostic information.1,2 in sub-saharan africa, the need for quality diagnostic services is apparent; nevertheless, access to quality and reliable laboratory services in the region has been a big challenge.3 there has been significant development in medical laboratory practice across the world with the adoption of state-of-the-art technology and methods and subsequent demand for more specialised skills in medical laboratory scientists.4 in sub-saharan africa, medical laboratory practice has also evolved, though significantly slower than in other countries in the west, except countries like the republic of south africa which has many accredited and technologically advanced laboratory institutions.5 since many sub-saharan africa countries are resource limited, financing of laboratory activities has not been of primary concern; hence, laboratory improvement activities have suffered a persistent shortage of funds, dragging the pace of advancement in medical laboratory practice.3 additionally, the slow growth of medical laboratory practice has also been facilitated by continued neglect of the medical laboratory profession leading to inadequately trained personnel in some parts and an unrecognised cadre.6 in malawi, medical laboratory practice has suffered similar setbacks. the laboratory profession has, nonetheless, achieved some milestones as we will discuss. however, some serious reforms, recommendations of, have to be implemented to tackle the challenges that seriously hamper efficient, high-quality, and technologically advanced diagnostic services as are needed not only in malawi but in sub-saharan africa as a region. we hereby present the current status of medical laboratory practice in malawi, from various dimensions, namely human resources, equipment and technology, funding, as well as strengthening policy. as a background, the laboratory is structured in four ascending tiers, namely health centre laboratories, district hospital laboratories, central hospital laboratories and the national reference laboratory.7 a lower laboratory refers to the next level laboratory where advanced tests are required. laboratory falls under the health technical support services, a directorate under the ministry of health. the health technical support services is responsible for ensuring the provision of quality diagnostic capacity, monitoring drug efficacy, and patient management services. human resources malawi is one of the countries in sub-saharan africa where the ratio of healthcare workers to patients is high, reflecting a shortage of healthcare staff. since the 1990s, however, there has been a significant effort from the government to train more healthcare personnel.8 historically, malamulo college of health sciences was the first to offer certificates in medical laboratory sciences in 1968 and later in 1978 started offering diplomas. since then, malawi has progressed to offering laboratory science degrees at three accredited institutions namely the university of malawi, malawi adventist university and mzuzu university today. as per malawi association of medical laboratory scientists (mamls) unpublished records for 2021, there were approximately 2069 trained laboratory personnel registered with the medical council of malawi: 677 laboratory technologists, 1073 laboratory technicians and 320 laboratory assistants. currently, there are over 468 medical laboratory technologists and technicians who are unemployed. a commonly given reason is that there are no posts available as per government establishment despite the country facing a huge gap in laboratory personnel in many facilities. there is no doubt that the laboratory is an essential service, although the practice and utilisation of the service have generally been suboptimal for the past years with an evident need for infrastructural and capacity development.9 good-quality laboratory services are largely dependent on adequate, appropriately trained, and qualified laboratory personnel, yet laboratory professionals are prominently among neglected health cadres in malawi and across most sub-saharan african countries.10 services offered are affected by insufficient staff even though colleges are producing many graduates, as there are not enough formal posts currently. in addition, services are affected by lack of specialist qualifications and almost non-existent career progression opportunities since the laboratory profession appears not to be a primary area of concentration for professional improvement and recognition in the country. this understandably lowers the motivation of laboratory professionals. those employed by the ministry of health are often working in underfunded, poorly equipped facilities with low safety standards and unmotivating environments. technology upgrades and investments the malawi health sector has gradually expanded and improved tests available to patients. this has happened due to the availability and increased coverage of several modern machines such as the genexpert (cepheid, sunnyvale, california, united states) and full blood count machines purchased through the global fund mainly at district and central hospitals.11 in addition, the country has integrated tests on the existing machines to fully utilise the existing technology. for example, targeted viral load testing is now being done on genexpert platforms in most facilities, an upgrade from just running tuberculosis specimens. this is a huge investment that has saved money since the procurement is only focusing on procuring the reagents instead of the new machines. currently, the laboratory system is still struggling to provide high-quality diagnostic services. frequent shortages in supplies and reagents challenge sustainable service provision. additionally, factors such as poor equipment maintenance systems, poor laboratory infrastructure and limited backup testing services exacerbate the operational inefficiencies of testing services. despite the presence of equipment service contracts for government laboratories in malawi, the provision of both emergency and routine services for machine breakdowns and maintenance has been significantly slow. this, in part, is due to the availability of a few professionally trained biomedical engineers locally. consequently, these delays interrupt diagnostic service delivery.9 it has been observed that many clinicians often doubt the laboratory results of their patients. this leads to the repetition of tests, since the clinical presentation of the patients is at times inconsistent with results from laboratory investigations.9 the observations in this study could be attributed to task shifting, the use of non-laboratory trained personnel to perform tests in point-of-care settings failure to calibrate equipment, usage of expired reagents, lack of external quality control and refresher courses as well as total disregard of laboratory quality management systems.10 funding and financing in resource-limited countries, allocation of resources to diagnostic services is barely a priority.3 inadequate funding has downgraded the laboratory profession leading to poor infrastructures failing to meet the demand of its specialty to the growing population. lack of representation in key decision-making bodies is one of the top contributing factors leading to poor laboratory services in malawi. this has led to the underperformance of instrument maintenance services and a zero integrated supply chain for laboratory consumables.11 the position of laboratory manager is not an established one at the district level and hence it is not represented in the district health management team. most of the projects in malawi and sub-saharan africa at large are donor driven and hence are somewhat disease specific. this has led to a lack of cross-sector laboratory capacity, fragmentation of laboratory services and diversion of scarce resources.12 poor salary structures have also aggravated the migration of highly skilled individuals to the private sector and research institutions, further derailing the system. strengthening policy as part of laboratory professional practice improvement, medical laboratory professionals in malawi have revived its previously dormant body, mamls. formed in 1998, mamls was not active until february 2020, when a group of medical laboratory scientists, in collaboration with the international federation of clinical chemistry and laboratory medicine, facilitated the hosting of laboratory professionals from across malawi and guests from canada, the united states, egypt and the united kingdom, to the first ever medical laboratory scientific conference, where a task force dedicated to revamping mamls was formed. in december 2021, a second scientific conference was held in the country’s capital, lilongwe.13 laboratorians have often complained about the lack of a body specifically formulated to have regulatory oversight over ethical conduct, performing objective quality assurance and accreditation of medical laboratories in the country as well as representing professional interests at the policy level.14 the revamping of mamls aims at promoting and safeguarding the interests of professional medical laboratory science practice which ultimately safeguards patients who access laboratory services. continued lobbying by mamls focuses on establishing an independent medical laboratory regulatory body that will ensure a robust diagnostic representation at the policy level. recommendations define clear laboratory networks a strong laboratory organisational infrastructure in sub-saharan africa is necessary to improve access to quality healthcare.10 similarly, in malawi, such a clear definition of function, authority and responsibility of the laboratory system is necessary as a baseline for improving laboratory standards. the ministry of health in liaison with the laboratory leadership in malawi should define these networks. laboratory networks should include a multilevel systematic integration of functions with an enhanced referral system where laboratories with less testing capacity at the bottom of the system can refer advanced tests to laboratories at higher levels with more testing capacity with ease and within acceptable expected turnaround times. additionally, laboratories at all levels should adhere to national and international quality systems.5 the call for a regulatory body specific for laboratory practice in the strengthening policy section serves this purpose as one of the duties of the body. establish regulatory body for medical laboratories to curtail challenges faced by laboratories and laboratory personnel to provide quality service, the malawi government should draft a medical laboratory regulatory act to lead the way in addressing chronic challenges affecting ethical, professional and legal laboratory practice and policy. establishing a regulatory body will ensure that medical laboratories are objectively regulated for laboratory quality assurance, help shape laboratory policy and improve quality service delivery expected by patients and hospitals. the regulatory act and legal mandate will prevent encroachment and imposition from other departmental mandates.15 designate specialised laboratory posts the availability of adequate and trained human resources is one of the key elements to achieving quality diagnostics services. the absence of such, or the presence of staff who have no formally defined roles or positions, compromises the efficiency of achieving such quality as they lack direction and motivation. designating managerial and non-managerial laboratory-based positions in the medical laboratory setup through the directorate of human resources in ministry of health will map out career development prospects as well as equip particular departments with necessary skills depending on individual previous experiences to ensure competence and skills in handling jobs. include medical laboratory professionals in policy boards and regulatory bodies including medical laboratory professionals in boards and regulatory bodies by the appointing authorities will ensure the implementation of policy that promotes the welfare of laboratory personnel as well as promotion of the ever-evolving standard quality and harmonised laboratory practice, reshaping regulation and helping to redefine professionalism. regulation should be done by those vested with the dogma, qualifications, philosophy and understanding of the current problems affecting the profession and trends of medical laboratory science and practice around the globe. conclusion it is evident that there has been significant progress in the laboratory profession in malawi and generally in sub-saharan africa since colleges started training professionals locally. however, with the current health demands in modern medical practice that require efficient and quality diagnostic services, the laboratory profession is facing new challenges.15 our recommendations on defining clear laboratory networks, enacting a medical laboratory regulatory act, designation of administrative and specialised posts for laboratory professionals at the district and central levels, and the inclusion of laboratory professionals in decision-making bodies will contribute to strengthening laboratory practice in malawi. strong laboratory systems will ensure reliable diagnostic services, a contributor to access to quality health services.10 regionally, it is essential to have reliable laboratory systems in sub-saharan africa as this not only serves the individual countries but helps to strengthen regional interdependence as countries will now trust each other’s services, leading to the formation of regional networks for advanced and more specialised tests. acknowledgements competing interests the authors do not have an association with a body, entity or organisation that might pose any conflict of interest. the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions s.f.n., p.m., s.m. and p.p. were involved in the conceptualisation, investigation and formal analysis of findings. they were involved in writing the manuscript draft and reviewing and editing the final version of the manuscript. d.e.l.-p. was involved in the activities listed for all the other authors and also supervision of the whole work. ethical considerations no ethical approval was sought for this opinion paper as there was no requirement. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability data sharing does not apply to this article as no new data were created or analysed in this study. disclaimer the view and opinions expressed in this article are those of the authors and do not in any way represent the official policy or stand of the authors’ affiliated agencies or institutions. references forsman rw. why is the laboratory an afterthought for managed care organizations? clin chem. 1996;42(5):813–816. https://doi.org/10.1093/clinchem/42.5.813 hallworth mj. the ‘70% claim’: what is the evidence base? ann clin biochem. 2011 nov 1;48(6):487–488. https://doi.org/10.1258/acb.2011.011177 petti ca, polage cr, quinn tc, ronald ar, sande ma. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006 feb 1;42(3):377–382. https://doi.org/10.1086/499363 miller wd, kalfoglou a, leroy l. technology trends in the clinical laboratory industry. in: wolman dm, kalfoglou al, leroy l, editors. medicare laboratory payment policy: now and in the future. 1st ed. washington, dc: national academies press (us), 2000; p. 58–71. schroeder lf, amukele t. medical laboratories in sub-saharan africa that meet international quality standards. am j clin pathol. 2014;141(6):791–795. https://doi.org/10.1309/ajcpq5ktkagsscfn schneidman m, dacombe rj, carter j. laboratory professionals in africa: the backbone of quality diagnostics [homepage on the internet]. health, nutrition and population (hnp) discussion paper series. world bank group, 2014 [cited 2022 may 14]; p. 1–52. available from: https://openknowledge.worldbank.org/handle/10986/21115 butao d, felling b, msipa p. malawi: laboratory services and supply chain assessment. task order 1. arlington, va: usaid, 2009; p. 1–104. o’neil m, jarrah z, nkosi l, et al. evaluation of malawi’s emergency human resources programme. cambridge: ehrp; 2010. moyo k, porter c, chilima b, et al. use of laboratory test results in patient management by clinicians in malawi. afr j lab med. 2015 may 13;4(1):277. https://doi.org/10.4102/ajlm.v4i1.277 davies j, abimiku a, alobo m, et al. sustainable clinical laboratory capacity for health in africa. lancet glob health. 2017 mar 1;5(3):e248–e249. https://doi.org/10.1016/s2214-109x(17)30024-4 mtonya b, chizimbi s. systemwide effects of the global fund in malawi: final report. washington, dc: global fund; 2006. zere e, walker o, kirigia j, zawaira f, magombo f, kataika e. health financing in malawi: evidence from national health accounts. bmc int health hum rights. 2010 nov 10;10(1):1–11. https://doi.org/10.1186/1472-698x-10-27 chipofya e. international federation of clinical chemistry and laboratory medicine. n° 1/2 – january/february 2021 [cited 2022 may 14]. 2021; p. 33–35. available from: https://www.ifcc.org/ifcc-news/2010-2021-archive/2021-archive/2021-02-10-ifcc-enews-no-1-2-january-february-2021/ chidzaye rw. assessing barriers to medical laboratory diagnostic service delivery in mzuzu city. int j biomed sci [cited 2022 may 14]. 2019;15(1):32–56. available from: http://www.embase.com/search/results?subaction=viewrecord&from=export&id=l2001804557 bossuyt x, verweire k, blanckaert n. laboratory medicine: challenges and opportunities. clin chem. 2007 oct 1;53(10):1730–1733. https://doi.org/10.1373/clinchem.2007.093989 abstract introduction methods results discussion acknowledgements references about the author(s) nalia ismael instituto nacional de saúde, maputo, mozambique orvalho augusto faculty of medicine, eduardo mondlane university, maputo, mozambique adolfo vubil instituto nacional de saúde, maputo, mozambique sofia o. viegas instituto nacional de saúde, maputo, mozambique fernanda miambo instituto nacional de saúde, maputo, mozambique instituto superior de ciência e tecnologia de moçambiq, maputo, mozambique patrina chongo instituto nacional de saúde, maputo, mozambique nédio mabunda instituto nacional de saúde, maputo, mozambique citation ismael n, augusto o, vubil a, et al. external quality assessment programme for early infant diagnosis of hiv-1, mozambique, 2011–2014. afr j lab med. 2018;7(1), a664. https://doi.org/10.4102/ajlm.v7i1.664 brief report external quality assessment programme for early infant diagnosis of hiv-1, mozambique, 2011–2014 nalia ismael, orvalho augusto, adolfo vubil, sofia o. viegas, fernanda miambo, patrina chongo, nédio mabunda received: 01 aug. 2017; accepted: 09 apr. 2018; published: 11 oct. 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract this study evaluated a national external quality scheme program for early infant diagnosis of hiv. fourteen laboratory technicians participated and nine testing panel cycles were sent between 2011 and 2014. the response rate was 100% for the first eight panels, and the number of technicians with a test score of 100% increased during the first three panels. based on the evaluations of the technicians, the quality of testing for early infant diagnosis of hiv improved over time in the laboratories. introduction early infant diagnosis (eid) of hiv infection assures early access to antiretroviral therapy for infected children, which significantly improves survival rates and provides substantial benefits.1,2 the antibody-specific methods, such as enzyme immunological assays used to diagnose hiv infection in adults, are not recommended for infant diagnosis, due to the passive transfer of antibodies by the mother to the baby in the uterus until the age of 18 months. nucleic acid tests such as hiv rna and dna polymerase chain reaction (pcr) assays are recommended to diagnose hiv in newborns.3,4 in settings that lack adequate infrastructure and cold-chain transportation systems to process whole blood, dried blood spot (dbs) samples offers many advantages. dried blood spot testing has simplified hiv screening in newborns, because samples are easy to collect often via heel prick or finger stick and no cold chain transportation system is required as dbs samples are stable at room temperature.5,6,7,8,9,10,11 participation in an external quality assurance (eqa) programme has been shown to detect possible errors during testing, trace possible corrective actions and improve the quality of testing, and is thus an important tool for laboratory quality assurance.12,13,14,15,16,17,18 available molecular tests for newborns have high sensitivity and specificity when performed correctly but when the procedures recommended by the manufacturers are not correctly performed it can result in incorrect results, which has serious consequences. incorrect results may lead to wrong clinical follow-up and a delay in treatment initiation.19,20 to ensure the accuracy and reliability of laboratory test results, participation in an eqa programme is crucial.21 since 2006, mozambique has been using pcr-based methods with dbs samples to diagnose hiv in exposed newborns. in 2011, the instituto nacional de saúde of mozambique, through the national eqa program, introduced a voluntary eqa scheme designated for each laboratory technician performing pcr-based eid on dbs samples. this report describes the results of four years of evaluation of the national eqa program scheme in mozambique. methods ethical considerations the ethics committee of the instituto nacional de saúde, maputo, mozambique approved the study (study number: 162/cibs-ins/2017). implementation of external quality assurance hiv-1 dna programme between 2011 and 2014, all laboratory technicians performing eid for hiv using dbs were informed about the availability of proficiency testing panels provided by the national eqa program of mozambique and were encouraged to enroll and participate. the panels were provided free of charge and sent to each technician for testing within four weeks of receipt. to maintain confidentiality, a unique code was assigned to each technician. panel description and preparation the proficiency testing panels consisted of 20 blinded dbs samples a mixture of negative and positive samples. negative dbs samples were prepared from a negative blood bag collected by the blood bank at hospital central de maputo. positive dbs samples were prepared from an hiv-positive individual’s blood used for cd4 testing at the cellular immunology laboratory in the same hospital. the positive and negative samples were retested using the amplicor hiv-1 dna pcr test, version 1.5 (roche molecular systems, branchburg, new jersey, united states), according the manufacturer’s instructions. proficiency panel description and sample organisation four series were prepared, and each series consisted of 20 blinded dbs samples in a different sample arrangement. negative and positive specimens were prepared separately, dried overnight, then encoded with a sample and serial number. each series contained 20 dbs specimens packed into a sealable plastic storage bag stored at 2°c – 8°c until shipment. all four series were sent to each laboratory, and each laboratory technician had to test one of the series sent. panel validation to validate the panel, each series was selected randomly and all 20 dbs samples within the series were retested using the amplicor hiv-1 dna pcr test, version 1.5 (roche, germany) according to the manufacturer´s instructions, by two different technicians. the results were crosschecked and both results had to match. following validation, the proficiency panels were sent and made available to all the laboratories across the country performing eid diagnosis. sample shipment, analysis, results and reporting a carrier company shipped the proficiency panels at room temperature three times during 2011 and twice between 2012 and 2014. the technicians had to report the results within 30 days after panel reception. all technicians that did not report their results within 30 days after receiving the panels were considered to be non-responders. test scores were reported in percentages according to the number of correct results out of the 20 tests per cycle. technicians were evaluated according to the concordance of their results with expected results established previously. after each panel, each technician was sent a report containing (1) the test scores (percentage of concordant results), (2) reported results, as well as expected results and possible causes in cases of discordant results. for technicians with results below 100%, corrective actions were conducted, including technical support through laboratory visits, emails or telephone calls. statistical analysis frequencies and proportions were used to describe the data. trends between 2011 and 2014 were studied by means of graphs or tables. to study the association between the proportion of technicians scoring 100% on a panel and concordance among technicians, we use a beta-binomial regression with a logit link. we chose this regression to keep the fitted concordance within the 0 to 1 range and to respect the uncaptured geographic heterogeneity (over dispersion) of the data. based on the akaike information criterion and bayesian information criterion, the proportion of technicians scoring 100% was included as linear and quadratic transformed. the quadratic terms were included to detect u-shaped relationships in the data. stata 14 (statacorp. 2015. stata: release 14. statistical software; statacorp lp, college station, texas, united states) was used to perform all analysis. results from 2011 to 2014, nine proficiency panels were sent to each technician. the overall number of technicians that participated increased from 10 in 2011 to 12 in 2014 (table 1). during the course of the study, there were some withdrawals and replacements for all laboratories with the exception of laboratory a, where the same technicians were evaluated during the whole course of the study (table 1). for all panels, results were sent within 30 days of receipt of the panels (78.5% – 100%; average: 97.6%), with the exception of one test panel (2014-b) because three technicians did not respond. table 1: overall number of technicians that participated in the national external quality assessment program for polymerase chain reaction of hiv dna across the nine panels, mozambique, 2011–2014. for laboratory d, 18 errors were observed, and 100% of the technicians had at least one error during the nine panels (table 2). laboratory b had the least errors with 2, followed by laboratory c with 10. at least one error occurred for 16.6% of technicians in laboratory b and 75% of technicians in laboratory c. laboratory a had the second-highest number of errors and 20% of technicians had at least one error. table 2: error characterisation of technicians per laboratory during the nine cycles. the number of false positives increased during the first three panels, while no false positives were observed during the last three panels (2013-b, 2014-a and 2014-b) (figure 1). on the other hand, the number of false negatives was constant for panels 2011-a, 2011-b, 2011-c, 2013-b, 2014-a and 2014-b. invalid results were observed during the second panels and re-emerged during the last panel (2014-b). during the first three panels, the proportion of technicians scoring 100% increased, but decreased by 2% during the last two panels (figure 2). figure 1: percentage of discordant results among all participating technicians. discordant results included false positives, false negatives and invalids. figure 2: percentage of technicians scoring 100% versus their concordance during each test panel from 2011–2014. the line is a fit of a beta-binomial regression with linear and quadratic terms of technicians scoring 100%. discussion during the expansion of eid testing and treatment programmes in mozambique, it was necessary to optimise the quality testing for hiv dna pcr. we report the successful implementation of the hiv-1 eid eqa program in mozambique during the period of 2011 to 2014. our results show that the quality testing slightly improved over time after the eid eqa program was implemented. if progress is to be maintained, eid eqa program should be sustained. the number of technicians participating increased during the first eight panels of the programme, with a response rate of 100%, suggesting a positive response and growing interest in the programme. initially, training on eid for hiv conducted by the laboratory that provided the proficiency testing panels and the clear explanation of the objectives and benefits of the programme to its participants may have accounted for the increased participation. for each panel, technicians with discordant results had direct site supervision and indirect technical support (via email or telephone). the decrease in the response rate during the last panel was most likely related to annual leave during the period that the panel was sent. as the number of samples tested in the country for eid hiv-1 infection increased, the number of technicians participating in the proficiency testing increased over time. although corrective actions such as technical support, training and discussions with the laboratory manager were performed, the number of errors for laboratory d, which accounted for most of the errors during the nine panels, did not decrease. for laboratory c, most technicians had at least one error over the nine panels. during the retraining and visits, simplification of the procedures was observed, because of the large number of samples being tested. however, for laboratory a, retraining a technician who was observed making errors helped to overcome some practical issues. corrective actions after each eqa panel and retraining of all technicians contributed to a significant reduction of errors over time. technical assistance decreased in 2013 because of logistical problems faced by the reference laboratory. as a consequence, the number of errors during this period increased, showing the importance of technical assistance to ensure quality testing. these results reinforce observations made by other studies that training and experience significantly affects the accuracy of proficiency panel testing over time.22,23,24 furthermore, benefits such as relationship strengthening and communication improvement within the laboratories was observed after the implementation of this eqa programme, as observed in other studies.25 one of the limitations of our study is that we were not able to follow up with the technicians during all the panels and new technicians were enrolled later in the programme, which might have contributed to the increased number of errors. no sociodemographic data, such as gender, education or how long the technician had been familiar with the procedure and number of panels tested were collected. thus, our study could not assess the association of such factors with errors observed over the time that the programme was evaluated. a continuous national quality assessment control programme that includes international panels is important for control of the analytical aspects of eid testing for hiv. this eqa programme ensures quality testing at both the technician and laboratory levels. as indicated by this evaluation, improvement in the quality of testing for eid of hiv for the laboratories from 2011 to 2014 was observed. our results show that both the national eqa program for eid of hiv testing model of evaluating technicians, as well as the laboratory, are successful in ensuring the quality of results. acknowledgements the authors would like to thank all the laboratories that participated in the national quality scheme program for early infant diagnosis of hiv. in addition, they would also like to thank the staff members of the molecular virology laboratory and the national programme of external quality scheme of the instituto nacional de saúde of mozambique. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. sources of support none. authors’ contributions n.i. supported the planning and implementation of the programme and wrote the manuscript; o.a. was responsible for the statistical analysis, and revised the manuscript; a.v. and s.o.v. supported the implementation of the programme and made conceptual contributions; f.m. performed the data analysis; p.c. and n.m led the implementation of the programme and reviewed the manuscript. references berk dr, falkovitz-halpern ms, hill dw, albin c, arrieta a, bork jm, et al. temporal trends in early clinical manifestations of perinatal hiv infection in a population-based cohort. jama. 2005 may 11;293(18):2221–31. violari a, cotton mf, gibb dm, babiker ag, steyn j, madhi sa, et al. early antiretroviral therapy and mortality among hiv-infected infants. n engl j med. 2008 nov 20;359(21):2233–44. jani iv, sabatier j, vubil a, subbarao s, bila d, de sousa a, et al. evaluation of a high-throughput diagnostic system for detection of hiv-1 in dried blood spot samples from infants in mozambique. j clin microbiol. apr;50(4):1458–60. stevens w, sherman g, downing r, parsons lm, ou cy, crowley s, et al. role of the laboratory in ensuring global access to arv treatment for hiv-infected children: consensus statement on the performance of laboratory assays for early infant diagnosis. open aids j. 2008;2:17–25. sherman gg, stevens g, jones sa, horsfield p, stevens ws. dried blood spots improve access to hiv diagnosis and care for infants in low-resource settings. j acquir immune defic syndr. 2005 apr 15;38(5):615–7. nyambi pn, fransen k, de beenhouwer h, chomba en, temmerman m, ndinya-achola jo, et al. detection of human immunodeficiency virus type 1 (hiv-1) in heel prick blood on filter paper from children born to hiv-1-seropositive mothers. j clin microbiol. 1994 nov;32(11):2858–60. cassol s, salas t, gill mj, montpetit m, rudnik j, sy ct, et al. stability of dried blood spot specimens for detection of human immunodeficiency virus dna by polymerase chain reaction. j clin microbiol. 1992 dec;30(12):3039–42. cassol sa, lapointe n, salas t, hankins c, arella m, fauvel m, et al. diagnosis of vertical hiv-1 transmission using the polymerase chain reaction and dried blood spot specimens. j acquir immune defic syndr. 1992;5(2):113–9. biggar rj, miley w, miotti p, taha te, butcher a, spadoro j, et al. blood collection on filter paper: a practical approach to sample collection for studies of perinatal hiv transmission. j acquir immune defic syndr hum retrovirol. 1997 apr 1;14(4):368–73. comeau am, pitt j, hillyer gv, landesman s, bremer j, chang bh, et al. early detection of human immunodeficiency virus on dried blood spot specimens: sensitivity across serial specimens. women and infants transmission study group. j pediatr. 1996 jul;129(1):111–8. boyle ds, lehman da, lillis l, peterson d, singhal m, armes n, et al. rapid detection of hiv-1 proviral dna for early infant diagnosis using recombinase polymerase amplification. mbio. apr 2;4(2). hannon wh, lewis ds, jones wk, powell mk. a quality assurance program for human immunodeficiency virus seropositivity screening of dried-blood spot specimens. infect control hosp epidemiol. 1989 jan;10(1):8–13. hofherr lk, peddecord km, benenson as, garfein rs, francis dp, ferran kl, et al. methods for a model blind proficiency testing system. clin lab sci. 1992 may-jun;5(3):160–4. jackson jb, drew j, lin hj, otto p, bremer jw, hollinger fb, et al. establishment of a quality assurance program for human immunodeficiency virus type 1 dna polymerase 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hiv rapid testing in developing countries: comprehensive approach for implementing quality assurance. am j clin pathol. oct;134(4):573–84. mashauri fm, siza je, temu mm, mngara jt, kishamawe c, changalucha jm. assessment of quality assurance in hiv testing in health facilities in lake victoria zone, tanzania. tanzan health res bull. 2007 may;9(2):110–4. hertzberg ms, mammen j, mccraw a, nair sc, srivastava a. achieving and maintaining quality in the laboratory. haemophilia. 2006 jul;12 suppl 3:61–7. learmonth km, mcphee da, jardine dk, walker sk, aye tt, dax em. assessing proficiency of interpretation of rapid human immunodeficiency virus assays in nonlaboratory settings: ensuring quality of testing. j clin microbiol. 2008 may;46(5):1692–7. peeling rw, smith pg, bossuyt pm. a guide for diagnostic evaluations. nat rev microbiol. 2006 dec;4(12 suppl):s2–6 chang d, learmonth k, dax em. hiv testing in 2006: issues and methods. expert rev anti infect ther. 2006 aug;4(4):565–82. chalermchan w, pitak s, sungkawasee s. evaluation of thailand national external quality assessment on hiv testing. int j health care qual assur. 2007;20(2–3):130–40. abstract introduction methods results discussion acknowledgements references about the author(s) leonard mutema department of haematological pathology, tygerberg hospital, national health laboratory service and stellenbosch university, cape town, south africa department of internal medicine, university of kwazulu-natal, durban, south africa zivanai chapanduka department of haematological pathology, tygerberg hospital, national health laboratory service and stellenbosch university, cape town, south africa fungai musaigwa department of haematological pathology, tygerberg hospital, national health laboratory service and stellenbosch university, cape town, south africa nomusa mashigo department of haematological pathology, tygerberg hospital, national health laboratory service and stellenbosch university, cape town, south africa citation mutema l, chapanduka z, musaigwa f, mashigo n. in-depth investigation of turn-around time of full blood count tests requested from a clinical haematology outpatient department in cape town, south africa. afr j lab med. 2021;10(1), a1318. https://doi.org/10.4102/ajlm.v10i1.1318 original research in-depth investigation of turn-around time of full blood count tests requested from a clinical haematology outpatient department in cape town, south africa leonard mutema, zivanai chapanduka, fungai musaigwa, nomusa mashigo received: 27 june 2020; accepted: 06 jan. 2021; published: 29 apr. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the performance of laboratories can be objectively assessed using the overall turn-around time (tat). however, tat is defined differently by the laboratory and clinicians; therefore, it is important to determine the contribution of all the different components making up the laboratory test cycle. objective: we carried out a retrospective analysis of the tat of full blood count tests requested from the haematology outpatient department at tygerberg academic hospital in cape town, south africa, with an aim to assess laboratory performance and to identify critical steps influencing tat. methods: a retrospective audit was carried out, focused on the full blood count tests from the haematology outpatient department within a period of 3 months between 01 february and 30 april 2018. data was extracted from the national health laboratory service laboratory information system. the time intervals of all the phases of the test cycle were determined and total tat and within-laboratory (intra-lab) tat were calculated. results: a total of 1176 tests were analysed. the total tat median was 275 (interquartile range [iqr] 200.0–1537.7) min with the most prolonged phase being from authorisation to review by clinicians (median 114 min; iqr: 37.0–1338.5 min). the median intra-lab tat was 55 (iqr 40–81) min and 90% of the samples were processed in the laboratory within 134 min of registration. conclusion: our findings showed that the intra-lab tat was within the set internal benchmark of 3 h. operational phases that were independent of the laboratory processes contributed the most to total tat. keywords: audit; turn-around time; full blood count; haematology clinic. introduction the increase in the number of patients accessing the public health system and the disproportionate patient to healthcare provider ratio in developing countries necessitates more efficient and cost-effective approaches to healthcare provision.1,2 the efficiency of healthcare providers is evaluated in part by how rapidly diagnoses are made and patients are prioritised for treatment.3 in addition, the rate at which laboratory results are made accessible to clinicians impacts both the patient outcome and the overall performance of a diagnostic laboratory.4 a prolonged turn-around time (tat) results in delayed diagnosis and impacts the management of patients. this may lead to prolonged hospital stay and increased costs.5,6 the consequences of prolonged tat are more pronounced in an emergency service setting and in outpatient departments, where a narrow window of opportunity exists to make key management decisions.7 therefore, constant monitoring and improvement of the tat in this setting is important to ensure the efficiency of laboratory services.8 the tat is crucial in clinical practice and is used as a measure of the performance of laboratories.5 however, the tat definition often varies between laboratories and clinicians, often resulting in unrealistic expectations.5,6,9,10 total tat is categorised into preanalytical, analytical and post-analytical stages and some define it as time from collection of the sample to when results are available for review by clinicians.9 clinicians tend to view tat as the time between requisition of a test and when they view a result.11 laboratories are inclined to exclude components of the test cycle that they have no control over, such as phlebotomy and specimen transport12,13 because it is difficult for laboratories to address delays in these phases.5 therefore, in laboratories, the tat is usually defined as a measure of the time taken from sample registration to authorisation of results, also known as intra-lab tat.5 although this approach is not all inclusive of the phases of total tat, it is generally accepted as the best representation of those elements of tat that the laboratory controls.12 to assess the hypothesis that our laboratory performance was within set local benchmarks and comparable with the widely accepted international benchmark of completion of 90% of the sample processing within 60 min, we performed a retrospective analysis to evaluate the respective contribution of the various phases of the test cycle to the total tat at tygerberg academic hospital (tbh) in cape town, south africa.14 methods ethical considerations this retrospective study was approved by the stellenbosch university research ethics committee (s18/10/232) and performed according to the declaration of helsinki. a waiver of consent was obtained, and patient confidentiality was maintained. moreover, patient-level data was not accessed. study design and setting a retrospective audit was conducted over a 3-month period between 01 february and 30 april 2018 at the haematological pathology laboratory of the tertiary referral tbh in cape town, south africa. the laboratory and its reception operate on a 24-h service and have three shifts: regular shift (08:00 to 16:30), evening shift (16:30 to 20:00) and night shift (20:00 to 08:00). the laboratory provides diagnostic pathology services to regional hospitals and clinics in the western cape, south africa. at tbh laboratory reception, samples go through the processes of sorting, registration and labelling before being transported to the haematology laboratory for analysis where samples are loaded into a siemens advia 2120i haematology analyser (siemens healthcare diagnostics, erlangen, germany). this analyser has the capacity of performing 120 full blood counts (fbcs) and differentials per hour. from the haematology analyser the preliminary results are entered into the laboratory information system (lis) at which point the results are available to clinicians as provisional results. the laboratory fast-tracks all urgent samples and aims to release the results within 3 h. samples from the haematology clinic are treated as urgent and are colour coded and given preference over other routine samples. the lis (intersystems trakcare® lab enterprise, cambridge, massachusetts, united states) tracks the processing of samples at various time points (preanalytical, analytical, and post-analytical). the haematology clinic is housed in a separate building from the main building that houses the haematology laboratory. at this clinic, tests are requested through paper forms which are then placed alongside the samples into transporting bags and transported to the laboratory reception by porters or, rarely, by doctors or nurses. the clinic operates monday to friday from 07:00 to 16:00. clinicians start seeing patients at 08:00 and most of the patient consultations are usually complete by 14:00, after which time the clinicians leave for the ward. patients who arrive late are seen by one clinician until 16:00 when the clinic closes. data collection we retrieved and extracted data from the lis to determine the tat reflecting all the operational phases. reports with two or more missing entries of the date or time points on the lis were excluded. full blood counts done as part of bone marrow examination requests were also excluded as they are not reported separately but as part of the full bone marrow report. four main phases of the test cycles were identified and evaluated. the first phase is between collection of samples (phlebotomy) and registration of the same in the laboratory. this is followed by the phase between registration of samples, processing by the haematology analyser, and loading of preliminary results into the lis. the third phase is when preliminary results are reviewed by laboratory staff and then authorised, and the final phase is from authorisation of results to when clinicians view these results. in this study, the total tat was defined as the time taken from specimen collection to the review of the results by the requesting clinician, while the intra-laboratory tat was defined as the time taken from specimen registration to authorisation of results after review of preliminary results by the laboratory staff. total tat was therefore calculated, for each sample, by adding the time taken in the four phases which are: collection to registration, registration to acquisition into lis, acquisition into lis to authorisation and, finally, authorisation to review by clinicians. intra-lab tat was calculated by adding the time taken in the two phases which are: registration to acquisition into lis and acquisition into lis to authorisation of results. we also carried out a sub-analysis of samples collected in the month of april. the operation phase from the collection of samples to registration was divided into two phases: transportation time (collection to receipt in the laboratory) and sorting time (from receipt in the laboratory to registration of samples). the relative contribution of these two phases were determined and compared to that of a similar study.12 statistical analysis data items which included sample collection, registration, acquisition to lis, authorisation and review by clinicians were extracted from the lis onto microsoft® office excel (redmond, washington, united states) 2016 (version 16.0). assessment of normality was done using the kolmogorov-smirnov test at p < 0.05 significance level.15 descriptive statistics, including median, percentages and interquartile range (iqr) were used. intra-lab and total tat were expressed as median with iqr and time to complete 90% of tests (90% completion time).16 all time intervals were calculated in minutes. the chi-square test was used to compare the expected total tat with the observed total tat and to compare the sorting time of our laboratory with that of a study carried out at new york presbyterian hospital12; values of p < 0.05 were considered statistically significant. using the percentage contribution of each phase for all 1176 samples, median percentages were calculated. the same was done for the intra-lab tat for each of the 1176 samples. all data analyses were performed using statistica software version 8 (tibco software inc., palo alto, california, united states). results a total of 1505 fbcs were requested between 01 february and 30 april 2018 from the haematology clinic, tbh, cape town. we performed an analysis on a subset of the retrieved fbc reports over the study period. a total of 329 (22%) patient reports were excluded either due to missing data (2 or more missing data points) or being reports for fbcs requested as part of a bone marrow biopsy (figure 1). in all, this retrospective study included a total of 1176 fbc reports. delays in registration of samples after receipt by the laboratory would not be reflected on the intra-laboratory tat but on the total tat. therefore, a sub-analysis of the phase between collection and registration was performed for the month of april 2018. this comprised 412 samples for the phase between collection of samples to receipt in the laboratory and 420 samples for the phase from receipt to registration. seventy percent of the 1176 samples were collected by 9:00 and 90% by 11.00. considering the closing time of the clinic, the clinicians had a window of 7 h and 5 h to receive results and make clinical decisions for 70% and 90% of patients. figure 1: screening and selection of reports of full blood counts requested from the haematology outpatient department at tygerberg academic hospital in cape town, south africa, over the period february 2018 to april 2018. intra-laboratory turn-around time and total turn-around time the majority of the fbc samples (93.3%) had an intra-lab tat of less than 3 h and most of them (80.1%) were processed within 90 min (table 1). the median intra-lab tat was 55 (iqr 40–81) min and had a median percentage of 17.1% of the total tat (table 2). ninety percent of the results were ready for viewing by clinicians within 134 min of registration. the median total tat was 275 (iqr 200.0–1537.7) min. the phases within the preanalytical and post-analytical phases contribute the most to the total tat, with collection time to registration and time to review results by clinicians representing a median of 32.5% (iqr 7.8–48.7) and 44.3% (iqr 18.1–87.0). the total tat was significantly delayed compared to the calculated expected tat (p < 0.05). table 1: contribution of the different phases of sample processing to total turn-around time and intra-lab turn-around time. table 2: summary of turn-around times across phases of workflow for 1176 full blood count requests from the haematology outpatient department at tygerberg academic hospital in cape town, south africa, over the period february 2018 to april 2018. preanalytical phase the preanalytical phase, defined in this study as the time from collection of samples to registration, had a median of 96 min (table 2). ninety percent of the samples completed this phase within 218 min. a sub-analysis of this phase during the month of april revealed a median of 60 (iqr 45–85) min for the period between collection of the sample to receipt in the laboratory (transportation) and a median of 39 (iqr 23.35–58.00) min from receipt to registration (sorting) (table 3). most of the samples (90%) spent less than 95 min in the sorting area. our laboratory performed significantly worse when we compared the time spent in the sorting area for each sample in april with the median of 15 min at new york presbyterian hospital, another tertiary hospital described in literature, using the chi-square test.12 table 3: summary of phases between collection to registration of full blood count samples at the haematology outpatient department for the month of april 2018 at tygerberg academic hospital in cape town, south africa. analytical phases we categorised the analytical phase into two independent intervals (interval 2 and interval 3) (table 1). our analysis showed that most samples (73.3%) spent less than 60 min in interval 2 while 85.2% of samples were authorised within 30 min in interval 3. notably, a few samples, 26.6 % and 6.3%, took more than 1 h in interval 2 and interval 3. the authorisation of reports is also impacted by the battery of laboratory tests requested and this may explain the minority of samples authorised after an hour. post-analytical phase we further evaluated the time taken by clinicians to review the authorised reports (interval 4). we observed a significant number of reports (48.9%) that were accessed after 2 h, while only 22.7% of the reports were viewed within 30 min (table 1). notably, 38.6% of reports were viewed after 4 h. the median time for this phase was 114 (iqr 37.0–1338.5) min and the time to view 90% of the reports was 10.78 days (table 2). discussion the constant increase in medical care cost and the growing demand for improved quality of care has necessitated the monitoring of determinants of the quality of healthcare services.17,18 the tat is one of the determinants that is commonly measured, as it is both objective and easily measured.19 however, different views exist regarding the definition of what the composition of the tat should be. these are often compounded by the lack of an interface for dialogue between the laboratory staff and clinicians. howanitz et al. showed that there was also no agreement among clinicians as to the definition of tat, with 56% using test requisition as the first step of tat, while 44% used the time of phlebotomy as the initial step. although most studies use reporting of results as the endpoint of tat,17 this study defined total tat as the time period from the time of phlebotomy to the time the results are reviewed by clinicians, whereas we considered the intra-laboratory tat as the time from registration of the sample to reporting of results (authorisation). this was done in efforts to determine the relative contribution of different phases of the laboratory process to tat and to identify phases requiring the most attention in alleviating delays. this study showed that the phases outside of the laboratory environment needed the most attention and that the laboratory was meeting its predetermined mandate. samples from the haematology outpatient department at tbh are regarded as urgent because for most patients, fbcs are required to make decisions on chemotherapy, transfusion and other interventions before leaving the clinic. the results of this study were therefore compared to findings of studies on tat of emergency departments. our findings were similar to those previously reported.5,20 the preanalytical and post-analytical phases accounted for a greater proportion of the total tat, with a median of 60 min transportation time (preanalytical phase) and 48.9% of the post-analytical phase taking more than 120 min. in contrast, most of the samples were processed in the laboratory within 90 min. the median intra-lab tat was 55 (iqr 40–81) min and 90% of samples were authorised within 134 min. therefore, our findings suggest that the laboratory is performing within the set threshold of 3 h as stated by the tbh laboratory standard operating procedure hae1813 for tests from the haematology clinic. this highlights the need to further interrogate the phases that the laboratory has no control over, that is, the preanalytical and post-analytical phases. in addition to the lack of consensus on the definition of tat, there is also no specific benchmark for tat parameters.21 however, databases used to benchmark tat thresholds can be obtained from the college of american pathologists q-probes and q-tracks programmes.16 we therefore used these databases and experiences from other international hospitals to assess our performance. the 1998 college of american pathologists q-probes study of emergency department tats showed a 90% completion time for haemoglobin testing of 55 min or less.22 in this study 90% completion time of 60 min or less is recommended,22 as opposed to that of tbh which is 134 min. in a survey carried out in nigeria involving 109 doctors, 91.3% of the respondents considered a tat of less than 2 h as ideal for their laboratory, although their median tat in emergency rooms was 5.12 h.23 in a chinese national survey the median intra-lab tat for white blood cells was 44.7 min.24 although our laboratory performance was within the local threshold, our laboratory performed below the expectations when compared to other laboratories in a similar setting.5,7,23,24 the april sub-analysis exposed the sorting area as a problematic area that is responsible for prolongation of the intra-lab tat and ultimately total tat. few studies focus on the analysis of this phase as the time of receipt of samples in the laboratory is often missing; therefore, the study at new york presbyterian hospital, which analysed total tat with emphasis on quantification of sorting time, was best suited for comparison with this study.12 as with this study, sorting time was also prolonged at new york presbyterian hospital. opening of sample bags, generation and printing of barcodes and decoding handwritten requests were responsible for the delays in the sorting area, which is also partly the case at tbh.12 electronic requesting of tests as well as the generation of barcodes at the time of requesting tests were suggested as solutions to this delay.12 total tat was significantly prolonged compared to the expected tat, with phases outside the laboratory causing the most delay. the time taken to view results by clinicians was the longest with a median time of 114 (iqr 37.0–1338.5) min. this could be due to the limited period during which clinicians can view results. once this window has elapsed the results can only be viewed on the patient’s next appointment which can vary from a day to several months later. this is a limitation of total tat in assessing laboratory performance, which explains in part why laboratories resort to the use of intra-lab tat.12 these delays can be improved by ensuring patients’ punctuality and having the laboratory contact the clinicians with results as soon as they are authorised. the other cause of delay for total tat was the time taken to transport samples from the clinic to the laboratory as well as sorting time. porters tend to wait for samples to pile up before transporting them. having a set time for phlebotomy and ensuring that patients avail themselves at this time may mitigate this problem. in addition, the use of pneumatic tube systems results in faster and reliable transportation of samples.25,26 as it is noted that a pragmatic approach is to set tat goals locally, informed by both the published literature and by local expectations,5 we made the following recommendations: establishment of point-of-care testing at the haematology clinic, employment of additional staff for the sorting area or the use of an automated barcode system, and the use of pneumatic tube systems. we also recommend the use of an interactive tat dashboard, a recently described system that offers information to enable the review of performance in real time.27 this may help in ensuring timely response to changes in performance. however, solutions requiring extra funding may take time to implement due to economic challenges facing the health sector in south africa.2,28 as noted earlier, some results are only reviewed on the patient’s next visit and this may pose serious challenges to patient care. therefore, the use of other communication avenues such as short message services, whatsapp, or emails to send authorised results to clinicians is recommended. not only will this improve the total tat, but it will also allow clinicians to act on results immediately rather than waiting for the patient’s next appointment. another suggestion is for clinicians to employ a clerk to review results even after the clinicians have left the clinic. for the laboratory, we recommend that considerations be made to incorporate phlebotomy and specimen transportation services into its armamentarium of service delivery. this will increase laboratory influence on preanalytical processes and subsequently improve total tat. limitations this study was limited by the absence of an interventional process, and we therefore recommend a follow-up audit after implementing some of the proposed solutions. the results from this study cannot be extrapolated to all hospitals and departments but only to those with a similar setting, as they are solely based on the experience of the haematology clinic at tbh. the collection times were based on what was reported by the nurses at the clinic; therefore, the accuracy of the recorded time following each phlebotomy cannot be ascertained. this is not the case for other time points retrieved from the lis as they are computer generated in real time. in addition, the laboratory receipt time for samples was not available for the entire study period. as noted earlier, 22% of the data had 2 or more missing data points which were critical in the calculation of intra-lab tat and total tat. this has the potential of introducing bias, which is likely if the proportion of missing data is greater than 10%.29 these missing data points were random occurrences on the extracted lis data and tats could not be computed; therefore, the samples were removed in a non-biased manner by listwise deletion. due to the random nature of these missing points, a case is therefore made that the generalisability of the study is not affected. the statistical method of handling missing data used in this case was complete-case analysis, which only includes participants or variables that are complete on all waves of data collection.30 conclusion this study demonstrated that tbh laboratory was compliant with its intra-lab tat benchmark and that the 90% completion time target was achieved for samples from the haematology outpatient clinic. the phases outside the control of the laboratory were primarily responsible for prolonged total tats. the monitoring of tat is a powerful tool for assessing a laboratory service’s performance and contributes to patient care. however, as demonstrated in this study, monitoring and process improvement requires measurement of tats for individual phases of the test cycle. acknowledgements the authors would like to thank ms fazlin kolia, the laboratory manager, for providing us with permission to use lis data and standard operation procedures. we also would like to extend our gratitude to mr wessel kleinhans for his critical role in data mining from the lis. lastly, we would like to thank prof. bongani nkambule for assistance in analysis of the data. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions n.m. and z.c. supervised the study by providing leadership and oversight. l.m. conceptualised the study, conducted the research and wrote the initial manuscript. f.m. and l.m. conducted data analysis. all authors contributed to final manuscript development. sources of support the authors received no financial support for the research, authorship or publication of this article. data 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chika k. onwuamah1 olumuyiwa b. salu1 fehintola a. ige1 emily meshack1 maureen aniedobe1 olufemi s. amoo1 azuka p. okwuraiwe1 florence okhiku1 chika l. okoli1 emmanuel o. fasela1 ebenezer. o. odewale1 roseline o. aleshinloye1 micheal olatunji1 emmanuel o. idigbe1 affiliations: 1human virology laboratory, nigerian institute of medical research, lagos, nigeria correspondence to: rosemary audu postal address: nigerian institute of medical research, 6, edmond crescent, p.m.b. 2013, yaba, lagos, nigeria dates: received: 09 dec. 2011 accepted: 05 sept. 2012 published: 29 oct. 2012 how to cite this article: audu ra, sylvester-ikondu u , onwuamah ck, et al. experience of quality management system in a clinical laboratory in nigeria. afr j lab med. 2012;1(1), art. #18, 5 pages. http://dx.doi.org/10.4102/ ajlm.v1i1.18 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. experience of quality management system in a clinical laboratory in nigeria in this lessons from the field... open access • abstract • introduction • description • lessons learned • recommendation • acknowledgement    • competing interests    • authors’ contributions • references abstract top ↑ issues: quality-management systems (qms) are uncommon in clinical laboratories in nigeria, and until recently, none of the nation’s 5 349 clinical laboratories have been able to attain the certifications necessary to begin the process of attaining international accreditation. nigeria’s human virology laboratory (hvl), however, began implementation of a qms in 2006, and in 2008 it was determined that the laboratory conformed to the requirements of iso 9001:2000 (now 2008), making it the first diagnostic laboratory to be certified in nigeria. the hvl has now applied for the world health organization (who) accreditation preparedness scheme. the experience of the qms implementation process and the lessons learned therein are shared here.description: in 2005, two personnel from the hvl spent time studying quality systems in a certified clinical laboratory in dakar, senegal. following this peer-to-peer technical assistance, several training sessions were undertaken by hvl staff, a baseline assessment was conducted, and processes were established. the hvl has monitored its quality indicators and conducted internal and external audits; these analyses (from 2007 to 2009) are presented herein. lessons learned: although there was improvement in the pre-analytical and analytical indicators analysed and although data-entry errors decreased in the post-analytical process, the delay in returning laboratory test results increased significantly. there were several factors identified as causes for this delay and all of these have now been addressed except for an identified need for automation of some high-volume assays (currently being negotiated). internal and external audits showed a trend of increasing non-conformities which could be the result of personnel simply becoming lax over time. application for laboratory accreditation, however, could provide the renewed vigour needed to correct these non-conformities. recommendation: this experience shows that sustainability of the qms at present is a cause for concern. however, the tiered system of accreditation being developed by who–afro may act as a driving force to preserve the spirit of continual improvement. introduction top ↑ laboratories play a pivotal role in all disease control and prevention programmes by providing timely and accurate information for patient management and disease surveillance.1 clinical laboratories specifically, provide accurate diagnosis of present, recent or past infections for appropriate case management. 1 though laboratories form the backbone of health systems, providing health care workers with critical test results for numerous deadly diseases, yet in sub-saharan africa, which carries a huge disease burden, laboratories are among the world’s most ill-equipped and poorly resourced facilities. as of 2009, there were 340 diagnostic laboratories in sub-saharan africa that were accredited and 312 of these (92%) were found in south africa.2 in nigeria, there are 5349 diagnostic laboratories, of which only the human virology laboratory (hvl) has iso certification.in 2000, the ford foundation provided a grant to the nigerian institute of medical research for the establishment of a national reference laboratory with capacities for basic, applied and operational research on hiv and aids in the country. the grant, which was worth $250 000, was meant for the construction of a laboratory facility, the purchase of equipment, and training of personnel. subsequently, an additional grant of $150 000 was awarded for two years for sustainability activities, such as revolving funds for purchase of kits and reagents. prior to this, there was no laboratory with facilities for hiv molecular diagnostics in nigeria, requiring clinical samples from people living with hiv to be sent out of the country to laboratories abroad for relevant tests and analyses. with the grant funding, two new large laboratory facilities were built and fully equipped with state-of-the-art equipment for hiv and aids research and services. setting up the quality management system at the hvl involved two major stages, namely, planning and implementation. the planning stage involved identifying and mapping out processes, risk assessment of the processes and appropriate management, as well as identifying quality indicators and setting targets for monitoring. implementing this process approach to management facilitated bottom-up communication, ownership and participation, thereby improving the quality of services provided. in order to meet international standards, the laboratory worked to conform to the requirement of nis iso 9001:2000 and has in fact converted to the iso 9001:2008 standard, making it the first diagnostic laboratory to be so certified in nigeria. the major difference between the two standards is that the iso 9001:2008 standard has a broader interpretation and application of its requirements. because iso 15890 requirements are more relevant to diagnostic laboratories, the hvl has applied for the who accreditation scheme through the efforts of the president’s emergency plan for aids relief (pepfar), which uses this standard. the effort of obtaining certification originated with the then-director general, a fact which facilitated securing resources and approvals. despite the high pre-assessment non-conformities, this ‘buy-in’ from staff at the highest levels of management was key to the success of this endeavour. implementing the qms was not without its challenges, however. one major challenge was getting the other staff to buy into this vision. this was achieved through involving every staff member, regular awareness creation at staff meetings, ensuring good staff morale and recognition of staff for exemplary performance. there is a clearly defined need for attracting and retaining qualified, motivated personnel within the public health sector. in the hvl, the majority of laboratory personnel were either recent graduates completing compulsory service to the nation or on internships with the laboratory and, as such, they typically maintained a high level of performance. a few of the longer-serving staff were determined to be performing at an unsatisfactory level and were posted out of the unit. this reduced the staff to 7 research fellows, 12 laboratory scientists and 12 support staff of varying cadres. an important aspect of addressing shortcomings in staff performance was the requirement that all personnel undergo basic training in performance standards. initially, documentation of non-conformities among staff was challenging as this was frequently misunderstood as a means of ‘reporting an individual’ for an infraction. staff members were informed that this documentation was not for any punitive purpose but was intended as a means of tracking and improving the system. the need to address this misperception among staff has been ongoing. another factor related to laboratory staffing had to be addressed as well. though nigeria, unlike many african nations, is not facing a crisis with respect to shortage of health workers, ‘brain circulation syndrome’ is quite high, with personnel frequently moving from one job to another in search of better wages or conditions of service. the hvl employed a group of highly skilled and dedicated staff who were committed to the vision of adopting the qms and working toward accreditation after having undergone several training sessions on qms. however, because of the hvl’s unique status as a self-sustaining laboratory within the government establishment, personnel were not guaranteed job security and this fact adversely affected the sustainability of the laboratory. moreover, the who recommends training of health workers to ensure quality service delivery and the retention of these trained workers because essential health services cannot be provided by people working on a voluntary basis if the service is to be sustainable. it is also recommended that trained health workers who are providing essential health services receive adequate wages and appropriate and commensurate incentives.3 hence, the need to ‘absorb’ hvl’s staff as permanent ministry of health employees was identified as a solution to this issue. in july 2008, 20 project staff of the hvl were absorbed as permanent government staff and the laboratory was made a unit in the microbiology division of the nigerian institute of medical research (nimr), which is a parastatal institution under the ministry of health. an additional challenge was the lack of a basic quality manual to establish guidelines for the hvl that were codified and easily accessible for personnel. with assistance from a trainer from the standard organisation of nigeria (son), this quality manual was eventually developed and adopted by the laboratory. the quality manual, which was written in line with the requirements of the standard, contained the structure of the quality system and its policies. but because son was not typically involved with developing capacity for qms in clinical settings, this made the overall expenditure on training higher, because assistance had to be sought first from senegal. similarly, the laboratory found it necessary to adopt a quality policy which drives its vision as well as to establish a top management committee, in line with the iso requirement, which serves as the management advisory committee to the laboratory. description top ↑ in november 2005, two personnel from the hvl were sent to the bio 24 laboratory in dakar, senegal, which was an iso 9001:2000 certified clinical laboratory, to study the implementation of a qms there. by july 2006, a consultant from senegal and the laboratory manager from the certified laboratory had trained the hvl staff on a basic implementation course, and a baseline assessment had been conducted. the laboratory then defined its processes and began to implement some basic qms structures such as developing of policies, proper archiving of records, and selection of quality indicators for monitoring the system. we also introduced regular and quality-focused staff meetings, which included training and retraining. by june 2007, the son was identified as an appropriate organisation to train staff on the iso 9000 basic and auditors’ course; 13 staff members qualified as internal auditors as a result of this training. twenty staff members were again trained on iso/iec 17025 in august 2008 and later undertook the iso 15189:2007 laboratory accreditation and auditors’ course in march 2009. both of these training courses were also conducted by the son. the laboratory also successfully completed a revalidation course for iso 9001:2008. though the process of obtaining certification and ultimately accreditation is an expensive one, the hvl project was able to address some of the costs through income generated from the services rendered by the laboratory. because nimr management saw the value of the improvements in laboratory personnel performance, the resources required have been perceived as a worthy investment and provided to continue the process. since 2007, the hvl’s quality indicators have been monitored, customer surveys have been performed, and internal and external audits have been conducted as part of the ongoing process. various methods were used for the customer survey conducted twice a year. the commonest method was the use of questionnaires administered to patients, clinicians and other laboratories that patronised the hvl. these questionnaires were designed and analysed by staff members. other occasional methods used included in-depth interviews with clinicians, focused group discussion with patients and the use of questionnaires administered to suppliers. suggestions from these surveys were used to improve the system. some staff of the laboratory who had qualified as internal auditors carried out internal audits twice a year. they prepared checklists using the iso 9001:2000 standards and the laboratory quality manual; corrective actions were taken for all reported non-conformities. external audits were however carried out twice a year by the standards organisation of nigeria to ensure the qms was still functional. this is a mandatory requirement, if the certification was to be maintained. reports of all of these activities were presented to the management during annual reviews. lessons learned top ↑ by december 2007, the laboratory was deemed to be in compliance with the iso 9001:2000 standard and was awarded its certificate in february 2008. by 2010, the hvl had conformed to iso 9001:2008. the quality indicators for the operational processes and the audit analyses from 2007 to 2009 are presented below. table 1: performance of some indicators in the operational system between 2007 and 2009 for all assays in hvl. table 1 shows the performance of some indicators in the operational system for all assays within the period reported. there was an improvement in the indicators for the pre-analytical and analytical processes over the years. it was observed earlier that there was a gap in awareness on how to collect samples for the various tests that were rendered in the laboratory. continuous education is needed for all stakeholders involved in laboratory testing to improve the quality of the pre-analytical phase of the total-testing process.4 the laboratory prepared a clients’ handbook which contained information on time of operation, test offered, specimen type, test turnaround time, packaging, storage and transportation of samples. this was used to educate clinicians and other laboratory scientists who send samples to hvl. these clients were also invited to the laboratory and were further trained on proper sample collection, storage and transportation. there was a continuous decline in the number of unanalysed samples and external controls exceeding specification, which is a positive development for the qms. westgard rules applied to a levey-jennings chart as practiced in the hvl are programmed to determine when an analytical run should be rejected. these rules are applied carefully so that true errors are detected while false rejections are minimised. the rules applied to high volume chemistry and hematology instruments produce low false rejection rates.5 this was achieved in the hvl; however, values recorded for the external controls were higher than values reported in other studies,6 hence more rigorous monitoring is required. during the monitoring, it was observed that a few personnel still had challenges in using the tool while another staff member had not fully understood the benefit of complying strictly with the rules, hence training and retraining on application and benefits of the westgard rule is now a regular practice in the hvl. there was an improvement in maintaining environmental temperatures within acceptable ranges. scores obtained from participation in external quality assessment were within an acceptable range.an improvement was also observed in the reduction of errors in data entry in the post-analytical process (table 1). the delay in reporting results became significantly higher in 2009 in spite of the fact that implementing the qms is expected to improve turnaround time.7 determining the root cause of this delay in order to eliminate it is a necessary approach.8 several factors contributed to the delay of results such as issues in supply chain management (unavailability of test kits provided by partners), failure of quality control for two assays (westgard rules), network problems (support infrastructure), and staff rotation. another major challenge was the daily manual testing of large numbers of samples for some of the assays. because the unavailability of kits was beyond the control of the laboratory, discussions were held with the supporting partner providing the kits to correct the challenges faced with supplies. there was also a change required in the brand of test kit because of repeated failure of the internal quality control, and an agreement for the maintenance of the database network was signed. in terms of staff rotation, an improved competency evaluation was established. appeals are still being made to the supporting partner for automation of these assays particularly in a reference laboratory such as the hvl. comparison of internal audits by staff and external audits by son over the three years showed an increase in the number of non-conformities, from 157 to 192 for internal audit and 39 to 55 for external audits – perhaps an indication that laboratory personnel were becoming lax in implementing the system. it is presumed that the process of applying for laboratory accreditation with its stringent technical demands could stimulate staff to sustain and even improve the system.9 this is most likely because of the stepwise accreditation preparedness approach of the world health organisation regional office for africa (who-afro). this programme is intended to provide an interim pathway for measuring, monitoring and recognising improvement toward the realization of international laboratory standards and subsequent application to full iso 15189 accreditation.2 recommendation top ↑ for qms to be implemented effectively there must be support from the highest levels of management; therefore strong advocacy to management is a necessary starting point. for laboratories planning to obtain accreditation, considerable training is required and this must be taken into account. automation of laboratory systems has advantages such as reduction in staff time and supply costs, reduced turnaround times, as well as reduction in operational errors .6 therefore it is recommended that clinical laboratories performing high volume testing in africa adopt automation of their systems as they prepare for possible who assisted approach for laboratory accreditation.the hvl audit experience shows there was an improvement in 2008 – the year that certification was obtained – but that, soon after, these gains diminished, suggesting that sustainability will continue to be a major challenge. the who’s accreditation preparedness scheme for african medical laboratories utilises a tiered system under which laboratories complying with the iso 15189 standards are audited and awarded recognition from one to five stars (in the form of a certificate). this may serve to provide the necessary impetus for continual quality improvement for african laboratories. under this program, audits are conducted annually and laboratories that fall behind will have their certificate of recognition withdrawn. only a few of africa’s laboratories are currently accredited to international standards, but, through this five-star accreditation preparedness process, laboratories will be able to be recognised for their improvement efforts using the who afro slipta checklist – and eventually apply for accreditation by a nationally-, regionally-, or internationally-recognised accreditation body. this is critical because good laboratory support leads to better health care delivery through correct diagnoses of diseases and monitoring of drug resistance. this is crucially important in nigeria (and across africa) to monitor patients infected with hiv, tb, malaria, and a host of other diseases. in addition, an efficient laboratory also can dramatically reduce waiting time to get results – allowing patients who often travel a day or more for testing to receive the laboratory results sooner. studies have shown that when patients need to return for a second visit to a hospital or clinic for test results, significant percentages fail to do so.10 in spite of the challenges inherent in this process and the continued need for improvement, the hvl can attest that implementing qms has brought about reliability and reproducibility of results as confirmed by its numerous clients. also there has been a widespread recognition of the laboratory such that corporate and private clients seek services, which has resulted in improved profit. consequently, most of the assays have now been automated. the introduction of the qms in nigerian medical laboratories – and in africa generally – truly represents a major step forward and the beginning of a whole new era for the continent’s public health systems. acknowledgement top ↑ the authors wish to acknowledge the commitment of the management of the nigerian institute of medical research, particularly prof oni idigbe, the immediate past director general, for nursing the idea and bringing it to reality. the entire management have supported this vision. the staff of the human virology laboratory, who have worked tirelessly, are highly appreciated. we also wish to acknowledge our implementing partners (aids prevention initiative in nigeria, apin) as well as our clients and suppliers who have supported and cooperated with us respectively. finally, we wish to acknowledge erik friedly, who assisted with the preparation of this manuscript. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this paper. authors’ contributions r.a.a. was the project leader, responsible for the project design and also wrote the manuscript. u.s. and c.k.o. performed internal audits and data management, they also monitored processes. o.b.s., f.a.i., e.m., m.a., o.s.a., a.p.o., e.o.f., e.o.o. and r.o.a. performed internal audits and monitored processes. f.o., c.l.o. and m.o. monitored processes, while e.o.i. made conceptual contributions. references top ↑ 1. strengthening public health laboratories in the who african region: a critical need for disease control. in: 58th session of the who regional committee for africa (afr/rc58/r2). yaounde: world health organization regional office for africa; 2008. 2. gershy-damet gm, rotz p, cross d, et al. the world health organization african region laboratory accredited process: improving the quality of laboratory systems in the african region. am. j. clin. pathol. 2010;134:393–400. http://dx.doi.org/10.1309/ajcptuuc2v1wjqbm 3. who/pepfar/unaids. task shifting: global recommendations and guidelines; 2008, isbn 978 92 4 159631 2. 4. simundic a, nikolac n. the prevalence of preanalytical errors in a croatian iso 15189 accredited laboratory. clin. chem. lab. med. 2010;48(7):1009–1014, issn (online) 1437-4331, issn (print) 1434-6621, doi: 10.1515/cclm.2010.221 5. westgard, jo, barry pl. cost-effective quality control: managing the quality and productivity of analytical processes. aacc press; 1986. 6. llopis ma, trujillo g, llovet mi, et al. quality indicators and specifications for key analytical-extranalytical processes in the clinical laboratory. five years’ experience using the six sigma concept. clin chem lab med. 2011 jan 31; [epub ahead of print]. http://dx.doi.org/10.1515/cclm.2011.067 7. markin rs, what a. laboratory automation: trajectory, technology and tactics. clin. chem. 2000; 46: 764–771. 8. howanitz jh, howanitz pj. laboratory results. timeliness as a quality attribute and strategy. am. soc. clin. path. 2001;116:311–5. http://dx.doi.org/10.1309/h0dy-6vtw-nb36-u3l6 9. aoyagi t. iso 15189 medical laboratory accreditation. rinsho byori. 2004 oct;52(10):860–865. 10. cdc laboratory accreditation success stories, global hiv/aids; 2009. http://www.cdc.gov/globalaids/success-stories/lab-accreditation.html abstract introduction methods results discussion acknowledgements references about the author(s) marli van heerden national health laboratory service, johannesburg, south africa faculty of health sciences, charlotte maxeke johannesburg academic hospital, university of the witwatersrand, johannesburg, south africa jaya a. george national health laboratory service, johannesburg, south africa faculty of health sciences, charlotte maxeke johannesburg academic hospital, university of the witwatersrand, johannesburg, south africa siyabonga khoza national health laboratory service, johannesburg, south africa faculty of health sciences, charlotte maxeke johannesburg academic hospital, university of the witwatersrand, johannesburg, south africa citation van heerden m, george ja, khoza s. the application of sigma metrics in the laboratory to assess quality control processes in south africa. afr j lab med. 2022;11(1), a1344. https://doi.org/10.4102/ajlm.v11i1.1344 note: additional supporting information may be found in the online version of this article as online supplementary document 1. original research the application of sigma metrics in the laboratory to assess quality control processes in south africa marli van heerden, jaya a. george, siyabonga khoza received: 26 july 2020; accepted: 30 mar. 2022; published: 22 june 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: laboratories use quality control processes to monitor and evaluate analytical performance in terms of precision and bias. sigma metrics provide an objective assessment of laboratory quality using the total allowable error as an additional parameter. objective: this study aimed to determine the sigma metrics of analytes when using different total allowable error guidelines. methods: a retrospective analysis was performed on 19 general chemistry analytes at charlotte maxeke johannesburg academic hospital in south africa between january 2017 and december 2017. sigma metrics were calculated on two identical analysers, using internal quality control data and total allowable error guidelines from the ricos biological variation database and three alternative sources (the royal college of pathologists of australasia, the clinical laboratory improvements amendment, and the european federation of clinical chemistry and laboratory medicine). results: the sigma performance was similar on both analysers but varied based on the guideline used, with the clinical laboratory improvements amendment guidelines resulting in the best sigma metrics (53% of analytes on one analyser and 46% on the other had acceptable sigma metrics) and the royal college of pathologists of australia guidelines being the most stringent (21% and 23%). sodium and chloride performed poorly across all guidelines (sigma < 3). there were also month-to-month variations that may result in acceptable sigma despite poor performance during certain months. conclusion: the sigma varies greatly depending on the total allowable error, but could be a valuable tool to save time and decrease costs in high-volume laboratories. sigma metrics calculations need to be standardised. keywords: quality control; diagnostic errors; laboratories; clinical chemistry tests; sigma metrics; six sigma; chemical pathology. introduction medical laboratories strive to produce accurate reproducible results as physicians rely on these for diagnosis, monitoring, and prognostication of patients.1 to produce results with no errors, medical laboratories thus monitor and evaluate analytical processes using several different quality control (qc) processes. in practice, there are no processes with zero defects.2 the performance of analytical procedures is typically evaluated in terms of precision and accuracy (bias). this is determined using qc procedures performed at intervals as determined by laboratory policy. a high standard deviation indicates poor precision, instability, and high random error.3 most south african laboratories use levey-jennings control charts and westgard qc rules to determine whether a qc run is acceptable based on an algorithm with specified limits. this approach might not be ideal, as one set of rules cannot be applied to all tests due to varying precision and goals.4 the number of qc levels and the frequency of qc runs varies greatly between laboratories.5 the national accreditation board for testing and calibration laboratories guidelines report that two-level controls should be run at a peak hour and, subsequently, one level every 8 h for laboratories that run continuously.6 different rules may be applied to determine if the qc values are acceptable or not. most laboratories use 12s as a warning rule. this implies that a single control measurement exceeding two standard deviations from the mean (in any direction) may indicate a problem.5 however, when this rule is used as a control rule, it can cause a false rejection rate of up to 14%.5 internal qc policies regard the 13s, r4s and 22s rules as criteria for rejection, while ten consecutive observations on one side of the mean (10 × rule) require further investigation. combinations of rules (multirules) are sometimes employed to reduce the rate of false rejections and to save time and effort by incorporating rules that are sensitive to both random and systematic errors. six sigma further elaborates on this by individualising control rules based on the analytical performance of the test.7 six sigma is a qc strategy where a statistical calculation is performed to evaluate the effectiveness of laboratory processes. the sigma scale provides an objective manner to assess and compare laboratory quality by incorporating both the imprecision and bias observed in a laboratory’s performance.8 the sigma metric is based on three parameters: total allowable error (tea), bias and imprecision. the tea guidelines from various sources are associated with significantly different sigma metrics for the same assay.9,10 six sigma can be used to decide on the best westgard rule by judging the performance of a process against a reference method and assessing the quality of laboratory processes, thereby identifying processes needing improvement. as demonstrated by litten,7 the implementation of a six sigma-designed qc programme can result in fewer controls per run, fewer false rejections, simpler westgard rules, and a 45% saving on laboratory reagents and supplies. our laboratory runs two identical analysers in parallel and currently does not use sigma metrics to manage qc. there has been a big shift in focus towards quality laboratory improvement, especially in developing countries. there are limited studies on sigma metric performance in south african laboratories. this study thus aimed to determine the variations in sigma metrics of selected analytes with different tea guidelines and to assess if sigma differed between two identical analysers. methods ethical considerations ethical approval, in the form of a waiver, was obtained from the university of the witwatersrand human research ethics committee (number: w-cbp-180216-01). the results of quality control samples were utilised and therefore no patient consent was required. study setting this retrospective study was conducted in a national health laboratory service laboratory at a large academic tertiary hospital in south africa, which is accredited to international organization for standardization standard 15189. there are two identical cobas® 8000 chemistry analyser (roche diagnostics, mannheim, germany) systems (referred to as analyser 1 and analyser 2) running in parallel in the laboratory, with some tests run on both analysers. study design a retrospective analysis of 19 analytes was performed using internal qc data obtained from the roche cobas® 8000 chemistry analyser it middleware system (roche diagnostics, mannheim, germany) over 12 months (january 2017 – december 2017). these analytes were chosen as they are routinely analysed and can be compared to studies in the literature. the laboratory used roche qc materials to perform the internal qc testing. two levels of internal qc are performed for each analyte, one in the normal range and one in the abnormal (high) range. the qc materials used include roche cobas® precicontrol clinchem multi 1 and 2, precicontrol tumor marker, and precicontrol universal. a single lot of qc materials, except for prostate-specific antigen (psa) and thyroid-stimulating hormone (tsh), was used throughout the 12 months. the following tests were included in our study: alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase (ast), calcium, chloride, cholesterol, creatine kinase (ck), creatinine, direct bilirubin (dbil), glucose, high-density lipoprotein cholesterol, potassium, sodium, tsh, total bilirubin, total protein, psa, urea, and uric acid. the reagents, qc materials and calibrator materials were all provided by roche diagnostics, mannheim, germany. data collection and analysis using internal qc means and standard deviations, we calculated the bias and coefficient of variation (cv). the bias was determined by subtracting the packing insert target value (mean) from the observed qc mean. the cv was determined using the following formula: outliers (values exceeding 13s) were excluded using the cobas it middleware. for our study, the biological variation (bv) database from ricos and colleagues, which was last updated by the spanish society of laboratory medicine in 2014,11 was used to determine the desirable test-specific quality requirements. this database was compared to the tea guidelines from the clinical laboratory improvement amendments (clia),12 the royal college of pathologists of australasia (rcpa),13 and the european federation of clinical chemistry and laboratory medicine (eflm).14 tea values given as percentages were converted to units with the following calculation: sigma metrics were calculated for each analyte on two levels as follows: thereafter, the average annual sigma metric was obtained. the quality goal index (qgi) indicates the possible source of error and represents the relative degree to which bias and precision meet their quality goals.15 the qgi was determined as follows: a qgi score of < 0.8 indicates imprecision, qgi scores between 0.8 and 1.2 indicate both imprecision and inaccuracy, and a score of > 1.2 indicates inaccuracy.15 meta-analysis data for within(cvi) and between(cvg) subject variation were obtained from the eflm biological variation database.14 the desirable specifications for imprecision, bias, and tea were calculated as follows: the allowable limits of performance for total bilirubin, dbil, calcium, and uric acid were obtained from the westgard website based on eflm data.16 the normalised method decision charts were created by calculating and then plotting the observed inaccuracy (bias% / tea%) and the observed imprecision (cv% / tea%). data capture and statistical analyses were performed using windows® 10, microsoft excel (microsoft corporation, redmond, washington, united states). the analyses performed include calculation of bias, cv, tea, sigma metrics, and the qgi, as specified above. results nine analytes (alanine aminotransferase, ast, total bilirubin, ck, urea, dbil, uric acid, psa, and tsh) achieved sigma ≥ 3 on both analysers, while one analyte (uric acid) had a sigma of ≥ 3 on analyser 1 and < 3 on analyser 2 (figure 1). figure 1: average annual sigma of analytes tested on two chemistry analysers, johannesburg, south africa, january 2017 – december 2017. (a) analyser 1; (b) analyser 2. performance was very similar across both analysers for each guideline. using the ricos database, ten analytes had a sigma of < 3 across both analysers, and four analytes had a sigma of > 6 on both analysers. three analytes achieved sigma > 6 on both control levels, namely dbil, psa, and tsh on analyser 1, as well as ck, dbil, and psa on analyser 2 using ricos (online supplementary document table 1). the sigma values for each analyte have been grouped according to performance for both analysers based on different sources of tea and concentration of control material. the poorly performing analytes (with sigma values < 3) all had qgi values < 0.8, indicating imprecision as a possible cause. several analytes had sigma < 3 for both qc levels across both analysers. these included sodium, chloride (online supplementary document figure 1), glucose, and cholesterol. only ast achieved a sigma of ≥ 3 with all guidelines and at both analyte concentrations, but this was seen only on analyser 1. aspartate aminotransferase and urea achieved sigma metrics between 3 and 6 on both analysers. no analyte achieved a sigma of > 6 across all guidelines and for two control levels. there were also large variations in performance from month-to-month. many analytes achieved an acceptable annual average sigma despite poor or marginal performance during certain months. examples include tsh, ast, total bilirubin and alanine aminotransferase. conversely, crea, ck level 1 and cholesterol level 2 failed to achieve acceptable sigma values, despite having sigma values ≥ 3 during certain months. alanine aminotransferase, total bilirubin and ast level 1 on analyser 1 (module 702) displayed similar patterns, with improvements noted during april, may, july, september, and november 2017. when comparing the sigma performance based on the different tea guidelines, the clia bv guidelines resulted in the best sigma metrics, with 46% (analyser 1) and 53% (analyser 2) of analytes achieving sigma values ≥ 3 (figure 2). using the ricos bv database, 43% of analytes on analyser 1 and 36% on analyser 2 had sigma values ≥ 3. sixteen percent of analytes on analyser 1 and 20% of analytes on analyser 2 had sigma values > 6. with the eflm guidelines, 39% of analytes on analyser 1 and 32% on analyser 2 had sigma values ≥ 3. the worst sigma metrics were obtained using rcpa guidelines: 21% of analytes had sigma ≥ 3 on analyser 1 and 23% on analyser 2. no analyte on analyser 2 had sigma values > 6 using the rcpa guidelines. figure 2: sigma performance of analytes tested on two chemistry analysers based on different total allowable error guidelines, johannesburg, south africa, january 2017 – december 2017. (a) sigma % per tea guideline – analyser 1; (b) sigma % per tea guideline – analyser 2. based on the method decision charts for specific analytes on analyser 1, more analytes were classified as excellent performers using the ricos guidelines compared to rcpa (online supplementary document figure 2). the same pattern was noted for tsh on this analyser, with the eflm guidelines also resulting in good sigma performance (figure 3). there were also variations in performance between qc levels, with performance generally better in level 2. the ricos and clia guidelines appear to be the most lenient, resulting in better performance for ck on analyser 2. in general, the eflm guidelines resulted in good sigma performance, while the rcpa guidelines, which are stricter, resulted in poor performance. high-density lipoprotein cholesterol generally performed poorly on analyser 2, except when the clia guidelines were applied. figure 3: normalised method decision chart for dbil, psa, tsh, ck, and hdl based on different total allowable error guidelines (ricos, clia, rcpa, eflm), johannesburg, south africa, january 2017 – december 2017. (a) dbil normalised method decision (analyser 1); (b) psa normalised method decision (analyser 1); (c) tsh normalised method decision (analyser 1); (d) ck normalised method decision (analyser 2); (e) hdl normalised method decision (analyser 2). discussion the analytical performance of a laboratory, as assessed by sigma metrics, has practical implications such as the design of qc programmes. in this study, qc data for 19 chemistry analytes collected over 12 months were analysed, and sigma was calculated for each analyte to objectively evaluate analytical performance. this study demonstrated that six sigma values vary depending on the tea guidelines used, with analyte concentration, and from month-to-month. there are no universally accepted tea guidelines, and published data show that sigma metrics vary with the tea guidelines used.8,10,17 there are several ways to address this. for example, sharkawy et al.17 created a harmonisation protocol for sigma calculation to allow the comparison of sigma metrics across laboratories by using similar tea guidelines. in this study, there were fewer differences across the two analysers using the same tea guidelines compared to the sigma variations observed with different tea guidelines. another proposed approach is to assess the effect of tea on patient outcomes. researchers in china18 assessed the ‘severity of harm’ caused by tea being exceeded in 36 analytes. a risk priority number was assigned by multiplying the sigma metric by the score of the intended use. the authors suggested that tea should be defined by the highest possible hierarchical model and recommended that tests with negligible risks to patients be allowed to reach lower sigma metrics.18 in our study, the clia bv guidelines resulted in the best sigma metrics. this may change in the future as clia has proposed new limits that appear to be less lenient.19 the rcpa guidelines appeared to be the most stringent, which is in keeping with the results of a 2018 study performed in china.20 recently, the eflm has established the working group on biological variation and the task and finish group for the biological variation database to assess the quality of existing bv data and to compile global estimates in an attempt to harmonise analytical performance specifications worldwide.21 as proposed by varela and pacheco,22 another option would be to use an algorithm that standardises the procedure for the selection of the most appropriate tea for evaluating analytical performance. in addition to tea, the analyte concentration is crucial when determining sigma metrics.8 in a study that investigated the performance of verified versus non-verified reagents, cao et al.23 observed that sigma varied with analyte concentration and suggested that different rules be used for different analyte concentrations. the observed changes in sigma value with analyte concentration may be due to changes in precision and/or bias.8 there are different approaches to the calculation of bias and cv, which may both influence the final sigma calculation. for example, bias may be determined from external quality assessment reports rather than from internal qc data as done in this study. guo et al.9 showed that both methods can be used for the determination of sigma metrics and suggest that laboratories evaluate sigma metrics multiple times to optimise qc schedules. alternatively, bias may also be obtained from package inserts or can be derived from a group mean. the month-to-month variation noted with sigma was due to changes in cv and bias over these months. the cv, which is a measure of imprecision, was based on results over a 12-month period and therefore would be influenced by changes in reagents, calibrators, or personnel. it is therefore expected to be wider than cv determined over shorter periods and expected to have consequently lower sigma metrics.24 current methods for the determination of sigma metrics have been criticised. according to oosterhuys,25 the inclusion of bias in the calculation will result in an underestimation of analyte performance due to the short-term bias already reflected in the analytical imprecision. westgard found that the assumption that bias should be ‘corrected or eliminated’ was invalid for many analytes as bias still exists despite attempts at standardisation.25,26 to address the variability of sigma performance between different qc levels, months and analysers, the average sigma or lowest sigma metric could be used for determining qc procedures.27 westgard multirules28 will be easy to implement for well-performing analytes such as psa and dbil. it will however be complicated for analytes with low sigma metrics such as the electrolytes, as well as those performing differently on the different qc levels. the goal is to achieve 90% error detection and a 5% false rejection rate while using the lowest possible number of control rules.7 operating specifications charts can be used to determine the number of control rules and the number of controls needed to achieve this goal.29 the lower the error detection, the more likely it is that more than one qc run will be required to detect a critical shift in performance and that erroneous patient results may be reported before the problem is detected.30 moving average qcs have been suggested for high-volume analytes with low sigma metrics to decrease the risk of reporting erroneous results between scheduled qc runs.31 our study demonstrated the need to increase internal qc and calibration frequency for some poorly performing analytes such as the electrolytes (sodium, potassium and chloride) regardless of the bv source used. the poor sigma performance of electrolytes is however not unique to our laboratory. potassium, sodium, and chloride have low bv, and tight quality specifications are expected to give low sigma results.27,32 we used the qgi to investigate the reasons for low sigma performance (sigma values < 3) and showed that the main problem was imprecision. a search for new and improved calibration methods may improve the precision and, subsequently, the sigma metrics.33 when imprecision is poor relative to analytical goals, good error detection is hard to attain, regardless of the qc rules used.30 ideally, sigma metrics should assist in decreasing operating costs by decreasing the amount of qc materials and reagents used and reducing unnecessary recalibrations.29,34 in addition, staff morale can improve by decreasing time spent troubleshooting and investigating false rejections.29 zhou et al.35 compared new qc procedures (based on recommended error detection and false rejection criteria) with previous procedures adopted in their laboratory and found that for analytes with sigma values > 6, there is cost reduction and increased efficiency. as poor-performing analytes will require the maximum number of control rules and control measurements per run, this can prove to be too expensive in our setting. westgard found that for analytes with sigma values < 3, a full multirule procedure with at least four control measurements per run is required.29,36 some authors find these stricter qc procedures to be unpractical because of the significant increase in the number of runs, especially when multiple analytes demonstrate sigma values < 4. however, when the quality of clinical results and benefits to patients are considered, the associated costs can be justified if reasonable.35 one of the strengths of this study is the 12-month duration; this gave a good reflection of the data as estimations of accuracy and imprecision are expected to improve with more data points.30 we also looked at the sigma performance of multiple analytes on two levels of qc to assess different modules on the instrument and to determine if the control level is a contributing factor. as the same lot of qc materials was used in this study, performance-related changes could be attributed to other factors. comparing two identical analysers allowed us to assess the performance of analysers operating under the same environmental conditions and improved the consistency of our findings. laboratories should explore the practicality and feasibility of introducing sigma metrics as part of routine qc practice and for the review of poorly performing methods. we also recommend the careful selection of tea guidelines and the standardisation of sigma metric calculations in the future. limitations one limitation of this study is that no third-party qc materials were used. the recommendation is that, when possible, third-party qc materials should be used rather than control materials supplied by the manufacturer.30 when qc materials are different from calibrator materials, it ensures an independent, unbiased assessment of the measurement procedure’s performance.37 conclusion laboratory results are crucial in the diagnosis, monitoring and prognostication of patients, and further action often relies on the value of one test result. laboratories should therefore aim to minimise errors that can affect patient outcomes. the sigma metrics tool has the potential to be a valuable quality management tool for monitoring analytical performance in comparison to world-class standards. however, it is important to set up standardised protocols for the determination of sigma metrics, including choosing the appropriate tea guidelines and approach to calculating bias. acknowledgements we thank dr h.e. van deventer for his support and assistance; as well as mrs l. ntlebi for her assistance in retrieving internal quality control data for the study period. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions m.v.h., j.a.g. and s.k. contributed to the design and implementation of the research, the analysis of the results and to the writing of the manuscript. m.v.h collected and analysed the data and drafted the first manuscript. j.a.g and s.k. contributed by supervision and revision of the manuscript in preparation for submission. sources of support the authors received no financial support for the research, authorship or publication of this article. data availability the data that support the findings of this study are available from the corresponding author, m.v.h., on request. disclaimer the views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the affiliated institution. references jordan b, mitchell c, anderson a, farkas n, batrla r. the clinical and health economic value of clinical laboratory diagnostics. ejifcc. 2015;26(1):47–62. charuruks n. sigma metrics across the total testing process. clin lab med. 2017;37(1):97–117. https://doi.org/10.1016/j.cll.2016.09.009 cooper g. basic lessons in laboratory quality control. hercules: bio-rad laboratories; 2008. westgard jo. internal quality control: 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laboratory standards institute; 2016. abstract introduction methods results discussion acknowledgements references about the author(s) abdulrazaq a. jimoh department of anatomic pathology and forensic medicine, faculty of basic clinical sciences, federal university of health sciences, azare, nigeria zainab a. adamu department of pathology, faculty of basic clinical sciences, ahmadu bello university, kaduna, nigeria mumini w. rasheed department of anatomic pathology, faculty of basic clinical sciences, federal university dutse, jigawa, nigeria samuel k. richard department of pathology, faculty of basic clinical sciences, university of abuja, gwagwalada, abuja, nigeria citation jimoh aa, adamu za, rasheed mw, richard sk. expression of cyclooxygenase-2 (cox-2) in colorectal adenoma in an indigenous african population in northern nigeria. afr j lab med. 2025;14(1), a2613. https://doi.org/10.4102/ajlm.v14i1.2613 original research expression of cyclooxygenase-2 (cox-2) in colorectal adenoma in an indigenous african population in northern nigeria abdulrazaq a. jimoh, zainab a. adamu, mumini w. rasheed, samuel k. richard received: 24 aug. 2024; accepted: 07 apr. 2025; published: 30 july 2025 copyright: © 2025. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the clinical significance of adenoma is as a result of being a precancerous lesion with long latency, harbouring of invasive carcinoma, bearing similar clinical features with colorectal cancer, and as part of hereditary colorectal cancer syndromes. over-expression of the cyclooxygenase-2 (cox-2) enzyme has been noticed in adenomas with unfavourable features. however, this information is limited in africa. objective: this study aimed to assess the proportion of adenomas in northern nigeria that over-express cox-2. methods: this 5-year retrospective, descriptive, hospital-based study examined the cox-2 immunohistochemistry of all histologically diagnosed colorectal adenomas in aminu kano teaching hospital, kano, nigeria, between 01 january 2015 and 31 december 2019. age, sex, site, diagnosis, and grade were obtained from the kano cancer registry and slide reviews of cases. results: there were cases of 29 adenoma (male, n = 20; female, n = 9). adenoma occurred more commonly among male patients (m:f, 2.2:1), in the age group 40–79 years, and included tubular adenomas (62.1%), tubulovillous adenomas (27.6%), and villous adenomas (10.3%). over-expression of cox-2 was observed in 3.4%. there was no association between cox-2 expression and age, sex, site, histological subtype, or grade. conclusion: over-expressed cox-2 was observed in only 3.4% of adenomas, which may indicate its early involvement in the spectrum of adenoma-carcinoma sequence. what this study adds: it provides key information about cox-2 expression in adenoma in an african population, which may serve as a rationale for other studies regarding cox-2 targets for chemoprevention and therapy in adenoma and colorectal cancer. keywords: cyclooxygenase-2; expression; colorectal adenoma; nigeria; africa. introduction colorectal adenomas or adenomatous polyps, which are the major sub-types of colorectal polyps, are benign glandular neoplasms of colorectal epithelium that occur either sporadically or as part of a polyposis syndrome, with some having malignant potential. in nigeria, the proportion of colorectal polyps that are adenomatous ranges from 28.8% to 47.5%.1,2,3,4,5 the distinct parameters that make adenoma relevant clinically include being a precursor lesion, harbouring invasive carcinoma, long latency period of malignant transformation, having similar clinical features with colorectal cancer (crc), and as part of hereditary crc syndrome.6,7,8,9 emerging evidence implicates the role of the cyclooxygenase-2 (cox-2) enzyme in adenoma. carriers of the cox-2 a-1195g ag genotype are at a twofold increased risk of developing colorectal adenomas, and 2.9 times increased risk of developing cancer, suggesting a role of cox-2 as a potential biomarker for cancer risk.10 contrastingly, the carriers of cox-2 a-1195g aa genotype have 1.8 times reduced risk of developing adenomas, and 3.1 times reduced risk of developing crc.10 chronic use of cyclooxygenase antagonists such as nonsteroidal anti-inflammatory drugs, particularly aspirin, is associated with reduced adenoma incidence, aggressiveness, size, and recurrence.11,12,13,14 inhibitors of cyclooxygenases such as nonsteroidal anti-inflammatory drugs are useful in the chemoprevention and management of adenoma, and they exert their effects by inhibiting cyclooxygenases (cyclooxygenase-1 and cox-2), which are enzymes required for the metabolism of arachidonic acid to prostaglandins.15,16,17 the expression of cox-2 is highly induced by tumour promoters, mitogens, and cytokines.15 over-expression of cox-2 is associated with inflammation, initiation of cancer, cancer progression, proliferation of surviving cancer stem cells, activation of vascular endothelial growth factor, angiogenesis, invasion, metastasis, and tumour relapse after therapy.16,17 the rate of cox-2 over-expression increases during the course of crc development from adenoma to crc in situ to invasive crc, thus recapitulating events in the adenoma-carcinoma progression.18 it may, therefore, imply that cox-2 over-expression may be a marker of the progression of crcs from adenomas.18 however, studies on cox-2 and its antagonists in colorectal adenomas are limited in africa, as most studies are from asia and western countries. this study examined cox-2 expression in adenoma in an african population, and it may serve as a rationale for other studies on cox-2 and its target in colorectal adenoma and carcinoma chemoprevention and therapy. this study aimed to assess the proportion of adenomas in northern nigeria that over-express cox-2 and establish any relationship between cox-2 over-expression with clinicopathological features such as age, sex, histological subtype, and tumour grade. methods ethical considerations ethical approval for this study was obtained from the health and research ethics committee of aminu kano teaching hospital, kano (ethical review reference number: akth/mac/sub/12a/p-3/v1/2904). additional consent of individuals whose samples were used in this study was waived, as is the institutional ethical committee’s policy for retrospective studies involving non-human subjects. patients’ identities were concealed at all times, and records were kept under lock. study design and setting this retrospective, descriptive, hospital-based study was carried out in the histopathology laboratory of the department of histopathology, aminu kano teaching hospital, kano (north-western nigeria). aminu kano teaching hospital is a tertiary hospital with over 700-bed capacity. the histopathology department of aminu kano teaching hospital receives an average of 5500 histological samples per annum, and renders services including cytology, histology, autopsy, and immunohistochemistry. inclusion and exclusion criteria adenoma cases included in this study were all cases that were histologically diagnosed with colorectal adenoma in the study centre from 01 january 2015 to 31 december 2019. cases with insufficient clinical information, particularly biodata, missing or damaged blocks, and tissue blocks with insufficient tissue were excluded (in this regard, excluded case is zero). data collection data, such as age, site of lesion, histologic diagnosis, and grade of the disease was obtained from kano cancer registry, pathology request forms, patients’ case notes, duplicate copies of histopathological reports, and slide reviews of cases. the patient’s identity was concealed at all times. the cases were classified and graded according to the world health organization classification of tumours of the colon and rectum.19 the two-tiered system of grading was used based on prognosis: low grade and high grade.19 immunohistochemistry for cyclooxygenase-2 expression immunohistochemical staining was performed using anti-cox-2 rabbit polyclonal antibody (elabscience, houston, texas, united states; catalogue no. e-ab-70031), used at a 1:500 dilution according to standard immunohistochemical staining protocols. the antibody was stored at −20 °c (using −25 °c freezer 110 l, biobase scientific, jinan, shandong, china) and it was centrifuged (using lc-4k-2 centrifuge, biobase scientific, jinan, shandong, china) before opening to ensure complete recovery of contents. a kidney sample (an autopsy sample) with intact renal tubules was used as a positive control, while a negative control was obtained by replacing the primary antibody with distilled water. tissue preparations (formalin-fixed paraffin-embedded tissue blocks) formalin-fixed paraffin-embedded tissues retrieved were cut or sectioned at 3 µm using a bk-2218 microtome (biobase scientific, jinan, shandong, china). the tissue slides were left to dry in an oven for 2 h at 60 °c after microtomy. then, the slides were dewaxed in xylene (obtained from central drug house ltd, daryaganji, new delhi, india) for 1 min, and then passed through decreasing concentrations of alcohol at 100%, 95% and 70% for 1 min each for rehydration of sections. antigen retrieval or unmasking of antigen sites antigen retrieval was done by placing the slides in 100 ml of citrate buffer (ph 6) (obtained from central drug house ltd, daryaganji, new delhi, india), and then heating using the microwave (tp-j1-8 microwave, chongqing top oil purifier co. ltd, jiulongpo, chongqing, china) heating retrieval method at 100 °c for 15 min – 20 min. slides were then washed in distilled water for another 2 min and then washed twice with phosphate buffer (obtained from central drug house ltd, daryaganji, new delhi, india) for 3 min each. they were then rinsed in distilled water for 2 min; 3% of hydrogen peroxide was added and left for 10 min. slides were then washed in running distilled water for 2 min and incubated with a blocking reagent (serum; elabscience, houston, texas, united states) for 10 min. blockage of endogenous peroxidase to block non-specific antigen sites, tissue sections were incubated for 1 h in 1.5% bovine serum albumin (obtained from central drug house ltd, daryaganji, new delhi, india) at room temperature.20 immunostaining procedure each slide was wiped with cotton wool to remove excess blocking solution. incubation with the primary antibodies was carried out at room temperature for 30 min with 200 µl of anti-cox-2 (elabscience, houston, texas, united states; catalogue no. e-ab-70031). following the primary antibody incubation step, a secondary antibody from a streptavidin-biotin complex peroxidase kit (lsab+ kit, dako, copenhagen, denmark) was then incubated by the manufacturer’s instructions. peroxidase activity was also developed with the substrate 3,3-diaminobenzidine tetrahydrochloride (dako) by incubating the tissue sections in 3,3-diaminobenzidine tetrahydrochloride for a period of 3 min.20 the tissue slides were washed in running water for a period of 3 min, and then counterstained with haematoxylin (obtained from central drug house ltd, daryaganji, new delhi, india). the tissue sections were then dehydrated in increasing concentrations of alcohol. they were cleared in xylene and the application of cover slip was done using distyrene plasticizer xylene (obtained from central drug house ltd, daryaganji, new delhi, india) and they were allowed to dry. the slides were viewed under the light microscope (dm 500 binocular leica microscope, leica microsystems, wetzlar, hessen, germany), brown cytoplasmic and membranous staining was interpreted as positive staining, and was scored semi-quantitatively using the immunoreactive score system, a final score that is a product of the intensity and distribution of cox-2 immunoreactivity.20,21,22 the intensity of staining was scored as 0 for no staining, 1 for weak staining, 2 for moderate, and 3 for strong staining. the percentage of positive tumour cells was scored: 0 indicating no cell with a positive reaction, 1 indicating 1% – 10% of cells with a positive reaction, 2 indicating 11% – 50% of cells with a positive reaction, 3 indicating 51% – 80% of cells with a positive reaction, and 4 indicating greater than 80% of cells with a positive reaction. the final immunoreactive score obtained by multiplying the distribution and intensity for each tumour was graded as follows: 9–12 strongly positive, 5–8 moderately positive, 1–4 weakly positive, and 0 negative. cyclooxygenase-2 was considered over-expressed if the immunoreactive score is moderate to strong (that is a score of 5 to 12) (table 1). table 1: cyclooxygenase-2 immunoreactive scoring system at aminu kano teaching hospital, kano, nigeria, january 2015 – december 2019. statistical analysis statistical analysis was performed using statistical package for social sciences version 25 (ibm® corp, armonk, new york, united states). chi-square was used with statistical significance set at p < 0.05 to test the association between the cox-2 expression and clinicopathological parameters such as age, sex, histological subtype, and grade. proportions and central tendencies of nominal and continuous variables were obtained by descriptive statistics of frequency, mean and median. results age distribution of colorectal adenomas twenty-nine adenoma cases were analysed, and 26 cases were endoscopic biopsies. the age distribution of colorectal adenoma cases ranged from 12 years to 80 years with a mean age of 53.4 (standard deviation = 16.0) years. the largest proportion of cases (23 of 29 cases; 79.3%) clustered within the category of patients aged 40 years to 79 years and peaked in the 6th decade. eleven cases of adenomas (37.9%) occurred before age 50 years while only six cases (20.7%) occurred at age 70 years or more (table 2). table 2: age distribution of adenomas with sex, histological sub-types, grade, and site at aminu kano teaching hospital, kano, nigeria, january 2015 – december 2019. gender distribution of adenomas there were 20 male cases and nine female cases, giving a male-to-female ratio of 2.2:1. male cases occurred in individuals aged 12 years to 75 years, with a mean age of 51.7 years. the nine cases that were observed among the female patients occurred from 39 years to 80 years, with a mean age of 57.2 years (table 2). histological types of adenomas eighteen (62.1%) cases of colorectal adenoma were tubular adenomas; of these, eight cases were high-grade lesions. eight (27.6%) cases were tubulovillous adenomas (five of which were high-grade), and only three (10.3%) cases were villous adenomas (including one high-grade lesion, figure 1). there were no significant associations between histological type and the age group (χ2 = 15.8538 degrees of freedom [df] = 14, p = 0.3224) or sex (χ2 = 0.2529, df = 2, p = 0.8812). figure 1: haematoxylin and eosin and cyclooxygenase-2 immunohistochemistry photomicrographs of adenoma cases at aminu kano teaching hospital, kano, nigeria. (a) low-grade tubular adenoma, haematoxylin and eosin ×40, (b) high-grade tubular adenoma, haematoxylin and eosin ×40, (c) low-grade tubulovillous adenoma, haematoxylin and eosin ×100, (d) cyclooxygenase-2 positive control (renal tubules) ×400, (e) cyclooxygenase-2 positive (low grade tubular adenoma) ×100, and (f) cyclooxygenase-2 negative, (low grade tubular adenoma) ×100. grading of adenomas fifteen cases (51.7%) of adenoma were high-grade adenomas (table 2). this comprised nine cases of tubular, five cases of tubulovillous, and one case of villous adenoma. fourteen cases (48.3%) were low-grade. among the female patients, there were only five cases (33.3%) of high-grade adenoma, and four cases (28.6%) in the low-grade tumours category. there were no statistically significant associations between the grade of tumour and age (χ2 = 8.8808, df = 7, p = 0.2613) or sex (χ2 = 0.0762, df = 1, p = 0.7818). site of adenomas thirteen cases (44.8%) of adenoma were colonic in origin, while the remaining cases were rectal tumours (table 2). there were no statistically significant associations between the site of tumour and age (χ2 = 5.2217, df = 7, p = 0.6329). cyclooxygenase-2 expression in adenomas the only case that showed cox-2 positivity (strong positive staining) was a low-grade tubular adenoma in a 60-year-old man (figure 1). there was no statistical association between cox-2 over-expression and age, sex, or histological type of the tumour. discussion adenomas occurred more commonly among male patients (m:f, 2.2:1), in the age group 40–79 years, and they were composed of tubular adenomas (62.1%), tubulovillous adenomas (27.6%), and villous adenomas (10.3%). over-expression of cox-2 was observed in 3.4%. there was no association between cox-2 expression and age, sex, histological subtype, or grade. colorectal adenomas are known precursor lesions for crc, usually through adenoma-carcinoma sequence, and they also share similar risk factors with crc.6,7,8,9 they, therefore, provide a window for prevention of crc through screening and early detection of these premalignant lesions.6 early detection of colorectal adenomas through a surveillance screening programme is therefore essential in interrupting the possibility of progression to colorectal cancer. the adenoma occurrence is more commonly observed in a relatively young population in our society, similar to the age pattern of crc as demonstrated by a mean age of 53.4 (standard deviation = 16.00) and peak age in the 5th decade in this study. ibrahim and dahiru have demonstrated a mean age of 47.3 years among adenomatous polyps in kano, nigeria.3 a mean age of 47 years has also been observed in china, although the peak age at which adenomas was observed was 65 years.22 adenomas are more frequent in the western world than in africa and asia, but they occur more in older populations in the united states and the united kingdom, and a mean age as high as 64 years and 68 years has been reported, thereby mirroring the age pattern of crc in these regions.23,24 the possible reason for higher occurrence in the areas may be largely because of exposure to modifiable risk factors such as the western-type diet similar to that of the crc. the male preponderance of colorectal adenomas in this study mirrors that of the crc and has also been reported worldwide.8,9,25 it is difficult to explain the higher male affectation but possible reasons have been attributed to differences in access to crc screening, differences in risk factors exposure, hormonal factors, and socioeconomic factors.8,9 it is more likely for men to deposit visceral fat than women, which is a risk factor for both adenoma and crc.26,27 men are also more likely to have a high consumption of red meat, processed meat, heavy alcohol, and cigarette smoking.27,28,29 for histological sub-types and grade, the predominant histological sub-types were tubular adenoma (62.1%), and for the grade, high-grade colorectal adenoma accounted for 51.7% of all grades. similar findings have also been documented.3,25 cyclooxygenase-2 expression in adenomas in the present study, cox-2 expression in the adenomas was 3.4% (1 out of 29 cases). the available data demonstrate relatively lower cox-2 expression in adenoma than in the crc. the possible reason why cox-2 expression is higher in crcs than in adenomas may be that although cox-2 expression is involved in tumour initiation, it is also a marker for advanced lesions (from dysplasia in adenomas to frank malignancy and invasiveness in carcinomas).30,31 for example, it has been shown that cox-2 expression has a positive correlation with adenoma size, dysplasia, and grade but not with the tumour location or configuration, and the expression is higher in the crc.30,32,33,34,35 this may also explain why the expression of cox-2 in colorectal adenomas demonstrates a wide variation similar to that of the crc, from 38% to 100%, because of adenoma size, presence of dysplasia, histological subtype, and key genetic mutation.35,36 early adenomas are usually small and the progression of most small adenomas is slow, over several years, even though not all adenomas progress. some may remain stable, regress or new ones may form. the progression in the spectrum of the adenoma-carcinoma sequence may involve more genetic mutations and molecular interplay, including over-expression of cox-2. to illustrate this, immunohistochemical studies have shown that 37.9% of 39 cases of adenoma were cox-2 positive; 90.9% of these positive cases had a kirsten rat sarcoma mutation that correlates with tumour size (larger tumour), and the positivity is more frequent in tubulovillous (63.3%) than tubular adenoma (36.4%).36 contrary to this assertion that the cox-2 expression is a marker of progression, a cox-2 immunohistochemical study of nine advanced crc and 12 adenoma cases in japan by bamba et al. showed a higher expression in adenomas than in crcs.37 bamba et al.’s contrary observation is difficult to decode, but it may stem from the low sample size in their study.37 the cox-2 expression (3.4%, 1 out of 29 cases) in this study confirms the assertion that cox-2 is a marker of progression in the adenoma-carcinoma spectrum, but it is relatively lower than other studies. it is difficult to determine the reasons, but the possibilities may include racial factors, as most studies involve non-african subjects, genetic make-up – particularly cox-2 gene polymorphism, and the fact that most adenomas analysed in this current study were colonoscopic biopsies, which may not represent the entire tumour heterogeneity, even though the adenomas from three colectomy cases did not express cox-2 as well. more studies are needed for africa to explain the possible racial disparity in cox-2 expression in adenomas, because most available cox-2 studies on colorectal adenomas and crcs are from america and asian countries. carriers of the cox-2 a-1195g ag polymorphism have increased risk for crc and adenoma, and mutation in the adenomatous polyposis coli (apc) gene has been shown to correlate with cox-2 expression in both adenomas and crcs.10,38,39 more studies are needed to establish the role of race in cox-2 expression in adenoma. implications and recommendations colorectal adenomas are known precursor lesions of crc with similar risk factors.6,7,8 thus efforts are required to interrupt the possibility of the progression of adenomas to colorectal cancer.6 the expression of cox-2 provides vital information for the uses of nonsteroidal anti-inflammatory drugs in the prevention and treatment of adenoma and hurting adenoma progression into carcinoma. thus, in terms of the prognosis and cox-2 targeted therapy in the near future, this study provides a strong rationale for additional studies in nigeria and africa at large. limitations the limitation of this study is the inability to include rare histological sub-types of adenoma and relatively low cases of adenomas in this study. to improve this, a multicentre study across africa will give a better reflection of cox-2 expression in colorectal adenoma in africa. conclusion adenomas occurred twice as commonly in men as in women. tubular adenomas constituted 62.1% of all adenomas, and 51.7% of adenomas are high-grade adenomas. the rate of over-expression of cox-2 in adenomas is 3.4%, and this may imply its early involvement in colorectal tumourigenesis. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions a.a.j. conceptualised the idea and developed the protocol and the first draft of the article. a.a.j., z.a.a., m.w.r., and s.k.r. participated in data collection, methodology, supervision, writing, and analysis. a.a.j., z.a.a., m.w.r. and s.k.r. reviewed and provided overall guidance in the entire write-up and approved the final version prior to submission. a.a.j., z.a.a., m.w.r., and s.k.r. are accountable for all aspects of this article. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability all relevant data that support the findings of this study are available within the article. disclaimer the views and opinions expressed in this article are those of the authors and are the product of professional research. the article does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the authors are responsible for this article’s results, findings, and content. references ray-offor e, abdulkareem fb. screening colonoscopy in port harcourt, nigeria. gastroenterol insights. 2019;10(1):7987. https://doi.org/10.4081/gi.2019.7987 oluyemi a, awolola n, oyedeji o. clinicopathologic review of polyps biopsied at colonoscopy in lagos, nigeria. pan afr med j. 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microenvironment. inflamm. regen. 2017;37:4. https://doi.org/10.1186/s41232-017-0036-7 bamba h, ota s, kato a, adachi a, itoyama s, matsuzaki f. high expression of cyclooxygenase-2 in macrophages of human colonic adenoma. jpn int j cancer. 1999;83(4):470–475. https://doi.org/10.1002/(sici)1097-0215(19991112)83:4<470::aid-ijc6>3.0.co;2-f dimberg j, hugander a, sirsjo a, soderkvist p. enhanced expression of cyclooxygenase-2 and nuclear beta-catenin are related to mutations in the apc gene in human colorectal cancer. anticancer res. 2001;21(2a):911–915. fujita m, fukui h, kusaka t, et al. relationship between cyclooxygenase-2 expression and k-ras gene mutation in colorectal adenomas. j gastroenterol hepatol. 2000;15(11):1277–1281. abstract introduction methods results discussion acknowledgements references about the author(s) reuben n. abednego microbiology laboratory, national public health laboratory, dar es salaam, united republic of tanzania vitus silago department of microbiology and immunology, weill bugando school of medicine, catholic university of health and allied sciences, mwanza, united republic of tanzania citation abednego rn, silago v. antibacterial activity of soil-isolated bacillus altitudinis/pumilus complex against methicillin-resistant staphylococcus aureus from mwanza, tanzania. afr j lab med. 2023;12(1), a2167. https://doi.org/10.4102/ajlm.v12i1.2167 brief report antibacterial activity of soil-isolated bacillus altitudinis/pumilus complex against methicillin-resistant staphylococcus aureus from mwanza, tanzania reuben n. abednego, vitus silago received: 25 jan. 2023; accepted: 15 may 2023; published: 18 july 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract antimicrobial resistance in methicillin-resistant staphylococcus aureus and beta-lactamase-producing gram-negative bacteria is a global health concern necessitating research and the development of effective antimicrobial agents. this study, conducted in may 2020 in mwanza, tanzania, aimed to determine the antibacterial activity of metabolites from soil-isolated bacillus species against clinical bacterial pathogens. one soil-isolated bacillus species, identified as bacillus altitudinis/pumilus complex, showed antibacterial activity against gram-positive cocci, including a methicillin-resistant s. aureus strain with inducible clindamycin resistance, previously isolated from a patient with osteomyelitis. bacillus altitudinis/pumilus complex metabolites may be a potential source of antimicrobial agents against multidrug-resistant bacteria. what this study adds: the study supports existing research on the discovery and development of new antimicrobial agents against multi-drug-resistant bacteria. we report the antimicrobial activity of metabolites extracted from soil-isolated bacillus altitudinis/pumilus complex strains against gram-positive bacteria, including a methicillin-resistant staphylococcus aureus strain with inducible clindamycin resistance. keywords: bacillus altitudinis/pumilus complex; gram-positive cocci; methicillin-resistant staphylococcus aureus; discovery of new antibiotic agent; soil bacteria. introduction the upsurge of multidrug-resistant (mdr) infections has led to an increase in healthcare costs and mortalities. other negative impacts of mdr infections include prolonged days of hospitalisation and lack of prophylactic protection.1,2 extended-spectrum beta-lactamase-producing gram-negative bacteria, methicillin-resistant staphylococcus aureus (mrsa) and inducible clindamycin-resistant s. aureus are common mdr bacteria reported globally.3,4,5 according to estimates, by 2050, mdr infections could cost up to $100 trillion united states dollars annually and cause approximately 10 million deaths per year.6,7 moreover, a predictive statistical modelling study published in 2022 estimated that 1.27 million deaths were directly attributable to antimicrobial resistance (amr), and 4.95 million deaths were associated with amr globally in 2019.8 these deaths were largely due to mdr strains of escherichia coli, s. aureus, and klebsiella pneumoniae.8 these projections have led to urgent calls by the world health organization to guide and promote research and development of new effective antimicrobials against mdr strains.9 since 1987 to date, no new class of antibiotics has been discovered and successfully introduced for clinical use, particularly for the treatment of infections caused by mdr bacteria such as extended-spectrum beta-lactamase-producing gram-negative bacteria, mrsa and inducible clindamycin-resistant s. aureus strains.10 currently, scientists are struggling to discover potential sources of antibiotics with activity against mdr bacteria. various potential sources have been explored, including reptile blood,11 soil microorganisms,12 marine microorganisms,13 and plants.14 certain microorganisms produce antimicrobial compounds such as antibiotics, antifungals, and antivirals as a means of gaining a competitive advantage in their environment where resources such as food are limited.15 the majority of antibiotics in clinical use today were sourced from microorganisms.16 antimicrobials such as penicillin g (sourced from penicillium notatum), vancomycin (amycolatopsis orientalis), gentamicin (micromonospora purpurea), and lincosamides (streptomyces lincolnensis) are some examples that have been derived from microorganisms.16 this study aimed to test the antibacterial activity of bacillus species isolated from soil samples in mwanza, tanzania, against medically important pathogenic bacteria, including mdr strains such as extended-spectrum beta-lactamase-producing gram-negative bacteria, mrsa, and inducible clindamycin-resistant s. aureus isolated from clinical samples. methods ethical considerations this study was approved by the joint catholic university of health and allied sciences and bugando medical centre research ethics and review committee with research clearance certificate number: crec/298/2023. the current study used bacteria species previously isolated from clinical samples at bugando medical centre and stored at –80 °c in the microbiology laboratory at catholic university of health and allied sciences in mwanza, tanzania. participants’ informed consent forms were not applicable because archived bacteria were used. moreover, patient-related information such as socio-demographic data were not used in the current study, to ensure data confidentiality. study design, duration, sampling and setting this was a laboratory-based study conducted in may 2020 in mwanza, tanzania. surface soil samples were collected in sterile falcon™ 50 ml conical tubes (corning, glendale, arizona, united states) from 20 different locations of the same site at bugando hill in mwanza, tanzania. soil samples were sent to the microbiology laboratory at the catholic university of health and allied sciences and processed within 1 h of collection. isolation of bacillus species from soil samples soil samples were serially diluted in 0.85% sterile saline as described previously17 before being inoculated onto 5% sheep blood agar (ba; himedia, mumbai, india) plates. briefly, 1 g of soil sample was suspended in 5 ml of sterile saline, which was then vortexed for 15 s and allowed to settle for 5 min. one millilitre of the resulting supernatant was mixed with 4 ml of sterile saline, vortexed for 15 s, and then allowed to settle for 5 min. this step was repeated three times to obtain five-fold serial dilutions. from the fifth dilution, 1 ml of supernatant was inoculated on a ba plate using the pour plate technique. plates were incubated aerobically at 37 °c for 20 h. presumptive bacillus species colonies (large-sized, dry, flat, greyish to whitish, and beta-haemolytic colonies) were selected and sub-cultured on fresh ba plates to obtain pure colonies. these plates were incubated aerobically at 37 °c for 20 h. gram staining was used for the preliminary identification of bacillus species before further analysis. extraction of bacterial metabolites and antibacterial activity testing a loopful (10 µl) of each presumptive bacillus isolate was suspended in 1 ml of sterile nuclease-free water in an eppendorf tube (1.5 ml safe lock microcentrifuge tube; sigma-aldrich chemie gmbh, taufkirchen, germany). the tubes were incubated at 50 °c in a heating block for 30 min with intermittent vortex-mixing for 15 s every 10 min. we selected 50 °c for the extraction of bacterial metabolites because this temperature is insufficient for the destruction of metabolites. after incubation, the tubes were centrifuged at 12 000 revolutions per minute for 10 min, after which 0.8 ml of the supernatant from each tube was transferred into sterile eppendorf tubes. the supernatants containing metabolites from soil-isolated presumptive bacillus species were used immediately for antibacterial activity determination. the well diffusion method, as described by yilmaz and colleagues,18 was used to test the antibacterial activity of the extracted metabolites against one isolate each of extended-spectrum beta-lactamase-producing and non-producing e. coli and k. pneumoniae, mrsa and methicillin-susceptible s. aureus, inducible clindamycin-resistant s. aureus, coagulase-negative staphylococci, and streptococcus pyogenes. these bacterial pathogens were isolated from clinical samples of previous studies conducted in the same setting19,20 and archived at –80 °c in 20% glycerol stocks. the recovery of these bacterial strains was performed by subculture on ba plates which were incubated at 37 °c for 24 h. bacterial suspensions of these test strains were prepared using sterile saline and adjusted to 0.5 mcfarland standard solution (remel, lenexa, kansas, united states). the bacterial suspensions were then inoculated onto the surface of mueller hinton agar (mha; himedia, mumbai, india) plates. within 15 min of inoculation, wells of 6 mm in diameter were created in the inoculated plates using a cork borer (sigma aldrich, darmstadt, germany), and 100 µl of the suspension containing the extracted metabolites was then pipetted into the bored wells. the inoculated mha plates were incubated in an upright position in ambient air at 37 °c for 20 h. the diameters of zones of inhibitions were measured in millimetres from the edge of the inhibition zone to the margin of bacillus species metabolites. the complete absence of inhibition was referred to as ‘negative antibacterial activity’ and the presence of a clear zone of inhibition was referred to as ‘positive antibacterial activity’. this experiment was performed in duplicate to ensure the validity of our results. in cases of positive antibacterial activity, an average zone of inhibition was calculated and recorded as the final result. identification of bacillus species with positive antibacterial activity only one soil-isolated presumptive bacillus species with a clear zone of inhibition (‘positive antibacterial activity’) was further identified at the species level. we first conducted biochemical identification tests, including catalase production, coagulase production, oxidase production, urease production, dnase production, indole production, citrate utilisation, lactose fermentation, hydrolysis of bile aesculin, and motility tests. we also tested the capacity of the isolates to grow on ba supplemented with 7% nacl, as well as at higher incubation temperatures of 50 °c and 70 °c. the biochemical identification tests were not conclusive. for further species identification, we also used the vitek ms (biomérieux, baden, germany) system, which is an automated microbial identification system that uses the matrix assisted laser desorption ionization time-of-flight technology. the latter was performed at the microbiology laboratory, national public health laboratory in dar es salaam, tanzania. data analysis laboratory data were documented and analysed using microsoft excel (microsoft corporation, redmond, washington, united states). categorical data were expressed as percentages. results a total of 20 soil samples were collected and processed for the isolation of bacillus species. out of the 20 soil samples processed, 16 (80.0%) showed growth of presumptive bacillus species. of the 16 presumptive bacillus species isolated, only one (6.3%) isolate – s9d – showed antibacterial activity against gram-positive bacteria only, including the mrsa strain, which was also inducible clindamycin-resistant (table 1 and figure 1). figure 1: inhibition of methicillin-resistant staphylococcus aureus by bacillus altitudinis/pumilus complex isolated from soil in mwanza, tanzania, may 2020. the zone of growth inhibition is indicated with black arrows. table 1: antibacterial activity of bacillus species isolates from soil samples against clinical bacterial isolates in mwanza, tanzania, may 2020. the presumptive bacillus species isolate with positive antibacterial activity was gram-positive and rod-shaped with central spores on microscopic examination after gram staining. the isolate was also β-haemolytic on 5% sheep ba and demonstrated catalase production, sugar fermentation, growth on ba supplemented with 7% nacl, and growth at 50 °c (table 2). using vitek ms, the isolate was identified as bacillus altitudinis/pumilus complex with a confidence level of 99.9%. table 2: biochemical characteristics of a soil-isolated bacillus altitudinis/pumilus complex isolate with positive antibacterial activity against gram-positive bacteria isolated from clinical samples, mwanza, tanzania, may 2020. discussion in this study, we report a bacillus altitudinis/pumilus complex isolate with antimicrobial activity against clinical gram-positive bacterial strains, including an mrsa strain that was also inducible clindamycin-resistant. due to the presence of several closely related species in the bacillus genus, the vitek ms system used in this study could not identify the bacillus altitudinis/pumilus complex isolate at the species level.21 the study finding is similar to a previous study in brazil in 2020,22 where marine-isolated bacillus altitudinis/pumilus was shown to possess antimicrobial activity against mdr bacterial strains. similarly, another study in thailand in 2007 also reported that a soil-isolated bacillus pumilus showed antibacterial activity against gram-positive bacteria, including mrsa and vancomycin-resistant enterococcus faecalis.23 the potential antimicrobial activity of bacillus altitudinis/pumilus complex isolates has been linked to the production of bacilysins and bacteriocins.22 the report from thailand in 2007 documented that pumilicin 4 is a bacteriocin produced by a bacillus pumilus strain and had bactericidal activity against mrsa strains.23 we observed no antibacterial activity against gram-negative bacteria, indicating that bacillus altitudinis/pumilus complex may only be effective against gram-positive bacteria. similar findings were reported in brazil in 2021 where a bacillus altitudinis isolate from wetland sediment showed no antibacterial activity against gram-negative bacteria.21 the presence of the outer membrane in gram-negative bacteria limits cellular permeability,24 and may thus result in poor diffusion of antimicrobial metabolites into the cytoplasmic membrane for potential inhibition of bacterial growth.25 limitations due to limited resources and funds, we were not able to identify the presumptive bacillus species with negative antibacterial activity beyond the genus level. we also could not isolate, purify, or characterise the active metabolites from the bacillus altitudinis/pumilus complex isolate with positive antibacterial activity. conclusion our findings suggest that metabolites from soil-isolated bacillus altitudinis/pumilus complex can be a potential source of antimicrobial agents with activity against gram-positive bacteria, including mrsa and inducible clindamycin-resistant strains. acknowledgements authors would like to express their sincerely appreciation to the microbiology laboratory at catholic university of health and allied sciences in mwanza, tanzania, and microbiology laboratory at national public health laboratory in dar es salaam, tanzania. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions v.s. conceptualised the article. both v.s. and r.n.a. performed sample collection, laboratory procedures, and prepared the article. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability all data presented in this article are 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wapb4. curr microbiol. 2007;55(4):308–313. https://doi.org/10.1007/s00284-006-0632-2 delcour ah. outer membrane permeability and antibiotic resistance. biochim biophys acta. 2009;1794(5):808–816. https://doi.org/10.1016/j.bbapap.2008.11.005 garcia-gutierrez e, mayer mj, cotter pd, narbad a. gut microbiota as a source of novel antimicrobials. gut microbes. 2019;10(1):1–21. https://doi.org/10.1080/19490976.2018.1455790 introduction conception and development promises and prospects covid-19: a novel challenge recent developments the african ‘single-cell data gap’ predictions and outlook acknowledgements references about the author(s) enahoro s. abhulimen college of medicine, university of ibadan, ibadan, nigeria citation abhulimen es. the human cell atlas: promises, recent developments, and bridging the african single-cell data gap. afr j lab med. 2024;13(1), a2583. https://doi.org/10.4102/ajlm.v13i1.2583 opinion paper the human cell atlas: promises, recent developments, and bridging the african single-cell data gap enahoro s. abhulimen received: 24 july 2024; accepted: 25 oct. 2024; published: 19 dec. 2024 copyright: © 2024. the author licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction the human genome project changed everything. the 13-year-long campaign to create a reference human genome finally ended with the announcement of an essentially complete draft of the sequence in 2003. advances in dna sequencing technology, such as the development of automated sanger sequencing machines like the abi prism range manufactured by applied biosystems, paved the way for the realisation of the mega project 2 years ahead of schedule.1 the creation of ambitious initiatives such as this is constrained by available technology; therefore, evolving genomic technologies have led to the development of several other large-scale genomics initiatives. conception and development the human genome project produced a reference genome sequence, which is identical in nearly all of the 37 trillion cells in the body. however, there is a wide diversity of cell types in the body, which arises from differences in the activated genes within these cell phenotypes. recent revolutions in genomics, such as developments in single-cell rna sequencing (scrna-seq) technologies, for example the introduction of in situ barcoding, have enabled the evaluation of gene expression at cellular resolution for hundreds of thousands of cells in parallel.2 additionally, advancements in spatial transcriptomics methods now allow for the mapping of gene expression, while retaining spatial information about the distribution of cells within tissue samples.2 coupled with modern computational biology methods necessary for analysing this data, these developments have made it possible to create a reference atlas of all the cell types in the human body, which would mark the completion of the 150-year-old effort to categorise these cell types. these advances have led to the conception of a human cell atlas, as they made realising the mammoth undertaking both feasible and economically viable. therefore, in 2016, sarah teichmann (wellcome sanger institute, cambridge, united kingdom) and aviv regev (broad institute, massachusetts institute of technology, cambridge, massachusetts, united states) co-founded the human cell atlas (hca), an open, large-scale, scientist-led consortium aimed at mapping out in detail the cellular architecture of the human body.2,3 global researchers convened for an inaugural planning meeting in london in 2016, and since then, membership of the consortium has grown to more than 3000 members from over 99 countries, spread across 18 biological networks.3 these biological networks were created for organisational efficiency, with each network composed of scientific experts in various systems and organs, working together from around the world to map out these systems. according to the hca’s white paper,4 the first draft of the atlas is expected to contain 30–100 million cells from all major human organs and systems. the plan to structure this ambitious project into shorter-term, achievable intermediate goals reflects insights from the past, mirroring the approach adopted during the human genome project, which led to the completion of the project years ahead of expected projections.5 the consortium has now mapped tens of millions of individual cells across the 18 biological networks, encompassing male and female individuals of varied ethnic backgrounds, and published over 190 scientific papers revealing new insights into human biology and disease.3 the data from these networks are stored in the hca data portal,6 a publicly accessible cloud-based platform, enabling scientists from around the world to access all the collected single-cell data. the complete atlas is expected to map at least 10 billion cells4 across all human tissues, organs, and systems, including developmental cells and cells from individuals with various diseases, with the projected time for its completion spanning another estimated 5–10 years. promises and prospects the hca promises to transform our understanding of human biology, thereby revolutionising how we treat diseases. the applications of a completed atlas range from advancements in our diagnostic capabilities to understanding drug efficacy and toxicity, as well as facilitating drug target discovery and advancements in regenerative medicine. a proposed diagnostic application of the atlas is the creation of essentially a ‘complete blood count (cbc) 2.0’, which is a more complex characterisation of the blood cells in the routine test. this involves genomic profiling of the various blood cell components giving a better understanding of the experiences the cells have undergone and their associations with various diseases, such as autoimmune diseases and malignancies.4,7 for histology, this technology aims to transform primitive haematoxylin and eosin (h&e) staining by integrating genomic profiling and spatial technology, thereby creating an ‘h&e 2.0’ approach that offers a molecular view of tissues.7 another promise of the complete atlas is improved knowledge of why drugs may or may not be functional or predict off-target locations where they may express their effects, by studying gene expression similarities between healthy and diseased cells. also, its application in regenerative medicine provides the opportunity to create precise organoid models and in vitro cells, which could enable targeted cell therapies.4 eukaryotic cells are already being synthesised, such as in the synthetic yeast genome project (sc2.0),8 indicating the potential for the creation of synthetic human cells that could be used to study disease models or for therapeutic purposes. the hca data could therefore be leveraged to design more accurate human cells, thereby revolutionising the emerging field of synthetic genomics. these promises may seem ambitious, but the prospect of even a few of these innovations materialising would create a paradigm shift, effectively transforming healthcare as we know it. notwithstanding, one would have to consider the practicability of these potential applications of the atlas data. for example, the promise of a ‘cbc 2.0’ and ‘h&e 2.0’ approach would be exciting to a researcher, but full adoption of these technologies may take several years, as the current technology is still adequately functional, and there might be clinician hesitation to pivot from existing reliable technology. also, the affordability or availability of the required technology to carry out these new tests in lowand middle-income countries, where there is still limited access to current technology, are other factors that may limit widespread adoption. furthermore, concerning the applications of the hca in synthetic genomics, the hca data may not be directly responsible for the revolution in currently available technology as improvements in our genetic engineering capabilities are still required to create major advancements in this field.7 however, as the era of personalised medicine approaches, the atlas could be used to make the therapeutic applications of synthetic genomics more precise. covid-19: a novel challenge in 2019, the emergence of the coronavirus disease 2019 (covid-19) pandemic allowed for new and unprecedented applications of the hca data. the lung biological network, which is focused on creating a complete atlas of the human lung and, eventually, the broader hca community, mobilised efforts to tackle this disease, subsequently resulting in the creation of a covid-19 cell atlas. initially, efforts were aimed at using the available hca data to understand the underlying biology of this disease, with subsequent studies using data from samples of covid-19 patients. studies from the consortium compared atlas data from healthy tissues with data from samples taken from postmortem tissue of patients with the disease, leading to the discovery of viral targets, and isolation of genes related to severe covid-19 infections.7 the ace2 receptors and tmprss2 protease were found to be the major receptors facilitating viral entry. research showed that the ace2 receptor binding affinity was found to influence viral replication rates and the severity of the disease.9 thus, expression of the ace2 and tmprss2 genes across various tissues was investigated using scrna-seq data sets from the hca. both genes were expressed in a wide variety of tissues, with the tmprss2 gene having a wider distribution among the tissues sampled, suggesting that the expression of the ace2 gene was a rate-limiting factor for viral infection. the study also found the highest expression rate of these genes in nasal ciliated and goblet cells.9 these findings provided insight into the most likely sites of entry and reservoir for the virus, a discovery that was crucial in shaping mask-wearing policy by helping us understand the routes of viral transmission in the early days of the pandemic. furthermore, analysis of scrna-seq data of immune cells in individuals who received the covid-19 vaccines helped researchers to better analyse the efficacy of these new vaccines.7 the adaptability displayed by the hca community, alongside the wide range of practical implementations of the atlas data during the pandemic, illustrates the potential for the diverse applications of the hca. this is especially impressive considering that the initiative was barely 3 years old at the start of the pandemic, leading us to wonder about the possible capabilities of a completed atlas. recent developments several studies published by the hca have transformed our understanding of human biology and disease. for instance, the human development biological network used scrna-seq and spatial transcriptomics to conduct a spatial analysis of gene expression patterns in human limb development across different gestational ages. this study10 has corrected previously conceived expectations about the pattern of growth of the limb digits, and also highlighted distinct anatomical separation between genes associated with brachydactyly and polysyndactyly, providing new insight into the development of these conditions. the various biological networks of the hca have also been involved in creating roadmaps that outline plans to ensure diversity in the samples collected, identify potential obstacles, and establish goals for creating a draft atlas of their respective systems. a few of these roadmaps have already been published for the skin, gut, lung, human development, and oral and craniofacial cell atlas. with the hca data sets rapidly expanding, these networks have pivoted to creating integrated atlases, such as the development of the first integrated single-cell atlas of the human lung. the integrated human lung cell atlas consists of 2.4 million cells from 49 data sets consisting of healthy lung data and data from more than 10 lung diseases, which were re-annotated to create a consistent cell type reference.11 the results obtained from merging these data sets have already begun to expand our understanding of various lung disease states. for example, the discovery of similar gene activity in profibrotic spp1+ monocyte-derived macrophages in lung carcinoma, covid-19, and idiopathic pulmonary fibrosis has led to the discovery of potential therapeutic targets for these conditions.11 the development of integrated atlases by the different biological networks will represent a defining milestone in the consortium’s effort, as the eventual goal is the creation of essentially an integrated atlas of the whole human body. therefore, it is expected that we will see several more of these atlas integrations in the near future. the african ‘single-cell data gap’ the hca is similar to the human genome project in many ways, as they are both ambitious large-scale initiatives, with the latter inspiring the conception of the former.5 however, a major difference between the two initiatives is that while the goal of the human genome project was to create a single dna sequence, the hca aims to create a data set of diverse cell types. this necessitates adequate representation in terms of age, gender, ethnicity, geographical location, and disease states. regardless, 65% of the data from the integrated human lung cell atlas belonged to individuals of european ethnicity,11 indicating a need for increased representation in the biospecimens obtained by the consortium. there is already a general underrepresentation of minorities in single-cell data and the hca may further widen this disparity, leading to comparatively worse healthcare outcomes within these populations.12 overcoming this challenge will therefore be an important determinant of the hca’s eventual success. the disproportionate representation of minority populations, such as africans, in the hca data seems to follow a similar trend observed in most current large-scale genomic studies. for example, african data constituted 1.1% of the world’s genome-wide association studies data in 2021, despite africa making up about 17% of the world’s population.13 the hca’s equity working group has devised strategies to ensure equity in its efforts, such as increasing global participation and collaboration in the initiative, especially in lowand middle-income countries, where adequate knowledge about or facilities for scrna-seq might be scarce.14 therefore, efforts must be made to prevent further widening of, or to bridge, any existing ‘single-cell data gaps’ that may have developed due to a lack of participation by african institutions. several organisations, such as the african centre of excellence for genomics of infectious diseases, 54gene, inqaba biotec, and the human heredity and health in africa consortium, are already working to bridge africa’s genomic data gap, and could be partnered with to advance the hca on the continent. another potential approach could involve prioritising obtaining samples from local ethnic minorities by member institutes, while also making efforts to secure both local and international funding for infrastructure development in lowand middle-income countries, such as those within the african continent. predictions and outlook nevertheless, from the author’s perspective, the initiative has already started to deliver on its promises, considering the numerous insights it has made into our understanding of human biology10,11 and diseases,7,9,11 even though it is still at least 5–10 years from its completion. however, some of the initial promises of the atlas, such as a ‘cbc 2.0’ or ‘h&e 2.0’, may require several years for development and widespread adoption. therefore, it is anticipated that the true promise of the atlas lies in its adaptability and all the unforeseen, practical ways in which we will come to use it, just as we did during the covid-19 pandemic. it is also expected that over time, as genomic technologies continue to advance, the initiative may evolve to map cells with even greater resolution while observing interactions between individual cells at the genomic level. this could involve the use of multi-omics-based technologies to observe heterogeneity among cell populations we currently believe to be homogeneous, further enhancing the potential of the completed atlas. acknowledgements competing interests the author declares that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. author’s contributions e.s.a. declares that they are the sole author of this research article. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the author and are the product of professional research. this article does not necessarily reflect the official policy or position of any affiliated institution, funder, agency, or that of the publisher. the author is responsible for this article’s results, findings, and content. references heather jm, chain b. the sequence of sequencers: the history of sequencing dna. genomics. 2015;107(1):1–8. https://doi.org/10.1016/j.ygeno.2015.11.003 aldridge s, teichmann sa. single cell transcriptomics comes of age. nat commun. 2020;11:1–4. https://doi.org/10.1038/s41467-020-18158-5 about the human cell atlas [homepage on the internet]. 2024 [cited 2024 apr 02]. available from: 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